Design and synthesis of 2-pyridones as novel inhibitors of the Bacillus anthracis enoyl-ACP reductase

Article abstract of DOI:10.1016/j.bmcl.2008.05.004

Enoyl-ACP reductase (ENR), the product of the FabI gene, from Bacillus anthracis (BaENR) is responsible for catalyzing the final step of bacterial fatty acid biosynthesis. A number of novel 2-pyridone derivatives were synthesized and shown to be potent inhibitors of BaENR.

Full text of DOI:10.1016/j.bmcl.2008.05.004

Bioorganic & Medicinal Chemistry Letters 18 (2008) 3565–3569
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of 2-pyridones as novel inhibitors of the Bacillus anthracis
enoyl-ACP reductase
Suresh K. Tipparaju a, Sipak Joyasawal a, Sara Forrester b, Debbie C. Mulhearn b, Scott Pegan b,
a,b,
a,
*
Michael E. Johnson b, , Andrew D. Mesecar
, Alan P. Kozikowski
*
*
a Drug Discovery Program, Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, 833 S. Wood Street, Chicago, IL 60612, USA
b Center for Pharmaceutical Biotechnology, University of Illinois at Chicago, 900 S. Ashland Avenue, Chicago, IL 60607, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Enoyl-ACP reductase (ENR), the product of the FabI gene, from Bacillus anthracis (BaENR) is responsible for
catalyzing the final step of bacterial fatty acid biosynthesis. A number of novel 2-pyridone derivatives
were synthesized and shown to be potent inhibitors of BaENR.
Received 20 March 2008
Revised 28 April 2008
Accepted 1 May 2008
Available online 4 May 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keyword:
Anthrax
Fatty acids are an essential source of energy for organisms from
all taxa. Fatty acid synthesis in mammals is substantially different
from that in bacteria. In mammals, the fatty acid synthesis involves
a single multifunctional enzyme–acyl carrier protein (ACP) com-
plex. In bacteria, the synthesis utilizes several small monofunction-
al enzymes that operate in conjunction with ACP-associated
substrates.1 Thus, it is possible to selectively target key enzymes
in the bacterial fatty acid biosynthesis.2
The final and rate-determining step of chain elongation in the
bacterial fatty acid biosynthesis is the reduction of enoyl-ACP to
an acyl-ACP, which is catalyzed by the enzyme–enoyl–acyl carrier
protein reductase. Considerable research over the past few years
has shown that ENR is the target of a number of known antibacte-
rial agents, including isoniazid,3 diazaboranes,4 and triclosan.5 Tar-
geting ENR is an attractive approach for the development of novel
antibacterials, which has been validated by the recent discovery of
several other small molecule inhibitors,6 including those that exhi-
bit potent in vitro activity against clinical isolates of methicillin-
resistant Staphylococcus epidermidis and Staphylococcus aureus.7
As part of an on-going program to develop novel therapeutics
for anthrax, we recently reported that the diphenyl ether-triclosan
and a number of its aryl ether derivatives inhibit BaENR.8 Although
triclosan is a potent inhibitor of BaENR (IC50 = 0.5 lM, MIC = 3.1 lg/
mL),9 a major caveat in developing triclosan or its derivatives as
drugs is the metabolic liability of the phenolic hydroxyl group.10
In addition, triclosan-like diphenyl ethers are fairly lipophilic mol-
ecules and pose serious solubility problems. In order to overcome
these structural drawbacks, it is important to design compounds
in which the phenolic group of ring A of triclosan is replaced by
a more metabolically stable functionality. We report herein our ef-
forts toward addressing these issues by developing novel scaffolds
that replace the phenolic ring of triclosan with other heterocyclic
rings that retain the essential structural features of triclosan re-
quired for interaction with ENR, such as p-stacking of the aromatic
ring with NAD+, hydrogen bonding of the phenolic OH group with
the ribose, as well as optimal flexibility of the two rings in order to
allow complementary interactions with the active site.9
Our structure optimization studies were based on predicted
binding interactions of 2-pyridones with the enzyme active site.
