Pyridones as glucokinase activators: Identification of a unique metabolic liability of the 4-sulfonyl-2-pyridone heterocycle

Article abstract of DOI:10.1016/j.bmcl.2009.04.107

A promising area of novel anti-diabetic therapy involves identification of small molecule activators of the glucokinase enzyme to reduce blood glucose and normalize glucose stimulated insulin secretion. Herein, we report the identification and optimizatio

Full text of DOI:10.1016/j.bmcl.2009.04.107

Bioorganic & Medicinal Chemistry Letters 19 (2009) 3247–3252
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Pyridones as glucokinase activators: Identification of a unique metabolic
liability of the 4-sulfonyl-2-pyridone heterocycle
b
a
a
Jeffrey A. Pfefferkorn a, , Jihong Lou , Martha L. Minich , Kevin J. Filipski
*
Mingying He b, Ru Zhou b, Syed Ahmed a, John Benbow a, Angel-Guzman Perez a
Meihua Tu a, John Litchfield a, Raman Sharma a, Karen Metzler a, Francis Bourbonais a
Cong Huang a, David A. Beebe a, Peter J. Oates a
a Pfizer Global Research & Development, Groton Laboratories, Eastern Point Road, Groton, CT 06344, USA
b Pfizer Global Research & Development, La Jolla Laboratories, 10770 Science Center Drive, San Diego, CA 92121, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A promising area of novel anti-diabetic therapy involves identification of small molecule activators of the
glucokinase enzyme to reduce blood glucose and normalize glucose stimulated insulin secretion. Herein,
we report the identification and optimization of a series of 4-sulfonyl-2-pyridone activators. The activa-
tors were evaluated for in vitro biochemical activation and pharmacokinetic properties. As part of these
efforts, a unique metabolic liability of the 4-sulfonyl-2-pyridone ring system was identified wherein this
heterocycle readily undergoes conjugation with glutathione under non-enzymatic conditions.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 18 March 2009
Revised 16 April 2009
Accepted 21 April 2009
Available online 3 May 2009
Keywords:
Glucokinase
Diabetes
Enzyme activation
Pyridone
Reactive metabolite
Type 2 diabetes mellitus (T2DM) is a rapidly expanding public
health problem affecting over 150 million people worldwide.1
The disease is characterized by elevated fasting plasma glucose
(FPG), insulin resistance, abnormally elevated hepatic glucose pro-
duction and reduced glucose stimulated insulin secretion (GSIS).2
While several classes of diabetic therapies are available for clinical
use, there still remains a significant need for new therapies with
improved efficacy and safety to help patients achieve their treat-
ment goals.3
A promising area of current research involves the use of small
molecule allosteric activators of the glucokinase (GK) enzyme to
lower blood glucose and normalize insulin secretion.4 Glucokinase
is responsible for the conversion of glucose to glucose-6-phosphate
(G-6-P), and it functions as a key regulator of glucose homeostasis.5
In the liver, glucokinase regulates hepatic glucose utilization and
output whereas in the pancreas it functions as a glucostat estab-
lishing the threshold for b-cell glucose-stimulated insulin secre-
tion.5 Glucokinase is also found in glucose sensing neurons of the
ventromedial hypothalamus where it regulates the counter regula-
tory response (CRR) to hypoglycemia.6 Finally, glucokinase is
reportedly expressed in the endocrine K and L cells where it may
help regulate incretin release.7 Therapeutically, it is anticipated
that activation of glucokinase in the liver and pancreas would be
an effective strategy for lowering blood glucose by up regulating
hepatic glucose utilization, down regulating hepatic glucose output
and normalizing glucose stimulated insulin secretion.4
Glucokinase (hexokinase IV) is unique among the members of
the hexokinase family given its low substrate binding affinity
(S0.5ꢀ8 mM), positive substrate cooperativity and lack of product
inhibition.8 As a monomeric enzyme, glucokinase achieves this
cooperativity through equilibration between multiple conforma-
tions.8 In pioneering work, Grimsby and co-workers demon-
strated that small molecule activators were capable of binding
to glucokinse at an allosteric site 20 Å remote from the active site
and influencing the enzyme’s kinetic profile by modulating both
9
S0.5 and Vmax
.
