Synthesis and evaluation of fluorogenic 2-amino-1,8-naphthyridine derivatives for the detection of bacteria

Article abstract of DOI:10.1039/c2ob06986e

Several novel fluorogenic N-aminoacylnaphthyridine substrates were synthesized in good yield and tested for their ability to detect pathogenic bacteria in agar-based cell culture. Simple 2-N-(β-alanyl)amino-5,7- dialkylnaphthyridine substrates were selectively hydrolysed by β-alanylaminopeptidase expressing bacteria, but were subject to diffusion in the agar medium. Diffusion was reduced in the 2-N-(β-alanyl)amino-7- alkylnaphthyridine substrates with longer alkyl chains, but inhibition of growth was increased. 2-N-(β-Alanyl)amino-7-octylnaphthyridine inhibited the growth of all species tested, except for strains resistant to colistin/polymyxin, providing a rationale for the development of substrates for the selective detection of drug resistant species in clinical samples. The Royal Society of Chemistry 2012.

Full text of DOI:10.1039/c2ob06986e

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PAPER
Synthesis and evaluation of fluorogenic 2-amino-1,8-naphthyridine
derivatives for the detection of bacteria†
a
a
a
a
b
c
Linda Váradi, Mark Gray, Paul W. Groundwater,‡ Andrew J. Hall,§ Arthur L. James, Sylvain Orenga,
d
a
John D. Perry and Rosaleen J. Anderson*
Received 25th November 2011, Accepted 17th January 2012
DOI: 10.1039/c2ob06986e
Several novel fluorogenic N-aminoacylnaphthyridine substrates were synthesized in good yield and tested
for their ability to detect pathogenic bacteria in agar-based cell culture. Simple 2-N-(β-alanyl)amino-5,7-
dialkylnaphthyridine substrates were selectively hydrolysed by β-alanylaminopeptidase expressing
bacteria, but were subject to diffusion in the agar medium. Diffusion was reduced in the 2-N-(β-alanyl)
amino-7-alkylnaphthyridine substrates with longer alkyl chains, but inhibition of growth was increased. 2-
N-(β-Alanyl)amino-7-octylnaphthyridine inhibited the growth of all species tested, except for strains
resistant to colistin/polymyxin, providing a rationale for the development of substrates for the selective
detection of drug resistant species in clinical samples.
1
Introduction
units results in enzyme specificity and quenching of the colour
or fluorescence.
The number of multidrug resistant nosocomial bacterial species
is continually increasing, thus diagnosis and treatment of infec-
tions caused by these ‘superbugs’ provides a significant chal-
lenge. As early isolation and treatment of infected patients is
essential, there is an emerging need for rapid detection and
identification of pathogenic bacteria. One method for the
identification of specific bacterial species involves the use of
chromogenic or fluorogenic substrates, which offers rapid,
simple and reliable screening of clinical samples and is relatively
inexpensive, requiring no specialized analytical equipment.
These tests are ideally based on an off-to-on switching mechan-
ism, Fig. 1. The ‘switched-off’ substrate is represented by a chro-
mophore or fluorophore attached to a targeting molecule. The
chromophore or fluorophore provides a means for visualization,
whilst the targeting molecule makes the substrate specific for a
certain enzymatic activity. Covalent bonding between the two
Often the targeting molecule is an amino acid or sugar, which
is susceptible to enzymatic cleavage from the chromophore/
fluorophore. Less commonly, a redox active residue, such as a
nitro group, may be employed to take advantage of oxidoreduc-
tase activity. If a sufficient quantity of the molecule is taken into
the bacterial cells and a specific enzyme is present, the colour or
fluorescence is ‘switched-on’ when the bond between the target-
ing molecule and the visualizing unit is broken, allowing
extended conjugation throughout the molecule.
Chromogenic substrates allow the rapid and easy identification
of microorganisms in clinical or food samples. Such substrates,
when utilised in a suitable cell culture medium, can reduce the
need for subcultures and further biochemical tests. For example,
we have previously shown that β-alanine-1-pentylresorufamine
1
2
(
β-Ala-1-PRF) 1, Scheme 1, can be used to distinguish between3
Pseudomonas aeruginosa and other non-fermenting bacteria.
β-Alanylaminopeptidase activity, which results in the cleavage
of the pale yellow coloured β-alanine-1-PRF 1 to release the red/
purple 1-PRF 2, is present in all Pseudomonas aeruginosa
strains, but not in Burkholderia gladioli, Acinetobacter sp.,
a
Sunderland Pharmacy School, University of Sunderland, Sunderland,
SR1 3SD, UK. E-mail: roz.anderson@sunderland.ac.uk; Tel: +44 (0)
1
91 5152591
b
School of Life Sciences, Northumbria University, Newcastle upon Tyne,
NE1 8ST, UK
c
Department of Microbiology, bioMérieux, La-Balme-les-Grottes,
3
8390, France
d
Department of Microbiology, Freeman Hospital, Newcastle upon Tyne,
NE7 7DN, UK
†
1
‡
Electronic supplementary information (ESI) available. See DOI:
0.1039/c2ob06986e
Now at Faculty of Pharmacy, University of Sydney, Sydney, NSW2006
Australia.
Now at Medway School of Pharmacy, Universities of Greenwich and
Kent at Medway, Chatham, ME4 4TB, UK.
§
Fig. 1 Off-to-on switching mechanism for the identification of specific
bacterial species.
2578 | Org. Biomol. Chem., 2012, 10, 2578–2589
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Scheme 1 Specific bacterial aminopeptidase action visualised by the
3
release of the highly coloured chromophore, 1-pentylresorufamine 2.
Scheme 3 Detection of Pseudomonas aeruginosa by the β-alanine
aminopeptidase activity on fluorogenic 2-N-(β-alanyl)amino-1,8-
naphthyridines 6a–g producing the fluorescent 2-amino-1,8-naphthyri-
dine derivatives 3a–g.
Amino acid derivatives of aminonaphthyridines have pre-
viously been reported by Nakatani et al. and studied as DNA
sequence dependent binding probes using their hydrogen
bonding and accepting patterns as imprints for the specific recog-
5
nition of particular sequences. Investigations in this area are still
of interest, particularly with a view to improving selectivity; for
example, increasing the binding constants of H-bond recognition
6
by the use of a polyhedral oligomeric silsesquioxane core.
Although the naphthyridine structure has received attention as a
possible scaffold for novel antibacterial molecules, the amino-
naphthyridines have not previously been studied in the context
of bacterial detection. The inherent fluorescence of the 2-amino-
Scheme 2 Quantitative analysis of nitrite by 2-amino-5,7-dimethyl-
naphthyridine 3a.
4
1
,8-naphthyridines, and the potential for N-acylation to cause
Stenotrophomonas maltophilia, Brevundimonas sp. or Chryseo-
bacterium meningosepticum.
quenching due to a reduction of electron delocalization through-
out the π-system, offers an opportunity to evaluate these struc-
tures for their potential in detecting specific bacterial
aminopeptidase activity. Analogous to the quenched chromo-
genic substrate β-alanine-1-pentylresorufamine (β-Ala-1-PRF) 1
and the corresponding highly coloured 7-aminoresorufamine 2,
we report here the preparation of novel fluorogenic 2-N-
The disadvantage of detection techniques that rely on the visu-
alization of colour, even with significant colour changes such as
those seen in the example in Scheme 1, is the requirement for at
least 24 hours of incubation for conclusive results. Since the
detection of fluorescence is inherently more sensitive than the
perception of colour change, a smaller degree of enzymatic clea-
vage of the substrate is required, which would improve upon
existing chromogenic sensors for these strains, in terms of both
rapidity and sensitivity. Some fluorescent probes already exist as
commercial products, but there are few amino substituted fluoro-
gens that would enable the detection of bacterial aminopepti-
dases. The aim of the current work was the synthesis of
fluorogenic substrates that would allow the specific detection of
commonly encountered multidrug resistant bacteria through their
aminopeptidase activity. As P. aeruginosa is a common nosoco-
mial pathogen that requires improved detection for rapid treat-
ment, β-alanine was chosen as the reactable moiety in order to
target β-alanylaminopeptidase, an unusual aminopeptidase with
strong expression and activity in P. aeruginosa, for selective
(
β-alanyl)amino-1,8-naphthyridines 6a–g, Scheme 3, and their
first evaluation as potential bacterial detection agents.
To investigate the requirements for binding to bacteria and the
effect of substituents on the fluorescence, the substituents R and
R were varied; simple symmetrical alkyl substituted naphthyri-
dines 3a,b,
the initial scaffolds. The bis(trifluoromethyl) analogue 3c was
expected to have increased lipophilicity and fluorescence, as is
observed for other fluorinated fluorogens. As we have already
1
2
11,12
which have previously been reported, served as
10
3
observed that aliphatic substituents result in better adhesion to
the bacterial colonies through an increase in the lipophilicity of
the chromophores, the pentynyl 3d, octynyl 3e, pentyl 3f, and
octyl 3g analogues were also synthesized.
3
cleavage of the fluorogenic substrate.
The 2-amino-1,8-naphthyridines are fluorescent molecules
with an emission wavelength between 350–490 nm in
2
Results and discussion
4
–9
solution
and have potential in the detection of chemical and
2
.1 Chemistry
biological species. 2-Amino-5,7-dimethylnaphthyridine 3a has
been proposed as a fluorescent reagent for the detection of
Synthesis of the 2-aminonaphthyridine substrates 6a–g was
achieved in 2 steps: the synthesis of the 2-aminonaphthyridine
fluorogen and the coupling to the amino acid.
4
nitrite under acidic conditions, Scheme 2; the fluorescence of
3
a was quenched by nitrite in a linear relationship through diazo-
tization and reaction with water to form 2-hydroxy-5,7-dimethyl-
1
1
,8-naphthyridine 4, which tautomerises to the corresponding
,8-naphthyrid-2-one 5 with loss of fluorescence.
2
.1.1 Preparation
of
2-amino-1,8-naphthyridines. Two
different strategies were used for the synthesis of the fluorogenic
This journal is © The Royal Society of Chemistry 2012
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substituted 2-amino-1,8-naphthyridines 3a–g. For 5,7-disubsti-
tuted aminonaphthyridines 3a–c, an adapted Combes quinoline
case of 13b, the level of reduction appeared to be very sensitive
to the activity of the catalyst and the reaction time, as long reac-
tion times resulted in over-reduction to the corresponding dihy-
dronaphthyridine 14c. To avoid this over-reduction, the reaction
was stopped at an early stage and 14b was used without further
purification as a mixture with the partially unsaturated derivative.
Hydrolysis of the acetamido groups of 13a,b and 14a,b gave the
fluorophores 3d,e and 3f,g, respectively.
