Analysis of the inhibition of mammalian carboxylesterases by novel fluorobenzoins and fluorobenzils

Article abstract of DOI:10.1016/j.bmc.2007.03.012

We have synthesized and assessed the ability of symmetrical fluorobenzoins and fluorobenzils to inhibit mammalian carboxylesterases (CE). The majority of the latter were excellent inhibitors of CEs however unexpectedly, the fluorobenzoins were very good enzyme inhibitors. Positive correlations were seen with the charge on the hydroxyl carbon atom, the carbonyl oxygen, and the Hammett constants for the derived K_i values with the fluorobenzoins.

Full text of DOI:10.1016/j.bmc.2007.03.012

Bioorganic & Medicinal Chemistry 15 (2007) 3801–3817
Analysis of the inhibition of mammalian carboxylesterases by novel
fluorobenzoins and fluorobenzils
Latorya D. Hicks,a Janice L. Hyatt,a Teri Moak,b Carol C. Edwards,a Lyudmila Tsurkan,a
Monika Wierdl,a Antonio M. Ferreira,c Randy M. Wadkinsb and Philip M. Pottera,*
aDepartment of Molecular Pharmacology, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA
bDepartment of Chemistry and Biochemistry, University of Mississippi, University, MS 38677, USA
cHartwell Center for Bioinformatics and Biotechnology, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA
Received 29 January 2007; revised 23 February 2007; accepted 8 March 2007
Available online 12 March 2007
Abstract—We have synthesized and assessed the ability of symmetrical fluorobenzoins and fluorobenzils to inhibit mammalian carb-
oxylesterases (CE). The majority of the latter were excellent inhibitors of CEs however unexpectedly, the fluorobenzoins were very
good enzyme inhibitors. Positive correlations were seen with the charge on the hydroxyl carbon atom, the carbonyl oxygen, and the
Hammett constants for the derived Ki values with the fluorobenzoins.
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
As CEs are responsible for the metabolism and activa-
tion of a host of different clinically useful agents, we
have hypothesized that selective inhibitors of these
enzymes may be useful in modulating the biological
activity of these drugs. For example, for compounds
that are inactivated by CEs (e.g., flestolol), addition of
an inhibitor may prolong the period of time for which
the drug is active.5 Conversely for drugs that are acti-
vated by these enzymes (e.g., CPT-11), specific CE
inhibitors may be useful in ameliorating the toxicity
associated with these agents.6 Therefore, we have
screened for compounds that have selective inhibitory
activity toward CEs. This was performed using
Telik’s Target Related Affinity Profiling (TRAP) tech-
nology.7–9 Following the identification of compounds
that demonstrated activity toward three mammalian
CEs (human intestinal CE (hiCE), human liver CE
(hCE1), and rabbit liver CE (rCE)), these chemicals
were then assessed for their inhibition of human acetyl-
cholinesterase (AChE). Molecules that inhibited the
latter enzyme were discarded. This process identified
benzil as a general, selective inhibitor of mammalian
CEs.10 More recent studies have shown that the
characteristics for a good CE inhibitor are the presence
of: (i) aromatic rings or increasing hydrophobicity, (ii) a
1,2-dione moiety, and (iii) substitution which does not
impede access of the compound reaching the active site
of the enzyme.10,11 These studies also demonstrated
that benzoin (2-hydroxy-1,2-diphenylethanone), an
Carboxylesterases (CEs) are ubiquitous enzymes respon-
sible for the detoxification of xenobiotics.1 It has been
reported that CEs can metabolize a wide variety of ester
containing compounds including clinical drugs such as,
meperidine, flumazenil, procaine, oxybutynin, and the
anticancer prodrugs capecitabine and CPT-11.1–4 Con-
sistent with their proposed function, CEs are expressed
in high levels in human tissues such as the liver, lung,
small intestine, and kidney. All CEs examined to date
maintain a catalytic triad of amino acids (serine, histi-
dine, and glutamic acid) that are essential for hydrolytic
activity.
Abbreviations: AChE, acetylcholinesterase; AcTCh, acetylthiocholine;
BChE, butyrylcholinesterase; BuTCh, butyrylthiocholine; CE, carb-
oxylesterase; CPT-11, irinotecan, 7-ethyl-10-[4-(1-piperidino)-1-piperi-
dino]carbonyloxycamptothecin; EDG, electron donating group;
EWG, electron withdrawing group; hCE1, human carboxylesterase
1; hiCE, human intestinal carboxylesterase; Ki, inhibition constant;
o-NPA, o-nitrophenyl acetate; q2, cross validation coefficient; QSAR,
quantitative structure–activity relationship; rCE, rabbit liver carboxy-
lesterase; TRAP, Target Related Affinity Profiling.
Keywords: Fluorobenzil; Fluorobenzoin; Carboxylesterase; Inhibitor.
*
Corresponding author. Tel.: +1 901 495 3440; fax: +1 901 495
4293; e-mail: phil.potter@stjude.org
0968-0896/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.03.012

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L. D. Hicks et al. / Bioorg. Med. Chem. 15 (2007) 3801–3817
intermediate in the synthesis of benzil from benzalde-
hyde, was a poor inhibitor of CEs, consistent with the
hypothesis that the 1,2-dione chemotype is important
in enzyme inhibition.
assessed them for CE inhibition using hiCE, hCE1,
and rCE. Results presented here indicate that fluorine
substitution within the benzene rings generates benzoin
analogs that are potent inhibitors of mammalian CEs.
We believe that the benzils are potent inhibitors, in part,
because the 1,2-dione structure mimics the ester chemo-
type allowing for the initiation of the nucleophilic addi-
tion-elimination reactions that are observed for this
class of compounds (Fig. 1a). Abortive nucleophilic
attack by the active site serine on one of the carbonyl
carbons would yield a tetrahedral intermediate that
would be unlikely to undergo C–C cleavage, the next
step in the reaction. Therefore, in the presence of benzil,
repetitive attack and release by the serine residue on the
carbonyl groups would occur, resulting in enzyme
inhibition. This hypothesis suggests that decreasing the
electron density around the carbonyl carbon atom
would make this atom more susceptible to nucleophilic
attack by the serine oxygen. Therefore, appropriate
inclusion of electron withdrawing groups (EWG) should
increase the likelihood of attack and presumably the
potency of the inhibitors (Fig. 1b). In this series of
studies, we also assessed the ability of the benzoin to
inhibit the mammalian CEs. Benzoins (a-hydroxy
ketones) were chosen because they are key intermediates
in the synthesis of benzils from the aldehydes and they
possess similar structural characteristics to benzil, that
is, aromatic rings, carbonyl groups, etc. (see Tables 1
and 2). While we have previously demonstrated that
the benzoins are poor inhibitors of CEs,10 modification
of the electron density associated with the dione carbon
atom, might have the potential to produce compounds
that demonstrate inhibitory activity.
2. Results
2.1. Analysis of carboxylesterase inhibition by
fluorobenzils
In a previous paper, we hypothesized that nucleophilic
attack at one of the carbonyl groups within the 1,2-
dione moiety of benzil (2) by the catalytic serine was,
in part, responsible for enzyme inhibition (Fig. 1a).10
We therefore surmised that substituted benzene rings,
which would withdraw electron density from these car-
bon atoms, might improve the potency of CE inhibition
(Fig. 1b). To assess the validity of this hypothesis, we
synthesized a panel of fluorobenzoins and fluorobenzils
for use in enzyme inhibition studies. The structures of
the compounds used for these assays are shown in
Tables 1 and 2.
The ability of these compounds to inhibit hiCE, hCE1,
rCE, human AChE, and human BChE was then deter-
mined and the Ki values for enzyme inhibition are re-
ported in Tables
3
and 4. As indicated, the
fluorobenzil analogs were all relatively good inhibitors
of CEs with Ki values ranging from 3 nm to 2.8 lM.
The most potent inhibitor was 1,2-bis(2,3-difluorophe-
nyl)ethane-1,2-dione (23), yielding
a Ki value of
3.3 nM with rCE. Interestingly, the inhibition constants
were as high as 2.84 lM with 1,2-bis[2,4-bis(trifluorom-
ethyl)phenyl]ethane-1,2-dione (32), with hiCE. In
general, inhibitors that were potent for one enzyme
demonstrated similar levels of activity toward the other
To test these hypotheses, we have synthesized a panel of
fluorobenzoins and their analogous fluorobenzils, and
Figure 1. (a) Proposed mechanism of interaction of the benzil analogs with the catalytic amino acids of CEs. A serine nucleophile is generated by
proton transfer to a glutamic acid via a histidine residue, and the resulting oxygen atom attacks one of the carbonyl groups within the dione moiety.
The tetrahedral intermediate that is generated is relatively stable, due to the increased strength of the C–C bond as compared to the C–O bond
present in esters. Therefore the former bond is not cleaved resulting in inhibition of the enzyme. (b) Increasing or decreasing the electron density
surrounding the carbonyl carbon atoms by introducing either electron withdrawing groups (EWG) or electron donating groups (EDG) within the
molecules should make the compounds better and poorer enzyme inhibitors, respectively.

