Synthesis and preliminary assessment of the anticancer and Wnt/β-catenin inhibitory activity of small amide libraries of fenamates and profens

Article abstract of DOI:10.1007/s00044-017-2001-z

As part of an ongoing program to study the anticancer activity of non-steroidal anti-inflammatory drugs (NSAIDs) through generating diversity libraries of multiple NSAID scaffolds, we synthesized a series of NSAID amide derivatives and screened these sets against three cancer cell lines (prostate, colon and breast) and Wnt/β-catenin signaling. The evaluated amide analog libraries show significant anticancer activity/cell proliferation inhibition, and specific members of the sets show inhibition of Wnt/β-catenin signaling.

Full text of DOI:10.1007/s00044-017-2001-z

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-2001-z
ORIGINAL RESEARCH
Synthesis and preliminary assessment of the anticancer and
Wnt/β-catenin inhibitory activity of small amide libraries of
fenamates and profens
Bini Mathew1 Judith V. Hobrath2 Wenyan Lu1 Yonghe Li1
●
●
●
●
3
Robert C. Reynolds
Received: 9 May 2017 / Accepted: 20 July 2017
© The Author(s) 2017. This article is an open access publication
Abstract As part of an ongoing program to study the
anticancer activity of non-steroidal anti-inflammatory drugs
(NSAIDs) through generating diversity libraries of multiple
NSAID scaffolds, we synthesized a series of NSAID amide
derivatives and screened these sets against three cancer cell
lines (prostate, colon and breast) and Wnt/β-catenin sig-
naling. The evaluated amide analog libraries show sig-
nificant anticancer activity/cell proliferation inhibition, and
specific members of the sets show inhibition of Wnt/β-
catenin signaling.
Introduction
Non-steroidal anti-inflammatory drugs (NSAIDs) are the
most widely used class of drugs for the treatment of pain
and inflammation. The anti-inflammatory mechanism of the
NSAIDs is attributed to the inhibition of the cycloox-
ygenases (COXs) and reducing the synthesis of pros-
taglandin signaling molecules (Vane 1971). There are two
major isoforms of the COXs, COX-1 and COX-2. COX-1 is
constitutively expressed in most tissues and plays an
important role in tissue homeostasis, while COX-2 is
induced as part of the acute inflammatory pathway. Epi-
demiological, preclinical and clinical studies have demon-
strated the chemopreventive efficacy of NSAIDs by
reducing cancer incidence in the general population by up to
50% (Thun et al. 2002; Chan 2002; Reeder et al. 2004; Soh
and Weinstein 2003). The depletion of physiologically
important prostaglandins through chronic COX (COX-1 or
COX-2) inhibition, however, can have significant life
threatening side effects including gastrointestinal, renal, and
cardiovascular toxicity (Cannon and Cannon 2012; Yu et al.
2012; Mukherjee 2002; Vane and Botting 1998; Vane et al.
1998). Unfortunately, these side effects limit the utility of
NSAIDs for cancer chemoprevention, which tends to
require high dosages and chronic treatment. NSAIDs are
most commonly believed to display their anticancer effects
through inhibition of COX-2, as this isozyme is thought to
play a role in carcinogenesis and is often over expressed in
human premalignant and malignant tissues (Brown and
DuBois 2005; Husain et al. 2002; Eberhart et al. 1994). On
the other hand, certain studies indicate that NSAIDs also
promote apoptosis through mechanisms that are indepen-
dent of COX inhibition. This proposition is further sup-
ported by the fact that compounds, structurally similar to
NSAIDs but not significantly inhibiting COX isozymes,
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Keywords NSAIDs Fenamates Amides Wnt/β-catenin
Cancer
Abbreviations
COX
HLM
MW
Cyclooxygenase
Human liver microsome
Molecular weight
NSAIDs Non-steroidal anti-inflammatory drugs
SSA Sulindac sulfide amide
Electronic supplementary material The online version of this article
(doi:10.1007/s00044-017-2001-z) contains supplementary material,
which is available to authorized users.
