Substituted pyridazino[3,4-b][1,5] benzoxazepin-5(6H)ones as multidrug-resistance modulating agents

Article abstract of DOI:10.1021/jm040803n

Pyridazino[3,4-b][1,5]benzoxazepin-5(6H)ones substituted with propylene-linked basic side chains were synthesized and investigated for the ability to reverse multidrug resistance (MDR) at vincristine-pretreated HeLa-MDR1 cells. The substances were found t

Full text of DOI:10.1021/jm040803n

J . Med. Chem. 2004, 47, 4627-4630
4627
Brief Articles
Su bstitu ted P yr id a zin o[3,4-b][1,5]ben zoxa zep in -5(6H)on es a s
Mu ltid r u g-Resista n ce Mod u la tin g Agen ts
Ingo Ott,† Brigitte Kircher,‡ Gottfried Heinisch,† and Barbara Matuszczak*,†
Department of Pharmaceutical Chemistry, Institute of Pharmacy, University of Innsbruck, Innrain 52a,
A-6020 Innsbruck, Austria, and Laboratory for Tumor and Immunobiology, Division of Hematology and Oncology,
Medical University Hospital of Innsbruck, Anichstrasse 35, A-6020 Innsbruck, Austria
Received March 2, 2004
Pyridazino[3,4-b][1,5]benzoxazepin-5(6H)ones substituted with propylene-linked basic side
chains were synthesized and investigated for the ability to reverse multidrug resistance (MDR)
at vincristine-pretreated HeLa-MDR1 cells. The substances were found to be effective
chemosensitizers with activity comparable to that of the known MDR modulator verapamil.
The observed antiproliferative effects were not caused by direct drug cytotoxicity.
In tr od u ction
Multidrug resistance (MDR) is defined as the capabil-
ity of leukemic or tumor cells to survive continuous
exposure to a broad variety of chemotherapeutic drugs.
The cells may constitutively display mechanisms to
confer MDR or acquire resistance during drug admin-
istration. Resistance can result from decreased drug
influx, activation of DNA repair mechanisms, defective
apoptotic pathways (e.g., decreased ceramide levels) or
activation of detoxifying systems (e.g., cytochrome P450).
An altered membrane transport resulting in lower
intracellular concentrations of cytotoxic drugs is con-
sidered as major event of MDR.1 The effect is mediated
by the overexpression of a variety of proteins belonging
to a family of ATP-binding cassette transporters such
as P-glycoprotein (Pgp)2 and multidrug resistance pro-
teins3 that act as ATP-dependent extrusion pumps. Pgp
is a 170 KDa membrane protein encoded by the MDR1
gene localized to chromosome 7.4 Pgp and MDR1 mRNA
have been shown to be expressed in acute myeloid
leukemia,5 breast cancer,6 ovarian cancer,7 and lung
F igu r e 1. Structures of MDR modulators.
cancer.8
A number of compounds, so-called chemosensitizers,
are able to reverse the effect of Pgp on MDR.9 However,
some MDR modulators such as verapamil were associ-
ated with many side effects.10 Compounds containing
heterocyclic rings and basic side chains, like N-substi-
tuted quinoline,11 thioxanthene,12,13 phenothiazine,12,14,15
and dibenzo[b,f][1,4]oxazepin-11(10H)one derivatives,16
are known to modulate MDR. Figure 1 shows relevant
examples of known potent MDR-modulating agents
developed in the past years.17 Compounds such as MS-
209, LY-335979, and the dibenzo[b,f][1,4]oxazepin-11-
(10H)ones are substances sharing common structural
features. An N-containing di- or tricyclic ring system is
connected by a short (C2 or C3) linker to a basic side
chain with at least one tertiary nitrogen.
Because related compounds are the subject of our
research, we focused our interests on substances struc-
turally close to this kind of MDR reversing agent. The
target compounds are characterized by a substituted
pyridazino[3,4-b][1,5]benzoxazepin-5(6H)one core. This
tricyclic ring system is closely related to that of the
dibenzo[b,f][1,4]oxazepin-11(10H)ones. These compounds
exhibit carboxamid nitrogens; the exocyclic one is
replaced by the pyridazino-N in the pyridazino[3,4-b]-
[1,5]benzoxazepin-5(6H)ones. Hydrophobic substituents
such as Cl and CF3 were of advantage in the dibenzo-
[b,f][1,4]oxazepin-11(10H)ones and were kept at the
corresponding positions in the target structures. The
essential basic substructure represents a substituted
piperazine that is linked at the tricyclic system via an
* To whom correspondence should be addressed. Phone: 43-512-
507-5262. Fax: 43-512-507-2940. E-mail: barbara.matuszczak@
uibk.ac.at.