L. D. Hicks et al. / Bioorg. Med. Chem. 15 (2007) 3801–3817
Table 1. Structure of the fluorobenzoins used in this study
3803
R
R
2
1
O
R
R
6
7
R
3
R
8
R
R
R
4
5
R
R
10
9
R = H unless otherwise indicated
ID
Name
R
R1
F
R2
R3
R4
R5
R6
F
R7
F
R8
R9
R10
1
3
Benzoin (1,2-diphenyl-2-hydroxy-ethanone)
1,2-Bis-(2-fluorophenyl)-2-hydroxyethanone
1,2-Bis-(3-fluorophenyl)-2-hydroxyethanone
1,2-Bis(4-fluorophenyl)-2-2-hydroxyethanone
1,2-Bis(2,4-difluorophenyl)-2-hydroxyethanone
1,2-Bis-(2,6-difluorophenyl)-2-hydroxyethanone
1,2-Bis(3,4-difluorophenyl)-2-hydroxyethanone
1,2-Bis(2,3-difluorophenyl)-2-hydroxyethanone
1,2-Bis(2,5-difluorophenyl)-2-hydroxyethanone
1,2-Bis(3,5-difluorophenyl)-2-hydroxyethanone
1,2-Bis-(2,3,5-trifluorophenyl)-2-hydroxyethanone
1,2-Bis-(2,3,4-trifluorophenyl)-2-hydroxyethanone
1,2-Bis-(3,4,5-triflu0rophenyl)-2-hydroxyethanone
1,2-Bis-[4-(trifluoromethyl)phenyl]-2-hydroxyethanone
1,2-Bis-[3-(trifluoromethyl)phenyl]-2-hydroxyethanone
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
4
F
5
F
F
F
F
6
F
F
F
F
7
F
F
8
F
F
F
F
F
F
9
F
F
F
F
10
11
12
13
14
15
16
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
CF3
F
F
CF3
F
CF3
CF3
Table 2. Structure of the fluorobenzils used in this study
R
R
R
2
1
5
O
R
R
6
7
9
R
3
R
8
R
R
4
R
R
10
R = H unless otherwise indicated
ID Name
R
R1
F
R2
R3
R4
R5 R6
R7
F
R8
R9
R10
2
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
Benzil (diphenylethane-1,2-dione)
@O
@O
@O
@O
@O
@O
@O
@O
@O
@O
@O
@O
@O
@O
@O
@O
1,2-Bis-(2-fluorophenyl)-ethane-1,2-dione
1,2-Bis-(3-fluorophenyl)-ethane-1,2-dione
1,2-Bis(4-fluorophenyl)-ethane-1,2-dione
1,2-Bis(2,4-difluorophenyl)-ethane-1,2-dione
1,2-Bis(2,6-difluorophenyl)-ethane-1,2-dione
1,2-Bis(3,4-difluorophenyl)-ethane-1,2-dione
1,2-Bis(2,3-difluorophenyl)-ethane-1,2-dione
1,2-Bis(2,5-difluorophenyl)-ethane-1,2-dione
1,2-Bis(3,5-difluorophenyl)-ethane-1,2-dione
1,2-Bis(2,3,6-trifluorophenyl)-ethane-1,2-dione
1,2-Bis(2,3,5-trifluorophenyl)-ethane-1,2-dione
1,2-Bis(2,3,4-trifluorophenyl)-ethane-1,2-dione
1,2-Bis(3,4,5-trifluorophenyl)-ethane-1,2-dione
1,2-Bis[4-(trifluoromethyl)phenyl]-ethane-1,2-dione
1,2-Bis[3(trifluoromethyl)phenyl]-ethane-1,2-dione
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
CF3
F
CF3
CF3
CF3
CF3
CF3
1,2-Bis[2,4-bis(trifluoromethyl)phenyl]-ethane-1,2-dione @O CF3
1,2-Bis[3,5-bis(trifluoromethyl)phenyl]-ethane-1,2-dione @O
1,2-Bis[2,5-bis(trifluoromethyl)phenyl]-ethane-1,2-dione @O CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
two CEs. Overall, these results demonstrate that these
fluorobenzils are good inhibitors; however, fluorine
substitution did not dramatically increase the inhibitory
potency of these compounds as compared to benzil.
The latter compound demonstrates Ki values of
15.1, 45.1, and 103 nM for hiCE, hCE1, and rCE,
respectively.
The trifluoromethyl analogs were less effective CE
inhibitors than the fluorobenzils. For example, only
the para-substituted compound (30) inhibited all three
mammalian enzymes. Indeed, none of the other trifluo-
romethyl analogs that we analyzed inhibited hCE1 and
compounds 32–34 also failed to inhibit rCE. We believe
that this is likely due to steric constraints afforded by the

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L. D. Hicks et al. / Bioorg. Med. Chem. 15 (2007) 3801–3817
Table 3. Ki values for enzyme inhibition using the benzoin analogs. Values for CEs were determined using o-NPA as a substrate, and those for AChE
and BChE using AcTCh or BuTCh as a substrate, respectively
ID
hiCE Ki SE (nM)
hCE1 Ki SE (nM)
rCE Ki SE (nM)
AChE Ki (nM)
BChE Ki (nM)
1
3
2670 1010
250 23
640 80
7210 2410
990 100
3130 470
>100,000
480 30
>100,000
91.4 44.0
660 45
>100,000
140 18
61.8 2.9
220 22
200 39
48.3 1.7
18.4 1.0
31.7 3.9
8.3 0.4
85.7 4.6
12.9 2.9
42.4 4.4
>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
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
4
5
>100,000a
390 44
6
7
120
9
190
5
8
150 17
260 15
55.2 3.7
71.0 9.0
99.5 9.2
25.7 3.1
1040 50
400 35
220 17
1300 210
730 60
9
10
11
12
13
14
15
16
230
7
170 16
665 30
260 13
1000 50
870 390
>100,000
a >100,000 indicates less than 50% enzyme inhibition at an inhibitor concentration of 100 lM, that is, essentially no inhibition.
CF3 group(s), since we have previously demonstrated
that the entrance to the active site of hCE1 is consider-
ably smaller and less flexible than the other CEs.12 As
the trifluoromethyl compounds are more bulky than
the other benzils assayed in these studies, it is likely that
steric hindrance prevents access to hCE1 and rCE active
sites, and hence they do not inhibit these proteins. The
benzoin analogs of compounds 32–34 were not pro-
duced due to their direct oxidation to the benzil deriva-
tive under the reaction conditions employed. This is
likely due to the electron-withdrawing effects of the tri-
fluoromethyl groups resulting in increased reactivity of
the oxygen atoms and hence facile oxidation to the cor-
responding benzil.
ence between these analogs is the position of the
single fluorine atom in the benzene ring. Our results
suggest, therefore, that bond polarization plays a ma-
jor role in mono-substituted aromatic rings causing
inductive effects to prevail when influencing the elec-
tron density associated with the a carbon atom. This
inductive effect is likely responsible for the differential
inhibition of the three mammalian CEs across the
mono-substituted series.
In the disubstituted analogs, it was apparent that reso-
nance effects could account for the differences in the
inhibition constants for the mammalian CEs. For exam-
ple compound 11 demonstrated lower Ki values than 6
or 9, probably due to withdrawal of electrons from the
dione moiety via the fluorine atoms at the 3- and 5-posi-
tions on the benzene ring. Due to the distances involved,
this is unlikely to be mediated by inductive effects, but
rather due to the stabilization of resonance structures
where the p electrons are conjugated via the benzene
rings. Substitution of fluorine atoms at the 3- and 5-
positions will enhance the stability of the resonance
structures, yielding a more electron deficient environ-
ment at the dione carbon atoms. This would make them
more susceptible to attack by the serine nucleophile and
therefore better CE inhibitors.
2.2. Analysis of carboxylesterase inhibition by
fluorobenzoins
Benzoin, 1, is not a good inhibitor of mammalian CEs; it
demonstrates Ki values of 2.7 and 7.2 lM for hiCE and
hCE1, respectively, and is inactive toward rCE.10 How-
ever, the addition of fluorine atoms to the benzene rings
in the benzoins resulted in compounds that were very
potent inhibitors of CEs (Table 4). The majority of the
fluorobenzoin analogs had Ki values ranging from
8 nM to 1.3 lM, with the most potent inhibitor being
1,2-bis(2,3,4-trifluorophenyl)-2-hydroxyethanone (13),
yielding a Ki value of 8.3 nM with rCE. With some
exceptions, the fluorobenzoins were more potent inhibi-
tors than benzoin. In contrast to the benzil analogs,
fluorine substitutions on the benzene rings resulted in
marked increases in the inhibitory potency of the
benzoins.
2.3. Assessment of electronic parameters for the fluoro-
benzils and fluorobenzoins
Having demonstrated that fluorine substitution
increased the potency of the fluorobenzoin analogs, we
decided to evaluate the electron distribution within these
compounds using computational methods. Therefore,
we calculated a variety of electronic parameters for
the fluorobenzoins and fluorobenzils using density
functional theory (Table 5). These included the atomic
charges of the oxygen and carbon atoms within the
dione (or the hydroxy-ethanone) group, the pKa of the
hydroxyl group of the benzoins, and Hammett
substituent constants. The results were then analyzed
and compared to the Ki values for each compound, with
each mammalian CE.
For example, compound 11 demonstrated Ki values
of 71.1, 170, and 18.4 nM for hiCE, hCE1, and
rCE, respectively. These values are 37- to 5400-fold
lower than that observed for the unsubstituted ben-
zoin (1) for the same enzymes. However, there were
examples where the patterns of enzyme inhibition
were very different. As an example, compounds 3
and 4 inhibited all three enzymes, but 5 did not inhi-
bit any of the enzymes (see Table 3). The only differ-

