Glycogen phosphorylase inhibitory effects of 2-oxo-1,2-dihydropyridin-3-yl amide derivatives

Article abstract of DOI:10.1016/j.bmc.2009.04.049

Glycogen phosphorylase (GP) plays a crucial role in the conversion of glycogen to glucose-1-phosphate (and in turn glucose) and is a promising target for therapeutic intervention in diabetes. In this study we synthesized new derivatives of 2-oxo-1,2-dihyd

Full text of DOI:10.1016/j.bmc.2009.04.049

Bioorganic & Medicinal Chemistry 17 (2009) 4724–4733
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Glycogen phosphorylase inhibitory effects of 2-oxo-1,2-dihydropyridin-3-yl
amide derivatives
a
a,b
N. David Karis a, Wendy A. Loughlin a,b, , Ian D. Jenkins , Peter C. Healy
*
a Eskitis Institute for Cell and Molecular Therapies, Nathan Campus, Griffith University, Brisbane, QLD 4111, Australia
b School of Biomolecular and Physical Sciences, Nathan Campus, Griffith University, Brisbane, QLD 4111, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
Glycogen phosphorylase (GP) plays a crucial role in the conversion of glycogen to glucose-1-phosphate
(and in turn glucose) and is a promising target for therapeutic intervention in diabetes. In this study
we synthesized new derivatives of 2-oxo-1,2-dihydropyridin-3-yl amides using a facile aminolysis reac-
tion, in which different alkyl and aryl esters and amides are substituted at N-1 and C-3 of the heterocyclic
ring. The in vitro inhibitory activity of compounds against glycogen phosphorylase was evaluated. From
Received 31 March 2009
Revised 22 April 2009
Accepted 23 April 2009
Available online 3 May 2009
this series the most potent compound exhibits good GPa inhibition (IC50 = 6.3 lM). A preliminary study of
these compounds showed that anti-GP activity was decreased by the incorporation of a C3–N carbonyl
group and favored by increased lipophilicity.
Keywords:
Glycogen phosphorylase
Pyridone amide
Inhibition
Ó 2009 Elsevier Ltd. All rights reserved.
X-ray structure
1. Introduction
Discovery of new structural diversity is important to identifying
new potent inhibitors of GP that are likely to be progressed
Glycogen phosphorylase (GP) is a therapeutic target for treating
hyperglycemia,1–4 as it plays a crucial role in the breakdown of gly-
cogen to glucose-1-phosphate in liver and skeletal muscle. Interest
in GP as a therapeutic target has increased since it was validated in
diabetic ob/ob mice.5 Recent work has also shown that blocking the
interaction of GPa with the C-terminus of GL-PP-1 increases glyco-
gen synthesis in primary rat hepatocytes.6 Of the known GP bind-
ing sites, the majority of inhibitors target either the catalytic site3
which binds glucose,7,8 glycogen and analogues of these;9 the allo-
steric (AMP) site which binds adenosine monophosphate (AMP),
inosine 50-monophosphate, adenosine tri-phosphate, and glucose-
6-phosphate;1 the purine nucleoside site (an allosteric site also
referred to as ‘the inhibitor site’), which is active toward purine
derivatives, such as caffeine;10 and a range of heterocyclic com-
pounds; or the indole site (another allosteric site) at the enzyme
dimer interface which primarily binds indoles.11–14 Recently a pos-
sible novel binding site on the surface of the protein located
roughly 32 Å from any other binding site has been reported.15 Most
compounds that inhibit GP have been screened in vitro, with some
inhibitors also screened in vivo. Comprehensive reviews on a range
of GP inhibitors have been reported elsewhere.1,2,4,5,16,17
through the drug discovery process. We have opted for an innova-
tive chemogenomics strategy to identify a new molecular scaffold
for inhibitor design. This approach is focused on the C-terminus of
the hepatic glycogen-binding subunit GL (encoded by the gene
PPP1R3B) of protein phosphatase-1 (PPP1). GPa binds to residues
269–284 at the C-terminus end of the GL-subunit of PPP-1 (GL
(PPP1R3B18). This C-terminus sequence is absent from the other
glycogen-targeting
subunits,
GM
(PPP1R3A),19
R5/PTG
(PPP1R3C),20,21 and R6 (PPP1R3D),22 (PPP1R3E)23 and (PPP1R3F),24
and (PPP1R3G).24 The binding site does not vary between mamma-
lian species (rat, mouse, human), which suggests that allosteric
regulation of GPa activity by GL-PPP1 is important in mammals25,26
and that GL has the highest glycogenic potency.27 In addition GL
binding occurs in the presence of caffeine and glucose. As caffeine
and glucose bind to unique sites that are homologous in muscle
and liver phosphorylases, GL is not binding at the same site. During
the course of our work, other studies provided evidence that a GP
inhibitor can block the interaction of GPa with the GL C-termi-
nus6,28,29 suggesting that this interaction could be targeted in the
development of a GPa inhibitor. In the present work, a chemoge-
nomics strategy was implemented based on the reported analysis
of the homology of amino acids between rat GL, rabbit GM and
human GM (Fig. 1).18
Recent inhibitors of GP are primarily glucose, purine, or indole
structures, that target the catalytic site, the AMP site and the indole
site, and in some cases appear to have reached a limit in potency.
Using this comparison (Fig. 1) and in a de novo approach, we
identified the amino acid sections that were unique within the
human GL C-terminus binding sequence (Fig. 1). Considering
synthetic accessibility and flexibility for generation of a library of
* Corresponding author. Tel.: +61 07 3735 7567; fax: +61 07 3735 7656.
E-mail address: w.loughlin@griffith.edu.au (W.A. Loughlin).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.04.049

N. D. Karis et al. / Bioorg. Med. Chem. 17 (2009) 4724–4733
4725
amines with lipophilic groups (e.g., ethyl, propyl, butyl, isopropyl
groups; 6–9a, 7–9d, 9g–j, Scheme 1), hydrophilic groups (e.g.,
hydroxyl group; 7u–v, 8–9e, 9k, 9u–v, Scheme 1), aromatic groups
(e.g., benzyl group; 6–9b, 6t, 7s–x, 9r–w, 9y, Scheme 1), a basic
amino group (9l, Scheme 1), or containing a ring system (e.g., pyr-
rolidine, cyclohexyl group; 6–9c, 8f, Scheme 1) and included priv-
ileged drug-like structures,33 such as piperidine and morpholine
(9m, 9o, Scheme 1) that were also attached with extension linkers
such as ethylene and propylene (9n, 9p, 9q, Scheme 1). The major-
ity of right hand modifications were carried out on easily purified
and stable pyridone ester 5. In the aminolysis procedure, amino
alcohols reacted only at the amine end to give the pyridone amide
(7u–v, 8e, 9e, 9k, 9u–v, Scheme 1). In the case of ethylenediamine
(9l, Scheme 1), dimer formation was avoided by use of excess eth-
ylenediamine, and mono pyridone amide 9l was obtained exclu-
sively. These compounds were purified by recrystallization, and
characterized (Microanalysis, mass spectrometry, IR, 1H, and 13C
NMR spectroscopy). To facilitate discussion, these derivatives will
be referred to by the left, then right hand side substituents; for
example, benzyl propyl pyridone 7a.
Figure 1. Comparison of the amino acid sequence homology of rat liver GL and
human liver GL, with the N-terminal regions of GM from rabbit and human skeletal
muscle. Homologous residues are shown in bold. The sequences are taken from
Figure 2 in Ref. 18.
compounds we selected as our scaffold a pyridone ring, a mimetic
of the Leu-Gly residues contained within the C-terminal residues
associated with activity6,28,29 (Fig. 2). As the scaffold is to be
extended to form derivatives it needs to be located along from
the end C-terminal residues. Formation of derivatives (Fig. 2) by
substitution of the C3 amino group, and aminolysis of the N1-ester
side chain was used to generate a library of pyridone derivatives.
The inhibition activity of the synthesized compounds was evalu-
ated against GPa. Herein we report on a discovery library, with
an excellent hit rate, and identify a 2-oxo-1,2-dihydropyridin-3-
yl amide, bearing appropriate side chains, as a new lead compound
for in vitro GPa inhibition.
3. X-ray structures of 7u, 7v, and 9k
Pyridones 7u, 7v, and 9k formed crystals suitable for single
crystal X-ray diffraction studies (see Supplementary data for repre-
sentative ORTEP plots). The solid state conformation was of inter-
est to see whether N–H groups from one pyridone molecule (in the
crystal lattice) establish hydrogen bonds with the C@O groups in
another pyridone molecule (in the same crystal lattice), reflective
of, for example, a ‘beta-strand-like’ peptide conformation. The re-
sults of this work showed 7u and 7v to crystallize as hydrated, iso-
morphous structures. The water molecules occupy hydrophilic
regions within the crystal lattice, forming a strong inter-molecular
O–HꢀꢀꢀO(carbonyl), O–HꢀꢀꢀO(water), and OꢀꢀꢀH–O(hydroxyl), OꢀꢀꢀH–N(amine)
hydrogen bonding network as illustrated in Figure 3. The amide
proton forms chains of N–HꢀꢀꢀO hydrogen bonds with the hydroxyl
rather than with the amide carbonyl group as usually found for
amide systems. The C3–N3 bond lengths of 1.357(7) Å (7u) and
1.366(7) Å (7v) are similar to those observed in the structures of
secondary anilines34 and reflect conjugation of this bond with
the pyridone ring. By comparison, the structure of 9k crystallized
with two independent molecules in the crystal lattice. For both
molecules the amino substituent group is approximately co-planar
with the pyridone ring. The structure of this fragment is character-
2. Chemistry
The synthesis of 2-pyridone derivatives 6a–c, 6t, 7a–d, 7s–x,
8a–f, 8t, 9a–e, 9g–w, and 9y was achieved following the steps out-
lined in Scheme 1. Aminopyridone 130,31 was obtained from com-
mercially available 2-hydroxy-3-nitropyridine by treatment with
sodium hydride and ethyl bromoacetate, followed by reduction
with hydrogen in the presence of 10% Pd/C in EtOAc.32 Reductive
amination of amine 1 with propionaldehyde gave 2 (67%), and with
benzaldehyde gave 3 (98%). Alternatively, acylation of amine 1
with acetyl chloride gave 4 (98%), and with benzoyl chloride gave
5 (99%).32 Esters 2–5 were converted to amide derivatives by using
direct aminolysis (e.g., excess of amine (5 lL/mg) at a reaction
temperature of 22, 90, or 120 °C, respectively, for 15 min for up
to 4 h), which was previously found to be a mild and atom efficient
procedure.32 Our previous work had shown that the aminolysis
proceeded readily with primary amines, and some secondary
amines. This influenced the choice of amines used in the aminoly-
sis of esters 2–5 and the selection of derivatives based on the pyri-
done scaffold 1. Pyridone esters (2–5) were reacted with a range of
ized by the presence of intermolecular
pꢀꢀꢀp interactions between
the pyridone rings and a relative absence of hydrogen bonding
interactions by the carbonyl and amine groups with intermolecular
OꢀꢀꢀH–N bonding observed only for the amide carbonyl in the first
molecule and the pyridone carbonyl in the second. In this struc-
ture, the change in the nitrogen atom from an amine to an amide
results in an increase in the C3–N3 bond lengths to 1.403(3) and
1.398(3) Å. The amide groups within the two molecules of 9k are
oriented at right angles to the pyridone plane and exhibit strong
intermolecular hydrogen bonding interactions between the car-
bonyl, amide and hydroxyl groups.