11–14
synthesis was employed,
using 2,6-diaminopyridine 7 and
the appropriate diketone 8a–c, Scheme 4. In order to introduce a
chlorine atom into these systems (to allow for further substitution
of the naphthyridine core), 2-amino-7-hydroxy-1,8-naphthyri-
dine 9 was prepared by the condensation of 2,6-diaminopyridine
1
1
7
and malic acid 10, Scheme 5. Before undertaking any reac-
tions on the hydroxyl group, the amino functionality of 2-amino-
-hydroxy-1,8-naphthyridine 9 was first protected by N-acety-
7
2.1.2 Amide bond formation. Each of the aminonaphthyri-
dines 3a–g could be readily converted into the aminoacyl
derived substrate 6a–g for a specific bacterial aminopeptidase,
11
lation, to give the amide 11. Chlorination using POCl gave
3
1
2, and the possibility of further structural elaboration involving
11
t
the chloro group.
through coupling to a protected amino acid, such as Boc-
Among the many possibilities offered by cross coupling strat-
egies, a Sonogashira-type reaction was chosen to give the
alkyne-substituted amide derivatives, 13a and 13b, with the aim
of improving adherence to the bacterial cell wall. The unsatu-
rated derivatives were also reduced, using 10% Pd/C catalyst and
hydrogen, to give the alkyl substituted analogues 14a,b. In the
β-alanine, followed by deprotection. The aminoacylation of the
t
2-aminonaphthyridines 3a–g with Boc-β-Ala-OH 17 was facili-
5
tated through the stable pentafluorophenyl ester 19, formed
t
from Boc-β-Ala-OH 17 and pentafluorophenol 20 with dicyclo-
hexylcarbodiimide as the dehydrating agent. Reaction of the
t
Boc-β-alanylpentafluorophenol ester 19 with the 2-amino-1,8-
naphthyridine derivatives 3a–g provided the protected aminoa-
cyl-substituted naphthyridines 21a–g in good yield, Table 1.
Deprotection gave the 2-N-(β-alanyl)aminonaphthyridines, 6a–g
in moderate to good yield, for microbiological evaluation,
Scheme 6 and Table 1.
2
.2 Fluorescent properties
The fluorescence spectra of the compounds prepared were
obtained, as the project uses off-to-on fluorescence (before and
after enzymatic activity) for bacterial detection. Practical evalu-
ation of clinical samples is based on the visual inspection of agar
plates, upon irradiation with UV light at 365 nm. Solutions of
enzyme substrates 6 and fluorogens 3 were visually observed
under UV light, followed by recording of the emission spectra at
Scheme 4 Synthesis of symmetrical 2-amino-5,7-disubstituted-1,8-
11–13
naphthyridines 3a–c.
3 4
Reagents and conditions: (i) H PO , reflux.
Scheme 5 Synthesis of 7-alkynyl and 7-alkylsubstituted naphthyridines 3d–g. Reagents and conditions: (i) conc. H
2
SO
4 2
, 110 °C, 3 h; (ii) Ac O,
reflux, 3 h; (iii) POCl , 95 °C, 2 h; (iv) Pd(Ph ) Cl , CuI, Et N, DMF, N , 80 °C; (v) H , 10 mol% 10 w/w% Pd on charcoal, MeOH; (vi) NaOH,
3 3 2 2 3 2 2
MeOH.
2580 | Org. Biomol. Chem., 2012, 10, 2578–2589
This journal is © The Royal Society of Chemistry 2012

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t
Table 1 Yields of (i) Boc protected N-aminoacylnaphthyridines 21a–g and (ii) final N-aminoacylnaphthyridine substrates 6a–g
Starting amines
R
1
R
2
(i) Protected fluorogens
Yield (%)
(ii) N-Aminoacyl naphthyridines
Yield (%)
3
3
3
a
b
c
Me
Et
Me
Et
21a
21b
21c
21d
21e
21f
86
62
31
86
94
89
54
6a
6b
6c
6d
6e
6f
83
76
84
77
87
65
59
CF
3
CF
H
3
3
d
1-Pentynyl
1-Octynyl
1-Pentyl
1-Octyl
3
3
e
f
H
H
H
3
g
21g
6g
Fig. 3 Emission spectra of 3d (blue solid line), 6d (blue dashed line),
e (green solid line), 6e (green dashed line), 3f (orange solid line), 6f
orange dashed line), 3g (purple solid line), 6g (purple dashed line) (8 ×
3
(
1
−8
0
2
M in MeOH–H O) (intensities are shown in arbitrary units).
Scheme 6 Peptide bond formation and deprotection to provide fluoro-
genic N-aminoacylnaphthyridine substrates 6a–g. Reagents and con-
ditions: (i) DIPEA, DMF, 40 °C; (ii) TFA.
(
3c) disubstituted derivatives, presumably due to the increasing
electronegativity of the substituents, thus promoting a greater
delocalization effect.
When comparing the naphthyridines bearing longer alkynyl
6
d, 6e and alkyl 6f, 6g substituents, an increase in the emission
intensity and wavelength was noticeable, perhaps through the
positive inductive effect of the substituents. This feature is ben-
eficial as the emission of the released fluorophores must be dif-
ferentiated from the background fluorescence of standard
endogenous media ingredients. The largest difference in fluor-
escence intensities between a 2-N-(β-alanyl)aminonaphthyridine
substrate and its corresponding fluorophore was observed for 3e
and 6e.
It has been reported previously that naphthyridines can form
complexes with certain transition metal and s-block ions,
depending on the substituents on the donor naphthyridines and
Fig. 2 Emission spectra of 3a (green solid line), 6a (green dashed
15,16
line), 3b (orange solid line), 6b (orange dashed line), 3c (blue solid line)
favourable acceptor ion sizes;
upon binding to these ions,
and 6c (blue dashed line) (8 × 10− M in MeOH–H
8
O) (intensities are
2
the fluorescent properties of the naphthyridines can significantly
change. Taking into account possible toxicity to bacterial cells
and convenience for use in a detection plate, fluorescent emis-
sions in the presence of Fe(III), Mg(II) and Ca(II) were also
assessed for each derivative, using ion concentrations in the
shown in arbitrary units).
fixed excitation wavelength (350 nm), Fig. 2 and 3. The emis-
sion spectra supported the conclusions reached from the obser-
vation of the solutions under UV; the bigger the increase in the
intensity of emitted light caused by specific enzymatic activity,
the more reliable the detection. In the case of 3a, 3c and 3d, the
presence of the aminoacyl moiety was found to have insufficient
effect on the emission to distinguish the acylated form 3a,c,d
from the amine 6a,c,d. As expected, the emission intensity
slightly increased across the CH (3a) < CH CH (3b) < CF
range of practical standards on agar plates (1.79 × 10− M).
3
17
Once again, the octynyl substituted naphthyridines 3e and 6e
showed the most desirable properties; complexation to Mg(II)
and Ca(II), in particular, resulted in an increase in the intensity of
the fluorescence, which would be advantageous for bacterial
differentiation, Fig. 4. The presence of Fe(III) quenched light
emission in each case, presumably due to its paramagnetic prop-
erties. Thus, octynyl substrate 6e and its corresponding
3
2
3
3
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 2578–2589 | 2581

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diffusion away from bacterial colonies. Adherence to the bac-
terial colonies, another desirable property for a detection
method, was improved with the introduction of more lipophilic
substrates. The individual colonies highlighted by the hydrolysis
of substrate 6f testify to the localization of the fluorogen 3f once
released; by comparison, the fluorescence released by hydrolysis
of 6a and 6b diffused across the plate, preventing visualization
of individual colonies, Fig. 5.
3
Conclusion
Novel fluorescent 2-amino-1,8-naphthyridines 3a–g were syn-
thesized, using simple procedures, in reliably good yields from
commercially available, inexpensive starting materials. These
amines were then converted into their β-alanyl analogues 6a–g
which were subjected to biological evaluation to assess their
potential for use in agar plate based diagnostic methods. It was
established that 6a–c and 6f are specific substrates for β-alanyla-
minopeptidase, releasing the expected fluorogens. Diffusion of
the fluorescent dyes was decreased by the use of more lipophilic
substrates, as in 6c–g, but this resulted in a loss of specificity for
Fig. 4 Emission spectra of 3e (continuous line) and 6e (dotted line) in
−6
the presence of chosen ions (0.8 × 10 M 3e/6e in MeOH–H
2
O; 2 ×
−4
1
0
M Ca(II)/Mg(II)/Fe(III) concentration, respectively) (intensities are
shown in arbitrary units).
fluorophore 3e were established as most suitable for the aims of
this project. In samples containing Mg(II) and Ca(II) ions, the
intensity of the fluorescence was increased, as was the wave-
length of the emitted light, the latter not significantly.
6
d, or an increase in toxicity. The growth inhibitory effect of
substrates 6e and 6g against many bacteria, and their detection
of colistin/polymyxin resistant bacterial strains, support the case
for development of agents for selective detection of these strains.
2
.3 Biological evaluation
The substrates incorporated in a commercially available Colum-
bia medium were subjected to microbiological evaluation against
a range of clinically relevant microorganisms.
4 Experimental
4
.1 Chemistry
All of the substrates were tested on ten Gram negative
4
.1.1 General. NMR spectra were obtained on a Bruker
(
1
2
Table 2 entries 1–10), eight Gram positive (Table 2 entries
1–18) microorganisms and two yeasts (Table 2 entries 19 and
0). The results are shown in Table 2 and Fig. 5. The intended
1
Ultrashield 300 spectrometer (at 300 MHz for H and at 75 MHz
for C spectra). The chemical shifts are shown in ppm
downfield from tetramethylsilane, using residual chloroform
δ = 7.26 in H NMR) or the middle peak of the CDCl carbon
triplet (δ = 77.23 in C NMR) as an internal standard. Melting
points were obtained using a Reichart-Kofler hot-stage micro-
scope apparatus and are uncorrected. Infrared spectra were
recorded using a Perkin Elmer Spectrum BX FT-IR instrument.
Low resolution mass spectra were recorded on a Bruker Esquire
13
specificity was achieved by substrates 6a, 6b, 6c, and 6f, as
these derivatives were hydrolyzed only by strains possessing
β-alanylaminopeptidase activity. Under long-wave UV light, the
colonies of strains hydrolyzing the substrates are highly fluor-
escent in comparison to the negative ones and the dark blue
background of the medium. Substrate 6d, with a pentynyl substi-
tuent, was hydrolyzed by all of the Gram negative bacteria,
making it unsuitable for use to distinguish β-alanylaminopepti-
dase activity, as this enzyme is only expressed in selected Gram
negative bacteria, indicating non-specific substrate hydrolysis. In
such detection methods, fluorogens that do not inhibit bacterial
growth are generally preferred and substrates 6a–d and 6f were
non-growth inhibitory. However in the presence of substrates 6e
and 6g (with longer saturated side chains) only Burkholderia
cepacia and Morganella morganii grew, indicating greater toxi-
city, perhaps due to the disruption of the bacterial cell membrane
through interaction of the long chain alkyl groups of 3e and 3g.
