Toward enediyne mimics: Methanolysis of azoesters and a bisazoester

Article abstract of DOI:10.1021/jo990750v

Enediyne anticancer antibiotics have attracted tremendous interest in the past decade. The inherent difficulty in synthesizing these structurally complex natural products with the strained enediyne moiety has motivated a search for simpler molecules that mimic enediyne chemistry. The ultimate objective is to identify molecules that produce 1,4-benzenoid diradicals, which are known to induce DNA cleavage in the natural products. Toward this goal, several aromatic azoesters have been synthesized, and EPR reveals the presence of radical intermediates in their methanolysis. A 1,4-bisazoester has also been synthesized, and its methanolysis products have been studied by reversed-phase HPLC. The formation of 1,2-dicyanobenzene from the 1,4- bisazoester is consistent with the existence of a 1,4-diradical intermediate.

Full text of DOI:10.1021/jo990750v

5644
J . Org. Chem. 1999, 64, 5644-5649
Tow a r d En ed iyn e Mim ics: Meth a n olysis of Azoester s a n d a
Bisa zoester
Veeraraghavan Srinivasan, David J . J ebaratnam,† and David E. Budil*
Department of Chemistry, Northeastern University, Boston, Massachusetts 02115
Received May 5, 1999
Enediyne anticancer antibiotics have attracted tremendous interest in the past decade. The inherent
difficulty in synthesizing these structurally complex natural products with the strained enediyne
moiety has motivated a search for simpler molecules that mimic enediyne chemistry. The ultimate
objective is to identify molecules that produce 1,4-benzenoid diradicals, which are known to induce
DNA cleavage in the natural products. Toward this goal, several aromatic azoesters have been
synthesized, and EPR reveals the presence of radical intermediates in their methanolysis. A 1,4-
bisazoester has also been synthesized, and its methanolysis products have been studied by reversed-
phase HPLC. The formation of 1,2-dicyanobenzene from the 1,4-bisazoester is consistent with the
existence of a 1,4-diradical intermediate.
Sch em e 1
In tr od u ction
With the discovery of neocarzinostatin in 1985,1 DNA
cleavage by the so-called “enediyne” natural products has
emerged as an important research area.2 In addition to
impressive advances in the total synthesis of these
natural products,3 many structurally simpler enediyne
analogues capable of generating 1,4-benzenoid diradicals
and hence cleaving DNA under physiological conditions
have also been reported.4 A few years ago, intrigued by
the fact that most attempts to generate 1,4-benzenoid
diradicals have relied on enediyne precursors (Scheme
1, approach 1),5 we initiated research on a nonenediyne
strategy (Scheme 1, approach 2). Our work has identified
several new classes of compounds,6-9 including azoesters
that cleave DNA under physiologically relevant condi-
tions.7,8
One question that remains to be answered is the extent
to which such compounds actually do mimic the mech-
anism of DNA cleavage by enediynes. Kosower’s observa-
tion that azoesters cause membrane damage in cells that
is attributable to phenyl radicals10 has led us to speculate
that radical intermediates might also be involved in the
observed cleavage of DNA by azoesters. However, we
have obtained no direct, independent evidence for the
existence of radical intermediates in the DNA-cleavage
reaction.
* To whom correspondence should be addressed. Phone: (617) 373-
2369. Fax: (617) 373-8795. E-mail: dbudil@neu.edu.
† Original principal investigator.
(1) Edo, K.; Mizugaki, M.; Koide, Y.; Seto, H.; Furihata, K.; Otake,
N.; Ishida, N. Tetrahedron Lett. 1985, 26, 331.
(2) For recent reviews, see: (a) Borders, D. B., Doyle, T. W., Eds.
Enediyne Antibiotics As Antitumor Agents; Marcel Dekker Inc.: New
York, 1995. (b) Sugiyama, H.; Yamashita, K.; Fujiwara, T.; Saito, I.
Tetrahedron 1994, 50, 1311.
Even if radicals are involved in azoester cleavage of
DNA, the probability is very low for two C-Y bonds (cf.
Scheme 1) to break simultaneously. In other words,
compounds represented by 1 (Scheme 1) are presumed
to generate monoradicals 2 first, rather than generating
the diradicals 3 directly; thus, they are not strictly
analogous to enediyne sytems, which generate 1,4-
benzenoid diradicals in a concerted fashion. On the other
hand, DNA cleavage itself is almost certainly not a
concerted reaction and should not depend on whether the
1,4 benzenoid diradical involved was generated via a
concerted mechanism. Therefore, it should be possible to
mimic the action of enediynes by generating a diradical
through sequential formation of two radical sites. This
(3) (a) Danishefsky, S. J .; Shair, M. D. J . Org. Chem. 1996, 61, 16.
