Studies on Quinazolinones as Dual Inhibitors of Pgp and MRP1 in Multidrug Resistance

Article abstract of DOI:10.1016/S0960-894X(01)00804-6

The syntheses and SAR studies of various quinazolinone compounds are described for the dual inhibition of Pgp and MRP1 in multidrug resistance.

Full text of DOI:10.1016/S0960-894X(01)00804-6

Bioorganic & Medicinal Chemistry Letters 12 (2002) 571–574
Studies on Quinazolinones as Dual Inhibitors
of Pgp and MRP1 in Multidrug Resistance
Shouming Wang,a,* Hamish Ryder,a Ian Pretswell,a Paul Depledge,b
a
John Milton,a Timothy C.Hancox, Ian Dale,b Wendy Dangerfield,b
Peter Charlton,b Richard Faint,b Rory Dodda and Stephanie Hassanb
aDepartment of Medicinal Chemistry, Xenova Ltd., 957 Buckingham Avenue, Slough, Berkshire SL1 4NL, UK
bDepartment of Pharmacology, Xenova Ltd., 957 Buckingham Avenue, Slough, Berkshire SL1 4NL, UK
Received 21 September 2001; accepted 26 November 2001
Abstract—The syntheses and SAR studies of various quinazolinone compounds are described for the dual inhibition of Pgp and
MRP1 in multidrug resistance. # 2002 Elsevier Science Ltd.All rights reserved.
The appearance of tumor cells resistant to a range of
cytotoxic drugs is a serious problem in cancer chemo-
therapy.This phenomenon is called multidrug resis-
tance (MDR).One form of MDR can be caused by
members of the ATP-binding cassette (ABC) family of
transport proteins.1 These are large polytopic mem-
brane proteins that actively transport drugs out of cells,
resulting in a decreased intracellular drug concentra-
tion.In humans, two ABC transporters have been
identified that cause resistance in tumor cells: P-glyco-
protein (Pgp) (MDR1)2 and the multidrug resistance
associated protein (MRP1).3 Pgp transports drugs in an
unmodified form, whereas MRP1 transports drugs
either conjugated to the anionic ligands glutathione
(GSH), glucuronide, or sulfate,4 or transports them in
an unmodified form, probably together with GSH.5
Among those cytotoxics transported by Pgp are various
natural product oncolytics, such as vinca alkaloids, epi-
podophyllotoxins, anthracyclines and taxanes,6 most of
which are also substrates for MRP1 transport,7
although taxanes are apparently not subject to MRP1
mediated resistance.8
The correlation between drug resistance and expression
of the drug efflux pumps, Pgp and MRP1, has spurred
considerable efforts in the development of inhibitors of
Pgp and MRP1.12
Among the specific potent inhibitors of Pgp entered into
clinical evaluation are GF120918,13 LY335979,14
OC144-09315 and XR9576.16 However, the development
of specific MRP1 modulators is still in its infancy,12b
although Eli Lilly has reported the raloxifene analogues
(1)17 and isooxazoloquinoline analogues (2)18 to be
selective MRP1 inhibitors.
Due to the functional similarity between Pgp and
MRP1, many of the reported Pgp modulators have been
evaluated for their ability to inhibit the function of
MRP1.Several compounds such as cyclosporin and
VX-710 (3) have been found to have dual activity and
VX-710 (3) has entered clinical trial as a dual inhibitor
of Pgp and MRP.19 The objective of our MDR pro-
gramme was to identify a potent, selective inhibitor of
Pgp and this resulted in XR9576, which displays no
activity against MRP1.Nevertheless, we have also
identified a quinazolinone molecule (4) which possessed
dual inhibitory activities against both Pgp and MRP1.
The differential expression and tissue/tumor specificities
of Pgp and MRP1 have been reviewed recently,9,10
although it is also known that Pgp and MRP can be
over-expressed at the same time in drug resistant cells.11
*Corresponding author.Te:l. +44-1753-706768; fax: +44-1753-
706607; e-mail: shouming_wang@xenova.co.uk
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd.All rights reserved.
PII: S0960-894X(01)00804-6

