Synthesis and first biological evaluation of 1-aza-9-oxafluorenes as novel lead structures for the development of small-sized cytostatics

Article abstract of DOI:10.1016/S0960-894X(01)00769-7

A first series of novel 1-aza-9-oxafluorenes has been prepared from 3-carbonyl substituted 1,4-dihydropyridines and p-benzoquinone as small-sized cytostatics. Biological evaluation has been carried out in various cancer cell-lines. First structure-activity relationships proved the 4-phenyl substituent to be more favorable than the 4-methyl substituent. Cytostatic properties are discussed.

Full text of DOI:10.1016/S0960-894X(01)00769-7

Bioorganic & Medicinal Chemistry Letters 12 (2002) 411–413
Synthesis and First Biological Evaluation of
1-Aza-9-oxafluorenes as Novel Lead Structures for the
Development of Small-Sized Cytostatics
Kristin Brachwitz and Andreas Hilgeroth*
Institute of Pharmaceutical Chemistry, Department of Pharmacy, Martin-Luther-University, Halle-Wittenberg,
Wolfgang-Langenbeck-Str. 4, 06120 Halle, Germany
Received 16 August 2001; revised 12 October 2001; accepted 13November 2001
Abstract—A first series of novel 1-aza-9-oxafluorenes has been prepared from 3-carbonyl substituted 1,4-dihydropyridines and p-
benzoquinone as small-sized cytostatics. Biological evaluation has been carried out in various cancer cell-lines. First structure–
activity relationships proved the 4-phenyl substituent to be more favorable than the 4-methyl substituent. Cytostatic properties are
discussed. # 2002 Elsevier Science Ltd. All rights reserved.
Among the diverse number of important cytostatic
agents which have been introduced in clinical practice,
the DNA-intercalating inhibitors of topoisomerase I or
II are one of the most perspective classes of anticancer
drugs.1 One important feature for potential intercal-
ating properties of such drug candidates is a planar
aromatic system. Almost all developed intercalating agents
have polycyclic structures consisting of more than three
annelated rings. Indolo- or quinolo-annelated systems
predominate within the promising candidates like
intoplicine 1, nitidine 2 (Fig. 1) or rebeccamycin-derived
indolopyrazole compounds.1
solubility. The reported number of small-sized tricyclic
cytostatic compounds like mitoxanthrone with promis-
ing better solubility is poor.
Low cytostatic activities of recently reported b-carbo-
line compounds as azacarbazole derivatives with DNA-
intercalating properties could be increased by an alkyl-
amine side chain that showed additional binding affi-
nities to the DNA-helix groove.4 Among a series of a-
carboline derivatives 4 (Fig. 1) only some candidates
One main problem of the polycyclic systems that con-
tributed to clinical failure of cytostatically potent ellip-
ticine 3 and derivatives was poor solubility of the
substances so that absorption rates were insufficient for
an oral therapy.2 Among the series of highly potent tet-
racyclic anthracycline compounds, cardiotoxicity is one
additional problem besides poor solubility.1 Tricyclic
anthracendione derivative mitoxanthrone shows better
solubility and less cardiotoxicity.3 Idarubicin shows
satisfying oral bioavailability.3 As was shown for the
class of anthracycline compounds, one strategy within
the development of novel cytostatic agents is an
improvement of oral absorption rates due to a better
*Corresponding author. Tel.:+49-345-55-25168; fax:+49-345-55-
27026; e-mail: hilgeroth@pharmazie.uni-halle.de
Figure 1.
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00769-7

