Benzothiazole amphiphiles promote the formation of dendritic spines in primary hippocampal neurons

Article abstract of DOI:10.1074/jbc.M115.701482

The majority of excitatory synapses in the brain exist on dendritic spines. Accordingly, the regulation of dendritic spine density in the hippocampus is thought to play a central role in learning and memory. The development of novel methods to control spine density could, therefore, have important implications for treatment of a host of neurodegenerative and developmental cognitive disorders. Herein, we report the design and evaluation of a new class of benzothiazole amphiphiles that exhibit a dosedependent response leading to an increase in dendritic spine density in primary hippocampal neurons. Cell exposure studies reveal that the increase in spine density can persist for days in the presence of these compounds, but returns to normal spine density levels within 24 h when the compounds are removed, demonstrating the capability to reversibly control spinogenic activity. Time-lapse imaging of dissociated hippocampal neuronal cultures shows that these compounds promote a net increase in spine density through the formation of new spines. Biochemical studies support that promotion of spine formation by these compounds is accompanied by Ras activation. These spinogenic molecules were also capable of inhibiting a suspected mechanism for dendritic spine loss induced by Alzheimer-related aggregated amyloid-? peptides in primary neurons. Evaluation of this new group of spinogenic agents reveals that they also exhibit relatively low toxicity at concentrations displaying activity. Collectively, these results suggest that small molecules that promote spine formation could be potentially useful for ameliorating cognitive deficiencies associated with spine loss in neurodegenerative diseases such as Alzheimer disease, and may also find use as general cognitive enhancers.

Full text of DOI:10.1074/jbc.M115.701482

JBC Papers in Press. Published on March 28, 2016 as Manuscript M115.701482
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M115.701482
[Benzothiazole amphiphiles promote spinogenesis]
Benzothiazole amphiphiles promote the formation of dendritic spines in
primary hippocampal neurons
Jessica L. Cifelli,† Lara Dozier, ‡ Tim Chung,† Gentry N. Patrick‡ and Jerry Yang†,*
†
‡
From the Department of Chemistry and Biochemistry and the Section of Neurobiology in the Division
of Biological Sciences, University of California, San Diego, La Jolla, California 92093-0358
Running title: Benzothiazole amphiphiles promote spinogenesis
To whom correspondence should be addressed: Prof. Jerry Yang, Department of Chemistry and
Biochemistry, University of California, 9500 Gilman Drive, La Jolla, California, 92093-0358, Telephone:
858-534-6006; Fax: 858-534-4554; Email: jerryyang@ucsd.edu
Keywords: Drug design, Benzothiazole, Spinogenic, Dendritic spine, RasGRF1 Neurodegenerative
disease, Alzheimer’s disease
ABSTRACT
The majority of excitatory synapses in the
spinogenic agents reveals that they also exhibit
relatively low toxicity at concentrations displaying
activity. Collectively, these results suggest that
small molecules that promote spine formation
could be potentially useful for ameliorating
cognitive deficiencies associated with spine loss in
neurodegenerative diseases such as Alzheimer’s
disease, and may also find use as general cognitive
enhancers.
brain exist on dendritic spines. Accordingly, the
regulation of dendritic spine density in the
hippocampus is thought to play a central role in
learning and memory. The development of novel
methods to control spine density could, therefore,
have important implications for treatment of a host
of neurodegenerative and developmental cognitive
disorders. Herein, we report the design and
evaluation of a new class of benzothiazole
amphiphiles that exhibit
a
dose-dependent
Dendritic
spines
are
specialized
response leading to an increase in dendritic spine
density in primary hippocampal neurons. Cell
exposure studies reveal that the increase in spine
density can persist for days in the presence of
these compounds, but returns to normal spine
density levels within 24 hours when the
compounds are removed, demonstrating the
capability to reversibly control spinogenic activity.
Time-lapse imaging of dissociated hippocampal
neuronal cultures shows that these compounds
promote a net increase in spine density through the
formation of new spines. Biochemical studies
support that promotion of spine formation by these
compounds correlates with Ras activation. These
spinogenic molecules were also capable of
inhibiting a suspected mechanism of the dendritic
spine loss induced by Alzheimer’s-related
aggregated amyloid-β peptides in primary
protrusions responsible for receiving excitatory
synaptic inputs, providing an important function in
communication between neurons (1–3). The
morphology of dendritic spines and their overall
density correlates with synaptic function and
strongly implicated in memory and learning (1, 4,
5). Consequently, alteration or misregulation of
dendritic spines can influence synaptic function
and plays a major role in various neurological and
psychiatric disorders such as autism, fragile X
syndrome, Parkinson’s Disease (PD) and
Alzheimer’s Disease (AD) (4, 6–12). For example,
in AD there is mounting evidence suggesting
deficits begin with alterations of hippocampal
synaptic function correlated with accumulation of
aggregated amyloid-β (Aβ) peptide prior to
neuronal loss (13–16). Therefore, treatment
strategies that target the initial synaptic loss, rather
than late stage disease intervention, may provide a
neurons.
Evaluation of this new group of
1
Copyright 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

[Benzothiazole amphiphiles promote spinogenesis]
better prognosis for the treatment of AD.
Furthermore, since most cognitive disorders elicit
abnormalities in the form and function of dendritic
spines, it would be desirable to target them
directly using a small molecule to alter or alleviate
these spine changes.
and, using BAM1-EG6 (1) as a representative
example, we demonstrate that the increase in spine
density is reversible by removal of the compound
from the cellular environment. Time-dependent
imaging studies of primary neurons treated with
BAM1-EG6 reveal that these benzothiazoles can
increase spine density through promoting the
formation of new spines. Signal transduction
studies support that these molecules promote spine
formation by involving the activation of the Ras
and do not directly affect overall F/G actin ratios
or ARP2 expression levels in primary neurons.
Taken together, these results demonstrate that
these BAM agents represent new potential tools to
study the relationship between dendritic spines and
cognitive behavior and may open up a new avenue
to explore the use of spinogenic agents for the
treatment of neurodegenerative and other spine-
related cognitive disorders.
We previously reported the design,
synthesis, and evaluation of two oligo(ethylene
glycol) derivatives of benzothiazole aniline
(BTA), BTA-EG6 and BTA-EG4, which exhibited
a variety of advantageous properties for the
potential treatment of neurodegenerative diseases
such as AD (17–19). Interestingly, BTA-EG4
showed the capability to improve memory and
learning in cognitive performance tests in both
wild-type mice and in a mouse model for AD (18,
19). This in vivo activity of BTA-EG4 was also
accompanied by a phenotypic increase in dendritic
spine density (18, 19). Due to the scarcity of small
molecules known to increase dendritic spine
density, this rare feature of benzothiazole
amphiphiles is of particular interest and could be
utilized as a tool to help study the relationship
between spines and cognitive function.
EXPERIMENTAL PROCEDURES
Materials—3-(4,5-dimethylthiazolyl-2)-2,
5-diphenyltetrazolium bromide (MTT) cell
proliferation assay (Product No: 30-1010K) was
purchased from American Type Culture Collection
(ATCC) (Manassas, VA). Pierce™ BCA Assay kit
(#23225), Active Ras Pull-down and Detection Kit
(#16117), and Pierce™ phosphatase inhibitor
tablets (#88667) were purchased from Thermo
Scientific. G-actin/F-actin In Vivo Assay Kit
(#BK037) was purchased from Cytoskeleton, Inc.
Protease inhibitor tablets (# 05892791001) were
purchased from Roche. Primary antibodies used
were: rabbit anti-Synapsin (EMD Millipore
AB1543), mouse anti-PSD95 (EMD Millipore
CP35), mouse anti-RasGRF1 (BD 610149), mouse
anti-Ras (Thermo #1862335), mouse anti-GADPH
(Sigma G8795), rabbit anti-Actin (Cytoskeleton
#AAN01), rabbit anti-Arp2 (Santa Cruz sc-
15389), mouse anti-Aβ (6E10) (Biolegend®
#803001), rabbit anti-p-Cofilin (Santa Cruz sc-
12912-R) and rabbit anti-Cofilin (Santa Cruz sc-
33779). Secondary antibodies used were as
follows: goat anti-mouse AlexaFluor 568
(Invitrogen A110040), goat anti-rabbit AlexaFluor
647 (Invitrogen A21244), ECL™ Horseradish
Peroxidase (HRP) linked anti-mouse (GE
#NA931) and anti-rabbit (GE #NA934).
Amersham™ ECL™ Prime Western Blotting
Detection Reagent (RPN2232) was purchased
from GE Healthcare. Diphytanoylphosphatidyl-
choline (DiPhyPC) was purchased from Avanti
Polar Lipids, Inc. Synthetic Aβ(1-42) peptide was
The in vivo results of BTA-EG4 suggest
that it may provide broad therapeutic benefits for
improving cognitive function in AD as well as in
other dendritic spine-related diseases (20–22).
However, we also observed toxicity of this
compound in SH-SY5Y neuroblastoma cells that
correlated with its ability to partition in
membranes and induce membrane lysis (23).
Toxicity is one of the biggest issues at every stage
of drug development (24) and the toxicity of
BTA-EG4 precluded our capability to adequately
evaluate the extent of its spinogenic biological
activity.
In order to further evaluate the ability of
the benzothiazole agents to promote spinogenesis,
herein we designed and characterized three novel
structural variants of the BTA compounds. These
new benzothiazole amphiphiles (BAMs) (1-3,
Figure 1) exhibit substantially less toxicity
compared to the parent BTA compound. We show
that these new BAM agents are capable of
promoting an increase in dendritic spine density
and can serve as either a pre- or co-treatment to
negate the overall net spine loss induced by the
presence of aggregated Aβ peptides. Additionally,
these compounds are capable of directly inhibiting
aggregated Aβ from inducing spine loss mediated
by a Cofilin-dependent pathway. This spinogenic
activity was dose-dependent in primary neurons,
2

