Antibacterial activity in serum of the 3,5-diamino-piperidine translation inhibitors

Article abstract of DOI:10.1016/j.bmcl.2008.04.023

Translation inhibitors of the 3,5-diamino-piperidine series act as aminoglycoside mimetics that inhibit bacterial growth. Here we show antibacterial SAR in the presence and absence of serum with a particular focus toward Pseudomonas aeruginosa.

Full text of DOI:10.1016/j.bmcl.2008.04.023

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 3369–3375
Antibacterial activity in serum of the
3,5-diamino-piperidine translation inhibitors
Yuefen Zhou, Chun Chow, Douglas E. Murphy, Zhongxiang Sun, Thomas Bertolini,
Jamie M. Froelich, Stephen E. Webber, Thomas Hermannà and Daniel Wall*
Anadys Pharmaceuticals, Inc., San Diego, USA
Received 18 February 2008; revised 9 April 2008; accepted 10 April 2008
Available online 13 April 2008
Abstract—Translation inhibitors of the 3,5-diamino-piperidine series act as aminoglycoside mimetics that inhibit bacterial growth.
Here we show antibacterial SAR in the presence and absence of serum with a particular focus toward Pseudomonas aeruginosa.
Ó 2008 Elsevier Ltd. All rights reserved.
The increasing prevalence of antibiotic resistance repre-
sents a global medical threat. In the United States about
two million patients are infected from hospital visits.1
The majority of these nosocomial pathogens are resis-
tant to at least one antibiotic and cause over 90,000
deaths per year. Pseudomonas aeruginosa has been iden-
tified as a particularly problematic pathogen.2 It is an
invasive, Gram-negative bacteria that causes a wide
range of severe infections including; a leading cause of
hospital acquired pneumonias, bloodstream and surgi-
cal wound infections and infections of immunocompro-
mised patients. In cystic fibrosis patients P. aeruginosa
incites inflammation that destroys lung tissue and leads
to respiratory failure. Compounding the problem of
causing life-threatening infections, P. aeruginosa has a
strong propensity to develop resistance to virtually any
antibiotic. For example, clinical isolates of P. aeruginosa
are frequently resistant to commonly used antibiotics
including fluoroquinolones (33%), imipenem (22%),
and ceftazidime (30%).2 Some isolates are resistant to
all FDA clinical approved antibiotics. For these reasons,
there is an urgent clinical need to develop new antibiot-
ics that work by novel mechanisms to treat P.
aeruginosa.
To address this important clinical need, we used a ra-
tional approach to develop new antibiotics for the treat-
ment of severe and drug resistant bacterial infections.
This approach was based on three-dimensional structural
information of aminoglycosides interactions with the
ribosomal decoding site, or A-site, to develop mimetics
that are amenable to rapid optimization by parallel syn-
thesis.3 Structural studies showed that 2-deoxystrepta-
mine (2-DOS), the common core scaffold among
aminoglycoside antibiotics, binds in a conserved configu-
ration regardless of the 4,5- or 4,6-disubstitutions found
in the neomycin or gentamicin families, respectively.4
The cis-1,3- amino groups of 2-DOS are involved in base
recognition via conserved hydrogen bonds with A1493,
G1494, and U1495 of the 16S rRNA. These interactions
anchor the aminoglycoside scaffold within the A-site
internal loop and displace residues A1492 and A1493
from the RNA interior. These two adenine residues act
as a molecular switch that is involved in securing the fidel-
ity of translation by interacting with the first two base
pairs of the mRNA–tRNA codon–anticodon hybrid.
Thus, aminoglycoside binding impairs the ribosome’s
ability to discriminate against near-cognate tRNA–
mRNA pairings, which causes an accumulation of defec-
tive peptides and, ultimately, cell death.
Keywords: Aminoglycosides; Antibiotics; Translation inhibitors;
2-Deoxy-streptamine; Ribosome; Decoding site; 3,5-Diamino-
piperidine.
*
Corresponding author at present address: Molecular Biology
Department, University of Wyoming, 1000 E. University Avenue,
Laramie, WY 82071, USA. Tel.: +1 307 766 3542; fax: +1 307 766
3875; e-mail: dwall2@uwyo.edu
Present address: CytRx Corporation, 3030 Bunker Hill Street, Suite
101, San Diego, CA 92109, USA.
While 2-DOS is recognized as a key pharmacophore for
binding the rRNA target, its synthesis and modification
is challenging due to the five contiguous stereogenic
à
Present address: Department of Chemistry and Biochemistry, Uni-
versity of California, San Diego, La Jolla, CA 92093-0358, USA.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.04.023

