A Tobramycin Vector Enhances Synergy and Efficacy of Efflux Pump Inhibitors against Multidrug-Resistant Gram-Negative Bacteria

Article abstract of DOI:10.1021/acs.jmedchem.7b00156

Drug efflux mechanisms interact synergistically with the outer membrane permeability barrier of Gram-negative bacteria, leading to intrinsic resistance that presents a major challenge for antibiotic drug development. Efflux pump inhibitors (EPIs) which block the efflux of antibiotics synergize antibiotics, but the clinical development of EPI/antibiotic combination therapy to treat multidrug-resistant (MDR) Gram-negative infections has been challenging. This is in part caused by the inefficiency of current EPIs to penetrate the outer membrane and resist efflux. We demonstrate that conjugation of a tobramycin (TOB) vector to EPIs like NMP, paroxetine, or DBP enhances synergy and efficacy of EPIs in combination with tetracycline antibiotics against MDR Gram-negative bacteria including Pseudomonas aeruginosa. Besides potentiating tetracycline antibiotics, TOB-EPI conjugates can also suppress resistance development to the tetracycline antibiotic minocycline, thereby providing a strategy to develop more effective adjuvants to rescue tetracycline antibiotics from resistance in MDR Gram-negative bacteria.

Full text of DOI:10.1021/acs.jmedchem.7b00156

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Article
A tobramycin vector enhances synergy and efficacy of efflux pump
inhibitors against multidrug-resistant Gram-negative bacteria
Xuan Yang, Sudeep Goswami, Bala Kishan Gorityala, Ronald Domalaon,
Yinfeng Lyu, Ayush Kumar, George G Zhanel, and Frank M. Schweizer
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b00156 • Publication Date (Web): 11 Apr 2017
Downloaded from http://pubs.acs.org on April 18, 2017
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A tobramycin vector enhances synergy and efficacy of efflux pump inhibitors
against multidrugꢀresistant Gramꢀnegative bacteria
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Xuan Yang , Sudeep Goswami , Bala Kishan Gorityala , Ronald Domalaon , Yinfeng Lyu , Ayush
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Kumar , George G. Zhanel and Frank Schweizer
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Department of Chemistry, University of Manitoba, Winnipeg, MB, R3T 2N2, Canada.
Department of Microbiology, University of Manitoba, Winnipeg, MB R3T 2N2, Canada.
Department of Medical Microbiology, University of Manitoba, Winnipeg, MB R3T 1R9, Canada
Institute of Animal Nutrition, Northeast Agricultural University, Harbin, Heilongjiang, 150030, P.R. China
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Abstract
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Drug efflux mechanisms interact synergistically with the outer membrane permeability barrier of Gramꢀ
negative bacteria leading to intrinsic resistance that presents a major challenge for antibiotic drug
development. Efflux pump inhibitors (EPIs) which block the efflux of antibiotics synergize antibiotics but
the clinical development of EPI/antibiotic combination therapy to treat multidrugꢀresistant (MDR) Gramꢀ
negative infections has been challenging. This is in part caused by the inefficiency of current EPIs to
penetrate the outer membrane and resist efflux. We demonstrate that conjugation of a tobramycin (TOB)
vector to EPIs like NMP, paroxetine or DBP enhances synergy and efficacy of EPIs in combination with
tetracycline antibiotics against MDR Gramꢀnegative bacteria including Pseudomonas aeruginosa. Besides
potentiating tetracycline antibiotics TOBꢀEPI conjugates can also suppress resistance development to the
tetracycline antibiotic minocycline thereby providing a strategy to develop more effective adjuvants to
rescue tetracycline antibiotics from resistance in MDR Gramꢀnegative bacteria.
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INTRODUCTION
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Multidrugꢀresistant (MDR) Gramꢀnegative bacterial infections pose a global threat to human health as our
arsenal to treat infections is quickly drying up with no novel drug classes against Gramꢀnegative pathogens
1
,2
in clinical development Among Gramꢀnegative bacteria, infections caused by Pseudomonas aeruginosa
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are especially challenging as this organism is intrinsically resistant to many classes of antibiotics . The
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Infectious Disease Society of America includes P. aeruginosa in its list of “ESKAPE” pathogens that pose
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the greatest threat to human health . The molecular basis of intrinsic resistance in P. aeruginosa is the
presence of a highly impermeable outer membrane with expression of multiple efflux pumps that effectively
5
reduce the intracellular concentration of the given drug . The inability to discover new antibacterials with
novel modes of action against Gramꢀnegative bacteria in general and P. aeruginosa in particular over the
past five decades demands strategies capable of restoring meaningful activity in antibiotics against resistant
pathogens. One such approach is the use of small moleculeꢀbased adjuvants capable of overcoming
6–8
resistance in Gramꢀnegative bacteria . Examples of adjuvants include βꢀlactamase inhibitors which prevent
inactivation of βꢀlactam antibiotics, membrane permeabilizers which destabilize the outer membrane in
bacteria and efflux pump inhibitors (EPIs). So far only βꢀlactamase inhibitors have been approved as
1
adjuvants for clinical use .
EPIs block the function of efflux pumps in Gramꢀnegative bacteria by competing with the antibiotic
binding site or by perturbation of the outer membrane channel or assembly of the tripartite protein complex
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of ResistanceꢀNodulationꢀDivision (RND) pumps . Alternatively, perturbation or disruption of the proton
9,10
motive force (PMF) which energize efflux pumps can also be used to block the function of RND pumps
.
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12
Several EPIs have been described including 1ꢀ(1ꢀnaphthylmethyl)ꢀpiperazine (NMP) , paroxetine
1
1,13
14
(PAR)
and dibasic peptides like DBP (Figure 1). However, demonstration of efficacy of EPI/antibiotic
1
5,16
combinations has only rarely been documented in animal models of infection
. NMP is a broad spectrum
EPI which synergizes with multiple classes of antibiotics including tetracyclines, fluoroquinolones,
macrolides, penicillins and rifampicin against certain clinically relevant Gramꢀnegative pathogens like E.
1
2,17–19
coli, Acinetobacter baumannii and Klebsiella pneumoniae but not P. aeruginosa
. PAR is a weak EPI
but potentiates tetracyclines and fluoroquinolones in Gramꢀpositive and Gramꢀnegative bacteria excluding P.
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aeruginosa . PAR is a selective serotonin uptake inhibitor and clinically used to treat depression and
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multiple types of mental disorders . DBP is an analogue of dibasic dipeptide DꢀAlaꢀDꢀhPheꢀ
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aminoquinoline (MCꢀ04,124 ) a former drug candidate that strongly synergizes fluoroquinolone antibiotics
1
4,15
14,15,21
in P. aeruginosa
and displays combined EPIꢀ and membraneꢀdestabilizing effects
. Mechanistic
studies with NMP and DBP analogs using the E. coli AcrB efflux pump indicated that both EPIs bind to
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their respective binding sites resulting in a distortion of the binding pocket that prevents effective binding of
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the antibiotic . Although, EPIs such as DBP, NMP and PAR inhibit efflux of tetracycline and
fluoroquinolone antibiotics in certain Gramꢀnegative bacteria they are still subject to intrinsic resistance in
organisms like P. aeruginosa. As a result, strategies that enhance outer membrane penetration and reduce
efflux of EPIs or the combination of EPI/antibiotic are expected to provide more effective adjuvants. To
reduce intrinsic resistance, we decided on a strategy that utilizes a tobramycin (TOB) vector to deliver the
EPI through the outer membrane of P. aeruginosa. It was anticipated that joining a TOBꢀbased vector to an
EPI would facilitate penetration through the outer membrane of P. aeruginosa by the selfꢀpromoted uptake
23
24–28
of aminoglycosides and amphiphilic aminoglycosides
. In addition, the resultant amphiphilic TOBꢀEPI
conjugate was expected to reduce efflux as aminoglycosides are considered to be poor substrates for most
11
29
efflux pumps in P. aeruginosa except the MexXYꢀOprM pump . Moreover, we anticipated that the
amphiphilic nature of the TOBꢀEPI conjugate may perturb the proton motive force of Gramꢀnegative
bacteria resulting in reduced susceptibility to efflux as recently reported for tobramycinꢀfluorquinolone
23,24
conjugates
.
RESULTS
Synthesis and Antibacterial Properties of Tobramycin-NMP Conjugates 1a-f, Tobramycin-
paroxetine Conjugate 2 and Tobramycin-DBP Conjugate 3. With this in mind, we prepared a series of
TOBꢀNMP conjugates 1a-f differing in the length of the tether conjoining TOB and NMP (Figure 1). NMP
was selected as it does not potentiate effluxꢀprone antibiotics against P. aeruginosa but instead inhibits
1
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efflux pumps in E. coli and A. baumannii . The absence of EPI function in P. aeruginosa by NMP is likely
the result of intrinsic resistance. The secondary amino function of the piperazine ring in NMP was linked to
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the Cꢀ5 position in tobramycin as this position was expected to promote outer membrane penetration in
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TOBꢀNMP conjugates 1aꢀf .
The synthesis of TOBꢀNMP conjugates 1b-f (Scheme 1) were performed as follows. Tobramycin was
treated with diꢀtertꢀbutyl dicarbonate and triethylamine to yield the Bocꢀprotected tobramycin intermediate 4.
Silyl protection of the hydroxyl groups, all except at position C5, was achieved by treating 4 with
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TBDMSCl and 1ꢀmethylimidazole in DMF, to provide intermediate 5 in excellent yield
. Reaction of
compound 5 with 1,n dibromoalkanes gave the bromoalkylated tobramycin derivatives 6b-f, which were
further coupled to NMP in the presence of potassium carbonate to produce protected conjugates 7b-f. After
deprotection, the desired TOBꢀNMP conjugates 1b-f bearing four carbon to twelve carbon tethers were
obtained. However, we were unable to prepare conjugate 1a following this protocol. Conjugate 1a bearing a
C2 tether was prepared as outlined in Scheme 2. Protected tobramycin intermediate 4 was treated with allyl
bromide to generate allylꢀC5 linkedꢀtobramycin intermediate 8. Dihydoxylation of the double bond was
performed to yield intermediate 9 followed by oxidative cleavage to generate aldehyde 10. The protected
conjugate 7a was prepared via reductive amination of aldehyde 10 with NMP. Deblocking was achieved by
exposure to methanolic HCl to afford desired, unprotected TOBꢀNMP conjugate 1a.
The antibacterial activity using the minimal inhibitory concentration (MIC) of TOBꢀNMP conjugates 1aꢀf
were evaluated against a panel of clinically relevant pathogens but none of the conjugates demonstrated
potent Gramꢀpositive (MIC > 8 µg/mL) or Gramꢀnegative (MIC > 32 µg/mL) activity (Supplementary Table
1
). We then assessed the adjuvant functions of 1aꢀf by using the fractional inhibitory concentration (FIC)
30
index as a measure of the interaction between two agents. FIC indices of > 0.5 – < 4, ≤ 0.5, and ≥ 4
3
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indicate no interaction, synergy and antagonism, respectively . We performed combination studies of 1aꢀf
with the tetracycline antibiotic minocycline which has been shown to be a substrate of P. aeruginosa RND
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efflux pumps . Moreover, minocycline was selected as it inhibits preferentially the biosynthesis of envelope
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proteins which may further compromise the intrinsic resistance barrier. We observed synergistic effects of
conjugates 1bꢀf with minocycline (FIC index 0.13 – 0.28) in P. aeruginosa PAO1 but no synergistic effects
with 1a (FIC index > 0.5) or tobramycin (FIC index = 1.06), indicating that a tether > 2 carbon atoms is
required for synergy (Table 1). As expected, no synergy was observed with a combination of NMP and
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minocycline (FIC index > 1.0). These results indicate that TOBꢀNMP conjugates can overcome the intrinsic
resistance of the pump inhibitor NMP in P. aeruginosa PAO1. Although TOBꢀNMP conjugates 1dꢀf show
comparable FIC indices, adjuvant 1f bearing a C12 tether between TOB and EPI required the lowest
concentration to achieve optimal synergy with minocycline (FIC index = 0.19). For instance, a fixed
concentration of 1f (8 µg/mL (7.2 µM)) achieved a 16ꢀfold reduction of the MIC of minocycline (MIC = 8
µg/mL reduced to 0.5 µg/mL) against P. aeruginosa PAO1 (Table 1).
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Intrigued by the potent adjuvant function of TOBꢀNMP 1f we also prepared TOBꢀPAR conjugate 2 and
TOBꢀDBP conjugate 3 both bearing a C12 tether and explored their synergistic effects with minocycline.
The synthesis of TOBꢀPAR conjugate 2 (Scheme 3) were performed as follows. The bromoalkylated
tobramycin intermediate 6f was treated with commercially available paroxetine to yield TOBꢀPAR
intermediate 11. Deblocking of the Boc and TBDMS protecting groups was achieved by exposure to
methanolic HCl to afford TOBꢀPAR conjugate 2. The preparation of TOBꢀDBP conjugate 3 (Scheme 4) was
performed as follows. The bromine substituent in intermediate 6f was converted to alcohol 12. Oxidation of
primary alcohol 12 using PCC produced aldehyde 13 in good yield. Aldehyde 13 was coupled to dipeptideꢀ
based amine 14 via reductive amination (see supporting information for detailed procedures of synthesis 14)
to afford the TOBꢀDBP intermediate 15. Deprotection under acidic condition followed by catalytic
hydrogenation provided desired TOBꢀDBP conjugate 3.
