Discovery of azetidine based ene-amides as potent bacterial enoyl ACP reductase (FabI) inhibitors

Article abstract of DOI:10.1016/j.ejmech.2014.07.036

A novel and potent series of ene-amides featuring azetidines has been developed as FabI inhibitors active against drug resistant Gram-positive pathogens particularly staphylococcal organisms. Most of the compounds from the series possessed excellent biochemical inhibition of Staphylococcus aureus FabI enzyme and whole cell activity against clinically relevant MRSA, MSSA and MRSE organisms which are responsible for significant morbidity and mortality in community as well as hospital settings. The binding mode of one of the leads, AEA16, in Escherichia coli FabI enzyme was determined unambiguously using X-ray crystallography. The lead compounds displayed good metabolic stability in mice liver microsomes and pharmacokinetic profile in mice. The in vivo efficacy of lead AEA16 has been demonstrated in a lethal murine systemic infection model.

Full text of DOI:10.1016/j.ejmech.2014.07.036

European Journal of Medicinal Chemistry 84 (2014) 382e394
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Discovery of azetidine based ene-amides as potent bacterial enoyl ACP
reductase (FabI) inhibitors*
Mohamed Takhi a, Kandepu Sreenivas a, Chandrashekar K. Reddy a,
Mahadari Munikumar a, Kolakota Praveena a, Pabolu Sudheer a, Bandaru N.V.M. Rao a,
Gollamudi Ramakanth a, Jampala Sivaranjani a, Shardaprasad Mulik a, Yeruva R. Reddy a,
Krishnamurthy Narasimha Rao b, Rentala Pallavi b, Anirudha Lakshminarasimhan b,
Sunil K. Panigrahi b, Thomas Antony b, Iskandar Abdullah c, Yean K. Lee c,
Murali Ramachandra b, Rohana Yusof d, Noorsaadah A. Rahman c,
,
*
Hosahalli Subramanya b
a Aurigene Discovery Technologies Ltd, Bollaram Road, Miyapur, Hyderabad 500 049, India
b Aurigene Discovery Technologies Ltd, 39-40, KIADB Industrial Area, Phase II, Electronic City, Hosur Road, Bangalore 560 100, India
c Department of Chemistry, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia
d Department of Molecular Medicine, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel and potent series of ene-amides featuring azetidines has been developed as FabI inhibitors active
against drug resistant Gram-positive pathogens particularly staphylococcal organisms. Most of the
compounds from the series possessed excellent biochemical inhibition of Staphylococcus aureus FabI
enzyme and whole cell activity against clinically relevant MRSA, MSSA and MRSE organisms which are
responsible for significant morbidity and mortality in community as well as hospital settings. The
binding mode of one of the leads, AEA16, in Escherichia coli FabI enzyme was determined unambiguously
using X-ray crystallography. The lead compounds displayed good metabolic stability in mice liver mi-
crosomes and pharmacokinetic profile in mice. The in vivo efficacy of lead AEA16 has been demonstrated
in a lethal murine systemic infection model.
Received 31 January 2014
Received in revised form
9 July 2014
Accepted 10 July 2014
Available online 11 July 2014
Keywords:
FabI inhibitor
Enoyl ACP reductase
Antibacterial
Azetidine
© 2014 Elsevier Masson SAS. All rights reserved.
Ene-amide
1. Introduction
action to combat bacterial resistance. In recent times, there has
been a great deal of interest in targeting bacterial fatty acid
The emergence of bacterial resistance continues to hamper the
effectiveness of existing antibacterial therapies and poses serious
threat worldwide in community and nosocomial settings.
Multidrug-resistant Gram-positive bacteria such as methicillin-
resistant Staphylococcus aureus (MRSA), Staphylococcus epi-
dermidis (MRSE) and vancomycin-resistant S. aureus (VRSA) are of
major concern [1]. This challenge is further exacerbated by the
reduced interest of major pharmaceutical companies in antibacte-
rial drug discovery leading to paucity in new antibiotics pipeline
[2]. Hence, there is a pressing need for increased and accelerated
efforts to identify new therapeutics based on novel mechanism of
biosynthesis (FAB) as a viable strategy to develop novel antibacte-
rial agents.
The bacterial fatty acid synthase system (FASII) utilizes discrete
monofunctional enzymes that operate in conjunction with acyl
carrier protein (ACP)-associated substrates. In mammalian fatty
acid synthase (FASI) pathway, lipid biosynthesis is mediated by a
single multifunctional enzyme-ACP complex. The differences in
prokaryote and eukaryote fatty acid biosynthesis provide an op-
portunity for selective FASII inhibition [3,4]. The inhibition of FASII
pathway causes the breakdown of cell wall and disruption of cell
membrane demonstrating its importance for bacterial survival [5].
FabI is an enoyl-ACP reductase that catalyses the ultimate and
rate limiting step of chain elongation in each cycle in FASII pathway.
The reaction involves the conjugate reduction of an enoyl-ACP to
the corresponding acyl-ACP using the cofactor NADH or NADPH as a
hydride source as shown in Scheme 1 [3e6]. FabI is an essential
*
Aurigene Publication No H-05-0201-2012.
* Corresponding author.
E-mail address: hosahalli_s@aurigene.com (H. Subramanya).
http://dx.doi.org/10.1016/j.ejmech.2014.07.036
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

M. Takhi et al. / European Journal of Medicinal Chemistry 84 (2014) 382e394
383
Scheme 1. Fabl biosynthetic reaction.
enzyme for the viability of several bacteria and shows low degree of
sequence homology with mammalian enzymes [3]. Several iso-
forms (FabI, FabK, FabL and FabV) of enoyl-ACP reductases (FabI
isozymes) have been reported in literature [3,7,8]. Different bacte-
ria have been reported to have one or more of these isoforms. For
example, single isoform, FabI is found in S. aureus and Escherichia
coli, and FabK is present in Staphylococcus pneumoniae. However,
two isoforms FabI and FabV are present in Pseudomonas aeruginosa
and FabI and FabL are present in Bacillus subtilis. These isoforms of
enoyl ACP-reductases have high sequence and structural similar-
ities. However, there are differences in the active site of these en-
zymes. Therefore, development of inhibitors that act on all the
isoforms leading to broad spectrum activity could be challenging.
The novel mechanism of action with a potential for activity
against drug resistant staphylococcal bacteria has led to the
discovery of several FabI inhibitors based on different chemo-
types. These include indole naphthyridinones [9], 2,34,5-
tetrahydro-1H-pyrido[2,4-b and e] [1,4] diazepines [10], spiro-
naphthyridinone piperidines [11], 1,2,3,4-tetrahydropyrido-in-
doles [12], 4-pyridones [13,14], biphenyl ethers [15] and ami-
nopyridines [16]. Representative FabI inhibitors are shown in
Fig. 1. AFN-1252 [17], MUT-056399 (FAB-001) [18] and CG-
400549 [19] have advanced to clinical trials for the treatment of
Gram-positive bacterial infections caused by MSSA, MRSA and
MRSE pathogens. Triclosan, a widely used biocide in a variety of
consumer products, is known to act via FabI inhibition [20].
In pursuit of our antibacterial drug discovery programs [21,22],
we were interested in developing novel and potent FabI inhibitors
for the treatment of drug resistant staphylococcal infections. The
efforts in this direction led to the development of a novel series of
FabI inhibitors featuring azetidine ene-amides (AEAs) showing
excellent in vitro and in vivo profile. This paper outlines the design,
synthesis, SAR, metabolic stability, in vivo pharmacokinetic profile
and in vivo antibacterial activity of a series of azetidine ene-amides
(AEAs).
Fig. 1. Fabl inhibitors.
open chain amides (Fig. 3) [24,25]. We envisaged that such modi-
fication could engender compounds with good metabolic stability
and pharmacokinetic profile while retaining the potency. Indeed,
the initial compounds AEA2 and AEA3 did show S. aureus FabI
enzyme inhibition when tested at 1
Fig. 3.
mM concentration as shown in
2.2. Design, synthesis and SAR of substituted azetidine ene amides
(AEAs) with naphthyridinon-one on right hand side
Encouraged by the biochemical data of initial compounds AEA2
and AEA3 we set out to investigate further the effect of azetidines
on SAR paradigm of ene-amide scaffold through iterative medicinal
chemistry approach. We initiated our efforts by varying different
substitutions on azetidine moiety on the left hand side (LHS) fol-
lowed by the modification of cyclic amide ring on right hand side
(RHS) as shown in Fig. 3.
The first iteration of SAR was focussed on the left hand side,
exploring the hydrophobic pocket in the FabI active site. Accord-
ingly, several analogues with structurally diverse and lipophilic
substitutions at the 3-position of azetidine as exemplified by aze-
tidine ene-amides AEA4-21 were designed and synthesized. We
hypothesized that these LHS substitutions on azetidine could
optimize interactions in the hydrophobic pocket and thereby
enhance biochemical and in vitro antibacterial activity.
The synthesis of azetidine ene-amides AEA1-12 is delineated in
Scheme 2. The key carboxylic acid intermediate 1 has been
conveniently prepared according to the literature procedure [9].
The compound AEA1 [25] was made by coupling acid 1 and
2. Results and discussion
2.1. Initial design of FabI inhibitors
We used structure-guided approach based on X-ray structure of
AFN-1252 bound to E. coli FabI determined in-house [23]. Critical
interactions observed between AFN-1252 and E. coli FabI are shown
in Fig. 2. The pyridine nitrogen and amidic NH makes two H-bonds
with residue Ala-95 while the carbonyl oxygen in the ene-amide
moiety is engaged in H-bond interaction with NADH and side
chain of Tyr-156 residue. The benzofuran ring occupies a hydro-
phobic pocket formed by the residues Tyr-146, Met-159 and Phe-
203 and makes a favourable
p-stacking interaction with residue
Tyr-146. The methyl substitution on benzofuran moiety at 3-
position further enhances this stacking interaction.
The crystal structure of AFN-1252 bound to E. coli FabI revealed
that ample space is available around the ene-amidic nitrogen for
the design of novel FabI inhibitors. Keeping these observations in
view, we designed novel FabI inhibitors by introducing azetidine
ring which is capable of producing conformationally rigid amides
that are relatively resistant to enzymatic hydrolysis compared to
Fig. 2. X-ray structure of AFN-1252 (shown in yellow) in complex with E. coli FabI.
NADH is shown in cyan colour. Hydrogen bond interactions are shown in dotted lines.

