Novel tertiary sulfonamides as potent anti-cancer agents

Article abstract of DOI:10.1016/j.bmc.2018.07.042

For adult women in the United States, breast cancer is the most prevalent form of cancer. Compounds that target dysregulated signal transduction can be efficacious anti-cancer therapies. A prominent signaling pathway frequently dysregulated in breast canc

Full text of DOI:10.1016/j.bmc.2018.07.042

Accepted Manuscript
Novel Tertiary Sulfonamides as Potent Anti-Cancer Agents
Karl J. Okolotowicz, Mary Dwyer, Daniel Ryan, Jiongjia Cheng, Emily A.
Cashman, Stephanie Moore, Mark Mercola, John R. Cashman
PII:
S0968-0896(18)31225-2
https://doi.org/10.1016/j.bmc.2018.07.042
BMC 14479
DOI:
Reference:
Bioorganic & Medicinal Chemistry
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Accepted Date:
4 July 2018
23 July 2018
24 July 2018
Please cite this article as: Okolotowicz, K.J., Dwyer, M., Ryan, D., Cheng, J., Cashman, E.A., Moore, S., Mercola,
M., Cashman, J.R., Novel Tertiary Sulfonamides as Potent Anti-Cancer Agents, Bioorganic & Medicinal
Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.07.042
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Novel Tertiary Sulfonamides as Potent Anti-Cancer Agents
Karl J. Okolotowicza, Mary Dwyera, Daniel Ryana, Jiongjia Chenga, Emily A. Cashmana, Stephanie Moorea, Mark
Mercolab and John R. Cashmana,*
aHuman BioMolecular Research Institute, 5310 Eastgate Mall, San Diego, CA 92121, USA;
bCardiovascular Institute and Department of Medicine, Stanford University, 300 Pasteur Dr., MC-5501, Stanford,
CA, USA 94305
Keywords: anti-proliferative, Wnt inhibitors, p53 activator, apoptosis, sulfonamides, breast cancer, Doxorubicin
synergy, anti-cancer
Abstract. For adult women in the United States, breast cancer is the most prevalent form of cancer. Compounds
that target dysregulated signal transduction can be efficacious anti-cancer therapies. A prominent signaling
pathway frequently dysregulated in breast cancer cells is the Wingless-related integration site (Wnt) pathway.
The purpose of the work was to optimize a “hit” from a screening campaign. 76,000 compounds were tested in a
Wnt transcription assay and revealed potent and reproducible “hit,” compound 1. Medicinal chemistry
optimization of 1 led to more potent and drug-like molecules, 19, 24 and 25 (i.e., Wnt pathway IC50 values = 11,
18 and 7 nM, respectively). The principal results showed compounds 19, 24 and 25 were potent anti-proliferative
agents in breast cancer cell lines, MCF-7 (i.e., IC50 values = 10, 7 and 4 nM, respectively) and MDA-MB 231
(i.e., IC50 values = 13, 13 and 16 nM, respectively). Compound 19 synergized anti-proliferation with
chemotherapeutic Doxorubicin in vitro. A major conclusion was that compound 19 enhanced anti-proliferation
of Doxorubicin in vitro and in a xenograft animal model of breast cancer.
Corresponding Author. John R. Cashman, 5310 Eastgate Mall, San Diego, CA, 92121; Telephone: (858) 458-
9305; Fax: (858) 458-9311; Email: JCashman@HBRI.org
Abbreviations. Wnt, Wingless-related integration site gene, p53, tumor protein p53, Pd/C, palladium on
carbon, HCl, hydrochloric acid, (ER+), estrogen receptor-positive, (ER-), estrogen receptor-negative, (PR-),
progesterone receptor-negative, (HER2-), human epidermal growth factor receptor 2-negative, (DMEM),
Dulbecco's Modified Eagle's cell culture medium, (FBS), fetal bovine serum
1

1.
Introduction.
In the United States, breast cancer is the most prevalent form of cancer and second most prevalent cause of death
for adult women1, 2. Common anti-breast cancer therapeutic modalities typically use a combination of surgery,
radiation and chemotherapeutics. Currently used chemotherapies include anthracyclines and taxanes (e.g.,
Doxorubicin and paclitaxel) and are usually used in combination with other chemotherapeutics including
fluorouracil and cyclophosphamide3.
Therapies that target protein components of dysregulated signal transduction pathways can be efficacious anti-
cancer therapies with minimal adverse effects4. Prominent among dysfunctional breast cancer signal transduction
pathways is the Wingless-related integration site (Wnt) pathway5. Wnt is named after Int1, a mouse mammary
proto-oncogene and Wingless (Wg), a gene from the Drosophila essential for wing development6. In adult women,
genes for a dysfunctional Wnt pathway have been established in causal association with breast cancer7. Currently,
targeting the Wnt pathway has been pursued for drug development, but only a few small molecules that modulate
the Wnt pathway are approved for treatments, (i.e., Pyrvinium, Celecoxib and Sulindac)8.
In addition to the Wnt pathway, dysregulation of the tumor protein p53 (p53) pathway is linked to the complexity
of cancer biology9. Therapies that have targeted p53 have focused on restoration of functional activity of p53, but
increases in endogenous p53 causes problems with cell adhesion and maintenance of tissue structure10, 11
.
Previously, a screen of 76,000 small molecules12 was conducted for inhibition of Wnt transcription13, 14. On the
basis of results from this screen, one selective and reproducible “hit”, (i.e., compound 1) was identified15, 16. In
contrast to other Wnt inhibitors described in the literature that inhibit signaling at the Axin level13, 14, 17, 1 was
shown to inhibit Wnt signaling far downstream in the pathway15. Herein, we describe the medicinal chemistry
and development of potent Wnt inhibitors derived from “hit” 1. The compounds elaborated were evaluated for
Wnt inhibition, inhibition of breast cancer cell proliferation and induction of p53 transcription in vitro. Compound
2

1 was optimized for both potency of inhibition of Wnt and for improvement in physiochemical properties. Based
on the results for Wnt inhibition and inhibition of breast cancer cell proliferation, a structure-activity relationship
(SAR) arose for the compounds examined. The results led to synthesis of significantly more potent Wnt inhibitors
with much improved anti-cancer and physicochemical properties. From SAR optimization of 1, several
compounds emerged (i.e., 19, 24 and 25) that showed increased Wnt potency and improved physicochemical
properties. Compounds 19, 24 and 25 were shown to be effective inhibitors of breast cancer cell (i.e., MCF-7
and MDA-MB 231) proliferation. In the presence of Doxorubicin, compound 19 showed increased inhibition of
proliferation of breast cancer cells in vitro and in in vivo xenograft studies. Compounds 19, 24 and 25 are new,
potent small molecules with potent anti-breast cancer functional activity.
2.
Results and Discussion
I
II
III
B
O
O
N
S N
O
A
1
Figure 1. “Hit” 1 identified from a screen for inhibition of Wnt transcription. Regions I, II and III conceptually
represent the regions of modification of “hit” 1 for optimization of the molecule.
2.1. Chemical Synthesis
Sulfonamides 4 – 25 were prepared using the synthetic route described in Scheme 1. In a typical synthesis, 1-
phenylpyrrolidinone, 2, was directly converted to the para-substituted sulfonyl chloride 3 using chlorosulfonic
acid. Generally, the crude solid material was isolated in sufficient purity to use without further purification.
Sulfonamides 4 - 25 were prepared by addition of the requisite amine to sulfonyl chloride 3.
Scheme 1. General synthesis of pyrrolidinone phenylsulfonamides 4 - 25a.
3

O
O
O
R1
R2
O
S N
O
ii
i
N
N
N
SO2Cl
R1R2NH
+
2
3
4 - 25
aReagents and conditions: i) chlorosulfonic acid, 25 °C, 15 h, ii) triethylamine, acetonitrile, 130 °C, 30 min.
For the synthesis of meta-substituted sulfonamide 30, a modified synthesis was used that followed literature
procedures18, 19 (Scheme 2). 3-Nitroaniline, 26, was converted to γ-lactam 27 by treatment with 4-chlorobutyryl
chloride that was added slowly by addition funnel. In a second step, the crude reaction product was added to a
freshly prepared solution of sodium ethoxide in ethanol to afford cyclization and produced a yellow-orange solid,
27. Hydrogenation of 27 in the presence of Pd/C gave aniline 28. Aniline 28 was converted to a diazonium salt
with sodium nitrite under acidic conditions followed by addition of sulfur dioxide gas and copper (II) chloride
dihydrate to afford 29. Sulfonyl chloride 29 was combined with the requisite amine to provide the meta-
substituted sulfonamide 30 in a similar manner as described in Scheme 1.
Scheme 2. Synthesis of meta-substituted phenylpyrrolidinone sulfonamide 30a.
aReagents and conditions: i) sodium phosphate dibasic, chloroform, 25 °C, 15 h.; sodium ethoxide, ethanol, 25
°C, 15 h, ii) H2, Pd/C, ethyl acetate, 25 °C, 15 h., iii) sodium nitrite, acetic acid/HCl, acetonitrile 0 °C, 1 h.; SO2(g)
,
4

