Requirements for mammalian carboxylesterase inhibition by substituted ethane-1,2-diones

Article abstract of DOI:10.1016/j.bmc.2011.06.012

Carboxylesterases (CE) are ubiquitous enzymes found in both human and animal tissues and are responsible for the metabolism of xenobiotics. This includes numerous natural products, as well as a many clinically used drugs. Hence, the activity of these agents is likely dependent upon the levels and location of CE expression. We have recently identified benzil is a potent inhibitor of mammalian CEs, and in this study, we have assessed the ability of analogues of this compound to inhibit these enzymes. Three different classes of molecules were assayed: One containing different atoms vicinal to the carbonyl carbon atom and the benzene ring [PhXC(O)C(O)XPh, where X = CH₂, CHBr, N, S, or O]; a second containing a panel of alkyl 1,2-diones demonstrating increasing alkyl chain length; and a third consisting of a series of 1-phenyl-2-alkyl-1,2-diones. In general, with the former series of molecules, heteroatoms resulted in either loss of inhibitory potency (when X = N), or conversion of the compounds into substrates for the enzymes (when X = S or O). However, the inclusion of a brominated methylene atom resulted in potent CE inhibition. Subsequent analysis with the alkyl diones [RC(O)C(O)R, where R ranged from CH₃ to C₈H₁₇] and 1-phenyl-2-alkyl-1,2-diones [PhC(O)C(O)R where R ranged from CH₃ to C₆H₁₃], demonstrated that the potency of enzyme inhibition directly correlated with the hydrophobicity (c log P) of the molecules. We conclude from these studies that that the inhibitory power of these 1,2-dione derivatives depends primarily upon the hydrophobicity of the R group, but also on the electrophilicity of the carbonyl group.

Full text of DOI:10.1016/j.bmc.2011.06.012

Bioorganic & Medicinal Chemistry 19 (2011) 4635–4643
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Requirements for mammalian carboxylesterase inhibition by substituted
ethane-1,2-diones
Elizabeth I. Parkinson a,b, M. Jason Hatfield a, Lyudmila Tsurkan a, Janice L. Hyatt a, Carol C. Edwards a,
Latorya D. Hicks a, Bing Yan a, Philip M. Potter a,
⇑
a Department of Chemical Biology and Therapeutics, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105-2794, USA
b Rhodes College, 2000 N Parkway, Memphis, TN 38112, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Carboxylesterases (CE) are ubiquitous enzymes found in both human and animal tissues and are
responsible for the metabolism of xenobiotics. This includes numerous natural products, as well as a many
clinically used drugs. Hence, the activity of these agents is likely dependent upon the levels and location of
CE expression. We have recently identified benzil is a potent inhibitor of mammalian CEs, and in this study,
we have assessed the ability of analogues of this compound to inhibit these enzymes. Three different clas-
ses of molecules were assayed: One containing different atoms vicinal to the carbonyl carbon atom and the
benzene ring [PhXC(O)C(O)XPh, where X = CH2, CHBr, N, S, or O]; a second containing a panel of alkyl 1,2-
diones demonstrating increasing alkyl chain length; and a third consisting of a series of 1-phenyl-2-alkyl-
1,2-diones. In general, with the former series of molecules, heteroatoms resulted in either loss of inhibi-
tory potency (when X = N), or conversion of the compounds into substrates for the enzymes (when
X = S or O). However, the inclusion of a brominated methylene atom resulted in potent CE inhibition. Sub-
sequent analysis with the alkyl diones [RC(O)C(O)R, where R ranged from CH3 to C8H17] and 1-phenyl-2-
alkyl-1,2-diones [PhC(O)C(O)R where R ranged from CH3 to C6H13], demonstrated that the potency of
enzyme inhibition directly correlated with the hydrophobicity (c log P) of the molecules. We conclude
from these studies that that the inhibitory power of these 1,2-dione derivatives depends primarily upon
the hydrophobicity of the R group, but also on the electrophilicity of the carbonyl group.
Received 21 March 2011
Revised 27 May 2011
Accepted 2 June 2011
Available online 4 July 2011
Keywords:
Carboxylesterase
Inhibitor
Ethane-1,2-diones
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
nous substrates have not been definitively identified, the patterns of
expression are consistent with them playing a protective role. Fur-
Carboxylesterases (CE ) are enzymes found in a wide range of
organisms, from humans to bacteria.1 CEs are known to be involved
in the hydrolysis of ester-containing xenobiotics1 using a catalytic
serine within a Ser-His-Glu triad to initiate hydrolysis of the mole-
cule. The products that result from this reaction are the respective
alcohol and carboxylic acid.2,3 Two major CEs exist in humans, hu-
man liver CE (hCE1; CES1) and human intestinal CE (hiCE; CES2)2,3
with hCE1 being primarily expressed in the liver, while hiCE is found
in both the liver and the small intestine. A third human CE, hBr3
(CES3), has been described, but very little is known about the levels
of expression and/or the substrate specificity of this enzyme.4 While
the exact role of these proteins in mammals is unclear, and endoge-
thermore, since the carboxylic ester chemotype is present in numer-
ous agents including natural products, pesticides and clinically used
drugs, de facto they a substrates for these enzymes.5–10 Hence drug
hydrolysis, which can result in either activation or inactivation of the
molecule, is dependent upon the levels of CE expressed in exposed
tissues and the substrate specificity of the protein.
One such chemotherapeutic agent that is metabolized by CEs is
the anticancer drug irinotecan (CPT-11, 7-ethyl-10-[4-(1-piperidi-
no)-1-piperidino]carbonyloxycamptothecin6,9,11). CPT-11 is a car-
bamate-derived prodrug that is hydrolyzed by hiCE into its active
metabolite, SN-38 (7-ethyl-10-hydroxycamptothecin).12,13 The lat-
ter is a potent topoisomerase I poison which exerts its toxicity at
low nanomolar concentrations. We have previously demonstrated
the efficient activation of CPT-11 by a rabbit liver CE (rCE) and used
this enzyme to modulate tumor cells sensitivity to this drug.14–16
The development of clinical approaches using this technology is
currently underway. However, the toxicity of CPT-11 (delayed diar-
rhea) is in part due to high levels of hiCE that are expressed in the
intestine.6,17 Therefore, identifying specific hiCE inhibitors which
could be used in combination with CPT-11 to ameliorate the delayed
diarrhea, may have clinical utility.2,3 Previously, we determined that
⇑
Corresponding author. Tel.: +1 901 595 2825; fax: +1 901 595 4293.
