Pyridinylquinazolines selectively inhibit human methionine aminopeptidase-1 in cells

Article abstract of DOI:10.1021/jm400227z

Methionine aminopeptidases (MetAPs), which remove the initiator methionine from nascent peptides, are essential in all organisms. While MetAP2 has been demonstrated to be a therapeutic target for inhibiting angiogenesis in mammals, MetAP1 seems to be vital for cell proliferation. Our earlier efforts identified two structural classes of human MetAP1 (HsMetAP1)-selective inhibitors (1-4), but all of them failed to inhibit cellular HsMetAP1. Using Mn(II) or Zn(II) to activate HsMetAP1, we found that 1-4 could only effectively inhibit purified HsMetAP1 in the presence of physiologically unachievable concentrations of Co(II). In an effort to seek Co(II)-independent inhibitors, a novel structural class containing a 2-(pyridin-2-yl)quinazoline core has been discovered. Many compounds in this class potently and selectively inhibited HsMetAP1 without Co(II). Subsequently, we demonstrated that 11j, an auxiliary metal-dependent inhibitor, effectively inhibited HsMetAP1 in primary cells. This is the first report that an HsMetAP1-selective inhibitor is effective against its target in cells.

Full text of DOI:10.1021/jm400227z

Article
pubs.acs.org/jmc
Pyridinylquinazolines Selectively Inhibit Human Methionine
Aminopeptidase‑1 in Cells
Feiran Zhang,†,‡ Shridhar Bhat,†,‡ Sandra B. Gabelli,●,§,⊥ Xiaochun Chen,†,◆ Michelle S. Miller,§,¶
Benjamin A. Nacev,†,# Yim Ling Cheng,∥ David J. Meyers,† Karen Tenney,▽ Joong Sup Shim,†
Phillip Crews,▽ L. Mario Amzel,§ Dawei Ma,○ and Jun O. Liu*,†,⊥
§
∥
†Departments of Pharmacology and Molecular Sciences, ●Medicine, Biophysics and Biophysical Chemistry, Cell Biology, and
⊥Oncology, and Medical Scientist Training Program, Johns Hopkins University School of Medicine, 725 North Wolfe Street,
#
Baltimore, Maryland 21205, United States
▽Department of Chemistry and Biochemistry, University of California, Santa Cruz, 1156 High Street, Santa Cruz, California 95064,
United States
○State Key Laboratory of Bioorganic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of
Sciences, 354 Fenglin Road, Shanghai 200032, China
S
* Supporting Information
ABSTRACT: Methionine aminopeptidases (MetAPs), which
remove the initiator methionine from nascent peptides, are
essential in all organisms. While MetAP2 has been demonstrated
to be a therapeutic target for inhibiting angiogenesis in
mammals, MetAP1 seems to be vital for cell proliferation. Our
earlier efforts identified two structural classes of human MetAP1
(HsMetAP1)-selective inhibitors (1−4), but all of them failed to
inhibit cellular HsMetAP1. Using Mn(II) or Zn(II) to activate
HsMetAP1, we found that 1−4 could only effectively inhibit
purified HsMetAP1 in the presence of physiologically unachiev-
able concentrations of Co(II). In an effort to seek Co(II)-
independent inhibitors, a novel structural class containing a 2-
(pyridin-2-yl)quinazoline core has been discovered. Many
compounds in this class potently and selectively inhibited HsMetAP1 without Co(II). Subsequently, we demonstrated that
11j, an auxiliary metal-dependent inhibitor, effectively inhibited HsMetAP1 in primary cells. This is the first report that an
HsMetAP1-selective inhibitor is effective against its target in cells.
Only one type of MetAP, either type 1 or type 2, can be found
in a prokaryotic cell. Archaea contain type 2 MetAPs,5b and
eubacteria contain type 1 MetAPs.8 However, all eukaryotic
genomes studied to date encode at least a cytosolic type 1
MetAP (MetAP1) and a cytosolic type 2 MetAP (MetAP2).
Extensions at the N-terminus of the catalytic domain of
eukaryotic cytosolic MetAPs distinguish them from their
prokaryotic counterparts.9,10 The sequence identity between
type 1 and type 2 MetAPs is very limited,9,10 however their
catalytic domains adopt the same pita-bread fold,3 with both
metal binding residues and the shape of methionine-binding
pocket highly conserved.3,11
NME accounts for the major source of N-terminal diversity
in mature proteins.12 NME is involved in the creation of N-
degrons for the N-end rule pathway.12 NME also prepares the
nascent peptides for the subsequent N-α-acetylation which
facilitates protein folding and protein−protein interaction and
INTRODUCTION
■
Almost all cellular protein syntheses begin with either an
initiator N-formylmethionine (ifMet) or an initiator methionine
(iMet).1 The ifMet or iMet in a significant number of proteins
(e.g., about 60% in Escherichia coli2) will be proteolytically and
irreversibly removed through N-terminal methionine excision
(NME). In archaea and eukaryotes, NME is catalyzed by
methionine aminopeptidases (MetAPs), a family of evolutio-
narily conserved metalloproteases.3 In the NME of eubacteria,
mitochondria, and plastids, MetAPs remove iMet after ifMet is
converted to iMet by peptide deformylases.4 MetAPs utilize
one or two divalent metal ions5 to catalyze the removal of iMet,
and they only cleave iMet from nascent peptides in which the
N-terminal P1 residues are methionine and the side chains of
the P1′ residues are small and uncharged (Ala, Gly, Pro, Ser,
Cys, Thr, or Val).6,7 Amino acid residues at the P2′ position
and beyond also contribute to the efficiency of cleavage.6c,7
MetAPs can be classified into type 1 and type 2. When
compared with type 1 MetAPs, type 2 enzymes contain a
characteristic insertion (∼60 aa) in their catalytic domain.3
Received: February 13, 2013
Published: May 1, 2013
© 2013 American Chemical Society
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Figure 1. (A) Structures of HsMetAP inhibitors. (B) A stereo view of the active site in the X-ray crystal structure of N-terminally truncated
HsMetAP1 in complex with Co(II) and compound 1 (PDB code 2NQ621). Hot-pink spheres are Co(II) ions. Surrounding residues are shown as
sticks (carbon white), water molecules as small red spheres, and metal interactions as dashed lines. Compound 1 shown as sticks (carbon green)
largely depends on an auxiliary Co(II) (M3) to bind and inhibit HsMetAP1. For all noncarbon atoms, nitrogen is blue, oxygen is red, and sulfur is
yellow.
dictates protein half-life.13,14 In addition, NME is a prerequisite
for N-terminal myristoylation which contributes to the
membrane localization and the function of some important
proteins.15 Furthermore, there is a family of enzymes that
require NME to expose a mature N-terminal residue other than
methionine for catalytic activity.16
In addition, because of the selectivity of fumagillin and its
analogues for type 2 MetAP enzymes, they were widely used as
tools to study the functions of MetAP2 in higher
eukaryotes.18,39
In contrast to vigorous research on HsMetAP2, the
understanding of the physiological functions of HsMetAP1 is
very limited and the efficacy of anti-MetAP1 therapy in the
treatment of neoplasia remains unknown, largely due to the
lack of MetAP1-selective inhibitors that are active in cells.
Peptidyl hydroxamic acids and barbiturate-based compounds
were shown to inhibit purified HsMetAP1 in vitro.40,41
However, their selectivity for HsMetAP1 was either low or
undetermined, and their activities in cells remain unclear.
Pyridine-2-carboxamides can potently inhibit the activities of
purified type 1 MetAPs from bacteria, yeast, and human.21,42,43
Later, Hu et al. reported that several analogues in this class had
great selectivity for HsMetAP1 over HsMetAP2, and 3-tert-
butoxycarbonylaminopyridine-2-carboxylic acid thiazole-2-yla-
mide (1, Figure 1A) diminished the activity of HsMetAP1 in
HeLa cells.21 In addition to pyridine-2-carboxamides, 2-pyridin-
2-ylpyrimidines were uncovered as a novel class of inhibitors of
human MetAPs.44 Hu et al. showed that these pyridinylpyr-
imidines potently inhibited both HsMetAPs, and 5-chloro-6-
methyl-N-(2-phenylethyl)-2-pyridin-2-ylpyrimidine-4-amine (2,
Figure 1A) could inhibit HsMetAPs in HeLa cells as well.44
Recently, we communicated our systematic medicinal chemistry
effort at improving the selectivity and potency of pyridinylpyr-
imidine class of HsMetAP1 inhibitors (e.g., 3 and 4, Figure
1A).45
The X-ray crystal structures of the N-terminally truncated
HsMetAP1 in complex with Co(II) and MetAP1 inhibitors
(PDB codes 2NQ6, 2G6P, and 4HXX) indicated that, instead
of the two cobalt ions (M1 and M2) chelated at the bottom of
the active site, each of 1−3 requires an auxiliary cobalt ion
(M3) to bind HsMetAP1 (Figure 1B).21,44,45 Besides 1−3, four
structurally distinct inhibitors of bacterial type 1 MetAP have
been uncovered independently by three research groups.
Crystallographic analysis demonstrated that all four inhibitors
mainly depended on the chelation of an auxiliary Co(II) or
Mn(II) to inhibit two eubacterial MetAPs.46 Being reminiscent
of the cases in HsMetAP1, both bacterial type 1 MetAPs
contacted the auxiliary metal atom solely through an imidazole
nitrogen from His178 (equivalent to His310 in HsMetAP1)46a
or His79 (equivalent to His212 in HsMetAP1).46b−d In living
NME is an essential cellular process. Mycoplasma genitalium
contains the smallest genome for cellular growth in pure
medium, and its 382 essential genes include one (map) that
encodes a MetAP.17 The disruption of the single map gene in
Escherichia coli or Salmonella typhimurium resulted in lethality.8
In Saccharomyces cerevisiae, deletion of either map1 or map2
gene generated a slow growth phenotype, while the deletion of
both became lethal.9 Cytosolic MetAP1 and MetAP2s are
functionally interchangeable in Arabidopsis thaliana, but a
complete blockade of NME halted the development of the
plant after germination.18 The disturbance of MetAP2 in
animals generated organism-dependent and tissue-specific
defects in development.19 When either human MetAP1
(HsMetAP1) or MetAP2 (HsMetAP2) was downregulated by
small-interfering RNA (siRNA) in human umbilical vein
endothelial cells (HUVEC), the growth of these primary cells
were greatly inhibited.20 Furthermore, it has been shown that
the downregulation of MetAP1 by siRNA duplexes slowed the
cell cycle progression of synchronized HeLa cells.21 Benga-
mides (Figure 1A), isolated from Jaspidae marine sponges,
display a broad antitumor activity in vitro22,23 and in vivo.24 An
extensive proteome analysis revealed that bengamides inhibit
both HsMetAP1 and HsMetAP2,25 but unfortunately, the
clinical study of a synthetic bengamide analogue LAF-389 in
patients with advanced cancer was terminated due to its
cardiovascular toxicity,26 which might be caused by the
simultaneous inhibition of HsMetAP1 and HsMetAP2.
Fumagillin and its synthetic analogue TNP-470 (also called
AGM-1470) (Figure 1A) were revealed as potent inhibitors of
angiogenesis more than two decades ago.27,28 Later, we and
others found that fumagillin and TNP-470 bind to MetAP2
covalently,29 leading to an irreversible inhibition.30,31 Because
the decreased enzymatic activity of MetAP2 preferentially
affects the proliferation of endothelial cells,32,33 TNP-470 has
been shown to inhibit angiogenesis and suppress the growth of
solid tumors in various animal models.34,35 Fumagillin
analogues also showed efficacy in the treatment of arthritis,36
nutritionally induced obesity,37 and pulmonary hypertension.38
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a
Scheme 1.
a
Reagents and conditions: (a) Na/MeOH, rt, then anthranilic acid, MeOH, 85 °C, 18 h; (b) PhNEt2, POCl3, 100 °C; (c) iPr2NEt, 1,4-dioxane, 110
°C; (d) TBSCl/Et3N/DCM; (e) Boc2O/Et3N/DCM; (f) allyl bromide, K2CO3, THF, then 5% TFA/DCM; (g) NaBH4/MeOH; (h) 2 N NaOH/
MeOH/THF; (i) (n-Bu)4NF/THF; (j) p-TsCl/Et3N/DCM; (k) aq NaOH/THF, AcCl; (l) HBTU, iPr2NEt, MeCN.
cells, the concentration of free transition metals (especially
cobalt) are believed to be several orders of magnitude lower
than the concentrations at which transition metals were
routinely used in the in vitro MetAP assays and crystallography.
Given that the ternary complexes of MetAP−Co(II)−inhibitor
may not exist in cells,46b it is not difficult to understand why
several Co(II)-dependent inhibitors that were very potent in
vitro lack antibacterial activities in vivo.42,46b
In the present work, we carefully assessed the cellular
activities of the previously developed MetAP1-selective
inhibitors using the NME of 14-3-3γ protein as a readout.
We found that 14-3-3γ is a shared substrate between
HsMetAP1 and HsMetAP2 in HUVEC, and the complete
NME of 14-3-3γ relies on the activities of both enzymes.
Surprisingly, pyridine-2-carboxamide 1 and pyridinylpyrimi-
dines 2−4 failed to inhibit the cellular activity of either
HsMetAPs in HUVEC appreciably at the highest tolerated
concentrations (just below the severe cytotoxicity threshold).
In addition, the reported inhibition of HsMetAP1 by 1 and 2 in
HeLa cells21,44 could not be reproduced using either previously
synthesized or freshly synthesized compounds. To assess the
relevance of metal ions, we applied two in vitro assays to
determine the potency of 1−4 and two chelating agents
(thiabendazole and 2,2′-bipyridine) against purified HsMetAP1
in the presence of Co(II), Mn(II), or Zn(II). As expected, 1−4,
as well as thiabendazole and 2,2′-bipyridine, could only inhibit
HsMetAP1 potently with the auxiliary Co(II). In the course of
the SAR studies, a novel class of HsMetAP1 inhibitors
containing a 2-(pyridin-2-yl)quinazoline emerged, which served
as a new lead for additional SAR studies. One analogue, 11j,
strongly inhibited the activity of MetAP1 in human cells.
Besides its superior inhibitory potency and selectivity for
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HsMetAP1, 11j and related analogues also exhibited rarely seen
“relaxed metal selectivity”, which appears to be a key feature for
a successful inhibition of MetAP1 in cells.47 Finally, we solved
the X-ray crystal structure of 11j in complex with Co(II) and
N-terminally truncated HsMetAP1. To the best of our
knowledge, this is the first report of a small molecule inhibitor
which can potently and selectively inhibit MetAP1 in human
cells. The present work also provided compelling evidence that
the auxiliary metal-mediated inhibitor identified in vitro can be
equally effective against the same target in cells.
CHEMISTRY
■
The syntheses of 2-(pyridin-2-yl)quinazoline derivatives (7a,
10a−10c, 11a−11m, and 13−15) with various C4 side chains
and C5′ substituents were carried out as shown in Scheme 1.
