1,4-Disubstituted imidazoles are potential antibacterial agents functioning as inhibitors of enoyl acyl carrier protein reductase (FabI)

Article abstract of DOI:10.1016/S0960-894X(01)00404-8

1,4-Disubstituted imidazole inhibitors of Staphylococcus aureus and Escherichia coli enoyl acyl carrier protein reductase (FabI) have been identified. Crystal structure data shows the inhibitor 1 bound in the enzyme active site of E. coli FabI.

Full text of DOI:10.1016/S0960-894X(01)00404-8

Bioorganic& Medicinal Chemistry Letters 11 (2001) 2061–2065
1,4-Disubstituted Imidazoles are Potential Antibacterial
Agents Functioning as Inhibitors of Enoyl Acyl
Carrier Protein Reductase (FabI)
Dirk A. Heerding,* George Chan, Walter E. DeWolf, Jr., Andrew P. Fosberry,
Cheryl A. Janson, Deborah D. Jaworski, Edward McManus, William H. Miller,
Terrance D. Moore, David J. Payne, Xiayang Qiu, Stephen F. Rittenhouse,
Courtney Slater-Radosti, Ward Smith, Dennis T. Takata, Kalindi S. Vaidya,
Catherine C. K. Yuan and William F. Huffman
GlaxoSmithKline Pharmaceuticals, Antibacterials and Host Defense, 1250 S. Collegeville Road, Collegeville, PA 19426, USA
Received 12 February 2001; accepted 11 May 2001
Abstract—1,4-Disubstituted imidazole inhibitors of Staphylococcus aureus and Escherichia coli enoyl acyl carrier protein reductase
(FabI) have been identified. Crystal structure data shows the inhibitor 1 bound in the enzyme active site of E. coli FabI. # 2001
Elsevier Science Ltd. All rights reserved.
Introduction
were screened against S. aureus FabI and imidazole 1
The emergence of pathogens resistant to known anti-
biotic therapy is becoming a serious global healthcare
problem.1 Enoyl acyl carrier protein reductase (FabI)
was identified as a novel antibacterial target. This
enzyme catalyzes the final reaction in bacterial fatty acid
synthesis. It has recently been shown that FabI2a cata-
lyzes this reaction in Staphylococcus aureus and Escher-
ichia coli and that FabK catalyzes the reaction in other
pathogens, such as Streptococcus pneumoniae.2b Eukar-
yotes produce fatty acids via a FAS I system, where the
lipids are synthesized using a multifunctional enzyme
complex in which all of the catalytic domains reside on
one or two polypeptide chains.3 The ACP is an integral
part of this complex. In contrast, prokaryotes utilize the
FAS II system where the enzymes which catalyze the
individual steps are found on seperate polypeptide
chains and the ACP is a discreet protein.4 Therefore,
there is considerable potential for selective inhibition of
bacterial fatty acid biosynthesis. Here we describe a
series of novel FabI inhibitors.
(Table 1) was identified as a lead (IC50=1.24 mM).
Herein we describe the results of our preliminary
investigation into the structure–activity relationship
(SAR) of a series of 1,4-disubstituted imidazoles rela-
ted to 1 as FabI inhibitors. The in vitro anti-bacterial
activity of selected compounds against a representative
Gram-positive and Gram-negative organism is also
reported.
Chemistry
The 1,4-disubstituted imidazoles were prepared using
one of two methods illustrated in Scheme 1. p-Anis-
aldehyde was condensed with tosylmethyl isocyanide
(TOSMIC) to give the corresponding oxazoline 3. The
crude oxazoline was heated with excess 4-methylbenzyl-
amine to give 4 (method A).5 Alternatively, 3-thiophe-
neboronic acid was coupled to 4-iodoimidazole6 under
Suzuki conditions7 to give 7. Treatment of 7 with 3-
nitrobenzylbromide in the presence of K2CO3 gave 8
(method B).8
Compounds from our proprietary compound collection
The regio-isomericimidazole 11 was prepared according
to the method shown in Scheme 2. The bromoketone 9
was condensed with the amidine 10 to give 11.9
*Corresponding author. Tel.: +1-610-917-7944; fax: +1-610-917-
4206; e-mail: dirk_a_heerding@sbphrd.com
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00404-8