Similarly to TOBꢀNMP 1f, both TOBꢀPAR conjugate 2 (FIC index = 0.19) and TOBꢀDBP conjugate 3
(FIC index = 0.09) demonstrated strong synergism with minocycline against P. aeruginosa PAO1 (Table 1).
As expected no synergy was observed with paroxetine (FIC index = 1) while strong synergy was observed
with the P. aeruginosaꢀactive pump inhibitor DBP (FIC index = 0.13) as shown in Table 1. Comparing the
adjuvant properties of DBP with TOBꢀDBP (3) to potentiate minocycline shows that TOBꢀDBP shows
enhanced potency than DBP. For instance, using a fixed concentration (8 µg/mL) of DBP (12.1 µM) or
TOBꢀDBP (5.8 µM) resulted in 8ꢀ or 64ꢀfold reduction in MIC of minocycline in P. aeruginosa PAO1,
respectively indicating that the presence of a TOB vector in DBP enhances the adjuvant properties (Table 1).
Besides potentiating minocycline, we also demonstrated that adjuvants 1f, 2 and 3 strongly synergized (0.13
≤ FIC index ≤ 0.25) with other members of the tetracycline class of antibiotics including doxycycline and
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tigecycline leading to a 8ꢀ to 64ꢀfold MIC reduction at a fixed concentration (≤ 8 µg/mL) in P. aeruginosa
PAO1 (Supplementary Table 2). It is noteworthy that synergistic potentiation of minocycline in P.
aeruginosa PAO1 could not be achieved with detergentꢀlike outer membrane permeabilizing agents such as
benzethonium chloride (FIC index > 1) and cetrimonium bromide (FIC index = 0.75) and only marginal
synergy was observed with outer membrane permeabilizing colistin (FIC index = 0.5) (Supplementary Table
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3).
Next we assessed the effect of the TOBꢀEPI conjugates 1f, 2 and 3 in combination with minocycline
against a panel of 8 clinical P. aeruginosa isolates including six MDR (nonꢀsusceptible or resistant to > 3
chemically unrelated antiꢀpseudomonal classes), and six extremelyꢀdrug resistant (XDR) (nonꢀsusceptible or
resistant to > 5 chemically unrelated antiꢀpseudomonal classes (Supplementary Table 4), obtained from
different Canadian hospitals. The panel also included two colistinꢀnonꢀsusceptible or resistant P. aeruginosa
strains. The results indicate that all three TOBꢀEPI conjugates demonstrated strong synergistic effects (0.02
<
FIC index < 0.38) with minocycline against the selected panel of MDR and XDR P. aeruginosa isolates
(Table 2). The following FIC index ranges for the TOBꢀEPI conjugates against the selected 8 MDR or XDR
P. aeruginosa isolates were observed: TOBꢀNMP (0.02 < FIC index < 0.28); TOBꢀPAR (0.02 < FIC index <
0
.38) and TOBꢀDBP (0.03 < FIC index < 0.31) as shown in Table 2. We also measured the absolute MIC of
minocycline against the 8 MDR/XDR P. aeruginosa isolates in the presence of conjugates 1f, 2 and 3 at a
fixed concentration (< 8 µg/mL, < 0.25 × MIC). These results show that all three conjugates lower the MIC
of minocycline from 8 ꢀ 256 fold against the 8 selected MDR or XDR P. aeruginosa isolates. Importantly, in
96 % of cases, conjugates 1f, 2 and 3 at a concentration of < 8 µg/mL, < 0.25 × MIC reached minocycline
susceptibility (MIC < 1 µg/mL) against the 8ꢀselected MDR or XDR P. aeruginosa isolates (Table 2).
To demonstrate that the adjuvant properties of the conjugates translate into a measurable in vivo effect, we
selected the Galleria mellonella larvae infection model that is an established in vivo model to study the
16,33
efficacy of antimicrobial therapy in P. aeruginosa . In pilot studies, we determined that conjugates 1f and
cause ≤ 5% hemolysis of ovine erythrocytes at 1000 µg/mL (Supplementary Figure 1) and show low
2
cytotoxicity (CC50 > 30 µM) against cancer cell lines while increased toxicity was noted for conjugate 3
(Supplementary Figure 2). Tolerability studies in G. mellonella using a dosage of 200 mg/kg of 1f or 2 or
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NMP or PAR showed no toxic effects up to 96 hours while a dose of 100 mg/kg of 3 or DBP resulted in
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100% or 80% killing of the larvae after 24 hours (Supplementary Figure 3). The toxicity of conjugate 3 or
DBP in the larvae prevented further use of this compound in the insect model. Efficacy studies were
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performed by infecting the larvae with a lethal dose of (1.0 × 10 CFUs) of XDR P. aeruginosa strain
#101856 followed by injection of the drug combination 2 hours post infection. Monotherapy with a single
dose (75 mg/kg) of minocycline or 1f (75 mg/kg) or NMP (75 mg/kg) resulted in 100% killing of the larvae
within 24 hours indicating that monotherapy was not able to provide protection of the larvae. In contrast,
combination therapy (37.5 mg/kg 1f + 37.5 mg/kg minocycline or 75 mg/kg 1f + 75 mg/kg minocycline)
resulted in 10% or 77% survival of the larvae, respectively, after 24 hours (Figure 2a). Similarly, efficacy
was seen for conjugate 2. For instance, single dose combination therapy (75 mg 2 + 75 mg minocycline)
resulted in 56% survival of the larvae while single dose monotherapy with minocycline (75 mg/kg) or
conjugate 2 (75 mg/kg) resulted in 100% killing after 24 hours (Figure 2b). Moreover, combination studies
of NMP or PAR (75 mg/kg) with minocycline (75 mg/kg) resulted in 100 % deaths of the larvae after 24
hours (Figure 2a and Figure 2b). These results suggest that combinations of minocycline/1f and
minocycline/2 possess therapeutic potential.
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6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Besides P. aeruginosa, we also studied the effect of the conjugates 1f, 2 and 3 to synergize minocycline in
other clinically relevant MDR Gramꢀnegative pathogens such as Klebsiella pneumoniae and Enterobacter
cloacae (Supplementary Table 5). Our results show that all three conjugates were able to synergize
minocycline in these pathogens leading to a 4ꢀ to 256ꢀfold reduction in MIC of minocycline at a fixed
concentration (≤ 8 µg/mL) of the conjugates (Table 3). In both pathogens the potency of the adjuvants 1f, 2
and 3 was superior when compared to NMP, PAR or DBP. These results indicate that the adjuvant function
of the conjugates is not limited to P. aeruginosa and can be extended to other clinically relevant Gramꢀ
negative pathogens. However the properties of the adjuvants appear to be optimal for P. aeruginosa.
Mechanistic Studies. To gain insight into the protective function of the conjugates, we performed a series
of mechanistic studies with P. aeruginosa PAO1. At first, we demonstrated that combinations of
bacteriostatic minocycline (0.5 × MIC) and 1f (0.5 × MIC) become bactericidal and result in complete
eradication of the pathogen within 24 hours (Supplementary Figure 4). Similar killing rates were observed
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Journal of Medicinal Chemistry
for minocycline/2 while a combination of minocycline/3 resulted in complete killing of the organism after 9
hours. We demonstrated that conjugate 1f permeabilizes the outer membrane of PAO1 in a doseꢀdependent
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
15
manner using the wellꢀestablished outer membrane permeability NPN (1ꢀNꢀphenylnapthylamine) assay
(Figure 2c). Similar doseꢀdependent permeability was seen for conjugates 2 and 3 (Supplementary Figure 5).
Next, we assessed whether the combination of conjugates 1f, 2 or 3 with outer membrane impermeable
antibiotics are synergistic in P. aeruginosa PAO1. For all three conjugates we observed strong synergy: with
rifampicin (FIC index < 0.05), novobiocin (FIC index < 0.1), chloramphenicol (FIC index < 0.1),
trimethoprim (FIC index < 0.1), vancomycin (FIC index < 0.15), erythromycin (FIC index < 0.2) indicating
that the conjugates enhance cellular uptake of these antibiotics into P. aeruginosa PAO1 (Figure 2d). As
rifampicin is not a substrate for P. aeruginosa RND efflux pumps these results must reflect enhanced
membrane penetration (Supplementary Table 6).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
We also assessed the ability of conjugates 1f, 2 or 3 to depolarize the cytoplasmic membrane in P.
aeruginosa PAO1 using the membrane potentialꢀsensitive dye 3,3′ꢀdipropylthiadicarbocyanine iodide
2
8
(DiSC (5)) . The results demonstrate that the three conjugates induce doseꢀdependent depolarization of the
3
cytoplasmic membrane in a comparable manner to colistin (Figure 2e and Supplementary Figure 6).
Significant depolarization of the cytoplasmic membrane was seen for conjugates 1f, 2 and 3 at a
concentration ≥ 8 µg/mL. Moreover, we also assessed the effect of the conjugates 1f, 2 or 3 on the
flagellumꢀdependent swimming motility of P. aeruginosa PAO1 and observed a strong and concentrationꢀ
dependent reduction in motility at sub MIC concentration. For instance, greatly reduced motility was
observed for all three conjugates at 1/64 × MIC (Figure 2f and Supplementary Figure 7). Reduction in
motility was further enhanced by addition of minocycline which has no effect on motility by itself
3
4
(
Supplementary Figure 8). As flagellar function requires an intact proton motive force (PMF) , our results
suggest that conjugates 1f, 2 or 3 dissipate the PMF at sub MIC concentration that perhaps also results in a
reduction in efflux pump activity leading to an increase in intracellular concentration of minocycline. We
would also like to point out that the synergy of minocycline activity by conjugates 1f, 2 or 3 was maximal
when RND efflux pumps are present, and that there is a decrease in synergy in strains lacking RND efflux
pumps. This observation is based on testing 1f, 2 or 3 in P. aeruginosa PAO200 (PAO1:ꢁmexABꢀoprM) as
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well as P. aeruginosa PAO750 (ꢁmexABꢀoprM, ꢁmexCDꢀoprJ, ꢁmexEFꢀoprN, ꢁmexXY, ꢁmexJK,
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
35
ꢁ
opmH) (Supplementary Table 6) . The decrease in synergy in absence of RND efflux pumps suggests
that, in addition to dissipating the PMF, conjugates 1f, 2 or 3 perhaps also inhibit the activity of RND
pumps, particularly that of the minocyclineꢀrelevant MexABꢀOprM, in a more specific fashion. The effect of
conjugates 1f, 2 or 3 on the uptake of tetracycline was also investigated in P. aeruginosa PAO1 using an
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
36
uptake assay for Gramꢀnegative bacteria This assay relies on monitoring the enhancement of tetracycline
as it enters the cell (Figure 3). Our results indicate that conjugates 1f, 2 or 3 enhance the uptake of
tetracycline in a concentrationꢀdependent fashion. Comparable enhancements in tetracycline uptake were
also observed with colistin while CCCP an uncoupler of oxidative phosphorylation which disrupts the
3
7
proton gradient of the bacterial membranes results in decreased uptake of tetracycline antibiotics . In
contrast, NMP and PAR where unable to enhance the uptake of tetracycline in PAO1. However, DBP
showed a slight increase in tetracycline uptake at the highest concentration tested consistent with the dual
effluxꢀinhibitory/membrane destabilizing effect of this compound.
We also assessed the potential of adjuvants 1f, 2 or 3 to delay the occurrence of resistance in P.
aeruginosa after 25 serial passages in the presence of subinhibitory concentrations (MIC/2) of
adjuvant/minocycline mixture (1:1 mass ratio). Our results indicate that all three adjuvants reduce the
potential of resistance development when coꢀadministered with minocycline. After 25 days of exposure, the
most effective adjuvants 2 and 3 led to a 1ꢀfold increase in the relative MIC of adjuvant/minocycline
mixture compared to MIC obtained for the firstꢀtime exposure while monotherapy with minocycline,
tobramycin or colistin resulted in 16ꢀfold, 256ꢀfold and >1024ꢀfold increase of their MIC, respectively. In
comparison, the combination of DBP + minocycline also delayed resistance development but required a 4ꢀ
fold higher mass ratio of both components (8ꢀfold higher stoichiometric quantity of 3) to select for a 1ꢀfold
increase in the relative MIC (Figure 2g).
DISCUSSION
Infections caused by P. aeruginosa are difficult to treat. Our current armamentarium to combat P.
aeruginosa infections is limited to select penicillins (eg. piperacillin/tazobactam), cephalosporins (eg.
