Synthetic analogs of stryphnusin isolated from the marine sponge: Stryphnus fortis inhibit acetylcholinesterase with no effect on muscle function or neuromuscular transmission

Article abstract of DOI:10.1039/c6ob02120d

The marine secondary metabolite stryphnusin (1) was isolated from the boreal sponge Stryphnus fortis, collected off the Norwegian coast. Given its resemblance to other natural acetylcholinesterase antagonists, it was evaluated against electric eel acetylcholinesterase and displayed inhibitory activity. A library of twelve synthetic phenethylamine analogs, 2a-7a and 2b-7b, containing tertiary and quaternary amines respectively were synthesized to investigate the individual structural contributions to the activity. Compound 7b was the strongest competitive inhibitor of both acetylcholinesterase and butyrylcholinesterase with IC₅₀ values of 57 and 20 μM, respectively. This inhibitory activity is one order of magnitude higher than the positive control physostigmine, and is comparable with several other marine acetylcholinesterase inhibitors. The physiological effect of compound 7b on muscle function and neuromuscular transmission was studied and revealed a selective mode of action at the investigated concentration. This data is of importance as the interference of therapeutic acetylcholinesterase inhibitors with neuromuscular transmission can be problematic and lead to unwanted side effects. The current findings also provide additional insights into the structure-activity relationship of both natural and synthetic acetylcholinesterase inhibitors.

Full text of DOI:10.1039/c6ob02120d

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Synthetic analogs of stryphnusin isolated from the marine sponge
Stryphnus fortis inhibit acetylcholinesterase with no effect on
muscle function or neuromuscular transmission
Lindon W. K. Moodiea, Monika C. Žužekb, Robert Frangežb Jeanette H. Andersenc, Espen
Hansenc, Elisabeth K. Olsenc, Marija Cergoljd,e, Kristina Sepčićd, Kine Ø. Hansenc,*, Johan
Svenson a,f,*
The marine secondary metabolite stryphnusin (1) was isolated from the boreal sponge
Stryphnus fortis, collected off the Norwegian coast. Given its resemblance to other natural
acetylcholinesterase antagonists, it was evaluated against electric eel acetylcholinesterase
and displayed inhibitory activity. A library of twelve synthetic phenethylamine analogs, 2a-7a
and 2b-7b, containing tertiary and quaternary amines respectively were synthesized to
investigate the individual structural contributions to the activity. Compound 7b was the
strongest competitive inhibitor of both acetylcholinesterase and butyrylcholinesterase with
IC50 values of 57 and 20 µM, respectively. This inhibitory activity is one order of magnitude
higher than the positive control physostigmine, and is comparable with several other marine
acetylcholinesterase inhibitors. The physiological effect of compound 7b on muscle function
and neuromuscular transmission was studied and revealed a selective mode of action at the
investigated concentration. This data is of importance as the interference of therapeutic
acetylcholinesterase inhibitors with neuromuscular transmission can be problematic and
lead to unwanted side effects. The current findings also provide additional insights into the
structure-activity relationship of both natural and synthetic acetylcholinesterase inhibitors.
aDepartment of Chemistry, UiT The Arctic University of Norway, Breivika, N-9037, Tromsø,
Norway. Present address for LWKM: Department of Chemistry, University of Umeå, SE-901
87, Umeå, Sweden
bInstitute of Preclinical Sciences, Veterinary faculty, University of Ljubljana, Ljubljana,
Slovenia
cMarbio, UiT The Arctic University of Norway, Breivika, N-9037, Tromsø, Norway
1

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dDepartment of Biology, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia
eDepartment of Biotechnology, University of Rijeka, Rijeka, Croatia
fDepartment of Chemistry, Materials and Surfaces SP Technical Research Institute of Sweden,
Box 857, SE-501 15 Borås, Sweden
*Authors to whom correspondence should be addressed: Johan Svenson: email:
johan.svenson@sp.se; and Kine Østnes-Hansen: email: kine.o.hanssen@uit.no
†Electronic Supplementary Information (ESI) available: 1H and 13C NMR spectra for synthetic
compounds not previously reported and additional neuromuscular experiments.
Keywords: Acetylcholinesterase inhibitor, Stryphnus fortis, marine natural products,
neuromuscular transmission, Alzheimer’s disease
Introduction
The diverse molecular scaffolds displayed by natural products hold great promise for drug
development and have already inspired a number of clinically used drugs1, 2. Historically,
most compounds have been isolated from terrestrial sources but, as a result of technological
advancements, the last 50 years have seen a rise in the number of isolated marine natural
products, with some 25000 reported in the scientific literature2, 3. Approximately 500 new
marine natural products are reported each year4. To date, the Food and Drug Administration
(FDA) has approved seven drugs of marine origin which illustrates their potential5. Natural
products generally expand into a broader chemical realm than the synthetic libraries
screened by the pharmaceutical industry6 and screening success has been shown to increase
when including natural-product-like scaffolds7.