Appropriately substituted 2-cyanopyridines (5) were treated
with sodium in methanol and methyl pyridine-2-carboximidate
generated in situ was condensed with anthranilic acid to yield
3,4-dihydroquinazolin-4-one (6). 4-Chloroquinazoline (7)
synthesized by dehydrohalogenation of quinazolin-4-one (6)
with phosphoryl chloride served as a starting material for all the
quinazoline derivatives discussed in this work. Thus, 4-
chloroquinazoline (7) was treated with various amines
(including 9) while refluxing in 1,4-dioxane with N,N-
diisopropylethylamine (iPr2NEt) as the base to afford the
final products (10a−10c, 11a−11m) and intermediate 12.
Quinazoline 11a was obtained by reducing the corresponding
acetophenone with sodium borohydride. Derivatives 11c and
11e were the saponification products from 11b and 11d,
respectively. In the cases of 13−15, the product after the
chloride displacement reaction was 4-(piperazin-1-yl)-2-(pyr-
idin-2-yl)quinazoline (12), which was converted to the desired
final products (13−15) by amide coupling with appropriate
acids.
Figure 2. Inhibition of iMet processing of 14-3-3γ by MetAP inhibitors
in human cell lines. Cells were treated with vehicle (DMSO),
bengamide A, bengamide B, or TNP-470 for 24 h. Cell lysates were
resolved by SDS-PAGE, transferred to nitrocellulose membrane, and
immunoblotted (IB) with antibodies specific for β-actin, α-tubulin,
total 14-3-3γ, or the iMet unprocessed form of 14-3-3γ (iMet 14-3-3γ).
The levels of iMet 14-3-3γ were quantified by densitometry and then
normalized to total 14-3-3γ. The IB of β-actin and α-tubulin served as
loading controls. (A) HUVEC, human umbilical vein endothelial cells;
Jurkat E6-1, T lymphocytes from human acute T cell leukemia; HeLa,
epithelial cells from human cervical adenocarcinoma; HEK293T,
human embryonic kidney epithelial cells constitutively expressing
SV40 T antigen. (B) PC-3, epithelial cells from human prostate
adenocarcinoma; MCF-7, epithelial cells from human breast
adenocarcinoma.
and TNP-470, respectively (Figure 2A), in agreement with
previous observations.25 The accumulation of unprocessed 14-
3-3γ in HUVEC had maximal responses to bengamide A and
TNP-470 at concentrations around 10 and 1 nM, respectively
(data not shown). Nevertheless, in all five neoplastic cell lines,
the treatment with 100 nM bengamide A/B merely increased
the ratio of unprocessed versus total 14-3-3γ by no more than
2.6-fold, while the treatment with 100 nM TNP-470 increased
the ratio by no more than 1.6-fold (Figure 2). For this reason,
we chose HUVEC as the optimal cell type for the detection of
MetAP inhibition instead of HeLa cells.
It was noticeable that 100 nM bengamide A/B induced larger
increases in the ratio of unprocessed versus total 14-3-3γ than
100 nM TNP-470 in HUVEC, insinuating that HsMetAP2
might not be the only human MetAP responsible for the NME
of 14-3-3γ. To test this possibility, siRNA knockdown of
HsMetAP1 or HsMetAP2 was performed in HUVEC (Figure
3). In comparison with the scrambled siRNA control, 20 nM
MetAP2 siRNA oligo decreased the protein level of HsMetAP2
about 90% and brought a 7-fold increase in the ratio of
unprocessed versus total 14-3-3γ, in agreement with the earlier
report in the literature.25 With a 30% decrease in the protein
level of HsMetAP1 upon siRNA treatment, there was a 3.2-fold
increase in the ratio of unprocessed versus total 14-3-3γ. Under
the same conditions, there was no obvious change in the
protein level of either HsMetAP2 or 14-3-3γ. Moreover, when a
higher concentration (100 nM) of the siRNA oligo was used,
an 80% reduction of HsMetAP1 protein led to an increase in
the ratio by nearly 5-fold. These pieces of evidence indicated
RESULTS AND DISCUSSION
■
The Retention of the Initiator Methionine in 14-3-3γ
Is a Cellular Indicator for the Inhibition of HsMetAP1
and/or HsMetAP2. In primary (HUVEC) and transformed
(H1299) human endothelial cells, inhibition of the enzymatic
activity of HsMetAP2 by a type 2-selective MetAP inhibitor
(fumagillin) or siRNA mediated down-regulation of the protein
level of HsMetAP2 both led to strong signals of unprocessed
14-3-3γ protein with intact iMet in the immunoblot.25
Therefore, 14-3-3γ protein is a substrate of at least MetAP2
in human endothelial cells. But whether 14-3-3γ is also a
substrate of MetAP1 in primary human cells remained unclear.
To identify a human cell line in which the iMet retention in
14-3-3γ caused by MetAP inhibition can be readily detected by
immunoblot, we treated one primary (HUVEC) and five
neoplastic cell lines (Jurkat E6-1, HeLa, HEK293T, PC-3, and
MCF-7) with either a nonselective MetAP inhibitor (benga-
mide A/B) or a type 2-selective MetAP inhibitor (TNP-470)
(Figure 2). In vehicle (dimethyl sulfoxide, DMSO) treated
HUVEC, the unprocessed form of 14-3-3γ could barely be
detected by a monoclonal antibody raised against a peptide
from the unprocessed N-terminus of 14-3-3γ (Figure 2A). On
the contrary, unprocessed 14-3-3γ could readily be detected in
the vehicle controls of all five neoplastic cell lines (especially in
Jurkat E6-1, HeLa, and HEK293T) (Figure 2). In HUVEC,
after 24 h treatment, the ratio of unprocessed 14-3-3γ versus
total 14-3-3γ was increased 8-fold and 4.5-fold by bengamide A
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tAP1 and HsMetAP2 disfavored large P1′ (Val and Thr) and
acidic P2′ residues (Asp and Glu),7 marking 14-3-3γ a slow
substrate for either HsMetAPs. Purified HsMetAP2 was more
active than purified HsMetAP1 toward several Met-Val peptides
in vitro.7,32 However, a Met-Val peptide derived from the N-
terminal sequence of human hemoglobin was a better substrate
for HsMetAP1 than HsMetAP2 in vitro.32 It is thus possible
that both HsMetAPs are required for the complete NME in
some if not all Met-Val substrates.
A Pyridine-2-carboxamide Derivative and Type 1-
Selective Pyridinylpyrimidines Failed to Inhibit HsMe-
tAP1 in Cells. Hu et al. showed that two cobalt-dependent
MetAP1 inhibitors, pyridine-2-carboxamide 1 and pyridinylpyr-
imidine 2, inhibited the enzymatic activity of HsMetAP1 in
HeLa cells, indicated by the enhanced signals of N-terminal
unprocessed 14-3-3γ protein in the immunoblots.21,44 To
investigate whether our recently identified MetAP1-selective
inhibitors based on pyridinylpyrimidine could target HsMetAP1
inside cells, we treated HUVEC with 2−4 for 24 h and then
examined the iMet status of 14-3-3γ by immunoblot with a
monoclonal antibody25 specific for the unprocessed form of 14-
3-3γ (Figure 4A). When HUVEC were treated with 0.1 μM
TNP-470 (a cytostatic dose without cytotoxic effects48),
compared to the vehicle (DMSO) controls, the ratios of
unprocessed 14-3-3γ versus total 14-3-3γ increased 4.3-fold and
5.9-fold (Figure 4A). However, when HUVEC were treated
with escalating doses of 4 or 3, no increase could be observed in
the ratios of unprocessed versus total 14-3-3γ at the doses up to
20 μM for 4 and up to 4 μM for 3 (Figure 4A, left). However,
compound 2 which was reported to potently inhibit HsMetAPs
at the concentrations from 1 to 10 μM in HeLa cells,44 merely
raised the ratios of unprocessed versus total 14-3-3γ by no more
than 2.1-fold at the concentrations from 0.1 to 90 μM in
HUVEC (Figure 4A, right). More surprisingly, compound 1,
Figure 3. Downregulation of the protein level of either MetAP1 or
MetAP2 inhibited the iMet processing of 14-3-3γ in HUVEC. A
scrambled control siRNA and siRNAs specific for MetAP1 or MetAP2
were introduced into HUVEC using either HiPerFect or TransIT-
TKO transfection reagent. Then 48 h after transfection, cell lysates
were resolved by SDS-PAGE, transferred to nitrocellulose membrane,
and immunoblotted (IB) with antibodies specific for α-tubulin,
MetAP1, MetAP2, total 14-3-3γ, or iMet 14-3-3γ. The levels of
MetAP1 and MetAP2 were quantified by densitometry and then
normalized to α-tubulin. The levels of iMet 14-3-3γ were quantified by
densitometry and then normalized to total 14-3-3γ.
that 14-3-3γ is also a substrate of HsMetAP1, and the NME in
14-3-3γ requires the activities of both HsMetAPs.
The first 10 amino acid residues of nascent human 14-3-3γ
are Met-Val-Asp-Arg-Glu-Gln-Leu-Val-Gln-Lys. Both HsMe-
Figure 4. Auxiliary metal-mediated human MetAP1 inhibitors 1−4 did not inhibit the iMet processing of 14-3-3γ in human primary cells. (A) The 2-
(pyridin-2-yl)pyrimidine derivatives 2−4 failed to inhibit the NME of 14-3-3γ in HUVEC. (B) The pyridine-2-carboxamide derivative 1 (freshly
prepared) failed to inhibit the NME of 14-3-3γ in HUVEC. Cells were treated with vehicle (DMSO), TNP-470, bengamide B, or compound 1−4 for
24 h. Cell lysates were resolved by SDS-PAGE, transferred to nitrocellulose membrane, and immunoblotted (IB) with antibodies specific for α-
tubulin, total 14-3-3γ, or iMet 14-3-3γ. The IB of α-tubulin served as a loading control. The levels of iMet 14-3-3γ were quantified by densitometry
and then normalized to total 14-3-3γ.
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Table 1. Inhibition of Different Metalloforms of Purified Human MetAP1 and Cell Proliferation by Metal Coordinating
Inhibitors
a
b
inhibition of HsMetAP1 (EC50, μM)
inhibition of cell proliferation (EC50, μM)
HUVEC HDFa HeLa
2.0 0.9 (B = 10%) 0.79 0.15 2.3 0.4 (B = 27%)
compd
Co(II)
0.35 0.10
Zn(II)
Mn(II)
c
d
d
1 (DJM)
>40
>40
>100
>100
c
d
d
1 (JL)
0.36 0.10
0.19 0.04
0.43 0.08
0.28 0.05
0.10 0.01
1.5 0.6 (B = 11%) 0.65 0.09 2.0 0.6 (B = 21%)
d
d
2
0.71 0.20 (B = 35%) 15 12 (B = 32%)
11 2.1
3.6 1.5
7.1 0.9
e
3
>30
>100
>200
ND
0.76 0.11 0.97 0.25
4
>100
4.3 1.0
1.9 0.29
1.9 0.7
>250
10 5.9
5.2 1.2
d
d
11j
1.1 0.3 (B = 16%)
5.1 0.8 (B = 12%) 1.6 0.4
d
e
thiabendazole
3.4 0.4 (B = 15%)
>250
>100
>100
>250
10
ND
d
f
e
2,2′-bipyridine 0.32 0.07 (B = 16%) >100
1
10−30
ND
a
EC50 values are shown as mean SD of at least three determinations; 0.15 μM purified recombinant HsMetAP1 activated by 10 μM CoCl2, 1 μM
ZnCl2 or 10 μM MnCl2 were used in the experiments. Representative inhibitory dose−response curves can be found in Supporting Information
b
c
Figure S3. EC50 values were shown as mean SD of at least three experiments. For compound 1, the batch “JL” was previously prepared21 and the
d
batch “DJM” was freshly prepared. B: the bottom value of the inhibitory dose−response curve. It has been noted in parentheses only when B >
e
f
10%. ND, not determined. The inhibitory dose−response curves for 2,2′-bipyridine were extremely steep between 10 and 30 μM (the Hill slopes
were around −14); 10 μM 2,2′-bipyridine did not inhibit the cell proliferation of HDFa, but 30 μM 2,2′-bipyridine inhibited 94 2% of cell
proliferation.
which was reported by Hu et al. to potently inhibit HsMetAP1
at the concentrations of 1 and 10 μM in HeLa cells,21 failed to
increase the ratio of unprocessed versus total 14-3-3γ in
HUVEC, HeLa, and human dermal fibroblast-adult (HDFa)
cells at the concentrations of 0.1, 1, and 10 μM (Supporting
Information Figure S1). To address this issue comprehensively,
we resynthesized pyridine-2-carboxamide 1 following the same
synthetic route as previously described,21 characterized it fully,
and found it was indistinguishable from the previously
synthesized sample in terms of both in vitro MetAP enzymatic
assays and cell proliferation assays (Table 1). Unfortunately,
the freshly prepared 1 at the same concentrations still could not
increase the ratio of unprocessed versus total 14-3-3γ in
HUVEC, HeLa, and HDFa cells (Figure 4B and Supporting
Information Figure S1B).
The accumulation of unprocessed MetAP substrates relies on
not only the inhibition of cellular MetAPs but also the
continuous production of nascent peptides by ribosomes.
Therefore, a MetAP inhibitor which is capable of inhibiting
translation may not increase the ratio of unprocessed versus
total 14-3-3γ in cells. To investigate this possibility, we treated
HUVEC with the combination of TNP-470 and one of our
pyridinylpyrimidines. When 0.1 μM TNP-470 was applied
together with 20 μM compound 4, the ratio of unprocessed
versus total 14-3-3γ became 2.5 times higher than the ratio in
the vehicle control and was 5 times higher than the ratio in
HUVEC treated with 20 μM compound 4 alone (Figure 4A,
left). A 3.5-fold increase in the ratio has been observed when
TNP-470 was supplemented to the cells treated with 30 μM
compound 2 (Figure 4A, right). A similar result for compound
1 was obtained in HUVEC as well (Supporting Information
Figure S1A). Therefore, 10 μM 1, 20 μM 2, or 30 μM 4 did not
completely inhibit translation, and compounds 1−4 could not
effectively inhibit either HsMetAPs in HUVEC at the highest
concentrations which did not lead to severe cytotoxicity.