These efforts resulted in the identification of a
phenylacetamide series of activators represented by 1 and 2. A re-
lated series of activators represented by 3 was later reported by
Fyfe et al.10 Subsequently, a variety of other structurally diverse
glucokinase activators have been identified, including aryl amides
4 and 5.11,12 These and other small molecule activators of gluco-
kinase have been recently reviewed.4 Encouragingly, these small
molecule glucokinase activators have been shown to effectively
lower blood glucose in a variety of diabetic animal models. Fur-
thermore, 2 was advanced to Phase 2 clinical studies and found
to effectively lower fasting and postprandial glucose in T2DM pa-
tients (Fig. 1).13
* Corresponding author. Tel.: +1 860 686 3421; fax: + 1 860 715 4608.
E-mail address: jeffrey.a.pfefferkorn@pfizer.com (J.A. Pfefferkorn).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.04.107

3248
J. A. Pfefferkorn et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3247–3252
O
OEt
OEt
O
O
O
O
a
c
Cl
Cl
Cl
Cl
H
N
8
9
H
N
S
Cl
N
N
b
O
N
O
S
O
S
O
(EtO)2HC
O
O
O
O
1: RO-28-1675
EC50 = 0.69 µM
Max Activation = 81%
2: RO-1440
CO2Et
CO2Et
EtO
EC50 = 1.42 µM
Max Activation = 148%
EtO
P O
EtO
11
10
O
d
O
S
H
S
F
N
OEt
OEt
N
N
H
N
S
N
F
EtO
MeS
EtO
O
NH2
O
N
CO2R1
S
e
h
OEt
MeO2S
f
O
O
3
4
13: R1 = Et
14: R1 = H
EC50 = 0.18 µM
Max Activation = 96%
EC50 = 0.62 µM
Max Activation = 108%
12
CO2H
g
O
O
N
N
H
H
5
N
N
O
EC50 = 0.045 µM
N
N
CO2Bn
O
Max Activation = 23%
O
S
O
S
O
O
O
O
O
Figure 1. Structures and biochemical properties of representative glucokinase
activators. EC50 and percent maximum activation (above control) were measured at
6.5 mM glucose and are reported as the mean of n > 2 determinations.
7
15
Scheme 1. Synthetic method A. Reagents and conditions: (a) Ethyl vinyl ether,
Et2O, 0 °C, 1 h; (b) (i) EtOH, 0?5 °C; (ii) Et3N, 10–25 °C, 0.5 h, 63% over two steps;
(c) (i) NaH, THF, 0 °C, 10 min; (ii) (EtO)2POCl, THF, 12 h, 89%; (d) NaSMe, NH4Cl, THF,
À60?25 °C, 12 h, 76%; (e) mCPBA, CH2Cl2, 0–25 °C, 1 h, 91%; (f) LiOH, MeOH, THF,
H
N
H
N
H2O, 25 °C, 0.5 h, 100%; (g) (i) L
-cyclopentylalanine benzyl ester, PyBrOPÒ, DIPEA,
X
Y
N
CH2Cl2, 0–25 °C, 12 h; (ii) THF, HCl (aq), 80 °C, 43%; (h) 2-amino-5-picoline, AlMe3,
1,2-DCE, 0?25 °C, 12 h, 76%.
N
N
Het
R1
O
O
R2
S
O
S
O
6
O
O
O
7
EC50 = 0.56 µM
Max Activation = 58%
was then added to enol phosphate 11 to afford sulfide 12 which
was treated with mCPBA to afford smooth conversion to the corre-
sponding sulfone 13. The ester of 13 was saponified to carboxylic
Figure 2. Strategy of evaluating N-linked heterocyclic glucokinase activators
affording pyridone 7.
acid 14 which was subsequently coupled to L-cyclopentylalanine
benzyl ester to produce the corresponding amide. This amide
was then treated with aqueous 1 N HCl in THF and heated to reflux
resulting in cyclization to generate the desired 4-sulfonyl-2-pyri-
done 15. Finally, AlMe3-mediated transamidation of 15 with 2-
amino-5-picoline provided glucokinase activator 7.21 Substitution
of structurally varied thiols, amino acid esters and heterocyclic
amines into this synthetic route enabled preparation of a diverse
set of activators.
