The relationship between physicochemical properties, in vitro activity and pharmacokinetic profiles of analogues of diamine-containing efflux pump inhibitors

Article abstract of DOI:10.1016/j.bmcl.2003.07.030

Following the optimization of diamine-containing efflux pump inhibitors with respect to in vitro potentiation activity, in vivo stability and acute toxicity, we addressed the question of how to control the pharmacokinetic properties of the series. Upon in

Full text of DOI:10.1016/j.bmcl.2003.07.030

Bioorganic & Medicinal Chemistry Letters 13 (2003) 4241–4244
The Relationship between Physicochemical Properties, In Vitro
Activity and Pharmacokinetic Profiles of Analogues of
Diamine-Containing Efflux Pump Inhibitors
William J. Watkins,* Yakira Landaverry, Roger Leger, Renee Litman, Thomas E. Renau,
Nicole Williams, Rose Yen, Jason Z. Zhang, Suzanne Chamberland, Deidre Madsen,
David Griffith, Vrushali Tembe, Keith Huie and Michael N. Dudley
Essential Therapeutics, Inc., 850 Maude Ave., Mountain View, CA 94043, USA
Received 8 April 2003; accepted 14 July 2003
Abstract—Following the optimization of diamine-containing efflux pump inhibitors with respect to in vitro potentiation activity, in
vivo stability and acute toxicity, we addressed the question of how to control the pharmacokinetic properties of the series. Upon
intravenous administration in the rat, tissue levels of MC-04,124 (the lead compound) were high and prolonged compared to those
in the serum. The lipophilicity and basicity of analogues of this compound were systematically varied, and effects on potency and
pharmacokinetics explored. The ratio of drug levels in tissue versus serum was not significantly reduced in any of the active ana-
logues examined.
# 2003 Published by Elsevier Ltd.
A number of recent publications have described the
discovery and characterization of the biological, micro-
biological and pharmacological properties of a novel
class of broad-spectrum bacterial RND efflux pump
inhibitors (EPIs).1À4 Their optimization with respect to
potentiation of the activity of levofloxacin verses Pseu-
domonas aeruginosa (both in vitro and in vivo) and sta-
bility have been described, and analogues have been
reported with significantly reduced acute toxicity. This
effort culminated in MC-04,124 (1, Fig. 1) as the opti-
mal agent.3
The pharmacokinetic profiles of 1 in the rat following
single and multiple doses are summarized in Figures 2
and 3. In contrast to serum, tissue levels following a
single dose remained high for prolonged periods, and
this led to significant accumulation of the drug in a
variety of organs upon repeated dosing.
Accumulation of polycationic compounds within acidic
organelles such as lysosomes can lead to toxicity.5À7 The
observation of high levels of 1 in a variety of tissues,
particularly the kidney, therefore, was a concern.
Figure 1. MC-04,124 (1).
Figure 2. Serum and tissue concentrations of 1 in rats. Two-hour iv
infusion of 8.4 mg/kg in three rats; serum data are shown for each rat,
and tissue concentrations are the mean. CL=1.6 L/h/kg, Vss=0.5 L/
kg, T1/2=0.73 h, protein binding=63%.
*Corresponding author. Fax: +1-650-428-3534; e-mail: wwatkins@
etrx.com
0960-894X/$ - see front matter # 2003 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2003.07.030