* Robert C. Reynolds
rcr12lkt@uab.edu
1
Drug Discovery Division, Southern Research Institute, 2000 Ninth
Avenue South, Birmingham, AL 35205, USA
2
Drug Discovery Unit, College of Life Sciences, University of
Dundee, Dundee DD1 5EH, UK
3
Division of Hematology and Oncology, The University of
Alabama at Birmingham, Birmingham, Alabama 35294, USA

Med Chem Res
Fig. 1 General structures and
substitutions of synthetic
libraries
may have chemopreventive and proapoptotic properties
(Piazza et al. 2010; Elder et al. 1997; Hanif et al. 1996;
Alberts et al. 1995).
The Wnt/β-catenin signaling pathway is a crucial player
in the management of cell proliferation, migration, and
differentiation, thus making it a powerful regulator of
embryonic development and tumorigenesis (Barker and
Clevers 2006). Wnt proteins are secreted glycoproteins that
bind to the low-density lipoprotein receptor-related pro-
tein5/6 and Frizzled to activate Wnt/β-catenin signaling. A
large body of evidence suggests that there may be direct
effects of NSAIDs on the Wnt/β-catenin signaling pathway
(Giardiello et al. 1993; Koehne and DuBois 2004; Jolly
et al. 2002; Yang et al. 2003; Mahmoud et al. 1998; Boon
et al. 2004; Gala and Chan 2015; Egashira et al. 2017;
Preisner et al. 2015; Sareddy et al. 2013; Stein et al. 2011;
Lu et al. 2009; Bombardo et al. 2017). For example, it was
found that aspirin and indomethacin attenuate the tran-
scription of β-catenin/TCF-responsive genes, by modulating
T-cell factor (TCF) activity without disrupting β-catenin/
TCF complex formation (Dihlmann et al. 2001).
Scheme 1 General synthetic scheme to prepare NSAID amides
acid, flufenamic acid, diclofenac and fenoprofen, replacing
the carboxylate with an amide functional group as in SSA in
order to examine the anticancer activity of the amide
libraries against three cancer cell lines (prostate, colon and
breast). These compounds were further screened against
Wnt/β-catenin signaling to elucidate COX-independent
mechanisms of their effects.
Materials and methods
Chemistry
Fenamate NSAIDs, including tolfenamic, mefenamic and
flufenamic acid have been derived from anthranilic acid, a
close isosteric analog of salicylic acid. Salicylic acid is a
metabolite of salicin and the active ingredient of the earliest
anti-inflammatory herbal medicines, first isolated from
willow bark. Interestingly, members of the NSAID fena-
mates and profens have been also shown effective for
cancer prevention (Basha et al. 2011; Kang et al. 2012;
Woo et al. 2004; Somchit et al. 2009; Lovering et al. 2004;
Zhu et al. 1999; Mayorek et al. 2010; Marjanovic et al.
2007). We previously reported that a relatively simple
alteration to sulindac in the form of sulindac sulfide amide
(SSA) demonstrated excellent anticancer activity compared
to the parent compound sulindac sulfide in vitro as well as
having in vivo xenograft activity (Piazza et al. 2009). These
findings inspired us to synthesize a series of libraries from
other NSAID scaffolds, such as tolfenamic acid, mefenamic
Figure 1 shows the general structures of the amide diversity
series: (1) tolfenamic amides; (2) mefenamic amides; (3)
flufenamic amides; (4) diclofenac amides; and (5) and
fenoprofen amides.
These compounds were prepared from the corresponding
commercially available fenamates and fenoprofen by
coupling with an amine set (a–o) using HATU (Carpino
1993) as the amide coupling reagent to afford compounds
(1–5)a–(1–5)o in good yields (Scheme 1).
Biological studies
Quantitative high-throughput screen (qHTS) cell
proliferation assays
All target compounds were screened against three cancer
cell lines (prostate, colon and breast) using a qHTS format.

Med Chem Res
In brief, liquid handling was performed on a Biomek FX
with a 384-multichannel head. In 384 well plates, com-
pounds were arrayed in columns 3–22 leaving 32 wells for
positive and negative controls. All compounds were diluted
together in a plate to plate transfer. Cells were then added to
assay plates containing diluted compound using a Matrix/
Thermo wellmate. Cells were incubated with compound for
three cell doublings. Due to differences in growth rates
between cell lines, the incubation period for PC3 and HT-29
cells was 72 h, but was increased to 96 h for MDA-MB-231
cells. Plates were incubated for the appropriate time (72 or
96 h) and cell viability was determined using Cell Titer Glo
(Promega).
