Synthesis, DNA cleavage, and cytotoxicity of a series of bis(propargylic) sulfone crown ethers

Article abstract of DOI:10.1016/S0968-0896(01)00150-X

Compound that couple molecular recognition of specific alkali metal ions with DNA damage may display selective cleavage of DNA under condition of elevated alkali metal ion level reported to exist in certain cancer cell . We have prepared a homologous serie of compound in which a DNA reactive moiety, a bis (propargylic) sulfone, is incorporated into an alkali metal ion binding crown ether ring. Using the alkali metal ion pricrate extraction assay, the ability of these crown ether to bind Li⁺, Na⁺, and K⁺, ions was determined. For the series of crown ethers , the association constant for Li⁺ ions are generally low (< 2 x 10⁴M^-1). Only two of the bis(propargylic) sulfone crown ethers associate with Na⁺ or K⁺ ions (K_a 4 8 x 10⁴M^-1), with little discrimination between Na⁺ or K⁺ ions. The ability of these compounds to cleave supercoiled DNA at pH 7.4 in the presence of Li⁺, Na⁺ , and K⁺ ions was determined. The two crown ethers that bind Na⁺ and K⁺ display a modest increase in DNA cleavage efficiency in the presence of Na⁺ or K⁺ ions as compared to Li⁺ ions. These two bis (propargylic) sulfone crown ethers are also more cytotoxic against a panel of human cancer cell line when compared to a non-crown ether macrocyclic bis(propargylic) sulfone. Copyright

Full text of DOI:10.1016/S0968-0896(01)00150-X

Bioorganic & Medicinal Chemistry 9 (2001) 2809–2818
Synthesis, DNA Cleavage, and Cytotoxicity of a Series of
Bis(propargylic) Sulfone Crown Ethers
Mark M. McPhee and Sean M. Kerwin*
Division of Medicinal Chemistry, College of Pharmacy, The University of Texas at Austin, Austin, TX 78712, USA
Received 14 March 2001; accepted 24 April 2001
Abstract—Compounds that couple molecular recognition of specific alkali metal ions with DNA damage may display selective
cleavage of DNA under conditions of elevated alkali metal ion levels reported to exist in certain cancer cells. We have prepared a
homologous series of compounds in which a DNA reactive moiety, a bis(propargylic) sulfone, is incorporated into an alkali metal
ion binding crown ether ring. Using the alkali metal ion pricrate extraction assay, the ability of these crown ethers to bind Li+,
Na+, and K+ ions was determined. For the series of crown ethers, the association constants for Li+ ionsare generally low
(<2ꢀ104 Mꢁ1). Only two of the bis(propargylic) sulfone crown ethers associate with Na+ or K+ ions( Ka 4–8ꢀ104 Mꢁ1), with little
discrimination between Na+ or K+ ions. The ability of these compounds to cleave supercoiled DNA at pH 7.4 in the presence of
Li+, Na+, and K+ ionswasdetermined. The two crown ethersthat bind Na + and K+ display a modest increase in DNA cleavage
+
efficiency in the presence of Na+ or K+ ionsascompared to Li
more cytotoxic against a panel of human cancer cell lines when compared to a non-crown ether macrocyclic bis(propargylic) sul-
ions. These two bis(propargylic) sulfone crown ethers are also
fone. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
the cyclization of bis-g,g-dimethylallenyl sulfones to the
corresponding diradicals,2 a more recent report by
Braverman and coworkersdemontsratesthat certain
bis(propargylic) sulfones (e.g., 1, R¼Ph) do undergo a
facile reaction involving the diradical intermediate 3.7
Nicolaou and coworkers have described bis(pro-
pargylic) sulfone-containing molecules (e.g., 1, Scheme 1)
1
asnovel DNA-cleaving agent.s
These agents were
designed with the capacity to enter two manifolds in
which two putative DNA-cleaving intermediatescould
be generated (Scheme 1). In both paths, isomerization
to a bis(allenic) species, 2, isrequired. Thisreactive
intermediate waspostulated to act either asan electro-
phile to alkylate DNA (path a) or asa precuros r to a
diradical intermediate via a Braverman cyclization
reaction2 (path b). Electrophilic alkylation of DNA can
result in DNA strand cleavage through a variety of
routes.3 Diradical intermediates(path b) may be cap-
able of abstracting hydrogen atoms from the DNA
backbone, resulting in strand scission.4 We,5 along with
Nicolaou and coworkers,6 have shown data that sug-
gests that the alkylation mechanism (path a) rather than
the diradical mechanism (path b) is the predominant or
exclusive mode of DNA cleavage for these compounds.
While thisfinding isin accord with Braverman’searlier
reportsof the relatively high temperature required for
Incorporation of the bis(propargylic) sulfone moiety
into a macrocyclic ring might affect the efficiency of
DNA cleavage through ring strain effects on the iso-
merization to the bis(allenylic) sulfone or the partition-
ing between electrophilic and free radical pathways
*Corresponding author. Tel.: +1-512-471-5074; fax: +1-512-232-
02696; e-mail: skerwin@mail.utexas.edu
Scheme 1. DNA cleavage by bis(propargylic) sulfones.
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00150-X

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M. M. McPhee, S. M. Kerwin / Bioorg. Med. Chem. 9 (2001) 2809–2818
(Scheme 1). The original DNA cleavage studies of
Nicoloau revealed that the cyclic bis(propargylic) sul-
fones display an interesting relationship between ring
size and DNA cleavage potency. The 11-membered ring
compound 6 (Scheme 2) wasfound to be slightly more
potent than either the 10-membered ring compound 5 or
the 13-membered ring compound 7.1 Later, Basak and
Khamrai reported the ‘locking’ effect of the b-lactam
facility of DNA cleavage by virtue of the conforma-
tional changesbrought about by such complexation or
by increased affinity of the cationic metal ion complexes
with the polyanionic backbone of DNA.9
Dai and coworkers have reported the synthesis, DNA
cleavage, and cytotoxicity of a number of propargylic
sulfone-armed
lariat
benzo-15-crown-5
crown
moiety in macrocyclic bis(propargylic)sulfone
8
ethers.10,11 In this series of compounds, there was no
evidence of metal-ion enhanced DNA cleavage or
increased cancer cell cytotoxicity relative to non-crown-
containing analogues; however, the metal ion binding
ability of these lariat crown ethers was not reported. We
have recently reported the synthesis and alkali metal
ion-regulated DNA cleavage by a distinct series of pro-
pargylic sulfone-armed lariat crown ethers.12 These
compoundscleave DNA more readily in the presence of
K+ than Li+ ions, with selectivities ranging from 2- to
10-fold. While these results are encouraging, the need
for higher degreesof eslectivity and Na +/K+ ion
decsrimination have led usto examine compoundsin
which the bis(proparglyic) sulfone DNA cleavage moi-
ety incorporated within a metal ion binding crown ether
ring. In this paper, we report the synthesis, alkali metal
ion binding, DNA cleavage studies, and cancer cell
cytotoxicity of a series of macrocyclic bis(propargylic)
sulfone crown ethers 10a–d (Scheme 3). For those com-
pounds in this series that bind sodium and potassium
ions, there is a modest enhancement of the DNA clea-
vage ability in the presence of these alkali metal ions.
