Structure guided development of novel thymidine mimetics targeting Pseudomonas aeruginosa thymidylate kinase: From hit to lead generation

Article abstract of DOI:10.1021/jm201349f

Thymidylate kinase (TMK) is a potential chemotherapeutic target because it is directly involved in the synthesis of an essential component, thymidine triphosphate, in DNA replication. All reported TMK inhibitors are thymidine analogues, which might retard their development as potent therapeutics due to cell permeability and off-target activity against human TMK. A small molecule hit (1, IC₅₀ = 58 μM), which has reasonable inhibition potency against Pseudomonas aeruginosa TMK (PaTMK), was identified by the analysis of the binding mode of thymidine or TP₅A in a PaTMK homology model. This hit (1) was cocrystallized with PaTMK, and several potent PaTMK inhibitors (leads, 46, 47, 48, and 56, IC₅₀ = 100-200 nM) were synthesized using computer-aided design approaches including virtual synthesis/screening, which was used to guide the design of inhibitors. The binding mode of the optimized leads in PaTMK overlaps with that of other bacterial TMKs but not with human TMK, which shares few common features with the bacterial enzymes. Therefore, the optimized TMK inhibitors described here should be useful for the development of antibacterial agents targeting TMK without undesired off-target effects. In addition, an inhibition mechanism associated with the LID loop, which mimics the process of phosphate transfer from ATP to dTMP, was proposed based on X-ray cocrystal structures, homology models, and structure-activity relationship results.

Full text of DOI:10.1021/jm201349f

Article
pubs.acs.org/jmc
Structure Guided Development of Novel Thymidine Mimetics
Targeting Pseudomonas aeruginosa Thymidylate Kinase: From Hit to
Lead Generation
Jun Yong Choi,† Mark S. Plummer,*,‡ Jeremy Starr,‡ Charlene R. Desbonnet,§ Holly Soutter,∥
Jeanne Chang,∥ J. Richard Miller,§ Keith Dillman,∥ Alita A. Miller,§ and William R. Roush*,†
†Department of Chemistry, Scripps Florida, Jupiter, Florida 33458, United States
§
∥
‡Antibacterials Medicinal Chemistry, Antibacterials Research Unit, and Structural Biology and Biophysics, Pfizer Global Research
and Development, Groton, Connecticut 06340, United States
S
* Supporting Information
ABSTRACT: Thymidylate kinase (TMK) is a potential chemotherapeutic target because it is directly involved in the synthesis
of an essential component, thymidine triphosphate, in DNA replication. All reported TMK inhibitors are thymidine analogues,
which might retard their development as potent therapeutics due to cell permeability and off-target activity against human TMK.
A small molecule hit (1, IC50 = 58 μM), which has reasonable inhibition potency against Pseudomonas aeruginosa TMK
(PaTMK), was identified by the analysis of the binding mode of thymidine or TP5A in a PaTMK homology model. This hit (1)
was cocrystallized with PaTMK, and several potent PaTMK inhibitors (leads, 46, 47, 48, and 56, IC50 = 100−200 nM) were
synthesized using computer-aided design approaches including virtual synthesis/screening, which was used to guide the design of
inhibitors. The binding mode of the optimized leads in PaTMK overlaps with that of other bacterial TMKs but not with human
TMK, which shares few common features with the bacterial enzymes. Therefore, the optimized TMK inhibitors described here
should be useful for the development of antibacterial agents targeting TMK without undesired off-target effects. In addition, an
inhibition mechanism associated with the LID loop, which mimics the process of phosphate transfer from ATP to dTMP, was
proposed based on X-ray cocrystal structures, homology models, and structure−activity relationship results.
is the last specific enzyme in the pathways for the synthesis of
thymidine triphosphate (dTTP), which is an essential
component in DNA synthesis.7 Therefore, targeting bacterial
TMK has been the subject of recent investigation with
inhibitors of Mycobacterium tuberculosis,8−12 Staphylococcus
aureus,13 and Bacillus anthracis14 TMKs reported in conjunction
with their cognate protein crystal structures.
However, most of the reported TMK inhibitors are
thymidine derived (Figure 1).15−21 Although inhibitor design
has been enhanced by utilizing protein structures,21 computer-
aided design,20 and quantitative structure−activity relationship
(QSAR) methods,19 the thymine headgroup of the inhibitors
always remains (Figure 1). The thymine headgroup was viewed
as a hindrance for designing inhibitors that penetrate the
complex cell membrane and avoid efflux pumps prevalent in Pa.
INTRODUCTION
■
Most classes of antibacterial agents currently employed in
clinical use were discovered over 45 years ago, with the
exception of the oxazolidinone class (Linezolid), introduced in
2000 to treat Gram-positive infections. New antibacterial
therapeutics that utilize new mechanisms of action are urgently
needed to combat growing resistance to existing antibacterial
agents for both Gram-positive and Gram-negative infections.
Although the discovery of new antibacterial classes is
extraordinarily difficult,1 the need is especially high for Gram-
negative organisms prevalent in the hospital and in particular
for infections caused by Pseudomonas aeruginosa (Pa), for which
treatment options are often limited.2−4
Thymidylate kinase (TMK) has emerged as an attractive
therapeutic target because inhibiting TMK functions blocks
DNA synthesis in replicating organisms.5 TMK phosphorylates
thymidine monophosphate (dTMP) to thymidine diphosphate
(dTDP), using ATP as a phosphoryl donor.6 In addition, TMK
Received: October 8, 2011
Published: January 13, 2012
© 2012 American Chemical Society
852
dx.doi.org/10.1021/jm201349f | J. Med. Chem. 2012, 55, 852−870

Journal of Medicinal Chemistry
Article
Therefore, small molecule TMK inhibitors, or nonsubstrate
analogues, are necessary to develop antibacterial therapeutics.
Herein, we report a nonthymidine inhibitor (1) targeting
PaTMK, the cocrystal structures of 1, and the evolved inhibitor
analogue 17 with PaTMK and structure-guided development of
TMK inhibitors. To our knowledge, this is the first report of
TMK inhibitors dispossessing the thymidine moiety and its
cocrystal structure with PaTMK. In addition, we describe the
use of computer-aided design, including virtual synthesis and
screening, with the cocrystal structures to expedite rational
design and synthesis of more potent PaTMK inhibitors.
RESULTS AND DISCUSSION
■
Hit Generation. Structural analysis of a PaTMK homology
model built based on cocrystal structure (PDB ID: 4TMK) of
Escherichia coli TMK22 with the thymidine-based dual substrate
inhibitor P1-(5′-adenosyl)-P5-(5′-thymidyl)pentaphosphate
(TP5A) led us to intimately understand the interactions
required for thymidine binding to its binding site of PaTMK
(Figure 2a). Furthermore, we found that a commercial
compound, 1-methyl-6-phenyl imidazopyridinone (1), has
similar chemical properties to thymidine, although their two-
dimensional structures have low similarity. Flexible alignment
Figure 1. Known TMK inhibitors.
Figure 2. Key interactions of dTMP and 1 in PaTMK. (a) Thymidine interactions in the active site of PaTMK in the homology model are
represented in two-dimensional view. (b) The result of flexible alignment of 1 to dTMP shows a clear pharmacophore match between 1 and dTMP.
The structure of dTMP is represented as a red line, and that of 1 is as stick with atom type color (gray for carbon, blue for nitrogen, red for oxygen,
and orange for phosphorus). (c) The overall X-ray cocrystal structure of PaTMK and 1 shows that 1 binds where dTMP bound (left panel). The
structure of TP5A was merged from the reported X-ray structure (PDB ID: 4TMK) to compare the similarity of the binding site of 1 and TP5A. The
binding mode of 1 in PaTMK at the X-ray cocrystal structure shows that the binding pose of 1 at PaTMK is identical to that of dTMP. The α-helix is
red, the β-sheet is yellow, and the loop is green (left panel). The water molecule is represented with a red sphere, 1 and amino acids associated with
the binding of 1 are represented as sticks with atom type color, and H-bond interactions are represented with black lines. π-Cation stacking between
the phenyl ring of 1 and Arg96 is not marked. Hydrogen atoms are omitted for clarity. Figures are generated with the Pymol program.
853
dx.doi.org/10.1021/jm201349f | J. Med. Chem. 2012, 55, 852−870

Journal of Medicinal Chemistry
Article
a
Table 1.
a
Each reported IC50 was determined at least in duplicate and is an average of the individual values determined. Each individual IC50 value in a single
determination was the result of a 20 point IC50 curve with each point determined in duplicate.
of 1 to thymidine structure was performed to recognize their
geometrical similarity using the Molecular Operating Environ-
ment program (MOE).23 The result showed that 1 and
thymidine are well overlapped with an identical pharmacophore
(Figure 2b). In the enzyme assay, compound 1 proved to be an
inhibitor of PaTMK with approximately 3-fold less potency
than the known TMK inhibitor, deoxy-fluorothymidine
(dFTM) (IC50 = 58 μM vs 20 μM in Table 1).
X-ray Structure Analysis. To elucidate the binding mode
of 1 to facilitate the structure-based development of TMK
inhibitors, 1 was cocrystallized with PaTMK, as described in the
Supporting Information, yielding cocrystals that diffracted to
1.91 Å. Gratifyingly, many of the interactions predicted in our
homology model and flexible alignment are identical to those in
the X-ray cocrystal structure (Figure 2c). Especially important
for molecular recognition is the ability of the cyclic
imidazopyridinone functionality to satisfy the hydrogen bond
donors and acceptors of the typical thymidine substrate:
specifically accepting hydrogen bonds from Arg74 and Thr101,
while both donating and accepting hydrogen bonds with
Gln105 (Figure 2c). Other important interactions with the
distal phenyl ring are the boxlike face to face and edge to face
aromatic interactions24 with Phe155 and Tyr104, which are
complemented by the π-cation interaction with Arg96.25−27
Also apparent is a structural water molecule that bridges
between the phenolic −OH group of Tyr104 and the amide
backbone (−NH) of Glu12. Although this bridging water does
not offer a favorable binding interaction with the inhibitor, it is
located only 3.8 Å from the meta-position of the distal phenyl
ring. Thus, the bridging structural water suggests a design
opportunity for increasing potency via either displacement or
additional interaction through hydrogen bonding.28
854
dx.doi.org/10.1021/jm201349f | J. Med. Chem. 2012, 55, 852−870

Journal of Medicinal Chemistry
Article
a
Scheme 1.
a
Reagents and conditions: (a) ClCO2Bn, Pyridine, 0 °C to room temperature, 5 h, 48%. (b) LiAlH4, Et2O, 0 °C to room temperature, 6 h, 99%. (c)
Triphosgene, THF, room temperature to reflux, 4 h, 83%. (d) Ph3CCl, Et3N, CH2Cl2, room temperature, 1 h, 84%. (e) Aryl boronic acid, 1 mol %
Pd2(dba)3, 2 mol % PCy3, K3PO4, dioxane, 100 °C (microwave), 1 h. (f) TFA, CH2Cl2, room temperature, 30 min, 50−99% (over two steps).
Synthesis of 1 and Its Analogues. The synthesis of
analogues of 1 was accomplished using 6 as a key intermediate
(Scheme 1). Briefly, 5-bromo 2,3-diaminopyridine 2 was
treated with benzyl chloroformate to give benzyl carbamate
3.29 The urethane was reduced to a methyl group with lithium
aluminum hydride, and the diamine was cyclized to the urea 5,
utilizing triphosgene.30 Palladium-mediated coupling reactions
of 5 were accomplished efficiently when the acidic urea NH was
protected with a trityl group as in 6. The coupling reaction of 6
with phenyl boronic acid provided 7 in good yield when
performed under microwave conditions.31−34 Removal of the
trityl group with trifluoroacetic acid provided the final inhibitor,
1. Various aryl boronic acids proposed by computational
studies were coupled to the intermediate 6 to produce the first
inhibitor library.
Scheme 2
Virtual Synthesis/Screening of First Round PaTMK
Inhibitors. As described in the analysis of the cocrystal
structure, the phenyl ring of 1 can be further modified to
increase inhibition potency by replacing it with electron-rich
aromatic rings and/or by incorporating functional groups
replacing the water molecule near Tyr104. In terms of
synthesis, aryl boronic acids can be coupled with intermediate
6 followed by trityl deprotection to produce 1 and its analogues
(Scheme 1). Therefore, to increase the diversity of inhibitors
being designed, virtual synthesis of inhibitors followed by
docking and scoring was applied as outlined in Scheme 2. First,
5000 commercially available boronic acids were computation-
ally attached to the 6-position of imidazopyridine-2-one 5 with
the combi-gen program in MOE. The virtually synthesized
5000 compounds were docked into the active site of PaTMK
with the water molecule removed using Glide 5.5.35 Hits were
ranked and sorted with docking score function ranging from
−11.5 to −2.3 kcal/mol. In this virtual screening, the lead
inhibitor 1 ranked 1933th with a score of −8.7 kcal/mol. The
virtual compounds with scores lower than 1 were visually
inspected using the maestro9.0 program;36 most aryl
appendages targeted nearby amino acid residues such as the
hydroxyl group of Tyr104, the carboxyl groups of Asp153 and
Glu156, and the guanidine group of Arg50. About 20
compounds were selected for synthesis, each of which had
small aryl groups that offered favorable interactions with the
protein, along with polar functionality to generally improve
physical properties while preserving the hydrogen-bonding
network of the imidazopyridinone. This structure-guided design
and synthesis resulted in the discovery of 17 (IC50 = 0.44 μM)
and 19 (IC50 = 2.2 μM), which have 130- and 30-fold improved
inhibition over 1, respectively.
Analysis of First Round PaTMK Inhibitors and X-ray
Cocrystal Structure of 17 with PaTMK. The tactic of
displacing the water molecule near Tyr104 with simple H-bond
donors and acceptors was explored using the docking result as a
guide. The assay results showed good consistency with the
rationale proposed with the X-ray structure and the modeling
technique with minor differences. The phenol substitution in
12 provided about a 4-fold increase in potency, while the
cyano-group in 14 resulted in loss of activity. Not only was the
cyano-compound 14 inactive, it also lacked the H-bond-
donating ability of the phenol in 12; therefore, follow-up
focused on incorporating hydrogen bond-donating groups that
were larger than the cyano-group. The meta-aminomethyl
group in 15 was chosen as there are several acidic protein
residues in the vicinity of the inhibitor binding site such as
Glu12, Glu156, and Asp153. The lack of activity observed for
this analogue contrasts with the good potency of para-
aminomethyl analogue 25, indicating that the para-amino
methyl group must extend toward the complementary acidic
residues providing a favorable protein−ligand interaction. The
similar in size but uncharged meta-hydroxymethyl group in 16
provided a 37 μM inhibitor, a 2-fold increase in potency over 1,
suggesting that the positive charge in 15 was problematic.
855
dx.doi.org/10.1021/jm201349f | J. Med. Chem. 2012, 55, 852−870

Journal of Medicinal Chemistry
Article
Figure 3. X-ray cocrystal structure of 17 and PaTMK. Key interactions of 17 in the active site of PaTMK are described as 2D (up) and 3D (down)
views. For clarity, interactions associated with imidazopyridinone moiety and hydrogen atoms are omitted. Furthermore, water molecules, which are
not involved in binding of 17, are omitted. The water molecule is represented as a red sphere, 17 and amino acids are represented as sticks with atom
type color, and H-bond interactions are represented with black lines.
a
Scheme 3.
a
Reagents and conditions: (a) 3-Carboxyphenylboronic acid, 1 mol % Pd2(dba)3, 2 mol % PCy3, K3PO4, dioxane, 100 °C (microwave), 1 h, then
aqueous HCl, 99%. (b) Pentafluorophenyl trifluoroacetate, N(i-Pr)2Et, CH2Cl2, 0 °C to room temperature, 1 h, 81%. (c) Primary amines, N(i-
Pr)2Et, CH2Cl2, room temperature, 1 h. (d) TFA, CH2Cl2, room temperature, 30 min, 100−56% (over two steps).
In an opposite tactic to displacing the bridging structural
water molecule, interactions through this water molecule were
probed by incorporation of pyridine, pyrimidine, and fluorine
groups as potential hydrogen bond acceptors as in 9−11. Only
the pyridine-containing inhibitor 10 retained the meaningful
activity of the parent compound 1 (Table 1). As described, the
phenyl ring of 1 forms a π-cation interaction with Arg96.25−27
Therefore, not surprisingly, compounds with electron-with-
drawing groups such as 9, 11, 14, 18, and 26 significantly lost
electronic potential at the phenyl ring followed by inhibition
potency (IC50 > 200). However, interestingly, 17 has
dramatically increased inhibition potency (130-fold over initial
lead 1 and a 60-fold over a standard inhibitor dFTM), although
it possesses an electron-poor aryl ring; this is also the case with
19, 21, and 22, which, however, are less potent than 17. These
results can be rationalized in the context of the solved X-ray
cocrystal structure of 17 and PaTMK (Figure 3).
Inhibitor 17 was cocrystallized with PaTMK and provided a
1.7 Å structure. The binding pose of 17 in virtual synthesis/
screening procedure is very consistent with that of the X-ray
structure. Indeed, the structural water targeted in the design
was clearly displaced with the carboxamide of 17, and a direct
856
dx.doi.org/10.1021/jm201349f | J. Med. Chem. 2012, 55, 852−870

Journal of Medicinal Chemistry
Article
a
Table 2.
a
Each reported IC50 was determined at least in duplicate and is an average of the individual values determined. Each individual IC50 value in a single
determination was the result of a 20 point IC50 curve with each point determined in duplicate.
hydrogen bond interaction between the carbonyl oxygen of 17
present was a water molecule-mediated H-bond network that
and the phenolic oxygen of Tyr104 is apparent (Figure 3). Also
further hydrates the carboxamide of 17 and could surround the
857
dx.doi.org/10.1021/jm201349f | J. Med. Chem. 2012, 55, 852−870