2062
D. A. Heerdinget al. / Bioorg. Med. Chem. Lett. 11 (2001) 2061–2065
Table 1. S. aureus FabI and E. coli FabI inhibition data
No.
Structure
Method
S. aureus FabI
IC50 (mM)
E. coli FabI
IC50 (mM)
R1
R2
1
4
8
2-Thienyl
4-MeO–C6H4–
3-Thienyl
3-Thienyl
3-Thienyl
3-Thienyl
3-Thienyl
3-Thienyl
3-Thienyl
3-Thienyl
3-Thienyl
4-MeO–C6H4–
4-MeO–C6H4–
4-CF3–C6H4–
2,3-Cl2–C6H3–
4-Me–C6H4–CH2–
4-Me–C6H4–CH2–
3-O2N–C6H4–CH2–
4-H2N–C6H4–CH2–
4-MeO–C6H4–CH2–
3-(HO2C)–C6H4–CH2–
4-Me–C6H4–CH2–
CH3–
3-Me–C6H4–CH2–
2-Me–C6H4–CH2–
4-(C6H5)–C6H4–CH2–
C6H5–CH2–
4-CF3–C6H4–CH2–
C6H5–CH2–
4-Me–C6H4–CH2–
A
A,B
B
A
A,B
B
A
A
A
A
1.24Æ0.03
0.36Æ0.17
14.7Æ0.65
2.03Æ0.04
1.90Æ0.08
>100
13.7Æ1.7
3.92Æ0.02
26.9Æ3.7
5.14Æ0.42
9.40Æ0.35
>100
20
21
22
23
24
25
26
27
28
29
30
31
0.25Æ0.25
6.44Æ2.9
>100
—
4.24Æ0.86
36.5Æ7.9
7.66Æ0.23
1.13Æ0.15
4.07Æ0.50
>100
12.2Æ0.63
65.4Æ0.3
1.18Æ0.21
5.28Æ0.05
28.3Æ4.9
—
B
A
A
A
A
>10
—
11
15
16
19
32
33
66.2Æ24.4
>100
>100
>100
>100
>100
51.0Æ12.2
>100
>100
>100
50.7Æ16.9
1.10Æ0.02
>100
Triclosan
0.43Æ0.036
The oxazolines (15 and 32) and the oxazole 16 were
prepared by condensing 3-thiophenecarboxylic acid
with 2-amino-3-phenylpropan-1-ol using EDC to give
the amide 14. Cyclodehydration of 14 using Burgess’
reagent10 gave the corresponding oxazoline 15. The
oxazole 16 was prepared by oxidizing the oxazoline with
DDQ (Scheme 3).
NADH using crotonoyl CoA as the substrate. The IC50
values were determined by monitoring the consumption
of NADH.12 Triclosan, a commercial antibacterial
agent and inhibitor of FabI, was included in all assays
as a positive control.13a
Whole-cell antimicrobial activity was determined by a
broth microdilution procedure.14 Compounds were tes-
ted in serial 2-fold dilutions ranging from 0.06 to 64 mg/
mL. The minimum inhibitory concentration (MIC) was
determined as the lowest concentration of compound
that inhibited visible growth.
The oxadiazoles 19 and 33 were prepared according to
the method of Bedford et al.11 This is illustrated in
Scheme 4 for the preparation of 19. Condensation and
cyclodehydration of the acyl chloride 17 with the
amidoxime 18 gave the oxadiazole 19.
Results and Discussion
Biology
Solution-phase arrays were used to probe the scope of
allowable substituents at the 1- and 4-positions of the
imidazole lead 1. The variable aromaticand aliphatic
substituents at the 1- and 4-positions were chosen using
The FabI enzyme inhibition assays were carried out
with varying concentrations of test compound and
either S. aureus FabI or E. coli FabI in the presence of

