Design, synthesis, and biological evaluation of sulfonyl acrylonitriles as novel inhibitors of cancer metastasis and spread

Article abstract of DOI:10.1021/jm501437v

The spread of intra-abdominal cancers is a vexing clinical problem for which there is no widely effective treatment. We discovered previously that (2E)-3-[(4-tert-butylphenyl)sulfonyl]acrylonitrile (1) induced cancer cell apoptosis during adhesion to normal mesothelial cells which line the peritoneum. We recently demonstrated that the sulfonylacrylonitrile portion of 1 and hydrophobic aryl substitution were essential for pro-apoptotic activity in cancer cells. Here we synthesized a diverse series of analogues of 1 in order to improve the efficacy and pharmaceutical properties. Analogues and 1 were compared in their ability to cause cancer cell death during adhesion to normal mesothelial cell monolayers. Potent analogues identified in the in vitro assay were validated and found to exhibit improved inhibition of intra-abdominal cancer in two clinically relevant murine models of ovarian and pancreatic cancer spread and metastasis, highlighting their potential clinical use as an adjunct to surgical resection of cancers.

Full text of DOI:10.1021/jm501437v

Subscriber access provided by SELCUK UNIV
Article
Design, synthesis and biological evaluation of sulfonyl
acrylonitriles as novel inhibitors of cancer metastasis and spread.
YI SHEN, Craig A. Zificsak, Jill E Shea, Xuegang Lao, Oana Bollt, Xiufen Li, Joseph G Lisko, Jay P.
Theroff, Courtney L Scaife, Mark A. Ator, Bruce A. Ruggeri, Bruce D. Dorsey, and Scott Ken Kuwada
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm501437v • Publication Date (Web): 12 Jan 2015
Downloaded from http://pubs.acs.org on January 21, 2015
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 87
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Design, synthesis and biological evaluation of sulfonyl acrylonitriles as novel
inhibitors of cancer metastasis and spread.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
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
a
b
c
a
a
a
Yi Shen, * Craig A. Zificsak, * Jill E. Shea, Xuegang Lao, Oana Bollt, Xiufen Li,
b
b
c
b
b
Joseph G. Lisko, Jay P. Theroff, Courtney L. Scaife, Mark A. Ator, Bruce A. Ruggeri,
b
a
Bruce D. Dorsey, Scott K. Kuwada
a
John A. Burns School of Medicine, University of Hawaii, Honolulu, HI 96813, USA
bTeva Branded Pharmaceutical Products R&D, Inc., 145 Brandywine Parkway, West
Chester, PA 19380, USA
cDepartment of Surgery, University of Utah School of Medicine, Salt Lake City, UT
8
4132, USA
∗
These co-authors contributed equally to this paper.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 87
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Abstract
The spread of intra-abdominal cancers is a vexing clinical problem for which there is
no widely effective treatment. We discovered previously that (2E)-3-[(4-tert-
Butylphenyl)sulfonyl]acrylonitrile (1) induced cancer cell apoptosis during
adhesion to normal mesothelial cells, which line the peritoneum. We recently
demonstrated that the sulfonylacrylonitrile portion of 1 and hydrophobic aryl
substitution were essential for pro-apoptotic activity in cancer cells. Here we
synthesized a diverse series of analogs of 1 in order to improve the efficacy and
pharmaceutical properties. Analogs and 1 were compared in their ability to cause
cancer cell death during adhesion to normal mesothelial cell monolayers. Potent
analogs identified in the in vitro assay were validated and found to exhibit improved
inhibition of intra-abdominal cancer spread in two clinically relevant murine
models of ovarian and pancreatic cancer spread and metastasis, highlighting their
potential clinical use as an adjunct to surgical resection of cancers.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Introduction
Peritoneal carcinomatosis is caused by cancer cell implantation of the peritoneal
lining of the abdominal cavity. The peritoneum is a thin tissue that lines the
abdominal organs and abdominal wall. The mesothelial cells form a monolayer of
cells on the surface of the peritoneum and produce peritoneal fluid that serves to
lubricate the peritoneal surfaces, significantly reducing friction between the
abdominal organs and cavity. Malignant gastrointestinal and gynecological tumors,
ACS Paragon Plus Environment

Page 3 of 87
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
such as advanced pancreatic cancer and ovarian cancer, commonly spread to the
peritoneum.1-6 Approximately 45,220 new cases of and 38,460 deaths from
pancreatic cancer occurred in 2013 in the United States, and 22,240 new cases of
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
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
7
and 14,030 deaths from ovarian cancer were estimated. As treatment options,
surgery, radiation therapy and chemotherapy may extend survival and/or relieve
symptoms in many patients, but seldom produce a cure for advanced cases. 8, 9
During the initial steps of peritoneal carcinomatosis, tumor cells are first shed from
intra-abdominal cancers or dislodged during surgical resection, and gain access to
the peritoneal cavity.10-15 The successful implantation of the mesothelium is the key
initial step in peritoneal carcinomatosis,16, 17 and treatments that inhibit this step
may significantly decrease recurrence and peritoneal carcinomatosis.
Our previous work showed that the small molecule 1 could inhibit peritoneal
carcinomatosis in a murine model.18 1 was found to inhibit c-FLIP (FLICE Inhibitory
Protein), a key inhibitor of death receptor-mediated apoptosis, which is commonly
overexpressed in cancer cells.19 Many cancer cells express death receptors of the
TNFα superfamily yet evade death receptor-mediated apoptosis through the
overexpression of survival proteins such as c-FLIP.19 The over expression of death
receptors by cancer cells appears to be conserved as they have been shown to
mediate cancer cell proliferation.20 Many normal tissues contain cells, such as
mesothelial cells, that express cognate death receptor ligands.21 We showed that
when cancer cells adhere to mesothelial cells, the death receptor ligands expressed
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 87
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
by mesothelial cells can activate death receptors expressed on cancer cells in a
juxtacrine fashion which results in apoptosis if 1 is present.19 Thus, 1-mediated
inhibition of c-FLIP expression restores death receptor-mediated cancer cell
apoptosis during their adhesion to normal human mesothelial cells.19
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
In a recent study, several analogs of 1 were synthesized to determine the role of the
sulfonylacrylonitrile moiety and assess the potential of an amide replacement for
the tert-butyl fragment of 1.22 While studies of these analogs pointed out the
importance of the functional groups to the pro-apoptotic efficacy of 1, few of the
analogs demonstrated increased efficacy in pancreatic, ovarian, or colon cancer cells
compared with the parent compound. In addition, although 1 was effective in
inhibiting peritoneal carcinomatosis in preclinical models, its relative insolubility in
water required the use of DMSO as a delivery vehicle, which impairs its potential
clinical use. The aims of the present study were to improve the efficacy of the
parent compound through structural modification strategies outlined (Figure 1)
and test lead analogs for efficacy in clinically relevant in vivo models of intra-
abdominal cancers. The incorporation of additional polarity was a priority in the
design of new analogs in the hope of increasing potency and drug like character.
Results and Discussion
2
2
Design. In our preliminary report, analogs of 1 wherein a methyl of the t-butyl
group is replaced by an amide were designed and evaluated for their ability to
induce apoptosis in pancreatic and ovarian cancer cells during adhesion to
ACS Paragon Plus Environment