1
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Journal of Medicinal Chemistry
ceftolozane/tazobactam), carbapenems (eg. imipenem), fluoroquinolones (eg. ciprofloxacin),
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
aminoglycosides (eg. tobramycin) but resistance to these agents is steadily increasing with no new novel
1,2
antiꢀpseudomonal agents in clinical development . Our inability to develop antipseudomonal agents with
novel modes of action demands to explore alternative strategies. One such strategy is the search for small
moleculeꢀbased adjuvants which when used in combination with legacy antibiotics rescue the antibiotic
from resistance. Several principal modes of action by which an adjuvant rescues an antibiotic from
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
38
resistance are possible : (1) an adjuvant prevents the degradation or modification of an antibiotic; (2) an
adjuvant allows the accumulation of an antibiotic by enhancing uptake; (3) an adjuvant allows the retention
of an antibiotic by inhibiting the efflux pumps; (4) an adjuvant inhibits the intrinsic repair pathway or
tolerance mechanism of cells to the antibiotic and (5) an adjuvant affects the physiological state of bacteria
for instance by preventing the formation of biofilm. Clinical use of adjuvants is currently limited to
combinations of βꢀlactam antibiotics/βꢀlactamase inhibitors which prevent degradation of βꢀlactam
antibiotics. Adjuvants which inhibit efflux pumps and/or enhance cell penetration in Gramꢀnegative bacteria
3
9,40
are known and have recently been reviewed
but their clinical efficacy/safety has not been demonstrated.
Contemporary examples of adjuvants which enhance outer membrane permeability include polymyxin
41
analogs such polymyxin B nonapeptide , octanoylꢀThrꢀAbuꢀcyclo[DabꢀDabꢀDPheꢀLeuꢀDabꢀDabꢀThr]
3
9
39
(
SPR7061 ) and acetylꢀThrꢀDSerꢀcyclo[DabꢀDabꢀDPheꢀLeuꢀDabꢀDabꢀThr] (SPR741 ), cationic steroid
42
43
antibiotics and oligoꢀacylꢀlysyls but also dicationic EPIs such as NMP, DBP and the recently optimized
4
4
45,46
4
ꢀoxoꢀ4Hꢀpyrido[1,2ꢀa]pyrimidine analog and pyranopyridine analogs . Most of these adjuvants have in
common to possess a polycationic (≥ +2 charge). A potential drawback of polycationic adjuvants is the risk
4
7
to induce nephrotoxicity and to concentrate in acidic vesicles as seen for certain DBPs . However, a number
of polycationic molecules are devoid of nephrotoxicity in clinical settings. These include among others the
4
8
antibiotic plazomicin and the dibasic macrolides suggesting that nephrotoxicity may not be an inherent
property of polycationic molecules.
In this project, we proposed that the adjuvant properties of known EPIs such as NMP, paroxetine and
DBP could be enhanced by linking them to a tobramycin vector. We hypothesized that (a) tobramycinꢀEPI
conjugates should facilitate outer membrane uptake of the EPI via the selfꢀpromoted uptake mechanism of
1
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aminoglycosides; (b) reduce efflux as aminoglycosides are poor substrates for most RND pumps and (c)
depolarize the cytoplasmic membrane because of the cationic amphiphilic nature of the conjugates. Our
results demonstrate that the adjuvant properties of three EPIs is greatly improved by linking them via a 12
carbon tether to a tobramycin vector. The resultant TOBꢀEPI conjugates 1f, 2 or 3 strongly synergize with
outer membrane impermeable agents including novobiocin, rifampicin, vancomycin, erythromycin,
chloramphenicol and trimethoprim against wildꢀtype P. aeruginosa PAO1 and enhance outer membrane
penetration in the NPN assay. Some of these outer membrane impermeable agents including rifampicin
which shows the highest synergy are not substrates of RND efflux pumps suggesting that the TOBꢀEPI
conjugates destabilize the outer membrane in P. aeruginosa. This result is consistent with related
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
3,24,26,27,58
amphiphilic tobramycin analogs described by us and other research groups.
However, the membrane effects of adjuvants 1f, 2 or 3 are not limited to the outer membrane but also
involve the cytoplasmic membrane as observed in the inner membrane depolarization, motility assays and
tetracycline uptake assays. These assays indicate that adjuvants 1f, 2 or 3 decrease the electrical component
of the proton motive force in P. aeruginosa PAO1. In order to counter this effect and maintain ATP
synthesis levels bacteria increase the pH gradient across the inner membrane that results in enhanced uptake
of tetracycline antibiotics in gramꢀnegative bacteria as previously observed for the antidiarrheal drug
3
6
loperamide . This interpretation is consistent with (i) the additive (nonꢀsynergistic) effect of the conjugates
1f, 2 and 3 with the aminoglycosides tobramycin and gentamicin (2.5 > FIC >1.5) (data not shown) which
4
9
require the electrical component of the proton motive force for uptake and (ii) the decrease in tetracycline
37
uptake by CCCP which reduces the pH component of the PMF . The inhibitory effect of the conjugates on
the efflux pumps is less clear but it is possible that the observed effects of the adjuvants on the PMF may
also compromise the function of efflux pumps leading to reduced efflux of antibiotics which are substrates
of RND efflux pumps including tetracycline antibiotics.
CONCLUSIONS
The discovery that linking a tobramycin vector to the efflux pump inhibitors NMP, PAR or DBP
generates adjuvants capable of rescuing tetracycline antibiotics against MDR and XDR Gramꢀnegative
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Journal of Medicinal Chemistry
pathogens including P. aeruginosa isolates opens up opportunities to develop novel and optimized adjuvants.
The aminoglycoside vector strategy is attractive because it induces multimodal effects in the adjuvant
involving both outer and inner membranes, affecting the proton motive force and possibly the function of
the efflux pumps. The overall multimodal effects of the adjuvant/antibiotic combination on the bacterial
membrane are expected to reduce the likelihood of resistance development as observed for other membrane
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
50,51
active or multiꢀtargeting antibiotics . As such the outlined development of adjuvant/tetracycline
combination therapy may preserve the tetracycline class of antibiotics against MDR Gramꢀnegative
7
pathogens which has the potential to fill the current antibiotic discovery void .
EXPERIMENTAL SECTION
Synthetic Chemistry General Comments. Synthesis of compounds were not performed under anoxic or
anhydrous conditions unless specifically noted. Reagents and solvents were purchased from commercially
available sources and used without purification, unless otherwise noted. Normalꢀ or reverseꢀphase flash
chromatography was of respectively. Reactions were monitored by thinꢀlayer chromatography (TLC) on
silica gel 60 F254 (0.25 mm, Merck), to which the compounds were visualized using ultraviolet light and/or
1
13
stain with ninhydrin solution (ninhydrin and acetic acid in ethanol). 1D and 2D ( H, C, DEPT, COSY,
HSQC, HMBC) NMR characterization experiments were performed on either Bruker AMXꢀ500 or Bruker
AMXꢀ300 spectrometer in the noted deuterated solvents. Chemical shifts (δ) are reported in parts per million
with CHCl (7.26 ppm), DHO (4.79 ppm) and CD HOH (3.31 ppm) used as internal standards. Electrospray
3
2
Ionization (ESI) Mass Spectrometry (MS) experiments were carried out on a Varian 500 MS Ion Trap Mass
Spectrometer. Matrixꢀassisted Laser Desorption Ionization (MALDI) MS experiments were performed on a
Bruker Daltonics Ultraflex MALDI TOF/TOF mass spectrometer. Analytical Highꢀperformance Liquid
Chromatography (HPLC) was carried out on Breeze HPLC Waters with 2998 PDA detector (1.2 nm
®
resolution) connected to a Kinetex 5 µm reverseꢀphase C18 100 Å LC column (150 × 4.6 mm,
TM
Phenomenex) or a Synergi 4 µm PolarꢀRP 80 Å LC column (50 × 4.6 mm, Phenomenex). Yields are
given following purification, unless otherwise stated. All of the biologically tested compounds are at least
95% pure as estimated by HPLC.
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Chemistry. Detailed experimental procedures of compounds 4, 5, 6bꢀf, 14 and DBP are described in the
supporting Information.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
General Synthetic Procedure A: Final Deprotection of Compounds 1a-d, 1f, 2 and 16. Boc and TBDMS
protected compounds were treated with 40% HCl in MeOH (2:3, v/v) and stirred at room temperature for 3
h. MethanolicꢀHCl was removed under reduced pressure and purified by reverseꢀphase flash
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
chromatography on a C18ꢀsilica (elution with 100% deionized water or from 100% deionized water to
24–26
deionized water/MeOH = 1:1, v/v) to give analytically pure products
.
General Synthetic Procedure B: Synthesis of Boc and TBDMS Protected TOBꢀNMP Derivatives (7b-f). 1ꢀ
(1ꢀnaphthylmethyl)ꢀpiperazine (NMP) (1.5 equiv) and K CO (3.0 equiv) were subsequently added to stirred
2
3
solutions of 6b-f (1.0 equiv) in anhydrous DMF under N gas. The reaction mixture was heated to 75 ºC and
2
stirred for 8 h. The solvent was removed in vacuo followed by purification using flash chromatography
(elution with hexanes/ethyl acetate = 8:1 ~1:2, v/v) to afford the desired compounds.
5ꢀOꢀ((2ꢀ(4ꢀ(naphthalenꢀ1ꢀylmethyl)piperazinꢀ1ꢀyl)ethyl)ꢀtobramycin 7×HCl (1a). Synthesized following
general procedure A from 7a (50 mg, 0.03 mmol), 40% HCl (1 mL) and MeOH (1.5 mL). The crude product
was purified by reverseꢀphase flash chromatography on a C18ꢀsilica (elution with 100% deionized water).
1
The product was isolated as a white solid. Yield: 16 mg (55%). H NMR (500 MHz, deuterium oxide) δ 8.26
(
d, J = 8.5 Hz, 1H), 8.14 – 8.06 (m, 2H), 7.79 – 7.58 (m, 4H), 5.45 (d, J = 2.9 Hz, 1H, anomeric CH of Hꢀ
1'), 5.23 (d, J = 3.4 Hz, 1H, anomeric CH of Hꢀ1''), 4.64 (s, 2H, CH of naphthylmethyl), 4.23 – 4.10 (m,
H, CH of Hꢀ5', CH of Hꢀ4, CH of Hꢀ6), 4.10 – 3.31 (m, 21H, CH of Hꢀ2'', CH of Hꢀ4', CH of Hꢀ6'', CH of
2
3
2
Hꢀ3'', CH of Hꢀ5'', CH of Hꢀ4'', OCH of linker, CH of Hꢀ5, CH of Hꢀ2', 4×CH of piperazine, CH of Hꢀ1,
2
2
CH of Hꢀ3), 3.31 – 3.25 (m, 2H, CH
2
2
of Hꢀ6'), 3.08 – 2.98 (m, 2H, piperazineꢀCH of linker), 2.52 – 2.42
(m, 1H, CHH of Hꢀ2), 2.28 – 2.20 (m, 1H, CHH of Hꢀ3'), 2.17 – 2.08 (m, 1H, CHH of Hꢀ3'), 1.96 – 1.86 (m,
13
1H, CHH of Hꢀ2). C NMR (126 MHz, deuterium oxide) δ 133.74, 132.07, 130.93, 130.32, 129.09, 128.99,
1
6
27.29, 126.59, 125.57, 123.56, 100.71 (anomeric Cꢀ1''), 92.60 (anomeric Cꢀ1'), 82.45, 81.59, 76.84, 73.66,
8.62, 65.73, 63.68, 60.37, 57.03, 56.49, 54.53, 50.41, 49.75, 48.39, 47.46, 39.01, 28.61. MALDIꢀTOFꢀMS
+
m/e calcd for C35
H
58
N
O
7 9
[M+H] : 720.430, found: 720.441.
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Journal of Medicinal Chemistry
5
ꢀOꢀ((4ꢀ(4ꢀ(Naphthalenꢀ1ꢀylmethyl)piperazinꢀ1ꢀyl)butyl)ꢀtobramycin 7×HCl (1b). Synthesized following
general procedure A from 7b (27 mg, 0.016 mmol), 40% HCl (1 mL) and MeOH (1.5 mL). The crude
product was purified by reverseꢀphase flash chromatography on a C18ꢀsilica (elution with 100% deionized
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
water). The product was isolated as a white solid. Yield: 10 mg (63%). H NMR (500 MHz, deuterium
oxide) δ 8.23 (d, J = 8.5 Hz, 1H), 8.14 (d, J = 8.3 Hz, 1H), 8.09 (dd, J = 8.2, 1.3 Hz, 1H), 7.80 – 7.72 (m,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
H), 7.72 – 7.62 (m, 2H), 5.41 (d, J = 2.7 Hz, 1H, anomeric CH of Hꢀ1'), 5.25 (d, J = 3.5 Hz, 1H, anomeric
CH of Hꢀ1''), 4.91 (s, 2H, CH of naphthylmethyl), 4.32 – 4.27 (m, 1H, CH of Hꢀ5'), 4.27 – 4.22 (m, 1H, CH
of Hꢀ4), 4.04 – 3.51 (m, 22H, CH of Hꢀ6, CH of Hꢀ2'', CH of Hꢀ4', CH of Hꢀ6'', CH of Hꢀ3'', CH of Hꢀ5'',
CH of Hꢀ4'', OCH of linker, CH of Hꢀ5, CH of Hꢀ2', 4×CH of piperazine, CH of Hꢀ1, CH of Hꢀ3), 3.48
2
2
2
2
(dd, J = 14.0, 9.1 Hz, 1H, CHH of Hꢀ6'), 3.38 – 3.27 (m, 3H, CHH of Hꢀ6', piperazineꢀCH of linker), 2.60 –
2
2
.52 (m, 1H, CHH of Hꢀ2), 2.38 – 2.30 (m, 1H, CHH of Hꢀ3'), 2.28 – 2.20 (m, 1H, CHH of Hꢀ3'), 2.08 –
.98 (m, 1H, CHH of Hꢀ2), 1.93 – 1.79 (m, 2H, NꢀCH ꢀCH of linker), 1.78 – 1.70 (m, 2H, OꢀCH ꢀCH of
1
2
2
2
2
13
linker). C NMR (126 MHz, deuterium oxide) δ 133.76, 131.68, 131.42, 131.21, 129.20, 127.63, 126.78,
125.59, 124.95, 123.02, 100.91 (anomeric Cꢀ1''), 92.74 (anomeric Cꢀ1'), 82.19, 81.48, 76.87, 75.78, 73.13,
7
2
2.10, 68.55, 64.84, 63.17, 59.36, 57.49, 56.74, 54.81, 49.54, 49.11, 48.91, 48.39, 47.30, 38.53, 28.03,
+
7.69, 26.44, 20.09. MALDIꢀTOFꢀMS m/e calcd for C37
H
62
N
7
O
9
[M+H] : 748.461, found: 748.472.
