Total synthesis of isoprekinamycin: Structural evidence for enhanced diazonium ion character and growth inhibitory activity toward cancer cells

Article abstract of DOI:10.1021/ol0712374

The structurally novel diazobenzo[a]fluorene antibiotic isoprekinamycin (IPK) has been synthesized for the first time employing a Suzuki coupling of a brominated AB ring synthon with a boronate ester representing the D ring, followed by anionic cyclizatio

Full text of DOI:10.1021/ol0712374

ORGANIC
LETTERS
2007
Vol. 9, No. 15
2915-2918
Total Synthesis of Isoprekinamycin:
Structural Evidence for Enhanced
Diazonium Ion Character and Growth
Inhibitory Activity toward Cancer Cells
Wei Liu,† Matthew Buck,† Nan Chen,† Muhong Shang,† Nicholas J. Taylor,†,‡
Jalil Asoud,† Xing Wu,§ Brian B. Hasinoff,§ and Gary I. Dmitrienko*,†
Department of Chemistry, UniVersity of Waterloo, Waterloo,
Ontario, Canada, N2L 3G1, and Faculty of Pharmacy,
UniVersity of Manitoba, Winnipeg, Manitoba, Canada, R3T 2N2
dmitrien@uwaterloo.ca
Received May 31, 2007
ABSTRACT
The structurally novel diazobenzo[a]fluorene antibiotic isoprekinamycin (IPK) has been synthesized for the first time employing a Suzuki
coupling of a brominated AB ring synthon with a boronate ester representing the D ring, followed by anionic cyclization and appropriate
functional group manipulations. The first indication that the diazobenzo[a]fluorene system exhibits in vitro anticancer activity is provided and
X-ray crystallographic evidence for enhancement of diazonium ion character as a consequence of intramolecular H-bonding is described.
The kinamycins, first believed to be N-cyanobenzo[b]-
carbazoles 1,1 but now known to be diazobenzo[b]fluorenes
2,2,3 are of current interest because of their antibacterial
activity and an in vitro cytotoxicity profile against cancer
cells suggestive of a mode of action different than that of
anticancer agents in current clinical use.4 The discovery of
the lomaiviticins 6, which are dimeric analogues of the
kinamycins possessing potent cytotoxicity against a range
of cancer cell lines, has heightened interest in these unusual
natural products.5 A number of synthetic strategies have been
disclosed,6 most notably a total synthesis of the biosynthetic
precursor 7,7 model studies toward a total synthesis of
lomaiviticin B,8 and recent total syntheses of kinamycin C
(2C)9 and its methyl ether.10
Isoprekinamycin (IPK), first assigned structure 3,11 is now
recognized to be the diazobenzo[a]fluorene 4.12 The previ-
(5) He, H.; 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, 5362-5363.
* Address correspondence to this author.
† University of Waterloo.
(6) Gould, S. J. Chem. ReV. 1997, 97, 2499-2509.
‡ Deceased, November 2006.
(7) (a) Hauser, F.; Zhou, M. J. J. Org. Chem. 1996, 61, 5722-5723. (b)
Birman, V. B.; Zhao, Z.; Guo, L. Org. Lett. 2007, 9, 1223-1225.
(8) Nicolaou, K. C.; Denton, R. M.; Lenzen, A.; Edmonds, D. J.; Li, A.;
Milburn, R. R.; Harrison, S. T. Angew. Chem., Int. Ed. 2006, 45, 2076-
2081.
§ University of Manitoba.
(1) Omura, S.; Nakagawa, A.; Yamada, H.; Hata, T.; Furusaki, A. Chem.
Pharm. Bull. 1973, 21, 931-940.
(2) Gould, S. J.; Tamayo, N.; Melville, C. R.; Cone, M. C. J. Am. Chem.
Soc. 1994, 116, 2207-2208.
(9) Lei, X.; Porco, J. A., Jr. J. Am. Chem. Soc. 2006, 128, 14790-14791.
(10) Kumamoto, T.; Kitani, Y.; Tsuchiya, H.; Yamaguchi, K.; Seki, H.;
Ishikawa, T. Tetrahedron 2007, 63, 5189-5199.
(11) Seaton, P. J.; Gould, S. J. J. Antibiot. 1989, 42, 189-197.
(12) Proteau, P. J.; Li, J.; Williamson, T. R.; Gould, S. J.; Laufer, R. S.;
Dmitrienko, G. I. J. Am. Chem. Soc. 2000, 122, 8325-8326.
(3) Mithani, S.; Weeratunga, G.; Taylor, N. J.; Dmitrienko, G. I. J. Am.
Chem. Soc. 1994, 116, 2209-2210.
(4) Hasinoff, B. B.; Wu, X.; Yalowich, J. C.; Goodfellow, V.; Laufer,
R. S.; Adedayo, O.; Dmitrienko, G. I. Anticancer Drugs 2006, 17, 825-
837.
10.1021/ol0712374 CCC: $37.00
© 2007 American Chemical Society
Published on Web 06/22/2007