4. Biology
GP activity of compounds 2–5, 6a–c, 6t, 7a–d, 7s–x, 8a–f, 8t,
9a–e, 9g–w, and 9y was measured, using the in vitro GP screen
reported in other recent studies,28,35 in the direction of glycogen
synthesis11 by the formation of inorganic phosphate from glu-
cose-1-phosphate.36,37 The results for compounds that displayed
levels of inhibition of GP are listed in Table 1. By comparison, an
Figure 2. Pyridone mimetic of Leu-Gly residues in the C-terminus (residues 269–
284) of human GL.
IC50 = 283 10 lM was obtained for the caffeine standard. The

4726
N. D. Karis et al. / Bioorg. Med. Chem. 17 (2009) 4724–4733
Scheme 1. Reagents and conditions: (i) For 2, propionaldehyde, NaBH(OAc)3, CH2Cl2, rt, 16 h, 67%; for 3, benzaldehyde, NaBH(OAc)3, CH2Cl2, rt, 16 h, 98%; (ii) for 4, acetyl
chloride, Et3N, CH2Cl2, rt, 4 h, 98%; for 5, benzoyl chloride, Et3N, CH2Cl2, rt, 4 h, 99%; (iii) amine, 22, 90, or 120 °C, 15 min, 2, 4, or 16 h.
compounds not included in Table 1 (compounds 2–5, 6a, 6c, 7a,
7c–d, 7s, 8a–f, 9a–e, 9g–s, 9u–w, and 9y), did not inhibit GPa at
the maximal concentration used in the assay (i.e., <20% at 222 lM).
led to a decrease in the activity against GPa, whereas inhibition of
GPa activity was generally favored by increased lipophilicity (Log P
>1.3, Table 1; e.g., vis-à-vis 7b and 7t).
5. Calculation of molecular physicochemical properties
6. Results and discussion
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 2.1,38 and are listed for the
active compounds 6–7b, 6–9t, 7u–w, and 7x (Table 1). The com-
pounds in Table 1 have a predicted Log P <3.52 in accordance with
Lipinski’s rules,39,40 and another study,41 have molecular weights
<500 and a PSA under 120 Å2,42 and none violate the ‘rule of five’
(#OHNH 6 5; #ON <10; #RB <8); with only 7u having a number of
rotatable bonds (an important predictor of good oral bioavailabil-
ity42–44) >8. Compounds could be classified into those with lower
calculated solubility from the interval between 4 and 100 mg/L
(compounds 6t, 7b, 7t, 7x, 8t, 9b, and 9t), and those with calculated
solubility from the interval between 0.1 and 3.5 g/L sufficient for fast
adsorption, (compounds 6b, 7u–w, and 8b). Overall, it appears that
incorporation of a C3–N carbonyl group (e.g., vis-à-vis 7t and 9t)
Initially, 34 compounds (2–5, 6a–c, 7a–d, 8a–f, 9a–e, and 9g–r,
Scheme 1) were synthesized and tested for their inhibition activities
against GPa. Compounds 2–5, 6a, 6c, 7a, 7c–d, 8a–f, 9a–e, and 9g–r
did not inhibit GPa at the maximal concentration used in the assay
(222
pyridone 7b inhibited GPa with estimated IC50 values of 343 and
34.2 M, respectively. These values compared favorably to the value
obtained for the caffeine standard (IC50 = 283 10 M). The estIC50
lM). However, propyl benzyl pyridone 6b and benzyl benzyl
l
l
value for 6b could be the result of non-specific inhibition of GPa,
whereas the lower value obtained for 7b is more likely associated
with specificinhibition of GPa. We noted that a carbonylgroup at po-
sition a (Fig. 4) appeared to lead to non-inhibition of GPa (cf. propyl
benzyl pyridone 6b (estIC50 = 343
lM) and acetyl benzyl pyridone
8b (not active); benzyl benzyl pyridone 7b (estIC50 = 34.2
l
M) and
benzoyl benzyl pyridone 9b (not active)). Furthermore, when the
benzyl and propyl group side chains were swapped (bMc, Fig. 4),

N. D. Karis et al. / Bioorg. Med. Chem. 17 (2009) 4724–4733
4727
Figure 4. A preliminary analysis for pyridone amide derivatives; position a = car-
bonyl group not favored; position b = position c = aromatic group favored.
bonding site; 7u, 7v), a methoxy group (to change the electronic
distribution; 7x),
a pyridine or amine group (to increase
basicity; 7s, 7w), and a different linker (to alter the steric/posi-
tion of aromatic ring; 7u–w). Benzoyl pyridones, 9s–w, were
synthesized to provide some ‘structural controls’ for the GPa
inhibition assay.
Compounds 7s, 9s, 9–w, and 9y did not inhibit GPa at <222
lM.
Benzyl pyridones 7t–x and benzoyl pyridone 9t inhibited GPa as
reported in Table 1. The hit rate from the initial discovery library
(46 compounds) was high with six compounds (hit rate 13%) dis-
playing estIC50 values <350
identified (compound7t, estIC50 = 6.3
l
M. In addition, an acceptable hit was
M), indicatingthatthe initial
l
design approach shows promise. The activity observed for benzyl
dichlorobenzyl pyridone 7t appeared to support the preference for
aromatic groups at positions b and c (Fig. 4). Where a direct struc-
tural comparison could be made between a benzyl or benzoyl group
at position b (Fig. 4) inhibitory activity was lost (vis-à-vis 7u and 9u;
7v and 9v; 7w and 9w) or significantly reduced (vis-à-vis 7t and 9t)
in the presence of the benzoyl group. This observation appeared to
further support the earlier observation that a carbonyl group at posi-
tion a (Fig. 4) was detrimental to the inhibitory activity of these pyri-
done derivatives against GPa. However, it was intriguing that one
Figure 3. Representation of the H-bonding structure of 7v.
benzyl propyl pyridone 7a did not inhibit GPa, whereas propyl ben-
zyl pyridone 6b displayed moderate inhibition of GPa
(estIC50 = 343 lM). From the initial compound set (compounds 2–
5, 6a–c, 7a–d, 8a–f, 9a–e, and 9g–r, Scheme 1), benzyl benzyl pyri-
done 7b represented the only compound with aromatic functional
groups at both positions b and c (Fig. 4). The above results suggested
that an aromatic functionality in position c in combination with an
aromatic group at position b (such as benzyl) was a favorable struc-
tural combination for inhibition of GPa. Thus, we selected benzyl
benzyl pyridone 7b for structural modification of the right-hand
amide aromaticsubstituent(positionc), whilstmaintainingtheben-
zyl group at position b.
A further 12 compounds (7s–x, 9s–w, 9y) were synthesized
(Scheme 1). Modification of the amide substituent at position c
was readily achieved by changing the amine used in the
aminolysis reaction, but within the constraints of the require-
ment for an aromatic substituent at position c, availability of
the amine and compatibility with the aminolysis procedure
(which excludes, e.g., carboxylic acid groups). Included in the
second set of compounds were aromatic amines with halogen
atoms (to increase lipophilicity; 7t), with an aliphatic hydroxyl
group (to increase hydrophobicity and introduce a hydrogen
benzoyl analogue, compound 9t (IC50 = 162
tory activity toward GPa. It was also notable that the direct benzyl
analog of 9t, dichloropyridone 7t (estIC50 = 6.3 M), was the most
lM), displayed inhibi-
l
potent compound, and also contained the lipophilic dichlorobenzyl
group at position c (Fig. 4). Two final compounds were synthesized
(propylamine dichlorobenzyl pyridone 6t and acetyl dichlorobenzyl
pyridone 8t; Scheme 1), in which the dichlorobenzyl ligand at posi-
tion c was combined with position b ligands (Fig. 4) used previously
in this study. Compounds 6t and 8t inhibited GPa (Table 1) at poorer
levels than for 7t and 9t. Benzyl dichlorobenzyl pyridone 7t was in
the order of 25-, 60- and 300-fold more potent than 9t, 8t, and 6t,
respectively (Table 1). Preliminary analysis show that inclusion of
a carbonyl group at position c (Fig. 4) was detrimental to inhibitory
activity. The inhibitory activity of pyridone amide derivatives
against GPa appears to be favored by aromatic ligands at positions
a and b (Fig. 4).
Table 1
Calculated physical (ALOGPS 2.1) and GP inhibition data for 6–7b, 6–9t, 7u–w, and 7x.
R1
R2
MW
GPa % inhibitiona or estIC50
(lM)
Log P
Log Sc (g/L)
#RB
#ON
#OHNH
TPSA
b
#
6b
6t
7b
7t
7u
7v
7w
7x
8t
Pr
Pr
NHBn
NHCH2(3,4-diClC6H4)
NHBn
NHCH2(3,4-diClC6H4)
NHCH(Bn)CH2OH
NHCH(Ph)CH2OH
NHCH(–CH2CH2–)2NBn
CHCH2(2-OCH3C6H4)
NHCH2(3,4-diClC6H4)
NHCH2(3,4-diClC6H4)
299
368
347
416
391
377
430
377
368
430
343 (80% at 222)
1770 (40% at 222)
34.2 (96% at 111)
6.3 (94% at 222)
94% at 222
120.4 (40% at 222)
813 (45% at 222)
308 (38% at 222)
372 (45% at 222)
162 (45% at 222)
1.63 0.44
2.85 0.43
2.25 0.51
3.52 0.49
1.98 0.50
1.71 0.50
2.78 0.68
2.34 0.60
1.33 0.86
3.00 0.71
0.824
0.051
0.096
0.004
0.167
0.273
0.201
0.067
0.054
0.005
7
7
7
7
9
8
8
8
5
6
5
5
5
5
6
6
6
6
6
6
2
2
2
2
3
3
2
2
2
2
63.1
63.1
63.1
63.1
83.3
83.4
66.4
72.4
80.2
80.2
Bn
Bn
Bn
Bn
Bn
Bn
Me
Ph
9t
a
b
c
Average values from 4 determinations (duplicates on two separate occasions) with a Hill slope between 0.5 and 3.0 and Z0 values of ꢁ0.8.
Estimated IC50 reported with % GPa inhibition observed at maximal concentration.
Calculated from Log S value determined from ALOGPS 2.1.