Nearly half of the new marine natural products reported are isolated from the Porifera
(sponges) taxon which is attributed to a high content of both opportunistic and symbiotic
microorganisms4, 8-10. Marine microorganisms are the source of many highly potent natural
products which include approved drugs and compounds in clinical trials1, 5. The marine
microbes are particularly challenging to cultivate and therefore the collection of larger
marine benthic organisms remains highly warranted for the continued discovery of novel
compounds of microbial origin.
2

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Our recent studies of Arctic marine organisms have led to the characterization of a range of
acetylcholinesterase (AChE) inhibitors11. During these investigations, we have reported the
isolation and AChE-inhibitory properties of the halogenated tyrosine derivatives pulmonarin
A and B, isolated from the ascidian Synoicum pulmonaria12, 13. These small, dibrominated
compounds displayed AChE inhibition in the pharmaceutically relevant range (Ki = 90 and 20
µM respectively) and represent interesting marine hits for further studies13.
Four brominated indole derivatives have also recently been isolated from the boreal sponge
Geodia barretti. A library of 22 synthetic compounds was synthesized in order to establish
the structure-activity relationship (SAR) against AChE and the role of indole bromination14.
The most potent natural compounds from that study were the 2,5-diketopiperazines
barettin and 8,9-dihydrobarettin which displayed significant inhibition of AChE, (inhibition
constants of 29 and 19 µM respectively) and butyrylcholinesterase (BChE; inhibition
constants of 14 and 48 µM respectively) via a reversible noncompetitive mechanism14.
Our previously isolated compounds bear resemblance to other natural AChE inhibitors such
as bufotenine, isolated from the mushroom Amanita mappa15 and from a range of frog skin
excretions16, and deformylflustrabromine originally isolated from the North sea bryozoan
Flustra foliacea17, 18. In addition, similar marine compounds, such as 6-bromohypaphorine
has been show to display agonistic effects on nicotinic acetylcholine receptors19
The chronic neurodegenerative condition known as Alzheimer’s disease (AD) is characterized
by progressive degeneration of cholinergic neurons and is the most common cause of
dementia20. AChE (E.C 3.1.1.7) is the key enzyme for termination of neurotransmission in
3

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cholinergic pathways via the rapid hydrolysis of the neurotransmitter acetylcholine following
its presynaptic release21, 22. Therefore, AChE inhibition is a promising approach for
symptomatic treatment of AD23. Recent studies also indicate that AChE inhibitor binding to
the peripheral anionic site of AChE can be beneficial for the inhibition of the amyloid cascade
and offer protection of neural cells against free radical induced damage24. In addition,
patients diagnosed with AD show a progressive increase in the activity of the related
cholinesterase enzyme, BChE (E.C. 3.1.1.8). This enzyme is found mainly in the blood
plasma25, and serves as a ‘‘back-up’’ when AChE activity is compromised or absent26. Both
enzymes represent relevant therapeutic targets for ameliorating the symptoms of the AD.
The FDA and European Medicines Agency have approved three compounds addressing the
cognitive impairment of AD patients: donepezil, rivastigmine and galanthamine21. The latter
two drugs are strongly affiliated with natural products chemistry. Rivastigmine was
developed from physostigmine, an alkaloid naturally occurring in the Calabar bean27 while
galanthamine was isolated from the bulb of Galanthus woronowii28, 29. All three compounds
inhibit AChE in a reversible manner, and interact directly with the active site or adjacent
binding pockets21. Several marine natural products have been shown to display neurological
activities although they have yet to reach the market30. The current state of neurologically
active marine natural products was recently reviewed by Sakai and Swanson31.
In the present report, we describe the isolation and evaluation of stryphnusin (1), a
brominated marine phenethylamine derivative isolated from the Arctic sponge Stryphnus
fortis (Vosmaer 1885). S. fortis is a large, smooth sponge which is found in dense colonies in
the northern Atlantic Ocean and is common to the Norwegian coast. S. fortis is known for
containing the bioactive secondary metabolite ianthelline which displays both antifouling
and cytotoxic bioactivities32, 33. However, the actual primary producer of ianthelline was
recently suggested to be the Hexadella dedritifera sponge which commonly grows on S.
fortis34. No attempts were made to search for H. dedritifera in the current S. fortis material.
The current study represents an extension of our continued search for novel cholinesterase
inhibitors of marine origin. Compound
1 is structurally related to the marine AChE inhibitors
isolated from S. pulmonaria and G. baretti and was evaluated for its ability to inhibit AChE.