Scheme 1. Synthesis of a Simplified Model of IPK
Figure 1. Structures of the kinamycins, lomaiviticin, isopreki-
namycin, and fluostatins.
ously rare benzo[a]fluorene class of natural products now
boasts several other members (e.g., 8) with various degrees
of oxygenation in ring D but lacking a diazo group.13 In this
group, studies suggesting a substantial diazonium ion char-
acter in the diazo group of IPK, which might play a role in
its bioactivity, have inspired us to design a practical synthesis
of IPK.14 To date there have been no reports of the total
synthesis of any of the benzo[a]fluorene natural products.
Reported herein are the first total synthesis of IPK, structural
data supporting the proposal that intramolecular H-bonding
enhances its diazonium ion character, and the first indication
that IPK is a potent cytotoxic agent, likely representing a
variant of the same pharmacophore as the diazobenzo[b]-
fluorenes.
Our synthetic strategy assumed that a precursor 11,
incorporating the AB and D rings of IPK, might be cyclized
to the full benzo[a]fluorene system followed by selective
functional group manipulations to provide the delicate diazo
group. In practice, 10 was constructed from the commercially
available dihydrocoumarin, via the known indanone 9.15
Dibromination followed by DBU-induced dehydrobromina-
tion gave 10.
Although Suzuki coupling of 10 with arylboronic acids,
under basic conditions with Pd(PPh3)4 as catalyst, gave the
arylation products 11a-c, nonbasic conditions, as developed
by Fu and co-workers,16 with [(t-Bu)3PH]BF4, KF, and
Pd2(dba)3‚CHCl3 were needed to generate 11d in good yield
by reaction of 10 with the appropriate commercially available
pinacol boronate.
The hope that the potential competing intramolecular aldol
reaction of deprotonated 11d would be impeded by ring strain
was realized and a good yield of the benzo[a]fluorene 12
was achieved via the desired Dieckman-like reaction. Cyano
group hydrolysis to the amide 13 followed by a modified
Hoffmann rearrangement in the presence of methanol17
provided the carbamate 14. Saponification of 14 gave the
aniline 15, which was diazotized to provide the IPK model
16, the structure of which was confirmed by a single-crystal
X-ray diffraction study.
(13) (a) Akiyama, T.; Nakamura, K. T.; Takahashi, Y.; Naganawa, H.;
Muraoka, Y.; Aoyagi, T.; Takeuchi, T. J. Antibiot. 1998, 51, 586-588. (b)
Schneider, K.; Nicholson, G.; Stro¨bele, M.; Baur, S.; Niehaus, J.; Fiedler,
H.-P.; Su¨ssmuth, R. D. J. Antibiot. 2006, 59, 105-109. (c) Baur, S.; Niehaus,
J.; Karagouni, A. D.; Katsifas, E. A.; Chalkou, K.; Meintanis, C.; Jones,
A. L.; Goodfellow, M.; Ward, A. C.; Beil, W.; Schneider, K.; Su¨ssmuth,
R. D.; Fiedler, H.-P. J. Antibiot. 2006, 59, 293-297. (d) Fondja, C. B.;
Schiebel, M.; Helmke, E.; Anke, H.; Laatsch, H. Z. Naturforsch. 2006, 61b,
320-325.
Adaptation of this approach to the preparation of IPK
required access to an appropriate arylboronate, to serve as a
D-ring synthon. The known o-iodobenzylic alcohol 1715,18
(16) Netherton, M. R.; Fu, G. C. Org. Lett. 2001, 3, 4295-4298.
(17) Moriarity, R. M.; Chany, C. J.; Vaid, R. K.; Prakash, O.; Tuldhar,
S. M. J. Org. Chem. 1993, 58, 2478-2482.
(14) Laufer, R. S.; Dmitrienko, G. I. J. Am. Chem. Soc. 2002, 124, 1854-
1855.
(15) See the Supporting Information for details.
(18) Qabaja, G.; Jones, G. B. Tetrahedron Lett. 2000, 41, 5317-5320.
2916
Org. Lett., Vol. 9, No. 15, 2007