(b) Myers, A. G.; Fraley, M. E.; Tom, N. J .; Cohen, S. B.; Madar, D. J .
J . Chem. Biol. 1995, 2, 33. (c) Hitchcock, S. A.; Boyer, S. H.; Chu-
Moyer, M. Y.; Olson, S. H.; Danishefsky, S. J . Angew. Chem., Int. Ed.
Engl. 1994, 33, 858. (d) Nicolaou, K. C.; Hummel, C. W.; Pitsinos, E.
N.; Nakada, M.; Smith, A. L.; Shibayama, K.; Saimoto, H. J . Am. Chem.
Soc. 1992, 114, 8082. (e) Nicolaou, K. C. Angew. Chem., Int. Ed. Engl.
1993, 32, 1377.
(4) For some notable examples, see: (a) Nicolaou, K. C.; Dai, W.
M.; Tsay, S. C.; Estevez, V. A.; Wrasidlo, W. Science 1992, 256, 1172.
(b) Semmelhack, M. F.; Gallagher, J . J .; Ding, W. D.; Krishnamurthy,
G.; Babine, R.; Ellestad, G. A. J . Org. Chem. 1994, 59, 4357.
(5) For some recent exceptions. see: (a) Wender, P. A.; Touami, S.
M.; Alayarc, C.; Philipp, U. C. J . Am. Chem. Soc. 1996, 118, 6522. (b)
Bregant, T. M.; Groppe, J .; Little, R. D. J . Am. Chem. Soc. 1994, 116,
3635. (c) Sullivan, R. W.; Coghlan, V. M.; Maunk, S. A.; Reed, M. W.;
Moore, H. W. J . Org. Chem. 1994, 59, 2276. (d) Griffiths, J .; Murphy,
J . A. J . Chem. Soc., Chem. Commun. 1992, 24. (e) Schottelius, M. J .;
Chen, P. J . Am. Chem. Soc. 1996, 118, 4896.
(6) Arya, D. P.; Warner, P. W.; J ebaratnam, D. J . Tetrahedron Lett.
1993, 34, 7823.
(7) J ebaratnam, D. J .; Arya, D. P.; Chen, H.; Kugabalasooriar, S.;
Vo, D. BioMed. Chem. Lett. 1995, 5, 1191.
(8) J ebaratnam, D. J .; Kugabalasooriar, S.; Chen, H.; Arya, D. P.
Tetrahedron Lett. 1995, 36, 3123.
(9) J ebaratnam, D. J .; Arya, D. P. Tetrahedron Lett. 1995, 36, 4369.
(10) (a) Huang, P. C.; Kosower, E. M. J . Am. Chem. Soc. 1968, 90,
2354. (b) Huang, P. C.; Kosower, E. M. J . Am. Chem. Soc. 1968, 90,
2362. (c) Huang, P. C.; Kosower, E. M. J . Am. Chem. Soc. 1968, 90,
2367. Wax, R.; Rosenberg, E.; Kosower, N. S.; Kosower, E. M. J .
Bacteriol. 1970, 81, 892. Huang, P. C.; Kosower, E. M. Biochim.
Biophys. Acta 1968,165, 483.
10.1021/jo990750v CCC: $18.00 © 1999 American Chemical Society
Published on Web 07/09/1999

Toward Enediyne Mimics
Sch em e 2
J . Org. Chem., Vol. 64, No. 15, 1999 5645
Sch em e 3
mechanism would require the first radical formed to be
relatively long-lived.
A strategy for generating diradicals in this way is
suggested by recent investigations of the heteroatom
version of the Bergman11 cyclization. In particular,
Chen12 has observed that radicals and diradicals gener-
ated from nitrogen-containing heterocycles abstract hy-
drogen more slowly and hence may be more selective in
DNA cleavage. The possibility that ring nitrogens could
exert a stabilizing influence leading to longer radical
lifetimes has stimulated our interest in heteroaromatic
azoesters as potential enediyne mimics.
Sch em e 4
In this paper, we describe the synthesis of a new series
of compounds aimed at clarifying our understanding of
DNA cleavage by azoesters. The ultimate goal of this
work is to investigate whether radical intermediates play
an essential role in DNA cleavage. On the basis of earlier
findings,10c we started with phenyl azoester 4 to verify
its ability to generate aryl radicals upon methanolysis
in chloroform.10c To investigate the effect of ring nitrogens
on radical stability as proposed by Chen,12 we also
synthesized and methanolyzed the azoesters of pyridine
5 and pyrimidine 6.
