Synthesis and characterization of a small analogue of the anticancer natural product leinamycin

Article abstract of DOI:10.1016/j.bmc.2012.10.021

Leinamycin (1) is a Streptomyces-derived natural product that displays nanomolar IC₅₀ values against human cancer cell lines. In the work described here, we report the synthesis and characterization of a small leinamycin analogue 19 that closely resembles the 'upper-right quadrant' of the natural product, consisting of an alicyclic 1,2-dithiolan-3-one 1-oxide heterocycle connected to an alkene by a two-carbon linker. The results indicate that this small analogue contains the core set of functional groups required to enable thiol-triggered generation of both redox active polysulfides and an episulfonium ion intermediate via the complex reaction cascade first seen in the natural product leinamycin. The small leinamycin analogue 19 caused thiol-triggered oxidative DNA strand cleavage in a manner similar to the natural product, but did not alkyate duplex DNA effectively. This highlights the central role of the 18-membered macrocycle of leinamycin in driving efficient DNA alkylation by the natural product.

Full text of DOI:10.1016/j.bmc.2012.10.021

Bioorganic & Medicinal Chemistry 21 (2013) 235–241
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and characterization of a small analogue of the anticancer
natural product leinamycin
Kripa Keerthi a, Anuruddha Rajapakse a, Daekyu Sun c, Kent S. Gates a,b,
⇑
a Department of Chemistry, University of Missouri, 125 Chemistry Building Columbia, MO 65211, United States
b Department of Biochemistry, University of Missouri, 125 Chemistry Building Columbia, MO 65211, United States
c Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, BIO5 Institute, Room 102, 1657 E. Helen Street, Tucson, AZ 85721, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 July 2012
Revised 15 October 2012
Accepted 20 October 2012
Available online 27 October 2012
Leinamycin (1) is a Streptomyces-derived natural product that displays nanomolar IC50 values against
human cancer cell lines. In the work described here, we report the synthesis and characterization of a
small leinamycin analogue 19 that closely resembles the ‘upper-right quadrant’ of the natural product,
consisting of an alicyclic 1,2-dithiolan-3-one 1-oxide heterocycle connected to an alkene by a two-
carbon linker. The results indicate that this small analogue contains the core set of functional groups
required to enable thiol-triggered generation of both redox active polysulfides and an episulfonium ion
intermediate via the complex reaction cascade first seen in the natural product leinamycin. The small
leinamycin analogue 19 caused thiol-triggered oxidative DNA strand cleavage in a manner similar
to the natural product, but did not alkyate duplex DNA effectively. This highlights the central role
of the 18-membered macrocycle of leinamycin in driving efficient DNA alkylation by the natural
product.
Keywords:
DNA alkylation
Antitumor agent
Natural product
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
episulfonium ion (5) that efficiently alkylates guanine residues in
duplex DNA (Scheme 1).18,23–25 The resulting leinamycin–guanine
Natural products have played a central role in the discovery of
anticancer, antifungal, and antibacterial drugs. Between 60% and
80% of the clinically used drugs in these categories are natural
products or are derived from natural product leads.1–4 In the fields
of organic, medicinal, and natural product chemistry there is a rich
history surrounding the preparation of small synthetic analogues
of bioactive natural products.5–10 In particular, studies of small,
less functionalized—‘stripped down’—analogues can help define
the core functional groups that confer function upon the natural
product and can facilitate the development of new derivatives with
improved properties.5–10 Here we describe the synthesis and char-
acterization of a small analogue of the anticancer natural product
leinamycin.
Leinamycin (1) is a Streptomyces-derived natural product that
displays nanomolar IC50 values against human cancer cell
lines.11–16 Reaction of biological thiols with the 1,2-dithiolan-
3-one 1-oxide heterocycle of leinamycin is thought to produce a
sulfenic acid intermediate 2 that cyclizes17 to eject a persulfide
intermediate (3) that, in turn, generates reactive oxygen species
(Scheme 1).18–22 The 1,2-oxathiolan-5-one intermediate (4)
formed in this process undergoes further rearrangement to an
adduct 6 undergoes rapid depurination to generate abasic sites in
cellular DNA.26–30
Previous studies showed that the small leinamycin analogues
10–12 replicate the natural product’s ability to generate reactive
oxygen species upon reaction with thiols.19,20,29 In the work de-
scribed here, we report the synthesis and characterization of a
small leinamycin analogue that closely resembles the upper right
quadrant of the natural product (1, Scheme 1), consisting of an ali-
cyclic 1,2-dithiolan-3-one 1-oxide heterocycle connected to an al-
kene moiety by a two-carbon linker. We felt this analogue was of
interest because it nominally contains the minimum set of func-
tional groups required to enable both the release of redox-active
polysulfur species and generation of an episulfonium ion via the
complex reaction cascade first seen in the natural product
leinamycin.
2. Results and discussion
2.1. Synthesis of the leinamycin analogue 19
Our synthesis began with conjugate addition of benzyl mer-
captan to commercially available geranic acid 14 in refluxing
piperidine to give 15 in 95% yield (Scheme 2). Removal of the
⇑
Corresponding author. Tel.: +1 573 882 6763; fax: +1 573 882 2754.
E-mail address: gatesk@missouri.edu (K.S. Gates).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.10.021

236
K. Keerthi et al. / Bioorg. Med. Chem. 21 (2013) 235–241
O
O
OH O
S
H
O
CH3
CO2
OH
–
S
OH
H
H
H
+
S
H
O–
N
N
O
N
H
O
N
H
S
S
9
Leinamycin (1)
RSH
Nu:
O
O
OH
CH3
C7
O
O
OH O
C6
–
C8
CO2
S+
SSR
SOH
OH
O
S
H
OH
H
H
OH
H
H
H
HO
H
N
N
N
O
O
N
H
O
N
H
N
H
S
S
S
5
2
4
+
RSSH
3
Nu
RSSR
RSH
CH3
O
O2
–
CO2
S
OH
H
H
Oxidative
Stress
H2O2
O2•–
HO
H
HO•
N
O
N
H
RSSxSR
S
8
6: Nu=N7-Guanine, 7: Nu=OH
Scheme 1.
