Computationally motivated synthesis and enzyme kinetic evaluation of N-(β-d-glucopyranosyl)-1,2,4-triazolecarboxamides as glycogen phosphorylase inhibitors

Article abstract of DOI:10.1039/c4md00335g

Following our recent study of N-(β-d-glucopyranosyl)oxadiazolecarboxamides (Polyk et al., Biorg. Med. Chem. 2013, 21, 5738) revealed as moderate inhibitors of glycogen phosphorylase (GP), in silico docking calculations using Glide have been performed on N-(β-d-glucopyranosyl)-1,2,4-triazolecarboxamides with different aryl substituents predicting more favorable binding at GP. The ligands were subsequently synthesized in moderate yields using N-(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)-tetrazole-5-carboxamide as starting material. Kinetics experiments against rabbit muscle glycogen phosphorylase b (RMGPb) revealed the ligands to be low μM GP inhibitors; the phenyl analogue (K_i = 1 μM) is one of the most potent N-(β-d-glucopyranosyl)-heteroaryl-carboxamide-type inhibitors of the GP catalytic site discovered to date. Based on QM and QM/MM calculations, the potency of the ligands is predicted to arise from favorable intra- and intermolecular hydrogen bonds formed by the most stable solution phase tautomeric (t2) state of the 1,2,4-triazole in a conformationally dynamic system. ADMET property predictions revealed the compounds to have promising pharmacokinetic properties without any toxicity. This study highlights the benefits of a computationally led approach to GP inhibitor design. This journal is

Full text of DOI:10.1039/c4md00335g

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Computationally motivated synthesis and enzyme
kinetic evaluation of N-(b-D-glucopyranosyl)-1,2,4-
triazolecarboxamides as glycogen phosphorylase
inhibitors†
Cite this: DOI: 10.1039/c4md00335g
a
b
c
c
a
*
Jaida Begum, Gergely Varga, Tibor Docsa, Pal Gergely, Joseph M. Hayes,
´
b
b
*
*
Laszlo Juhasz and Laszlo Somsak
´
´
´
´
´
´
Following our recent study of N-(b-D-glucopyranosyl)oxadiazolecarboxamides (Polyak et al., Biorg. Med.
´
Chem. 2013, 21, 5738) revealed as moderate inhibitors of glycogen phosphorylase (GP), in silico docking
calculations using Glide have been performed on N-(b-D-glucopyranosyl)-1,2,4-triazolecarboxamides
with different aryl substituents predicting more favorable binding at GP. The ligands were subsequently
synthesized in moderate yields using N-(2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl)-tetrazole-5-
carboxamide as starting material. Kinetics experiments against rabbit muscle glycogen phosphorylase b
(RMGPb) revealed the ligands to be low mM GP inhibitors; the phenyl analogue (Ki ¼ 1 mM) is one of the
most potent N-(b-D-glucopyranosyl)-heteroaryl-carboxamide-type inhibitors of the GP catalytic site
discovered to date. Based on QM and QM/MM calculations, the potency of the ligands is predicted to
arise from favorable intra- and intermolecular hydrogen bonds formed by the most stable solution phase
tautomeric (t2) state of the 1,2,4-triazole in a conformationally dynamic system. ADMET property
predictions revealed the compounds to have promising pharmacokinetic properties without any toxicity.
This study highlights the benefits of a computationally led approach to GP inhibitor design.
