Synthesis of retinoid enhancers based on 2-aminobenzothiazoles for anti-cancer therapy

Article abstract of DOI:10.1016/j.bmc.2012.09.035

Indole-3-amides and dipeptides were produced from 2-aminobenzothiazoles using the PyBop peptide coupling reagent. These analogues were tested in anti-cancer cell viability assays against SH-SY5Y neuroblastoma and MDA-MB-231 breast adenocarcinoma cell lines, and were found to exhibit cytotoxic activities at concentrations ranging from 0.1 to 20 μM. These compounds were also found to act additively with a low dosage of 13-cis-retinoic acid in neuroblastoma cells. Then, using neuroblastoma cells transfected to stably overexpress the RARβ₂ gene, a SAR was developed for the indole-3-amides. Real-time PCR was also used to demonstrate their RARβ₂ agonistic activity.

Full text of DOI:10.1016/j.bmc.2012.09.035

Bioorganic & Medicinal Chemistry 20 (2012) 6877–6884
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of retinoid enhancers based on 2-aminobenzothiazoles
for anti-cancer therapy
Christopher R. Gardner a,b, Belamy B. Cheung b, Jessica Koach b, David StC. Black a, Glenn M. Marshall b,c
,
Naresh Kumar a,
⇑
a School of Chemistry, The University of New South Wales, Sydney, NSW 2052, Australia
b Children’s Cancer Institute Australia for Medical Research, Lowy Cancer Research Centre, The University of New South Wales, Sydney, NSW 2052, Australia
c Centre for Children’s Cancer and Blood Disorders, Sydney Children’s Hospital, Randwick, NSW 2031, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
Indole-3-amides and dipeptides were produced from 2-aminobenzothiazoles using the PyBop peptide
coupling reagent. These analogues were tested in anti-cancer cell viability assays against SH-SY5Y neu-
roblastoma and MDA-MB-231 breast adenocarcinoma cell lines, and were found to exhibit cytotoxic
Received 13 June 2012
Revised 6 September 2012
Accepted 14 September 2012
Available online 3 October 2012
activities at concentrations ranging from 0.1 to 20 lM. These compounds were also found to act addi-
tively with a low dosage of 13-cis-retinoic acid in neuroblastoma cells. Then, using neuroblastoma cells
transfected to stably overexpress the RARb2 gene, a SAR was developed for the indole-3-amides. Real-
time PCR was also used to demonstrate their RARb2 agonistic activity.
Keywords:
Benzothiazole
Neuroblastoma
Retinoid
Ó 2012 Elsevier Ltd. All rights reserved.
Indole
Dipeptide
1. Introduction
standard therapy for patients with high risk neuroblastoma in their
first remission. However, due to the inherent toxicity associated
Neuroblastoma, an embryonal malignancy of the sympathetic
nervous system, is the most common extracranial solid tumor in
the pediatric population, accounting for approximately 7% of all
childhood cancers.1 Almost 50% of patients are diagnosed between
the ages of 1 and 4, with a further 30% diagnosed before the age of
1. Approximately 80% of patients over 18 months present with
metastatic disease and despite intensive treatment, including
high-dose chemotherapy, surgery, radiotherapy, autologous bone
marrow transplantation, retinoids and more recently immunother-
apy, have a dismal 3-year event-free survival (EFS) rate of 38%.2
Retinoids (vitamin A derivatives) have preventative and thera-
peutic activity for many types of human cancers. The effective
use of retinoids as differentiation reagents and tumor growth
inhibitors has been well documented since the first use of all-
trans-retinoic acid in the treatment of patients with acute promy-
elocytic leukemia (APL) in the mid 1980s.3 Since then, a number of
clinical trials have shown that treatment with natural and syn-
thetic retinoids can induce tumor cell differentiation and reduce
the occurrence of secondary malignancies.4 Furthermore, treat-
ment with isotretinoin (13-cis-retinoic acid) is currently the
with natural derivatives of vitamin A, the use of retinoids as che-
motherapeutics is somewhat limited. Therefore, it is highly desir-
able to develop compounds which act synergistically with
retinoids to enhance their activity in cancer cell lines, whilst min-
imizing teratogenic and mucocutaneous toxicities.5
The activity of retinoids is mediated mainly through their inter-
actions with the nuclear retinoic acid receptors (RARs) and retinoid
X receptors (RXRs). Each of these receptors has three subtypes (
a, b
and ), with each subtype having a number of isoforms with differ-
c
ing affinities to retinoids and different biological functions. It has
been demonstrated that a loss of RARb2 expression is associated
with tumorigenesis and retinoid resistance. The observation that
RARb2 was upregulated in patients after treatment with 13-cis-
RA, and that increased expression of RARb2 was correlated with
clinical response, suggested that RARb2 has an important role in
suppressing carcinogenesis.6 Recently developed receptor-specific
retinoids and rexinoids have been shown to be less toxic and have
enhanced anti-cancer effects.
In light of these recent results, our group performed an in silico
pilot screen to identify potential RARb2 agonists and retinoid
enhancers. In this paper, we discuss the synthesis, characterization
and in vitro biological evaluation of a number of 2-substituted
benzothiazoles based on structures identified as potential RARb
specific ligands from in silico screening.
⇑
Corresponding author. Tel.: +61 2 9385 4698; fax: +61 2 9385 6141.
E-mail address: n.kumar@unsw.edu.au (N. Kumar).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.09.035

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C. R. Gardner et al. / Bioorg. Med. Chem. 20 (2012) 6877–6884
Me
N
2. Results and discussion
Me
O
N
O
N
S
2.1. Analysis of the ligands proposed via in silico methods
S
N
NH
NH HN
T5688216
Me
Me
N
H
O
A subset of the Enamine small molecule library was screened
using in silico methods, in order to identify small molecules capa-
ble of binding to the ligand binding domain of RARb2. This screen-
ing returned a series of 26 hits that were proposed as suitable
ligands for RARb2. These ligands were then purchased from Enam-
ine, and screened for in vitro activity against BE(2)-C neuroblas-
toma cells with the full-length human cDNA for RARb cloned into
the PvuII multi-cloning site of the pMEP4 episomal Epstein–Barr
virus-based expression vector, as previously reported.7,8 Briefly,
cells were allowed to attach for 24 h in 96 well culture plates be-
T5497721
Figure 2. Structure of two compounds from in silico screening.
benzimidazole or benzothiazole motif, as well as the amide bonds
that were prominent in the 8 most effective compounds. Further-
more, T5688216 shows striking resemblance to a glycine-phenylal-
anine dipeptide derivative. This structural similarity to a dipeptide
was of interest as it offers many interesting possibilities for further
functionalization using the many methods for peptide synthesis.
fore being exposed to the ligands at a concentration of 5 lM for
96 h. Comparative values for cell viability in each well were deter-
mined by a Wallac 1420 Victor III spectrophotometer, which mea-
sured light absorbance in each well at 570 nm. The mean and
standard error of mean (SEM) values of each experiment were
determined after at least three replicates. Of the 26 compounds,
8 showed favorable in vitro activity, reducing the cell viability of
the RARb2 transfected cells by at least 20% more than the empty
vector (E.V.) control cells (Fig. 1). Of the eight compounds that
showed favorable in vitro activity, compound T5497721 displayed
the highest level of activity, reducing cell viability by 20% more
than the next best compound.