384
M. Takhi et al. / European Journal of Medicinal Chemistry 84 (2014) 382e394
Fig. 3. Design of azetidine ene-amide series and initial hits.
commercially available azetidine 2 under EDC·HCl and HOBt con-
ditions in the presence of Hunig's base. The substituted azetidine
counterparts have been accessed by different routes following re-
ported procedures and coupled with the carboxylic acid 1 to pro-
duce final molecules. Compounds AEA2 and AEA3 have been made
starting from readily available amines 3 and 4 respectively [25]. The
azetidine amides AEA4-11 were obtained by coupling acid 1 and
the amines 5e12 made following literature procedures [26]. The
base promoted O-arylation of alcohol 13 with 2-chloro benzothia-
zole in the presence of sodium hydride furnished 14 which in turn
was treated with trifluoroacetic acid followed by coupling with acid
1 to yield AEA12.
substituted azetidines that have moieties interacting with hy-
drophobic pocket demonstrated good biochemical and cell based
activities. Among the azetidinyl ethers AEA3 and AEA4,
a
noticeable increase in FabI potency and whole cell activity was
observed when oxygen is in conjugation with phenyl moiety
with
(IC50 ¼ 0.265
a
methylene spacer as exemplified by AEA4
g/mL). The fluoro phenyl de-
m
M, MIC ¼ 0.25e1
m
rivatives AEA5-7, in general, showed good FabI enzyme activity
and the p-fluoro phenyl analogue AEA7 showed superior anti-
bacterial activity with MICs in the range of 0.25e1
mg/mL
compared to other fluoro phenyl analogues. In the case of methyl
substituted phenyl analogues AEA8-10, ortho derivative AEA8
The synthesis of carbon linked azetidines (AEA13e21) was
executed as represented in Scheme 3. Benzothiazole nucleus was
introduced to the azetdine 15 [27] employing the Minisci reaction
conditions [28]. The radical mediated oxidative coupling of ben-
zothiazole and 15 in the presence of hydrogen peroxide and ferrous
sulphate produced the azetidine derivative 16 which subsequently
furnished AEA13. Similarly, azetidine intermediates 18e24 were
synthesized by reacting iodide 17 [28] with corresponding heter-
oaryls under the Minisci reaction conditions. The transformation of
these intermediates to final compounds AEA14e20 followed
routine procedures. The compound AEA21 was smoothly obtained
by coupling commercially available phenyl-3-azetidine with acid 1
under standard amide coupling conditions.
possessed potent enzyme activity (IC50 ¼ 0.177
m
M) which
g/mL)
translated into good whole cell activity (MIC ¼ 0.25e1
m
compared to other isomers. Introduction of biaryl moieties on
azetidine through oxymethylene (AEA11), oxygen (AEA12) and
methylene (AEA13) spacers yielded compounds with poor to
moderate potency which could be due to unfavourable geometry
leading to the loss of
p-stacking interaction with Tyr-146. In
contrast, direct linkage of biaryl moiety to azetidine exemplified
by AEA14 led to dramatic improvement in FabI inhibition
(IC50 ¼ 0.373
m
M) as well as cell based activity against staphy-
g/mL). Encouraged by this SAR
lococcal strains (MIC ¼ 0.25e1
m
finding, a few more analogues (AEA15e21) were made wherein
an aryl or a biaryl moiety is directly attached to azetidine at the
3-position through carbonecarbon linkage as mentioned in
Scheme 3.
The final compounds were screened in
a S. aureus FabI
biochemical assay followed by whole cell assay comprising a panel
of MSSA, MRSA and MRSE organisms. As shown in Table 1, intro-
duction of azetidine resulted in potent FabI inhibitors illustrating a
range of tolerable substitutions on azetidine ring.
Unsubstituted analogue AEA1 is inactive probably because of
the lack of a group occupying the hydrophobic pocket. However,
In a gratifying result, installation of 3-methyl benzoxazole on
azetidine afforded the compound AEA16 with improved FabI
enzyme inhibition (IC50 ¼ 0.141
tivity against various staphylococcal organisms (MIC ¼ 0.06e0.5
mL). The 5-methoxy-benzoxazole AEA17 and 5-chloro benzoxazole
mM) and the best antibacterial ac-
mg/
Scheme 2. Reagents and conditions: (i) EDC·HCl, HOBt, DIPEA, r.t, overnight; (ii) NaH, DMF, 100 ꢀC, overnight; (iii) TFA, CH2Cl2, r.t, 2 h. R1 ¼ H, 2, AEA1; R1 ¼ PhO, 3, AEA2;
R1 ¼ PhCH2O, 4, AEA3; R1 ¼ PhOCH2, 5, AEA4; R1 ¼ o-fluoro-C6H4-OCH2, 6, AEA5; R1 ¼ m-fluoro-C6H4-OCH2, 7, AEA6; R1 ¼ p-fluoro-C6H4-OCH2, 8, AEA7; R1 ¼ o-methyl-C6H4-
OCH2, 9, AEA8; R1 ¼ m-methyl-C6H4-OCH2, 10, AEA9; R1 ¼ p-methyl-C6H4-OCH2, 11, AEA10; R1 ¼ 2-napthyl-OCH2, 12, AEA11.

M. Takhi et al. / European Journal of Medicinal Chemistry 84 (2014) 382e394
385
Scheme 3. Reagents and conditions: (i) FeSO4$7H2O, H2SO4, H2O2, DMSO, r.t, 16 h; (ii) (a) TFA, CH2Cl2, r.t, 2 h (b) 1, EDC$HCl, HOBt, DIPEA, r.t, overnight.
AEA18 exhibited good in vitro antibacterial profile. However, quin-
oline AEA19 and pyridine AEA20 exhibited poor to moderate whole
cell activity, presumably due to poor cell permeability of molecule
and/or efflux barriers present in bacterial membrane.
Above SAR results indicate that right combination of groups on
both sides of ene-amide scaffold is essential for achieving good FabI
potency as well as in vitro antibacterial activity. Further, in addition
to biochemical potency, appropriate balance of physicochemical
properties such as clogP, molecular weight and polarity are critical
to overcome bacterial cell membrane permeability barriers [32].
Therefore, the variation in MIC values of these compounds can be a
consequence of differences in their aforementioned properties.
Among these compounds, best in vitro antibacterial activity
2.3. Design, synthesis and SAR of benzofuranyl-azetidine ene
amides with variation on right hand side
Having established SAR on left hand side, we turned our
attention to the right hand side (RHS) part of the ene-amide scaf-
fold. In this iteration, modifications were carried out on the potent
compound AEA16 keeping the 2-benzfuranyl azetidine moiety
constant. Substitutions on RHS occupy a hydrophilic pocket and
compounds with basic functionalities in this region have been
previously reported [10,11]. Accordingly, a cross section of ana-
logues with variations (AEA22e28) on pyridine ring have been
made and screened.
CompoundsAEA22e24 have been prepared by coupling amine
25, obtained from 20, with corresponding acids 26 [9], 27 [29] and
28 [30] as shown in Scheme 4. The azetidine ene-amides
AEA25e28 were synthesized in a convergent manner as delin-
eated in Scheme 5. The bromo-diazepinone intermediates 30 [10],
31 [10], 32 [31], 33 [10] have been obtained according to literature
procedures and coupled to acrylamide 29 under Heck reaction
conditions to furnish corresponding products AEA25e28.
These analogues were also tested in biochemical and whole cell
assays. The results are summarized in Table 2. Although its enzyme
activity is retained, the acetamide AEA22 showed inferior cell
based activity compared to its cyclic congener AEA16. Surprisingly,
1,2,3-triazole substituted acetamide AEA23 showed poor
biochemical and antibacterial activities which could be due to the
disturbance in a hydrogen bonding network between the backbone
carbonyl and amide hydrogen of Ala-95 from S. aureus FabI enzyme.
The substitution of N-alkyl morpholine onto cyclic urea yielded
highly potent FabI inhibitor AEA24 with IC50 of 58 nM. This
observation is reminiscent to the results reported by the Affinium
group wherein the basic appendage can occupy the solvent pocket
away from the target site [11]. The N-methyl diazepinone AEA26
was found to be equipotent to its corresponding NeH diazepinone
(MIC ¼ 0.06
mg/mL) is obtained against MRSA and MSSA organisms
for AEA16 which possess a naphthyridinone ring on the right hand
side and benzofuranyl moiety on left hand side. These structural
features are similar to AFN-1252 as shown in Fig. 4.
An X-ray crystal structure of one of the leads AEA16 bound to
E. coli FabI enzyme (Fig. 5) in the presence of NADH was determined
to a resolution of 3.2 Å [33] and it was found to be similar to other
crystal structures reported from the ene-amide series [9].
A superposition of AEA16, AFN-1252 and triclosan X-ray struc-
tures (PDB ID: 1C14) is shown in Fig. 6. It is evident that critical
interactions observed in AFN-1252 are conserved in AEA16 bound
E. coli FabI complex. Benzofuran moieties in the AEA16 and AFN-
1252, and chlorine atom in triclosan occupy the same hydrophobic
pocket. Hydroxyl moiety of triclosan is hydrogen bonded to Tyr-156
and NADH whereas oxygen atoms of carbonyls of AFN-1252 and
AEA16 in the ene-amide moiety are engaged in hydrogen boding
with the same residues. This key hydrogen bond interaction with
Tyr-156 residue is also reported in several known FabI inhibitors
[9,17,34,35].
2.4. Metabolic stability and in vivo pharmacokinetic profile of
selected compounds
Selected compounds possessing good cell based activity were
tested for metabolic stability in mice liver microsomes. The com-
pounds showing good metabolic stability were further evaluated
for in vivo pharmacokinetic properties in mice by oral route. The
results are summarized in Table 3. Most of these azetidine ene-
amides, except AEA26, were found to have high metabolic stabil-
ity. The good metabolic stability of these analogues could be due to
enzymatic stability of cyclic and conformationally restricted azeti-
dine amide. The compound AEA26 appears to be metabolically less
stable likely because of demethylation of diazepinone ring by liver
enzymes [36]. AFN-1252 was found to be less stable compared to
azetidine ene-amide analogues presumably due to the presence of
open chain amide as discussed. Most of these compounds
AEA25 and showed MICs in the range of 0.25e0.5 mg/mL. Extended
substitutions on diazepinone yielded less potent AEA27 compared
to AEA24 which could be due to inability of this morpholine group
to access the solvent pocket. The compound AEA28 showed almost
equal biochemical potency and cell based activity to its positional
isomer AEA25.