O
O
O
O
S
SH
OH
OH
Ph
S
S
+
O–
+
S
S
O–
piperidine
reflux, 20 h
10
11
15
14
O
O
Na, liq. NH3
THF
HO
S
S
+
O–
+
S
S
O
O–
O
DCC, DMAP
THF, RT
OH
SH
12
S
R
R
13 R = CH3 or H
17
16
1. H2S, TEA, – 35°C RT
2. FeCl3, MeOH
O
O
O
benzyl group from 15 using sodium in liquid ammonia gave the
thiol derivative 16 in good yield (80%). Activation of the carbox-
ylic acid in 16 with dicyclohexylcarbodiimide and DMAP in tetra-
hydrofuran converted this material to the thiolactone 17 in 75%
yield. Treatment of 17 with H2S and triethylamine, followed by
oxidation with FeCl3 in methanol gave the 1,2-dithiolan-3-one
compound 18 in 60% yield for the two steps. Oxidation of this
material with dimethyldioxirane gave a 1:1.7 diastereomeric mix-
ture of the desired leinamycin analogue 19 in 80% yield
(Scheme 2). Our approach represents a modified version of the
route pioneered by Pattenden and Shuker for the synthesis of
simple 1,2-dithionlan-3-one 1-oxides.31 A similar route was used
by Lee et al. for the preparation of analogues containing a pen-
dent alkene.32
O
O
S
S
S
S
S
S
+
+
acetone
O–
O–
18
19
Scheme 2.
2.2. Reaction of 19 with mercaptans
We examined the reaction of 19 with several different thiols.
Compound 19 was used as the mixture of diastereomers obtained
in the synthesis above. A similar spectrum of products was

K. Keerthi et al. / Bioorg. Med. Chem. 21 (2013) 235–241
237
O
O
O
O
S
O
SSR
SOH
OH
OH
RSH
OH
S
O
S
+
S
+
H2O
S
O–
RSH
20
21
25
19
26
+
OH
+
O
O
RSSH
22
S
S
SSR
RSSxSR
18
23
24
Scheme 3.
generated in reactions of 19 with 2-mercaptoethanol, ethanethiol,
and benzyl mercaptan, although the relative yields of each product
varied somewhat. The reaction with benzyl mercaptan is described
here as a representative example. Compound 19 (20 mM) was
mixed with benzyl mercaptan (100 mM) in a 1:1 mixture of so-
dium phosphate buffer (50 mM, pH 7)/acetonitrile. After 3 h, the
mixture was treated with diazomethane to methylate carboxylic
acid-containing products.
13,23,35 the formation of 26 is envisioned to proceed via an epis-
ulfonium ion intermediate 25 (Scheme 3) generated by reaction
of the alkene with the electrophilic sulfur in the oxathiolanone
ring.
2.3. Thiol-triggered DNA damage by 19
We employed a plasmid-based assay to examine the DNA-
damaging properties of 19. In this assay, single-strand cleavage
converts supercoiled plasmid DNA (form I) to the open-circular
form (form II).36–38 The two forms of plasmid DNA are then sepa-
rated using agarose gel electrophoresis, the gel stained with a
DNA-binding dye such as ethidium bromide, and the relative
amounts of cleaved and intact plasmid quantitatively determined
by digital image analysis. The direct strand breaks (not requiring
thermal or basic workup) monitored in this type of experiment
typically arise via hydrogen atom abstraction from the 20-deoxyri-
bose residues in the backbone of DNA,39–45 although the assay also
can be used to detect abasic sites that arise via DNA alkylation
processes.46,47
We found that 19 (used in this first experiment as the mixture
of diastereomers) generated direct strand breaks when incubated
with double-stranded plasmid DNA in the presence of 1.5 equiv
of 2-mercaptoethanol at 24 °C in pH 7 sodium phosphate buffer
(lane 4, Fig. 1). In the absence of thiol, 19 induced relatively little
strand cleavage (lane 2, Fig. 1). Other control reactions showed
that DNA in buffer alone, or DNA plus 2-mercaptoethanol was
not significantly cleaved (lanes 1 and 3, respectively in Fig. 1).
The reaction of 19 with benzyl mercaptan generated the
deoxygenated product 18 in 15% isolated yield (Scheme 3). An
analogous deoxygenation product was observed previously in
the reaction of 10 with propanethiol.18 Sulfoxides can be deoxy-
genated by reaction with thiols,33,34 and 18 could arise via this
route or by mechanisms unique to the 1,2-dithiolan-3-one 1-oxide
heterocycle. The disulfide 24 was produced in 10% yield and the
cyclized product 26 in 35% yield. In a separate reaction with 2-
mercaptoethanol, GC/MS was used to detect the formation of
polysulfides 23. The products 18, 23, and 24 are analogous to
those seen previously in the reactions of 10 with thiol.18 In line
with the conclusions from the early studies with 10, we envision
that the attack of a thiol group on the central, sulfenyl sulfur of
the 1,2-dithiolan-3-one 1-oxide heterocycle in 19 gives rise to
an 1,2-oxathiolan-5-one 21 and a persulfide 22 via the sulfenic
acid intermediate 20 (Scheme 3).17 Attack of a second equivalent
of mercaptan on the 1,2-oxathiolan-5-one provides the disulfide
24, while the persulfide 22 decomposes to yield a mixture of poly-
sulfides 23.21 The alkene residue in 19 is clearly involved in the
generation of the cyclized product 26. Analogous to the reactions
seen for leinamycin (1) and small synthetic leinamycin analogues
Figure 2. DNA strand cleavage by 10 or 19 in the presence of 2-mercaptoethanol.
