Synthesis and biological evaluation of sulfonyl acrylonitriles as novel inhibitors to peritoneal carcinomatosis

Article abstract of DOI:10.1016/j.bmcl.2012.01.085

The vast majority of cancer patients die from metastasis, the process by which cancer cells spread to secondary tissues through body fluids. Peritoneal carcinomatosis is a type of metastasis in which cancer cells gain access to the intra-abdominal cavity and then implant in the peritoneum, the thin tissue that lines the abdominal wall and internal organs. Unfortunately, peritoneal carcinomatosis can occur following surgical resection of intra-abdominal malignancies. We previously reported proapoptotic activity of (2E)-3-[[4-(1,1-dimethylethyl)phenyl]sulfonyl]-2-propenenitrile (BAY 11-7085, 1) on colon and pancreatic cancer cells during adhesion and demonstrated that this compound could significantly inhibit peritoneal carcinomatosis in mice. ^1,2 In order to determine the chemical basis of the anti-metastatic properties of BAY 11-7085, a series of analogs were synthesized and evaluated for their ability to induce apoptosis in pancreatic and ovarian cancer cells during adhesion to mesothelial cells, which line the surface of the peritoneum. The co-culture assay results were validated using a murine peritoneal carcinomatosis model. These analogs may greatly benefit patients undergoing surgical resections of colorectal, pancreatic, and ovarian cancers depending on their tolerability.

Full text of DOI:10.1016/j.bmcl.2012.01.085

Bioorganic & Medicinal Chemistry Letters 22 (2012) 1850–1853
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of sulfonyl acrylonitriles as novel
inhibitors to peritoneal carcinomatosis
⇑
,
Craig A. Zificsak a, , Yi Shen b, , Joseph G. Lisko a, Jay P. Theroff a, Xuegang Lao b, Oana Bollt b, Xiufen Li b,
Bruce D. Dorsey a, Scott K. Kuwada b
a Worldwide Discovery Research, Cephalon, Inc., 145 Brandywine Parkway, West Chester, PA 19380, USA
b Department of Medicine, University of Hawaii, Honolulu, HI 96813, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The vast majority of cancer patients die from metastasis, the process by which cancer cells spread to sec-
ondary tissues through body fluids. Peritoneal carcinomatosis is a type of metastasis in which cancer cells
gain access to the intra-abdominal cavity and then implant in the peritoneum, the thin tissue that lines
the abdominal wall and internal organs. Unfortunately, peritoneal carcinomatosis can occur following
surgical resection of intra-abdominal malignancies. We previously reported proapoptotic activity of
(2E)-3-[[4-(1,1-dimethylethyl)phenyl]sulfonyl]-2-propenenitrile (BAY 11-7085, 1) on colon and pancre-
atic cancer cells during adhesion and demonstrated that this compound could significantly inhibit peri-
toneal carcinomatosis in mice.1,2 In order to determine the chemical basis of the anti-metastatic
properties of BAY 11-7085, a series of analogs were synthesized and evaluated for their ability to induce
apoptosis in pancreatic and ovarian cancer cells during adhesion to mesothelial cells, which line the sur-
face of the peritoneum. The co-culture assay results were validated using a murine peritoneal carcinoma-
tosis model. These analogs may greatly benefit patients undergoing surgical resections of colorectal,
pancreatic, and ovarian cancers depending on their tolerability.
Received 29 November 2011
Revised 20 January 2012
Accepted 23 January 2012
Available online 30 January 2012
Keywords:
Peritoneal carcinomatosis
BAY 11-7085
Ó 2012 Elsevier Ltd. All rights reserved.
Peritoneal carcinomatosis, a uniformly fatal form of metastasis,
results from the peritoneal implantation of cancer cells, which are
shed from intra-abdominal cancers or dislodged during surgical
resection. At the time patients with abdominal malignancies un-
dergo resection of their tumors, seeding of the peritoneal cavity
and the circulatory system can occur.3,4 Perhaps the best evidence
for the iatrogenic spread of cancer cells to the peritoneum is from
studies demonstrating seeding of tracts created in the abdominal
wall to allow laparoscopic resection of intra-abdominal cancers.5
lining the peritoneum express the cognate ligands of these death
receptors, which activate death receptor-mediated signaling.2 Inhi-
bition of c-FLIP allows activated death receptors to recruit caspase
8 and 10, which in turn initiate the cleavage of downstream ‘exe-
cutioner’ caspases that effect apoptosis. Although restoration of
death receptor-mediated apoptosis has been demonstrated in can-
cer cell cultures, we were the first to demonstrate this occurs in a
juxtacrine fashion when cancer cells come into contact with nor-
mal human mesothelial cells.2
The small molecule BAY 11-7085 (1, Fig. 1), a known NF-
j
B
As a first study, we designed analogs of 1 wherein a methyl of the
t-butyl group is replaced by a carboxylic acid, or alkyl, aryl or benzyl
amide. An in vitro model of peritoneal tumor implantation was uti-
lized in which pancreatic or ovarian cancer cells in suspension were
allowed to adhere to confluent monolayers of mesothelial cells. The
cancer cells were transduced with a constitutively expressing firefly
inhibitor, was shown to induce apoptosis of colon and pancreatic
cancer cells during cell adhesion and reduce colon and pancreatic
cancer cell implantation in a murine peritoneal seeding model.1,2
The molecular mechanism of BAY 11-7085 relates to its ability to
inhibit the expression of c-FLIPL (FLICE Inhibitory Protein) which
restores death receptor signaling in cancer cells.2 c-FLIPL is com-
monly overexpressed in cancer cells of many types and inhibits
the recruitment of caspase 8 to activated death receptors of the
O O
S
O O
S
TNFa
superfamily.6–8 Many types of cancer cells paradoxically ex-
CN
CN
press death receptors which mediate pleiotropic effects, such as
R
proliferation, despite what their name implies.9,10 Mesothelial cells
1
2
⇑
R = CONR1R2
Corresponding author. Tel.: +1 610 738 6745; fax: +1 610 738 6643.
E-mail address: czificsa@cephalon.com (C.A. Zificsak).
BAY 11-7085
These co-authors contributed equally to this paper.
Figure 1. BAY 11-7085 (1) and novel inhibitors 2.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2012.01.085

