Synthesis and DNA cleavage reaction characteristics of enediyne prodrugs activated via an allylic rearrangement by base or UV irradiation

Article abstract of DOI:10.1016/j.bmc.2005.12.040

A number of enediyne prodrugs 1-5 possessing an (E)-3-hydroxy-4-(2′- hydroxy-1′-phenylethylidene)cyclodeca-1,5-diyne scaffold have been synthesized via the Sonogashira coupling and an intramolecular Nozaki-Hiyama-Kishi reaction as the key steps. Upon incu

Full text of DOI:10.1016/j.bmc.2005.12.040

Bioorganic & Medicinal Chemistry 14 (2006) 3199–3209
Synthesis and DNA cleavage reaction characteristics of enediyne
prodrugs activated via an allylic rearrangement by base or
UV irradiationq
Yukihiro Tachi,a Wei-Min Dai,b,* Kazuhito Tanabea and Sei-ichi Nishimotoa,*
aDepartment of Energy and Hydrocarbon Chemistry, Graduate School of Engineering, Kyoto University, Katsura Campus,
Nishikyo-ku, Kyoto 615-8510, Japan
bDepartment of Chemistry and Open Laboratory of Chirotechnology of The Institute of Molecular Technology for Drug Discovery
and Synthesis, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong SAR, China
Received 15 November 2005; revised 15 December 2005; accepted 17 December 2005
Available online 18 January 2006
Abstract—A number of enediyne prodrugs 1–5 possessing an (E)-3-hydroxy-4-(20-hydroxy-10-phenylethylidene)cyclodeca-1,5-diyne
scaffold have been synthesized via the Sonogashira coupling and an intramolecular Nozaki–Hiyama–Kishi reaction as the key steps.
Upon incubation with enediyne prodrugs 4 and 5 possessing a free hydroxymethyl group on the exocyclic double bond, circular
supercoiled DNA (Form I) underwent single strand cleavage into circular relaxed DNA (Form II) in buffer solution at pH 8.5, while
the silylated analogs 1–3 showed very weak DNA cleavage activity. Alternatively, the silylated analogs 1–3 could be activated by UV
irradiation via a photochemical alkene isomerization followed by an allylic rearrangement to form the putative epoxy enediyne,
resulting in efficient DNA cleavage similar to the level observed with the prodrugs 4 and 5.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
After disclosure of the mechanism of DNA cleavage by
a family of naturally occurring enediynes, a great num-
ber of efforts were devoted to design and synthesize var-
ious types of enediyne-mimic compounds.6 The most
importance in designing enediyne model compounds is
how to regulate the reactivity using a simple triggering
device, by which a stable precursor can be transformed
in situ to a highly reactive enediyne ready for cycloaro-
matization under physiological conditions. A large num-
ber of activation strategies have been hitherto proposed,
involving the formation of enyne–allenes or enyne–
cumulenes via nucleophilic attack, photolysis or elimina-
tion of a substituent.7
Naturally occurring enediyne antitumor antibiotics1,2
possessing either a 9- or 10-membered ring enediyne
core structure have shown potent antitumor activity that
was believed to result from DNA cleavage.3,4 The cyclic
enediyne core could be activated toward cycloaromati-
zation under physiological conditions, furnishing a 1,4-
benzenoid diradical species that abstracts hydrogen
atoms from DNA backbone to induce DNA strand scis-
sion responsible for lethal damages of cells. More
recently, antitumor activity of enediyne antibiotics has
been demonstrated to be attributable to not only
DNA cleavage but also RNA cleavage, protein agglom-
eration, and apoptosis.5
In our previous studies,8 we have established methodol-
ogies of generating enediynes in situ via rearrangement
of an allylic double bond under neutral,9 acidic,10 and
lanthanide (III) catalysis11,12 conditions, respectively.
We also have developed three synthetic routes to the
10-membered ring enediyne precursors: (a) intramolecu-
lar acetylide addition toward aldehyde;10c (b) intramo-
lecular Sonogashira cross-coupling reaction;13 and (c)
intramolecular Nozaki–Hiyama–Kishi reaction.12b,c
The precursors prepared by these synthetic routes were
confirmed to be converted into 10-membered ring
Keywords: Allylic rearrangement; DNA cleavage; Enediyne; Prodrug;
UV irradiation.
q
The Institute of Molecular Technology for Drug Discovery and
Synthesis is supported by the Areas of Excellence Scheme (AoE/P-10/
01) established under the University Grants Committee of Hong
Kong SAR, China.
*
Corresponding authors. Tel.: +852 2358 7365; fax: +852 2358 1594
(W.-M.D.); tel.: +81 75 383 2500; fax: +81 75 383 2501 (S.N.); e-mail
addresses: chdai@ust.hk; nishimot@scl.kyoto-u.ac.jp
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.12.040

3200
Y. Tachi et al. / Bioorg. Med. Chem. 14 (2006) 3199–3209
enediynes via an allylic cation intermediate in buffer
solution.14 A more remarkable result was that introduc-
tion into enediyne precursors with a neighboring nucle-
ophilic group could assist in the rearrangement of allylic
esters to cyclic enediynes. These enediyne precursors
would represent a synthetic model system that mimics
the intramolecular allylic rearrangement involved in
the activation of maduropeptin chromophore artifacts.8
mixture of the desired cross-coupling product and the
debromination by-product 8 (as E and Z mixtures)
was obtained from the pure (E)-9 in 14–38% combined
yields in THF, MeCN or DMF in the presence of
Et3N or K2CO3 at 22–80 ꢁC.17 On the other hand, the
cross-coupling of the minor isomer (Z)-9 with 1,7-octa-
diyne under similar conditions afforded the desired
product in 54–75% yields, which was free of the debro-
mination by-product 8. Reduction of (E)-9 with Dibal-
H afforded the alcohol (E)-10 in 96% yield. We tried
to reduce (Z)-9 by Dibal-H in a similar manner, but suc-
cessful result was not obtained due to decomposition of
the materials. From the bromo alcohol 10 the acyclic
enediyne 11 was obtained in 75% yield via the Sonogash-
ira coupling of 10 with 1,7-octadiyne. Unexpectedly,
isomerization of the double bond occurred during the
cross-coupling reaction and 11 was obtained as a 46:54
mixture of E and Z isomers. Conversion of 11 into the
iodoalkyne 12 was achieved in 90% yield by treatment
of the terminal alkyne with iodine and morpholine in
PhMe at 60 ꢁC.18 The E and Z isomers of the iodoalk-
yne 12 were separated and the pure (E)-12 was oxidized
to the aldehyde (E)-13 in a quantitative yield by treating
with PDC. In contrast, (Z)-12 decomposed into uniden-
tified materials in a similar PDC oxidation. The double
bond configurations of compounds 8–13 described
above were determined according to the NOE experi-
ments (see Figures S1–S6 in Supplementary data). The
intramolecular Nozaki–Hiyama–Kishi reaction of the
aldehyde with the iodoalkyne moiety within (E)-13 was
carried out with 3 equiv of CrCl2 and 1 equiv of
NiCl212b,c,16,18a in THF at room temperature under high
dilution conditions to give the exocyclic enediyne alco-
hol 1 in 24% yield. The rest of the materials was decom-
posed and could not be identified. Acetylation of the
alcohol 1 was performed with Ac2O–DMAP to give
the acetate 2 in 84% yield. Alternatively, treatment of
1 with MeOCH2CO2H under the DCC–DMAP condi-
tions furnished the methoxyacetate 3 in 52% yield.
Finally, desilylation with PPTS in MeOH at room tem-
perature transformed both silyl ethers 2 and 3 into the
corresponding alcohols 4 and 5, respectively, in excellent
yields.
As an extension of our work on the development of
antitumor prodrugs through activation into enediyne
formation, we synthesized the enediyne prodrugs 1–5
(Fig. 1) possessing an exocyclic 20-hydroxy-10-phenyle-
thylidene unit and evaluated their cytotoxicity and
DNA cleavage potency. Some results on the synthesis
and cytotoxicity were communicated in a short letter
before15 and the details on synthesis and DNA cleav-
age reactions are reported here. Among the enediyne
prodrugs synthesized, compounds 4 and 5 possessing
a free hydroxy group showed potent DNA cleavage
activity, which was induced by spontaneous allylic
rearrangement in basic buffer solutions, while the silyl
ether-protected analogs 1–3 could be activated by phot-
oirradiation to undergo an alkene isomerization–allylic
rearrangement process, resulting in efficient DNA
cleavage reaction as observed for the enediyne pro-
drugs 4 and 5.
