Optimization and determination of the absolute configuration of a series of potent inhibitors of human papillomavirus type-11 E1-E2 protein-protein interaction: A combined medicinal chemistry, NMR and computational chemistry approach

Article abstract of DOI:10.1016/j.bmc.2007.01.036

We have previously reported the discovery and initial SAR optimization of the first series of inhibitors of the human papillomavirus type-11 (HPV11) E1-E2 protein-protein interaction. These inhibitors featured an indandione system spiro-fused onto an all

Full text of DOI:10.1016/j.bmc.2007.01.036

Bioorganic & Medicinal Chemistry 15 (2007) 2690–2700
Optimization and determination of the absolute configuration
of a series of potent inhibitors of human papillomavirus type-11
E1–E2 protein–protein interaction: A combined medicinal
chemistry, NMR and computational chemistry approach
a,
a,à
a
Nathalie Goudreau,a, Dale R. Cameron, Robert Deziel, Bruno Hache,
Araz Jakalian,a Eric Malenfant,a Julie Naud,a William W. Ogilvie,a,§
Jeff O’Meara,a Peter W. Whiteb and Christiane Yoakima
*
´
´
aDepartment of Chemistry, Boehringer Ingelheim (Canada) Ltd, Research & Development,
2100 Cunard Street, Laval, Que., Canada H7S 2G5
bDepartment of Biological Sciences, Boehringer Ingelheim (Canada) Ltd, Research & Development,
2100 Cunard Street, Laval, Que., Canada H7S 2G5
Received 11 December 2006; revised 15 January 2007; accepted 19 January 2007
Available online 24 January 2007
Abstract—We have previously reported the discovery and initial SAR optimization of the first series of inhibitors of the human pap-
illomavirus type-11 (HPV11) E1–E2 protein–protein interaction. These inhibitors featured an indandione system spiro-fused onto
an all syn substituted tetrahydrofuran ring. In this paper, we report new SAR efforts which have led to the identification of the first
low nanomolar inhibitor of the HPV11 E1–E2 protein–protein interaction. In addition, we report a combined NMR and compu-
tational chemistry approach which allowed the successful determination of the absolute stereochemistry of the active species
originating from the initial racemic lead.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
nata) and are the most common ‘low-risk’ types,
whereas HPV16, -18, and -31 represent the most preva-
lent ‘high-risk’ types and are the major cause of cervical
cancer.1 Interestingly, the recent approval of a new vac-
cine targeting HPV types 6, 11, 16, and 18, referred to as
Gardasilꢁ, represents a significant breakthrough in
women’s health. Indeed, this vaccine was shown to be
efficacious in decreasing the incidence of HPV infection,
and thus of both genital warts and cervical cancer.2
However, therapies to treat HPV-induced lesions in-
clude a variety of ablative or cytodestructive procedures
as well as the use of the immunostimulator imiqui-
mod.3,4 These treatments are far from ideal and no spe-
cific small molecule antivirals are yet available for the
treatment of HPV infections.
Papillomaviruses are a family of small double-stranded
DNA viruses associated with a broad spectrum of dis-
eases ranging from cutaneous and genital warts to ano-
genital cancer.1 More than 25 types of human
papillomaviruses (HPV) infect the anogenital region
and these HPV types have been classified as ‘low-risk’
or ‘high-risk’ types depending on the particular pathol-
ogies they are associated with. For example, HPV6
and 11 cause benign genital warts (condyloma acumi-
Keywords: Human papillomavirus; Protein–protein interaction; Indan-
dione, NMR.
*
Corresponding author. Tel.: +1 450 682 4640; fax: +1 450 682
4189; e-mail: ngoudreau@lav.boehringer-ingelheim.com
Present addresses: Migenix; BC Research Building, 3650 Wesbrook
mall, Vancouver, BC, Canada V6S 2L2.
´
Methylgene, 7220 Frederick Banting #200, Montreal, Que., Canada
H4S 2A1.
The HPV genome encodes eight well-characterized pro-
teins, only two of which, E1 and E2, are required for
DNA replication.5 Viral DNA replication is initiated
by the cooperative binding of E1 and E2 to specific
DNA sequences within the HPV viral origin. Assembly
of this E1–E2-origin complex is dependent on the
à
§
Department of chemistry, University of Ottawa, 10 Marie Curie,
Ottawa, Ont., Canada K1N 6N5.
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.01.036

N. Goudreau et al. / Bioorg. Med. Chem. 15 (2007) 2690–2700
2691
binding of E2 to high affinity sites in the origin as well as
on a critical protein–protein interaction between E1 and
E2.5,6 The essentiality of E1 and E2 in HPV DNA rep-
lication makes them attractive targets for novel antiviral
therapies.
the 4-chlorophenyl moiety of the inhibitor and showed
that a 20-fold increase in potency could be obtained
by the addition of another chloro substituent, as in 6
(Table 1).7 In this paper, we will report further improve-
ment to this series of inhibitors through novel SAR
studies. In addition, we will describe the use of a com-
bined NMR and computational chemistry approach
which allowed the successful determination of the abso-
lute configuration of the active enantiomer originating
from the initial racemic lead.
By screening our corporate compound collection we
identified 1, a molecule capable of antagonizing the pro-
tein–protein interaction between the E1 and E2 proteins
of HPV11 (Fig. 1).7,8 Specifically, this inhibitor featured
an indandione system spiro-fused onto a substituted tet-
rahydrofuran ring and had an IC50 of 11 lM in our in vi-
tro E1–E2-ori-complex formation assay.9 Upon
resynthesis and separation of the resulting diastereoiso-
mers, the inhibitory activity of the original hit 1 was
found to reside in the syn/syn isomer 2 (Fig. 1).7 In addi-
tion, isothermal titration calorimetry and fluorescence
studies showed that this series of inhibitors act by bind-
ing to the N-terminal transactivation domain (TAD) of
E2, the region of the protein that interacts with E1.6,8
Furthermore, these inhibitors were found inactive in a
mechanistically related specificity assay that measures
binding of SV40 large T antigen to its cognate origin.8
2. Chemistry
The syntheses of compounds 2–21 were carried out
according to Scheme 1, as previously described.7,10 In
brief, Knoevenagel condensation of indan-1,3-dione with
the appropriately substituted benzaldehydes (di-chloro or
di-bromo) in the presence of piperidine gave the corre-
sponding benzylidene intermediates, which were treated
with peroxide under basic conditions to give the corre-
sponding epoxide derivatives A. Dipolar cycloaddition
with different maleimide derivatives B in refluxing toluene
gave a 1:1 mixture ofthe syn/synadducts C andthe syn/anti
compounds D, which were readily separated by flash
chromatography. The syn/syn derivatives C were finally
converted to the corresponding sodium salts E by the slow
addition of aqueous NaOH.