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S. Wang et al. / Bioorg. Med. Chem. Lett. 12 (2002) 571–574
anthranilic amides (12) were converted into the corre-
sponding iminophosphorane derivatives (13) with tri-
phenyl phosphine/hexachloroethane/triethyl amine.21
The subsequent aza-Wittig reactions and heterocycliz-
ations with acid chlorides (10) in refluxing toluene gave
the quinazolinones (7) in moderate yields.
Biology Assays
Herein, we describe our syntheses and preliminary SAR
studies on the analogues derived from lead molecule (4)
for their dual inhibitory activity of Pgp and MRP1.
Drug accumulation assay (MRP)
Compounds were assayed for inhibition of MRP1-
dependent transport of the radiolabeled cytotoxic agent
and MRP substrate, daunomycin.CORL. 23/R (MRP
expressing non-small cell lung carcinoma MDR subline)
cells were seeded 48 h prior to assay into 96-well opaque
culture plates.Compounds were serially diluted over a
range of concentrations from 100 to 0.015 mM in assay
medium containing tritiated daunomycin at 0.3 mCi/mL
and incubated with COR.L23/R cells at 37 ꢀC for 2 h
before washing and determination of cell associated
Chemistry
The general quinazolinone analogues were synthesized
according to either one of the routes modified from lit-
erature methods as shown in Schemes 120 and 2.21
radioactivity.Results are expressed as an IC for dau-
50
nomycin accumulation, where 100% accumulation is
that observed in the presence of the known MDR
modulator verapamil at a concentration of 100 mM.
Potentiation assay
The ability of modulators to potentiate the cytotoxicity
of doxorubicin was evaluated in the COR.L23/R cell
line as outlined previously.22 IC50 values for doxorubicin
(concentration resulting in 50% inhibition of cell growth)
was calculated from plotted results using untreated cells
Scheme 1. Reagents: (a) R1COCl (10), TEA or Pyr, DCM, 0 ꢀC; (b)
R2NH2 (11), Tol., pTSA; (c) AcOH/H2SO4 (9:1), 100 ꢀC; (d)
.
SnCl2 2H2O, EtOH, reflux; (e) R COCl, TEA, CHCl3.
as 100%.EC
values for modulators (concentration
50
0
required to give 50% of full reversal) were obtained
from graphs of potentiation index (ratio of IC50 of
cytotoxic drug alone/IC50 of cytotoxic drug in the pre-
sence of modulator) plotted against concentration of
modulator.
Similar protocols22 were applied for Pgp assays with the
use of Pgp expressing EMT6/AR1.0 subline.
Results and Discussion
With compound 4 as the lead, SAR was examined at the
N-3 position of the quinazolinone template with the
preparation of a library of compounds at this position,
while the rest of the molecule was kept intact.Our initial
aim was to quickly screen a variety of N-3 substituents
bearing different functional and lipophilic groups.The
accumulation assay activity of a selection of compounds
is shown in Table 1.
Scheme 2. Reagents: (a) PPh3, Cl3CCCl3, benzene, TEA, reflux; (b)
R1COCl (10), TEA, tol.
Thus, when anthranilic acids (5) were reacted with acid
chlorides (10) in pyridine at 0 ꢀC, benzooxazinones (6)
were obtained as crystalline products.The subsequent
reflux with amines (11) and catalytic amount of pTSA in
toluene gave a mixture of quinazolinones (7) and uncy-
clized diamides (8), which were converted to products
(7) by heating in acetic acid and concentrated sulfuric
acid (9:1) at 100 ꢀC for 15 min.Further derivatizations
were also carried out with the substituents of the ben-
zoquinazolinones (7), such as the reduction of the nitro
group and subsequent amide couplings, yielding a series
of analogues (9) (R=NH2, NHCOR0).In Scheme 2,
Compounds with 3-substitutents bearing no basic cen-
tres show reduced or no activities against Pgp or MRP1
(14–16).Introduction of a pyridyl group in the side
chain restores the activities in both resistant cell lines
(17), compared with the lead compound (4).After the
first round of screening, the most potent compound (18)
was found to exhibit dual inhibitory activity against