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Y. Zhou et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3369–3375
centers.5 Therefore, we developed simplified synthetic
mimetics of 2-DOS, one of which is cis-3,5-diamino-
piperidine (DAP).4b,6 The DAP ring retains the charac-
teristic cis-1,3-diamine configuration of 2-DOS which is
required for RNA recognition. Typically, two symmet-
ric DAP moieties are attached to a triazine core (termed
DAPT).6 To optimize antibacterial activity, the so-
called ‘tailpiece’ substituents were attached at the tri-
azine 4-position via an N-linkage.7 Prior studies
described the synthesis and structure–activity relation-
ships of the DAP series as inhibitors of in vitro transla-
tion and antibacterial potency.3,6,7 Here, we describe the
potent antibacterial activity against P. aeruginosa and
the effect of serum on MIC (minimal inhibitory
concentration).
strate that the DAP series exhibits exceptional anti-P.
aeruginosa activity that justifies further exploration.
Although many DAP compounds show potent antibac-
terial activity, their potencies are significantly reduced
by serum. Figure 1 plots the fold shift in MIC values
for a set of 45 compounds incubated in the presence
or absence of 50% serum against Escherichia coli and
P. aeruginosa. These results show that serum dramati-
cally increased MIC values. Moreover, serum had a dif-
ferential effect between organisms; E. coli has
approximately a 10-fold mean MIC shift, while P. aeru-
ginosa has a 50-fold mean shift with serum. The reason
for the species difference is not clear, though it may re-
flect different properties in outer membrane composi-
tion. Similarly, the aminoglycosides tobramycin and
gentamicin exhibited a more pronounce serum shift
against P. aeruginosa (4- to 8-fold, respectively) com-
pared to E. coli (1- to 2-fold). Although antibiotic inter-
actions with serum proteins are usually attributed to
albumin binding,8 this protein appears to have a mini-
mal effect on the DAP series as incubations with 8%
albumin typically resulted in modest MIC changes.
Interestingly, some compounds actually exhibited a syn-
ergistic effect that lowered E. coli MIC values with ser-
um. Such results were reproducible and titratable.
While serum protein binding can have positive effects
on antibiotic pharmacokinetics,8 we found that the com-
pounds with high serum MIC shifts correlated with no
or very poor efficacy in mice. In contrast, a DAP com-
pound that was not inhibited or shifted by serum fully
protected mice from an E. coli septicemia infection.3
For these reasons, we sought to understand serum
SAR to improve activity.
Table 1 shows the antibacterial activity of N-{4-[4,6-bis-
(3,5-diamino-piperidin-1-yl)-[1,3,5]triazin-2-ylamino]-2-
hydroxy-phenyl}-4-chloro-benzamide (compound 1)3a
against 53 clinical isolates of P. aeruginosa. For compar-
ison, the activity of seven diverse clinical antibiotics,
most of which are used to treat P. aeruginosa infections,
are shown as well. Compound 1 shows a reasonable dis-
tribution in activity with the highest MIC value as good
as or better than any other antibiotic. Fluoroquinolones,
such as ciprofloxacin, can show potent activity toward
P. aeruginosa, but as discussed above, resistance is com-
mon and consequently the MIC90 value of ciprofloxacin
is poor. In contrast, compound 1 shows robust and
broadly consistent activity against a panel of clinical iso-
lates and has the most potent MIC80 and MIC90 values
among the eight antibiotics tested. These results demon-
Table 1. Antibacterial activity against 53 clinical P. aeruginosa isolates
Table 2 shows that simple addition of a nitrogen atom in
aromatic rings of the tailpiece substituent could signifi-
cantly reduce serum effect on MIC for two bacterial spe-
cies (E. coli and P. aeruginosa). This modest structural
change resulted in a 32-fold increase in serum MIC
potencies (compare compounds 2 and 4). The position
of the nitrogen atom in the aromatic ring had little im-
pact on antibacterial activity (compare 3 and 4). Similar
SAR were found in other cases (compare 7 and 8 or 9,
NH2
NH2
N
N
N
H2N
NH2
O
N
N
HN
N
H
OH
Cl
1
10
P. aeruginosa
Antibiotic
MICa (lg/ml)
8
6
4
2
0
E. coli
Distribution Mean MIC50 MIC80 MIC90
Compound 1
Amikacin
Tetracycline
1
1
1
32
128
4
6
4
4
4
8
8
32
>128 32
>128 >128 >128
27
64
64
Chloramphenicol 64
Ciprofloxacin
Imipenemb
>128
32
16
>128
64
16
0.06
128
32
1
3
0.25
0.5
1
2
8
2
Piperacillinc
Ceftazidime
128
32
12
4
32
8
64
16
0.25 0.5
1
2
4
8
16
32
64
128
0.5
MIC fold shift
Susceptibility test was done by agar dilution using Mueller–Hinton
(MH) agar by Fujisawa Pharmaceuticals Co., Ltd.
a Minimum inhibitory concentration. Subscript designation of 50, 80
or 90 refers to percent of isolates inhibited at a given concentration.
b Imipenem/cilastatin sodium.
Figure 1. The effect of 50% mouse serum on a set of 45 DAP
compounds. MIC (lg/ml) values were determined by microdilution
method in MH media according to National Committee for Clinical
Laboratory Standards. Strains: E. coli ATCC 25922 and P. aeruginosa
ATCC 27853.
c Piperacillin/tazobactam.

