Membrane active vancomycin analogues: A strategy to combat bacterial resistance

Article abstract of DOI:10.1021/jm500270w

The alarming growth of antibiotic resistant superbugs such as vancomycin-resistant Enterococci and Staphylococci has become a major global health hazard. To address this issue, we report the development of lipophilic cationic vancomycin analogues possessing excellent antibacterial activity against several drug-resistant strains. Compared to vancomycin, efficacy greater than 1000-fold was demonstrated against vancomycin-resistant Enterococci (VRE). Significantly, unlike vancomycin, these compounds were shown to be bactericidal at low concentrations and did not induce bacterial resistance. An optimized compound in the series, compared to vancomycin, showed higher activity in methicillin-resistant Staphylococcus aureus (MRSA) infected mouse model and exhibited superior antibacterial activity in whole blood with no observed toxicity. The remarkable activity of these compounds is attributed to the incorporation of a new membrane disruption mechanism into vancomycin and opens up a great opportunity for the development of novel antibiotics.

Full text of DOI:10.1021/jm500270w

Article
pubs.acs.org/jmc
Membrane Active Vancomycin Analogues: A Strategy to Combat
Bacterial Resistance
Venkateswarlu Yarlagadda, Padma Akkapeddi, Goutham B. Manjunath, and Jayanta Haldar*
Chemical Biology and Medicinal Chemistry Laboratory, New Chemistry Unit, Jawaharlal Nehru Centre for Advanced Scientific
Research, Jakkur, Bengaluru 560064, Karnataka India
*
S Supporting Information
ABSTRACT: The alarming growth of antibiotic resistant
superbugs such as vancomycin-resistant Enterococci and
Staphylococci has become a major global health hazard. To
address this issue, we report the development of lipophilic
cationic vancomycin analogues possessing excellent antibacte-
rial activity against several drug-resistant strains. Compared to
vancomycin, efficacy greater than 1000-fold was demonstrated
against vancomycin-resistant Enterococci (VRE). Significantly,
unlike vancomycin, these compounds were shown to be
bactericidal at low concentrations and did not induce bacterial
resistance. An optimized compound in the series, compared to
vancomycin, showed higher activity in methicillin-resistant
Staphylococcus aureus (MRSA) infected mouse model and
exhibited superior antibacterial activity in whole blood with no observed toxicity. The remarkable activity of these compounds is
attributed to the incorporation of a new membrane disruption mechanism into vancomycin and opens up a great opportunity for
the development of novel antibiotics.
INTRODUCTION
toward the development of glycopeptide analogues to combat
■
1
0−23
the resistance.
Semisynthetic glycopeptides such as
Since their discovery in mid 1950s, glycopeptide antibiotics
have been a vital weapon in the arsenal of antibiotics to treat
pathogenic Gram-positive bacterial infections. In fact, a
oritavancin, dalbavancin, and telavancin containing hydro-
phobic groups were shown to exhibit high antibacterial activity
1
24
against resistant strains with excellent pharmacological profile.
member of their family, vancomycin, has been considered as
the “drug of last resort” to treat lethal infections caused by
methicillin-resistant Staphylococcus aureus (MRSA), which
makes up for 60% of total ICU infections, in developed
It has also been shown that oritavancin and telavancin impart
an additional mechanism of action, namely bacterial membrane
2
5,26
disruption.
Recently, Boger et al. synthesized vancomycin
2
aglyconamidine, which showed potent antibacterial activity
countries like USA. Emergence of virulent species such as
2
7,28
(
0.31 μg/mL) against vancomycin-resistant enterococci.
The perennial persistence of vancomycin resistance calls for
vancomycin-resistant Enterococci and Staphylococci has
3
,4
currently become major global health concern. Treatment
of these drug-resistant infections is limited by the lack of
an urgent need to develop more potent analogues having
additional mode of action, which would make bacterial
resistance difficult to develop. Bacteria are known to develop
slow resistance against cell membrane disrupting agents like
5
,6
effective antibiotics.
Vancomycin is known to impart its action by acting on the
bacterial cell wall precursors. It forms a tight complex with D-
Ala−D-Ala termini of peptidoglycan precursors present in the
29,30
antimicrobial peptides and peptidomimetics.
Hence, a
1
compound which combines the inherent mode of action of
vancomycin along with membrane disrupting effect is expected
to have not only increased antibacterial potency but also
counter the development of bacterial resistance. We have come
up with a design wherein a lipophilic cationic quaternary
ammonium moiety is appended to the vancomycin molecule to
produce a series of lipophilic cationic vancomycin analogues
developing cell wall. The tight binding of the antibiotic
through five hydrogen bonds prevents transpeptidation, thus
arresting cell wall biosynthesis and maturation, and eventually
1
leading to cell death. Over the years, bacteria recognizing this
vancomycin stress have remodeled their cell wall precursors
from D-Ala−D-Ala to D-Ala−D-Lac and have become
vancomycin resistant (VanA and VanB phenotypes). This
modification is exhibited by five van genes and is sufficient to
lessen the binding affinity of vancomycin to its target by 1000-
fold, and as a consequence its antibacterial activity goes down
by >1000 times (VanA phenotype). Many semisynthetic
approaches have been adopted by various groups including us
7
(
Scheme 1). These analogues possess strong, broad-spectrum
antibacterial activity and are about 1000-fold more effective
8,9
Received: December 16, 2013
©
XXXX American Chemical Society
A
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a
Scheme 1. General Synthetic Scheme for the Preparation of New Vancomycin Analogues
a
Where (Boc) O, di-tert-butyldicarbonate; DIPEA, N,N-diisopropylethylamine; HBTU, N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)uronium
2
hexafluorophosphate.
Table 1. In Vitro Antibacterial Activity of the Compounds
(
MIC, μM)
a
b
c
d
e
compd
MSSA
MRSA
VISA
VSE
VRE
750
vancomycin
0.63 ± 0.01
0.62 ± 0.02
0.39 ± 0.01
0.37 ± 0.04
0.31 ± 0.02
0.31 ± 0.03
1.25 ± 0.4
5.03 ± 0.52
0.55 ± 0.05
0.3
0.63 ± 0.02
0.61 ± 0.02
2.42 ± 0.07
0.6 ± 0.02
0.31 ± 0.01
0.31 ± 0.05
0.65 ± 0.03
13 ± 2.0
1.47 ± 0.5
1.3 ± 0.1
1.14 ± 0.02
0.4 ± 0.02
0.3 ± 0.01
0.36 ± 0.05
0.92 ± 0.2
1.0 ± 0.1
0.6 ± 0.02
0.63 ± 0.01
0.45 ± 0.03
0.31 ± 0.05
0.15 ± 0.03
0.2 ± 0.01
0.12 ± 0.02
1
2
3
4
5
6
7
>100
>100
>100
12.5 ± 0.25
3 ± 0.5
0.7 ± 0.02
1.9 ± 0.05
5 ± 0.5
4.0
f
f
ND
ND
13
0.61 ± 0.02
0.3
0.4 ± 0.02
0.15
g
telavancin
h
dalbavancin
0.04
0.04
0.08
>18
a
b
c
Methicillin-sensitive S. aureus (MTCC 737). Methicillin-resistant S. aureus (ATCC 33591). Vancomycin-intermediate-resistant S. aureus was
d
e
generated from MRSA (ATCC 33591) after treating with vancomycin. Vancomycin-sensitive E. faecium (ATCC 19634). Vancomycin-resistant E.
f
g
31
h
32
faecium (VanA phenotype, ATCC 51559). Not determined. Taken from literature. Taken from literature. MIC90 values were mentioned for
telavancin and dalbavancin. Three separate experiments were done to determine the MIC values of compounds 1−7, 13 as well as vancomycin, and
expressed as mean ± standard deviation.
than vancomycin against VRE (Table 1). Unlike vancomycin,
these compounds were found to be bactericidal and did not
induce the development of bacterial resistance. An optimized
compound, compared to vancomycin, showed higher in vivo
antibacterial activity against MRSA thigh infection model.
