Hypoxia activated prodrugs of a 9-aza-anthrapyrazole derivative that has promising anticancer activity

Article abstract of DOI:10.1021/jm200984x

Mono- and bis-N-oxides of a 9-aza-anthrapyrazole derivative having two 2-(dimethylamino)ethyl appendages were prepared by using a mild oxaziridine reagent. Biochemical and cell culture assays indicate that the bis-oxide is an inactive prodrug that readily converts to the active parent molecule under hypoxic conditions that are analogous to those present within certain tumors.

Full text of DOI:10.1021/jm200984x

Brief Article
pubs.acs.org/jmc
Hypoxia Activated Prodrugs of a 9-Aza-anthrapyrazole Derivative
That Has Promising Anticancer Activity
Mohammad H. El-Dakdouki,†,§ Nicholas Adamski,‡ Lecia Foster,‡ Miles P. Hacker,*,‡
and Paul W. Erhardt*,†
†Center for Drug Design and Development, College of Pharmacy and Pharmaceutical Sciences, The University of Toledo, Toledo,
Ohio 43606, United States
‡Department of Pharmacology, College of Pharmacy and Pharmaceutical Sciences, The University of Toledo, Toledo, Ohio 43606,
United States
S
* Supporting Information
ABSTRACT: Mono- and bis-N-oxides of a 9-aza-anthrapyrazole derivative having two 2-(dimethylamino)ethyl appendages were
prepared by using a mild oxaziridine reagent. Biochemical and cell culture assays indicate that the bis-oxide is an inactive prodrug
that readily converts to the active parent molecule under hypoxic conditions that are analogous to those present within certain
tumors.
INTRODUCTION
■
Human solid tumors grow within a unique microenvironment
that, among other features, is characterized by significantly
lower oxygen levels compared to those found in normal
tissues.1 The median oxygen tension for all human tumors
ranges from 1.3% to 3.9%, with most of the measurements in
solid tumors being less than 0.3%. Alternatively, values for
normal cells are in the range 3.1−8.7%.2 The hypoxia in solid
tumors can be attributed to its vasculature which is diminished
and abnormal in architecture, leading to insufficient supply of
oxygen and nutrients. Low oxygen tension, in turn, is associated
with low intracellular pH, low glucose concentrations, and high
lactate concentrations because glycolysis, the oxygen-independ-
ent metabolic pathway, becomes the primary mechanism to
generate ATP.3,4 There is also convincing evidence that hypoxia
promotes metastasis5 and angiogenesis6 and may led to the
selection of cells with a more malignant phenotype.7
Treatment of hypoxic tumors has been limited by systemic
toxicity, inefficient delivery of drugs to cells that are distant
from blood vessels, and reduced uptake of drugs by nondividing
cancer cells. These features have rendered hypoxic tumor cells
intrinsically more resistant to conventional treatments by
ionizing radiation8 and chemotherapeutic drugs.9 Alternatively,
from a drug design standpoint, the same morphological and
physiological differences between hypoxic tumor cells and
normal cells may be able to be exploited to selectively target
tumor cells.10,11
The anthracycline antitumor and antibiotic agents doxor-
ubicin and daunorubicin (Figure 1) have been used for the
treatment of several types of cancer such as breast and ovarian
cancers, acute nonlymphocytic leukemia in adults, and acute
lymphocytic leukemia in adults and children.12 Anthracyclines
intercalate between DNA bases, thus blocking DNA synthesis
and transcription. They also inhibit the activity of top-
oisomerase II (Topo II), leading to breaks in genomic
DNA.13 However, these anticancer agents possess serious side
effect toxicity, especially cardiotoxicity. Although mitoxantrone
Figure 1. Structures of clinically useful anthracyclines.
(Figure 1), a structurally similar anticancer agent, was found to
have an improved tolerance profile, it is still not devoid of
serious side effects that are associated with myelosuppression
and cardiotoxicity. Moreover, cell histotypes that developed
resistance to doxorubicin due to the overexpression of P-
glycoprotein (Pgp) also showed resistance to mitoxantrone.14
The biochemical basis for cardiotoxicity is not fully understood,
but it is thought that the in vivo reduction of the quinone
moiety plays an important role. The addition of an electron to
the quinone leads to the formation of a semiquinone free
radical that, in turn, transfers an electron to molecular oxygen
to generate superoxide radical anions. The so-formed radical
anions lead to hydroxyl radicals that can damage cardiac tissue.