Journal of Medicinal Chemistry
Article
a
Scheme 4.
a
Reagents and conditions: (a) Monopropagylamine hydrochloride, N(i-Pr)2Et, CH2Cl2, room temperature to 50 °C, 1 h, 98%. (b) Aryl bromide, 10
mol % Pd(PPh3)4, NEt3, 5 mol % CuI, DMF, 80 °C (microwave), 1 h, 73%. (c) H2, Pd/C, MeOH, 12 h, 63%. (d) TFA, CH2Cl2, room temperature,
30 min, 46%. (e) 10% KOH(aq), MeOH, 60 °C, 12 h, then TFA, CH2Cl2, room temperature, 30 min, 22% (over four steps).
Figure 4. Homology model of PaTMK and 46 based on the X-ray structure of PaTMK and 17. The blue loop is the generated LID loop in
homology modeling. In the close view (right panel), the terminal phenyl ring of 46 occupies the hydrophobic pocket composed of Pro11 (facing to
the plane of the phenyl ring), Phe163, Val139, Leu137, Ile141, and Leu143 (surround the terminal phenyl ring of 46). Other residues in the loop are
oriented toward a solvent-accessible area such as Pro138, Glu140, and Gly142, which were omitted for clarity. In addition, interactions associated
with imidazopyridinone are the same as those in the previous X-ray cocrystal structure complexed with 1 or 17 as in Figure 2c; thus, these
interactions and hydrogen atoms are omitted for a clear view.
entrance of the inhibitor (or dTMP) binding site to make the
release of 17 slow. As a combined effect of H-bond interactions
with Tyr104 and the H-bond network, PaTMK inhibition by
17 was significantly increased as compared to 1: ca. 1000-fold
increase over 26 (p-carboxamide), which possesses an electron-
withdrawing group but without the aforementioned cooperative
hydrogen-bonding interactions. The effect of the H-bond
interactions mediated by a network of water molecules appears
to be much more important than H-bond interactions with
Tyr104 because of decreased potency of 19 and 20. The
activity of inhibitor 19 (N-methylamide), which presumably
does not support the extensive H-bond network seen in 17, was
decreased 5-fold as compared with 17. The importance of the
hydrogen bonds with the structural water molecules is
supported by the significant, 240-fold decreased inhibition
observed for 20 (N,N-dimethylamide), which likely would lose
the H-bond network while retaining a H-bond interaction with
Tyr104. Consequently, as rationalized in the computer-guided
design approach, the π-cation interaction of an electron-rich
phenyl ring with Arg96 and H-bond interactions of the
carbonyl oxygen of the carboxamide with Tyr104 are important
for inhibition of PaTMK. In addition, the X-ray cocrystal
structure of 17 provided critical clues, specifically the water-
mediated H-bond network, which provides dramatically
increased potency of 17, although possessing an electron-
withdrawing group. Because the secondary amide 19 retained
reasonable potency and intermediate 28 (Scheme 3) was
conducive to rapid analogue production, this vector was
explored with the aim of increasing potency.
Virtual Synthesis/Screening of Second Round PaTMK
Inhibitors. A second round of virtual synthesis was designed to
efficiently expand diversity on lead 19. Virtual synthesis was
performed with 1188 commercially available primary amines to
obtain various alkyl-tethered benzamide derivatives that were
then virtually screened by docking into the active site of
PaTMK using Glide 5.5. About 900 hits were ranked and sorted
with docking scores ranging from −14.6 to −0.9 kcal/mol.
Among them, inhibitor 19 ranked 390th with a score of −11.4
kcal/mol. With a focus on improving potency, 20 compounds
were selected for synthesis by visually inspecting the top-
858
dx.doi.org/10.1021/jm201349f | J. Med. Chem. 2012, 55, 852−870

Journal of Medicinal Chemistry
Article
Figure 5. Comparison of the active sites of PaTMK (left) and hTMK (right). Amino acids associated with the interaction of 46 in PaTMK and their
corresponding amino acids in hTMK are represented as sticks with atom type color. The structure of 46 and PaTMK was obtained from the docking
study with the X-ray cocrystal structure of PaTMK complexed with 17. The structure of 46 and hTMK was obtained through structure-based
multiple sequence alignment followed by merging the coordinate of 46 to hTMK structure (PDB ID: 1E2F) from PaTMK structure. H-bond
interactions are highlighted with dashed black lines. Edge to face hydrophobic interactions and π-cation stacking are annotated with black arrows.
scoring compounds followed by ranking for size and enhanced
interaction with the protein while maintaining the integrity of
the imidazopyridinone hydrogen-bonding interactions (Table
2).
polar group increased potency. Exploring extension of the
hydrophobic group in the series 40 to 42 to 46/47/57
indicated that a propyl spacer between the amide was optimal
with inhibitor 46 having 0.12 μM inhibitory activity, an 18-fold
improvement over the methyl amide 19. Inhibitors 47 and 48
both retain a high level of potency, indicating that electronic
characteristics and steric bulk can be varied. The addition of
polar groups in 59−63 to the phenyl ring resulted in a 4−20-
fold loss of inhibition as compared with 46. Introduction of the
carboxyl group into the propyl side chain, making hybrids of 31
and 46 provided racemic compound 52, lost significant
potency. However, the hybrid of 35 and 46 giving racemic
inhibitor 53 with a carboxyl group proved to be a useful
combination where a good level of potency was retained.
Analysis of Second Round PaTMK Inhibitors. The trend
of the second round SAR is that compared to 19, ca. 18-fold
increased potency of inhibitors 46, 47, 48, 53, and 57, which
possess hydrophobic aromatic rings linked with an aliphatic
propyl chain. However, substitution with small functional
groups in 30−38 and an aromatic ring linked with a short
aliphatic chain in 40−45 did not provide significantly increased
inhibition of PaTMK. For rationalization of these results,
homology modeling has been performed to fill out the missing
amino acids (Val139 to Arg151, a part of the LID loop22,37) in
the X-ray cocrystal structure of PaTMK and 17, because the
terminal phenyl ring of 46 is oriented toward the region where
the LID loop is involved (Figure 4). Interestingly, the results of
the homology modeling showed that the terminal phenyl ring
of 46 occupies a hydrophobic pocket composed of residues
such as Pro11, Leu137, Val139, Ile141, Leu143, and Phe163.
Therefore, the favorable hydrophobic contact between the
terminal phenyl ring of 46 and the hydrophobic pocket may be
the main reason for the increased potency of 46. Furthermore,
47, 57, and 58 with p-fluoro, p-trifluoromethyl, and o-
methoxycarbonyl groups, respectively, have very similar
potency to 46. However, a terminal aryl ring with charged
functional groups as in 59−63 lost about 10-fold inhibition
potency. The LID loop is not clearly solved in most X-ray
structures of TMK because it is highly flexible. However, it is
known that this highly adaptable loop undergoes substantial
conformational changes to close the ATP and dTMP binding
sites when the phosphoryl donor, ATP, binds to the enzyme for
phosphoryl transfer. In addition, TP5A, which is a phosphoryl
Synthesis of 19 and Its Analogues. Synthesis of
analogues of 19 was achieved using 28 as a key intermediate
(Scheme 3). Intermediate 6 in Scheme 1 was coupled with 3-
carboxylphenylboronic acid to provide 27 in near quantitative
yield. The acid in 27 was activated as the pentafluorophenyl
ester 28, which was stable to storage (0.1 M solution of
CH2Cl2). About 0.2 mmol of 28 (2 mL solution of CH2Cl2)
was treated with the various primary amines and diisopropy-
lethylamine to provide the desired amides. Removal of the trityl
group with TFA provided the final inhibitors. In addition, for
modification of the terminal phenyl ring of 46, intermediate 28
was treated with propargylamine to produce 54 (Scheme 4).
The terminal alkyne of 54 was coupled with various aryl
bromides mediated by Pd(0) in the presence of a catalytic
amount of copper(I) iodide to give 55. Hydrogenation of 55
provided arylpropyl amides 56 from which the trityl group was
removed with TFA to give the desired inhibitors 57−63.
Assay Results of Second Round PaTMK Inhibitors. A
number of small polar secondary amides were synthesized to
explore functionality accommodated by the binding pocket
30−38 (Table 2). The glycinamide and glycine analogues 30
and 31 proved to be the most potent polar analogues
synthesized. Each has submicromolar activity and improved
potency about 2.5-fold over secondary amide 19, although they
were both less potent than the parent amide analogue 17. Both
negatively and positively charged groups as in 31, 32, 35, and
37 also were accommodated without large changes in potency
when compared with 19.
The X-ray cocrystal structure of 17 indicated that the area
immediately adjacent to the m-carboxamide might also have
hydrophobic character (Figure 4), so small hydrophobic
extensions of the amide were investigated as in compounds
40−47 (Table 2). Inhibitors 40 and 41 both retained the
potency of the parent methylamide 19 showing that larger
groups could be accommodated in this area. Because potency
did not increase in a manner commensurate with the increase
in size, 40 and 41 were deemed inefficient binding ligands.
More polar heterocycles as in 43−45 lost potency as compared
with 19, which was in contrast to the glycinamide 30 where a
859
dx.doi.org/10.1021/jm201349f | J. Med. Chem. 2012, 55, 852−870

Journal of Medicinal Chemistry
Article
transfer mimetic, has high binding affinity due to LID loop
closure (Kd = 20 nM).22 Therefore, the LID loop is directly
involved in the mechanism of phosphoryl transfer by covering
the active site when both ATP and dTMP are bound. On the
basis of our structural model and SAR results, it can be
proposed that the terminal phenyl ring of 46 forces shielding of
the active site by the LID loop via forming direct hydrophobic
contacts between the inhibitor and the hydrophobic pocket of
PaTMK, resulting in increased binding affinity of 46.
Biological Evaluation of Inhibitors. Each compound in
Tables 1 and 2 was tested against P. aeruginosa PA01 and
PAO280 (MexABoprM, MexXY, MexZ efflux pump knock out)
and were found to have minimum inhibitory concentrations
(MICs) >256 μg/mL. Inhibitors 46 (and also 47/48/57) have
nanomolar enzyme inhibition and have comparable enzyme
inhibition to the cellularly active TMKmt inhibitor [5′-deoxy-5′-
N-arylthiourea α-thymidine derivatives (thiourea-α-TM) in
Figure 1: Ki = 1.0 μM (against TMKmt) and MIC (M. bovis)
= 25 μg/mL]. Therefore, lacking cellular activity in an efflux
pump knock out strain suggests that penetration of these
inhibitors into the cytosol is probably poor due to the complex
cell membrane of Gram-negative bacteria.38
Sequence Similarity and Selectivity with Human TMK.
Structure-based multiple sequence alignment of PaTMK with
other species including human has been performed to elucidate
the selectivity of inhibitors over human TMK (21% sequence
identity between P. aeruginosa and human TMKs). As indicated
in Figure 5, the important amino acids directly involved in the
binding of 1, 17, and 46 at PaTMK are significantly different
from those of hTMK:39 Arg74, Arg96, Thr101, Tyr104,
Gln105, and Phe163 of PaTMK are equivalent to Arg76,
Arg97, Gly102, Phe105, Thr106, and Gln157, respectively. We
envisage Arg76 and Arg97 of hTMK, having a weak H-bond
interaction with imidazopyridinone moiety and π-cation
interaction with the central phenyl ring of 46. Importantly,
the hTMK interactions with 1 are missing key H-bond
interactions of imidazopyridinone moiety with Thr101 and
Gln105 of PaTMK. In addition, the Phe105 of hTMK
corresponds to Tyr104 of PaTMK, which eliminates the
phenolic oxygen, which is involved in the potency boosting H-
bond interaction with carboxamide oxygen of 17 and 46.
Furthermore, the Gln157 of hTMK matches to Phe163 of
PaTMK, which is associated with LID closing and hydrophobic
contacts with 46. Collectively, the inhibitors presented here
would be predicted to have reduced activity toward human
TMK.
and 124 μM, and % inhibition of 43, 58, and 55%, respectively,
at 200 μM). Consequently, these inhibitors have over 1000-fold
selectivity for PaTMK over hTMK. That 46, 47, and 57 exhibit
very weak inhibition of hTMK at a high concentration is
consistent with the fact that the hydrophobic pocket formed by
LID closing is highly conserved between PaTMK and hTMK
[the hydrophobic residues of Val14, Leu135, Leu137, Ala140,
and Phe146 of hTMK (PDB ID: 1E2F) are oriented toward the
area where the terminal phenyl ring of 46, 47, or 57 occupies,
and these amino acids are at the equivalent positions of Pro11,
Leu137, Val139, Ile141, and Leu143 of PaTMK in Figure 4].
Therefore, because of the hydrophobic contacts of the terminal
phenyl ring of 46, 47, or 57, these inhibitors are able to inhibit
hTMK but only at a high concentration. In addition, these
results indirectly support the proposed inhibition mechanism
via hydrophobic contacts assisted by LID loop closing.
CONCLUSION
■
Visual inspection of the binding of the thymidine mimetic
TP5A in a homology model of PaTMK derived from E. coli led
to the identification of a novel thymidine mimetic, 1, which had
an IC50 of 58 μM. Inhibitor 1 was cocrystallized with PaTMK
to give the first P. aeruginosa TMK cocrystal structure. The
binding orientation of 1 is identical to what we expected by the
modeling approaches. Affinity optimization of 1 using both
structure-based design and its directed library build-up
provided inhibitors with nanomolar potency (IC50 = ca. 100−
200 nM). The SAR results are fully rationalized with the X-ray
cocrystal structures and modeling structures. In particular, the
water molecule-mediated H-bond network in 17, which was not
recognized in the modeling approach, plays a significant role for
high potency toward PaTMK. In addition, LID loop closing
mediated by hydrophobic contacts between the terminal phenyl
ring of 46 and the hydrophobic pocket of the PaTMK could be
the main reason for high inhibition of 46 toward PaTMK.
There have been many reports of TMK inhibitors using
thymidine analogues targeting M. tuberculosis, S. aureus, and B.
anthracis. Therefore, we anticipate that the lead compounds 1,
17, and 46 that we developed and the knowledge from
structure-based SAR will be useful for the development of
inhibitors targeting the TMKs of these other species because of
high sequence identity within the bacterial TMKs. In particular,
the amino acids directly involved in interaction of 1, 17, or 46
are very similar within the bacterial species but not in human
TMK. Therefore, because of the low similarity of the amino
acid residues in the active sites of PaTMK and hTMK,
particularly the residues associated with inhibitor binding, the
inhibitors described here should enable off-target effects to be
avoided. Therefore, the present set of inhibitors has great
potential as leads for the development of novel antibacterial
agents.
As a verification of this prediction, five compounds 1, 17, 46,
47, and 57 were assayed toward human TMK. As shown in
Table 3, 1 and 17 are inactive toward hTMK even at high
Table 3. Activity of Selected Inhibitors toward hTMK
Unfortunately, all of the TMK inhibitors described here were
inactive against P. aeruginosa bacterial strains. The complex cell
membrane of P. aeruginosa is an effective barrier for preventing
cellular activity of these new TMK inhibitors because growth
inhibition did not occur even in an efflux pump knockout
strain. It has been proposed that quinolones penetrate the outer
membrane of Gram-negative bacteria by translocation through
the OmpF channel (in case of P. aeruginosa, OprD40) due to
their unique zwitterionic nature;41 the inner membrane is
crossed, and the cytosol is accessed due to the substantial
amount of uncharged properties in the zwitterions.42 Therefore,
incorporating zwitterions into the inhibitors is a plausible next
compd
IC50 (μM)
% inhibition at 200 μM
DTBN
1
1.53
>200
>200
>200
123
17
12
43
58
55
17
46
47
57
124
concentration (% inhibition at 200 μM = 17 and 12%,
respectively). In addition, 46, 47, and 57 possess meaningful
inhibition only at very high concentrations (IC50 = >200, 123,
860
dx.doi.org/10.1021/jm201349f | J. Med. Chem. 2012, 55, 852−870