C. A. Zificsak et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1850–1853
1851
O
O
S
Br
O
O
S
Br
Br
O
a
SO2Na
CN
O
R
R
HO
HO
a
6
7a, R = H
4
3
7b, R = Me
7c, R = Ph
b
7d, R = CONH2
7e, R = CO2Et
O
O
S
O
O
S
S
CN
CN
CN
O
R1
b
N
R2
1
8
5a-j
Scheme 2. (a) DMF, 80 °C, 3–15% yield; (b) 4-t-BuPhSH, Et3N, CHCl3, 97%.
Scheme 1. (a) (i) PhLi; (ii) n-BuLi; (iii) SO2; (iv) 2-chloroacrylonitrile; AcOH; (v)
Et3N; (b) amine, EDCI, HOBT, MeCN or DCM.
Table 1
Biological data for compounds 1, 4, 5a–j
O O
S
CN
R
Compd
R
Efficacya of compounds by co-culture assay
Su86
BxPC3
A2780
SKOV3
1
4
Me
CO2H
0.13 0.01
0.89 0.03
0.07 0.03
0.92 0.03
0.04 0.00
1.08 0.04
0.32 0.02
1.07 0.03
O
5a
5b
0.43 0.02
0.52 0.02
0.22 0.03
0.22 0.02
0.20 0.01
0.17 0.00
0.60 0.04
0.55 0.04
N
H
O
O
N
H
5c
5d
5e
0.95 0.03
0.94 0.09
0.51 0.00
0.98 0.05
0.85 0.11
0.25 0.00
0.95 0.01
0.66 0.04
0.08 0.01
1.09 0.03
1.01 0.07
0.51 0.06
N
N
O
N
O
O
N
O
N
H
5f
0.43 0.05
0.27 0.02
0.15 0.04
0.63 0.02
Cl
O
O
5g
5h
5i
0.29 0.05
0.65 0.05
0.65 0.04
0.24 0.02
0.62 0.05
0.64 0.14
0.05 0.00
0.55 0.01
0.63 0.01
0.47 0.08
0.98 0.04
0.93 0.06
N
H
O
N
MeO
H
N
H
O
N
5j
0.29 0.05
0.24 0.02
0.05 0.00
0.47 0.08
N
H
a
The final concentration of all compounds was defined at 10
lM. Luminescence of cancer cells = living cancer cell number. Efficacy = luminescence of cancer cells treated
with analog/luminescence of cells treated with DMSO; the data are presented as mean SEM, n = 3.

