Identification, synthesis, and characterization of new glycogen phosphorylase inhibitors binding to the allosteric AMP site

Article abstract of DOI:10.1021/jm031121n

Inhibition of glycogen phosphorylase (GP) has attracted considerable attention during the last five to 10 years as a means of treating the elevated hepatic glucose production seen in patients with type 2 diabetes. Several different GP inhibitors binding t

Full text of DOI:10.1021/jm031121n

J . Med. Chem. 2004, 47, 3537-3545
3537
Id en tifica tion , Syn th esis, a n d Ch a r a cter iza tion of New Glycogen P h osp h or yla se
In h ibitor s Bin d in g to th e Alloster ic AMP Site
Marit Kristiansen,*,† Birgitte Andersen,‡ Lars Fogh Iversen,§ and Niels Westergaard‡,|
Novo Nordisk A/ S, Novo Nordisk Park, DK-2760 Måløv, Denmark
Received December 3, 2003
Inhibition of glycogen phosphorylase (GP) has attracted considerable attention during the last
five to 10 years as a means of treating the elevated hepatic glucose production seen in patients
with type 2 diabetes. Several different GP inhibitors binding to various binding sites of the
GP enzyme have been reported in the literature. In this paper we report on a novel class of
compounds that have been identified as potent GP inhibitors. Their synthesis, mode of binding
to the allosteric AMP site as well as in vitro data on GP inhibition are shown. The most potent
inhibitor was found to be 4-[2,4-bis-(3-nitrobenzoylamino)phenoxy]phthalic acid (4j) with an
IC50 value of 74 nM. This compound together with a closely related analogue was further
characterized by enzyme kinetics and in primary rat hepatocytes.
In tr od u ction
Type 2 diabetes is a heterogeneous disorder charac-
terized by hyperglycaemia, which is the result of both
impaired insulin secretion and insulin resistance in
target tissues.1 Numerous studies have shown that
hepatic glucose production (HGP) is increased both
under postabsorptive (fasted) and postprandial (fed)
conditions, and, moreover, it is now increasingly recog-
nized that elevated HGP is the major contributor to
hyperglycaemia of the diabetic state.2-4 Using improved
tracer methods, most studies find that basal HGP is
elevated by 10-30% in patients with fasting blood
glucose of 8-9 mM and even higher in patients with
higher fasting blood glucose due to significant renal
F igu r e 1. Examples of GP inhibitors that bind at the different
sites of the GP enzyme.
glucose loss.5,6 Hepatic glucose can be produced from
either gluconeogenesis (the synthesis of glucose from
3-carbon precursors) or from glycogenolysis (the break-
cascade of events, resulting in covalent modification of
down of glycogen). Which of these two pathways are the
GP by phosphorylation (at Ser14) into its active form,
most predominant for the production of hepatic glucose
which is termed GPa.16 Dephosphorylation of GPa to
in the diabetic state has been a matter of controversy7
its inactive form, termed GPb, is mediated by protein
resulting in various approaches for pharmacological
phosphatases.17,18 Both GPa and GPb can exist in an
intervention targeting gluconeogenesis8,10 and glyco-
inactive conformation, the T-state, and an active con-
genolysis.8,10
formation, the R-state.19-21 Allosteric effectors, espe-
Hepatic insulin resistance is suggested to lead to lack
cially AMP, are also important for the activity of muscle
of depression on glycogenolysis9 and therefore glycogen
GPb, which can be activated to 80% of the GPa activity
phosphorylase (GP) inhibitors have attracted consider-
by stabilizing the more active R-state.22,23
able attention during the last five to 10 years because
There are several known binding sites in the GP-
of their potential use for the treatment of type 2
enzyme: the catalytic site, the inhibitor (or nucleoside)
diabetes.7,10 This has been shown both in vitro using
site, the AMP allosteric (or nucleotide) site, the glycogen
cultured hepatocytes11,12 and in type 2 diabetic animal
storage site, and a new allosteric site.7,10,24,25 The first
models13,14 and humans.15 Glycogen phosphorylase ca-
class of nonphysiological inhibitors of GP described were
talyses the phosphohydrolysis of glycogen to yield
the glucose analogues.26-29 These inhibitors bind to the
glucose-1-phosphate and this is the rate-limiting step
catalytic site and one of the most potent compounds is
in the overall process of glycogen degradation. In the
a spirohydantoin (Figure 1), which inhibits rabbit
liver, glucagon stimulates glycogen degradation via a
muscle GPb with a Ki value of 3 µM.26 Other inhibitors
binding at the catalytic site are the iminosugars. Earlier
* To whom the correspondence should be addressed. Phone: +45
we reported a group of piperidines within this series as
4443 4855. Fax: +45 4466 3939. E-mail: mkri@novonordisk.com.
potent inhibitors of GP;30 however, the most potent
compound, 1,4-dideoxy-1,4-imino-D-arabinitol, DAB (Fig-
ure 1), inhibits liver GPa with a Ki value of 400 nM.13
Caffeine and other heteroaromatic compounds bind to
† Medicinal Chemistry Research II.
‡ Hepatic Biochemistry.
§ Protein Science.
| Present address: LEO Pharma A/S, Industriparken 55, DK-2750
Ballerup, Denmark.
10.1021/jm031121n CCC: $27.50 © 2004 American Chemical Society
Published on Web 06/05/2004

3538 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14
Kristiansen et al.
Sch em e 1a
laboratories, since several species, tissue isoforms, phos-
phorylation states, and GP activity assay (glycogenolysis
or glycogen synthesis) have been used to evaluate
inhibitors. Especially the role of glucose and AMP for
the potency of these inhibitors needs to be explored. For
example, glucose is known to potentiate the inhibitory
action of caffeine,37 whereas AMP is known to increase
the Ki value for glucose.38 This suggests that the
simultaneous presence of both AMP and glucose could
be determining for the potency of various inhibi-
tors.12,14,27,28,39
In this paper we report structure-activity relation-
ship (SAR) and kinetic data of a series of compounds
that have been identified as GP inhibitors and which
bind to the AMP-binding site as shown by protein
crystallography. In addition, the effect of glucose and
AMP on the potency of compound 4a was investigated.
Ch em istr y
a
Reagents and conditions: (a) K2CO3, DMF, 100 °C. (b) H2, Pd/
Compound 4a was identified as a GPa inhibitor by
high-throughput screening and was synthesized as
outlined in Scheme 1 (R1 ) NO2, R2 ) R3 ) R4 ) H).
The phenoxyphthalate 1a was prepared by reaction of
dimethyl 4-hydroxyphthalate with 2-fluoronitrobenzene.