The ability of the three colistin/polymyxin resistant bacterial
strains, B. cepacia, M. morganii and Providencia rettgeri, to
survive on the plates containing 6g, Fig. 5, lends support to this
argument—these strains are resistant to antibacterial agents
1
(
3
13
3
000plus analyser using electrospray source in positive ion
mode. High resolution mass spectra were obtained on a LTQ
Orbitrap XL instrument in nanospray ionization mode. Elemental
analyses were carried out using an Exeter Analytical CE-440
Elemental Analyzer. Excitation and emission spectra were
recorded on a Perkin Elmer LS50B Luminescence Spectrometer.
All commercially available reagents and solvents were obtained
from Sigma-Aldrich, Alfa-Aesar, Fisher Scientific and Riedel-
de-Haan and were used without any further purification. Thin
layer chromatography was carried out on Merck silica gel plates
(
60F-254).
4.1.2 Preparation of 2-amino-5,7-disubstituted-1,8-naphthyr-
idines 3a–c by condensation
12
4.1.2.1 2-Amino-5,7-dimethyl-1,8-naphthyridine3a Amixture
of 2,4-pentanedione 8a (0.50 mL, 4.85 mmol) and 2,6-diamino-
pyridine 7 (0.50 g, 4.58 mmol) in phosphoric acid (2.50 mL)
were heated at reflux for 2 hours. After cooling to room tempera-
ture, the resulting mixture was poured over ice and neutralized
with 10% aqueous NaOH solution; the solid formed was
1
8
known to disrupt the integrity of the cell wall. These latter sub-
strates, 6e and 6g, offer potential as selective inhibitors of bac-
terial growth—an alternative method for bacterial identification.
Upon hydrolysis of the substrates 6a–g, the release of the
fluorophores gave purple fluorescence (374–406 nm) with some
2582 | Org. Biomol. Chem., 2012, 10, 2578–2589
This journal is © The Royal Society of Chemistry 2012

Table 2 Microbiological evaluation of the bacterial detection and identification ability and fluorogenic properties of the substrates 6a–g
Control
6a
6b
6c
6d
6e
6f
6g
a
Microbes
Growth
Fl.
Growth
Fl.
Growth
Fl.
Growth
Fl.
Growth
Fl.
Growth
Fl.
Growth
Fl.
Growth
Fl.
1
2
3
4
5
6
7
8
9
1
Eschericia coli
++
++
++
+
++
++
++
++
++
++
−
−
−
−
−
−
−
−
−
−
++
++
++
++
+
++
++
++
++
+
−
++
++
++
++
+
++
++
++
++
Traces
−
+
+
+
+
++
+
+
+
++
+
−
+
+
++
++
++
++
+
++
++
++
++
+
+/−
+/−
+/−
+/−
+/−
−
+/−
+/−
+/−
+/−
−
−
−
++
−
−
−
+
n/a
n/a
n/a
+
n/a
n/a
n/a
n/a
n/a
n/a
+
+
+
+
+
+
+
+
+
+
−
−
−
−
+
−
−
−
+
n/a
n/a
n/a
−
Serratia marcescens
Pseudomonas aeruginosa
Burkholderia cepacia
Yersinia enterocolitica
Salmonella typhimurium
Citrobacter freundii
Morganella morganii
Enterobacter cloacae
Providencia rettgeri
+/− [D]
+/− [D]
+/−
+/−
+/−
−
+/− [D]
+/− [D]
+/− [D]
+/− [D]
+
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
n/a
n/a
n/a
−
−
−
+/−
+/−
—
−
−
−
+
n/a
−
0
1
1
Bacillus subtilis
+
+
+
+
+
+
+
+
−
−
−
−
−
−
−
−
−
−
+
+
+
+
+
+
+
+
−
−
−
−
−
−
−
−
−
−
+
+
+
+
+
+
+
+
−
−
−
−
−
−
−
−
−
−
+
+
+
+
+
+
+
+
−
−
−
−
−
−
−
−
−
−
+
+
+
+
+
+
+
+
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a −
n/a
n/a
+
+
+
+
+
+
+
+
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
12
13
14
15
16
17
18
19
20
Enterococcus faecalis
Enterococcus faecium
Streptococcus epidermidis
Staphylococcus aureus
b
MRSA
Streptococcus pyogenes
Listeria monocytogenes
Candida albicans
+/−
Traces
+/−
Traces
+/−
Traces
+
+/−
Traces
+
Candida glabrata
Traces
Traces
Background fluorescence
—
None
−
−
−
−
−
+/−
−
+/−
−
+/−
−
−
−
−
a
Fl.: fluorescence; ++ very good/strong; + medium; − none/weak; [D] diffusion of fluorescence in the medium; Microbes 1–10: Gram negative bacteria; 11–18: Gram positive bacteria; 19 and 20:
b
yeasts. MRSA: methicillin resistant Staphylococcus aureus.

View Online
13
4
.1.2.3 2-Amino-5,7-di(trifluoromethyl)-1,8-naphthyridine 3c
A mixture of 2,6-diaminopyridine 7 (4.90 g, 45.00 mmol),
1
4
1
6
,1,1,5,5,5-hexafluoropentane-2,4-dione
8c
(10.00
g,
8.00 mmol) and 85% phosphoric acid (25 mL) was stirred at
20 °C overnight. After standing at room temperature for
hours, the reaction mixture was poured into ice water and 10%
NaOH was added until pH 7 was reached. The white precipitate
obtained was filtered and dried over PCl to yield 3c (6.30 g,
5
13
2
2.00 mmol, 50%); mp 211–212 °C [lit. mp 204–206 °C];
[
4
(
Found: C, 42.47; H, 1.88; N, 14.93. C H F N requires C,
10 5 6 3
−
1
2.72; H, 1.79; N, 14.95%]; νmax/cm 3335 (NH), 1642, 1113
1
C-F); H NMR (300 MHz, DMSO-d ) δ 7.18 (1H, d, J = 9.3
6 H
Hz, CH-3), 7.56 (2H, br s, NH ), 7.81 (1H, s, CH-6), 8.16 (1H,
2
13
dd, J = 9.3 and 1.8 Hz, CH-4); C NMR (75 MHz, DMSO-d6)
δC 109.4 (CH, m, J = 2.4 Hz, CH-6), 114.3 (quat., C-4a), 118.7
(
CH-3), 121.4 (CF , q, J = 273 Hz, C7-CF ), 123.2 (CF , q, J =
3 3 3
2
74 Hz, C5-CF ), 133.0 (CH-4), 135.8 (quat., q, J = 32.1 Hz,
3
C-5), 148.3 (quat., q, J = 34.5 Hz, C-7), 157.5 (quat., C-8a),
+
Fig. 5 Fluorescence visible after the culture of various bacterial
species with fluorogenic substrates 6a–g (λex = 365 nm). Dotted cultures
correspond to the numbered plate; numbers on the agar plates corre-
spond to the entries in Table 2. Streaked cultures are P. aeruginosa
samples.
1
62.3 (quat., C-2); MS (ESI) m/z 282.0 (MH ).
4.1.3 Preparation of key intermediate 2-acetylamino-7-
chloro-1,8-naphthyridine 12
.1.3.1 2-Amino-7-hydroxy-1,8-naphthyridine
was prepared by the previously published method (96%); mp
19
4
9
Compound
9
>
19
−1
300 °C (deg.) [lit. mp >350 °C]; νmax/cm 3377, 3157
collected by vacuum filtration. The desired product 3a was
obtained (0.33 g, 1.94 mmol, 40%) as a white solid after purifi-
cation by column chromatography (10% methanol, 90% dichloro-
methane) followed by recrystallization from water; mp
1
(NH), 1615; H NMR (300 MHz, DMSO-d ) δ 6.11 (1H, d,
6 H
J = 9.3 Hz, CH), 6.36 (1H, d, J = 8.4 Hz, CH), 6.94 (2H, br s,
13
NH ), 7.64 (2H, m, 2 × CH), 11.79 (1H, br s, OH); C NMR
2
1
2
−1
(75 MHz, DMSO-d
137.7 (CH), 140.0 (CH), 150.9 (quat.), 160.9 (quat.), 164.1
quat.).
.1.3.2 2-Acetylamino-7-hydroxy-1,8-naphthyridine 11 Com-
6 C
) δ 105.4 (quat.), 105.5 (CH), 115.4 (CH),
2
(
26–227 °C [lit. mp 216–218 °C]; νmax/cm 3245, 3179
1
NH), 1588; H NMR (300 MHz, CDCl ) δ 2.54 (3H, s, C5-
3 H
(
CH ), 2.62 (3H, s, C7-CH ), 4.95 (2H, br s, NH ), 6.69 (1H, d,
3
3
2
11
4
J = 8.7 Hz, CH-3), 6.91 (1H, s, CH-6), 8.01 (1H, d, J = 8.7 Hz,
13
pound 9 was converted into 11 using the known method to give
a yellow powder (80%); mp >300 °C [lit. mp ∼300 °C]; νmax/
CH-4); C NMR (75 MHz, CDCl ) δC 17.9 (CH , C5-CH ),
3
3
3
2
5.3 (CH , C7-CH ), 110.7 (CH-3), 115.2 (quat., C-4a), 120.1
3 3
−1
cm 3171 (broad NH and/or OH), 1653 (CvO), 1583 (amide
(CH-6), 134.5 (CH-4), 145.4 (quat., C-5), 156.2 (quat., C-2),
1
I), 1522 (amide II); H NMR (300 MHz, TFA-d ) δ 3.30 (3H,
1
H
1
58.7 (quat., C-8a), 161.7 (quat., C-7); MS (ESI) m/z 174.0
+
s, CH ), 7.78 (1H, d, J = 9.6 Hz, CH-3), 8.19 (1H, d, J = 8.7
3
(MH ).