5
ꢀOꢀ((6ꢀ(4ꢀ(Naphthalenꢀ1ꢀylmethyl)piperazinꢀ1ꢀyl)hexyl)ꢀtobramycin 7×HCl (1c). Synthesized following
general procedure A from 7c (52 mg, 0.03 mmol), 40% HCl (1 mL) and MeOH (1.5 mL). The crude product
was purified by reverseꢀphase flash chromatography on a C18ꢀsilica (elution with 100% deionized water).
1
The product was isolated as a white solid. Yield: 20 mg (65%). H NMR (500 MHz, deuterium oxide) δ 8.22
(
d, J = 8.5 Hz, 1H), 8.16 (d, J = 8.3 Hz, 1H), 8.10 (dd, J = 8.2, 1.3 Hz, 1H), 7.83 – 7.75 (m, 2H), 7.73 – 7.63
m, 2H), 5.41 (d, J = 2.7 Hz, 1H, anomeric CH of Hꢀ1'), 5.22 (d, J = 3.5 Hz, 1H, anomeric CH of Hꢀ1''), 5.03
(
(s, 2H, CH
2
of naphthylmethyl), 4.33 – 4.28 (m, 1H, CH of Hꢀ5'), 4.28 – 4.22 (m, 1H, CH of Hꢀ4), 4.02 –
3
.59 (m, 22H, CH of Hꢀ6, CH of Hꢀ2'', CH of Hꢀ4', CH of Hꢀ6'', CH of Hꢀ3'', CH of Hꢀ5'', CH of Hꢀ4'',
2
OCH of linker, CH of Hꢀ5, CH of Hꢀ2', 4×CH of piperazine, CH of Hꢀ1, CH of Hꢀ3), 3.46 (dd, J = 14.0,
2
2
9
.2 Hz, 1H, CHH of Hꢀ6'), 3.37 – 3.28 (m, 3H, CHH of Hꢀ6', piperazineꢀCH
2
of linker), 2.60 – 2.54 (m, 1H,
CHH of Hꢀ2), 2.37 – 2.30 (m, 1H, CHH of Hꢀ3'), 2.29 – 2.22 (m, 1H, CHH of Hꢀ3'), 2.12 – 2.02 (m, 1H,
1
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Journal of Medicinal Chemistry
Page 16 of 50
2 2 2 2
CHH of Hꢀ2), 1.80 (p, J = 7.5 Hz, 2H, NꢀCH ꢀCH of linker), 1.74 – 1.63 (m, 2H, OꢀCH ꢀCH of linker),
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
13
1
1
8
.47 – 1.36 (m, 4H, 2×CH
2
of linker). C NMR (126 MHz, deuterium oxide) δ 133.76, 131.94, 131.70,
31.64, 129.28, 127.84, 126.88, 125.61, 123.56, 122.84, 101.19 (anomeric Cꢀ1''), 92.66 (anomeric Cꢀ1'),
1.95, 81.66, 76.52, 75.57, 73.28, 73.08, 68.57, 64.81, 63.32, 59.27, 57.29, 56.95, 54.79, 49.72, 48.67,
48.51, 48.47, 47.36, 38.61, 29.24, 28.15, 27.68, 25.87, 24.64, 23.38. MALDIꢀTOFꢀMS m/e calcd for
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
C
39
H
66
N
7
O
9
[M+H] : 776.492, found: 776.507.
5ꢀOꢀ((8ꢀ(4ꢀ(Naphthalenꢀ1ꢀylmethyl)piperazinꢀ1ꢀyl)octyl)ꢀtobramycin 7×HCl (1d). Synthesized following
general procedure A from 7d (31 mg, 0.018 mmol), 40% HCl (1 mL) and MeOH (1.5 mL). The crude
product was purified by reverseꢀphase flash chromatography on a C18ꢀsilica (elution with 100% deionized
1
water). The product was isolated as a white solid. Yield: 13 mg (70%). H NMR (500 MHz, deuterium
oxide) δ 8.23 (d, J = 8.5 Hz, 1H), 8.15 (d, J = 8.3 Hz, 1H), 8.12 – 8.08 (m, 1H), 7.80 – 7.73 (m, 2H), 7.72 –
7
.63 (m, 2H), 5.40 (d, J = 2.6 Hz, 1H, anomeric CH of Hꢀ1'), 5.20 (d, J = 3.5 Hz, 1H, anomeric CH of Hꢀ1''),
.93 (s, 2H, CH of naphthylmethyl), 4.33 – 4.28 (m, 1H, CH of Hꢀ5'), 4.25 – 4.19 (m, 1H, CH of Hꢀ4), 4.00
4
2
– 3.56 (m, 22H, CH of Hꢀ6, CH of Hꢀ2'', CH of Hꢀ4', CH of Hꢀ6'', CH of Hꢀ3'', CH of Hꢀ5'', CH of Hꢀ4'',
2
OCH of linker, CH of Hꢀ5, CH of Hꢀ2', 4×CH of piperazine, CH of Hꢀ1, CH of Hꢀ3), 3.44 (dd, J = 14.0,
2
2
9
.0 Hz, 1H, CHH of Hꢀ6'), 3.34 (dd, J = 14.0, 3.8 Hz, 1H, CHH of Hꢀ6'), 3.29 – 3.24 (m, 2H, piperazineꢀ
CH of linker), 2.60 – 2.53 (m, 1H, CHH of Hꢀ2), 2.33 – 2.22 (m, 2H, CH of Hꢀ3'), 2.06 – 1.98 (m, 1H,
CHH of Hꢀ2), 1.80 – 1.72 (m, 2H, NꢀCH ꢀCH of linker), 1.72 – 1.61 (m, 2H, OꢀCH ꢀCH of linker), 1.42 –
2
2
2
2
2
2
1
3
1
1
8
4
.30 (m, 8H, 4×CH of linker). C NMR (126 MHz, deuterium oxide) δ 133.76, 131.66, 131.54, 131.34,
2
29.22, 127.68, 126.80, 125.59, 124.60, 122.94, 101.32 (anomeric Cꢀ1''), 92.72 (anomeric Cꢀ1'), 81.84,
1.79, 76.67, 75.75, 73.73, 73.12, 68.53, 64.76, 63.19, 59.23, 57.44, 57.03, 54.76, 49.74, 48.83, 48.43,
7.30, 38.52, 29.46, 28.80, 28.26, 28.07, 27.70, 25.66, 25.15, 23.37. MALDIꢀTOFꢀMS m/e calcd for
+
C
41
H
70
N
7
O
9
[M+H] : 804.524, found: 804.537.
5ꢀOꢀ((10ꢀ(4ꢀ(Naphthalenꢀ1ꢀylmethyl)piperazinꢀ1ꢀyl)docyl)ꢀtobramycin 7×TFA (1e). TBAF solution (1M
in THF, 0.67 mL) was added to a stirred solution of 7e (120 mg, 0.067 mmol) in THF (2mL). The reaction
mixture was stirred at room temperature for 2 h. It was then concentrated in vacuo. The residue was purified
by flash chromatography (elution with DCM/MeOH = 20:1 ~ 5:1) to afford a white solid (77 mg) to which
1
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Journal of Medicinal Chemistry
2
was further treated with TFA (90% TFA of H O, 5 mL) and stirred at room temperature for 1 h. The above
1
2
solution was concentrated under reduced pressure and purified by reverseꢀphase flash chromatography on a
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
C18ꢀsilica (elution with 100% deionized water) to give the desired product 1e as a white solid. Yield: 81 mg
1
(
74%, two steps). H NMR (500 MHz, deuterium oxide) δ 8.20 (d, J = 8.4 Hz, 1H), 8.13 – 8.05 (m, 2H),
7.79 – 7.57 (m, 4H), 5.38 (d, J = 2.6 Hz, 1H, anomeric CH of Hꢀ1'), 5.16 (d, J = 3.5 Hz, 1H, anomeric CH of
Hꢀ1'' ), 4.75 (s, 2H, CH of naphthylmethyl ), 4.28 (m, 1H, CH of Hꢀ5'), 4.17 – 4.09 (m, 1H, CH of Hꢀ4),
.99 – 3.69 (m, 12H, CH of Hꢀ6, CH of Hꢀ2'', CH of Hꢀ4', CH of Hꢀ6'', CH of Hꢀ3'', CH of Hꢀ5'', CH of Hꢀ
'', OCH of linker, CH of Hꢀ5, CH of Hꢀ2'), 3.68 – 3.43 (m, 10H, 4×CH of piperazine, CH of Hꢀ1, CH of
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
3
4
2
2
2
Hꢀ3), 3.42 – 3.37 (m, 1H, CHH of Hꢀ6'), 3.31 (dd, J = 14.0, 3.9 Hz, 1H, CHH of Hꢀ6'), 3.24 – 3.15 (m, 2H,
piperazineꢀCH of linker), 2.59 – 2.51 (m, 1H, CHH of Hꢀ2), 2.31 – 2.22 (m, 2H, CH of Hꢀ3'), 1.99 – 1.88
2
2
(m, 1H, CHH of Hꢀ2 ), 1.76 – 1.68 (m, 2H, NꢀCH
2
ꢀCH
2
2 2
of linker), 1.68 – 1.57 (m, 2H, OꢀCH ꢀCH of
13
2
linker), 1.43 – 1.23 (m, 12H, 6×CH of linker). C NMR (126 MHz, deuterium oxide) δ 133.74, 131.68,
131.02, 130.87, 129.12, 127.45, 126.68, 125.82, 125.54, 123.05, 101.38 (anomeric Cꢀ1''), 92.74 (anomeric
Cꢀ1'), 81.91, 81.84, 76.89, 75.92, 73.82, 73.15, 68.51, 64.71, 63.07, 59.17, 57.60, 56.98, 54.77, 49.70, 49.25,
48.95, 48.34, 47.24, 38.35, 29.46, 29.00, 28.78, 28.58, 28.22, 28.01, 27.68, 25.65, 25.30, 23.36. MALDIꢀ
+
TOFꢀMS m/e calcd for C43
H
73
N
7
O
9
Na [M+Na] : 854.537, found: 854.531.
5ꢀOꢀ((12ꢀ(4ꢀ(Naphthalenꢀ1ꢀylmethyl)piperazinꢀ1ꢀyl)dodecyl)ꢀtobramycin 7×HCl (1f). Synthesized
following general procedure A from 7f (38 mg, 0.021 mmol), 40% HCl (1 mL) and MeOH (1.5 mL). The
crude product was purified by reverseꢀphase flash chromatography on a C18ꢀsilica (elution with 100%
1
deionized water). The product was isolated as a white solid. Yield: 16 mg (69%). H NMR (500 MHz,
deuterium oxide) δ 8.21 (d, J = 8.5 Hz, 1H), 8.16 (d, J = 8.3 Hz, 1H), 8.12 – 8.07 (m, 1H), 7.84 – 7.73 (m,
2
H), 7.72 – 7.62 (m, 2H), 5.40 (d, J = 2.3 Hz, 1H, anomeric CH of Hꢀ1'), 5.21 – 5.17 (m, 1H, anomeric CH
of Hꢀ1''), 5.03 (s, 2H, CH of naphthylmethyl), 4.33 – 4.28 (m, 1H, CH of Hꢀ5'), 4.25 – 4.19 (m, 1H, CH of
Hꢀ4), 4.00 – 3.70 (m, 16H, CH of Hꢀ6, CH of Hꢀ2'', CH of Hꢀ4', CH of Hꢀ6'', CH of Hꢀ3'', OCH of linker,
2
2
2
CH of Hꢀ5'', CH of Hꢀ4'', CH of Hꢀ5, CH of Hꢀ2', 2×CH of piperazine), 3.70 – 3.44 (m, 6H, 2×CH of
2
2
piperazine, CH of Hꢀ1, CH of Hꢀ3), 3.43 – 3.39 (m, 1H, CHH of Hꢀ6'), 3.36 – 3.26 (m, 3H, CHH of Hꢀ6',
piperazineꢀCH of linker), 2.65 – 2.50 (m, 1H, CHH of Hꢀ2), 2.35 – 2.20 (m, 2H, CH of Hꢀ3'), 2.10 – 1.95
2
2
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(
2 2 2 2
m, 1H, CHH of Hꢀ2), 1.82 – 1.71 (m, 2H, NꢀCH ꢀCH of linker), 1.70 – 1.58 (m, 2H, OꢀCH ꢀCH of
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
13
linker), 1.41 – 1.25 (m, 16H, 8×CH
2
of linker). C NMR (126 MHz, deuterium oxide) δ 133.77, 131.91,
1
31.70, 131.62, 129.28, 127.82, 126.88, 125.59, 123.56, 122.78, 101.35 (anomeric Cꢀ1''), 92.71 (anomeric
Cꢀ1'), 81.84, 81.80, 76.71, 75.83, 73.85, 73.14, 68.52, 64.77, 63.18, 59.22, 57.30, 57.11, 54.75, 49.76, 48.67,
48.44, 47.28, 38.49, 29.47, 29.04, 28.95, 28.86, 28.77, 28.62, 28.22, 28.05, 27.70, 25.61, 25.34, 23.34.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
MALDIꢀTOFꢀMS m/e calcd for C45
H
78
N
7
O
9
[M+H] : 860.5861, found: 860.5874.