Finally, a 1,4-bisazoester 7 (Y ) azoester in Scheme
1, approach 2) was synthesized in an effort to generate a
1,4-diradical from a precursor other than an enediyne.
We find evidence from EPR that radicals are indeed
formed by methanolysis of each of the monoazoesters
4-6. Although no EPR signal was observed from metha-
nolysis of the bisazoester 7, the reaction mixture was
analyzed by HPLC for possible degradation products of
a diradical. Diradicals similar to 8 have been proposed
as intermediates in cycloaromatization reactions, as
shown in Scheme 2.13 Evidence suggests that they are
quite unstable and readily undergo retro-Bergman cy-
clization to form the corresponding nitriles in very high
yields.13a-c The analogous decomposition product for the
diradical that would be formed by methanolysis of 7 is
1,2-dicyanobenzene 9, which is found in the reaction
mixture in 25% yield. Taken together, these results
suggest that a 1,4-diradical may be formed in significant
amounts by methanolysis of the bisazoester.
procedure.10 The basic methanolysis of the azoester was
carried out in an EPR tube, and the EPR spectrum was
recorded immediately at 200 K. The pyridine and pyrim-
idine azoesters 5 and 6 were synthesized from readily
available starting materials in very high yields (Schemes
3 and 4), and their methanolyses were carried out in an
analogous fashion.
Parts a-c of Figure 1 show the EPR spectra obtained
after methanolysis of phenylazoester 4, pyridyl-2-azoester
5, and pyrimidyl-2-azoester 6, respectively. By analogy
with the mechanism proposed by Huang and Kosower,10c
these reactions should produce sp2 radicals of six-
membered aromatic rings, respectively, containing 0, 1,
and 2 nitrogens adjacent to the radical site. However,
we found that these spectra were not directly comparable
to published spectra of the phenyl and pyridyl radicals.
EPR spectra of phenyl and 2-pyridyl free radicals
formed in matrixes at 4 K do allow assignments of the
hyperfine constants of the ring hydrogens.14,15 The spec-
trum of the 2-pyridyl radical further exhibits the hyper-
fine interaction with the ring nitrogen, which produces
an overall triplet with a splitting of approximately 27 G.
The two equivalent ring nitrogens of the 2-pyrimidyl
radical should presumably produce a 1:2:3:2:1 quintet
with a splitting comparable to that of the 2-pyridyl
radicals, although we are unaware of any published
spectrum of the 2-pyrimidyl radical.
Resu lts a n d Discu ssion
Phenyl azoester 4 was synthesized in two steps from
the corresponding hydrazine 10 following the published
The spectra shown in Figure 1 are not comparable to
published low-temperature spectra for a number of
(11) Bergmann, R. G. Acc. Chem. Res. 1973, 6, 625.
(12) (a) (a) Logan, C. F.; Chen, P. J . Am. Chem. Soc. 1996, 118, 2313.
(b) Chen, P. Angew. Chem., Int. Ed. Engl. 1996, 35, 1478.
(13) (a) Shi, C.; Wang, K. K. J . Org. Chem. 1998, 63, 3517. (b) David,
W. M.; Kerwin, S. M. J . Am. Chem. Soc. 1997, 119, 1464. (c) Nam, H.
H.; Leroi, G. E.; Harrison, J . F. J . Phys. Chem. 1991, 95, 6514.
(14) Kasai, P. H.; Hedaya, E.; Whipple, E. B. J . Am. Chem. Soc.
1969, 91, 4364.
(15) Kasai, P. H.; McLeod, D., J r. J . Am. Chem. Soc. 1972, 94, 720.

5646 J . Org. Chem., Vol. 64, No. 15, 1999
Srinivasan et al.
Sch em e 5
EPR signal was seen when 0. 1 mL of 0.27 M NaOMe
was added to neat chloroform in the absence of azoesters.
However, signals similar to those shown in Figure 1 were
observed when 0.1 mL of 0.27 M NaOMe was added to a
200 mM solution of each azoester in acetonitrile. These
results indicate that radical formation depends on the
presence of the azoester precursor but does not depend
on the solvent present. Since a phenyl azoester very
similar to 4 has been shown to produce radical inter-
mediates,10c the mere observation of radicals upon meth-
anolysis of the other aryl azoesters strongly suggests that
they cleave via a similar mechanism.