Hz, CH-6), 8.93 (1H, d, J = 9.6 Hz, CH-4), 9.42 (1H, d, J = 8.7
13
11
Hz, CH-5); C NMR (75 MHz, TFA-d ) δ 22.6 (CH ), 109.7
4
.1.2.2 2-Amino-5,7-diethyl-1,8-naphthyridine 3b A mixture
1
C
3
(CH-6), 113.2 (quat., C-4a), 121.9 (CH-3), 139.8 (quat., C-8a),
of 3,5-heptanedione 8b (20.00 mL, 0.15 mol) and 2,6-diamino-
pyridine 7 (15.00 g, 0.14 mol) were heated in phosphoric acid
for 2 hours. The mixture was poured into ice and neutralized
1
40.6 (CH-4), 146.9 (CH-5), 147.7 (quat., C-7), 164.2 (quat.,
+
C-2), 177.2 (quat., CvO); MS (ESI) m/z 204.0 (MH ), 226.0
+
(MNa ).
with 28% NH OH. After filtration, the precipitated solid was
4
11
continuously extracted with dichloromethane. The solvent was
removed in vacuo and the crude product was purified by column
chromatography (10% methanol, 90% dichloromethane). Recrys-
tallization from ethyl acetate gave a pale yellow solid 3b (1.66 g,
4.1.3.3 2-Acetylamino-7-chloro-1,8-naphthyridine 12
A
mixture of naphthyridine 11 (1.00 g, 0.05 mol) and POCl3
(17.50 mL) was heated at 95 °C for 1.5 hours, in the presence of
a catalytic amount of DMF. The resulting solution was cooled to
room temperature and poured carefully into ice water. The solu-
tion was made basic (pH 8) with 10% NaOH resulting in for-
mation of a brown precipitate. The solid was collected by
vacuum filtration, air dried and continuously extracted with
chloroform for 12 hours. The water layer was extracted with
chloroform. The solvent of the combined organic layers was
removed by vacuum filtration, and the crude product was
purified by column chromatography to give the title compound
12 after recrystallization from ethyl acetate (3.30 g, 15.00 mmol,
30%); mp 238–241 °C [lit. mp 250–252 °C]; [Found: C, 54.66;
H, 3.74; N, 18.99. C H ClN O requires C, 54.19; H 3.64; N,
11
8
.28 mmol, 6%); mp 192–195 °C [lit. mp 187–190 °C];
[
Found: C, 71.44; H, 7.45; N, 20.70. C H N requires C,
1.61; H, 7.51; N, 20.88%]; νmax/cm 3252, 3122 (NH), 1582;
1
2 15 3
−1
7
1
H NMR (300 MHz, DMSO-d ) δ 1.20–1.29 (6H, m, 2 ×
6
H
CH ), 2.77 (2H, q, J = 7.5 Hz, C7-CH ), 2.89 (2H, q, J = 7.5
3
2
Hz, C5-CH ), 6.64 (2H, s, NH ), 6.79 (1H, d, J = 9.0 Hz,
2
2
13
CH-3), 6.91 (1H, s, CH-6), 8.07 (1H, d, J = 9.0 Hz, CH-4);
C
NMR (75 MHz, DMSO-d ) δ 13.9 (C7-CH ), 15.1 (C5-CH ),
6
C
3
3
2
4.4 (C5-CH ), 31.8 (C7-CH ), 112.1 (CH-3), 113.8 (quat.,
2 2
C-4a), 116.1 (CH-6), 133.9 (CH-4), 150.8 (quat., C-5), 157.1
quat., C-8a), 160.7 (quat., C-2), 165.2 (quat., C-7); MS (ESI)
(
1
0
8
3
+
−1
m/z 202.1 (MH ).
18.96%]; νmax/cm 3189 (NH), 1694 (CvO), 1603 (amide I),
2584 | Org. Biomol. Chem., 2012, 10, 2578–2589
This journal is © The Royal Society of Chemistry 2012

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1
1
484 (amide II); H NMR (300 MHz, DMSO-d ) δ 2.19 (3H,
(CH-5), 138.7 (CH-4), 147.4 (quat., C-7), 154.3 (quat., C-2),
6
H
+
s, CH ), 7.56 (1H, d, J = 8.4 Hz, CH-6), 8.39–8.48 (3H, m,
CH-3 and CH-4 and CH-5), 11.11 (1H, br s, NH); C NMR
154.5 (quat., C-8a), 169.9 (CvO); MS (ESI) m/z 296.2 (MH ),
3
1
3
+
+
318.2 (MNa ); HRMS (NSI) calcd for (C H N O) 296.1757,
18 22 3
(
75 MHz, DMSO-d ) δ 24.7 (CH ), 115.6 (CH), 119.4 (quat.),
found 296.1756.
6
C
3
1
21.9 (CH-6), 140.1 (CH), 140.8 (CH), 153.2 (quat.), 154.6
4
.1.5 General procedure for the reduction of 2-acetylamino-
(
(
quat.), 155.6 (quat.), 170.8 (quat., CvO); MS (ESI) m/z 222.0
MH ), 244.0 (MNa ).
+
+
7-(alk-1-yn-1-yl)-1,8-naphthyridine 13. The catalyst, 10 mol%
10 w/w%) Pd on charcoal, was added to a solution of 2-acetyla-
mino-7-(alk-1-yn-1-yl)-1,8-naphthyridine 13 in MeOH and the
mixture was stirred under H (1 atm) at ambient temperature.
After H absorption was complete, the catalyst was removed by
(
4
.1.4 General procedure for the synthesis of 2-acetylamino-
7
-(alk-1-yn-1-yl)-1,8-naphthyridine 13. To a solution of 2-acetyl-
2
amino-7-chloro-1,8-naphthyridine 12 in anhydrous DMF, an
2
excess of Et N was added, followed by 4 mol% Pd(PPh ) Cl
2
filtration and the filtrate was concentrated to dryness. Trituration
with diethylether gave the saturated product 14 as a solid.
3
3 2
and 4 mol% CuI under N at room temperature. The resulting
2
mixture was stirred for 15 minutes at 80 °C. One equivalent of
4.1.5.1 2-Acetylamino-7-pentyl-1,8-naphthyridine 14a. 2-Acet-
ylamino-7-(pent-1-yn-1-yl)-1,8-naphthyridine 13a (1.26 g,
5.00 mmol) was reduced to give the product as a yellow solid
after trituration with diethyl ether (1.07 g, 4.20 mmol, 84%); mp
127–130 °C; [Found: C, 69.65; H, 7.40; N, 16.25. C H N O
1
-alkyne in DMF was then added dropwise. The resulting solu-
tion was stirred at 80 °C for 24 hours and then the volatiles were
removed under reduced pressure. The residue was dissolved in
dichloromethane and passed through a short plug of Celite
which was washed with ethyl acetate. The organic phase was
evaporated to dryness and the residue purified by column chrom-
atography (50% petroleum ether, 50% ethyl acetate) to give 13.
15 19 3
−1
requires C, 70.01; H, 7.44; N, 16.33%]; νmax/cm 3176, 2926
1
(NH), 1701 (CvO), 1605 (amide I), 1503 (amide II); H NMR
(300 MHz, DMSO-d ) δ 0.88 (3H, t, J = 6.9 Hz, CH -5′), 1.33
6
H
3
4
.1.4.1 2-Acetylamino-7-(pent-1-yn-1-yl)-1,8-naphthyridine 13a.
(4H, m, CH
2
-3′ and 4′), 1.79 (2H, quint., J = 7.5 Hz, CH
-2′),
2
From the reaction of 2-acetylamino-7-chloro-1,8-naphthyridine
2.18 (3H, s, CH
3
CO), 2.92 (2H, t, J = 7.5 Hz, CH -1′), 7.40
2
1
0
0
2 (0.20 g, 0.90 mmol), Et N (1.90 mL), Pd(PPh ) Cl (25 mg,
.04 mmol), CuI (7 mg, 0.04 mmol) and 1-pentyne (61 mg,
.90 mmol), after column chromatography (33% petroleum
(1H, d, J = 8.1 Hz, CH-6), 8.24 (1H, d, J = 8.1 Hz, CH-5), 8.31
(1H, d, J = 8.7 Hz, CH-3), 8.36 (1H, d, J = 8.7 Hz, CH-4),
3
3 2
2
13
10.98 (1H, s, NH); C NMR (75 MHz, DMSO-d
(CH , C-5′), 21.9 (CH , C-4′), 24.1 (CH , CH CO), 28.1 (CH
C-2′), 30.9 (CH , C-3′), 38.1 (CH , C-1′), 113.8 (CH-3), 118.1
) δ 13.9
6
C
ether, 66% ethyl acetate) and recrystallization, 13a was isolated
as a white powder (0.19 g, 0.73 mmol, 81%); mp 182–184 °C;
3
2
3
3
,
2
2
2
[
7
(
Found: C, 70.91; H, 6.03; N, 16.61. C H N O requires C,
(quat., C-4a), 120.7 (CH-6), 136.7 (CH-5), 139.1 (CH-4), 153.9
(quat., C-2), 154.4 (quat., C-8a), 165.9 (quat., C-7), 169.9 (quat.,
CvO); MS (ESI) m/z 258.1 (MH ).
1
5 15 3
−1
1.13; H, 5.97; N, 16.59%]; νmax/cm 2957 (broad NH), 2217
1
+
CuC), 1702 (CvO), 1596 (amide I), 1499 (amide II);
H
NMR (300 MHz, CDCl ) δH 1.12 (3H, t, J = 7.2 Hz, CH -5′),
4.1.5.2 2-Acetylamino-7-octyl-1,8-naphthyridine14b. 2-Acetyl-
3
3
1
2
8
8
.73 (2H, sextet, J = 7.2 Hz, CH -4′), 2.37 (3H, s, CH CO),
amino-7-(oct-1-yn-1-yl)-1,8-naphthyridine
13b
(0.91
g,
2
3
.51 (2H, t, J = 7.2 Hz, CH -3′), 7.47 (1H, d, J = 8.7 Hz, CH-6),
3.10 mmol) was reduced by the method above using 10% Pd on
charcoal (0.66 g). The white product 14b was obtained as a
mixture after trituration with diethyl ether (0.75 g, 2.51 mmol,
81%), and was used without further purification; mp 86–88 °C;
[Found: C, 72.36; H, 8.03; N, 13.97. C H N O requires C,
2
.07 (1H, d, J = 8.7 Hz, CH-5), 8.19 (1H, d, J = 8.7 Hz, CH-4),
1
3
.55 (1H, d, J = 8.7 Hz, CH-3), 9.71 (1H, br s, NH); C NMR
(75 MHz, CDCl ) δ 13.6 (CH , C-5′), 21.5 (CH , C-3′), 21.8
3 C 3 2
(CH , C-4′), 25.1 (CH , CH CO), 81.2 (quat., C-1′), 93.8 (quat.,
2
3
3
18 25 3
−1
C-2′), 115.7 (CH-3), 119.5 (quat., C-4a), 124.1 (CH-6), 136.4
CH-5), 138.9 (CH-4), 147.5 (quat., C-7), 154.6 (2 × quat., C-2
72.21; H, 8.42; N, 14.03%]; νmax/cm 3189, 2925 (NH), 1697
(CvO), 1603 (amide I), 1500 (amide II); MS (ESI) m/z 298.2,
(
+
+
and C-8a), 170.3 (quat., CvO); MS (ESI) m/z 254.1 (MH ),
300.2 (MH ).
+
2
76.1 (MNa ).
.1.4.2 2-Acetylamino-7-(oct-1-yn-1-yl)-1,8-naphthyridine 13b.
Using 2-acetylamino-7-chloro-1,8-naphthyridine 12 (1.00 g,
4
.1.5.3 2-Acetylamino-7-octyl-5,6-dihydro-1,8-naphthyridine 14c.
