Oxa-enediynes: Probing the electronic and stereoelectronic contributions to the bergman cycloaromatization

Article abstract of DOI:10.1021/jo0256888

Efficient routes to three classes of 10-membered oxa-enediynes are presented. The electronic and stereoelectronic contributions to half-lives are supported by density functional theory calculations. One member of this class cyclizes to give an isochroman which binds to and degrades the aryl hydrocarbon receptor (AhR).

Full text of DOI:10.1021/jo0256888

Oxa -En ed iyn es: P r obin g th e Electr on ic a n d Ster eoelectr on ic
Con tr ibu tion s to th e Ber gm a n Cycloa r om a tiza tion
Graham B. J ones,*,† J ustin M. Wright,† George Hynd,† J ustin K. Wyatt,† Philip M. Warner,†
Robert S. Huber,‡ Aiwen Li,‡ Michael W. Kilgore,‡ Robert P. Sticca,‡ and Richard S. Pollenz§
Department of Chemistry, Northeastern University, Boston, Massachusetts 02115, GHS Medicinal
Chemistry Laboratory, Department of Chemistry, Clemson University, Clemson, South Carolina 29634,
and Department of Biology, University of South Florida, Tampa, Florida 33620
Gr.J ones@neu.edu
Received March 6, 2002
Efficient routes to three classes of 10-membered oxa-enediynes are presented. The electronic and
stereoelectronic contributions to half-lives are supported by density functional theory calculations.
One member of this class cyclizes to give an isochroman which binds to and degrades the aryl
hydrocarbon receptor (AhR).
The enediynes are a promising class of antitumor
agents, with spectacular biological profiles.1 Over 20
natural enediynes have been discovered, and currently,
clinical development of neocarzinostatin analogues con-
tinues in J apan and Europe. U.S. trials of a monoclonal
antibody (MoAb) conjugate of calicheamicin (Mylotarg)
are entering phase III, and esperamicin is entering phase
II trials.2 Interest in calicheamicin was heightened
because the MoAb conjugate induced remission in up to
40% of patients with acute myeloid leukemia (AML) who
had previously relapsed following other chemotherapies.
While the in vitro and in vivo effectiveness of enediynes
against certain cancers is unquestioned, the exact mech-
anism(s) of biological activity are complex and remain
to be fully understood.1 Enediynes per se are biologically
inactive but undergo cycloaromatization reactions which
give rise to cytotoxic diradicals, which are capable of
inducing DNA strand scission at low concentration.1 The
basic pharmacophore of many of the natural enediynes
is a cyclodec-3-ene-1,5-diyne unit (1, X ) CH2), biological
or thermal activation of which results in Bergman
cyclization of the core to yield the reactive diyl radical
† Northeastern University.
‡ Clemson University.
§ University of South Florida.
(1) Xi, Z.; Goldberg, I. DNA Damaging Enediyne Compounds. In
species 2, which then participates in atom-transfer
Comprehensive Natural Products Chemistry; Barton, D. H. R., Nakan-
chemistry.3 In a cellular environment, these atom-
ishi, K., Eds.; Pergamon: Oxford, 1999; Vol. 7, p 553. Smith, A. L.;
Nicolaou, K. C. J . Med. Chem. 1996, 39, 2103. Grissom, J . W.;
Gunawardena, G. U.; Klingberg, D.; Huang, D. Tetrahedron 1996, 52,
6453. Maier, M. E. Synlett 1995, 13.
(2) Schor, N. F. In Cancer Therapeutics: Experimental and Clinical
Agents; Teicher, B., Ed.; Humana Press: Totowa, NJ , 1996; pp 229-
240. Schmitt, D. A.; Kisanuki, K.; Kimura, S.; Oka, K.; Pollard, R. B.;
Maeda, H.; Suzuki, F. Anticancer Res. 1992, 12, 2219. Takahashi, T.;
Yamaguchi, T.; Kitamura, K.; Noguchi, A.; Mitsuyo, H.; Otsuji, E. J pn.
