Synthesis and structure-function analysis of Fe(II)-form-selective antibacterial inhibitors of Escherichia coli methionine aminopeptidase

Article abstract of DOI:10.1016/j.bmcl.2009.01.011

Methionine aminopeptidase (MetAP) is a promising target for the development of novel antibacterial, antifungal and anticancer therapy. Based on our previous results, catechol derivatives coupled with a thiazole or thiophene moiety showed high potency and selectivity toward the Fe(II)-form of Escherichia coli MetAP, and some of them clearly showed antibacterial activity, indicating that Fe(II) is likely the physiologically relevant metal for MetAP in E. coli and other bacterial cells. To further understand the structure-function relationship of these Fe(II)-form selective MetAP inhibitors, a series of catechol derivatives was designed and synthesized by replacement of the thiazole or thiophene moiety with different five-membered and six-membered heterocycles. Inhibitory activities of these newly synthesized MetAP inhibitors indicate that many five- and six-membered rings can be accommodated by MetAP and potency on the Fe(II)-form can be improved by introducing substitutions on the heterocyles to explore additional interactions with the enzyme. The furan-containing catechols 11-13 showed the highest potency at 1 μM on the Fe(II)-form MetAP, and they were also among the best inhibitors for growth inhibition against E. coli AS19 strain. These findings provide useful information for the design and discovery of more effective MetAP inhibitors for therapeutic applications.

Full text of DOI:10.1016/j.bmcl.2009.01.011

Bioorganic & Medicinal Chemistry Letters 19 (2009) 1080–1083
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and structure-function analysis of Fe(II)-form-selective
antibacterial inhibitors of Escherichia coli methionine aminopeptidase
*
Wen-Long Wang, Sergio C. Chai, Qi-Zhuang Ye
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, 635 Barnhill Drive, Indianapolis, IN 46202, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Methionine aminopeptidase (MetAP) is a promising target for the development of novel antibacterial,
antifungal and anticancer therapy. Based on our previous results, catechol derivatives coupled with a thi-
azole or thiophene moiety showed high potency and selectivity toward the Fe(II)-form of Escherichia coli
MetAP, and some of them clearly showed antibacterial activity, indicating that Fe(II) is likely the physi-
ologically relevant metal for MetAP in E. coli and other bacterial cells. To further understand the struc-
Received 18 November 2008
Revised 5 January 2009
Accepted 6 January 2009
Available online 10 January 2009
ture-function relationship of these Fe(II)-form selective MetAP inhibitors,
a series of catechol
Keywords:
derivatives was designed and synthesized by replacement of the thiazole or thiophene moiety with dif-
ferent five-membered and six-membered heterocycles. Inhibitory activities of these newly synthesized
MetAP inhibitors indicate that many five- and six-membered rings can be accommodated by MetAP
and potency on the Fe(II)-form can be improved by introducing substitutions on the heterocyles to
explore additional interactions with the enzyme. The furan-containing catechols 11–13 showed the high-
Methionine aminopeptidase
Drug discovery
Metalloform
Catechol
Antibacterial
Protease
est potency at 1 lM on the Fe(II)-form MetAP, and they were also among the best inhibitors for growth
inhibition against E. coli AS19 strain. These findings provide useful information for the design and discov-
ery of more effective MetAP inhibitors for therapeutic applications.
Ó 2009 Elsevier Ltd. All rights reserved.
Methionine aminopeptidase (MetAP) plays an important role in
removing the N-terminal methionine from nascent proteins in all
types of cells and is one of the essential enzymes required for bac-
terial survival.1–3 Inhibitors of MetAPs are of considerable interest
as potential antibacterial, antifungal and anticancer agents.4,5 All
MetAPs require a divalent metal ion for activation, such as Co
(II), Mn (II), Ni (II), Zn(II), or Fe (II), but it is uncertain which of
these ions is the most important in vivo.6–8 Most of current MetAP
inhibitors show potent activity in vitro but often fail to show po-
tency in vivo .9–11 Although other factors, such as difficulty in
cell-wall penetration, should be considered, it is possible that the
lack of cellular efficacy for MetAP inhibitors may be partly due to
a disparity between the metalloform of MetAP tested in vitro and
the one that is physiologically important in cells. For developing
MetAP inhibitors as therapeutics, it is critical to clarify the divalent
metal ion that activates MetAP in a cellular environment and make
sure that the MetAP inhibitors are effective in inhibiting the phys-
iologically relevant metalloform of MetAP.
throughput screening of a large diverse chemical library of small or-
ganic compounds, we have identified several MetAP inhibitors with
high potency and superb selectivity toward either the Co(II)-form or
the Mn(II)-form of Escherichia coli MetAP.12 Recently, we discovered
additional inhibitors with selectivity for the Fe(II)-form of E. coli Me-
tAP.13,14 A unique structural feature for these Fe(II)-form selective
inhibitors is the requirement of a catechol moiety for their inhibitory
activity. Initial structure-function studies with a series of thiazole
and thiophene derivatives lead to the conclusion that Fe(II) is the
likely metal used by MetAP in bacterial cellular environment.14 We
also obtained an X-ray structure of E. coli MetAP in complex with
one of the inhibitors and confirmed that these inhibitors directly
interact with MetAP at the active site with the catechol moiety che-
lating with the catalytic metal ions.14 In this paper, we report our ex-
tended structure-function studies, in which we kept the essential
catechol moiety but included additional five- and six-membered
heterocyles in place of the thiazole and thiophene moieties. We ob-
served that some of these derivatives showed improved potency on
the Fe(II)-form of purified MetAP and displayed considerable anti-
bacterial activity.
Our own work in this field has been focused on discovering un-
ique MetAP inhibitors that can distinguish different metalloforms
of MetAP as research tools for the clarification and developing these
inhibitors as early leads for antibacterial compounds.11–14 By high
Synthesis of compound 1 is outlined in Scheme 1. The commer-
cially available 2,3- dihydroxybenzoic acid 14 was coupled with
Gly-OMe in the presence of HOBt and EDCI to yield compound
15, followed by dehydration in the presence of POCl3 to produce
compound 1 in 30% yield.
* Corresponding author. Tel.: +1 317 278 0304; fax: +1 317 274 4686.
E-mail address: yeq@iupui.edu (Q.-Z. Ye).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.01.011