Based on the initial observed inhibitory activity of
4
1 against electric eel AChE, a library of

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simplified synthetic analogs were prepared and evaluated. Although the structure of 1 was
originally reported in 2000 after isolation from the Caribbean sponge Verongula gigantea35,
and again in 2010, from the Mediterranean phlebobranchiate ascidian Ciona edwardsii36,
only limited bioactivity data has been reported. The effect on BChE, and the physiological
effect on neuromuscular transmission and muscle function were evaluated for the most
active synthetic analog. The data reported expands the knowledge of marine cholinesterase
inhibitors and represents the first study of their effect on muscle function.
Results and Discussion
The monobrominated 1 was found in organic phase of the S. fortis extract and was isolated
using mass guided preparative HPLC. The compound was identified based on spectroscopic
analysis35. Compound 1 was evaluated as inhibitor of electric eel AChE and was found to
exhibit a moderate inhibitory activity (Table 1.). Based on the initially observed activity, and
in order to supplement our previously obtained structure activity relationship (SAR) data, a
range of synthetic analogs were prepared and tested, affording six tertiary (2a-7a) and six
quaternary amines (2b-7b) (Scheme 1). The degree and position of phenyl ring bromine,
hydroxyl and methoxy substituents was incorporated by consideration of the appropriate
phenethylamine starting materials. The tertiary amines 2a-7a were prepared via reductive
amination, and further alkylation by methyl iodide yielded the corresponding quaternary
amines 2b-7b. The starting materials for compounds 6a37 and 7a38 were prepared using
reported methods.
Scheme 1. The synthesis of stryphnusin analogs 2a-7a and 2b-7b.
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The kinetics of the in vitro inhibition of electric eel AChE were assessed by employing the
colorimetric assay developed by Ellman39 and the data is presented in Table 1.
Table 1. Inhibition of electric eel acetylcholinesterase and horse serum butyrylcholinesterase by natural
compounds and their synthetic analogs (1-7b).
Compound
IC50 (µM)a Ki (µM)
AChE
1
232
1675
1513
1395
968
774
163
1096
1387
1287
293
235
n.d.
n.d.
n.d.
n.db
n.d.
202
n.d.
n.d.
n.d.
n.d.
n.d.
51
2a
3a
4a
5a
6a
7a
2b
3b
4b
5b
6b
7b
444
57
physostigmine
pulmonarin Ac
pulmonarin Bc
6-bromoconicaminc
barettinc
3
150
36
230
36
4
90
20
90
29
BChE
7b
6-bromoconicaminc
barettinc
20
14
26
n.d.
11
14
a
IC50 is determined as the concentration of the compound inducing 50%
inhibition of the enzyme activity
bKi not determined for compounds displaying an IC50 > 250 µM
cData taken from ref 13 and14
The in vitro AChE inhibition of 1 was modest with an IC50 of 232 µM (Figure 1.), which is
similar to the recently reported 6-bromoconicamin. The prepared synthetic analogs
generally demonstrated weaker inhibition with the exception of 7a and 7b, which displayed
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IC50s of 163 and 57 µM respectively. Compound 7b was also evaluated as an inhibitor of
BChE and displayed an IC50 value of 20 µM as shown in Figure 1.
Figure 1. Inhibition of electric eel acetylcholinesterase (solid symbols) and horse serum butyrylcholinesterase
(open symbols) by compounds 1 (triangles) and 7b (circles). The IC50 values towards AChE was determined to
be 232 μM for 1 and 57 μM for 7b, and the IC50 value for 7b towards BChE was determined to be 20 μM.
For those compounds displaying IC50s < 250 µM the inhibitory constants Ki were also
determined using Dixon plot analysis, as shown in Figure 2 for 1 and 7b. All the examined
compounds were shown to be reversible competitive AChE inhibitors, suggesting their
binding to the active site of the free enzyme.
Figure 2. Determination of electric eel AChE inhibition type and the inhibition constants (Ki) for 1 (left graph),
and 7b (right graph) by Dixon plot analysis. The concentrations of the substrate acetylthiocholine were 0.125
(●), 0.25 (□), and 0.50 mM (■). Ki towards AChE was determined to be 235 μM for 1 and 51 μM for 7b.
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Compound 7b was the most active competitive inhibitor with a Ki of 51 µM in our assays.
This was superior to pulmonarin A and 6-bromoconicamin and comparable to pulmonarin B
and barettin13, 14. When compared to the natural product 1, it appears that the additional
bromination and phenolic methylation are sufficient to increase the inhibitory activity. The
reported Ki of the FDA approved AChE inhibitor galanthamine ranges from 2-10 µM28 and
there is generally a wide concentration range in which AChE inhibitors effectively exerts their
mode of action40. AChE inhibitors also often yield different affinities depending on the
enzyme source and experimental setup. The positive control in our study, physostigmine
displayed an IC50 of 3 µM which is relatively high yet comparable to other reported IC50
values against electric eel AChE (0.028–6.45 μM)41, 42. That implies that the inhibitory
activities observed for 7b is near the pharmaceutically relevant concentration range.