J . Cancer Res. 1993, 84, 976. Oda, M.; Sato, F.; Maeda, H. J . Natl.
Cancer Inst., 1987, 79, 1205.
(3) Bergman, R. G. Acc. Chem. Res. 1973, 6, 25.
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transfer events can result in formation of ribosyl radicals
on DNA,1,4 and it has also been suggested that they may
participate in the generation of peptide radicals,5 in part
10.1021/jo0256888 CCC: $22.00 © 2002 American Chemical Society
Published on Web 06/28/2002
J . Org. Chem. 2002, 67, 5727-5732
5727

J ones et al.
SCHEME 1. BLYP An a lysis of Cycloa r om a tiza tion
Ba r r ier for Oxa -En ed iyn e 4
accounting for the typically high biological activity of
most cyclic enediynes. Since the Bergman cycloaromati-
zation reaction is the key triggering event in the biologi-
cal cascade, a great deal of effort has been spent studying
the process and devising efficient means to control the
process.1 The relief of strain experienced when the C-10
ring undergoes cyclization has been examined and de-
termined to play a dominant role and has been exploited
in the form of triggering devices.6 Additionally, a rule of
thumb has been adopted by correlating the intra-acety-
lenic distance in the enediyne (the so-called “c-d”
distance) with experimentally determined half-lives.7
Other contributing factors involve the electronics of the
process, and several reports point to subtle effects
observed with substituted enediynes.8 For our own stud-
ies, we wished to examine the effects of heteroatoms in
the process, since (i) they may impart subtle strain and/
or electronic contributions, (ii) the effects could then be
modulated further by appropriate substitution R to the
heteroatom, and (iii) synthesis of the agents could be
expected to be straightforward, invoking disconnections
adjacent to the heteroatom.9,10
TABLE 1. Com p a r ison of In tr a -Acetylen ic Dista n ce a n d
En th a lp ic Ba r r ier s to Cycloa r om a tiza tion a
r1,6
-
∆Hrel
-
t
1/2(obsd)b
(h)
entry substrate
(diyne)
r1,6(ts) (diyne) ∆Hrel(ts)
1
2
3
4
1, X ) CH2 3.413
1.973
0.0
0.0
0.0
0.0
23.7
22.4
21.3
16.0
18
17
15
1
4
3.365
3.346
3.170
1.985
1.990
2.002
20
28
a
All enthalpies in kcal/mol based on BLYP/6-311+G**//BLYP/
6-31-G* electronic energies and BLYP/6-31G* rovibrational cor-
rections. 37 °C, 0.1 M 1,4-cyclohexadiene.
b
For an initial test case, we elected to study oxa-
enediyne 4 and its expected conversion to diyl 6 through
transition state 5. Prior to embarking on synthetic
studies, we probed the cyclization barrier using compu-
tational analyses and examined the intra-acetylenic “1-
6” distance in the ground state and transition state
(Scheme 1).11The results indicated a shortening of the
1-6 distance for 4 and a consequent earlier transition
state (longer 1-6 distance) and decreased enthalpy of
activation relative to that of the carbocyclic parent
enediyne 1, X ) CH2 (Table 1). The structural differences
between 4 and 1, X ) CH2, are due to the shorter CO
versus CC bond distances. One could expect the ∆H#
difference to translate to an experimental half-life of less
than the 18 h/37 °C reported for 1, X ) CH2,12 and
accordingly, 4 became a synthesis target.
Initially, the synthesis of 4 was investigated using
popular Pd-mediated coupling chemistry, and two inde-
pendent routes were examined, as shown in Scheme 2.