Triclosan and synthesized pyridones were docked into the BaENR
crystal structure9 using the GOLD-docking program.11 It was ob-
served that the 2-pyridones dock to BaENR in the same binding
pocket as that of triclosan, and maintain similar H-bonding inter-
actions with the residues in the active site. Figure 1 shows the sim-
ilarities in the binding geometry of triclosan and a representative
2-pyridone, 2. Figure 2 shows the crystal structure of triclosan
bound to BaENR and the GOLD docking conformations of com-
pounds 2 and 35 in the active site. The X-ray structure of triclosan
bound to BaENR shows that the phenolic hydroxyl group on ring A
is involved in two hydrogen bonds, one with Tyr157 (OH) and the
other to the 20-hydroxyl group of nicotinamide ribose (shown in
Fig. 2A).9 These interactions appear to be preserved in the GOLD-
docking conformation of the pyridones as well, and show that
the oxygen on the carbonyl group of ring A is involved in a hydro-
gen bonding interaction with Tyr157 (OH) and the NAD+ (shown in
Fig. 2B and C). As seen in Figure 1, the ring A pocket of the
* Corresponding authors. Tel.: +1 312 996 9114; fax: +1 312 413 9303 (M.E.J.);
tel.: +1 312 996 1877; fax: +1 312 413 9303(A.D.M.); tel.: +1 312 996 7577; fax: +1
312 413 0577 (A.P.K.).
E-mail addresses: mjohnson@uic.edu (M.E. Johnson), mesecar@uic.edu (A.D.
Mesecar), kozikowa@uic.edu (A.P. Kozikowski).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.05.004

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S. K. Tipparaju et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3565–3569
B
NAD+-
sugar
A
NAD+-
Tyr157 sugar
Tyr157
Ala97
NAD+-
nicotinamide
Ala97
NAD+-
nicotinamide
Pro192
O
Cl
B
OH
A
Cl
B
1'
O
N1
A
Pro192
4'
3
O
Cl
Cl
Val154
Val154
Leu102
Val201
Leu102
Val201
Arg99
Arg99
Phe204
Phe204
Ile207
Ile207
Figure 1. Schematic representation of residues in the binding pockets of rings A and B of (A) triclosan and (B) compound 2.
Figure 2. (A) Crystal structure of triclosan bound to BaENR. ENR atoms are colored by atom type, NAD+ is green, and triclosan is magenta. (B) GOLD-docking conformation of 2
against the crystal structure of BaENR. ENR atoms are colored by atom type, NAD+ is green, and 2 is cyan. Distances between atoms that are close enough to be within
hydrogen bonding range are shown in green. (C) GOLD-docking conformation of 35 against the crystal structure of BaENR. ENR atoms are colored by atom type, NAD+ is green,
and 35 is coral.
pyridones is surrounded by Val 154, Val 201, Ile 207, and Phe 204,
sulted in the carboxylic acid 6, hydrolysis in the presence of hydro-
gen peroxide gave the carboxamide 7. Isoxazole 10 was prepared
by treating 9 with ethyl chlorooximidoacetate in the presence of
a base.13 Rapid and selective O-debenzylation of 4-benzyloxy-
2(1H)-pyridone derivatives occurred when treated with Pd/C un-
der atmospheric pressure of hydrogen to give compounds 11 and
12. Compound 13 was obtained by treatment of 12 with BBr3.
The above 4-hydroxy-pyridones were elaborated into a number
of derivatives via a series of alkylation reactions (Scheme 2). Com-
pound 14 was prepared by benzylation of 4-hydroxy-pyridone
with 4-cyanobenzyl bromide. Naphthyl derivatives 15 and 16 were
obtained by using a similar procedure. The common synthetic
intermediates 17 were prepared by alkylating the 4-hydroxy-pyri-
dones with 1,3-dibromopropane. Compounds 18–20 were pre-
pared in turn by N-alkylation of carbazole with these
intermediates. Compounds 21–23 were synthesized using a similar
protocol.