2. Results and discussion
2.1. Chemistry
Synthesis of the enediyne prodrugs 1–5 from the com-
mercially available starting material, 2-hydroxyacetoph-
enone (6), is outlined in Scheme 1. Following protection
of the hydroxyl group of 6, the resultant silyl ether 7 was
subjected to a Horner–Wadsworth–Emmons reaction to
give the unsaturated ester 8 in a quantitative yield as a
40:60 mixture of E and Z isomers. After column chro-
matographic separation, the pure (E)-8 was treated with
bromine to form a dibromo adduct, which, without fur-
ther purification, was subjected to a base-induced elimi-
nation of HBr to give the vinyl bromide 9 as a 70:30
mixture of E and Z isomers. Unfortunately, (Z)-8 could
not be transformed into 9 with recovery of the starting
materials quantitatively. Our initial attempts were made
to carry out the cross-coupling of 9 with 1,7-octadiyne
under the catalysis of Pd(0) and Cu(I). However, a
2.2. DNA cleavage
The DNA cleavage activity of enediyne prodrugs 1–5
was evaluated by monitoring the conversion of circular
supercoiled DNA (Form I) to circular relaxed (Form
II) or linear (Form III) DNA. UX174 RFI supercoiled
DNA (Form I) was incubated with various enediyne
prodrugs in TAE buffer solution (pH 8.5) containing
20% DMSO at 37 ꢁC for 72 h, and then the reaction
mixtures were analyzed by gel electrophoresis over 1%
agarose gel (ethidium bromide stain). Figure 2 shows
the representative gel picture (A) observed at various
drug concentrations and the corresponding scanning
densitometry results (B). The enediyne prodrugs 1–3
with a silyl-protected hydroxy group were generally very
weak in causing DNA damage. In contrast, the enediyne
prodrugs 4 and 5 exhibited DNA cleaving activity in a
concentration-dependent manner. About 49% and 41%
net DNA strand scissions were observed for the acetate
Figure 1. Molecular structures of enediyne prodrugs 1–5.

Y. Tachi et al. / Bioorg. Med. Chem. 14 (2006) 3199–3209
3201
Scheme 1. Synthesis of enediyne prodrugs 1–5.
4 and the methoxyacetate 5, respectively, at 1.0 mM
drug concentration. However, formation of Form III
DNA was not observed for the action of the enediyne
prodrugs 1–5.
It was reported that irradiation of 2,5-dihydrofuran in-
duced a photochemical [1,3]-shift of an oxygen substitu-
ent to form 3,4-epoxybut-1-ene.19a A similar [1,3]-shift
of allylic hydroxyl or methoxy group occurred under
photochemical conditions in 8-hydroxy- and 8-meth-
oxy-germacrene B.19b,19c The medium-sized ring of the
germacrene B derivatives was crucial for the photochem-
ical [1,3]-shifts. Photochemistry of the 4-ethyl-4-methyl
disubstituted 3-alkylidene-2-naphthalenol derivatives
was reported20a but the results of the photochemical
[1,3]-OH shift were not reproduced.20b As compared to
[1,3]-shifts of allylic alcohol photochemical isomeriza-
tion of E and Z configurations of alkenes was known
to be much faster.19d,20 We anticipated that if a similar
photochemical [1,3]-shift of the oxygenated group X in
1–5 (Fig. 1) or a photochemical isomerization of the
exocyclic double bond takes place (vide infra), enhanced
DNA cleavage potency would be observed under UV
irradiation. For examination of the photoinduced
DNA cleavages by the enediyne prodrugs 1–5, we car-
ried out the comparative experiments without and with
photoirradiation at 365 nm. The mixtures of UX174
RFI DNA with 1.0 mM prodrugs 1–5 in TAE buffer
solution containing 20% DMSO were irradiated for 4
and 8 h, respectively, followed by incubation along with
the non-irradiated controls for 72 h. The representative
gel picture and the corresponding scanning densitometry
results are shown in Figure 4. It was confirmed that the
Figure 3 illustrates the time dependency of DNA
cleavage by the enediyne prodrugs 1–5 evaluated with
1.0 mM drug concentration under the identical condi-
tions as described in Figure 2. The extents of DNA
cleavage by 4 and 5 increased with incubation time
(12%, 30%, and 37% for 4; 18%, 27%, and 31% for
5 after 24, 48, and 72 h incubation, respectively) and
significant cleavages of about 30% were observed after
incubation for 48 h. Although the cleavage values are
slightly lower than those given in Figure 2, the relative
DNA cut potency is consistent with the fact that the
acetate 4 is more active than the methoxyacetate 5
in pH 8.5 buffer at 37 ꢁC for 72 h. Moreover, DNA
strand breakage by the silyl-protected analogs 1–3
was negligible at different incubation durations. On
the basis of the results shown in Figures 2 and 3,
we can conclude that the enediyne prodrugs 1–3 are
not activated in basic buffer at pH 8.5 while the ened-
iyne prodrugs 4 and 5 may undergo a self-assisted
allylic rearrangement in pH 8.5 buffer to migrate the
exocyclic double bond into the endocyclic position,
resulting in formation of a reactive 10-membered
enediyne (vide infra).

3202
Y. Tachi et al. / Bioorg. Med. Chem. 14 (2006) 3199–3209
Figure 4. (A) Gel picture of UV-promoted DNA cleavage by enediyne
prodrugs 1–5. (B) Scanning densitometry results of the gel picture
shown in (A). UX174 RFI DNA (50 lM/bp) was incubated without
drug (controls) or with 1.0 mM of 1–5 for 72 h at 37 ꢁC without UV
irradiation (lanes 1, 4, 7, 10, 13, and 16) or with UV irradiation for 4 h
(lanes 2, 5, 8, 11, 14, and 17) and 8 h (lanes 3, 6, 9, 12, 15, and 18),
respectively, in TAE buffer (pH 8.5) containing 20% DMSO and
analyzed by gel electrophoresis (1% agarose gel, ethidium bromide
stain). The percentage of net DNA cleavage was calculated in the same
manner as given in Figure 2. The net DNA cleavage values are 5.4%,
25.6%, and 29.4% for 1, 3.1, 34.7 and 34.7% for 2, 7.5%, 26.8%, and
27.7% for 3, 33.6%, 31.0%, and 23.3% for 4 and 28.3%, 39.9%, and
41.3% for 5, respectively, without UV irradiation and with UV
irradiation for 4 and 8 h.
Figure 2. (A) Gel picture of concentration-dependent DNA cleavage
by enediyne prodrugs 1–5. (B) Scanning densitometry results of the gel
picture shown in (A). UX174 RFI DNA (50 lM/bp) was incubated for
72 h at 37 ꢁC without drug (control) or with 0.1 mM (lanes 2, 5, 8, 11,
and 14), 0.5 mM (lanes 3, 6, 9, 12, and 15) and 1.0 mM (lanes 4, 7, 10,
13, and 16) of 1–5 in TAE buffer (pH 8.5) containing 20% DMSO and
analyzed by gel electrophoresis (1% agarose gel, ethidium bromide
stain). Form I is the supercoiled DNA. Form II and Form III bands
represent the single- and double-strand cleaved DNA products,
respectively (see Ref. 24). The percentage of net DNA cleavage was
calculated by the following equation: {[(Form II)s/[(Form I)s + (Form
II)s] · 100}ꢁ{(Form II)c/[(Form I)c + (Form II)c] · 100}. The sub-
scripts ‘s’ and ‘c’ refer to as the samples and control(s), respectively.
The net DNA cleavage values are 9.7%, 30.3%, and 49.0% for 4 and
11.4%, 26.2%, and 40.8% for 5, respectively, at 0.1, 0.5, and 1.0 mM
drug concentrations. TAE buffer = Tris + acetic acid + EDTA.
enediyne prodrugs 1–3 with a silyl-protected hydroxy
group could be activated by UV irradiation and the
DNA cleavage values are 26%, 35%, and 27% (4 h UV
irradiation) and 29%, 35%, and 28% (8 h UV irradia-
tion) for 1–3, respectively. The results indicate that 4 h
UV irradiation prior to incubation in pH 8.5 buffer is
sufficient for activating 1–3, resulting in about 30% net
DNA strand scission comparable to those of the ened-
iyne prodrugs 4 and 5. We also observed some interest-
ing behaviors of the enediyne prodrugs 4 and 5 under
photoirradiation. Without UV irradiation, the acetate
4 caused 34% net DNA cleavage which is higher than
28% damaged by the methoxyacetate 5. However, the
potency of 4 decreased to 31% and 24% of net DNA
cut after 4 and 8 h UV exposure, respectively. In con-
trast, the methoxyacetate 5 produced enhanced DNA
cleavage potency to 40% and 41% after UV irradiation
for 4 and 8 h. Therefore, the DNA scission potency with
4 h UV irradiation follows the order of 5 (40%) > 2
(35%) > 4 (31%) > 3 (27%) ꢀ 1 (26%).
Figure 3. (A) Gel picture of time-dependent DNA cleavage by
enediyne prodrugs 1–5. (B) Scanning densitometry results of the gel
picture shown in (A). UX174 RFI DNA (50 lM/bp) was incubated
without drug (controls) or with 1.0 mM of 1–5 at 37 ꢁC for 1 day (lanes
1, 4, 7, 10, 13, and 16), 2 days (lanes 2, 5, 8, 11, 14, and 17) and 3 days
(lanes 3, 6, 9, 12, 15, and 18) in TAE buffer (pH 8.5) containing 20%
DMSO and analyzed by gel electrophoresis (1% agarose gel, ethidium
bromide stain). The percentage of net DNA cleavage was calculated in
the same manner as given in Figure 2. The net DNA cleavage values
are 12.3%, 30.0%, and 37.2% for 4 and 18.4%, 26.8%, and 30.8% for 5,
respectively, at 24, 48, and 72 h incubation time.