While most of the synthetic analogs were not active enough to motivate their detailed Ki
analysis, the link between degree of substitution and the inhibitory potency of the
compounds was still evident. The quaternary amines also generally displayed a higher
inhibitory activity in relation to their tertiary structural counterparts. By dissecting the
molecules into individual chemical constituents it was possible to assess both the charged
contribution as well as the Connolly solvent excluded volume of the substituted ethylphenyl
part of the molecules as presented in Table 2 and Figure 3.
Table 2. Correlation between hydrophobic volume, amine substitution and the AChE inhibitory activity of
compounds 1-7b.
Solvent excluded
volume (Å3)a
Log Pa
IC50 tertiary
(”a”) (µM)
IC50 quaternary
Compound
(”b”) (µM)
1
2
3
4
5
6
7
130.2
104.6
111.2
126.5
141.7
150.2
167.6
3.38
2.94
2.81
2.55
3.64
4.21
4.47
n.a.
1675
1513
1395
968
232
1096
1387
1287
293
774
163
444
57
aCalculated using ChemBio3D Ultra 14.0 disregarding the substituted and ionized nitrogen atom, hence only the
contribution from the substituted ethylphenyl moiety.
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Figure 3. Correlation plot illustration the influence of the hydrophobic volume on the inhibitory effect of the
different types of analogs. -×- are the tertiary amines, 2a-7a and -•-represent the quaternary amines 1, 2b-7b.
The correlation between the bulk of the molecules and their inhibitory activity is clear and
this trend is also evident when examining the influence of log P on activity. The quaternary
amines generally displayed higher inhibitory activities in comparison to their tertiary amine
counterparts. Although all molecules of the current study can be regarded as basic in a
physiological context, it is obvious that the constant positive charge of the quaternary
compounds 1 and 2b-7b is beneficial. This is not surprising given that this functionality is
chemically analogous with the natural substrate of AChE43. Isolated 1 and its methoxy
analogue 5b, demonstrate similar activities (232 and 293 µM, respectively), suggesting that
these compounds do not engage in any crucial hydrogen bond formation with groups within
the active site. It is of interest though, that the dibromo methoxy 7b (57 µM) is significantly
more active than its phenolic counterpart 6b (444 µM). Considering the data as a whole, we
propose that compounds containing large, hydrophobic substituents on the phenyl group, in
addition to the quaternary amine, display the most effective inhibitory behavior. These
findings contrast our recent study of bromotryptamines where no obvious trend between
the hydrophobicity and AChE inhibition was seen14.
The cytotoxicity of the compounds was also evaluated employing human MRC- 5 fibroblast.
None the included compounds displayed any significant toxic effects at concentrations up to
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150 µM (data not included). Compound 1 has previously been tested against rat PC 12 cells
and shown to display no toxicity at 10 µg/mL36. The use of AChE inhibitors can have several
drawbacks including unwanted muscle contraction and neuromuscular transmission44, 45
.
Given that 7b was the most potent molecule from our initial studies, an extensive
physiological evaluation of its effects on muscle contraction employing isolated mouse
hemidiaphragm was conducted.
The effects of 7b at the concentration which significantly reduced the AChE activity by 20%
(20 µM) on both nerve evoked and directly elicited single twitch and tetanic contractions in
isolated mouse hemidiaphragm preparation were studied. AChE inhibition in hippocampus
CA1, CA3 and striatum produced by ethanol extract from Ptychopetalum olacoides was
shown to be 33%, 20% and 17%, respectively, and these levels of inhibition significantly
improved cognitive abilities in old rats46. In line with this, we performed the experiments on
muscle function and neuromuscular transmission employing a 20 µM concentration.
Reversible AChE inhibitor neostigmine methylsulfate (3 µM) (Sigma–Aldrich, USA) was
employed as a positive control. At this concentration neostigmine inhibits AChE in mouse
diaphragm muscle by 96%47. In the muscle contraction experiment neostigmine induced
characteristic facilitation of neuromuscular transmission associated with anticholinesterase
treatment followed by a decrease in indirectly elicited muscle twitches. Neostigmine
produced the complete block of tetanic contractions evoked by repetitive nerve stimulation.
7b appeared to have no effect on directly and indirectly evoked muscle twitch amplitude and
the amplitude of directly and indirectly evoked tetanic muscle contraction (Figures 4 and 5).
The potential extended effects of 7b on indirectly evoked muscle twitch amplitude were also
investigated by incubating 7b for 60 min with the mouse hemidiaphragm preparation (Figure
6). Compound 7b behaved in a similar fashion to the negative control (methanol) in the
time-course study and appeared to have little effect on muscle contraction, an
advantageous property for AChE drugs.