It has been observed that small molecule inhibitors with high
potency against purified type 1 MetAPs failed to inhibit the
corresponding enzymes in cells. Because the single MetAP
(EcMetAP) is essential in Escherichia coli,8a inhibition of
EcMetAP slowed down the growth of bacterial cells.49 Within
the past decade, three distinct classes of MetAP inhibitors have
been reported which potently inhibited purified EcMetAP in
vitro but none were active in vivo. First, several pyridine-2-
carboxamides were reported for their potent inhibition of
EcMetAP (0.15 μM) with IC50 values between 0.13 and 0.33
μM in vitro.42 However, the minimum inhibitory concen-
trations (MICs) of these compounds against an enter-
opathogenic Escherichia coli strain were 1527−3338 times
higher than their IC50 values against purified EcMetAP.42 In an
independent study, thiabendazole, a fungicide and parasiticide
(Figure 1A), was found to be inactive against the growth of
Escherichia coli even at 1 mM, a concentration 2500 times
higher than the Ki value of thiabendazole against purified
EcMetAP.46b More recently, Chai et al. showed that a thiazol-2-
yl-oxalamide derivative50 failed to halt the growth of three
Escherichia coli strains at 1 mM, a concentration almost 15000
times higher than this compound’s IC50 value against Co(II)-
loaded EcMetAP in vitro.47 Intriguingly, the crystallographic
analyses demonstrated that all of these inhibitors depend on an
auxiliary Co(II) to interact with an imidazole nitrogen from
His79 in the active site of EcMetAP.46b,47 Historically, the lack
of activity in cells for these inhibitors has been attributed to the
assumed poor pharmacokinetic properties,42 the identity of the
physiologically relevant metal in the active site of EcMetAP,42
or the limited availability of free Co(II) in cells.46b
The capabilities of our pyridinylpyrimidine derivatives (2−4)
to affect cell proliferation were measured using a 30-h [3H]-
thymidine incorporation assay (Table 1). No solubility
problems were seen for any compounds at their highest
doses tested (90 μM for 2, 40 μM for 3, and 40 μM for 4).
Compound 2 inhibited the proliferation of HUVEC and HDFa
cells with IC50 values of 11 and 3.6 μM, respectively. Although
these values are 58 and 19 times higher than the IC50 value of 2
against purified HsMetAP1, they are comparable to the
reported IC50 value (9.6 μM) of 2 against the proliferation of
HeLa cells.44 The proliferation of HUVEC, HDFa, and HeLa
cells were inhibited by 4, with IC50 values of 4.3, 1.9, and 10
μM, respectively. Compound 3 was the most potent at
inhibiting the proliferation of HDFa and HeLa cells with IC50
values below 1 μM. Compound 2 at 30 μM and compound 4 at
20 μM concentrations inhibited more than 90% of HUVEC
proliferation within 30 h. Compound 3 at 10 μM exhibited
severe cytotoxicity, as most cells were floating after 24 h.
Because compounds 1−4 could not diminish the activity of any
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Figure 5. Activation of full-length human MetAP1 by divalent metal ions. The metal activation assays were carried out in a 100 μL mixture of (A)
150 nM MetAP1, 600 μM Met-Pro-p-nitroanilide, and 15 U/mL Bacillus coagulans proline iminopeptidase or (B) 150 nM MetAP1, 600 μM Met-
Ala-Ser, 1 U/mL L-amino acid oxidase (LAO), 0.1 mg/mL horseradish peroxidase, 0.2 mg/mL o-dianisidine (3,3′-dimethoxybenzidine), and one of
the divalent metal ions in MetAP assay buffer. Initial rates are expressed as the mean (symbol) and standard deviation (error bar) of two (A) or three
(B) determinations. Invisible error bars are included in the symbols.
HsMetAP effectively in cells, the inhibition of cell proliferation
is likely to be due to unknown off-target effects. Unless their
antiproliferative effects were mediated by changes in the
property of plasma membrane or transmembrane signaling
transduction, compounds 1−4 should have no pharmacokinetic
problems to stay inside the cells.
supplementation of 1−100 μM transition metals. Furthermore,
to obtain crystals of MetAP holo enzymes in complex with
inhibitors, the final concentrations of transition metals used in
the experiments could easily exceed 1 mM. But due in part to
their cytotoxicity, transition metals are tightly regulated in living
cells.51 Co(II) is the optimal cofactor to activate the enzymatic
activities of most MetAPs in vitro. As a result, Co(II) has been
widely used in various MetAP assays and most X-ray crystal
structures of MetAP enzymes have been obtained with Co(II).
However, except for the Co(II) tightly coordinated by the
corrin ring in vitamin B12, cobalt is relatively rare and it is not
known to be stringently required for the biological function of
any cytosolic enzyme.52 Consequently, if a small molecule
inhibitor strictly requires an auxiliary Co(II) to bind to and
inhibit a MetAP, it will have little chance to be effective in cells
due to the inaccessibility of the free metal ion.
HsMetAP1 Can Be Activated Not Only by Co(II), but
Also by Mn(II) and Zn(II). Before we investigated whether
auxiliary Co(II)-mediated HsMetAP1 inhibitors can utilize any
other metal to bind the cellular enzyme, it was deemed critical
to determine the effects of other metals on HsMetAP1. It has
been previously reported that Co(II), Mn(II), Fe(II), Zn(II),
and Ni(II) can all activate bacterial type 1 MetAP in vitro.53
When a tripeptide (Met-Ala-Ser) was used as the substrate and
L-amino acid oxidase (LAO) and horseradish peroxidase (HRP)
were used as the coupling enzymes, recombinant HsMetAP1
was activated by Co(II) and Mn(II) but inhibited by Zn(II),
Ni(II), and Cu(II).54 However, when Met-SCH2CO-Phe was
used as the substrate, both Co(II) and Zn(II) activated purified
recombinant HsMetAP1, while Mn(II) and Ni(II) did not.43
When Met-Pro-p-nitroanilide was used as the substrate and
Bacillus coagulans proline iminopeptidase (BcProIP) was used
as the coupling enzyme, HsMetAP1 was activated by Co(II)
and a very high concentration (10 mM) of Zn(II) but inhibited
by Mn(II) and Ni(II).11 We employed the assay with Met-Pro-
p-nitroanilide as the substrate and BcProIP as the coupling
enzyme to further assess the effects of different metal ions on
HsMetAP1 (Figure 5A). Full-length HsMetAP1 was expressed
as a GST-fusion protein in Escherichia coli after IPTG induction.
Upon purification, the N-terminal GST tag was cleaved by
The active site of HsMetAP1 is shallow and mostly
hydrophobic.11 At the bottom of the active site, two divalent
transition metals (M1 and M2) are coordinated by five highly
conserved residues (one His, two Glu, and two Asp) (Figure
1B). M1 adopts distorted trigonal bipyramidal coordination
geometry and is coordinated by one His, two Glu, one Asp, and
one water (W4), whereas M2 adopts distorted octahedral
geometry and is coordinated by one shared Glu, one shared
Asp, one unique Asp, and two water molecules (W4 and W5).
The three crystal structures of N-terminally truncated
HsMetAP1 in complex with Co(II) and an inhibitor revealed
that compounds 1−3 all utilize an auxiliary Co(II) (M3) to
bind to the active site of HsMetAP1 (Figure 1B).21,44,45 In
addition, the pyridinylpyrimidine core of 4 also binds
HsMetAP1 in a similar way (personal communication with
Dr. Anthony Addlagatta). It is likely that MetAP inhibitors
which directly bind the active site metal(s) must inhibit cellular
MetAPs in their physiologically relevant metalloforms to be
active in cells. However, the M3 utilized by the aforementioned
compounds was not one of the “regular” metals (M1 and M2)
in the active site. This auxiliary metal only contacted the
enzyme through an imidazole nitrogen from a catalytically
important histidine (His212). It is known that the identity of
M1 and M2 in the active site did not result in significant
changes to the MetAP structures.50 Moreover, the structures of
HsMetAP1 containing 1−3 transition metal ions were
essentially identical.11 Therefore, if the physiologically relevant
metals in the active site of HsMetAP1 are not Co(II), the
inhibitory potency of auxiliary metal-dependent inhibitors in
cells should not be crippled.
Like many other metalloenzymes, activation of purified
MetAP in vitro requires relatively high concentrations of
transition metal ions. For example, the assays to measure the
activity of purified MetAP in vitro often require the
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PreScission protease. Purified recombinant HsMetAP1 dis-
played a basal activity about 10 μM·min−1·μM−1 (Figure 5),
which was close to the previously reported values.43,54 It is well-
known that one or two divalent metal ions are required to
activate each molecule of MetAP in vitro. The activities of all
MetAPs tested could be inhibited by metal chelators such as
EDTA. Thus, the basal activity observed in the HsMetAP1
assays must come from the copurified metal(s) in the active
site. As shown in Figure 5A, Co(II) was the best cofactor to
activate HsMetAP1. In agreement with the literature,43
HsMetAP1 was most active (about 45 μM·min−1·μM−1) with
Co(II). However, the highest enzymatic activity was found in
the presence of 10 μM Co(II), instead of 100 μM Co(II)
reported previously,54 which later was used by several other
groups.11,21,41,44 Excess metals are known to inhibit metal-
lopeptidases,55 thus it is not surprising that higher concen-
trations of Co(II) decreased MetAP activity. Mn(II) was also
an activator of HsMetAP1 (Figure 5A). On the contrary,
Cu(II), Ni(II), and Zn(II) inhibited HsMetAP1 at the
concentrations of 10 μM and above. Two physiologically
abundant divalent metals, Mg(II) and Ca(II), did not affect the
activity of HsMetAP1 over a wide concentration range (from
0.1 nM to 1 mM).
Because BcProIP was used as the coupling enzyme in the
assay, all compounds and metal ions that inhibited the readout
of the assay were counter screened against BcProIP. As negative
controls, Co(II) and Mn(II) did not inhibit BcProIP at
concentrations up to 1 mM (Supporting Information Figure
S2). It has been previously pointed out that 500 μM ZnCl2
abrogated the activity of BcProIP.56 Indeed, 1 μM Zn(II)
decreased the activity of BcProIP by about 45% and 10 μM
Zn(II) led to a complete inhibition (Supporting Information
Figure S2). In addition, more than 95% of the ProIP activity
was inhibited by Cu(II) at 0.1 μM. In contrast, 100 μM Ni(II)
only reduced the activity of BcProIP by less than 20%. On the
basis of the fact that the amount of BcProIP routinely used in
our MetAP assay (15 U/ml) was at least 100 times higher than
the amount required to saturate the coupling reaction, there is
no doubt that under the assay conditions, Ni(II) is an inhibitor
of HsMetAP1.
The incompatibility of Zn(II) and the BcProIP-coupled
MetAP assay prompted us to turn to another assay to measure
MetAP activity in the presence of Zn(II). When we used LAO
and HRP as the coupling enzymes and Met-Ala-Ser as the
substrate,6d the basal activity of purified recombinant
HsMetAP1 was close to 10 μM·min−1·μM−1 (Figure 5B). In
this LAO and HRP-coupled MetAP assay, 10 μM Co(II) and 1
μM Zn(II) increased the activity of HsMetAP1 to 56 and 19
μM·min−1·μM−1, respectively (Figure 5B). Both 0.1 and 10 μM
Zn(II) consistently activated HsMetAP1 as well. Moreover, at
concentrations higher than 1 μM, Cu(II) began to inhibit
HsMetAP1 in the LAO and HRP-coupled HsMetAP1 assay,
similar to its effect in the BcProIP-coupled assay. In summary,
we demonstrated that Co(II), Mn(II), and Zn(II) can activate
HsMetAP1 in vitro.
Type 1-Selective MetAP Inhibitors 1−4 are Selectively
Dependent on Co(II) for Their Inhibition of HsMetAP1 in
Vitro. As both Zn(II) and Mn(II) can activate purified
HsMetAP1 in vitro and these two metals are more abundant
and more stable than Co(II) in living cells,51,52 we measured
the IC50 (or EC50) values of the pyridine-2-carboxamide
derivative 1 and pyridinylpyrimidine derivatives 2−4 against
HsMetAP1 supplemented with either Zn(II) or Mn(II) (Table
1 and Supporting Information Figure S3). When 10 μM Co(II)
in the MetAP assay was replaced by 1 μM Zn(II), a 3.7-fold
increase in the EC50 value of 2 was observed. The inhibition of
HsMetAP1 by 2 reached a plateau at 65% even at
concentrations up to 100 μM. The IC50 values of compounds
1, 3, and 4 against HsMetAP1 were greater than 40, 30, and 100
μM, respectively. Similarly, when Co(II) in the MetAP assay
was replaced by the same concentration of Mn(II), the
inhibitory potency of 1−4 were also greatly compromised.
The EC50 value of 2 for the inhibition of HsMetAP1 was
increased about 80 times from 0.19 μM with Co(II) to 15 μM
with Mn(II), and the maximal level of inhibition induced by 2
was merely 68% at the concentrations from 10 nM to 100 μM.
The IC50 values of 1, 3, and 4 against HsMetAP1 were greater
than 100 μM, 100 μM and 200 μM, respectively. The “metal
selectivity” of compounds 1−4 (Table 1) was consistent with
the known preference of N,N-donor ligands to coordinate soft
metals like Co(II).47 Thus, the inhibition of purified HsMetAP1
by compound 1, 3, and 4 was strictly Co(II)-dependent, and in
the absence of Co(II), compound 2 could not inhibit purified
HsMetAP1 effectively. Their high activities against the Co(II)
form of HsMetAP1 in vitro mainly come from the stability of
their complexes with Co(II).
The tight regulation of cobalt ion likely offers a plausible
explanation for why inhibitors 1−4 do not work in cells.
However, it is conceivable that one of the two less tightly
bound metal ions in the active site of MetAP1 may become
available to facilitate the formation of an inhibitor−enzyme
complex. It is well-known that the first transition metal ion
binds MetAP much more tightly than the second. For example,
Co(II) binds EcMetAP with a Kd of 0.3 μM at the first site
(most likely to be the M1 site) and with a Kd of 2.5 mM at the
second site (most likely to be the M2 site).5a More than 3
orders of magnitude difference in the binding affinities raised a
possibility that auxiliary metal-dependent inhibitors might
competitively chelate a Co(II) directly from the active site of
MetAP. Although Co(II) remains the best cofactor to activate
nearly all MetAPs in vitro, it is uncertain whether Co(II) is the
physiologically relevant cofactor of MetAPs in cells. Rather than
Co(II), Fe(II) was reported as the native cofactor of
EcMetAP,49 and Zn(II) is believed to be the native cofactor
of the MetAP1 in Saccharomyces cerevisiae.52 Moreover, in
humans, the physiologically relevant cofactor of MetAP2 turned
out to be Mn(II).54 The utilization of physiologically abundant
metals other than Co(II) could also enable metal-dependent
inhibitors to form complexes with MetAP in cells. It has been
demonstrated that a quinolinyl sulfonamide derivative inhibited
EcMetAP in vivo at a sublethal concentration, and the addition
of extra 1 mM Mg(II) or 100 μM Ca(II) moderately increased
the potency of this compound to inhibit the Mn(II) form of
EcMetAP.47 However, when we activated purified HsMetAP1
with 1 μM Zn(II), and supplemented with extra Mg(II) or
Ca(II) to form a ternary complex of HsMetAP1 with an
auxiliary metal ion, none of the compounds 1−4 registered a
significant improvement in the inhibitory activities (Supporting
Information Figure S4A). Thus compounds 1−4 appear to
stringently require an auxiliary Co(II) for their tight binding to
HsMetAP1, hence function as inhibitors of HsMetAP1. This
perhaps is the reason for the lack of inhibitory activities of these
compounds against HsMetAP1 in cells.
