Synthesis and antibacterial activity of conjugates between norfloxacin and analogues of the siderophore vanchrobactin

Article abstract of DOI:10.1016/j.bmc.2012.10.028

From synthetic functionalized analogues of vanchrobactin, a siderophore produced by the fish pathogenic bacteria Vibrio anguillarum serotype O2, several vanchrobactin analogues-norfloxacin conjugates were obtained and their antimicrobial activities against the wild-type and mutant strains of Vibrio anguillarum serotype O2 have been determined.

Full text of DOI:10.1016/j.bmc.2012.10.028

Bioorganic & Medicinal Chemistry 21 (2013) 295–302
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and antibacterial activity of conjugates between norfloxacin
and analogues of the siderophore vanchrobactin
Alba Souto a, Marcos A. Montaos a, Miguel Balado b, Carlos R. Osorio b, Jaime Rodríguez a,
,
⇑
Manuel L. Lemos b, Carlos Jiménez a,
⇑
a Departamento de Química Fundamental, Facultade de Ciencias, Universidade de A Coruña, Campus da Zapateira, A Coruña 15071, Spain
b Department of Microbiology and Parasitology, Institute of Aquaculture, University of Santiago de Compostela, Campus Sur, Santiago de Compostela 15782, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 6 July 2012
Revised 12 October 2012
Accepted 13 October 2012
Available online 29 October 2012
From synthetic functionalized analogues of vanchrobactin, a siderophore produced by the fish pathogenic
bacteria Vibrio anguillarum serotype O2, several vanchrobactin analogues–norfloxacin conjugates were
obtained and their antimicrobial activities against the wild-type and mutant strains of Vibrio anguillarum
serotype O2 have been determined.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Vibrio anguillarum
Norfloxacin
Antibacterial compounds
Vanchrobactin
Siderophores
Trojan horse strategy
1. Introduction
ions to form a complex which is recognized by outer membrane
receptors and transported into the cell by an intricate system of
Vibriosis is a highly fatal hemorrhagic septicaemia affecting
marine and freshwater fish species throughout the world which
is mainly caused by some strains of the fish pathogen Vibrio anguil-
larum. This disease results in considerable economic losses in
aquaculture farming worldwide.1,2 The abusive and indiscriminate
use of broad spectrum antibiotics in aquaculture is causing serious
problems such as the emergence of pathogenic bacteria resistant to
most of the drugs, threats to human health via the food chain and
environmental problems due to the presence of antibiotic residues
in aquaculture products for human consumption. For all these rea-
sons, the search for more effective and specific treatments against
pathogenic bacteria is a priority in aquaculture.3,4
Iron is one of the most essential nutrients for all microorgan-
isms. However, iron bioavailabity is limited due to the low solubil-
ity of iron(III) at physiological pH. Thus, iron concentration in the
open sea is among the lowest observed in earth due to the slightly
basic pH of seawater.5 In response to the restricted access to solu-
ble iron, bacteria evolved high-affinity iron uptake pathways com-
prised of ferric iron specific carriers, termed siderophores. They are
excreted to the extracellular environment where they bind ferric
membrane-associated proteins. Finally, ferric reductases enzymes
can then liberate the internalized iron.6
In order to develop suitable targets for antimicrobial therapies,
several approaches have being applied using well known iron up-
take pathways.7–10 One of them, the siderophore-mediated drug
delivery (Trojan Horse strategy) facilitates the penetration of anti-
biotics into the bacterial cells transporting the antimicrobial drugs
across the bacterial membranes.11,12 This methodology, inspired in
the natural sideromycins,13,14 consists in the conjugation of a
known antibiotic to a siderophore analogue through a linker to en-
ter bacterial cells via active iron uptake pathways.15,16, In some
cases, the drug must be cleaved from the siderophore moiety.17
In previous studies, we identified vanchrobactin (1)18 as the
second siderophore-mediated system of V. anguillarum encoded
by a chromosomal gene cluster and its structure as well as a syn-
thesis19/regulation20 scheme was established. The other sidero-
phore-mediated iron uptake system previously described in V.
anguillarum is a plasmid (pJM1)-encoded siderophore named
anguibactin which is uniquely found in pJM1-containing serotype
O1 strains.21 The synthesis of a series of vanchrobactin analogues
allowed us to identify two of them, compounds 2 and 3, which
maintain the siderophore activity to V. anguillarum and bear an
appropriate functionality (an amino group) that can be used as an-
chor group to attach it to other bioactive agents (Fig. 1). In this
⇑
Corresponding authors. Tel.: +34 981 167000; fax: +34 981 167065.
E-mail addresses: jaime.rodriguez@udc.es (J. Rodríguez), carlos.jimenez@udc.es
(C. Jiménez).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.10.028

296
A. Souto et al. / Bioorg. Med. Chem. 21 (2013) 295–302
the piperazinyl ring that facilitates its conjugation. We have se-
lected the vanchrobactin analogues DHBA- -ornithine (2) and
DHBA- -ornithine– -serine (3) because they keep the siderophore
activity when they were tested for growth promotion under iron
deficient conditions in the wild-type V. anguillarum strain RV22,
and also they can be anchored through the amino group to norflox-
acin.12 Secondly, compounds 2 and 3 promote the growth of V.
OH
OH
D
OH
OH
NH2
NH2
D
L
H
H
N
(R)
N
N
H
NH3
O
O
O
NH
O
NH
COO
HO
HO
(S)
COO
anguillarum serotype O2 mutant strain containing the
DvabB muta-
2
1
tion (deletion of vabB prevents production of vanchrobactin in V.
anguillarum) while they were not able to promote the growth of
OH
OH
OH
OH
V. anguillarum serotype O2 double mutant strain containing
DvabB
H
H
D
fvtA mutations (further deletion of fvtA abolish production of
D
N
N
NH3
vanchrobactin receptor in V. anguillarum). This last fact was indic-
ative that FvtA is the sole route of entry for these compounds into
the bacteria.13 An acetate linker was used as space arm to attach
the drug to the vanchrobactin analogues.
NH2
O
O
O
O
4
3
In order to find a simpler analogue of vanchrobactin, we have
synthesized aminochelin (4), one of the siderophores produced
by the Gram-negative free-living soil bacterium Azotobacter vine-
landii.24 Although aminochelin (4) displayed siderophore activity
in V. anguillarum serotype O2, it is not using the FvtA receptor as
the route of entry since it was able to promote the growth of both
Figure 1. Structures of vanchrobactin (1), vanchrobactin analogues 2 and 3, and
aminochelin (4).
way, we have considered them as potential antibiotic vectors for
the delivery of antibiotics via the bacterial iron uptake systems
(Trojan horse strategy).22
a
D
vabB mutant strain and a
DvabB DfvtA double mutant strain.
Later on, we have found that FvtA protein is the outer mem-
brane transporter for vanchrobactin (1) and its analogues 2 and 3
in V. anguillarum. The gene fvtA encoding the vanchrobactin recep-
tor and most vanchrobactin biosynthesis genes are ubiquitous in
both vanchrobactin and anguibactin producing V. anguillarum
strains. These findings reinforced the utility of the iron scavenging
system mediated by vanchrobactin as an appropriate candidate for
a Trojan horse strategy to develop new drugs potentially effective
against fish vibriosis.23
Herein we describe the synthesis of several vanchrobactin ana-
logues–norfloxacin conjugates, compounds 5–8, with the aim of
exploring their antimicrobial activity against the fish pathogen Vib-
rio anguillarum.
The synthesis of aminochelin–norfloxacin conjugate 5 started
with the coupling of 2,3-diisopropyloxybenzoic acid (9)12 with
tert-butyl(4-aminobutyl)carbamate (10) using TBTU as the cou-
pling agent to give 11 in very good yield. The carbamate of 11
was cleaved quantitatively with trifluoroacetic acid (TFA), afford-
ing the free amine 12 which was coupled with chloroacetyl chlo-
ride to afford 13. Coupling reaction between the functionalized
vanchrobactin analogue 13 and norfloxacin was carried out in
dry DMF in the presence of DIPEA and KI to give compound 14.