The dose response format employed a cross-plate method
rather than an in-plate method, allowing for more efficient
compound dilution and addition to assay plates. Two-fold
dilutions of the compound mother plate were aliquoted to a
series of 384-well plates using a stacked plate (or cross-
plate) format. Object manager was used to create the assay
plates by replicating the compound mother plate and
assigning concentration values to the assay plates. Lumi-
nescence values were read on the envision plate reader for
each of the assay plates. The entire experiment of assay
plates was set up in a single day with a complete read of all
plates occurring at 72 h and or at 96 h as required for the cell
lines. Data were imported and analyzed within 24 h of the
endpoint read. From set up to final report, all data points
were generated and reported within one week. Therefore,
only a single passage was required for each cell line,
eliminating potential variation due to passage and cell
count.
Data were analyzed using Activity Base software
(IDBS). Data were imported directly into the database and
calculated using an ActivityBase XE template where the
Virtual Plate functionality was employed to maintain the
link between the assay plates and the compound mother
plate from which they were created. For each plate the
median, standard deviations, coefficient of variations and Z
values were calculated for the control wells. These values
were used to assure quality and consistency across all test
plates and to normalize percent cell viability for each well.
XLFit and MathIQ were used within the ActivityBase XE
template to plot the dose response curves and calculate
CC50 values. The CC50s were calculated by plotting the cell
viability relative to the mean of the cell control at each of
the tested compound concentration. Compounds that caused
cell viability <80% were considered active. Values were
calculated only for active compounds using a 4-parameter
Levenburg-Marquardt algorithm (XLFit #205), with the
maximum and minimum locked at 0 and 100 respectively.
Data and graphical results were then reported and compared
across the three cell lines.
Wnt/β-catenin signaling screens
The effects of all target compounds on Wnt/β-catenin sig-
naling were examined with the Wnt reporter luciferase
assay in HEK293 cells as previously described (Lu et al.
2011; Lu et al. 2012). In brief, cells were plated into 24-well
plates. After overnight culture, cells were transiently
transfected with LRP6 plasmid (kindly provided by Dr.
Christof Niehrs, Deutsches Krebsforschungszentrum, Hei-
delberg, Germany) along with the Wnt signaling reporter
construct Super8XTOPFlash (kindly provided by Dr. Ran-
dall T. Moon, University of Washington, Seattle) and β-
galactosidase-expressing vector (Promega) by FuGENE HD
(Roche). After 24 h incubation, cells were treated with each
individual compound at the indicated concentration. Cells
were then lysed 24 h later and both luciferase and β-galac-
tosidase activities were determined. The luciferase activity
was normalized to the β-galactosidase activity. Initial
activity was measured at 100 µM, and, typically for those
compounds that showed <50% activity relative to the
control level and cancer cell line inhibition, a dose of 10 µM
was tested in order to determine if Wnt/β-catenin activity
was dose-dependent.
Results and discussion
Screening results
Table 1 lists the anticancer activity of tolfenamic amide
analogs 1 against colon, prostate, and breast cancer cell
lines as well as their Wnt/β-catenin signaling data. Benzyl
amide derivative of tolfenamic acid 1a displayed significant
anticancer activity in all the three assays. 1-Phenylpropyl
amide 1b exhibited moderate activity in colon, prostate and
breast cancer assays with CC50 values of 15.92, 25.37 and
18.08 µM, respectively. Compound 1c with a furan-2-
ylmethyl group at the amide linker showed better inhibitory
potency than 1b, but was less potent than 1a. Interestingly,
compounds 1a, 1b and 1c at 10 µM significantly inhibited
Wnt/β-catenin signaling in HEK293 cells in a dose-
dependent manner as signaling activity decreased at the
higher dose of 100 µM, suggesting that the inhibition of
Wnt/β-catenin signaling could contribute to anticancer
activity.