However, even in the presence of the cognate alkali
metal ions, the DNA cleavage ability of these com-
pounds is in general worse than a simple macrocyclic
bis(propopargylic) sulfone that is unable to bind metal
ions. The cytotoxicity of the sodium and potassium
binding bis(propargylic) sulfone crown ethers is greater
than a macrocyclic bis(propargylic) sulfone that does
not incorporate a crown ether ring.
(Scheme 2), that, unlike the catecol-derived compound
9, does not undergo isomerization to the bis(allenylic)
sulfone.8
We have attempted to modulate the DNA cleavage of
bis(propargylic) sulfones by incorporating this moiety
into crown ether rings. In addition to inherent differ-
encesin DNA cleavage efficiency dictated by the crown
ether ring size, we hypothesized that complexation of
the crown by an appropriate metal ion might affect the
Scheme 2. Structures of cyclic bis(proparglic) sulfones.
Scheme 3. Synthesis of crown ethers.

M. M. McPhee, S. M. Kerwin / Bioorg. Med. Chem. 9 (2001) 2809–2818
2811
Results
Synthesis of crown ethers
the small Li+ cation best within the ion-binding cavity.
Affinity for Na+ and K+ ionsfollow similar trendsin
the bis(propargylic) sulfone crown ether series; as the
crown ether ring size increases, so too does the affinity
for these metal ions. Only the two largest crown ethers,
10c and 10d, bind Na+ and K+ ionswell. In general,
these crowns do not distinguish well between Na+ and
K+ ions. As expected, the model bis(propargylic) sul-
fone 9, which lacksa crown ether metal ion recognition
moiety, doesnot bind alkali metal ionsto any
appreciable extent.
Our previously published route5 to the bis(propargylic)
sulfone crown ether 10a involved addition of 4-bromo-
but-2-yn-4-ol to triethylene glycol. Effortsto produce
the homologousesriesof crown ethers
10b–d by this
route were hampered by difficultiesencountered in the
alkylation of higher ethylene glycol homologues. Based
upon our successful route to the thiocrown ether 14c, an
intermediate in the syntheses of the corresponding ene-
diyne crown ether,13 we adopted an alternative route to
the bis(propargylic) sulfone crown ethers 10a–d, which
isshown in Scheme 3. The appropriate ethylene glycols
were first alkylated with propargyl bromide. The resul-
tant bis(propargylic) ethers 11a–d were subjected to
deprotonation with nBuLi in the presence of TMEDA,
followed by quenching with paraformaldehyde to afford
the diols 12a–d in modest yield. Treatment of diols 12a–
d with in situ generated PPh3Br2 afforded the desired
dibromides 13a–d. Dibromides 13a–c underwent mac-
rocyclization in the presence of alumina-supported
Na2S to afford the thiocrown ethers 14a–d. Oxidation
with Oxone114 afforded excellent yieldsof the deisred
bis(propargylic) sulfone crown ethers, which, in most
cases, were analytically pure after workup. We also
prepared the previously reported8 compound 9 (Scheme
2) as a control bis(propargylic) sulfone lacking a crown
ether ring. Compound 9 (Scheme 2) had been prepared
as a mixture along with the corresponding bis(allenylic)
sulfone by mCPBA oxidation of the corresponding sul-
fide. We found that Oxone1 oxidation of the sulfide
affords the catecol-derived bis(propargylic) sulfone 9
free from any contaminating bis(allenylic) material.
DNA cleavage studies
Initial DNA cleavage studies utilizing supercoiled,
Form I ꢀX174 phage DNA at pH 8.5 demonstrated the
ability of bis(propargylic) sulfone crown ether 10a to
produce frank strand breakage.5 However, we were
most interested in the ability of alkali metal ions to
influence the degree of DNA cleavage exhibited by these
compounds at physiological pH. Stock solutions of
supercoiled pGAD424 plasmid, isolated using a QIA-
prep Miniprep kit (QIAGEN Inc.), were diluted with
sterile, pH 7.4 lithium, sodium, or potassium phosphate
buffers (20 mM alkali metal ions) to afford DNA solu-
tionsthat contained primarily a isngle alkali metal
cation species. These alkali metal ion-enriched DNA
preparationsare referred to asM
+DNAs(e.g.,
Li+DNA, Na+DNA, K+DNA). DNA cleavage
experimentswere performed by adding freshly prepared
solutions of the compounds in DMSO to solutions of
M+DNA followed by incubation at 25 ꢂC for 18 h. Few
frank DNA strand breaks were evident under these
DNA cleavage reaction conditions; however, when
DNA cleavage incubationswere followed by brief (90 s)
heating at 70 ꢂC, much more DNA cleavage wasevi-
dent. The DNA cleavage productswere quantified by
agarose gel electrophoresis followed by ethidium bro-
mide staining and quantification by a fluorimager. An
image of the separated, stained DNA fragments from a
typical DNA cleavage reaction involving compound 10b
is shown in Figure 1. DNA cleavage results are pre-
sented as EC25 values, the concentration of each com-
pound required to produce 25% normalized DNA
cleavage, which were determined by interpolation of
concentration versus normalized DNA cleavage curves.
Metal ion binding
The complex association constants, Ka, for the alkali
metal ion–host complexes were determined by picrate
extraction techniques as described previously,15 and the
average values of these determinations are presented in
Table 1. For hosts that did not display measurable affi-
nity for a particular metal ion, the lower limit of the Ka
value that could be determined ispresented.
Inspection of Table 1 reveals that bis(propargylic) sul-
fone crown ether 10a displays a weak affinity for Li+
ions. This is not surprising considering that this agent
has the smallest ring size and thus might accommodate
The results of the DNA cleavage assays for the bis(pro-
pargylic)sulfone crown ethers 10a–d and model bis(pro-
pargylic)sulfone 9 are presented in Table 2. All of the
compoundsexamined here are modets DNA cleavage
Table 1. Complex association constants, Ka, for Li+, Na+, and K+
ions with bis(propargylic) sulfone hosts
agentsat pH 7.4, with EC
valuesin the millimolar
25
range. Nicolaou and coworkershave demonstrated that
propargylic sulfones cleave DNA optimally at a pH of
8.5. Under optimal pH conditions(pH 8.5) bi(spro-
parglyic) sulfone crown ether 10a cleavesDNA more
efficiently, with an EC25 of 200 mM,5 which iscompar-
able to the cleavage efficiency reported for other mac-
rocyclic bis(propargylic) sulfones.1 While most of the
bis(propargylic) sulfone crown ethers did not show a
strong dependence of DNA cleavage ability on alkali
metal ion identity, compounds 10c and 10d both show a
modest enhancement of cleavage of Na+DNA and
Host
Initial concn (mM)a
Ka (ꢀ10ꢁ3 Mꢁ1
)
Li+
Na+
K+
<3
10a
10b
10c
10d
9
15
30
13
10
30
15ꢃ4
<3
<2
6ꢃ2
38ꢃ4
40ꢃ10
<2
14ꢃ1
44ꢃ1
80ꢃ10
4ꢃ4
<5
<2
<2
aInitial concentration of the host in the organic phase. The initial
concentration of alkali metal ion picrate in the aqueousphaes was
3 mM.