1852
C. A. Zificsak et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1850–1853
Table 2
Biological data for compounds 7a–e, 8
Compd
R
Efficacya of tested compounds by co-culture assay
Su86
BxPC3
A2780
SKOV3
1
CN
H
Me
Ph
CONH2
CO2Et
CN (sulfide)
0.13 0.01
0.80 0.03
0.99 0.04
0.97 0.02
0.59 0.03
0.52 0.01
1.03 0.04
0.07 0.03
0.84 0.11
0.99 0.05
1.01 0.10
0.44 0.08
0.20 0.01
1.08 0.05
0.04 0.00
1.16 0.02
1.15 0.01
1.13 0.02
0.77 0.01
0.39 0.04
1.06 0.08
0.32 0.02
1.05 0.06
1.08 0.02
1.17 0.05
0.50 0.03
0.67 0.02
0.92 0.04
7a
7b
7c
7d
7e
8
a
The final concentration of all compounds was defined at 10
lM. Luminescence of cancer cells = living cancer cell number. Efficacy = luminescence of cancer cells treated
with analog/luminescence of cells treated with DMSO; the data are presented as mean SEM, n = 3.
luciferase plasmid, which allowed the cancer cells to be detected by
bioluminescence in the presence of substrate ( -luciferin). The ana-
in vivo model. As previously published,1 athymic 5-week-old fe-
male nu/nu mice were randomized to IP (intraperitoneal) injection
with 5 mg/kg of drug or an equal volume of vehicle (4:1 PEG400:E-
tOH) 4 h before IP injection of 1.5 million suspended A2780-luc
ovarian cancer cells. The mice continued to receive vehicle or drug
every three days for 14 days total. The peritoneal tumor implants
were examined weekly using a bioluminescence detection system
and monitored out to 14 days. The results are shown in Figure 2.
Significant inhibition of intraperitoneal implantation was ob-
served in the groups treated with BAY 11-7085 and analog 5g com-
pared with the control mice which received vehicle only. Analog 5b
inhibited peritoneal carcinomatosis compared with the controls,
D
logs were added to the cancer cell suspensions and then the mixture
overlaid on the mesothelial cell monolayer. The cancer cells that
were able to adhere to the mesothelial cells and survive were de-
tected by bioluminescence which correlates with the number of
surviving and adherent cancer cells present.
The synthesis of the amide analogs began with bromophenyl
isobutyric acid 3,11 which was treated sequentially with phenyl-
and n-butyl lithium and the resultant lithiated salt was quenched
with sulfur dioxide (Scheme 1). The sulfinic acid was then coupled
with 2-chloroacrylonitrile and after workup, triethylamine-in-
duced elimination12 of HCl generated the E-sulfonyl acrylonitrile
acid 4. The target amides 5a–j were then prepared following stan-
dard peptide amide coupling with EDCI and HOBT in low to accept-
able yields (17%–82%).13
Table 3
Dose response data for active compounds
Several amide analogs of 1 were prepared to assess functional
group tolerability. The free acid 4 was inactive in the cellular
screening assays, but once coupled to an amine, the resultant
amides showed comparable activity to 1 (Table 1). Secondary
Compd
IC50 (l
M)a by co-culture assay
Su86
BxPC3
A2780
SKOV3
1
5.9 0.2
7.5 0.4
8.7 0.1
7.6 0.1
7.3 0.1
3.7 0.1
4.1 0.0
4.1 0.1
4.2 0.2
4.1 0.1
2.6 0.1
2.7 0.1
3.4 0.0
3.3 0.1
2.4 0.1
7.6 0.5
9.9 0.2
11.4 0.2
12.9 0.1
10.9 0.2
5b
5f
5g
5h
amides 5a–b showed good activity when screened at 10 lM across
the four cell lines. Tertiary amides 5c and 5d showed a dramatic
decrease in activity, but when piperidine amide 5e was assessed,
activity returned, highlighting the preference for non-polar func-
tionality. Additional secondary amides 5f–i showed moderate to
good potency in the four cell lines, while pyridine-containing ana-
log 5j maintained potency.
a
IC50 values are reported as the average of three separate determinations.