Page 5 of 87
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
mesothelial cell monolayers. Secondary and tertiary amide analogs displayed
comparable activity to 1 in co-culture assay across four pancreatic and ovarian
cancer cell lines. Any modification to the sulfonyl acrylonitrile fragment resulted in
reduced potency in the co-culture assays, highlighting the need of a strong Michael
acceptor for activity. To establish a more complete picture of the structure-activity
relationship (SAR) and identify compounds with improved in vivo potency versus
compound 1, a systematic approach to modifying compound 1 was undertaken
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
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
(Figure 1). Modification of the t-butyl or phenyl ring of 1 resulted in the synthesis
of five different classes of compounds. Based on promising in vivo activity
demonstrated in a luciferase tagged A2780 ovarian peritoneal carcinomatosis
2
2
mouse model, additional aromatic, aliphatic and benzylic amides were prepared to
expand the Series 1 amide analogs. Bioisosteric replacement of the amide moiety in
Series 1 provided the heterocyclic analogs of Series 2. Keeping the amide fragment
in place and tethering the remaining geminal methyl groups of 1 into a series of
cyclic products furnished Series 3. Appending the amide fragment to the
phenylsulfonyl group through an ether linkage, while migrating the t-butyl
substituent to the meta-position afforded the phenoxy amide Series 4. Lastly, the
phenyl ring was replaced with either a pyridine or quinoline ring to afford the Series
5 heterocyclic analogs.
Chemistry. 4-Bromophenyl-2-propanoic acid (2) served as the starting point for all
of the amides of Series 1 (Scheme 1). Deprotonation and metalation, followed by a
sulfur dioxide quench furnished the intermediate sulfinic acid, which was converted
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 87
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
to the arylsulfonyl acrylonitrile in modest yield. Routine coupling of aliphatic and
aromatic amines with 3 furnished amides 4a-t as shown in Table 1. The Series 2
heterocyclic analogs also utilized 3 as a starting material (Scheme 2); oxadiazole
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
3
analogs were prepared via a direct coupling with substituted thiosemicarbazides
(5a-b) or via a two step coupling/dehydration with acyl hydrazines (5c-d). Amide
analogs 4u-w were cyclodehydrated under acidic conditions to afford the
benzoxazole analogs 6a-b and benzimidazole 6c. Propargyl amide 4x was similarly
24
cyclized, under gold catalysis, to afford the methyl oxazole 7.
Scheme 1. Synthesis of amide analogsa
O
O
O
O
Br
S
S
O
a
O
CN
b
O
CN
1
R
HO
HO
N
R
2
2
3
4a-w
a
Reagents and conditions: (a) i: PhLi, n-BuLi, −80 °C; ii: SO
2
, −80 °C to rt; iii: 2-chloroacrylonitrile,
AcOH, MeOH, rt; iv: Et
3
N, 43% (b) amine, EDCI, HOBT, rt, 22-67%.
Scheme 2. Synthesis of heterocyclic amide isosteresa
O
O
O
O
S
S
a
N
O
O
CN
N
CN
R
HO
3
5a-d
R
R
O
O
O
O
O
O
S
S
b
c
S
S
O
O
CN
CN
N
N
CN
CN
N
H
A
AH
4u-w
6a-c
O
O
O
N
H
4x
7
ACS Paragon Plus Environment

Page 7 of 87
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
a
Reagents and conditions: (a) RCSNHNH
HOBT, MeCN, rt, 4h; ii: dioxane, POCl , 90 °C, 2h, 72-76%; (b) MsOH, 1,4-dioxane, 90 °C, 24h, 55% or
AcOH, 70-105 °C, 1-3 d, 11-24%; (c) AuCl , MeCN, 4 d, 30%.
2
, 3 equiv EDCI, DCM, rt, 11-19%; or i: RCONHNH
2
, EDCI,
3
3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
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
The synthesis of the cyclic amide analogs 10a-h (Scheme 3) followed the same
sequence as the Series 1 amides, simply starting with the appropriately tethered
cyclic carboxylic acid 8a-c (X = direct bond, CH
2
or –CH
2
OCH –). The phenoxy amide
2
25
analogs of Series 4 utilized bromide 11 as a precursor to the aryl sulfonyl
acrylonitrile derivatives 13a-e (Scheme 4).
Scheme 3. Synthesis of cyclic amide analogsa
O
O
O
O
Br
S
S
O
a
O
CN
b
O
CN
R
HO
HO
N
H
8
a-c
9a-c
10a-h
X
X
X
a
Reagents and conditions: (a) i: PhLi, n-BuLi, −80 °C; ii: SO
2
, −80 °C to rt; iii: 2-chloroacrylonitrile,
AcOH, MeOH, rt; iv: Et N, 10-27% (b) amine, EDCI, HOBT, MeCN or DCM, rt, 28-69%.
3
Scheme 4. Synthesis of phenoxy amide analogsa
O
O
t-Bu
O
Br
t-Bu
O
S
CN
a
b
HO C
HO C
2
2
11
12
O
S
O
O O
S
t-Bu
O
t-Bu
c
R
CN
CN
HN
N
O
O
13a-d
O
14
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 87
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a
Reagents and conditions: (a) i: PhLi, n-BuLi, −80 °C; ii: SO
2
, −80 °C to rt; iii: 2-chloroacrylonitrile,
AcOH, MeOH, rt; iv: Et
3
N, 32% (b) amine, EDCI, HOBT, MeCN or DCM, rt, 38-79%; (c) MsOH, 1,4-
dioxane, 90 °C, 11 h, 50%.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Lastly, the phenyl ring of 1 was replaced with either a quinoline to afford 17 or a
pyridine, with a pendant cyclohexyl amide, to afford 20. Quinoline 16 was prepared
by a Friedländer condensation of bromide 15 with pinacolone; subsequent
formation of the sulfonyl acrylonitrile as earlier shown provided 17. The reversal of
the order of the amidation and sulfone formation steps was necessary to prepare
pyridine 20 to avoid decomposition observed in reactions of carboxylic acid 18
with n-butyllithium. The reduced sulfide 21, readily prepared from 1, was used as a
negative control, highlighting the need for the sulfone oxidation state in active
analogs.
Scheme 5. Synthesis of heteroaryl analogs and a negative controla
H
O
O
Br
Br
S
O
a
b
CN
H N
N
O
N
2
15
16
17
O
O
Br
Br
S
O
CN
c
d
HO C
2
N
N
N
N
N
H
H
18
19
20
O
O
S
S
CN
e
CN
1
21
a
Reagents and conditions: (a) t-BuCOCH
3
, KOH, water, 100 °C, 3 h, 64%; (b) i: PhLi, n-BuLi, −80 °C; ii:
N, 59%; c) cyclohexylamine, Et3N,
SO , −80 °C to rt; iii: 2-chloroacrylonitrile, AcOH, MeOH, rt; iv: Et
2
3
ACS Paragon Plus Environment

