2-Oxo-1,2-dihydropyridinyl-3-yl amide-based GPa inhibitors: Design, synthesis and structure-activity relationship study

Article abstract of DOI:10.1016/j.ejmech.2016.01.031

Glycogen phosphorylase (GP), which plays a crucial role in the conversion of glycogen to glucose-1-phosphate, is a target for therapeutic intervention in diabetes. In this study, we report the design and synthesis of 29 new derivatives of 2-oxo-1,2-dihydro pyridin-3-yl amides, as potential inhibitors of GP. The hit rate (45%) was high with 13 compounds inhibiting GPa (between 33% at 4.40 mM and an IC₅₀ of 1.92 μM). Two lead compounds were identified as compounds exhibiting good GPa inhibition (IC₅₀ = 2.1 and 1.92 μM). SAR analysis of these compounds revealed sensitivity of GPa to the length of the 2-oxo-1,2-dihydro pyridin-3-yl amide derivative and a preference for inclusion of a 3,4-dichlorobenzyl moiety.

Full text of DOI:10.1016/j.ejmech.2016.01.031

Accepted Manuscript
2-Oxo-1,2-dihydropyridinyl-3-yl amide-based GPa inhibitors: Design, synthesis and
structure-activity relationship study
Wendy A. Loughlin, Ian D. Jenkins, N. David Karis, Stephanie S. Schweiker, Peter C.
Healy
PII:
S0223-5234(16)30032-0
10.1016/j.ejmech.2016.01.031
EJMECH 8321
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 8 December 2015
Revised Date: 18 January 2016
Accepted Date: 18 January 2016
Please cite this article as: W.A. Loughlin, I.D. Jenkins, N.D. Karis, S.S. Schweiker, P.C. Healy, 2-
Oxo-1,2-dihydropyridinyl-3-yl amide-based GPa inhibitors: Design, synthesis and structure-activity
relationship study, European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.01.031.
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Graphical Abstract

ACCEPTED MANUSCRIPT
European Journal of Medicinal Chemistry
journal homepage: www.elsevier.com
2
-Oxo-1,2-dihydropyridinyl-3-yl amide-based GPa inhibitors: Design, synthesis and
structure-activity relationship study
a,∗
b
b
b,
ο
a,c
Wendy A. Loughlin , Ian D. Jenkins , N. David Karis , Stephanie S. Schweiker , Peter C. Healy
a
School of Natural Sciences, Griffith University, Brisbane, Qld, 4111, Australia
Eskitis Institute for Drug Discovery, Griffith University, Brisbane, Qld, 4111, Australia
Central Analytical Research Facility, Queensland University of Technology, Brisbane, Qld, 4001, Australia
b
c
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Glycogen phosphorylase (GP), which plays a crucial role in the conversion of glycogen to
glucose-1-phosphate, is a target for therapeutic intervention in diabetes. In this study, we report
the design and synthesis of 29 new derivatives of 2-oxo-1,2-dihydro pyridin-3-yl amides, as
potential inhibitors of GP. The hit rate (45%) was high with 13 compounds inhibiting GPa
(between 33% at 4.40 mM and an IC50 of 1.92 µM). Two lead compounds were identified as
compounds exhibiting good GPa inhibition (IC50 = 2.1 and 1.92 ꢀM). SAR analysis of these
compounds revealed sensitivity of GPa to the length of the 2-oxo-1,2-dihydro pyridin-3-yl
amide derivative and a preference for inclusion of a 3,4-dichlorobenzyl moiety
Available online
Keywords:
Glycogen phosphorylase inhibitors
Pyridone amides
2
009 Elsevier Ltd. All rights reserved.
Peptide mimetic
Structure Activity Relationship
Type 2 diabetes
—
——
∗
Corresponding author:
E-mail address: w.loughlin@griffith.edu.au
ο
Present address: Faculty of Health Science and Medicine, Bond University, Gold Coast, Queensland, 4229, Australia

1
. Introduction
previous study [32], the derivatives were designed to include
ACCEPTED MANUSCRIPT
functional groups that had apparent pharmacophoric features
aromatic ring and aromatic hydrophobic moiety (positions b and
c, Figure 1); and lack of a hydrogen bond acceptor at C3-N
position a, Figure 1). In particular the derivatives were designed
Current medication for Type 2 diabetes commonly has
(
adverse side effects, which has identified a need for new and
better treatments. Control of glycogenolysis (the breakdown of
glycogen to glucose) is a promising approach to control of blood
glucose levels. Glycogen Phosphorylase (GP) is a key enzyme in
the glycogenolysis pathway and plays a crucial role in
carbohydrate metabolism, catalysing the phosphorolysis of
glycogen to form glucose-1-phosphate which can be utilised to
form ATP in muscle cells or in the case of hepatocytes, to form
glucose for maintaining blood glucose levels. This is reflected in
the distribution of three main isoforms of GP in the body;
muscle, liver, brain. Studies have confirmed the efficacy of
inhibitors of GP on hepatic glycogen balance and blood glucose
control, thus making GP a molecular therapeutic target for the
design of compounds that could treat hyperglycaemia associated
with Type II Diabetes Mellitus [1-7]. GP is a highly regulated
allosteric enzyme with multiple binding sites for inhibitors which
include the catalytic site which binds glucose derivatives [8,9],
the glycogen storage site [10] and binding sites [11] for allosteric
(
to explore (a) diversity at the C3-amino group through use of (i)
readily available aldehydes which explored the optimal position
1
for the chlorine substituent on the aromatic ring (R = a-d,
1
Scheme 1), (ii) readily available heterocyclic aromatic rings (R
=
e-h, Scheme 1), and (iii) para-toluenesulfonyl as another
1
aromatic functional group (R = i, Scheme 1); (b) combinations
of chlorobenzyl substituents at both the C3-amino group and the
N1 of the pyridine (Scheme 1) and (c) the structural length of the
derivatives, and thus placement of the aromatic moiety, through
shortening or extending the substituent at the N1 of the pyridone
and/or the C3-amino group (Schemes 1 and 2). Design and
choice of derivatives was guided further by (i) previous results
where benzyl 3,4-dichlorobenzyl pyridone 1 and benzyl benzyl
pyridone 2 displayed an order of magnitude of difference in
inhibition of GPa, (ii) iterative GPa assay results obtained during
the present study and (iii) commercial availability of aldehyde
and amine precursors.
1
effectors (purine site [12], indole site at the enzyme dimer
interface [13,14], allosteric (AMP) site [1,15] and new allosteric
site [16]). Glucose derivatives, which bind to the catalytic site of
GP, represent the most widely studied group of inhibitory
molecules [17-19], However, other structural classes of inhibitors
show promise; for example GPa has been validated as a target in
diabetic ob/ob mice [20] for indole inhibitors. Comprehensive
reviews on a range of GP inhibitors have been reported in the
patent literature [21,22] and elsewhere [1-7, 18, 23-31].
Reductive amination of aminopyridone ester 4 [36] (Scheme
), with a range of readily available aldehydes, gave pyridone
1
esters 5-11 and 13 in good to excellent yields (84-99%).
Interestingly, whereas reductive animation of aminopyridone
ester 4 with N-methyl pyrrole aldehyde was unsuccessful,
introduction of an electron withdrawing group led to reductive
amination of 4 with N-tosylpyrrole-2-carbaldehyde [37] and gave
tosylpyrrole pyridine ester 12 (78%). With esters 5-13 in hand,
conversion to a library of amides was carried out using direct
aminolysis, which was previously found to be a mild and atom-
efficient procedure [36]. Pyridone esters 5-13 were reacted with
Previously, we reported a chemogenomics strategy for GP
inhibitor design [32], based on studies which showed that a GP
inhibitor can block the interaction of the C-terminus of the
hepatic glycogen-binding subunit GL (encoded by the gene
PPP1R3B) of protein phophatase-1 (PP-1) [33-35]. A pyridone
ring was established as a useful mimetic of the Leu-Gly residues
of the C-terminus residues identified with GP activity. Arising
from this work, a ‘first generation’ library of 2-oxo-1,2-
dihydropyridin-3-yl amides was synthesized and screened against
GPa. Benzyl 3,4-dichlorobenzyl pyridone 1 (est. IC50 = 6.3 µM)
was identified as the lead GPa inhibitor, in the context of the
other 2-oxo-1,2-dihydropyridin-3-yl amide inhibitors that were
identified from the same study [32]; for example: benzyl
3
,4-dichlorobenzylamine to incorporate the apparent favored
moiety and derivatives 14-17 and 20-24 were obtained in
excellent yields (93-100%). Two further examples were prepared
to facilitate SAR comparison at positions (b) and (c) in Figure 1.
Aminolysis of 3,4-dichlorobenzyl aminopyridone ester 8 with
benzylamine and propylamine gave the derivatives 18 (95%) and
1
9 (97%), respectively. In addition selected benzoyl derivatives
were chosen to confirm the difference in activity between the
carbonyl or methylene moiety at position (a) in Figure 1.
Acylation of aminopyridone ester 4 with 2-chlorobenzoyl
chloride and phenyl isocyanate gave 25 and 26 (89-97%). Esters
5 and 26 were converted to amide derivatives 27, 28 and 29 (88-
00%) (Scheme 1) by aminolysis with 3,4-dichlorobenzylamine,
benzylamine, and propylamine, respectively. Attempted
formation of the corresponding phenylurea 3,4-dichlorobenzyl
pyridone from ester 26 and 3,4-dichlorobenzylamine resulted in
decomposition (see supplementary data).
benzylpyridone
2 (est. IC50 = 34.2 µM) and benzoyl
dichlorobenzyl pyridone 3 (est. IC50 = 162 µM). A preliminary
analysis showed that inhibitory activity of a compound appeared
to be favoured by aromatic ligands (positions (b) and (c), Figure
2
1
1
), whereas inclusion of a carbonyl group adjacent to C3-N
(position (a), Figure 1) was detrimental to potency [32].
A GP-1 ligand X-Ray structure has yet to be obtained to
establish the binding site of the 2-oxo-1,2-dihydropyridin-3-yl
amides to GP. Herein, we report on a second-generation library
consisting of a range of derivatives of benzyl 3,4-dichlorobenzyl
pyridone 1. We maintained the use of the pyridone moiety as the
primary scaffold, as it provides diversity points through the C3
amino group and the N1 of the pyridone ring. An excellent hit
rate was obtained and the inhibitory activity of the library of
pyridone derivatives provided useful Structure-Activity
Relationships (SAR) for the 2-oxo-1,2-dihydropyridin-3-yl amide
inhibitors of GPa.
Alteration of the spacing of the aromatic moieties and
flexibility of the alkyl linking chains at the C3-amino group and
the N1 of the pyridine was explored through insertion or deletion
of an ethylene group. This required a different synthetic approach
(Scheme 2). 2-Hydroxy-3-nitropyridine 30 was N-alkylated with
the corresponding bromoacetophenones 31-36 generated from
bromoacetyl bromide and an amine (aniline, 3,4-dichloroaniline,
phenylethylamine,
3,4-dichlorobenzylamine,
3,4-dichloro
phenylethylamine, 2,4-dichlorophenylethylamine) in moderate to
excellent yields (30-93%). Two methods were used for the
generation
of
nitropyridones
37-42.
Addition
of
2
. Chemistry
bromoacetophenones 31-34 to a mixture of NaH and 2-hydroxy-
3-nitropyridine 30 in THF and then irradiation of the solution
under microwave conditions (150 C, 60 mins) gave
The synthesis of 2-oxo-1,2-dihydropyridin-3-yl amide
o
derivatives with variations at the C3-amino group and the N1 of
the pyridone is outlined in Schemes 1 and 2. Based on our
nitropyridones 37-40 in good to excellent yields (68-100%).

