Discovery and structural modification of inhibitors of methionine aminopeptidases from Escherichia coli and Saccharomyces cerevisiae

Article abstract of DOI:10.1021/jm0300532

A series of pyridine-2-carboxylic acid derivatives were synthesized according to the leads from the screening, and potent inhibitors have been obtained by structural modification. They have shown submicromolar inhibition of the enzymes (for example, for 9

Full text of DOI:10.1021/jm0300532

J . Med. Chem. 2003, 46, 2631-2640
2631
Discover y a n d Str u ctu r a l Mod ifica tion of In h ibitor s of Meth ion in e
Am in op ep tid a ses fr om Esch er ich ia coli a n d Sa cch a r om yces cer evisia e
Qun-Li Luo,† J ing-Ya Li,† Zhi-Ying Liu,‡ Ling-Ling Chen,† J ia Li,† Zhen Qian,† Qiang Shen,† Yu Li,‡
Gerald H. Lushington,# Qi-Zhuang Ye,*,†,§ and Fa-J un Nan*,†
Chinese National Center for Drug Screening, Shanghai Institute of Materia Medica, Shanghai Institutes for Life Sciences,
Chinese Academy of Sciences, 189 Guo Shou J ing Road, Zhangjiang Hi-Tech Park, Shanghai 201203, China, Department of
Chemistry, Lanzhou University, 216 Tian Shui Road, Lanzhou, 730000, China, and High-Throughput Screening Laboratory
and Molecular Graphics and Modeling Laboratory, University of Kansas, Lawrence, Kansas 66047
Received J anuary 31, 2003
A series of pyridine-2-carboxylic acid derivatives were synthesized according to the leads from
the screening, and potent inhibitors have been obtained by structural modification. They have
shown submicromolar inhibition of the enzymes (for example, for 9n , IC50 ) 130 nM for
EcMetAP1 and IC50 ) 380 nM for ScMetAP1). They represent small-molecule MetAP inhibitors
with novel structures different from alkylating fumagillin derivatives and peptidic bestatin-
based MetAP inhibitor.
In tr od u ction
in only a slow-growth phenotype.2 Therefore, the MetAP
enzymes present good targets for new antibiotic drug
discovery, and inhibitors against MetAPs offer hope for
a new treatment of bacterial and fungal infections.3a
Although there are intensive efforts in searching for
MetAP inhibitors in both academia and industry, natu-
ral product fumagillin and its derivatives4 with specific-
ity for type II MetAPs are the only potent MetAP
inhibitors described in the literature so far, and they
are alkylating agents and covalently modify MetAPs.
A peptidic inhibitor AHHpA-Ala-Leu-Val-Phe-OMe de-
signed on the basis of leucine peptidase inhibitors with
a bestatin moiety showed only weak activity against
EcMetAP1 (IC50 ) 5.0 µM),5 and there are few inhibitors
reported for type I MetAPs,6 although it is the only and
essential enzyme in bacteria. Clearly, there are needs
for potent inhibitors of bacterial and fungal MetAPs for
antibiotic applications.
Emerging bacterial resistance to current antibiotic
drugs has driven the search for novel prokaryotic targets
and for novel small molecules to modulate the activity
of these targets. The methionine aminopeptidases
(MetAPs) are a unique class of cobalt-containing met-
alloproteinases that facilitate the removal of the N-
terminal initiator methionine from nascent polypeptides
in a nonprocessive manner.1 MetAPs exist in both
prokaryotic and eukaryotic cells, and their gene se-
quences have been determined in several clinically
important pathogens, such as Mycobacterium tubercu-
losis in tuberculosis, Enterococcus faecalis in urinary
tract infection and endocarditis, Streptococcus pneumo-
niae in pneumonia, Haemophilus influenzae in respira-
tory tract infection, and Helicobacter pylori in ulcers.
The pathogenic Escherichia coli strain O157:H7 has the
exact MetAP sequence as the E. coli laboratory strain
K12.
There are two types (types I and II) of MetAP that
have been identified. Eubacteria (for example, E. coli,
Bacillus subtilis, and Salmonella typhimurium) have
only type I MetAPs, while archaea (for example, Metha-
nobacterium thermoautotrophicum, Sulfolobus solfa-
taricus, and Pyrococcus furiosis) have only type II
MetAPs. Eukaryotic cells contain both type I and type
II MetAPs.2 The presence of MetAPs is essential for cell
viability, and disruption of the gene for MetAP in E.
coli (EcMetAP1) or Salmonella typhimurium is a lethal
event.3 Since the yeast Saccharomyces cerevisiae has
both type I and type II MetAPs (ScMetAP1 and Sc-
MetAP2), both genes need to be blocked to affect cell
viability, and disruption of either gene alone resulted
In the present work, we report our discovery of
inhibitors of EcMetAP1 through high-throughput screen-
ing (HTS) and the structural modifications based on the
screening leads. Inhibitors with submicromolar inhibi-
tory potencies in vitro against the bacterial EcMetAP1
and the yeast ScMetAP1 have been obtained.
Resu lts a n d Discu ssion
The starting point of this work was a random screen-
ing of a diverse small molecule library of 4500 com-
pounds using a recombinant EcMetAP1 expressed and
purified in our laboratory. Compound 1 (Figure 1), with
an IC50 of 5.0 µM, was identified along with several
other leads. For understanding the structure-activity
relationship, we initially prepared compounds 2-4, in
which we replaced the N atom in the pyridine ring of
compound 1 with CH, the S atom in the thiazole ring
by its isosteric ethylenic group, or inserted an NH
between the pyridine ring and carbonyl group (Figure
1). None of these compounds showed any activities to
the EcMetAP1 up to 100 µM. These results indicated
that the N atom in the pyridine ring, the direct connec-
tion of the carbonyl group to the pyridine ring, and the
* To whom correspondence should be addressed. For F.-J .N.: phone,
+86-21-50801313, extension 231; fax, +86-21-50800721; e-mail,
fjnan@mail.shcnc.ac.cn. For Q.-Z.Y.: phone, 785-330-4330; fax, 785-
330-4332; e-mail, qye@ku.edu.
† Chinese National Center for Drug Screening.
‡ Lanzhou University.
#
Molecular Graphics and Modeling Laboratory, University of
Kansas.
§ High-Throughput Screening Laboratory, University of Kansas.
10.1021/jm0300532 CCC: $25.00 © 2003 American Chemical Society
Published on Web 05/22/2003

2632 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13
Luo et al.
F igu r e 1. Compound 1 and prepared compounds 2-10.
N and/or S atoms in the thiazole ring are essential for
effective inhibition of EcMetAP1.
To further understand the key structural scaffold,
compounds 5 and 6 were synthesized, which changed
the substitute position of the carbonyl group in the
pyridine ring from the 2- to the 3- and 4-positions.
Surprisingly, neither 5 nor 6 showed any activity.
Pyridine-2-carboxylic acid 7 also showed no activity.
These results strongly suggested that pyridine-2-car-
boxylic acid thiazol-2-ylamide (PCAT) is a key structural
scaffold for effective inhibition of the enzyme activity.
With this assumption, we reasoned that to improve
its inhibitory potency, an increase of the van der Waals
contacts was needed between the ligand and the hydro-
phobic surface of the MetAP, such as the S1 pocket,
which serves as the specificity pocket for the NH2
terminal methionine side chain of natural substrates.
We introduced a number of hydrophobic substitutions
at various positions on the pyridine ring of 1, and a
small library with a general structure as that for 8 was
designed. In this series of compounds, we focused our
attention on 3-substituted pyridine-2-carboxylic acid
thiazol-2-ylamides 9 and 10, because we observed
inhibitory activity from the initial derivatives on Ec-
MetAP1 and ScMetAP1.
F igu r e 2. (A) Active site of interactions of EcMetAP1 with a
bestatin-based inhibitor. (B) Docked structure of molecule 10k
inside the EcMetAP1 model (cut-away Connolly surface with
polar regions shown in blue; hydrophobic in gold) determined
from X-ray crystallography.7 Rendered in ball-and-stick form,
the predicted conformation of 10k is contrasted with the
bestatin analogue (wire model, oriented according to the
crystal structure7).
the most potent EcMetAP1 and ScMetAP1 inhibitors
described in the literature to date (Table 1).
When compounds 9 and 10 with the same R group
(9g-l, 9q-t vs 10d -i, 10k -n ) were compared, the 3-N-
substituted derivatives 9 were often more active than
the 3-O-substituted derivatives against EcMetAP1,
indicating that an N is preferred at that position.
However, it is just opposite against ScMetAP1, and the
3-O-substituted derivatives 10 were often more active
than the 3-N-substituted derivatives.