2.3. Mechanisms of action
We considered that the enediyne prodrugs 4 and 5 were
converted into the epoxy enediyne 14 in basic buffer via
an intramolecular SN20 substitution reaction with the
departure of acetate or methoxyacetate anion (Scheme
2). Conversion of 4 and 5 into 14 is also possible to in-
volve an allylic cation intermediate, which is trapped
intramolecularly by the free hydroxy group.14 Accord-
ing to the pKa values of acetic acid (4.76) and methoxy-

Y. Tachi et al. / Bioorg. Med. Chem. 14 (2006) 3199–3209
3203
Scheme 2. Proposed mechanisms of base- and UV-promoted DNA cleavage by enediyne prodrugs 1–5.
acetic acid (3.53), the methoxyacetate 5 should be easily
converted into 14 than the acetate 4. It seems to be not
consistent with a higher potency of 4 than that of 5 in
DNA strand scission and cytotoxicity.15 One possibility
is that the acetate 4 slowly releases the reactive species,
while the methoxyacetate 5 serves as a fast-acting
DNA cleaver. Non-DNA cutting decomposition of the
epoxy enediyne 14 might occur in the action of 5.
conversion of 1–5 into a 10-membered ring enediyne
of the structure 18 [Y = OH, OAc, and OC(O)CH2OMe;
Z = t-BuMe2SiO] under photochemical conditions but
we are confident that it is not a main reaction pathway
due to a fast alkene isomerization under photoirradia-
tion.19,20 The diminished activity of 4 with UV activa-
tion is unusual according to our discussion given
above. We suspect that a non-DNA cutting pathway
might be competing with formation of 14 and the details
are not known.
According to our previous studies on allylic rearrange-
ment of acyclic 1,2-dialkynylallyl alcohols,10d cleavage
of X in 1–3 to form the less stable sickle allylic cation
16 is difficult as compared to formation of the more sta-
ble sickle allylic cation 17 from the isomeric precursors
15. It accounts for the inactivity of the silyl-protected
analogs 1–3 in pH 8.5 buffer. Upon UV irradiation, a
fast isomerization of the exocyclic double bond should
occur to convert 1–3 into the alkene isomers 15.19d,20
The latter decomposed in buffer to the sickle allylic cat-
ion 17 followed by attack of the neighboring silyloxy
group with migration of the double bond into the endo-
cyclic position. After loss of the silyl group from the
intermediate, the epoxy enediyne 14 is formed. It was
found that the acetate 2 was slightly more potent than
the methoxyacetate 3 under photochemical conditions.
The results are parallel to those of non-UV-activated
DNA damage by 4 and 5 shown in Figure 3. Involve-
ment of a common reactive species 14 for DNA cleavage
of 1–5 under UV irradiation is reasonably supported.
We cannot definitely rule out the possibility of direct
We made a lot of effort in the isolation of the epoxy
enediyne 14 but without success due to its instability
during purification over silica gel. Therefore, we tried
to detect its presence in solution by mass spectrometry.
The compound 4 (1.0 mM) in TAE buffer solution (pH
8.5) containing 20% DMSO was incubated at 37 ꢁC for
72 h in the absence of a DNA substrate and the mixture
was subjected to mass analysis under the +FAB condi-
tions. Figures 5 and 6 show the recorded mass spectrum
of the crude reaction mixture and the possible fragment
ions, respectively. The peak m/z 249 (M+H+) corre-
sponds to the ion III (the protonated epoxy enediyne
14) or II (formed from the protonated 4 by loss of
HOAc) as shown in Figure 6. We carefully examined
the mass spectrum of 4 obtained under +FAB condi-
tions (spectrum not shown) and found, in addition to
the base peak m/z 249 (M+H+ꢁHOAc), the molecular
ion m/z 309 (M+H+, 10% of relative intensity). Howev-
er, in the mass spectrum shown in Figure 5, the ion m/z

3204
Y. Tachi et al. / Bioorg. Med. Chem. 14 (2006) 3199–3209
Figure 5. Mass spectrum of the reaction mixture of the enediyne prodrug 4 (1.0 mM) in TAE buffer (pH 8.5) containing 20% DMSO after incubated
at 37 ꢁC for 72 h. The reaction mixture was analyzed with a JEOL JMX-SX 102 A spectrometer under +FAB conditions. The structures of the ions
II–V are found in Figure 6.
products 18a,b, which then undergo a similar thermal
cycloaromatization described for 14 in Scheme 2.
Although reactions of epoxy compounds with DNA
have been extensively reported in the literature,22 we
have not experimentally confirmed for 14 and the relat-
ed precursors in this study. Nevertheless, the epoxy
enediyne 14 represents a synthetic model system for
mimicking the intramolecular allylic rearrangement for
the activation of the maduropeptin chromophore
artifacts.23
Cytotoxicity of compounds 1–5 was evaluated against
P388 cancer cell line.15 Compounds 1–3 with a silyl-pro-
tected hydroxy group exhibited similar IC50 values of
8.6–17.2 lM, while compounds 4 and 5 with a free
hydroxyl group gave IC50 values of 39.0–54.2 lM. Obvi-
ously, the cytotoxicity data are opposite to the observed
DNA cleavage activity of these compounds in the ab-
sence of UV activation (Figs. 2 and 3). One possibility
is that the protected compounds 1–3 converted into
the active enediyne species slowly than compounds 4
and 5, resulting in a long-lasting action on the cancer
cells. However, the nature of the difference in two assays
for compounds 1–5 is not clear.
Figure 6. Proposed structures for the molecular and fragment ions
seen in the mass spectra of 4 (not shown) and 14 (given in Fig. 5).
3. Conclusion
309 was not seen. The results imply that the ion of m/z
249 in Figure 5 is for the structure III. The mass peaks
m/z 231 and m/z 221 are the fragments of the ions II
and III, and they were observed in all mass spectra of
1–5 measured under +CI conditions by using CH4 and
NH3 as the ion source. In combination with the DNA
cleavage data presented in Figures 2 and 3, we suggest
that compounds 4 and 5 incubated at pH 8.5 underwent
a base-promoted allylic rearrangement to form the
epoxy enediyne 14. The latter thus generated in situ
can undergo thermal cycloaromatization to form 1,4-
benzenoid diradical species 19, thereby resulting in
DNA strand cleavage through abstraction of hydrogen
atoms from the sugar-phosphate backbone (Scheme
2).1a,3 On the other hand, there is a possibility for the
epoxy enediyne 14 damaging DNA via base alkylation
through the epoxy moiety21 to form the DNA alkylation
We have designed and synthesized the enediyne pro-
drugs 1–5 possessing an (E)-3-hydroxy-4-(20-hydroxy-
10-phenylethylidene)cyclodeca-1,5-diyne scaffold and
evaluated their DNA cleavage reactions in pH 8.5 buffer
without and with UV irradiation. The enediyne pro-
drugs 4 and 5 exhibited concentration- and time-depen-
dent DNA strand scission in basic buffer (pH 8.5) at
37 ꢁC, while the silyl-protected analogs 1–3 are almost
inactive. The results suggest that a base-promoted intra-
molecular allylic rearrangement takes place to convert 4
and 5 into a reactive epoxy enediyne 14, which can cause
DNA strand cleavage most likely via hydrogen atom
abstraction from the sugar-phosphate backbone. Under
photochemical conditions, the enediyne prodrugs 1–3
could be activated to enter the alkene isomerization–al-
lylic rearrangement cascade of reactions, leading to for-
mation of the same epoxy enediyne 14. An enhanced

Y. Tachi et al. / Bioorg. Med. Chem. 14 (2006) 3199–3209
3205
potency for the methoxyacetate 5 with UV irradiation
was observed although the acetate 4 gave the opposite
result for some unknown reasons. With the help of mass
spectrometry, the putative epoxy enediyne 14 was
detected in the incubation mixture of the acetate 4 under
the conditions used for DNA cleavage experiments.
Based on the results, the enediyne prodrugs 1–3 are
identified to be potentially useful for photoactivated
prodrugs. Thus, enediyne precursors 1–5 are potential
prototype compounds for a new class of antitumor
prodrugs.
4.2.2. (E)- and (Z)-4-(tert-Butyldimethylsilyloxy)-3-phe-
nylbut-2-enoic acid methyl ester [(E)- and (Z)-8]. To a
solution of trimethyl phosphonoacetate (2.8 mL,
17.1 mmol) in dry THF (30 mL) cooled in a dry ice–ac-
etone bath (ꢁ78 ꢁC) was added n-BuLi (1.6 M in hex-
ane, 13.3 mL, 21.3 mmol) followed by stirring at the
same temperature for 30 min. To the resultant mixture
was added a solution of 7 (3.563 g, 14.2 mmol) in dry
THF (20 mL). The resultant mixture was then allowed
to warm up to room temperature and stirred for 5 h at
the same temperature. The reaction was quenched with
saturated aqueous NH4Cl (10 mL) and extracted with
EtOAc (3· 10 mL). The combined organic layer was
washed with brine, dried over anhydrous MgSO4, fil-
tered, and concentrated under reduced pressure. The
residue was purified by flash column chromatography
(silica gel, 1% EtOAc–hexane) to give (Z)-8 (1.729 g,
60%) and (E)-8 (2.613 g, 40%).
