Anthranilimide-based glycogen phosphorylase inhibitors for the treatment of Type 2 diabetes: 2. Optimization of serine and threonine ether amino acid residues

Article abstract of DOI:10.1016/j.bmcl.2008.11.084

Optimization of the amino acid residue of a series of anthranilimide-based glycogen phosphorylase inhibitors is described leading to the identification of serine and threonine ether analogs. t-Butylthreonine analog 20 displayed potent in vitro inhibition of GPa, low potential for P450 inhibition, and excellent pharmacokinetic properties.

Full text of DOI:10.1016/j.bmcl.2008.11.084

Bioorganic & Medicinal Chemistry Letters 19 (2009) 981–985
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Anthranilimide-based glycogen phosphorylase inhibitors for the treatment
of Type 2 diabetes: 2. Optimization of serine and threonine ether amino
acid residues
*
Steven M. Sparks , Pierette Banker, David M. Bickett, Daphne C. Clancy, Scott H. Dickerson,
Dulce M. Garrido, Pamela L. Golden, Andrew J. Peat, Lauren R. Sheckler, Francis X. Tavares,
Stephen A. Thomson, James E. Weiel
Metabolic Center for Excellence in Drug Discovery, GlaxoSmithKline, 5 Moore Drive, PO Box 13398, Research Triangle Park, NC 27705, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Optimization of the amino acid residue of a series of anthranilimide-based glycogen phosphorylase inhib-
itors is described leading to the identification of serine and threonine ether analogs. t-Butylthreonine
analog 20 displayed potent in vitro inhibition of GPa, low potential for P450 inhibition, and excellent
pharmacokinetic properties.
Received 6 October 2008
Revised 17 November 2008
Accepted 19 November 2008
Available online 27 November 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Glycogen phosphorylase
Type 2 diabetes
Therapies targeting the reduction of hepatic glucose production
(HGP) as a method to control the hyperglycemia associated with
Type 2 diabetes have recently attracted considerable attention.1
Gluconeogenesis, the de novo synthesis of glucose from lactate,
amino acids, and glycerol, and glycogenolysis, the release of mono-
meric glucose from its polymeric storage form glycogen, are the
two processes which contribute to HGP. Pharmacological interven-
tions aimed at inhibiting both processes have been investigated
with the goal of reducing endogenous glucose production.2
Among biological targets which directly inhibit both gluconeo-
genesis and glycogenolysis, antagonists of the glucagon receptor3
and inhibitors of glucose-6-phosphatase translocase4 have been
examined. The selective inhibition of gluconeogenesis has been
studied with the development of inhibitors for fructose 1,6-bis-
phosphatase5 and phosphoenolpyruvate carboxykinase,2b while
inhibition of glycogen phosphorylase6 (GP) has been studied for
the selective inhibition of glycogenolysis. A variety of targets have
also been studied for the indirect reduction of HGP. These include
inhibitors of 11-b-hydroxysteroid dehydrogenase-1,1b dipeptidyl
peptidase-IV,7 and pyruvate dehydrogenase kinase,8 in addition
to activators of glucokinase9 and antagonists of the glucocorticoid
receptor.10
cogen phosphorylase (GP) inhibitors leading to the identification of
orally bioavailable compounds 1 and 2 with diminished activity at
CYP2C9 (Fig. 1).11 In this communication we report further optimi-
zation of the amino acid residue.
Early lead exploration efforts resulting from a high throughput
synthesis campaign revealed that anthranilimides with acyclic
amino acid residues provided submicromolar enzyme potency
Me
OH
OH
HN
HN
O
O
O
NH
O
NH
O
N
O
N
H
H
2
1
IC50 (+glu) = 5 nM
IC50 (cell) = 498 nM
CYP2C9 IC50 > 33,000 nM
28 %F (rat)
IC50 (+glu) = 10 nM
IC50 (cell) = 350 nM
CYP2C9 IC50 = 9,200 nM
37 %F (rat)
In the preceding communication we described the optimization
of the amino acid fragment of a series of anthranilimide based gly-
* Corresponding author. Tel.: +1 919 483 1104.
E-mail address: Steven.M.Sparks@gsk.com (S.M. Sparks).
Figure 1. In vitro profile of anthranilimides 1 and 2.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.11.084