W.-L. Wang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1080–1083
1081
OMe
O
OMe
O
O
O
HO
HO
HO
HO
HO
HO
a
b
N
OH
N
H
15
14
1
Scheme 1. Reagents and conditions: (a) EDCI, HOBt, Gly-OMe, DMF, 70%; (b) POCl3, 90 °C, 30%.
HO
HO
COOH
BnO
BnO
COOH
BnO
BnO
COCl
a
c
b
14
16
17
O
O
O
N
N
e
BnO
BnO
f
N
BnO
BnO
BnO
BnO
COOH
NHR
3a 4a
COOEt
O
18
19
,
g
d
O
O
N
N
HO
HO
HO
HO
COOEt
NHR
O
2
3 4
,
Scheme 2. Reagents and conditions: (a) BnBr, K2CO3, Acetone, reflux overnight, 90%; then NaOH, H2O, MeOH, reflux, 2 h, 90%; (b) oxalyl chloride, DMF, DCM; (c) Ethyl
isocyanoacetate, Et3N, THF, 70%; (d) H2, Pd/C, MeOH, 90%; (e) LiOH, MeOH, H2O, 100%; (f) EDCI, amine, DMAP, DCM, 50–60%; (g) BCl3, DCM, À78 °C–rt, 40–50%.
a-
Compounds 2–4 were synthesized by the route illustrated in
Scheme 2. The acid 14 was first bis-benzylated with three equiva-
lents of benzyl bromide in acetone to obtain free carboxylic acid
1615, followed by treatment of oxalyl chloride with DMF as catalyst
to form compound 17. Compound 18 was obtained by the reaction
1,2-dibenzyloxybenzene 20 in 80% yield, followed by iodination
in the presence of iodine activated by mercuric oxide to yield
21.17 Subsequent standard Sonogashira coupling reaction employ-
ing TMS-Acetylene (TMS = trimethylsilyl) led to the formation of
TMS-protected alkyne 22 in 70% yield.18 The desired building block
23 was obtained in 80% yield by deprotection of 22 with K2CO3 in
methanol at room temperature.19 Then the mixture of benzyl bro-
mide, sodium azide and phenylacetylene 23 in distilled water in
the presence of CuI was heated at 65 °C (oil bath) to form com-
pound 24 in 85% yield, followed by hydrogenolysis to yield final
compound 5 in 80% yield.20
of methyl a-isocyanoacetate with compound 17 in the presence of
triethylamine.16 Hydrogenolysis of compound 18 produced com-
pound 2. Further basic hydrolysis of 18 gave compound 19, fol-
lowed by condensation with appropriate amine in the presence
of EDC in DMF, afforded 3a–4a, which were transferred into com-
pounds 3–4, respectively.
Compound 5 was produced by the route shown in Scheme 3.
The preparation commenced with the reaction of catechol with
benzyl bromide and potassium carbonate in acetone, providing
Synthesis of compounds 6–13 was outlined in Scheme 4. Suzuki
coupling of 3,4-dimethoxyphenylboronic acid with appropriate hal-
ogenated compounds with a five-membered or six-membered het-
HO
HO
BnO
BnO
BnO
BnO
BnO
a
b
c
d
I
BnO
SiMe3
20
BnO
BnO
21
22
BnO
BnO
HO
HO
f
e
N
N
N
N
N
Ph
Ph
N
23
24
5
Scheme 3. Reagents and conditions: (a) BnBr, K2CO3, Acetone, 80%; (b) I2, HgO, DCM, rt, 15 h, 85%; (c) iPr2NH, 10 mol% (PPh3)2PdCl2, 5 mmol% CuI, TMS-acetylene, reflux, 70%;
(d) K2CO3, MeOH, 80%; (e) BnBr, NaN3, 0.1 mol% CuI, H2O, 65 °C, 85%; (f) H2, 10% Pd/C, MeOH, 80%.
MeO
MeO
B(OH)2
MeO
MeO
R
HO
HO
R
a
b
6a - 13a
6 - 13
Scheme 4. Reagents and conditions: (a) Pd(PPh3)4, 2 N Na2 CO3, RBr, DMF, 80 °C, 40–85%; (b) 1 M BCl3, DCM, À78 °C–rt, 30–60% .

