From COX-2 inhibitor nimesulide to potent anti-cancer agent: Synthesis, in vitro, in vivo and pharmacokinetic evaluation

Article abstract of DOI:10.1016/j.ejmech.2011.11.012

Cyclooxygenase-2 (COX-2) inhibitor nimesulide inhibits the proliferation of various types of cancer cells mainly via COX-2 independent mechanisms, which makes it a good lead compound for anti-cancer drug development. In the presented study, a series of ne

Full text of DOI:10.1016/j.ejmech.2011.11.012

European Journal of Medicinal Chemistry 47 (2012) 432e444
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
From COX-2 inhibitor nimesulide to potent anti-cancer agent: Synthesis, in vitro,
in vivo and pharmacokinetic evaluation
,
,
Bo Zhong a 1, Xiaohan Cai a 1, Snigdha Chennamaneni a, Xin Yi a, Lili Liu c, John J. Pink d, Afshin Dowlati c,
Yan Xu a, Aimin Zhou a b, Bin Su a
,
,b,
*
a Department of Chemistry, College of Sciences and Health Professions, Cleveland State University, 2121 Euclid Avenue, Cleveland, OH 44115, USA
b Center for Gene Regulation in Health and Disease, College of Sciences & Health Professions, Cleveland State University, 2121 Euclid Avenue, Cleveland, OH 44115, USA
c Division of Hematology and Oncology, Case Western Reserve University School of Medicine, 10900 Euclid Avenue, Cleveland, OH 44106, USA
d Division of General Medical Sciences-Oncology, Case Western Reserve University School of Medicine, 10900 Euclid Avenue, Cleveland, OH 44106, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Cyclooxygenase-2 (COX-2) inhibitor nimesulide inhibits the proliferation of various types of cancer cells
mainly via COX-2 independent mechanisms, which makes it a good lead compound for anti-cancer drug
development. In the presented study, a series of new nimesulide analogs were synthesized based on the
structureefunction analysis generated previously. Some of them displayed very potent anti-cancer
activity with IC50s around 100 nMe200 nM to inhibit SKBR-3 breast cancer cell growth. CSUOH0901
(NSC751382) from the compound library also inhibits the growth of the 60 cancer cell lines used at
National Cancer Institute Developmental therapeutics Program (NCIDTP) with IC50s around 100 nM
e500 nM. Intraperitoneal injection with a dosage of 5 mg/kg/d of CSUOH0901 to nude mice suppresses
HT29 colorectal xenograft growth. Pharmacokinetic studies demonstrate the good bioavailability of the
compound.
Received 10 August 2011
Received in revised form
2 November 2011
Accepted 5 November 2011
Available online 15 November 2011
Keywords:
COX-2 inhibitor
Nimesulide
Anti-cancer
Xenograft
Published by Elsevier Masson SAS.
Pharmacokinetics
1. Introduction
prostaglandin cannot rescue the cells from growth inhibition caused
by COX-2 inhibitors [17e22]. Therefore, it is speculated that COX-2-
Numerous studies have demonstrated the overexpression of
cyclooxygenase-2 (COX-2) in solid malignancies [1e9]. Epidemio-
logical, clinical, and preclinical investigations also provide compel-
ling evidence that COX-2 inhibitors could act as chemopreventive
agents [8,10e12]. The anti-cancer effects of COX-2 inhibitors are
based on the assumption that prostaglandins generated by COX-2
promote tumor growth in an autocrine and/or paracrine manner
[13,14].
Theoretically, COX-2 inhibitors exhibit all the anti-cancer or
cancer preventive activity by blocking COX-2, thereby decrease the
concentration of prostaglandins inside the tumor. However, these
small molecules may also target other growth pathway, which
may lead to cell growth inhibition, apoptosis or necrosis [8,15,16].
Many COX-2 inhibitors can suppress the growth of non-COX-2
expressing tumor cells, while supplementation with exogenous
independent effects may contribute to or even be fully responsible
for the anti-cancer properties of some COX-2 inhibitors. Further-
more, the relative potency of COX-2 inhibitors to inhibit COX-2
enzyme does not match their potency to inhibit cancer cell growth
[19]. In addition to the lack of correlation between COX-2 inhibition
and anti-cancer activities, the required concentrations of these COX-
2 inhibitors to inhibit tumor cell growth significantly exceed the
concentrations required to inhibit COX-2. This phenomenon
suggests that the COX-2 inhibitors mainly target other pathways
which need much higher concentration for COX-2 inhibitors to block
[19,23e26]. The strongest evidence for a COX-independent mecha-
nism is that some non-COX-2 inhibitory derivatives of certain COX-2
inhibitors still exhibit significant anti-cancer activity [27,28].
The COX-2 selective inhibitor nimesulide, N-(2-phenoxy-4-
nitrophenyl)methanesulfonamide, is a promising lead compound
for anti-cancer drug discovery. In several in vivo experiments,
nimesulide exhibits chemopreventive activity against 2-amino-1-
methyl-6- phenylimidazo [4,5-b] pyridine-induced mammary
carcinogenesis in rats and against the post-initiation development
of squamous cell carcinomas in 4-nitroquinoline-1-oxide-induced
rat tongue carcinogenesis [29e31]. In addition, nimesulide is
* Corresponding author. Department of Chemsitry, College of Sciences and Health
Professions, Cleveland State University, 2121 Euclid Avenue, Cleveland, OH 44115,
USA. Tel.: þ1 216 687 9219; fax: þ1 216 687 9298.
E-mail address: b.su@csuohio.edu (B. Su).
Equal contribution.
1
0223-5234/$ e see front matter Published by Elsevier Masson SAS.
doi:10.1016/j.ejmech.2011.11.012

B. Zhong et al. / European Journal of Medicinal Chemistry 47 (2012) 432e444
433
shown to protect against N-nitrosobis(2-oxopropyl)amine-induced
pancreatic tumors in hamster [32]. In some in vitro experiments,
nimesulide is able to inhibit the proliferation and to increase the
apoptosis rate of various types of cancer cells [18,25,33e38].
However, the nimesulide concentrations used in these studies are
mixture was stirred at room temperature overnight to obtain the
N,N-bimethanesulfonamido b. Compound b was hydrolyzed with
10% NaOH solution to generate c as a monomethanesulfonamido
compound. Compound c was treated with sodium hydride and
methyl iodine in DMF at room temperature to obtain compound d.
Then the nitro group was reduced to an amine group to obtain
compound e. Compound e was treated with different substituted
acetyl chloride and K2CO3 to generate the substituted benzamides
1e39, respectively. Structures of all the synthesized compounds
were determined by 1H NMR, MS, and their purity was confirmed
by HPLC with two mobile phases.
ranged from 200 to 500 mM, which greatly exceed the concentra-
tion necessary to inhibit COX-2 activity. These facts suggest that
nimesulide inhibits cancer cell growth and induces cancer cell
apoptosis independent of COX-2.
JCC76 is a non-COX-2 active nimesulide analog (Fig. 1) [39e41],
and it inhibits SKBR-3 breast cancer cell growth with an IC50 of
1.38
mM, which is about 100 fold more active than nimesulide
(Table 1). The N-Methylation of JCC76 blocks the ionization of the
sulfonamide group, which abolishes the potential COX-2 activity
[42e44]. In addition, the aromatic nitro group is converted to an
amide moiety on the structure of JCC76, which could diminish the
potential hepatotoxicity, since nimesulide shows hepatotoxicity
due to the multistep nitroreductive bioactivation that produces the
hazardous nitroanion radical and nitroso intermediate [45]. Based
on the structure of JCC76, more analogs were designed and
synthesized in the current studies. Some new analogs such as
CSUOH0901 (NSC751382) inhibited SKBR-3 breast cancer cell
2.2. Biological evaluation of the new analogs with breast cancer cell
line SKBR-3
The compounds were tested for the inhibition of SKBR-3 breast
cancer cell growth (Table 1). Subsequently, a detailed structure
activity analysis was performed based on the structure and the
anti-cancer activity. The new derivatives have same core structure
as JCC76, the different activity is correlated with the different amide
moieties.
For the substituted benzamide moiety in the structure, para
position bulky halogen substituted benzamide such as 9 (bromine)
with an IC50 of 0.22 mM and 29 (iodine) with an IC50 of 0.13 mM
show better potency than JCC76, whereas small halogen chlorine at
para position (28) slightly decreases the activity with an IC50 of
2.15 mM. It is also possible that the activity is related to the elec-
tronegativity or electron density of the halogens. Iodine has the
lowest electronegativity and highest electron density, which may
contribute to the potent anti-cancer activity. 2, 8, 25, 20, and 27,
which have single meta substitution groups, display weaker
growth with IC50s around 0.1 mMe0.2 mM, which is about 10 fold
more active than JCC76 and almost 1000 fold more active than
nimesulide. In addition, CSUOH0901 inhibited the growth of
a broad range of cancer cell lines with IC50s of 0.2 mM to 0.5 mM. It
also inhibited the growth of HT29 colorectal xenograft in nude mice
as well. All the studies suggest that the newly developed JCC76
derivatives are promising anti-cancer drug candidates.
2. Results and discussion
inhibitory activity with IC50s ranging from 1.97 mM to 7.34 mM. It
2.1. Compound design and parallel synthesis of JCC76 derivatives
seems that single meta substitution is predominated by steric
effect. The activity is decreased no matter the substitution is
electron-donating or withdrawing group. Dual chlorine substitu-
tion (4 and 14) slightly increases the activity compared to JCC76,
In previous studies, systematic modification was performed on
the structure of nimesulide to improve the anti-cancer activity and
erase the COX-2 inhibitory activity [41,46]. SAR result suggests that
the dimethyl benzyl and methylsulfonamide moieties are critical
for the nimesulide analogs to inhibit cancer cell growth (Fig. 1).
Further, the conversion of the nitro group to a bulky amide moiety
generated novel anti-cancer agent JCC76 [39e41,46]. In the current
study, dimethyl benzyl and methylsulfonamide groups which are
important for the anti-cancer activity of the derivatives were
maintained, and we focused on the modification of the amide
moiety of JCC76. Various chemical structures including alkyl amide
groups, electron-donating or withdrawing group substituted ben-
zamides, bulky or small group substituted benzamides, and
heterocyclic amides were introduced at this moiety.
with IC50 of 0.91
34, which have electron-withdrawing groups as substitution,
exhibit IC50s among 1.0 Me3.0 M. The results suggest that
mM and 0.80 mM respectively. 1, 2, 3, 7, 8, 15, 21 and
m
m
electron-withdrawing groups such as nitro, cynao, multi-fluoro,
and trifluoro-methyl substituted benzamide do not increase the
activity. 26 shows very weaker activity with an IC50 of 55.35 mM,
which possibly is due to the very strong electron-withdrawing
effect and the meta position steric effect of the di-trifluoro-
methyl groups. Most of the strong electron-donating groups such
as methoxyl and multi-methoxyl substituted benzamide (10, 11, 16,
18 and 19) increase the activity with IC50s among 0.19
However, para-methyl benzamide (33) and para-ethyl benzamide
(36) show lesser potency with IC50s of 2.48 M and 2.82 mM
mMe0.68 mM.
The synthesis is described in Scheme 1. The starting material 2-
amino-5-nitrophenol was refluxed with K2CO3 and 2, 5-dimethyl
benzyl chloride to obtain compound a. Sodium hydride and
methanesulfonyl chloride were added to compound a in dry
dimethylformamide (DMF) at room temperature and the reaction
m
respectively, although both methyl and ethyl groups are electron-
donating substitution. It suggests that para position steric effect
reduces the activity, but strong electron-donating group at para
position overall increase the activity because the electron-donating
H
N
H
N
O2N
O
O
O
O
O
NH
N
N
O
S O
O
S O
O
S O
O
JCC76
First generation of Nimesulide
analog as anti-cancer agent
O
Nimesulide
CSUOH0901
Second generation
Fig. 1. Anti-cancer drug development based on COX-2 inhibitor nimesulide.