Pyridinylquinazoline Derivatives are Novel HsMe-
tAP1-Selective Inhibitors with Relaxed Metal Selectivity.
For MetAP inhibitors to work in vivo, it is vital that they
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Table 2. Inhibition of Purified Human MetAPs by 2-(Pyridin-2-yl)Quinoline and 2-(Pyridin-2-yl)Quinazoline Derivatives
a
EC50 (μM)
compd
R
HsMetAP1 Co(II)
HsMetAP1 Mn(II)
>100
HsMetAP2 Mn(II)
7a
Cl
1.5 0.2
0.94 0.12
1.6 0.2
>100
>100
>300
>300
>100
>100
>100
>100
>100
10a
10b
10c
16
NHCH2CH2Ph
24
5
NHCH2CH2NHPh
NHCH2CH2-(3-1H-isoindole)
N-piperidinyl
>100
b
0.12 0.02
0.11 0.01
0.049 0.004
0.39 0.08
0.41 0.09
5.3 0.7 (B = 19%)
b
7.6 1.3 (B = 14%)
17
4-morpholinyl
41 33
>100
18
N-piperidinyl
19
4-morpholinyl
>100
20
4-methylpiperazin-1-yl
0.87 0.43
>100
a
b
EC50 values are shown as mean SD of at least three determinations. B: the bottom value of the inhibitory dose−response curve. It has been
noted in parentheses only when B > 10%.
effectively bind MetAPs in their physiologically relevant
metalloforms.49 For an inhibitor which mainly relies on an
auxiliary metal to inhibit MetAP, we reasoned that its ability to
use one of the physiologically abundant metal ions could be a
solution. It has been reported that the NME of recombinant
glutathione-S-transferase in E. coli was inhibited by an auxiliary
metal-mediated inhibitor.47 In addition to Co(II), this inhibitor
could also utilize Mn(II) and Fe(II) to inhibit EcMetAP
effectively in vitro.47 In retrospect, this was the first hint that
the relaxed metal selectivity of an auxiliary metal-mediated
MetAP inhibitor is the key for its activity in cells.
Because both pyridine-2-carboxamides and pyridinylpyrimi-
dines have stringent metal requirement for Co(II) and lack
cellular activity, we began to search for novel auxiliary metal-
mediated MetAP inhibitors with both good selectivity over
HsMetAP2 and relaxed metal selectivity for the inhibition of
HsMetAP1. We tested six 2-(pyridin-2-yl)quinazoline deriva-
tives (7a, 10a−10c, 16, and 17) and three 2-(pyridin-2-
yl)quinoline derivatives (18−20) that contain new structural
scaffolds (Table 2). Seven of the nine compounds displayed
submicromolar IC50 values against HsMetAP1 in the presence
of 10 μM Co(II). None of them showed appreciable inhibition
of HsMetAP2 at concentrations below 100 μM. In comparison
to compound 2, 10a contains the same C-4 side chain (shown
as the “R” group in Table 2) and 2-pyridinyl group and is about
5-fold less potent against HsMetAP1 than 2. Unlike 2, 10a
could completely inhibit HsMetAP1 in the presence of 10 μM
Mn(II). In addition, the IC50 value of 10a against HsMetAP1
with Mn(II) was 24 μM, smaller than that of 2 (30 μM; the
EC50 value of 2 was 14 μM). Thus, the 2-(pyridin-2-
yl)quinazolines displayed more relaxed metal selectivity than
the 2-(pyridin-2-yl)pyrimidines. When the C4 side chain was
extended, the resultant 10b completely lost the inhibitory
activity against HsMetAP1 in the presence of Mn(II), while its
potency against HsMetAP1 with Co(II) was retained. Changing
the phenyl group at the end of the C4 side chain of 10a to a 3-
1H-isoindole led to 7.8-fold and 4.5-fold increases in the
potency of 10c against HsMetAP1 with Co(II) and Mn(II),
respectively. Swapping the long C4 side chain of 10c for a
shorter and more rigid N-piperidinyl group afforded 16 with
similar potency. Interestingly, when a 4-morpholinyl group was
installed on the 2-(pyridin-2-yl)quinazoline core, 17 became 19
times more potent against HsMetAP1 with Co(II) compared
with 10a. But the IC50 value of 17 against HsMetAP1 with
Mn(II) was not improved. Furthermore, when the large C4
side chain was replaced by a chloro substitution, 7a completely
lost the inhibitory activity against HsMetAP1 in the absence of
Co(II). At last, in contrast to 16 and 17, all three 2-(pyridin-2-
yl)quinoline derivatives (18−20) had no effect on Mn(II)-
bound HsMetAP1 (Table 2). Therefore, the 2-(pyridin-2-
yl)quinazoline core is necessary but not sufficient for the
relaxed metal selectivity of our novel HsMetAP1-selective
inhibitors.
On the basis of the structure of 16, sixteen more 2-(pyridin-
2-yl)quinazoline derivatives were synthesized. After analyzing
the inhibitory potencies of these compounds against both
HsMetAP1 and HsMetAP2 (Table 3), three SAR trends
became evident. (1) The 4-piperazin-1-yl-2-(pyridin-2-yl)-
quinazoline core seemed to guarantee considerable selectivity
for HsMetAP1 over HsMetAP2. Except 11e and 11l, the other
14 derivatives all had submicromolar IC50 values against
HsMetAP1 with Co(II), while their IC50 values against
HsMetAP2 were above 100 μM. (2) In N4′′ side chains (R2
groups in Table 3) of 11 derivatives (11a−11j, and 11m),
various substituents at the para-position of the phenyl group
were well tolerated, thus retaining the inhibitory potency
against HsMetAP1 in the presence of Mn(II), with two
exceptions (11c and 11e). Both 11c and 11e contained a free
carboxyl group at the very end of the C4 side chains. The IC50
values of both compounds against HsMetAP1 with Mn(II)
were higher than 100 μM. Neutralization of the free carboxyl
group via the formation of an ethyl ester immediately restored
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Table 3. Inhibition of Purified Human MetAPs by 4-(Piperazin-1-yl)-2-(Pyridin-2-yl)Quinazoline Derivatives
a
b
EC50 values are shown as mean SD of at least three determinations. B: the bottom value of the inhibitory dose−response curve. It has been
noted in parentheses only when B > 10%.
the potency against HsMetAP1 with either Co(II) or Mn(II), as
seen in 11b and 11d. (3) Unlike the trend in 2-(pyridin-2-
yl)pyrimidine based MetAP1 inhibitors,45 the installation of
halogen substituents at C5′ position of 11j failed to enhance
the potency against HsMetAP1. To the contrary, compared to
11j, 11k with a chloro substituent and 11l with a bromo
substituent were actually less potent against HsMetAP1 with
either Co(II) or Mn(II).
As shown in Table 3 and Supporting Information Figure
S3D, the EC50 value of 11j (5.1 μM) for HsMetAP1 with 10
μM Mn(II) was 51-fold higher than its IC50 value (0.10 μM)
against HsMetAP1 with 10 μM Co(II). However, in the
presence of Mn(II), 10 and 100 μM 11j managed to inhibit
about 62% and 86% activity of purified HsMetAP1, respectively.
Because Zn(II) could activate HsMetAP1, we also determined
the EC50 value of 11j for the inhibition of HsMetAP1 in the
presence of 1 μM Zn(II) (Table 1 and Supporting Information
Figure S3D). In the presence of Zn(II), 11j had an EC50 value
about 1.1 μM and it inhibited about 84% activity of purified
HsMetAP1 at a concentration of 100 μM. Interestingly, the
IC50 value of 11j (0.13 μM) against HsMetAP1 with both 10
μM Mn(II) and 10 μM Co(II) was much closer to its IC50
value (0.10 μM) against HsMetAP1 with Co(II) alone than its
IC50 value (5.1 μM) against HsMetAP1 with Mn(II) alone
(Supporting Information Figure S3D). This indicates that 11j
prefers to utilize Co(II) to inhibit HsMetAP1 over Mn(II).
To investigate whether physiologically abundant divalent
metals could be utilized by 11j to inhibit HsMetAP1, 0.15 μM
purified HsMetAP1 was activated by 10 μM Mn(II). The in
vitro enzymatic activity of HsMetAP1 in the presence of 1 μM
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Figure 6. The iMet processing of 14-3-3γ in human primary cells was inhibited by (A) 11j but not (B) thiabendazole. Cells were treated with vehicle
(DMSO), bengamide G, TNP-470, 11j, 2,2′-bipyridine, or thiabendazole for 24 h. Cell lysates were resolved by SDS-PAGE, transferred to
nitrocellulose membrane, and immunoblotted (IB) with antibodies specific for β-actin, α-tubulin, total 14-3-3γ, or iMet 14-3-3γ. The IB of β-actin
and α-tubulin served as loading controls. The levels of iMet 14-3-3γ were quantified by densitometry and then normalized to total 14-3-3γ.
11j was monitored after the supplementation of extra divalent
metals to the reaction (Supporting Information Figure S4B).
Mg(II) and Ca(II) are both relatively abundant in cells and
they did not dramatically affect the activity of HsMetAP1 at the
concentrations from 0.1 nM to 1 mM in vitro (Figure 5). As
shown in Supporting Information Figure S4B, 11j’s inhibitory
potency against HsMetAP1 was not significantly enhanced by
10−1000 μM MgCl2. The highest concentration (1 mM) of
CaCl2 only increased the potency of 11j by less than 8% (p-
value = 0.036). Meanwhile, despite the fact that it would further
activate HsMetAP1, 10 μM Co(II) improved the potency of 1
μM 11j by 80% (p-value < 0.0001). Therefore, in contrast to
the previously reported quinolinyl sulfonamide based EcMetAP
inhibitor (21) whose potency was increased by both Mg(II)
and Ca(II),47 the relaxed metal selectivity of 11j was only
limited to some divalent transition metals.
A 4-Piperazin-1-yl-2-(pyridin-2-yl)quinazoline Deriva-
tive (11j) Inhibits HsMetAP1 in Cells. To determine
whether 11j could eventually inhibit the activity of HsMetAP1
inside the cells, we determined its effect on the NME of 14-3-3γ
protein by immunoblot in two primary cell lines (Figure 6). As
a positive control, a 24 h treatment with TNP-470 in HUVEC
increased the ratio of unprocessed 14-3-3γ versus total 14-3-3γ
more than 6 times. A dose-dependent inhibition of the NME of
14-3-3γ by 11j was observed (Figure 6A, left). 11j started to
inhibit MetAP activity at a concentration of 0.4 μM, and a 7.1-
fold increase in the ratio was achieved at the highest
concentration. The siRNA mediated down-regulation of
HsMetAP1 and HsMetAP2 proved that 14-3-3γ was also a
shared substrate of both HsMetAPs in normal HDFa (data not
shown). After 24 h incubation, TNP-470 and bengamide G
caused 3.4-fold and 7.0-fold increases, respectively, in the ratio
of unprocessed versus total 14-3-3γ in HDFa cells (Figure 6A,
right). Reminiscent of the observation in HUVEC, 11j dose-
dependently inhibited the NME of 14-3-3γ in HDFa cells.
Therefore, 11j brought appreciable increases in the ratio of
unprocessed versus total 14-3-3γ in both human primary cell
lines at single-digit micromolar concentrations, which are very
close to its IC50 values for the inhibition of cell proliferation as
well as its EC50 values against HsMetAP1 bound to Zn(II) or
Mn(II) in vitro (Table 1).
4-(1H-1,3-Benzodiazol-2-yl)-1,3-thiazole (thiabendazole), a
compound frequently used as a fungicide and parasiticide, has
been reported as a potent inhibitor of EcMetAP with a Ki value
of 0.4 μM.46b EcMetAP is essential for the growth of E. coli.8a
But thiabendazole was inactive against the proliferation of this
bacteria at concentrations up to 1 mM.46b As shown in Table 1,
thiabendazole inhibited 0.15 μM HsMetAP1 in the presence of
Co(II) with an EC50 value of 3.4 μM. In the absence of Co(II),
thiabendazole failed to inhibit the activity of HsMetAP1 at
concentrations up to 250 μM (Supporting Information Figure
S3E). The X-ray crystal structure of thiabendazole in complex
with EcMetAP and Co(II) (PDB code 1YVM) revealed that
thiabendazole mainly depended on an auxiliary Co(II) to
inhibit EcMetAP.46b Although thiabendazole was reported as a
vascular disrupting agent and it could disrupt the tube
formation in cultured HUVEC,57 this compound did not
show any appreciable inhibition of the proliferation of HUVEC,
as well as HDFa cells, at concentrations up to 250 μM (Table
1). If the relaxed metal selectivity is crucial for the cellular
activities of auxiliary metal-mediated MetAP inhibitors, an
auxiliary Co(II)-dependent inhibitor may fail to inhibit cellular
HsMetAPs. Indeed, 3−250 μM thiabendazole did not affect the
NME of 14-3-3γ in two human primary cell lines (Figure 6B).
2,2′-Bipyridine is a cell-permeable bidentate chelating agent
which can form complexes with many transition metals, and its
structure is closely related to the 2-(pyridin-2-yl)pyrimidine
core of compound 2−4. 2,2′-Bipyridine inhibited purified
HsMetAP1 with Co(II) as potently as 2−4, but it inhibited the
activity of HsMetAP1 in the absence of Co(II) by no more than
30% at 100 μM (Table 1 and Supporting Information Figure
S3F). As shown in Figure 6B, the ability of 2,2′-bipyridine to
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block the NME of 14-3-3γ in cells was at least 30 times lower
than 11j. Together, these results demonstrate that among
similar pharmacophores of pyridinylpyrimidine, pyridinylquino-
line, pyridinylquinazoline, benzodiazolylthiazole, and 2,2′-
bipyridine, only the 2-(pyridin-2-yl)quinazoline core offers
the relaxed metal selectivity to auxiliary metal-mediated
HsMetAP1 inhibitors.
not be a substrate of any MetAP. TXNL1 contains an N-
terminal sequence of MVGVKPV. An oligopeptide
(MVGVKPY) sharing the first six residues with TXNL1 was
preferably processed by HsMetAP2 in vitro.7 BTF3L4-3xFlag or
TXNL1-3xFlag was immunoprecipitated from the whole cell
extract using anti-Flag-conjugated resin. The unprocessed iMet
at the N-terminus of partially purified TXNL1-3xFlag was
released by incubation with both purified recombinant
HsMetAP1 and HsMetAP2 (Figure 7). The resin containing
Accumulation of N-terminal unprocessed MetAP substrates
in cells depends not only on the inhibition of cellular MetAP(s)
but also the active translation producing nascent peptides. In
theory, accumulation of iMet 14-3-3γ caused by any MetAP
inhibitor should be time-dependent. To test this hypothesis, we
determined the time-course of the iMet processing of 14-3-3γ
in HUVEC treated with 11j. Cell lysates were collected 1−7 h
after the addition of 10 μM 11j to the cell culture. With no
appreciable change in the levels of total 14-3-3γ, the level of
unprocessed 14-3-3γ increased in a time-dependent manner
(Supporting Information Figure S5A). To assess the
importance of translation in the accumulation of unprocessed
14-3-3γ, we treated HUVEC with cycloheximide prior to the
addition of MetAP inhibitors. Cycloheximide is known to
inhibit the elongation step of eukaryotic translation through
binding to a pocket in the E-site of the 60S ribosomal subunit.58
In the absence of cycloheximide, all MetAP inhibitors
(bengamide A, TNP-470, and 11j) resulted in elevated levels
of unprocessed 14-3-3γ (Supporting Information Figure S5B,
left), but in the presence of cycloheximide, none of the MetAP
inhibitors could generate a detectable signal of unprocessed 14-
3-3γ. Therefore, the accumulation of N-terminal unprocessed
14-3-3γ induced by 11j relied on active translation as expected.