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S. M. Sparks et al. / Bioorg. Med. Chem. Lett. 19 (2009) 981–985
and micromolar cellular potency.12 Thus, the primary goals of
these efforts were to identify acyclic amino acid residues which pro-
vided increased potency at the enzyme and cellular levels while
maintaining the pharmacokinetic profile of anthranilimides 1 and 2.
The general synthesis of the anthranilimide inhibitors is shown
in Scheme 1. Amide bond formation between the appropriate ami-
no acid ester and 2-aminonaphthoic acid provided the amide
derivatives I, which were then condensed with substituted 2,4,6-
trimethylphenyl-isocyanate and saponified to provide the desired
anthranilimide analogs II.
Our initial studies revealed that the enzyme preferred anthra-
nilimide inhibitors with nonpolar lipophilic amino acid residues.11
Based on this understanding, we surveyed several commercially
available amino acids with acyclic lipophilic residues (Table 1).
The compounds were evaluated in an enzyme assay utilizing hu-
man liver glycogen phosphorylase and subsequently progressed
to a cellular assay measuring inhibition of forskolin induced
glycogenolysis.11,16
Analogs derived from valine, tert-leucine, and norvaline (3–5) re-
vealed that increasing chain length provided a 10-fold increase in
Custom amino acid esters were prepared via several methods
detailed in Scheme 2. Dehydroamino esters were prepared by the
condensation of aldehydes with phosphorylglycine esters and sub-
sequently reduced to provide the racemic amino acid esters.13 In
select cases, the racemic anthranilimides derived from this method
were separated by chiral SFC to deliver enantiomerically pure final
products. Serine and threonine ether derivatives were prepared by
the Lewis acid assisted ring opening of N-acylated aziridines with
alcohols14 and C-4 substituted threonine analogs were prepared
utilizing the methods of Schöllkopf.15
enzyme potency while maintaining cellular potency near 1 lM (Ta-
ble 1). Examination of methyl substitution (6–8) revealed that both
b-substitution (isoleucine 6) and chain elongation (norleucine 8)
provided inhibitors with submicromolar potency in the cellular as-
say. Further modifications to the terminal atom of the residue
including elongation (9 and 10) produced a 3- to 4-fold reduction
in cellular potency. Having determined that a 4-atom residue was
optimal for both enzyme and cellular potency, d-substitution (11
and 12) was evaluated which provided excellent enzyme and cellu-
lar potency, but also increased CYP2C9 inhibition (8 vs 11 and 12).
R
CO2Me
HN
R
CO2H
a
O
CO2Me
H2N
+
NH2
NH2
I
R
O
CO2H
HN
b, c
NH
O
N
H
II
Scheme 1. General Syntheses of the anthranilimide core. Reagents and conditions: (a) HATU, iPrNEt2, DMF, rt (b) 2,4,6-trimethylphenyl isocyanate, pyridine, rt (c) 2 M LiOH,
MeOH, THF.
R
a, b
R
CHO
H2N
CO2Me
R
O
R
O
R
H
R
R
b
c
Cbz
BnO
N
N
H
CO2Me
Cbz
CO2Me
N
H
CO2Me
O
R
OH
OBn
R
e
O
N
d
N
O
O
N
H2N
O
N
O
O
Scheme 2. General syntheses of the amino acid esters. Reagents and conditions: (a) N-Cbz-2-(diethoxyphosphoryl)glycine methyl ester, DBU, DCM (b) H2, 10% Pd/C (c) ROH,
BF3ꢀEtO2, CHCl3 (d) n-BuLi, THF, ꢁ78ꢀC; RCHO (e) NaH, BnBr, DMF; aq HCl, CH3CN.

