Novel flavonoids with antiproliferative activities against breast cancer cells

Article abstract of DOI:10.1021/jm101440r

A series of flavone analogues were synthesized and evaluated for their antiproliferation activity against breast cancer cells. The IC₅₀ of compound 10 and 24 were determined to be at 5 μM. These compounds were used as baits to screen breast cancer cDNA expression phage display proteome library. DNA sequencing of the binding phages suggests that eEF1A1 is a target protein for 10 and 24. Further optimization of these compounds led to the discovery of 39 with higher cytotoxic potency (IC₅₀ = 1 μM) and binding to eEF1A2. Biological and biochemical data suggest that eEF1A2 might be a therapeutic target and that 39 is an excellent lead compound for further development.

Full text of DOI:10.1021/jm101440r

ARTICLE
pubs.acs.org/jmc
Novel Flavonoids with Antiproliferative Activities against Breast
Cancer Cells
Nianhuan Yao,†,^ Chao-Yu Chen,†,^ Chun-Yi Wu,† Kiyomi Motonishi,† Hsing-Jien Kung,†,§ and
Kit S. Lam*,†,‡,§
†Department of Biochemistry and Molecular Medicine, ‡Division of Hematology & Oncology, §UC Davis Cancer Center,
University of California Davis, Sacramento, California 95817, United States
S Supporting Information
b
ABSTRACT: A series of flavone analogues were synthesized
and evaluated for their antiproliferation activity against breast
cancer cells. The IC50 of compound 10 and 24 were determined
to be at 5 μM. These compounds were used as baits to screen
breast cancer cDNA expression phage display proteome library.
DNA sequencing of the binding phages suggests that eEF1A1 is
a target protein for 10 and 24. Further optimization of these compounds led to the discovery of 39 with higher cytotoxic potency
(IC50 = 1 μM) and binding to eEF1A2. Biological and biochemical data suggest that eEF1A2 might be a therapeutic target and that
39 is an excellent lead compound for further development.
’ INTRODUCTION
and ER positive breast cancer cell lines. The molecular targets of
these lead compounds were identified by using them as bait to
screen cDNA expression phage display proteome library. Further
optimization of the lead compounds resulted in the development
of a relatively potent antiproliferative compound that selectively
bind to eukaryotic elongation factor 2A (eEF1A2).
Breast cancer is the most common malignant tumor and the
second most lethal cancer among women.1 Women have a 1 in 8
lifetime risk of developing breast cancer and 1 in 35 risk of breast
cancer causing death in the US. Many breast tumors express
higher levels of estrogen receptors than normal breast tissues.2
Estrogen receptor (ER)/progesterone receptor (PR) positive
breast cancers often respond to hormonal therapy. Conventional
chemotherapeutic drugs are used for the treatment of ER/PR
negative breast cancer. Herception is useful for the treatment of
Her-2/neu positive breast cancer. Despite of combination hor-
monal therapy, chemotherapy, and targeted therapy,3,4 most
metastatic breast cancer eventually becomes refractory to such
treatments. There is an urgent need to explore new drug
candidates with novel mechanisms of action.
Many current anticancer drugs are either natural products or
their derivatives.5,6 Natural products have also served as useful
scaffolds for chemical diversification in the context of drug
discovery.7 Flavonoids are the most explored class of natural
products because they are widely distributed among various
plants and common components of the human diet.8 They
probably play an important role in cancer prevention by inter-
fering with cell proliferation, survival, cell signaling, and regulat-
ing the immune system.9ꢀ11 Additional studies have also
indicated that some flavonoids exhibit aromatase inhibitory
activity12,13 and tyrosinase inhibitory activity.14 We have pre-
viously developed several novel flavonoid scaffolds with three
diversification points.15 Here, based on the flavone template, a
series of flavonoid derivatives were synthesized on solid phase
and evaluated for their antiproliferative activities in breast cancer
cell line MDA-MB-231 and MCF-7. Two compounds were
found to exhibit potent cytotoxic effect in both ER negative
’ RESULTS AND DISCUSSION
A number of flavone scaffolds were developed with three
functional groups (carboxy, fluoro, and nitro). Heterocycles or
other pharmacophores can be readily introduced to the flavo-
none scaffolds. In our previous study, we reported the use of solid
phase method to prepare several flavone derivatives (Figure 1,
1ꢀ6).15 As part of ongoing search for anticancer agents, we used
MTT assay to evaluate their antiproliferactive activity in breast
cancer cell line MDA-MB-231(ERꢀ) and MCF-7(ERþ). A
compound was considered active if the IC50 was e10 μM during
the initially screening. Only 5 showed cytotoxic activity in both
breast cancer cell lines (Figure 2).
Design, Synthesis, and Biological Evaluation of Flavone
Analogues 7ꢀ32. To optimize 5, we designed and synthesized
26 flavone analogues (7ꢀ32) according to our published method
(Scheme 1).15 Various Fmoc amino acids, such as polar, acidic,
and hydrophobic amino acids (R1 group in Table 1) were first
introduced to Rink resin. After Fmoc deprotection, 4-(7-fluoro-
6-nitro-4-oxo-4H-chromen-2-yl)-benzoic acid as a functionalized
flavone scaffold was attached to the resin. The fluorine in the
resin-bound flavone scaffold was subsequently replaced by a
number of structurally diverse primary amines, such as aliphatic
Received: November 19, 2010
Published: May 20, 2011
r
2011 American Chemical Society
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Figure 1. Structure of flavone model compounds 1ꢀ6.
and aromatic amines (R2 group in Table 1). Reduction of the
nitro group then afforded the o-phenylenediamine intermediates.
The resin bound o-phenylenediamine flavone was treated with
different isothiocyanates (R3 group in Table 1) in the presence of
1, 3-diisopropylcarbodiimide (DIC) to provide 7ꢀ17 and
18ꢀ20 (without introducing R1 group). To increase structural
diversities, the resin bound o-phenylenediamine flavone was
reacted with various aliphatic aldehydes and substituted aromatic
aldehydes (R3 group in Table 1) to yield 21ꢀ28 and 29ꢀ32
(without introducing R1 group). Compounds 7ꢀ32 were tested
for in vitro anticancer activity against two breast cancer cell lines
MDA-MB-231(ERꢀ) and MCF-7(ERþ). Cells were treated
with 10 μM concentration of each analogue for three days. 10
and 24 were among the most active compounds (Figure 3).
Further MTT assays were carried out to test 10 and 24 in varying
concentration, ranging from 0.08 μM to 50 μM (Supporting
Figure 2. Cytotoxicity of flavone analogues 1ꢀ6.
the bound phage clones were analyzed via NCBI blast. In the
panning experiment with 10, 4 out of 12 phage clones encoded
DNA matched with known human proteins, two clones encoded
eukaryotic elongation factor 1A1 (eEF1A1), one clone encoded
apolipoprotein 3 (APOL3), and one encoded tropomysin 2
(TMP2) (sequence shown in Supporting Information). Eukaryotic
elongation factor 1A 1 (eEF1A1) was also recovered in the panning
experiment with 24. To verify eEF1A1 as the protein target for 10
and 24, biotinylated 10 and 24 were synthesized (Supporting
Information Scheme 2), and incubated with MDA-MB-231 cell
lysate. Neutravidin beads were then used to pull down proteins
bound to 10 and 24. Subsequent Western blot analysis demon-
strated that eEF1A did indeed bind to 10 and 24. (Figure 4)
StructureꢀActivity Relationships (SAR) and Surface Plas-
mon Resonance (SPR) Analysis. To discover more active
flavone derivatives and to establish preliminary SAR, 10 and 24
were further optimized. Exchange of R1, R2, and R3 groups of 10
and 24, afforded 33ꢀ37 (Figure 5). Systematic structural
simplification of 10 and 24 by removal of R1 and R3 group,
Information, Figure 1). Both 10 and 24 had high efficacy (IC50
=
5.0 μM) in MDA-MB-231cells. Additionally, 10 (IC50 = 5.0 μM)
was higher than 24 (IC50 = 8.0 μM) in MCF-7 cells.
Identification of eEF1A1 as a Binding Protein for Com-
pound 10 and 24. Phage display technology enables one to express
and display recombinant peptides or proteins on the phage surface.