Of the 26 hit compounds from the virtual screen, four contained
either benzimidazole or benzothiazole moieties, with two of those
also being included in the 8 with favorable activity. Benzothiazoles
are a class of heterocyclic compounds found throughout nature in
both plants and animals. Benzothiazole was first isolated from the
volatile constituents of cranberries (Vaccinium macrocarpon), and
since then many analogues have been isolated from bacteria, mar-
ine sponges and fireflies.9,10 The biological properties, including
anti-cancer activities, of 2-substituted benzothiazoles have been
well documented.11–14 Due to this precedent, the 2-substituted
benzothiazole motif was incorporated as a conserved feature
throughout our synthesis program.
2.2. Synthesis of peptide-based ligands
Due to the structural resemblance of compound T5682216 to a
dipeptide motif, this structure was utilized as a general scaffold for
the development of a series of dipeptide analogues. Solution phase
peptide syntheses involving 2-aminobenzothiazole 1a and similar
systems utilizing a range of different conditions and coupling re-
agents have been reported. Luo et al. have used N,N0-dicyclohexyl-
carbodiimide (DCC) in the presence of N-hydroxybenzotriazole
(HOBt) and N-methylmorpholine (NMM) to couple 2-aminobenzo-
thiazole 1a to N-Boc-glycine, followed by deprotection with dilute
HCl in EtOAc.16 Moriarty et al. reported the use of (Benzotriazol-1-
yloxy)tripyrrolidino-phosphonium hexafluorophosphate (PyBop)
in the presence of N,N-diisopropylethylamine (DIPEA) to couple
benzothiazole 1a to an N-Boc-protected amino acid.17 The latter
method was selected for this work due to its comparative ease as
well as the reported success of PyBOP in difficult couplings.18
Hence, 2-aminobenzothiazole 1a was coupled to N-Boc-pro-
tected glycine or L-alanine by PyBOP coupling in the presence of DI-
PEA in DCM to give the corresponding protected monopeptides 2a–
b in yields of 81% and 91% respectively. Cleavage of the Boc group
was then achieved by stirring in a 4 M HCl in EtOAc solution to af-
ford the amines 3a–b as their hydrochloride salts in 90% and 91%
yields, respectively. These systems were then further extended into
dipeptides via the same PyBOP coupling method. Peptide 3a was
Further analysis of the structures of the 8 favourable ligands re-
vealed highly conserved linker functionality. Amide bonds were
present in 4 of the 8 compounds, while 1,3,4-oxadiazoles, which
are frequently used as isosteres for amides, were present in three
more structures.15 Compounds T5497721 and T5688216 (Fig. 2.)
were selected from the 8 most effective compounds as leads for
structural optimization. This selection was based upon the
similarity between their structures, as they both contained the
coupled to N-Boc-
L-phenylalanine to afford 4a in 61% yield, while
peptide 3b was coupled to N-Boc-L-tryptophan to give 4b in 50%
yield. Finally, dipeptides 4a–b were de-protected under the same
conditions as stated previously to afford the hydrochloride salts
5a–b in 92% and 89% yield, respectively, (Scheme 1).
The 1H NMR spectrum of (S)-2-amino-N-(2-(benzothiazol-2-
ylamino)-2-oxoethyl)-3-phenylpropanamide hydrochloride 5a
(DMSO-d6) exhibited a triplet at d 9.21 (J = 5.6 Hz, 1H) correspond-
ing to the phenylalanine amide proton and a doublet at d 8.36
(J = 3.0 Hz, 3H) corresponding to the protonated amine group. For
the benzothiazole ring, two doublets at d 7.98 (J = 7.2 Hz, 1H) and
d 7.76 (J = 7.6 Hz, 1H) corresponded to H7 and H4, respectively,
while two doublets of triplets at d 7.44 (J = 1.3, 7.8 Hz, 1H) and d
7.30 (J = 1.1, 8.4 Hz, 1H) corresponded to H5 and H6 respectively.
It was observed that the other dipeptides gave similar coupling
patterns for the benzothiazole ring and peptide chain. Further-
more, the DEPT-135 and broadband-decoupled 13C NMR spectra
of dipeptides 5a–b displayed the appropriate number of CH and
CH2 resonances, confirming the production of the dipeptides.
Empty Vector
RARβ
2
120
100
80
60
40
20
0
79
61
21
76
88
16
40
69
2.3. Synthesis of indole amides
SO
96
19
77
00
83
22
24
89
DM 44
T5
48
49
59
62
68
82
02
T5
T5
T5
T5
T5
T5
T6
Indole 6 and its derivatives form a class of heterocyclic com-
pounds that are found widely throughout nature. Both the natu-
rally occurring molecules, as well as their synthetic analogues,
Figure 1. Cell viability of BE(2)-C transfected cells relative to empty vector control
after 96 h treatment with 5 M ligand.
l

C. R. Gardner et al. / Bioorg. Med. Chem. 20 (2012) 6877–6884
6879
OH
O
O
N
N
S
HO2C
R1
N
S
a
n
NHBoc
NH2
+
NH2
+
S
N
H
NHBoc
R1
R
1a
N
H
1a R = H
1b R = Cl
1c R = OEt
2a:
2b:
R
R
1 = H
9a n = 1
9b n = 2
1 = Me
b
NH
R
O
N
R2
a
O
N
O
N
H
N
a
NH3+Cl-
S
N
H
S
NHBoc
n
N
H
S
N
R1
O
H
R1
10a: R = H, n = 1
10b: R = Cl, n = 1
10c: R = OEt, n = 1
10d: R = H, n = 2
1 = H, R2 = CH2Ph
3a:
3b:
1 = H
4a:
4b:
R
R
R
R
1 = Me, R2 = -3-CH2-indole
1 = Me
b
Scheme 3. Reagents and conditions: (a) PyBOP, DIPEA, DCM, 2 h, rt.
O
R2
N
H
N
S
NH3+Cl-
N
H
coupling procedure as described previously (Scheme 3). Table 1
summarizes the yields of the indole amides.
R1
O
R
R
1 = H, R2 = CH2Ph
5a:
5b:
The 1H NMR spectrum of indole 10a was similar to that of 8,
with the same aromatic patterns for both the indole and benzothi-
azole rings, but with the addition of a singlet at d 3.93 (2H) corre-
sponding to the CH2 group of the amide linker. Indoles 10b–c
displayed different splitting patterns for the peaks of the benzothi-
azole ring due to the substitution at the 6-position. In the 1H NMR
spectra of 10b and 10c, the benzothiazole H4 proton was observed
as a doublet at d 7.39 and 7.54 ppm, respectively, the H5 proton ap-
peared as a doublet at d 7.65 and 6.98 ppm, respectively, while the
1 = Me, R2 = -3-CH2-indole
Scheme 1. Reagents and conditions: (a) DIPEA, PyBOP, DCM, 2 h, rt; (b) 4 M HCl/
EtOAC, 4 h, rt.
have been shown to possess a vast range of biological functions
and properties, including anti-cancer activities. Due to the well-
documented biological properties of indole, as well as our group’s
continuing interest in indole-related chemistry, we proceeded to
develop 2-substituted benzothiazole derivatives that incorporated
this heterocycle. Indole amides and glyoxylamides were explored
due to their structural similarity to the initial 26 hit compounds.