Precipitation of the product from solution requ
use of 1.1 equivalents of NaH. In the alternative method, NaH
A
ire
C
d t
C
he
E
c
P
on
T
tr
E
oll
D
ed MALE
N
a
U
nd
S
L
C
E
R
LPIPfo
T
r compounds 1, 2, 14-18, 20, 23, 56, 59, and 63
are shown in Table 4.
and 2-hydroxy-3-nitropyridine 30 were reacted in THF, then the
5
. Calculation of molecular physiochemical properties
solvent
was
removed.
The
resultant o solid
and
bromoacetophenones 35 or 36 were heated at 140 C for 20 hrs to
give nitropyridones 41-42 in moderate yield (44-63%)
Compounds (5-29, 49-63) (Schemes 1 and 2) were converted
to their SMILES-codes. Numerical values for lipophilicity (Log
P), solubility (Log S), polar surface area (PSA), number of H-
bond donors (#OHNH), number of oxygen and nitrogen atoms
(#ON), and number of rotatable bonds (#RB) were calculated
with ALOGPS 2.1 [44], and Molinspiration [45] are listed for the
compounds 1-3, 7, 8, 12, 14-24, 49-63 (Tables 1-3 and Table S1).
The compounds in Tables 1-3 and Table S1 have a predicted Log
P <5 in accordance with Lipinski’s rules [46,47], and LogP < 3.5
Reduction of nitropyridones 37-42 by hydrogenation over
palladium on carbon at atmospheric pressure gave
aminopyridones 43-48, which were unstable and used directly in
the next step. Reductive amination using NaBH(OAc)3,
aminopyridones 43-48, and an aldehyde (benzaldehyde, 3,4-
dihlorobenzaldehyde, ortho-chlorobenzaldehyde, meta-chloro
benzaldehyde,
para-chlorobenzaldehyde,
dihydrocinnam
(compounds 2, 3, 7, 8, 12, 18, 19, 20, 21, 22, 24, 49, 50) the
aldehyde; whereas cinnamaldehyde remained unreacted) gave the
target compounds 50-63 in low to moderate yields (5-32%). This
was due to the high instability of the intermediate
aminopyridones 43-48. Again a selected benzoyl derivative was
prepared. Benzoylation of aminopyridone 44 with benzoyl
chloride in the presence of triethylamine gave 49 (30%).
preferred value for lead–like compounds [48]. All compounds in
Tables 1-3 and Table S1 have molecular weights <500 (except
for 23) and a TPSA under 120 Å [49]. The majority of
compounds do not violate the ‘rule of five’ (#OHNH ≤ 5; #ON
<
10; #RB <8); with 12, 22, 23, 52, 53, 56-58, 62, and 63 having
the number of rotatable bonds (an important predictor of good
oral bioavailability [49-51]) ≥8. Compounds could be classified
into those with lower calculated solubility from the interval
between 4 and 100 mg/L [50] (compounds 1-3, 7, 8, 12, 18-24,
All compounds were purified by chromatography and
recrystallization and 1 characterized (Microanalysis, mass
13
spectrometry, FTIR, H, and C NMR spectroscopy). To
facilitate discussion, these derivatives will be referred to by the
left (LHS), followed by the right hand side (RHS) substituents;
for example, 4-chlorobenzyl 3,4-dichlorobenzyl pyridone 16.
4
9, 51-54, 56, 62 and 63), and those with lower calculated
solubility of < 4 mg/L (compounds 14-17, 55, 57-61). However
apparent solubility observed in the bioassay (Table 1) varied
from the LogP predictions. It can also be noted that there were no
correlations between TPSA and GPa inhibition levels, illustrated
by compounds 19 (TPSA 63.01; 67% at 222 µM), 18 (TPSA
63.01; IC50 = 10.2µM), and 23 (TPSA 102.2; IC50 = 3.4 µM)
(Tables 1 and 2).
3
. X-ray Structures of 13 and 37
In conjunction with the synthetic work, pyridone ester 13 (not
active against GPa), and nitro pyridone 37 formed crystals
suitable for single crystal X-ray diffraction studies (see
supplementary data). The ORTEP-3 diagram of the molecular
structures of pyridone ester 13 and nitro pyridone 37 are shown
in Figures 2 and 3.
6
. Results and discussion
The preliminary lead compound 1 [32] upon which this study
was based suggested that inclusion of a 3,4-dichlorobenzyl group
at position (c) (Figure 1) and a methylene group at position (a)
(Figure 1) were favored moieties for GPa inhibitory activity. A
library of 29 compounds was generated which explored structural
variations of compound 1 and were multiply grouped as sub-sets
based on:
4
. Biology
GP activity of compounds 5-29, 49-63 was measured, using
the in vitro GP screen reported in other recent studies [34,38], in
the direction of glycogen synthesis [39] by the formation of
inorganic phosphate from glucose-1-phosphate [40,41]. The
results for compounds that displayed levels of inhibition of GP
are listed in Tables 1-3 and Table S1 (supplementary data). By
comparison, a typical IC50 of 229 ± 2, 283 ± 10 or 477 ± 14 µM
was obtained for the caffeine standard. In addition to compounds
(i)
Creation of analogues of 1 with a 3,4-dichlorobenzyl
group at position (c) and explore aryl variation at position
(b): Compounds 20-24.
2
1 (Table 2), 50 (Table 1) and 61 (Table 3), the compounds not
(ii) Selected benzoyl analogs (27-29 and 49) as ‘structural
included in Tables 1-3 and Table S1 (compounds 5, 6, 9-11, 13,
and 25-29), did not inhibit GPa at a maximal concentration used
in the assay (i.e., <20% at 222 µM). An apparent IC50 inhibitory
concentration was recorded at 50% inhibition for assays reaching
controls’ for the GPa inhibition assay
(
iii) Lengthening the methylene linker at position (b) or
position (c): Compounds 52, 53, 56-58, 62 and 63.
≥
90% inhibition, where compounds with a Hillslope > 5.0 were
(iv) Shortening the methylene linker at position (b) or position
included in Table S1 (compounds 22, 49, 55 and 57;
supplementary data). The high Hillslope values suggest that
analogues 22, 49, 55 and 57 may have formed precipitates in the
assay (not observable by eye) leading to turbidity and creating
false positives due to light scattering interference in the
colorimetric inhibition assay. Thus these compounds were
excluded from Tables 1-3 and only referenced in the SAR
analysis.
(c): Compounds 50, 51, 54, 55, 59, 60 and 61.
(
v)
Variation of the aryl chloro substitution at position (b) or
c) or (b) and (c): Compounds 14-24 and 51-61.
(
The library of 29 target compounds gave rise to 23 compounds
that inhibited GPa (between 33% at 4.40 mM and an IC50 of 1.92
µM); an initial hit rate of 79% for the library.
In the previous study, the 3,4-dichlorobenzyl substituent had
only been used at position (c) with benzyl at position (b). The
,4-dichlorobenzyl substituent is present in successful drugs,
such as the antidepressant Zoloft (Sertraline), the most prescribed
antidepressant in the U.S. in 2013 [52]. In the present study,
analogs of 1 with the 3,4-dichlorobenzyl substituent at position
The compounds with defined activity against GPa and
meeting acceptable Hillslope values were analysed for ligand
efficiency (LE) [42] and ligand-efficiency-dependent lipophilicity
3
(
LELP) [43] to provide further assessment of the quality of the
hits obtained from the GPa screening. The calculated values for
(c) and a range of aryl substituents at position (b) (compounds

2
0-24) (Table 2) were screened against GPa.
by these derivatives was variable; for example N-tosyl-pyrrole
3, displayed a potent activity with an IC50 of 3.4 µM, whereas
A
Inh
C
ib
C
iti
E
on
P
o
T
f
E
G
D
Pa MAEl
N
se
U
wh
S
er
C
e a
R
l
I
o
P
wer limit of LE (0.3) and a range of -10 < LELP <
T
10 for LELP has been discussed [42]. Upon consideration of the
values for LE and LELP in combination with the potency
observed, as compared to the previous lead 1 (IC50 = 6.3 µM),
two tiers of hits were proposed for the compounds in Table 4.
Compounds 15, 16 and 17 were considered to be Tier One hits
(IC50 < 6.3 µM and LE ≥ 0.25), with compounds 1, 14, 18, 20
and 23 being Tier Two hits (IC50 < 20 µM and LE ≤ 0.25). The
remaining compounds in Table 4 had an IC50 > 20 µM. Thus the
lead compounds with improved potency were narrowed to para-
chlorobenzyl 3,4-dichlorobenzyl 16 and 3,4-dichlorobenzyl 3,4-
dichlorobenzyl 17 (Tier One hits).
2
pyridyl 21 was inactive, perhaps as a result of protonation of the
pyridyl nitrogen. We noted that the structural controls, benzoyl
(27 and 28) and urea (29) analogs were inactive or gave a
possible false positive result (49; see section 4 biology). This
result confirmed the previous SAR that incorporation of a C3-N
carbonyl group generally resulted in a decrease in inhibition of
GPa. Similarly, incorporation of other polar groups such as
sulfonyl at C3-N also resulted in a significant reduction in
inhibition of GPa (cf 1, IC50 6.3 µM and 24, 37% @ 222 µM)
It was interesting to note that the apparent length of the
pyridone analogs, as a function of shortening or lengthening the
methylene linkers between the substituent and N or C3-N of the
pyridone ring, had an impact on the potency of GPa inhibition.
Approximations for an extended conformation (ChemBio3D
Ultra 11.0) were used to aid comparison to benzyl 3,4-
dichlorobenzyl pyridone (1; est IC50 6.3 µM), which was ~19.5Å
in length. Increased length of an analog (eg. 52; ~20 Å and 62;
7
. Conclusion
The design, synthesis and testing of a second generation of 29
new pyridone amide derivatives resulted in 23 compounds that
inhibited GPa (between 33% at 4.4 mM and an IC50 of 1.92 µM).
The hit rate from this second generation library was high with 13
compounds (hit rate 45%) displaying IC50 values < 70 µM. Two
new lead compounds 16 and 17 (IC =2.1 and 1.92 µM
respectively) from the dichlorobenzyl pyridone class of GPA
inhibitors have been identified. Both were more potent that the
50
~
21.5 Å) gave decreased inhibition levels of GPA values to > 60
µM (63; IC50 = 68µM) or negligible inhibition of GPa (52, 53, 58
and 62; 22-93%) (Tables 1 and 2). Similarly, shorter analogs (eg.
previously reported 1 (IC =6.3 µM). The sensitivity of GPa to
50
5
1; ~ 16 Å) decreased GPa inhibition values to >20 µM (59),
negligible (51, 54 and 60; 19-41%) or not active (50 and 61)
Tables 1-3). However, specific non-extended conformations of a
the length of the analogues suggests that the binding site has
limited spatial tolerance. The SAR suggests that GPa has a
sufficiently large hydrophobic binding pocket to accommodate
two 3,4-dichlorobenzyl substituents, or there are two separate
hydrophobic binding pockets, each of which can accommodate a
dichlorobenzyl group. The improvement in potency and
increased SAR understanding supports further studies on
dichlorobenzyl pyridones as a class of GPa inhibitors.
(
compound are likely to further promote favorable interactions
with GPa in situ, where the length/floppiness of a compound will
impact accordingly.
In conjunction with the screening of the final target compounds,
ester intermediates 5-12 and 25 (Scheme 1) were also screened.
Interestingly, three of the ester intermediates showed marginal
inhibitory activity against GPa; 7 (75% @ 222µM), 8 (43% @
8. Experimental Section
8
.1 General
2
22µM), and 12 (75% @ 222µM; Table S1). The left-hand side
functional groups were present in target compounds with GPa
inhibition (e.g. compounds 16, 17 and 23). The left hand side of
esters 7, 8 and 12 may be favoured for either their lipophilic
properties, possible π-stacking with an aromatic ring, or their
steric fit with GPa.
All reagents were purchased from commercial suppliers and
used without further purification. Column chromatography was
performed using silica gel 60 Å (0.040-0.063 mm). Analytical
thin layer chromatography (TLC) was performed using
aluminium plates coated with silica gel 69 F254 (0.2 mm), and
visualized by means of ultra-violet irradiation (254 nm) or
vanillin dip. Melting points were measured on a variable
temperature apparatus by the capillary o method and are
uncorrected. Temperatures are reported in C. High resolution
mass spectroscopy (HRMS) was performed on a Fourier
Transform Mass Spectrometer equipped with an electrospray
source (ESI-FTMS). Mass spectra 1were recorded using
Testing the preference for the 3,4-dichlorobenzyl substituent at
positions (b) and (c) was illustrated by comparison of 3,4-
dichlorobenzyl propyl 19 (67% inhibition @ 222µM) and 3,4-
dichlorobenzyl benzyl 18 (IC50 = 10.2 µM) to benzyl 3,4-
dichlorobenzyl 1 (IC50 = 6.3 µM) and benzyl benzyl 2 (IC 34.2
50
µM) (Table 1). Incorporation of the 3,4-dichlorobenzyl moiety at
the RHS (position c) improved inhibition levels of the
compounds against GPa. Further exploration of the chloro
substitution at the aryl substituent at the LHS (position b)
revealed a preference by GPa for derivatives with meta 15, para
13
electrospray as the ionization technique. H and C NMR spectra
were recorded in DMSO-d at 300 or 400 MHz. Coupling
6
constants J are valued in Hertz (Hz). Chemical shifts are
reported in parts per million, using the appropriate signal for
solvent as a reference. IR spectra were recorded on a FT-IR
Spectrometer as KBr discs for solids or as neat sample for
1
6 or meta and para 17, rather than ortho chloro substituents
(
Table 3). The improved activity of the bis-dichloro analogue 17
suggests either the GPa binding pocket is sufficiently large to
accommodate two dichlorobenzyl substituents (for a folded
conformation of 17; Figure 4A), or there are two separate
hydrophobic binding pockets (for an extended conformation of
-
1
oils. Absorption maxima are reported in wavenumbers (cm ).
Where given, systematic compound names are those
generated by ChemDraw Ultra 11.0 following IUPAC
conventions (Supplementary Data; Note on compound
nomenclature).
1
7; Figure 4B). The even larger N-(p-tosyl)-pyrrole is also
readily accommodated (23, IC50 = 3.4 µM). Despite the ability of
the hydrophobic pocket(s) to accommodate relatively large
substituents, the sensitivity of GPa to the length of the analogues
indicates an analogue-enzyme interaction that has specific spatial
constraints.
The synthesis of the following compounds are reported in the
Supplementary data: Esters 5-13, 25 and 26, Bromoacetamides
1-36; 3-Nitropyridones 37-42; 3-Aminopyridones 43-48.
3
8
.2 Experimental Procedures
The Ligand Efficiency (LE) and Ligand-Efficiency-dependent
Lipophilicity (LELP) were calculated for the compounds in
Tables 1-3 and compared with the IC50 values (Table 4).