The iso-propyl protecting group in 14 was removed with BCl3 in
MeOH to afford desired conjugate 5 which was purified by RP-
HPLC (Scheme 1).
For the synthesis of DHBA-
and 7, Nd-Cbz- -ornithine-OtBu (15), prepared from the commer-
cially available Nd-carboxybenzoyl-
-ornithine, was coupled with
D-ornithine-norfloxacin conjugates 6
D
2. Results and discussion
D
2,3-diisopropyloxybenzoic acid (9)25 using TBTU as the coupling
agent to give the corresponding ester 16 in very good yield. Re-
moval of the Cbz group of fully protecting 16 by catalytic hydroge-
nation gave the free amine 17. Following the same procedure as
before, the free amine 17 was coupled with chloroacetyl chloride
and the resulting chloride 18 was reacted with norfloxacin to give
the conjugate 19. The catechol iso-propyl group and the carboxylic
2.1. Chemistry: synthesis of vanchrobactin analogues–
norfloxacin conjugates
In the preparation of the conjugates, the antibiotic norfloxacin
was chosen because it showed a strong antimicrobial activity
against V. anguillarum and it has a secondary amine function in
OiPr
OiPr
OiPr
OiPr
OH
c
a
+
H2N
H
NHBoc
N
NHR
O
O
11 R = Boc
9
10
b
12 R = H
O
OiPr
F
CO2H
OR
OiPr
OR
d
O
O
N
N
H
H
N
Cl
N
N
NH
N
H
O
O
14 R = iPr
5 R = H
13
e
Scheme 1. Synthesis of conjugate 5. Reagents and conditions: (a) TBTU, Et3N, DMF, rt, 67%; (b) TFA/CH2Cl2 (1:2.3), 3 h, quant; (c) chloroacetyl chloride, Et3N, CH2Cl2, 0 °C to rt,
53%; (d) norfloxacin, DIPEA, KI, DMF, rt, 36 h, quant; (e) 1 M BCl3/CH2Cl2 then MeOH, 43%.

A. Souto et al. / Bioorg. Med. Chem. 21 (2013) 295–302
297
OiPr
OiPr
c
a
+
9
H2N
H
NHCbz
N
NHR
COOtBu
O
COOtBu
15
16 R = Cbz
17 R = H
b
O
N
F
CO2H
OiPr
OR1
OiPr
OR1
d
O
O
N
H
H
N
Cl
N
N
N
H
N
COOtBu
H
O
O
COOR2
18
6
R1 = H, R2 = Me
19 R1 = iPr, R2 = tBu
R1 = R2 = H
e
f
7
Scheme 2. Synthesis of conjugates 6 and 7. Reagents and conditions: (a) TBTU, Et3N, DMF, rt, 53%; (b) H2, Pd–C, MeOH, overnight, quant; (c) chloroacetyl chloride, Et3N,
CH2Cl2, 0 °C to rt, 97%; (d) norfloxacin, DIPEA, KI, DMF, rt, overnight, 64%; (e) 1 M BCl3/heptanes then MeOH, 15%; (f) 1 M BCl3/heptanes then H2O, 30%.
tert-butyl groups in 19 could be removed by the treatment with
BCl3 in MeOH. However, in these conditions the carboxylic acid
was methylated giving the corresponding methyl ester 6. When re-
moval of the protecting groups in 19 was performed with BCl3 in
H2O the expected conjugate diacid 7 was obtained. Both products
were purified by RP-HPLC (Scheme 2).
N-Cbz-
barium hydroxide, giving the acid 22 in a quantitative yield. This
acid 22 was coupled with O-tert-butyl- -serine tert-butyl ester
(23), which was prepared from -serine with treatment with HClO4
D
-ornithine.12 The resulting ester 21 was hydrolyzed with
L
L
in AcOtBu, to give the dipeptide 24. Following a similar scheme as
before, removal of the Cbz group giving the free amine 25, coupling
with chloroacetyl chloride to afford 26, reaction with norfloxacin
to obtain 27 and deprotection with BCl3 in H2O gave the conjugate
8, which was finally purified by RP-HPLC (Scheme 3).
The synthesis of last conjugate 8 started with coupling of 2,3-
diisopropyloxybenzoic acid (9) and Nd-Cbz-
D
-ornithine-OMe (20),
which was prepared by esterification of the commercially available
t
OiPr
CO2 Bu
tBuO
OiPr
23
NH2
c
a
+
H2N
9
H
NHCbz
N
NHCbz
COOMe
O
COOR
20
21 R = Me
22 R = H
b
OiPr
OiPr
OiPr
OiPr
O
H
H
e
N
f
N
Cl
NHR
N
H
O
O
O
O
NH
NH
t BuO
t BuO
CO2t Bu
t
CO2 Bu
24 R = Cbz
25 R = H
26
d
O
F
CO2H
OR1
OR1
N
N
O
H
27 R1 = iPr, R2 = tBu
R1 = R2 = H
N
N
g
N
8
H
O
O
NH
R2O
COOR2
Scheme 3. Synthesis of conjugate 8. Reagents and conditions: (a) TBTU, Et3N, DMF, rt, 63%; (b) Ba(OH)2, THF/H2O, quant; (c) TBTU, Et3N, DMF, rt, 70%; (d) H2, Pd–C, MeOH,
overnight, quant; (e) chloroacetyl chloride, Et3N, CH2Cl2, 0 °C to rt, 59%; (f) norfloxacin, DIPEA, KI, DMF, rt, overnight, 85%; (g) 1 M BCl3/heptanes then H2O, 13%.

298
A. Souto et al. / Bioorg. Med. Chem. 21 (2013) 295–302
Table 1
indicate that terminal amino group in vanchrobactin analogues
2–4 may be critical for recognition by FvtA.
Antibiotic activities of conjugates 5–8 and of norfloxacin against the three Vibrio
anguillarum O2 strains: RV22 (wild-type strain), the fvtA mutant MB12 (non
vanchrobactin-producing strain) and vabB mutant MB84 (vanchrobactin receptor
D
D
4. Experimental
4.1. Chemistry
mutant). The CAS test gives negative values: higher numbers indicate higher iron
chelating activity
Compound
MIC (
lg/mL)
CAS test
RV22/MB12/MB84
Nuclear magnetic resonance spectra (proton and carbon) were
recorded on Bruker AC200 F, and 300 or 500 Advance spectrome-
ters at the University of A Coruña, using CDCl3 and CD3OD as the
solvents and internal standards. Multiplicities of 13C signals were
obtained by DEPT. Medium-pressure chromatographic separations
were carried out on silica gel 60 (230–400 mesh). Optical rotations
were determined on a JASCO DIP-1000 polarimeter, with Na
(589 nm) lamp and filter. LREIMS and HRESIMS were measured
on Applied Biosystems QSTAR Elite. HPLC separations were carried
out on an Agilent HP1100 liquid chromatography system equipped
with a solvent degasser, quaternary pump and an UV detector (Agi-
lent Technologies, Waldbronn, Germany). A Scharlau HPLC column
Norfloxacin
5
6
7
0.006
0.097
25
12.5
3.125
ꢀ0.541
NT
ꢀ0.612
ꢀ0.562
ꢀ0.632
8
Vanchrobactin
NT = not tested.