386
M. Takhi et al. / European Journal of Medicinal Chemistry 84 (2014) 382e394
Table 1 (continued)
Table 1
SAR on LHS part of ene-amide.
Compound
no
A
S. aureus FabI
inhibition
MIC (
m
g/mL)a
%@1
87%
mM IC50(m
M) MSSAb MRSAc MRSEd
AEA20
0.242
0.488
4
1
8
2
16
2
Compound
no
A
S. aureus FabI
inhibition
MIC (
m
g/mL)a
AEA21
80%
%@1
15%
mM IC50(m
M) MSSAb MRSAc MRSEd
a
MIC ¼ Minimum inhibitory concentration.
AEA1
AEA2
NDe
NDe
>32
>32
>32
b
c
MSSA e methicillin-sensitive Staphylococcus aureus ATCC 29213.
MRSA e methicillin-resistant Staphylococcus aureus ATCC 33591.
MRSE e methicillin-resistant Staphylococcus epidermidis AUCC 704.
ND ¼ Not determined.
d
e
35%
69%
4
4
16
AEA3
AEA4
0.376
0.265
1
2
2
1
possessed excellent pharmacokinetic profile with good systemic
exposures as exemplified by AEA7, AEA14, AEA16 and AEA18.
Among these compounds AEA8, AEA15 and AEA25 showed rela-
tively lower AUC and Cmax values compared to the rest of the
molecules. Higher rate of clearance due to lower metabolic stability
of AEA8 and AEA15 could be contributing to lower AUC and Cmax
values compared to other compounds. Despite the good metabolic
stability, AEA25 showed suboptimal pharmacokinetics which could
be a consequence of poor absorption. AFN-1252 displayed a poor
pharmacokinetic profile compared to these azetidine-ene amides
presumably due to its poor metabolic stability in mice liver
microsomes.
81%
99%
89%
92%
80%
47%
67%
68%
16%
41%
0.25
0.25
AEA5
0.378
0.438
0.316
0.177
ND
1
1
2
AEA6
1
2
2
AEA7
0.25
0.25
2
0.5
0.5
2
1
2.5. In vivo efficacy of compound AEA16
AEA8
1
Based on potent cell based activity (MIC ¼ 0.06
mg/mL) and
favourable pharmacokinetic profile, compound AEA16 was
advanced to in vivo efficacy studies in a lethal murine systemic
infection model by oral route, employing MRSA as the infectious
organism. The results are presented in Table 4. AEA16 protected the
mice from infection at the ED50 dose of 0.90 mg/kg/day which was
superior to that of Linezolid (ED50 ¼ 2.8 mg/kg/day), a current
standard of care for the treatment of MRSA infections. The potent
in vitro antibacterial activity of lead AEA16 coupled with good
pharmacokinetic profile led to impressive in vivo efficacy.
AEA9
2
AEA10
AEA11
AEA12
AEA13
0.921
1.41
1
1
2
4
4
>32
>32
2
NDe
>32
>32
3. Conclusions
In summary, we have developed a potent and promising series
of ene-amides bearing azetidines as FabI inhibitors active against a
panel of resistant staphylococcal organisms. Structure-guided SAR
in iterative approach through the introduction of structurally
diverse azetidines followed by right hand side modifications
afforded potent compounds with excellent FabI inhibition
NDe
2
4
AEA14
AEA15
83%
89%
0.373
0.709
0.25
0.25
1
2
(IC50 ꢁ 0.058
mM) and antibacterial activity (MICs ꢁ0.06
mg/mL)
0.125 0.25
against MSSA, MRSA and MRSE pathogens. Excellent metabolic
stability and pharmacokinetic properties have been observed for
the compounds from this series. The lead AEA16 demonstrated
very good in vivo efficacy in a murine systemic infection model. The
key SAR insights in this program will be utilized for further opti-
mization of ene-amide series to identify a clinical candidate.
AEA16
AEA17
96%
NDe
0.141
NDe
0.06
0.5
0.06
1
0.5
2
4. Experimental
AEA18
AEA19
82%
84%
0.204
0.242
0.25
32
0.25
0.5
4.1. General
All chemicals were purchased from SigmaeAldrich, Lancaster
and Combiblock and were used without further purification. Re-
actions were monitored by TLC performed on silica gel aluminium
>32
>32

M. Takhi et al. / European Journal of Medicinal Chemistry 84 (2014) 382e394
387
Scheme 4. Reagents and conditions: (i) TFA, CH2Cl2, r.t, 2 h; (ii) EDC$HCl, HOBt, DIPEA, r.t, overnight.
Scheme 5. Reagents and conditions: (i) Acryloyl chloride, Et3N, CH2Cl2, r.t, 16 h; (ii) Pd(OAc)2, P(o-tolyl)3, DIPEA, Propionitrile, reflux, 6 h.
plates containing 60 F254. Column chromatography was performed
with Merck 100e200 mesh silica gel. 1H NMR and 13C NMR were
recorded on Varian Mercury Plus (400 MHz) or Varian Unity Inova
(500 MHz) instruments. Chemical shifts are reported in parts per
stirred at the same temperature for 16 h, then diluted with H2O (50
volumes) and extracted with ethyl acetate (3 ꢃ 50 volumes). The
combined organic layers were washed with brine (50 volumes),
dried over Na2SO4, filtered and volatiles were removed under
vacuum. The crude residue was purified by column chromatog-
raphy to furnish the desired products AEA1-24.
million (d in ppm) relative to the peak for tetramethylsilane (TMS)
as internal standard and the coupling constants are reported in
Hertz (Hz). High resolution mass spectra (HRMS) were recorded on
a Waters LCT-Premier mass spectrometer. IR spectra were recorded
4.2.1. (E)-6-(3-(azetidin-1-yl)-3-oxoprop-1-en-1-yl)-3,4-dihydro-
1,8-naphthyridin-2(1H)-one (AEA1)
on a FT-IR spectrometer and only major peaks are reported in cmꢂ1
.
Yield: 9%; White solid; mp: 278e279 ꢀC; IR (KBr, cmꢂ1): 3535,
2883, 1654, 1598, 1462, 1300, 1197, 983, 852, 530; 1H NMR
(400 MHz, DMSO-d6): dH 10.64 (s, 1H), 8.33 (d, J ¼ 1.5 Hz, 1H), 8.00
(s, 1H), 7.38 (d, J ¼ 15.9 Hz, 1H), 6.69 (d, J ¼ 15.9 Hz, 1H) 4.28 (t,
J ¼ 7.6 Hz, 2H), 3.94 (t, J ¼ 7.6 Hz, 2H), 2.91 (t, J ¼ 7.6 Hz, 2H),
2.58e2.52 (m, 2H), 2.28e2.20 (m, 2H); 13C NMR (500 MHz, DMSO-
d6): dC 14.8, 23.2, 30.0, 47.6, 49.8, 116.1, 119.0, 125.3, 133.5, 136.3,
4.2. General procedure for the synthesis of AEA1-24
Diisopropyl ethyl amine (3 equiv) was added to a stirred so-
lution of azetidine or substituted azetidine-TFA salt (1.1 equiv),
carboxylic acid 1 (1 equiv), HOBt (2 equiv) and EDC·HCl (2 equiv) in
DMF (10 volumes) at room temperature. The reaction mixture was

