Lead optimization of COX-2 inhibitor nimesulide analogs to overcome aromatase inhibitor resistance in breast cancer cells

Article abstract of DOI:10.1016/j.bmcl.2009.09.109

A series of COX-2 selective inhibitor nimesulide derivatives were synthesized. Their anti-cell proliferation activities were evaluated with a long-term estrogen deprived MCF-7aro (LTEDaro) breast cancer cell line, which is the biological model of aromatas

Full text of DOI:10.1016/j.bmcl.2009.09.109

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6733–6735
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Lead optimization of COX-2 inhibitor nimesulide analogs to overcome
aromatase inhibitor resistance in breast cancer cells
*
Bin Su, Shiuan Chen
Division of Tumor Cell Biology, Beckman Research Institute of the City of Hope, 1500 E Duarte Road, Duarte, CA 91010, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of COX-2 selective inhibitor nimesulide derivatives were synthesized. Their anti-cell proliferation
activities were evaluated with a long-term estrogen deprived MCF-7aro (LTEDaro) breast cancer cell line,
which is the biological model of aromatase inhibitor resistance for hormone-dependent breast cancer.
Received 11 August 2009
Revised 24 September 2009
Accepted 29 September 2009
Available online 3 October 2009
Compared to nimesulide which inhibited LTEDaro cell proliferation with an IC50 at 170.30
new compounds showed IC50 close to 1.0 M.
lM, several
l
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Breast cancer
Aromatase inhibitor
Resistance
Nimesulide
About two-thirds of breast cancers are hormone dependent,
which contain estrogen receptors (ER) and require estrogen for tu-
mor growth. These patients are therefore suitable candidates for
hormonal therapy, which aims to block estrogen stimulation of
breast cancer cell growth.1 Tamoxifen has been the mainstay of
hormonal therapy in both early and advanced breast cancer pa-
tients for approximately three decades. However, aromatase inhib-
itors (AIs) are now proving to be more effective and to increase
survival more than antiestrogens.2–5 Over recent years AIs have be-
come the first-line endocrine therapy for ER positive patients with
advanced breast cancer.6 However, after prolonged endocrine ther-
apy, acquired resistance to AIs is expected to occur in a majority of
breast cancer patients.7–10 The possible resistance mechanism has
been investigated in preclinical models in our laboratory and oth-
ers.8–11
The long-term estrogen deprivation (LTED) system has been
used as a model for AI resistance in several laboratories, mainly
due to its lack of a hormone environment that mimics the aroma-
tase inhibition effect.8,12–15 It has been reported that the activation
of the growth factor signaling pathways in LTED cell lines such as
HER2 and insulin-like growth factor I receptor, which crosstalk
with the ER signaling pathway resulting in an activation of various
MAPKs and PI3K/AKT, is responsible for the cell survival and prolif-
eration.8–10 Although ER is still functional in LTED cells, the trans-
activation potential of ER is altered which suggests that ER
transcriptional regulation function was partially lost. LTEDaro cell
line was generated using an aromatase-overexpressing MCF-7 cell
line and was suggested to be a late stage endocrine resistance
model. Nevertheless, from drug discovery point of view, LTEDaro
is a good model for the evaluation of potential compounds to over-
come AI resistance.15
Nonsteroidal anti-inflammatory drugs (NSAIDs) are beneficial
in breast cancer treatment.16 It has been reported that COX-2
inhibitor nimesulide suppressed the development of 2-amino-1-
methyl-6-phenylimidazo [4,5-b] pyridine (PhIP)-induced mam-
mary gland carcinogenesis in rats.17 Other research demonstrated
that nimesulide also suppressed aromatase activity and expression
in several breast cancer cell lines.18 Nimesulide derivatives which
do not have COX-2 inhibitory activity were more active than
nimesulide to target aromatase.19,20 Further study reveals that sev-
eral nimesulide analogs were able to selectively inhibit Her2 over-
expressing breast cancer cell proliferation, which suggests that
they are potentially able to overcome AI resistant breast cancer cell
growth.21 Consequent investigations demonstrated that the com-
pounds induce LTEDaro cell apoptosis, which exhibited that they
can overcome AI resistance for hormone-dependent breast cancer.