copper (II) chloride dihydrate, 0 - 25 °C, 15 h., iv) 1,2,3,4-tetrahydroquinoline, triethylamine, acetonitrile, 130
°C, 30 min.
The synthesis of aryl-substituted compounds (e.g., 34) was done beginning with synthesis of key intermediate 32
(i.e., 2-methylphenyl pyrrolidinone) according to a literature method19 (Scheme 3). Aryl-substituted compound
34 was prepared by combining the sulfonyl chloride with an amine in a similar manner as described in Scheme
1.
Scheme 3. Synthesis of aryl-substituted sulfonamide 34a.
aReagents and conditions: i) sodium phosphate dibasic, chloroform, 25 °C, 15 h.; sodium ethoxide, ethanol, 25
°C, 15 h., ii) chlorosulfonic acid, 25 °C, 15 h, iii)1,2,3,4-tetrahydroquinoline, triethylamine, acetonitrile, 130 °C,
30 min.
Analogs were synthesized to examine the effect of substituents on the heterocyclic ring of the pyrrolidine moiety
shown in Region I (Figure 1). For example, to prepare 1,2,4-triazole analog 36 (Scheme 4a), phenylhydrazine
hydrochloride and formamide were combined in the absence of solvent in a microwave vial and heated at 175 °C
to afford 1-phenyl-1,2,4-triazole, 35.20 Intermediate 35 was directly converted to para-substituted sulfonyl
chloride by treatment with chlorosulfonic acid. Likewise, dihydropyrazole analog 38 (Scheme 4b), was prepared.
Phenylhydrazine and dibromopropane were combined in the absence of solvent in a microwave vial and heated
at 175 °C to provide the dihydropyrazole 37 following a literature procedure.21, 22 Intermediate 37 was converted
to its para-substituted sulfonyl chloride by treatment with chlorosulfonic acid. Sulfonamides 36 and 38 were
prepared by combining sulfonyl chlorides formed from 35 and 37 with 1,2,3,4-tetrahydroisoquinoline according
to the same procedure shown in Scheme 1.
Scheme 4. Synthesis of heterocyclic sulfonamides 36 and 38a.
5

a.
NHNH2
O
S N
O
N
N
N
i
O
iii
N
N
+
+
N
N
H2N
H
35
36
b.
NHNH2
ii
O
S N
O
N
N
iii
N
Br Br
37
38
aReagents and conditions: i)175 °C, microwave 1.5 h., ii)130 °C, microwave 30 min, iii) chlorosulfonic acid, 25
°C, 15 h.;1,2,3,4-tetrahydroquinoline, triethylamine, acetonitrile, 130 °C, 30 min.
Pyrrolidine analogs (e.g., compound 39) were prepared by direct reduction of pyrrolidinone 1 with zinc /
triethoxysilane23 as shown in Scheme 5.
Scheme 5. Synthesis of phenylpyrrolidine sulfonamide 39a.
O
O
O
i
N
S N
O
N
S N
O
1
39
aReagents and conditions: i) Zn(OAc)2 (10 mol %), HSi(OEt)3, THF, 70 °C, 30 h.
3.
Biological studies
3.1.
Effect of Sulfonamides on the Wnt Pathway. A diverse set of 76,000 compounds24 were tested for
inhibition of Wnt transcription in a cell-based assay in HEK293 cells, where cells were transfected with a Wnt
luciferase plasmid to over-express Wnt25. From a screen for inhibition of Wnt, one reproducible “hit” was found
(i.e., compound 1, IC50 = 25 nM). Counterscreens showed none of the compounds examined inhibited luciferase.
Based on the structure of “hit” 1, synthetic analogs 4-25, 30, 34, 36, 38 and 39 were synthesized and tested for
inhibition of Wnt transcription. Each compound was tested in a dose-response study (i.e., 0 – 5 µM, nine
6

concentrations, in triplicate) to determine an IC50 for inhibition of Wnt transcription. To systematically determine
a structure-activity relationship (SAR) for inhibition of Wnt transcription and quantify the effect of modification
of 1, the molecule was conceptually divided into three exploratory regions: (I) the pyrrolidinone region, (red),
(II) the central aryl region, (black) and (III) the 1,2,3,4-tetrahydroquinoline region, (blue). The 1,2,3,4-
tetrahydroquinoline ring (III) was further conceptually divided into A and B rings (Figure 1). Initially, a small
group of approximately 20 compounds directed at structural modifications of the 1,2,3,4-tetrahydroquinoline
portion of 1 (i.e., Region III) were synthesized and tested for inhibition of Wnt transcription (Table 1). The results
of these studies showed that a sulfonyl-1,2,3,4-tetrahydroquinoline moiety was essential to maintain potency. For
example, compared to 1, ring-opening and ring contraction of the A ring of Region III (i.e., 4 and 5, respectively,
IC50 > 5,000 nM) resulted in loss of potency. Various other unsaturated ring-systems (e.g., 6, IC50 > 5,000 nM)
or saturated ring-systems (e.g., 7 and 8, IC50 >5,000 nM, respectively) showed that other ring systems in Region
III of the molecule decreased potency. To observe the effect of the regio orientation of the 1,2,3,4-
tetrahydroquinoline nitrogen in ring system III, analog 9 was prepared and tested. Compared to 1, 1,2,3,4-
tetrahydroisoquinoline analog 9 gave decreased potency (IC50 = 634 nM). This indicated that the position of the
nitrogen atom in the 1,2,3,4-tetrahydroquinoline ring system was essential to maintain maximal potency. The data
for 4 – 9, Table 1, showed the 1,2,3,4-tetrahydroquinoline moiety was necessary to maintain potency. Finally,
to explore the effect of the sulfonamide moiety on inhibition of Wnt transcription, the analogous amide analog of
1 was prepared (i.e., 10). Replacement of the sulfonamide moiety with an amide moiety resulted in loss of potency
(i.e., 10 had an IC50 > 5,000 nM). This data showed that the sulfonamide moiety of 1 was essential for the potency
of the molecule.
To further examine the SAR around region III, substituted 1,2,3,4-tetrahydroquinolines were prepared (i.e.,
compounds 11 – 16). The effect of small substituents on the aryl portion of the 1,2,3,4-tetrahydroquinoline ring
of 1 on inhibition of Wnt transcription showed that both electron-withdrawing or electron-donating substituents
showed similar potency (i.e., 11 – 14, IC50 = 7, 6, 13, 54 nM, respectively). Large bulky groups, (e.g., t-butyl-
substituted compound, 15, IC50 = 660 nM) showed decreased potency compared to 1. This suggested the potency
7

of inhibition of Wnt transcription was sensitive to steric constraints and large bulk at this position resulted in low
potency. Compared to 1, a racemic methyl group ortho to the sulfonamide moiety on the 1,2,3,4-
tetrahydroquinoline was quite potent (i.e., 16, IC50 = 11 nM). This showed ortho substituents were well-tolerated
at this position (Table 1).
Table 1. Effect of substituents of Region III of 1 on inhibition of Wnt transcription.
IC50 Std.
IC50 Std.
Compound
Compound
Dev. (nM)a
Dev. (nM)a
25
3
> 5,000
1
10
> 5,000
> 5,000
7
6
2
2
4
5
11
12
O
O
b
S N
O
Cl
6
7
> 5,000
> 5,000
13
54
2
13
14
O
S N
O
CF3
1
O
S N
O
8

8
9
> 5,000
660
11
2
15
16
O
S N
O
O
S N
O
O
634
4
3
S N
O
aIC50 is the mean of three determinations Std Dev. bCommercially available.
Compounds 17 and 19 (i.e., IC50 = 10 nM and 11 nM, respectively, Table 2) were more potent than 1.
Compared to 1, compounds 17 and 19 increased the number of potential H-bond donors and increased the
aqueous solubility approximately 10-fold. By comparison, the N-desmethyl analog, 18, and 4-hydroxyethyl
analog, 20, showed poor potency for inhibition of Wnt transcription (Table 2). Compared to 1, compound 21
with a 1,5-naphthyridine substructure showed loss of potency (i.e., IC50 > 5,000 nM). In summary, compared to
1, the results from Table 2 and Supporting Table S1 showed that incorporation of tertiary amines into the
1,2,3,4-tetrahydroquinoline ring increased aqueous solubility of the molecule more than 10-fold . Data
suggested that compared to 1, compounds 17 and 19 possessed superior physicochemical properties. For
example, (i.e., solubility (i.e., 9.5 and 10.5 µg/mL), logD3.0/logD7.0 (1.7/1.7 and 1.9/1.9) and Calc. tPSA (i.e.,
66.9 and 60.9 Å2) for 17 and 19, respectively, were superior to compound 1 solubility (2.8 µg/mL),
logD3.0/logD7.0 (2.5/2.5) and Calc. tPSA (i.e., 57.7 Å2). A small group, (i.e., N-methyl), in the 4-position of the
1,2,3,4-tetrahydroquinoline, (i.e., 19, IC50 = 11 nM), afforded considerable potency while improving compound
solubility (Supporting Table S1). By contrast, 1,5-naphthyridine 21 resulted in loss of potency. However,
solubility alone does not explain all the results. For example, compounds 18 and 19 have similar solubilities but
vastly different potency. It may be that the nature of the substituent (H-bond donor versus H-bond acceptor) and
its interaction with the biological target(s) may play an important role in the overall potency.
9