388
M. Takhi et al. / European Journal of Medicinal Chemistry 84 (2014) 382e394
Table 2
SAR on RHS part of ene-amide.
Compound
no
B
S. aureus FabI
inhibition
MIC
m
g/mLa
%@1 mM IC50(m
M) MSSAb MRSAc MRSEd
AEA22
AEA23
97%
66%
0.161
0.843
1
1
8
>32
>32
>32
AEA24
105%
0.058
1
1
2
AEA25
AEA26
83%
99%
0.384
0.112
0.25
0.25
0.25
0.25
1
Fig. 5. X-ray structure of AEA16 (shown in yellow) in complex with E. coli FabI. NADH
is shown in cyan colour. Hydrogen bonded interaction is shown in dotted line.
0.5
170.9; HRMS m/z Calcd for C20H19N3O3 [MþH]þ: 350.1505, Found:
350.1489.
AEA27
AEA28
100%
0.289
1
1
2
4.2.3. (E)-6-(3-(3-(benzyloxy)azetidin-1-yl)-3-oxoprop-1-en-1-yl)-
3,4-dihydro-1,8-naphthyridin-2(1H)-one (AEA3)
Yield ¼ 22%; Off-white solid; mp: 205e206 ꢀC; IR (KBr, cmꢂ1):
3485, 2924, 1687, 1591, 1452, 1357, 1296, 1192, 974, 846, 698, 530;
1H NMR (400 MHz, DMSO-d6) dH 10.65 (brs, 1H), 8.30 (s, 1H), 8.00
(s, 1H), 7.39 (d, J ¼ 15.2 Hz, 1H), 7.37e7.31 (m, 5H), 6.70 (d,
J ¼ 15.6 Hz, 1H), 4.47e4.44 (m, 4H), 4.14e4.10 (m, 2H), 3.73 (d,
J ¼ 7.8 Hz, 1H), 2.91 (t, J ¼ 7.6 Hz, 2H), 2.58e2.52 (m, 2H); 13C NMR
(400 MHz, DMSO-d6): dC 23.3, 30.0, 54.9, 57.0, 66.9, 70.0, 116.3,
119.0, 125.2, 127.7, 127.9, 128.3, 133.5, 136.6, 137.5, 147.2, 152.4, 165.1,
170.9; HRMS m/z Calcd for C21H21N3O3 [MþH]þ: 364.1661, Found:
364.1666.
92%
0.316
0.029
0.25
0.5
1
AFN-1252
100%
0.008 0.008 0.008
a
MIC ¼ Minimum inhibitory concentration.
b
c
MSSA e methicillin-sensitive Staphylococcus aureus ATCC 29213.
MRSA- methicillin-resistant Staphylococcus aureus ATCC 33591.
MRSE e methicillin-resistant Staphylococcus epidermidis AUCC 704.
d
147.1, 152.3, 164.8, 170.9; HRMS m/z Calcd for C14H15N3O2 [MþH]þ:
258.1243, Found: 258.1246.
4.2.4. (E)-6-(3-oxo-3-(3-(phenoxymethyl)azetidin-1-yl)prop-1-en-
1-yl)-3,4-dihydro-1,8-naphthyridin-2(1H)-one (AEA4)
4.2.2. (E)-6-(3-oxo-3-(3-phenoxyazetidin-1-yl)prop-1-en-1-yl)-
3,4-dihydro-1,8-naphthyridin-2(1H)-one (AEA2)
Yield ¼ 17%; Pale yellow solid; mp: 207e208 ꢀC; IR (KBr, cmꢂ1):
3199, 2875, 1701, 1492, 1236, 989, 761, 5281H NMR (400 MHz,
DMSO-d6): dH 8.58 (brs,1H), 8.33 (d, J ¼ 1.5 Hz,1H), 7.64 (s, 1H), 7.60
(d, J ¼ 15.6 Hz, 1H), 7.31 (d, J ¼ 7.6 Hz, 1H), 7.29 (d, J ¼ 7.3 Hz, 1H),
6.98 (t, J ¼ 7.3 Hz, 1H), 6.91 (d, J ¼ 8.0 Hz, 2H), 6.45 (d, J ¼ 15.6 Hz,
1H), 4.45 (t, J ¼ 8.4 Hz, 1H), 4.26 (dd, J ¼ 8.6 Hz, 17.3 Hz, 1H),
4.23e4.18 (m, 1H), 4.17e4.12 (m, 2H), 4.10e3.99 (m, 1H), 3.64 (t,
J ¼ 6.1 Hz, 1H), 2.99 (t, J ¼ 7.3 Hz, 2H), 2.69 (t, J ¼ 7.3 Hz, 2H); 13C
NMR (500 MHz, DMSO-d6): dC 23.3, 27.6, 30.0, 49.9, 52.5, 69.0,
114.5,116.2, 119.0,120.7, 125.3,129.4,133.5,136.4,147.1, 152.3, 158.3,
165.1, 170.9; HRMS m/z Calcd for C21H21N3O3 [MþH]þ: 364.1661,
Found: 364.1660.
Yield ¼ 17%; White solid; mp: 244e245 ꢀC; IR (KBr, cmꢂ1): 3061,
2873, 1658, 1600, 1489, 1354, 1236, 1192, 1024, 842, 754, 692; 1H
NMR (400 MHz, DMSO-d6): dH 10.65 (brs, 1H), 8.35 (d, J ¼ 1.9 Hz,
1H), 8.02 (s, 1H), 7.42 (d, J ¼ 15.6 Hz, 1H), 7.35e7.31 (m, 2H), 7.00 (t,
J ¼ 7.3 Hz, 1H), 6.88 (d, J ¼ 7.8 Hz, 2H), 6.76 (d, J ¼ 15.6 Hz, 1H),
5.11e5.08 (m, 1H), 4.77e4.73 (m, 1H), 4.42 (dd, J ¼ 7.0 Hz, 10.5 Hz,
1H), 4.22 (dd, J ¼ 3.1 Hz, 9.5 Hz, 1H), 3.90 (dd, J ¼ 3.1 Hz, 10.9 Hz,
1H), 2.90 (t, J ¼ 7.8 Hz, 2H), 2.53 (d, J ¼ 7.8 Hz, 2H); 13C NMR
(400 MHz, DMSO-d6): dC 23.3, 29.9, 54.6, 57.0, 65.3, 114.6, 116.2,
119.0, 121.3, 125.2, 129.7, 133.6, 136.9, 141.2, 152.4, 156.2, 165.1,
Fig. 4. Structural similarities between AFN-1252 and AEA16.

M. Takhi et al. / European Journal of Medicinal Chemistry 84 (2014) 382e394
389
Fig. 6. Superposition of X-ray structure of AEA16 (yellow), AFN-1252 (cyan) and triclosan (green) in complex with E. coli FabI. Hydrogen bonding interaction is shown in dotted line.
Table 3
4.2.6. (E)-6-(3-(3-((3-fluorophenoxy)methyl)azetidin-1-yl)-3-
oxoprop-1-en-1-yl)-3,4-dihydro-1,8-naphthyridin-2(1H)-one
Metabolic stability and in vivo pharmacokinetic parameters of selected compounds.
Compound
Metabolic stabilitya
In vivo pharmacokineticsb
(AEA6)
Yield ¼ 22%; Cream colour solid; mp: 228e229 ꢀC; IR (KBr,
15 min
60 min
AUC
g/mL*h)
Cmax
g/mL)
t1/2
(h)
Tmax
(h)
cmꢂ1): 2877, 1591, 1489, 1136, 950, 842,528; 1H NMR (400 MHz,
DMSO-d6): dH 10.65 (s, 1H), 8.34 (d, J ¼ 1.5 Hz, 1H), 8.03 (s, 1H), 7.40
(d, J ¼ 15.7 Hz, 1H), 7.32 (q, J ¼ 8.3 Hz, 1H), 6.87e6.78 (m, 3H), 6.74
(d, J ¼ 15.6 Hz, 1H), 4.43 (t, J ¼ 8.8 Hz, 1H), 4.20e4.06 (m, 4H),
3.78e3.74 (m, 1H), 3.12e3.06 (m, 1H), 2.91 (t, J ¼ 7.8 Hz, 2H),
2.55e2.51 (m, 2H); 13C NMR (500 MHz, DMSO-d6): dC 23.3, 27.5,
30.0, 49.9, 52.4, 69.5, 102.0, 107.4, 110.9, 116.2, 119.0, 125.2, 130.6,
130.7, 133.5, 136.5, 147.1, 152.3, 159.9, 165.1, 170.9; HRMS m/z Calcd
for C21H20FN3O3 [MþH]þ: 382.1567, Found: 382.1602.
(
m
(
m
AEA7
AEA8
91.7%
82.1%
84.0%
65.5%
79.4%
81.0%
96.2%
25.6%
55.4%
83.4%
44.4%
59.9%
36.1%
55.7%
80.2%
76.4%
6.6%
95.2
38.5
75.9
35.5
63.5
86.8
9.0
15.1
7.60
14.4
2.79
3.23
4.74
2.17
4.11
3.20
2.00
8.5
2.0
2.0
2.0
4.0
2.0
1.0
0.5
NDc
1.00
AEA14
AEA15
AEA16
AEA18
AEA25
AEA26
AFN-1252
8.95
8.10
2.25
NDc
NDc
NDc
1.84
17.5%
2.20
0.44
a
Metabolic stability study was carried in the presence of mice liver microsomes
and measured percentage of compound remaining after 15 min and 60 min.
4.2.7. (E)-6-(3-(3-((4-fluorophenoxy)methyl)azetidin-1-yl)-3-
oxoprop-1-en-1-yl)-3,4-dihydro-1,8-naphthyridin-2(1H)-one
(AEA7)
b
Pharmacokinetic experiments were performed in mice using single dose
(10 mg/kg) through oral route of administration.
c
ND ¼ not determined.
Yield ¼ 21%; White solid; mp: 98e99 ꢀC; IR (KBr, cmꢂ1): 2879,
1687, 1604, 1506, 1203, 829, 750; 1H NMR (400 MHz, DMSO-d6): dH
8.55 (brs, 1H), 8.34 (d, J ¼ 1.9 Hz, 1H), 7.64 (d, J ¼ 1.6 Hz, 1H), 7.58 (d,
J ¼ 15.6 Hz, 1H), 7.01e6.97 (m, 2H), 6.87e6.84 (m, 2H), 6.45 (d,
J ¼ 15.6 Hz, 1H), 4.47e4.43 (m, 1H), 4.27e4.20 (m, 2H), 4.19e4.10
(m, 2H), 4.08e3.98 (m, 1H), 3.14e3.11 (m, 1H), 2.99 (t, J ¼ 7.6 Hz,
2H), 2.72e2.68 (m, 2H); 13C NMR (400 MHz, DMSO-d6): dC 23.3,
27.6, 30.0, 49.9, 52.4, 69.7, 115.7, 115.8, 115.8, 115.9, 116.2, 119.0,
125.3, 133.5, 136.4, 147.1, 152.3, 154.8, 157.7, 165.0, 170.9; HRMS m/z
Calcd for C21H20FN3O3 [MþH]þ: 382.1567, Found: 382.1581.
4.2.5. (E)-6-(3-(3-((2-fluorophenoxy)methyl)azetidin-1-yl)-3-
oxoprop-1-en-1-yl)-3,4-dihydro-1,8-naphthyridin-2(1H)-one (AEA5)
Yield ¼ 13%; Pale yellow solid; mp: 186e187 ꢀC; IR (KBr, cmꢂ1):
3041, 1649, 1589, 1504, 1282, 1193, 1109,842, 748; 1H NMR
(400 MHz, DMSO-d6): dH 10.65 (s, 1H), 8.34 (s, 1H), 8.03 (s, 1H), 7.40
(d, J ¼ 15.7 Hz, 1H), 7.22e7.12 (m, 3H), 6.98e6.94 (m, 1H), 6.74 (d,
J ¼ 15.6 Hz, 1H), 4.43 (t, J ¼ 8.8 Hz, 1H), 4.26e4.06 (m, 4H),
3.82e3.76 (m,1H), 3.16e3.06 (m,1H), 2.92e2.88 (m, 2H), 2.55e2.53
(m, 2H); 13C NMR (500 MHz, DMSO-d6): dC 23.2, 27.6, 30.0, 49.8,
52.4, 70.3, 115.4, 116.0, 116.2, 119.0, 121.3, 124.8, 125.3, 133.5, 136.5,
146.3, 147.1, 150.8, 152.3, 165.1, 170.9; HRMS m/z Calcd for
4.2.8. (E)-6-(3-oxo-3-(3-((o-tolyloxy)methyl)azetidin-1-yl)prop-1-
en-1-yl)-3,4-dihydro-1,8-naphthyridin-2(1H)-one (AEA8)
C
21H20FN3O3 [MþH]þ: 382.1567, Found: 382.1569.
Yield ¼ 27%; Yellow solid; mp: 220e221 ꢀC; IR (KBr, cmꢂ1):
2877, 1654, 1591, 1494, 1365, 1246, 1192, 989, 750, 528; 1H NMR
(400 MHz, DMSO-d6): dH 10.64 (brs, 1H), 8.34 (s, 1H), 8.03 (s, 1H),
7.40 (d, J ¼ 15.7 Hz, 1H), 7.15e7.12 (m, 2H), 6.96 (d, J ¼ 8.3 Hz, 2H),
6.85 (d, J ¼ 7.3 Hz, 1H), 6.75 (d, J ¼ 15.7 Hz, 1H), 4.44e4.40 (m, 1H),
4.22e4.12 (m, 2H), 4.10e4.06 (m, 1H), 3.88e3.83 (m, 1H), 3.14e3.04
Table 4
In vivo efficacy of AEA16 in murine systemic infection
model.
Compound
ED50 (mg/kg)
AEA16
Linezolid
0.90 (0.5e1.9)a
2.30 (1.2e4.6)a
(m, 1H), 2.90 (t, J ¼ 7.8 Hz, 2H), 2.54e2.52 (m, 2H), 2.13 (s, 3H); 13
C
NMR (500 MHz, DMSO-d6): dC 15.7, 23.2, 27.8, 30.0, 49.8, 52.3, 68.7,
a
Values in brackets denote confidence range.
111.5, 116.2, 119.0, 120.4, 125.3, 125.7, 126.9, 130.3, 133.5, 136.4, 147.1,