Peptide and protein epitope for antibodies,16 EGFR,17,18 and
binding proteins for bioactive small molecules19ꢀ21 have been
successfully identified by such technology. To identify the target
proteins for 10 and 24, these chemical compounds were synthesized
on TentaGel beads (polystyrene with grafted polyoxyethylene)
(Supporting Information Scheme 1) and used as bait to screen
cDNA expression phage display proteome library constructed from
human breast tumor cDNA. TentaGel resin was used because of its
favorable swelling characteristics both in organic and aqueous
media, its uniform size, and its nonstick property.22 After four
rounds of biopanning, 12 phage clones from each compound were
randomly selected, amplified and sequenced. The DNA sequence of
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Scheme 1a
a Reagents and conditions: (a) Amino Acid (3 equiv), HOBt/DIC (3 equiv), DMF, r.t., 2 h; (b) 20% piperidine/DMF, 15 min, twice; (c) 4-(7-fluoro-6-
nitro-4-oxo-4H-chromen-2-yl)-benzoic acid (2 equiv), HOBt/DIC (3 equiv), DMF, r.t., 3 h; (d) 1 M amine, 25% DIPEA/DMF, r.t., overnight; (e) 2 M
SnCl2/DMF, r.t., overnight; (f) 1 M isothiocyanate, 1 M DIC, DMF, r.t., overnight; (g) aldehyde (10 equiv), 5% HOAc/DMF, r.t. overnight; (h) TFA/
TIS/H2O (95%:2.5%:2.5%), r.t., 3 h. (i) 10% Acetic anhydride/10% pyridine/DMF, 30 min.
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Table 1. Building Blocks of Compounds 7ꢀ32
Figure 3. Cytotoxicity of flavone analogues 7ꢀ32.
reduction of the nitro group, and subsequently capping with
acetyl group, yielded 38ꢀ44 (Figure 5). 33ꢀ44 were tested for
antiproliferation against MDA-MB-231 cells(ERꢀ) (Figure 5).
In the case of modification of 10, replacement of Leu with Phe
(33), (R)-(ꢀ)-1-cyclohexylethylamine with benzylamine (34),
and both Leu with Phe and (R)-(ꢀ)-1-Cyclohexylethylamine
with benzylamine (35), resulted in a significant loss of anticancer
activity. In contrast, optimization of 24 using the same strategy
(36 and 37) showed the equal potency as parent compound 24.
Compared to 10 (IC50 = 5 μM), 39 in absence of an R3 group
showed a 5-fold decrease in IC50. However, reduction of the nitro
group to an amino group (40) and subsequently capping amino
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Figure 4. Western blot analysis. Biotinylated 10, 24, and 39 were
incubated with MDA-MB-231cell lysate and pulled down using neu-
travidin beads. Linker-Lys-biotin was used as negative control. The
membrane was blotted with anti-eEF1A antibody.
group with acetyl group (41) resulted in a marked increase in
IC50, suggesting that the nitro group may be critical for MDA-
MB-231 cell killing. Additionally, removal of the R3 group in 24
(41), reduction of the nitro group to an amino group (42), and
capping the amino group with acetyl group (43), all resulted in a
significant loss of anticancer activity. Consequently, 39,
(simplified from 10) with deletion of the R3 group and posses-
sion of two chiral centers in Leu(R1) and (R)-(ꢀ)-1-
cyclohexylethylamine(R2), was found to have higher potency
against MDA-MB-231 cells (IC50 = 1 μM). Encouraged by this
result, we further synthesized the optical isomers of 39 (45ꢀ47),
and derivatives of 39 by replacing Leu with Phe (48) and D-Phe
(49) (Figure 5). MTT assay was carried on 45ꢀ49 using MDA-
MB-231 cells (Figure 5). The result, however, indicated that
further modifications of 39 led to a reduction in the antiproli-
ferative activity.
In comparison to 10 and 24, 39 lacked an imidazole ring. To
explore whether eEF1A1 was also a binding protein to 39, the
same pull down approach described above was performed on
biotinylated 39. Interestingly, Western blot analysis showed a
slight difference between 39, 10, and 24, (Figure 4, Supporting
Information, Figure 2) which may be due to the two isomers
present in eukaryotic elongation factor (eEF1A1 and eEF1A2),
sharing 92% amino acid identity.23 Therefore, we hypothesize
that 39 may selectively bind to one of eukaryotic elongation
factors. To test this assumption, we attempted to use surface
plasmon resonance (SPR) to evaluate their binding affinity
between eukaryotic elongation factor and 10, 24, or 39. Biotio-
nylated 10, 24, and 39 were immobilized on the streptavidin
(SA) sensor chip via biotin. Different concentrations of eEF1A1
and eEF1A2 were each infused over this sensor chip (Supporting
Information, Figure 3). It is clear that both eEF1A1 and eEF1A2
did bind to these flavonoids. Unfortunately because of the lack of
sufficient amount of target proteins to cover the entire concen-
tration range, the apparent KDs obtained from the SPR studies
were unreliable. Furthermore, 10, 24, and 39 were subjected to
MTT assay with immortalized normal breast cells MCF10A. The
result showed that 10 and 24 killed normal MCF10A cell in a
similar manner as cancerous MCF-7 and MDA-MB-213 cells;
whereas, 39 only killed about 65% of normal MCF10A cells at 50
μM. The preferential anticancer effects of 39 could be explained
by its selective binds to eEF1A2, which was overexpressed in
breast tumor but not normal breast tissue. eEF1A2 might be the
therapeutic target for the anticancer activity of 39.
Validation of Selective Binding of 39 to eEF1A2. eEF1A
proteins are one of the key enzymes in protein synthesis, which
carries aminoacyl-tRNA into ribosome and catalyzes the first step
of peptide elongation.24 Besides protein synthesis, eEF1A is also
involved in several cell regulatory processes, including cytoske-
letal remodeling, apoptosis, and ubiquitin mediated protein
degradation.24 Recently, eEF1A2 (eukaryotic elongation factor
1A2), has been reported to be a putative oncoprotein.25 eEF1A2
has been shown to be overexpressed in 30ꢀ60% of ovarian, lung
Figure 5. Structure and cytotoxicity of 10 and 24 derivatives.
and breast cancers.25ꢀ28 Expression of eEF1A2 in fibroblast cells
show tumorigenicity and increase the growth of xenograft in
nude mice.25
To investigate the effect of 39 binding to eEF1A1 and eEF1A2,
we first tested whether it could inhibit protein translation.
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showed that 39 was able to suppress phosphorylation of serine 473
and threonine 308 in Akt (Figure 7a). Moreover, eEF1A2 is known
to involve in actin remodeling and could increase cell migration.29,30
To test whether 39 inhibits cell migration, cells were treated with 39
for 4 h, and then subjected to a transwell migration assay. 39 was
found to show nearly 50% inhibition in cell migration (Figure 7b).
Taken together, the inhibitory effects of 39 on cell proliferation,
protein translation, Akt phosphorylation and cell migration of
MDA-MB-231 breast cancer cells, can be explained by its inhibitory
effect on eEF1A2. However, it would be very difficult to prove that
the cellular and biochemical effects caused by 39 are mediated
entirely through eEF1A2. More studies will be needed to fully
elucidate the mechanisms of action of this anticancer flavonoid
compound.
Figure 6. Cells were treated with DMSO, 2, 4, and 8 μM of 39 for 8 h
followed by depletion of methionine for 1 h. Before lysing the cells,
35S-labeled methionine were incubated with the cells for 3 h. Cell lysate
were resolved by SDS-PAGE. (a) 35S labeled proteins were detected by
phosphoimager. A decrease of 35S-methionine incorporation into pro-
tein was shown. (b) Commassie blue staining of the same gel was shown
as loading control.
’ CONCLUSION
A series of flavone analogues were synthesized and evaluated
against breast cancer cells. The primary compounds (10 and 24)
had an imidazole ring attached on the flavone and were found to
exhibit modest activity against breast cancer cells (MDA-MB-
231(ERꢀ) and MCF-7 ERþ)). Furthermore, eEF1A1 was
demonstrated to be a target protein of 10 and 24 through
screening T7 phage display (human breast tumor cDNA ex-
pression) proteome library. Importantly, subsequent SAR study
indicated that simplification of 10 resulted in 39 with more
potent anticancer activity, suggesting that the imidazole ring is
not needed for the desired activity; whereas, the original nitro
group is necessary for anticancer activity. Additionally, 39 was
found to selectively bind to eEF1A2. Overall, our results establish
that 39 is an excellent drug lead with novel mechanisms against
breast cancer.
