Microwave-assisted synthesis of 4-chloro-N-(naphthalen-1-ylmethyl)-5-(3- (piperazin-1-yl)phenoxy)thiophene-2-sulfonamide (B-355252): A new potentiator of nerve growth factor (NGF)-induced neurite outgrowth

Article abstract of DOI:10.1016/j.tet.2010.09.028

The synthesis of 4-chloro-N-(naphthalen-1-ylmethyl)-5-(3-(piperazin-1-yl) phenoxy)thiophene-2-sulfonamide (B-355252) using an MW-assisted nucleophilic aromatic substitution (S_NAr) reaction will be discussed. Utilization of this method allowed for the rapid generation of B-355252 heteroaryl ether core structure in the presence of cesium carbonate in dimethylformamide or tripotassium phosphate in N-methyl-2-pyrrolidone in 94% yield. Evaluation of B-355252 enhancement of nerve growth factor's ability to stimulate neurite outgrowths was determined using NS-1 cells.

Full text of DOI:10.1016/j.tet.2010.09.028

Tetrahedron 66 (2010) 9577e9581
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Microwave-assisted synthesis of 4-chloro-N-(naphthalen-1-ylmethyl)-5-(3-
(piperazin-1-yl)phenoxy)thiophene-2-sulfonamide (B-355252): a new
potentiator of nerve growth factor (NGF)-induced neurite outgrowth
,
b
,
b
b
b
Alfred L. Williams a , Srinivasa R. Dandepally , Nailya Gilyazova , Sam M. Witherspoon ,
*
,
b
Gordon Ibeanu a
a Department of Pharmaceutical Sciences, North Carolina Central University, Durham, NC 27707, USA
b Biomanufacturing Research Institute and Technology Enterprise, North Carolina Central University, Durham, NC 27707, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 10 July 2010
Received in revised form 4 September 2010
Accepted 7 September 2010
Available online 21 September 2010
The synthesis of 4-chloro-N-(naphthalen-1-ylmethyl)-5-(3-(piperazin-1-yl)phenoxy)thiophene-2-sul-
fonamide (B-355252) using an MW-assisted nucleophilic aromatic substitution (SNAr) reaction will be
discussed. Utilization of this method allowed for the rapid generation of B-355252 heteroaryl ether core
structure in the presence of cesium carbonate in dimethylformamide or tripotassium phosphate in
N-methyl-2-pyrrolidone in 94% yield. Evaluation of B-355252 enhancement of nerve growth factor’s
ability to stimulate neurite outgrowths was determined using NS-1 cells.
Keywords:
Ó 2010 Elsevier Ltd. All rights reserved.
Microwave synthesis
Nucleophilic aromatic substitution
Nerve growth factor
Neurite outgrowth
1. Introduction
alternative for enhancing neurite outgrowth by potentiating NGF-
dependent activity in neuronal cells.
Nerve growth factor (NGF) is a member of a family of proteins
called neurotrophins.1 Other members of this family of proteins
includes brain-derived neurotrophic factor (BDNF), neurotrophin
(NT)-3, and NT-4/5. NGF is involved in the survival, development
and function of neurons located in the central, and peripheral
nervous system.2 In the central nervous system, NGF is synthesized
predominantly by neurons under physiological conditions3 and has
been shown to upregulate cholinergic function and to prevent
degeneration of basal forebrain neurons.4 The neuroprotective role
displayed by NGF and the observations of reversing atrophy and
age-related cognitive impairment made in animal models of neu-
rodegeneration helped to establish the use of NGF as a potential
treatment for a variety of neurological diseases like Alzheimer’s
disease (AD), Parkinson’s disease (PD) and Amyotrophic lateral
sclerosis (ALS).5 Unfortunately, delivery of exogenous NGF failed to
demonstrate efficacy in several human clinical trials.6 The in-
effectiveness of NGF as a treatment modality is hampered by the
challenges of developing peptides as clinical candidates, which
includes penetrating the bloodebrain barrier, metabolic instability
and ineffective protein delivery.7 To overcome these hurdles, efforts
have been focused on identifying small organic molecules as an
Overthe years, avariety of small molecules have been reported to
enhance neurite outgrowth by potentiating NGF dependent activity
in neuronal cells.8 Natural products like verbenachalcone (VC) and
a host of other small molecules, such as the potent acetylcholines-
terase (AChE) inhibitor donepezil, were reported to stimulate
neurite outgrowth (Fig. 1).9,10 A recent primary screen of our library
collection11 led us tothe discoveryof N-(naphthalen-1-ylmethyl)-5-
Fig. 1. Compounds that stimulate neurite outgrowth in neuronal cells.
