Design, Synthesis, and Biological Evaluation of Novel Selenium (Se-NSAID) Molecules as Anticancer Agents

Article abstract of DOI:10.1021/acs.jmedchem.5b01503

The synthesis and anticancer evaluation of novel selenium-nonsteroidal anti-inflammatory drug (Se-NSAID) hybrid molecules are reported. The Se-aspirin analogue 8 was identified as the most effective agent in reducing the viability of different cancer cell

Full text of DOI:10.1021/acs.jmedchem.5b01503

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Article
Design, Synthesis, and Biological Evaluation of Novel
Selenium (Se-NSAID) Molecules as Anticancer Agents
Daniel Plano, Deepkamal Karelia, Manoj Pandey, Julian
E. Spallholz, Shantu G. Amin, and Arun Kumar Sharma
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01503 • Publication Date (Web): 09 Jan 2016
Downloaded from http://pubs.acs.org on January 12, 2016
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Design, Synthesis, and Biological Evaluation of
Novel Selenium (Se-NSAID) Molecules as
Anticancer Agents
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‡
, ϕ
ϕ,†
†
§
Daniel Plano , Deepkamal N. Karelia , Manoj K. Pandey , Julian E. Spallholz , Shantu
†
†,
Amin , and Arun K. Sharma
†
‡
Department of Pharmacology; Penn State Hershey Cancer Institute, CH72; Penn State College
of Medicine, 500 University Drive, Hershey, PA 17033, USA.
Department of Organic and Pharmaceutical Chemistry, University of Navarra, Irunlarrea 1, E-
3
1008 Pamplona, Spain.
§
Department of Nutrition; Texas Tech University, Lubbock, TX 79430, USA.
Authors contributed equally.
ϕ

Author to whom correspondence should be addressed
Arun K. Sharma, Ph.D.
Department of Pharmacology
Penn State College of Medicine
Penn State Hershey Cancer Institute, CH72
Penn State Milton S. Hershey Medical Center
500 University Drive
Hershey, PA 17033
Phone: 717-531-4563
717-531-0244
E-mail: aks14@psu.edu
Fax:
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ABSTRACT
The synthesis and anti-cancer evaluation of novel Selenium-Non-steroidal anti-inflammatory
drugs (Se-NSAIDs) hybrid molecules are reported. The Se-aspirin analog 8, was identified as
the most effective agent in reducing viability of different cancer cell lines, particularly colorectal
cancer (CRC) cells, was more selective towards cancer cells than normal cells, and was >10
times more potent than 5-FU, the current therapy for CRC. Compound 8 inhibits CRC growth
via inhibition of cell cycle in G1 and G2/M phase, and reduces the cell cycle markers like cyclin
E1 and B1 in a dose dependent manner; the inhibition of the cell cycle may be dependent on the
ability of 8 to induce p21 expression. Furthermore, 8 induces apoptosis by activating caspase 3/7
and PARP cleavage, and its longer exposure causes increase in intracellular ROS levels in CRC
cells. Taken together, 8 has the potential to be developed further as a chemotherapeutic agent for
CRC.
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INTRODUCTION
A cohort of studies supports the indisputable promise of aspirin (ASA) and other non-steroidal
anti-inflammatory drugs (NSAIDs) as chemo-preventive agents, especially in colorectal cancer
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(
CRC). Meta-analyses and systematic reviews suggest that ASA and other NSAIDs use is
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associated with a decreased incidence of colonic adenomas, CRC, and metastatic CRC.
Furthermore, several other studies suggest that daily dosing of ASA decreases the risk of great
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variety of cancer types, including lung, breast, skin, pancreas and ovarian cancers.
Although
routine ASA use may not be warranted in the general population, an international panel
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advocated additional research in high-risk populations. In fact, a randomized clinical trial in
carriers of hereditary CRC, named CAPP2 (Colorectal Adenoma/carcinoma Prevention
Program), has demonstrated a substantial protection by ASA against CRC and other Lynch
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syndrome cancers. The long-term results showed that 600 mg of ASA per day for a mean of 25
months substantially reduced cancer incidence after 55.7 months in Lynch syndrome patients,
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which is the major form of hereditary CRC.
Additionally, NSAIDs have demonstrated their intestinal antineoplastic effects in animal
models, showing their effects in more than 90% of the 110 individual studies of various animal
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intestinal cancer models. A key mechanism for NSAIDs efficacy is cyclooxygenase (COX)
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inhibition and reduced production of prostaglandins. Nonetheless, several other mechanisms of
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action have been proposed, including induction of apoptosis,
inhibition of angiogenesis,
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activation of nuclear factor kappa B (NF-κB),
modulation of insulin-related neoplastic
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pathways, inhibition of the mammalian target of rapamycin (mTOR) pathway and modulation
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of cellular metabolism through the AMP-activated protein kinase (AMPK).
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Several modifications, which maintained the anti-inflammatory effect of the parental
NSAIDs with favorable gastrointestinal tolerance or with combined immune-modulatory and
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antioxidant activities, have been carried out over different NSAID scaffolds.
The
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combination of reactive nitrogen species, e.g. nitric oxide (NO) and nitroxyl (HNO), with
different NSAIDs, particularly ASA, has been widely studied and characterized. The rationale
behind the generation of these molecules is that the combined effect of both the scaffolds will
exceed those of each structural component individually, along with a reduction of the renal and
hepatic toxicities associated with chronic NSAID use. Several reports have demonstrated that the
NO- and HNO-releasing NSAIDs possess stronger cytotoxicity and chemo-preventive effects,
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mainly against CRC, together with a lower toxicity than the corresponding NSAID alone.
Notably, hydrogen sulfide-releasing NSAIDs (HS-NSAIDs) are formed by the conjugation of the
parent NSAID with a dithiolethione moiety, which releases hydrogen sulfide. These scaffolds
were less toxic to the GI tract and exhibited a marked increase in the anti-inflammatory activity
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and potency in a murine model of colitis than their parental NSAIDs.
Additionally, several
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HS-NSAIDs evinced growth inhibitory effects in a wide variety of human cancer cells.
Furthermore, dual NO- and HS-releasing ASA derivatives have been synthesized, showing
potent anti-inflammatory activity and inhibition of cancer cell growth in vitro along with a
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marked reduction of tumor volume in colon cancer xenograft models.
Other modified
NSAIDs extensively reported in the literature are the phospho-NSAIDs, which have shown
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preclinical chemotherapeutic and chemopreventive properties in several cancer models.