Given the promise of glucokinase activation for treating T2DM,
we have been investigating structurally novel small molecule
activators. Of particular interest is the replacement of the aryl
ring of the phenylacetamide series with N-linked heterocycles
in an effort to improve potency and reduce lipophilicity. Previous
literature reports suggested that heterocycles such as hydanto-
ins,14 isoindolin-1-ones15 and indazoles16 are tolerated at this po-
sition. Through evaluation of a variety of other heterocycles, we
identified the 4-sufonyl-2-pyridone as an effective replacement
as illustrated by 7 (Fig. 2).17 Herein, we describe the evaluation
of a series of glucokinase activators based upon this template.
As highlighted in Schemes 1 and 2, two general synthetic strat-
egies (A and B) were employed to construct the activators utilized
in the current studies. The first approach (Scheme 1) utilized a
cyclization reaction to construct pyridone heterocycles on amino
acid derived substrates. Complete synthetic details of this 4-sulfo-
nyl-pyrid-2-one forming reaction have recently been reported.18
Briefly, this approach required initial construction of a linear pyri-
done precursor which commenced with reaction of malonyl chlo-
ride (8) with ethyl vinyl ethers to form intermediate 9 which
was subsequently treated with ethanol followed by triethylamine
to generate ketoester 10.19 The enolate of ketoester 10 was then
generated by treatment with sodium hydride and reacted with
diethyl chlorophosphate to afford enol phosphate 11 as an inconse-
quential mixture of geometric isomers.20 Sodium thiomethoxide
For analogs not accessible via the above approach (particularly
those bearing poly-substituted pyridone rings), a complementary
pyridone N-alkylation strategy was employed as shown in Scheme
2. This route commenced with conversion of amino acid D-cyclo-
pentylalanine (16) to the corresponding hydroxy ester 17 via a
diazatization reaction that proceeded with net retention of stereo-
chemistry.22 Carboxylic acid 17 was then converted to the corre-
sponding methyl ester 18 via treatment with thionyl chloride
and methanol. In preparation for a pyridone alkylation reaction,
triflate 19 was prepared from 18 via treatment with 2,6-lutidine
and trifluoromethanesulfonic anhydride. Separately, the pyridone
21 coupling partner was prepared from 4,5-dichloro-2-amino-pyr-
idine (20) via diazatization and subsequent hydrolysis. Alkylation
of the newly formed pyridone 21 was accomplished via deprotona-
tion with sodium hydride and reaction with triflate 19 to afford the
desired N-alkylated product 22 in modest yield with inversion of
stereochemistry along with the O-alkylated side product. Installa-
tion of the 4-sulfone moiety was accomplished by reaction of

J. A. Pfefferkorn et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3247–3252
3249
All analogs were initially evaluated in a biochemical activation
assay with human glucokinase (hexokinase IV) measuring potency
and percentage maximum activation (above control) at 6.5 mM
glucose.23 Initially, structure–activity relationships of the pyridone
ring were evaluated as highlighted in Table 1. Increasing lipophil-
icity via steric bulk at the sulfone position afforded increasing po-
tency as illustrated with 29 (R2 = cyclobutyl) being 10-fold more
potent that 7 (R2 = Me). The changes at the R2 had a negligible im-
pact on the maximum activation of these analogs. Consistent with
previous reports from the phenylacetamide series, addition of
small substituents adjacent to the sulfone (R1 or R3) afforded slight
improvements in potency with the most notable being 33 (R3 = Cl)
which was three fold more potent than its unsubstituted compar-
ator 27 (R3 = H). Interestingly, in the case of methyl sulfones 25 and
30, addition of either Cl or Me at the R1 position afforded a signif-
icant increase in maximum activation with only a marginal effect
on potency.
