Synthesis and DNA cleaving activity of water-soluble non-conjugated thienyl tetraynes

Article abstract of DOI:10.1016/j.bmc.2008.04.009

Water-soluble non-conjugated thienyl tetraynes (3-6) were synthesized and their DNA cleaving activity was evaluated using electrophoresis, atomic force microscopy (AFM) and Escherichia coli (E. coli) transformation techniques. The amino-functionalized compound 4 was shown to possess an activity to cleave plasmid DNA by both electrophoresis and E. coli transformation techniques. AFM also showed a cleavage of the circular DNA into a linear form with a formation of burst-star-shaped architectures, which were envisaged to be cross-linked DNA oligomers.

Full text of DOI:10.1016/j.bmc.2008.04.009

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 5441–5451
Synthesis and DNA cleaving activity of water-soluble
non-conjugated thienyl tetraynes
Kohei Torikai,a,*, Yoichi Otsuka,a Makoto Nishimura,a Megumi Sumida,a
Tomoji Kawai,a Kiyotoshi Sekiguchib and Ikuo Uedaa
aThe Institute of Scientific and Industrial Research, Osaka University, Mihogaoka, Ibaraki, Osaka 567-0047, Japan
bInstitute for Protein Research, Osaka University, Yamadaoka, Suita, Osaka 565-0871, Japan
Received 14 February 2008; revised 7 April 2008; accepted 8 April 2008
Available online 11 April 2008
Abstract—Water-soluble non-conjugated thienyl tetraynes (3–6) were synthesized and their DNA cleaving activity was evaluated
using electrophoresis, atomic force microscopy (AFM) and Escherichia coli (E. coli) transformation techniques. The amino-func-
tionalized compound 4 was shown to possess an activity to cleave plasmid DNA by both electrophoresis and E. coli transformation
techniques. AFM also showed a cleavage of the circular DNA into a linear form with a formation of burst-star-shaped architectures,
which were envisaged to be cross-linked DNA oligomers.
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
there have been many reports on the design and synthe-
sis of artificial ene–diyne compounds,2c,4 but to date,
there have been few studies to change the conjugated
ene–diyne core structure. As a new entry to DNA cleav-
ing molecules, we focused on non-conjugated ene–yne
systems with an interest to investigate the reactivity of
ene–polyyne systems,5 and as expected, non-conjugated
aryl (phenyl and pyridyl) tetraynes (TYs) were found to
undergo CA by the way of biradical species with the
breakage of a single chain of duplex DNA.6
Cyclization reactions of conjugated polyyne systems
such as ene–diyne, enyne–allene, and enyne–ketene,
forming biradical species as intermediates, have been re-
ported in the 1960s–1970s in the field of structural or-
ganic chemistry.1 On the other hand, from the 1980s
to date, many antitumor natural products possessing
ene–diyne structures, as shown in Figure 1, have been
isolated,2 and their molecular target was identified as
DNA.2,3 These natural products share some structural
and functional similarities. One fragment of a structure
is responsible for the recognition and transport, another
part acts as a molecular trigger while the third, the reac-
tive ene–diyne unit, undergoes a cycloaromatization
(CA) to cleave the backbone of DNA. Although mem-
bers of the ene–diyne family are already in clinical use
to treat various cancers, more general use is limited by
their complex structures. Therefore, in order to develop
more simple, selective, and potent anticancer agents,
Despite their potential importance in therapeutic appli-
cation, the TYs we developed have suffered from obsta-
cles in their use under physiological conditions due to
their hydrophobicity and lack of a triggering system to
generate active biradical species. Thus, to solve these
problems, we planned to introduce hydrophilic func-
tionalities and to set a trigger on aryl TYs. Fortunately,
during the course of our study to investigate the reactiv-
ity of aryl TYs, we recently have found that the CA
reaction of thienyl TY A does not proceed even at
37 °C, but that of the corresponding ketone derivative
B proceeds smoothly to form E (Scheme 1).7,8 Since this
property was envisaged to be useful as a triggering de-
vice, thienyl TY was selected as a core structure.
According to the proposed mechanism of the CA of
non-conjugated thienyl TYs8 (Scheme 1), two active spe-
cies capable of cleaving DNA can be proposed. One is
biradical C1, which breaks the DNA backbone as a
Keywords: Ene–yne; Non-conjugated; DNA cleavage; Thermal cycli-
zation; Cycloaromatization; Escherichia coli (E. coli) transformation;
Atomic force microscopy (AFM).
*
Corresponding author. Tel.: +81 3 5452 5496; fax: +81 3 5452
5751; e-mail: torikai@cbl.rcast.u-tokyo.ac.jp
Present address: Research Center for Advanced Science and Technol-
ogy, The University of Tokyo, 4-6-1, Komaba, Meguro-Ku, Tokyo
153-8904, Japan.
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.04.009

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K. Torikai et al. / Bioorg. Med. Chem. 16 (2008) 5441–5451
Figure 1. Ene–yne antitumor agents from natural resources.
Scheme 1. Proposed mechanism for thermal cycloaromatization of thienyl tetraynes and hypothesis of dual DNA cleaving pathway.
result of abstraction of a hydrogen atom from deoxyri-
bose-phosphate chains, and the other is cation D which
alkylates DNA bases to cleave. There have been few re-
ports of ene–yne compounds which generate both radi-
cal and cationic active species simultaneously,9 hence,
if the reactivity, recognition, and transport could be well
controlled, thienyl TY might be a good candidate as a
new ene–yne antitumor agent, for instance, to cure the
multi-drug resistant cancers. Herein, we report the syn-
thesis of water-soluble thienyl TYs and the evaluation of
their DNA damaging abilities via electrophoresis, atom-
ic force microscopy (AFM), and Escherichia coli (E. coli)
transformation techniques (Scheme 2).
nyl terminus was changed from the originally reported
trimethylsilyl group7,8 into a tert-butyl group in order
to improve the stability; (ii) as hydrophilic functional-
ities, dimethylamino and carboxy groups, which are pos-
itively and negatively charged, respectively, in
physiological conditions, were selected for a facile struc-
ture–activity relationship (SAR) study; (iii) the above
functionalities were introduced onto the thiophene ring
at locations where these were believed to least likely af-
fect the reactivity of the thermal cyclization of the TY
moiety as well as to be more readily synthesized. In
addition, alcohols (1, 3, and 5), whose thermal CA does
not occur at 37 °C, were synthesized to demonstrate
whether DNA damage is definitely induced via thermal
CA reactions.
2. Results and discussion
First of all, the synthesis of hydrophobic thienyl TY 2
commenced with the previously reported aldehyde 7.8
Aldehyde 7 was treated with diethynyllithium, which
was generated in situ from butadiyne 810 andMeLi–LiBr
in refluxing methyl tert -butyl ether, to give racemic
2.1. Design and synthesis of thienyl tetraynes
We designed water-soluble thienyl TYs (2, 4, and 6) tak-
ing into account the following points (Fig. 2): (i) diethy-

K. Torikai et al. / Bioorg. Med. Chem. 16 (2008) 5441–5451
5443
Scheme 2. Reagents and conditions: (a) 8, MeLi, MeO-t-Bu, reflux, 3.5 h, then 7, À70 to À55 °C, 1.5 h, 88%; (b) MnO2, Na2SO4, CH2Cl2, 0–7 °C,
15.5 h, quant.; (c) ethyleneglycol, PPTS, benzene, Dean-Stark trap, reflux, 21 h, 99%; (d) n-BuLi, THF, À78 °C, 5 min, then DMF, À78°C–rt, 2.5 h,
84%; (e) HNMe2HCl, Et3N, Ti(O-i-Pr)4, EtOH, rt, 19 h, then NaBH4, rt, 3.5 h; (f) PPTS, acetone, H2O, reflux, 2 days, 91% (two steps); (g) t-Bu–
C„C–C„C–Li, MeO-t-Bu, Et2O, THF, À70 to À55 °C, 1.5 h, 66%; (h) HCl/MeOH, CHCl3, 0 °C, 5 min, quant.; (i) MnO2, Na2SO4, 0–7 °C,
CH2Cl2, 16 h; (j) HCl/MeOH, CH2Cl2, CHCl3, 0 °C, 56% (two steps); (k) n-BuLi, THF, À78 °C, 5 min, then ClCO2Me, À78 °C, 30 min, 80%; (l)
PPTS, acetone, H2O, reflux, 29 h, 92%; (m) 0.1 M aqueous NaOH, THF, rt, 70 min, quant.; (n) TIPSCl, imidazole, CH2Cl2, rt, 1 h, 92%; (o) t-Bu–
C„C–C„C–Li, MeO-t-Bu, Et2O, À72 to 0 °C, 1.5 h, 40%; (p) K2CO3 (0.5 equiv), MeOH,0–4 °C, 41 h, 75%; (q) MnO2, Na2SO4, CH2Cl2,0–7 °C,
27 h, 98%; (r) K2CO3 (0.5 equiv), MeOH, ether, CHCl3, 0 °C, 20 min, 77%.