Following formation of the unsymmetrical dialkynyl
ether 7, we had hoped to effect direct conversion to 4 via
in situ tandem vinylation. Unfortunately, this coupling
gives rise to a mixture of products 8/9, and even under
ideal conditions (stoichiometry, solvent, addition rates),
not even a trace of 4 is formed. In an effort to effect
closure in a stepwise manner, 7 was converted to masked
alkyne 10, and monovinylation was effected, followed by
deprotection to give substrate 11. Again, under a variety
of different conditions, direct conversion to 4 could not
be effected; however, over extended periods, polymeric
materials were recovered which may have resulted from
cyclization and in situ cycloaromatization. This suggested
that a Pd-mediated route to 4 may not be desirable, and
we sought a more rapid means to effect closure. Remedy
was found in the form of a Williamson ether synthesis
(Scheme 3). Chlorovinylation of 12 to give 13 was
followed by propargylation/deprotection to give 14. Bro-
mination then gave 15 in good yield and was followed by
mild unmasking to give bromo alcohol 16. Exposure to
base and tetrabutylammonium sulfate gave 4 directly;
however, because of the predicted thermal instability of
4, we elected to protect the enediyne as its cobalt carbonyl
(5) Zein, N.; Solomon, W.; Cassazza, A. M.; Kadow, J . F.; Krishnan,
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Bdour, H. M.; Shain, J . C.; Mandal, S.; Rudra, K. R.; Nag, S. Bioorg.
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(9) For communication describing synthesis of 4 and 20 and bioassay
of 20, see: J ones, G. B.; Kilgore, M. W.; Pollenz, R. S.; Li, A.; Mathews,
J . E.; Wright, J . M.; Huber, R. S.; Tate, P. L.; Price, T. L.; Sticca, R. P.
Bioorg. Med. Chem. Lett. 1996, 6, 1971.
(10) Related heterocyclic enediynes: Sakai, Y.; Nishiwaki, E.; Shi-
shido, K.; Shibuya, M.; Kido, M. Tetrahedron Lett. 1991, 32, 4363.
Basak, A.; Khamrai, U. K. Tetrahedron Lett. 1996, 37, 2475. Basak,
A.; Khamrai, U. K.; Mallik, U. J . Chem. Soc., Chem. Commun. 1996,
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Khamrai, U. K.; Basak, A.; Tetrahedron Lett. 1997, 38, 6067.
(11) J ones, G. B.; Warner, P. M. J . Am. Chem. Soc. 2001, 123, 2134.
(12) J ones, G. B.; Wright, J . M.; Plourde, G. W., II; Hynd, G.; Huber,
R. S.; Mathews, J . E. J . Am. Chem. Soc. 2000, 122, 1937.
5728 J . Org. Chem., Vol. 67, No. 16, 2002

Oxa-Enediynes
SCHEME 2. Attem p ted P d -Med ia ted Rou tes to Oxa -En ed iyn e 4
SCHEME 3. Refin ed Rou tes to Oxa -En ed iyn e 4 a n d Its Coba lt Ca r bon yl Com p lex 17
SCHEME 4. BLYP An a lysis of Bicyclic Oxa -En ed iyn e 20
derivative 17, which was stable for extended periods. As
needed, smooth decomplexation was achieved by expo-
sure to TBAF in THF.13 Though this was effective, we
wished to develop an even more direct route to 4 and
succeeded; bis hydroxymethylation of 7 gave 18 in high
yield, which was then followed by bis bromination to give
19. Intramolecular carbenoid coupling/elimination fol-
lowed by in situ complexation gave 17 in high yield.14
Overall, the carbenoid route via dibromide 19 proved far
superior in terms of both efficiency and cost-effectiveness,
and could be conducted effortlessly on a multigram scale.
With quantities of 4 in hand, Bergman cyclization was
then investigated under controlled conditions; incubation
of 4 at 37 °C in the presence of 1,4-cyclohexadiene
(30 equiv) gave a nearly quantitative (>90%) yield of
isochroman (3, X ) O, R ) H), presumably via the
intermediacy of diyl 2, X ) O. Significantly, the half-life
for the Bergman cycloaromatization was in agreement
with that predicted by the computational studies (Table
1).