2.2. Biological evaluations
The antibacterial activities (MIC expressed in
l
g/mL) of the four
conjugates 5–8 were evaluated in vitro against three Vibrio anguil-
larum O2 strains: the wild-type strain (RV22), the fvtA mutant
(MB12) and vabB mutant (MB84). As a control, the experiment
Nucleosil 120 C18 10
separations.
l
m 250 ꢁ 4 mm was used in the HPLC
D
D
All moisture-sensitive reactions were carried out under an
atmosphere of argon in flame-dried glassware closed by rubber
septum, unless otherwise noted. Solvents were distilled prior to
use under argon atmosphere and dried according to standard pro-
cedures using the following desiccants: Na/benzophenone for THF
and Et2O, CaH2 for dichloromethane, pyridine and triethylamine;
magnesium for methanol and anhydrous CaSO4 for acetone. DMF
was distilled over CaH2 and was kept over molecular Sieves 4 Å un-
der argon atmosphere. Solutions and solvents were added via syr-
inge or cannula. Thin layer chromatography was performed using
sílica gel GF-254 Merck, spots were revealed employing UV light
(254 nm) and/or by heating the plate pretreated with an ethanolic
solution of phosphomolibdic acid, a solution of cerium sulphate or
a solution of ninhydrine in BuOH–AcOH–H2O. CRYOCOOL appara-
tus was used for low-temperature reactions.
was repeated with siderophore-free norfloxacin (Table 1).
Although the four conjugates showed clear antibacterial activity,
this activity was lower than that of the corresponding unconju-
gated norfloxacin. Conjugate 5 showed the highest antibiotic activ-
ity among the conjugates synthesized being less active than
norfloxacin by an order of magnitude. The activity of ester conju-
gate 6 was lower than the corresponding acid conjugate 7. The four
conjugates displayed identical growth inhibitions against the wild-
type strain as well as against the
DfvtA mutant and DvabB mutant
and they also were comparable in presence and absence of iron
(III). Taken together these results demonstrate that conjugates 5–
8 do not employ the siderophore uptake system (i.e. FvtA) for entry
into V. anguillarum and thus do not operate by a Trojan Horse strat-
egy. While disappointing these results do show that introduction
of the siderophore moiety on the piperazinyl ring is tolerated since
conjugate 5 possesses nearly identical activity as norfloxacin.26
One explanation for the lack of FvtA-mediated uptake by conju-
gates 5–8 is that the terminal amino group in vanchrobactin ana-
logues 2–4 may be critical for recognition by FvtA.
The relative iron complexing capacities of the conjugates were
evaluated by reactivity with chrome azurol-S (CAS) assay,27 and
the results are shown in Table 1. All of them gave a positive reac-
tion in this test with a range of values from ꢀ0.562 for conjugate 8
to ꢀ0.612 for conjugate 7 and they were similar to that of vanch-
robactin (ꢀ0.632). These results suggest that the binding of the
vanchrobactin analogs to norfloxacin through the amino function-
ality did not affect the chelating ability of these compounds. Thus,
the reduced inhibitory activities of the conjugates compared to
that of the norfloxacin alone, indicated that the drug conjugation
interferes in their action mode as antibiotics.
4.1.1. Synthesis of conjugate 5
4.1.1.1. Compound 11.
2,3-Diisopropoxybenzoic acid (9) (1 g,
4.19 mmol) was coupled to tert-butyl(4-aminobutyl)carbamate
(10) (0.79 g, 4.19 mmol) in DMF (25 mL) and triethylamine
(1.46 mL, 10.49 mmol) using TBTU as coupling agent (2.02 g,
6.29 mmol). The solvent was removed in vacuo and the residue
was pre-absorbed onto silica and purified by column chromatogra-
phy (silica gel, CH2Cl2/MeOH 1%) to yield tert-butyl 4-(2,3-diiso-
propoxybenzamido)butylcarbamate (11) as
a light yellow oil
(1.147 g) in a 67% yield: 1H NMR (300 MHz, CDCl3) dH: 1.27 (d,
J = 6.2 Hz, 6H, i-Pr); 1.34 (d, J = 6.1 Hz, 6H, i-Pr); 1.41 (s, 9H, t-
Bu); 1.59 (m, 4H); 3.14 (m, 2H); 3.43 (dd, J = 12.7, 6.7 Hz, 2H);
4.53 (m, 1H, i-Pr); 4.64 (m, 1H, i-Pr); 6.97 (dd, J = 7.9, 1.7 Hz,
1H); 7.05 (t, J = 7.9 Hz, 1H); 7.63 (dd, J = 7.9, 1.7 Hz, 1H); 8.03 (br
t, J = 5.1 Hz, NH). 13C NMR (75 MHz, CDCl3) dC: 22.18 (2 CH3, i-
Pr); 22.46 (2 CH3, i-Pr); 27.14 (CH2); 27.87 (CH2); 28.50 (CH, t-
Bu); 39.33 (CH2); 40.32 (CH2); 71.20 (CH, i-Pr); 76.36 (CH, i-Pr);
118.32 (CH); 122.90 (CH); 123.81 (CH); 128.61 (CH); 146.05 (C);
150.86 (C); 156.09 (C); 162.63 (C, CO); 165.96 (C, CO). (+)-LREIMS
m/z (%): 409 ([M+H]+, 100).
3. Conclusions
In summary, four vanchrobactin analogue–norfloxacin conju-
gates (5–8) were synthesized using acetate as a spacer arm. The
biological assays of these compounds against a wild-type strain
of Vibrio anguillarum, a
D
fvtA mutant and a
D
vabB mutant showed
4.1.1.2. Compound 12.
Compound 11 (0.814 g, 1.994 mmol)
that the four conjugates displayed antimicrobial activity. The lower
antibiotic activity displayed by conjugates 5–8 in relation to
unconjugated norfloxacin against the same strains indicates that
they are not working as Trojan horse conjugates. The fact that
the conjugate 5 shows a similar antibiotic activity as norfloxacin
suggests that introduction of the siderophore moiety on the piper-
azinyl ring is tolerated.28–30 Furthermore, these results seem to
was dissolved in anhydrous CH2Cl2 (14 mL), then TFA (6 mL) was
added and the mixture was stirred at room temperature. After
3 h, the solvent was concentrated under reduced pressure to afford
N-4-aminobutyl-2,3-diisopropoxybenzamide (12) as an orange oil
(0.615 g) in a quantitative yield, which was used without further
purification: 1H NMR (500 MHz, CDCl3) dH: 1.26 (d, J = 6.2 Hz, 6H,
i-Pr); 1.35 (d, J = 6.1 Hz, 6H, i-Pr); 1.70 (m, 2H); 1.78 (m, 2H);

A. Souto et al. / Bioorg. Med. Chem. 21 (2013) 295–302
299
3.09 (br s, 2H); 3.42 (m, 2H); 4.54 (m, 1H, i-Pr); 4.68 (m, 1H, i-Pr);
7.02 (dd, J = 7.9, 1.6 Hz, 1H); 7.06 (t, J = 7.9 Hz, 3H); 7.49 (dd, J = 7.9,
1.6 Hz, 1H); 8.44 (br t, NH). 13C NMR (126 MHz, CDCl3) dC: 21.80 (2
CH3, i-Pr); 22.08 (2 CH3, i-Pr); 24.57 (CH2); 26.43 (CH2); 38.51
(CH2); 38.97 (CH2); 71.13 (CH, i-Pr); 76.39 (CH, i-Pr); 118.51
(CH); 121.96 (CH); 124.18 (CH); 127.68 (C); 145.96 (C); 150.79
(C); 166.90 (C, CO). (+)-LREIMS m/z (%): 309 ([M+H]+, 100).