Page 9 of 87
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
propylphosphonic anhydride, DMF, rt, 51%; d) i: PhLi, n-BuLi, −80 °C; ii: SO
2
, −80 °C to rt; iii: 2-
, 97%.
chloroacrylonitrile, AcOH, MeOH, rt; iv: Et
3
N, 15%; e) 4-t-BuPhSH, Et
3
N, CHCl
3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
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
Biological Evaluation of Inhibitors
Screening was conducted against two pancreatic cancer cell lines (SU.86 and BxPC-
) and two ovarian cancer cell lines (A2780 and SKOV3) that were transduced with
3
a constitutively expressing firefly luciferase plasmid which allowed for specific
bioluminescent detection of living cancer cells. The cancer cells were suspended and
then allowed to adhere to human mesothelial cell monolayers in the presence of
vehicle or drug. Each series of analogs was tested at a single concentration (10 µM)
and efficacy was calculated as the fraction of adherent cells which remained alive as
compared to vehicle (DMSO). Surviving cancer cells were quantified by firefly
luciferase activity in a luminometer. In Series 1 (Table 1), benzyl amide 4b
showed better potency than the phenyl- (4a) or phenethyl (4c) amides, and 4b had
potency close to that of 1. Substitution of the benzyl amide was further explored;
neither stereoisomer of α-methyl benzylamide (4d or 4e) showed a marked
improvement in potency. Aromatic substitutions were investigated and it was found
that strongly polar fragments were not tolerated (sulfone 4f or sulfonamide 4g). A
methoxy group at either the ortho- (4h) or meta- (4i) position provided highly
potent analogs. In an attempt to aid aqueous solubility, the para- methoxy-ethoxy
substituent was incorporated (4j) but potency against all four cell lines was
diminished. Fluorine substitution was tolerated at all three positions (4k-m) and
resulted in improved efficacy in the ovarian cell lines as compared to 4b. A weakly
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 87
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
basic heterocyclic substituent showed diminished activity (4n) as did the
difluoroethyl amide (4o). The aminocaprolactam containing analog 4p was largely
inactive, again pointing at the need for non-polar substitution of the amide. The
phenylpropargyl amide 4q exhibited nearly identical activity as the amide without
the acetylenic spacer. A pair of N-methyl benzyl amides, 4r and 4s, as well as the
piperidine amide 4t, showed that disubstituted amides were equally efficacious in
the in vitro assay.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The heterocyclic amide bioisosteres prepared in Scheme 2 followed similar polarity
based trends in activity (Table 2). Phenylamino-oxadiazole 5a was more potent
than the ethyl derivative 5b, but both were less potent than the aryl/alkyl
substituted oxadiazoles 5c-d. Benzoxazoles 6a and 6b were among the most potent
analogs from all series whereas the benzimidazole 6c lacked potency in all cell lines.
The smaller methyloxazole 7 was also active, but not as potent as the benzoxazole
analogs.
The Series 3 analogs, wherein the gem-dimethyl group of the amide analogs is tied
back into a cyclic derivative, were assayed across the four cell lines (Table 3). The
cyclopropane (X = direct bond, 10a-b) and cyclobutane (X = CH , 10c-e) analogs
2
were roughly equipotent with the corresponding amide analogs from Series 1. A
significant drop in activity was observed for the tetrahydropyran analogs 10f-h (X =
CH
2
OCH ).
2
ACS Paragon Plus Environment

Page 11 of 87
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
For the Series 4 analogs, a more dramatic change in structure was implemented
(Table 4). Shifting of the tert-butyl group from para to meta on the aryl sulfone was
paired with a glycolic ether linkage. The glycolic acid derivative 12 was inactive as
expected. The amide derivatives 13a-d were potent in all four cell lines, particularly
the BxPC-3 and A2780 cells. Taking a cue from the highly potent benzoxazole
analogs of Series 2, the benzoxazole derivative 14 was prepared and found to be
slightly less potent than the corresponding phenolic amide 13d. For a direct
comparison to 1, meta derivative 13e was synthesized and found to be equipotent.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
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
Lastly, the heterocyclic derivatives 17 and 20 were assessed in the co-culture assay
(Table 5). Quinoline replacement for phenyl (17 vs 1) showed equivalent potency
in the ovarian cell lines, but a drop in activity in the pancreatic cell lines was
observed. A pyridyl-for-phenyl replacement showed mid-range activity in the four
cell lines and was comparable with the cyclohexyl amide analog previously
disclosed.22
For active analogs, 4b, 5d, 6a, 6b, 10e, 13a and 13d, in addition to 1, a series of
concentrations were used to determine half-maximal effective concentration (EC50).
All of these analogs decreased cancer cell viability in the co-culture assay in the
micromolar range (Table 6).
As we previously showed, compound 1 rapidly and strongly induces apoptosis of
cancer cells during adhesion.19 The most active analogs in the co-culture assay were
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 87
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
tested for their ability to induce apoptosis of A2780 ovarian cancer cells by
demonstrating increased cellular levels of cleaved PARP and cleaved caspase 3,
which are indicators of apoptosis. While all of the analogs induced apoptosis to a
degree, analogs 6b and 13a showed similar levels of apoptotic induction as
compound 1 (Figure 2). The reduced sulfide 21 served as a negative control and
showed no apoptotic effect.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Due to our previous studies showing that both the sulfone and nitrile groups are
essential for the apoptotic activity of 1 on cancer cells, the ability of 1 and its
analogs to bind glutathione (GSH) was examined. Active analogs 4k, 4r, along with
1, strongly reduced detectable GSH levels, while two inactive analogs,, 22 and 23
2
2
(Figure 3F), that were previously reported to be inactive compared to 1 , did not
Figure 3A). To determine if the compounds could diminish GSH in cancer cells in
vivo, 1, 4k, and 4r and inactive analogs (22, 23), were tested in HT29 cells. Only 1,
k, and 4r could decrease cellular GSH levels in vivo while 22 and 23 could not
Figure 3B). The assay used to measure cellular glutathione levels in vivo could not
(
4
(
distinguish if 1 and its active analogs diminished cellular glutathione levels by
inhibiting its synthesis or through covalent modification. Therefore, transiently
suspended HT-29 cancer cells were exposed to 1, in the absence or presence of N-
acetylcysteine (NAC), during co-culture with mesothelial cell monolayers. NAC was
able to completely inhibit the apoptotic activity of 1 on adhering HT-29 cells (Figure
3C). Furthermore, mixing of NAC and 1 before exposure to cancer cells completely
inactivated the apoptotic activity of 1 on cancer cells (not shown) indicating that 1
ACS Paragon Plus Environment