Selenium (Se) compounds have attracted a vast interest in the last decades as promising
chemo-preventive agents and several epidemiological studies have reported an inverse
association between the nutritional Se status and cancer risk, although considerable controversy
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has arisen mainly due to the negative outcome of the SELECT trial.
Contrary to the
skepticism generated by the diverse clinical trials on Se as a chemo-preventive agent, a rising
interest in its potential cancer therapeutic effects has been developed in the past few years.
Several organoselenium compounds have been shown to inhibit cancer cell growth in various
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xenograft rodent models for different cancers types,
as well as to have synergistic effects in
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combination with chemotherapeutic drugs.
The mechanisms of action by which seleno
compounds exert their anti-tumor effects differ with the overall structure of the molecule and
have not been completely characterized. However, some of the representative anti-cancer
mechanisms of seleno compounds are induction of apoptosis, inhibition of angiogenesis and
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modulation of AKT and COX pathways.
To our knowledge, the only NSAID framework
modified to date with Se functionalities is the celecoxib scaffold, yielding greater anti-
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inflammatory and cancer cell growth inhibition; for the novel selenocoxib derivatives.
Further development of these selenocoxib derivatives afforded the glutathione analog of
selenocoxib-1, which elicited potent inhibition of tumor development in melanoma xenografted
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mice.
Considering the aforementioned chemo-preventive effects of NSAIDs and the biological
effects of organoselenium compounds, along with the previous reports that support the
modification of NSAID scaffolds to obtain more potent and less systemically toxic antitumor
agents, we designed novel hybrid Se-NSAID derivatives. This drug design is additionally
supported by the reported protective effects of Se compounds against gastric toxicity induced by
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indomethacin,
along with the potential synergistic effects of Se and NSAIDs.
In the present study, we report the design and synthesis of several Se-NSAID hybrid
compounds, their cytotoxic activity in a panel of cancer cell lines, and the preliminary
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characterization of the mechanism of action for the lead compound (8) in the HCT116 CRC cell
line. Two shared mechanisms implicated in the biological effects of ASA and seleno compounds
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on cancer cells are apoptosis
and inhibition of inflammatory pathways.
In this paper, we
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focused on the anti-proliferative effects of the lead compound 8 and report insights into its
mechanism of action in inhibiting CRC cell growth.
RESULTS AND DISCUSSION
Design. Several studies have pointed out the chemo-preventive effects of NSAIDs and the anti-
tumor and chemo-preventive effects of multiple Se species in various cancers. In addition,
several Se compounds seem to possess a protective effect against indomethacin-induced gastric
toxicity, as well as a synergistic effect with sulindac to inhibit intestinal tumorigenesis.
Considering all the above mentioned, we hypothesized that assembly of both groups in the same
molecule could be a valid approach in the development of potent and safe cancer preventive and
therapeutic agents. Therefore, novel molecules were designed by the incorporation of an
appropriate Se moiety into various NSAIDs according to the general structure shown in Figure 1.
In order to identify the NSAID that formed the most effective anti-cancer hybrid molecule with
Se, three different NSAIDs [ASA, ibuprofen (Ibu) and naproxen (Nap)] where selected for Se
incorporation. Based on our past experience and literature evidences on the structure-activity
relationship of organoselenium compounds, 2-selenocyanoethanamine (1) was used as an
appropriate Se moiety to introduce into ASA, Ibu and Nap scaffolds. The in vitro cytotoxic
results in a panel of four cancer cell lines (Table 1) indicated that only in the case of ASA the
IC50 value of the resulting hybrid Se-NSAID derivative (compound 8) was decreased
significantly as compared to the parent NSAID scaffold. Accordingly, the hybrid Se-ASA (8)
was selected for further structural modifications with various Se moieties
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In a second approach, several structural modulations were performed in two different
regions of the Se-ASA scaffold: i) The functional group used to link the ASA with the Se moiety
was modulated, being amide, ester or selenoester as the selected conjugate groups (Figure 1); and
ii) The functional groups bearing the Se atom were selected among selenocyanate (-SeCN),
monoselenide (-Se-ASA), and methylselenocysteinyl (-CO H-CH SeCH ) (Figure 1). The
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methylselenocysteine moiety was incorporated into ASA structure to evaluate the influence of its
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anti-cancer properties
as well as the demonstrated selective protection it offers against organ-
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specific toxicity induced by clinically active agents. Taking into account the IC50 values
obtained for the six Se-ASA hybrid molecules (8, 10, 12-15), compound 8 emerged as the most
potent and promising anti-cancer agent. To gain further knowledge about the importance of the
Se atom in this scaffold, its isosteric sulfur analog (16) was also synthesized and tested.
Chemistry. The Se derivatives of Ibu (4) and Nap (5) were prepared from the corresponding
acyl chloride and 2-selenocyanoethanamine (1) by a nucleophilic addition/elimination reaction in
the presence of triethylamine (TEA) to neutralize the hydrochloric acid formed in the reaction
mixture (Scheme 1). The SeCN derivative 1 was obtained by the nucleophilic substitution of -Br
atom in 2-bromoethanamine by -SeCN, using KSeCN as nucleophilic donor, in acetonitrile as
solvent and under a nitrogen atmosphere (Scheme 1). The acyl chloride (3) of Ibu was
synthesized by the reaction of (RS)-2-(4-(2-methylpropyl)phenyl)propanoic acid (2) with oxalyl
chloride in methylene chloride.
Surprisingly, the reaction of the acyl chloride derivative of ASA (6) with 2-
selenocyanoethanamine (1), under the reaction conditions used for compounds 4 and 5, did not
yield the expected ASA analog (8) (Scheme 1). Modifications of the reaction conditions
including changes of solvent, reaction temperature, and time failed to yield compound 8. The
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chemical reasons for this surprising behavior are unknown. We hypothesize that an
intramolecular cyclization is occurring for this specific reaction, however, further investigations
are warranted to prove this hypothesis. An alternative synthetic route was thus developed for
compound 8. In this case, the SeCN group was inserted in 2-((2-bromoethyl)carbamoyl)phenyl
acetate (7), obtained by the reaction of 2-bromoethylamine hydrobromide with acid chloride 6,
by refluxing in dry acetonitrile with KSeCN with a 41% yield (Scheme 1).
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The hybrid SeCN-ASA derivative (10) with the ester linker was synthesized by reaction
of the aspirinyl chloride (6) and 2-selenocyanatoethanol (9) in the presence of TEA under a
nitrogen atmosphere at room temperature in methylene chloride with a 79% yield (Scheme 2).
Compound 9 was synthesized by the replacement of the bromine atom in 2-bromoethanol by a
SeCN group using KSeCN in dry acetonitrile (Scheme 2).