1082
W.-L. Wang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1080–1083
Table 1
Inhibition of different metalloforms of purified E. coli MetAP and inhibition of growth of E. coli AS19 cells by catechol-containing MetAP inhibitorsa
b
Compound
Structure
Inhibition of metal-activated MetAP enzymes, IC50
,
lM
Inhibition of E. coli cell growth, IC50 (lM)
Co(II)
Mn(II)
Fe(II)
OMe
O
O
1
>100
>100
11.2
228 (164)
HO
HO
N
N
HO
HO
2
3
4
5
6
7
8
9
>100
>100
>100
24.6
>100
>100
>100
19.5
14.0
11.0
13.4
16.1
25.5
10.6
19.3
>100
120 (99)
165 (199)
97.2 (99)
97.7 (79)
>250
OEt
O
O
N
HO
HO
N
H
O
O
N
HO
HO
N
H
O
N
N
N
Bn
HO
HO
N
HO
HO
>100
52.6
>100
>100
>100
>100
N
N
F
N
N
HO
HO
206 (80)
211 (106)
>250
HO
HO
>100
>100
N
HO
HO
N
O
NH
NH
O
O
10
11
>100
22.5
>100
14.8
55.0 (26)
43.9 (29)
HO
HO
O
7.9
1.2
HO
HO

