Discovery and preclinical evaluation of a novel class of cytotoxic propynoic acid carbamoyl methyl amides (PACMAs)

Article abstract of DOI:10.1021/jm101655d

Herein, we discovered a series of propynoic acid carbamoyl methyl-amides (PACMAs) with potent cytotoxicity against a panel of cancer cell lines. These compounds interrupted cell cycle progression at low micromolar concentrations and induced early and late stage apoptosis. A representative compound suppressed tumor growth without apparent toxicity in an MDA-MB-435 mouse xenograft model. We used a Kinexus 628-antibody microarray and the Ingenuity Pathway Analysis (IPA) bioinformatics tools to better understand their mechanisms. The IPA analysis revealed the initiation of Nrf2-mediated oxidative stress through modulating the expression of SOD1 and STIP1 by compound 1. The involvement of the oxidative stress pathway was further validated by measuring the levels of the PACMA-induced mitochondrial superoxide species. To our knowledge, this is the first report on the discovery and biological evaluations of PACMAs as anticancer agents. Their broad-spectrum in vitro cytotoxicity, possibly through an oxidative stress-mediated pathway, and in vivo efficacy warrant further preclinical investigations.

Full text of DOI:10.1021/jm101655d

ARTICLE
pubs.acs.org/jmc
Discovery and Preclinical Evaluation of a Novel Class of Cytotoxic
Propynoic Acid Carbamoyl Methyl Amides (PACMAs)
Roppei Yamada,† Xuefei Cao,† Alexey N. Butkevich,z Melissa Millard,† Srinivas Odde,† Nick Mordwinkin,§
Rambabu Gundla,† Ebrahim Zandi,#,‡ Stan G. Louie,§,‡ Nicos A. Petasis,z,‡ and Nouri Neamati*,†,‡
†Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, zDepartment of Chemistry and Loker Hydrocarbon
Research Institute, USC College, §Department of Clinical Pharmacy and Pharmaceutical Economics & Policy, School of Pharmacy,
#Department of Molecular Microbiology and Immunology, Keck School of Medicine, and ‡Norris Comprehensive Cancer Center, Keck
School of Medicine, University of Southern California, Los Angeles, California, 90089
S Supporting Information
b
ABSTRACT: Herein, we discovered a series of propynoic acid
carbamoyl methyl-amides (PACMAs) with potent cytotoxicity
against a panel of cancer cell lines. These compounds inter-
rupted cell cycle progression at low micromolar concentrations
and induced early and late stage apoptosis. A representative
compound suppressed tumor growth without apparent toxicity
in an MDA-MB-435 mouse xenograft model. We used a
Kinexus 628-antibody microarray and the Ingenuity Pathway
Analysis (IPA) bioinformatics tools to better understand their
mechanisms. The IPA analysis revealed the initiation of Nrf2-mediated oxidative stress through modulating the expression of SOD1
and STIP1 by compound 1. The involvement of the oxidative stress pathway was further validated by measuring the levels of the
PACMA-induced mitochondrial superoxide species. To our knowledge, this is the first report on the discovery and biological
evaluations of PACMAs as anticancer agents. Their broad-spectrum in vitro cytotoxicity, possibly through an oxidative stress-
mediated pathway, and in vivo efficacy warrant further preclinical investigations.
’ INTRODUCTION
proteomic data for in-depth understanding of the molecular
mechanisms of diseases and drug actions.4,5
Conventional cytotoxic chemotherapies have proven effective
in curing and reducing the risk of recurrence for certain types of
cancers. Despite great strides achieved in the design and dis-
covery of novel anticancer agents, there is still a dire need to
develop highly effective and safe anticancer medications.1,2 The
early phase drug discovery process usually includes lead identi-
fication, lead optimization, target identification/validation, and
preclinical evaluations.3 The initial focus of lead identification
through random screening is to select compounds with desired
in vitro biological activities and drug-like properties from a
chemically diverse library of small molecules. Evaluation of
pharmacokinetics (PK), pharmacodynamics (PD), and metabo-
lism of the active compounds by calculating selective physico-
chemical properties important for absorption, distribution,
metabolism, excretion, and toxicity (ADMET) is also essential
to ensure the drug discovery process to be successful and cost-
effective. It is noteworthy that a major hurdle to overcome in
random screening is to identify lead compounds with cancer cell-
specific toxicity and selective molecular targets. An emerging
technology in elucidating the molecular targets and possible
mechanisms of action of a pharmacological agent is the applica-
tion of bioinformatics tools that allow detailed pathway analysis.
It is now widely accepted that bioinformatics technology plays a
vital role in the interpretation and exploitation of genomic and
In an effort to discover novel lead compounds for potential
anticancer drugs, we carried out cell-based high-throughput
screening (HTS) of our diverse in-house library of 10 000
compounds using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-
trazolium bromide (MTT) assays. Using this method, we
identified two cytotoxic propynoic acid carbamoyl methyl amides
(PACMAs), compounds 1 and 2, as potential leads. To develop a
preliminary structureꢀactivity relationship (SAR), we further
performed a substructure search within our multiconformational
small molecule database containing 10 million compounds.
Seventeen additional compounds were found to possess chemi-
cal structures similar to the original leads. An additional 61
compounds lacking the propynamide moiety were also selected
from the similarity search and found to be inactive, implying the
essential role of the propynamide group in eliciting cytotoxicity,
and not further pursued. Importantly, all active compounds met
desirable ADMET criteria and possessed favorable PK character-
istics as indicated by our computational simulations, suggesting
that human clinical trials may be successful with these
compounds.
Received: December 31, 2010
Published: March 28, 2011
r
2011 American Chemical Society
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In our current study, we evaluated the therapeutic potential of
these PACMA compounds in a panel of cancer cell lines and
explored the molecular pharmacology of selected active compounds.
Our results indicated that compound 4 interrupted cell cycle
progression and induced apoptosis in MDA-MB-435 cells. Addi-
tionally, we also evaluated the in vivo therapeutic potential of
compound 2 in an MDA-MB-435 mouse xenograft model. To
understand the mechanisms underlying their cytotoxicity, we ana-
lyzed compound 1-treated cell lysates using a Kinexus 628-antibody
microarray and the Ingenuity Pathway Analysis (IPA) bioinformatics
platform in search of potential signaling transduction networks.
Scheme 1. General Method for the Synthesis of PACMAs (E)
Scheme 2. PACMA Compounds 1ꢀ19
’ RESULTS AND DISCUSSION
Chemistry. The propynoic acid carbamoyl methyl amide
compounds (PACMAs) investigated herein, were conveniently
prepared in a single step as shown in Scheme 1 by employing the
four-component Ugi reaction.6ꢀ8 Each of the corresponding
PACMA compounds 1ꢀ19 (Scheme 2) were obtained in good
yields by reacting equimolar amounts of a primary amine or
aniline component A, an aryl or heteroaryl aldehyde B, propynoic
acid C, and a cycloalkyl or benzyl isocyanide D in methanol at
room temperature over 48 h, followed by purification via
chromatography and recrystallization.
Drug-like Properties of the PACMA Compounds. Oral
bioavailability is a desirable property of investigational com-
pounds in the drug discovery process. Lipinski’s rule-of-five is a
simplified model to predict the absorption and intestinal perme-
ability of a compound.9,10 In this model, compounds are con-
sidered likely to be well absorbed when they possess cLogP < 5,
molecular weight < 500, number of H-bond donors < 5, and
number H-bond acceptors < 10. Earlier studies by Palm et al.11ꢀ13
and Kelder et al.14 recommend that compounds with polar
surface area > 140 Å2 will likely have poor absorption (<10%),
whereas compounds with polar surface area < 60 Å2 are predicted
to show complete absorption (>90%). The calculated atom-
based LogP (Sþ logP) values of the PACMAs range from 2.2 to
4.4, and H-bond donor and acceptor counts are <5 and <10,
respectively. As shown in Figure 1 and Table 1, all active (except
4 and 9) PACMA compounds fall within the desirable range
favoring excellent oral bioavailability, whereas the reference
compounds doxorubicin and paclitaxel have maximum polar
surface areas of >150 Å2, and camptothecin has a moderate polar
surface area of 103 Å2. In conclusion, all PACMA compounds
demonstrate desirable physicochemical properties and, there-
fore, are potentially promising for further optimization and
experimental evaluations.
PACMAs Exhibit Cytotoxicity at Low Micromolar Concen-
trations in a Panel of Human Cancer Cell Lines. The 19
PACMA compounds were tested in eight human cancer cell lines
derived from different tumor origins, including the MDA-MB-
435 breast cancer, HCT116 p53þ/þ, HCT116 p53ꢀ/ꢀ, and
HT29 colon cancer, HEY and doxorubicin-resistant NCI/
ADR-RES ovarian cancer, and UMUC3 and 5637 bladder
cancer cell lines (Table 2 and Table S1, Supporting In-
formation). Remarkably, all compounds showed potent cyto-
toxicity in the NCI/ADR-RES cell line, and in fact, this cell line
exhibited the greatest sensitivity to these compounds (Table 2;
Figure S1B, Supporting Information). NCI/ADR-RES cells
have been shown to overexpress multidrug resistance transpor-
ters and p-glycoproteins,15,16 which represents the most com-
mon mechanisms for acquisition of resistance by cancer cells.