ketone 2 in excellent yield (quantitative). Since we
successfully constructed a common core structure, we
moved on to the synthesis of dimethylamino-functional-
ized thienyl TY 4, starting with the protection of
common intermediate 7, as acetal 9 (99%). Due to the
fact that previous attempts at the one-step introduction
of a dimethylaminomethyl group to the thiophene ring
of 9 via a Mannich-type reaction had failed,11 a sequen-
tial method was examined. Lithiation of 9 with n-butyl-
lithium, followed by treatment with DMF furnished
formylthiophene 10 (84%), which was converted to dime-
thylaminomethylthiophene 11 via titanium tetraisoprop-
oxide mediated reductive amination.12 Removal of the
Figure 2. Structures of functionalized thienyl tetraynes.
acetal from 11 with pyridinium p-toluenesulfonate
(PPTS), followed by the addition of diethynyllithium
alcohol 1 in 88%. The final oxidation required to be
conducted at around 0 °C, without chromatographic
purification if possible, as thermal cyclization of ketone
2 was expected to proceed even at room temperature.
Considerable experimentations revealed that MnO2 in
the presence of Na2SO4 was the best oxidant to furnish
to the resulting aldehyde gave TY 12 in 60% for three
steps. Although the final oxidation of 12 using MnO2
proceeded smoothly to give the corresponding ketone,
the resulting amino-free ketone after drying, was found
to be unstable, even at À20 °C in an argon atmosphere.
Therefore, amino-free alcohol 12 was oxidized with

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K. Torikai et al. / Bioorg. Med. Chem. 16 (2008) 5441–5451
MnO2 to the ketone, and converted immediately to the
corresponding hydrochloride 4 using 10% HCl–MeOH,
followed by evaporation at 0 °C. As a result, synthesis of
the desired ketone 4, which was clarified to be isolable,
was achieved.
the dimethylaminomethyl group may increase the DNA
cleaving activity of 4, not only by enhancing the electro-
static interaction with DNA, but also by providing an
electron-withdrawing effect. On the other hand, treat-
ment with 4 also gave an intriguing smear at the higher
molecular weight region, concomitant with form II and
III DNA (lane 4). For the rational explanation of this
unexpected observation, we next examined directly the
molecular shape of DNA after treatment with 4 using
an atomic force microscope.
The final task, synthesis of the carboxy-functionalized
thienyl TY 6, commenced with acetal 9. Lithiation of
9 followed by treatment with methyl chloroformate gave
methoxycarbonyl thiophene 13 in 80%. Removal of the
acetal from 13 with PPTS (14, 92%), followed by sapon-
ification, gave carboxylic acid 15 in quantitative yield.
Carboxylic acid 15 was protected as triisopropylsilyl
(TIPS) ester (16, 92%), and subjected to the addition
of diethynyllithium to give TY 17 (40%).13 Finally,
MnO2-oxidation of 17, followed by the removal of the
TIPS group using 0.5 equivalent of potassium carbonate
afforded potassium salt 6 in 75% for two steps. The cor-
responding alcohol 5 was also prepared (75%) as a neg-
ative control in experiments.14
2.2.2. Atomic force microscopy. Recently, AFM has at-
tracted much attention in the field of nano-biotechnol-
ogy.16 Because AFM is a powerful tool in that it
enables the direct observation of a single biomolecule
without special treatments, we envisaged that AFM is
the most suitable method to accurately visualize the
state of DNA,17 after treatment with 4, which gave an
aberrant electrophoretic pattern. Figure 4a and b are
AFM images of intact PB plasmid DNA showing that
almost all the PB molecules exist in a supercoiled form
I. Contrary to these, images after treatment of 4
(1.0 mM, Fig. 4c–e) indicated both the cleavage of circu-
lar DNA into a linear form (form III, Fig. 4d) and the
formation of burst-star-shaped architectures (Fig. 4e),
that are consistent with the results obtained by electro-
phoresis. Although the precise structure of the burst-
star-shaped architectures was unclear, these can be con-
sidered as DNA oligomers resulting from intermolecular
conjunction between linear DNA radicals. This unique
topological change of DNA by ketone 4, which differs
from that by CPA (treatment with CPA resulted in only
the scission of PB, which was confirmed by both electro-
phoresis and AFM, data not shown), inspired us to eval-
uate the functional change of PB using a biological
method.
2.2. Evaluation of DNA cleaving activities of thienyl
tetraynes
2.2.1. Electrophoresis. With functionalized thienyl TYs
in hand, we turned our attention to the evaluation of
their DNA cleaving abilities. First of all, after treatment
with thienyl TYs15 in the presence of DMSO (3%),
pBluescript II KS+ (PB) plasmid DNA was analyzed
by agarose gel electrophoresis (Fig. 3a). As shown in
lane 1, intact PB exists mostly as a supercoiled form
(form I). When PB was treated with cyclophosphamide
(CPA, 10 mM), a clinically used anticancer DNA cleav-
ing agent by alkylation, a smear at the lower molecular
weight (ca. 700 bp) region was observed, indicative of
the cleavage of DNA to small pieces (lane 2). On the
other hand, although only a slight or almost no change
was observed by treating with alcohol 3, 5, and carboxy-
functionalized ketone 6 (1.0 mM, lane 3, 5, and 6,
respectively), a significant decrease in form I with a
concomitant increase in form II (nicked) and form III
(linear) was observed, when treated with amino-func-
tionalized ketone 4 (lane 4). Since carboxy-functional-
ized ketone 6 was supposed to be less accessible to
negatively charged DNA due to its anionic properties,
it was reasonable to find that ketone 6 was inactive, even
though it undergoes CA under the conditions examined.
Contrary to 6, amino-functionalized ketone 4, which is
considered to interact more potently with DNA owing
to Coulomb force, exhibited DNA cleaving activity, as
evidenced by a significant conversion of the circularized
form I to the nicked form II and/or the linearized form
III. Furthermore, electrophoretic analysis of the above
DNA after treatment with 4 at the lower concentrations
(0.1, 0.3, 0.6, and 1 mM, respectively, Fig. 3b) revealed
that the DNA cleaving activity of 4 is dose-dependent,
and ca. 100 lM (lane 2) would be a minimum concentra-
tion to elicit the activity. Meanwhile, a rough compari-
son of the half-lives of ketones 2, 4, and 6 by
measuring the time-dependent changes of 1H NMR
indicated that the rate of the CA reaction was
4 > 2 > 6, an order, which corresponds to the electron-
poverty of the thienyl moiety of each ketone. Therefore,
2.2.3. Evaluation of functional damage of PB via trans-
formation of E. coli. Since PB is a designed plasmid
DNA routinely used in cloning application, transforma-
tion by PB renders E. coli resistant to ampicillin, an anti-
biotic agent. We postulated that if the function of PB
was destroyed by ketone 4, E. coli transformed by dam-
aged PB would not survive in ampicillin-containing
media.