L. D. Hicks et al. / Bioorg. Med. Chem. 15 (2007) 3801–3817
Table 4. Ki values for enzyme inhibition using the benzil analogs
3805
ID
hiCE Ki SE (nM)
hCE1 Ki SE (nM)
rCE Ki SE (nM)
AChE Ki (nM)
BChE Ki (nM)
2
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
15.1 2.0
55.9 3.4
25.9 5.1
170 12
45.1 3.4
1
103 20
47.8 1.4
36.2 7.3
400 59
>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
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
1270 100
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
185
100 27
230 11.8
370 109
5650 420
270 41
830 63
150 17
98.0 28.8
1170 90
49.3 6.3
3.34 0.02
60.8 9.0
22.8 3.5
33.8 8.2
28.9 10.1
3.3 0.6
300
8
4.98 0.16
61.4 2.7
30.6 3.4
40.7 3.8
31.9 8.8
13.6 3.4
360 62
34.7 2.7
260 24
97.2 8.5
320 33
140 28
91.3 12.3
470 113
170 45
>100,000
>100,000
>100,000
>100,000
67.9 4.5
12.7 6.0
150 38
180 91
250 55
2840 950
550 170
2370 630
>100,000
>100,000
>100,000
Values for CEs were determined using o-NPA as a substrate, and those for AChE and BChE using AcTCh or BuTCh as a substrate, respectively.
As can be seen in Table 6, no correlations were observed
between the charges present on the carbonyl carbon or
oxygen atoms with the benzils and the observed Ki val-
ues for CE inhibition. In addition, poor Spearman r cor-
relation coefficients were seen with the predicted charge
on the hydroxyl oxygen atom in the benzoins, when
Table 5. Calculated electronic parameters for the compounds used in this study
ID
ChargeaC–OH
ChargeaC@O
ChargeaC–OH
ChargeaC@O
Benzoin OH pKa
Hammett d
1
2
0.019
0.411
0.342
0.389
0.412
0.411
0.410
0.386
0.414
0.395
0.373
0.410
0.401
0.412
0.418
0.347
0.418
0.317
0.348
0.343
0.318
0.337
0.281
0.321
0.321
0.353
0.341
0.326
0.354
0.352
0.346
0.349
0.318
0.357
0.298
ꢀ0.531
ꢀ0.444
ꢀ0.462
ꢀ0.444
ꢀ0.442
ꢀ0.452
ꢀ0.444
ꢀ0.426
ꢀ0.445
ꢀ0.439
ꢀ0.394
ꢀ0.436
ꢀ0.433
ꢀ0.438
ꢀ0.439
ꢀ0.452
ꢀ0.440
ꢀ0.416
ꢀ0.456
ꢀ0.465
ꢀ0.419
ꢀ0.400
ꢀ0.376
ꢀ0.411
ꢀ0.410
ꢀ0.449
ꢀ0.395
ꢀ0.405
ꢀ0.445
ꢀ0.453
ꢀ0.452
ꢀ0.453
ꢀ0.414
ꢀ0.447
ꢀ0.400
7.909
0
0
3
0.029
0.022
0.017
0.034
0.041
0.020
0.027
0.026
0.028
0.025
0.034
0.022
0.029
0.021
ꢀ0.531
ꢀ0.539
ꢀ0.539
ꢀ0.545
ꢀ0.534
ꢀ0.539
ꢀ0.529
ꢀ0.544
ꢀ0.538
ꢀ0.526
ꢀ0.544
ꢀ0.538
ꢀ0.539
ꢀ0.538
7.487
7.214
7.209
6.668
7.230
7.713
7.052
6.719
8.435
6.855
7.079
8.342
10.764
10.784
1.060
0.674
0.124
0.592
1.060
0.399
0.867
0.867
0.674
1.204
0.929
0.736
1.080
0.860
1.060
0.674
0.124
0.867
1.060
0.399
0.867
0.867
0.674
1.397
1.024
0.929
0.736
1.080
0.860
0.750
0.860
0.640
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
a The charge on the respective atom is indicated in bold.

3806
L. D. Hicks et al. / Bioorg. Med. Chem. 15 (2007) 3801–3817
Table 6. Correlation coefficients for the atomic charges on the hydroxyl and the carbonyl oxygen and carbon atoms in the fluorobenzoins and the
fluorobenzils, with the observed Ki values for CE inhibition
Compound
Atoma
Linear regression (r2)
Spearman correlation coefficient
(Spearman r)
p value for Spearman
correlationb
hiCE
hCE1
rCE
hiCE
hCE1
rCE
hiCE
hCE1
rCE
Benzoins
C–OH
C@O
C–OH
C@O
C@O
C@O
0.16
0.02
0.02
0.23
0.07
0.03
0.22
0.08
0.03
0.11
0.0
0.25
0.04
0.00
0.20
0.03
0.02
ꢀ0.470
0.176
ꢀ0.757
0.533
ꢀ0.533
0.223
0.077
0.53
0.48
0.006
0.63
0.64
0.001
0.04
0.60
0.007
0.53
0.31
0.041
0.43
0.96
0.05
0.50
0.87
ꢀ0.198
ꢀ0.672
ꢀ0.118
0.115
ꢀ0.146
ꢀ0.665
ꢀ0.152
0.245
ꢀ0.013
ꢀ0.518
ꢀ0.166
ꢀ0.041
Benzils
0.0
a Charge on specific atom is indicated in bold.
b p values 60.05 are indicated in bold.
Table 7. Correlation coefficients for the pKa values for the hydroxyl proton in the fluorobenzoins with the observed Ki values for CE inhibition
Compound
Linear regression (r2)
Spearman correlation coefficient
(Spearman r)
p value for Spearman correlation
hiCE
0.02
hCE1
0.13
rCE
0.01
hiCE
0.232
hCE1
0.336
rCE
hiCE
0.41
hCE1
0.22
rCE
0.55
Benzoins
ꢀ0.170
Table 8. Correlation coefficients for the Hammett substituent constants for the fluorobenzoins and the fluorobenzils, with the observed Ki values for
CE inhibition
Compound
Linear regression (r2)
Spearman correlation coefficient
(Spearman r)
p value for Spearman correlationa
hiCE
hCE1
rCE
hiCE
hCE1
rCE
hiCE
hCE1
rCE
Benzoins
Benzils
0.26
0.0
0.11
0.0
0.63
0.0
ꢀ0.489
ꢀ0.041
ꢀ0.462
ꢀ0.703
0.065
0.87
0.083
0.98
0.004
0.08
0.006
0.407
a p values 60.05 are indicated in bold.
compared to the enzyme inhibition data. However, we
observed significant p values (<0.05) for the correlation
analyses when the charge on the carbonyl oxygen atom
of the fluorobenzoins was compared to the Ki values for
the mammalian CEs (Table 6). Additionally, a correla-
tion (p = 0.077 with hiCE at the 10% level) was seen
between the charge on the hydroxyl carbon atom and
the Ki values for the inhibition of hCE1 and rCE with
these compounds. Overall, these results indicate that the
relative efficiency of inhibition by the benzoins is related
to the electron density surrounding these atoms within
the hydroxy-ethanone moiety. Perhaps not surprisingly,
no obvious correlations were apparent when using linear
regression analysis of the datasets (Table 6).
indicated that the charge distribution within the central
carbon atoms may be an important factor in CE inhibi-
tion, we performed 3D-QSAR analysis of the data using
Quasar software. These experiments generated linear
correlation coefficient (r2) values ranging from 0.65 to
0.89 for the observed versus predicted Ki values for
CE inhibition (Table 9; Fig. 2). In addition, excellent
cross correlation coefficients (q2) were obtained from
these analyses. Since these values, and q2/r2 ratios, are
all close to unity, these results suggest that the models
have considerable predictive power in assessing CE inhi-
bition by the benzoins and the benzils.
Graphic representations of the 3D-QSAR models for
hCE1, hiCE, and rCE are depicted in Figure 3. No sim-
ple descriptor of the active site gorge would explain the
inhibitory activity of the benzils and benzoins toward
No correlations were seen between the Ki values for
enzyme inhibition and the pKa value of the hydroxyl
proton in the benzoins (Table 7). Interestingly, a
significant p value was seen for the correlation between
the Hammett constants and the Ki values for rCE with
the benzoins, but not, however, with the benzils (Table
8). This is likely a consequence of the lack of polarizabil-
ity in the C–OH bond relative to the C@O bond.
Table 9. Correlation coefficients for the QSAR models
Compound Enzyme Observed versus Cross
predicted
q2/r2
correlation
coefficient (q2)
Ki values (r2)
Benzoins
Benzils
hiCE
hCE1
rCE
0.816
0.914
0.898
0.751
0.861
0.826
0.92
0.94
0.92
2.4. QSAR analysis of fluorobenzoin- and fluorobenzil-
mediated inhibition of carboxylesterases
hiCE
hCE1
rCE
0.686
0.918
0.885
0.648
0.887
0.848
0.94
0.97
0.96
Since the results obtained from the comparisons of the
enzyme inhibition data and the electronic parameters

L. D. Hicks et al. / Bioorg. Med. Chem. 15 (2007) 3801–3817
3807
Figure 2. Graphs depicting the observed versus the predicted Ki values for the inhibition of the CEs following QSAR analysis. Data points used to
construct the model are shown in black, and the test values are indicted in red.
each enzyme. Rather, as we previously observed with
other benzil analogs,10 it is the overall hydrophobic
and electrostatic milieu of the binding site that contrib-
utes to the affinity of each analog, and these character-
istics are slightly different for each enzyme.
their inhibitory potency, the corresponding benzoin
compounds were considerably more potent CE inhibi-
tors (Table 4). This effect was most pronounced when
the fluorine atoms were either at the ortho- or the
meta-positions. For example, compounds 3 (ortho-
substituted) and 4 (meta-substituted) demonstrated
inhibition toward all CEs, whereas 5 (para-substituted)
displayed no inhibitory effect toward any of the enzymes
(see Table 4). The lack of increase in the potency of the
fluorobenzil analogs may be due to the fact that the elec-
trons in both the r and the p orbitals in the carbonyl
bonds are principally distributed toward the more elec-
tronegative O atom. Therefore, any attempt to reduce
the electron density surrounding the carbon atom (by
substituting EWG groups in the benzene ring) will actu-
ally redistribute the electrons within the polarizable p
bond toward the carbon atom. This would reduce the
overall positive charge on this atom (consistent with
the results seen in Table 5) rendering it less susceptible
to nucleophilic attack. This is also consistent with our
experimental results (e.g., most of the fluorobenzils are
poorer inhibitors of the human CEs than benzil; Table
4). The change in the electron density, due to polariza-
tion of the C–OH bond in the fluorobenzoins by addi-
tion of fluorine atoms, would be much less
pronounced and hence this effect is unlikely to be ob-
served with these compounds. Again, this is born out
3. Discussion
In this paper, we have examined the fluorine substitu-
ent effects on the inhibitory potency of a panel of flu-
orobenzoins and fluorobenzils toward mammalian
CEs. These studies were initiated since we had previ-
ously hypothesized that the electron density surround-
ing the carbonyl carbon atoms in the 1,2-dione moiety
may be an important factor in inhibitor potency.10 If
nucleophilic attack on these atoms, by the catalytic
serine in the active site of the CE, is necessary for
enzyme inhibition (Fig. 1), then reducing the electron
density around the carbonyl carbons may make the
inhibitors more potent. We reasoned that this could
be achieved by introducing EWG (e.g., fluorine atoms)
as substituents to the benzene rings of these
compounds.
While inclusion of fluorine (or multiple fluorine atoms)
within the benzil analogs did not dramatically improve

