Extending the N-linked aminopiperidine class to the mycobacterial gyrase domain: Pharmacophore mapping from known antibacterial leads

Article abstract of DOI:10.1016/j.ejmech.2014.08.018

Bacterial DNA gyrase is a well-established and clinically validated target to develop novel antibacterial. Our effort was designated to search for synthetically better compounds with possibility of hit to lead development. With this as objective, a series of 1-(2-(4-aminopiperidin-1-yl)ethyl)-1,5- naphthyridin-2(1H)-one derivatives were designed by molecular hybridization strategy and synthesized following nine step reaction to yield activity in low nanomolar range and commendable antibacterial activities. Compound 1-(4-fluorophenyl)-3-(1-(2-(7-methoxy-2-oxo-1,5-naphthyridin-1(2H)-yl)ethyl) piperidin-4-yl)urea (35) emerged as the most promising inhibitor with an IC ₅₀ of 78 nM against Mycobacterium tuberculosis DNA gyrase enzyme, with MTB MIC of 0.62 μM, and not cytotoxic at 50 μM in eukaryotic cell line.

Full text of DOI:10.1016/j.ejmech.2014.08.018

European Journal of Medicinal Chemistry 85 (2014) 593e604
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Extending the N-linked aminopiperidine class to the mycobacterial
gyrase domain: Pharmacophore mapping from known antibacterial
leads
Karyakulam Andrews Bobesh, Janupally Renuka, Variam Ullas Jeankumar,
Singh Kakan Shruti, Jonnalagadda Padma Sridevi, Perumal Yogeeswari,
Dharmarajan Sriram*
Department of Pharmacy, Birla Institute of Technology & Science-Pilani, Hyderabad Campus, Shameerpet, Jawaharnagar, RangaReddy District, Hyderabad
500 078, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Bacterial DNA gyrase is a well-established and clinically validated target to develop novel antibacterial.
Our effort was designated to search for synthetically better compounds with possibility of hit to lead
development. With this as objective, a series of 1-(2-(4-aminopiperidin-1-yl)ethyl)-1,5-naphthyridin-
2(1H)-one derivatives were designed by molecular hybridization strategy and synthesized following nine
step reaction to yield activity in low nanomolar range and commendable antibacterial activities. Com-
Received 19 June 2014
Received in revised form
5 August 2014
Accepted 6 August 2014
Available online 7 August 2014
pound
1-(4-fluorophenyl)-3-(1-(2-(7-methoxy-2-oxo-1,5-naphthyridin-1(2H)-yl)ethyl)piperidin-4-yl)
urea (35) emerged as the most promising inhibitor with an IC50 of 78 nM against Mycobacterium
Keywords:
DNA gyrase
tuberculosis DNA gyrase enzyme, with MTB MIC of 0.62
m
M, and not cytotoxic at 50
mM in eukaryotic cell
line.
Tuberculosis
Aminopiperidine
Antimycobacterial
Supercoiling
Cytotoxicity
© 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
trapping the gyrase-DNA complex which further resulted in
oxidative damage that ultimately led to the bacterial cell death and
has been found effective against both replicating and non-
replicating, persistent mycobacterium strains [4]. The reports on
moxifloxacin, also suggested that DNA gyrase may be a good target
for reducing the length of TB treatment regimens. However, in spite
of the quite successful progress made by the FQ class of analogues,
the emerging resistance to the FQ leads to grave concern and drives
the quest for newer, safer, and more effective TB treatment options
[5]. Novel bacterial type II topoisomerase inhibitors (NBTIs) are
promising class of topoisomerase inhibitor that are structurally and
mechanistically different from the FQ class and hence may not be
impacted by target mutations that cause resistance to FQ [6]. These
classes of drugs have made significant progress in the recent years
exhibiting broad spectrum antibacterial potency with many
promising candidates being evaluated at various stages of drug
discovery program. Their overall chemical structure, topology in-
corporates an N-linked ‘4-aminopiperidine’ linker fused between a
bicyclic left hand nucleus (LHS) and an aryl/heteroaryl right-hand-
core (RHS). NBTIs, though well explored as a probable class for
developing newer antibacterial leads; scarcely anything have been
Human negligence and lack of novel therapeutic agents have
allowed Mycobacterium tuberculosis (Mtb), the etiological agents of
Tuberculosis (TB) to resurge in more deadly drug resistant forms,
with the recently identified totally drug resistant strains rendering
complete resistance to currently available drug regime [1].The
current TB drug discovery pipeline, though presently insufficient to
address the unmet need of treatment, has few promising reports on
leads that have the potential to become future drug candidate. The
ones in the most advanced stages include the fluoroquinolones
(FQ), specifically gatifloxacin and moxifloxacin, which are currently
being evaluated in phase 2 and 3 clinical trials respectively [2]. This
class of drugs acts by inhibiting DNA gyrase, the sole topoisomerase
in Mtb that introduce negative supercoils into DNA and regulate the
super helical state of the bacterial chromosomes [3]. These drugs
predominately bind to the GyrA subunit of DNA gyrase, thereby
* Corresponding author.
E-mail addresses: dsriram@hyderabad.bits-pilani.ac.in, drdsriram@yahoo.com
(D. Sriram).
http://dx.doi.org/10.1016/j.ejmech.2014.08.018
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

594
K.A. Bobesh et al. / European Journal of Medicinal Chemistry 85 (2014) 593e604
achieved with regard to exploiting this class as a potential lead for
developing novel antimycobacterial agents [7]. Recent success in
the design of many mycobacterial gyrase inhibitors in the earlier
reports on antibacterial analogues of the same chemical class
encouraged us to evaluate the inhibitory potential of NBTIs towards
the mycobacterial gyrase domain. This is in consideration to the fact
that these compounds act on a clinically proven target through a
novel mechanism of inhibition with potency against
fluoroquinolone-resistant isolates. In this study we disclose a new
class of mycobacterial DNA gyrase inhibitor developed via molec-
ular hybridization of previously reported antibacterial leads [6e7].
The designed molecules were subsequently synthesized and eval-
uated in vitro for their ability to inhibit DNA gyrase enzyme and
whole cell MTB as important steps towards the derivation of
structureeactivity relationship.
right hand core to increase stability and also to evaluate the steric
and electronic effects on the antimycobacterial potency. The N-
linked aminopiperidine based analogues have made significant
progress in recent years as potential antibacterial leads. Research
group from GSK have also found success by extending the above
antibacterial aminopiperidine class to the antimicrobial target as
well, with many compounds exhibiting promising inhibition of
MTB [10].
Synthesis of the compounds started with the construction of 7-
fluoro1-5-naphthyridin-2(1H)-one via a Heck coupling reaction of
2-chloro-5-fluoropyridin-3-amine (5) with butyl acrylate. Though
various literatures have explored a variety of conditions and re-
agents to afford the Heck product, the protocol reported by Voight
et al. was the most beneficial as it underwent an in-situ cyclization
of the so obtained Heck product butyl 3-(3-amino-5-fluoropyridin-
2-yl)acrylate to give the desired 7-fluoro1,5-naphthyridin-2(1H)-
one (6) in good yield [11]. The ethyl bridge that connected the
naphthyridinone core to the aminopiperidine linker was intro-
duced at N-1 position by alkylating the so obtained 7-fluoro-1,5-
naphthyridin-2(1H)-one (6) with bromoethanol in presence of
Cs2CO3. Small amounts of the O-alkylated product obtained were
removed by column chromatography. Compound 7 on further
treatment with trifluoromethanesulfonic anhydride and pyridine
afforded the corresponding triflate (8) in good yield. This was
further condensed with 4-N-Boc-aminopiperidine via a SnAr
displacement to obtain the nitrogen-linked analog (9). Subsequent
Boc deprotection afforded the scaffold 11 in good yields. Concor-
dantly, the so obtained tert-butyl (1-(2-(7-fluoro-2-oxo-1,5-
naphthyridin-1(2H)-yl)ethyl)piperidin-4-yl)carbamate (9) was
also treated with sodium methoxide in methanol under reflux to
introduce the methoxy substituent at the 7th position of 1,5-
naphthyridin-2(1H)-one core via nucleophilic displacement of the
fluoro group. This was subsequently subjected to Boc deprotection
in a similar fashion to the fluoro analogue to give the desired
product (12). The final library was then assembled by treating the
obtained scaffolds 11and 12 with the desired isocyanates/iso-
thiocyanates to afford compounds 13e52 in excellent yields.
2. Resultsand discussion
2.1. Design and synthesis
With target-based/phenotypic screening approaches offering
few tangible successes in discovering novel antitubercular drugs,
the concept of molecular hybridization could be of significant use to
generate newer scaffold as potential antimycobacterial leads [8].
Molecular hybridization approach is an emerging structural
modification tool involving adequate fusion of the two or more
pharmacophoric units derived from previously reported leads/
drugs in the design of new hybrid architecture that could maintain
preselected characteristics of the original template [9]. We there-
fore envisaged that re-engineering the previously reported NBTIs
could deliver a new scaffold/lead with better antimycobacterial
activity via inhibition of the gyrase domain. The design strategy
utilized for developing the inhibitor has been sketched in Fig. 1. It
was decided to retain the 1-(2-(4-aminopiperidin-1-yl)ethyl)-1,5-
naphthyridin-2(1H)-one scaffold in our initial structure-activity
exploration as it was understood to be an important requisite in
retaining the gyrase inhibitory potential. Since fluoro and methoxy
groups at 7th position of the 1,5-naphthyridin-2(1H)-one core
significantly improved the gyrase inhibition of the previously re-
ported antibacterial NBTIs, these were also retained in our studies.
Various carbamide/thiocarbamide derivatives were introduced as
Fig. 1. Strategy employed for designing the lead. Chemical structure of previously reported synthetic inhibitors of DNA gyrasebearing1,5-naphthyridin-2(1H)-one core (1),
carbamide side chain (2e3) and the inhibitor designed through molecular hybridization (4).