390
M. Takhi et al. / European Journal of Medicinal Chemistry 84 (2014) 382e394
152.3, 156.4, 165.1, 170.9; HRMS m/z Calcd for C22H23N3O3 [MþH]þ:
(m, 1H), 7.43 (d, J ¼ 7.3 Hz, 1H), 7.39 (d, J ¼ 15.8 Hz, 1H), 6.74 (d,
J ¼ 15.8 Hz, 1H), 4.46 (t, J ¼ 8.5 Hz, 1H), 4.13 (t, J ¼ 8.7 Hz, 2H), 3.79
(dd, J ¼ 5.5 Hz, 10.1 Hz, 1H), 3.49 (d, J ¼ 7.9 Hz, 2H), 3.22e3.16 (m,
1H), 2.89 (t, J ¼ 7.6 Hz, 2H), 2.64e2.52 (m, 2H); 13C NMR (500 MHz,
DMSO-d6): dC 23.2, 27.8, 30.0, 37.1, 52.5, 54.8, 116.3, 119.0, 122.0,
122.2, 124.9, 125.3, 126.1, 133.5, 134.6, 136.4, 147.2, 152.3, 152.7,
165.09, 169.0, 170.9; HRMS m/z Calcd for C22H20N4O2S [MþH]þ:
405.1385, Found: 405.1386.
378.1818, Found: 378.1843.
4.2.9. (E)-6-(3-oxo-3-(3-((m-tolyloxy)methyl)azetidin-1-yl)prop-
1-en-1-yl)-3,4-dihydro-1,8-naphthyridin-2(1H)-one (AEA9)
Yield ¼ 27%; Yellow Solid; mp: 208e209 ꢀC; IR (KBr, cmꢂ1):
3537, 2877, 1687, 1602, 1454, 1292, 1159, 977, 771, 688; 1H NMR
(400 MHz, DMSO-d6): dH 10.64 (s, 1H), 8.34 (d, J ¼ 2.0 Hz, 1H), 8.03
(s, 1H), 7.40 (d, J ¼ 16.2 Hz, 1H), 7.16 (t, J ¼ 7.8 Hz, 1H), 6.79e6.72 (m,
4H), 4.43e4.38 (m, 1H), 4.15e4.04 (m, 4H), 3.78e3.73 (m, 1H),
3.07e3.04 (m, 1H), 2.92e2.87 (m, 2H), 2.54e2.50 (m, 2H), 2.27 (s,
3H); 13C NMR (500 MHz, DMSO-d6): dC 21.0, 23.3, 27.6, 30.0, 49.9,
52.4, 68.9, 111.5, 115.1, 116.2, 119.0, 121.4, 125.3, 129.2, 133.5, 136.4,
138.9, 147.1, 152.3, 158.4, 165.0, 170.9; HRMS m/z Calcd for
4.2.14. (E)-6-(3-(3-(benzo[d]thiazol-2-yl)azetidin-1-yl)-3-oxoprop-
1-en-1-yl)-3,4-dihydro-1,8-naphthyridin-2(1H)-one (AEA14)
Yield ¼ 25%; Brown solid; mp: 258e259 ꢀC; IR (KBr, cmꢂ1):
3061, 2879, 1602, 1452, 1359, 1294, 1192, 1120, 979, 844, 761, 530;
1H NMR (400 MHz, DMSO-d6): dH 10.66 (brs, 1H), 8.37 (s, 1H), 8.12
(d, J ¼ 7.6 Hz, 1H), 8.05 (s, 1H), 8.02 (d, J ¼ 7.9 Hz, 1H), 7.55e7.52 (m,
1H), 7.46 (s, 1H), 7.45 (d, J ¼ 15.9 Hz, 1H), 6.80 (d, J ¼ 15.9 Hz, 1H),
4.85e4.75 (m, 1H), 4.75e4.55 (m, 1H), 4.50e4.35 (m, 2H),
4.25e4.15 (m, 1H), 2.91 (t, J ¼ 7.4 Hz, 2H), 2.55e2.49 (m, 2H); 13C
NMR (400 MHz, DMSO-d6): dC 23.3, 30.0, 31.6, 53.6, 55.6, 116.0,
119.0, 122.3, 122.5, 125.2, 126.3, 133.6, 134.7, 136.9, 137.8, 147.3,
152.4, 152.5, 165.3, 170.9, 171.5; HRMS m/z Calcd for C21H18N4O2S
[MþH]þ: 391.1229, Found: 391.1173.
C
22H23N3O3 [MþH]þ: 378.1818, Found: 378.1774.
4.2.10. (E)-6-(3-oxo-3-(3-((p-tolyloxy)methyl)azetidin-1-yl)prop-
1-en-1-yl)-3,4-dihydro-1,8-naphthyridin-2(1H)-one (AEA10)
Yield ¼ 32%; Pale brown solid; 210e211 ꢀC; IR (KBr, cmꢂ1): 2875,
1689, 1602, 1510, 1359, 1292, 1242, 1193, 977, 815, 526; 1H NMR
(400 MHz, DMSO-d6): dH 10.65 (s,1H), 8.34 (d, J ¼ 1.9 Hz,1H), 8.02 (s,
1H), 7.39 (d, J ¼ 15.6 Hz,1H), 7.09 (t, J ¼ 8.4 Hz, 2H), 6.85 (d, J ¼ 8.8 Hz,
2H), 6.74 (d, J ¼ 15.6 Hz, 1H), 4.40 (t, J ¼ 8.8 Hz, 1H), 4.13e4.09 (m,
4H), 3.77e3.74 (m, 1H), 3.10e3.02 (m, 1H), 2.92e2.88 (m, 2H),
2.55e2.53 (m, 2H), 2.23 (s, 3H); 13C NMR (500 MHz, DMSO-d6): dC
20.0, 23.3, 27.6, 30.0, 40.9, 52.5, 69.1, 114.3, 116.2, 119.0, 125.3, 129.4,
129.8, 133.5, 136.4, 147.1, 152.3, 156.3, 165.0, 170.9; HRMS m/z Calcd
for C22H23N3O3 [MþH]þ: 378.1818, Found: 378.1855.
4.2.15. (E)-6-(3-(3-(3-methylbenzo[b]thiophen-2-yl)azetidin-1-yl)-
3-oxoprop-1-en-1-yl)-3,4-dihydro-1,8-naphthyridin-2(1H)-one
(AEA15)
Yield ¼ 23%; White solid; mp: 255e256 ꢀC; IR (KBr, cmꢂ1): 3018,
1656, 1462, 1215, 1190, 756, 669; 1H NMR (400 MHz, DMSO-d6): dH
10.66 (brs, 1H), 8.37 (s, 1H), 8.04 (s, 1H), 7.94 (d, J ¼ 7.8 Hz, 1H), 7.72
(d, J ¼ 7.8 Hz, 1H), 7.46 (d, J ¼ 15.6 Hz, 1H), 7.41e7.33 (m, 2H), 6.80
(d, J ¼ 15.6 Hz, 1H), 4.82 (d, J ¼ 7.8 Hz, 1H), 4.51e4.41 (m, 2H), 4.35
(d, J ¼ 8.3 Hz, 1H), 4.00e3.99 (m, 1H), 2.94e2.89 (m, 2H), 2.66e2.53
(m, 2H), 2.32 (s, 3H); 13C NMR (400 MHz, DMSO-d6): dC 11.4, 23.3,
27.4, 30.0, 55.4, 57.5, 113.8, 116.1, 116.6, 119.1, 121.7, 122.5, 124.3,
125.3, 128.1, 133.79, 137.0, 147.2, 152.4, 158.2, 158.6, 165.2, 171.0;