Assays were performed as described in the Section 4. Briefly, supercoiled double-
stranded DNA (30 lM bp) was incubated with the indicated amounts of 10 or 19
Figure 1. Thiol-dependent DNA strand cleavage by 19. Assays were performed as
described in the Section 4. Briefly, supercoiled double-stranded DNA (30 lM bp)
and thiol in sodium phosphate buffer (50 mM, pH 7) containing acetonitrile (10% v/
v) for 15 h at 37 °C. Form I and II plasmid DNA were resolved on an agarose gel and
stained with ethidium bromide. The DNA was visualized by UV-transillumination
and the amounts measured by digital image analysis. The number of single strand
breaks per plasmid DNA molecule (S) was calculated using the equation S = ꢀln f1
where f1 is the fraction of plasmid present as form I.64 The numbers in the
parentheses following the description of each lane indicate the number of strand
breaks per plasmid molecule. Lane 1: DNA alone (0.21), Lane 2: 2-mercaptoethanol
was incubated with 19 and 2-mercaptoethanol in sodium phosphate buffer
(50 mM, pH 7) containing acetonitrile (10% v/v) for 3 h at 37 °C. Form I and II
plasmid DNA were resolved on an agarose gel and stained with ethidium bromide.
The DNA was visualized by UV-transillumination and the amounts measured by
digital image analysis. The number of single strand breaks per plasmid DNA
molecule (S) was calculated using the equation S = ꢀln f1 where f1 is the fraction of
plasmid present as form I.64 The numbers in the parentheses following the
description of each lane indicate the number of strand breaks per plasmid molecule
unless indicated otherwise. Lane 1: DNA in buffer alone (S = 0.14 0.), Lane 2: 19
(375
Lane 4: 10 (250
Lane 5: minor isomer of 19 (250
minor isomer of 19 (250 M) + 2-mercaptoethanol (375
mL) (0.27), Lane 7: major isomer of 19 (250 M) + 2-mercaptoethanol (375
M) + 2-mercaptoethanol (375 M) + 100 l
l
M) alone (0.2), Lane 3: 10 (250
M) + 2-mercaptoethanol (375
M) + 2-mercaptoethanol (375
M) + catalase (100 l
l
M) + 2-mercaptoethanol (375
M) + catalase (100 g/mL) (0.25),
M) (1.23), Lane 6:
g/
M)
g/
lM) (0.89),
l
l
l
l
l
alone (250
lM) (0.22 0.09), Lane 3: 2-mercaptoethanol alone (375
l
M)
l
l
(0.26 0.08), Lane 4: 19 (250
l
M) + 2-mercaptoethanol (375
lM) (100% form II, S
l
l
>4.6), Lane 5: 19 (250
lM) + 2-mercaptoethanol (375
lM) + catalase (100
lg/mL)
(1.35), Lane 8: major isomer 19 (250
l
l
(0.30 0.02).
mL catalase (0.25).

238
K. Keerthi et al. / Bioorg. Med. Chem. 21 (2013) 235–241
Importantly, thiol-triggered strand cleavage by 19 was strongly
inhibited by addition of the peroxide-destroying enzyme catalase
(lane 5, Fig. 1). This provided evidence that thiol-dependent
strand cleavage by 19 involves the generation of reactive oxygen
species (O2Åꢀ, H2O2, and HOÅ).19,48 Yields of thiol-triggered strand
cleavage by the minor diastereomer of 19 (lane 7, Fig. 2) and
major diastereomer of 19 (lane 5, Fig. 2) were very similar and
were comparable to that generated by the leinamycin model
compound 10 (lane 3, Fig. 2) that lacks a pendent alkene.19
Thiol-triggered strand cleavage by 10 and the two diastereomers
of 19 was strongly inhibited by addition of the peroxide-
destroying enzyme catalase. This confirms previous results show-
ing that strand cleavage by 10 was inhibited by the presence of
catalase in the reaction mixture and is consistent with the
involvement of reactive oxygen species.19 The thiol-dependent
generation of reactive oxygen species in the case of 10 was previ-
ously attributed to thiol-driven redox cycling of the persulfide/
polysulfide couple as shown in Scheme 1.20 The extent to which
catalase inhibited thiol-triggered strand cleavage was nearly iden-
tical for the two diastereomers of 19 and 10 (lanes 4, 6, and 8,
Fig. 2). The presence of catalase brings strand cleavage yields al-
most down to the level of control lanes containing DNA incubated
with buffer alone (lane 1, Fig. 2) or DNA incubated with just thiol
(lane 2, Fig. 2). This result, coupled with the observation that
polysulfides were generated in the reaction of thiol with 19, sug-
gested that thiol-dependent release of redox active polysulfur
species from 19 generated DNA-cleaving reactive oxygen species
as described previously for 1, 10, and 11 and shown in the
unbalanced Equations 1–5, where M represents an adventitious
transition metal ion such as iron or copper.18
The reaction of thiols with leinamycin generates two
different types of DNA-damaging reactive intermediates: an
episulfonium ion alkylating agent and redox-active polysulfides
(Scheme 1).20,23,24 Our findings suggest that the small leinamycin
analogue 19 emulates both of these reaction manifolds. Specifi-
cally, the reaction of 19 with thiol leads to the generation of both
polysulfides 23 and an episulfonium-derived product 26
(Scheme 3). In analogy with mechanisms proposed to explain the
products arising from the reaction of thiols with leinamycin, we
suggest that 26 arises via intramolecular reaction of the pendent
alkene in 19 with the electrophilic sulfur atom in the putative
1,2-oxathiolan-5-one intermediate 21 (Scheme 3). Bimolecular
versions of this process, involving reaction of an alkene with an
O-acylsulfenic acid have been reported previously.50–53 The yield
of the episulfonium ion-derived product 26 (35%) generated in
the reaction of 19 with 2-mercaptoethanol was somewhat lower
than that for the analogous product generated from 13 (50%) under
the same conditions.35 Yields of the episulfonium ion-derived
products from leinamycin are quite high (75% isolated yields).23
Overall, the results may illustrate the role of leinamycin’s 18-mem-
bered macrocycle in properly positioning the alkene of leinamycin
for reaction with the electrophilic sulfur in 4. In this regard, the
solution structures of 1 and 4 deserve further consideration. DNA
clearly is not required for episulfonium ion formation, as the
hydrolysis product 7 forms efficiently in the absence of DNA;23
nonetheless it may be interesting to consider how the noncovalent
association of activated leinamycin with DNA affects episulfonium
ion formation, position of the 5/9 equilibrium, or the rate at which
5 undergoes hydrolysis.