4
13b (0.81 g, 2.76 mmol) was reduced using 10% Pd/C
(0.7 g) in MeOH–EtOAc mixture under 1.8 bar H2 pressure
overnight. The catalyst was removed by filtration through Celite
and washing with MeOH. After evaporation of the solvent, the
crude product was purified by gradient column chromatography
(20% ethyl acetate, 80% petroleum ether to 50% ethyl acetate,
50% petroleum ether) to obtain a pale yellow sticky solid
4
0
4
.50 mmol), Et N (9.40 mL), Pd(PPh ) Cl (0.15 g,
3 3 2 2
.22 mmol), CuI (42 mg, 0.22 mmol) and 1-octyne (0.49 g,
.50 mmol), 13b was obtained (1.18 g, 4.00 mmol, 89% yield);
−1
mp 148–150 °C; νmax/cm 3327, 2928 (NH), 2226 (CuC),
1
1
679 (CvO), 1597 (amide I), 1503 (amide II); H NMR
1
(300 MHz, CDCl ) δH 0.83 (3H, t, J = 6.9 Hz, CH -8′), 1.25
(0.60 g, 1.98 mmol, 71%); H NMR (300 MHz, CDCl ) δ 0.83
3
3
3
H
(
4H, m, CH -6′ and 7′), 1.42 (2H, quint., J = 6.9 Hz, CH -5′),
(3H, t, CH ), 1.27–1.82 (14H, m, 7 × CH ), 1.90–1.96 (2H, m,
3 2
2
2
1
2
8
8
.59 (2H, quint., J = 6.9 Hz, CH -4′), 2.27 (3H, s, CH CO),
CH ), 2.11 (3H, s, CH CO), 2.65–2.69 (2H, m, CH ), 3.31–3.39
2 3 2
2
3
.42 (2H, t, J = 6.9 Hz, CH -3′), 7.42 (1H, d, J = 8.4 Hz, CH-6),
(1H, m, CH), 4.59 (1H, br, NH), 7.15 (1H, d, J = 8.1 Hz, CH),
2
13
.02 (1H, d, J = 8.1 Hz, CH-5), 8.14 (1H, d, J = 8.8 Hz, CH-4),
7.32 (1H, br, CH), 7.67 (1H, br, C(O)NH); C NMR (75 MHz,
13
.51 (1H, d, J = 8.1 Hz, CH-3), 9.82 (1H, broad, NH);
C
CDCl ) δC 13.9 (CH ), 22.5 (CH ), 24.5 (CH , CH CO), 24.9
3
3
2
3
3
NMR (75 MHz, CDCl ) δ 13.9 (CH , C-8′), 19.4 (CH , C-3′),
(CH ), 25.5 (CH ), 27.4 (CH ), 29.1 (CH ), 29.4 (CH ), 29.5
2 2 2 2 2
3
C
3
2
2
2.4 (CH , C-7′), 24.9 (CH , CH CO), 28.1 (CH , C-4′), 28.5
(CH ), 31.7 (CH ), 36.4 (CH ), 51.4 (CH), 101.9 (CH), 111.8
2 2 2
2
3
3
2
(
CH , C-5′), 31.2 (CH , C-6′), 80.9 (quat., C-1′), 93.7 (quat.,
(quat.), 138.1 (CH), 147.8 (quat.), 154.4 (quat.), 167.9 (quat.),
168.0 (quat., CvO); MS (ESI) m/z 304.5 (MH ).
2
2
+
C-2′), 115.3 (CH-3), 119.3 (quat., C-4a), 123.9 (CH-6), 136.2
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 2578–2589 | 2585

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+
4
.1.6 General procedure for the synthesis of 2-amino-7-(alk-
-yn-1-yl)-1,8-naphthyridine 3d–g. The N-acetyl protected ami-
nonaphthyridines 13, 14 were stirred in MeOH with an excess of
M NaOH for 3–5 hours at room temperature. After concen-
72%); HRMS (NSI) calcd for (C H N ) 258.1965, found
16 24 3
1
258.1964; crude 3g was used directly for the preparation of 6g.
t
4
.1.7 Preparation of activated ester butyl-2-((perfluorophe-
noxy)carbonyl)ethylcarbamate 19. Boc-β-alanine (1.89 g,
0.00 mmol) and pentafluorophenol (2.00 g, 11.00 mmol) were
dissolved in ethyl acetate and cooled in an ice bath. To the
stirred solution, N,N-dicyclohexyl carbodiimide (2.30 g,
1
5
t
tration under vacuum, the residue was taken up into ethyl acetate
and extracted with water. The organic layer was dried over
MgSO and evaporated to dryness. The isolated material was
recrystallized from ethyl acetate and petrol.
4
1
4
1
1.00 mmol) was added. After 1 hour at 0 °C, the precipitated N,
.1.6.1 2-Amino-7-(pent-1-yn-1-yl)-1,8-naphthyridine 3d. The
N-dicyclohexylurea was collected by filtration. The filtrate was
concentrated in vacuo and the resulting white solid was treated
with petroleum ether. After standing at low temperature, colour-
less crystals were collected by filtration (3.00 g, 8.50 mmol,
deprotection of 2-acetylamino-7-(pent-1-yn-1-yl)-1,8-naphthyri-
dine 13a (0.14 g, 0.55 mmol) gave 3d as a white powder after
recrystallization (0.09 g, 0.44 mmol, 80%); mp 162–164 °C;
−1
1
νmax/cm 3468, 3117 (NH), 2223 (CuC); H NMR (300 MHz,
CDCl ) δ 1.05 (3H, t, J = 7.2 Hz, CH -5′), 1.66 (2H, sextet, J
−1
8
1
2
5%); mp 62–64 °C; νmax/cm 2357, 1789, 1681 (CvO),
3
H
3
1
517; H NMR (300 MHz, CDCl ) δ 1.49 (9H, s, (CH ) C),
=
7.2 Hz, CH -4′), 2.44 (2H, t, J = 7.2 Hz, CH -3′), 5.85 (2H, br
3
H
3 3
2
2
.96 (2H, t, J = 6.3 Hz, CH CO ), 3.56 (2H, q, J = 6.3 Hz,
s, NH ), 6.80 (1H, d, J = 8.7 Hz, CH-3), 7.19 (1H, d, J = 8.1
2
2
2
13
CH NH), 5.03 (1H, br s, NH); C NMR (75 MHz, CDCl ) δC
Hz, CH-6), 7.72 (1H, d, J = 8.7 Hz, CH-4), 7.78 (1H, d, J = 8.1
2
3
13
2
8.3 ((CH ) C), 33.9 (CH , d, J = 8.8 Hz, CH CO ), 36.1 (CH ,
3 3 2 2 2 2
Hz, CH-5); C NMR (75 MHz, CDCl ) δC 13.6 (CH , C-5′),
3
3
CH NH), 79.8 (quat., C(CH ) ), 124.9 (quat., m, C ), 137.9
2
1.5 (CH , C-3′), 21.9 (CH , C-4′), 81.4 (quat., C-1′), 92.4
2
3 3
Ar
2
2
(
quat., m, C ), 141.3 (quat., m, C ), 142.9 (quat., m, C ),
(quat., C-2′), 113.1 (CH-3), 116.5 (quat., C-4a), 121.5 (CH-6),
Ar Ar Ar
1
55.8 (quat., NHCO ), 168.4 (quat., CH CO ).
2 2 2
1
36.2 (CH-5), 137.7 (CH-4), 146.2 (quat., C-7), 156.1 (quat.,
+
C-8a), 160.1 (quat., C-2); MS (ESI) m/z 212.1 (MH ); HRMS
+
4.1.8 General procedure for the coupling of the substituted
(NSI) calcd for (C H N ) 212.1182, found 212.1181.
13 14 3
t
2
-amino-1,8-naphthyridine 3 and Boc-β-alanine. To a solution
4
.1.6.2 2-Amino-7-(oct-1-yn-1-yl)-1,8-naphthyridine 3e. 2-
Acetylamino-7-(oct-1-yn-1-yl)-1,8-naphthyridine 13b (1.47 g,
.00 mmol) was deprotected using the method above and
resulted in 3e as a white powder (1.10 g, 4.40 mmol, 88%); mp
12–114 °C; [Found: C, 75.81; H, 7.59; N, 16.70. C H N
6 19 3
of the activated ester 19 in dry DMF, one equivalent of substi-
tuted 2-amino-1,8-naphthyridine 3 and 1 equivalent of N,N-dii-
sopropylethylamine were added. The mixture was stirred at
5
4
0 °C for 24 hours. The solvent was evaporated to dryness and
1
1
−
1
the crude residue was purified by silica gel column chromato-
graphy. Recrystallization from ethyl acetate and petroleum ether
gave the protected amino acid derivatives 21 as a solid.
requires C, 75.85; H, 7.56; N, 16.59%]; νmax/cm 3479, 3282,
3
1
117, 2959 (NH), 2221(CuC), 1628, 1388;
H NMR
(
300 MHz, CDCl ) δH 0.89 (3H, t, J = 6.9 Hz, CH -8′), 1.30
3
3
t
4
.1.8.1 2-N-(N′-( Butoxycarbonyl)-β-alanyl)-amino-5,7-dimethyl-
(
4H, m, CH -6′ and 7′), 1.47 (2H, quint., J = 6.9 Hz, CH -5′),
2
2
5
1
(
3
(
,8-naphthyridine 21a By the reaction of activated ester 19
1.00 g, 2.80 mmol), 2-amino-5,7-dimethyl-1,8-naphthyridine
(0.48 g, 2.80 mmol) and N,N-diisopropylethylamine
0.48 mL, 2.80 mmol) the title product was obtained as a white
solid (0.83 g, 2.40 mmol, 86%); mp 205–208 °C; [Found: C,
1
.64 (2H, quint., J = 6.9 Hz, CH -4′), 2.52 (2H, t, J = 6.9 Hz,
2
CH -3′), 6.56 (2H, br, NH ), 7.01 (1H, d, J = 8.7 Hz, CH-3),
2
2
13
a
7
.18 (1H, d, J = 7.8 Hz, CH-6), 7.74 (2H, m, CH-5 and 4);
C
NMR (75 MHz, CDCl ) δ 14.1 (CH , C-8′), 19.6 (CH , C-3′),
3
C
3
2
2
2.6 (CH , C-7′), 28.4 (CH , C-6′), 28.7 (CH , C-5′), 31.4 (CH ,
2 2 2 2
6
7
2.76; H, 7.07; N, 16.06. C H N O requires C, 62.77; H,
18 24 4 3
C-4′), 80.7 (quat., C-1′), 93.8 (quat., C-2′), 113.9 (CH-3), 116.3
−1
.02; N, 16.27%]; νmax/cm 3206, 3121, 2927 (NH), 1678
(
(
quat., C-4a), 121.7 (CH-6), 136.3 (CH-5), 137.8 (CH-4), 145.9
1
(CvO), 1599 (amide I), 1508 (amide II); H NMR (300 MHz,
quat., C-7), 151.3 (quat., C-8a), 159.9 (quat., C-2); MS (ESI)
+
CDCl ) δ 1.43 (9H, s, (CH ) C), 2.63 (3H, s, CH ), 2.67 (3H,
m/z 254.1 (MH ).