Table 2. Effect of modifications of the 1,2,3,4-tetrahydroquinoline moiety on inhibition of Wnt transcription.
IC50 Std. Dev. (nM)a
Compound
10
1
17
18
19
>5000
11
3
> 5000
>5000
20
21
aIC50 is mean of three determinations Std. Dev.
To further explore the effects of the SAR of analogs of 1, several analogs were prepared to study the synergistic
effects of multiple substituents, for the most potent compounds listed in Tables 1 and 2. Table 3 summarizes the
results for 22 – 25. The 6-methoxy substituent of 12 was combined with the 1,2,3,4-tetrahydroquinoxaline moiety
of 19 to provide 22. Compound 22 was shown to be equipotent as 1 (i.e., IC50 = 17 nM for 22 vs IC50 = 25 nM
for 1). Compound 23 (i.e., IC50 = 9 nM), the conceptual hybridization of 16 and 19, showed an improvement in
potency compared to 1. The results from 22 and 23 showed C-2 substitutions maintained potency whereas C-6
substitutions of 1 afforded slightly less potency. The conceptual hybridization of 38 and 39 with 19 resulted in
24 and 25, respectively. Compound 24 was shown to be equipotent to 1, (i.e., IC50 = 18 nM for 24 vs IC50 = 25
10

nM for 1). In contrast, 25 was shown to be equipotent to 19 (i.e., IC50 = 7 nM for 25 vs IC50 = 11 nM for 19).
These results showed that a saturated ring in Region I was important for potency.
Table 3. Effect of pyrrolidinone and tetrahydroquinoline functionality on inhibition of Wnt transcription.
Structure
IC50 Std. Dev. (nM)a
Compound
O
N
O
17
2
22
O
S N
N
O
O
O
9
2
23
24
N
S N
O
N
O O
S
N
N
N
18
2
N
N
O
S
O
N
N
7
1
25
aIC50 is mean of three determinations Std. Dev.
To explore the effect of substitutions of the aryl ring of Region II, two analogs were synthesized (i.e., 30 and 34).
Compound 30 (Scheme 2) was prepared to investigate the effect of regio-orientation of the aryl Region II on
potency. The meta-substituted sulfonamide 30 was weakly potent (i.e., IC50 > 5,000 nM). We conclude that para-
but not meta-orientation of the sulfonamide group was required for potency. Compound 34 (Scheme 3) showed
very low potency (i.e., IC50 > 5,000 nM). The loss of potency of 34 could be due to an ortho-methyl group effect
on Region II. Compared to 1, the methyl group of 34 may “lock” the molecule in a suboptimal conformation for
binding to its biological target due to potential steric interactions between the ortho-methyl group and the carbonyl
11

of the pyrrolidinone ring. In summary, para-substituted sulfonamides were required for potency and aryl ortho-
substituents abolished potency. Accordingly, synthesis and testing of other aryl-substituted compounds was not
extensively pursued.
Based on the potency of 1, 4 and 7, the 1,2,3,4-tetrahydroquinoline ring was used as a starting point to investigate
modification of the pyrrolidinone ring system of Region I (Table 4). Replacement of the pyrrolidinone ring with
a 1,2,4-triazole (i.e., 36) resulted in loss of potency (i.e., IC50 > 5,000 nM). The partially reduced pyrazole, (i.e.,
dihydropyrazole 38, IC50 = 15 nM) was a potent inhibitor of Wnt transcription. Further work showed chemical
reduction of the carbonyl of pyrrolidinone 1 to afford the pyrrolidine 39 maintained potency (i.e., IC50 = 20 nM).
Together, this data showed the carbonyl of the pyrrolidinone ring was not essential for Wnt inhibition potency.
The apparent potency of 38 and 39 prompted the preparation of additional ring-opened and ring-closed nitrogen-
containing compounds of Region I to examine the effect of heterocycle structure in Region I on inhibition of Wnt
transcription. Ring expansion of the 5-membered lactam 1 to a 6-membered lactam (40) resulted in loss of potency
(i.e., 40 = IC50 > 5,000 nM). Synthesis and testing of the ring-opened tertiary amide analog 41 (i.e., IC50 > 5,000
nM) resulted in loss of potency. The results from 40 and 41 showed the necessity of the 5-membered ring system
for inhibition of Wnt transcription. Replacement of the pyrrolidine ring in 39 with a pyrazole ring (i.e., 42)
afforded a compound with a significant decrease in potency (i.e., IC50 = 1,650 nM). These results showed that,
compared to 1, introduction of more conjugated systems, (i.e., 36 and 42), resulted in loss of potency, whereas
compounds with saturated or partially saturated ring systems, (i.e., 38 and 39), showed equal or greater potency.
In contrast, ring- expanded or ring-opened analogs in Region I of 1 (i.e., 40 and 41, respectively) resulted in loss
of potency (Table 4).
Table 4. Effect of modifications of Region I on inhibition of Wnt transcription.
IC50 Std. Dev. (nM)a
O
Compound
S N
O
12

> 5000
36
38
39
14
20
2
2
> 5000.
> 5000
40
b
41
42
1650
3
aIC50 is mean of three determinations Standard Deviation (Std. Dev.). bCommercially available.
Synthesis and testing of structural analogs centered around Regions I – III showed: 1) for Region I, 5-membered
aliphatic heterocycles afforded considerable potency, 2) for Region II, a para-substitution pattern was essential
for potency and 3) for Region III, a 1,2,3,4-tetrahydroquinoline moiety gave significant potency. In addition to
pharmacological properties, modulation of physicochemical properties were conducted to improve the overall
properties of the most potent compounds. For example, the solubility of 1 was increased by incorporation of a
tertiary amine HCl salt as in compound 19 and this resulted in increased potency (i.e., IC50 = 11 nM, Table 2). In
addition, compared to 1, the A ring of Region III of the 1,2,3,4,-tetrahydroquinoline nucleus permitted small
substitutions (e.g., 16, IC50 = 11 nM, Table 1) provided that the ring system of the 1,2,3,4-tetrahydroquinoline
remained intact.
13

3.1.1. SAR Summary. In summary, a systematic SAR was developed around analogs of 1 to examine inhibition
of Wnt transcription in a cell-based assay. The structural features for potent inhibition of Wnt transcription
included: 1) a 5-membered aliphatic heterocycle for Region I, 2) a para-substituted arylsulfonamide for Region
II and 3) a 1,2,3,4-tetrahydroquinoline or a 4-methyl-1,2,3,4-tetrahydroquinoxaline ring with small substituents
at the 4- or 6-positions of Region III. We elected to examine highly potent Wnt inhibitors (i.e., 19, 24 and 25) in
further in vitro and in vivo breast cancer studies.
3.2. Effects of sulfonamides on Proliferation of Breast Cancer cell lines.
Dysfunction of the Wnt pathway has been linked to proliferation of several types of cancers including colon26,
pancreatic27 and breast cancer6, 7,24. Two human breast cancer cell lines with a dysfunctional Wnt pathway were
used for cell-based proliferation studies (i.e., MCF-7 and MDA-MB 231)28, 29. MCF-7 breast cancer cells are
estrogen receptor-positive (ER+) and human epidermal growth factor receptor 2-negative (HER2-). In contrast,
MDA-MB 231 breast cancer cells are ER- and HER2-30. MCF-7 cells are one of the most commonly used cell
lines in breast cancer research31 because of its hormone sensitivity due to expression of the estrogen receptor32.
MDA MB-231 cells are an example of a triple negative cell line33 where cells are ER- and progesterone receptor
(PR-) and HER2-. Wnt proteins are known to be expressed in human breast cancer tissue34, 35 and for adult
women, dysregulated Wnt pathways have been associated with breast cancer7. Based on the effect of sulfonamides
on Wnt transcription inhibition described above, a group of 11 sulfonamides ranging from very potent (i.e., IC50
= 11 nM) to less potent (i.e., IC50 > 5000 nM) were tested for their effect on cell proliferation in both MDA-MB
231 and MCF-7 breast cancer cell lines. The results showed sulfonamides with potent inhibition of Wnt
transcription (i.e., 19, 24, 25 with IC50 = 11, 18, 7 nM, respectively), were potent anti-proliferation agents against
the MCF-7 (i.e., IC50 = 10, 7, 4 nM, respectively, Table 5). In good agreement, in MDA-MB 231 cells, potent
inhibition of Wnt transcription was also directly correlated to potent inhibition of proliferation of MDA-MB 231
breast cancer cell lines (i.e., 19, 24, 25 with IC50 = 13, 13, 16 nM, respectively, Table 5).
14