5ꢀOꢀ(dodecylparoxetine)ꢀtobramycin 6×HCl (2). Synthesized following general procedure A from 11 (97
mg, 0.05 mmol), 40% HCl (2 mL) and MeOH (3 mL). The crude product was purified by reverseꢀphase
flash chromatography on a C18ꢀsilica (elution with 100% deionized water). The product was isolated as a
1
white solid. Yield: 36 mg (61%). H NMR (500 MHz, deuterium oxide) δ 7.33 – 7.23 (m, 2H), 7.11 – 7.04
(
m, 2H), 6.73 – 6.66 (m, 1H), 6.43 – 6.36 (m, 1H), 6.25 – 6.16 (m, 1H), 5.89 (s, 2H, OꢀCH
paroxetine,), 5.42 (d, J = 2.5 Hz, 1H, anomeric CH of Hꢀ1'), 5.21 (d, J = 3.4 Hz, 1H, anomeric CH of Hꢀ1''),
.36 – 4.30 (m, 1H, CH of Hꢀ5'), 4.28 – 4.17 (m, 1H, CH of Hꢀ4), 4.01 – 3.55 (m, 18H, CH of Hꢀ6, CH of
Hꢀ2'', CH of Hꢀ4', CH of Hꢀ6'', CH of Hꢀ3'', CH of Hꢀ5'', CH of Hꢀ4'', OCH of linker, OꢀCH ꢀCH of
2
ꢀO of
4
2
2
2
paroxetine, CH of Hꢀ5, CH of Hꢀ2', NꢀCHHꢀCH of piperidine, NꢀCHHꢀCH of piperidine, CH of Hꢀ1, CH of
2
Hꢀ3), 3.44 (dd, J = 14.0, 9.2 Hz, 1H, CHH of Hꢀ6'), 3.35 (dd, J = 14.0, 3.9 Hz, 1H, CHH of Hꢀ6'), 3.26 –
3
.15 (m, 2H, NꢀCH
2.98 – 2.90 (m, 1H, NꢀCH
NꢀCH ꢀCH of piperidine), 2.33 – 2.24 (m, 2H, CH of Hꢀ3'), 2.17 – 1.99 (m, 3H, NꢀCH ꢀCH CHH of Hꢀ2),
2
of linker), 3.15 – 3.06 (m, 2H, NꢀCHHꢀCH
2
of piperidine, NꢀCHHꢀCH of piperidine),
2
ꢀCH ꢀCH of piperidine), 2.61 – 2.54 (m, 1H, CHH of Hꢀ2), 2.54 – 2.45 (m, 1H,
2
2
2
2
2,
1
8
1
.84 – 1.72 (m, 2H, NꢀCH ꢀCH of linker), 1.71 – 1.61 (m, 2H, OꢀCH ꢀCH of linker), 1.42 – 1.24 (m, 16H,
2 2 2 2
13
×CH
2
of linker). C NMR (126 MHz, deuterium oxide) δ 161.65 (d, J = 242.9 Hz, CF), 153.51, 147.76,
41.58, 137.37, 137.35, 129.23, 129.16, 115.68, 115.51, 108.24, 106.42, 101.38 (anomeric Cꢀ1''), 101.35,
98.22, 92.70 (anomeric Cꢀ1'), 81.87, 81.83, 76.70, 75.84, 73.89, 73.18, 68.55, 68.20, 64.82, 63.25, 59.29,
5
2
7.58, 54.77, 54.66, 52.92, 49.78, 48.47, 47.32, 40.93, 39.82, 38.55, 30.78, 29.50, 29.06, 28.98, 28.90,
8.82, 28.67, 28.34, 28.09, 27.72, 25.91, 25.36, 23.52. MALDIꢀTOFꢀMS m/e calcd for C H FN O
12
49
80
6
+
[
M+H] : 963.5818, found: 963.5819.
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Journal of Medicinal Chemistry
TOBꢀDBP Conjugate (3). AcOH (5 mL) and 10% Pd/C were added to a stirred solution of compound 16
1
2
(
2
86 mg, 0.07 mmol) in MeOH (4 mL) and H O (1 mL). The reaction flask was subjected to catalytic
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
hydrogenation via hydrogen balloon overnight at room temperature. The reaction mixture was filtered
®
through a bed of Celite and washed with methanolic HCl. The solvents were removed under reduced
pressure to afford a crude product which was purified by reverseꢀphase flash chromatography on C18ꢀsilica
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(elution with a gradient from 100% 0.1% TFA of deionized water to 20% MeOH and 80% 0.1% TFA of
1
deionized water) to give the desired product as a white solid. Yield: 61 mg (77%). H NMR (500 MHz,
deuterium oxide) δ 8.03 – 7.86 (m, 4H, aromatic H), 7.65 – 7.54 (m, 2H, aromatic H), 7.50 (dd, J = 8.8, 2.2
Hz, 1H, aromatic H), 7.42 – 7.37 (m, 2H, aromatic H), 7.36 – 7.28 (m, 3H, aromatic H), 5.39 (d, J = 2.6 Hz,
1H, anomeric CH of Hꢀ1'), 5.19 (d, J = 3.4 Hz, 1H, anomeric CH of Hꢀ1''), 4.60 – 4.49 (m, 2H, CH of Proα,
CH of Homophe
α
), 4.33 – 4.29 (m, 1H, CH of Hꢀ5'), 4.24 – 4.18 (m, 1H, CH of Hꢀ4), 4.04 (dd, J = 11.4, 6.8
of Hꢀ6'', OCH of
Hz, 1H, CHH of Proδ1), 4.00 – 3.68 (m, 11H, CH of Hꢀ4', CH of Hꢀ2'', CH of Hꢀ6, CH
2
2
linker, CH of Hꢀ4'', CH of Hꢀ5, CH of Hꢀ5'', CH of Hꢀ2'), 3.65 – 3.52 (m, 3H, CH of Hꢀ1, CH of Hꢀ3'', CH
of Hꢀ3), 3.44 – 3.11 (m, 7H, , CH of Hꢀ6', Pro CH ꢀNH , NꢀCH of linker, CHH of Pro ), 2.96 – 2.89 (m,
2
γꢀ
2
2
2
δ2
1
H, CHH of Homophe ), 2.85 – 2.73 (m, 2H, CHH of Homophe , CH of Pro ), 2.59 – 2.54 (m, 1H, CHH of
γ
γ
γ
Hꢀ2), 2.54 – 2.42 (m, 2H, CH
2
of Pro
β
), 2.36 – 2.24 (m, 4H, CH
ꢀCH
2
of linker). C NMR (126 MHz, deuterium oxide) δ 171.84, 168.58, 140.52, 134.01, 133.22,
2
of Homophe
β
, CH
2
of Hꢀ3'), 2.06 – 1.96
(m, 1H, CHH of Hꢀ2), 1.74 – 1.61 (m, 4H, NꢀCH
2
of linker, OꢀCH ꢀCH of linker), 1.38 – 1.22 (m,
2 2 2
13
16H, 6×CH
131.01, 129.03, 128.86, 128.69, 127.73, 127.64, 127.02, 126.57, 126.06, 121.29, 118.99, 101.39 (anomeric
Cꢀ1''), 92.71 (anomeric Cꢀ1'), 81.88, 81.82, 76.76, 75.87, 73.92, 73.18, 68.52, 65.85, 64.75, 63.16, 59.23,
5
2
7.72, 55.87, 54.75, 54.40, 49.74, 48.42, 47.28, 40.32, 38.47, 34.90, 33.30, 32.51, 31.39, 29.49, 29.06,
8.99, 28.89, 28.84, 28.57, 28.36, 28.04, 27.72, 25.72, 25.36, 25.33. MALDIꢀTOFꢀMS m/e calcd for
+
C
56
H
89
N
9
O
11Na [M+Na] : 1086.6579, found: 1086.6592.
5
ꢀOꢀ((2ꢀ(4ꢀ(naphthalenꢀ1ꢀylmethyl)piperazinꢀ1ꢀyl)ethyl)ꢀ1,3,2
butoxycarbonyl)ꢀ4 ,2′′,4′′,6′′ꢀtetraꢀOꢀTBDMSꢀtobramycin (7a). Five mL of DCM was added to
a flask containing compound 10 (65 mg, 0.04 mmol) and 1ꢀ(1ꢀnaphthylmethyl)ꢀpiperazine (NMP) (12 mg,
.05 mmol) under N gas. Acetic acid (2 µL, 0.04 mmol) was then added to the solution at room temperature
′,6′,3′′ꢀpentaꢀNꢀ(tertꢀ
′
0
2
1
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and stirred for 1 h. The reaction mixture was cooled down to 0 ºC followed by the addition of NaBH(OAc)
3
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
38 mg, 0.18 mmol). It was then stirred at room temperature for 18 h before being quenched with saturated
NaHCO solution at 0 ºC. Water (3 mL) was added and the resulting mixture was extracted with DCM (3 ×
mL). The combined organic layers were washed with saturated brine and dried over anhydrous sodium
3
3
sulfate. The solvent was removed in vacuo followed by purification using flash chromatography (elution
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
with a gradient of hexanes/ethyl acetate = 10:1 ~ 1:1) to give product 7a as a pale yellow oil. Yield: 50 mg
1
(
75%). H NMR (500 MHz, chloroformꢀd) δ 8.33 – 8.25 (m, 1H), 7.84 (dd, J = 7.8, 1.6 Hz, 1H), 7.76 (d, J =
7.9 Hz, 1H), 7.59 – 7.34 (m, 4H), 5.34 – 5.14 (m, 2H, anomeric CH of Hꢀ1', anomeric CH of Hꢀ1''), 4.32 –
4.06 (m, 3H), 3.89 (s, 2H), 3.86 – 3.74 (m, 2H), 3.74 – 3.03 (m, 14H), 2.68 – 2.26 (m, 10H), 2.09 – 1.95 (m,
1
3
1H), 1.64 – 1.22 (m, 47H), 1.14 – 1.02 (m, 1H), 1.02 – 0.78 (m, 36H), 0.22 – ꢀ0.05 (m, 24H). C NMR (126
MHz, chloroformꢀd) δ 155.91, 155.65, 155.18, 154.91, 154.78, 134.42, 133.94, 132.71, 128.46, 127.87,
1
27.32, 125.82, 125.67, 125.25, 124.89, 97.87 (anomeric C), 96.72 (anomeric C), 85.62, 79.98, 79.53, 79.42,
9.34, 79.16, 75.56, 72.94, 71.64, 70.67, 68.12, 67.31, 63.47, 61.24, 61.10, 59.07, 57.37, 54.23, 53.67,
7
53.51, 50.70, 49.51, 48.98, 41.81, 36.01, 35.46, 29.84, 28.80, 28.75, 28.67, 28.64, 28.58, 26.30, 26.18,
26.15, 25.95, 18.59, 18.47, 18.25, 18.07, ꢀ3.19, ꢀ3.55, ꢀ4.04, ꢀ4.67, ꢀ4.73, ꢀ4.85, ꢀ4.91, ꢀ4.93. MS (ESI) m/e
+
calcd for C84
H
154
N
7
O19Si
4
[M+H] : 1678.5, found: 1677.8.