E-mail address: phil.potter@stjude.org (P.M. Potter).
Abbreviations: CE, carboxylesterase CPT-11 - Irinotecan, 7-ethyl-10-[4-(1-piperi-
dino)-1-piperidino]carbonyloxycamptothecin; hAChE, human acetylcholinesterase;
hBChE, human butyrylcholinesterase; hBr3, human brain carboxylesterase; hCE1,
human carboxylesterase 1; hiCE, hCE2, human intestinal carboxylesterase; HPLC, high
performance liquid chromatography; 4-MUA, 4-methylumbelliferone acetate; o-NPA,
nitrophenyl acetate; rCE, rabbit liver carboxylesterase; SN-38, 7-ethyl-10-
hydroxycamptothecin.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.06.012

4636
E. I. Parkinson et al. / Bioorg. Med. Chem. 19 (2011) 4635–4643
small molecules containing the ethane 1,2-dione moiety were po-
tent inhibitors of CEs.18–21 These compounds demonstrated no
activity toward human acetyl- or butyrylcholinesterase, and one
class, the benzils, inhibited hiCE intracellularly and modulated cel-
lular response to CPT-11.22 Preliminary studies indicated that the
planarity of the ethane-1,2-dione group could determine specificity
of enzyme inhibition and that that inhibitor potency was increased
when phenyl groups were present within the molecule.
tane-2,3-dione (8), hexane-3,4-dione (9), and 1-phenyl-1,2-prop-
anedione (17) were obtained from Sigma Aldrich. Diphenyl
ethanedioate (4), 1,4-diphenylbutane-2,3-dione (5), 1,4-dibromo-
1,4-diphenylbutane-2,3-dione (6) and 1-bromo-1,4-diphenylbu-
tane-2,3-dione (7) were purchased from MDPI (Basel, Switzerland).
Octane-4,5-dione (10) was obtained from the Developmental Ther-
apeutics Program at NIH and 1,2-dicyclohexylethane-1,2-dione
(16) was synthesized as previously described.20,23 The structures
of the compounds described in this paper are illustrated in Table 1.
For compounds synthesized as part of these studies, melting
points were determined using a Mel-temp (Barnstead Interna-
tional, Dubuque, IA) and purity and structures were assessed by
TLC, NMR, HRMS and total C, H, N, O, S analysis (as appropriate).
Complete physical data is presented as Supplementary data.
In this study, we sought to determine the chemical require-
ments for inhibition of CEs by ethane 1,2-diones and to assess
whether nucleophilic attack by the serine Oc atom within the ac-
tive site is the mechanism by which enzyme inhibition occurs. This
included evaluating the role of the atoms immediately adjacent to
the carbonyl groups towards inhibitor potency and to assess the
necessity for the inclusion of the phenyl rings. Our results indicate
that the atoms bonded to the carbonyl groups in the 1,2-diones
play a major role towards inhibitor potency, both by moderating
carbonyl electrophilicity, and compound hydrophobicity. Indeed,
molecules with c log P >2.75 were much potent inhibitors of mam-
malian CEs than more hydrophilic compounds (c log P <2.75). Fur-
thermore, aromaticity within the molecule is not a requirement for
enzyme inhibition.
2.2. Enzymes
Pure hCE1, hiCE, and rabbit liver CE (rCE) were obtained by
purification from baculovirus infected Spodoptera frugiperda cells
as previously described.24 Human acetylcholinesterase (hAChE)
and butyrylcholinesterase (hBChE) were obtained from Sigma Bio-
chemicals (St. Louis, MO).
2.3. Synthesis of oxanilide (N,N0-diphenylethanediamide (2))
2. Materials and methods
Oxanilide was synthesized from aniline and oxalyl chloride as
previously described.25 Physical parameters were consistent with
that reported previously. Identity was confirmed by melting point,
NMR, and elemental analysis.
2.1. Chemicals and general chemistry
All solvents and starting materials were purchased from Sigma
Aldrich (St. Louis, MO) and were ACS grade or better. Benzil (1), bu-
Table 1
Structures, enzyme inhibition constants and kinetic parameters for the aromatic ethane-1,2-diones
ID Name
Structure
c log P
Ki (nM)
hiCE irreversible
inhibition (%)
hCE1
hiCE
rCE
hAChE
hBChE
O
1
Benzil
3.02
15 1.9a
45 3.2a
103 19a
>100,000 >100,000 <8
O
O
NH
2
3
4
5
Oxanilide
2.24
3.62
3.39
3.19
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000 >100,000 ND
>100,000 >100,000 ND
>100,000 >100,000 ND
>100,000 >100,000 ND
NH
O
O
S
S1,S2-Diphenyl ethane bis(thioate)
Diphenyl ethanedioate
1,4-Diphenylbutane-2,3-dione
S
O
O
O
O
O
O
3080 230 21,610 2270 >100,000
O
Br
O
1,4-Dibromo-1,4-diphenyl butane-
2,3-dione
6
4.07
3.71
107 21
334 25
20.5 3.3
>100,000 >100,000 <5
O
Br
O
Br
1-Bromo-1,4-diphenyl butane-
2,3-dione
7
2630 70
267 38
3680 320 >100,000 >100,000 ND
O
a
Data taken from Wadkins et al.21

E. I. Parkinson et al. / Bioorg. Med. Chem. 19 (2011) 4635–4643
4637
2.4. Synthesis of S1,S2-diphenyl ethanebis(thioate) (3)
2.9. Irreversible enzyme inhibition assays
The named compound was synthesized by condensation of
thiophenol with oxalyl chloride in dichloromethane.26 Identity
was confirmed by melting point, NMR, and elemental analysis.
To assess irreversible enzyme inhibition by selected com-
pounds, inhibitors were added to hiCE at a final concentration of
10 lM in Hepes pH 7.4 and incubated on ice for 40 min. At this
time, samples were diluted 1:500 and enzyme activity was deter-
mined using o-NPA as a substrate.