2812
M. M. McPhee, S. M. Kerwin / Bioorg. Med. Chem. 9 (2001) 2809–2818
Figure 1. Cleavage of supercoiled (Form I) Li+-, Na+-, and K+DNA by bis(propargylic) sulfone crown ether 10b. Solutionsof pH 7.4 M +DNA
(23 mM base pairs) containing 12.5% v/v DMSO were incubated for 18 h at 25 ꢂC with compound 10b. Sampleswere heated at 70 ꢂC for 90 sthen
mixed with loading buffer, and the nicked DNA products (form II) separated from supercoiled (form I) DNA by electrophoresis (0.7% agarose,
TBE buffer, 45 V, 3 h) followed by ethidium bromide fluorescence imaging. Lane 1, Li+DNA control; lane 2, Na+DNA control; lane 3, K+DNA
control; lane 4, 10b (7.5 mM)+ Li+DNA; lane 5, 10b (10 mM)+Li+DNA; lane 6, 10b (15 mM)+Li+DNA; lane 7, 10b (7.5 mM)+Na+DNA; lane
8, 10b (10 mM)+Na+DNA; lane 9, 10b (15 mM)+Na+DNA; lane 10, 10b (7.5 mM)+K+DNA; lane 11, 10b (10 mM)+K+DNA; lane 12, 10b
(15 mM)+K+DNA.
K+DNA ascompared to Li +DNA. Asexpected, the
model sulfone 9 hasno alkali metal ion preference in its
DNA cleavage reactions.
affinity for Na+ or K+ ionsasdetermined by the
picrate extraction method. Using a DNA cleavage assay
at physiologically relevant pH conditions, all of the
bis(propargylic) sulfones examined cleave double-stran-
ded DNA to afford products of single-strand DNA
scission, with significantly more cleavage observed after
brief heat treatment of the DNA cleavage reactions.
These results are commensurate with an electrophilic
DNA cleavage mechanism (Scheme 1, path a). We
noted an effect of the crown ether ring size on the DNA
cleavage ability in this series of bis(propargylic) sulfone
crown ethers. Figure 2 demonstrates the relationships
between DNA cleavage efficiency, alkali metal ions, and
ring size for bis(propargylic) sulfone crown ethers 10a–d
and the model bis(propargylic) sulfone compound 9.
Examination of Figure 2 demonstrates that the DNA
cleavage ability of model compound 9 (ring size 13) and
the crown ethers 10a (ring size 19) and 10b (ring size 22)
isinesnsitive to the nature of the alkali metal ion pre-
sent, and that these three compounds cleave DNA with
approximately the same efficiency. In contrast, the
higher crown ether homologues 10c (ring size 25) and
10d (ring size 28) are much less efficient DNA cleavage
Cytotoxicity
Two of the bis(propargylic) sulfone crown ethers, com-
pounds 10c and 10d, displayed the highest sodium and
potassium metal ion binding in the series, and showed a
modest enhancement in DNA cleavage in the presence
of sodium and potassium. These two bis(propargylic)
sulfone crown ethers were assayed against a range of
cancer cell linesfrom nine different panelsby the NCI.
The results of these assays, summarized in Table 3,
demonstrate that both of these crown ethers are more
growth inhibitory than the non-crown ether bis(pro-
pargylic) sulfone 6. In particular, compound 10c isa
moderately strong inhibitor (GI50<10 mM) against two
leukemia cell lines, one non-small cell lung cancer cell
line, two colon cell lines, and two melanoma cell lines.
Discussion
We have prepared a homologous series of bis(pro-
pargylic) sulfone crown ethers in order to explore the
role of alkali metal ion binding on the DNA cleavage
efficiency and cancer cell cytotoxicity in these com-
pounds. Only two of the compounds in this series,
crown ethers 10c and 10d, showed any significant
Table 2. Cleavage of Li+-, Na+-, and K+-DNA by bis(propargylic)
sulfones
Compound
EC25 (mM)a
Na+DNA
Li+DNA
K+DNA
10a
10b
10c
10d
9
4.6ꢃ0.5
3.2ꢃ0.9
8.7ꢃ0.9
11.8ꢃ0.8
1.9ꢃ0.5
3.7ꢃ0.9
3.0ꢃ0.4
4.1ꢃ0.5
6.6ꢃ0.9
1.7ꢃ0.4
3.1ꢃ0.4
1.9ꢃ0.2
4.2ꢃ0.5
8.3ꢃ0.6
2.4ꢃ0.7
Figure 2. DNA cleavage efficiency, expressed as EC25, the concentra-
tion of compound require to effect 25% normalized cleavage of
supercoiled DNA, as a function of ring size and alkali metal ion for
bis(propargylic) sulfones 9 and 10a–d.
aConcentration required to effect 25% normalized cleavage of super-
coiled DNA at pH 7.4, 25 ꢂC for 18 h. All values are expressed as the
meanꢃSD for three separate determinations.