1H); 6.87 (d, J = 7.8 Hz, 1H); 7.13 (d, J = 7.1 Hz, 1H, norfloxacin);
7.19 (br d, J = 8.0 Hz, 1H); 7.99 (d, J = 13.3 Hz, 1H, norfloxacin);
8.89 (s, 1H, norfloxacin). 13C NMR (75 MHz, CDCl3) dC: 14.72
(CH3, norfloxacin); 27.89 (CH2); 27.96 (CH2); 39.63 (CH2); 40.07
(CH2); 50.83 (CH2, norfloxacin); 50.87 (CH2, norfloxacin); 50.95
(CH2, norfloxacin); 54.20 (2 CH2, norfloxacin); 62.24 (CH2);
106.51 (CH); 108.46 (C); 112.89 (CH); 116.76 (C); 118.53 (CH);
119.52 (CH); 121.36 (CH); 138.95 (C); 147.37 (C); 149.45 (C);
150.32 (C); 151.90 (C); 154.07 (CH); 156.06 (C); 169.79 (C, CO);
171.55 (C, CO); 172.62 (C, CO); 178.36 (CO). (+)-LREIMS m/z (%):
584 ([M+H]+, 100); (+)-HRESIMS: m/z 584.2518 [M+H]+ (calcd. for
4.1.1.3. Compound 13.
was dissolved in anhydrous CH2Cl2 (3 mL) and stirred at 0 °C, then
chloroacetyl chloride (46 L, 0.576 mmol) and Et3N (80 L,
Compound 12 (0.071 g, 0.230 mmol)
l
l
0.576 mmol) were added and the mixture was stirred at this tem-
perature during 1 h and for another 2 h at room temperature. After
that, the mixture was extracted with CH2Cl2 and the organic layer
was washed with 1 N HCl, water and brine, dried with anhydrous
MgSO4, filtered and concentrated under reduced pressure. The
crude was purified by column chromatography (silica gel, CH2Cl2/
MeOH 95:5) to afford N-(4-(2-chloroacetamido)butyl)-2,3-diiso-
propoxybenzamide (13) as a yellow oil (0.047 g) in a 53% yield:
1H NMR (300 MHz, CDCl3) dH: 1.29 (d, J = 6.3 Hz, 6H, i-Pr); 1.36
(d, J = 6.0 Hz, 6H, i-Pr); 1.66 (m, 4H); 3.36 (m, 2H); 3.44 (m, 2H);
4.03 (s, 2H); 4.55 (m, 1H, i-Pr); 4.67 (m, 1H, i-Pr); 7.00 (dd,
J = 8.0, 1.9 Hz, 1H); 7.08 (dd, J = 8.0, 7.8 Hz, 1H); 7.64 (dd, J = 7.8,
C
29H35N5O7F, 584.2515).
4.1.2. Synthesis of conjugates 6 and 7
4.1.2.1. Compound 16.
(0.993 g, 4.14 mmol) was coupled to (R)-tert-butyl 2-amino-5-
(((benzyloxy)carbonyl)amino)pentanoate
2,3-Diisopropoxybenzoic acid (9)
(15)
(1.343 g,
4.17 mmol) in DMF (37 mL) and triethylamine (1.45 mL,
10.4 mmol) using TBTU as coupling agent (2.010 g, 6.26 mmol).
The solvent was removed in vacuo and the residue was pre-ab-
sorbed onto silica and purified by column chromatography (silica
gel, hexanes/ethyl acetate 1:5) to yield (R)-tert-butyl 5-(((benzyl-
oxy)carbonyl)amino)-2-(2,3-diisopropoxybenzamido)pentanoate
1.9 Hz, 1H); 8.10 (t, J = 5.1 Hz, NH); 8.19 (t, J = 5.1 Hz, NH).13
C
16 as a light yellow oil (1.251 g) in a 53% yield: ½a D31
ꢂ
: +1.74 (CHCl3,
NMR (75 MHz, CDCl3) dC: 22.21 (2 CH3, i-Pr); 22.52 (2 CH3, i-Pr);
26.92 (CH2); 27.36 (CH2); 39.13 (CH2); 39.69 (CH2); 42.79 (CH2–
Cl); 71.25 (CH, i-Pr); 76.48 (CH, i-Pr); 118.43 (CH); 122.84 (CH);
123.90 (CH); 128.45 (C); 146.06 (C); 150.90 (C); 166.14 (C, CO);
166.49 (C, CO). (+)-LREIMS m/z (%): 385 ([M+H]+, 67).
c = 1.11). 1H NMR (300 MHz, CDCl3) dH: 1.25 (d, J = 6.6 Hz, 3H, i-Pr);
1.33 (d, J = 6.0 Hz, 3H, i-Pr); 1.34 (d, J = 6.0 Hz, 3H, i-Pr); 1.36 (d,
J = 6.6 Hz, 3H, i-Pr); 1.49 (s, 9H, t-Bu); 1.51–1.67 (m, 2H); 1.68–
1.82 (m, 1H); 1.82–1.96 (m, 1H); 3.12–3.27 (m, 2 H); 4.52 (m, 1
H, i-Pr); 4.65 (dt, J = 6.6, 6.0 Hz, 1H); 4.77 (m, 1H, i-Pr); 5.05 (s,
2H, Cbz); 6.96–7.08 (m, 2H); 7.21–7.36 (m, 4H); 7.66 (dd, J = 7.1,
2.2 Hz, 1H); 8.68 (d, J = 7.7 Hz, 1H). 13C NMR (300 MHz, CDCl3)
dC: 14.15; 22.03; 22.10; 22.19; 25.83; 27.98; 29.64; 30.43; 31.36;
36.41; 38.56; 40.56; 52.81; 60.32; 66.43; 71.24; 76.17; 81.85;
119.03; 123.01; 123.42; 127.70; 127.92; 128.40; 136.67; 146.52;
150.74; 156.38; 162.49; 171.24. (+)-LREIMS m/z (%): 543
([M+H]+, 75%), 565 ([M+Na]+, 100%); (+)-HRESIMS: m/z 543.3067
[M+H]+ (calcd. for C30H43N2O4, 543.3064).
4.1.1.4. Compound 14.
A mixture of norfloxacin (0.048 g,
0.151 mmol) and DIPEA (0.058 g, 0.451 mmol) in DMF (2 mL)
was added to a mixture of compound 13 (0.058 g, 0.151 mmol)
and KI (0.006 g) also dissolved in DMF (2 mL) and stirred at room
temperature over 36 h. Then, the mixture was concentrated under
reduced pressure to dryness to afford 100 mg of compound 14 as
yellow oil in a quantitative yield: 1H NMR (300 MHz, CDCl3) dH:
1.26 (d, J = 6.2 Hz, 6H, i-Pr); 1.33 (d, J = 6.1 Hz, 6H, i-Pr); 1.53 (t,
J = 7.2 Hz, 3H, norfloxacin); 1.64 (m, 4H); 2.77 (m, 4H, norfloxacin);
3.11 (s, 2H); 3.35 (m, 4H, norfloxacin); 3.43 (m, 4H); 4.32 (q,
J = 7.2 Hz, 2H, norfloxacin); 4.52 (m, 1H, i-Pr); 4.65 (m, 1H, i-Pr);
6.89 (d, J = 6.9 Hz, 1H, norfloxacin); 6.98 (dd, J = 7.5, 2.8 Hz, 1H);
7.04 (t, J = 7.5 Hz, 1H); 7.61 (br d, J = 7.5 Hz, 1H); 8.06 (d,
J = 13.9 Hz, 1H, norfloxacin); 8.64 (s, 1H, norfloxacin). 13C NMR
(75 MHz, CDCl3) dC: 14.57 (CH3, norfloxacin); 22.13 (2 CH3, i-Pr);
22.45 (2 CH3, i-Pr); 25.91 (CH2); 27.30 (CH2); 38.76 (CH2); 39.62
(CH2); 49.83 (CH2, norf); 49.85 (2 CH2, norf); 53.18 (2 CH2, norf);
61.40 (CH2); 70.72 (CH, i-Pr); 76.43 (CH, i-Pr); 104.30 (CH);
108.35 (C); 112.94 (CH); 118.08 (CH); 119.26 (C); 122.58 (CH);
123.80 (CH); 128.26 (C); 137.20 (C); 145.91 (C); 145.93 (C);
146.00 (CH); 147.24 (C); 150.87 (C); 166.06 (C, CO); 167.34 (C,
CO); 169.53 (C, CO); 177.06 (CO). (+)-LREIMS m/z (%): 668
([M+H]+, 100).
