Diazo group electrophilicity in kinamycins and lomaiviticin A: Potential insights into the molecular mechanism of antibacterial and antitumor activity

Article abstract of DOI:10.1021/ja0167809

Theoretical and chemical studies of the reactivity of isoprekinamycin, the kinamycins, and the lomaiviticins support the proposal that these natural products exhibit enhanced diazonium salt character and may owe their antitumor antibiotic properties to th

Full text of DOI:10.1021/ja0167809

Published on Web 02/08/2002
Diazo Group Electrophilicity in Kinamycins and Lomaiviticin A: Potential
Insights into the Molecular Mechanism of Antibacterial and Antitumor Activity
Radoslaw S. Laufer and Gary I. Dmitrienko*
Department of Chemistry, UniVersity of Waterloo, Waterloo, Ontario, Canada, N2L 3G1
Received August 7, 2001
Scheme 1 a
Microbial secondary metabolism has yielded a wealth of
compounds with unusual structures and novel modes of action as
antimicrobial and anticancer agents.1 In this context, the structural
novelty of the diazobenzo[b]fluorene ring system assigned to the
kinamycins, 1,2 and the diazobenzo[a]fluorene structure assigned
to isoprekinamycin, 2,3 is of considerable interest. This interest is
heightened by the very recent report of the related lomaiviticin A,
5, which exhibits very potent activity against a broad spectrum of
cancer cell lines and against Gram positive bacteria.4
a (a) HN(i-Pr)2, Et2O, rt. (b) (1) s-BuLi, TMEDA, THF, -78 °C. (2)
B(OMe)3, -78 °C f rt. (c) (1) n-BuLi, THF, rt. (2) BrCH2CH2Br, -78 °C
f rt. (d) Pd(PPh3)4, 2 M aq Na2CO3, DME, Vv. (e) NBS, DMF, rt. (f) LDA,
THF, 0 °C f rt (85% X ) H or 49% X ) Br). (g) 48% HBr, AcOH, Vv.
Although much is known about the biosynthesis of the kinamy-
cins,5 surprisingly little information exists concerning the chemical
(h) NaBH4, MeOH, MeONa, 0 °C f rt. (i) TBSOTf, 2,6-lutidine, CH2Cl2,
0 °C f rt. (j) Pd2(dba)3‚CHCl3, BINAP, PhMe, t-BuOK, BnNH2, 80 °C.
(k) 10% Pd-C, H2 (1atm), AcOH, rt. (l) (1) NaNO2, HCl, EtOH, 0-5 °C.
(2) NaHCO3. (m) (1) n-BuLi, THF, rt. (2) B(OMe)3 -78 °C f rt. (3) 19
then step (d). (o) CH3SO3H, 65 °C.
basis for their biological activity. It has been suggested that the
diazo group in the kinamycins may serve as a precursor for carbene
intermediates in vivo and experiments which demonstrate thermally6
and photochemically7 induced cleavage of DNA by 9-diazofluo-
renes, and heterocyclic analogues in the presence of metal ions (e.g.
Cu2+) in vitro have been presented as models for a possible mode-
of-action for these antitumor antibiotics. More recently, DNA
cleavage by 5 under reductive conditions has been observed but
the details of these studies have yet to be disclosed.4
We report herein an enhanced reactivity of the diazo group in
these natural products that may play a role in their antitumor and
antibacterial activities. In particular, we disclose the synthesis of 4
and a comparison of its susceptibility to nucleophilic attack with
that of isoprekinamycin, 2, which indicates that this natural product
is rendered more diazonium ion-like by virtue of the intramolecular
H-bonding network present. Furthermore, we report the results of
ab initio molecular orbital calculations which support not only the
notion that 2 might act in vivo as a diazonium-like species but
also that related and still greater diazonium ion-like character should
be expected for the kinamycins, 1, and lomaiviticin A, 5.
benzylamine11 also resulted in desilylation and oxidation to give
ketone 17. Hydrogenolysis gave 18, which underwent diazotization
and demethylation with HNO2 to form 4.
The problem arising from the poor nucleophilicity of the benzo-
[a]fluorenone ring was solved better by electrophilic substitution
prior to cyclization. The amide 11 was brominated smoothly at C-5
(NBS/DMF), but the strong base used for anionic cyclization caused
debromination of 12. Thus, the Suzuki cross coupling strategy was
modified to allow us to produce a brominated biaryl system suitable
for cyclization under Friedel-Crafts rather than anionic conditions.
Reaction of o-deprotonated 2-methoxynaphthalene with trimethyl
borate gave the corresponding dimethyl aryl boronate, which reacted
smoothly with ethyl 2-bromobenzoate in situ under Keay’s modified
Suzuki conditions12 to give the biaryl 20. Bromination with NBS
followed by cyclization in methanesulfonic acid gave the bromi-
nated benzo[a]fluorenenone 22 in 91% yield. Amination produced
17, which was converted into 4 as before.
Our synthetic strategy to the diazobenzo[a]fluorene 48 was based
on a Suzuki coupling.9,10 Pd-catalyzed coupling of boronic acid 8
and aryl bromide 10 afforded the biaryl 11. Anionic cyclization of
11 and demethylation gave 14. Introduction of the diazo group
proved to be problematic. Neither diazo transfer nor bromination
could be effected at C-5 of 14, presumably because of the electron-
withdrawing effect of the keto group. Borohydride reduction of 13
followed by silylation yielded 15, which was brominated smoothly
at C-5 with NBS/DMF. Palladium-catalyzed amination of 16 with
With the model 4 in hand, we explored its properties in
comparison with those of isoprekinamycin, 2, in the hope that
insights into the chemical properties of 2, of importance to the
mode-of-action of these diazo natural products, would emerge.
The available data concerning the biological activity of simpler
o- and p-quinodiazides13 and the related aryldiazonium ions14,15
suggests that such compounds elicit some of their biological effects
by chemical modification of nucleic acids by mechanisms involving
aryl radicals. In the case of aryl diazonium ions, nucleophilic attack
9
1854 VOL. 124, NO. 9, 2002 J. AM. CHEM. SOC.
10.1021/ja0167809 CCC: $22.00 © 2002 American Chemical Society

