Design, synthesis and biological evaluation of novel pyrenyl derivatives as anticancer agents

Article abstract of DOI:10.1016/j.ejmech.2014.09.072

Polycyclic aromatic hydrocarbons are widespread in nature with a toxicity range from non-toxic to extremely toxic. A series of pyrenyl derivatives has been synthesized following a four-step strategy where the pyrene nucleus is attached with a basic heterocyclic moiety through a carbon linker. Virtual screening of the physicochemical properties and druggability has been carried out. The cytotoxicity of the compounds (1-8) have been evaluated in vitro against a small panel of human cancer cell lines which includes two liver cancer (HepG2 and Hepa 1-6), two colon cancer (HT-29 and Caco-2) and one each for cervical (HeLa) and breast (MCF-7) cancer cell lines. The IC₅₀ data indicate that compound 6 and 8 are the most effective cytotoxic agents in the present set of pyrenyl derivatives, suggesting that having a 4-carbon linker is more effective than a 5-carbon linker and the presence of amide carbonyl groups in the linker severely reduces the efficacy of the compound. The compounds showed selectivity toward cancer cells at lower doses (<51/4M) when compared with the normal hepatocytes. The mechanism of action supports the cell death through apoptosis in a caspase-independent manner without cleavage of poly (ADP-ribose) polymerase (PARP), even though the compounds cause plasma membrane morphological changes. The compounds, whether highly cytotoxic or mildly cytotoxic, localize to the membrane of cells. The compounds with either a piperidine ring (6) or an N-methyl piperazine (8) in the side chain were both capable of circumventing the drug resistance in SKOV3-MDR1-M6/6 ovarian cancer cells overexpressing P-glycoprotein. Qualitative structure-activity relationship has also been studied.

Full text of DOI:10.1016/j.ejmech.2014.09.072

Accepted Manuscript
Design, synthesis and biological evaluation of novel pyrenyl derivatives as anticancer
agents
Debasish Bandyopadhyay, Jorge L. Sanchez, Adrian M. Guerrero, Fang-Mei Chang,
Jose C. Granados, John D. Short, Bimal K. Banik
PII:
S0223-5234(14)00891-5
10.1016/j.ejmech.2014.09.072
EJMECH 7379
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 19 July 2014
Revised Date: 19 September 2014
Accepted Date: 23 September 2014
Please cite this article as: D. Bandyopadhyay, J.L. Sanchez, A.M. Guerrero, F.-M. Chang, J.C.
Granados, J.D. Short, B.K. Banik, Design, synthesis and biological evaluation of novel pyrenyl
derivatives as anticancer agents, European Journal of Medicinal Chemistry (2014), doi: 10.1016/
j.ejmech.2014.09.072.
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Graphical abstract
Design, synthesis and biological evaluation of novel pyrenyl derivatives as anticancer agents
Debasish Bandyopadhyay, Jorge L. Sanchez, Adrian M. Guerrero, Fang-Mei Chang, Jose C. Granados, John D.
Short, and Bimal K. Banik

ACCEPTED MANUSCRIPT
Design, synthesis and biological evaluation of novel pyrenyl derivatives as
anticancer agents
1
1,2
1
2
Debasish Bandyopadhyay , Jorge L. Sanchez , Adrian M. Guerrero , Fang-Mei Chang ,
2
2,3,
1,
Jose C. Granados , John D. Short *, and Bimal K. Banik *
1
Department of Chemistry, The University of Texas-Pan American, 1201 West University Drive,
Edinburg, Texas 78539, USA
2
University of Texas Health Science Center at San Antonio-Regional Academic Health Center,
Medical Research Division, 1214 West Schunior Street, Edinburg, TX 78541, USA
3
Department of Pharmacology, University of Texas Health Science Center at San Antonio,
7
703 Floyd Curl Drive, San Antonio, TX 78229, USA
*
Corresponding authors, Ph: +1(956)6658741, Fax: +1(956)3845006, E-mails: banik@utpa.edu
or shortj@uthscsa.edu
ABSTRACT
Polycyclic aromatic hydrocarbons are widespread in nature with a toxicity range from non-
toxic to extremely toxic. A series of pyrenyl derivatives has been synthesized following a four-
step strategy where the pyrene nucleus is attached with a basic heterocyclic moiety through a
carbon linker. Virtual screening of the physicochemical properties and druggability have been
carried out. The cytotoxicity of the compounds (1−8) have been evaluated in vitro against a
small panel of human cancer cell lines which includes two liver cancer (HepG2 and Hepa 1−6),
two colon cancer ( HT−29 and Caco−2) and one each for cervical (HeLa) and breast (MCF-7)
cancer cell lines. The IC data indicate that compound 6 and 8 are the most effective cytotoxic
5
0
agents in the present set of pyrenyl derivatives, suggesting that having a 4-carbon linker is more
effective than a 5-carbon linker and the presence of amide carbonyl groups in the linker severely
reduces the efficacy of the compound. The compounds showed selectivity toward cancer cells at
lower doses (< 5 ꢀM) when compared with the normal hepatocytes. The mechanism of action

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supports the cell death through apoptosis in a caspase-independent manner without cleavage of
poly (ADP-ribose) polymerase (PARP), even though the compounds cause plasma membrane
morphological changes. The compounds, whether highly cytotoxic or mildly cytotoxic, localize
to the membrane of cells. The compounds with either a piperidine ring (6) or an N-methyl
piperazine (8) in the side chain were both capable of circumventing the drug resistance in
SKOV3-MDR1-M6/6 ovarian cancer cells overexpressing P-glycoprotein. Qualitative structure-
activity relationship has also been studied.
KEYWORDS
Polycyclic, PAH, Pyrene, Anticancer, Cytotoxicity, Apoptosis, Drug resistance.