S. Wang et al. / Bioorg. Med. Chem. Lett. 12 (2002) 571–574
573
both Pgp and MRP1.Further modifications of the side
chain of this compound (18) were carried out by inves-
tigating different carbon chain lengths and con-
formationally restricted and unrestricted ring systems
and various lipophilic groups attached to the basic cen-
ters.Most of these compounds show dramatically
decreased activity against MRP1 while the correspond-
ing Pgp activity is either retained (19–21) or enhanced
(22).In compound 22, the Pgp inhibitory activity is
nearly 100-fold greater than that of MRP1.
As shown in Table 2, removal of 4-N,N-dimethylamino
group results in decrease of activity in both cell lines
(23).In contrast, the 3- N,N-dimethylamino analogue
(24) and 3-n-propyl and 4-isopropyl analogues (27, 25)
show very little variation from compound 18 in Pgp and
MRP activities, the activity of 25 is remarkable as the
change from N,N-dimethyl to isopropyl is a very sig-
nificant one in electronic terms, whilst the steric aspects
are maintained.However, the slightly larger 4- N,N-
diethlyamino substituted analogue (26) shows greatly
decreased MRP activity but retains the Pgp activity.A
small ring fusion at the 3,4-position (28) retains most of
the activity, which lends support for the above findings
that small non-planar groups at the 3- and 4-positions
seem to be tolerated for dual inhibitory activities, but
the 2-hydroxy substituted analogue (29) results in dra-
matic loss of MRP activity (only one example).Inter-
estingly, large lipophilic group substituted analogues
(30, 31) retain the dual inhibitory activities against both
cell lines; this may suggest that planar conformation is
tolerated, while concomitant increase of lipophilicity is
not detrimental to activity.
Having achieved one micromolar potency in both Pgp
and MRP1 expressing cell lines, we then examined the
SAR at the 2-position of the quinazolinone template
while maintaining the active 3-substituent of compound
18.Table 2 illustrates the accumulation assay data for
some of the substituted phenyl analogues.
Table 1. Inhibitory activity of 3-substituted compounds in accumu-
lation assays for Pgp (EMT6/AR1.0) and MRP1 (L23/R)
After evaluating the active analogues prepared so far in
terms of their dual activity and physicochemical prop-
erties, we decided to stay with compound 18 for further
SAR studies on the fused benzene ring of the quinazo-
linone template.Table 3 illustrates the activity data
from the accumulation assays on some of the selected
examples.
Compd
R
IC50
IC50
(mM)/Pgp (mM)/MRP1
4
CH2-N-Tetrahydroisoquinoline
n-Pentyl
Cyclohexyl
5.0
40.0
14.0
41.0
10.0
3.20
>100
10.0
>100
3.33
1.05
14
15
16
17
18
Ph
CH2-4-pyridyl
Table 3. Inhibitory activity for benzo-substituted quinazolinone
compounds in accumulation assays for Pgp (EMT6/AR1.0) and
MRP1 (L23/R)
CH2-N-(6,7-dimethoxy)-
tetrahydroisoquinoline
CH2-N-Piperazine-N-Bn
CH2N(CH3)–(CH2)2–
(3,4-dimethoxy-phenyl)
CH2-N-Piperazine-N-(3,4,5-
trimethoxy)-benzoyl
CH2-N-Piperazine-N-2-pyrimidine
1.07
19
20
2.10
1.90
39.0
37.0
21
22
4.20
0.39
7.20
36.0
Table 2. Inhibitory activity of 2-(substituted)phenyl compounds in
accumulation assays for Pgp (EMT6/AR1.0) and MRP1 (L23/R)
Compd
R
IC50 (mM)/Pgp
IC50 (mM)/MRP1
18
32
33
34
35
36
37
38
39
40
41
H
5-Cl
6-Cl
7-Cl
1.07
0.16
1.70
1.35
1.06
0.63
0.05
1.10
7.20
0.60
2.0
1.05
3.84
1.56
1.33
2.91
6.35
15.1
2.02
18.57
0.40
7.60
8-Cl
8-CH3
8-OCH3
7-NO2
7-NH2
6-NO2
6-NH2
Compd
R
IC50 (mM)/Pgp
IC50 (mM)/MRP1
18
23
24
25
26
27
28
29
30
31
4-N(CH3)2
H
3-N(CH3)2
4-iPr
4-N(Et)2
3-nPr
3,4-(OCH2O)
2-OH
4-Ph
1.07
8.0
1.60
1.0
1.30
0.80
3.70
nda
1.03
0.85
1.05
2.80
4.50
0.50
>10.0
1.0
1.50
95.0
1.0
Table 4. Inhibitory activity of compound 18 and VX-710 in Pgp and
MRP potentiation assays
Compd
Pgp EC50 (mM)
MRP EC50 (mM)
18
VX-710a
3.30
5.0
3.01
5.18
0.90
aSynthesized by a modified procedure23 developed in-house.
and, not determined.