(3-(piperazin-1-yl)phenoxy)thiophene-2-sulfonamide, B-355252
(Fig 1), which amplified neurite outgrowth in NS-1 cells in the
presence of NGF. This finding launched the exploration into the
* Corresponding author. E-mail address: awilliams@nccu.edu (A.L. Williams).
0040-4020/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2010.09.028

9578
A.L. Williams et al. / Tetrahedron 66 (2010) 9577e9581
Table 1
resynthesis of this new compound to confirm its structure and bi-
ological activity.
Optimization of reaction conditions
Basea
Solvent
Temperature
MW, 120 ꢀC
80 ꢀC
T (min)
Yieldb (%)
To assemble the heteroaryl ether core structure of B-355252, we
recently developed a nucleophilic aromatic substitution (SNAr)
methodology, which involved reacting substituted phenols with
p-methoxybenzyl (PMB) protected 4,5-chloro-N-(aryl/alkyl) thio-
phene-2-sulfonamides.12 Under conventional heating, this method
requires the use of a PMB protecting group for this reaction to pro-
ceed in 3e6 h. Although this reaction proceeds well using thermal
heating, we want to develop a methodology, which could possibly
eliminate the use of PMB protection and also develop a way to
rapidly generate many diverse analogs of B-355252 by shortening
the reaction times for synthesizing its heteroaryl ether core. One
approach, which can be used accomplish this would be to heat the
reaction using microwave (MW) irradiation. Due to its ability to in-
crease reaction rates and to produce cleaner reactions in higher
yields, microwave assisted synthesis is widely used to improve re-
action conditions in organic synthesis.13 Herein we report an MW-
assisted SNAr approach for the synthesis of B-355252 and evaluation
of its enhancement of NGF activity on neurite outgrowths.
Entry
1
2
3
4
5
6
7
8
Cs2CO3
Cs2CO3
K2CO3
Na2CO3
K3PO4
Cs2CO3
Cs2CO3
Cs2CO3
Cs2CO3
K3PO4
K3PO4
K3PO4
K3PO4
K3PO4
K3PO4
DMF
DMF
DMF
DMF
DMF
DMF
DMF
DMSO
DMA
NMP
NMP
NMP
NMP
NMP
NMP
25
2.5 h
25
30
30
10
5
10
10
20
10
10
6
94
94
MW, 120 ꢀC
MW, 120 ꢀC
MW, 120 ꢀC
MW, 150 ꢀC
MW, 200 ꢀC
MW, 150 ꢀC
MW, 200 ꢀC
MW, 120 ꢀC
MW, 140 ꢀC
MW, 160 ꢀC
MW, 180 ꢀC
MW, 200 ꢀC
140 ꢀC
50c
10c
60c
60c
MPd
60c
20c
60c
85c
97
9
10
11
12
13
14
15
91
94
97
4
40
a
Amount of base used, Cs2CO3, K2CO3, and Na2CO3: 1.5 equiv; K3PO4: 3 equiv
b
c
Isolated yields.
Conversion based on LC/MS.
Multiple products.
d
reaction temperature above 120 ꢀC in DMF, DMSO or DMA only
gave reduced yields and multiple products (Table 1, entries 5e8).