was converted to the nitrile 18, and then to the target boronate
19 via a palladium-induced coupling with pinacol borane.19
Suzuki coupling of 19 and 10 to give 20 proceeded well
but, very disappointingly, attempted cyclization proceeded
contrary to the favorable results of the model study and
provided the aldol product 22 almost exclusively, clearly
indicating the subtle energetics associated with the interaction
of the D-ring methoxy group with the carbomethoxy group
and with the keto group.
24, which was treated with LDA in situ. Aqueous workup
provided a mixture of the cyclized product 25 and the ketone
21, formed by partial air oxidation. The most efficient route
to amide 27 involved O-methylation of the mixture of 21
and 25 and hydrolysis of the cyano group to the amide with
H2O2/K2CO3, which also led to oxidation to the ketone. The
synthesis then paralleled that of 16 to give 30, which upon
demethylation with BCl3 gave IPK.15,22
Slow crystallization of IPK provided crystals for an X-ray
diffraction study that confirms the assigned structure.12
Comparison of structures of model 16 and IPK reveals
increased diazonium ion character in IPK as evidenced by
the alternating shorter N-N (1.104 versus 1.116 Å), longer
C5-N (1.362 versus 1.334 Å), shorter C5-C6 (1.424 versus
1.457 Å) ,and longer C6-O (1.246 versus 1.227 Å) bond
lengths which are entirely consistent with an increased
diazonium ion contribution to the resonance hybrid repre-
senting the o-quinodiazide functionality in IPK relative to
that 16.14
Scheme 2. Total Synthesis of Isoprekinamycin
The increased availability of IPK opens the door to more
extensive biological studies. We have now found that IPK,
previously shown to exhibit modest antibacterial activity,11
significantly inhibits the growth of CHO (IC50 ) 5.8 µM)
and K562 human leukemia cells (IC50 ) 6.4 µM).15,23
Superimposition of the crystal structure or a computed
model of 4 on a model of 7 reveals that the oxygen and
Figure 2. Structural comparison of the diazobenzo[a]fluorene and
the diazobenzo[b]fluorene systems.
nitrogen atoms on the periphery align well despite the
rearranged carbon skeleton, suggesting that the diazobenzo-
[a]- and diazobenzo[b]fluorenes are variants of the same
To avoid the aldol reaction, 20 was reduced with boro-
hydride but cyclization of 23 gave the desired Claisen product
21 and the aldol product 22 in low yields, likely because of
regeneration of the keto group at the expense of degradation
of the starting material.
(20) Lithium trialkylaluminumhydrides are known to reduce ketones, but
the concomitant use of the trialkylaluminate for protection of the alcohol,
as in 26, is new and offers advantages over trialkylsilyl protection when
the alcohol is base-sensitive. Attempts at silylation of the alcohol 23 or its
conjugate base led to oxidation and complex byproducts.
(21) Kim, S.; Ahn, K. H. J. Org. Chem. 1984, 49, 1717-1724.
(22) The synthesis of IPK is also a formal synthesis of prefluostatin (ref
13d) since we have generated it previously from natural IPK (ref 14).
(23) The clinically useful anticancer agent etoposide exhibits IC50 ) 1.4
and 3.4 µM, respectively, in the same assays.
Reduction of 20, using a mixture of n-butyllithium and
DIBAL at low temperature,20,21 gave the trialkylaluminate
(19) Baudoin, O.; Gue´nard, D.; Gue´ritte, F. J. Org. Chem. 2000, 65,
9268-9271.
Org. Lett., Vol. 9, No. 15, 2007
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pharmacophore. Consistent with this hypothesis is our finding
that IPK-diacetate 5, which is more soluble than IPK, inhibits
the topoisomerase IIR-catalyzed decatenation of kDNA (IC50
) 9.7 µM) just as we observed recently for 2A and 2C (IC50
) 43 and 3.2 µM, respectively).4,15,24
The ease of synthesis of IPK relative to the kinamycins
and lomaiviticins makes the diazobenzo[a]fluorene system
attractive for generation of synthetic analogues. Efforts to
create other benzo[a]fluorenes and congeners of IPK with
improved solubility and drug-like properties are in progress.
of M. Buck, University of Waterloo, 2003. We thank Dr. O.
Adedayo and V. Goodfellow (University of Waterloo) for
isolation of natural IPK and assistance with HPLC experi-
ments. The Canadian Institutes for Health Research (CIHR)
and the Natural Sciences and Engineering Research Council
of Canada (NSERC) are thanked for support of this research
by means of a Canada Research Chair to B.B.H. and an
NSERC Discovery Grant to G.I.D., respectively
Supporting Information Available: Full experimental
details for all reactions and bioassays and CIF files for IPK
and 16. This material is available free of charge via the
Internet at http://pubs.acs.org.
Acknowledgment. Dedicated to the memory of Dr.
Nicholas Justin Taylor. Taken, in part, from the M.Sc. thesis
(24) Unlike etoposide, 5 and the kinamycins inhibit topoisomerase IIR
but do not act as topoisomerase II poisons (ref 4).
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