The syntheses of 3-N-acyl-substituted compounds 9
could be accomplished by the well-precedented conden-
sation of 3-tert-butoxycarbonylaminopyridine-2-carboxy-
lic acid with 2-aminothiazole in the presence of DCC in
DMF, followed by the amine deprotection and acylation
with various carboxylic acids or chlorides under neutral
or basic conditions (Scheme 1). By employment of a
similar procedure, the 3-O-acyl-substituted compounds
10 were obtained from 3-hydroxypyridine-2-carboxylic
acid in good yields.
Forty-one 3-substituted compounds 9 and 10 were
synthesized and evaluated for their inhibitory activity
against both EcMetAP1 and ScMetAP1. Of the 41
compounds tested, all of compounds showed inhibition
of EcMetAP1 with IC50 values of less than 10 µM, and
most of them showed better activity than compound 1
(Table 1). In particular, compounds 9n , 9m , and 9c are
the most active against EcMetAP1, with IC50 values of
130, 150, and 180 nM, respectively, and compounds 9o
and 10g were the most active against ScMetAP1, with
IC50 values of 140 and 200 nM, respectively. They were
It seems that EcMetAP1 is less sensitive to the
substitution at the 3-position of the pyridine ring, and
compounds with either an aliphatic or an aromatic
group often showed submicromolar potency. This could
not hold true for ScMetAP1 though, and we observed a
much bigger variation of IC50 values on ScMetAP1.
None of the four compounds 9 with aromatic groups
directly connected to the carbonyl carbon (9q, 9r , 9s,
and 9t) showed measurable activity against ScMetAP1.
However, the aromatic substitutions at this position for
compounds 10 gave mixed results, and compounds 10q
and 10s even showed submicromolar potencies. Among
compounds 10 with the aromatic substitutions, Sc-
MetAP1 prefers a group at the ortho position on the
aromatic ring to meta or para positions (10l, 10q, 10s,
for example).
The X-ray structure of EcMetAP1 complexed with the
bestatin-based inhibitor AHHpA-Ala-Leu-Val-Phe-OMe
has been reported, and the peptidic inhibitor interacts
with the two cobalt ions (Figure 2A).7 From molecular
docking studies carried out via the AutoDock program8

Inhibitors of Methionine Aminopeptidases
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13 2633
Sch em e 1. Syntheses of Pyridine-2-carboxylic Acid Thiazol-2-ylamides 9 and 10
for a sample set of the PCAT scaffold (compounds 1, 9a ,
9d , 9n , 10a , 10b, and 10k given in Table 1) bound to
the EcMetAP1, one finds that the inhibitors consistently
occupy a region spatially similar to that chosen by the
bestatin-based ligand. For example, Figure 2B showed
the docked structure of molecule 10k inside the Ec-
MetAP1 model (cut-away Connolly surface with polar
regions shown in blue; hydrophobic in gold) determined
from X-ray crystallography.7 Rendered in ball-and-stick
form, the predicted conformation of 10k is contrasted
with the bestatin analogue (wire model, oriented ac-
cording to the crystal structure7), suggesting affinity for
the same spatial region. In both cases, the presence of
the two Co2+ ions (rendered as magenta space filling
spheres at the bottom of the cavity) appears to provide
a strong electropositive environment capable of fixing
the ligand’s most electronegative features. Not all
molecules in the PCAT scaffold have the shape and
flexibility necessary to direct three electronegative
groups toward the ions; many instead orient two in that
direction. Compounds 1, 9n , and 10g all appear to bind
optimally by orienting their pyridine N and central (i.e.,
non side chain) carbonyl O toward the cobalts, 10a
employs the central O and thiazol S for this purpose,
and compounds 9a and 10b couple via their side chain
electronegative (amino and ester, respectively) atoms.
Compound 9d is unusual in that it appears to only bind
to the Co’s via a single atom, the pyridine N. Although
this information partially supported our assumption, the
exact binding mode of the PCAT derivative with MetAPs
should be obtained from X-ray crystal experiments.
The differences in sensitivity to the substitution at
the 3-position of the pyridine ring may reflect the
differences at the active sites of EcMetAP1 and Sc-
MetAP1 and the differences in the interaction between
the inhibitors and the MetAPs. From docking studies,
it appears that the substituted group at this position
can influence the molecule’s docked conformation, but
the relative similarity among docking free energies
among the PCAT analogues (all between -8.7 to -11.9
kcal/mol) agrees with the relative similarity among IC50
values and indicated that side chain substitutions may
not greatly influence binding efficacy within the Ec-
MetAP1 receptor. Without a corresponding crystal
structure for the ScMetAP1 receptor, we are unable at
this point to offer sound speculation on the reason for
the much greater variation in IC50 data for this latter
system.
The ratio of the IC50 of EcMetAP1 to the IC50 of
ScMetAP1 (Table 1) is another indication of the differ-
ences in the inhibitor binding to the enzymes. Com-
pound 9r (IC50 ) 280 nM for EcMetAP1; IC50 > 100 µM
for ScMetAP1; ratio, <0.0028) is the most selective for
EcMetAP1, and compound 10c (IC50 ) 5.4 µM for
EcMetAP1; IC50 ) 380 nM for ScMetAP1; ratio, 14) is
the most selective for ScMetAP1. The selectivity for
either EcMetAP1 or ScMetAP1 gives us confidence in
obtaining the antibiotic drugs specific for the targeting
microorganisms, and the selectivity will become more
important if we want to develop antibiotics without
affecting human MetAPs for low toxicity.
We only tested a few compounds for antibacterial
activity (Table 2). Like most antibiotics that target
protein synthesis, these are bacteriostatic agents. Com-
pound 1 was active against Gram-positive bacteria,
including Staphylococcus aureus and Enteropathogenic

2634 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13
Ta ble 1. Inhibition of EcMetAP1 and ScMetAP1a
Luo et al.

Inhibitors of Methionine Aminopeptidases
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13 2635
Ta ble 1. (Continued)

2636 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13
Ta ble 1. (Continued)
Luo et al.
a
The asterisk (/) indicates the positive control. Similar potency was reported as AHHpA-Ala-Leu-Val-Phe-OMe.5
Ta ble 2. Antibacterial Activities of Pyridine-2-carboxylic Acid
Derivatives
the metal-substituted EcMetAP1s with the hope of
improving their in vivo potencies.
a
MIC (µg/mL)
Bacteria have only one MetAP and its inhibition could
be sufficient in controlling bacterial infections. Fungi
as eukaryotic cells have type II MetAPs in addition to
the type I MetAPs, such as ScMetAP1. Fumagillin and
its analogues are MetAP inhibitors specific for type II
MetAPs. However, they are alkylating agents, and other
type II MetAP inhibitors need to be developed. Com-
bined use of the inhibitor for type I MetAPs and the
inhibitor for type II MetAPs is required for antifungal
applications. Alternatively, a dual inhibitor for MetAPs
of both types is needed.
cipro-
floxacin
strain
1
9n
10c 9b
9r
Staphylococcus aureus
ATCC25923
2.0 62.5 125 62.5 15.6 0.125
Enteropathogenic E. coli
12.5 125
250 125
500 500
250
250
0.06
ATCC25922
Pseudomonas aeruginosa 250
ATCC27853
500
0.125
a
MIC ) minimum inhibitory concentration, determined after
24 h.
E. coli. Compounds 9n , 9b, and 9r also showed moder-
ate activity against these strains. We were puzzled that
although we have improved their inhibitory potencies
for the MetAP inhibitors by structural modification, we
did not see improvement in in vivo activity against these
bacteria. One possibility is the differences of these
inhibitors in their ability in penetrating the bacterial
cell wall, which we have not tested. However, the failure
in correlating the in vitro and in vivo activities may
point to the big question in MetAP research: Which is
the physiologically relevant metal in the enzyme?
MetAPs have been traditionally studied in vitro as Co-
(II) enzymes, and the EcMetAP1 we used for screening
and characterization was a Co(II) enzyme. Recent data
suggest that the MetAPs may have a metal other than
Co(II) at their active sites, such as Zn(II),9 Fe(II),10 or
Mn(II),11 and our preliminary data showed that the
metal substitution could cause significant changes in
specificity toward substrates and inhibitors. We are in
the process of assessing these and other inhibitors on
Con clu sion
In summary, pyridine-2-carboxylic acid thiazol-2-
ylamide 1 was discovered as an EcMetAP1 inhibitor
through HTS, and various pyridine-2-carboxylic acid
thiazol-2-ylamide analogues 9 and 10 were synthesized
and identified as EcMetAP1 and ScMetAP1 inhibitors.
