1,2-Bis(methylsulfonyl)-1-(2-chloroethyl)-2-[[1-(4-nitrophenyl)ethoxy]carbonyl]hydrazine (KS119): A Cytotoxic Prodrug with Two Stable Conformations Differing in Biological and Physical Properties

Article abstract of DOI:10.1111/j.1747-0285.2011.01193.x

The anticancer prodrug 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[[1-(4-nitrophenyl)ethoxy]carbonyl]hydrazine (KS119) selectively releases a short-lived cytotoxin following enzymatic reduction in hypoxic environments found in solid tumors. KS119, in add

Full text of DOI:10.1111/j.1747-0285.2011.01193.x

ª 2011 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2011.01193.x
Chem Biol Drug Des 2011; 78: 513–526
Research Article
1,2-Bis(methylsulfonyl)-1-(2-chloroethyl)-2-
[[1-(4-nitrophenyl)ethoxy]carbonyl]hydrazine
(KS119): a Cytotoxic Prodrug with Two Stable
Conformations Differing in Biological and
Physical Properties
Philip G. Penketh1,*, Raymond P.
Abbreviations: 90CE, 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydra-
zine; CIP, calf intestinal alkaline phosphatase; DPP, differential
Baumann1, Krishnamurthy Shyam1,
pulse polarography; E1 ⁄ 2, polarographic half-wave reduction potential;
Hugh S. Williamson2, Kimiko Ishiguro1,
G-C ethane cross-link, 1-(N3-cytosinyl),-2-(N1-guaninyl)ethane; KS119,
Rui Zhu1, Emma S. E. Eriksson3, Leif A.
1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[[1-(4-nitrophenyl)ethoxy]carbon
yl]hydrazine; KS119W, 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[[1-(3-
Eriksson3 and Alan C. Sartorelli1
phospho-4-nitrophenyl)ethoxy]carbonyl]hydrazine;
bis(methylsulfonyl)-1-(2-chloroethyl)-2-[[1-(3-hydroxy-4-nitrophenyl)eth-
oxy]carbonyl]hydrazine; PNBC, 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-
[(4-nitrobenzyloxy)carbonyl]hydrazine.
KS119WOH,
1,2-
1Department of Pharmacology, Yale University School of Medicine,
New Haven, CT 06520, USA
2BP Exploration, Chertsey Road, Sunbury-on-Thames, Middlesex,
TW16 7LN, UK
Received 7 January 2011, revised 12 July 2011 and accepted for publi-
cation 14 July 2011
3School of Chemistry, National University of Ireland – Galway,
University Road, Galway, Ireland
*Corresponding author: Philip G. Penketh, philip.penketh@yale.edu
or philip.penketh@gmail.com
The anticancer prodrug 1,2-bis(methylsulfonyl)-1-
(2-chloroethyl)-2-[[1-(4-nitrophenyl)ethoxy]car-
1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[[1-(4-nitrophenyl)ethoxy]-
carbonyl]hydrazine (KS119) is a prodrug that requires an oxygen-
deficient reductive environment to liberate a cytotoxin within
malignant tumors (1). To accomplish this, KS119 and related mole-
cules are composed of three domains: a 'trigger' region consisting
of a nitrobenzyl moiety, an oxycarbonyl 'linker' region, and a cyto-
toxic 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine DNA inter-
strand cross-linking agent (90CE) which is considered the
'therapeutic warhead' region (Figure 1) (1,2). 1,2-Bis(methylsulfonyl)-
bonyl]hydrazine (KS119) selectively releases
a
short-lived cytotoxin following enzymatic reduc-
tion in hypoxic environments found in solid
tumors. KS119, in addition to two enantiomers,
has two stable atropisomers (conformers differing
in structure owing to hindered bond rotation) that
interconvert at 37 ꢀC in aqueous solution by first-
order kinetics with t1 ⁄ 2 values of ꢀ50 and ꢀ64 h.
The atropisomers differ in physical properties
such as partition coefficients that allow their chro-
matographic separation on non-chiral columns. A
striking difference in the rate of metabolism of
the two atropisomers occurs in intact EMT6 mur-
ine mammary carcinoma cells under oxygen-defi-
cient conditions. A structurally related molecule,
1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[[1-(3-
hydroxy-4-nitrophenyl)ethoxy]carbonyl]hydrazine
(KS119WOH), was also found to exist in similar sta-
ble atropisomers. The ratio of the atropisomers of
KS119 and structurally related agents has the
potential to impact the bioavailability, activation,
and therapeutic activity. Thus, thermally stable
atropisomers ⁄ conformers in small molecules can
result in chemically and enantiomerically pure com-
pounds having differences in biological activities.
1-(2-chloroethyl)-2-[[1-(4-nitrophenyl)ethoxy]carbonyl]hydrazine
was
designed to exploit the fact that hypoxic ⁄ anoxic areas are found in
solid tumors (Figure 1). These oxygen-deficient regions often con-
found conventional therapeutic strategies involving radiation and
chemotherapeutic agents, because the low oxygen tension in solid
tumors renders these cells highly resistant to x-irradiation and the
poor blood supply to these tumor areas can result in decreased
delivery to and activity of cytotoxic drugs at these malignant sites
(3–6). In addition, the low oxygen concentration of these cells also
encourages metastatic spread (7). These unfavorable properties
from a therapeutic point of view are, however, counterbalanced by
the fact that oxygen-deficient regions can be exploited to gain
specificity by the design of reductively activated prodrugs (1,2,8).
The agents that we are developing, after an initial one electron
reduction by enzymes, such as NADPH:cytochrome P450 reductase,
NADH:cytochrome b5 reductase and xanthine oxidase, to yield a
nitro radical anion, are readily reoxidized by molecular oxygen back
Key words: atropisomers, chemotherapy, conformers, cytotoxicity,
hypoxia, KS119, prodrug, targeting
513

Penketh et al.
A
C
B
Figure 1: Background summarization figure: (A) The distribution of capillaries and oxygen in different regions of a solid tumor and normal
tissue. (B) The mechanism underlying the inverse oxygen concentration dependence of net KS119 reduction. (C) The mechanism of
fragmentation to generate a chloroethylating species upon reduction and the subsequence formation of a G-C ethane cross-link in DNA by
these species.
to the parental drug (Figure 1), thereby protecting normoxic normal
tissue. However, upon further reduction of the nitro radical anion to
either the amino or hydroxylamino functionality, which only occurs
efficiently when the oxygen concentration is low, the molecule rela-
tively rapidly fragments to release the 'therapeutic warhead', a
short-lived (t1 ⁄ 2 = ꢀ30 seconds, at pH 7.4 and 37 ꢀC) alkylating
species (90CE) that ultimately results in the formation of a highly
toxic DNA G-C ethane interstrand cross-link (1,2,9,10). The short
half-life of 90CE permits little diffusional escape to normal tissue,
and thus, the bulk of the alkylations are delivered close to the site
of parental drug reduction.
the methylene carbon, resulting in the release of the cytotoxic moi-
ety in the absence of reduction (1,2). The addition of the shielding
methyl group of KS119 blocked this secondary mode of activation,
thereby increasing the hypoxic selectivity and contributing at
least in part to the five log hypoxic ⁄ oxic cell kill differential
against EMT6 carcinoma cells in vitro, previously observed with
KS119 (2).
The addition of the methyl group in the linker region introduces a
chiral center resulting in two KS119 optical isomers. Surprisingly,
the addition of the methyl group also results in significant changes
in the polarographic properties of the molecule and the existence
of two peaks by HPLC analysis over extended elution times on a
non-chiral C18 reverse-phase column (Figure 3). Because such col-
umns (non-chiral) are incapable of separating optical isomers, other
structural features must be responsible for this behavior. Our stud-
ies indicate that two major distinct and stable conformational forms
or atropisomers (conformers differing in structure owing to hindered
bond rotation) of KS119 exist (Figure 4) which possess different
physical and biological properties.
1,2-Bis(methylsulfonyl)-1-(2-chloroethyl)-2-[[1-(4-nitrophenyl)ethoxy]car-
bonyl]hydrazine is a modified version of 1,2-bis(methylsulfonyl)-1-
(2-chloroethyl)-2-[(4-nitrobenzyloxy)carbonyl]hydrazine
(PNBC),
a
structurally related agent of lower hypoxic selectivity (1,2). PNBC
lacks the shielding methyl group on the methylene carbon adjacent
to the linker region (Figure 2) and as such, in addition to reductive
activation, is subject to an additional activation mechanism via an
SN2 nucleophilic attack by glutathione ⁄ glutathione-S-transferase at
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Chem Biol Drug Des 2011; 78: 513–526