The synthesis of the aspirinyl selenoester derivatives (12-14) was carried out by first
reacting aspirinyl chloride (6) with an aqueous solution of sodium hydrogen selenide, following
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a previously published method, followed by the treatment of the resulting sodium salt of
aspirinyl selenoester (11) in situ with 1,2-dibromoethane in a mixture of distilled water and THF,
yielding a mixture of the mono- and di-substituted derivatives (12 and 13, respectively) (Scheme
3
). Both of the compounds were easily isolated by silica gel column chromatography and the
ratio for both compounds could be modulated using different proportions of 1,2-dibromoethane
and different reaction times. Finally, the selenocyanate derivative 14 was obtained by the
reaction of compound 12 with KSeCN in dry acetonitrile at room temperature with a 72% yield
(Scheme 3).
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The methylseleno derivative 15 was obtained by a nucleophilic addition/elimination
reaction between the aspirinyl chloride (6) and Se-(methyl)selenocysteine hydrochloride
(Scheme 4) in the presence of TEA. The synthesis of the sulfur analog (16) of the lead Se-ASA
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compound (8) was carried out by following the similar methods as described in Scheme 1 using
NH SCN instead of KSeCN (Scheme 5).
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Evaluation of the anti-proliferative activities of the novel molecules.
The three NSAID-SeCN compounds (4, 5, and 8), the corresponding parent NSAIDs (Ibu, Nap,
and ASA), and all the Se- (8, 10, and 12-15) and S- (16) derivatives of ASA were examined for
their effects on cancer cell viability. To determine the effect of –NH-CH CH -SeCN
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incorporation into the various NSAIDs, compounds 4, 5, and 8 were screened against a panel of
four different cancer cell lines: HT-29 (human colon carcinoma), MDA-MB-231 (human breast
adenocarcinoma), PANC-1 (human pancreatic carcinoma) and UACC903 (human malignant
melanoma); using an MTT assay. As reported in Table 1, parent NSAIDs (Ibu, Nap, and ASA)
had no effect on cancer cell viability up to the maximum tested dose of 50 µM. The IC values
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obtained for the hybrid SeCN derivatives 4, 5 and 8, compared to their corresponding parent
NSAIDs, showed that only the introduction of the Se moiety in ASA, resulted in a significantly
more potent analog 8, while similar modification of Ibu and Nap had no effect on cancer cell
viability (Table 1). Cell viability curves for 8 at 48 h time point are shown in Figure 2. This
clearly demonstrated that the enhanced cytotoxicity may not simply be the result of incorporation
of the Se-moiety into the NSAID, but a unique combination of the Se moiety with ASA is
required for cytotoxicity. Based on this observation, we generated several Se analogs of ASA
and screened them against same panel of the cells as used in Table 1. Table 2 summarizes the
effects of all agents on cancer cell viability, which were designed by further modifying 8.
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An overview analysis of the IC values obtained and summarized in Table 2 evinced that
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was effective at 48 and 72 h time points, while 15 and 16 were moderately cytotoxic only to
PANC-1 cells. Compounds 8 and 13 showed a potent effect on cancer cell viability in all cancer
cell lines; with 8 being the most potent (Table 2, and Figure 2). Notably, in comparison to
selenocoxib-1 and its glutathione conjugate (selecoxib-1-GSH), the only Se-NSAID hybrid
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compounds reported in the literature,
as compared to selecoxib-1-GSH after 48 and 72 h of treatment against UACC903 cells.
Compound 8 also exhibited IC values comparable with selenocoxib-1 in prostate cancer cells,
compound 8 possessed up to 5-fold lower IC values
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the cytotoxic assays, however, were performed in different cell lines (DU145 for 8 and PC3M
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for selenocoxib-1).
Considering the potency of these Se-ASA hybrid molecules on cancer cell viability, some
structure-activity relationship (SAR) can be inferred: 1) the most favorable functional group used
to link the ASA scaffold with the Se is the amide group (8); the replacement of the amide group
forming an ester (10) or selenoester (14) reduced the potency of the compounds on cancer cell
viability; 2) the introduction of a carboxylic acid group to the methylene group adjacent to the
amide, along with the replacement of selenocyanate by a methylseleno group yielded inactive
compound 15 in most of the cancer cell lines tested, with the exception of PANC-1 where 15
displayed a moderate effect on cancer cell viability; 3) the Se atom in the hybrid ASA analogs
seems to be necessary for the anti-cancer activity of the Se-ASA derivatives. This conclusion is
inferred from the fact that the isosteric substitution of the Se atom with S causes a dramatic
reduction in or the total loss of effect on cancer cell viability of the ASA-modified scaffold (8
versus 16; Table 2).
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Based on the potency of 8 in reducing cancer cell viability, it was selected as the lead
compound for further evaluation in six different cancer cell lines [DU 145 (human prostate
carcinoma), NCI-H460 (human large-cell lung carcinoma), HEPG2 (human liver hepatocellular
carcinoma), RKO and HCT116 (human colon carcinoma), and SKOV3 (human ovarian
carcinoma)]. Compound 8 was effective in reducing cell viability of all of the above mentioned
cancer cell lines with IC values in the low μM range at 48 and 72 h of treatment (Table 3 and
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Figure 3). The cytotoxic effect of 8 was particularly striking in all three colon cancer cell lines,
HT29, RKO and HCT116, where it showed cytotoxicity as early as at 24 h time point (Figure
3
E-G). We further confirmed this observation by using another method known as Live and Dead
assay. HCT116 cells were treated with 15 µM of 8 for 24 h and were compared with 1 mM of
ASA. As shown in Figure 3H, ASA had no significant effect on population of dead cells even at
1
mM concentration, while 8 increased dead cells population to about 51%. These data were in
concordance with the MTT assay. It is interesting to note that even at nearly 67 times lower dose,
is >5 times more potent in causing HCT116 cell death than parent ASA.
8
Compound 8 was more selective towards cancer cells in reducing cell viability and showed
greater potency compared to current CRC therapy.