[Benzothiazole amphiphiles promote spinogenesis]
purchased from PL Lab. All chemical reagents
were purchased and used as is from Sigma Aldrich
or Fisher unless otherwise stated.
BAM2-EG6, (Ex/Em 365/428 nm), and BAM3-
EG6 (Ex/Em 335/398 nm). Each experiment was
repeated at least three separate times and error bars
denote standard deviation from the mean. Data
were processed using Origin 7.0 (MicroCal
Software, Inc., Northampton, MA).
Compounds—BTA-EG6 was synthesized
as previously reported (25). The general synthetic
procedures we used to prepare benzothiazole
amphiphiles (BAMs 1-3) are outlined in Figure 1b.
For the synthesis of the benzothiazole core for
BAM2, commercially available 4-hydroxy
benzaldehyde (4) was alkylated with 2-chloro-N-
methylacetamide (5) via an in situ Finklestein
reaction (26). The aryl ether (6) underwent a
rearrangement under basic conditions to yield 4-N-
(methylamino) benzaldehyde (7) (27). Microwave
irradiation in ionic liquid ([pmim]Br) (28) of 2-
aminothiophenol (8) with benzaldehyde (7)
afforded benzothiazole 9. An analogous
microwave-assisted reaction (29) between 8 and
12 gave 2-(4-(methylthio)phenyl)benzo[d]thiazole
(13) in good yield. The methylthiol group on 13
was then oxidized to the sulfoxide via mCPBA
oxidation to yield 2-(4-(methylsulfinyl)phenyl)-
benzo[d]thiazole (14). Pummerer rearrangement
(30, 31) of compound 16 gave the α-acyloxy-
thioether (16), which was converted to the thiol
(17). Compounds 9, 10 (commercially available),
and 16 were then reacted with EG6-Iodide (11)
(23) under standard nucleophilic substitution
conditions to yield BAM1-EG6 (1), BAM2-EG6
(2), and BAM3-EG6 (3), respectively. Extended
Estimation of Log
accessible surface area (SASA)—Log P values
were calculated using molinspiration
P
and solvent
cheminformatics software and solvent accessible
surface area (SASA) values were calculated with
PyMOL.
Neuronal
Cultures—Rat
dissociated
hippocampal neurons from postnatal day
1
Sprague Dawley® outbred rats of both sexes were
plated at a density of 45,000 cells/cm2 onto poly-
D-lysine-coated coverslips or 96-well plates.
Neurons were maintained in B27 supplemented
Neurobasal media (Invitrogen) until days in vitro
(DIV) 18-23 as previously described (33, 34).
Overall neuronal health was monitored visually
weekly and throughout the experimental process.
Measurement of Cell Viability in the
Presence of BAMs 1-3 and BTA-EG6— An MTT
cell viability assay was performed. Briefly, 21
DIV rat dissociated hippocampal neurons in 96-
well plates were dosed with 100 µL of various
samples solutions of either BTA-EG6 or BAMs 1-
3 with final concentrations of 0 to 250 µM. Cells
were exposed to these solutions for 24 h at 37 °C,
5% CO2. An MTT cell viability kit (ATCC,
Product No: 30-1010K) was then used to
determine cell viability. Briefly, 20 µL of the
provided MTT reagent was added per well and
cells were placed in the incubator for 3 h. The
insoluble intracellular purple formazan was then
dissolved by the addition of 100 µL of detergent
reagent provided and let solubilize overnight at
detailed
descriptions
of
syntheses
and
characterization of compounds can be found in the
supplemental information.
Measurement of Fluorescence Emission
Spectra—The emission spectra of benzothiazoles
in different environments was evaluated as
previously described (23). Briefly, BAMs 1-3 and
BTA-EG6 were diluted to a final concentration of
50 µM in deionized H2O, pure octanol, and a
liposome suspension. The liposomes were
prepared from a total lipid concentration of 10 mM
of diphytanoylphosphatidylcholine (DiPhyPC) in
water by the gentle dehydration/rehydration
method followed by tip sonication (32). 200 µL of
each sample was transferred to a cuvette (Helma
Analytics, Quartz SUPRASIL (QS), 10mm) and
the fluorescence emission spectrum was measured
in a PTI spectrofluorometer (0.5 nm step size) in
water, octanol and an aqueous liposome
suspension for BAMs 1-3 and BTA-EG6. Maximal
excitation and emission values (λmax) for all
compounds were as follows: BTA-EG6 (Ex/Em
355/420 nm), BAM1-EG6 (Ex/Em 355/420 nm),
room temperature.
The cell viability was
determined by measuring the absorbance at 570
nm using a Spectramax 190 microplate reader
(Molecular Devices). All results are presented as
percent reduction of MTT relative to untreated
cells (100% viability), and all wells were blanked
with absorbance values from the wells containing
medium, MTT reagent and detergent only.
Western Blot Analysis— Rat dissociated
neurons were lysed with RIPA buffer containing
both protease and phosphatase inhibitors. Protein
concentration was determined by BCA assay and
proteins were separated by SDS-PAGE followed
by transfer onto nitrocellulose membranes.
Membranes were blocked with either 5% BSA or
3