The potent cytotoxicity of these compounds in NCI/ADR-RES
cells indicates their promising therapeutic potential for treatment
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Table 1. Physicochemical Properties of PACMA Compounds
Calculated by Computational Simulationa
of human cancers that have developed resistance to standard
chemotherapeutic agents, such as doxorubicin and paclitaxel. In
addition to NCI/ADR-RES cells, Rb-null 5637 cells were also
sensitive to these compounds, whereas Rb-expressing UMUC3
cells were the least sensitive among the cell lines tested
(Table 2). When tested in MDA-MB-435 cells, compound 9
appeared to be the most potent analogue (Figure 2A). Among
the selected cell lines, 4 showed remarkable potency in 5637
cells (Figure 2B). It has been reported that 5637 bladder cancer
cells lack a functional Rb protein and are highly metastatic.17,18
On the basis of observed Rb-dependency by these compounds,
they are predicted to have potential applications in highly
malignant bladder cancers.19,20 To further confirm the MTT
compound
MW
MLogP Sþ logP
PSA
HBD HBA
1
2
3
4
5
6
7
8
9
450.5
444.5
412.5
446.5
412.5
382.5
412.5
404.5
428.5
368.4
436.5
432.4
434.5
410.5
444.5
442.5
429.3
366.5
396.5
2.54
3.70
2.26
3.91
2.26
2.55
2.26
2.62
3.54
2.39
2.33
4.38
3.88
4.11
3.32
3.86
4.11
3.07
4.17
1.67
ꢀ0.82
0.54
3.62
4.20
2.83
2.81
2.86
2.99
2.74
2.97
2.63
2.27
3.23
4.67
4.53
4.93
3.41
4.26
4.11
3.35
4.24
1.60
0.54
3.66
55.18
40.65
49.91
122.06
44.69
49.85
45.72
51.90
129.21
43.08
54.79
32.99
39.73
37.34
57.05
42.75
27.71
31.53
31.57
103.63
233.93
156.48
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
6
4
7
5
6
7
6
5
6
6
7
10
11
12
13
14
15
16
17
18
19
5
7
4
4
4
6
4
4
4
4
camptothecin 348.4
5
doxorubicin
543.5
12
15
paclitaxel
839.9
a MW, molecular weight; MLogP, Moriguchi octanolꢀwater partition
coefficient; Sþ logP, SimulationsPlus, Inc., model of logP; PSA, three-
dimensional polar surface area; HBD, hydrogen-bond donor; HBA,
hydrogen-bond acceptor.
Figure 1. Prediction of drug absorption by computational simulation.
3-D Polar surface area in Å2 versus partition coefficient (Sþ logP)
calculated by ADMET Predictor.
Table 2. In Vitro Cytotoxicity of Compounds 1ꢀ19 in a Panel of Human Cancer Cell Lines
IC50a (μM)
compound
HT29
HCT116 p53þ/þ
HCT116 p53ꢀ/ꢀ
HEY
MDA-MB-435
UMUC3
>20
5637
NCI/ADR-RES
1
6.3 ( 1.5
7.2 ( 0.2
5.5 ( 4.2
1.8 ( 1.7
2.3 ( 0.3
2.3 ( 0.3
1.6 ( 1.0
3.2 ( 1.2
1.5 ( 1.1
12.5 ( 2.1
>10
11.0
4.4 ( 3.0
5.2 ( 3.4
3.4 ( 1.5
1.5 ( 0.8
4.1 ( 3.4
1.9 ( 0.7
1.5 ( 1.1
4.1 ( 3.4
1.9 ( 0.3
4.3 ( 3.9
2.3 ( 0.4
2.4 ( 0.2
5.2 ( 3.6
1.7 ( 1.1
5.4 ( 2.9
6.3 ( 1.1
2.0 ( 0.1
1.4 ( 0.2
1.7 ( 0.5
1.7 ( 0.4
0.5 ( 0.3
1.8 ( 0.6
0.6 ( 0.3
1.7 ( 0.6
2.4 ( 0.1
5.2 ( 1.2
6.9 ( 0.8
7.3 ( 0.0
>10
2.2 ( 0.2
0.6 ( 0.4
2.0 ( 0.7
0.7 ( 0.5
1.7 ( 0.8
0.3 ( 0.0
0.3 ( 0.0
1.9 ( 0.6
0.4 ( 0.1
2.9 ( 0.8
1.9 ( 0.6
0.5 ( 0.3
1.6 ( 0.7
0.5 ( 0.3
2.8 ( 1.1
1.2 ( 1.1
1.8 ( 0.1
3.5 ( 1.4
2.1 ( 0.0
0.3 ( 0.0
0.2 ( 0.1
0.9 ( 0.8
1.0 ( 0.3
0.4 ( 0.0
0.3 ( 0.0
0.3 ( 0.0
0.3 ( 0.0
1.0 ( 0.7
1.2 ( 0.4
0.3 ( 0.0
0.4 ( 0.0
0.4 ( 0.0
0.3 ( 0.0
1.3 ( 1.0
0.7 ( 0.6
2.2 ( 0.2
1.3 ( 0.4
0.4 ( 0.1
2
8.8 ( 0.4
9.6 ( 4.9
5.5 ( 0.7
7.0 ( 0.7
5.6 ( 0.8
4.7 ( 2.3
>20
>20
>20
3
7.7 ( 1.9
6.3 ( 1.8
6.9 ( 0.6
7.2 ( 0.2
5.7 ( 0.6
17.3 ( 1.1
7.2 ( 0.2
10.5
4
6.3 ( 1.0
>20
6.9 ( 1.2
5.7 ( 1.5
>20
7.8 ( 1.8
>20
>20
8.0 ( 1.4
5
6
7
8
9
5.7 ( 0.9
13.0 ( 2.8
>20
10
11
12
13
14
15
16
17
18
19
>10
2.1 ( 0.4
4.8 ( 3.2
6.0 ( 1.4
4.9 ( 3.4
15.0
7.5 ( 0.7
8.3 ( 1.1
6.8 ( 1.8
>10
6.9 ( 0.6
10.4 ( 0.2
7.4 ( 0.8
>20
>20
>20
>20
>20
>20
>20
>20
>10
>10
7.3 ( 0.3
5.4 ( 3.0
9.0 ( 1.4
7.7 ( 0.5
14.0
16.1 ( 5.6
7.3 ( 1.4
>20
7.1 ( 2.6
5.8 ( 1.3
4.2 ( 3.0
8.2 ( 0.2
>10
>10
7.7 ( 0.5
>10
5.6 ( 2.1
11.9 ( 0.1
10.1 ( 2.9
6.5 ( 2.1
a Cytotoxic concentration (IC50) is defined as drug concentration causing a 50% decrease in cell population using MTT assay as described in the
Experimental Section.
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Figure 2. Cytotoxicity of selected PACMA compounds in a panel of cancer cell lines. (A) The IC50 values of 1, 2, 4, 6, and 9 in MDA-MB-435 cells range
from 0.6 to 6.3 μM. (B) The IC50 values of compound 4 range between 1 and 2.5 μM in tested cell lines using MTT assays.
drug-related death or body weight loss (Figure 4B). Specifically,
the untreated control mice had an average weight of 23 ( 2 g
(mean ( SD) before the experiment and 28 ( 2 g after the
experiment. Mice treated with 2 had a comparable average weight
of 29 ( 2 g after treatment. Furthermore, histopathological
examinations of the organs derived from at least three mice in the
treatment group showed no histological evidence of organ
toxicity (data not shown).
Compound 4 Induces S and G2/M Cell Cycle Arrest in
MDA-MB-435 Cells. Compound 4 is one of the most active
compounds in the MDA-MB-435 cell line (Table 2). Hence, we
selected this compound for further examination to determine its
ability to induce cell cycle perturbation in MDA-MB-435 cells.
Cells were treated with 4 for 6, 12, and 24 h. The analysis of DNA
profiles by flow cytometry demonstrated that 4 was able to block
cell cycle progression at both S and G2/M phases (Figure 5). In
particular, at 24 h, there was a 10% and 7% increase in the number
of cells retained in S and G2/M phases, respectively, when
compared with untreated control cells. The property of com-
pound 4 to disrupt cell cycle progression makes it an interesting
agent for combination treatments with drugs acting at different
stages of cell cycle.
Compounds 1, 2, and 4 Trigger Apoptosis. Most cytotoxic
anticancer drugs induce apoptosis.21 To understand the mecha-
nisms underlying the cytotoxicity of PACMAs, we measured the
percentage of early and late apoptotic cells in MDA-MB-435 cells
treated with compounds 1, 2, and 4, respectively, by flow
cytometry. An early event in apoptotic cell death is the transloca-
tion of phosphatidyl-serine residues to the outer cell membrane.
This event precedes nuclear breakdown, DNA fragmentation,
and appearance of most apoptosis-associated molecules and is
readily distinguished from the late apoptotic processes by
annexin V/propidium iodide binding assay. As shown in Figure 6,
the percentage of early apoptotic cells reached 24% after 48 h
exposure to compound 4 and the ratio of late apoptotic cells was
significantly increased to 27% and 62% after 24 and 48 h
treatment, respectively. The original lead compounds, 1 and 2,
failed to induce early apoptosis at 24 h, and the early apoptotic
cells were slightly increased by 8% after 48 h treatment.
However, the percentage of late apoptotic cells reached 35%
and 37% for 1 and 2, respectively, after 48 h treatment,
suggesting a less marked and delayed induction of apoptosis
by these two compounds. Taken together, these observations
implied that apoptosis might be one of the mechanisms utilized
by these compounds to elicit cytotoxicity. It is known that
apoptosis signaling pathways have a profound effect on both
cancer progression and response to chemotherapy.22ꢀ24
Figure 3. Colony formation assay of the active PACMA compounds in
MDA-MB-435 cells. MDA-MB-435 cells were treated with 1 μM
PACMA compounds. The colony formation observations were consis-
tent with the MTT results. Abbreviations are as follows: PTX, paclitaxel;
CDDP, cisplatinum; CONT, vehicle (DMSO) control.
observations, we performed colony formation assays on all
compounds at 1 μM in MDA-MB-435 cells (Figure 3). The
results were conclusive and consistent with the MTT observa-
tions. In addition to these 19 compounds, we also tested 61
compounds without the propynamide functional group in MDA-
MB-435 cells, and 13 compounds exhibited moderate cytotoxi-
city around 40% at 10 μM (data not shown). However, when
tested in NCI/ADR-RES cells, they failed to inhibit colony
formation (Figure S1A, Supporting Information), suggesting
the critical function of the propynamide moiety in inducing
cytotoxicity of this class of compounds.
