Quinazolinone-based fungal efflux pump inhibitors. Part 1: Discovery of an (N-methylpiperazine)-containing derivative with activity in clinically relevant Candida spp.

Article abstract of DOI:10.1016/j.bmcl.2004.07.070

The discovery of a series of quinazolinone-based fungal efflux pump inhibitors by high-throughput screening for potentiation of fluconazole in C. albicans is described. Attempts to improve the aqueous solubility of screening hits led to the discovery of an analog with greatly improved physical properties and activity against clinically-relevant Candida spp.

Full text of DOI:10.1016/j.bmcl.2004.07.070

Bioorganic & Medicinal Chemistry Letters 14 (2004) 5127–5131
Quinazolinone-based fungal efflux pump inhibitors.
Part 1: Discovery of an (N-methylpiperazine)-containing derivative
with activity in clinically relevant Candida spp.
a,
a
a,
Remy C. Lemoine, * Tomasz W. Glinka, William J. Watkins, Aesop Cho,a Jessie Yang,a
Nadeem Iqbal,b Rajeshwar Singh,b Deidre Madsen,a Karen Lolans,a Olga Lomovskaya,a
Uma Ozaa and Michael N. Dudleya
´
aEssential Therapeutics, Inc., 850 Maude Ave., Mountain View, CA 94043, USA
bNAEJA Pharmaceutical Inc., #2 4290-91 A St., Edmonton, Alberta, Canada T6E 5V2
Received 18 May 2004; accepted 28 July 2004
Available online 20 August 2004
Abstract—The discovery of a series of quinazolinone-based fungal efflux pump inhibitors by high-throughput screening for poten-
tiation of fluconazole in C. albicans is described. Attempts to improve the aqueous solubility of screening hits led to the discovery of
an analog with greatly improved physical properties and activity against clinically-relevant Candida spp.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
inhibitors using mode of action assays designed in these
laboratories. 6 Several structurally-diverse classes of hits
were obtained, some of which inhibited pumps from
more than one family simultaneously. However, our
attention was drawn to a series of quinazolinones which,
although apparently specific for inhibition of CDR1 in
C. albicans, represented attractive starting points for
medicinal chemistry due to their potency and their rela-
tive ease of synthesis.
In recent years there has been a marked increase in the
incidence of systemic fungal infections due to organisms
that are not susceptible to commonly-used antifungal
agents such as fluconazole.1 Intrinsic and acquired
resistance to azoles in Candida spp.2 and Aspergillus
fumigatus3 has been associated with the overproduction
of multidrug transporters of the ABC and Major Facil-
itator superfamilies. The precedent for the potentiation
of antimicrobial agents through the inhibition of efflux
pumps in bacteria by a variety of molecular classes is
now well established,4 and the potential for the analo-
gous potentiation of antifungal agents through combi-
nation with fungal efflux pump inhibitors is, therefore,
readily apparent.
Two representative hits (1, 2) are illustrated in Figure 1,
and their activity in vitro is summarized in Table 1. The
fluconazole potentiation activity is conveniently ex-
pressed as the lowest concentration of FEPI achieving
an 8-fold reduction in fluconazole MIC (Minimum
O
Recently, we reported the isolation and characterization
of the first fungal efflux pump inhibitors (FEPIs).5 The
molecules were identified in a high-throughput screen
for potentiation of fluconazole versus C. albicans, and
were subsequently validated as fungal efflux pump
1
2
O
O
N
R =
R
N
H
N
N
N
O
Cl
3
*
N
Corresponding author at present address: Inflammatory and Viral
O
Diseases Unit, Roche Palo Alto, 3431 Hillview Avenue, R6E-3, Palo
Alto, CA 94304, USA. Tel.: +1 650 855 5774; fax: +1 650 852
1311; e-mail: remy.lemoine@roche.com
O
Figure 1. Structures of 1, 2, and 3.
E-mail: wwatkins@gilead.com
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.07.070