D. A. Heerdinget al. / Bioorg. Med. Chem. Lett. 11 (2001) 2061–2065
2063
the Topliss analysis decision tree as a guide.15 This semi-
empirical method is designed to rapidly optimize biolo-
gical activity by systematically probing electronic and
stericeffects of substituents. These studies revealed that
electron-rich benzyl rings at the 1-position of the imi-
dazole ring (R2) are required for FabI inhibition (4, 20,
and 21 vs 8, 22, and 29, Table 1). Additionally, small
electron-donating groups, such as methyl and methoxy
groups, are preferred at the 4-position of the aromatic
ring (21 and 23). Larger groups, such as a phenyl group,
were not tolerated at this position (27). Moving the
methyl group from the 4-position to the 2- and 3-posi-
tions gave less active inhibitors (25 and 26). These
studies also suggested that electron-rich aromatic
groups are preferred at the 4-position (R1) of the
imidazole ring (23 and 28 vs 30 and 31).
These findings are consistent with results from X-ray co-
crystallization studies with 1 bound to E. coli FabI/
NAD+. The crystal structure has been solved to 2.8
A resolution (R 19.1%, Rf 27.3%) and shows that 1 is
bound in the enzyme active site.16 The thiophene ring of
the inhibitor occupies a hydrophobic pocket and is
engaged in a p-stacking interaction with the electron-
poor nicotinamide ring of NAD+. Electron-rich aro-
maticrings at the 4-position of the imidazole are
expected to favorably influence the stacking interaction,
while electron-poor rings should destabilize the com-
plex. The unsubstituted imidazole nitrogen appears to
be engaged in a hydrogen-bonding interaction with the
phenolichydroxyl of Tyr 156 from the enzyme (Fig. 1).
Scheme 1. Preparation of the 1,4-disubstituted imidazoles: (a) TOS-
MIC, NaCN, EtOH, rt, 30 min, quantitative; (b) 4-methylbenzyl-
amine, toluene, reflux, 24 h, 38%; (c) Pd(PPh3)4, Na2CO3, DMF,
80 ꢀC, 85%; (d) K2CO3, 3-nitrobenzyl bromide, DMF, 16%.
Scheme 3. Preparation of the oxazolines and oxazoles: (a) EDC,
HOBt, Et3N, DMF, 63%; (b) Burgess’ reagent, 81%; (c) DDQ, 38%.
Scheme 2. Preparation of the 2,4-disubstituted imidazoles: (a)
CH2Cl2, rt, 18 h, 75%.
Scheme 4. Preparation of the oxadiazole: (a) THF, rt; (b) NaH, THF,
reflux, 66% (two steps).
Figure 1. Stereoview of 1 and NAD+ bound in the active site of E. coli FabI. For clarity, only the side chains of the residues which define the
hydrophobicpocket of FabI have been shown. The thiophene ring and part of the imidazole ring of 1 were revealed by the electron-density map. The
4-methylbenzyl moiety represents a model since there was no electron density observed to define this part of the inhibitor.