We found that the small, synthetic leinamycin analogue 19 gen-
erates strand cleavage in the presence of the thiol, 2-mercap-
toethanol. Our evidence indicates that the strand cleavage process
proceeds via reactive oxygen species (O2Åꢀ, H2O2, and HOÅ). This is
consistent with previous work showing that the reaction of thiols
with 1, 10, 11, and 12 leads to the release of redox active polysulfur
species that generate DNA-cleaving reactive oxygen species via re-
dox cycling (Scheme 1) and the release of H2S.8,18–21,54
RSSH ꢀ RSS— þ Hþ
ð1Þ
ð2Þ
RSS— þ Mðnþ1Þþ ! RSSꢁ þ Mnþ
M
nþ þ O2 ! Mðnþ1Þþ þ O2ꢁ—
ð3Þ
ð4Þ
O—2ꢁ þ HO2 ! H2O2 þ O2
Our results further provided evidence that the incubation of 19
with duplex DNA in the presence of thiol did not result in efficient
alkylation of the DNA substrate. Upon first consideration, the fail-
ure of 19 to carry out thiol-triggered DNA alkylation may seem sur-
prising given the evidence for thiol-triggered generation of an
episulfonium ion intermediate from this compound (Scheme 3).
However, this result can easily be rationalized in light of existing
data. First, small episulfonium ions are typically very inefficient
DNA-alkylating agents because they primarily undergo hydrolysis
in aqueous solution.55,56 Second, the macrocycle of leinamycin
plays critical roles in DNA alkylation by the natural product. The
18-membered macrocycle of leinamycin associates noncovalently
with the double helix in a manner that drive efficient DNA alkyl-
ation by 5/9 (Scheme 1).24,49,57 In addition, the DNA-alkylating
episulfonium ion of leinamycin may be stabilized against hydroly-
sis via an equilibrium involving the C8 alcohol, in what can be clas-
sified as a reversible thia-Payne rearrangement (Scheme 1).23
Finally, the macrocycle of leinamycin may stabilize the 1,2-dithio-
lan-3-one 1-oxide residue against hydrolytic degradation.58,59 The
simple leinamycin analogues synthesized and characterized here
enjoy none of the benefits imparted by the 18-membered macrocy-
cle found in the natural product and, therefore, are incapable of
effective DNA alkyation.
ꢁ
H2O2 þ Mnþ ! HOꢁ þ HO— þ Mðnþ1Þþ
ð5Þ
The observation that the peroxide-destroying enzyme catalase
inhibits DNA strand cleavage by 19, was consistent with the
involvement of reactive oxygen species; however, alkylating
agents also have the potential to generate strand cleavage in the
plasmid assay.47 Indeed, a separate control experiment showed
that the DNA-alkylating49 epoxide, glycidol (250
lM), generates
measurable amounts of strand breaks (0.34 0.2 above back-
ground) under our reaction conditions. However, strand cleavage
by glycidol was not significantly altered by the presence of catalase
(100 lg/mL; 0.42 0.27 breaks above background). Thus, the
observation that strand cleavage by 19 is almost completely
quenched in the presence of catalase suggests that this compound
damages DNA primarily via the generation of reactive oxygen spe-
cies and not via DNA alkylation.
3. Conclusion
In this work, we prepared a simple analogue of leinamycin con-
sisting of a 1,2-dithiolan-3-one 1-oxide bearing a pendent alkene.
Our synthesis expands the scope of a route developed by Pattenden
and Shuker for simple 1,2-dithiolan-3-one 1-oxides such as 11 and
12 related to leinamycin.31 A similar approach was used by Lee
et al. to prepare a leinamycin analogue similar to 19 bearing a pen-
dent alkene; however, these researchers did not report the reac-
tions of their product with thiols or DNA.32
The results presented here, along with our previous work,35
provide evidence that the 1,2-dithiolan-3-one 1-oxide heterocycle
with a pendent alkene represents the minimal functional unit
required to enable thiol-triggered generation of both redox active
polysulfides and an episulfonium ion intermediate via the
sequence of rearrangement reactions first seen in the natural

K. Keerthi et al. / Bioorg. Med. Chem. 21 (2013) 235–241
239
product leinamycin. However, the relatively low yields of
episulfonium-derived products from 19 and the failure of this
simple analogue to carry out thiol-triggered DNA alkylation
highlight the central roles played by the 18-membered macrocy-
cle of leinamycin in driving efficient DNA alkylation by the natural
product.
d 10.00 (br s, 1H), 5.06 (m, 1H), 2.68 (s, 2H), 2.16–2.08 (m, 2H),
1.74–1.70 (m, 2H), 1.69 (s, 3H), 1.60 (s, 3H), 1.47 (s, 3H); 13C
NMR (62.9 MHz, CDCl3) d 176.7, 132.2, 123.3, 48.2, 44.9, 43.8,
29.8, 25.6, 23.6, 17.6; MS-(ESI-) C10H18O2S (MꢀH) calcd for
202.10, found 201.11. If left in solution this compound cyclizes
as described previously for 2-mercapto allylbenzene63 and this
material was used immediately in the next step without further
purification.