To determine whether the elevated level of the iMet
unprocessed 14-3-3γ caused by 11j can be counteracted by
upregulated MetAP activity in cells, HsMetAP1 was ectopically
expressed in HUVEC via a construct transduced by lentivirus.
Lentiviruses containing either an empty pLenti6m vector
(control virus) or the coding sequence of full-length HsMetAP1
(overexpression virus) were produced in human embryonic
kidney 293T (HEK293T) cells. Concentrated lentiviruses were
used to transduce HUVEC. The transduced cells were pooled
to preclude the possible clonal differences in response to 11j.
As shown in Supporting Information Figure S6, the protein
level of HsMetAP1 was dramatically upregulated in HUVEC
infected by the overexpression virus. In HUVEC infected by the
control virus, the signals of endogenous HsMetAP1 were barely
visible, as the exposure time was greatly decreased to avoid
saturation. While there was no obvious change in the levels of
total 14-3-3γ, compared with the HUVEC transduced by the
control virus, the ectopic expression of HsMetAP1 decreased
the ratio of unprocessed 14-3-3γ versus total 14-3-3γ up to 40%
in the cells treated with 11j. Interestingly, in the absence of 11j,
HsMetAP1 overexpression reduced the background level of
unprocessed 14-3-3γ as well.
Figure 7. Both 11j and TNP-470 caused the retention of the initiator
methionine of TXNL1 in HEK293T cells. BTF3L4 and TXNL1
contain the N-terminal sequences of MNQEKLA and MVGVKPV,
respectively. HEK293T cells overexpressing C-terminally 3xFlag-
tagged BTF3L4 and TXNL1 were treated with vehicle (0.2%
DMSO), 11j (10 μM), TNP-470 (100 nM), or a combination of
11j and TNP-470, and subsequently pulsed with [35S]-Met. Then 4.5
h later, cells were lysed and the immunoprecipitated BTF3L4-3xFlag
and TXNL1-3xFlag proteins were washed and aliquoted for [35S]-Met
scintillation counting after incubation with MetAP reaction buffer
(buffer only) or after in vitro processing by purified HsMetAP1 and
HsMetAP2 (MetAP1 + 2). The percentage of [35S]-Met released from
anti-Flag-conjugated resin was calculated. For each compound
treatment, the percentage in the “buffer only” was subtracted from
the percentage in “MetAP1 + 2”, and the resulted number was
normalized to the vehicle control. Error bars are the standard deviation
of three biological independent determinations. * p = 0.03 (unpaired
two-tailed t test).
TXNL1-3xFlag purified from the TNP-470 treated cells
released 2.2 times more [35S]-Met into the solution than the
resin from the vehicle control, recapitulating earlier findings.7
Surprisingly, unlike compound 1,7 the HsMetAP1-selective 11j
increased the release of [35S]-Met as well (40 17% more than
the vehicle). Then 100 nM of a covalent inhibitor in the
fumagillin family was believed to be high enough to abolish the
enzymatic activity of cellular HsMetAP2.20 In addition, [35S]-
Met released from the immunoprecipitated TXNL1-3xFlag in
the TNP-470 and 11j cotreatment group was significantly more
abundant than the group treated only with TNP-470 (2.20-fold
versus 2.73-fold of the vehicle control, p = 0.03). As expected,
for BTF3L4-3xFlag, there was no appreciable increase in its
iMet retention after the treatment of 11j, TNP-470, or a
combination of the two (Figure 7). Together, these results
suggest that TXNL1 serves as a substrate of both HsMetAP1
and HsMetAP2, similar to 14-3-3γ.
Human thioredoxin-like protein 1 (TXNL1) was previously
shown to be a specific substrate of HsMetAP2 in HEK293T
cells.7 However, this conclusion was based on the unrepeatable
finding of HsMetAP1 inhibition by compound 1 in human
cells.21 To reinvestigate the role of HsMetAP1 in the removal of
the iMet of TXNL1, HEK293T cells expressing C-terminally
Flag-tagged BTF3L4 or TXNL1 were treated with vehicle
(0.2% DMSO), 10 μM 11j, 100 nM TNP-470, or the
combination of the latter two, and half an hour later, newly
synthesized proteins were labeled by [35S]-Met. The BTF3L4
protein has MNQEKLA at its N-terminus. Therefore, it should
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The X-ray Crystal Structure of N-Terminally Truncated
HsMetAP1 in Complex with 11j. The crystal structure of N-
terminally truncated (Δ1−80) human MetAP1 (tHsMetAP1)
in complex with Co(II) and 11j was determined at a resolution
of 1.90 Å (Table 4). The structure revealed the presence of
Table 4. X-ray Data Collection and Refinement Statistics of
the N-terminal Truncated (Δ1−80) Human MetAP1
(tHsMetAP1) in Complex with 11j
tHsMetAP1 with Co(II) and 11j (PDB code 4IU6)
space group
P21
cell dimensions
a (Å)
47.3
77.3
47.8
91.5
b (Å)
c (Å)
β (deg)
X-ray data collection statistics
X-ray source
FR-E+/Raxis IV
1.54178
wavelength (Å)
resolution range (Å) (HighRes shell)
collected reflections
unique reflections
I/σ
50.00−1.90 (1.97−1.90)
74840
Figure 8. The X-ray crystal structure of N-terminally truncated human
MetAP1 in complex with 11j. (A) and (C) tHsMetAP1 in complex
with 11j, PDB code 4IU6 (orange). (B) and (D) A superimposition of
4IU6 to the crystal structures of tHsMetAP1 with 2 (purple) and 3
(cyan) (PDB codes 2G6P44 and 4HXX45). A HEPES molecule
(carbon purple) in 2G6P was from the buffer. The stereo views of the
active site of tHsMetAP1 in (A) and (B) show that all three
compounds (sticks) mainly depend on an auxiliary Co(II) (Co3, green
sphere) to bind the H212 residue (sticks) of HsMetAP1. Water
molecules are shown as red spheres, and metal interactions are shown
as dashed lines. The surface-filling views of tHsMetAP1 (surface
colored gray) in (C) and (D) show the cavity in the active site which
accommodates 11j, 2, or 3. The C4 side chains of the three
compounds interact with different parts of the enzyme. For all
noncarbon atoms, nitrogen is blue, oxygen is red, sulfur is yellow, and
chlorine is green.
24981
28.8 (3.3)
completeness (%)
Rmerge (%)
91.6 (59.7)
5.5 (27.6)
refinement statistics
Rcryst (%)
0.17 (0.25)
0.22 (0.33)
Rfree (%)
rmsd
bond length (Å)
angle (deg)
0.016
1.432
1
monomer in ASU
total atoms
2731
2411
276
protein atoms
water molecules
ligand
34
B-factor (ΔHsMetAP1) (Å2)
B-factor (11j) (Å2)
B-factor (H2O) (Å2)
29.1
33.0
39.7
side chain of 11j is projected out toward the opening of the
active site cavity and the α-helix 125−133. In the C4 side chain
of 11j, the 4-methoxyphenyl group forms a stacking interaction
with the side chain of Tyr196, and the terminal methoxy group
may participate in an indirect ionic interaction with Gln129
through a water molecule.
three Co(II) in the active site: Co1 and Co2 found in all
members of the MetAP1 family and Co3 coordinating a
quinazoline nitrogen (N1) and the pyridine nitrogen (N1′) of
11j (Figure 8A). The octahedral coordination of Co3 is
completed by His212 and three water molecules. Similar
coordination has been described for the structures of
tHsMetAP1 in complex with 2 (PDB code 2G6P) or 3 (PDB
code 4HXX) in the presence of Co(II) (Figure 8B).44,45 The 2-
pyridinyl group of 11j occupies a hydrophobic pocket formed
by Pro192, Tyr195, Phe198, Cys203, Phe309, and Trp353
(Figure 8C). This pocket is the binding site for the side chain of
the iMet residue when a substrate polypeptide is being
processed by MetAP, and it was occupied by an acetate ion
in the structure of holoenzyme of tHsMetAP1 (PDB code
2B3K).11 The relative positions of the 2-pyridinyl group in the
methionine side chain binding pocket are similar for 2 and 11j
(Figure 8B). The distances from C5′ of the pyridine ring to C1
and C4 of the phenyl group in Phe198 are 3.87 and 4.64 Å,
respectively, in 2G6P; and the distances are 4.02 and 5.70 Å,
respectively, in 4IU6. The quinazoline part of 11j displays a
tight stacking with the imidazole ring of His310. The pyridine
ring and the quinazoline ring of 11j are nearly coplanar. The C4
From a structural perspective, there seems to be two critical
elements contributing to the interactions between 11j and
tHsMetAP1. The first element is the 2-(pyridin-2-yl)-
quinazoline part of 11j, which despite its simplicity is able to
(1) perfectly coordinate the third metal, (2) occupy the
hydrophobic binding cavity for the substrate, and (3) stack with
the conserved His310 at the same time (Figure 8A). The
second element is the 4-methoxyphenyl group in the C4 side
chain of 11j , which stacks with the side chain of Tyr196. The
adequate spacing between the pyridinylquinazoline core and
the 4-methoxyphenyl group is achieved by using a piperazine
ring as a linker. Piperazine provides enough flexibility without
an onerous increase in degrees of freedom. Various additions to
the C4 side chain of 11j beyond the phenyl group failed to
significantly improve the inhibitory potency against purified
HsMetAP1, as evidenced by 11a−11i and 11m (Table 3). This
may be partially due to the fact that the 4-methoxyphenyl group
of 11j is very close to the surface of tHsMetAP1, because at the
end of the C4 side chain which points toward the aqueous
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solution, it would be quite difficult to introduce any new
interaction between the inhibitor and the enzyme. To further
increase the interactions between an inhibitor and HsMetAP1,
one option is to simultaneously increase the length and the
flexibility of the C4 side chain but at the expense of losing the
stacking interaction with Tyr196. The chloro group at C5′
position of 3 improved its potency against HsMetAP1 with
Co(II).45 If 11k and 11l adopt the same binding mode as 3
binding to HsMetAP1 with Co(II) (Figure 8B), in order to
accommodate the C5′ halogen, the 2-(pyridin-2-yl)quinazoline
moiety of 11k and 11l may need to move up to 1 Å out of the
hydrophobic pocket for methionine side chain. Unlike the
flexible C4 side chain of 3, the C4 side chains of both 11k and
11l contain a relatively rigid piperazine ring and 4-
methoxyphenyl group. Therefore, the stacking interaction of
the 4-methoxyphenyl group to the side chain of Tyr196 and the
potential indirect ionic interaction between the terminal
methoxy group and Gln129 are very likely to be disrupted
because of the halogen substituents in 11k and 11l. This may
help to explain why the introduction of chloro or bromo groups
at C5′ position decreased the activities of 11k and 11l against
HsMetAP1 with Co(II) or Mn(II).
(Supporting Information Table S1 and Figure S7). It was
speculated that the small angle (76.0°) formed from the two
coordinating nitrogen atoms of a quinolinyl sulfonamide based
EcMetAP inhibitor to the auxiliary Mn(II) and the long
distances (2.32, 2.22 Å) from the two nitrogen atoms of the
inhibitor to the auxiliary Mn(II) might provide the structural
basis to the relaxed metalloform selectivity of that inhibitor.47
However, we do not think that the coordinating geometry of an
auxiliary metal is a reliable indicator for the relaxed metal
selectivity of a MetAP inhibitor.
CONCLUSION
■
In this work, we demonstrated that 14-3-3γ is a shared substrate
between MetAP1 and MetAP2 in HUVEC. The complete
excision of the iMet in 14-3-3γ relies on the activities of both
HsMetAP1 and HsMetAP2. Using the retention of the iMet in
14-3-3γ as an indicator of cellular MetAP inhibition, we found
that one pyridine-2-carboxamide derivative (1) and two
pyridinylpyrimidines (3−4) do not inhibit any HsMetAP in
cells and the pyridinylpyrimidine derivative 2 cannot potently
decrease the cellular activities of HsMetAPs. Purified
recombinant HsMetAP1 can be reliably activated by not only
Co(II) but also Mn(II) and Zn(II). In the absence of Co(II),
compounds 1−4, thiabendazole, and 2,2′-bipyridine no longer
can inhibit purified HsMetAP1 potently in vitro. In an effort to
seek HsMetAP1-selective inhibitors with relaxed metal
selectivity, we succeeded in finding a novel class of inhibitors
containing a 2-(pyridin-2-yl)quinazoline. Many compounds in
this class can potently and selectively inhibit HsMetAP1 with
relaxed metalloform selectivity. Among them, a 4-piperazin-1-
yl-2-(pyridin-2-yl)quinazoline derivative 11j can utilize at least
Co(II), Zn(II), and Mn(II) to potently inhibit HsMetAP1 in
vitro, without affecting HsMetAP2. In addition, 11j can dose-,
time-, and translation-dependently induce the accumulation of
the N-terminal unprocessed form of 14-3-3γ in human primary
cells, and this accumulation can be partially reversed by ectopic
expression of HsMetAP1. Lastly, crystallographic analysis
confirmed that 11j is an auxiliary metal-mediated MetAP
inhibitor. 11j may serve as a useful molecular probe to facilitate
the understanding of the physiological functions of HsMetAP1.
The crystal structure of tHsMetAP1 in complex with 11j was
superimposed on the crystal structure of HsMetAP2 in complex
with L-methionine (PDB code 1KQ959) (Figure 9). Given the
Figure 9. A superimposition of the crystal structures of N-terminally
truncated human MetAP1 in complex with 11j and human MetAP2
with L-methionine. HsMetAP1 is shown as gray surface, and 11j is
shown as orange sticks. The PDB code of HsMetAP2 structure is
1KQ9.60 The backbone of HsMetAP2 is shown as teal wires and
ribbons, and the residues of HsMetAP2 are shown as teal sticks. The
numbering of the residues is based on HsMetAP2. The insertion
(382−444) in the catalytic domain of HsMetAP2 generates a tight
entrance to the active site. In this superimposition, the Y444 of
HsMetAP2 clashed with 11j. D442 and H339 on the other side of 11j
narrow the binding site as well. The L-methionine and the surface
filling model of HsMetAP2 are not shown for the sake of clarity. For all
noncarbon atoms, nitrogen is blue and oxygen is red.