Early stability studies indicated however that compound
2 was readily converted in the binding assay buffer (sodi-
um phosphate, pH 7.4) to other products. The structures
of these new products were elucidated and the mechanism
by which they were produced was determined using NMR
and mass spectrometry techniques.10 In particular, it was
demonstrated that dissolution of inhibitor 2 in aqueous
solutions at pH 7.0 or above resulted in the rapid and reg-
ioselective hydrolysis of its maleimide functionality to
generate the corresponding carboxylate 4. Moreover, this
process was found to involve neighboring-group partici-
pation of the indandione ketone moiety via the formation
of the intermediate hydroxylactone 5 (Fig. 2). Similar to
the maleimide 2, this latter derivative was also found
to rapidly convert in the assay buffer to the carboxylate
species 4, in agreement with the comparable activity
measured for all three compounds.10
The epoxide derivative K necessary for the synthesis of
compounds 22 and 23 is highlighted in Scheme 2.
Cl
O
Cl
O
O
O
H
O
N
HO
O
O
NaO
O
O
HN
O
O
O
O
O
4
5
IC50 = 7.8 μM
IC50 = 6.6 μM
Having established that the active form of our original
hit was represented by 4, we then undertook an initial
SAR study. This first optimization process focused on
Figure 2. Structures and biological activities of the hydrolysis products
of inhibitor 2. Compounds are racemic.
Cl
Cl
Cl
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
N
N
N
O
O
O
O
O
O
1
2
3
IC50 > 80.0 μM
IC50 = 8.3 μM
IC50 = 11.0 μM
syn/syn
syn/anti
relative configuration
relative configuration
Figure 1. Structures of the initial lead inhibitors of E1–E2 protein–protein interaction. All compounds are racemic.

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N. Goudreau et al. / Bioorg. Med. Chem. 15 (2007) 2690–2700
Table 1. Biological activities in the HPV11 in vitro E1–E2-ori-complex
formation assay8
in good yield. Further elaboration of this intermediate
to inhibitors 22 and 23 was accomplished as highlighted
in Scheme 1.
Cl
Cl
O
O
3. Results and discussion
O
O
NaO
Compound 6 was selected as the starting point of this
new optimization, which mainly focused on the amide
portion of the inhibitor. As shown in Table 1, replace-
ment of the original 1,3-benzodioxol-5-yl amide moiety
by a simple phenyl amide resulted in only a 2-fold loss
in potency (7). However a more drastic loss in potency
(6- or 10-fold) was observed when a tert-butyl group
(8) or a benzyl amide derivative (9) was introduced.
These early results suggested that anilide derivatives
were overall preferred. Thus, starting from the anilide
derivative 7, a variety of substituents were next intro-
duced onto its phenyl ring. The general trends revealed
by this exercise can be summarized with the results of
compounds 10, 11, and 12. First, addition of substitu-
ents at the ortho position of the phenyl ring was found
to be highly detrimental, as exemplified by the almost
100-fold loss in potency observed following the intro-
duction of an o-methyl amide substituent (10). This
observation was also true for a wide number of ortho
substituents (not shown). Introduction of substituents
at the meta position was in contrast better tolerated,
as illustrated by the similar potency displayed by 11.
However, it is at the para position that substituents dis-
played the most beneficial effect, as exemplified with
compounds 12, 13, 14, and 15. In addition, this position
was found to be quite permissive since a wide variety of
substituents were tolerated. Overall, the most significant
gain was observed when different heterocycles were
introduced such as the thiadiazole substituent (14)
which displayed a 4-fold improvement in potency as
compared to the unsubstituted reference analog 7.
Finally, the best para substituent we identified consisted
in the morpholino moiety (15) which provided a further
improvement in potency.
HN R
O
Compounda
R
IC50 (lM)
O
O
6
0.35
7
0.68
2.1
8
9b
3.5
O
10
11
65.0
0.74
N
H
O
NH
12
13
14
15
0.44
0.38
0.15
0.088
NH
O
O
N
It is worth mentioning that all the inhibitors had been
synthesized as racemic mixtures. We were therefore very
interested to find out if both enantiomers were able to
antagonize the E1–E2 protein–protein interaction to
the same extent. To address this important issue, we first
introduced on our current series of inhibitors an addi-
tional chiral center in order to generate a pair of diaste-
reoisomers. Specifically, analogs of 9 containing a
(R)-methyl benzyl amide derivative were prepared and
successfully separated to provide two diastereoisomers
(16 and 17) with the all-syn furan stereochemistry; the
‘all-down’ (2S,3S,4R,5S) and the ‘all-up’ (2R,3R,4S,5R)
furan configuration, respectively (Fig. 3). Interestingly,
only one of the two resulting diastereoisomers (17) was
found to be active, displaying an IC50 of 6.8 lM. At this
point, the absolute configuration of this active derivative
remained unknown and we were consequently very
interested in elucidating this matter.
N
S
N
O
a All compounds are racemic.
b Prepared as the hydroxylactone form.
Homologation of commercially available aldehyde F
with malonic acid followed by catalytic hydrogenation
of the exocyclic double bond provided acid H. Cycliza-
tion under acidic condition gave intermediate I in 35%
yield. Compound I was condensed with 3,4-dibromo-
benzaldehyde under acidic condition in benzene to gen-
erate compound J in moderate yield. Oxidation to the
corresponding diketone followed by epoxidation of the
double bond provided the required epoxy-diketone K
In a previous study, we had demonstrated that under
acidic conditions our inhibitors could be isolated as

N. Goudreau et al. / Bioorg. Med. Chem. 15 (2007) 2690–2700
2693
O
O
O
O
O
a,b
+
H
X'
O
X
X'
A
X
X
X
c
O
O
X'
X'
O
O
+
O
O
N
O
O
R
O
O
O
O
N
R
N
R
B
C
D
d
X
O
O
X'
O
O
N
O
NaO
H
R
E
Scheme 1. Reagents and condition: (a) EtOH, piperidine (cat.); (b) H2O2, NaOH, MeOH; (c) toluene, reflux; (d) CH3CN, NaOH slow addition;
X = X0 = Cl or Br.
a
b
CO2H
CO2H
CHO
S
S
S
F
G
H
O
O
c
d
e,f
Br
Br
O
S
S
H
I
J
Br
Br
O
O
S
Br
O
Br
K
Scheme 2. Reagents and conditions: (a) malonic acid, piperidine/pyridine, reflux, H2O, HCl, 100% yield; (b) Pd(OH)2, EtOH, H2, 93% yield; (c)
polyphosphoric acid, 75 ꢂC, 35% yield; (d) p-toluenesulfonic acid, benzene, reflux, 49% yield; (e) CrO3, CH2Cl2, tert-butylhydroperoxide, 60% yield;
(f) H2O2, NaOH, MeOH, 90% yield.
hydroxylactone intermediates (5, Fig. 2) through the
involvement of one of the neighboring indandione ke-
tones.10 Although these intermediates were shown to
rapidly convert in the assay buffer into their carboxylate
counterparts, they were nevertheless stable enough to al-
low their isolation and full characterization in organic
solvents.10 Moreover, one interesting feature of these
intermediates was the level of asymmetry they displayed,
at least from an NMR viewpoint. Therefore, in order
to establish the absolute configuration of the active

2694
N. Goudreau et al. / Bioorg. Med. Chem. 15 (2007) 2690–2700
(Fig. 4). This was in contrast to what was observed for
"all-down"
"all-up"
O
analog 17, for which the 3,4-dichlorophenyl ring
protons displayed specific weak NOEs to those of the
methyl group but not to those of the benzyl moiety
(supplementary material). Altogether, these results
clearly demonstrated the different conformational
behavior that these two diastereoisomers exhibit in
solution.