RESULTS
■
Synthesis and Characterization. Cationic lipophilic
vancomycin analogues (1−7) were prepared by coupling the
carboxylic group of vancomycin with lipophilic cationic
moieties through amide coupling using N,N,N′,N′-tetrameth-
yl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate
B
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Figure 1. Bactericidal activities of new vancomycin analogues. (A) Bactericidal properties of compounds 4, 6, and vancomycin against MRSA in
media. Stars correspond to <50 CFU/mL. (B) Antibacterial activity of vancomycin and compounds 4 and 6 after 3 h incubation in 90% human
whole blood against MRSA. Stars correspond to <50 CFU/mL.
(
HBTU) (Scheme 1) with 70−80% yield. To prepare
>1000-fold higher activity than vancomycin against VRE. The
MIC values of telavancin and dalbavancin against VRE (VanA
vancomycin carboxamides (1−7), vancomycin was dissolved
in 1:1 dry DMF:DMSO and HBTU solution in DMF was
added dropwise at 0 °C. Subsequently, the desired amine
90
phenotype) were found to be 4 and 18 μM, respectively, which
3
1,32
are similar or less active than compound 6 (Table 1).
(
12a−12f) was added to the vancomycin solution and the
To assess the role of installed cationic feature (permanent
positive charge), we have evaluated the antibacterial activity of
compound 13 having a cationic lipophilic moiety bearing
secondary amine (which becomes cationic under physiological
conditions) and compared the results with corresponding
vancomycin derivative 6 (bearing a quaternary ammonium
group having permanent positive charge). The antibacterial
activity of compound 13 was found to be similar to compound
6 against sensitive bacteria (Table 1). When tested against
VRE, the activity of compound 6 was found to be 7-fold higher
than compound 13 (Supporting Information Figure S1A). Our
results suggest that the appended permanent positive charge
along with lipophilicty afforded substantial increase in
antibacterial activity against VRE.
reaction mixture was stirred at room temperature for 2 h. All
the derivatives of vancomycin were purified by reverse phase
HPLC to more than 95% purity and characterized by H NMR
and MALDI-MS. To synthesize the lipophilic cationic moieties
1
1
1
(
12a−12g), N ,N -dimethylpropan-1,3-diamine was protected
using di-t-butylpyrocarbonate, and then the tertiary amine
group was quaternized by various alkyl bromides (ethyl to
octadecyl) followed by deprotection of primary amine group
under acidic conditions (Scheme 1). An important vancomycin
analogue (compound 13) was also prepared wherein
1
vancomycin was conjugated to N -tetradecylpropan-1,3-dia-
mine, which does not have permanent cationic charge
(Supporting Information Scheme S1).
In Vitro Antibacterial Activities. The antibacterial
To evaluate the activity of cationic lipophilic portion alone
(without vancomycin), we synthesized compound 14 (Support-
ing Information Scheme S2) as a control. The antibacterial
activity of compound 14 was found to be 225-fold lower than
the compound 6 against VRE (Supporting Information Figure
S1B). We also evaluated the activity of a physical mixture of
vancomycin and compound 14. The physical mixture was
found to be inactive, even up to their individual concentrations
of 7 μM against VRE, whereas compound 6 showed MIC of 0.7
μM (Supporting Information Figure S2).
Bactericidal Activity (Time-Kill Kinetics and Whole
Blood Study). Next we carried out in vitro time-kill assay with
compounds 4, 6, and vancomycin against MRSA (starting
bacterial concentration of 8 log10 CFU/mL) at two different
concentrations (1 × MIC and 6 × MIC). Our results portrayed
an impressively rapid bactericidal activity with compounds 4
and 6, which increased with increasing concentration.
Compound 4 produced ∼3 log10 CFU/mL reduction in
bacterial count at 6 × MIC, whereas at 1 × MIC it was limited
activities of vancomycin and its derivatives 1−7 were
determined against vancomycin-sensitive strains of Staph-
ylococci (MSSA and MRSA) and Enterococci (VSE), as well
as against vancomycin-resistant strains of Staphylococci (VISA)
and Enterococci (VRE). The results are summarized in Table 1.
The compounds 4 (octyl chain) and 5 (decyl chain) showed
the best activities against MRSA (MIC = 0.31 μM) and
compound 6 (tetradecyl chain) against VSE (MIC = 0.12 μM).
In case of resistant strain VISA, most of these derivatives were
found to be ∼40-fold more active than vancomycin, the lowest
MIC being in the range of 0.3−0.4 μM (Table 1). When tested
against pathogenic VRE, these derivatives exhibited MIC in the
range of 0.7 to >100 μM. Against the strain VRE, a gradual
decrease in MIC was observed with increasing chain length,
with compound 6 demonstrating an outstanding MIC value of
0
.7 μM. With further increase in chain length, however, the
MIC values showed an increasing trend. We found that
compound 6 possesses an optimum chain length and showed
C
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to being bacteriostatic (∼8 log CFU/mL). Meanwhile,
displaced into the solution. The membrane potential
dissipation of MRSA (Figure 3A) and VRE (Figure 3B) was
observed in less than 3 min by the compounds 4 (octyl chain)
to 7 (octadecyl chain) at 10 μM. This membrane depolarization
effect increased in a concentration dependent manner
(Supporting Information Figure S3). On the other hand
compounds 1−3 (butyl chain) and vancomycin were
ineffective.
1
0
compound 6 was rapidly bactericidal at 1 × MIC and 6 ×
MIC (>5 log10 CFU/mL reduction) and persisted its
bactericidal activity until 24 h at 6 × MIC. Conversely,
bacteriostatic effect was observed at 24 h for compound 6 at 1
×
MIC. In the case of vancomycin, even though concentration
dependent action was seen from 1 × MIC to 6 × MIC, it was
restricted to being bacteriostatic, unlike compounds 4 and 6
(
Figure 1A).
The effect of the new compounds on bacterial membrane
integrity was also confirmed by studying the kinetics of bacterial
cytoplasmic membrane permeabilization by measuring the
uptake of the fluorescent probe propidium iodide (PI). This
dye enters only membrane-compromised cells and fluoresces
upon binding to nucleic acids. Unlike vancomycin and
compounds 1−3, which did not cause significant membrane
permeability, compounds 4−7 showed strong ability to
permeabilize the cytoplasmic membrane of MRSA (Figure
4A) and VRE (Figure 4B) at a concentration of 10 μM.
Membrane permeability increased with increase in concen-
tration and observed within 5 min (Supporting Information
Figure S4).
Also, we performed whole blood assay to compare the
abilities of these compounds to retain activity in complex
media. Compounds 4 and 6 showed rapid bactericidal activity
against MRSA after incubation for 3 h in 90% human whole
blood, whereas vancomycin showed slow killing (Figure 1B).
Propensity to Induce Bacterial Resistance. The growth
of drug resistance in bacteria led us to assess the potential
emergence of bacterial resistance against these compounds. The
propensity of bacteria to generate resistance can be evaluated
35
33
using serial exposure of organisms to antimicrobial agents.
We exposed MRSA to serial passages of compounds 4, 6, and
vancomycin and monitored the changes in MIC values over a
period of 52 days. Even after 52 serial passages, the MIC of
compounds 4 and 6 remained the same (Figure 2). However, in
the case of vancomycin, MIC value started increasing after 20
passages and the value increased to >20-fold after 52 passages
It is known that release of potassium ion from the bacterial
36
cell occurs upon disruption of the membrane potential. We
further investigated the release of potassium ion caused by the
compounds (10 μM) using potassium ion sensitive fluoro-
phore, PBFI-AM against MRSA (Figure 5A) and VRE (Figure
(Figure 2).