We then switched to using the base and solvent combination of
K3PO4 in NMP. Initial heating at 120 ꢀC for 20 min gave only a 60%
conversion to 6 (Table 1, entry 10) but as the temperature was in-
creased, the reaction times decreased and the yield of 6 increased
(Table 1, entries 11e14). The best results were obtained when the
reaction was heated to 200 ꢀC for 4 min to produce 6 in a yield of
94% (Table 1, entry 14). In addition, we also observed that K3PO4 in
NMP substantially reduced the reaction time when heated ther-
2. Results and discussion
To examine the use of MW irradiation toward synthesizing the
heteroaryl ether core of B-355252, we first synthesized the SNAr
precursor, PMB protected 4,5-dichlorothiophene sulfonamide 4, by
reacting commercially available 4,5-dichlorothiophene-2-sulfonyl
chloride 1 with 1-napthlymethyl amine 2 to generate 4,5-dichlor-
othiophene sulfonamide 3 in 91% yield. We then reacted 3 with
NaH in DMF, p-methoxybenzyl bromide and a catalytic amount of
TBAI to obtain compound 4 in 91% yield (Scheme 1).
Scheme 1.
With 4 now in hand, we began investigating the optimal con-
ditions to use in our MW assisted SNAr reaction using a variety of
reaction conditions (Table 1). When we initiated our search using
Cs2CO3 in DMF as the base and solvent, we observed nucleophilic
displacement of the C-5 chlorine of 4 with N-Boc protected 3-
(piperazin-1-yl)phenol 5 to produce N,N0-diprotected 5-(3-(piper-
mally. Under these conditions, 6 was generated in an excellent yield
of 97% at 140 ꢀC after heating for only 40 min (Table 1, entry 15).
This enhancement of reaction times using both MW and thermal
heating reveals the versatility of this base and solvent system in our
SNAr reactions.
We next turned our attention toward determining whether MW
irradiation can be used to overcome the need for using a PMB
protecting group in our SNAr reaction (Scheme 2).
Using both Cs2CO3 in DMF and K3PO4 in NMP, we microwave
irradiated unprotected 4,5-dichlorothiophene sulfonamide 3 in the
presence of phenol 5 (Table 2). We found that we were only able to
achieve 3e6% conversion to compound 7 after 30 min when we
azin-1-yl)phenoxy)thiophene-2-sulfonamide
6 in an excellent
yield of 94% after only 25 min with MW heating (Table 1, entry 1).
When compared to thermal heating, this reaction required 2.5 h to
produce 6 in the same yield (Table 1, entry 2). The use of other
bases, such as K2CO3, Na2CO3, and K3PO4 in DMF resulted in lower
yields of 6 (Table 1, entries 3e4 and 9). Attempts to elevate the

A.L. Williams et al. / Tetrahedron 66 (2010) 9577e9581
9579
Scheme 2.
Table 2
complete our synthesis, both PMB and Boc protecting groups of
compound 6 were simultaneously removed using TFA in DCM (1:1)
to give us B-355252 in 97% yield (Scheme 3).
Attempted reaction conditions
Entry
Base (equiv)
Conditions
Yield (%)a
DMF, MW, 120 ꢀC, 30 min
DMF, MW, 140 ꢀC, 30 min
DMF, MW, 160 ꢀC, 30 min
NMP, MW, 160 ꢀC, 20 min
NMP, MW, 180 ꢀC, 20 min
NMP, MW, 200 ꢀC, 20 min
NMP, 140 ꢀC, 40 h
3
3
6
With B-355252 in hand, we commenced assessing the com-
pounds enhancement of NGF-primed neurite outgrowth (NOG) in
a Neuroscreen-1 (NS-1) cell line.14 We used verbenachalcone (VC) as
a positive control compound for biological activity potentiation of
NGF in NS-1 cells. The ratio of neutite bearing cells to total cells was
detected using a Beckton Dickinson (BD) Pathway 855 High-Content
automated imaging platform and Cellomics NOG kit. The data was
analyzed with a BD AttoVision neurite outgrowth software version
1.6 (BD Biosciences, Franklin Lakes, NJ). In the presence of 2 ng/mL
NGF, VC shows a dose-dependent enhancement of NGF’s effects with
1
2
3
4
5
6
7
Cs2CO3 (1.5)
Cs2CO3 (1.5)
Cs2CO3 (1.5)
K3PO4 (3)
K3PO4 (3)
K3PO4 (3)
12b
14b
16b
26b
K3PO4 (3)
a
Based on LC/MS analysis.