Cl
Cl
Cl
Cl
O
R
O
S
O
R
S
R
S
S
R
H
R
H
S
HO
N
HO
R
O
N
O
R
O
O
O
O
The two hydroxylactone derivatives were also studied by
molecular mechanics in order to determine their confor-
mational preferences in-silico and establish a correlation
with our experimental data. To begin, a systematic
conformational search around key single bonds was
performed for each compound using the MMFF94
forcefield within the MOE software package (Chemical
Computing Group Inc., Montreal, Canada), which
allowed the identification of a series of distinct low ener-
gy conformations (Table 2). The lowest energy confor-
mation found for each diastereoisomer is shown in
Figure 5. In addition, the distances between the center
of the 3,4-dichlorophenyl ring (A) and both the centers
of the benzyl ring (B) and of the methyl moiety (C) were
measured for the five lowest energy conformations of
each diastereoisomer (Table 3). Specifically for the ‘all-
down’ diastereoisomer conformations, the distance be-
tween A and B was found to be shorter on average
16
17
IC50 > 75.0 μM
IC50 = 6.8 μM
Figure 3. Structures and biological activities of the two chiral
inhibitors used in the combined NMR and computational chemistry
study.
diastereoisomer 17, we carried out a combined NMR and
computational chemistry approach that took advantage
of the asymmetry and rigidity of the hydroxylactones,
as well as to the presence of the remote chiral center of
known stereochemistry. Indeed, we believed that this
chiral center would confer sufficiently distinct conforma-
tional preferences to each of the diastereoisomers to
allow their absolute configuration to be established.
The simple comparison of the 1D proton NMR spectra
of the two hydroxylactone derivatives 16 and 17 in
DMSO highlighted specific chemical shift differences.
Most noticeable was the 0.5 ppm difference observed be-
tween their methyl resonances, as shown in Figure 4.
This observation reflected the distinct local environment
that this specific methyl group is experiencing in each
compound, most probably as a result of their overall dif-
ferent conformational behavior. This difference was fur-
ther confirmed through the recording of 1D NOE and
2D ROESY experiments. In case of compound 16, we
were able to observe that its 3,4-dichlorophenyl ring
protons were having weak NOEs to those of the benzyl
ring moiety, but not to those of the methyl group
˚
˚
(4.7 A) than the distance between A and C (6.2 A). In
contrast to the ‘all-up’ diastereoisomer conformations,
the distance between A and C was found to be shorter
˚
on average (4.8 A) than the average distance between
˚
A and B (6.7 A). These data clearly highlighted the dif-
ferent conformational preferences that these two diaste-
reoisomers exhibit in-silico. Interestingly when the
computational and NMR results were compared, specif-
ic correlations could be established. For instance, the
lowest energy conformation determined in-silico for
the ‘all-down’ isomer was found to be consistent with
the NOE information obtained for inhibitor 16 (inac-
tive), while the lowest energy conformation of the
Cl
O
O
CH3
Cl
NOE s
H
HO
N
O
16
O
O
3.0
2.6
2.2
1.8
1.4
1.0
0.6
0.2
No
(ppm)
NOE
shielding of 0.5 ppm
for methyl resonance
Cl
O
O
No
CH3
Cl
NOE
H
N
HO
O
O
O
17
3.0
2.6
2.2
1.8
1.4
1.0
0.6
0.2
NOEs
(ppm)
Figure 4. Subregion of 1H NMR spectra and ROESY information of inhibitors 16 and 17.

N. Goudreau et al. / Bioorg. Med. Chem. 15 (2007) 2690–2700
2695
Table 2. Systematic conformational search of the two hydroxylactone derivatives 16 and 17
"all-down"
"all-up"
Cl
Cl
Cl
Cl
O
b
O
b
O
R
O
S
R
S
S
R
S
R
R
H
N
R
S
H
N
c
HO
c
HO
O
a
O
a
O
d
d R
O
O
O
e
e
˚
Me–Phe distance (A)
˚
Phe–Phe distance (A)
Conformers
Energy (kcal/mol)
‘all-down’
1
2
3
4
5
67.8175
67.9376
69.2578
69.3113
69.6694
6.59
6.30
5.98
6.71
5.43
4.15
4.82
4.19
6.08
4.49
‘all-up’
1
2
3
4
5
69.0609
69.1739
69.3271
70.1207
70.2856
3.85
3.95
6.43
4.75
4.91
6.54
6.78
4.12
7.99
8.13
Table 3. Biological activities in the HPV11 in vitro E1–E2-ori-complex
formation assay
Br
O
O
R
Br
N
H
N
O
NaO
O
O
Figure 5. Lowest energy conformations of the two hydroxylactone
diastereosiomers. For clarity some of the hydrogen atoms are omitted.
O
‘all-up’ isomer was found to be consistent with the NOE
information obtained for inhibitor 17 (active). In addi-
tion, these conformations were also consistent with the
0.5 ppm up-field shift observed for the methyl resonanc-
es of 17, since in the ‘all-up’ representative conformation
the methyl moiety is directly positioned in the anisotrop-
ic shielding cone of the 3,4-dichlorophenyl ring (Fig. 5).
Therefore, based on this combined NMR and computa-
tional chemistry study, we predicted that the active
enantiomer 17 had the 2R,3R,4S,5R furan absolute con-
figuration (‘all-up’; Fig. 3). Subsequent to this study we
obtained an X-ray co-crystal structure of inhibitor 18,
bound to the HPV11 E2 transactivation domain
(Fig. 6).11 In addition to identifying the binding pocket
of this series of inhibitors, this X-ray structure unambig-
uously confirmed that the active enantiomer had the
3R,4S,5R furan absolute stereochemistry (‘all-up’) as
correctly predicted (Fig. 6).