434
B. Zhong et al. / European Journal of Medicinal Chemistry 47 (2012) 432e444
Table 1 (continued)
Table 1
Inhibition of cancer cell growth of the JCC76 anlogs.
Entry
IC50 against
SKBR-3 breast
cancer cell growth
H
N
Entry
IC50 against
SKBR-3 breast
cancer cell growth
H
N
O
R
O
R
N
S
N
S
O
O
O
O
O
O
CF3
15
2.78 ꢀ 0.29
m
M
R =
R=
JCC76
1.38 ꢀ 0.10
1.13 ꢀ 0.10
1.97 ꢀ 0.21
m
m
m
M
M
M
O
O
R =
OCH3
1
2
R =
R =
16
17
18
0.19 ꢀ 0.14
34.02 ꢀ 2.01
0.15 ꢀ 0.05
m
m
m
M
M
M
NO2
OCH3
OCH3
O
NO2
R =
R =
R =
O
OCH3
O
O
NO2
Cl
R =
R =
3
4
5
3.35 ꢀ 0.40
0.91 ꢀ 0.05
0.21 ꢀ 0.01
mM
mM
mM
OCH3
O
O
OCH3
Cl
Cl
19
20
21
0.68 ꢀ 0.32
6.88 ꢀ 3.18
2.16 ꢀ 1.08
m
m
m
M
M
M
O
O
O
R =
R =
OCH3
R =
R =
O
F
F
6
7
2.28 ꢀ 0.09
mM
OCH3
F
O
R =
O
1.46 ꢀ 0.06
3.01 ꢀ 0.12
m
M
M
22
23
24
25
51.24 ꢀ 4.49
11.21 ꢀ 4.47
m
m
M
M
R =
CN
CN
O
O
8
9
m
R =
R =
R =
R =
O
O
O
0.22 ꢀ 0.01
0.20 ꢀ 0.01
m
M
M
>100 mM
Br
O
O
10 (CSUOH0901;
NSC751382)
O
O
R =
R =
m
R =
3.95 ꢀ 2.09
m
M
OCH3
OCH3
O
11
12
0.30 ꢀ 0.02
m
M
M
CF3
R =
26
27
55.35 ꢀ 4.11
7.34 ꢀ 3.99
m
m
M
M
OCH3
CF3
O
O
43.27 ꢀ 7.38
m
R =
R =
Br
R =
O
O
13
14
11.05 ꢀ 4.76
0.80 ꢀ 0.01
m
3M
M
O
14
2.15 ꢀ 0.94
m
M
R =
28
m
R =
Cl
Cl
Cl

B. Zhong et al. / European Journal of Medicinal Chemistry 47 (2012) 432e444
435
Table 1 (continued )
effect of meta substitution. 6 (para-phenyl benzamide) shows
weaker activity with an IC50 of 2.28 M. Because the phenyl group
cannot form super conjugation with the benzamide due to the
steric effect of the two phenyl rings. It makes the phenyl substi-
tution predominated mainly by steric effect. Substituted Benzyla-
m
Entry
IC50 against
SKBR-3 breast
cancer cell growth
H
N
O
R
N
S
O
O
O
mide, namely 17 and 24, display much weaker activity with IC50
above 30 M. Heterocyclic amides (37, 38, 39) decrease the activity,
with IC50s of 16.65 M, 20.12 M, and 21.88 M respectively. The
s
m
m
m
m
results suggest that benzamide moiety is better for the activity
compared to the heterocyclic amides.
R =
R =
R =
29
30
0.13 ꢀ 0.07
0.66 ꢀ 0.32
m
M
M
For the alkyl-substituted amide, lead compound JCC76 is still the
most potent one with an IC50 of 1.38
ethyl (12), 3-pentyl (22), and cyclopentyl (23) greatly decrease the
activity, and the corresponding IC50 are 43.27 M, 51.24 M, and
11.21 M respectively. Very bulky alkyl group such as hexadacanyl
(13) decreasesthe activityas well, with an IC50 of11.05 M. Itappears
mM. Small alkyl groups such as
I
O
m
m
m
m
m
SCH3
that the best alkyl substitution is a middle size and closed ring.
Overall, para position of the benzamide is very critical for the
anti-cancer activity of the JCC76 analogs. Strong electron-donating
groups at para position significantly increase the activity. Single
meta substituted benzamide greatly decreases the activity, no
matter it is electron- donating or withdrawing groupas substitution.
Severalverypotent analogs(5, 9,10,16,18, 29), which areabout 5e10
fold more active than JCC76, share some structure similarity: para
position substituted benzamide, or para and meta both electron-
donating group substituted benzamide. 10, discovered earlier in
the study, was named CSUOH0901 and submitted to the Develop-
mental Therapeutic Program at the National Cancer Institute for
screening against 60 human tumor cell lines. It was also investigated
for the anti-cancer mechanism and in vivo activity in the study.
O
31
1.01 ꢀ 0.52
mM
N
O
R =
R =
32
33
0.41 ꢀ 0.03
2.48 ꢀ 1.44
m
M
M
OC2H5
O
m
C2H5
O
2.3. CSUOH0901 caused cell population concentrated at sub-G1
and G2 phase
R =
R =
R =
34
35
36
37
1.20 ꢀ 0.59
0.58 ꢀ 0.29
2.82 ꢀ 1.51
16.65 ꢀ 2.26
m
m
m
m
M
M
M
M
CF3
Compound CSUOH0901 significantly inhibited SKBR-3 breast
cancer cell growth. It is important to elucidate the anti-cancer
mechanisms of the compound. When SKBR-3 cells were treated
O
O
OCF3
with 0.5
mM and 5
mM of CSUOH0901, cells were accumulated at G2/
M and subG1 phase after 12 h. As for 0.25
mM treatment, this
phenomenon was observed after 24 h (Table 2). The results
demonstrate that CSUOH0901 was able to inhibit cell mitosis and
induce cell apoptosis at similar dosage to inhibit cell growth. JCC76
has been shown in previous studies to induce cell apoptosis via
cytochrome c release [39,40]. It is not surprising that the more potent
JCC76 analog CSUOH0901 exhibits better potency to induce cell
apoptosis. However, JCC76 did not show good potency to cause cell
cycle arrest in previous studies [39,40]. CSUOH0901 obviously
exhibited the cell G2/M phase arresting activity. Therefore, the anti-
mitosis potency is significantly increased by the structure modifi-
CH3
O
O
R =
O
O
S
38
39
20.12 ꢀ 5.28
21.88 ꢀ 5.08
m
M
M
R =
R =
cation. 0.1 mM CSUOH0901 clearly induced subG1 cell accumulation
after 24 h treatment, but no G2/M cell accumulation was observed,
suggesting that the compound at low concentrations was able to
induce cell apoptosis without affecting the cell replication. It is
difficult to determine if the cell apoptosis and cell growth arrested by
the compound are correlated or not, since the specific molecular
targets of CSUOH0901 still remain unclear. Further investigation is
needed to identify the anti-cancer molecular targets of these
compounds.
O
m
N
Cells were treated with indicated compounds at various concentrations by six
replicates for 48 h and cell viability was measured by MTT assay as described in the
experimental section.
effect overcomes the steric effect. 2-Naphthyl amide (5) increases
the activity with an IC50 of 0.21
mM, but 1-Naphthyl amide (25)
decreases the activity with an IC50 of 3.95
m
M. Because 2-Naphthyl
2.4. Multi-cancer cell lines growth inhibition and animal acute
toxicity studies of CSUOH0901
group is more like a dual para and meta position substituted ben-
zamide, and the substitution has very strong electron-donating
effect at para position due to the super conjugation effect. 1-
Naphthyl group is more like a dual meta and ortho position
substituted benzamide, which decreases the activity due to steric
Due to the structure novelty and potency of CSUOH0901, it was
submitted tothe Developmental Therapeutic Program at the National
CancerInstitute(assignedwithcodeNSC751382), andwasselectedby