a
c
H2N
CO2H
HO
CO2R1
TfO
CO2Me
17: R1 = OH
16
20
19
b
18: R1 = OMe
Cl
Cl
Cl
d
N
NH
e
NH2
Cl
O
21
f
Cl
Cl
Cl
N
CO2Me
N
CO2Me
S
O
O
Structure–activity studies next examined the effect of modifica-
tion of the cycloalkyl ring of these pyridone analogs as illustrated
in Table 2. Utilizing a relatively consistent set of sulfone and amide
23
22
g
substituents, the cyclopentyl (27, EC50 = 0.23
lM) and cyclohexyl
ring (36, EC50 = 0.24 M) were shown to offer optimal potency.
l
The smaller cyclobutyl ring of 35 as well as the acyclic isopropyl
of 34 were tolerated but tended to be several fold less potent than
the cyclopentyl comparator 27. Introduction of fluorine (e.g., 37) or
an ether linkage (e.g., 38) into the cyclohexyl ring of 37 resulted in
a loss of overall activation. Finally, the evaluation of an aryl ring
(e.g., 39) revealed it to be 14-fold less active than the correspond-
ing saturated cyclohexyl analog 36.
H
h
Cl
Cl
N
N
N
CO2Me
N
O
S
O
O
S
O O
O
O
24
25
Scheme 2. Synthetic method B. Reagents and conditions: (a) NaNO2, H2SO4, H2O,
0?25 °C, 19 h, 92%; (b) SOCl2, MeOH, 65 °C, 0.5 h, 72%; (c) trifluoromethanesulfonic
anhydride, 2,6-lutidine, CH2Cl2, 0 °C, 0.5 h, 96%; (d) NaNO2, HCl, H2O, 25 °C, 0.5 h,
83%; (e) NaH, THF, 25 °C, 2 h, 67%; (f) NaSMe, THF, 25 °C, 16 h, 57%; (g) OxoneÒ, THF,
H2O, 48 h, 71%.; (h) 2-amino-5-picoline, AlMe2Cl, 1,2-DCE, 0?25 °C, 3 h, 73%.
The final area of structural modification was the amino hetero-
cycle of the template’s amide moiety. Previous precedent has
established that a 2-amino nitrogen containing heterocycle is re-
quired to act as a donor-acceptor engaging the glucokinase protein
in a pair of hydrogen bonding interactions.4 Consistent with these
reports, preliminary efforts (analogs not shown) to remove this
motif in this series resulted in complete loss of biological activity.
Hence, a variety of 2-amino heterocycles were examined as high-
dichloride 22 with sodium thiomethoxide affording selective dis-
placement to generate thioether 23. Oxidation of this thioether
with OxoneÒ then afforded sulfone 24 in good yield. Finally, Al-
Me2Cl-mediated transamidation20 of ester 24 with 2-amino-5-pic-
oline provided glucokinase activator 25. Substitution of alternative
amino acids, pyridones and heterocyclic amines into this synthetic
route enabled preparation of a diverse set of activators.
Table 2
Biochemical properties of glucokinase activators 27, 34–39
R1
H
N
N
N
R2
O
S
O
Table 1
O O
Biochemical properties of glucokinase activators 7, 25–33
R1
R2
EC50
0.51
(lM)
Maximum activation (%)
37
H
34
35
iPr
R1
S
N
N
N
R2
O
O
iPr
iPr
0.67
0.23
72
65
R3
O O
R1
R2
R3 Synthetic method EC50
(lM) Maximum activation (%)
27
7
H
H
H
H
H
Me
Et
iPr
cPr
cBu
Me
H
H
H
H
H
H
H
H
H
Cl
A
A
A
A
A
B
A
B
A
B
0.56
0.50
0.23
0.11
0.06
0.48
0.44
0.26
0.16
0.07
58
73
65
76
67
122
103
69
48
57
26
27
28
29
X = CH2
X = CF2
X = O
36
37
38
iPr
iPr
Et
0.24
0.16
1.5
46
15
26
X
25 Cl
30 Me Me
31 Cl iPr
32 Me iPr
33 iPr
39
iPr
3.3
30
H
EC50 and percent maximum activation (above control) were measured at 6.5 mM
glucose and are reported as the mean of n > 2 determinations. All compounds were
prepared by synthetic Method A.