P h th a la zin e-1,4-bisa zoester (7). The 1,4-bisazoester
7 was prepared from the corresponding dichloride 11 in
two steps (Scheme 5). The dichloride was reacted with
ethylcarbazate to provide roughly equal yields of the
mono- and bishydrazo esters, from which the disubsti-
tuted compound was separated. This was immediately
oxidized to the bisazoester in the usual manner in very
high yield, and its methanolysis was carried out in the
EPR tube as described above for the monoazoesters.
No EPR signal was observed after methanolysis of the
bisazoester 7 at temperatures as low as 145 K. It is
unclear whether a spectrum characteristic of a diradical
intermediate should in fact have been observable. For a
triplet diradical ground state, one expects an EPR
spectrum that is severely broadened by strong electronic
dipolar interactions. Hirama et al. have reported observ-
ing such a spectrum, which they attribute to p-benzyne,
in a footnote found in refs 17 and 18, but the spectrum
has not been published to the best of our knowledge.
Moreover, a recent computational study19 has concluded
F igu r e 1. EPR spectra of the hydrolysis products of (a)
phenylazoester 4, (b) pyridine-2-azoester 5, and (c) pyrimidine-
2-azoester 6 at 200 K.
reasons. First, the radicals are generated chemically and
trapped by freeze-quenching instead of being formed in
situ at low temperatures. The very short lifetime of the
radical intermediate thus requires relatively high con-
centrations of reactants to produce sufficient amounts of
the radical to be observable by EPR. Second, the available
experimental apparatus only allowed spectra to be ob-
tained down to about 145 K. Thus, the spectra shown in
Figure 1 are significantly narrower than those reported
at 4 K, most likely because of spectral averaging due to
rotation of the molecule in the ring plane. The line width
we observe for the phenyl radical is consistent with the
known narrowing of the phenyl radical spectrum at
higher temperatures.16 Presumably, similar narrowing
would occur in the other two radicals under the same
experimental conditions. However, although the EPR
spectra are consistent with previous observations of aryl
radicals at higher temperatures, they are not sufficiently
well-resolved to assign a structure to the observed
species, and we cannot rule out even the possibility that
we are observing paramagnetic products of secondary
radical reactions.
Nevertheless, the EPR results do confirm that radicals
are formed under the experimental conditions, and
control experiments allow us to attribute radical forma-
tion to the methanolysis of the azoesters. No detectable
(17) Hirama, M. Pure Appl. Chem. 1997, 69, 525.
(18) Iida, K.; Hirama, M. J . Am. Chem. Soc. 1995, 117, 8875.
(19) Cramer, C. J .; Nash, J . J .; Squires, R. R. Chem. Phys. Lett. 1997,
277, 311
(16) Ohnishi, S. I.; Tanei, T.; Nitta, I. J . Chem. Phys. 1962, 37, 2402.

Toward Enediyne Mimics
J . Org. Chem., Vol. 64, No. 15, 1999 5647
Although such a significant yield of 1,2 dicyanobenzene
is consistent with the putative intermediate 8, its ap-
pearance could conceivably be explained by other mech-
anisms that do not involve radical intermediates. For
example, formation of a diazene from the azoester
precursor can in some cases lead to the corresponding
carbanion, as summarized in ref 10c, with subsequent
rearrangement to form the nitrile. One may also imagine
consecutive concerted shifts of the hydrogens on the
diazene groups via six-membered-ring transition states
that ultimately produce the bisnitrile.21
Although such mechanisms can explain the formation
of the bisnitrile, they do not account for the EPR signals
observed upon methanolysis of the corresponding mono-
azoesters. Since the methanolysis of the bisazoester was
carried out under the same conditions that produce EPR
signals from the analogous monoazoesters, it is most
likely that the methanolysis of each individual azoester
in this reaction proceeds by a similar mechanism, i.e.,
produces a radical site. Thus, although our results do not
conclusively rule out alternative mechanisms, the EPR
evidence that azoester cleavage produces radicals, com-
bined with the observation of a bisnitrile product from
diazeoster methanloysis, does give a reasonable indica-
tion that a 1,4-diradical species may be formed in
appreciable amounts. We are presently carrying out EPR
spin-trapping studies that will allow direct spectroscopic
identification of the radical intermediates, which will be
reported shortly.
F igu r e 2. HPLC trace of the hydrolysis products of phthala-
zine-1,4-bisazoester 7: (a) crude reaction mixture after hy-
drolysis; (b) dicyanobenzene fraction after isolation.
that all three isomers of benzyne should be singlet ground
states, which would make them EPR-inactive.