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1
. Introduction
According to NASA’s announcement on February 21, 2014, more than 20% percent of the
carbon in the universe is confined in polycyclic aromatic hydrocarbons (PAHs) [1], some of
which are considered potent atmospheric pollutants [2]. These PAHs are widely distributed in
nature. For example, significant amounts of PAHs are found in natural crude oil and coal
deposits, and they are also found in processed fossil fuels, tar and various edible oils [3].
Nevertheless, some PAHs have been identified as carcinogenic, mutagenic or teratogenic [4].
PAHs are soluble in lipid, can be metabolized by cytochrome P450 enzymes, and can interact
with cellular components like protein and nucleic acid. Because of this metabolic activation of
PAHs, they are considered as indirect acting carcinogens [5]. The toxicity of polycyclic aromatic
hydrocarbons (PAHs) is structure-dependent. For example, different PAHs with the same
formula and number of rings can vary from being nontoxic to extremely toxic [6]. A key factor
in PAH toxicity is the formation of reactive metabolites, and not all PAHs are of the same
toxicity because of differences in structure that affect metabolism.
Alternatively, certain PAHs have been identified as antitumoral. For example, the antitumoral
anthracyclines (daunorubicin and doxorubicin) and the synthetic anthracene-9,10-dione
(mitoxantrone) are potent agents in clinical use today with broad application in the treatment of
several leukemias, lymphomas and solid tumors, as well as in combination chemotherapy of
solid tumors [7,8]. Additionally, some PAHs have been identified as DNA intercalating agents,
an important class of antitumoral DNA binders that are characterized by the insertion of their
planar aromatic or heteroaromatic rings between DNA base pairs. For example, anthracyclines,
acridines, and ellipticines are DNA intercalating agents [9,10] that are thought to poison
topoisomerases I and II [11].
Cancer is a leading cause of death worldwide and accounted for 8.2 million deaths in 2012
among which 70% occur in low- and middle-income countries. According to the World Health
Organization the annual cancer cases will rise from 14 million in 2012 to 22 within the next two
decades [12,13]. Therefore, a better understanding of how PAHs induce or inhibit carcinogenesis
could lower tumor incidence or help fight this disease. Cancer cells often adapt to develop
resistance to chemotherapeutic agents, in part through increased expression of protein efflux
pumps, which translocate drugs out of the cell [14]. Therefore, it is also important to develop

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novel chemotherapeutic agents, including PAHs, which are more potent tumor-selective
cytotoxic agents and can circumvent drug resistant cancer cells.
We reported the novel synthesis of chrysenyl or pyrenyl compounds coupled to side chains
using bismuth nitrate pentahydrate and microwave irradiation [15], an emerging field of research
in synthetic organic chemistry. In fact, we have demonstrated the synthetic uses of trivalent
bismuth nitrate pentahydrate in a number of examples, resulting in various methods which
includes but are not limited to, nitration of aromatic systems [16,17], Paal-Knorr synthesis of
pyrroles [18−21], electrophilic substitution of indoles [22], synthesis of α-aminophosphonates
[23], Hantzsch synthesis of 1,4-dihydropyridines [24], anticancer quinoxalines [25], bioactive
benzimidazoles [26] etc. Microwave-induced synthesis of bioactive molecules has also been
reported from our laboratory [27−30]. The previous reported pyrenyl compounds exhibited
autofluorescent properties and were moderately cytotoxic against selected cancer cell lines
(Table 3; Compound 1 and ref 15). However, the cellular and molecular effect(s) of these
molecules was not examined in our previous study. Herein, we have used organo-bismuth
chemistry to synthesize even more effective anticancer pyrenyl-coupled compounds, and we
have examined the molecular and cellular mechanisms by which these compounds cause
cytotoxicity. Importantly, we have shown these novel pyrenyl compounds to selectively target
cancer cell lines and are capable of circumventing MDR-1-mediated drug resistance in cancer
cells. A few analogous compounds derived from chrysene were reported previously [31−33]. The
rationale of synthesizing the pyrenyl derivatives reported in this paper is to introduce more
effective (against cancer cells) and less toxic (against normal cells) anticancer agents following
the U.S. Environmental Protection Agency (EPA) guidelines of polycyclic aromatic
hydrocarbons, since chrysene belongs to the list of “probable human carcinogens” while pyrene
belongs to “not classifiable as to human carcinogenicity” list [34].
2
. Results and Discussion
2
.1. Chemistry
The first step of the present synthesis involves bismuth nitrate induced nitration of pyrene
under microwave irradiation. The product 1-nitropyrene was successfully reduced to the
corresponding amine with excellent yield. Various linkers were introduced following a coupling
reaction between 1-aminopyrene and corresponding long chain acid. In the final step, the

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carbonyl group (both in amide and ester) was reduced by refluxing with borane in anhydrous
tetrahydrofuran (Scheme 1).
NO2
NH2
Bi(NO ) .5H O
3
3
2
In/H O/EtOH/NH Cl
2
4
KSF clay, MWI
X
N
O
X
CH Cl (Dry)
2
2
(
)n
O
+
O
Refulx, 4-6 h
N
(
)n
O
H
O
OH
–
0 to 5 ºC
and then RT
4 h
X = CH , NCH
n = 1, 2
(in dry CH Cl )
2
3
2
2
+
2
Et N
3
and then
O
CH3
CH3
in dry CH Cl )
Cl
O
(
2
2
O
X
X
N
N
(
)n
( )n
HN
HN
O
1.0 M Borane/THF
3
: n=1, X=CH2
Reflux, 24 h
4
: n=2, X=CH2
6
7
8
: n=1, X=CH2
5: n=1, X=NCH3
: n=2, X=CH2
: n=1, X=NCH3
+
O
CH3
CH3
HN
O
HN
O
CH3
CH3
1.0 M Borane/THF
Reflux, 24 h
(
2)
(1)
Scheme 1: Synthesis of the pyrenyl derivatives (1˗8)

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Here it is important to be mentioned that in an earlier investigation [32] it was found that the
anticancer activity of 3-carbon linker PAH derivatives was less than 5-carbon linker analogs. It
was reported that 5-carbon linker PAH derivatives were more active anticancer agents than 3-
carbon linker analogs but less active than 4-carbon linker analogs. Taken together, the activity of
3
-, 4-, and 5-carbon linker PAH derivatives were as: 4-carbon linker > 5-carbon linker > 3-
carbon linker. Based on this background information, the synthesis and anticancer screening of
the corresponding 3-carbon linker pyrene analogs were excluded in the present investigation.
2
2
.2. Biology
.2.1. Virtual screening for druggability
The computed molecular properties of the N-pyrenyl derivatives (1−8) are presented in Table
. The lipophilicity (fat-liking, miLogP), total polar surface area (TPSA), number of hydrogen
1
bond acceptor, number of hydrogen bond donor, number of rotatable bonds and molecular
volume have been calculated using Molinspiration online property calculation toolkit (version
2
013.09) [35] to validate the druggability [36−38] of the pyrenyl derivatives (1−8). The drug-
likeness model score (a collective property of physicochemical parameters, pharmacokinetics
and pharmacodynamics of a compound is represented by a numerical value) has been calculated
by MolSoft software [39]. The predicted bioactivities such as the ability of the pyrenyl
compounds (1−8) to act as G protein-coupled receptor (GPCR) ligand, ion channel modulator,
kinase inhibitor, nuclear receptor ligand, protease inhibitor and overall enzyme inhibitor have
also been calculated using the same toolkit (version 2011.06) [35]. The results are summarized in
Table 2.
2
.2.2. Analysis of spectral emission
A previous study from our lab showed that either chrysenyl- or pyrenyl-coupled derivatives,
including compounds 1 and 2, exhibited autofluorescent properties whereby addition of an alkyl
side chain to pyrene caused a red-shift in the spectral emission properties [15]. Therefore, to
determine whether the novel pyrenyl derivatives described in Scheme 1 also exhibited
autofluorescent properties, compounds were analyzed by fluorescence spectroscopy. Similar to
compound 1 and 2, compounds 6−8 exhibited peak fluorescent wavelengths ranging from 430
nm to greater than 450 nm, whereas the peak fluorescence of compounds 3−5 exhibited a peak