Having established a high yielding and economical
route to oxa-enediynes of type 1, we sought to incorporate
the method in the syntheses of more complex systems
which would allow us to gauge the impact of other effects
on cycloaromatization. We envisioned that bicyclic oxa-
enediyne 20 would provide an opportunity to gauge the
effect of strain on the cycloaromatization and is also a
target that might be accessible using the carbenoid
method of synthesis. Accordingly, the BLYP analysis of
20 and its barrier to transition state 21 en route to diyl
22 was examined (Scheme 4). It was revealed that the
barrier to cyclization was less than that for 4, in accord
with the shortened 1-6 distance; again, the transition
state occurred earlier (Table 1).
(13) J ones, G. B.; Wright, J . M.; Rush, T. M.; Plourde, G. W., II;
Kelton, T. F.; Mathews, J . E.; Huber, R. S.; Davidson, J . P. J . Org.
Chem. 1997, 62, 9379.
(14) J ones, G. B.; Huber, R. S. U.S. Patent 5,436,361. J ones, G. B.;
Huber, R. S.; Mathews, J . E. J . Chem. Soc., Chem. Commun. 1995,
1791. J ones, G. B.; Huber, R. S. Tetrahedron Lett. 1994, 35, 2655. Hynd,
G.; J ones, G. B.; Plourde, G. W.; Wright, J . W. Tetrahedron Lett. 1999,
40, 4481.
J . Org. Chem, Vol. 67, No. 16, 2002 5729

J ones et al.
SCHEME 5. Ca r ben oid Cou p lin g Rou te to Bicyclic
Oxa -En ed iyn e
Finally, we elected to examine the contribution that
arylation of the heteroatom would have on the cycloaro-
matization process. Enediyne 28 was identified as a
target, in part, on the basis of computational analyses of
its conversion to 29 (Table 1, Scheme 6). Surprisingly,
the barrier proved minimal, although the relative effects
of increased strain and electronics are not readily as-
sessed. However, it became clear that any intended
synthesis would require careful handling of the product.
Two complimentary synthetic strategies were envisioned,
either with a carbenoid coupling from 34 or, possibly,
with a Williamson type etherification from 38 (Scheme
7). Both sequences began with Pd-mediated couplings of
an alkynyl substrate to produce 33 and 37. Substrate 33
was elaborated by etherification with trimethylsilyl pro-
pargyl bromide to give a bis TMS protected intermediate.
Deprotection with TBAF followed by hydroxymethylation
gave the corresponding diol, which was converted to
dibromide 34 by employing mild bromination conditions
with MsCl and LiBr. All attempts to prepare 28 and then
conduct an in situ complexation to give 35 were unsuc-
cessful, since the enediyne decomposed rapidly to give a
complex mixture of products. However, trace quantities
of 28 were obtained, allowing spectroscopic analysis and
determination of its half-life, which, as predicted, was
short (Table 1). The alternate method, achieved via
bromination of 37 and then Williamson closure on 38,
likewise allowed isolation of trace quantities of 28, but
again, it proved impossible to prepare quantities of its
derived complex 35. Clearly, computational analyses had
successfully predicted the thermal instability of this
enediyne (Table 1).
The synthesis of 20 commenced from cyclohexene oxide
which was ring opened with lithiotriethylsilyl acetylide
(Scheme 5). Acetylene desilylation proved more effective
at this stage and was then followed by propargylation to
give bis alkyne 25. Hydroxymethylation, followed by
bromination under mild conditions, then provided ene-
diyne precursor 26. The intramolecular carbenoid addi-
tion/elimination reaction was again employed, which gave
a high yield of 20 directly. For practical purposes,
however, the cyclization was followed by immediate
enediyne protection using dicobalt octacarbonyl to give
27 that was stable at 25 °C for extended periods.
Deprotection of 27 to liberate 20 was again easily
achieved using TBAF. The half-life of 20 was found to
be approximately 15 h at physiological temperature, in
good empirical agreement with its calculated intra-
acetylenic (c-d) distance of 3.346 Å. Incubation of 20 in
the presence of 1,4-cyclohexadiene (30 equiv) led to
conversion (>80%) to tricyclic product 23,15 presumably
via the intermediacy of diradical 22. As such, the process
represents an efficient route to substituted isochromans.