Journal of Medicinal Chemistry
Article
DHFR from the 1 complex as a search model in PHASER.49
Calculation of the Matthew's coefficient indicated that this crystal form
contained four molecules in the asymmetric unit. The correlation
coefficient and R factor from the molecular replacement solutions
indicated that the correct space group was P21. Rigid body and
restrained refinement were performed in REFMAC45 at 3.0 and 1.9 Å,
respectively. Five percent of randomly selected reflections was
designated as test reflections for use in the free-R cross-validation
method46 and used throughout the refinement. dFTM was modeled
into the SigmaA-weighted47 |Fo| − |Fc| electron density maps
contoured at 2σ. Three hundred seventy-seven water molecules and
four magnesium atoms were added. The model was refined to a final
Rcryst/Rfree of 17.5/23.6% in REFMAC (Table 1 in the Supporting
Information).
The structure of Pa TMK in complex with 17 was solved by
molecular replacement at 3 Å resolution using the structure of Pa
DHFR from the 1 complex as a search model in Molrep.44 Calculation
of the Matthew's coefficient indicated that this crystal form contained
two molecules in the asymmetric unit. The correlation coefficient and
R factor from the molecular replacement solutions indicated that the
correct space group was P2. Rigid body and restrained refinement
were performed in BUSTER50 at 3.0 and 1.7 Å, respectively. Five
percent of randomly selected reflections was designated as test
reflections for use in the free-R cross-validation method46 and used
throughout the refinement. Compound 17 was modeled into the
SigmaA-weighted47 |Fo| − |Fc| electron density maps contoured at 2σ.
Two hundred water molecules were added. The model was refined to
a final Rcryst/Rfree of 19.2/21.2% in BUSTER50 (Table 1 in the
Supporting Information).
strategy for penetrating the complex cell membrane of Gram-
negative bacteria and to inhibit P. aeruginosa bacteria cell
growth and division.
EXPERIMENTAL SECTION
■
Cloning and Expression of P. aeruginosa TMK. Pa TMK was
subcloned in the pET15b vector (EMD Biosciences) and expressed in
E. coli BL21 DE3. Cells were grown in 30 L batches using fermentation
at 37 °C in LB broth supplemented with 150 mg/L amplicillin. Cells
were grown to late log phase, pelleted by centrifugation, and stored at
−80 °C.
Purification. Cell pellets were resuspended in lysis buffer (50 mL
of 50 mM NaH2PO4, 2 mM TCEP, 5 mM imidazole, pH 7.5, and 300
mM NaCl per 10 g cells). One Roche EDTA-free protease inhibitor
tablet was added per 50 mL of lysate. The solution was brought up to
0.002% Benzonase and 10 mM MgCl2 and lysed by sonication on ice.
The lysate was clarified by centrifugation at 20200g for 1 h. The
clarified lysate was bound to a HisTrap HP (GE Life Sciences) column
that had been equilibrated in 50 mM NaH2PO4, 2 mM TCEP, 5 mM
imidazole, pH 7.5, and 300 mM NaCl. The bound material was
washed with 5 column volumes of 50 mM NaH2PO4, 2 mM TCEP, 5
mM imidazole, pH 7.5, and 300 mM NaCl followed by 5 column
volumes of 50 mM NaH2PO4, 2 mM TCEP, 30 mM imidazole, pH
7.5, and 300 mM NaCl. The bound protein was eluted with a gradient
of 5−50% 50 mM NaH2PO4, 2 mM TCEP, 500 mM imidazole, pH
7.5, and 300 mM NaCl for 20 column volumes followed by 100% 50
mM NaH2PO4, 2 mM TCEP, 500 mM imidazole, pH 7.5, and 300
mM NaCl for 10 column volumes. Fractions containing Pa TMK were
pooled and dialyzed against 8 L of 25 mM HEPES, pH 7.5, 2 mM
TCEP, and 300 mM NaCl overnight at 4 °C. The dialyzed material
was concentrated for loading onto a Superdex 200 column (GE Life
Sciences) that had been equilibrated with 25 mM HEPES, pH 7.5, 2
mM TCEP, and 300 mM NaCl. Fractions containing Pa TMK were
pooled and concentrated to 10 mg/mL for crystallization.
Crystallization. Pa TMK at 10 mg/mL was mixed with 2 mM
inhibitor (from a 100 mM stock in DMSO) and incubated on ice for
30 min. The precipitated material was removed by centrifugation at
12000g for 5 min. The 1, dFTM, and 17 complexes were crystallized
by hanging drop vapor diffusion. The protein was mixed 1:1 with a
reservoir solution containing 30% PEG 4000, 0.2 M MgCl2, and 0.1 M
Tris, pH 7.5−8.5, and incubated at 22 °C. Crystals were prepared for
data collection by cryoprotection in a mother liquor solution
containing an additional 20% ethylene glycol and flash freezing in
liquid nitrogen.
Coordinates and structure factors of PaTMK complexed with 1, 17,
and dFTM are available from the Protein Data Bank with accession
codes 3UWK, 3UWO, and 3UXM, respectively.
Thymidylate Kinase Kinetic Assay Materials and Methods.51
TMK catalyzes the phosphorylation of thymidine-5′-monophosphate
(dTMP) to form thymidine-5′-diphosphate (dTDP) in both de novo
and salvage pathways of dTTP (thymidine-5′-triphosphate) synthesis
and is the last unique enzyme in the pathway specific for dTMP. A
384-well enzyme assay was performed in which dTDP production is
enzymatically coupled to NADH oxidation. This permits the rate of
TMK turnover to be monitored by the decrease in absorbance at 340
nm. The amounts of each coupling enzyme were optimized to ensure
that TMK turnover is rate limiting. The assay utilized 40 data points
(absorbance readings) over a 20 min time period to calculate the initial
rates produced in the presence of compound relative to that of the
inhibited (enzyme in the presence of EDTA) and uninhibited controls
(enzyme alone).
Data Collection. Diffraction data were collected at 100 K by the
rotation method (360 frames, 0.5° oscillation per frame) at beamline
17-ID (λ = 1.0 Å) at the Advance Photon Source. The data were
processed with HKL2000.43
The assay component consisted of 2 mM adenosine 5′-triphosphate
(ATP), 2 mM phosphoenolpyruvate (PEP), 5 U/mL pyruvate kinase
(PK), 5 U/mL lactate dehydrogenase (LDH), 1.2 U/mL nucleotide
diphosphate kinase (NDP kinase), 0.22 mM (nicotinamide adenine
dinucleotide, reduced form (NADH), 54 μM thymidine-5′-mono-
phosphate (TMP) in 40 mM HEPES, pH 8.0, 80 mM KCl, and 1.6
mM MgCl2. P. aeruginosa TMK enzyme was cloned, expressed, and
purified using standard molecular biology techniques and diluted in
enzyme diluent buffer containing 25 mM Tris, pH 7.8, 250 mM NaCl,
5% glycerol, 1 mM DTT, and 0.0025% Triton X-100. Test compounds
were solvated in 100% DMSO at 30 mM concentration, and 20 1.67-
fold serial dilutions starting at a concentration of 200 or 100 μM (final
concentration) were performed in 96-well polypropylene plates. The
Biomek FX was used to dilute and spot 5 μL = solution containing
compound in duplicate into Corning #3702 384-well clear flat bottom
polystyrene microplates. The 50 μL reaction was initiated by the
addition of 40 nM enzyme, and the oxidation of NADH was
monitored for 20 min at 25 °C at absorbance 340 nm using a
Molecular Devices Spectramax Plus plate reader. The initial rates from
the kinetic time−course absorbance data for duplicates of each
compound concentration were exported into an XLfit4 Excel-based
plug-in spreadsheet, which allowed curve fitting and statistical analysis
to determine IC50 values for each compound tested. Compounds that
Structure Solution and Refinement. The structure of Pa TMK
in complex with 1 was solved by molecular replacement at 3 Å
resolution using the structure of S. pneumo TMK as a search model in
Molrep.44 Calculation of the Matthew's coefficient indicated that this
crystal form contained two molecules in the asymmetric unit. The
correlation coefficient and R factor from the molecular replacement
solutions indicated that the correct space group was P21212. Rigid
body and restrained refinement were performed in REFMAC45 at 3.0
and 1.9 Å, respectively. Five percent of randomly selected reflections
was designated as test reflections for use in the free-R cross-validation
method46 and used throughout the refinement. During the refinement,
residues that differed in identity between the Pa and the S. pneumo
enzymes were mutated to Ala and then built into SigmaA-weighted47
|
Fo| − |Fc| electron density maps contoured at 2σ using the visualization
and model building program Coot.48 Compound 1 was modeled into
the SigmaA-weighted |Fo| − |Fc| electron density maps contoured at 2σ.
One hundred water molecules and one magnesium atom were added.
The model was refined to a final Rcryst/Rfree of 19.5/24.2% in REFMAC
(Table 1 in the Supporting Information).
The structure of Pa TMK in complex with dFTM was solved by
molecular replacement at 3 Å resolution using the structure of Pa
861
dx.doi.org/10.1021/jm201349f | J. Med. Chem. 2012, 55, 852−870

Journal of Medicinal Chemistry
Article
resulted in single-digit micromolar potency were retested to confirm
activity.
moisture or oxygen were conducted under an argon atmosphere using
flame-dried (under vacuum) or oven-dried glassware (overnight). The
removal of solvents was accomplished on a rotary evaporator under
reduced pressure in the water bath below 35 °C followed by using high
vacuum pump. Microwave-assisted reactions were performed using a
Biotage Initiator microwave reactor.
Human Thymidylate Kinase Assay. The Human Thymidylate
Kinase Assay Kit Plus (Catalog no. HTMK500KE at Profoldin) was
used for measurement of the inhibition IC50 values. The kinase assay is
based on detection of ADP generated by the kinase reaction in the
presence of the kinase substrate dTMP. The total volume of each assay
reaction mixture was 50 μL. In a black 96-well plate, 1 μL of inhibitor,
27 μL of H2O, 5 μL of 10 × assay buffer, 2.5 μL of 1 mM ATP, 2 μL of
10 mM dTMP, and 2.5 μL of 1 μM human thymidylate kinase were
mixed. The reaction mixture was incubated at room temperature for 2
min. Then, 5 μL of 10 × MUK Reagent A and 5 μL of 10 × MUK
Reagent B were added. The reaction was incubated at room
temperature for 30 min. Finally, 50 μL of the fluorescence dye was
added, and the fluorescence at 535 nm with excitation at 485 nm was
measured. The final concentrations were 50 mM Tris-HCl, pH 8.0, 3
mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 50 mM NaCl, 0.003% Brij-
35, 50 μM ATP, 400 μM dTMP, 50 nM human thymidylate kinase,
and 0.39−200 μM inhibitor. Negative controls (no enzyme control):
The assay reactions without the kinase were used as negative controls
to observe the fluorescence of the compounds at each concentration.
The fluorescence reading without the kinase reaction without
compound was used as the 100% inhibition value. Assay controls:
The assay reactions with the product of the kinase (5 μM ADP) were
used as assay controls to observe the assay interference of the
compounds at each concentration. The data from the assay controls
were used for correction of the assay values. The percentage inhibition
values were calculated from the corrected assay values and used for
IC50 curve fitting.
Virtual Synthesis/Screening. The in-house cocrystal structure of
PaTMK and 1 was initially applied for docking studies. Protein
Preparation Wizard in maestro9.0 was employed to refine the structure
for Glide docking by deleting unnecessary metals and water molecules
followed by optimizing hydrogen-bonding network. After three water
molecules near 1 were removed, a grid was generated using default
values except constraints such as the aforementioned three H-bond
interactions of 1 with Arg74 and Gln105. The structures of
commercially available aryl boronic acids (ca. 5000) were obtained
from ZINC database. Virtual inhibitors (5000 analogues of 1)
possessing various aryl groups at the 6-position of 5 in Scheme 1
were generated using 'Combigen' in the MOE program package. These
were docked into the active site of PaTMK using Glide 5.0 with XP
mode and three predefined constraints. The docking results were
scored and sorted based on docking scores. The virtual screening
results were visually inspected in maestro9.0, and 20 inhibitors were
selected for synthesis and screening.
Similarly, the cocrystal structure of PaTMK and 17 was refined
using Protein Preparation Wizard in maestro9.0, and a grid was
generated for Glide docking. The structures of primary alkyl and aryl
amines were obtained from Sigma-Aldrich, Acros, and Maybridge
Websites. 'Combigen' in MOE was applied to generate 1188 virtual
inhibitors (analogues of 19 in Scheme 3), which were docked into the
active site of PaTMK using Glide 5.0. The docking results were scored
and visually inspected to select amine reagents for synthesis and
screening.
Loop Searching by Homology Modeling. The cocrystal
structure of PaTMK with 17 was applied to fill in the missing loop
region (Glu140 ∼ Gly150). The complete sequence of P. aeruginosa
TMK was obtained from Uniprot database (ID: Q9HZN8) and
aligned to that of the cocrystal structure using blosum62 algorithm and
the tree-based build-up method. The final model was obtained by
generating 100 intermediates and averaging these intermediates
followed by minimization using Amber99 forcefield with 0.1 rms
gradient. The coordination of 46 was docked into the final model by
superimposing the model and the result of Glide docking.
Proton nuclear magnetic resonance (1H NMR) spectra and carbon
13 (13C) NMR spectra were recorded on a commercial 400 MHz
Bruker NMR spectrometer. Chemical shifts are reported in ppm (δ)
relative to tetramethylsilane as an internal standard. Coupling
constants (J) are reported in hertz (Hz).
Analytical thin-layer chromatography (TLC) was performed on
glass plates precoated with a 0.25 mm thickness of silica gel. The TLC
plates were visualized with UV light. Column chromatography was
performed using a Biotage Isolera flash purification system using
Biotage SNAP HP-SIL cartridge (30 μm silica, 10−100 g size) and
SNAP C18 cartridges. Unless noted otherwise, all compounds isolated
by chromatography were sufficiently pure by 1H NMR analysis for use
in subsequent reactions.
All final compounds were further introduced to high-performance
liquid chromatography (HPLC, Varian 1100 series) on a reverse phase
ZORBAX Eclipse XDB-C18 column (4.6 mm × 150 mm, 5 μm). A
linear gradient elution was performed ranging from 2 to 98% CH3CN
and H2O (containing 0.1% TFA and 1% CH3CN) at 1.5 mL/min. The
purity of all final compounds (typically ≥96%) was assayed at 254 nm
wavelength.
Benzyl (2-Amino-5-bromopyridin-3-yl)carbamate (3). To a
solution of 5-bromopyridine-2,3-diamine (8.0 g, 42 mmol) and
pyridine (12 mL) in dry THF (200 mL) was added benzyl
chloroformate (11 mL) using a syringe pump (rate 2 mL/h) at 0
°C. After the addition of benzyl chloroformate was complete, the
reaction mixture was stirred at room temperature for 12 h. The excess
of ethyl acetate was added to the crude mixture, which was washed
with saturated aqueous NaHCO3 and brine. The organic layer was
dried over magnesium sulfate, filtered, and concentrated in vacuo.
Purification of the crude product by flash chromatography eluting with
a linear gradient ranging from 12 to 100% EtOAc−hexane provided
6.6 g (48%) of 3 as a yellow solid. 1H NMR (400 MHz, DMSO-d6): δ
9.00 (s, 1H), 7.90 (s, 1H), 7.80 (d, J = 2.0 Hz, 1H), 7.45−7.32 (m,
5H), 6.11 (s, 2H), 5.17 (s, 2H). 13C NMR (100 MHz, DMSO-d6): δ
154.03, 151.04, 142.76, 136.32, 128.45, 128.16, 128.11, 120.16, 104.65,
66.29. MS (ESI) 322, 324 m/z [M + H]+.
5-Bromo-N3-methylpyridine-2,3-diamine (4). To a solution of
LiAlH4 (3.2 g) in dry ether was added 3 (6.6 g, 20 mmol) at 0 °C. The
reaction mixture was stirred for 15 min, then was warmed to ambient
temperature, and stirred for 18 h. The mixture was cooled to 0 °C, and
then, 3.2 mL of water, 3.2 mL of 10% NaOH(aq), and 3.2 mL of water
were sequentially added. The precipitated white solid was filtered off,
and the filtrate was concentrated with a rotary evaporator. The crude
product was purified by flash chromatography eluting with a linear
gradient ranging from 12 to 100% EtOAc−hexane yielded 4.1 g (99%)
1
of 4 as a white solid. H NMR (400 MHz, DMSO-d6): δ 7.28 (d, J =
2.4 Hz, 1H), 6.56 (d, J = 2.0 Hz, 1H), 5.64 (s, 2H), 5.22 (d, J = 4.8 Hz,
1H), 2.69 (d, J = 4.8 Hz, 3H). 13C NMR (100 MHz, DMSO-d6): δ
147.35, 133.47, 133.01, 114.06, 107.14, 29.49. MS (ESI) 202, 204 m/z
[M + H]+.
6-Bromo-1-methyl-1H-imidazo[4,5-b]pyridin-2(3H)-one (5). To a
solution of triphosgene (27 g) in dry toluene (200 mL) was added a
solution of 4 (10 g, 50 mmol) in dry THF (20 mL) dropwise at room
temperature. The reaction mixture was stirred at 100 °C for 5 h. The
solution was cooled to ambient, and then, the solvent was removed
under reduced pressure. Ethyl acetate and 10% NaOH(aq) were added
to the product mixture. The aqueous layer was collected and treated
with 1 N HCl(aq) to neutralize it, and a yellow solid was formed. The
product was obtained by filtering, and further purification by flash
chromatography eluting with a linear gradient ranging from 16 to
Synthesis. General Methods. All reaction solvents were purified
before use. Dichloromethane, tetrahydrofuran, dimethylformamide,
and toluene were purified by passing through a solvent column
composed of activated A-1 alumina. All other reagents purchased from
commercial suppliers were used as received. All reactions sensitive to
1
100% EtOAc−hexane yielded 5 (9.2 g, 80%) as a yellow solid. H
NMR (400 MHz, DMSO-d6): δ 11.71 (s, 1H), 7.96 (d, J = 2.0 Hz,
1H), 7.70 (d, J = 2.0 Hz, 1H), 3.27 (s, 3H). 13C NMR (100 MHz,
DMSO-d6): δ 153.79, 142.53, 139.57, 126.42, 116.18, 111.32, 26.50.
862
dx.doi.org/10.1021/jm201349f | J. Med. Chem. 2012, 55, 852−870