436
B. Zhong et al. / European Journal of Medicinal Chemistry 47 (2012) 432e444
O2N
OH
O2N
O
O2N
O
NaH, DMF
CH3SO2Cl
K2CO3, DMF
NaOH
aq
O
S
O
2, 5-dimethyl
Benzyl Chloride
NH2
N
S
NH2
O
O
a
b
Zn/FeCl3/H2O/DMF
NaH, DMF, CH3I
O2N
O
O2N
O
H2N
O
N
S
NH
N
S
O
O
O
S O
e
O
O
d
c
H
1,4-dioxane
O
N
O
Acyl chloride
R
N
S
O
O
20 R= 3-Methoxy-phenyl (91%)
21 R= 3-Methoxy-2,4,5-trifluorophenyl (98%)
22 R= 3-Pentyl (96%)
23 R= Cyclopentyl (93%)
24 R= Benzyl (99%)
1
R= 4-Nitro-phenyl (88%)
R= 3-Nitro-phenyl (86%)
R= 4-Chloro-3-nitro-phenyl (82%)
R= 3,4-Dichloro-phenyl (69%)
R= 2-Naphthyl (72%)
2
3
4
5
6
7
8
9
25 R= 1-Naphthyl (83%)
R= 4-Biphenyl (73%)
26 R= 3,5-Bis(trifluoromethyl)-phenyl (93%)
27 R= 3-Bromophenyl (99%)
28 R= 4-Chlorophenyl (99%)
29 R= 4-Iodophenyl (96%)
30 R= 4-Methylsulfanyl-phenyl (92%)
31 R= 4- Dimethylamino-phenyl (66%)
32 R= 4-Ethoxy-phenyl (70%)
33 R= 4-Ethyl-phenyl (64%)
34 R= 4-Trifluoromethyl-phenyl (84%)
35 R= 4-Trifluoromethoxy-phenyl(79%)
36 R= 4-Methyl-phenyl (69%)
37 R= 2-Furyl (66%)
R= 4-Cyano-phenyl (75%)
R= 3-Cyano-phenyl (71%)
R= 4-Bromo-phenyl (80%)
10 R= 5-Benzo[1,3]dioxolyl(71%)
11 R= 3,4,5-Trimethoxy-phenyl (93%)
12 R= Propionyl (88%)
13 R= Hexadecanyl (98%)
14 R= 2,4-Dichloro-phenyl (80%)
15 R= 3-Trifluoromethyl-phenyl (93%)
16 R= 3,4-Dimethoxy-phenyl (93%)
17 R= 3,4-Dimethoxy-benzyl (95%)
18 R= 4-Methoxy-phenyl (96%)
19 R= 2-Methoxy-phenyl (97%)
38 R= 2-Thiophenyl (72%)
39 R= 5-Isoxazolyl(99%)
Scheme 1. Synthesis of JCC76 analogs.
Table 2
Summary of altered cell cycle distribution in response to treatment with CSUOH0901 (NSC751382). SKBR-3 cells were treated for different time period with the indicated
concentrations of compound. Cells were processed for FACS using propidium iodide staining as described. Percent distribution of cells in each cell cycle phase was displayed.
Time
3 h
Concentrations
of CSUOH0901
Sub-G1%
G1%
S%
G2/M%
0.10 (
0.25 (
0.50 (
5.0 (
mM)
mM)
mM)
mM)
mM)
mM)
mM)
mM)
mM)
mM)
mM)
mM)
mM)
mM)
mM)
mM)
mM)
mM)
mM)
mM)
3.12
3.23
3.43
2.81
3.60
3.49
4.03
3.42
5.27
6.85
6.36
9.76
6.04
8.89
8.47
8.31
11.78
24.95
27.94
27.62
70.84
69.63
67.14
69.09
71.43
72.21
67.10
64.76
73.14
73.62
63.15
63.71
75.43
73.42
61.19
63.55
67.90
41.27
31.77
31.87
17.20
16.34
17.76
16.49
15.84
14.95
14.73
14.61
8.17
6.14
5.94
5.99
8.71
6.81
7.28
6.73
8.91
10.87
11.21
11.53
8.83
10.77
11.54
11.57
9.07
6 h
0.10 (
0.25 (
0.50 (
5.0 (
9.32
13.90
17.19
12.05
13.35
24.26
20.54
9.71
10.57
22.27
21.80
11.23
22.53
28.14
28.29
12 h
18 h
24 h
0.10 (
0.25 (
0.50 (
5.0 (
0.10 (
0.25 (
0.50 (
5.0 (
0.10 (
0.25 (
0.50 (
5.0 (

B. Zhong et al. / European Journal of Medicinal Chemistry 47 (2012) 432e444
437
NCIDTP for screening against 60 human tumor cell lines, representing
leukemia, melanoma, andcancers ofthelung, colon, CNS, ovary, renal,
prostate, and breast. After 48 h treatment, CSUOH0901 does-depen-
dently inhibited the growth of sixtycell lines from each class of tumor
cells. Three dose response parameters are calculated for the experi-
mental agent. Growth inhibition of 50% (GI50) is the drug concen-
tration resulting in a 50% reduction in the net protein increase
compared with control cells during the drug incubation. Total growth
inhibition (TGI) shows the drug concentration that causes a 100%
reduction in the net protein increase during the drug incubation. The
LC50 is the concentration of drug resulting in a 50% reduction in
protein at the end of the drug treatment as compared to the protein
amount present at the time of drug addition. Values are calculated for
each of these three parameters if the requisite level of activity is
achieved. The values of these parameters among the 60 different cell
lines after 48 h treatment are as follows: concentration resulting in
Table 3
Growth inhibitory effect of CSUOH0901 on 60 human tumor cell lines. Growth
inhibition of 50% (GI50), total growth inhibition (TGI), and LC50 are listed in the
table.
Panel/Cell line
GI50
TGI
LC50
Leukemia
CCRF-CEM
HL-60(TB)
K-562
4.85E-8
2.69E-8
4.03E-8
1.63E-7
2.90E-7
>1.00E-4
9.67E-8
>1.00E-4
>1.00E-4
>1.00E-4
>1.00E-4
>1.00E-4
>1.00E-4
>1.00E-4
MOLT-4
RPMI-8226
Non-small cell lung cancer
A549/ATCC
EKVX
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
CNS cancer
SF-268
SF295
SF539
SNB-19
SNB-75
2.78E-7
5.98E-7
3.29E-7
3.22E-6
6.86E-7
1.62E-7
4.68E-7
1.84E-7
3.35E-8
1.36E-5
>1.00E-4
>1.00E-4
>1.00E-4
>1.00E-4
>1.00E-4
>1.00E-4
>1.00E-4
>1.00E-4
>1.00E-4
> 1.00E-4
> 1.00E-4
> 1.00E-4
> 1.00E-4
> 1.00E-4
> 1.00E-4
7.44E-7
50% growth inhibition (GI50), 0.03
mMe0.5
mM; concentration
resulting in total growth inhibition (TGI), 0.2
m
Me2.0 M (in
m
approximately one half of the cell lines); concentrations resulting in
a 50% reduction in the measured protein level at the end of drug
treatment (LC50), above 20 mM in only 3 cell lines (Table 3).
The compound was also tested for the acute toxicity to deter-
mine the maximum tolerated dose in nude mice. After a single dose
of 400 mg/kg, 200 mg/kg, or 100 mg/kg, the nude mice were
observed for a period of 2 weeks. No body weight lost was found in
the three mice tested, indicating that the animals were highly
tolerable to CSUOH0901.
2.40E-7
3.18E-7
1.20E-7
7.53E-8
2.89E-7
7.72E-8
8.55E-8
1.12E-7
1.07E-6
3.92E-7
1.02E-6
>1.00E-4
4.19E-7
1.13E-6
>1.00E-4
>1.00E-4
2.97E-6
>1.00E-4
>1.00E-4
>1.00E-4
>1.00E-4
>1.00E-4
2.88E-7
9.83E-8
1.25E-7
5.37E-7
2.66E-7
2.66E-7
>1.00E-4
6.26E-7
>1.00E-4
>1.00E-4
>1.00E-4
>1.00E-4
>1.00E-4
>1.00E-4
2.5. In vivo investigation
>1.00E-4
To determine whether the high activity of our compounds at cell
culture would be translated into in vivo active anti-cancer agents,
CSUOH0901 as a representative one was investigated in the tumor
xenograft nude mice model. SKBR-3 cells do not readily form
xenografts in nude mice, therefore we used a colon cancer HT29
xenograft model to investigate the in vivo activity of the compound,
since it significantly inhibited HT29 cell growth in the in vitro study
(Developmental Therapeutics Program at National Cancer Institute)
U251
2.19E-5
Melanoma
LOXIMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian cancer
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
Renal cancer
786e0
6.66E-8
2.29E-7
7.57E-8
2.85E-8
8.97E-8
6.92E-7
2.53E-7
1.44E-7
4.14E-7
>1.00E-4
>1.00E-4
6.69E-7
9.14E-8
6.16E-7
>1.00E-4
1.65E-6
>1.00E-4
>1.00E-4
>1.00E-4
>1.00E-4
>1.00E-4
>1.00E-4
>1.00E-4
>1.00E-4
3.41E-5
with an IC50 of 0.42
CSUOH0901 in HT29 cells with an MTT cell proliferation assay, and
obtained an IC50 of 0.46 M. HT29 xenograft is a well established
mM (Table 3). We confirmed the activity of
>1.00E-4
>1.00E-4
m
in vivo tumor model, and has been used to test the activity of many
anti-cancer drug candidates. Nude mice (three mice per group, two
tumors per mouse) bearing HT29 xenografts were given daily (5
times per week) intraperitoneal injections (5 mg/kg) of CSUOH0901
for three weeks after the tumor reached a measurable size.
CSUOH0901 treatment significantly decreased the size of the
HT29 tumors compared to the control group (Fig. 2A). Weights of
the mice were not affected by the treatment (Fig. 2B), suggesting
the low toxicity of the compound. After the treatment, the tumors
were removed and weighed. CSUOH0901 significantly decreased
the tumor weights as well (Fig. 2C). The results reveal that
CSUOH0901 is active in vivo and could be a promising anti-cancer
drug candidate.
2.37E-7
3.96E-8
>1.00E-4
1.66E-7
>1.00E-4
>1.00E-4
>1.00E-4
4.07E-7
>1.00E-4
>1.00E-4
>1.00E-4
>1.00E-4
>1.00E-4
>1.00E-4
5.38E-7
2.65E-7
5.62E-8
2.85E-7
>1.00E-4
1.91E-7
1.22E-7
6.87E-7
1.26E-7
1.28E-7
4.72E-7
3.54E-7
3.95E-7
1.11E-6
8.53E-7
>1.00E-4
>1.00E-4
>1.00E-4
>1.00E-4
>1.00E-4
>1.00E-4
>1.00E-4
>1.00E-4
A498
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
>1.00E-4
>1.00E-4
4.03E-7
>1.00E-4
>1.00E-4
>1.00E-4
Prostate cancer
PC-3
DU-145
6.77E-8
1.59E-7
>1.00E-4
4.57E-7
>1.00E-4
>1.00E-4
2.6. Pharmacokinetic study
Breast cancer
MCF7
MDA-MB-231/ATCC
HS 578T
BT-549
CSUOH0901 exhibited potent in vitro and in vivo anti-tumor
activity. To support future further pharmacological and toxicolog-
ical study, a pharmacokinetic study of the compound was also
performed. CSUOH0901 was administrated to rats intraperitoneally
1.20E-7
2.56E-7
8.70E-8
6.56E-7
6.82E-7
1.55E-7
>1.00E-4
9.26E-7
>1.00E-4
>1.00E-4
>1.00E-4
>1.00E-4
>1.00E-4
1.99E-5
>1.00E-4
>1.00E-4
4.01E-7
T-47D
MDA-MB-468
at a dose of 20 mg/kg. Blood samples (150 mL each) were collected
from the saphenous veins and femoral veins into heparized tubes at
0 h (before drug administration) and at 0.25, 0.5, 1, 2, 4, 8 and 24 h
after dosing. The peripheral blood drug level was then determined