4728
N. D. Karis et al. / Bioorg. Med. Chem. 17 (2009) 4724–4733
1H NMR (400 MHz, CDCl3)
d 0.85 (3H, t, J = 7.2 Hz, 1-
7. Conclusion
NHCH2CH2CH3), 0.99 (3H, t, J = 7.2 Hz, 30-NHCH2CH2CH3), 1.47
(2H, tq, J = 7.2, 7.2 Hz, 1-NHCH2CH2), 1.67 (2H, tq, J = 7.2, 7.2 Hz,
30-NHCH2CH2), 3.03 (2H, t, J = 7.2 Hz, 30-NHCH2), 3.15 (2H, dt,
J = 7.0, 6.8 Hz, 1-NHCH2), 4.56 (2H, s, H2), 6.20 (1H, dd, J = 6.6,
6.6 Hz, H50), 6.24 (1H, m, H40), 6.71 (1H, dd, J = 6.6, 1.8 Hz, H60),
The design, synthesis and testing of a range of pyridone amides
led to the identification of selected oxo-1,2-dihydropyridin-3-yl
amide derivatives as a new ‘type’ of GPa inhibitor. The X-ray struc-
ture determination of three representative examples (7u, 7v, 9k)
confirmed strong intermolecular H-bonding patterns were possible
in such pyridone derivatives. The hit rate from the initial discovery
library (46 compounds) was high with six compounds (hit rate
13%) displaying estIC50 values <350
hit was identified (compound 7t, estIC50 = 6.3
considerably more active than caffeine (283 M), the commonly
used standard, indicating that the initial design approach shows
promise. Inhibitory activity of a compound appeared to be favored
by aromatic ligands, whereas inclusion of a C3–N carbonyl group
was detrimental to the potency of a compound. In the future, a
more detailed SAR is required to shed insight on the positioning
of specific functional groups within a defined pyridone amide.
We view benzyl dichlorobenzyl pyridone 7t as a promising hit
compound for a new class of GPa inhibitors, deserving of further
studies.
6.94 (1H, br s, Wh ꢁ 16 Hz, 1-NH), NH not observed; 13C NMR
½
(100 MHz, CDCl3)
d
11.2 (1-NHCH2CH2CH3), 11.6 (30-
NHCH2CH2CH3), 21.9 (30-NHCH2CH2), 22.5 (1-NHCH2CH2), 41.2
(1-NHCH2), 45.2 (30-NHCH2), 54.0 (C2), 107.4 (C40), 108.3 (C50),
123.2 (C60), 138.5 (C30), 157.8 (C20), 167.4 (C1); MS (ESI) m/z (%):
274.2 ([M+Na]+, 100); 258.2 ([M+Li]+, 100). Anal. Calcd for
C13H21O2N3: C, 62.13; H, 8.42; N, 16.72. Found: C, 61.80; H, 8.34;
N, 16.57.
l
M. In addition, an acceptable
l
M) which was
l
8.2.2. N-Benzyl-2-[20-oxo-30-(propylamino)pyridin-10(2H)-
yl]acetamide (6b)
Compound
2 (0.300 g, 1.26 mmol), and neat benzylamine
(1.5 mL) were treated at 120 °C for 2 h using method A. Compound
6b was obtained (0.347 g, 92%). Mp 153–155 °C; 1H NMR
(400 MHz, DMSO-d6): d 0.90 (3H, t, J = 7.2 Hz, CH3), 1.56 (2H, tq,
J = 7.2, 7.2 Hz, CH3CH2), 2.97 (2H, dt, J = 6.8, 6.8 Hz, 30-NHCH2),
4.30 (2H, d, J = 6.0 Hz, CH2), 4.57 (2H, s, H2), 5.24 (1H, t,
J = 6.0 Hz, NH), 6.09 (1H, dd, J = 6.8, 6.8 Hz, H50), 6.17–6.19 (1H,
m, H40), 6.82 (1H, dd, J = 6.8, 1.2 Hz, H60), 7.22–7.33 (5H, m, Ph),
8.59 (1H, t, J = 6.0 Hz, 1-NH); 13C NMR (100 MHz, DMSO-d6): d
11.5 (CH3), 21.3 (CH3CH2), 42.1 (CH2), 44.2 (30-NHCH2), 51.4 (C2),
105.5 (C40), 105.9 (C50), 124.4 (C60), 126.8 (p-Ph), 127.2 (o-Ph),
128.2 (m-Ph), 138.2 (C30), 139.2 (i-Ph), 157.0 (C20), 167.0 (C1);
MS (ESI) m/z (%): 322.2 ([M+Na]+, 100), 300.2 ([M+H]+, 20); 306.3
([M+Li]+, 100). Anal. Calcd for C17H21N3O2: C, 68.21; H, 7.07; N,
14.04. Found: C, 68.14; H, 6.88; N, 13.78.
8. Experimental
8.1. Chemistry general
In general, reagents and solvents were purchased and used
without further purification. 1H NMR (400 MHz) and 13C NMR
(100 MHz) spectra were recorded in the deuterated solvents indi-
cated. Chemical shifts are reported in parts per million (d units,
relative to the reported solvent as an internal standard (e.g., CDCl3
13C, 77.0 ppm). 1H NMR and 13C NMR spectral assignments are sup-
ported by gCOSY, gHSQC, and gHMBC analysis. Mass spectral data,
MS were obtained by electrospray ionization (ESI). For preparative
scale chromatography, silica gel (60–200 mesh) was used. Melting
points are uncorrected. Compounds 1, 4, 5, 8a–f, 9a–e, 9k, 9m, 9o,
and 9u were prepared as previously reported.32
8.2.3. 1-[20-Oxo-20-(pyrrolidin-100-yl)ethyl]-3-
(propylamino)pyridin-2(1H)-one (6c)
Compound
2 (0.300 g, 1.26 mmol), and neat pyrrolidine
(1.5 mL) were treated at 90 °C for 4 h using method A. Compound
6c was obtained (0.311 g, 94%). Mp 113–114 °C; 1H NMR
(400 MHz, CDCl3) d 0.98 (3H, t, J = 7.2 Hz, CH3), 1.66 (2H, tq,
J = 7.2, 6.6 Hz, CH3CH2), 1.86 (2H, tt, J = 6.8, 6.8 Hz, H300 or H400),
2.00 (2H, tt, J = 6.8, 6.8 Hz, H300 or H400), 3.02 (2H, t, J = 7.2 Hz, 3-
NHCH2), 3.50 (2H, t, J = 6.8 Hz, H200 or H500), 3.59 (2H, t, J = 6.8 Hz,
H200 or H500), 4.66 (2H, s, H10), 6.16 (1H, dd, J = 7.0, 7.0 Hz, H5),
6.23 (1H, m, H4), 6.68 (1H, m, H6), NH not observed; 13C NMR
(100 MHz, CDCl3) d 11.9 (CH3), 22.2 (CH3CH2), 24.3 (C300 or C400),
26.4 (C300 or C400), 45.5 (CH2NH), 46.3 (C200 or C500), 46.4 (C200 or
C500), 50.9 (C10), 107.5 (C4, C5), 124.2 (C6), 138.6 (C3), 157.9 (C2),
165.2 (C20); MS (ESI) m/z (%): 286.3 ([M+Na]+, 45); 270.3
([M+Li]+, 100). Anal. Calcd for C14H21N3O2: C, 63.85; H, 8.04; N,
15.96. Found: C, 63.73; H, 8.24; N, 15.68.
8.2. General procedure for the preparation of compounds 6a–c,
6t, 7a–d, 7s, 7t, 7w, 7x, 8t, 9g–j, 9l, 9n, 9p–q, 9s–t, 9w
Method A: Aminolysis was carried out in a conical micro scale
reaction vessel by mixing amine (5 lL amine per mg of ester 4 or
5) and ester 4 or 5 (0.02–1.0 g). The reaction mixture was heated
for 2, 4, 16, or 48 h at 90 or 120 °C; Method B: Aminolysis was car-
ried out according to method A with the reaction mixture stirred at
room temperature for 15 min or 16 h. Workup: Excess amine was
removed under vacuum when possible, or 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 h). 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 solu-
tion was cooled in a freezer overnight forming a white solid precip-
itate. The white precipitate was filtered, washed with diethyl ether
and dried to give the required compounds 6a–c, 6t, 7a–d, 7s, 7t,
7w, 7x, 8t, 9g–j, 9l, 9n, 9p–q, 9s–t, 9w as colorless solids, in 95–
99% purity, as determined by 1H NMR spectroscopy.
8.2.4. N-(30,40-Dichlorobenzyl)-2-[200-oxo-300-
(propylamino)pyridin-100(2H)-yl]acetamide (6t)
Compound 2 (0.300 g, 1.26 mmol), and neat 3,4-dichlorobenzyl-
amine (1.5 mL) were treated at 120 °C 4 h using method A. Com-
pound 6t was obtained (0.423 g, 91%). Mp 174–176 °C (dec); 1H
NMR (400 MHz, DMSO-d6) d 0.90 (3H, t, J = 7.4 Hz, CH3), 1.56 (2H,
tq, J = 7.2, 7.2 Hz, CH3CH2), 2.96 (2H, dt, J = 6.8, 6.8 Hz, 30-NHCH2),
4.29 (2H, d, J = 6.0 Hz, 1-NHCH2), 4.57 (2H, s, H2), 5.21 (1H, br t,
J = 6 Hz, 30-NH), 6.09 (1H, dd, J = 7.0, 7.0 Hz, H50), 6.18 (1H, dd,
J = 7.2, 1.6 Hz, H40), 6.83 (1H, dd, J = 6.8, 1.6 Hz, H60), 7.27 (1H,
dd, J = 8.4, 2.0 Hz, H600), 7.54 (1H, d, J = 2.0 Hz, H200), 7.57 (1H, d,
J = 8.0 Hz, H500), 8.66 (1H, br t, J = 6.0 Hz, 1-NH); 13C NMR
(100 MHz, DMSO-d6) d 12.2 (CH3), 22.1 (CH3CH2), 41.7 (1-NHCH2),
44.9 (30-NHCH2), 52.3 (C2), 106.3 (C50), 106.7 (C40), 125.1 (C60),
128.2 (C600), 129.8 (C200), 129.9 (C300), 131.1 (C500), 131.6 (C400),
8.2.1. 2-[20-Oxo-30-(propylamino)pyridin-10(2H)-yl]-N-
propylacetamide (6a)
Compound
2 (0.300 g, 1.26 mmol), and neat propylamine
(1.5 mL) were treated at room temperature for 15 min using meth-
od B. Compound 6a was obtained (0.307 g, 97%). Mp 135–136 °C;

N. D. Karis et al. / Bioorg. Med. Chem. 17 (2009) 4724–4733
4729
138.9 (C30), 141.3 (C100), 157.7 (C20), 168.0 (C1); MS (ESI) m/z
(%):374.2 ([M+Li]+, 100). Anal. Calcd for C17H19Cl2N3O2: C, 55.45;
H, 5.20; N, 11.41. Found: C, 55.43; H, 5.03; N, 11.17.