’ EXPERIMENTAL SECTION
Materials and Methods. TentaGel S NH2 resin (90 μm, 0.26
mmol/g) was purchased from Rapp Polymere GmbH (T€ubingen,
Germany). Rink amide MBHA resin (0.5 mmol/g), amino acid deriva-
tives, HOBt, and DIC were purchased from GL Biochem (Shanghai,
China). 4-(7-Fluoro-6-nitro-4-oxo-4H-chromen-2-yl)-benzoic acid was
synthesized in our laboratory.15 All solvents and other chemical reagents
were purchased from Aldrich (Milwaukee, WI) and were analytical
grade. Analytical HPLC analyses (Vydac column; 4.6 mm ꢁ 250 mm;
5 μm; 300 Å; C18; 1.0 mL/min; 25-min gradient from 100% aqueous
H2O (0.1% TFA) to 100% CH3CN (0.1% TFA); 214, 220, 254 and
280 nm) were performed on a Beckman System Gold HPLC system
(Fullerton, CA), or on Waters 2996 photodiode array Detector, a
Waters 2525 Binary Gradient Module, a Waters 2767 Sample Manager
equipped with a 4.6 ꢁ 150 mm Waters Xterra MS C18 5.0 μm column
employing a 20 min gradient from 100% aqueous H2O (0.1% TFA) to
100% CH3CN (0.1% TFA) at a flow rate of 1.0 mL/min. NMR was
recorded on a Bruker DRX spectrometer (Billerica, MA) in DMSO-d6 at
25 ꢀC (500 MHz for 1H NMR, 125 MHz for 13C NMR spectra). The
purity of all the compounds was assessed by RP-HPLC under 254 nm.
All final compounds were confirmed to be g95% purity by analysis of
their peak area. ESIMS was performed with Finnigan LCQ. The
dissociation constants were obtained using Biacore 3000 system
(Biacore Inc. Piscataway, NJ). MDA-MB-231 cells and MCF-7 cells
were obtained from American Type Culture Collection (Manassas,VA).
T7 phage expressing human breast tumor cDNA, T7 up primer, T7
down primer, and NovaTaq Hot Start DNA Polymerase were purchased
from Novagen (Madison, WI). ExoSAP-IT PCR cleanup kit was
purchased from GE Healthcare Bio-Sciences Corp.(Piscataway, NJ)
Figure 7. (a) MDA-MB-231 cells were treated with DMSO and 2 μM
of 39 for 6 and 12 h, respectively. Total protein was extracted, and
subjected to Western blot analysis using antiphospho-Akt (Ser473 and
Thr308), and anti-β actin. (b) MDA-MB-231 Cells were treated with
DMSO or 2 μM of 39 for 4 h, and then seeded into a transwell. After
48 h, cell migration to the bottom well was monitored, and cells were
counted and expressed as numbers of cells per field. The experiments
were carried out at least twice with triple counts. * indicate p < 0.05.
35S-methionine was used to monitor protein translation in cell
culture. Cells treated with 39 showed decreased 35S-methionine
incorporation into cellular proteins in a dose-dependent manner,
indicating that 39 could indeed inhibit protein biosynthesis
(Figure 6). Recently, Amiri A. et al. reported that overexpression
of eEF1A2 could lead to Akt activation in phosphoinositide
3-kinase (PI3K)-dependent manner, enhance filopodia forma-
tion and increase cell invasion, suggesting that eEF1A2 does play
a critical role in tumor development and metastasis.29 To determine
whether 39 could inhibit both eEF1A2 function and Akt activation,
MDA-MB-231 cells were treated with 39. Western blot analysis
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Anti-eEF1A antibody was purchased from Cell signaling Technology,
Inc.(Danvers, MA). Tagged eEF1A1 and eEF1A2 proteins were pur-
chased for, Abcam, Inc. (Cambridge, MA).
Compound 13, yellow solid, Purity, 97%; 1H NMR (500 MHz,
DMSO-d6) δ 8.53(d, J = 10 Hz, 1H), 8.24(d, J = 5 Hz, 2H), 7.98(d, J =
5 Hz, 2H), 7.86(brs, 1H), 7.59(brs, 1H), 7.41ꢀ7.45(m, 4H), 7.21(brs,
1H), 7.13(d, J = 10 Hz, 2H), 7.00(s, 1H), 6.97(brs, 1H), 6.65(d, J =
10 Hz, 2H), 4.92(m, 1H), 4.61(m, 1H), 3.05(m, 1H), 2.91(m, 1H),
2.25(m, 1H), 1.72(m, 1H), 1.60(d, J = 5 Hz, 3H), 1.48(m, 1H), 0.98(d,
J = 5 Hz, 3H), 0.87(d, J = 5 Hz, 3H). ESI-MS m/z: 728.5 [M þ H]þ.
Compound 14, yellow solid, Purity, 96%; 1H NMR (500 MHz,
DMSO-d6) δ8.52(d, J = 5 Hz, 1H), 8.23(d, J = 10 Hz, 2H), 8.10(d, J =
10 Hz, 2H), 7.87(s, 1H), 7.73(s, 1H), 7.70(d, J = 5 Hz, 2H), 7.64(brs,
2H), 7.37(s, 1H), 7.05(s, 1H), 7.00(brs, 1H), 6.68(d, J = 5 Hz 1H),
6.61(d, J = 10 Hz, 1H), 6.53(dd, J = 10, 5 Hz, 1H), 4.52(t, J = 5 Hz, 2H),
4.44(m, 1H), 2.90(t, J = 5 Hz, 2H), 2.35(t, J = 10 Hz, 2H), 2.10(m, 1H),
1.97(m, 1H). ESI-MS m/z: 788.4 [M þ H]þ.
Preparation of Compounds 7ꢀ32. Synthesis of Compounds
7ꢀ17. For each compound, 200 mg of Rink-MBHA resin was swollen in
DMF for 2 h. The resin was treated with 20% piperidine (v/v) in DMF
(2 ꢁ 15 min). The resin was then thoroughly washed with DMF,
MeOH, DCM, and DMF. A mixture of Fmoc-AA-OH (3equiv), HOBt
(3equiv), and DIC (3equiv) was added to the resin, the final mixture was
shaken until a negative Kaiser Test was obtained. The resin was washed
with DMF, MeOH, DCM and DMF, followed by Fmoc deprotection. A
solution of 4-(7-fluoro-6-nitro-4-oxo-4H-chromen-2-yl)-benzoic acid
(2equiv), HOBt (3equiv), and DIC (3equiv) was added to the resin.
The final mixture was shaken until the Kaiser Test was negative. The
resin was washed with DMF, MeOH, DCM, and DMF, and then added a
solution of various amines (1M) in 25% DIEPA/DMF. The mixture was
shaken overnight, and the supernatant was drained off. The resin was
washed with DMF, MeOH, and DMF. 2 M SnCl2/DMF (3 mL) was
added to the resin. The mixture was shaken overnight. After washing
with DMF, MeOH, and DMF, the resin was added a solution of various
Isothiocyanates (1 M) and DIC (1 M) in DMF. The mixture was shaken
overnight, and the supernatant was removed. The resin was washed with
DMF, MeOH, and DCM. After TFA cleavage, the crude product was
purified by HPLC.
Compound 15, yellow solid, Purity, 98%; 1H NMR (500 MHz,
DMSO-d6) δ 8.53(d, J = 10 Hz, 1H), 8.29(d, J = 5 Hz, 2H), 8.10(d, J =
5 Hz, 2H), 8.06(s, 1H), 7.90(s, 1H), 7.70(s, 1H), 7.53(brs, 1H),
7.34ꢀ7.38(m, 2H), 7.09(s, 1H), 7.00ꢀ7.04(m, 2H), 4.94(m, 1H),
4.45(m, 1H), 2.52(s, 3H), 2.35(t, J = 5 Hz, 2H), 2.24(m, 1H),
2.11(m, 1H), 1.98(m, 1H), 1.80(m, 1H), 1.69(d, J = 10 Hz, 3H),
1.34(m, 1H), 0.93(t, J = 5 Hz, 3H), 0.82(t, J = 5 Hz, 3H). ESI-MS m/z:
656.5 [M þ H]þ.
Compound 16, yellow solid, Purity, 99%; 1H NMR (500 MHz,
DMSO-d6) δ 8.31(d, J = 5 Hz, 2H), 8.10(d, J = 5 Hz, 2H), 8.07(d, J =
5 Hz, 1H), 8.02(s, 1H), 7.94(s, 1H), 7.83(brs, 1H), 7.57(d, J = 10 Hz,
1H), 7.41(d, J = 5 Hz, 1H), 7.37(brs, 1H), 7.07(s, 1H), 7.04(brs, 1H),
6.88(td, J = 10, 5 Hz, 1H), 4.80(m, 1H), 4.40(m, 1H), 4.13(m, 1H),
2.21(m, 1H), 2.03(m, 1H), 1.68(d, J = 5 Hz, 3H), 1.15(d, J = 5 Hz, 3H),
0.82(t, J = 5 Hz, 3H). ESI-MS m/z: 572.4 [M þ H]þ.
