False positives in a reporter gene assay: Identification and synthesis of substituted N-pyridin-2-ylbenzamides as competitive inhibitors of firefly luciferase

Article abstract of DOI:10.1021/jm8004509

Luciferase reporter-gene assays are a commonly used technique in high-throughput screening campaigns. In this study, we report on a luciferase inhibitor (1), which emerged from an antagonistic G protein-coupled receptor luciferase reporter-gene assay scre

Full text of DOI:10.1021/jm8004509

4724
J. Med. Chem. 2008, 51, 4724–4729
False Positives in a Reporter Gene Assay: Identification and Synthesis of Substituted
N-Pyridin-2-ylbenzamides as Competitive Inhibitors of Firefly Luciferase
Laura H. Heitman,† Jacobus P. D. van Veldhoven,† Annelien M. Zweemer, Kai Ye, Johannes Brussee, and
Adriaan P. IJzerman*
DiVision of Medicinal Chemistry, Leiden/Amsterdam Center for Drug Research, Leiden UniVersity, P.O. Box 9502,
2300 RA Leiden, The Netherlands
ReceiVed April 21, 2008
Luciferase reporter-gene assays are a commonly used technique in high-throughput screening campaigns.
In this study, we report on a luciferase inhibitor (1), which emerged from an antagonistic G protein-coupled
receptor luciferase reporter-gene assay screen. Instead of displaying receptor activity, compound 1 was shown
to potently inhibit luciferase in an in vitro enzymatic assay with an IC50 value of 1.7 ( 0.1 µM. In addition,
1 was a competitive inhibitor with respect to the substrate luciferin. A database search yielded another
inhibitor (3) with a similar N-pyridin-2-ylbenzamide core. Subsequently, several analogues were prepared
to investigate the structure-activity relationships of these luciferase inhibitors. This yielded the most potent
inhibitor of this series (6) with an IC50 value of 0.069 ( 0.01 µM. Further molecular modeling studies
suggested that 6 can be accommodated in the luciferin binding site. This paper is meant to alert users of
luciferase reporter-gene assays for possible false positive hits including highly “druglike” molecules due to
direct luciferase inhibition.
Scheme 1. Luciferase-Catalyzed Reactiona
Introduction
Firefly luciferase has a long history of use in biology. This
enzyme catalyzes the formation of luciferyl adenylate from the
substrates luciferin and ATPa (Scheme 1).1 The luciferyl
adenylate is oxidized and converted to an electronically excited
state of oxyluciferin. Return to the ground state results in the
emission of visible light with a wavelength of approximately
562 nm. The cloning of firefly luciferase in 1985 generated a
great deal of interest in possible applications of the gene as a
tool in scientific research.2,3 For example, luciferase has been
proposed as a model for the µ-opioid receptor, as there are
structural similarities between the catalytic site of the enzyme
and the opioid binding site of the receptor.4 Nowadays,
luciferase is applied widely as a reporter gene in high throughput
a In the presence of ATP, luciferin is activated to luciferyl-AMP, which
is oxidized by O2 to produce an excited state of oxyluciferin (/). On return
to the ground state, light is emitted.
screening processes because of its high sensitivity and ease of
use.5 Reporter assays couple the biological activity of a target
to the expression of a variety of readily detected enzymes and
thereby provide a highly amplified signal. It should be noted
that the luciferin-luciferase reaction has been shown to be
inhibited strongly by the products oxyluciferin and AMP.6,7 In
addition, many substrate-like compounds such as luciferin8 and
ATP analogues,7 but also dissimilar compounds such as
pifithrin-R,9 lipoic acid,10 and N-tosylphenylalanine chloro-
methyl ketone (TPCK),11 have been shown to inhibit luciferase
activity.
study, we report that compound 1, which emerged from that
screen, is a highly potent competitive luciferase inhibitor with
respect to one of the substrates, luciferin. Very recently,
luciferase inhibitors in the Pubchem database were described
in a paper by Auld and co-workers.12,13 That library contained
several structural analogues of 1 of which one compound was
a highly potent luciferase inhibitor (3). Therefore, different
N-pyridin-2-ylbenzamide analogues (4-12, 22-39) were pre-
pared to shed light on the molecular requirements for luciferase
inhibition. In addition, the most potent inhibitor (6) was docked
into the crystal structure of luciferase at the luciferin binding
site, suggestive of its competitive nature. Since these compounds
are druglike molecules, it should be taken into account that “false
positives” can easily emerge when luciferase activity is used
as a readout in high-throughput screens.