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R. C. Lemoine et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5127–5131
Table 1. In vitro activity of two representative screening hits
No.
Concn required for 50% pump inhibition in S. cerevisiae (lg/mL)a
MPC8 (lg/mL)
C. glabrata
CDR1
CDR2
CgCDR1
CgCDR2
C. albicans
1
2
0.5
0.125
>32
>32
>32
>32
>32
>32
1
0.5
>32
>32
a See Materials and methods.
Potentiation Concentration, or MPC8). The spectrum of
pump inhibition of each compound is also characterized
using isogenic strains of Saccharomyces cerevisiae (de-
rived from YKKB-13) in which PDR5 is deleted and
individual CDR pumps are over-expressed under the
control of a common promoter.7
stereoisomers since atropoisomerism has already been
reported in quinazolinones.9
The limited aqueous solubility of 1 and 2 (<1lg/mL at
pH = 7) was a cause for concern. As these analogs differ
only at R1, we explored the introduction of ionizable
groups at this position. Gratifyingly, the solubility of
the piperazine-containing compound 3 (see Fig. 1) was
dramatically improved (130lg/mL at pH = 7; 2mg/mL
in water as the mesylate salt).
In this paper, we describe the discovery of 3, the first
example of the series with fluconazole potentiation
activity against a wide range of clinically relevant Can-
dida spp. and with dramatically improved aqueous
solubility.
Quite unexpectedly, 3 displayed a second important
advantage: it inhibited pumps in both C. glabrata as
well as C. albicans (Table 2). Subsequent studies
showed that 3 also potentiates the activity of FLU
versus C. tropicalis, C. parapsilosis and, to a lesser
extent, C. krusei, although no activity was seen versus
strains of S. cerevisiae over-expressing efflux pumps
other than from the ABC superfamily (data not shown).
The broader spectrum of activity of 3 was of great
significance to the program, given the prevalence of
these species in fluconazole-resistant yeast infections.
Other analogs bearing an N-methylpiperazinyl substitu-
ent on any of the aryl rings rather than at R1 lacked
activity versus C. glabrata, indicating that the basic
nitrogen at R1 was critical for the broad-spectrum
activity of 3.
New compounds were initially synthesized as racemates
according to the general synthetic scheme shown in
Scheme 1.
The quinazolinone bicyclic system was readily derived
from anthranilic acid, via the intermediacy of the isatoic
anhydride formed by condensation with propionic anhy-
dride;8 this route was particularly conducive to facile
variation at R1. Following bromination, displacement
by the relatively nucleophilic 2,4-dimethoxyaniline
was achieved under mild conditions. Finally, reaction
with 3-chlorophenylisocyanate introduced the urea
functionality.
At room temperature, the proton NMR spectra of final
products were more complex than expected. However,
we showed, that at 100ꢁC, all the protons gave discrete
and readily-interpretable signals. We suggest that at
room temperature the quinazolinones are composed of
a set of conformational isomers that interconvert on
the NMR time scale. This set could include atropodia-
Given the presence of an asymmetric center in 3, and
noting the spectral evidence for rotational barriers, we
systematically explored the influence of methyl substitu-
ents at this position upon activity. The enantiomers of 3
were ultimately derived from the requisite lactic acid10
(Scheme 2).
O
O
N
O
R1
OH
NH2
(a)
(b)
O
N
N
(c)
O
N
O
O
N
R1
R1
N
N
(e)
Cl
(d)
R1
Br
N
N
H
N
N
O
NH
O
O
O
O
Scheme 1. General racemic synthesis. Reagents and conditions: (a) (EtCO)2O, 70ꢁC; (b) R1-NH2, AcOH, 70ꢁC; (c) Br2, NaOAc, AcOH, rt; (d) 2,4-
dimethoxyaniline, Na2CO3, DMF, 80ꢁC; (e) 3-chlorophenylisocyanate, CH2Cl2, 40ꢁC.