The results are depicted in Table 1. When intact PB was
introduced into E. coli (DH5a or JM109 competent
cells), E. coli became resistant to ampicillin, yielding
many (more than 1000) colonies on Luria–Bertani
(LB) agar plates-containing ampicillin. On the other
hand, when PB was pretreated with CPA (10 mM), an
alkylative DNA cleaving agent, no colonies were ob-
served. As for TYs (1.0 mM), contrary to the observa-
tion of many colonies using PB pretreated with 3, 5,
and 6, treatment with amino-functionalized ketone 4
yielded only eight colonies indicating the functional dis-
ruption of PB. In addition, due to the fact that treatment
with both amino-functionalized alcohol 3 and trimethyl-
amine hydrochloride did not affect ampicillin resistance,
the functional change is not a result of aggregation of
DNA due to an ionic interaction between the dimethyl-
amino group and phosphate (of DNA) neutralizing the

K. Torikai et al. / Bioorg. Med. Chem. 16 (2008) 5441–5451
5445
Figure 3. Agarose gel electrophoresis of pBluescript II KS+ plasmid DNA in the presence of DNA cleaving agents. (a) pBluescript II KS+ (2 lg) alone
(lane 1); in the presence of cyclophosphamide (CPA, 10 mM, lane 2); in the presence of alcohol 3 (1.0 mM, lane 3); in the presence of ketone 4
(1.0 mM, lane 4); in the presence of alcohol 5 (1.0 mM, lane 5); in the presence of ketone 6 (1.0 mM, lane 6); (b) pBluescript II KS+ (2 lg) alone (lane
1); in the presence of 4 (0.1 mM, lane 2; 0.3 mM, lane 3; 0.6 mM, lane 4; 1 mM, lane 5, respectively). DNA was visualized via treatment of ethydium
bromide, followed by the irradiation of UV.
Figure 4. Direct observation of pBluescript II KS+ plasmid DNA by atomic force microscopy. (a) Intact pBluescript II KS+ on a flat mica plate;
(b) a representative image of a PB plasmid DNA molecule in supercoiled form from (a); (c) PB after treatment of amino-functionalized ketone 4
(1.0 mM, 37 °C, 6 h); (d) a representative image of cleaved pieces of PB molecule from (c); (e) a representative image of the burst-star-shaped
architecture from (c).
Table 1. Numbers of E. coli colonies transformed by PBs after treatment with compounds (1.0 mM for tetraynes and trimethylamine hydrochloride,
10 mM for CPA) in ampicillin-containing LB media
Compound
None
CPA
0
3
4
5
6
Me3N HCl
>1000
Colonies/platea
>1000
>1000
8
>1000
>1000
a Incubated at 37 °C for 15 h on LB plates.

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K. Torikai et al. / Bioorg. Med. Chem. 16 (2008) 5441–5451
charge, but from the active species via CA reaction of an
enediyne core. Furthermore, other radical species, for
example, hydroxyl radical, derived from hydrogen
abstraction from water by biradical intermediates,
would not be involved in damaging DNA, because the
CA reaction of ketone 6 did not cause DNA damage.
These findings implied that TYs bound to DNA to exert
their activities. A facile SAR study of thienyl TYs in this
work should provide important information for improv-
ing affinity to DNA and setting a better triggering de-
vice, which are expected to be achieved in the near
future, to lead TYs to a new class of ene–yne antibiotics.
(77.0), CD3OD (49.0), CD2Cl2 (53.1). The following
abbreviations are used to designate the multiplicities:
s = singlet, d = doublet, t = triplet, q = quartet, m = mul-
tiplet. Mass spectra were recorded on a JEOL JMS600
under FAB conditions using m-nitrobenzyl alcohol
(NBA) as a matrix. Combustion elemental analyses were
performed usingPerkin-Elmer 240C. Melting point of so-
lid samples was measured on a device from Gallenkamp.
4.2. Synthesis of thienyl tetraynes
4.2.1. 1-{3-[4-(2-Methoxymethyl-phenyl)-buta-1,3-diy-
nyl]-thiophen-2-yl}-6,6-dimethyl-hepta-2,4-diyn-1-ol (1).
In argon atmosphere, to a solution of asymmetric but-
adiyne 8 (975 mg, 5.47 mmol) in methyl tert-butyl ether
(20 mL) was addedmethyllithium–lithium bromide com-
plex (1.5 M, in ether, 1.9 mL, 2.7 mmol) at room tem-
perature. The mixture was stirred under reflux for
3.5 h, and the resulting alkynyl lithium solution was
added to a solution of aldehyde 7 (300 mg, 1.07 mmol)
in ether (10 mL) at À78 °C. After being stirred at
À78 °C for 1.5 h, the mixture was treated with satd
NH4Cl aq to quench the reaction, and diluted with ethyl
acetate. The organic layer was separated, washed with
brine, dried over Na2SO4, filtered and the filtrate was
concentrated under reduced pressure. The residue was
purified by silica gel column chromatography (hexane/
ethyl acetate = 5:1) to afford alcohol 1 (363 mg, 88%)
3. Conclusion
We synthesized water-soluble thienyl tetraynes and eval-
uated their DNA cleaving activities via electrophoresis,
atomic force microscopy and E. coli transformation tech-
niques. As a result, it was found that positively charged
dimethylamino-fuctionalized thienyl tetrayne 4 cleaves
pBluescript II KS+ plasmid DNA whereas carboxylate
6 is ineffective. Furthermore, concomitant with topolog-
ical change, the biological function of DNA, as observed
in an E. coli transformation assay, was also abrogated.
The burst-star-shaped architectures of plasmid DNA,
which were visualized by atomic force microscopy
(AFM), are believed to be covalently cross-linked
DNA oligomers, and are a unique feature of 4, suggest-
ing the involvement of biradical species, additionally to
that of previously reported cationic species8 to cleave
DNA. Because of the dual DNA cleaving species, thienyl
tetrayne 4 is potentially a lead compound for a novel
family of DNA cleaving agents. Further investigations
on DNA cleaving cycloaromatization of non-conjugated
ene–tetrayne system are now in progress.
1
as a yellow oil: H NMR (400 MHz, CDCl3) d 7.54 (d,
J = 7.6 Hz, 1H), 7.49 (d, J = 7.8 Hz, 1H), 7.40 (dd,
J = 7.6, 7.6 Hz, 1H), 7.25–7.29 (m, 2H), 7.07 (d,
J = 5.1 Hz, 1H), 5.99 (d, J = 5.4 Hz, 1H), 4.66 (s, 2H),
3.48 (s, 3H), 2.46 (d, J = 5.4 Hz, 1H), 1.24 (s, 9H); 13C
NMR (100 MHz, CDCl3) d 149.5, 141.5, 133.1, 130.2,
129.5, 127.5, 127.4, 125.3, 120.1, 118.8, 90.7, 80.3,
77.8, 77.7, 75.4, 74.5, 72.4, 71.4, 62.9, 59.3, 58.6, 30.3,
28.0; IR (ether, CDCl3): 3424, 2401, 2251 cmÀ1; FAB-
MS (NBA) m/z 369 [(MÀOH)+]; Anal. Calcd for
C25H22O2S: C, 77.69; H, 5.74; S, 8.30. Found: C,
77.44; H, 5.97; S, 8.67.