4. Experimental
4.1. General methods
All reactions were carried out under a dry nitrogen
atmosphere using freshly distilled solvents unless other-
wise noted. Reagents were purchased from suppliers and
used without further purification. Tetrahydrofuran
(THF) and toluene were distilled over sodium and ben-
zophenone. Dichloromethane, acetonitrile, and DMF
were distilled over calcium hydride. Reactions were
monitored by thin-layer chromatography (TLC) on E.
Merck silica gel plates (Silica Gel 60 F254) using UV
light and 7% ethanolic phosphomolybdic acid and heat
as the visualizing methods. Flash column chromatogra-
phy was carried out on E. Merck silica gel (230–400
mesh). NMR Spectra were recorded on a 300 MHz spec-
trometer at ambient temperature. Chemical shifts are
reported in ppm related to residual chloroform
Compound (E)-8. A colorless oil; Rf = 0.46 (10%
EtOAc–hexane); IR (neat) 2953, 2857, 1716, 1633,
1
1257, 1171, 1098 cmꢁ1; H NMR (300 MHz, CDCl3) d
7.50–7.46 (m, 2H), 7.37–7.33 (m, 3H), 6.05 (t,
J = 1.0 Hz, 1H), 5.19 (d, J = 1.2 Hz, 2H), 3.77 (s, 3H),
0.77 (s, 9H), 0.02 (s, 6H); 13C NMR (75.5 MHz, CDCl3)
d 166.3, 158.8, 139.0, 128.6, 127.9 (2·), 127.6 (2·), 117.1,
59.7, 51.4, 25.8 (3·), 18.2, ꢁ5.2 (2·); MS (+CI) m/z (rel-
ative intensity) 307 (M+H+, 100); HRMS (+FAB) calcd
for C17H27O3Si (M+H+) 307.1729, found 307.1734.
Compound (Z)-8. A colorless oil; Rf = 0.60 (10%
EtOAc–hexane); IR (neat) 2954, 2858, 1732, 1659,
1
1
(d = 7.26 in H NMR and d = 77.0 in 13C NMR). Mul-
1223, 1158 cmꢁ1; H NMR (300 MHz, CDCl3) d 7.37–
tiplicity is designated as singlet (s), doublet (d), doublet
of doublet (dd), triplet (t), triplet of doublet (td), or mul-
tiplet (m). IR spectra were measured on a FT-IR spec-
trophotometer. Mass spectra were recorded under +CI
or +FAB conditions.
7.33 (m, 3H), 7.19–7.16 (m, 2H), 6.23 (t, J = 2.3 Hz,
1H), 4.35 (d, J = 1.8 Hz, 2H), 3.58 (s, 3H), 0.97 (s,
9H), 0.13 (s, 6H); 13C NMR (75.5 MHz, CDCl3) d
166.4, 158.2, 137.2, 127.9, 127.9 (2·), 127.2 (2·), 114.5,
66.9, 51.1, 26.0 (3·), 18.5, ꢁ5.3 (2·); MS (+CI) m/z (rel-
ative intensity) 307 (M+H+, 100); HRMS (+FAB) calcd
for C17H27O3Si (M+H+) 307.1729, found 307.1735.
4.2. Synthesis of enediyne prodrugs
4.2.1. 2-(tert-Butyldimethylsilyloxy)acetophenone (7). To
a
4.2.3. (E)- and (Z)-2-Bromo-4-(tert-butyldimethylsilyl-
oxy)-3-phenylbut-2-enoic acid methyl ester [(E)- and
(Z)-9]. To a solution of (E)-8 (2.622 g, 8.6 mmol) in
dry CH2Cl2 (43 mL) cooled in a dry ice–acetone bath
(ꢁ78 ꢁC) was added bromine (0.88 mL, 17.1 mmol) in
dry CH2Cl2 (10 mL) through a dropping funnel, fol-
lowed by stirring at the same temperature for 12 h. To
the resultant mixture was added triethylamine (6.0 mL,
42.8 mmol), followed by stirring at the same tempera-
ture for 38 h. The reaction was then quenched with sat-
urated aqueous NH4Cl (20 mL) and extracted with
CH2Cl2 (3· 20 mL). The combined organic layer was
washed with saturated aqueous Na2S2O3 (50 mL) and
brine, dried over anhydrous MgSO4, filtered, and con-
centrated under reduced pressure. The residue was puri-
fied by flash column chromatography (silica gel, 1%
EtOAc–hexane) to give a 70:30 mixture of (E)-9 and
(Z)-9 (2.326 g, 71%).
solution of 2-hydroxyacetophenone (2.000 g,
14.7 mmol) and imidazole (1.500 g, 22.0 mmol) in dry
DMF (30 mL) cooled in an ice-water bath (0 ꢁC)
was added tert-butyldimethylsilyl chloride (2.657 g,
17.6 mmol) followed by stirring the resultant mixture
at room temperature for 30 min. The reaction was
quenched with cold water (250 mL) and extracted with
EtOAc (3· 80 mL). The combined organic layer was
washed with brine, dried over anhydrous MgSO4, fil-
tered, and concentrated under reduced pressure. The
residue was purified by flash column chromatography
(silica gel, 5% EtOAc–hexane) to give 7 (3.563 g, 97%)
as a colorless oil; Rf = 0.51 (10% EtOAc–hexane); IR
(neat) 2930, 2857, 1707, 1255, 1155 cmꢁ1 1H NMR
;
(300 MHz, CDCl3) d 7.92 (d, J = 6.9 Hz, 2H), 7.57 (t,
J = 7.4 Hz, 1H), 7.45 (t, J = 7.5 Hz, 2H), 4.93 (s, 2H),
0.95 (s, 9H), 0.15 (s, 6H); 13C NMR (75.5 MHz, CDCl3)
d 197.2, 134.8, 133.1, 128.5 (2·), 127.8 (2·), 67.5, 25.9
(3·), 18.6, ꢁ5.2 (2·); MS (+CI) m/z (relative intensity)
251 (M+H+, 31), 235 (100); HRMS (+FAB) calcd for
C14H23O2Si (M+H+) 251.1468, found 251.1483.
Compound (E)-9. A yellow oil; Rf = 0.54 (10% EtOAc–
hexane); IR (neat) 2953, 2930, 2857, 1734, 1232,
1101 cmꢁ1 1H NMR (300 MHz, CDCl3) d 7.33–7.28
;

3206
Y. Tachi et al. / Bioorg. Med. Chem. 14 (2006) 3199–3209
(m, 3H), 7.20–7.15 (m, 2H), 4.67 (s, 2H), 3.51 (s, 3H),
0.76 (s, 9H), ꢁ0.03 (s, 6H); 13C NMR (75.5 MHz,
CDCl3) d 164.9, 149.0, 137.6, 127.9, 127.9 (2·), 127.7
(2·), 109.6, 66.2, 52.7, 25.7 (3·), 18.2, ꢁ5.4 (2·); MS
(+CI) m/z (relative intensity) 387 (M+2+H+, 81Br, 30),
385 (M+H+, 79Br, 29), 255 (M++2ꢁOSiMe2t-Bu, 81Br,
100), 253 (M+ꢁOSiMe2t-Bu, 79Br, 100); HRMS
(+FAB) calcd for C17H26BrO3Si (M+H+, 79Br)
385.0835, found 385.0830.
sure. The residue was purified by flash column chroma-
tography (silica gel, 5% EtOAc–hexane) to give a 46:54
mixture of (E)-11 and (Z)-11 (0.357 g, 75%).
Compound (E)-11. A yellow oil; Rf = 0.34 (20% EtOAc–
hexane); IR (neat) 3406, 2930, 2858, 2216, 2118, 1463,
1
1256, 1096 cmꢁ1; H NMR (300 MHz, CDCl3) d 7.35–
7.28 (m, 3H), 7.18–7.15 (m, 2H), 4.69 (s, 2 H), 4.01 (s,
2H), 2.52–2.48 (m, 2H), 2.30–2.25 (m, 2H), 1.98 (t,
J = 2.7 Hz, 1H), 1.79–1.72 (m, 4H), 0.79 (s, 9H), ꢁ0.04
(s, 6H); 13C NMR (75.5 MHz, CDCl3) d 147.3, 137.8,
128.6 (2·), 127.8 (2·), 127.3, 121.0, 96.8, 83.9, 77.9,
68.7, 65.7, 62.6, 27.7, 27.7, 26.0 (3·), 19.3, 18.3, 18.1,
ꢁ5.3 (2·); MS (+CI) m/z (relative intensity) 383
(M+H+, 18), 381 (MꢁH+, 26), 365 (M+ꢁOH, 85), 251
(M+ꢁOSiMe2t-Bu, 58), 233 (100); HRMS (+FAB) calcd
for C24H33O2Si [(MꢁH)+] 381.2250, found 381.2249.