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Figure 4. Effects of 7b on contractions in isolated mouse hemidiaphragm preparation. (A) Representative
control tracing. Arrow (MeOH) indicates superfusion of methanol solution in 0.35 v/v% final concentration. (B)
‘Positive control’ with AChE inhibitor neostigmine (3 µM). (C) 7b (20 µM). N — denotes nerve evoked muscle
contraction; D — denotes directly elicited muscle contraction; Tn — denotes nerve evoked tetanic contraction;
Td — denotes directly elicited tetanic contraction; W — wash out.
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Figure 5. The time-course effects of 7b on indirectly evoked muscle twitch of isolated mouse hemidiaphragm
preparation. Each point represents the mean value ± SEM obtained from 2–3 different nerve muscle
preparations. Graphs including the positive control neostigmine can be found in the Supporting information.
Figure 6. Effects of 7b on both, nerve evoked (A) and directly elicited tetanic (B) contractions of isolated mouse
hemidiaphragm preparation. Note that 7b (20 µM) have no effect on the amplitude of directly and indirectly
evoked tetanic muscle contraction. Graphs including the positive control neostigmine can be found in the
Supporting information.
Inhibition of AChE in the neuromuscular junction is associated with the inability to sustain a
tetanic contraction produced by the repetitive high frequency stimulation of the motor
nerve48. The effect of 7b on the maximal amplitude of nerve evoked tetanic contraction was
thus also established. 7b did not influence the tetanic contractions and tetanic fade was only
seen for the positive control neostigmine in our studies (Figure S11 in supporting
information).
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Finally the depolarization effect of 7b on the skeletal muscle end plate potentials (EPPs) was
investigated. For the EPPs experiments the mouse hemidiaphragm preparations were
pretreated for 30 min with a 2 µM solution of conotoxin GIIIB, and all experiments were
further performed in the presence of 2 µM conotoxin GIIIB to record full sized EPPs and to
prevent muscle twitches. Compound 7b displayed no prominent activity towards the EPPs
and induced no changes in evoked neurotransmitter release (A) or EPPs half decay (B) after
both 30 and 60 minutes exposure of the neuromuscular preparation to the compound
(Figure 7).
Figure 7. Effects of 7b on EPPs amplitude and EPPs half decay. Mouse hemidiaphragm preparations were
pretreated for 30 min with 2 µM conotoxin GIIIB, and all experiments were performed in the presence of 2 µM
conotoxin GIIIB to record full sized EPPs and to prevent muscle twitches. Each point represents the mean value
± SEM obtained from 8-12 muscle fibers of each from 2–3 different nerve muscle preparations. Evoked
neurotransmitter release (A) and EPPs half decay (B) were determined after 30 and 60 min.
From the neuromuscular data we can conclude that 7b, our most potent analog of
stryphnusin, inhibits AChE without also inflicting unwanted collateral physiological responses
in the neuromuscular system. Of the prepared compounds, only compound 7b was
investigated due to its relatively high inhibitory activity and its structural similarity with both
natural and synthetic phenethylamine analogs. Intrigued by this selectivity, and to further
investigate the generality of these findings, an analogous synthetic compound, a
monobrominated tryptamine from our recent study (compound “9a” in ref14) was also
included14. The brominated tryptamine behaved in a very similar fashion (see supporting
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information Figures S8-S13 for comparison with 7b and neostigmine) in the neuromuscular
experiments. This illustrates that both these types of compounds exert their AChE inhibition
without side effects on muscular transmission. To the best our knowledge, this is the first
reported neuromuscular investigation of these types of small, halogenated AChE inhibitors.
Several natural AChE inhibitors such as the bufotenins and related compounds display a
similar size, degree of substitution and distribution of functionalities and these results
illustrate that this structural motif can be used to generate small selective reversible AChE
inhibitors.
Conclusions
The marine natural product stryphnusin (1) was isolated from an organic extract of the
marine sponge S. fortis. 1 shares structural features with known AChE inhibitors and the
natural substrate and was therefore evaluated for inhibitory activity against electric eel
AChE, displaying moderate inhibitory properties. In order to identify analogues with greater
activity and to develop a pharmacophore model, a library of 12 compounds was synthesized.
The majority of the synthetic compounds were less active than the natural product but 7b,
which contained an additional bromine and methyl functionality, displayed inhibitory
properties comparable to several other marine AChE inhibitors. SAR analysis of the library
highlighted that both phenyl ring substituents that contribute to steric bulk and
hydrophobicity, and that analogues bearing a quaternary amine improved activity, thus
providing new insights into the structure-activity relationship of AChE inhibitors. Our most
promising compound, 7b, and a structurally related tryptamine, were employed in
neuromuscular transmission studies and showed no significant effect; a desirable property
when developing therapeutic AChE inhibitors.