M. M. McPhee, S. M. Kerwin / Bioorg. Med. Chem. 9 (2001) 2809–2818
2813
Table 3. Cancer cell growth inhibition by bis(propargylic) sulfone crown ethers
Panel/cell line
Compound 10c
GI50 (mM)a
Compound 10d
GI50 (mM)a
Compound 6
GI50 (mM)a
Leukemia
HL-60 (TB)
K-562
MOLT-4
RPMI-8226
SR
13.5
11.2
3.7
ndb
8.2
20.9
23.9
19.9
17.4
18.9
50.1
63.1
39.8
ndb
39.8
Non-small cell lung cancer
A-549/ATCC
EKVX
HOP-62
HOP-92
NCI-H226
NCI-H322M
NCI-H460
NCI-H522
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
20.2
15.1
21.7
10.9
39.7
17.5
19.3
2.86
61.7
20.9
21.0
19.6
86.9
32.3
38.0
10.6
>100
>100
>100
31.6
ndb
>100
>100
20.0
7.2
19.1
8.1
15.5
13.8
16.2
10.4
14.7
17.7
15.3
32.7
18.7
20.7
14.3
>100
63.1
>100
>100
>100
>100
39.8
HT29
KM12
SW-620
CNS cancer
SF-268
SF-295
SF-539
SNB-19
24.2
32.0
11.1
18.2
19.9
16.9
30.5
81.2
26.2
27.9
32.6
18.3
ndb
>100
>100
ndb
79.4
>100
SNB-75
U251
Melanoma
LOX IMVI
MALME-3M
M14
11.1
2.7
19.9
14.6
16.9
17.3
17.9
18.1
21.2
17.1
63.1
20.0
39.8
>100
79.4
>100
>100
>100
ndb
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian cancer
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
Renal cancer
786-0
A498
ACHN
CAKI-1
RXF 393
SN12C
TK-10
16.1
18.9
16.8
11.6
0.28
16.3
15.8
19.8
17.1
14.2
36.0
44.6
19.7
27.3
18.0
33.3
32.2
39.8
>100
>100
>100
>100
>100
11.0
16.9
13.5
12.2
15.1
13.2
13.9
18.4
54.7
22.4
21.2
27.0
18.8
23.0
>100
>100
>100
ndb
>100
>100
>100
Prostate cancer
PC-3
DU-145
18.6
32.8
43.4
>100
ndb
ndb
Breast cancer
MCF7
MCF7/ADR-RES
MDA-MB-231/ ATCC
HS 578T
MDA-MB-435
MDA-N
BT-549
10.1
25.7
20.0
40.7
13.3
14.2
14.1
20.6
22.0
>100
23.5
ndb
ndb
ndb
ndb
ndb
ndb
ndb
ndb
52.8
20.4
18.9
20.0
T-47D
24.5
aConcentration of compound required to inhibit cell line growth by 50%, as determined by the sulforhodamine B assay.
bNot determined.

2814
M. M. McPhee, S. M. Kerwin / Bioorg. Med. Chem. 9 (2001) 2809–2818
+
agentsin the presence of Li
ions. However, these two
although the effect of the nature of the alkali metal ion
crown ethers, which are the only two in the series that
bind Na+ and K+ ionswith appreciable affinity, di-s
play slightly enhanced DNA cleavage efficiency in the
presence of Na+ and K+ ions. Because compounds
10a–d do not bind Li+ ionswith appreciable affinity,
the general trend of decreasing DNA cleavage in the
presence of Li+ ions with increasing ring size in this
series must be due to ring size effects independent of
metal ion coordination effects. It appears that metal ion
coordination with Na+ or K+ ionscan esrve to par-
tially rescue the larger crown ethers 10c and 10d from
thisdeleteriousring isze effect; however, even in the
presence of Na+ or Li+ ions, 10c and 10d do not cleave
on DNA cleavage is modest. These two crown ethers
also inhibit the growth of a series of human cancer cell
lines, and are more potent than the non-crown ether
cyclic bis(propargylic) sulfone 6. Studiesto elucidate the
molecular nature of the metallo-regulated DNA clea-
vage ability and the role of metal ion binding in the
cytotoxicity of these bis(propargylic) sulfone crown
ethersare underway, and will be reported in due
course.
Experimental19
DNA aswell asthe catechol-baesd compound
9. Dai
General procedure for bis(alkylation) using propargyl
bromide. 1,8-Bis(2-propyn-1-oxy)-3,6-dioxaoctane (11a).5
To a cooled (0 ꢂC) suspension of t-BuOK (3.77 g, 95%
w/w, 32 mmol) in 28 mL of THF under argon wasadded
a solution of triethylene glycol (2.13 g, 14.2 mmol) in
3 mL of THF. The resulting glycol dialkoxide was
allowed to warm to room temperature and wasthen
added under argon via cannula to an ice-water bath
cooled solution of propargyl bromide (6.3 mL, 80% w/
w, 56.5 mmol) in 114 mL of THF. The reaction mixture
was stirred with a mechanical stirrer for an additional
18 h asthe ice-water bath melted and the reaction was
allowed to warm to room temperature. The reaction
mixture wasdiluted with 75 mL of 3:1 brine/water and
the aqueouslayer wasextracted with EtOAc (3 ꢀ50 mL).
The combined organic layerswere wahs ed with 40 mL
of 1:1 brine/water and 65 mL brine. The residue upon
drying and concentration waspurified by flash chroma-
tography on silica gel (50% hexanes in EtOAc) to afford
bis(propargyl ether) 11a (2.37 g, 74%) asa light golden
oil: Rf 0.46 (50% hexanesin EtOAc); 1H NMR d 2.40 (t,
J=2.5 Hz, 2H), 3.63–3.72 (m, 12H), 4.18 (d, J=2.5 Hz,
4H); 13C NMR d 58.05, 68.77, 70.08, 70.26, 74.35,
79.39; IR 2879, 2117, 1099 cmꢁ1; MS 227 (MH+), 171,
127; HR-MS m/e calcd for C12H19O4: 227.1283, found
227.1288.
and coworkers have discussed the role of intercalation
in the DNA cleavage efficiency of proparglyic sul-
fones.11 It is possible that the cleavage efficiency of
compound 9 isenhanced by intercalation interactions
between the catechol ring of 9 and the DNA. The alkali
metal ion enhanced DNA cleavage due to 10c and 10d,
although modest, appears to be due to specific interac-
tionsbetween thees crown ethersand the alkali metal
ions. Weak metal ion binders like 10a–b, and 9 do not
show any difference in DNA cleavage ability in the pre-
sence of different alkali metal ions, ruling out metal ion-
induced changesin the DNA asthe origin of the effect.
The two bis(propargylic) sulfone crown ethers, 10c–d,
that demonstrated some degree of alkali metal ion-
dependent DNA cleavage were examined for their
growth inhibitory effectsagaints a variety of human
cancer cell lines. Both compounds are moderately effec-
tive at inhibiting the growth of all cell linesexamined,
with little selectivity for any particular panel of cell
lines. Crown ether 10c, the more potent of the two
compounds, is also the more efficient DNA cleavage
agent; however, the concentrationsrequired for growth
inhibition are orders of magnitude less than those
required in the DNA cleavage assays. A similar dis-
crepancy hasbeen noted in the DNA cleavage veruss
cytotoxicity of enediyne antitumor compounds.16 In
comparison, the previously reported bis(propargylic)
sulfone 6 ismuch lessgrowth inhibitory than crown
ethers 10c–d, displaying weak inhibitory activity mainly
against the leukemia cell lines. Alkali metal ion con-
centrationswithin cellsare regulated during the cell
cycle17 and may be significantly altered in cancer cells
versus normal cells.18 The anticancer activity of 10c and
10d relative to the macrocyclic bis(propargylic) sulfone
6 may reflect the ability of 10c and 10d to cleave DNA
better under the conditionsof elevated osdium or
potassium levels that may exist, at least transiently, in
cancer cells. Further investigation with more potent
non-metal ion-binding DNA cleavage agents, such as
compound 9, isrequired in order to addresthe role of
alkali metal ion binding in the anticancer activity of
these crown ethers.