5
B), considering valinomycin as a positive control. Compounds
4
−7 caused significant leakage of intracellular potassium like
valinomycin, whereas vancomycin and compounds 1−3 were
ineffective (Figure 5A and 5B). Considering 100% K leakage
for valinomycin, the percentage of K leakage was found to vary
+
+
from 43% for compound 4 to 99% for compound 7
(
Supporting Information Figure S5). Hence, bacterial exposure
to compounds 4−7 resulted in increased membrane perme-
ability, perturbation of cell membrane potential, and finally
+
leakage of intracellular K , while vancomycin and compounds
1
−3 showed no such effect.
To ascertain the role of permanent positive charge, we have
carried out membrane disruption studies with compound 13
having secondary amine which becomes cationic at pH 7.4),
(
and the results were compared with corresponding vancomycin
derivative 6 (quaternary ammonium group having permanent
positive charge). Our results emphasize that compound 13 did
not show any membrane disruption effect at 10 μM, whereas
corresponding vancomycin derivative 6 showed substantial
membrane disruption effect at 10 μM against VRE (Figures 3B,
Figure 2. Bacterial resistance studies. MIC values of vancomycin and
compounds 4 and 6 against MRSA after the number of serial passages
indicated.
4
B, and 5B). This indicates that installed cationic feature
(
permanent positive charge in compound 6) along with
Disruption of Bacterial Membrane Integrity: A New
Mechanism of Action. To confirm our hypothesis that these
compounds act by disrupting the bacterial cell membrane
integrity, we investigated the molecular mechanism of
antibacterial action of all cationic lipophilic vancomycin
derivatives (1−7) using fluorescence spectroscopy against
MRSA and VRE. First, we examined the abilities of these
compounds to depolarize bacterial membranes, using the
lipophilicity plays a vital role in producing membrane
disruption effect thereby having high antibacterial activity.
Also, we have carried out membrane disruption studies of
compound 14 (cationic lipophilic portion alone) and a physical
mixture of vancomycin with compound 14 against VRE. None
of them showed any membrane disruption effect at 10 μM,
which was also reflected in their antibacterial activity against
VRE (Figures 3B, 4B, and 5B). This implies that lipophilic
cationic moiety should be conjugated to vancomycin to impart
membrane disruption properties at low concentrations.
membrane potential sensitive dye DiSC (5) ((3,3′-dipropylth-
3
iadicarbocyanine iodide)). In this experiment, the dye was
added to bacterial cells, and the change in fluorescence intensity
was monitored. As the dye accumulates in the membranes, the
In Vitro Toxicity (Hemolysis and Cytotoxicity). As these
derivatives were shown to disrupt the bacterial cell membrane
integrity, we studied the toxicity of compounds 4 and 6 by
measuring cytotoxicity (EC ) against mammalian cells (HeLa)
34
fluorescence intensity decreases because of self-quenching.
Upon disruption of the membrane potential by the compound,
50
an increase in fluorescence is observed due to DiSC (5) being
and hemolytic activity (HC ) against human RBC (Supporting
3
50
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Figure 3. (A) Cytoplasmic membrane depolarization ability of vancomycin, compounds 1−7 at 10 μM against MRSA (B) Cytoplasmic membrane
depolarization ability of compounds 4−7, control compounds 13 and 14, and physical mixture of vancomycin and 14 at 10 μM against VRE.
Figure 4. (A) Cytoplasmic membrane permeabilization of vancomycin, compounds 1−7 at 10 μM against MRSA (B) Cytoplasmic membrane
permeabilization of compounds 4−7, control compounds 13 and 14, and physical mixture of vancomycin and 14 at 10 μM against VRE.
Figure 5. (A) Intracellular potassium ion leakage ability of vancomycin, compounds 1−7 at 10 μM against MRSA. (B) Intracellular potassium ion
leakage ability of compounds 4−7, control compounds 13 and 14, and physical mixture of vancomycin and 14 at 10 μM against VRE. Valinomycin
used as a positive control in both the experiments at 10 μM.
E
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Information Table S1). None of the derivatives showed
significant toxicity toward either of the cells even up to 100
μM concentration. Notably, compound 4 did not show any
hemolytic activity, even up to 1000 μM. The selectivity (HC /
MIC) of control cationic lipophilic compound 14 was found to
be ≤5, whereas corresponding vancomycin analogue 6 showed
selectivity >140, which indicates the selective toxicity of
compound 6 against bacterial cells compared to control
compound 14.
to the existing drug. The antibacterial activities of these
derivatives were seen to be dependent on the chain length of
the lipophilic moiety as well as on the bacterial strains. In the
case of vancomycin sensitive strains such as MSSA, MRSA, and
VSE, presumably the effect of vancomycin predominates. The
compounds have similar to slightly better efficacy compared to
vancomycin, and the differences in activities among the
compounds are not very dramatic. However, a more definite
trend in activity versus chain length was observed in the case of
the resistant strains such as VISA and VRE, where the cationic
lipophilic unit contributes more significantly to the antibacterial
action. Compounds 1−3 had low (VISA) to no (VRE) activity,
whereas compound 4 onward the antibacterial activity started
to increase, thus successfully overcoming vancomycin resist-
ance. The in vitro antibacterial activities of these new
derivatives were comparable to second generation of
glycopeptides such as oritavancin, telavancin, and dalbavancin
against sensitive bacteria. However, against VRE, compound 6
showed 7-fold, 25-fold, and >1000-fold more activity than
telavancin, dalbavancin, and vancomycin, respectively. The
enhanced antibacterial activity of these cationic lipophilic
vancomycin derivatives may be attributed to their increased
cationic charge and lipophilicity compared to vancomycin,
which facilitates better interaction of the compounds with the
negatively charged bacterial membrane. This increased
association with the bacterial membranes presumably serves
to anchor the drug, thereby allowing it to stay for a longer time
at the cell wall region and disrupt the membrane.
50
In Vivo Antibacterial Efficacy. A widely used animal
model for evaluating antimicrobial activity of preclinical
compounds is the thigh burden model, in which the thigh
muscle of neutropenic mice is inoculated with bacteria,
3
7
followed by iv administration of the compound. To
demonstrate the potential of these compounds for in vivo
applications, we chose the most nontoxic compound 4 as an
example for further studies. The in vivo efficacy of compound 4
was evaluated in a neutropenic mouse thigh infection model. In
6
this study, mice were infected with MRSA (10 CFU/mL) in
the thigh. After 1 h of infection, the mice were treated with a
dual dose (q12 h) of intravenously administered vancomycin
−1
(
12 mg kg ) and compound 4, respectively, saline being used
as control (Figure 6A). After 24 h of the initial treatment,
The molecular mechanism of antibacterial action of these
vancomycin derivatives was evaluated against MRSA and VRE.
The compounds 4−7 (bearing octyl chain and above) caused
rapid membrane depolarization, showed strong ability to
permeabilize the cytoplasmic membrane, and displayed
significant leakage of intracellular potassium. On the other
hand, vancomycin and compounds 1−3 (bearing either no alkyl
chain or smaller alkyl chain length than octyl) did not cause any
membrane disruption. Our results emphasize that octyl chain is
the minimal hydrophobic moiety required to cause bacterial
membrane disruption. It was also found that the membrane
disruption action increased with increase in length of alkyl
chain from octyl chain onward. The detailed mechanistic
studies substantiate that the addition of lipophilic cationic
moieties to vancomycin impart a new mode of action to the
drug, namely that of bacterial membrane disruption. This mode
of action contributes to the rapid bactericidal activity of these
compounds, whereas vancomycin showed bacteriostatic action.
Most significantly, the introduction of this new mechanism of
action to vancomycin had a significant impact on stalling the
development of bacterial resistance to the drug because of the
complexity in remodeling bacterial membrane in a way that is
compatible with bacterial survival.