Small Molecule Conformation and Activity
Determination of KS119 and KS119WOH by
HPLC
HPLC measurements of the concentration of KS119 were performed
using a Beckman 127P solvent module and a Beckman 168 UV ⁄ vis
detector (Beckman, Fullerton, CA, USA); two HPLC methods were
used. A standard curve for the estimation of total KS119 was estab-
lished by analyzing known concentrations of KS119 over a range of
0–200 lM, and a linear relationship between concentration and the
area under the curve was established. The first method (method A)
was used to determine the concentration of KS119 conformers when
relatively rapid analysis was required. Samples in 30% v ⁄ v acetoni-
trile were separated on a five micron 220 · 4.6 mm Applied Biosys-
tems RP-18 C-18 reverse phase column (Applied Biosystems,
Carlsbad, CA, USA) by elution with 34% acetonitrile in buffer (0.03 M
KH2PO4, 1.0 mM NaN3, pH 5.4) for 5 min followed by a 34–70% ace-
tonitrile linear gradient in buffer, at a flow rate of 0.6 mL ⁄ min from 5
to 35 min. After this point, the concentration of acetonitrile was
maintained at this level for 5 min then returned to the starting con-
centration over an additional 5 min. Absorbance was monitored at
280 nm using a Beckman 168 UV ⁄ vis detector. KS119 eluted as a split
peak at approximately 35 min. KS119WOH eluted as a split peak at
approximately 32 min. To more accurately resolve the proportions of
the different conformers and for separation purposes, a second
method was developed (method B) by eluting the column with a sol-
vent of constant composition consisting of ꢀ39% acetonitrile and
buffer, at a flow rate of 0.6 mL ⁄ min. Under these conditions, the con-
formers of KS119 eluted as two partly overlapping peaks at approxi-
mately 83 and 86 min, while the conformers of KS119WOH eluted as
two partly overlapping peaks at approximately 69 and 73 min.
Figure 2: Chemical structures of PNBC, KS119, KS119W, and
KS119WOH, and the chiral carbon configurations in KS119-R,
KS119WOH-R, KS119-S, and KS119WOH-S.
In this study, we have examined the thermal interconversion of the
KS119 conformers ⁄ atropisomers, their octanol:buffer partition coeffi-
cients, their polarographic reduction and their metabolism by
NADPH:cytochrome P450 reductase, xanthine oxidase, and EMT6
carcinoma cells under oxygenated and oxygen deficient conditions.
The possible therapeutic implications of these conformational differ-
ences are discussed.
Separation of conformers
Materials and Methods
Relatively pure samples of the early- and late-eluting conformers of
both KS119 and KS119WOH were obtained at purities of >98% by
collecting fractions from HPLC (method B) corresponding to the ini-
tial rising portion of the early peak and the falling portion of the
late peak (Figure 5). The concentration and purity of the conformer
preparations were then determined by repeat HPLC. For partition
studies, these samples were diluted fourfold with 100 mM potas-
sium phosphate, pH 7.4, and were stored at 0 ꢀC or lower prior to
use. This gives aqueous buffer conformer solutions which contain
approximately 9.5% acetonitrile and 82 mM potassium phosphate,
with the pH value remaining at about 7.3–7.4.
Chemicals and reagents
1,2-Bis(methylsulfonyl)-1-(2-chloroethyl)-2-[[1-(4-nitrophenyl)ethoxy]car-
bonyl]hydrazine (racemic mixture) and PNBC were synthesized as
previously described (1). Enantiomerically pure KS119-R and KS119-
S were produced by chiral synthesis from their respective chiral
nitrobenzyl alcohols by Vion Pharmaceuticals Inc. (New Haven, CT.
USA) and were supplied by the company in very limited quantities.
KS119W, 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[[1-(3-phospho-4-
nitrophenyl)ethoxy]carbonyl]hydrazine
(enantiomers
and
ra-
cemic mixtures) were also provided by Vion Pharmaceuticals.
KS119WOH, 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[[1-(3-hydroxy-
4-nitrophenyl)ethoxy]carbonyl]hydrazine (enantiomers and racemic
mixtures) were produced in solution immediately prior to HPLC anal-
ysis and separation, from KS119W (enantiomers and racemic mix-
tures) by the enzymatic removal of the phosphate group using calf
intestinal alkaline phosphatase (CIP) from New England Biolabs, Ips-
wich, MA, USA. Briefly, 50 lL of 10 mM KS119W dissolved in
DMSO was added to 0.95 mL of 50 mM NaCl, 25 mM Tris–HCl,
5 mM MgCl2, 0.5 mM dithiothreitol, pH 7.9 buffer containing
20 units of CIP. This mixture was then incubated at 37 ꢀC for
20 min, and stored on ice until used. The aforesaid agents were all
greater than 95% purity by HPLC analysis. All other chemicals were
purchased from the Sigma-Aldrich Chemical Company, St Louis, MO,
USA.
Preparation of purified KS119A and KS119B for
polarographic and cell cytotoxicity studies
To prepare conformer samples for cytotoxicity studies, the conform-
ers were separated as described earlier (HPLC, method B) except
that the eluting solutions contained no buffer components or NaN3,
and consisted of equivalent mixtures of acetonitrile and distilled
water. The fractions corresponding to the early- and late-eluting
conformers were collected and diluted threefold with ice-cold dis-
tilled water, then shaken with 5% by volume of dichloromethane.
The mixture was allowed to separate and the lower dichlorome-
thane layer containing the conformers was then collected. The
dichloromethane layer was evaporated in an ice bath with a stream
of dry nitrogen gas and the residual material dissolved in a small
Chem Biol Drug Des 2011; 78: 513–526
515