(1H, dd, J = 7.2, 1.6 Hz, H40), 6.77 (1H, dd, J = 6.8, 1.6 Hz, H60),
7.19–7.24 (1H, m, Ph), 7.28–7.32 4H, (m, Ph), 8.00 (1H, br d,
J = 7.6 Hz, CONH); 13C NMR (100 MHz, DMSO-d6)
d
23.1
(2 ꢂ CH3), 41.3 (CH), 46.8 (CH2NH), 51.8 (C2), 106.2 (C50), 107.1
(C40), 125.6 (C60), 127.4 (p-Ph), 127.7 (o-Ph), 129.0 (m-Ph), 138.5
(C30), 140.1 (i-Ph), 157.6 (C20), 166.5 (C1); MS (ESI) m/z (%): 322.2
([M+Na]+, 100), 300.2 ([M+H]+, 35); 306.3 ([M+Li]+, 100). Anal.
Calcd for C17H21N3O2: C, 68.21; H, 7.07; N, 14.04. Found: C,
68.17; H, 7.01; N, 13.82.
8.2.5. 2-[30-(Benzylamino)-20-oxopyridin-10(2H)-yl]-N-
propylacetamide (7a)
Compound 3 (0.200 g, 0.698 mmol), and neat propylamine
(1.0 mL) were treated at room temperature for 15 min using meth-
od B. Compound 7a was obtained (0.201 g, 96%). Mp 162–165 °C
(dec); 1H NMR (400 MHz, DMSO-d6) d 0.85 (3H, t, J = 7.4 Hz, CH3),
1.41 (2H, tq, J = 7.4, 7.2 Hz, CH3CH2), 3.02 (2H, dt, J = 6.6, 6.0 Hz,
1-NHCH2), 4.27 (2H, d, J = 4.0 Hz, PhCH2), 4.49 (2H, s, H2), 5.94–
6.00 (2H, m, H50, NH), 6.05 (1H, dd, J = 7.0, 1.8 Hz, H40), 6.77 (1H,
dd, J = 7.0, 1.8 Hz, H60), 7.19–7.24 (1H, m, Ph), 7.28–7.32 (4H, m,
Ph), 8.07 (1H, br t, J = 6.0 Hz, CONH); 13C NMR (100 MHz, DMSO-
d6) d 11.5 (CH3), 22.8 (CH3CH2), 41.4 (1-NHCH2), 47.8 (PhCH2),
54.2 (C2), 108.3 (C50), 108.7 (C40), 124.1 (C60), 127.5 (o-Ph), 127.6
(p-Ph), 128.9 (m-Ph), 138.1 (C30), 138.5 (i-Ph), 158.0 (C20), 167.5
(C1); MS (ESI) m/z (%): 322.1 ([M+Na]+, 55); 306.2 ([M+Li]+, 100).
Anal. Calcd for C17H21N3O2: C, 68.21; H, 7.07; N, 14.04. Found: C,
68.21; H, 7.14; N, 13.82.
8.2.9. 2-[30-(Benzylamino)-20-oxopyridin-10(2H)-yl]-N-(pyridin-
200-ylmethyl)acetamide (7s)
Compound
3 (0.300 g, 1.05 mmol), and neat 2-(amino-
methyl)pyridine (1.5 mL) were treated at 120 °C for 4 h using
method A. Compound 7s was obtained (0.321 g, 88%). Mp 158–
161 °C (dec); 1H NMR (400 MHz, DMSO-d6)
d 4.28 (2H, d,
J = 6.0 Hz, 30-NHCH2), 4.39 (2H, 1d, J = 6.0 Hz, –NHCH2), 4.62 (2H,
s, H2), 5.98–6.03 (2H, m, H50, 30-NH), 6.07 (1H, dd, J = 7.2, 1.8 Hz,
H40), 6.83 (1H, dd, J = 6.8, 1.8 Hz, H60) 7.20–7.36 (7H, m, o, m, p-
Ph, H400, H600), 7.75 (1H, ddd, J = 7.7, 7.7, 1.9 Hz, H500), 8.48–8.50
(1H, m, H300), 8.71 (1H, br t, J = 5.8 Hz, 1-NH); 13C NMR
(100 MHz, DMSO-d6) d 44.2 (1-NHCH2), 46.1 (30-NHCH2), 51.5
(C2), 105.7 (C50), 106.5 (C40), 120.9 (C600), 122.1 (C400), 124.8 (C60),
126.7 (p-Ph), 127.0 (o-Ph), 128.3 (m-Ph), 136.7 (C500), 137.8 (C30),
139.4 (i-Ph), 148.8 (C300), 157.1 (C20), 158.3 (C100), 167.3 (C1); MS
(ESI) m/z (%): 371.2 ([M+Na]+, 30), 349.2 ([M+H]+, 20), 241.0
8.2.6. N-Benzyl-2-[30-(benzylamino)-20-oxopyridin-10(2H)-
yl]acetamide (7b)
Compound 3 (0.210 g, 0.733 mmol), and neat benzylamine
(1.1 mL) were treated at 120 °C for 4 h using method A. Compound
7b was obtained (0.243 g, 95%). Mp 184–185 °C; 1H NMR
(400 MHz, CDCl3) d 4.33 (2H, s, 30-NHCH2), 4.45 (2H, d, J = 5.6 Hz,
([MꢃNHCH2C5H4N]+,
100);
355.2
([M+Li]+,
80),
241.0
([MꢃNHCH2C5H4N]+, 100). HRMS: calcd for [M+Na]+ C20H20N4O2Na
1-NHCH2), 4.64 (2H, s, H2), 5.45 (1H, br s, Wh ꢁ 21 Hz, 30-NH),
371.1478, found 371.1472.
½
6.19 (1H, dd, J = 7.0, 7.0 Hz, H50), 6.28 (1H, dd, J = 7.0, 1.6 Hz,
H40), 6.79 (1H, dd, J = 7.0, 1.6 Hz, H60), 7.24–7.36 (11H, m, 30-
NHCH2Ph, 1-NHCH2Ph, 1-NH); 13C NMR (100 MHz, DMSO-d6) d
43.7 (1-NHCH2), 48.0 (30-NHCH2), 54.3 (C2), 108.5 (C50), 109.0
(C40), 124.5 (C60), 127.6, 127.7, 128.9 (o, m, p-30-NHCH2Ph, o, m,
p-1-NHCH2Ph), 137.8 (i-30-NHCH2Ph, i-1-NHCH2Ph), 138.1 (C30),
158.1 (C20), 167.5 (C1); MS (ESI) m/z (%): 370.2 ([M+Na]+, 100),
348.2 ([M+H]+, 85); 354.2 ([M+Li]+, 100). Anal. Calcd for
C21H21N3O2: C, 72.60; H, 6.09; N, 12.09. Found: C, 72.72; H, 6.17;
N, 12.09.
8.2.10. 2-[30-(Benzylamino)-20-oxopyridin-10(2H)-yl]-N-(300,400-
dichlorobenzyl)acetamide (7t)
Compound 3 (0.100 g, 0.349 mmol), and neat 3,4-dichloroben-
zylamine (0.5 mL) were treated at 120 °C for 4 h using method A.
Compound 7t was obtained (0.135 g, 93%). Mp 182–183 °C (dec);
1H NMR (400 MHz, DMSO-d6) d 4.28 (2H, d, J = 6.4 Hz, PhCH2NH),
4.30 (2H, d, J = 6.0 Hz, 1-NHCH2), 4.58 (2H, s, H2), 5.96 (1H, br t,
J = 6.4 Hz, 30-NH), 6.01 (1H, dd, J = 7.0, 7.0 Hz, H50), 6.07 (1H, dd,
J = 7.2, 1.6 Hz, H40), 6.82 (1H, dd, J = 6.8, 1.6 Hz, H60), 7.19–7.33
(6H, m, o, m, p-Ph, H600), 7.54 (1H, d, J = 2.4 Hz, H200), 7.57 (1H, d,
J = 8.0 Hz, H500), 8.68 (1H, br t, J = 6.0 Hz, 1-NH); 13C NMR
(100 MHz, DMSO-d6) d 41.1 (1-NHCH2), 46.1 (PhCH2NH), 51.5
(C2), 105.7 (C50), 106.5 (C40), 124.8 (C60), 126.7 (C600), 127.0 (o-
Ph), 127.5 (p-Ph), 128.3 (m-Ph), 129.1 (C500), 129.2 (C400), 130.4
(C200), 130.9 (C300), 137.8 (C30), 139.4 (C100), 140.5 (i-Ph), 157.0
(C20), 167.3 (C1); MS (ESI) m/z (%): 422.2 ([M+Li]+, 100). Anal. Calcd
for C21H19Cl2N3O2: C, 60.59; H, 4.60; N, 10.09. Found: C, 60.42; H,
4.54; N, 10.27.
8.2.7. 1-[20-Oxo-20-(pyrrolidin-100-yl)ethyl]-3-
(benzylamino)pyridin-2(1H)-one (7c)
Compound 3 (0.300 g, 1.05 mmol) and neat pyrrolidine (1.5 mL)
were treated at 90 °C for 4 h using method A. Compound 7c was
obtained (0.323 g, 99%). Mp 137–139 °C (dec); 1H NMR
(400 MHz, CDCl3) d 1.89 (3H, tt, J = 7.0, 6.8 Hz, H300 or H400), 2.02
(2H, tt, J = 7.0, 6.8 Hz, H300 or H400), 3.52 (2H, t, J = 7.0 Hz, H200 or
H500), 3.66 (2H, t, J = 7.0 Hz, H200 or H500), 4.32 (2H, d, J = 5.6 Hz,
CH2NH), 4.68 (2H, s, H10), 5.36–5.42 (1H, m, NH), 6.12 (1H, dd,
J = 7.0, 7.0 Hz, H5), 6.17 (1H, dd, J = 7.4, 1.8 Hz, H4), 6.69 (1H, dd,
J = 6.6, 1.8 Hz, H6), 7.24–7.28 (1H, m, 1H, Ph), 7.31–7.38 (4H, m,
8.2.11. 2-[30-(Benzylamino)-20-oxopyridin-10(2H)-yl]-N-(100-
benzylpiperidin-400-yl)acetamide (7w)
13
Ph); C NMR (100 MHz, CDCl3) d 24.3 (C300 or C400), 26.4 (C300 or
Compound 3 (0.300 g, 1.05 mmol), and neat 4-amino-1-ben-
zylpiperidine (1.5 mL) were treated at 120 °C for 4 h using method
A. Compound 7w was obtained (0.366 g, 85%). Mp 176–178 °C; 1H
NMR (400 MHz, DMSO-d6) d 1.36–1.45 (2H, m, H200, H600), 1.70–
1.73 (2H, m, H200, H600), 1.99 (2H, br t, J = 10.4 Hz, H300, H500),
2.71–2.74 (2H, m, H300, H500), 3.43 (2H, s, 400-CH2), 3.49–3.56 (1H,
m, H100), 4.26 (2H, d, J = 6.4 Hz, CH2), 4.48 (2H, s, H2), 5.95–5.99
(2H, m, H50, NH), 6.04 (1H, dd, J = 7.2, 1.6 Hz, H40), 6.76 (1H, dd,
J = 6.8, 1.6 Hz, H60), 7.18–7.33 (10H, m, Ph, 400-CH2Ph), 8.07 (1H,
C400), 46.3 (C200 or C500), 46.4 (C200 or C500), 48.0 (CH2) 50.9 (C10),
107.4 (C5), 108.6 (C4), 124.8 (C6), 127.4 (p-Ph), 127.5 (o-Ph),
128.8 (m-Ph), 138.2 (i-Ph), 138.4 (C3), 157.9 (C2), 165.2 (C20); MS
(ESI) m/z (%): 334.0 ([M+Na]+, 45); 318.0 ([M+Li]+, 100). HRMS:
calcd for [M+Na]+ C18H21N3O2Na 334.1526, found 334.1542.