The resulting nitrophenoxy-phthalate was reduced with
hydrogen in the presence of Pd/C in methylene chloride
to give the aminophenoxy-phthalate 2a , which was
reacted with 3-nitrobenzoyl chloride to give 3a . Hy-
drolysis of 3a with 2 N sodium hydroxide in dioxane
gave compound 4a (R1 ) NO2, R2 ) R3 ) R4 ) H).
A library of analogues of 4a having different substit-
uents in the B- or the C-phenyl ring was prepared using
a solid-phase method starting from the commercially
available Wang resin 5 as described in Scheme 2.
Successive 1,3- diisopropylcarbodiimide (DIC)-mediated
coupling with 4-hydroxyphthalic acid, reaction with
different analogues of o-fluoronitrobenzene, reduction
of the nitro group with tin(II) chloride, DIC-mediated
coupling with different benzoic acids, and finally tri-
C, CH2Cl2, room temp. (c) TEA, acetone. (d) 2 N NaOH, dioxane,
room temp.
the inhibitor site. Flavopiridol (Figure 1) is one of the
most potent compounds within this class and inhibits
rabbit muscle GPb with an IC50 value of 1 µM.31
Glycogen phosphorylase inhibitors that bind to the
allosteric site have also been reported. BAY-W1807
(Figure 1) was until recently the most potent in this
class with a Ki value, in the absence of AMP, of 11 nM32
(muscle GPa); however, a phthalic acid derivative with
an IC50 (human liver GPa) of 1 nM has just been
published.33 A few years ago a new allosteric site was
discovered. Different indole derivatives, e.g., CP-320,626
(Figure 1), were found to bind to this new site, and IC50
values were reported to be in the low nM range (human
liver GPa).14,34-36 It still remains unclear which of the
preciously described binding sites that will be the most
beneficial for pharmacological intervention.12 Also, one
has to be cautious, when comparing data from various
Sch em e 2a
a
Reagents and conditions: (a) DIC, DMAP, DMF, room temp. (b) 0.5 M potassium bis(trimethylsilyl)amide, DMF. (c) Tin(II) chloride,
NMP. (d) DIC, DMF, CH2Cl2. (e) CH2Cl2, TFA (1:1).

Glycogen Phosphorylase Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14 3539
Ta ble 1. IC50 Values of 4a -i Using Pig Liver GP and Rabbit
Muscle GP
GPa liver
GPb muscle
compd
R1
NO2
R2
(µM)
(µM)
4a
4b
4c
4d
4e
4f
4g
4h
4i
H
H
1.3
41
0.9
6
Cl
OCH3
COCH3
H
COOH
Cl
H
H
H
H
Cl
Cl
CH3
∼40
∼70
>200
>100
>50
>200
>30
2.4
50
50
∼100
>200
H
H
CP-320,626a
0.5
a
CP-320,626 from Pfizer was used as control.
Ta ble 2. IC50 Values for 4j-u in Pig Liver GPa
F igu r e 2. 4a in the allosteric AMP binding pocket of GPb.
(A) The 2Fo - Fc electron density map for 4a found in the
allosteric AMP site contoured at one sigma level in yellow and
at three sigma in red. (B) The interaction of compound 4a with
the various residues in the AMP site. Hydrogen bonds are
indicated (in white) and van der Waals interactions are in
yellow.
known as an activation site, the GP inhibitor 4a ,
however, clearly binds in a different mode compared to
AMP (see Figure 3A and 3B). 4a thus induces confor-
mational changes upon binding to the GPb enzyme;
hence, the enzyme was found in the inactive T-confor-
mation (not shown), in a similar way as reported for
the GP inhibitor BAY-W1807 from Bayer.40
Table 1 shows the ability of compound 4a -i to inhibit
the activity of pig liver GPa and rabbit muscle GPb
measured in the direction of glycogenolysis. Data for the
known GP inhibitor, CP-320,62612 (Figure 1), is included
for comparison. Modifications in the C-ring resulted in
loss of potency, especially when using the pig liver GPa
isozyme. This correlated well with the structural data,
as the C-ring is buried in the AMP site. The B-ring,
however, was found to be located at the outer part of
the AMP binding pocket with the R3 and R4 position
available for substitution and with direction out of the
AMP binding pocket and pointing toward the other GP
molecule in the GP homodimer. Thus, when a substitu-
ent in the B-ring was introduced (Table 2), the potency
increased by severalfold with compound 4j being the
most potent one having an IC50 value of 74 nM.
fluoroacetic acid (TFA) resin cleavage gave the desired
products. Compounds 4b-u (Tables 1 and 2) were
prepared by either of the two methods (Scheme 1 and
Scheme 2).
Resu lts a n d Discu ssion
As compound 4a was identified by high-throughput
screening, no binding site data existed for this type of
compound. To identify the binding site and interactions
with the enzyme, GPb was cocrystallized with compound
4a . A single electron density peak was identified in a
difference Fourier map which showed that compound
4a occupied the allosteric AMP binding pocket of GP
(see Figure 2A and 2B). Compound 4a was found not to
bind elsewhere. Although the allosteric AMP site is

3540 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14
Kristiansen et al.
F igu r e 4. Compound 4j (shown in standard colors) in the GPb
homodimer. In magenta the GPb monomer where compound
4j binds such as compound 4a (identical interactions as shown
in Figure 2B). Residues 40-43 of the second monomer in the
GPb homodimer are depicted (carbon atoms in light green and
nitrogen/oxygen in standard colors). The novel contacts (as
compared to compound 4a ) are shown.
F igu r e 3. AMP in the allosteric AMP binding pocket of GPb.
The structures of 4a and AMP (PDB code 8GPB) were
superimposed and the AMP site depicted (4a in standard colors
and AMP in light green). (A) 4a and AMP alone. (B) The full
AMP site; the major movements in the pocket are indicated
with white arrows.
Compound 4j was also cocrystallized with GPb and
as expected the R4 substituent was extending outward
from the AMP binding pocket, making contact with the
other GP molecule in the GP homodimer (Figure 4).
Hence, compound 4j makes contacts to both GP mol-
ecules in the homodimer and this could explain the
increase in potency of 4j compared to, for example, 4a .