Penketh et al.
Figure 3: HPLC traces of PNBC,
KS119, KS119W, and KS119WOH
under two different HPLC
protocols; fast elution protocol
(30–36-min elution time) and slow
elution protocol (69–86-min elution
time) and LCMS of KS119. Panel
A, HPLC traces of (left panel)
KS119 (racemic mixture) using the
fast elution protocol: (right panel)
KS119 (racemic mixture) using the
slow elution protocol. Panel B,
HPLC traces of KS119 optical
isomers using the slow elution
protocol (left panel) KS119-R; (right
panel) KS119-S. Panel C, HPLC
traces of PNBC using the fast
elution protocol (left panel); PNBC
using the slow elution protocol
(right panel). Panel D, HPLC traces
of KS119WOH racemic mixture and
separate optical isomers using the
slow elution protocol (left panel)
KS119WOH (racemic mixture);
KS119WOH-R (central panel), and
KS119WOH-S (right panel). Panel
E, LCMS of the early (KS119A)-
and late (KS119B)-eluting peaks of
KS119 (racemic mixture).
volume of a 70:30 DMSO ⁄ water mixture (freezing point )70 ꢀC)
and stored at )20 ꢀC until required. The concentration and purity
of the conformer preparations were checked by repeat HPLC prior
to use. Conformer preparations stored in this manner maintained a
high level of purity (>90%) for greater than a month.
mass spectroscopy using a chromatographic system consisting of
an Agilent 1200 series HPLC system, including a binary pump
(Model G1312B), a vacuum degasser (Model G1379B), an autosam-
pler (Model G1367C), and a column oven (Model G1316B). The
mass spectrometer was an Applied Biosystems Sciex 4000 Q-
trapꢁ mass spectrometer (Applied Biosystems Sciex, Foster, CA,
USA). Data acquisition was carried out by ANALYST 1.4.2ꢁ
software on a DELL computer. A declustering potential of 50 V,
excitation energy of 100 V and a collision energy of 10 V were
utilized.
LCMS of KS119A and KS119B
Material comprising the rising portion of the early (KS119A) peak
and the falling portion of the late (KS119B) peak was analyzed by
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Chem Biol Drug Des 2011; 78: 513–526

Small Molecule Conformation and Activity
tubes to completely hydrolyze the KS119. The samples were then
diluted with an equal volume of 100 mM KH2PO4 solution. The
resultant solutions, containing the equivalent of 25 lM hydrolyzed
KS119 conformers were then analyzed using HPLC protocol A and
compared with an authentic 25 lM solution of the 1-(4-nitrophe-
nyl)ethanol synthesized as previously described (1). A sharp peak
at ꢀ12 min corresponding to the expected generation of the 1-
(4-nitrophenyl)ethanol was observed with both KS119A and
KS119B.
A
B
n-Octanol:buffer partition coefficient
determination for conformers of KS119 and
KS119WOH
To determine the octanol:buffer partition coefficients, 20 lL of n-
octanol was added to a 1 mL volume of purified conformer of
known concentration determined by HPLC in ice-cold 82 mM potas-
sium phosphate buffer containing 9.5% acetonitrile, pH ꢀ7.4. This
mixture was vortexed for 30 seconds and then placed on ice for 30
additional sec, and the process was repeated three times. This mix-
ture was then centrifuged at 10 000 g for 30 seconds to separate
the phases, and the lower aqueous phase was analyzed by HPLC
method A to determine the remaining conformer concentration.
From the decrease in conformer concentration in the aqueous
phase, and the known volumes of the phases, the partition coeffi-
cient was calculated. A much smaller volume of octanol compared
with the aqueous phase was used as the KS119 and KS119WOH
conformers greatly favored the octanol phase; therefore, for accu-
rate measurement by this method, the concentration in the aqueous
phase must not be reduced more than several fold.
C
Polarographic determination of half-wave
reduction potentials
Differential pulse polarography (DPP) voltagrams of PNBC, KS119,
KS119A, and KS119B were obtained using a pH 7.0 buffer com-
posed of 100 mM potassium chloride and 50 mM potassium phos-
phate as the supporting electrolyte. Compounds were added as
2 mM solutions in DMSO or in 70:30 DMSO ⁄ water mixture to
give final concentrations of 20 lM. The samples were purged
with nitrogen to remove dissolved oxygen, and DPP voltagrams
were obtained using a Princeton Applied Research electrochemi-
cal trace analyzer model 394, linked to a model 303A static mer-
cury drop electrode (Princeton Applied Research, Oak Ridge, TN,
USA). Scans were performed from 0 to )900 mv (2 mv ⁄ second)
using a platinum counter electrode versus an Ag ⁄ AgCl reference
electrode (saturated KCl ⁄ AgCl electrolyte). A pulse amplitude of
50 mv was used and the polarographic half-wave reduction
potential E1 ⁄ 2 was calculated from the peak current potential
(EP) according to the following equation; E1 ⁄ 2 = EP – pulse ampli-
tude ⁄ 2 (11).
Figure 4: Scheme proposed to account for the existence of sta-
ble conformers of KS119 but not of PNBC based upon rotational
restriction in the linker region. (A) Two-dimensional planar represen-
tation scheme showing relatively free rotation of the nitrobenzyl
trigger domain with respect to the 1,2-bis(sulfonyl)-1-(2-chloroeth-
yl)hydrazine warhead domain in PNBC. (B) Two-dimensional planar
representation scheme showing restricted rotation of the nitrobenzyl
trigger domain with respect to the 1,2-bis(sulfonyl)-1-(2-chloroeth-
yl)hydrazine warhead domain in KS119 confining the molecule to
particular relative orientations. (C) Space filling three-dimensional
representation showing the locking action of the methyl group on
the free rotation of the nitrobenzyl trigger domain with respect to
the 1,2-bis(sulfonyl)-1-(2-chloroethyl)hydrazine warhead domain in
KS119.
Hydrolysis of KS119A and KS119B
Ten microlitre samples of 5 mM purified KS119A or KS119B solu-
tions (concentration determined by HPLC assuming both conformers
have the same extinction at 280 nm as the equilibrium mixture)
were added to 990 lL of 10 mM NaOH to give 50 lM solutions.
These solutions were then heated at 100 ꢀC for 30 min in sealed
Hypoxic ⁄ oxic reduction of KS119 conformers
by NADPH:cytochrome P450 reductase and
xanthine oxidase
In these experiments, oxygen deficiency was enzymatically gener-
ated as previously described (12). Briefly, glucose ⁄ glucose oxidase
Chem Biol Drug Des 2011; 78: 513–526
517