An in-house antagonistic luciferase reporter-gene assay screen
resulted in a high amount of false positive hits. In the present
* To whom correspondence should be addressed. Phone: +31- (0)71
527 4651. Fax: +31- (0)71 527 4565. E-mail address: ijzerman@
lacdr.leidenuniv.nl.
†
Both authors contributed equally to this study.
a Abbreviations: AID, assay identifier number in PubChem data reposi-
tory; AMP, adenosine 5′-monophosphate; ATP, adenosine 5′-triphosphate;
BSA, bovine serum albumin, CID, compound identifier number in PubChem
data repository; DCM, dichloromethane; DMF, dimethylformamide; EDTA,
ethylenediaminetetraacetic acid; GPCR, G-protein-coupled receptor; KM,
substrate concentration at half-maximal reaction rate; IC50, inhibitory
concentration (50%); rmsd, root-mean-square deviation; SEM, standard error
of the mean; TLC, thin-layer chromatography; Vmax, maximal reaction rate.
Results and Discussion
Chemistry. The initial active compound, N-quinolin-2-
ylbenzamide (1), was obtained from the reaction of 2-amino-
quinoline with benzoyl chloride in pyridine.14 On the basis of
the interesting behavior of quinoline 1 and the more active
10.1021/jm8004509 CCC: $40.75
2008 American Chemical Society
Published on Web 07/23/2008

Benzamides as Inhibitors of Firefly Luciferase
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4725
Scheme 2. Synthetic Route to the Compoundsa
Table 1. Inhibition of Luciferase Activity by Compounds 4-12,
Expressed as IC50 Values or as % Inhibition at 10 µM
a (a) 3-7, 9, 11, and 12: acid chlorides, pyridine. (b) 8: benzyl chloride,
pyridine, MW, 180 °C, 2 h. (c) 10: phenyl isocyanate, dioxane.
Scheme 3. Synthetic Route to the N-(5-Phenylpyridin-2-yl)benza-
mides 22-39a
a (a) (subst) phenylboronic acid, Na2CO3, Pd(OAc)2, DMF, H2O, 110°C;
(b) (subst) benzoyl chloride, pyridine.
pyridine 313 in the luciferase assay, N-pyridin-2-ylbenzamide
analogues (3-12, 22-39) were synthesized (Schemes 2 and
3). A Suzuki coupling between phenylboronic acid and 2-amino-
5-bromopyridine gave the 2-amino-5-phenylpyridine (2a).15 The
2-amino-5-phenylpyridine (2a), the commercially available
2-amino-6-phenylpyridine (2b), 2-amino-5-methylpyridine (2c),
and 2-aminopyridine (2d) were benzoylated in pyridine at room
temperature to the desired N-pyridin-2-ylbenzamides (3-7). A
small library was designed around the privileged N-(5-phe-
nylpyridin-2-yl)benzamide (6). The benzamide function of 6 was
replaced by a benzylamine (8), phenylurea (10),16 phenyl
carbamate (9), butanamide (11), and cyclohexanecarboxamide
(12). The benzylamine 8 was obtained from the reaction of
2-aminopyridine 2a and benzyl chloride in the microwave
reactor at 180 °C. A reaction with phenyl isocyanate and 2a
yielded the phenylurea analogue (10). Compounds 9, 11, and
12 were obtained by the reaction of 2a with the appropriate
acid chlorides. Introduction of substituents on the phenyl rings
of 6 according to the Topliss system of substitution17 was done
by Suzuki coupling and acylation as described above, resulting
in compounds 22-39 (Table 2).