438
B. Zhong et al. / European Journal of Medicinal Chemistry 47 (2012) 432e444
compound recommends further drug development of these small
molecule anti-cancer agents.
A
2500
2000
1500
1000
500
Control
CSU0901
3. Conclusion
The structural modifications of nimesulide have been effective
in abolishing its COX-2 inhibiting property and reducing its hepa-
totoxicity. Among the new derivatives synthesised, compounds 5,
9, 10 (CSUOH0901), 16, 18 and 29 have exhibited good growth
inhibitory activity against SK-BR-3 breast cancer cells at low-
nanomolar concentrations. CSUOH0901 displayed good potency
to inhibit the growth of a broad range of cancer cell lines and
demonstrated minor animal toxicity. The in vivo tumor suppression
activity and pharmacokinetic results of CSUOH0901 suggest that
the drug candidate has great clinical application potential.
0
5
8
3
11
14
17
20
2
26
29
Days
B
32
30
28
26
24
Control
CSU0901
4. Experimental section
4.1. Chemistry
Chemicals were commercially available and used as received
without further purification unless otherwise noted. Moisture
sensitive reactions were carried out under a dry argon atmosphere
in flame-dried glassware. Solvents were distilled before use under
argon. Thin-layer chromatography was performed on precoated
silica gel F254 plates (Whatman). Silica gel column chromatog-
raphy was performed using silica gel 60A (Merck, 230-400 Mesh),
and hexane/ethyl acetate was used as the elusion solvent. Mass
spectra were obtained on the Micromass QTOF Electrospray mass
spectrometer at Cleveland State University MS facility Center.
Melting point was recorded with a Mel-Temp melting point appa-
ratus. All the NMR spectra were recorded on a Varian 400 MHz in
6
9
12
15
18
21
24
27
30
Days
C
P=0.0136
0.6
0.4
0.2
0.0
either DMSO-d6 or CDCl3. Chemical shifts (
d
) for 1H-NMR spectra
are reported in parts per million to residual solvent protons. The IR
spectra were obtained on a Bruker ALPHA FT-IR spectrometer with
ATR module.
For the HPLC analysis, a 1.00 mg/mL stock solution of each
standard was prepared in either methanol or acetonitrile. The HPLC
system consists of two LC-20AD pumps, a DGU-20A3 degasser,
a SIL-20AC autosampler, and a CBM-20A module (Shimazu, Tokyo,
Japan). The chromatographic separation was performed on a Luna
*
C18 column (2.0 mm ꢁ 150 mm, 5
mm) with a guard column
m) from Phenomenex (Torrance, CA, USA) at
(2 mm ꢁ 40 mm, 5
m
Control
CSU0901
room temperature with a flow rate of 0.2 mL/min. Two mobile
phases (10 mM ammonium acetate in 90% methanol or acetonitrile)
were employed to run 15 min. An injection volume of 5e15 mL was
used. The UV detector was set up at 290 and 256 nM.
Fig. 2. In vivo anti-cancer evaluation of CSUOH0901. A. Nude mice bearing HT29
xenograft after the treatment. The treatment group is significantly smaller than the
control group (n ¼ 6); B Mice weight changing (n ¼ 3); C HT29 tumor weight
comparison (n ¼ 6). *P < 0.05 vs control.
Compounds aee were prepared as described by Su et al. [41,46].
4.1.1. General procedure for the preparation of the substituted
benzamide 1e39
with LC-MS/MS. The mean CSUOH0901 concentration in plasma
versus time profile was presented in Fig. 3. Peak drug concentra-
tions were observed at 2 h after administration and reached
nearly1500 ng/mL. The pharmacokinetic parameters were calcu-
lated by using non-compartmental model. The estimated phar-
macokinetic parameters including the terminal phase elimination
half-life (T1/2), the area under the plasma concentration time
curve (AUC) from time 0 to time of the last measurable concen-
tration (AUC(0-t)), the volume of distribution (Vz/F), the total body
clearance (Cl/F), and the mean residence time (MRT) from time 0 to
time of the last measurable concentration (MRT(0-t)) are listed in
Table 4. The half life and volume of distribution of the compound is
relatively lower compared with more hydrophobic anti-cancer
drugs such as Taxol [47], suggesting the compound has better
drug like characters than Taxol. The good bioavailability of the
K2CO3 (5 mmol, 5eq) and substituted acyl chloride (1.2 mmol,1.2
eq) were successively added to a solution of the aniline e (1.0 mmol,
1.0 eq) in 3 mL dry 1, 4 -dioxane and the mixture was stirred at
room temperature overnight. 10 mL H2O and 3 mL saturated
aqueous Na2CO3 was added to the mixture and it was stirred at
room temperature overnight. The precipitated solid was collected
by filtration and purified by silica gel column chromatography.
4.1.1.1. N-[3-(2,5-dimethyl-benzyloxy)-4-(methanesulfonyl-methyl-
amino)-phenyl]-4-nitro-benzamide (1). 4-Nitro-benzoyl chloride
was used and it was stirred at room temperature overnight. Pale
yellow solid, melting point 206e209 ꢂC, yield 88%: 1H NMR
(400 MHz, DMSO-d6)
(2H, d, J ¼ 8.2 Hz), 7.75 (1H, s), 7.44 (1H, d, J ¼ 8.4 Hz), 7.33(2H, m),
d
10.68 (1H, s), 8.41 (2H, d, J ¼ 8.0 Hz), 8.21

B. Zhong et al. / European Journal of Medicinal Chemistry 47 (2012) 432e444
439
Fig. 3. CSUOH0901 rats blood concentrations vs time. CSUOH0901 was administrated to rats (n ¼ 3) intraperitoneally with 20 mg/kg. Blood samples of 150
m
L each were collected
from the saphenous veins and femoral veins into heparized tubes at 0 h (before drug administration) and at 0.25, 0.5, 1, 2, 4, 8 and 24 h after dosing. The blood drug concentration
was then determined with LC-MS/MS.
7.15(1H, d, J ¼ 7.8 Hz), 7.09(1H, d, J ¼ 7.8 Hz), 5.11 (2H, s), 3.12 (3H, s),
2.88 (3H, s), 2.32 (3H, s), 2.28 (3H, s); HRMS calculated for
C24H26N2NaO4S [M þ Na]þ 461.1511, found: 461.1511.
4.1.1.5. Naphthalene-2-carboxylic acid [3-(2,5-dimethyl-benzyloxy)-4-
(methanesulfonyl-methyl-amino)-phenyl]-amide (5). Naphthalene-
2-carbonyl chloride was used and it was stirred at room temperature
for three days. White solid, melting point 133e136 ꢂC, yield 72%: IR
4.1.1.2. N-[3-(2,5-dimethyl-benzyloxy)-4-(methanesulfonyl-methyl-
amino)-phenyl]-3-nitro-benzamide (2). 3-Nitro-benzoyl chloride was
used and it was stirred at room temperature overnight. White solid,
melting point 163e165 ꢂC, yield 86%: 1H NMR (400 MHz, DMSO-d6)
n
¼ 3314 (NeH), 1649 (C]O), 1595, 1509 (aromatic C]C). 1H NMR
(400 MHz, DMSO-d6)
d 10.56 (1H, s), 8.60 (1H, s), 8.12 (4H, m), 7.82
(1H, s), 7.67 (2H, m), 7.50 (1H, d, J ¼ 8.5 Hz), 7.33(2H, m), 7.16(1H, d,
J ¼ 7.8 Hz), 7.10 (1H, d, J ¼ 7.3 Hz), 5.13 (2H, s), 3.13 (3H, s), 2.88 (3H,
d
10.70 (1H, s), 8.80 (1H, s), 8.48 (1H, d, J ¼ 8.0 Hz), 8.43 (1H, d,
s), 2.34 (3H, s), 2.29 (3H, s). 13C NMR (100 MHz, DMSO-d6)
d 165.581,
J ¼ 8.0 Hz),7.89 (1H, dd, J ¼ 7.8, 7.8 Hz), 7.75 (1H, s), 7.45 (1H,d,
J ¼ 8.6 Hz),7.34(2H, m), 7.15(1H, d, J ¼ 7.4 Hz), 7.09 (1H, d, J ¼ 7.5 Hz),
5.12 (2H, s), 3.12 (3H, s), 2.88 (3H, s), 2.33 (3H, s), 2.28 (3H, s). ESI-MS
(m/z) calculated for C24H24N3O6S [M þ H]þ: 482.1, found: 481.9.
155.169, 148.112, 140.164, 134.702, 134.229, 134.084, 133.100,
131.925, 130.956, 130.003, 129.133, 128.874, 128.660, 127.989,
127.951, 127.836, 127.600, 126.829, 124.663, 124.297, 112.289,
105.348, 68.106, 37.623, 37.509, 20.520,17.873. ESI-MS calculated for
C28H27N2O4S [M þ H]þ: 487.2, found: 486.9.
4.1.1.3. 4-Chloro-N-[3-(2,5-dimethyl-benzyloxy)-4-(methanesulfonyl-
methyl-amino)-phenyl]-3-nitro-benzamide (3). 4-Chloro-3-nitro-be-
nzoyl chloride was used and it was stirred at room temperature
overnight. White solid, melting point 204e206 ꢂC, yield 82%: 1H
4.1.1.6. Biphenyl-4-carboxylic acid [3-(2,5-dimethyl-benzyloxy)-4-
(methanesulfonyl-methyl-amino)-phenyl]-amide
(6). Biphenyl-4-
carbonyl chloride was used and it was stirred at room tempera-
NMR (400 MHz, DMSO-d6)
d
10.65 (1H, s), 8.65 (1H, s), 8.28 (1H, d,
ture for three days. White solid, melting point 136e139 ꢂC, yield
J ¼ 8.4 Hz), 8.01 (1H, d, J ¼ 8.4 Hz), 7.72 (1H, s), 7.42 (1H, d,
J ¼ 8.4 Hz), 7.33(2H, m), 7.15(1H, d, J ¼ 7.7 Hz), 7.09 (1H, d, J ¼ 7.4 Hz),
5.11 (2H, s), 3.12 (3H, s), 2.87 (3H, s), 2.32 (3H, s), 2.28 (3H, s). ESI-MS
calculated for C24H23ClN3O6S [M þ H]þ: 516.1, found: 515.8.
73%: 1H NMR (400 MHz, DMSO-d6)
d 10.42 (1H, s), 8.09 (2H, d,
J ¼ 7.9 Hz), 7.88 (2H, d, J ¼ 7.8 Hz), 7.81 (1H, s), 7.79 (2H, d,
J ¼ 7.2 Hz), 7.54(4H, m), 7.33 (2H, m), 7.16(1H, d, J ¼ 7.7 Hz), 7.10 (1H,
d, J ¼ 7.5 Hz), 5.12 (2H, s), 3.12 (3H, s), 2.87 (3H, s), 2.33 (3H, s), 2.28
(3H, s). ESI-MS calculated for C30H29N2O4S [M þ H]þ: 513.2, found:
512.9.
4.1.1.4. 3,4-Dichloro-N-[3-(2,5-dimethyl-benzyloxy)-4-(methanesulf-
onyl-methyl-amino)-phenyl]-benzamide (4). 3,4-Dichloro-benzoyl
chloride was used and it was stirred at room temperature over-
night. White solid, melting point 177e179 ꢂC, yield 69%: 1H NMR
4.1.1.7. 4-Cyano-N-[3-(2,5-dimethyl-benzyloxy)-4-(methanesulfonyl-
methyl-amino)-phenyl]-benzamide (7). 4-Cyano-benzoyl chloride
was used and it was stirred at room temperature overnight. White
solid, melting point 183e187 ꢂC, yield 75%: 1H NMR (400 MHz,
(400 MHz, DMSO-d6)
d 10.51 (1H, s), 8.23 (1H, s), 7.97 (1H, d,
J ¼ 8.4 Hz), 7.85 (1H, d, J ¼ 8.4 Hz), 7.73 (1H, s), 7.42 (1H, d,
J ¼ 7.6 Hz), 7.31(2H, m), 7.15(1H, d, J ¼ 7.7 Hz), 7.09 (1H, d,
J ¼ 7.6 Hz), 5.10 (2H, s), 3.12 (3H, s), 2.87 (3H, s), 2.32 (3H, s), 2.28
(3H, s). ESI-MS calculated for C24H23Cl2N2O4S [M þ H]þ: 505.1,
found: 504.8.
DMSO-d6)
d
10.60 (1H, s), 8.13 (2H, d, J ¼ 8.4 Hz), 8.06 (2H, d,
J ¼ 8.2 Hz), 7.75 (1H, d, J ¼ 1.7 Hz), 7.43 (1H, dd, J ¼ 1.9, 8.5 Hz), 7.32
(2H, m), 7.15(1H, d, J ¼ 7.7 Hz), 7.09 (1H, d, J ¼ 7.7 Hz), 5.11 (2H, s),
3.12 (3H, s), 2.87 (3H, s), 2.32 (3H, s), 2.28 (3H, s). ESI-MS calculated
for C25H24N3O4S [M þ H]þ: 462.1, found: 461.9.
Table 4
The pharmacokinetic parameters of CSUOH0901.
4.1.1.8. 3-Cyano-N-[3-(2,5-dimethyl-benzyloxy)-4-(methanesulfonyl-
methyl-amino)-phenyl]-benzamide (8). 3-Cyano-benzoyl chloride
was used and it was stirred at room temperature overnight. White
solid, melting point 166e170 ꢂC, yield 71%: 1H NMR (400 MHz,
Parameters
Rat 1
Rat 2
Rat 3
Mean
S.D.
T1/2 (h)
AUC(0-t) (ng*h/mL)
Vz/F (L/kg)
Cl/F (L/h)
MRT(0-t) (h)
2.44
16466
1.51
0.42
5.95
3.71
22648
1.65
0.32
6.43
3.21
9718
3.33
0.70
5.62
3.12
16277
2.16
0.48
6.00
0.64
6467
1.01
0.20
0.40
DMSO-d6)
d
10.54 (1H, s), 8.42 (1H, s), 8.27 (1H, d, J ¼ 7.8 Hz), 8.10
(1H, d, J ¼ 7.7 Hz), 7.80 (2H, m), 7.43 (1H, d, J ¼ 8.5 Hz), 7.32(2H, m),
7.15(1H, d, J ¼ 7.7 Hz), 7.09 (1H, d, J ¼ 7.8 Hz), 5.11 (2H, s), 3.12 (3H,

