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