Structure-Activity Relationships. In an initial screen,
several compounds were tested for activity at a certain GPCR
using the firefly luciferase reporter-gene system (data not
shown). This resulted in a high amount of apparent receptor
antagonists. We wondered whether some of these compounds
were luciferase inhibitors rather than receptor antagonists,
yielding false positive hits, an observation that has also been
reported in a high-throughput screen for antibacterials with the
same reporter gene.18 Therefore, we tested the “active” com-
pounds in an in vitro enzymatic luciferase assay as described
in the Experimental Section. This led to the discovery of 1 that
displayed significant enzyme inhibition with an IC50 value of
1.7 ( 0.1 µM (Figures 1 and 2).
a Inhibition of luciferase activity (IC50 ( SEM (µM), n ) 3, duplicate)
or % inhibition at 10 µM concentrations (n ) 2, duplicate).
Table 2. Inhibition of Luciferase Activity by Compounds 6, 22-39,
Expressed as IC50 Values or as % Inhibition at 10 µM
compd
R4
R5
IC50 (µM) or % inha
6
H
H
H
H
H
H
H
H
H
H
H
0.069 ( 0.01
0.56 ( 0.02
0.31 ( 0.02
51% (47-55)
38% (34-42)
14% (12-16)
38% (27-48)
49% (46-53)
39% (35-42)
4% (0-9)
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
4-Cl
4-OMe
4-Me
3-Cl
4-N(Me)2
4-OiPr
4-tBu
4-CF3
3,4-diCl
H
H
H
H
H
H
H
H
H
4-Cl
4-OMe
4-Me
0% (0-0)
28% (27-28)
18% (17-19)
0.16 ( 0.01
13% (11-16)
26% (22-30)
1.2 ( 0.05
3-Cl
3,4-diCl
2,4-diOMe
4-NH2
4-OiPr
4-N(Me)2
12% (8-17)
35% (29-42)
a Inhibition of luciferase activity (IC50 ( SEM (µM), n ) 3, duplicate)
or % inhibition at 10 µM concentrations (n ) 2, duplicate).
Because the luciferase assay is based on a readout of light
production at 562 nm, quenching could cause false positive hits.
Therefore, the absorbance spectra (240-600 nm) were deter-
mined for D-luciferin, 1, and “Nile red”, which was identified
as a luciferase inhibitor in the antibacterial screen mentioned
above.18 As expected from its structure (and naming), the latter
compound was a quencher of the luciferase signal with an
absorption peak around 550 nm. On the other hand, both the
endogenous substrate and compound 1 did not show any
absorption at 350 nm and higher (data not shown).
Further studies were undertaken to investigate the pharma-
cological characteristics of compound 1. Therefore, the
Michaelis-Menten kinetics of luciferin in the absence and
presence of two concentrations of 1 were examined (Figure 3a).
Saturation of luciferase activity by increasing concentrations
of luciferin resulted in a KM value of 12 ( 2 µM in the absence

4726 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Heitman et al.
indicated that compound 1 competitively inhibited the action
of luciferin. As luciferase has a second catalytic site for ATP,
the Michaelis-Menten kinetics of ATP in the absence and
presence of compound 1 were examined as well (Figure 3b).
The presence of 1 µM 1 resulted in a significantly decreased
Vmax value (Table 3), proof for a noncompetitive inhibition of
ATP. Hence, 1 appears to solely compete for the luciferin
binding site at luciferase. Notably, the ATP saturation curves
were best analyzed by a two-site binding model (p < 0.0001).
These results support earlier reports of two different ATP
binding sites in the enzyme.7,19 At one of these sites the
Mg-ATP complex is bound and at the other ATP, where the
latter is thought to promote the release of product.20 The second
site was therefore described as an allosteric site with positive
cooperativity.
In our search for further evidence of direct luciferase
inhibitors, we analyzed screening data deposited at the PubChem
database (luciferase profiling assay AID 411).13 Interestingly,
one of the most active compounds (CID 649849) also contained
an N-pyridin-2-ylbenzamide core and had an IC50 value of 0.25
µM in their assay (3, Figure 1). This compound was synthesized
by us and tested in the luciferase assay described here, which
yielded a similar IC50 value of 0.61 ( 0.09 µM (Figure 2). For
compounds 1 and 3 the Hill coefficients of the inhibition curves
were 1.07 ( 0.02 and 1.02 ( 0.02, respectively, which indicates
that the binding of these ligands is independent of the presence
of any other substrates.