Next, whole blood assay was used to evaluate the
antimicrobial activities of these derivatives under rigorous
conditions, potentially relevant to their function as therapeutic
agents in vivo. The ability of the compounds to retain
bactericidal activity as demonstrated in whole blood assay
indicates that these derivatives can potentially maintain
antibacterial activity in vivo with insignificant loss to non-
specific interactions with tissue components such as blood.
Further, absence of significant in vitro toxicity against hRBC
and HeLa cells suggests that these compounds interact more
specifically to more negatively charged bacterial cells compared
to mammalian cells.
Figure 6. In vivo antibacterial activity of vancomycin and compound 4
in thigh infection model against MRSA. (A) Experimental design (B)
Experimental result.
antibacterial activity was determined by finding the bacterial
titer in the infected thighs. Compound 4 showed more activity
(ED3−log kill) compared to vancomycin (ED1−log kill) against
MRSA (Figure 6B).
DISCUSSION
■
In this report, permanent cationic (quaternary ammonium
group) hydrophobic moiety was appended to vancomycin using
a facile synthetic methodology to impart a new mode of action
F
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6H), 1.69−1.62 (m, 2H), 1.43 (s, 9H). 13C NMR (100 MHz,
The superior selectivity of the compounds toward bacteria
led us to evaluate the in vivo activity of most nontoxic
compound 4. Compound 4 showed excellent in vivo activity
against MRSA infected mouse model. A plausible explanation
for the compound 4 having high efficacy may be the presence
of cationic lipophilic moiety in the compound causing bacterial
membrane disruption, which leads to its rapid bactericidal
activity.
CD OD) δ/ppm: 157.2, 78.43, 57.32, 45.784, 38.8, 26.2.
3
3
-((tert-Butoxycarbonyl)amino)-N,N-dimethyl-N-alkylpropan-1-
aminium Bromide (11a−11f). Compound 10 (1 g, 4.92 mmol) was
dissolved in dry ethanol (10 mL) in a sealed tube, and appropriate
alkyl bromide (9.84 mmol) was added to it. The reaction mixture was
refluxed for 48 h. Then the required compounds were purified by
column chromatography (CHCl /CH OH) using Silica gel to afford
3
3
quaternized derivatives 11a−11f in 60−65% yield.
3
-((tert-Butoxycarbonyl)amino)-N,N-dimethyl-N-ethylpropan-1-
1
aminium Bromide (11a). H NMR (400 MHz, CDCl ) δ/ppm: 5.76
3
CONCLUSIONS
■
(bs, 1H), 3.64−3.59 (m, 4H), 3.43−3.41 (t, 2H), 3.29 (s, 6H), 3.26−
The clinical translation of such efficient antibiotics is likely to
be enormous. This strategy paves way for a rational approach in
the development of antibiotics for the treatment of
vancomycin-resistant bacterial infections. The incorporation
of lipophilic moiety into vancomycin along with increased
positive charge makes these compounds distinct from other
existing derivatives in their ability to cause strong bacterial
membrane disruption. This is significantly portrayed in the high
antibacterial activity of the compounds against VISA, VRE, and
MRSA and their ability to stall the development of bacterial
resistance. An optimized derivative showed high in vivo
antibacterial efficacy against MRSA and no observed toxicity.
We believe that these derivatives would be a beneficial
extension to the therapeutic armamentarium for the treatment
of infections compelled by drug resistant bacteria.
3.21 (q, 2H), 2.06−1.99 (t, 3H), 1.38 (s, 9H).
3
-((tert-Butoxycarbonyl)amino)-N,N-dimethyl-N-butylpropan-1-
1
aminium Bromide (11b). H NMR (400 MHz, CDCl ) δ/ppm: 5.78
3
(
3
0
bs, 1H), 3.62−3.58 (m, 2H), 3.47−3.40 (t, 4H), 3.29 (s, 6H), 3.24−
.20 (q, 2H), 2.05−1.98 (q, 2H), 1.72−1.64 (m, 2H), 1.39 (s, 9H),
.97−0.93 (t, 3H).
3
-((tert-Butoxycarbonyl)amino)-N,N-dimethyl-N-octylpropan-1-
1
aminium Bromide (11c). H NMR (400 MHz, CDCl ) δ/ppm: 5.65−
3
5.62 (t, 1H), 3.71−3.67 (t, 2H), 3.44−3.39 (t, 2H), 3.33 (s, 6H),
3.29−3.24 (q, 2H), 2.08−2.01 (m, 2H), 1.73−1.67 (m, 2H), 1.35−
13
1
.25 (m, 10H), 1.43 (s, 9H), 0.89−0.86 (t, 3H). C NMR (100 MHz,
CDCl ) δ/ppm: 156.2, 64.67, 62.31, 51.31, 31.59, 29.1, 28.98, 28.4,
3
2
6.3, 23.5, 22.8, 22.5, 14.01.
-((tert-Butoxycarbonyl)amino)-N,N-dimethyl-N-decylpropan-1-
3
1
aminium Bromide (11d). H NMR (400 MHz, CDCl ) δ/ppm: 5.78
3
(
bs, 1H), 3.62−3.61 (t, 2H), 3.41−3.37 (t, 2H), 3.29 (s, 6H), 3.24−
3
.23 (t, 2H), 2.47−2.5 (m, 2H), 2.1−2.06 (m, 2H), 1.68 (m, 2H), 1.39
13
(
s, 9H), 1.28−1.22 (m, 14H), 0.86−0.82 (t, 3H). C NMR (100
MHz, CDCl ) δ/ppm: 156.18, 80.79, 64.26, 61.92, 48.51, 45.00, 31.90,
EXPERIMENTAL SECTION
3
■
2
1
9.63, 29.59, 29.49, 29.38, 29.33, 29.00, 28.29, 26.67, 26.10, 22.66,
4.08.
Materials and Instrumentation. All reagents were purchased
from Sigma-Aldrich and SD Fine and used without further purification.
Analytical thin layer chromatography (TLC) was performed on E.
Merck TLC plates precoated with silica gel 60 F254 (250 μm
thickness). Visualization was accomplished using UV light and Iodine.
Column chromatography was performed on silica gel (60−120 Å pore
size). All final compounds were purified by reverse phase HPLC using
3
-((tert-Butoxycarbonyl)amino)-N,N-dimethyl-N-tetradecylpro-
1
pan-1-aminium Bromide Bromide (11e). H NMR (400 MHz,
CDCl ) δ/ppm: 5.68 (bs, 1H), 3.68−3.64 (t, 2H), 3.43−3.39 (t, 2H),
3
3
2
.33 (s, 6H), 3.29−3.23 (m, 2H), 2.07−2.00 (m, 2H), 1.71−1.7 (m,
13
H), 1.42 (s, 9H), 1.33−1.23 (m, 22H), 0.88−0.84 (t, 3H). C NMR
(100 MHz, CDCl ) δ/ppm: 156.21, 79.5, 64.57, 62.25, 51.34, 37.52,
3
0
.1% trifluoroacetic acid (TFA) in water/acetonitrile (0−100%) as
3
2
1.89, 29.64, 29.61, 29.55, 29.44, 29.36, 29.32, 29.18, 28.41, 26.28,
3.46, 22.77, 22.65, 14.08.
mobile phase to more than 95% purity. HPLC analysis was performed
on a Shimadzu-LC 8 Å liquid chromatography instrument (C18
column, 10 mm diameter, 250 mm length) with UV detector
3-((tert-Butoxycarbonyl)amino)-N,N-dimethyl-N-octadecylpro-
1
pan-1-aminium Bromide (11f). H NMR (400 MHz, CDCl ) δ/
3
monitoring at 270 nm. Nuclear magnetic resonance spectra were
recorded on Bruker (AV-400) 400 MHz spectrometer in deuterated
solvents. MALDI mass spectra (MALDI-MS) were obtained using
Bruker Ultraflex II MALDI/TOF mass spectrometer. Bacterial strains,
Staphylococcus aureus MTCC 737 purchased from MTCC (Chandi-
garh, India) and MRSA ATCC 33591, Enterococcal strains were
obtained from ATCC (Rockvillei, Md). Tryptic-soy agar media was
used for Staphylococci and sheep blood agar plates were used for
Enterococci. Eppendorf 5810R centrifuge was used. TECAN (Infinite
series, M200 pro) plate reader was used to measure absorbance.