440
B. Zhong et al. / European Journal of Medicinal Chemistry 47 (2012) 432e444
s), 2.87 (3H, s), 2.32 (3H, s), 2.28 (3H, s). ESI-MS calculated for
7.64 (1H, d, J ¼ 8.2 Hz), 7.59 (1H, d, J ¼ 8.4 Hz), 7.29 (3H, br), 7.14(1H,
d, J ¼ 7.4 Hz), 7.08 (1H, d, J ¼ 7.4 Hz), 5.09 (2H, s), 3.12 (3H, s), 2.87
(3H, s), 2.31 (3H, s), 2.27 (3H, s). ESI-MS calculated for
C24H23Cl2N2O4S [M þ H]þ: 505.1, found: 504.8.
C25H24N3O4S [M þ H]þ: 462.1, found: 461.9.
4.1.1.9. 4-Bromo-N-[3-(2,5-dimethyl-benzyloxy)-4-(methanesulfonyl-
methyl-amino)-phenyl]-benzamide (9). 4-Bromo-benzoyl chloride
was used and it was stirred at room temperature overnight. White
4.1.1.15. N-[3-(2,5-dimethyl-benzyloxy)-4-(methanesulfonyl-methyl-
amino)-phenyl]-3-trifluoromethyl-benzamide (15). 3-Trifluorome-
thyl-benzoyl chloride was used and it was stirred at room
temperature overnight. White solid, melting point 179e181 ꢂC,
solid, melting point 196e197 ꢂC, yield 80%: IR
n
¼ 3338 (NeH), 1654
(C]O), 1593, 1507 (aromatic C]C). 1H NMR (400 MHz, DMSO-d6)
d
10.43 (1H, s), 7.93 (2H, d, J ¼ 8.2 Hz), 7.79 (2H, d, J ¼ 8.5 Hz), 7.75
(1H, s), 7.40 (1H, m), 7.31 (2H, m), 7.15(1H, d, J ¼ 7.7 Hz), 7.09 (1H, d,
J ¼ 7.7 Hz), 5.10 (2H, s), 3.11 (3H, s), 2.87 (3H, s), 2.32 (3H, s), 2.28
yield 93%: 1H NMR (400 MHz, DMSO-d6)
d 10.61 (1H, s), 8.30 (2H,
m), 8.00 (1H, d, J ¼ 7.6 Hz), 7.81 (2H, m), 7.44 (1H, d, J ¼ 8.5 Hz),
7.32(1H, s), 7.32 (1H, d, J ¼ 8.4 Hz), 7.15(1H, d, J ¼ 7.6 Hz), 7.09 (1H, d,
J ¼ 7.6 Hz), 5.11 (2H, s), 3.12 (3H, s), 2.87 (3H, s), 2.32 (3H, s), 2.28
(3H, s). ESI-MS calculated for C25H24F3N2O4S [M þ H]þ: 505.1,
found: 504.9.
(3H, s). 13C NMR (100 MHz, DMSO-d6)
d 164.551, 155.146, 139.874,
134.702, 134.046, 133.657, 133.100, 131.368, 130.948, 130.003,
129.720,129.141,128.668, 125.456,124.815,112.335,105.393, 68.114,
37.623, 37.486, 20.520, 17.866. ESI-MS calculated for C24H24BrN2O4S
[M þ H]þ: 515.1, found: 514.8.
4.1.1.16. N-[3-(2,5-dimethyl-benzyloxy)-4-(methanesulfonyl-methyl-
4.1.1.10. Benzo[1,3]dioxole-5-carboxylic acid [3-(2,5-dimethyl-ben-
zyloxy)-4-(methanesulfonyl-methyl-amino)-phenyl]-amide (10). 1,3-
Dihydro-isobenzofuran-5-carbonyl chloride was used and it was
stirred at room temperature overnight. White solid, melting point
amino)-phenyl]-3,4-dimethoxy-benzamide
(16). 3,4-Dimethoxy-
benzoyl chloride was used and it was stirred at room temperature
overnight. White solid, melting point 184e186 ꢂC, yield 93%: IR
n
¼ 3352 (NeH), 1649 (C]O), 1599, 1505 (aromatic C]C). 1H NMR
159e165 ꢂC (decomposed), yield 71%: IR
n
¼ 3354 (NeH), 1653 (C]
(400 MHz, CDCl3)
d
8.025 (2H, m), 7.510 (1H, J ¼ 2 Hz), 7.434 (1H, dd,
O), 1596, 1506 (aromatic C]C). 1H NMR (400 MHz, DMSO-d6)
J ¼ 8.4, 2 Hz), 7.309 (1H, J ¼ 8.8 Hz), 7.162 (1H, s), 7.109 (2H, m),
6.919 (1H, d, J ¼ 8.4 Hz), 6.832 (1H, dd, J ¼ 2, 8.4 Hz), 5.067 (2H, s),
3.957 (3H, s), 3.952 (3H, s), 3.195 (3H, s), 2.707 (3H, s), 2.335 (3H, s),
d
10.18 (1H, s), 7.75 (1H, s), 7.60 (1H, d, J ¼ 8.1 Hz), 7.52 (1H, s), 7.41
(1H, dd, J ¼ 1.5, 8.4 Hz), 7.31 (1H, s), 7.28 (1H, d, J ¼ 8.5 Hz), 7.15(1H,
d, J ¼ 7.6 Hz), 7.09 (2H, d, J ¼ 8.0 Hz), 6.15 (2H, s), 5.09 (2H, s), 3.11
(3H, s), 2.86 (3H, s), 2.32 (3H, s), 2.28 (3H, s). 13C NMR (100 MHz,
2.320 (3H, s). 13C NMR (100 MHz, DMSO-d6)
d 164.933, 155.138,
151.682, 148.242, 140.278, 134.709, 134.099, 133.130, 130.865,
130.010, 129.194, 128.683, 126.616, 124.457, 120.948, 112.312,
110.955, 110.749, 105.355, 68.114, 55.588, 55.527, 37.608, 37.516,
20.520, 17.873. ESI-MS calculated for C26H29N2O6S[M þ H]þ: 497.2,
found: 496.9.
DMSO-d6)
d 164.475, 155.100, 150.088, 147.311, 140.186, 134.686,
134.068, 133.107, 130.857, 129.995, 129.171, 128.660, 128.362,
124.472, 122.832, 112.206, 107.873, 107.598, 105.271, 101.770,
68.076, 37.585, 37.493, 20.513, 17.858. ESI-MS calculated for
C25H25N2O6S [M þ H]þ: 481.1, found: 480.8.
4.1.1.17. N-[3-(2,5-dimethyl-benzyloxy)-4-(methanesulfonyl-methyl-
amino)-phenyl]-(3,4-dimethoxyphenyl)-acetamide (17). (3,4-Dime-
thoxy-phenyl)-acetyl chloride was used and it was stirred at
room temperature overnight. White solid, melting point
4.1.1.11. N-[3-(2,5-dimethyl-benzyloxy)-4-(methanesulfonyl-methyl-
amino)-phenyl]-3,4,5-trimethoxy-benzamide (11). 3,4,5-Trimethoxy-
benzoyl chloride was used and it was stirred at room temperature
overnight. White solid, melting point 187e189 ꢂC, yield 93%: 1H
149e152 ꢂC, yield 95%: 1H NMR (400 MHz, CDCl3)
d 7.844 (1H, d,
NMR (400 MHz, DMSO-d6)
d
10.26 (1H, s), 7.76 (1H, s), 7.36 (5H, m),
J ¼ 2.4 Hz), 7.240 (2H, m), 7.141 (1H, s), 7.103 (2H, m), 6.893 (2H, m),
6.825 (1H, d, J ¼ 1.6 Hz), 6.555 (1H, dd, J ¼ 2.4, 8.4 Hz), 5.029 (2H, s),
3.907 (3H, s), 3.898 (3H, s), 3.696 (2H, s), 3.157 (3H, s) 2.683 (3H, s),
2.322 (3H, s), 2.312 (3H, s). ESI-MS calculated for C27H31N2O6S
[M þ H]þ: 511.2, found: 510.9.
7.13(1H, d, J ¼ 7.7 Hz), 7.09 (1H, d, J ¼ 7.7 Hz), 5.11 (2H, s), 3.89 (6H, s),
3.75 (3H, s), 3.12 (3H, s), 2.87 (3H, s), 2.32 (3H, s), 2.28 (3H, s). ESI-MS
calculated for C27H31N2O7S [M þ H]þ: 527.2, found: 526.9.
4.1.1.12. N-[3-(2,5-dimethyl-benzyloxy)-4-(methanesulfonyl-methyl-
amino)-phenyl]-propionamide (12). Propionoyl chloride was used
and it was stirred at room temperature overnight. White solid,
melting point 168e170 ꢂC, yield 88%: 1H NMR (400 MHz, DMSO-d6)
4.1.1.18. N-[3-(2,5-dimethyl-benzyloxy)-4-(methanesulfonyl-methyl-
amino)-phenyl]-4-methoxy-benzamide
chloride was used and it was stirred at room temperature over-
night. White solid, melting point 175e178 ꢂC, yield 96%: IR
¼ 3345
(NeH), 1649 (C]O),1602,1506 (aromatic C]C). 1H NMR (400 MHz,
CDCl3)
(18). 4-Methoxy-benzoyl
d
10.00 (1H, s), 7.58 (1H, s), 7.28 (1H, s), 7.23 (1H, d, J ¼ 8.5 Hz),
n
7.16(1H, s), 7.14(1H, d, J ¼ 7.6 Hz), 7.08 (1H, d, J ¼ 7.6 Hz), 5.06 (2H,
s), 3.08 (3H, s), 2.84 (3H, s), 2.36 (2H, dd, J ¼ 7.5, 6.4 Hz), 2.30 (3H, s),
2.27 (3H, s), 1.10 (3H, dd, J ¼ 7.5, 7.6 Hz). ESI-MS calculated for
C20H25N2O4S [M þ H]þ: 389.2, found: 388.9.
d
8.022 (1H, d, J ¼ 2.4 Hz), 7.864 (3H, m), 7.330 (1H, d,
J ¼ 8.4 Hz), 7.175 (1H, s), 7.113 (2H, m), 6.995 (2H, m), 6.823 (1H, dd,
J ¼ 2.4, 8.4 Hz), 5.086 (2H, s), 3.886 (3H, s), 3.198 (3H, s), 2.712 (3H,
s), 2.345 (3H, s), 2.325 (3H, s). 13C NMR (100 MHz, DMSO-d6)
4.1.1.13. Hexadecanoic acid [3-(2,5-dimethyl-benzyloxy)-4-(meth-
anesulfonyl-methyl-amino)-phenyl]-amide (13). Hexadecanoyl chlo-
ride was used and it was stirred at room temperature overnight.
White solid, melting point 134e140 ꢂC(decomposed), yield 98%: 1H
d 164.887, 161.920, 155.107, 140.324, 134.694, 134.091, 133.107,
130.857, 129.995, 129.552, 129.171, 128.660, 126.600, 124.380,
113.541, 112.190, 105.264, 68.083, 55.351, 37.593, 37.509, 20.513,
17.866. ESI-MS calculated for C25H27N2O5S [M þ H]þ: 467.2, found:
466.9.
NMR (400 MHz, DMSO-d6)
d 10.03 (1H, s), 7.59 (1H, s), 7.28 (1H, s),
7.20 (4H, m), 5.06 (2H, s), 3.08 (3H, s), 2.84 (3H, s), 2.30 (3H, s), 2.27
(3H, s), 1.58 (2H, br), 1.23 (26H, br), 0.87 (3H, dd, J ¼ 5.1, 6.6 Hz).
4.1.1.19. N-[3-(2,5-dimethyl-benzyloxy)-4-(methanesulfonyl-methyl-
amino)-phenyl]-2-methoxy-benzamide
(19). 2-Methoxy-benzoyl
4.1.1.14. 2,4-Dichloro-N-[3-(2,5-dimethyl-benzyloxy)-4-(meth-
anesulfonyl-methyl-amino)-phenyl]-benzamide (14). 2,4-Dichloro-
benzoyl chloride was used and it was stirred at room temperature
overnight. White solid, melting point 163e166 ꢂC, yield 80%: 1H
chloride was used and it was stirred at room temperature over-
night. White solid, melting point 147e149 ꢂC, yield 97%: 1H NMR
(400 MHz, CDCl3)
(1H, d, J ¼ 2.4 Hz), 7.535 (1H, m), 7.342 (1H, d, J ¼ 8.4 Hz), 7.106 (5H,
d
9.932 (1H, s), 8.283 (1H, dd, J ¼ 2, 8 Hz), 8.202
NMR (400 MHz, DMSO-d6)
d 10.68 (1H, s), 7.79 (1H, s), 7.61 (1H, s),
m), 6.759 (1H, dd, J ¼ 2, 8.4 Hz), 5.119 (2H, s), 4.079 (3H, s), 3.201