Figure 5 shows the dose-response curves for inhibi-
tion of pig liver GPa and rabbit muscle GPb when
measured in the direction of glycogenolysis using com-
pounds 4j and 4a . As can be seen, both compounds are
potent inhibitors with 4j being 18 and 41 times more
potent than 4a in inhibiting liver GPa and muscle GPb,
respectively. To study the effect of glucose on the
potency of these types of compounds dose-response
curves were generated for 4a in the presence of 0-6 mM
glucose and measured in the direction of glycogen
synthesis. It has been reported previously that BAY-
W180732 acts in synergism with glucose similar to what
has been shown for caffeine.37 Likewise it can be seen
(Figure 6A) that glucose synergistically increased the
potency of 4a using pig liver GPa. The maximal syner-
gistic effect was obtained at 5-6 mM glucose where the
IC50 value was 50% of the initial value. This is thus
consistent with the findings for BAY-W1807. Since 4a
F igu r e 5. Pig liver GPa (A) and rabbit muscle GPb (B) were
measured in the direction of glycogen degradation using 2 mg/
mL glycogen as the substrate in the presence of increasing
concentrations of 4j (9) or 4a (1). The IC50 values (µM) for 4j
and 4a were calculated to be 0.074 ( 0.015 µM (3) and 1.3 (
0.2 µM (5) on pig liver GPa and 0.87 ( 0.13 µM (3) and 0.21
µM (1) on rabbit muscle GPb, respectively. Results are aver-
ages ( SEM of the number of experiments given in parenthe-
ses.
was found to bind to the AMP binding site, dose-
response curves were generated in order to investigate
the effect of AMP on the potency of 4a both in the
presence and in absence of 6 mM glucose. Figure 6B
shows that AMP increased the IC50 values of 4a in a
dose-dependent fashion, i.e., the potency decreased.
However, in the presence of 6 mM glucose this effect
was less profound. In this context it should be noted

Glycogen Phosphorylase Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14 3541
F igu r e 6. Pig liver GPa was assayed in the direction of
glycogen synthesis. (A) The effect of increasing concentrations
of glucose on the potency of 4a on pig liver GPa was measured.
100% corresponds to an IC50 value of 34 ( 2 nM. (B) The
potency of 4a was measured with increasing concentrations
of AMP in the presence (2) or absence (9) of 6 mM glucose.
Results are average of three individual experiments ( SEM.
F igu r e 7. Effect of 4a and 4j on basal and glucagon-induced
glycogenolysis. (A) Basal (9, 0) and glucagon-induced (0.5 nM)
(b, O) glucose production were measured over 70 min. Open
symbols 4a , closed symbols 4j. Basal glucose production was
set to 100% and equals 269 ( 12 nmol glucose/mg protein ×
70 min. (B) Glycogen levels after basal (9, 0) and glucagon-
induced (b, O) glycogenolysis were determined. Open symbols
4a , closed symbols 4j. Glycogen level before start of the
experiment was set to 100% and equals 2604 ( 367 nmol
glycosyl unit/mg protein. All results are means ( SEM of four
separate hepatocyte isolation.
that the IC50 value is severalfold lower compared to
when the inhibition is measured in the direction of
glycogenolysis. This is also found for other types of GP
inhibitors12 binding to various binding sites, and there-
fore caution must be exercised when the potency of GP
inhibitors is compared.
after incubation with the two compounds are shown in
Figure 7B. The glycogen content of the hepatocytes was
measured to 2600 nM ( 370 nmol glycosyl unit/mg
protein before initiation of glycogenolysis, and this is
set to 100% in Figure 7B. The IC50 values calculated
from Figure 7B are not significantly different from the
IC50 values calculated from Figure 7A.
In summary, we have discovered a class of GP
inhibitors that bind to the allosteric AMP binding site.
On the basis of SAR and crystallography data, the
potency of this class of compounds was increased by
creating an interaction with both GP monomers in the
GP homodimer, leading to compound 4j. Furthermore,
compound 4j inhibits glycogenolysis in primary rat
hepatocytes.
The best binding site for pharmacological intervention
of GP inhibitors is difficult to predict. However, as
shown in this paper, the intracellular concentrations of
glucose and AMP have to be taken into consideration
for GP inhibitors binding to the AMP binding site. Thus
future studies will show if 4j can lower blood glucose in
an animal model of type 2 diabetes as well.
To characterize the effect of the two compounds, 4a
and 4j, in a cell model system, the inhibitors were tested
in cultured primary rat hepatocytes. The effect of the
two compounds on glycogenolysis with respect to glucose
and glycogen is showed in Figure 7. Glucagon (0.5 nM)
increased the glucose production 2.1-fold (Figure 7A).
4j was also found to be the most potent compound in
the hepatocytes. From Figure 7A, the IC50 values for 4j
for inhibition of basal and glucagon-induced glyco-
genolysis were calculated to be 1.6 ( 0.2 µM and 4.7 (
0.3 µM, respectively. The IC50 values for 4a for inhibi-
tion of basal and glucagon-induced glucose production
were calculated (Figure 7A) to be 9.8 ( 2.4 µM and 30.0
( 4.5 µM, respectively. The IC50 values for inhibition
of glucagon-induced glycogenolysis is increased ap-
proximately 3-fold compared to inhibition of basal
glycogenolysis. This is in agreement with the increased
AMP level found when cells are stimulated with gluca-
gon. The increase in cAMP caused by glucagon will
spontaneously break down to AMP by phosphodi-
esterases. The intracellular concentration of AMP in a
basal state has been reported to approximately 500 µM
in rat liver.39 At this concentration of AMP, GP is fully
saturated39,41 and inhibitors binding to the AMP site
are expected to be less potent compared to the situation
where AMP is absent (Figure 6). This may explain why
the potency of 4a and 4j was less profound using
cultured hepatocytes. This also has to be considered
when GP inhibitors are designed for pharmacological
intervention. The glycogen levels in the hepatocytes
Exp er im en ta l Section
Ma ter ia ls. Reagents and solvents were obtained from
commercial suppliers and used without further purification.
Solvents used were either AR or HPLC grade. Dry DMF was
prepared and stored over molecular sieves (3 Å).
Gen er a l Ch em istr y. Melting points (uncorrected) were
measured on a Bu¨chi B545 apparatus. 1H and 13C NMR spectra
were recorded on a Bruker Advance DPX 200 MHz instrument
unless otherwise noted. Chemical shifts are given in ppm (δ)
relative to TMS and coupling constants are in Hz. The HPLC-

3542 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14
Kristiansen et al.