3
H
3 3
3
s, CH ), 2.80 (2H, br, CH -2′), 3.53 (2H, br, CH -3′), 5.44 (1H,
4
.1.6.3 2-Amino-7-(pentyl)-1,8-naphthyridine
3f. 2-Acetyl-
3
2
2
br s, NHCO ), 7.08 (1H, s, CH-6), 8.28 (1H, d, J = 9.0 Hz,
amino-7-(pentyl)-1,8-naphthyridine 14a (0.56 g, 2.20 mmol)
was deprotected, resulting in 3f as a white powder (0.43 g,
2
CH-3), 8.43 (1H, d, J = 9.0 Hz, CH-4), 10.1 (1H, br s, NHCO);
13
−
1
C NMR (75 MHz, CDCl ) δC 18.0 (CH ), 25.4 (CH ), 28.5
2
1
.00 mmol, 92%); mp 111–113 °C; νmax/cm 3146, 2923 (NH),
3
3
3
1
(
(
(
(CH ) C), 36.3 (CH , C-2′ or 3′), 37.4 (CH , C-3′ or 2′), 79.3
3 3 2 2
595, 1509, 1381; H NMR (300 MHz, CDCl ) δ 0.87 (3H, t,
3
H
quat., C(CH ) ), 114.0 (CH-3), 118.6 (quat., C-4a), 122.4
J = 7.2 Hz, CH -5′), 1.33–1.38 (4H, m, CH -4′ and 3′), 1.80
3 3
3
2
CH-6), 135.6 (CH-4), 145.3 (quat., C-5), 153.4 (quat., C-2),
(
2H, quint., J = 7.5 Hz, CH -2′), 2.90 (2H, t, J = 7.5 Hz, CH -
2
2
t
1
54.4 (quat., CvO, Boc), 155.9 (quat., C-8a), 162.9 (quat.,
1
′), 6.50 (2H, br, NH ), 6.94 (1H, d, J = 8.7 Hz, CH-3), 7.07
2
+
13
C-7), 171.8 (quat., C-1′); MS (ESI) m/z 345.2 (MH ), 367.2
(
(1H, d, J = 8.1 Hz, CH-6), 7.76–7.82 (2H, m, CH-4 and 5);
C
+
MNa ).
NMR (75 MHz, CDCl ) δ 14.0 (CH , C-5′), 22.6 (CH , C-4′),
3
C
3
2
t
2
9.5 (CH , C-2′), 31.6 (CH , C-3′), 38.8 (CH , C-1′), 112.6
2 2 2
4.1.8.2 2-N-(N′-( Butoxycarbonyl)-β-alanyl)-amino-5,7-diethyl-
(
(
CH-3), 115.3 (quat., C-4a), 118.7 (CH-6), 136.5 (CH-5), 138.3
1,8-naphthyridine 21b. By the reaction of activated ester 19
(1.00 g, 2.80 mmol), 2-amino-5,7-diethyl-1,8-naphthyridine 3b
(0.56 g, 2.80 mmol) and N,N-diisopropylethylamine (0.48 mL,
2.80 mmol), the title product was obtained as a white solid
CH-4), 154.7 (quat., C-8a), 159.4 (quat., C-2), 166.2 (quat.,
+
C-7); MS (ESI) m/z 216.2 (MH ); HRMS (NSI) calcd for
(
+
C H N ) 216.1495, found 216.1491.
13 18 3
−
1
4
.1.6.4 2-Amino-7-(octyl)-1,8-naphthyridine 3g. The deprotec-
(0.64 g, 1.70 mmol, 62%); mp 183–186 °C; νmax/cm 3359,
3201, 2967 (NH), 1677 (CvO), 1594 (amide I), 1507 (amide
tion of 14b (0.21 g, 0.70 mmol) gave 3g as a yellow oil, which
was purified by trituration with diethyl ether (0.13 g, 0.50 mmol,
1
II); H NMR (300 MHz, CDCl ) δ 1.28–1.35 (6H, m, 2 ×
3
H
2586 | Org. Biomol. Chem., 2012, 10, 2578–2589
This journal is © The Royal Society of Chemistry 2012

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t
CH ), 1.36 (9H, s, (CH ) C), 2.69 (2H, t, J = 6.0 Hz, CH -2′),
4.1.8.5 2-N-(N′-( Butoxycarbonyl)-β-alanyl)-amino-(7-oct-1-yn-
1-yl)-1,8-naphthyridine 21e. The reaction of 2-amino-7-(oct-1-
yn-1-yl)-1,8-naphthyridine 3e (0.50 g, 1.97 mmol) and activated
ester 19 (0.70 g, 1.97 mmol) resulted in a pale yellow solid after
recrystallization from ethyl acetate (0.79 g, 1.85 mmol, 94%);
3
3 3
2
2
.87–3.02 (4H, m, 2 × CH ), 3.46 (2H, br, CH -3′), 5.25 (1H, br
2 2
s, NHCO ), 7.07 (1H, s, CH-6), 8.28 (1H, d, J = 9.0 Hz, CH-3),
2
13
8
.36 (1H, d, J = 9.0 Hz, CH-4), 9.32 (1H, br s, NHCO);
C
NMR (75 MHz, CDCl ) δ 13.4 (CH ), 14.3 (CH ), 24.7 (CH ),
3
C
3
3
2
−1
2
7
8.4 ((CH ) C), 32.2 (CH ), 36.3 (CH , C-3′), 37.5 (CH , C-2′),
mp 123–125 °C; νmax/cm 3369, 2928 (NH), 2225 (CuC),
1702 (CvO), 1675 (CvO), 1596 (amide I), 1499 (amide II); H
3
3
2
2
2
1
9.4 (quat., C(CH ) ), 113.9 (CH-3), 118.0 (quat., C-4a), 119.4
3
3
(
CH-6), 135.3 (CH-4), 151.3 (quat., C-5), 154.6 (quat., C-2 or
NMR (300 MHz, DMSO-d
8′′), 1.33 (4H, m, CH -5′′ and 6′′), 1.38 (9H, s, (CH
(2H, m, CH -7′′), 1.62 (2H, m, CH -4′′), 2.53 (2H, m, CH
2.65 (2H, t, J = 6.6 Hz, CH -2′), 3.28 (2H, q, J = 6.6 Hz, CH
3′), 6.83 (1H, br, NHCO ), 7.51 (1H, d, J = 8.1 Hz, CH-3),
.31–8.42 (3H, m, CH-4, 5 and 6), 10.99 (1H, br s, NHCO);
NMR (75 MHz, DMSO-d ) δC 14.4 (CH , C-8′′), 19.1 (CH ,
6
) δ
H
0.89 (3H, t, J = 6.9 Hz, CH
C), 1.46
-3′′),
3
-
t
C-8a), 155.9 (2 × quat., C-2 or C-8a and CvO, Boc), 167.9
(
3
3
2
)
3 3
+
quat., C-7), 171.5 (quat., C-1′); MS (ESI) m/z 373.3 (MH ),
2
2
2
+
+
95.3 (MNa ); HRMS (NSI) calcd for (C H O N )
2
-
2
2
0 29 3 4
73.2234, found 373.2233.
2
13
8
C
t
4
.1.8.3 2-N-(N′-( Butoxycarbonyl)-β-alanyl)-amino-5,7-ditrifluoro-
6
3
2
methyl-1,8-naphthyridine 21c. From the reaction of 2-amino-(5,7-
ditrifluoromethyl-1,8-naphthyridine) 3c (2.00 g, 7.10 mmol),
C-3′′), 22.5 (CH , C-7′′), 28.3 (CH , C-5′′), 28.5 (CH , C-4′′),
2
2
2
2
8.7 ((CH ) C), 31.2 (CH , C-6′′), 36.8 (CH , C-3′), 37.3 (CH ,
3 3 2 2 2
t
Boc-β-alanylpentafluorophenol ester 19 (2.80 g, 7.90 mmol)
C-2′), 78.1 (quat., C(CH ) ), 81.9 (quat., C-1′′), 93.4 (quat.,
3
3
and N,N-diisopropylethylamine (2.23 mL, 12.90 mmol), the
title compound was obtained after gradient column chromato-
graphy (66.6% petroleum ether, 33.3% ethyl acetate) giving a
yellow oil. Upon trituration with diethyl ether, the product 21c
was obtained as a white powder (1.00 g, 2.20 mmol, 31%); mp
1
1
(
(
C-2′′), 115.7 (CH-3), 119.6 (quat., C-4a), 124.1 (CH-6), 137.8
CH-5), 139.6 (CH-4), 146.8 (quat., C-7), 155.0 (quat., C-2 or
(
t
C-8a), 155.2 (quat., C-2 or C-8a), 156.0 (quat., CvO, Boc),
1
4
+
71.9 (quat., C-1′); HRMS (NSI) calcd for (C H O N )
24 33 3 4
25.2547, found 425.2547.
−1
27–128 °C; νmax/cm 3342 (NH), 1689 (CvO), 1675 (CvO),
t
1
4.1.8.6 2-N-(N′-( Butoxycarbonyl)-β-alanyl)-amino-7-pentyl-1,8-
587 (amide I), 1512 (amide II), 1271, 1140 (C–F); H NMR
naphthyridine 21f. Starting with 2-amino-7-pentyl-1,8-naphthyri-
dine 3f (0.35 g, 1.63 mmol), activated ester 19 (0.58 g,
300 MHz, DMSO-d ) δH 1.37 (9H, s, (CH ) C), 2.67
6 3 3
2H, t, J = 6.6 Hz, CH -2′), 3.28 (2H, q, J = 6.6 Hz, CH -3′),
2
2
1
1
.63 mmol) and N,N-diisopropylethylamine (0.28 mL,
.63 mmol), the title compound was obtained, after purification,
6
9
.79 (1H, br s, NHCO ), 8.26 (1H, s, CH-6), 8.65 (1H, d, J =
2
.3 Hz, CH-3), 8.71 (1H, d, J = 9.3 Hz, CH-4), 11.41 (1H, br s,
13
as a white solid (0.56 g, 1.45 mmol, 89%); mp 59–61 °C; νmax/
NHCO); C NMR (75 MHz, DMSO-d ) δC 28.7 ((CH ) C),
3
1
(
6
3 3
−1
cm 3372, 2925 (NH), 1709 (CvO), 1675 (CvO), 1606
6.6 (CH ), 37.47 (CH ), 78.1 (quat., C(CH ) ), 113.9 (CH-6),
2 2 3 3
1
(
amide I), 1506 (amide II); H NMR (300 MHz, CDCl ) δ 0.88
3 H
16.9 (quat.), 119.6 (CH-3), 121.7 (CF , q, J = 274 Hz), 122.9
3
(
3H, t, J = 7.2 Hz, CH -5′′), 1.25–1.40 (4H, m, CH -4′′ and 3′′),
3
2
CF , q, J = 273 Hz), 135.7 (CH-4), 136.8 (quat., q, J = 32.55
3
1
.43 (9H, s, (CH ) C), 1.84–1.88 (2H, m, CH -2′′), 2.76 (2H, t,
3 3 2
Hz, C-5 or 7), 149.6 (quat., q, J = 35.25, C-5 or 7), 154.8
quat.), 155.9 (quat.), 156.7 (quat., CvO), 172.4 (quat., CvO);
J = 6.0 Hz, CH -2′), 3.02 (2H, t, J = 7.8 Hz, CH -1′′), 3.53 (2H,
2
2
(
+
q, J = 6.0 Hz, CH
2
-3′), 5.17 (1H, br, NHCO
2
), 7.33 (1H, d, J =
HRMS (NSI) calcd for (C H N O F ) 453.1356, found
1
8 19 4 3 6
8
9
.1 Hz, CH-6), 8.08 (1H, d, J = 8.1 Hz, CH-5), 8.18 (1H, d, J =
.0 Hz, CH-3), 8.46 (1H, d, J = 9.0 Hz, CH-4), 9.54 (1H, br,
4
53.1355.