Received 1st August 2014
Accepted 14th September 2014
DOI: 10.1039/c4md00335g
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inhibitor, glycogen storage, benzimiadazole6 and the very
recently identied quercetin binding site.7 Signicant efforts
Introduction
have been afforded to the design of GP inhibitors in recent
years, with catalytic site inhibitors the most explored.4,8,9 The
physiological inhibitor of GP is a-D-glucose (Ki ¼ 1.7 mM), but b-
substitutions at the anomeric carbon of D-glucose have led to
the most effective GP catalytic site inhibitors and have
demonstrated blood sugar lowering effects in vivo.10,11
Type 2 diabetes mellitus (T2DM) is characterized by hypergly-
cemia and peripheral insulin resistance, and represents more
than 90% of all diabetic cases. The signicant increase in the
global incidence of diabetes is a major cause for concern,
having doubled over the previous 3 decades to 347 million
people in 2008.1 Without adequate control of blood glucose
levels, T2DM has several long term complications such as
neuropathy and nephropathy, as well as an increased risk of
blindness and cardiovascular disease.2 Glycogen phosphorylase
(GP; EC 2.4.1.1) is a validated target for T2DM having a direct
inuence on blood glucose levels through the glycogenolysis
pathway.3 It is an allosteric enzyme with a number of different
binding sites,4,5 the catalytic, allosteric, new allosteric,
An evaluation of the current status of b-D-glucopyranosyl
analogues as GP inhibitors can be found in recent reviews.4,8
The four most potent discovered to date are shown in Fig. 1, all
of which possess a 2-naphthyl moiety exploiting favorable
interaction of aryl groups in the catalytic b-cavity, a pocket lined
by both polar and non-polar groups. In general, the 2-naphthyl
moiety has proved the most effective in terms of potency, but
our recent studies on N-(b-D-glucopyranosyl)-oxadiazole-car-
boxamides with linkers 2–4 (Table 1) have revealed that this is
not always the case.16 However, these ligands only demon-
strated moderate potency at best, 2b and 3c the most potent
with Ki's ꢀ 30 mM. The 1,2,4-triazole moiety in the form of 3-(b-D-
glucopyranosyl)-5-substituted-1,2,4-triazoles has very recently
revealed some of the most potent inhibitors of the GP catalytic
site,13,14 with the 2-naphthyl derivative (1c in Fig. 1) the most
potent. In the current work, we have investigated N-(b-D-gluco-
pyranosyl)-3-substituted-1,2,4-triazole-5-carboxamides,
aCentre for Materials Science, Division of Chemistry, University of Central Lancashire,
Preston PR1 2HE, UK. E-mail: jhayes@uclan.ac.uk; Fax: +44 (0)172894981; Tel: +44
(0)172894334
bDepartment of Organic Chemistry, University of Debrecen, POB 20, H-4010 Debrecen,
Hungary. E-mail: juhasz.laszlo@science.unideb.hu; somsak.laszlo@science.unideb.
hu; Fax: +36 52512744; Tel: +36 52512900 ext. 22474; +36 52512900 ext. 22348
cDepartment of Medical Chemistry, Medical and Health Science Centre, University of
´
Debrecen, Egyetem ter 1, H-4032 Debrecen, Hungary
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c4md00335g
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Results & discussion
In silico binding studies
All ligands in Table 1 were prepared for calculations using
Maestro and the LigPrep 2.5 program18 generating minimized
favorable tautomeric and ionization states of all ligands at pH ¼
7 ꢁ 2. All ligands were assigned as neutral, but three tautomeric
forms (t1, t2 and t3) of the 1,2,4-triazole (linker 5) were
produced, with t1 and t2 the most probable based on LigPrep
with Epik (Table 2).18 Monte Carlo conformational searches on
models of ligands 5 and its three tautomers t1–t3 were per-
formed followed by Jaguar 8.0 density functional theory (DFT)
with M06-2X19 and the 6-31+G* basis set20,21(M06-2X/6-31+G*),
and yielded the conformations shown in Table 2. Higher level
M06-2X/cc-pVTZ++22 single point energy (SPE) calculations in
gas and solution phase at these geometries revealed that while
t1 is the most favored in the gas phase by ꢀ6 kcal molꢂ1, t2 is
clearly preferred in solution (by ꢀ9 kcal molꢂ1). This is in
contrast to the tautomeric probabilities from LigPrep/Epik,
highlighting the value of a QM approach to such analysis.