The introduction of the 3-N-acyl group (compounds 9)
or 3-O-acyl group (compounds 10) greatly enhanced the
activity against EcMetAP1 and ScMetAP1. This is the
first identification of small-molecule MetAP inhibitors
with structural units totally different from known
fumagillin or bestatin-based MetAP inhibitors. A few
compounds were tested and showed moderate antibac-
terial activity. These results open a new avenue to
finding new MetAP inhibitors with potential antibiotic
activity. Further efforts in modifying these and other
lead structures with the aim of improving potency as
well as specificity in vitro and efficacy in vivo are in
progress.

Inhibitors of Methionine Aminopeptidases
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13 2637
tion of starting material. The solvents were removed, and then
the residue was dissolved in saturated aqueous NaHCO3 and
extracted with CH2Cl2. The combined organic phases were then
processed in the usual way to yield 9a (940 mg, 91%). 1H NMR
(CDCl3, 300 MHz): δ 11.32 (br, 1H), 7.94 (d, J ) 4.2 Hz, 1H),
7.51 (d, J ) 3.6 Hz, 1H), 7.24 (dd, J ) 4.2, 8.1 Hz, 1H), 7.05
(d, J ) 8.1 Hz, 1H), 7.00 (d, J ) 3.6 Hz, 1H), 5.97 (br, 2H).
EI-MS m/z: 220 (M+). HREI-MS: exact mass calcd for C9H8N4-
OS (M+), 220.0419; found, 220.0421. HPLC purity, 99.29%.
Exp er im en ta l Section
1
Gen er a l Meth od s. H (300 MHz) and 13C (75 MHz) NMR
spectra were recorded on a Varian Mercury-VX300 Fourier
transform spectrometer. The chemical shifts were reported in
δ (ppm) using the δ 7.26 signal of CDCl3 (1H NMR) and the δ
77.23 signal of CDCl3 (13C NMR) as internal standards. Low-
resolution mass spectra were obtained on a SHIMADZU
GCMS-QP5050A spectrometer, and high-resolution MS data
were obtained on a MAT-711 mass spectrometer, an APEXIII
7.0 TESLA FTMS mass spectrometer, or a Concept 1H series
mass spectrometer. HPLC analyses were performed on an
HP1100 series LC system (HP ChemStation A.06.03; column,
Zorbax SB-C18, 4.6 × 150 mm, 5 µm; mobile phase, MeOH/
H2O, 80:20; flow rate, 1.0 mL/min; UV wavelength, 254 nm;
temperature, ambient temperature; injection volume, 10 µL).
The reaction mixture was generally poured into water, and
the separated aqueous phase was then thoroughly extracted
with the specified solvent. After the mixture was washed with
10% aqueous HCl and/or NaHCO3 (if required), water, and
saturated aqueous NaCl, the combined organic phases were
dried over anhydrous Na2SO4 or MgSO4 and then filtered and
concentrated under reduced pressure to yield the crude reac-
tion product.
P yr id in e-2-ca r boxylic Acid Th ia zol-2-yla m id e (1). To
a mixture of pyridine-2-carboxylic acid (1.23 g, 10 mmol),
thiazol-2-ylamine (1.01 g, 10 mmol), and HOBT (1.45 g, 10.75
mmol) in dry DMF (10 mL) was added DCC (2.16 g, 10.05
mmol). The reaction mixture was stirred at room temperature
for 24 h. The reaction mixture was diluted with 50 mL of
EtOAc and 20 mL of H2O. The aqueous phase was extracted
with EtOAc. The combined organic phases were then processed
in the usual way and chromatographed (3:1 petroleum ether/
3-F or m yla m in op yr id in e-2-ca r boxylic Acid Th ia zol-2-
yla m id e (9b). A mixture of 1 mL of Ac2O and 0.5 mL of
HCOOH was heated at 60 °C under N2 for 2 h, and then 1.2
mL of HOAc was added. The solution was cooled to 40 °C, and
9a (40 mg, 0.18 mmol) was added. The mixture was stirred at
60 °C for an additional 2 h before 2 g of ice was added. The
aqueous phase was neutralized to pH 7 with saturated aqueous
NaHCO3 and then extracted with EtOAc (3 × 10 mL). The
combined organic phases were then processed in the usual way
to yield the crude product, which was recrystallized from
1
EtOAc give pure 9b (9 mg, 20%). H NMR (CDCl3, 300 MHz):
δ 11.50 (s, 1H), 11.00 (br. 1H), 9.17 (d, J ) 8.1 Hz, 1H), 8.60
(s, 1H), 8.36 (d, J ) 3.3 Hz, 1H), 7.55 (m, 2H), 7.09 (d, J ) 3.3
Hz, 1H). EI-MS m/z: 248(M+). HREI-MS: exact mass calcd
for C10H8N4O2S (M+), 248.0368; found, 248.0366. HPLC purity,
98.63%.
Gen er a l P r oced u r e for P r ep a r a tion of Com p ou n d s 9d ,
9f-k , a n d 9q-t. To a solution of 9a (0.1 mmol) in 3 mL of a
1:1 EtOAc/H2O mixture was added excess NaHCO3 (160 mg,
1.9 mmol). The appropriate acid chloride (0.15 mmol) was
added at 0 °C. The mixture was stirred for 2-24 h with
gradual warming to room temperature. The reaction mixture
was diluted with 5 mL of EtOAc and 2 mL of H2O. The aqueous
phase was extracted with EtOAc. The combined organic phases
were then processed in the usual way and chromatographed
to yield the desired products.
1
EtOAc) to yield 1 (1.50 g, 73%). H NMR (CDCl3, 300 MHz):
δ 11.20 (br, 1H), 8.66 (dd, J ) 0.9, 4.2 Hz, 1H), 8.29 (dd, J )
0.6, 7.5 Hz, 1H), 7.94 (ddd, J ) 0.9, 7.5, 7.8 Hz, 1H), 7.53 (m,
2H), 7.05 (d, J ) 3.3 Hz, 1H). EI-MS m/z: 205 (M+). HREI-
MS: exact mass calcd for C9H7N3OS (M+), 205.0304; found,
205.0306. HPLC purity, 99.88%.
3-iso-Bu toxycar bon ylam in opyr idin e-2-car boxylic Acid
Th ia zol-2-yla m id e (9d ). Yield 38%. 1H NMR (CDCl3, 300
MHz): δ 11.49 (br, 1H), 10.90 (s, 1H), 8.93 (dd, J ) 1.5, 8.7
Hz, 1H), 8.26 (dd, J ) 1.5, 4.5 Hz, 1H), 7.55 (d, J ) 3.6 Hz,
1H), 7.51 (dd, J ) 4.5, 8.7 Hz, 1H), 7.07 (d, J ) 3.6 Hz, 1H),
4.01 (d, J ) 6.9 Hz, 2H), 2.03 (m, 1H), 1.01 (d, J ) 6.9 Hz,
6H). EI-MS m/z: 320 (M+). HREI-MS: exact mass calcd for
3-ter t-Bu toxycar bon ylam in opyr idin e-2-car boxylic Acid
(12). 3-Aminopyridine-2-carboxylic acid (5.02 g, 36 mmol) was
suspended in 60 mL of dry DMF, and Et3N (15.2 mL, 108
mmol) was added dropwise at room temperature. To the
resulting brown solution was added Boc2O (11.80 g, 54 mmol).
After being stirred for 10 min, the mixture was heated at 40-
50 °C overnight. The reaction mixture was poured into water
and was then extracted with EtOAc (2 × 50 mL). The aqueous
phase was acidified to pH 4-5 with 2 M aqueous HCl and then
extracted with CH2Cl2 (3 × 50 mL). The combined organic
phases were then processed in the usual way and chromato-
graphed (13:1 CHCl3/MeOH) to yield 12 (4.2 g, 49%). 1H NMR
(CDCl3, 300 MHz): δ 10.27 (br, 1H), 8.96 (d, J ) 8.7 Hz, 1H),
8.23 (d, J ) 4.2 Hz, 1H), 7.55 (dd, J ) 4.4, 8.9 Hz, 1H), 1.53
(s, 9H).
3-ter t-Bu toxycar bon ylam in opyr idin e-2-car boxylic Acid
Th ia zol-2-yla m id e (9c). To a mixture of 12 (2.51 g, 10.5
mmol), thiazol-2-ylamine (1.05 g, 10.5 mmol), and HOBT (1.61
g, 11.9 mmol) in dry DMF (20 mL) was added DCC (3.36 g,
16.3 mmol). The reaction mixture was stirred at room tem-
perature for 24 h. The reaction mixture was diluted with 50
mL of EtOAc and 20 mL of H2O. The aqueous phase was
extracted with EtOAc. The combined organic phases were then
processed in the usual way and chromatographed (4:1 petro-
C
14H16N4O3S (M+), 320.0943; found, 320.0971. HPLC purity,
99.93%.