.1.8.4 2-N-(N′-( Butoxycarbonyl)-β-alanyl)-amino-7-(pent-1-
yn-1-yl)-1,8-naphthyridine 21d. By the reaction of 2-amino-7-
pent-1-yn-1-yl)-1,8-naphthyridine 3d (0.10 g, 0.47 mmol) and
the activated ester 19 (0.17 g, 0.47 mmol), the title compound
1d was obtained as a yellow solid (0.15 g, 0.40 mmol, 86%);
mp 149–151 °C; [Found: C, 65.81; H, 6.80; N, 14.57.
t
13
4
NHCO); C NMR (75 MHz, CDCl ) δ 13.9 (CH , C-5′′), 22.5
3 C 3
(CH , C-4′′), 28.4 ((CH ) C), 29.1 (CH , C-2′′), 31.6 (CH ,
2
3 3
2
2
(
C-3′′), 36.7 (CH , C-3′), 37.8 (CH , C-2′), 38.8 (CH , C-1′′),
2 2 2
79.5 (quat., C(CH ) ), 114.6 (CH-3), 118.8 (quat., C-4a), 121.5
3
3
2
(CH-6), 137.1 (CH-5), 139.7 (CH-4), 153.1 (quat., C-2), 153.3
t
(quat., C-8a), 155.9 (quat., CvO, Boc), 167.5 (quat., C-7),
−1
+
+
C H N O requires C, 65.95; H, 6.85; N, 14.65%]; νmax/cm
3
1
172.5 (quat., C-1′); MS (ESI) m/z 387.3 (MH ), 409.3 (MNa );
2
1 26 4 3
+
210, 3120, 2966, 2933 (NH), 2227 (CuC), 1700 (CvO),
HRMS (NSI) calcd for (C H O N ) 387.2391, found
21 31 3 4
1
684 (CvO), 1597 (amide I), 1498 (amide II); H NMR
387.2384.
(
300 MHz, DMSO-d ) δ 1.09 (3H, t, J = 7.5 Hz, CH -5′′), 1.42
6 H 3
4
.1.9 General procedure for the preparation of the substi-
(9H, s, (CH ) C), 1.68 (2H, sextet, J = 7.1 Hz, CH -4′′), 2.56
3
3
2
tuted 3-amino-N-(1,8-naphthyridin-2-yl)propanamide trifluoro-
acetate 22. To the Boc protected derivatives 21 (1.00 mmol),
(2H, t, J = 7.1 Hz, CH -3′′), 2.69 (2H, t, J = 6.9 Hz, CH -2′),
2
2
t
3
.31 (2H, q, J = 6.9 Hz, CH -3′), 6.87 (1H, br t, NHCO ), 7.56
2 2
neat TFA (8 mL) was added. After stirring at room temperature
for an hour, the reaction mixture was concentrated under reduced
pressure and the residue was taken up into methanol. The solid
crude product was obtained by trituration with diethyl ether at
low temperature.
(
1H, d, J = 8.1 Hz, CH-3), 8.37 (1H, d, J = 8.1 Hz, CH-4), 8.42
2H, 2 × d, J = 8.7 Hz, CH-5 and CH-6), 11.04 (1H, s, NHCO);
(
1
3
C NMR (75 MHz, DMSO-d ) δC 13.8 (CH , C-5′′), 21.0
6
3
(
CH , C-4′′), 21.8 (CH , C-3′′), 28.7 ((CH ) C), 36.7 (CH ,
2 2 3 3 2
C-3′), 37.3 (CH , C-2′), 78.1 (quat., C(CH ) ), 82.1 (quat.,
2
3 3
C-1′′), 93.2 (quat., C-2′′), 115.7 (CH-3), 119.6 (quat., C-4a),
4.1.9.1 3-Amino-N-(5,7-dimethyl-1,8-naphthyridin-2-yl)propan-
amide trifluoroacetate 6a. Deprotection of derivative 21a (0.34 g,
1.00 mmol) resulted in the title compound 6a (0.30 g,
0.83 mmol, 83%) after recrystallization from isopropyl alcohol;
mp 238–239 °C; [Found: C, 49.97; H, 4.69; N, 15.52.
1
24.1 (CH-6), 137.8 (CH-5), 139.6 (CH-4), 146.8 (quat., C-7),
1
55.0 (quat., C-2), 155.2 (quat., C-8a), 155.9 (quat., CvO,
t
+
Boc), 171.9 (quat., C-1′); MS (ESI) m/z 383.2 (MH ), 405.2
+
(MNa ).
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 2578–2589 | 2587

View Online
C H F N O requires C, 50.28; H, 4.78; N, 15.64%]; νmax/
DMSO-d ) δC 13.8 (CH -5′′), 21.0 (CH -3′′), 21.8 (CH -4′′),
34.2 (CH -2′), 35.1 (CH -3′), 82.0 (quat., C-1′′), 93.4 (quat.,
2 2
1
5
−
17
3
4
3
6
3
2
2
1
cm 3263, 3084 (NH), 1674 (CvO), 1596 (amide I), 1508
1
(
amide II); H NMR (300 MHz, DMSO-d ) δ 2.63 (3H, s,
C-2′′), 115.6 (CH-3), 119.7 (quat., C-4a), 124.3 (CH-6), 137.9
(CH-5), 139.9 (CH-4), 146.9 (quat., C-7), 154.9 (quat., C-2),
155.0 (quat., C-8a), 170.8 (quat., CvO); MS (ESI) m/z 283.1
6
H
CH ), 2.65 (3H, s, CH ), 2.9 (2H, t, J = 6.9 Hz, CH -2′), 3.16
3
3
2
+
(
2H, br, CH -3′), 7.3 (1H, s, CH-6), 7.93 (3H, br s, NH ), 8.3
2 3
+
+
(
1
1H, d, J = 9.0 Hz, CH-2), 8.54 (1H, d, J = 9.0 Hz, CH-3),
(MH ); HRMS (NSI) calcd for (C H N O) 283.1553, found
16 19 4
1
3
1.18 (1H, br s, NHCO); C NMR (75 MHz, DMSO-d ) δC
283.1557.
6
1
8.1 (CH ), 25.2 (CH ), 34.10 (CH , C-2′), 35.14 (CH , C-3′),
4.1.9.5 3-Amino-N-(7-(oct-1-yn-1-yl)-1,8-naphthyridin-2-yl)pro-
panamide trifluoroacetate 6e. Deprotection of derivative 21e
(0.52 g, 1.23 mmol) resulted in the title compound 6e as a
brown solid (0.47 g, 1.07 mmol, 87%); mp 189–191 °C; νmax/
3
3
2
2
1
1
14.1 (CH-3), 118.5 (quat., C-4a), 122.5 (CH-6), 136.6 (CH-4),
46.9 (quat., C-5), 154.0 (quat., C-2 or C-8a), 154.3 (quat., C-2
or C-8a), 162.3 (quat., C-7), 170.6 (quat., CvO); MS (ESI) m/z
+
+
−1
2
45.3 (MH ), 267.0 (MNa ).
.1.9.2 3-Amino-N-(5,7-diethyl-1,8-naphthyridin-2-yl)propan-
amide trifluoroacetate 6b. Deprotection of derivative 21b (0.37 g,
.00 mmol) resulted in the title compound 6b as a yellow solid
after recrystallization from isopropyl alcohol (0.29 g, 0.76 mmol,
cm 3235, 2924 (NH), 2224 (CuC), 1672 (CvO), 1596
1
4
(amide I), 1503 (amide II); H NMR (300 MHz, DMSO-d ) δ
6
H
0.89 (3H, t, J = 6.9 Hz, CH -8′′), 1.33 (4H, m, CH -6′′ and 7′′),
3
2
1
1.47 (2H, m, CH -4′′ or 5′′), 1.60 (2H, m, CH -4′′ or 5′′), 2.53
2 2
(2H, t, J = 6.9 Hz, CH -3′′), 2.91 (2H, t, J = 6.6 Hz, CH -2′),
2
2
−
1
7
6%); mp 227–229 °C; νmax/cm 3253, 2970 (NH), 1677
3.15 (2H, broad, CH -3′), 7.51 (1H, d, J = 8.1 Hz, CH-6), 7.87
2
1
+
(CvO), 1597 (amide I), 1510 (amide II); H NMR (300 MHz,
(3H, broad, NH ), 8.33 (2H, m, CH-3 and 5), 8.42 (1H, d, J =
3
13
DMSO-d ) δH 1.19–1.26 (6H, m, 2 × CH ), 2.76–2.86 (4H, m,
9.0 Hz, CH-4), 11.24 (1H, s, NHCO); C NMR (75 MHz,
6
3
2
× CH ), 2.97 (2H, q, J = 7.5 Hz, CH ), 3.06 (2H, t, J = 6.9
DMSO-d ) δ 13.9 (CH ), 18.6 (CH , C-3′′), 21.9 (CH , C-6′′ or
7′′), 27.7 (CH , C-4′′ or 5′′), 27.9 (CH , C-4′′ or 5′′), 30.7 (CH ,
2 2 2
2
2
6
C
3
2
2
+
Hz, CH -3′), 7.19 (1H, s, CH-6), 7.77 (3H, br, NH ), 8.21 (1H,
2
3
d, J = 9.0 Hz, CH-3), 8.49 (1H, d, J = 9.0 Hz, CH-4), 11.11
C-7′′ or 6′′), 33.7 (CH , C-2′), 34.6 (CH , C-3′), 81.4 (quat.,
2 2
13
(
1H, br s, NHCO); C NMR (75 MHz, DMSO-d ) δC 13.3
C-1′′), 93.1 (quat., C-2′′), 115.1 (CH-3), 119.2 (quat., C-4a),
123.8 (CH-6), 137.4 (CH-5), 139.4 (CH-4), 146.4 (quat., C-7),
154.4 (quat., C-2), 154.5 (quat., C-8a), 170.3 (quat., CvO); MS
6
(
CH ), 14.7 (CH ), 24.4 (CH ), 31.53 (CH ), 34.1 (CH , C-2′),
3
3
2
2
2
3
5.1 (CH , C-3′), 114.1 (CH-3), 117.8 (quat., C-4a), 119.6
2
+
+
(CH-6), 136.2 (CH-4), 152.3 (quat., C-2 or C-8a), 153.9 (quat.,
(ESI) m/z 325.2 (MH ), 347.2 (MNa ); HRMS (NSI) calcd for
+
C-5), 154.6 (quat., C-2 or C-8a), 166.9 (quat., C-7), 170.5 (quat.,
(C H N O) 325.2023, found 325.2022.