4. Experimental section
4.1. Materials and methods
4.4. 4-Methyl-4-(4-methyl-pent-3-enyl)-thietan-2-one (17)
Reagents were of highest purity available and were used with-
out further purification unless otherwise noted. Materials were
purchased from the following suppliers: solvents, Fisher; silica
gel 60 (0.04–0.063 mm pore size) for column chromatography,
Merck; TLC plates coated with general purpose silica containing
UV254 fluorophore, Aldrich Chemical Company; catalase from Sig-
ma–Aldrich Chemical Co.; agarose from Seakem, bromophenol
blue, sucrose, and other chemicals were purchased from Aldrich
Chemical Company. Water was distilled, deionized and glass redis-
tilled. All reactions were carried out under an atmosphere of nitro-
gen, unless otherwise noted. Dimethyl dioxirane60 (DMD) and
diazomethane (CAUTION: explosion hazard)61 were freshly pre-
pared. The plasmid PGL2BASIC was prepared using standard
methods.62
To a stirred solution of 16 (3 g, 14.8 mmol) in dry, distilled THF
(30 mL) under nitrogen, dicyclohexyl carbodiimide (3.7 g,
17.8 mmol) and a catalytic amount of 4-dimethylaminopyridine
(181 mg, 1.5 mmol) were added. The reaction was stirred for
48 h and the solvent evaporated under reduced pressure. The
resulting white, oily suspension was mixed with acetone and the
dicyclohexylurea precipitate (white solid) removed by filtration.
The filtrate was evaporated under reduced pressure to give a pale
yellow oil. Flash column chromatography on silica gel eluted with
20:1 pentane:ether gave 17 as a colorless liquid (1.63 g, 60% yield,
Rf = 0.54 in 10:1 pentane:ether): 1H NMR (500 MHz, CDCl3) d 5.13
(m, 1H), 3.68 (d, 17 Hz, 1H), 3.60 (d, 17 Hz, 1H), 2.14–1.94 (m, 4H),
1.75 (s, 3H), 1.70 (s, 3H), 1.63 (s, 3H); 13C NMR (125.75 MHz, CDCl3)
d 190.7, 132.8, 122.5, 66.1, 44.4, 43.7, 28.7, 25.6, 17.6; HRMS (EI)
calcd for C10H16OS (M+) 184.0922, found 184.0928.
4.2. 3-Benzylsulfanyl-3,7-dimethyl-oct-6-enoic acid (15)
4.5. 5-Methyl-5-(4-methyl-pent-3-enyl)-[1,2]dithiolan-3-one
(18)
Benzyl mercaptan (9 g, 8.4 mL, 71 mmol) was added to geranic
acid (14, 10 g, 10.3 mL, 59 mmol) in freshly distilled piperidine
(15 mL). This mixture was heated at reflux under nitrogen for
12 h. The resulting mixture was cooled in an ice bath followed by
addition of 10% HCl until the pH of the solution reached ꢂ2–3
(ꢂ20 mL). The resulting suspension was extracted with diethyl
ether (3 ꢃ 30 mL). The combined organic extracts were washed
with brine (2 ꢃ 20 mL), dried over anhydrous sodium sulfate, and
concentrated under reduced pressure to give a pale yellow oil.
Flash column chromatography on silica gel eluted with 8:1 hex-
ane:ethyl acetate, followed by 6:1 hexane:ethyl acetate gave 15
(13 g, 75%, Rf = 0.37, in 4:1 hexane:ethyl acetate, a streaky spot)
as a thick colorless oil: 1H NMR (250 MHz, CDCl3) d 7.36–7.23
(m, 5H), 5.08 (m, 1H), 3.77 (s, 2H), 2.68 (s, 2H), 2.20–2.05 (m,
2H), 1.75–1.71 (m, 2H), 1.69 (s, 3H), 1.64 (s, 3H), 1.48 (s, 3H); 13C
NMR (62.9 MHz, CDCl3) d 175.7, 137.4, 132.0, 129.0, 128.5, 127.0,
123.5, 47.5, 45.1, 39.8, 32.8, 25.9, 25.6, 23.0, 17.6; HRMS (ESI)
A vigorously-stirred solution of 17 (1.5 g, 8.1 mmol) in carbon
tetrachloride (25 mL) at –35 °C was saturated with a stream of
hydrogen sulfide gas for 30 min (CAUTION: hydrogen sulfide is
highly toxic and the gas stream vented from the reaction should
be scrubbed and the reaction performed in a well ventilated hood).
Freshly distilled triethylamine (1.3 mL, 9.7 mmol) was then added
dropwise. The resulting solution was maintained at ꢀ35 °C and
bubbling with hydrogen sulfide gas was continued for 8 h. The
resulting mixture was allowed to warm to room temperature and
stirred for another 5 h. Hydrogen sulfide and solvent was removed
by blowing a stream of nitrogen gas over the solution to give a pale
yellow solid. This solid was dissolved in methanol (25 mL) and an
aqueous solution of ferric chloride (0.05 M, 25 mL) was added over
30 min with vigorous stirring at room temperature (24 °C). The
resulting orange solution was stirred for an additional hour and
then extracted with diethyl ether (3 ꢃ 20 mL). The organic extract
was washed with water (2 ꢃ 15 mL) and brine (2 ꢃ 15 mL) before
drying over anhydrous sodium sulfate. Evaporation of the solvent
under reduced pressure gave a pale yellow oil. Flash column chro-
matography on silica gel eluted with 20:1 hexane:ethyl acetate
afforded 18 as a pale yellow oil (1.63 g, 60% yield, Rf = 0.67 in 9:1
hexane:ethyl acetate): 1H NMR (500 MHz, CDCl3) d 5.10 (m, 1H),
2.83 (d, 16 Hz, 1H), 2.75 (d, 16 Hz, 1H), 2.18–2.11 (m, 2H), 1.95–
1.89 (m, 1H), 1.83–1.75 (m, 1H), 1.70 (s, 3H), 1.63 (s, 3H), 1.59 (s,
3H); 13C NMR (125.75 MHz, CDCl3) d 206.3, 133.1, 122.6, 56.9,
56.3, 39.4, 25.6, 24.2, 24.1, 17.6 d ppm; HRMS (EI) C10H16OS2
(M+) calcd for 216.0643, found 216.0647.
C
17H24O2SNa+ calcd for 315.1389, found 315.1379.