4. Experimental
4.1. General methods and instruments for organic
synthesis
4.2.2. 1-{3-[4-(2-Methoxymethyl-phenyl)-buta-1,3-diynyl]-
thiophen-2-yl}-6,6-dimethyl-hepta-2,4-diyn-1-one (2). To
a solution of alcohol 1 (200 mg, 0.518 mmol) in CH2Cl2
(10 mL) was added Na2SO4 (300 mg, 2.11 mmol) and ac-
tive c-MnO2 (4.0 g, 46 mmol) at 0 °C. After being vigor-
ously stirred at 0–7 °C for 15.5 h, the heterogeneous
mixture was filtered and the filtrate was concentrated at
0 °C to give ketone 2 (199 mg, quantitative) as a yellow
powder: mp 90.7 °C (dec); 1H NMR (400 MHz, CD2Cl2)
d 7.65 (d, J = 5.1 Hz, 1H), 7.55 (d, J = 7.8 Hz, 1H), 7.49
(d, J = 7.8 Hz, 1H), 7.42 (ddd, J = 1.2, 7.3, 7.8 Hz, 1H),
7.29 (m, 2H), 4.65 (s, 2H), 3.45 (s, 3H), 1.21 (s, 9H); 13C
NMR (100 MHz, CD2Cl2) d 166.6, 145.7, 141.8, 133.5,
133.2, 132.8, 129.4, 127.2, 127.0, 125.2, 119.4, 98.6, 81.7,
80.1, 78.2, 77.4, 75.9, 72.0, 71.9, 62.3, 58.1, 29.3, 28.1; IR
(CD2Cl2): 2304, 2224, 2199, 1602 cmÀ1; FAB-MS (NBA)
m/z 385 [(M+H)+].
Anhydrous diethyl ether, methyl tert-butyl ether, tetrahy-
drofuran (THF), and N,N-dimethylformamide (DMF)
were purchased from Kanto Chemical Co. Inc. or Wako
Pure Chemical Industries Ltd and used without further
drying. Other solvents were dried over activated molecu-
lar sieves 4A (3A for methanol). All the other chemicals
were obtained from local venders, and used as supplied
unless otherwise stated. Analytical thin-layer chromatog-
raphy (TLC) was performed using E. Merck silica gel 60
F254 pre-coated plates (0.25-mm thickness). For column
chromatography, silica gel 60 (70–270 mesh ASTM,
Merck), aluminum oxide 90 (Merck), and florisilÒ (75–
150 lm, Wako) were used. For high performance liquid
chromatography (HPLC), JEOL RI-2031 UV-2075 PU-
2086 system was used. IR spectra were recorded on a Shi-
madzu FT-IR-8300. NMR spectra were recorded on a
JEOL JNM-LA400 (400 MHz). Chemical shifts were re-
ported in ppm from tetramethylsilane with reference to
internal residual solvent [1H NMR, CHCl3 (7.26),
CHD2OD (4.78), CHDCl2 (5.32); 13C NMR, CDCl3
4.2.3. 2-{3-[4-(2-Methoxymethyl-phenyl)-buta-1,3-diy-
nyl]-thiophen-2-yl}-[1,3]dioxolane (9). To a solution of
aldehyde 7 (8.25 g, 29.4 mmol) in benzene (400 mL)
was added ethyleneglycol (8.2 mL, 147 mmol) and

K. Torikai et al. / Bioorg. Med. Chem. 16 (2008) 5441–5451
5447
pyridinium
p-toluenesulfonate
(PPTS,
740 mg,
ous ammonia with vigorous stirring for 20 min. The
resulting slurry was filtered and the filtrate was extracted
with ethyl acetate. The combined organic layer was
washed with brine, dried over Na2SO4, filtered and the fil-
trate was concentrated under reduced pressure. The resid-
ual oil was roughly purified by alumina column
chromatography (hexane/ethyl acetate = 7:1) to give a
crude product 11 which was used in the next reaction
without further purification. To a solution of the above
crude 11 in a mixed solvent of acetone/H2O = (20 mL:3
mL), was added PPTS (4.00 g, 15.9 mmol) at room tem-
perature. After being stirred under reflux for 46 h, the
reaction mixture was cooled to room temperature and
the reaction was quenched with satd NaHCO3 aq and
the resulting mixture was diluted with ethyl acetate. The
organic layer was separated, washed with brine, dried
over Na2SO4, filtered and the filtrate was concentrated
under reduced pressure. The residual oil was purified by
alumina column chromatography (hexane/ethyl ace-
tate = 5:1) to afford aldehyde (1.15 g, 91% for two steps)
2.95 mmol). The mixture was stirred under reflux for
37 h in a glassware equipped with a Dean-Stark trap,
and the resulting mixture was treated withsatd NaHCO3
aq at room temperature to quench the reaction and
diluted with ethyl acetate. The organic layer was sepa-
rated, washed with brine, dried over Na2SO4, filtered
and the filtrate was concentrated under reduced pres-
sure. The residue was purified by silica gel column chro-
matography (hexane/ethyl acetate = 7:1) to afford acetal
9 (9.07 g, 95%) as a pale yellow powder: mp. 73.2–73.6
1
°C; H NMR (400 MHz, CDCl3) d 7.52 (d, J = 7.6 Hz,
1H), 7.47 (d, J = 7.9 Hz, 1H), 7.38 (dd, J = 7.6, 7.8 Hz,
1H), 7.27 (d, J = 5.1 Hz, 1H), 7.26 (dd, J = 7.8, 7.9 Hz,
1H), 7.10 (d, J = 5.1 Hz, 1H), 6.31 (s, 1H), 4.66
13
C
(s, 2H), 4.17 (m, 2H), 4.06 (m, 2H), 3.47 (s, 3H);
NMR (100 MHz, CDCl3) d 147.1, 141.6, 133.1, 130.5,
129.4, 127.4, 127.3, 125.7, 120.1, 120.1, 98.9, 80.0,
77.9, 77.0, 75.6, 72.4, 65.5, 58.6; IR (CDCl3):
2401 cmÀ1; FAB-MS (NBA) m/z 325 [(M+H)+]; Anal.
Calcd for C19H16O3S: C, 70.35; H, 4.97; S, 9.88. Found:
C, 70.20; H, 5.19; S, 9.84.
1
as orange oil: H NMR (400 MHz, CDCl3) d 10.10 (s,
1H), 7.55 (d, J = 7.6 Hz, 1H), 7.49 (d, J = 8.1 Hz, 1H),
7.42 (dd, J = 7.6, 7.6 Hz, 1H), 7.29 (dd, J=7.6, 8.1 Hz,
1H), 7.06 (s, 1H), 4.66 (s, 2H), 3.63 (s, 2H), 3.48 (s, 3H),
2.31 (s, 6H); 13C NMR (100 MHz, CDCl3) d 182.1,
153.3, 145.3, 141.8, 133.2, 129.8, 129.6, 128.6, 127.6,
127.4, 119.5, 81.8, 79.5, 77.3, 74.5, 72.3, 58.5, 58.3, 45.2;
IR (CDCl3): 2252, 2217, 1666 cmÀ1; FAB-MS (NBA) m/
z 338 [(M+H)+], 307 [(MÀOMe)+]; Anal. Calcd for
C20H19NO2S: C, 71.19; H, 5.68; N, 4.15; S, 9.50. Found:
C, 71.41; H, 5.73; N, 4.04; S, 9.40.