3-P r op ion yla m in op yr id in e-2-ca r boxylic Acid Th ia zol-
2-yla m id e (9f). Yield 18%. 1H NMR (CDCl3, 300 MHz): δ
11.52 (s, 1H), 9.20 (dd, J ) 1.5, 8.7 Hz, 1H), 8.30 (dd, J ) 1.5,
4.5 Hz, 1H), 7.56 (d, J ) 3.6 Hz, 1H), 7.51 (dd, J ) 4.5, 8.7 Hz,
1H), 7.07 (d, J ) 3.6 Hz, 1H), 2.57 (q, J ) 7.5 Hz, 2H), 1.28 (t,
J ) 7.5 Hz, 3H). EI-MS m/z: 276 (M+). HREI-MS: exact mass
calcd for C12H12N4O2S (M+), 276.0681; found, 276.0681. HPLC
purity, 98.81%.
3-(2,2-Dim eth ylp r op ion yla m in o)p yr id in e-2-ca r boxyl-
ic Acid Th ia zol-2-yla m id e (9g). Yield 57%. 1H NMR (CDCl3,
300 MHz): δ 11.80 (br, 2H), 9.23 (dd, J ) 1.5, 8.7 Hz, 1H),
8.30 (dd, J ) 1.5, 4.5 Hz, 1H), 7.56 (d, J ) 3.6 Hz, 1H), 7.50
(dd, J ) 4.5, 8.7 Hz, 1H), 7.07 (d, J ) 3.6 Hz, 1H), 1.38 (s,
9H). EI-MS m/z: 304 (M+). HREI-MS: exact mass calcd for
C
14H16N4O2S (M+), 304.0994; found, 304.0988. HPLC purity,
99.76%.
3-Hexa n oyla m in op yr id in e-2-ca r boxylic Acid Th ia zol-
2-yla m id e (9h ). Yield 40%. 1H NMR (CDCl3, 300 MHz): δ
11.49 (s, 1H), 9.20 (dd, J ) 1.2, 8.7 Hz, 1H), 8.29 (dd, J ) 1.2,
4.2 Hz, 1H), 7.55 (d, J ) 3.6 Hz, 1H), 7.50 (dd, J ) 4.2, 8.7 Hz,
1H), 7.07 (d, J ) 3.6 Hz, 1H), 2.51 (t, J ) 7.5 Hz, 2H), 1.79
(m, 2H) 1.39 (m, 4H), 0.90 (t, J ) 6.3 Hz, 3H). EI-MS m/z:
318 (M+). HREI-MS: exact mass calcd for C15H18N4O2S (M+),
318.1150; found, 318.1136. HPLC purity, 97.52%.
1
leum ether/EtOAc) to yield 9c (1.56 g, 44%). H NMR (CDCl3,
300 MHz): δ 11.50 (br, 1H), 10.66 (br, 1H), 8.92 (dd, J ) 1.2,
8.7 Hz, 1H), 8.23 (dd, J ) 1.2, 4.2 Hz, 1H), 7.55 (d, J ) 3.6 Hz,
1H), 7.47 (dd, J ) 4.2, 8.7 Hz, 1H), 7.06 (d, J ) 3.6 Hz, 1H),
1.55 (s, 9H). EI-MS m/z: 320 (M+). HREI-MS: exact mass calcd
for
C
14H16N4O3S (M+), 320.0943; found, 320.0972. HPLC
purity, 99.76%.
3-(Cycloh exa n eca r b on yla m in o)p yr id in e-2-ca r b oxyl-
ic Acid Th ia zol-2-yla m id e (9i). Yield 61%. 1H NMR (CDCl3,
300 MHz): δ 11.49 (br, 1H), 9.23 (dd, J ) 1.2, 8.4 Hz, 1H),
8.30 (dd, J ) 1.2, 4.5 Hz, 1H), 7.60 (d, J ) 3.6 Hz, 1H), 7.50
(dd, J ) 4.5, 8.4 Hz, 1H), 7.08 (d, J ) 3.6 Hz, 1H), 2.41 (m,
1H), 2.04 (m, 2H), 1.86 (m, 2H), 1.76 (m, 1H), 1.58 (m, 2H),
3-Am in op yr id in e-2-ca r boxylic Acid Th ia zol-2-yla m id e
(9a ). To a stirred solution of 9c (1.56 g, 4.6 mmol) in CH2Cl2
(10 mL) at -10 °C was added TFA (4.0 mL) dropwise. The
reaction mixture was stirred for 4 h with gradual warming to
room temperature. TLC monitoring shows complete consump-

2638 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13
Luo et al.
1.33 (m, 3H). EI-MS m/z: 330 (M+). HREI-MS: exact mass
calcd for C16H18N4O2S (M+), 330.1150; found, 330.1144. HPLC
purity, 99.76%.
enoyl bromide (50 µL) in 0.5 mL of CH2Cl2 was added at 0 °C.
The mixture was stirred for 8 h with gradual warming to room
temperature. The reaction mixture was diluted with 5 mL of
EtOAc and 2 mL of H2O. The aqueous phase was extracted
with EtOAc. The combined organic phases were then processed
in the usual way and chromatographed (50:10:1:1 petroleum
ether/EtOAc/acetone/HOAc) to yield compound 9m (3.2 mg,
3-P h en yla cetyla m in op yr id in e-2-ca r boxylic Acid Th ia -
zol-2-yla m id e (9j). Yield 52%. 1H NMR (CDCl3, 300 MHz):
δ 11.57 (br, 2H), 9.18 (dd, J ) 1.2, 0.7 Hz, 1H), 8.28 (dd, J )
1.2, 7.2 Hz, 1H), 7.54 (d, J ) 3.6 Hz, 1H), 7.49 (dd, J ) 4.2,
8.7 Hz, 1H), 7.41 (m, 5H), 7.07 (d, J ) 3.6 Hz, 1H), 3.84 (s,
1H). EI-MS m/z: 338 (M+). HREI-MS: exact mass calcd for
1
11%). H NMR (CDCl3, 300 MHz): δ 11.62 (s, 1H), 9.18 (d, J
) 9.0 Hz, 1H), 8.31 (d, J ) 4.5 Hz, 1H), 7.55 (d, J ) 3.6 Hz,
1H), 7.52 (dd, J ) 4.5, 9.0 Hz, 1H), 7.07 (d, J ) 3.6 Hz, 1H),
5.11 (d, J ) 8.7 Hz, 1H), 3.24 (s, 2H), 1.89 (s, 3H). 13C NMR
(CDCl3, 75 MHz): δ 171.01 (C), 164.88 (C), 158.92 (C), 142.73
(CH), 139.18 (C), 138.99 (C), 137.01 (CH), 130.98 (C), 129.12
(CH), 129.05 (CH), 116.73 (CH2), 114.28 (CH), 48.29 (CH2),
22.74 (CH3). EI-MS m/z: 302 (M+), HREI-MS: exact mass
calcd for C14H14N4O2S (M+), 302.0837; found, 302.0833. HPLC
purity, 95.21%.
C
17H14N4O2S (M+), 338.0837; found, 338.0833. HPLC purity,
99.24%.
3-(2-Met h ylb u t -2-en oyla m in o)p yr id in e-2-ca r b oxylic
Acid Th ia zol-2-yla m id e (9k ). Yield 29%. 1H NMR (CDCl3,
300 MHz): δ 11.86 (br, 1H), 9.25 (dd, J ) 1.2, 8.7 Hz, 1H),
8.29 (dd, J ) 1.2, 4.5 Hz, 1H), 7.55 (d, J ) 3.6 Hz, 1H), 7.51
(dd, J ) 4.5, 8.7 Hz, 1H), 7.06 (d, J ) 3.6 Hz, 1H), 6.90 (m,
1H), 2.04 (s, 3H), 1.90 (d, J ) 3.0 Hz, 3H). EI-MS m/z: 302
(M+). HREI-MS: exact mass calcd for C14H14N4O2S (M+),
302.0837; found, 302.0827. HPLC purity, 96.66%.
Gen er a l P r oced u r e for P r ep a r a tion of Com p ou n d s 9e
a n d 9n -p . Pyridine (0.2 mL) was added to a solution of 2,2-
dimethylpropionyl chloride in 1.5 mL of dry benzene at 0 °C.