EXPERIMENTAL SECTION
■
General. Unless stated otherwise, all nonaqueous reactions were
carried out under ambient atmosphere in oven-dried glassware.
Indicated reaction temperatures refer to those of the reaction bath,
while room temperature (rt) is noted as 25 °C. All the solvents were of
reagent grade purchased from Fisher Scientific or VWR, and they were
used as received. Commercially available starting materials and
reagents were purchased from Acros, Aldrich, or TCI America and
were used as received.
Analytical thin layer chromatography (TLC) was performed on
Analtech Uniplates (silica gel HLF, W/UV254, 250 μm). Visualization
was achieved using 254 nm UV light or additionally by staining with
iodine or ceric ammonium molybdate stain. Crude products were
purified by air-flashed column chromatography over silica gel (0.06−
0.2 mm, 60 Å, from Acros) using indicated eluents. Melting points
were recorded on a Mel-Temp II apparatus and are uncorrected. NMR
data were collected on a Varian Unity-400 (400 MHz 1H, 100.6 MHz
13C), Inova-500 (500 MHz 1H, 125 MHz 13C), or Bruker-Spectrospin
400 MHz spectrometers. Chemical shifts are reported in ppm (δ) with
the solvent resonance or 0.1% tetramethylsilane contained in the
deuterated solvent as the internal reference. Data are reported as
follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet,
q = quartet, dd = doublet of doublet, m = multiplet, br = broad, app =
apparent, exch = exchangeable), coupling constants (J, reported in
present conformation, it is obvious that the piperazine ring in
the C4 side chain of 11j suffers a severe clash with the side
chain of Tyr444 if 11j were to bind the active site of
HsMetAP2. This steric clash may help to explain why 4-
piperazin-1-yl-2-(pyridin-2-yl)quinazoline derivatives (11a−
11m and 13−15) have great selectivity for HsMetAP1 over
HsMetAP2.
With four crystal structures of Co(II)-bound HsMetAP1 in
complex with auxiliary metal-dependent inhibitors in hand, we
analyzed the angle formed from the two coordinating atoms of
an inhibitor to the auxiliary metal ion and the distances from
the two coordinating atoms to the auxiliary metal ion
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Hertz, Hz), and number of protons. Low resolution mass spectra were
acquired on a Thermo-Finnigan MAT, LCQ Classic ESI-mass
spectrometer or Voyager DE-STR, MALDI-TOF (Applied Biosys-
tems) instruments. The MALDI samples were prepared by mixing the
droplets of the sample solutions in chloroform or methanol and 2,5-
dihydroxybenzoic acid solution in acetone, where the latter served as
the matrix. Data are reported in the form m/z (molecular ion).
If needed, products were purified using a JASCO HPLC fitted with
a semipreparative VyDAC C18 column (7 μm, 10 mm × 250 mm). A
mixture of water, acetonitrile, and 2-propanol was used as a mobile
phase while eluting at a flow rate of 3 mL/min. Samples were analyzed
for purity on a JASCO HPLC equipped with a Phenomenex Luna C18
column (3 μm, 4.6 mm × 150 mm). The mobile phase set at a flow
rate of 1.0 mL/min was programmed for a gradient elution starting
from a 90:5:5 mixture of H2OMeCNMeOH to 50:45:5 over 6
min. Thereafter, the gradient was ramped up to 10:80:10 H2O
MeCNMeOH over 5 min, maintained with the same gradient for 4
more min, and was reverted back to the starting gradient of 90:5:5
H2OMeCNMeOH, making the whole run 16 min long.
Detection was achieved using JASCO MD-2010 photodiode array
detector while monitoring the absorption at 254, 275, and 296 nm
wavelengths. Purity of the final compounds was determined to be
>95%, using a 10 μL injection (approximately 1 mM in acetonitrile)
with quantitation by area under the curve (AUC) at 254, 275, and 296
nm (JASCO diode array detector). The retention time (tR) is reported
in minutes with the AUC given in parentheses.
150.46, 137.21, 135.14, 129.66, 129.19, 125.85, 125.22, 124.43, 123.09.
MALDI-TOF: m/z 243 (M + H)+, 265 (M + Na)+.
N-Phenethyl-2-(pyridin-2-yl)quinazolin-4-amine (10a).63
1
Yield: 72%. tR: 8.440 min (98.6%). H NMR (400 MHz, CDCl3): δ
8.81 (ddd, J = 8.4, 1.6, and 0.8 Hz, 1H), 8.06 (d, J = 8.0 Hz, 1H), 8.63
(d, J = 8.4 Hz, 1H), 7.84 (ddd, J = 8.4, 7.6, and 1.6 Hz, 1H), 7.73 (d, J
= 8.4 Hz, 1H), 7.34 (m, 2H), 7.66 (t, J = 7.6 Hz, 1H), 7.29 (m, 2H),
7.23 (m, 2H), 6.47 (br t, 1H), 3.99 (dt, J = 12.4, 6.8 Hz, 2H), 3.02 (t, J
= 7.6 Hz, 2H). 13C NMR (125 MHz, CD3OD): δ 161.85, 160.65,
157.00, 150.78, 150.28, 141.02, 138.55, 134.11, 130.04, 129.57, 128.77,
127.51, 127.37, 126.13, 125.25, 123.33, 115.71, 44.06, 36.43. MALDI-
TOF: m/z 327 (M + H)+, 349 (M + Na)+.
N1-Phenyl-N2-[2-(pyridin-2-yl)quinazolin-4-yl]ethane-1,2-di-
amine (10b). Yield: 82%. tR: 8.840 min (95.1%). 1H NMR (400
MHz, acetone-d6): δ 8.83 (m, 1H), 8.6 (app d, J = 8.4 Hz, 1H), 8.37
(m, 1H), 7.93 (m, 3H), 7.51 (m, 2H), 7.13 (m, 2H), 6.73 (m, 2H),
6.55 (t, J = 7.6 Hz, 1H), 4.13 (br s, 2H), 3.71 (m, 4H). 13C NMR (125
MHz, CD3CN): δ 164.32, 156.88, 155.02, 150.45, 149.31, 148.43,
137.26, 132.93, 128.25, 122.58, 121.29, 120.89, 118.91, 112.58, 48.77,
47.83. MALDI-TOF: m/z 342 (M + H)+, 364 (M + Na)+.
N-[2-(1H-indol-3-yl)ethyl]-2-(pyridin-2-yl)quinazolin-4-
amine (10c). Yield: 88%. tR: 8.520 min (96.9%). 1H NMR (500 MHz,
methanol-d4): δ 8.63 (d, J = 4.3 Hz, 1H), 8.23 (d, J = 7.8 Hz, 1H), 8.00
(d, J = 8.4 Hz, 1H), 7.85−7.78 (m, 2H), 7.69 (td, J = 7.7 and 0.9 Hz,
1H), 7.56 (dt, J = 8.0 and 0.9 Hz, 1H), 7.45−7.38 (m, 2H), 7.22 (dt, J
= 8.2 and 0.9 Hz, 1H), 7.02−6.98 (m, 2H), 6.88 (td, J = 6.7 and 0.9,
1H), 3.99 (t, J = 7.2 Hz, 2H), 3.27 (m, 2H), 3.13 (t, J = 7.4 Hz, 2H).
13C NMR (125 MHz, CD3OD): δ 162.77, 160.47, 155.97, 151.04,
Commercially available 2-(pyridin-2-yl)quinazolines 16 (CAS no.
57768−72−0) and 17 (CAS no. 836652−57−4) were purchased from
́
Ambinter (Orleans, France), and 2-(pyridin-2-yl)quinoline 18 (CAS
149.11, 139.43, 138.98, 135.44, 129.70, 128.65, 127.78, 127.40, 126.08,
124.43, 123.19, 120.42, 120.30, 116.07, 114.29, 113.13, 44.38, 27.04.
MALDI-TOF: m/z 366 (M + H)+, 388 (M + Na)+.
no. 633199−21−5), 19 (CAS no. 133698−98−3), and 20 (CAS no.
156094−96−1) were purchased from Maybridge (Cambridge, United
Kingdom), Sigma-Aldrich (St. Louis, MO), and Ambinter, respec-
tively. The masses of the five compounds were confirmed by ESI-MS
(Agilent Technologies 6120 Quadrupole LC/MS system). Alter-
natively, quinazolines 16 and 17 may also be synthesized using the
general procedure described below and the syntheses of commercially
available quinolines 18, 19, and 20 have also been described in the
literature.60
1-(4-{4-[2-(Pyridin-2-yl)quinazolin-4-yl]piperazin-1-yl}-
phenyl)ethanol (11a). This was obtained by reducing the
corresponding ketone using sodium borohydride in methanol. Yield:
93%. tR: 7.373 min (97.6%). 1H NMR (400 MHz, CDCl3): δ 8.87 (d, J
= 4 Hz, 1H), 8.60 (dt, J = 6.0 and 2.8 HZ, 1H), 8.21 (d, J = 8.4 Hz,
1H), 7.97 (m, 2H), 7.82 (m, 2H), 7.49 (m, 2H), 7.40 (m, 1H), 7.00
(m, 2H), 4.86 (q, J = 6.4 Hz, 1H), 4.04 (m, 4H), 3.44 (m, 4H), 1.49
(d, J = 6.0 Hz, 3H). 13C NMR (100 MHz, CDCl3): δ 157.40, 151.42,
143.87, 137.72, 134.78, 126.69, 124.09, 116.40, 85.84, 50.01, 49.40,
17.33. MALDI-TOF: m/z 412 (M + H)+, 434 (M + Na)+.
Bengamides A, B, and G were provided by Dr. Philip Crews (UC
Santa Cruz). Fumagillin, thiabendazole, 2,2′-bipyridine, 2-(pyridin-2-
yl)pyrimidines 2 and 4 were acquired from commercial sources. TNP-
470 was prepared starting from the commercially available fumagillin
analogous to the procedure described by Arico-Muendel et al.61 The
synthesis of pyridinylpyrimidine 3 has been communicated recently.45
Synthesis of t-Butyl 2-(thiazol-2-ylcarbamoyl)pyridin-3-yl-
carbamate (1). The synthesis of compound 1 followed the previously
described procedure.21 The characterization of the final product
matched with the literature report.21
Ethyl 4-{4-[2-(Pyridin-2-yl)quinazolin-4-yl]piperazin-1-yl}-
1
benzoate (11b). Yield: 78%. tR: 8.000 min (96.1%). H NMR (400
MHz, CDCl3): δ 8.25 (d, J = 1.6 Hz, 1H), 7.96 (d, J = 8.4 Hz, 1H),
7.56 (d, J = 8.4 Hz, 1H), 7.29 (m, 4H), 6.82 (m, 3H), 6.23 (m, 2H),
4.17 (q, J = 4.6 Hz, 2H), 3.70 (m, 4H), 3.57 (m, 4H), 0.93 (t, J = 4.5
Hz, 3H). 13C NMR (100 MHz, CDCl3): δ 166.86, 155.47, 152.02,
150.37, 148.88, 136.98, 133.76, 131.28, 129.03, 127.48, 124.21, 123.59,
122.13, 113.78, 60.52, 49.37, 47.81, 14.60. MALDI-TOF: m/z 440 (M
+ H)+.
4-{4-[2-(Pyridin-2-yl)quinazolin-4-yl]piperazin-1-yl}benzoic
Acid (11c). This benzoic acid was obtained after performing a
saponification reaction on the ethyl ester. Yield: 89%. tR: 10.013 min
(96.3%). 1H NMR (400 MHz, CDCl3): δ 11.02 (br s, 1H), 8.77 (d, J =
1.6 Hz, 1H), 8.59 (dd, J = 7.6 and 1.5 Hz, 1H), 8.27 (dd, J = 7.5 and
1.5 Hz, 1H), 8.11 (m, 2H), 7.85 (m, 4H), 7.67 (m, 1H), 7.59 (dt, J =
7.8 and 1.2 Hz, 2H), 3.79 (m, 4H), 3.65 (m, 4H). 13C NMR (125
MHz, CD3OD): δ 171.42, 165.85, 164.56, 151.96, 151.04, 150.69,
139.76, 136.85, 135.94, 133.60, 129.36, 128.48, 128.18, 124.14, 123.94,
122.36, 108.39, 52.88, 50.48. MALDI-TOF: m/z 411 (M)+, 434 (M +
Na)+.
General Procedure for the Synthesis of 2-(Pyridin-2-
yl)quinazolin-4-amine Derivatives. According to the procedure
described by Flanagan et al.62 suitably substituted cyanopyridine was
treated with sodium metal in methanol and refluxed in methanol with
anthranilic after an hour of stirring at room temperature.
Quinazolinone 6 was collected by filtering and cooled reaction
mixture, and a second crop was obtained after letting the mother
liquor stand overnight. Quinazolinone 6 was pure by NMR and was
directly converted to the 4-chloroquinazoline (7) by refluxing with
diethylaniline and phosphorus oxychloride in benzene for 4 h.
Diethylaniline was distilled off under high vacuum and the crude
product was washed with saturated NaHCO3 and the concentrated
reaction mixture was purified by column chromatography over silica
gel (eluent: hexanes and then neat DCM). The chloro group in 7 was
replaced with various amines by heating at 110 °C overnight.
Substituted quinazolines 8 were purified by column chromatography
over silica gel.
Ethyl 2-(4-{4-[2-(Pyridin-2-yl)quinazolin-4-yl]piperazin-1-yl}-
phenoxy)acetate (11d). The amine used in the final step was ethyl
2-[4-(piperazin-1-yl)phenoxy]acetate. Yield: 72%. tR: 8.560 min
1
4-Chloro-2-(pyridin-2-yl)quinazoline (7a).62 Yield: 69% (over
two steps). 1H NMR (400 MHz, CDCl3): δ 8.91 (d, J = 4.5 Hz, 1H),
8.63 (d, J = 7.5 Hz, 1H), 8.31 (m, 2H), 7.99 (dt, J = 8.5, 1.6 Hz, 1H),
7.89 (dt, J = 8.1, 1.7 Hz, 1H), 7.74 (t, J = 8.1 Hz, 1H), 7.41 (m, 1H).
13C NMR (100 MHz, CDCl3): δ 163.33, 158.91, 154.01, 151.82,
(95.5%). H NMR (400 MHz, CDCl3): δ 8.64 (d, J = 2.6 Hz, 1H),
8.57 (t, J = 8.0 Hz, 1H), 8.33 (d, J = 7.6 Hz, 1H), 7.89 (m, 4H), 7.49
(m, 1H), 6.95 (m, 4H), 4.58 (s, 2H), 4.25 (q, J = 7.2 Hz, 2H), 4.01
(m, 4H), 3.44 (m, 4H), 1.29 (t, J = 6.8 Hz, 3H). 13C NMR (400 MHz,
CDCl3): δ 161.59, 153.03, 150.28, 148.89, 148.53, 137.70, 134.76,
4010
dx.doi.org/10.1021/jm400227z | J. Med. Chem. 2013, 56, 3996−4016

Journal of Medicinal Chemistry
Article
128.19, 127.47, 126.92, 126.43, 122.15, 118.46, 115.60, 66.26, 61.50,
50.60, 50.03, 14.35. MALDI-TOF: m/z 470 (M + H)+, 508 (M + K)+.