† University of Innsbruck.
‡ Medical University Hospital of Innsbruck.
10.1021/jm040803n CCC: $27.50 © 2004 American Chemical Society
Published on Web 08/04/2004

4628 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18
Brief Articles
Sch em e 1. Synthesis of the Target Compounds of Type
ence of a base at 60-100 °C led to a mixture of products
but did not result in the formation of the target 7. This
conclusion is based on the 1H NMR spectra of the
isolated reaction products. In all cases the signal of the
H4 (pyridazine-H), which appears at ∼8 ppm, was
missing. Efforts are underway to elucidate the struc-
tures of these isolated compounds, and the results will
be published elsewhere. Moreover, 3-chloropyridazino-
[3,4-b][1,5]benzoxazepin-5(6H)one derivatives bearing a
6-(2-bromoethyl) or a 6-(4-iodobutyl) substituent did not
represent useful precursors for the target compounds
of type 7.
7a
These results prompted us to investigate an alterna-
tive approach. It consists of reaction of the tricycles 4
with N-(ω-halogenpropyl)-substituted piperazine deriva-
tives 6. The derivatives were prepared by treatment of
a 1-substituted piperazine (i.e., 1-phenylpiperazine,
1-benzylpiperazine, and 1-(diphenylmethyl)piperazine)
with 1,3-dibromopropane and a base in dry acetone at
room temperature in analogy to a method described in
the literature.22 GC/MS determination showed that the
alkylpiperazines formed represent mixtures of the
bromo and chloro derivatives that may result from
halogen exchange reaction during the workup. These
mixtures were used for subsequent reaction without
separation. For the preparation of the target compounds
the N-(ω-halogenalkyl)-substituted piperazines 6 were
reacted with a (substituted) 3-chloropyridazino[3,4-b]-
[1,5]benzoxazepin-5(6H)one 4 in a dry solvent (e.g. N,N-
dimethylformamide) in the presence of base at room
temperature or elevated temperature. The crude com-
pounds 7 thus obtained were purified by column chro-
matography and subsequent recrystallization. The struc-
tures of the novel compounds were confirmed by
a
Reagents and conditions: (i) triethylamine, CH2Cl2, 0 °C f
room temp; (ii) NaH, 1,4-dioxane, 100 °C; (iii) KOH, DMSO,
X-CH2(CH2)n-X′, room temp; (iv) 1-substituted piperazine, NaI
or Na2CO3, DMF, 60-80 °C; (v) K2CO3, acetone; (vi) dry solvent,
base.
alkylene spacer (in accordance to the literature [quino-
lines] preferably propylene). Furthermore, the pipera-
zine ring is connected to lipophilic substituents (phenyl,
benzyl, or diphenylmethylene), a concept that seems
promising because of the results obtained with com-
pounds such as MS-209 and LY-335979.
To evaluate the activity of the new target compounds,
comparative studies of multidrug-resistant HeLa (HeLa-
MDR1) cells and the corresponding wild-type HeLa S3
cells (HeLa-WT) were conducted.
1
elemental analyses and IR and H NMR spectroscopy.
Ch em istr y
In Vitr o Activity
The synthesis of the target compounds of type 7 is
summarized in Scheme 1. (Substituted) 2-aminophe-
nol(s) were acylated with 3,6-dichloropyridazine-4-car-
boxylic acid chloride (1) to give the corresponding
carboxamides 3. The carboxamides were then cyclized
by treatment with sodium hydride in refluxing 1,4-
dioxane based on a method developed by our group.18
The trifluoromethyl-substituted aminophenols (2 with
R ) CF3)19 became accessible by reduction of the
corresponding 2-nitrophenol derivatives using ammo-
nium formate as a hydrogen source and palladium on
charcoal as catalyst. The nitro compounds were pre-
pared as described in the literature by nitration of
3-hydroxybenzotrifluoride20 or by diazotation of 4-tri-
fluoromethyl-2-nitroanilin and subsequent reaction in
aqueous CuSO4 solution.21
The key step to the synthesis of the target 7 was the
introduction of the propylene-linked basic side chain.