Page 13 of 87
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
is more than likely binding covalently to glutathione and perhaps other thiol-
containing molecules. When amide analog 4b was incubated with glutathione, the
formation of the sulfinic acid elimination product as well as glutathione-conjugated
acrylonitrile was observed. Unfortunately, attempts to identify acrylonitrile-
conjugated protein from cell studies failed to identify a similar conjugate. These and
our previous studies show the importance of the Michael acceptor functional
acrylonitrile group to the anti-cancer activity of 1 and its active analogs.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
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
To determine if glutathione depletion may be involved in the apoptotic mechanism
of 1 and its active analogs, the apoptotic activity of buthionine-sulfoximine (BSO), an
irreversible inhibitor of γ-glutamylcysteine synthetase, which is the rate-limiting
enzyme for glutathione (GSH) synthesis, was tested. BSO did decrease GSH levels in
HT29 cells within 4 hours (Figure 3D), but did not affect cancer cell viability when
cultured on mesothelial cell monolayers (Figure 3E). These results suggested that
diminution of intracellular GSH levels per se was unlikely to be involved in the
apoptotic activity of 1 and its active analogs during cell adhesion.
In order to validate the in vitro co-culture assay results, an in vivo murine peritoneal
carcinomatosis model was performed using A2780 and SKOV3 ovarian cancer cells.
Athymic 5-week-old female nu/nu mice were randomized to intraperitoneal
injection with 5 mg/kg of test compound or an equal volume of vehicle (4:1 PEG400
:
EtOH) four hours before IP injection of 1.5 million A2780-luc or 2 million SKOV3-
luc ovarian cancer cells (n = 7 for each arm). The strategy to dose prior to injection
of cancer cells was designed to mimic the shedding of tumor cells during
intraperitoneal surgery with concomitant use of a chemotherapeutic lavage. Dosing
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 87
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
was continued every three days for 14 days total. The peritoneal tumor implants
were examined weekly using a bioluminescence detection system that detects
luciferase activity in the tumors (Figure 4).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
For A2780 (Figure 4A) and SKOV3 (Figure 4B) ovarian cancer cells, significant
inhibition of intraperitoneal implantation was observed in the groups treated with 1
and analogs 4b, 6b, 13a and 10e compared with the control mice (p<0.05) which
received vehicle only. Benzoxazole 6b showed significantly greater inhibition of
peritoneal carcinomatosis than compound 1 for A2780 but not SKOV-3 ovarian
cancer cells. None of the other analogs showed significantly greater inhibition of
peritoneal carcinomatosis than compound 1. Some degree of weight loss was
initially observed in all mice but this corrected over the 14-day treatment period.
A selection of the active analogs was assayed in a second in vivo model of pancreatic
cancer metastasis. MIA PaCa-2 cells that stably expressed red fluorescent protein
(
RFP) were surgically implanted in the pancreatic tail. Surgical resection of the
tumor 6-8 weeks post implantation was combined with IP treatment with analogs
b, 5d, 6a, 10e, 1 or vehicle (4:1 PEG400:EtOH). Animals were treated at the time of
4
surgery then on days 3, 5 and 7. After 8 weeks, local and metastatic recurrence was
assessed via fluorescent imaging of the mice. Control mice treated with vehicle alone
exhibited 67% recurrence (local in the remaining pancreas or metastatic to the
peritoneum and mesenteric lymph nodes). Mice treated with 1 displayed a
reduction in total recurrence to 38% of the study animals (Table 7). Benzyl amide
ACS Paragon Plus Environment

Page 15 of 87
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
4
b showed a strong reduction in recurrence, with only one of eighteen subjects
exhibiting both local and metastatic recurrence. The remaining three analogs,
phenyl oxadiazole 5d, benzoxazole 6a and the cyclohexyl-cyclobutane amide 10e,
displayed in vivo activity comparable or slightly less effective than 1, with 40-50%
recurrence either locally or metastatic.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
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
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 87
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
CONCLUSIONS:
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
In these studies, we have shown that a variety of structural modifications to 1 are
permitted while maintaining in vitro co-culture efficacy. In vivo efficacy in two
models of cancer spread can be improved with incorporation of a benzyl amide or
benzoxazole in place of a methyl group present in 1. Activity in the co-culture
assays is correlated with the overall polarity of the test compounds and compounds
sufficiently more polar than 1 begin to lose activity. The requirement for more
hydrophobic group substitutions for efficacy is likely related to cell penetration.
Overall potency of active compounds in the co-culture assay as measured by EC50
ranged between 2 and 8 µM and doses of 5 mg/kg were found efficacious in two in
vivo models of peritoneal carcinomatosis. Importantly, the requirement for DMSO
for solubility of 1 and the active analogs of 1 was obviated by the use of
polyethylene glycol and ethanol, which makes the clinical use of these drugs in
humans more tenable.
Based on the structure and presumed covalent nature of our active analogs, we
performed additional glutathione binding studies. Our studies show that the
activity of 1 and its active analogs against cancer cells correlates directly with their
ability to bind glutathione thus making it likely that these drugs work on cancer
cells through covalent binding. While glutathione depletion has been shown to
ACS Paragon Plus Environment