4.3. 3-Mercapto-3,7-dimethyl-oct-6-enoic acid (16)
A solution of 15 (5 g, 17.1 mmol) in distilled THF (5 mL) was
added to a dry flask equipped with a stir bar, calcium chloride dry-
ing tube, dry ice condenser, and an ammonia inlet. The flask was
cooled in a ꢀ78 °C dry ice-acetone bath. Liquid ammonia (15 mL)
was condensed into the flask and finely divided sodium metal
(1 g, 43 mmol) was added. A deep blue color developed and the
mixture was stirred at ꢀ78 °C for 1 h, followed by addition of solid
ammonium chloride (5 g). The ammonia and THF was removed by
blowing a stream of nitrogen gently on the solution. To the result-
ing white residue 10% HCl was added until the pH of the solution
reached ꢂ3 (10–15 mL). The mixture was then extracted with
diethyl ether (3 ꢃ 20 mL), followed by washing the pooled ether
extracts with brine (2 ꢃ 15 mL). The ether extract was further
dried over anhydrous sodium sulfate and then concentrated under
reduced pressure to afford 16 as a colorless oil (3.3 g, 95%, Rf = 0.34,
in 3:1 hexane:ethyl acetate, a streaky spot faintly visible under UV
and strongly visible using iodine stain): 1H NMR (250 MHz, CDCl3)
4.6. 5-Methyl-5-(4-methyl-pent-3-enyl)-1-oxo-[1,2]dithiolan-3-
one (19)
To a vigorously-stirred solution of dithiolanone 18 (276 mg) in
HPLC grade acetone (3 mL) freshly prepared dimethyldioxirane
(5 mL of an approximately 0.09 M solution in acetone) was added
dropwise. The disappearance of the starting material was closely
monitored by TLC, which revealed the appearance of two products.

240
K. Keerthi et al. / Bioorg. Med. Chem. 21 (2013) 235–241
When the starting material was consumed, the mixture was evap-
orated under reduced pressure to give a pale yellow oil. Flash col-
umn chromatography on silica gel eluted with 12:1 hexane:ethyl
acetate afforded 19 as a 1:1.7 ratio of diastereomers. The isomers
were separated by column chromatography on silica gel to give
the minor isomer (100 mg, 30%, Rf = 0.35 in 4:1 hexane:ethyl ace-
tate) and the major isomer (150 mg, 50%, Rf = 0.43 in 4:1 hex-
ane:ethyl acetate), both as colorless oils. The minor isomer of 19:
1H NMR (250 MHz, CDCl3) d 5.04 (m, 1H), 3.38 (d, 1H), 2.96 (d,
1H), 2.17–2.08 (m, 2H), 1.77–1.74 (m, 2H), 1.71 (s, 3H), 1.42 (s,
6H); 13C NMR (62.9 MHz, CDCl3) d 200.8, 133.7, 121.9, 68.4, 48.2,
34.7, 25.5, 23.1, 18.8, 17.7; the major isomer of 19: 1H NMR
(250 MHz, CDCl3) d 5.13 (m, 1H), 3.40 (d, 1H), 2.87 (d, 1H), 2.23–
2.10 (m, 2H), 1.99–1.95 (m, 2H), 1.70 (s, 3H), 1.61 (s, 3H), 1.42 (s,
3H); 13C NMR (62.9 MHz, CDCl3) d 201.0, 133.7, 122.1, 67.6, 49.3,
34.4, 25.6, 22.8, 18.9, 17.7; HRMS (EI) calcd for HRMS (EI)
33.9, 25.6, 25.0, 23.1, 17.6, 14.3; HRMS (EI) C13H24O2S2 (M+) calcd
for 276.1218, found 276.1217.
4.8. DNA-cleavage by the major and minor isomers of 19
In a typical assay, the major or minor isomer of 19 (2
2.5 mM stock solution in acetonitrile) was added to a solution con-
taining 2-mercaptoethanol (2 L of a 3.75 mM freshly prepared
stock solution in water) and supercoiled plasmid DNA (2 L of a
0.25 g/
L solution in 1ꢃ TE buffer) and incubated for 3–15 h at
37 °C (final concentrations: 19, 250 M; 2-mercaptoethanol,
375 M; sodium phosphate, 50 mM pH 7.0; acetonitrile, 10% v/v).
In assays containing catalase, the enzyme was added (2 L of
1 mg/mL freshly prepared stock in water) prior to 19. Following
incubation, glycerol loading buffer (5 L) containing 0.25% bromo-
lL of
l
l
l
l
l
l
l
l
phenol blue and 40% sucrose was added. The samples were then
agitated on a vortex mixer for 1 s, spun in a bench-top centrifuge
for 5 s, and loaded onto a 0.9% agarose gel. The gel was electropho-
resed at 80 V for 3 h in 1ꢃ TAE buffer (40 mM Tris base, 20 mM
acetate, 1 mM EDTA, pH 8.0) and then stained in an aqueous ethi-
C10H26O2S2 calcd for 232.0630, found 232.0589.
4.7. Reaction of 19 with benzyl mercaptan in aqueous buffered
solution
dium bromide solution (0.2 lg/mL) for 6–8 h. The DNA in the gel
In a typical reaction, compound 19 (1.27 mL of a 500 mM stock
solution in acetonitrile) was added to a solution of sodium phos-
phate buffer (0.5 mL of 500 mM, pH 7 solution), water (2 mL),
and benzyl mercaptan (1.23 mL of 620 mM stock solution in aceto-
nitrile; final concentrations: 19, 127 mM; sodium phosphate,
50 mM, pH 7; thiol, 152.5 mM; acetonitrile 50% v/v). The mixture
was stirred for 3 h at room temperature, acidified with 5% HCl to
a final pH of 2–3, and extracted with ethyl acetate (3 ꢃ 2 mL).