8.2.8. 2-[30-(Benzylamino)-20-oxopyridin-10(2H)-yl]-N-
isopropylacetamide (7d)
13
Compound 3 (0.300 g, 1.05 mmol) and neat isopropylamine
(1.5 mL) were treated at room temperature for 16 h using method
B. Compound 7d was obtained (0.285 g, 91%). Mp 173–174 °C
(dec); 1H NMR (400 MHz, DMSO-d6) d 1.06 (6H, d, J = 7.2 Hz,
CH3), 3.81 (1H, dsept {8 lines}, J = 7.0, 6.8 Hz, CH), 4.27 (2H, d,
J = 6.4 Hz, CH2), 4.46 (2H, s, H2), 5.98 (2H, m, H50, CH2NH), 6.05
d, J = 7.6 Hz, 1-NH); C NMR (100 MHz, DMSO-d6) d 31.6 (C200
and C600), 46.1 (PhCH2), 46.2 (C100), 51.2 (C2), 51.8 (C300 and C500),
62.1 (400-CH2), 105.5 (C50), 106.4 (C40), 124.9 (C60), 126.7 (p-Ph or
p-400-CH2Ph), 126.8 (p-Ph or p-400-CH2Ph), 127.0 (m-Ph or m-400-
CH2Ph), 128.1 (o-Ph), 128.3 (m-Ph or m-400-CH2Ph), 128.7 (o-400-
CH2Ph), 137.8 (C30), 138.6 (i-400-CH2Ph), 139.4 (i-Ph), 156.9 (C20),

4730
N. D. Karis et al. / Bioorg. Med. Chem. 17 (2009) 4724–4733
166.0 (C1); MS (ESI) m/z (%): 431.3 ([M+H]+, 100); 437.3 ([M+Li]+,
45), 431.3 ([M+H]+, 100). Anal. Calcd for C26H30N4O2: C, 72.53; H,
7.02; N, 13.01. Found: C, 72.63; H, 6.95; N, 12.96.
3.07 (2H, dt, J = 6.8, 5.6 Hz, NHCH2), 4.61 (2H, s, H10), 6.33 (1H,
dd, J = 7.2, 7.2 Hz, H5), 7.40 (1H, dd, J = 7.2, 2.4 Hz, H6), 7.52–
7.56 (2H, m, m-Ph), 7.59–7.63 (1H, m, p-Ph), 7.89–7.91 (2H, m,
o-Ph), 8.17 (1H, br t, J = 5.6 Hz, 20-NH), 8.27 (1H, dd, J = 7.2,
8.2.12. 2-[30-(Benzylamino)-20-oxopyridin-10(2H)-yl]-N-(200-
methoxybenzyl)acetamide (7x)
2.4 Hz, H4), 9.28 (1H, br s, Wh ꢁ 5 Hz, CONH); 13C NMR
½
(100 MHz, DMSO-d6)
d
13.6 (CH3), 19.5 (CH2CH3), 31.1
Compound 3 (0.300 g, 1.05 mmol), and neat 2-methoxybenzyl-
amine (1.5 mL) were treated at 120 °C 4 h using method A. Com-
pound 7x was obtained (0.358 g, 90%). Mp 158–160 °C (dec); 1H
NMR (400 MHz, DMSO-d6) d 3.70 (3H, s, CH3), 4.25 (2H, d,
J = 5.6 Hz, 1-NHCH2), 4.27 (2H, d, J = 5.6 Hz, PhCH2), 4.59 (2H, s,
H2), 5.98–6.02 (2H, m, H50, NH), 6.06 (1H, dd, J = 7.2, 1.6 Hz, H40),
6.85 (1H, dd, J = 6.8, 1.6 Hz, H60), 6.90 (1H, ddd, J = 7.4, 7.4,
1.6 Hz, H500), 6.95–6.98 (1H, m, H600), 7.22–7.23 (7H, m, Ph, 300,
400), 8.42 (1H, t, J = 5.6 Hz, 1-NH); 13C NMR (100 MHz, DMSO-d6)
d 37.3 (1-NHCH2), 46.1 (PhCH2), 51.4 (C2), 55.3 (CH3), 105.7 (C50),
106.4 (C40), 110.4 (C300), 120.1 (C500), 124.9 (C60), 126.4 (C100),
126.7 (C400), 127.0 (o-Ph), 127.8 (C600), 128.1 (p-Ph), 128.3 (m-Ph),
137.8 (C30), 139.4 (i-Ph), 156.6 (C200), 157.1 (C20), 167.0 (C1); MS
(ESI) m/z (%): 400.2 ([M+Na]+, 100), 378.2 ([M+H]+, 15); 384.2
([M+Li]+, 100). Anal. Calcd for C22H23N3O3: C, 70.01; H, 6.14; N,
11.13. Found: C, 70.24; H, 5.78; N, 11.16.
(NHCH2CH2), 38.3 (NHCH2), 51.6 (C10), 104.8 (C5), 123.6 (C4),
127.1 (o-Ph), 128.0 (C3), 128.8 (m-Ph), 132.0 (p-Ph), 133.7 (C6),
133.9 (i-Ph), 157.1 (C2), 164.8 (CO), 166.1 (C20); MS (ESI) m/z
(%): 334.3 ([M+Li]+, 100). HRMS: calcd for [M+Na]+ C18H21N3O3-
Na 350.1475, found 350.1451.
8.2.16. N-{1-[20-(Isobutylamino)-20-oxoethyl]-2-oxo-1,2-
dihydropyridin-3-yl}benzamide (9i)
Compound 5 (0.500 g, 1.66 mmol), and neat isobutylamine
(2.5 mL) were treated at room temperature for 16 h using method
B. Compound 9i was obtained (0.499 g, 92%). Mp 200–201.5 °C; 1H
NMR (400 MHz, DMSO-d6) d 0.85 (6H, d, J = 6.4 Hz, CH3), 1.69 (1H,
m, CH), 2.91 (2H, dd, J = 6.4, 6.4 Hz, NHCH2), 4.64 (2H, s, H10), 6.33
(1H, dd, J = 7.0, 7.0 Hz, H5), 7.40 (1H, dd, J = 7.0, 1.6 Hz, H6), 7.52–
7.56 (2H, m, m-Ph), 7.59–7.63 (1H, m, p-Ph), 7.89–7.91 (2H, m, o-
Ph), 8.18 (1H, br t, J = 5.6 Hz, 20-NH), 8.28 (1H, dd, J = 7.2, 1.6 Hz,
H4), 9.27 (1H, s, CONH); 13C NMR (100 MHz, DMSO-d6) d 20.1
(2 ꢂ CH3), 28.1 (CH), 46.2 (NHCH2), 51.6 (C10), 104.8 (C5), 123.6
(C4), 127.1 (o-Ph), 128.0 (C3), 128.8 (m-Ph), 132.0 (p-Ph), 133.8
(i-Ph), 133.9 (C6), 157.1 (C2), 164.8 (CO), 166.3 (C20); MS (ESI) m/
z (%): 350.3 ([M+Na]+, 100), 328.3 ([M+H]+, 10); 334.3 ([M+Li]+,
100). Anal. Calcd for C18H21N3O3: C, 66.04; H, 6.47; N, 12.84.
Found: C, 65.87; H, 6.54; N, 12.82.
8.2.13. 2-(30-Acetamido-20-oxopyridin-10(2H)-yl)-N-(300,400-
dichlorobenzyl)acetamide (8t)
Compound 4 (0.100 g, 0.420 mmol), and neat 3,4-dichloroben-
zylamine (0.5 mL) were treated at 120 °C for 4 h using method B.
Compound 8t was obtained (0.145 g, 94%). Mp 220–225 °C (dec);
1H NMR (400 MHz, DMSO-d6) d 2.10 (3H, s, CH3), 4.30 (2H, d,
J = 6.0 Hz, CH2), 4.65 (2H, s, H2), 6.23 (1H, dd, J = 7.2, 7.2 Hz, H50),
7.27 (1H, dd, J = 8.4, 2.0 Hz, H600), 7.35 (1H, dd, J = 7.2, 1.6 Hz,
H60), 7.53 (1H, d, J = 2.0 Hz, H200), 7.58 (1H, d, J = 8.4 Hz, H500),
8.18–8.20 (1H, m, H40), 8.74 (1H, br t, J = 6.0 Hz, 1-NH), 9.19 (1H,
s, CONH); 13C NMR (100 MHz, DMSO-d6) d 24.0 (CH3), 41.1 (CH2),
51.8 (C2), 104.8 (C50), 122.8 (C40), 127.6 (C600), 128.7 (C30), 129.2
(C500), 129.3 (C400), 130.4 (C200), 130.9 (C300), 132.8 (C60), 140.5
(C100), 156.8 (C20), 164.8 (C1), 166.2 (CO); MS (ESI) m/z (%): 390.1
([M+Na]+, 100), 368.1 ([M+H]+, 90); 374.1 ([M+Li]+, 100). Anal.
Calcd for C16H15Cl2N3O3: C, 52.19; H, 4.11; N, 11.41. Found: C,
52.19; H, 4.08; N, 11.16.