The mixture was stirred for 5 min, and the appropriate acid
(1 mmol) was added. After 15 min, a solution of 9a (0.1 mmol)
in 1 mL of dry DMF was added. The reaction mixture was
stirred at room temperature for 8 h. The mixture was
partitioned between H2O (20 mL) and EtOAc (20 mL). The
phases were separated, and the aqueous phase was extracted
with EtOAc. The combined organic phases were then processed
in the usual way and chromatographed to yield the desired
products.
3-(2,2,2-Tr iflu or oa cet yla m in o)p yr id in e-2-ca r b oxylic
Acid Th ia zol-2-yla m id e (9e). Yield 22%. 1H NMR (CDCl3,
300 MHz): δ 12.70 (s, 1H), 9.13 (d, J ) 8.7 Hz, 1H), 8.51 (d,
J ) 4.5 Hz, 1H), 7.64 (dd, J ) 4.5, 8.7 Hz, 1H), 7.59 (d, J )
3.6 Hz, 1H), 7.14 (d, J ) 3.6 Hz, 1H). EI-MS m/z: 316 (M+).
HREI-MS: exact mass calcd for C11H7F3N4O2S (M+), 316.0242;
found, 316.0263. HPLC purity, 95.05%.
3-Ben zoyla m in op yr id in e-2-ca r boxylic Acid Th ia zol-2-
yla m id e (9q). Yield 60%. 1H NMR (CDCl3, 300 MHz): δ 12.49
(s, 1H), 11.55 (br, 1H), 9.40 (dd, J ) 1.5, 8.4 Hz, 1H), 8.36 (dd,
J ) 1.5, 4.5 Hz, 1H), 8.12-8.09 (m, 2H), 7.62-7.55 (m, 5H),
7.09 (d, J ) 3.6 Hz, 1H). EI-MS m/z: 324 (M+). HREI-MS:
exact mass calcd for C16H12N4O2S (M+), 324.0681; found,
324.0688. HPLC purity, 99.72%.
3-(2-F lu or oben zoyla m in o)p yr id in e-2-ca r boxylic Acid
Th ia zol-2-yla m id e (9r ). Yield 19%. 1H NMR (CDCl3, 300
MHz): δ 12.37 (s, 1H), 9.37 (dd, J ) 0.9, 8.7 Hz, 1H), 8.38 (dd,
J ) 0.9, 4.2 Hz, 1H), 8.07 (dd, J ) 1.8, 7.8 Hz, 1H), 7.60-7.57
(m, 2H), 7.55 (d, J ) 3.6 Hz, 1H), 7.29 (m, 2H), 7.06 (d, J )
3.6 Hz, 1H). EI-MS m/z: 342 (M+). HREI-MS: exact mass calcd
for C16H11FN4O2S (M+), 342.0587; found, 342.0561. HPLC
purity, 99.47%.
3-(3-F lu or oben zoyla m in o)p yr id in e-2-ca r boxylic Acid
Th ia zol-2-yla m id e (9s). Yield 18%. 1H NMR (CDCl3, 300
MHz): δ 12.52 (s, 1H), 11.54 (s, 1H), 9.36 (d, J ) 8.4 Hz, 1H),
8.39 (d, J ) 4.5 Hz, 1H), 7.88 (d, J ) 7.8 Hz, 1H), 7.80 (d, J )
9.3 Hz, 1H), 7.63-7.53 (m, 3H), 7.32 (dt, J ) 8.4, 6.0 Hz, 1H),
7.11 (d, J ) 3.6 Hz, 1H). EI-MS m/z: 342 (M+). HREI-MS:
exact mass calcd for C16H11FN4O2S (M+), 342.0587; found,
342.0583. HPLC purity, 99.57%.
3-(4-F lu or oben zoyla m in o)p yr id in e-2-ca r boxylic Acid
Th ia zol-2-yla m id e (9t). Yield 22%. 1H NMR (CDCl3, 300
MHz): δ 12.49 (s, 1H), 11.38 (br, 1H), 9.36 (dd, J ) 0.9, 8.4
Hz, 1H), 8.36 (dd, J ) 0.9, 4.5 Hz, 1H), 8.14-8.10 (m, 2H),
7.61-7.57 (m, 2H), 7.28-7.22 (m, 2H), 7.10 (d, J ) 3.6 Hz,
1H). 13C NMR (CDCl3, 75 MHz) δ 167.27-163.91 (J ) 252.8
Hz), 165.53, 165.12, 157.06, 142.79, 139.75, 139.47, 138.62,
131.52, 130.33, 130.21, 129.17-129.04 (J ) 9.8 Hz), 116.45-
116.16 (J ) 21.8 Hz), 114.46. EI-MS m/z: 342 (M+). HREI-
MS: exact mass calcd for C16H11FN4O2S (M+), 342.0587; found,
342.0583. HPLC purity, 99.81%.
3-(3-Met h ylb u t -2-en oyla m in o)p yr id in e-2-ca r b oxylic
Acid Th ia zol-2-yla m id e(9l). To a solution of 9a (78 mg, 0.35
mmol) in 4 mL of CH2Cl2 was added Et3N (0.16 mL, 1.15
mmol). A solution of 3-methyl-but-2-enoyl bromide (110 µL)
in 1 mL of CH2Cl2 was added at 0 °C. The mixture was stirred
for 2 h with gradual warming to room temperature. The
reaction mixture was diluted with 5 mL of EtOAc and 2 mL
of H2O. The aqueous phase was extracted with EtOAc. The
combined organic phases were then processed in the usual way
to yield the crude product, which was recrystallized from
EtOAc/petroleum ether to give compound 9l (12.5 mg, 12%).
1H NMR (CDCl3, 300 MHz): δ 11.38 (s, 1H), 9.26 (dd, J ) 1.5,
9.0 Hz, 1H), 8.28 (dd, J ) 1.5, 4.5 Hz, 1H), 7.56 (d, J ) 3.6 Hz,
1H), 7.49 (dd, J ) 4.5, 9.0 Hz, 1H), 7.07 (d, J ) 3.6 Hz, 1H),
5.90 (s, 1H), 2.25 (s, 3H), 1.97 (s, 3H). EI-MS m/z: 302 (M+).
HREI-MS: exact mass calcd for C14H14N4O2S (M+), 302.0837;
found, 302.0825. HPLC purity, 97.80%.
3-(Bu t-3-en oyla m in o)p yr id in e-2-ca r boxylic Acid Th ia -
1
zol-2-yla m id e (9n ). Yield 76%. H NMR (CDCl3, 300 MHz):
δ 11.59 (s, 1H), 11.50 (br, 1H), 9.18 (d, J ) 8.7 Hz, 1H), 8.30
(d, J ) 4.2 Hz, 1H), 7.55 (d, J ) 3.6 Hz, 1H), 7.51 (dd, J ) 4.2,
8.7 Hz, 1H), 7.07 (d, J ) 3.6 Hz, 1H), 6.08 (m, 1H), 5.39 (d, J
) 4.8 Hz, 1H), 5.35 (s, 1H), 3.30 (d, J ) 6.6 Hz, 2H). 13C NMR
(CDCl3, 75 MHz): δ 170.97, 164.88, 142.84, 142.57, 139.07,
138.37, 131.28, 130.25, 129.12, 128.81, 120.53, 114.23, 43.55.
EI-MS m/z: 288 (M+), HREI-MS: exact mass calcd for
C
13H12N4O2S (M+), 288.0681; found, 288.0683. Anal. (C15H16
N4O2S) C, H, N, S.