Compound 2 Exhibits Promising in Vivo Efficacy in an
MDA-MB-435 Mouse Xenograft Model. To investigate the
in vivo efficacy of these compounds, we selected compound 2 and
evaluated its anticancer activity in a nude mouse xenograft model
using MDA-MB-435 cells. Animals were treated with daily intraper-
itoneal (i.p.) injections of DMSO in sesame oil (vehicle control) or
compound 2 at 10 mg/kg for 9 days, followed by 20 mg/kg for 5
days and concluding with 40 mg/kg for 12 days. After 26 days of
dosing, the drug treatment was discontinued. Mice were mon-
itored daily for an additional 17 days, and tumors were measured
twice per week. Figure 4A summarizes the tumor volume (mean
( SEM) of the treatment groups throughout the observation
time. Compound 2 significantly reduced tumor burden in this
xenograft model. The possible toxicity elicited by 2 was also
evaluated by following the body weight of the mice over the
course of treatment and histological examination of the organs.
The treatment was well-tolerated and did not result in any
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Figure 4. Compound 2 showed in vivo efficacy in an MDA-MB-435 mouse xenograft model. (A) Compound 2 reduced tumor burden. Athymic nude
mice implanted with MDA-MB-435 cells were treated with indicated doses of 2 by i.p. administration 5 times weekly. Values represent the tumor
volumes (mean ( SEM) for each group. (B) Compound 2 treatment did not cause weight loss. This compound was well-tolerated and did not result in
any drug-related deaths or changes in body weight.
Figure 5. Cell cycle profiles of MDA-MB-435 cells treated with compound 4. Cells were treated with 5 μM compound 4 for 6, 12, and 24 h, collected,
stained with propidium iodide, and analyzed for perturbation of cell cycle by flow cytometry. Compound 4 induced both S and G2/M arrest.
A great number of genes are involved in apoptosis and have
been found to be defective in a high percentage of human
malignancies.25 Thus, reactivation of apoptosis pathways by
modulating apoptosis-related proteins is an attractive and
growing trend for anticancer drug discovery. Currently, we
are in the process of identifying the apoptosis-related proteins
involved and elucidating the specific apoptotic signaling path-
ways initiated by these compounds.
Compound 1 Targets Pro-apoptotic p53 and Caspase-9.
Among the proteins associated with apoptosis signaling, p53 and
caspase-9 are the most commonly activated pro-apoptotic pro-
teins by apoptosis-inducing agents. Thus, as a preliminary
mechanistic investigation, we examined the cellular effect of
the original lead compound 1 on apoptotic signaling through
the protein expression changes of p53 and caspase-9. As shown in
Figure 7A, the protein level of p53 was up-regulated by 1 after 24
h treatment and the activation of p53 was sustained for 72 h
(lanes 4ꢀ6 versus lane 1), suggesting the involvement of p53 in
compound 1-mediated cytotoxicity. Several lines of evidence
have demonstrated the activation of caspases during p53-
mediated apoptosis in both cell-based and cell-free systems.26,27
We, therefore, measured the protein levels of full-length caspase-
9 in response to compound 1 treatment by Western blotting. A
2-fold decrease in the full-length caspase-9 protein level was
observed after 24 h, and the change was maintained up to 72 h
(Figure 7B). These observations led us to hypothesize that the
decrease in full-length caspase-9 protein level is due to its
cleavage and activation in response to apoptotic signaling
initiated by compound 1. This suggests that compound 1 elicits
cytotoxicity through activating p53 as well as the downstream
signaling cascades of p53-mediated apoptotic events.
Signaling Network Analysis Using the Ingenuity Pathway
Analysis (IPA) Platform. To better understand the mechanisms
of action of this class of novel PACMA compounds, we subjected
cell lysates from untreated and compound 1-treated MDA-MB-
435 cells to the Kinexus 628-antibody microarray analysis in
search of potential signaling molecules; 350 pan-specific and 270
phospho-site antibodies were included to track changes in
protein expression and phosphorylation status. The cell lysates
were run in parallel on the Kinexus antibody chip and the
differential binding of dye-labeled proteins was detected and
quantified. Proteins with significant expression changes were
then identified and used as the basis for signaling pathway
interpretation.
To visualize and understand the signaling networks affected by
compound 1, we uploaded the sets of significantly altered pan-
specific proteins to the IPA bioinformatics platform. All proteins
uploaded were recognized by the IPA software as being eligible
for pathway analysis. Several pathways were proposed as a result
of the knowledge-based data curation (Figure 8A). The major
putative canonical pathway suggested by the IPA was the
oxidative stress signaling pathway mediated by Nrf2 (nuclear
factor erythroid 2-related factor 2).28ꢀ30 This pathway had a
p-value of 5.82 ꢁ 10ꢀ5, based on the expression changes of several
kinases and transcription factors identified in the Nrf2-mediated
oxidative stress pathway, including protein kinase C (PKC),
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Figure 6. Compounds 1, 2, and 4 induce apoptosis. Cells were treated with compounds 1, 2, and 4 for 24 and 48 h, stained with annexin V/propidium
iodide, and analyzed by flow cytometry. Untreated control cells (48 h) were also included in the analysis. Cells in the bottom left quadrant (annexin
V-negative, propidium iodide-negative) are viable, whereas cells in the top left quadrant (annexin V-positive, propidium iodide-negative) are in the early
stages of apoptosis, and the cells in the top right quadrant (annexin V-positive, propidium iodide-positive) are in later stages of apoptosis and necrosis.
STIP1, MAPK3, and superoxide dismutase 1 (SOD1) (Figure 8B).
Reactive oxygen species (ROS), such as superoxide and hydro-
gen peroxide, are metabolites transiently formed during normal
cellular metabolism. However, elevated production of ROS
damages most cellular components, and the perturbation of
cellular homeostasis, in turn, results in the propagation of more
ROS. The positive-feedback loop between ROS production and
homeostasis disturbance ultimately causes cell death. Nrf2 is
widely expressed in a number of organs and functions as a key
sensor for cellular response to oxidative stress, by means of its
regulatory function on the transcription of a number of genes
encoding antioxidant proteins and related enzymes.31ꢀ34 SOD1
is one of two antioxidant isozymes responsible for scavenging
cellular superoxide radicals and was found to be up-regulated by
compound 1 in our antibody microarray assay. SOD1 is a
downstream target of Nrf2, and it has been reported that Nrf2
activates the transcription of SOD1,28 which then phosphory-
lates STAT1 to execute antioxidant responses.35 In addition to
Figure 7. p53 and caspase-9 protein expression in MDA-MB-435 cells
treated with 1 μM compound 1. (A) p53 was up-regulated by 1 in a time-
dependent manner. (B) The full-length caspase-9 was decreased in
response to 1 treatment. Ratios of p53 to GAPDH and full-length
caspase-9 to GAPDH were determined using the ImageQuant (GE
Healthcare).
SOD1, Nrf2 may also respond to oxidative stress by activating the
mRNA transcription of STIP1.36 STIP1 functions as a molecular
chaperone for heat shock protein 70 (HSP70) and heat shock
protein 90 (HSP90). Under nonstress conditions, HSP90, acting
as a molecular chaperone, facilitates protein folding and protein
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Figure 8. Prediction of compound 1-mediated signaling pathways using IPA bioinformatics tool. (A) Ingenuity pathway networks of compound 1-
induced cell signaling (score 41) obtained on a set of altered pan-specific proteins. Proteins with their ratio changes and corresponding Swiss-Prot
accession numbers were uploaded to IPA software. (—) Direct interaction; (ꢀ ꢀ ꢀ) Indirect interaction. Blue bars represent the ꢀlog(p-value) of each
representative pathway, with the threshold for statistical significance denoted by the thin gray line. Points connected by the solid line represent the ratio
of affected molecules versus the total number of molecules in a given pathway. (B) Canonical pathway analysis. Fifteen canonical pathways of interest
were selected and presented. (C) Quantification of mitochondrial superoxide production upon drug treatment in MDA-MB-435 cells. Cells were treated
with compounds 1, 2, 4, and 18 for 24 h. The untreated and treated cells were incubated with 5 μM MitoSOX for 10 min at 37 °C, collected in Hank’s
balanced salt solution (HBSS), and subjected to flow cytometric analysis. (D) Examination of mitochondrial superoxide production in response to 1, 2,
9, and 18 treatment in MDA-MB-435 cells. Cells were treated with indicated compounds for 24 h. Untreated and treated cells were incubated with 5 μM
of MitoSOX for 10 min at 37 °C and observed under a fluorescence microscope.
degradation to maintain cellular homeostasis. In addition to
these functions, HSP90 was recently found to confer antiapop-
totic functions by regulating several apoptosis-related
molecules.37,38 Therefore, it is conceivable that down-regulat-
ing HSP90 protein expression could abolish HSP90-mediated
cell survival. ROS were reported to be able to mediate HSP90
protein cleavage, and this ROS-dependent HSP90 cleavage was
involved in arsenic- and MMA-induced apoptosis.39 On the
basis of these reports, it is plausible that compound 1-induced
down-regulation of STIP1 may decrease the amount of HSP90
and, hence, induce apoptosis. Taken together, we hypothesize
that compound 1 elicits cytotoxicity by inducing ROS produc-
tion and further alters the expression of oxidative stress-related
proteins, such as SOD1, STIP1, and HSP90. The oxidative
stress sensor, Nrf2, may be one of the early proteins activated in
response to compound 1 treatment.
Compounds 1, 2, 4, and 18 Induce the Accumulation of
Mitochondrial Superoxide. As a preliminary validation of the
pathway proposed by IPA, we examined mitochondrial super-
oxide production by compounds 1, 2, 4, and 18 in MDA-MB-435
cells. Mitochondrial superoxide species was detected using
MitoSOX Red mitochondrial superoxide live cell indicator. Cells
were treated with these compounds at their respective IC50 or
3 ꢁ IC50 concentrations for 24 h. All compounds were found to
induce significant amounts of superoxide upon 24 h treatment
(Figure 8C). The flow cytometry observation was further con-
firmed by examining the staining pattern of mitochondrial
superoxide in response to compound treatment by fluorescence
microscopy. Consistently, all compounds, including the less
active compound 18, remarkably enhanced the intensity of
MitoSOX staining (Figure 8D). Interestingly, paclitaxel only
induced a minor amount of mitochondrial superoxide even at
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ARTICLE
Table 3. PK Parameters of Compounds 4 and 9 after i.p.