Journal of Medicinal Chemistry
Article
MS (ESI) 228, 230 m/z [M + H]+. HRMS (ESI) calcd for
C7H7BrN3O [M + H]+, 227.9772; found, 227.9773.
124.23, 112.20, 26.47. MS (ESI) 227.3 m/z [M + H]+. Purity (≥98%,
tr = 3.74 min).
6-Bromo-1-methyl-3-trityl-1H-imidazo[4,5-b]pyridin-2(3H)-one
(6). To a solution of 5 (9.2 g, 40 mmol) in dry CH2Cl2 (150 mL) were
added trityl chloride (13 g, 46 mmol) and NEt3 (10 mL) at room
temperature. The reaction mixture was stirred overnight (ca. 16 h),
and the solvent was removed under reduced pressure. The product
mixture was diluted with ethyl acetate and washed with saturated
NaHCO3(aq) and brine. The organic layer was dried over magnesium
sulfate, filtered, and concentrated in vacuo. Purification of the crude
product by flash chromatography eluting with a linear gradient ranging
from 6 to 50% EtOAc−hexane provided 17 g (88%) of 6 as a white
1-Methyl-6-(pyrimidin-5-yl)-1H-imidazo[4,5-b]pyridin-2(3H)-one
(11). The general procedure A was followed using 6 (202 mg, 431
μmol) and pyrimidine-5-boronic acid to provide 11 as a yellow solid
(78 mg, 346 μmol, 80%). 1H NMR (400 MHz, DMSO-d6): δ 11.75 (s,
1H), 9.19−9.18 (m, 3H), 8.36 (d, J = 2.0 Hz, 1H), 7.94 (d, J = 2.0 Hz,
1H), 3.35 (s, 3H). 13C NMR (100 MHz, DMSO-d6): δ 157.07, 154.47,
154.02, 144.11, 138.37, 131.49, 125.61, 123.03, 111.99, 26.52. MS
(ESI) 228.4 m/z [M + H]+. Purity (≥98%, tr = 4.48 min).
6-(3-Hydroxyphenyl)-1-methyl-1H-imidazo[4,5-b]pyridin-2(3H)-
one (12). The general procedure A was followed using 6 (192 mg, 408
μmol) and 3-hydroxyphenylboronic acid to provide 12 as a dime
1
solid. H NMR (400 MHz, CDCl3): δ 7.91 (d, J = 2.0 Hz, 1H), 7.63
1
yellow solid (66 mg, 247 μmol, 67%). H NMR (400 MHz, DMSO-
(d, J = 7.6 Hz, 6H), 7.32 (t, J = 7.4 Hz, 6H), 7.23 (t, J = 7.4 Hz, 3H),
7.17 (d, J = 2.0 Hz, 1H), 3.26 (s, 3H). 13C NMR (100 MHz, CDCl3):
δ 153.68, 142.52, 139.80, 129.03, 127.38, 126.62, 125.94, 115.20,
112.72, 74.84, 26.88. MS (ESI) 243 m/z [decomposition of trityl
group, M + H]+. HRMS (ESI) calcd for C26H21BrN3O [M + H]+,
470.0868; found, 470.0876.
d6): δ 11.58 (s, 1H), 9.68 (br, 1H), 8.13 (d, J = 2.0 Hz, 1H), 7.66 (d, J
= 1.6 Hz, 1H), 7.25 (t, J = 7.8 Hz, 1H), 7.09−7.06 (m, 2H), 6.78 (ddd,
J = 8.0, 2.0, 0.8 Hz, 1H), 3.34 (s, 3H). 13C NMR (100 MHz, DMSO-
d6): δ 158.02, 154.19, 143.13, 139.49, 137.98, 129.99, 129.79, 125.42,
117.45, 114.39, 113.63, 112.25, 105.86, 26.43. MS (ESI) 242.2 m/z [M
+ H]+. Purity (96%, tr = 5.48 min).
General Procedure A for Synthesis of Aryl-Substituted
Analogues (1 and 9−26) in Scheme 1. 1-Methyl-6-phenyl-3-
trityl-1H-imidazo[4,5-b]pyridin-2(3H)-one (7). A reaction mixture of
6 (0.11 g, 0.23 mmol), phenyl boronic acid (36 mg, 0.30 mmol),
Pd2(dba)3 (5 mg, 2 mol %), PCy3 (5 mg, 5 mol %), and K3PO4 (1 M,
1 mL) in dioxane (4 mL) was stirred under microwave heating (100
°C) for 1 h. The palladium catalyst was removed by filtration. The
product mixture was diluted with ethyl acetate (30 mL) and washed
with water (10 mL) and brine (10 mL). The organic layer was dried
over magnesium sulfate, filtered, and concentrated in vacuo. The
resulting product was purified by flash chromatography eluting with a
linear gradient ranging from 6 to 50% EtOAc−hexane to afford 7 (0.12
6-(3-Aminophenyl)-1-methyl-1H-imidazo[4,5-b]pyridin-2(3H)-
one (13). The general procedure A was followed using 6 (121 mg, 255
μmol) and 3-aminobenzeneboronic acid to provide 13 as a yellow
1
solid (61 mg, 254 μmol, 100%). H NMR (400 MHz, DMSO-d6): δ
11.71 (s, 1H), 10. 46 (br, 2H), 8.19 (d, J = 2.0 Hz, 1H), 7.75−7.72 (m,
2H), 7.68 (d, J = 1.6 Hz, 1H), 7.59 (t, J = 8.0 Hz, 1H), 7.38 (dd, J =
8.0, 1.2 Hz, 1H), 3.36 (s, 3H). 13C NMR (100 MHz, DMSO-d6): δ
154.08, 143.55, 139.56, 137.98, 133.16, 130.38, 128.24, 126.16, 125.56,
121.96, 121.09, 112.15, 26.45. MS (ESI) 241.2 m/z [M + H]+. Purity
(≥98%, tr = 4.25 min).
3-(1-Methyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-6-yl)-
benzonitrile (14). The general procedure A was followed using 6 (480
mg, 1.02 mmol) and 3-cyanophenylboronic acid to provide 14 as a
1
g) as a yellow solid. H NMR (400 MHz, CDCl3): δ 7.91 (d, J = 2.0
Hz, 1H), 7.46−7.44 (m, 6H), 7.41−7.39 (m, 2H), 7.33−7.30 (m, 2H),
7.23 (t, J = 7.2 Hz, 1H), 7.17−7.06 (m, 10H), 3.25 (s, 3H). 13C NMR
(100 MHz, CDCl3): δ 154.37, 143.50, 142.99, 138.58, 138.41, 130.72,
129.35, 129.13, 127.62, 127.48, 127.12, 126.69, 125.26, 111.29, 74.91,
27.04. MS (ESI) 243 m/z [decomposition of trityl group, M + H]+.
HRMS (ESI) calcd for C32H26N3O [M + H]+, 468.2076; found,
468.2090.
1
white solid (265 mg, 537 μmol, 53%). H NMR (400 MHz, DMSO-
d6): δ 11.68 (s, 1H), 8.31 (d, J = 2.0 Hz, 1H), 8.22 (t, J = 1.6 Hz, 1H),
8.08 (ddd, J = 8.0, 2.0, 1.2 Hz, 1H), 7.88 (d, J = 2.0 Hz, 1H), 7.82 (dt,
J = 8.0, 1.2 Hz, 1H), 7.67 (t, J = 8.0 Hz, 1H), 3.36 (s, 3H). 13C NMR
(100 MHz, DMSO-d6): δ 154.10, 143.75, 139.23, 138.41, 131.24,
130.79, 130.15, 130.07, 127.28, 125.51, 118.79, 112.27, 112.14, 26.47.
MS (ESI) 251.1 m/z [M + H]+. Purity (≥98%, tr = 6.32 min).
6-(3-(Aminomethyl)phenyl)-1-methyl-1H-imidazo[4,5-b]pyridin-
2(3H)-one Hydrochloride (15). The general procedure A was followed
using 6 (790 mg, 1.66 mmol) and 3-N-Boc-aminomethylphenylbor-
onic acid (0.88 g, 3.09 mmol) to provide 15 as a white solid (0.23 mg,
0.791 mmol, 48%). 1H NMR (400 MHz, D2O): δ 7.85 (d, J = 2.0 Hz,
1H), 7.39−7.26 (m, 4H), 7.15 (d, J = 1.6 Hz, 1H), 4.11 (s, 2H), 3.06
(s, 3H), 13C NMR (100 MHz, D2O): δ 154.92, 141.02, 137.70, 136.97,
133.13, 129.65, 129.13, 127.74, 126.43, 125.99, 124.79, 112.94, 42.92,
26.09, MS (ESI) 255 m/z [M + H]+, Purity (≥98%, tr = 4.50 min).
6-(3-(Hydroxymethyl)phenyl)-1-methyl-1H-imidazo[4,5-b]-
pyridin-2(3H)-one (16). The general procedure A was followed using
6 (127 mg, 270 μmol) and 3-(hydroxymethyl)phenylboronic acid to
1-Methyl-6-phenyl-1H-imidazo[4,5-b]pyridin-2(3H)-one (1). To a
solution of 7 (0.12 g) in CH2Cl2 (5 mL) was added trifluoroacetic acid
(1 mL) at room temperature. The reaction mixture was stirred for 30
min. The solvent and trifluoroacetic acid were removed in vacuo. The
product 8 was purified by flash chromatography eluting with a linear
gradient ranging from 12 to 100% EtOAc−hexane as a white solid (49
1
mg, 93% over 2 steps). H NMR (400 MHz, DMSO-d6): δ 11.58 (s,
1H), 8.21 (d, J = 2.0 Hz, 1H), 7.74 (d, J = 2.0 Hz, 1H), 7.72−7.69 (m,
2H), 7.48 (t, J = 7.6 Hz, 2H), 7.37 (tt, J = 7.4, 1.2 Hz, 1H), 3.36 (s,
3H). 13C NMR (100 MHz, DMSO-d6): δ 154.09, 143.09, 138.05,
129.49, 128.95, 127.27, 126.65, 125.40, 112.21, 26.39. MS (ESI) 226
m/z [M + H]+. HRMS (ESI) calcd for C13H12N3O [M + H]+,
226.0980; found, 226.0981. Purity (≥98%, tr = 6.58 min).
1
provide 16 as a white solid (44 mg, 170 μmol, 63%). H NMR (400
6-(3-Fluorophenyl)-1-methyl-1H-imidazo[4,5-b]pyridin-2(3H)-
one (9). The general procedure A was followed using 6 (185 mg, 393
μmol) and 3-fluorophenylboronic acid to give 9 as a white solid (76
MHz, CDCl3): δ 11.25 (br, 1H), 8.28 (d, J = 1.2 Hz, 1H), 7.57−7.33
(m, 5H), 5.41 (s, 2H), 3.46 (s, 3H). 13C NMR (100 MHz, CDCl3): δ
154.84, 143.42, 139.06, 139.06, 134.38, 130.63, 129.88, 128.26, 128.05,
127.62, 126.01, 112.71, 69.57, 26.98. MS (ESI) 256.4 m/z [M + H]+.
Purity (95%, tr = 5.35 min).
3-(1-Methyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-6-yl)-
benzamide (17). The general procedure A was followed using 6 (208
mg, 443 μmol) and 3-carbamoylphenylboronic acid to provide 17 as a
white solid (51 mg, 190 μmol, 43%). 1H NMR (400 MHz, DMSO-d6):
δ 11.63 (s, 1H), 8.30 (d, J = 2.0 Hz, 1H), 8.18 (t, J = 1.6 Hz, 1H), 8.09
(br, 1H), 7.87 (t, J = 1.6 Hz, 1H), 7.85 (t, J = 2.0 Hz, 1H), 7.81 (d, J =
2.0 Hz, 1H), 7.55 (t, J = 2.0 Hz, 1H), 7.45 (br, 1H), 3.37 (s, 3H). 13C
NMR (100 MHz, DMSO-d6): δ 167.72, 154.09, 143.30, 138.26,
138.01, 135.01, 129.31, 128.95, 128.90, 126.42, 125.51, 125.45, 112.21,
26.42. MS (ESI) 269.1 m/z [M + H]+. HRMS (ESI) calcd for
C14H13N4O2 [M + H]+, 269.1039; found, 269.1041. Purity (≥98%, tr =
5.00 min).
1
mg, 314 μmol, 80%). H NMR (400 MHz, DMSO-d6): δ 11.65 (s,
1H), 8.26 (d, J = 2.0 Hz, 1H), 7.79 (d, J = 2.0 Hz, 1H), 7.59−7.55 (m,
2H), 7.52−7.46 (m, 1H), 7.18 (tdd, J = 8.4, 2.4, 1.2 Hz, 1H), 3.35 (s,
3H). 13C NMR (100 MHz, DMSO-d6): δ 163.93, 161.51, 154.10,
143.52, 140.57, 140.49, 138.26, 130.90, 125.43, 122.60, 122.57, 114.03,
113.82, 113.40, 113.18, 112.23, 26.44. MS (ESI) 244.2 m/z [M + H]+.
Purity (96%, tr = 6.85 min).
1-Methyl-6-(pyridin-3-yl)-1H-imidazo[4,5-b]pyridin-2(3H)-one
(10). The general procedure was followed using 6 (194 mg, 412 μmol)
and pyridine-3-boronic acid to provide 13 as a white solid (97 mg, 431
μmol, 100%). 1H NMR (400 MHz, DMSO-d6): δ 11.69 (s, 1H), 8.98
(d, J = 1.6 Hz, 1H), 8.61 (d, J = 3.6 Hz, 1H), 8.29 (d, J = 2.0 Hz, 1H),
8.21 (dt, J = 4.0, 2.4 Hz, 1H), 7.86 (d, J = 2.0 Hz, 1H), 7.57 (dd, J =
8.0, 4.8 Hz, 1H), 3.36 (s, 3H). 13C NMR (100 MHz, DMSO-d6): δ
154.06, 147.41, 146.64, 143.73, 138.34, 134.98, 134.03, 125.97, 125.54,
863
dx.doi.org/10.1021/jm201349f | J. Med. Chem. 2012, 55, 852−870