The target indole amides were synthesized by two routes. In the
first method, following the well-established procedure of Shaw,
oxalyl chloride was added to a solution of indole 6 in Et2O at 0 °C
H7 proton appeared as
respectively.
a singlet at d 7.98 and 7.43 ppm,
2.4. Determination of the compounds in vitro cytotoxicity
The anti-proliferative activity of dipeptide 5a, indoles 8 and 10a
was determined in the retinoid-sensitive SH-SY5Y neuroblastoma
and retinoid-resistant MDA-MB-231 breast adenocarcinoma cell
lines, using the Alamar blue (Resazurin) assay described ear-
lier.20,21 Briefly, cells were allowed to attach for 24 h in 96 well cul-
ture plates before being exposed to dilutions of the compounds for
96 h. Comparative values for cell viability in each well were deter-
mined by a Wallac 1420 Victor III spectrophotometer which mea-
sured light absorbance in each well at 570 nm. The mean and
standard error of mean (SEM) values of each experiment were
determined after at least three replicates. Figure 3 shows the
dose-dependent response of the compounds on cell viability in
each of the cell lines, relative to a DMSO treated control.
under a nitrogen atmosphere to produce a-oxo-1H-indole-3-acetyl
chloride 7.19 This was subsequently condensed with benzothiazole
1a in the presence of NEt3 in DCM to afford the desired glyoxyla-
mide N-(benzo[d]thiazol-2-yl)-2-(1H-indol-3-yl)-2-oxo acetamide
8 in 75% yield (Scheme 2).
The 1H NMR spectrum of glyoxylamide 8 displayed three sing-
lets at d 12.86 (1H), d 12.46 (1H) and d 8.68 (1H) corresponding
to the indole NH, glyoxylamide NH and H2 respectively. Two mul-
tiplets at d 8.26 (J = Hz, 1H) and d 7.58 (J = Hz, 1H) corresponded to
H4 and H7, respectively, while a third multiplet at d 7.33 (m, 2 H)
were assigned to H5 and H6. Furthermore, two doublets at d 8.07
(J = 7.9 Hz, 1H) and d 7.84 (J = 7.7, 1H) corresponded to H70 and
H40, respectively, while two doublets of triplets at d 7.49 (J = 1.3,
7.2 Hz, 1H), d 7.38 (J = 1.1, 7.9 Hz, 1H) corresponded to H5 and
H6, respectively.
Among the compounds tested for their effect on cell viability,
indole 10a was found to be the most effective at reducing cell via-
bility. At a concentration of 1 lM, indole 10a reduced the cell via-
bility of the SH-SY5Y and MDA-MB-231 cell lines by 55% and 58%,
respectively. Interestingly, indole 8 was ineffective in the SH-SY5Y
cell line, suggesting that the simple transition from a carboxylic
amide (10a) to its corresponding glyoxylamide (8) results in
diminished activity against cell viability. This suggests that the
additional carbonyl on the linking chain may be restricting the li-
gands rotation to conformations that are less favorable for interac-
tions with the RARb2 ligand binding domain. Similarly to indole
10a, dipeptide 5a demonstrated consistent, but lower levels of
activity in both cell lines. Dipeptide 5a displayed a reduction in
In order to access the indole amides, a second route was estab-
lished in which benzothiazoles 1a–c were coupled to indole-3-ace-
tic acid 9a and indole-3-propionic acid 9b following the PyBOP
Cl
5'
7'
4'
4a'
O
1
N 3'
O
2
NH
6'
O
7a
a
7
7a' S 2'
1'
3
N
H
N
H
4a
the cell viability by approximately 50% at 20 lM in both cell lines.
6
N
O
H
6
4
In order to examine the relationship between structure, the ef-
fect on cell viability, and potential RARb2 activation, indole amides
10a–d were used to treat the pMEP4 cloned BE(2)-C neuroblas-
toma cells used in the screening of the initial 26 ligands. The
5
7
8
Scheme 2. Reagents and conditions: (a) oxalyl chloride, Et2O, 2 h, 0 °C; (b)
benzothiazole 1a, NEt3, DCM, 4 h, rt.

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C. R. Gardner et al. / Bioorg. Med. Chem. 20 (2012) 6877–6884
Table 1
A
B
C
D
120
100
80
60
40
20
0
Indole amides based on 2-aminobenzothiazoles
Empty Vector
Indole amide
R
n
Yieldsa (%)
RARβ
10a
10b
10c
10d
H
Cl
OEt
H
1
1
1
2
68
70
71
73
a
Yields of isolated pure product.
A
120
100
80
60
40
20
0
DMSO
DMSO
DMSO
DMSO
1 M 10a
μ
120
100
80
60
40
20
0
Empty Vector
RARβ
0.01
0.1
Concentration (µM)
10a
1
10
100
5a
8
20 M 10b
μ
120
100
80
60
40
20
0
B
120
100
80
60
40
20
0
Empty Vector
RARβ
20 M 10c
μ
0.01
0.1
1
10
100
Concentration (µM)
120
100
80
60
40
20
0
Empty Vector
RARβ
5a
10a
Figure 3. Cell viability of treated (A) SH-SY5Y and (B) MDA-MB-231 cell lines as a
percentage of DMSO negative control after 96 h exposure.
screening was performed in a similar manner to that described ear-
lier for the SH-SY5Y and MDA-MB-231 cell lines. Figure 4 shows a
comparison of cell viability between the empty vector (E.V.) and
RARb2 overexpressing cell line for a given concentration.
Indole 10a showed quite promising results, maintaining a level
of activity in the BE(2)-C cell lines similar to what was observed in
the SH-SY5Y and MDA-MB-231 cell lines. In the empty vector cell
line, indole 10a reduced the cell viability by 38%, while it reduced
the cell viability by 50% in the RARb2 overexpressing clone. It was
interesting to observe that upon addition of a bulky, hydrophobic
chlorine at the C60 position to give indole 10b, the effect on cell via-
bility is reduced, giving reductions of 43% and 48% in the empty
10 M 10d
μ
Figure 4. Cell viability of transfected BE(2)-C cell lines after treatment with (A)
indole 10a, (B) indole 10b, (C) indole 10c and (D) indole 10d, as a percentage of
negative control after 72 h exposure.
Additionally, when the linker of indole 10a is increased in length
by one CH2 unit to give the corresponding propanamide 10d, the
effect on cell viability is again reduced. Indole 10d displays 38%
and 53% reductions in the cell viability of the empty vector and
vector and RARb2 cell lines respectively, at 20 lM. This is in con-
trast to previous findings made by other groups, where the addi-
tion of halogens at certain positions have increased RARb
selectivity.22 Furthermore, upon substitution of an ethoxy group
at the same position, giving indole 10c, the activity is reduced fur-
ther. Indole 10c reduced the cell viability of the empty vector and
RARb2 cell lines by 29% and 39%, respectively, suggesting that the
C60 position is not tolerant to modifications, particularly those
which increase the bulk or hydrophobicity of the molecule.
RARb2 cell lines, respectively, at 10 lM. Whilst this increase in
chain length does reduce the activity, it is not as dramatic as the
reduction seen for substitution at the C60 position.