Administration
PK parameters
compound 4
compound 9
Cmax (ng/mL)
Tmax (h)
344
1.0
6.2
5
Kel (hꢀ1
)
0.206
3.5
0.0
9.3
124.9
t1/2 (h)
AUC (ng h/mL)
520
3
1.5 μM (Figure 8D). In addition to mitochondrial superoxide, we
also measured the production of other types of reactive oxygen
species using 2,7-dichlorodihydrofluorescein (DCFH) staining,
and no change was observed (data not shown). Based on these
observations, we propose that superoxide induction by PACMAs
is a unique mechanism by which these compounds elicit
cytotoxicity.
Figure 9. The pharmacokinetics distribution of compounds 4 and 9 in
mice. Mice were administered 24 mg/kg of 4 and 9 via i.p. administra-
tion. Blood samples were collected at 1, 2, 5, and 24 h for 4 and 0.5, 1, 2,
5, and 24 h for 9. Plasma concentrations of 4 and 9 are plotted against
time. The plasma concentration was determined using a validated LC-
MS method where the lowest level of sensitivity is 1 ng/mL.
Compounds 4 and 9 Show Favorable Pharmacokinetic
Profiles. To assess the efficacy, tolerability, and pharmaco-
kinetics of these novel compounds following i.p. administration,
the pharmacokinetic profile of compounds 4 and 9 was evaluated
(Table 3). Mice were treated with 4 and 9 via i.p. injection and
serial blood sampling was determined at indicated times after
administration. The plasma concentration was determined using
a validated liquid chromatographyꢀmass spectrometry (LC-MS)
methodwhere thelowestlevel ofquantification was 1 ng/mL. One
hour following i.p. administration of 4 and 9, their concentrations
in the blood were 344 and 6.2 ng/mL, respectively. Sequential
plasma analysis revealed an apparent two compartmental phar-
macokinetic elimination (Figure 9). The half-lives of compounds
4 and 9 via i.p. administration were determined to be approxi-
mately 3.5 and 9.3 h, respectively, when a noncompartmental
pharmacokinetics analysis was used. The favorable pharmacoki-
netics profile of 4 and 9 is consistent with the ADMET predic-
tions described previously.
In conclusion, the reported propynoic acid carbamoyl methyl
amides (PACMAs) are promising anticancer agents with a
number of desirable features and presumably operate through
an oxidative stress-mediated pathway. Identification of their
direct cellular target is beyond the scope of this paper. Of special
note is their ability to show significant cytotoxicity in various
drug-resistant cell lines. Their broad-spectrum in vitro cytotoxi-
city and in vivo efficacy warrant further mechanistic and pre-
clinical investigations. The synthesis of these compounds is quite
simple, amenable to parallel as well as large-scale synthesis. We
have synthesized numerous other analogs to better understand
their pharmacological properties and to carry out in-depth
toxicological studies. Results of these latter studies will be
reported elsewhere.
(99.9% D, Cambridge Isotope Laboratories) were used in all experi-
ments as indicated. Chemical shifts are reported as parts per million (δ)
relative to tetramethylsilane. CF multiplets in 13C NMR spectra are
interpreted wherever possible.
General Method for the Synthesis of PACMA Compounds
(1ꢀ19). A solution of the required aniline or benzylamine A (2 mmol),
aryl or heteroaryl aldehyde B (2 mmol), propynoic acid C (2.2 mmol),
and isocyanide D (2.2 mmol) in methanol (2 mL) was stirred at room
temperature for 48 h. The products that precipitated from the reaction
mixture were filtered off, washed with aqueous methanol (1:1), and
recrystallized from ethyl acetateꢀhexane. Alternatively, the reaction
mixture was evaporated to dryness and subject to column chromatog-
raphy in ethyl acetateꢀhexane. The fractions containing the target
compound were evaporated to dryness, and the product E was recrys-
tallized from ethyl acetateꢀhexane.
N-[2-(Cyclohexylamino)-1-(4-methoxyphenyl)-2-oxoethyl]-N-(3,5-
1
dimethoxyphenyl) Propynamide (1). Yield 241 mg (54%). H NMR
(400 MHz, CDCl3): δ 7.07ꢀ7.13 (m, 2H), 6.71ꢀ6.77 (m, 2H),
6.30ꢀ6.45 (m, 3H), 5.86 (s, 1H), 5.53 (br.d, J = 8.3 Hz, 1H), 3.76 (s,
3H), 3.74ꢀ3.85 (m, 1H), 3.66 (s, 6H), 2.83 (s, 1H), 1.80ꢀ1.96 (m, 2H),
1.51ꢀ1.75 (m, 3H), 1.23ꢀ1.40 (m, 2H), 0.96ꢀ1.17 (m, 3H). 13C NMR
(100 MHz, CDCl3): δ 167.7, 160.3, 159.8, 153.5, 140.8, 131.6, 125.8,
113.9, 108.7, 101.3, 80.1, 76.1, 64.8, 55.5, 55.3, 48.8, 32.8, 25.5,
24.8, 24.7.
N-[2-(Cyclopentylamino)-1-(3-methoxyphenyl)-2-oxoethyl]-N-[3-
1
(trifluoromethyl) phenyl] Propynamide (2). Yield 828 mg (93%). H
NMR (400 MHz, CDCl3): δ 7.24ꢀ7.60 (m, 4H), 7.09 (t, J = 7.9 Hz,
1H), 6.74 (d, J = 7.9 Hz, 1H), 6.68 (d, J = 7.5 Hz, 1H), 6.58 (s, 1H), 6.06
(s, 1H), 5.88 (br.d, J = 7.1 Hz, 1H), 4.20 (sextet, J = 6.6 Hz, 1H), 3.60 (s,
3H), 2.82 (s, 1H), 1.83ꢀ2.02 (m, 2H), 1.47ꢀ1.65 (m, 4H), 1.33ꢀ1.45
(m, 1H), 1.19ꢀ1.31 (m, 1H). 13C NMR (100 MHz, CDCl3): δ 167.9,
159.5, 153.3, 139.2, 134.7, 134.5, 130.6 (quartet, J = 33.1 Hz), 129.3,
128.9, 128.0 (quartet, J = 3.7 Hz), 125.1 (quartet, J = 3.7 Hz), 123.3
(quartet, J = 273.6 Hz), 122.5, 115.2, 114.9, 81.1, 75.6, 64.1, 55.1, 51.7,
32.7, 32.6, 23.59, 23.57. 19F NMR (376 MHz, CDCl3): δ ꢀ62.72.
N-[2-(Cyclopentylamino)-2-oxo-1-thien-2-ylethyl]-N-(3,4-dimetho-
xyphenyl) Propynamide (3). Yield 706 mg (86%). 1H NMR (400 MHz,
CDCl3): δ 7.19 (dd, J = 4.9 Hz, J = 1.2 Hz, 1H), 6.91 (br.d, J = 3.3 Hz,
1H), 7.19 (dd, J = 4.9 Hz, J = 3.7 Hz, 1H), 6.60ꢀ6.75 (m, 3H), 6.23 (br.
d, J = 7.5 Hz, 1H), 6.20 (s, 1H), 4.15 (sextet, J = 6.6 Hz, 1H), 3.77 (s,
3H), 3.65 (s, 3H), 2.84 (s, 1H), 1.82ꢀ1.98 (m, 2H), 1.45ꢀ1.63 (m, 4H),
1.28ꢀ1.43 (m, 2H). 13C NMR (100 MHz, CDCl3): δ 167.1, 153.5,
’ EXPERIMENTAL SECTION
Chemistry. All commercially available compounds were used with-
out further purification. 3-Methyl-4-(1H-tetrazol-1-yl)aniline was
synthesized from commercially available 2-methyl-4-nitroaniline accord-
ing to the reported procedure.40 Flash chromatography was performed
using Sorbent Technologies 60 Å silica gel (40ꢀ63 μm). All final
compounds were purified to >95% purity as determined by Varian
HPLC instrument with UV detection at 254 nm. Proton (1H), fluorine
(19F), and carbon (13C) nuclear magnetic resonance (NMR) spectra
were recorded on a Varian Mercury 400 MHz NMR spectrometer.
CDCl3 (99.8% D, Cambridge Isotope Laboratories) and DMSO-d6
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Journal of Medicinal Chemistry
ARTICLE
148.9, 148.2, 135.0, 131.1, 129.7, 127.8, 126.2, 122.7, 113.0, 109.9, 80.6,
75.8, 59.2, 55.7, 55.5, 51.5, 32.6, 32.5, 23.50, 23.46.
5.96 (d, J = 6.6 Hz, 1H), 4.22 (sextet, J = 6.6 Hz, 1H), 2.93 (s, 1H), 2.09
(s, 1H), 1.86ꢀ2.04 (m, 2H), 1.50ꢀ1.68 (m, 4H), 1.25ꢀ1.48 (m, 2H).
13C NMR (100 MHz, CDCl3): 167.9, 153.1, 142.9, 140.9, 134.4, 133.8,
133.4, 132.5, 130.1, 129.9, 129.0, 128.7, 125.5, 81.2, 75.8, 64.3, 51.7,
32.68, 32.65, 23.58, 23.56, 17.6.
N-[2-(Cyclopentylamino)-1-(2-fluorophenyl)-2-oxoethyl]-N-[3-methyl-
4-(1H-tetraazol-1-yl)phenyl] Propynamide (4). Yield 532 mg (60%).