The observed uniform cytotoxicity of 8 to various cancer cell lines warranted its evaluation on
normal cells to determine if it was selectively toxic to cancer cells. Since cytotoxicity towards
healthy cells is frequently associated with unwanted side effects, it was essential to test 8 against
normal cells as a surrogate experiment to access its systemic toxicity in vivo. A human dermal
fibroblast (nHDF) and mouse embryonic fibroblasts (MEFs) were used as a model of normal
cells for the cytotoxicity study. Interestingly, nHDF and MEFs were not affected at doses of 8,
which are toxic towards CRC cells at 48 h (Figure 4A), and for that matter, all other cancer cells
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tested (see Tables 1 and 2). These data demonstrated that 8 is selectively toxic to cancer cells but
not transformed cells. Furthermore, we compared 8 with the current therapy for CRC, 5-FU, in a
cell viability assay. Compound 8 was more effective in inhibiting CRC cell viability than 5-FU
or ASA (Figure 4B). Therefore, 8 represents a truly novel Se-ASA molecule, which is more
potent than the parent compound ASA and the current CRC therapy, at inhibiting cancer cell
viability. In addition, 8 showed high selectivity towards cancer cells suggesting that this
compound may have a very high therapeutic window.
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Compound 8 inhibited CRC cell growth by inducing cell cycle arrest.
To explore the underlying mechanism for the reduced cell viability of CRC cells by 8, we
investigated effect of 8 on the cell cycle by Flow cytometry. Treatment of HCT116 cells with 8
effectively arrested cells in G1 and G2/M phase of cell cycle. The population of cells in S phase
was drastically reduced compared to vehicle (DMSO) treated cells (Figure 5A-B).
The cyclins B1, E1 and p21 play important roles in regulation of the cell cycle. Cyclin B1
is required for cells to enter the mitosis phase, while cyclin E1 is required for cell to transit from
G1 to the synthesis (S phase) phase. The activation of cyclin-dependent kinase inhibitor, p21 has
70
been associated with G1/S and G2/M cell cycle arrest. Hence, the effect of 8 on these cell
regulatory proteins was investigated. Treatment of CRC cells with 8 for 24 h, showed an
increasing p21 expression in a dose dependent manner (Figure 5C). Both Cyclins B1 and E1
were also inhibited at similar doses where p21 expression was induced. These results suggest
that 8 up-regulates p21 expression, which leads to inhibition of Cyclin B1 and E1 expression,
and arresting CRCs in the G1 and G2/M phases of the cell cycle.
Compound 8 induced apoptosis in CRC cells.
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Since CRC cells treated with 8 showed a profound G1 and G2/M arrest accompanied by
activation of p21, we investigated whether the inhibition in the cell cycle leads to apoptosis.
HCT116 cells were treated with indicated concentrations (Figure 6) of compound 8 for 24 h.
Apoptosis was measured by using Muse Annexin V & Dead cell assay kit following
manufactures protocol. As shown in Figure 6A, cells treated with vehicle (DMSO), were mostly
in bottom left quadrant (BLQ). As compound 8 dose was increased, cells shifted from healthy
state to apoptotic. At 2.5 µM concentration of compound 8, >20% cells were apoptotic, and as
the dose increased, cells shift from bottom right quadrant (BRQ) to upper right quadrant (URQ).
More than 60% cells were detected to be apoptotic at 5 µM concentration of compound 8 and at
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0 µM >80% cells were apoptotic. Further, none of the cells were necrotic as there were no cells
detected in upper left quadrant (ULQ), which were 7-ADD positive, while Annexin-V and
Caspase 3/7 negative. In a similar manner, 8 increased caspase 3/7 activity in a dose dependent
manner (Figure 6B). Figures 6C and 6D quantify the amount of total percent of apoptotic cells
determined by both Annexin-V and caspase 3/7 assays, respectively.
We further confirmed caspase 3/7 activity by subjecting the whole cell lysates of treated
HCT116 cells with compound 8 to western bot analysis. As shown in Figure 6E, compared to the
vehicle treated cells, exposure to 8 significantly induced the cleavage of PARP, which is one of
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the main cleavage targets of caspase 3, and a marker of cells undergoing apoptosis. Overall, our
results confirm that compound 8 inhibits growth of CRC cells by inducing apoptosis.
Compound 8 increased ROS production in CRC cells in a time dependent manner.
Studies have shown that anti-cancer effect of Se containing molecules are associated with
7
2
increase in total ROS levels. To investigate whether induced cytotoxic effect of 8 is related
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with ROS production, we measured total ROS levels in 8 treated CRC cells following a
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previously reported method. The CRC cells (HCT116) were treated with 8 for 3, 6, and 24 h
and total ROS level was measured. As shown in the Figure 7, the total ROS levels did not change
significantly with the treatment of 8 at early time points (3 and 6 h), however, at 24 h ROS levels
increased to about 66% in 8 treated cells. These results strongly suggest that Se containing 8 is
not able to induce ROS production at early time points. However, longer exposure of cells with 8
induces ROS production which is correlated with an increase in apoptosis, suggesting that the
induction of cell death by 8 may be due the ROS production.
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Conclusions
In conclusion, addition of Se moieties into NSAID structures led to the discovery of novel Se-
ASA analogs as potential cancer therapeutics. Of the new Se-ASA derivatives, 8 emerged as the
most potent anti-cancer agent, particularly against CRC cells. Compound 8 showed more
selectivity towards cancer cells and was >10 fold potent than current CRC therapeutic agent, 5-
FU. Our studies revealed that 8 inhibited CRC growth via cell cycle arrest leading to apoptosis.
In addition, 8 induced apoptotic cell death evident by Caspase 3/7 activity and positivity of
phosphatidylserine (PS) on outer membrane of CRC cells. Further, 8 also increased ROS levels
in correlation with apoptosis at 24 h. Overall, 8 was identified as a potential selenium containing
cancer therapeutic compound with a solid mechanistic basis of its activity. While developing
ASA related analogs as therapeutics, it is important to consider the major adverse effect of ASA,
which is gastrointestinal bleeding. However, these effects are generally associated with long term
74
and high dosage use of ASA. Such issues with compound 8 are not anticipated since (i) it is
several fold more potent than ASA and therefore much lower concentration of 8 would be
required, and (ii) it is being developed for cancer patients who currently have very low survival
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rates. Thus the ASA like adverse effects, if any, related to long-term usage of 8 would be a
negligible problem as compared to the benefits. Future studies warrant evaluation of the in vivo
efficacy, toxicity, and greater detailed mechanism of action of lead compound 8.
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EXPERIMENTAL SECTION
Chemistry
General. All reactions were conducted in round-bottom flasks equipped with a Teflon-coated
magnetic stirring bar. Experiments involving moisture and/or air sensitive components were
performed under a nitrogen atmosphere in oven-dried glassware. Reagents, starting materials,
and anhydrous solvents were purchased from commercial suppliers and were used as received.