Compound (Z)-9. A yellow oil; Rf = 0.50 (10% EtOAc–
hexane); IR (neat) 2953, 2930, 2857, 1732, 1252,
1106 cmꢁ1 1H NMR (300 MHz, CDCl3) d 7.42–7.30
;
(m, 3H), 7.23–7.19 (m, 2H), 4.65 (s, 2H), 3.88 (s, 3H),
0.79 (s, 9H), ꢁ0.05 (s, 6H); 13C NMR (75.5 MHz,
CDCl3) d 164.7, 150.8, 139.1, 127.9, 127.8 (2·), 127.8
(2·), 110.3, 64.6, 53.1, 25.8 (3·), 18.3, ꢁ5.5 (2·); MS
(+CI) m/z (relative intensity) 387 (M+2+H+, 81Br, 87),
385 (M+H+, 79Br, 91), 255 (M++2ꢁOSiMe2t-Bu, 81Br,
100), 253 (M+ꢁOSiMe2t-Bu, 79Br, 100); HRMS
(+FAB) calcd for C17H26BrO3Si (M+H+, 79Br)
385.0835, found 385.0819.
Compound (Z)-11. A yellow oil; Rf = 0.40 (20% EtOAc–
hexane); IR (neat) 3430, 2930, 2858, 2216, 2118, 1463,
1
1256, 1109, 1074 cmꢁ1; H NMR (300 MHz, CDCl3) d
7.41–7.27 (m, 5H), 4.52 (s, 2 H), 4.36 (s, 2H), 2.27–
2.23 (m, 2H), 2.13–2.07 (m, 2H), 1.95 (t, J = 2.7 Hz,
1H), 1.53–1.38 (m, 4H), 0.88 (s, 9H), ꢁ0.04 (s, 6H);
13C NMR (75.5 MHz, CDCl3) d 145.8, 140.9, 128.3
(2·), 127.7 (2·), 127.2, 123.8, 94.6, 84.1, 80.3, 68.4,
63.7, 62.7, 27.3, 27.2, 25.9 (3·), 19.1, 18.3, 18.0, ꢁ5.3
(2·); MS (+CI) m/z (relative intensity) 383 (M+H+,
18), 381 (MꢁH+, 44), 365 (M+ꢁOH, 43), 251
(M+ꢁOSiMe2t-Bu, 87), 233 (94); HRMS (+FAB) calcd
for C24H33O2Si [(MꢁH)+] 381.2250, found 381.2255.
4.2.6. (E)- and (Z)-2-[20-(tert-Butyldimethylsilyloxy)-10-
phenylethylidene]-10-iododeca-3,9-diyn-1-ol [(E)- and
(Z)-12]. To a solution of iodine (5.002 g, 19.7 mmol) in
toluene (100 mL) was added morpholine (4.6 mL,
52.6 mmol). The resultant mixture was heated at 60 ꢁC
with stirring for 40 min. To the resultant mixture was
added a 46:54 mixture of (E)-11 and (Z)-11 (2.422 g,
6.6 mmol) in toluene (100 mL), followed by stirring at
60 ꢁC for 18 h. The reaction mixture was allowed to cool
down and purified directly by flash column chromatog-
raphy (silica gel, hexane then 20% EtOAc–hexane) to
give a 46:54 mixture of (E)-12 and (Z)-12 (2.925 g, 90%).
4.2.4. (E)-2-Bromo-4-(tert-butyldimethylsilyloxy)-3-phe-
nylbut-2-en-1-ol [(E)-10]. To
a solution of (E)-9
(1.782 g, 4.6 mmol) in dry CH2Cl2 (23 mL) cooled in a
dry ice–acetone bath (ꢁ78 ꢁC) was added Dibal-H (1.0
M in THF, 23.1 mL, 23.1 mmol), followed by stirring
at the same temperature for 1 h. The reaction was then
quenched with methanol (20 mL) at ꢁ78 ꢁC, followed
by warming to room temperature. Saturated aqueous
NH4Cl (20 mL) was added and the resultant mixture
was stirred at room temperature for 1 h and extracted
with CH2Cl2 (3· 30 mL). The combined organic layer
was washed with brine, dried over anhydrous MgSO4,
filtered, and concentrated under reduced pressure. The
residue was purified by flash column chromatography
(silica gel, 5% EtOAc–hexane) to give (E)-10 (1.589 g,
96%) as a pale yellow oil; Rf = 0.49 (20% EtOAc–hex-
ane); IR (neat) 3391, 2930, 2858, 1945, 1875, 1803,
1728, 1644, 1471, 1256, 1086, 1004 cmꢁ1 1H NMR
;
(300 MHz, CDCl3) d 7.34–7.30 (m, 3H), 7.22–7.20 (m,
2H), 4.62 (s, 2H), 4.47 (s, 2H), 0.87 (s, 9H), 0.00 (s,
6H); 13C NMR (75.5 MHz, CDCl3) d 142.2, 141.3,
128.0, 128.0 (2·), 127.4 (2·), 127.2, 65.5, 64.2, 25.8
(3·), 18.3, ꢁ5.4 (2·); MS (+CI) m/z (relative intensity)
359 (M+2+H+, 81Br, 11), 357 (M+H+, 79Br, 16), 341
(M++2ꢁOH, 81Br, 100), 339 (M+ꢁOH, 79Br, 100);
HRMS (+FAB) calcd for C16H26BrO2Si (M+H+, 79Br)
357.0886, found 357.0898.
Compound (E)-12. A yellow oil; Rf = 0.34 (20% EtOAc–
hexane); IR (neat) 3401, 2929, 2857, 2215, 1255, 1095,
1005 cmꢁ1 1H NMR (300 MHz, CDCl3) d 7.34–7.27
;
(m, 3H), 7.19–7.13 (m, 2H), 4.69 (s, 2H), 4.01 (s, 2H),
2.52–2.42 (m, 4H), 1.80–1.68 (m, 4H), 0.80 (s, 9H),
ꢁ0.03 (s, 6H); 13C NMR (100 MHz, CDCl3) d 147.3,
137.8, 128.6 (2·), 127.8 (2·), 127.3, 121.0, 96.7, 94.0,
78.0, 68.7, 65.7, 62.6, 27.8, 27.7, 25.9 (3·), 20.5, 19.3,
18.3, ꢁ5.2 (2·); MS (+CI) m/z (relative intensity) 509
(M+H+, 7), 491 (M+ꢁOH, 68), 345 (100); HRMS
(+FAB) calcd for C24H32IO2Si [(MꢁH)+] 507.1216,
found 507.1220.
4.2.5. (E)- and (Z)-2-[20-(tert-Butyldimethylsilyloxy)-10-
phenylethylidene]deca-3,9-diyn-1-ol [(E)- and (Z)-11]. To
a suspension of Pd(PPh3)4 (0.144 g, 0.12 mmol) and
CuI (0.047 g, 0.25 mmol) in degassed CH3CN (0.8 mL)
were added (E)-10 (0.445 g, 1.20 mmol), triethylamine
(1.2 mL), and 1,7-octadiyne (0.5 mL, 3.70 mmol) in de-
gassed CH3CN (4.0 mL). The reaction mixture was re-
fluxed with stirring in dark for 18 h. The reaction was
quenched with saturated aqueous NH4Cl (5 mL) and
extracted with EtOAc (3· 10 mL). The combined organ-
ic layer was washed with brine, dried over anhydrous
MgSO4, filtered, and concentrated under reduced pres-
Compound (Z)-12. A yellow oil; Rf = 0.40 (20% EtOAc–
hexane); IR (neat) 3419, 2929, 2857, 2216, 2117, 1256,
1
1109, 1073, 1005 cmꢁ1; H NMR (300 MHz, CDCl3) d
7.41–7.26 (m, 5H), 4.52 (s, 2H), 4.36 (d, J = 5.1 Hz,
2H), 2.28–2.22 (m, 4H), 1.50–1.35 (m, 4H), 0.88 (s,

Y. Tachi et al. / Bioorg. Med. Chem. 14 (2006) 3199–3209
3207
9H), 0.04 (s, 6H); 13C NMR (100 MHz, CDCl3) d 145.9,
140.9, 128.2 (2·), 127.7 (2·), 127.2, 123.8, 94.5, 94.2,
80.4, 68.4, 63.7, 62.7, 27.3, 27.2, 25.9 (3·), 20.4, 19.1,
18.3, ꢁ5.3 (2·); MS (+CI) m/z (relative intensity) 509
(M+H+, 9), 491 (M+ꢁOH, 65), 250 (100); HRMS
(+FAB) calcd for C24H32IO2Si [(MꢁH)+] 507.1216,
found 507.1194.
was filtered through a short plug of silica gel with rinsing
by CH2Cl2. The filtrate was concentrated under reduced
pressure and the residue was purified by flash column
chromatography (silica gel, 5% EtOAc–hexane) to give
2 (44.4 mg, 84%) as a pale brown oil; Rf = 0.60 (20%
1
EtOAc–hexane); IR (neat) 2930, 1220, 1092 cmꢁ1; H
NMR (300 MHz, CDCl3) d 7.35–7.23 (m, 3H), 7.12–
7.06 (m, 2H), 5.53 (s, 1H), 4.71 and 4.64 (ABq,
J = 12.3 Hz, 2H), 2.58–2.15 (m, 4H), 1.95 (s, 3H),
1.86–1.64 (m, 4H), 0.78 (s, 9H), ꢁ0.07 (s, 6H); 13C
NMR (75.5 MHz, CDCl3) d 169.3, 149.0, 137.0, 128.6,
128.2 (2·), 127.8 (2·), 127.6, 119.8, 102.4, 92.3, 79.3,
66.6, 65.4, 27.7 (2·), 25.8 (3·), 21.8, 21.0, 20.8, 18.3,
ꢁ5.3 (2·); MS (+CI) m/z (relative intensity) 423
(M+H+, 5), 363 (M+ꢁOAc, 100), 249 (10), 233 (14),
231 (10); HRMS (+FAB) calcd for C26H35O3Si
(M+H+) 423.2355, found 423.2352.