Figure 1. Chemical structures of compounds 1, 3, and 6.
Subsequently, analogues of N-pyridin-2-ylbenzamide (4-12)
were synthesized and tested for luciferase inhibition to explore
the structure-activity relationships of this compound class
(Table 1). Replacement of the quinoline ring system of 1 for a
pyridine ring (4) resulted in a dramatic loss of potency.
Subsequent introduction of a 2-phenyl ring (5) did not improve
the potency. Substitution with a 3-phenyl ring, however, resulted
in a highly potent luciferase inhibitor (6) (Figure 2). This
compound had an IC50 value of 0.069 ( 0.01 µM that was 10-
fold lower than that of compound 3. Interestingly, introduction
of a p-methyl substituent as found in 3 decreased the potency
by 100-fold (7, IC50 ) 6.4 ( 0.4 µM). Apparently, either the
2,4-dimethoxy substituted phenyl ring on the right-hand side
or the phenyl-substituted pyridine ring was important for high
potency. In addition, the effect of different linkers between the
pyridine and the phenyl ring was studied. As follows from Table
1, neither the amine (8) nor the carbamic acid ester (9) nor the
urea (10) linker resulted in an increase in potency. On the
contrary, compounds 8-10 showed negligible inhibition, if any.
With the amide as the preferred linker, two alkyl substituents
were tested. A cyclohexyl group (12), but not an n-propyl group
(11), also resulted in a potent luciferase inhibitor, although
approximately 4-fold less potent than compound 3 (Table 1).
Apparently, a larger substituent, either alkyl (12) or aryl (6), is
preferred in the binding pocket of the enzyme.
Figure 2. Inhibition of luciferase activity by compounds 1, 3, and 6.
The IC50 values of 1, 3, and 6 were 1.7 ( 0.1, 0.61 ( 0.09, and 0.069
( 0.01 µM, respectively. Representative graphs are from one of three
experiments performed in duplicate.
The design of other analogues was based on a Topliss
approach in which either one of the aromatic rings was modified
with various substituents (Table 2).17 In general, modifications
both on the 5-phenyl ring (22-30) and on the 2-phenylamide
group (31-39) of the pyridine ring resulted in a loss of potency
in comparison to 6. Apparently, the binding pocket does not
tolerate any substituents on either one of the phenyl rings,
indicating steric hindrance. Four compounds, however, showed
inhibition of luciferase in the high nanomolar to low micromolar
concentration range. On the 5-phenyl ring, introduction of a
4-chloro (22) or 4-methoxy (23) substituent resulted in IC50
values of 0.56 ( 0.02 or 0.31 ( 0.02 µM, respectively.
Figure 3. Saturation of luciferase activity by (a) luciferin or (b) ATP
in the absence (control) or presence of 0.1 or 1 µM 1. Representative
graphs are from one experiment performed in duplicate (see Table 3
for KM and Vmax values).
of inhibitor (Table 3). In the presence of 0.1 or 1 µM 1, the KM
value significantly increased while the Vmax was unchanged. This

Benzamides as Inhibitors of Firefly Luciferase
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4727
Table 3. Saturation of Luciferase Activity by Luciferin and ATP in the Absence and Presence of 0.1 or 1 µM Compound 1, Represented by KM and
Vmax Valuesa
luciferinb
ATPc
KM (µM)
Vmax (%)
KM1 (µM)
Vmax1 (%)
KM2 (µM)
Vmax2 (%)
control
+0.1 µM 1
+1 µM 1
12 ( 1
16 ( 0.8*
53 ( 4***
100 ( 2
96 ( 0.8
95 ( 6
3.1 ( 0.3
ND
3.8 ( 0.5
100 ( 39
ND
87 ( 29
138 ( 16
ND
94 ( 4**
100 ( 22
ND
56 ( 13*
a Values are the mean ((SEM) of three separate assays performed in duplicate: (/) p < 0.05; (//) p < 0.01, (///) p < 0.001 versus control. b Saturation
of luciferase activity by increasing concentrations of luciferin at 90 µM ATP. c Saturation of luciferase activity by increasing concentrations of ATP at 150
µM luciferin. ND: not determined.