As it was found to be the most effective of the tested compounds,
indole 10a was further tested for toxicity to the MRC-5 normal hu-
man lung fibroblast cell line (Fig. 5.). At a concentration of 1 lM,

C. R. Gardner et al. / Bioorg. Med. Chem. 20 (2012) 6877–6884
6881
MDA-MB-231
SH-SY5Y
DMSO (-)
100
AC261066 (+)
13-cis-RA (+)
10a
100
80
60
40
20
0
BE(2)-C - E.V.
BE(2)-C - RARβ
10
1
MRC-5
10d
0.1
Empty Vector
RARbeta
10a
13-cis-RA
Figure 5. Comparative toxicity of indole 10a and 13-cis-retinoic acid (13-cis-RA) in
the tested cell lines at 1 M after 96 h exposure.
Figure 6. Comparative fold changes in RARb2 mRNA expression in pMEP4 cloned
BE(2)-C cells after 24 h exposure to controls and indoles 10a and 10d.
l
In order to determine the effect treatment with indole amides
had on RARb2 mRNA expression, pMEP4 cloned BE(2)-C cells were
allowed to attached to duplicate wells of 6 well culture plates for
24 h. The cells were then exposed to a DMSO solvent control, 13-
indole 10a reduced MRC-5 cell viability by 20%, which is at least
27% lower than its effects in any of the cancerous cell lines. How-
ever, it was observed that in the MRC-5 normal cell line, treatment
with 13-cis-retinoic acid, also at 1 lM, produced only a 10% reduc-
cis-retinoic acid (10
agonist, indole 10a (1
mRNA expression levels in the pMEP4 cloned BE(2)-C cell lines
were assayed using quantitative real time PCR technique
(Fig. 6). Total RNA was isolated from cell lines using PureLink Mini
Kit (Invitrogen). For each RT-PCR, 2 g of total RNA was converted
into cDNA using SuperScript III reverse transcriptase (Invitrogen).
L of the reverse transcription reaction was then quantified by
l
l
M), AC261066 (10 nM), an RARb2 selective
tion in cell viability. This degree of specificity suggests that there is
a therapeutic window, within which these indole derivatives may
be utilized, however, further development is necessary to improve
this specificity reduce their level of toxicity to normal cells.
M) or indole 10d (10
l
M), for 24 h.23 RARb2
a
l
2.5. Examination for enhancement of the retinoid signaling
pathway
2
l
quantitative real time PCR using API-7500 Sequence Detection Sys-
tem and SYBR Green PCR Master Mix (Applied Biosystems). The
quantitative results of RARb2 mRNA were normalized to b2 M
mRNA from the same sample.
We next sought to determine if the newly synthesized com-
pounds were able to enhance the retinoid signaling pathway. In or-
der to examine this, two sets of experiments were envisaged. In the
first, combinations of isotretinoin and the synthesized molecules
would be examined for additive or synergistic enhancement of ret-
inoid therapy, while the second examined the effect of the synthe-
sized ligands on the level of RARb2 mRNA expression.
In order to determine whether these compounds acted to en-
hance the efficacy of retinoid treatment, dipeptide 5a and indole
10a were administered in combination with 13-cis-retinoic acid.
SH-SY5Y cells were allowed to attach for 24 h in 96 well culture
plates before relevant wells were exposed to 13-cis-retinoic acid
Upon treatment with indoles 10a and 10d, the level of RARb2
expression was observed to increase by 2.1 and 1.5 fold, respec-
tively, in the RARb2 overexpressed cell line. This was much less
than the 9.3 fold increase observed with 13-cis-retinoic acid treat-
ment, but quite close to the 2.7 fold increase observed for the
known RARb2 agonist AC261066. It was also interesting to note
that the indole acetamide 10a showed higher levels of RARb2
induction than the propanamide 10d, which correlates to the ob-
served difference in their effects on cell viability. This suggests that
the indole amide derivatives are capable of causing RARb2 activa-
tion, and that their effect on cell viability may be linked to this acti-
vation. Examination of the levels of RARb2 activation at other
concentrations is necessary to determine their maximal effect, as
at a concentration of 0.1
sure of relevant wells to dipeptide 5a (10
(0.1 M) for 96 h. Comparative values for cell viability in each well
l
M. This was followed 24 h later by expo-
lM) or indole 10a
l
were determined by a Wallac 1420 Victor III spectrophotometer
which measured light absorbance in each well at 570 nm. The
mean and standard error of the mean (SEM) values of each exper-
iment were determined after at least three replicates (Table 2).
It was observed that the combination of dipeptide 5a and iso-
tretinoin caused a reduction in cell viability of 57%, which was
greater than the required additive value of 41%. This suggests that
dipeptide 5a is enhancing the efficacy of the retinoid treatment in
the SH-SY5Y cell line. It was also observed that indole 10a, in com-
bination with isotretinoin, caused a reduction in cell viability of
40%, which was approximately equal to the required additive value
of 39%. This suggests that combination therapies with the indole
derivative may act additively with low doses of isotretinoin.
is measurement of RARa, RARc and other RARb isotype mRNA lev-
els to determine their isotype selectivity.
3. Conclusion
The benzothiazole motif was identified as a promising struc-
tural scaffold for RARb2 agonists using virtual screening. A series
of 2-substituted benzothiazoles was developed via simple peptide
coupling reactions to amino acids and indol-3-yl acids. The indole
amides displayed moderate effects on cell viability, and showed
some selectivity towards cancerous cell lines. Furthermore, these
compounds also increased levels of RARb2 mRNA production com-
pared to a negative control. This data suggests that this class of
compounds warrants further development.
Table 2
Combination treatment of 5a (10
(0.1 M; 13-cis-RA)
lM) or 10a (0.1 lM) with 13-cis-retinoic acid
l
4. Experimental section
Compound Dose
M)
Reduction in cell viability (%)
(
l
Compound
alone
13-cis-RA
alone
Combination
treatment
4.1. Materials and methods
5a
10a
10
0.1
13
12
4
3
28
27
2
2
57
40
2
1
All reagents and solvents were obtained from commercial
sources and purified if necessary. Melting points were measured

6882
C. R. Gardner et al. / Bioorg. Med. Chem. 20 (2012) 6877–6884
using a Mel-Temp melting point apparatus and are uncorrected. 1H
and 13C NMR spectra were obtained on Bruker DPX300 (300 MHz).
Mass spectra were recorded on either a Bruker FT-ICR MS (EI) or a
Micromass ZQ2000 (ESI). Infrared spectra were recorded with a
Thermo Nicolet 370 FTIR Spectrometer using KBr discs. Column
chromatography was carried out using Merck 230–400 mesh ASTM
silica gel, and preparative thin layer chromatography was per-
(+ESI) m/z Calcd for C
23H27N4O4S (M+1)+ 455.1748. Found
455.1733.
4.1.5. (S)-tert-Butyl-1-(2-(benzo[d]thiazol-2-ylamino)-2-
oxoethylamino)-3-(1H-indol-3-yl)-1-oxopropan-2-ylcarbamate
(4b)
White solid, yield; 49%; mp 117–119 °C; UV (MeOH): kmax 293
formed using Merck silica gel 7730 60GF254
.