Penketh et al.
A
B
Figure 5: Separation ⁄ purification of the early- and late-eluting conformers of KS119 and KS119WOH and their thermal re-equilibration.
Panel A (left panel) HPLC of racemic KS119 showing the collected fractions corresponding to the initial rising portion of the early-eluting con-
former peak and the falling portion of the late-eluting conformer peak. The upper and lower central panels of panel A show the purity of the
collected late KS119B and early KS119A eluting conformer fractions, respectively. The upper and lower right hand panels of panel A show
the thermal equilibration of the collected pure KS119B and KS119A conformer fractions, respectively, to give the equilibrium mixture of con-
formers. Panel B (left panel) HPLC of racemic KS119WOH showing the collected fractions corresponding to the initial rising portion of the
early-eluting conformer KS119WOHA peak and the falling portion of the late-eluting conformer KS119WOHB peak. The upper and lower cen-
tral panels of panel B show the purity of the collected KS119WOHB and KS119WOHA conformer fractions, respectively. The upper and lower
right hand panels of panel B show the thermal equilibration of the collected pure KS119WOHB and KS119WOHA eluting conformer fractions,
respectively, to give the equilibrium mixture of conformers.
was used to rapidly consume the available free oxygen, and cata-
lase was employed to remove generated hydrogen peroxide. In the
absence of other components, this system itself does not measur-
ably reduce KS119 in the time frames employed in these experi-
ments. The reaction mixtures in a total volume of 0.5 mL were as
follows: 100 mM potassium phosphate buffer, pH 7.4, containing
of the conformers of KS119, when 20 lM KS119, consisting of
the normal equilibrium mixture of conformers, was incubated under
oxygenated conditions (shaken in a shallow layer in T25 flasks) and
oxygen-deficient conditions (0.7 mL aliquots in microfuge tubes plus
the enzymes required to create oxygen deficient conditions) with
107 EMT6 carcinoma cells ⁄ mL for 3 h in Dulbecco's Minimal Essen-
tial Medium (DMEM) supplemented with 10% fetal bovine serum
and 10 mM glucose. At 0, 1, 2 and 3 h, aliquots were mixed with
an equal volume of acetonitrile, in which KS119 is very soluble, to
quench the enzymatic reactions, lyse the cells, precipitate the
enzymes and other macromolecules and extract the remaining
KS119. These samples were allowed to stand at room temperature
for 15 min to complete the precipitation then centrifuged at
10 000 g for 10 min. The supernatant was then analyzed using
HPLC method A.
10 mM glucose, 5 lL of
a
solution of glucose oxidase
(200 units ⁄ mL) and catalase (12 000 units ⁄ mL), 20 lM KS119
(added as a 100· solution in DMSO) plus either 5 lL of
104 units ⁄ mL of NADPH:cytochrome P450 reductase (Sigma, human
recombinant C8113) plus 1 mM NADPH or 0.16 units ⁄ mL of xanthine
oxidase (Sigma, bovine milk, X4500-5UN) plus 1 mM xanthine. The
aerobic reactions were identical except that the glucose needed for
the oxygen deficiency system to operate was omitted. Furthermore,
the aerobic reaction volumes were scaled up 10-fold and the mix-
ture incubated as a shallow layer with shaking in sealed T25 cul-
ture flasks to ensure that full air saturation was maintained and
evaporation prevented. The reaction mixtures were incubated at
37 ꢀC and samples were removed at 0, 1, 2 and 3 h after initiation,
mixed with an equal volume of CH3CN and centrifuged at 10 000 g
for 10 min to sediment any precipitated protein. The supernatant
was then analyzed using HPLC method A for KS119 content and
conformer composition.
Clonogenic cytotoxicity assays
Clonogenic survival assays were performed essentially as described
previously (13). O6-Alklyguanine-DNA alkyltransferase-deficient
EMT6 tumor cells were seeded into 25-cm2 plastic tissue culture
flasks at 5–8 · 105 cells per flask. When confluent, cells were
treated with either the early (KS119A)- or late (KS119B)-eluting con-
former of KS119 dissolved in 70% v ⁄ v DMSO aqueous mixture in a
total volume of 10 mL of medium for 4 h at 37 ꢀC. For oxygen-defi-
cient conditions, cells were incubated with KS119 conformers in
the presence of 2 units ⁄ mL of glucose oxidase (Sigma G6641),
120 units ⁄ mL of catalase (Sigma, C1345) in high glucose Dulbecco's
Reduction of KS119 by EMT6 cells
These experiments were performed in a manner similar to the enzy-
matic studies. They involved following the time course of the loss
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Chem Biol Drug Des 2011; 78: 513–526

Small Molecule Conformation and Activity
modified Eagle medium (Invitrogen, Carlsbad, CA, USA). Flasks were
flushed with nitrogen for 10 seconds and caps were screwed on
tightly. This facilitates oxygen depletion of the medium by glucose
oxidase through the removal of residual oxygen containing air and
denial of the entry of additional air. After a 4-h treatment, monolay-
ers were rinsed, and cells were detached by trypsinization, sus-
pended in culture medium, counted and sequential cell dilutions
were plated in duplicate into 6-well plates at a density of 1 · 102,
1 · 103, and 1 · 104 cells per well. Ten days later, colonies were
fixed, stained with 0.25% crystal violet in 80% methanol and quan-
tified. All analyses were corrected for plating efficiency in the pres-
ence of the DMSO vehicle at concentrations equivalent to those
used for exposure to the test agent. DMSO concentrations were
£0.5% and non-toxic. In the absence of cells, no measurable direct
metabolism of KS119 was detected in the presence of the glucose
oxidase and catalase enzyme components of the oxygen deficiency
generating system (12).
Confirmation of equivalent absorbancies at
280 nm
Purified samples of KS119A and KS119B quantified by HPLC,
assuming that both forms had extinction coefficients at 280 nm that
were identical to the KS119 equilibrium mixture standard, were
subjected to alkaline hydrolysis and both forms gave essentially
identical molar yields of 1-(4-nitrophenyl)ethanol. The measured
yields of 1-(4-nitrophenyl)ethanol from KS119A and KS119B were
100 € 2% and 98 € 2%, respectively. These findings verified the
assumption that both forms have equivalent or very close to equiva-
lent optical absorbancies at 280 nm. Therefore, the relative HPLC
peak areas at 280 nm directly correspond to the relative quantities
of the two forms.
HPLC analysis of pure KS119 optical isomers
The addition of the methyl group in KS119 to the methylene carbon
within the linker region introduces a chiral center, resulting in two
optical isomers. Very small quantities of enantiomerically pure opti-
cal isomers of KS119 synthesized from their respective enantiomeri-
cally pure chiral alcohols supplied by Vion Pharmaceuticals Inc.
were analyzed under identical HPLC conditions. Both the R and S
isomers of KS119 gave identical, two peak, HPLC traces indistin-
guishable from that produced by the racemic mixture (Figure 3).
HPLC traces of the racemic mixture, enantiomerically pure R-form
and enantiomerically pure S-form KS119WOH also gave equivalent
double peak HPLC traces (Figure 3).
Computational methodology for molecular
conformation prediction
A range of conformations of KS119S corresponding to different
energy minima were geometry-optimized using the M06 functional
(14) in combination with the 6-311 + G(2d,2p) basis set. The geome-
try optimizations were performed in aqueous bulk solvent modeled
using the C-PCM formalism (15,16). This is the implementation of
the conductor-like screening model COSMO (17) in the PCM frame-
work, with atomic radii optimized for COSMO-RS. Frequency calcu-
lations were performed at the same level of theory in bulk
solvation and it was confirmed that the optimized geometries corre-
spond to energy minima (no imaginary frequencies). In the evalua-
tion of data, the correction to the Gibbs free energy at 298 K was
added to the minimum energies in solvent. All calculations were
carried out in the GAUSSIAN 09 program (18).
Thermal equilibration
Purified KS119A, KS119B, KS119WOHA, and KS119WOHB were
heated in dilute solution. This resulted in the purified components
re-establishing the two peak HPLC pattern previously observed
under the appropriate conditions (Figure 5). Thus, heating either
KS119A or KS119B for 30 min at 100 ꢀC gave the equilibrium ratio
of KS119A:KS119B of approximately 43–57 regardless of the initial
concentration in the range tested (1–50 lM). This heat treatment
was too intense for the less chemically stable KS119WOH, as it
resulted in the complete decomposition of this compound. The pH
was decreased from ꢀ7.4 to 4.6 to chemically stabilize KS119WOH
and the experiment repeated. Equilibration of KS119WOHA and
KS119WOHB (the early- and late-eluting forms of KS119WOH,
respectively) was then observed with an approximate 45% loss of
material because of chemical decomposition. A study of the inter-
conversion of the conformers at physiologically relevant tempera-
tures and pH values was then undertaken. These studies involved
incubating the purified KS119A and KS119B in a largely aqueous
buffer of 82 mM potassium phosphate, pH ꢀ7.4 at 37 ꢀC, for peri-
ods of up to 10 days and following the proportions of each form by
HPLC; under these conditions, approximately 28–30 h were required
to reach the half equilibrium point in the case of KS119. The low
chemical stability of KS119WOH under these conditions complicated
the experiments which were not further pursued in detail; however,
the kinetics for the interconversion of the KS119WOH conformers
appeared to be similar to those of KS119. Time points from six sep-
arate KS119 equilibration experiments (three each for the conver-
sion of KS119A to KS119A ⁄ KS119B equilibrium mixture and
KS119B to KS119A ⁄ KS119B equilibrium mixture) were combined
Results
HPLC analysis of KS119
HPLC analysis of samples of chemically pure KS119 by elemental
analyses, while giving a single peak under short elution time con-
ditions (<15 min), gave a split peak under extended elution times
(35 min), and two partially overlapping peaks (at 83 and 86 min)
under relatively long elution times. The two components had rela-
tive peak areas of approximately 43 and 57 for the earlier eluting
(KS119A) and later eluting (KS119B) components, respectively (Fig-
ure 3). Material from the rising portion of the early peak and the
falling portion of the late peak was analyzed by mass spectros-
copy. The resulting mass spectra recorded for KS119A and KS119B
were essentially identical and contained the expected KS119
molecular ion sodium adduct peak of 466.25 (443.25 + 23), con-
firming the chemical equivalence of these two components (Fig-
ure 3).
Pure samples of PNBC synthesized in an identical manner, except
for the use of 4-nitrobenzyl alcohol in place of 1-(4-nitrophenyl)etha-
nol as a starting material, gave a single peak in all cases using
identical HPLC conditions (Figure 3).
Chem Biol Drug Des 2011; 78: 513–526
519