Compounda
R
IC50(lM)
O
21
0.028
O
O
S
S
22
23
0.46
O
O
0.006
O
a Compounds are enantiomerically pure.
that most of the activity resided in one enantiomer, we
then undertook the separation of racemic 19 using chiral
HPLC in order to obtain the two enantiomerically pure
inhibitors, the 3R,4S,5R (‘all-up’) and the 3S,4R,5S
(‘all-down’) analogs, respectively. Again, we were quite
pleased to see that isomer 20 (‘all-down’) was found to
Further SAR investigations also allowed us to demon-
strate that the 3,4-dichlorophenyl moiety could be re-
placed by its 3,4-dibromo counterpart giving rise to a
moderate improvement in potency, as illustrated with
19 (IC50 = 0.051 lM, Fig. 7) when compared to 15
(IC50 = 0.088 lM, Table 1). In addition, since we knew

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N. Goudreau et al. / Bioorg. Med. Chem. 15 (2007) 2690–2700
Cl
Cl
O
R
O
S
R
S
H
N
O
O
O
N
ONa
N
S
18
IC50 = 0.18 µM
All-up
Figure 6. Bound conformation of inhibitor 18 to the HPV11 E2 transactivation domain derived from the X-ray co-crystal structure.11
(data not shown). The synthesis, purification, and char-
acterization of these particular compounds became how-
ever quite challenging as the dipolar cycloaddition
reaction yielded each time a mixture of two regioisomers
(at the level of the indandione) in combination with a
mixture of syn/syn and syn/anti tetrahydrofuran adducts
(analogs C and D, Scheme 1) giving raise to a total of
four possible diasteroisomeric pairs (each being race-
mic). The only substantial gain in potency that was ob-
served during this exercise was when the original
indandione portion was replaced by a cyclopentathioph-
ene derivative, as shown in Table 3. Indeed, compound
23 (which is enantiomerically pure) displayed an IC50 of
0.006 lM which represented a 4.5-fold improvement
over reference compound 21. It is also interesting to
note that the thiophene regiochemistry was found to
be extremely important for the activity since regioisomer
22 was found to be 75-fold less potent than its regio-
counterpart 23. This again further highlights the specific
nature of the HPV11 E1–E2 protein–protein interaction
that this class of inhibitors is antagonizing. Compound
23 also represents the first low nanomolar small mole-
cule inhibitor of HPV11.
Br
Br
O
O
O
H
N
O
O
NaO
N
19
IC50 = 0.051 μM
O
syn/syn relative configuration
(racemic)
Chiral HPLC
All-down
All-up
Br
Br
Br
Br
O
O
R
S
O
S
O
R
R
S
H
N
H
N
O
O
O
O
O
NaO
O
NaO
N
N
4. Conclusion
O
O
IC50 = 15.0 μM
IC50 = 0.028 μM
21
20
An extensive SAR study allowed us to improve the poten-
cy of our inhibitors by approximately 58-fold, as
compared to the starting compound 6. Moreover, com-
pound 23 represents the first low nanomolar inhibitor of
the HPV11 E1–E2 protein–protein interaction. In addi-
tion, using a combined NMR and computational chemis-
try approach, we were able to successfully determine the
absolute stereochemistry of our active enantiomer and
demonstrate that the activity resided in the 3R,4S,5R
(‘all-up’) furan absolute configuration. Thiswas later con-
firmed by X-ray co-crystallization studies.11
Figure 7. Structures and biological activities of the two enantiomer-
ically pure inhibitors. The absolute configuration was assigned based
on the X-ray co-crystal structure of analog 18.10
be significantly less active, displaying an IC50 of 15 lM,
as compared to the other enantiomer 21 (‘all-up’) which
was found to be almost 2-fold more potent than the
racemic mixture, with an IC50 of 0.028 lM. These data
definitively illustrated the specific nature of the HPV11
E1–E2 protein–protein interaction that this class of
inhibitors is able to antagonize.
5. Experimental
5.1. General
Compound 21 was then selected as the starting point of
a final optimization process, which focused on the
indanedione portion of the inhibitor. Introduction of
single small substituents (F, Me) at the different posi-
tions of its indane ring resulted in compounds that were
either less potent or at best only marginally more potent
All reactions were carried out under argon. Solvents and
reagents were used as purchased. 1H and 13C NMR
spectra were recorded at 400 and 100 MHz, respectively,

N. Goudreau et al. / Bioorg. Med. Chem. 15 (2007) 2690–2700
2697
in DMSO-d6 on a Bruker instrument and chemical shifts
are referenced relative to internal DMSO set at
2.50 ppm (1H) and 39.51 ppm (13C). ES mass spectra
were recorded on a Micromass Quattro II mass spec-
trometer. Column chromatography was performed
either on silica gel (10–40 lm or 230–400 mesh ASTM,
Merck) or preparative HPLC using a Partisil 10 ODS-
3, C18 preparative column (50 cm · 22 mn). Analytical
HPLC gradient chromatography was performed with
acetonitrile and water containing 0.06% trifluoroacetic
acid (TFA) using a Vydac C18 column (10 lm,
4.6 · 250 mm). Preparative chiral HPLC gradient chro-
matography was performed with acetonitrile and water
containing 0.06% trifluoroacetic acid (TFA) using a Chi-
ralcel OD column (10 lm, 20 · 250 mm).
(1.0 g, 2.51 mmol) was added. After stirring for 18 h at
room temperature the solution was diluted with CH2Cl2
and water, and extracted three times with small portions
of CH2Cl2. The combined organics were dried over
MgSO4 and concentrated in vacuo. Trituration of the
resulting solid with ether provided the corresponding
diketone (0.61 g, 60%). The material so obtained
(0.45 g) was dissolved in EtOH (15 mL) to which were
added 30% H2O2 (0.38 mL) and one drop of 1 N NaOH.
After stirring for 3 h the solution was filtered to give K
as a yellow solid (421 mg, 90%).
5.7. Compound characterization
1
5.7.1. Compound 2. H NMR (400 MHz, DMSO-d6) d
(ppm): 8.13–8.10 (m, 4H), 7.49 (d, J = 8.5 Hz, 2H),
7.43 (d, J = 8.5 Hz, 2H), 7.03 (d, J = 8.1 Hz, 1H), 6.67
(d, J = 1.9 Hz, 1H), 6.65 (dd, J = 8.1, 1.9 Hz, 1H), 6.09
(s, 2H), 6.08 (d, J = 8.0 Hz, 2H), 4.26 (dd, J = 7.9,
7.9 Hz, 1H), 4.16 (d, J = 8.0 Hz, 1H); 13C NMR
(DMSO-d6) d: 197.8, 194.6, 173.4, 172.6, 147.3, 140.7,
139.0, 137.7, 137.6, 135.3, 132.5, 128.1, 128.0, 125.2,
124.5, 123.8, 120.6, 108.2, 107.5, 101.8, 84.1, 83.0,
52.1, 51.2; MS (ESꢀ) m/z: 500.0 (MꢀH)ꢀ, 518.1
(M+18-H)ꢀ, 474.1; IR (KBr) m 1788, 1713 cmꢀ1 Anal.