2.5. Synthesis of alkyl diones and phenyl alkyl diones
2.10. Enzyme inhibition assays and determination of Ki values
using CPT-11 as a substrate
Alkyl diones (11–15) were synthesized by oxidation of the cor-
responding alkyne using potassium permanganate in an acetone/
water phase transfer system.27 Routinely, 10.8 mM 1-phenyl-1-al-
kyne dissolved in 420 ml acetone was added to 240 ml aqueous
32 mM NaHCO3 and 22 mM MgSO4. Potassium permanganate
(6.65 g, 42 mmol) was added in one portion and the reaction stir-
red for 4 hours at rt. Small portions of NaNO2 and 10% H2SO4 were
added until the reaction was clear (ꢀ5 g and 50 ml, respectively)
and solid NaCl was then added to achieve saturation. Samples
were worked up using standard approaches and compound iden-
tity was confirmed by 1H NMR, elemental analysis (Microlab Inc.,
Norcross, GA), and HRMS (UIUC School of Chemical Sciences, Ur-
bana, IL). Typically yields were greater than 70% and compound
physical parameters were consistent with those previously
reported.
CPT-11 (20
lM) and inhibitor concentrations ranging from 0 to
100 M were pre-incubated for 2 min at 37 °C in 50 mM HEPES pH
l
7.4. Purified hiCE (25 U) was then added and the sample was incu-
bated at 37 °C for 5 min. The reaction was terminated by addition
of 100 ll of acid methanol and the amount of SN-38 in the sample
was determined using high performance liquid chromatogra-
phy.14,36 Ki values were then determined using Prism software as
described earlier.
2.11. Determination of ability of compounds to act as substrates
Compounds 2, 3, or 4 (200
varying time periods (1, 5, or 20 min) at either rt or 37 °C in a total
volume of 200 l of 50 mM Hepes pH 7.4 and reactions were termi-
lM) were incubated with enzyme for
l
2.6. c log P calculations
nated by addition of acetonitrile. Samples were then evaluated by
reverse-phase high-performance liquid chromatography and prod-
ucts were identified by UV detection. These results were compared
to samples without enzyme and to samples with marker com-
pounds consisting of aniline, thiophenol, or phenol.
c log P values were calculated using ChemSilico Predict v2.0
software (ChemSilico LLC, Tewksbury, MA).
2.7. Determination of Ki values using o-nitrophenyl acetate
(o-NPA) as a substrate
2.12. Intracellular CE inhibition assay
Inhibition of CEs was assessed using a multiwell plate spectro-
Intracellular CE inhibition was assessed using the substrate 4-
methylumbelliferone acetate (4-MUA) as previously described.22
Briefly, 106 cells were suspended in PBS with stirring in a cuvette
photometric assay with 3 mM o-NPA as a substrate.21,28 Inhibitors
(100 lM) and substrate were aliquoted into wells of a 96-well
plate and enzyme was added. The rate of change in absorbance
at 420 nm was recorded at 15 s intervals for 5 min and data was
compared to wells without inhibitor. Data was expressed as the
yield of o-nitrophenol per minute per milligram of protein. Com-
pounds demonstrating greater than 50% reduction of CE activity
and were pre-incubated with inhibitor (10
lM) for 1 h. 4-MUA
was then added (100 M) and fluorescence was determined using
l
a Hitachi F-2000 fluorimeter, with an excitation wavelength of
365 nm and an emission wavelength of 460 nm. Data was collected
over 2 min and the percentage of inhibition of product formation
as compared to cell not treated with inhibitor was determined by
assessing the difference in fluorescence intensity at 30 s.
were re-screened with dilutions ranging from 1 pM to 100
Data was fitted to the following equation to determine the mode
of inhibition29
lM.
:
2.13. Growth inhibition assay
½Iꢁf½sꢁð1 ꢂ bÞ þ Ksð
a
ꢂ bÞg
Ksg
i ¼
½Iꢁf½sꢁ þ Ksg þ Kif ½sꢁ þ
a
a
a
Growth inhibition assays using inhibitors and CPT-11 were
undertaken as previously described.15,22,37 Briefly, cells were pla-
ted in 6-well plates (50,000 cells/well) and allowed to adhere over-
where i is the fractional inhibition, [I] is the inhibitor concentration,
[s] is the substrate concentration, is the change in affinity of the
a
substrate for enzyme, b is the change in the rate of enzyme sub-
strate complex decomposition, Ks is the dissociation constant for
the enzyme substrate complex, and Ki is the inhibitor constant.
night. Following pre-incubation with inhibitor (10
cells were treated for 2 h with both inhibitor and varying concen-
trations of CPT-11 (0–100 M). Cells were then washed and al-
lM) for 1 h,
l
Examination of the curve fits where
a
ranged from 0 to 1 and b ran-
lowed to grow in drug-free medium for 4 days (ꢀ3 cell
doublings), prior to harvesting and counting using a Coulter Z2
counter (Beckman Coulter, Fullerton, CA). Routinely, all concentra-
tions were conducted in triplicate and the percentage of surviving
cells was determined for each treatment condition and compared
to control cells not exposed to inhibitor. Survival curves were gen-
erated and IC50 values were calculated using Prism software
(GraphPad, San Diego, CA).
ged from 0 to 1 was performed using GraphPad Prism software and
Perl data language.21,28 Curves were generated and analyzed using
Akaike’s information criteria.30,31 Using the equation generated by
Prism considered to be the best fit for the datasets, Ki values were
computed.21,28
2.8. Inhibition of acetylcholinesterase and
butyrylcholinesterase
2.14. Molecular dynamics simulations
Inhibition of hAChE and hBChE by compounds was determined
as previously described using 1 mM of either acetylthiocholine
(AcTCh) or butyrylthiocholine (BuTCh), respectively, as sub-
strates.32–35
Molecular dynamics simulations to evaluate the dihedral angle
of the 1,2-dione bond [(O)CC(O)] were performed as previously de-
scribed.19 Briefly, analyses were run using the MM3 module of Al-

4638
E. I. Parkinson et al. / Bioorg. Med. Chem. 19 (2011) 4635–4643
chemy (Tripos Inc., St. Louis, MO) with angle measurements re-
corded every 10 fs, over a total time frame of 15 ps, using a time
step of 1 fs.
It is possible the bromine analogues may change the mecha-
nism of enzyme inhibition by allowing nucleophilic attack on the
methylene atom rather than the carbonyl carbon. This would then
result in irreversible inhibition of the protein. Therefore, we evalu-
ated whether compound 6 could inactivate hiCE following incuba-
tion with the protein. As indicated in Table 1 however, no
irreversible inhibition of the enzyme was observed with this
analogue.