1,11-Bis(2-propyn-1-oxy)-3,6,9-trioxaundecane (11b).13
Following the general procedure (see compound 11a),
tetraethylene glycol (2.51 g, 12.9 mmol) gave a residue
after workup that waspurified by flash chromatography
on silica gel (35% hexanes in EtOAc) to afford bis(pro-
pargyl ether) 11b (2.67 g, 77%) asa light yellow oil: Rf
0.45 (35% hexanesin EtOAc); 1H NMR d 2.37 (t,
J=2.4 Hz, 2H), 3.53–3.63 (m, 16H), 4.10 (d, J=2.4 Hz,
4H); 13C NMR d 58.11, 68.83, 70.13, 70.31, 70.33,
74.38, 79.43; IR 2872, 2117, 1101 cmꢁ1; MS 271
(MH+), 215, 171, 127; HR-MS m/e calcd for C14H23O5:
271.1545, found 271.1542.
1,17-Bis(2-propyn-1-oxy)-3,6,9,12,15-pentaoxaheptade-
cane (11d). Following the general procedure (see com-
pound 11a), hexaethylene glycol (2.89 g, 10.2 mmol)
gave a residue after workup that was purified by flash
chromatography (5% MeOH on EtOAc) to afford
bis(propargyl ether) 11d (3.11 g, 85%) asa yellow oil: Rf
0.53 (5% MeOH in EtOAc); 1H NMR d 2.38 (t,
J=2.4 Hz, 2H), 3.55–3.66 (m, 24H), 4.14 (d, J=2.4 Hz,
4H); 13C NMR d 58.22, 68.95, 70.24, 70.42 (4C), 74.42,
In conclusion, we have shown that within a series of
bis(propargylic) sulfone crown ethers, compounds 10c
and 10d, which are the best ligands for Na+ and K+
ions, display alkali metal ion-regulated DNA cleavage,

M. M. McPhee, S. M. Kerwin / Bioorg. Med. Chem. 9 (2001) 2809–2818
2815
79.52; IR 2872, 2116, 1107 cmꢁ1; MS 359 (MH+), 259,
215, 171, 127; HR-MS m/e calcd for C18H31O7:
359.2070, found 359.2060.
4 mL of CH2Cl2 under argon wasadded Br (87 mL,
2
1.7 mmol). After an additional 5 min, a solution of diol
12a (202 mg, 0.707 mmol) in 1.9 mL of CH2Cl2 was
added dropwise via cannula over 3 min, and the result-
ing reaction mixture wasstirred for an additional 5 h as
the reaction mixture wasallowed to warm to room
temperature. The reaction mixture wasdiluted with
40 mL of EtOAc and washed with 15 mL of saturated
aqueousNaHCO 3. The aqueouslayer wasextracted
with EtOAc (3ꢀ10 mL), and the combined organic lay-
erswere wahsed with 20 mL of brine, dried, and con-
centrated to a residue that was purified by flash
chromatography on silica gel (3% hexanes in EtOAc) to
afford dibromide 13a (239 mg, 82%) asa pale yellow oil:
Rf 0.81 (3% hexanesin EtOAc); 1H NMR d 3.59–3.65
(m, 12H), 3.90 (t, J=2.0 Hz, 4H), 4.20 (t, J=2.0 Hz,
4H); 13C NMR d 14.18, 58.56, 69.14, 70.29, 70.48,
81.30, 82.79; IR 617 cmꢁ1; MS 411 (MH+), 263, 219,
175; HR-MS m/e calcd for C14H21Br2O4: 410.9807,
found 410.9802.
General procedure for hydroxymethylation. 1,8-Bis(4-
hydroxy-2-butyn-1-oxy)-3,6-dioxaoctane (12a).5 An
argon-flushed 100 mL three-neck round bottom flask
equipped with a mechanical stirrer was charged with a
solution of bis(propargyl) ether 11a (593 mg, 2.62 mmol)
in 22 mL of THF and TMEDA (4.0 mL, 26.5 mmol).
The mixture wascooled with stirring to ꢁ78 ꢂC, and n-
BuLi (2.7 mL, 2.37 M, 6.39 mmol) wasadded dropwise
with stirring. After an additional 5 min, a stirring sus-
pension of paraformaldehyde (1.7 g, 56.6 mmol formal-
dehyde equivalents) in 7 mL of THF under argon was
added quickly via cannula. After an additional 5 min the
cooling bath wasremoved and the reaction mixture was
allowed to warm to room temperature. The reaction
mixture wastsirred an additional 1 h and diluted with
90 mL of EtOAc and 60 mL of saturated aqueous
NaH2PO4. The aqueouslayer wasextracted with EtOAc
(3ꢀ45 mL), and the combined organic layerswere
washed with 60 mL of saturated NaHCO3 and 65 mL of
brine. The residue upon drying and concentration was
purified by flash chromatography (2% MeOH in
EtOAc) to afford diol 12a (293 mg, 39%) asa pale yel-
low solid: mp 33–34 ꢂC; Rf 0.34 (2% MeOH in
EtOAc); 1H NMR d 2.95 (s(br), 2H), 3.60–3.73 (m,
12H), 4.20 (t, J=1.8 Hz, 4H), 4.27 (t, J=1.8 Hz, 4H);
13C NMR d 50.76, 58.62, 68.99, 70.37, 70.50, 81.27,
85.05; IR 3400 cmꢁ1; MS 287 (MH+), 269, 201, 157,
113; HR-MS m/e calcd for C14H23O6: 287.1495, found
287.1485.
1,11-Bis(4-bromo-2-butyn-1-oxy)-3,6,9-trioxaundecane (13b).
Following the general procedure (see compound 13a),
diol 12b (220 mg, 0.668 mmol) gave a residue after
workup that waspurified by flahs chromatography on
silica gel (EtOAc) to afford dibromide 13b (275 mg,
1
90%) asa pale yellow oil: Rf 0.73 (EtOAc); H NMR d
3.55–3.65 (m, 16H), 3.90 (t, J=2.0 Hz, 4H), 4.20 (t,
J=2.0 Hz, 4H); 13C NMR d 14.15, 58.54, 69.13, 70.27,
70.45, 70.48, 81.28, 82.78; IR 614 cmꢁ1; MS 456
(MH+), 377, 309, 265, 221, 177; HR-MS m/e calcd for
C16H25Br2O5: 455.0069, found 455.007.