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Experimental
General Experimental Procedures
The preparatory HPLC system used to isolate 1 consisted of a 600 pump, a 2996 Photodiode
Array UV detector, a 3100 Mass Detector, and a 2767 sample manager (Waters, Milford, MA,
USA). NMR spectra were acquired on either a Varian VNMRS 600 MHz or a Varian 7000e 400
MHz spectrometer. Carbon resonances were either acquired directly or derived from gHMBC
experiments and the chemical shifts were referenced to the residual solvent peaks. HRMS
was recorded on an LTQ Orbitrap XL Hybrid Fourier Transform mass spectrometer from
Thermo Scientific and the Thermo Scientific Accela HPLC-LTQ Ion Trap-Orbitrap Discovery
system was used to determine accurate mass of the synthetic compounds. Infrared spectra
were recorded on an Avatar 320 FT-IR spectrometer from Nicolet. Solvents, reagents and
compound 2a were acquired from commercial sources and used without further purification.
The starting materials for compounds 6a37 and 7a38 were prepared using reported methods.
Spectroscopic data is included for novel compounds, or those lacking characterization in the
literature. For the neuromuscular investigation adult male Balb/C mice were used.
Isolation and characterization of 1
Specimens of S. fortis were collected northwest off Spitsbergen (79°33`N, 8°53`E) at 333 m
depth using an Agassiz dredge trawl in September 2007. The sample was stored at -23 °C
until use. A subsample is kept at the Norwegian National Marine Biobank (Marbank,
reference number M10037), UiT The Arctic University of Norway, Tromsø. Frozen sponge
material (2.0 kg) was extracted as previously described yielding 34.62 g of organic extract33.
The organic extract (2 g) was partitioned between n-hexane (150 mL) and 90% MeOH (100
mL). The 90% MeOH fraction was dried under vacuum and further purified by mass guided
prep-HPLC using a XTerra RP18 HPLC column employing a linear gradient from 5 to 10 %
acetonitrile in ultra-pure water (both containing 0.1% formic acid) at a flow rate of 6 mL/min
over 13 min resulting in the isolation of 1. The structure of 1 was confirmed using MS, 1D
and 2D NMR (COSY) spectroscopic techniques and comparison with literature data35.
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General procedure for reductive amination
3-bromo-N,N-dimethyl-4-methoxyphenethylamine (5a).
A
solution of 3-bromo-4-
methoxyphenethylamine (137 mg, 0.44 mmol) in methanol (5 mL) was treated with
formaldehyde (330 µL, 4.4 mmol, 37% solution in water) and sodium cyanoborohydride (277
mg, 4.4 mmol). The reaction was stirred for 15 hours and then concentrated under reduced
pressure. The resulting thick oil was dissolved in ethyl acetate, washed with saturated
sodium bicarbonate, water and brine. After drying with sodium sulfate and removal of
solvent, the resulting residue was purified by column chromatography (CHCl3 – MeOH) to
1
afford 5a (50 mg, 44%). IR (neat) νmax 2941, 2766, 1497, 1254, 1054, 807 cm-1; H NMR (600
MHz , CD3OD) δ 7.40 (1H, d, J = 2.1 Hz), 7.16 (1H, dd, J = 8.4, 2.1 Hz), 6.95 (1H, d, J = 8.4 Hz),
3.84 (3H, s), 2.74 – 2.70 (2H, m), 2.57 – 2.51 (2H, m), 2.31 (6H, s); 13C NMR (151 MHz ,
CD3OD) δ 155.9, 134.7, 134.3, 129.9, 113.4, 112.4, 62.2, 56.7, 45.3, 33.3; HRMS m/z
258.0488 (calcd for C11H1779BrNO [M + H]+: 258.0489).
The spectral data of compounds 3a (55%, 0.38 mmol)49, 4a (88%, 0.52 mmol)50, and 6a (73%,
0.1 mmol)51 were consistent with previous reports.
3,5-dibromo-N,N-dimethyl--4-methoxyphenethylamine (7a). (63% , 0.23 mmol) amorphous
solid; IR (neat) νmax 2926, 1471, 1260, 993, 737 cm-1; 1H NMR (600 MHz, CDCl3) δ 7.35 (2H, s),
3.86 (3H, s), 2.76 – 2.72 (2H, m), 2.60 – 2.55 (2H, m), 2.34 (6H, s); 13C NMR (151 MHz, CDCl3)
δ 152.6, 132.9, 131.0, 118.1, 60.7, 60.7, 45.3, 29.9; HRMS m/z 335.9596 (calcd for
C11H1679Br2NO [M + H]+: 335.9593).