MS analyses were performed on a PE Sciex API 100 LC/MS
system using a Waters 3 mm × 150 mm, 3.5 µm, C-18
Symmetry column and a positive ion spray with a flow rate of
20 µL/min. The column was eluted with a linear gradient 10%
to 100% A, in 7.5 min at a flow rate of 1.5 mL/min (Solvent A
) acetonitrile, 0.01% TFA). Detection: 210 nm. The elemental
composition of the compounds agrees to within (0.4% of the
calculated value. The HPLC purifications were performed on
a Gilson instrument with two Gilson 306 pumps equipped with
25 mL SC pump heads, Gilson 806 manometer, and Gilson
811c dynamic mixing chamber. UV detection was performed
with Gilson 119 UV/VIS detector. Gilson 215 Nebula was used
as combined injector and fraction collector. Analytical HPLC
was performed on a Merck-Hitachi with a Waters Xterra MS,
3 mm × 50 mm, 3.5 µm, C-18 column. The column was eluted
with a linear gradient 0% to 100% B, in 7 min at a flow rate
of 1.2 mL/min (Solvent A ) 5% acetonitrile and 0.01% TFA
diluted with water to 100%; solvent B ) acetonitrile). Detec-
tion: 210 nm.
7.65 (2H, m), 7.8 (1H, J ) 2.0 Hz, t), 7.83 (1H, J ) 8.5 Hz, d),
8.27 (1H, br s), 8.55 (1H, J ) 1.8 and 8.5 Hz, dd).
4-(2-Ben zoyla m in op h en oxy)p h th a lic Acid Dim eth yl
Ester (3e). Yield: 100% crude product. LC/MS: m/z: 375 (M
1
- CH3O), 406 (MH+). H NMR (CDCl3) in ppm: δ 3.9 (6H, 2
× s), 6.98 (1H, J ) 1.8 and 8.7 Hz, dd), 7.08-7.2 (2H, m), 7.23-
7.35 (2H, m), 7.4-7.6 (3H, m), 7.75-7.78 (2H, m), 7.83 (1H, J
) 8.7 Hz, d), 8.3 (1H, br s), 8.63 (1H, J ) 1.8 and 8.4 Hz, dd).
4-[2-(3-E t h o x y c a r b o n y lb e n z o y la m i n o )p h e n o x y ]-
p h th a lic Acid Dim eth yl Ester (3f). Yield: 100% crude
1
product. H NMR (CDCl3) in ppm: δ 1.4 (3H, J ) 6.9 Hz, t),
3.9 (6H, 2 × s), 4.40 (2H, J ) 6.9 Hz, q), 6.98 (1H, J ) 1.8 and
8.7 Hz, dd), 7.10-7.33 (4H, m), 7.56 (1H, J ) 8.0 Hz, t), 7.83
(1H, J ) 8.7 Hz, d), 7.97 (1H, J ) 1.6 and 8.0 Hz, dt), 8.20
(1H, J ) 1.4 and 8.3 Hz, dt), 8.30 (1H, br s), 8.45 (1H, J ) 1.6
Hz, t), 8.60 (1H, J ) 1.8 and 8.3 Hz, dd).
4-[2-(3-Nitr oben zoylam in o)ph en oxy]ph th alic Acid (4a).
Sodium hydroxide (2 N, 12 mL) was added to a solution of 4-[2-
(3-nitrobenzoylamino)phenoxy]phthalic acid dimethyl ester, 3a
(1.15 g, 2.83 mmol) in 1,4-dioxane (12 mL) at room tempera-
ture. The mixture was stirred for 2 h and cooled to <10 °C
and the pH adjusted to 2-3 with 1 N HCl (approximately 24
mL). The resulting crystals were filtered off and washed water.
The crystals were dried to give beige crystals of 4a (Yield: 0.82
g (76%)). LC-MS m/z 423 (MH+). 1H NMR (DMSO-d6) in ppm:
δ 10.3 (1H, s), 8.58 (1H, m), 8.38 (1H, m), 8.18 (1H, J ) 7.8
Hz, d), 7.6-7.8 (3H, m), 7.3-7.4 (2H, m), 7.18 (1H, J ) 1.9
and 7.6 Hz, dd), 7.1 (1H, m), 7.07 (1H, J ) 2.5 and 8.5 Hz,
dd). 13C NMR (DMSO-d6) in ppm: δ 116.6, 118.4, 120.8, 122.3,
125.0, 126.1, 127.3, 127.5, 129.4, 130.0, 133.0, 133.8, 135.5,
136.0, 147.6, 148.6, 158.8, 164.5, 167.3, 168.1. mp: 200-202
°C. Anal. (C21H14N2O8) C, H, N.
4-(2-Nitr op h en oxy)p h th a lic Acid Dim eth yl Ester (1a ).
A mixture of 4-hydroxyphthalic acid dimethyl ester (1.25 g,
5.95 mmol), o-fluoronitrobenzene (0.81 mL, 7.64 mmol), and
potassium carbonate (1.66 g, 12 mmol) in dry DMF (25 mL)
was heated at 100 °C for 1 h. The mixture was cooled to room
temperature and filtered and the filtrate evaporated to dryness
in vacuo to give the crude product of 1a as a yellow oil (Yield:
1.97 g (100%)). The compound was used in the next step
1
without further purification. H NMR (CDCl3) in ppm: δ 3.9
(6H, 2 × s), 7.1-7.2 (2H, m), 7.2 (1H, J ) 2.9 Hz, d), 7.3-7.4
(1H, m), 7.6-7.7 (1H, m), 7.84 (1H, J ) 8.2 Hz, d), 8.03 (1H,
J ) 1.8 and 8.2 Hz, dd). 13C NMR (CDCl3) in ppm: δ 168.1
(CO), 167.0 (CO), 159.5, 148.6, 142.4, 135.8, 135.2, 132.0, 126.4,
126.4, 125.8, 123.1, 119.8, 117.8, 53.2, 53.0.
P r ep a r a tion of Com p ou n d s 4b, 4e, 4f. The compounds
were prepared in a similar way as described for compound 4a .
4-[2-(3-Ch lor ob en zoyla m in o)p h en oxy]p h t h a lic Acid
(4b). Yield: 69%. mp: 194-195 °C. LC-MS: m/z 412 (MH+),
4-(2-Am in oph en oxy)ph th alic Acid Dim eth yl Ester (2a).
4-(2-Nitrophenoxy)phthalic acid dimethyl ester, 1a (10 g, 30.2
mmol), was dissolved in methylene chloride (500 mL). 10%
Pd/C (450 mg) was added, and the mixture was hydrogenated
for 19 h at 30 psi. The reaction mixture was filtered and
evaporated to dryness in vacuo. 2a was obtained as an oil
1
394 (MH+ - H2O). H NMR (DMSO-d6) in ppm: δ 10.0 (1H,
s), 8.1 (1H, br s), 7.7 (4H, m), 7.58 (1H, J ) 8.0 Hz, d), 7.45
(1H, m), 7.27 (2H, m), 7.1 (1H, J ) 7.5 Hz, d), 7.02 (1H, J )
2.5 and 8.5 Hz, dd). Anal. (C21H14ClNO6) C, H, N.