W.-L. Wang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1080–1083
1083
Table 1 (continued)
b
Compound
Structure
Inhibition of metal-activated MetAP enzymes, IC50
,
lM
Inhibition of E. coli cell growth, IC50 (lM)
Co(II)
Mn(II)
Fe(II)
O
O
NH
NH
O
O
12
30.1
11.8
0.9
23.4 (19)
HO
HO
13
21.2
6.7
1.0
50.8 (39)
HO
HO
a
Relative standard derivations are <20% in all values.
Numbers in parenthesis are MIC values in lg/mL.
b
erocycle yielded compound 6a–13a (40–85% yield).21 Demethyla-
tion in the presence of BCl3 yielded compounds 6–13 (30–60% yield).
Evaluation of inhibitory activity of these compounds (Table 1)
was performed by using purified apoenzyme of E. coli MetAP that
was activated by Co(II), Mn(II) or Fe(II) during activity assays.14
Previously, we coupled thiazole or thiophene moieties to the cate-
chol moiety14, and here we present findings on other catechol
derivatives with different five- or six-membered heterocyclic rings.
It is clear that when the catechol moiety is intact, different hetero-
cyclic rings can be substituted to maintain or improve potency and
selectivity on MetAP. The four five-membered oxazole analogs (1–
4) maintained the potency and selectivity on the Fe(II)-form of
E. coli MetAP, so were the three six-membered pyridine and pyrim-
idine analogs (6–8). The outliers were the triazole derivative 5 and
the imidazole derivative 9. The former (5) maintained potency on
the Fe(II)-form but gained activity on the Co(II)- and Mn(II)-forms,
and therefore it lost selectivity. The latter (9) did not show activity
on all three metalloforms tested. The reason for the inactivity of 9
is not clear, because it is small enough, comparing with 2–4, to fit
into the active site pocket. For 5, the addition of benzyl group may
introduce extra binding interaction to tilt the catechol moiety
slightly, affecting its interaction with the metal ions. The four fur-
an-containing compounds 10–13 with an amide group attached to
the furan ring are very interesting. By changing substitution on the
amide nitrogen, the potency on the Fe(II)-form increased from
showed that the growth inhibition correlates with MetAP
inhibition.13
In conclusion, the structure-function analysis of derivatives of
initial inhibitors of the Fe(II)-form of E. coli MetAP clearly indicates
that various five- and six-membered rings can be accommodated
by MetAP and potency on the Fe(II)-form, which is the physiolog-
ically relevant metalloform13, can be enhanced by introducing
additional groups to the heterocyclic rings. These findings provide
a new starting point for the design and discovery of more potent
antibacterial MetAP inhibitors.
Acknowledgment
This research was supported by NIH Grant AI065898 (Q.-Z.Y.).
References and notes
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14.8 lM for 10 to around 1 lM for 11–13. This potency is better
than thiazoles or thiophenes we reported previously.14 Compounds
11–13 also showed considerable potency on the Co(II)- and Mn(II)-
forms. Likely, the side chain groups found additional binding inter-
actions at or near the active site.
MetAP is an essential enzyme in bacteria, and gene knockout
experiments showed lethal phenotype when the functional MetAP
gene was absent.2,3 Conceivably, inhibition of MetAP will lead to
growth inhibition of bacterial cells. To minimize complications of
cell penetration by these inhibitors, we used E. coli strain AS19 to
test our MetAP inhibitors, which has unspecified mutations on its
cell membrane that make it more permeable to small organic com-
pounds.22 Consistent with the data from enzyme inhibition, the
better inhibitors of the Fe(II)-form MetAP showed better growth
inhibition of the E. coli cells, and the best inhibitors for both the en-
zyme activity and cell growth are 11–13. Previous experiments by
monitoring N-terminal processing of a recombinant GST protein
19. Lee, J.; Cha, J. Tetrahedron 2000, 56, 10175.
20. Wang, Z. X.; Zhao, Z. G. J. Heterocyclic. Chem. 2007, 44, 89.
21. Pereira, R.; Iglesias, B.; de Lera, A. R. Tetrahedron 2001, 57, 7871.
22. Zorzopulos, J.; de Long, S.; Chapman, V.; Kozloff, L. M. FEMS Microbiol. Lett.
1989, 52, 23.