K.A. Bobesh et al. / European Journal of Medicinal Chemistry 85 (2014) 593e604
595
2.2. Pharmacological evaluations
100 mM concentration by using 4,5-dimethylthiazol-2-yl-2,5-
diphenyltetrazoliumbromide (MTT) assay [14]. All 40 molecules
showed lesser cytotoxicity within a range of 19e45% as shown in
Table 1. The most promising anti-TB compound 35 showed only
31.5% cytotoxicity with selectivity index of >161. The R and R0
dimethoxy substituted groups comparatively showed lesser
toxicity when compared to other compounds in the series. Novo-
biocin was used as standard that exhibited 29.44% inhibition.
All the forty compounds synthesized were screened for their
enzyme inhibition studies using MTB DNA gyrase kit (Inspiralis,
Norwich) [12]. Preliminary screening was performed at concen-
trations of 500, 125, 31.3, 7.8, and 1.95
active were further tested at 250, 62.5, 15.6, 3.9 and 0.97
centrations. Finally molecules that exhibited more than 60%
inhibitory activity at 0.97 M were further screened at lower con-
centrations of 0.48, 0.24, 0.12, and 0.06 M to ensure the activity
profile. Among the entire series of forty compounds, thirteen
compounds showed IC50s less than 0.97 M. While the most active
mM and those which were
mM con-
m
m
3. Conclusion
m
By combining molecular hybridization strategy with biological
assays we could successfully re-engineer the previously reported
antibacterial leads that exhibited promising attributes of synthetic
accessibility, excellent in vitro enzyme inhibition profiles, and
antitubercular activity. With the urgent requirement of new anti-TB
agents, we believe that the present class of DNA gyrase inhibitors
reported in this paper could provide an interesting potential for
further optimization to yield novel drugs aimed at combating the
ever-increasing bacterial infections Scheme 1.
compound 35 showed an IC50 of 78 nM which possessed electro-
negative group (fluorine) and the chlorine compound 36 exhibited
an IC50 of 92 nM which indicated that the substitution of electro-
negative groups at the para position of the phenyl ring and the most
electropositive methoxy group at 7th position of 1,5-naphthyridin-
2(1H)-one could ensure excellent inhibitory property. Compounds
34 and 40 with hydrogen and methyl group at para position of
phenyl group exhibited IC50 of about 10 mM and lost their inhibitory
potential by a factor of hundred, thus marking the importance of
the electronegative groups at this position. Eleven other com-
pounds (13, 16, 20, 22e24, 26, 39, 43, 46 and 51) showed IC50s in
4. Experimental section
between 0.2 and 0.6
m
M which signified the importance of amino-
4.1. Chemistry
piperidine moiety in the inhibition of DNA gyrase enzyme. Novo-
biocin and moxifloxacin were considered as positive controls as
they have been shown to be potent inhibitors of DNA supercoiling
of mycobacterial DNA gyrase. All the compounds in this study
showed better enzyme supercoiling inhibitions compared to stan-
4.1.1. General
All commercially available chemicals and solvents were used
without further purification. TLC experiments were performed on
alumina-backed silica gel 60 F254 plates (Merck, Darmstadt, Ger-
many). Homogeneity of the compounds was monitored by thin
layer chromatography (TLC) on silica gel 60 F254 coated on
aluminium plates, visualized by UV light and KMnO4 treatment. All
1H and 13C NMR spectra were recorded on a Bruker AM-300
(300.12 MHz, 75.12 MHz) NMR spectrometer, Bruker BioSpin
dard moxifloxacin drug whose IC50 was 11.2
mM and has been
considered as one of the potent DNA gyrase inhibitor till date.
Similarly the other broad spectrum standard drug novobiocin
showed an IC50 of 46 nM. Dose-dependent inhibition profile of the
most active compound 35 at different inhibitor concentrations
along with standard novobiocin is illustrated in Fig. 2.
Corp, Germany. Chemical shifts were reported in ppm (d) with
The compounds were further screened for their in vitro anti-
mycobacterial activity against M. tuberculosis H37Rv strain by
microplate Alamar blue assay method [13]. As these compounds
were mostly hydrophilic, the in vitro antimycobacterial activities
reference to the internal standard TMS. The signals were desig-
nated as follows: s, singlet; d, doublet; t, triplet; m, multiplet.
Molecular weights of the synthesized compounds were checked by
LCMS 6100B series Agilent Technology. Elemental analyses were
carried out on an automatic Flash EA 1112 Series, CHN Analyzer
(Thermo).
were in commendable range of 0.49e30.3
15.31 M), isoniazid (MIC: 0.66 mM), moxifloxacin (MIC: 1.2
and novobiocin (MIC: >200 M) were considered as standard drugs
mM. Ethambutol (MIC:
m
mM)
m
for comparison in this assay. Compared to the first-line antituber-
cular drugs like ethambutol and isoniazid, the most active com-
4.1.2. General procedure for synthesis of 7-fluoro-1,5-naphthyridin-
2(1H)-one (6)
pounds 35 showed a better MIC of 0.62
16, 20, 22, 26, 29, 36, 42e43, 46, 48 and 51) exhibited good anti-
mycobacterial inhibitory profiles with MIC less than 2 M. These
compounds could be considered as promising antitubercular leads
with best in vitro DNA gyrase enzyme inhibitory profile.
The eukaryotic cell safety profile of all the compounds was
observed by testing their in vitro cytotoxicity against RAW
264.7 cell (Mouse leukaemic monocyte macrophage cell line) at
m
M. Twelve compounds (13,
To a stirred mixture of substituted2-chloro-5-fluoropyridin-3-
amine (5) (2 g, 13.65 mmol) and butyl acrylate (2.09 g,
16.38 mmol) in cumene (10 mL) at room temperature, was added
N-methyldicyclohexylamine (7.99 g, 40.94 mmol), followed by
catalytic amounts of tri tert-butyl phosphoniumtetrafluroborate
(0.158 g, 0.546 mmol). This reaction mixture was purged for 5 min
with argon followed by the addition of Pd(OAc)2 (0.061 g,
0.273 mmol). The reaction mixture was heated at 150 ꢀC for 4 h
(monitored by TLC & LCMS for completion), and the reaction
mixture was filtered through celite bed and solvent was evaporated
under reduced pressure. The reaction mixture was further extrac-
ted with ethyl acetate (3 ꢁ 40 mL). The combined organic extracts
were washed with brine (2 ꢁ 40 mL) and water (3 ꢁ 50 mL), dried
(MgSO4) and concentrated under reduced pressure. The crude
residue was purified by silica gel column chromatography using
hexane: ethyl acetate as eluent to give 7-fluoro-1, 5-naphthyridin-
2(1H)-one (6) (1.23 g, 53%) as brown solid. M.p: 282e284 ꢀC. 1H
m
NMR [300 MHz, DMSO-d6]: dH12.31 (s, 1H), 8.21e6.95 (m, 4H). 13
C
NMR [100 MHz, DMSO-d6] dc: 163.5, 157.9, 142.8, 140.5, 132.3, 127.1,
123.2, 110.2 ESI-MS m/z 165 (MþH)þ. Anal Calcd for C8H5FN2O: C,
58.54; H, 3.07; N, 17.07; Found: C, 58.58; H, 3.12; N, 17.03.
Fig. 2. Depicting the supercoiling assay picture of compound 35 at four different
concentrations of 0.31, 0.15, 0.07, 0.06
mM and novobiocin as standard where R-Relaxed
DNA substrate þDMSO; C-Relaxed DNA substrate.

596
K.A. Bobesh et al. / European Journal of Medicinal Chemistry 85 (2014) 593e604
Table 1
mixture was then filtered through celite and washed with
dichloromethane. The filtrate was concentrated under reduced
pressure. The reaction mixture was further extracted with ethyl
acetate (3 ꢁ 40 mL). The combined organic extracts were washed
with brine (2 ꢁ 40 mL) and water (3 ꢁ 50 mL), dried (MgSO4) and
concentrated under reduced pressure. The crude product was pu-
rified by silica gel column chromatography using hexane: ethyl
acetate as eluent to give the corresponding 7-fluoro-1-(2-
hydroxyethyl)-1,5-naphthyridin-2(1H)-one (7) (0.953 g, 75%) as
pale yellow solid. M.p: 241e243 ꢀC. 1H NMR [300 MHz, DMSO-d6]
In vitro inhibitory activities of synthesized compounds.
N
dH: 8.14e6.52 (m, 4H), 3.74e3.47 (m, 5H). 13C NMR [DMSO-d6] dC
:
162.3, 157.8, 147.5, 140.1, 132.3, 125.6, 123.2, 110.1, 64.5, 46.2. ESI-MS
m/z 209 (MþH)þ. Anal Calcd for C10H9FN2O2: C, 57.69; H, 4.36; N,
13.46; Found: C, 57.72; H, 4.38; N, 13.45.
Cmpd. R1
X R2
MTB
supercoiling MIC
assay (IC50
MTB
RAW 264.7
cytotoxicity at
Selective
index
4.1.4. General procedure for synthesis of 2-(7-fluoro-2-oxo-1,5-
naphthyridin-1(2H)-yl)ethyl trifluoromethanesulfonate (8)
m
M
)
100
m
M (% inhib.) (CC50/MIC)
mM
A
solution of 7-fluoro-1-(2-hydroxyethyl)-1,5-naphthyridin-
2(1H)-one (7) (1 g, 4.80 mmol) in dichloromethane (10 mL) and
pyridine (1.45 g, 14.41 mmol)was cooled to ꢂ20 ꢀC. To this was
added a solution of trifluromethanesulphonic anhydride (1.49 g,
5.28 mmol) in dichloromethane (10 mL) drop wise. The resultant
reaction mixture was stirred at the same temperature for half an
hour. Completion of the reaction was confirmed by TLC. Reaction
mixture was then warmed to room temperature and given sodium
bicarbonate (3 ꢁ 20 mL), water (3 ꢁ 20 mL) and brine (3 ꢁ 20 mL)
washes. The organic layer was dried over magnesium sulphate and
concentrated under reduced pressure to get a brownish thick
liquid. The crude product was purified by silica gel column chro-
matography using hexane: ethyl acetate as eluent to give the cor-
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
F
F
F
F
F
F
F
F
F
F
F
F
F
F
S
S
S
S
S
S
S
O H
O F
O Cl
O NO2
O COCH3
O OCH3
O CH3
S
S
S
S
S
S
S
H
F
Cl
NO2
0.5 0.22
7.1 0.35
3.72 0.25
0.28 0.14
1.83 41.43
6.80 38.54
8.52 40.46
0.91 39.57
21.22 36.49
1.71 30.83
3.54 38.58
1.90 42.95
3.64 39.58
1.29 25.69
3.77 38.56
2.91 40.65
3.23 25.49
1.84 29.46
7.14 37.56
3.42 38.45
1.65 32.57
6.46 35.78
6.89 38.56
13.19 41.47
3.45 38.83
14.82 45.78
0.62 31.54
1.85 28.46
3.94 38.56
6.60 39.45
3.78 29.45
30.30 39.56
7.10 46.56
1.64 43.67
1.66 41.32
7.37 27.45
3.70 42.67
1.89 32.11
7.50 33.64
1.60 29.45
12.97 19.44
13.42 28.34
1.65 40.88
3.35 39.52
1.2 ND
>54.64
>14.70
>11.73
>109.89
>4.71
COCH3 11.3 0.67
OCH3
CH3
1.8 0.34
3.6 0.72
0.44 0.1
3.12 0.66
0.3 0.05
0.25 0.09
0.2 0.15
2.2 0.55
0.67 0.12
5.2 0.89
4.1 0.43
2.3 0.69
3.9 0.25
3.1 0.42
8.6 0.24
3.8 0.17
10.4 0.44
0.078 0.02
0.092 0.05
1.91 0.3
>58.48
>28.24
>52.63
>27.47
>77.51
>26.52
>34.0.36
>30.95
>54.34
>14.00
>29.23
>60.60
>15.47
>14.51
>7.58
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3 O H
OCH3 O F
OCH3 O Cl
OCH3 O NO2
OCH3 O COCH3 11.6 0.78
OCH3 O OCH3
OCH3 O CH3
F
F
H
F
Cl
NO2
COCH3
OCH3
CH3
responding
2-(7-fluoro-2-oxo-1,5-naphthyridin-1(2H)-yl)ethyl
trifluoromethanesulfonate (8) (01.23 g, 73%) as Yellow oil. 1H NMR
[DMSO-d6] dH: 8.23e6.95 (m, 4H), 3.81e3.64 (m, 4H). 13C NMR
[DMSO-d6] d: 162.8, 157.8, 145.2, 140.2, 132.9, 125.6, 123.5, 119.2,
110.1, 61.2, 42.5. ESI-MS m/z: 341 (MþH)þ. Anal Calcd for
>28.95
>6.74
C11H8F4N2O4S: C, 38.83; H, 2.37; N, 8.23; Found: C, 38.85; H, 2.35; N,
8.27.
>161.29
>54.05
>25.38
>15.15
>26.45
>3.30
>14.08
>60.97
>60.24
>13.56
>27.02
>52.91
>13.33
>62.50
>7.71
4.1.5. General procedure for synthesis of tert-butyl (1-(2-(7-fluoro-
2-oxo-1,5-naphthyridin-1(2H)-yl)ethyl)piperidin-4-yl)carbamate
(9)
0.2 0.06
10.9 0.72
1.56 0.31
1.9 0.77
0.4 0.43
1.5 0.31
2.9 0.23
0.41 0.2
1.87 0.54
1.53 0.22
3.2 0.81
1.87 0.26
0.45 0.04
2.76 0.35
11.2 0.23
S
S
S
O H
O Cl
H
Cl
OCH3
To
a
solution of corresponding 2-(7-fluoro-2-oxo-1,5-
F
F
F
F
naphthyridin-1(2H)-yl)ethyl trifluoromethanesulfonate (8) (1 g,
2.93 mmol) in dry N,N0 dimethyl formamide (10 mL) was added
K2CO3 (1.22 g, 8.82 mmol) and followed by tert-butyl piperidin-4-
ylcarbamate (0.706 g, 3.52 mmol) and was heated in sealed tube
at 100 ꢀC for 4 h (monitored by TLC & LCMS for completion). The
reaction mixture was then filtered through celite and washed with
dichloromethane. The filtrate was concentrated under reduced
pressure. The reaction mixture was further extracted with ethyl
acetate (3 ꢁ 40 mL). The combined organic extracts were washed
with brine (2 ꢁ 40 mL) and water (3 ꢁ 50 mL), dried (MgSO4) and
concentrated under reduced pressure. The crude product was pu-
rified by silica gel column chromatography using hexane: ethyl
acetate as eluent to give the corresponding tert-butyl (1-(2-(7-
fluoro-2-oxo-1,5-naphthyridin-1(2H)-yl)ethyl)piperidin-4-yl)
carbamate (9) (0.724 g, 63%) as pale yellow solid. M.p: 210e212 ꢀC.
1H NMR [DMSO-d6] dH: 10.32 (s, 1H), 8.21e6.73 (m, 4H), 4.63e1.39
O OCH3
OCH3
OCH3
OCH3
OCH3 O H
OCH3 O Cl
OCH3 O OCH3
S
S
S
H
Cl
OCH3
>7.45
>60.60
>29.85
ND
ND
ND
Moxifloxacin
Novobiocin
Ethambutol
46 0.24 >200
ND 9.84 ND
29.44
ND indicated not determined.
4.1.3. General procedure for synthesis of 7-fluoro-1-(2-
hydroxyethyl)-1,5-naphthyridin-2(1H)-one (7)
To a solution corresponding 7-fluoro-1,5-naphthyridin-2(1H)-
one (6) (1 g, 6.09 mmol) in dry N,N0 dimethyl formamide (10 mL)
was added Cs2CO3 (4.95 g, 15.23 mmol) and followed by 2-bro-
moethanol (0.913 g, 7.31 mmol) and heated in sealed tube at 120 ꢀC
for 4 h (monitored by TLC & LCMS for completion). The reaction
(m, 13H), 1.38 (s, 9H). 13C NMR [DMSO-d6]
d: 162.3, 157.8, 156.2,
146.2, 140.1, 132.5, 125.6, 123.1, 110.2, 80.2, 55.2, 52.3 (2C), 50.2,
49.5, 30.2 (2C), 27.6 (3C).ESI-MS m/z: 391 (MþH)þ. Anal Calcd for
C20H27FN4O3: C, 61.52; H, 6.97; N, 14.35; Found: C, 61.55; H, 6.93; N,
14.37.