Compound 7, yellow solid, Purity, 96%; 1H NMR (500 MHz,
DMSO-d6) δ 8.77(t, J = 5 Hz, 1H), 8.19(d, J = 5 Hz, 2H), 8.05(d,
J = 5 Hz, 2H), 7.94(brs, 1H), 7.74(s, 1H), 7.58(brs, 1H), 7.38ꢀ7.44
(m, 3H), 7.33(brs, 1H), 7.29(t, J = 10 Hz, 1H), 7.03(s, 2H), 6.94(brs,
1H), 6.67(dd, J = 10, 5 Hz, 1H), 5.64(s, 2H), 3.87(d, J = 5 Hz, 2H),
3.80(s, 3H). ESI-MS m/z: 610.4 [M þ H]þ.
Compound 17, yellow solid, Purity, 97%; 1H NMR (500 MHz,
DMSO-d6) δ8.26(d, J = 10 Hz, 2H), 8.10(d, J = 10 Hz, 2H), 8.06(d, J =
10 Hz, 1H), 7.99(brs, 1H), 7.82(s, 1H), 7.81(d, J = 10 Hz, 2H), 7.37(brs,
1H), 7.07(s, 1H), 7.05(brs, 1H), 4.40(m, 2H), 4.13(m, 1H),
1.64ꢀ1.68(m, 3H), 1.15(d, J = 5 Hz, 3H), 0.98(d, J = 5 Hz, 6H). ESI-
MS m/z: 593.5 [M þ H]þ.
Compound 8, yellow solid; Purity, 98%; 1H NMR (500 MHz,
DMSO-d6) δ 8.79(t, J = 5 Hz, 1H), 8.23(d, J = 10 Hz, 2H), 8.08(d,
J = 10 Hz, 2H), 7.92(brs, 1H), 7.78(dd, J = 10, 5 Hz, 1H), 7.69(brs, 1H),
7.61(t, J = 10 Hz, 1H), 7.36ꢀ7.40(m, 3H), 7.14(s, 1H), 7.03(s, 1H),
6.97(brs, 1H), 6.70(s, 1H), 6.62(d, J = 5 Hz, 1H), 6.55(d, J = 5 Hz, 1H),
4.55(brs, 2H), 3.87(t, J = 5 Hz, 2H), 2.91(t, J = 5 Hz, 2H). ESI-MS m/z:
658.5 [M þ H]þ.
Synthesis of Compounds 18ꢀ20. Two hundred milligrams of Rink-
MBHA resin was swollen in DMF for 2 h. The resin was treated with
20% piperidine (v/v) in DMF (2 ꢁ 15 min). After thorough washing
with DMF, MeOH, DCM, and DMF, a mixture of 4-(7-fluoro-6-nitro-4-
oxo-4H-chromen-2-yl)-benzoic acid (2equiv), HOBt (3equiv), and DIC
(3equiv) was added to the resin. The final mixture was shaken until a
negative Kaiser Test was obtained. The resin was washed with DMF,
MeOH, and DCM, and a solution of various amines (1 M) in 25%
DIEPA/DMF was added. The mixture was shaken overnight, and the
supernatant was drained off. The resin was washed with DMF, MeOH,
and DMF. 2 M SnCl2/DMF (3 mL) was added to the resin. The mixture
was shaken overnight. The resin was added a solution of various
isothiocyanates (1 M) and DIC (1 M) in DMF followed by washing
with DMF, MeOH, and DMF. The mixture was shaken overnight, and
the supernatant was removed. The resin was washed with DMF, MeOH,
and DCM. After TFA cleavage, the crude product was purified
by HPLC.
Compound 9, yellow solid, Purity, 97%; 1H NMR (500 MHz, DMSO-
d6) δ8.70(d, J= 10 Hz, 1H), 8.20(d, J= 10 Hz, 2H), 8.05(d, J=10Hz, 2H),
7.79(s, 1H), 7.73(brs, 1H), 7.32(brs, 1H), 7.06(s, 2H), 6.90(brs, 1H),
6.88(s, 1H), 6.79(d, J = 5 Hz, 1H), 5.98(s, 2H), 5.34(brs, 2H), 4.79(m,
2H), 4.79(m, 1H), 3.45(q, J = 5 Hz, 2H), 2.64ꢀ2.85(m, 2H), 1.68(m, J =
5 Hz, 2H), 0.95(t, J = 5 Hz, 3H). ESI-MS m/z: 612.4 [M þ H]þ.
Compound 10, yellow solid, Purity, 99%; 1H NMR (500 MHz,
DMSO-d6) δ 8.51(d, J = 10 Hz, 1H), 8.29(d, J = 10 Hz, 2H), 8.09(d, J =
10 Hz, 3H), 7.84(s, 1H), 7.61(d, J = 10 Hz, 2H), 7.36(brs, 1H), 7.09(s,
1H), 7.05(d, J = 10 Hz, 2H), 6.92(brs, 1H), 4.51(m, 2H), 3.81(s, 3H),
2.30(m, 1H), 2.03(m, 1H), 1.83(m, 1H),1.75(m, 1H), 1.70(d, J = 5 Hz,
3H), 1.59ꢀ1.62(m, 3H), 1.35(m, 1H), 1.07ꢀ1.21(m, 4H), 0.95(d, J = 5
Hz, 3H), 0.93(d, J = 5 Hz, 3H). ESI-MS m/z: 650.5 [M þ H]þ.
Compound 11, yellow solid, Purity, 96%; 1H NMR (500 MHz,
DMSO-d6) δ 8.49(d, J = 10 Hz, 1H), 8.18(d, J = 5 Hz, 2H), 8.08(d, J = 5
Hz, 2H), 7.81(s, 1H), 7.76(s, 1H), 7.35(brs, 1H), 7.04(s, 1H), 6.97(s,
1H), 6.90(d, J = 10 Hz, 1H), 6.89(s, 2H), 5.98(s, 2H), 5.40(s, 2H),
4.50(m, 1H), 4.31(brs, 1H), 4.19(q, J = 5 Hz, 2H), 1.69ꢀ1.72(m, 2H),
1.59(m, 1H), 1.23(t, J = 5 Hz, 3H), 0.93(d, J = 5 Hz, 3H), 0.91(d, J = 5
Hz, 3H). ESI-MS m/z: 654.4 [M þ H]þ.
Compound 18, yellow solid, Purity, 99%; 1HNMR(500MHz, DMSO-
d6) δ 8.21(d, J = 10 Hz, 2H), 8.17(s, 1H), 8.08(d, J = 10 Hz, 2H), 7.86(s,
1H), 7.74(s, 1H), 7.65(brs, 2H), 7.57(s, 1H), 7.44(t, J = 5 Hz, 2H), 7.33(t,
J = 5 Hz, 1H), 7.18(m, 1H), 7.10(m, 2H), 7.09(s, 1H), 4.67(t, J = 7.5 Hz,
2H), 3.16(t, J = 7.5 Hz, 2H). ESI-MS m/z: 519.5 [M þ H]þ.
Compound 12, yellow solid, Purity, 98%; 1H NMR (500 MHz,
DMSO-d6): δ8.54(d, J = 10 Hz, 1H), 8.27(d, J = 5 Hz, 2H), 8.03(s, 1H),
7.99(d, J = 5 Hz, 2H), 7.91(brs, 2H), 7.45(brs, 1H), 7.40(d, J = 10 Hz,
2H), 7.14(s, 1H), 7.13(brs, 2H), 7.06(s, 1H), 7.00(brs, 1H), 6.65(d, J =
10 Hz, 2H), 4.80(m, 1H), 4.60(m, 1H), 3.05(m, 1H), 2.91(m, 1H),
2.21(m, 1H), 2.04(m, 1H), 1.69(d, J = 10 Hz, 3H), 0.83(t, J = 5 Hz, 3H).
ESI-MS m/z: 700.5 [M þ H]þ.
Compound 19, yellow solid, Purity, 96%; 1H NMR (500 MHz,
DMSO-d6) δ 8.29(brs, 2H), 8.23(d, J = 10 Hz, 2H), 8.17(s, 1H), 8.07(d,
J = 10 Hz, 2H), 7.92(s, 1H), 7.88(s, 1H), 7.64(t, J = 10 Hz, 1H), 7.56(s,
1H), 7.40(d, J = 10 Hz, 1H), 7.09(s, 1H), 4.57(t, J = 7.5 Hz, 2H), 3.75(t,
J = 7.5 Hz, 2H), 3.26(s, 3H). ESI-MS m/z: 523.5 [M þ H]þ.