8.2.17. N-{1-[20-(300-Methoxypropylamino)-20-oxoethyl]-2-oxo-
1,2-dihydropyridin-3-yl}benzamide (9j)
Compound 5 (0.500 g, 1.66 mmol), and neat 3-methoxypropan-
1-amine (2.5 mL) were treated at room temperature for 16 h using
method B. Compound 9j was obtained (0.537 g, 94%). Mp 169–
171 °C; 1H NMR (400 MHz, DMSO-d6) d 1.64 (2H, tt, J = 6.8,
6.6 Hz, H200), 3.12 (2H, dt, J = 6.8, 6.4 Hz, H100), 3.21 (3H, s, CH3),
3.33 (t, J = 6.4 Hz, 2H, 300), 4.61 (2H, s, H10), 6.33 (1H, dd, J = 7.2,
7.2 Hz, H5), 7.40 (1H, dd, J = 7.2, 1.8 Hz, H6), 7.52–7.56 (2H, m,
m-Ph), 7.59–7.63 (1H, m, p-Ph), 7.89–7.91 (2H, m, o-Ph), 8.19
(1H, br t, J = 5.6 Hz, 20-NH), 8.28 (1H, dd, J = 7.2, 1.8 Hz, H4), 9.28
13
8.2.14. N-{1-[20-(Ethylamino)-20-oxoethyl]-2-oxo-1,2-
(1H, s, CONH); C NMR (100 MHz, DMSO-d6) d 29.1 (C200), 36.0
dihydropyridin-3-yl}benzamide (9g)
(C100), 51.6 (C10), 57.9 (CH3), 69.4 (C300), 104.9 (C5), 123.6 (C4),
127.1 (o-Ph), 128.0 (C3), 128.8 (m-Ph), 132.0 (p-Ph), 133.7 (C6),
133.9 (i-Ph), 157.1 (C2), 164.8 (CO), 166.3 (C20); MS (ESI) m/z (%):
366.3 ([M+Na]+, 100), 344.3 ([M+H]+, 15); 350.3 ([M+Li]+, 100).
Anal. Calcd for C18H21N3O4: C, 62.96; H, 6.16; N, 12.24. Found: C,
62.91; H, 6.15; N, 12.08.
Compound
5 (0.300 g, 0.999 mmol), and neat ethylamine
(5.0 mL, 70% w/w in water) were treated at room temperature
for 15 min using method B. The water was removed by freeze-dry-
ing prior to recrystallization. Compound 9g was obtained (0.296 g,
100%). Mp 214–217 °C; 1H NMR (400 MHz, DMSO-d6) d 1.03 (3H, t,
J = 7.2 Hz, CH3), 3.10 (2H, dq, J = 7.2, 5.6 Hz, CH2), 4.60 (2H, s, H10),
6.33 (1H, dd, J = 7.2, 7.2 Hz, H5), 7.40 (1H, dd, J = 7.2, 1.8 Hz, H6),
7.52–7.56 (2H, m, m-Ph), 7.59–7.63 (1H, m, p-Ph), 7.89–7.91 (2H,
m, o-Ph), 8.20 (1H, br t, J = 5.2 Hz, 20-NH), 8.28 (1H, dd, J = 7.2,
1.8 Hz, H4), 9.28 (1H, s, CONH); 13C NMR (100 MHz, DMSO-d6) d
17.5 (CH3), 36.5 (CH2), 54.5 (C10), 107.7 (C5), 126.5 (C4), 130.0
(o-Ph), 130.9 (C3), 131.7 (m-Ph), 134.9 (p-Ph), 136.6 (C6), 136.8
(i-Ph), 160.0 (C2), 167.7 (CO), 168.9 (C20); MS (ESI) m/z (%): 322.2
([M+Na]+, 100); 306.2 ([M+Li]+, 100). HRMS: calcd for [M+H]+
C16H18N3O3 300.1343, found 300.1344.
8.2.18. N-{1-[20-(200-Aminoethylamino)-20-oxoethyl]-2-oxo-1,2-
dihydropyridin-3-yl}benzamide (9l)
Compound 5 (0.300 g, 0.999 mmol), and neat ethylenediamine
(1.5 mL) were treated at room temperature for 15 min using meth-
od B. Compound 9l was obtained (0.273 g, 87%). Mp 190–192 °C
(dec); 1H NMR (400 MHz, DMSO-d6) d 1.60 (2H, br s, Wh ꢁ 45 Hz,
½
NH2) 2.57 (2H, t, J = 6.4 Hz, H200), 3.07 (2H, dt, J = 6.4, 6.0 Hz, H100),
4.63 (2H, s, H10), 6.33 (1H, dd, J = 7.0, 7.0 Hz, H5), 7.40 (1H, dd,
J = 6.8, 1.8 Hz, H6), 7.52–7.56 (2H, m, m-Ph), 7.59–7.63 (1H, m, p-
Ph), 7.89–7.91 (2H, m, o-Ph), 8.18 (1H, br t, J = 5.6 Hz, 20-NH),
8.2.15. N-{1-[20-(Butylamino)-20-oxoethyl]-2-oxo-1,2-
dihydropyridin-3-yl}benzamide (9h)
8.28 (1H, dd, J = 7.2, 1.8 Hz, H4), 9.28 (1H, br s, W ꢁ 17 Hz,
½
13
CONH); C NMR (100 MHz, DMSO-d6) d 41.3 (C200), 42.5 (C100),
Compound
5
(0.200 g, 0.666 mmol), and neat butylamine
51.7 (C10), 104.8 (C5), 123.6 (C4), 127.1 (o-Ph), 128.0 (C3), 128.8
(m-Ph), 132.0 (p-Ph), 133.7 (C6), 133.9 (i-Ph), 157.1 (C2), 164.8
(CO), 166.4 (C20); MS (ESI) m/z (%): 315.3 ([M+H]+, 100); 321.3
([M+Li]+, 100). Anal. Calcd for C16H18N4O3: C, 61.14; H, 5.77; N,
17.82. Found: C, 60.91; H, 5.90; N, 18.02.
(3.0 mL) were treated at room temperature for 16 h using meth-
od B. Compound 9h was obtained (0.192 g, 88%). Mp 201–
205 °C; 1H NMR (400 MHz, DMSO-d6) d 0.87 (3H, t, J = 7.0 Hz,
CH3), 1.26–1.32 (2H, m, CH2CH3), 1.36–1.43 (2H, m, NHCH2CH2),

N. D. Karis et al. / Bioorg. Med. Chem. 17 (2009) 4724–4733
4731
8.2.19. N-(2-Oxo-1-{20-oxo-20-[200-(piperidin-1000-
(1H, dd, J = 6.8, 1.6 Hz, H6), 7.52–7.56 (2H, m, m-Ph), 7.59–7.63
(1H, m, p-Ph), 7.77 (1H, ddd, J = 7.6, 7.6, 1.8 Hz, H500), 7.90–
7.92 (2H, m, o-Ph), 8.29 (1H, dd, J = 7.2, 1.6 Hz, H4), 8.49–8.51
(1H, m, H300), 8.83 (1H, br t, J = 6.0 Hz, 20-NH), 9.30 (1H, s,
yl)ethylamino]ethyl}-1,2-dihydropyridin-3-yl) benzamide (9n)
Compound 5 (0.300 g, 0.999 mmol), and neat 1-(2-amino-
ethyl)piperidine (1.5 mL) were treated at room temperature for
16 h using method B. Compound 9n was obtained (0.620 g,
95%). Mp 199–202 °C; 1H NMR (400 MHz, DMSO-d6) d 1.30–
1.37 (2H, m, H4000), 1.44–1.50 (4H, m, H3000, H5000), 2.28–2.32
(6H, m, H200, H2000, H6000), 3.17 (2H, dt, J = 6.8, 6.0 Hz, H100), 4.63
(2H, s, H10), 6.33 (1H, dd, J = 7.2, 7.2 Hz, H5), 7.40 (1H, dd,
J = 7.0, 1.6 Hz, H6), 7.52–7.56 (2H, m, m-Ph), 7.59–7.63 (1H, m,
p-Ph), 7.89–7.91 (2H, m, o-Ph), 8.09 (1H, br t, J = 5.4 Hz, 20-
NH), 8.28 (1H, dd, J = 7.2, 1.6 Hz, H4), 9.28 (1H, s, CONH); 13C
NMR (100 MHz, DMSO-d6) d 24.0 (C4000), 25.5 (C3000, C5000), 36.4
(C100), 51.6 (C10), 54.0 (C2000, C6000), 57.6 (C200), 104.9 (C5), 123.6
(C4), 127.1 (o-Ph), 128.0 (C3), 128.8 (m-Ph), 132.0 (p-Ph), 133.6
(C6), 133.9 (i-Ph), 157.1 (C2), 164.8 (CO), 166.2 (C20); MS (ESI)
m/z (%): 405.3 ([M+Na]+, 30), 383.3 ([M+H]+, 100); 389.3
([M+Li]+, 20), 383.3 ([M+H]+, 100). HRMS: calcd for [M+H]+
C21H27N4O3 383.2078, found 383.2063.
13
CONH); C NMR (100 MHz, DMSO-d6) d 44.3 (CH2), 51.8 (C10),
105.0 (C5), 121.1 (C600), 122.2 (C400), 123.8 (C4), 127.1 (o-Ph),
128.1 (C3), 128.8 (m-Ph), 132.1 (p-Ph), 133.7 (C6), 133.9 (i-Ph),
136.8 (C500), 148.9 (C300), 157.2 (C2), 158.2 (C100), 164.9 (CO),
166.8 (C20); MS (ESI) m/z (%): 369.2 ([M+Li]+, 100). Anal. Calcd
for C20H18N4O3: C, 66.29; H, 5.01; N, 15.46. Found: C, 66.44; H,
4.99; N, 15.40.
8.2.23. N-{1-[20-(300,400-Dichlorobenzylamino)-20-oxoethyl]-2-
oxo-1,2-dihydropyridin-3-yl}benz amide (9t)
Compound 5 (0.100 g, 0.333 mmol), and neat 3,4-dichloroben-
zylamine (0.5 mL) were treated at 120 °C 4 h using method A. Com-
pound 9t was obtained (0.143 g, 100%). Mp 261–262 °C; 1H NMR
(400 MHz, DMSO-d6) d 4.32 (2H, d, J = 5.6 Hz, CH2), 4.71 (2H, s,
H10), 6.34 (1H, dd, J = 7.0, 7.0 Hz, H5), 7.27 (1H, dd, J = 8.4, 2.0 Hz,
H600), 7.45 (1H, dd, J = 7.0, 1.8 Hz, H6), 7.54–7.64 (5H, m, m, p-Ph,
H200, H500), 7.89–7.92 (2H, m, o-Ph), 8.30 (1H, dd, J = 7.4, 1.8 Hz,
H4), 8.80 (1H, br t, J = 5.6 Hz, 20-NH), 9.30 (1H, s, CONH); 13C
NMR (100 MHz, DMSO-d6) d 41.1 (CH2), 52.0 (C10), 105.1 (C5),
123.6 (C4), 127.0 (o-Ph), 127.5 (C600), 128.0 (C3), 128.8 (m-Ph),
129.1 (C200), 129.3 (C300 or C400), 130.4 (C500), 130.9 (C300 or C400),
132.2 (p-Ph), 133.6 (C6), 133.9 (i-Ph), 140.4 (C100), 157.1 (C2),
164.8 (CO), 166.8 (C20); MS (ESI) m/z (%): 452.1 ([M+Na]+, 100);
436.1 ([M+Li]+, 100). Anal. Calcd for C21H17Cl2N3O3: C, 58.62; H,
3.98; N, 9.77. Found: C, 58.34; H, 3.66; N, 9.59.