o
8
.2.1 General procedure for Aminolysis at 120 C: Preparation of
8.2.1.4 N-(3'',4''-Dichlorobenzyl)-2-[3'-[(3''',4'''-dichlorobenzyl)
amino]-2'-oxopyridin-1'(2H)-yl]acetamide 17 Colourless solid
ACCEPTED MANUSCRIPT
compounds 14-18, 20-24, 27, and 28.
Aminolysis was carried out in a conical micro scale reaction
vessel by mixing amine (5 ꢀL amine per mg of ester) and ester
0.15-0.6 g). The reaction mixture was heated for 4 hours at 120
(
3
0.410 g, 100%). M.p 186-190 °C. FTIR (KBr) ν: 3354, 3256,
072, 1650, 1601, 1556, 719 cm . H NMR (400 MHz, DMSO-
-
1 1
(
d ) δ: 4.29 (t, 4H, J = 6.2 Hz, 1-NHCH , 3'-NHCH ), 4.59 (s, 2H,
6
2
2
o
C. Excess amine was removed under vacuum when possible, or
H2), 6.01 (dd, 1H, J = 6.8, 6.8 Hz, H5'), 6.06 (dd, 1H, J = 7.2, 1.6
Hz, H4'), 6.20 (t, 1H, J = 6.2 Hz, 3'-NH), 6.84 (dd, 1H, J = 6.8,
the reaction mixture was transferred while still warm to a conical
flask and diluted with diethyl ether. The diethyl ether mixture
was cooled in a freezer overnight (16 hours). Excess amine was
separated from the crude solid product by filtration and washing
with additional diethyl ether. The crude solid was recrystallized
by dissolving in hot acetone and then adding small amounts of
diethyl ether. The resulting solution was cooled in a freezer
overnight forming a white solid precipitate. The white precipitate
was filtered and washed with diethyl ether.
1
.6 Hz, H6'), 7.26-7.32 (m, 2H, H6'', H6'''), 7.53-7.58 (m, 4H,
13
H2'', H2''' and H5'', H5'''), 8.69 (t, 1H, J = 6.2 Hz, 1-NH).
C
NMR (100 MHz, DMSO-d ) δ: 41.1 (1-NHCH ), 44.8 (3'-
6
2
NHCH ), 51.5 (C2), 105.7 (C5'), 106.7 (C4'), 125.2 (C6'), 127.3
2
(
(
C6'' or C6'''), 127.5 (C6'' or C6'''), 128.9 (C2'' or C2'''), 129.0
C3'' or C3''' or C4'' or C4'''), 129.1 (C2'' or C2'''), 129.3 (C3'' or
C3''' or C4'' or C4'''), 130.3 (C5'' or C5'''), 130.4 (C5'' or C5'''),
30.88 (C3'' or C3''' or C4'' or C4'''), 130.9 (C3'' or C3''' or C4'' or
C4''') 137.4 (C3'), 140.5 (C1'''), 141.1 (C1''), 157.0 (C2'), 167.3
1
8
.2.1.1
2-[3'-[(2'''-Chlorobenzyl)amino]-2'-oxopyridin-1'(2H)-
yl]-N-(3'',4''-dichlorobenzyl)acetamide 14 Colourless solid
+
(
C1). ESI-MS m/z 492.2 (M+Li ). Anal. Calc. for C H O N Cl :
21 17 2 3 4
C, 51.99; H, 3.53; N, 8.66%. Found: C, 51.97; H, 3.34; N, 8.60%.
(
3
0.281 g, 100%). M.p 184-188 °C (dec.). FTIR (KBr) ν: 3399,
-
1
1
8
.2.1.5 N-Benzyl-2-[3'-[(3'',4''-dichlorobenzyl)amino]-2'-oxo
265, 3089, 1659, 1642, 1605, 756, 727 cm . H NMR (400
pyridin-1'(2H)-yl]acetamide 18 Colourless solid (0.334 g, 95%).
M.p 214-216 °C (dec.). FTIR (KBr) ν: 3334, 3269, 1663, 1642,
MHz, DMSO-d ) δ: 4.31 (d, 2H, J = 6.0 Hz, 1-NHCH ), 4.35 (d,
H, J = 6.0 Hz, 3'-NHCH ), 4.60 (s, 2H, H2), 5.99-6.06 (m, 3H,
H4', H5', NH), 6.86 (dd, 1H, J = 6.0, 2.4 Hz, H6'), 7.25-7.31 (m,
H, H3''', H4''', H5''', H6''), 7.42-7.46 (m, 1H, H6'''), 7.55 (d, 1H,
J = 2.0 Hz, H2''), 7.57 (d, 1H, J = 8.4 Hz, H5''), 8.70 (t, 1H, J =
.0 Hz, 1-NH). C NMR (100 MHz, DMSO-d ) δ: 41.1 (1-
NHCH ), 43.9 (3'-NHCH ), 51.5 (C2), 105.7 (C5'), 106.6 (C4'),
25.2 (C6'), 127.2 (C4'''), 127.5 (C6''), 128.5 (C3''' or C5'''), 128.6
C3''' or C5'''), 129.1 (C2''), 129.2 (C6'''), 129.3 (C4''), 130.4
6
2
2
2
-
1
1
1
4
1
597, 731, 694 cm . H NMR (400 MHz, DMSO-d ) δ: 4.28-
6
.31 (m, 4H, 3'-NHCH , 1-NHCH ), 4.58 (s, 2H, H2), 6.00 (dd,
H, J = 6.8, 6.8 Hz, H5'), 6.05 (dd, 1H, J = 7.8, 1.6 Hz, H4'), 6.23
4
2
2
13
(brt, 1H, J = 6.0 Hz, NH), 6.84 (dd, 1H, J = 6.8, 1.6 Hz, H6'),
6
6
7
.21-7.34 (m, 6H, Ph, H6''), 7.54-7.57 (m, 2H, H2'', H5''), 8.61
2
2
13
(brt, 1H, J = 6.0 Hz, 1-NH). C NMR (100 MHz, DMSO-d ) δ:
1
6
4
2.1 (3'-NHCH ), 44.8 (1-NHCH ), 51.4 (C2), 105.6 (C5'), 106.7
2 2
(
(
(
(
(
(
C4'), 125.2 (C6'), 126.8 (p-Ph), 127.2 (o-Ph), 127.3 (C6''), 128.2
m-Ph), 128.9 (C2''), 129.1 (C4''), 130.4 (C5''), 130.9 (C3''), 137.4
C3'), 139.1 (i-Ph), 141.1 (C1''), 157.0 (C2'), 167.0 (C1). ESI-MS
C5''), 130.9 (C3'') 132.1 (C1'''), 136.0 (C2'''), 137.6 (C3'), 140.5
C1''), 157.0 (C2'), 167.3 (C1). ESI-MS m/z 456.1 (M+Li ). Anal.
Calc. for C H O N Cl : C, 55.96; H, 4.03; N, 9.32%. Found: C,
+
21
18
2
3
3
+
+
+
m/z 438.1 (M+Na ), 416.2 (M+H ), 422.2 (M+Li ). Anal. Calc.
5
6.11; H, 3.92; N, 9.17%.
for C H O N Cl : C, 60.59; H, 4.60; N, 10.09%. Found: C,
21
19
2
3
2
8
.2.1.2 2-[3'-[(3'''-Chlorobenzyl)amino]-2'-oxopyridin-1'(2H)-
yl]-N-(3'',4''-dichlorobenzyl)acetamide 15 Colourless solid
0.402 g, 95%). M.p 175-178 °C (dec.). FTIR (KBr) ν: 3322,
252, 3052, 1642, 1585, 780, 727, 682 cm . H NMR (400 MHz,
DMSO-d ) δ: 4.29-4.31 (m, 4H, 1-NHCH , 3'-NHCH ), 4.60 (s,
H, H2), 6.00-6.07 (m, 2H, H5', H4'), 6.14 (t, 1H, J = 6.4 Hz, 3'-
NH), 6.83-6.85 (m, 1H, H6'), 7.25-7.34 (m, 5H, H6'', H2''', H4''',
H5''', H6'''), 7.54 (d, 1H, J = 1.0 Hz, H2''), 7.57 (d, 1H, J = 8.4
Hz, H5''), 8.70 (t, 1H, J = 6.0 Hz, 1-NH); C NMR (100 MHz,
DMSO-d ) δ: 41.1 (1-NHCH ), 45.4 (3'-NHCH ), 51.5 (C2),
05.7 (C5'), 106.6 (C4'), 125.0 (C6'), 125.7 (C2''' or C6'''), 126.6
C4''' or C5'''), 126.7 (C2''' or C6'''), 127.5 (C6''), 129.1 (C2''),
29.3 (C4''), 130.1 (C4''' or C5'''), 130.3 (C5''), 130.9 (C3''), 133.1
C3''') 137.6 (C3'), 140.5 (C1''), 142.3 (C1'''), 157.0 (C2'), 167.3
C1). ESI-MS m/z 456.1 (M+Li ). Anal. Calc. for C H O N Cl :
C, 55.96; H, 4.03; N, 9.32%. Found: C, 55.78; H, 3.92; N, 9.26%.
6
0.63; H, 4.66; N, 10.08%.
8
.2.1.6 N-(3'',4''-Dichlorobenzyl)-2-[2'-oxo-3'-[(2'''-thienyl
(
3
-
1 1
methyl)amino]pyridin-1'(2H)-yl]acetamide 20 Colourless solid
(
3
0.273 g, 96%). M.p 190-191 °C (dec.). FTIR (KBr) ν: 3314,
6
2
2
-1 1
265, 1646, 1585, 756, 698 cm . H NMR (400 MHz, DMSO-d )
2
6
δ: 4.29 (d, 2H, J = 6.0 Hz, 1-NHCH ), 4.46 (d, 2H, J = 6.0 Hz, 3'-
2
NHCH ), 4.58 (s, 2H, H2), 5.91 (t, 1H, J = 6.0 Hz, 3'-NH), 6.06
2
13
(dd, 1H, J = 6.8, 6.8 Hz, H5'), 6.25 (d, 1H, J = 6.8 Hz, H4'), 6.86
(d, 2H, J = 6.0 Hz, H6'), 6.95 (dd, 1H, J = 5.0, 3.6 Hz, H4'''), 7.03
(d, 1H, J = 3.6 Hz, H3'''), 7.27 (dd, 1H, J = 8.0, 1.2 Hz, H6''),
6
2
2
1
(
1
(
7
.36 (d, 1H, J = 5.0 Hz, H5'''), 7.54 (d, 1H, J = 1.2 Hz, H2'') 7.57
13
(d, 1H, J = 8.0 Hz, H5''), 8.70 (t, 1H, J = 6.0 Hz, 1-NH).
C
NMR (100 MHz, DMSO-d ) δ: 41.0 (1-NHCH ), 41.5 (3'-
6
2
+
NHCH ), 51.5 (C2), 105.6 (C5'), 107.0 (C4'), 124.6 (C5'''), 125.0
(
2
21
18
2
3
3
(
(
(
C3'''), 125.3 (C6'), 126.7 (C4'''), 127.5 (C6''), 129.1 (C2''), 129.2
C4''), 130.4 (C5''), 130.9 (C3''), 137.5 (C3'), 140.5 (C1'') 143.3
8
.2.1.3 2-[3'-[(4'''-Chlorobenzyl)amino]-2'-oxopyridin-1'(2H)-
yl]-N-(3'',4''-dichloro benzyl)acetamide 16 Colourless solid
0.277 g, 99%). M.p 213-218 °C (dec.). FTIR (KBr) ν: 3334,
260, 1650, 1597, 829, 702 cm . H NMR (400 MHz, DMSO-d )
δ: 4.27 (d, 2H, J = 6.4 Hz, 3'-NHCH ), 4.30 (d, 2H, J = 5.6 Hz, 1-
NHCH ), 4.58 (s, 2H, H2), 5.98-6.09 (m, 3H, H4', H5', 3'-NH),
.85 (dd, 1H, J = 6.4, 2.0 Hz, H6'), 7.28 (dd, 1H, J = 8.4, 2.0 Hz,
H6''), 7.31-7.37 (m, 4H, H2''', H3''', H5''', H6'''), 7.54 (d, 1H, J =
.0 Hz, H2''), 7.57 (d, 1H, J = 8.4 Hz, H5''), 8.68 (brt, 1H, J = 5.6
Hz, 1-NH). C NMR (100 MHz, DMSO-d ) δ: 41.1 (1-NHCH ),
5.3 (3'-NHCH ), 51.5 (C2), 105.7 (C5'), 106.6 (C4'), 125.0
C6'), 127.5 (C6''), 128.2 (C2''', C6'''), 128.8 (C3''', C5'''), 129.1
C2''), 129.2 (C4''), 130.4 (C5''), 130.9 (C3''), 131.2 (C4'''), 137.6
+
C2'''), 157.0 (C2'), 167.3 (C1). ESI-MS m/z 428.2 (M+Li ).
Anal. Calc. for C H O N SCl : C, 54.04; H, 4.06; N, 9.95%.
19
17
2
3
2
(
3
Found: C, 53.72; H, 4.17; N, 9.86%.
-1 1
6
8
.2.1.7 N-(3'',4''-Dichlorobenzyl)-2-[2'-oxo-3'-[(pyridin-2'''-yl
2
methyl)amino]pyridin-1'(2H)-yl]acetamide 21 Colourless solid
2
(
3
0.271 g, 93%). M.p 165-169 °C (dec.). FTIR (KBr) ν: 3412,
248, 3072, 1675, 1589, 760 cm . H NMR (400 MHz, DMSO-
6
-
1 1
d ) δ: 4.30 (d, 2H, J = 6.0 Hz, 1-NHCH ), 4.35 (d, 2H, J = 6.0
2
6
2
13
Hz, 3'-NHCH ), 4.60 (s, 2H, H2), 6.05 (dd, 1H, J = 7.0, 7.0 Hz,
2
6
2
H5'), 6.12 (dd, 1H, J = 7.2, 1.6 Hz, H4'), 6.20 (t, 1H, J = 6.0 Hz,
4
2
3
'-NH), 6.86 (dd, 1H, J = 6.8, 1.6 Hz, H6'), 7.24-7.27 (m, 1H,
(
(
(
H6''), 7.29 (d, 1H, J = 2.0 Hz, H5'''), 7.32 (d, 1H, J = 7.6 Hz,
H3'''), 7.55 (d, 1H, J = 1.6 Hz, H2''), 7.57 (d, 1H, J = 8.4 Hz,
H5''), 7.73 (ddd, 1H, J = 7.6, 7.6, 2.0 Hz, H4'''), 8.52-8.54 (m,
C3'), 138.5 (C1''') 140.6 (C1''), 157.0 (C2'), 167.3 (C1); ESI-MS
+
m/z 458.2 (M+Li ). Anal. Calc. for C H O N Cl : C, 55.96; H,
21
18
2
3
3
13
1
H, H6'''), 8.70 (t, 1H, J = 6.0 Hz, 1-NH). C NMR (100 MHz,
4
.03; N, 9.32%. Found: C, 55.96; H, 3.93; N, 9.22%.