Reaction courses were monitored by thin-layer chromatography (TLC) on pre-coated silica gel
6
0 F254 aluminum sheets (Merck, Darmstadt, Germany), and the spots were visualized under UV
light. The crude reaction products were purified by silica gel column chromatography using
silica gel 60 Ȧ (Merck, 230-400 mesh), and hexane/ethyl acetate was used as the elution solvent.
1
13
H-NMR and C-NMR spectra were recorded on a Bruker Advance 500 instrument in either
D O or CDCl , operating at 500 and 125 MHz, respectively. Chemical shifts are reported in δ
2
3
values (ppm) and J values are reported in hertz (Hz). The signals are quoted as s (singlet), d
doublet), t (triplet), sept (septet), q (quadruplet), m (multiplet), dd (doublet of doublets), and td
(
(triplet of doublets). High resolution (ESI) MS were carried out at the Chemistry Instrumentation
Center, State University of New York at Buffalo, NY. The purity of the final compounds (≥98%)
was quantified by high performance liquid chromatography analysis.
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Synthesis of 2-selenocyanatoethanamine (1). KSeCN (3.0 g, 14.64 mmol) was added to a
mixture of 2-bromoethylamine hydrobromide (2.1 g, 14.64 mmol) in anhydrous acetonitrile (40
mL) under a nitrogen atmosphere. The reaction mixture was stirred at room temperature for 14 h
and the solid formed in the reaction was filtered, washed with diethyl ether and dried under
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vacuum. The isolated solid was used without purification for further reactions. Yield, 91%. H
NMR (500 MHz, D O) δ 3.59 (t, 2H, J = 7.5 Hz, CH -SeCN); 3.95 (d, 2H, J = 7.5 Hz, CH -NH).
2
2
2
General procedure for the preparation of NSAID-SeCN derivatives (4 and 5). To a mixture
of 2-selenocyanatoethanamine (1, 0.45 g, 3.0 mmol) and TEA (0.68 ml, 4.9 mmol) in anhydrous
methylene chloride (25 mL) was added the corresponding acyl chloride (1.96 mmol). The
reaction mixture was stirred at room temperature under a nitrogen atmosphere for 18-24 h, and
then filtered. The filtrate was concentrated in vacuo and partitioned between water and
methylene chloride. The organic layer was dried with magnesium sulfate and concentrated in
vacuo. The crude product was purified using silica gel chromatography with an hexane/ethyl
acetate gradient (starting from 10% up to 40% ethyl acetate) to afford the desired product.
2-(4-isobutylphenyl)-N-(2-selenocyanatoethyl)propanamide (4). The title compound was
synthesized from 1 and the acyl chloride 3 (0.4 g, 1.96 mmol) according to the general procedure
1
described above. Yield 44%; mp 145−147 °C. H NMR (500 MHz, CDCl ) δ 0.92 (d, 6H, J = 6.5
3
Hz, 2-CH ); 1.55 (d, 3H, J = 7.5 Hz, CH ); 1.87 (sept, 1H, J = 7.0 Hz, CH); 2.47 (d, 1H, J = 7.0
3
3
Hz, CH -Ph); 3.18 (td, 2H, J = 8.0 and 1.0 Hz, CH -SeCN); 3.68 (q, 1H, J = 7.5 Hz, CH); 3.84
2
2
(
td, 2H, J = 8.0 and 2.5 Hz, CH -NH); 7.13 (d, 2H, J = 8.0 Hz, H-3, H-5); 7.20 (d, 2H, J = 8.0
2
1
3
Hz, H-2, H-6). C NMR (125 MHz, CDCl ) δ 18.4 (CH ), 22.4 (2CH ), 26.7 (CH), 30.1 (CH -
3
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3
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Se), 45.0 (CH -NH), 47.4 (CH), 55.7 (CH -Ph), 127.4, 129.8, 137.2, 141.1 (aryl), 158.4 (SeCN),
2
2
+
1
75.6 (C=O). HRMS (ESI) calcd for C H N OSe [M + H] : 339.0970. Found: 339.0971.
16 23 2
(S)-2-(6-methoxy-2-naphthyl)-N-(2-selenocyanatoethyl)propanamide (5). The title compound
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was synthesized from 1 and (S)-2-(6-methoxy-2-naphthyl)propionyl chloride (0.49 g, 1.96
1
mmol) according to the general procedure described above. Yield 29%; mp 157−158 °C. H
NMR (500 MHz, CDCl ) δ 1.64 (d, 3H, J = 7.0 Hz); 3.18 (td, 2H, J = 7.5 and 3.0 Hz); 3.81 (m,
3
2
H); 3.87 (q, 1H, J = 7.5 Hz); 3.95 (s, 3H); 7.13 (d, 1H, J = 2.5 Hz); 7.17 (dd, 1H, J = 9.0 and
1
3
2.5 Hz); 7.38 (dd, 1H, J = 8.5 and 1.5 Hz); 7.71 (m, 3H). C NMR (125 MHz, CDCl ) δ 18.3
3
(
CH ), 26.2 (CH -Se), 47.8 (CH -NH), 54.6 (CH), 55.3 (OCH ), 105.7 (SeCN), 119.2, 126.1,
3 2 2 3
1
26.4, 127.7, 129.0, 129.3, 133.9, 135.2, 157.8, 160.5 (aryl), 176.1 (C=O). HRMS (ESI) calcd
+
for C H N O Se [M + H] : 363.0606. Found: 363.0609.
1
7
18
2
2
Synthesis of 2-((2-bromoethyl)carbamoyl)phenyl acetate (7). To a mixture of 2-
bromoethylamine hydrobromide (2.94 mmol, 1.5 equiv) and TEA (4.9 mmol, 2.5 equiv) in
anhydrous methylene chloride (25 mL) was added O-acetylsalicyloyl chloride (6, 1.96 mmol, 1
equiv). The reaction mixture was stirred at room temperature under a nitrogen atmosphere for 6
h. The mixture was then filtered. The filtrate was concentrated in vacuo and then partitioned
between water and methylene chloride. The organic layer was dried over magnesium sulfate and
concentrated in vacuo. The crude product was purified using silica gel chromatography with an
hexane/ethyl acetate gradient (starting from 10% up to 60% ethyl acetate) to afford the desired
1
product in 51% yield. H NMR (500 MHz, CDCl ) δ 2.42 (s, 3H, CH ), 3.62 (t, 2H, J = 6.0 Hz,
3
3
CH ); 3.89 (q, 2H, J = 6.0 Hz, CH ); 6.83 (br s, 1H, NH); 7.16 (d, 1H, J = 8.0, H-6); 7.36 (dt,
2
2
1
2
H, J = 7.5 and 1.0 Hz, H-5); 7.52 (dt, 1H, J = 7.5 and 1.5 Hz, H-4); 7.78 (dd, 1H, J = 8.0 and
.0 Hz, H-3).