Y. Zhou et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3369–3375
3371
Table 2. DAPT serum SAR with nitrogen heterocycle tailpieces
NH2
N
NH2
N
N
H2N
NH2
N
N
R1
R1
IVT IC50 (lM)
E. coli
Serum fold shift
P. aeruginosa
Serum fold shift
a
Compound
MICb
MIC
H
N
O
OH
2
24
1
32
60.5
P128
N
H
H
N
O
O
OH
3
4
NAc
4
4
2
2
1
16
N
N
H
H
N
OH
N
5
1
8
N
H
H
N
O
O
OH
N
5
6
24
4
1
1
4
1
1
8
8
N
H
Cl
H
N
OH
N
5
N
H
OH
H
N
O
7
8
9
11
10
4
4
8
8
4
2
1
2
32
N
H
OH
OH
HO
H
N
O
4
8
N
N
H
HO
H
N
O
60.5
P32
N
H
OH
HO
N
H
N
O
10
11
9
3
4
4
8
2
1
P64
P32
Cl
N
H
OH
OH
HO
H
N
O
N
60.5
Cl
H
HO
N
a Coupled in vitro transcription-translation assay with E. coli extracts.3
b MICs (lg/ml) as described in Figure 1.
c NA, not available.

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Y. Zhou et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3369–3375
Table 3. Quinolone tailpieces and serum shift
NH2
N
NH2
N
N
H2N
NH2
O
N
N
HN
N
H
R2
Compound
R2
IVT IC50 (lM)
E. coli
Serum fold shift
P. aeruginosa
Serum fold shift
MIC
16
MIC
8
O
N
F
12
1
2
2
16
8
N
N
O
O
O
O
13
14a
15
3
1
4
1
2
2
N
O
2
4
4
N
N
O
N
F
0.4
64
0.06
2
N
HN
O
F
16
1.3
60.5
P4
2
8
N
N
N
O
N
17
18
0.9
32
0.13
1
1
4
4
N
N
N
O
N
HN
HN
F
2
60.5
1
N
a The corresponding nalidixic acid molecule has an IVT IC50 > 100.
10 and 11), prompting us to synthesize a small focused
library that contained nitrogen heterocycles as tailpieces
(Table 3). Since at least two aromatic rings in the tail-
piece imparted an optimal configuration for activity,7
we chose to make a focused library using quinolones
as tailpieces. Table 3 indeed shows that nitrogen con-
taining heterocycles from quinolones result in potent
DAPT compounds that had modest serum MIC shifts.
In some cases the E. coli MIC values were dramatically
reduced by serum (compounds 15 and 17), suggesting
synergistic antibacterial effects with serum, which is con-
sistent with known antibacterial activity in serum.8
These results show that nitrogen containing heterocycles
as tailpieces lead to potent in vitro translation inhibitors
that have no or modest shifts in antibacterial activity in
the presence of serum.
Aside from tailpiece SAR, we also explored substitution
of the triazine core. As shown in Table 4, when the DAP
and tailpiece moieties were kept constant, a variety of
core substitutions including benzene, pyridine, pyrimi-
dine and purine rings had a minimal effect on translation

Y. Zhou et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3369–3375
3373
Table 4. Core SAR
DAP
NH2
N
NH2
Core
N
N