Penketh et al.
into a single figure (Figure 6) and compared with calculated curves.
These curves were derived from equations describing the concentra-
tion of KS119A and KS119B versus time, starting with pure KS119A
or pure KS119B, and assuming the conversion of KS119A to
KS119B and KS119B to KS119A were both first-order processes. The
first-order rate constants can be calculated from the time required to
reach the half equilibrium point. At pH ꢀ7.4 and 37 ꢀC, the calculated
first-order rate constants Ka and Kb were approximately 0.33 and
0.26 ⁄ day, respectively; these values corresponded to half lives of
50.4 h for KS119A and 64 h for KS119B. To obtain approximate val-
ues for the activation energies for these transitions, using the Arrhe-
nius equation, a similar series of experiments were performed at
65 ꢀC where half lives of 1.7 h for KS119A and 2.3 h for KS119B
were calculated (first-order rate constants Ka and Kb were approxi-
mately 0.4 and 0.3 ⁄ h, respectively, at 65 ꢀC). Activation energies of
ꢀ103 and ꢀ105 kJ ⁄ mol were calculated for the KS119A to KS119B
and KS119B to KS119A reactions, respectively. From the equilibrium
position, we determined the DGꢀ under the reaction conditions
employed for the KS119A to KS119B conversion to be )0.73 kJ ⁄ mol.
Comparison of the half-wave reduction
potentials (E1 ⁄ 2) of KS119 conformers and
PNBC
Differential pulse polarography (DPP) voltagrams of PNBC and
KS119 were also recorded; PNBC gave a single peak corresponding
to a E1 ⁄ 2 of about )453 mV, while two peaks were observed in the
case of KS119, corresponding to E1 ⁄ 2 values of approximately )415
and )575 mV (Figure 7). Both purified conformers gave almost iden-
tical two peak traces.
Relative conformer enzymic reduction rates
To determine whether the conformers of KS119 differed substan-
tially in their rates of reduction by two commonly used nitro reduc-
tase enzymes, NADPH:cytochrome P450 reductase and xanthine
oxidase, equilibrium mixtures of the conformers were incubated
with each enzyme system under oxygen-deficient conditions, and
samples were withdrawn at various time intervals and analyzed by
HPLC. Even at time points at which the total KS119 was largely
consumed, no significant changes were observed in the ratios of
the conformers in the remaining KS119 (Figure 8).
Partition coefficient differences between
conformers
The octanol:buffer partition coefficients were determined for
KS119A and KS119B and values of 139.9 € 1.2 (SEM) and
159.4 € 1.8 (SEM), respectively, were obtained. A proportionally lar-
ger difference was found in the partition coefficients of
KS119WOHA and KS119WOHB of 20.4 € 0.8 (SEM) and 24.2 € 0.9
(SEM), respectively.
Relative conformer reduction rates by intact
EMT6 cells
The conformer preference of intact EMT6 tumor cells under oxygen-
ated and oxygen-deficient conditions was also studied. This cell line
has a high level of total nitro reductase activity. Under oxygen-defi-
cient conditions, net KS119 loss occurred at ꢀ3.3· the oxygenated
Figure 6: Calculated KS119 conformer proportion curves versus time at 37 ꢀC assuming first-order kinetics for the interconversion of the
conformers (Ka = 0.33 ⁄ day and Kb = 0.26 ⁄ day corresponding to t1 ⁄ 2 values of 50.4 and 64 h for KS119A and KS119B, respectively) starting
with 100% KS119A conformer (early-eluting conformer) panel A, and 100% KS119B conformer (late-eluting conformer). Panel B. Experimental
points from six experiments (three starting with 100% KS119A and three experiments starting with 100% KS119B) are overlaid on these cal-
culated curves. Panel C, derivation of expressions for the concentrations of KS119A (A) and KS119B (B) versus time (starting with 100%
KS119B) and Ka from the half equilibrium time.
520
Chem Biol Drug Des 2011; 78: 513–526