B. Zhong et al. / European Journal of Medicinal Chemistry 47 (2012) 432e444
441
(3H, s), 2.706 (3H, s), 2.361 (3H, s), 2.329 (3H, s). ESI-MS calculated
(trifluoro-methyl)benzoyl chloride was used and it was stirred at
for C25H27N2O5S [M þ H]þ: 467.2, found: 466.9.
room temperature overnight. White solid, melting point
191e192 ꢂC, yield 93%: 1H NMR (400 MHz, CDCl3)
d 8.348 (3H, s),
4.1.1.20. N-[3-(2,5-dimethyl-benzyloxy)-4-(methanesulfonyl-methyl-
8.067 (1H, s), 7.892 (1H, s), 7.289 (1H, d, J ¼ 10.8 Hz), 7.173 (1H, s),
7.108 (2H, m), 6.908 (1H, d, J ¼ 8.4 Hz), 5.051 (2H, s), 3.193(3H, s),
2.754 (3H, s), 2.342 (3H, s), 2.309 (3H, s). ESI-MS calculated for
C26H23F6N2O4S [M þ H]þ: 573.1, found: 572.9.
amino)-phenyl]-3-methoxy-benzamide
(20). 3-Methoxy-benzoyl
chloride was used and it was stirred at room temperature over-
night. White solid, melting point 143e144 ꢂC, yield 91%: 1H NMR
(400 MHz, CDCl3)
d
8.021 (1H, d, J ¼ 2.4 Hz), 7.938 (1H, s), 7.445 (1H,
m), 7.406 (2H, m), 7344 (1H, d, J ¼ 8.4 Hz), 7.177 (1H, s), 7.121 (3H,
m), 6.848 (1H, dd, J ¼ 2.4, 8.4 Hz), 5.093 (2H, s), 3.885 (3H, s), 3.201
(3H, s), 2.714 (3H, s), 2.349 (3H, s), 2.327 (3H, s). ESI-MS calculated
for C25H27N2O5S [M þ H]þ: 467.2, found: 466.9.
4.1.1.27. N-[3-(2,5-dimethyl-benzyloxy)-4-(methanesulfonyl-methyl-
amino)-phenyl]-3-bromobenzamide (27). 3-Bromobenzoyl chloride
was used and it was stirred at room temperature overnight. White
solid, melting point 176e178 ꢂC, yield 99%: 1H NMR (400 MHz,
CDCl3)
d
8.022 (2H, m), 7.947 (1H, d, J ¼ 2.4 Hz)), 7.810 (1H, d,
4.1.1.21. N-[3-(2,5-dimethyl-benzyloxy)-4-(methanesulfonyl-methyl-
amino)-phenyl]-3-methoxy-2,4,5-trifluorobenzamide (21). 3-Meth-
oxy-2,4,5-trifluorobenzoyl chloride was used and it was stirred at
room temperature overnight. White solid, melting point
J ¼ 8 Hz), 7.698 (1H, d, J ¼ 8 Hz), 7.404 (1H, t, J ¼ 8 Hz), 7.320 (1H, d,
J ¼ 8.4 Hz), 7.172 (1H, s), 7.114 (2H, m), 6.867 (1H, dd, J ¼ 2.4, 8.4 Hz),
5.072 (2H, s), 3.199 (3H, s), 2.726 (3H, s), 2.343 (3H, s), 2.322 (3H, s).
ESI-MS calculated for C24H24BrN2O4S [M þ H]þ: 515.1, found: 514.8.
166e169 ꢂC, yield 98%: 1H NMR (400 MHz, CDCl3)
d 8.381 (1H, d,
J ¼ 14.8 Hz), 7.927 (1H, s), 7.680 (1H, m), 7.362 (1H, d, J ¼ 8.4 Hz),
7.180 (1H, s), 7.115 (2H, m), 6.880 (1H, d, J ¼ 8.4 Hz), 5.094 (2H, s),
4.106 (3H, s), 3.199 (3H, s), 2.719 (3H, s), 2.358 (3H, s), 2.328 (3H, s).
ESI-MS calculated for C25H24F3N2O5S [M þ H]þ: 521.1, found: 520.9.
4.1.1.28. N-[3-(2,5-dimethyl-benzyloxy)-4-(methanesulfonyl-methyl-
amino)-phenyl]-4-chlorobenzamide (28). 4-Chloro-benzoyl chlo-
ride was used and it was stirred at room temperature overnight.
White solid, melting point 171e174 ꢂC, yield 99%: 1H NMR
(400 MHz, CDCl3)
d
7.967 (1H, d, J ¼ 2.4 Hz), 7.951 (1H, s), 7.838 (2H,
4.1.1.22. N-[3-(2,5-dimethyl-benzyloxy)-4-(methanesulfonyl-methyl-
amino)-phenyl]-2-ethyl- butyramide (22). 2-Ethyl-butyryl chloride
was used and it was stirred at room temperature overnight. White
solid, melting point 151e154 ꢂC, yield 96%: 1H NMR (400 MHz,
m), 7.491 (2H, m), 7.335 (1H, d, J ¼ 8.4 Hz), 7.176 (1H, s), 7.111 (2H,
m), 6.850 (1H, dd, J ¼ 2.4, 8.4 Hz), 5.084 (2H, s), 3.204 (3H, s), 2.725
(3H, s), 2.348 (3H, s), 2.326 (3H, s). ESI-MS calculated for
C24H24ClN2O4S [M þ H]þ: 471.1, found: 470.8.
CDCl3)
d
8.049 (1H, d, J ¼ 2.4 Hz), 7.296 (1H, s), 7.275 (1H, s), 7.157
(1H, s), 7.108 (2H, m), 6.673 (1H, dd, J ¼ 2.4, 8.8 Hz), 5.064 (2H, s),
3.172 (3H, s), 2.691(3H, s), 2.339 (3H, s), 2.318 (3H, s), 2.08 (1H, m),
1.70 (4H, m), 0.970 (6H, t, J ¼ 7.6 Hz). ESI-MS calculated for
C23H31N2O4S [M þ H]þ: 431.2, found: 430.9.
4.1.1.29. N-[3-(2,5-dimethyl-benzyloxy)-4-(methanesulfonyl-methyl-
amino)-phenyl]-4-iodobenzamide (29). 4-Iodo-benzoyl chloride
was used and it was stirred at room temperature overnight. White
solid, melting point 207e209 ꢂC, yield 96%: IR
n
¼ 3308 (NeH), 1653
(C]O), 1594, 1518 (aromatic C]C). 1H NMR (400 MHz, DMSO-d6)
4.1.1.23. N-[3-(2,5-dimethyl-benzyloxy)-4-(methanesulfonyl-methyl-
amino)-phenyl]-cyclopentanecarboxamide (23). Cyclopentane carb-
onyl chloride was used and it was stirred at room temperature
overnight. White solid, melting point 170e172 ꢂC, yield 93%: 1H
d
10.408 (1H, s), 7.939 (2H, m), 7.748 (3H, m), 7.398 (1H, dd, J ¼ 2.4,
8.8 Hz), 7.291 (2H, m), 7.124 (2H, m), 5.090 (2H, s), 3.105(3H, s),
2.858 (3H, s), 2.311 (3H, s), 2.270 (3H, s). 13C NMR (100 MHz, DMSO-
d6)
d 164.811, 155.130, 139.912, 137.204, 134.686, 134.046, 133.954,
NMR (400 MHz, CDCl3)
d
7.998 (1H, d, J ¼ 2 Hz), 7.357 (1H, s), 7.265
133.092, 130.926, 129.995, 129.514, 129.133, 128.660, 124.762,
112.328, 105.386, 99.474, 68.106, 37.615, 37.486, 20.513, 17.858. ESI-
MS calculated for C24H24IN2O4S [M þ H]þ: 563.1, found: 562.8.
(1H, d, J ¼ 8.4 Hz), 7.149 (1H, s), 7.106 (2H, m), 6.641 (1H, dd, J ¼ 2.4,
8.4 Hz), 5.045 (2H, s), 3.169 (3H, s), 2.703 (1H, m), 2.690 (3H, s),
2.327 (3H, s), 2.318 (3H, s), 1.921 (4H, m), 1.80 (2H, m), 1.641(2H, m).
ESI-MS calculated for C23H29N2O4S [M þ H]þ: 429.2, found: 428.9.
4.1.1.30. N-[3-(2,5-dimethyl-benzyloxy)-4-(methanesulfonyl-methyl-
amino)-phenyl]-4-methylsulfanyl-benzamide (30). 4-Methylsulfanyl-
benzoyl chloride was used and it was stirred at room temperature
overnight. White solid, melting point 195e197 ꢂC, yield 92%: 1H NMR
4.1.1.24. N-[3-(2,5-dimethyl-benzyloxy)-4-(methanesulfonyl-methyl-
amino)-phenyl]-2-phenyl-acetamide (24). Phenylacetyl chloridewas
used and it was stirred at room temperature overnight. White solid,
melting point 185e186 ꢂC, yield 99%: 1H NMR (400 MHz, CDCl3)
(400 MHz, CDCl3)
d
8.017 (1H, d, J ¼ 2.4 Hz), 7.868 (1H, s), 7.799 (2H,
m), 7.327 (3H, m), 7.177 (1H, s), 7.116 (2H, m), 6.834 (1H, dd, J ¼ 2,
8.4 Hz), 5.093 (2H, s), 3.200 (3H, s), 2.715 (3H, s), 2.543 (3H, s), 2.348
(3H, s), 2.326 (3H, s). ESI-MS calculated for C25H27N2O4S2 [M þ H]þ:
483.1, found: 483.0.
d
7.867 (1H, d, J ¼ 2.4 Hz), 7.399 (5H, m), 7.239 (1H, d, J ¼ 8.4 Hz), 7.187
(1H, s), 7.102 (3H, m), 6.535 (1H, dd, J ¼ 3, 9 Hz), 5.026 (2H, s), 3.757
(2H, s), 3.156 (3H, s), 2.679 (3H, s), 2.320 (3H, s), 2.312 (3H, s). ESI-MS
calculated for C25H27N2O4S [M þ H]þ: 451.2, found: 450.9.
4.1.1.31. N-[3-(2,5-dimethyl-benzyloxy)-4-(methanesulfonyl-methyl-
amino)-phenyl]-4-dimethylamino-benzamide (31). 4- Dimethylamino-
benzoyl chloride was used and it was stirred at room temperature
overnight. White solid, melting point 171e172 ꢂC, yield 66%: 1H NMR
4.1.1.25. N-[3-(2,5-dimethyl-benzyloxy)-4-(methanesulfonyl-methyl-
amino)-phenyl]-1-naphthalenecarboxyamide
(25). 1-Naphthoyl
chloride was used and it was stirred at room temperature over-
night. White solid, melting point 164e168 ꢂC, yield 83%: 1H NMR
(400 MHz, CDCl3)
d
8.086 (1H, d, J ¼ 2.4 Hz), 7.823 (1H, s), 7.789 (2H, d,
(400 MHz, CDCl3)
d
8.350 (1H, d, J ¼ 8 Hz), 8.105 (1H, s), 7.992 (1H,
J ¼ 9.2 Hz), 7.321 (1H, d, J ¼ 8.4 Hz), 7.174 (1H, s), 7.110 (2H, m), 6.790
(1H, dd, J ¼ 2, 8.4 Hz), 6.720 (2H, d, J ¼ 9.2 Hz), 5.089 (2H, s), 3.194 (3H,
s), 3.064 (6H, s), 2.703 (3H, s), 2.343 (3H, s), 2.324 (3H, s). ESI-MS
calculated for C26H30N3O4S [M þ H]þ: 480.2, found: 479.9.
d, J ¼ 8.4 Hz), 7.921 (1H, m), 7.865 (1H, s), 7.741 (1H, d, J ¼ 6.4 Hz),
7.543 (3H, m), 7.345 (1H, d, J ¼ 8.4 Hz), 7.199 (1H, s), 7.116 (2H, m),
6.845 (1H, dd, J ¼ 2, 8.4 Hz), 5.129 (2H, s), 3.208 (3H, s), 2.711 (3H, s),
2.365 (3H, s), 2.336 (3H, s). ESI-MS calculated for C28H29N2O4S
[M þ H]þ: 489.2, found: 489.1.
4.1.1.32. N-[3-(2,5-dimethyl-benzyloxy)-4-(methanesulfonyl-methyl-
amino)-phenyl]-4-ethoxy-benzamide (32). 4-Ethoxy-benzoyl chlo-
ride was used and it was stirred at room temperature overnight.
White solid, melting point 184e185 ꢂC, yield 70%: 1H NMR
4.1.1.26. N-[3-(2,5-dimethyl-benzyloxy)-4-(methanesulfonyl-methyl-
amino)-phenyl]-3,5-bis(trifluoro-methyl)benzamide
(26). 3,5-Bis