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Synthesis of 2-((2-selenocyanatoethyl)carbamoyl)phenyl acetate (8). To a mixture of
potassium selenocyanate (1.15 mmol, 1 equiv) in anhydrous acetonitrile (25 mL) was added 2-
((2-bromoethyl)carbamoyl)phenyl acetate (7, 1.15 mmol, 1 equiv). The reaction mixture was
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stirred at room temperature under a nitrogen atmosphere for 16 h and filtered. The filtrate was
concentrated in vacuo and then partitioned between water and methylene chloride. The organic
layer was dried with magnesium sulfate and concentrated in vacuo. The crude product was
purified using silica gel chromatography with a hexane/ethyl acetate gradient (starting from 10%
1
up to 40% ethyl acetate) to afford the desired product. Overall yield 41%; mp 103-104 °C. H
NMR (500 MHz, CDCl ) δ 2.38 (s, 3H, CH ); 3.33 (t, 2H, J = 6.2 Hz, CH ); 3.89 (q, 2H, J = 6.1
3
3
2
Hz, CH ); 6.81 (s, 1H, NH); 7.15 (dd, 1H, J = 8.2 and 1.0 Hz, H-6); 7.34 (m, 1H, H-5); 7.53 (m,
2
1
3
1H, H-4); 7.78 (dd, 1H, J = 7.7 and 1.7 Hz, H-3). C NMR (125 MHz, CDCl ) δ 21.2 (CH ),
3
3
2
1
3
8.9 (CH -Se), 40.3 (CH -NH), 101.2 (SeCN), 123.3, 126.3, 127.3, 129.5, 132.4, 148.2 (aryl),
2 2
+
66.5, 169.0 (C=O). HRMS (ESI) calcd for C H N NaO Se [M + Na] : 334.9905. Found:
12 12
2
3
34.9905.
Synthesis of 2-selenocyanatoethyl 2-acetoxybenzoate (10). The title compound was
synthesized from 2-selenocyanatoethan-1-ol (9, 0.38 g, 2.5 mmol) and O-acetylsalicyloyl
chloride (6, 0.50 g, 2.5 mmol) according to the procedure described above for the preparation of
1
NSAID-SeCN derivatives, stirring the reaction mixture for 22 h. Overall yield 79%; mp 48-50 °C. H
NMR (500 MHz, CDCl ) δ 2.37 (s, 3H, CH ); 3.39 (t, 2H, J = 6.5 Hz, CH ); 4.69 (t, 2H, J = 6.5
3
3
2
Hz, CH ); 7.14 (dd, 1H, J = 8.0 and 1.0 Hz, phenyl H-3); 7.36 (td, 1H, J = 8.0 and 1.0 Hz, phenyl
2
H-5); 7.62 (td, 1H, J = 8.0 and 1.5 Hz, phenyl H-4); 8.07 (dd, 1H, J = 8.0 and 1.5 Hz, phenyl H-
1
3
6
). C NMR (125 MHz, CDCl ) δ 21.0 (CH ), 27.1 (CH -Se), 63.1 (CH -O), 100.5 (SeCN),
3 3 2 2
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22.3, 124.0, 126.2, 131.8, 134.5, 151.6 (aryl), 163.8, 169.6 (C=O). HRMS (ESI) calcd for
+
C H NNaO Se [M + Na] : 335.9746. Found: 335.9747.
12 11
4
General procedure for the preparation of selenoester derivatives (12 and 13). To a water
solution, previously filtered through celite, of sodium 2-acetoxybenzoselenate (11, 3.36 mmol, 1
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equiv), obtained in situ following the procedure reported by Sanmartin et al., 1,2-
dibromoethane (3.36 mmol, 1 equiv) dissolved in THF (20 mL) was added. The reaction mixture
was stirred at room temperature for 30 min, filtered through celite and extracted with methylene
chloride (3 x 20 mL). The organic layers were dried with magnesium sulfate and concentrated in
vacuo. The crude product contained a mixture of the compounds 12 and 13, which was separated
and purified using silica gel chromatography with a hexane/ethyl acetate gradient (starting from
5
% up to 15% ethyl acetate).
2
-(((2-Bromoethyl)selenyl)carbonyl)phenyl acetate (12). Overall yield 31%; mp 156−158 °C.
1
H NMR (500 MHz, CDCl ) δ 2.37 (s, 3H, CH ); 3.45 (t, 2H, J = 10.0 Hz, CH -Se); 3.61 (t, 2H,
3
3
2
J = 10.0 Hz, CH -Br); 7.14 (d, 1H, J = 8.0 Hz, phenyl H-3); 7.35 (t, 1H, J = 8.0 Hz, phenyl H-5);
2
1
3
7
.59 (td, 1H, J = 8.0 and 1.5 Hz, phenyl H-4); 7.89 (dd, 1H, J = 8.0 and 1.5 Hz, phenyl H-6). C
NMR (125 MHz, CDCl ) δ 21.2 (CH ), 27.1 (CH -Se), 30.7 (CH -Br), 124.1, 126.4, 129.8,
3
3
2
2
1
31.3, 134.2, 147.4 (aryl), 169.1, 190.6 (C=O). HRMS (ESI) calcd for C H O BrNaSe [M +
11 11 3
+
Na] : 372.8949. Found: 372.8950.
((Ethane-1,2-diylbis(selanediyl))bis(carbonyl))bis(2,1-phenylene) diacetate (13). Overall
1
yield 3%; mp 154-158 °C. H NMR (500 MHz, CDCl ) δ 2.38 (s, 6H, 2(CH )); 3.39 (s, 4H,
3
3
2
(CH )); 7.15 (dd, 1H, J = 8.0 and 1.0 Hz, phenyl H-3); 7.37 (td, 1H, J = 8.0 and 1.0 Hz, phenyl
2
H-5); 7.59 (td, 1H, J = 8.0 and 1.5 Hz, phenyl H-4); 7.93 (dd, 1H, J = 8.0 and 1.5 Hz, phenyl H-
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2
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2
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3
3
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13
6
). C NMR (125 MHz, CDCl ) δ 21.2 (CH ), 26.1 (CH -Se), 124.1, 126.3, 129.8, 131.7, 133.9,
3 3 2
+
1
47.3 (aryl), 169.2, 191.6 (C=O). HRMS (ESI) calcd for C H O NaSe [M + Na] : 536.9326.
20 18 6 2
Found: 536.9328.