HRMS m/z Calcd for C23H21N3O2S [MþH]þ: 404.1433, Found:
404.1394.
4.2.11. (E)-6-(3-(3-(naphthalen-2-yloxy)azetidin-1-yl)-3-oxoprop-
1-en-1-yl)-3,4-dihydro-1,8-naphthyridin-2(1H)-one (AEA11)
Yield ¼ 23%; Brown solid; 250e251 ꢀC; IR (KBr, cmꢂ1): 3197,
3059, 2875, 1681, 1598, 1442, 1215, 844, 756, 528; 1H NMR
(400 MHz, DMSO-d6): dH 10.64 (brs, 1H), 8.35 (d, J ¼ 1.0 Hz, 1H),
8.03 (s, 1H), 7.83 (d, J ¼ 8.8 Hz, 1H), 7.80 (d, J ¼ 8.3 Hz, 2H), 7.45 (d,
J ¼ 7.8 Hz, 1H), 7.41 (d, J ¼ 15.6 Hz, 1H), 7.39e7.37 (m, 2H), 7.18 (dd,
J ¼ 2.4 Hz, 8.8 Hz, 1H), 6.76 (d, J ¼ 15.6 Hz, 1H), 4.50e4.42 (m, 1H),
4.31 (d, J ¼ 6.3 Hz, 2H), 4.22e4.10 (m, 2H), 3.88e3.80 (m, 1H),
3.20e3.10 (m, 1H), 2.91 (t, J ¼ 7.9 Hz, 2H), 2.56e2.53 (m, 2H); 13C
NMR (500 MHz, DMSO-d6): dC 23.3, 27.6, 30.0, 50.0, 52.5, 69.1,106.9,
116.2, 118.5, 119.0, 123.6, 125.3, 126.4, 126.6, 127.4, 128.5, 129.3,
133.5, 134.2, 136.5, 147.1, 152.3, 156.3, 165.1, 170.9; HRMS m/z Calcd
for C25H23N3O3 [MþH]þ: 414.1818; Found: 414.1840.
4.2.16. (E)-6-(3-(3-(3-methylbenzofuran-2-yl)azetidin-1-yl)-3-
oxoprop-1-en-1-yl)-3,4-dihydro-1,8-naphthyridin-2(1H)-one
(AEA16)
Yield ¼ 20%; White solid; mp: 236e237 ꢀC; IR (KBr, cmꢂ1): 3128,
2881, 1691, 1651, 1454, 1192, 979, 748, 675, 526; 1H NMR (400 MHz,
DMSO-d6): dH 10.66 (brs,1H), 8.37 (s,1H), 8.03 (s,1H), 7.56e7.53 (m,
2H), 7.47 (d, J ¼ 15.6 Hz, 1H), 7.28e7.24 (m, 2H), 6.80 (d, J ¼ 15.6 Hz,
1H), 4.75e4.62 (m, 1H), 4.54e4.48 (m, 1H), 4.40e4.32 (m, 1H),
4.31e4.21 (m, 1H), 4.18e4.10 (m, 1H), 2.91 (t, J ¼ 7.9 Hz, 2H),
2.56e2.52 (m, 2H), 2.19 (s, 3H); 13C NMR (400 MHz, DMSO-d6): dC
7.5, 23.3, 24.6, 30.0, 52.4, 54.4, 110.8, 111.4, 116.1, 119.1, 119.3, 122.5,
124.1, 125.3, 129.6, 133.7, 136.9, 147.3, 151.6, 152.5, 153.4, 165.2,
171.0; HRMS m/z Calcd for C23H21N3O3 [MþH]þ: 388.1661, Found:
388.1685.
4.2.12. (E)-6-(3-(3-(benzo[d]thiazol-2-yloxy)azetidin-1-yl)-3-
oxoprop-1-en-1-yl)-3,4-dihydro-1,8-naphthyridin-2(1H)-one
(AEA12)
Yield ¼ 36%; White solid; mp: 275e276 ꢀC; IR (KBr, cmꢂ1): 3460,
2922, 1656, 1527, 1442, 1253, 1043, 761; 1H NMR (400 MHz, DMSO-
d6): dH 10.65 (brs,1H), 8.35 (s,1H), 8.04 (s,1H), 7.94 (d, J ¼ 7.9 Hz,1H),
7.69 (d, J ¼ 7.9 Hz, 1H), 7.43 (d, J ¼ 15.6 Hz, 1H), 7.43 (s, 1H), 7.32 (d,
J ¼ 7.6 Hz, 1H), 6.78 (d, J ¼ 15.6 Hz, 1H), 5.63 (t, J ¼ 3.2 Hz, 1H),
4.83e4.79 (m, 1H), 4.45e4.16 (m, 2H), 4.10e4.08 (m, 1H), 2.90 (t,
J ¼ 7.6 Hz, 2H), 2.54e2.50 (m, 2H); 13C NMR (500 MHz, DMSO-d6): dC
23.3, 29.9, 54.4, 57.2, 70.1,116.2,119.0,120.7,122.2,124.0,125.2,126.3,
131.5, 133.6, 136.9, 147.3, 148.6, 152.4, 165.2, 170.6, 170.9; HRMS m/z
Calcd for C21H18N4O3S [MþH]þ: 407.1178, Found: 407.1165.
4.2.17. (E)-6-(3-(3-(5-methoxy-3-methylbenzofuran-2-yl)azetidin-
1-yl)-3-oxoprop-1-en-1-yl)-3,4-dihydro-1,8-naphthyridin-2(1H)-
one (AEA17)
Yield ¼ 16%; White solid; mp: 238e239 ꢀC; IR (KBr, cmꢂ1): 3745,
2924, 1656, 1462, 1192, 833, 723; 1H NMR (400 MHz, DMSO-d6): dH
10.66 (brs,1H), 8.37 (s,1H), 8.03 (s,1H), 7.46 (d, J ¼ 15.6 Hz,1H), 7.44
(d, J ¼ 8.8 Hz,1H), 7.04 (d, J ¼ 2.4 Hz,1H), 6.85 (dd, J ¼ 2.5 Hz, 8.8 Hz,
1H), 6.79 (d, J ¼ 15.6 Hz, 1H), 4.67 (t, J ¼ 8.6 Hz, 1H), 4.49e4.45 (m,
1H), 4.33 (t, J ¼ 9.2 Hz, 1H), 4.26e4.19 (m, 1H), 4.12e4.08 (m, 1H),
3.79 (s, 3H), 2.91 (t, J ¼ 7.6 Hz, 2H), 2.55e2.50 (m, 2H), 2.16 (s, 3H);
13C NMR (400 MHz, DMSO-d6): dC 7.5, 23.3, 24.7, 30.0, 52.4, 54.4,
55.6, 111.3, 111.6, 112.3, 113.7, 116.2, 116.6, 119.1, 125.3, 130.3, 133.8,
4.2.13. (E)-6-(3-(3-(benzo[d]thiazol-2-ylmethyl)azetidin-1-yl)-3-
oxoprop-1-en-1-yl)-3,4-dihydro-1,8-naphthyridin-2(1H)-one
(AEA13)
Yield ¼ 23%; Pale brown solid; mp: 78e79 ꢀC; IR (KBr, cmꢂ1):
3537, 3118, 2870, 1691, 1656, 1444, 1284, 1182, 773, 524; 1H NMR
(400 MHz, DMSO-d6): dH 10.64 (brs, 1H), 8.33 (d, J ¼ 1.5 Hz, 1H),
8.07 (d, J ¼ 7.9 Hz,1H), 8.03 (s, 1H), 7.95 (d, J ¼ 7.9 Hz,1H), 7.52e7.48