-
3-(4-Met h ylp en t -3-en oyla m in o)p yr id in e-2-ca r b oxylic
Acid Th ia zol-2-yla m id e (9o). Yield 63%. 1H NMR (CDCl3,
300 MHz): δ 11.65 (br, 2H), 9.21 (dd, J ) 1.5, 8.7 Hz, 1H),
8.28 (dd, J ) 1.5, 4.5 Hz, 1H), 7.55 (d, J ) 3.3 Hz, 1H), 7.49
(dd, J ) 4.5, 8.7 Hz, 1H), 7.06 (d, J ) 3.3 Hz, 1H), 5.45 (t, J )
7.2 Hz, 1H), 3.23 (d, J ) 7.2 Hz, 2H), 1.97 (s, 3H), 1.78 (s,
3H). 13C NMR (CDCl3, 75 MHz): δ 172.16, 164.65, 157.35,
142.48, 139.75, 139.06, 138.43, 131.39, 128.97, 128.77, 115.64,
114.15, 38.13, 26.14, 18.28. EI-MS m/z: 316 (M+). HREI-MS:
exact mass calcd for C15H16N4O2S (M+), 316.0994; found,
316.0999. Anal. (C15H16N4O2S) C, H, N, S.
3-(5-Met h ylh ex-4-en oyla m in o)p yr id in e-2-ca r b oxylic
Acid Th ia zol-2-yla m id e (9p ). Yield 47%. 1H NMR (CDCl3,
300 MHz): δ 11.50 (br, 2H), 9.19 (dd, J ) 1.2, 8.7 Hz, 1H),
8.28 (dd, J ) 1.2, 4.5 Hz, 1H), 7.55 (d, J ) 3.3 Hz, 1H), 7.50
(dd, J ) 4.5, 8.7 Hz, 1H), 7.07 (d, J ) 3.3 Hz, 1H), 5.18 (t, J )
7.2 Hz, 1H), 2.56 (m, 3H), 2.48 (m, 3H), 1.69 (s, 3H), 1.65 (s,
3H). 13C NMR (CDCl3, 75 MHz): δ 173.02, 164.87, 157.20,
142.43, 139.24, 138.48, 133.76, 130.95, 129.09, 128.92, 122.35,
114.27, 38.85, 25.94, 24.14, 17.97. EI-MS m/z: 330 (M+). HREI-
MS: exact mass calcd for C16H18N4O2S (M+), 330.1150; found,
330.1170. HPLC purity, 98.71%.
3-Hyd r oxyp yr id in e-2-ca r boxylic Acid Th ia zol-2-yla m -
id e (10a ). A mixture of 11 (1.39 g, 10 mmol), HOBT (1.35 g,
10 mmol), DCC (2.27 g, 11 mmol), and Et3N (0.4 mL, 30 mmol)
in dry DMF (20 mL) was stirred at room temperature for 20
min, and then thiazol-2-ylamine (1.05 g, 10.5 mmol) was
3-(3-Met h ylb u t -3-en oyla m in o)p yr id in e-2-ca r b oxylic
Acid Th ia zol-2-yla m id e (9m ). To a solution of 9a (22 mg,
0.1 mmol) in 1 mL of CH2Cl2 was added excess of Et3N (0.5
mL, 3.5 mmol). A solution of newly prepared 3-methyl-but-2-

Inhibitors of Methionine Aminopeptidases
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13 2639
added. The reaction mixture was stirred at room temperature
for 2 h and was then diluted with 100 mL of EtOAc and 40
mL of H2O. The aqueous phase was extracted with EtOAc. The
combined organic phases were then processed in the usual way
and chromatographed (3:1 petroleum ether/EtOAc) to yield 10a
(1.41 g, 64%). 1H NMR (CDCl3, 300 MHz): δ 8.17 (dd, J ) 1.5,
4.2 Hz, 1H), 7.56 (d, J ) 3.6 Hz, 1H), 7.44 (dd, J ) 4.2, 8.4 Hz,
1H), 7.39 (dd, J ) 1.5, 8.4 Hz, 1H), 7.05 (d, J ) 3.6 Hz, 1H).
13C NMR (CDCl3, 300 MHz): δ 166.43, 158.45, 156.84, 140.59,
138.56, 130.11, 130.00, 126.83, 114.58. EI-MS m/z: 221 (M+).
HREI-MS: exact mass calcd for C9H7N3O2S (M+), 221.0259;
found, 221.0235. HPLC purity, 98.46%.
Gen er a l P r oced u r e for P r ep a r a tion of Com p ou n d s
10b-u . To solution of 10a (0.1 mmol) in 2 mL of acetone was
added excess of anhydrous K2CO3 (0.5 g). The appropriate acid
chloride (0.15 mmol) was added at 0 °C. The mixture was
stirred for 2-24 h with gradual warming to room temperature.
The mixture was concentrated under reduced pressure to
remove acetone, and the residue was diluted with 5 mL of
EtOAc and 2 mL of H2O. The aqueous phase was extracted
with EtOAc. The combined organic phases were then processed
in the usual way and chromatographed to yield the desired
products.
2H), 7.51 (d, J ) 3.6 Hz, 1H), 7.24 (m, 1H), 7.01 (d, J ) 3.6
Hz, 1H), 2.02 (s, 3H), 1.94 (d, J ) 7.2 Hz, 3H). EI-MS m/z:
303(M+). HREI-MS: exact mass calcd for C14H13N3O3S (M+),
303.0678; found, 303.0661. HPLC purity, 97.49%.
3-(3-Meth ylbu t-2-en oyloxy)p yr id in e-2-ca r boxylic Acid
Th ia zol-2-yla m id e (10i). Yield 56%. 1H NMR (CDCl3, 300
MHz): δ 11.40 (br, 1H), 8.52 (dd, J ) 3.0, 3.3 Hz, 1H), 7.58 (d,
J ) 3.3 Hz, 2H), 7.50 (d, J ) 3.6 Hz, 1H), 6.99 (d, J ) 3.6 Hz,
1H), 6.10 (s, 1H), 2.22 (s, 3H), 2.03 (s, 3H). EI-MS m/z: 303-
(M+). HREI-MS: exact mass calcd for C14H13N3O3S (M+),
303.0678; found, 303.0686. HPLC purity, 97.94%.
3-[3-(2-Meth oxyp h en yl)a cr yloyloxy]pyr idin e-2-ca r box-
ylic Acid Th ia zol-2-yla m id e (10j). Yield 28%. 1H NMR
(CDCl3, 300 MHz): δ 11.24 (br, 1H), 8.55 (dd, J ) 1.5, 4.2 Hz,
1H), 8.25 (d, J ) 15.9 Hz, 1H), 7.69-7.59 (m, 3H), 7.51 (d, J
) 3.6 Hz, 1H), 7.40 (dt, J ) 1.5, 8.1 Hz, 1H), 7.03-6.94 (m,
2H), 6.99 (d, J ) 3.6 Hz, 1H), 6.89 (d, J ) 16.2 Hz, 1H), 3.92
(s, 3H). EI-MS m/z: 381 (M+). HREI-MS: exact mass calcd
for
C
19H15N3O4S (M+), 381.0778; found, 381.0777. HPLC
purity, 99.63%.
3-Ben zoyloxyp yr id in e-2-ca r b oxylic Acid Th ia zol-2-
yla m id e (10k ). Yield 47%. 1H NMR (CDCl3, 300 MHz): δ
11.33 (br, 1H), 8.60 (d, J ) 4.5 Hz, 1H), 8.29 (d, J ) 7.5 Hz,
2H), 7.75-7.63 (m, 3H), 7.56 (t, J ) 4.5 Hz, 2H), 7.50 (d, J )
3.0 Hz, 1H), 7.05 (d, J ) 3.0 Hz, 1H). EI-MS m/z: 325 (M+).
HREI-MS: exact mass calcd for C16H11N3O3S (M+), 325.0521;
found, 325.0541. HPLC purity, 97.40%.
3-Acet yloxyp yr id in e-2-ca r b oxylic Acid Th ia zol-2-yl-
a m id e (10b). Yield 23%. 1H NMR (CDCl3, 300 MHz): δ 11.24
(br, 1H), 8.54 (dd, J ) 1.8, 4.2 Hz, 1H), 7.59 (d, J ) 4.2 Hz,
1H), 7.58 (d, J ) 1.8 Hz, 1H), 7.52 (d, J ) 3.6 Hz, 1H), 7.02 (d,
J ) 3.6 Hz, 1H), 2.47 (s, 3H). 13C NMR (CDCl3, 75 MHz): δ
169.57, 160.40, 157.67, 148.38, 145.98, 139.71, 138.20, 133.71,
128.78, 113.39, 21.28. EI-MS m/z: 263 (M+). HREI-MS: exact
mass calcd for C11H9N3O3S (M+), 263.0365; found, 263.0357.
HPLC purity, 95.79%.
3-(2-Flu or oben zoyloxy)pyr idin e-2-car boxylic Acid Th i-
a zol-2-yla m id e (10l). Yield 52%. 1H NMR (CDCl3, 300
MHz): δ 11.38 (br, 1H), 8.60 (dd, J ) 1.6, 4.5 Hz, 1H), 8.27
(ddd, J ) 1.6, 7.2, 7.8 Hz, 1H), 7.74-7.60 (m, 3H), 7.50 (d, J
) 3.6, 1H), 7.36-7.20 (m, 2H), 6.98 (d, J ) 3.6 Hz, 1H). EI-
MS m/z: 343 (M+). HREI-MS: exact mass calcd for C16H10
-
3-P r op ion yloxyp yr id in e-2-ca r boxylic Acid Th ia zol-2-
yla m id e (10c). Yield 80%. 1H NMR (CDCl3, 300 MHz): δ
11.30 (br, 1H), 8.55 (dd, J ) 1.8, 4.2 Hz, 1H), 7.58 (dd, J )
2.7, 4.2 Hz, 2H), 7.52 (d, J ) 3.6 Hz, 1H), 7.02 (d, J ) 3.6 Hz,
1H), 2.80 (q, J ) 7.5 Hz, 2H), 1.33 (t, J ) 7.5 Hz, 3H). EI-MS
m/z: 277 (M+). HREI-MS: exact mass calcd for C12H11N3O3S
(M+), 277.0521; found, 277.0514. HPLC purity, 98.23%.