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fluorescent wavelength of approximately 400 nm, (Ref 15, Figure 1 and Supplemental Figure 1
of this article). These data indicate that each of the novel pyrenyl derivatives with either
piperidine or N-methyl piperazine exhibited autofluorescent properties, with the addition of the
various long chains to pyrene causing a red-shift in spectral emission when compared to pyrene,
since previous reports have identified that the peak emission wavelength of pyrene is less than
4
00 nm, with a shoulder for that emission peak at approximately 420 nm [40,41].
2
.2.3. In vitro cytotoxicity evaluation
To determine whether the novel pyrenyl-coupled derivatives with side chains containing
piperidine or N-methyl piperazine rings were cytotoxic, a small panel of cancer cell lines,
including liver cells (HepG2 and Hepa1-6), colon cancer cells (HT-29 and Caco-2), a cervical
cancer cell line (HeLa) and a breast cancer cell line (MCF-7) were treated with the eight novel
compounds (1-8) described in Scheme 1. While two of the compounds, 3 and 4, were not very
cytotoxic, the other compounds exhibited cytotoxicity against each of the cell lines tested (Table 1).
Furthermore, compounds 6 and 8 exhibited potent cytotoxicity against the cell lines tested with IC50
values of less than 5 µM (Table 3). These data indicate that compound 6 and 8 are the most
effective cytotoxic molecules of this set of pyrenyl derivatives, suggesting that having a 4-carbon
linker is more effective than a 5-carbon linker (compare compound 6 with compound 7) and that
a more polar side chain is more effective than having hydrophilic groups such as ketones
(compare compounds 6 and 8 with compounds 3 and 5, respectively) present in the side chain.
We also investigated the time at which the more potent compounds, 6 and 8, caused cytotoxicity
in cancer cells. Both compounds 6 and 8 caused cytotoxicity at a similar time-point in HepG2
cells when cells were treated with a dose that was two times greater than the IC value for the
5
0
indicated cell type, causing toxicity between six and 12 hours of treatment (Figure 2).
2
.2.4. In vitro evaluation of selective cytotoxicity
Upon identifying the cytotoxic pyrenyl derivatives containing piperidine or N-methyl
piperazine rings, we sought to determine whether these compounds were selectively cytotoxic
against a mouse cancer cell line (Hepa 1-6) in vitro when compared with normal hepatocytes. At
very high doses (50 µM), compound 6 and compound 8 killed both the cancer cells and the
normal hepatocytes (Figure 2); however, at lower doses (less than 5 µM) the pyrenyl compounds

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killed the cancer cell line much more effectively when compared with the normal hepatocytes
(
Figure 2). These data suggest that pyrenyl derivatives containing a four carbon linker with either
a piperidine or an N-methyl piperazine ring are capable of selectively killing certain cell types.
2
.2.5. Apoptosis through caspase-independent manner
To determine the mechanism by which compounds 6 and 8 caused cytotoxicity, the
morphology of HeLa cells was examined for the presence of membrane blebbing, an indicator of
cellular apoptosis. Membrane blebbing was detected in HeLa cells that were treated for 12 hours
with either compound 6 or compound 8 (Figure 3A, white arrows), and similar morphology was
detected when cells were treated for either 8 hours or 15 hours (data not shown). These data
suggest that the cytotoxic pyrenyl-coupled derivatives containing a piperidine or an N-methyl
piperazine ring cause cytotoxicity by apoptosis. However, there were also some cells with a
rounded and swollen morphology in the presence of either compound 6 or compound 8,
suggesting that these cells were undergoing necrosis (Figure 3A, black arrows).
To further determine whether compound 6 or 8 was inducing apoptosis, we examined
cleavage of poly (ADP-ribose) polymerase (PARP), a known proteolytic target of caspase
proteins during progression of apoptosis [42]. Surprisingly, neither compound 6 nor 8 induced
cleavage of PARP in cells that were treated with dosages less than 10 µM for 8 to 18 hours
(
(
Figure 3B and data not shown). Only when cells were treated with higher doses of compounds
10 µM) could a minimal level of cleaved PARP be detected (Figure 3B). These data suggest that
most of the cytotoxicity induced by compound 6 or compound 8 occurs in a caspase-independent
manner, even though the compounds are causing plasma membrane morphological changes.
2
.2.6. Localization to plasma membrane
Our group has previously shown that compound 2, which is not as cytotoxic as compounds 6
or 8 (see Table 3), is localized outside the cell nucleus in a punctate pattern, when examined by
epifluorescent microscopy [15]. To determine whether the cytotoxic pyrenyl-coupled derivatives
containing a four carbon linker with either a piperidine or an N-methyl piperazine ring were
capable of penetrating the cell and localizing to the nucleus, we compared the localization of
these compounds by epifluorescent microscopy. Similar to our previous study, compound 2
exhibited a punctate pattern that was not localized to the nucleus (Figure 4A). Similarly,

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compounds 6 and 8 exhibited a punctate pattern that appeared to be localized to the cytoplasmic
membrane (Figure 4A). However, some diffuse staining could also be detected throughout the
cell (Figure 4A), suggesting that some compound was getting into the nucleus. Therefore, we
next analyzed the localization of compounds along with BODIPY TR-conjugated ceramide
analogs, which localize to membrane fractions, by confocal microscopy. Each compound (2, 6,
and 8) was detected in a punctate pattern that was predominantly co-localized with BODIPY TR-
conjugated ceramide analogs, and the nucleus could be seen as an unstained component of the
labeled cells (Figure 4B). Taken together, these data indicate that novel pyrenyl derivatives,
whether highly cytotoxic or mildly cytotoxic, are localized to the membrane of cells.
Cells that were labeled with the fluorescent pyrenyl compounds and BODIPY TR-conjugated
TM
ceramide analogs were also subjected to biochemical fractionation using an OptiPrep density
gradient. Both ceramide analogs and the pyrenyl compounds were predominantly detected in the
less dense fractions (fractions 2−4), although some of the ceramide could be detected in heavier
fractions (predominantly fractions 8−9) as well (Figure 5). Fractions were also analyzed by
western blotting for two proteins that have previously been shown to localize to lipid rafts, Lyn
and Thy-1 [43], as well as Glyceraldehyde Phosphate Dehydrogenase (GAPDH). Interestingly,
Thy-1, which is a glycosylphosphatidylinositol (GPI)-anchored protein that binds to the outer
membrane leaflet of cells [44], was detected in fractions that contained much of the pyrenyl
compound as well as ceramide; however, both Lyn and GAPDH were detected in the denser
fractions (Figure 5). These data suggest that these novel pyrenyl compounds are localized to the
plasma membrane and are most likely located in inositol-rich regions within the membrane.
2
.2.7. Circumvention of P-glycoprotein-mediated drug resistance
Because the cytotoxic pyrenyl derivatives containing a piperidine or an N-methyl piperazine
ring (compound 6 and 8, respectively) were localized to the cytoplasmic membrane and not to
the cytoplasm, we hypothesized that these compounds might circumvent P-glycoprotein-
mediated drug resistance in tumor cells. Therefore, cell viability of SKOV3 cells or a retrovirally
transduced and subcloned SKOV3 cell line overexpressing P-Glycoprotien-1, SKOV3-MDR1-
M6/6 [45,46] was assessed after treatment with low doses (0-4 ꢀM) or high doses (0-25 ꢀM) of
Paclitaxel, 0-25 ꢀM compound 6, or 0-25 ꢀM compound 8 for 48 hours. These SKOV3-MDR1-
M6/6 cells have previously been shown to be resistant to cytotoxicity induced by anticancer