Journal of Medicinal Chemistry
Article
3-(1-Methyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-6-yl)-
benzenesulfonamide (18). The general procedure A was followed
using 6 (233 mg, 490 μmol) and benzenesulfonamide-3-boronic acid
pinacol ester to provide 18 as a white solid (18 mg, 59 μmol, 12%). 1H
NMR (400 MHz, DMSO-d6): δ 11.69 (s, 1H), 8.26 (d, J = 2.0 Hz,
1H), 8.15 (t, J = 1.6 Hz, 1H), 7.94 (ddd, J = 7.6, 1.6, 0.8 Hz, 1H), 7.82
(ddd, J = 8.0, 1.6, 1.2 Hz, 1H), 7.77 (d, J = 2.0 Hz, 1H), 7.67 (t, J = 7.6
Hz, 1H), 7.41 (br, 2H), 3.37 (s, 3H). 13C NMR (100 MHz, DMSO-
d6): δ 154.11, 144.91, 143.65, 138.84, 138.25, 129.92, 129.69, 128.18,
125.56, 124.24, 123.59, 112.08, 26.47. MS (ESI) 305.1 m/z [M + H]+.
Purity (≥98%, tr = 5.07 min).
N-(3-(1-Methyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-6-
yl)phenyl)acetamide (21). The general procedure A was followed
using 6 (487 mg, 1.04 mmol) and 3-acetamidophenylboronic acid to
provide 21 as a yellow solid (105 mg, 373 μmol, 36%). 1H NMR (400
MHz, DMSO-d6): δ 11.63 (s, 1H), 10.04 (s, 1H), 8.13 (d, J = 2.0 Hz,
1H), 7.86 (s, 1H), 7.65 (d, J = 1.6 Hz, 1H), 7.59 (d, J = 7.6 Hz, 1H),
7.41−7.33 (m, 2H), 3.35 (s, 3H), 2.06 (s, 3H). 13C NMR (100 MHz,
DMSO-d6): δ 168.43, 154.10, 143.16, 139.90, 138.58, 137.89, 129.64,
129.35, 125.44, 121.56, 118.00, 117.29, 112.16, 26.39, 24.04. MS (ESI)
283.2 m/z [M + H]+. Purity (98%, tr = 5.44 min).
3-(1-Methyl-2-oxo-3-trityl-2,3-dihydro-1H-imidazo[4,5-b]pyridin-
6-yl)benzoic Acid (27). A reaction mixture of 6 (6.22 g, 13.1 mmol),
phenyl boronic acid (2.81 g, 16.9 mmol), Pd2(dba)3 (99 mg, 1 mol %),
PCy3 (108 mg, 2 mol %), and K3PO4 (2 M, 12 mL) in dioxane (28
mL) was stirred under microwave heating (100 °C) for 1 h. The
palladium catalyst was removed by filtration. The filtrate was diluted
with ethyl acetate (100 mL) and treated with 1 N HCl(aq) to acidify.
The aqueous layer was removed, and then, the organic layer was
washed with water (20 mL) and brine (20 mL). The organic layer was
dried over magnesium sulfate, filtered, and concentrated in vacuo.
Purification of the crude product by flash chromatography eluting with
a linear gradient ranging from 25 to 100% EtOAc−hexane provided
6.76 g (99%) of 27 as a yellow solid. 1H NMR (400 MHz, DMSO-d6):
δ 13.07 (s, 1H), 8.18 (t, J = 1.2 Hz, 1H), 8.06 (d, J = 2.0 Hz, 1H),
7.92−7.90 (m, 2H), 7.80 (d, J = 2.0 Hz, 1H), 7.56 (t, J = 8.0 Hz, 1H),
7.51 (d, J = 7.6 Hz, 6H), 7.23 (t, J = 7.6 Hz, 6H), 7.15 (d, J = 7.2 Hz,
3H), 3.34 (s, 3H). 13C NMR (100 MHz, DMSO-d6): δ 167.17, 153.32,
142.96, 137.95, 136.81, 131.59, 130.98, 129.27, 128.82, 128.53, 128.21,
127.34, 127.27, 126.23, 125.40, 112.06, 73.78, 26.84. MS (ESI) 510.4
m/z [M − H]−. HRMS (ESI) calcd for C33H26N3O3 [M + H]+,
512.1974; found, 512.1978.
6-(3-Acetylphenyl)-1-methyl-1H-imidazo[4,5-b]pyridin-2(3H)-one
(22). The general procedure A was followed using 6 (82 mg, 161
μmol) and 3-acetyphenylboronic acid to provide 22 as a white solid
(29 mg, 108 μmol, 67%). 1H NMR (400 MHz, DMSO-d6): δ 11.64 (s,
1H), 8.29 (d, J = 2.0 Hz, 1H), 8.22 (t, J = 1.6 Hz, 1H), 7.98−7.93 (m,
2H), 7.83 (d, J = 2.0 Hz, 1H), 7.63 (t, J = 8.0 Hz, 1H), 3.37 (s, 3H),
2.70 (s, 3H). 13C NMR (100 MHz, DMSO-d6): δ 198.00, 154.09,
143.41, 138.54, 138.32, 137.56, 131.29, 129.38, 128.66, 126.91, 126.31,
125.46, 112.32, 26.95, 26.47. MS (ESI) 288.2 m/z [M + H]+. Purity
(≥98%, tr = 6.19 min).
6-(4-Hydroxyphenyl)-1-methyl-1H-imidazo[4,5-b]pyridin-2(3H)-
one (23). The general procedure A was followed using 6 (205 mg, 430
μmol) and 4-hydroxyphenylboronic acid to provide 23 as a dark
yellow solid (12 mg, 52 μmol, 12%). 1H NMR (400 MHz, DMSO-d6):
δ 11.49 (s, 1H), 9.53 (s, 1H), 8.11 (d, J = 2.0 Hz, 1H), 7.63 (d, J = 2.0
Hz, 1H), 7.50 (dt, J = 8.8, 2.0 Hz, 2H), 6.85 (dt, J = 8.4, 2.0 Hz, 2H),
3.36 (s, 3H). 13C NMR (100 MHz, DMSO-d6): δ 156.98, 154.08,
142.34, 137.36, 129.70, 128.77, 127.75, 125.31, 115.75, 111.73, 26.33.
MS (ESI) 242.2 m/z [M + H]+. Purity (≥98%, tr = 5.17 min).
1-Methyl-6-(pyridin-4-yl)-1H-imidazo[4,5-b]pyridin-2(3H)-one
(24). The general procedure A was followed using 6 (149 mg, 317
μmol) and pyridine-4-boronic acid to provide 24 as a light yellow solid
(56 mg, 246 μmol, 78%). 1H NMR (400 MHz, DMSO-d6): δ 11.81 (s,
1H), 8.71 (s, 2H), 8.47 (d, J = 2.0 Hz, 1H), 7.97 (d, J = 2.0 Hz, 1H),
7.95 (d, J = 6.0 Hz, 2H), 3.37 (s, 3H). 13C NMR (100 MHz, DMSO-
d6): δ 154.06, 148.23, 147.26, 144.84, 139.10, 125.65, 125.58, 121.39,
112.00, 26.53. MS (ESI) 227.2 m/z [M + H]+. Purity (≥98%, tr = 3.80
min).
Pentafluorophenyl 3-(1-Methyl-2-oxo-3-trityl-2,3-dihydro-1H-
imidazo[4,5-b]pyridin-6-yl)benzoate (28). To a solution of 27
(0.502 g, 0.96 mmol) and diisopropylethyl amine (0.35 mL) in dry
CH2Cl2 (10 mL) was added pentafluorophenyl trifluoroacetate (0.25
mL, 1.45 mmol) dropwise at 0 °C. The mixture was stirred for 10 min,
then was warmed to the ambient temperature, and stirred for 1 h. The
solvent was removed under reduced pressure, and then, purification of
the crude product by flash chromatography eluting with a linear
gradient ranging from 6 to 50% EtOAc−hexane yielded 0.528 g (81%)
of 28 as viscous yellow oil. A solution of 28 (0.1 M) in dry CH2Cl2
was prepared for the following amide formation reaction and stored at
room temperature. 1H NMR (400 MHz, CDCl3): δ 8.39 (t, J = 1.6 Hz,
1H), 8.22 (dt, J = 7.6, 1.2 Hz, 1H), 8.11 (d, J = 2.0 Hz, 1H), 7.87 (ddd,
J = 8.0, 1.6, 0.8 Hz, 1H), 7.66−7.60 (m, 7H), 7.34 (d, J = 2.0 Hz, 1H),
7.31−7.28 (m, 6H), 7.25−7.21 (m, 3H), 3.42 (s, 3H). 13C NMR (100
MHz, CDCl3): δ 162.57, 154.30, 144.09, 142.85, 139.63, 138.39,
133.18, 129.79, 129.67, 129.30, 129.21, 129.06, 127.85, 127.50, 126.74,
125.45, 111.10, 75.03, 27.12. MS (ESI) 243 m/z [decomposition of
trityl group, M + H]+. HRMS (ESI) calcd for C39H25F5N3O3 [M +
H]+, 678.1816; found, 678.1814.
General Procedure B for the Synthesis of the Substituted
Benzamide Analogues (19, 20, and 30−53) in Scheme 3. To a
solution of 28 (0.1 M) and diisopropylethyl amine (ca. 3 equiv) was
added the appropriate primary amine (1.2 equiv) at 0 °C. The reaction
mixture was warmed to the ambient temperature and stirred for 1 h.
The solvent was removed by reduced pressure. The crude product was
diluted with ethyl acetate (30 mL) and washed with saturated aqueous
NaHCO3 (10 mL) and brine (10 mL). The organic layer was dried
over magnesium sulfate, filtered, and concentrated in vacuo.
Purification of the crude product by flash chromatography eluting
with a linear gradient ranging from 25 to 100% EtOAc−hexane
provided trityl protected benzamide analogues 29. To a solution of 29
in CH2Cl2 (5 mL) was added trifluoroacetic acid (1 mL) at room
temperature. The reaction mixture was stirred for 30 min. After the
solvent and trifluoroacetic acid were removed in vacuo, the desired
product (19, 20, and 30−53) was purified by flash chromatography
eluting with a linear gradient ranging from 0 to 20% MeOH/EtOAc.
N-Methyl-3-(1-methyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-b]-
pyridin-6-yl)benzamide (19). The general procedure B was followed
using 28 (216 mg, 318 μmol) and methylamine to provide 19 as a
white solid (86 mg, 305 μmol, 96%). 1H NMR (400 MHz, DMSO-d6):
δ 11.64 (s, 1H), 8.59 (d, J = 4.4 Hz, 1H), 8.29 (d, J = 2.0 Hz, 1H), 8.14
(t, J = 1.6 Hz, 1H), 7.86−7.81 (m, 3H), 7.55 (t, J = 8.0 Hz, 1H), 3.37
(s, 3H), 2.82 (d, J = 4.8 Hz, 3H). 13C NMR (100 MHz, DMSO-d6): δ
166.49, 154.11, 143.33, 138.27, 138.07, 135.29, 129.14, 129.01, 128.94,
126.10, 125.46, 125.12, 112.23, 26.43, 26.23. MS (ESI) 283.0 m/z [M
+ H]+. HRMS (ESI) calcd for C15H15N4O2 [M + H]+, 283.1195;
found, 283.1194. Purity (≥98%, tr = 5.28 min).
6-(4-(Aminomethyl)phenyl)-1-methyl-1H-imidazo[4,5-b]pyridin-
2(3H)-one Hydrochloride (25). The general procedure A was followed
using 6 (613 mg, 1.29 mmol) and 4-(N-Boc-aminomethyl)-
phenylboronic acid to provide 25 as a white solid (71 mg, 279
μmol, 22%), which was purified by flash chromatography using reverse
phase C13 column eluting with a linear gradient ranging from 0 to
1
100% acetonitrile/water. H NMR (400 MHz, D2O): δ 7.98 (s, 1H),
7.51 (s, 1H), 7.41−7.37 (m, 4H), 4.13 (s, 2H), 3.18 (s, 3H). 13C NMR
(100 MHz, D2O): δ 154.89, 140.05, 134.48, 132.42, 129.83, 129.43,
126.76, 115.44, 42.61, 26.54. MS (ESI) 255 m/z [M + H]+. Purity
(≥98%, tr = 4.32 min).
4-(1-Methyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-6-yl)-
benzamide (26). The general procedure A was followed using 6 (200
mg, 421 μmol) and 4-carbamoylphenylboronic acid to provide 26 as a
white solid (54 mg, 200 μmol, 48%). 1H NMR (400 MHz, DMSO-d6):
δ 11.65 (s, 1H), 8.30 (d, J = 2.0 Hz, 1H), 8.04 (br, 1H), 7.98 (d, J =
8.4 Hz, 2H), 7.83 (d, J = 2.0 Hz, 1H), 7.81 (d, J = 8.8 Hz, 2H), 7.38
(br, 1H), 3.64 (s, 3H). 13C NMR (100 MHz, DMSO-d6): δ 167.47,
154.10, 143.51, 140.68, 138.36, 132.85, 128.48, 128.17, 126.25, 125.48,
112.20, 26.44. MS (ESI) 269.1 m/z [M + H]+. Purity (≥98%, tr = 4.80
min).
864
dx.doi.org/10.1021/jm201349f | J. Med. Chem. 2012, 55, 852−870

Journal of Medicinal Chemistry
Article
N,N-Dimethyl-3-(1-methyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-b]-
pyridin-6-yl)benzamide (20). The general procedure B was followed
using 28 (123 mg, 182 μmol) and dimethylamine to provide 20 as a
3-(3-(1-Methyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-6-yl)-
benzamido)propanoic Acid (35). The general procedure B was
followed using a 0.1 M solution of 28 (3 mL, 300 μmol) and β-alanine
benzylester hydrochloride, and then, benzyl ester deprotection was
performed with Pd/C and H2 in MeOH (10 mL) to provide 35 as a
white solid (33 mg, 97 μmol, 32%). 1H NMR (400 MHz, DMSO-d6):
δ 12.52 (s, 1H), 11.64 (s, 1H), 8.67 (t, J = 5.6 Hz, 1H), 8.30 (d, J = 1.6
Hz, 1H), 8.14 (s, 1H), 7.87−7.80 (m, 3H), 7.56 (t, J = 8.0 Hz, 1H),
3.50 (q, J = 6.8 Hz, 2H), 3.37 (s, 3H), 2.55 (t, J = 6.8 Hz, 2H). 13C
NMR (100 MHz, DMSO-d6): δ 172.92, 166.11, 154.10, 143.34,
138.32, 138.09, 135.12, 129.33, 129.02, 128.92, 126.20, 125.46, 125.21,
112.23, 35.65, 33.79, 26.46. MS (ESI) 339 m/z [M − H]−. Purity
(72%, tr = 5.75 min).
1
white solid (26 mg, 89 μmol, 50%). H NMR (400 MHz, CDCl3): δ
11.05 (s, 1H), 8.27 (d, J = 2.0 Hz, 1H), 7.63 (t, J = 1.6 Hz, 1H), 7.59
(dt, J = 8.0, 1.6 Hz, 1H), 7.46 (t, J = 7.6 Hz, 1H), 7.38 (dt, J = 7.6, 1.6
Hz, 1H), 7.35 (d, J = 2.0 Hz, 1H), 3.43 (s, 3H), 3.13 (s, 3H), 3.01 (s,
3H). 13C NMR (100 MHz, CDCl3): δ 171.48, 154.77, 143.34, 139.08,
138.89, 137.40, 130.59, 129.21, 128.33, 126.24, 126.08, 125.95, 112.71,
39.85, 35.62, 26.96. MS (ESI) 297.3 m/z [M + H]+. Purity (≥98%, tr =
5.58 min).
N-(2-Amino-2-oxoethyl)-3-(1-methyl-2-oxo-2,3-dihydro-1H-
imidazo[4,5-b]pyridin-6-yl)benzamide (30). The general procedure B
was followed using a 0.1 M solution of 28 (5 mL, 500 μmol) and
glycine benzyl ester and then followed by heating with 2.0 M NH3 in
MeOH (5 mL) at 100 °C for 5 h to provide 30 as a white solid (87
N-(2-Hydroxyethyl)-3-(1-methyl-2-oxo-2,3-dihydro-1H-imidazo-
[4,5-b]pyridin-6-yl)benzamide (36). The general procedure B was
followed using 28 (114 mg, 261 μmol) and ethanolamine to provide
1
1
mg, 268 μmol, 54%). H NMR (400 MHz, DMSO-d6): δ 11.64 (s,
36 as a white solid (22 mg, 69 μmol, 26%). H NMR (400 MHz,
1H), 8.81 (t, J = 5.6 Hz, 1H), 8.32 (d, J = 2.0 Hz, 1H), 8.21 (t, J = 1.6
Hz, 1H), 7.87 (t, J = 8.0 Hz, 1H), 7.81 (d, J = 2.0 Hz, 1H), 7.58 (t, J =
7.6 Hz, 1H), 7.40 (s, 1H), 7.06 (s, 1H), 3.86 (d, J = 6.0 Hz, 2H), 3.37
(s, 3H). 13C NMR (100 MHz, DMSO-d6): δ 170.99, 166.29, 154.09,
143.34, 138.31, 138.00, 134.85, 129.37, 128.99, 128.89, 126.38, 125.46,
125.41, 112.17, 42.44, 26.44. MS (ESI) 326.3 m/z [M + H]+. Purity
(66%, tr = 4.78 min).
DMSO-d6): δ 11.63 (s, 1H), 8.56 (t, J = 5.6 Hz, 1H), 8.30 (d, J = 2.0
Hz, 1H), 8.16 (t, J = 1.6 Hz, 1H), 7.86−7.83 (m, 2H), 7.81 (d, J = 2.0
Hz, 1H), 7.56 (t, J = 7.6 Hz, 1H), 4.75 (br, 1H), 3.54 (t, J = 5.6 Hz,
2H), 3.39−3.35 (m, 5H). 13C NMR (100 MHz, DMSO-d6): δ 166.18,
154.09, 143.32, 138.32, 138.02, 135.30, 129.21, 128.96, 128.94, 126.24,
125.44, 125.23, 112.21, 59.76, 42.23, 26.45. MS (ESI) 313 m/z [M +
H]+. Purity (≥98%, tr = 4.95 min).
2-(3-(1-Methyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-6-yl)-
benzamido)acetic Acid (31). The general procedure B was followed
using a 0.1 M solution of 28 (3 mL, 300 μmol) and glycine benzyl
ester and followed by debenzylation using H2 and Pd/C in MeOH (10
mL) to provide 31 as a pale yellow solid (98 mg, 300 μmol, 100%). 1H
NMR (400 MHz, DMSO-d6): δ 8.37 (t, J = 5.6 Hz, 1H), 8.28 (d, J =
2.0 Hz, 1H), 8.14 (t, J = 1.6 Hz, 1H), 7.84−7.80 (m, 3H), 3.70 (d, J =
5.2 Hz, 2H), 3.36 (s, 3H). 13C NMR (100 MHz, DMSO-d6): δ 171.63,
165.41, 154.13, 143.33, 138.26, 138.07, 135.53, 129.10, 129.00, 128.97,
126.03, 125.45, 125.15, 112.25, 43.57, 26.53. MS (ESI) 327.2 m/z [M
+ H]+. Purity (64%, tr = 5.52 min).
N-(2-Aminoethyl)-3-(1-methyl-2-oxo-2,3-dihydro-1H-imidazo-
[4,5-b]pyridin-6-yl)benzamide Hydrochloride (37). The general
procedure B was followed using a 0.1 M solution of 37 (3 mL, 300
μmol) and N-Boc-ethylenediamine. After 1 N HCl(aq) was added,
purification with reverse phase flash chromatography eluting with a
linear gradient ranging from 5 to 100% acetonitrile/water provided 37
as a white solid (30 mg, 96 μmol, 28%). 1H NMR (400 MHz, DMSO-
d6): δ 11.65 (s, 1H), 9.00 (t, J = 5.6 Hz, 1H), 8.33 (d, J = 2.0 Hz, 1H),
8.31 (s, 1H), 8.13 (br, 2H), 7.96 (d, J = 2.0 Hz, 1H), 7.91−7.88 (m,
2H), 7.58 (t, J = 8.0 Hz, 1H), 3.57 (q, J = 6.0 Hz, 2H), 3.38 (s, 3H),
3.03 (q, J = 6.0 Hz, 2H). 13C NMR (100 MHz, DMSO-d6): δ 166.59,
154.09, 143.29, 138.13, 137.96, 134.74, 129.35, 129.01, 128.79, 126.49,
125.52, 125.43, 112.44, 38.62, 37.17, 26.55. MS (ESI) 312 m/z [M +
H]+. Purity (≥98%, tr = 4.63 min).
2-(3-(1-Methyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-6-yl)-
benzamido)propanoic Acid (38). The general procedure B was
followed using 28 (213 mg, 314 μmol) and D/L-alanine benzylester
and followed by debenzylation under H2 and Pd/C in MeOH (10 mL)
to provide 38 as a white solid (51 mg, 151 μmol, 48%). 1H NMR (400
MHz, DMSO-d6): δ 11.65 (s, 1H), 8.80 (d, J = 7.2 Hz, 1H), 8.58 (br,
1H), 8.32 (d, J = 2.0 Hz, 1H), 8.20 (s, 1H), 7.89−7.86 (m, 2H), 7.81
(d, J = 2.0 Hz, 1H), 7.58 (d, J = 7.6 Hz, 1H), 4.47 (p, J = 7.2 Hz, 1H),
3.37 (s, 3H), 1.43 (d, J = 7.6 Hz, 3H). 13C NMR (100 MHz, DMSO-
d6): δ 174.20, 165.96, 154.12, 143.37, 138.36, 138.11, 134.63, 129.57,
129.02, 128.92, 126.50, 125.47, 125.42, 112.24, 48.23, 26.47, 12.33.
MS (ESI) 341 m/z [M + H]+. Purity (34%/66%, tr = 5.32 and 5.90
min).
2-(3-(1-Methyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-6-yl)-
benzamido)ethanesulfonic Acid (32). The general procedure B was
followed using a 0.1 M solution of 28 (3 mL, 300 μmol) and taurine to
1
provide 32 as a pale yellow solid (83 mg, 221 μmol, 74%). H NMR
(400 MHz, DMSO-d6): δ 11.68 (s, 1H), 8.63 (t, J = 5.6 Hz, 1H), 8.28
(d, J = 1.6 Hz, 1H), 8.10 (s, 1H), 7.85 (d, J = 7.6 Hz, 1H), 7.81 (d, J =
2.0 Hz, 1H), 7.78 (d, J = 8.0 Hz, 1H), 7.56 (d, J = 7.6 Hz, 1H), 3.57
(q, J = 7.6 Hz, 2H), 3.37 (s, 3H), 2.74 (t, J = 7.6 Hz, 2H). 13C NMR
(100 MHz, DMSO-d6): δ 165.62, 154.08, 143.22, 138.10, 137.94,
135.36, 129.29, 129.09, 128.95, 125.91, 125.59, 125.13, 112.40, 50.28,
36.22, 26.48. MS (ESI) 332.3 m/z [M + H]+. Purity (96%, tr = 4.42
min).
N-(Cyanomethyl)-3-(1-methyl-2-oxo-2,3-dihydro-1H-imidazo-
[4,5-b]pyridin-6-yl)benzamide (33). The general procedure B was
followed using 28 (229 mg, 526 μmol) and aminoacetonitrile to
1
provide 33 as a white solid (29 mg, 95 μmol, 18%). H NMR (400
MHz, DMSO-d6): δ 11.65 (s, 1H), 9.31 (t, J = 5.6 Hz, 1H), 8.30 (d, J
= 1.6 Hz, 1H), 8.18 (t, J = 1.6 Hz, 1H), 7.92 (d, J = 8.0 Hz, 1H), 7.86
(d, J = 8.0 Hz, 1H), 7.82 (d, J = 2.0 Hz, 1H), 7.61 (t, J = 8.0 Hz, 1H),
4.37 (d, J = 5.6 Hz, 2H), 3.37 (s, 3H). 13C NMR (100 MHz, DMSO-
d6): δ 166.52, 154.09, 143.42, 138.35, 138.32, 133.57, 130.09, 129.29,
128.68, 126.34, 125.47, 125.40, 117.64, 112.21, 27.75, 26.45. MS (ESI)
308.2 m/z [M + H]+. Purity (93%, tr = 5.46 min).
N-(2-(1H-Imidazol-5-yl)ethyl)-3-(1-methyl-2-oxo-2,3-dihydro-1H-
imidazo[4,5-b]pyridin-6-yl)benzamide (34). The general procedure B
was followed using a 0.1 M solution of 28 (3 mL, 300 μmol) and
histamine to provide 34 as a white solid (113 mg, 300 μmol, 100%).
1H NMR (400 MHz, DMSO-d6): δ 14.39 (br, 1H), 11.65 (s, 1H), 8.99
(d, J = 1.2 Hz, 1H), 8.75 (t, J = 5.6 Hz, 1H), 8.28 (d, J = 2.0 Hz, 1H),
8.10 (s, 1H), 7.86 (d, J = 8.0 Hz, 1H), 7.80−7.78 (m, 2H), 7.56 (t, J =
8.0 Hz, 1H), 7.48 (s, 1H), 3.60 (q, J = 6.8 Hz, 2H), 3.37 (s, 3H), 2.95
(t, J = 6.8 Hz, 2H). 13C NMR (100 MHz, DMSO-d6): δ 166.29,
154.11, 143.36, 138.29, 138.13, 135.07, 133.84, 131.11, 129.42, 129.03,
128.90, 126.19, 125.46, 125.21, 116.24, 112.19, 38.18, 26.45, 24.50.
MS (ESI) 363 m/z [M + H]+. Purity (≥98%, tr = 4.84 min).
3-(1-Methyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-6-yl)-N-
(3-morpholinopropyl)benzamide (39). The general procedure B was
followed using a 0.1 M solution of 28 (1 mL, 100 μmol) and N-(3-
aminopropyl)morpholine to provide 39 as a pale yellow solid (34 mg,
1
86 μmol, 86%). H NMR (400 MHz, DMSO-d6): δ 11.65 (s, 1H),
8.75 (br, 1H), 8.30 (d, J = 2.0 Hz, 1H), 8.15 (s, 1H), 7.89−7.83 (m,
2H), 7.81 (d, J = 2.0 Hz, 1H), 7.58 (t, J = 7.6 Hz, 1H), 3.76−3.56 (m,
4H), 3.41−3.33 (m, 5H), 3.15−3.10 (m, 4H), 1.92−1.91 (br, 2H),
1.25 (q, J = 6.4 Hz, 2H). 13C NMR (100 MHz, DMSO-d6): δ 166.33,
154.10, 143.36, 138.28, 138.12, 135.05, 129.41, 129.05, 128.87, 126.23,
125.46, 125.20, 112.20, 74.12, 63.78, 53.53, 26.46, 18.04, 16.71. MS
(ESI) 396 m/z [M + H]+. Purity (≥98%, tr = 4.93 min).
N-Benzyl-3-(1-methyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-b]-
pyridin-6-yl)benzamide (40). The general procedure B was followed
using 28 (88 mg, 203 μmol) and benzylamine to provide 40 as a white
1
solid (106 mg, 295 μmol, 99%). H NMR (400 MHz, DMSO-d6): δ
11.62 (s, 1H), 9.15 (t, J = 6.0 Hz, 1H), 8.31 (d, J = 2.0 Hz, 1H), 8.21
(t, J = 1.2 Hz, 1H), 7.90−7.86 (m, 2H), 7.81 (d, J = 2.0 Hz, 1H), 7.58
(t, J = 7.6 Hz, 1H), 7.36−7.31 (m, 4H), 7.27−7.23 (m, 1H), 4.53 (d, J
865
dx.doi.org/10.1021/jm201349f | J. Med. Chem. 2012, 55, 852−870

