Design and synthesis of a biotinylated probe of COX-2 inhibitor nimesulide analog JCC76

Article abstract of DOI:10.1016/j.bmcl.2011.07.025

JCC76 is a derivative of cyclooxygenase-2(COX-2) selective inhibitor nimesulide and exhibits potent anti-breast cancer activity. It selectively induces apoptosis of Her2 positive breast cancer cells. However, the specific molecular targets of JCC76 still

Full text of DOI:10.1016/j.bmcl.2011.07.025

Bioorganic & Medicinal Chemistry Letters 21 (2011) 5324–5327
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of a biotinylated probe of COX-2 inhibitor nimesulide
analog JCC76
Bo Zhong a, Rati Lama a, Kerri M. Smith a, Yan Xu a, Bin Su a,b,
⇑
a Department of Chemistry, College of Sciences and Health Professions, Cleveland State University, 2121 Euclid Ave., Cleveland, OH 44115, USA
b Center for Gene Regulation in Health and Disease, College of Sciences and Health Professions, Cleveland State University, 2121 Euclid Ave., Cleveland, OH 44115, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
JCC76 is a derivative of cyclooxygenase-2(COX-2) selective inhibitor nimesulide and exhibits potent anti-
breast cancer activity. It selectively induces apoptosis of Her2 positive breast cancer cells. However, the
specific molecular targets of JCC76 still remain unclear, which significantly withdraw the further drug
development of JCC76. To identify the molecular targets of JCC76, a six carbon linker and biotin conju-
gated JCC76 probe was designed and synthesized. The anti-proliferation activity of the probe and its ana-
logs was evaluated.
Received 10 May 2011
Revised 28 June 2011
Accepted 6 July 2011
Available online 14 July 2011
Keywords:
COX-2
Published by Elsevier Ltd.
Nimesulide
JCC76
Biotin
Probe
Numerous studies have demonstrated overexpression of cyclo-
oxygenase-2 (COX-2) in solid malignancies including breast, pros-
tate, colon, pancreas, nonsmall-cell lung, bladder, and endo
metrium.1–5 Prostaglandin-2 (PGE2), the product of COX-2, pro-
motes tumor invasiveness, angiogenesis, and progression in various
cancers.1,4,5 It is therefore reasonable to conclude that inhibition of
COX-2 would arrest carcinogenesis and thus, (1) prevent cancer
development and (2) regress cancer once developed. Corresponding
to these observations and theory, epidemiological, clinical, and pre-
clinical studies provide compelling evidence that the use of COX-2
selective inhibitors may reduce the incidence of mammary cancer
and colorectal cancer.6–9 Theoretically, COX-2 inhibitors achieve
all the anti-cancer or cancer preventive activity by targeting COX-
2. However, these small molecules may also block other cellular
machineries to affect the cancer cell function, which may lead to
cell growth inhibition, apoptosis or necrosis. With the extensive
studies of COX-2 inhibitors about their anti-cancer or cancer pre-
vention activity, molecular targets of the drugs beyond COX-2
seems play a very important role for their antineoplastic activity.
Many researchers even suggested that COX-independent effects
might be fully responsible for the anti-cancer properties of some
COX-2 inhibitors.10,11 The phenomenon is more like an off-target ef-
fect, which also underscores the needs to explore targets beyond
COX-2, especially in view of emerging toxicity of the COX-2 inhib-
itors. Developing nonCOX-2 inhibitory analogs as new anti-cancer
drug based on COX-2 inhibitors as lead compound is feasible and
practicable. In fact, this principle has been successfully applied in
the discovery of some new anti-cancer drugs. For instance, COX-2
inhibitor Celecoxib weakly inhibits PDK-1 kinase in prostate cancer
cells. It has been used as a molecular scaffold to develop more po-
tent PDK-1 inhibitors (no COX-2 inhibitory activity) which block
PI3 K/AKt pathway. The generated new analog, such as OSU-
03012 is 20-fold more active than Celecoxib to inhibit tumor cell
proliferation and to induce cell apoptosis, which is currently in a
phase I clinical trial.12,13 Nimesulide (4-nitro-2-phenoxymethane-
sulfoanilide) is a nonsteroidal anti-inflammatory drug with a pref-
erential COX-2 inhibitory activity and is available in some Asian and
European countries since 1985. Studies suggest that nimesulide
could induce apoptosis in liver and lung cancer cells; it also
suppressed the development of 2-amino-1-methyl-6-phenylimi-
dazo[4,5-b]pyridine (PhIP)-induced mammary gland carcinogene-
sis in rats.14–17 NonCOX-2 active nimesulide derivatives have
been synthesized. Some analogs were much more active than
nimesulide in suppressing breast cancer cell growth.18 Interest-
ingly, the most potent analog JCC76 was selectively potent in
Her2 overexpressed breast cancer cells.19,20 Both in vitro and
in vivo anticancer properties of JCC76 make it a potential new drug
candidate or drug lead for Her2 overexpressed breast cancer.
To rational design more potent analogs of JCC76 based on its
binding pocket and optimize the structure–activity relationship
(SAR), it is a prerequisite to identify the specific molecular target(s)
of JCC76. Although JCC76 exhibited good selectivity to Her2 over-
expressed breast cancer cells, it did not show any significant block-
⇑
Corresponding author. Tel.: +1 216 687 9219; fax: +1 216 687 9298.
E-mail address: B.su@csuohio.edu (B. Su).
0960-894X/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2011.07.025