DMSO-d ) δ: 41.1 (1-NHCH ), 47.9 (3'-NH
A
CH ),
C
C
E
51
P
.5
T
(
E
C
D
2), MA8.
N
2.1
oxoethyl}-2'-oxo-1',2'-dihydropyridin-3-yl)benzamide
Colourless solid (0.407 g, 98%). M.p 232-234 °C. FTIR (KBr) ν:
U
.1
S
1
C
2-
R
C
I
h
P
lo
T
ro-N-(1'-{2''-[(3''',4'''-dichlorobenzyl)amino]-2''-
6
2
2
1
1
1
1
05.8 (C5'), 106.7 (C4'), 121.2 (C3'''), 122.2 (C5'''), 125.1 (C6'),
27.5 (C6''), 129.1 (C2''), 129.2 (C3''), 130.4 (C5''), 130.9 (C4''),
36.7 (C4'''), 137.8 (C3'), 140.6 (C1'') 148.8 (C6'''), 157.0 (C2'),
27
-
1
1
3326, 3273, 3056, 2912, 1638, 1532, 768, 743, 657 cm . H
NMR (400 MHz, DMSO-d ) δ: 4.31 (d, 2H, J = 5.6 Hz, 2''-
NHCH ), 4.69 (s, 2H, H1''), 6.34 (dd, 1H, J = 6.8, 6.8 Hz, H5'),
7.26 (d, 1H, J = 8.4 Hz, H6'''), 7.42-7.57 (m, 6H, H3, H4, H5,
H6', H2''', H5'''), 7.63 (d, 1H, J = 7.2 Hz, H6), 8.32 (d, 1H, J =
+
58.2 (C2'''), 167.3 (C1). ESI-MS m/z 417.2 (M+Na ), 242.3 (M-
6
+
NHCH C H Cl ). HRMS +m/z: 417.0878; calc. for
C H O N Cl : 417.0880 (M+H ).
2
6
3
2
2
20
19
2
4
2
8
2
9
2
.2.1.8 N-(3'',4''-Dichlorobenzyl)-2-[3'-[(4'''-nitrobenzyl)amino]-
'-oxopyridin-1'(2H)-yl]acetamide 22 Yellow solid (0.197 g,
4%). M.p 191-193 °C (dec.). FTIR (KBr) ν: 3326, 3265, 3072,
6
.8 Hz, H4'), 8.78 (brt, 1H, J = 5.6 Hz, 2''-NH), 9.45 (s, 1H, 3'-
13
NH). C NMR (100 MHz, DMSO-d ) δ: 41.1 (2''-NHCH ), 51.9
(C1''), 104.8 (C5'), 124.0 (C4'), 127.3 (C3), 127.5 (C6'''), 128.0
6
2
-
1
1
929, 1663, 1646, 1597, 1520, 1344, 1250, 723 cm . H NMR
(
(
(
C3'), 129.1 (C2'''), 129.3 (C4'''), 129.5 (C6), 129.7 (C2), 129.9
C5'''), 130.3 (C5), 130.9 (C3'''), 131.7 (C4), 134.0 (C6') 135.4
C1), 140.4 (C1'''), 156.9 (C2'), 164.7 (3'-NHCO), 166.8 (C2'').
(
400 MHz, DMSO-d ) δ: 4.30 (d, 2H, J = 6.0 Hz, 1-NHCH ),
6
2
4
.43 (d, 2H, J = 6.4 Hz, 3'-NHCH ), 4.59 (s, 2H, H2), 5.97-6.02
2
(
=
1
(
m, 2H, H4', H5'), 6.28 (t, 1H, J = 6.4 Hz, 3'-NH), 6.84 (dd, 1H, J
6.0, 2.8 Hz, H6'), 7.28 (dd, 1H, J = 8.0, 2.0 Hz, H6''), 7.54 (d,
H, J = 2.0 Hz, H2''), 7.57 (d, 2H, J = 8.8 Hz, H2''', H6'''), 7.58
d, 1H, J = 8.0 Hz, H5''), 8.17 (d, 2H, J = 8.8 Hz, H3''', H5'''),
+
ESI-MS m/z 470.1 (M+Li ). Anal. Calc. for C H O N Cl : C,
21
16
3
3
3
5
4.27; H, 3.47; N, 9.04%. Found: C, 54.34; H, 3.33; N, 8.95%.
8.2.1.12 N-{1'-[2''-(Benzylamino)-2''-oxoethyl]-2'-oxo-1',2'-
dihydro pyridin-3'-yl}-2-chlorobenzamide 28 Colourless solid
(0.311 g, 88%). M.p 196-198 °C. FTIR (KBr) ν: 3330, 3273,
13
8
.69 (t, 1H, J = 6.0 Hz, 1-NH). C NMR (100 MHz, DMSO-d6)
δ: 41.1 (1-NHCH ), 45.5 (3'-NHCH ), 51.5 (C2), 105.6 (C5'),
2
2
-
1
1
1
06.7 (C4'), 123.5 (C3''', C5'''), 125.2 (C6'), 127.5 (C6''), 128.0
3074, 1646, 1589, 1524, 743, 694, cm . H NMR (400 MHz,
(C2''', C6'''), 129.1 (C2''), 129.2 (C4''), 130.4 (C5''), 130.9 (C3''),
DMSO-d ) δ: 4.21 (d, 2H, J = 5.6 Hz, CH ), 4.69 (s, 2H, H1''),
6
2
1
1
37.5 (C3'), 140.6 (C1''), 146.4 (C4'''), 148.1 (C1'''), 157.0 (C2'),
6.33 (dd, 1H, J = 7.2, 7.2 Hz, H5'), 7.23-7.33 (m, 5H, Ph), 7.42-
7.45 (m, 2H, H6', H4), 7.48-7.55 (m, 2H, H3, H5), 7.63 (dd, 1H,
J = 7.6, 1.6 Hz, H6), 8.32 (d, 1H, J = 6.4 Hz, H4'), 8.70 (brt, 1H,
+
+
67.3 (C1). ESI-MS m/z 483.1 (M+Na ), 461.1 (M+H ), 467.1
+
(M+Li ). HRMS m/z: 461.0794; calc. for C H O N Cl :
21
19
4
4
2
+
13
4
61.0778 (M+H ).
J = 5.6 Hz, 2''-NH), 9.48 (s, 1H, 3'-NH). C NMR (100 MHz,
DMSO-d ) δ: 42.2 (CH ), 51.7 (C1''), 104.7 (C5'), 124.0 (C4'),
6
2
8
.2.1.9 N-(3'',4''-Dichlorobenzyl)-2-[2'-oxo-3'-(1-{[(para-methyl
1
26.8 (p-Ph), 127.2 (o-Ph), 127.3 (C3), 128.0 (C3'), 128.3 (m-
Ph), 129.5 (C6), 129.7 (C2), 129.8 (C5), 131.6 (C4), 134.0 (C6')
35.5 (C1), 139.0 (i-Ph), 156.9 (C2'), 164.8 (3'-NHCO), 166.5
phenyl)sulfonyl]-1H-pyrrol-2'''-ylmethyl}amino)pyridine-1'(2H)-
yl]acetamide 23 Colourless solid (0.781 g, 100%). M.p 129-133
1
°
C (dec.). FTIR (KBr) ν: 3334, 3220, 3056, 1679, 1581, 1360,
+
+
-
1
1
(C2''). ESI-MS m/z 418.1 (M+Na ), 396.1 (M+H ); 402.3
1
3
6
168, 735, 700 cm ; H NMR (400 MHz, DMSO-d ) δ: 2.35 (s,
+
6
(M+Li ). Anal. Calc. for C H O N Cl: C, 63.72; H, 4.58; N,
21 18 3 2
H, CH ), 4.30 (d, 2H, J = 6.0 Hz, 1-NHCH ), 4.34 (d, 2H, J =
3
2
1
0.62%. Found: C, 63.76; H, 4.50; N, 10.51%.
.4 Hz, 3'-NHCH ), 4.57 (s, 2H, H2), 5.70 (t, 1H, J = 6.4 Hz, 3'-
2
NH), 5.90 (dd, 1H, J = 7.2, 1.6 Hz, H4'), 5.95 (dd, 1H, J = 7.0,
.0 Hz, H5'), 6.16-6.17 (m, 1H, H4'''), 6.23 (dd, 1H, J = 3.2, 3.2
Hz, H3'''), 6.83 (dd, 1H, J = 6.8, 1.6 Hz, H6'), 7.27 (dd, 1H, J =
8.2.2 General procedure for Aminolysis at room temperature:
Preparation of compounds 19 and 29.
7
Aminolysis was carried out in a conical micro scale
reaction vessel by mixing amine (5 ꢀL amine per mg of ester)
and ester (0.2 or 1.18g). The reaction mixture was stirred for 1
hour at room temperature. Excess amine was removed under
vacuum when possible, or the reaction mixture was transferred to
a conical flask and diluted with diethyl ether. The diethyl ether
mixture was cooled in a freezer overnight (16 hours). Excess
amine was separated from the crude solid product by filtration
and washing with additional diethyl ether. The crude solid was
recrystallized by dissolving in hot acetone and then adding small
amounts of diethyl ether, the resulting solution was cooled in a
freezer overnight forming a white solid precipitate. The white
precipitate was filtered and washed with diethyl ether.
8
2
.0, 2.0 Hz, H6''), 7.34 (dd, 1H, J = 3.6, 2.0 Hz, H5'''), 7.40 (d,
H, J = 8.4 Hz, m-Ph), 7.54 (d, 1H, J = 2.0 Hz, H2''), 7.56 (d, 1H,
J = 8.0 Hz, H5''), 7.79 (d, 2H, J = 8.4 Hz, o-Ph), 8.69 (t, 1H, J =
1
3
6
4
1
1
.0 Hz, 1-NH); C NMR (100 MHz, DMSO-d ) δ: 21.0 (CH ),
6 3
1.0 (3'-NHCH ), 41.0 (1-NH-CH ), 51.5 (C2), 105.5 (C5'),
2
2
06.8 (C4'), 111.9 (C4'''), 114.0 (C3'''), 123.1 (C5'''), 125.3 (C6'),
26.7 (o-Ph), 127.5 (C6''), 129.1 (C2''), 129.2 (C4''), 130.3 (m-
Ph), 130.4 (C5''), 130.9 (C3''), 131.7 (C2'''), 135.2 (i-Ph), 137.1
C3'), 140.5 (C1''), 145.4 (p-Ph), 156.9 (C2'), 167.3 (C1). ESI-
(
+
+
+
MS m/z 581.1 (M+Na ), 559.1 (M+H ); 565.1 (M+Li ). HRMS
m/z: 559.0979; calc. for C H O N SCl : 559.0968 (M+Na ).
+
26
25
4
4
2
8
.2.1.10
sulfonyl]amino}-2'-oxopyridin-1'(2H)-yl]acetamide
Colourless solid (0.411 g, 100%). M.p 248-250 °C (dec.). FTIR
N-(3''',4'''-Dichlorobenzyl)-2-[3'-{[(4''-methylphenyl)
24
8.2.2.1
2-[3'-[(3'',4''-Dichlorobenzyl)amino]-2'-oxopyridin-
1'(2H)-yl]-N-propylacetamide 19 Colourless solid (0.201 g,
97%). M.p 193-195 °C (dec.). FTIR (KBr) ν: 3322, 3256, 3081,
-
(
KBr) ν: 3403, 3126, 1691, 1646, 1581, 1152, 882, 821, 751 cm
1
1
-1 1
.
H NMR (400 MHz, DMSO-d ) δ: 2.32 (s, 3H, CH ), 4.27 (d,
2968, 1646, 1581, 1483, 756 cm . H NMR (400 MHz, DMSO-
) δ: 0.85 (t, 3H, J = 7.4 Hz, 1-NHCH CH CH ), 1.41 (tq, 2H, J
= 7.2, 7.2 Hz, 1-NHCH CH ), 3.02 (dt, 2H, J = 6.8, 6.0 Hz, 1-
NHCH ), 4.28 (d, 2H, J = 6.4 Hz, 3'-NHCH ), 4.49 (s, 2H, H2),
6
3
2
7
4
1
1
H, J = 6.0 Hz, NHCH ), 4.56 (s, 2H, H2), 6.16 (dd, 1H, J = 7.2,
d
6
2
2
3
2
.2 Hz, H5'), 7.25 (dd, 1H, J = 8.0, 2.0 Hz, H6'''), 7.30-7.34 (m,
H, H4', H6', H3'', H5''), 7.50 (d, 1H, J = 2.0 Hz, H2'''), 7.56 (d,
H, J = 8.4 Hz, H5'''), 7.75 (d, 2H, J = 8.0 Hz, H2'', H6''), 8.73 (t,
2
2
2
2
5.98 (dd, 1H, J = 6.8, 6.8 Hz, H5'), 6.04 (dd, 1H, J = 7.2, 1.6 Hz,
H4'), 6.19 (t, 1H, J = 6.4 Hz, 3'-NH), 6.79 (dd, 1H, J = 6.8, 1.6
Hz, H6'), 7.30 (dd, 1H, J = 8.0, 1.6 Hz, H6''), 7.55 (d, 2H, J = 8.0
Hz, H5''), 7.56 (d, 1H, J = 1.6 Hz, H2''), 8.07 (t, 1H, J = 5.4 Hz,
13
H, J = 6.0 Hz, 3'-NH), 9.00 (brs, 1H, Wh ~ 16 Hz, 1-NH).
C
½
NMR (100 MHz, DMSO-d ) δ: 21.0 (CH ), 41.1 (NHCH ), 51.6
6
3
2
(C2), 104.3 (C5'), 124.1 (C4'), 126.8 (C2'', C6''), 127.3 (C3'),
1
3
1
27.5 (C6'''), 129.1 (C2'''), 129.3 (C4'''), 129.6 (C3'', C5''), 130.4
1-NH).
NHCH
(3'-NHCH
C
NMR (100 MHz, DMSO-d
CH ), 22.3 (1-NHCH CH ), 40.4 (1-NHCH
), 51.2 (C2), 105.5 (C5'), 106.6 (C4'), 125.2 (C6'),
2
6
)
δ: 11.4 (1-
(
C5'''), 130.9 (C3'''), 134.5 (C6'), 137.0 (C1''), 140.4 (C1''') 143.4
2
CH
2
3
2
2
2
), 44.8
+
(C4''), 156.9 (C2'), 166.7 (C1). ESI-MS m/z 502.0 (M+Na ),
+
4
4
80.1 (M+H ). HRMS m/z: 480.0523; calc. for C H O N Cl S:
127.3 (C6''), 128.9 (C2''), 129.1 (C4'') 130.4 (C5''), 130.9 (C3''),
137.4 (C3'), 141.1 (C1''), 156.9 (C2'), 166.7 (C1). ESI-MS m/z
21
20
4
3
2
+
80.0546 (M+H ).