1,17-Bis(4-bromo-2-butyn-1-oxy)-3,6,9,12,15-pentaoxa-
heptadecane (13d). Following the general procedure
(see compound 13a), diol 12d (427 mg, 1.02 mmol) gave
a light tan solid after workup that contained dibromide
13d (1.17 g, 43% w/w 13d, 90%) and Ph3PO asan inse-
parable mixture. Analytical data for dibromide 13d: Rf
1,11-Bis(4-hydroxy-2-butyn-1-oxy)-3,6,9-trioxaundecane
(12b). Following the general procedure (see compound
12a), compound 11b (769 mg, 2.85 mmol) gave a residue
after workup that waspurified by flash chromatography
on silica gel (5% MeOH in EtOAc) to afford diol 12b
(450 mg, 48%) asa pale yellow oil: Rf 0.3 (5% MeOH in
EtOAc); 1H NMR d 2.86 (s(br), 2H), 3.60–3.70 (m,
16H), 4.20 (t, J=1.9 Hz, 4H), 4.26 (t, J=1.9 Hz, 4H);
13C NMR d 50.75, 58.60, 68.96, 70.37, 70.49 (2C), 81.28,
85.07; IR 3400 cmꢁ1; MS 331 (MH+), 313, 201, 157,
113; HR-MS m/e calcd for C16H27O7: 331.1757, found
331.1756.
1
0.56 (5% MeOH in EtOAc); H NMR d 3.55–3.70 (m,
24H), 3.93 (t, J=2.2 Hz, 4H), 4.24 (t, J=2.2 Hz, 4H);
13C NMR d 14.20, 58.66, 69.26, 70.39, 70.60 (4C), 81.39,
82.91; MS 544 (MH+), 307; HR-MS m/e calcd for
C20H33Br2O7: 543.0593, found 543.0583.
.
.
Na2S Al2O3 reagent. To Na2S 9H2O (7.1 g, 0.03 mmol)
that had been rinsed with a small amount of distilled,
deionized water and placed in a flask under argon was
added 18 mL of warm, distilled, deionized water that
had been boiled to remove CO2. The resultant solution
waspoured into a flaks containing Al 2O3 (neutral,
Brockmann Activity I, 80–200 mesh, 8.7 g, 0.085 mmol),
and the water wasremoved in vacuo via rotary eva-
porator with gentle heating in a warm water bath. The
material wasthen activated by heating in vacuo (95 ꢂC,
0.1 torr) for 1.5 h until the material (21.2% w/w Na2S)
wasa free-flowing pink powder. The reagent wasstored
under argon and used shortly after it was prepared.
1,17-Bis(4-hydroxy-2-butyn-1-oxy)-3,6,9,12,15-pentaoxa-
heptadecane (12d). Following the general procedure (see
compound 12a), compound 11d (755 mg, 2.11 mmol)
gave a residue after workup that was purified by flash
chromatography on silica gel (10% MeOH in EtOAc) to
afford compound 12d (357 mg, 40%) asa pale yellow
1
oil: Rf 0.22 (10% MeOH in EtOAc); H NMR d 2.70
(s(br), 2H), 3.58–3.70 (m, 24H), 4.20 (t, J=1.9 Hz, 4H),
4.26 (t, J=1.9 Hz, 4H); 13C NMR d 50.78, 58.61, 69.01,
70.38 (4C), 70.51, 81.32, 85.03; IR 3442 cmꢁ1; MS 419
(MH+), 401, 289, 245, 201; HR-MS m/e calcd for
C20H35O9: 419.2281, found 419.2284.
General procedure for macrocyclization. 6,9,12,15-Tetra-
oxa-1-thiacyclononadeca-3,17-diyne (14a).5 To a solu-
tion of dibromide 13a (150 mg, 0.364 mmol) in 24 mL of
5:1 CH2Cl2/EtOH wasadded in one portion
General procedure for bromination. 1,8-Bis(4-bromo-2-
butyn-1-oxy)-3,6-dioxaoctane (13a). To an ice-water
bath cooled solution of PPh3 (457 mg, 1.74 mmol) in

2816
M. M. McPhee, S. M. Kerwin / Bioorg. Med. Chem. 9 (2001) 2809–2818
Na2Sꢄ Al2O3 (0.311 g, 22% w/w Na2S, 0.877 mmol). The
reaction mixture wasallowed to tsir under argon at
room temperature for 3 days. The heterogeneous reac-
tion mixture wasfiltered through Celite and the os lids
were washed with EtOAc. The combined eluant was
concentrated, and the residue was purified by flash
chromatography on silica gel (3% hexanes in EtOAc) to
afford sulfide 14a (85 mg, 82%) as a colorless solid: mp
42–43 ꢂC; Rf 0.52 (3% hexanesin EtOAc); 1H NMR d
3.49 (t, J=1.9 Hz, 4H), 3.58–3.75 (m, 12H), 4.25 (t,
J=1.9 Hz, 4H); 13C NMR d 19.13, 58.64, 68.78, 70.35,
70.51, 79.70, 81.23; IR 1142, 1100 cmꢁ1; MS 285
(MH+); HR-MS m/e calcd for C14H21O4S: 285.1161,
found 285.1164.
were washed with 55 mL of water and 65 mL of brine.
Concentration of the organic layer afforded sulfone 10a
(80.2 mg, 93%) as a white solid. An analytical sample
recrystallized from 40% EtOAc in hexanes gave the
following: mp 132–133 ꢂC; Rf 0.5 (5% MeOH in
EtOAc); 1H NMR d 3.58–3.74 (m, 12H), 4.12 (t,
J=1.8 Hz, 4H), 4.27 (t, J=1.8 Hz, 4H); 13C NMR d
43.84, 58.55, 69.29, 70.32, 70.51, 73.35, 84.76; IR
1328 cmꢁ1; MS 317 (MH+); HR-MS m/e calcd for
C14H21O6S: 317.1059, found 317.1054.
6,9,12,15,18-Pentaoxa-1-thiacyclodocosa-3,20-diyne 1,1-
dioxide (10b). Following the general procedure (see
compound 10a), sulfide 14b (29.3 mg, 89.3 mmol) gave,
after workup, sulfone 10b (29.9 mg, 93%) asa colorles
solid. An analytical sample recrystallized from 40%
EtOAc in hexanesgave the following: mp 72.5–73.5 ꢂC;
6,9,12,15,18-Pentaoxa-1-thiacyclodocosa-3,20-diyne (14b).
Following the general procedure (see compound 14a),
dibromide 13b (563 mg, 1.24 mmol) gave a residue after
workup that waspurified by flahs chromatography on
silica gel (EtOAc) to afford sulfide 14b (285 mg, 70%) as
1
Rf 0.54 (10% MeOH in EtOAc); H NMR d 3.57–3.72
(m, 16H), 4.10 (t, J=1.8 Hz, 4H), 4.26 (t, J=1.8 Hz,
4H); 13C NMR d 43.64, 58.51, 69.22, 70.34, 70.43,
70.72, 73.45, 84.57; IR 1338 cmꢁ1; MS 361 (MH+), 317;
HR-MS m/e calcd for C16H25O7S: 361.1321, found
361.1324.