Table 5. Effect of Sulfonamides on Wnt Inhibition and Inhibition of Proliferation of Breast Cancer Cells In Vitro.
IC50 Std. Dev. (nM)a IC50 Std. Dev. (nM)a
Compound
Compound
Wnt
MDA-
Wnt
MDA-
MCF-7
MCF-7
Inhibition MB 231
Inhibition MB 231
25
11
7
3
3
1
2
2
15
13
16
13
17
2
2
2
2
2
17
10
4
2
6
2
20
14
2
2
25
14
1
2
1
12
22
4
1
17
2
19
25
24
11
1
> 5000
> 5000
> 5000
318
> 5000
18
7
2
660
2
608
2
3
15
7
15
1
> 5000
> 5000
36
aIC50 is mean of three determinations Std. Dev. bWnt inhibition described in Methods. cMDA-MB 231 and
MCF-7 data refer to IC50 values for inhibition of proliferation.
Figure 2. Plot of log of IC50 of inhibition of proliferation of MCF-7 breast cancer cells versus the log of the IC50
values for inhibition of Wnt transcription in HEK 293 cells for compounds 1, 4, 11, 12, 15, 19, 22, 24, 25 and 36.
Correlation coefficient, r2 = 0.93.
15

A plot of IC50 values of the compounds in Table 5 for inhibition of MCF-7 cell proliferation versus IC50 values
for inhibition of Wnt transcription (Figure 2) showed a large, positive correlation (i.e., r2 = 0.93). Similarly, a
plot of the IC50 values for inhibition of MDA-MB 231 cell proliferation versus inhibition of Wnt transcription
(Supporting Figure S1) also showed a large, positive correlation (i.e., r2 = 0.97). Thus, for the sulfonamides
examined, the more potent the degree of inhibition of Wnt transcription, the more potent the inhibition of breast
cancer cell proliferation.
3.3. Effect of Sulfonamides on p53 Transcription in HEK-293 cells.
p53 is a unique protein that plays a role in several physiological processes including cell metabolism36, stem cell
maintenance37 and cell adhesion38. In cancer cells, p53 is often inactivated and this results in cancer cells unable
to conduct apoptotic function39, 40. In cancer cells, a decrease in p53 function has been linked to increased Wnt
functional activity41. As a consequence of increased Wnt functional activity and a decrease in p53 in cancer cells,
a decrease in apoptosis occurs. Conversely, activation or stabilization of p53 helps a cancer cell regulate the
physiological processes controlled by p5342 and increases apoptotic function. To test the function of sulfonamides
described herein on p53 transcription, sulfonamides listed in Table 5 were examined for their effects on p53
transcriptional activity using a p53 transcriptional reporter assay43. A plot of IC50 values of the compounds listed
in Table 5 for inhibition of MCF-7 cell proliferation versus the IC50 values for activation of p53 (Supporting
Table S2, Figure S2) showed a large, positive correlation (i.e., r2 = 0.61). This is in good agreement with
inhibition of MCF-7 and MDA-MB 231 cell proliferation described above (Table 5). Western blot analysis of
MCF-7 cell lysates treated with 19 showed a significant increase in total p53 protein with an EC50 = 18 nM
(Supporting Figure S3). This result showed that the sulfonamides described herein that are potent inhibitors of
cancer cell proliferation are also potent activators of the p53 pathway. Also, the data showed that the more potent
inhibitors of Wnt transcription were also more potent activators of p53. That potent Wnt inhibitors and p53
activators (i.e., 19, 24 and 25) also potently induced Caspase-Glo-3/7 (Supporting Table S3) showed that these
compounds promote apoptosis in breast cancer cells.
3.4.
Synergistic effects of 19 and Doxorubicin on the proliferation of MCF-7 and MDA-MB-231 cells.
16

Combination drug treatments have shown promising results as cancer treatments44. One of the most common
chemotherapeutics for treatment of breast cancer is Doxorubicin3 and is usually given in combination with other
chemotherapeutics, including paclitaxel or 5-fluorouracil45. Based on the overall potent anti-proliferative effects
of 19 on MCF-7 and MDA-MB 231 cells (i.e., IC50 = 10 nM and 13 nM, respectively, Table 5), 19 was examined
in the presence of Doxorubicin to inhibit MCF-7 or MDA-MB 231 cell proliferation. The results from these
studies showed 19 and Doxorubicin had a synergistic effect on the inhibition of proliferation of either MCF-7 or
MDA-MB 231 breast cancer cells. Compared to Doxorubicin alone, inhibition of proliferation for Doxorubicin
in the presence of 19 was 2-3-fold greater (p < 0.05) in both MCF-7 and MDA-MB-231 cells (Supporting Figure
S4). In the presence of Doxorubicin, 19 synergized inhibition of proliferation of MCF-7 and MDA-MB-231 cells
and showed a low “combination index” value (CI < 1) by Chou-Talalay synergism analysis46 (Supporting Table
S4). Compared to treatments in MCF-7 cells, drug combination studies done in MDA-MB-231 cells showed
greater synergism (Supporting Table S4, ED90).
3.5.
In vivo effects of 19 in the presence of absence of Doxorubicin on breast cancer xenografts.
Pharmaceutical studies showed 19 was suitable for further studies. For example, viability studies in mammalian
cells showed that 19 did not cause any detectable cytotoxicity at the concentrations examined15. Compound 19
did not cause any detectable inhibition of hERG up to 50 µM. No detectable off-target inhibition of kinases
(CEREP panel) was observed15.
As a prelude to efficacy studies of 19, in vitro and in vivo DMPK studies were conducted15. Incubations with
human or rat liver microsomes (+ NADPH) showed 19 was metabolically stable for > 60 mins as judged by
HPLC. The only minor metabolite observed was the N-demethylation metabolite, 18, that was not potent (Table
2). No apparent inhibition of human cytochrome P-450 (i.e., testosterone hydroxylase) by 19 (50 µM) was
observed in vitro15. Pharmacokinetic studies in rats showed 19 possessed acceptable bioavailability (i.e., 15-
20%)15. Accordingly, in vivo efficacy in xenograft studies with mice were done.
Human breast tumors were implanted subcutaneously (1 million MDA-MB-231 cells) in female nu/nu
mice and assessed by daily caliper measurements. After the tumors were established (on day 35, average size,
17

75.5 12 mm3), test compounds were administered by i.p. injection. The design of the xenograft study was to
compare treatment of mice in four groups (i.e., vehicle, Doxorubicin, 19, and 19 plus Doxorubicin, n = 9) at the
end of 16 days of treatment. Compared to vehicle-treated animals, Doxorubicin (5 mg/kg/week) decreased human
breast cancer tumor volume by 84.3%, 19 (20 mg/kg, 16 consecutive days) decreased breast cancer volume by
91.5% and 19 (20 mg/kg, 16 consecutive days) plus Doxorubicin (5 mg/kg/week) decreased breast cancer volume
96.2%. On day 51, animals treated with 19 plus Doxorubicin decreased tumor volume on average to 24.7 5.9
mm3, a 6.2.-fold decrease in tumor volume compared to the volume of tumor on day 35 (i.e., 154.3 37 mm3) (P
< 0.01). In contrast, vehicle-treated animals grew tumors to an average volume of 653 192 mm3 or a 4.2-fold
increase in tumor volume at day 51 compared to tumor volume of vehicle-treated animals on day 35 (Figure 3B).
Thus, after tumors were established, treatment with 19 and Doxorubicin for 16 days dramatically decreased tumor
volume compared to vehicle-treated mice. Compared to vehicle-treated animals, at the end of the 16-day study,
weights of excised tumors for animals treated with 19 plus Doxorubicin were significantly lower (i.e., 93.7%
lower) (P < 0.01) (Figure 3C).
Figure 3. Effect of Doxorubicin, compound 19 or Doxorubicin plus 19 on MDA-MB-231 xenograft tumor growth
in nu/nu mice. A. The effect of vehicle (solid circle, n=7), 5 mg/kg/week Doxorubicin (Dox) (open square, n=8),
20 mg/kg/day 19 (solid triangle, n=6) or 19 (20 mg/kg/day) plus Doxorubicin (5 mg/kg/week) (open circle, n=5)
on tumor volume of subcutaneous (back) MDA-MB-231 breast cancer xenografts in mice. Treatment was
administered for 16 days starting on day 35 by intraperitoneal injection. B. Tumor volume and C. tumor weights
at day 51 from the same animals as A. White bar, vehicle, n=7; grey bar, Doxorubicin, n=8; black bar, 19, n=6
18