The amino acid analog 1d led to a complete loss of
potency in the proliferation assays although modest activity
was seen at 100 µM in the Wnt/β-catenin signaling assay.
Acyclic basic amide derivatives of tolfenamic acid 1e–1k
displayed various levels of anticancer activity and CC50
values ranged from 6 µM to >50 µM. N,N-diethylethyla-
mide derivative 1f and N,N-diethylpropylamide derivative
1g demonstrated similar activity against the three cell lines.

Med Chem Res
Table 1 Anticancer and Wnt/β-
catenin signaling data of
Cpd
Cell Line - CC50 (µM)
Wnt/β-catenin
tolfenamic amides 1a–1o
HT29
PC3
MDA-MB-231
% Control (100 µM)
% Control (10 µM)
1a
1b
1c
1d
1e
1f
0.99 0.35
15.92 2.14
3.11 0.49
>50
5.89 2.50
25.37 4.38
9.43 1.16
>50
7.27 6.61
18.08 4.64
6.21 3.20
>50
4.78 0.84
13.23 3.89
8.40 0.74
51.15 3.41
66.61 7.70
N/A
30.66 2.88
56.55 3.55
47.12 4.65
ND
18.93 1.29
6.58 0.58
8.74 1.14
12.04 1.24
20.02 1.83
17.33 1.64
6.05 0.58
7.15 0.73
6.69 0.43
11.31 1.31
5.43 0.28
26.42 2.86
7.92 0.87
13.29 1.56
35.05 1.96
>50
26.81 1.98
10.67 0.85
16.57 1.97
>50
ND
148.21 0.63
ND
1g
1h
1i
N/A
69.26 9.56
157.05 3.74
81.87 4.04
N/A
ND
>50
ND
1j
>50
>50
ND
1k
1l
14.51 1.74
9.56 0.83
8.19 0.77
29.12 3.22
8.12 0.89
16.68 1.82
10.39 1.15
10.21 0.93
21.11 1.30
14.15 1.03
128.71 13.66
163.73 7.58
179.39 6.78
129.82 7.08
95.84 0.30
N/A
1m
1n
1o
N/A
16.24 2.39
N/A
N/A not available due to cell toxicity at this concentration, ND not done
Table 2 Anticancer and Wnt/β-
catenin signaling data of
Cpd
Cell line - CC50 (µM)
Wnt/β-catenin
mefenamic amides 2a–2o.
HT29
PC3
MDA-MB-231
% Control (100 µM)
% Control (10 µM)
2a
2b
2c
2d
2e
2f
5.93 0.95
31.50 3.63
12.06 1.21
>50
17.69 2.91
>50
17.35 9.25
25.83 3.97
10.78 2.65
>50
8.15 1.73
42.15 4.53
2.25 0.70
47.19 6.36
255.95 59.07
N/A
64.46 0.36
73.55 3.55
33.91 4.86
>50
59.64 7.64
ND
>50
>50
>50
ND
11.52 0.88
15.14 0.41
19.68 0.84
39.45 2.33
28.55 0.88
12.98 0.39
6.08 0.38
8.27 0.45
19.51 0.94
8.73 0.61
20.55 2.39
33.82 4.77
>50
19.21 2.08
27.44 1.64
>50
163.22 37.56
2g
2h
2i
196.32 9.61
178.13 9.46
189.74 10.96
179.79 15.34
18.84 2.24
N/A
ND
ND
>50
>50
ND
2j
>50
>50
ND
2k
2l
29.06 2.98
10.41 1.29
9.82 1.11
>50
30.81 1.73
11.32 1.35
13.20 0.43
>50
ND
132.88 10.51
174.42 37.71
ND
2m
2n
2o
N/A
144.22 7.82
100.10 8.28
20.45 2.55
>50
ND
N/A not available due to cell toxicity at this concentration, ND not done
The introduction of an ethyl or a methyl group at the amide
nitrogen of 1f decreased the inhibitory activity as shown by
1h and 1i. In the case of 1j, replacement of one ethyl group
at the terminal nitrogen with a hydrogen and introduction of
an ethyl group at the amide nitrogen of 1g reduced the
inhibitory activity by two-fold in HT-29 cells. Replacing
both ethyl groups of 1j by two isopropyl groups (1k)
improved the activity by three-fold. Compounds 1l–1o are
the cyclic basic amide analogs of tolfenamic acid. Among
these four compounds, 1-piperidinylethyl analog 1l and 1-
ethylpyrrolidinylmethyl analog 1m have similar activity and
4-pyridylpiperazine derivative 1o displayed slightly better
anticancer activity than the other three examples. Notably,
compounds 1d–1o displayed weak activity at best against
Wnt/β-catenin signaling in HEK293 cells.