4.2.4. 5-[1,3]Dioxolan-2-yl-4-[4-(2-methoxymethyl-phe-
nyl)-buta-1,3-diynyl]-thiophene-2-carbaldehyde (10). In
argon atmosphere, to a solution of acetal 9 (320 mg,
0.983 mmol) in THF (15 mL) was added n-butyllithium
(1.5 M in hexane, 0.80 mL, 1.2 mmol) dropwise at
À78 °C. The mixture was stirred for 5 min, and to the
resulting bright blue solution was added DMF
(0.12 mL, 1.6 mmol) and stirred for 2 h at temperature
elevated to À50 °C. The reaction mixture was quenched
with satd NH4Cl aq and the resulting mixture was di-
luted with ethyl acetate. The organic layer was sepa-
rated, washed with brine, dried over Na2SO4, filtered
and the filtrate was concentrated under reduced pres-
sure. The residual oil was purified by silica gel column
chromatography (hexane/ethyl acetate = 3:1) to afford
aldehyde 10 (306 mg, 88%) as an orange powder: mp
In argon atmosphere, to a solution of asymmetric diyne
8 (1.3 g, 6.7 mmol) in methyl tert-butyl ether (35 mL)
was added methyllithium–lithium bromide complex
(1.4 M in ether, 2.5 mL, 3.5 mmol) at room tempera-
ture. The reaction mixture was stirred under reflux for
3.5 h, and the resulting diethynyl lithium reagent was
cooled to 0 °C. In argon atmosphere, at À70 °C, to
the solution of the aldehyde (300 mg, 0.889 mmol) in
THF (15 mL) was added the above diethynyllithium
solution. After being stirred at À70 to À55 °C for
1.5 h, the reaction mixture was treated with satd NH4Cl
aq to quench the reaction, and the resulting mixture was
diluted with ethyl acetate. The organic layer was sepa-
rated, washed with brine, dried over Na2SO4, filtered
and the filtrate was concentrated under reduced pres-
sure. The residual oil was purified by silica gel column
chromatography (hexane/ethyl acetate = 1:1) to afford
alcohol 12 (263 mg, 66%) as a pale yellow powder: mp
1
66.8–68.2 °C; H NMR (400 MHz, CDCl3) d 9.87 (s,
1H), 7.74 (s, 1H), 7.54 (d, J = 7.7 Hz, 1H), 7.49 (d,
J = 7.6 Hz, 1H), 7.40 (dd, J = 7.3, 7.7 Hz, 1H), 7.27
(dd, J = 7.6, 7.3 Hz, 1H), 6.31 (s, 1H), 4.66 (s, 2H),
4.18 (m, 2H), 4.08 (m, 2H), 3.48 (s, 3H); 13C NMR
(100 MHz, CDCl3) d 182.6, 156.0, 142.4, 141.7, 138.8,
133.2, 129.7, 127.5, 127.4, 120.8, 119.7, 98.6, 80.8,
77.9, 77.4, 73.8, 72.4, 65.7, 58.6; IR (CDCl3): 2254,
1680 cmÀ1; FAB-MS (NBA) m/z 353 [(M+H)+], 321
+
[(MÀOMe) ]; Anal. Calcd for C20H16O4S: C, 68.16; H,
4.58; S, 9.10. Found: C, 68.21; H, 4.93; S, 8.79.
1
124.5 °C (dec); H NMR (400 MHz, CDCl3) d 7.53 (d,
4.2.5. 1-{5-Dimethylaminomethyl-3-[4-(2-methoxymethyl-
phenyl)-buta-1,3-diynyl]-thiophen-2-yl}-6,6-dimethyl-hep-
ta-2,4-diyn-1-ol (12). In argon atmosphere, to a solution
of aldehyde 10 (1.317 g, 3.72 mmol) in ethanol (50 mL)
were added triethylamine (2.60 mL, 18.6 mmol), dimeth-
ylamine hydrochloride (1.50 g, 18.4 mmol), and titanium
tetra i-propoxide (2.2 mL, 7.5 mmol) at room tempera-
ture. After being stirred at room temperature for 20 h,
the reaction mixture was cooled to 0 °C, followed by the
addition of sodium borohydride (700 mg, 18.5 mmol).
The mixture was stirred at room temperature for 5.5 h,
and the reaction was quenched with concentrated aque-
J = 7.6 Hz, 1H), 7.48 (d, J = 7.8 Hz, 1H), 7.39 (ddd,
J = 1.2, 7.6, 7.6 Hz, 1H), 7.26 (dd, J = 7.6, 7.8 Hz,
1H), 6.86 (s, 1H), 5.93 (s, 1H), 4.66 (s, 2H), 3.57 (s,
2H), 3.48 (s, 3H), 2.86 (broad s, 1H), 2.29 (s, 6H),
1.24 (s, 9H); 13 C NMR (100 MHz, CDCl3) d 149.9,
141.6, 141.5, 133.1, 129.4, 128.7, 127.4, 127.3, 120.1,
117.9, 90.4, 80.2, 77.9, 77.2, 75.8, 74.9, 72.4, 71.1,
63.1, 59.3, 58.6, 57.9, 44.8, 30.3, 28.0; IR (CDCl3):
3422, 2401, 2254 cmÀ1; FAB-MS (NBA) m/z 442
[(MÀH)+], 426 [(MÀOH)+]; Anal. Calcd for C28H29NO2
S: C, 75.81; H, 6.59; N, 3.19; S, 7.23. Found: C, 75.51;
H, 6.70; N, 3.12; S, 7.00.

5448
K. Torikai et al. / Bioorg. Med. Chem. 16 (2008) 5441–5451
4.2.6. 1-{5-Dimethylaminomethyl-3-[4-(2-methoxymethyl-
phenyl)-buta-1,3-diynyl]-thiophen-2-yl}-6,6-dimethyl-hep-
ta-2,4-diyn-1-ol hydrochloride (3). At 0 °C, to a solution
of alcohol 12 (108 mg, 0.243 mmol) in CHCl3 (5 mL)
was added HCl–methanol solution (10%, 0.17 mL,
0.36 mmol). The reaction mixture was stirred at 0 °C
for 5 min, and the solvent was removed in vacuo. To
the residual oil was then added ether and the resulting
gummy slurry was irradiated by ultrasound to solidify.
The solvent was removed to dryness to afford HCl salt
3 (118 mg, quantitative) as a colorless powder: mp
109.3 °C (dec); 1H NMR (400 MHz, CD2Cl2) d 11.93
(broad s, 1H), 7.53 (d, J = 7.6 Hz, 1H), 7.45 (d,
J = 7.6 Hz, 1H), 7.39 (m, 2H), 7.27 (dd, J = 7.3,
7.6 Hz, 1H), 5.95 (s, 1H), 5.73 (broad s, 1H), 4.61 (s,
2H), 4.44 (broad s, 2H), 3.43 (s, 3H), 2.84 (s, 6H), 1.20
(s, 9H); 13C NMR (100 MHz, CDCl3) d 154.3, 141.5,
135.0, 132.8, 129.2, 128.5, 127.3, 127.1, 119.5, 117.5,
90.3, 80.4, 77.7, 77.1, 74.7, 74.5, 72.0, 70.3, 62.5, 58.7,
58.1, 54.0, 41.5, 29.7, 27.6; IR (CDCl3 ): 3232, 2304,
methyl ester 13 (2.355 g, 80%) as a yellow powder: mp
69.5–71.0 °C; 1H NMR (400 MHz, CDCl3) d 7.67 (s,
1H), 7.45 (d, J = 7.6 Hz, 1H), 7.40 (d, J = 7.8 Hz, 1H),
7.31 (dd, J = 7.6, 7.6 Hz, 1H), 7.18 (dd, J = 7.6,
7.8 Hz, 1H), 6.20 (s, 1H), 4.57 (s, 2H), 4.03 (m, 4H),
3.81 (s, 3H), 3.39 (s, 3H); 13C NMR (100 MHz, CDCl3)
d 161.7, 153.3, 141.6, 136.2, 133.1, 132.8, 129.6, 127.4,
127.3, 120.1, 119.8, 98.6, 80.4, 77.5, 77.4, 74.3, 72.3,
65.6, 58.6, 52.5; IR (CDCl3): 2254, 1717 cmÀ1; FAB-
MS (NBA) m/z 382 [M+]; Anal. Calcd for C21H18O5S:
C, 65.95; H, 4.74; S, 8.38. Found: C, 65.93; H, 4.77; S,
8.55.