and hatched bar, Doxorubicin plus 19, n=5. Data are mean s.e.m. in A, B and C. The P-value was estimated by
Student’s t-test test in B and C (*P< 0.05, **P< 0.01, ****P< 0.0001).
At the end of the study, serum concentrations of 19 from 19-treated animals were determined to be ~ 0.2
µg/ml by HPLC (see Supporting Information) and confirmed by LCMS. Tumors excised from animals upon
termination of the study (i.e., Day 51) and analyzed for amounts of p53, phospho Ser15-p53 protein and percent
PARP cleavage by Western blot analysis showed, compared to vehicle-treated animals, animals treated with 19
plus Doxorubicin had approximately 1.5- and 3.0-fold greater p53 and phospho Ser15-p53 protein, respectively,
and 30.0-fold greater PARP cleavage (P < 0.05) (Supporting Figure S5).
4.
Conclusion. From a screen of 76,000 compounds for inhibition of Wnt transcription, a selective and potent
“hit” compound 1 was observed. From over 120 medicinal chemical analogues of compound 1, several lead
compounds were developed. For example, compared to 1, compounds 19, 24 or 25 had improved potency for Wnt
inhibition and improved potency of inhibition of breast cancer cell proliferation and increased aqueous solubility.
Compounds 19, 24 or 25 were shown to have potent anti-proliferative effects in two breast cancer cell lines with
dysregulated Wnt signaling. A large positive correlation between inhibition of Wnt transcription and breast cancer
cell proliferation in both MCF-7 and MDA-MB 231 breast cancer cell lines was observed. This indicated that for
the compounds examined, Wnt transcription inhibition and inhibition of breast cancer cell proliferation go hand
in hand. In addition, an increase in p53 transcriptional functional activity also correlated to inhibition of Wnt
transcription and anti-proliferation effects. The results suggested that compounds 19, 24 or 25 inhibit Wnt and
induce apoptosis in breast cancer cell lines. Finally, in an in vitro study, 19 was observed to synergize with breast
cancer chemotherapy drug Doxorubicin used at a relatively low dose to afford improved cancer cell anti-
proliferative effects. In vivo, compared to Doxorubicin alone, 19 and Doxorubicin markedly decreased breast
cancer tumor size and weight in xenografts. It may be that using non-toxic 19 in conjunction with lower doses of
chemotherapeutics could afford a combination treatment with overall lower toxicity. Thus, from this study,
19

compounds 19, 24, and 25 emerged as potent inhibitors of Wnt transcription and anti-breast cancer compounds
with excellent physiochemical properties.
5.
Experimental Section
5.1. General. Reagents, starting materials and solvents were purchased in the highest purity available from
commercial suppliers and used as received. Compounds 26, 31, 41, 43 and 44 were commercially available
from Sigma-Aldrich (St. Louis, MO). Chemical names were created with ChemDraw Ultra version 11
(PerkinElmer, Waltham, MA). Microwave reactions were conducted using a Biotage Initiator microwave
synthesizer (Biotage, Uppsala, Sweden). Microwave reactions were run under ambient pressure and variable
power to reach the indicated temperature. Reaction products were purified, when necessary, using an Isco
Combiflash Rf flash chromatography system (Teledyne-Isco, Lincoln, NE) with the solvent systems indicated.
Nuclear magnetic resonance (NMR) data were recorded on a Varian Mercury 300 MHz Spectrometer (Agilent,
Santa Clara, CA) using TMS as an internal standard and CDCl3 as a solvent except where indicated.
Electrospray ionization (ESI) mass spectral data was obtained using an Agilent 1100 LC/MS (Agilent, Santa
Clara, CA). Final test compounds had a purity greater than 95% on the basis LCMS analysis using UV-Vis
detection at 275 nM and 220 nM. Compounds were prepared as hydrochloride salts unless otherwise noted.
Hydrochloride salts were prepared by dissolution of the appropriate compound in a minimum amount of
dichloromethane and addition of excess 2 M HCl in ether. The solvent was evaporated and hydrochlorides were
used directly for evaluation. Aqueous solubilities were determined by preparing a DMSO stock solution of the
desired compound at 10 mM and then diluting the stock solution in distilled water to the desired final
concentration or until turbidity was observed. Dulbecco's Modified Eagle's cell culture medium (DMEM) was
purchased from Lonza (Allendale, NJ) and fetal bovine serum (FBS) was purchased from Atlanta Biologicals
(Flowery Branch, GA). The reagents used in the transfection assay, OptiMEM and lipofectamine 2000, as well
as the phosphate buffered saline (PBS) and SYBR Green used in cell proliferation assays were purchased from
Life Technologies (Carlsbad, CA). The Dual Luciferase Assay Kit to measure the Wnt and p53 transcriptional
activity and the Caspase-Glo 3/7 assay kit to measure the cell apoptosis were purchased from Promega
20

(Fitchburg, WI). Fluorescence was determined using a Tecan SPECTRAFluor Plus plate reader (Tecan, San
Jose, CA). Luminescence was recorded on a Wallac Victor plate reader (PerkinElmer Inc., Waltham, MA).
4-(2-Oxopyrrolidin-1-yl)benzene-1-sulfonyl chloride, 3. Two g of phenylpyrrolidinone was added to 20 mL of
chlorosulfonic acid. The mixture was stirred overnight at room temperature. The mixture was stopped by slow
addition to ice. The aqueous mixture was extracted with 4 volumes of dichloromethane. The organic layers were
combined, dried over Na2SO4 and concentrated in vacuo to give an off white solid. The sulfonyl chloride was
used without further purification, (88 % yield). 1H NMR (CDCl3): 2.19 – 2.30 (m, 2H), 2.69 (t, J = 8.0 Hz, 2H),
3.93 (t, J = 7.2 Hz, 2H), 7.91 (d, J = 9.4 Hz, 2H), 8.01 (d, J = 7.4 Hz, 2H).
General synthesis of pyrrolidinone sulfonamides. To a microwave vial was added 30 mg of the sulfonyl chloride,
1.5 eq. of the appropriate amine, 1.2 eq. of triethylamine, and 300 µL of acetonitrile. The vial was sealed and
heated at 130 °C for 30 min. The mixture was diluted with dichloromethane and the organic phase was washed
2 times with water and brine. The organic layer was dried over Na2SO4 and concentrated in vacuo. Crude
sulfonamide products were purified either by recrystallization from ethanol or by flash chromatography 0 – 75 %
EtOAc/Hexanes.
1-(4-(3,4-Dihydroquinolin-1(2H)-ylsulfonyl)phenyl)pyrrolidin-2-one, 1. ESI/MS: calculated C19H20N2O3S m/z =
1
356.4, found m/z = 357.0 [M+1]. H NMR (CDCl3): 1.60-1.70 (m, 2H), 2.12-2.22 (m, 2H), 2.44 (t, J = 6.6 Hz,
2H), 2.63 (t, J = 6.6 Hz, 2H), 3.78-3.86 (m, 4H), 6.97-7.19 (m, 2H), 7.54 (d, J = 9.1 Hz, 2H), 7.69 (d, J = 9.1 Hz,
2H), 7.77 (d, J = 8.8 Hz, 2H).
1-(4-(6-Methyl-3,4-dihydroquinolin-1(2H)-ylsulfonyl)phenyl)pyrrolidin-2-one,
4.
ESI/MS:
calculated
C20H22N2O3S m/z = 370.1, found m/z = 371.0 [M+1]. 1H NMR (CDCl3): 1.56-1.65 (m, 2H), 2.14-2.21 (m, 2H),
2.29 (s, 3H), 2.39 (t, J = 6.6 Hz, 2H), 2.64 (t, J = 6.6 Hz, 2H), 3.76-3.88 (m, 4H), 6.79 (bs, 1H), 6.98 (d, J = 9.1
Hz, 1H), 7.55 (d, J = 9.1 Hz, 2H), 7.65-7.72 (m, 4H).
1-(4-(Octahydroquinolin-1(2H)-ylsulfonyl)phenyl)pyrrolidin-2-one, 5. ESI/MS: calculated C19H26N2O3S m/z =
362.2, found m/z = 363.0 [M+1]. 1H NMR (CDCl3): 0.87-1.77 (m, 14H), 2.14-2.24 (m, 2H), 2.37-2.49 (m, 1H),
2.65 (t, J = 6.6 Hz, 2H), 3.89 (t, J = 6.6 Hz, 2H), 4.04-4.11 (m, 1H), 7.72-7.82 (m, 4H).
21