EC50 and percent maximum activation (above control) were measured at 6.5 mM
glucose and are reported as the mean of n > 2 determinations.

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J. A. Pfefferkorn et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3247–3252
Table 3
Biochemical properties of glucokinase activators 27, 40–51
H
N
R1
S
N
Het
R2
O
O
O
O
R1
R2
Heterocycle
Heterocycle substituent
Synthetic method
EC50
(lM)
Maximum activation (%)
40
27
41
42
H
H
H
Cl
iPr
iPr
iPr
Me
X = H
A
A
A
B
0.60
0.23
0.33
0.14
43
65
35
82
N
X = Me
X = CF3
X = Cl
X
43
44
45
46
H
H
Cl
H
cPr
cBu
iPr
X = H
X = H
X = Me
X = Me
A
A
B
A
1.3
94
66
104
101
N
N
0.59
0.70
3.5
X
X
Me
47
48
49
50
H
H
H
H
iPr
iPr
cPr
cBu
X = H
A
A
A
A
0.72
0.91
1.5
50
57
84
67
N
N
X = Me
X = Me
X = Me
0.26
N
S
51
H
iPr
A
1.3
42
EC50 and percent maximum activation (above control) were measured at 6.5 mM glucose and are reported as the mean of n > 2 determinations.
lighted in Table 3. Generally, 2-amino pyridine amides (27 and 40–
42) afforded the greatest potency while the 2-amino pyrazoles
(47–50) exhibited similar levels of activation but were several fold
less potent. Relative to their pyridine comparators, compounds
containing 2-aminopyrazine amides (43–46) were generally two
fold less potent but offered increased levels of maximum
activation.
liver microsomes. As shown, the analogs exhibited good stability
in human microsomes and moderate stability in rat microsomes.
We next selected two representative compounds (46 and 48)
for evaluation in in vivo PK experiments as shown in Table 5.
Somewhat unexpectedly, both 46 and 48 exhibited very high clear-
ance (Cl > 100 mg/min/kg), short half-lives (T1/2 < 0.5 h) and poor
bioavailabilities (F% < 10%).
Having evaluated the structure activity relationships of this ser-
ies and identified a set of analogs with promising biochemical
activity, we next sought to evaluate the ADME properties of se-
lected analogs. Generally, compounds in this series exhibited high
permeability (data not shown) and reasonable aqueous solubility
The apparent disconnect between the moderate predicted clear-
ance (based on in vitro microsomal data) for compound 46 and its
very high in vivo clearance prompted us to more closely examine
the metabolism of this compound. Biotransformation studies of
46 were conducted in both microsomes and hepatocytes to exam-
ine Phase 1 and Phase 2 metabolism, respectively.24 First, incuba-
tion of 46 in both human and rat microsomes revealed minor
hydroxylation of the cyclopentyl ring or the 5-methyl group of
the pyrazine consistent with the low microsomal clearance ob-
served for this compound (i.e., Table 4). Subsequently, as high-
lighted in Figure 3, incubation of 46 with rat hepatocytes
resulted in complete consumption of parent and the formation of
a single major metabolite which was confirmed to be glutathione
conjugate 52 via extracted ion chromatography (Fig. 4).
(>50 lM). The microsomal metabolic stability of a representative
set of analogs is shown in Table 4 examining both human and rat
Table 4
Metabolic stability of selected glucokinase activators in human and rat liver
microsomes
Structure
HLM
RLM
Clh (mL/min/kg)
T1/2 (min)
CLh (mL/min/kg)
T1/2 (min)
To better understand this propensity for bioconjugate forma-
tion, in a separate experiment, 46 was incubated directly with
aqueous glutathione (5 mM) at 37 °C for 1.0 h and the same conju-
gate 52 was observed in the absence of any metabolic activation.