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S. Wang et al. / Bioorg. Med. Chem. Lett. 12 (2002) 571–574
Substitution by a chlorine atom at each position gives
dramatically different results; the 5-chloro analogue (32)
is a potent Pgp selective inhibitor with 24-fold selectivity
over MRP, while the 8-chloro analogue (35) shows a
moderate Pgp selectivity over MRP and good activity in
both cell lines.The 6- and 7-chloro analogues ( 33, 34)
display dual activity against Pgp and MRP with
retained potency.We then examined the electronic
effects at each position, in particular, the 8-position.
Thus, the 8-methyl analogue (36) gives slightly better
Pgp activity but worse MRP activity, while the more
electron donating 8-methoxy analogue (37) is a Pgp
inhibitor with 50 nM potency and is 300-fold selective
with respect to MRP.Introduction of electron-with-
drawing groups at the 7- and 6-positions retain or
improve the dual activity (38, 40), while the more elec-
tron-donating groups at these positions give reduced
activity, especially in the MRP cell line (39, 41).These
results suggest that electron-withdrawing groups are, in
general, better tolerated than electron-donating groups
for good dual inhibitory activity, particularly at the 6-
and 7-positions.Further derivatizations of the 6- and 7-
amino analogues (41, 39) to the corresponding amides
(9), as shown in Scheme 1, failed to give any compounds
with better dual activity against Pgp and MRP1.
References and Notes
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To further demonstrate the dual inhibitory activities of
these quinazolinone compounds, a selection of potent
inhibitors were subjected to the potentiation assays.22
Table 4 illustrates the data (EC50 values) of the com-
pound 18 in comparison with those of VX-710 in our
Pgp and MRP potentiation assays.
16. Roe, M.; Folkes, A.; Ashworth, P.; Brumwell, J.; Chima,
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Compound 18 exhibits equal potentiation activity in
both assays and appears to be slightly more active than
VX-710 in reversal of Pgp and MRP1 mediated drug
resistance.
In summary, we have identified a series of quinazo-
linone analogues with potent dual inhibitory activities
against both Pgp and MRP1.Although the clinical
benefit for developing the dual inhibitors of Pgp and
MRP1 remains to be proven, the pursuit of dual inhi-
bitory activity of Pgp and MRP1 inevitably sacrificed
the potency levels in comparison with that achieved for
selective inhibition of Pgp.16 Nevertheless, our SAR
study in this series of compounds has been useful in our
ongoing development of selective MRP1 inhibitors
which will be reported in due course.
21. Okawa, T.; Toda, M.; Eguchi, S.; Kakehi, A. Synthesis
1998, 1467.
22. Dale, I. L.; Tuffley, W.; Callaghan, R.; Holmes, J. A.;
Martin, K.; Luscombe, M.; Mistry, P.; Ryder, H.; Stewart,
A. J.; Charlton, P.; Twentyman, P. R.; Bevan, P. Br. J. Cancer
1998, 78, 885.
Acknowledgements
23. Armistead, D. M.; Saunders, J. O.; Boger, J. S. PCT/US
93/09145.
The authors would like to thank Mr.Lal Chima for his
contribution to this project.