Small Molecule Conformation and Activity
the KS119B conformer was also suggested, with the conformer
ratio increasing slightly to 0.80 over 3 h.
Relative cytotoxicity of KS119A and KS119B
toward EMT6 cells under oxygen-deficient
conditions
The cytotoxicities of the purified conformers against EMT6 cells
were measured in a clonal assay after a relatively short period of
drug exposure (4 h) to minimize problems of equilibration. These
experiments indicated that the more avidly reduced KS119B con-
former was more cytotoxic than the less readily reduced KS119A
conformer (Figure 10).
Computational chemistry-based conformations
Figure 11 displays two out of several conformations found for
KS119S. These two conformations display the largest differences in
structure (open ⁄ closed) and likely display the largest differences in
physical properties. Several other conformations corresponding to
additional energy minima on the potential energy surface were also
found, with energies between these two extreme cases.
Figure 7: Differential pulse polarograms of KS119 and PNBC.
Panel A, differential pulse polarogram of an equilibrium mixture of
KS119. Panel B, differential pulse polarogram of PNBC. Panel C, dif-
ferential pulse polarogram of KS119A (early-eluting conformer).
Panel D, differential pulse polarogram of KS119B (late-eluting con-
former).
Discussion
HPLC traces of pure KS119 displayed two peaks under slow elution
conditions (Figure 3). This was not observed with PNBC, a molecule
differing only by the absence of the methyl group that produces the
chiral center (Figure 2). Non-chiral columns, such as the C18
reverse-phase column used, are incapable of separating optical iso-
mers (19); therefore, features not solely relating to the chiral carbon
must be responsible. This was confirmed by HPLC of the pure opti-
cal isomers, both of which gave two peak traces identical to the
rate, with a strong preference for the late-eluting KS119B con-
former over the KS119A conformer (Figure 9). The starting
KS119A:KS119B ratio was approximately 0.75, but after 3 h under
oxygen-deficient conditions with 107 EMT6 cells ⁄ mL this ratio
increased to 3.25. Under oxygenated conditions, a preference for
A
B
Figure 8: The reduction of KS119 and lack of conformer preference exhibited by xanthine oxidase and NADPH:cytochrome P450 reductase
under oxygen-deficient conditions. Panel A, reduction of total KS119 and the contributions of KS119A (early-eluting conformer) and KS119B
(late-eluting conformer) to the total versus time by xanthine oxidase under oxygen-deficient conditions. The concentration ratio of the con-
formers at the different time points is given in the included table. Panel B, reduction of total KS119 and the contributions of KS119A (early-
eluting conformer) and KS119B (late-eluting conformer) to the total versus time by NADPH:cytochrome P450 reductase under oxygen-deficient
conditions. The concentration ratio of the conformers at the different time points is given in the included table.
Chem Biol Drug Des 2011; 78: 513–526
521

Penketh et al.
A
B
C
Figure 9: Conformer selective metabolism of KS119 by EMT6 carcinoma cells. Panel A, upper row shows typical HPLC traces of samples
taken from oxygenated incubations of 20 lM KS119 with EMT6 cells at 107 cells ⁄ mL at 0, 1, 2 and 3 h; lower row shows typical HPLC traces
of samples taken from oxygen-deficient incubations of 20 lM KS119 with EMT6 cells at 107 cells ⁄ mL at 0, 1, 2, and 3 h. Panels B and C are
graphical representations of the data in panel A. Panel B, plots of the concentrations (mean of three experiments) of total KS119 (initial con-
centration ꢀ20 lM) and the contributions of KS119A (early-eluting conformer) and KS119B (late-eluting conformer) to the total versus time
under oxygenated conditions during incubations with EMT6 cells at 107 cells ⁄ mL. Panel C, plots of the concentrations (mean of three experi-
ments) of total KS119 (initial concentration ꢀ20 lM) and the contributions of KS119A (early-eluting conformer) and KS119B (late-eluting con-
former) to the total versus time under oxygen-deficient conditions during incubations with EMT6 cells at 107 cells ⁄ mL.
racemic mixture (Figure 3). Identical phenomena were observed with
the optical isomers and the racemic mixture of a related molecule,
KS119WOH (Figure 3). LCMS gave identical mass spectra for
KS119A and KS119B confirming their equivalent composition and
that the two peaks were not a consequence of the reversible for-
mation of an unanticipated covalent adduct with a solvent or buffer
component. Furthermore, purified KS119A and KS119B thermally re-
equilibrated at moderate temperatures (Figure 5). This could not
occur with chiral carbon–based optical isomers of the structure type
present in KS119 and KS119WOH. If equilibration was possible, a
50:50 rather than a 43:57 ratio would result, as optical isomers
have identical thermodynamic properties (20). The kinetics of equili-
bration closely matched our model, which assumes that both com-
ponents interconvert with first-order kinetics, with half-lives at
37 ꢀC and pH 7.4 of 50.4 and 64 h for KS119A and KS119B,
respectively (Figure 6).
The chemical stability of KS119 and KS119WOH is partly dependent
upon the electron withdrawing effects of the nitro group; reduction
to an electron-donating group results in molecular fragmentation
(Figure 1) (1). The phenolic hydroxyl group in KS119WOH exists lar-
gely as an anion at physiological pH, with the negative charge
being delocalized to the nitro group attenuating its stabilizing
actions. At pH 7.4 and 37 ꢀC, the chemical half-life of KS119WOH
is less than that for the conformational interconversions. Dropping
the pH to 4.6 stabilizes KS119WOH sufficiently to observe confor-
mational equilibration (Figure 5).
Space filling molecular models of PNBC allow free rotation of the
nitrobenzyl and chloroethyl bis(sulfonyl)hydrazine moieties with
respect to each other and the linker domain, whereas in models of
KS119, it is more hindered because of the steric interference pro-
duced by the bulky methyl group in the linker region (Figure 4). This
522
Chem Biol Drug Des 2011; 78: 513–526

Small Molecule Conformation and Activity
may help confine KS119 to two major spatial conformations or
atropisomers. Atropisomers are usually the consequence of hindered
rotation around a bond because of the presence of bulky neighbor-
ing substituents and or the bond possessing partial double bond
character. To be defined as an atropisomer, the conformational
forms must have half-lives of greater than 1000 seconds under
ambient conditions (21–23). Atropisomerism is becoming an
emerging problem for the pharmaceutical industry as examples with
half-lives as long as 1000 years, differing in biological activity by
hundreds of fold have been discovered (24,25). The consequences
of hindered rotation can be somewhat similar to traditional chiral
centers and can give rise to geometrical diastereoisomers which
have different physical properties. Symmetrization can be used to
eliminate chiral axes (as well as chiral centers) and diastereoiso-
meric atropisomers can be frequently prevented using this strategy
(24). A loss of diastereoisomeric atropisomerism as a consequence
of symmetrization probably explains the single HPLC peak observed
for PNBC. Peptide type bonds can have partial double bond charac-
ter owing to the lone electron pair on the amino group being delo-
calized to the carbonyl group; this produces two possible
conformations corresponding to cis and trans isomers (26). In
KS119, a carbonyl group is attached to a hydrazine nitrogen and a
Figure 10: Cytotoxicity of KS119A and KS119B to EMT6 carci-
noma cells after a 4-h exposure under conditions of oxygen defi-
ciency. Cells were treated with either 0, 5, or 10 lM KS119A or
KS119B conformer in medium for 4 h at 37 ꢀC under conditions of
oxygen deficiency. After 4 h of treatment, the cells were washed
free of agent, detached by trypsinization, and plated at densities of
1 · 102, 1 · 103, and 1 · 104 cells per well. Ten days later, colo-
nies were quantified and the surviving percentage calculated. The
data represent the arithmetic means of three experiments € the
standard error values.
A
B
Figure 11: Computer-predicted
open and closed conformations of
KS119 and schematic representing
the different KS119 forms and
conformations. Panel A, computer-
predicted three-dimensional
conformations of the open and
closed forms of KS119S
corresponding to calculated stable
energy minima. Panel B, Schematic
of the four forms of KS119
indicating the mirror image pairs
(1,2) and (3,4) with identical
physical properties and the non-
superimposable pairs (1,3) and (2,4)
with dissimilar physical properties.
Chem Biol Drug Des 2011; 78: 513–526
523