Page 17 of 87
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
cause apoptosis in cancer cells, treatment of HT-29 cells with various concentrations
of buthionine sulphoximine did not induce apoptosis when they were allowed to
adhere to mesothelial cell monolayers. However, pre-incubation of cancer cells with
N-acetylcysteine inhibited the apoptotic activity of 1, suggesting that 1 may bind
other thiol-containing molecules.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
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
Traditionally, covalent inhibitors have been shunned due to their broad reactivity
(
and subsequent off-target effects) and perceived toxicity in humans. However, an
increasing proportion of clinically effective drugs, including carfilzomib, dimethyl
fumarate, afatinib, and omeprazole, act through covalent binding of cellular targets
and with acceptable safety profiles.28 Covalent inhibitors allow sustained inhibition
of cellular targets affording them potential advantages vital to cancer treatment: 1)
lower and less frequent dosing, 2) dissociation of pharmacokinetics and
pharmacodynamics since the latter is dependent on protein resynthesis, and 3)
prevention of drug resistance due to longer target suppression.30
We previously showed that 1 and its active analogs rapidly diminish cellular c-FLIP
protein, an important inhibitor of death receptor-mediated caspase
activation.19 Previous studies showed that c-FLIP protein is regulated by JNK-
dependent activation of ITCH, a ubiquitin ligase, that targets c-FLIP for proteasomal
degradation.30 Activation of the JNK pathway by 1 suggests a potential mechanism
for the inhibition of c-FLIP and a focus for future studies.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 87
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
The first step leading to peritoneal carcinomatosis is the shedding or dislodgement
of cancer cells from intra-abdominal cancers. Peritoneal seeding by cancer cells
occurs in up to 35% of patients following curative resections of pancreatic
cancers.26 Iatrogenic seeding is a paradoxical event in that resection is the mainstay
of treatment of localized intra-abdominal cancers. In fact, the detection of
intraperitoneal cancer cells during the resection of intra-abdominal cancers
portends a poor survival.27-32
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
While it can be difficult to know if occult peritoneal carcinomatosis is already
present by the time some patients undergo curative resection of intra-abdominal
cancers, there is evidence that this condition may be due to seeding following
medical interventions. The most obvious examples are cancer seeding of port-sites
used to remove cancers laparoscopically from the abdominal cavity.33, 34 In one
study, patients with nonmetastatic pancreatic cancer who all underwent
neoadjuvant chemoradiation had either percutaneous radiologically guided or
endoscopic ultrasound guided final needle aspirates of their pancreatic cancers. 35
At the end of treatment, all patients underwent restaging with computed
tomography followed by attempted surgical resection. At the time of surgery, 16.3%
and 2.2% of patients who underwent percutaneous or endoscopic needle biopsies,
respectively, developed peritoneal carcinomatosis. However, other studies have not
reflected an increased risk of peritoneal carcinomatosis in pancreatic cancer
patients who underwent or did not undergo percutaneous of their cancers. 36
However, in a large randomized controlled trial, intraperitoneal chemotherapy was
ACS Paragon Plus Environment

Page 19 of 87
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
shown to significantly increase patient survival following curative resection of
3
4
ovarian cancer suggesting that inhibition of peritoneal cancer spread may
improve survival. The problem with most of these and other studies regarding this
controversy is that the majority of studies is retrospective and small, and, show
modest benefit making their clinical import questionable. A drug that specifically
and rapidly inhibited implantation of cancer cells in the peritoneum at the time of
curative resection would permit randomized controlled trials to resolve the
controversy of whether or not biopsies or surgical resection of intra-abdominal
cancers results in peritoneal seeding.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
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
The two pre-clinical murine models used for these studies measured different
outcomes and clinical scenarios. The ovarian cancer model simply tested the ability
of the drugs to inhibit ovarian cancer cells from seeding the peritoneum. The
pancreatic cancer model tested the ability of the drugs to inhibit local recurrence of
pancreatic cancer at the site of resection as well as distant metastasis to lymph
nodes and the liver following pancreatic cancer resection. For certain cancers such
as pancreatic cancer, loco-regional recurrence after surgical cancer resection is a
large clinical problem for which there is no effective therapy.
All lead analogs and 1 showed significant reductions in peritoneal carcinomatosis in
the ovarian cancer mouse model. One analog, 6b, was significantly more effective at
inhibiting peritoneal carcinomatosis by A2780 ovarian cancer cells than 1, thus
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 87
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
achieving the primary endpoint of designing and synthesizing an analog with
greater potency than the parent compound.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
For the pancreatic cancer mouse model, 4b emerged as the most efficacious analog
compared with vehicle and 1. Although 4b was significantly better at inhibiting
peritoneal carcinomatosis from ovarian cancer cells than vehicle, it was not
significantly more effective than 1. These results may reflect tissue-specific
differences for the various lead analogs.
The mechanism by which these drugs work appears be due to the ability of 1 to
rapidly inhibit the expression of survival proteins that are overexpressed in cancer
cells.18, 19 One key intracellular protein target identified in our previous studies is c-
FLIP, which is commonly overexpressed in cancer cells and capable of inhibiting
death receptor-mediated apoptosis in cancer cells. 19 Investigations on how 1 and
its active analogs inhibit c-FLIP are presently underway.
In summary, these results suggest that these unique agents could be administered in
the immediate peri-operative period to inhibit the spread of intra-abdominal
cancers following surgical resection of ovarian and pancreatic cancers. The pre-
clinical cancer assays developed and utilized for this study may be useful in
developing compounds that prevent the spread of cancer cells.
ACS Paragon Plus Environment

Page 21 of 87
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
EXPERIMENTAL PROCEDURES
Synthetic Procedures and Analytical Data:
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
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
All reagents and solvents were obtained from commercial sources and used as
received. All final products were purified to >95% purity as determined by HPLC
and LCMS. Analytical HPLC was run using a Zorbax Eclipse XDB-C8 3.5 ꢀm 4.6x75
mm column eluting with a mixture of acetonitrile and water containing 0.1%
trifluoroacetic acid with a 5 minute gradient of 10-100%. LCMS results were
obtained on either of two instruments. A Waters Acquity Ultra Performance LC with
a Waters Aquity UPLC BEH C18 1.7 ꢀm 2.1x50 mm column was paired with a
Micromass-ZQ 2000 quadrupole mass spectrometer with electrospray ionization.
Alternatively, an Agilent 1100 series HPLC with a Zorbax Eclipse XDB-C8 3.5 ꢀm
2.1x30mm column was paired with a Bruker Esquire 2000 mass spectrometer with
1
13
electrospray ionization. H-NMR and C-NMR spectra were obtained on a Bruker
Avance at 400 MHz and 100 MHz, respectively, in the solvent indicated with
tetramethylsilane as an internal standard. High resolution LCMS was performed on
a Waters Acquity UPLC using a Phenomenex Kinetex C18 1.7 μm 2.1x50 mm column
and interfaced to a Waters Synapt G2 Q-TOF mass spectrometer using positive
electrospray with Leu-enkephalin as a lock-mass standard. The mobile phases were
0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) using a
gradient of 2%B to 95%B in 3 minutes to elute the compounds. Preparative HPLC
was performed on a Gilson HPLC using a Phenomenex Gemini NX 5 ꢀm C18,
21.2x100 mm column with UV detection or a Waters HPLC using a Waters XBridge
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 87
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
PrepC8 5ꢀm OBD 30x75 mm column with MS detection on a Micromass-ZQ 2000
quadrupole mass spectrometer with electrospray ionization. Gradient silica gel
column chromatography was performed on a CombiFlash Companion or Rf
instrument (Teledyne Isco Inc.).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
-[4-((E)-2-Cyano-ethenesulfonyl)-phenyl]-N-phenyl-isobutyramide (4a)
2-[4-((E)-2-Cyano-ethenesulfonyl)-phenyl]-2-methyl-propionic acid (300.0 mg,
.074 mmol), aniline (0.098 mL, 1.07 mmol), EDCI (206 mg, 1.07 mmol) and HOBt
1
(72.6 mg, 0.537 mmol) were dissolved in tetrahydrofuran (30 mL) and the reaction
mixture was allowed to stir overnight at room temperature. The reaction mixture
was poured over saturated sodium bicarbonate and organics were extracted with
dichloromethane/ethyl acetate. Combined extracts were then dried over sodium
sulfate, filtered and concentrated. The crude reaction mixture was purified on silica
gel, eluting with hexanes/ethyl acetate to afford 2-[4-((E)-2-Cyano-ethenesulfonyl)-
phenyl]-N-phenyl-isobutyramide as a foam (171 mg, 45%). LCMS (ESI): 355
+
1
(
(
(
M+H) ; H-NMR (DMSO-d
6
, 400 MHz) δ 9.22 (s, 1H), 8.23 (d, 1H, J = 15.7 Hz), 7.90
d, 2H, J = 7.8 Hz), 7.68 (d, 2H, J = 7.8 Hz), 7.57 (d, 2H, J = 7.7 Hz), 7.27 (m, 2H), 7.05
1
3
m, 1H), 6.90 (d, 1H, J = 15.7 Hz), 1.60 (s, 6H); C-NMR: (DMSO-d
6
, 100 MHz) δ
73.7, 153.2, 149.2, 139.0, 135.5, 128.4, 128.2, 127.6, 123.5, 120.4, 114.6, 112.0,
7.9, 26.5.
1
4
N-Benzyl-2-[4-((E)-2-cyano-ethenesulfonyl)-phenyl]-isobutyramide (4b)
2
-[4-((E)-2-Cyano-ethenesulfonyl)-phenyl]-2-methyl-propionic acid (300.0 mg,
.074 mmol), benzylamine (0.115 g, 1.07 mmol), EDCI (206 mg, 1.07 mmol) and
1
ACS Paragon Plus Environment