C O M M U N I C A T I O N S
Scheme 2 a,21
at the terminal nitrogen of the diazonium group is an obligatory
step prior to radical formation by loss of N2.16
Ar-N2+ + :Nu- a [Ar-NdN-Nu] f Ar• + N2 + •Nu
In the reactions of aryldiazonium ions with DNA, radical
formation is preceded by nucleophilic attack by the C-2 amino group
of guanine residues and by the C-6 amino group of adenine to form
labile triazenes. Such intermediates, which are isolable in some
cases,17 decompose with loss of N2 to form aryl radicals which
combine with the purine ring radical to form C-8 aryl purines and
also react in other ways with the initially modified DNA molecule
to form depurinated sites as well as to cleave the phosphodiester
backbone.
a (a) â-naphthol, Cs2CO3, THF, 0 °C f rt. (b) H2O (pH 9), EtOH, Vv.
niation and azo coupling reactions involve nucleophilic attack on
the diazo group, this observation is a clear indication of a much
higher degree of electrophilic character for the diazo group of 2.23
It is clear that much remains to be done before the modes-of-
action of the kinamycins, isoprekinamycin, and lomaiviticin A are
fully understood. We suggest, however, that recognition of the en-
hanced diazonium ion character of the diazo groups in these natural
products may be a significant step toward an understanding of the
molecular basis for their potent antitumor and antibacterial activity.
Acknowledgment. We thank V. Goodfellow and C. Beshara
for isolating 2, J. Venne for NMR studies, N. J. Taylor for X-ray
crystallographic analysis, and NSERC for financial support.
Supporting Information Available: Synthetic procedures, and ab
initio MO results (PDF) and crystallographic data for 28 (CIF). This
material is available free of charge via the Internet at http://pubs.acs.org.
Table 1. Calculated C-N2 Frequencies and N-N Bond Lengths
-1
compound
calcd ν (cm
)
calcd N−N (Å)
9-diazofluorene
1906
2056
2087
2101
2125
2139
2188
2212
2212
1.133
1.111
1.108
1.107
1.105
1.103
1.099
1.097
1.100
2,1-naphthoquinodiazide
4
24
25
2
kinamycin B (1)
23
Ph-NtN+ Cl-
References
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In this context, we were intrigued by the IR stretching frequency
assignable to the diazo group in the model compound 4 (2105
cm-1), which was 57 cm-1 lower than that for isoprekinamycin
(2) (2162 cm-1). We have found that ab initio MO calculations
reproduce this difference well (calculated ∆ν ) 52 cm-1) and reveal
a shortening of the N-N bond in 2 relative to that in 4.18
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calculated for related systems indicate a trend of increasing
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naphthoquinodiazide < 4 < 24 < 25 < 2 (Table 1). This trend
indicates that the diazonium ion character of the diazo group in 2
is enhanced both by the keto group in ring B and by the
intramolecular H-bonding network. As indicated by the higher
computed C-N2 frequency for 25 versus 24, the diazonium ion
character is influenced most strongly by the H-bond that stabilizes
the partial negative charge on the quinodiazide oxygen atom (26).
Similar calculations indicate even higher degrees of diazonium ion
character in the N-N bond in the kinamycins and in the simplified
model (23) of lomaiviticin A paralleling the trend in antitumor and
antibiotic activities (2 < 1 e 5).
Thus, we conclude that isoprekinamycin, the kinamycins, and
the lomaiviticins should be activated toward attack by nucleophiles
at the diazo group. To probe this predicted enhanced reactivity in
the case of 2, the model 4 and isoprekinamycin 2 were reacted
with â-naphthol. At room temperature in the presence of Cs2CO3
as base, 4 gave the azo adduct 27 whereas 2 gave a 1:2 mixture of
the adduct 29 and the hydrodediazonization product 30 (Scheme
2).19,20
At 0 °C, it was possible to test the relative electrophilicities of
the diazo group of 2 and that of 4. Under such conditions, complete
conversion of isoprekinamycin to a mixture of 29 and 30 was
observed in 9 h, whereas negligible reaction of 4 was observed
after 17 h.18,22 Since the mechanisms of both the hydrodediazo-
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(18) See the Supporting Information.
(19) The structure of 27 was established by X-ray analysis of the diacetate 28,
and that of 29 by spectrometric comparison with 27 (ref 18).
(20) The greater tendency of 2 to undergo hydrodediazoniation is compatible
with its higher diazonium character. Product 30 is expected to be formed
via 31 (ref 16). The higher partial positive charge on the diazo group of
2 favors reaction with the harder site (O) in â-naphthoxide. Hydrodedia-
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(pH 9), EtOH, Vv), in the absence of â-naphthoxide, likely via 32 (ref 18).
(21) Yields for small scale reactions (2-5 mg) are only approximate (ref 18).
(22) Incubation of such solutions (48 h, rt) gave 27 (ref 18).
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under hypoxic conditions (ref 18).
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