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drugs, including Daunorubicin and Paclitaxel, when compared with SKOV3 cells [45,46]. As
expected, SKOV3 cells were extremely susceptible to Paclitaxel-induced cell death, with and
IC value of less than 1 nM (Figure 6A, left panel). In contrast, SKOV3-MDR1-6/6 displayed a
5
0
clear resistance to Paclitaxel, which killed only 40% of cells, even at the highest dosage
administered (Figure 6A and 6B, right panels). In contrast, the novel cytotoxic pyrenyl
derivatives, compound 6 and 8, killed SKOV3 and SKOV3-MDR1-6/6 cells similarly, having
both a comparable IC value and maximum cell death percentage in each cell type (Figure 6C
5
0
and 6D). This comparative cytotoxicity data indicates that these novel pyrenyl derivatives with
either a piperidine or an N-methyl piperazine ring in their side chain bypasses the P-
glycoprotein-mediated resistance of SKOV3-MDR1-M6/6 cells.
2
.2.8. Qualitative structure-activity relationship
Our group has previously shown that coupling of nitrated PAHs, including nitrated pyrene,
with isobutyl chloroformate results in compounds that are autofluorescent and moderately
cytotoxic against certain cancer cell lines [15]. In an effort to improve the cytotoxic potency of
pyrenyl-coupled compounds, we have coupled side chains to nitrated pyrene that contain either
piperidine or N-methyl piperazine in this study. We found that either piperidine (compound 6) or
N-methyl piperazine (compound 8) coupled to nitrated pyrene through a four carbon linker
lacking keto groups were most potent at causing cytotoxicity in cancer cells. We have also
shown that these compounds can selectively ‘kill’ cancer cells when compared with primary
cells and circumvent P-glycoprotein-mediated drug resistance in cancer cells in vitro, possibly
because the mechanism of action for these molecules occurs through their interaction with the
cellular membrane.
Other anticancer agents that contain either a piperidine or an N-methyl piperazine ring include
Raloxifene (a selective estrogen receptor modulator), Imatinib (a tyrosine kinase inhibitor), and
Bosutinib (a Src kinase inhibitor) [47−49]. In fact, the piperidine ring of Raloxifene is critical for
its antagonistic effect on Estrogen Receptor activity [50]. The piperidine ring of Raloxifene is
coupled to its adjacent benzene ring through an ester bond along with a 2-carbon linker [48],
while the N-methyl piperazine of Imatinib or Bosutinib is linked through a 1-carbon or an ester
along with a 3-carbon linker, respectively [47,49]. Our data indicate the a four carbon linker
connecting piperidine to the pyrenyl backbone is more potent when compared to compounds

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with a five carbon linker (Table 3; compare compound 6 with 7) and that the presence of amide
carbonyl groups in the linker severely reduces the efficacy of the compound (Table 3; compare
compound 3 with 6 or compound 5 with 8). These highly cytotoxic compounds were also
selectively cytotoxic, killing rapidly growing cancer lines at much lower doses than non-dividing
primary cells (Figure 2). Perhaps reducing the number of carbons in the linker between the
piperidine or N-methyl piperazine ring and the pyrenyl backbone even further will render future
compounds even more potent or selective.
Cytotoxicity can be caused through multiple processes, including apoptosis, apoptosis-like
programmed cell death (e.g., autophagy, mitotic catastrophe, and paraptosis), necrosis, or
necrosis-like programmed cell death [51]. A major difference between apoptosis and other types
of cell death is that caspase proteins are activated during progression of apoptosis [51]. Both
Raloxifene, which contains a piperidine moiety, and Imatinib, which contains an N-methyl
piperazine moiety, have been shown to induce caspase activation in various cancer cell types,
including HeLa cells [52−58]. In addition, a previous study has shown that pyrenyl coupled with
a maleimide moiety causes cell death through caspase activation in T-cell leukemia-derived
Jurkat cells [59]. However, the most potent cytotoxic compounds examined in this study (6 and
8
) caused cellular membrane blebbing, which is indicative of apoptosis, but did not induce
caspase activation in HeLa cells (Figure 3). These data suggest that pyrenyl-coupled cytotoxic
agents may kill different cell types through different mechanisms or that the moiety that is
coupled to the pyrenyl may change the cytotoxic mechanism of the compound. Furthermore,
these data suggest that pyrenyl coupled with a side chain with either a piperidine or N-methyl
piperazine ring causes caspase-independent cell death, which has been shown to occur when
certain cancer cell types were treated with other types of cytotoxic agents including Cladribine,
Paclitaxel, or Vitamin D [51]. Thus, it is possible that the novel cytotoxic pyrenyl-coupled
compounds described here induce apoptosis-like programmed cell death, cellular necrosis,
necrosis-like programmed cell death, or a combination of these cell death mechanisms. This is a
plausible scenario, since Imatinib has previously been shown to induce cell death in leukemic
cells through two separate mechanisms: (i) apoptosis and (ii) a caspase-independent, necrosis-
like manner that was blocked by a serine protease inhibitor [60].
In addition to killing several different types of cancer cell lines, the novel pyrenyl-coupled
compounds with either a piperidine ring (6) or an N-methyl piperazine (8) in the side chain were