Page 23 of 87
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
HOBt (72.6 mg, 0.537 mmol) were dissolved in tetrahydrofuran (30 mL) and the
reaction mixture was allowed to stir overnight at room temperature. The reaction
mixture was poured over saturated sodium bicarbonate and organics were
extracted with dichloromethane/ethyl acetate. Combined extracts were then dried
over sodium sulfate, filtered and concentrated. The crude reaction mixture was
purified on silica gel, eluting with hexanes/ethyl acetate to afford N-Benzyl-2-[4-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
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
((E)-2-cyano-ethenesulfonyl)-phenyl]-isobutyramide as a white powder (173 mg,
+ 1
4
4%). LCMS (ESI): 369 (M+H) ; H-NMR (DMSO-d , 400 MHz) δ 8.24 (d, 1H, J =
6
15.6 Hz), 8.06 (t, 1H, J = 5.6 Hz), 7.86 (d, 2H, J = 7.6 Hz), 7.63 (d, 2H, J = 7.6 Hz), 7.27
(
m, 2H), 7.21 (m, 1H), 7.11 (d, 2H, J = 7.3 Hz), 6.90 (d, 1H, J = 15.6 Hz), 4.24 (d, 2H, J =
1
3
5
.6 Hz), 1.52 (s, 6H); C-NMR: (DMSO-d , 100 MHz) δ 174.6, 153.4, 149.2, 139.6,
6
135.3, 128.0, 127.9, 127.6, 126.7, 126.5, 114.5, 111.9, 46.6, 42.3, 26.3; HRMS-TOF-
+
MS-ES+ (m/z): [M+H] calcd for C20
H
21
N
2
O S, 369.1273; found, 369.1274.
3
2-[4-((E)-2-Cyano-ethenesulfonyl)-phenyl]-N-phenethyl-isobutyramide (4c)
-[4-((E)-2-Cyano-ethenesulfonyl)-phenyl]-2-methyl-propionic acid (75.0 mg, 0.268
2
mmol), phenylethylamine (33.7 µL, 0.268 mmol), EDCI (51.5 mg, 0.268 mmol) and
HOBt (18.1 mg, 0.134 mmol) were dissolved in methylene chloride (7.5 mL) and
the reaction mixture was allowed to stir overnight at room temperature. The
reaction was concentrated under reduced pressure and the residue was taken up in
1
.2 mL of acetonitrile. Purification by mass-directed HPLC using a gradient of 25-
5% acetonitrile:water both containing 0.1% TFA as the eluting solvent afforded 2-
7
[4-((E)-2-Cyano-ethenesulfonyl)-phenyl]-N-phenethyl-isobutyramide as a white
+
1
foam (39 mg, 38%). LCMS (ESI): 383 (M+H) ; H-NMR (CDCl , 400 MHz) δ 7.79 (d,
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 87
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
H, J = 8.6 Hz), 7.49 (d, 2H, J = 8.6 Hz), 7.29-7.21 (m, 3H), 7.19 (d, 1H, J = 15.7 Hz),
7.07-7.02 (m, 2H), 6.55 (d, 1H, J = 15.7 Hz), 5.20-5.08 (m, 1H), 3.53-3.46 (m, 2H),
13
2
1
3
.76 (t, 2H, J = 6.7 Hz), 1.54 (s, 6H); C-NMR: (CDCl
3
, 100 MHz) δ 175.2, 153.3,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
48.9, 138.4, 135.6, 128.8, 128.67, 128.65, 127.9, 126.7, 113.3, 110.7, 47.4, 40.7,
+
5.3, 26.8; HRMS-TOF-MS-ES+ (m/z): [M+H] calcd for C21
H
23
N
2
O S, 383.1429;
3
found, 383.1425.
2-[4-((E)-2-Cyano-ethenesulfonyl)-phenyl]-N-((S)-1-phenyl-ethyl)-isobutyramide
(4d)
2-[4-((E)-2-Cyano-ethenesulfonyl)-phenyl]-2-methyl-propionic acid (75.0 mg, 0.268
mmol), (S)-1-phenylethylamine (34.3 µL, 0.268 mmol), EDCI (51.5 mg, 0.268 mmol)
and HOBt (18.1 mg, 0.134 mmol) were dissolved in methylene chloride (7.5 mL)
and the reaction mixture was allowed to stir overnight at room temperature. The
reaction was concentrated under reduced pressure and the residue was taken up in
1
.3 mL of acetonitrile. Purification by mass-directed HPLC using a gradient of 25-
5% acetonitrile:water both containing 0.1% TFA as the eluting solvent afforded 2-
7
[4-((E)-2-Cyano-ethenesulfonyl)-phenyl]-N-((S)-1-phenyl-ethyl)-isobutyramide as
+
1
an amorphous white solid (66 mg, 64%). LCMS (ESI): 383 (M+H) ; H-NMR (CDCl
3
,
400 MHz) δ 7.84 (d, 2H, J = 7.6 Hz), 7.55 (d, 2H, J = 7.6 Hz), 7.37-7.25 (m, 3H), 7.24-
7.14 (m, 3H), 6.53 (d, 1H, J = 15.7 Hz), 5.59 (d, 1H, J = 7.1 Hz), 5.16-5.06 (m, 1H), 1.61
+
(
s, 3H), 1.60 (s, 3H), 1.43 (d, 3H, J = 6.8 Hz); HRMS-TOF-MS-ES+ (m/z): [M+H] calcd
S, 383.1429; found, 383.1430.
for C21
H
23
N
O
2 3
ACS Paragon Plus Environment