Journal of Medicinal Chemistry
Article
1
= 6.0 Hz, 2H), 3.37 (s, 3H). 13C NMR (100 MHz, DMSO-d6): δ
166.03, 154.08, 143.34, 139.60, 138.33, 138.15, 135.03, 129.41, 129.07,
128.90, 128.28, 127.24, 126.75, 126.30, 125.44, 125.26, 112.21, 42.66,
26.44. MS (ESI) 359 m/z [M + H]+. Purity (≥98%, tr = 6.90 min).
3-(1-Methyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-6-yl)-N-
(2-(pyridin-4-yl)ethyl)benzamide (41). The general procedure B was
followed using a 0.1 M solution of 28 (2 mL, 200 μmol) and 4-(2-
aminoethyl)pyridine to provide 41 as a white solid (64 mg, 171 μmol,
56%). H NMR (400 MHz, DMSO-d6): δ 11.61 (s, 1H), 8.60 (t, J =
5.6 Hz, 1H), 8.30 (d, J = 2.0 Hz, 1H), 8.14 (t, J = 1.6 Hz, 1H), 7.86−
7.82 (m, 2H), 7.80 (d, J = 2.0 Hz, 1H), 7.56 (t, J = 8.0 Hz, 1H), 7.30−
7.15 (m, 5H), 3.37−3.29 (m, 5H), 2.65 (t, J = 7.6 Hz, 2H), 1.86 (p, J =
7.6 Hz, 2H). 13C NMR (100 MHz, DMSO-d6): δ 166.04, 154.10,
143.32, 141.75, 138.32, 138.07, 135.42, 129.21, 128.99, 128.97, 128.31,
128.28, 126.20, 125.73, 125.45, 125.21, 112.24, 38.96, 32.67, 30.89,
26.46. MS (ESI) 387.3 m/z [M + H]+. HRMS (ESI) calcd for
C23H23N4O2 [M + H]+, 387.1821; found, 387.1818. Purity (≥98%, tr =
7.58 min).
1
86%). H NMR (400 MHz, DMSO-d6): δ 11.65 (s, 1H), 8.71 (t, J =
5.6 Hz, 1H), 8.66 (br, 2H), 8.27 (d, J = 2.0 Hz, 1H), 8.08 (t, J = 1.6
Hz, 1H), 7.85 (dd, J = 7.6, 1.2 Hz, 1H), 7.79−7.77 (m, 2H), 7.64 (d, J
= 5.6 Hz, 2H), 7.56 (t, J = 7.6 Hz, 1H), 3.63 (q, J = 6.8 Hz, 2H), 3.37
(s, 3H), 3.05 (t, J = 6.8 Hz, 2H). 13C NMR (100 MHz, DMSO-d6): δ
166.20, 154.36, 154.09, 145.52, 143.35, 138.27, 138.12, 135.08, 129.38,
129.05, 128.88, 126.12, 125.81, 125.45, 125.16, 112.18, 34.67, 26.44.
MS (ESI) 374 m/z [M + H]+. Purity (≥98%, tr = 4.98 min).
N-(3-(4-Fluorophenyl)propyl)-3-(1-methyl-2-oxo-2,3-dihydro-1H-
imidazo[4,5-b]pyridin-6-yl)benzamide (47). The general procedure B
was followed using a 0.1 M solution of 28 (2 mL, 200 μmol) and 3-(4-
fluorophenyl)propan-1-amine to provide 47 as a white solid (56 mg,
1
137 μmol, 69%). H NMR (400 MHz, DMSO-d6): δ 11.63 (s, 1H),
8.58 (t, J = 5.6 Hz, 1H), 8.30 (d, J = 2.0 Hz, 1H), 8.13 (t, J = 1.6 Hz,
1H), 7.86−7.80 (m, 2H), 7.80 (d, J = 2.0 Hz, 1H), 7.56 (t, J = 7.6 Hz,
1H), 7.30−7.25 (m, 2H), 7.13−7.07 (m, 2H), 3.37 (s, 3H), 3.31 (q, J
= 7.2 Hz, 2H), 2.64 (t, J = 7.6 Hz, 2H), 1.84 (p, J = 7.6, 7.2 Hz, 2H).
13C NMR (100 MHz, DMSO-d6): δ 166.04, 154.09, 143.32, 138.31,
3-(1-Methyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-6-yl)-N-
phenethylbenzamide (42). The general procedure B was followed
using a 0.1 M solution of 28 (2 mL, 200 μmol) and phenethylamine to
1
provide 42 as a white solid (63 mg, 170 μmol, 85%). H NMR (400
MHz, DMSO-d6): δ 11.64 (s, 1H), 8.68 (t, J = 5.6 Hz, 1H), 8.29 (d, J
= 2.0 Hz, 1H), 8.12 (t, J = 1.6 Hz, 1H), 7.87−7.79 (m, 3H), 7.56 (t, J
= 7.6 Hz, 1H), 7.33−7.18 (m, 5H), 3.52 (q, J = 6.8 Hz, 2H), 3.37 (s,
3H), 2.88 (t, J = 7.2 Hz, 2H). 13C NMR (100 MHz, DMSO-d6): δ
166.01, 154.09, 143.33, 139.51, 138.28, 138.06, 135.35, 129.22, 129.00,
128.92, 128.66, 128.33, 126.13, 126.09, 125.45, 125.17, 112.19, 40.94,
35.12, 26.44. MS (ESI) 373 m/z [M + H]+. Purity (98%, tr = 7.15
min).
138.07, 137.85, 137.82, 135.40, 130.06, 129.98, 129.20, 128.97, 128.96,
126.18, 125.44, 125.20, 114.99, 114.79, 112.22, 38.81, 31.74, 30.93,
26.45. MS (ESI) 405.2 m/z [M + H]+. HRMS (ESI) calcd for
C23H22FN4O2 [M + H]+, 405.1727; found, 405.1727. Purity (≥98%, tr
= 7.66 min).
N-(3-(1H-Indol-3-yl)propyl)-3-(1-methyl-2-oxo-2,3-dihydro-1H-
imidazo[4,5-b]pyridin-6-yl)benzamide (48). The general procedure B
was followed using a 0.1 M solution of 28 (2 mL, 200 μmol) and 3-
(1H-indol-3-yl)propan-1-amine hydrochloride to provide 48 as a white
N-(2-(1H-Benzo[d]imidazol-2-yl)ethyl)-3-(1-methyl-2-oxo-2,3-di-
hydro-1H-imidazo[4,5-b]pyridin-6-yl)benzamide (43). The general
procedure B was followed using a 0.1 M solution of 28 (2 mL, 200
μmol) and 2-(1H-benzimidazol-2-yl)ethanamine hydrochloride to
1
solid (51 mg, 119 μmol, 59%). H NMR (400 MHz, DMSO-d6): δ
11.63 (s, 1H), 10.76 (s, 1H), 8.61 (t, J = 5.6 Hz, 1H), 8.30 (d, J = 2.0
Hz, 1H), 8.15 (t, J = 1.6 Hz, 1H), 7.86−7.82 (m, 2H), 7.80 (d, J = 2.0
Hz, 1H), 7.56 (t, J = 7.6 Hz, 1H), 7.52 (d, J = 8.0 Hz, 1H), 7.33 (d, J =
8.0 Hz, 1H), 7.17 (d, J = 2.0 Hz, 1H), 7.06 (td, J = 7.2, 1.2 Hz, 1H),
6.96 (td, J = 8.0, 0.8 Hz, 1H), 3.40−3.33 (m, 5H), 2.76 (t, J = 7.2 Hz,
2H), 1.94 (p, J = 7.2 Hz, 2H). 13C NMR (100 MHz, DMSO-d6): δ
166.02, 154.09, 143.31, 138.31, 138.07, 136.32, 135.49, 129.17, 128.97,
127.13, 126.19, 125.44, 125.20, 122.27, 120.81, 118.26, 118.07, 114.00,
112.23, 111.31, 29.83, 26.45, 22.23. MS (ESI) 426.2 m/z [M + H]+.
HRMS (ESI) calcd for C25H24N5O2 [M + H]+, 426.1930; found,
426.1928. Purity (≥98%, tr = 7.38 min).
1
provide 43 as a white solid (24 mg, 57 μmol, 29%). H NMR (400
MHz, DMSO-d6): δ 11.65 (s, 1H), 8.89 (t, J = 5.6 Hz, 1H), 8.28 (d, J
= 2.0 Hz, 1H), 8.14 (s, 1H), 7.86 (d, J = 7.6 Hz, 1H), 7.82−7.80 (m,
2H), 7.58−7.54 (m, 3H), 7.23 (dd, J = 6.0, 3.2 Hz, 2H), 3.79 (q, J =
6.8 Hz, 2H), 3.36 (s, 3H), 3.21 (t, J = 6.8 Hz, 2H). 13C NMR (100
MHz, DMSO-d6): δ 166.33, 158.19, 157.89, 154.10, 152.75, 143.34,
138.28, 138.07, 135.12, 129.34, 129.01, 128.88, 126.24, 125.45, 125.28,
122.41, 114.27, 112.21, 37.85, 28.37, 26.46. MS (ESI) 413 m/z [M +
H]+. Purity (≥98%, tr = 5.42 min).
3-(1-Methyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-6-yl)-N-
(pyridin-2-ylmethyl)benzamide (44). The general procedure B was
followed using a 0.1 M solution of 28 (2 mL, 200 μmol) and 2-
(aminomethyl)pyridine to provide 44 as a white solid (53 mg, 148
3-(1-Methyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-6-yl)-N-
(3-(pyridin-3-yl)propyl)benzamide (49). The general procedure B was
followed using a 0.1 M solution of 28 (2 mL, 200 μmol) and 3-
(pyridin-3-yl)propan-1-amine hydrochloride to provide 49 as a white
1
1
μmol, 74%). H NMR (400 MHz, DMSO-d6): δ 11.64 (s, 1H), 9.26
solid (55 mg, 141 μmol, 71%). H NMR (400 MHz, DMSO-d6): δ
(t, J = 6.0 Hz, 1H), 8.54 (d, J = 4.8 Hz, 1H), 8.32 (d, J = 5.0 Hz, 1H),
8.25 (s, 1H), 7.92−7.88 (m, 2H), 7.82−7.77 (m, 2H), 7.59 (t, J = 7.6
Hz, 1H), 7.39 (d, J = 7.6 Hz, 1H), 7.30 (dd, J = 7.2, 4.8 Hz, 1H), 4.63
(d, J = 5.6 Hz, 2H), 3.36 (s, 3H). 13C NMR (100 MHz, DMSO-d6): δ
166.25, 158.59, 154.09, 148.57, 143.36, 138.34, 138.17, 137.10, 134.87,
129.50, 129.11, 128.88, 126.34, 125.46, 125.32, 122.23, 121.19, 112.21,
44.63, 26.45. MS (ESI) 360 m/z [M + H]+. Purity (≥98%, tr = 4.94
min).
11.64 (s, 1H), 8.67−8.63 (m, 2H), 8.54 (d, J = 4.4 Hz, 1H), 8.30 (d, J
= 2.0 Hz, 1H), 8.14 (s, 1H), 7.98 (d, J = 8.0 Hz, 1H), 7.86−7.82 (m,
2H), 7.80 (d, J = 2.0 Hz, 1H), 7.58−7.54 (m, 2H), 3.37−3.31 (m,
5H), 2.75 (t, J = 7.2 Hz, 2H), 1.90 (p, J = 7.2 Hz, 2H). 13C NMR (100
MHz, DMSO-d6): δ 166.12, 154.10, 146.76, 144.51, 143.33, 139.35,
138.70, 138.31, 138.08, 135.34, 129.24, 128.98, 128.95, 126.20, 125.45,
125.22, 124.58, 112.23, 38.61, 30.26, 29.48, 26.45. MS (ESI) 388.2 m/
z [M + H]+. Purity (≥98%, tr = 5.12 min).
N-((1H-Benzo[d]imidazol-2-yl)methyl)-3-(1-methyl-2-oxo-2,3-di-
hydro-1H-imidazo[4,5-b]pyridin-6-yl)benzamide (45). The general
procedure B was followed using a 0.1 M solution of 28 (2.5 mL, 250
μmol) and (1H-benzimidazol-2-ylmethyl)amine hydrochloride to
provide 45 as a white solid (65 mg, 164 μmol, 66%). 1H NMR
(400 MHz, DMSO-d6): δ 11.66 (s, 1H), 9.49 (t, J = 5.2 Hz, 1H), 8.33
(d, J = 2.0 Hz, 1H), 8.29 (s, 1H), 7.93 (dd, J = 7.6, 1.6 Hz, 1H), 7.81
(d, J = 2.0 Hz, 1H), 7.68−7.60 (m, 3H), 7.36 (dd, J = 6.0, 3.2 Hz, 2H),
4.88 (d, J = 5.6 Hz, 2H), 3.37 (s, 3H). 13C NMR (100 MHz, DMSO-
d6): δ 166.76, 154.09, 152.37, 143.41, 138.30, 138.15, 134.16, 129.81,
129.16, 128.76, 126.54, 125.54, 125.48, 123.66, 114.35, 112.11, 36.98,
26.45. MS (ESI) 399 m/z [M + H]+. Purity (88%, tr = 5.35 min).
3-(1-Methyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-6-yl)-N-
(3-phenylpropyl)benzamide (46). The general procedure B was
followed using a 0.1 M solution of 28 (2 mL, 200 μmol) and 3-
phenylpropylamine to provide 46 as a white solid (43 mg, 111 μmol,
N-(3-(1H-Imidazol-4-yl)propyl)-3-(1-methyl-2-oxo-2,3-dihydro-
1H-imidazo[4,5-b]pyridin-6-yl)benzamide (50). The general proce-
dure B was followed using a 0.1 M solution of 28 (2 mL, 200 μmol)
and 3-(1H-imidazol-4-yl)propan-1-amine hydrochloride to provide 50
as a white solid (43 mg, 113 μmol, 57%). 1H NMR (400 MHz,
DMSO-d6): δ 14.24 (br, 1H), 11.65 (s, 1H), 8.96 (d, J = 1.2 Hz, 1H),
8.70 (t, J = 5.6 Hz, 1H), 8.30 (d, J = 2.0 Hz, 1H), 8.15 (s, 1H), 7.87 (d,
J = 8.0 Hz, 1H), 7.83 (d, J = 7.6 Hz, 1H), 7.81 (d, J = 1.6 Hz, 1H),
7.57 (t, J = 7.6 Hz, 1H), 7.47 (s, 1H), 3.37−3.32 (m, 5H), 2.72 (t, J =
7.2 Hz, 2H), 1.90 (p, J = 7.2 Hz, 2H). 13C NMR (100 MHz, DMSO-
d6): δ 166.19, 154.10, 143.35, 138.29, 138.11, 135.22, 133.71, 133.06,
129.32, 129.03, 128.91, 126.20, 125.46, 125.18, 115.58, 112.21, 38.37,
28.03, 26.46, 21.55. MS (ESI) 377.3 m/z [M + H]+. Purity (96%, tr =
5.02 min).
N-(3-Cyclopentylpropyl)-3-(1-methyl-2-oxo-2,3-dihydro-1H-
imidazo[4,5-b]pyridin-6-yl)benzamide (51). The general procedure B
866
dx.doi.org/10.1021/jm201349f | J. Med. Chem. 2012, 55, 852−870