Con clu sion
In lieu of any direct EPR evidence, we searched for
chemical indications of a diradical intermediate in the
methanolysis of 7. The bisazoester was methanolyzed
under the same conditions that produced EPR-observable
radical intermediates from the monoazoester, and the
reaction mixture was examined by reversed-phase HPLC
in a specific attempt to identify degradation products
consistent with a diradical intermediate.
Figure 2 shows the HPLC trace of the crude reaction
mixture. Although we did not individually characterize
the many reaction products found, the results are gener-
ally consistent with the generation of reactive radicals
followed by various secondary reactions such as hydrogen
abstraction, formation of solvent adducts, and radical
recombination. Since presumed intermediates similar to
8 have proved difficult to trap chemically,20 it is likely
that the diradical undergoes a concerted decomposition
that is rapid compared with competing radical reactions
(cf. Scheme 2). We therefore focused on searching for
possible decomposition products consistent with a diradi-
cal intermediate rather than attempting to characterize
all of the reaction products.
One possibility for this decomposition is a rapid retro-
Bergman reaction to form the corresponding bisnitrile,
namely 1,2-dicyanobenzene.13a,c,20 1,2-Dicyanobenzene
was identified as a product in the reaction mixture by
spiking experiments with commercially available bisni-
trile. The corresponding fraction was separated by re-
versed-phase HPLC, collected, and characterized further
by GC-MS. This was then quantified as described in the
Experimental Section, and the calculated yield of the
bisnitrile was 25%.
A series of heteroatomic aryl azoesters with different
numbers of ring nitrogens adjacent to the azoester group
have been synthesized and their methanolysis in chlo-
roform studied. EPR spectroscopy confirms that that
these azoesters produce radicals under the given experi-
mental conditions. By analogy with earlier findings,10c
the most likely products are the corresponding sp2 aryl
radicals with 0, 1, and 2 nitrogens adjacent to the radical
site.
A 1,4-bisazoester has also been synthesized and its
methanolysis studied under the same experimental
conditions as the monoazoesters. The degradation prod-
uct 1,2-dicyanobenzene was found in significant yield,
consistent with a 1,4-diradical intermediate that under-
goes a retro-Bergman cyclization. Although alternative
mechanisms for the formation of this product are pos-
sible, the observation that azoesters do produce radicals
under the given conditions suggests that a significant
amount of diradical is formed from the bisazoester.
Having established DNA cleavage by azoesters, and the
possibility that bisazoesters generate 1,4-diradicals, our
results indicate that such compounds are good candidates
for diradical generators based on a nonenediyne strategy
(Scheme 1, approach 2). Efforts to make a more direct
spectroscopic identification of the radical intermediates
are underway and will be reported shortly.
Exp er im en ta l Section
Gen er a l Meth od s. All reactions were performed under a
positive pressure of dry nitrogen atmosphere. 1H NMR spectra
were obtained in a 300 MHz spectrometer and 13C NMR at
(20) Hoffner, J .; Schottelius, M. J .; Feichtinger, D.; Chen, P. J . Am.
Chem. Soc. 1998, 120, 376.
(21) We acknowledge one of the reviewers of this manuscript for
pointing out this possibility.

5648 J . Org. Chem., Vol. 64, No. 15, 1999
Srinivasan et al.
75.2 MHz. Chemical shifts are relative to TMS (when used)
or DMSO-d6. Splitting patterns are designated as singlet (s),
doublet (d), triplet (t), quartet(q), broad (br), and multiplet (m).
EPR spectra were recorded on an ER-200 spectrometer at a
frequency of 9.438 GHz over a 100 G field range centered on
3370 G. For all EPR spectra shown in this paper, the field
sweep time was 100 s, and a gain of 2.5 × 105 was used with
a modulation amplitude of 0.10 G. GC-MS was carried using
helium as the carrier gas. Solvents were freshly distilled prior
to use: pyridine, ethanol, and dichloromethane from calcium
hydride. Reaction product solutions were concentrated by using
a rotary evaporator.
room temperature for 2 h. Excess ethanol was removed at the
pump, and the residue was partitioned between water and
ethyl acetate. The aqueous layer was further extracted with
ethyl acetate and the combined organic layer dried over
anhydrous sodium sulfate. After most of the ethyl acetate was
removed by concentration in vacuo, traces were removed at
the pump to leave behind a yellow oil. This was recrystallized
from ether to give pyridine-2-hydrazoester 13 (0.24 g, 85%)
as yellow solid: 1H NMR (DMSO) δ 9.0 (br, 1H), 8.2 (br, 1H,),
8.0 (m, 1H), 7.5 (m, 1H), 6.6 (m, 1H), 6.5 (m, 1H), 4.0 (q, 2H,
J ) 7.0 Hz), 1.0 (t, 3H, J ) 7.0 Hz); 13C NMR (DMSO) δ 159.9,
156.9, 147.6, 137.4, 114.3, 105.9, 60.3,14.6; IR (KBr) cm-1 3380
(NH), 3200 (NH), 1740 (carbonyl).