S. M. Sparks et al. / Bioorg. Med. Chem. Lett. 19 (2009) 981–985
983
Table 1
In vitro GPa inhibition: acyclic amino acids 3–11
R
HN
O
NH
O
N
H
Compound
R
GPa IC50, nMa
GPa (cell) IC50, nMa
CYP2C9 IC50, nM
12,000
3
387 (118)
1170 (89)
~
CO2H
4
311 (14)
39 (8)
1138 (194)
1019 (177)
688 (70)
2300
8400
1940
nt
~
~
CO2H
CO2H
5
6
72 (16)
101 (21)
23 (4)
~
CO2H
7
1427 (131)
501 (84)
~
~
CO2H
CO2H
8
3000
nt
CF3
9
123 (46)
114 (14)
9
2003 (450)
1533 (288)
452 (67)
~
~
CO2H
10
11
nt
CO2H
740
450
~
~
CO2H
CO2H
12
25 (2)
311 (21)
a
Values are means of three experiments, standard error is given in parentheses.
To further explore the SAR around the acyclic amino acids,
incorporation of oxygen into the residues examined (Table 2). Ser-
ine and threonine ethers were targeted with the goal of further
improving potency and incorporating polar functionality to de-
crease CYP2C9 inhibition.17 Serine ethers (13–17) were initially
prepared and confirmed that d-substitution provided excellent
cellular potency (14 and 15) and revealed that incorporation of
oxygen into the residue provided a greater than 10-fold reduction
in CYP2C9 activity (12 vs 15). Incorporation of a b-methyl group in
the form of threonine ethers (18–23) provided further improve-
ments in enzyme and cellular potency along with additional reduc-
tions in CYP2C9 inhibition. t-Bu-Thr analog 20 was identified as
having the optimal combination of enzyme (5 nM) and cellular
(139 nM) activity along with reduced potential for CYP2C9 inhibi-
tion. Substitution off of the threonine
c
-carbon was explored with
analogs 24 and 25, which afforded a decrease in both enzyme and
cellular activity and a dramatic increase in CYP2C9 inhibition.
With potent GP inhibitors incorporating acyclic amino acid res-
idues in hand, pharmacokinetic (PK) studies were undertaken (Ta-
ble 3). Analogs 8, 15, and 20 were evaluated in rat PK experiments.
The PK profiles were characterized by moderate oral exposures
with low to moderate clearances and low steady-state volumes
of distribution. Half-lives ranged from 2 to 4 h. Of the analogs
examined, t-butylthreonine analog 20 showed superior bioavail-
ability (40%) and improved oral exposure. These parameters cou-
pled with the compound’s superior cellular activity
(IC50 = 139 nM) and reduced CYP2C9 inhibition (IC50 = 10
tated selection of the compound for in vivo efficacy studies.
lM) dic-

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S. M. Sparks et al. / Bioorg. Med. Chem. Lett. 19 (2009) 981–985
Table 2
In vitro GPa inhibition: serine and threonine analogs 12–24
R
O
HN
NH
O
N
H
Compound
R
GPa IC50 (nM)a
GPa (cell) IC50 (nM)a
CYP2C9 IC50 (nM)
3300
O
13
56
4467 (396)
~
~
CO2H
O
14
15
16
17
18
19
20
21
22
23
24
25
148 (94)
44 (18)
79 (31)
95 (28)
298 (54)
17 (2)
7 (2)
451 (95)
387 (70)
949 (103)
1139 (141)
2457 (711)
156 (22)
139 (41)
434 (64)
159 (17)
420 (15)
309 (19)
613 (111)
6920
6000
4700
2800
32,000
14,000
10,000
2100
7230
1900
680
CO2H
O
~
CO2H
O
~
~
CO2H
O
CO2H
O
~
~
~
~
CO2H
O
CO2H
O
CO2H
O
17 (5)
CO2H
O
6
~
~
CO2H
O
Ph
16 (3)
24 (4)
73 (18)
CO2H
O
Ph
~
CO2H
O
Ph
50
~
CO2H
aValues are means of three experiments, standard error is given in parentheses.
In summary, systematic modifications to the amino acid res-
idue of a series of anthranilimide based glycogen phosphorylase
inhibitors led to the identification of a series of serine and thre-
onine ethers with excellent potency and attenuated CYP2C9 inhi-
bition. Anthranilimide 20, containing the t-butylthreonine
residue, was identified for further progression into in vivo effi-
cacy models, the results of which will be reported in the follow-
ing communication.

S. M. Sparks et al. / Bioorg. Med. Chem. Lett. 19 (2009) 981–985
985
Table 3
Rat pharmacokinetic data for anthranilimide GPIs
Compound
AUC0?1,po(ng.h/mL)a
Clb (mL/min/kg)
Vss (L/kg)
T1/2, b(h)
F(%)
po
8
15
20
320
256
514
4
26
13
0.6
1.9
0.5
2.8
4.0
3.0
8
17
40
a
Dose adjusted oral AUC normalized to 1 mg/kg.
Sprague–Dawley rats (n = 2), Oral dose = 10 mg/kg, IV dose = 2.5 mg/kg.
b
10. Mohler, M. L.; He, Y.; Wu, Z.; Hong, S.-S.; Miller, D. D. Expert Opin. Ther. Patents
2007, 17, 59.
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