To further investigate the possible mechanism of action, a series
of analogs with alkene modifications were prepared (Scheme
2)14,15 and screened (Table 2). All of the replacements of the nitrile
fragment present in 1 showed significant reduction in antitumor
activity by co-culture assay in all four cell lines assessed. An addi-
tional modification, vinyl sulfide 8,16 the reduced analog of 1, was
screened as well and found to be weakly active. The lack of activity
highlights the need for an electron-deficient Michael acceptor to
effect apoptosis of cancer cells during adhesion to mesothelial cells
in the co-culture assay. Sulfonyl acrylonitriles are known to readily
undergo Michael addition/elimination reactions in the litera-
ture,17,16 and it is postulated that this is the mechanism by which
the active analogs are affecting the cancer cells.
Concentration–response studies of the active analogs 5b, 5f, 5g
and 5h were performed in the co-culture assay. All of these analogs
showed inhibition in the micromolar range, similar to the activity
of 1 (Table 3). Additionally, treatment of SKOV3.luc cells with 1, 5b
Figure 2. BAY 11-7085 and active analogs inhibit peritoneal carcinomatosis. The
mice were pre-treated intraperitoneally (IP) with drug or vehicle. Four hours later,
1.5 Â 106 A2780-luciferase ovarian cancer cells were injected IP into the mice. The
mice subsequently underwent treatment (5 mg/kg, IP) every three days for 14 days.
The intraperitoneal tumor implants were detected and quantified using a biolumi-
or 5f–h at a concentration of 7.5 lM during their adhesion to mes-
othelial cell monolayers resulted in the formation of detached
apoptotic cells for all five compounds, relative to vehicle.2,18
In order to validate the in vitro co-culture assay results, an
in vivo murine peritoneal carcinomatosis model was employed.
Analogs were chosen based on their activity in the co-culture as-
says in order to determine if they performed similarly in an
nescence imager following IP D-luciferin administration. The graph shows abdom-
inal bioluminescence output for the mice at day 14. The horizontal bars represent
the medians.ÃÃ, p <0.01 compared with control mice that were treated with vehicle
alone.

C. A. Zificsak et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1850–1853
1853
but the effect was not statistically significant, which demonstrates
References and notes
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importance of the sulfonyl group on the anti-cancer activity of
these analogs. Some degree of weight loss was initially observed
in the mice treated with the active analogs but this corrected over
the 14-day treatment period.
In summary, seventeen analogs of 1 with t-butyl or alkene mod-
ifications were synthesized and their antitumor activities evalu-
ated in vitro with in vivo analysis of three select analogs. Analog
5g showed similar activity to BAY 11-7085 in co-culture assay
and greater inhibition of peritoneal carcinomatosis in mice sug-
gesting that specific modifications of the t-butyl group could im-
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Through the synthesis of analogs of 1, we have defined a group
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the mesothelial cells. If the tolerability and intraperitoneal expo-
sure of these compounds is acceptable, they may be useful when
administered during the resection of intraabdominal cancers, a
time when tumors are disrupted and cancer cells shed into the per-
itoneal cavity.
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2012.01.085.