1
(Yield: 8.5 g (93%)). H NMR (CDCl3) in ppm: δ 3.86 (3H, s),
4-(2-Ben zoyla m in op h en oxy)p h th a lic Acid (4e). Yield:
42% (97.4% purity by HPLC). LC-MS: m/z 378 (MH+), 360
(MH+ - H2O). 1H NMR (DMSO-d6) in ppm: δ 9.8 (1H, s), 8.16
(1H, J ) 8.5 Hz, d), 7.77 (1H, m), 7.71-7.74 (3H, m), 7.52 (1H,
J ) 7.0 Hz, t), 7.43 (2H, J ) 7.5 Hz, t), 7.26 (2H, m), 7.11 (1H,
m), 7.05 (1H, J ) 2.5 and 8.5 Hz, dd).
4-[2-(3-Ca r b oxyp h en ylca r b a m oyl)p h en oxy]p h t h a lic
Acid (4f). Yield: 47%. (97.1% purity by HPLC). mp: 235-
237 °C. LC/MS: m/z 467 (M+2Na), 404 (MH+- H2O). 1H NMR
(DMSO-d6) in ppm: δ 10.2 (1H, s), 8.4 (1H, J ) 1.5 Hz, t), 8.1
(1H, J ) 7.6 Hz, d), 8.02 (1H, J ) 7.6 Hz, d), 7.65-7.8 (2H,
m), 7.6 (1H, J ) 8.1 Hz, t), 7.3 (2H, m), 7.2 (1H, m), 7.13 (1H,
J ) 2.5 and 8.6 Hz, dd).
P r epar ation of 4-[2-(3-Meth oxyben zoylam in o)ph en oxy]-
p h th a lic Acid (4c). To a suspension of Wang resin (1 g, 0.92
mmol, Bachem, loading: 0.92 mmol/g) in a mixture of meth-
ylene chloride (5 mL) and DMF (5 mL) were added 4-hydroxy-
phthalic acid (1.7 g, 9.3 mmol), 1,3-diisopropylcarbodiimide (1.4
mL, 9.0 mmol), and 4-(dimethylamino)pyridine (66 mg, 0.54
mmol). The mixture was shaken for 20 h and filtered. The resin
was washed with DMF (4 × 10 mL), methylene chloride (4 ×
10 mL), methanol (4 × 10 mL), and methylene chloride (4 ×
10 mL). The resin was dried in vacuo. o-Fluoronitrobenzene
(0.4 mL, 3.77 mmol) and 0.5 M potassium bis(trimethylsilyl)-
amide (1.2 mL, 0.6 mmol) were added to a suspension of the
dried resin in DMF (4 mL). The mixture was shaken for 24 h
under a nitrogen atmosphere and filtered. The resin was
washed with DMF (4 × 5 mL), methylene chloride (4 × 5 mL),
DMF (4 × 5 mL), and N-methylpyrrolidone (NMP) (4 × 5 mL).
A solution of tin(II) chloride dihydrate (0.9 g, 3.99 mmol) in
NMP (4 mL) was added to the resin. The mixture was shaken
for 24 h, filtered, and washed with NMP (4 × 5 mL), methylene
chloride (4 × 5 mL), and methanol (4 × 5 mL). The resin was
dried in vacuo to give approximately 244 mg of material.
3.90 (3H, s), 6.7-6.97 (3H, m), 7.0-7.1 (2H, m), 7.15 (1H, J )
2.0 Hz, d), 7.78 (1H, J ) 8.3 Hz, d). 13C NMR (CDCl3) in ppm:
δ 52.9; 53.2; 116.5; 117.4; 118.2; 119.4; 121.6; 124.4; 126.7;
132.1; 136.1; 139.2; 141.6; 160.8; 167.1; 168.8.
4-[2-(3-Nitr oben zoyla m in o)p h en oxy]p h th a lic Acid Di-
m eth yl Ester (3a ). A solution of 3-nitrobenzoyl chloride (0.43
g, 2.3 mmol) in dry acetone (2 mL) was added slowly to a
solution of 4-(2-aminophenoxy)phthalic acid dimethyl ester, 2a
(0.63 g, 2.09 mmol) and TEA (0.35 mL, 2.5 mmol) in dry
acetone (2 mL) at 0 °C. The mixture was stirred at room
temperature for 20 h and filtered and the filtrate evaporated
to dryness in vacuo. The crude product was purified on silica
gel (Eluent: methylene chloride: ethyl acetate (95:5)) to give
1
3a as a golden oil (Yield: 1.19 g (47%)). H NMR (CDCl3) in
ppm: δ 3.9 (6H, 2 × s), 7.02 (1H, J ) 1.6 and 8.5 Hz, dd),
7.1-7.25 (2H, m), 7.25-7.35 (2H, m), 7.7 (1H, J ) 8.0 Hz, t),
7.83 (1H, J ) 8.5 Hz, d), 8.1 (1H, J ) 1.1 and 8.0 Hz, dt), 8.3
(1H, br s), 8.4 (1H, m), 8.54 (1H, J ) 1.6 and 8.0 Hz, dd), 8.65
(1H, m). 13C NMR (CDCl3) in ppm: δ 53.1, 53.3, 118.0, 119.6,
119.8, 122.6, 123.2, 125.9, 126.0, 126.3, 126.9, 129.9, 130.5,
132.2, 133.2, 136.1, 136.6, 145.2, 148.7, 159.4, 163.5, 166.9,
168.1. If needed, the compound could be further purified by
precipitation as a mixed hydrochloride and acetate salt. Anal.
(C16H15NO5‚HCl‚1/2HOAc) C, H, N.
P r ep a r a tion of Com p ou n d s 3b, 3e, 3f. The compounds
were prepared in a similar way as described for compound 3a .
The crude products were isolated and used as such in the next
step.