[Benzothiazole amphiphiles promote spinogenesis]
milk in Tris-Buffered Saline with Tween 20
(TBST), followed by incubation with primary
antibodies overnight at 4 °C with shaking. Proteins
were visualized using the appropriate HRP-labeled
secondary by ECL and detection was carried out
on film (Freedom Imaging, SRX-101A). The
intensity of each band was then quantified using
ImageJ software.
ECM 600 electroporator (BTX, Holliston, MA) at
220V, 129Ω, and 1050µF. After 24 h, virion was
collected and concentrated by centrifugation at
20,000 rpm for 90 min using a Beckman Coulter
Optima MAX Ultracentrifuge (Beckman Coulter,
Indianapolis, IN). The treated neurons were
infected with palGFP expressing sindbis 18 h prior
to fixation. The palGFP (membrane targeting)
signal was imaged directly in fixed neurons.
Measurement of Cellular Active Ras
Levels— To analyze the levels of active Ras in the
rat primary neuronal lysates treated with BTA-
EG6, BAMs 1-3 or control, we used an Active-Ras
Pulldown and Detection Kit (Thermo) according
to the manufacturers guidelines.
Confocal Microscopy and Dendritic Spine
Analysis—For all imaging of neurons, we used a
Leica DMI6000 inverted microscope outfitted
with a Yokogawa Spinning disk confocal head, an
Orca ER High Resolution CCD camera (6.45
µm/pixel at 1×) (Hamamatsu), Plan Apochromat
63×/1.4 na objective, and PerkinElmer solid-state
laser with 488nm excitation. Confocal z-stacks
were acquired in all experiments and all imaging
was acquired in the dynamic range of 8-bit
acquisition (0–255 pixel intensity units,
respectively) with Volocity (PerkinElmer) imaging
software. Imaged dendrites from one secondary
dendrite per cell (after 1 branch) at a distance of
40-80 µm from the soma were straightened using
ImageJ. We estimated spine density as the number
of manually counted spines (length <2 x width)
divided by dendrite segment length. The analyzer
was blind to treatment and statistical significance
was determined between experimental conditions
by either unpaired t tests (two groups) or by
ANOVA and indicated post hoc multiple-
comparison test (>2 experimental conditions).
Real Time Imaging of Spine Changes in
Rat Primary Hippocampal Neurons—For this
study, 21 DIV neurons plated in 35 mm dishes
(MatTek) were rinsed 3x with an excess of HBSS
and then left in HBSS for the duration of imaging.
For live imaging we kept cells at 37°C and used a
Leica DMI6000 inverted microscope outfitted
with a Yokogawa Spinning disk confocal head, an
Orca ER High Resolution CCD camera (6.45
µm/pixel at 1×) (Hamamatsu), Plan Apochromat
63×/1.4 na objective, and PerkinElmer solid-state
laser with 488nm excitation. The spine changes on
the same neuron were monitored 1 h before dosing
(-60 min) and up to 3 h after dosing (+180 min).
Dosing occurred at t=0 and consisted of either 0
(for control) or 5 µM BAM1-EG6. For each
condition, neurons from three different neuronal
preparations (prep) were used and two neurons per
prep were monitored. Confocal z-stacks were
acquired in all experiments and all imaging was
acquired in the dynamic range of 8-bit acquisition
Measurement of Cellular F-Actin/G-Actin
Ratio— To analyze the levels of filamentous (F-
actin) and globular (G-actin) in rat primary
neuronal lysates treated with BTA-EG6, BAMs 1-3
or control, we used a G-actin/F-actin In Vivo
Assay Kit (Cytoskeleton) according to the
manufacturers guidelines.
Neuronal Treatments—For all neuronal
treatments, the following general protocol was
followed: Briefly, 18-23 DIV rat dissociated
hippocampal neurons were dosed with various
concentrations (0-50 µM final concentration) of
BAMs 1-3 or BTA-EG6 (with 0.1% final DMSO
concentration) for various incubation times (24-72
h depending on the experiment). After dosing and
at the desired time point, the medium was
removed and cells were rinsed with PBS-MC
(phosphate-buffered saline, 1 mM MgCl2 and 0.1
mM CaCl2). Following rinsing, cells were fixed
with 4% paraformaldehyde (PFA)/sucrose in PBS
for 10 min at RT. After fixation, coverslips were
carefully rinsed (3 x PBS-MC) and then mounted
onto slides (Polysciences Inc., 18606) for imaging.
Colocalization of PSD95 and Synapsin —
After following the general procedure for neuronal
treatment, fixed cells were then permeabilized,
blocked in 5% BSA/PBS-MC, and incubated
overnight at 4°C with anti-Synapsin (1:1000) and
anti-PSD95 (1:1000). After incubation with
Alexafluor conjugated secondary antibodies, the
coverslips were mounted for imaging.
Sindbis Production—PalGFP SinRep5
DNA was obtained as a generous gift from
Takahiro Furuta (Kyoto University, Kyoto, Japan)
(35). Recombinant Sindbis virion production was
accomplished through RNA transcription using the
SP6 mMessage mMachine kit (Ambion, Austin,
TX). Electroporation of RNA into baby hamster
kidney cells (BHK) was completed using a BTX
4

[Benzothiazole amphiphiles promote spinogenesis]
(0–255 pixel intensity units, respectively) with
Volocity (PerkinElmer) imaging software. For
analysis, imaged dendrites were straightened using
ImageJ and the same dendrite length was analyzed
for each condition. Spines gained were counted as
any new spines found at each respective time point
and spines lost were counted as spines that
disappeared from the analyzed segment. The
analyzer was blind to treatment.
fixed with 4% paraformaldehyde (PFA)/sucrose in
PBS for 10 min at RT, and mounted onto slides
(Polysciences Inc., 18606) for imaging. All
analysis was done blinded and each experiment
was repeated at least three separate times from
neurons from three different preparations.
RESULTS
Design and evaluation of benzothiazole
Preparation and Characterization of
amphiphiles (BAMs) 1-3 with decreased
partitioning in membranes compared to BTA-
EG6—The toxicity of BTA-EG4,6 agents were
previously reported to correlate with their
capability to partition in membranes (23). We
hypothesized that altering the hydrophobic core of
these molecules would decrease their energetic
driving force to partition into membranes, thereby
reducing their concomitant toxicity. To test this
hypothesis, we used BTA-EG6 as a lead compound
for the design of three new compounds 1-3 (Figure
1a). We designed these new benzothiazoles to test
whether moving the 6-methyl group in BTA-EG6
to the aniline nitrogen or complete removal of the
6-methyl group would reduce the hydrophobicity
of the compounds enough to significantly reduce
membrane partitioning and toxicity to cells. We
also tested the importance of retaining the aniline
nitrogen in 1 by modifying it to a sulfur group as
in BAM3-EG6 (3).
Aggregated Aβ(1-42)—Aggregated Aβ(1-42) was
prepared as previously described (36). Briefly,
Aβ(1-42) was initially solubilized in 100%
1,1,1,3,3,3,-hexafluoro-2-propanal (HFIP) to 1
mM concentration at RT for 21 h with shaking.
The solution was sonicated and vortexed before it
was diluted in cold nanopure water (2:1
H2O:HFIP). Aliquoted fractions were lyophilized
for 2 days, followed by storage at -80 °C until use.
Solutions of Aβ were obtained by dissolving Aβ in
sterile PBS to a concentration of 100 µM and
incubated at 37 °C for 3 days before use. Western
blot analysis of the three day incubated Aβ was
carried out to determine composition. We
estimated the relative abundance of monomers,
oligomers, and protofibrils of Aβ using ImageJ
and percentage of each composition was
calculated by dividing the intensity of each
aggregation state over the total intensity for Aβ in
the lane. This preparation of Aβ lead to a
composition of ~12% monomers, ~16% low MW
oligomers, and ~72% mixture of soluble
protofibrils/fibrils (data not shown). Aggregated
Aβ was also characterized by EM, MALDI-TOF,
and binding by Thioflavin T (data not shown).
Rescue of Net Aβ-Induced Spine Loss in
Calculations (Figure 2c) suggested that
small modifications to BTA-EG6 could affect its
hydrophobic character as determined by their
octanol-water partitioning coefficient (log P) and
their solvent accessible surface area (SASA)
without the need to modify the core benzothiazole
structure, which is presumably required to impart
spinogenic properties. We, therefore, examined
the hydrophobic character of the new BAMs 1-3
relative to the parent compound, BTA-EG6, by
taking advantage of the solvatochromic nature of
these compounds. In this assay, the fluorescence
emission spectra were measured for each
compound in octanol, water and an aqueous
suspension of liposomes to mimic cell membranes.
All compounds exhibited a shift of maximum
fluorescence emission to shorter wavelengths in a
more hydrophobic environment (i.e., in octanol
compared to water) (Figure 2a,c). The emission
maximum in an aqueous suspension of liposomes
was measured for all compounds and it was found
that compounds 1-3 exhibited λmax that reflected a
more polar, water-like environment compared to
BTA-EG6, with changes of emission max of 29-
Rat
Primary
Hippocampal
Neurons—We
followed the same general dosing procedure as
described for neuronal treatment, except with the
following noted changes. For Aβ co-treatment, 18
DIV rat dissociated hippocampal neurons were
dosed with a final concentration of 1 µM of
aggregated Aβ(1-42) with or without the presence
of 1 or 5 µM of BAMs 1-3 or BTA-EG6 for 3
days. Control cells were treated with vehicle
control only (0.1% DMSO) for the three-day
period. For Aβ pre-treatment, rat dissociated
hippocampal neurons were dosed with 5 µM of
BAMs 1-3 or BTA-EG6 for 24 h. After a wash out
of compounds, neurons were dosed with a final
concentration of 1 µM of aggregated Aβ(1-42) for
3 days. For both treatments, after dosing medium
was removed, cells were rinsed with PBS-MC,
5