(e
46700 cmꢁ1 Mꢁ1) 299 (61700) nm; IR (KBr): mmax 3281, 3059,
2977, 1654, 1532, 1442, 1366, 1269, 1166, 1093, 1014, 962, 864,
4.1.1. General procedure for the synthesis of N-Boc protected
peptides (2a–d, 4a–d)
743, 692 cmꢁ1 1H NMR (DMSO-d6): d 12.45 (s, 1H, N1H), 10.81
;
(s, 1H, N2H), 8.39 (d, J = 6.4 Hz, 1H, CH3CHNH), 7.98 (d, J = 7.7 Hz,
1H, H4), 7.76 (d, J = 7.8 Hz, 1H, H7), 7.65 (d, J = 7.7 Hz, 1H, H40),
7.44 (t, J = 7.4 Hz, 1H, H6), 7.32 (d, J = 7.6 Hz, 1H, H70), 7.29 (t,
J = 6.3 Hz, 1H, H5), 7.16 (s, 1H, H30), 7.05 (t, J = 7.1 Hz, 1H, H60)
6.97 (t, J = 7.5 Hz, 1H, H50), 4.59 (p, J = 6.9 Hz, 1H, CH3CH), 4.36
(p, J = 7.3 Hz, 1H, CHCH2), 3.02 (m, 2H, CHCH2), 1.38 (d, J = 6.9 Hz,
3H, CH3CH), 1.30 (s, 9H, 3 ꢀ CH3); 13C NMR (75.6 MHz, DMSO-
d6): d 172.6 (CO), 170.8 (CO), 158.2 (C2), 155.6 (COO), 149.0
(C7a), 136.5 (C7a0), 131.9 (C4a), 127.9 (C4a0) 126.6 (C5), 124.3
(C6), 124.1 (C3), 122.2 (C7), 121.3 (C60), 121.1 (C50), 119.1 (C40),
118.6 (C4), 111.7 (C70), 110.4 (C30), 78.5 (C–O) 60.2 (CH2CH), 46.3
(CHCH3), 26.3 (CH2), 28.1 (3 ꢀ CH3), 18.0 (CHCH3); HRMS (+ESI)
m/z Calcd for C26H30N5O4S (M+1)+ 508.2013. Found 508.2001.
Benzothiazole 1 (1.1 mmol), the N-protected amino acid deriv-
ative (1 mmol) and PyBop (1 mmol) were dissolved in DCM (1 mL),
and DIPEA (2 mmol, or 3 mmol if HCl salt) was added dropwise.
The reaction mixture was stirred at rt for 1 h, and the solvent
was evaporated. The residue was redissolved in EtOAc (20 mL)
and washed with 5% KHSO4 (2 ꢀ 50 mL), brine (50 mL), 5% NaHCO3
(2 ꢀ 50 mL), again with brine (50 mL), dried over anhydrous
Na2SO4, filtered and concentrated under reduced pressure. The
product was used in the next step without further purification.
4.1.2. tert-butyl 2-(Benzo[d]thiazol-2-ylamino)-2-
oxoethylcarbamate (2a)
White solid yield: 91%; mp 177–179 °C; UV (MeOH): kmax 269
(
e
4100 cmꢁ1 Mꢁ1), 297 (2400) nm; IR (KBr): mmax 3433, 3177,
4.1.6. General procedure for deprotection of the N-Boc group
(3a-d, 5a-d)
3064, 2976, 2931, 2860, 1698, 1598, 1536, 1500, 1435, 1367,
1268, 1243, 1163, 1052, 1022, 871, 762 cmꢁ1 1H NMR (300 MHz,
;
A solution of 4 mol/L HCl/EtOAc (20 mL) was prepared by dilut-
ing a stock solution of 10 mol/L HCl with EtOAc. The N-protected
product was added to the solution and stirred at rt for 4 h. Excess
EtOAc and hydrogen chloride gas was removed under reduced
pressure to give the peptide as the hydrochloride salt. The oily
product formed was washed with Et2O to yield the title compound
as an off-white solid.
DMSO-d6): d 12.38 (br s, 1H, NH), 7.98 (d, J = 7.8 Hz, 1H, H4),
7.74 (d, J = 8.1 Hz, 1H, H7), 7.43 (dt, J = 1.2, 8.7 Hz, 1H, H5), 7.30
(dt, J = 1.2, 8.4 Hz, 1H, H6), 7.19 (t, J = 1.8 Hz, 1H, CH2NH), 3.90
(d, J = 6.3 Hz, 2H, CH2), 1.40 (s, 9H, 3 ꢀ CH3); 13C NMR (75.6 MHz,
DMSO-d6): d 166.4 (C@O), 157.2 (C2), 148.3 (C3a), 131.4 (C7a),
126.4 (C5), 123.9 (C6), 121.9 (C7), 120.8 (C4), 78.3 (C–O), 43.2
(CH2), 28.2 (CH3); HRMS (+ESI) m/z Calcd For C14H18N3O3S
(M+H)+ 308.1063. Found 308.1058.
4.1.7. 2-Amino-N-(benzo[d]thiazol-2-yl)acetamide
hydrochloride (3a)
4.1.3. (S)-tert-Butyl 1-(benzo[d]thiazol-2-ylamino)-1-
oxopropan-2-ylcarbamate (2b)
White solid, yield: 90%; mp 177–179 °C; UV (MeOH): kmax 289
(e
102700 cmꢁ1 Mꢁ1), 297 (9900) nm; IR (KBr): mmax 2964, 2921,
White solid, yield: 81%; mp 181–183 °C; UV (MeOH): kmax 274
2582, 1731, 1589, 1568, 1460, 1325, 1191, 895 cmꢁ1 1H NMR
;
(e
4500 cmꢁ1 Mꢁ1) 298 (3200) nm; IR (KBr): mmax 3258, 3076,
(300 MHz, DMSO-d6): d 8.52 (br s, 3 H, NH3Cl), 8.02 (d, J = 7.2 Hz,
1H, H4), 7.78 (d, J = 7.6 Hz, 1H, H7), 7.46 (dt, J = 1.3, 7.8 Hz, 1H,
H5), 7.33 (dt, J = 1.2, 7.4 Hz, 1H, H6), 3.97 (d, J = 5.7 Hz, 2H, CH2);
13C NMR (75.6 MHz, DMSO-d6): d 166.4 (CO), 157.2 (C2), 148.3
(C3a), 131.4 (C7a), 126.3 (C5), 123.9 (C6), 121.9 (C7), 120.8 (C4),
40.6 (CH2); HRMS (+ESI) m/z Calcd. for C9H10N3OS (M+H)+
208.0539. Found 208.0981.
2984, 2932, 1680, 1602, 1475, 1392, 1287, 1166, 1047, 1018,
961, 869, 750, 727 cmꢁ1 1H NMR (300 MHz, DMSO-d6): 12.39
;
(br s, 1H, NH), 7.97 (d, J = 7.8 Hz, 1H, H4), 7.74 (d, J = 7.7 Hz, 1H,
H7), 7.43 (dt, J = 1.1, 7.6 Hz, 1H, H5), 7.30 (dt, J = 0.9, 7.1, Hz, 1H,
H6), 4.28 (p, 1H, J = 7.1 Hz, CH), 1.37 (s, 9 H, 3 ꢀ CH3), 1.28 (d,
J = 7.2 Hz, 3H, CH3); 13C NMR (75.6 MHz, DMSO-d6): d 173.1
(C@O), 158.0 (C2), 155.3 (O-), 148.5 (C3a), 131.5 (C7a), 126.1
(C5), 123.6 (C6), 121.7 (C7), 120.5 (C4), 78.3 (C–O), 49.9 (CH),
29.1 (3 ꢀ CH3), 17.5 (CH3); HRMS (+ESI) m/z Calcd for
C15H19N3NaO3S (M+Na)+ 344.1045. Found 344.1034.