Several published studies have discussed structural mod-
ification of mitoxantrone in attempts to reduce its adverse side
effects.15−17 These studies led to the discovery of 9-aza-
anthrapyrazole analogues whose general structure is shown in
Figure 2. Because they were expected to be less prone to
undergo bioreduction, the anthrapyrazole analogues were
designed to have increased antitumor activity and reduced
cardiotoxicity. In addition, the presence of two amine-
containing side chains (e.g., R1 and R2 being 2-
(dimethylamino)ethyl in Figure 2) can enhance their DNA
Received: July 22, 2011
Published: October 19, 2011
© 2011 American Chemical Society
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Journal of Medicinal Chemistry
Brief Article
Scheme 1. Synthesis of 9-Aza-anthrapyrazole N-Oxide
a
Analogues
Figure 2. General structure of 9-aza-anthrapyrazole.
binding affinity and cytotoxicity, which is believed to be
achieved through topoisomerase inhibition. However, selective
targeting of such drugs to cancer cells remains an important
issue because these analogues still have the capability to
intercalate with the DNA of normal cells and that of cancer
cells.18
One approach to achieve selective targeting is to mask the
chemotherapeutic agent as a prodrug that becomes unmasked
at the cancer site.19 This strategy has been described previously
by Cerecetto et al., Lee et al., and Nishida et al.11,20,21 relative to
the analogous anthracene system. Herein, we describe the
synthesis of 9-aza-anthrapyrazole N-oxide analogues as
“bioreductive-dependent agents and as potential hypoxia-
selective cytotoxins for cancer therapy”.20 The lower pH and
oxygen-deprived (reductive) nature of hypoxic cancer cells may
be able to reduce these inactive prodrugs to their active amine
forms. Cellular reduction of the N-oxide functionalities will
then result in “bis-armed” anthrapyrazole amines that regain
DNA binding affinity and cytotoxicity. Such anthrapyrazole
moieties are unique DNA-complexing agents that may then
exhibit reduced or no cardiotoxicity because of their diminished
tendency to form semiquinone free radicals at sites other than
hypoxic tumor cells.18
a
Reagents and conditions: (i) K2CO3, reflux, then NaOH, 62%; (ii)
DIPEA, THF, room temp, 75%; (iii) [2-(dimethylamino)ethyl]amine,
anhydrous pyridine, 90 °C, 60%; (iv) 2.5 equiv of 9, CH2Cl2/MeOH
(3:1), 0−5 °C, then HCl (g), 87%; (v) 1 equiv of 9, CH2Cl2/MeOH
(3:1), 0−5 °C, 49%.
anthrapyrazole mono-N-oxide adducts in a 1:1 mixture of 10
and 11 as determined by NMR. These spectra displayed eight
distinct peaks corresponding to the eight different methylene
groups in 10 and 11, while there were only four peaks in
anthrapyrazole bis-N-oxide analogue 8. The oxaziridine-
containing, mild oxidizing reagent 9 was synthesized according
to a literature procedure.24
CHEMISTRY
■
Our syntheses of the anthrapyrazole N-oxide analogues were
adapted from the route previously reported by Krapcho et al.15
who used it within the analogous anthrapyrazole system. We
began by preparing the substituted hydrazine 3 that is essential
for constructing the pyrazole ring via the reaction of hydrazine
monohydrate 1 and dimethylaminoethyl chloride 2 under basic
conditions (Scheme 1).22 Regioselective construction of the
pyrazole ring with an N-substituent can be achieved by treating
anthraquinone 4 with hydrazine 3.15 Two aspects merit
highlight in this respect. First, on the basis of previous SAR
studies where regioisomers bearing nitrogen atoms at position
7, 8, 9, or 10 of the anthrapyrazole chromophore have been
prepared;23 the most active 9-aza-regioisomer has been selected
for this synthesis. Second, the regioselective construction of the
pyrazole ring was induced by choosing the fluorine and chlorine
atoms as substituents at C-6 and C-9 of anthraquinone 4,
respectively, because of the difference in their electrophilicity
during SNAr displacements.23
BIOLOGY AND DISCUSSION
■
Preliminary biological experiments using the regioisomeric
mixture of “partial prodrug” mono-N-oxides 10 and 11
compared to using the “full prodrug” bis-N-oxide 8 indicated
that more consistent behavior was observed for the latter. Thus,
a focus was placed on characterizing the profile of 8. Toward
that end, parent compounds 7 and 8 were first studied in a
biochemical assay for inhibition of Topo II. A gel electro-
phoresis for the control runs in these experiments shows a
single band for DNA, whereas multiple bands result when
Topo II is added. Compound 7 was able to inhibit Topo II’s
fragmentation of DNA at 10 μM, while its prodrug 8 remained
inactive up to 50 μM. Interactions with DNA were likewise
assessed in a biochemical assay wherein displacement of the
standard ligand, ethidium bromide, can be readily measured by
the change in fluorescence. In this case, mitoxantrone was also
tested as a standard. Mitoxantrone’s effective concentration to
inhibit ethidium bromide binding by 50% (IC50) was 4.5 μM.
The IC50 for 7 was reasonably similar (6.8 μM), while prodrug
8 was unable to displace ethidium bromide up to 50 μM.