One of the goals of our synthesis program is to explore
new molecular targets for enediynes, and we have been
SCHEME 6. BLYP An a lysis of Ben zofu sed Oxa -En ed iyn e 28
SCHEME 7. Com p lim en ta r y Rou tes to Oxa -En ed iyn e 28 a n d Its Coba lt Ca r bon yl Com p lex 35
5730 J . Org. Chem., Vol. 67, No. 16, 2002

Oxa-Enediynes
F IGURE 1. Protein (AhR) expression as a function of expo-
sure to enediyne 20 in MCF-7 cells. Cells were incubated with
either DMSO (control) or 20 at indicated concentrations (M)
for 16 h. Cells were then harvested, lysed, and subjected to
Western blot analysis.
F IGURE 2. Effect of incubation time in the degradation of
AhR by enediyne 20. Triplicate plates of MCF-7 cells were
treated with 2 × 10-5 M 20 for 4, 8, 16, or 24 h. Cells were
then harvested, resolved by SDS-PAGE, blotted, and stained
for AhR and actin.19
actively developing enediyne mimics of transcription
factors, including the estrogen receptor.16 Another inter-
esting target is the orphan receptor referred to as the
aryl hydrocarbon receptor (AhR). The AhR is a ligand-
activated transcription factor which is responsible for
regulation of the production of a number of cytochromes
P-450.17 The highest affinity ligands for the AhR are
oxygenated heterocycles, including chlorodibenzofuran
and TCDD. Because of the structural similarity with
oxoenediyne products 23/31, we sought means to inves-
tigate the potential interaction of the parent enediynes
with this receptor. A number of natural and synthetic
enediynes are reported to possess proteolytic activity, and
it is very likely that diradicals derived from the enediynes
contribute to the observed in vitro effects by interacting
with specific protein targets.5 Because of the instability
of oxoenediyne 28, we opted to restrict our initial study
of biological targets to enediyne 20.
First, we conducted experiments to monitor AhR
expression in MCF-7 breast cancer cells over a range of
concentrations of 20. The results showed a nearly 60%
reduction in protein at 10-5 M and a correlation between
enediyne and protein at lower concentrations (Figure 1).18
Though AhR degradation mediated by 20 might be
expected to yield various protein fragments, no smaller
fragments were identified on Western analysis (N.B. the
antibody used is against the first 416 amino acids of this
805aa protein and is thus incapable of detecting a
C-terminal fragment19), suggesting that the enediyne
behaves in a manner similar to that of xenobiotics which
F IGURE 3. Antiproliferative effects of 20 versus T47-D and
T47-D-Y breast cancer cells. Cells received estradiol (E2) at 1
× 10-9 M or enediyne 20 at indicated concentrations (M). All
cells received 3H thymidine (1 × 10-6 M); after 6 h, nucleii
3
were recovered and incorporated H thymidine was measured.
degrade the AhR, including TCDD.18 The current model
for this degradation pathway involves AhR ubiquitination
and subsequent breakdown via the 26S proteasome,
resulting in complete conversion to small oligopeptides.
Significantly, preformed 23 was incapable of inducing
AhR degradation even at the highest concentration used
(within standard error margin). Thus, it is reasonable
to assume that a 1,4-diyl (or potentially an oxa-allene
intermediate) derived from 20 is responsible for the
observed degradation.
(15) Negishi, E.; Nguyen, T.; O’Connor, B.; Evans, J . M.; Silveira,
A. Heterocycles 1989, 28, 55.
(16) J ones, G. B.; Purohit, A.; Wright, J . W.; Hynd, G.; Wyatt, J .;
Swamy, N.; Ray, R. Tetrahedron Lett. 2001, 42, 8579. J ones, G. B.;
Huber, R. S.; Mathews, J . E.; Li, A. Tetrahedron Lett. 1996, 37, 3643.