3808
L. D. Hicks et al. / Bioorg. Med. Chem. 15 (2007) 3801–3817
Figure 3. 3D-QSAR pseudoreceptor models for the inhibition of CEs by the fluorobenzoins (upper panel) and the fluorobenzils (lower panel). In
each case, the models for hiCE (a), hCE1 (b), and rCE (c) are shown as colored spheres on a hydrophobic gray grid. Areas that are hydrophobic are
indicated in gray, with blue spheres representing regions that are positively charged and hydrophobic (+0.1e), and light blue spheres corresponding to
hydrogen bond donors. Orange spheres indicate hydrogen bond acceptors and orange-red spheres correspond to areas that are negatively charged
and hydrophobic (ꢀ0.1e). The structure of benzil or benzoin is shown in black. The figure was created using Raster3D and Molscript.32
by both the MO calculations (Table 5) and the inhibi-
tion assays (Table 3).
consistent with the reduced inductive effects that would
be exerted by the fluorine atoms in the para-position as
compared to those in the meta-position.
The substituent effects exerted by the fluorine atoms
could be achieved by any one (or a combination) of
three different properties. These include resonance,
inductive, and/or field effects. Being highly electronega-
tive, it is likely that the inductive effects produced by
these atoms would be sufficient to modulate the inhibi-
tory activity of the benzoin analogs. The results we
obtained are consistent with the inductive effects exerted
by the fluorine atoms via the sigma bonds in the
molecule. Hence, when the fluorine groups are closer
to the hydroxy-ethanone moiety (e.g., in the ortho-posi-
tion), they have a more pronounced effect on the charge
on the carbon atoms, and hence the Ki values are lower.
In compounds where the fluorine group is further from
the hydroxy-ethanone chemotype (e.g., the meta- or
Additionally, intramolecular hydrogen bonding between
fluorine in the ortho-position and the benzoin hydroxyl
hydrogen atom may influence inhibitor potency. Such
a bond would generate a relatively stable 6-membered
ring that may minimize or impede rotation of the ben-
zene ring in the benzoin. Such an interaction might be
favorable, for example, if the inhibitor is locked in a
conformation that is more potent at enzyme inhibition,
or unfavorable, if the reverse is true. Comparison of the
Ki values for different ortho-substituted benzoin analogs
versus their meta-substituted isomers (e.g., 2 vs 3, 6 vs 8
or 7 vs 11) however, revealed no clear pattern of enzyme
inhibition. This may be due to the fact that all of the
biochemical assays are performed in aqueous solution
(ꢁ55 M H2O), and hence the formation of a stable
Fꢂ ꢂ ꢂH–O–C intramolecular hydrogen bond would be
unlikely.
para-substituted analogs), there is
a considerable
reduction in the potency of these molecules as CE inhib-
itors. As a consequence, an order for the Ki values for
the single substituted fluorobenzoins was observed
where ortho-< meta- < para-(3 < 4 < 5). Similarly, with
the di-substituted analogs, the ortho-meta-substituted
benzoin (10) was more potent at CE inhibition than
the ortho-para-analog (6). Again, these data are all
Attempts to correlate the predicted charge on the car-
bon atoms within the dione moiety of the benzils with
the Ki values for CE inhibition proved unsuccessful (Ta-
ble 6). However, similar analyses indicated that for the

L. D. Hicks et al. / Bioorg. Med. Chem. 15 (2007) 3801–3817
3809
benzoins, the charge on the hydroxyl carbon highly cor-
related with the inhibition constants. Indeed, Spearman
r coefficients of ꢀ0.470, ꢀ0.757, and ꢀ0.533, and p val-
ues of 0.077, 0.001, and 0.041 were obtained for hiCE,
hCE1, and rCE, respectively. Additionally, we observed
similar correlation with the charges on the carbonyl
oxygen atom and the Ki values (Table 6). This suggests
that the initiating event in enzyme inhibition in the case
of the benzoins is the interaction of the serine
nucleophile with the hydroxyl carbon (Fig. 4a). Since
the inhibition is reversible, the formation of any diol
intermediate (Fig. 4a) is presumably transient and
presumably repetitive removal and release of the proton
on the hydroxyl carbon atom would occur. This mecha-
nism would account for the inhibition of the CEs.
the inhibition of CEs can be obtained. In addition, we
have previously performed similar analyses for a variety
of different CE inhibitors, generating highly predictive
3D-pseudoreceptor site models for the different mam-
malian proteins.6,10
Analysis of the inhibition data for the benzoins and the
benzils using Quasar software yielded good r2 values for
the observed versus predicted Ki values (Table 9; Fig. 2).
In addition, cross correlation coefficients (q2) ranging
from 0.65 to 0.89 were obtained for the datasets. Since
q2 provides a measure of the predictive power of the
model, and values greater than 0.4 are generally as-
sumed to be suitable for use in biological systems,13
our results suggest that the 3D-pseudoreceptor site mod-
els will be useful in the design of novel fluorine-based
benzoin and benzil CE inhibitors.
Alternatively, since the acidity and hence the lability of
the hydroxyl proton is mediated in part by the electro-
negativity of the carbon atom, it is possible that the
Oꢀ present within the serine may remove this proton
to regenerate the amino acid (Fig. 4b). Again, this would
likely be in rapid equilibrium such that upon removal of
the inhibitor, free active protein would be obtained. It is
currently unknown whether either of the above mecha-
nisms is correct, but both chemical and structural stud-
ies are underway to assess the validity of these scenarios.
Six inhibitors with known Ki values were used to test
the ability of the benzoin models to predict Ki values,
while seven were used to test the benzil models. In
general, prediction was accurate (as indicated in
Fig. 2). However, in all of the benzoin models, com-
pound 15, having symmetric p-trifluoromethyl groups
on the phenyl rings, was systematically predicted to
be a better inhibitor than was observed experimentally.
Likewise, the inhibition constants for the benzil analog
31 were mis-predicted for hCE1 and rCE. Analysis of
the models and data did not reveal any obvious reason
why these inhibitors should have lower biological
activity than was predicted. We hypothesize that these
inhibitors may be interacting with sites on the surface
of the CEs that cannot be accounted for in the Ki
data, particularly the opening to the active site
gorge.12 We have demonstrated that the entrance to
the CE active site can influence substrate turnover
and hence it is likely that the same holds true for
enzyme inhibitors.
Since we did not identify one single parameter that
would reflect the biological activity of all of the benzoins
and the benzils, we used 3D-QSAR analysis of the data-
sets, to derive suitable relationships that would correlate
the chemical structures of the inhibitors with their bio-
logical potency. The advantage of the 3D approach is
that multiple parameters can be simultaneously evalu-
ated and hence models that more accurately represent
For example, 31 was correctly predicted by the QSAR
model to have good inhibitory activity toward rCE. As
rCE has a demonstrably larger opening to the entrance
to the active site gorge on the surface of the enzyme, this
presumably allows access of bulkier inhibitors to the cat-
alytic amino acids. Since both hiCE and hCE1 have
more constrained active site entrances,12 potentially fac-
ile access of compound 31 to the catalytic amino acids
would be impeded. This would not be predicted in the
current models. In contrast, the biological activity of
compound 30 was accurately predicted by the QSAR
models. Since 30 and 31 are isomers, we believe that it
is unlikely that the low experimental Ki values of 31
would be so significantly different from those obtained
with analog 30 unless interactions outside the active site
were responsible.
Figure 4. Proposed mechanism of inhibition of CEs by the fluor-
obenzoins. (a) The serine nucleophile (Prot-Oꢀ) removes the proton on
the hydroxy carbon atom to yield an intermediate that rearranges to
form the ethylene diol analog. However, this species is probably in
equilibrium with the benzoin and hence the reaction is readily
reversible. This repetitive transfer of the proton likely inhibits the
CE. (b) In a reversible process, the proton on the oxygen atom of the
hydroxyl group of the benzoin is transferred to the serine oxygen atom.
This results in the formation of the hydroxyl moiety on the serine
residue. However, this would likely be attacked by the oxygen atom in
the benzoin to reform the starting materials.
Consistent with out previous studies, none of the benzo-
ins or the benzils demonstrated any inhibitory activity
toward AChE or BChE, with the exception of com-
pound 23. This fluorobenzil demonstrated reasonably
good inhibition of BChE. The exact reason for this is
unclear, especially since this compound failed to inhibit
AChE. Interestingly, we have seen inhibition of both of