Calcd for C27H16ClNO7: C, 64.62; H, 3.21; N, 2.79.
Found: C, 64.50; H, 3.20; N, 2.88.
5.2. Preparation of intermediate G
A solution of F (9.5 g, 75.4 mmol), malonic acid (15.7 g,
151 mmol), and piperidine (1.3 mL) in pyridine (40 mL)
was refluxed overnight. The resulting mixture was al-
lowed to cool to room temperature whereupon water
(200 mL) was added. The mixture was acidified by the
addition of concentrated HCl and allowed to stir for
1 h. The mixture was filtered and the solid washed with
water. Drying under vacuum gave G as a yellow powder
(12.8 g, 100%).
1
5.3. Preparation of intermediate H
5.7.2. Compound 3. H NMR (400 MHz, DMSO-d6) d
(ppm): 8.18–8.09 (m, 4H), 7.58 (d, J = 8.0 Hz, 2H),
7.55 (d, J = 8.0 Hz, 2H), 7.07 (d, J = 8.3 Hz, 1H), 6.80
(d, J = 1.9 Hz, 1H), 6.74 (dd, J = 8.3, 1.9 Hz, 1H), 6.12
(s, 2H), 5.73 (d, J = 7.2 Hz, 1H), 4.27 (d, J = 9.8 Hz,
1H), 3.96 (dd, J = 9.8, 7.2 Hz, 1H); 13C NMR
(DMSO-d6) d: 198.0, 194.2, 174.1, 173.1, 148.2, 141.3,
141.1, 137.3, 137.2, 137.0, 134.5, 129.0, 127.3, 124.7,
124.6, 124.3, 120.7, 108.5, 107.9, 101.9, 84.2, 83.0,
55.0, 51.1; MS (ESꢀ) m/z: 500.0 (MꢀH)ꢀ, 518.0
(M+18-H)ꢀ, 474.1 (M+18-44)ꢀ; IR (KBr) m 1743,
1708, 1687 cmꢀ1; Anal. Calcd for C27H16ClNO7: C,
64.62; H, 3.21; N, 2.79. Found: C, 64.46; H, 3.27; N,
2.82.
A mixture of G (7.5 g, 44.6 mmol) and Pd(OH)2
(500 mg) in ethanol was stirred under an atmosphere
of hydrogen for 18 h. Filtering through glass microfiber
and removal of solvent gave H as a white solid (7.0 g,
93%). Removal of solvent under reduced pressure gave
H as a brown solid (3.72 g, 62%).
5.4. Preparation of intermediate I
Solid H (1 g, 5.88 mmol) was added in small portions
to warm (75 ꢂC) polyphosphoric acid (8.5 g). Heating
was continued at 75 ꢂC for 1 h after the addition
was complete. Cooling to room temperature was fol-
lowed by dilution with water and extraction with
CH2Cl2 (3·). The combined organics were dried over
MgSO4 and concentrated. Flash chromatography
(50% EtOAc in hexanes) gave I as a white solid
(0.31 g, 35%).
1
5.7.3. Compound 4. H NMR (400 MHz, DMSO-d6) d
(ppm): 9.91 (s, 1H), 8.04–7.96 (m, 4H), 7.38 (d,
J = 8.5 Hz, 2H), 7.31 (d, J = 8.5 Hz, 2H), 7.09 (d,
J = 1.6 Hz, 1H), 6.89 (dd, J = 8.4, 1.6 Hz, 1H), 6.83
(d, J = 8.4 Hz, 1H), 5.96 (s, 2H), 5.71 (d, J = 5.9 Hz,
1H), 4.15 (d, J = 8.7 Hz, 1H), 3.65 (dd, J = 8.7, 5.9 Hz,
1H); 13C NMR (DMSO-d6) d: 202.0, 199.8, 169.1,
167.5, 146.8, 143.8, 142.7, 138.8, 137.2, 136.1, 135.8,
133.5, 131.8, 127.7, 123.4, 112.5, 107.8, 102.0, 100.8,
85.1, 83.0, 62.0, 56.7; MS (ESꢀ) m/z: 518.1 (MꢀH)ꢀ,
(ES+) m/z: 520.0 (M+H)+, 542.0 (M+Na)+, 502.0
(MꢀNa+2H)+; mp 204.2 ꢂC; IR (KBr) m 1744, 1707,
5.5. Preparation of intermediate J
A solution of I (1.06 g, 6.97 mmol), 3,4-dibromobenzal-
dehyde (1.84 g, 6.97 mmol), and p-toluenesulfonic acid
(100 mg) in benzene (25 mL) was refluxed for 24 h with
azeotropic removal of water. Upon cooling and addition
of ether (25 mL), a solid precipitated which was filtered
to give J as a tan solid (1.35 g, 49%).
1660 cmꢀ1
.
1
5.7.4. Compound 5. H NMR (400 MHz, DMSO-d6) d
(ppm): 10.15 (s, 1H), 7.98 (dd, J = 7.4, 7.4 Hz, 1H),
7.94 (d, J = 7.4 Hz, 1H), 7.87 (d, J = 7.4 Hz, 1H), 7.77
(dd, J = 7.4, 7.4 Hz, 1H), 7.58 (s, 1H), 7.49 (d,
J = 8.4 Hz, 2H), 7.39 (d, J = 8.4 Hz, 2H), 6.76 (d,
J = 8.43 Hz, 1H), 6.71 (d, J = 1.7 Hz, 1H), 6.51 (dd,
5.6. Preparation of intermediate K
To a solution of CrO3 (50 mg, 0.50 mmol) in CH2Cl2
(15 mL) was added tert-butylhydroperoxide (2.6 mL of
a 70% solution in water). After stirring for 2 min, J

2698
N. Goudreau et al. / Bioorg. Med. Chem. 15 (2007) 2690–2700
J = 8.3, 1.7 Hz, 1H), 6.02 (d, J = 5.4 Hz, 1H), (m, 1H),
5.96 (s, 2H), 4.32 (d, J = 8.8 Hz, 1H), 3.98 (dd, J = 8.8,
5.4 Hz, 1H); 13C NMR (DMSO-d6) d: 200.0, 171.4,
168.4, 148.2, 146.9, 144, 137.4, 134.4, 133.8, 132.6,
131.8, 131.1, 127.9, 124.8, 123.6, 113.6, 107.8, 105.6,
102.4, 101.1, 88.4, 88.0, 53.0, 52.7; MS (ESꢀ) m/z:
517.9 (MꢀH)ꢀ, 473.9 (MꢀCO2)ꢀ; mp 157.6 ꢂC; IR
5.7.10. Compound 11. 1H NMR (400 MHz, DMSO-d6) d
(ppm): 10.18 (s, 1H), 9.80 (s, 1H) 8.06–7.96 (m, 4H),
7.64 (s, 1H), 7.59 (d, J = 1.6 Hz, 1H), 7.48 (d,
J = 8.3 Hz, 2H), 7.35 (dd, J = 8.3, 1.6 Hz, 1H), 7.17–
7.11 (m, 2H), 5.69 (d, J = 5.7 Hz, 1H), 4.14 (d,
J = 8.6 Hz, 1H), 3.72 (dd, J = 8.6, 5.7 Hz, 1H), 2.01 (s,
3H); MS (ESꢀ) m/z: 565.1, 567.1; HRMS (FAB) Calcd
for C28H20Cl2N2O7 (M+Na)+: 589.0545. Found:
589.0545. IR (KBr) m 3567, 3267, 3189, 3071, 1742,
(KBr) m 3296–2900, 1746, 1710, 1655 cmꢀ1
.