General procedure for quaternary amine formation
(3-bromo-4-methoxyphenethyl)trimethylammonium iodide (5b). Compound 5a (34 mg,
0.13 mmol) was dissolved in methanol (2 mL) and treated with methyl iodide (33 µL, 0.53
mmol). After 12 hours, the reaction was concentrated under reduced pressure, the resulting
solid washed with cold methanol, providing the product 5b (48 mg, 91%) (iodide salt) as an
1
amorphous solid. IR (neat) νmax 2970, 1498, 1255, 1054, 953 cm-1; H NMR (400 MHz,
(CD3)2SO) δ 7.59 (1H, d, J = 2.2 Hz), 7.31 (1H, dd, J = 8.4, 2.2 Hz), 7.10 (1H, d, J = 8.4 Hz), 3.83
(3H, s), 3.54 – 3.45 (2H, m), 3.13 (9H, s), 3.03 – 2.96 (2H, m); 13C NMR (101 MHz, (CD3)2SO) δ
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154.3, 133.3, 129.8, 129.6, 112.8, 110.6, 65.7, 56.2, 52.3, 27.1; HRMS m/z 272.0647 (calcd
for C12H1979BrNO [M]+: 272.0645).
The spectral data of compounds 6b (82%, 0,04 mmol)52 was consistent with those reported.
Compounds 2b53, 3b54, 4b55 and 7b56 have been reported but lack full characterization data.
(phenethyl)trimethylammonium iodide (2b). (84%, 0.57 mmol) amorphous solid; IR (neat)
νmax 1689, 1479, 1201, 1054, 740, 699 cm-1; 1H NMR (600 MHz, CD3OD) δ 7.38 – 7.32 (4H, m),
7.29 – 7.26 (1H, m), 3.64 – 3.58 (2H, m), 3.25 (9H, s), 3.18 – 3.12 (2H, m), 13C NMR (151 MHz,
CD3OD) δ 136.9, 130.1, 130.0, 128.4, 68.4*, 53.8*, 30.3; HRMS m/z 164.1431 (calcd for
C11H18N [M]+: 164.1434). *Present as triplets.
(4-hydroxyphenethyl)trimethylammonium iodide (3b). (88%, 0.08 mmol) amorphous solid;
1
; IR (neat) νmax 3237, 2413, 1608, 1511, 1213, 836 cm-1; H NMR (400 MHz, CD3OD) δ 7.15
(2H, d, J = 8.4 Hz), 6.76 (2H, d, J = 8.5 Hz), 3.57 – 3.49 (2H, m), 3.22 (9H, s), 3.08 – 2.99 (2H,
m); 13C NMR (101 MHz, CD3OD) δ 157.9, 131.1, 127.3, 116.7, 68.8*, 53.7*, 29.5; HRMS m/z
180.1383 (calcd for C11H18NO [M]+: 180.1383). *Present as triplets.
(4-methoxyphenethyl)trimethylammonium iodide (4b). (88%, 0.20 mmol) amorphous solid;
; IR (neat) νmax 1610, 1513, 1246, 1179, 823 cm-1; 1H NMR (600 MHz, CD3OD) δ 7.27 (2H, d, J
= 8.7 Hz), 6.90 (2H, d, J = 8.7 Hz), 3.77 (3H, s), 3.60 – 3.53 (2H, m), 3.24 (9H, s), 3.11 – 3.03
(2H, m); 13C NMR (151 MHz, CD3OD) δ 160.4, 131.2, 128.6, 115.4, 68.6*, 55.7, 53.8*, 29.5;
HRMS m/z 194.1540 (calcd for C12H20NO [M]+: 194.1539). *Present as triplets.
(3,5-dibromo-4-methoxyphenethyl)trimethylammonium iodide (7b). (84%, 0.09 mmol)
1
amorphous solid; ; IR (neat) νmax 2928, 1467, 1259, 958, 737 cm-1; H NMR (400 MHz,
CD3OD) δ 7.63 (2H, s), 3.85 (3H, s), 3.60 – 3.54 (2H, m), 3.22 (9H, s), 3.15 – 3.07 (2H, m); 13C
NMR (101 MHz, CD3OD) δ 154.8, 134.6, 133.6, 119.3, 67.7, 61.1*, 53.8*, 28.8.; HRMS m/z
349.9762 (calcd for C12H1879Br2NO [M]+: 349.9750). *Present as triplets.
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Cholinesterase Inhibition Assay
AChE activity was measured by Ellman’s method, using acetylthiocholine chloride (0.125,
0.25, and 0.5 mM, respectively) as a substrate in 100 mM potassium phosphate buffer pH
7.4 at 25°C, and electric eel AChE, or horse serum BChE as enzyme sources (Sigma, final
concentration in the test 0.0075 U/mL). Hydrolysis of acetylthiocholine chloride was
followed on a VIS microplate reader (Dynex Technologies, USA) at 405 nm. AChE or BChE
inhibition was monitored for 5 minutes at 20°C for each compound (prepared from a 2
mg/mL stock in methanol and then progressively diluted in 100 mM potassium phosphate
buffer pH 7.4). The positive control (physostigmine, Sigma) was prepared in ethanol (at a 10
mM final concentration) and progressively diluted in the same buffer. The effect of the pure
methanol or ethanol on enzyme inhibition was also checked, and all readings were corrected
for their appropriate blanks. Every measurement was repeated at least three times.