Page 25 of 87
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
2
-[4-((E)-2-Cyano-ethenesulfonyl)-phenyl]-N-((R)-1-phenyl-ethyl)-isobutyramide
(4e)
2-[4-((E)-2-Cyano-ethenesulfonyl)-phenyl]-2-methyl-propionic acid (75.0 mg, 0.268
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
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
mmol), (R)-1-phenylethylamine (34.2 µL, 0.268 mmol), EDCI (51.5 mg, 0.268 mmol)
and HOBt (18.1 mg, 0.134 mmol) were dissolved in methylene chloride (7.5 mL)
and the reaction mixture was allowed to stir overnight at room temperature. The
reaction was concentrated under reduced pressure and the residue was taken up in
1
.3 mL of acetonitrile. Purification by mass-directed HPLC using a gradient of 25-
5% acetonitrile:water both containing 0.1% TFA as the eluting solvent afforded 2-
7
[4-((E)-2-Cyano-ethenesulfonyl)-phenyl]-N-((R)-1-phenyl-ethyl)-isobutyramide as
+
1
an amorphous white solid (69 mg, 67%). LCMS (ESI): 383 (M+H) ; H-NMR (CDCl
3
,
400 MHz) δ 7.84 (d, 2H, J = 7.3 Hz), 7.54 (d, 2H, J = 7.3 Hz), 7.37-7.26 (m, 3H), 7.23-
7.15 (m, 3H), 6.54 (d, 1H, J = 15.6 Hz), 5.68 (d, 1H, J = 6.8 Hz), 5.16-5.06 (m, 1H), 1.62
1
3
(s, 3H), 1.60 (s, 3H), 1.44 (d, 3H, J = 6.8 Hz); C-NMR: (CDCl , 100 MHz) δ 174.4,
3
153.5, 148.9, 142.7, 135.7, 128.9, 128.7, 127.8, 127.6, 126.0, 113.3, 110.8, 49.1, 47.4,
+
2
6.9, 21.4; HRMS-TOF-MS-ES+ (m/z): [M+H] calcd for C21
H
23
N
2
O S, 383.1429;
3
found, 383.1429.
-[4-((E)-2-Cyano-ethenesulfonyl)-phenyl]-N-(4-methanesulfonyl-benzyl)-
isobutyramide (4f)
-Methanesulfonyl-benzylamine hydrochloride (59.5 mg, 0.268 mmol) was placed
2
4
in methylene chloride (10 mL) and macroporous carbonate resin (3.16 mmol/g
loading, 255 mg, 0.806 mmol) was added. The reaction was stirred at room
temperature for 30 minutes and then filtered to remove the resin. The solution was
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 87
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
added to a vial containing 2-[4-((E)-2-Cyano-ethenesulfonyl)-phenyl]-2-methyl-
propionic acid (75.0 mg, 0.268 mmol), HOBt (18.1 mg, 0.134 mmol), and EDCI (51.5
mg, 0.268 mmol). The reaction was stirred at 40 °C overnight and was then
concentrated under reduced pressure. The residue was taken up in 1.6 mL of DMSO
and purified by preparative HPLC using a gradient of 10-45% acetonitrile:water
both containing 0.1% TFA as the eluting solvent to afford 2-[4-((E)-2-Cyano-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ethenesulfonyl)-phenyl]-N-(4-methanesulfonyl-benzyl)-isobutyramide as a white
+ 1
foam (33 mg, 28%). LCMS (ESI): 447 (M+H) ; H-NMR (CDCl , 400 MHz) δ 7.87 (d,
3
2
H, J = 8.4 Hz), 7.80 (d, 2H, J = 8.2 Hz), 7.62 (d, 2H, J = 8.4 Hz), 7.32 (d, 2H, J = 8.2 Hz),
.22 (d, 1H, J = 15.6 Hz), 6.57 (d, 1H, J = 15. 6 Hz), 5.99 (t, 1H, J = 5.6 Hz), 4.49 (d, 2H,
7
J = 6.0 Hz), 3.03 (s, 3H), 1.66 (s, 6H).
2-[4-((E)-2-Cyano-ethenesulfonyl)-phenyl]-N-(4-sulfamoyl-benzyl)-isobutyramide
(4g)
4-Aminomethyl-benzenesulfonamide hydrochloride (59.8 mg, 0.268 mmol) was
placed in methylene chloride (10 mL) and macroporous carbonate resin (3.16
mmol/g loading, 255 mg, 0.806 mmol) was added. The reaction was stirred at room
temperature for 30 minutes and then filtered to remove the resin. The solution was
added to a vial containing 2-[4-((E)-2-Cyano-ethenesulfonyl)-phenyl]-2-methyl-
propionic acid (75.0 mg, 0.268 mmol), HOBt (18.1 mg, 0.134 mmol), and EDCI (51.5
mg, 0.268 mmol). The reaction was stirred at 40 °C overnight and then concentrated
under reduced pressure. The residue was taken up in 1.6 mL of DMSO and purified
by preparative HPLC using a gradient of 10-45% acetonitrile:water both containing
0.1% TFA as the eluting solvent to afford 2-[4-((E)-2-Cyano-ethenesulfonyl)-
ACS Paragon Plus Environment