4.2.7. (E)-2-[20-(tert-Butyldimethylsilyloxy)-10-phenyle-
thylidene]-10-iododeca-3,9-diynal [(E)-13]. To a suspen-
˚
sion of (E)-12 (0.782 g, 1.5 mmol) and powdered 4A
molecular sieves in dry CH2Cl2 (7.7 mL) was added
PDC (0.578 g, 3.1 mmol), followed by stirring at room
temperature for 4 h. The reaction mixture was filtered
through a short plug of silica gel with rinsing by
CH2Cl2. The filtrate was concentrated under reduced
pressure and the residue was purified by flash column
chromatography (silica gel, 3% EtOAc–hexane) to give
(E)-13 (0.776 g, 100%) as a yellow oil; Rf = 0.60 (20%
EtOAc–hexane); IR (neat) 2930, 2858, 2225, 2118,
4.2.10. (E)-4-[20-(tert-Butyldimethylsilyloxy)-10-phenyle-
thylidene]-3-(methoxyacetoxy)-cyclodeca-1,5-diyne (3).
To a solution of 1 (50.2 mg, 0.13 mmol), DCC
(0.272 g, 1.3 mmol), and DMAP (0.161 g, 1.3 mmol) in
dry CH2Cl2 (1.3 mL) was added methoxyacetic acid
(0.10 mL, 1.3 mmol), followed by stirring at room tem-
perature for 3 days. The reaction mixture was filtered
through a short plug of silica gel with rinsing by
CH2Cl2. The filtrate was concentrated under reduced
pressure and the residue was purified by flash column
chromatography (silica gel, 5% EtOAc–hexane) to give
3 (30.9 mg, 52%) as a colorless oil; Rf = 0.46 (20%
EtOAc–hexane); IR (neat) 2929, 2856, 2236, 2212,
1683, 1586, 1257, 1111 cmꢁ1 1H NMR (300 MHz,
;
CDCl3) d 9.36 (s, 1H), 7.38–7.36 (m, 3H), 7.26–7.23
(m, 2H), 4.90 (s, 2H), 2.56–2.51 (m, 2H), 2.46–2.42 (m,
2H), 1.76–1.70 (m, 4H), 0.76 (s, 9 H), ꢁ0.02 (s, 6H);
13C NMR (75.5 MHz, CDCl3) d 190.5, 165.6, 134.6,
129.6 (2·), 128.9, 127.8 (2·), 122.6, 100.7, 93.9, 73.5,
68.6, 65.3, 27.7, 27.6, 25.7 (3·), 20.5, 19.4, 18.2, ꢁ5.3
(2·); MS (+CI) m/z (relative intensity) 507 (M+H+,
100); HRMS (+FAB) calcd for C24H30IO2Si [(MꢁH)+]
505.1060, found 505.1073.
4.2.8. (E)-4-[20-(tert-Butyldimethylsilyloxy)-10-phenyle-
thylidene]cyclodeca-1,5-diyn-3-ol (1). To a suspension
of anhydrous CrCl2 (0.134 g, 1.10 mmol) and NiCl2
(0.048 g, 0.37 mmol) in dry THF (74 mL) was added
(E)-13 (0.187 g, 0.37 mmol) in dry THF (10 mL), fol-
lowed by stirring at room temperature for 8 h. The reac-
tion was quenched with cold water (30 mL) and brine,
and extracted with EtOAc (3· 50 mL). The combined
organic layer was washed with brine, dried over anhy-
drous MgSO4, filtered, and concentrated under reduced
pressure. The residue was purified by flash column chro-
matography (silica gel, 10% EtOAc–hexane) to give 1
(34.2 mg, 24%) as a yellow oil; Rf = 0.46 (20% EtOAc–
hexane); IR (neat) 3436, 2928, 2856, 2213, 1462, 1361,
1761, 1463, 1254, 1178, 1126 cmꢁ1
;
1H NMR
(300 MHz, CDCl3) d 7.31–7.24 (m, 3H), 7.10–7.07 (m,
2H), 5.65 (d, J = 2.1 Hz, 1H), 4.70 and 4.63 (ABq,
J = 12.3 Hz, 2H), 3.96 and 3.84 (ABq, J = 16.5 Hz,
2H), 3.38 (s, 3H), 2.55–2.14 (m, 4H), 1.86–1.50 (m,
4H), 0.78 (s, 9H), ꢁ0.07 (s, 6H); 13C NMR
(75.5 MHz, CDCl3) d 168.5, 149.3, 136.9, 128.1 (2·),
127.9 (2·), 127.7, 119.5, 102.5, 92.8, 79.3, 78.9, 69.5,
66.6, 65.6, 59.4, 27.7 (2·), 25.8 (3·), 21.7, 20.8, 18.3,
ꢁ5.3 (2·); MS (+CI) m/z (relative intensity) 470
(M+NH4+, 21), 380 [M–C(O)CH2OCH3+H+, 100], 363
[M+ꢁOC(O)CH2OCH3, 81], 250 (27), 233 (24); HRMS
(+FAB) calcd for C27H37O4Si (M+H+) 453.2462, found
453.2448.
1
1254, 1095 cmꢁ1; H NMR (400 MHz, CDCl3) d 7.38–
7.27 (m, 5H), 5.42 (s, 1H), 4.71 and 4.64 (ABq,
J = 12.3 Hz, 2H), 2.58–2.18 (m, 4H), 1.85–1.64 (m,
4H), 0.78 (s, 9H), ꢁ0.04 (s, 6H); 13C NMR (100 MHz,
CDCl3) d 146.1, 137.2, 128.8 (2·), 127.8 (2·), 127.4,
124.1, 101.7, 91.8, 82.7, 79.5, 66.6, 64.0, 27.7 (2·), 25.8
(3·), 21.7, 20.7, 18.3, ꢁ5.2 (2·); MS (+CI) m/z (relative
intensity) 381 (M+H+, 10), 363 (M+ꢁOH, 95), 249
(M+ꢁOSiMe2t-Bu, 100), 233 (15), 231 (18), 221 (37);
HRMS (+FAB) calcd for C24H33O2Si (M+H+)
381.2250, found 381.2248.
4.2.11.
ene)cyclodeca-1,5-diyne (4). To
(E)-3-Acetoxy-4-(20-hydroxy-10-phenylethylid-
solution of
a
2
(32.9 mg, 7.8 · 10ꢁ2 mmol) in MeOHꢁH2O (10:1,
1.1 mL) was added PPTS (2.0 mg, 7.8 · 10ꢁ3 mmol), fol-
lowed by stirring at room temperature for 16 h. The
reaction mixture was concentrated under reduced pres-
sure. The residue was purified by flash column chroma-
tography (silica gel. 30% EtOAc–hexane) to give 4
(22.0 mg, 92%) as a colorless oil; Rf = 0.57 (50%
EtOAc–hexane); IR (neat) 3445, 2929, 2859, 2237,
2215, 1739, 1371, 1221 cmꢁ1 1H NMR (300 MHz,
;
4.2.9. (E)-3-Acetoxy-4-[20-(tert-butyldimethylsilyloxy)-
10-phenylethylidene]cyclodeca-1,5-diyne (2). To a solu-
tion of 1 (47.7 mg, 0.13 mmol) and DMAP (15.3 mg,
0.13 mmol) in dry CH2Cl2 (1.3 mL) was added acetic
anhydride (24 lL, 0.25 mmol), followed by stirring at
room temperature for 50 min. The reaction mixture
CDCl3) d 7.35–7.29 (m, 3H), 7.18–7.12 (m, 2H), 5.57
(s, 1H), 4.69 and 4.63 (ABq, J = 12.9 Hz, 2H), 2.60–
2.16 (m, 4H), 1.96 (s, 3H), 1.90–1.66 (m, 4H); 13C
NMR (75.5 MHz, CDCl3) d 169.2, 148.4, 136.6, 128.4
(2·), 128.1, 127.8 (2·), 120.7, 103.2, 92.5, 79.1, 66.5,
65.3, 27.6 (2·), 21.7, 20.9, 20.8; MS (+CI) m/z (relative

3208
Y. Tachi et al. / Bioorg. Med. Chem. 14 (2006) 3199–3209
intensity) 291 (M+ꢁOH, 30), 249 (M+–OAc, 100), 233
(17), 231 (47), 221 (86); MS (+FAB) m/z (relative inten-
sity) 309 (M+H+, 10), 249 (M+ꢁOAc, 100), 231 (85),
221 (100); HRMS (+FAB) calcd for C20H21O3
(M+H+) 309.1491, found 309.1486.