1
a pale yellow oil: Rf 0.36 (EtOAc); H NMR d 3.47 (t,
J=2.2 Hz, 4H), 3.62–3.73 (m, 16H), 4.23 (t, J=2.2 Hz,
4H); 13C NMR d 19.05, 58.69, 68.80, 70.41 (2C), 70.82,
79.60, 81.34; IR 1135, 1101 cmꢁ1; MS 329 (MH+);
HR-MS m/e calcd for C16H25O5S: 329.1423, found
329.1423.
6,9,12,15,18,21-Hexaoxa-1-thiacyclopentacosa-3,23-diyne
1,1-Dioxide (10c). Following the general procedure (see
compound 10a), sulfide 14c (57.4 mg, 0.154 mmol) gave
a residue after workup that was purified by flash chro-
matography on silica gel (10% MeOH in EtOAc) to
afford sulfone 10c (60.4 mg, 97%) as colorless solid: mp
6,9,12,15,18,21-Hexaoxa-1-thiacyclopentacosa-3,23-diyne
(14c).13 Following the general procedure (see compound
14a), dibromide 13c (382 mg, 0.764 mmol), prepared
previously,13 gave a residue after workup that was pur-
ified by flash chromatography on silica gel (5% MeOH
in EtOAc) to afford sulfide 14c (213 mg, 75%) asa pale
yellow oil.
63–64 ꢂC; Rf 0.44 (10% MeOH in EtOAc); H NMR d
1
3.56–3.73 (m, 20H), 4.12 (t, J=2.0 Hz, 4H), 4.26 (t,
J=2.0 Hz, 4H); 13C NMR d 43.63, 58.55, 69.66, 70.45,
70.71, 70.76, 70.82, 73.57, 84.66; IR 1337 cmꢁ1; MS 405
(MH+), 361, 341; HR-MS m/e calcd for C18H29O8S:
405.1583, found 405.1593.
6,9,12,15,18,21,24-Heptaoxa-1-thiacyclooctacosa-3,26-
diyne (14d). Following the general procedure (see com-
pound 14a), dibromide 13d (379 mg, 43% w/w mixture
with Ph3PO, 0.3 mmol) gave a residue after workup that
was purified by flash chromatography on silica gel (10%
MeOH in EtOAc) to afford 30.9 mg of pure sulfide 14d
as a colorless oil. Preparative TLC (1 mm silica gel
plate, 10% MeOH in EtOAc) of 14d-containing frac-
tionsthat were contaminated with Ph 3PO yielded an
additional 20.7 mg of pure 14d (41% for combined
material). Analytical data for compound 14d: Rf 0.33
6,9,12,15,18,21,24-Heptaoxa-1-thiacyclooctacosa-3,26-
diyne 1,1-dioxide (10d). Following the general proce-
dure (see compound 10a), sulfide 14d (41.5 mg,
99.8 mmol) gave sulfone 10d (40.8 mg, 91%) after
workup. An analytical sample recrystallized from 40%
EtOAc in hexanesgave the following: mp 87–88 ꢂC; Rf
1
0.32 (10% MeOH in EtOAc); H NMR d 3.52–3.71 (m,
24H), 4.11 (s(br), 4H), 4.24 (s(br), 4H); 13C NMR d
43.56, 58.45, 69.17, 70.42, 70.60 (4C), 73.57, 84.53; IR
1355 cmꢁ1; MS 449 (MH+), 404, 384, 361; HR-MS m/e
calcd for C20H33O9S: 449.1845, found 449.1849.
1
(10% MeOH in EtOAc); H NMR d 3.46 (t, J=2.2 Hz,
4H), 3.62–3.70 (m, 24H), 4.23 (t, J=2.2 Hz, 4H); 13C
NMR d 19.2, 58.69, 68.91, 70.43, 70.61 (2C), 70.67 (2C),
79.37, 81.43; IR 1115, 1097 cmꢁ1; MS 417 (MH+); HR-
MS m/e calcd for C20H33O7S: 417.1947, found 417.1944.
Alkali metal picrate extraction
The general procedure employed wasasprevioulsy
described.12 Briefly, in a typical extraction, 350 mL of
3.0 mM aqueousmetal picrate and 350 mL of 15.0 mM
host in CHCl3 were placed in a 0.5 gram vial. The vials
were then vortexed for 1 min and centrifuged for 15 min
to effect complete phase separation. A 50 mL aliquot of
the CHCl3 layer wasremoved and diluted to 1.0 mL
with MeCN. Absorbance of this solution at 380 nm was
used to calculated the association constants (Ka’s) by
the method of Cram.15 The valuespresented in Table 1
represent the mean plus or minus one standard deviation
from two separate extractions.
General procedure for sulfone formation. 6,9,12,15-Tet-
raoxa-1-thiacyclononadeca-3,17-diyne
1,1
Dioxide
(10a).5 To an ice-water bath cooled solution of sulfide
14a (77.7 mg, 0.274 mmol) in 2.6 mL of MeOH was
added dropwise a suspension of Oxone1 (1.086 g,
0.877 mmol) in 2.6 mL of water and 1.24 mL of 2.5 M,
pH 5 aqueous potassium citrate. Stirring was continued
for an additional 18 h asthe reaction mixture warmed to
room temperature. The reaction mixture wasdiluted
with 85 mL of water in a separatory funnel and extrac-
ted with 3ꢀ60 mL of CHCl3. The chloroform extracts

M. M. McPhee, S. M. Kerwin / Bioorg. Med. Chem. 9 (2001) 2809–2818
2817
Preparation of alkali metal ion DNA
Cytotoxicity assay
The cytotoxicity assays were conducted at the NCI and
Supercoiled DNA wasprepared asprevioulsy repor-
ted.12 Briefly, DH5a Escherichia coli cellstranfsected
with that pGAD424 were grown, collected, and lysed.
The plasmid DNA was isolated with a QIAGEN mini-
prep spin kit according to the manufacturer’s instruc-
tions, eluting the DNA from the column with distilled
water. Aliquots(10 mL) of thisoslution were gently
mixed with 212.2 mL of sterile, pH 7.4 aqueous alkali
metal phosphate solution. The plasmid DNA so
obtained wasdetermined psectrophotometrically to be
17 mM base pair and typically contained 75–85%
supercoiled (Form I) DNA. For EC25 experiments, the
concentration of alkali metal ion before addition of the
aqueousDNA solution was20 mM.
20
were performed asdescribed.
Briefly, cell suspensions
that were diluted according to the particular cell type
and the expected target cell density (5000–40,000 cells
per well based on cell growth characteristics) were
added by pipet (100 mL) into 96-well microtiter plates.