2-(4-{4-[2-(Pyridin-2-yl)quinazolin-4-yl]piperazin-1-yl}-
phenoxy)acetic Acid (11e). This was prepared by alkylation of
SB11882B with ethyl bromoacetate followed by alkaline hydrolysis.
8.8 Hz, 1H), 8.18 (d, J = 8.8 Hz, 1H), 8.01 (d, J = 2.8 Hz, 1H), 7.99
(m, 1H), 7.86 (app t, J = 7.2 Hz, 1H), 7.60 (app t, J = 7.2 Hz, 1H),
7.03 (app d, J = 9.2 Hz, 2H), 6.88 (app d, J = 9.2 Hz, 2H), 4.06 (m,
4H), 3.75 (s, 3H), 3.64 (m, 4H). 13C NMR (125 MHz, CD3OD): δ
166.43, 158.51, 156.12, 149.38, 146.93, 138.39, 134.50, 129.72, 127.53,
126.70, 126.32, 120.11, 115.65, 56.10, 52.34, 51.07. MALDI-TOF: m/z
433 (M + H)+, 455 (M + Na)+, 494 (M + K)+.
2-(5-Bromopyridin-2-yl)-4-[4-(4-methoxyphenyl)piperazin-
1-yl]quinazoline (11l). This was synthesized starting from 4-bromo-
2-cyanopyridine. Yield: 53% (over 3 steps). tR: 9.147 min (98.6%). 1H
NMR (400 MHz, acetone-d6): δ 8.84 (d, J = 2.4 Hz, 1H), 8.55 (d, J =
8.8 Hz, 1H), 8.16 (m, 2H), 7.99 (dd, J = 7.2 and 2.3 Hz, 1H), 7.86 (dt,
J = 8.8 and 2.4 Hz, 1H), 7.60 (dt, J = 8.8 and 2.4 Hz, 1H), 7.02 (dd, J
= 7.2 and 2.4 Hz, 2H), 6.88 (dd, J = 6.8 and 2.0 Hz, 2H), 4.06 (m,
2H), 3.75 (s, 3H), 3.36 (m, 4H). 13C NMR (125 MHz, CD3OD): δ
166.72, 157.12, 151.38, 145.91, 136.31, 134.03, 129.67, 127.11, 126.51,
126.02, 119.66, 115.19, 62.64, 51.91, 50.62. MALDI-TOF: m/z 476
(M + H)+, 500 (M + Na)+.
1
Yield: 66% (over two steps). tR: 10.027 min (98.2%). H NMR (400
MHz, CDCl3): δ 11.05 (br s, 1H), 8.67 (d, J = 4.8 Hz, 1H), 8.58 (app
d, J = 8.0 Hz, 1H), 8.36 (app d, J = 8.4 Hz, 1H), 7.90 (m, 3H), 7.80
(m, 4H), 7.04 (m, 2H), 6.85 (m, 2H), 4.62 (s, 2H), 3.66 (m, 4H), 3.10
(m, 4H). 13C NMR (100 MHz, CDCl3): δ 171.80, 154.85, 149.00,
148.71, 137.75, 135.42, 134.89, 128.24, 127.58, 127.00, 126.49, 123.20,
122.57, 120.92, 119.89, 115.80, 65.77, 51.91, 49.11. MALDI-TOF: m/z
441 (M+).
4-{4-[2-(Pyridin-2-yl)quinazolin-4-yl]piperazin-1-yl}phenol
(11f). In the final step, displacement reaction was performed using 1-
{4-[(tert-butyldimethylsilyl)oxy]phenyl}piperazine and the silyl group
was deprotected using 1 M tetrabutylammonium flouoride in THF.
1
Yield: 76% (over two steps). tR: 6.720 min (96.5%). H NMR (400
MHz, CDCl3): δ 8.79 (s, 1H), 8.58 (app d, J = 6.8 Hz, 1H), 8.10 (app
d, J = 7.2 Hz, 1H), 7.90 (m, 1H), 7.87 (m, 1H), 7.73 (m, 1H), 7.41
(m, 2H), 6.79 (m, 4H), 4.02 (m, 4H), 3.18 (m, 4H). 13C NMR (100
MHz, CDCl3): δ 165.06, 158.17, 155.50, 152.63, 151.78, 149.69,
144.60, 137.33, 132.94, 129.59, 125.94, 125.03, 124.85, 124.26, 119.11,
116.34, 115.76, 51.33, 49.92. MALDI-TOF: m/z 385 (M + H)+, 407
(M + Na)+.
4-[4-(3,4-Dichlorophenyl)piperazin-1-yl]-2-(pyridin-2-yl)-
1
quinazoline (11m). Yield: 91%. tR: 9.987 min (99.0%). H NMR
(500 MHz, CDCl3): δ 3.32 (m, 4H), 3.91 (m, 4H), 6.68 (dd, J = 9, 2.9
Hz, 1H), 6.92 (d, J = 2.84 Hz, 1H), 7.21 (d, J = 8.3 Hz, 1H), 7.30
(ddd, J = 7.3, 4.7, and 1.2 Hz, 1H), 7.41 (td, J = 8.6 and 1.2 Hz, 1H),
7.70 (td, J = 8.4 and 1.4 Hz, 1H), 7.78 (ddd, J = 8.27, 7.7, and 1.8 Hz,
1H), 7.86 (dd, J = 8.4, and 0.9 Hz), 8.16 (d, J = 8.5 Hz, 1H), 8.51 (pair
of t’s, J = 1.0 Hz, 1H). 8.82 (dq, J = 4.7 and 0.8 Hz. 13C NMR (125
MHz, CDCl3): δ 165.01, 158.41, 155.77, 152.94, 150.45, 136.84,
132.95, 132.85, 130.65, 130.17, 126.04, 124.63, 124.03, 122.59, 117.34,
115.45, 49.48, 48.56. MALDI-TOF: m/z 437 (M + H)+.
4-[4-(4-Nitrophenyl)piperazin-1-yl]-2-(pyridin-2-yl)-
1
quinazoline (11g). Yield: 81%. tR: 8.400 min (99.1%). H NMR
(400 MHz, CDCl3): δ 8.87 (d, J = 4.4 Hz, 1H), 8.57 (t, J = 10.4 Hz,
1H), 8.19 (m, 2H), 7.97 (t, J = 10.3 Hz, 1H), 7.81 (m, 2H), 7.51 (m,
1H), 7.38 (m, 1H), 7.27 (m, 1H), 6.85 (m, 2H), 4.08 (m, 4H), 3.70
(m, 4H). 13C NMR (100 MHz, CDCl3): δ 164.91, 158.48, 155.76,
154.68, 153.06, 150.35, 138.97, 136.97, 133.05, 130.35, 126.20, 124.70,
124.61, 124.06, 115.83, 112.72, 49.12, 46.78. MALDI-TOF: m/z 413
(M + H)+, 435 (M + Na)+.
4-[4-(4-Methylbenzenesulfonyl)piperazin-1-yl]-2-(pyridin-2-
1
yl)quinazoline (13). Yield: 77%. tR: 8.547 min (98.4%). H NMR
(400 MHz, CDCl3): δ 8.83 (m, 1H), 8.55 (d, J = 8.2 Hz, 1H), 8.21 (d,
J = 8.2 Hz, 1H), 7.84 (m, 1H), 7.63 (m, 4H), 7.29 (m, 4H), 3.97 (m,
4H), 3.21 (m, 4H), 2.41 (s, 3H). 13C NMR (125 MHz, CD3CN): δ
166.36, 159.74, 157.16, 153.85, 150.94, 145.75, 145.55, 138.17, 134.44,
133.98, 133.84, 131.28, 129.26, 127.49, 126.55, 125.26, 116.97, 51.40,
50.37, 47.61, 47.34, 21.99. MALDI-TOF: m/z 446 (M + H)+, 468 (M
+ Na)+.
1-(4-{4-[2-(Pyridin-2-yl)quinazolin-4-yl]piperazin-1-yl}-
1
phenyl)methanone (11h). Yield: 81%. tR: 7.773 min (98.3%). H
NMR (400 MHz, CDCl3): δ 8.87 (d, J = 4.0 Hz, 1H), 8.58 (d, J = 7.6
Hz, 1H), 8.21 (d, J = 8.4 Hz, 1H), 7.96 (d, J = 8.4 Hz, 1H), 7.91 (app
d, J = 9.2 Hz, 2H), 7.83 (app t, J = 8.0 Hz, 1H), 7.76 (app t, J = 8.2 Hz,
1H), 7.50 (app t, J = 8.0 Hz, 1H), 7.37 (m, 1H), 6.91 (app d, J = 8.8
Hz, 2H), 4.05 (m, 4H), 3.63 (m, 4H0, 2.53 (s, 3H). 13C NMR (100
MHz, CDCl3): δ 196.76, 165.05, 158.48, 155.80, 153.97, 153.03,
150.31, 136.93, 132.94, 130.64, 130.27, 128.16, 126.10, 124.64, 124.60,
124.06, 115.89, 113.54, 49.40, 47.23, 26.38. MALDI-TOF: m/z 410
(M + H)+.
N-[(2S)-1-Oxo-3-phenyl-1-{4-[2-(pyridin-2-yl)quinazolin-4-
yl]piperazin-1-yl}propan-2-yl)acetamide (14). This was prepared
by coupling Ac-Phe-OH to 4-(piperazin-1-yl)-2-(pyridin-2-yl)-
quinazoline using HBTU and DIEA. Yield: 67%. tR: 7.080 min
1
(98.8%). H NMR (400 MHz, CDCl3): δ 8.89 (d, J = 4.0 Hz, 1H),
8.79 (d, J = 8.0 Hz, 1H), 8.40 (d, J = 8.4 Hz, 1H), 8.36 (d, J = 8.4 Hz,
1H), 8.25 (t, J = 6.0 Hz, 1H), 8.14 (t, J = 7.2 Hz, 1H), 784 (m, 2H),
7.50 (d, J = 7.6 Hz, 1H), 7.28 (m, 5H), 7.21 (m, 1H), 5.11 (dt, J = 8.0
and 6.8 Hz, 1H), 3.90 (m, 4H), 3.70 (m, 4H), 2.93 (d, J = 6.8 Hz,
2H), 1.91 (s, 3H). 13C NMR (125 MHz, CD3CN): δ 171.83, 170.74,
164.59, 153.90, 151.04, 148.20, 140.30, 138.62, 137.75, 130.97, 129.84,
128.96, 128.23, 127.18, 125.38, 113.84, 51.65, 45.29, 42.19, 39.67,
23.25. MALDI-TOF: m/z 481 (M + H)+, 503 (M + Na)+.
4-{4-[4-(Allyloxy)phenyl]piperazin-1-yl}-2-(pyridin-2-yl)-
quinazoline (11i). This was prepared by displacement of the chloro
group on 7a using a known 4-allyloxyphenylpiperazin, which in turn
was prepared in two steps starting from 4-hydroxypiperazine. Yield:
1
65% (over 3 steps). tR: 9.987 min (96.4%). H NMR (400 MHz,
CDCl3): δ 8.65 (d, J = 1.2 Hz, 1H), 8.61 (d, J = 3.6 Hz, 1H), 8.36 (d, J
= 3.5 Hz, 1H), 7.93 (m, 2H), 7.81 (m, 2H), 7.53 (m, 3H), 6.84 (m,
2H), 5.93 (m, 1H), 5.25 (dd, J = 16.1 and 10 Hz, 2H), 3.58 (m, 4H),
3.45 (m, 4H), 3.01 (m, 2H). 13C NMR (125 MHz, CD3CN): δ
162.42, 154.48, 151.05, 150.66, 150.54, 150.09, 139.65, 136.25, 135.75,
129.50, 128.86, 128.20, 127.86, 124.20, 123.32, 120.00, 116.92, 70.43,
52.24, 51.91. MALDI-TOF: m/z 424 (M + H)+, 462 (M + K)+.
4-[4-(4-Methoxyphenyl)piperazin-1-yl]-2-(pyridin-2-yl)-
N-[4-(2-Oxo-2-{4-[2-(pyridin-2-yl)quinazolin-4-yl]piperazin-
1-yl}ethyl)thiazol-2-yl]acetamide (15). This was prepared by
coupling 2-(2-acetamidothiazol-4-yl)acetic acid to 4-(piperazin-1-yl)-
2-(pyridin-2-yl)quinazoline using HBTU and DIEA. Yield: 71%. tR:
1
7.160 min (95.9%). H NMR (400 MHz, CDCl3): δ 8.58 (d, J = 4.4
Hz, 1H), 8.67 (d, J = 7.6 Hz, 1H), 8.26 (d, J = 8.4 Hz, 1H), 8.12 (m,
1H), 8.01 (t, J = 6.8 Hz, 1H), 7.73 (m, 3H), 6.87 (m, 2H), 4.87 (s,
2H), 3.65 (m, 4H), 3.31 (m, 4H), 2.18 (s, 3H). 13C NMR (125 MHz,
CD3CN): δ 169.87, 164.67, 159.28, 153.93, 151.04, 148.24, 146.27,
142.25, 140.28, 137.73, 130.00, 129.90, 128.96, 125.39, 121.59, 113.86,
111.33, 45.99, 42.25, 37.76, 23.58. MALDI-TOF: m/z 474 (M + H)+,
496 (M + Na)+.
Materials. Acrylamide/bis solution (37.5:1) 30%, ammonium
persulfate, and N,N,N′,N′-tetramethylethlenediamine (TEMED) for
SDS-PAGE was purchased from Bio-Rad Laboratories (Hercules, CA).
CoCl2·6H2O, MnCl2·4H2O, ZnCl2, NiCl2·6H2O, and CuCl2·2H2O
were purchased from Sigma-Aldrich (St. Louis, MO). CaCl2·2H2O was
purchased from Fisher Scientific (Hampton, NH). MgCl2·6H2O was
purchased from Avantor Performance Materials (Center Valley, PA).