[Benzothiazole amphiphiles promote spinogenesis]
34% from water (relative to λmax in pure octanol)
compared to a 50% change in λmax for BTA-EG6
(Figure 2a,c). These results demonstrate that the
novel structural modifications in BAMs 1-3
decreased their membrane partitioning compared
to BTA-EG6.
increase in the density of colocalized PSD95-
Synapsin puncta in neurons dosed with BAM1-
EG6 over the control (Figure 4).
The cumulative distribution of spine
length and width was also measured for neurons
dosed with BAM1-EG6. No observable difference
in average spine length or width was found
compared to cells treated with vehicle control
(Figure 5a,b).
BAMs 1-3 exhibit a decreased toxicity
compared to BTA-EG6—An MTT cell proliferation
assay was performed to compare the toxicity of
compounds 1-3 to the parent compound, BTA-
EG6. In this assay, BTA-EG6 exhibited moderate
toxicity to rat primary neurons with an IC50 of 9.8
µM after 24 h exposure (Figure 2b,c). Satisfyingly,
we found that all BAMs 1-3 were significantly less
toxic than BTA-EG6, with IC50’s ranging from 44-
67 µM (Figure 2b,c).
To evaluate the maximum effect of the
BAM agents on spine density increase, we dosed
primary neurons for 24 h in the presence of 1-25
µM BAM1-EG6. The maximum observed increase
in spine density was ~20%, occurring with a dose
of 5 µM with no further increase at higher
concentrations (Figure 5c).
Effects of BAMs 1-3 on dendritic spine
A time course of spinogenic activity was
density— BTA-EG6 was first used to assess
increases in spine density in primary hippocampal
neurons as a control due to its previously
published ability to increase spine density (37). In
order to visualize all spines, we used a virally
transfected membrane-targeting palGFP that is
known to reliably fluorescently label dendritic
spines (38–40). Moreover, expression was limited
to less than 18 h to minimize any artifacts from
viral transduction. After confirming an observed
increase in dendritic spine density in BTA-EG6-
treated neurons over treatment with vehicle control
(Figure 3), neurons were next treated with 1 or 5
µM concentrations of benzothiazoles 1-3. All new
compounds 1-3 showed a dose-dependent increase
in spine density after a 24 h exposure (Figure 3).
In addition, compounds 1-3 were able to produce a
statistically significant increase in net spine
density at a lower concentration compared to
BTA-EG6, suggesting the structural differences
(and possibly the decreased hydrophobic
character) of 1-3 compared to BTA-EG6 results in
overall increased spinogenic activity. There was
no observed change in spine density when the cells
were treated with the vehicle control (0.1%
DMSO) (41).
also examined in three separate experiments: In
the first experiment, BAM1-EG6 was exposed to
primary neurons at a constant concentration (5
µM) in the culture medium for up to 72 h. At
various time points, we fixed cells and measured
spine density (as estimated by spine number per
µm). This experiment revealed a trend of
increasing average spine density between 2 and 24
hours, with statistically significant spine density
increase reached after a 12 h exposure of the
neurons to BAM1-EG6 (Figure 5d). The
spinogenic activity of the benzothiazoles reached
equilibrium within 24 h, and this maximal increase
of ~20% in spine density levels (compared to
treatment with vehicle) persisted for up to 3 days
upon exposure to a constant concentration of the
BAM1-EG6.
In a second experiment, we evaluated
whether the spine density increases induced by the
benzothiazoles persisted after the compounds have
been removed from the culture medium. Primary
hippocampal neurons were dosed for 24 h with 5
µM BAM1-EG6, resulting in the expected ~20%
increase in spine density levels compared to
treatment with vehicle alone (0.1% DMSO). The
cells were then rinsed and the culture medium was
replaced with compound-free medium, and we
monitored the average spine density on the cells
over an additional 48 h. The initial spine increase
after 24 h exposure to BAM1-EG6 did not persist
once we removed the compound, with the density
of spines returning back to normal levels (i.e., to
the spine density observed in control cells) within
24 h of removal of BAM1-EG6 (Figure 5e).
Using BAM1-EG6 as a representative
compound for this class of benzothiazoles, we next
examined the effects of BAM agents on the
density of neuronal puncta containing pre- and
post-synaptic markers. We dosed neurons for 24 h
with and without BAM1-EG6 (5 µM) and then
examined the colocalization of PSD95 (post-
synaptic marker) and Synapsin (pre-synaptic
marker). In addition to the increase in dendritic
spine density (Figure 3), we also observed an
In a third experiment, we monitored the
effect on spine density in primary neurons by
6

[Benzothiazole amphiphiles promote spinogenesis]
adding fresh aliquots of BAM1-EG6 every 24 h to
the culture medium over a 72 h period. We
incubated primary neurons initially in culture
medium containing a final concentration of 5 µM
BAM1-EG6 (1x dose). At 24 hours (2x dose) and
48 hours (3x dose) of incubation, an additional 2
µL of a 5 mM BAM1-EG6 DMSO stock (final
concentration 5 µM, 0.1% DMSO) was added to
the culture medium. We found that further
addition of BAM1-EG6 every 24 h (which
putatively increased the final concentration of
compound after every addition) did not result in
further increases in dendritic spine density above
the original observed increase of ~20% after 24 h
exposure of 5 µM BAM1-EG6 (Figure 5f).
effects of these compounds on the expression level
of both RasGRF1 and active Ras in rat dissociated
primary neurons. When we exposed primary
neurons to 5 µM concentrations of compounds 1-3
or BTA-EG6, we observed about a 2-fold increase
in RasGRF1 expression levels (Figure 7a).
Additionally, about a ~1.5 fold increase in active
Ras/ total Ras was also observed for compounds 1-
3 or BTA-EG6 compared to control cells (Figure
7b).
BAMs 1-3 do not directly affect overall
cellular cytoskeletal actin dynamics or actin-
related protein-2 (ARP2) —Due to the rapid
changes in dendritic spine dynamics as seen in
both live and fixed cell imaging, we next sought to
examine the effects of BAMs on cytoskeletal
rearrangement. The actin-related protein -2/3
(ARP2/3) complex plays a central role in the
regulation of actin organization (43). Therefore,
we first inspected whether our BAM agents
affected ARP2 expression levels in primary
neurons. We did not find a statistical difference in
ARP2 expression between the control cells and
cells treated with 5 µM of BAMs 1-3 or BTA-EG6
(Figure 7c).
BAMs 1-3 promote the formation of new
dendritic spines—An observed increase in
dendritic spine density by benzothiazoles 1-3
could arise either by promoting the formation of
new spines or by increasing the stability of
previously formed dendritic spines. In order to
help elucidate which mechanistic pathway BAMs
promotes dendritic spine density alterations, we
monitored the changes in spine number in real
time by periodically capturing live confocal
images of primary neurons over a 4 h time period.
To account for baseline changes in spine
dynamics, neurons were monitored 1 h prior to
dosing. Live imaging then continued up to 3 h
after dosing with either compound 1 (5 µM) or the
vehicle control to gain insight into the spine
changes induced by compound 1. We observed
that dosing with compound 1 led to a statistical
increase in new spines compared to the control 60
min after dosing, while no significant change in
spine loss was observed over the same time period
(Figure 6).
We next examined whether the BAM
agents had an effect on overall actin
polymerization within neurons. We utilized a G-
actin/F-actin assay kit, which enabled us to probe
both F- and G-actin levels from primary lysates
that had been dosed with 5 µM of BAMs 1-3 and
BTA-EG6 for 24 h. We did not observe a
significant difference in the ratio of F-actin over
total actin levels in neurons treated with any of the
compounds compared to control neurons (Figure
7d).
BAMs 1-3 inhibit Aβ-induced Cofilin
activation and negate Aβ-induced net spine loss—
Since BAMs 1-3 were able to increase dendritic
spine density in primary hippocampal neurons
(Figure 3), we examined whether co- or pre-
treatment of neurons with these compounds could
negate net spine loss in neurons exposed to
aggregated amyloid-β (Aβ 1-42), the toxic peptide
cleavage product of the amyloid precursor protein
(APP) associated with AD.
BAMs 1-3 promote Ras signaling—Ras
and RasGRF1, a guanine nucleotide exchange
factor involved in Ras signaling, are important
intermediates in the regulation of spine density
(42). Previous work has reported that BTA-EG4
and BTA-EG6 could promote spine density
increases in vitro in murine primary hippocampal
neurons (37) and BTA-EG4 could promote spine
density increases in vivo in the hippocampus of wt
mice and a 3x tg mouse model for AD (18, 19).
The increase in spine density in neurons by BTA-
EG4,6 correlated with an increase in expression of
RasGRF1 compared to control cells (37). In order
to test whether the spinogenic activity induced by
For co-treatment, we treated primary
neurons for 3 days with medium containing
aggregated Aβ (1-42) with or without the presence
of BAMs 1-3 or BTA-EG6. We observed around a
20% decrease in spine density in primary neurons
that were incubated in the presence of 1 µM
aggregated Aβ (1-42) alone for 3 days (Figure 8).
compounds 1-3 operated along
a
similar
mechanistic path as BTA-EG4,6, we analyzed the
7