442
B. Zhong et al. / European Journal of Medicinal Chemistry 47 (2012) 432e444
(400 MHz, CDCl3)
d
8.014 (1H, d, J ¼ 2 Hz), 7.925 (1H, s), 7.847 (2H,
4.1.1.39. N-[3-(2,5-dimethyl-benzyloxy)-4-(methanesulfonyl-methyl-
amino)-phenyl]-isoxazole-5-carboxamide (39). Isoxazole-5-carbonyl
chloride was used and it was stirred at room temperature over-
night. White solid, melting point 130e138 ꢂC(decomposed), yield
m), 7.316 (1H, d, J ¼ 8.4 Hz), 7.168 (1H, s), 7.110 (2H, m), 6.972 (2H,
m), 6.822 (1H, dd, J ¼ 2.4, 8.4 Hz), 5.074 (2H, s), 4.111 (2H, q,
J ¼ 7.2 Hz), 3.195 (3H, s), 2.708 (3H, s), 2.340 (3H, s), 2.320 (3H, s),
1.455 (3H, t, J ¼ 7.2 Hz). ESI-MS calculated for C26H29N2O5S
[M þ H]þ: 481.2, found: 480.9.
99%: 1H NMR (400 MHz, CDCl3)
d
8.418 (1H, d, J ¼ 2 Hz), 8.360 (1H, s),
7.849 (1H, dd, J ¼ 2.4 Hz), 7.391 (1H, d, J ¼ 8.4 Hz), 7.181 (1H, s), 7.126
(2H, m), 7.060 (1H, d, J ¼ 2 Hz), 7.005 (1H, dd, J ¼ 2.4, 8.4 Hz), 5.095
(2H, s), 3.211 (3H, s), 2.741 (3H, s), 2.357 (3H, s), 2.329 (3H, s). ESI-MS
calculated for C21H22N3O5S [M þ H]þ: 428.1, found: 427.8.
4.1.1.33. N-[3-(2,5-dimethyl-benzyloxy)-4-(methanesulfonyl-methyl-
amino)-phenyl]-4-ethyl-benzamide (33). 4-Ethyl-benzoyl chloride
was used and it was stirred at room temperature overnight. Pale
yellow solid, melting point 178e181 ꢂC, yield 64%: 1H NMR
4.2. Pharmacological studies
(400 MHz, CDCl3)
d
8.038 (1H, d, J ¼ 2.4 Hz), 7.987 (1H, s), 7.814 (2H,
m), 7.327 (3H, m), 7.173 (1H, s), 7.112 (2H, m), 6.834 (1H, dd, J ¼ 2.4,
8.4 Hz), 5.083 (2H, s), 3.197 (3H, s), 2.733 (2H, q, J ¼ 7.6 Hz), 2.708
(3H, s), 2.343 (3H, s), 2.324 (3H, s), 1.276 (3H, t, J ¼ 7.6 Hz). ESI-MS
calculated for C26H29N2O4S[M þ H]þ: 465.2, found: 464.9.
4.2.1. Cell culture
SKBR-3, HT29 cells were obtained from ATCC (Rockville, MD). All
cells were maintained in RPMI1640 medium supplemented with
10% fetal bovine serum (FBS), 2 mmol/L L-Glutamine, 1 mmol/L
sodium pyruvate, 100 U/mL penicillin-streptomycin. FBS was heat
inactivated for 30 min in a 56 ꢂC water bath before use. Cell cultures
were grown at 37 ꢂC, in a humidified atmosphere of 5% CO2 in
a Hereaus CO2 incubator.
4.1.1.34. N-[3-(2,5-dimethyl-benzyloxy)-4-(methanesulfonyl-methyl-
amino)-phenyl]-4-trifluoromethyl-benzamide (34). 4-Trifluorome-
thyl-benzoyl chloride was used and it was stirred at room
temperature overnight. White solid, melting point 215e218 ꢂC,
yield 84%: 1H NMR (400 MHz, DMSO-d6)
d
10.590 (1H, s), 8.157 (2H,
4.2.2. Cell viability analysis
d, J ¼ 8 Hz), 7.941 (2H, d, J ¼ 8 Hz), 7.753 (1H, d, J ¼ 2.4 Hz), 7.419
(1H, dd, J ¼ 2.4, 8.8 Hz), 7.307 (2H, m), 7.129 (2H, m), 5.107 (2H, s),
3.116 (3H, s), 2.871 (3H, s), 2.319 (3H, s), 2.275 (3H, s). ESI-MS
calculated for C25H24F3N2O4S [M þ H]þ: 505.1, found: 504.9.
The effect of nimesulides derivatives on SKBR-3 and HT29 cell
viability was assessed by using the 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyl-2H-tetrazolium bromide assay in six replicates. Cells
were grown in RPMI1640 medium in 96-well, flat-bottomed plates
for 24 h, and were exposed to various concentrations of JCC76
derivatives dissolved in DMSO (final concentration ꢃ0.1%) in media
for 48 h. Controls received DMSO vehicle at a concentration equal to
that in drug-treated cells. The medium was removed, replaced by
4.1.1.35. N-[3-(2,5-dimethyl-benzyloxy)-4-(methanesulfonyl-methyl-
amino)-phenyl]-4-trifuoromethoxy-benzamide (35). 4-Trifluorome-
thoxy-benzoyl chloride was used and it was stirred at room
temperature overnight. White solid, melting point 194e197 ꢂC,
200 ml of 0.5 mg/mL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
yield 79%: 1H NMR (400 MHz, CDCl3)
d
7.954 (4H, m), 7.350 (3H, m),
2H-tetrazolium bromide in fresh media, and cells were incubated in
the CO2 incubator at 37 ꢂC 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 ml/
well DMSO. Absorbance at 570 nm was determined on a plate
reader.
7.177 (1H, s), 7.117 (2H, m), 7.848 (1H, dd, J ¼ 2.4, 8.4 Hz), 5.088 (2H,
s), 3.204 (3H, s), 2.724 (3H, s), 2.349 (3H, s), 2.324 (3H, s). ESI-MS
calculated for C25H24F3N2O5S [M þ H]þ: 521.1, found: 520.9.
4.1.1.36. N-[3-(2,5-dimethyl-benzyloxy)-4-(methanesulfonyl-methyl-
amino)-phenyl]-4-methyl-benzamide (36). 4-Methyl-benzoyl chlo-
ride was used and it was stirred at room temperature overnight.
White solid, melting point 170e173 ꢂC, yield 69%: 1H NMR
4.2.3. Flow cytometry analysis
For all the assays, cells were treated for the indicated time. To
analyze the cell cycle profile, treated cells were fixed overnight with
70% EtOH at ꢄ20 ꢂC and stained with propidium iodide buffer
(400 MHz, CDCl3)
d
8.032 (1H, d, J ¼ 2 Hz), 7.956 (1H, s), 7.786 (2H,
m), 7.339 (1H, s), 7.309 (2H, d, J ¼ 7.6 Hz), 7.175 (1H, s), 7.113 (2H, m),
6.834 (1H, dd, J ¼ 2, 8.4 Hz), 5.084 (2H, s), 3.198 (3H, s), 2.710 (3H, s),
2.438 (3H, s), 2.344 (3H, s), 2.324 (3H, s). ESI-MS calculated for
C25H27N2O4S [M þ H]þ: 451.2, found: 450.9.
[38 mM sodium citrate (pH 7.5), 69
mM propidium iodide, and
120
mg/mL RNase A]. Samples were mixed gently and incubated at
room temperature in the dark for 15 min. Immediately before
analysis by flow cytometry, 400 mL binding buffer was added to each
4.1.1.37. N-[3-(2,5-dimethyl-benzyloxy)-4-(methanesulfonyl-methyl-
amino)-phenyl]-furan-2-carboxamide (37). 2-Furoyl chloride was
used and it was stirred at room temperature overnight. White solid,
melting point 156e157 ꢂC, yield 66%: 1H NMR (400 MHz, CDCl3)
sample. A total of 1.2 ꢁ 104 cells were acquired for each sample and
a maximum of 1 ꢁ104 cells within the gated region were analyzed.
4.2.4. In vivo xenograft studies
d
8.159 (1H, s), 8.004 (1H, d, J ¼ 2.4 Hz), 7.551 (1H, m), 7.352 (1H, d,
Five- to six-week-old BALB/c nu/nu, athymic, mice were
purchased from Charles River Laboratories, Inc. (Wilmington, MA).
Subconfluent HT29 cells were harvested from monolayer culture
and resuspended in an equal volume of Matrigel (BD Biosciences,
San Jose, CA) to a final concentration of 1 ꢁ107/0.2 mL. At 10 weeks
of age, each animal received s.c. inoculations in two sites per flank
J ¼ 8.4 Hz), 7.268 (1H, m), 7.180 (1H, s), 7.119 (2H, m), 6.875 (1H, dd,
J ¼ 2.4, 8.8 Hz), 6.595 (1H, dd, J ¼ 2, 3.6 Hz), 5.091 (2H, s), 3.202 (3H,
s), 2.720 (3H, s), 2.351 (3H, s), 2.328 (3H, s). ESI-MS calculated for
C22H23N2O5S [M þ H]þ: 427.1, found: 426.8.
4.1.1.38. N-[3-(2,5-dimethyl-benzyloxy)-4-(methanesulfonyl-methyl-
amino)-phenyl]-thiophene-2-carboxamide (38). 2-Thiophene carbonyl
chloride was used and it was stirred at room temperature overnight.
White solid, melting point 163e164 ꢂC, yield 72%: 1H NMR (400 MHz,
with 200 mL of HT29 cell suspension. Six animals were randomly
grouped into two. Tumors were measured twice weekly with
calipers, and tumor volume was calculated by the following
formula: (width)2 ꢁ length/2. Treatments began when the tumors
reached a measurable size (w200 mm3). Treated group was intra-
peritoneal administrated 5 mg/kg/d of CSUOH0901 five times
a week (1%DMSO, sesame oil as vehicle). The control group received
the vehicle treatment. Body weights were monitored weekly as an
indicator of the animals’ overall health. After three weeks of
CDCl3)
d
7.986 (1H, d, J ¼ 2.4 Hz), 7.877 (1H, s), 7.668 (1H, dd, J ¼ 1.2,
3.6 Hz), 7.589 (1H, dd, J ¼ 1.2, 5.2 Hz), 7.323 (1H, d, J ¼ 8.4 Hz), 7.144
(4H, m), 6.816 (1H, dd, J ¼ 2.4, 8.8 Hz), 5.072 (2H, s), 3.197 (3H, s),
2.718 (3H, s), 2.341(3H, s), 2.324 (3H, s). ESI-MS calculated for
C22H23N2O4S2[M þ H]þ: 443.1, found: 442.8.