Penketh et al.
similar situation could be envisioned resulting in restricted rotation
at this bond. However, unlike the peptide bond case, the nitrogen
lone pair is not freely available for delocalization to the carbonyl
oxygen because of a highly electron withdrawing methylsulfonyl
neighboring group. An illustration of this effect is the fact that the
N–H group in 90CE is a weak acid (9) rather than being basic. The
bond attaching the oxygen to the opposite side of the carbonyl
group with respect to the nitrogen has the potential to exhibit par-
tial double bond character because of electron lone pair delocaliza-
tion, permitting two stable axial configurations which could give
rise to diastereoisomeric atropisomers in the case of KS119. In this
case, the electron-deficient nitrogen would promote this effect. We
have performed some computer-based conformational modeling con-
sistent with this hypothesis. Two out of several predicted conforma-
tions were of particular interest as these conformations displayed
the largest differences in stable structure (open ⁄ closed) which
would likely result in measurable differences in partition and HPLC
behavior. Other conformations corresponding to additional energy
minima on the potential energy surface were also found, with ener-
gies and structure in-between the two favored predictions. These
calculated open ⁄ closed conformations are illustrated in Figure 11,
together with a graphic illustrating how a KS119 racemic mixture
and both its pure R and S forms could all appear to be mixtures of
molecules with the same two sets of dissimilar physical properties.
Although additional stabilization because of partial double bond
character is not possible at the N–N bond of the hydrazine moiety,
making conformational forms at this position less likely, sterically
restricted bond rotation because of the presence of four bulky sub-
stituents could still be possible, resulting in diastereoisomeric atrop-
isomers because of the presence of the chiral carbon in KS119. If
the two forms represented an equilibrium between slowly dissociat-
ing dimers and slowly associating monomers, the equilibrium posi-
tion would be expected to change with concentration rather than
be fixed, and only the dissociation would be expected to show
first-order kinetics. Furthermore, upon heating, dissociation would
be likely, resulting in an increase in monomer concentration, rather
than a re-equilibration. A much larger HPLC separation would also
be expected. Therefore, we believe the two distinct forms of KS119
are atropisomers arising purely because of the large activation
energy of ꢀ100 kJ ⁄ mol required to circumvent steric ⁄ electronic
hindrance barriers to rotation probably around the oxygen to car-
bonyl moiety bond. X-Ray crystallography of the purified atropisom-
ers may be required to determine unambiguously the precise
structural differences between these forms.
the transition would also be influenced by the solvent. As the octa-
nol:water partition coefficient has a major influence on absorption,
distribution, target binding, metabolism, and excretion, conformers
would exhibit some differences in biological activity in vivo purely
from these differences. The influence of the octanol:water partition
coefficient on the therapeutic index of nitrosourea alkylating agents
has been reported and the nitrosoureas with the greatest partition
coefficients tended to have the greatest therapeutic indices (27).
The DPP voltagram of PNBC gave a single peak corresponding to
an E1 ⁄ 2 of )453 mV, whereas that of KS119 gave two peaks corre-
sponding to E1 ⁄ 2 values of approximately )415 and )575 mV (Fig-
ure 7). Initially, we suspected that these two peaks corresponded to
the two conformations, with the large difference in their E1 ⁄ 2 val-
ues being because of the nitro groups being in very different local
environments. However, this did not appear to be the case, as both
purified conformers of KS119 gave almost identical two peak
traces. The E1 ⁄ 2 values of approximately )415 and )453 mV
undoubtedly correspond to the reduction of the nitro group of
KS119 and PNBC, respectively. The additional peak (E1 ⁄ 2 )575 mV)
seen with KS119 is not the expected consequence of the addition
of an electrochemically inert methyl group on the linker region, but
could correspond to the reduction of a linker component favored by
steric strain.
The enzymes NADPH:cytochrome P450 reductase and xanthine oxi-
dase did not appear to differ in their KS119 conformer preference.
Even when the total KS119 was largely consumed, no significant
change in the ratio of the conformers in the residual KS119 was
seen (Figure 8). This contrasts sharply with the EMT6 cell experi-
ments where a strong preference (>sevenfold) for KS119B was
observed. The starting KS119A:KS119B ratio was approximately
0.75, but after 3 h under oxygen-deficient conditions, this ratio
increased to 3.25 compared to 0.80 under oxygenated conditions,
where the net metabolism was much less because of back oxida-
tion. Several factors may be involved in this preference: (i) selective
uptake of KS119B or export of KS119A, leading to a greater con-
centration of KS119B within the cell and increased enzymatic reduc-
tion ⁄ metabolism, (ii) selective enzymatic reduction of the KS119B;
even though xanthine oxidase and NADPH:cytochrome P450 reduc-
tase showed no real conformer preference, other reducing systems
with conformer preference might be operative, or (iii) a combination
of these effects. If these agents entered cells solely by passive dif-
fusion, the more hydrophobic KS119B would be expected to enter
cells somewhat faster than KS119A and this could account for at
least a portion of the selectivity.
Differences in the octanol:buffer partition coefficients of approxi-
mately 14% and 19% were measured for the conformers of KS119
and KS119WOH, respectively. The proportionally greater difference
in the partition coefficients of KS119WOH conformers probably
explains their superior HPLC separation. These differences must
reflect a change in the relative solvent exposed hydrophilic and
hydrophobic surface areas between the two conformations and
would be consistent with the computer-predicted forms, with
KS119A and KS119B corresponding to the closed and open struc-
tures, respectively. Such differences also mean that the equilibrium
position would be somewhat dependent on the solvent (something
we have observed); furthermore, if the transition state involved a
change in the exposed hydrophobic areas, the activation energy for
A mechanism involving the direct conversion of KS119B to KS119A
by the cell is thought to be unlikely, as the ratio changed little
under oxygenated conditions when there was little net metabolism.
The concentration and ratio changes appear to be consistent with
the preferential loss of KS119B. The preferential reductive activa-
tion of KS119B would be expected to result in KS119B expressing
more cytotoxicity than KS119A. Therefore, we measured the cyto-
toxicity to EMT6 cells of the purified conformers in a clonal assay
(Figure 10). It is clear that KS119B is significantly more cytotoxic
than KS119A; this difference is particularly apparent at the highest
concentrations. Based upon the conformer preference observed in
524
Chem Biol Drug Des 2011; 78: 513–526