Interestingly, the same substituents on the other side of the
pyridine ring (almost) completely abolished the activity. This
may indicate that these luciferase inhibitors bind in a certain
pocket with specific sites of interaction. In addition, introduction
of a 3-chloro (34) or 4-amino (37) substituent on the 2-pheny-
lamide group resulted in IC50 values of 0.16 ( 0.01 or 1.2 (
0.05 µM, respectively.
Finally, compound 6 was docked into a homology model of
firefly luciferase based on its crystal structure including AMP
and oxyluciferin.21 From Figure 4, it becomes clear that AMP
and oxyluciferin bind in two different pockets at the enzyme.
Compound 6 was docked into the AMP and the oxyluciferin
pocket. Interestingly, the best model was obtained when the
binding pocket of compound 6 overlapped with that of oxylu-
ciferin. There are two reasons for that. First, the binding pocket
of AMP is curved while the pocket of oxyluciferin is flat. A
largely planar compound as 6 is therefore accommodated best
by the latter pocket. Second, when the phenyl ring attached to
the amide group is superimposed on the thiazole ring of
oxyluciferin, the “other” phenyl ring of 6 extends into an
available pocket in the enzyme. The results obtained with this
Figure 4. Docking results obtained using Autodock, showing firefly
docking study therefore correspond with the competitive and
noncompetitive inhibition of 1 that was found with respect to
luciferin and ATP, respectively (Figure 3). In addition, from
Figure 4 it follows that the further introduction of substituents
on both phenyl rings would cause steric hindrance, which may
explain the fact that the unsubstituted compound 6 is the most
potent inhibitor.
luciferase homology model (cyan) with AMP (green), oxyluciferin
(yellow), and compound 6 (magenta). For details of the modeling
procedure, see the Experimental Section.
q ) quartet, m ) multiplet, br ) broad, Ar ) aromatic protons.
Elemental analyses were performed by the Leiden Institute of
Chemistry and are within 0.4% of the theoretical values unless
otherwise stated. Reactions were routinely monitored on TLC using
Merck silica gel F254 plates. Microwave reactions were performed
in an Emrys Optimizer (Biotage AB, formerly Personal Chemistry).
Wattage was automatically adjusted to maintain the desired
temperature. The yields of all products were not optimized. All
the final products were purified by column chromatography.
General Procedure for the Preparation of 1, 3-7, 9, 11,
12, and 22-39.24 The appropriate acid chloride (1.1 mmol) was
added to a solution of 2-amino-5-phenylpyridine (1.0 mmol) in
pyridine (4 mL) at room temperature under a nitrogen atmosphere.
According to TLC the reaction went to completion after 2 h. The
organic material was extracted with DCM, dried over MgSO4, and
evaporated under reduced pressure. The crude product was purified
by column chromatography, eluting with a mixture of 0.5-2%
methanol and chloroform or 0.5-2% methanol and dichlo-
romethane, resulting in a yield of 31-75% of the desired products.
N-Quinolin-2-ylbenzamide (1). Starting from 2-aminoquino-
line.1 Yield 31%; white solid, recrystallized from ethanol. 1H NMR
δ (CDCl3): 8.99 (br s, 1H, NH), 8.59 (d, 1H, J ) 9.1 Hz, quinoline-
H), 8.21 (d, 1H, J ) 8.8 Hz, quinoline-H), 8.01-7.96 (m, 2H,
phenyl-H), 7.84-7.77 (m, 2H, quinoline-H), 7.66 (dd, 1H, J1 )
8.4 Hz, J2 ) 1.5 Hz, quinoline-H), 7.56-7.41 (m, 4H, phenyl-H
+ quinoline-H). 13C NMR δ (CDCl3): 151.1, 146.5, 141.0, 138.5,
134.1, 132.3, 129.9, 129.2, 128.7, 127.5, 127.2, 125.1, 114.3, 114.1.
Anal. (C16H12N2O·0.05EtOH) C, H, N.