Page 27 of 87
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
phenyl]-N-(4-sulfamoyl- benzyl)-isobutyramide as a white foam (17 mg, 14%).
LCMS = 448 (M+H); HNMR (d6-DMSO, 400 MHz) δ 8.24 (d, 1H, J = 15.6 Hz), 8.16 (t,
1H, J = 5.8 Hz), 7.88 (d, 2H, J = 8.4 Hz), 7.73 (d, 2H, J = 8.1 Hz), 7.64 (d, 2H, J = 8.4 Hz),
7.35-7.26 (m, 4H), 6.91 (d, 1H, J = 15.6 Hz), 4.28 (d, 2H, J = 5.8 Hz), 1.52 (s, 6H)
2-[4-((E)-2-Cyano-ethenesulfonyl)-phenyl]-N-(2-methoxy-benzyl)-isobutyramide
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
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
(4h)
2-[4-((E)-2-Cyano-ethenesulfonyl)-phenyl]-2-methyl-propionic acid (75.0 mg, 0.268
mmol), 2-methoxy-benzylamine (34.7 ꢀL, 0.268 mmol), EDCI (51.5 mg, 0.268 mmol)
and HOBt (18.1 mg, 0.134 mmol) were dissolved in acetonitrile (1.0 mL) and the
reaction mixture was allowed to stir for 2 hours at room temperature. The reaction
was concentrated under reduced pressure and the residue was taken up in 1.6 mL of
DMSO. Purification by mass-directed HPLC using a gradient of 25-75%
acetonitrile:water both containing 0.1% TFA as the eluting solvent afforded 2-[4-
((E)-2-Cyano-ethenesulfonyl)-phenyl]-N-(2-methoxy-benzyl)-isobutyramide (48
+ 1
mg, 45%) as a white lyophilate. LCMS (ESI): 399 (M+H) ; H-NMR (CDCl , 400
3
MHz) δ 7.81 (d, 2H, J = 8.7 Hz), 7.53 (d, 2H, J = 8.7 Hz), 7.30-7.23 (m, 1H), 7.23-7.16
(
m, 2H), 6.94-6.87 (m, 1H), 6.85-6.80 (m, 1H), 6.54 (d, 1H, J = 15.7 Hz), 5.89-5.80 (m,
1
3
1
H), 4.38 (d, 2H, J = 5.8 Hz), 3.69 (s, 3H), 1.58 (s, 6H); C-NMR (CDCl , 100 MHz) δ
3
175.0, 157.4, 153.7, 148.9, 135.5, 129.9, 129.1, 128.7, 127.9, 125.7, 120.8, 113.2,
+
1
C
2
10.7, 110.3, 55.2, 47.5, 40.4,26.8; HRMS-TOF-MS-ES+ (m/z): [M+H] calcd for
21
H
23
N
2 4
O S, 399.1379; found, 399.1381.
-[4-((E)-2-Cyano-ethenesulfonyl)-phenyl]-N-(3-methoxy-benzyl)-isobutyramide
(4i)
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 87
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
-[4-((E)-2-Cyano-ethenesulfonyl)-phenyl]-2-methyl-propionic acid (75.0 mg, 0.268
mmol), 3-methoxy-benzylamine (34.7 ꢀL, 0.268 mmol), EDCI (51.5 mg, 0.268 mmol)
and HOBt (18.1 mg, 0.134 mmol) were dissolved in acetonitrile (1.0 mL) and the
reaction mixture was allowed to stir for 2 hours at room temperature. The reaction
was concentrated under reduced pressure and the residue was taken up in 1.6 mL of
DMSO. Purification by mass-directed HPLC using a gradient of 25-75%
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
acetonitrile:water both containing 0.1% TFA as the eluting solvent afforded 2-[4-
(
(E)-2-Cyano-ethenesulfonyl)-phenyl]-N-(3-methoxy-benzyl)-isobutyramide (40
+
1
mg, 37%) as a white lyophilate. LCMS (ESI): 399 (M+H) ; H-NMR (CDCl
3
, 400
MHz) δ 7.85 (d, 2H, J = 8.6 Hz), 7.61 (d, 2H, J = 8.6 Hz), 7.25-7.16 (m, 2H), 6.84-6.78
(
m, 1H), 6.77-6.72 (m, 1H), 6.69-6.65 (m, 1H), 6.54 (d, 1H, J = 15.7 Hz), 5.57-5.48 (m,
13
1H), 4.38 (d, 2H, J = 5.7 Hz), 3.77 (s, 3H), 1.64 (s, 6H); C-NMR (CDCl
3
, 100 MHz) δ
175.1, 159.9, 153.3, 148.9, 139.5, 135.8, 129.8, 128.9, 127.9, 119.7, 113.4, 113.3,
+
112.8, 110.7, 55.3, 47.5, 43.9, 26.9; HRMS-TOF-MS-ES+ (m/z): [M+H] calcd for
C
21
H
23
N
2 4
O S, 399.1379; found, 399.1378.
2-[4-((E)-2-Cyano-ethenesulfonyl)-phenyl]-N-[4-(2-methoxy-ethoxymethyl)-
phenyl]-isobutyramide (4j)
a) Potassium hydroxide (0.682 g, 10.3 mmol) was added to a suspension of p-
nitrobenzylbromide (2.048 g, 9.480 mmol) in 2-methoxyethanol (20 mL, 254 mmol)
and the mixture was heated in a vial at 105 °C. After 90 min, the mixture was cooled
to room temperature. The mixture was poured into EtOAc (100 mL) and washed
with water (3X10 mL), brine (20 mL) and dried over sodium sulfate. Concentration
in vacuo after filtration gave an oil, which was purified on ISCO (25g preload
ACS Paragon Plus Environment

Page 29 of 87
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
w/DCM, 80g column, 5-40% EtOAc:Hex). Pure fractions were combined and conc. to
afford 1-(2-Methoxy-ethoxymethyl)-4-nitro-benzene (1.245 g, 62%) as an orange
oil.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
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
b) 1-(2-Methoxy-ethoxymethyl)-4-nitro-benzene (200 mg, 0.947 mmol was
dissolved in methanol (9.5 mL) and 5% platinum on carbon, sulfided (0.5%) (36.9
mg, 0.00947 mmol) was added. The reaction was hydrogenated at 50 psi for 60
minutes and then filtered through celite to remove the catalyst. The filtrate was then
concentrated under reduced pressure to afford 4-(2-Methoxy-ethoxymethyl)-
phenylamine as a yellow oil.
c) 2-[4-((E)-2-Cyano-ethenesulfonyl)-phenyl]-2-methyl-propionic acid (75.0 mg,
0.268 mmol), 4-(2-Methoxy-ethoxymethyl)-phenylamine (48.7 mg, 0.268 mmol),
EDCI (51.5 mg, 0.268 mmol) and HOBt (18.1 mg, 0.134 mmol) were dissolved in
methylene chloride (7.5 mL) and the reaction mixture was allowed to stir overnight
at room temperature. The reaction was concentrated under reduced pressure and
the residue was taken up in 1.6 mL of DMSO. Purification by mass-directed HPLC
using a gradient of 25-75% acetonitrile:water both containing 0.1% TFA as the
eluting solvent to afford 2-[4-((E)-2-Cyano-ethenesulfonyl)-phenyl]-N-[4-(2-
methoxy-ethoxymethyl)-phenyl]-isobutyramide as an orange oil (57 mg, 48%).
LCMS = 465 (M+Na); HNMR (d6-DMSO, 400 MHz) δ 9.23 (s, 1H), 8.23 (d, 1H, J = 15.7
Hz), 7.89 (d, 2H, J = 8.6 Hz), 7.67 (d, 2H, J = 8.6 Hz), 7.54 (d, 2H, J = 8.6 Hz), 7.22 (d,
2
H, J = 8.6 Hz), 6.0 (d, 1H, J = 15.7 Hz), 4.40 (s, 2H), 3.53-3.43 (m, 4H), 3.24 (s, 3H),
.60 (s, 6H).
1
ACS Paragon Plus Environment