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Synthesis of 2-(((2-selenocyanatoethyl)selenyl)carbonyl)phenyl acetate (14). The title
compound was synthesized by reacting potassium selenocyanate (0.11 g, 0.76 mmol) and 12
(
0.25 g, 0.71 mmol) according to the procedure described for the preparation of compound 8,
1
stirring the reaction mixture for 20 h. Overall yield 72%; mp 34−35 °C. H NMR (500 MHz,
CDCl ) δ 2.36 (s, 3H, CH ); 3.36 (dd, 2H, J = 10.0 and 6.0 Hz, CH -SeCN); 3.48 (dd, 2H, J =
3
3
2
1
0.0 and 6.0 Hz, CH -Se-C(=O)); 7.14 (d, 1H, J = 8.0 Hz, phenyl H-3); 7.35 (t, 1H, J = 8.0 Hz,
2
phenyl H-5); 7.59 (td, 1H, J = 8.0 and 1.5 Hz, phenyl H-4); 7.88 (dd, 1H, J = 8.0 and 1.5 Hz,
1
3
phenyl H-6). C NMR (125 MHz, CDCl ) δ 21.2 (CH ), 27.1 (CH -SeCN), 28.9 (CH -Se),
3
3
2
2
1
00.8 (SeCN), 124.2, 126.4, 129.8, 131.0, 134.4, 147.5 (aryl), 169.1, 190.6 (C=O). HRMS (ESI)
+
calcd for C H O NNaSe [M + Na] : 399.8962. Found: 399.8959.
1
2
11
3
2
Synthesis of 2-(2-acetoxybenzamido)-3-(methylselenyl)propanoic acid (15). The title
compound was synthesized from Se-(methyl)selenocysteine hydrochloride (1.09 g, 5.0 mmol)
and O-acetylsalicyloyl chloride (6, 1.00 g, 5.0 mmol) according to the procedure described for
the preparation of NSAID-SeCN derivatives, stirring the reaction mixture for 32 h. Overall yield
1
1
0%; mp 105−107 °C. H NMR (500 MHz, CDCl ) δ 2.05 (m, 3H, SeCH ); 2.45 (s, 3H, CH );
3
3
3
3.16 (t, 2H, J = 6.0 Hz, CH -Se); 5.12 (dd, 1H, J = 11.0 and 6.0 Hz, CH-NH); 7.18 (d, 1H, J =
2
8
4
.0 Hz, phenyl H-3); 7.35 (td, 1H, J = 8.0 and 1.0 Hz, phenyl H-5); 7.52-7.56 (m, 1H, phenyl H-
1
3
); 7.60 (d, J = 7.0 Hz, 1H, COOH); 7.96 (dd, 1H, J = 8.0 and 1.5 Hz, phenyl H-6). C NMR
(
125 MHz, CDCl ) δ 5.5 (Se-CH ), 21.3 (CH ), 26.7 (CH -Se), 52.6 (CH-NH), 123.4, 126.2,
3 3 3 2
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26.4, 130.7, 132.6, 148.3 (aryl), 165.2, 169.1, 174.2 (C=O). HRMS (ESI) calcd for
+
C H O NNaSe [M + Na] : 368.0008. Found: 368.0005.
13 15
5
Synthesis of 2-((2-thiocyanatoethyl)carbamoyl)phenyl acetate (16). The title compound was
synthesized from ammonium thiocyanate (0.08 mg, 1.1 mmol) and 7 (0.31 g, 1.1 mmol)
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11
12
13
14
15
16
17
18
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according to the procedure described for the preparation of compound 8, stirring the reaction
1
mixture for 18 h. Overall yield 54%; mp 37−40 °C. H NMR (600 MHz, CDCl ) δ 2.39 (s, 3H,
3
CH ); 3.25 (t, 2H, J = 6.0 Hz, CH -S); 3.84 (dd, 2H, J = 12.0 and 6.0 Hz, CH -NH); 6.79 (s, 1H,
3
2
2
NH); 7.16 (d, 1H, J = 8.0 Hz, phenyl H-3); 7.35 (t, 1H, J = 8.0 Hz, phenyl H-5); 7.51-7.55 (m,
13
1
2
H, phenyl H-4); 7.81 (dd, 1H, J = 8.0 and 1.5 Hz, phenyl H-6). C NMR (125 MHz, CDCl ) δ
3
1.2 (CH ), 33.2 (CH -S), 39.5 (CH -NH), 111.6 (SCN), 123.3, 126.3, 127.4, 129.6, 133.3, 148.2
3
2
2
+
(aryl), 163.3, 169.0 (C=O). HRMS (ESI) calcd for C H O N NaS [M + Na] : 287.0461. Found:
12 12 3 2
2
87.0461.
Reagents and antibodies for biological evaluation. Antibodies for western blot purpose were
ordered from following sources: Cell signaling (anti-PARP (9542s), cyclin E1 (4129), cyclin B1
(12231), caspase 3 (9668s), Mcl-1 (5453s) and Bcl-xL (2764s)); Abcam (Bcl-2 (ab59348));
Sigma-Aldrich (β-actin (A5316)); and Santa Cruz Biotechnology (p21 (sc6246)). Thiazolyl Blue
Tetrazolium Bromide (MTT) (M5655-500MG), Triton X-100 (93443), RNase A (R6513), and
Propidium iodide (PI) (P4170) were purchased from Sigma-Aldrich.
Cell culture conditions. MDA-MB-231, PANC-1, BxPC3, MEFs and 1205Lu cell lines were
maintained in DMEM medium; UACC903, DU145, and H460 cells were maintained in RPMI
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640 medium; HT29, SKOV3, HCT116 and RKO cells were maintained in McCoy's 5A
medium; HEPG2 cells were maintained in Eagle's Minimum Essential Medium (EMEM)
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supplemented with 10% fetal bovine serum (FBS) and 100 units/ml of penicillin and
streptomycin at 37 °C and 5% CO . nHDFs were grown in Fibroblast Basal Medium (ATCC
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PCS-201-030) supplemented with Fibroblast Growth Kit–Serum-Free (ATCC PCS-201-040) and
Penicillin-Streptomycin-Amphotericin B Solution (ATCC PCS-999-002). All the cell lines were
obtained from ATCC.