First, the appropriate N-ω-halogenalkyl derivatives
were considered as suitable precursors for the target
compounds. Such precursors of type 5 can be prepared
by reaction of (substituted) 3-chloropyridazino[3,4-b]-
[1,5]benzoxazepin-5(6H)ones 4 with an excess of 1,3-
dibromopropane in dry dimethyl sulfoxide in the pres-
ence of powdered potassium hydroxide. Treatment of
derivatives of type 5 with N-nucleophiles [N-methylpi-
perazine, N-benzylpiperazine, or 1-(diphenylmethyl)-
piperazine] in dry N,N-dimethylformamide in the pres-
The MDR effect was analyzed by incubating the cells
with concentrations of vincristine sulfate (VCR) ranging
from 1 pg/mL to 10 ng/mL. A concentration of 1 ng/mL
VCR inhibited proliferation of HeLa-WT cells from 100%
(in the absence of all compounds) to 75.7 ( 23.9% (p is
not significant) and a concentration of 10 ng/mL to 21.3
( 3.7% (p ) 0.0022). The HeLa-MDR1 cell line was more
resistant to VCR. Proliferation was inhibited from 100%
to 89.0 ( 1.7% by 1 ng/mL (p ) 0.0239) and to 44.7 (
8.4% by 10 ng/mL VCR (p ) 0.022). The effectiveness
of the compounds in inhibiting the MDR effect was
tested on the ability to modulate proliferation of VCR-
pretreated HeLa-WT and HeLa-MDR1 cells.
An anti-MDR effect was already observed with 1 µM
of either compound. The VCR (10 ng/mL) reduced
proliferation of HeLa-MDR1 cells was further decreased
from 44.7 ( 8.4% to 16.0 ( 3.6% by 7a (p ) 0.0497), to
19.7 ( 3.8% by 7b (p ) 0.031), to 19.3 ( 4.6% by 7c (p
) 0.029), and to 19.0 ( 5.3% by 7d (p ) 0.0241) (Figure
2). This decrease was identical to that of 1 µM verapamil
(19.3 ( 4.9%, p ) 0.0641; Figure 2). Higher compound
concentrations almost blocked proliferation of HeLa-
MDR1 cells. Proliferation was inhibited by 5 µM 7a from
44.7 ( 8.3% to 6.7 ( 1.2% (p ) 0.0576), by 7b to 2.7 (
0.7% (p ) 0.0344), by 7c to 2.7 ( 0.7% (p ) 0.0344),
and by 7d to 2.3 ( 0.3% (p ) 0.0353) (Figure 2).
Verapamil at 5 µM reduced the proliferation to 6.0 (
1.0% (p ) 0.0496; Figure 2).

Brief Articles
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18 4629
Exp er im en ta l Section
Gen er a l P r oced u r e for th e Syn th esis of 6-P ip er a zin -
yla lk yl Su bstitu ted 3-Ch lor op yr id a zin o[3,4-b][1,5]ben z-
oxa zep in -5(6H)on es of Typ e 7. To a solution of 1 equiv of
the substituted 3-chloropyridazino[3,4-b][1,5]benzoxazepin-
5(6H)one of type 4 (1.5-2.0 mmol) in 25 mL of an appropriate
solvent (dimethyl sulfoxide, acetone, or N,N-dimethylforma-
mide) was added 2 equiv of base (potassium hydroxide or
sodium carbonate). After the mixture was stirred for 1 h, 1.1
equiv of the appropriate alkylating agent (4-substituted 1-(3-
halogenpropyl)-piperazine) were added and stirring was con-
tinued until the starting material was completely alkylated
(TLC monitoring, dichloromethane/ethyl acetate 9/1, or ethyl
acetate).
Then the mixture was poured into cold water (50 mL) and
the mixture was extracted with dichloromethane. The com-
bined organic phases were washed with water and saturated
NaCl solution, dried over anhydrous Na2SO4, and evaporated.
Pure products could be obtained after column chromatography
and recrystallization.
F igu r e 2. Effect of compounds 7a -d and verapamil on the
proliferation of VCR-pretreated HeLa-MDR1 cells.
3-Ch lor o-8,10-d im eth yl-6-[3-(4-p h en ylp ip er a zin -1-yl)-
pr opyl]pyr idazin o[3,4-b][1,5]ben zoxazepin -5(6H)on e (7a).