Subsequently, analogs 26, 27, 28, 30 and 42 were evaluated under
these same conditions. These additional analogs contained increas-
ing steric bulk either directly on the sulfone or adjacent to it thus
enabling an evaluation as to whether or not steric hindrance might
mitigate this conjugation liability. Unfortunately, all of these ana-
logs also underwent facile reactions with glutathione suggesting
that steric considerations were not necessarily important and that
this was a general liability inherent to the 4-sulfonyl-2-pyridone
motif of these activators. This liability prevented further evaluation
of this series of glucokinase activators in models of diabetic effi-
cacy. Interestingly, a review of the literature did not afford similar
reports of the instability of the 4-sulfonyl-2-pyridone ring sys-
tem25 despite the use of this heterocycle in other chemotypes
7
Table 1
Table 2
Table 2
Table 3
Table 3
5
8
<5
<5
10
>120
73
>120
>120
93
58
36
<18
31
58
9
37
38
46
48
42
>120
56
8
Human liver (HLM) and rat liver (RLM) microsomal stability was determined via
incubation of 1.0 lM of compound with 0.8 mg/mL of protein.
Table 5
Pharmacokinetic parameters for compounds 46 and 48
F (%)
T1/2 (h)
Vdss (mL/kg)
Cl (mL/min/kg)
PPB Fu
46
48
2
8
0.2
0.2
4.6
2.0
337
164
0.35
0.59
Pharmacokinetic parameters expressed as geometric mean (n = 2) of Sprague–
Dawley rats dosed 0.5 mg/kg iv and 5 mg/kg po. Rat plasma protein binding (PPB) is
reported as fraction unbound.

J. A. Pfefferkorn et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3247–3252
3251
22.14
Rat Hepatocyte (t = 0 h)
46
30000
20000
10000
0
16.30
Rat Hepatocyte (t = 4 h)
52
15000
10000
5000
0
10
11
12
N
13
14
15
16
17
18
19
O
20
21
22
23
46: R1 = SO2Me
52: R1
NH2
=
H
N
S
N
H
CO2H
N
NH
HO2C
O
R1
O
N
O
Figure 3. Results of rat hepatocyte incubation with compound 46.
16.44
H
100
50
N
N
S
N
NH2
NH
O
O
S
N
N
N
O
Cl
F
N
O
F
O
O
Me
O
53: NHE-3 inhibitor
54: CB2 agonist
0
0
5
10
15
20
N
N
N
NH
nPr
Time (min)
N
Me
523.1855
S
O
O
100
O
O
O
HO
S
N
523.1857
O
55: Angiotensin II antagonist
Figure 5. Representative examples of other 4-sulfonyl-2-pyridones.
NH
394.1431
HN
O
O
NH
N
50
N
OH
O
Acknowledgment
H2N
We would like to thank Amit Kalgutkar for helpful discussions
regarding the biotransformation data.
394.1433
0
200 250 300 350 400 450 500 550 600 650
m/z
Supplementary data
Figure 4. Confirmation of the structure of glutathione conjugate 52 by extracted
ion chromatography. Extracted ion chromatogram of GSH conjugate 52 (top panel)
and exact mass product ion spectra obtained by CID of the MH+ ion (m/z 632) with
the origins of the diagnostic ions are as indicated.
Supplementary data (experimental procedures for the glucoki-
nase biochemical activation assay and metabolite identification
experiments) associated with this article can be found, in the on-
line version, at doi:10.1016/j.bmcl.2009.04.107.
including NHE-3 inhibitors (53, Fig. 5), CB2 agonists (54) and
angiotensin II receptor antagonists (55).26–28 Mechanistically, this
transformation can be viewed as a nucleophilic addition–elimina-
References and notes
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a,b-unsaturated amide. While the local elec-
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propensity for such a displacement reaction, our observations sug-
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