4.2.9. 5-Formyl-4-[4-(2-methoxymethyl-phenyl)-buta-1,3-
diynyl]-thiophene-2-carboxylic acid methyl ester (14). To
a solution of acetal 13 (3.00 g, 7.84 mmol) in a mixed
solvent of acetone/H2O = (130 mL:13 mL) was added
PPTS (6.50 g, 25.9 mmol) at room temperature. After
being stirred under reflux for 41 h, the reaction mixture
was cooled to room temperature and the reaction mix-
ture was quenched with satd NaHCO3 aq and the result-
ing mixture was diluted with ethyl acetate. The organic
layer was separated, washed with brine, dried over
MgSO4, filtered and the filtrate was concentrated under
reduced pressure. The residual oil was purified by silica
gel column chromatography (hexane/ethyl acetate = 5:1)
to afford aldehyde 14 (2.532 g, 95%) as a yellowpowder:
+
2198 cmÀ1; FAB-MS (NBA) m/z 444 [(MÀCl) ]; Anal.
Calcd for C28H30ClNO2 S: C, 70.05; H, 6.30; N, 2.92.
Found: C, 70.06; H, 6.34; N, 2.88.
4.2.7. 1-{5-Dimethylaminomethyl-3-[4-(2-methoxymethyl-
phenyl)-buta-1,3-diynyl]-thiophen-2-yl}-6,6-dimethyl-hep-
ta-2,4-diyn-1-one hydrochloride (4). At 0 °C, to a solution
of alcohol 12 (150 mg, 0.338 mmol) in a mixed solvent
(CH2Cl2/CHCl3 = 4.5 mL:3.0 mL) were added Na2SO4
(300 mg, 2.11 mmol) and active c-MnO2 (2.9 g,
33 mmol). After being vigorously stirred at 0–7 °C for
16 h, the heterogeneous mixture was filtered. To the fil-
1
mp 69.7–71.1 °C; H NMR (400 MHz, CDCl3) d 10.19
(s, 1H), 7.85 (s, 1H), 7.56 (d, J = 7.8 Hz, 1H), 7.50 (d,
J = 7.8 Hz, 1H), 7.43 (dd, J = 7.6, 7.8 Hz, 1H), 7.29
(dd, J = 7.6, 7.8 Hz, 1H), 4.66 (s, 2H), 3.95 (s, 3H),
3.48 (s, 3H); 13 C NMR (100 MHz, CDCl3) d 182.8,
161.2, 149.3, 141.9, 139.9, 136.7, 133.4, 130.1, 128.3,
127.7, 127.5, 119.3, 82.4, 80.4, 76.9, 73.2, 72.4, 58.7, 53.0;
IR (CDCl3): 2255, 1723, 1675 cmÀ1; FAB-MS (NBA) m/
z 339 [(M+H)+]; Anal. Calcd for C19H14O4 S: C, 67.44;
H, 4.17; S, 9.48. Found: C, 67.42; H, 4.21; S, 9.43.
trate was added
HCl–methanol solution (10%,
0.24 mL, 0.51 mmol) and the solvent was removed at
0 °C in vacuo. The residue was washed with ether and
hexane, followed by drying at 0 °C to afford HCl salt 4
1
(90 mg, 56%) as a yellow powder: mp 83.5 °C (dec); H
NMR (400 MHz, CD2Cl2) d 13.32 (broad s, 1H), 7.74
(s, 1H), 7.55 (d, J = 7.6 Hz, 1H), 7.48 (d, J = 7.6 Hz,
1H), 7.42 (dd, J = 7.3, 7.6 Hz, 1H), 7.28 (dd, J = 7.3,
7.6 Hz, 1H), 4.63 (s, 2H), 4.45 (s, 2H), 3.44 (s, 3H),
2.82 (s, 6H), 1.19 (s, 9H); 13C NMR (100 MHz, CD2Cl2)
d 166.3, 147.4, 141.9, 137.9, 137.0, 132.9, 129.6, 127.2,
127.0, 125.3, 119.1, 99.4, 82.3, 80.9, 79.4, 77.1, 75.0,
72.0, 71.6, 62.2, 58.1, 53.5, 41.6, 29.2, 28.1; IR (CDCl3):
2304, 2223, 2198, 1604 cmÀ1; FAB-MS (NBA) m/z 442
[(MÀCl)+].
4.2.10. 5-Formyl-4-[4-(2-methoxymethyl-phenyl)-buta-1,3-
diynyl]-thiophene-2-carboxylic acid (15). In argon atmo-
sphere, to a solution of methyl ester 14 (2.55 g,
7.54 mmol) in THF (100 mL) was added 1 M NaOH aq
(11 mL, 11 mmol). The mixture was stirred at room tem-
perature for 70 min, and the reaction mixture was
quenched with 1 M HCl aq (20 mL, 20 mmol) at 0 °C.
The resulting mixture was diluted with ethyl acetate
and the organic layer was separated, washed with brine,
dried over Na2SO4, decolorized with charcoal, filtered
and the filtrate was concentrated under reduced pressure
to give carboxylic acid 15 (2.44 g, quantitative) as an or-
4.2.8. 5-[1,3]Dioxolan-2-yl-4-[4-(2-methoxymethyl-phe-
nyl)-buta-1,3-diynyl]-thiophene-2-carboxylic acid methyl
ester (13). In argon atmosphere, to a solution of acetal 9
(2.50 g, 7.71 mmol) in THF (80 mL) at À78 °C was
added n-butyllithium (1.4 M in hexane, 6.5 mL,
9.4 mmol). The mixture was stirred at À78 °C for
10 min, to the resulting bright blue solution was added
methyl chloroformate (1.8 mL, 23 mmol) and the reac-
tion mixture was stirred at À78 °C for 30 min. The reac-
tion mixture was quenched with satd NH4Cl aq and
diluted with ethyl acetate. The organic layer was sepa-
rated, washed with brine, dried over MgSO4, filtered
and the filtrate was concentrated under reduced pres-
sure. The residual oil was purified by silica gel column
chromatography (hexane/ethyl acetate = 5:1) to afford
1
ange powder: mp 128.4–134.7 °C; H NMR (400 MHz,
CDCl3) d 10.18 (s, 1H), 7.89 (s, 1H), 7.58 (d,
J = 7.6 Hz, 1H), 7.49 (d, J = 7.8 Hz, 1H), 7.43 (dd,
J = 7.6, 7.6 Hz, 1H), 7.31 (dd, J = 7.6, 7.8 Hz, 1H), 4.69
(s, 2H), 3.48 (s, 3H); 13C NMR (100 MHz, CDCl3) d
182.8, 164.5, 150.0, 141.5, 139.6, 137.6, 133.5, 130.1,
128.4, 128.1, 127.7, 119.6, 80.6, 77, 73.1, 72.4, 60.7,
58.4; IR (CDCl3): 3422, 2256, 1679 cmÀ1; FAB-MS
(NBA) m/z 325 [(M+H)+].
4.2.11. 5-Formyl-4-[4-(2-methoxymethyl-phenyl)-buta-1,3-
diynyl]-thiophene-2-carboxylic acid triisopropylsilyl ester
(16). To a solution of carboxylic acid 15 (2.4 g,

K. Torikai et al. / Bioorg. Med. Chem. 16 (2008) 5441–5451
5449
7.4 mmol) in CH2Cl2 (40 mL) were added imidazole
(600 mg, 8.81 mmol) and triisopropylsilyl chloride
(TIPSCl, 1.9 mL, 9.0 mmol). After being stirred at room
temperature for 3 h, the reaction mixture was diluted
with CH2Cl2 and H2O. The organic layer was separated,
washed with brine, dried over MgSO4, filtered, and the
filtrate was concentrated under reduced pressure. 4
wt% of the residual crude oil (150 mg) was purified by
florisil (deactivated with 20% of H2O) column chroma-
tography (hexane/ether = 8:1) to afford TIPS ester 16
(98 mg, ca. 70%) as an organge wet powder: mp 55.5–
mixture was diluted with H2O, acidified with 1 M HCl
aq (0.15 mL, 0.15 mmol), and extracted with ether.
