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

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

[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-EG₆reveal 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-EG₆and 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-EG₄was 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-EG₄suggest

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-EG₄precluded 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-EG₆was 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/cm²onto 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-EG₆or 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-EG₆were 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 (λ_max) 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 MgCl₂and 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

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[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-EG_4,6agents 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-EG₆as 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-EG₆could 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 λ_maxthat 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-EG₆for 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-EG₆for 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 λ_maxin pure octanol)

compared to a 50% change in λ_maxfor 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-

EG₆over the control (Figure 4).

The cumulative distribution of spine

length and width was also measured for neurons

dosed with BAM1-EG_6.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-EG₆exhibited moderate

toxicity to rat primary neurons with an IC₅₀of 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-EG₆was 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-EG₆results 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-EG₆was 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-EG₆did 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-EG₆as 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-EG₆every 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-EG₆DMSO stock (final

concentration 5 µM, 0.1% DMSO) was added to

the culture medium. We found that further

addition of BAM1-EG₆every 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-EG₆compared 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-EG₆for 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-EG₆could promote spine density

increases in vitro in murine primary hippocampal

neurons (37) and BTA-EG₄could 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-

EG_4,6correlated 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-EG_4,6, 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-EG₆were ~ 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-EG₆and benzothiazoles 1-3

effectively protects cells from an overall net

decrease in dendritic spine density induced by

aggregated Aβ (1-42) peptides.

EG_4,6

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-EG₆with 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-EG₆and 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-EG₆together 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-EG_4,6to

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-EG₆was 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-EG₆showed 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-

EG₆were 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-EG₆was 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-EG_4,6compounds

(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-EG₆returned 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-EG₄on 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-EG₆in water, octanol, or an aqueous solution of liposomes. (B) Viability of primary neurons as

a function of increasing concentration of BTA-EG₆or BAMs 1-3. (C) Table of calculated hydrophobic parameters,

measured membrane partitioning properties, and IC₅₀values 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-EG₆and BAMs 1-3 observed in rat primary hippocampal neurons. (A)

Representative spine segments (23 microns) for BTA-EG₆and 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-EG₆compared 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-EG₆compared to control. (D) Quantitative representation of amount of colocalized PSD95 and Synapsin

puncta per micron for neurons treated with BAM1-EG₆compared 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-EG₆observed 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-EG₆on spine density. (D) Kinetics of spine density increase in cells exposed to BAM1-EG₆compared 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-EG₆on dendritic spine number after treatment of cells for 24 h. After 24 h, BAM1-EG₆was

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-EG₆is indistinguishable from control cells. (F) Effect on spine

density increases of adding additional doses of BAM1-EG₆every 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-EG₆or 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-EG₆and 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-EG₆and 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-EG₆or 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-EG₆or 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-EG₆or 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.

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[Benzothiazole amphiphiles promote spinogenesis]

Figure 2.

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[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.

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[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 130^oC.

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 N₂for 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 N₂for 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 (R_f=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

Supporting Information References:

1.

Ranu, B. C., and Jana, R. (2006) Ionic Liquid as Catalyst and Reaction Medium – A Simple, Efficient and Green Procedure

for Knoevenagel Condensation of Aliphatic and Aromatic Carbonyl Compounds Using a Task-Specific Basic Ionic Liquid.

European J. Org. Chem. 2006, 3767–3770

2.

3.

Namboodiri, V. V., and Varma, R. S. (2002) Solvent-Free Sonochemical Preparation of Ionic Liquids. Org. Lett. 4, 3161–

3163

Prangkio, P., Rao, D. K., Lance, K. D., Rubinshtein, M., Yang, J., and Mayer, M. (2011) Self-assembled, cation-selective ion

channels from an oligo(ethylene glycol) derivative of benzothiazole aniline. Biochim. Biophys. Acta 1808, 2877–85

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

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