Mimicking the biological activity of diazobenzo[b]fluorene natural products with electronically tuned diazofluorene analogs

Article abstract of DOI:10.1016/j.bmcl.2006.07.024

Under appropriate electronic modulation, simple diazofluorene analogs recapitulate the DNA cleavage activity of kinamycin D under thiol-based reducing conditions. Achieving DNA cleavage under these reducing conditions is key to anticancer activity, as the most active compound, 1-methoxydiazofluorene, inhibits the proliferation of HeLa cells.

Full text of DOI:10.1016/j.bmcl.2006.07.024

Bioorganic & Medicinal Chemistry Letters 16 (2006) 5148–5151
Mimicking the biological activity of diazobenzo[b]fluorene
natural products with electronically tuned diazofluorene analogs
Wei Zeng, T. Eric Ballard, Alexander G. Tkachenko,
Virginia A. Burns, Daniel L. Feldheim and Christian Melander*
Department of Chemistry, North Carolina State University, Raleigh, NC 27695-8204, USA
Received 1 July 2006; accepted 6 July 2006
Available online 25 July 2006
Abstract—Under appropriate electronic modulation, simple diazofluorene analogs recapitulate the DNA cleavage activity of
kinamycin D under thiol-based reducing conditions. Achieving DNA cleavage under these reducing conditions is key to anticancer
activity, as the most active compound, 1-methoxydiazofluorene, inhibits the proliferation of HeLa cells.
Ó 2006 Elsevier Ltd. All rights reserved.
The kinamycins are a family of microbial metabolites
that are characterized by an uncommon diazo-
benzo[b]fluorene skeleton.1,2 In addition to their atypical
architecture, they have been reported to possess both
antitumor and antibiotic activities.3,4 Interest in this
class of natural products has been further enhanced by
the recent disclosure of lomaiviticin A by researchers
at Wyeth-Ayerst.5 Lomaiviticin A is a glycosylated
homodimeric diazobenzo[b]fluorene that was reported
to possess potent anticancer activity against a wide
range of cancer types (0.01–98 ng/mL), as well as activ-
Figure 1. Kinamycins A–D and lomaiviticin A.
ity against Gram-positive bacteria.5 Lomaiviticin A was
reported to cleave DNA under reducing conditions, and
its cytotoxicity profile in the 24-cancer cell line panel was
unique when compared to known DNA damaging
agents (adriamycin and mitomycin C)5 (Fig. 1).
ing ability of a diazobenzo[b]fluorene natural product
under reducing conditions. All previously reported cases
have relied upon photochemical6,7,9,10 or oxidative8 acti-
vation of the diazo group. Since the reactivity of a diazo
compound is dictated by its electronic properties,11 we
posited that electronically tuned diazofluorene analogs
would demonstrate similar cleavage activity as a dia-
zobenzo[b]fluorene natural product, and that this activ-
ity would translate into antiproliferative activity. To test
this hypothesis, we synthesized a range of diazofluorene
analogs (Fig. 2) that were under differential electronic
modulation, and compared their DNA cleaving activity
to that of kinamycin D. Compounds that recapitulated
the DNA cleavage activity of kinamycin D were then
assayed for antiproliferative activity against HeLa cells.
We were attracted to this class of natural products
because of its ability to mediate DNA cleavage under
reducing conditions. Given this biological activity, and
that reducing conditions are prevalent in intracellular
space, there is potential to harness synthetic diazo com-
pounds that cleave DNA under reducing conditions as
cancer chemotherapeutics. There have been a number
of reports that document the DNA cleavage activity of
synthetic, diazo-based compounds;6–10 however, none
of these compounds have recapitulated the DNA cleav-
Keywords: Kinamycins; Lomaiviticins; DNA cleavage; Diazofluorenes;
Antiproliferative activity.
The diazofluorene parent was synthesized as previously
described.12 Our synthesis of the 1-substituted and
4-substituted diazofluorenes is outlined in Scheme 1.
*
Corresponding author. Tel.: +1 919 513 2960; fax: +1 919 515
5079; e-mail: Christian_Melander@NCSU.edu
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.07.024