Conclusion
In summary, we have shown that (druglike) compounds, such
as 1 or 6, are competitive inhibitors of luciferase with respect
to luciferin. In addition, these compounds are noncompetitive
inhibitors with respect to ATP. The inclusion of similar
compounds would result in a high number of “false positives”
in screening campaigns that rely on luciferase reporter-gene
assays, an otherwise robust screening approach with good signal-
to-noise ratio. Notably, we learned that the same structure of 1
has been patented for osteoporosis treatment.22,23 However, also
in these disclosures a luciferase reporter-gene assay was used
to identify the compounds. The data presented in this paper are
therefore meant to warn researchers of direct luciferase inhibitors
that also have druglike properties. Such compounds may be
“flagged” after their evaluation in an assay with, for example,
purified luciferase.
Experimental Section
Chemistry: Material and Methods. All reagents used were
obtained from commercial sources, and all solvents were of
analytical grade. 1H and 13C NMR spectra were recorded on a
Bruker AC 200 (1H NMR, 200 MHz; 13C NMR, 50.29 MHz)
spectrometer with tetramethylsilane as an internal standard. Chemi-
cal shifts are reported in δ (ppm), and the following abbreviations
are used: s ) singlet, d ) doublet, dd ) double doublet, t ) triplet,
2,4-Dimethoxy-N-(5-methylpyridin-2-yl)benzamide (3). Start-
1
ing from 2-amino-5-methylpyridine. Yield 54%; white solid. H
NMR δ (CDCl3): 10.17 (br s, 1H, NH), 8.31 (d, 1H, J ) 8.4 Hz,
pyridine-H), 8.24 (d, 1H, J ) 9.1 Hz, Ar-H), 8.13 (s, 1H, pyridine-

4728 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Heitman et al.
H), 7.52 (d, 1H, J ) 8.4 Hz, pyridine-H), 6.63 (dd, 1H, J1 ) 8.8
Hz, J2 ) 2.2 Hz, Ar-H), 6.53 (d, 1H, J ) 2.2 Hz, Ar-H), 4.04 (s,
3H, OCH3), 3.86 (s, 3H, OCH3), 2.29 (s, 3H, CH3). 13C NMR δ
(CDCl3): 163.7, 162.9, 158.6, 149.7, 147.5, 138.4, 133.8, 128.3,
114.0, 113.8, 105.0, 98.2, 55.9, 55.2, 17.5. Anal. (C15H16N2O3) C,
H, N.
Data Analysis. All enzymatic data were analyzed using the
nonlinear regression curve-fitting program GraphPad Prism, version
5.00 (GraphPad Software Inc., San Diego, CA). Inhibitory binding
constants (IC50) were directly obtained from the concentration-effect
curves. The KM and Vmax values of luciferin and ATP in the absence
or presence of 1 were obtained by computer analysis of one- or
two-site saturation curves, respectively. All values obtained are the
mean values of three independent experiments performed in
duplicate.
Docking Studies. The crystal structure of Luciola cruciata
complexed with oxyluciferin and AMP was retrieved from the
Brookhaven Protein Databank (PDB entry 2D1R).21 Sequence
alignment of Luciola cruciata and Luciola mingrelica was per-
formed using CLUSTALW.26 The structural homology models were
created using InsightII 98 (San Diego, CA).
N-(5-Phenylpyridin-2-yl)benzamide (6). Yield 46%; white
1
solid. H NMR δ (CDCl3): 8.83 (br s, 1H, NH), 8.50-8.46 (m,
2H, pyridine-H), 8.00-7.93 (m, 3H, pyridine-H + phenyl-H),
7.58-7.38 (m, 8H, phenyl-H). 13C NMR δ (CDCl3): 166.3, 150.9,
145.7, 137.0, 136.6, 134.5, 132.5, 131.9, 128.8, 128.5, 127.6, 127.4,
126.4, 114.1. Anal. (C18H14N2O·0.04CHCl3) C, H, N.