The combined organic layer was washed with brine,
dried over Na2SO4, filtered, and the filtrate was concen-
trated under reduced pressure. The residual solid was
washed with hexane and then, with a mixed solvent of
hexane/CHCl3 = 10:1 for several times to afford carbox-
ylic acid (50 mg, 85%) as an orange powder: mp 158.8–
165.0 °C (dec); 1H NMR (400 MHz, CD3OD) d 7.69 (s,
1H), 7.54 (d, J = 7.6 Hz, 1H), 7.47 (d, J = 7.8 Hz, 1H),
7.40 (dd, J = 7.6, 7.8 Hz, 1H), 7.30 (dd, J = 7.6,
7.6 Hz, 1H), 5.84 (s, 1H), 4.62 (s, 2H), 3.45 (s, 3H),
1.22 (s, 9H); 13C NMR (100 MHz, CD3OD) d 164.1,
158.7, 142.7, 136.8, 134.6, 134.2, 130.8, 129.2, 128.9,
121.5, 119.4, 91.1, 81.5, 78.6, 78.3, 75.6, 75.5, 73.6,
71.6, 64.1, 60.2, 58.9, 30.7, 29.1; IR (CD2Cl2 ): 3500–
2300 (broad), 2254, 1685 cmÀ1; FAB-MS (NBA) m/z
413 [(MÀOH)+], 399 [(MÀOMe)+].
1
62.0 °C; H NMR (400 MHz, CDCl3) d 10.19 (s, 1H),
7.81 (s, 1H), 7.56 (d, J = 7.8 Hz, 1H), 7.50 (d,
J = 7.8 Hz, 1H), 7.43 (dd, J = 7.5, 7.8 Hz, 1H), 7.29
(dd, J = 7.5, 7.8 Hz, 1H), 4.66 (s, 2H), 3.48 (s, 3H),
1.40 (m, 3H), 1.14 (d, J = 7.6 Hz, 18H); 13 C NMR
(100 MHz, CDCl3) d 182.9, 159.8, 149.3, 142.3, 142.0,
136.8, 133.5, 130.1, 128.5, 127.8, 127.6, 119.4, 82.5,
80.3, 73.5, 72.5, 58.6, 31.7, 13.7, 12.0; IR (CDCl3):
2254, 2219, 1700, 1675 cmÀ1; FAB-MS (NBA) m/z 481
[(M+H)+]; Anal. Calcd for C27H32O4 SSi: C, 67.46; H,
6.71; S, 6.67. Found: C, 67.55; H, 6.64; S, 6.70.
To a solution of the above carboxylic acid (133 mg,
0.309 mmol) in MeOH (5 mL) was added K2CO3
(21.5 mg, 0.156 mmol) at 0 °C. The mixture was stirred
at 0–4 °C for 41 h, and the solvent was removed in va-
cuo. To the residual viscous oil was added ether to solid-
ify to afford potassium salt 5 (127 mg, 88%) as a greenish
yellow powder: 1H NMR (400 MHz, CD3OD) d 7.55 (d,
J = 7.6 Hz, 1H), 7.47 (d, J = 7.3 Hz, 1H), 7.44 (s, 1H),
7.41 (dd, J = 7.3, 7.6 Hz, 1H), 7.31 (dd, J = 7.6,
7.6 Hz, 1H), 5.79 (s, 1H), 4.63 (s, 2H), 3.46 (s, 3H),
1.23 (s, 9H); IR (CDCl3): 3362 (broad), 2254, 1588,
1365 cmÀ1; FAB-MS (NBA) m/z 429 [(MÀK)+].
4.2.12. 5-(1-Hydroxy-6,6-dimethyl-hepta-2,4-diynyl)-4-
[4-(2-methoxymethyl-phenyl)-buta-1,3-diynyl]-thiophene-
2-carboxylic acid triisopropylsilyl ester (17). In argon
atmosphere, to a solution of asymmetric butadiyne 8
(1.48 g, 8.30 mmol) in methyl tert-butyl ether (35 mL)
was added methyllithium–lithium bromide complex
(1.4 M, in ether, 3.0 mL, 4.2 mmol) at room tempera-
ture. The mixture was stirred under reflux for 3 h, and
the resulting alkynyl lithium solution was cooled to
0 °C and added to a solution of aldehyde 16 (500 mg,
1.04 mmol) in ether (14 mL) at À72 °C. After being stir-
red at À72 to À48 °C for 2 h, the reaction mixture was
treated with satd NH4Cl aq to quench the reaction
and diluted with ethyl acetate. The organic layer was
separated, washed with brine, dried over Na2SO4, fil-
tered, and the filtrate was concentrated under reduced
pressure. The residue was roughly purified by deacti-
vated silica gel (by adding 25wt% of H2O) column chro-
matography (hexane/ether = 5:1) to give crude alcohol
17. The above crude (40 mg) was purified by HPLC
(CHCl3) to give pure 17 (23 mg, ca. 40%) as yellow
oil: 1H NMR (400 MHz, CDCl3) d 7.69 (s, 1H), 7.54
(d, J = 7.6 Hz, 1H), 7.48 (d, J = 7.8 Hz, 1H), 7.40 (dd,
J = 7.3, 7.8 Hz, 1H), 7.27 (dd, J = 7.3, 7.6 Hz, 1H),
5.95 (s, 1H), 4.66 (s, 2H), 3.48 (s, 3H), 2.74 (broad s,
1H), 1.38 (m, 3H), 1.24 (s, 9H), 1.13 (d, J = 7.6 Hz,
18H); 13C NMR (100 MHz, CDCl3) d 160.5, 155.5,
141.7, 136.1, 134.9, 133.2, 129.7, 127.6, 127.4, 119.9,
119.2, 91.3, 80.8, 78.2, 77.5, 74.4, 73.6, 72.4, 72.1, 62.8,
59.8, 58.6, 30.3, 28.1, 17.8, 12.0; IR (CDCl3): 3360,
1689 cmÀ1; FAB-MS (NBA) m/z 586 [M+]; Anal. Calcd
for C35H42O4SSi: C, 71.63; H, 7.21; S, 5.46. Found: C,
71.37; H, 7.16; S, 5.62.
4.2.14. 5-(6,6-Dimethyl-hepta-2,4-diynoyl)-4-[4-(2-methoxy-
methyl-phenyl)-buta-1,3-diynyl]-thiophene-2-carboxylic acid
potassium salt (6). To a solution of alcohol 17 (100 mg,
0.170 mmol) in CH2Cl2 (10 mL) was added Na2SO4
(142 mg, 1.00 mmol) and active c-MnO2 (1.5 g, 17 mmol)
at 0 °C. After being vigorously stirred at 0–7 °C for 27 h,
the heterogeneous mixture was filtered and the filtrate was
concentrated at 0 °C to give ketone (98 mg, 98%) as an or-
ange oil: 1H NMR (400 MHz, CD2Cl2) d 7.81 (s, 1H), 7.56
(d, J = 7.6 Hz, 1H), 7.49 (d, J = 7.8 Hz, 1H), 7.42 (dd,
J = 7.6, 7.8 Hz, 1H), 7.29 (dd, J = 7.6, 7.6 Hz, 1H), 4.64
(s, 2H), 3.45 (s, 3H), 1.41 (m, 3H), 1.21 (s, 9H), 1.14 (d,
J = 7.6 Hz, 18H); 13 C NMR (100 MHz, CD2Cl2) d
167.6, 160.1, 149.8, 142.6, 141.8, 138.5, 133.6, 130.3,
128.0, 127.8, 125.5, 120.0, 100.3, 82.8, 81.0, 80.3, 77.9,
75.9, 72.7, 72.5, 63.0, 58.8, 30.0, 28.9, 17.9, 12.4; IR
(CD2Cl2): 2220, 1701, 1609 cmÀ1; FAB-MS (NBA) m/z
585 [(M+H)+].