5
ꢀOꢀ((4ꢀ(4ꢀ(Naphthalenꢀ1ꢀylmethyl)piperazinꢀ1ꢀyl)butyl)ꢀ1,3,2
′,6′,3′′ꢀpentaꢀNꢀ(tertꢀ
butoxycarbonyl)ꢀ4 ,2′′,4′′,6′′ꢀtetraꢀOꢀTBDMSꢀtobramycin (7b). Synthesized following general
′
2 3
procedure B from 6b (48 mg, 0.03 mmol), NMP (11 mg, 0.05 mmol) and K CO (13 mg, 0.09 mmol). The
1
product was isolated as a white solid. Yield: 27 mg (52%). H NMR (500 MHz, chloroformꢀd) δ 8.29 (d, J =
8.2 Hz, 1H), 7.82 (dd, J = 7.9, 1.6 Hz, 1H), 7.75 (d, J = 8.0 Hz, 1H), 7.54 – 7.35 (m, 4H), 5.23 – 5.17 (m,
1H, anomeric H), 5.09 – 4.97 (m, 2H), 5.16 – 5.10 (m, 1H, anomeric), 4.31 – 4.03 (m, 3H), 3.89 (s, 2H),
3.85 – 3.73 (m, 2H), 3.73 – 3.64 (m, 2H), 3.64 – 3.05 (m, 11H), 2.69 – 2.35 (m, 8H), 2.34 – 2.18 (m, 2H),
2.04 – 1.93 (m, 1H), 1.58 – 1.35 (m, 50 H), 1.27 – 1.22 (m, 1H), 1.10 – 1.01 (m, 1H), 0.98 – 0.79 (m, 36H),
13
0.18 – ꢀ0.03 (m, 24H). C NMR (75 MHz, chloroformꢀd) δ 155.89, 155.84, 155.61, 154.79, 154.67, 134.15,
33.88, 132.70, 128.29, 127.77, 127.36, 125.57, 125.53, 125.19, 124.87, 97.77 (anomeric C), 96.62
anomeric C), 85.74, 79.58, 79.40, 79.36, 79.03, 75.39, 72.98, 72.93, 71.52, 67.91, 66.99, 67.85, 63.22,
1
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Journal of Medicinal Chemistry
6
2
5
1.09, 59.50, 58.74, 57.28, 53.38, 53.23, 50.48, 49.01, 48.34, 41.70, 35.71, 30.74, 30.12, 28.52, 27.67,
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
7.63, 26.92, 26.01, 26.00, 24.38, 18.53, 18.30, 18.19, 18.01, ꢀ3.42, ꢀ3.81, ꢀ4.12, ꢀ4.79, ꢀ4.88, ꢀ4.91, ꢀ5.06, ꢀ
+
.20. MS (ESI) m/e calcd for C84
H
158
N
7
O19Si
4
[M+H] : 1706.6, found: 1706.9.
5ꢀOꢀ((6ꢀ(4ꢀ(Naphthalenꢀ1ꢀylmethyl)piperazinꢀ1ꢀyl)hexyl)ꢀ1,3,2
′,6′,3′′ꢀpentaꢀNꢀ(tertꢀ
butoxycarbonyl)ꢀ4 ,2′′,4′′,6′′ꢀtetraꢀOꢀTBDMSꢀtobramycin (7c). Synthesized following general
′
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2 3
procedure B from 6c (183 mg, 0.12 mmol), NMP (39 mg, 0.17 mmol) and K CO (48 mg, 0.35 mmol). The
1
product was isolated as a white solid. Yield: 52 mg (26%). H NMR (500 MHz, chloroformꢀd) δ 8.29 (d, J =
8.0 Hz, 1H), 7.83 (dd, J = 7.7, 1.7 Hz, 1H), 7.75 (d, J = 8.0 Hz, 1H), 7.55 – 7.35 (m, 4H), 5.26 – 5.18 (m,
1H, anomeric H), 5.18 – 5.11 (m, 1H, anomeric H), 4.32 – 4.01 (m, 3H), 3.90 (s, 2H), 3.84 – 3.09 (m, 15H),
2.68 – 2.51 (m, 4H), 2.51 – 2.35 (m, 4H), 2.35 – 2.22 (m, 2H), 2.06 – 1.96 (m, 1H), 1.61 – 1.35 (m, 50H),
13
1.35 – 1.17 (m, 5H), 1.09 – 1.00 (m, 1H), 1.00 – 0.79 (m, 36H), 0.21 – ꢀ0.04 (m, 24H). C NMR (75 MHz,
chloroformꢀd) δ 155.99, 155.85, 155.67, 154.85, 154.71, 134.35, 133.94, 132.72, 128.45, 127.93, 127.42,
1
7
5
25.77, 125.64, 125.23, 124.93, 97.95 (anomeric C), 96.61 (anomeric C), 85.93, 80.07, 79.53, 79.36, 79.28,
9.12, 75.44, 73.37, 72.86, 71.68, 68.14, 67.02, 66.10, 63.29, 61.24, 59.00, 57.43, 53.48, 53.13, 50.82,
0.67, 49.06, 48.47, 41.83, 35.83, 31.04, 30.86, 28.79, 28.65, 28.57, 28.28, 27.11, 26.28, 26.17, 26.13,
25.93, 18.63, 18.47, 18.24, 18.06, ꢀ3.22, ꢀ3.63, ꢀ4.05, ꢀ4.73, ꢀ4.79, ꢀ4.90, ꢀ5.01, ꢀ5.06. MS (ESI) m/e calcd
+
for C H N O Si [M+H] : 1734.6, found: 1734.6.
88
162
7
19
4
5
ꢀOꢀ((8ꢀ(4ꢀ(Naphthalenꢀ1ꢀylmethyl)piperazinꢀ1ꢀyl)octyl)ꢀ1,3,2
butoxycarbonyl)ꢀ4 ,2′′,4′′,6′′ꢀtetraꢀOꢀTBDMSꢀtobramycin (7d). Synthesized following general
procedure B from 6d (61 mg, 0.04 mmol), NMP (13 mg, 0.06 mmol) and K CO (16 mg, 0.11 mmol). The
′,6′,3′′ꢀpentaꢀNꢀ(tertꢀ
′
2
3
1
product was isolated as a white solid. Yield: 31 mg (48%). H NMR (500 MHz, chloroformꢀd) δ 8.28 (d, J =
8.1 Hz, 1H), 7.81 (dd, J = 7.7, 1.7 Hz, 1H), 7.74 (d, J = 8.2 Hz, 1H), 7.56 – 7.31 (m, 4H), 5.27 – 5.17 (m,
1
2
1
1
H, anomeric H), 5.17 – 5.10 (m, 1H, anomeric H), 4.35 – 3.98 (m, 3H), 3.88 (s, 2H), 3.85 – 3.07 (m, 15H),
.73 – 2.34 (m, 8H), 2.34 – 2.23 (m, 2H), 2.06 – 1.94 (m, 1H), 1.64 – 1.34 (m, 50H), 1.34 – 1.16 (m, 9H),
1
3
.12 – 1.01 (m, 1H), 1.00 – 0.78 (m, 36H), 0.26 – ꢀ0.10 (m, 24H). C NMR (126 MHz, chloroformꢀd) δ
55.75, 155.61, 155.61, 154.80, 154.64, 134.25, 133.88, 132.65, 128.39, 127.88, 127.35, 125.71, 125.58,
125.15, 124.87, 97.95 (anomeric C), 96.59 (anomeric C), 85.83, 79.97, 79.44, 79.28, 78.91, 75.38, 73.43,
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7
4
2
2.77, 71.65, 68.11, 66.93, 65.56, 63.19, 61.31, 61.19, 61.12, 58.90, 57.34, 53.52, 53.42, 53.07, 50.61,
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
9.02, 48.45, 43.85, 41.76, 36.02, 35.75, 30.72, 30.10, 29.72, 28.73, 28.59, 28.49, 27.82, 27.02, 26.23,
6.11, 26.08, 25.88, 18.57, 18.55, 18.42, 18.18, 18.00, ꢀ3.32, ꢀ3.70, ꢀ4.10, ꢀ4.78, ꢀ4.85, ꢀ4.97, ꢀ5.07, ꢀ5.13.
+
MS (ESI) m/e calcd for C H N O Si [M+H] : 1762.7, found: 1762.3.
90 166
7
19
4
5ꢀOꢀ((10ꢀ(4ꢀ(Naphthalenꢀ1ꢀylmethyl)piperazinꢀ1ꢀyl)docyl)ꢀ1,3,2
butoxycarbonyl)ꢀ4 ,2′′,4′′,6′′ꢀtetraꢀOꢀTBDMSꢀtobramycin (7e). Synthesized following general
procedure B from 6e (220 mg, 0.13 mmol), NMP (45 mg, 0.2 mmol) and K CO (55 mg, 0.40 mmol). The
′,6′,3′′ꢀpentaꢀNꢀ(tertꢀ
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
′
2
3
1
product was isolated as a white solid. Yield: 120 mg (50%). H NMR (500 MHz, chloroformꢀd) δ 8.32 –
8.24 (m, 1H), 7.83 (dd, J = 7.7, 1.7 Hz, 1H), 7.75 (d, J = 8.0 Hz, 1H), 7.54 – 7.34 (m, 4H), 5.27 – 5.18 (m,
1
H, anomeric H), 5.18 – 5.12 (m, 1H, anomeric H), 4.33 – 4.21 (m, 1H), 4.20 – 4.12 (m, 1H), 4.12 – 4.02
(m, 1H), 3.90 (s, 2H), 3.86 – 3.05 (m, 15H), 2.71 – 2.35 (m, 8H), 2.35 – 2.27 (m, 2H), 2.05 – 1.96 (m, 1H),
13
1.65 – 1.35 (m, 50H), 1.35 – 1.13 (m, 13H), 1.00 – 0.78 (m, 36H), 0.20 – ꢀ0.04 (m, 24H). C NMR (126
MHz, chloroformꢀd) δ 155.72, 155.58, 155.52, 154.73, 154.55, 134.14, 133.79, 132.55, 128.30, 127.79,
1
27.28, 125.62, 125.50, 125.07, 124.76, 97.83 (anomeric C), 96.52 (anomeric C), 85.71, 79.89, 79.36, 79.20,
5.27, 73.37, 72.67, 71.53, 68.00, 66.82, 63.09, 61.21, 61.07, 60.34, 58.84, 57.27, 53.32, 53.29, 52.97,
7
50.52, 48.95, 48.36, 43.76, 41.66, 36.69, 35.90, 35.63, 30.63, 30.03, 29.69, 29.64, 29.62, 28.63, 28.50,
2
1
8.48, 28.40, 27.70, 26.91, 26.19, 26.13, 26.01, 25.98, 25.92, 25.78, 24.73, 22.62, 21.01, 18.48, 18.32,
170 7 4
8.09, 17.91, ꢀ3.43, ꢀ3.80, ꢀ4.20, ꢀ4.88, ꢀ4.95, ꢀ5.08, ꢀ5.18, ꢀ5.24. MS (ESI) m/e calcd for C92H N O19Si
+
[
M+H] : 1790.7, found: 1790.5.
5ꢀOꢀ((12ꢀ(4ꢀ(Naphthalenꢀ1ꢀylmethyl)piperazinꢀ1ꢀyl)dodecyl)ꢀ1,3,2′,6′,3′′ꢀpentaꢀNꢀ(tertꢀ
butoxycarbonyl)ꢀ4
′,2′′,4′′,6′′ꢀtetraꢀOꢀTBDMSꢀtobramycin (7f). Synthesized following general
2 3
procedure B from 6f (59 mg, 0.04 mmol), NMP (12 mg, 0.05 mmol) and K CO (15 mg, 0.11 mmol). The
1
product was isolated as a white solid. Yield: 38 mg (60%). H NMR (500 MHz, chloroformꢀd) δ 8.29 (d, J =
8
.1 Hz, 1H), 7.83 (d, J = 7.9 Hz, 1H), 7.76 (d, J = 8.0 Hz, 1H), 7.58 – 7.33 (m, 4H), 5.27 – 5.18 (m, 1H,
anomeric H), 5.18 – 5.12 (m, 1H, anomeric H), 4.35 – 4.22 (m, 1H), 4.22 – 4.13 (m, 1H), 4.13 – 4.02 (m,
H), 3.90 (s, 2H), 3.86 – 3.07 (m, 15H), 2.72 – 2.36 (m, 8H), 2.35 – 2.26 (m, 2H), 2.07 – 1.95 (m, 1H), 1.61
– 1.35 (m, 50H), 1.35 – 1.16 (m, 17H), 1.12 – 1.00 (m, 1H), 1.01 – 0.77 (m, 36H), 0.22 – ꢀ0.04 (m, 24H).