K.A. Bobesh et al. / European Journal of Medicinal Chemistry 85 (2014) 593e604
597
Scheme 1. Synthetic protocol adopted to achieve the designed ligands.
4.1.6. General procedure for synthesis of tert-butyl (1-(2-(7-
methoxy-2-oxo-1,5-naphthyridin-1(2H)-yl)ethyl)piperidin-4-yl)
carbamate (10)
A solution of corresponding tert-butyl (1-(2-(7-fluoro-2-oxo-
1,5-naphthyridin-1(2H)-yl)ethyl)piperidin-4-yl)carbamate
(2.0 g, 5.12 mmol) in methanol (10 mL) at room temperature was
added sodium methoxide (0.553 g, 10.2 mmol). The reaction
mixture was heated at 65 ꢀC for 3 h (monitored by TLC & LCMS for
completion), and solvent evaporated under reduced pressure. The
reaction mixture was further extracted with ethyl acetate
(3 ꢁ 40 mL). The combined organic extracts were washed with
brine (2 ꢁ 40 mL) and water (3 ꢁ 50 mL), dried (MgSO4) and
concentrated under reduced pressure. The crude residue was
purified by silica gel column chromatography using hexane: ethyl
acetate as eluent to give the corresponding tert-butyl (1-(2-(7-
methoxy-2-oxo-1,5-naphthyridin-1(2H)-yl)ethyl)piperidin-4-yl)
carbamate (10) (1.35 g, 65%) as pale yellow solid. M.p: 232e234 ꢀC.
1H NMR [DMSO-d6]: 10.32 (s, 1H), 8.21e6.73 (m, 4H), 4.63e1.39 (m,
16H), 1.38 (s, 9H). 13C NMR [DMSO-d6] dc: 161.5, 156.7, 155.2, 146.2,
136.1, 132.5, 125.6, 119.1, 105.2, 80.3, 56.2, 55.2, 52.3 (2C), 50.2, 49.8,
30.2 (2C), 27.6 (3C).ESI-MS m/z: 403 (MþH)þ. Anal Calcd for
(9)
C
21H30N4O4: C, 62.67; H, 7.51; N, 13.92; Found: C, 62.61; H, 7.56; N,
13.90.

598
K.A. Bobesh et al. / European Journal of Medicinal Chemistry 85 (2014) 593e604
4.1.7. General procedure for synthesis of substituted 1-(2-(4-
aminopiperidin-1-yl)ethyl)-1,5-naphthyridin-2(1H)-one (11e12)
A solution of corresponding substituted tert-butyl (1-(2-(2-oxo-
1,5-naphthyridin-1(2H)-yl)ethyl)piperidin-4-yl)carbamate (9e10)
(1 eq) in dichloromethane (10 mL) was cooled to 0 ꢀC followed by
drop wise addition of HCl in dioxane (2 mL) and stirred for 1 h. After
completion of the reaction (monitored by TLC & LCMS), quenched
with ice and concentrated under reduced pressure. The residue was
partitioned between ethyl acetate (20 mL) and water (20 mL).
Aqueous layer was basified with sodium carbonate solution and
extracted with ethyl acetate (3 ꢁ 20 mL) and washed with water
(3 ꢁ 10 mL) and brine (3 ꢁ 10 mL). Organic layer was dried over
anhydrous sodium sulphate, filtered, and concentrated under
reduced pressure to give the corresponding 1-(2-(4-
aminopiperidin-1-yl)ethyl)-1,5-naphthyridin-2(1H)-one (11e12)
in good yield.
165e168 ꢀC. 1H NMR [DMSO-d6] dH: 10.33 (b, 1H), 7.98e6.69 (m,
9H), 4.33 (t, J ¼ 6.6 Hz, 2H), 4.05 (b, 1H), 2.81e1.22 (m, 11H). 13C
NMR [DMSO-d6] dc: 178.1, 160.2, 157.9, 146.2, 140.3, 139.5, 133.8,
130.3 (2C), 129.2, 127.7 (2C), 125.2, 124.8,109.5, 56.6, 54.2, 52.8 (2C),
47.6, 30.3 (2C). ESI-MS m/z: 426 (MþH)þ. Anal Calcd for
C22H24FN5OS: C, 62.10; H, 5.68; N, 16.46. Found: C, 62.11; H, 5.66; N,
16.45.
4.1.8.2. 1-(1-(2-(7-Fluoro-2-oxo-1,5-naphthyridin-1(2H)-yl)ethyl)
piperidin-4-yl)-3-(4-fluorophenyl)thiourea (14). The compound was
synthesized according to the general procedure using 1-(2-(4-
aminopiperidin-1-yl)ethyl)-7-fluoro-1,5-naphthyridin-2(1H)-one
(11) (0.1 g, 0.344 mmol) and 4 fluro phenyl isothiocyanate (0.063 g,
0.413 mmol) to afford 14 (0.112 g, 70%) as pale yellow solid. M.p:
184e186 ꢀC. 1H NMR [DMSO-d6] dH: 10.37 (b, 1H), 7.95e6.67 (m,
8H), 4.35 (t, J ¼ 6.9 Hz, 2H), 4.06 (b, 1H), 2.92e1.24 (m, 11H). 13C
NMR [DMSO-d6] d: 178.9, 162.5, 161.7, 157.8, 145.3, 140.2, 135.6,
4.1.7.1. 1-(2-(4-Aminopiperidin-1-yl)ethyl)-7-fluoro-1,5-
naphthyridin-2(1H)-one (11). The compound was synthesized ac-
cording to the general procedure described above by using tert-
butyl(1-(2-(7-fluoro-2-oxo-1,5-naphthyridin-1(2H)-yl)ethyl)piper-
idin-4-yl)carbamate (9) (2.0 g, 5.12 mmol) and HCl in dioxane
(10 mL) to afford 11 (1.25 g, 84%) as pale yellow solid. M.p:
232e234 ꢀC. 1H NMR [DMSO-d6] dH: 8.26e6.72 (m, 4H), 6.56 (s,
132.8, 130.8 (2C), 125.6, 123.8, 116.2 (2C), 109.5, 56.8, 54.5, 52.9 (2C),
47.5, 30.6 (2C). ESI-MS m/z: 444 (MþH)þ. Anal Calcd for
C22H23F2N5OS: C, 59.58; H, 5.23; N, 15.79. Found: C, 59.60; H, 5.20;
N, 15.80.
4.1.8.3. 1-(4-Chlorophenyl)-3-(1-(2-(7-fluoro-2-oxo-1,5-
naphthyridin-1(2H)-yl)ethyl)piperidin-4-yl)thiourea (15). The com-
pound was synthesized according to the general procedure using 1-
(2-(4-aminopiperidin-1-yl)ethyl)-7-fluoro-1,5-naphthyridin-
2(1H)-one (11) (0.1 g, 0.344 mmol) and 4-chloro phenyl isothio-
cyanate (0.069 g, 0.413 mmol) to afford 15 (0.102 g, 64%)as brown
solid. M.p: 188e190 ꢀC. 1H NMR [DMSO-d6] dH: 10.37 (b, 1H),
7.92e6.55 (m, 8H), 4.30 (t, J ¼ 6.6 Hz, 2H), 4.12 (b, 1H), 2.88e1.29
(m, 11H). 13C NMR [DMSO-d6] dc: 178.1, 161.7, 157.8, 145.9, 140.2,
137.2, 134.5, 132.8, 131.9 (2C), 130.3 (2C), 125.6, 123.8, 109.6, 56.8,
54.5, 52.3 (2C), 47.5, 30.3 (2C).ESI-MS m/z: 460 (MþH)þ. Anal Calcd
for C22H23ClFN5OS: C, 57.45; H, 5.04; N, 15.23. Found: C, 57.47; H,
5.07; N, 15.29.
2H), 4.35e1.38 (m, 13H). 13C NMR [DMSO-d6]
d: 162.8, 155.9, 145.6,
140.2, 132.9, 125.1, 123.4, 109.7, 55.2, 52.6 (2c), 50.3, 45.2, 33.5 (2C).
ESI-MS m/z: 291 (MþH)þ. Anal Calcd for C15H19FN4O: C, 62.05; H,
6.60; N, 19.30; Found: C, 62.01; H, 6.64; N, 19.31.
4.1.7.2. 1-(2-(4-Aminopiperidin-1-yl)ethyl)-7-methoxy-1,5-
naphthyridin-2(1H)-one (12). The compound was synthesized ac-
cording to the general procedure by utilizing tert-butyl (1-(2-(7-
methoxy-2-oxo-1,5-naphthyridin-1(2H)-yl)ethyl)piperidin-4-yl)
carbamate (10) (2.0 g, 4.97 mmol) and HCl in dioxane (10 mL) to
afford 12 (1.35 g, 88%) as Yellow solid. M.p: 225e227 ꢀC. 1H NMR
[DMSO-d6] dH: 8.11e6.63 (m, 4H), 6.42 (s, 2H), 4.48 (t, J ¼ 7.2 Hz,
2H), 3.92 (s, 3H), 3.65e1.36 (m, 11H). 13C NMR [DMSO-d6]
d: 162.3,
155.6, 145.7, 136.2, 132.8, 125.4, 119.2, 107.2, 56.2, 54.5, 52.3 (2C),
4.1.8.4. 1-(1-(2-(7-Fluoro-2-oxo-1,5-naphthyridin-1(2H)-yl)ethyl)
piperidin-4-yl)-3-(4-nitrophenyl)thiourea (16). The compound was
synthesized according to the general procedure using 1-(2-(4-
aminopiperidin-1-yl)ethyl)-7-fluoro-1,5-naphthyridin-2(1H)-one
(11) (0.1 g, 0.344 mmol) and 4-nitro phenyl isothiocyanate (0.074 g,
0.413 mmol) to afford 16 (0.138 g, 85%)as yellow solid. M.p:
205e207 ꢀC. 1H NMR [300 MHz, DMSO-d6]: dH9.97 (b, 1H),
8.57e6.82 (m, 8H), 4.34 (t, J ¼ 6.3 Hz, 2H), 4.09 (b, 1H), 2.93e1.28
58.5, 46.2, 33.2 (2C) ESI-MS m/z: 303 (MþH)þ. Anal Calcd for
C
16H22N4O2: C, 63.55; H, 7.33; N, 18.53; Found: C, 63.58; H, 7.31; N,
18.55.
4.1.8. General procedure for the synthesis of substituted 1-(1-(2-(7-
fluoro-2-oxo-1,5-naphthyridin-1(2H)-yl)ethyl)piperidin-4-yl)-3-
phenylthiourea (13e19)
A solution of 1-(2-(4-aminopiperidin-1-yl)ethyl)-7-fluoro-1,5-
naphthyridin-2(1H)-one (11) (0.344 mmol) in dichloromethane
(3 mL) was cooled to 0 ꢀC followed by the addition of triethylamine
(1.03 mmol). Corresponding substituted phenyl isothiocyanate was
added to the reaction mixture and was stirred in room temperature
for 12 h (monitored by TLC & LCMS for completion). The reaction
mixture was then washed with water (3 ꢁ 5 mL) and brine
(3 ꢁ 5 mL). Organic layer was dried (MgSO4) and concentrated
under reduced pressure. The crude product was purified by silica
gel column chromatography using hexane: ethyl acetate as eluent
to give the corresponding substituted1-(1-(2-(7-fluoro-2-oxo-1,5-
naphthyridin-1(2H)-yl)ethyl)piperidin-4-yl)-3-phenylthiourea
(13e19)in good yields.
(m, 11H). 13C NMR [DMSO-d6]
d: 178.9, 160.7, 157.3, 146.4, 142.3,
142.1, 141.6, 139.9, 132.8, 124.4 (2C), 123.9 (2C), 120.1, 109.3, 54.94,
53.16, 52.2 (2C), 50.6, 30.7 (2C). ESI-MS m/z: 471 (MþH)þ. Anal
Calcd for C22H23FN6O3S: C, 56.16; H, 4.93; N, 17.86; Found: C, 56.18;
H, 4.90; N, 17.87.
4.1.8.5. 1-(4-Acetylphenyl)-3-(1-(2-(7-fluoro-2-oxo-1,5-
naphthyridin-1(2H)-yl)ethyl)piperidin-4-yl)thiourea (17). The com-
pound was synthesized according to the general procedure using 1-
(2-(4-aminopiperidin-1-yl)ethyl)-7-fluoro-1,5-naphthyridin-
2(1H)-one (11) (0.1 g, 0.344 mmol) and 4-acetyl phenyl isothiocy-
anate (0.073 g, 0.413 mmol) to afford 17 (0.122 g, 76%)as white solid.
M.p: 188e190 ꢀC. 1H NMR [DMSO-d6] dH: 10.49 (b, 1H), 8.38e6.75
(m, 8H), 4.62 (t, J ¼ 7.5 Hz, 2H), 4.15 (b, 1H), 3.63e3.02 (m, 3H), 2.52
(s, 3H), 2.43e1.18 (m, 8H). 13C NMR [DMSO-d6] dc: 197.5, 178.9,
160.1, 157.8, 145.3, 143.4, 140.2, 138.3, 132.5, 130.2 (2C), 127.8 (2C),
125.3, 109.8, 55.8, 54.2, 53.5, 52.9 (2C), 47.8, 30.2 (2C), 25.8. ESI-MS
m/z: 468 (MþH)þ. Anal Calcd for C24H26FN5O2S: C, 61.65; H, 5.60; N,
14.98; Found: C, 61.63; H, 5.57; N, 14.97.
4.1.8.1. 1-(1-(2-(7-Fluoro-2-oxo-1,5-naphthyridin-1(2H)-yl)ethyl)
piperidin-4-yl)-3-phenylthiourea (13). The compound was synthe-
sized according to the general procedure using 1-(2-(4-
aminopiperidin-1-yl)ethyl)-7-fluoro-1,5-naphthyridin-2(1H)-one
(11) (0.1 g, 0.344 mmol) and phenyl isothiocyanate (0.055 g,
0.413 mmol) to afford 13 (0.085 g, 58%) as white solid. M.p:

K.A. Bobesh et al. / European Journal of Medicinal Chemistry 85 (2014) 593e604
599
4.1.8.6. 1-(1-(2-(7-Fluoro-2-oxo-1,5-naphthyridin-1(2H)-yl)ethyl)
8.63e6.84 (m, 8H), 4.36e1.42 (m, 13H). 13C NMR [DMSO-d6] dc
:
161.8,160.7,155.8,154.9,145.9,140.2,136.7,132.2,125.5,123.7,120.2
(2C), 116.2 (2C), 109.8, 54.5, 52.3 (2C), 47.8, 46.7, 30.3 (2C). ESI-MS
m/z: 428 (MþH)þ. Anal Calcd for C22H23F2N5O2: C, 61.82; H, 5.42;
N, 16.38; Found: C, 61.83; H, 5.44; N, 16.43.
piperidin-4-yl)-3-(4-methoxyphenyl)thiourea (18). The compound
was synthesized according to the general procedure using 1-(2-(4-
aminopiperidin-1-yl)ethyl)-7-fluoro-1,5-naphthyridin-2(1H)-one
(11) (0.1 g, 0.344 mmol) and 4 methoxyphenyl isothiocyanate
(0.068 g, 0.413 mmol) to afford 18 (0.126 g, 80%) as pale yellow
solid. M.p: 130e132 ꢀC. 1H NMR [DMSO-d6] dH: 10.45 (b, 1H),
8.25e6.75 (m, 8H), 4.33 (t, J ¼ 6.0 Hz, 2H), 4.05 (b, 1H), 3.85 (s, 3H),
2.84e1.31 (m, 11H). 13C NMR [DMSO-d6] dc: 178.2, 161.2, 160.5,
157.8, 145.7, 140.2, 132.3, 129.5, 128.2 (2C), 125.6, 123.5, 115.1 (2C),
110.1, 56.2, 55.9, 52.5, 51.9 (2C), 48.5, 30.3 (2C). ESI-MS m/z: 456
(MþH)þ. Anal Calcd for C23H26FN5O2S: C, 60.64; H, 5.75; N, 15.37.
Found: C, 60.69; H, 5.70; N, 15.35.
4.1.9.3. 1-(4-Chlorophenyl)-3-(1-(2-(7-fluoro-2-oxo-1,5-
naphthyridin-1(2H)-yl)ethyl)piperidin-4-yl)urea (22). The com-
pound was synthesized according to the general procedure using 1-
(2-(4-aminopiperidin-1-yl)ethyl)-7-fluoro-1,5-naphthyridin-
2(1H)-one (11) (0.1 g, 0.344 mmol) and 4-chloro phenyl isocyanate
(0.063 g, 0.413 mmol) to afford 22 (0.133 g, 87%) as brown solid.
M.p: 275e277 ꢀC. 1H NMR [DMSO-d6] dH: 10.11 (b,1H),10.05 (b,1H),
8.65e6.82 (m, 8H), 4.33e1.37 (m, 13H). 13C NMR [DMSO-d6] dc
:
4.1.8.7. 1-(1-(2-(7-Fluoro-2-oxo-1,5-naphthyridin-1(2H)-yl)ethyl)
piperidin-4-yl)-3-(p-tolyl)thiourea (19). The compound was syn-
thesized according to the general procedure using 1-(2-(4-
aminopiperidin-1-yl)ethyl)-7-fluoro-1,5-naphthyridin-2(1H)-one
(11) (0.1 g, 0.344 mmol) and 4-methyl phenyl isothiocyanate
(0.062 g, 0.413 mmol) to afford 19 (0.118 g, 78%) as white solid. M.p:
181e183 ꢀC. 1H NMR [DMSO-d6] dH: 10.42 (b, 1H), 7.94e6.58 (m,
8H), 4.31 (t, J ¼ 6.9 Hz, 2H), 4.15 (b, 1H), 2.92e1.31 (m, 14H). 13C
NMR [DMSO-d6] dc: 178.1, 160.2, 157.8, 145.8, 140.2, 138.5, 136.7,
132.8,130.3 (2C),126.7 (2C),125.2,123.8,109.5, 56.8, 54.2, 52.8 (2C),
47.6, 30.3 (2C), 22.4. ESI-MS m/z: 440 (MþH)þ. Anal Calcd for:
160.7, 157.8, 155.8, 145.9, 140.2, 138.6, 134.7, 132.3, 130.4 (2C), 125.6,
123.7, 120.5 (2C), 109.7, 54.2, 52.8 (2C), 47.9, 46.5, 30.4 (2C) ESI-MS
m/z: 444 (MþH)þ. Anal Calcd for C22H23ClFN5O2: C, 59.53; H, 5.22;
N, 15.78; Found: C, 59.57; H, 5.20; N, 15.76.
4.1.9.4. 1-(1-(2-(7-Fluoro-2-oxo-1,5-naphthyridin-1(2H)-yl)ethyl)
piperidin-4-yl)-3-(4-nitrophenyl)urea (23). The compound was
synthesized according to the general procedure using 1-(2-(4-
aminopiperidin-1-yl)ethyl)-7-fluoro-1,5-naphthyridin-2(1H)-one
(11) (0.1 g, 0.344 mmol) and 4-nitro phenyl isocyanate (0.067 g,
0.413 mmol) to afford 23 (0.097 g, 62%) as pale yellow solid. M.p:
261e263 ꢀC. 1H NMR [DMSO-d6] dH: 10.12 (b, 1H), 9.99 (b, 1H),
C
23H26FN5OS: C, 62.85; H, 5.96; N,15.93. Found: C, 62.87; H, 5.95; N,
15.92.
8.63e6.83 (m, 8H), 4.36e1.42 (m, 13H). 13C NMR [DMSO-d6] dc
:
160.9, 155.2, 154.8, 144.3, 143.3, 142.9, 139.7, 132.8, 125.2, 124.9 (2C),
123.8, 120.2 (2C), 109.5, 54.5, 52.8 (2C), 47.8, 46.9, 30.2 (2C). ESI-MS
m/z: 455 (MþH)þ. Anal Calcd forC22H23FN6O4: C, 58.14; H, 5.10; N,
18.49; Found: C, 58.17; H, 5.13; N, 18.45.
4.1.9. General procedure for the synthesis of substituted 1-(1-(2-(7-
fluoro-2-oxo-1,5-naphthyridin-1(2H)-yl)ethyl)piperidin-4-yl)-3-
phenylurea (20e26)
A solution of 1-(2-(4-aminopiperidin-1-yl)ethyl)-7-fluoro-1,5-
naphthyridin-2(1H)-one (11) (0.344 mmol) in dichloromethane
(3 mL) was cooled to 0 ꢀC followed by the addition of triethylamine
(1.03 mmol). Corresponding substituted phenyl isocyanate
(0.413 mmol) was added to the reaction mixture and was stirred in
4.1.9.5. 1-(4-Acetylphenyl)-3-(1-(2-(7-fluoro-2-oxo-1,5-
naphthyridin-1(2H)-yl)ethyl)piperidin-4-yl)urea (24). The com-
pound was synthesized according to the general procedure using 1-
(2-(4-aminopiperidin-1-yl)ethyl)-7-fluoro-1,5-naphthyridin-
2(1H)-one (11) (0.1 g, 0.344 mmol) and 4-acetyl phenyl isocyanate
(0.066 g, 0.413 mmol) to afford 24 (0.104 g, 67%)as white solid. M.p:
224e226 ꢀC. 1H NMR [DMSO-d6] dH: 10.56 (b, 1H), 10.44 (b, 1H),
8.39e6.72 (m, 8H), 4.68 (t, J ¼ 7.8 Hz, 2H), 3.78e3.09 (m, 3H), 2.53
(s, 3H), 2.49e1.17 (m, 8H). 13C NMR [DMSO-d6] dc: 196.1, 161.2,
156.8, 154.2, 145.1, 143.2, 140.1, 136.7, 131.3, 129.1 (2C), 124.5, 123.8,
120.2 (2C), 109.8, 54.5, 51.4 (2C), 47.6, 45.5, 29.6 (2C), 26.2. ESI-MS
m/z: 452 (MþH)þ. Anal Calcd for C24H26FN5O3: C, 63.85; H, 5.80; N,
15.51; Found: C, 63.86; H, 5.88; N, 15.49.
room temperature for 12
h (monitored by TLC &LCMS for
completion). The reaction mixture was then washed with water
(3 ꢁ 5 mL) and brine (3 ꢁ 5 mL). Organic layer was dried (MgSO4)
and concentrated under reduced pressure. The crude product was
purified by silica gel column chromatography using hexane: ethyl
acetate as eluent to give the corresponding substituted1-(1-(2-(7-
fluoro-2-oxo-1,5-naphthyridin-1(2H)-yl)ethyl)piperidin-4-yl)-3-
phenylurea (20e26) in good yield.
4.1.9.1. 1-(1-(2-(7-Fluoro-2-oxo-1,5-naphthyridin-1(2H)-yl)ethyl)
piperidin-4-yl)-3-phenylurea (20). The compound was synthesized
according to the general procedure using 1-(2-(4-aminopiperidin-
1-yl)ethyl)-7-fluoro-1,5-naphthyridin-2(1H)-one (11) (0.1 g,
0.344 mmol) and phenyl isocyanate (0.049 g, 0.413 mmol) to afford
20 (0.107 g, 76%) as Pale yellow solid. M.p: 212e214 ꢀC. 1H NMR
[DMSO-d6] dH: 10.36 (b, 1H), 10.28 (b, 1H), 7.95e6.67 (m, 9H),
4.64e1.38 (m, 13H). 13C NMR [DMSO-d6] dc: 160.7, 157.8, 155.6,
145.3, 140.2, 138.9, 132.9, 130.3 (2C), 129.2, 125.6, 123.4, 120.6 (2C),
109.8, 54.6, 52.8 (2C), 47.9, 46.8, 30.2 (2C). ESI-MS m/z: 410 (MþH)þ.
Anal Calcd for C22H24FN5O2: C, 64.53; H, 5.91; N, 17.10. Found: C,
64.55; H, 5.96; N, 17.07.
4.1.9.6. 1-(1-(2-(7-Fluoro-2-oxo-1,5-naphthyridin-1(2H)-yl)ethyl)
piperidin-4-yl)-3-(4-methoxyphenyl)urea (25). The compound was
synthesized according to the general procedure using 1-(2-(4-
aminopiperidin-1-yl)ethyl)-7-fluoro-1,5-naphthyridin-2(1H)-one
(11) (0.1 g, 0.344 mmol) and 4-methoxyphenyl isocyanate (0.062 g,
0.413 mmol) to afford 25 (0.117 g, 77%) as white solid. M.p:
196e198 ꢀC. 1H NMR [DMSO-d6] dH: 10.16 (b, 1H), 10.03 (b, 1H),
8.57e6.73 (m, 8H), 4.37 (t, J ¼ 6.6 Hz, 2H), 3.81 (s, 3H), 4.32e1.38
(m, 11H). 13C NMR [DMSO-d6] dc: 160.7, 159.7, 157.8, 155.3, 145.2,
140.1, 131.7, 130.8, 125.6, 123.8, 120.2 (2C), 115.6 (2C), 109.5, 56.2,
54.2, 52.3 (2C), 47.8, 46.7, 30.3 (2C). ESI-MS m/z: 440 (MþH)þ. Anal
Calcd for C23H26FN5O3: C, 62.86; H, 5.96; N, 15.94; Found: C, 62.88;
H, 5.95; N, 15.99.
4.1.9.2. 1-(1-(2-(7-Fluoro-2-oxo-1,5-naphthyridin-1(2H)-yl)ethyl)
piperidin-4-yl)-3-(4-fluorophenyl)urea (21). The compound was
synthesized according to the general procedure using 1-(2-(4-
aminopiperidin-1-yl)ethyl)-7-fluoro-1,5-naphthyridin-2(1H)-one
(11) (0.1 g, 0.344 mmol) and 4-fluro phenyl isocyanate (0.056 g,
0.413 mmol) to afford 21 (0.122 g, 82%) as pale yellow solid. M.p:
239e241 ꢀC. 1H NMR [DMSO-d6] dH: 10.12 (b, 1H), 9.99 (b, 1H),
4.1.9.7. 1-(1-(2-(7-Fluoro-2-oxo-1,5-naphthyridin-1(2H)-yl)ethyl)
piperidin-4-yl)-3-(p-tolyl)urea (26). The compound was synthe-
sized according to the general procedure using 1-(2-(4-
aminopiperidin-1-yl)ethyl)-7-fluoro-1,5-naphthyridin-2(1H)-one
(11) (0.1 g, 0.344 mmol) and 4-methyl phenyl isocyanate (0.054 g,