1
quinazoline (11j). Yield: 90%. tR: 10.133 min (98.9%). H NMR
(400 MHz, CDCl3): δ 8.89 (m, 1H), 8.6 (d, J = 8.0 Hz, 1H), 8.21 (d, J
= 9.2 Hz, 1H), 7.98 (d, J = 7.6 Hz, 1H), 7.86 (m, 1H), 7.8 (m, 1H),
7.5 (m, 1H), 7.39 (m, 1H), 7.01 (m, 2H), 6.9 (m, 2H), 4.05 (m, 4H),
3.79 (s, 3H), 3.34 (m, 4H). 13C NMR (100 MHz, CD3OD): δ 166.43,
159.50, 156.77, 156.07, 153.71, 150.66, 146.98, 138.95, 134.55, 129.83,
127.53, 126.79, 126.66, 125.58, 120.08, 116.97, 115.74, 56.23, 52.29,
51.14. MALDI-TOF: m/z 398 (M + H)+, 420 (M + Na)+.
2-(5-Chloropyridin-2-yl)-4-[4-(4-methoxyphenyl)piperazin-
1-yl]quinazoline (11k). This was synthesized starting from 4-chloro-
2-cyanopyridine. Yield: 47% (over 3 steps). tR: 9.000 min (98.7%). 1H
NMR (400 MHz, acetone-d6): δ 8.74 (d, J = 2.8 Hz, 1H), 8.62 (d, J =
4011
dx.doi.org/10.1021/jm400227z | J. Med. Chem. 2013, 56, 3996−4016

Journal of Medicinal Chemistry
Article
Restriction enzymes were purchased from New England BioLabs
(Ipswich, MA).
or HDFa (2000 cells/well) were seeded into 96-well plates with 190
μL of corresponding growth medium. Cells were allowed to adhere at
37 °C overnight. Next day, serial dilutions of the compounds in vehicle
(DMSO) or vehicle itself were diluted 50 times with the growth
medium before they were added to the cells. Final concentration of
DMSO never exceeded 0.1%. The cells were treated with DMSO or
the compounds for 24 h. Cells were pulsed with 1 μCi [3H]-thymidine
(5 Ci per mmol) (American Radiolabeled Chemicals, St. Louis, MO)
for 6 h and then treated with trypsin-EDTA (Life Technologies). The
suspended cells were harvested onto Filtermat A glass fiber filters
(PerkinElmer, Waltham, MA) and washed at least seven times with
dH2O using a Harvester 96 cell harvester (Tomtec, Hamden, CT).
The glass fiber filters were dried in a ventilated hood overnight. Next
day, the [3H]-thymidine counts on the glass fiber filters were measured
using a MicroBeta plate reader (PerkinElmer) with Betaplate Scint
Wallac HiSafe cocktail (PerkinElmer).
ProIP-Coupled MetAP Activity Assay. This assay was performed
as described.45 In brief, HsMetAP1 (0.15 μM) or HsMetAP2 (0.1 μM)
was incubated with either vehicle (1% DMSO) or test compounds in
MetAP assay buffer (100 mM NaCl, 40 mM HEPES, pH 7.5)
containing 15 U/mL BcProIP and 10 μM CoCl2 or MnCl2 at room
temperature for 20 min. The enzymatic reaction was initiated by the
addition of 600 μM Met-Pro-pNA. Absorbance at 405 nm (ε = 1.06 ×
104 M−1 cm−1) was recorded every 30 s within the first 30 min of
reaction by the FLUOstar OPTIMA microplate reader. The initial rate
was then calculated from the early linear portion (usually between 2
and 15 min after the reaction started) of the curve when absorbance
values were plotted versus the reaction time. In addition, to rule out
the possibility that any inhibitor found by the BcProIP-coupled MetAP
assay might work simply because it inhibited the coupling enzyme,
compounds showing inhibitory effects were counter screened against
0.1 U/mL BcProIP, 600 μM Pro-pNA, and 10 μM CoCl2 or MnCl2 in
the MetAP assay buffer.
LAO and HRP-Coupled MetAP Activity Assay. This assay was
performed as described.54 MetAP activity was measured by
continuously monitoring the production of free L-methionine with L-
amino acid oxidase (LAO, from Sigma) and horseradish peroxidase
(HRP, from Sigma). The lyophilized powder of HRP was
reconstituted in MetAP assay buffer to 5 mg/mL. The substrate
Met-Ala-Ser (Sigma) and o-dianisidine (3,3′-dimethoxybenzidine,
from Sigma) were dissolved in ddH2O. The test compounds were
dissolved in 100% DMSO. The total reaction volume was 100 μL, and
the reaction was carried out in 96-well plates at room temperature.
HsMetAP1 (0.15 μM) was incubated with either vehicle (DMSO) or
test compounds in MetAP assay buffer containing 1 U/mL LAO, 0.1
mg/mL HRP, 0.2 mg/mL o-dianisidine, and 1 μM ZnCl2 at room
temperature for 20 min. The enzymatic reaction was initiated by the
addition of 600 μM Met-Ala-Ser. Absorbance at 450 nm (ε = 1.53 ×
104 M−1 cm−1) was recorded every 30 s within the first 30 min of
reaction by the FLUOstar OPTIMA microplate reader. After
subtracting the background hydrolysis determined in the absence of
MetAP, the net increases in the absorbance were plotted versus the
reaction time and the slope of the early linear portion (usually between
2 and 20 min after the reaction started) of the curve was determined
by the function “linear regression analysis” in GraphPad Prism 5
(GraphPad Software, La Jolla, CA). The initial rate was then calculated
from the slope.
Metal Activation Assay. All buffer solutions, ddH2O (used to
dissolve and dilute metals), substrates, and enzymes (except
HsMetAP1) in the metal activation assays were pretreated with
Chelex 100 resin (Bio-Rad). Except (1) the indicated concentration of
hydrochloride salts of divalent metals were used in the metal activation
assays, instead of fixed concentrations of CoCl2, MnCl2, or ZnCl2 and
(2) there was no incubation with vehicle or compounds, anything else
in the metal activation assays were the same as ProIP-coupled or LAO
and HRP-coupled MetAP activity assays. The initial rate was calculated
from the slope of the early linear portion of the enzymatic reaction
curve and expressed as μmol/L of methionine release per minute per
μmol/L of human MetAP1.
Cell Lines and Cell Culture. Human umbilical vein endothelial
cells (HUVEC) (Lonza, Allendale, NJ) were grown in endothelial cell
growth medium-2 (EGM-2) using the EGM-2 bullet kit (Lonza) per
manufacturer’s instructions. Normal human fibroblasts-adult (HDFa)
(Lifeline Cell Technology, Frederick, MD) were grown in FibroLife-
S2 cell growth medium using the FibroLife-S2 LifeFactors kit (Lifeline
Cell Technology) per manufacturer’s instructions. The phenotypes of
HUVEC and normal HDFa were verified by the manufacturers based
on morphological observation. Jurkat E6-1 human acute T cell
leukemia lymphocytes (ATCC TIB-152, Manassas, Virginia), PC-3
human prostate adenocarcinoma epithelial cells (ATCC CRL-1435),
and MCF-7 human breast adenocarcinoma epithelial cells (ATCC
HTB-22) were grown in Roswell Park Memorial Institute (RPMI)-
1640 medium (Life Technologies, Carlsbad, CA), supplemented with
10% fetal bovine serum (FBS) (Life Technologies) and 100 units/mL
penicillin plus 100 μg/mL streptomycin (Life Technologies). HeLa
human cervical adenocarcinoma epithelial cells (ATCC CCL-2) and
HEK293T human embryonic kidney epithelial cells expressing SV40 T
antigen (ATCC CRL-11268) were grown in Dulbecco’s Modified
Eagle Medium (DMEM) with low glucose (Life Technologies),
supplemented with 10% FBS and 100 units/mL penicillin plus 100
μg/mL streptomycin. The genotypes of the five transformed cell lines
were verified by ATCC. All cell lines were maintained in a humidified
incubator at 37 °C in an atmosphere of 5% CO2. High Five (BTI-TN-
5B1-4) cells (Life Technologies) used to overexpress human MetAP2
were grown in Grace’s insect cell medium (Life Technologies)
supplemented with 10% FBS and were maintained in a nonhumidified
incubator at 27 °C as per manufacturer’s instructions.
Primary and Secondary Antibodies. Mouse antihuman
unprocessed 14-3-3γ (iMet 14-3-3γ) monoclonal antibody (mAb)
(NB100−407) was purchased from Novus Biologicals (Littleton, CO).
Rabbit antihuman total 14-3-3γ mAb (5522) was purchased from Cell
Signaling (Danvers, MA). Mouse antihuman MetAP1 mAb
(MAB3537) was purchased from R&D Systems (Minneapolis, MN).
Mouse antihuman MetAP2 mAb was generated as described
previously.21 Mouse antihuman α-tubulin mAb (sc-5286), goat
antihuman GAPDH pAb (sc-20357), and goat antimouse IgM
antibody conjugated with horseradish peroxidase (HRP) (sc-2064)
were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
Mouse antihuman β-actin (JLA20) mAb was purchased from
Developmental Studies Hybridoma Bank (DSHB) at the University
of Iowa (Iowa City, IA). Sheep antimouse IgG antibody conjugated
HRP (NA931 V) and donkey antirabbit IgG antibody conjugated with
HRP (NA934 V) was purchased from GE Healthcare (Pittsburgh,
PA).
Transfection of HUVEC with siRNAs against MetAP1 and
MetAP2. A duplex oligonucleotides encoding siRNA against human
MetAP1, a siRNA against human MetAP2, and a scrambled control
siRNA were purchased from Qiagen (Valencia, CA). The sequences of
the sense strands of the siRNAs were as follows: MetAP1 siRNA, 5′-
GGCCAGUGCCAAGUUAUAU-dTdT-3′, targeting the bases 290−
308 in the ORF of MetAP1 mRNA; MetAP2 siRNA, 5′-
GACUGUUCACGCAAGUUAA-dTdT-3′, targeting the bases 604−
622 in the ORF of MetAP2 mRNA; and the scrambled control siRNA,
5′-UUCUCCGAACGUGUCACGU-dTdT-3′. HUVEC 7.5 × 104
were seed into one well of 6-well plates with 2 mL of EGM-2 growth
medium. Cells were allowed to adhere at 37 °C overnight. Next day,
cells were transfected with scrambled control siRNA (20 and 100 nM),
MetAP1 siRNA (20 and 100 nM), or MetAP2 siRNA (20 nM) diluted
in serum free EBM-2 basal medium (Lonza) using either HiPerFect
(Qiagen) or TransIT-TKO (Mirus Bio, Madison, WI) transfection
reagent per manufacturer’s instructions. Then 48 h after transfection,
cell lysates were prepared and boiled in 1× Laemmli buffer for
immunoblot analysis.
[3H]-Thymidine Incorporation Assay. To determine the IC50
values of the compounds against cell proliferation, seven different
concentrations and one vehicle (DMSO) control were used for each
compound. HUVEC (2500 cells/well), HeLa cells (3000 cells/well),
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Article
Initiator Methionine Release Assay. This assay was performed
as described.54 In brief, HEK293T cells were transiently transfected
with Superfect transfection reagent (Qiagen) and the appropriate
plasmid DNA (p3xFLAG-CMV-14-BTF3L4 or p3xFLAG-CMV-14-
TXNL1). Then 19.5 h after transfection, cells were treated with either
vehicle (DMSO), 11j, TNP-470, or the combination of the latter two.
Thirty minutes later, the cells were incubated with 0.2 mCi of [35S]-
methionine (PerkinElmer) for 4 h. BTF3L4-3xFlag or TXNL1-3xFlag
in the whole cell extract of HEK293T cells was immunoprecipitated by
anti-Flag-conjugated resin (Sigma), washed extensively, resuspended
in MetAP assay buffer, and split into two equal aliquots (A and B). A
was incubated with a 50 μL mixture of recombinant HsMetAP1 and
HsMetAP2 (5 μM each) in MetAP assay buffer, and B was incubated
with the buffer only. After incubation overnight (∼16 h) at room
temperature, the [35S]-methionine released from the resin into the
solution was determined by scintillation counting (scintillation fluid,
from PerkinElmer) on a 1450 MicroBeta apparatus (Wallac) for both
A and B. The percentage of [35S]-methionine release was calculated
from the ratio of counts in the assay buffer to the sum of these counts
and the counts from the corresponding resin sample. The percentage
of [35S]-methionine release in B was subtracted from that in A, and the
resulting numbers in inhibitor-treated groups were normalized to the
number in the corresponding vehicle control.
S.B.G., M.S.M., and F.Z. performed the crystallographic
analysis.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by National Institutes of Health
(NIH) grant R01 CA078743 (to J. O. L.) and NIH Medical
Scientist Training Program grant T32GM07309 (to B.A.N.).
The mouse antihuman β-actin monoclonal antibody (JLA20)
developed by Lin, J.J.-C. was obtained from the Developmental
Studies Hybridoma Bank (DSHB) developed under the
auspices of the NICHD and maintained by The University of
Iowa, Department of Biology, Iowa City, IA. Molecular graphics
and analyses were performed in part with the UCSF Chimera
package. Chimera is developed by the Resource for
Biocomputing, Visualization, and Informatics at the University
of California, San Francisco, funded by grants from the NIH
National Center for Research Resources (2P41RR001081) and
National Institute of General Medical Sciences
(9P41GM103311).
Statistical Analysis. The statistical significance of the difference
between two experimental groups was determined by two-tailed
unpaired Student’s t-test using GraphPad Prism 5 software.
ABBREVIATIONS USED
■
BcProIP, Bacillus coagulans proline iminopeptidase; DMSO,
dimethyl sulfoxide; EC50, half-maximal effective concentration;
EcMetAP, Escherichia coli MetAP; HDFa, human dermal
fibroblasts−adult; HRP, horseradish peroxidase; HsMetAP1,
human cytosolic MetAP type 1; HsMetAP2, human cytosolic
MetAP type 2; HUVEC, human umbilical vein endothelial
cells; IC50, half maximal inhibitory concentration (IC50 equals
to EC50 when the bottom value of an inhibitory dose−response
curve is zero); ifMet, initiator N-formylmethionine; iMet,
initiator methionine; Ki, dissociation constant for inhibitor
binding; map, gene encoding prokaryotic methionine amino-
peptidase; LAO, L-amino acid oxidase; map1, gene encoding
MetAP1; map2, gene encoding MetAP2; MetAP, methionine
aminopeptidase; MetAP1, eukaryotic cytosolic MetAP type 1;
MetAP2, eukaryotic cytosolic MetAP type 2; NME, N-terminal
methionine excision; SAR, structure−activity relationship; SDS-
PAGE, sodium dodecyl sulfate−polyacrylamide gel electro-
phoresis; siRNA, small-interfering RNA
ASSOCIATED CONTENT
* Supporting Information
■
S
Figures, table, and experimental procedures for immunoblot-
ting, expression, and purification of HsMetAP1, tHsMetAP1,
HsMetAP2, and BcProIP, determination of the activity of
BcProIP and the coupling condition, determination of the EC50
or IC50 values of inhibitors, construction of HsMetAP1
expression vector, lentivirus production, transduction of
human primary endothelial cells, crystallization of tHsMetAP1
in complex with 11j, data collection and processing, and
structure determination and refinement. This material is
available free of charge via the Internet at http://pubs.acs.org.
Accession Codes
PDB ID code for compound 11j is 4IU6.
AUTHOR INFORMATION
Corresponding Author
*Phone: 410-955-4619. E-mail: joliu@jhu.edu.
■
REFERENCES
Present Addresses
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◆X.C.: Department of Pediatrics, University of Maryland
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Author Contributions
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