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both capable of circumventing the drug resistance in SKOV3-MDR1-M6/6 ovarian cancer cells
overexpressing P-glycoprotein (Figure 6). These cells were previously shown to exhibit a robust
resistance to Paclitaxel (Figure 6 and ref [46]). P-glycoprotein is one of nine Multidrug Resistant
Proteins (MRPs), and MRPs are expressed in various tissues of the body and capable of
transporting numerous physiological substrates or anticancer drugs out of cells [61]. Thus, it
would be interesting to assess whether the pyrenyl-coupled compounds described here or
additional pyrenyl-coupled compounds circumvented the multidrug resistance mediated by
MDR1 and other MDR proteins in vitro and in vivo.
One possibility for the mechanism by which the novel pyrenyl-coupled compounds described
here kill cancer cells potently and selectively, as well as circumvent the P-glycoprotein-mediated
drug resistance, is that these compounds localize to the plasma membrane of target cells and do
not enter the cytoplasm or intracellular organelles of the cell to cause cytotoxicity. Both
compound 6 and 8 co-localized with a BODIPY-labeled sphingolipid at punctate regions on the
cell surface when examined by microscopy (Figure 4) and in similar subcellular fractions when
examined by biochemical fractionation of cells. These BODIPY-labeled sphingolipids have been
reported to localize along the endocytic pathway, including the plasma membrane, endosomes,
lysosomes, and the Golgi apparatus [62]. However, the lipid is primarily integrated in the plasma
membrane when incubated at cold temperatures, as described here [62].
3
. Conclusion
The cytotoxic pyrenyl-coupled compounds were primarily detected in cellular fractions
containing the Thy-1 protein (CD90), a GPI-anchored protein found on the outer leaflet of the
lipid bilayer [44], but not with GAPDH, an intracellular protein that has been shown to interact
with phosphatidylserine [63]. Thus, our data suggest that the cytotoxic pyrenyl-coupled
compounds are localized to the outer leaflet of the plasma membrane of target cells. However,
the presence of pyrenyl-coupled cytotoxic compounds in endosomes or lysosomes cannot be
ruled out by these results, since Thy-1 has been observed to localize at the plasma membrane and
within endolysosomes [64]. Nonetheless, if these novel pyrenyl-coupled compounds potently and
selectively kill cancer cells and circumvent drug resistance by binding to the plasma membrane
or lysosome and causing a caspase-independent programmed cell death event, then they could
become chemotherapeutic drugs with clinical usefulness.

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4
4
4
. Experimental
.1. Chemistry
.1.1. General
Unless otherwise stated, all materials were obtained from commercially available sources and
were used without purification. Pyrene, bismuth nitrate pentahydrate, montmorillonite KSF clay,
indium, ammonium chloride, isobutyl chloroformate, 1.0 M borane in tetrahydrofuran, and
dimethylsulfoxide (DMSO) were purchased from (Sigma-Aldrich Corporation, St. Louis, MO)
while other solutions like phosphate-buffered saline (PBS), Dulbecco’s Modified Eagle’s
Medium (DMEM), Fetal Bovine Serum (FBS), and McCoy’s media were purchased from
Invitrogen (Carlsbad, CA). All other chemicals were purchased from Sigma-Aldrich Corporation
(
analytical grade). Throughout the project solvents were purchased from Fisher-Scientific
Pittsburgh, PA). Deionized water was used for the preparation of all aqueous solutions.
Melting points were determined in a Fisher Scientific electrochemical Mel-Temp* manual
(
melting point apparatus (Model 1001) equipped with a 300°C thermometer. FT-IR spectra were
TM
registered on a Thermo Nicolet NEXUS 470 FT-IR E.S.P. spectrophotometer as KBr discs.
1
13
H-NMR (600 MHz) and C-NMR (150 MHz) spectra were obtained at room temperature with
TM
Bruker superconducting Ultrashield Plus 600 MHz NMR spectrometer with central field 14.09
Tesla, coil inductance 89.1 Henry and magnetic energy 1127.2 kJ using CDCl as solvent.
3
Elemental analyses (C, H, N) were conducted using the Perkin-Elmer 2400 series II elemental
analyzer, their results were found to be in good agreement (± 0.2%) with the calculated values
for C, H, N.
4
4
.1.2. Synthesis of the compounds 1−8
.1.2.1. Nitration of pyrene
Pyrene (1 mmol) and montmorillonite KSF clay (500 mg, Aldrich) were added to a
suspension of bismuth nitrate pentahydrate (1 equiv.) in anhydrous dichloromethane (10 mL).
The solvent was then evaporated under reduced pressure and the reaction mixture was irradiated
using microwave irradiation for 6 min (2x3 min). A 500 mL glass beaker full with ice was used
as heat sink. Every after 2 min the reaction was monitored by TLC. After completion of the

ACCEPTED MANUSCRIPT
reaction the reaction mixture was washed with dichloromethane (3x 5mL) and the solvent was
evaporated by reduced pressure distillation. The pure product (1-nitropyrene) was isolated by
crystallization from dichloromethane/hexanes mixture in excellent yield (~ 80%).
4
.1.2.2. Reduction of 1-nitropyrene to the 1-aminopyrene
-Nitropyrene (1 mmol) and indium (570 mg), 2.5 mL ethanol and 2.5 mL 20% aqueous
1
ammonium chloride solution was refluxed vigorously for 24 hrs (monitored by TLC). After
completion of the reaction it was filtered through Büchner funnel and the filtrate was extracted
with dichloromethane (2x 3 mL). The dichloromethane layer was washed with brine and water
successively and dried over anhydrous sodium sulfate. The pure amine was isolated by
crystallization from dichloromethane/hexanes mixture in excellent yield (86%).
4
.1.2.3. Synthesis of the acidic chains
The anhydride (n=1, succinic anhydride and n=2, glutaric anhydride) and piperidine (1:1
molar ratio) were dissolved in anhydrous dichloromethane (10 mL) and the mixture was refluxed
for 4―6 hrs; cooled down at room temperature. The crystals were filtered through vacuum,
washed successively with diethyl ether and hexanes and dried in desiccator.
4
.1.2.4. Coupling of 1-aminopyrene with the acidic chains in the presence of isobutyl
chloroformate
1-Aminopyrene (1 mmol) was stirred with dry triethylamine (3 mmol) in anhydrous
dichloromethane (5 mL) at a temperature –5 to 0°C and isobutyl chloroformate (1.8 mmol in 2
mL anhydrous dichloromethane) was added drop wise during an hour. After the addition was
over, the mixture was stirred for 24 hours at room temperature. After completion of the reaction
(monitored by TLC), the mixture was washed with saturated solution of sodium bicarbonate,
brine and water successively. The pure product was isolated after column chromatography over
silica gel (>70% yield).
4
.1.2.5. Reduction of the diamides (3―5) to diamines (4―6)
mmol of the diamide was refluxed with 6 mL of 1.0 M borane/tetrahydrofuran solution for
6 hours and then 5 mL of 4% aqueous hydrochloric acid solution was added and the mixture
1
3