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Journal of Medicinal Chemistry
1
3
C NMR (126 MHz, chloroformꢀd) δ 155.59, 155.52, 154.73, 154.72, 154.55, 133.80, 132.54, 129.46,
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
7
5
28.33, 127.85, 127.32, 125.65, 125.52, 125.09, 124.76, 97.82 (anomeric C), 96.55 (anomeric C), 85.71,
9.90, 79.36, 79.20, 75.28, 73.40, 72.68, 71.53, 68.00, 67.75, 66.82, 63.10, 61.05, 58.79, 57.28, 53.28,
0.53, 48.96, 48.38, 41.65, 38.90, 35.94, 35.64, 32.80, 31.91, 30.63, 30.56, 30.05, 29.73, 29.71, 29.68,
29.65, 29.63, 28.97, 28.64, 28.51, 28.49, 28.40, 27.66, 26.87, 26.19, 26.14, 26.02, 25.99, 25.79, 23.98,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
22.95, 22.67, 18.48, 18.33, 18.10, 17.92, ꢀ3.41, ꢀ3.80, ꢀ4.20, ꢀ4.87, ꢀ4.94, ꢀ5.07, ꢀ5.18, ꢀ5.23. MS (ESI) m/e
+
calcd for C94
H
174
N
7
O19Si
4
[M+H] : 1818.8, found: 1818.5. 5ꢀOꢀallylꢀ1,3,2′,6′,3′′ꢀpentaꢀNꢀ(tertꢀ
butoxycarbonyl)ꢀ4′,2′′,4′′,6′′ꢀtetraꢀOꢀTBDMSꢀtobramycin (8). Compound 4 (0.78 g, 0.55 mmol) was
dissolved in anhydrous toluene (5 mL). Allyl bromide (95 µL, 1.1 mmol) and a catalytic amount of
tetrabutylammonium hydrogensulfate (TBAHS) (19 mg, 0.06 mmol) were added into this solution
subsequently, followed by KOH (84 mg, 1.5 mmol). This reaction mixture was stirred at room temperature
2
under N gas for 8 h and then concentrated under reduced pressure. The residue was purified via flash
chromatography (elution with a gradient of hexanes/ethyl acetate = 10:1 ~ 4:1) to give the desired product as
1
a white solid. Yield: 0.64 g (79%). H NMR (500 MHz, chloroformꢀd) δ 5.95 – 5.80 (m, 1H, CH=CH ), 5.29
2
(d, J = 17.2 Hz, 1H, CH=CHH), 5.24 – 5.12 (m, 2H, anomeric H), 5.11 – 5.04 (m, 1H, CH=CHH), 4.63 –
4
.47 (m, 1H), 4.28 – 4.05 (m, 3H), 3.87 – 3.24 (m, 13H), 3.23 – 3.07 (m, 1H), 2.54 – 2.40 (m, 1H), 1.99 (m,
H), 1.62 – 1.50 (m, 1H), 1.50 – 1.29 (m, 45H), 1.18 – 1.02 (m, 1H), 1.02 – 0.78 (m, 36H), 0.21 – ꢀ0.04 (m,
1
13
24H). C NMR (126 MHz, chloroformꢀd) δ 155.88, 155.69, 154.96, 154.89, 154.75, 134.48, 117.38, 97.99
(anomeric C), 96.85 (anomeric C), 85.14, 80.02, 79.53, 79.38, 79.21, 75.39, 73.79, 72.74, 71.62, 68.13,
6
2
7.25, 63.43, 57.42, 50.70, 49.22, 48.54, 41.78, 36.03, 35.70, 28.76, 28.63, 28.61, 28.54, 26.23, 26.14,
5.92, 18.61, 18.43, 18.23, 18.04, ꢀ3.35, ꢀ3.63, ꢀ4.07, ꢀ4.75, ꢀ4.78, ꢀ4.98, ꢀ5.01, ꢀ5.05. MS (ESI) m/e calcd
+
for C70
H
137
N
5
O19Si
4
Na [M+Na] : 1488.2, found: 1487.0.
5ꢀOꢀ(2,3ꢀdihydroxypropyl)ꢀ1,3,2
′
,6
′,3′′ꢀpentaꢀNꢀ(tertꢀbutoxycarbonyl)ꢀ4
′,2′′,4′′,6′′ꢀ
tetraꢀOꢀTBDMSꢀtobramycin (9). Compound 8 (250 mg, 0.17 mmol), OsO
4
(69 µL, 6.8 µmol, 2.5 wt. %
solution in tertꢀbutanol) and 2,6ꢀlutidine (40 µL, 0.34 mmol) were added to a stirred solution of 1,4ꢀdioxane
(6 mL) at room temperature. The reaction mixture was stirred at 60 ºC overnight, quenched by saturated
2 2 3
Na S O solution (2 mL) and extracted with ethyl acetate (5 mL × 3), subsequently. The organic layer
2
3
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2 4
extracts were combined, washed with saturated brine (8 mL) and dried over anhydrous Na SO . It was then
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
concentrated in vacuo to afford 150 mg crude product as a yellow oil, to which was used in the next step
+
without further purification. MS (ESI) m/e calcd for C70
H N
139 5
O
21Si
4
Na [M+Na] : 1522.2, found: 1521.2.
5
ꢀOꢀacetaldehydeꢀ1,3,2′,6′,3′′ꢀpentaꢀNꢀ(tertꢀbutoxycarbonyl)ꢀ4′,2′′,4′′,6′′ꢀtetraꢀOꢀTBDMSꢀtobramycin
(10). Compound 9 (150 mg crude product from last step) was dissolved in 1,4ꢀdioxane (15 mL) at room
temperature. NaIO (107 mg, 0.5 mmol) solution in H O (5 mL) was added and stirred at 55 ºC overnight.
The reaction mixture was concentrated under reduced pressure and purified via flash chromatography
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
2
(elution with a gradient of hexanes/ethyl acetate = 10:1 ~ 2:1) to give the desired product as a colorless oil.
1
Yield: 65 mg (26% two steps). H NMR (500 MHz, chloroformꢀd) δ 9.53 (s, 1H, CHO), 5.18 – 5.10 (m, 1H,
anomeric H), 5.10 – 5.04 (m, 1H, anomeric H), 4.80 (d, J = 17.5 Hz, 1H), 4.37 – 4.23 (m, 1H), 4.23 – 3.98
(m, 2H), 3.86 – 3.24 (m, 13H), 3.24 – 3.02 (m, 1H), 2.55 – 2.36 (m, 1H), 2.01 – 1.87 (m, 1H), 1.61 – 1.32
13
(
m, 46H), 1.13 – 1.02 (m, 1H) 1.00 – 0.73 (m, 36H), 0.21 – ꢀ0.05 (m, 24H). C NMR (126 MHz,
chloroformꢀd) δ 197.58 (CHO), 155.88, 155.64, 154.90, 154.82, 154.75, 98.22 (anomeric C), 97.32
(anomeric C), 86.68, 80.21, 79.80, 79.66, 79.50, 79.23, 78.63, 77.41, 77.16, 76.91, 75.76, 72.76, 71.44,
6
2
1
7.82, 67.25, 63.42, 57.25, 50.56, 49.24, 48.56, 41.58, 36.79, 36.01, 35.44, 32.03, 29.80, 29.46, 28.73,
8.61, 28.59, 28.54, 28.48, 26.24, 26.18, 26.14, 25.94, 25.89, 24.82, 22.79, 18.62, 18.38, 18.28, 18.22,
8.15, 18.06, 18.01, 14.23, ꢀ3.41, ꢀ3.64, ꢀ4.08, ꢀ4.75, ꢀ4.78, ꢀ4.96, ꢀ5.02, ꢀ5.05. MS (ESI) m/e calcd for
+
C
69
H
135
N
5
O
20Si
4
Na [M+Na] : 1489.2, found: 1489.2.
5
ꢀOꢀ(dodecylparoxetine)ꢀ1,3,2
tetraꢀOꢀTBDMSꢀtobramycin (11). To a stirred solution of paroxetine hydrochloride (33 mg, 0.09 mmol) in
MeOH (2 mL) and H O (1 mL) was added K CO (41 mg, 0.3 mmol). The mixture was concentrated under
′,6′,3′′ꢀpentaꢀNꢀ(tertꢀbutoxycarbonyl)ꢀ4′,2′′,4′′,6′′ꢀ
2
2
3
reduced pressure to generate a white solid. A solution of compound 6f (100 mg, 0.06 mmol) in DMF (3 mL)
was added into the above white solid mixture and heated up to 80 ºC. The reaction was stirred at this
temperature for 12 h. DMF was removed under reduced pressure. Then, DCM (8 mL) and MeOH (2 mL)
were added to the residue. It was then filtered to afford a clear solution to which was concentrated in vacuo.
The crude was purified by flash chromatography (elution with a gradient of hexanes/ethyl acetate = 10:1 ~
1
1
:1) to give a desired product as a white solid. Yield: 97 mg (84%). H NMR (500 MHz, chloroformꢀd) δ
2
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Journal of Medicinal Chemistry
7
1
4
2
.19 – 7.08 (m, 2H), 7.01 – 6.88 (m, 2H), 6.60 (d, J = 8.5 Hz, 1H), 6.33 (d, J = 2.5 Hz, 1H), 6.15 – 6.08 (m,
H) 5.86 (s, 2H), 5.26 – 5.19 (m, 1H, anomeric H), 5.19 – 5.11 (m, 1H, anomeric H), 4.35 – 4.21 (m, 1H),
.21 – 4.13 (m, 1H), 4.13 – 3.99(m, 1H), 3.85 – 3.65 (m, 4H), 3.65 – 2.97 (m, 16H), 2.58 – 2.31 (m, 4H),
.31 – 2.13 (m, 1H), 2.13 – 1.97 (m, 3H), 1.85 – 1.76 (m, 1H), 1.62 – 1.36 (m, 50H), 1.35 – 1.16 (m, 17H),
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
3
1.08 – 1.00 (m, 1H), 1.00 – 0.76 (m, 36H), 0.23 – ꢀ0.04 (m, 24H). C NMR (126 MHz, chloroformꢀd) δ
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
161.44 (d, J = 244.2 Hz, CF), 155.51, 154.71, 154.54, 154.39, 148.08, 141.48, 139.82, 128.81, 128.75,
115.39, 115.23, 107.77, 105.55, 101.00, 97.96, 97.93, 96.53, 85.70, 79.88, 79.35, 79.18, 75.27, 73.40, 72.67,
71.52, 69.67, 68.00, 66.81, 63.08, 59.25, 57.75, 57.26, 54.13, 50.52, 48.95, 48.37, 44.22, 42.11, 41.66,
35.91, 35.63, 34.40, 30.62, 30.05, 29.74, 29.71, 29.68, 29.66, 28.62, 28.50, 28.48, 28.39, 27.77, 27.09,
26.19, 26.12, 26.00, 25.98, 25.77, 18.47, 18.32, 18.09, 17.90, ꢀ3.43, ꢀ3.81, ꢀ4.21, ꢀ4.89, ꢀ4.96, ꢀ5.08, ꢀ5.19, ꢀ
+
5.24. MS (ESI) m/e calcd for C98
H
176FN
6
O
22Si
4
[M+H] : 1921.8, found: 1921.7.
5
ꢀOꢀ(12ꢀhydroxydodecyl)ꢀ1,3,2′,6′,3′′ꢀpentaꢀNꢀ(tertꢀbutoxycarbonyl)ꢀ4′,2′′,4′′,6′′ꢀtetraꢀOꢀTBDMSꢀ
tobramycin (12). DMF (15 mL) and H O (3 mL) were added to a flask containing compound 6f (628 mg,
0.38 mmol) and K CO (104 mg, 0.75 mmol). The reaction mixture was heated to 80 ºC and stirred for 24 h.
2
2
3
Water (15 mL) was added and the resulting mixture was extracted with ethyl acetate (3 × 10 mL). The
organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. The solvent was
removed in vacuo followed by purification using flash chromatography (elution with a gradient of
1
hexanes/ethyl acetate = 20:1 ~ 2:1) to give product 12 as a white solid. Yield: 260 mg (43%). H NMR (500
MHz, chloroformꢀd) δ 5.25 – 5.19 (m, 1H, anomeric H), 5.12 (m, 1H, anomeric H), 4.32 – 4.21 (m, 1H),
4
1
3
.21 – 4.12 (m, 1H), 4.13 – 4.02 (m, 1H), 3.82 – 3.13 (m, 16H), 2.47 (d, J = 12.6 Hz, 1H), 2.06 – 1.95 (m,
H), 1.62 – 1.52 (m, 3H), 1.52 – 1.38 (m, 48H), 1.38 – 1.17 (m, 17H), 0.98 – 1.13 (m, 1H), 1.00 – 0.78 (m,
13
6H), 0.21 – ꢀ0.02 (m, 24H). C NMR (126 MHz, chloroformꢀd) δ 155.70, 155.52, 154.75, 154.70, 154.57,
97.85 (anomeric C), 96.52 (anomeric C), 85.73, 79.90, 79.22, 77.20, 75.29, 73.37, 72.65, 71.55, 68.01,
6
6.82, 63.11, 63.01, 57.27, 50.52, 48.95, 48.37, 41.67, 35.89, 35.63, 32.81, 30.61, 29.95, 29.57, 29.52,
2
9.38, 18.48, 18.32, 18.09, 17.91, ꢀ3.42, ꢀ3.80, ꢀ4.20, ꢀ4.89, ꢀ4.95, ꢀ5.09, ꢀ5.19, ꢀ5.24. MS (ESI) m/e calcd
+
for C79
H
157
N
5
O20Si
4
Na [M+Na] : 1632.5, found: 1631.4.