The synthesis of the 3-substituted 2-pyridones and N-oxide
derivatives is shown in Scheme 3. Pyridones 33–35 were synthe-
sized by acetic anhydride mediated rearrangement of the corre-
sponding N-oxides, followed by acidic hydrolysis.14 The N-oxides
were synthesized from the corresponding pyridines by m-CPBA
oxidation. While the ethers 27 and 28 were prepared by standard
coupling reactions, intermediate 29 was obtained by treating 2-
benzoyloxy-5-bromopyridine with benzyltrimethyltin under Stille
reaction conditions.
and thus appears to be dominated by
hydrophobicity.
a high amount of
The synthesis of compounds 1–3, 5, and 9 involved selective N-
alkylation of the commercially available 4-benzyloxy-2(1H)-pyri-
done with the corresponding benzyl halides according to the pro-
cedure described by Conreaux et al. (Scheme 1).12 Reduction of 3
gave the amino compound 4, which was further converted to the
acetamide 8. While alkaline hydrolysis of the benzonitrile 5 re-
O
O
R1
NH
N
N
a
BnO
BnO
BnO
R2
1, R1 = R2 = H
2, R1 = Cl, R2 = H
f
O
3, R1 = Cl, R2 = NO2
4, R1 = Cl, R2 = NH2
5, R1 = Cl, R2 = CN
6, R1 = Cl, R2 = CO2H
7, R1 = Cl, R2 = CONH2
b
c
e
d
9
g
8, R1 = Cl, R2 = NHCOCH3
R1
10
R1
O
O
N
N
h
HO
R2
BnO
R2
11, R1 = R2 = H
The compounds synthesized were evaluated for their BaENR
inhibitory and anitibacterial activities.15 Inhibitor 2 appears to be
the best compound in this series and was considered as a lead.
As expected from the interactions of hydrophobic residues located
around the 3-position of the pyridone ring in the active site, replac-
ing the benzyloxy group at this position with a more polar hydroxy
group led to a substantial decrease in the ENR inhibitory activity
(Table 1. compounds 11–13). A four–fold improvement in the en-
12, R1 = H, R2 = OMe
13, R1 = H, R2 = OH
i
Scheme 1. Reagents and conditions: (a) KOtBu, TBAI, THF, ArCH2X, 0–25 °C, 16 h;
(b) NaBH4, Cu(OAc)2, THF, 0 °C, 40 min; (c) 25% NaOH, EtOH, reflux, 20 h; (d) 35%
H2O2, 3 N NaOH, EtOH, 30 °C, 20 h; (e) Ac2O, DMAP, Et3N, CH2Cl2, 0–25 °C, 3 h; (f)
KOtBu, TBAI, THF, propargyl bromide, 0–25 °C, 16 h; (g) ethyl chlorooximidoacetate,
Et3N, rt, 5 h; (h) Pd/C, H2, MeOH, rt, 10–15 min; (i) BBr3, CH2Cl2, ꢀ78 °C to rt, 12 h.

S. K. Tipparaju et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3565–3569
R1
3567
O
O
R1
N
a
N
O
R2
14, R1 = Cl, R2 = H
R1
c
R3
O
R2
O
NC
18, R1 = Cl, R2 = H, R3 = carbazolyl
19, R1 = H, R2 = OMe, R3 = carbazolyl
20, R1 = R2 = H, R3 = carbazolyl
O
R1
N
b
e
N
Br
O
R2
O
R1
17a, R1 = H, R2 = H or OMe
17b, R1 = Cl or H, R2 = H
HO
R2
N
O
R1
d
R3
O
R2
N
21, R1 = Cl, R2 = H, R3 = indolyl
22, R1 = Cl, R2 = H, R3 = bezotriazolyl
23, R1 = Cl, R2 = H, R3 = imidazolyl
R3
O
R2
15, R1 = Cl, R2 = H, R3 = 1-naphthyl
16, R1 = Cl, R2 = H, R3 = 2-naphthyl
Scheme 2. Reagents and conditions: (a) NaH, DMF/THF, 4-CN-PhCH2Br, 0–25 °C, 12 h; (b) NaH, DMF, 1,3-dibromopropane, 0–25 °C, 6 h; (c) NaH, DMF, carbazole, 0–25 °C,
12 h; (d) For 21: NaH, DMF, indole, rt, 16 h; for 22: benzotriazole, DMF, K2CO3, rt, 12 h; for 23: imidazole, KOH, THF, 85 °C, 16 h; (e) NaH, DMF/THF, 1- or 2-bromomethyl
naphthalene, 0–25 °C, 12 h.