1H NMR (400 MHz, CDCl3): δ 8.81 (s, 1H), 7.23ꢀ7.60 (m, 3H),
7.10ꢀ7.21 (m, 2H), 6.94ꢀ7.08 (m, 2H), 6.38 (s, 1H), 6.22 (br.d, J = 7.1
Hz, 1H), 4.26 (sextet, J = 6.6 Hz, 1H), 2.93 (s, 1H), 2.12 (s, 3H),
1.90ꢀ2.07 (m, 2H), 1.43ꢀ1.74 (m, 5H), 1.31ꢀ1.42 (m, 1H). 13C NMR
(100 MHz, CDCl3): 167.4, 160.7 (d, J = 248.5 Hz), 153.0, 142.9, 140.5,
133.9 (d, J = 14.2 Hz), 132.5, 131.2 (d, J = 9.0 Hz), 131.1, 129.5, 125.6,
124.4 (d, J = 2.6 Hz), 120.7 (d, J = 12.9 Hz), 115.4 (d, J = 21.9 Hz), 81.2,
75.6, 70.1, 57.5, 51.8, 32.72, 32.65, 23.6, 17.7. 19F NMR (376 MHz,
CDCl3): δ ꢀ115.35.
N-[2-(Cyclopentylamino)-1-(2-fluorophenyl)-2-oxoethyl]-N-(2-furyl-
methyl) Propynamide (10). Yield 586 mg (80%). In CDCl3, mixture of
two rotameric forms in 70:30 ratio. 1H NMR (400 MHz, CDCl3): δ 7.44
(td, J = 7.5 Hz, J = 1.2 Hz, 0.7H), 7.29ꢀ7.37 (m, 2 ꢁ 0.3H), 7.22ꢀ7.29
(m, 0.7H), 7.13ꢀ7.19 (m, 2 ꢁ 0.3Hþ0.7H), 7.09 (td, J = 7.5 Hz, J = 1.2
Hz, 0.7H), 6.97ꢀ7.05 (m, 0.3H), 6.87ꢀ6.94 (m, 0.7H), 6.34 (s, 0.3H),
6.17 (dd, J = 2.9 Hz, J = 1.7 Hz, 0.3H), 6.09ꢀ6.14 (m, 0.3Hþ0.7H), 6.07
(s, 0.7H), 6.02 (br.d, J = 7.1 Hz, 0.7H), 5.95 (d, J = 3.3 Hz, 0.3H), 5.85
(d, J = 3.3 Hz, 0.7H), 4.90 (d, J = 17.0 Hz, 0.7H), 4.76 (d, J = 17.0 Hz,
0.7H), 4.61 (d, J = 15.8 Hz, 0.3H), 4.27 (d, J = 15.8 Hz, 0.3H), 4.19
(sextet, J = 7.1 Hz, 0.3H), 4.13 (sextet, J = 7.1 Hz, 0.7H), 3.26 (s, 0.3H),
3.18 (s, 0.7H), 1.85ꢀ1.99 (m, 2 ꢁ 0.3Hþ2 ꢁ 0.7H), 1.47ꢀ1.64 (m, 4 ꢁ
0.3Hþ4 ꢁ 0.7H), 1.19ꢀ1.35 (m, 2 ꢁ 0.3Hþ2 ꢁ 0.7H). 13C NMR (100
MHz, CDCl3): δ 167.4, 167.2, 161.4 (d, J = 249.8 Hz), 161.1 (d, J =
249.8 Hz), 156.3, 154.3, 154.2, 149.9, 149.8, 141.9, 141.6, 131.1, 131.0,
130.92, 130.90, 130.6 (d, J = 9.0 Hz), 124.4 (d, J = 2.6 Hz), 124.1 (d, J =
2.6 Hz), 121.3 (d, J = 14.2 Hz), 115.7 (d, J = 20.6 Hz), 115.3 (d, J = 21.9
Hz), 110.6, 110.3, 108.7, 108.0, 80.9, 80.0, 75.5, 74.8, 60.4, 55.23, 55.21,
51.5, 51.4, 44.6, 39.4, 32.64, 32.60, 23.6. 19F NMR (376 MHz, CDCl3):
ꢀ114.62 (0.3F), ꢀ114.75 (0.7F).
N-[2-(Cyclopentylamino)-2-oxo-1-thien-2-ylethyl]-N-(2,4-dimetho-
xyphenyl) Propynamide (5). Yield 453 mg (55%). In CDCl3, mixture of
two rotameric forms in 60:40 ratio. 1H NMR (400 MHz, CDCl3): δ 7.34
(d, J = 8.7 Hz, 0.6H), 7.12ꢀ7.22 (m, 0.6Hþ0.4Hþ0.4H), 7.01 (d, J =
3.3 Hz, 0.4H), 6.91 (d, J = 3.3 Hz, 0.6H), 6.86 (dd, J = 5.4 Hz, J = 3.7 Hz,
0.4H), 6.82 (dd, J = 5.4 Hz, J = 3.7 Hz, 0.6H), 6.53 (d, J = 8.7 Hz, 0.4H),
6.37ꢀ6.46 (m, 0.6Hþ0.6Hþ0.4H), 6.27 (d, J = 2.5 Hz, 0.6H), 6.22 (dd,
J = 8.7 Hz, J = 2.5 Hz, 0.4H), 6.12 (s, 0.4H), 5.99 (s, 0.6H), 4.15ꢀ4.27
(m, 0.6Hþ0.4H), 3.82 (s, 1.2H), 3.78 (s, 1.8H), 3.75 (s, 1.2H), 3.55 (s,
1.8H), 2.74 (s, 0.4H), 2.72 (s, 0.6H), 1.89ꢀ2.06 (m, 2 ꢁ 0.6Hþ2 ꢁ
0.4H), 1.31ꢀ1.75 (m, 6 ꢁ 0.6Hþ6 ꢁ 0.4H). 13C NMR (100 MHz,
CDCl3): δ 167.5, 167.2, 161.2, 161.0, 157.1, 157.0, 154.7, 154.3, 135.6,
134.5, 132.0, 131.9, 129.7, 129.5, 127.7, 127.5, 126.0, 125.5, 120.9, 120.0,
104.3, 103.9, 99.0, 98.8, 78.4, 78.2, 76.2, 76.1, 60.4, 60.0, 55.7, 55.4, 55.3,
51.6, 51.3, 32.89, 32.86, 32.8, 23.8, 23.8, 23.7.
N-[2-(Cyclopentylamino)-2-oxo-1-thien-2-ylethyl]-N-(3-methoxy-
phenyl) Propynamide (6). Yield 533 mg (70%). 1H NMR (400 MHz,
CDCl3): δ 7.23 (d, J = 5.0 Hz, 1H), 7.13 (t, J = 7.9 Hz, 1H), 6.96 (d, J =
3.3 Hz, 1H), 6.72ꢀ6.89 (m, 4H), 6.10ꢀ6.17 (m, 2H), 4.19 (sextet, J =
6.6 Hz, 1H), 3.68 (s, 3H), 2.84 (s, 1H), 1.86ꢀ2.00 (m, 2H), 1.48ꢀ1.66
(m, 4H), 1.32ꢀ1.45 (m, 2H). 13C NMR (100 MHz, CDCl3): δ 167.0,
159.5, 153.2, 139.8, 135.0, 129.9, 129.1, 128.0, 126.3, 122.2, 115.00,
114.97, 80.7, 75.7, 60.0, 55.2, 51.7, 32.7, 23.6, 23.5.
N-[2-(Cyclopentylamino)-2-oxo-1-thien-2-ylethyl]-N-(2,5-dimethoxy-
phenyl) Propynamide (7). The reaction was run in 2,2,2-trifluoroetha-
nol (2 mL) for 1 h. Yield 207 mg (25%). In CDCl3, mixture of two
rotameric forms in 50:50 ratio. 1H NMR (400 MHz, CDCl3): δ 7.26 (br.
d, J = 6.2 Hz, 0.5H), 7.21 (dd, J = 5.0 Hz, J = 1.0 Hz, 0.5H), 7.18 (dd, J =
5.0 Hz, J = 1.0 Hz, 0.5H), 7.05 (d, J = 2.9 Hz, 0.5H), 7.03 (d, J = 3.3 Hz,
0.5H), 6.94 (d, J = 3.3 Hz, 0.5H), 6.87 (dd, J = 5.4 Hz, J = 3.7 Hz, 0.5H),
6.73ꢀ6.86 (m, 4 ꢁ 0.5H), 6.69 (d, J = 9.1 Hz, 0.5H), 6.51 (br.d, J = 7.1
Hz, 0.5H), 6.18 (d, J = 2.9 Hz, 0.5H), 6.16 (s, 0.5H), 5.89 (s, 0.5H), 4.22
(sextet, J = 7.1 Hz, 0.5H), 4.20 (sextet, J = 7.1 Hz, 0.5H), 3.85 (s, 3 ꢁ
0.5H), 3.74 (s, 3 ꢁ 0.5H), 3.61 (s, 3 ꢁ 0.5H), 3.52 (s, 3 ꢁ 0.5H), 2.73 (s,
0.5H), 2.70 (s, 0.5H), 1.90ꢀ2.05 (m, 4 ꢁ 0.5H), 1.32ꢀ1.75 (m, 12 ꢁ
0.5H). 13C NMR (100 MHz, CDCl3): δ 167.3, 167.1, 154.2, 153.6,
153.2, 150.4, 150.2, 135.7, 134.6, 129.8, 129.5, 128.3, 127.7, 127.4, 127.2,
126.2, 125.5, 116.4, 116.2, 116.1, 115.8, 112.5, 112.0, 78.3, 78.0, 76.1,
75.9, 60.6, 60.3, 56.2, 55.8, 55.7, 55.6, 51.6, 51.2, 32.8, 32.7, 23.75,
23.71, 23.6.