4.1.2.2. Compound 17.
Compound 16 (1.177 g, 2.102 mmol)
was dissolved in methanol (20 mL) and the flask was flushed with
argon. 10% Pd/C (100 mg) was added and the flask was again
flushed with argon. The reaction mixture was flushed with hydro-
gen and stirred under a hydrogen atmosphere overnight. The reac-
tion mixture was filtered through Celite and the solvent was
removed in vacuo to afford (R)-tert-butyl 5-amino-2-(2,3-diiso-
propoxybenzamido)pentanoate (17) as a colorless oil (0.955 g) in
a quantitative yield, which was used without further purification:
½
a 3D1: +7.02 (CHCl3, c = 0.225); 1H NMR (300 MHz, CDCl3) dH: 1.27
ꢂ
(d, J = 6.0 Hz, 3H, i-Pr); 1,33 (d, J = 6.0 Hz, 3H, i-Pr); 1.35 (d,
J = 6.0 Hz, 3H, i-Pr); 1.38 (d, J = 6.6 Hz, 3H, i-Pr); 1.47 (s, 9H, t-
Bu); 1.47–1.64 (br s, 1H); 1.68–1.83 (m, 1H); 1.83–2.00 (m, 2H);
2.64–2.80 (br s, 2 H); 4.53 (m, 1H, i-Pr); 4.66 (dt, J = 6.0, 6.6 Hz,
1H); 4.78 (m, 1H, i-Pr); 6.93–7.09 (m, 2H); 7.67 (dd, J = 1.7,
7.7 Hz, 1H); 8.66 (d, J = 7.1 Hz, 1H, NH). 13C NMR (300 MHz, CDCl3)
dC: 21.70; 22.05; 22.13; 22.23; 28.03; 29.66; 30.38; 30.88; 38.57;
41.66; 53.03; 71.26; 76.14; 81.67; 118.99; 123.07; 123.43;
127.88; 146.50; 150.75; 165.23; 171.43. (+)-LREIMS m/z (%): 409
([M+H]+, 97); (+)-HRESIMS: m/z 409.2692 [M+H]+ (calcd. for
4.1.1.5. Conjugate 5.
To a solution of compound 14 (100 mg,
0.151 mmol) in anhydrous CH2Cl2 (5 mL), 1.05 mL (1.05 mmol) of
1 M BCl3 in CH2Cl2 was added at ꢀ78 °C and stirred overnight.
Then, the reaction was quenched by the addition of MeOH
(3 mL), allowed to reach room temperature and concentrated in
vacuo to afford a yellow oil residue. Part of this residue (3 mg)
was purified by HPLC (Scharlau C18, flow rate 1.5 mL/min, H2O/
MeOH, 1:4 containing 0.1 % TFA, k = 254 nm) to give 1.2 mg of con-
jugate 5 (retention time of 11 min) as a colorless oil (43% yield): 1H
NMR (300 MHz, CD3OD) dH: 1.55 (t, J = 7.1 Hz, 3H, norfloxacin);
1.64 (m, 4H); 2.75 (m, 4H, norfloxacin); 3.11 (s, 2H); 3.17–3.47
(m, 8H); 4.52 (q, J = 7.1 Hz, 2H, norfloxacin); 6.68 (t, J = 8.0 Hz,
C22H37N2O5, 409.2696).
4.1.2.3. Compound 18.
Compound 17 (0.909 g, 2.225 mmol)
was dissolved in CH2Cl2 (20 mL) and stirred at 0 °C, then chloroace-
tyl chloride (0.44 mL, 5.56 mmol) and Et3N (0.78 mL, 5.56 mmol)
were added. After 4 h, the mixture was distributed between CH2Cl2
and aqueous 5% HCl. The organic layer was washed with water and
brine, dried with MgSO4, filtered and concentrated under reduced

300
A. Souto et al. / Bioorg. Med. Chem. 21 (2013) 295–302
pressure. The crude was purified by column chromatography (sil-
MeOH, 55:45 containing 0.05% TFA) to give 7 mg of conjugate 7
ica gel, CH2Cl2/MeOH, 96:4) to afford (R)-tert-butyl 5-(2-chloroace-
(retention time of 22.13 min) as a white solid in a 30% yield:
tamido)-2-(2,3-diisopropoxybenzamido)pentanoate (18) as
a
½
a 3D0: +20.27 (MeOH, c = 0.225). 1H NMR (300 MHz, D2O) dH: 1.63
ꢂ
brown oil (1.046 g) in a 97% yield: ½a D30
ꢂ
: +1.55 (CHCl3, c = 2.075).
(t, J = 7.2 Hz, 3H); 1.75 - 1.94 (m, 2H); 1.94–2.11 (m, 1H); 2.11–
2.23 (m, 1H); 3.49 (br t, 2H); 3.62–3.85 (m, 8H); 4.21 (s, 2H);
4.60 (q, J = 7.2 Hz, 2H); 4.70 (m, 1H); 6.90 (t, J = 8.0 Hz, 1H); 7.05
(d, J = 8.0 Hz, 1H); 7.23 (d, J = 6.9 Hz, 2H); 7.35 (d, J = 8.0 Hz, 1H);
7.92 (d, J = 12.9 Hz, 1H); 8.90 (s, 1H). 13C NMR (500 MHz, D2O)
dC: 13.64; 24.59; 27.88; 38.79; 46.29; 46.32; 50.37; 52.01; 52.75;
56.91; 106.18; 106.41; 111.36; 115.13; 117.45; 119.17; 119.40;
119.64; 137.26; 144.26; 144.51; 146.65; 148.24; 148.27; 162.83;
164.21; 169.73; 175.69; 176.19. (ꢀ)-LREIMS m/z (%): 626 ([MꢀH]ꢀ,
31); (ꢀ)-HRESIMS: m/z 626.2296 [MꢀH]ꢀ (calcd. for C30H33N5O9F,
626.2267).
1H NMR (300 MHz, CDCl3) dH: 1.28 (d, J = 6.6 Hz, 3H, i-Pr); 1.37
(d, J = 6.0 Hz, 3H, i-Pr); 1.38 (d, J = 6.0 Hz, 3H, i-Pr); 1.41 (d,
J = 6.6 Hz, 3H, i-Pr); 1.50 (s, 9H, t-Bu); 1.60–1.86 (m, 1H); 1.87–
2.01 (m, 1H); 3.34 (m, 2H); 4.04 (s, 2H); 4.56 (m, 1H, i-Pr); 4.71
(q, J = 6.6 Hz, 1H); 4.80 (m, 1H, i-Pr); 6.77–6.95 (br s, 1H); 6.99–
7.13 (m, 4H); 7.67 (d, J = 2.2 Hz, 1H, NH); 8.76 (d, J = 7.1 Hz, 1H,
NH). 13C NMR (300 MHz, CDCl3) dC: 21.69; 22.05; 22.12; 25.12;
28.01; 29.66; 30.58; 39.31; 40.97; 42.58; 52.17; 71.32; 76.33;
82.17; 119.20; 122.97; 123.52; 127.40; 146.63; 150.79; 165.65;
166.35; 169.11; 171.07. (+)-LREIMS m/z (%): 507 ([M+Na]+, 27);
485 ([M+H]+, 100); (+)-HRESIMS: m/z 485.2414 [M+H]+ (calcd. for
C24H38N2O6Cl35, 485.2412).