Compound 20, yellow solid, Purity, 98%; 1H NMR (500 MHz,
DMSO-d6) δ 8.22(d, J = 10 Hz, 2H), 8.17(s, 1H), 8.07(d, J = 10 Hz,
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ARTICLE
2H), 7.56(s, 1H), 7.47(m, 2H), 7.25(m, 1H), 7.10(s, 1H), 4.42(t, J =
7.5 Hz, 2H), 2.90(t, J = 7.5 Hz, 2H). ESI-MS m/z: 553.5 [M þ H]þ.
Synthesis of Compounds 21ꢀ28. Two hundred milligrams of Rink-
MBHA resin was swollen in DMF for 2 h. The resin was incubated with
20% 4-methylpiperidine(v/v) in DMF (2 ꢁ 15 min). The resin was then
thoroughly washed with DMF, MeOH, DCM and DMF. A mixture of
Fmoc-AA-OH (3equiv), HOBt (3equiv), and DIC (3equiv) was added
to the resin, the final mixture was shaken until the Kaiser Test was
negative. The resin was washed with DMF, MeOH, DCM and DMF,
followed by Fmoc deprotection. A solution of 4-(7-Fluoro-6-nitro-4-
oxo-4H-chromen-2-yl)-benzoic acid(2equiv), HOBt(3equiv) and DIC-
(3equiv) was added to the resin. The final mixture was shaken until the
Kaiser Test was negative. A solution of amines (1M) in 25% DIEPA/
DMF was added to the resin. The mixture was shaken overnight, and the
supernatant was drained off. The resin was washed with DMF, MeOH, and
DMF. 2 M SnCl2/DMF (3 mL) was added to the resin. The mixture was
shaken overnight. The resin was then washed with DMF, MeOH, and
DMF. A solution of aldehydes (10 equiv) in 5%HOAc/DMF (3 mL) was
added to the resin. The mixture was shaken overnight, and the supernatant
was removed. The resin was washed with DMF, MeOH, and DCM. After
TFA cleavage, the crude product was purified by HPLC.
Compound 27, yellow solid, Purity, 98%; 1H NMR (500 MHz,
DMSO-d6) δ 8.25(d, J = 5 Hz, 2H), 8.23(s, 1H), 8.14(d, J = 5 Hz, 1H),
8.00(d, J = 5 Hz, 2H), 8.02(s, 1H), 7.44(brs, 1H), 7.36(m, 1H), 7.24(m,
1H), 7.07(s, 1H), 7.07(brs, 1H), 6.77(brs, 1H), 5.70(s, 2H), 4.36(dd, J =
10, 5 Hz, 1H), 4.10(m, 1H), 2.52(m, 1H), 1.28(d, J = 10 Hz, 6H),
1.09(d, J = 10 Hz, 3H). ESI-MS m/z: 575.5 [M þ H]þ.
Compound 28, yellow solid, Purity, 97%; 1H NMR (500 MHz,
DMSO-d6) δ 8.35(s, 1H), 8.33(d, J = 5 Hz, 2H), 8.24(s, 1H), 8.17(d, J =
5 Hz, 1H), 8.12(d, J = 5 Hz, 2H), 7.46(brs, 1H), 4.60(t, J = 5 Hz, 2H),
4.37(dd, J = 10, 5 Hz, 1H), 4.11(m, 1H), 3.88(s, 9H), 3.85(t, J = 5 Hz,
2H), 3.78(d, J = 10 Hz, 6H), 1.13(d, J = 5 Hz, 3H). ESI-MS m/z: 631.6
[M þ H]þ.
Synthesis of Compounds 29ꢀ32. Two hundred milligrams of Rink-
MBHA resin was swollen in DMF for 2 h. The resin was retreated with
20% piperidine (v/v) in DMF (2 ꢁ 15 min). The resin was then
thoroughly washed with DMF, MeOH, DCM, and DMF. A mixture of
4-(7-fluoro-6-nitro-4-oxo-4H-chromen-2-yl)-benzoic acid (2equiv),
HOBt (3equiv), and DIC (3equiv) was added to the resin. The final
mixture was shaken until the Kaiser Test was negative. The resin was
washed with DMF, MeOH, and DCM, and added a solution of various
amines (1 M) in 25% DIEPA/DMF. The mixture was shaken overnight,
and the supernatant was drained off. The resin was washed with DMF,
MeOH, and DMF. 2 M SnCl2/DMF (3 mL) was added to the resin. The
mixture was shaken overnight. After washed with DMF, MeOH, and
DMF, the resin was added a solution of aldehyde (10 equiv) in 5%
HOAc/DMF (3 mL). The mixture was shaken overnight, and the
supernatant was removed. The resin was washed with DMF, MeOH, and
DCM. After TFA cleavage, the crude product was purified by HPLC.
Compound 29, yellow solid, Purity, 99%; 1H NMR (500 MHz,
DMSO-d6) δ 8.32(s, 1H), 8.27(d, J = 10 Hz, 2H), 8.26(s, 1H), 8.18(brs,
1H), 8.09(d, J = 10 Hz, 2H), 8.06(d, J = 10 Hz, 2H), 7.91(d, J = 10 Hz,
2H), 7.81(d, J = 10 Hz, 2H), 7.58(brs, 1H), 7.54(t, J = 10 Hz, 2H),
7.45(m, 1H), 7.15(s, 1H), 4.59(t, J = 5 Hz, 2H), 3.90(t, J = 5 Hz, 2H),
3.39(t, J = 5 Hz, 2H), 3.33(t, J = 5 Hz, 2H). ESI-MS m/z: 546.5
[M þ H]þ.
Compound 21, yellow solid, Purity, 98%; 1H NMR (500 MHz,
DMSO-d6) δ 8.89(d, J = 10 Hz, 1H), 8.74(d, J = 5 Hz, 1H), 8.37
(s, 1H), 8.31(d, J = 5 Hz, 1H), 8.28(d, J = 5 Hz, 1H), 8.22(s, 1H), 8.19
(s, 1H), 8.18(d, J = 5 Hz, 1H), 8.09(d, J = 10 Hz, 1H), 8.07(d, J = 5 Hz,
1H), 8.01(d, J = 5 Hz, 1H), 7.89(t, J = 5 Hz, 1H), 7.16(brs, 1H), 7.13
(s, 1H), 6.95(brs, 1H), 4.80(m, 1H), 4.40(t, J = 10 Hz, 2H), 2.82(m,
1H), 2.64(m, 1H), 1.66(q, J = 5 Hz, 2H), 1.54(m, 1H), 0.81(d, J = 5 Hz,
6H). ESI-MS m/z: 635.5 [M þ H]þ.
Compound 22, yellow solid, Purity, 96%; 1H NMR (500 MHz,
DMSO-d6) δ 8.69(d, J = 5 Hz, 1H), 8.42(s, 1H), 8.33(d, J = 10 Hz, 2H),
8.33(s, 1H), 8.00(d, J = 10 Hz, 2H), 7.94(d, J = 10 Hz, 2H), 7.82(d, J =
10 Hz, 2H), 7.80(d, J = 10 Hz, 2H), 7.59(brs, 1H), 7.54(t, J = 10 Hz,
2H), 7.45(m, 1H), 7.19(s, 1H), 7.15(d, J = 10 Hz, 2H), 7.11(brs, 1H),
6.67(d, J = 10 Hz, 2H), 4.51ꢀ4.68(m, 2H), 3.05(m, 1H), 2.90(m, 1H),
2.25(m, 1H), 1.99(m, 1H), 1.78(d, J = 10 Hz, 3H), 0.58(t, J = 5 Hz, 3H).
ESI-MS m/z: 677.5 [M þ H]þ.
Compound 30, yellow solid, Purity, 96%; 1H NMR (500 MHz,
DMSO-d6) δ 8.26(d, J = 10 Hz, 2H), 8.18(s, 2H), 8.12(brs, 1H), 8.06(d,
J = 10 Hz, 2H), 7.57(brs, 1H), 7.11(s, 1H), 4.18(d, J = 5 Hz, 2H),
1.88(m, 1H), 1.68(m, 2H), 1.62(m, 1H), 1.54(m, 2H), 1.37(d, J = 5 Hz,
6H), 1.16(m, 6H). ESI-MS m/z: 444.4 [M þ H]þ.
Compound 23, yellow solid, Purity, 97%; 1H NMR (500 MHz,
DMSO-d6) δ 8.68(d, J = 5 Hz, 1H), 8.39(s, 1H), 8.33(d, J = 10 Hz, 2H),
8.28(s, 1H), 8.00(d, J = 10 Hz, 2H), 7.69(d, J = 10 Hz, 2H), 7.57(brs,
1H), 7.15(d, J = 10 Hz, 2H), 7.15(s, 1H), 7.10(brs, 1H), 6.64(d, J = 10
Hz, 2H), 4.71(m, 1H), 4.60(m, 1H), 3.88(s, 3H), 3.02(m, 1H), 2.90(m,
1H), 2.18(m, 1H), 1.78(d, J = 5 Hz, 3H), 1.73(m, 1H), 1.10(m, 1H),
0.65(d, J = 5 Hz, 3H), 0.58(d, J = 5 Hz, 3H). ESI-MS m/z: 659.5
[M þ H]þ.