Acknowledgments
This work is supported in part by the Department of
Chemistry, HKUST and The Areas of Excellence
Scheme (AoE/P-10/01) established under The University
Grants Committee of Hong Kong SAR, China.
4.2.12. (E)-4-(20-Hydroxy-10-phenylethylidene)-3-(meth-
oxyacetoxy)cyclodeca-1,5-diyne (5). To a solution of 3
(20.5 mg, 4.5 · 10ꢁ2 mmol) in MeOHꢁH2O (10:1,
1.7 mL) was added PPTS (1.1 mg, 4.5 · 10ꢁ3 mmol),
followed by stirring at room temperature for 16 h.
The reaction mixture was concentrated under reduced
pressure. The residue was purified by flash column
chromatography (silica gel, 30% EtOAc–hexane) to
give 5 (15.3 mg, 100%) as a colorless oil; Rf = 0.49
(50% EtOAc–hexane); IR (neat) 3436, 2930, 2860,
Supplementary data
Figures S1–S6 for the NOE data of the compounds 8–
13. Supplementary data associated with this article can
be found, in the online version, at doi:10.1016/
j.bmc.2005.12.040.
References and notes
2236, 2212, 1757, 1183, 1124 cmꢁ1
;
1H NMR
1. (a) Nicolaou, K. C.; Dai, W.-M. Angew. Chem., Int. Ed.
Engl. 1991, 30, 1387–1530, Selected recent reviews; (b) Xi,
Z.; Goldberg, I. H.. In Barton, D. H. R., Nakanishi, K.,
Meth-Cohn, O., Eds.; Comprehensive Natural Products
Chemistry; Elesiver: New York, 1999; Vol. 7, pp 553–592;
(c) Thorson, J. S.; Sievers, E. L.; Ahlert, J.; Shepard, E.;
Whitwam, R. E.; Onwueme, K. C.; Ruppen, M. Curr.
Pharm. Des. 2000, 6, 1841–1879; (d) Jones, G. B.; Fouad,
F. S. Curr. Pharm. Des. 2002, 8, 2415–2440; (e) Wenk, H.
H.; Winkler, M.; Sander, W. Angew. Chem., Int. Ed. 2003,
42, 502–528.
2. (a) Borders, D. B.; Doyle, T. W., Eds.; Enediynes
Antibiotics as Antitumor Agents; Marcel Dekker: New
York, 1995.(b) Meunier, B., Ed.; DNA and RNA Cleavers
and Chemotherapy of Cancer and Viral Diseases; Kluwer
Academic Publishers: Dordrecht, 1996; pp 1–73.; (c)
Maeda, H.; Edo, K.; Ishida, E., Eds.; Neocarzinostatin:
The Past, Present, and Future of an Anticancer Drug;
Springer: New York, 1997.
3. (a) Jones, R. R.; Bergman, R. G. J. Am. Chem. Soc.
1972, 94, 660–661; (b) Bergman, R. G. Acc. Chem. Res.
1973, 6, 25–31; (c) Darby, N.; Kim, C. V.; Salaun, J. A.;
Shelton, K. W.; Takada, S.; Masamune, S. J. Chem.
Soc., Chem. Commun. 1971, 1516–1517; (d) Wong, H.
N. C.; Sondheimer, F. Tetrahedron Lett. 1980, 21, 217–
220.
4. (a) Kappen, L. S.; Ellenberger, T. E.; Goldberg, I. H.
Biochemistry 1987, 26, 384–390; (b) Lee, M. D.; Ellestad,
G. A.; Borders, D. B. Acc. Chem. Res. 1991, 24, 235–243;
(c) Zein, N.; McGahren, W. J.; Morton, G. O.; Ashcroft,
J.; Ellestad, G. A. J. Am. Chem. Soc. 1989, 111, 6888–
6890; (d) Sugiura, Y.; Arakawa, T.; Uesugi, M.; Shiraki,
T.; Ohkuma, H.; Konishi, M. Biochemistry 1991, 30,
2989–2992.
5. (a) Xi, Z.; Goldberg, I. H.. In Barton, D. H. R.,
Nakanishi, K., Eds.; Comprehensive Natural Products
Chemistry; Pergamon: Oxford, 1999; Vol. 7, pp 392–553;
(b) Zein, N.; Solomon, W.; Casazza, A. M.; Kadow, J. F.;
Krishnan, B. S.; Tun, M. M.; Vyas, D. M.; Doyle, T. W.
Bioorg. Med. Chem. Lett. 1993, 3, 1351–1356; (c) Jones, G.
B.; Plourde, G. W.; Wright, J. M. Org. Lett. 2000, 2, 811–
813; (d) Jones, G. B.; Wright, J. M.; Hynd, G.; Wyatt, J.
K.; Yancisin, M.; Brown, M. A. Org. Lett. 2000, 2, 1863–
1866.
6. (a) Nicolaou, K. C.; Smith, A. L.; Wendeborn, S. V.;
Hwang, C. K. J. Am. Chem. Soc. 1991, 113, 3106–3114;
(b) Matsumoto, Y.; Kuwatani, Y.; Ueda, I. Tetrahedron
Lett. 1995, 36, 3197–3200; (c) Takahashi, T.; Tanaka, H.;
Matsuda, A.; Doi, T.; Yamada, H. Bioorg. Med. Chem.
(300 MHz, CDCl3). d 7.40–7.30 (m, 3H), 7.15–7.13
(m, 2H), 5.69 (s, 1H), 4.67 and 4.61 (ABq,
J = 12.6 Hz, 2H), 3.96 and 3.84 (ABq, J = 16.5 Hz,
2H), 3.38 (s, 3H), 2.55–2.13 (m, 4H), 1.83–1.64 (m,
4H); 13C NMR (75.5 MHz, CDCl3) d 168.5, 148.6,
136.5, 128.5 (2·), 128.3, 127.7 (2·), 120.4, 103.3, 93.1,
79.0, 78.7, 69.4, 66.5, 65.5, 59.4, 27.6 (2·), 21.7,
20.8; MS (+CI) m/z (relative intensity) 356
(M+NH4+, 18), 266 [MꢁC(O)CH2OCH3+H+, 100],
249 [M+ꢁOC(O)CH2OCH3, 43], 233 (28), 231 (8).
221 (6); HRMS (+FAB) calcd for C21H23O4 (M+H+)
339.1596, found 339.1604.
4.3. DNA cleaving assay
Photoirradiation at 365 nm was carried out using a
ULTRA-VIOLET PRODUCTS NTFL-40 transillumi-
nator. An ATTO sequencing gel electrophoresis appa-
ratus was used for agarose gel electrophoresis. The
gels were analyzed by scanning densitometry with an
ATTO Lane Analyzer (version 3). DNA cleavage stud-
ies on the enediyne prodrugs 1–5 were performed by
the use of supercoiled, covalently closed, circular
UX174 RFI double-stranded DNA (Form I). Solution
of 50.0 lM/bp (micromolar per base pair) of UX174
RFI DNA and the enediyne prodrugs at various con-
centrations in TAE buffer (pH 8.5) containing 20%
DMSO (total volume 10 lL) was incubated at 37 ꢁC
for the indicated times without or with UV-irradiation
using a transilluminator (kex = 365 nm). The resultant
mixtures were then analyzed by gel electrophoresis
(1% agarose gel, ethidium bromide stain). DNA cleav-
age was determined by the formation of relaxed circu-
lar DNA (Form II) and linearized DNA (Form III).
The gels were placed on a UV transilluminator
(kex = 365 nm) and photographed with Polaroid 667
film. The relative densities of various DNA bands on
the gel pictures were quantified by scanning
densitometry with an ATTO Lane Analyzer. Since
Form III DNA was not observed in this study, the per-
centage of net DNA cleavage was calculated by the fol-
lowing equation: {[(Form II)s/[(Form I)s + (Form
II)s] · 100} ꢁ {(Form
II)c/[(Form
I)c + (Form
II)c] · 100}. The subscripts ‘s’ and ‘c’ refer to as the
samples and control(s), respectively. The results are
given in Figures 2–4.
´ `
Lett. 1998, 8, 3299–3302; (d) Rodrıguez, G.; Rodrıguez,

Y. Tachi et al. / Bioorg. Med. Chem. 14 (2006) 3199–3209
3209
´
D.; Lopez, M.; Castedo, L.; Domınguez, D.; Saa, C.
Synlett 1998, 1282–1284; (e) Ferri, F.; Bruckner, R.;
´
´
19. (a) Cristol, S. J.; Lee, G. A.; Noreen, A. L. Tetrahedron
Lett. 1971, 12, 4175–4178; (b) Fransen, H. R.; Buck, H.