H2N
NH2
O
N
N
HN
OH
N
H
R
Compound
Product
IVT IC50 (lM)
E. coli
Serum fold shift
P. aeruginosa
Serum fold shift
MIC
1
MIC
N
DAP
DAP
19
24
32
60.5
P128
N
N
R
DAP
DAP
DAP
DAP
DAP
DAP
20
21
22
23
12
16
26
14
2
2
2
2
P32
32
2
P32
P128
64
R
60.5
N
R
N
16
1
1
R
N
DAP
Cl
DAP
Cl
32
P64
R
DAP
N
DAP
24
18
18
60.5
P16
8
P8
N
R
DAP
6
H
25a
1
8
2
32
N
N
2
DAP
8
N
N
R
a A mixture of 2 regio-isomers (other: 2,6-di-DAP-8-R-purine) as intermediates of 6,8-di-DAP-2-Cl-purine and 2,6-di-DAP-8-Cl-purine were not
isolated. Synthesis follows either method A or B as described in supplementary material.
inhibition, suggesting that the aromatic core acts as a
scaffold for the DAP functional groups to bind at the
A-site RNA.3,6 With respect to MIC in the presence of
serum, a purine core (compound 25) resulted in a mod-
erate improvement as compared with 19. The replace-
ment of the triazine core with a pyridine core
(compounds 22 and 23) may also have a modest effect
on the MIC shift with serum.
Summary and perspective: This work expands on our ef-
forts toward rationally designed 2-DOS mimetics.3,9 The
active DAP pharmacophore was derived from structural
information of the molecular recognition between the
conserved 2-DOS scaffold of aminoglycosides and the
A-site target RNA.3,6 After the initial design of DAPT,
the series then evolved through an iterative process of
parallel synthesis and high-throughput testing that led
to promising lead compounds. Rapid medicinal chemis-
try elaboration can now be used to potentially generate
a clinical candidate that may improve on the pharma-
ceutical properties of aminoglycosides, including persist-
ing resistance, poor bioavailability and undesirable
toxicity.
To explore possible synergistic or additive effects be-
tween core and tailpiece substitutions, a selected number
of compounds were synthesized. As shown in Table 5,
the combination of a triazine core replacement with a
purine or pyridine core with nitrogen-containing hetero-
cycle tailpieces resulted in potent translation inhibitors
with improved serum MICs. Of particular note, addition
of a nitrogen atom in the tailpiece aromatic ring of a
pyridine compound 28 (Table 5) resulted in a significant
improvement in serum MIC as compared to compound
22 (Table 4).
This letter focused on antibacterial optimization in the
presence of serum of our novel DAP translation inhib-
itors. Improvements in potency correlated to com-
pound structure were achieved with nitrogen
containing heterocycle tailpieces. Substitutions of the