2
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5
ꢀOꢀ(12ꢀdodecanal)ꢀ1,3,2′,6′,3′′ꢀpentaꢀNꢀ(tertꢀbutoxycarbonyl)ꢀ4′,2′′,4′′,6′′ꢀtetraꢀOꢀTBDMSꢀtobramycin
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
13). PCC (pyridinium chlorochromate, 388 mg, 1.8 mmol) and NaOAc (5 mg, 0.06 mmol) were added to a
stirred solution of compound 12 (0.965 g, 0.6 mmol) in dry DCM (20 mL). The reaction mixture was stirred
at room temperature under N gas for 1 h. When a TLC analysis shows that most of the starting alcohol is
2
consumed, the chromium species were removed by filtration through a pad of silica gel and washed with
ethyl acetate. The collected organic phase was concentrated under reduced pressure to afford the crude
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
which was then purified via flash chromatography (elution with a gradient of hexanes/ethyl acetate = 1:1 ~
1
1
00% ethyl acetate) to give the desired product 13 as a white solid. Yield: 0.897 g (93%). H NMR (500
MHz, chloroformꢀd) δ 9.74 (s, 1H, CHO), 5.24 – 5.15 (m, 1H, anomeric), 5.16 – 5.10 (m, 1H, anomeric),
4.33 – 4.19 (m, 1H), 4.19 – 4.10 (m, 1H), 4.09 – 3.99 (m, 1H), 3.84 – 3.28 (m, 12H), 3.28 – 3.03 (m, 3H),
2
1
.52 – 2.42 (m, 1H), 2.42 – 2.35 (m, 2H), 2.02 – 1.94 (m, 1H), 1.63 – 1.57 (m, 2H), 1.55 – 1.33 (m, 48H),
13
.33 – 1.14 (m, 15H), 1.09 – 0.99 (m, 1H), 0.99 – 0.75 (m, 36H), 0.21 – ꢀ0.07 (m, 24H). C NMR (126
MHz, chloroformꢀd) δ 202.8 (CHO), 155.67, 155.49, 154.70, 154.68, 154.53, 97.82 (anomeric C), 96.48
(anomeric C), 85.70, 79.85, 79.33, 79.17, 78.77, 75.25, 73.33, 72.64, 71.52, 67.99, 66.81, 63.08, 57.24,
5
–
5
0.50, 48.91, 48.34, 43.87, 41.64, 35.89, 35.61, 30.59, 29.97, 29.63, 29.58, 29.56, 29.39, 29.31, 29.15, 28.80
28.17, 26.28 – 25.50, 22.06, 18.45, 18.30, 18.07, 17.88, ꢀ3.45, ꢀ3.82, ꢀ4.23, ꢀ4.92, ꢀ4.98, ꢀ5.11, ꢀ5.21, ꢀ
+
.27. MS (ESI) m/e calcd for C79
Compound 15. K CO
H
155
N
5
O20Si
4
Na [M+Na] : 1630.5, found: 1631.1.
2
3
solution (25 mg, 0.18 mmol in 3 mL of H
2
O) was added to a flask containing
compound 14 (TFA salt, 350 mg, 0.22 mmol) and stirred for 5 min at room temperature. The mixture was
extracted with ethyl acetate (3 × 3 mL), dried over anhydrous sodium sulfate and concentrated under
reduced pressure to give a pale yellow solid. The resulting solid was mixed with aldehyde 13 (350 mg, 0.22
mmol), followed by the addition of dry DCM (10 mL) and AcOH (10 µL, 0.018 mmol) under N
2
gas. The
reaction mixture was stirred at room temperature for 7 h before NaBH(OAc) (153 mg, 0.72 mmol) was
3
added at 0 ºC. The solution was gradually warmed to room temperature while stirring overnight. The
reaction mixture was cooled to 0 ºC and quenched carefully by the dropꢀwise addition of saturated NaHCO3
solution (10 mL). The solution was then extracted with DCM (3 × 5 mL). The organic layer was washed
with brine, dried over anhydrous sodium sulfate and concentrated in vacuo. The crude product was purified
2
6
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Journal of Medicinal Chemistry
by flash chromatography (elution with a gradient of hexanes/ethyl acetate = 10:1 ~ 1:2) to afford the desired
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
product 15 as a white solid. Yield: 0.28 g (72%). H NMR (500 MHz, methanolꢀd
4
) δ 8.24 (d, J = 2.2 Hz,
1
H), 7.80 – 7.71 (m, 3H), 7.56 (dd, J = 8.8, 2.1 Hz, 1H), 7.46 – 7.40 (m, 1H), 7.40 – 7.35 (m, 1H), 7.34 –
.27 (m, 4H), 7.27 – 7.21 (m, 3H), 7.20 – 7.10 (m, 3H), 5.47 – 5.34 (m, 2H, anomeric), 5.05 (s, 2H, ,
7
OCH Ph), 4.67 – 4.61 (m, 1H), 4.21 – 4.12 (m, 1H), 3.98– 3.20 (m, 17H), 3.19 – 3.02 (m, 3H), 2.75 – 2.64
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(m, 2H), 2.63ꢀ2.51(m, 1H), 2.51ꢀ2.41(m, 1H), 2.41ꢀ2.29 (m, 1H), 2.29 – 2.14 (m, 1H), 2.14 – 2.01 (m, 3H),
1
0
.98ꢀ1.82 (m, 2H), 1.68 – 1.52 (m, 4H), 1.52 – 1.36 (m, 47H), 1.36 – 1.06 (m, 18H), 0.99 – 0.81 (m, 36H),
13
4
.17 – 0.01 (m, 24H). C NMR (126 MHz, methanolꢀd ) δ 177.18, 171.66, 158.88, 158.17, 157.99, 157.47,
157.03, 156.98, 142.28, 138.37, 137.02, 135.20, 132.13, 129.62, 129.57, 129.46, 129.42, 128.92, 128.88,
128.72, 128.64, 128.61, 127.51, 127.16, 126.07, 121.09, 117.90, 96.78 (anomeric C), 95.41 (anomeric C),
8
6
3
6.59, 86.36, 80.64, 80.56, 80.33, 80.16, 79.54, 78.28, 75.20, 74.86, 73.87, 73.54, 72.60, 71.89, 68.80,
8.68, 67.38, 65.06, 58.90, 57.65, 57.31, 54.44, 52.98, 49.89, 44.26, 42.15, 39.66, 36.68, 35.99, 35.62,
3.02, 31.92, 31.31, 30.90, 30.88, 30.84, 30.82, 30.78, 30.12, 29.30, 29.25, 29.12, 28.97, 28.92, 28.90,
28.63, 27.71, 27.00, 26.92, 26.73, 26.68, 26.65, 26.53, 19.50, 19.10, 18.96, 18.90, ꢀ3.24, ꢀ3.79, ꢀ3.93, ꢀ4.15,
+
ꢀ
4.20, ꢀ4.41, ꢀ4.63, ꢀ4.85. MALDIꢀTOFꢀMS m/e calcd for C H N O Na [M+Na] : 2177.303, found:
1
13 191
9
23
2
177.592.
Compound 16. Synthesized following general procedure A from 15 (0.28 g, 0.13 mmol), 40% HCl (4 mL)
and MeOH (6 mL). The compound was purified by reverseꢀphase flash chromatography on C18ꢀsilica
(elution with a gradient from 100% deionized water to deionized water/MeOH = 1:1, v/v) to give the desired
1
product as a white solid. Yield: 86 mg (52%). H NMR (500 MHz, methanolꢀd ) δ 8.20 (d, J = 2.1 Hz, 1H),
4
7
.83 – 7.73 (m, 3H), 7.57 (d, J = 8.9 Hz, 1H), 7.47 – 7.22 (m, 11H), 7.420 – 7.13 (m, 1H), 5.46 (d, J = 3.0
Hz, 1H, anomeric), 5.18 (d, J = 3.4 Hz, 1H, anomeric), 5.06 (s, 2H, , OCH Ph), 4.65 – 4.58 (m, 1H), 4.42 –
4.34 (m, 1H), 4.31 – 4.22 (m, 1H), 4.22 – 4.14 (m, 1H), 3.29 – 3.22 (m, 4H), 3.97 – 3.84 (m, 2H), 3.84 –
2
3
2
.48 (m, 13H), 3.19 – 3.06 (m, 2H), 2.95 – 2.69 (m, 3H), 2.59 – 2.50 (m, 1H), 2.51 – 2.41 (m, 1H), 2.40 –
13
.28 (m, 1H), 2.28 – 2.05 (m, 6H), 1.71 – 1.55 (m, 4H), 1.40 – 1.19 (m, 16H). C NMR (75 MHz,
methanolꢀd
4
) δ 171.86, 169.47, 159.12, 142.11, 138.24, 136.98, 135.19, 132.23, 129.63, 129.54, 129.07,
128.84, 128.63, 128.56, 127.57, 127.31, 126.18, 121.24, 118.09, 102.34 (anomeric C), 93.89 (anomeric C),
2
7
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Journal of Medicinal Chemistry
Page 28 of 50
8
5
3
3.41, 82.74, 77.06, 76.33, 75.40, 73.99, 70.53, 67.95, 67.69, 66.87, 65.58, 61.39, 59.18, 56.86, 56.41,
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1.06, 50.04, 49.90, 49.61, 49.33, 49.21, 49.05, 48.88, 48.76, 48.48, 48.20, 42.72, 40.51, 39.07, 35.21,
4.36, 33.33, 31.30, 31.08, 30.94, 30.84, 30.71, 30.56, 30.30, 28.98, 27.60, 27.14, 26.97. MS (ESI) m/e
+
calcd for C H N O [M+H] : 1199.5, found: 1199.2.
64 96
9
13
Biology. Clinical Isolates. Bacterial isolates were obtained as part of the Canadian National Intensive
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
2
53,54
Care Unit (CANꢀICU) study and Canadian Ward Surveillance (CANWARD) studies
. Nineteen
medical centers from all regions of Canada with active intensive care units (ICUs) participated on the CANꢀ
ICU study. Each participating centre collected a maximum of 300 consecutive bacterial isolates recovered
from blood, urine, wound/tissue, and respiratory specimens (one pathogen per cultured site per patient) of
ICU patients from September 2005 to June 2006, inclusive. The 4180 bacterial isolates obtained represented
2
580 patients (or 1.62 isolates per patient). Note that each participating study sites were requested to only
collect “clinically significant” specimens from patients with a presumed infectious disease. All isolates were
transported to the reference laboratory (Health Sciences Centre, Winnipeg, Canada) on Amies charcoal
swabs, subcultured onto an appropriate media, and stocked in skim milk at ꢀ80°C until antimicrobial
susceptibility testing was carried out.
Antimicrobial Susceptibilities. Following two subcultures from frozen stock, the in vitro antibacterial
activities of agents were determined by microbroth dilution susceptibility test in accordance with the
Clinical and Laboratory Standards Institute (CLSI) guidelines. All pathogens obtained from the CANꢀICU
and CANWARD studies have been obtained from these national Health Canada endorsed studies that have
received Ethics approval from the University of Manitoba Ethics Committee. In addition, individual
hospitals obtain ethics approval in order to submit isolates. The minimum inhibitory concentration (MIC) of
the antimicrobial agents for the bacterial isolates were determined using 96ꢀwell plates containing doubling
antimicrobial dilutions of cation adjusted MuellerꢀHinton broth (MHB) and inoculated to achieve a final
5
concentration of approximately 5 × 10 CFU/mL. We used stock concentration of either 10.24 mg/mL or
5
.12 mg/mL in deionized water or DMSO depending on the solubility of the compounds. The plates were
then incubated in ambient air for 18 ~ 24 hours prior to reading. Reference bacterial strains including
Staphylococcus aureus ATCC 29213, methicillinꢀresistant S. aureus (MRSA) ATCC 33592, Enterococcus
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Page 29 of 50
Journal of Medicinal Chemistry
faecalis ATCC 29212, Enterococcus faecium ATCC 27270, Streptococcus pneumoniae ATCC 49619,
Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853 and Klebsiella pneumoniae ATCC
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13883 were acquired from the American Type Culture Collection (ATCC) and were used as a quality
control strains. The clinical isolates methicillinꢀresistant Staphylococcus epidermidis (MRSE cefazolin MIC
> 32 ꢂg/mL) CANꢀICU 61589, gentamicinꢀresistant E. coli CANꢀICU 61714, amikacinꢀresistant (MIC = 32
ꢂg/mL) E. coli CANꢀICU 63074, gentamicinꢀresistant P. aeruginosa CANꢀICU 62584, Strenotrophomonas
maltophilia CANꢀICU 62584 and Acinetobacter baumannii CANꢀICU 63169 were obtained from hospitals
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across Canada as a part of the CANꢀICU study . Methicillinꢀsusceptible S. epidermidis (MSSE)
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CANWARDꢀ2008 81388 was obtained from the 2008 CANWARD study while gentamicinꢀresistant
tobramycinꢀresistant ciprofloxacinꢀresistant [aminoglycoside modifying enzyme aac(3)ꢀIIa present] E. coli
CANWARDꢀ2011 97615 and gentamicinꢀresistant tobramycinꢀresistant P. aeruginosa CANWARDꢀ2011
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6846 were obtained from the 2011 CANWARD study .
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Antibacterial Combination Screening. Checkerboard method : The checkerboard consists of columns in
which each well contains the same amount of the antibiotic being twoꢀfold diluted along the x axis of 96ꢀ
well plate. Rows in which each well contains the same amount of the synthetic compound being twoꢀfold
diluted on the y axis. The result is that each well in the checkerboard contains a unique combination of the
two agents being tested. Overnight bacterial culture was standardized in saline using the 0.5 McFarland
turbidity standard and diluted 1:50 in MHB. Fifty microliter of standardized culture was added to each of the
wells and the plate was incubated at 37 ˚C for 18 h. Fractional inhibitory concentration index (FICI)
determination: FICs were determined by checkerboard method. The MIC for each drug was the lowest
concentration showing no bacterial growth. The FIC for the compounds and antibiotics were calculated as
the [MIC of agents in combination] / [MIC of agent alone]. The FICI is the sum of the FIC of the compound
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and the antibiotic . The combination is considered synergistic when the FICI is ≤ 0.5, no interaction when
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the FICI is > 0.5 to < 4.0, and antagonistic when the FICI is ≥ 4.0 .
Hemolytic Assay. The capability of the synthesized agents to lyse eukaryotic red blood cells were
determined using ovine erythrocytes (obtained from a slaughter house) following Institutional biosafety
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rules using a modified protocol . First, the erythrocytes were washed in Trisꢀbuffered saline (10 mM Tris,
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