Fluorescence measurements were obtained using a PerkinElmer
spectrofluorometer. Human RBCs were used for hemolytic assay,
and HeLa cells (NCCS Pune) were used for cytotoxic studies. Human
whole blood was used for in vitro whole blood assay. Animal studies
were performed according to protocols approved by Animal Ethics
Committee, JNCASR, and Animal Disease Monitoring and
Surveillance, Bangalore, India. CD-1 female mice of six−eight weeks
old were used for all the experiments.
ppm: 5.61−5.59 (t, 1H), 3.72−3.68 (t, 2H), 3.44−3.40 (t, 2H), 3.34
(s, 6H), 3.3−3.25 (q, 2H), 2.09−2.02 (m, 2H), 1.81−1.67 (m, 2H),
13
1
.43 (s, 9H), 1.34−1.25 (m, 30H), 0.89−0.86(t, 3H). C NMR (100
MHz, CDCl ) δ/ppm: 156.1, 79.57, 64.65, 62.3, 51.28, 37.57, 31.9,
3
2
2
9.68, 29.66, 29.63, 29.56, 29.44, 29.36, 29.33, 29.18, 28.4, 26.28,
3.47, 22.78, 22.66, 14.08.
3
-Amino-N-alkyl-N,N-dimethylpropan-1-aminium Chloride
(12a−12f). Quaternized NH-Boc compounds 11a−11f were dissolved
in MeOH and 37% HCl. The reaction mixture was stirred at room
temperature for 5 h and dried in vacuum to afford 3-amino-N-alkyl-
N,N-dimethylpropan-1-aminium chloride derivatives 12a−12f in
quantitative yield.
3-Amino-N-ethyl-N,N-dimethylpropan-1-aminium Chloride
(12a). 1H NMR (400 MHz, D O) δ/ppm: 3.5−3.45 (m, 6H),
2
13
3.15−3.13 (m, 2H), 3.13 (s, 6H), 3.09−3.05 (t, 3H). C NMR (100
MHz, D O) δ/ppm: 61.56, 50.9, 37.8, 28.71, 22.2, 8.5. MALDI-MS:
m/z 130.99 (observed); 131.25 (calculated for M ).
2
+
3-Amino-N-butyl-N,N-dimethylpropan-1-aminium Chloride
1
Synthesis and Characterization. tert-Butyl (3-
(12b). H NMR (400 MHz, CD
3
OD) δ/ppm: 3.54−3.49 (m, 2H),
1
1
(
Dimethylamino)propyl)carbamate (10). N ,N -Dimethylpropan-
3.43−3.38 (m, 4H), 3.17 (s, 6H), 3.11−3.0 (t, 2H), 1.48−1.39 (m,
13
1
,3-diamine (3.27 g, 31.9 mmol) was dissolved in 1 M NaOH
4H), 1.05−1.01 (t, 3H). C NMR (100 MHz, CD OD) δ/ppm: 65.8,
3
solution, and 2 equiv of (Boc) O (27.92 g, 127 mmol) was added to it.
61.9, 51.46, 51.42, 37.67, 25.41, 22.07, 20.05, 13.8. MALDI-MS: m/z
2
+
The reaction mixture was stirred vigorously at room temperature for
160.31 (observed); 159.06 (calculated for M ).
1
0 h. Then the reaction was stopped, and compound 10 was extracted
3-Amino-N-octyl-N,N-dimethylpropan-1-aminium Chloride
1
into the organic layer using chloroform. The resultant organic solution
(12c). H NMR (400 MHz, CD OD) δ/ppm: 3.61−3.56 (q, 2H),
3
was evaporated and dried to afford colorless oily tert-butyl (3-
3.54−3.48 (t, 2H), 3.42−3.38 (t, 2H), 3.18 (s, 6H), 3.3−3.1 (t, 2H),
1
(
dimethylamino)propyl)carbamate with 70% yield. H NMR (400
2.27−2.21 (m, 2H), 1.85−1.8 (m, 2H), 1.39−1.31 (m, 10H), 0.91−
MHz, CD OD) δ/ppm: 3.07−3.04 (t, 2H), 2.4−2.33 (q, 2H), 2.26 (s,
0.88 (t, 3H). 13C NMR (100 MHz, CD OD) δ/ppm: 66.1, 62.0, 61.9,
3
3
G
dx.doi.org/10.1021/jm500270w | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
5
1.4, 45.66, 37.7, 30.2, 27.28, 23.54, 23.52, 22.06, 14.35. MALDI-MS:
7.89 (s, 1H), 7.77−7.48 (m, 8H), 7.42 (s, 1H), 7.3−7.1 (m, 3H),
6.85−6.72 (m, 3H), 6.39−6.38 (d, 1H), 5.9 (bs, 1H), 5.76 (bs, 1H),
5.43−5.18 (m, 9H), 4.85 (s, 1H), 4.68 (s, 1H), 4.45 (s, 3H), 4.19−
4.16 (d, 2H), 4.01−4.0 (d, 2H), 3.71−3.67 (m, 2H), 3.41 (s, 6H),
3.2−2.8 (m, 5H), 2.68−2.66 (m, 3H), 2.32 (s, 2H), 1.73−1.65 (m,
4H), 1.29 (s, 3H), 1.08−1.06 (m, 3H), 0.92−0.86 (m, 8H). MALDI-
+
m/z 216.23 (observed); 216.41 (calculated for M ).
-Amino-N-decyl-N,N-dimethylpropan-1-aminium Chloride
3
1
(
3
2
12d). H NMR (400 MHz, CD OD) δ/ppm: 3.48−3.44 (t, 3H),
3
.37−3.34 (m, 2H), 3.14 (s, 6H), 3.07−3.04 (t, 2H), 2.14−2.2 (m,
13
H), 1.83−1.78 (m, 2H), 1.4−1.29 (m, 16H), 0.91−0.88 (t, 3H).
C
+
NMR (100 MHz, CD OD) δ/ppm: 66.15, 61.99, 51.36, 37.71, 33.01,
MS: 1561.634 (observed), 1561.479 (calculated for M ).
3
3
0.68, 30.59, 30.54, 30.4, 30.23, 27.39, 23.60, 14.36. MALDI-MS: m/z
N,N-Dimethyl-N-(3-(butylamino)propyl)-vancomycin Carboxa-
+
1
2
44.4324 (observed); 244.552 (calculated for M ).