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W. J. Watkins et al. / Bioorg. Med. Chem. Lett. 13 (2003) 4241–4244
Figure 3. Serum and tissue concentrations of 1 following multiple
doses in rats. Daily 10-min iv infusion of 25 mg/kg at 0, 24, 48, 72, and
96 h. Each point represents one rat.
Scheme 2. Synthesis of 3, 4, and 5. Reagents and conditions: (a) see ref
9; (b) NaN3, Bu4N+ClÀ, DMF; (c) SnCl2, PhSH, Et3N, CH3CN;
(d) BOC2O, aq NaOH, 1,4-dioxane; (e) H2, Pd/C, MeOH; (f) BOC-
Gly-OH, iPr2NEt, PyBrop, THF; (g) LiOH, THF, MeOH, H2O;
(h) C6F5OH, DCC, EtOAc, then amine A or B; (i) TFA; (j) H2, Pd/C,
BOC2O, EtOH; (k) Et3N, EtOCOCl, THF; CH2N2; PhCO2ÀAg+
.
Scheme 1. Synthetic routes to amine intermediates.
Mindful also of the need to control the pharmacokinetic
profile in order to achieve maximum synergy with an
antibacterial agent in vivo, a study of the physicochem-
ical characteristics of the series with respect to both
levofloxacin potentiation activity and pharmacokinetic
properties was instigated. The results are reported here.
The partition coefficient at a given pH (logD) of a
dibasic compound is a function of the partition coeffi-
cient (logP) and the ionization constants (pKa) of both
basic groups,8 and may therefore be varied by changes
in all three parameters. Initially, we were interested to
explore compounds in which the substituted proline
moiety was varied. Through incorporation of electron-
withdrawing groups adjacent to the basic entities, and
through juxtaposition of the basic groups themselves,
we were able to modulate the ionization constants quite
widely within the range of interest. The synthesis of 1
and 2 has been described previously.3 The synthetic
routes by which the remaining analogues in this study
were generated are shown in Schemes 1–4.
Scheme 3. Synthesis of 6 and 7. Reagents and conditions: (a) see ref
10; (b) NaN3, Bu4N+ClÀ, DMF; (c) LiOH, THF, MeOH, H2O;
(d) amine A or B, EDAC, HOBT, CH2Cl2; (e) H2, Pd/C, EtOH;
(f) TFA; (g) NaCN, DMSO; (h) BOC-Gly-OH, Et3N, EtOCOCl,
CH2Cl2; (i) PtO2, BOC2O, aq NaOH.
because of their greater lipophilicity (logP increased by
0.3–0.4). The activity is tolerant of considerable struc-
tural diversity in the diamine moiety, both with regard
to the incorporation of extra amide bonds or hydroxyl
groups, as well as the position of the two amines relative
to each other and to the rest of the molecule: compare,
for example, the similar potency of 1 and 7, or 2 and 5.
This allows deliberate modulation of the ionization
constants, for which comparison of the amine-contain-
Table 1 displays analogues in which the ionization con-
stants are systematically varied, together with their
potentiation activity. Analogues containing a 3-quinolyl
substituent were marginally more potent than their
6-quinolyl comparators (cf. 2 vs 1 and 4 vs 3)—perhaps

W. J. Watkins et al. / Bioorg. Med. Chem. Lett. 13 (2003) 4241–4244
4243
Table 1. Physicochemical parameters and potentiation activity of
analogues of 1
No.
R
X
Y
pKa
pKa
MPC8
(mg/mL)
1
2
1
2
CH
N
N
9.6
9.6
6.9
6.8
10
5
CH
Scheme 4. Synthesis of 8 and 9. Reagents and conditions: (a) BOC2O
(1 equiv), aq NaOH, 1,4-dioxane, then BnOCOCl; (b) CH2N2, THF;
(c) H2, Pd/C, MeOH; (d) BOC-Gly-OH, iPr2NEt, PyBrop, THF;
(e) KOH, THF, MeOH, H2O; (f) amine A, EDAC, CH2Cl2; (g) TFA;
(h) BOC2O (2.2 equiv), aq NaOH, 1,4-dioxane; (i) BOC-Gly-OH,
EDAC, CH2Cl2.
3
4
5
6
7
CH
N
N
8.2
8.1
9.3
8.8
9.0
7.2
7.1
6.3
5.2
7.7
20
10
CH
CH
CH
N
ing substituent at the pyrrolidine 4-position in 2, 4, and
6 is illustrative. In 2, this primary amine is the most
basic. In 4, the methylene unit connecting this amine to
the pyrrolidine ring is deleted. Consequently, the amine
is closer to electron-withdrawing amide bonds, render-
N
5
ing it less basic; the observed pK is typical of an
a1
N
>40
10
N-terminal glycine and is probably due to protonation
of the other amine in the molecule. In 6, however, the
primary amine at the 4-position becomes the most basic
again. Once ionized, its proximity to the ring has a
profound effect on the ionization constant of the sec-
ondary amine. We attribute the lack of activity of 6 to
the inadequate basicity of this secondary amine; incor-
poration of a methylene linker between it and the adja-
cent amide, as in 5, raises pKa and restores potency.
Similarly, the differing activity 2of isomers 8 and 9 is
CH
8
9
N
N
CH
CH
8.2
8.5
6.3
5.4
20
attributable to the lower pKa in 9.
2
>40
Compounds 1–9 encompass examples in which the logD
is altered primarily by variation in the ionization con-
stant. The tissue pharmacokinetics of a representative
subset of these analogues, together with two additional
examples (10 and 11), are summarized in Table 2. In 10,
the amido-quinolyl substituent is replaced by a much
more lipophilic benzimidazole,4 resulting in good activ-
ity (MPC8 5 mg/mL). In 11, one amine is removed alto-
gether, resulting in an inactive analogue that is
nonetheless an important comparator for establishing
the structural features of the series that result in high
drug levels in tissues.
benzimidazole moiety. For the (inactive) analogue 6, the
relative lack of basicity is such that at physiological pH
it is calculated to exist largely (ca. 95%) as the mono-
cation, although at pH 5 the dicationic form would
predominate (75%).8 In this case the kidney level is
somewhat reduced compared to other diamines. The
trend is emphasized by the monoamine 11, which dis-
plays tissue levels that are greatly reduced relative to all
the other compounds. Taken together, the data strongly
implicates the diamine moiety, which is essential for EPI
activity, in giving rise to the pharmacokinetic profile
seen in 1.
Despite the variation of logD from À0.5 to 1.9, no dia-
mine analogues with markedly different tissue distribu-
tion profiles from that seen with 1 were found. For 3, 4,
and 7 lung levels were reduced, perhaps reflecting a
somewhat lower logP, but the propensity for distribu-
tion to the liver and kidney remained. The profile of 10
appears even more extreme than other diamine analo-
gues—perhaps because of the additional weakly basic
While it is unclear from these studies whether the dis-
tribution to tissues is attributable to pH differences
within organelles or to some more specific binding phe-
nomenon, none of the active analogues investigated in
this study displayed the pharmacokinetic profile we
desired. Furthermore, based on the results herein show-