8.2.20. N-{1-[20-(Morpholinoamino)-20-oxoethyl]-2-oxo-1,2-
dihydropyridin-3-yl}benzamide (9p)
Compound 5 (0.300 g, 0.999 mmol), and neat 4-aminomorpho-
line (1.5 mL) were treated at room temperature for 16 h using
method B. Compound 9p was obtained (0.316 g, 89%). Mp 258–
260 °C (dec); 1H NMR (400 MHz, DMSO-d6) d 2.60–3.00 (4H, m,
2 ꢂ NCH2), 3.40–3.90 (4H, m, 2 ꢂ CH2O), 5.74 (2H, s, H10), 6.33
(1H, dd, J = 7.0, 7.0 Hz, H5), 7.40 (1H, dd, J = 6.6, 1.8 Hz, H6),
7.52–7.55 (2H, m, m-Ph), 7.59–7.63 (1H, m, p-Ph), 7.89–7.91 (2H,
m, o-Ph), 8.27–8.30 (1H, m, H4), 8.98 (1H, s, 20-NH), 9.28 (1H, s,
13
CONH); C NMR (100 MHz, DMSO-d6) d 50.1 (C10), 54.8 (NCH2),
8.2.24. N-{1-[20-(100-Benzylpiperidin-400-ylamino)-20-oxoethyl]-
2-oxo-1,2-dihydropyridin-3-yl}benz amide (9w)
55.7 (NCH2), 65.8 (2 ꢂ CH2O), 104.8 (C5), 123.4 (C4), 127.1 (o-
Ph), 128.0 (C3), 128.8 (m-Ph), 132.1 (p-Ph), 133.7 (C6), 133.8 (i-
Ph), 157.1 (C2), 164.8 (CO), 168.5 (C20); MS (ESI) m/z (%): 379.2
([M+Na]+, 100), 357.2 ([M+H]+, 10); 363.2 ([M+Li]+, 100). Anal.
Calcd for C18H20N4O4: C, 60.67; H, 5.66; N, 15.72. Found: C,
60.68; H, 5.70; N, 15.71.
Compound 5 (0.300 g, 0.999 mmol), and neat 4-amino-1-ben-
zylpiperidine (1.5 mL) were treated at 120 °C for 4 h using method
A. Compound 9w was obtained (0.383 g, 86%). Mp 191–194 °C; 1H
NMR (400 MHz, DMSO-d6) d 1.36–1.46 (2H, m, H200, H600), 1.70–
1.73 (2H, m, H200, H600), 1.97–2.02 (2H, m, H300, H500), 2.72–2.75
(2H, m, H300, H500), 3.43 (2H, s, 400-CH2), 3.49–3.58 (1H, m, H100),
4.62 (2H, s, H10), 6.32 (1H, dd, J = 7.0, 7.0 Hz, H5), 7.21–7.32 (5H,
m, CH2Ph), 7.39 (1H, dd, J = 6.8, 1.6 Hz, H6), 7.51–7.55 (2H, m, m-
Ph), 7.58–7.62 (1H, m, p-Ph), 7.89–7.91 (2H, m, o-Ph), 8.19 (1H,
d, J = 7.6 Hz, 20-NH), 8.27 (1H, dd, J = 7.4, 1.6 Hz, H4), 9.28 (1H, s,
8.2.21. N-{1-[20-(300-Morpholinopropylamino)-20-oxoethyl]-2-
oxo-1,2-dihydropyridin-3-yl}benz amide (9q)
Compound 5 (0.300 g, 0.999 mmol), and neat 3-morpholinopro-
pylamine (1.5 mL) were treated at room temperature for 16 h
using method B. Compound 9q was obtained (0.356 g, 89%). Mp
165–169 °C (dec); 1H NMR (400 MHz, DMSO-d6) d 1.56 (2H, tt,
J = 7.2, 7.2 Hz, H200), 2.26–2.32 (6H, m, H300, 2 ꢂ NCH2), 3.10 (2H,
dt, J = 6.8, 5.6 Hz, H100), 3.53–3.56 (4H, m, 2 ꢂ CH2O), 4.61 (2H, s,
H10), 6.33 (1H, dd, J = 7.0, 7.0 Hz, H5), 7.40 (1H, dd, J = 6.8, 1.6 Hz,
H6), 7.54–7.56 (2H, m, m-Ph), 7.59–7.63 (1H, m, p-Ph), 7.87–7.91
(2H, m, o-Ph), 8.19 (1H, br t, J = 5.4 Hz, 20-NH), 8.28 (1H, dd,
J = 7.6, 2.0 Hz, H4), 9.28 (1H, s, CONH); 13C NMR (100 MHz,
DMSO-d6) d 25.8 (C200), 36.9 (C100), 51.6 (C10), 53.2 (2 ꢂ NCH2),
55.5 (C300), 66.1 (2 ꢂ CH2O), 104.7 (C5), 123.5 (C4), 127.0 (o-Ph),
127.9 (C3), 128.7 (m-Ph), 132.0 (p-Ph), 133.6 (C6), 133.8 (i-Ph),
157.0 (C2), 164.7 (CO), 166.1 (C20); MS (ESI) m/z (%): 421.3
([M+Na]+, 10), 399.3 ([M+H]+, 100); 405.3 ([M+Li]+, 25), 399.3
([M+H]+, 100). Anal. Calcd for C21H26N4O4: C, 63.30; H, 6.58; N,
14.06. Found: C, 63.16; H, 6.69; N, 14.09.
13
CONH); C NMR (100 MHz, DMSO-d6) d 31.6 (C200, C600), 46.3
(C100), 51.5 (C10), 51.8 (C300, C500), 62.1 (400-CH2), 104.7 (C5), 123.6
(C4), 126.8 (p-Bn), 127.1 (o-Ph), 128.0 (C3), 128.1 (o-Bn), 128.7
(m-Bn), 128.8 (m-Ph), 132.0 (p-Ph), 133.8 (C6), 133.9 (i-Ph), 138.6
(i-Bn), 157.1 (C2), 164.8 (CO), 165.5 (C20); MS (ESI) m/z (%): 445.3
([M+H]+, 100); 451.3 ([M+Li]+, 30), 445.3 ([M+H]+, 100). Anal. Calcd
for C26H28N4O3: C, 70.25; H, 6.35; N, 12.60. Found: C, 70.21; H,
6.35; N, 12.62.
8.3. General procedure for the preparation of compounds 7u–v,
9r, 9v, and 9y
Method C: A 1:1 equimolar mixture of ester 4 and solid amine
was melted together and transferred to a conical micro-scale
reaction vessel. The resulting solid mixture was placed in a
pre-heated oil bath at 120 °C. Heating was carried out without
stirring until a solution was formed and then with stirring until
all eliminated ethanol was evaporated and a solid formed, or 4 h
if no solid was formed. The crude reaction mixture was trans-
ferred to a conical flask and re-dissolved in warm acetone. Small
amounts of diethyl ether were added and the resulting solution
cooled in a freezer overnight forming a white solid. The required
compounds 7u–v, 8t, 9r, 9v, and 9y were obtained as colorless
solids.
8.2.22. N-{2-Oxo-1-[20-oxo-20-(pyridin-200-
ylmethylamino)ethyl]-1,2-dihydropyridin-3-yl}benz amide (9s)
Compound
5 (0.300 g, 0.999 mmol), and neat 2-(amino-
methyl)pyridine (1.5 mL) were treated at 120 °C for 4 h using
method A. Compound 9s was obtained (0.336 g, 93%). Mp 206–
208 °C (dec); 1H NMR (400 MHz, DMSO-d6)
d 4.41 (2H, d,
J = 6.0 Hz, CH2), 4.76 (2H, s, H10), 6.35 (1H, dd, J = 7.0, 7.0 Hz,
H5), 7.25–7.28 (1H, m, H400), 7.35 (1H, d, J = 7.6 Hz, H600), 7.45

4732
N. D. Karis et al. / Bioorg. Med. Chem. 17 (2009) 4724–4733
8.3.1. (S)-2-[30-(Benzylamino)-20-oxopyridin-10(2H)-yl]-N-(100-
hydroxy-300-phenylpropan-200-yl) acetamide (7u)
7.21–7.33 (5H, m, 100-Ph), 7.39 (1H, dd, J = 6.8, 1.6 Hz, H6), 7.51–
7.55 (2H, m, m-Ph), 7.59–7.62 (1H, m, p-Ph), 7.88–7.90 (2H, m, o-
Ph), 8.26 (1H, dd, J = 7.2, 1.6 Hz, H4), 8.69 (1H, d, J = 8.4 Hz, 20-
NH), 9.27 (1H, s, CONH); 13C NMR (100 MHz, DMSO-d6) d 51.5
(C10), 55.2 (C100), 64.6 (C200), 104.8 (C5), 123.6 (C4), 126.8 (p0-Ph),
127.0 (o0-Ph), 127.1 (o-Ph), 128.0 (C3), 128.1 (m0-Ph), 128.8 (m-
Ph), 132.0 (p-Ph), 133.8 (i-Ph), 133.9 (C6), 140.9 (i0-Ph), 157.1
(C2), 164.8 (CO), 166.1 (C20); MS (ESI) m/z (%): 414.3 ([M+Na]+,
100), 392.3 ([M+H]+, 15); 398.3 ([M+Li]+, 100). Anal. Calcd for
C22H21N3O4: C, 67.51; H, 5.41; N, 10.74. Found: C, 67.54; H, 5.20;
N, 10.74.
Compound 3 (0.300 g, 1.05 mmol), and neat (S)-2-amino-3-phe-
nyl-1-propanol (0.158 g, 1.05 mmol) were treated at 120 °C for 1 h
using method C. Compound 7u was obtained (0.344 g, 84%). Mp
156–158 °C; 1H NMR (400 MHz, DMSO-d6) d 2.64 (1H, dd, J = 7.8,
13.4 Hz, H300), 2.83 (1H, dd, J = 5.8, 13.8 Hz, H300), 3.28–3.39 (2H,
m, H100), 3.83–3.91 (1H, m, H200), 4.27 (2H, d, J = 6.4 Hz, CH2),
4.48 (2H, ABq, J = 15.2 Hz, H2), 4.77 (1H, t, J = 5.4 Hz, OH), 5.95–
6.03 (2H, m, H50, 30-NH), 6.04 (1H, dd, J = 7.2, 1.6 Hz, H40), 6.65
(1H, dd, J = 6.8, 1.6 Hz, H60), 7.15–7.31 (10H, m, Ph, 300-Ph), 8.04
(1H, d, J = 8.4 Hz, 1-NH); 13C NMR (100 MHz, DMSO-d6) d 36.5
(C300), 46.1 (CH2), 51.1 (C2), 52.7 (C200), 62.2 (C100), 105.6 (C50),
106.3 (C40), 124.6 (C60), 125.9 (p-Ph or p-300-Ph), 126.7 (p-Ph or p-
300-Ph), 127.0 (o-Ph), 128.1 (m-Ph), 128.3 (m-300-Ph), 129.1 (o-300-
Ph), 137.8 (C30), 137.8 (i-300-Ph), 139.4 (i-Ph), 156.9 (C20), 166.5
(C1); MS (ESI) m/z (%): 414.2 ([M+Na]+, 100), 392.2 ([M+H]+, 10);
398.2 ([M+Li]+, 100). Anal. Calcd for C23H25N3O3: C, 70.57; H,
6.44; N, 10.73. Found: C, 70.58; H, 6.36; N, 10.81.