W. Zeng et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5148–5151
5149
To access either the 1- or 4-nitrofluorenone, we oxidized
1- or 4-aminofluorenone with Tf2O/H2O2.13 The chloro-
fluorenones were synthesized from the respective amino-
fluorenone under Sandmeyer conditions (NaNO2/HCl/
CuCl/H2O),14 while the methoxyfluorenone derivatives
were synthesized by converting the aminofluorenones
to hydroxyfluorenones (NaNO2, H2SO4, H2O)15 fol-
lowed by methylation (either NaH/MeI or NaOH/
Me2SO4). Finally, the diazo group was installed by
either: (1) condensing the fluorenone with hydrazine fol-
lowed by oxidation with mercuric oxide, or (2) condens-
ing the fluorenone with tosyl hydrazine followed by
elimination of the tosyl group under basic conditions.
The 1,4-substituted compounds were synthesized as out-
lined in Scheme 2. Briefly, 1,4-dimethoxybenzene and
2-iodobenzoic acid were coupled under the action of
triflic anhydride.16 Pd(II)-mediated coupling then
delivered 1,4-dimethoxyfluorenone (11) for further elab-
oration.16 To access the 1,4-dimethoxydiazofluorene 9,
11 was condensed with tosyl hydrazine and subjected
to base-mediated elimination with sodium methoxide.
To access the 1,4-diazoquinone derivative 10, 11 was
Figure 2. Diazofluorene probes.
17
quantitatively demethylated with BBr3 and condensed
with hydrazine. The resulting crude hydrazone was
treated with Fetizon’s reagent (Ag2CO3/celite),17 which
affected the tandem oxidation of the hydrazone and
1,4-hydroquinone to the target quinone 10.
First, we established the conditions under which kina-
mycin D-mediated DNA cleavage. There have been a
limited number of reports that address the mechanism
of action (MOA) of the diazobenzo[b]fluorenes. Lomai-
viticin A was reported to cleave DNA under reducing
conditions;5 however, no experimental details were doc-
umented. Using the diazobenzo[a]fluorene isoprekina-
mycin as a model substrate, Laufer and Dmitrienko18
postulated that the electrophilicity of the diazo group
would mediate formation of a nucleophilic adduct that
would subsequently decompose to form a diradical
and induce DNA damage (Scheme 3A). Using prekina-
mycin as their model substrate and guided by the estab-
lished biological chemistry of other quinone natural
products,19 Feldman and Eastman20 have postulated
that bioreduction of the quinone to a hydroquinone
would destabilize the diazo moiety, resulting in decom-
position to a carbon-based radical with concomitant
release of N2 (Scheme 3B).
Scheme 1. Reactions and conditions: (a) NH2NHTs, EtOH, HCl,
reflux; (b) NaOMe, MeOH, reflux; (c) NaNO2, H2SO4, H2O; (d) NaH,
DMF then MeI; (e) NH2NHTs, THF, HCl, reflux; (f) NaNO2, HCl,
CuCl, H2O; (g) N2H4, n-butanol, reflux; (h) HgO, toluene, reflux; (i)
Tf2O, H2O2, CH2Cl2; (j) 50% NaOH, dioxane, 50 °C; (k) NaOH,
Me2SO4, EtOH, reflux.
Scheme 2. Reactions and conditions: (a) Tf2O, TFA, reflux; (b)
Pd(OAc)2, NaOAc, DMA, 125 °C; (c) NH2NHTs, THF, HCl, reflux;
(d) NaOMe, MeOH, reflux; (e) BBr3, CH2Cl2; (f) N2H4, EtOH, reflux;
(g) Fetizon’s reagent (Ag2CO3 on celite), TEA, CH2Cl2.