4-[2-(3-Ch lor ob en zoyla m in o)p h en oxy]p h t h a lic Acid
Dim eth yl Ester (3b). Yield: 100% crude product. LC/MS:
1
m/z: 408 (M - CH3O), 440 (MH+). H NMR (CDCl3) in ppm:
δ 3.9 (6H, 2 × s), 6.98 (1H, J ) 1.8 and 8.0 Hz, dd), 7.1-7.2
(2H, m), 7.23-7.35 (2H, m), 7.4 (1H, J ) 8.0 Hz, d), 7.48-

Glycogen Phosphorylase Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14 3543
To a suspension of this resin (50 mg, 0.046 mmol) in a
mixture of DMF (0.5 mL) and methylene chloride (0.5 mL)
were added 3-methoxybenzoic acid (80 mg, 0.526 mmol) and
1,3-diisopropylcarbodiimide (40 µL, 0.257 mmol). The mixture
was shaken for 24 h and filtered and the resin washed with
DMF (4 × 4 mL), THF (4 × 4 mL), and methylene chloride (4
× 4 mL). The resin was shaken with methylene chloride:TFA
(1:1)(1 mL) for 20 min. The mixture was filtered and the resin
washed with carbon tetrachloride (4 mL), and the collected
filtrates were evaporated to dryness in vacuo to give 4c (20
mg, yield: 96%) as a brownish oil. The oil was further purified
on HPLC: The oil was dissolved in 2200 µL of acetonitrile to
which one drop of DMSO was added. HPLC purification was
performed on Nucleosil C-18 7 µm 8 × 100 mm columns
(packed by Grom). A standard gradient with water/acetonitrile
with 0.01% TFA was used. Flow rate 9 mL/min starting at
10% organic modifier ending after 18 min on 100% modifier.
This condition was kept for 1 min. Fractions of 4 mL were
collected in a deep-well collection plate. Evaporation of the
solvent in vacuo gave 4c (98% purity by HPLC). LC-MS: m/z
408 (MH+), 390 (MH+ - H2O). 1H NMR (DMSO-d6) in ppm: δ
9.95 (1H, s), 7.73 (2H, m), 7.27-7.40 (5H, m), 7.19 (1H, m),
7.11 (2H, m), 7.09 (1H, J ) 2.5 and 8.6 Hz, dd), 3.8 (3H, s).
P r ep a r a tion of Com p ou n d s 4d a n d 4g-u . The com-
pounds were prepared in a similar way as described for
compound 4c.
4-[2-(3-Acetylben zoylam in o)ph en oxy]ph th alic Acid (4d).
LC-MS: m/z 420 (MH+), 402 (MH+ - H2O). 1H NMR (CD3OD)
in ppm: δ 8.24 (1H, br s), 8.11 (1H, J ) 7.6 Hz, d), 7.89 (1H,
J ) 8.1 Hz, d), 7.80 (1H, J ) 2.0 and 7.6 Hz, dd), 7.72 (1H, J
) 8.6 Hz, d), 7.62 (1H, J ) 8.1 Hz, t), 7.35 (2H, m), 7.16-7.22
(2H, m), 7.09 (1H, J ) 2.5 and 8.6 Hz, dd), 2.57 (3H, s). 96%
purity by HPLC.
4-[2-(3,4-Dich lor oben zoylam in o)ph en oxy]ph th alic Acid
(4g). LC-MS: m/z 428/430 (MH+ - H2O). 1H NMR (DMSO-
d6) in ppm: δ 10.02 (1H, s), 7.96 (1H, br s), 7.73-7.76 (3H,
m), 7.71 (1H, J ) 1.5 and 7.1 Hz, dd), 7.34 (2H, m), 7.19 (1H,
J ) 2.0 and 7.6 Hz, dd), 7.11 (1H, m), 7.09 (1H, J ) 2.5 and
8.6 Hz, dd). 98% purity by HPLC.
4-[2-(4-Ch lor ob en zoyla m in o)p h en oxy]p h t h a lic Acid
(4h ). LC-MS: m/z 412 (MH+), 394 (MH+ - H2O). 1H NMR
(DMSO-d6) in ppm: δ 9.9 (1H, s), 8.07 (1H, m), 7.80 (2H, J )
8.5 Hz, d), 7.65-7.8 (2H, m), 7.50 (2H, J ) 8.5 Hz, d), 7.26
(2H, m), 7.10 (1H, J ) 7.5 Hz, d), 7.02 (1H, J ) 2.5 and 9.0
Hz, dd). 96% purity by HPLC.
4-[2-(4-Met h ylb en zoyla m in o)p h en oxy]p h t h a lic Acid
(4i). LC-MS: m/z 392 (MH+), 374 (MH+ - H2O). 1H NMR
(DMSO-d6) δ 9.8 (1H, s), 7.76 (2H, m), 7.69 (2H, J ) 8.1 Hz,
d), 7.31 (2H, m), 7.27 (2H, J ) 8.1 Hz, d), 7.16 (2H, m), 7.08
(1H, J ) 2.5 and 8.6 Hz, dd). 96% purity by HPLC.
4-[2,4-Bis-(3-n itr oben zoylam in o)ph en oxy]ph th alic Acid
(4j). LC-MS: m/z 587 (MH+), 569 (MH+ - H2O). 1H NMR
(DMSO-d6) δ 10.8 (1H, s), 10.45 (1H, s), 8.83 (1H, br s), 8.58
(1H, br s), 8.4 (3H, m), 8.24 (1H, J ) 1.8 Hz, d), 8.19 (1H, J )
8.1 Hz, d), 7.86 (1H, J ) 8.1 Hz, t), 7.76 (3H, m), 7.25 (1H, J
) 8.1 Hz, d), 7.09 (1H, J ) 2.5 and 8.6 Hz, dd), 7.08 (1H, s).
mp: 184-186 °C. Anal. (C28H18N4O11) C, H, N.
4-[4-Ben zoyla m in o-2-(3-n itr oben zoyla m in o)p h en oxy]-
p h th a lic Acid (4k ). LC-MS: m/z 524 (MH+ - H2O) 1H NMR
(DMSO-d6) in ppm: δ 10.42 (1H, s), 10.38 (1H, s), 8.5 (1H, s),
8.37 (1H, J ) 8.1 Hz, d), 8.18 (1H, J ) 2.5 Hz, d), 8.15 (1H, J
) 7.6 Hz, d), 7.96 (2H, J ) 7.1 Hz, d), 7.73 (2H, m), 7.67 (1H,
J ) 8.3 Hz, d), 7.5-7.62 (3H, m), 7.2 (1H, J ) 8.8 Hz, d), 7.02-
7.1 (2H, m). 100% purity by HPLC.
d), 8.33 (1H, J ) 8.6 Hz, d), 8.1 (1H, J ) 8.1 Hz, d), 7.82 (1H,
J ) 8.1 Hz, t), 7.66-7.2 (3H, m), 7.08-7.13 (3H, m). 100%
purity by HPLC.