O
N
Cl
Cl
Cl
O
O
Br
N
O
N
Br
O
b
f
d
N
N
MeO
X1
Cl
Cl
Cl
Y
27, X1 = H
28, X1 = Cl
d
36
Cl
37
Cl
24,Y= OMe
25,Y= F
26,Y= OBn
c
a
X1
X2
Z
O
Y
Z
d
e
HN
N
N
X2
X1
Y
BnO
33, Y = OMe, X1 = H, X2 = Cl, Z = O
34, Y = OMe, X1 = X2 = Cl, Z = O
35, Y = OBn, X1 = X2 = H, Z = CH2
30, Y = OMe, X1 = H, X2 = Cl, Z = O
31, Y = OMe, X1 = X2 = Cl, Z = O
32, Y = OBn, X1 = X2 = H, Z = CH2
29
Scheme 3. Reagents and conditions: (a) benzyl alcohol, NaH, THF, rt, 12 h; (b) for 27: KOH-CuCO3, 2-chlorophenol, 180 °C, 15 h; For 28: 2,4-dichlorophenol, KOtBu, DMF, rt,
3 h, then at 45 °C under vacuum, Cu(OTf)2ꢁPhCH3, DMF, reflux, 16 h; (c) trimethylbenzylstannane, Pd(PPh3)4, DMF, 80 °C, 12 h; (d) m-chloroperbenzoic acid, CHCl3, rt, 12 h; (e)
Ac2O, reflux, 5 h, 1 N HCl, 100 °C, 12 h; (f) KOH–CuCO3, 2,4-dichlorophenol, 180 °C, 15 h.
zyme inhibitory activity was observed when a chlorine atom is
present at the 20-position on the aromatic ring B (cf. 1 vs 2). A sim-
ilar improvement in the binding affinity of triclosan derivatives to
the BaENR active site was recently observed by us.8 Attachment of
an electron withdrawing cyano group to the aromatic ring of the
benzyloxy moiety resulted in 14, which showed an ENR inhibitory
activity lower than the lead compound. Thus, we explored the
activities of compounds with other hydrophobic substitutents at
the 3-position of the pyridone ring. Introduction of 1- and 2-naph-
thyl groups resulted in compounds 15 and 16 whose enzymatic
activity was comparable to the lead compound. Introduction of a
bulkier carbazole unit, tethered with a three-carbon chain on the
other hand, decreased the BaENR inhibitory activity (Table 1, com-
pounds 18–20). Again, the importance of having a chlorine atom at
the 20-position on ring B to improve the inhibitory activity of these
compounds is evident by comparing the activities of 18 and 20.
Replacing the carbazole unit with a smaller indole moiety resulted
in twofold improvement in the ENR inhibitory activity (compound
21), while a benzotriazolyl substitution resulted in the compound
(22) with an ENR inhibitory activity comparable to that of the lead
compound. Introduction of an imidazolyl unit did not improve the
activity.
compounds 3–8 with various functional groups at the 40-position of
the ring B. Compound 4, bearing an amino group at the 40-position
turned out to be the best compound with an IC50 of 0.8 lM. Conver-
sion of the amino functionality into an acetamide (compound 8) re-
duced the BaENR inhibitory activity by half. Thus, it appears that the
presence of an electron-donating group at the 40-position is able to
enhance the interaction of ligands with the enzyme active site. At-
tempts to replace the aromatic ring B of these 2-pyridones with an
acetylene (compound 9) or an isoxazole (compound 10) were not
successful in improving the activity (Table 2).
We briefly explored the activities of C-substituted 2-pyridones
that are structurally similar to the N-substituted 2-pyridones dis-
cussed above (compounds 33–35). These C-substituted pyridones
are capable of existing in their enol form as hydroxypyridines, and
thus closely mimic triclosan in structure. The activities of these com-
pounds are shown in Table 2. It is gratifying to note that the novel C-
substituted 2-pyridone, 35 showed a 10-fold improvement in ENR
inhibitory activity over its N-substituted analog 1. The GOLD-dock-
ing conformation of 35 inFigure 2C suggests a nearly identical orien-
tation of the C-substituted 2-pyridones compared to the N-
substituted pyridones. Although the origin of improved activity of
compound 35 is not completely clear at this stage, the pyridone
NH and the nicotinamide ring are about 3.6 Å apart and thus ligand
binding stabilization from an N–Hꢁ ꢁ ꢁp interaction cannot be ruled
out.16 Moderate ENR inhibition was observed by the 3-phenoxy-2-
pyridones 33 and 34. Among the pyridine N-oxides, compound 37
exhibited modest ENR inhibition, while compound 30 was inactive.