4.1.8. (S)-2-Amino-N-(benzo[d]thiazol-2-yl)propanamide
hydrochloride (3b)
White solid, yield: 91%; mp 181–183 °C; UV (MeOH): kmax 203
(e
12100 cmꢁ1 Mꢁ1), 221 (17100), 261 (6300) nm; IR (KBr): mmax
4.1.4. (S)-tert-Butyl (1-((2-(benzo[d]thiazol-2-ylamino)-2-
oxoethyl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate (4a)
3388, 2868, 1727, 1595, 1562, 1455, 1342, 1309, 1184, 1103,
760, 721, 654 cmꢁ1 1H NMR (300 MHz, DMSO-d6): d 8.70 (bs,
;
White solid, yield; 60%; mp 82-84 °C; UV (MeOH): kmax 293 (
e
3H, NH2), 8.01 (d, J = 7.8 Hz, 1H, H4), 7.77 (d, J = 7.8 Hz, 1H, H7),
7.45 (t, J = 6.9 Hz, 1H, H5), 7.33 (t, J = 6.9 Hz, 1H, H6), 4.24 (p,
J = 6.9 Hz, 1H, CH), 1.52 (d, J = 6.9 Hz, 3H, CH3); 13C NMR
(75.6 MHz, DMSO-d6): d 169.7 (CO), 157.4 (C2), 148.2 (C3a),
131.5 (C7a), 126.4 (C5), 124.0 (C6), 121.9 (C7), 120.7 (C4), 48.6
(CH), 16.7 (CH3); HRMS (+ESI) m/z Calcd. for C10H12N3OS (M+H)+
222.0696. Found 222.0690.
66200 cmꢁ1 Mꢁ1) 298 (76400) nm; IR (KBr): mmax 3278, 3061,
2974, 2869, 1709, 1603, 1550, 1455, 1443, 1366, 1269, 1167,
1086, 1016, 757, 730, 699 cmꢁ1 1H NMR (DMSO-d6): d 12.28 (s,
;
1H, NH), 8.41 (t, J = 5.5 Hz, 1H, CH2NH), 7.98 (d, J = 7.2 Hz, 1H,
H4), 7.75 (d, J = 7.7 Hz, 1H, H7), 7.44 (dt, J = 1.3, 8.7 Hz, 1H, H5),
7.31 (dt, J = 1.2, 8.4 Hz, 1H, H6), 7.24 (m, 5H, H20, H30, H40, H50,
H60), 4.24 (m, 1H, CH), 4.10 (d, 2H, J 5.0 Hz, COCH2NH), 3.08 (m,
2H, CHCH2), 1.30 (s, 9H, 3 ꢀ CH3); 13C NMR (75.6 MHz, DMSO-
d6): d 172.1 ((C@O)CH2), 157.8 ((C@O)CH), 155.5 ((C@O)O), 148.5
(C3a), 137.5 (C10), 131.5 (C7a), 129.3 (C30, C50), 128.1 (C20, C60),
126.4 (C40), 126.1 (C5), 123.6 (C6), 121.7 (C7), 120.5 (C4), 78.3
(C(CH3)3), 56.2 (CH), 45.8 (CH2), 36.8 (CHCH2), 28.1 (CH3); HRMS
4.1.9. (S)-2-Amino-N-(2-(benzo[d]thiazol-2-ylamino)-2-
oxoethyl)-3-phenylpropanamide hydrochloride (5a)
White solid, yield: 90%; mp 186–188 °C; UV (MeOH): kmax 286
(e
12300 cmꢁ1 Mꢁ1), 288 (12600), 297 (11900) nm; IR (KBr): mmax
3434, 3210, 2925, 2621, 1697, 1680, 1570, 1519, 1346, 1202,

C. R. Gardner et al. / Bioorg. Med. Chem. 20 (2012) 6877–6884
6883
1168, 753 cmꢁ1
;
1H NMR (300 MHz, DMSO-d6): d 9.21 (t, 1H, J
7.28 (t, J = 7.2 Hz, 1H, H7), 7.08 (t, J = 6.9 Hz, 1H, H50), 7.00 (t,
J = 6.8 Hz, 1H, H60), 3.93 (s, 2H, CH2); 13C NMR (75.6 MHz, DMSO-
d6): d 170.7 (CO), 158.1 (C2), 148.5 (C3a), 136.1 (C7a0), 131.4
(C7a), 127.1 (C3a), 126.1 (C5), 124.3 (C6), 123.5 (C20), 121.7 (C7),
121.1 (C60), 120.5 (C50), 118.6 (C4, C40), 111.5 (C70), 107.2 (C30),
32.4 (CH2); HRMS (+ESI) m/z Calcd. for C17H14N3OS (M+H)+
308.0858. Found 308.0849.
5.6 Hz, NH), 8.36 (s, 3H, NH2), 7.98 (d, 1H, J 7.2 Hz, H4), 7.76 (d,
1H, J 7.6 Hz, H7), 7.44 (dt, 1H, J 7.8, 1.3 Hz, H5), 7.31 (m, 6H, H6,
H20, H30, H40, H50, H60), 4.12 (d, 3H, J 5.6 Hz, COCH2NH, CH), 3.14
(m, 2 H, CHCH2); 13C NMR (DMSO-d6): d 168.7 (CO), 168.4 (CO),
157.6 (C2), 148.5 (C7a), 135.0 (C10), 131.5 (C4a), 129.7 (C30, C50),
128.5 (C20, C60), 127.1 (C50), 126.2 (C6), 123.7 (C5), 121.8 (C4),
120.6 (C7), 53.4 (CH), 42.2 (COCH2), 36.8 (CHCH2); HRMS (+ESI):
m/z Calcd. For C18H19N4O2S (M+H)+ 355.1229. Found 355.1222.
4.1.15. N-(6-Chlorobenzo[d]thiazol-2-yl)-2-(1H-indol-3-yl)
acetamide (10b)
4.1.10. (S)-2-Amino-N-(2-(benzo[d]thiazol-2-ylamino)-2-
oxoethyl)-3-(1H-indol-3-yl)propanamide hydrochloride (5b)
White solid, yield: 66%; mp 138–140 °C; UV (MeOH): kmax 289
Brown solid, yield: 70%; mp 199-201 °C. UV (MeOH): kmax 291
(e
25400 cmꢁ1 Mꢁ1) 304 (17700) nm; IR (KBr): mmax 3349, 3177,
3057, 2968, 2914, 1685, 1595, 1533, 1446, 1267, 1099, 743 cmꢁ1
;
(e
10000 cmꢁ1 Mꢁ1) nm; IR (KBr): mmax 3225, 3057, 2976, 2914,
1H NMR (300 MHz, d6-acetone): d 10.23 (br s, 1H, NH), 7.98 (dd,
J = 0.4, 2.2, Hz, 1H, H7), 7.69 (s, 1H, H20), 7.65 (dd, J = 0.5 8.2 Hz,
1H, H5) 7.37 (m, 2H, H40, H70), 7.39 (dd, J = 2.2, 6.3 Hz, 1H, H4),
7.12 (dt, J = 1.3, 7.6 Hz, 1H, H50), 7.04 (dt, J = 1.2, 6.9 Hz, 1H, H60)
4.08 (d, J = 0.8 Hz, 2H, CH2); 13C NMR (75.6 MHz, d6-acetone): d
171.4 (CO), 159.6 (C2), 148.8 (C3a), 137.6 (C7a0), 134.7 (C7a),
129.2 (C3a0), 127.3 (C5), 125.2 (C30), 122.7 (C60), 122.5 (C7), 121.9
(C50), 119.9 (C40), 119.5 (C4), 112.3 (C70), 108.3 (ArC), 33.7 (CH2);
HRMS (+ESI) m/z Calcd. for C17H13ClN3OS (M+H)+ 342.0462. Found
342.0454.