Journal of Medicinal Chemistry
Article
was followed using a 0.1 M solution of 28 (2.0 mL, 200 μmol) and 3-
cyclopentylpropan-1-amine to provide 51 as a white solid (32 mg, 84
mmol, 42%). H NMR (400 MHz, DMSO-d6): δ 11.63 (s, 1H), 8.55
microwave for 1 h. The product mixture was diluted with ethyl acetate
(30 mL) and washed with water (10 mL) and brine (10 mL). The
combined organic layer was dried over magnesium sulfate, filtered, and
concentrated in vacuo. The resulting product was purified by flash
chromatography eluting with a linear gradient ranging from 25 to
100% EtOAc−hexane to afford 55a as a pale yellow solid (0.15 g,
0.215 mmol), which was directly used for the next step. To a solution
of 55a in MeOH (10 mL) was added 10% Pd/C (ca. 30 mg) at room
temperature. After air was removed from the flask using a vacuum
pump, hydrogen gas was introduced using a balloon. The reaction
mixture was stirred for 1 h, and the flask was evacuated under vacuum
and refilled with hydrogen gas. This procedure was repeated three
times, and the reaction mixture was stirred overnight at ambient
temperature. Palladium on carbon was removed by filtration through a
Celite pad. The filtrate was collected and evaporated to give the crude
product 56a, from which the trityl group was removed upon treatment
with TFA (1 mL) in CH2Cl2 (10 mL) for 1 h. The TFA and solvent
were then removed under reduced pressure. The resulting product was
purified by flash chromatography eluting with a linear gradient ranging
from 0 to 20% MeOH/EtOAc to afford 57 (67 mg, 0.148 mmol, 34%
over three steps) as a white solid. 1H NMR (400 MHz, DMSO-d6): δ
11.64 (s, 1H), 8.61 (t, J = 5.6 Hz, 1H), 8.30 (d, J = 2.0 Hz, 1H), 8.13
(s, 1H), 7.86 (d, J = 8.0 Hz, 1H), 7.83 (d, J = 8.0 Hz, 1H), 7.80 (d, J =
1.6 Hz, 1H), 7.65 (d, J = 8.0 Hz, 2H), 7.57 (d, J = 7.6 Hz, 1H), 7.49
(d, J = 8.0 Hz, 2H), 3.38−3.31 (m, 5H), 2.76 (t, J = 7.6 Hz, 2H), 1.90
(p, J = 7.2 Hz, 2H). 13C NMR (100 MHz, DMSO-d6): δ 166.07,
154.08, 146.80, 143.32, 138.30, 138.07, 135.36, 129.21, 129.15, 129.15,
128.96, 128.94, 126.17, 125.44, 125.19, 125.10, 125.06, 112.21, 32.39,
30.46, 26.44. MS (ESI) 455 m/z [M + H]+. HRMS (ESI) calcd for
C24H22F3N4O2 [M + H]+, 455.1695; found, 455.1694. Purity (≥98%,
tr = 8.32 min).
1
(d, J = 5.6 Hz, 1H), 8.29 (d, J = 2.0 Hz, 1H), 8.13 (d, J = 1.6 Hz, 1H),
7.86−7.81 (m, 2H), 7.80 (d, J = 2.0 Hz, 1H), 7.55 (t, J = 7.6 Hz, 1H),
3.37 (s, 3H), 3.28 (q, J = 6.8 Hz, 2H), 1.81−1.69 (m, 3H), 1.60−1.44
(m, 6H), 1.36−1.30 (m, 2H), 1.10−1.04 (m, 2H). 13C NMR (100
MHz, DMSO-d6): δ 165.86, 154.09, 143.32, 138.31, 138.06, 135.44,
129.16, 128.97, 126.17, 125.45, 125.17, 112.23, 39.37, 33.02, 32.22,
28.41, 26.45, 24.70. MS (ESI) 379.4 m/z [M + H]+. Purity (≥98%, tr =
8.39 min).
2-(3-(1-Nethyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-6-yl)-
benzamido)-4-phenylbutanoic Acid (52). The general procedure was
followed using a 0.1 M solution of 28 (3 mL, 300 μmol) and benzyl 2-
amino-4-phenylbutanoate followed by debenzylation using Pd/C, H2,
and MeOH (10 mL) for 12 h at room temperature to provide 52 as a
1
white solid (47 mg, 110 μmol, 37% over three steps). H NMR (400
MHz, DMSO-d6): δ 12.66 (s, 1H), 11.64 (s, 1H), 8.81 (d, J = 7.6 Hz,
1H), 8.33 (d, J = 2.0 Hz, 1H), 8.21 (s, 1H), 7.90 (dt, J = 7.6, 2.0 Hz,
2H), 7.82 (d, J = 2.0 Hz, 1H), 7.60 (t, J = 7.6 Hz, 1H), 7.31−7.17 (m,
5H), 4.42−4.37 (m, 1H), 3.38 (s, 3H), 2.80−2.65 (m, 2H), 2.16−2.08
(m, 2H). 13C NMR (100 MHz, DMSO-d6): δ 173.70, 166.46, 154.10,
143.36, 141.06, 138.36, 138.14, 134.71, 129.61, 129.02, 128.92, 128.40,
128.33, 126.51, 125.91, 125.52, 125.47, 112.25, 52.14, 32.43, 31.84,
26.46. MS (ESI) 429.4 m/z [M − H]−. Purity (53%/47%, tr = 7.00 and
7.71 min).
2-Benzyl-3-(3-(1-methyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-b]-
pyridin-6-yl)benzamido)propanoic Acid (53). The general procedure
B was followed using a 0.1 M solution of 28 (2 mL, 200 μmol) and
methyl 3-amino-2-benzylpropanoate hydrochloride followed by
hydrolysis using 10% KOH(aq) (10 mL) and MeOH (5 mL) at 60
°C for 12 h to provide 53 as a white solid (69 mg, 160 μmol, 80%). 1H
NMR (400 MHz, DMSO-d6): δ 12.30 (s, 1H), 11.65 (s, 1H), 8.73 (t, J
= 5.6 Hz, 1H), 8.31 (d, J = 2.0 Hz, 1H), 8.14 (s, 1H), 7.87 (d, J = 7.6
Hz, 1H), 7.83 (d, J = 8.0 Hz, 1H), 7.80 (d, J = 1.6 Hz, 1H), 7.57 (d, J
= 8.0 Hz, 1H), 7.31−7.18 (m, 5H), 3.55−3.42 (m, 2H), 3.38 (s, 3H),
3.00 (p, J = 7.6 Hz, 1H), 2.91−2.81 (m, 2H). 13C NMR (100 MHz,
DMSO-d6): δ 174.87, 166.28, 154.10, 143.35, 139.17, 138.32, 138.07,
135.14, 129.34, 129.01, 128.91, 128.77, 128.25, 126.24, 126.17, 125.46,
125.26, 112.21, 46.74, 41.26, 35.52, 26.46. MS (ESI) 429.4 m/z [M −
H]−. HRMS (ESI) calcd for C24H23N4O4 [M + H]+, 431.1719; found,
431.1724. Purity (60%/24%, tr = 6.66 and 7.43 min).
Methyl 2-(3-(3-(1-Methyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-b]-
pyridin-6-yl)benzamido)propyl)benzoate (58). The general proce-
dure B was followed using a 0.1 M solution of 28 (2 mL, 0.2 mmol)
and methyl 2-(3-aminopropyl)benzoate hydrochloride to provide 58
1
as a white solid (47 mg, 0.105 mmol, 52%). H NMR (400 MHz,
DMSO-d6): δ 11.63 (s, 1H), 8.65 (t, J = 5.6 Hz, 1H), 8.29 (d, J = 2.0
Hz, 1H), 8.15 (t, J = 2.0 Hz, 1H), 7.86−7.83 (m, 2H), 7.79 (d, J = 2.0
Hz, 1H), 7.65 (dd, J = 7.6, 1.6 Hz, 1H), 7.58−7.50 (m, 2H), 7.16 (d, J
= 7.6 Hz, 1H), 7.01 (td, J = 7.6, 0.8 Hz, 1H), 4.13 (t, J = 6.0 Hz, 2H),
3.79 (s, 3H), 3.51 (q, J = 6.8 Hz, 2H), 3.36 (s, 3H), 2.02 (p, J = 6.4
Hz, 2H). 13C NMR (100 MHz, DMSO-d6): δ 166.25, 166.20, 157.66,
154.09, 143.31, 138.30, 138.06, 135.37, 133.51, 130.69, 129.22, 128.96,
126.22, 125.44, 125.26, 120.16, 120.09, 113.58, 112.22, 66.24, 51.75,
36.47, 28.88, 26.44. MS (ESI) 444.3 m/z [M + H]+. Purity (≥98%, tr =
7.23 min).
N-(3-(4-(Aminomethyl)phenyl)propyl)-3-(1-methyl-2-oxo-2,3-di-
hydro-1H-imidazo[4,5-b]pyridin-6-yl)benzamide Hydrochloride
(59). The general procedure C was followed using 54 (0.19 g, 0.349
mmol), tert-butyl 4-bromobenzyl carbamate (0.27, 0.94 mmol),
Pd(PPh3)4 (40 mg, 10 mol %), NEt3 (0.5 mL), and CuI (5 mg) in
DMF (5 mL) to provide 55b (0.17 g, 0.254 mmol, 73%), which was
hydrogenated to give 56b (0.12 g, 0.159 mmol, 63%). To a solution of
56b (0.12 g, 0.159 mmol) in CH2Cl2 (5 mL) was added trifluoroacetic
acid (ca. 1 mL). The reaction mixture was stirred for 1 h at room
temperature. After evaporation of the solvent and trifluoroacetic acid, 1
N aqueous HCl was added to the product mixture. The resulting
product was purified by reverse phase flash chromatography eluting
with a linear gradient ranging from 5 to 100% acetonitrile−water to
provide 59 (33 mg, 0.074 mmol, 46%) as a white solid. 1H NMR (400
MHz, DMSO-d6): δ 11.64 (s, 1H), 8.68 (t, J = 5.6 Hz, 1H), 8.30 (d, J
= 2.0 Hz, 1H), 8.28 (br, 3H), 8.17 (t, J = 1.6 Hz, 1H), 7.87−7.83 (m,
3H), 7.56 (t, J = 7.6 Hz, 1H), 7.40 (d, J = 8.0 Hz, 2H), 7.29 (d, J = 8.0
Hz, 2H), 3.98 (s, 2H), 3.37 (s, 3H), 3.3−3.29 (m, 2H), 2.67 (t, J = 7.6
Hz, 2H), 1.86 (p, J = 7.2 Hz, 2H). 13C NMR (100 MHz, DMSO-d6): δ
166.04, 154.10, 143.32, 142.19, 138.29, 138.06, 135.37, 131.42, 129.20,
128.98, 128.94, 128.92, 128.57, 126.23, 125.45, 125.22, 112.28, 41.98,
32.32, 30.82, 26.48. MS (ESI) 416 m/z [M + H]+. Purity (≥98%, tr =
5.57 min).
General Procedure C for the Synthesis of the Substituted
Benzamide Analogues (57−63) in Scheme 4. 3-(1-Methyl-2-
oxo-3-trityl-2,3-dihydro-1H-imidazo[4,5-b]pyridin-6-yl)-N-(prop-2-
yn-1-yl)benzamide (54). To a solution of 28 (0.1 M CH2Cl2, 5 mL,
5.0 mmol) were added monopropagylamine hydrochloride (79 mg)
and diisopropylethylamine (1.0 mL) at room temperature. The
reaction mixture was stirred at 50 °C for 1 h. The mixture was cooled
to ambient, and then, solvent was removed by evaporation. The crude
mixture was diluted with ethyl acetate (40 mL) and was washed with
saturated aqueous NaHCO3 (10 mL) and brine (10 mL). The organic
layer was dried over magnesium sulfate, filtered, and concentrated in
vacuo. Purification of the crude product by flash chromatography
eluting with a linear gradient ranging from 12 to 100% EtOAc−hexane
1
provided 0.27 g (98%) of 54 as a white solid. H NMR (400 MHz,
DMSO-d6): δ 8.99 (t, J = 5.6 Hz, 1H), 8.17 (d, J = 2.0 Hz, 1H), 8.14
(t, J = 1.2 Hz, 1H), 7.87−7.82 (m, 3H), 7.56−7.50 (m, 7H), 7.26−
7.22 (m, 6H), 7.17−7.13 (m, 3H), 4.09 (dd, J = 5.6, 2.4, 2H), 3.33 (s,
3H), 3.15 (t, J = 2.4, 1H). 13C NMR (100 MHz, DMSO-d6): δ 165.64,
153.31, 142.95, 142.92, 137.54, 137.02, 134.41, 129.46, 129.12, 128.85,
128.53, 127.26, 126.55, 126.23, 125.36, 125.01, 111.81, 81.17, 73.78,
72.99, 28.51, 26.84. MS (ESI) 243 m/z [decomposition of trityl group,
M + H]+. HRMS (ESI) calcd for C36H29N4O2 [M + H]+, 549.2291;
found, 549.2297.
3-(1-Methyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-6-yl)-N-
(3-(4-(trifluoromethyl)phenyl)propyl)benzamide (57). A mixture of
54 (0.17 g, 0.310 mmol), 1-bromo-4-(trifluoromethyl)benzene (0.110
g, 0.431 mmol), Pd(PPh3)4 (40 mg, 10 mol %), NEt3 (0.5 mL), and
CuI (5 mg) in DMF (5 mL) was stirred under heating (80 °C) by
867
dx.doi.org/10.1021/jm201349f | J. Med. Chem. 2012, 55, 852−870