Cytotoxicity testing
The potential cytotoxicity of compounds 1 – 7b was evaluated against human MRC-5 normal
lung fibroblasts, using the tetrazolium based (MTS) CellTiter 96® Aqueous One Solution Cell
Proliferation Assay. Percent cell survival was calculated by comparing exposed cells to
untreated cells and cells treated with Triton X-100 (0.01 %), as previously described33.
Muscle contraction experiments
Mice were sacrificed by cervical dislocation, followed by immediate exsanguination. The
diaphragm with corresponding phrenic nerves was dissected and used.
Hemidiaphragm was tightly pinned to the Rhodorsil coated organ bath containing
oxygenated standard Krebs-Ringer solution composed of (in mM): 154 NaCl, 2 CaCl2 , 5 KCl, 1
MgCl2, 5 HEPES and 11 D-glucose, pH 7.4, at 22–24°C. The tendinous side of the
hemidiaphragm was attached with a steel hook via silk thread to an isometric force
displacement transducer FT 03 (Grass instruments, West Warwick, RI, USA). Nerve-evoked
single isometric twitches were recorded as follows: the motor nerve of isolated
neuromuscular preparation was stimulated with a square pulse S-48 stimulator (Grass
instruments, West Warwick, RI, USA) via a suction electrode with pulses of 0.1 ms duration,
0.1 Hz stimulation rate and with the supramaximal voltage of 8–10 V. Directly evoked single
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isometric twitches were evoked by stimulating hemidiaphragm preparation with a platinum
electrode assembly placed along the organ bath with pulses of 0.1 ms in duration, with a 0.1
Hz stimulation rate and with the supramaximal voltage of 60–80 V. Directly or nerve-evoked
tetanic muscle contraction recordings were obtained by stimulating the hemidiaphragm with
train of pulses (1000 ms duration at 80 Hz). Each hemidiaphragm preparation was then left
to equilibrate for 20 min to achieve stable resting tension before beginning the experiments.
Electrical signals were amplified by a P122 strain gage amplifier (Grass instruments, West
Warwick, RI, USA) and then digitized at a sampling rate of 1 kHz using a data acquisition
system (Digidata 1440A; Molecular Devices, Sunnyvale, CA, USA)57. The effect of 7b on the
neuromuscular hemidiaphragm preparation was measured for 60 min.
Recordings of end plate potentials (EPPs)
The experiments were performed at 22–24°C on oxygenated mouse hemidiaphragm
preparations, pretreated for 30 min with 2 µM µ-conotoxin GIIIB, an inhibitor of muscle
sodium channels, to record full-sized endplate potentials without contracting the muscle.
The resting membrane potentials and endplate potentials (EPPs) were recorded from
endplate regions in superficial muscle fibres using intracellular borosilicate microelectrodes
filled with 3 M KCl and pulled with a P-97 Flaming/Brown microelectrode puller (Sutter
Instruments, Novato, CA, USA). Microelectrodes with resistance from 10–20 MΩ were used.
Recordings were performed before, 30 and 60 min after application of 7b, and 15 min after
washing-out the 7b. EPPs were evoked by stimulating the phrenic nerve with supramaximal
square pulses of 0.1 ms duration and with a frequency of 1 Hz. EPP and MEPP recordings
were digitized using Digidata 1440A and the pClamp 10 software. Data were analyzed using
the pClamp-Clampfit 10 program. Amplitudes of EPPs were normalized to a membrane
potential of -70 mV using the formula formula Vc = V0 x (-70)/E, where Vc is the normalized
amplitude of EPPs, V0 is the recorded amplitude and E is the resting membrane potential.
Data analysis and statistics
Data were statistically analysed using SigmaPlot for Windows 11.0 (Systat Software Inc.,
Germany). The results are presented as the mean ± SEM. Data were firstly tested for
normality (Shapiro–Wilk) and equal variance for assignment to parametric or non-parametric
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analysis. For the statistical analysis of the data, a two-tailed Student t-test was used and P
value ≤ 0.05 was considered to be statistically significant.
Acknowledgements
This work was partly supported with grants from the Norwegian research council (ES508288)
and JS and LM are grateful for the support. The Slovenian authors wish to thank the
Slovenian research agency for financial support (Grant P1-0207 and P4-0053), and the
ERASMUS Student mobility programme for financial support of MC.
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Table of content graphic
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