1665, 1601, 1544, 1456, 1442, 1267, 1152, 1098, 746 cmꢁ1 1H
;
NMR (300 MHz, DMSO-d6): d 11.13 (s, 1H, N1H), 9.28 (t, 1H, J
5.4 Hz, NH), 8.30 (br s, 2 H, NH2), 7.98 (d, 1 H, J 7.8 Hz, H4), 7.75
(d, 2 H, J 7.5 Hz, H7, H40), 7.44 (t, 1H, J 7.2 Hz, H5), 7.33 (m, 3 H,
H6, H20, H70), 7.08 (t, 1H, J 6.9 Hz, H50), 6.99 (t, 1H, J 7.2 Hz, H60),
4.55, (m, 2 H, COCH2NH), 4.17 (d, 1H, J 5.4 Hz, CH), 3.21 (m, 2H,
CHCH2). 13C NMR (DMSO-d6): d 172.0 (CO), 168.5 (CO), 157.8
(C2), 148.5 (C7a), 136.3 (C7a0), 131.5 (C4a), 127.4 (C4a0), 126.2
(C6), 125.2 (C5), 123.7 (C20), 121.8 (C4), 121.1 (C60), 120.6 (C50),
118.5 (C40), 118.3 (C7), 111.5 (C70), 106.7 (C30), 52.4 (CHNH2),
48.9 (CHCH3), 25.9 (CH2) 17.3 (CH3); HRMS (+ESI): m/z Calcd. For
4.1.16. N-(6-Ethoxybenzo[d]thiazol-2-yl)-2-(1H-indol-3-
yl)acetamide (10c)
C
21H22N5O2S (M+H)+ 408.1489. Found 408.1479.
Brown solid, yield: 71%; mp 184–186 °C. UV (MeOH): kmax 308
4.1.11.
a
-Oxo-1H-Indole-3-acetyl chloride (8)
(e
17900 cmꢁ1 Mꢁ1) nm; IR (KBr): mmax 3393, 3177, 3048, 2976,
Yellow solid, yield: 86%; mp 116–118 °C, lit. 117–119 °C.24
2926, 1688, 1605, 1548, 1458, 1263, 1220, 1152, 1061, 742 cmꢁ1
;
1H NMR (300 MHz, d6-acetone): d 10.22 (bs, 1H, N1H), 7.68 (d,
J = 7.3 Hz, 1H, H40), 7.54 (d, J = 8.5 Hz, 1H, H4), 7.43 (s, 1H, H7),
7.41 (d, J = 5.3 Hz, 1H, H70), 7.12 (dt, J = 1.1, 7.5 Hz, 1H, H60), 7.04
(dt, J = 1.1, 7.6 Hz, 1H, H50), 6.98 (dd, J = 2.58, 8.84 Hz, 1H, H5),
4.08 (q, J = 6.9 Hz, 2 H, CH3CH2) 4.04 (s, 2H, CH2), 1.38 (t,
J = 6.9 Hz, 3H, CH3CH2); 13C NMR (75.6 MHz, d6-acetone): d 169.9
(CO), 156.4 (C60), 155.7 (C4a0) 136.7 (C7a), 131.1 (C7a0) 126.9
(C4a), 124.4 (C6), 123.3 (C5), 121.4 (C4), 120.8 (C50), 118.5 (C40),
115.9 (C2), 111.8 (C7), 105.2 (C70), 64.3 (CH2CH3), 33.6 (CH2),
15.0 (CH3);. HRMS (+ESI) m/z Calcd. for C19H18N3O2S (M+H)+
352.1114. Found 352.1105.
4.1.12. N-(Benzo[d]thiazol-2-yl)-2-(1H-indol-3-yl)-2-
oxoacetamide (8)
Brown solid, yield: 75%; mp 294-296 °C. UV (DMSO): kmax 336
(e
18200 cmꢁ1 Mꢁ1) nm; IR (KBr): mmax 3373, 3281, 1681, 1630,
1524, 1441, 1419, 1238, 1124, 823, 729 cmꢁ1 1H NMR (300 MHz,
;
DMSO-d6): d 12.86 (bs, 1H, N1H), 12.46 (br s, 1H, CONH), 8.68 (s,
1H, H2), 8.26 (m, 1 H, H4), 8.07 (d, J = 7.9 Hz, 1H, H40), 7.84 (d,
J = 7.7 Hz, 1H, H70), 7.58 (m, 1H, H7), 7.49 (dt, J = 1.3, 7.2 Hz, 1H,
H50), 7.38 (dt, J = 1.1, 7.9 Hz, 1H, H60), 7.33 (m, 2 H, H5, H6); 13C
NMR (75.6 MHz, DMSO-d6): d 180.2 (NHCO), 163.9 (CO), 157.8
(C2), 149.0 (C4a), 139.2 (C30), 137.1 (C20), 132.3 (C7a0), 126.8
(C5), 126.3 (C7a), 124.5 (C6), 124.3 (C7), 123.4 (C4a0), 122.3 (C50),
121.7 (C60), 121.4 (C4), 113.3 (C70), 112.4 (C40); HRMS (+ESI) m/z
Calcd for C17H11N3NaO2S (M+Na)+ 344.0470. Found 344.0459.
4.1.17. N-(Benzo[d]thiazol-2-yl)-3-(1H-indol-3-yl)propanamide
(10d)
White solid, yield: 73%; mp 224–226 °C. UV (MeOH): kmax 295
(e
73900 cmꢁ1 Mꢁ1) 299 (79000) nm; IR (KBr): mmax 3374, 3141,
4.1.13. General procedure for the synthesis of indole amides
(10a–c)
2960, 2922, 1706, 1598, 1541, 1492, 1454, 1444, 1348, 1270,
1232, 1190, 1154, 1106, 754, 735 cmꢁ1 1H NMR (300 MHz,
;
Benzothiazole 1a–c (1 mmol), indole-3-acetic acid 9 (1 mmol)
and PyBop (1 mmol) were dissolved in DCM (1 mL), and DIPEA
(3 mmol) was added dropwise. The reaction mixture was stirred
at rt for 2 h. The solvent was evaporated and the residue was redis-
solved in EtOAc (20 mL) and washed with 5% KHSO4 (2 ꢀ 50 mL),
brine (50 mL), 5% NaHCO3 (2 ꢀ 50 mL), again with brine (50 mL),
dried over anhydrous Na2SO4, filtered and concentrated under re-
duced pressure. The solid so obtained was purified by flash column
chromatography with 2:3 EtOAc:hexane as the eluent.