1-(4-(Piperidin-1-ylsulfonyl)phenyl)pyrrolidin-2-one, 6. ESI/MS: calculated C15H20N2O3S m/z = 308.1, found m/z
= 309.0 [M+1]. 1H NMR (CDCl3): 1.36-1.44 (m, 2H), 1.60-1.67 (m, 4H), 2.16-2.26 (m, 2H), 2.66 (t, J = 6.6 Hz,
2H), 2.96 (t, J = 6.6 Hz, 2H), 3.90 (t, J = 6.6 Hz, 2H), 7.70-7.82 (m, 4H).
4-(2-Oxopyrrolidin-1-yl)-N-phenyl-N-propylbenzenesulfonamide, 7. ESI/MS: calculated C19H22N2O3S m/z =
358.1, found m/z = 359.0 [M+1]. 1H NMR (CDCl3): 0.9 (t, J = 7.4 Hz, 3H), 1.38 – 1.48 (m, 2H), 2.16 - 2.27 (m,
2H), 2.67 (t, J = 8.4 Hz 2H), 3.47 – 3.52 (m, 2H), 3.86 (t, J = 6.9 Hz, 2 H), 7.02 – 7.06 (m, 2H), 7.28 – 7.32 (m,
2H), 7.56 (d, J = 9.0 Hz, 2 H), 7.74 (d, J = 9.0 Hz, 2 H).
N-(Naphthalen-1-yl)-4-(2-oxopyrrolidin-1-yl)benzenesulfonamide, 8. ESI/MS: calculated C20H18N2O3S m/z =
366.1, found m/z = 367.0 [M+1].1H NMR (CDCl3): 2.10-2.21 (m, 2H), 2.62 (t, J = 6.6 Hz, 2H), 3.80 (t, J = 6.6
Hz, 2H), 7.08 (bs, NH), 7.32-7.47 (m, 3H), 7.63-7.92 (m, 8H).
1-(4-(1,2,3,4-Tetrahydroquinoline-1-carbonyl)phenyl)pyrrolidin-2-one, 9. ESI/MS: calculated C20H20N2O2 m/z =
320.2, found m/z = 321.1 [M+1]. 1H NMR (CDCl3) 2.07 (quintet, J = 6.3 Hz, 2H),2.19 (quintet, J = 7.7 Hz, 2H),
2.64 (t, J = 8.3 Hz, 2H), 2.86 (t, J = 6.3 Hz, 2H), 3.86 (t, J = 6.8 Hz, 2H), 3.92 (t, J = 6.3 Hz, 2H), 6.71 (m, 1H),
6.88 (m, 1H), 7.00 (m, 1H), 7.16 (m, 1H), 7.39 (m, 2H), 7.58 m, 2H).
1-(4-(3,4-Dihydroisoquinolin-2(1H)-ylsulfonyl)phenyl)pyrrolidin-2-one, 11. ESI/MS: calculated C19H20N2O3S
m/z = 356.1, found m/z = 357.0 [M+1]. 1H NMR (CDCl3): 2.16 – 2.27 (m, 2H), 2.66 (t, J = 8.0 Hz 2H), 2.90 (t, J
= 6.0 Hz 2H), 3.36 (t, J = 6.0 Hz 2H), 3.90 (t, J = 7.0 Hz 2H), 4.25 (s, 2 H), 7.00 – 7.15 (m, 4H) 7.82 (s, 4H).
1-(4-(2-Methyl-3,4-dihydroquinolin-1(2H)-ylsulfonyl)phenyl)pyrrolidin-2-one,
12.
ESI/MS:
calculated
C20H22N2O3S m/z = 370.1, found m/z = 371.0 [M+1]. 1H NMR (CDCl3): 1.3 (d, J = 6.6 Hz, 3 H), 1.26 – 1.40 (m,
2H), 1.77 – 1.88 (m, 2H), 2.13 – 2.23 (m, 2H), 2.35 – 2.45 (m, 2H), 2.64 (t, J = 8.3 Hz 2H), 3.82 – 3.87 (m, 2 H),
4.32 – 4.42 (m, 1 H), 6.95 – 6.97 (m, 1H), 7.07 – 7.12 (m, 1H), 7.19 – 7.25 (m, 1H), 7.46 (d, J = 9.0 Hz, 2 H),
7.66 (d, J = 9.0 Hz, 2 H), 7.72 – 7.77 (m, 1H).
1-(4-(6-Chloro-3,4-dihydroquinolin-1(2H)-ylsulfonyl)phenyl)pyrrolidin-2-one,
13.
ESI/MS:
calculated
C19H19ClN2O3S m/z = 390.1, found m/z = 391.0 [M+1]. 1H NMR (CDCl3): 1.51 – 1.66 (m, 2H), 2.15 – 2.25 (m,
22

2H), 2.43 (t, J = 6.1 Hz 2H), 3.77 – 3.81 (m, 2H), 3.86 (t, J = 8.3 Hz 2H), 3.82 – 3.87 (m, 2 H), 6.97 – 6.99 (m,
1H), 7.12 – 7.16 (m, 1H), 7.56 (d, J = 9.0 Hz, 2 H), 7.73 (d, J = 9.0 Hz, 2 H), 7.74 – 7.77 (m, 1H).
1-(4-(6-Methoxy-3,4-dihydroquinolin-1(2H)-ylsulfonyl)phenyl)pyrrolidin-2-one, 14. ESI/MS: calculated
1
C20H22N2O4S m/z = 386.1, found m/z = 387.0 [M+1]. H NMR (CDCl3): 1.53 – 1.62 (m, 2H), 2.14 – 2.25 (m,
2H), 2.35 (t, J = 6.9 Hz 2H), 2.64 (t, J = 8.5 Hz 2H), 3.75 – 3.78 (m, 2H), 3.78 (s, 3 H), 3.86 (t, J = 7.2 Hz 2H),
6.50 – 6.51 (m, 1H), 6.73 – 6.77 (m, 1H), 7.52 (d, J = 9.1 Hz, 2 H), 7.67 – 7.72 (m, 3H).
1-(4-(6-(Trifluoromethyl)-3,4-dihydroquinolin-1(2H)-ylsulfonyl)phenyl)pyrrolidin-2-one,
15.
ESI/MS:
calculated C20H19F3N2O3S m/z = 424.1, found m/z = 425.0 [M+1]. 1H NMR (CDCl3): 1.67 – 1.75 (m, 2H), 2.13
– 2.25 (m, 2H), 2.56 – 2.67 (m, 4H), 3.83 – 3.89 (m, 4H), 7.38 – 7.42 (m, 1H), 7.61 (d, J = 8.8 Hz, 2H), 7.75 (d,
J = 8.8 Hz, 2 H), 7.90 – 7.93 (m, 1H), 8.00 – 8.04 (m, 1H).
Racemic 1-(4-(6-tert-Butyl-3,4-dihydroquinolin-1(2H)-ylsulfonyl)phenyl)pyrrolidin-2-one, 16. ESI/MS:
calculated C23H28N2O3S m/z = 412.2, found m/z= 413.0 [M+1]. 1H NMR (CDCl3): 1.30 (s, 9H), 1.59 – 1.68 (m,
2H), 2.14 – 2.24 (m, 2H), 2.48 (t, J = 6.6, 2H), 2.64 (t, J = 8.3, 2H), 3.77 – 3.80 (m, 2H), 3.86 (t, J = 6.9, 2H),
6.98 (d, J = 2.5 Hz, 1H), 7.19 (dd, J = 2.5 Hz and 8.8 Hz, 1H), 7.59 (d, J = 9.1 Hz, 2H), 7.67 (d, J = 8.8 Hz, 1 H),
7.70 (d, J = 9.1 Hz, 2 H).
1-(4-(2H-Benzo[b][1,4]oxazin-4(3H)-ylsulfonyl)phenyl)pyrrolidin-2-one, 17. ESI/MS: calculated C18H18N2O4S
m/z = 357.1, found m/z = 358.0 [M+1]. 1H NMR (CDCl3): 2.16 – 2.21 (m, 2 H), 2.64 (t, J = 8.3 Hz, 2H), 2.93 –
2.96 (m, 2H), 3.79 – 3.87 (m, 4H), 6.43 – 6.46 (m, 1H), 6.66 – 6.71 (m, 1H), 6.94 – 6.99 (m, 1H), 7.54 (d, J = 8.8
Hz, 2H), 7.64 – 7.68 (m, 1 H), 7.70 (d, J = 9.0 Hz, 2 H).
1-(4-(4-Methyl-3,4-dihydroquinoxalin-1(2H)-ylsulfonyl)phenyl)pyrrolidin-2-one, 18. ESI/MS: calculated
C19H21N3O3S m/z = 371.1, found m/z = 372.0 [M+1]. 1H NMR (CDCl3): 2.14 – 2.24 (m, 2 H), 2.64 (t, J = 8.5 Hz,
2H), 2.66 (s, 3H), 2.88 (t, J = 5.5 Hz, 2H), 3.83 – 3.89 (m, 4H), 6.55 – 6.58 (m, 1H), 6.65 – 6.71 (m, 1H), 6.55 –
6.58 (m, 1H), 7.50 (d, J = 9.0 Hz, 2H), 7.59 – 7.62 (m, 1 H), 7.69 (d, J = 9.0 Hz, 2 H).
1-(4-(4-Methyl-3,4-dihydroquinoxalin-1(2H)-ylsulfonyl)phenyl)pyrrolidin-2-one, 19. ESI/MS: calculated
C19H21N3O3S m/z = 371.1, found m/z = 372.0 [M+1]. 1H NMR (CDCl3): 2.14 – 2.24 (m, 2 H), 2.64 (t, J = 8.5 Hz,
23