3810
L. D. Hicks et al. / Bioorg. Med. Chem. 15 (2007) 3801–3817
the cholinesterases by some nitrogen-containing fused
ring diones (Hyatt et al., manuscript in preparation).
These results suggest that this heteroatom is important
for interaction of such molecules with the active sites
of these proteins. This is perhaps not surprising since
the choline group that is hydrolyzed from the choline
esters contains a quaternary nitrogen that interacts with
negatively charged amino acid residues within the
cholinesterase active sites. This so-called anionic site is
critical for efficient hydrolysis of these esters. Since none
of the phenylethane-1,2-diones, or their hydroxy-etha-
none analogs that we have assayed, can form stable
positively charged quaternary compounds, we believe it
is unlikely that these inhibitors would demonstrate
activity toward AChE or BChE. The data presented here,
and in previous publications,10,11 support this hypothesis.
on ice, crude product was filtered and washed with ice
cold 75% ethanol. Material was re-crystallized from eth-
anol, and purity and structures were assessed by melting
point, TLC, NMR, and total elemental analyses. All
physical data for the synthesized compounds are shown
in Table 10.
4.2.1. 1,2-Bis(2-fluorophenyl)-2-hydroxyethanone (3). 1,2-
Bis(2-fluorophenyl)-2-hydroxyethanone was synthesized
from 2-fluorobenzaldehyde. Physical and NMR param-
eters of 3 were consistent with those found in the
literature.15
4.2.2. 1,2-Bis(3-fluorophenyl)-2-hydroxyethanone (4). 1,2-
Bis(3-fluorophenyl)-2-hydroxyethanone was synthesized
by the condensation of 3-fluorobenzaldehyde. Physical
and NMR parameters of 4 were consistent with those
previously reported.15
Overall our studies suggest that the mechanism of CE
inhibition by benzil and its analogs works via abortive
nucleophilic attack of the active site serine toward one
of the carbonyl groups within the molecule (Fig. 1). Sub-
stitution of fluorine within the benzene rings did not
alter benzil potency, but resulted in a significant increase
in the inhibitory activity of the corresponding benzoins.
This is likely due to a decrease in the electron density
surrounding the target carbon atom, resulting in more
facile attack by the serine nucleophile. In summary,
these studies should allow the design of more potent
selective CE inhibitors.
4.2.3. 1,2-Bis(4-fluorophenyl)-2-hydroxyethanone (5). 1,2-
Bis(4-fluorophenyl)-2-hydroxyethanone was synthesized
from 4-fluorobenzaldehyde to give white crystals in 75%
yield.
4.2.4. 1,2-Bis(2,4-difluorophenyl)-2-hydroxyethanone (6).
1,2-Bis(2,4-difluorophenyl)-2-hydroxyethanone was syn-
thesized from 2,4-difluorobenzaldehyde to give white
crystals in 67% yield.
4.2.5. 1,2-Bis(2,6-difluorophenyl)-2-hydroxyethanone (7).
1,2-Bis(2,6-difluorophenyl)-2-hydroxyethanone was syn-
thesized from 2,6-difluorobenzaldehyde to give white
crystals in 72% yield.
4. Experimental
4.1. General
4.2.6. 1,2-Bis(3,4-difluorophenyl)-2-hydroxyethanone (8).
1,2-Bis(3,4-difluorophenyl)-2-hydroxyethanone was syn-
thesized from 3,4-difluorobenzaldehyde to give white
crystals in 70% yield.
Thiamine, copper acetate, ammonium nitrate, and flu-
orobenzaldehydes were purchased from Sigma–Aldrich
(St. Louis, MO). Product formation was monitored by
thin layer chromatography TLC using pre-coated sil-
ica gel GF plates (Analtech Inc., Newark, DE) and
visualized using UV light (254 nm). Melting points
were determined using a Mel-temp (Barnstead Inter-
national, Dubuque, IA) and are reported uncorrected.
NMR spectra (1H and 13C) were obtained using a
Bruker DPX-400 (400 MHz) spectrometer using
CDCl3 (Aldrich) as a solvent and chemical shifts are
reported in d units (ppm) using tetramethylsilane as
an internal standard with coupling constants (J) indi-
cated in Hertz (Hz). Elemental analyses were per-
formed by either Atlantic Microlabs (Norcross, GA)
or Galbraith Laboratories (Knoxville, TN). Mass
spectra were recorded on a VG 70-VSE(B) instrument
(UIUC, Urbana-Champagne, IL) using EI or CI
techniques.
4.2.7. 1,2-Bis(2,3-difluorophenyl)-2-hydroxyethanone (9).
1,2-Bis(2,3-difluorophenyl)-2-hydroxyethanone was syn-
thesized from 2,3-difluorobenzaldehyde to give white
crystals in 73% yield.
4.2.8. 1,2-Bis(2,5-difluorophenyl)-2-hydroxyethanone (10).
1,2-Bis(2,5-difluorophenyl)-2-hydroxyethanone was syn-
thesized from 2,5-difluorobenzaldehyde to give white crys-
tals in 72% yield.
4.2.9. 1,2-Bis(3,5-difluorophenyl)-2-hydroxyethanone (11).
1,2-Bis(3,5-difluorophenyl)-2-hydroxyethanone was syn-
thesized from 3,5-difluorobenzaldehyde to give white crys-
tals in 72% yield.
4.2.10. 1,2-Bis(2,3,5-trifluorophenyl)-2-hydroxyethanone
(12). 1,2-Bis(2,3,5-trifluorophenyl)-2-hydroxyethanone
was synthesized from 2,3,5-trifluorobenzaldehyde to
give white crystals in 74% yield.
4.2. Synthesis of fluorobenzoins
Fluorinated benzoins (1,2-diphenyl-2-hydroxy-ethanon-
es) were synthesized by condensation of the correspond-
ingly substituted fluorobenzaldehyde (0.04 mol) in the
presence of thiamine hydrochloride (0.002 mol) in eth-
anolic sodium hydroxide (0.005 mol).14 Routinely reac-
tions were run at 50 °C for 48 h and following cooling
4.2.11. 1,2-Bis(2,3,4-trifluorophenyl)-2-hydroxyethanone
(13). 1,2-Bis(2,3,4-trifluorophenyl)-2-hydroxyethanone
was synthesized from 2,3,4-trifluorobenzaldehyde to
give white crystals in 72% yield.

Table 10. Physical and NMR parameters for the compounds synthesized in this paper
ID
Mp
1H NMR
13C NMR
MS m/z
Elemental analysis
5
81–82
d 7.95 (m, J = 7.9 Hz, 2H),
d 7.15 (m, J = 7.3 Hz, 2H),
d 7.0 (m, J = 7.0 Hz, 4H),
d 5.95 (s, 1H) d 4.2 (bs, 1H)
d 197.20 (C@O), d 167.38
(Ar, C–F), d 161.56 (Ar, C–F),
d 134.83 (Ar, C–C@O),
248
Anal. (C14H10F2O2); Calcd (%): C,
67.74; H, 4.06;
F, 15.31. Found (%): C, 67.81; H,
4.03; F, 15.44
d 133.92 (Ar, C–C–OH),
d 131.70 (Ar, C–H), d 129.26
(Ar, C–H), d 116.37
(Ar, C–H), d115.99 (Ar, C–H),
d 88.36 (C–OH)
6
83–85
d 7.95 (m, J = 7.9 Hz, 1H),
d 7.75 (m, J = 7.5 Hz, 1H),
d 7.15 (m, J = 7.2 Hz, 2H),
d 7.05 (m, J = 7.0 Hz, 2H),
d 6.1 (s, 1H), d 4.3 (bs, 1H)
d 188.57 (C@O), d 165.4
284
Anal. (C14H8F4O2); Calcd (%): C,
59.16; H, 2.84;
(Ar, C–F), d 164.49 (Ar, C–F),
d 162.60 (Ar, C–F), d 162.47
(Ar, C–F), d 132.84 (Ar, C–H),
d 132.12 (Ar, C–H), d 118.01
(Ar, C–C–OH), d 117.90
F, 26.74. Found (%): C, 59.22; H,
2.73; F, 26.78
(Ar, C–C@O), d 112.69 (Ar, C–H),
d 111.99 (Ar, C–H),
d 111.68 (Ar, C–H), d 105.15
(Ar, C–H), d 78.62 (C–OH)
d 197.2 (C@O), d 162.70
(Ar, C–F), d 159.26 (Ar, C–F),
d 133.86 (Ar, C–H), d 130.13
(Ar, C–H), d 115.23
7
71–73
d 7.51(m, J = 7.6 Hz, 1H),7.30
(m, J = 7.4 Hz, 1H),
284
Anal. (C14H8F4O2); Calcd (%): C,
59.16; H, 2.84;
d 7.20 (m, J = 7.2 Hz, 2H),
d 7.10 (m, J = 7.1 Hz, 2H),
d 6.01 (s, 1H) d 4.2 (bs, 1H)
F, 26.74. Found (%): C, 59.23; H,
2.63; F, 26.75
(Ar, C–C@O), d 112.54 (Ar, C–H),
d 112.17 (Ar, C–H),
d 110.12 (Ar, C–C–OH),
d 76.31 (C–OH)
8
62–64
d 7.85 (m, J = 7.9 Hz, 4H),
d 7.41 (m, J = 7.4 Hz, 2H),
d 6.00 (s, 1H), d 4.2 (bs, 1H)
d 191.23 (C@O), d 155.21
(Ar, C–F), d 151.61 (Ar, C–F),
d 150.12 (Ar, C–F), d 146.33
(Ar, C–F), d 132.99 (Ar, C–C@O),
d 132.01 (Ar, C–C–OH), d 127.54
(Ar, C–H), d 126.92 (Ar, C–H),
d 124.12 (Ar, C–H), d 121.36
(Ar, C–H), d 119.62 (Ar, C–H),
d 117.35 (Ar, C–H), d 82.41
(Ar, C–OH)
284
Anal. (C14H8F4O2); Calcd (%): C,
59.16; H, 2.84;
F, 26.74. Found (%): C,
59.15; H, 2.66; F, 26.67
9
54–56
d 7.60 (m, J = 7.6 Hz, 1H),
d 7.35 (m, J = 7.4 Hz, 1H),
d 7.10 (m, J = 7.1 Hz, 4H)
d 6.10 (s, 1H), d 4.48 (bs, 1H)
d 195.74 (C@O), d 156.96
(Ar, C–F), d 150.14 (Ar, C–F),
d 147.23 (Ar, C–F), d 139.85
(Ar, C–F), d 127.66 (Ar, C–C–OH),
d 127.25 (Ar, C–C@O), d 125.36
(Ar, C–H), d 125.33 (Ar, C–H),
d 124.93 (Ar, C–H), d 122.7
(Ar, C–H), d 120.52
284
Anal. (C14H8F4O2); Calcd (%): C,
59.16; H, 2.84;
F, 26.74. Found (%): C, 59.10; H,
2.80; F, 26.75
(Ar, C–H), d 117.96
(Ar, C–H), d 78.65 (C–OH)
(continued on next page)