M. Takhi et al. / European Journal of Medicinal Chemistry 84 (2014) 382e394
391
136.9, 147.1, 152.4, 155.5, 158.6, 165.22, 171.0; HRMS m/z Calcd for
C
8.14e8.08 (m, 1H), 8.11 (m, 1H), 7.56e7.53 (m, 2H), 7.49 (d,
J ¼ 15.7 Hz, 1H), 7.28e7.24 (m, 2H), 6.84 (d, J ¼ 15.7 Hz, 1H),
4.71e4.67 (m, 1H), 4.52e4.48 (m, 1H), 4.37e4.33 (m, 1H), 4.30e4.23
(m, 1H), 4.15e4.11 (m, 1H), 2.18 (s, 3H), 2.10 (s, 3H); 13C NMR
(400 MHz, DMSO-d6): dC 7.4, 23.9, 24.6, 52.4, 54.4,110.8,111.3,112.8,
116.7, 119.2, 122.4, 124.1, 126.1, 129.6, 136.5, 136.6, 148.5, 151.5, 152.7,
24H23N3O4 [MþH]þ: 418.1767, Found: 418.1731.
4.2.18. (E)-6-(3-(3-(5-chloro-3-methylbenzofuran-2-yl)azetidin-1-
yl)-3-oxoprop-1-en-1-yl)-3,4-dihydro-1,8-naphthyridin-2(1H)-one
(AEA18)
Yield ¼ 14%; White solid; mp: 261e262 ꢀC; IR (KBr, cmꢂ1): 2949,
1689, 1602, 1444, 1190, 977, 750, 526; 1H NMR (400 MHz, DMSO-
d6): dH 10.66 (brs, 1H), 8.37 (s, 1H), 8.03 (s, 1H), 7.63 (s, 1H), 7.59 (d,
J ¼ 8.8 Hz, 1H), 7.46 (d, J ¼ 15.8 Hz, 1H), 7.30 (dd, J ¼ 1.6 Hz, 8.5 Hz,
1H), 6.78 (d, J ¼ 15.8 Hz, 1H), 4.68 (t, J ¼ 8.5 Hz, 1H), 4.51e4.46 (m,
1H), 4.37e4.25 (m, 2H), 4.14e4.10 (m, 1H), 2.91 (t, J ¼ 7.5 Hz, 2H),
2.54e2.51 (m, 2H), 2.17 (s, 3H); 13C NMR (500 MHz, DMSO-d6): dC
7.3, 23.3, 24.6, 30.0, 52.3, 54.3, 111.4, 112.4, 116.1, 118.9119.0, 123.9,
125.2,127.0,131.3,133.6,136.9,147.2,151.9,152.4,153.5,165.1,170.9;
HRMS m/z Calcd for C23H20ClN3O3 [MþH]þ: 422.1271, Found:
422.1289.
153.4, 165.0, 169.4; HRMS m/z Calcd for
C22H21N3O3
[MþH]þ:376.1661, Found: 376.1678.
4.2.23. (E)-N-(5-(3-(3-(3-methylbenzofuran-2-yl)azetidin-1-yl)-3-
oxoprop-1-en-1-yl)pyridin-2-yl)-2-(1H-1,2,3-triazol-1-yl)
acetamide (AEA23)
Yield ¼ 24%; Pale yellow solid; mp: 227e228 ꢀC; IR (KBr, cmꢂ1):
3321, 2951, 1697, 1537, 1390, 1305, 744; 1H NMR (400 MHz, DMSO-
d6): dH 11.21 (s, 1H), 8.68 (d, J ¼ 1.9 Hz, 1H), 8.20 (dd, J ¼ 2.5 Hz,
8.8 Hz, 1H), 8.16 (s, 1H), 8.03 (d, J ¼ 8.3 Hz, 1H), 7.77 (s, 1H),
7.56e7.54 (m, 2H), 7.51 (d, J ¼ 15.6 Hz, 1H), 7.30e7.22 (m, 2H), 6.89
(d, J ¼ 15.6 Hz, 1H), 5.46 (s, 2H), 4.72e4.67 (m, 1H), 4.52e4.48 (m,
1H), 4.38e4.33 (m, 1H), 4.29e4.23 (m, 1H), 4.16e4.12 (m, 1H), 2.19
(s, 3H); 13C NMR (500 MHz, DMSO-d6): dC 7.4, 24.6, 51.9, 52.4, 54.4,
110.8, 111.3, 113.1, 117.2, 119.2, 122.4, 124.1, 126.5, 126.8, 129.6, 133.1,
136.4, 136.8, 148.7, 151.5, 152.0, 153.4, 164.9, 165.4; HRMS m/z Calcd
for C24H22N6O3 [MþH]þ:443.1832, Found: 443.1816.
4.2.19. (E)-6-(3-(3-(4-methylquinolin-2-yl)azetidin-1-yl)-3-
oxoprop-1-en-1-yl)-3,4-dihydro-1,8-naphthyridin-2(1H)-one
(AEA19)
Yield ¼ 18%; Brown solid; mp: 223e224 ꢀC; IR (KBr, cmꢂ1): 3061,
1651, 1598, 1489, 1290, 1190, 972, 848, 769, 526; 1H NMR (400 MHz,
DMSO-d6): dH 10.65 (brs, 1H), 8.36 (d, J ¼ 1.5 Hz, 1H), 8.08 (d,
J ¼ 7.8 Hz, 1H), 8.05 (s, 1H), 8.00 (d, J ¼ 8.3 Hz, 1H), 7.82e7.73 (m,
1H), 7.63e7.59 (m, 1H), 7.46e7.41 (m, 2H), 6.81 (d, J ¼ 15.6 Hz, 1H),
4.73e4.63 (m, 2H), 4.41e4.37 (m, 1H), 4.24e4.15 (m, 2H), 2.91 (t,
J ¼ 7.6 Hz, 2H), 2.69 (s, 3H), 2.55e2.53 (m, 2H); 13C NMR (500 MHz,
DMSO-d6): dC 18.1, 23.3, 30.0, 34.4, 52.9, 54.7, 116.3, 119.0, 121.0,
124.1, 125.3, 126.0, 126.7, 129.0, 129.4, 133.6, 136.6, 145.0, 147.0,
147.2, 152.4, 160.5, 165.2, 170.9; HRMS m/z Calcd for C24H22N4O2
[MþH]þ: 399.1821, Found: 399.1833.
4.2.24. (E)-6-(3-(3-(3-methylbenzofuran-2-yl)azetidin-1-yl)-3-
oxoprop-1-en-1-yl)-3-(2-morpholinoethyl)-3,4-dihydropyrido[2,3-
d]pyrimidin-2(1H)-one (AEA24)
Yield ¼ 13%; Off-white solid; mp: 218e219 ꢀC; (KBr, cmꢂ1):
2949, 2818, 1672, 1593, 1492, 1450, 1408, 1296, 1114, 746; 1H NMR
(400 MHz, DMSO-d6): dH 9.88 (brs, 1H), 8.35 (s, 1H), 7.93 (s, 1H),
7.56e7.53 (m, 2H), 7.45 (d, J ¼ 15.8 Hz, 1H), 7.30e7.23 (m, 2H), 6.74
(d, J ¼ 15.5 Hz, 1H), 4.67 (t, J ¼ 8.5 Hz, 1H), 4.54 (s, 2H), 4.50e4.47
(m, 1H), 4.36e4.33 (m, 1H), 4.29e4.25 (m, 1H), 4.14e4.11 (m, 1H),
3.60e3.50 (m, 4H), 3.45 (t, J ¼ 6.1 Hz, 2H), 2.41e2.35 (m, 6H), 2.19 (s,
3H); 13C NMR (500 MHz, DMSO-d6): dC 7.4, 24.6, 40.0, 46.9, 52.4,
53.2, 54.3, 55.4, 65.9, 110.8, 111.3, 113.4, 115.5, 119.2, 122.4, 124.1,
124.5, 129.6, 131.7, 136.9, 148.2, 151.2, 151.5, 152.8, 153.4, 165.1;
4.2.20. (E)-6-(3-oxo-3-(3-(pyridin-2-yl)azetidin-1-yl)prop-1-en-1-
yl)-3,4-dihydro-1,8-naphthyridin-2(1H)-one (AEA20)
Yield ¼ 23%; Brown solid; mp: 222e223 ꢀC; IR (KBr, cmꢂ1):
3007, 2877, 2677, 1687, 1597, 1433, 1357, 1188, 779, 530; 1H NMR
(400 MHz, DMSO-d6): dH 10.67 (brs, 1H), 8.61 (d, J ¼ 4.4 Hz, 1H),
8.36 (s, 1H), 8.03 (s, 1H), 7.80e7.76 (m, 1H), 7.42 (d, J ¼ 15.6 Hz, 1H),
7.37 (d, J ¼ 7.8 Hz, 1H), 7.30 (dd, J ¼ 5.1 Hz, 7.1 Hz, 1H), 6.77 (d,
J ¼ 15.6 Hz, 1H), 4.65 (t, J ¼ 8.0 Hz, 1H), 4.48e4.45 (m, 1H), 4.46 (t,
J ¼ 8.8 Hz, 1H), 4.10e3.99 (m, 2H), 3.09 (dt, J ¼ 7.3 Hz, 12.2 Hz, 2H),
2.90 (t, J ¼ 7.6 Hz, 2H); 13C NMR (400 MHz, DMSO-d6): dC 23.3, 30.0,
33.9, 53.4, 55.4, 116.2, 119.0, 122.2, 122.4, 125.3, 133.6, 136.5, 136.8,
147.1, 149.4, 152.4, 160.3, 165.1, 170.9; HRMS m/z Calcd for
HRMS m/z Calcd for
502.2432.
C
28H31N5O4 [MþH]þ: 502.2454, Found:
4.3. General procedure for the synthesis of AEA25e28
To a stirred suspension of bromo derivative 30 or 31 or 32 or 33
(1 equiv), acrylamide 29 (1.3 equiv) in propionitrile:DMF (4:1 vol-
umes) was added diisopropyl ethyl amine (3 equiv) and the reac-
tion mixture was degassed with nitrogen for 10 min. Then Pd(OAc)2
(0.2 equiv), P(o-tolyl)3 (0.4 equiv) were added, again degassed with
nitrogen for another 10 min and the reaction mixture was allowed
to stir at 110 ꢀC for 16 h. The reaction mixture was cooled to room
temperature, filtered through celite and filtrate was evaporated
under vacuum. The resulting residue was diluted with H2O (50
volumes) and extracted with ethyl acetate (3 ꢃ 50 volumes). The
combined organic layers were washed with brine (50 volumes),
dried over Na2SO4, filtered and volatiles were removed under
vacuum. The crude residue was purified by column chromatog-
raphy to furnish the desired products AEA25e28.
C
19H18N4O2 [MþH]þ: 335.1508, Found: 335.1515.
4.2.21. (E)-6-(3-oxo-3-(3-phenylazetidin-1-yl)prop-1-en-1-yl)-3,4-
dihydro-1,8-naphthyridin-2(1H)-one (AEA21)
Yield ¼ 50%; Off-white Solid; mp: 268e269 ꢀC; IR (KBr, cmꢂ1):
3034, 2875, 1656, 1591, 1365, 1290, 1193, 758, 700; 1H NMR
(400 MHz, DMSO-d6): dH 10.65 (s, 1H), 8.35 (d, J ¼ 2.0 Hz, 1H), 8.02
(d, J ¼ 0.9 Hz, 1H), 7.43 (d, J ¼ 15.7 Hz, 1H), 7.38e7.36 (m, 4H),
7.35e7.25 (m, 1H), 6.77 (d, J ¼ 15.6 Hz, 1H), 4.71 (t, J ¼ 8.3 Hz, 1H),
4.39e4.27 (m, 2H), 3.95e3.89 (m, 2H), 2.93e2.89 (m, 2H),
2.56e2.51 (m, 2H); 13C NMR (400 MHz, DMSO-d6): dC 23.3, 30.0,
32.6, 54.8, 56.9, 116.3, 119.0, 125.3, 126.7, 128.6, 133.5, 136.5, 136.8,
142.1147.1, 147.7, 165.1, 170.9; HRMS m/z Calcd for C20H19N3O2
[MþH]þ: 334.1556, Found: 334.1557.
4.3.1. (E)-7-(3-(3-(3-methylbenzofuran-2-yl)azetidin-1-yl)-3-
oxoprop-1-en-1-yl)-4,5-dihydro-1H-pyrido[2,3-e][1,4]diazepin-
2(3H)-one (AEA25)
4.2.22. (E)-N-(5-(3-(3-(3-methylbenzofuran-2-yl)azetidin-1-yl)-3-
oxoprop-1-en-1-yl)pyridin-2-yl)acetamide (AEA22)
Yield ¼ 10%; Pale yellow solid; mp: 204e205 ꢀC; IR (KBr, cmꢂ1):
3280, 3062, 2912, 2883, 1651, 1454, 1328, 977, 856, 748; 1H NMR
(400 MHz, DMSO-d6): dH 10.07 (brs, 1H), 8.45 (d, J ¼ 2.0 Hz, 1H),
7.98 (s, 1H), 7.56e7.53 (m, 2H), 7.47 (d, J ¼ 15.7 Hz, 1H), 7.30e7.22
(m, 2H), 6.83 (d, J ¼ 16.2 Hz, 1H), 4.69 (t, J ¼ 8.5 Hz, 1H), 4.52e4.48
Yield ¼ 15%; White solid; mp: 226e227 ꢀC; IR (KBr, cmꢂ1): 3255,
2949, 2883, 1651, 1519, 1444, 1301, 975, 837, 744, 624; 1H NMR
(400 MHz, DMSO-d6): dH 10.67 (s, 1H), 8.61 (d, J ¼ 1.9 Hz, 1H),