ACCEPTED MANUSCRIPT
was again refluxed for another 24 hrs. After completion of the reaction (monitored by TLC), the
pH of the solution was changed to ~7.0 by 10% aqueous sodium hydroxide solution and the
mixture was extracted with ethyl acetate for three times (3x3 mL). The combined organic layer
was washed with brine and water successively. The pure diamine was isolated after column
chromatography (10% methanolic ethyl acetate) over silica gel (~70% yield).
4
.1.2.6. Reduction of (1) to (2):
Compound (1) was formed as a byproduct during diamide synthesis. Following the previous
protocol of diamide reduction it was reduced to compound (2) and finally purified by column
chromatography (15% ethyl acetate in hexanes) over silica gel (~60% yield).
4
.1.2.7. Spectroscopic data for characterization of the compounds
The spectral and analytical data of the compounds 1 and 2 are already reported [15] from our
laboratory. The data for the compounds (3−8) are as follows:
4
.1.2.7.1. 4-Oxo-4-(piperidin-1-yl)-N-(pyren-4-yl)butanamide (3): Brown crystalline solid
-1 1
(
74%); mp 163−164 °C; IR (KBr) 3265, 2926, 1670, 1624, 1508, 838, 711 cm ; H NMR (600
MHz, CDCl ) δ 1.46 (m, 6H), 2.75 (t, J = 6.36 Hz, 2H), 2.89 (t, J = 5.70 Hz, 2H), 3.32 (m, 2H),
3
3
9
1
1
7
4
.56 (t, J = 5.40 Hz, 2H), 7.98 (m, 7H), 8.14 (d, J = 9.18 Hz, 1H), 8.44 (d, J = 8.28 Hz, 1H),
1
3
.59 (s, 1H); C NMR (150 MHz, CDCl
3
) 26.41, 25.60, 26.29, 29.70, 33.08, 43.22, 46.51,
20.92, 121.41, 122.88, 124.69, 124.81, 125.09 (2C), 125.16, 125.97, 126.39, 127.36, 127.52,
28.49, 130.91 (2C), 131.39, 170.50, 171.99. Anal. Calcd for C25
.29. Found: C, 78.01; H, 6.23; N, 7.20.
24 2 2
H N O : C, 78.10; H, 6.29; N,
.1.2.7.2. 5-Oxo-5-(piperidin-1-yl)-N-(pyren-4-yl)pentanamide (4): White amorphous solid
71%); mp 102-104 °C; IR (KBr) 3232, 2933, 1685, 1647, 1555, 1521, 1437, 1266, 843, 710
(
-1 1
cm ; H NMR (600 MHz, CDCl ) δ 1.53 (m, 8H), 2.07 (q, J = 6.84, 13.62 Hz, 2H), 2.59 (t, J =
3
6
.96 Hz, 2H), 3.40 (t, J = 7.02 Hz, 2H), 3.52 (t, J = 5.34 Hz, 2H), 7.87 (t, J = 7.56 Hz, 2H), 7.91
(d, J = 10.56 Hz, 1H), 8.02 (m, 4H), 8.12 (d, J = 9.12 Hz, 1H), 8.32 (d, J = 8.22 Hz, 1H), 9.06 (s,
1
3
1
H); C NMR (150 MHz, CDCl ) 21.85, 24.49, 25.65, 26.59, 32.12, 36.68, 42.83, 46.82,
3
1
20.87, 121.99, 123.37, 124.75, 124.80, 125.11 (2C), 125.17, 126.00, 126.56, 127.32, 127.65,
28.78, 130.88, 131.06, 131.35, 171.02, 172.21. Anal. Calcd for C H N O : C, 78.36; H, 6.58;
1
2
6
26
2
2
N, 7.03. Found: C, 78.17; H, 6.41; N, 7.09.

ACCEPTED MANUSCRIPT
4
.1.2.7.3. 4-(4-Methylpiperazin-1-yl)-4-oxo-N-(pyren-4-yl)butanamide (5): Brownish white
amorphous solid (68%); mp 160−161 °C; IR (KBr) 3289, 2929, 1646, 1555, 1526, 1429, 1287,
-
1 1
1
3
007, 831, 705 cm ; H NMR (600 MHz, CDCl ) δ 1.91 (broad s, 1H), 2.27 (s, 3H), 2.37 (m,
3
H), 2.86 (m, 2H), 3.00 (m, 2H), 3.50 (broad s, 2H), 3.73 (broad s, 2H), 7.99 (m, 3H), 8.06 (d, J
=
9.00 Hz, 1H), 8.15 (m, 3H), 8.21 (d, J = 9.06 Hz, 1H), 8.50 (d, J = 7.92 Hz, 1H), 9.48 (s, 1H);
1
3
3
C NMR (150 MHz, CDCl ) 29.49, 32.84, 41.98, 45.30, 45.95, 54.61, 54.88, 120.83, 121.60,
1
1
6
4
23.05, 124.77, 125.15 (2C), 126.02, 126.50, 127.34, 127.60, 128.63, 130.87, 131.18, 131.37,
70.73, 171.76. Anal. Calcd for C H N O : C, 75.16; H, 6.31; N, 10.52. Found: C, 75.01; H,
2
5
25
3
2
.40; N, 10.37.
.1.2.7.4. N-(4-(piperidin-1-yl)butyl)pyren-4-amine (6): Yellowish green crystalline solid
-1 1
(
69%); mp 184−185 °C; IR (KBr) 3339, 2933, 1601, 1528, 1283, 839, 724 cm ; H NMR (600
MHz, d -DMSO) δ 1.34 (m, 1H), 1.80 (m, 8H), 3.01 (m, 3H), 3.20 (t, J = 7.56 Hz, 1H), 3.26 (dd,
6
J = 6.48 Hz, 1H), 3.41 (m, 4H), 6.85 (t, J = 5.40 Hz, 1H), 7.05 (d, J = 8.70 Hz, 1H), 7.32 (m,
1
H), 7.44 (m, 1H), 7.64 (m, 1H), 7.88 (m, 1H), 8.01 (m, 1H), 8.05 (d, J = 8.46 Hz, 1H), 8.46 (d,
1
3
J = 9.30 Hz, 1H), 10.63 (s, 1H); C NMR (150 MHz, d -DMSO) 20.89, 21.44, 22.21, 25.57,
6
2
1
7.50, 42.40, 51.74, 51.77, 55.48, 108.20, 115.52, 121.10, 121.64, 121.95, 122.51, 122.97,
24.40, 125.08, 125.54, 125.95, 126.75, 127.74, 131.52, 132.08, 143.51. Anal. Calcd for
C H N : C, 84.23; H, 7.92; N, 7.86. Found: C, 84.04; H, 7.86; N, 7.77.
25
28
2
4
.1.2.7.5.
N-(5-(piperidin-1-yl)pentyl)pyren-4-amine (7): Brown solid (67%); mp 65-67 °C;
-1 1
IR (KBr) 3347, 2928, 1620, 1525, 1461, 1266, 1042, 759 cm ; H NMR (600 MHz, d -DMSO)
6
δ 1.40 (m, 12H), 1.63 (m, 1H), 2.20 (m, 4H), 2.98 (t, J = 8.16 Hz, 1H), 3.20 (m, 2H), 7.03 (d, J =
8
7
8
2
1
8
4
.64 Hz, 1H), 7.28 (d, J = 8.46 Hz, 1H), 7.32 (d, J = 7.02 Hz, 1H), 7.39 (d, J = 8.76 Hz, 1H),
.44 (t, J = 7.26 Hz, 1H), 7.58 (d, J = 8.76 Hz, 1H), 7.67 (m, 1H), 7.87 (m, 1H), 7.98 (m, 1H),
1
3
.39 (d, J = 9.30, 1H); C NMR (150 MHz, d
6
-DMSO) 23.23, 24.20, 24.81, 26.31, 27.52, 28.86,
9.78, 43.26, 54.10, 58.66, 108.14, 112.00, 114.67, 121.81, 122.12, 122.40, 126.08, 126.32,
26.65, 126.74, 126.86, 127.74, 128.10, 128.62, 134.33, 143.79. Anal. Calcd for C26
4.28; H, 8.16; N, 7.56. Found: C, 84.13; H, 8.04; N, 7.39.
30 2
H N : C,
.1.2.7.6.
N-(4-(4-methylpiperazin-1-yl)butyl)pyren-4-amine (8): Dark green solid (64%);
mp 72−73 °C; IR (KBr) 3403, 2925, 2783, 1593, 1515, 1441, 1277, 1162, 1131, 1001, 820, 756,
-
1 1
7
7
32 cm ; H NMR (600 MHz, d -DMSO) δ 1.53 (m, 2H), 1.64 (m, 2H), 2.28 (s, 3H), 2.30 (m,
6
H), 2.98 (t, J = 7.80 Hz, 2H), 3.38 (m, 3H), 7.06 (d, J = 8.52 Hz, 1H), 7.32 (m, 1H), 7.39 (d, J =