R. C. Lemoine et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5127–5131
5129
Table 2. In vitro activity of 3, 4, 5, 3-(S), and 3-(R)
O
N
N
N
N
N
(S)-3: R1=H, R2=Me
(R)-3: R1=Me, R2=H
R1
R2
NH
4:
5:
R1=R2=H
Cl
R1=R2=Me
O
O
O
No.
Concn required for 50% pump inhibition in S. cerevisiae (lg/mL)
MPC8 (lg/mL)
C. glabrata
CDR1
CDR2
CgCDR1
CgCDR2
C. albicans
3
4
2
>16
>32
>32
>32
>32
8
4
>16
>32
>32
32
1
1
4
1
(S)-3
(R)-3
4
>32
32
>32
>32
>32
>32
16
>32
16
5
>32
>32
>32
>32
O
O
O
OH
NH
(a), (b), (c)
(d)
O
Cl
OH
O
OAc
(e), (f), (g)
O
OAc
(h)
N
N
O
N
N
O
N
N
N
N
N
N
N
(i)
N
NH
O
N
O
OH
S
NO2
O2
O
O
O2N
O
N
(j)
N
N
(S)-3
N
H
N
N
O
Cl
O
O
Scheme 2. Homochiral synthesis of (S)-3. Reagents and conditions: (a) LiOH, MeOH, rt; (b) AcOH, H2SO4, reflux; (c) SOCl2, reflux; (d) anthranilic
acid, aqNaHCO 3, AcOEt, 0ꢁC; (e) Ac2O, 70ꢁC. (f) 1-amino-4-methyl-piperazine, AcOH, 70ꢁC; (g) Et3N, MeOH, H2O, rt. (h) N-(2,4-
dimethoxyphenyl)-(2,4-dinitrobenzenesulfonamide), PPh3, DIAD, THF, rt; (i) HSCH2COOH, Et3N, CH2Cl2, rt; (j) 3-chlorophenylisocyanate,
CH2Cl2, rt.
Racemization during the alkylation of the aniline was
minimized by resorting to a Mitsunobu-type displace-
ment, and the optical purity of the final product was
shown by chiral HPLC to be >95% in each case.
methyl substituent on the asymmetric carbon is to serve
as a conformational lock, rendering the piperazine and
the three aryl residues suitably disposed for interaction
with the pump. A global computational search by
semi-empirical methods failed to identify a single low-
energy conformer for 3; three discrete sets in which the
relative orientations of the aryl groups and the pipera-
zine ring differ significantly were observed within
2kcal/mol of the global minimum (Fig. 2).
The synthesis of the des-methyl compound 4 was analo-
gous to that of 3, replacing propionic anhydride with
acetic anhydride; the dimethyl analog 5 was made as
shown (Scheme 3).
All the activity of 3 is seen to reside in the (S) enantio-
mer, which is markedly more active than any other ana-
log in this set (see Table 2). The proton NMR of 4 at
room temperature showed discrete signals indicating a
much faster interconversion rate between the conforma-
tional isomers (or possible atropoisomers) in 4 as com-
pared to 3. These data suggest that the role of the
The population of low-energy conformers of 4 and 5 dif-
fered markedly from 3, supporting the notion that the
methyl group, while important for activity, may not be
in direct contact with the pump.
Attempts to improve the aqueous solubility of screening
hits led to the discovery of 3, the first quinazolinone-based

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R. C. Lemoine et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5127–5131
O
O
O
OH
NH
OH
NH2
OH
NH
(a)
(b)
O
(c)
O
NH
O
O
Br
O
OH
NH
O
N
O
N
N
N
O
N
(e)
Cl
(d)
Cl
O
H
N
N
O
Cl
H
H
N
N
O
N
N
O
5
O
O
O
O
O
O
Scheme 3. Synthesis of 5. Reagents and conditions: (a) BrC(CH3)2COCl, aqNaHCO 3, AcOEt, 0ꢁC; (b) 2,4-dimethoxyaniline, K2CO3, EtOH, reflux;
(c) 3-chlorophenylisocyanate, CH2Cl2, rt; (d) Ac2O, 80ꢁC; (e) 1-amino-4-methylpiperazine, AcOH, 70ꢁC.
2.2. Solubility assays
Compounds were suspended in 0.05M potassium phos-
phate buffer (pH 7.0), and sonicated for 15s. After an
hour on a shaker, the sample was centrifuged and the
amount of compound in solution was quantified by
HPLC (using an Agilent Zorbax 4.6 · 150mm column,
eluting with an acetonitrile–water gradient at 0.5mL/
min) against a standard curve. While similar solubility
studies were not performed in microbiological medium,
no precipitation was observed at the test concentrations
employed.
Figure 2. Low-energy conformers of 3. The global minimum is
depicted in CPK colors and the two other conformers within 2kcal/
mol in magenta and green.
2.3. In vitro potentiation of fluconazole
FEPI showing fluconazole potentiation in clinically rel-
evant Candida spp. Investigation into the role of the
methyl substituent on the asymmetric center demon-
strated that all the activity in 3 resides in the (S) enanti-
omer, and supports the notion that the primary role of
the substituent is to rigidify the structure, leaving the
piperazine moiety and the three aryl residues suitably
disposed for efficient pump inhibition.
Fluconazole (FLU) MICs in the presence or absence
of inhibitors were determined in RPMI using a
broth microdilution version of the NCCLS reference
method.12 MICs were measured in YEM 15, a FLU-
resistant (MIC = 128lg/mL) laboratory strain of Can-
dida albicans over-expressing the ABC transporters
CDR1 and CDR2, and in YEM 19, a strain of C. glab-
rata overexpressing CgCDR1 and CgCDR2. The activ-
ity of the inhibitors was quantified by the term MPC8,
which is the minimum concentration (lg/mL) of inhibi-
tor required to decrease (potentiate) the MIC of FLU
8-fold. None of the compounds described herein showed
any intrinsic antifungal activity. Not all of the analogs
reported in this study were assessed in full checkerboard
format; an abbreviated method of evaluation that in-
volved testing varying concentrations of efflux pump
inhibitor in the presence of fixed concentrations of fluc-
onazole equivalent to 1/8 and 1/32 MIC. This method
was validated on a set of eight analogs by comparison
with the MPC8 values derived from full checkerboard
studies; the results were identical.
In vitro SAR for pump inhibition and optimization of
the pharmacokinetic characteristics of the series will be
the subject of further publications.11
2. Materials and methods
2.1. Chemicals
All FEPIs described herein were purified by chromato-
graphy on silica gel, eluting with varying proportions
of ethyl acetate and hexanes or methanol and dichloro-
methane. The structural identity of each compound was
confirmed by 1H NMR and MS. Optical purity of
homochiral compounds was measured by chiral chro-
matography on a Chiralpack AD 250 · 4.6mm column,
eluting with 30% 2-propanol in hexanes at 1mL/min.
Absolute stereochemistry was assigned by analogy with
relative retention times of both enantiomers.
The inhibition of specific ABC transporters from C. alb-
icans and C. glabrata was assessed using defined strains
of S. cerevisiae: YEM139 (PDR5 deleted; FLU MIC
0.5lg/mL), YEM172 (CDR1 over-expressed; FLU
MIC 128lg/mL), YEM171 (CDR2 over-expressed;