Compound 31, yellow solid, Purity, 98%; 1H NMR (500 MHz,
DMSO-d6) δ 8.40(s, 1H), 8.35(d, J = 10 Hz, 2H), 8.29(s, 1H), 8.20
(s, 1H), 8.08(d, J = 10 Hz, 2H), 7.58(brs, 1H), 7.17(s, 1H), 7.08(s, 1H),
6.91(s, 1H), 4.14(m, 1H), 3.93(s, 3H), 3.84(s, 3H), 3.74(s, 3H),
2.15(m, 1H), 1.93(m, 1H), 1.68(d, J = 10 Hz, 3H), 0.57(m, 3H). ESI-
MS m/z: 528.4 [M þ H]þ.
Compound 24, yellow solid, Purity, 99%; 1H NMR (500 MHz,
DMSO-d6) δ 8.72(d, J = 10 Hz, 1H), 8.24(s, 1H), 8.19(d, J = 10 Hz,
2H), 8.09(s, 1H), 7.97(d, J = 10 Hz, 2H), 7.60(brs, 1H), 7.35(d, J =
10 Hz, 4H), 7.25(t, J = 10 Hz, 2H), 7.13(m, 4H), 7.10(s, 1H), 5.67
(s, 2H), 4.66(m, 1H), 3.36(m, 1H), 3.13(dd, 1H), 3.00(dd, J = 10, 5 Hz,
1H), 1.78(dd, J = 10, 10 Hz, 1H), 1.28(d, J = 5 Hz, 6H). ESI-MS m/z:
585.5 [M þ H]þ.
Compound 32, yellow solid, Purity, 97%; 1H NMR (500 MHz,
DMSO-d6) δ 8.57(s, 2H), 8.43(s, 1H), 8.39(s, 1H), 8.32(s, 1H), 8.29(d,
J = 10 Hz, 2H), 8.19(brs, 1H), 8.09(d, J = 10 Hz, 2H), 7.58(brs, 1H),
7.17(s, 1H), 4.40(t, J = 5 Hz, 2H), 1.70(m, 2H), 1.59(m, 1H), 0.82(d, J =
5 Hz, 6H). ESI-MS m/z: 588.5 [M þ H]þ.
MTT Assay. Cells (MDA-MB-231 and MCF-7) were seeded in a 96-
well plate (5000 cells/well) for 24 h. Cells were then treated with 10 μM
of compounds or varying concentrations of compounds for 72 h. Each
compound or each concentration was tested as a triplicate. After 72 h
incubation, cells were washed with PBS for three times and then MTT
was added for 4hours. Purple crystal formed was then dissolved in 0.04N
HCl in isopropanol. The plates were read under 570 nm.
Compound 25, yellow solid, Purity, 96%; 1H NMR (500 MHz,
DMSO-d6) δ 8.91(t, J = 5 Hz, 1H), 8.30(d, J = 5 Hz, 1H), 8.29(s, 1H),
8.27(s, 1H), 8.09(d, J = 10 Hz, 2H), 7.85(d, J = 10 Hz, 2H), 7.44(brs,
1H), 7.18(d, J = 5 Hz, 2H), 7.15(s, 1H), 7.08(brs, 1H), 4.36(d, J = 5 Hz,
2H), 3.88(s, 3H), 3.86(t, J = 5 Hz, 2H), 1.11(m, 1H), 0.41(m, 2H),
0.24(m, 2H). ESI-MS m/z: 523.4 [M þ H]þ.
Compound 26, yellow solid, Purity, 98%; 1H NMR (500 MHz,
DMSO-d6) δ 8.92(t, J = 5 Hz, 1H), 8.37(s, 1H), 8.36(d, J = 5 Hz, 2H),
8.34(s, 1H), 8.09(d, J = 5 Hz, 2H), 7.85(m, 2H), 7.69(d, J = 5 Hz, 1H),
7.60(t, J = 5 Hz, 1H), 7.44(brs, 1H), 7.17(s, 1H), 7.08(brs, 1H), 4.20(m,
1H), 3.87(d, J = 5 Hz, 2H), 2.24(m, 2H), 2.02(m, 2H), 0.68(t, J = 5 Hz,
6H). ESI-MS m/z: 587.4 [M þ H]þ.
Phage Panning. TentaGel resins bound compound 10 and 24
were swelled in PBSTB (8 mM Na2HPO4, 1.5 mM KH2PO4, 137 mM
NaCl, 27 mM KCl, 0.1%(V/V) Tween 20, 0.1% (W/V) BSA, and 0.05%
(W/V) NaN3, pH 7.5 overnight, respectively. 10 μL of each beads were
incubated with 105 of phage in 1 mL PBSTB for 2 h. The beads were then
washed extensively with PBSTB for 5 times and 50 μL of log phase E. coli
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Journal of Medicinal Chemistry
ARTICLE
(strain BLT5403) were added and shaken for 30 min at 37 ꢀC. The
infected E. coli were then added to 1 mL log phase E. coli, and shaken for
3 h at 37 ꢀC until all the E. coli were lysed. The E. coli debris was then
spun down at 14,000 rpm for 10 min. The amplified phage was used for a
second round of panning. The panning was repeated three more times.
Finally, the phage was mixed with log phase E. coli in soft agar for single
colony on ampicilin plate. Single colony were picked and subjected to
PCR for DNA sequencing. 1 μL of amplified phage were used for PCR
with NovaTaq Hot Start DNA Polymerase. T7 down primer (50-
AACCCCTCAAGACCCGTTTA-30), and T7 up primer (50-GGA-
GCTGTCGTATTCCAGTC-30) were used as primer pair. The PCR
conditions were 95 ꢀC for 7 min and then repeat 34 cycles of 94 ꢀC for
1 min, 55 ꢀC for 1 min, and 72 ꢀC for 2 min. The last cycle was extended
for 1 more minute at 72 ꢀC. The PCR product were cleaned up using
ExoSAP-IT kit, and finally subjected to DNA sequencing.
Pull Down Assay. MDA-MB-231 were grown to 85% confluence
and lysed with lysis buffer (PBS, protease inhibitor cocktail, DNase,
RNase, and 0.1% TritonX-100). 1 mM of biotinylated 10 and 24 were
added to the cell lysate overnight in 4 ꢀC. Then the compounds
were pulled down by incubating with avidin beads for 2 h at 4 ꢀC. Beads
were washed with PBS for 3 times and boiled with sampler buffer for
10 min at 95 ꢀC. Samples were then run on SDS-PAGE and transferred
on PVDF membrane following blotting with anti-eEF1A antibody (1:1000
dilution) and antirabbit HRP conjugate (Cell Signaling Technology, MA).
Western Blot Analysis. MDA-MB-231 were grown to 80%
confluence and treated with compound for 16 h. Cells were then lysed
with lysis buffer (25 mM tris HCl, 10 mM MgCl2, 1 mM Na3VO4,
50 mM β-glycerol phosphate, 0.1% Triton-X 100, and protease inhibitor
cocktail). The cell lysate was then separated using SDS-PAGE and
transferred to PVDF membrane. The membrane was blotted using anti-
phosohoAkt (Ser 437 and Thr 308) (1:1000 dilution) and antirabbit
HRP conjugate (Cell Signaling Technology, MA).
Surface Plasmon Resonance (SPR) Analysis. One micromolar
of biotinylated compounds 10, 24, and 39 in Biacore HBS-EP buffer
were individually immobilized on 3 of the 4 flow cells on a streptavidin
sensor chip (Biacore SA Chip) at 5uL/min for 10 min each. The
remaining one empty flow cell was assigned as a reference flow cell and
was immobilized with Linker-Lysin-Biotin only. eEF1A1 and GST were
2-fold serial diluted in HBS-EP buffer from 20 nM to 1.25 nM. eEF1A2
was 2-fold serial diluted in HBS-EP buffer from 100 nm to 6.25 nM. The
protein at each concentration was then injected at 30uL/min for 3 min
and dissociated for 5 min. Kon, Koff, and KD were evaluated by using
BiaEvaluation software to fit binding curves with proper binding models.