+
3
3
74.2 (M+Li ). HRMS m/z: 368.0918; calc. f
A
or
C
C
C
H
EP
O
T
N
E
C
D
l : MA
H
N
6')
U
, 7
S
.2
C
0
R
(t,IP1H
T
, J = 5.5 Hz, p-C H ), 7.20-7.25 (m, 1H, p-
1
7
20
2
3
2
6 5
+
68.0927 (M+H ).
CH C H ), 7.30-7.40 (m, 5H, m-C H , m-CH C H , o-CH C H )
2
6
5
6
5
2
6
5
2
6
5
1
3
7
.45-7.62 (m, 3H, o-CH C H , o-C H ) 10.30 (s, 1H, CONH).
C
2
6
5
6
5
8
-
1
1
3
.2.2.2 2-[3'-[(Anilinocarbonyl)amino]-2'-oxopyridin-1'(2H)-yl]
N-propylacetamide 29 Colourless solid (1.228 g, 100%). M.p
93-196 °C (dec.). FTIR (KBr) ν: 3326, 3293, 1646, 1581, 1548,
499, 751, 694 cm . H NMR (400 MHz, DMSO-d ) δ: 0.85 (t,
H, J = 7.4 Hz, CH ), 1.42 (tq, 2H, J = 7.2, 7.2 Hz, 1-
NHCH CH ), 3.04 (dt, 2H, J = 6.8, 6.0 Hz, NHCH ), 4.58 (s, 2H,
H2), 6.23 (dd, 1H, J = 7.2, 7.2 Hz, H5'), 6.94-6.97 (m, 1H, p-Ph),
.20 (dd, 1H, J = 6.8, 1.6 Hz, H6'), 7.26 (t, 2H, J = 7.8 Hz, m-
Ph), 7.43 (d, 2H, J = 8.0 Hz, o-Ph), 8.07 (dd, 1H, J = 7.6, 1.6 Hz,
H4'), 8.14 (brt, 1H, J = 6.0 Hz, 1-NH), 8.55 (s, 1H, 3'-NH), 9.51
s, 1H, 3'-NHCONH). C NMR (100 MHz, DMSO-d ) δ: 11.3
CH ), 22.3 (1-NHCH CH ), 40.4 (1-NHCH ), 51.6 (C2), 105.1
C5'), 117.9 (o-Ph), 119.1 (C4'), 121.9 (p-Ph), 128.8 (m-Ph),
29.7 (C3'), 130.8 (C6'), 139.6 (i-Ph), 152.4 (CO), 156.7 (C2'),
66.4 (C1). ESI-MS m/z 351.3 (M+Na ); 329.3 (M+H ); 335.3
M+Li ). Anal. Calc. for C H O N : C, 62.18; H, 6.14; N,
NMR (100 MHz, DMSO-d ) δ: 46.0 (3'-NHCH ), 51.9 (C2),
6
2
1
05.7 (C4'), 106.6 (C5'), 118.9 (o-C H ), 123.3 (p-C H ), 124.9
6 5 6 5
-
1
1
(C6'), 126.7 (p-CH C H ), 127.0 (o-CH C H ) 128.3 (m-C H ),
2 6 5 2 6 5 6 5
6
1
28.8 (m-CH C H ), 137.8 (C3'), 138.8 (i-C H ), 139.4 (i-
2 6 5 6 5
+
3
CH C H ), 157.0 (C2'), 165.7 (C1). ESI-MS m/z 355.9 (M+Na ).
2
6
5
2
2
2
+
HRMS m/z: 356.1352; calc. for C H N O : 356.1369 (M+Na ).
20
19
3
2
7
8.2.4.2 2-(3'-(Benzylamino)-2'-oxopyridin-1' (2''H)-yl)-N-(3',4'-
dichlorophenyl)acetamide 51 Pale blue solid (80 mg, 18 %). M.p.
195-196 °C. FTIR (KBr) ν: 3290, 3100, 1659, 1524, 760, 698,
623 cm . H NMR (400 MHz, DMSO-d ) δ: 4.30 (d, J = 5.0 Hz,
2H, 3'-NHCH ), 4.75 (s, 2H, H2), 6.00-6.10 (m, 3H, H4', H5', 3'-
NH), 6.83 (d, 1H, J = 5.0 Hz, H6’), 7.20-7.25 (m, 1H, p-
CH C H ), 7.30-7.40 (m, 4H, o-CH C H , m-CH C H ), 7.45-
7.60 (m, 2H, H5'', H6'') 7.95-8.99 (m, 1H, H2''), 10.63 (s, 1H, 1''-
13
-1 1
(
(
(
1
1
6
6
3
2
2
2
2
2
6
5
2
6
5
2
6
5
+ +
+
13
(
NH). C NMR (100 MHz, DMSO-d ) δ: 46.0 (3'-NHCH ), 52.0
17
20
3
4
6
2
1
7.06%. Found: C, 62.27; H, 6.18; N, 17.16%.
(C2), 105.8 (C4'), 106.6 (C5'), 118.9 (C''), 120.1 (C''), 124.7 (C''),
25.1 (C6'), 126.7 (p-CH C H ), 126.9 (o- CH C H ), 128.3 (m-
1
2
6
5
2
6
5
8
.2.3 Preparation of N-{1-[2'-(3'',4''-Dichlorophenylamino)-2'-
CH C H ), 130.8 (C''), 131.0 (C''), 137.8 (C3'), 138.8 (C''), 139.4
2
6
5
oxoethyl]-2-oxo-1,2-dihydropyridin-3-yl}benzamide 49
(
1
4
i-CH C H ), 157.0 (C2’'), 166.3 (C1). ESI-MS m/z 123.83,
2 6 5
+
Benzoyl chloride (0.14 mL, 1.5 equiv) was added to a solution
25.84 (M+Na ). HRMS m/z: 402.0766; calc. for C H Cl N O :
20 18 2 3 2
+
of compound 44 (250 mg, 1.0 equiv) and Et N (0.17 mL, 1.5
3
02.0771 (M+H ).
equiv) in DCM (anhydrous) under nitrogen. The resulting
solution was stirred 16 hrs at rt, and then concentrated under
reduced pressure. The crude product was purified by silica gel
8.2.4.3 N-(3',4'-dichlorophenethyl)-2-(3''-(benzylamino)-2''-oxo
pyridin-1'' (2H)-yl)acetamide 52 Pale green solid (90 mg, 20 %).
M.p. 140-146 °C. FTIR (KBr) ν: 3246, 3392, 3077, 1642, 1529,
column chromatography (DCM/Et N, 99.5:0.5).
3
-
1
1
7
1
64, 692, 624 cm . H NMR (300 MHz, DMSO-d ) δ: 1.60 (s,
6
8
.2.3.1
N-{1-[2'-(3'',4''-Dichlorophenylamino)-2'-oxoethyl]-2-
H, 3''-NH), 2.72 (t, 2H, J = 6.8 Hz, 1-NHCH CH ), 3.25-3.30
2
2
oxo-1,2-dihydropyridin-3-yl}benzamide 49 Pale yellow gum (100
mg, 30 %). FTIR (KBr) ν: 3249, 3070, 1640, 1528, 764, 690, 623
cm . H NMR (400 MHz, DMSO-d ) δ: 4.85 (s, 2H, 1'CH ), 6.40
(
m, 2H, 1-NHCH ), 4.30 (d, 2H, J = 4.3 Hz, 3''-NHCH ), 4.50 (s,
2
2
-
1
1
2H, H2), 5.97-6.11 (m, 2H, H4'', H5''), 6.75 (d, 1H, J = 4.0 Hz,
6
2
H6'), 7.18-7.29 (m, 2H, H2', H5'), 7.30-7.40 (m, 5H, C H ), 7.53
6
5
(t, 1H, J = 5.5 Hz, H4), 7.40-7.65 (m, 6H, p-C H , m-C H , H5,
13
6
5
6
5
(d, 1H, J = 6.0 Hz, H6''), 8.30-8.40 (m, 1H, 1-NH). C NMR (75
H6, H6''), 7.80-8.00 (m, 2H, o-C H , H''), 8.30 (dd, 1H, J = 0.6,
7
6
5
1
3
MHz, DMSO-d ) δ: 33.9 (1-NHCH CH ), 38.8 (1-NHCH ), 46.0
6
2
2
2
.5 Hz, H2'') 9.30 (s, 1H, 3-NH), 10.75 (s, 1H, 1''-NH). C NMR
(
3''-NHCH ) 51.1 (C2), 105.6 (C4''), 106.3 (C5''), 124.7 (C6'),
2
(100 MHz, DMSO-d ) δ: 52.5 (C2'), 105.1 (C4), 119.0 (C5),
6
1
1
1
26.7 (C2'), 126.9 (o-C H ), 127.3 (p-C H ), 128.3 (m-C H ),
28.7 (C5'), 129.3 (C4'), 130.3 (C3'), 130.7 (C6''), 137.8 (C1'),
6
5
6
5
6
5
1
20.2 (C6''), 124.9 (C2''), 127.1 (C6), 128.0 (C4'') 128.4 (p-
CH C H ), 128.7 (o-CH C H ), 129.2 (m-CH C H ), 130.8 (C5''),
1
2
6
5
2
6
5
2
6
5
39.3 (C3''), 140.7 (i-C H ), 156.9 (C2''), 166.9 (C1). ESI-MS
6
5
32.6 (C3''), 133.6 (C3), 138.7 (C1''), 157.1 (i-CH C H ), 164.8
+
2
6
5
m/z 452.05, 454.05 (M+Na ). HRMS m/z: 468.0658; calc. for
C H21Cl N O : 468.0642 (M+Na ).
(C2), 165.9 (3-NHC=O), 167.3 (C1'). ESI-MS m/z 437.81
+
+
+
22 2 3 2
(M+Na ), 415.82 (M+H ). HRMS m/z: 438.0367; calc. for
+
C H Cl N O : 438.0383 (M+Na ).
8.2.4.4 N-(2',4'-Dichlorophenethyl)-2-(3''-(benzylamino)-2''-oxo
pyridin-1'' (2H)-yl)acetamide 53 Pale blue solid (99 mg, 21 %).
M.p. 166-170 °C. FTIR (KBr) ν: 3246, 3392, 3077, 1642, 1529,
20
15
2
3
3
8
.2.4 General procedure for reductive amination: Preparation of
-
1 1
7
64, 692, 624 cm . H NMR (300 MHz, DMSO-d ) δ: 2.76-2.92
6
compounds 50-63
(
m, 2H, 1-NHCH CH ), 3.23-3.40 (m, 2H, 1-NHCH ), 4.28 (d,
The amine (1.0 equiv) was dissolved in DCE (anhydrous) and
the aldehyde (1.1 equiv) was added under an atmosphere of
nitrogen. The solution was stirred at rt for 16 hrs then
NaBH(OAc) (2 equiv) was added. The resulting suspension was
stirred for 16 hrs. The reaction was quenched with K CO (1 M)
and stirred vigorously for 30 mins before the phases were
separated. The aqueous phase was extracted with DCM. The
combined organic layers were washed (brine), dried (anhydrous
MgSO ) and concentrated under reduced pressure. The crude
product was purified by silica column chromatography (as
2
2
2
2
H, J = 6.6 Hz, 3''-NHCH ), 4.50 (s, 2H, H2), 5.75-6.15 (m, 3H,
2
H4'', H5'', H6'), 6.76 (d, 1H, J = 7.3 Hz, H5'), 7.18-7.44 (m, 6H,
H3', C H ), 7.50-7.64 (m, 1H, H6''), 8.20-8.30 (m, 1H, 1-NH).
6
5
3
13
C NMR (75 MHz, DMSO-d ) δ: 32.2 (1-NHCH CH ), 38.2 (1-
6
2
2
2
3
NHCH ), 46.0 (3''-NHCH ) 51.2 (C2), 105.6 (C4''), 106.3 (C5''),
2
2
1
24.7 (C5'), 126.6 (C3'), 126.9 (o-C H ), 127.3 (p-C H ), 128.2
6 5 6 5
(
m-C H ), 128.5 (C6'), 131.7 (C4'), 132.5 (C2'), 133.9 (C6''),
6 5
1
35.7 (C3''), 137.8 (C1'), 139.3 (i-C H ), 156.9 (C2''), 166.9 (C1).
6 5
+
4
ESI-MS m/z 452.05, 454.05 (M+Na ). HRMS m/z: 430.1079;
calc. for C H Cl N O : 430.1084 (M+H ).
+
indicated below; 50, 60, 61, 63) or recrystallized from
24 25
2
3
2
dichloromethane
(52,
53,
56,
57,
58,
62),
or
dichloromethane/hexane (51, 54, 55, 59).
8
.2.4.5 2-(3'-(3'',4''-Dichlorobenzylamino)-2'-oxopyridin-1' (2''H)
8
.2.4.1 2-(3'-(Benzylamino)-2'-oxopyridin-1' (2''H)-yl)-N-phenyl
-yl)-N-phenylacetamide 54 Pale blue solid (122 mg, 22 %). M.p.
acetamide 50 Chromatography (ethyl acetate/hexane: 80/20).
Pale blue solid (100 mg, 32 %). M.p. 193-197 °C. FTIR (KBr) ν:
2
6
2
12-214 °C. FTIR (KBr) ν: 3300, 3070, 1650, 1599, 760, 698,
-
1 1
23 cm . H NMR (400 MHz, DMSO-d ) δ: 4.24 (d, J = 5.0 Hz,
6
-
1
1
3
318, 3070, 1659, 1600, 760, 698 cm . H NMR (400 MHz,
H, 3'-NHCH ), 4.76 (s, 2H, H2), 6.00-6.10 (m, 2H, H4', H5'),
2
DMSO-d ) δ: 4.30 (d, J = 5.0 Hz, 2H, 3'-NHCH ), 4.75 (s, 2H,
6
2
6.20 (t, 1H, J = 5.5 Hz, 3'-NH), 6.83 (d, 1H, J = 5.5 Hz, H6'),
.18 (t, 1H, J = 5.5 Hz, p-C H ), 7.30-7.38 (m, 1H, 3H, H6'', m-
H2), 5.90-6.10 (m, 3H, H4', H5', 3'-NH), 6.84 (d, 1H, J = 5.5 Hz,
7
6
5