M. J. Chem. Soc., Chem. Commun. 1982, 786–787; (c)
Fransen, H. R.; Dormans, G. J. M.; Bezemer, G. J.; Buck,
H. M. Recl. Trav. Chim. Pays-Bas 1984, 103, 115–118; (d)
Dormans, G. J. M.; Fransen, H. R.; Buck, H. M. J. Am.
Chem. Soc. 1984, 106, 1213–1216.
20. (a) Peijnenburg, W. J. G. M.; Buck, H. M. Tetrahedron
1988, 44, 4821–4836; (b) Miesen, F. W. A. M.; Vandon-
gen, J. L. J.; Meijer, E. W. Recl. Trav. Chim. Pays-Bas
1994, 113, 307–317.
¨
Herges, R. New J. Chem. 1998, 531–545; (f) Bekele, T.;
Brunette, S. R.; Lipton, M. A. J. Org. Chem. 2003, 68,
8471–8479; (g) Suzuki, I.; Shigenaga, A.; Nemoto, H.;
Shibuya, M. Tetrahedron Lett. 2004, 45, 1955–1959.
7. (a) Kappen, L. S.; Goldberg, I. H. Nucleic Acids Res. 1978,
5, 2959–2967; (b) Hirama, M.; Fujiware, K.; Shigematsu,
K.; Fukazawa, Y. J. Am. Chem. Soc. 1989, 111, 4120–
4122; (c) Uesawa, Y.; Kuwahara, J.; Sugiura, Y. Biochem.
Biophys. Res. Commun. 1989, 164, 903–911; (d) Kawata,
S.; Oishi, T.; Hirama, M. Tetrahedron Lett. 1994, 35,
4591–4598; (e) Maier, M. E. Synlett 1995, 13–26; (f)
Grissom, J. W.; Gunawardena, G. U.; Huang, D. Tetra-
hedron 1996, 52, 6453–6518; (g) Alabugin, I. V.; Kov-
alenko, S. V. J. Am. Chem. Soc. 2002, 124, 15141; (h)
Alabugin, I. V.; Manoharam, M. J. Phys. Chem. A 2003,
107, 3363–3371; (i) Poloukhtine, A.; Popik, V. V. J. Org.
Chem. 2005, 70, 1297–1305, Also see activation of
enediyne via transannular cyclization; (j) Basak, A.;
Roy, S. K.; Mandal, S. Angew. Chem., Int. Ed. 2005, 44,
132–135.
8. Dai, W.-M. Curr. Med. Chem. 2003, 10, 2265–2283.
9. Dai, W.-M.; Fong, K. C.; Danjo, H.; Nishimoto, S.
Angew. Chem., Int. Ed. Engl. 1996, 35, 779–781.
10. (a) Dai, W.-M.; Fong, K. C. Tetrahedron Lett. 1996, 37,
8413–8416; (b) Dai, W.-M.; Lee, M. Y. H. Tetrahedron
Lett. 1998, 39, 8149–8152; (c) Dai, W.-M.; Fong, K. C.;
Lau, C. W.; Zhou, L.; Hamguchi, W.; Nishimoto, S. J.
Org. Chem. 1999, 64, 682–683; (d) Dai, W.-M.; Wu, J.;
Fong, K. C.; Lee, M. Y. H.; Lau, C. W. J. Org. Chem.
1999, 64, 5062–5082.
11. (a) Shull, B. K.; Sakai, T.; Koreeda, M. J. Am. Chem. Soc.
1996, 118, 11690–11691; (b) Dai, W.-M.; Mak, W. L.; Wu,
A. Tetrahedron Lett. 2000, 41, 7101–7105; (c) Dai, W.-M.;
Mak, W. L. Chin. J. Chem. 2003, 21, 772–783.
12. (a) Dai, W.-M.; Lee, M. Y. H. Tetrahedron Lett. 1998, 39,
2397–2400; (b) Dai, W.-M.; Wu, A.; Hamaguchi, W.
Tetrahedron Lett. 2001, 42, 4211–4214; (c) Dai, W.-M.;
Wu, A.; Lee, M. Y. H.; Lai, K. W. Tetrahedron Lett. 2001,
42, 4215–4218.
13. Dai, W.-M.; Wu, A. Tetrahedron Lett. 2001, 42, 81–83.
14. Dai, W.-M.; Lai, K. W.; Wu, A.; Hamaguchi, W.; Lee, M.
Y. H.; Zhou, L.; Ishii, A.; Nishimoto, S. J. Med. Chem.
2002, 45, 758–761.
15. Dai, W.-M.; Tachi, Y.; Nishimoto, S.; Zhong, X.; Guo, Z.
Lett. Drug Des. Discov. 2004, 1, 69–72.
21. Dai, W.-M.; Wu, J.; Wu, A. Tetrahedron Lett. 1998, 39,
4091–4094.
22. (a) LePla, R. C.; Landreau, C. A. S.; Shipman, M.;
Hartley, J. A.; Jones, G. D. D. Bioorg. Med. Chem. Lett.
2005, 15, 2861–2864; (b) Heald, R. A.; Dexheimer, T. S.;
Vankayalapati, H.; Siddiqui-Jain, A.; Szabo, L. Z.;
Gleason-Guzman, M. C.; Hurley, L. H. J. Med. Chem.
2005, 48, 2993–3004; (c) Michel, S.; Gaslonde, T.; Tille-
quin, F. Eur. J. Med. Chem. 2004, 39, 649–655; (d)
Hashimoto, M.; Matsumoto, M.; Yamada, K.; Terashi-
ma, S. Tetrahedron 2003, 59, 3089–3097; (e) Coleman, R.
S.; Burk, C. H.; Navarro, A.; Brueggemeier, R. W.; Diaz-
Cruz, E. S. Org. Lett. 2002, 4, 3545–3548; (f) Guilbaud,
N.; Leonce, S.; Tillequin, F.; Koch, M.; Hickman, J. A.;
Pierre, A. Anticancer Drugs 2002, 13, 445–449; (g) Naka-
tani, K.; Matsuno, T.; Adachi, K.; Hagihara, S.; Saito, I.
J. Am. Chem. Soc. 2001, 123, 5695–5702; (h) Hodgkinson,
T. J.; Kelland, L. R.; Shipman, M.; Suzenet, F. Bioorg.
Med. Chem. Lett. 2000, 10, 239–241; (i) Zang, H.; Gates,
K. S. Biochemistry 2000, 39, 14968–14975; (j) Okamoto,
A.; Nakamura, T.; Yoshida, K.; Nakatani, K.; Saito, I.
Org. Lett. 2000, 2, 3249–3251; (k) Prakash, A. S.; Tran,
H.-P.; Peng, C.; Koyalamudi, S. R.; Dameron, C. T.
Chem. Biol. Interact. 2000, 125, 133–150; (l) Nakatani, K.;
Okamoto, A.; Matsuno, T.; Saito, I. J. Am. Chem. Soc.
1998, 120, 11219–11225; (m) Tretyakova, N. Y.; Sangaiah,
R.; Yen, T. Y.; Swenberg, J. A. Chem. Res. Toxicol. 1997,
10, 779–785; (n) Bolt, H. M.; Capellmann, M.; Jelitto, B.;
Hindermeier, U.; Kirkovsky, L. I. Toxicology 1996, 113,
294–296; (o) Ciuffetti, L. M.; Kim, S. D.; Knoche, H. W.;
Dunkle, L. D. Physiol. Mol. Plant Pathol. 1995, 46, 61–70;
(p) Prakash, A. S.; Moore, A. G.; Murray, V.; Matias, C.;
McFadyen, W. D.; Wickham, G. Chem. Biol. Interact.
1995, 95, 17–28; (q) Segerback, D.; Ostermangolkar, S.;
Molholt, B.; Nilsson, R. Regul. Toxicol. Pharma. 1994, 20,
1–14; (r) Adam, W.; Sauter, M. Lieb. Ann. Chem. 1994,
689–693.
16. Suffert, J.; Toussaint, D. Tetrahedron Lett. 1997, 38, 5507–
5510.
17. (a) Uenishi, J.; Matsui, K. Tetrahedron Lett. 2001, 42,
4353–4355; (b) Uenishi, J.; Matsui, K.; Ohmiya, H. J.
Organomet. Chem. 2002, 653, 141–149.
23. Schroeder, D. R.; Colson, K. L.; Klohr, S. E.; Zein, N.;
Langley, D. R.; Lee, M. S.; Matson, J. A.; Doyle, T. W. J.
Am. Chem. Soc. 1994, 116, 9351–9352.
24. Lown, J. W. The Chemistry of DNA Damage by
Anticancer Drugs. In Molecular Aspects of Anticancer
Drug Action; Neidle, S., Waring, M. J., Eds.; Verlag
Chemie: Weinheim, 1983; pp 283–314.
´
18. (a) Crevisy, C.; Beau, J.-M. Tetrahedron Lett. 1991, 32,
3171–3174; (b) Southwick, P. L.; Kirchner, J. R. J. Org.
Chem. 1962, 27, 3305–3308.