In addition to unreacted 3, several minor products (LC/MS) also present.
b
used Cs2CO3 in DMF (Table 2, entries 1e3). MW irradiation for
longer than 30 min resulted in compound decomposition. When
we attempted to use K3PO4 in NMP, the conversion to compound 7
was slightly improved but multiple products were detected by LC/
MS (Table 2, entries 4e6). When we heated the reaction thermally,
we saw a 26% conversion to product 7 (Table 2, entry 7). Previous
attempts using a variety different bases and solvents resulted in no
product formation.12a Although using K3PO4 in NMP gave product 7,
the reaction was not clean and required heating for 40 h. To
a peak enhancement at 15 mM when compared to NGF treatment
alone (Fig. 2, panel A). This result is consistent with what was ob-
served by Li et al. using PC-12 cells.9a We next examined B-355252 at
various concentrations in the presence of 1 ng/mL NGF. This lower
NGF concentration was used to better observe the neurite outgrowth
and elongation effects of B-355252. As shown (Fig. 2, panel B), co-
treatment of NS-1 with 1 ng/mL NGF and B-355252 was accompa-
nied by a marked dose-dependent increase neutite bearing cells,
with a peak enhancement of approximately 2-fold observed in the
Scheme 3.
Fig. 2. Resynthesized B-355252 potentiates NGF dependent neurite outgrowth and elongation in a neuronal cell model. Panel A shows the response of control VC assay in 2 ng/mL
NGF while panel B represents the dose dependent activity of B-355252 for NOG in the presence of 1 ng/mL NGF. Each data point was acquired in triplicate from two separate
experiments.

9580
A.L. Williams et al. / Tetrahedron 66 (2010) 9577e9581
presence of 1.33
m
M B-355252 when compared to NGF treatment
extracted with CH2Cl2 (100 mL), washed with brine, dried (Na2SO4),
and concentrated under vacuo. The residue was purified by re-
crystallization from CH2Cl2/hexane to afford 3 (1.350 g, 91%) as
a white crystalline product; mp:136e138 ꢀC; 1H NMR (500 MHz,
alone. B-322525 was devoid of neurite promoting properties in the
NS-1 neuronal cell model in the absence of NGF (data not shown).
From the generated dose response curve (Fig. 1, panel B), B-355252
was determined to display an EC50 of w1.0
m
M. The EC50 of resyn-
CDCl3)
d
(ppm) 4.66 (d, 2H, J¼4.0 Hz), 4.98 (br s, 1H), 7.21 (s, 1H),
thesized B-355252 is essentially the same as the value obtained from
the original hit compound in our initial primary screen and this
confirmed our compounds structure and activity.
7.34e7.40 (m, 2H), 7.49e7.56 (m, 2H), 7.79e7.83 (m,1H), 7.85 (d,1H,
J¼2.5 Hz), 7.90 (d, 1H, J¼3.0 Hz); 13C NMR (125 MHz, CDCl3)
d (ppm)
45.8, 122.9, 124.6, 125.1, 126.2, 126.9, 127.4, 128.9, 129.5, 130.4, 131.0,
131.1, 133.8, 137.2; HRMS (ESI) m/z calcd for C15H11Cl2NO2S2Na
[MþNa]þ 393.9500, obsd: 393.9503.