[Benzothiazole amphiphiles promote spinogenesis]
In contrast, when we treated primary neurons with
1 µM Aβ (1-42) and 1 or 5 µM concentrations of
BAMs 1-3 or BTA-EG6, we observed a net
increase in dendritic spine density by ~ 20%
compared to control (Figure 8a). Furthermore, the
observed net increase in spine density in cells
treated simultaneously with Aβ (1-42) and BAMs
1-3 or BTA-EG6 were ~ 40% higher than in cells
treated with Aβ (1-42) alone.
the formation of new dendritic spines. The
capability to promote increases in spine density
through external administration of a drug could
lead to a better understanding of the underlying
circuitry affecting cognitive behavior, and,
ultimately, to novel approaches for treatment of
cognitive disorders.
We
previously
reported
that
oligo(ethylene glycol) derivatives of benzothiazole
could insert into planar lipid bilayers and induce
membrane lysis (23). The concentration required
to observe lysis in membranes roughly correlated
with the observed cytotoxicity of the compounds
in human SH-SY5Y neuroblastoma cells (IC50 ~60
µM), suggesting lysis of cells as the significant
factor for the apparent toxicity (46) of the BTA-
For the pre-treatment regiment, we pre-
treated primary neurons for 1 day in medium
containing BAMs 1-3 or BTA-EG6 (or vehicle
control). This pre-treatment presumably increased
the dendritic spine density in cells exposed to
compounds (Figure 3). Next, we replaced the
medium and added in 1 µM Aβ (1-42) for 3
additional days. When we pre-treated primary
neurons with 5 µM concentrations of BAMs 1-3 or
BTA-EG6, we observed no statistically difference
in overall spine number compared to neurons that
were not exposed to compounds or Aβ peptide
(Figure 8b). These results demonstrate that pre-
treatment with BTA-EG6 and benzothiazoles 1-3
effectively protects cells from an overall net
decrease in dendritic spine density induced by
aggregated Aβ (1-42) peptides.
EG4,6
compounds
at
high
micromolar
concentrations (23). Hence, we hypothesized that
altering the hydrophobic core of these molecules
would decrease their energetic driving force to
partition into membranes, thereby reducing their
toxicity.
With a goal of generating structural
analogs of BTA-EG6 with reduced hydrophobic
character, we designed and synthesized
benzothiazole analogs 1-3 (Figures 1). This new
set of benzothiazoles exhibited reduced
partitioning in membranes and lower overall
toxicity in primary neurons (Figure 2) compared to
parent BTA-EG6. Interestingly, we did not find a
correlation between the calculated log P values (a
typical measure of hydrophobicity) and toxicity.
Instead, we found a positive correlation between
solvent-accessible surface area (SASA) and
toxicity in BTA-EG6 and all of the BAM
derivatives. The SASA could, therefore, represent
We next examined whether the BAM
agents could specifically inhibit a mechanistic
pathway for Aβ-induced spine loss. Cofilin
promotes actin depolymerization and its activation
is promoted through dephosphorylation of p-
Cofilin (44). Shatz and coworkers recently
reported that Aβ aggregates induce spine loss
through a pathway that involves activation of
Cofilin-dependent depolymerization of actin upon
binding of Aβ aggregates to the PirB receptor (45).
a
more useful and quantifiable alternative
We observed
a
statistical decrease in p-
parameter to Log P for guiding the development of
additional members of this class of spinogenic
compounds with low toxicity.
Cofilin/total Cofilin levels in primary neurons that
had been dosed with aggregated Aβ alone (Figure
8c), which agrees well with the previous report by
Shatz. However, when neurons had been dosed
with BAMs 1-3 or BTA-EG6 together with
aggregated Aβ, the ratio of p-Cofilin/total Cofilin
was the same as cells that were not exposed to
aggregated Aβ peptides (Figure 8c). This results
supports that the BAM agents are able to block the
Aβ-induced activation of Cofilin, thereby blocking
a specific mechanism for Aβ-induced spine loss.
While reducing toxicity of benzothiazole
agents is an important step towards improving
their biocompatibility, it is also important to assess
whether the new BAMs 1-3 retain the potential
beneficial biological activity of the parent
compound. The capability of BTA-EG4,6 to
promote an increase in dendritic spine density is a
distinctive and extremely rare property for any
small molecule reported to date. The results
demonstrate that the new benzothiazoles 1-3 are
indeed capable of promoting dose-dependent
increases in dendritic spine density in primary
hippocampal neurons (Figure 3), with maximal
DISCUSSION
Many
cognitive
disorders
are
accompanied with loss of dendritic spines, yet
there are few examples of molecules that promote
8