mide (3). Yield 78% (52.3 μmol). H NMR (400 MHz, DMSO-d )
6
3
-Amino-N-tetradecyl-N,N-dimethylpropan-1-aminium Chloride
δ/ppm: 9.35 (s, 1H), 9.09−9.03 (d, 2H), 8.75 (bs, 1H), 8.56 (bs, 1H),
8.18 (bs, 1H), 7.88−7.83 (m, 4H), 7.72−7.21 (m, 6H), 7.12 (bs, 1H),
6.77−6.71 (m, 2H), 6.52 (bs, 1H), 6.38−6.37 (d, 1H), 6.22 (s, 1H),
5.78 (s, 1H), 5.55−5.45 (d, 1H), 5.43−5.19 (m, 4H), 4.92 (s, 1H),
4.72 (s, 1H), 4.55 (s, 1H), 4.29 (s, 1H), 4.28 (bs, 1H), 4.17 (s, 1H),
3.95 (bs, 1H), 3.72−3.43 (m, 6H), 3.19 (s, 6H), 3.17−3.16 (m, 6H),
3.0−2.86 (m, 3H), 2.69−2.62 (m, 2H), 2.38 (s, 1H), 2.20 (S, 1H),
1.95−1.88 (m, 2H), 1.69−1.65 (m, 5H), 1.31−1.24 (m, 2H), 1.08 (s,
2H), 0.92−0.85 (m, 9H). MALDI-MS: 1589.646 (observed), 1589.532
1
(
3
1
(
3
2
12e). H NMR (400 MHz, CD OD) δ/ppm: 3.51−3.45 (t, 2H), 3.4−
3
.35 (m, 2H), 3.15 (s, 6H), 3.09−3.05 (t, 2H), 2.22−2.14 (m, 2H),
13
.84−1.78 (m, 2H), 1.4−1.28 (m, 22H), 0.91−0.88 (t, 3H). C NMR
100 MHz, CD OD) δ/ppm: 66.17, 62.02, 51.42, 49.83, 37.83, 37.74,
3
3.0, 30.72, 30.68, 30.67, 30.59, 30.54, 30.4, 30.22, 27.38, 23.66, 23.62,
2.16, 14.39. MALDI-MS: m/z 300.89 (observed); 300.58 (calculated
+
for M ).
3
-Amino-N-octadecyl-N,N-dimethylpropan-1-aminium Chloride
1
(
3
1
12f). H NMR (400 MHz, CD OD) δ/ppm: 3.51−3.46 (t, 2H),
+
3
(calculated for M ).
.4−3.36 (m, 2H), 3.15 (s, 6H), 3.09−3.05 (t, 2H), 2.2−2.16 (m, 2H),
N,N-Dimethyl-N-(3-(octylamino)propyl)-vancomycin Carboxa-
13
1
.84−1.78 (m, 2H), 1.41−1.28 (m, 30H), 0.91−0.88 (t, 3H).
C
mide (4). Yield 80% (54 μmol). H NMR (400 MHz, DMSO-d )
6
NMR (100 MHz, CD OD) δ/ppm: 66.17, 62.02, 51.41, 37.74, 33.03,
δ/ppm: 9.35 (s, 1H), 9.09−9.03 (d, 2H), 8.75 (bs, 1H), 8.56 (bs, 1H),
8.18 (bs, 1H), 7.88−7.83 (m, 4H), 7.72−7.21 (m, 6H), 7.12 (bs, 1H),
6.77−6.71 (m, 2H), 6.52 (bs, 1H), 6.38−6.37 (d, 1H), 6.22 (s, 1H),
5.78 (s, 1H), 5.55−5.45 (d, 1H), 5.43−5.19 (m, 4H), 4.92 (s, 1H),
4.72 (s, 1H), 4.55 (s, 1H), 4.29 (s, 1H), 4.28 (bs, 1H), 4.17 (s, 1H),
3.95 (bs, 1H), 3.72−3.43 (m, 6H), 3.19 (s, 6H), 3.17−3.16 (m, 4H),
3.0−2.86 (m, 3H), 2.69−2.62 (m, 2H), 2.38 (s, 1H), 2.20 (S, 1H),
1.95−1.88 (m, 5H), 1.69−1.65 (m, 5H), 1.31−1.24 (m, 10H), 1.08 (s,
2H), 0.92−0.85 (m, 9H). MALDI-MS: 1645.734 (observed), 1645.638
3
3
1
0.74, 30.71, 30.62, 30.57, 30.42, 30.24, 27.4, 23.69, 23.63, 22.18,
4+.41. MALDI-MS: m/z 356.53 (observed); 356.68 (calculated for
M ).
tert-Butyl 3-(Tetradecylaminopropyl) Carbamate (13a). tert-Butyl
-aminopropylcarbamate (1 g, 5.73 mmol) was dissolved in dry
dichloromethane (10 mL) in a sealed tube, and tetradecyl bromide
1.58 g, 5.73 mmol) was added to it. The reaction mixture was refluxed
for 24 h. Then the required compounds were purified by column
chromatography (CHCl /CH OH) using silica gel to afford
3
(
+
3
3
(calculated for M ).
1
compound 13a in 40% yield. H NMR (400 MHz, CDCl ) δ/ppm:
N,N-Dimethyl-N-(3-(decylamino)propyl)-vancomycin Carboxa-
3
1
5
2
3
.77 (bs, 1H), 3.15−3.01 (m, 6H), 2.11−2.0 (m, 2H), 1.65−1.53 (Q,
mide (5). Yield 75% (50.3 μmol). H NMR (400 MHz, DMSO-d )
6
H), 1.4 (s, 9H), 1.3−1.28 (m, 22H), 0.85−0.83 (t, 3H). HR-MS: m/z
δ/ppm: 9.33 (s, 1H), 9.07−9.0 (d, 2H), 8.75 (bs, 1H), 8.56 (bs, 1H),
8.18 (bs, 1H), 7.82−7.01 (m, 9H), 6.8−6.7 (m, 2H), 6.52 (bs, 1H),
6.38−6.37 (d, 1H), 5.76−5.62 (m, 2H), 5.48−5.45 (d, 1H), 5.43−5.19
(m, 4H), 4.92 (s, 1H), 4.72 (s, 1H), 4.55 (s, 1H), 4.29 (s, 1H), 4.28
(bs, 1H), 4.17 (s, 1H), 3.95 (bs, 1H), 3.72−3.43 (m, 6H), 3.19 (s,
6H), 3.17−3.16 (m, 4H), 3.0−2.86 (m, 3H), 2.69−2.62 (m, 2H), 2.38
(s, 1H), 2.20 (S, 1H), 1.95−1.88 (m, 5H), 1.69−1.65 (m, 5H), 1.31−
1.24 (m, 14H), 1.08 (s, 3H), 0.92−0.86 (m, 9H). MALDI-MS:
+
70.36 (observed); 370.61 (calculated for M ).
3
-(Tetradecylamino)propan-1-aminium Chloride (13b). Com-
pound 13a was dissolved in MeOH and 37% HCl. The reaction
mixture was stirred at room temperature for 5 h and dried in vacuum
to afford 3-(tetradecylamino)propan-1-aminium chloride (13b) in
1
quantitative yield. H NMR (400 MHz, CDCl ) δ/ppm: 3.18−3.06
3
(
2
(
m, 6H), 2.14−2.05 (m, 2H), 1.73−1.66 (Q, 2H), 1.38−1.29 (m,
+
2H), 0.89−0.86 (t, 3H). HR-MS: m/z 343.392 (observed); 343.42
1673.031(observed), 1673.745 (calculated for M ).
+
calculated for M ).
N,N-Dimethyl-N-(3-(tetradecylamino)propyl)-vancomycin Car-
3
8
1
Vancomycin Carboxamides Derivatives (1−7 and 13).
Vancomycin hydrochloride (100 mg, 67 μmol) was dissolved in dry
dimethylformamide (1 mL) and dry dimethyl sulfoxide (1 mL). To
boxamide (6). Yield 72% (48.2 μmol). H NMR (400 MHz,
DMSO-d ) δ/ppm: 9.34 (s, 1H), 9.08−9.02 (d, 2H), 8.75 (bs, 1H),
6
8.56 (bs, 1H), 8.18 (bs, 1H), 7.88−7.83 (m, 4H), 7.72−7.21 (m, 6H),
7.12 (bs, 1H), 6.77−6.71 (m, 2H), 6.52 (bs, 1H), 6.38−6.37 (d, 1H),
6.22 (s, 1H), 5.78 (s, 1H), 5.55−5.45 (d, 1H), 5.43−5.19 (m, 4H),
4.92 (s, 1H), 4.72 (s, 1H), 4.55 (s, 1H), 4.29 (s, 1H), 4.28 (bs, 1H),
4.17 (s, 1H), 3.95 (bs, 1H), 3.72−3.43 (m, 6H), 3.15 (s, 6H), 3.17−
3.16 (m, 4H), 3.0−2.86 (m, 3H), 2.69−2.62 (m, 2H), 2.38 (s, 1H),
2.20 (S, 1H), 1.91−1.63 (m, 8H), 1.31−1.24 (m, 22H), 1.08 (s, 3H),
0.92−0.83 (m, 9H). MALDI-MS: 1729.908 (observed), 1730.238
1
1
this, 2 equiv of compounds bearing primary amine group [N ,N -
dimethylpropan-1,3-diamine), 3-amino-N-octadecyl-N,N-dimethylpro-
pan-1-aminium chloride (12a−12f), and 3-(tetradecylamino)propan-
1
-aminium chloride (13b)] in 1 mL of dry dimethylformamide was
added. The reaction mixture was cooled to 0 °C, and 0.22 mL (1.5
equiv) of 0.45 M HBTU solution in DMF and 58 μL (5.0 equiv) of
diisopropylethylamine (DIPEA) were added to the reaction mixture.