1
5.7.5. Compound 6. H NMR (400 MHz, DMSO-d6) d
(ppm): 10.02 (s, 1H), 8.07–7.96 (m, 4H), 7.59 (d,
J = 1.6 Hz, 1H), 7.52 (d, J = 8.6 Hz, 1H), 7.35 (dd,
J = 8.3, 1.6 Hz, 1H), 7.06 (d, J = 1.9 Hz, 1H), 6.83 (d,
J = 8.3 Hz, 1H), 6.73 (dd, J = 8.6, 1.9 Hz, 1H), 5.96 (s,
2H), 5.70 (d, J = 5.7 Hz, 1H), 4.13 (d, J = 8.6 Hz, 1H),
3.66 (dd, J = 8.9, 6.0 Hz, 1H); 13C NMR (DMSO-d6)
d: 201.7, 199.5, 168.7, 167.3, 146.7, 143.7, 142.7, 138.8,
138.5, 137.0, 135.7, 133.3, 130.4, 139.8, 129.7, 127.8,
126.2, 123.4, 123.3, 112.6, 107.7, 102.0, 100.7, 84.3,
83.0, 61.9, 56.2; MS (ESꢀ) m/z: 552.1 (MꢀH)ꢀ; HRMS
(FAB) Calcd for C27H17Cl2NO8 (M+Na)+: 576.0228.
Found: 576.0228.
1707, 1666, 1592, 1551, 1483, 1393 cmꢀ1
.
5.7.11. Compound 12. 1H NMR (400 MHz, DMSO-d6) d
(ppm): 10.12 (s, 1H), 9.85 (s, 1H) 8.04–8.00 (m, 4H),
7.60 (s, 1H), 7.51–7.44 (m, 3H), 7.36–7.35 (m, 2H),
5.70 (br d, 1H), 4.16 (d, J = 8.6 Hz, 1H), 3.68 (m, 1H),
2.00 (s, 3H); MS (ES+) m/z: 567.0, 569.1; HRMS
(FAB) Calcd for C28H20Cl2N2O7 (M+Na)+: 589.0545.
Found: 589.0545.
5.7.12. Compound 13. 1H NMR (400 MHz, DMSO-d6) d
(ppm): 10.53 (s, 1H), 8.10–7.97 (m, 4H), 7.89 (d,
J = 8.6 Hz, 2H), 7.63 (d, J = 8.9 Hz, 2H), 7.58–7.57
(m, 1H), 7.48 (d, J = 8.3 Hz, 1H), 7.35–7.33 (m, 1H),
5.72 (d, J = 5.7 Hz, 1H), 4.17 (d, J = 8.6 Hz, 1H), 3.80
(dd, J = 8.6, 5.7 Hz, 1H), 2.45 (s, 3H); MS (ES+) m/z:
552.1, 554.1; HRMS (FAB) Calcd for C28H19Cl2NO7
(M+Na)+: 574.0436. Found: 574.0436. IR (KBr) m
1
5.7.6. Compound 7. H NMR (400 MHz, DMSO-d6) d
(ppm): 10.1 (s, 1H), 8.07–7.97 (m, 4H), 7.60 (d, J = 2.0
Hz, 1H), 7.50 (t, J = 8.2 Hz, 3H), 7.36 (dd, J = 8.5,
2.0 Hz, 1H), 7.27 (t, J = 7.9 Hz, 2H), 7.01 (t,
J = 7.5 Hz, 1H), 5.72 (d, J = 6.0 Hz, 1H), 4.16 (d,
J = 8.0 Hz, 1H), 3.71 (dd, J = 8.7, 5.0 Hz, 1H); MS
(ES+) m/z: 509.9, 511.9; HRMS (FAB) Calcd for
C26H17Cl2NO6 (M+Na)+: 532.0330. Found: 532.0328.
2900, 1710, 1591 cmꢀ1
.
5.7.13. Compound 14. 1H NMR (400 MHz, DMSO-d6) d
(ppm): 10.34 (s, 1H), 9.51 (s, 1H), 8.11–7.96 (m, 6H),
7.66 (d, J = 8.4 Hz, 2H), 7.61 (d, J = 1.8 Hz, 2H), 7.50
(d, J = 8.4 Hz, 1H), 7.37 (dd, J = 8.4, 1.8 Hz, 1H), 5.74
(d, J = 5.7 Hz, 1H), 4.19 (d, J = 8.6 Hz, 1H), 3.76 (dd,
J = 8.6, 5.7 Hz, 1H); MS (ESꢀ) m/z: 592.0, 594.0;
HRMS (FAB) Calcd for C28H17Cl2N3O6S (M+Na)+:
616.0112. Found: 616.0112.
1
5.7.7. Compound 8. H NMR (400 MHz, DMSO-d6) d
(ppm): 8.16–8.03 (m, 4H), 7.85 (s, 1H), 7.72 (d, J = 1.9
Hz, 1H), 7.68 (d, J = 8.3 Hz, 1H), 7.47 (dd, J = 8.3,
1.9 Hz, 1H), 5.73 (d, J = 6.3 Hz, 1H), 4.10 (d,
J = 8.9 Hz, 1H), 3.53 (dd, J = 8.7, 6. Hz, 1H), 1.22 (s,
9H); MS (ESꢀ) m/z: 488.1, 490.0, 491.1; HRMS
(FAB) Calcd for C24H20Cl2NO6 (M+Na)+: 512.0643.
Found: 512.0645.
5.7.14. Compound 15. 1H NMR (400 MHz, DMSO-d6) d
(ppm): 9.90 (s, 1H), 8.05–7.95 (m, 4H), 7.60 (d,
J = 1.9 Hz, 1H), 7.50 (d, J = 8.3 Hz, 1H) 7.40–7.30 (m,
1H), 7.30 (d, J = 9 Hz, 2H), 6.85 (d, J = 9 Hz, 2H),
5.70 (d, J = 5.7 Hz, 1H), 4.13 (d, J = 8.6 Hz, 1H),
3.74–3.67 (m, 4H), 3.62–3.60 (m, 1H), 3.04–3.02 (m,
4H); MS (ESꢀ) m/z: 593.0, 595.0; HRMS (FAB) Calcd
for C30H24Cl2N2O7 (M+Na)+: 617.0858. Found:
617.0858.