[Benzothiazole amphiphiles promote spinogenesis]
spine density increases of ~20% after 24 hours of
exposure to cells.
h (Figure 6 and 5d). The surprising speed at which
we observe changes in spine dynamics suggests
that cytoskeletal reorganization (48, 49) plays an
important role in the mechanism of action of the
BAM agents. While we did not find a statistical
difference in either the global F-/G-actin ratios or
ARP2 expression levels in neurons exposed to the
BAM agents (Figure 7c,d), these results do not
rule out the possibility that the BAM agents act on
more localized cytoskeletal machinery that affects
dendritic spine dynamics.
The activity of BAM1-EG6 was further
examined as a representative for this class of
compounds to further evaluate the spinogenic
effects of this class of benzothiazoles. Analysis of
the colocalization of both pre- and post-synaptic
markers, Synapsin and PSD95, in neurons exposed
to BAM1-EG6 showed both an increase in the
spines density and an increase in the density of
colocalized PSD95/Synapsin puncta (Figure 4).
This result supports that the promotion of new
dendritic spines may correlate with an increase in
the number of synapses formed in neurons
exposed to the BAM agents. Furthermore, the
analysis of the cumulative distribution of spine
width and length of cells exposed to BAM1-EG6
showed no difference compared to control cells
(Figure 5a,b), demonstrating that the increase in
spine density by BAM agents does not affect the
overall distribution of spine morphology in the
cells (47).
Finally, we showed that co- and pre-
treatment of neurons with BAMs 1-3 and BTA-
EG6 were able negate the net dendritic spine loss
observed in the presence of aggregated Aβ (Figure
8a,b). Importantly, we found that the BAM agents
could block Aβ-induced activation of Cofilin
(Figure 8c), which has been implicated as a
specific mechanism for Aβ-induced synaptic
dysfunction in AD through the binding of
aggregated Aβ to the PirB receptor (45). Since
BTA-EG6 was previously shown to bind to
aggregated Aβ and act as a general inhibitor of
protein-amyloid interactions (17, 25), we
hypothesize that the BAM agents inhibit Aβ-
induced spine loss by binding to Aβ and inhibiting
its interaction with the PirB receptor (45) (and,
thus, lead to the downstream inhibition of Cofilin
activation by aggregated Aβ). Collectively, these
results support a dual mode of action of BTA-EG6
and the BAM agents on the spine density of
primary neurons exposed to aggregated Aβ: 1)
these compounds are capable of directly inhibiting
Aβ from inducing spine loss through a Cofilin-
dependent pathway, and 2) these compounds can
act on an Aβ-independent pathway to promote the
formation of dendritic spines. These results,
therefore, demonstrate that BAMs 1-3 have
potential to counteract one of the earliest observed
pathological events associated with AD (16, 50).
In conclusion, we used rational design to
develop a novel set of benzothiazole amphiphiles
1-3 with improved biocompatibility compared to
the previously reported BTA-EG4,6 compounds
(17–19). These new compounds were capable of
1) promoting dose-dependent increases in
dendritic spine density, 2) temporally and
reversibly controlling elevated spine levels, and 3)
protecting against Aβ-induced dendritic spine loss.
Current efforts are focused on identifying the
cellular target for the BAM agents and elucidating
additional mechanistic details leading to the
spinogenic activity of these compounds. These
Temporal studies showed that the spine
density increased within 12 h in neurons and
stably persisted for 72 h in the presence of BAM1-
EG6. However, the spine density increase in cells
exposed to BAM1-EG6 returned to basal levels
(i.e., levels comparable to control neurons) within
24 h of removal of this compound from the
medium (Figure 5d,e). This capability of BAM
agents to reversibly control the magnitude of spine
density changes in primary neurons may be very
attractive as a tool for further studies on the
relationship between dendritic spines and other
parameters related to neural circuitry.
Live cell imaging and biochemical studies
support that these benzothiazoles promote the
formation of new dendritic spines in neurons
(Figure 6) through
a
mechanism that is
accompanied by an increase in both RasGRF1
expression and active Ras levels (Figure 7a,b).
Previous studies showed that shRNA knockdown
of RasGRF1 in primary neurons completely
blocked the effect of BTA-EG4 on spine density
increases (18), further supporting that Ras
signaling is essential for the spinogenic activity of
the BAM agents.
The kinetic data gained from the live cell
imaging and from imaging of fixed cells after
various short exposures to the BAM agents
indicate that changes in spine dynamics begin
within the first couple hours, with significant net
increases in dendritic spine density observed by 12
9

[Benzothiazole amphiphiles promote spinogenesis]
novel benzothiazoles represent a significant step
towards the development of new tools to study and
treat spine related disorders, and may also lead to a
new class of general cognitive enhancers.
Acknowledgments: This work was partially supported by the UCSD Alzheimer’s Disease Research
Center (NIH 3P50 AG005131). We would also like to thank Dr. Yongxuan Su for use of the molecular
mass spectrometry facility (MMSF) at UCSD.
Conflict of interest: The authors declare that they have no conflicts of interest with the contents of this
article.
Author contributions: JLC, GNP and JY designed the research. JLC and TC synthesized all compounds.
JLC evaluated and analyzed compound physical and toxic properties. LD and GNP prepared and
maintained neuronal cultures; JLC and LD performed and analyzed all dendritic spine experiments. JLC
and JY wrote the paper. All authors reviewed and approved the final version of the manuscript.
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FOOTNOTES
The content is solely the responsibility of the authors and does not necessarily represent the official views
of the National Institutes of Health.
FIGURE LEGENDS
FIGURE 1. Design and synthesis of benzothiazole amphiphiles (BAMs) (1-3).(A) Benzothiazole amphiphiles
(BAMs) (1-3) were designed to exhibit decreased hydrophobicity and hydrogen-bonding capabilities compared to
the parent compound, BTA-EG6. (B) Synthetic scheme for the preparation of BAM 1-3. Abbreviations for all
synthetic steps are as follows: potassium carbonate (K2CO3), potassium iodide (KI), potassium hydroxide (KOH),
ionic liquid or 1-pentyl-3-methylimidazolium bromide ([pmIm]Br, microwave (MW), tetrahydrofuran (THF), meta-
chloroperoxybenzoic acid (mCPBA), dichloromethane (DCM), trifluoroacetic anhydride (TFAA), sodium
hydroxide (NaOH), methanol (MeOH), sodium hydride (NaH), dimethylformamide (DMF), 17-iodo-3,6,9,12,15-
pentaoxaheptadecan-1-ol (EG6-I).
12

[Benzothiazole amphiphiles promote spinogenesis]
FIGURE 2. Physical and toxic properties of BAMs 1-3 and BTA-EG6. (A) Fluorescence emission properties of
BAMs 1-3 and BTA-EG6 in water, octanol, or an aqueous solution of liposomes. (B) Viability of primary neurons as
a function of increasing concentration of BTA-EG6 or BAMs 1-3. (C) Table of calculated hydrophobic parameters,
measured membrane partitioning properties, and IC50 values of toxicity in rat dissociated primary neurons of BAMs
a
b
1-3 and BTA-EG6. log P values were calculated from molinspirations cheminformatics software. SASA values
were calculated with PyMOL.
FIGURE 3. Spinogenic properties of BTA-EG6 and BAMs 1-3 observed in rat primary hippocampal neurons. (A)
Representative spine segments (23 microns) for BTA-EG6 and BAMs 1-3 compared to control (0.1% DMSO). (B)
Quantitative representation of spine number per micron for all compounds compared to control. Data are expressed
as mean values ± SEM, n ≥ 54, *p <0.001, **p, <0.0001 as determined by unpaired t-test compared to control.
FIGURE 4. Analysis of the localization of synaptic proteins of neurons exposed to BAM1-EG6. (A)
Representative images of dendrites (GFP), PSD95 (post-synaptic marker) and Synapsin (pre-synaptic marker) from
neurons dosed with control (0.1% DMSO) or BAM1-EG6. Scale bar = 20 microns (B) Representative images of
spine segments (20 microns) for cells treated with BAM1-EG6 compared to control (0.1% DMSO) labeled with pal-
GFP (green), PSD95 (red) and Synapsin (blue). (C) Quantitative representation of spine number per micron for
BAM1-EG6 compared to control. (D) Quantitative representation of amount of colocalized PSD95 and Synapsin
puncta per micron for neurons treated with BAM1-EG6 compared to control. Data are expressed as mean values ±
SEM, n ≥ 48, **p, <0.0001 as determined by unpaired t-test compared to control.
FIGURE 5. Examination of the spinogenic properties of BAM1-EG6 observed in rat primary hippocampal
neurons. Cumulative distribution of spine length (A) or width (B) of control cells versus cells treated with
compound BAM1-EG6 (1 µM). (C) Concentration-dependent effects of neurons dosed for 24 h with 1-25 µM of
BAM1-EG6 on spine density. (D) Kinetics of spine density increase in cells exposed to BAM1-EG6 compared to
vehicle control (0.1% DMSO). Neurons were dosed and then fixed at 2, 4, 12, 24, 48 and 72 h. (E) Effects of
removal of BAM1-EG6 on dendritic spine number after treatment of cells for 24 h. After 24 h, BAM1-EG6 was
washed out (w.o.) and spine changes were monitored for an additional 24 and 48 h (48 and 72 h total time). The
dendritic spine density 24 h after removal of BAM1-EG6 is indistinguishable from control cells. (F) Effect on spine
density increases of adding additional doses of BAM1-EG6 every 24 h over a total incubation time of 72 hours.
Neurons were dosed at 24 h (1x), 48 h (2x) and 72 h (3x) with no observable additional increase of dendritic spine
density compared to the 1x dose. Data are expressed as mean values ± SEM, n ≥ 54. **p ≤ 0.0001, n.s. = not
significant, as determined by unpaired t-test compared to control. Arrows denote time points when aliquots of cells
were fixed and analyzed.
FIGURE 6. Live cell imaging showing the increase in formation of new spines upon dosing with BAM1-EG6
(compound 1) (A) Representative segments (20 microns) of live cells before (-time) and after dosing (+time) with
BAM1-EG6 (5 µM) or vehicle control (0.1% DMSO). * denotes new spines. (B) Quantitative representation of the
total dendritic spines gained or lost per 20 micron segments for either BAM1-EG6 or vehicle control. N= 6, *p <0.01
compared to control at same time point by unpaired t-test.
FIGURE 7. Analysis of protein expression, activation, and dynamics in primary neurons exposed to benzothiazole
agents. Relative expression levels of A) RasGRF1, B) active Ras and C) ARP2 in primary neurons upon dosing with
5 µM of BTA-EG6 and BAMs 1-3. D) Ratio of F-actin (filamentous actin) over total actin (F- plus G-actin (globular
actin)) in primary neurons treated with BTA-EG6 and BAMs 1-3 (5 µM) or vehicle control. Data are expressed as
mean values ± SEM, n= 3 or more for each concentration. n.s. = not significant compared to control *, p <0.05
compared to control **, p <0.01 compared to control as determined by unpaired t test.
FIGURE 8. Compounds counteract Aβ associated net spine loss in rat primary hippocampal neurons. (A)
Representative spine segments (23 microns) of primary neurons dosed with Aβ, or Aβ plus BTA-EG6 or BAMs 1-3
compared to control (0.1% DMSO) and quantitative representation of spine number per micron for all dosing
experiments compared to control. (B) Representative spine segments (23 microns) of primary neurons pre-treated
with 5 µM BTA-EG6 or BAMs 1-3 for 24 h followed by addition of aggregated Aβ. (C) Relative expression levels
of p-Cofilin/total Cofilin in primary neurons treated with Aβ alone or Aβ plus BTA-EG6 or BAMs 1-3 compared to
13