Journal of Medicinal Chemistry
Article
N-(3-(3-(Aminomethyl)phenyl)propyl)-3-(1-methyl-2-oxo-2,3-di-
hydro-1H-imidazo[4,5-b]pyridin-6-yl)benzamide Hydrochloride
(60). The procedure for the synthesis of 59 was followed using 54
(144 mg, 262 μmol) and tert-butyl 3-bromobenzyl carbamate to
provide 60 as a white solid (35 mg, 78 μmol, 30% over four steps). 1H
NMR (400 MHz, DMSO-d6): δ 11.64 (s, 1H), 8.76 (t, J = 5.6 Hz,
1H), 8.35 (br, 2H), 8.31 (d, J = 2.0 Hz, 1H), 8.20 (s, 1H), 7.87−7.84
(m, 3H), 7.56 (t, J = 7.6 Hz, 1H), 7.38 (s, 1H), 7.36−7.25 (m, 3H),
3.99 (d, J = 5.2 Hz, 2H), 3.38−3.31 (m, 5H), 2.67 (t, J = 7.6 Hz, 2H),
1.89 (p, J = 7.6, 7.2 Hz, 2H). 13C NMR (100 MHz, DMSO-d6): δ
166.03, 154.10, 143.31, 142.15, 138.28, 138.05, 135.34, 133.97, 129.17,
128.99, 128.92, 128.88, 128.56, 128.43, 126.28, 126.24, 125.46, 125.21,
112.32, 42.20, 32.55, 30.72, 26.50. MS (ESI) 416 m/z [M + H]+.
Purity (≥98%, tr = 5.64 min).
Accession Codes
Coordinates and structure factors of PaTMK complexed with 1,
17, and dFTM are available from the Protein Data Bank with
accession codes 3UWK, 3UWO, and 3UXM, respectively.
AUTHOR INFORMATION
Corresponding Author
■
*(M.P.) Tel: 860-339-5319. Fax: 860-339-5319. E-mail:
marksplummer@gmail.com. (W.R.R.) Tel: 561-228-2450. Fax:
561-228-3052. E-mail: roush@scripps.edu.
ACKNOWLEDGMENTS
■
Support by the Pfizer-Scripps Florida postdoctoral research
fellow program to J.Y.C. is gratefully acknowledged. Additional
financial support for J.Y.C. and W.R.R. at Scripps Florida was
provided by NIH Grant U54MH084512, Hugh Rosen,
Principal Investigator. We also thank Lisa Mullins and Joe
Penzien of Pfizer for performing preliminary antibacterial
assays.
3-(3-(3-(1-Methyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-6-
yl)benzamido)propyl)benzoic Acid (61). The general procedure C
were followed using 54 (153 mg, 278 μmol) and methyl 3-
bromobenzoate to provide 56d. A solution of 56d in MeOH (5
mL) and 10% KOH(aq) (10 mL) was stirred at 60 °C for 24 h. The
reaction mixture was cooled to ambient, and then, 1 N HCl was added
to acidify the product mixture, and a white solid precipitated. The
mixture was diluted with ethyl acetate (30 mL) and washed with water
(10 mL) and brine (10 mL). The organic layer was dried over
magnesium sulfate, filtered, and concentrated in vacuo. Purification of
the crude product by flash chromatography eluting with a linear
gradient ranging from 0 to 30% MeOH/EtOAc provided 61 (26 mg,
ABBREVIATIONS USED
■
TP5A, P1-(5′-adenosyl)-P5-(5′-thymidyl)pentaphosphate;
dFTM, deoxy-fluorothymidine; dFTM, 5′-fluorodeoxythymi-
dine; thiourea-α-TM, 5′-deoxy-5′-N-arylthiourea α-thymidine
derivatives; DTBN, 5,5′-dithio-bis (2-nitrobenzoic acid); MIC,
minimum inhibitory concentration; IC50, half-maximal inhib-
itory concentration
1
61 μmol, 22%, four steps). H NMR (400 MHz, DMSO-d6): δ 12.90
(br, 1H), 11.63 (s, 1H), 8.60 (t, J = 5.6 Hz, 1H), 8.30 (d, J = 1.6 Hz,
1H), 8.14 (s, 1H), 7.86−7.82 (m, 3H), 7.80 (d, J = 2.0 Hz, 1H), 7.77
(d, J = 7.6 Hz, 1H), 7.56 (t, J = 7.6 Hz, 1H), 7.48 (d, J = 7.6 Hz, 1H),
7.40 (t, J = 7.6 Hz, 1H), 3.37−3.30 (m, 5H), 2.72 (t, J = 7.6 Hz, 2H),
1.88 (p, J = 7.2 Hz, 2H). 13C NMR (100 MHz, DMSO-d6): δ 166.08,
154.09, 143.31, 142.07, 138.31, 138.07, 135.41, 129.19, 129.09, 128.96,
128.40, 128.37, 126.81, 125.44, 125.22, 112.23, 32.40, 30.77, 26.45.
MS (ESI) 429 m/z [M − H]−. Purity (95%, tr = 6.58 min).
REFERENCES
■
(1) Payne, D. J.; Gwynn, M. N.; Holmes, D. J.; Pompliano, D. L.
Drugs for bad bugs: Confronting the challenges of antibacterial
discovery. Nat. Rev. Drug Discovery 2007, 6 (1), 29−40.
(2) Obritsch, M. D.; Fish, D. N.; MacLaren, R.; Jung, R. Nosocomial
infections due to multidrug-resistant Pseudomonas aeruginosa:
Epidemiology and treatment options. Pharmacotherapy 2005, 25
(10), 1353−1364.
4-(3-(3-(1-Methyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-6-
yl)benzamido)propyl)benzoic Acid (62). The procedure for the
synthesis of 61 was followed using 54 (0.16 g, 0.293 mmol) and
methyl 4-bromobenzoate to provide 62 as a white solid (26 mg, 60.8
μmol, 21% over four steps). 1H NMR (400 MHz, DMSO-d6): δ 12.77
(br, 1H), 11.63 (s, 1H), 8.62 (t, J = 5.6 Hz, 1H), 8.30 (d, J = 2.0 Hz,
1H), 8.14 (s, 1H), 7.88−7.80 (m, 5H), 7.56 (t, J = 7.6 Hz, 1H), 7.41−
7.36 (m, 2H), 3.37−3.30 (m, 5H), 2.73 (t, J = 7.6 Hz, 2H), 1.89 (p, J =
7.6 Hz, 2H). 13C NMR (100 MHz, DMSO-d6): δ 167.24, 166.05,
154.08, 147.17, 143.31, 138.30, 138.06, 135.37, 129.37, 129.19, 128.94,
128.51, 128.38, 126.18, 125.43, 125.20, 112.23, 32.60, 30.47, 26.44.
MS (ESI) 429 m/z [M − H]−. Purity (≥98%, tr = 6.44 min).
(3) Rice, L. B. The clinical consequences of antimicrobial resistance.
Curr. Opin. Microbiol. 2009, 12 (5), 476−481.
(4) Rice, L. B. Emerging issues in the management of infections
caused by multidrug-resistant gram-negative bacteria. Cleve. Clin. J.
Med. 2007, 74 (Suppl. 4), S12−S20.
(5) Kandeel, M.; Kato, A.; Kitamura, Y.; Kitade, Y. Thymidylate
kinase: The lost chemotherapeutic target. Nucleic Acids Symp. Ser.
(Oxford) 2009, 53, 283−284.
2-(3-(3-(1-Methyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-6-
yl)benzamido)propyl)benzoic Acid (63). The procedure for the
synthesis of 61 was followed using 58 (0.16 g, 0.360 mmol) to provide
(6) Anderson, E. Nucleoside and nucleotide kinases. In the Enzymes,
3rd ed.; Academic Press: New York, 1973; Vol. 8, pp 49−96.
(7) Van Calenbergh, S. Structure-aided design of inhibitors of
Mycobacterium tuberculosis thymidylate kinase. Verh. K Acad. Geneeskd.
Belg. 2006, 68 (4), 223−248.
(8) Fioravanti, E.; Adam, V.; Munier-Lehmann, H.; Bourgeois, D.
The crystal structure of Mycobacterium tuberculosis thymidylate kinase
in complex with 3′-azidodeoxythymidine monophosphate suggests a
mechanism for competitive inhibition. Biochemistry 2005, 44 (1),
130−137.
(9) Gasse, C.; Douguet, D.; Huteau, V.; Marchal, G.; Munier-
Lehmann, H.; Pochet, S. Substituted benzyl-pyrimidines targeting
thymidine monophosphate kinase of Mycobacterium tuberculosis:
Synthesis and in vitro anti-mycobacterial activity. Bioorg. Med. Chem.
2008, 16 (11), 6075−6085.
(10) Gasse, C.; Huteau, V.; Douguet, D.; Munier-Lehmann, H.;
Pochet, S. A new family of inhibitors of Mycobacterium tuberculosis
thymidine monophosphate kinase. Nucleosides Nucleotides Nucleic Acids
2007, 26 (8−9), 1057−1061.
(11) Vanheusden, V.; Munier-Lehmann, H.; Froeyen, M.; Busson, R.;
Rozenski, J.; Herdewijn, P.; Van Calenbergh, S. Discovery of bicyclic
thymidine analogues as selective and high-affinity inhibitors of
1
63 as a white solid (0.12 g, 0.279 mmol, 78%). H NMR (400 MHz,
DMSO-d6): δ 12.55 (br, 1H), 11.63 (s, 1H), 8.66 (t, J = 5.6 Hz, 1H),
8.29 (d, J = 2.0 Hz, 1H), 8.14 (s, 1H), 7.86−7.79 (m, 2H), 7.79 (d, J =
2.0 Hz, 1H), 7.64 (dd, J = 7.6, 2.0 Hz, 1H), 7.55 (d, J = 7.6 Hz, 1H),
7.48 (td, J = 8.4, 2.0 Hz, 1H), 7.13 (d, J = 8.4 Hz, 1H), 6.99 (td, J =
7.6, 0.8 Hz, 1H), 4.13 (d, J = 6.4 Hz, 2H), 3.50 (d, J = 6.4 Hz, 2H),
3.36 (s, 3H), 2.03 (p, J = 6.4 Hz, 2H). 13C NMR (100 MHz, DMSO-
d6): δ 167.38, 166.22, 157.41, 154.09, 143.32, 138.31, 138.07, 135.33,
132.94, 130.63, 129.23, 128.96, 126.22, 125.44, 125.25, 121.63, 120.08,
113.53, 112.23, 66.20, 36.43, 30.66, 28.87, 26.44. MS (ESI) 429 m/z
[M − H]−. Purity (≥98%, tr = 6.50 min).
ASSOCIATED CONTENT
■
S
* Supporting Information
Data collection and refinement statistics for the X-ray
cocrystallization of PaTMK with 1, 17, and dFTM. Illustration
of the four reactions that comprise the assay system. This
material is available free of charge via the Internet at http://
pubs.acs.org.
868
dx.doi.org/10.1021/jm201349f | J. Med. Chem. 2012, 55, 852−870

Journal of Medicinal Chemistry
Article
Mycobacterium tuberculosis thymidine monophosphate kinase. J. Med.
Chem. 2004, 47 (25), 6187−6194.
(12) Vanheusden, V.; Van Rompaey, P.; Munier-Lehmann, H.;
Pochet, S.; Herdewijn, P.; Van Calenbergh, S. Thymidine and
thymidine-5′-O-monophosphate analogues as inhibitors of Mycobacte-
rium tuberculosis thymidylate kinase. Bioorg. Med. Chem. Lett. 2003, 13
(18), 3045−3048.
(30) Savelli, F.; Boido, A.; Vazzana, I.; Sparatore, F.
Tetrahydrocyclopenta[e]pyrido[3,2-b][1,4]diazepin and -cyclopenta-
[e]pyrido[2,3-b][1,4]diazepine derivatives. J. Heterocycl. Chem. 1987,
24, 1709−1716.
(31) Kudo, N.; Perseghini, M.; Fu, G. C. A versatile method for
Suzuki cross-coupling reactions of nitrogen heterocycles. Angew.
Chem., Int. Ed. Engl. 2006, 45 (8), 1282−1284.
(13) Kotaka, M.; Dhaliwal, B.; Ren, J.; Nichols, C. E.; Angell, R.;
Lockyer, M.; Hawkins, A. R.; Stammers, D. K. Structures of S. aureus
thymidylate kinase reveal an atypical active site configuration and an
intermediate conformational state upon substrate binding. Protein Sci.
2006, 15 (4), 774−784.
(14) Byun, Y.; Vogel, S. R.; Phipps, A. J.; Carnrot, C.; Eriksson, S.;
Tiwari, R.; Tjarks, W. Synthesis and biological evaluation of inhibitors
of thymidine monophosphate kinase from Bacillus anthracis. Nucleo-
sides Nucleotides Nucleic Acids 2008, 27 (3), 244−260.
(15) Andrade, C. H.; Pasqualoto, K. F.; Ferreira, E. I.; Hopfinger, A.
J. 3D-Pharmacophore mapping of thymidine-based inhibitors of
TMPK as potential antituberculosis agents. J. Comput.-Aided Mol.
Des. 2010, 24 (2), 157−172.
(16) Cui, H.; Ruiz-Perez, L. M.; Gonzalez-Pacanowska, D.; Gilbert, I.
H. Potential application of thymidylate kinase in nucleoside analogue
activation in Plasmodium falciparum. Bioorg. Med. Chem. 2010, 18
(20), 7302−7309.
(17) Familiar, O.; Munier-Lehmann, H.; Ainsa, J. A.; Camarasa, M. J.;
Perez-Perez, M. J. Design, synthesis and inhibitory activity against
Mycobacterium tuberculosis thymidine monophosphate kinase of acyclic
nucleoside analogues with a distal imidazoquinolinone. Eur. J. Med.
Chem. 2010, 45 (12), 5910−5918.
(18) Frecer, V.; Seneci, P.; Miertus, S. Computer-assisted
combinatorial design of bicyclic thymidine analogs as inhibitors of
Mycobacterium tuberculosis thymidine monophosphate kinase. J.
Comput.-Aided Mol. Des. 2011, 25 (1), 31−49.
(19) Andrade, C. H.; Pasqualoto, K. F.; Ferreira, E. I.; Hopfinger, A.
J. Rational design and 3D-pharmacophore mapping of 5′-thiourea-
substituted alpha-thymidine analogues as mycobacterial TMPK
inhibitors. J. Chem. Inf. Model. 2009, 49 (4), 1070−1078.
(20) Douguet, D.; Munier-Lehmann, H.; Labesse, G.; Pochet, S.
LEA3D: A computer-aided ligand design for structure-based drug
design. J. Med. Chem. 2005, 48 (7), 2457−2468.
(21) Van Daele, I.; Munier-Lehmann, H.; Froeyen, M.; Balzarini, J.;
Van Calenbergh, S. Rational design of 5′-thiourea-substituted alpha-
thymidine analogues as thymidine monophosphate kinase inhibitors
capable of inhibiting mycobacterial growth. J. Med. Chem. 2007, 50
(22), 5281−5292.
(22) Lavie, A.; Ostermann, N.; Brundiers, R.; Goody, R. S.; Reinstein,
J.; Konrad, M.; Schlichting, I. Structural basis for efficient
phosphorylation of 3′-azidothymidine monophosphate by Escherichia
coli thymidylate kinase. Proc. Natl. Acad. Sci. U.S.A. 1998, 95 (24),
14045−14050.
(23) MOE; Chemical Computing Group Inc.: Montreal, 2009.10.
(24) Bissantz, C.; Kuhn, B.; Stahl, M. A medicinal chemist's guide to
molecular interactions. J. Med. Chem. 2010, 53 (14), 5061−5084.
(25) Ma, J. C.; Dougherty, D. A. The Cation-pi Interaction. Chem.
Rev. 1997, 97 (5), 1303−1324.
(26) Mecozzi, S.; West, A. P. Jr.; Dougherty, D. A. Cation-pi
interactions in aromatics of biological and medicinal interest:
Electrostatic potential surfaces as a useful qualitative guide. Proc.
Natl. Acad. Sci. U.S.A. 1996, 93 (20), 10566−10571.
(27) Dougherty, D. A. Cation-pi interactions in chemistry and
biology: a new view of benzene, Phe, Tyr, and Trp. Science 1996, 271
(5246), 163−168.
(32) Barder, T. E.; Walker, S. D.; Martinelli, J. R.; Buchwald, S. L.
Catalysts for Suzuki-Miyaura coupling processes: Scope and studies of
the effect of ligand structure. J. Am. Chem. Soc. 2005, 127 (13), 4685−
4696.
(33) Arvela, R. K.; Leadbeater, N. E. Suzuki coupling of aryl chlorides
with phenylboronic acid in water, using microwave heating with
simultaneous cooling. Org. Lett. 2005, 7 (11), 2101−2104.
(34) Leadbeater, N. E. Fast, easy, clean chemistry by using water as a
solvent and microwave heating: the Suzuki coupling as an illustration.
Chem. Commun. (Cambridge) 2005, 23, 2881−2902.
(35) Glide 5.5; Schrodinger Inc.: New York.
(36) Maestro9.0; Schrodinger Inc.: New York.
(37) Lavie, A.; Konrad, M.; Brundiers, R.; Goody, R. S.; Schlichting,
I.; Reinstein, J. Crystal structure of yeast thymidylate kinase complexed
with the bisubstrate inhibitor P1-(5′-adenosyl) P5-(5′-thymidyl)
pentaphosphate (TP5A) at 2.0 A resolution: Implications for catalysis
and AZT activation. Biochemistry 1998, 37 (11), 3677−3686.
(38) Cabeen, M. T.; Jacobs-Wagner, C. Bacterial cell shape. Nat. Rev.
Microbiol. 2005, 3 (8), 601−610.
(39) Ostermann, N.; Schlichting, I.; Brundiers, R.; Konrad, M.;
Reinstein, J.; Veit, T.; Goody, R. S.; Lavie, A. Insights into the
phosphoryltransfer mechanism of human thymidylate kinase gained
from crystal structures of enzyme complexes along the reaction
coordinate. Structure 2000, 8 (6), 629−642.
(40) Biswas, S.; Mohammad, M. M.; Patel, D. R.; Movileanu, L.; van
den Berg, B. Structural insight into OprD substrate specificity. Nat.
Struct. Mol. Biol. 2007, 14 (11), 1108−1109.
(41) Mach, T.; Neves, P.; Spiga, E.; Weingart, H.; Winterhalter, M.;
Ruggerone, P.; Ceccarelli, M.; Gameiro, P. Facilitated permeation of
antibiotics across membrane channelsInteraction of the quinolone
moxifloxacin with the OmpF channel. J. Am. Chem. Soc. 2008, 130
(40), 13301−13309.
(42) Nikaido, H.; Thanassi, D. G. Penetration of lipophilic agents
with multiple protonation sites into bacterial cells: tetracyclines and
fluoroquinolones as examples. Antimicrob. Agents Chemother. 1993, 37
(7), 1393−1399.
(43) Carter, J. C. W.; Sweet, R. M. Macromolecular Crystallography,
part A; Academic Press: New York, 1997; Vol. 276, pp 307−326.
(44) The CCP4 suite: Programs for protein crystallography. Acta
Crystallogr., Sect. D: Biol. Crystallogr. 1994, 50 (Part 5), 760−763.
(45) Murshudov, G. N.; Vagin, A. A.; Dodson, E. J. Refinement of
macromolecular structures by the maximum-likelihood method. Acta
Crystallogr., Sect. D: Biol. Crystallogr. 1997, 53 (Part 3), 240−255.
(46) Brunger, A. T. Free R value: A novel statistical quantity for
assessing the accuracy of crystal structures. Nature 1992, 355 (6359),
472−475.
(47) Read, R. J. Improved fourier coefficients for maps using phases
from partial structures with errors. Acta Crystallogr., Sect. A 1986, 42,
140−149.
(48) Emsley, P.; Cowtan, K. Coot: Model-building tools for
molecular graphics. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2004,
60 (Part 12, Part 1), 2126−2132.
(49) Read, R. J. Pushing the boundaries of molecular replacement
with maximum likelihood. Acta Crystallogr., Sect. D: Biol. Crystallogr.
2001, 57 (Part 10), 1373−1382.
(28) Michel, J.; Tirado-Rives, J.; Jorgensen, W. L. Energetics of
displacing water molecules from protein binding sites: consequences
for ligand optimization. J. Am. Chem. Soc. 2009, 131 (42), 15403−
15411.
(50) Bricogne, G.; Blanc, E.; Brandl, M.; EFlensburg, C.; Keller, P.;
Paciorek, W.; Roversi, P.; Smart, O.; Vonrhein, C.; Womack, T.
BUSTER, version 2.8.0; Global Phasing Ltd.: Cambridge, United
Kingdom, 2009.
(29) Grivas, S.; Lindstrom, S. Palladium(0)-catalyzed phenylation of
(51) Petit, C. M.; Koretke, K. K. Characterization of Streptococcus
imidazo[4,5-b]pyridines. J. Heterocycl. Chem. 1995, 32, 467−471.
pneumoniae thymidylate kinase: Steady-state kinetics of the forward
869
dx.doi.org/10.1021/jm201349f | J. Med. Chem. 2012, 55, 852−870

Journal of Medicinal Chemistry
Article
reaction and isothermal titration calorimetry. Biochem. J. 2002, 363
(Part 3), 825−831.
870
dx.doi.org/10.1021/jm201349f | J. Med. Chem. 2012, 55, 852−870