3-(2,2-Dim et h ylp r op ion yloxy)p yr id in e-2-ca r b oxylic
FN3O3S (M+), 343.0421; found, 343.0429. HPLC purity, 99.55%.
3-(3-Flu or oben zoyloxy)pyr idin e-2-car boxylic Acid Th i-
a zol-2-yla m id e (10m ). Yield 34%. 1H NMR (CDCl3, 300
MHz): δ 11.38 (br, 1H), 8.62 (dd, J ) 1.4, 4.3 Hz, 1H), 8.07 (d,
J ) 7.8 Hz, 1H), 7.96 (ddd, J ) 1.5, 2.5, 8.7 Hz, 1H), 7.73 (dd,
J ) 1.7, 8.0 Hz, 1H), 7.60 (dd, J ) 4.5, 8.4 Hz, 1H), 7. 57-7.52
(m, 1H), 7.50 (d, J ) 3.6 Hz, 1H), 7.38 (ddd, J ) 1.9, 7.8 Hz,
8.2 Hz, 1H), 6.98 (d, J ) 3.6 Hz, 1H). EI-MS m/z: 343(M+).
HREI-MS: exact mass calcd for C16H10FN3O3S (M+), 343.0421;
found, 343.0423. HPLC purity, 99.22%.
1
Acid Th ia zol-2-yla m id e (10d ). Yield 97%. H NMR (CDCl3,
300 MHz): δ 11.50 (br, 1H), 8.55 (dd, J ) 1.5, 4.2 Hz, 1H),
7.57 (m, 2H), 7.51 (d, J ) 3.6 Hz, 1H), 7.01 (d, J ) 3.6 Hz,
1H), 1.46 (s, 9H). EI-MS m/z: 305 (M+). HREI-MS: exact mass
calcd for C14H15N3O3S (M+), 305.0829; found, 305.0829. HPLC
purity, 99.42%.
3-(4-Flu or oben zoyloxy)pyr idin e-2-car boxylic Acid Th i-
a zol-2-yla m id e (10n ). Yield 33%. 1H NMR (CDCl3, 300
MHz): δ 11.29 (br, 1H), 8.59 (dd, J ) 1.5, 4.5 Hz, 1H), 8.33-
8.28 (m, 2H), 7.71 (dd, J ) 1.5, 8.4 Hz, 1H), 7.65 (dd, J ) 4.5,
8.4 Hz, 1H), 7.50 (d, J ) 3.6 Hz, 1H), 7.22 (dt, J ) 4.2, 8.4 Hz,
2H), 6.97 (d, J ) 3.6 Hz, 1H). EI-MS m/z: 343(M+). EI-MS
m/z: 343 (M+). HREI-MS: exact mass calcd for C16H10FN3O3S
(M+), 343.0421; found, 343.0417. HPLC purity, 99.78%.
3-(2-Nitr oben zoyloxy)p yr id in e-2-ca r boxylic Acid Th i-
a zol-2-yla m id e (10o). Yield 42%. 1H NMR (CDCl3, 300
MHz): δ 8.64 (dd, J ) 1.2, 4.2 Hz, 1H), 8.31 (dd, J ) 0.9, 7.5
Hz, 1H), 8.16 (d, J ) 7.8 Hz, 1H), 7.88 (t, J ) 7.5 Hz, 2H),
7.77-7.68 (m, 2H), 7.54 (d, J ) 3.6 Hz, 1H), 7.04 (d, J ) 3.6
Hz, 1H). EI-MS m/z: 370 (M+). HREI-MS: exact mass calcd
3-Hexa n oyloxyp yr id in e-2-ca r boxylic Acid Th ia zol-2-
yla m id e (10e). Yield 62%. 1H NMR (CDCl3, 300 MHz): δ 8.53
(dd, J ) 1.2, 4.2 Hz, 1H), 7.61-7.51 (m, 3H), 7.01 (dd, J ) 0.9,
3.6 Hz, 1H), 2.76 (t, J ) 7.5 Hz, 2H), 1.82 (m, 2H), 1.45-1.40
(m, 4H), 0.95 (t, J ) 6.9 Hz, 3H). 13C NMR (CDCl3, 75 MHz):
δ 172.36, 160.44, 157.79, 148.45, 145.83, 139.85, 138.13,
133.71, 128.66, 113.84, 34.27, 31.42, 24.34, 22.57, 14.16. EI-
MS m/z: 319 (M+). HREI-MS: exact mass calcd for C15H17N3O3S
(M+), 319.0985; found, 319.0984. HPLC purity, 98.03%.
3-(Cycloh exan ecar bon yloxy)pyr idin e-2-car boxylic Acid
Th ia zol-2-yla m id e (10f). Yield 82%. 1H NMR (CDCl3, 300
MHz): δ 8.55 (dd, J ) 1.8, 4.2 Hz, 1H), 7.58 (d, J ) 4.2 Hz,
1H), 7.55 (d, J ) 3.9 Hz, 1H), 7.52 (d, J ) 3.6 Hz, 1H), 7.02 (d,
J ) 3.6 Hz, 1H), 2.70 (m, 1H), 2.19 (m, 2H), 1.85 (m, 2H), 1.65
(m, 3H), 1.35 (m, 3H). EI-MS m/z: 331 (M+). HREI-MS: exact
mass calcd for C16H17N3O3S (M+), 331.0985; found, 331.0985.
HPLC purity, 99.11%.
for
C
16H10N4O5S (M+), 370.0372; found, 370.0367. HPLC
purity, 96.19%.
3-(3-Nitr oben zoyloxy)p yr id in e-2-ca r boxylic Acid Th i-
a zol-2-yla m id e (10p ). Yield 15%. 1H NMR (CDCl3, 300
MHz): δ 11.28 (br, 1H), 9.12 (dd, J ) 1.5, 1.8 Hz, 1H), 8.64
(dd, J ) 1.5, 4.5 Hz, 1H), 8.60 (dt, J ) 1.5, 7.5 Hz, 1H), 8.54
(ddd, J ) 1.5, 2.4, 8.1 Hz, 1H), 7.76 (m, 2H), 7.69 (dd, J ) 4.5,
8.1 Hz, 1H), 7.50 (d, J ) 3.6HZ, 1H), 6.97 (d, J ) 3.6 Hz, 1H).
EI-MS m/z: 370 (M+). HREI-MS: exact mass calcd for
3-P h en yla cetyloxyp yr id in e-2-ca r boxylic Acid Th ia zol-
1
2-yla m id e (10g). Yield 77%. H NMR (CDCl3, 300 MHz): δ
11.40 (br, 1H), 8.55 (dd, J ) 1.5, 4.2 Hz, 1H), 7.57 (d, J ) 4.2
Hz, 1H), 7.54 (d, J ) 3.6 Hz, 1H), 7.52 (d, J ) 3.6 Hz, 1H),
7.45 (m, 2H), 7.36 (m, 3H), 7.05 (d, J ) 3.6 Hz, 1H), 4.10 (s,
2H). EI-MS m/z: 339 (M+). HREI-MS: exact mass calcd for
C
16H10N4O5S (M+), 370.0366; found, 370.0364. HPLC purity,
96.58%.
3-(2-Meth oxyben zoyloxy)p yr id in e-2-ca r boxylic Acid
17H13N3O3S (M+), 339.0678; found, 339.0668. HPLC purity,
Th ia zol-2-yla m id e (10q). Yield 56%. 1H NMR (CDCl3, 300
MHz): δ 11.23 (br, 1H), 8.56 (dd, J ) 1.5, 4.2 Hz, 1H), 8.32
(dd, J ) 1.5, 7.8 Hz, 1H), 7.72 (dd, J ) 1.5, 8.4 Hz, 1H), 7.64-
7.56 (m, 2H), 7.50 (d, J ) 3.6 Hz, 1H), 7.12 (dt, J ) 1.2, 7.8
Hz, 1H), 7.05 (d, J ) 8.4 Hz, 1H), 6.97 (d, J ) 3.6 Hz, 1H),
C
96.57%.