N-[2-(Cyclopentylamino)-1-(4-methoxyphenyl)-2-oxoethyl]-N-(3,5-
1
dimethoxyphenyl) Propynamide (11). Yield 62 mg (7%). H NMR
(400 MHz, CDCl3): δ 7.04ꢀ7.14 (m, 2H), 6.70ꢀ6.77 (m, 2H),
6.30ꢀ6.45 (m, 3H), 5.86 (s, 1H), 5.53 (br.d, J = 7.1 Hz, 1H), 4.20
(sextet, J = 6.6 Hz, 1H), 3.74 (s, 3H), 3.64 (s, 6H), 2.83 (s, 1H),
1.85ꢀ2.02 (m, 2H), 1.47ꢀ1.63 (m, 4H), 1.21ꢀ1.41 (m, 2H). 13C NMR
(100 MHz, CDCl3): δ 168.2, 160.2, 159.7, 153.4, 140.6, 131.5, 125.6,
113.8, 108.7, 101.2, 80.1, 76.1, 64.6, 55.4, 55.2, 51.6, 32.8, 32.7,
23.62, 23.60.
N-[2-(Cyclopentylamino)-1-(4-fluorophenyl)-2-oxoethyl]-N-[3-(triflu-
oromethyl)phenyl] Propynamide (12). Yield 522 mg (60%). 1H NMR
(400 MHz, CDCl3): δ 7.24ꢀ7.54 (m, 4H), 7.09 (dd, J = 8.7 Hz, J = 5.4
Hz, 2H), 6.88 (t, J = 8.7 Hz, 2H), 6.09 (s, 1H), 5.87 (br.s, 1H), 4.21
(sextet, J = 7.1 Hz, 1H), 2.83 (s, 1H), 1.79ꢀ2.05 (m, 2H), 1.48ꢀ1.68
(m, 4H), 1.35ꢀ1.47 (m, 1H), 1.21ꢀ1.32 (m, 1H). 13C NMR (100
MHz, CDCl3): δ 167.9, 162.8 (d, J = 249.8 Hz), 153.4, 138.9, 134.7,
132.1 (d, J = 9.0 Hz), 130.8 (quartet, J = 33.5 Hz), 129.1 (d, J = 2.6 Hz),
129.0, 128.1 (quartet, J = 3.9 Hz), 125.3 (quartet, J = 3.9 Hz), 123.3
(quartet, J = 271.7 Hz), 115.6 (d, J = 20.6 Hz), 81.3, 75.5, 63.3, 51.7,
31.73, 32.71, 23.63, 23.59. 19F NMR (376 MHz, CDCl3): δ ꢀ62.80,
ꢀ111.77.
N-[2-(Cyclohexylamino)-2-oxo-1-thien-2-ylethyl]-N-[3-(trifluoromethyl)
phenyl] Propynamide (13). Yield 579 mg (67%). 1H NMR (400 MHz,
CDCl3): δ 7.46ꢀ7.55 (m, 2H), 7.35ꢀ7.43 (m, 2H), 7.23 (dd, J = 5.0 Hz,
J = 0.8 Hz, 1H), 6.92 (d, J = 3.3 Hz, 1H), 6.85 (dd, J = 5.0 Hz, J = 3.3 Hz,
1H), 6.25 (s, 1H), 6.16 (br.d, J = 7.1 Hz, 1H), 4.20 (sextet, J = 6.7 Hz,
1H), 2.85 (s, 1H), 1.87ꢀ2.03 (m, 2H), 1.50ꢀ1.67 (m, 4H), 1.31ꢀ1.46
(m, 2H). 13C NMR (100 MHz, CDCl3): δ 167.0, 153.0, 139.1, 134.5,
134.0, 131.9 (quartet, J = 33.5 Hz), 130.1, 129.1, 128.2, 127.4 (quartet, J
= 3.9 Hz), 126.6, 125.4 (quartet, J = 3.9 Hz), 123.3 (quartet, J = 273.0
Hz), 81.4, 75.4, 59.3, 51.8, 32.7, 23.63, 23.58. 19F NMR (376 MHz,
CDCl3): δ ꢀ62.69.
N-[2-(Cyclopentylamino)-2-oxo-1-phenylethyl]-N-(2,3-dihydro-1,4-
1
benzodioxin-6-yl) Propynamide (8). Yield 697 mg (86%). H NMR
(400 MHz, CDCl3): δ 7.17ꢀ7.25 (m, 3H), 7.10ꢀ7.16 (m, 2H),
6.52ꢀ6.80 (m, 3H), 5.93 (s, 1H), 5.74 (d, J = 7.5 Hz, 1H), 4.10ꢀ4.25
(m, 5H), 2.84 (s, 1H), 1.84ꢀ2.00 (m, 2H), 1.46ꢀ1.62 (m, 4H),
1.21ꢀ1.41 (m, 2H). 13C NMR (100 MHz, CDCl3): δ 168.0, 153.9,
143.6, 142.8, 133.7, 132.1, 130.1, 128.6, 128.4, 123.8, 119.5, 116.5, 80.5,
76.1, 64.9, 64.1, 63.9, 51.5, 32.72, 32.67, 23.60, 23.57.
N-[2-(Cyclopentylamino)-2-oxo-1-phenylethyl]-N-[3-methyl-4-(1H-
tetraazol-1-yl)phenyl] Propynamide (9). Yield 461 mg (54%). 1H NMR
(400 MHz, CDCl3): δ 8.81 (s, 1H), 7.13ꢀ7.35 (m, 8H), 6.13 (s, 1H),
N-[2-(Cyclopentylamino)-2-oxo-1-phenylethyl]-N-(2-naphthyl) Pro-
pynamide (14). Yield 652 mg (82%). 1H NMR (400 MHz, DMSO-d6):
δ 8.24 (d, J = 6.6 Hz, 1H), 7.88 (br.s, 1H), 7.74ꢀ7.84 (m, 2H),
7.60ꢀ7.73 (m, 1H), 7.40ꢀ7.50 (m, 2H), 6.99ꢀ7.17 (m, 5H), 6.10 (s,
1H), 4.11 (s, 1H), 4.08 (sextet, J = 6.6 Hz, 1H), 1.71ꢀ1.90 (m, 2H),
1.38ꢀ1.68 (m, 5H), 1.21ꢀ1.33 (m, 1H). 13C NMR (100 MHz, DMSO-
d6): δ 168.2, 152.8, 136.2, 134.3, 132.2, 131.9, 130.01, 129.96, 128.7,
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127.9, 127.4, 127.3, 126.5, 126.2, 83.1, 76.8, 63.6, 50.7, 32.1, 31.8,
23.44, 23.40.
N-(1,3-Benzodioxol-5-ylmethyl)-N-[2-(benzylamino)-1-(2-fluorophenyl)-
161.0 (d, J= 249.8 Hz), 160.8 (d, J= 248.5 Hz), 159.2 (d, J= 249.8 Hz), 159.1
(d, J= 251.1 Hz), 153.8, 153.6, 133.2, 131.7, 131.5, 131.2, 130.8, 130.7, 130.65,
130.57, 126.4 (d, J = 11.6 Hz), 126.0 (d, J = 12.9 Hz), 124.1, 124.0 (d, J = 3.9
Hz), 123.4 (d, J= 3.9 Hz), 121.3 (d, J= 14.2 Hz), 120.1 (d, J= 14.2 Hz), 115.9
(d, J = 20.6 Hz), 115.2 (d, J = 20.6 Hz), 79.9, 79.2, 75.4, 75.1, 58.58, 58.56,
2-oxoethyl] Propynamide (15). Yield 814 mg (92%). In DMSO-d6,
1
mixture of two rotameric forms in 60:40 ratio. H NMR (400 MHz,
DMSO-d6): δ 8.99 (t, J = 5.8 Hz, 0.4H), 8.85 (t, J = 5.8 Hz, 0.6H),
7.22ꢀ7.38 (m, 6H), 7.18 (td, J = 7.5 Hz, J = 1.2 Hz, 0.4H), 7.11 (td, J =
7.5 Hz, J = 1.2 Hz, 0.6H), 6.97ꢀ7.03 (m, 0.4H), 6.84ꢀ6.91 (m, 0.6H),
6.63 (d, J = 7.9 Hz, 0.6H), 6.57 (d, J = 7.9 Hz, 0.4H), 6.46 (s, 0.4H), 6.42
(d, J = 1.2 Hz, 0.6H), 6.34 (dd, J = 7.9 Hz, J = 1.2 Hz, 0.6H), 6.28 (d, J =
1.2 Hz, 0.4H), 6.25 (s, 0.6H), 6.19 (dd, J = 7.9 Hz, J = 1.2 Hz, 0.4H),
5.88ꢀ5.95 (m, 2 ꢁ 0.6Hþ2 ꢁ 0.4H), 5.07 (d, J = 17.0 Hz, 0.6H), 4.93
(d, J = 15.8 Hz, 0.4H), 4.81 (s, 0.4H), 4.62 (s, 0.6H), 4.51 (d, J = 17.0 Hz,
0.6H), 4.26ꢀ4.44 (m, 2 ꢁ 0.6Hþ2 ꢁ 0.4H), 4.15 (d, J = 15.8 Hz, 0.4H).
13C NMR (100 MHz, DMSO-d6): δ 168.6, 161.4 (d, J = 247.2 Hz),
161.2 (d, J = 247.2 Hz), 154.7, 154.6, 151.7, 147.1, 147.0, 146.2, 146.0,
139.3, 139.2, 131.7, 131.56, 131.48, 131.40, 131.37, 131.32, 130.88,
130.86, 130.62, 130.60, 128.8, 128.7, 127.8, 127.7, 127.4, 127.3, 124.9 (d,
J = 2.6 Hz), 124.7 (d, J = 2.6 Hz), 122.9 (d, J = 14.2 Hz), 122.3 (d, J =
14.2 Hz), 120.5, 119.7, 115.6 (d, J = 20.6 Hz), 115.3 (d, J = 20.6 Hz),
108.0, 107.9, 107.7, 106.9, 101.2, 101.1, 84.2, 83.1, 76.7, 75.9, 59.0, 54.5,
50.6, 47.4, 43.0, 42.8. 19F NMR (376 MHz, DMSO-d6): δ ꢀ114.70
(0.6F), ꢀ115.24 (0.4F).