2.15. Computational chemistry
All computational analyses were performed using Gaussian 03
software (Gaussian, Wallingford, CT). Molecules were constructed
using Gauss-View and geometry optimization was performed
using the density functional theory method using the B3LYP hybrid
functional38 with the 6-31G(d,p) basis set.39 Charge distribution
was then visualized using Molden (Centre for Molecular and Bio-
molecular Informatics, Radboud University Nijmegen Medical Cen-
tre, Nijmegen, Netherlands).
3.1.3. Flexibility of the (O)C–C(O) bond
In previous studies, we have seen a correlation between the
flexibility of rotation around the 1,2-dione bond and the inhibitory
potency of the molecules. Typically, compounds that adopted a
more rigid structure tended to be more potent CE inhibitors.19
Therefore using molecular dynamics approaches, we evaluated
the rotation of this bond for benzil and compound 5. As indicated
in Figure 1, the 1,2-dione in benzil rapidly adopted a stable confor-
mation with a dihedral angle of ꢀ230°. In contrast, with 1,4-
diphenylbutane-2,3-dione, rotation around the dione continued
to oscillate over the entire course of the dynamics run. Since com-
pound 5 is a poorer inhibitor than benzil, these results are consis-
tent with those seen previously for a panel of heteroaromatic
ethane-1,2-diones, where increased oscillation was observed with
weaker molecule potency.19
3. Results
3.1. Analysis of CE inhibition by 1,4-diphenylbutane-2,3-dione
analogues
To assess the role of the atom vicinal to the carbonyl carbon
atoms in the molecule towards enzyme inhibition, we synthesized
a series of 1,4-diphenylbutane-2,3-dione analogues containing a
variety of different moieties in the a-position. In general, symmet-
rical compounds were used to ensure that that any inhibitory ef-
fects would not be as a result of competitive inhibition by two
different sites of nucleophilic attack by the protein.
3.2. Synthesis and enzyme inhibition by alkyl-1,2-diones
Since the active sites of the mammalian CEs are highly hydro-
phobic,2 we hypothesized that small molecules with larger c log P
values would be more potent enzyme inhibitors. Therefore, we
obtained, or generated, a series of alkyl-1,2-diones ranging from
butane-2,3-dione to octadecane-9,10-dione. Synthesis was accom-
plished by oxidation of the corresponding alkyne using potassium
permanganate in an acetone/water phase transfer system. Yields
were routinely greater than 70% and most compounds were pale
yellow crystalline solids. Chemical and physical properties for
these compounds are presented as Supplementary data.
CE inhibition by the alkyl-1,2-diones was assessed for both
hCE1 and hiCE, as well as rCE, using o-NPA as a substrate (Table 2).
As can be seen, as the compounds become larger, the molecules be-
come more potent inhibitors. Since this is likely due to the increase
in the hydrophobicity of the compounds, we calculated the c log P
of the alkyl diones using commercially available software. As indi-
3.1.1. Oxanilide, S1,S2-diphenyl ethanebis(thioate) and diphenyl
ethanedioate
Oxanilide (2) was not an inhibitor of hCE1, hiCE or rCE (Table 1)
and also lacked activity towards the cholinesterases. Similarly,
analogues containing either sulfur (3; S1,S2-diphenyl ethanebis(thi-
oate)) or oxygen (4; diphenyl ethanedioate) yielded molecules that
were ineffective at enzyme inhibition (Table 1). However, unlike
oxanilide, the latter two compounds underwent spontaneous
hydrolysis in aqueous solution liberating either thiophenol or phe-
nol, respectively (data not shown). Furthermore, this reaction was
not enhanced by the addition of CE. While detailed kinetics were
not determined, greater than 40% of each compound was hydro-
lyzed within 5 min of addition to buffered aqueous (pH 7.4) solu-
tion. While this was expected for
3 (as it is an activated
thioester), it was unclear whether compound 4 would be subject
to hydrolysis. Our results confirm that the ester is also labile under
these conditions.
3.1.2. 1,4-Diphenylbutane-2,3-dione, 1-bromo-1,4-
diphenylbutane-2,3-dione and 1,4-dibromo-1,4-
diphenylbutane-2,3-dione
Insertion of a carbon atom in the a-position (5; 1,4-diphenylbu-
tane-2,3-dione) resulted in a molecule that was effective at CE
inhibition, but the potency was ꢀ200- to 480-fold weaker than
that seen with benzil (Table 1). The mode of enzyme inhibition
was deemed to be partially competitive (i.e., indicating that while
the small molecule structurally resembled a CE substrate, they
were unable to reduce substrate hydrolysis to 0%, even at infinite
inhibitor concentrations). The brominated derivatives, 1,4-dibro-
mo-1,4-diphenylbutane-2,3-dione (compound 6) and 1-bromo-
1,4-diphenylbutane-2,3-dione (compound 7), were considerably
more active, with the dibromo analogue ꢀ30- to 5000-fold more
potent inhibitor than 5. These results suggest that the activation
of the carbonyl group was a major effector in improving inhibitor
function. While compound 7 was generally more potent than 5,
dramatic improvements in the inhibition constants for this mole-
cule were only observed with hiCE (Table 1).
Figure 1. Plot of the dihedral angle for the 1,2-dione group [(O)CC(O)] versus time
for benzil (black line) and 1,4-diphenylbutane-2,3-dione (5; blue line) for the
molecular dynamics simulations.