5150
W. Zeng et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5148–5151
Figure 4. Plasmid cleavage assay, top band is type II nicked DNA, the
bottom band is type I supercoiled DNA. Conditions: all lanes contain
714 ng of plasmid DNA, final [DTT] = 1.0 M, final [com-
pound] = 1.0 mM. Lane 1, 2; lane 2, 9; lane 3, 5; lane 4, 10; lane 5,
kinamycin D; lane 6, DNA only.
Scheme 3. Diazo decomposition. (A) Nucleophile-mediated; (B)
reduction-mediated.
In the latter two cases, extrapolation of the reported
reaction conditions to a cellular environment remains
ambiguous. Dmitrienko utilized b-naphthol as a nucleo-
phile in THF,18 while Feldman employed AIBN/
Bu3SnH in refluxing benzene as the source of a one-elec-
tron reductant.20 Neither mechanistic study addressed
DNA cleavage.
Figure 5. Plasmid cleavage assay, top band is type II nicked DNA, the
bottom band is type I supercoiled DNA. Conditions: all lanes contain
714 ng of plasmid DNA. Final [compound] = 1.0 mM. Lane 1, DNA
only; lane 2, 2/DNA; lane 3, 5/DNA; lane 4, 0.5 M NADPH/DNA;
lane 5, 2, 0.5 M NADPH/DNA; lane 6, 1.0 M NaCN/DNA; lane 7, 2,
1.0 M NaCN/DNA.
Based upon these two hypotheses, we explored kinamy-
cin D cleavage of the supercoiled plasmid pBR322 under
a variety of nucleophilic and reductive conditions. Kina-
mycin D was first incubated with pBR322 (Fig. 3, lane
2), and no DNA cleavage was noted, even after 3 days.
Kinamycin D was then incubated with pBR322 and
either dithiothreitol (DTT), nicotinamide adenine dinu-
cleotide phosphate (NADPH), or sodium cyanide
(NaCN). Only in the presence of DTT (Fig. 3, lane 4)
did we observe any appreciable DNA damage. We also
conducted a time course of kinamycin D-mediated
DNA cleavage in the presence of DTT (data not shown)
and noted maximal cleavage after 2 days.
absence of DTT. Under these conditions, 4-aminodiazo-
fluorene 5 retained DNA cleavage activity, while
1-methoxydiazofluorene 2 displayed minimal activity.
1-Methoxydiazofluorene 2 did not cleave DNA in the
presence of either NaCN or NADPH (Fig. 5). We also
confirmed (data not shown) that the diazo group was
essential for DNA cleavage activity by assaying for the
ability of 1-methoxyfluorenenone and 4-aminofluore-
none to induce DNA damage. Neither compounds pos-
sessed DNA cleavage activity.
We quantitated the extent of conversion from type I
supercoiled DNA to type II nicked DNA for each com-
pound. This is summarized in Table 1. In the presence of
DTT, 1-methoxydiazofluorene 2 exhibited 150% of the
cleavage potential of kinamycin D, while 4-aminodiazo-
fluorene 5 exhibited 64% activity in comparison to
kinamycin D. Interestingly, 4-aminodiazofluorene 5
exhibited increased cleavage activity in the absence of
DTT. We are conducting extensive mechanistic studies
to delineate the differential reactivity between 1-meth-
oxydiazofluorene 2 and 4-aminodiazofluorene 5.
Under these optimized conditions (DNA, DTT, 2 days),
we assayed each of our diazofluorene derivatives for
their ability to cleave DNA. Despite our best efforts,
1-aminodiazofluorene was unstable and decomposed
(even storing at À80 °C). We observed that the 1-meth-
oxydiazofluorene and the 4-aminodiazofluorene deriva-
tives cleaved DNA with similar efficiency as kinamycin
D (Fig. 4, lanes 1 and 3, compounds 9 and 10 are includ-
ed for reference). All other diazo compounds, including
the diazofluorene parent, did not induce extensive dam-
age to the supercoiled plasmid.
Table 1. Quantitation of DNA cleavage (%) by kinamycin D and
diazofluorene analogs
Next, we assayed for the ability of 1-methoxydiazofluo-
rene 2 and 4-aminodiazofluorene 5 to cleave DNA in the
Compound
No promoter
DTT
Kinamycin D
Diazo parent
<5
<5
nd
28
<5
nd
41
<5
<5
18
13
<5
<5
7
6
1-Aminodiazofluorene (1)
1-Methoxydiazofluorene (2)
1-Chlorodiazofluorene (3)
1-Nitrodiazofluorene (4)
4-Aminodiazofluorene (5)
4-Methoxydiazofluorene (6)
4-Chlorodiazofluorene (7)
4-Nitrodiazofluorene (8)
1,4-Dimethoxydiazofluorene (9)
Diazoquinofluorene (10)
11
9
7
<5
<5
44
Figure 3. Plasmid cleavage assay, top band is type II nicked DNA, the
bottom band is type I supercoiled DNA. Conditions: all lanes contain
714 ng of plasmid DNA. Lanes 2, 4, 6, and 8, final [kinamycin
D] = 1.0 mM. Lane 1, DNA; lane 2, kinamycin D + DNA; lane 3,
DNA/DTT (1.0 M); lane 4, kinamycin D + DNA/DTT (1.0 M); lane 5,
DNA/NADPH (0.5 M); lane 6, kinamycin D + DNA/NADPH
(0.5 M); lane 7, DNA/NaCN (1.0 M); lane 8, kinamycin D + DNA/
NADPH (0.5 M).
8
6
<5
<5
<5
<5
<5
9
10
8
9
nd, not determined.