Table 10 (continued)
ID
Mp
1H NMR
13C NMR
MS m/z
Elemental analysis
10
91–93
d 7.8 (m, J = 7.8 Hz, 2H)
d 7.50 (m, J = 7.5 Hz, 2H),
d 7.20 (m, J = 7.3 Hz, 2H),
d 6.2 (s, 1H), d 4.55 (bs, 1H)
d 188.44 (C@O), d 159.91(Ar, C–F),
d 157.73 (Ar, C–F), d 157.70
(Ar, C–F),
284
Anal. (C14H8F4O2); Calcd (%): C, 59.16; H, 2.84;
F, 26.74. Found (%): C, 59.06; H, 2.70; F, 26.73
d 156.82 (Ar, C–F), d 123.87
(Ar, C–C–OH), d 123.62 (Ar, C–C@O),
d 122.09 (Ar, C–H), d 118.38 (Ar, C–H),
d 118.25 (Ar, C–H), d 117.93 (Ar, C–H),
d 116.39 (Ar, C–H), d 115.37
(Ar, C–H), d 79.26 (C–OH)
d 192.3 (C@O), d 164.50
(Ar, C–F), d 161.3 (Ar, C–F),
d 139.23 (Ar, C–C@O),
d 138.86 (Ar, C–C–OH), d 113.06
(Ar, C–H) d 110.26 (Ar, C–H),
d 109.19
11
78–80
d 7.25 (m, J = 7.4 Hz, 2H),
d 7.05 (m, J = 7.0 Hz, 1H),
d 6.95 (m, J = 6.9 Hz, 2H),
d 6.98 (m, 6.8 Hz, 1H),
284
Anal. (C14H8F4O2); Calcd (%): C, 59.16; H, 2.84;
F, 26.74. Found (%): C, 59.18; H, 2.71; F, 26.6
d 5.95 (s, 1H), d 4.15 (bs, 1H)
(Ar, C–H) d 102.12 (Ar, C–H),
d 86.32 (C–OH)
12
73–75
d 7.35 (d, J = 7.4 Hz, 1H),
d 7.15 (d, J = 7.2 Hz, 1H),
d 6.9 (d, J = 6.9 Hz, 1H),
d 6.65 (d, J = 6.7 Hz, 1H),
d 6.01 (s, 1H), d 4.5 (bs, 1H)
d 192.23 (C@O), d 160.10 (Ar, C–F),
d 157.89 (Ar, C–F), d 157.32 (Ar, C–F),
d 149.65 (Ar, C–F), d 140.23 (Ar, C–F),
d 133.26 (Ar, C–F), 125.26 (Ar, C–C–OH),
d 124.45 (Ar, C–C@O), d 113.66 (Ar, C–H),
d 112.19 (Ar, C–H), d 110.87(Ar, C–H),
d 102.70 (Ar, C–H), d 81.23 (C–OH)
d 191.23 (C@O), d 157.32 (Ar, C–F),
d 151.56 (Ar, C–F), d 148.0 (Ar, C–F),
d 143.65 (Ar, C–F), d 140.95
(Ar, C–F), d 139.99 (Ar, C–F),
d 130.26 (Ar, C–H),
320
Anal. (C14H6F6O2); Calcd (%): C, 52.52; H, 1.89;
F, 35.60. Found (%): C, 52.56; H, 1.71; F, 35.43
13
63–65
d 7.75 (m, J = 7.7 Hz, 1H),
d 7.50(m, J = 7.6 Hz, 1H),
d 7.35 (m, J = 7.3 Hz, 1H)
d 7.25 (m, J = 7.3 Hz, 1H)
d 6.0 (s, 1H), d 4.50 (bs, 1H)
320
Anal. (C14H6F6O2); Calcd (%): C, 52.52; H, 1.89;
F, 35.60. Found (%): C, 52.45; H, 1.70; F, 35.51
129.26 (Ar, C–H), d 123.45
(Ar, C–C–OH), d 122.65
(Ar, C–C@O), d 118.23
(Ar, C–H), d 113.66
(Ar, C–H), d 83.23 (C–OH)
d 197.67(C@O), d 161.34
(Ar, C–F), d 152.36
14
75–77
d 7.25 (s, 2H), d 7.95 (s, 2H),
d 5.9 (s, 1H), d 4.1 (bs, 1H)
348
Anal. (C14H6F6O2); Calcd (%): C, 52.52; H, 1.89;
F, 35.60. Found (%): C, 52.44; H, 1.71; F, 35.51
(Ar, C–F), d 145.27 (Ar, C–F),
d 140.28 (Ar, C–F), d 133.96
(Ar, C–C@O), d 133.72
(Ar, C–C–OH), d 115.09
(Ar, C–H), d 113.36
(Ar, C–H), d 85.62 (C–OH)

15
87–89
d 8.0 (d, J = 8.0 Hz, 2H),
d 7.85 (d, J = 7.7 Hz, 2H),
d 7.30 (d, J = 7.5 Hz, 2H),
d 7.2 (d, J = 7.3 Hz, 2H),
d 6.01 (s, 1H), d 4.42 (bs, 1H)
d 197.68 (C@O),
d 141.95 (Ar, C–C@O),
d 138.98 (Ar, C–C–OH), d 135.61
(Ar, C–CF3), d 129.40
348
Anal. (C16H10F6O2); Calcd (%): C, 55.18; H, 2.89;
F, 32.73. Found (%): C, 55.20; H, 2.84; F, 33.00
(Ar, C–CF3), d 128.10
(Ar, C–H), d 126.84
(Ar, C–H),
d 125.94 (Ar, C–H),
d 124.12 (Ar, C–H),
d 123.69 (CF3), d 85.12 (C–OH)
d 191.32 (C@O), d 136.89
(Ar, C–C@O), d 136.55
16
56–58
d 8.0 (s, 2H), d 7.70 (d, J = 8.0 Hz, 2H),
d 7.60 (m, J = 7.9 Hz, 2H),
348
Anal. (C16H10F6O2); Calcd (%): C, 55.18; H, 2.89;
F, 32.73. Found (%): C, 55.38; H, 2.85; F, 32.92
d 7.4 (d, J = 7.7 Hz, 2H), d 6.01 (s, 1H),
d 4.42 (bs, 1H)
(Ar, C–C–OH), d 133.65
(Ar, C–H), d 132.86
(Ar, C–H), d 130.46 (Ar, C–CF3),
d 129.55 (Ar, C–CF3),
d 128.23 (Ar, C–H), d 127. 56
(Ar, C–H), d 126.71 (Ar, C–H),
d 125.91 (Ar, C–H),
d 124.0 (Ar, C–H), d 123.99 (CF3)
d 123.92 (CF3), d 85.18 (C–OH)
d 190.21 (C@O), d 163.0 (Ar, C–F),
d 136.72 (Ar, C–H), d 130.89 (Ar, C–H),
d 125.00 (Ar, C–H), d 122.32 (Ar, C–C@O)
17
18
103–105
97–99
d 8.05 (d, J = 8.0 Hz, 2H),
d 7.50 (m, J = 7.9 Hz, 2H),
d 7.05 (m, J = 7.7 Hz, 2H),
d 6.85 (m, J = 7.7 Hz, 2H)
d 7.74 (d, J = 8.0 HZ, 2H),
d 7.71 (d, J = 7.8 Hz, 2H),
d 7.51 (m, J = 7.5 Hz, 2H)
d 7.39 (d, J = 7.4 Hz, 2H)
d 8.05 (m, J = 7.9 Hz, 4H),
d 7.50 (m, J = 7.5 Hz, 4H)
246
246
Anal. (C14H8F2O2); Calcd (%): C, 68.30; H, 3.28;
F, 15.43. Found (%): C, 68.15; H, 3.18; F, 15.66
d 192.20 (C@O), d 162.89 (Ar, C–F),
d 138.68 (Ar, C–C@O), d 130.89 (Ar, C–H),
d 126.05 (Ar, C–H), d 122.26 (Ar, C–H),
d 116.21 (Ar, C–H)
d 192.23 (C@O), d 168.17 (Ar, C–F),
d 132.82 (Ar, C–H), d 128.36
(Ar, C–C@O), d 116.47 (Ar, C–H)
d 188.45 (C@O), d 169.48 (Ar, C–F),
d 165.32 (Ar, C–F), d 134.38
(Ar, C–H), d 118.38 (Ar, C–C@O),
d 115.71 (Ar, C–H), d 110.67 (Ar, C–H)
d 185.07 (C@O), d 162.37 (Ar, C–F),
d 135.86 (Ar, C–H), d 112.39
(Ar, C–H), d 111.84 (Ar, C–C@O)
d 190.20 (C@O), d 156.30 (Ar, C–F),
d 150.37 (Ar, C–F), d 133.70
(Ar, C–C@O), d 127.70 (Ar, C–H),
d 126.30 (Ar, C–H), d 118.50 (Ar, C–H)
d 188.60 (C@O), d 157.50 (Ar, C–F),
d 150.0 (Ar, C–F), d 128.50
Anal. (C14H8F2O2); Calcd (%): C, 68.30; H, 3.28;
F, 15.43. Found (%): C, 68.17; H, 3.25; F, 15.59
19
20
118–119
91–93
246
282
Anal. (C14H8F2O2); Calcd (%): C, 68.30; H, 3.28;
F, 15.43. Found (%): C, 68.51; H, 3.20; F, 15.7
d 8.49 (d, J = 8.0 Hz, 2H),
d 7.40 (m, J = 7.7 Hz, 2H),
d 7.20 (m, J = 7.8 Hz, 2H)
Anal. (C14H6F4O2); Calcd (%): C, 59.59; H, 2.14;
F, 26.93. Found (%): C, 59.32; H, 2.05; F, 26.77
21
22
163–165
107–109
d 7.95 (m, J = 7.9 Hz, 2H),
d 7.45 (m, J = 7.4 Hz, 4H)
282
282
Anal. (C14H6F4O2); Calcd (%): C, 59.59; H, 2.14;
F, 26.93. Found (%): C, 59.59; H, 2.05; F, 27.13
d 8.05 (d, J = 8.1 Hz, 2H),
d 7.85 (d, J = 7.9 Hz, 2H),
d 7.70 (m, J = 7.7 Hz, 2H)
Anal. (C14H6F4O2); Calcd (%): C, 59.59; H, 2.14;
F, 26.93. Found (%): C, 59.32; H, 2.08; F, 26.63
23
91–93
d 7.85 (d, J = 8.0 Hz, 2H),
d 7.45 (m, J = 7.5 Hz, 2H),
d 7.25 (m, J = 7.4 Hz, 2H)
282
Anal. (C14H6F4O2); Calcd (%): C, 59.59; H, 2.14;
F, 26.93. Found (%): C, 59.45; H, 2.03; F, 26.98
(Ar, C–H), d 126.50 (Ar, C–H),
d 125.10 (Ar, C–C@O), d 123.00 (Ar, C–H)
(continued on next page)