1
5.7.8. Compound 9. H NMR (400 MHz, DMSO-d6) d
(ppm): 9.02 (dd, J = 7.3, 4.4 Hz, 1H), 8.00 (s, 1H),
7.97 (d, J = 7.6 Hz, 1H), 7.95 (t, J = 7.6 Hz, 1H), 7.87
(d, J = 7.6 Hz, 1H), 7.78 (t, J = 7.6 Hz, 1H), 7.66 (s,
1H), 7.60 (d, J = 8.5 Hz, 1H), 7.40 (d, J = 8.5 Hz, 1H),
7.22–7.15 (m, 3H), 6.67 (d, J = 7.7 Hz, 2H), 5.95 (d,
J = 5.7 Hz, 1H), 4.28 (d, J = 8.6 Hz, 1H), 4.22 (dd,
J = 15.1, 7.3 Hz, 1H), 3.94 (dd, J = 8.6, 5.7 Hz, 1H),
3.88 (dd, J = 15.1, 4.4 Hz, 1H); MS (ES+) m/z: 524.0,
526.0; HRMS (FAB) Calcd for C27H19Cl2NO6
(M+H)+: 524.0667. Found: 524.0667.
1
5.7.15. Compound 16. H NMR (400 MHz, DMSO-d6)
d (ppm): 8.98 (d, J = 8.1 Hz, 1H), 7.97 (s, 1H), 7.99–
7.90 (m, 2H), 7.85 (d, J = 7.1 Hz, 1H), 7.76 (t,
J = 7.1 Hz, 1H) 7.57 (d, J = 1.6 Hz, 1H), 7.47 (d,
J = 8.3 Hz, 1H), 7.32 (dd, J = 8.3, 1.6 Hz, 1H), 7.20–
7.19 (m, 3H), 6.64 (d, J = 7.1 Hz, 2H), 5.91 (d,
J = 5.3 Hz, 1H), 4.69 (m, 1H), 4.26 (d, J = 8.9 Hz,
1H), 3.92 (dd, J = 8.9, 5.3 Hz, 1H), 1.23 (d,
J = 7.0 Hz, 3H); MS (ESꢀ) m/z: 536.1, 538.1; HRMS
(FAB) Calcd for C28H21Cl2NO6 (M+H)+: 538.0824.
Found: 538.0826.
1
5.7.9. Compound 10. H NMR (400 MHz, DMSO-d6) d
(ppm): 9.56 (s, 1H), 9.50 (s, 1H), 8.18 (d, J = 8.3 Hz,
1H), 8.09–8.00 (m, 4H),7.70 (s, 1H), 7.65 (d,
J = 8.3 Hz, 1H), 7.47 (d, J = 8.3 Hz, 1H), 7.15 (t,
J = 7.6 Hz, 1H), 6.95 (t, J = 7.6 Hz, 1H), 6.70 (d,
J = 7.6 Hz, 1H), 5.78 (d, J = 6.0 Hz, 1H), 4.23 (d,
J = 8.9 Hz, 1H), 3.73 (dd, J = 8.9, 6.0 Hz, 1H), 2.09 (s,
3H); MS (ESꢀ) m/z: 565.0, 567.0; HRMS (FAB) Calcd
for C28H20Cl2N2O7 (M+Na)+: 589.0545. Found:
589.0545.
5.7.16. Compound 17. 1H NMR (400 MHz, DMSO-d6) d
(ppm): 8.85 (d, J = 8.1 Hz, 1H), 7.96 (t, J = 7.3 Hz, 1H),
7.92 (s, 1H), 7.90 (d, J = 7.3 Hz, 1H), 7.85 (d,

N. Goudreau et al. / Bioorg. Med. Chem. 15 (2007) 2690–2700
2699
J = 7.6 Hz, 1H), 7.76 (t, J = 7.6 Hz, 1H) 7.70 (d,
5.8. NMR methods
J = 8.7 Hz, 1H), 7.68 (s, 1H), 7.44 (d, J = 8.6 Hz, 1H),
7.29 (t, J = 7.2 Hz, 2H), 7.23 (d, J = 7.2 Hz, 1H), 7.17
(d, J = 7.2 Hz, 2H), 5.98 (d, J = 5.1 Hz, 1H), 4.60 (m,
1H), 4.23 (d, J = 8.7 Hz, 1H), 3.95 (dd, J = 8.7, 5.2 Hz,
1H), 0.74 (d, J = 7.0 Hz, 3H); MS (ES+) m/z: 538.1,
540.1; HRMS (FAB) Calcd for C28H21Cl2NO6
(M+H)+: 538.0824. Found: 538.0826.
1D NOE and 2D COSY, ROESY, HMQC, and HMBC
experiments were recorded at 300 K on a Bruker
DRX400 spectrometer equipped with a gradient triple
resonance probe using the standard pulse sequences pro-
vided by the manufacturer. The ROESY experiments for
compounds 16 and 17 were recorded with a 300 ms spin-
lock period. The 2D data sets were typically acquired
with 2048 points in t2, 256 points in t1, and 128 scans.
The data were processed and analyzed using
XWinNMR software (Bruker Canada, Milton,
Ontario).
5.7.17. Compound 18. 1H NMR (400 MHz, DMSO-d6) d
(ppm): 10.4 (s, 1H), 9.51 (s, 1H), 8.05 (d, J = 8.3 Hz,
1H), 8.00–7.60 (m, 6H), 7.53 (d, J = 8.4 Hz, 1H), 7.37
(d, J = 8.4 Hz, 1H), 5.74 (br s, 1H), 4.17 (br d,
J = 8.3 Hz, 1H), 3.76 (br s, 1H), 2.61 (s, 3H); MS
(ESꢀ) m/z: 606.1, 608.0, 609.0; HRMS (FAB) Calcd
for C29H19Cl2N3O6S (M+Na)+: 630.0269. Found:
630.0269.
5.9. Computational chemistry methods
The two hydroxylactone derivatives 16 and 17 were
studied by molecular mechanics in order to determine
their conformational preferences in-silico. A systematic
conformational search around key single bonds (a–e,
shown below) was performed for each compound using
the MMFF94 forcefield within the MOE software pack-
age (Chemical Computing Group Inc., Montreal, Cana-
da). The calculations were performed in vacuo using a
distance dependent dielectric constant and without non-
bonded cutoffs. The different angles (a–e) were rotated
systematically while the remaining parameters were al-
lowed to relax. Specifically, angle a was varied by
120ꢂ, angle b by 60ꢂ, angle c by 15ꢂ, angle d by 15ꢂ,
and angle e by 120ꢂ to generate ultimately 5393 points.