Cell Migration Assay. MDA-MB-231 cells were grown to 80%
confluence and treated with compound 39 (2 μM) for 4 h; 0.2 mL of
cells (5 ꢁ 105cell/mL) in serum free medium were seeded into the top
chambers of 6.5-mm Corning Costar transwells (Corning, NY, New
York, USA), and the bottom wells were loaded with complete medium.
After 48 h incubation, the images of cells in the bottom wells were
captured and the cell number was counted.
1H), 7.39(d, J = 10 Hz, 2H), 7.30ꢀ7.36(m, 4H), 7.25(t, J = 10 Hz, 2H),
7.13ꢀ7.19(m, 3H), 7.09(s, 1H), 7.06(d, J = 10 Hz, 2H), 5.62(s, 2H),
4.67(m, 1H), 3.79(s, 3H), 3.14(dd, J = 10, 10 Hz, 1H), 2.99(dd, J = 10, 5
Hz, 1H). ESI-MS m/z: 664.7 [M þ H]þ.
Compound 34, yellow solid, Purity, 98%; 1H NMR (500 MHz,
DMSO-d6) δ 8.58(d, J = 10 Hz, 1H), 8.20(d, J = 10 Hz, 2H), 8.07(d, J =
10 Hz, 2H), 7.87(s, 1H), 7.83(brs, 1H), 7.66(d, J = 5 Hz, 2H), 7.44(brs,
1H), 7.39(d, J = 10 Hz, 2H), 7.34(m, 3H), 7.11(s, 1H), 7.06(d, J = 5 Hz,
2H), 7.00(s, 1H), 5.66(s, 2H), 4.48(m, 1H), 3.79(s, 3H), 1.61ꢀ1.73(m.
2H), 1.55(m, 1H), 0.92(d, J = 10 Hz, 3H), 0.89(d, J = 10 Hz, 3H). ESI-
MS m/z: 630.7 [M þ H]þ.
Compound 35, yellow solid, Purity, 98%; 1H NMR (500 MHz,
DMSO-d6) δ 8.76(d, J = 10 Hz, 1H), 8.29(d, J = 10 Hz, 2H), 8.24(s,
1H), 7.98(d, J = 10 Hz, 2H), 7.83(s, 1H), 7.63(brs, 1H), 7.56(d, J = 5 Hz,
2H), 7.37(d, J = 10 Hz, 2H), 7.26(t, J = 10 Hz, 2H), 7.19(t, J = 10 Hz,
1H), 7.15(brs, 2H), 7.11(d, J = 5 Hz, 2H), 4.68(m, 1H), 4.55(m, 1H),
3.82(s, 3H), 3.13(dd, J = 10, 10 Hz, 1H), 3.00(dd, J = 10, 5 Hz, 1H),
2.29(m, 1H), 2.04(m, 1H), 1.83(m, 1H), 1.72(d, J = 5 Hz, 3H), 1.62(m,
2H), 1.00ꢀ1.36(m, 6H). ESI-MS m/z: 684.6 [M þ H]þ.
Compound 36, yellow solid, Purity, 97%; 1H NMR (500 MHz,
DMSO-d6) δ 8.59(d, J = 10 Hz, 1H), 8.32(s, 1H), 8.23(d, J = 10 Hz,
2H), 8.09(s, 1H), 8.08(d, J = 10 Hz, 2H), 7.45(brs, 1H), 7.34(t, J =
10 Hz, 2H), 7.31(s, 1H), 7.29(t, J = 10 Hz, 1H), 7.15(d, J = 10 Hz, 2H),
7.14(s, 1H), 7.00(s, 1H), 5.84(s, 2H), 4.48(m, 1H), 3.39(m, 1H),
1.63ꢀ1.74(m. 2H), 1.55(m, 1H), 1.29(d, J = 5 Hz, 6H), 0.92(d, J =
10 Hz, 3H), 0.89(d, J = 10 Hz, 3H). ESI-MS m/z: 551.7 [M þ H]þ.
Compound 37, yellow solid, Purity, 97%; 1H NMR (500 MHz,
DMSO-d6) δ 8.76(d, J = 10 Hz, 1H), 8.37(brs, 1H), 8.31(d, J = 10 Hz,
2H), 8.22(s, 1H), 7.98(d, J = 10 Hz, 2H), 7.62(s, 1H), 7.36(d, J = 5 Hz,
2H), 7.26(t, J = 5 Hz, 2H), 7.18(t, J = 5 Hz, 1H), 7.17(s, 1H), 4.68(m,
1H), 4.41(m, 1H), 3.54(m, 1H), 3.15(dd, J = 10, 10 Hz, 1H), 3.08(dd,
J = 10, 5 Hz, 1H), 2.29(m, 1H), 2.06(m, 1H), 1.84(m, 1H), 1.69(d, J =
5 Hz, 3H), 1.62(m, 2H), 1.52(1, 1H), 1.45(d, J = 10 Hz, 3H), 1.33(d, J =
10 Hz, 3H), 0.88ꢀ1.22(m, 6H). ESI-MS m/z: 605.7 [M þ H]þ.
Compound 38, yellow solid, Purity, 99%; 1H NMR (500 MHz,
DMSO-d6) δ 8.31(d, J = 10 Hz, 2H), 8.26(brs, 1H), 8.20(brs, 1H),
8.07(d, J = 10 Hz, 2H), 7.83(s, 1H), 7.59(s, 1H), 7.54(d, J = 10 Hz, 2H),
7.16(s, 1H), 7.11(d, J = 10 Hz, 2H), 4.55(m, 1H), 3.82(s, 3H), 2.29(m,
1H), 2.04(m, 1H), 1.83(m, 1H), 1.72(d, J = 5 Hz, 3H), 1.62(m, 2H),
0.99ꢀ1.36(m, 6H). ESI-MS m/z: 537.6 [M þ H]þ.
Compound 39, yellow solid, Purity, 98%; 1H NMR (500 MHz,
DMSO-d6) δ 8.70(s, 1H), 8.61(d, J = 5 Hz, 1H), 8.23(d, J = 10 Hz, 2H),
8.08(d, J = 10 Hz, 2H), 7.45(brs, 1H), 7.36(s, 1H), 7.06(s, 1H), 7.00(s,
1H), 4.48(m, 1H), 3.81(m, 1H), 1.87(m, 1H), 1.55ꢀ1.75(m, 10H),
1.24(d, J = 10 Hz, 3H), 1.03ꢀ1.15(m, 3H), 0.93(d, J = 10 Hz, 3H),
0.89(d, J = 10 Hz, 3H). ESI-MS m/z: 549.7 [M þ H]þ.
Compound 40, yellow solid, Purity, 99%; 1H NMR (500 MHz,
DMSO-d6) δ 8.70(s, 1H), 8.23(d, J = 10 Hz, 2H), 8.14(brs, 1H), 8.10(d,
J = 10 Hz, 1H), 8.05(d, J = 10 Hz, 2H), 7.58(brs, 1H), 7.37(s, 1H),
7.05(s, 1H), 3.80(m, 1H), 1.59ꢀ1.89(m, 7H), 1.23(d, J = 5 Hz, 3H),
1.00ꢀ1.18(m, 4H). ESI-MS m/z: 436.6 [M þ H]þ.
35S-Methionine Labeled Protein Translation Assay. Cells
were treated with DMSO or different concentration of 39 for 8 h. Cell
culture medium was then changed to methionine free medium for 1 h.
To start the translation labeling, 5 μL of 35S-methionine (specific
activity >1000 Ci/mMole) (PerkinElmer, Waltham, MA) were added to
the cells and incubated for 3 h. Cells were then harvested and lysed.
Proteins were separated on SDS-PAGE and stained with commassie
blue. The same gel was then dried and the 35S-methionine was detected
using phosphoimager.
Compound 41, yellow solid, Purity, 96%; 1H NMR (500 MHz,
DMSO-d6): δ9.22(s, 1H), 8.57(d, J = 10 Hz, 1H), 8.17(d, J = 10 Hz,
2H), 8.06(d, J = 10 Hz, 2H), 7.80(s, 1H), 7.45(brs, 1H), 6.99(brs, 1H),
6.92(s, 1H), 6.85(s, 1H), 5.63(d, J = 10 Hz, 1H), 4.48(m, 1H), 3.52(m,
1H), 2.11(s, 3H), 1.87(m, 1H), 1.54ꢀ1.74(m, 8H), 1.16(d, J = 10 Hz,
3H), 0.98ꢀ1.23(m, 5H), 0.91(d, J = 10 Hz, 3H), 0.89(d, J = 10 Hz, 3H).
ESI-MS m/z: 561.8 [M þ H]þ.