Table 10 (continued)
ID
Mp
1H NMR
13C NMR
MS m/z
Elemental analysis
24
110–112
d 8.49 (d, J = 8.1 Hz, 2H) d 7.38
(m, J = 7.8 Hz, 2H),
d 188.50 (C@O), d 160.20 (Ar, C–F),
d 157.75 (Ar, C–F), d 124.75
282
Anal. (C14H6F4O2); calc. (%):
C, 59.59; H, 2.14;
d 7.39 (m, J = 7.7 Hz, 2H)
(Ar, C–C@O), d 122.10 (Ar, C–H),
d 118.20 (Ar, C–H), d 116.50 (Ar, C–H)
d 183.55 (C@O), d 158.50 (Ar, C–F),
d 155.00 (Ar, C–F), d 148.55
(Ar, C–F), d 122.80 (Ar, C–H), d 113.30
(Ar, C–CH), d 112.0 (Ar, C–C@O)
d 186.80 (C@O), d 158.30 (Ar, C–F),
d 157.75 (Ar, C–F), d 146.70
(Ar, C–F), d 123.0 (Ar, C–C@O),
d 111.70 (Ar, C–H), d 110.50 (Ar, C–H)
d 194.38 (C@O), d 157.32 (Ar, C–F),
d 151.32 (Ar, C–F), d 138.39
(Ar, C–F), d 127.29 (Ar, C–H), d 121.25
(Ar, C–H), d 120.42 (Ar, C–C@O)
d 191.90 (C@O), d 136.40 (Ar, C–CF3),
d 135.25 (Ar, C–C@O),
F, 26.93. Found (%): C, 59.42; H,
2.08; F, 27.13
26
27
28
30
31
124-126
98–100
94–96
d 7.45 (m, J = 7.4 Hz, 2H),
d 7.00 (m, J = 7.0 Hz, 2H)
318
318
318
346
346
Anal. (C14H4F6O2); Calcd (%): C,
52.85; H, 1.27;
F, 35.83. Found (%): C, 52.62; H,
1.15; F, 35.49
d 7.55 (m, J = 7.5 Hz, 2H), d 7.30 (m, 2H)
Anal. (C14H4F6O2); Calcd (%): C,
52.85; H, 1.27;
F, 35.83. Found (%): C, 52.68; H,
1.16; F, 35.72
d 7.86 (m, J = 7.9 Hz, 2H), d 7.23
(m, J = 7.2 Hz, 2H)
Anal. (C14H4F6O2); Calcd (%): C,
52.85; H, 1.27;
F, 35.83. Found (%): C, 52.72; H,
1.21; F, 36.03
134–136
101–103
d 8.10 (d, J = 8.1 Hz, 4H), d 7.78
(d, J = 7.8 Hz, 4H)
Anal. (C16H8F6O2); Calcd (%): C,
55.50; H, 2.33;
d 130.37 (Ar, C–H), d 126.18 (Ar, C–H),
d 124.63 (CF3)
d 191.32 (C@O), d 139.05 (Ar, C–C@O),
d 132.14 (Ar, C–CF3),
d 131.58 (Ar, C–H), d 130.25 (Ar, C–H),
d 129.85 (Ar, C–H),
F, 32.92. Found (%): C, 55.28; H,
2.20; F, 33.2
d 8.30 (s, 2H), d 8.18
Anal. (C16H8F6O2); Calcd (%): C,
55.50; H, 2.33;
(d, J = 8.2 Hz, 2H), d 7.95
(d, J = 7.9 Hz, 2H), d 7.78
(m, J = 7.7 Hz, 2H)
F, 32.92. Found (%): C, 55.31; H,
2.23; F, 33.21
d 127.69 (Ar, C–H), d 124.70 (CF3)
d 188.45 (C@O), d 136.68 (Ar, C–CF3),
d 133.70 (Ar, C–C@O),
32
103–105
d 8.10 (s, 2H), d 8.0
(d, J = 8.0 Hz, 2H),
483 (M+H)+1
Anal. (C18H6F12O2); Calcd (%): C,
44.83; H, 1.25;
d 7.78 (d, J = 7.7 Hz, 2H)
d 130.58 (Ar, C–H), d 129.57 (Ar, C–CF3),
d 128.79 (Ar, C–H),
F, 47.28. Found (%): C, 44.76; H,
1.18; F, 47.55
d 124.29 (Ar, C–H), d 123.78 (Ar, CF3),
d 121.43 (Ar, CF3)
d 188.11 (C@O), d 135.23 (Ar, C–C@O),
d 132.42 (Ar, C–CF3),
d 129.03 (Ar, C–H), d 128.10 (Ar, C–H),
d 124.11 (CF3)
d 188.09 (C@O), d 134.38 (Ar, C–CF3),
d 132.41 (Ar, C–CF3),
33
34
130–132
135–137
d 8.00 (s, 4H), d 7.80 (s, 2H)
483 (M+H)+1
483 (M+H)+1
Anal. (C18H6F12O2); Calcd (%): C,
44.83; H, 1.25;
F, 47.28. Found (%): C, 44.83;
H, 1.14; F, 47.47
d 8.08 (s, 2H), d 7.71
(d, J = 8.0Hz, 2H),
Anal. (C18H6F12O2); Calcd (%): C,
44.83; H, 1.25;
d 7.79 (d, J = 7.7Hz, 2H)
d 131.33 (Ar, C–H), d 130.01
(Ar, C–C@O), d 127.31(Ar, C–H),
d 126.58 (Ar, C–H),
F, 47.28. Found (%): C, 44.86; H,
1.09; F, 47.35
d 124.17(CF3), d 120.36 (CF3)