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M. Takhi et al. / European Journal of Medicinal Chemistry 84 (2014) 382e394
(m, 1H), 4.35 (t, J ¼ 9.3 Hz, 1H), 4.30e4.23 (m, 1H), 4.15e4.11 (m,
1H), 3.90 (s, 2H), 3.63 (s, 2H), 3.06 (brs, 1H), 2.19 (s, 3H); 13C NMR
(400 MHz, DMSO-d6): dC 7.4, 24.6, 50.3, 52.4, 54.4, 55.1, 110.8, 111.3,
116.7, 119.3, 122.4, 124.1, 125.2, 126.5, 129.6, 135.4, 136.4, 147.1, 151.0,
151.5, 153.4, 165.1, 174.5; HRMS m/z Calcd for C23H22N4O3 [MþH]þ:
403.1770, Found: 403.1757.
enzyme activity was determined. Test compounds were initially
screened at 1 M, in duplicate. Doseeresponse studies were sub-
m
sequently carried out for active compounds. IC50 values were
determined by fitting the activity data at different concentrations of
the compound to sigmoidal dose response (variable slope) curve
fitting program using GraphPad Prism software V5. The standard
error in IC50 values estimated from the curve fitting was <15%.
4.3.2. (E)-4-methyl-7-(3-(3-(3-methylbenzofuran-2-yl)azetidin-1-
yl)-3-oxoprop-1-en-1-yl)-4,5-dihydro-1H-pyrido[2,3-e][1,4]
diazepin-2(3H)-one (AEA26)
4.5. In vitro antibacterial screen
Yield ¼ 44%; Pale green solid; mp: 218e219 ꢀC; IR (KBr, cmꢂ1):
3062, 2945, 2879, 1651, 1454, 1344, 850, 742, 663; 1H NMR
(400 MHz, DMSO-d6): dH 10.38 (brs, 1H), 8.54 (s, 1H), 8.11 (s, 1H),
7.56e7.54 (m, 2H), 7.50 (d, J ¼ 15.6 Hz, 1H), 7.30e7.22 (m, 2H), 6.86
(d, J ¼ 15.6 Hz, 1H), 4.70 (t, J ¼ 8.6 Hz, 1H), 4.53e4.49 (m, 1H), 4.36
(t, J ¼ 9.0 Hz, 1H), 4.29e4.26 (m, 1H), 4.15e4.12 (m, 1H), 3.80 (s, 2H),
3.44 (s, 2H), 2.37 (s, 3H), 2.19 (s, 3H); 13C NMR (400 MHz, DMSO-
d6): dC 7.4, 24.6, 42.3, 52.4, 54.4, 56.8, 60.3, 110.8, 111.3, 117.2, 119.2,
122.4,123.0,124.1,126.1,129.6,136.2,137.0, 148.0,151.5, 151.8, 153.4,
165.0, 171.4; HRMS m/z Calcd for C24H24N4O3 [MþH]þ:417.1927,
Found: 417.1877.
Minimum Inhibitory Concentration (MIC) was determined by
broth microdilution method as per CLSI guidelines. Serial two-fold
dilution of the compounds was made using MHB in 96 well
microtitre plates at twice the desired final concentration. 50 mL of
the adjusted inoculum suspension was dispensed into each well to
give a final inoculum density of 5 ꢃ 105 CFU/mL. Broth, compound
and organism controls were set up in duplicate. S. aureus ATCC
29213 was incorporated as a quality control strain in the study.
Plates were incubated at 35
2
ꢀC for 16e20 h in an ambient air
incubator. After the incubation period, growth of organism in the
wells was detected by unaided eye facilitated by a viewing device.
The amount of growth in the wells containing the antibiotic was
compared with the amount of growth in organism control wells (no
antibiotic) to help in determining the end point. MIC was the lowest
concentration of the antibiotic/compound which inhibits bacterial
4.3.3. (E)-7-(3-(3-(3-methylbenzofuran-2-yl)azetidin-1-yl)-3-
oxoprop-1-en-1-yl)-4-(2-morpholinoethyl)-4,5-dihydro-1H-pyrido
[2,3-e][1,4]diazepin-2(3H)-one (AEA27)
Yield ¼ 23%; Pale yellow solid; mp: 240e241 ꢀC; IR (KBr, cmꢂ1):
3059, 2947, 2812, 1658, 1454, 1114, 854, 746; 1H NMR (400 MHz,
DMSO-d6): dH 10.32 (s, 1H), 8.52 (d, J ¼ 2.0 Hz, 1H), 8.10 (d,
J ¼ 2.0 Hz,1H), 7.56e7.53 (m,1H), 7.49 (d, J ¼ 15.9 Hz,1H), 7.26e7.24
(m, 2H), 6.86 (d, J ¼ 15.9 Hz, 1H), 4.68e4.64 (m, 1H), 4.52e4.48 (m,
1H), 4.39e4.36 (m, 2H), 4.28e4.20 (m, 1H), 4.18e4.16 (m, 2H), 3.91
(s, 2H), 3.53 (s, 2H), 3.52e3.51 (m, 3H), 3.29 (s, 2H), 2.67e2.65 (m,
2H), 2.42e2.40 (m, 2H), 2.33e2.31 (m, 2H), 2.19 (s, 3H); 13C NMR
(400 MHz, DMSO-d6): dC 7.4, 24.6, 50.2, 52.4, 53.4, 54.4, 55.2, 56.3,
59.1, 66.0, 110.8, 111.3, 117.1, 119.2, 122.4, 123.1, 124.1, 125.9, 129.6,
136.3, 137.1, 147.9, 151.5, 151.7, 153.4, 165.0, 172.1; HRMS m/z Calcd
for C29H33N5O4 [MþH]þ: 516.2611, Found: 516.2665.
growth as detected by unaided eye and expressed as mg/mL.
4.6. Protein expression, purification and co-crystallization of E. coli
FabI [37]
Gene sequence corresponding to full length E. coli FabI was
cloned into pET28a vector. Transformants of E. coli BL21DE3 con-
taining pET28a-Ec FabI were grown to an optical density (595 nm)
of 0.8 and induced with 0.5 mM IPTG at 18 ꢀC for 18 h. Cell pellet
was resuspended in lysis buffer containing 50 mM Tris pH 7.5,
500 mM NaCl, 1 mM DTT, 50
mg/mL Lysozyme, cells lysed with
sonication and clarified lysate passed through Ni-NTA column.
Protein was eluted with buffer containing 50 mM Tris pH 7.5,
500 mM NaCl, 1 mM DTT and 250 mM imidazole. Fractions con-
taining E. coli FabI were pooled and passed through Superdex-75
gel filtration column. Fractions containing pure FabI protein were
incubated with 5 M excess of inhibitor and 2 M excess of NADH for
1 h at 4 ꢀC. After, incubation protein was concentrated to 16 mg/mL
for crystallization experiments. Crystals were grown at 20 ꢀC using
hanging drop vapour diffusion method. Crystallization well buffer
contained 0.1 M sodium acetate pH 4.6, 12% PEG 4000 and 200 mM
ammonium acetate. One microlitre protein solution was mixed
with one microlitre of crystallization well buffer containing 0.1 M
sodium acetate pH 4.6, 12% PEG 4000 and 200 mM ammonium
acetate. Crystals with the dimensions of 0.1 ꢃ 0.05 ꢃ 0.05 mm were
obtained overnight.
4.3.4. (E)-8-(3-(3-(3-methylbenzofuran-2-yl)azetidin-1-yl)-3-
oxoprop-1-en-1-yl)-2,3-dihydro-1H-pyrido[2,3-b][1,4]diazepin-
4(5H)-one (AEA28)
Yield ¼ 31%; Pale brown solid; mp: 301e302 ꢀC; IR (KBr, cmꢂ1):
3302, 3066, 2947, 2881, 1656, 1585, 1452, 1382, 850, 744; 1H NMR
(400 MHz, DMSO-d6): dH 9.75 (s, 1H), 7.99 (d, J ¼ 1.5 Hz, 1H),
7.56e7.52 (m, 2H), 7.39 (d, J ¼ 15.6 Hz, 1H), 7.36 (d, J ¼ 1.9 Hz, 1H),
7.30e7.22 (m, 2H), 6.65 (d, J ¼ 15.6 Hz, 1H), 6.07 (t, J ¼ 3.6 Hz, 1H),
4.68 (t, J ¼ 8.5 Hz, 1H), 4.51e4.46 (m, 1H), 4.35 (t, J ¼ 9.3 Hz, 1H),
4.30e4.24 (m, 1H), 4.13 (dd, J ¼ 5.9 Hz, 9.3 Hz, 1H), 3.44e3.40 (m,
2H), 2.61 (t, J ¼ 5.4 Hz, 2H), 2.19 (s, 3H); 13C NMR (500 MHz, DMSO-
d6): dC 7.4, 24.6, 37.7, 42.2, 52.4, 54.3, 110.8, 111.3, 116.3, 119.2, 122.4,
122.7, 124.1, 126.5, 129.6, 135.0, 136.9, 137.4, 140.0, 151.4, 153.3,
164.9, 172.1; HRMS m/z Calcd for C23H22N4O3 [MþH]þ: 403.1770,
Found: 403.1803.
4.7. X-ray crystallography
4.4. S. aureus FabI biochemical assay
The co-crystals were flash frozen at 100 K using 20% glycerol as
cryo-protectant. The diffraction data sets were collected using in-
house Rigaku RU300 X-ray generator with R-AXIS IVþþ detector to
a maximum resolution of3.2 Å. Data indexing, integration and scaling
wereperformedusingDENZOandSCALEBPCK[38]. Thestructurewas
solved by molecular replacement (MR) method using the PDB code
1C14 as the template search model. Alternate cycles of restrained
refinement and manual rebuilding were performed with the pro-
grams REFMAC 5.2.0001 [39,40] and Coot [41] respectively. 5% of the
reflections were randomly excluded from the refinement to monitor
the free residual-factor (Rfree). A summary of the data reduction and
The enzymatic assay [17] is based on the decrease in absorbance
at 340 nm resulting from the oxidation of NADPH accompanying
the reduction of enoyl e ACP, catalysed by FabI enzyme. The assay
was carried out in 100 mM Sodium ADA (N-[2-Acetamido] imino-
diacetic acid) buffer, pH 6.5. FabI enzyme (2400 ng/assay) was pre-
incubated with NADH (375
the reaction was started by adding Crotonoyl CoA (250
total reaction volume was 100 L. After 2 h incubation at room
temperature, the absorbance at 340 nm was measured and the
mM) and test compounds for 30 min and
mM). The
m

M. Takhi et al. / European Journal of Medicinal Chemistry 84 (2014) 382e394
393
structure refinementstatisticsisprovidedin supplementary material.
Acknowledgements
Coordinates of the X-ray structure have been deposited in the Protein
Data Bank with accession codes 4JQC (AFN-1252) and 4JX8 (AEA16).
Figures showing structural models were rendered using PyMOL [42].
Inhibitors could be modelled into the electron density unambigu-
ously near the active site. Figure depicting the electron density map is
shown in the supplementary information.
We appreciate the services extended by the Analytical Research
department of Aurigene Discovery Technologies for having carried
out all the analytical work. NAR, RY, IA and YKL acknowledge the
support from the University of Malaya, Malaysia.
Appendix A. Supplementary data
4.8. Metabolic stability study in mice liver microsomes
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.07.036.
In vitro metabolism of compounds was studied in mouse liver
microsomes to assess the metabolic stability at 1 mM concentration.
The typical reaction mixture consists of incubation buffer (KH2PO4,
pH 7.4), protein (liver microsomes, assay concentration: 0.3 mg/
mL), NADPH (assay concentration: 1 mM) and test compound. Re-
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