B. Zhong et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5324–5327
5325
cer cells. In an effort to identify the bio-molecule(s) that bind to
JCC76, we planned to use biotin–streptavidin affinity purification.
For this purpose, biotinylated derivative of JCC76 with consider-
able bioactivity should be designed and synthesized. Generally, a
biotinylated linker can be incorporated at the suitable position of
JCC76, so that the biological activity of the small molecule can be
conserved. Moreover, a linker between small molecule and biotin
should have appropriate length in order to provide enough space
for biotin to interact with streptavidin (Fig. 1).
Figure 1. JCC76 biotinylated probe design.
The previous structure–activity analysis indicated that the inhi-
bition of breast cancer cell growth by nimesulide derivatives re-
quires a hydrophobic group substituted bulky phenyl ring, a
methanesulfonamide and a hydrophobic carboxamide moiety.18
Those three moieties might not be suitable for incorporation of
biotinylated linker. N-methyl group on the sulfonamide group is
the only position feasible for the introduction of biotin (Fig. 2).
Replacement of N-methyl group with hydrogen or alkyl chains
of different length generated a series of novel derivatives. The syn-
thetic approach used is shown in Scheme 1. The key intermediate 5
was prepared according to the procedure previously used for syn-
thesis of JCC76.18 The starting material 1 was treated with 2,5-dim-
ethylbenzylbromide in the presence of potassium carbonate to
afford compound 2, followed by mesylation and hydrolysis to gen-
erate compound 3. Instead of N-methylation of sulfonamide group,
compound 3 was directly reduced in the presence of Zn/FeCl3 to
generate substituted aniline 4. Coupling with cyclohexanecarbonyl
Bulky phenyl ring
Carboxamide moiety
H
O
N
O
Position suitable for Biotin
N
S
O
O
Methanesulfonamide
Figure 2. JCC76 structure analysis.
ing effects on the well-documented Her2 downstream kinases.19,20
It is difficult to elucidate the molecular targets of JCC76 by check-
ing the signal transduction net in Her2 overexpressed breast can-
O2N
OH
O2N
O
a
c
O2N
O
b
NH2
NHMs
NH2
1
2
3
H
N
H
N
O
O
O
O
H2N
O
e
d
NR
Ms
NHMs
NHMs
4
5
6a-c
6a, R = n-C6H13
6b, R = (CH2)2O(CH2)2OCH3
6c, R = (CH2)6OH
H
N
O
O
N
H
N
g
f
O
O
NHCOR
NH2
N
Ms
Ms
8a-b
8a, R = n-C5H11
7c
h
8b, R = 2-Naphthyl
H
N
O
O
H
NH
S
H
N
N
Ms
N
H
H
O
O
8c
Scheme 1. Regents and conditions: (a) 2,5-dimethylbenzylbromide, K2CO3, DMF; (b) (1) MsCl, NaH, DMF; (2) NaOH, MeOH; (c) FeCl3, Zn, DMF/H2O; (d) cyclohexanecarbonyl
chloride, K2CO3, 1,4-dioxane; (e) RX (X = Br or I), K2CO3, DMF; (f) (1) MsCl, Et3N, DCM; (2) NH3ÁH2O, EtOH; (g) RCOCl, K2CO3, 1,4-dioxane; (h), (+)- -biotin, PyBOP, Et3N, DMF.
D