2-Eth ylp yr id yld ia zen eca r boxyla te (5). Pyridine-2-hy-
drazoester 13 (0.15 g, 0.83 mmol) was dissolved in 10 mL of
dichloromethane. It was then cooled to 0 °C in an ice bath,
and 0.36 g (0.83 mmol) of Pb(OAc)4 was added. A red color
characteristic of pyridine-2-azoester 5 was observed immedi-
ately. The reaction mixture was stirred at 0 °C for an extra
30 min and then filtered to remove solid lead salts, and the
filtrate partitioned between water and dichloromethane. The
aqueous layer was further extracted with dichloromethane,
and the combined organic layers dried over anhydrous sodium
sulfate and concentrated in vacuo to obtain pyridine-2-azoester
5 (0.14 g, 94%) as a red oil judged to be pure by NMR
analysis: 1H NMR (CDCl3) δ 8.6 (m, 1H), 7.9 (m, 1H), 7.7 (m,
1H), 7.4 (m, 1H), 4.4 (q, 2H, J ) 7.1 Hz), 1. 4 (t, 3H, J ) 7.1
Hz); 13C NMR (CDCl3) δ 162.1, 161.2, 149.6, 138.5, 127.1,
117.7, 64.5, 13.9; IR (CHCl3) cm-1 3040 (aromatic CH), 1754
(carbonyl).
2-Hyd r a zin op yr im id in e (16). 2-Chloropyrimidine 17 (0.7
g, 6.1 mmol) was dissolved in 15 mL of pyridine. Anhydrous
hydrazine (2.53 g, 78.9 mmol) followed by an extra 8 mL of
pyridine were added dropwise. After 1.5 h at room tempera-
ture, pyridine was removed by concentration in vacuo and the
residue suspended in 4 mL of water. Filtration followed by
washing of the solid with methanol (2 × 25 mL) and drying at
the pump gave pure 2-hydrazinopyrimidine 15 (0.64 g, 95%)
as a white solid: mp ) 109-110 °C (lit.22 mp 110-111 °C); 1H
NMR (DMSO) δ 8.3 (d, 2H, J ) 4.6 Hz), 8. 1 (br, 1H), 6.6 (t,
1H, J ) 4.8 Hz), 4.1 (br, 2H); 13C NMR (DMSO) δ 164.4, 157.8,
18.4; IR (KBr) cm-1 3400-2400 (br).
EP R Mea su r em en ts. Solutions (200 mM) of all azoesters
were made in chloroform and thoroughly degassed by freeze-
thaw-pump cycles (four to five times) on a Schlenk line using
a glass adapter with a septum access. The solutions were then
cooled to ∼200 K, 0. 1 mL of 0.27 M NaOMe (made from Na
metal and methanol) was added via
a syringe, and the
resulting reddish brown solutions were thoroughly mixed. The
reaction was performed in the EPR tube and then transferred
to the cavity, and the spectrum was recorded immediately.
HP LC An a lysis. An HPLC system equipped with a UV
detector and a strip chart recorder using a reversed-phase C-18
analytical column was employed for all HPLC experiments.
For making up the samples, a prefiltration of the crude
mixture on a C-18 cartridge followed by ultracentrifugation
was carried out. This crude mixture was subjected to a
gradient elution using an acetonitrile/water mixture. Samples
of commercially available 1,2-dicyanobenzene were used for
spiking experiments and for GC-MS analyses. A calibration
curve was used to estimate the amount of 1,2-dicyanobenzene
formed from the methanolysis of phthalazine-1,4-bisazoester
7.