2064
D. A. Heerdinget al. / Bioorg. Med. Chem. Lett. 11 (2001) 2061–2065
Table 2. Antibacterial profile of 1, 4, 20, 21, 23, 25, and 28
Conclusion
MIC (ug/mL)
We have shown that appropriately functionalized 1,4-
disubstituted imidazoles are effective inhibitors of FabI
and that representative compounds have antibacterial
activity against Gram-positive and Gram-negative bac-
teria. These results provide further validation of FabI as
an antibacterial target.
No.
S. aureus Oxford
M. catarrhalis 1502
1
4
20
21
23
128
8
>64
16
—
32
>64
64
32
32
8
25
28
>64
64
64
Triclosan
0.03
0.06
References and Notes
1. (a) Chu, D. T. W.; Plattner, J. J.; Katz, L. J. Med. Chem.
1996, 39, 3853. (b) Ehlert, K. Curr. Pharm. Des. 1999, 5, 45.
2. (a) Heath, R. J.; Rock, C. O. J. Biol. Chem. 1995, 270,
26538. (b) Heath, R. J.; Rock, C. O. Nature 2000, 406, 145.
3. Smith, S. FASEB J. 1994, 8, 1248.
The remainder of the inhibitor was disordered in the
crystal structure. The interactions observed between
1 and E. coli FabI/NAD+ are similar to the inter-
actions observed between the known FabI inhibitor,
triclosan, and E. coli FabI/NAD+.17 Namely, a p-
stacking interaction is observed between the phenol of
triclosan and the nicotinamide ring of NAD+. A
hydrogen bond is also observed between the phenolic
hydroxyl group of triclosan and the hydroxyl group of
Tyr 156.
4. Fulco, A. J. Prog. Lipid Res. 1983, 22, 133.
5. Horne, D. A.; Yakushijin, K.; Buechi, G. Heterocycles
1994, 39, 139.
6. Cliff, M. D.; Pyne, S. G. Synthesis 1994, 681.
7. Kawasaki, I.; Katsuma, H.; Nakayama, Y.; Yamashita,
M.; Ohta, S. Heterocycles 1998, 48, 1887.
8. Antonini, I.; Cristalli, G.; Franchetti, P.; Grifantini, M.;
Martelli, S. Synthesis 1983, 47.
9. Baldwin, J. J.; Christy, M. E.; Denny, G. H.; Habecker,
C. N.; Freedman, M. B.; Lyle, P. A.; Ponticello, G. S.; Varga,
S. L.; Gross, D. M.; Sweet, C. S. J. Med. Chem. 1986, 29,
1065.
10. Wipf, P.; Miller, C. P. J. Org. Chem. 1993, 58, 1575.
11. Bedford, C. D.; Howd, R. A.; Dailey, O. D.; Miller, A.;
Nolen, H. W.; Kenley, R. A., III; Kern, J. R.; Winterle, J. S. J.
Med. Chem. 1986, 29, 2174.
12. The E. coli and S. aureus FabI enzymes were cloned,
overexpressed and purified as described previously.17a Assays
were carried out in half-area, 96-well microtitre plates. Com-
pounds were evaluated in 150-mL assay mixtures containing
100 mM NaADA, pH 6.5 (ADA=N-[2-acetamido]-2-imino-
diacetic acid), 4% glycerol, 0.25 mM crotonoyl CoA, 50 mM
NADH, and an appropriate dilution of E. coli Fab I (usually
60 nM). Inhibitors were typically varied over the range of
0.01–10 mM. The consumption of NADH was monitored for
20 min at 30 ꢀC by following the change in absorbance at
340 nm (e=5.28 mMÀ1). Initial velocities were estimated from
an exponential fit of the non-linear progress curves repre-
sented by the slope of the tangent at t=0 min. IC50 values were
estimated from a fit of the initial velocities to a standard 4-
parameter model and are typically reported as the meanÆSD
of duplicate determinations. Triclosan was included in all
assays as a positive control. IC50 determinations with S. aureus
FabI were determined in a similar fashion.
13. (a) McMurry, L. M.; Oethinger, M.; Levy, S. B. Nature
1998, 394, 531. (b) McDonnell, G.; Pretzer, D. ASM News
1998, 64, 670. (c) Russell, A. D. J. Appl. Microbiology 1997,
83, 155.
14. Compounds in DMSO were diluted 1:10 with water giving
a 256 mg/mL solution. This solution (50 mL) was serially dilu-
ted into cation adjusted Mueller Hinton broth. A 50 mL ali-
quot of bacteria (ca. 1Â106 cfu/mL) was added to each well.
Inoculated plates were incubated at 35 ꢀC for 24 h. Minimum
inhibitory concentration (MIC)=the lowest concn of com-
pound that inhibited visible growth.
Another array was prepared to assess the chain
length requirements for the aryl groups attached to
the central imidazole core. FabI inhibition was
optimal when the aryl group at the 4-position (R1)
was directly attached to the imidazole core and
when the aryl group at the 1-position (R2) was sepa-
rated from the imidazole nitrogen by a single methylene
group (data not shown).
The 1,4-disubstituted imidazole ring appears to be
critical for FabI activity. The regio-isomeric 2,4-
disubstituted analogues are no longer effective
inhibitors of FabI (4 vs 11). Oxazolines (15 and 32),
oxazoles (16) and oxadiazoles (19 and 33) are also
not tolerated as replacements for the 1,4-disubstituted
imidazole ring. At this time we are unable to
explain these results. There are no obvious unfavor-
able steric or electronic interactions when 11, 15, 16,
19, 32 and 33 are docked into the enzyme active site
using the X-ray crystal structure solved for 1 bound to
FabI.
Compounds with IC50<5 mM against S. aureus FabI
were tested against a Gram-positive bacteria (S. aureus
Oxford) and a Gram-negative bacteria (Moraxella
catarrhalis 1502). The results show a correlation
between the IC50 values in the enzyme assay and
the MIC values against whole cell bacteria (Table 2).
(Triclosan gives much lower MIC values than would
be predicted from its enzyme IC50 level. This is pre-
sumably due to the multiple modes of action ascribed
to Triclosan.13b,c) In addition, the antibacterial activity
of 23 was evaluated in a S. aureus strain engineered to
overexpress FabI. A greater than 8-fold increase in MIC
values was observed in the overexpressing strain when
compared to wild type. These results suggest that the
antibacterial activity is primarily due to inhibition of
FabI.
15. Topliss, J. G. J. Med. Chem. 1977, 20, 463.
16. The atomicco-ordinates have been deposited at the Pro-
tein Data Bank (PDB) (accession number 1I2Z). The condi-
tions used to crystalize the protein and solve the X-ray
structure are similar to those described in ref 17a.

D. A. Heerdinget al. / Bioorg. Med. Chem. Lett. 11 (2001) 2061–2065
2065
17. (a) Qiu, X.; Janson, C. A.; Court, R. I.; Smyth, M. G.;
Payne, D. J.; Abdel-Meguid, S. S. Protein Sci. 1999, 8, 2529.
(b) Roujeinikova, A.; Levy, C. W.; Rowsell, S.; Sedelnikova,
S.; Baker, P. J.; Minshull, C. A.; Mistry, A.; Colls, J. G.;
Camble, R.; Stuitje, A. R.; Slabas, A. R.; Rafferty, J. B.;
Pauptit, R. A.; Viner, R.; Rice, D. W. J. Mol. Biol. 1999, 294,
527. (c) Heath, R. J.; Rubin, J. R.; Holland, D. R.; Zhang, E.;
Snow, M. E.; Rock, C. O. J. Biol. Chem. 1999, 274, 11110.