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B. Zhong et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5324–5327
Table 1
IC50 of inhibition of SKBR-3 breast cancer cells growth by JCC76 probes
H
N
O
O
N
R
Ms
Compound
R
Inhibition of SKBR-3 cell growth (IC50 lM)
JCC76
5
Me
H
3.43 1.57
83.68 42.77
6a
6b
81.68 31.18
12.95 4.92
O
O
H
N
8a
6.00 2.52
O
H
8b
45.28 12.47
N
O
H
NH
S
H
N
8c
2.21 0.78
N
H
O
O
H
SKBR-3 cells were treated with indicated compounds at various concentrations by triplicates for 48 h and cell viability was measured by MTT assay.22
chloride afforded 5. N-alkylation of sulfonamide group with alkyl
bromide/iodide in the presence of potassium carbonate yielded
6a–c. Conversion of hydroxyl group in 6c to amino group followed
by coupling with alkyl or aromatic carbonyl chloride generated 8a–
b.21
prove that JCC76 was the moiety that cause the anti-proliferation
activity of the biotinylated probe.
In conclusion, we synthesized a biotin-tagged probe of anti-
proliferative agent JCC76 in this work. The biotin moiety was
successfully introduced in the molecule without loss of the biologi-
cal activity of the parental compound. JCC76 is nonCOX-2 active
agent developed based on COX-2 inhibitor nimesulide as a lead
compound. It selectively inhibits Her2 over expressed breast cancer
cell proliferation,19,20 but its specific anti-cancer molecular target(s)
still remain unclear. There are numerous studies demonstrate that
COX inhibitors exhibit anti-cancer activity via COX independent
mechanism(s).1–5 However, it is difficult to elucidate the specific
nonCOX targets of these small molecules. Our work can provide a
feasible approach for the identification of the new molecular targets
of the COX inhibitors. We currently focus on the purification and
identification of JCC76 targeted protein(s) by protein pull down
and proteomic approach based on the biotin-tagged JCC76 probe.
The anti-proliferation effect of compounds 5, 6a–b, and 8a–b
was evaluated against breast cancer cell line SKBR-3. The results
are summarized in Table 1. It can be seen that alkyl group on the
nitrogen of sulfonamide appreciably affects the biological activity
of JCC76 derivatives. Replacement of methyl group with hydrogen
or hydrophobic alkyl chain n-C6H13 almost abolishes the inhibitory
activity. Replacement of methyl group with hydrophilic (CH2)2O
(CH2)2OCH3 to afford 6b and results in an about fourfold loss in
inhibitory potency. It seems that hydrophilic linker is better than
hydrophobic linker. However, it is difficult to further modify the
end structure of the hydrophilic linker 6b. Extension of the linear
C6 chain with another alky chain or an aromatic ring through for-
mation of an amide bond restores the biological activity to some
extent. Compared to 6a, compound 8b is about twofold more po-
tent and compound 8a even exhibits 14-fold more potency. Notice-
ably, the inhibitory potency of compound 8a is comparable to that
of JCC76, which indicates that the long chain in 8a is well tolerated.
A survey of several JCC76 derivatives with different N-alkyl chains
shows that an appropriate alkyl group can be introduced in lead
compound JCC76 without jeopardizing the biological activity.
Based on the information obtained from this preliminary SAR
study, we designed and synthesized compound 8c, a novel biotin-
ylated derivative of JCC76. PyBOP-mediated coupling of intermedi-
ate 7c with biotin efficiently yielded compound 8c.21 This
compound has a very similar long chain as in compound 8a. Hypo-
thetically, with this long chain the biotin moiety should protrude
for the interaction with streptavidin. The anti-proliferation effect
of 8c on SKBR-3 was evaluated and its IC50 value was determined
Acknowledgment
This work was supported by a start up grant from Cleveland
State University.
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to be 2.21 0.78
cell growth inhibition potency with lead compound JCC76. Biotin
did not show any inhibitive activity even at 100 M, which further
lM. This biotinylated derivative has comparable
l