Screening data for the mefenamic amide series 2 are
shown in Table 2. In general, compounds (2a–2o) showed
modestly decreased cell growth inhibition potency

Med Chem Res
Table 3 Anticancer and Wnt/β-
catenin signaling data of
Cpd
Cell Line - CC50 (µM)
Wnt/β-catenin
flufenamic amides 3a–3o
HT29
PC3
MDA-MB-231
% Control (100 µM)
% Control (10 µM)
3a
3b
3c
3d
3e
3f
7.75 1.29
35.64 4.04
15.11 1.17
>50
19.79 3.03
>50
22.55 9.86
32.07 3.34
17.91 3.61
>50
4.56 0.08
9.10 0.72
6.89 7.98
33.31 0.43
152.99 13.15
N/A
62.33 7.32
74.87 8.54
27.38 2.60
>50
70.49 8.01
ND
17.82 0.73
8.16 0.48
16.58 0.68
23.55 1.14
39.92 2.45
38.76 2.60
10.63 0.26
7.06 0.65
7.83 0.35
22.82 0.74
11.17 0.47
41.33 6.50
15.45 1.78
33.89 2.63
>50
41.44 2.07
14.78 1.25
29.88 2.24
>50
ND
ND
3g
3h
3i
95.15 7.59
124.87 3.64
156.09 3.79
119.75 5.61
39.83 5.51
N/A
ND
ND
>50
>50
ND
3j
>50
>50
ND
3k
3l
25.25 2.98
13.21 1.69
10.44 0.99
>50
24.12 2.35
15.71 0.89
12.02 0.53
>50
ND
153.34 8.53
131.68 2.86
ND
3m
3n
3o
N/A
80.49 6.54
5.31 1.92
17.14 1.45
30.39 3.00
ND
N/A not available due to cell toxicity at this concentration, ND not done
compared to the tolfenamic amide series 1a–1o. Among this
series, benzyl amide derivative 2a, 1-piperidinylethyl deri-
vative 2l, 1-ethylpyrrolidinylmethyl derivative 2m, and
4-pyridylpiperazine derivative 2o are the most active com-
pounds with CC50 values ranging from 5 to 9 µM against
the HT-29 cell line. Moreover, benzyl amide derivative 2a
also suppressed Wnt/β-catenin signaling in HEK293 cells
with activity that was modestly less than the related tolfe-
namic amides 1a. Similar results were seen with analog 2c
as compared to the related compound 1c.
be more active than other compounds in the cyclic amide
series. Notably, compounds 4a–4o displayed no or weak
activity against Wnt/β-catenin signaling in HEK293 cells.
We next examined the anticancer activity of corre-
sponding fenoprofen analogs 5 (Table 5). None of these
compounds, except N-isopropylpropyl amide analog 5k and
4-pyridylpiperazine amide analog 5o, exhibited good
activity. Furthermore, compounds 5a–5o displayed weak
activity at best against Wnt/β-catenin signaling in HEK293
cells.
Our results for the flufenamic amides 3 are summarized
in Table 3. The activity pattern of these compounds (3a–3o)
is very similar to the mefenamic amide series 2a–2o.
Compounds 3a (benzyl amide), 3f(N,N-diethylethyl amide),
3k (N-isopropylpropyl amide), 3l (1-piperidinylethyl
amide), 3m (1-ethylpyrrolidinylmethyl amide) and 3o (4-
pyridylpiperazine amide) showed moderate activity in all
the three assays. However, compounds 3a–3o displayed
modest to little activity against Wnt/β-catenin signaling in
HEK293 cells although the amides 3a–3c consistently were
higher in activity as in tolfenamic amides (1) and mefe-
namic amide (2) series.