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Y. Zhou et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3369–3375
Table 5. Purine and pyridine core optimization
Compound
Product
IVT IC50 (lM)
E. coli
P. aeruginosa
Serum fold shift
MIC
1
Serum fold shift
MIC
1
DAP
H
N
H
N
N
26a
NA
8
2
16
O
O
OH
N
N
N
DAP
N
H
DAP
H
N
H
N
N
27a
0.4
8
16
4
OH
N
N
DAP
DAP
N
N
H
N
DAP
28
2.2
P64
60.06
60.5
P4
HN
O
OH
N
H
N
DAP
Cl
N
DAP
Cl
HN
O
O
N
29
0.8
8
2
8
4
F
N
H
N
NH
a Compounds 26 and 27 were synthesized with a different method than 25 in Table 4. Synthesis follows either method A or B as described in
supplementary material.
triazine core with a purine or pyridine ring also
showed clear SAR. A promising feature of the DAP
derivatives10 is their potent antimicrobial activity
against clinical P. aeruginosa isolates (Table 1). This
pathogen is notorious for developing antibiotic resis-
tance.2 Our description of the novel DAP series that
exhibits exceptional MIC90 value against a panel of
clinically resistant P. aeruginosa is significant. How-
ever, enthusiasm for the series was initially diminished
by the loss of antibacterial activity in the presence of
serum. Our current results show that this obstacle
can be addressed and that certain compounds have
acceptable antibacterial activity with serum. We be-
lieve that these and other findings warrant further
development of the DAP series as antibiotics.
References and notes
1. National Institute of Allergy and Infectious Disease Fact
Sheet, April 2006.
2. Talbot, G. H.; Bradley, J.; Edwards, J. E.; Gilbert, D.;
Scheld, M.; Bartlett, J. G. Clin. Infect. Dis. 2006, 42, 657.
3. (a) Zhou, Y.; Gregor, V. E.; Sun, Z.; Ayida, B. K.;
Winters, G. C.; Murphy, D.; Simonsen, K. B.; Vourlo-
umis, D.; Fish, S.; Froelich, J. M.; Wall, D.; Hermann, T.
Antimicrob. Agents Chemother. 2005, 49, 4942; (b) Zhou,
Y.; Vourloumis, D.; Gregor, V. E.; Winters, G. C.;
Hermann, T.; Ayida, B.; Sun, Z.; Murphy, D. E.;Simon-
sen, K. B. US patent application US7,223,759 B2,2007.
This patent describes the general syntheses for analogs
containing triazine, pyridine, pyrimidine, benzene, and
purine core structures; (c) There are two general methods
(A & B in supplementary material) most frequently used
for the synthesis of purine analogs. Only method A is
described in Ref. 3b.
Acknowledgment
4. (a) Pfister, P.; Hobbie, S.; Vicens, Q.; Bo¨ttger, E. C.;
Westhof, E. ChemBioChem 2003, 4, 1078; (b) Simonsen,
K. B.; Ayida, B. K.; Vourloumis, D.; Winters, G. C.;
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ChemBioChem 2003, 4, 886; (c) Vicens, Q.; Westhof, E.
Biopolymers 2003, 70, 42.
This work was supported in part by a grant from the
National Institute of Allergy and Infectious Disease
(AI51105).
5. Busscher, G. F.; Rutjes, F. P. J. T.; van Delft, F. L. Chem.
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Supplementary data
6. Zhou, Y.; Gregor, V. E.; Ayyida, B. K.; Winters, G. C.;
Sun, Z.; Murphy, D.; Haley, G.; Bailey, D.; Froelich, J.
M.; Fish, S.; Webber, S. E.; Hermann, T.; Wall, D. Bioorg.
Med. Chem. Lett. 2007, 17, 1206.
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.
2008.04.023.

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10. All the DAP derivatives described in this work are HCl
salts.