B. Zhong et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5324–5327
5327
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6.686 (1H, d, J = 8.4 Hz), 5.677 (1H, b), 5.024 (2H, s), 3.487 (2H, b), 3.184 (2H, d,
J = 6.4 Hz), 2.691 (3H, s), 2.312 (7H, m), 2.178–1.290 (26H, m), 0.878 (3H, t,
J = 6.8 Hz); Compound 8b: White powder, 1H NMR (400 MHz, CDCl3) d 8.294
(1H, s), 8.006 (1H, s), 7.854 (5H, m), 7.528 (2H, m), 7.185 (1H, d, J = 8.4 Hz),
7.077 (3H, m), 6.696 (1H, dd, J = 2, 8.4 Hz), 6.589 (1H, s), 4.997 (2H, s), 3.455
(4H, m), 2.680 (3H, s), 2.289 (7H, m), 1.931–1.192 (18H, m); Compound 8c:
White powder, 1H NMR (400 MHz, CDCl3) d 8.201 (1H, s), 8.085 (1H, s), 7.203
(1H, d, J = 8.4 Hz), 7.101 (3H, m), 6.763 (1H, dd, J = 2, 8.4 Hz), 6.186 (1H, s),
6.115 (1H, t, J = 6 Hz), 5.223 (1H, s), 5.040 (2H, s), 4.498 (1H, m), 4.314 (1H, m),
3.488 (2H, m), 3.149 (3H, m), 2.900 (1H, dd, J = 4.8, 128 Hz), 2.717 (1H, d,
J = 124 Hz), 2.684 (3H, s), 2.314 7H, m), 2.195 (2H, t, J = 7.2 Hz), 1.951–1.283
(24H, m); HRMS calculated for
756.3847.
C
39H58N5O6S2 (M+H)+ 756.3828, found
20. Chen, B.; Su, B.; Chen, S. Biochem. Pharmacol. 2009, 77, 1787.
21. Compound 5: White powder, 1H NMR (400 MHz, CDCl3)
d
7.970 (1H, d,
22. The effect of JCC76 probes on SKBR-3 breast cancer cell viability was assessed by
using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide
assay (MTT) in triplicates. Cells were grown in custom medium in 96-well, flat-
bottomed plates for 24 h, and were exposed to various concentrations of
nimesulide derivatives dissolved in DMSO (final concentration 60.1%) in media
for 72 h. Controls received DMSO vehicle at a concentration equal to that in
J = 2 Hz), 7.453 (1H, d, J = 8.4 Hz), 7.232 (1H, s), 7.126 (3H, m), 6.696 (1H, dd,
J = 2, 8.4 Hz), 6.555 (1H, s), 5.047 (2H, s), 2.834 (3H, s), 2.336 (3H, s), 2.313 (3H,
s), 2.242 (1H, tt, J = 3.6, 12 Hz), 1.245–1.985 (10H, m); Compound 6a: White
powder, 1H NMR (400 MHz, CDCl3) d 7.995 (1H, d, J = 2.4 Hz), 7.410 (1H, s),
7.210 (1H, d, J = 8.4 Hz), 7.094 (3H, m), 6.643 (1H, dd, J = 2.4, 8.4 Hz), 5.036 (2H,
s), 3.482 (2H, b), 2.693 (3H, s), 2.317 (6H, s), 2.258 (1H, tt, J = 3.6, 12 Hz), 1.970–
1.174 (18H, m), 0.842 (3H, t, J = 6.8 Hz); Compound 6b: White powder, 1H
NMR (400 MHz, CDCl3) d 7.902 (1H, d, J = 2.4 Hz), 7.509 (1H, s), 7.230 (1H, d,
J = 8.4 Hz), 7.178 (1H, s), 7.091 (2H, m), 6.696 (1H, dd, J = 2.4, 8.8 Hz), 5.001
(2H, s), 3.471 (8H, m), 3.338 (3H, s), 2.792 (3H, s), 2.317 (3H, s), 2.311 (3H, s),
2.253 (1H, tt, J = 3.6, 12 Hz), 1.956–1.254 (10H, m); Compound 8a: White
powder, 1H NMR (400 MHz, CDCl3) d 8.027 (1H, s), 7.883 (1H, s), 7.134 (4H, m),
drug-treated cells. The medium was removed, replaced by 200 ll of 0.5 mg/ml
of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide in fresh
media, and cells were incubated in the CO2 incubator at 37 °C for 2 h.
Supernatants were removed from the wells, and the reduced 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium
bromide
dye
was
solubilized in 200
plate reader.
ll/well DMSO. Absorbance at 570 nm was determined on a