Because of the unique character of nimesulide derivatives, we pro-
pose that the modification of the structure might change the drug
from a COX-2 inhibitor to an anti-cancer agent.20 Furthermore,
these new analogs selectively target Her2 overexpressing breast
cancer cells which makes them good candidates to overcome AI
resistance.
We try to further optimize the structure of nimesulide using the
combinatorial strategies to modify the four positions depicted in
Figure 1. Previous study demonstrated that B position as proton,
or methyl group, is the best fit for the analogs to inhibit cancer cell
growth. For C position, small methyl sulfonamide or acetyl groups
* Corresponding author. Tel.: +1 626 256 4673x63454; fax: +1 626 301 8972.
E-mail address: Schen@coh.org (S. Chen).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.09.109

6734
B. Su, S. Chen / Bioorg. Med. Chem. Lett. 19 (2009) 6733–6735
benzyls. D will be kept as pyridine or hydrophobic groups (Scheme
1). These compounds and their biological activity will enable us to
identify the key pharmacophore of this scaffold on the suppression
of LTEDaro breast cancer cell growth.
A
O2N
O
D
The results suggest that A position as 2,5-dimethyl or dichloro
benzyl is the best fit. Compounds 33–36 and 58–61 are relatively
more active, except compounds 37 and 62 (Table 1). It seems that
D position as a picolinyl group harms the biological activity. Only
one methyl group substituted benzyl group at A position definitely
decreases the activity. Compounds 38–47 show much lower activ-
ity compared with compounds 33–36. Compounds 48–57 are not
as active as compounds 33–36, which suggests that the methyl
group at 2,5 position of benzyl at A position is very critical for
the activity. Tri-methyl groups clearly do not increase the activity,
which has been demonstrated by relatively low activity of com-
pounds 53–57. 4-Isopropyl benzyl group or hexyl group at A posi-
tion does not help the activity based on the biological results of
compounds 63–72. However, 2,5-dichloro benzyl group at A posi-
tion can slightly increase the activity. Compounds 58 and 59 show
better activity compared with compounds 33 and 34.24 Overall,
nitrogen-containing aromatic group at D position does not increase
NH
S
B
O
O
C
Figure 1. Chemical structure of nimesulide.
is the best fit. Bulky groups will decrease the pharmacological
activity.20 For A position, methyl group substituted benzyl is better
for the activity.20 For D position, we will try pyridine group in this
study. Since nitrogen-containing heterocyclics can increase aroma-
tase inhibition activity, according to several other reports.22,23 In
the newly designed derivatives, we will keep B position as methyl
group and C position as methyl sulfonamide. A was modified by
using different positions and numbers of methyl group substituted
O2N
O
R1
R1X, DMF,
RT or Heat
O2N
O