600
K.A. Bobesh et al. / European Journal of Medicinal Chemistry 85 (2014) 593e604
0.413 mmol) to afford 26 (0.120 g, 82%) as brown solid. M.p:
242e244 ꢀC. 1H NMR [DMSO-d6] dH: 10.39 (b, 1H), 10.26 (b, 1H),
Anal Calcd for C23H26ClN5O2S: C, 58.53; H, 5.55; N, 14.84. Found: C,
58.57; H, 5.50; N, 14.89.
7.88e6.65 (m, 8H), 4.40 (t, J ¼ 7.2 Hz, 2H), 3.05e1.36 (m, 14H). 13
C
NMR [DMSO-d6] dc: 160.7, 157.2, 155.4, 145.8, 140.2, 137.9, 137.2,
132.7, 130.2 (2C), 125.6, 123.8, 120.8 (2C), 109.8, 54.6, 52.5 (2C), 47.8,
46.5, 30.2 (2C), 20.8. ESI-MS m/z 424 (MþH)þ. Anal Calcd for:
4.1.10.4. 1-(1-(2-(7-Methoxy-2-oxo-1,5-naphthyridin-1(2H)-yl)
ethyl)piperidin-4-yl)-3-(4-nitrophenyl)thiourea (30). The com-
pound was synthesized according to the general procedure using 1-
(2-(4-aminopiperidin-1-yl)ethyl)-7-methoxy-1,5-naphthyridin-
2(1H)-one (12) (0.1 g, 0.330 mmol) and 4-nitro phenyl isothiocya-
nate (0.071 g, 0.397 mmol) to afford 30 (0.099 g, 62%)as yellow
solid. M.p: 229e231 ꢀC. 1H NMR [DMSO-d6] dH: 10.03 (b, 1H),
8.62e6.87 (m, 8H), 4.41 (t, J ¼ 6.3 Hz, 2H), 4.18 (b, 1H), 3.95 (s, 3H),
3.05e1.42 (m, 11H). 13C NMR [DMSO-d6] dc: 178.9, 160.7, 157.3,
146.4, 142.3, 142.1, 141.6, 139.9, 132.8, 124.4 (2C), 123.9 (2C), 120.1,
109.3, 55.9, 54.94, 53.16, 52.2 (2C), 50.6, 30.7 (2C). ESI-MS m/z: 483
(MþH)þ. Anal Calcd for C23H26N6O4S: C, 57.25; H, 5.43; N, 17.42.
Found: C, 57.21; H, 5.42; N, 17.38.
C
23H26FN5O2: C, 65.23; H, 6.19; N, 16.54. Found: C, 65.25; H, 6.18; N,
16.55.
4.1.10. General procedure for the synthesis of substituted 1-(1-(2-
(7-methoxy-2-oxo-1,5-naphthyridin-1(2H)-yl)ethyl)piperidin-4-
yl)-3-phenylthiourea (27e33)
A solution of 1-(2-(4-aminopiperidin-1-yl)ethyl)-7-methoxy-
1,5-naphthyridin-2(1H)-one (12) (0.33 mmol) in dichloromethane
(1 mL) was cooled to 0 ꢀC followed by the addition of triethylamine
(0.99 mmol). Corresponding substituted phenyl isothiocyanate
(0.397 mmol) was added to the reaction mixture and was stirred in
room temperature for 12 h (monitored by TLC & LCMS for
completion). The reaction mixture was washed with water
(3 ꢁ 5 mL) and brine (3 ꢁ 5 mL). Organic layer was dried (MgSO4)
and concentrated under reduced pressure. The crude product was
purified by silica gel column chromatography using hexane: ethyl
acetate as eluent to give the corresponding substituted1-(1-(2-(7-
methoxy-2-oxo-1,5-naphthyridin-1(2H)-yl)ethyl)piperidin-4-yl)-
3-phenylthiourea (27e33)in good yields.
4.1.10.5. 1-(4-Acetylphenyl)-3-(1-(2-(7-methoxy-2-oxo-1,5-
naphthyridin-1(2H)-yl)ethyl)piperidin-4-yl)thiourea (31). The com-
pound was synthesized according to the general procedure using 1-
(2-(4-aminopiperidin-1-yl)ethyl)-7-methoxy-1,5-naphthyridin-
2(1H)-one (12) (0.1 g, 0.330 mmol) and 4-acetyl phenyl isothiocy-
anate (0.070 g, 0.397 mmol) to afford 31 (0.11 g, 69%) as white solid.
M.p: 194e196 ꢀC. 1H NMR [DMSO-d6] dH: 10.35 (b, 1H), 8.25e6.62
(m, 8H), 4.41 (t, J ¼ 7.8 Hz, 2H), 4.18 (b, 1H), 3.85 (s, 3H), 3.54e2.99
(m, 3H), 2.56 (s, 3H), 2.44e1.23 (m, 8H). 13C NMR [DMSO-d6] dc
:
4.1.10.1. 1-(1-(2-(7-Methoxy-2-oxo-1,5-naphthyridin-1(2H)-yl)ethyl)
piperidin-4-yl)-3-phenylthiourea (27). The compound was synthe-
sized according to the general procedure using 1-(2-(4-
aminopiperidin-1-yl)ethyl)-7-methoxy-1,5-naphthyridin-2(1H)-
one (12) (0.1 g, 0.330 mmol) and phenyl isothiocyanate (0.055 g,
0.397 mmol) to afford 27 (0.142 g, 82%) as white solid. M.p:
178e180 ꢀC. 1H NMR [DMSO-d6] dH: 10.33 (b, 1H), 7.92e6.65 (m,
9H), 4.34 (t, J ¼ 6.6 Hz, 2H), 4.09 (b, 1H), 3.82 (s, 3H), 2.86e1.23 (m,
11H). 13C NMR [DMSO-d6] dc: 178.2, 160.5, 157.8, 145.2, 141.3, 138.5,
134.8, 130.3 (2C), 129.7, 126.7 (2C), 125.2, 124.8, 110.5, 56.6, 55.3,
53.2, 52.8 (2C), 47.6, 30.3 (2C). ESI-MS m/z: 438 (MþH)þ. Anal Calcd
for C23H27N5O2S: C, 63.13; H, 6.22; N, 16.01. Found: C, 63.11; H, 6.25;
N, 16.09.
197.8, 177.5, 162.3, 155.3, 143.9, 143.5, 138.5, 136.2, 132.8, 130.2 (2C),
125.2 (2C), 124.9, 119.2, 105.8, 56.5, 54.2, 53.5, 51.5 (2C), 47.5, 30.2
(2C), 25.6.ESI-MS m/z: 480 (MþH)þ. Anal Calcd for C25H29N5O3S: C,
62.61; H, 6.09; N, 14.60; Found: C, 62.66; H, 6.11; N, 14.56.
4.1.10.6. 1-(1-(2-(7-Methoxy-2-oxo-1,5-naphthyridin-1(2H)-yl)
ethyl)piperidin-4-yl)-3-(4-methoxyphenyl)thiourea (32). The com-
pound was synthesized according to the general procedure using 1-
(2-(4-aminopiperidin-1-yl)ethyl)-7-methoxy-1,5-naphthyridin-
2(1H)-one (12) (0.1 g, 0.330 mmol) and 4-methoxyphenyl isothio-
cyanate (0.065 g, 0.397 mmol) to afford 32 (0.125 g, 81%) as brown
solid. M.p: 121e123 ꢀC. 1H NMR [DMSO-d6] dH: 10.32 (b, 1H),
7.83e6.58 (m, 8H), 4.49 (t, J ¼ 6.3 Hz, 2H), 4.12 (b, 1H), 3.85 (s, 3H),
3.75 (s, 3H), 2.93e1.41 (m, 11H). 13C NMR [DMSO-d6] dc: 178.3,
163.5, 160.2, 155.8, 143.3, 136.8, 132.3, 131.6, 128.1 (2C), 125.6, 119.2,
115.6 (2C), 105.2, 55.8 (2C), 55.2, 54.3, 52.8 (2C), 50.2, 30.5 (2C). ESI-
MS m/z: 468 (MþH)þ. Anal Calcd for C24H29N5O3S: C, 61.65; H, 6.25;
N, 14.98. Found: C, 61.63; H, 6.27; N, 14.97.
4.1.10.2. 1-(4-Fluorophenyl)-3-(1-(2-(7-methoxy-2-oxo-1,5-
naphthyridin-1(2H)-yl)ethyl)piperidin-4-yl)thiourea (28). The com-
pound was synthesized according to the general procedure using 1-
(2-(4-aminopiperidin-1-yl)ethyl)-7-methoxy-1,5-naphthyridin-
2(1H)-one (12) (0.1 g, 0.330 mmol) and 4-fluro phenyl isothiocya-
nate (0.060 g, 0.397 mmol) to afford 28 (0.108 g, 72%) as yellow
solid. M.p: 165e167 ꢀC. 1H NMR [DMSO-d6] dH: 10.35 (b, 1H),
7.98e6.65 (m, 8H), 4.33 (t, J ¼ 7.2 Hz, 2H), 4.05 (b, 1H), 3.84 (s, 3H),
2.97e1.26 (m,11H). 13C NMR [DMSO-d6] dc: 178.9,162.5,161.7,157.8,
145.3, 140.2, 135.6, 132.8, 130.8 (2C), 125.6, 123.8, 116.2 (2C), 109.5,
56.8, 55.7, 54.5, 52.9 (2C), 47.5, 30.6 (2C). ESI-MS m/z: 456 (MþH)þ.
Anal Calcd for C23H26FN5O2S: C, 60.64; H, 5.75; N, 15.37. Found: C,
60.68; H, 5.72; N, 15.39.
4.1.10.7. 1-(1-(2-(7-Methoxy-2-oxo-1,5-naphthyridin-1(2H)-yl)
ethyl)piperidin-4-yl)-3-(p-tolyl)thiourea (33). The compound was
synthesized according to the general procedure using 1-(2-(4-
aminopiperidin-1-yl)ethyl)-7-methoxy-1,5-naphthyridin-2(1H)-
one (12) (0.1 g, 0.330 mmol) and 4-methyl phenyl isothiocyanate
(0.059 g, 0.397 mmol) to afford 33 (0.115 g, 77%) as white solid. M.p:
134e136 ꢀC. 1H NMR [DMSO-d6] dH: 10.37 (b, 1H), 7.89e6.52 (m,
8H), 4.29 (t, J ¼ 6.9 Hz, 2H), 4.11 (b, 1H), 3.75 (s, 3H), 2.92e1.31 (m,
14H). 13C NMR [DMSO-d6] dc: 180.2, 163.3, 155.6, 142.9, 136.2, 135.3,
134.9, 132.1, 130.3 (2C), 126.8 (2C), 124.4, 119.2, 104.3, 56.4, 55.8,
53.8, 53.1 (2C), 50.1, 30.8 (2C), 22.6. ESI-MS m/z 452 (MþH)þ. Anal.
Calcd. for: C24H29N5O2S: C, 63.83; H, 6.47; N, 15.51. Found: C, 63.82;
H, 6.45; N, 15.55.
4.1.10.3. 1-(4-Chlorophenyl)-3-(1-(2-(7-methoxy-2-oxo-1,5-
naphthyridin-1(2H)-yl)ethyl)piperidin-4-yl)thiourea (29). The com-
pound was synthesized according to the general procedure using 1-
(2-(4-aminopiperidin-1-yl)ethyl)-7-methoxy-1,5-naphthyridin-
2(1H)-one (12) (0.1 g, 0.330 mmol) and 4-chloro phenyl isothio-
cyanate (0.067 g, 0.397 mmol) to afford 29 (0.121 g, 77%) as brown
solid. M.p: 167e169 ꢀC. 1H NMR [DMSO-d6] dH: 10.46 (b, 1H),
8.01e6.71 (m, 8H), 4.30 (t, J ¼ 6.3 Hz, 2H), 4.15 (b, 1H), 3.91 (s, 3H),
2.91e1.35 (m, 11H). 13C NMR [DMSO-d6] dc: 178.1, 161.7, 157.8, 145.9,
140.2, 137.2, 134.5, 132.8, 131.9 (2C), 130.3 (2C), 125.6, 123.8, 109.6,
56.8, 55.4, 54.5, 52.3 (2C), 47.5, 30.3 (2C).ESI-MS m/z: 473 (MþH)þ.
4.1.11. General procedure for the synthesis of substituted 1-(1-(2-
(7-methoxy-2-oxo-1,5-naphthyridin-1(2H)-yl)ethyl)piperidin-4-
yl)-3-phenylurea (34e40)
A solution of 1-(2-(4-aminopiperidin-1-yl)ethyl)-7-methoxy-
1,5-naphthyridin-2(1H)-one (12) (0.33 mmol) in dichloromethane
(1 mL) was cooled to 0 ꢀC followed by the addition of triethylamine