Eth yl P h en ylh yd r a zoca r boxyla te (12). A 1.0 g (0.91 mL,
0.0092 mol) sample of phenylhydrazine 10 was dissolved in
10 mL of pyridine. The solution was cooled in an ice bath, and
1.0035 g (0.88 mL, 0.0092 mol) of ethyl chloroformate was
added dropwise over a period of 30 min. The mixture was
stirred at 0 °C for 15 min and at room temperature for an
additional 30 min. After addition of water (20 mL) and ether
(100 mL), the ether layer was separated and concentrated (to
a volume of approximately 10 mL). Phenyl hydrazoester began
to crystallize upon leaving the concentrated solution in the
refrigerator for a few hours. The crystals were collected by
filtration, washed with ether, and dried under vacuum to
obtain pure phenyl hydrazoester 12 (1.29 g, 78%): mp ) 71-
2-Eth yl P yr im id ylh yd r a zoca r boxyla te (18). To a solu-
tion of 2-hydrazinopyrimidine 16 (0.14 g, 1.27 mmol) in 8 mL
of anhydrous ethanol was added sodium carbonate (0.067 g,
0.635 mmol), and the solution was cooled to -15 °C. A solution
of ethyl chloroformate (0.138 g, 0.121 mL, 1.27 mmol) in 8 mL
of anhydrous ethanol was then added dropwise over a period
of 25 min via an addition funnel. The resulting mixture was
stirred at -15 °C for 2 h and at room temperature for an
additional 2 h. After removal of ethanol in vacuo, the residue
was partitioned between water and ethyl acetate and the
aqueous layer extracted further with ethyl acetate. The
combined organic layers were dried over anhydrous sodium
sulfate, and ethyl acetate was distilled off at reduced pressure
to leave behind a white solid. The crude product was recrystal-
lized from ether to obtain pyrimidine-2-hydrazoester 18 (0.188
1
72 °C; H NMR (DMSO) δ 8.9 (br, 1H), 7.6 (br, 1H), 7.1 (m,
2H), 6.7 (m, 3H), 4.05 (q, 2H, J ) 7.1 Hz), 1. 18 (t, 3H, J ) 7.2
Hz); 13C NMR (DMSO) δ 157.11, 149.5, 128.8, 118.3, 111.7,
60.1, 14.5; IR (KBr) cm-1 3370 (NH), 3225 (NH), 3050
(aromatic CH), 2975 (aliphatic CH), 1745 (carbonyl).
Eth yl P h en yld ia zen eca r boxyla te (4). Phenyl hydrazo-
ester 12 (0.25 g, 1.39 mmol) was taken up in 10 mL of
dichloromethane. Lead tetraacetate (0.62 g, 1.39 mmol) was
added all at once to the above solution, which was maintained
at 0 °C. A red color, indicative of phenyl azoester 4, appeared
almost immediately. The reaction mixture was stirred at 0 °C
for an extra 30 min and then filtered to remove solid lead salts,
and the filtrate was partitioned between water and dichlo-
romethane. The aqueous layer was further extracted with
dichloromethane, and the combined organic layers were dried
over anhydrous sodium sulfate and concentrated in vacuo to
obtain phenyl azoester 4 (0.23 g, 91%) as a red oil judged to
be pure by NMR analysis: 1H NMR (CDCl3) δ 7.9 (m, 2H), 7.5
(m, 3H), 4.5 (q, 2H, J ) 7.1 Hz), 1.4 (t, 3H, J ) 7.1 Hz); 13C
NMR (CDCl3) δ 162.0, 151.4, 133.6, 129.1, 123.5, 64.2, 13.9;
IR (CHCl3) cm-1 3050 (aromatic CH), 2950 (aliphatic CH), 1750
(carbonyl).
2-E t h ylp yr id ylh yd r a zoca r b oxyla t e (13). 2-Hydrazino-
pyridine 14 (0.17 g, 1.56 mmol) was dissolved in 10 mL of
anhydrous ethanol to give a red solution. Anhydrous sodium
carbonate (0.082 g, 0.78 mmol) was added, and the mixture
was cooled to -15 °C. A solution of ethyl chloroformate (0.169
g, 0.149 mL, 1.56 mmol) in 8 mL of ethanol was added
dropwise and the reaction stirred at -15 °C for 1 h and at
I
g, 82%): mp ) 103-104 °C; H NMR (DMSO) δ 9.1 (br, 1H),
8.9 (br, 1H), 8.4 (d, 2H, J ) 4.7 Hz), 6.6 (t, 1H, J ) 4.6 Hz),
4.1 (q, 2H, J ) 7.2 Hz), 1.2 (t, 3H, J ) 6.9 Hz); 13C NMR
(DMSO) δ 163.3, 158.2, 156.9, 112.5, 60.2, 14.5; IR (KBr) cm-1
3450 (NH), 3250 (NH), 1745 (carbonyl).
2-Eth yl P yr im id yld ia zen eca r boxyla te (6). Pyrimidine-
2-hydrazoester 18 (0.2 g, 1.1 mmol) was dissolved in 10 mL of
dichloromethane. To this clear and colorless solution was
added 0.487 g (1.1 mmol) of Pb(OAc)4. The resulting yellow
solution was stirred for I h at 0 °C. After separation of the
byproducts (inorganic lead compounds) by filtration, the
filtrate was partitioned between dichloromethane and water.