Compound 7a was prepared by reaction of 3-chloro-8,10-
dimethylpyridazino[3,4-b][1,5]benzoxazepin-5(6H)one (4 with
R ) 8,10-(CH3)2) with 1-(3-bromopropyl)-4-phenylpiperazine
in DMSO in the presence of KOH (5 h). The crude product
was purified by column chromatography (eluent, ethyl acetate)
followed by recrystallization from diisopropyl ether to give 13%
yield of colorless crystals: mp 185-186 °C; IR (KBr) 1654 cm-1
;
MS m/z 478/480 (M+); H NMR (CDCl3) δ 7.94 (s, 1 H, H-4),
7.29-7.21 (m, 3 H), 6.96-6.81 (m, 4 H) (H-7, H-9, phenyl-H),
4.20 (“s”, br, 2 H, N-CH2), 3.13 (t, J ) 5.1 Hz, 4 H, 2 ×
piperazine-CH2), 2.57-2.41 (m, 9 H, 2 × piperazine-CH2,
N-CH2, CH3), 2.29 (s, 3 H, CH3), 2.00-1.86 (m, 2 H, CH2).
Anal. (C26H28ClN5O2) C, H, N.
1
F igu r e 3. Effect of compounds 7a -d and verapamil on the
proliferation of HeLa-WT cells.
3-Ch lor o-8-tr iflu or om eth yl-6-[3-(4-p h en ylp ip er a zin -1-
yl)p r op yl]p yr id a zin o[3,4-b][1,5]b e n zoxa ze p in -5(6H )-
on e (7b). Compound 7b was prepared by reaction of 3-chloro-
8-trifluoromethylpyridazino[3,4-b][1,5]benzoxazepin-5(6H)-
one (4 with R ) 8-CF3) with 1-(3-bromopropyl)-4-phenylpip-
erazine in acetone in the presence of KOH (48 h). The crude
product was purified by column chromatography (eluent, ethyl
acetate) followed by recrystallization from diisopropyl ether/
ethyl acetate to give 15% yield of colorless crystals: mp 160-
The proliferation of VCR-pretreated HeLa-MDR1
incubated with compound concentrations higher than
5 µM was similarly low as that of HeLaWT, indicating
that the drug resistance effect is completely removed
at higher concentrations (data not shown).
To exclude that the effect of the compounds on
proliferation of HeLa-WT and HeLa-MDR1 cells could
be due to toxicity, both cell lines were incubated with
the compounds in the absence of VCR. Low concentra-
tions (1-10 µM) of the compounds and verapamil
showed no significant antiproliferative activity on HeLa-
WT cells (Figure 3). At 100 µM, strong differences
between the compounds could be observed. The prolif-
eration was only slightly reduced from 100.0 ( 0.0% in
the absence of either compound to 87.3 ( 33.2% by 7c
(p ) 0.7395). 7a decreased the proliferation to 55.3 (
22.9% (p ) 0.1907) and 7b to 28.3 ( 14.3% (p ) 0.0376).
7d and verapamil blocked the proliferation (decrease to
1.3 ( 0.3% (p ) 0.0001) and 5.7 ( 1.9% (p ) 0.0004),
respectively). Similar results were obtained with HeLa-
MDR1 cells (data not shown).
1
162 °C; IR (KBr) 1658 cm-1; H NMR (CDCl3) δ 8.00 (s, 1 H,
H-4), 7.70-7.53 (m, 3 H), 7.29-7.21 (m, 2 H), 6.91-6.81 (m, 3
H) (H-7, H-9, H-10, phenyl-H), 4.27 (t, J ) 6.8 Hz, 2 H,
N-CH2), 3.10 (t, J ) 4.9 Hz, 4 H, 2 × piperazine-CH2), 2.55-
2.43 (m, 6 H, 2 × piperazine-CH2, N-CH2), 2.04-1.91 (m, 2
H, CH2). Anal. (C25H23ClF3N5O2) C, H, N.
6-[3-(4-Ben zylp ip er a zin -1-yl)p r op yl]-3-ch lor o-8-tr iflu o-
r om et h ylp yr id a zin o[3,4-b][1,5]b en zoxa zep in -5(6H )on e
(7c). Compound 7c was prepared by reaction of 3-chloro-8-
trifluoromethylpyridazino[3,4-b][1,5]benzoxazepin-5(6H)one (4
with R ) 8-CF3) with 4-benzyl-1-(3-bromopropyl)piperazine in
DMF in the presence of Na2CO3 and catalytic amounts of KI
(25 h at 60 °C, 3 h at 90 °C). The crude product was purified
by column chromatography (eluent 1, ethyl acetate; eluent 2,
dichloromethane/ethanol ) 19/1) followed by recrystallization
from diisopropyl ether to give 26% yield of colorless crystals:
mp 170-171 °C; IR (KBr) 1662 cm-1; 1H NMR (CDCl3) δ 7.99
(s, 1 H, H-4), 7.68-7.63 (m, 2 H, H-7, H-10), 7.54 (dd, J ) 8.3
Hz, J ) 1.7 Hz, 1 H, H-9), 7.31-7.22 (m, 5 H, phenyl-H), 4.22
(t, J ) 7.0 Hz, 2 H, N-CH2), 3.45 (s, 2 H, CH2), 2.41-2.35 (m,
10 H, 4 × piperazine-CH2, N-CH2), 1.98-1.84 (m, 2 H, CH2).
Anal. (C26H25ClF3N5O2) C, H, N.