In silico evaluation of lipophilicity/physicochemical
properties
Compounds that have attractive physicochemical properties,
such as cell permeability and metabolic stability are more
likely to succeed as lead candidates in early drop develop-
ment. LogD and the molecular weight (MW) have been
identified as properties showing correlation with perme-
ability and stability data available for large data sets
(Waring 2009; Johnson et al. 2009). Using Caco-2 perme-
ability data for 16,227 compounds and human liver
microsome (HLM) stability determined for 47,018 com-
pounds, optimal ranges for logD and MW have been
derived that describe compounds meeting both require-
ments, good permeability and HLM stability (Johnson et al.
2009). On a plot of MW versus logD these optimal ranges
cover a triangular area with baseline at MW 200 and logD
−2 to 5 and an apex at MW 450 between logD 1 to 2,
referred to as the Golden Triangle area. Several analogs
from each presented series map to the Golden Triangle area,
We next turned our attention to anticancer activity of
diclofenac amides 4a–4o (Table 4).
In this series, acyclic (4e–4k) and cyclic (4l–4o) basic
amide compounds exhibited significant inhibitory activity
against cancer. The activity of aromatic amides was rela-
tively weak. Among the acyclic series, N-isopropylpropyl
amide derivative 4k displayed relatively potent activity
against all three cell lines. Compound 4o, with a 4-
pyridylpiperazine group at the amide linker, was found to

Med Chem Res
Table 4 Anticancer and Wnt/β-
catenin signaling data of
Cpd
Cell Line - CC50 (µM)
Wnt/β-catenin
diclofenac amides 4a–4o
HT29
PC3
MDA-MB-231
% Control (100 µM)
% Control (10 µM)
4a
4b
4c
4d
4e
4f
>50
31.82 9.34
>50
30.75 7.76
>50
131.26 52.55
15.97 3.91
52.36 5.34
13.51 11.78
132.99 25.46
N/A
ND
>50
ND
>50
35.47 10.32
32.82 5.34
>50
49.33 6.53
42.07 7.57
42.21 4.98
19.09 2.52
18.71 0.85
25.15 2.68
29.67 1.61
19.16 1.59
9.11 0.32
14.97 1.16
13.51 1.12
16.64 0.93
16.57 1.33
ND
>50
117.56 4.50
ND
18.72 1.29
5.48 0.20
7.52 0.28
7.25 0.28
8.85 0.32
7.21 0.43
4.56 0.15
6.29 0.18
6.42 0.57
10.15 0.49
3.51 0.35
16.87 2.63
22.26 2.00
14.26 1.61
22.42 2.00
10.83 1.58
6.65 0.74
10.75 1.25
13.17 1.46
19.8 2.34
10.33 1.72
200.60 18.09
173.75 4.36
163.92 5.45
151.45 5.82
134.87 13.58
138.80 0.96
190.60 14.46
186.88 12.50
ND
4g
4h
4i
N/A
N/A
11.90 7.28
N/A
4j
4k
4l
N/A
N/A
4m
4n
4o
N/A
6.39 1.66
181.77 61.37
ND
N/A not available due to cell toxicity at this concentration, ND not done
Table 5 Anticancer and Wnt/β-
catenin signaling data of
Cpd
Cell Line—CC50 (µM)
Wnt/β-catenin
fenoprofen amides 5a–5o
HT29
PC3
MDA-MB-231
% Control (100 µM)
% Control (10 µM)
5a
5b
5c
5d
5e
5f
>50
>50
>50
9.60 1.54
109.27 12.08
>50
>50
>50
6.77 0.39
114.35 18.16
>50
>50
>50
71.24 10.54
4.76 43
ND
>50
>50
>50
79.12 6.12
>50
>50
>50
82.57 4.03
131.04 5.43
139.50 13.04
77.34 5.76
114.07 18.30
140.26 18.54
24.76 4.86
125.69 7.98
92.17 8.49
101.52 10.31
8.51 1.30
ND
29.6 2.04
35.72 9.18
15.07 1.57
23.11 3.60
17.57 1.28
7.46 0.63
20.37 1.82
28.51 3.16
13.83 1.30
7.9 0.84
>50
>50
ND
5g
5h
5i
>50
>50
ND
>50
>50
116.65 11.31
>50
>50
ND
5j
41.3 3.34
24.01 3.84
>50
>50
ND
5k
5l
30.8 7.06
>50
131.90 7.46
ND
5m
5n
5o
>50
>50
ND
>50
>50
ND
11.51 0.90
13.22 2.35
82.33 3.50
ND not done
identified with labeled data points in the MW versus logD
plot (Supplemental materials), where logD values were
computed using StarDrop (version 6.2.0). For example,
these optimal ranges are satisfied by analogs with promising
activities, such as 2m (logD 2.77, MW 351.5), 5k (logD
2.08, MW 382.5), 5o (logD 2.66, MW 387.5). These