K.A. Bobesh et al. / European Journal of Medicinal Chemistry 85 (2014) 593e604
601
(0.99 mmol). Corresponding substituted phenyl isocyanate
(0.397 mmol) was added to the reaction mixture and was stirred in
room temperature for 12 h (monitored by TLC & LCMS for
completion). The reaction mixture was washed with water
(3 ꢁ 5 mL) and brine (3 ꢁ 5 mL). Organic layer was dried (MgSO4)
and concentrated under reduced pressure. The crude product was
purified by silica gel column chromatography using hexane: ethyl
acetate as eluent to give the corresponding substituted1-(1-(2-(7-
methoxy-2-oxo-1,5-naphthyridin-1(2H)-yl)ethyl)piperidin-4-yl)-
3-phenylthiourea (34e40) in good yield.
4.1.11.5. 1-(4-Acetylphenyl)-3-(1-(2-(7-methoxy-2-oxo-1,5-
naphthyridin-1(2H)-yl)ethyl)piperidin-4-yl)urea (38). The com-
pound was synthesized according to the general procedure using 1-
(2-(4-aminopiperidin-1-yl)ethyl)-7-methoxy-1,5-naphthyridin-
2(1H)-one (12) (0.1 g, 0.330 mmol) and 4-acetyl phenyl isocyanate
(0.063 g, 0.397 mmol) to afford 38 (0.12 g, 78%) as white solid. M.p:
230e232 ꢀC. 1H NMR [DMSO-d6] dH: 10.59 (b, 1H). 10.42 (b, 1H),
8.34e6.70 (m, 8H), 4.68 (t, J ¼ 6.6 Hz, 2H), 4.05 (s, 3H), 3.73e3.05
(m, 3H), 2.51 (s, 3H), 2.45e1.15 (m, 8H). 13C NMR [DMSO-d6] dc
:
196.1, 161.0, 156.8, 154.2, 145.0, 140.8, 136.7, 135.1, 131.3, 129.6 (2C),
125.4, 120.9 (2C), 116.5, 104.9, 57.0, 52.0, 51.48 (2C), 51.0, 47.6, 29.2
(2C), 26.2. ESI-MS m/z: 464 (MþH)þ. Anal Calcd for C25H29N5O4: C,
64.78; H, 6.31; N, 15.11; Found: C, 64.77; H, 6.29; N, 15.14.
4.1.11.1. 1-(1-(2-(7-Methoxy-2-oxo-1,5-naphthyridin-1(2H)-yl)ethyl)
piperidin-4-yl)-3-phenylurea (34). The compound was synthesized
according to the general procedure using 1-(2-(4-aminopiperidin-
1-yl)ethyl)-7-methoxy-1,5-naphthyridin-2(1H)-one (12) (0.1 g,
0.330 mmol) and phenyl isocyanate (0.047 g, 0.397 mmol) to afford
34 (0.102 g, 73%) as yellow solid. M.p: 186e188 ꢀC. 1H NMR [DMSO-
d6] dH: 10.46 (b, 1H), 10.38 (b, 1H), 7.92e6.65 (m, 9H), 4.67 (t,
J ¼ 6.9 Hz, 2H), 3.85 (s, 3H), 3.01e1.42 (m, 11H). 13C NMR [DMSO-d6]
dc: 161.7, 156.8, 155.6, 145.3, 140.2, 136.9, 132.9, 130.3 (2C), 129.2,
125.6, 123.4 (2C), 120.6, 106.8, 56.2, 53.6, 52.8 (2C), 47.9, 46.8, 30.2
(2C). ESI-MS m/z: 422 (MþH)þ. Anal. Calcd. for C23H27N5O3: C,
65.54; H, 6.46; N, 16.62. Found: C, 65.55; H, 6.40; N, 16.67.
4.1.11.6. 1-(1-(2-(7-Methoxy-2-oxo-1,5-naphthyridin-1(2H)-yl)ethyl)
piperidin-4-yl)-3-(4-methoxyphenyl)urea (39). The compound was
synthesized according to the general procedure using 1-(2-(4-
aminopiperidin-1-yl)ethyl)-7-methoxy-1,5-naphthyridin-2(1H)-
one (12) (0.1 g, 0.330 mmol) and 4-methoxyphenyl isothiocyanate
(0.059 g, 0.397 mmol) to afford 39 (0.114 g, 76%) as yellow solid.
M.p: 217e219 ꢀC. 1H NMR [DMSO-d6] dH: 10.42 (b,1H),10.53 (b,1H),
7.88e6.63 (m, 8H), 4.51 (t, J ¼ 6.9 Hz, 2H), 3.85 (s, 3H), 3.81 (s, 3H),
3.27e1.39 (m, 11H). 13C NMR [DMSO-d6] dc: 162.7, 159.7, 157.8,
155.3, 145.2, 136.1, 131.7, 130.8, 125.6, 120.2 (2C), 119.5, 115.6 (2C),
106.5, 56.2 (2C), 54.2, 52.3 (2C), 47.8, 46.7, 30.3 (2C). ESI-MS m/z:
452 (MþH)þ. Anal Calcd for C24H29N5O4: C, 63.84; H, 6.47; N, 15.51.
Found: C, 63.88; H, 6.43; N, 15.55.
4.1.11.2. 1-(4-Fluorophenyl)-3-(1-(2-(7-methoxy-2-oxo-1,5-
naphthyridin-1(2H)-yl)ethyl)piperidin-4-yl)urea (35). The com-
pound was synthesized according to the general procedure using 1-
(2-(4-aminopiperidin-1-yl)ethyl)-7-methoxy-1,5-naphthyridin-
2(1H)-one (12) (0.1 g, 0.330 mmol) and 4-fluro phenyl isocyanate
(0.059 g, 0.397 mmol) to afford 35 (0.117 g, 81%) as pale yellow
solid. M.p: 209e211 ꢀC. 1H NMR [DMSO-d6] dH: 10.42 (b, 1H), 10.35
(b, 1H), 7.98e6.65 (m, 8H), 4.52 (t, J ¼ 6.6 Hz, 2H), 3.82 (s, 3H),
3.48e1.38 (m, 11H). 13C NMR [DMSO-d6] dc: 163.1, 160.7, 155.8,
154.9, 145.9, 136.7, 134.9, 132.2, 125.5, 120.2 (2C), 119.1, 116.2 (2C),
106.8, 56.5, 54.5, 52.3 (2C), 49.5, 46.7, 30.3 (2C). ESI-MS m/z: 440
(MþH)þ. Anal Calcd for C23H26FN5O3: C, 62.86; H, 5.96; N, 15.94.
Found: C, 62.82; H, 5.99; N, 15.91.
4.1.11.7. 1-(1-(2-(7-Methoxy-2-oxo-1,5-naphthyridin-1(2H)-yl)ethyl)
piperidin-4-yl)-3-(p-tolyl)urea (40). The compound was synthe-
sized according to the general procedure using 1-(2-(4-
aminopiperidin-1-yl)ethyl)-7-methoxy-1,5-naphthyridin-2(1H)-
one (12) (0.1 g, 0.330 mmol) and 4-methyl phenyl isocyanate
(0.053 g, 0.397 mmol) to afford 40 (0.103 g, 72%) as pale brown
solid. M.p: 225e227 ꢀC. 1H NMR [DMSO-d6] dH: 10.42 (b, 1H), 10.36
(b, 1H), 7.83e6.66 (m, 8H), 4.42 (t, J ¼ 7.5 Hz, 2H), 3.87 (s, 3H)
3.12e1.39 (m,14H). 13C NMR [DMSO-d6] dc: 161.7,157.2,155.4,145.8,
137.9, 137.2, 135.3, 132.7, 130.2 (2C), 125.6, 120.8 (2C), 119.2, 106.8,
56.2, 54.6, 52.5 (2C), 47.8, 46.5, 30.2 (2C), 20.8. ESI-MS m/z: 436
(MþH)þ. Anal Calcd for C24H29N5O3: C, 66.19; H, 6.71; N, 16.08.
Found: C, 66.22; H, 6.78; N, 16.12%.
4.1.11.3. 1-(4-Chlorophenyl)-3-(1-(2-(7-methoxy-2-oxo-1,5-
naphthyridin-1(2H)-yl)ethyl)piperidin-4-yl)urea (36). The com-
pound was synthesized according to the general procedure using 1-
(2-(4-aminopiperidin-1-yl)ethyl)-7-methoxy-1,5-naphthyridin-
2(1H)-one (12) (0.1 g, 0.330 mmol) and 4-chloro phenyl isocyanate
(0.061 g, 0.397 mmol) to afford 36 (0.128 g, 85%) as pale brown
solid. M.p: 206e208 ꢀC. 1H NMR [DMSO-d6] dH: 10.49 (b, 1H), 10.41
(b, 1H), 7.96e6.68 (m, 8H), 4.49 (t, J ¼ 6.3 Hz, 2H), 3.85 (s, 3H),
3.25e1.39 (m, 11H). 13C NMR [DMSO-d6] dc: 161.7, 156.8, 155.8,
145.9, 138.6, 135.7, 132.9, 132.3, 130.4 (2C), 125.6, 120.5 (2C), 119.5,
106.7, 56.2, 54.2, 52.8 (2C), 47.9, 46.5, 30.4 (2C). ESI-MS m/z: 456
(MþH)þ. Anal Calcd for C23H26ClN5O3: C, 60.59; H, 5.75; N, 15.36.
Found: C, 60.57; H, 5.78; N, 15.38.
4.1.12. General procedure for the synthesis of substituted 1-benzyl-
3-(1-(2-(7-fluoro-2-oxo-1,5-naphthyridin-1(2H)-yl)ethyl)piperidin-
4-yl)thiourea (41e43)
A solution of 1-(2-(4-aminopiperidin-1-yl)ethyl)-7-fluoro-1,5-
naphthyridin-2(1H)-one (11) (0.344 mmol) in dichloromethane
(3 mL) was cooled to 0 ꢀC followed by the addition of triethylamine
(1.02 mmol). Corresponding substituted benzyl isothiocyanate
(0.413 mmol) was added to the reaction mixture and was stirred in
room temperature for 12 h (monitored by TLC & LCMS for
completion). The reaction mixture was washed with water
(3 ꢁ 5 mL) and brine (3 ꢁ 5 mL). Organic layer was dried over
(MgSO4) and concentrated under reduced pressure. The crude
product was purified by silica gel column chromatography using
hexane: ethyl acetate as eluent to give the corresponding
substituted1-benzyl-3-(1-(2-(7-fluoro-2-oxo-1,5-naphthyridin-
1(2H)-yl)ethyl)piperidin-4-yl)thiourea (41e43) in good yields.
4.1.11.4. 1-(1-(2-(7-Methoxy-2-oxo-1,5-naphthyridin-1(2H)-yl)ethyl)
piperidin-4-yl)-3-(4-nitrophenyl)urea (37). The compound was
synthesized according to the general procedure using 1-(2-(4-
aminopiperidin-1-yl)ethyl)-7-methoxy-1,5-naphthyridin-2(1H)-
one (12) (0.1 g, 0.330 mmol) and 4-nitro phenyl isocyanate (0.065 g,
0.397 mmol) to afford 37 (0.092 g, 60%) as yellow solid. M.p:
221e223 ꢀC. 1H NMR [DMSO-d6] dH: 10.52 (b, 1H), 10.43 (b, 1H),
8.15e6.68 (m, 8H), 4.52 (t, J ¼ 6.9 Hz, 2H), 3.86 (s, 3H), 3.36e1.48
(m, 11H). 13C NMR [DMSO-d6] dc: 162.9, 155.2, 154.8, 144.3, 143.3,
142.9, 135.7, 132.8, 125.2, 124.9 (2C), 120.2 (2C), 119.2, 106.5, 56.2,
54.1, 52.8 (2C), 47.8, 46.9, 30.2 (2C). ESI-MS m/z: 467 (MþH)þ. Anal
Calcd for C23H26N6O5: C, 59.22; H, 5.62; N,18.02. Found: C, 59.25; H,
5.58; N, 18.08.
4.1.12.1. 1-Benzyl-3-(1-(2-(7-fluoro-2-oxo-1,5-naphthyridin-1(2H)-
yl)ethyl)piperidin-4-yl)thiourea (41). The compound was synthe-
sized according to the general procedure using 1-(2-(4-
aminopiperidin-1-yl)ethyl)-7-fluoro-1,5-naphthyridin-2(1H)-one
(11) (0.1 g, 0.344 mmol) and benzyl isothiocyanate (0.062 g,
0.413 mmol) to afford 41 (0.108 g, 71%) as pale yellow solid. M.p:

602
K.A. Bobesh et al. / European Journal of Medicinal Chemistry 85 (2014) 593e604
174e176 ꢀC. 1H NMR [DMSO-d6] dH: 8.68e6.82 (m, 9H), 5.52 (b, 1H),
5.26 (b, 1H), 4.36e4.12 (m, 4H), 2.89e1.25 (m, 11H). 13C NMR
[DMSO-d6] dc: 179.8, 160.7, 157.3, 145.3, 139.8, 137.2, 132.7, 128.3
(2C), 127.8 (2C), 127.5, 124.6, 123.8, 109.8, 54.2, 53.4, 52.8 (2C), 47.3,
43.5, 30.2 (2C). ESI-MS m/z: 440 (MþH)þ. Anal Calcd for
4.1.13.2. 1-(4-Chlorobenzyl)-3-(1-(2-(7-fluoro-2-oxo-1,5-
naphthyridin-1(2H)-yl)ethyl)piperidin-4-yl)urea (45). The com-
pound was synthesized according to the general procedure using 1-
(2-(4-aminopiperidin-1-yl)ethyl)-7-fluoro-1,5-naphthyridin-
2(1H)-one (11) (0.1 g, 0.344 mmol) and 4-chloro benzyl isocyanate
(0.069 g, 0.413 mmol) to afford 45 (0.105 g, 66%) as yellow solid.
M.p: 215e217 ꢀC. 1H NMR [DMSO-d6] dH: 8.77e6.86 (m, 8H), 6.11 (b,
1H), 5.89 (b, 1H), 4.32e4.11 (m, 4H), 2.88e1.21 (m, 11H). 13C NMR
[DMSO-d6] dc: 160.7, 158.6, 157.4, 144.5, 139.9, 138.7, 137.5 (2C),
132.9, 132.7, 128.3 (2C), 124.8, 123.5, 109.8, 53.3, 52.8 (2C), 52.5,
46.5, 42.6, 31.3 (2C). ESI-MS m/z: 458 (MþH)þ. Anal Calcd for
C
23H26FN5OS: C, 62.85; H, 5.96; N, 15.93; Found: C, 62.82; H, 5.95;
N, 15.95.
4.1.12.2. 1-(4-Chlorobenzyl)-3-(1-(2-(7-fluoro-2-oxo-1,5-
naphthyridin-1(2H)-yl)ethyl)piperidin-4-yl)thiourea (42). The com-
pound was synthesized according to the general procedure using 1-
(2-(4-aminopiperidin-1-yl)ethyl)-7-fluoro-1,5-naphthyridin-
2(1H)-one (11) (0.1 g, 0.344 mmol) and 4-chloro benzyl isothiocy-
anate (0.075 g, 0.413 mmol) to afford 42 (0.132 g, 80%) as yellow
solid. M.p: 163e165 ꢀC. 1H NMR [DMSO-d6] dH: 8.65e6.66 (m, 8H),
C23H25ClFN5O2: C, 60.33; H, 5.50; N, 15.29; Found: C, 60.35; H, 5.53;
N, 15.27.
4.1.13.3. 1-(1-(2-(7-Fluoro-2-oxo-1,5-naphthyridin-1(2H)-yl)ethyl)
piperidin-4-yl)-3-(4-methoxybenzyl)urea (46). The compound was
synthesized according to the general procedure using 1-(2-(4-
aminopiperidin-1-yl)ethyl)-7-fluoro-1,5-naphthyridin-2(1H)-one
(11) (0.1 g, 0.344 mmol) and 4-methoxy benzyl isocyanate (0.067 g,
0.413 mmol) to afford 46 (0.125 g, 80%) as brown solid. M.p:
137e139 ꢀC. 1H NMR [DMSO-d6] dH: 8.67e6.81 (m, 8H), 6.84 (b, 1H),
5.51 (b, 1H), 5.32 (b, 1H), 4.27e4.07 (m, 4H), 2.76e1.19 (m, 11H). 13
C
NMR [DMSO-d6] dc: 179.8, 160.7, 157.8, 145.2, 140.3, 137.8, 135.2
(2C),132.9,132.5,129.5 (2C),125.6,123.5,109.8, 55.4, 53.5, 51.5 (2C),
47.8, 46.5, 30.2 (2C). ESI-MS m/z: 474 (MþH)þ. Anal Calcd for
C
23H25ClFN5OS: C, 58.28; H, 5.32; N, 14.78; Found: C, 58.30; H, 5.30;
N, 14.75.
5.81 (b, 1H), 4.34e4.08 (m, 4H), 3.71 (s, 3H), 2.83e1.14 (m, 11H). 13
C
4.1.12.3. 1-(1-(2-(7-Fluoro-2-oxo-1,5-naphthyridin-1(2H)-yl)ethyl)
piperidin-4-yl)-3-(4-methoxybenzyl)thiourea (43). The compound
was synthesized according to the general procedure using 1-(2-(4-
aminopiperidin-1-yl)ethyl)-7-fluoro-1,5-naphthyridin-2(1H)-one
(11) (0.1 g, 0.344 mmol) and 4-methoxy benzyl isothiocyanate
(0.074 g, 0.413 mmol) to afford 43 (0.128 g, 79%)as brown solid. M.p:
133e135 ꢀC. 1H NMR [DMSO-d6] dH: 8.52e6.73 (m, 8H), 5.34 (b, 1H),
NMR [DMSO-d6] dc: 160.6, 160.4, 158.0, 157.3, 139.9, 134.4, 133.1,
132.8 (2C), 132.7, 128.3, 123.9, 113.6 (2C), 109.3, 55.0, 53.1, 52.4 (2C),
52.2, 46.0, 42.2, 32.3 (2C). ESI-MS m/z: 454 (MþH)þ. Anal Calcd for
C24H28FN5O3: C, 63.56; H, 6.22; N, 15.44; Found: C, 63.59; H, 6.25;
N, 15.41.
4.1.14. General procedure for the synthesis of substituted 1-benzyl-
3-(1-(2-(7-methoxy-2-oxo-1,5-naphthyridin-1(2H)-yl)ethyl)
piperidin-4-yl)thiourea (47e49)
5.21 (b, 1H), 4.29e4.05 (m, 4H), 3.69 (s, 3H), 2.81e1.12 (m, 11H). 13
C
NMR [DMSO-d6] dc: 179.8, 160.6, 158.0, 157.3, 145.6, 139.9, 132.7,
129.8 (2C), 129.6, 125.6, 123.9, 113.6 (2C), 109.3, 55.7, 54.9, 53.6, 52.8
(2C), 47.8, 45.2, 30.3 (2C). ESI-MS m/z: 470 (MþH)þ. Anal Calcd
forC24H28FN5O2S: C, 61.39; H, 6.01; N, 14.91; Found: C, 61.35; H,
6.05; N, 14.93.
A solution of 1-(2-(4-aminopiperidin-1-yl)ethyl)-7-methoxy-
1,5-naphthyridin-2(1H)-one (12) (0.330 mmol) in dichloromethane
(1 mL) was cooled to 0 ꢀC followed by the addition of triethylamine
(0.99 mmol). Corresponding substituted benzyl isothiocyanate
(0.397 mmol) was added to the reaction mixture and was stirred in
room temperature for 12 h (monitored by TLC & LCMS for
completion). The reaction mixture was washed with water
(3 ꢁ 5 mL) and brine (3 ꢁ 5 mL). Organic layer was dried (MgSO4)
and concentrated under reduced pressure. The crude product was
purified by silica gel column chromatography using hexane: ethyl
acetate as eluent to give the corresponding substituted1-benzyl-3-
(1-(2-(7-methoxy-2-oxo-1,5-naphthyridin-1(2H)-yl)ethyl)piper-
idin-4-yl)thiourea (47e49) in good yield.
4.1.13. General procedure for the synthesis of substituted 1-benzyl-
3-(1-(2-(7-fluoro-2-oxo-1,5-naphthyridin-1(2H)-yl)ethyl)piperidin-
4-yl)urea (44e46)
A solution of 1-(2-(4-aminopiperidin-1-yl)ethyl)-7-fluoro-1,5-
naphthyridin-2(1H)-one (11) (0.344 mmol) in dichloromethane
(3 mL) was cooled to 0 ꢀC followed by the addition of triethylamine
(1.03 mmol). Corresponding substituted benzyl isocyanate
(0.413 mmol) was added to the reaction mixture and was stirred in
room temperature for 12 h (monitored by TLC & LCMS for
completion). The reaction mixture was washed with water
(3 ꢁ 5 mL) and brine (3 ꢁ 5 mL). Organic layer was dried over
(MgSO4) and concentrated under reduced pressure. The crude
product was purified by silica gel column chromatography using
hexane: ethyl acetate as eluent to give the corresponding
substituted1-benzyl-3-(1-(2-(7-fluoro-2-oxo-1,5-naphthyridin-
1(2H)-yl)ethyl)piperidin-4-yl)urea (44e46) in good yield.
4.1.14.1. 1-Benzyl-3-(1-(2-(7-methoxy-2-oxo-1,5-naphthyridin-
1(2H)-yl)ethyl)piperidin-4-yl)thiourea (47). The compound was
synthesized according to the general procedure using 1-(2-(4-
aminopiperidin-1-yl)ethyl)-7-methoxy-1,5-naphthyridin-2(1H)-
one (12) (0.1 g, 0.330 mmol) and benzyl isothiocyanate (0.059 g,
0.397 mmol) to afford 47 (0.108 g, 77%) as pale yellow solid. M.p:
149e151 ꢀC. 1H NMR [DMSO-d6] dH: 7.79e7.61 (m, 2H), 7.37 (b, 1H),
7.31 (b, 1H), 7.23e6.65 (m, 7H), 4.68 (s, 2H), 4.42 (t, J ¼ 6.6 Hz, 2H),
3.91 (s, 3H), 3.08e1.25 (m, 11H). 13C NMR [DMSO-d6] dc: 180.91,
161.9, 155.6, 146.2, 138.2, 136.2, 132.4, 129.1 (2C), 126.2 (2C), 125.9,
124.5, 119.1, 105.2, 56.2, 54.9, 54.1, 52.5 (2C), 51.2, 48.5, 30.2 (2C).
ESI-MS m/z: 452 (MþH)þ. Anal Calcd for C24H29N5O2S: C, 63.83; H,
6.47; N, 15.51. Found: C, 63.86; H, 6.44; N, 15.55.
4.1.13.1. 1-Benzyl-3-(1-(2-(7-fluoro-2-oxo-1,5-naphthyridin-1(2H)-
yl)ethyl)piperidin-4-yl)urea (44). The compound was synthesized
according to the general procedure using 1-(2-(4-aminopiperidin-
1-yl)ethyl)-7-fluoro-1,5-naphthyridin-2(1H)-one (11) (0.1 g,
0.344 mmol) and benzyl isocyanate (0.055 g, 0.413 mmol) to afford
44 (0.116 g, 80%) as yellow solid. M.p: 180e182 ꢀC. 1H NMR [DMSO-
d6] dH: 8.71e6.84 (m, 9H), 6.12 (b, 1H), 5.84 (b, 1H), 4.36e4.12 (m,
4H), 2.89e1.25 (m, 11H). 13C NMR [DMSO-d6] dc: 161.6, 158.1, 157.3,
144.2, 139.9, 137.2, 132.7, 128.3 (2C), 127.8 (2C), 127.2, 124.6, 123.9,
109.4, 53.1, 52.4 (2C), 51.2, 48.1, 45.5, 31.3 (2C). ESI-MS m/z: 424
(MþH)þ. Anal Calcd for C23H26FN5O2: C, 65.23; H, 6.19; N, 16.54;
Found: C, 65.21; H, 6.17; N, 16.53.
4.1.14.2. 1-(4-Chlorobenzyl)-3-(1-(2-(7-methoxy-2-oxo-1,5-
naphthyridin-1(2H)-yl)ethyl)piperidin-4-yl)thiourea (48). The com-
pound was synthesized according to the general procedure using 1-
(2-(4-aminopiperidin-1-yl)ethyl)-7-methoxy-1,5-naphthyridin-
2(1H)-one (12) (0.1 g, 0.330 mmol) and 4-chloro benzyl isothio-
cyanate (0.072 g, 0.397 mmol) to afford48 (0.114 g, 71%) as white