[Benzothiazole amphiphiles promote spinogenesis]
control expression levels. Data are expressed as mean values ± SEM, n = 42. ## p ≤ 0.01, as determined by unpaired
t-test compared to control. *p ≤ 0.001, *p ≤ 0.0001 as determined by unpaired t-test compared to cells treated with
Aβ alone.
14

[Benzothiazole amphiphiles promote spinogenesis]
Figure 1.
15

[Benzothiazole amphiphiles promote spinogenesis]
Figure 2.
16

[Benzothiazole amphiphiles promote spinogenesis]
Figure 3.
17

[Benzothiazole amphiphiles promote spinogenesis]
Figure 4.
18

[Benzothiazole amphiphiles promote spinogenesis]
Figure 5.
19

[Benzothiazole amphiphiles promote spinogenesis]
Figure 6.
20

[Benzothiazole amphiphiles promote spinogenesis]
Figure 7.
21

[Benzothiazole amphiphiles promote spinogenesis]
Figure 8.
22

Supplementary Information
for
Benzothiazole amphiphiles promote the formation of dendritic spines in primary
hippocampal neurons
Jessica L. Cifelli,† Lara Dozier, ‡ Tim Chung,† Gentry N. Patrick‡ and Jerry Yang†,*
†
‡
From the Department of Chemistry and Biochemistry and the Section of Neurobiology in the Division of Biological
Sciences, University of California, San Diego, La Jolla, California 92093-0358
To whom correspondence should be addressed: Prof. Jerry Yang, Department of Chemistry and Biochemistry, University
of California, 9500 Gilman Drive, La Jolla, California, 92093-0358, Telephone: 858-534-6006; Fax: 858-534-4554;
Email: jerryyang@ucsd.edu
S1

Chemical Synthesis of BAMs1-3:
Figure S1. Synthetic scheme for the preparation of BAM1-3.
Alkylation of 4-hydroxy benzaldehyde (6):
4-Hydroxy benzaldehyde 4 (2g, 16.5 mmole, 1.1 equiv.) and anhydrous potassium carbonate (K2CO3) (4.14g, 29.9 mmole, 2 equiv.)
were dissolved in acetone (30 mL) and let stir under nitrogen (N2) for 30 min. Then 2-chloro-N-methylacetamide 5 (1.61g, 15 mmole,
1 equiv.) and potassium iodide (KI) (249 mg, 1.5 mmole, 0.1 equiv.) were added and let reflux for 24 h. After cooling to room
temperature (RT), solids were filtered off and the solvent was removed and replaced with dichloromethane (DCM). Extraction was
done with 10% sodium hydroxide (NaOH) followed by column chromatography purification (95% DCM/ methanol (MeOH)) to yield
compound 6 as a white solid (2.4 g, 83% yield)
1H NMR (500 MHz, CDCl3): δ9.91 (s, 1H), 7.88(d, 2H), 7.04 (d, 2H), 6.50 (b, 1H), 4.57 (s, 2H), 2.93 (s, 3 H). ESI-MS (m/z):
194.12 [M+ H]+
Synthesis of 4-N-(methylamino) benzaldehyde (7):
To a round bottom with dry toluene, compound 6 (300 mg, 1.55 mmole, 1 equiv.) and potassium hydroxide (KOH) pellets (174 mg,
3.10 mmole, 2 equiv.) were added and let reflux for 24 h. After cooling to RT, the reaction was put on ice and water was added. The
S2

organic layer was washed 3x with water, dried, and concentrated. Column chromatography (50% ethyl acetate (EtOAc)/Hexanes)
yielded compound 7 as a red solid (64 mg, 30% yield).
1H NMR (500 MHz, CDCl3): δ9.72 (s, 1H), 7.71 (d, 2H), 6.61 (d, 2H), 4.41 (b, 1H), 2.91 (s, 3 H). ESI-MS (m/z): 136.19 [M+ H]+
Synthesis of Benzothiazole (9)(1):
A microwave vial was charged with 2-aminothiophenol 7 (45 mg, 0.36 mmole. 1 equiv.), followed by 1-pentyl-3-methylimidazolium
bromide ([pmIm]Br)(2) (29 mg, 0.18 mmole, 0.5 equiv.) and then 4-(methylamino)benzaldehyde 8 (49mg, 0.36 mmole, 1 equiv.). The
mixture was irradiated under MW conditions (150 °C, 4 min). The reaction mixture was extracted with ether/H2O (4x). The ether was
evaporated and the compound was purified by column chromatography (25%DCM/70%Hexanes/5%EtOAc), affording compound 9
as a light orange solid (55 mg, 64% yield).
1H NMR (500 MHz, CDCl3): δ8.02 (d, 1H), 7.96 (d, 2H), 7.84 (d, 1H), 7.44 (t, 1 H), 7.32 (t, 1 H), 6.66 (d, 2 H), 2.92 (s, 3 H). 13
C
NMR (400 MHz, CDCl3): δ169.05, 154.53, 151.82, 134.73, 129.32 (2C), 126.25, 124.50, 122.68, 122.53, 121.60, 112.24 (2C),
30.54. ESI-MS (m/z): 241.0 [M+ H]+
General protocol for (ethylene glycol)6 (EG6) addition:
Synthesis of 17-iodo-3,6,9,12,15-pentaoxaheptadecan-1-ol (EG6-I) was prepared as previously described(3). A microwave vial was
charged with EG6-I (1 equiv.), benzothiazole aniline 9 or 10 (2 equiv.), potassium carbonate (3 equiv.) and tetrahydrofuran (THF).
The mixture was irradiated under MW (125 °C, 2 h). The mixture was filtered, concentrated and normal phase column
chromatography (4% MeOH/EtOAc) followed by reverse phase column chromatography (3:1 MeOH/H2O) yielded compound 1 (285
mg, 48% yield) or compound 2 (13 mg, 30% yield).
BAM1-EG6 (1):
1H NMR (500 MHz, CDCl3): δ7.99 (d, 1H), 7.92 (d, 2H), 7.84 (d, 1H), 7.43 (t, 1 H), 7.30 (t, 1 H), 6.76 (d, 2 H), 4.97 (b, 1H), 3.73-
3.58 (m, 22H), 3.39 (t, 2H). 13C NMR (500 MHz, CDCl3): δ168.92, 154.51, 151.38, 134.74, 129.13 (2C), 126.24, 124.54, 123.20.
122.55, 121.60, 113.28 (2C), 71.68, 69.81-69.03 (69.81, 69.59, 69.45, 69.30, 69.24, 69.23, 69.03), 68.74, 60.44, 43.86. HR/MS (ESI
+): Calcd for [C25H34N2O6S + Na] 513.2030 found 513.2029 [M+Na]+
BAM2-EG6 (2):
S3