W. Zeng et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5148–5151
5151
Finally, we have assayed 1-methoxydiazofluorene 2, 4-
aminodiazofluorene 5, and the diazo parent for antipro-
liferative activity against HeLa cells. We chose this com-
pound set to provide validation that achieving DNA
cleavage under reducing conditions was the key to
achieving antiproliferative activity. Previous studies
have shown that the diazofluorene parent possesses
DNA cleavage activity; however, it is only active under
oxidizing conditions (Cu2+).8 Therefore, this set allows
us to assay compounds that achieve maximal DNA
cleavage under disparate reaction conditions: thiol-
mediated reducing conditions (diazofluorene 2), no
promoter (diazofluorene 5), and oxidizing conditions
(diazofluorene parent). All antiproliferative studies were
conducted at 100 nM, the solubility limit of the synthetic
diazo compounds in cell culture media. Although IC50
values could not be established due to this limited solu-
bility, we did observe that 1-methoxydiazofluorene 2
had the highest activity of the three diazo compounds.
1-Methoxydiazofluorene 2 demonstrated a time-depen-
dent inhibition of HeLa cell proliferation and inhibited
cell growth 35–40% at 12 h. This activity is similar to
the antiproliferative activity of the most active
kinamycins.3
NCSU Facility was obtained from the North Carolina
Biotechnology Center and the NSF. The authors also
thank Professor Phil Proteau (Oregon State University)
for providing authentic samples of kinamycin D.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2006.07.024.
References and notes
1. Marco-Contelles, J.; Molina, M. T. Curr. Org. Chem.
2003, 7, 1433.
2. Gould, S. J. Chem. Rev. 1997, 97, 2499.
3. Hata, T.; Omura, S.; Iwai, Y.; Nakagawa, A.; Otani, M.;
Ito, S.; Matsuya, T. J. Antibiot. 1971, 24, 353.
4. Ito, S.; Matsuya, T.; Omura, S.; Otani, M.; Nakagawa, A.;
Takeshim, H.; Iwai, Y.; Ohtani, M.; Hata, T. J. Antibiot.
1970, 23, 315.
5. He, H. T.; Ding, W. D.; Bernan, V. S.; Richardson, A. D.;
Ireland, C. M.; Greenstein, M.; Ellestad, G. A.; Carter, G.
T. J. Am. Chem. Soc. 2001, 123, 5362.
6. Maiya, B. G.; Ramana, C. V.; Arounaguiri, S.; Nagarajan,
M. Bioorg. Med. Chem. Lett. 1997, 7, 2141.
7. Nakatani, K.; Maekawa, S.; Tanabe, K.; Saito, I. J. Am.
Chem. Soc. 1995, 117, 10635.
8. Arya, D. P.; Jebaratnam, D. J. J. Org. Chem. 1995, 60,
3268.
9. Nielsen, P. E.; Jeppesen, C.; Egholm, M.; Buchardt, O.
Nucleic Acids Res. 1988, 16, 3877.
10. Eppley, H. J.; Isada, V. J.; Lato, S. M.; Huffman, J. C.;
Ellington, A. D.; Zaleski, J. M. J. Inorg. Biochem. 1999,
74, 124.
In conclusion, we have successfully identified 1-meth-
oxydiazofluorene 2 as a simplified diazo compound that
recapitulates the DNA cleaving activity of kinamycin D
under thiol-mediated conditions. In addition, we have
demonstrated that achieving DNA cleavage activity
under thiol-mediated conditions translates into antipro-
liferative activity, and we have identified simple diazo
compounds that have similar anticancer activity as the
structurally complex kinamycins. We are currently
working toward improving solubility and incorporating
these structural elements into simplified lomaiviticin A
analogs, and will report on their antiproliferative activ-
ity shortly.
11. Patai, S. The Chemistry of Diazonium and Diazo Groups; J.
Wiley, Chichester [Eng.]: New York, 1978.
12. Schonberg, A.; Awad, W.; Latif, N. J. Chem. Soc. 1951,
1368.
13. Manka, J. T.; Guo, F. L.; Huang, J. P.; Yin, H. Y.; Farrar,
J. M.; Sienkowska, M.; Benin, V.; Kaszynski, P. J. Org.
Chem. 2003, 68, 9574.
Acknowledgments
14. Tilly, D.; Samanta, S. S.; Faigl, F.; Mortier, J. Tetrahedron
Lett. 2002, 43, 8347.
15. Lukeman, M.; Wan, P. J. Am. Chem. Soc. 2002, 124, 9458.
16. Qabaja, G.; Jones, G. B. J. Org. Chem. 2000, 65, 7187.
17. Williams, W.; Sun, X.; Jebaratnam, D. J. Org. Chem.
1997, 62, 4364.
18. Laufer, R. S.; Dmitrienko, G. I. J. Am. Chem. Soc. 2002,
124, 1854.
19. Moore, H. W. Science 1977, 197, 527.
20. Feldman, K. S.; Eastman, K. J. J. Am. Chem. Soc. 2005,
127, 15344.
The authors thank North Carolina State University for
funding, NMR, and mass spectrometry. A.G.T. and
D.L.F. thank the W. M. Keck foundation and The Keck
Center for RNA-Mediated Evolutionary Materials
Chemistry at N.C. State University for partial support
of this work. D.L.F. also acknowledge the National
Institutes of Health for partial support of this work
(CA098194). Mass spectra were obtained at the Mass
Spectrometry Laboratory for Biotechnology at North
Carolina State University. Partial funding for the