E. I. Parkinson et al. / Bioorg. Med. Chem. 19 (2011) 4635–4643
4639
Table 2
Structure, log P and Ki values for enzyme inhibition for alkyl 1,2-diones
O
R
R
O
ID
Name
R
c log P
Ki (nM)
rCE
hCE1
hiCE
hAChE
hBChE
8
9
Butane-2,3-dione
Hexane 3,4-dione
Octane-4,5-dione
Decane-5,6-dione
Dodecane-6,7-dione
Tetradecane-7,8-dione
Hexadecane-8,9-dione
Octadecane-9,10-dione
1,2-Dicyclohexylethane-1,2-dione
CH3
C2H5
C3H7
C4H9
C5H11
C6H13
C7H15
C8H17
Cyc. C6H11
ꢂ0.47
0.75
1.89
2.92
3.94
4.89
5.68
6.03
3.93a
>100,000
>100,000
8060 330
46.6 9.3
3.0 0.4
>100,000
>100,000
4670 260
189 15
57.7 2.2
76.4 9.4
25.6 2.3
5.7 1.4
>100,000
>100,000
17,780 1290
944 75
38.6 1.6
15.1 0.9
6.5 1.9
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
10
11
12
13
14
15
16
4.8 0.3
7.6 1.6
0.84 0.28
9.0 3.8
27 7.7
5.0 0.7a
72 7.0a
a
Data taken from Hyatt et al.20
Table 3
Structure, log P and Ki values for enzyme inhibition for 1-phenyl-alkyl-1,2-diones
O
R
O
ID
Name
R
c log P
Ki (nM)
rCE
4930 1320
12,500 1980
2710 170
337 22
hCE1
hiCE
hAChE
hBChE
17
18
19
20
21
22
Phenyl-1,2-propanedione
Phenyl-1,2-butanedione
Phenyl-1,2-pentanedione
Phenyl-1,2-hexanedione
Phenyl-1,2-heptanedione
Phenyl-1,2-octanedione
CH3
1.07
1.66
2.25
2.83
3.42
4.01
5270 1730
1550 380
102 12
26.1 2.2
6.65 0.22
2.19 0.08
1840 260
2420 150
401 21
72.7 5.1
45.5 6.1
28.7 2.3
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
C2H5
C3H7
C4H9
C5H11
C6H13
110 10
28.1 1.8
cated in Table 2, both inhibitor potency and the c log P values in-
creased with increasing chain length. As an example, compound
15 containing eight carbon atoms in the alkyl group, was a very po-
tent inhibitor of the mammalian CEs (0.84 and 5.7 nM for hCE1 and
hiCE, respectively) and demonstrated a c log P value of 6.03. As be-
fore, the mode of enzyme inhibition by these compounds was
found to be partially competitive.
partially competitive, and inhibitor potency appeared to correlate
with the c log P of the compounds.
3.4. Correlation between c log P and inhibitory potency for the
1,2-diones
Since we saw a trend towards increased inhibitory potency with
higher c log P values for both the alkyl-1,2-diones, and 1-phenyl-
1,2-alkyldiones, we evaluated whether correlations could be deter-
mined for these parameters. As indicated in Figure 2, linear corre-
lations could be described for both individual datasets (panel A—
alkyl-1,2-diones; panel B—1-phenyl-2-alkyl-1,2-diones), as well
as the combined values for all compounds (panel C). The linear
regression analyses yielded r2 values ranging from 0.72 to 0.99,
depending upon both the enzyme and the class of compounds.
Overall, these data indicate that the c log P and Ki values for all of
the CEs and inhibitors that were evaluated appear to be related,
with increasing c log P resulting in more potent enzyme inhibition.
3.3. Carboxylesterase inhibition by 1-phenyl-2-alkyl-1,2-diones
Having observed potent inhibition by the alkyl-1,2-diones, we
next evaluated a series of phenyl alkyl derivatives. These were cho-
sen since our original data demonstrated that benzil (1) was an
excellent inhibitor and that potency, and potentially selectivity,
might be afforded by the alkyl chain. Therefore, we synthesized a
series of 1-phenyl-1,2-alkyldiones using similar methodology to
that described for the alkyl derivatives. Similar yields (ꢀ70%) were
obtained and all physical parameters were consistent with their
structure (see Supplementary data).
As indicated in Table 3, potent enzyme inhibition was observed
when the alkyl chain exceeded 4 carbon atoms. For example, 1-
phenyl-1,2-octanedione (22), the most potent compound synthe-
sized, yielded Ki values of 2.2, 28.7 and 28.1 nM for hCE1, hiCE
and rCE, respectively. In general, these molecules did not generate
inhibition constants as low as that seen for the alkyl-1,2-diones,
however these molecules were clearly effective CE inhibitors.
Again, the mode of enzyme inhibition was determined to be
3.5. Modulation of CPT-11 metabolism and intracellular
carboxylesterase inhibition
Having identified several inhibitors that could potently inhibit
hiCE, we sought to determine the Ki values for this enzyme when
using CPT-11 as a substrate. This was undertaken only with com-
pounds that demonstrated reasonable efficacy at inhibiting o-
NPA hydrolysis (compounds 11–15 and 18–22). As indicated in Ta-

4640
E. I. Parkinson et al. / Bioorg. Med. Chem. 19 (2011) 4635–4643
Table 4
Ki values for the inhibition of CPT-11 hydrolysis by mammalian CEs with alkyl- and
phenyl alkyl-1,2-diones and intracellular enzyme inhibition using 4-MUA as
a
substrate
ID
Alkyl chain length
(carbon atoms)
hiCE (CPT-11)
(nM)
Intracellular hiCE
inhibitiona (%)
11
12
13
14
15
18
19
20
21
22
4
5
6
7
8
2
3
4
140 18.0
193 26.8
271 42.7
66.8 9.75
24.5 2.35
2010 189
495 39
168 16.6
98.1 20.6
130 11.0
175 29b
85.0
90.0
91.2
92.9
80.4
70.3
86.1
93.0
93.6
95.1
95.1c
5
6
NA
Benzil (1)
a
b
c
Data represent the results obtained from a representative experiment.
Taken from Wadkins et al.21
Taken from Hyatt et al.22
the cell permeability of the molecules.22 As shown in Table 4, all
of the inhibitors were effective at inhibiting hiCE within U373MG
cells, with the most potent inhibition observed with compound
22 (1-phenyl-2-octane-1,2-dione).
To assess whether intracellular inhibition could modulate CPT-
11 cytotoxicity, we undertook drug survival assays in cells express-
ing hiCE, following exposure to selected inhibitors. As indicated in
Table 5, both benzil and 1-phenyl-2-octane-1,2-dione reduced the
toxicity of CPT-11 to U373MGhiCE cells by ꢀ6- and 3-fold, respec-
tively. Interestingly, compound 13 (tetradecane-7,8-dione) was
ineffective in this assay, despite demonstrating good in vitro bio-
chemical properties (Tables 2 and 4), and inhibiting hiCE by
ꢀ91% in the fluorometric assay.