4.1.3. Synthesis of conjugate 8
4.1.3.1. Compound 21.
2,3-Diisopropoxybenzoic acid (9)
4.1.2.4. Compound 19.
A mixture of compound 18 (152 mg,
(0.890 g, 3.74 mmol) was coupled to (R)-methyl 2-amino-5-(((ben-
zyloxy)carbonyl)amino)pentanoate (20) (1.119 g, 3.74 mmol) in
DMF (37 mL) and triethylamine (1.3 mL, 9.35 mmol) using TBTU
as coupling agent (1.804 g, 5.62 mmol). The solvent was removed
under reduced pressure and the obtained residue was pre-ab-
sorbed onto silica and purified by column chromatography (silica
gel, gradient of hexanes/ethyl acetate from 1:3 to 1:5) to give
1.136 g of (R)-methyl 5-(((benzyloxy)carbonyl)amino)-2-(2,3-
diisopropoxybenzamido)pentanoate (21) as a light yellow oil in a
0.372 mmol), norfloxacin (119 mg, 0.372 mmol) and KI (186 mg,
1.12 mmol) in DMF (7 mL) was stirred at rt overnight. Then, the
mixture was concentrated under reduced pressure, filtered
throught a C18 cartridge, and purified by HPLC (Scharlau C18, flow
rate 1.5 mL/min, H2O/MeOH, 1:4 containing 0.05 % TFA) to afford
5 mg of compound 19 (retention time of 13.2 min) as a yellow solid
(183 mg) in a 64% yield: ½a D30
ꢂ
: +46.57 (CHCl3, c = 0.07). 1H NMR
(500 MHz, CDCl3) dH: 1.28 (d, J = 6.5 Hz, 3H); 1.37 (d, J = 6.0 Hz,
3H); 1.36 (d, J = 6.5 Hz, 3H); 1.38 (d, J = 6.0 Hz, 3H); 1.50 (s, 9H);
1.54–1.62 (m, 2H); 1.67 (br s, 3H); 1.77–1.89 (m, 1H); 1.89–2.00
(m, 1H); 3.27 (m, 2H); 3.50–3.57 (m, 4H); 3.57–3.65 (m, 4H);
4.36 (br s, 2H); 4.55 (m, 1H); 4.61 (m, 1H); 4.80 (m, 1H); 7.01–
7.09 (m, 3H); 7.64 (d, J = 6.4 Hz, 1H); 8.12 (d, J = 12.3 Hz, 1H);
8.26 (br s, 1H); 8.72 (s, 1H); 8.97 (d, J = 7.1 Hz, 1H). 13C NMR
(300 MHz, CDCl3) dC: 14.07; 14.58; 21.72; 22.03; 22.13; 22.26;
28.06; 29.70; 38.71; 49.87; 49.91; 49.92; 49.95; 53.04; 53.22;
54.97; 71.18; 76.36; 82.21; 108.45; 113.14; 118.74; 122.59;
123.42; 123.46; 125.01; 127.48; 128.79; 130.89; 135.20; 146.51;
147.26; 150.83; 165.30; 167.17; 167.76; 171.13; 177.00. (+)-LRE-
IMS m/z (%): 768 ([M+H]+, 100); (+)-HRESIMS: m/z 768.3962
[M+H]+ (calcd. for C40H55N5O9F, 768.3978).
63% yield: ½a 3D0
ꢂ
: +6.1 (CHCl3, c = 1.15). 1H NMR (300 MHz, CDCl3)
dH: 1.28 (d, J = 6.0 Hz, 3H, i-Pr); 1.37 (d, J = 6.0 Hz, 3H, i-Pr); 1.38
(d, J = 6.0 Hz, 3H, i-Pr); 1.40 (d, J = 6.0 Hz, 3H, i-Pr); 1.54–1.73 (m,
2H); 1.73–1.89 (m, 1H); 1.89–2.06 (m, 1H); 3.25 (q, J = 6.0 Hz,
2H); 3.77 (s, 3H, methyl ester); 4.57 (m, 1H, i-Pr); 4.82 (m, 1H);
4.84 (m, 1H, i-Pr); 5.10 (s, 2H, Cbz); 6.99–7.11 (m, 2H, aromatic
protons), 7.28–7.40 (m, 5H, aromatic protons); 7.70 (dd, J = 7.6,
2.0 Hz, 1 H); 8.75 (d, J = 7.7 Hz, 1H, NH). 13C NMR (300 MHz, CDCl3)
dC: 14.19; 21.60; 22.06; 22.14; 22.31; 26.03; 30.22; 40.54; 52.16;
52.28; 66.61; 71.26; 76.58; 77.20; 99.98; 119.00; 123.09; 123.54;
127.43; 128.03; 127.47; 136.58; 146.43; 150.73; 156.34; 165.39;
172.61. (+)-LRESIMS m/z (%): 501 ([M+H]+, 100); (+)-HRESIMS: m/
z 501.2572 [M+H]+ (calcd. for C27H37N2O7, 501.2595).
4.1.2.5. Conjugate 6.
To a solution of compound 19 (90 mg,
4.1.3.2. Compound 22.
To a solution of compound (R)-methyl
0.0391 mmol) in CH2Cl2 (6 mL), 1.23 mL (1.23 mmol) of 1 M BCl3
in heptanes was added at ꢀ78 °C and allowed to reach ꢀ40 °C for
6 h. Then, the reaction was quenched by the addition of MeOH
(3 mL), allowed to reach rt and concentrated in vacuo. The residue
was purified by HPLC (Scharlau C18, flow rate 1.5 mL/min, H2O/
MeOH, 55:45 containing 0.05 % TFA) to give 11 mg of conjugate 6
(retention time of 37.00 min) as a white solid in a 15% yield.
5-(((benzyloxy)carbonyl)amino)-2-(2,3-diisopropoxybenzami-
do)pentanoate (21) (1.055 g, 2.11 mmol) in THF (25 mL) and water
(25 mL), 2.070 g (4.58 mmol) of barium hydroxide octahydrate was
added and the mixture was stirred at 50 °C overnight. Then, DOW-
EX-50 W(H+) was added to the solution to give a pH = 4 and the re-
sin was removed by filtration. The resulting filtrate was evaporated
under reduced pressure to give 1.018 g of compound (R)-5-(((ben-
zyloxy)carbonyl)amino)-2-(2,3-diisopropoxybenzamido)pentanoic
½
a 3D2: +20.32 (CHCl3, c = 0.37). 1H NMR (500 MHz, CD3OD) dH:
ꢂ
1.58 (t, J = 7.1 Hz, 3H); 1.67–1.77 (m, 2H); 1.85–1.97 (m, 1H);
2.01–2.12 (m, 1H); 3.37 (m, 2H); 3.50–3.64 (m, 4H); 3.64–3.83
(m, 4H); 3.78 (s, 3H); 4.03 (s, 2H); 4.59 (q, J = 7.1 Hz, 2H); 4.73
(m, 1H); 6.77 (t, J = 8.0 Hz, 1H); 6.97 (dd, J = 8.0, 1.4 Hz, 1H); 7.29
(d, J = 7.0 Hz, 1H); 7.35 (dd, J = 8.0, 1.4 Hz, 1H); 8.10 (d,
J = 12.9 Hz, 1H); 8.94 (s, 1H). 13C NMR (500 MHz, C6D6) dC: 14.57;
23.22; 29.88; 32.39; 49.82; 49.87, 49.95; 50.10; 52.48; 53.31;
53.33; 105.96; 108.92; 115.12; 115.92; 117.38; 134.93; 137.93;
142.35; 146.27; 148.31; 167.63; 167.69; 169.58; 173.45; 177.68.