O2N
OH
R1
1 CH3SO2Cl, DMF,NaH
NH
S
X=Cl, Br, or I
2 (3N)NaOH aq
NH2
O
O
NH2
R
1 is hydrophobic
1 R1=2,5-dimethylbenzyl (93%)
2 R1=2-methylbenzyl (94%)
3 R1=3-methylbenzyl (86%)
4 R1=2,6-dimethylbenzyl (85%)
5 R1=2,4,6-trimethylbenzyl (88%)
6 R1=2,5-dichlorobenzyl (90%)
7 R1=4-isopropylbenzyl (96%)
8 R1=hexyl (90%)
9
R1=2,5-dimethylbenzyl (93%)
group
10 R1=2-methylbenzyl (91%)
11 R1=3-methylbenzyl (96%)
12 R1=2,6-dimethylbenzyl (95%)
13 R1=2,4,6-trimethylbenzyl (70%)
14 R1=2,5-dichlorobenzyl (88%)
15 R1=4-isopropylbenzyl (98%)
16 R1=hexyl (98%)
O2N
O
H2N
O
R1 FeCl3, DMF, Zn
R2COCl, K2CO3
R1
O
CH3I, NaH, DMF
1, 4-Dioxane
N
S
N
S
O
O
O
17 R1=2,5-dimethylbenzyl (87%)
18 R1=2-methylbenzyl (81%)
19 R1=3-methylbenzyl (76%)
20 R1=2,6-dimethylbenzyl (79%)
21 R1=2,4,6-trimethylbenzyl (78%)
22 R1=2,5-dichlorobenzyl (76%)
23 R1=4-isopropylbenzyl (75%)
24 R1=hexyl (91%)
25 R1=2,5-dimethylbenzyl (78%)
26 R1=2-methylbenzyl (81%)
27 R1=3-methylbenzyl (75%)
28 R1=2,6-dimethylbenzyl (82%)
29 R1=2,4,6-trimethylbenzyl (41%)
30 R1=2,5-dichlorobenzyl (77%)
31 R1=4-isopropylbenzyl (81%)
32 R1=hexyl (82%)
H
33 R1=2,5-dimethylbenzyl; R2=cyclohexanyl (99%)
34 R1=2,5-dimethylbenzyl; R2=phenyl (97%)
35 R1=2,5-dimethylbenzyl; R2=isonicotinyl (96%)
36 R1=2,5-dimethylbenzyl; R2=nicotinyl (89%)
37 R1=2,5-dimethylbenzyl; R2=picolinyl (79%)
38 R1=2-methylbenzyl; R2=cyclohexanyl (92%)
39 R1=2-methylbenzyl; R2=phenyl (80%)
O
N
O
53 R1=2,4,6-trimethylbenzyl; R2=cyclohexanyl (87%)
54 R1=2,4,6-trimethylbenzyl; R2=phenyl (84%)
55 R1=2,4,6-trimethylbenzyl; R2=isonicotinyl (68%)
56 R1=2,4,6-trimethylbenzyl; R2=nicotinyl (79%)
57 R1=2,4,6-trimethylbenzyl; R2=picolinyl (71%)
58 R1=2,5-dichlorobenzyl; R2=cycl ohexanyl (93%)
59 R1=2,5-dichlorobenzyl; R2=phenyl (97%)
60 R1=2,5-dichlorobenzyl; R2=isonicotinyl (86%)
61 R1=2,5-dichlorobenzyl; R2=nicotinyl (92%)
62 R1=2,5-dichlorobenzyl; R2=picolinyl (94%)
63 R1=4-isopropylbenzyl; R2=cyclohexanyl (83%)
64 R1=4-isopropylbenzyl; R2=phenyl (86%)
65 R1=4-isopropylbenzyl; R2=isonicotinyl (79%)
66 R1=4-isopropylbenzyl; R2=nicotinyl (87%)
67 R1=4-isopropylbenzyl; R2=picolinyl (77%)
68 R1=hexyl; R2=cyclohexanyl (71%)
R1
R2
N
S
O
O
40 R1=2-methylbenzyl; R2=isonicotinyl (80%)
41 R1=2-methylbenzyl; R2=nicotinyl (87%)
42 R1=2-methylbenzyl; R2=picolinyl (81%)
43 R1=3-methylbenzyl; R2=cyclohexanyl (84%)
44 R1=3-methylbenzyl; R2=phenyl (75%)
45 R1=3-methylbenzyl; R2=isonicotinyl (74%)
46 R1=3-methylbenzyl; R2=nicotinyl (88%)
47 R1=3-methylbenzyl; R2=picolinyl (71%)
48 R1=2,6-dimethylbenzyl; R2=cyclohexanyl (90%)
49 R1=2,6-dimethylbenzyl; R2=phenyl (86%)
50 R1=2,6-dimethylbenzyl; R2=isonicotinyl (87%)
51 R1=2,6-dimethylbenzyl; R2=nicotinyl (90%)
52 R1=2,6-dimethylbenzyl; R2=picolinyl (82%)
69 R1=hexyl; R2=phenyl (93%)
70 R1=hexyl; R2=isonicotinyl (68%)
71 R1=hexyl; R2=nicotinyl (70%)
72 R1=hexyl; R2=picolinyl (83%)
Scheme 1. Synthesis of nimesulide analogs.