4-[4-Met h yl-2-(3-n it r ob en zoyla m in o)p h en oxy]p h t h a -
1
lic Acid (4n ). LC-MS: m/z 437 (MH+), 419 (MH+ - H2O). H
NMR (DMSO-d6) in ppm: δ 10.28 (1H, s), 8.55 (1H, s), 8.38
(1H, J ) 8.3 Hz, d), 8.16 (1H, J ) 7.8 Hz, d), 7.75 (1H, J ) 7.8
Hz, t), 7.7 (1H, J ) 8.8 Hz, d), 7.5 (1H, s), 7.17 (1H, J ) 8.0
Hz, d), 7.0-7.1 (3H, m), 2.8 (3H, s). 100% purity by HPLC.
4-[4-F lu or o-2-(3-n it r ob en zoyla m in o)p h en oxy]p h t h a -
1
lic Acid (4o). LC-MS: m/z 441 (MH+), 423 (MH+ - H2O). H
NMR (DMSO-d6) in ppm: δ 10.44 (1H, s), 8.54 (1H, s), 8.39
(1H, J ) 2.2 and 8.6 Hz, dd), 8.15 (1H, J ) 8.9 Hz, d), 7.76
(1H, J ) 8.9 Hz, t), 7.65-7.73 (2H, m), 7.27 (1H, m), 7.19 (1H,
m), 7.05-7.1 (2H, m). >98% purity by HPLC.
4-[4-Br om o-2-(3-n it r ob en zoyla m in o)p h en oxy]p h t h a -
lic Acid (4p ). LC-MS: m/z 483/485 (MH+ - H2O). 1H NMR
(DMSO-d6) in ppm: δ 10.46 (1H, s), 8.56 (1H, s), 8.4 (1H, J )
2.2 and 8.1 Hz, dd), 8.17 (1H, J ) 7.8 Hz, d), 7.96 (1H, J ) 2.5
Hz, d), 7.76 (1H, J ) 8.1 Hz, t), 7.72 (1H, J ) 8.3 Hz, d), 7.51
(1H, J ) 2.5 and 8.8 Hz, dd), 7.1-7.18 (3H, m). 100% purity
by HPLC.
4-[2-(3-Nit r ob e n zoyla m in o)-4-t r iflu or om e t h ylp h e -
n oxy]p h th a lic Acid (4q). LC-MS: m/z 491 (MH+), 473 (MH+
- H2O). 1H NMR (DMSO-d6) in ppm: δ 10.58 (1H, s), 8.64
(1H, s), 8.43 (1H, J ) 2.2 and 8.3 Hz, dd), 8.25 (1H, J ) 8.2
Hz, d), 8.17 (1H, s), 7.74-7.84 (2H, m), 7.68 (1H, J ) 2.0 and
8.8 Hz, dd), 7.31 (1H, J ) 8.8 Hz, d), 7.19-7.27 (2H, m). >98%
purity by HPLC.
4-[4-Acet yla m in o-2-(3-n it r ob en zoyla m in o)p h en oxy]-
p h th a lic Acid (4r ). LC-MS: m/z 480 (MH+), 462 (MH+
-
H2O). 1H NMR (DMSO-d6) in ppm: δ 10.31 (1H, s), 10.12 (1H,
s), 8.4 (1H, s), 8.31 (1H, J ) 9.0 Hz, d), 8.05 (1H, J ) 7.8 Hz,
d), 7.89 (1H, s), 7.67 (1H, J ) 8.1 Hz, t), 7.62 (1H, J ) 8.5 Hz,
d), 7.44 (1H, J ) 8.8 Hz, d), 7.1 (1H, J ) 8.8 Hz, d), 6.99 (1H,
J ) 2.5 and 8.8 Hz, dd), 6.95 (1H, s). > 98% purity by HPLC.
4-[4-Met h oxyca r b on yl-2-(3-n it r ob en zoyla m in o)p h e-
n oxy]p h th a lic Acid (4s). LC-MS: m/e 481 (MH+), 463 (MH+
- H2O). 1H NMR (DMSO-d6) in ppm: δ 10.50 (1H, s), 8.63
(1H, s), 8.42 (1H, J ) 7.8 Hz, d), 8.35 (1H, s), 8.24 (1H, J )
7.6 Hz, d), 7.90 (1H, J ) 2.2 and 8.6 Hz, dd), 7.72-7.82 (2H,
m), 7.18-7.26 (3H, m). 100% purity by HPLC.
4-[4-Cya n o-2-(3-n it r ob en zoyla m in o)p h en oxy]p h t h a -
lic Acid (4t). LC-MS: m/z 430 (MH+ - H2O). 1H NMR
(DMSO-d6) in ppm: δ 10.58 (1H, s), 8.6 (1H, s), 8.41 (1H, J )
8.1 Hz, d), 8.18-8.24 (2H, m), 7.78 (2H, J ) 8.3 Hz, d), 7.75
(1H, J ) 2.5 and 8.6 Hz, dd), 7.28 (1H, J ) 8.6 Hz, d), 7.2-
7.25 (2H, m). 100% purity by HPLC.
4-[5-Met h yl-2-(3-n it r ob en zoyla m in o)p h en oxy]p h t h a -
lic Acid (4u ). LC-MS: m/z 419 (MH+ - H2O).1H NMR
(DMSO-d6) in ppm: δ 10.26 (1H, s), 8.55 (1H, s), 8.38 (1H, J
) 8.3 Hz, d), 8.17 (1H, J ) 7.8 Hz, d), 7.78 (1H, J ) 7.8 Hz, t),
7.7 (1H, J ) 8.3 Hz, d), 7.58 (1H, J ) 8.1 Hz, d), 7.17 (1H, J
) 8.1 Hz, d), 7.05-7.1 (2H, m), 7.0 (1H, s), 2.8 (3H, s). >98%
purity by HPLC.
Biology. En zym e Assa y. Preparation of pig liver GPa
(GPb<10%) was performed according to literature.13 Rabbit
muscle GPb was obtained from Sigma Chemicals and was
activated by 25 µM AMP unless otherwise stated.
Glycogen phosphorylase activity was measured in the direc-
tion of glycogen breakdown from the photometrical determi-
nation of the rate of NADPH formation in an assay coupled to
phosphoglucomutase and glucose 6-phosphate dehydrogenase
(G-6-P-DH).13 The enzyme activity was assayed at pH 6.8 and
37 °C in a phosphate buffer containing KH2PO4 (18 mM),
Na2HPO4 (27 mM), MgCl2 (15 mM), EDTA (100 µM), NADP+
(340 µM), glucose 1,6-bisphosphate (4 µM), G-6-P DH (6000
U/L), and phosphoglucomutase (800 U/L) using a glycogen
concentration of 2 mg/mL.