56.67, 56.64, 48.9, 48.4, 32.48, 32.44, 25.33, 25.27, 24.7, 24.6, 24.52, 24.48. 19
F
NMR (376 MHz, CDCl3): δ ꢀ114.95 (0.75Fþ0.25F), ꢀ118.16 (0.25F),
ꢀ119.47 (0.75F).
Computational Simulation to Predict the Drug-like Prop-
erties of PACMA Compounds. After the required ligand structures
of a specified configuration were built, energy minimization was
performed using the Catalyst software package (Accelrys, Inc., San
Diego, CA). The lowest-energy conformation for each compound was
exported to ADMET Predictor to calculate ADME (absorption, dis-
tribution, metabolism, and excretion) properties and 3-D polar
surface areas.
Cell Culture. The human breast cancer cell line MDA-MB-435 and
the colon cancer cell line HT29 were purchased from the American Type
Cell Culture (Manassas, VA). Colon cancer cell lines HCT116 p53þ/þ
and HCT116 p53ꢀ/ꢀ were kindly provided by Dr. Bert Vogelstein
(Johns Hopkins Medical Institutions, Baltimore, MD). The human
ovarian carcinoma cell line (HEY), naturally resistant to cisplatin
(CDDP), was kindly provided by Dr. Louis Dubeau (USC Norris
Cancer Center).41,42 The bladder cancer cell lines were kindly provided
by Dr. Richard Cote (USC Keck School of Medicine). Cells were
maintained as monolayer cultures in the appropriate media: RPMI-1640
(HT29, HCT116 p53þ/þ, HCT116 p53ꢀ/ꢀ, and HEY cell lines) or
DMEM (MDA-MB-435 and UMUC3 cell lines) supplemented with
10% fetal bovine serum (FBS) (HyClone, Road Logan, UT) at 37 °C in
a humidified atmosphere of 5% CO2. To remove the adherent cells from
the flask for subculture and counting, cells were washed with PBS
without calcium or magnesium, incubated with a small volume of 1ꢁ
trypsinꢀEDTA solution (Sigma-Aldrich, St. Louis, MO) for 5 min,
resuspended with fresh culture medium, and centrifuged at 1200 rpm for
5 min. All experiments were performed using cells in exponential growth
phase. Cells were routinely checked for Mycoplasma contamination
using PlasmoTest kit (InvivoGen, San Diego, CA).
N-[2-(Benzylamino)-2-oxo-1-thien-2-ylethyl]-N-[3-(trifluoromethyl)-
phenyl] Propynamide (16). Yield 498 mg (56%). 1H NMR (400 MHz,
CDCl3): δ 7.58 (d, J = 7.5 Hz, 1H), 7.53 (br.d, J = 7.9 Hz, 1H), 7.47 (br.
s, 1H), 7.41 (t, J = 7.9 Hz, 1H), 7.22ꢀ7.35 (m, 6H), 6.95 (d, J = 3.3 Hz,
1H), 6.87 (dd, J = 5.0 Hz, J = 3.7 Hz, 1H), 6.78 (br.t, J = 5.4 Hz, 1H), 6.33
(s, 1H), 4.42ꢀ4.54 (m, 2H), 2.88 (s, 1H). 13C NMR (100 MHz,
CDCl3): δ 127.6, 153.0, 139.1, 137.6, 134.2, 133.9, 130.9 (quartet, J =
33.5 Hz), 130.3, 129.1, 128.5, 128.3, 127.39, 127.35, 127.30, 127.26,
126.6, 125.4 (quartet, J = 3.9 Hz), 123.3 (quartet, J = 273.0 Hz), 81.4,
75.3, 59.6, 43.7. 19F NMR (376 MHz, CDCl3): δ ꢀ62.57.
N-(2-Chlorobenzyl)-N-[2-(cyclopentylamino)-2-oxo-1-phenylethyl]
Propynamide (17). Yield 482 mg (61%). In CDCl3, mixture of two
rotameric forms in 80:20 ratio. 1H NMR (400 MHz, CDCl3): δ
7.05ꢀ7.33 (m, 9 ꢁ 0.8Hþ6 ꢁ 0.2H), 6.97ꢀ7.03 (m, 2 ꢁ 0.2H),
6.87ꢀ6.92 (m, 0.2H), 6.21 (s, 0.2H), 6.06 (br.d, J = 7.5 Hz, 0.2H), 5.99
(br.d, J = 7.1 Hz, 0.8H), 5.78 (s, 0.8H), 5.14 (d, J = 17.9 Hz, 0.8H), 4.95
(d, J = 17.0 Hz, 0.2H), 4.93 (d, J = 17.9 Hz, 0.8H), 4.64 (d, J = 17.0 Hz,
0.2H), 4.12ꢀ4.28 (m, 0.8Hþ0.2H), 3.40 (s, 0.2H), 3.04 (s, 0.8H),
1.87ꢀ1.99 (m, 2 ꢁ 0.8Hþ2 ꢁ 0.2H), 1.49ꢀ1.65 (m, 4 ꢁ 0.8Hþ4 ꢁ
0.2H), 1.27ꢀ1.40 (m, 2 ꢁ 0.8Hþ2 ꢁ 0.2H). 13C NMR (100 MHz,
CDCl3): δ 167.6, 167.5, 155.0, 154.4, 134.1, 133.7, 133.5, 131.9, 131.8,
129.4, 129.17, 128.88, 128.71, 128.62, 128.56, 128.02, 127.7, 127.6,
126.3, 126.2, 81.0, 79.6, 75.5, 75.4, 66.0, 62.2, 51.5, 51.4, 48.9, 45.1, 32.6,
32.5, 23.57, 23.54.
Compound Dilutions. Ten millimolar stock solutions of all
compounds were prepared in DMSO and stored at ꢀ20 °C. Further
dilutions were made fresh in cell-culture media.
Cytotoxicity Assays. Cytotoxicity was assessed by MTT assay as
described previously.43 In brief, cells were seeded in 96-well microtiter
plates and allowed to attach overnight. Cells were subsequently treated
with continuous exposure to corresponding drugs for 72 h. At the end of
treatments, an MTT solution (at a final concentration of 0.5 mg/mL)
was added to each well, and cells were incubated for 4 h at 37 °C. After
removal of the supernatant, DMSO was added, and the absorbance was
read at 570 nm. All assays were done in triplicate. The IC50 was then
determined for each drug from a plot of log(drug concentration) versus
percentage of cells killed.
Colony Formation Assay. Colony formation assays were per-
formed as previously described to confirm the activity of these
compounds.44 In brief, cells were plated in 24-well plates at a density
of 1000 cells per well and allowed to attach overnight. The next day,
1 μM concentration of each compound was added for 24 h. Cells were
washed with 1ꢁ PBS, and fresh media was then added to the cells. Cells
were incubated until colonies formed (8ꢀ10 days). Subsequently, cells
were washed, fixed with a 1% glutaraldehyde solution for 30 min, and
stained with a solution of crystal violet (2%) for 30 min. After staining,
cells were thoroughly washed with distilled water. Colonies were imaged
on a Chemi-Doc Imaging System (Bio-Rad, Hercules, CA) and counted
using the Quantity One software package (Bio-Rad, Hercules, CA). The
data reported is a representative of at least three independent
experiments.
N-[2-(Cyclohexylamino)-2-oxo-1-thien-2-ylethyl]-N-phenyl Propy-
1
namide (18). Yield 487 mg (66%). H NMR (400 MHz, CDCl3): δ
7.16ꢀ7.35 (m, 6H), 6.97 (d, J = 2.9 Hz, 1H), 6.85ꢀ6.91 (m, 1H), 6.24
(s, 1H), 6.15 (br.d, J = 7.9 Hz, 1H), 3.75ꢀ3.87 (m, 1H), 2.84 (s, 1H),
1.83ꢀ1.99 (m, 2H), 1.53ꢀ1.75 (m, 3H), 1.27ꢀ1.43 (m, 2H),
1.08ꢀ1.25 (m, 3H). 13C NMR (100 MHz, CDCl3): δ 166.6, 153.3,
138.7, 134.9, 130.0, 129.8, 128.8, 128.5, 127.9, 126.2, 80.8, 75.7, 59.7,
48.7, 32.5, 32.4, 25.3, 24.6, 24.5.
N-[2-(Cyclohexylamino)-1-(2-fluorophenyl)-2-oxoethyl]-N-(2-fluorophe-
nyl) Propynamide (19). Yield 548 mg (69%). In CDCl3, mixture of two
rotameric forms in 75:25 ratio. 1H NMR (400 MHz, CDCl3): δ 7.91 (td, J =
7.9 Hz, J = 1.7 Hz, 0.75H), 7.31 (br.t, J = 7.9 Hz, 0.25H), 7.10ꢀ7.25 (m, 2 ꢁ
0.75Hþ2ꢁ 0.25H), 6.84ꢀ7.09 (m, 3 ꢁ 0.75Hþ3ꢁ 0.25H), 6.68ꢀ6.82 (m,
2 ꢁ 0.75Hþ2 ꢁ 0.25H), 6.40ꢀ6.47 (m, 0.75Hþ0.25H), 6.15 (s, 0.25H),
6.09 (br.d, J = 7.9 Hz, 0.75H), 3.70ꢀ3.82 (m, 0.75Hþ0.25H), 2.80 (s,
0.25H), 2.71 (s, 0.75H), 1.49ꢀ1.98 (m, 5 ꢁ 0.75Hþ5 ꢁ 0.25H), 0.94ꢀ1.37
(m, 5 ꢁ 0.75Hþ5 ꢁ 0.25H). 13C NMR (100 MHz, CDCl3): δ 167.2, 165.9,
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Journal of Medicinal Chemistry
ARTICLE
In Vivo Mouse Xenograft Studies. Sixteen virgin female athymic
nude (nu/nu) mice (Simonsen Laboratory Inc., Gilroy, CA) were used for
in vivo efficacy testing. The animals were fed ad libitum and kept in
temperature controlled rooms at20 ( 2 °C with a 12 h lightꢀdark period.