C H ), 7.50-7.64 (m, 4H, H5'', H2'', o-C H )
A
5
C
1
C
0.3
E
0
P
(
T
s,
E
1
D
H, MA2H
NUSC
,
1-N
H
,
R
H5
I
'
P
''),
T
7.52-7.60 (m, 2H, H3', H4''), 8.15-8.26 (m, 1H,
6
5
6
13
13
CONH). C NMR (100 MHz, DMSO-d ) δ: 44.7 (3'-NHCH ),
H6''). C NMR (75 MHz, DMSO-d ) δ: 32.2 (1-NHCH CH ),
6
2
6
2
2
5
1.9 (C2), 105.6 (C4'), 108.8 (C5'), 118.8 (o-C H ), 123.2 (p-
38.2 (1-NHCH ), 44.7 (3''-NHCH ) 51.2 (C2), 105.5 (C4''), 110.2
2 2
6
5
C H ), 125.3 (C6'), 127.3 (C''), 128.7 (m-C H ), 128.8 (C''), 128.9
(C5''), 125.6 (C5'), 127.1 (C6'''), 127.3 (C3'), 128.5 (C5'''), 128.9
(C2'''), 129.0 (C6'), 130.4 (C4'''), 131.4 (C4'), 132.5 (C2'), 133.9
(C6''), 135.8 (C3''), 137.4 (C3'''), 138.1 (C1'), 141.0 (C1'''), 157.1
6
5
6
5
(
C''), 130.4 (C''), 130.9 (C''), 137.4 (C3'), 138.8 (i-C H ), 141.0
6 5
(
C''), 156.9 (C2'), 165.7 (C1). ESI-MS m/z 423.94, 425.89
+
+
(M+Na ). HRMS m/z: 402.0779; calc. for C H Cl N O :
(C2''), 167.0 (C1). ESI-MS m/z 519.96, 521.95, 523.95 (M+Na ).
20
18
2
3
2
+
4
02.0771 (M+H ).
HRMS m/z: 520.0107; calc. for C H Cl N O : 520.0124
22
19
4
3
2
+
(
M+Na ).
8
1
.2.4.6 2-(3'-(3'', 4''-Dichlorobenzylamino)-2'-oxopyridin-
(2''H)-yl)-N-(3',4'-dichlorophenyl)acetamide 55 Blue solid (40
8.2.4.10 2-(3'-(2'-Chlorobenzylamino)-2'-oxopyridin-1' (2''H)-
yl)-N-(3',4'-dichlorophenyl)acetamide 59 Pale blue solid (100
mg, 21 %). M.p. 130-134 °C. FTIR (KBr) ν: 3299, 1653, 1528,
mg, 8 %). M.p. 120-125 °C. FTIR (KBr) ν: 3285, 1653, 1524,
-
1 1
7
60, 698, 623 cm . H NMR (400 MHz, DMSO-d ) δ: 4.26 (d, J
6
-
1
1
=
5.0 Hz, 2H, 3'-NHCH ), 4.72 (s, 2H, H2), 6.00-6.10 (m, 2H,
765, 696, 626 cm . H NMR (400 MHz, DMSO-d ) δ: 4.36 (d,
2
6
H4', H5'), 6.22 (t, 1H, J = 5.9 Hz, 3'-NH), 6.87 (d, J = 5.9 Hz,
H, H6'), 7.28 (d, J = 8.0 Hz, 1H, H6'''), 7.43 (dd, 1H, J = 2.0, 5.0
Hz, 1H, H6''), 7.50-7.60 (m, 3H, H5'', H5''', H2''') 7.96 (d, J = 2.0
2H, J = 6.5 Hz, 3'-NHCH ), 4.75 (s, 2H, H2), 6.00-6.10 (m, 3H,
2
1
H4', H5', 3'-NH), 6.90 (d, 1H, J = 7.1 Hz, H6'), 7.21-7.30 (m, 3H,
H6''', H5''', H4'''), 7.40-7.50 (m, 2H, H6'', H3'''), 7.50 (d, 1H, J =
7.1 Hz, H5’'') 7.96 (d, J = 1.4 Hz, 1H, H2''), 10.65 (s, 1H, 1''-
13
Hz, 1H, H2''), 10.60 (s, 1H, 1''-NH). C NMR (100 MHz,
13
DMSO-d ) δ: 44.7 (3'-NHCH ), 52.0 (C2), 105.7 (C4'), 106.8
NH). C NMR (100 MHz, DMSO-d ) δ: 43.8 (3'-NHCH ), 52.0
6
2
6
2
(
(
1
1
C5'), 118.9 (C6''), 120.1 (C2''), 124.7 (C4''), 125.1 (C6’), 127.3
C6’”), 128.9 (C5''), 129.0 (C3'''), 130.4 (C5'''), 130.7 (C4'''),
(C2), 105.8 (C4'), 106.7 (C5'), 118.9 (C6''), 120.1 (C6''), 124.7
(C4''), 125.1 (C6'), 127.1 (C5'''), 128.4 (C5'''), 128.5 (C4'''), 129.2
(C3'''), 130.7 (C5''), 131.0 (C3''), 132.0 (C2''), 135.9 (C3'), 137.5
(C1''), 138.8 (C1'''), 156.9 (C2'), 166.3 (C1). ESI-MS m/z 157.78,
30.9 (C2'''), 131.0 (C3''), 137.4 (C3'), 138.8 (C1''), 141.0 (C1'''),
+
56.9 (C2'), 166.3 (C1). ESI-MS m/z 493.75 (M+Na ). HRMS
+
+
m/z: 491.9787; calc. for C H Cl N O : 491.9811 (M+Na ).
159.80 (M+Na ). HRMS m/z: 458.0191; calc. for C H Cl N O :
20
15
4
3
2
20 16
3
3
2
+
4
58.0200 (M+Na ).
8
.2.4.7 2-(3'-(3'',4''-Dichlorobenzylamino)-2'-oxopyridin-1' (2H)-
yl)-N-phenethylacetamide 56 Pale blue solid (254 mg, 32 %).
8.2.4.11 2-(3'-(3''-Chlorobenzylamino)-2'-oxopyridin-1' (2''H)-
M.p. 183-184 °C. FTIR (KBr) ν: 3249, 3300, 3070, 1640, 1528,
yl)-N-(3',4'-dichlorophenyl)acetamide 60 Chromatography
(DCM/Et N, 9:1) and recrystallisation (DCM/Hexane). Pale blue
solid (100 mg, 18 %). M.p. 175-178 °C. FTIR (KBr) ν: 3249,
-
1
1
7
2
4
64, 690, 623 cm . H NMR (400 MHz, DMSO-d ) δ: 2.72 (t,
3
6
H, J = 7.7 Hz, NHCH CH ), 3.24-3.32 (m, 2H, NHCH CH ),
2
2
2
2
-
1
1
.28 (d, 2H, J = 6.5 Hz, 3'-NHCH ), 4.50 (s, 2H, H2), 5.96-6.07
1649, 1523, 768, 690, 623 cm . H NMR (400 MHz, DMSO-
2
(m, 2H, H5', H4'), 6.22 (t, 1H, J = 6.5 Hz, 3'-NH), 6.78 (dd, 1H, J
d ) δ: 4.38 (d, 2H, J = 5.6 Hz, 3'-NHCH ), 4.72 (s, 2H, H2), 6.00-
6
2
=
3.0, 6.5 Hz, H6'), 7.15-7.34 (m, 6H, C H , H6''), 7.52-7.60 (m,
6.10 (m, 2H, H4', H5'), 6.22 (t, 1H, J = 7.4 Hz, 3'-NH), 6.89 (dd,
1H, J = 0.8, 7.4 Hz, H6'), 7.20-7.35 (m, 4H, H6''', H5''', H4''',
H2'''), 7.49 (dd, 1H, J = 1.6, 8.1 Hz, H6''), 7.55 (d, 1H, J = 8.1
6
5
13
2
H, H2'', H5''), 8.20 (t, 1H, J = 4.8 Hz, NH). C NMR (100
MHz, DMSO-d ) δ: 35.0 (NHCH CH ), 40.3 (NHCH CH ), 44.7
6
2
2
2
2
1
3
(3'-NHCH ) 51.2 (C2), 105.5 (C4'), 106.6 (C5'), 125.1 (C6'),
Hz, H5'') 7.92 (d, 1H, J = 1.6 Hz, H2''), 10.60 (s, 1H, 1''-NH).
C
2
1
1
1
26.0 (C4''), 127.3 (p-C H ), 128.3 (o-C H ), 128.6 (m-C H ),
28.9 (C6''), 129.0 (C2''), 130.4 (C5''), 130.9 (C3''), 137.4 (C3'),
NMR (100 MHz, DMSO-d ) δ: 45.3 (3'-NHCH ), 52.0 (C2),
6 5 6 5 6 5
6
2
105.8 (C4'), 106.7 (C5'), 118.9 (C6''), 120.1 (C2''), 124.7 (C4''),
124.9 (C6'), 125.6 (C6'''), 126.6 (C4'''), 126.7 (C2'''), 130.1 (C5'''),
130.8 (C5''), 131.0 (C3''), 133.0 (C3'''), 137.5 (C3'), 138.8 (C1''),
39.3 (C1''), 141.0 (i-C H ), 156.9 (C2'), 166.8 (C1). ESI-MS
6
5
+
m/z 429.93, 431.91 (M+H ). HRMS m/z: 452.0883; calc. for
C H Cl N O : 452.0903 (M+Na ).
+
22
21
2
3
2
142.3 (C1”’), 156.9 (C2'), 166.3 (C1). ESI-MS m/z 457.78,
+
4
4
59.80 (M+Na ). HRMS m/z: 458.0193; calc. for C H Cl N O :
20
16
3
3
2
8
.2.4.8 2-(3'-(3'',4''-Dichlorobenzylamino)-2'-oxopyridin-1' (2H)-
+
58.0200 (M+Na ).
yl)-N-(3''',4'''-dichlorophenethyl)acetamide 57 Pale green solid
(
1
164 mg, 23 %). M.p. 163-165 °C. FTIR (KBr) ν: 3283, 1653,
523, 761, 690, 622 cm . H NMR (300 MHz, DMSO-
8.2.4.12 2-(3'-(4''-Chlorobenzylamino)-2'-oxopyridin-1' (2''H)-
yl)-N-(3',4'-dichlorophenyl)acetamide 61 Chromatography
(DCM/Et N, 99:1) and recrystallisation (DCM/Hexane). Pale
blue solid (100 mg, 18 %). M.p. > 300 °C (dec.). FTIR (KBr) ν:
-
1
1
d ) δ: 2.66-2.78 (m, 2H, 1-NHCH CH ), 3.23-3.30 (m, 2H, 1-
NHCH ), 4.28 (d, 2H, J = 6.5 Hz, 3'-NHCH ), 4.45 (s, 2H, H2),
5
7
8
3
6
2
2
2
2
-
1
1
.95-6.04 (m, 2H, H4', H5'), 6.72-6.280 (m, 1H, H6'', H6'''), 7.15-
.40 (m, 3H, H2''', H2'', H5''), 7.50-7.60 (m, 2H, H5''', H6'), 8.15-
3253, 1644, 1524, 760, 698, 623 cm . H NMR (400 MHz,
DMSO-d ) δ: 4.33 (d, 2H, J = 5.6 Hz, 3'-NHCH ), 4.65 (s, 2H,
6
2
13
.23 (m, 1H, 1-NH). C NMR (75 MHz, DMSO-d ) δ: 33.9 (1-
H2), 6.00-6.10 (m, 2H, H4', H5'), 6.22 (t, 1H, J = 7.0 Hz, 3'-NH),
6.85 (dd, 1H, J = 0.8, 7.4 Hz, H6’), 7.25-7.40 (m, 4H, H6''', H5''',
H2''', H3'''), 7.45-7.60 (m, 2H, H6'', H5''), 7.90 (d, 1H, J = 1.6 Hz,
6
NHCH CH ), 35.0 (1-NHCH ), 44.7 (3'-NHCH ) 51.2 (C2),
2
2
2
2
1
1
1
1
05.5 (C4'), 106.6 (C5'), 125.0 (C6''), 126.8 (C6'''), 127.3 (C5'''),
28.2 (C2'''), 128.6 (C5''), 129.3 (C2''), 130.3 (C4'''), 130.6 (C3'''),
30.7 (C4''), 130.9 (C3''), 137.4 (C6'), 139.4 (C3'), 140.0 (C1'''),
13
H2''), 10.69 (s, 1H, 1''-NH). C NMR (100 MHz, DMSO-
d ) δ: 45.2 (3'-NHCH ), 52.0 (C2), 105.8 (C4'), 106.7 (C5'),
6
2
41.0 (C1''), 156.9 (C2'), 166.9 (C1). ESI-MS m/z 452.05, 454.05
118.9 (C6''), 120.1 (C2''), 124.7 (C4''), 124.9 (C6'), 127.9 (C2'''),
128.2 (C6'''), 128.8 (C5'''), 129.2 (C3'''), 130.7 (C5''), 131.0 (C3''),
+
(M+Na ). HRMS m/z: 520.0117; calc. for C H Cl N O :
22
19
4
3
2
+
5
20.0124 (M+Na ).
131.1 (C4'''), 137.6 (C3'), 138.5 (C1''), 138.9 (C1'''), 156.9 (C2'),
+
1
4
66.4 (C1). ESI-MS m/z 457.78, 459.78 (M+Na ). HRMS m/z:
8
.2.4.9 N-(2',4'-Dichlorophenethyl)-2-(3''-(3''',4'''-dichlorobenzyl
+
58.0212; calc. for C H Cl N O : 458.0200 (M+Na ).
20
16
3
3
2
amino)-2''-oxopyridin-1''(2H)-yl)acetamide 58 Pale blue solid
(
1
86 mg, 16 %). M.p. 119-123 °C. FTIR (KBr) ν: 3283, 1653,
523, 761, 690, 622 cm . H NMR (300 MHz, DMSO-
8.2.4.13 N-(3',4'-Dichlorobenzyl)-2-(3''-(3'''-phenylpropylamino)-
2''-oxopyridin-1'' (2H)-yl)acetamide 62 Pale green solid (82 mg,
24 %). M.p. 148-149 °C. FTIR (KBr) ν: 3247, 3390, 3074, 1640,
1528, 764, 690, 623 cm . H NMR (400 MHz, DMSO-
d6) δ: 1.69-1.97 (m, 2H, H2'''), 2.63 (t, 2H, J = 8.4 Hz, H3'''),
2.93-3.17 (m, 2H, H1'''), 4.25 (d, 2H, J = 6.0 Hz, 3''-NHCH2),
-
1
1
d ) δ: 2.75-2.86 (m, 2H, 1-NHCH CH ), 3.25-3.30 (m, 2H, 1-
NHCH ), 4.30 (d, 2H, J = 4.0 Hz, 3''-NH), 4.49-4.51 (m, 2H, 3''-
NHCH ), 5.00 (s, 2H, H2), 5.96-6.24 (m, 2H, H6', H5''), 6.40-
6
6
2
2
-
1
1
2
2
.46 (m, 2H, H5', H6'''), 6.76-6.82 (m, 1H, H2'''), 7.20-7.40 (m,