4. Discussion
Past publications have demonstrated the importance of the 1,2-
dione chemotype within molecules for the selective inhibition of
CE.19–21 Specifically, benzil was found to be an excellent inhibitor
of hiCE, hCE1 and rCE. We previously hypothesized that the mech-
anism of enzyme inhibition by these compounds was due to abor-
tive nucleophilic attack on one of the carbonyl carbon atoms of the
molecule by the catalytic serine.21 Since the tetrahedral intermedi-
ate that would be formed from this reaction would contain a C–C
bond (rather than the C–O bond in the ester), we postulated that
the enzyme would be unable to cleave this bond. Hence, an equi-
librium would be established between the free inhibitor and the
enzyme–inhibitor complex, resulting in reversible inhibition. We
reasoned that the simplest way to test this hypothesis would be
to generate compounds containing either deactivated (e.g., oxani-
lide) or activated (e.g., diphenyl ethanedioate, 1,4-dibromo-1,4-
diphenylbutane-2,3-dione) carbonyl groups, and then assess their
activity towards the CEs. Therefore the studies presented here de-
tail the interaction of the mammalian CEs with small molecules
containing either highly electrophilic, or essentially inactivated,
carbonyl carbon atoms.
Figure 2. Graphs demonstrating the correlation between c log P and Ki values for
the alkyl 1,2-diones (A), the 1-phenyl-1,2-alkyldiones (B) and composite results for
both classes of compounds (C). Data are shown for the three different enzymes hiCE
(blue), hiCE (red) and rCE (black) and the corresponding linear correlation
coefficient is indicated on the graph.
For oxanilide (2), where the
a-atom with respect to the car-
bonyl carbon is nitrogen, no enzyme inhibition was observed (Ta-
ble 1). This is likely due to the decreased electrophilicity of the
carbonyl. This renders the molecule less susceptible to nucleophilic
attack by the enzyme and as a consequence, oxanilide is unable to
inhibit the mammalian CEs. This would suggest that activated elec-
trophiles, such as the carbonyl groups present in compounds 3 and
4, would be effective CE inhibitors. However, both analogues were
both subject to spontaneous hydrolysis by water and this reaction
ble 4, the majority of these molecules were effective at inhibiting
hiCE-mediated CPT-11 metabolism, with compound 15 (octade-
cane-9,10-dione) yielding the lowest Ki value (ꢀ24 nM).
Having established that these inhibitors could modulate CPT-11
hydrolysis in vitro, we assessed the ability of these compounds to
inhibit hiCE intracellularly. This was determined using 4-MUA as a
substrate in a fluorometric assay and would provide a measure of

E. I. Parkinson et al. / Bioorg. Med. Chem. 19 (2011) 4635–4643
4641
Table 5
benzil was ꢀ230°, whereas for compound 5, the 1,2-dione was still
oscillating and never achieved a stable conformation at the end of
the dynamics simulations. The exact reason why this parameter
correlates with inhibitor potency is not clear, but may be due to
the alignment of the small molecule in a configuration such that
nucleophilic attack by the catalytic serine is facile.
Modulation of IC50 values for CPT-11 in U373MG cells expressing
hiCE by selected 1,2-dione inhibitors
a
Compound
IC50
(
lM)
CPT-11
3.6
Benzil (1)
CPT-11 + Benzil (1)
13
CPT-11 + 13
22
>100
22.9
>100
4.0
>100
10.2
Furthermore, as indicated in Figure 3, the stability of the confor-
mation adopted by benzil is presumably due to conjugation of the
p
electrons between the phenyl ring and the adjacent carbonyl
group. This accounts for the high frequency 1660 cmꢂ1 carbonyl
IR stretch seen with this compound.20 However, no conjugation oc-
curs between the carbonyl group themselves. With molecule 5, no
conjugation is observed between the phenyl group and the car-
bonyl moiety (the IR carbonyl wave number is 1709 cmꢂ1 40) and
hence the rotation around the carbon–carbon bonds within the bu-
tane moiety is less restricted. These observations are consistent
with the molecular dynamics simulations presented in Figure 1.
Also, as can be seen in Figure 3, the electron distribution on the
aromatic rings, coincident with the side of attack by a nucleophile
in benzil, is reduced as compared to compound 5. Hence, this may
make the carbonyl carbon atom in 1,4-diphenylbutane-2,3-dione
CPT-11 + 22
a
Data are representative values from several experiments.
was not enhanced by the addition of enzyme (data not shown).
Since oxygen and sulfur are both electron withdrawing in nature,
and each carbonyl group is further activated by the adjacent one,
the 1,2-dione in these compounds is highly reactive, to the extent
that even poor nucleophiles such as water, can readily hydrolyze
these molecules.
1,4-Diphenylbutane-2,3-dione (5) was a much poorer inhibitor
of the CEs than benzil (Table 1) indicating that the proximity of the
benzene ring is important towards biological activity. In benzil, the
phenyl group is directly attached to this moiety and it would be
anticipated that due to resonance effects, this would reduce the
electrophilicity of the carbonyl group. Furthermore with com-
pound 5, such an effect would be negated by the methylene atom,
and inductive effects from the phenyl ring would actually activate
this group. However, the biological data (Table 1) clearly demon-
strate that this is not the case. This suggests that other parameters
play a role in inhibitor potency, rather than just the electrophilicity
of the carbonyl carbon atom.
We have previously demonstrated the flexibility of the 1,2-
dione bond is an important parameter with respect to compound
potency.19 For a series of heteroaromatic ethane-1,2-diones, we
observed that the ability of the dione bond to adopt a relatively
stable conformation correlated with biological activity. In addition,
if the final dihedral angle that the molecule adopted was to close to
230°, it could be predicted that the compound would be a potent
CE inhibitor. As indicated in Figure 1, the final dihedral angle for
less prone to interaction with the serine Oc atom due to increased
repulsion from the charge localized on the benzene rings.
Alternatively, interaction of benzil with the enzyme may result
in a conformational change in the former that results in increased
affinity for the protein. Since both the molecular dynamics simula-
tions and the computational approaches (Figs. 1 and 3, respec-
tively), indicate that benzil and compound 5 adopt different
poses, it is possible that reaction of these molecules with the CEs,
would result in a significant change in the energetics of the system.
Since the biological data indicates that the interaction of benzil
with these proteins is clearly favored, as compared to 5, we are cur-
rently undertaking very detailed computational analyses of the
proposed enzyme–inhibitor complexes to assess whether signifi-
cant changes in small molecule conformation can be used to deter-
mine and/or predict inhibitor potency.
Activation of the carbonyl carbon by the addition of a bromine
atom on the vicinal carbon atom (compounds 6 and 7) resulted in
decreased Ki values for enzyme inhibition. Due to the electron
Figure 3. Charge distribution patterns in 1,4-diphenylbutane-2,3-dione (5; A) and benzil (1; B). Images are oriented with the oxygen atoms projecting either towards (left
panel) or away (right panel) from the viewer. Red indicates relative negative charge and blue relative positive charge.