L. D. Hicks et al. / Bioorg. Med. Chem. 15 (2007) 3801–3817
3815
4.2.12. 1,2-Bis(3,4,5-trifluorophenyl)-2-hydroxyethanone
(14). 1,2-bis(3,4,5-trifluorophenyl)-2-hydroxyethanone
was synthesized from 3,4,5-trifluorobenzaldehyde to
give white crystals in 70% yield.
4.3.10. 1,2-Bis(2,3,6-trifluorophenyl)ethane-1,2-dione (26).
1,2-Bis(2,3,6-trifluorophenyl)ethane-1,2-dione was synthe-
sized from 2,3,6-trifluorobenzaldehyde under the condi-
tions described for the benzoin reactions. This was
presumably due to immediate oxidation of the benzoin
to the corresponding benzil.
4.2.13. 1,2-Bis[4-(trifluoromethyl)phenyl]-2-hydroxyetha-
none (15). 1,2-Bis[4-(trifluoromethyl)phenyl]-2-hydroxyeth-
anonewassynthesizedfrom4-trifluoromethylbenzaldehyde
to give white crystals in 68% yield.
4.3.11. 1,2-Bis(2,3,5-trifluorophenyl)ethane-1,2-dione (27).
1,2-Bis(2,3,5-trifluorophenyl)ethane-1,2-dione was synthe-
sized from 12 to give yellow crystals in 90% yield.
4.2.14. 1,2-Bis[3-(trifluoromethyl)phenyl]-2-hydroxyetha-
none (16). 1,2-Bis[3-(trifluoromethyl)phenyl]-2-hydroxyeth-
anonewassynthesizedfrom3-trifluoromethylbenzaldehyde
to give white crystals in 69% yield.
4.3.12. 1,2-Bis(2,3,4-trifluorophenyl)ethane-1,2-dione (28).
1,2-Bis(2,3,4-trifluorophenyl)ethane-1,2-dione was synthe-
sized from the oxidation of 13 to give yellow crystals in
95% yield.
4.3. Synthesis of fluorobenzils
4.3.13. 1,2-Bis(3,4,5-trifluorophenyl)ethane-1,2-dione (29).
1,2-Bis(3,4,5-trifluorophenyl)ethane-1,2-dione was synthe-
sized from 14 to give yellow crystals in 94% yield. Physical
and NMR parameters of 29 were as previously
described.10
The substituted fluorobenzils were synthesized by oxida-
tion of the corresponding benzoin using copper acetate
(0.001 mol) and ammonium nitrate (0.006 mol) in 80%
acetic acid.16 Briefly, the benzoin was refluxed for
90 min and following cooling, the product appeared as
a solid yellow mass. After washing extensively with
water and cold 75% ethanol, the benzil was re-crystal-
lized from ethanol. Product purity was assessed as de-
scribed above for the benzoins.
4.3.14. 1,2-Bis[4-(trifluoromethyl)phenyl]ethane-1,2-dione
(30). 1,2-Bis[4-(trifluoromethyl)phenyl]ethane-1,2-dione
was synthesized from 15 to give yellow crystals in 97%
yield.
4.3.1. 1,2-Bis(2-fluorophenyl)ethane-1,2-dione (17). 1,2-
Bis(2-fluorophenyl)ethane-1,2-dione was synthesized
from 3 to give yellow crystals in 95% yield.
4.3.15. 1,2-Bis[3-(trifluoromethyl)phenyl]ethane-1,2-dione
(31). 1,2-Bis[3-(trifluoromethyl)phenyl]ethane-1,2-dione
was synthesized from 16 to give yellow crystals in 90%
yield.
4.3.2. 1,2-Bis(3-fluorophenyl)ethane-1,2-dione (18). 1,2-
Bis(3-fluorophenyl)ethane-1,2-dione was synthesized
from 4 to give yellow crystals in 98% yield.
4.4. Synthesis of 1,2-bis[2,4-bis(trifluoromethyl)phenyl]eth-
ane-1,2-dione (32), 1,2-bis[3,5-bis(trifluoromethyl)phenyl]-
ethane-1,2-dione (33), and 1,2-bis[2,5-bis(trifluoromethyl)-
phenyl]ethane-1,2-dione (34)
4.3.3. 1,2-Bis(4-fluorophenyl)ethane-1,2-dione (19). 1,2-
Bis(4-fluorophenyl)ethane-1,2-dione was synthesized
from 5 to give yellow crystals in 98% yield.
Synthesis of the above compounds was achieved by di-
rect condensation of the substituted benzaldehyde using
the method described for the benzoins (see above). Un-
der these conditions, the benzil analog was produced,
presumably via immediate oxidation of the benzoin
intermediate.
4.3.4. 1,2-Bis(2,4-difluorophenyl)ethane-1,2-dione (20).
1,2-Bis(2,4-difluorophenyl)ethane-1,2-dione was synthe-
sized from 6 to give yellow crystals in 95% yield.
4.3.5. 1,2-Bis(2,6-difluorophenyl)ethane-1,2-dione (21).
1,2-Bis(2,6-difluorophenyl)ethane-1,2-dione was synthe-
sized from 7 to give yellow crystals in 98% yield.
4.4.1. 1,2-Bis[2,4-bis(trifluoromethyl)phenyl]ethane-1,
2-dione (32). 1,2-Bis[2,4-bis(trifluoromethyl)phenyl]eth-
ane-1,2-dione was synthesized from bis-(2,4-trifluorom-
ethyl)benzaldehyde to give yellow crystals in 76% yield.
4.3.6. 1,2-Bis(3,4-difluorophenyl)ethane-1,2-dione (22).
1,2-Bis(3,4-difluorophenyl)ethane-1,2-dione was synthe-
sized from 8 to give yellow crystals in 98% yield.
4.4.2. 1,2-Bis[3,5-bis(trifluoromethyl)phenyl]ethane-1,
2-dione (33). 1,2-Bis[3,5-bis(trifluoromethyl)phenyl]eth-
ane-1,2-dione was synthesized from bis-(3,5-trifluorom-
ethyl)benzaldehyde to give yellow crystals in 76% yield.
4.3.7. 1,2-Bis(2,3-fluorophenyl)ethane-1,2-dione (23). 1,2-
Bis(2,3-fluorophenyl)ethane-1,2-dione was synthesized
from 9 to give yellow crystals in 93% yield.
4.4.3. 1,2-Bis[2,5-bis(trifluoromethyl)phenyl]ethane-1,
2-dione (34). 1,2-Bis[2,5-bis(trifluoromethyl)phenyl]eth-
ane-1,2-dione was synthesized from bis-(2,5-trifluorom-
ethyl)benzaldehyde to give yellow crystals in 76% yield.
4.3.8. 1,2-Bis(2,5-difluorophenyl)ethane-1,2-dione (24).
1,2-Bis(2,5-difluorophenyl)ethane-1,2-dione was synthe-
sized from 10 to give yellow crystals in 96% yield.
4.3.9. 1,2-Bis(3,5-difluorophenyl)ethane-1,2-dione (25). 1,2-
Bis(3,5-difluorophenyl)ethane-1,2-dione was synthesized
from 11 to give yellow crystals in 97% yield. Physical and
NMR parameters of 25 were as previously described.10
4.5. Enzymes
Pure rCE and hCE1 were prepared as described previ-
ously.17 hiCE was prepared by concentration of bacu-

3816
L. D. Hicks et al. / Bioorg. Med. Chem. 15 (2007) 3801–3817
lovirus media from Sf9 cells expressing a secreted form
of the protein. While not homogeneous, the prepara-
tion was at least 60% pure. Since no CE activity is
expressed or secreted from uninfected Sf9 cells, the
only CE present in the culture media was the recombi-
nant hiCE protein. The GenBank Accession Nos. of
the cDNAs used to generate the enzymes for this study
were as follows: hiCE, Y09616;18 hCE1, M73499;19
rCE, AF036930.20
compound was constructed using Gauss-View and
geometry optimizations were performed at the B3LYP/
6-31G(p,d) level of theory.26,27 Mulliken atomic charges
for atoms within the molecules were calculated from
these datasets. pKa values were predicted using ChemSil-
ico Predict v2.0 software (ChemSilico LLC, Tewksbury,
MA) and Hammett substituent constants were obtained
from previously published reports.28
4.10. Linear regression and Spearman correlation
analyses
Human acetylcholinesterase (AChE) and butyrylcholin-
esterase (BChE) were obtained from Sigma–Aldrich.
Datasets were analyzed using GraphPad Prism software.
This allowed for simultaneous calculation of both linear
regression correlates (r2) and Spearman r coefficients.
For the latter analyses, Spearman r values close to 1
or ꢀ1 indicate good correlations, whereas values closer
to 0 indicate a lack of statistical correlation.
4.6. Inhibition of carboxylesterases
CE inhibition was assessed using a spectrophotometric
multiwell plate assay using 3 mM o-nitrophenyl acetate
(o-NPA) as a substrate.6,11 Briefly, the test compound
and substrate (o-NPA) were aliquoted into duplicate
wells of a 96-well plate and enzyme was added using a
multiwell pipettor. The rate of change in absorbance
at 420 nm was measured at 15 s intervals for 5 min
and compared to wells containing no inhibitor. Rou-
tinely, inhibitor concentrations ranged from 1 nM to
100 lM. All assays were performed in duplicate and in-
cluded both positive (50 lM bis(4-nitrophenyl)phos-
phate) and negative controls (DMSO, no enzyme).
4.11. 3D-QSAR analysis
3D-QSAR analysis was performed as previously
described.6,10 Briefly, compounds were initially con-
structed using Chem3D and atom types were determined
using the antechamber module of AMBER7 (University
of California, San Francisco, CA). After assignment of
partial atomic charges using the bond charge correction
approach,29 compounds were analyzed using Quasar 4.0
software.29–31 This program generates a 3D-receptor-sur-
face model that contains the molecular properties of both
the receptor site and the ligand that will be docked into
this domain. Typically, 200 independent models are
generated for each dataset and these are evaluated to yield
7000 pseudoreceptor site models. Model evaluation was
then performed until the cross correlation coefficients
(q2) exceed 0.7 for the observed versus the predicted Ki
values. Routinely this produced correlation coefficients
(r2) of >0.9.
4.7. Inhibition of acetylcholinesterase and
butyrylcholinesterase
The ability of compounds to inhibit AChE and BChE
was performed as previously described using either
1 mM acetylthiocholine (AcTCh) or butyrylthiocholine
(BuTCh), respectively, as substrates.21,22
4.8. Determination of Ki values
Data obtained from the above assays were fitted to the fol-
lowing equation23 to determine the inhibition constant
(Ki)
Acknowledgments
2
2
^
^
^
½Iꢃf½sꢃð1 ꢀ I Þ þ KsðI ꢀ I Þg
This work was supported in part by NIH Grants
CA76202, CA79763, CA98468, CA108775, DA18116,
a Cancer Center Core Grant P30 CA 21765, and by
the American Lebanese Syrian Associated Charities.
i ¼
^
^
^
½Iꢃf½sꢃ þ I ꢄ Ksg þ KifI ꢄ ½sꢃ þ I ꢄ Ksg
where i is the fractional inhibition, [I] is the inhibitor
concentration, [s] is the substrate concentration, a is
the change in affinity of substrate for enzyme, b is
the change in the rate of enzyme substrate complex
decomposition, Ks is the dissociation constant for the
enzyme substrate complex, and Ki is the inhibitor con-
stant. Curve fits were generated (where a ranged from 0
to 1 and b ranged from 0 to 1) using GraphPad Prism
software (San Diego, CA) and those generating the
highest r2 values were further analyzed using Akaike’s
information criteria.24,25 After determination of the
best fit for the experimental data, Ki values were then
calculated using Prism.
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