ACCEPTED MANUSCRIPT
8
2
2
1
.70 Hz, 1H), 7.44 (t, J = 7.32 Hz, 1H), 7.59 (d, J = 8.64 Hz, 1H), 7.64 (dd, J = 10.74, 3.84 Hz
1
3
H), 7.88 (m, 1H), 8.00 (m, 1H), 8.39 (d, J = 9.30, 1H); C NMR (150 MHz, d -DMSO) 21.36,
6
3.96, 26.76, 27.52, 43.14, 45.77, 52.70, 54.80, 57.52, 112.08, 114.68, 121.06, 122.12, 123.89,
25.20, 126.09, 126.31, 126.65, 126.87, 128.10, 131.03, 134.35, 143.78 . Anal. Calcd for
C H N : C, 80.82; H, 7.87; N, 11.31. Found: C, 80.70; H, 7.71; N, 11.27.
25
29
3
4
4
.2. Biology
.2.1. Mammalian cell culture and viability assays
HepG2, Hepa1-6, HeLa, and MCF-7 cells were cultured in Dulbecco’s Modified Eagle’s
Medium (DMEM) (Invitrogen, Carlsbad, CA) containing 10% Fetal Bovine Serum (FBS,
Invitrogen); Caco-2 cells were cultured in DMEM containing 20% FBS, and HT-29 cells were
cultured in McCoy’s media (Invitrogen) containing 10% FBS. SKOV3, and SKOV3-MDR1-M6/6
cells were grown in Basal Medium Eagle (BME) media containing 10% FBS. All cell lines were
purchased from American Type Culture Collection (ATCC, Manassas, VA) except for MCF-7, which
were provided by Dr. R.K. Dearth (UTPA), as well as SKOV3 and SKOV3-MDR1-M6/6 cells, which
o
were provided by Dr. Susan Mooberry (UTHSCSA). All cells were incubated at 37 C with 5% CO
2
.
Cell viability assays were performed as described previously [15], and 10-ꢀM doxorubicin was
used as a positive control for cytotoxicity.
4
.2.2. Spectral analysis of the compounds (1―8)
The autofluorescent properties of compounds (2-8) were analyzed as described previously
[15]. Briefly, each compound was excited with light at a wavelength of 350 nm, and the
subsequent light emission was analyzed between the wavelengths of 350-700 nm using a
SpextraMaxM5 plate reader (Molecular Devices, Sunnyvale, CA).
4
.2.3. Apoptosis assays
To detect membrane blebbing, HeLa cells were plated onto 6-well dishes at a density of 1.5-
5
2
.0 x 10 cells/well. Cells were then treated with the compound 6 or 8 at the indicated dosages
and visualized after 12 hrs using an Olympus CKX31 culture microscope (Olympus Corp.,
Tokyo, Japan). Images were captured using a Nikon Coolpix E995 eyepiece camera (Nikon,
Japan).

ACCEPTED MANUSCRIPT
For PARP cleavage, HeLa cells were treated with the indicated dosages of compound 6 or 8
for 12-hrs, and cells were lysed using 0.5% SDS-Lysis Buffer (50mM Tris - pH 8.0, 0.5% SDS,
1
mM DTT, 2.5mM sodium pyrophosphate, 1mM β-glycerophosphate, 1mM Na VO , and 1X
3 4
Roche Complete Protease Inhibitors) . Lysates were analyzed by western blotting using an anti-
PARP antibody (Cell Signaling Technology, Danvers, MA), and Staurosporine was used as a
positive control for PARP cleavage.
4
.2.4. Subcellular localization
For localization of compounds, HepG2 cells were plated onto 6-well dishes (250,000
o
cells/well) containing 12-mm glass cover slips and incubated overnight at 37 C. The following
day, experimental groups were treated with compound 2 (50ꢀM) for 4 hrs, or with compound 6
or 8 (30ꢀM) for 3 hours. Treated cells were then fixed and visualized as described previously
[15]. Briefly, treated cells were visualized using a Zeiss AxioImager.Z1 epifluorescent
microscope (Carl Zeiss Microimaging, LLC, Thornwood, NY) with EGFP (Excitation – 470/40;
Emission – 525/50) and DAPI filters, and images were acquired using AxioVision Rel. 4.6
software (Zeiss).
For co-localization of compounds with ceramide, cells (200,000 cells/well) were plated onto
o
1
2-well dishes (Costar) containing 12-mm glass cover slips and incubated overnight at 37 C. The
following day, experimental groups were treated with compound 2 (50 ꢀM) for 4 hrs or with
compound 6 or 8 for 3 hours. After treatment, cells were rinsed several times with PBS
containing powdered Bovine Serum Albumin (BSA) (0.34 mg/mL) and then stained with 5-ꢀM
o
BODIPY TR-labeled sphingolipids (Molecular Probes) for 30 minutes at 4 C. Cells were then
fixed with PBS containing 4% paraformaldehyde for 25 minutes at room temperature and
permeabilized using PBS containing 0.2% Triton-X-100 for 5 minutes at room temperature.
Coverslips were then mounted onto glass slides using ProLong Gold Antifade mounting media
(
Invitrogen) and visualized using an Olympus Fluoview FV1000 Confocal microscope (Olympus
America Inc. Center Valley, PA, USA) with Cyanine 3.5 (Excitation – 543; Emission - 592) and
AlexaFluor405 (Excitation – 405; Emission - 415/75) channels engaged. Z-stacked composites
were generated across a 10 ꢀm field at 1ꢀm increments under 60x oil immersed objective
magnification and 4.0 digital zoom.