3. Conclusions
The synthesis of B-355252, N-(naphthalen-1-ylmethyl)-5-(3-
(piperazin-1-yl)phenoxy)thiophene-2-sulfonamide, was success-
fully completed using an MW assisted SNAr reaction. The generation
of the heteroaryl ether core structure of B-355252 was optimal in the
presence of Cs2CO3 in DMF or K3PO4 in NMP. Addition of N-Boc
protected phenol 5 to give N,N0-diprotected compound 6 occurs
more rapidly and in higher yields when 4,5-dichlorothiophene sul-
fonamide 4 is PMB protected compared to low product 7 conversion
and by-product formation without this protection. These results
stress the importance of having a PMB protecting group present in
this reaction. Resynthesized B-355252 enhancement of NGF’s effects
on neurite outgrowths was demonstrated in an NS-1 cell line thus
confirming its biological activity. The synthesis of the heteroaryl
ether core using our MW assisted SNAr methodology should allow
for the very rapid generation of many diverse analogs of B-355252 to
be used in future structureeactivity relationship (SAR) studies.
4.1.2. 4,5-Dichloro-N-(4-methoxybenzyl)-N-(naphthalen-1-yl-
methyl)thiophene-2-sulfonamide (4). Sodium hydride (0.081 g,
3.375 mmol) was slowly added in portions to a solution of 3 (1.250 g,
3.358 mmol) in anhydrous DMF (10 mL) at 0 ꢀC and stirred for
15 min. Then, 4-methoxybenzyl bromide (PMBBr) (0.675 g,
3.357 mmol) and a catalytic amount of TBAI (0.030 g, 0.081 mmol)
were added and stirred at room temperature for 2 h. After the
completion of the reaction, it was quenched by slow addition of
water (5 mL) and extracted with EtOAc (100 mL), washed with water
and brine, dried (Na2SO4), and concentrated under vacuo. The resi-
due purified by flash silica gel column chromatography (CombiflashÒ
Rf) using EtOAc/hexane (1:9) as eluant to afford 4 (1.500 g, 91%) as
a white solid; mp: 63e65 ꢀC; 1H NMR (500 MHz, CDCl3)
d (ppm) 3.71
(s, 3H), 4.28 (s, 2H), 4.83 (s, 2H), 6.61 (d, 2H, J¼8.5 Hz), 6.90 (d, 2H,
J¼8.5 Hz), 7.10 (s, 1H), 7.32e7.38 (m, 2H), 7.46e7.50 (m, 2H), 7.76 (d,
1H, J¼7.0 Hz), 7.78e7.82 (m, 1H), 8.02e8.05 (m, 1H); 13C NMR
(125 MHz, CDCl3)
d (ppm) 50.2, 51.1, 55.2, 113.6, 123.4, 124.7, 124.9,
4. Experimental
126.0, 126.5, 127.0, 127.7, 128.6, 129.2, 129.4, 129.7, 130.0, 130.7, 130.9,
131.6, 133.7, 136.9, 159.1; HRMS (ESI) m/z calcd for C23H19Cl2NO3S2Na
[MþNa]þ 514.0076, obsd: 514. 0078.
4.1. General experimental procedures
All solvents and reagents were obtained from commercial sour-
ces and used without further purification unless otherwise stated.
All reactions were performed in oven-dried glassware (either in RB
flasks or 10 mL microwave tubes equipped with a cap) under an
atmosphere of nitrogen and the progress of reactions was monitored
by thin-layer chromatography and LC/MS. All microwave irradiations
were performed using a CEM Explorer 24 sample microwave reactor.
Analytical thin-layer chromatography was performed on precoated
4.1.3. tert-Butyl 4-(3-(3-chloro-5-(N-(4-methoxybenzyl)-N-(naph-
thalen-1-ylmethyl)sulfamoyl)thiophen-2-yloxy)phenyl)piperazine-
1-carboxylate (6). A mixture of 4 (0.050 g, 0.101 mmol), tert-butyl
4-(3-hydroxyphenyl)piperazine-1-carboxylate
(5)
(0.034
g,
0.122 mmol), and Cs2CO3 (0.050 g, 0.152 mmol) in anhydrous DMF
(1 mL) was taken in a 10 mL microwave tube, and the tube sealed
with a pressure cap. The tube was submitted to microwave irradia-
tion for 25 min at 120 ꢀC. The solvent was evaporated under vacuo
and the residue purified by flash silica gel column chromatography
(CombiflashÒ Rf) using EtOAc/hexane (1:4) to afford 6 as a white
solid (0.070 g, 94%); mp: 116e118 ꢀC; 1H NMR (CDCl3, 500 MHz):
250 mm layer thickness silica gel 60 F254 plates (EMD Chemicals Inc.).