1H NMR (500 MHz, CDCl3): δ7.96 (d, 1H), 7.93 (d, 2H), 7.84 (d, 1H), 7.42 (t, 1 H), 7.29 (t, 1 H), 6.76 (d, 2 H), 3.72-3.28 (m, 24H),
3.07 (s, 3H). 13C NMR (500 MHz, CDCl3): δ168.94, 154.64, 151.39, 134.74, 129.17 (2C), 126.19, 124.40, 122.49, 121.57, 121.55,
111.80 (2C), 72.76, 71.0-70.50 (71.00, 70.88, 70.85, 70.80, 70.76, 70.75, 70.71, 70.50), 68.73, 61.93, 52.29, 39.26. HR/MS (ESI-
TOFMS +): Calcd for [C26H36N2O6S + Na] 527.2191 found 527.2187 [M+ Na]+
2-(4-(methylthio)phenyl)benzo[d]thiazole (14):
2-amino thiophenol 8 (376mg, 3 mmol, 1 equiv.), [pmIm]Br (400 mg, 0.5 equiv), 4-(methylthio)benzaldehyde 13 (457 mg, 3 mmol, 1
equiv.) were added respectively, into a 5 mL microwave tube with a stir bar. The reaction tube was microwaved for 4 min at 130oC.
The reaction mixture was dissolved in diethyl ether and extracted with water to remove the ionic liquid solution. The diethyl ether
was removed under reduced pressure and the crude solid 14 was purified by recrystallization in a 3:1 mixture of hexanes:EtOAc (547
mg, 71% yield).
1H NMR (400 MHz, CDCl3): δ 8.05 (d, 1H), 8.01 (d, 2H), 7.90 (d, 1H), 7.49 (t, 1H), 7.38 (t, 1H), 7.33 (d, 2H), 2.55 (s, 3H). 13
NMR (300 MHz, CDCl3): δ143.00, 127.99, 126.56, 125.31, 123.23, 121.81, 15.39. ESI-MS (m/z): 258.25 [M+H]+
C
2-(4-(methylsulfinyl)phenyl)benzo[d]thiazole (15):
2-(4-(methylthio)phenyl)benzo[d]thiazole 14 (300 mg, 1.1 mmol) was dissolved in 6 mL of DCM. meta-chloroperoxybenzoic acid (m-
o
CPBA) (242 mg, 1.4 mmol) was dissolved in 4 mL of DCM and added dropwise at 0 C to the methyl sulfide 14 solution over a
period of 20 min. NaHCO3 (80 mg) was added and the solution was let stir. The reaction mixture was monitored by TLC analysis
(100% EtOAc) until completion. The white precipitate was filtered away and the DCM was removed under reduced pressure to afford
a white solid. The solid was purified by recrystallization in 100% EtOAc to give product 15 (254 mg, 80% yield).
1H NMR (400 MHz, CDCl3): δ 8.26 (d, 2H), 8.11 (d, 1H), 7.94 (d, 1H), 7.78 (d, 2H), 7.53 (t, 1H), 7.44 (t, 1H), 2.79 (s, 3H). ESI-MS
(m/z): 274.17 [M+H]+, 296.10 [M+Na]+
((4-(benzo[d]thiazol-2-yl)phenyl)thio)methyl 2,2,2-trifluoroacetate (16):
2-(4-(methylsulfinyl)phenyl)benzo[d]thiazole (15) (50 mg, 0.18 mmol) was dissolved in 2 mL of freshly distilled DCM in an oven
dried 50 mL round bottom. Trifluoroacetic anhydride (TFAA) (0.15 mL) was added to the reaction flask and the reaction was gently
o
refluxed at 40 C for 2 h under N2. The solvent was removed under reduced pressure to afford the crude product 16 (72 mg,
approximately quantitative conversion). The product was taken on to the next step without further purification.
1H NMR (500 MHz, CDCl3): δ 8.07 (m, 3H), 7.92 (d, 1H), 7.58 (d, 8Hz, 2H), 7.53 (t, 1H), 7.43 (t, 1H), 5.70 (s, 2H)
S4

4-(benzo[d]thiazol-2-yl)benzenethiol (17):
((4-(benzo[d]thiazol-2-yl)phenyl)thio)methyl 2,2,2-trifluoroacetate (16) (72mg, 0.19 mmol) was dissolved in 3 mL of MeOH and 0.6
mL of 1M NaOH was added to the reaction flask and refluxed under N2 for 1 h. The reaction mixture was cooled and the solvent was
removed under reduced pressure. 0.6mL of 1M HCl was then added to the crude mixture and the product was extracted into EtOAc by
washing the aqueous layer with 3 x 2mL of EtOAc. The organic layer was washed with a saturated NaCl solution and dried
over Na2SO4. The EtOAc was removed under reduced pressure to afford the crude product 17 (44 mg, 93% crude yield).
1H NMR (500 MHz, CDCl3): δ 8.08 (d, 1H), 7.95 (d, 2H), 7.90 (d, 1H), 7.51 (t, 1H), 7.39 (m, 3H), 3.68 (s, 1H). ESI-MS (m/z):
244.28 [M+H]+
17-((4-(benzo[d]thiazol-2-yl)phenyl)thio)-3,6,9,12,15-pentaoxaheptadecan-1-ol (3):
In an oven dried 50 mL round bottom, solid sodium hydride (NaH) (2 mg, 0.074 mmol) was added and the round bottom was tightly
capped with a rubber septum. The round bottom was purged with N2. The crude 4-(benzo[d]thiazol-2-yl)benzenethiol (17) (12mg,
0.05 mmol, 1 equiv.) was dissolved in 1mL of freshly distilled dimethylformamide (DMF) and added dropwise to the round bottom
flask containing NaH. The reaction mixture was stirred for 30 min. 17-iodo-3,6,9,12,15-pentaoxaheptadecan-1-ol (EG6-I, 20 mg, 0.05
mmol, 1 equiv.) was dissolved in 1 mL of freshly distilled DMF in a separate vial and added dropwise into reaction mixture. The
reaction was then refluxed under N2 for 12 h. The reaction mixture was cooled to RT and the solvent was removed under reduced
pressure. The product was purified via silica gel flash chromatography (using a gradient of EtOAc:MeOH 0-4%) to afford the desired
product 3 as a yellow oil (Rf=0.24, 100% EtOAc). The yellow oil product was purified once more using a reverse-phase preparatory
plate (using a 3:1 mixture of MeOH:H2O as eluent) to give final product 3 (11mg, 44% yield).
BAM3-EG6 (3):
1H NMR (500 MHz, CDCl3): δ 8.04 (d, 1H), 7.99 (d, 2H), 7.89 (d, 1H), 7.48 (t, 1H), 7.41 (d, 2H), 7.37 (t, 2H), 3.74-3.70 (m, 4H),
3.64 (m, 16H), 3.60-3.58 (m, 2H), 3.20, (t, 2H). 13C NMR (500 MHz, CDCl3): δ167.42, 154.09, 140.57, 134.88, 130.83, 128.01,
127.86, 126.38, 125.18, 123.08, 121.62, 72.50, 70.64-70.30 (70.64, 70.59, 70.55, 70.53, 70.50, 70.30), 69.68, 61.74, 32.08. HR/MS:
calcd for C25H33NO6S2 [M+Na] 530.1641 found [M+Na] 530.1640
S5

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for Knoevenagel Condensation of Aliphatic and Aromatic Carbonyl Compounds Using a Task-Specific Basic Ionic Liquid.
European J. Org. Chem. 2006, 3767–3770
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S6

Benzothiazole Amphiphiles Promote the Formation of Dendritic Spines in Primary
Hippocampal Neurons
Jessica L. Cifelli, Lara Dozier, Tim Chung, Gentry N. Patrick and Jerry Yang
J. Biol. Chem. published online March 28, 2016
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