B. Su, S. Chen / Bioorg. Med. Chem. Lett. 19 (2009) 6733–6735
6735
Table 1
IC50 of inhibition of LTEDaro breast cancer cells growth by compounds 33–72
References and notes
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Compd
Inhibition of LTEDaro cell growth (IC50 lM)
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Nimesulide
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
173.30 20.30
2.66 0.57
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Coleman, R. E.; Fallowfield, L. J.; Mickiewicz, E.; Andersen, J.; Lonning, P. E.;
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4.68 0.54
2.37 0.44
1.69 0.25
174.20 79.33
28.46 5.74
175.60 94.37
11.76 2.34
71.49 23.07
93.89 30.52
44.18 16.04
16.07 3.65
16.08 3.08
93.63 59.03
14.89 2.08
39.38 13.88
16.24 3.32
19.91 5.58
22.53 6.50
41.37 15.70
18.49 2.75
10.30 3.10
13.18 2.23
10.36 2.42
51.27 14.91
1.00 0.39
2.15 0.54
7.64 1.67
14.05 4.16
23.58 8.78
16.06 4.94
7.93 2.85
11.46 2.75
8.26 3.04
11.41 4.11
6.98 2.93
12.04 2.36
9.56 1.90
12.07 2.86
7.68 2.45
24. Compound 36: White powder, 1H NMR (500 MHz, DMSO-d6) d 10.51 (1H, s),
9.08 (1H, s), 8.74 (1H, s), 8.27 (1H, d, J = 8.0 Hz), 7.72 (1H, s), 7.55 (1H, d,
J = 8.0 Hz), 7.37 (5H, m), 5.07 (2H, s), 3.08 (3H, s), 2.83 (3H, s), 2.28 (3H, s), 2.24
(3H, s); HRMS calculated for C23H26N3O4S (M+H)+ 440.1639, found 440.1638.
Compound 58: White powder, 1H NMR (500 MHz, DMSO-d6) d 9.91 (1H, s), 7.71
(1H, d, J = 2.5 Hz), 7.56 (2H, m), 7.47 (1H, m), 7.22 (2H, m), 5.13 (2H, s), 3.07
(3H, s), 2.86 (3H, s), 2.27 (1H, m), 1.76 (4H, m) 1.62 (1H, m), 1.37 (2H, m), 1.24
(3H, m); HRMS calculated for C22H27Cl2N2O4S (M+H)+ 485.1063, found
LTEDaro cells were treated with indicated compounds at various concentrations by
triplicates for 72 h and cell viability was measured by MTT assay.25
the biological activity, even though compound 36 is slightly more
potent than compounds 33 and 34.
In brief, we optimized nimesulide structure and developed sev-
eral more potent analogs, such as compounds 36, 58, and 59, which
485.1061. Compound 59: White powder, 1H NMR (500 MHz, DMSO-d6)
d
inhibit LTEDaro cell growth with IC50 of 1.69 0.25
1.00 0.39 M, and 2.15 0.54 M, respectively. Compared with
nimesulide with IC50 of 173.30 20.30 M, the new derivatives
lM,
10.34 (1H, s), 7.93 (2H, d, J = 1.0 Hz), 7.92 (2H, m), 7.59 (6H, m), 7.30 (1H, d,
J = 8.5 Hz), 5.18 (2H, s), 3.11 (3H, s), 2.89 (3H, s); HRMS calculated for
C22H20Cl2N2NaO4S (M+Na)+ 501.0413, found 501.0410.
l
l
l
25. The effect of nimesulides derivatives on LTEDaro 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 6 0.1%) in media for 72 h. Controls received DMSO vehicle at a
concentration equal to that in drug-treated cells. The medium was removed,
have much more potent pharmacological activity against LTEDaro
breast cancer cell growth. Structure–activity relationship study
suggests that A position needs 2,5-dimethyl or dichloro benzyl
group to increase the biological activity. The exact biological mech-
anism of the compound is still under investigation in our
laboratory.
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
Acknowledgment
dye was solubilized in 200
determined on a plate reader.
ll/well DMSO. Absorbance at 570 nm was
This work was supported by grants from the National Institutes
of Health CA44735 (S.C.), ES08528 (S.C.).