4642
E. I. Parkinson et al. / Bioorg. Med. Chem. 19 (2011) 4635–4643
withdrawing properties of the halogen, and the fact that bromine is
a relatively good leaving group, it is possible that nucleophilic at-
pounds maybe subjected to metabolic processes that may
inactivate them, but as yet we have not identified a specific mecha-
nism for the lack of activity of this molecule. However it is clear that
compounds of this type can be used to modulate intracellular levels
of CE activity and can be used as biochemical tools to evaluate en-
zyme function.
tack by the O
c within the catalytic serine could occur either at
the carbonyl, or the methylene, carbon atom. The latter would re-
sult in covalent bond formation and irreversible inhibition of the
enzyme. However, as indicated in Table 1, no loss of enzyme activ-
ity was observed following dilution of the inhibitor from the reac-
tion. These results imply that interaction of the protein with the
inhibitor occurred at the carbonyl carbon and not at the vicinal car-
bon atom. Additionally, since the kinetic analyses indicated that
the mode of enzyme inhibition was partially competitive, this in-
fers that these inhibitors bind to the same site within the CE, and
that they structurally resemble the substrate. This would argue
that a similar mechanism of interaction likely occurs between
the enzyme and the inhibitor/substrate. Again, these data support
the hypothesis that the catalytic serine undergoes nucleophilic at-
tack of the carbonyl carbon atom. Furthermore, the halogens also
increased the hydrophobicity of the molecules (Table 1), likely
allowing for a further increase inhibitor potency. Since the active
site gorges of CE are very hydrophobic,2,21 compounds demonstrat-
ing greater c log P values would preferentially localize within this
domain. Evidence to this effect has been noticed with a series of
isatin analogues.18 Overall, these results suggest that the electro-
philicity of the carbonyl carbon atom plays a role in determining
the potency of inhibition towards CEs.
To further examine the effect of small molecule hydrophobicity
on enzyme inhibition, we synthesized a series of alkyl-1,2-diones
and evaluated their inhibitory potency against the CEs. We reasoned
that by simply increasing the size of the alkyl group, we could effect
an increase in the c log P of the compounds, without significantly
altering the electrostatic configuration of the molecule. As indicated
in Table 2, increasing the length of the alkyl side chains resulted in
dramatically reduced Ki values for the inhibitors with the CEs. This
correlated well with the increasing c log P values of the compounds
(Fig. 2). This phenomenon was observed for all three mammalian
CEs, suggesting that the environment within the active site was sim-
ilar for each protein. As indicated above, since the active sites of
these enzymes are highly hydrophobic, this increase in potency is
likely due to preferred localization of the longer chain compounds
within this domain. Indeed, since good correlation coefficients were
observed between the Ki values for enzyme inhibition and the
c log P values of these molecules (Fig. 2), this suggests that this
parameter is an important factor in determining inhibitor potency.
Based upon our observations with the 1,4-diphenylbutane-2,3-
dione analogues and the alkyl-1,2-diones, we hypothesized that
compounds containing a phenyl group and alkyl chain on either side
of the dione moiety should yield very potent CE inhibitors. As dis-
played in Table 3, we saw selective enzyme inhibition by such mol-
ecules, with inhibitory potency correlating with the hydrophobicity
of the molecule (Fig. 2). These results are in agreement with those
described for the alkyl-1,2-diones and further reinforce the impor-
tance of hydrophobicity towards enzyme inhibition.
5. Conclusions
Overall, our studies have delineated the roles of selected atoms
within the 1,2-dione CE-specific inhibitors and demonstrated that
the electrophilicity of the carbonyl group, the conformation that
the molecules adopt, and the hydrophobicity of these compounds
play major roles in determining inhibitor potency. Our data further
support the hypothesis that nucleophilic attack by the protein on
the carbonyl carbon atom represents the most likely mechanism
of enzyme inhibition. This has yielded several series of small mol-
ecules that demonstrate potency and selectivity towards CE, and
elucidated the chemical requirements for the design of further
compounds. These studies are currently underway.
Acknowledgments
The authors wish to thank Drs. Thomas Webb and Kip Guy for
helpful discussion and critical reading of the manuscript. This work
was supported in part by NIH Grants CA108775, an NIH Cancer Cen-
ter Core Grant CA21765, and by the American Lebanese Syrian Asso-
ciated Charities and St. Jude Children’s Research Hospital (SJCRH).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.06.012.
References and notes
1. Cashman, J.; Perroti, B.; Berkman, C.; Lin, J. Environ. Health Perspect. 1996, 104,
23.
2. Potter, P. M.; Wadkins, R. M. Curr. Med. Chem. 2006, 13, 1045.
3. Redinbo, M. R.; Potter, P. M. Drug Discovery Today 2005, 10, 313.
4. Mori, M.; Hosokawa, M.; Ogasawara, Y.; Tsukada, E.; Chiba, K. FEBS Lett. 1999,
458, 17.
5. Fleming, C. D.; Bencharit, S.; Edwards, C. C.; Hyatt, J. L.; Tsurkan, L.; Bai, F.;
Fraga, C.; Morton, C. L.; Howard-Williams, E. L.; Potter, P. M.; Redinbo, M. R. J.
Mol. Biol. 2005, 352, 165.
6. Khanna, R.; Morton, C. L.; Danks, M. K.; Potter, P. M. Cancer Res. 2000, 60, 4725.
7. Ross, M. K.; Borazjani, A.; Edwards, C. C.; Potter, P. M. Biochem. Pharmacol. 2006,
71, 657.
8. Ross, M. K.; Potter, P. M.; Borazjani, A. Toxicol. Sci. 2005, 84, A1569.
9. Satoh, T.; Hosokawa, M.; Atsumi, R.; Suzuki, W.; Hakusui, H.; Nagai, E. Biol.
Pharm. Bull. 1994, 17, 662.
10. Zhang, J.; Burnell, J. C.; Dumaual, N.; Bosron, W. F. J. Pharmacol. Exp. Ther. 1999,
290, 314.
11. Humerickhouse, R.; Lohrbach, K.; Li, L.; Bosron, W.; Dolan, M. Cancer Res. 2000,
60, 1189.
12. Hsiang, Y. H.; Liu, L. F. Cancer Res. 1988, 48, 1722.
13. Tanizawa, A.; Fujimori, A.; Fujimori, Y.; Pommier, Y. J. Natl. Cancer Inst. 1994,
86, 836.
