Conformationally-restricted analogues of efflux pump inhibitors that potentiate the activity of levofloxacin in Pseudomonas aeruginosa

Article abstract of DOI:10.1016/S0960-894X(03)00556-0

Conformational restriction of the ornithine residue of the efflux pump inhibitor D-ornithine-D-homophenylalanine-3-aminoquinoline (MC-02,595, 2) furnished bioisosteric proline derivatives that were less toxic in vivo and as active as the lead in potentiat

Full text of DOI:10.1016/S0960-894X(03)00556-0

Bioorganic & Medicinal Chemistry Letters 13 (2003) 2755–2758
Conformationally-Restricted Analogues of Efflux Pump Inhibitors
that Potentiate the Activity of Levofloxacin in
Pseudomonas aeruginosa
Thomas E. Renau,a,* Roger Leger,a Lubov Filonova,a Eric M. Flamme,a Michael Wang,a
Rose Yen,a Deidre Madsen,a David Griffith,a Suzanne Chamberland,a Michael N. Dudley,a
Ving J. Lee,a Olga Lomovskaya,a William J. Watkins,a Toshiharu Ohta,b
Kiyoshi Nakayamab and Yohei Ishidab
aEssential Therapeutics, Inc., 850 Maude Avenue, Mountain View, CA 94043, USA
bNew Product Research Laboratories I, Daiichi Pharmaceutical Co. Ltd., 1-16-13, Kita-Kasai, Edogawa, Tokyo 134-8630, Japan
Received 29 July 2002; accepted 13 May 2003
Abstract—Conformational restriction of the ornithine residue of the efflux pump inhibitor d-ornithine-d-homophenylalanine-3-
aminoquinoline (MC-02,595, 2) furnished bioisosteric proline derivatives that were less toxic in vivo and as active as the lead in
potentiating the activity of the fluoroquinolone levofloxacin via the inhibition of efflux pumps in Pseudomonas aeruginosa.
# 2003 Elsevier Ltd. All rights reserved.
We recently described the identification and optimiza-
tion of a series of inhibitors of the RND efflux pumps in
Pseudomonas aeruginosa.1À4 The compounds, exempli-
fied by l-Phe-l-Arg-b-naphthylamine (MC-207,110, 1)
and d-Orn-d-hPhe-3-aminoquinoline (MC-02,595, 2)
(Chart1 ), produce an 8-fold reduction in the MIC of
the fluoroquinolone levofloxacin (LVFX) at concentra-
tions of the inhibitor as low as 5–10 mg/mL.5,6
Both analogues potentiate the activity of LVFX but are
toxic to mice, with LD50s<25 mg/kg for each com-
pound. The observed toxic effect was immediate fol-
lowing the iv bolus injection of the compounds. A
variety of structurally diverse compounds that similarly
potentiated the activity of LVFX in P. aeruginosa, such
as the N-Me analogue 3, the benzothiazole 4 and the
t-butyl quinoline derivative 5, also displayed acute toxi-
city profiles in mice similar to that observed for 1 and 2
(Table 1). To address the observed toxicity of the entire
class of EPIs, we began to probe whether the structural
features that elicited this effect could be uncoupled from
the potentiation activity.
As the optimization phase of the program progressed, it
became apparent that, when administered via intrave-
nous (iv) bolus injection, many of the key compounds
displayed appreciable toxic effects in mice and rats. A
concerted synthetic effort was undertaken to address
this problem and we describe herein our efforts in this
area. We demonstrate that conformational restriction of
a key residue furnishes efflux pump inhibitors (EPIs)
that are as potent as but significantly less toxic than
both 1 and 2.
We first examined the role that routes of compound
delivery had on toxicity and showed that iv infusion,
intraperitoneal (ip) or subcutaneous (sc) administration
significantly ameliorated the immediate toxic effects
(Table 2). For instance, the MLD (mg/kg) of 2 was
<200 versus 25 when the compound was administered
ip rather than iv, respectively. Less lipophilic analogues,
such as the Leu derivative 6, were less toxic but also less
potent. The basic residue of the ornithine group was
ultimately implicated as the major contributor to the
observed toxicity (compare, for example, the toxicity
profile of 7 with 2). Realizing that a basic functional
The in vitro activity and in vivo toxicity in mice of the
representative EPIs 1 and 2 are displayed in Table 1.
*Corresponding author. Fax: +1-650-852-1875; e-mail: thomas.
renau@roche.com
0960-894X/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00556-0