8.3.5. (R)-N-(1-{20-[(100-Benzyl-200-hydroxyethyl)amino]-20-
oxoethyl}-2-oxo-1,2-dihydropyridin-3-yl)benzamide (9y)
Compound 5 (0.300 g, 0.999 mmol), and neat (R)-2-amino-3-
phenyl-1-propanol (0.151 g, 0.999 mmol) were treated at 120 °C
for 1 hour using method C. Compound 9y was obtained (0.364 g,
90%). Mp 207–209 °C; 1H NMR (400 MHz, DMSO-d6) d 2.65 (1H,
dd, J = 13.6, 7.6 Hz, 100-CH2), 2.85 (1H, dd, J = 13.6, 6.0 Hz, 100-CH2),
3.29–3.40 (2H, m, H200), 3.83–3.91 (1H, m, H100), 4.61 (2H, ABq,
J = 15.6 Hz, H10), 4.80 (1H, t, J = 5.2 Hz, OH), 6.31 (1H, dd, J = 7.2,
7.2 Hz, H5), 7.16–7.31 (H6, m, 6H, CH2Ph), 7.52–7.56 (2H, m, m-
Ph), 7.59–7.63 (1H, m, p-Ph), 7.89–7.91 (2H, m, o-Ph), 8.17 (1H,
d, J = 8.0 Hz, 20-NH), 8.26 (1H, dd, J = 7.2, 1.6 Hz, H4), 9.28 (1H, s,
8.3.2. (S)-2-[30-(Benzylamino)-20-oxopyridin-10(2H)-yl]-N-(200-
hydroxy-100-phenylethyl)acetamide (7v)
Compound 3 (0.300 g, 1.05 mmol), and neat (S)-2-amino-2-
phenylethanol (0.144 g, 1.05 mmol) were treated at 120 °C for
4 h using method C. Compound 7v was obtained (0.336 g, 85%).
Mp 181–181.5 °C (dec); 1H NMR (400 MHz, DMSO-d6) d 3.58 (2H,
dd, J = 5.8, 5.8 Hz, H200), 4.26 (2H, d, J = 6.4 Hz, CH2), 4.61 (2H,
ABq, J = 15.4 Hz, H2), 4.81–4.88 (2H, m, H100, OH), 5.94–5.96 (2H,
m, H50, NH), 6.04 (1H, dd, J = 7.2, 1.6 Hz, H40), 6.77 (1H, dd,
J = 6.8, 1.6 Hz, H60), 7.18–7.34 (10H, m, Ph, 100-Ph), 8.55 (1H, d,
J = 8.4 Hz, 1-NH); 13C NMR (100 MHz, DMSO-d6) d 46.0 (CH2),
51.2 (C2), 55.1 (C100), 64.6 (C200), 105.6 (C50), 106.4 (C40), 124.8
(C60), 126.7 (p-Ph, or p-100-Ph), 126.8 (p-Ph, or p-100-Ph), 126.9 (o-
Ph), 127.0 (o-100-Ph), 128.0 (m-Ph), 128.3 (m-100-Ph), 137.8 (C30),
139.4 (i-Ph), 140.9 (i-100-Ph), 157.0 (C20), 166.5 (C1); MS (ESI) m/z
(%): 400.2 ([M+Na]+, 100), 378.2 ([M+H]+, 10); 384.2 ([M+Li]+,
100). Anal. Calcd for C22H23N3O3: C, 70.01; H, 6.14; N, 11.13.
Found: C, 69.75; H, 6.37; N, 11.03.
13
CONH); C NMR (100 MHz, DMSO-d6) d 37.2 (CH2Ph), 52.1 (C10),
53.6 (C100), 62.8 (C200), 105.5 (C5), 124.3 (C4), 126.7 (p-CH2Ph),
127.8 (o-Ph), 128.7 (C3), 128.9 (m-CH2Ph), 129.5 (m-Ph), 129.9
(o-CH2Ph), 132.7 (p-Ph), 134.3 (C6), 134.6 (i-Ph), 139.6 (i-CH2Ph),
157.8 (C2), 165.5 (CO), 166.7 (C20); MS (ESI) m/z (%): 428.3
([M+Na]+, 100), 406.3 ([M+H]+, 15); 412.3 ([M+Li]+, 100). Anal.
Calcd for C23H23N3O4: C, 68.13; H, 5.72; N, 10.36. Found: C,
68.07; H, 5.61; N, 10.30.
9. X-ray structure determination
A full cif deposition resides with the Cambridge Crystallo-
graphic Data Centre, CCDC for 9k, 7u, and 7v; 698940, 698941,
698942, respectively. Crystal data, ORTEP plots are described in
the Supplementary data.
8.3.3. N-[1-(20-{20-[4000-(Aminosulfonyl)phenyl]ethylamino}-20-
oxoethyl)-2-oxo-1,2-dihydro pyridine-3-yl]benzamide (9r)
Compound 5 (0.200 g, 0.666 mmol), and neat 4-(2-amino-
ethyl)benzenesulfonamide (0.133 g, 0.666 mmol) were treated at
120 °C for 1 h using method C. Compound 9r was obtained
(0.302 g, 100%). Mp 258–260 °C (dec); 1H NMR (400 MHz, DMSO-
d6) d 2.81 (2H, t, J = 7.0 Hz, H200), 3.32–3.36 (2H, m, H100 and
H2O), 4.62 (2H, s, H10), 6.34 (1H, dd, J = 7.0, 7.0 Hz, H5), 7.30 (2H,
s, NH2), 7.38–7.43 (3H, m, H6, H2000, H6000), 7.52–7.56 (2H,m, m-
Ph), 7.59–7.63 (1H, m, p-Ph), 7.76 (2H, d, J = 8.4 Hz, H3000, H5000),
7.91 (2H, d, J = 7.2 Hz, o-Ph), 8.29 (1H, m, H4), 8.35 (1H, t,
J = 5.4 Hz, 20-NH), 9.29 (1H, s, NH); 13C NMR (100 MHz, DMSO-d6)
d 34.7 (C200), 40.0 (C100), 51.6 (C10), 104.9 (C5), 123.7 (C4), 125.7
(C3000, C5000), 127.1 (o-Ph), 128.0 (C3), 128.8 (m-Ph), 129.2 (C2000,
C6000), 132.1 (p-Ph), 133.7 (C6), 133.9 (i-Ph), 142.1 (C4000), 143.5
(C1000), 157.1 (C2), 164.9 (CO), 166.5 (C20); MS (ESI) m/z (%): 477.2
([M+Na]+, 100), 455.2 ([M+H]+, 35); 461.3 ([M+Li]+, 100). HRMS:
calcd for [M+Na]+ C22H22O5N4SNa 477.1203, found 477.1192.
10. GPa inhibition assay
RMGPa (Rabbit Muscle Glycogen Phosphorylase a from Sigma)
(0.475 l
g/mL) activity was measured as described11,35 in the direc-
tion of glycogen synthesis by the formation of inorganic phosphate
from glucose-1-phosphate36 using a 384 well plate at 22 °C in
45 lL of buffer containing 50 mM Hepes (pH 7.2), 100 mM KCl,
2.5 mM EGTA, 2.5 mM MgCl2, 0.25 mM glucose-1-phosphate, and
1 mg/mL glycogen with a 30 min incubation time. Phosphate was
measured at 620 nm, 5 min after the addition of 150
containing 10 mg/mL ammonium molybdate and 0.38 mg/mL mala-
chite green.37 Test compoundswere added to theassay in 5
L of 14%
lL of 1 M HCl
l
DMSO. Compounds were tested against a caffeine standard in 11
point concentration–response curve in duplicate on two separate
occasions. DatawasanalyzedusingGraphPadPrismv.4.03. Anonlin-
ear regression (curve fit) analysis with a sigmoidal dose–response
equation (variable slope) was applied to generate IC50 and Hill slope
values. The reported IC50 had a Hill slope between 0.7 and 1.2 and a Z0
value of ꢁ0.8. Compounds were screened with maximal concentra-
8.3.4. (S)-N-(1-{20-[(200-Hydroxy-100-phenylethyl)amino]-20-
oxoethyl}-2-oxo-1,2-dihydropyridin-3-yl) benzamide (9v)
Compound 5 (0.300 g, 0.999 mmol), and neat (S)-2-amino-2-
phenylethanol (0.137 g, 0.999 mmol) were treated at 120 °C for
1 h using method C. Compound 9v was obtained (0.356 g, 91%).
Mp 207–210 °C; 1H NMR (400 MHz, DMSO-d6) d 3.58 (2H, dd,
J = 5.6, 5.6 Hz, H200), 4.74 (2H, s, H10), 4.84 (1H, dt, J = 8.0, 6.2 Hz,
H100), 4.91 (1H, t, J = 5.6 Hz, OH), 6.31 (1H, dd, J = 7.2, 7.2 Hz, H5),
tions of 222 lM. The assay was carefully monitored for signs of com-
pound insolubility. The results are presented as mean values from 4
determinations. Samples used in screening were of 98+% (1H NMR
purity; compounds 2, 4, 5, 7c, 7s, 8f, 9g–h, 9m–n, 9r) or 100% (micro-
analytical purity; compounds 3, 6a–c, 6t, 7a–b, 7d, 7t–x, 7g–j, 8a–e,
8t, 9a–e, 9i–l, 9o–q, 9s–w, 9y) purity.

N. D. Karis et al. / Bioorg. Med. Chem. 17 (2009) 4724–4733
4733
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Acknowledgments
The Financial support of (1) Diabetes Australia Research Trust,
(2) Griffith University, (2) Natural Product Discovery, Griffith Uni-
versity, (3) Eskitis Institute for Cell and Molecular Therapies, Grif-
fith University; (4) The award of an APAWS to N. D. Karis, (5) The
support of the Lead Discovery Biology section of Natural Product
Discovery, Griffith University, (6) Alan White for assistance with
X-ray data collection.
Supplementary data
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