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W. J. Watkins et al. / Bioorg. Med. Chem. Lett. 13 (2003) 4241–4244
Table 2. Comparative tissue levels of analogues of 1
bacterial activity (MIC >40 mg/mL).
No.
logP
logD
AUC0À24 h (mg h/kg)a
Serum and tissue pharmacokinetics were measured in 5
male Sprague–Dawley rats with femoral and jugular
venous catheters. EPIs were administered alone or in a
cassette of up to five compounds as a 2 h constant rate
infusion into the femoral vein at doses varying from 1 to
10 mg/kg. Blood samples were collected for up to 8 h
from the jugular catheter. At 0.25, 2, 8, 16, and 24 h
after the start of the infusion, a single rat was sacrificed
and liver, kidney, and lung tissue were harvested. Serum
and tissue homogenates were assayed using HPLC/MS/
MS. Pharmacokinetic data were analyzed using com-
partmental and non-compartmental methods. Tissue
AUC0À24 h were normalized to a dose of 1 mg/kg for
comparison.
Kidney
Liver
Lung
1
3
4
6
2.3
1.4
1.8
2.3
1.2
2.8
ND
0.5
0.5
0.9
1.1
56
96
79
25
100
133
22
12
20
13
15
4
3
14
4
5
ND
7
À0.5
26
176
5.5
10b
11c
1.9
ND
8.5
aNormalized to 1 mg/kg dose; serum AUC0À24 for all compounds
References and Notes
h
was ca. 1 mg h/L.
bSynthesized as for 3, but coupling with amine C.
cSynthesized by coupling of BOC-d-Ala-OH with amine B (iPr2NEt,
PyBrop, CH2Cl2), followed by treatment with TFA.
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All analogues were purified by reverse-phase MPLC
and tested as their bis-TFA salts. The structural identity
of each compound was confirmed by 1H NMR and MS.
Levofloxacin was provided by Daiichi Pharmaceutical
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The ionization constants, partition coefficients and
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the GLpKaTM Potentiometric System. LogP measure-
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Æ0.15. LogD values are reported at physiological pH
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Levofloxacin MICs for P. aeruginosa strain PAM 1723
(MexAB-OprM
overexpressed;
DmexCD-oprJ,
DmexEF-oprN)11 were measured in the presence or
absence of varying concentrations of efflux pump inhi-
bitor (checkerboard format). Drugs were tested in
Mueller–Hinton broth according to NCCLSreference
methods. The in vitro potency of the EPI is expressed as
the minimum potentiation concentration (MPC8),
which is the lowest concentration required for an 8-fold
reduction in levofloxacin MIC. Assay controls estab-
lished the variability of the MPC8 as Æ2-fold. All the
compounds reported in this work had no intrinsic anti-