(17) Chaloupka, K.; Krishnan, V.; Safe, S. Carcinogenesis 1992, 13,
2233. Wilhelmsson, A.; Whitelaw, M. L.; Gustafsson, J .-A.; Poellinger,
L. J . Biol. Chem. 1994, 269, 19028. Merchant, M.; Morrison, V.;
Santostefano, M.; Safe, S. Arch. Biochem. Biophys. 1992, 298, 389.
(18) Pollenz, R. S. Mol. Pharmacol. 1996, 49, 391. Pollenz, R. S.;
Santostefano, M. J .; Klett, E.; Richardson, V.; Necela, B.; Birnbaum,
L. S. Toxicol. Sci. 1998, 42, 117. Davarions, N. A.; Pollenz, R. S. J .
Biol. Chem. 1999, 274, 28708. Pollenz, R. S.; Barbour, E. R. Mol. Cell.
Biol. 2000, 20, 6094.
Since the enediyne 20 has a half-life of 15 h at
physiological temperature, additional assays were per-
formed at 10-5 M enediyne, with increased incubation
times before analysis for protein. The results showed
(Figure 2) that depletion levels off by 16 h of incubation
and AhR levels do not recover.
(19) Holmes, J .; Pollenz, R. S. Mol. Pharmacol. 1997, 52, 202.
J . Org. Chem, Vol. 67, No. 16, 2002 5731

J ones et al.
Since the enediyne clearly has an impact on receptor
levels, the ramifications of such degradation in the cell
cycle were then studied. It has been reported that ligands
with an affinity for AhR cause a decrease in nuclear
estrogen receptor (hER) levels and, as such, can behave
as antiestrogens.17 To investigate this, we studied the
cytotoxicity of 20 using two breast cancer cell lines: one
that is known to contain both AhR and functional human
estrogen receptor (T-47D) and one that is deficient of the
hER (T-47D-Y). Using a conventional thymidine uptake
assay, while the T-47D-Y cells grew at rates apparently
unaffected by enediyne concentrations 10-8 through 10-4
M, the T-47D cells were sensitive, with agent 20 proving
cytotoxic at concentrations as low as 10-6 M and causing
83% growth inhibition at 10-4 M (Figure 3). As expected,
when stimulated with estradiol (E2, 10-9 M) only the
T-47D cell line showed enhanced proliferation, suggesting
that the observed antiproliferative effects of 20 may be
due to its interaction with the transcriptional machinery.
that of the present example, as exemplified by our recent
findings on the degradation of the estrogen receptor by
designed estramycins.16 Such agents may be useful as
probes of transcriptional events or even as antitumoral
agents in their own right.20
In summary, a series of thermally activated oxa-
enediynes were produced using an efficient carbenoid
coupling/elimination procedure. The impact of the het-
eroatom on enediyne cycloaromatization showed good
correlation with computational analyses. Since even one
of the simple members of this class of enediynes is
capable of inducing receptor degradation, it is likely that
more advanced members can be designed for use as
biological modulators. It is expected that the interplay
of chemical synthesis, computational chemistry, and
bioassay will permit identification of optimal candidates
for in-depth evaluation.
Ack n ow led gm en t. This work was supported by
grants from the NIH (5RO1GM57123), the Elsa U.
Pardee Foundation, and the GHS-CU Biomedical Co-
operative research program.
Monitoring for AhR protein in MCF-7 cells showed a
clear correlation between enediyne concentration and the
depletion of cellular protein (Figure 1), and it is tempting
to speculate that this impacts cell proliferation (Figure
3). Using affinity-driven synthesis, it may be possible to
produce oxa-enediynes with an even higher activity than
Su p p or tin g In for m a tion Ava ila ble: Synthetic proce-
dures and spectroscopic data for the preparation of all com-
pounds and calculation coordinates for compounds 4, 20, and
28 (23 pages). This material is available free of charge via the
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(20) For enediyne-estrogen synthesis, see: Wang, J .; DeClercq, P.
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