DMSO-d6): d 12.36 (s, 1H, N1H), 10.79 (s, 1H, CONH), 7.97 (d,
J = 7.2 Hz, 1H, H4), 7.72 (d, J = 7.6 Hz, 1H, H7), 7.58 (d, J = 7.8 Hz,
1H, H 40), 7.42 (dt, J = 1.3, 7.8 Hz, 1H, H5), 7.31 (dt, J = 1.1, 7.9 Hz,
1H, H6), 7.28 (d, J = 6.1 Hz, 1H, H70), 7.13 (d, J = 2.3 Hz, 1H, H20),
7.06 (dt, J = 1.2, 7.5 Hz, 1H, H60), 6.97 (dt, J = 1.1, 7.4 Hz, 1H, H50),
3.07 (t, J = 7.1 Hz, 2H, COCH2), 2.87 (t, J 8.1 Hz, 2H, CH2); 13C
NMR (75.6 MHz, DMSO-d6): d 172.0 (CO), 157.9 (C2), 148.5 (C3a),
136.2 (C7a), 131.4 (C7a), 126.9 (C30a), 126.0 (C5), 123.4 (C6),
122.3 (C7), 121.7 (C60), 121.0 (C50), 120.4 (C4), 118.4 (C40), 118.2
(C70), 113.2 (C30), 111.4(C70), 36.2 (COCH2), 20.4(CH2); HRMS
4.1.14. N-(Benzo[d]thiazol-2-yl)-2-(1H-indol-3-yl)acetamide
(10a)
(+ESI) m/z Calcd. for C
18H16N3OS (M+H)+ 322.1009. Found
322.1004.
Brown solid, yield: 53%; mp 209–211 °C. UV (MeOH): kmax 289
(e
17100 cmꢁ1 Mꢁ1), 298 (14400) nm; IR (KBr): mmax 3373, 3163,
4.2. Biological experiments
3062, 2960, 1747, 1693, 1599, 1529, 1450, 1396, 1316, 1261,
752, 727 cmꢁ1
;
1H NMR (300 MHz, DMSO-d6): d 12.54 (s, 1H,
4.2.1. Cell biology techniques
N1H), 10.99 (s, 1H, NH), 7.95 (d, J = 7.2 Hz, 1H, H4), 7.74 (d,
J = 7.6 Hz, 1H, H7), 7.61 (d, J = 7.8 Hz, 1H, H40), 7.42 (t, J = 7.2 Hz,
1H, H5), 7.37 (d, J = 8.0 Hz, 1H, H70), 7.32 (d, J = 2.4 Hz, 1H, H20),
The retinoic acid (RA)-sensitive SH-SY5Y neuroblastoma cell
line was donated by Dr. J. Biedler (Memorial Sloan-Kettering
Cancer Center, New York). The RA-resistant MDA-MB-231 breast

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C. R. Gardner et al. / Bioorg. Med. Chem. 20 (2012) 6877–6884
cancer cell line was purchased from the American Type Culture
Collection. All cell lines were cultured under standard conditions
at 37 °C in 5% CO2 as an adherent monolayer in Dulbecco’s modi-
of its coinvestment in the National Collaborative Research Infra-
structure Strategy.
fied Eagle’s medium supplemented with
L
-glutamine (DMEM)
References and notes
(Invitrogen, USA) and 10% fetal calf serum (FCS) (Thermo Fisher
Scientific).
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4.2.2. Method for cell viability assays
Cell viability was measured by the standard Alamar blue assay,
as previously described.25 Briefly, cells were allowed to attach for
24 h in 6 replicate wells of 96-well culture plates. The cells were
then continuously exposed to serial dilutions of the benzothiazole
derivative for 96 h. Cell viability was determined by addition of
6. Lotan, R.; Xu, X.; Lippman, S.; Ro, J.; Lee, J.; Lee, J.; Hong, W. N. Engl. J. Med 1995,
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22 lL of Alamar blue reagent, recording comparative 0 h and 5 h
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values, using a Wallac 1420 Victor III spectrophotometer, which
measured light absorbance in each well at 570 nm. The cell viabil-
ity of each plate was calculated as a percentage compared to
matched DMSO controls (0.5%). The mean (+/ꢁ SEM) is shown for
three independent experiments.
12. Caleta, I.; Grdisa, M.; Mrvos-Sermek, D.; Cetina, M.; Tralic-Kulenovic, V.;
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4.2.3. Method for determining mRNA expression levels using
quantitative real-time PCR
Cells were allowed to attach to duplicate wells of 6 well culture
plates for 24 h before being exposed to a DMSO solvent control
(0.05%), 13-cis-retinoic acid (10
selective agonist, indole 10a (1
24 h.23 Total RNA was isolated from cell lines using PureLink Mini
Kit (Invitrogen). For each RT-PCR, 2 g of total RNA was converted
into cDNA using SuperScript III reverse transcriptase (Invitrogen).
L of the reverse transcription reaction was then quantified by
lM), AC261066 (10 nM) an RARb2
l
M) or indole 10d (10 M), for
l
16. Luo, Z.; Zeng, C.; Yang, D.; Huang, Y.; Wang, F.; Du, H.; Hu, L. C. J. Med. Chem.
2009, 27.
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Magolda, R.; Takahashi, H. Bioorg. Med. Chem. Lett. 2008, 18, 5537.
18. Frerot, E.; Coste, J.; Pantalom, A.; Dufour, M.; Joum, P. Tetrahedron 1991, 47,
259.
l
2
l
quantitative real time PCR using API-7500 Sequence Detection Sys-
tem and SYBR Green PCR Master Mix (Applied Biosystems). The
quantitative results of RARb2 mRNA were normalized to b2 M
mRNA from the same sample
19. Shaw, K. N. F. M. A.; Gudmundson, A. G.; Armstrong, M. D. J. Org. Chem. 1958,
23, 1171.
20. Cheung, B.; Yan, J.; Smith, S. A.; Nguyen, T.; Lee, M.; Kavallaris, M.; Norris, M.
D.; Haber, M.; Marshall, G. M. Int. J. Cancer 2003, 105, 856.
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Acknowledgments
22. de Lera, A. R.; Bourguet, W.; Altucci, L.; Gronemeyer, H. Nat. Rev. Drug Disc.
2007, 6, 811.
We thank the University of New South Wales, and the Austra-
lian Research Council (ARC) for Linkage Project (LP0455373) fund-
ing to NK and DB. We thank The Australian Chinese Charity
Foundation for purchasing 26 compounds. Mass spectrometric
analysis for this work was carried out at the Bioanalytical Mass
Spectrometry Facility, UNSW and was supported in part by infra-
structure funding from the New South Wales Government as part
23. Lund, B. W.; Piu, F.; Gauthier, N. K.; Eeg, A.; Currier, E.; Sherbukhin, V.; Brann,
M. R.; Hacksell, U.; Olsson, R. J. Med. Chem. 2005, 48, 7517.
24. Kamila, S.; Ankrti, H.; Biehl, E. R. ARKIVOC 2011, 94.
25. Marshall, G. M.; Bell, J. L.; Koach, J.; Tan, O.; Kim, P.; Malyukova, A.; Thomas,
W.; Sekyere, E. O.; Liu, T.; Cunningham, A. M.; Tobias, V.; Norris, M. D.; Haber,
M.; Kavallaris, M.; Cheung, B. B. Oncogene 2010, 29, 6172.