4
2
.60 (s, 2H, H2), 5.33 (t, 1H, J = 6.0 Hz, 3''-NH), 6.12-6.20 (m,
H, H4'', H5''), 6.82 (d, 1H, J = 8.4 Hz, H6''), 7.10-7.38 (m, 6H,
ACCEPTED MANUSCRIPT
1
1. Computational Studies
H6', C H ) 7.50-7.62 (m, 2H, H5', H2'), 8.70 (t, 1H, J = 6.0 Hz,
1
6
5
1
3
-NH). C NMR (100 MHz, DMSO-d ) δ: 29.6 (C2'''), 32.6
6
1
1.1 Ligand Efficiency (LE). Ligand Efficiency [42] was
(C3'''), 41.0 (C1''') 41.9 (1-NHCH ), 51.5 (C2), 105.6 (C4''),
2
calculated for a temperature of 300K using the following
equation.
1
05.9 (C5''), 124.4 (C6''), 125.7 (C4'), 126.1 (C6'), 127.4 (p-
C H ), 128.2 (m-C H ), 129.1 (o-C H ), 129.2 (C2'), 130.3 (C5'),
6
5
6
5
6
5
1
30.8 (C3'), 138.1 (C3''), 140.5 (C1'), 141.9 (i-C H ), 156.9
LE = ∆g = (∆G)/N
6
5
+
(C2''), 167.3 (C1). ESI-MS m/z 444.11, 446.12 (M+H ). HRMS
+
where ∆G = - RTln Kd and N is the number of non-hydrogen
atoms. The units used for LE were units kcal/mol per non-
hydrogen atom. Following the practice of substitution of pKd
with pIC50, LE can be expressed as LE = (1.37/HA) x pIC50
[42]. pIC50 values were calculated from the IC50 values [53].
The LE values were comparable as the assay conditions were the
same for all tested compounds.
m/z: 466.1044; calc. for C H Cl N O : 466.1059 (M+Na ).
23
23
2
3
2
8
.2.4.14
N-(3',4'-Dichlorophenethyl)-2-(3''-(3-phenylpropyl
amino)-2''-oxopyridin-1''(2H)-yl)acetamide 63 Chromatography
(ethyl acetate/hexane/Et N, 80:19.5:0.5). Pale blue/green gum
3
(30 mg, 5 %). FTIR (KBr) ν: 3243, 3391, 3074, 1641, 1520, 762,
-
1 1
6
90, 623 cm . H NMR (300 MHz, DMSO-d ) δ: 1.40 (s, 1H, 3''-
6
NH), 1.83-2.01 (m, 2H, 3''-NHCH CH ), 2.65-2.82 (m, 4H, 3''-
2
2
1
1.2 Ligand-Efficiency-dependent Lipophilicity (LELP).
NHCH CH CH 3''-NHCH ), 3.10 (t, 2H, J = 5.4 Hz, 1-
2
2
2,
2
Ligand-Efficiency-dependent Lipophilicity [43] was calculated
for compounds using the definition of LELP = the ratio of log P
and ligand efficiency (LE).
NHCH CH ), 3.62 (t, 2H, J = 5.4 Hz, 1-NHCH ), 4.60 (s, 2H, 2x
2
2
2
H2), 6.20-6.31 (m, 2H, H4'', H5''), 6.74 (dd, 1H, J = 1.9, 5.7 Hz,
H6'), 7.12-7.28 (m, 6H, H3', C H ), 7.36-7.44 (m, 2H, H5', H6''),
6
5
13
1
-NH not observed. C NMR (75 MHz, DMSO-d ) δ: 31.6 (3''-
Conflict of interest
None.
6
NHCH CH CH ), 34.2 (3''-NHCH CH ), 35.4 (1-NHCH CH ),
2
2
2
2
2
2
2
4
1
1.5 (1-NHCH ), 43.3 (3''-NHCH ), 53.3 (C2), 108.8 (C4''),
2
2
09.3 (C5''), 125.0 (C6'), 126.9 (C2'), 129.4 (p-C H ), 129.4 (o-
6
5
Acknowledgments
C H ), 129.5 (m-C H ), 129.9 (C5’), 131.1 (C4'), 131.5 (C3'),
6
5
6
5
1
31.9 (C6''), 139.9 (C1'), 141.3 (C3''), 143.0 (i-C H ), 159.8
6 5
+
This research was supported by the Eskitis Institute, Griffith
University and partially from a grant from the Diabetes Australia
Research Trust. N. D. Karis and S. S. Schweiker were both in
receipt of an Australian Postgraduate Award.
(C2''), 169.6 (C1). ESI-MS m/z 480.08, 482.09 (M+Na ). HRMS
+
m/z: 480.1198; calc. for C H Cl N O : 480.1216 (M+Na ).
24
25
2
3
2
9
. X-ray structure determination
Supplementary Data
A full cif deposition resides with the Cambridge Crystallographic
Data Centre, CCDC for 13, and 37; 1439368, and 1439367,
respectively. Crystal data, ORTEP plots are described in the
Supplementary data.
Supplementary data associated with this article can be found
in the online version, at
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25] E.D. Chrysina, The prototype of glycogen phosphorylase, Mini-
Rev. Med. Chem. 10 (2010) 1093-1101.
[47] C.A. Lipinski, F. Lombardo, B.W. Dominy, P.J. Feeney,
Experimental and computational approaches to estimate solubility
and permeability in drug discovery and development settings,
Adv. Drug Deliver. Rev. 23 (1997) 3-25.
[48] S.J. Teague, A.M. Davis, P.D. Leeson, T. Oprea, The design of
leadlike combinatorial libraries, Angew. Chem. Int. Ed. 38 (1999)
3743-3748.
[
[
26] W.A. Loughlin, Recent advances in the allosteric inhibition of
glycogen phosphorylase, Mini-Rev. Med. Chem. 10 (2010) 1139-
1
155.
[49] F. Darvas, G. Keseru, A. Papp, A.; Dorman, G.; L. Urge, P.
Krajcsi. In silico and ex silico ADME approaches for drug
discovery, Curr. Top. Med. Chem. 2 (2002) 1287-1304.
[50] D.F. Veber, S.R. Johnson, H-Y. Cheng, B.R. Smith, K.W. Ward,
K.D. Kopple. Molecular Properties That Influence the Oral
Bioavailability of Drug Candidates, J. Med. Chem. 45 (2002)
2615-2623.
[51] H.F.F. Refsgaard, B.F. Jensen, P.B. Brockhoff, S.B. Padkjær, M.
Guldbrandt, M.S. Christensen, In Silico Prediction of Membrane
Permeability from Calculated Molecular Parameters, J. Med.
Chem. 48 (2005) 805-811.
[
[
[
[
27] W-L. Li, C. Jian, M-H. Luo, L. Han, M. Li, W-L. Li, Recent
advances in design of glycogen phosphorylase inhibitors, Curr.
Enzyme Inhib. 7 (2011) 259-267.
28] B.R. Henke, Inhibition of glycogen phosphorylase as a strategy for
the treatment of type 2 diabetes, RSC Drug Discov. Series 27(New
Therapeutic Strategies for Type 2 Diabetes) (2012) 324-365.
29] J.P. Praly, S. Vidal, Inhibition of glycogen phosphorylase in the
context of type 2 diabetes, with focus on recent inhibitors bound at
the active site, Mini-Rev. Med. Chem. 10 (2010) 1102-1126.
30] J. M. Hayes, A. L. Kantsadi, D. D. Leonidas, Natural products and
their derivatives as inhibitors of glycogen phosphorylase: potential
[52] John M. Grohol (2014). "Top 25 Psychiatric Medication
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015.< http://psychcentral.com/lib/top-25-psychiatric-medication-
ACCEPTED MANUSCRIPT
prescriptions-for-2013/>.
[
53] C. Selvaraj, S.K. Tripathi, K. K. Reddy, S.K. Singh, Tool
development for Prediction of pIC50 values from the IC50 values
-
1
A pIC50 value calculator. Curr. Trends Biotech. Pharm. 5 (2011)
104-1109.

a
ACCEPTED MANUSCRIPT
O
N
R
R
N 3
N
1
H
H
O
b
c
1
2
R = Cl; GPa IC50 = 6.3 µM
R = H; GPa IC50 = 34.2 µM
O
O
N
Cl
Cl
N
N
H
H
O
3
GPa IC50 = 162 µM
Fig. 1. A preliminary analysis for pyridone amide derivatives; position a -
carbonyl group not favored; positions b, c - aromatic group favored.
.
Fig. 2. ORTEP-3 diagram of the molecular structure pyridone ester 13
Displacement ellipsoids for non-hydrogen atoms are drawn with 30%
probability.
.
Fig. 3. ORTEP-3 diagram of the molecular structure nitro pyridone 37
Displacement ellipsoids for non-hydrogen atoms are drawn with 30%
probability.