(+)-LREIMS m/z (%): 642 ([M+H]+, 100).
acid (22) as a brown oil in a quantitative yield: ½a D31
ꢂ
: +4.2 (CHCl3,
c = 0.195). 1H NMR (300 MHz, CDCl3) dH: 1.23 (d, J = 6.2 Hz, 3H, i-
Pr); 1.33 (d, J = 5.9 Hz, 3H, i-Pr); 1.35 (d, J = 6.2 Hz, 3H, i-Pr); 1.38
(d, J = 5.86 Hz, 3H, i-Pr); 1.46–1.73 (m, 2H); 1.71–2.13 (m, 2H);
3.02–3.31 (m, 2H); 4.41–4.64 (m, 1H, i-Pr); 4.69–4.90 (m, 2H);
5.05 (s, 2H, Cbz); 6.95–7.12 (m, 1H); 7.20–7.38 (m, 1H); 7.61–
7.73 (m, 5H); 7.65 (dd, J = 5.5, 3.6 Hz, 1 H); 8.76–8.94 (m, 1H,
NH). 13C NMR (300 MHz, CDCl3) dC: 21.58; 22.04; 22.12; 22.24;
25.85; 29.87; 30.94; 40.49; 52.35; 55.23; 66.59; 71.19; 78.21;
119.03; 122.94; 126.60; 127.21; 127.98; 128.45; 136.87; 146.46;
150.70;151.65; 165.00; 172.56; 174.28. (+)-LREIMS m/z (%): 487
([M+H]+, 100); (+)-HRESIMS: m/z 487.2420 [M+H]+ (calcd. for
4.1.2.6. Conjugate 7.
To a solution of compound 19 (30 mg,
0.039 mmol) in CH2Cl2 (1 mL), 0.41 mL (0.41 mmol) of 1 M BCl3
in heptanes was added at ꢀ78 °C and allowed to reach ꢀ40 °C for
6 h. Then, the reaction was quenched by the addition of H2O
(1 mL), allowed to reach rt and concentrated in vacuo. The residue
was purified by HPLC (Scharlau C18, flow rate 1.5 mL/min, H2O/
C
26H35N2O7, 487.2438).
4.1.3.3. Compound 23.
(S)-2-Amino-3-hydroxypropanoic
acid (1 g, 9.43 mmol) was suspended in AcOtBu (30 mL), HClO4
(0.9 mL, 70% in water, 10.4 mmol) was added and the mixture

A. Souto et al. / Bioorg. Med. Chem. 21 (2013) 295–302
301
was stirred for 4 h. After that time, the solution was poured onto a
saturated NaHCO3 aqueous (100 mL) and stirring for 0.5 h. Then,
the mixture was partitioned between water and CH2Cl2, the aque-
ous phase was extracted with CH2Cl2 and the organic layer was
washed with brine and dried over MgSO4, filtered and concen-
trated in vacuo to afford 1.65 g of (S)-tert-butyl 2-amino-3-tert-
168.93; 170.86. (+)-LREIMS m/z (%): 628 ([M+H]+, 100%); (+)-HRE-
SIMS: 628.3353 ([M+H]+ (calcd. for C31H50Cl35N3O8, 628.3359).
4.1.3.7. Compound 27.
Compound 26 (182 mg, 0.376 mmol)
was coupled with norfloxacin (93 mg, 0.290 mmol) in a similar
way as 13 and 18. The resulting mixture was concentrated in va-
cuo, filtered through a C18 cartridge and purified by HPLC (Schar-
lau C18, flow rate 1.5 mL/min, H2O/MeOH, 1:4 containing 0.05 %
TFA) to afford compound 27 (retention time of 14.33 min) as a yel-
butoxypropanoate (23) as a colorless oil in a 80% yield: ½a D31
ꢂ
:
ꢀ5.0 (CHCl3, c = 4.71). 1H NMR (300 MHz, CDCl3) dH: 1.15 (s, 9H);
1.45 (s, 9H); 1.97 (br s, 2H); 3.46 (br s, 1H); 3.51–3.66 (m, 2H).
13C NMR (300 MHz, CDCl3) dC: 27.55; 28.20; 55.69; 63.99; 72.98;
81.12; 173.39. (+)-LREIMS m/z (%): 218 ([M+H]+, 100); (+)-HRE-
SIMS: m/z 218.1721 [M+H]+ (calcd. for C11H24NO3, 218.1756).
low solid (224 mg) in a 85% yield: ½a D31
ꢂ
: +16.30 (CHCl3, c = 0.745).
1H NMR (300 MHz, CDCl3) dH: 1.08 (s, 9H); 1.27 (d, J = 6.2 Hz,
3H); 1.33 (d, J = 6.0 Hz, 9H); 1.39 (s, 9H); 1.53 (t, J = 7.2 Hz, 3H);
1.62–1.74 (m, 2H); 1.84 (m, 1H); 1.96 (m, 1H); 3.37 (m, 2H);
3.57 (dd, J = 9.0, 3.2 Hz, 1H); 3.61–3.70 (m, 4H); 3.74 (dd, J = 9.0,
3.2 Hz, 1H); 4.03 (br s, 2H); 4.36 (br s, 2H); 4.44–4.61 (m, 2H);
4.72–4.86 (m, 2H); 6.99 (m, 3H); 7.54 (t, J = 4.8 Hz, 1H); 7.95 (d,
J = 12.5 Hz, 1H); 8.41 (br s, 1H); 8.66 (br s, 1H); 8.88 (d,
J = 7.5 Hz, 1H). 13C NMR (300 MHz, CDCl3) dC : 14.45; 21.92;
22.00; 22.03; 22.07; 22.15; 24.64; 27.23; 27.92; 30.66; 39.06;
46.83; 46.86; 46.89; 49.85; 52.10; 53.03; 53.39; 57.41; 61.93;
71.31; 76.41; 77.22; 81.97; 105.37; 108.15; 112..73; 119.21;
121.46; 122.58; 123.43; 126.85; 137.00; 144.28; 146.60; 147.47;
150.79; 154.94; 163.65; 165.94; 167.32; 169.23; 171.21; 176.76.
(+)-LREIMS m/z (%): 911 ([M+H]+, 100%); (+)-HRESIMS: 911.4917
[M+H]+ (calcd. for C47H67FN6O11, 911.4924).
4.1.3.4. Compound 24.
(R)-5-(((Benzyloxy)carbonyl)amino)-
2-(2,3-diisopropoxybenzamido) pentanoic acid (22) (1 g,
2.13 mmol) was coupled to (S)-tert-butyl 2-amino-3-tert-butoxy-
propanoate (23) (0.460 g, 2.13 mmol) in anhydrous DMF (10 mL)
and triethylamine (0.7 mL, 5.33 mmol) using TBTU as coupling
agent (1.030 g, 3.20 mmol). The solvent was removed under re-
duced pressure and the residue was pre-absorbed onto silica and
purified by column chromatography (silica gel, gradient of hex-
anes/ethyl acetate from 1:1 to 1:2) to give 1.020 g of compound
24 as a light yellow oil in a 70% yield: ½a D31
ꢂ
: +17.3 (CHCl3,
c = 3.595). 1H NMR (200 MHz, CDCl3) dH: 1.08 (s, 9H); 1.26 (d,
J = 6.3 Hz, 3H); 1.34 (d, J = 6.3 Hz, 3H); 1.35 (d, J = 6.0 Hz, 3H); 1.