The reaction mixture was then allowed to warm to room temperature
and stirred for 8−12 h. The product was purified by preparative
+
(calculated for M ).
N,N-Dimethyl-N-(3-(octadecylamino)propyl)-vancomycin Car-
1
reverse-phase HPLC using 0.1% trifluoroacetic acid in H O/
boxamide (7). Yield 80% (54 μmol). H NMR (400 MHz, DMSO-
2
acetonitrile mixture and then lyophilized to afford tris(trifluoroacetate)
salts of final compounds with more than 95% purity (47−54 μmol,
d ) δ/ppm: 9.36 (s, 1H), 9.09−9.03 (d, 2H), 8.75 (bs, 1H), 8.56 (bs,
6
1H), 8.18 (bs, 1H), 7.88−7.83 (m, 4H), 7.72−7.21 (m, 6H), 7.12 (bs,
1H), 6.77−6.71 (m, 2H), 6.52 (bs, 1H), 6.38−6.37 (d, 1H), 6.22 (s,
1H), 5.78 (s, 1H), 5.55−5.45 (d, 1H), 5.43−5.19 (m, 4H), 4.92 (s,
1H), 4.72 (s, 1H), 4.55 (s, 1H), 4.29 (s, 1H), 4.28 (bs, 1H), 4.17 (s,
1H), 3.95 (bs, 1H), 3.72−3.43 (m, 6H), 3.19 (s, 6H), 3.17−3.16 (m,
4H), 3.0−2.86 (m, 3H), 2.69−2.62 (m, 2H), 2.38 (s, 1H), 2.20 (S,
1H), 1.95−1.88 (m, 5H), 1.69−1.65 (m, 5H), 1.31−1.24 (m, 30H),
1.08 (s, 3H), 0.92−0.85 (m, 9H). MALDI-MS: 1786.214 (observed),
7
0−80% yield).
N,N-Dimethyl-N-(3-aminopropyl)-vancomycin Carboxamide (1).
1
Yield 72% (48.2 μmol). H NMR (400 MHz, D O) δ/ppm: 7.76−7.61
2
(
(
(
1
1
m, 5H), 7.34−7.0 (m, 6H), 6.61−6.53 (d, 2H), 6.05 (bs, 1H), 5.85
bs, 1H), 5.58−5.37 (m, 8H), 4.93−4.92 (m, 2H), 4.65 (bs, 2H), 4.3
s, 2H), 4.17−4.16 (t, 2H), 3.85−3.5 (m, 5H), 2.85 (s, 6H), 2.63 (bs,
H), 2.16−2.10 (m, 3H), 1.88−1.68 (m, 4H), 1.47−1.37 (m, 4H),
+
.26−1.22 (m, 4H), 0.96−0.90 (m, 8H). MALDI-MS: mlz 1534.56
1786.638 (calculated for M ).
+
(
observed), 1534.518 (calculated for MH ).
3-(Tetradecylamino)propyl-vancomycin Carboxamide (13). Yield
1
N,N-Dimethyl-N-(3-(ethylamino)propyl)-vancomycin Carboxa-
mide (2). Yield 70% (47 μmol). H NMR (400 MHz, DMSO-d )
δ/ppm: 9.41 (s, 1H), 9.14−9.03 (d, 2H), 8.73 (bs, 1H), 8.56 (bs, 1H),
75% (50.3 μmol). H NMR (400 MHz, DMSO-d
6
) δ/ppm: 9.34 (s,
1
1H), 9.08−9.02 (d, 2H), 8.75 (bs, 1H), 8.56 (bs, 1H), 8.18 (bs, 1H),
7.88−7.83 (m, 4H), 7.72−7.21 (m, 6H), 7.12 (bs, 1H), 6.77−6.71 (m,
6
H
dx.doi.org/10.1021/jm500270w | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
2
5
4
3
H), 6.52 (bs, 1H), 6.38−6.37 (d, 1H), 6.22 (s, 1H), 5.78 (s, 1H),
.55−5.45 (d, 1H), 5.43−5.19 (m, 4H), 4.92 (s, 1H), 4.72 (s, 1H),
.55 (s, 1H), 4.29 (s, 1H), 4.28 (bs, 1H), 4.17 (s, 1H), 3.95 (bs, 1H),
.72−3.43 (m, 6H), 3.17−3.16 (m, 4H), 3.0−2.86 (m, 3H), 2.69−2.62
period, antibacterial activity was determined by finding the bacterial
titer in the infected blood.
42
Resistance Studies. MIC values of vancomycin and compounds 4
and 6 were determined against MRSA as described above. After the
initial MIC experiment, serial passaging was initiated by harvesting
bacterial cells growing at the highest concentration of the compound,
the OD620 of the one-half the MIC well of the previous MIC assay, and
inoculating into fresh yeast−dextrose broth. This inoculum was
subjected to another MIC assay. After 24 h incubation period, cells
growing in the highest concentration of the compound from the
previous passage were once again harvested and assayed for the MIC.
The process was repeated for 52 passages. The MIC value of the
compound was plotted against the number of passages, and the fold
increase in MIC was determined.
(m, 2H), 2.38 (s, 1H), 2.20 (S, 1H), 1.91−1.63 (m, 8H), 1.31−1.24
(m, 22H), 1.08 (s, 3H), 0.92−0.83 (m, 9H). MALDI-MS: 1702.03
(observed), 1701.74 (calculated for M ).
+
N-(3-(Dimethylamino)propyl)acetamide (14a). A mixture of
1
1
N ,N -dimethylpropan-1,3-diamine (4.86 g, 52.8 mmol) and acetic
anhydride (19.5 g, 190.9 mmol) was stirred at room temperature for 1
h, after which acetic anhydride was removed under reduced pressure
and the carboxylate salt was neutralized with sodium hydroxide and
extracted into chloroform. The chloroform was removed to yield
1
compound 14a (8.6 g, 79%) in the salt form. H NMR (400 MHz,
CDCl ) δ/ppm: 6.54 (bs, 1H), 3.32 (t, 2H), 2.78 (t, 2H), 2.52 (s, 6H),
43
Cytoplasmic Membrane Depolarization Assay. Mid log phase
bacterial cells (MRSA, VRE) were harvested, washed with 5 mM
HEPES and 5 mM glucose, and resuspended in 5 mM glucose, 5 mM
3
13
2
1
.01 (s, 3H), 1.86 (m, 2H). C NMR (100 MHz, CDCl ) δ/ppm:
70.9, 55.57, 43.3, 36.9, 24.99, 23.0.