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Cell viability assay. The effect of the compounds on cancer cell viability was determined by the
7
5
MTT assay uptake method as described before. Briefly, 3000 cells were incubated with
different concentrations of the test compounds in triplicate in a 96-well plate for 24 h, 48 h and
7
2 h at 37 °C. Three hours prior to experiment termination, MTT solution (20 µL of 5.0 mg/mL
solution) was added to each well and incubated at 37 °C. At the termination time point, the
resultant formazan crystals were dissolved in DMSO, and the optical densities were measured at
5
70 nm and 630 nm using a 96-well multiscanner (Dynex Technologies, MRX Revelation;
Chantilly, VA, USA). Graphpad Prism software was used to calculate the appropriate IC values
5
0
calculated by non-linear regression analysis.
Live and dead assay: To determine dead cell population, we used LIVE/DEAD
Viability/Cytotoxicity Kit (Life technologies, USA). HCT116 cells were treated with either
DMSO, ASA (1 mM) or 8 (15 µM) for 24 h. Cells were trypsinized and subjected to live and
dead analysis using LIVE/DEAD Viability/Cytotoxicity Kit according to manufactures protocol.
Kit contains two components, calcein-AM and ethidium homodimer-1. Green-fluorescent
calcein-AM indicates intracellular esterase activity (live cells), while red-fluorescent ethidium
homodimer-1 indicates loss of plasma membrane integrity (dead cells). Stained cells were
pipetted on the slides and covered with cover slip. Pictures were taken using a fluorescence
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microscope (Zeiss - Axio Scope.A1) at 20X objective lens. Percentage of live and dead cells
were calculated.
Cell cycle analysis. Cell cycle analyses were determined by following a flow cytometry protocol
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as described earlier. In brief, HCT116 cells were serum deprived for 72 h to get them
synchronized. At the end of 72 h, cells were supplied with fresh medium or fresh medium with 5
μM of 8 for 12 and 24 h time points. After each time point the cells were fixed in ice cold 70%
ethanol. Fixed cells were washed with 1X PBS and suspended in PI staining solution. PI staining
solution was made by adding 0.1% (v/v) triton X-100, 20 mg DNAse-free RNAse A and 2 mg of
PI in 100 mL PBS. After staining for 15 minutes, the cells were analyzed by a BD FACS Calibur
(
BD Biosciences) for total DNA content.
Western blot analysis. Whole-cell lysates were made by subjecting 8 treated CRC cells to lysis
in RIPA lysis buffer (Thermo Scientific, USA) containing protease (Roche, USA) and
7
7
phosphatase inhibitor cocktails (Sigma-Aldrich, USA) as described before. The resulting
lysates were spun at 15,000 rpm for a duration of 10 minutes to remove any insoluble debris. The
resulting supernatant was stored at -80 °C until use. NuPAGE gel 4-12% (Life Technologies,
Carlsbad, CA) was used to resolve the lysates. After electrophoresis/SDS page, the resolved
proteins were electro-transferred to PVDF membrane and blotted with the different antibodies.
Enhanced Chemiluminescent reagent (Life technologies, USA) was used to detect the protein of
interest. In order to save time and samples, consecutive detections of different proteins
wasachieved by using Restore Western blot stripping buffer (Thermo Scientific, USA).
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Annexin V assay: In order to evaluate the ability of 8 to induce apoptosis, HCT116 (5 x 10 )
cells were treated with increasing dose of compound 8 or DMSO for 24 h. Different stages of
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apoptosis, in both floating and adherent cells, were measured using the Muse Annexin V & Dead
Cell kit with Muse cell analyzer (EMD Millipore, Billerica, MA, USA), according to
manufacturer’s protocol. The Muse Annexin V & Dead cell assay uses Annexin V to identify
phosphatidylserine (PS) on the external surface of apoptotic cells. The kit also has a dead cell
marker, 7-amino-actinomycin D (7-ADD), which does not enter live, healthy and early apoptotic
cells, with intact cell membrane (Millipore, Catlog No, MCH100105). Data obtained from the
equipment were analyzed using Muse 1.4 software.
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Caspase 3/7 activity assay: Activation of caspase 3/7 in HCT116 cells treated with compound 8,
was detected using Muse Caspase-3/7 Assay kit with Muse cell analyzer (EMD Millipore,
Billercia, MA. USA) according to manufacturer’s protocol. In brief, the Muse Caspase 3/7 kit
uses a reagent namely NucView for detection of activated caspase 3/7. This reagent is cell
membrane permeable and possesses no known toxicity towards cells. This reagent contains a
DNA binding dye that is linked to a DEVD peptide substrate. DEVD inhibits the dye from
binding to the DNA. Upon cleavage of the DEVD in presence of active caspase 3/7, DNA
binding dye is released and hence gives high fluorescence (Ref Millipore, Catlog No.
MCH100108). The kit also contains a dead cell marker, 7-ADD, for detection of dead cells with
compromised cell membrane, but excludes cells with intact cell membrane. Data from Muse cell
analyzer were analyzed using Muse 1.4 software.
ROS measurement: Measurement of total ROS levels in HCT116 cells, treated with 8, was
performed by using the Muse Oxidative Stress Kit (EMD Millipore, Billerica, MA) as per
manufacturer’s protocol. The kit utilizes the Muse Oxidative Stress Reagent for detection of
ROS inside the cells. The kit identifies two different cell population ROS (-) cells and ROS (+)
cells. ROS (-) population represented by M1 peak, while ROS (+) cells represented by M2 peak
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in the graph. H O (0.9 M) treated HCT116 cells were used as positive control for identification
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of M1 and M2 peaks. Percentage of ROS (-) and ROS (+) cells were measured using Muse cell
analyzer.
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ASSOCIATED CONTENT
1
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Supporting Information. Copies of the H NMR and C NMR spectra for new compounds 4,
5
, 8, 10 and 12-16, and SMILES and IC data (CSV). This material is available free of charge
50
via the internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: 1-717-531-4563. Fax: 1-717-533-0244. E-mail: aks14@psu.edu
Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval
ϕ
to the final version of the manuscript. Authors contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
The authors thank the Department of Pharmacology, Penn State College of Medicine, and Penn
State Hershey Cancer Institute for financial support. The authors thank the Flow Cytometry Core
and Solution Phase NMR Facility (Dr. Jyh-Ming Lin) at Core Research Facilities of the
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Pennsylvania State University, College of Medicine. DP acknowledges the support of his post-
doctoral fellowship by Anabasid program granted by Navarra´s Government.
ABBREVIATIONS
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ASA, Aspirin; Ibu, Ibuprofen; Nap, Naproxen; CRC, colorectal cancer; mTOR, mammalian
target of rapamycin; AMPK, AMP-activated protein kinase; MEFs, mouse embryonic
fibroblasts; nHDF, human dermal fibroblast; PS, phospatidylserine; ROS, Reactive Oxygen
Species.
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