The combined ethyl acetate extracts were dried over anhydrous so-
dium sulfate and concentrated under reduced pressure to yield a
colorless oil. The product was treated with diazomethane
(CAUTION: explosion hazard)61 and the solvent removed by rotary
evaporation to yield a pale yellow oil. Flash column chromatogra-
phy on silica gel eluted with 4:1 hexane–ethyl acetate was used to
isolate the major products of the reaction. The S-deoxy analogue 18
was obtained in 15%, yield (20 mg). Spectral data for this com-
pound matched that for the material described above in the syn-
thesis of 19. In addition, several other products were obtained:
2-(5-(2-hydroxypropan-2-yl)-2-methyltetrahydrothiophen-2-yl)
acetic acid methyl ester (26) as an inseparable (ꢂ1:1) mixture of
diastereomers in 35% yield (22 mg): 1H NMR (500 MHz, CDCl3) d
3.698 (s, 3H), 3.693 (s, 3H), 2.73–2.45 (m, 6H), 2.19–1.83 (m, 8H),
1.58 (s, 3H), 1.50 (s, 3H), 1.22 (6H), 1.19 (6H); 13C NMR
(125.75 MHz, CDCl3) d 171.42, 171.38, 70.2, 69.9, 63.01, 62.98,
54.5, 54.0, 51.49, 51.45, 48.2, 47.2, 44.59, 44.53, 31.35, 31.32,
29.7, 29.4, 28.7, 26.0, 25.9; HRMS (EI) C11H20O3S (M+) calcd for
239.1133, found 239.1138. Using GC/MS equipped with an electron
impact detector, a mixture of polysulfides RSSxR (m/z 218, x = 4; m/
z 186, x = 3; m/z 154, x = 2) was detected in the reaction of 2-
mercaptoethanol with 19. The properties of these products
matched those characterized previously from the reaction of thiols
with 1, 10, and 11.20 The compound 3-benzyldisulfanyl-3,7-
dimethyl-oct-6-enoic acid methyl ester (methyl ester of 24,
R = CH2Ph) was obtained in 10% yield (31 mg): 1H NMR
(500 MHz, CDCl3) d 7.28 (m, 5H), 5.04 (m, 1H), 4.12 (m, 2), 3.78
(s, 3H), 2.89 (m, 2H), 2.06 (m, 2H), 1.69 (m, 2H), 1.67 (s, 3H),
1.54 (s, 3H), 1.30 (s, 3H); 13C NMR (125.75 MHz, CDCl3) d 195.6,
137.3, 132.5, 129.0, 128.8, 127.3, 123.2, 63.3, 56.4, 56.3, 44.9,
33.6, 32.8, 25.7, 22.3, 17.6. An analogous reaction of 19 with etha-
nethiol gave the corresponding disulfide, 3-ethyldisulfanyl-3,7-di-
methyl-oct-6-enoic acid methyl ester (methyl ester of 24,
R = CH2CH3) in 30% yield: 1H NMR (500 MHz, CDCl3) d 5.08 (m,
1H), 3.67 s, 3H), 2.74–2.65 (m, 4H), 2.12 (m, 1H), 1.73 (m, 2H),
1.67 (s, 3H), 1.62 (s, 3H), 1.43 (s, 3H), 1.29 (t, 7 Hz, 3H); 13C NMR
(500 MHz, CDCl3) d 171.0, 132.0, 123.5, 52.0, 51.4, 44.1, 38.6,
was then visualized by UV-transillumination and the amount of
DNA in each band was quantitatively measured by digital image
analysis.
Acknowledgement
We thank the National Institutes of Health for support of this
work (CA83925 and 119131).
References and notes
1. Cragg, G. M.; Grothaus, P. G.; Newman, D. J. Chem. Rev. 2009, 109, 3012.
2. Newman, D. J. J. Med. Chem. 2008, 51, 2589.
3. Newman, D. J.; Cragg, G. M. J. Nat. Prod. 2012, 75, 311.
4. Galm, U.; Shen, B. Chem. Biol. 2007, 14, 1098.
5. Morin, R. B.; Jackson, B. G.; Mueller, R. A.; Lavagnino, E. R.; Scanlon, W. B.;
Andrews, S. L. J. Am. Chem. Soc. 1963, 85, 1896.
6. Christensen, B. G.; Ratcliffe, R. W. Annu. Rep. Med. Chem. 1976, 11, 271.
7. Nicolaou, K. C.; Dai, W.-M. Angew. Chem., Int. Ed. Engl. 1991, 30, 1387.
8. Chatterji, T.; Gates, K. S. Bioorg. Med. Chem. Lett. 1998, 8, 535.
9. Stanley, T. H. J. Pain Sympt. Manag. 1992, 7, S3.
10. Martinez, E. J.; Owa, T.; Schreiber, S. L.; Corey, E. J. Proc. Nat. Acad. Sci. U.S.A.
1999, 96, 3496.
11. Gates, K. S. Chem. Res. Toxicol. 2000, 13, 953.
12. Hara, M.; Asano, K.; Kawamoto, I.; Takiguchi, T.; Katsumata, S.; Takahashi, K.;
Nakano, H. J. Antibiotics 1989, 42, 1768.
13. Hara, M.; Saitoh, Y.; Nakano, H. Biochemistry 1990, 29, 5676.
14. Hara, M.; Takahashi, I.; Yoshida, M.; Kawamoto, I.; Morimoto, M.; Nakano, H. J.
Antibiotics 1989, 42, 333.
15. Bassett, S.; Urrabaz, R.; Sun, D. Anticancer Drugs 2004, 15, 689.
16. Tang, G.-L.; Cheng, Y.-Q.; Shen, B. Chem. Biol. 2004, 11, 33.
17. Keerthi, K.; Gates, K. S. Org. Biomol. Chem. 2007, 5, 1595.
18. Behroozi, S. B.; Kim, W.; Gates, K. S. J. Org. Chem. 1995, 60, 3964.
19. Behroozi, S. J.; Kim, W.; Dannaldson, J.; Gates, K. S. Biochemistry 1996, 35, 1768.
20. Mitra, K.; Kim, W.; Daniels, J. S.; Gates, K. S. J. Am. Chem. Soc. 1997, 119, 11691.
21. Chatterji, T.; Keerthi, K.; Gates, K. S. Bioorg. Med. Chem. Lett. 2005, 15, 3921.
22. Breydo, L.; Gates, K. S. J. Org. Chem. 2002, 67, 9054.
23. Asai, A.; Hara, M.; Kakita, S.; Kanda, Y.; Yoshida, M.; Saito, H.; Saitoh, Y. J. Am.
Chem. Soc. 1996, 118, 6802.