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T. E. Renau et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2755–2758
Chart 1.
Table 1. In vitro activity and in vivo toxicity of selected analoguesa
of arginine, which itself is an isostere of ornithine.7 This
approach led to the identification of the constrained
derivative 8 (Fig. 1). The compound, which incorpor-
ated the two basic residues necessary for activity, was
less toxic (MLD 50 mg/kg, iv bolus) and more active
than 2. The positive toxicity results observed for 8 led us
to prepare many of the most compelling analogues from
our previous studies1À3 with the new, constrained pro-
line piece. A sampling of the diverse set of compounds
prepared in order to capitalize on this result is high-
lighted by compounds 9–11. In general the original
observation for 8 held; that is, analogues containing the
new proline scaffold were typically less toxic than the
corresponding compounds incorporating the arginine or
ornithine residue.
c
Compd
MICb
(mg/mL)
MPC8
(mg/mL)
MLDd, iv
(mg/kg)
LD50
(mg/kg)
1
2
3
4
5
8
>512
>512
256
128
64
>512
>512
128
>512
—
10
10
5
5
5
5
10
5
10
—
<25
<25
<25
25
35
50
<25
<25
—
—
—
—
—
—
—
9
10
11
LVFX
70
>150
>100
>100
>100
aSee text and refs 5 and 6 for a complete description of the potentia-
tion and toxicity testing.
bMIC, minimum concentration (mg/mL) of efflux pump inhibitor
required to inhibit the growth of PAM 1723.
cMPC8, minimum concentration (mg/mL) of efflux pump inhibitor
required to reduce (potentiate) the MIC of levofloxacin 8-fold.
dMLD, minimum dose (mg/kg) causing lethality to ꢁ66% of the ani-
mals tested.
One of the more compelling derivatives from this study
was MC-04,124 (11), which was >4-fold less toxic in
rodents but as potent as the earlier optimized lead 2.
Like 2, 11 displayed no activity against a strain of
P. aeruginosa lacking all three primary efflux pumps,
confirming that it was acting via the inhibition of these
pumps. The compound retained the broad-spectrum
pump inhibitory characteristics of the entire series,
which involves blockade of the MexAB-OprM,
MexCD-OprJ and MexEF-OprN RND efflux pumps.5,6
Pharmacological profiling showed that 11 had similar
ratproetin binding ( ꢀ65%) as 2 butimproved rat
pharmacokinetics [CLfree (L/h/kg) 1.7 vs 2.9, respec-
tively]. In addition, 11 demonstrated, in combination
with LVFX, efficacy in an in vivo model of infection
similar to the results obtained and previously reported
for 2.7
Table 2. The role of the di-amine in the acute toxicitya
Compd
X
Y
MLDa (mg/kg)
MPC8b (mg/mL)
iv
ip
2
6
7
Orn
Orn
Ala
hPhe
Leu
hPhe
<25
50
125
200
300
—
10
20
>40
aMLD, minimum dose (mg/kg) causing lethality to ꢁ66% of the ani-
mals tested.
Because of its interesting profile, an efficient synthesis of
11 was necessary so as to obtain sufficient material for
extensive in vitro and in vivo biological and tox-
icological studies. As shown in Scheme 1, the proline
moiety was prepared in seven steps using a modified
literature procedure8 and then was coupled to the free
base of d-hPhe-3-aminoquinoline, which was prepared
in two steps from N-Boc-d-hPhe. Deprotection and
purification led to the desired 11 in good yields.
bMPC8, minimum concentration (mg/mL) of efflux pump inhibitor
required to reduce (potentiate) the MIC of levofloxacin 8-fold.
group was the primary cause of the observed toxic effects
1
butknowing from our previous work that this same
group was crucial for potentiation activity led us to con-
sider various appropriate replacements for ornithine.
We evaluated an assortment of structural modifications to
address this problem, one of which was to con-
formationally restrict the amino group of ornithine to fur-
nish appropriately substituted proline derivatives.
Analogues such as these had been shown to be bioisosteres
In summary, conformational restriction of a residue
critical to the pharmacophore of this class of RND EPIs
was successful in uncoupling the observed toxic effects
in rats and mice from the potentiation activity. Results
of the extensive pharmacokinetic bio-profiling of 11 and

T. E. Renau et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2755–2758
2757
Figure 1. Conformational restriction of the ornithine group of 2.
Scheme 1. Synthesis of MC-04,124 (11).
related proline-containing analogues will be the subject
of a future publication.
(3–5/dose) were treated iv or ip with the test compound.
Endpoints were recorded as the Minimum Lethal Dose
(MLD), which is defined as the minimum dose causing
lethality to ꢁ66% of the animals tested.
Chemicals
The preparation of 1 was described in ref 1, and 2 and 3
were reported in ref 2. The peptidomimetics 4 and 5
were reported in ref 3. All analogues were purified by
reverse-phase MPLC and tested as their bis-TFA salts.
The structural identity of each compound was con-
In Vitro Potentiation assay
Compounds were assayed in the presence and absence of
LVFX against PAM 1723, a laboratory strain of
P. aeruginosa that overexpresses the MexAB-OprM
pump. The activity of the inhibitors was quantified by
the term MPC8, which is the minimum concentration
(mg/mL) of inhibitor required to decrease (potentiate) the
MIC of LVFX 8-fold. The MIC (mg/mL) of each com-
pound was also determined to ensure that the potentia-
tion effect observed was not due to intrinsic antibacterial
activity of the putative EPI. A more complete description
of the methods utilized to monitor the biological activity
of test compounds can be found in refs 5 and 6.
1
firmed by H NMR and MS. LVFX was provided by
Daiichi Pharmaceutical Co., Ltd. (Tokyo, Japan).
Toxicity Assays
LD50s were determined using standard protocols. To
minimize animal use, an alternative acute toxicity assay
was developed whereby male Swiss Webster mice

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T. E. Renau et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2755–2758
3. Renau, T. E.; Leger, R.; Yen, R.; She, M. W.; Flamme,
Acknowledgements
E. M.; Sangalang, J.; Gannon, C. L.; Chamberland, S.;
Lomovskaya, O.; Lee, V. J. Bioorg. Med. Chem. Lett. 2002,
12, 763.
4. Renau, T. E.; Lemoine, R. C. Drugs Future 2001, 26, 1171.
5. Lomovskaya, O.; Lee, A.; Hoshino, K.; Ishida, H.; Mistry,
A.; Warren, M. S.; Boyer, E.; Chamberland, S.; Lee, V. J.
Antimicrob. Agents Chemother. 1999, 43, 1340.
The authors thank Scott Hecker and Asfia Qureshi for
their review of the manuscript. This work was funded
principally by Daiichi Pharmaceuticals.
References and Notes
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