ACCEPTED MANUSCRIPT
hydrophobic
binding
pocket
N
HN
O
O
N
H
Cl
Cl
Cl
Cl
(
A) folded conformation of 17
O
N
Cl
Cl
Cl
Cl
N
N
H
H
O
(
B) extended conformation of 17
Fig. 4. Proposed (A) folded and (B) extended conformations of benzyl 3,4-
dichlorobenzyl 3,4-dichlorobenzyl pyridone 17 and interactions with a
hydrophobic binding pocket
.

ACCEPTED MANUSCRIPT
a
Table 1. GP inhibition data and calculated physical data values for RHS derivatives 18, 19, 50, 54 and 56 produced via Schemes
1
and 2 and compounds 1-3 [32].
#
Structure
MW
GPa inhibition
% at µM) or
IC50 (µM)
Log P
Log S
(g/L)
#RB
#ON
#OHN
H
TPSA
(
b,d
O
N
Cl
Cl
N
N
1
2
3
1
1
b
H
H
416
347
6.3
3.52±0.49
2.25±0.51
3.00±0.71
3.48±0.60
0.004
0.096
0.005
0.012
7
7
6
7
5
5
6
5
2
2
2
2
63.1
63.1
86.2
63.1
O
O
N
N
N
b
H
H
34.2
O
O
O
N
Cl
Cl
c
N
N
4
30
162
H
H
O
O
Cl
Cl
N
N
N
8
9
b
H
H
416
10.2
O
O
Cl
Cl
N
N
H
N
3
68
67%@222
not active
2.82±0.52
2.32±0.54
0.059
0.180
7
6
5
5
2
2
63.1
63.1
H
O
O
N
333
402
5
5
0
4
N
N
H
H
O
O
Cl
Cl
N
N
N
H
H
19%@440
3.56±0.59
3.80±0.60
0.007
0.009
6
8
5
5
2
2
63.1
63.1
O
O
Cl
Cl
N
b
5
6
N
N
430
31.3
H
H
O
a
Calculated with ALOPS 2.1 and Molinspiration
b
c
d
IC50 = Inhibition concentration at 50% inhibition for assays reaching ≥ 90% inhibition.
est IC5 reported with % GPa inhibition observed at maximal concentration; 45% at 222µM.
0
The caffeine standard IC was 283 ± 10 µM for 1-3; 229 ± 2 µM for 18-19 and 490 ± 14 µM for 54 and 56.
50

ACCEPTED MANUSCRIPT
a
Table 2. GP inhibition data and calculated physical data values for RHS derivatives 20, 21, 23, 24, 51-53, 62 and 63
produced via Schemes 1 and 2
#
Structure
MW
GPa
inhibition (%
at µM) or
Log P
Log S
(g/L)
#RB
#ON
#OHN
H
TPSA
b,c
IC50 (µM)
or
appIC50
d
(
µM)
O
N
Cl
Cl
c
2
0
N
N
422
417
19.5
3
.22±0.48
2.52±0.57
3.63±0.79
0.012
7
5
6
8
2
2
2
63.1
76.0
H
S
H
O
O
N
N
Cl
Cl
not active
0.070
0.020
7
9
2
2
1
3
N
N
H
H
O
O
N
Cl
b
N
N
559
480
3.4
102.2
N
H
H
O
Cl
Cl
Tos
O
O2
S
N
37%@222
3.24±0.62
3.56±0.59
0.013
0.007
7
6
7
5
2
2
97.3
63.1
2
5
4
1
N
N
H
H
O
H3C
Cl
Cl
O
N
402
430
41%@90
N
N
H
Cl
H
O
Cl
Cl
O
5
2
80%@444
3.81±0.60
0.009
0.009
8
8
5
5
2
2
63.1
63.1
N
N
N
H
H
O
Cl
O
4
30
93%@44
3
.80±0.60
5
6
3
2
N
N
H
N
H
O
Cl
O
N
Cl
Cl
N
N
H
e
444
458
33%@4.4
H
4.11±0.57
4.55±0.62
0.005
0.004
9
5
5
3
2
63.1
63.1
O
Cl
Cl
O
b
N
68
10
6
3
N
N
H
H
O
a
b
c
d
e
Calculated with ALOPS 2.1 and Molinspiration
IC50 = Inhibition concentration at 50% inhibition for assays reaching ≥ 90% inhibition.
The caffeine standard IC was 229 ± 2 µM for 20-24 and 490 ± 14 µM for 51-53 and 62-63.
app. IC50 = apparent IC50 inhibition concentration at 50% inhibition for assays reaching < 90% inhibition: 20 (73% at 222µM).
mM
50

ACCEPTED MANUSCRIPT
a
Table 3. GP inhibition data and calculated physical data values for compounds 14-17, 58-60 and 61 produced via
Schemes 1 and 2
#
Structure
MW
GPa inhibition
% at µM) or
Log P
Log S
(g/L)
#RB
#ON
#OHN
H
TPSA
(
b,c
IC50 (µM) or
d
appIC50 (µM)
Cl
O
1
1
4
5
N
Cl
Cl
c
N
H
N
H
451
18.5
3.94±0.58
0.002
7
5
2
63.1
O
O
Cl
N
Cl
Cl
b
N
H
N
H
451
451
485
499
437
5.66
3.94±0.59
4.08±0.64
4.49±0.64
4.97±0.73
4.15±0.66
7
7
7
8
6
5
5
5
5
5
2
2
2
2
2
63.1
63.1
63.1
63.1
63.1
0
.002
O
O
O
b
1
1
6
7
2.1
0.002
N
N
Cl
Cl
N
H
N
H
O
O
Cl
Cl
Cl
Cl
Cl
b
N
H
N
H
1.92
0
.0005
0.0004
0.002
Cl
O
5
5
8
9
22%@44
Cl
Cl
N
N
N
H
H
O
Cl
Cl
Cl
Cl
O
b
N
57
N
N
H
H
O
Cl
Cl
O
6
6
0
1
Cl
N
4
37
33%@20
not active
4.15±0.66
4.17±0.65
0.002
0.003
6
6
5
5
2
2
63.1
63.1
N
N
H
H
O
Cl
Cl
O
N
437
N
H
N
H
O
Cl
a
Calculated with ALOPS 2.1 and Molinspiration
b
c
d
IC50 = Inhibition concentration at 50% inhibition for assays reaching ≥ 90% inhibition.
The caffeine standard IC was 229 ± 2 µM for 14-17 and 490 ± 14 µM for 58-61.
50
app. IC50 = apparent IC50 inhibition concentration at 50% inhibition for assays reaching < 90% inhibition: 14 (77% at 222µM).

ACCEPTED MANUSCRIPT
Table 4. Calculated Ligand Efficiency (LE) and
Ligand-Efficiency-dependent Lipophilicity (LELP) of
compounds 1, 2, 14-18, 20, 23, 56, 59 and 63
a
b
Compound
IC50 (µM)
LE
cLogP
LELP
1
2
6.3
0.24
0.24
0.22
0.25
0.27
0.26
0.24
0.25
0.24
0.21
0.21
0.18
3.52
2.25
3.94
3.94
4.08
4.49
3.48
3.22
3.63
3.80
4.15
4.55
14.75
9.55
34.2
18.5
5.66
2.1
1
1
1
1
1
2
2
5
5
6
4
5
6
7
8
0
3
6
9
3
17.60
15.87
15.19
17.18
14.23
12.96
15.00
17.83
19.96
24.67
1.92
10.2
19.5
3.4
31.3
57
68
a
Values as reported in Tables 1-3
Calculated with ALOGPS 2.1
b

ACCEPTED MANUSCRIPT
O
O
(b)
R1
N
R1
N
R2
N
OEt
N
N
H
H
H
(a)
O
O
1
1
1
2
1
2
5
6
R = a 10 R = f
14 R = a, R = a' 20 R = e, R = a'
O
1
1
1
2
1
2
R = b 11 R = g
15 R = b, R = a' 21 R = f, R = a'
N
1
1
1
2
1
2
H N
2
OEt
7 R = c 12 R = h
16 R = c, R = a' 22 R = g, R = a'
1
1
1
2
1
2
O
8 R = d 13 R = i
17 R = d, R = a' 23 R = h, R = a'
4
1
1
2
1
2
9
R = e
18 R = d, R = b' 24 R = i, R = a'
1
2
1
9 R = d, R = c'
(c)
O
O
O
O
(d)
N
R2
N
R1
R1
N
N
N
OEt
H
H
H
O
1
O
2
1
1
27 R = j, R = a'
8 R = j, R = b'
9 R = k, R = c'
2
5 R = j 26 R = k
1
2
2
2
1
2
Cl
CH2
e
SO2
CH2
S
i
a
H C
3
N
CH2
CH2
Cl
CH2
CH2
CH2
f
b
Cl
R1
j
g
c
O N
2
Cl
Cl
k
PhNH
h
CH2
d
N
Cl
Tos
CH2
Cl
Cl
R2
b' CH Ph
2
a'
c' CH CH CH
2 2 3
o
Scheme 1. (a) aldehyde or p-toluenesulfonyl chloride, 2hrs, rt then NaBH(OAc)
(
3 3
, 16 hrs, rt. (b) amine, 4 hrs, 120 C; (c) 2-chlorobenzoyl chloride, Et N, 4 hrs, rt
o
compound 25) or phenyl isocyanate, 4hrs, rt (compound 26); (d) amine, 4 hrs, 120 C (compounds 27,28) or 1hr, rt (compound 29).

ACCEPTED MANUSCRIPT
O
(
a)
N
R2
O N
2
N
NH
O N
2
H
O
O
O
2
2
Cl
Cl
Br
R2
3
7 R = a' 40 R = d'
N
O
O
30
2
2
H
38 R = b' 41 R = e'
(b)
N
2
2
2
2
Ph
N
N
H
3
9 R = c' 42 R = f'
3
3
3
1 R = a' 34 R = d'
H
2
2
2 R = b' 35 R = e'
O
49
2
2
3 R = c' 36 R = f'
(c)
O
O
R1
N
R2
R2
(d)
N
N
H
N
H N
2
N
H
H
O
O
2
2
1
2
1
2
4
4
4
3 R = a' 46 R = d'
4 R = b' 47 R = e'
5 R = c' 48 R = f'
50 R = a, R = a' 57 R = b, R = e'
2
2
1
2
1
2
51 R = a, R = b' 58 R = b, R = f'
2
2
1
2
1
2
52 R = a, R = e' 59 R = c, R = b'
1
2
1
2
5
5
5
5
3 R = a, R = f' 60 R = d, R = b'
1
2
1
2
4 R = b, R = a' 61 R = e, R = b'
1
2
1
2
5 R = b, R = b' 62 R = f, R = d'
1
2
1
2
6 R = b, R = c' 63 R = f, R = e'
R1
R2
Cl
CH2
Cl
Cl
CH2
a
b
a'
b'
Cl
Cl
H C
Cl
Cl
2
CH2
Cl
CH2
CH2
H C
2
c
e
d
f
d'
c'
Cl
Cl
CH2
H C
2
H C
2
e'
f' Cl
Cl
Cl
o
o
Scheme 2. (a) NaH, THF, microwave 150 C, 60 min or NaH, 140 C, 20 hrs; (b) H
2
3 3
, Pd/C. CH OH, 6-48 hrs, rt; (c) benzoyl chloride, Et N, 16 hrs, rt; (d)
3
NaBH(OAc) , 16 hrs, rt.

ACCEPTED MANUSCRIPT
2
-Oxo-1,2-dihydropyridinyl-3-yl amide-based Glycogen Phosphorylase inhibitors: Design,
synthesis and structure-activity relationship study
Wendy A. Loughlin, Ian D. Jenkins, N, David Karis, Stephanie S. Schweiker, Peter C. Healy
Highlights
•
•
•
Design and synthesis of 29 new pyridone amide inhibitors of Glycogen Phosphorylase
Second generation library hit rate was 45%, with 13 compounds inhibiting GPa
Two lead compounds 16 and 17 with improved GPa inhibition (IC = 2.1 and 1.92
5
0
µM)
•
•
SAR analysis revealed sensitivity of GPa to the length of the pyridone amide
SAR analysis revealed a preference for inclusion of a 3,4-dichlorobenzyl moiety