38 (d, J = 6.0 Hz, 3H); 1.41 (s, 9H); 1.53–1.71 (m, 2H); 1.73–1.89
(m, 2H); 1.90–2.02 (m, 2H); 3.53 (dd, J = 8.8, 3.1 Hz, 1H); 3.74
(dd, J = 8.8, 2.9 Hz, 1H); 4.56 (m, 2H); 4.79 (m, 2H); 5.07 (s, 2H);
7.04 (t, J =7.3 Hz, 1H); 7.05 (dd, J = 7.3, 1.7 Hz, 1H); 7.68 (dd,
J = 7.3, 2.3 Hz, 1H); 8.76 (m, 1H). 13C NMR (200 MHz, CDCl3) dC:
20.56; 21.14; 21.38; 21.58; 21.62; 21.74; 21.85; 26.82; 27.49;
30.88; 38.12; 51.94; 52.84; 59.96; 65.78; 70.77; 75.77; 79.37;
81.04; 122.34; 122.49; 122.94; 123.08; 127.39; 127.43; 127.48;
127.93; 127.96; 146.08; 150.33; 156.29; 164.90; 168.66; 170.55;
172.18. (+)-LREIMS m/z (%): 686 ([M+H]+, 100).
4.1.3.8. Conjugate 8.
To a solution of compound 27 (50 mg,
0.055 mmol) in CH2Cl2 (3 mL), 0. 768 mL (0.768 mmol) of 1 M
BCl3 in heptanes was added at ꢀ78 °C and allowed to reach
ꢀ40 °C for 6 h. Then, the reaction was quenched by the addition
of H2O (3 mL), allowed to reach rt and concentrated in vacuo.
The residue was purified by HPLC (Scharlau C18, flow rate
1.5 mL/min, H2O/MeOH, 55:45 containing 0.05 % TFA) to give
5 mg of conjugate 8 (retention time of 21.43 min) as a white solid
in a 13% yield: ½a D32
ꢂ
: +29.71 (MeOH, c = 0.105). 1H NMR (500 MHz,
CD3OD) dH: 1.57 (t, J = 7.1 Hz, 3H), 1.64–1.79 (m, 2H), 1.79–1.95 (m,
1H), 1.95–2.12 (m, 1H), 3.45 (m, 2H), 3.60 (br s, 4H), 3.70 (br s, 4H),
3.88 (dd, J = 11.3, 3.8 Hz, 1H), 3.97 (dd, J = 11.3, 4.7 Hz, 1H), 4.04 (s,
2H), 4.53–4.63 (m, 4H), 6.78 (t, J = 7.9 Hz, 1H), 6.98 (dd, J = 7.9,
1.4 Hz, 1H), 7.27 (d, J = 7.0 Hz, 1H), 7.36 (dd, J = 7.9, 1.4 Hz, 1H),
8.05 (d, J = 12.9 Hz, 1H), 8.90 (s, 1H). 13C NMR (500 MHz, CD3OD)
dC: 13.45; 25.13; 29.14; 38.42; 46.63; 49.72; 52.35; 52.67; 53.35;
54.83; 56.91; 56.93; 61.29; 106.16; 107.18; 111.19; 111.63;
116.01; 118.45; 118.52; 118.66; 129.34; 137.39; 145.70; 148.37;
156.16; 156.28; 164.26; 168.19; 169.34; 172.00; 172.72; 176.92.
(ꢀ)-LREIMS m/z (%): 713 ([MꢀH]ꢀ, 100%); (ꢀ)-HRESIMS:
713.2581 [MꢀH]ꢀ (calcd. for C33H39FN6O11, 713.2588).
4.1.3.5. Compound 25.
Compound 24 (0.506 g, 0.738 mmol)
was hydrogenated in a similar way as 16 to afford 0.407 g of com-
pound 25 as a colorless oil in a quantitative yield, which was used
without further purification: ½a D31
ꢂ
: +13.9 (CHCl3, c = 0.245). 1H
NMR (300 MHz, CDCl3) dH: 1.15 (s, 9H); 1.31 (d, J = 6.2 Hz, 3H);
1.34 (d, J = 6.0 Hz, 3H); 1.35 (d, J = 6.0 Hz, 3H); 1.41 (d, J = 6.2 Hz,
3H); 1.47 (s, 9H); 1.52–1.68 (m, 2H); 1.87–2.02 (m, 2H); 2.67 (m,
2H); 3.53 (dd, J = 8.3, 3.6 Hz, 1H); 3.60 (dd, J = 8.3, 4.9 Hz, 1H);
4.54 (m, 2H); 4.76 (m, 2H); 7.02 (dd, J = 7.9, 1.8 Hz, 1H); 7.07 (t, J
=7.9 Hz, 1H); 7.69 (dd, J = 7.9, 1.8 Hz, 1H); 8.88 (d, J = 5.6 Hz, 1H).
13C NMR (300 MHz, CDCl3) dC: 21.24; 21.51; 22.15; 22.20; 22.23;
27.44; 27.48; 28.02; 29.76; 41.93; 51.10; 55.64; 62.22; 71.50;
76.51; 80.99; 81.74; 119.49; 123.20; 123.50;128.22; 146.84;
150.90; 165.86; 170.94; 171.52. (+)-LRESIMS m/z (%): 552
([M+H]+, 100%); (+)-HRESIMS: 552.3637 [M+H]+ (calcd. for
4.2. Biological activity
The antibacterial activity was evaluated by determining the
Minimum Inhibitory Concentration (MIC) for each compound.
MIC was defined as the lowest concentration at which no visible
growth was observed. MICs were calculated in a micro-well dilu-
tion assay as follows. Thirty-two serial twofold dilutions of each
assayed compound were prepared to obtain a final concentration
C
29H50N3O7 [M+H]+ 552.3643).
,
4.1.3.6. Compound 26.
Compound 25 (300 mg, 0544 mmol)
was coupled with chloroacetyl chloride (0.11 mL, 1.36 mmol) in a
similar way as 12 and 17. The crude was purified by column chro-
matography (silica gel, CH2Cl2/MeOH 96:4) to afford 203 mg of
range from 100 to 9.2 ꢁ 10ꢀ8
lg/mL in 50 lL of CM9 medium.
compound 26 as brown oil in a 59% yield: ½a D31
ꢂ
: +15.17 (CHCl3,
Overnight cultures of strains RV22, MB12 (non vanchrobactin-pro-
ducing strain) and MB84 (vanchrobactin receptor FvtA mutant) in
TSB medium were used as inocula. The cultures were diluted 1:20
c = 0.530). 1H NMR (200 MHz, CDCl3) dH: 1.07 (s, 9 h); 1.21–1.49
(m, 12H); FALTA 1tBu 1.57–2.04 (m, 4H); 2.24–2.95 (m, 2H);
3.17–3.56 (m, 3H); 3.66–3.85 (m, 1H); 4.02 (s, 2H); 4.44–4.63 (m,
2H); 4.65–4.88 (m, 1H); 6.83–7.16 (m, 3H); 7.61–7.71 (m, 1H);
8.70–8.80 (m, 1H). 13C NMR (300 MHz, CDCl3) dC: 21.90; 22.03;
22.10; 22.21; 25.34; 27.28; 27.93; 30.26; 38.58; 39.18; 42.61;
52.90; 53.22; 61.98; 71.29; 73.03; 76.07; 76.35; 78.55; 81.81;
119.22; 123.07; 123.46; 127.35; 146.53; 150.70; 165.56; 166.08;
in CM9 medium containing 80
dil to achieve iron limiting conditions for bacterial growth. Each
micro-plate well contained a final volume of 100 L of CM9 at a
40
M of 2,20-dipyridil. The growth assays were also performed
in parallel using CM9 medium plus 10
M FeCl3 instead 2,20-
dipyridil to obtain iron repleting growth conditions. The plates
l
M of the iron chelator 2,20-dipyri-
l
l
l

302
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the CAS (chrome-azurol S) spectrophotometric assay.27 This assay
is based in the change of color of CAS dye when it is bound to Fe(III)
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Acknowledgments
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This work was financially supported by Grants from the Minis-
try of Science and Innovation of Spain (CTQ2008-04024 and
AGL2009-12266-C02-02) and Xunta de Galicia (10PXIB235157PR).
We also thank to Servizos de Apoio a Investigación from University
of A Coruña (SAI-UDC) for the analytical support.
Supplementary data
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Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.10.028.
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