24. Fekry, M.; Szekely, J.; Dutta, S.; Breydo, L.; Zang, H.; Gates, K. S. J. Am. Chem. Soc.
2011, 132, 17641.
25. Zang, H.; Gates, K. S. Chem. Res. Toxicol. 2003, 16, 1539.
26. Nooner, T.; Dutta, S.; Gates, K. S. Chem. Res. Toxicol. 2004, 17, 942.
27. Shipova, K.; Gates, K. S. Bioorg. Med. Chem. Lett. 2005, 15, 2111.
28. Dutta, S.; Chowdhury, G.; Gates, K. S. J. Am. Chem. Soc. 2007, 129, 1852.
29. Viswesh, V.; Gates, K. S.; Sun, D. Chem. Res. Toxicol. 2010, 23, 99.
30. Gates, K. S.; Nooner, T.; Dutta, S. Chem. Res. Toxicol. 2004, 17, 839.
31. Pattenden, G.; Shuker, A. J. J. Chem. Soc., Perkin 1 1992, 1215.
32. Lee, A. H. F.; Chan, A. S. C.; Li, T. Tetrahedron 2003, 59, 833.
33. Epstein, W. W.; Sewwt, F. W. Chem. Rev. 1967, 67, 247.
34. Wallace, T. J.; Mahon, J. J. J. Org. Chem. 1966, 30, 1502.
35. Chatterji, T.; Kizil, M.; Keerthi, K.; Chowdhury, G.; Posposil, T.; Gates, K. S. J. Am.
Chem. Soc. 2003, 125, 4996.
36. Hintermann, G.; Fischer, H. M.; Crameri, R.; Hutter, R. Plasmid 1981, 5, 371.
37. Jonson, P. H.; Grossman, L. I. Biochemistry 1977, 16, 4217.

K. Keerthi et al. / Bioorg. Med. Chem. 21 (2013) 235–241
241
38. Mirabelli, C. K.; Huang, C. H.; Fenwick, R. G.; Crooke, S. T. Antimicrob. Agents
Chemother. 1985, 27, 460.
39. Greenberg, M. M. Org. Biomol. Chem. 2007, 5, 18.
52. Havlik, A. J.; Kharasch, N. J. Am. Chem. Soc. 1956, 78, 1207.
53. Putnam, R. E.; Sharkey, W. H. J. Am. Chem. Soc. 1957, 79, 6526.
54. Chatterji, T.; Gates, K. S. Bioorg. Med. Chem. Lett. 2003, 13, 1349.
40. Greenberg, M. M. Chem. Res. Toxicol. 1998, 11, 1235.
55. Ogston, A. G.; Holiday, E. R.; Philpot, J. S. L.; Stocken, L. A. Trans. Faraday Soc.
1948, 44, 45.
56. Yang, Y.-C.; Szafraniec, L. L.; Beaudry, W. T.; Ward, J. R. J. Org. Chem. 1988, 53,
3293.
57. Breydo, L.; Barnes, C. L.; Gates, K. S. Acta Cryst. C 2002, C58, 0447.
58. Wu, S.; Greer, A. J. Org. Chem. 2000, 65, 4883.
59. Sivaramakrishnan, S.; Bredyo, L.; Sun, D.; Gates, K. S. Bioorg. Med. Chem. Lett.
2012, 22, 3791–3794.
60. Adam, W.; Bialas, J.; Hadjiarapoglou, L. Chem. Ber. 1991, 124, 2377.
61. Aldrich, S. http://www.sigmaaldrich.com/aldrich/bulletin/AL-180.pdf 2003.
62. Sambrook, J.; Fritsch, E. F.; Maniatis, T. Molecular Cloning: A Lab Manual; Cold
Spring Harbor Press: Cold Spring Harbor, NY, 1989.
63. Kwart, H.; Evans, R. E. J. Org. Chem. 1966, 31, 413.
64. Hertzberg, R. P.; Dervan, P. B. Biochemistry 1984, 23, 3934.
41. Pogozelski, W. K.; Tullius, T. D. Chem. Rev. 1998, 98, 1089.
42. Breen, A. P.; Murphy, J. A. Free Rad Biol. Med. 1995, 18, 1033.
43. Pratviel, G.; Bernadou, J.; Meunier, B. Angew. Chem., Int. Ed. Engl. 1995, 34, 746.
44. Gates, K. S. In Reviews of Reactive Intermediates; Platz, M. S., Moss, R. A., Jones,
M. J., Eds.; John Wiley and Sons, Inc.: Hoboken, 2007; p 333.
45. Gates, K. S. Chem. Res. Toxicol. 2009, 22, 1747.
46. McHugh, P. J.; Knowland, J. Nucleic Acids Res. 1995, 23, 1664.
47. Zang, H.; Breydo, L.; Mitra, K.; Dannaldson, J.; Gates, K. S. Bioorg. Med. Chem.
Lett. 2001, 11, 1511.
48. Halliwell, B.; Gutteridge, J. M. C. Methods Enzymol. 1990, 186, 1.
49. Breydo, L.; Zang, H.; Gates, K. S. Tetrahedron Lett. 2004, 45, 5711.
50. Bryant, H. J.; Dardonville, C. Y.; Hodgkinson, T. J.; Hursthouse, M. B.; Malik, K.
M. A.; Shipman, M. J. Chem. Soc., Perkin Trans. 1 1998, 1249.
51. Morishita, T.; Furukawa, N.; Oae, S. Tetrahedron 1981, 37, 2539.