ACCEPTED MANUSCRIPT
4
.2.5. Biochemical fractionation
6
HepG2 cells were plated onto 10-cm dishes (1x10 cells/plate) and incubated overnight at 37°
C. The following day, cells were treated with compound 2 (5ꢀM) for 4 hrs, or cells were treated
with compound 6 or 8 (3ꢀM) for 2 hrs. After treatment, plates were washed with PBS containing
defatted BSA and stained with 5-ꢀM BODIPY TR-labeled sphingolipids (Molecular Probes) for
o
3
0 min at 4 C. After exposure to modified sphingolipids, cells were scraped from plates in PBS
containing 1X Protease Inhibitors (Roche) and pelleted by centrifugation for 5 min at 3,000 x g.
After thorough washing, cells were lysed with PBS containing 0.5% Brij-96 and protease
inhibitors for one hour. Protein lysates were then quantified by Bicinchoninic Acid Assay (BCA)
and normalized. Equal mass of protein lysate (600 µL) for each treatment was then loaded onto a
density gradient composed of 600-µL step-wise gradients (2.5%, 10%, 20%, and 30 %) of
OptiPrep (Sigma), and then separated by ultra-centrifugation at 125,000 g for 12 hrs with no
braking. Equal volume fractions (300 ꢀL) were then collected from the density gradient, and a
portion of each fraction was analyzed by spectral analysis using a SpectraMax M5
Spectrophotometer (Molecular Devices) to detect both compounds (Ex. 350 nm /Em. 450 nm)
and BODIPY TR-labeled sphingolipids (Ex. 589 nm; Em. 617 nm) in each fraction. In addition,
a portion of each fraction collected was used for western blotting analysis using an antibody for
Lyn or Thy-1 (Cell Signaling Technology), or an antibody for GAPDH (Santa Cruz Biotech.).
Acknowledgments
We gratefully acknowledge the funding support from National Cancer Institute (NIH/NCI-
P20, Grant# 5P20CA138022-02) and Kleberg Foundation of Texas.
Supporting data
The following information can be found online: (i) The fluorescence emission spectra of three
compounds (4, 5, and 7), (ii) the time-course for compounds 6 and 8) causing cytotoxicity of
HepG2 cells, and (iii) the infrared (IR), proton magnetic resonance (PMR) and carbon magnetic
resonance (CMR) spectra of the compounds 3−8
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Table 1. In silico physicochemical properties and drug-likeness of the N-pyrenyl derivatives
†
(1−8)
Compound miLogP* TPSA** Mol.
No. of H- No. of No. of
bond H-
Molecular Drug-
2
No.
(Å )
wt.
rotatable Volume
likeness
Model
Score
-0.14
-0.69
0.40
3
acceptor bond
donor
bond
(Å )
1
2
3
4
5
6
7
5.857
5.755
4.783
5.288
3.766
6.14
38.332
21.261
49.407
49.407
52.645
15.265
15.265
18.503
317.388 3
303.405 2
384.479 4
398.506 4
399.494 5
356.513 2
1
1
1
1
1
1
1
1
4
5
4
5
4
6
7
6
295.724
293.542
358.526
375.328
371.069
354.163
370.965
366.706
0.26
0.96
0.34
0.34
6.646
5.124
370.54
371.528 3
2
8
0.52
†
Calculated by Molinspiration property engine v2013.09
*
lipophilicity
*
*total polar surface area
‡
Table 2. In silico predicted bioactivity of the N-pyrenyl derivatives (1−8)
Compound GPCR
Ion channel
modulator
Kinase
inhibitor
Nuclear
receptor
ligand
0.12
-0.07
-0.16
-0.15
-0.20
-0.00
-0.00
Protease
inhibitor
Enzyme
inhibitor
No.
ligand
1
2
3
4
5
6
7
0.16
0.02
0.10
0.09
0.10
0.20
0.19
0.24
0.20
0.01
0.01
0.01
0.03
0.18
0.17
0.21
0.09
0.03
0.01
0.01
0.06
0.19
0.18
0.26
0.25
0.04
0.09
0.08
0.06
0.11
0.10
0.10
0.11
-0.06
0.00
0.00
-0.01
0.11
0.11
0.11
8
-0.05
‡
Calculated by Molinspiration bioactivity score v2011.06

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Table 3. IC50 values (ꢀM) of pyrenyl derivatives against a panel of cancer cell lines
HepG2
Hepa1-6
HT-29
HeLa
MCF7
Caco-2
Compounds Cells
†
†
†
†
†
†
1
2
3
4
5
6
7
8
30.4±5.0
39.6±7.1
>30
5.9±2.5
9.9±5.2
20.7±3.8 16.2±3.8 ND
36.3±3.6
>50
†
†
†
†
14.0±5.7 9.8±1.03 ND
>30
>30
14.8*
ND
>30
>30
>30
ND
>30
ND
ND
ND
ND
16.9±3.6
1.4±0.2
18.8±2.9
2.4±1.5
19.3±5.5 16.8*
14.1*
2.5±1.4
18.3±1.4
1.1±0.2
2.98*
2.6±1.2
1.1±0.01 ND
19.8*
18.2±1.4 14.1±0.9 ND
4.8±0.1
4.1±1.2
2.6±0.6 7.2266*
†
See reference [15]
*
Based on single dose curve

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Figure 1. Spectral emission analysis of novel pyrenyl derivatives (3, 6, and 8).
The fluorescence emission spectra of the indicated compound (50 µM) between
the wavelengths of 350-650 nm, as measured in relative fluorescent units (RFUs),
was determined upon excitation at 350 nm.