3
8
HEPES buffer, and 100 mM KCl solution in 1:1:1 ratio (10 CFU/
N-(3-Acetamidopropyl)-N,N-dimethyltetradecan-1-aminium Bro-
mL). Measurements were made in a cuvette containing 2 mL of
mide (14). A mixture of 14a and bromo tetradecane (3.63 g,13.1
mmol) was dissolved in acetone (30 mL). Potassium carbonate (1.68
g) was also added. The entire mixture was refluxed for 48 h. The
solution was filtered to remove insoluble potassium carbonate, after
bacterial suspension and 2 μM DiSC (5). The fluorescence of the dye
3
was monitored for 10 min at RT using a PerkinElmer spectro-
fluorometer at an excitation wavelength of 622 nm (slit width: 10 nm)
and emission wavelength of 670 nm (slit width: 5 nm). Dye uptake,
and resultant self-quenching, was modulated by the membrane
potential. After reaching the maximum uptake of the dye by bacteria,
which is indicated by a minimum in dye fluorescence (after 10 min),
test compounds were added to the cells, and the decrease in potential
was monitored by the increase in fluorescence for further 10 min.
which the compound was purified by column chromatography. Yield
1
4
0%. H NMR (400 MHz, CDCl ) δ/ppm: 7.9 (bs, 1H), 3.85 (t, 2H),
3
3
.38 (t, 2H), 3.33 (t, 2H), 3.2 (s, 6H), 2.08 (s, 3H), 2.01 (m, 2H), 1.7
13
(
m, 2H) 1.35−1.25 (m, 22H), 0.88 (t, 3H). C NMR (100 MHz,
CDCl ) δ/ppm: 171.7, 64.9, 63.0, 51.08, 36.11, 31.9, 26.29, 22.6−
2
3
9.65, 14.1.
44
Inner Membrane Permeabilization Assay. Mid log phase (grown
Biological Assays. In Vitro Antibacterial Activity. Minimum
3
9
for 6 h) bacterial cells (MRSA, VRE) were harvested (4000 rpm, 4 °C,
Inhibitory Concentration (MIC). All test compounds were assayed in
microdilution broth format following CLSI guidelines. The com-
pounds were serially diluted using autoclaved Millipore water. Bacteria,
to be tested, were grown for 6 h in the suitable media. Overnight
1
0 min), washed, and resuspended in PBS buffer of pH 7.2. Then test
compounds were added to a cuvette containing 2 mL of cells and 10
μM propidium iodide (PI). The fluorescence of the dye was
monitored for 10 min at RT using a PerkinElmer spectrofluorometer
at excitation wavelength of 535 nm (slit width: 10 nm) and emission
wavelength of 617 nm (slit width: 5 nm). The uptake of PI was
measured by the increase in fluorescence of PI for 10 min as a measure
of inner membrane permeabilization.
9
grown bacteria contained ∼10 CFU/mL (determined by spread
plating method), which was then diluted to 105 CFU/mL using
suitable media. Then 50 μL of serially diluted compound was added to
a 96-well plate containing 150 μL of media containing bacterial
solution. Two controls were made: one containing 150 μL of media
and 50 μL of compound of every concentration and the other
containing 50 μL of media and 150 μL of media containing bacterial
solution. The plate was then incubated at 37 °C, and the O.D. value
was measured at 600 nm using TECAN (Infinite series, M200 Pro)
plate reader. Each concentration had triplicate values, and the whole
experiment was done at least twice and the MIC value was determined
by taking the average of triplicate O.D. values for each concentration
and plotting it against concentration. The data was then subjected to
sigmoidal fitting. From the curve the MIC value was determined, as
the point in the curve where the O.D. is similar to that of control
having no bacteria.
Time-Kill Assay. The bactericidal activity was assessed by the
kinetics or the rate at which it affects the killing action of the
compound. Briefly, MRSA grown in yeast−dextrose broth was used for
time-kill kinetics. Test compounds (vancomycin, compounds 4 and 6)
having final concentrations of 1 × MIC and 6 × MIC (0.6 and 3.6 μM
for vancomycin, and compound 6 and 0.3 and 1.8 μM for compound
+
45
K Leakage Assay. Mid log phase (grown for 6 h) bacterial cells
(
MRSA, VRE) were harvested (4000 rpm, 4 °C, 10 min), washed
twice with 10 mM HEPES (pH 7.2) and 0.5% glucose, and were
resuspended in the same amount of 10 mM HEPES (pH 7.2) and
0
.5% glucose. The bacterial suspension (2 mL) was placed in a
fluorimeter cuvette. The fluorescence of the bacterial suspension was
measured and allowed to stabilize for 60 s at room temperature before
the addition of PBFI-AM dye (1 μM). Data were collected for an
additional 2 min to establish a baseline signal before the addition of
test compounds. The fluorescence signals were collected for each
sample over 1000 s. The fluorescence of the dye was monitored for 10
min at RT using a PerkinElmer spectrofluorometer at an excitation
40
wavelength of 346 nm (slit width: 10 nm) and emission wavelength of
+
5
05 nm (slit width: 5 nm). Percentage of K leakage was calculated
using the formula
+
%
of K leakage = 100[(I − I )/(I − I )]
t
0
∞
1
4
, respectively) was inoculated with MRSA suspensions having starting
Where I is I before the addition of compounds, I is the fluorescence
intensity before the addition of valinomycin, and I∞ is the fluorescence
0
t
1
8
inoculum of 1.6 × 10 CFU/mL. Bacterial suspension containing
specified concentrations of the compound along with control (which
contains only 0.9% saline) was incubated at 37 °C with shaking.
Aliquots (20 μL) were removed from the cultures at 0, 1, 2, 3, 6, and
intensity at 1000 s after the addition.
46
Hemolytic Assay. Erythrocytes were isolated from freshly drawn,
heparanized human blood and resuspended to 5 vol % in PBS (pH
2
4 h and were serially diluted 10-fold in 0.9% saline and plated onto
7
.4). In a 96-well microtiter plate, 150 μL of erythrocyte suspension
sterile yeast−dextrose agar medium. The number of viable cells was
determined by plating each sample onto yeast−dextrose agar medium.
Plates were then incubated for 24 h at 37 °C, CFU was counted, and
the total bacterial log10 CFU/mL was determined.
In Vitro Whole Blood Assay. To 30 μL of MRSA in saline (0.9%
NaCl; 106 CFU/mL), 10 μL of test compounds (vancomycin,
compounds 4 and 6 at 3.6 μM) and 270 μL of fresh human whole
blood were added and incubated at 37 °C for 3 h. After the incubation
was added to 50 μL of serially diluted compounds. Two controls were
made, one without compound and the other with 50 μL of 1 vol %
solution of Triton X-100. The plate was incubated for 1 h at 37 °C.
The plate was then centrifuged at 3500 rpm for 5 min, 100 μL of the
supernatant from each well was transferred to a fresh microtiter plate,
and A540 was measured. Percentage of hemolysis was determined as (A
− A )/(A − A ) × 100, where A is the absorbance of the test well,
4
1
0
total
0
A the absorbance of the negative controls (without compound), and
0
I
dx.doi.org/10.1021/jm500270w | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Atotal the absorbance of 100% hemolysis wells (with Triton X-100), all
sensitive E. faecium; VRE, vancomycin-resistant E. faecium;
at 540 nm.
MIC, minimum inhibitory concentration; HC , 50% hemolytic
50
4
7
Cytotoxicity Assay. Cytotoxicity of the vancomycin and
compounds 4 and 6 was assessed against an HeLa cell line. The
cells were grown in a 96-well plate in DMEM media supplemented
with 10% fetal bovine serum and 5% penicillin−streptomycin until
they reached 70−80% confluency. The cells were then treated with
serially diluted compounds. Two controls were made, one containing
no compound (untreated cells) and the other control being cells
treated with 10 vol % Triton-X 100 solution. Cells were incubated for
concentration; CFU, colony forming units; DiSC (5), 3,3′-
3
dipropylthiadicarbocyanine iodide; PI, propidium iodide; PBFI-
AM, potassium binding benzofuran isophtalate
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Corresponding Author
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jayanta@jncasr.ac.in.
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