Cell culture studies were used to assess cytotoxicity.25 These
investigations were conducted under oxygen rich (“oxic”) and
hypoxic (“anoxic”) conditions using three cell lines: LoVo from
colon cancer; MCF-7/S from breast cancer sensitive to
Thus, the most nucleophilic nitrogen of the hydrazine adduct
selectively displaced the C-9 fluorine atom, yielding inter-
mediate 5 which under the reaction conditions also underwent
spontaneous cyclization to yield anthrapyrazole 6. Refluxing 6
with N,N-dimethylaminoethylamine in anhydrous pyridine
under a nitrogen atmosphere displaced the C-5 chlorine atom
and resulted in the bis-substituted anthrapyrazole diamine 7.15
Oxidation of anthrapyrazole 7 using 2.5 equiv of the mild
oxidizing reagent oxaziridine 9 resulted in anthrapyrazole bis-N-
oxide 8.20 Oxidizing 7 with 1 equiv of 9 afforded the
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Journal of Medicinal Chemistry
Brief Article
doxorubicin; MCF-7/Dox from breast cancer that has become
resistant to doxorubicin. Mitoxantrone was again utilized as a
standard agent. Cells were cultured in 96-well plates for 74 h
with varying concentrations of test agent. Cytotoxicity was
determined by sulforhodamine B assay where viable cells
convert the colorless solution to blue. Gradations of color
versus test agent concentration allowed for calculation of IC50,
in this case the latter reflecting 50% inhibition of cultured cell
growth. The hypoxic medium was created by incubating cells in
an anerobic chamber flushed with 95% N2 and 5% CO2. As
shown in Table 1, in the LoVo and MCF-7/S cell lines,
Table 1. IC50 (μM) Derived from the Cytotoxicity Assay
a
under Oxic and Anoxic Conditions
Figure 3. Binding curves for prodrug 8 and sequentially increasing
concentrations of DNA without prior incubation with CYP450 (lower
panel) and after incubation with CYP450 (upper panel). Axes y and x
are absorbance and wavelength. See text for details.
cell line
LoVo
drug
oxic
0.1
anoxic
mitoxantrone
0.9
3.3
1.5
1.0
4.0
5.0
>10
1.2
1.3
7
1.3
8
>25
0.1
SUMMARY
■
MCF-7/S
mitoxantrone
The composite of our results suggests that 8 serves as an
effective prodrug for 7 by undergoing reduction under hypoxic
conditions. Thus, there may be an added degree of selectivity
that 8 will be able to display toward solid tumors compared to
healthy cells. Further studies are underway in advanced
pharmacological models to substantiate the additional promise
demonstrated by this prodrug.
7
0.9
8
>25
4.7
MCF-7/Dox
mitoxantrone
7
8
1.0
>25
a
Data represent the average of two determinations across at least three
different concentrations wherein the entire range of values for each
point was always within 10% of the average.
ASSOCIATED CONTENT
* Supporting Information
■
S
mitoxantrone and 7 showed higher activity under oxic
conditions compared to the hypoxic conditions. Alternatively,
8 proved to be significantly more active under hypoxic
conditions wherein it was able to achieve levels comparable
to those of mitoxantrone and 7. Interestingly, while
mitoxantrone’s activity fell off in the hypoxic MCF-7/Dox
panel, thus reflecting the multidrug resistance that this cell line
can display toward chemotherapeutics, 7 and 8 were able to
maintain their potency. Since one of the principle components
of multidrug resistance is overexpression of the P-glycoprotein
(Pgp) efflux transporter, it would appear that 7 and 8 are poor
substrates for Pgp.
To further evaluate 8 as a potential prodrug, we examined
the ability for the cytochrome P450 system (CYP450) to
perform the requisite reduction when under hypoxic con-
ditions. As part of these tests, 7 and 8 were incubated with the
S-9 fraction from rat liver in normal air or in 1% O2 for 2 h at
37 °C. After precipitation of proteins by ethanol, samples were
centrifuged and the supernatants used in the same tests for
Topo II activity and cell culture cytotoxicity under oxic
conditions. While 7 displayed comparable activity after either
pretreatment, 8 demonstrated activity only from the metabolic
incubations that were performed under hypoxic conditions.
Finally, 8 and hypoxic CYP450-treated 8 were studied side by
side in an alternative assay for DNA binding.26 These results are
depicted in Figure 3. While the scans for untreated 8 (lower
panel) did not change upon sequentially increasing the
concentrations of DNA, those for treated 8 (upper panel)
displayed increases in hypochromicity accompanied by a minor
bathochromic shift. These results further demonstrate that 8
does not interact significantly with DNA until it becomes
reduced, in this case by CYP450.
Experimental methods and details for synthesis of the oxidizing
reagent; biology testing methods and raw data for the
biochemical studies; and NMR spectra for all intermediates
and target compounds. This material is available free of charge
via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*For M.P.H.: phone, 419-383-1598; e-mail, miles.hacker@
utoledo.edu. For P.W.E.: phone, 419-530-2167; e-mail, paul.
erhardt@utoledo.edu.
Present Address
§Department of Chemistry, Michigan State University, East
Lansing, MI 48864, United States.
ABBREVIATIONS USED
CYP450, cytochrome P450; Pgp, P-glycoprotein; Topo II,
topoisomerase II
■
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