Symmetry was exploited when possible. Each point
was then gradient minimized and redundant structures
5.7.18. Compound 19. 1H NMR (400 MHz, DMSO-d6) d
(ppm): 10.12 (s, 1H), 8.12–7.69 (m, 6H), 7.4 (d,
J = 7.3 Hz, 1H), 7.00 (d, J = 8.5 Hz, 2H), 6.82 (d,
J = 8.5 Hz, 2H), 5.99 (br s, 1H), 4.30 (br d, J = 9.0 Hz,
1H), 4.00 (br s, 1H), 3.70 (m, 4H), 3.02–3.01 (m, 4H);
MS (ES+) m/z: 582.8, 584.8, 586.8; HRMS (FAB) Calcd
for C30H24Br2N2O7 (M+Na)+: 704.9847. Found:
704.9847.
5.7.19. Compound 20. 1H NMR (400 MHz, DMSO-d6) d
(ppm): 10.12 (s, 1H), 8.12–7.69 (m, 6H), 7.4 (d,
J = 7.3 Hz, 1H), 7.00 (d, J = 8.5 Hz, 2H), 6.81 (d,
J = 8.5 Hz, 2H), 6.0 (d, J = 5.4 Hz, 1H), 4.30 (d,
J = 8.6 Hz, 1H), 4.00 (dd, J = 8.6, 5.4 Hz, 1H), 3.70
(m, 4H), 3.01 (m, 4H); MS (ESꢀ) m/z: 681.0, 683.0,
685.0; HRMS (FAB) Calcd for C30H24Br2N2O7
(M+Na)+: 704.9847. Found: 704.9847.
˚
were eliminated (RMSD < 1 A), leading to the identifi-
cation of a series of distinct low energy conformations.
The five lowest energy conformations found for each
diastereoisomer were selected (supplementary materi-
als). In addition, the distances between the center of
the 3,4-dichlorophenyl ring and both the centers of the
benzyl ring and of the methyl moiety were measured
for these five lowest energy conformations and
tabulated.
5.7.20. Compound 21. 1H NMR (400 MHz, DMSO-d6) d
(ppm): 10.12 (br s, 1H), 8.14–7.65 (m, 6H), 7.4 (d,
J = 7.3 Hz, 1H), 7.00 (d, J = 8.9 Hz, 2H), 6.81 (d,
J = 8.9 Hz, 2H), 6.0 (d, J = 5.4 Hz, 1H), 4.30 (br d,
J = 9.0 Hz, 1H), 4.00 (br s, 1H), 3.70 (m, 4H), 3.01 (m,
4H); MS (ESꢀ) m/z: 681.0, 683.0, 684.0; HRMS
(FAB) Calcd for C30H24Br2N2O7 (M+Na)+: 704.9847.
Found: 704.9847.
5.10. In vitro assay
The activity of all the inhibitors was determined using a
E1–E2-ori-complex formation assay as previously de-
scribed.8 Each reported IC50 value represents the mean
of at least three determinations. In addition, all inhibi-
tors were tested and found inactive (data not shown)
in a mechanistically related scintillation proximity assay
(SPA) that measures binding of SV40 large T antigen to
its cognate origin, demonstrating that they do not inter-
fere with the scintillation proximity detection procedure
or bind promiscuously to DNA or protein.8
5.7.21. Compound 22. 1H NMR (400 MHz, DMSO-d6) d
(ppm): 9.25 (s, 1H), 7.72 (d, J = 2.0 Hz, 1H), 7.58 (d,
J = 8.3 Hz, 1H), 7.34 (s, 1H), 7.27 (dd, J = 2.0,
8.3. Hz, 1H), 7.04 (d, J = 9.2 Hz, 2H), 6.76 (d,
J = 9.2 Hz, 2H), 5.63 (d, J = 6.1 Hz, 1H), 4.35 (d,
J = 8.6, 1H), 3.81 (dd, J = 6.1, 8.6 Hz, 1H), 3.66 (br s,
4H), 2.96 (br s, 4H), 2.66 (s, 3H); MS (ESꢀ) m/z:
700.9, 702.9, 704.8.
5.7.22. Compound 23. 1H NMR (400 MHz, DMSO-d6) d
(ppm): 10.1 (s, 1H), 7.85 (s, 1H), 7.65 (d, J = 8.2 Hz,
1H), 7.40 (d, J = 8.2. Hz, 1H), 7.35 (s, 1H), 6.95 (d,
J = 9.0 Hz, 2H), 6.85 (d, J = 9.0 Hz, 2H), 5.95 (d,
J = 6.0 Hz, 1H), 4.31 (d, J = 8.5 Hz, 1H), 4.04 (dd,
J = 6.0, 8.5 Hz, 1H), 3.87 (br s, 4H), 3.05 (br s, 4H),
2.70 (s, 3H); MS (ESꢀ) m/z: 700.9, 702.9, 704.8; HRMS
(FAB) Calcd for C29H23Br2N2O7S (MꢀH)ꢀ: 700.9592.
Found: 700.9598.
Acknowledgments
The authors thank Norman Aubry, Colette Boucher,
Michael Little, and Serge Valois for excellent analytical
support. We also acknowledge Dr. Yong Wang and
´
Rene Coulombe for the crystallographic work, Louise
Thauvette for protein production and purification, and

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N. Goudreau et al. / Bioorg. Med. Chem. 15 (2007) 2690–2700
L.; Hougham, A. J.; Schmitt, K. A. Antimicrob. Agents
Chemother. 1998, 42, 789–794.
5. Howley, P. M. In Fields Virology, 3rd ed.; Lippincott-
Raven Publishers: Philadadelphia, 1996; pp 2045–2076.
6. Abbate, E. A.; Berger, J. M.; Botchan, M. R. Gene and
Development 2004, 18, 1981–1996.
Ewald Welchner for IC50 determination. Finally we ex-
tend our gratitude to Drs. Paul Anderson, Jacques
Archambault, Michael Bo¨s, and Michael Cordingley
for encouragement and support.
7. Yoakim, C.; Ogilvie, W. W.; Goudreau, N.; Naud, J.;
´
Supplementary data
Hache, B.; O’Meara, J. A.; Cordingley, M. G.; Archa-
mbault, J.; White, P. W. Bioorg. Med. Chem. Lett. 2003,
13, 2539–2541.
Supplementary data associated with this article can
be found, in the online version, at doi:10.1016/j.bmc.
2007.01.036.
8. White, P. W.; Titolo, S.; Brault, K.; Thauvette, L.;
Pelletier, A.; Welchner, E.; Bourgon, L.; Doyon, L.;
Ogilvie, W. W.; Yoakim, C.; Cordingley, M. G.; Archa-
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