We explored the SAR of the 2-pyridones by functionalizing ring B.
From the docking conformations of the lead compound into the
BaENR X-ray crystal structure (Fig. 2B), we anticipated that hydro-
gen bond donors/acceptors at the 40-position would be ideally posi-
tionedto interactwith eitherAla97orArg99. Hence, wesynthesized

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S. K. Tipparaju et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3565–3569
Table 1
Table 2
BaENR inhibitory activities of compounds
BaENR inhibitory activities of compounds
Compounda
Structure
IC50 (lM)
O
R2
O
N
R1
R3
9
>100b
N
N
BnO
BnO
Compound
R1
R2
R3
IC50 (lM)
>100a
O
O
11
12
13
1
OH
OH
OH
BnO
H
H
H
H
Cl
Cl
H
OMe
OH
H
H
H
>100a
10
35
33
34
30
>100b
>100a
OEt
O
6.8 0.8
1.5 0.1
2.7 0.4
N
2b
14
BnO
4-CN-PhCH2O
O
O
O
O
0.7 0.4
18.8 4.2
23.7 11.7
>100b
HN
BnO
15
16
Cl
Cl
H
H
1.5 0.8
1.1 0.1
Cl
Cl
Cl
O
O
O
O
HN
MeO
N
N
N
O
O
O
HN
18
19
20
Cl
H
H
H
>6c
MeO
Cl
O
N
OMe
>3d
MeO
O
Cl
N
O
37
21.4 7.7
H
>100a
Cl
Cl
a
MIC values against DANR B. anthracis were >100 lg/mL.
BaENR inhibition was less than 30% at 100 lM.
b
21
22
23
Cl
Cl
Cl
H
H
H
0.8 0.2
1.6 0.3
28.0 6.2
N
N
O
O
BaENR inhibition and improving antibacterial activities of these
compounds.
Acknowledgments
N
N
This work was funded by a grant from NIH/NIAID (U19
AI056575) and a contract from DOD (W81XWH-07-1-0445).
N
N
O
Supplementary data
5
BnO
BnO
BnO
BnO
BnO
BnO
Cl
Cl
Cl
Cl
Cl
Cl
CN
7.0 1.1
3.6 0.3
23.8 2.5
3.7 1.1
0.8 0.1
1.8 0.5
7
CONH2
COOH
NO2
NH2
NHAc
6b
3
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2008.05.004.
4
8
References and notes
a
BaENR inhibition was less than 30% at 100 lM.
b
MIC values against DANR B. anthracis15 for compound 2: 16 lg/mL, for com-
1. (a) Rock, C. O.; Jackowski, S. Biochem. Biophys. Res. Commun. 2002, 292, 1155;
(b) Wakil, S. J. Biochemistry 1989, 28, 4523.
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Science (New York, NY) 1998, 279, 98.
pound 6: 74 lg/mL. MICs for all other compounds in the table are >80 lg/mL.
c
The inhibitor precipitated at concentrations >6 lM.
The inhibitor precipitated at concentrations >3 lM.
d
4. Levy, C. W.; Baldock, C.; Wallace, A. J.; Sedelnikova, S.; Viner, R. C.; Clough, J. M.;
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In conclusion, we have identified certain 2-pyridone derivatives
as novel, small-molecule inhibitors of bacterial enoyl-ACP reduc-
tase (ENR) from B. anthracis. Compound 2 showed good ENR-inhib-
itory activity as well as reasonable antibacterial activity, thus
providing a lead compound for further development. Compounds
4 and 21 show nearly a twofold improvement in ENR inhibitory
activity over compound 2. Compound 35, a ‘reversed’ pyridone, is
also an encouraging lead for the development of a new class of
ENR inhibitors. Current efforts focus on further improvement of

S. K. Tipparaju et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3565–3569
3569
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using the default parameters and the ChemScore scoring function. GOLD
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