2H), 2.66 (s, 3H), 2.88 (t, J = 5.5 Hz, 2H), 3.83 – 3.89 (m, 4H), 6.55 – 6.58 (m, 1H), 6.65 – 6.71 (m, 1H), 6.55 –
6.58 (m, 1H), 7.50 (d, J = 9.0 Hz, 2H), 7.59 – 7.62 (m, 1 H), 7.69 (d, J = 9.0 Hz, 2 H).
1-(4-(4-(2-Hydroxyethyl)-3,4-dihydroquinoxalin-1(2H)-ylsulfonyl)phenyl)pyrrolidin-2-one,
20.
ESI/MS:
calculated C20H23N3O4S m/z = 401.1, found m/z = 402.0 [M+1]. 1H NMR (CDCl3): 2.15 – 2.25 (m, 2H), 2.63 (t,
J = 8.3, 2H), 3.00 (t, J = 5.1, 2H), 3.26 (t, J = 5.1, 2H), 3.46 – 3.50 (m, 2H), 3.80 – 3.88 (m, 4H), 6.58 (d, J = 8.2,
1H), 6.68 (t, J = 7.7, 1H), 7.05 – 7.10 (m, 1H), 7.44 – 7.51 (m, 4H), 7.62 (d, J = 8.0 Hz, 1H).
1-(4-(3,4-Dihydro-1,5-naphthyridin-1(2H)-ylsulfonyl)phenyl)pyrrolidin-2-one,
21.
ESI/MS:
calculated
1
C18H19N3O3S m/z = 357.1, found m/z = 358.0 [M+1]. H NMR (CDCl3): 1.65 – 1.74 (m, 2H), 2.15 – 2.24 (m,
2H), 2.62 – 2.73 (m, 4H), 3.79 – 3.88 (m, 4H), 7.15 (dd, J = 4.7 Hz and 8.3 Hz, 1H), 7.57 (d, J = 9.4 Hz,2H), 7.74
(d, J = 9.1 Hz, 2H), 8.16 (dd, J = 1.4 Hz and 8.3 Hz, 1H), 8.30 (dd, J = 1.4 Hz and 4.7 Hz, 1H).
1-(4-(6-Methoxy-4-methyl-3,4-dihydroquinoxalin-1(2H)-ylsulfonyl)phenyl)pyrrolidin-2-one,
22.
ESI/MS:
calculated C20H23N3O4S m/z = 401.1 found m/z = 402.0 [M+1]. 1H NMR (CDCl3): 2.14 – 2.25 (m, 2H), 2.62 (s,
3H), 2.63 – 2.67 (m, 2H), 2.81 – 2.85 (m, 2H), 3.80 (s, 3H), 3.83 – 3.89 (m, 4H), 6.13 (d, J = 2.8 Hz, 1H), 6.30
(dd, J = 2.8 Hz and 8.8 Hz, 1H), 7.48 (d, J = 9.4 Hz, 2H), 7.52 (d, J = 8.8 Hz, 1H), 7.69 (d, J = 9.1 Hz, 2H).
Racemic 1-(4-(2,4-Dimethyl-3,4-dihydroquinoxalin-1(2H)-ylsulfonyl)phenyl)pyrrolidin-2-one, 23. ESI/MS:
calculated C20H23N3O3S m/z = 385.2 found m/z = 386.0 [M+1]. 1H NMR (CDCl3): 1.15 (d, J = 6.9 Hz, 3H), 2.12
– 2.23 (m, 2H), 2.63 – 2.65 (m, 2H), 2.68 (s, 3H), 2.69 – 2.75 (m, 2H), 3.83 (t, J = 7.2 Hz, 2H), 4.41 – 4.51 (m,
1H), 6.56 (d, J = 8.3 Hz, 1H), 6.67 – 6.73 (m, 1H), 7.05 – 7.11 (m, 1H), 7.43 (d, J = 8.9 Hz, 2H), 7.63 – 7.67 (m,
1H), 7.66 (d, J = 8.9 Hz, 2H).
1-(3-(3,4-Dihydroquinolin-1(2H)-ylsulfonyl)phenyl)pyrrolidin-2-one, 30. ESI/MS: calculated C19H20N2O3S m/z
= 356.1 found m/z = 357.0 [M+1]. 1H NMR (CDCl3): 1.64 – 1.72 (m, 2H), 2.09 – 2.19 (m, 2H), 2.44 (t, J = 6.9,
2H), 2.60 (t, J = 8.0, 2H), 3.65 (t, J = 6.9, 2H), 3.81 – 3.85 (m, 2H), 6.98 – 7.09 (m, 2H), 7.16 – 7.21 (m, 1H),
7.34 – 7.42 (m, 2H), 7.49 (s, 1H), 7.78 (d, J = 8.3 Hz, 1H), 8.11 – 8.14 (m, 1H).
1-(4-(3,4-Dihydroquinolin-1(2H)-ylsulfonyl)-2-methylphenyl)pyrrolidin-2-one,
34.
ESI/MS:
calculated
C20H22N2O3S m/z = 370.1 found m/z = 371.0 [M+1]. 1H NMR (CDCl3): 1.64 – 1.69 (m, 2H), 2.18 – 2.23 (m, 2H),
24

2.26 (s, 3H), 2.45 (t, J = 6.6 Hz, 2H), 2.52 (t, J = 8.2 Hz, 2H), 3.61 (t, J = 7.1 Hz, 2H), 3.77 – 3.79 (m, 2H), 7.00
– 7.02 (m, 1H), 7.07 – 7.10 (m, 1H), 7.18 – 7.21 (m, 1H), 7.28 (d, J = 8.3 Hz, 1H), 7.36 – 7.40 (m, 2H), 7.78 (d,
J = 8.2, 1H).
1-Phenyl-1H-1,2,4-triazole, 35. To a 15 mL microwave vial 2 g (13.8 mmol) of phenyl hydrazine hydrochloride
was added to 10 mL of formamide. The vial was sealed and heated at 175°C for 1.5 h in a microwave reactor.
The mixture was poured into water and extracted 3 times with ethyl acetate. The organic layers were combined
and washed 3 times with water and once with brine. The organic phase was dried over sodium sulfate and
concentrated in vacuo. The crude material was purified by flash chromatography 0 – 50 % EtOAc/Hexanes and
a yellow oil was isolated that solidified upon standing, 1.2 g. ESI/MS: calculated C8H7N m/z = 145.1 found m/z
= 146.0 [M+1]. 1H NMR (CDCl3): 7.37 – 7.42 (m, 1H), 7.47 – 7.53 (m, 2H), 7.66 (d, J = 8.3 Hz, 2H), 8.10 (s,
1H), 8.54 (s, 1H).
1-(4-(1H-1,2,4-Triazol-1-yl)phenylsulfonyl)-1,2,3,4-tetrahydroquinoline, 36. To 10 mL of chlorosulfonic acid at
0 °C was added 1 g (6.9 mmol) of 1-phenyl-1H-1,2,4-triazole, 35. The mixture was stirred overnight and then
cooled to 0 °C. The solution was slowly added to ice and extracted 3 three times with dichloromethane. The
organic layers were combined, dried over sodium sulfate and concentrated to dryness in vacuo to give 1 g of a
tan solid. The crude sulfonyl chloride was used directly without further purification. To a microwave vial was
added 25 mg (0.095 mmol) of the sulfonyl chloride, 15 mg (0.11 mmol) of 1,2,3,4-tetrahydroquinoline, 14 mg
(0.014 mmol) of triethylamine and 300 µL of acetonitrile. The vial was sealed and heated at 130 °C for 30 min.
The mixture was diluted with dichloromethane and the organic phase was washed 2 times with water and brine.
The organic layer was dried over sodium sulfate and concentrated in vacuo. The crude sulfonamide was purified
by flash chromatography 0 – 75 % EtOAc/Hexanes. ESI/MS: calculated C17H16N4O2S m/z = 340.1 found m/z =
341.0 [M+1]. 1H NMR (CDCl3): 1.63 – 1.71 (m, 2H), 2.43 – 2.48 (m, 2H), 3.83 – 3.87 (m, 2H), 6.99 – 7.01 (m,
1H), 7.07 – 7.12 (m, 1H), 7.18 – 7.23 (m, 1H), 7.69 – 7.81 (m, 5H), 8.11 (s, 1H), 8.60 (1H).
1-Phenyl-4,5-dihydro-1H-pyrazole, 37. 1.4 g (10 mmol) of phenylhydrazine hydrochloride, 1 mL (10 mmol) 1,3-
dibromopropane, 1.4 g (10 mmol) potassium carbonate and 10 mL water were added to a 15 mL microwave vial
25

and heated at 130 °C for 30 min. After cooling, the mixture was extracted 4 times with ethyl acetate. The organic
layers were combined, dried over sodium sulfate and concentrated in vacuo to give a yellow residue. The crude
mixture was purified by flash chromatography 0 – 25 % EtOAc/Hexanes and 300 mg of a yellow solid was
isolated. ESI/MS: calculated C9H10N2 m/z = 146.1 found m/z = 147.0 [M+1]. 1H NMR (CDCl3): 2.93 (dt, J = 10.5
Hz and 1.7 Hz, 2H), 3.67 (t, J = 10.5 Hz, 2H), 6.82 – 6.88 (m, 2H), 7.03 (d, J = 7.7 Hz, 2H), 7.24 – 7.31 (m, 2H).
1-(4-(4,5-Dihydro-1H-pyrazol-1-yl)phenylsulfonyl)-1,2,3,4-tetrahydroquinoline, 38. To 1.8 mL (27 mmol) of
chlorosulfonic acid at 0 °C was added 200 mg (1.4 mmol) of 37. The mixture was stirred overnight and then
cooled to 0 °C. The solution was slowly added to ice and extracted 3 three times with dichloromethane. The
organic layers were combined, dried over sodium sulfate and concentrated to dryness in vacuo to give 150 mg of
an orange solid. The sulfonyl chloride was used without any further purification. To a microwave vial was added
50 mg (0.20 mmol) of the sulfonyl chloride, 30 mg (0.25 mmol) of 1,2,3,4-tetrahydroquinoline, 30 mg (0.3 mmol)
of triethylamine and 300 µL of acetonitrile. The vial was sealed and heated at 130 °C for 30 min. After cooling,
the mixture was diluted with dichloromethane and the organic layer was washed 2 times with water and brine.
The organic layer was dried over sodium sulfate and concentrated in vacuo. The crude sulfonamide was purified
by flash chromatography 0 – 75 % EtOAc/Hexanes to give 30 mg of a yellow solid. ESI/MS: calculated
C17H16N4O2S m/z = 341.1 found m/z = 342.0 [M+1]. 1H NMR (CDCl3): 1.62 – 1.68 (m, 2H), 2.45 – 2.48 (m, 2H),
2.97 – 3.02 (m, 2H), 3.66 – 3.71 (m, 2H), 3.78 – 3.80 (m, 2H), 6.90 – 6.92 (m, 3H), 6.98 – 6.99 (m, 1H), 7.03 –
7.06 (m, 1H), 7.16 – 7.19 (m, 1H), 7.46 (d, J = 9.3 Hz, 2H), 7.80 (d, J = 8.2 Hz, 1H).
1-(4-(Pyrrolidin-1-yl)phenylsulfonyl)-1,2,3,4-tetrahydroquinoline, 39. To a microwave vial was added 5 mg
(0.028 mmol) of zinc acetate. The vial was sealed and purged / filled three times with vacuum then argon. A
solution of 100 mg (0.28 mmol) of 1 and 137 mg (0.84 mmol) of triethoxysilane in 750 µL of tetrahydrofuran
was added. The mixture was heated at 80 °C for 15 h. To the mixture was added 1 M sodium hydroxide and
stirred at ambient temperature for 2 h. The aqueous fraction was extracted 3 times with ethyl acetate, the organic
phase dried over sodium sulfate and concentrated. The crude mixture was purified by flash chromatography, 0 –
15 % EtOAc/Hexanes to give 70 mg of a white solid. ESI/MS: calculated C19H22N2O2S m/z = 342.1 found m/z =
26