N-[5-(4-Chorophenyl)pyridin-2-yl]benzamide (22). Yield 53%;
white solid. 1H NMR δ (CDCl3): 8.86 (br s, 1H, NH), 8.48 (d, 1H,
J ) 8.76 Hz, pyridine-H), 8.43 (d, 1H, J ) 2.6 Hz, pyridine-H),
7.97-7.91 (m, 3H, pyridine-H + phenyl-H), 7.59-7.40 (m, 7H,
phenyl-H + Ar-H). 13C NMR δ (CDCl3): 165.8, 150.9, 145.6,
136.5, 135.5, 134.2, 133.8, 132.0, 131.4, 129.0, 128.6, 127.7, 127.2,
113.9. Anal. (C18H13ClN2O·0.04CHCl3) C, H, N.
N-[5-(4-Methoxyphenyl)pyridin-2-yl]benzamide (23). Yield
68%; white solid. 1H NMR δ (CDCl3): 8.80 (br s, 1H, NH),
8.47-8.43 (m, 2H, pyridine-H), 7.97-7.90(m, 3H, pyridine-H +
phenyl-H), 7.62-7.48 (m, 5H, phenyl-H + Ar-H), 7.00 (d, 2H, J
) 8.7 Hz, Ar-H), 3.86 (s, 3H, OCH3). 13C NMR δ (CDCl3): 165.8,
159.3, 150.2, 145.3, 136.1, 134.3, 132.4, 131.9, 129.5, 128.5, 127.5,
127.2, 114.3, 113.9, 55.1. Anal. (C19H16N2O2 ·0.06CHCl3) C, H,
N.
Docking simulations were performed with AutoDock 3.27 Grid
maps of 20 Å × 20 Å × 20 Å representing the protein were
calculated with AutoGrid. Docking simulations were carried out
using the Lamarckian genetic algorithm, with an initial popula-
tion of 100 individuals, a maximum number of 10 000 000
energy evaluations, and a maximum number of 50 000 genera-
tions.27 Resulting orientations lying within 1.5 Å in the rmsd
were clustered together. Finally the configuration with the most
favorable free energy of binding was further optimized by 1500
energy minimization steps with InsightII. PyMOL, version 1.0
(DeLano Scientific, Palo Alto, CA), was used to superimpose
and visualize the model.
3-Chloro-N-(5-phenylpyridin-2-yl)benzamide (34). Yield 59%;
white solid. H NMR δ (CDCl3): 8.74 (br s, 1H, NH), 8.51 (dd,
Supporting Information Available: Experimental details of the
synthesis of the compounds described in this paper, their 1H NMR
and 13C NMR spectroscopic data, and their elemental analysis
results. This material is available free of charge via the Internet at
http://pubs.acs.org.
1
1H, J1 ) 2.2 Hz, J2 ) 0.7 Hz, pyridine-H), 8.44 (dd, 1H, J1 ) 8.4
Hz, J2 ) 0.7 Hz, pyridine-H), 8.01 (d, 1H, J ) 2.6 Hz, pyridine-
H), 7.96 (dd, 1H, J1 ) 3.5 Hz, J2 ) 1.8 Hz, Ar-H), 7.83-7.78 (m,
1H, Ar-H), 7.60-7.35 (m, 7H, Ar-H + phenyl-H). 13C NMR δ
(DMSO): 164.7, 151.4, 145.8, 136.8, 133.3, 131.7, 130.3, 129.1,
128.0, 127.8, 126.8, 126.5, 114.7. Anal. (C18H13ClN2O) C, H, N.
4-Amino-N-(5-phenylpyridin-2-yl)benzamide (37). Yield 33%;
off-white solid. 1H NMR δ (CDCl3): 8.67 (br s, 1H, NH), 8.49-8.43
(m, 2H, pyridine-H), 7.95 (dd, 1H, J1 ) 8.6 Hz, J2 ) 2.2 Hz,
pyridine-H), 7.78 (d, 2H, J ) 8.4 Hz, Ar-H), 7.50-7.33 (m, 5H,
phenyl-H), 6.70 (d, 2H, J ) 8.8 Hz, Ar-H), 4.09 (br s, 2H, NH2).
13C NMR δ (DMSO): 165.7, 152.6, 152.1, 145.5, 137.0, 136.0,
130.9, 129.9, 129.2, 127.7, 126.4, 120.2, 114.3, 112.7. Anal.
(C18H15N3O) C, H, N.
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