To a solution of the above ketone (95 mg, 0.16 mmol) in a
mixed solvent (MeOH/CH2Cl2/ether = 10:1:1, 8.4 mL) was
at 0 °C added K2CO3 (12 mg, 0.087 mmol). The mixture
was stirred at 0 °C for 20 min, and the solvent was removed
at 0 °C. The residual amorphous was washed with cold ether
for several times to afford potassium salt 6 (58 mg, 77%) as a
1
4.2.13. 5-(1-Hydroxy-6,6-dimethyl-hepta-2,4-diynyl)-4-
[4-(2-methoxymethyl-phenyl)-buta-1,3-diynyl]-thiophene-
2-carboxylic acid potassium salt (5). To a solution of
TIPS ester 17 (80 mg, 0.14 mmol) in MeOH (3.5 mL)
was at 0 °C added K2CO3 (10 mg, 0.072 mmol). After
being stirred at room temperature for 5 h, the reaction
yellow powder: mp 73.0 °C (dec); H NMR (400 MHz,
CD3OD) d 7.58 (s, 1H), 7.56 (d, J = 7.3 Hz, 1H), 7.47 (d,
J = 7.8 Hz, 1H), 7.43 (dd, J = 7.6, 7.8 Hz, 1H), 7.32 (dd,
J = 7.3, 7.6 Hz, 1H), 4.64 (s, 2H), 3.45 (s, 3H), 1.18(s, 9H);
IR (CD2Cl2): 2304, 2224, 2198, 1604, 1359 cmÀ1; FAB-MS
(NBA) m/z 467 [(M+H)+].

5450
K. Torikai et al. / Bioorg. Med. Chem. 16 (2008) 5441–5451
4.3. Agarose gel electrophoresis
Acknowledgments
Stock solutions of thienyl tetraynes were prepared as
follows: thienyl tetraynes were dissolved in DMSO fol-
lowed by dilution with distilled deionized water
(DDW) till the final concentration of DMSO reached
to 5.0%. Before adding to DNA, the above stock solu-
tion was diluted with 5.0% DMSO. To a solution of
pBluescript II KS+ DNA (2.0 lg/7.5 lL) was added
the diluted tetrayne stock solution to set the final incu-
bation conditions: total volume 20 lL; final DMSO con-
centration 3.0%. The mixture was incubated at 37 °C
over night, DNA was precipitated by treatment with
3 M sodium acetate (2 lL) and ethanol (60 lL). After
being incubated for 20 min at À80 °C, the suspension
was centrifuged (15000 rpm, 4 °C, 10 min). The superna-
tant was removed and the residue was washed with 70%
ethanol, followed by centrifugation (15,000 rpm, 4 °C,
10 min) and the removal of supernatant again. The res-
idue was dried in vacuo, dissolved in H2O (8.0 lL), and
the resulting solution was incubated at 60 °C for 10 min,
at 37 °C for 30 min. To the above DNA solution was
added loading buffer (2.0 lL), the resulting solution
was developed on 0.7% agarose gel. After electrophore-
sis, DNA was visualized by treating the gel with ethydi-
um bromide solution for 10 min followed by the
irradiation of UV.
We are grateful to Dr. Yoko Mizuno, Mr. Tomo-
hiro Kozaki (Institute for Protein Research, Osaka
University), Dr. Patrick C. Reid (Research Center
for Advanced Science and Technology, the Univer-
sity of Tokyo), Dr. Tomikazu Kawano and Mr.
Hiroki Inai (our laboratory), for invaluable discus-
sion. Dr. Hiroyuki Ido and Ms. Yoshiko Takagi in
Institute for Protein Research, Osaka University,
are greatly acknowledged for preparing competent
cells of E. coli. We are also indebted to Mr. Hito-
shi Yamada and Ms. Sachiyo Nomura in the Mate-
rial Analysis Center of ISIR-Sanken for the
elemental analyses and the measurement of MS
spectra.
References and notes
1. (a) Wang, K. K. Chem. Rev. 1996, 96, 207; (b) Wenk, H.
H.; Winkler, M.; Sander, W. Angew. Chem., Int. Ed. 2003,
42, 502.
2. Reviews on early isolation and chemical biological studies
of ene–diyne antibiotics, see: (a) Nicolaou, K. C.; Dai,
W.-M. Angew. Chem., Int. Ed. Engl. 1991, 30, 1387; (b)
Grissom, J. W.; Gunawardena, G. U.; Klingberg, D.;
ˇ
Huang, D. Tetrahedron 1996, 52, 6453; (c) Gredicak, M.;
´
Jeric, I. Acta Pharm. 2007, 57, 133.
4.4. Atomic force microscopy
3. Enediyne Ene–diyneAntibiotics as Antitumor Agents; Bor-
der, D. B., Doyle, T. W., Eds.; Mercell Dekker: New
York, 1995.
4. (a) Nicolaou, K. C.; Dai, W. M.; Tsay, S. C.; Estevez, V.
A.; Wrasidlo, W. Science 1992, 256, 1172; (b) Kar, M.;
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5. (a) Miyawaki, K.; Suzuki, R.; Kawano, T.; Ueda, I.
Tetrahedron Lett. 1997, 38, 3943; (b) Ikemoto, C.;
Kawano, T.; Ueda, I. Tetrahedron Lett. 1998, 39,
5053; (c) Miyawaki, K.; Kawano, T.; Ueda, I.
pBluescript II KS+ DNA was treated with tetraynes
(1.0 mM, 37 °C, 6 h) and isolated by ethanol precip-
itation as described in Section 4.3. After ethanol pre-
cipitation, pellet was dissolved in 800 lL of H2O to
give a sample solution. DNA was settled on a mica
plate as follows: 10 lL of the above sample solution
was dropped on a flat mica plate, incubated at room
temperature for 1 min and unnecessary water was
flushed away with air. The surface of the mica plate
possessing DNA was visualized using an atomic
force microscope (Multimode atomic force micro-
scope (Nanoscope IV) from Veeco Instruments,
Inc., USA).
Tetrahedron Lett.
1998, 39, 6923; (d) Miyawaki,
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Miyawaki, K.; Sugane, T.; Sakurai, Y.; Wada, Y.; Futai,
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11. For example, aqueous formaldehyde, Me2NH, AcOH,
100 °C, 68 h, or Eschenmoser’s salt (iodide, chloride),
THF or CH3CN, reflux, 20 h, failed to provide 12.
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13. Although TLC analysis of the reaction showed clean
formation of desired product 17, higher yield could not be
achieved due to the loss during chromatographic
purification.
4.5. Escherichia coli transformation assay
pBluescript II KS+ DNA was treated with tetraynes
(1.0 mM) or other reagents, isolated by ethanol precip-
itation as described in Section 4.3. The resulting DNA
was dissolved in 8.0 lL of H2O, and the solution was
incubated at 60 °C for 10 min. To a solution of compe-
tent cell of E. coli (DH5a or JM109) at 0 °C was added
the above DNA solution (8.0 lL) and the mixture was
incubated at 42 °C for 1 min (heat shock). The result-
ing solution was immediately cooled to 0 °C and incu-
bated for 3 min at 0 °C, then Luria–Bertani (LB) media
(900 lL) was added. The mixture was incubated at
37 °C for 1 h (transformation), the resulting E. coli
solution (100 lL) was spread to an LB agar plate-con-
taining ampicillin (100 mg/L). The plate was incubated
at 37 °C for 15 h, and the number of colonies was
counted.

K. Torikai et al. / Bioorg. Med. Chem. 16 (2008) 5441–5451
5451
14. Treatment of 17 with excess amount of base resulted in
low yield owing to Meyer-Schuster rearrangement: Meyer,
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15. The DNA cleaving activities of thienyl TYs 1 and 2 could
not be evaluated due to their poor solubility.
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