Visualization was performed by ultraviolet light and/or by staining
with phosphomolybdic acid (PMA) or p-anisaldehyde. All the silica
gel column chromatography purifications were carried out by using
CombiflashÒ Rf (Teledyne Isco) either with EtOAc/hexane or MeOH/
CH2Cl2 mixtures as the eluants. Melting points were measured on
a MEL-TEMPÒ capillary melting point apparatus and are uncorrected.
Proton nuclear magnetic resonance (1H NMR) spectra and carbon
nuclear magnetic resonance (13C NMR) spectra were recorded on
d
(ppm) 1.48 (s, 9H), 3.16 (t, 4H, J¼5.0 Hz), 3.57 (t, 4H, J¼5.0 Hz), 3.71
(s, 3H), 4.27 (s, 2H), 4.82 (s, 2H), 6.52 (dd, 1H, J¼2.5, 8.5 Hz),
6.58e6.62 (m, 2H), 6.65 (t, 1H, J¼2.5 Hz), 6.75 (dd, 1H, J¼2.5, 8.5 Hz),
6.86e6.90 (m, 2H), 7.20 (s, 1H), 7.25 (t, 1H, J¼8.0 Hz), 7.31e7.37 (m,
2H), 7.45e7.50 (m, 2H), 7.75 (d, 1H, J¼8.0 Hz), 7.79e7.82 (m, 1H),
8.03e8.06 (m, 1H); 13C NMR (CDCl3, 125 MHz):
d (ppm) 28.4, 48.7,
a Varian VNMRS-500 (500 MHz) spectrometer. Chemical shifts (
d
)
49.9, 50.9, 55.2, 80.0, 105.6, 108.2, 111.8, 112.9, 113.5, 123.5, 124.9,
125.9, 126.5, 127.2, 127.5, 127.9, 128.6, 129.1, 129.7, 130.2, 130.5 (2),
131.7, 133.7, 152.9, 154.6, 158.3, 159.0; HRMS (ESI) m/z calcd for
C38H41ClN3O6S2 [MþH]þ 734.2120, obsd: 734.2117.
for proton are reported in parts per million (ppm) downfield from
tetramethylsilane and are referenced to it (TMS 0.0 ppm). Coupling
constants (J) are reported in hertz. Multiplicities are reported using
the following abbreviations: br¼broad; s¼singlet; d¼doublet;
t¼triplet; q¼quartet; m¼multiplet. Chemical shifts (
d
) for carbon are
4.1.4. 4-Chloro-N-(naphthalen-1-ylmethyl)-5-(3-(piperazin-1-yl)