Animal care and manipulation were in agreement with the University of
Southern California (USC) institutional guidelines, which are in accor-
dance with the Guidelines for the Care and Use of Laboratory Animals.
Human breast cancer MDA-MB-435 cells in logarithmic phase growth
from in vitro cell culture were inoculated subcutaneously on the rear flank
of the mice (1.5 ꢁ 106 cells/mouse) under aseptic conditions. Tumor
growth was assessed twice weekly by measuring tumor diameters with a
Vernier caliper (length ꢁ width). Tumor weight was calculated according
to the formula: TW (mg) = tumor volume (mm3) = d2 ꢁ D/2, where d
and D are the shortest and longest diameters, respectively. Tumors were
allowed to grow to an average volume of 100 mm3. Animals were then
randomly assigned to control and treatment groups (n = 8), to receive
vehicle control or compound 2 (10 mg/kg for 9 days, 20 mg/kg for 5 days,
and 40 mg/kg for 12 days, 2 was dissolved in 10% DMSO/90% sesame
oil) via i.p. injectiononcedaily. Treatment ofeachanimalwasbasedonthe
average body weight of the treatment group.
Tris-HCl, pH 7.5, 150 mM NaCl, and 1% NP-40) and pelleted by
centrifugation at 14 000 rpm for 20 min at 4 °C. Protein concentration
of the whole cell lysates was measured using BCA protein assay and
equal amounts of total protein were resolved on a 10% tris-glycine gel
via SDSꢀPAGE. The separated proteins were electroblotted onto
nitrocellulose membrane and blocked in 5% BSA/PBS for 1 h at room
temperature. The membrane was probed with anti-p53 (SC-126,
Santa Cruz Biotechnology Inc.), anticaspase 9 (Cat. no. 9508; Cell
Signaling Technology), anti-R-tubulin (SC-5286, Santa Cruz Bio-
technology Inc.), and anti-GAPDH (Y3322GAPDH, Biochain
Institute, Inc.) antibodies at 4 °C overnight. Horseradish peroxi-
dase-conjugated secondary antibodies (Invitrogen, Carlsbad, CA) in
combination with SuperSignal Dura (ThermoFisher, Rockford, IL)
enhanced chemiluminescence (ECL) solutions were used to visualize
proteins of interest with a ChemiDoc Imaging system (Bio-Rad,
Hercules, CA).
Kinexus Antibody Microarray. MDA-MB-435 cells were treated
with 0.1 μM compound 1 for 24 h. After treatment, cells were washed
with ice-cold PBS to remove residual medium. Cells were then lysed in
200 μL of lysis buffer (20 mM MOPS, pH 7.0, 2 mM EGTA, 5 mM
EDTA, 30 mM sodium fluoride, 60 mM β-glycerophosphate, pH 7.2,
20 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1 mM
phenylmethylsulfonylfluoride, 3 mM benzamidine, 5 μM pepstatin A,
10 μM leupeptin, 1% Triton X-100, 1 mM dithiothreitol) and collected
into a microcentrifuge tube. The cell lysates were sonicated four times for
10 s each with 15 s intervals on ice to rupture cell membrane and shear
chromosomal DNA. After sonication, the homogenates were centrifuged
at 90 000 ꢁ g for 30 min at 4 °C. The supernatants were then transferred to
a clean microcentrifuge tube, and the protein concentrations were measured
using BCA protein assay. Aliquots (250 μL) of the whole cell lysates were
submitted to Kinexus for the 628-antibody microarray analysis.
Ingenuity Pathway Analysis. Potential signaling pathways in-
duced by compound 1 were analyzed by Ingenuity Pathway Analysis
(IPA) software using the Kinexus 628-antibody microarray results. The
statistically significant up-regulated or down-regulated pan-specific
proteins with their corresponding Swiss-Prot accession numbers and
ratio changes were uploaded as an Excel spreadsheet file to the IPA
server. Compound 1-mediated signaling pathways were then analyzed
by IPA core analysis.
Detection of Mitochondrial Superoxide Production. MDA-
MB-435 cells were treated with compounds 1, 2, 4, and 18 at their IC50
or 3 ꢁ IC50 for 24 h. After drug treatments, a solution of 5 μM MitoSOX
Red mitochondrial superoxide indicator (Invitrogen, Carlsbad, CA) was
added, and cells were incubated for 10 min at 37 °C. Cells were then
washed three times with Hank’s balanced salt solution (HBSS) to
remove excess MitoSOX. After washing, cells were either observed
under the fluorescence microscope or collected by trypsinization,
washed twice with HBSS, and measured for superoxide production
using a BD LSRII flow cytometer (BD Biosciences, San Jose, CA, USA).
Data were analyzed with the BD FACSDiva software package (BD
Biosciences, San Jose, CA, USA).
Statistical Analysis. Eight mice were assigned to each group and
the results were expressed as the mean ( SEM. Statistical analysis and
p-value determination were done by two-tailed paired t-test with a
confidence interval of 95% for determination of the differences between
groups. A p-value of <0.05 was considered to be statistically significant.
ANOVA was used to test for significance among groups. The SAS
statistical software package (SAS Institute, Cary, NC) was used for
statistical analysis.
Cell Cycle Analysis. Cell cycle perturbations were analyzed by
propidium iodide DNA staining. Exponentially growing MDA-MB-435
cells were treated with 5 μM compound 4 for 6, 12, and 24 h. At the end of
treatment, cells were collected and washed with 1ꢁ PBS after a gentle
centrifugation at 1500 rpm for 5 min. Cells were then thoroughly
resuspended in 0.5 mL of PBS and fixed in 70% ethanol overnight at
4 °C. Ethanol-fixed cells were centrifuged at 3000 rpm for 5 min and
washedtwice in PBS to removeresidual ethanol. For cell cycle analysis, the
pellets were resuspended in 1 mL of PBS containing 0.02 mg/mL of
propidium iodide and 0.5 mg/mL of DNase-free RNase A and incubated
at 37 °C for 30 min. Cell cycle profiles were obtained using a BD LSRII
flow cytometer (BD Biosciences, San Jose, CA). Propidium iodide was
excited using a 488 nm blue argon laser. Resulting fluorescence was
measured using a PMT detector equipped with 550 nm long pass dichroic
mirror and 575/26 bandpass filter. Data were analyzed with the ModFit
LT software package (Verify Software House, Inc., Topsham, ME).
Apoptosis Assay. To quantify drug-induced apoptosis, annexin V/
propidium iodide staining was performed and analyzed by flow cyto-
metry. MDA-MB-435 cells were treated with 10 μM compound 1, 2, or 4
for 24 and 48 h. After treatment, both floating and adherent cells were
combined and subjected to annexin V/propidium iodide staining using
an annexin VꢀFITC apoptosis detection kit (Oncogene Research
Products, San Diego, CA) according to manufacturer’s recommenda-
tions. Untreated control cells (48 h) were maintained in parallel to drug-
treated groups. Double staining was used to distinguish between viable,
early apoptotic, and necrotic or late apoptotic cells. The resulting
fluorescence (FITC-A channel for green fluorescence and PE-A channel
for red fluorescence) was measured by flow cytometry using a BD LSRII
flow cytometer (BD Biosciences, San Jose, CA). According to this
method, the lower left quadrant shows the viable cells, the upper left
quadrant shows early apoptotic cells, the upper right quadrant shows late
apoptotic, and the lower right quadrant shows both necrotic cells and
cellular debris.
Pharmacokinetics Study. Preliminary pharmacokinetics of com-
pounds 4 and 9 were performed using 18 mice, each time point consisting
of two mice. Mice were stratified to various doses given as an i.p. injection
of 4 and 9 (24 mg/kg). At the specified time points following drug
administration, blood was collected with heparinized syringes. The
samples were allowed to settle in 500 μL eppendorf vials and then
centrifuged at 1500 rpm for 10 min. The plasma was removed, transferred
into new eppendorf tube, and frozen at ꢀ80 o C until analysis.
’ ASSOCIATED CONTENT
Western Blotting Analysis. Cells were detached using 1ꢁ
trypsinꢀEDTA solution and collected by centrifugation at 1200 rpm
for 5 min. Cells were then lysed in 80 μL of 1ꢁ cell lysis buffer (50 mM
S
Supporting Information. Structures, colony formation
b
assay for moderately active and inactive compounds, and selected
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Journal of Medicinal Chemistry
ARTICLE
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’ AUTHOR INFORMATION
Corresponding Author
*Mailing adress: Department of Pharmacology and Pharmaceu-
tical Sciences, University of Southern California, Los Angeles,
California, 90089. Phone: 323-442-2341. Fax: 323-442-1390.
E-mail: neamati@usc.edu.
’ ACKNOWLEDGMENT
This study was supported in part by the Sharon and William
Cockrell Endowed Cancer Research Fund (N.N.) and by the
USC Zumberge Research and Innovation Fund (N.A.P.).
’ ABBREVIATIONS
ADMET, absorption, distribution, metabolism, excretion, and
toxicity; DCFH, 2,7-dichlorodihydrofluorescein; DMSO, dimeth-
yl sulfoxide; HBA, hydrogen-bond acceptor; HBD, hydrogen-
bond donor; HSP, heat shock protein; HTS, high-throughput
screening; i.p., intraperitoneal; IPA, Ingenuity Pathway Analysis;
MMA, monomethylarsonous acid; MW, molecular weight; MLogP,
Moriguchi octanolꢀwater partition coefficient;Nrf2, nuclear fac-
tor erythroid 2-related factor 2;PACMAs, propynoic acid carba-
moyl methyl amides; PBS, phosphate-buffered saline;PK,
pharmacokinetics; PD, pharmacodynamics;PSA, polar surface
area, three-dimensional; ROS, reactive oxygen species; SAR, struc-
tureꢀactivity relationship; SEM, standard error of mean; SOD1,
superoxide dismutase 1;Sþ logP), SimulationsPlus, Inc., model
of log P
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