3-Ch lor o-9-tr iflu or om eth yl-6-[3-(4-d ip h en ylm eth ylp i-
p er a zin -1-yl)p r op yl]p yr id a zin o[3,4-b][1,5]ben zoxa zep in -
5(6H)on e (7d ). Compound 7d was prepared by reaction of
3-chloro-9-trifluoromethylpyridazino[3,4-b][1,5]benzoxazepin-
5(6H)one (4 with R ) 9-CF3) with 1-(3-bromopropyl)-4-(diphe-
nylmethyl)piperazine in DMF in the presence of Na2CO3 and
catalytic amounts of KI (18 h at 60 °C). The crude product
was purified by column chromatography (eluent 1, ethyl
Con clu sion
The compounds synthesized in this initial study are
potent MDR-modulating drugs. The drug resistance of
vincristine-pretreated HeLa-MDR1 cells was lowered to
the same extent as by the known chemosensitizer
verapamil. The absence of toxicity of 7c and the low
toxicity of 7a even at high concentrations (verapamil
blocks proliferation at this concentration) suggest that
these compounds are promising candidates for further
developments.

4630 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18
Brief Articles
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acetate/ethanol ) 10/1; eluent 2, ethyl acetate) followed by
recrystallization from diisopropyl ether to give 11% yield of
colorless crystals: mp 156-159 °C; IR (KBr) 1662 cm-1 1H
;
NMR (CDCl3) δ 7.97 (s, 1 H, H-4), 7.79 (“s”, 1 H), 7.57-7.52
(m, 2 H), 7.41-7.12 (m, 10 H) (H-7, H-8, H-10, phenyl-H),
4.22-4.14 (m, 4 H, 2 × piperazine-CH2), 2.42-2.29 (m, 9 H, 2
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Anal. (C32H29ClF3N5O2) C, H, N.
In Vitr o Activity. The human HeLa S3 cell line (HeLa-
WT) and the multidrug resistant MDR-overexpressing HeLa
cell line (HeLa-MDR1) were kindly provided by J . Hofman
(Institute of Medical Chemistry and Biochemistry, Medical
University Hospital of Innsbruck).
Both cell lines were grown in RPMI 1640 (Biochrom, Berlin,
Germany) supplemented with 2 mM glutamine (Biochrom),
100 U/mL penicillin (Biochemie GmbH, Vienna, Austria), 100
µg/mL streptomycin (Gru¨nenthal GmbH, Stolberg, Austria),
and 10% fetal calf serum (Life Technologies, Paisley, Scotland)
at 37 °C in 5%CO2/95% air. Logarithmically growing cells were
trypsinized, washed with phosphate buffered saline, resus-
pended in culture medium at 2 × 105 cells/mL, and plated in
triplicate in flat bottom microtiter plates (Falcon, Becton
Dickinson, Franklin Lakes, NY). Various concentrations of
vincristine sulfate (VCR, 0-10 ng/mL, Vincristin, Pharmacia
Austria, Vienna, Austria) were added 30 min thereafter. 7a -d
and verapamil (Institute of Pharmacy, University Innsbruck)
were prepared as stock solutions in DMSO (Sigma-Aldrich,
Vienna, Austria) and were adjusted to the final concentrations
(1-100 µM) with culture medium. The concentration of DMSO
did not exceed 0.1% (v/v). One hour after the addition of VCR,
the solutions of the compounds were added and continuously
exposed for 72 h. For the last 12-16 h of culture, cells were
pulsed with 2 µCi of 3[H]thymidine (40-60 Ci/mmol, Amer-
sham, Arlington Heights, IL) and harvested using a semiau-
3
tomated device and [H]thymidine uptake was measured in a
liquid scintillation counter (Beckman LS 1801, Galway, Ire-
land). The paired Student’s t-test was used to calculate
statistical differences. Results are shown as mean proliferation
( SE. Proliferation in the absence of all compounds was set
at 100%.
Su p p or tin g In for m a tion Ava ila ble: General remarks,
synthesis, and analytical data for 1-5. This material is
available free of charge via the Internet at http://pubs.acs.org.
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