The aqueous layer was further extracted with dichloromethane,
and the combined organic layers were dried over anhydrous
sodium sulfate. Concentration in vacuo gave pyrimidine-2-
(22) Vanderhaeghe, C. Bull. Soc. Chim. Belg. 1959, 68, 30.

Toward Enediyne Mimics
J . Org. Chem., Vol. 64, No. 15, 1999 5649
azoester 6 (0.185 g, 95%) judged to be pure as evidenced by
1H and 13C NMR analyses: IH NMR (CDCl3) δ 9.0 (d, 2H, J )
4.7 Hz), 7.5 (t, 1H, J ) 4.7 Hz), 4.5 (q, 2H, J ) 7.0 Hz), 1.5 (t,
3H, J ) 7.0 Hz); 13C NMR (CDCl3) δ 165.6, 161.7, 159.1, 122.5,
65.0, 14.1; IR (CHCl3) cm-1 3050 (aromatic CH), 1748 (carbo-
nyl).
virtually instantaneous. After the mixture was stirred at 0 °C
for 1 h, solid lead salts were removed by filtration and the
filtrate was partitioned with water (washed a number of times
with water to remove brown lead compound) and dichlo-
romethane. The aqueous layer was extracted further with
dichloromethane, and the combined organic layers were dried
over anhydrous sodium sulfate and concentrated in vacuo. The
red oily film was then chromatographed using hexanes/ethyl
acetate (Rf ) 0.5 in 1:1 hexanes/ethyl acetate) to obtain pure
phthalazine-1,4-bisazoester 7 as a dark red oil (0.096 g, 91%):
1H NMR (CDCl3) δ 8.6 (m, 2H), 8.2 (m, 2H), 4.6 (q, 4H, J ) 7
Hz), 1.5 (t, 6H, J ) 7 Hz); 13C NMR (CDCl3) δ 161.6, 160.6,
134.6, 125.2, 124.4, 65.6, 14.4; IR (CHCl3) cm-1 3025 (aromatic)
1745 (carbonyl).
1,4-Dieth yl P h th a la zin ylh yd r a zoca r boxyla te (20). 1,4-
Dichlorophthalazine 11 (0.275 g, 1.3 mmol) was added, all at
once, to a stirred solution of ethyl carbazate (0.28 g, 2.7 mmol)
in 15 mL of ethanol. After addition of another 8 mL of ethanol,
the reaction mixture was heated to 55 °C and stirred overnight
(12 h). TLC analysis of the crude mixture after 12 h showed
two spots corresponding to the mono- and bishydrazoesters.
The desired phthalazine-1,4-bishydrazoester 20 (0.21 g, 48%)
was obtained as an off-white solid upon chromatographic
purification (Rf ) 0. 13 in 95:5 ethyl acetate/methanol; mp )
185-186 °C). The yield of the corresponding monohydra-
zoester, a yellow solid, was 50%. Due to oxidative instability,
lead tetraacetate oxidation of phthalazine-1,4-bishydrazoester
20 had to be carried out immediately (described below): 1H
NMR (DMSO) δ 8.9 (br, 2H), 8.7 (br, 2H), 8.2 (br, 2H), 7.8 (br,
2H), 4.1 (q, 4H, J ) 7.0 Hz), 1.2 (t, 6H, J ) 7.0 Hz); 13C NMR
(DMSO) δ 157.1, 150.0, 131.2, 122.1, 118.7, 59.8, 14.5.
1,4-Dieth ylph th alazin yldiazen ecar boxylate(7). Phthala-
zine-1,4-bishydrazoester 20 (0.11 g, 0.32 mmol) was dissolved
in dichloromethane and the solution cooled in an ice bath to 0
°C. Lead tetraacetate (0.28 g, 0.64 mmol) was added, and the
appearance of deep red color characteristic of azoesters was
Ack n ow led gm en t. This work was supported by
grants NSF CHE-9406469 (D.J .) and NSF MCB-
9600940 (D.E.B.). We are grateful to Profs. David A.
Forsyth and Graham J ones for helpful discussions and
to Dr. Stephen V. Kolaczkowski for assistance with the
EPR freeze-quenching experiments.
Su p p or tin g In for m a tion Ava ila ble: A listing of the 75
MHz 13C NMR spectra of compounds 12, 4, 13, 5, 17, 18, 6,
20, and 7. This material is available free of charge via the
Internet at http://pubs.acs.org.
J O990750V