14. Danks, M. K.; Morton, C. L.; Krull, E. J.; Cheshire, P. J.; Richmond, L. B.; Naeve, C.
W.; Pawlik, C. A.; Houghton, P. J.; Potter, P. M. Clin. Cancer Res. 1999, 5, 917.
15. Potter, P. M.; Pawlik, C. A.; Morton, C. L.; Naeve, C. W.; Danks, M. K. Cancer Res.
1998, 52, 2646.
16. Wierdl, M.; Morton, C. L.; Weeks, J. K.; Danks, M. K.; Harris, L. C.; Potter, P. M.
Cancer Res. 2001, 61, 5078.
17. Hatfield, M. J.; Tsurkan, L.; Garrett, M.; Shaver, T.; Edwards, C. C.; Hyatt, J. L.;
Hicks, L. D.; Potter, P. M. Biochem. Pharmacol. 2011, 81, 24.
18. Hyatt, J. L.; Moak, T.; Hatfield, J. M.; Tsurkan, L.; Edwards, C. C.; Wierdl, M.;
Danks, M. K.; Wadkins, R. M.; Potter, P. M. J. Med. Chem. 2007, 50, 1876.
19. Hyatt, J. L.; Stacy, V.; Wadkins, R. M.; Yoon, K. J.; Wierdl, M.; Edwards, C. C.;
Zeller, M.; Hunter, A. D.; Danks, M. K.; Crundwell, G.; Potter, P. M. J. Med. Chem.
2005, 48, 5543.
20. Hyatt, J. L.; Wadkins, R. M.; Tsurkan, L.; Hicks, L. D.; Hatfield, M. J.; Edwards, C.
C.; Ii, C. R.; Cantalupo, S. A.; Crundwell, G.; Danks, M. K.; Guy, R. K.; Potter, P. M.
J. Med. Chem. 2007, 50, 5727.
Having identified the necessary properties for these molecules to
effect CE inhibition in vitro, we evaluated the ability of selected com-
pounds to modulate enzyme activity in cells and to determine
whether these inhibitors could alter CPT-11 cytotoxicity in cells
expressing hiCE. We confirmed that the majority of the alkyl and
phenylalkyl derivatives were cell permeable and were effective at
inhibitionhydrolysisof CPT-11by hiCE. Furthermore, in cell viability
studies, benzil and compound 22 were effective at modulating the
toxicity of this drug. Surprisingly, tetradecane-7,8-dione (13) was
ineffective at reducing CPT-11 toxicity in cells expressing hiCE. At-
tempts to identify a mechanism for this lack of activity (e.g. the
use of serum-free culture media to eliminate protein binding, the
use of glass culture dishes to minimize interaction with the plastic-
ware, etc) have so far been unsuccessful. Potentially, these com-

E. I. Parkinson et al. / Bioorg. Med. Chem. 19 (2011) 4635–4643
4643
21. Wadkins, R. M.; Hyatt, J. L.; Wei, X.; Yoon, K. J.; Wierdl, M.; Edwards, C. C.;
31. Akaike, H. IEEE Trans. Automatic Control 1974, AC-19, 716.
Morton, C. L.; Obenauer, J. C.; Damodaran, K.; Beroza, P.; Danks, M. K.; Potter, P.
M. J. Med. Chem. 2005, 48, 2905.
22. Hyatt, J. L.; Tsurkan, L.; Wierdl, M.; Edwards, C. C.; Danks, M. K.; Potter, P. M.
Mol. Cancer Ther. 2006, 5, 2281.
23. Babudri, F.; Fiandanese, V.; Marchese, G.; Punzi, A. Tetrahedron Lett. 1995, 36,
7305.
24. Morton, C. L.; Potter, P. M. Mol. Biotechnol. 2000, 16, 193.
25. Santana, M. D.; Garcia, G.; Julve, M.; Lloret, F.; Perez, J.; Liu, M.; Sanz, F.; Cano,
J.; Lopez, G. Inorg. Chem. 2004, 43, 2132.
26. Jones, H. O.; Tasker, H. S. J. Chem. Soc. 1909, 95, 1904.
27. Srinivasan, N. S.; Lee, D. G. J. Org. Chem. 1979, 44, 1574.
28. Wadkins, R. M.; Hyatt, J. L.; Yoon, K. J.; Morton, C. L.; Lee, R. E.; Damodaran, K.;
Beroza, P.; Danks, M. K.; Potter, P. M. Mol. Pharmacol. 2004, 65, 1336.
29. Webb, J. L. In Enzyme and Metabolic Inhibitors, General Principles of Inhibition;
Academic Press: New York, 1963; Vol. 1,
32. Doctor, B. P.; Toker, L.; Roth, E.; Silman, I. Anal. Biochem. 1987, 166, 399.
33. Ellman, G. L.; Courtney, K. D.; Anders, V.; Featherstone, R. M. Biochem.
Pharmacol. 1961, 7, 88.
34. Morton, C. L.; Wadkins, R. M.; Danks, M. K.; Potter, P. M. Cancer Res. 1999, 59,
1458.
35. Wadkins, R. M.; Potter, P. M.; Vladu, B.; Marty, J.; Mangold, G.; Weitman, S.;
Manikumar, G.; Wani, M. C.; Wall, M. E.; Von-Hoff, D. D. Cancer Res. 1999, 59,
3424.
36. Guichard, S.; Morton, C. L.; Krull, E. J.; Stewart, C. F.; Danks, M. K.; Potter, P. M.
Clin. Cancer Res. 1998, 4, 3089.
37. Danks, M. K.; Morton, C. L.; Pawlik, C. A.; Potter, P. M. Cancer Res. 1998, 58, 20.
38. Stephens, P. J.; Devlin, F. J.; Chabalowski, C. F.; Frisch, M. J. J. Phys. Chem. 1994,
98, 11623.
39. Petersson, G. A.; Bennett, A.; Tensfeldt, T. G.; Al-Laham, M. A.; Shirley, W. A.;
Mantzaris, J. J. Chem. Phys. 1988, 89, 2193.
30. Akaike, H. In Information Theory and An Extension of The Maximum Likelihood
Principle, Second International Symposium on Information Theory; Petrov, B. N.;
Csaki, F., Eds.; Budapest, Akademiai Kiado, 1973, pp 267–281.
40. Stetter, H.; Raemsch, R. Y. Synthesis 1981, 6, 477.