Inoculateswere allowed a preincubation period of 24 h
at 37 ꢂC for stabilization. Dilutions at twice the intended
test concentration were added at time zero in 100-mL
aliquotsto the microtiter plate well.s Tets compounds
were evaluated at five 10-fold dilutionswith the highest
well concentration being 100 mM. Incubationslasted for
48 h in 5% CO2 atmosphere and 100% humidity. The
cells were assayed by using the sulforhodamine B
assay.21,22 A plate reader wasuesd to read the optical
densities, and a microcomputer program was used to
process the optical densities into the GI50 concentration,
the concentration of test drug where 100ꢀ(TꢁT0)/
(CꢁT0)=50, where the optical density of the test well
after a 48-h period of exposure to test drug is T, the
optical density at time zero is T0, and the control optical
density is C.
Quantification of DNA cleavage by propargylic sulfones
In a typical cleavage experiment, 2 mL of a freshly pre-
pared solution of bis(propargylic) sulfone compound in
DMSO-d6 wasadded to a tserile Eppendorf tube fol-
lowed by 14 mL of an aqueousos lution of alkali metal
ion-containing DNA prepared asabove. The contents
were mixed by brief (5 s) centrifugation at 6000 rpm and
allowed to stand at room temperature for 18 h. Tubes
were then heated for 90 sat 70 ꢂC. Upon cooling, 2 mL
of 8ꢀ loading dye (0.13 g/mL each of Bromophenol
Blue and Xylene Cyanol in 30% aqueousglycerol) was
added to each tube, the contentswere gently homo-
genized, and 5 mL of the resulting solution was loaded
onto a 0.7% w/v agarose gel. The DNA cleavage pro-
ducts were separated by electrophoresis in 1ꢀ TBE
running buffer at 45 V for 2.25 h. The agarose gel was
stained for 15min in TBE buffer that contained 0.25mg/
mL ethidium bromide and then destained in distilled
water for 15–30 min. The gel wasscanned with a Molecu-
lar DynamicsFluorimager and the quantitiesof Forms
I, II and III DNA were assessed with the ImageQuaNT
software program. The degree of cleavage of Form I
DNA wasdetermined using eq 1.
Acknowledgements
Thiswork wassupported from grantsfrom the Robert
A. Welch Foundation, the American Association of
Collegesof Pharmacy, and the US Public Health Ser-
vice (GM-50892).
References and Notes
1. Nicolaou, K. C.; Skokotas, G.; Maligres, P.; Zuccarello,
G.; Schweiger, E. J.; Toshima, K.; Wenderborn, S. Angew.
Chem., Int. Ed. Eng. 1989, 28, 1272.
2. Braverman, S.; Duar, Y.; Segev, D. Tetrahedron Lett. 1976,
3181.
3. Gates, K. S. In Comprehensive Natural Products Chemistry;
Barton, D., Nakanishi, K., Meth-Cohn, O., Eds.; Pergamon:
Oxford 1999; Vol pp 491–552.
Percent cleavage
4. Pogozelski, W. K.; Tullius, T. D. Chem. Rev. 1998, 98,
1089.
5. Kerwin, S. M. Tetrahedron Lett. 1994, 35, 1023.
6. Nicolaou, K. C.; Wendeborn, S.; Maligres, P.; Isshikti,
M. K.; Zein, N.; Ellestad, G. Angew. Chem., Int. Ed. Engl.
1991, 30, 418.
ð2  ½Form IIIŠ þ ½Form IIŠ
¼
 100 ð1Þ
ð2  ½Form IIIŠ þ ½Form IIŠ þ ½Form IŠ
The reported, normalized percent cleavage accountsfor
cleavage in control samples under the reaction conditions
employed and thiswascalculated according to eq (2).
7. Braverman, S.; Zafrani, Y.; Gottlieb, H. E. Tetrahedron
Lett. 2000, 41, 2675.
8. Basak, A.; Khamrai, U. K. Tetrahedron Lett. 1995, 36,
7913.
9. Manning, G. S. Quart. Rev. Biophys. 1978, II, 179.
10. Dai, W.-M.; Fong, K. C. Tetrahedron Lett. 1995, 36, 5613.
11. Dai, W.-M.; Fong, K. C.; Danjo, H.; Nishimoto, S.-I.;
Solow, M.; Mak, W. L.; Yeung, M. L. Bioorg. Med. Chem.
Lett. 1996, 6, 1093.
12. McPhee, M. M.; Kern, J. T.; Hostler, B. C.; Kerwin, S. M.
Bioorg. Chem. 2000, 8, 98.
13. McPhee, M. M.; Kerwin, S. M. J. Org. Chem. 1996, 61,
9385.
Normalized percent cleavage
% cleavage ðdrugÞ ꢀ % cleavage ðcontrolÞ
¼
ð2Þ
100 ꢀ % cleavage control
The reported EC25 valueswere obtained by interpola-
tion from plotsof normalized percent cleavage veruss
concentration. The EC25 valuesreported repreesnt the
averageꢃstandard deviation for three or more separate
determinations.
14. Trost, B. M.; Curran, D. B. Tetrahedron Lett. 1981, 22,
1287.

2818
M. M. McPhee, S. M. Kerwin / Bioorg. Med. Chem. 9 (2001) 2809–2818
15. Helgeson, R. C.; Weisman, G. R.; Toner, J. L.; Tar-
nowski, T. L.; Chao, Y.; Mayer, J. M.; Cram, D. J. J. Am.
Chem. Soc. 1979, 101, 4928.
16. Wrasidlo, W.; Hiatt, A.; Mauch, S.; Merlock, R. A.;
Nicolaou, K. C. Acta Oncol. 1995, 34, 157.
20. Monks, A.; Scudiero, D.; Skehan, P.; Shoemaker, R.;
Paull, K.; Vistica, D.; Hose, C.; Langley, J.; Cronise, P.; Vai-
gro-Wolff, A.; Gray-Goodrich, M.; Campbell, H.; Mayo, J.;
Boyd, M. J. Nat. Cancer Inst. 1991, 83, 757.
21. Rubinstein, L. V.; Shoemaker, R. H.; Paull, K. D.; Simon,
R. M.; Tosini, S.; Skehan, P.; Scudiero, D. A.; Monks, A.;
Boyd, M. R. J. Natl. Cancer Inst. 1990, 82, 1113.
17. Stephen, J.; Osborne, M. P.; Spencer, A. J.; Warley, A.
Scanning Microsc. 1990, 4, 781.
18. Zs.-Nagy, I. Scanning Microsc. 1989, 3, 473. Roomans,
G.; Von Euller, A. Cell Biol. Int. 1996, 20, 103. Cameron, I. L.;
Smith, N. K. R. Magnesium 1989, 8, 31.
22. Skehan, P.; Storeng, R.; Scudiero, D. A.; Monks, A.;
McMahon, J.; Vistica, D. T.; Warren, J. T.; Bokesch, H.;
Kenny, F.; Boyd, M. R. J. Natl. Cancer Inst. 1990, 82,
1107.
19. For general procedures, see ref 13.