phenoxy)thiophene-2-sulfonamide (B-355252). To a solution 6
reported in parts per million (ppm) downfield from tetramethylsi-
lane and are referenced to residual solvent peaks: carbon (CDCl3
77.0 ppm). HRMS data were recorded on an Agilent Technologies
6210 LC-TOF.
(0.140 g, 0.191 mmol) in anhydrous CH2Cl2 (1 mL) was added TFA
(1 mL) and stirred at room temperature for 2 h. The solvent mixture
was removed under vacuo and the residue was re-dissolved in
CH2Cl2 (25 mL), washed with aqueous satd NaHCO3 followed by
brine, dried (Na2SO4), and concentrated under vacuo. The crude
product was purified by flash silica gel column chromatography
(CombiflashÒ Rf) using MeOH/CH2Cl2 (1:9) to afford B-355252 as
a white solid (0.095 g, 97%); mp: 142e144 ꢀC; 1H NMR (CDCl3,
4.1.1. 4,5-Dichloro-N-(naphthalen-1-ylmethyl)thiophene-2-sulfon-
amide (3). To a solution of 4,5-dichlorothiophene-2-sulfonyl chlo-
ride (1) (1.000 g, 4.002 mmol) in anhydrous CH2Cl2 (20 mL) was
added 1-naphthylmethylamine (2) (0.630 g, 4.007 mmol) followed
by Et3N (0.84 mL, 6.027 mmol) and stirred at room temperature for
2 h. The reaction mixture was diluted with water (20 mL) and
500 MHz):
d
(ppm) 2.81 (t, 4H, J¼5.0 Hz), 3.09 (t, 4H, J¼5.0 Hz), 4.56
(s, 2H), 6.43 (dd, 1H, J¼2.0, 8.0 Hz), 6.75 (t, 1H, J¼2.5 Hz), 6.83 (dd,

A.L. Williams et al. / Tetrahedron 66 (2010) 9577e9581
9581
1H, J¼2.5, 8.0 Hz), 7.26 (t, 1H, J¼8.0 Hz), 7.43e7.48 (m, 3H),
7.54e7.58 (m, 2H), 7.87 (dd, 1H, J¼1.5, 7.5 Hz), 7.93e7.96 (m, 1H),
8.06e8.09 (m, 1H); 13C NMR (CDCl3, 125 MHz):
(ppm) 44.5, 45.3,
48.6, 104.3, 106.7, 109.5, 112.1, 123.5, 125.2, 125.8, 126.2, 126.8, 128.3,
128.5, 129.0, 129.6, 130.5, 130.8, 132.2, 133.2, 153.3, 157.5, 157.8;
HRMS (ESI) m/z calcd for C25H25ClN3O3S2 [MþH]þ 514.1020, obsd:
514.1028.
Supplementary data
d
Supplementary data associated with this article can be found in
the online version at doi:10.1016/j.tet.2010.09.028.
References and notes
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4.2. Neurite outgrowth assay
Eds.; Lippincott/Williams
& Wilkins: New York, NY, 1998; Chapter 19,
pp 383e398; (b) Kaplan, D. R.; Miller, F. D. Curr. Opin. Neurobiol. 2000, 10,
381e391.
2. (a) Hempstead, B. L. Curr. Alzheimer Res. 2006, 3, 19e24; (b) Reichardt, L. F.
Philos. Trans. R. Soc., B 2006, 361, 1545e1564.
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5. Price, R. D.; Milne, S. A.; Sharkey, J.; Matsuoka, N. Pharmacol. Ther. 2007, 115,
292e306.
The enhancement of NGF-primed neurite outgrowth (NOG) by
resynthesized B-355252 was assessed in the Neuroscreen-1 (NS-1)
cell line, a clonal outgrowth of the rat pheochromocytoma PC12
neuronal cell model, using the Beckton Dickinson (BD) Pathway
855 High-Content automated imaging platform and Cellomics NOG
kit (Cellomics, Inc., Pittsburg, PA). NS-1 cells were plated at 1ꢁ103
cells/well in a 96-well imaging plate. Following overnight in-
cubation at 37 ꢀC in a humidified cell culture incubator, the wells
were co-treated with NGF (1 ng/mL) and various concentrations of
B-355252 in treatment media (RPMI-1640 supplemented with 1%
FBS, 2% horse serum, and 4 mM glutamine). The neurite bearing
cells as a percentage of total cells were detected and captured on
the BD bioimager after 48 h of compound treatment by staining the
cells with fluorescent labeled antibody neurite outgrowth reagent
kit (Cellomics, Pittsburg, PA) according to the manufacturer’s pro-
tocol. The image was analyzed with BD AttoVision neurite out-
growth software version 1.6 (BD Biosciences, Franklin Lakes, NJ).
Cells with neurite processes equal to or greater than twice the di-
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Acknowledgements
We gratefully acknowledge The Golden LEAF Foundation, Rocky
Mount, NC, for the financial support. We thank Dr. Li-An Yeh, Di-
rector, BRITE, for the encouragement. The project described was
supported by Award Number SC3GM081092 from the National
Institute of General Medical Sciences. The content is solely the re-
sponsibility of the authors and does not necessarily represent the
official views of the National Institute of General Medical Sciences
or the National Institutes of Health.
€
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