compounds are predicted to possess optimal values asso-
ciated with good permeability and metabolic stability
properties. Metabolically labile site predictions included in
Supplemental materials support that compound 5o has
minimal metabolic liabilities and predicts that it has a low
estimated efficiency of CYP3A4 metabolism as computed
based on all metabolic sites in the molecule. In the case of
compound 5k, a single labile site is predicted at the iso-
propylamine group suggesting redesign of this group to
achieve enhanced metabolic stability for this scaffold. The
remainder of labile sites in 5k are predicted to be relatively
stable or moderately stable to metabolism. Overall, both

Med Chem Res
compounds 5o and 5k have reduced metabolic liability and
vulnerability compared to other scaffolds, for example as
predicted for the lead NSAID analog SSA (please see
Supplemental materials).
are predicted to have acceptable physicochemical properties
for consideration in future studies. Among these, analogs
2m, 5k and 5o are highlighted for possible advancement
in vivo.
LogD values computed for compounds in series 1–3
(Tables 1–3) show linear correlation with suppression of
Wnt/β-catenin signaling at 10 µM (r2 equals 0.74), while at
100 µM drug concentration the analogous correlation is
weak (r2 equals 0.39). The correlation plot (included in
Supplemental materials) suggests that higher logD values
are associated with more potent Wnt/β-catenin activities (at
10 µM). Caco-2 cell permeability descriptors computed
using Schrödinger software predict that all compounds in
this study fall in the range associated with high perme-
ability, except for analogs containing substitution d in
Fig. 1, which are predicted moderately permeable. StarDrop
software tools predict all compounds permeable (human
intestinal absorption >30%). While the presented com-
pounds cover a wide range of logD (1.66 to 5.96), nearly all
compounds are predicted highly permeable. Further, we
found no correlation between the computed logD and can-
cer cell line data for compounds in series 1–3. For com-
pounds in series 4–5 (Tables 4–5), there was no notable
correlation between computed logD values and cancer cell
line or Wnt/β-catenin activities.
Acknowledgements We are grateful to Drs. Christof Niehrs and
Randall T. Moon for their generosity in providing reagents. This work
was supported by grants from the National Institutes of Health NCI
1R01CA131378 (RCR PI) and RO1CA124531 (Y. Li PI). We also
acknowledge DOD Era of Hope award W81XWH-07-1-0463 (RCR
PI) for supporting HTS cancer cell activity profiling. We thank the
University of Alabama at Birmingham, Department of Hematology
and Oncology for their support. We also thank both Lucile White and
Lynn Rasmussen for HTS support services.
Compliance with ethical standards
Conflict of interest The authors declare that they have no competing
interests.
Open Access This article is distributed under the terms of the
Creative Commons Attribution 4.0 International License (http://crea
tivecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided you give
appropriate credit to the original author(s) and the source, provide a
link to the Creative Commons license, and indicate if changes were
made.
Thus, computed logD may be correlated with Wnt/β-
catenin suppression for analogs in Tables 1–3, however, no
analogous correlation was found with cancer cell line
activities. Based on in silico predictions cell permeability
properties may not be linked to the predicted effect of logD
on Wnt/β-catenin activities.
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