4-[4-(4-Iod ob en zoyla m in o)-2-(3-n it r o-b en zoyla m in o)-
p h en oxy]p h th a lic Acid (4l). LC-MS: m/z 668 (MH+), 650
1
(MH+ - H2O). H NMR (DMSO-d6) in ppm: δ 10.47 (1H, s),
10.39 (1H, s), 8.56 (1H, s), 8.39 (1H, J ) 8.0 Hz, d), 8.15-8.21
(2H, m), 7.94 (2H, J ) 8.3 Hz, d), 7.68-7.8 (5H, m), 7.22 (1H,
J ) 8.8 Hz, d), 7.09 (2H, m). 100% purity by HPLC.
In some cases the GP activity was assayed in the direction
of glycogen synthesis by measuring the formation of inorganic
phosphate from glucose 1-phosphate in a buffer containing
N,N-bis[2-hydroxyethyl]-2-aminoethanesulfonic acid (BES) (50
mM), EDTA (1 mM), and glycogen (0.125%) at pH 6.8 and at
4-[5-Met h yl-2,4-b is-(3-n it r ob en zoyla m in o)p h en oxy]-
p h th a lic Acid (4m ). LC-MS: m/z 601 (M+1), 583 (MH+-
1
H2O). H NMR (DMSO-d6) in ppm: δ 10.35 (1H, s), 8.75 (1H,
s), 8.46 (1H, s), 8.42 (1H, J ) 8.1 Hz, d), 8.37 (1H, J ) 7.6 Hz,

3544 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14
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(4) Tappy, L. Regulation of hepatic glucose production in healthy
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Aubertin, J .; Gin, H. Measurement of post-absorptive glucose
kinetics in non-insulin-dependent diabetic patients: method-
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(6) Wajngot, A.; Chandramouli, V.; Schumann, W. C.; Ekberg, K.;
J ones, P. K.; Efendic, S.; Landau, B. R. Quantitative contribu-
tions of gluconeogenesis to glucose production during fasting in
type 2 diabetes mellitus. Metab., Clin. Exp. 2001 50, 47-52.
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phorylase inhibitors for treatment of type 2 diabetes mellitus.
Expert. Opin. Invest. Drugs 2001, 10, 439-454.
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S. J .; Rohde, J . J .; Souers, A. J .; Szczepankiewicz, B. G. Potential
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(9) Lewis, G. F.; Carpentier, A.; Vranic, M.; Giacca, A. Resistance
to insulin’s acute direct hepatic effect in suppressing steady-
state glucose production in individuals with type 2 diabetes.
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(10) McCormack, J . G.; Westergaard, N.; Kristiansen, M.; Brand, C.
L.; Lau, J . Pharmacological approaches to inhibit endogenous
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1,4-imino-D-arabinitol. Biochem. J . 1999, 342, 545-550.
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25 °C. The reaction was started with glucose 1-phosphate at
concentration of 0.5 mM. The rate of inorganic phosphate
formation was determined using a kit [EnzCheck, Molecular
probes] (see also ref 42).
Isola tion a n d Cu ltu r in g of Hep a tocytes. Primary rat
hepatocytes were isolated form Male Wistar rats (∼250 g) and
cultured in 24-well plates as described previously.11,43 The
hepatocytes were incubated in high glucose (15 mM) and high
insulin (10 nM) to induced glycogen synthesis. After intensive
wash in glucose-free buffer (117.6 mM NaCl, 5.4 mM KCl, 0.82
mM Mg2SO4, 1.5 mM KH2PO4, 20 mM HEPES, 9 mM
NaHCO3, 0.1% w/v HSA, and 2.25 mM CaCl2, pH 7.4), the
inhibitory effect of 4a and 4j on basal and glucagon induced
(0.5 nM glucagon) glycogenolysis were studied. Glucose re-
leased into the medium was measured using a glucose oxidase
kit. Glycogen levels were determined after two rapid washes
of cells with ice-cold 0.9% w/v NaCl by amyloglucosidase (exo-
1.4-R-D-glucosidase) digest as described by Gomez-Lechon et
al.44
Cocr ysta lliza tion of GP b a n d Com p ou n d s 4a a n d 4j.
P r otein P u r ifica tion . GPb was isolated from rabbit skeletal
muscle and prepared for crystallization as previously de-
scribed.45
Cr ysta lliza tion . An approximately 25 mg/mL GPb solution
consisting of 10 mM BES, 0.1 mM EDTA, 3 mM DTT, 1 mM
spermine, pH 6.7, was prepared for crystallization. Crystals
were grown by the hanging drop method using seeding as
nucleation. One mmol of compound 4a or 4j was added to the
protein solution before crystallization. Three microliters of
protein-ligand solution was used per drop. The reservoir
volume was 1 mL consisting of 10 mM BES, 0.1 mM EDTA, 3
mM DTT, 1 mM spermine, pH 6.7. Crystals grew to the size
of 1.0 × 0.2 × 0.2 mm over 2-4 days.
Da ta Collection . A GPb crystal (with either compound 4a
or 4j) of the size 1.0 × 0.2 × 0.2 mm was flash frozen at 100
K prior to data collection. The following cryo conditions were
used: the crystal was removed from the hanging drop and
transferred to 35% glycerol (containing 1.0 mmol inhibitor, 10
mM BES, and 0.1 mM EDTA). After a 45 s soak, the crystal
was flash frozen.
Data were collected on in-house equipment (RU300, Cu KR
50 kV/100 mA). The crystal to detector distance was 200 mm.
A 0.75° oscillation was used for 100 images; in total, 75° were
collected. Data were collected using a 5 min exposure per
image. See Supporting Information for data collection details.
The space group was determined to be P43212. Data processing
was performed using Denzo, Scalepack and the CCP4 program
suite.46,47
Refin em en ts. The 1a8i RCSB entry (1.7 Å data, and
isomorph with current data sets) was used as a starting model
for phasing with water and ligand molecules omitted from the
structure. All refinements were performed with X-PLOR
(version 3.851) (Accelrys Inc.). Interchanging cycles of model
building (QUANTA) and refinement (X-PLOR) were per-
formed; for details, see Supporting Information. The Fo - Fc
and 2Fo - Fc maps were inspected by the use of X-LIGAND
(QUANTA application) for densities that could correspond to
the structure of compound 4a or 4j. A well-suited density was
identified in the allosteric AMP binding pocket. No other
densities were identified to fit the inhibitors. The protein
structure was inspected and corrected by the use of X-BUILD
(Accelrys Inc.). Water molecules were inserted using the
X-SOLVATE program (Accelrys Inc.).
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of charge via the Internet at http://pubs.acs.org.
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