3-(2-Meth ylbu t-2-en oyloxy)p yr id in e-2-ca r boxylic Acid
Th ia zol-2-yla m id e (10h ). Yield 76%. 1H NMR (CDCl3, 300
MHz): δ 8.56 (dd, J ) 2.7, 4.2 Hz, 1H), 7.61 (d, J ) 3.3 Hz,

2640 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13
Luo et al.
3.95 (s, 3H). 13C NMR (CDCl3, 75 MHz): δ 163.55, 160.41,
160.38, 157.85, 148.54, 145.81, 140.12, 138.10, 134.88, 134.14,
133.27, 128.55, 120.62, 118.45, 113.75, 112.33, 56.29. EI-MS
m/z: 355 (M+). HREI-MS: exact mass calcd for C17H13N3O4S
(M+), 355.0621; found, 355.0621. HPLC purity, 99.81%.
3-(2-Acetyloxyben zoyloxy)p yr id in e-2-ca r boxylic Acid
Th ia zol-2-yla m id e (10r ). Yield 35%. 1H NMR (CDCl3, 300
MHz): δ 11.22 (br, 1H), 8.60 (dd, J ) 1.5, 4.2 Hz, 1H), 8.44
(dd, J ) 1.5, 7.8 Hz, 1H), 7.72-7.62 (m, 3H), 7.51 (d, J ) 3.6
Hz, 1H), 7.45 (t, J ) 7.8 Hz, 1H), 7.20 (d, J ) 7.8 Hz, 1H),
6.99 (d, J ) 3.6 Hz, 1H), 2.28 (s, 3H). 13C NMR (CDCl3, 75
MHz): δ 169.81, 162.65, 160.16, 157.77, 151.42, 148.16, 146.15,
139.84, 138.09, 134.94, 134.01, 132.95, 128.76, 126.51, 124.01,
122.34, 113.91, 21.24. EI-MS m/z: 383 (M+). HREI-MS: exact
mass calcd for C18H13N3O5S (M+), 383.0570; found, 383.0573.
HPLC purity, 99.51%.
30271528 (F.-J .N.) and 39725032 (Q.-Z.Y.), the Qi Ming
Xing Foundation of Shanghai Ministry of Science and
Technology Grant 02QB14013 (F.-J . N.), the 863 Hi-
Tech Program Grant 2001AA234011 (F.-J . N.), the NIH
COBRE Award 1 P20 RR15563, and matching support
from the State of Kansas (Q.-Z.Y.)
Refer en ces
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mation. J . Biol. Chem. 1990, 265, 19638-19643. (b) Huang, S.;
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2) is the common target for angiogenesis inhibitors AGM-1470
and ovalicin. Chem Biol. 1997, 4, 461-471. (b) Sin, N.; Meng,
L.; Wang, M. Q.; Wen, J . J .; Bornmann, W. G.; Crews, C. M.
The anti-angiogenic agent fumagillin covalently binds and
inhibits the methionine aminopeptidase, MetAP-2. Proc. Natl.
Acad. Sci. U.S.A. 1997, 94, 6099-6103.
(5) Keding, S. J .; Dales, N. A.; Lim, S.; Beaulieu, D.; Rich, D. H.
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4463-4470.
(6) Lim, S.; Rich, D. H.; Hodges, R. S.; Smith, J . A. Pept.: Chem.,
Struct. Biol. 1994, 625-627.
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mutant, and inhibited enzymes for the mechanism of catalysis.
Biochemistry 1999, 38, 7678-7688.
(8) Morris, G. M.; Goodsell, D. S.; Halliday, R. S.; Huey, R.; Hart,
W. E.; Belew, R. K.; Olson, A. J . Automated Docking Using a
Lamarckian Genetic Algorithm and Empirical Binding Free
Energy Function. J . Comput. Chem. 1998, 19, 1639-1662.
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dase I can utilize either Zn2+ or Co2+ as a cofactor: a case of
mistaken identity. Protein Sci. 1998, 7, 2684-2687.
3-(2-Iod oben zoyloxy)p yr id in e-2-ca r boxylic Acid Th ia -
1
zol-2-yla m id e (10s). Yield 41%. H NMR (CDCl3, 300 MHz):
δ 8.55 (dd, J ) 1.5, 4.2 Hz, 1H), 8.35 (dd, J ) 1.8, 7.5 Hz, 1H),
8.04 (d, J ) 7.8 Hz, 1H), 7.79-7.59 (m, 3H), 7.51 (d, J ) 3.6
Hz, 1H), 7.24 (dt, J ) 1.5, 8.1 Hz, 1H), 6.99 (d, J ) 3.6 Hz,
1H). EI-MS m/z: 451 (M+). HREI-MS: exact mass calcd for
C
16H10IN3O3S (M+), 450.9482; found, 450.9483. HPLC purity,
98.99%.
3-(3-Met h oxy-2-n it r ob en zoyloxy)p yr id in e-2-ca r b ox-
ylic Acid Th ia zol-2-yla m id e (10t). Yield 50%. 1H NMR
(CDCl3, 300 MHz): δ 8.73 (d, J ) 4.2 Hz, 1H), 8.03 (d, J ) 8.4
Hz, 1H), 7.91-7.81 (m, 4H), 7.56 (d, J ) 3.6 Hz, 1H), 7.31 (d,
J ) 3.6 Hz, 1H), 3.98 (s, 3H). EI-MS m/z: 400 (M+). HREI-
MS: exact mass calcd for C17H12N4O6S (M+), 400.0472; found,
400.0474. HPLC purity, 95.20%.
3-(3-Br om o-4-flu or ob en zoyloxy)p yr id in e-2-ca r b oxyl-
ic Acid Th ia zol-2-yla m id e (10u ). Yield 30%. 1H NMR
(CDCl3, 300 MHz): δ 11.30 (br, 1H), 8.62 (dd, J ) 1.5, 4.5 Hz,
1H), 8.51 (dd, J ) 2.1, 6.6 Hz, 1H), 8.27-8.21 (m, 1H), 7.75-
7.64 (m, 2H), 7.51 (d, J ) 3.6 Hz, 1H), 7.29 (dd, J ) 8.4, 8.7
Hz, 1H), 6.98 (d, J ) 3.6 Hz, 1H). 13C NMR (CDCl3, 75 MHz):
δ 172.5, 164.6-161.2 (J ) 254.6 Hz), 163.9, 160.3, 148.7, 146.2,
139.8, 138.2, 136.6-136.5 (J ) 1.7 Hz), 133.7, 132.2-132.0 (J
) 8.6 Hz), 128.8, 126.8-126.7 (J ) 3.6 Hz), 117.2-116.9 (J )
23.1 Hz), 113.9, 110.0-109.7 (J ) 21.6 Hz). EI-MS m/z: 421,
423 (M+). HREI-MS: exact mass calcd for C16H9BrFN3O3S
(M+), 420.9527; found, 420.9536. HPLC purity, 96.45%.
MetAP Activity Assa ys. The thiopeptolide Met-S-Gly-Phe-
OH was synthesized in this lab according to the literature
procedure,12 and the chromogenic assays using the thiopep-
tolide were carried out as described with minor modification.
The enzymatic assay was carried out in a 100 µL system
containing 50 mM MOPS (pH 7.0), 1 mM DTNB, 100 µM
thiopeptolide, and active enzyme (final protein concentrations
of 150 nM for EcMetAP1 and 330 nM for ScMetAP1). The
activation of EcMetAP1 or ScMetAP1 was carried out by
incubating the MetAP enzyme with 25 mM HEPES (pH 7.5),
150 mM KCl, 15 mM methionine, and 500 µM CoCl2 for 30
min prior to the assay. The hydrolysis of thiopeptolide was
monitored by a microplate reader SpectraMax 340 (Molecular
Devices, Sunnyvale, CA) at 412 nm for 60 s. In the IC50
determinations, the inhibitors were diluted from 10 µM to 1
nM.
(10) D’souza, V. M.; Holz, R. C. The methionyl aminopeptidase from
Escherichia coli can function as an iron(II) enzyme. Biochemistry
1999, 38, 11079-11085.
(11) D’souza, V. M.; Swierczek, S. I.; Cosper, N. J .; Meng, L.; Ruebush,
S.; Copik, A. J .; Scott, R. A.; Holz, R. C. Kinetic and structural
characterization of manganese(II)-loaded methionyl aminopep-
tidases. Biochemistry 2002, 41, 13096-13105.
(12) Zhou, Y.; Guo, X. C.; Yi, T.; Yoshimoto, T.; Pei, D. Two continuous
spectrophotometric assays for methionine aminopeptidase. Anal.
Biochem. 2000, 280, 159-165.
Ack n ow led gm en t. This work was supported by the
National Natural Science Foundation of China Grants
J M0300532