343.0 [M+1]. 1H NMR (CDCl3): 1.61 – 1.70 (m, 2H), 2.00 – 2.05 (m, 4H), 2.47 – 2.51 (m, 2H), 3.27 – 3.42 (m,
4H), 3.76 – 3.80 (m, 2H), 6.41 (d, J = 9.1 Hz, 2H), 6.95 – 7.06 (m, 2H), 7.13 – 7.19 (m, 1H), 7.39 (d, J = 9.1 Hz,
2H), 7.80 (d, J = 8.3 Hz, 1H).
1-(4-(1H-pyrazol-1-yl)phenylsulfonyl)-1,2,3,4-tetrahydroquinoline, 42. ESI/MS: calculated C18H17N3O2S m/z =
1
339.1 found m/z = 340.0 [M+1]. H NMR (CDCl3): 1.62 – 1.71 (m, 2H), 2.45 (t, J = 6.6, 2H), 3.82 – 3.86 (m,
2H), 6.50 – 6.51 (m, 1H), 6.99 (d, J = 7.4 Hz, 1H), 7.09 (t, J = 7.4 Hz, 1H), 7.20 (t, J = 7.4 Hz, 1H), 7.65 (d, J =
8.8 Hz, 2H), 7.73 (d, J = 8.8 Hz, 2H), 7.73 – 7.74 (m, 1H), 7.81 (d, J = 8.3, 1H), 7.94 (d, J = 2.8, 1H).
5.2. Biology. Effect of Sulfonamides on Wnt Transcriptional Activity. A Super(8x)TOPflash (STF) and a
Wnt3A-expression vector to activate the Wnt pathway were co-transfected with a TK-driven Renilla luciferase
plasmid (Rluc) (for transfection efficiency normalization) into HEK-293 cells (culture media: DMEM, 10% FBS).
After incubation at 37° C, 5% CO2 for 8 h, cells were counted and seeded on a 96-well plate at a density of 25,000
cells per well with 135 µL media. The cells were treated with sulfonamides at a final concentration of 1.6, 8, 40,
200, 1000, 5000 nM (1.6 nM to 5 µM) or DMSO (vehicle control) for 20-22 h. Each condition was run in triplicate.
After administration of each compound, media was thoroughly removed and 50 µL of passive cell lysis buffer
from a Dual Luciferase Assay Kit (Promega) was added. Luminescent signal was measured sequentially by adding
Firefly and Renilla luciferase substrate that was proportional to luciferases activity. Data was processed by
normalizing (dividing) the Firefly luciferase signal by the Renilla luciferase signal. The average and standard
deviation of relative Firefly luciferase luminescent signal for compound-treated cells (relative to DMSO treated
cells) for each concentration (sample ran in triplicate) was calculated and then analyzed by nonlinear regression
(GraphPad Prism, San Diego, CA) for IC50 determination of Wnt transcriptional activity.
5.2.1. Effect of Sulfonamides on p53 Transcriptional Activity. HEK-293 cells were transiently transfected with a
p53-Luciferase (p53-Luc) vector driven by a (13x) p53-binding consensus-firefly luciferase response element,
together with a TK-driven Renilla luciferase plasmid as an internal control to normalize the luminescence signal
for transfection efficiency13. The detailed protocol was similar to the Wnt transcriptional assay but the final serum
27

concentration was kept at 2.5% to minimize basal reporter activity of p53-Luc. Each condition was run in
triplicate. The transfected cells were treated with sulfonamides or DMSO for 20-22 h. Each condition was done
in triplicate. After administration of each compound, media was thoroughly removed and 50 µL of passive cell
lysis buffer in the Dual Luciferase Assay Kit (Promega) was added. Luminescent signal was measured
sequentially by adding Firefly and Renilla luciferase substrate reagent. Data was processed by normalizing
(dividing) the Firefly luciferase signal by the Renilla luciferase signal. The average and standard deviation of
relative Firefly luciferase luminescent signal for compound-treated cells (relative to DMSO-treated cells) for each
concentration (sample ran in triplicate) was calculated and then analyzed by nonlinear regression (GraphPad
Prism) for IC50 determination of p53 transcriptional activity.
5.2.2. Effect of sulfonamides on breast cancer cell proliferation. Cells were plated at a density of 500 cells per
well in eight 96-well plates with DMEM and 10% FBS culture media. After cells were attached to the plate (i.e.,
24 h at 37 °C 5% CO2), compounds to be tested were added to cells that afforded a concentration of 1.6 nM to 5
µM or DMSO (vehicle control). Seven plates were incubated at 37° C, 5% CO2 and every 24 h one plate was
harvested and stored at -20 °C for 7 days. Following harvest of all the plates (i.e., for 7 days), SYBR Green (1X
stock by diluting 10,000 X original stock in PBS) (Life Technologies) was added to the cell lysate. Cellular DNA
was detected by measuring the fluorescence (Ex495 nm, Em535 nm). The average standard deviation of relative
fluorescence for compound treated cells (relative to DMSO treated cells) for each concentration (sample ran in
triplicate) was calculated and analyzed by a nonlinear regression (GraphPad Prism) for each IC50 determination.
5.2.3. Effect of sulfonamides on cell apoptosis. Cells were plated at a density of 25,000 cells per well in 96-well
plates (culture media: DMEM, 10% FBS). Following 24 h incubation after plating, the cells were treated with
sulfonamides at a final concentration of 1.6 nM to 5 µM or DMSO (vehicle control) for 24 h. At the end of 24 h,
apoptosis was measured using a Caspase-Glo 3/7 reaction system (Promega) monitoring the activity of Caspase
3/7 according to the manufacturer’s protocol. Luminescent signal was measured by adding Caspase-Glo 3/7
reagent to each well. The average and standard deviation of relative luminescence for compound-treated cells
28

(relative to DMSO treated cells) for each concentration (sample ran in triplicate) was calculated and then analyzed
by a nonlinear regression (GraphPad Prism) for IC50 determination.
5.2.4. Synergistic effects of 19 and Doxorubicin on proliferation of MCF-7 and MDA-MB-231 cells. MCF-7 and
MDA-MB-231 cells (ATCC, Manassas, Virginia) were plated at a density of 2,000 cells per well in 96-well plates
(culture media: DMEM, 10% FBS) and incubated for 24 h at 37 °C 5% CO2. After 24 h, 19 was added to cells
that afforded a concentration of 1.6 nM to 2 µM in the presence or absence of Doxorubicin (Sigma Aldrich, St.
Louis, MO) (1.6 nM to 2 µM in MCF-7 cells and 4 nM to 5 µM in MDA-MB-231 cells) or DMSO as vehicle
control and incubated for 72 h at 37 °C 5% CO2. After 72 h, SYBR Green (1X stock by diluting 10,000 X original
stock in PBS) (Life Technologies) was added to the cell lysate. Cellular DNA was detected by measuring the
fluorescence (Ex495 nm, Em535 nm). The average and standard deviation of relative fluorescence for compound-
treated cells (relative to DMSO-treated cells) for each concentration (samples run in triplicate) was calculated and
analyzed by nonlinear regression (GraphPad Prism) for each IC50 determination and Chou-Talalay analysis46.
6. Supporting Information Available: Synthesis and 1H NMR of compounds 27 – 29, 32, 33 and synthesis and
1H NMR of 1,2,3,4-tetrahydroquinoxalines used for synthesis of 22 and 23 are provided. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*JCashman@hbri.org
Notes
The authors declare no competing financial interest.
Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to the final
version of the manuscript.
7.
ACKNOWLEDGMENT
29