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K. Brachwitz, A. Hilgeroth / Bioorg. Med. Chem. Lett. 12 (2002) 411–413
showed poor cytostatic activities.5 Introduction of a 6-
hydroxy function in the carboline scaffold was promis-
ing because 9-hydroxyellipticine showed improved
cytostatic activity compared to ellipticine.3 However,
the 6-hydroxycarbolines were found inactive because of
metabolic inactivation by dimerization via the indolo-
nitrogen.6
We present the synthesis and cytostatic evaluation of a
first series of novel 1-aza-9-oxafluorenes 10–13 as tri-
cyclic aromatic compounds with higher activity as were
reported for comparable tricyclic carboline systems.
Compared to a-carbolines, we formally replaced the
indolo-nitrogen by oxygen so that metabolic inactiva-
tion of the compounds via dimerization could be exclu-
ded. The compounds 10 and 11 showed cytostatic
activity in the range of ellipticine derivatives. Supported
by experimental data, the cytostatic action is discussed
as non-intercalative.
Synthesis
The N-acetyl 1,4-dihydropyridine starting compounds
5–8 were prepared from ethyl nicotinate and 3-acetyl
pyridine. First, acetylation was carried out with acetyl
chloride at low temperature (À15 ꢀC) in dried tetra-
hydrofurane. The intermediate N-acetyl pyridinium
salts were regioselectively arylated or alkylated at the 4-
position of pyridinium ring by Grignard reagents and
catalytic amounts of copper(I) iodide (Scheme 1).7
The isolated 4-substituted 1,4-dihydropyridines 5–8
were then stirred with equimolar amounts of p-benzo-
quinone in dioxane/perchloric acid (5%) at room tem-
perature. A regioselective formation of a primary
cycloaddition product tetrahydro-1-aza-9-oxafluorene 9
took place. After the cycloaddition reaction was com-
pleted, the mixture was treated with an excess of p-ben-
zoquinone leading to oxidized 1-aza-9-oxaflourenes 10–
13 as aromatic compounds besides hydroquinone
(Scheme 1). The structures of target compounds 10–13
were confirmed by IR and 1H NMR spectroscopy, mass
spectrometry and elemental analysis after their purifica-
tion by recrystallization from ethanol or by silica gel
column chromatography.
Scheme 1. Key: (i) CH3COCl, THF, À15 ꢀC; (ii) R2MgCl, CuI,
À15 ꢀC; (iii) dioxane/HClO4 (5%), rt.
Table 1. The GI50 values (mM) of compounds 10–13 to five cancer
cell-linesa
Compd SF-268 KM12 SNB-75 IGROVI NCI/ADR-RES
10
11
12
13
29.7
27.7
69.9
>100
5.1
40.8
70.326
—
16.5
2.6
2.5
5.0
4.7
19.6
86
41
—
—
—
aSRB protein cytotoxicity assay, primary screening method performed
at the National Cancer Institute, Bethesda, MD, USA, using a repor-
ted procedure.9,10
Cytostatic Activity
Cytotoxic evaluation was carried out in various cell-
lines as shown in Table 1. Highest activities in the range
of reported data for ellipticine of about GI50=1–10 mM
in severe cell-lines (KB,8 L12103) were found for the 4-
phenyl substituted compound 10 in KM12 (colon can-
cer), IGROVI (ovarian cancer) and NCI/ADR-RES
(breast cancer) with GI50=2.5–5 mM and for 11 in SNB-
75 (CNS cancer) and IGROVI with GI50=2.6–5 mM.
The 3-acetyl compound 13 was found almost inactive
with GI50>100 mM tested in just one cell-line.
Discussion and Conclusion
Obviously the 4-phenyl substituent plays an important
role in the cytostatic activity of the 1-aza-9-oxafluorenes
as a novel class of potent small-sized cytostatics. The
most decisive feature for intercalating property of
potential cytostatics is the already mentioned planar
aromatic system. The tricyclic 1-aza-9-oxafluorene scaffold,
A significant decrease in cytostatic activity was found
for the 4-methyl derivatives: compound 12 showed
relatively moderate inhibitory activity as was reported
for the alkyl chain substituted b-carboline derivatives.4

K. Brachwitz, A. Hilgeroth / Bioorg. Med. Chem. Lett. 12 (2002) 411–413
413
however, fulfils this requirement. Cytostatic activities of
the comparable tricyclic a-carboline systems in the mil-
limolar range11 are much lower than those found for the
4-phenyl-1-aza-9-oxafluorenes 10 and 11. So we ques-
tioned whether the 4-phenyl ring contributes to planar-
ity of the whole molecule. A coplanar 4-phenyl
substituent may result in an enhanced intercalation
potential of a quasi-tetracyclic aromatic system, thus
giving an explanation for the increased activity of our
compounds 10 and 11 compared to the reported tri-
cyclic a-carboline derivatives. We analyzed the 1H
NMR spectra of all target compounds 10–13 and of the
isolated tetrahydro-intermediates focusing on the che-
mical shifts of the 5-H, 7-H and 8-H: we found high-
field-shifts of the 5-H in 10 to 6.46 ppm and in 11 to
6.28 ppm compared to the resonances of 5-H in both 4-
phenyl tetrahydro-precursors 9 at 6.73and 6.66 ppm.
The resonances of 7-H and 8-H were found at higher
ppm values in 10 with 6.96 and 7.47 ppm and in 11 with
6.97 and 7.48 ppm due to the enhanced aromatic sys-
tems after complete molecule oxidation. The 5-H signals
of both 4-methyl derivatives 12 and 13 at 7.53and 7.56
ppm were found with comparably high ppm-values, like
7-H and 8-H between 7.03and 7.53ppm.
Finally, we presented a novel class of cytostatic agents.
Among the classes of tricyclic compounds they are more
potent than intercalative carboline systems. The 4-
phenyl derivatives show cytostatic activity like tetra-
cyclic ellipticine compounds. So 1-aza-9-oxafluorenes
are promising lead structures in the group of small-sized
cytostatics with potentially better bioavailability. In
present studies, we investigate the mode of cytostatic
action which may be novel within the group of tricyclic
cytostatics.
References and Notes
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5. Wieczorek, J.; Peczyska-Czoch, W.; Mordarski, M.; Kacz-
marek, L.; Nantka-Namirski, P. Arch. Immunol. Ther. Exp.
1986, 34, 315.
6. Peczyska-Czoch, W. Arch. Immunol. Ther. Exp. 1987, 35,
125.
7. Hilgeroth, A.; Baumeister, U. Angew. Chem. 2000, 112,
588. Hilgeroth, A.; Baumeister, U. Angew. Chem. Int. Ed.
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Pharm. (Weinheim) 1988, 321, 463.
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The significant highfield-shift of 5-H in 10 and 11 of more
than 1 ppm compared to the resonances of 5-H in the 4-
methyl derivatives is caused by a shielding effect of the
neighboring phenyl substituent that will consequently not
lie within the plane of the aromatic system. Otherwise the
resonances of both 5-protons would have been found at
higher ppm values. These experimental results suggest
that the novel cytostatics may not act as intercalators.