B. Zhong et al. / European Journal of Medicinal Chemistry 47 (2012) 432e444
443
treatment, the mice were euthanized and the tumors were
removed, weighed.
mean residence time (MRT) from time 0 to time of the last
measurable concentration (MRT(0-t)) were determined.
4.2.5. Preparation of calibration standards and quality controls
(QC) samples
Acknowledgements
The stock standard solutions of CSUOH0901 were prepared by
dissolving in DMSO it at 1 mg/mL and stored at ꢄ20 ꢂC. One set of
CSUOH0901 working solutions at 3,10, 20,100, 200 and 1000 ng/mL,
prepared by serial diluting stock solution with 50% acetonitrile in
water, was used for calibration standards. Another set of JCC76
working solutions at 3, 9, 90, and 900 ng/mL was made in the similar
way and used for QC samples. All of the working solutions were
freshly prepared before use. Calibration standards were prepared by
This research was supported by a startup fund from Cleveland
State University. The research was also supported by the Trans-
lational Research Core Facility and Athymic Animal & Xenograft
Core Facility of the Case Comprehensive Cancer Center (P30
CA43703). We thank the Developmental Therapeutic Program at
the National Cancer Institute for screening the compound against
60 human tumor cell lines and acute toxicity studies.
spiking 5
mL different CSUOH0901 working solutions into 50 mL
blank rat plasma to give the final concentration of CSUOH0901 at 0.3,
1, 2,10, 2 and 100 ng/mL. The QC samples were prepared in sameway
as the calibration standards at four different levels of 0.3, 0.9, 9 and
90 ng/mL, representing lower limit of quantitation (LLOQ), low QC
(LQC), middle QC (MQC) and high QC (HQC) of CSUOH0901 in
plasma. All of the calibration standards and QC samples were further
treated in the same preparation procedure.
Appendix. Supplementary material
Supplementary data related to this article can be found online at
doi:10.1016/j.ejmech.2011.11.012.
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