K.A. Bobesh et al. / European Journal of Medicinal Chemistry 85 (2014) 593e604
603
solid. M.p: 151e153 ꢀC. 1H NMR [DMSO-d6]: dH8.28e0.7.85 (m, 2H),
7.70 (b, 1H), 7.47 (b, 1H), 7.41e6.64 (m, 6H), 4.64 (s, 2H), 4.38 (t,
J ¼ 7.2 Hz, 2H), 3.98 (s, 3H), 2.91e1.33 (m, 11H). 13C NMR [DMSO-d6]
dc: 180.2, 161.5, 155.6, 146.2, 137.1, 136.5, 134.7 (2C), 132.5, 132.1,
129.2 (2C), 125.1, 119.2, 105.8, 56.2, 55.7, 54.5, 52.5 (2C), 51.2, 50.1,
30.2 (2C). ESI-MS m/z487 (MþH)þ. Anal Calcd for C24H28ClN5O2S: C,
59.31; H, 5.81; N, 14.41; Found: C, 59.41; H, 5.79; N, 14.45.
m/z: 470 (MþH)þ. Anal Calcd for C24H28ClN5O3: C, 61.34; H, 6.01;
N, 14.90; Found: C, 61.39; H, 6.01; N, 14.97.
4.1.15.3. 1-(1-(2-(7-Methoxy-2-oxo-1,5-naphthyridin-1(2H)-yl)
ethyl)piperidin-4-yl)-3-(4-methoxybenzyl)urea (52). The compound
was synthesized according to the general procedure using 1-(2-(4-
aminopiperidin-1-yl)ethyl)-7-methoxy-1,5-naphthyridin-2(1H)-
one (12) (0.1 g, 0.330 mmol) and 4-methoxy benzyl isocyanate
(0.064 g, 0.397 mmol) to afford52 (0.119 g, 77%)as brown solid. M.p:
185e187 ꢀC. 1H NMR [DMSO-d6] dH: 10.44 (b, 1H), 10.29 (s, 1H),
8.12e6.71 (m, 8H), 4.65 (s, 2H), 4.48 (t, J ¼ 6.6 Hz, 2H), 3.92 (s, 3H),
3.87 (s, 3H), 3.35e1.43 (m,11H). 13C NMR [DMSO-d6] dc: 162.1,159.7,
158.1, 155.6, 146.2, 136.6, 132.8, 130.7 (2C), 129.1, 124.8, 119.2, 115.2
(2C), 105.1, 56.2 (2C), 54.6, 52.5 (2C), 51.2, 48.6, 45.6, 30.2 (2C). ESI-
MS m/z: 466 (MþH)þ. Anal Calcd for C25H31N5O4: C, 64.50; H, 6.71;
N, 15.04; Found: C, 64.55; H, 6.69; N, 15.09.
4.1.14.3. 1-(1-(2-(7-Methoxy-2-oxo-1,5-naphthyridin-1(2H)-yl)
ethyl)piperidin-4-yl)-3-(4-methoxybenzyl)thiourea (49). The com-
pound was synthesized according to the general procedure using 1-
(2-(4-aminopiperidin-1-yl)ethyl)-7-methoxy-1,5-naphthyridin-
2(1H)-one (12) (0.1 g, 0.330 mmol) and 4-methoxy benzyl iso-
thiocyanate (0.071 g, 0.397 mmol) to afford 49 (0.122 g, 77%) as
brown solid. M.p: 184e186 ꢀC. 1H NMR [DMSO-d6]: dH8.2e7.87 (m,
2H), 7.58 (b, 1H), 7.43 (b, 1H), 7.45e6.61 (m, 6H), 4.57 (s, 2H), 4.35 (t,
J ¼ 6.6 Hz, 2H), 3.98 (s, 3H), 395 (s, 3H), 2.87e1.35 (m,11H). 13C NMR
[DMSO-d6] dc: 180.5, 161.5, 159.5, 155.1, 146.2, 135.3, 132.5, 130.2
(2C), 129.5, 125.3, 119.1, 115.2 (2C), 104.1, 56.2 (2C), 55.8, 54.5, 52.5
(2C), 51.2, 50.3, 30.2 (2C). ESI-MS m/z: 482 (MþH)þ. Anal Calcd for
4.2. M. tuberculosis DNA supercoiling assay
MTB DNA supercoiling assay was performed using hetero
tetramer gyrase enzyme containing GyrA and GyrB subunits
together, by using a MTB DNA supercoiling assay kit (Inspiralis
Limited, Norwich) [8].The assay was performed in 1.5 mL Eppen-
C
25H31N5O3S: C, 62.35; H, 6.49; N,14.54; Found: C, 62.38; H, 6.42; N,
14.56.
4.1.15. General procedure for the synthesis of substituted 1-benzyl-
3-(1-(2-(7-methoxy-2-oxo-1,5-naphthyridin-1(2H)-yl)ethyl)
piperidin-4-yl)urea (50e52)
dorf tubes in 30
mL reaction volume consisting of assay buffer
(50 mM HEPES. KOH (pH 7.9), 6 mM magnesium acetate, 100 mM
potassium glutamate, 4 mM DTT, 2 mM spermidine, 1 mM ATP, and
0.05 mg/mL of albumin). This mixture was together incubated with
A solution of 1-(2-(4-aminopiperidin-1-yl)ethyl)-7-methoxy-
1,5-naphthyridin-2(1H)-one (12) (0.33 mmol) in dichloromethane
(1 mL) was cooled to 0 ꢀC followed by the addition of triethylamine
(0.99 mmol). Corresponding substituted benzyl isocyanate
(0.397 mmol) was added to the reaction mixture and was stirred in
room temperature for 12 h (monitored by TLC & LCMS for
completion). The reaction mixture was washed with water
(3 ꢁ 5 mL) and brine (3 ꢁ 5 mL). Organic layer was dried over
(MgSO4) and concentrated under reduced pressure. The crude
product was purified by silica gel column chromatography using
hexane: ethyl acetate as eluent to give the corresponding
substituted1-benzyl-3-(1-(2-(7-methoxy-2-oxo-1,5-naphthyridin-
1(2H)-yl)ethyl)piperidin-4-yl)urea (50e52) in good yield.
1U of MTB DNA gyrase with 0.5 mg of relaxed pBR322, a substrate
and at varied test compound concentrations for 30 min at 37 ꢀC, to
calculate their IC50 value. Eventually the reaction was quenched by
the addition of 30 mL of chloroform: isoamyl alcohol in a ratio of
24:1 and STEB buffer (40% sucrose, 100 mM TriseHCl (pH 8.0),
100 mM EDTA and 0.5 mg/mL bromophenol blue) in equal volumes.
Later the samples were subjected to brisk vortex for obtaining a
homogenous state, centrifuged and loaded onto 1% Agarose gel in
1ꢁ TAE buffer. Staining with ethidium bromide continued for
10 min and de staining for 10 min with 1ꢁ TAE buffer was followed.
Further, samples were analysed using Image lab software (Bio-Rad)
and relative quantification was done based on the control sample.
Novobiocin which was used as a positive control in this assay.
4.1.15.1. 1-Benzyl-3-(1-(2-(7-methoxy-2-oxo-1,5-naphthyridin-
1(2H)-yl)ethyl)piperidin-4-yl)urea (50). The compound was syn-
thesized according to the general procedure using 1-(2-(4-
aminopiperidin-1-yl)ethyl)-7-methoxy-1,5-naphthyridin-2(1H)-
one (12) (0.1 g, 0.330 mmol) and benzyl isocyanate (0.052 g,
0.397 mmol) to afford 50 (0.105 g, 73%) as pale yellow solid. M.p:
184e186 ꢀC. 1H NMR [DMSO-d6] dH: 10.75 (b, 1H), 10.52 (s, 1H),
7.92e6.67 (m, 9H), 4.56 (s, 2H), 4.41 (t, J ¼ 7.2 Hz, 2H), 3.87 (s, 3H),
3.28e1.36 (m, 11H). 13C NMR [DMSO-d6] dc: 163.1, 157.8, 155.3,
145.7, 138.1, 136.1, 132.3, 129.1 (2C), 127.1 (2C), 126.8, 125.1, 119.2,
104.2, 56.2, 54.5, 52.3 (2C), 51.3, 48.5, 45.6, 30.1 (2C). ESI-MS m/z:
436 (MþH)þ. Anal Calcd for C24H29N5O3: C, 66.19; H, 6.71; N, 16.08.
Found: C, 66.22; H, 6.75; N, 16.05.
4.3. In vitro MTB MABA assay
All the compounds were further screened for their in vitro
antimycobacterial activity against M. tuberculosis H37Rv strain
(obtained from National Institute of Research in Tuberculosis,
Chennai, India) by microplate alamar blue assay method [9] from
25 to 0.78 mg/mL. In brief, the inoculum was prepared from fresh LJ
medium which was re-suspended in 7H9-S medium (7H9 broth,
0.1% casitone, 0.5% glycerol, supplemented oleic acid, albumin,
dextrose, and catalase [OADC]), adjusted to a McFarland tube No. 1,
and diluted 1:20; for each 100 mL was used as inoculum. Each drug
stock solution was thawed and diluted in 7H9-S by four-fold the
final highest concentration tested. Serial two-fold dilutions of each
drug were prepared directly in a sterile 96-well flat-bottomed
4.1.15.2. 1-(4-Chlorobenzyl)-3-(1-(2-(7-methoxy-2-oxo-1,5-
naphthyridin-1(2H)-yl)ethyl)piperidin-4-yl)urea (51). The com-
pound was synthesized according to the general procedure using 1-
(2-(4-aminopiperidin-1-yl)ethyl)-7-methoxy-1,5-naphthyridin-
2(1H)-one (12) (0.1 g, 0.330 mmol) and 4-chloro benzyl isocyanate
(0.066 g, 0.397 mmol) to afford 51 (0.129 g, 83%) as pale brown
solid. M.p: 169e171 ꢀC. 1H NMR [DMSO-d6] dH: 10.65 (b, 1H), 10.32
(s, 1H), 8.03e6.62 (m, 8H), 4.51 (s, 2H), 4.43 (t, J ¼ 6.3 Hz, 2H), 3.86
(s, 3H), 3.31e1.42 (m, 11H). 13C NMR [DMSO-d6] dc: 161.2, 158.2,
155.6, 145.6, 137.2, 135.9, 135.1 (2C), 132.5, 131.9, 129.5 (2C), 125.2,
119.1, 105.1, 56.2, 54.5, 52.5 (2C), 51.5, 48.5, 45.6, 30.2 (2C). ESI-MS
micro titre plates using 100 mL of 7H9-S. A growth control con-
taining no antibiotic and a sterile control were also prepared for
each plate. In order to avoid evaporation during incubation of 7
days, sterile water was added to all perimeter wells. The plate was
covered, sealed in plastic bags and incubated at 37 ꢀC in normal
atmosphere. After 7 days of incubation, 30 mL of alamar blue so-
lution was added to each well, and the plate was further re-
incubated overnight. Subsequently, a change in colour from blue
(oxidized state) to pink (reduced condition) indicated growth of

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4.4. In vitro cytotoxicity screening
Eukaryotic RAW 264.7 mouse macrophages cells were used to
test the cytotoxic activity of all the compounds [10]. Toxicity was
measured by incubating the test compounds in 96 flat-bottomed
well plates containing cell counts of 5 ꢁ 105 at different concen-
trations, with 5% CO2 and 95% O2atmosphere for 48 h at 37 ꢀC [15].
About 4 h before the end of incubation period 10 mL of MTT reagent
(10 mg mLꢂ1) was added, and the plate was centrifuged at 1200 rcf
for about 3 min to obtain a clear supernatant. The supernatant was
removed, and subsequently to each well 200 mL of DMSO was added
to dissolve the so formed formazan crystals. The absorbance was
measured at a wavelength of 560 nm on Perkin Elmer Victor X3
microplate reader against the blank after a span of 10 min. Assays
were performed in triplicates for each concentration of drug to
minimize the error rate. Cytotoxicity of each compound was
expressed as % inhibition at that particular concentration.
[10] (a) D. Barros, Recent advances in TB drug development-novel MTB DNA
Gyrase inhibitors, in: 40th Union World Conference on Lung Health, Cancun,
2009;
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[11] E.A. Voight, Hao Yin, S.V. Downing, S.A. Calad, Hayao Matsuhushi,
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DS is thankful to UGC, Government of India for the UGC research
award.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.08.018.
[14] M. Ferrari, M.C. Fornasiero, A.M. Isetta, J. Immunol. Methods 131 (1990)
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[15] D. Gerlier, N. Thomasset, J. Immunol. Methods 94 (1986) 57e63.
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