R. C. Lemoine et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5127–5131
5131
Chemotherapy, San Francisco, CA, Sept 1999; Abstract
447.
4. Renau, T. E.; Lemoine, R. C. Drugs of the Future 2001, 26,
1171.
FLU MIC 8lg/mL), Y170 (CgCDR1 over-expressed;
FLU MIC 128lg/mL), and Y218 (CgCDR2 over-ex-
pressed; FLU MIC 2lg/mL). An inoculum of 104 cells
of each strain was incubated at 30ꢁC in YPD in the pres-
ence of varying concentrations of inhibitor and a single
concentration of fluconazole corresponding to the geo-
metric mean of the MIC of the test strain and the
MIC of the parent pump-deleted strain (i.e., YEM172
was incubated with 8lg/ml fluconazole, which is the
geometric mean of 128 (MIC of YEM 172) and 0.5
(MIC of YEM167). MICs were determined as 80% inhi-
bition of growth after 48h.
5. Lomovskaya, O.; Warren, M.; Mistry, A.; Staley, J.;
Galazzo, J.; Fuernkranz, H.; Lee, M.; Miller, G.; Sang-
lard, D. In 39th Interscience Conference on Antimicrobial
Agents and Chemotherapy, San Francisco, CA, Sept 1999;
Abstract 1269; further publications on the biological
aspects of this work will be forthcoming.
6. Mistry, A.; Warren, M. S.; Blais, J.; Staley, A. L.;
Galazzo, J. L.; Fuernkranz, H.; Chamberland, S.; Lee,
M. D.; Watkins, W. J.; Lomovskaya, O.; Miller, G. H.;
Sanglard, D. In 40th Interscience Conference on Antimi-
crobial Agents and Chemotherapy, Toronto, ON, Sept
2000; Abstract 1500.
7. The authors are grateful to D. Sanglard for the provision
of the plasmids used in this study; see also Ref. 4.
8. Smith, K.; El-Hiti, G. A.; Abdel-Megeed, M. F.; Abdo,
M. J. Org. Chem. 1996, 61, 647, et op. cit.
9. Dai, X.; Wong, A.; Virgil, S. C. J. Org. Chem. 1998, 63,
2597.
10. Diastereomeric excesses were determined by reaction of
the acyl chloride with enantiomerically-pure 2-ethyl-
benzylamine and analysis of the products using the chiral
column described in Materials and Methods.
11. Watkins, W. J.; Lemoine, R. C.; Chong, L.; Cho, A.;
Renau, T. E.; Kuo, B.; Wong, V.; Ludwikow, M.; Garizi,
N.; Iqbal, N.; Barnard, J.; Jankowska, R.; Singh, R.;
Madsen, D.; Lolans, K.; Lomovskaya, O.; Oza, U.;
Dudley, M. N. Bioorg. Med. Chem. Lett. 2004, 14,
following paper in this issue. doi:10.1016/j.bmcl.2004.
07.071.
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