19 25 4
+
+
CvO); MS (ESI) m/z 273.1 (MH ), 295.1 (MNa ); HRMS
4.1.9.6 3-Amino-N-(7-pentyl-1,8-naphthyridin-2-yl)propanamide
trifluoroacetate 6f. The deprotection of 21f (0.34 g, 0.88 mmol)
resulted in the title compound 6f as a white solid (0.23 g,
0.57 mmol, 65%); mp 185–188 °C; [Found: C, 53.88; H, 5.87;
N, 13.88. C H F N O requires C, 53.99; H, 5.79; N,
+
(
NSI) calcd for (C H N O) 273.1710, found 273.1714.
15 21 4
4
.1.9.3 3-Amino-N-(5,7-ditrifluoromethyl-1,8-naphthyridin-2-yl)
propanamide trifluoroacetate 6c. By the deprotection of com-
pound 21c (0.50 g, 1.10 mmol) the title compound 6c was
obtained as a white powder after trituration with diethyl ether
1
8 23 3 4 3
−1
13.99%]; νmax/cm
(amide I), 1506 (amide II); H NMR (300 MHz, DMSO-d ) δ
3232, 2934 (NH), 1673 (CvO), 1610
−
1
1
(
(
(
0.43 g, 0.92 mmol, 84%); mp 162–169 °C; νmax/cm 3075
6
H
NH), 1673 (CvO), 1585 (amide I), 1512 (amide II), 1156
broad C-F); H NMR (300 MHz, D O) δ 3.06 (2H, t, J = 6.3
0.87 (3H, t, J = 6.6 Hz, CH -5′′), 1.30–1.35 (4H, m, CH -3′′ and
3
2
1
4′′), 1.79 (2H, quint., J = 7.2 Hz, CH -2′′), 2.85–2.94 (4H, m,
2
H
2
Hz, CH ), 3.42 (2H, t, J = 6.3 Hz, CH ), 8.25 (1H, s, CH-6),
CH -1′′ and 3′), 3.14 (2H, t, J = 6.6 Hz, CH -2′), 7.42 (1H, d, J
2
2
2
2
+
8
.31 (1H, d, J = 9.6 Hz, CH-3), 8.58 (1H, dd, J = 1.5 and 9.3
= 8.4 Hz, CH-6), 7.82 (3H, br, NH ), 8.25–8.30 (2H, m, CH-3
3
13
Hz, CH-4); C NMR (75 MHz, D O) δC 33.4 (CH ), 35.1
and 5), 8.38 (1H, d, J = 8.7 Hz, CH-4), 11.2 (1H, s, NHCO);
2
2
13
(
CH ), 114.9 (m, CH-6), 116.5 (quat., q, J = 273 Hz, CF ),
C NMR (75 MHz, DMSO-d ) δC 13.8 (CH , C-5′′), 21.9
6 3
2
3
117.5 (quat.), 118.8 (CH-3), 120.5 (quat., q, J = 273 Hz, CF3),
(CH , C-4′′), 28.1 (CH , C-2′′), 30.9 (CH , C-3′′), 33.6 (CH ,
2 2 2 2
1
36.3 (CH-4), 137.9 (quat., q, J = 33 Hz, C-5 or 7), 150.0
C-2′), 34.7 (CH , C-3′), 38.1 (CH , C-1′′), 113.8 (CH-3), 118.3
2 2
(
1
quat., q, J = 35 Hz, C-5 or 7), 153.8 (quat.), 154.9 (quat.),
(quat., C-4a), 120.9 (CH-6), 136.8 (CH-5), 139.3 (CH-4), 153.6
(quat., C-2 or 8a), 154.3 (quat., C-2 or 8a), 166.2 (quat., C-7),
170.0 (CvO); MS (ESI) m/z 287.3 (MH ).
+
+
71.8 (CvO); MS (ESI) m/z 353.1 (MH ), 375.1 (MNa );
+
+
HRMS (NSI) calcd for (C H N OF ) 353.0832, found
1
3
11
4
6
3
53.0832.
.1.9.4 3-Amino-N-(7-(pent-1-yn-1-yl)-1,8-naphthyridin-2-yl)
propanamide trifluoroacetate 6d. By the deprotection of 21d
1.24 g, 3.25 mmol) the title compound 6d was obtained as a
yellow solid (0.99 g, 2.51 mmol, 77%); mp 203–206 °C;
Found: C, 50.23; H, 4.40; N, 12.71. (C H N O) (CF COO)
4.1.9.7 3-Amino-N-(7-octyl-1,8-naphthyridin-2-yl)propanamide
trifluoroacetate 6g. Amine 3g (0.11g, 0.43 mmol) was dissolved
in DMF, N,N-diisopropylethylamine (0.074 mL, 0.43 mmol) was
added and the reaction mixture was stirred at 40 °C. The reaction
was quenched with water and extraction with ethyl acetate was
carried out. The organic layer was dried over Na SO , and the
4
(
[
1
6
19
4
2
3
3
2
4
−
1
t
requires C, 50.39; H, 4.23; N, 12.37%]; νmax/cm 3229, 3063
NH), 2231 (CuC), 1672 (CvO), 1597 (amide I), 1502 (amide
solvent was evaporated. The crude 2-N-(N′-( butoxycarbonyl)-
β-alanyl)-amino-7-octyl-1,8-naphthyridine 21g was obtained as
a yellow oil (0.10 g, 0.23 mmol, 54%) and used without further
purification. Deprotection of 21g resulted in title compound 6g
(0.06 g, 0.14 mmol, 59%) as a white powder; mp 216–220 °C;
[Found: C, 56.65; H, 6.66; N, 12.55. C H F N O requires C,
(
1
II); H NMR (300 MHz, DMSO-d ) δ 0.96 (3H, t, J = 7.2 Hz,
CH -5′′), 1.54 (2H, sextet, J = 7.2 Hz, CH -4′′), 2.41 (2H, t, J =
6
H
3
2
7
.2 Hz, CH -3′′), 2.82 (2H, t, J = 6.3 Hz, CH -2′), 3.06 (2H, br
2 2
t, J = 6.3 Hz, CH -3′), 7.45 (1H, d, J = 8.1 Hz, CH-6), 7.78 (3H,
br, NH ), 8.22–8.27 (2H, m, CH-3 and 5), 8.35 (1H, d, J = 9.0
Hz, CH-4), 11.15 (1H, br s, NHCO); C NMR (75 MHz,
2
21 29
−1
3 4 3
+
57.00; H, 6.61; N, 12.66%]; νmax/cm 3244, 2927 (NH), 1674
3
1
3
1
(CvO), 1609 (amide I), 1507 (amide II); H NMR (300 MHz,
2588 | Org. Biomol. Chem., 2012, 10, 2578–2589
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DMSO-d ) δH 0.87 (3H, m, CH -8′′), 1.10–1.34 (10H, m, 5 ×
to a suspension equivalent to 0.5 McFarland units using a densi-
tometer. 100 μl of this suspension was pipetted into the corre-
sponding wells of a multipoint inoculation device. Each set of
6
3
CH ), 1.79–1.83 (2H, m, CH -2′′), 2.78 (2H, t, J = 6.4 Hz, CH -
2
2
2
2
′), 2.94 (2H, t, J = 7.5 Hz, CH -1′′), 3.09 (2H, m, CH -3′), 7.43
2 2
+
5
(1H, d, J = 8.2 Hz, CH ), 7.81 (3H, NH ) 8.28 (1H, d, J = 8.2
plates received 1 μl of bacterial suspension, giving 1.5 × 10
Ar
3
Hz, CH ), 8.33 (1H, d, J = 8.8 Hz, CH ), 8.41 (1H, d, J = 8.8
organisms per spot on each inoculation. Twenty strains were
inoculated per plate and the plates were incubated for 18 h in air
at 37 °C.
Ar
Ar
1
3
Hz, CH ), 11.2 (1H, s, NHCO); C NMR (75 MHz, DMSO-
Ar
d6) δC 14.4 (CH , C-8′′), 22.5 (CH ), 28.9 (CH ), 29.1 (CH ),
3
2
2
2
2
3
9.2 (CH ), 29.3 (CH ), 31.7 (CH ), 36.2 (2 × CH , C-2′ and
2 2 2 2
4
.2.4 Activity determination. After incubation, the activity
′), 38.6 (CH ), 114.3 (CH-3), 118.6 (quat., C-4a), 121.3
2
of the microorganisms with the test substrates was determined by
observing the plates under UV irradiation at 365 nm and compar-
ing with the substrate-free control. The presence of fluorescence
emission was considered as positive evidence for the hydrolysis
of the substrate by β-alanylaminopeptidase.
(CH-6), 137.3 (CH-4 or 5), 139.7 (CH-4 or 5), 154.2 (quat., C-2
or 8a), 155.0 (quat., C-2 or 8a), 166.6 (quat., C-7), 171.3
+
+
(CvO); MS (ESI) m/z 329.3 (MH ), 351.3 (MNa ); HRMS
+
(NSI) calcd for (C H N O) 329.2336, found 329.2340.
19 29 4
4
.2 Biological testing
Acknowledgements
4
.2.1 Preparation of culture media containing substrates 6a–g.
We would like to show our appreciation for the support given by
bioMérieux through a PhD student scholarship (LV). We also
thank EPSRC National Mass Spectrometry Centre, University of
Wales, Swansea, for providing all accurate mass measurements.
1
L of Columbia agar was prepared as follows; 41 g of
Columbia agar (Oxoid Basingstoke, UK) was dissolved by
boiling in distilled water (1 L). The solution was then sterilized
by autoclaving at 116 °C for 20 min and left to cool at 50 °C.
1
0 mg of each substrate 6a–g to be tested was initially dissolved
in 200 μl DMSO and this was added to Columbia agar (100 mL)
and poured into sterile Petri dishes to give a final concentration
of 100 mg L− . Columbia agar plus DMSO was used as a
growth control. Solidified plates were surface dried in a warm air
cabinet for 5 min.
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This journal is © The Royal Society of Chemistry 2012
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