Small Molecule Conformation and Activity
Figure 9, one might have expected to see an even larger difference
in the relative cytotoxicities. However, these two experimental pro-
cedures have important differences; in the conformer preference
experiment, the conformers are competing for metabolism ⁄ uptake,
whereas in the differential cytotoxicity experiments, the cells were
exposed to each purified individual conformer in the absence of a
competitor. If uptake ⁄ metabolism of both conformers occurred by
the same proteins, it is likely that the uptake ⁄ metabolism of the
less preferred KS119A would be greater in the absence of compet-
ing KS119B, leading to a smaller fold differential in experiments
where the conformers were tested individually. Furthermore, some
degree of equilibration of the conformers will occur in these experi-
ments, resulting in a smaller differential.
oxycarbonyl derivatives of 1,2-bis(methylsulfonyl)-1-(2-chloroeth-
yl)hydrazines. J Med Chem;42:941–946.
2. Seow H.A., Penketh P.G., Shyam K., Rockwell S., Sartorelli A.C.
(2005) 1,2-Bis(methylsulfonyl)-1-(2-chloroethyl)-2-[[1-(4-nitrophe-
nyl)ethoxy]carbonyl]hydrazine: an anticancer agent targeting hyp-
oxic cells. Proc Natl Acad Sci USA;102:9282–9287. PMCID:
PMC1166587.
3. Vaupal P., Harrison L. (2004) Tumor hypoxia: causative factors,
compensatory mechanisms, and cellular response. Oncolo-
gist;9:4–9.
4. Moulder J.E., Rockwell S. (1987) Tumor hypoxia: its impact on
cancer therapy. Cancer Metastasis Rev;5:313–341.
5. Vaupel P.W., Frinak S., Bicher H.I. (1981) Heterogeneous oxygen
partial pressure and pH distribution in C3H mouse mammary
adenocarcinoma. Cancer Res;41:2008–2013.
Conclusions and Future Directions
6. Green S.L., Giaccia A.J. (1998) Tumor hypoxia and the cell cycle:
implications for malignant progression and response to therapy.
Cancer J Sci Am;4:218–223.
7. Bristow R, Hill R (2008) Hypoxia. DNA repair and genetic insta-
bility. Nature;8:180–192.
8. Workman P., Stratford I.J. (1993) The experimental development
of bioreductive drugs and their role in cancer therapy. Cancer
Metastasis Rev;12:73–82.
9. Penketh P.G., Shyam K., Sartorelli A.C. (1994) Studies on the
mechanism of decomposition and structural factors affecting the
aqueous stability of 1,2-bis(sulfonyl)-1-alkylhydrazines. J Med
Chem;37:2912–2917.
The unusually high stability (half-life >2 days at physiological pH
and temperature) of the two conformational forms ⁄ atropisomers of
KS119 allowed them to be treated and characterized as two distinct
compounds with differing physical and biological properties. Thus,
manipulation of the conformer ratio could conceivably have thera-
peutic benefits. For example, enrichment for KS119B may increase
potency if KS119 was normally cleared rapidly. Alternatively, enrich-
ing for the more poorly metabolized KS119A may provide more time
for distribution into oxygen-deficient regions where eventual equili-
bration could generate the more potent KS119B.
10. Penketh P.G., Baumann R.P., Ishiguro K., Shyam K., Seow H.A.,
Sartorelli A.C. (2008) Lethality to leukemia cell lines of DNA in-
terstrand cross-links generated by cloretazine derived alkylating
species. Leukemia Res;32:1546–1553. PMCID: PMC2888535.
11. Bianco P., Haladjian J. (1977) Study of two forms of ferredoxin
from desulfovibrio gigas by differential pulse polarography. Bio-
chem Biophys Res Commun;78:323–327.
12. Baumann R.P., Penketh P.G., Seow H.A., Shyam K., Sartorelli
A.C. (2008) Generation of oxygen deficiency in cell culture using
a two-enzyme system to evaluate agents targeting hypoxic
tumor cells. Radiation Res;170:651–660. PMCID: PMC2667281.
13. Belcourt M.F., Hodnick W.F., Rockwell S., Sartorelli A.C. (1998)
The intracellular location of NADH:cytochrome b5 reductase
modulates the cytotoxicity of the mitomycins to Chinese hamster
ovary cells. J Biol Chem;273:8875–8881.
14. Zhao Y., Truhlar D.G. (2008) The M06 suite of density functionals
for main group thermochemistry, thermochemical kinetics, nonco-
valent interactions, excited states, and transition elements: two
new functionals and systematic testing of four M06-class func-
tionals and 12 other functionals. Theor Chem Acc;120:215–241.
15. Barone V., Cossi M. (1998) Quantum calculation of molecular
energies and energy gradients in solution by a conductor solvent
model. J Phys Chem;102:1995–2001.
16. Cossi M., Rega N., Scalmani G., Barone V. (2003) Energies,
structures, and electronic properties of molecules in solution
with the C-PCM solvation model. J Comput Chem;24:669–681.
17. Klamt A., Schꢁꢁrmann G. (1993) COSMO: a new approach to
dielectric screening in solvents with explicit expressions for the
The role of different three-dimensional conformations in simple mol-
ecules is not usually given much consideration, largely because of
the normally rapid free rotation around single bonds and the lack of
strong stabilizing interactions between different regions of small
molecules precludes stable energy minima at normal temperatures.
While KS119 may represent an unusual case, the present work
supports the earlier findings of other examples of stable conforma-
tions with different biological activities in relatively small molecules
(24). In the case of KS119, small differences in their chromato-
graphic properties alerted us to their presence; however, atropisom-
ers ⁄ conformers could occur in other small molecules without readily
discernible differences in chromatographic properties under many
conditions. Thus, atropisomers ⁄ conformers could go largely unde-
tected despite potentially having large differences in their biological
properties that may contribute toward experimental variability or
biphasic ⁄ multiphasic responses.
Acknowledgments
This work was supported in part by US Public Health Service Grants
CA-090671, CA-122112 and CA-129186 from the National Cancer Insti-
tute, and a Grant from the National Foundation for Cancer Research.
References
screening energy and its gradient.
Trans;2:799–805.
J
Chem Soc Perkin
1. Shyam K., Penketh P.G., Shapiro M., Belcourt M.F., Loomis R.H.,
Rockwell S., Sartorelli A.C. (1999) Hypoxia-selective nitrobenzyl-
Chem Biol Drug Des 2011; 78: 513–526
525

Penketh et al.
18. Frisch M.J., Trucks G.W., Schlegel H.B., Scuseria G.E., Robb
M.A., Cheeseman J.R., Scalmani G. et al. (2009) Gaussian 09
Revision A.02. Wallingford, CT: Gaussian, Inc.
24. Clayden J., Moran W.J., Edwards P.J., LaPlante S.R. (2009) The
challenge of atropisomerism in drug discovery. Angew Chem Int
Ed;48:6398–6401.
19. Lochmꢁllera C.H., Soute R.W. (1975) Chromatographic resolution
of enantiomers : selective review. J Chromatogr A;113:283–302.
20. Stoker H.S. (2008) Chapter 18, Carbohydrates, In: Kennedy K.
and Berardy R., editors. General Organic and Biological Chemis-
try, 5th edition. Belmont, CA: Brooks ⁄ Cole , USA; p. 567–568.
21. Oki M. (1983) Application of dynamic NMR spectroscopy to
organic chemistry. Top Stereochem;14:1–81.
25. Eveleigh P., Hulme E.C., Schudt C., Birdsall N.J. (1989) The exis-
tence of stable enantiomers of telenzepine and their stereose-
lective interaction with muscarinic receptor subtypes. Mol
Pharmacol;35:477–483.
26. Plusquellic D.F., Pratt D.W. (2007) Probing the electronic struc-
ture of peptide bonds using methyl groups. J Phys Chem A;111:
7391–7397.
22. Bringmann G., Mortimer A.J.P., Keller P.A., Gresser M.J., Garner
J., Breuning M. (2005) Atroposelective synthesis of axially chiral
biaryl compounds. Angew Chem;117:5518–5563.
23. Bringmann G., Mortimer A.J.P., Keller P.A., Gresser M.J., Garner
J., Breuning M. (2005) Atroposelective synthesis of axially chiral
biaryl compounds. Angew Chem Int Ed;44:5384–5427.
27. Wheeler G.P., Bowdon B.J., Grimsley J.A., Lloyd H.H. (1974)
Interrelationships of some chemical, physicochemical and biolog-
ical activities of several 1-(2-haloethyl)-1-nitrosoureas. Cancer
Res;34:194–200.
526
Chem Biol Drug Des 2011; 78: 513–526