Compound 42, yellow solid, Purity, 97%; 1H NMR (500 MHz,
DMSO-d6) δ 8.25(s, 1H), 8.21(d, J = 10 Hz, 2H), 8.16(brs, 1H), 8.06(s,
1H), 8.05(d, J = 10 Hz, 2H), 7.55(brs, 1H), 7.36(t, J = 10 Hz, 2H),
7.29(t, J = 5 Hz, 1H), 7.19(t, J = 5 Hz, 1H), 7.15(d, J = 5 Hz, 2H), 7.12(s,
1H), 5.68(s, 2H), 3.37(m, 1H), 1.29(d, J = 5 Hz, 6H). ESI-MS m/z:
438.5 [M þ H]þ.
Preparation of Compounds 33ꢀ51. Compounds 33ꢀ51 were
synthesized in the same manner as above.
Compound 33, yellow solid, Purity, 96%; 1H NMR (500 MHz,
DMSO-d6) δ 8.72(d, J = 10 Hz, 1H), 8.19(d, J = 10 Hz, 2H), 7.96(d, J =
10 Hz, 2H), 7.86(s, 1H), 7.82(brs, 1H), 7.67(d, J = 10 Hz, 2H), 7.60(s,
4347
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Journal of Medicinal Chemistry
ARTICLE
Compound 43, orange solid, Purity, 99%; 1H NMR (500 MHz, DMSO-
d6) δ8.88(t, J = 5 Hz, 1H), 8.72(s, 1H), 8.71(d, J = 5 Hz, 1H), 8.12(d, J=10
Hz, 2H), 7.94(d, J = 10 Hz, 2H), 7.60(brs, 1H), 7.45(d, J = 10 Hz, 2H),
7.39(d, J= 10 Hz, 2H), 7.35(t, J= 10 Hz, 2H), 7.28(t, J =10 Hz, 1H), 7.25(t,
J = 10 Hz, 2H), 7.16(t, J = 10 Hz, 1H), 7.14(s, 1H), 7.13(brs, 1H), 7.14(s,
1H), 7.02(s, 1H), 4.74(d, J = 5 Hz, 2H), 4.66(m, 1H), 3.13(dd, J = 10, 10
Hz, 1H), 2.99(dd, J = 10, 5 Hz, 1H). ESI-MS m/z: 563.7 [M þ H]þ.
Compound 44, yellow solid, Purity, 97%; 1H NMR (500 MHz,
DMSO-d6) δ 9.33(s, 1H), 8.68(d, J = 5 Hz, 1H), 8.06(d, J = 10 Hz, 2H),
7.91(d, J = 10 Hz, 2H), 7.76(s, 1H), 7.58(brs, 1H), 7.43(d, J = 10 Hz,
2H), 7.36(d, J = 10 Hz, 2H), 7.34(t, J = 10 Hz, 2H), 7.23ꢀ7.27(m, 3H),
7.07ꢀ7.17(m, 3H), 6.88(s, 1H), 6.66(s, 1H), 4.65(m, 1H), 4.51(d, J = 5
Hz, 2H), 3.13(dd, J = 10, 10 Hz, 1H), 2.99(dd, J = 10, 5 Hz, 1H), 2.11(s,
3H). ESI-MS m/z: 575.7 [M þ H]þ.
’ ACKNOWLEDGMENT
This work was supported by U.S. Department of Defense
Breast Cancer Research Program’s Multidisciplinary Postdoctor-
al Training Award contract no. W81XWH-06-1-0447 (N. Yao),
NIH R01CA115483, and U19CA113298. We thank Mr. David
Olivos for editorial assistance.
’ ABBREVIATIONS USED
Fmoc, fluorenylmethoxycarbonyl; HOBt, N-hydroxybenzotria-
zole; DIC, N,N0-diisopropylcarbodiimide; DIEPA, N,N-diiso-
propylethylamine; TFA, trifluoroacetic acid; TIS, triisopropyl-
silane; HOAc, acetic acid; DMF, N,N-dimethylformamide; DCM,
dichloromethane; eEF1A, eukaryotic elongation factor 1 alpha;
ESI, electrospray ionization; NMR, nuclear magnetic resonance;
RP-HPLC, reverse-phase high performance liquid chromato-
graphy
1
Compound 45, orange solid, Purity, 96%; H NMR (500 MHz,
DMSO-d6) δ 8.71(s, 1H), 8.60(d, J = 10 Hz, 1H), 8.23(d, J = 10 Hz,
2H), 8.11(d, J = 5 Hz, 1H), 8.07(d, J = 10 Hz, 2H), 7.45(brs, 1H), 7.37(s,
1H), 7.06(s, 1H), 7.00(s, 1H), 4.47(m, 1H), 3.83(m, 1H), 1.55ꢀ1.90(m,
10H), 1.23(d, J = 5 Hz, 3H), 1.03ꢀ1.15(m, 4H), 0.93(d, J = 5 Hz, 3H),
0.89(d, J = 5 Hz, 3H). ESI-MS m/z: 549.7 [M þ H]þ.
1
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Compound 46, orange solid, Purity, 98%; H NMR (500 MHz,
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DMSO-d6) δ 8.71(s, 1H), 8.60(d, J = 10 Hz, 1H), 8.23(d, J = 10 Hz,
2H), 8.10(d, J = 5 Hz, 1H), 8.07(d, J = 10 Hz, 2H), 7.45(brs, 1H), 7.37(s,
1H), 7.06(s, 1H), 7.00(s, 1H), 4.47(m, 1H), 3.82(m, 1H),
1.55ꢀ1.90(m, 10H), 1.23(d, J = 5 Hz, 3H), 1.03ꢀ1.15(m, 4H),
0.93(d, J = 5 Hz, 3H), 0.89(d, J = 5 Hz, 3H). ESI-MS m/z: 549.7
[M þ H]þ.
1
Compound 47, orange solid, Purity, 96%; H NMR (500 MHz,
DMSO-d6) δ 8.70(s, 1H), 8.60(d, J = 10 Hz, 1H), 8.23(d, J = 10 Hz,
2H), 8.10(d, J = 5 Hz, 1H), 8.07(d, J = 10 Hz, 2H), 7.45(brs, 1H), 7.36(s,
1H), 7.06(s, 1H), 7.00(s, 1H), 4.47(m, 1H), 3.82(m, 1H),
1.54ꢀ1.89(m, 10H), 1.23(d, J = 5 Hz, 3H), 1.03ꢀ1.15(m, 4H),
0.93(d, J = 5 Hz, 3H), 0.89(d, J = 5 Hz, 3H). ESI-MS m/z: 549.7 [M
þ H]þ.
1
Compound 48, orange solid, Purity, 99%; H NMR (500 MHz,
DMSO-d6) δ 8.74(d, J = 10 Hz, 1H), 8.70(s, 1H), 8.22(d, J = 10 Hz,
2H), 8.11(d, J = 10 Hz, 1H), 7.96(d, J = 10 Hz, 2H), 7.61(s, 1H), 7.36(s,
1H), 7.35(d, J = 5 Hz, 2H), 7.26(t, J = 5 Hz, 2H), 7.17(t, J = 10 Hz, 1H),
7.13(brs, 1H), 7.03(s, 1H), 4.66(m, 1H), 3.80(m, 1H), 3.14(dd, J = 10,
5 Hz, 1H), 3.00(dd, J = 10, 10 Hz, 1H), 1.54ꢀ1.89(m, 7H), 1.23(d, J =
5 Hz, 3H), 1.00ꢀ1.15(m, 4H). ESI-MS m/z: 583.7 [M þ H]þ.
1
Compound 49, orange solid, Purity, 97%; H NMR (500 MHz,
DMSO-d6): δ8.74(d, J = 10 Hz, 1H), 8.70(s, 1H), 8.21(d, J = 10 Hz,
2H), 8.10(d, J = 10 Hz, 1H), 7.96(d, J = 10 Hz, 2H), 7.61(s, 1H), 7.36(s,
1H), 7.35(d, J = 5 Hz, 2H), 7.26(t, J = 5 Hz, 2H), 7.16(t, J = 10 Hz, 1H),
7.13(brs, 1H), 7.03(s, 1H), 4.66(m, 1H), 3.80(m, 1H), 3.14(dd, J = 10,
5 Hz, 1H), 3.00(dd, J = 10, 10 Hz, 1H), 1.59ꢀ1.89(m, 7H), 1.23(d, J = 5
Hz, 3H), 1.00ꢀ1.15(m, 4H). ESI-MS m/z: 583.7 [M þ H]þ.
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Supporting Information. Synthetic procedures, Surface
b
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’ AUTHOR INFORMATION
Corresponding Author
*Phone: (916) 734-0910. Fax: (916) 734-6415. E-mail: Kit.lam@
ucdmc.ucdavis.edu.
Notes
^These authors should be considered first authors.
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