Tautomer t2 also allows for approximately two equally stable
conformations (u ¼ 0ꢃ or 180ꢃ) in the free unbound solvated
state, as dened by the dihedral angle u dened in Table 2. The
molecular electrostatic potentials (MESPs) are shown in Fig. 2.
For tautomer t2, the two different conformations have little
Fig. 1 The four most potent glucose analogue inhibitors of GP
discovered to date (1a,12 1b,5 1c13,14 and 1d15) together with their Ki's for
RMGPb inhibition. All ligands possess
a 2-naphthyl substituent
exploiting favorable interactions in the GP catalytic site b-cavity.
replacing the oxadiazole moiety of linkers 2–4 with a 1,2,4-tri-
azole (5, Table 1) in an attempt to improve ligand potency. Prior
to undertaking synthesis, in silico calculations in the form of
Glide docking, quantum mechanics (QM) and quantum
mechanics/molecular mechanics (QM/MM) calculations were
performed to probe the binding potential of these ligands at GP.
Table 1 Inhibition constants (Ki [mM], RMGPb) and Emodel docking scores from Glide (in italics) for N-(b-D-glucopyranosyl)-oxadiazole-car-
boxamides 2–4, and N-(b-D-glucopyranosyl)-1,2,4-triazolecarboxamides 5. The normalized values of Emodel are also shown in parentheses, as
calculated using eqn (1)
Ar
Linker
Scaffold
a
b
c
Oxadiazoles
545b
30
172b
ꢂ92.31(ꢂ3.69)
ꢂ116.15(ꢂ4.01)
ꢂ112.17(ꢂ3.87)
2
3
4
136b
ꢂ100.68(ꢂ4.03)
N.I.a
ꢂ115.78(ꢂ3.99)
33
ꢂ117.65(ꢂ4.06)
104
N.I.a
ꢂ111.92(ꢂ3.86)
145b
ꢂ113.96(ꢂ3.93)
ꢂ100.15(ꢂ4.01)
1,2,4-Triazoles
1
9.2
—
ꢂ102.34(ꢂ4.09)
ꢂ116.52(ꢂ4.02)
ꢂ120.43(ꢂ4.15)
5
a
No inhibition. b Calculated from the IC50 values by the Cheng–Prusoff equation: Ki ¼ IC50/(1 + [S]/Km).17
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Table 2 Comparison of the relative energies (kcal molꢂ1) of different tautomers and conformations of the 1,2,4-triazole (linker 5) for models of
the N-(b-D-glucopyranosyl)-1,2,4-triazolecarboxamides, as calculated using density functional theory. For simplicity and speed, in the calcu-
lations the b-D-glucopyranose was replaced by a methyl group and a phenyl used for the Ar group. Geometries used in calculations were from
M06-2X/6-31+G* optimizationsa
Solution phasec,d
M06-2X/cc-pVTZ++
Gas phasec
M06-2X/6-31+G* M06-2X/cc-pVTZ++
Dihedral
LigPrep tautomer
probability
Tautomer
angle ub (ꢃ)
180
0.487
0.487
0.0(0.0)e
0.0
8.6
t1
0
180
6.5(7.2)e
5.9(6.9)e
5.9
5.3
0.5
0.0
t2
t3
0
0.027
5.9(6.4)e
5.5
9.0
a
Tautomeric states were determined using LigPrep.18 MacroModel 9.9 18 conformational searches were used to locate the above favorable
conformations of the compounds, which were then used in the QM optimizations. The dihedral angle u adopted by the atoms 1, 2, 3, 4 for
b
c
rotation around the 2,3 bond is key to the conformational properties. Values for the minima from the M06-2X/6-31+G* optimizations. Single
d
point energy calculations at the optimized M06-2X/6-31+G* geometries. Continuum treatment of solvation which involved accurate numerical
e
solution of the Poisson–Boltzmann (PBF) equation.24,25 Relative Gibbs free energies given in parentheses.
effect on the ESPs of the amide. However, the ESP in the space in the kinetics results for the N-(b-D-glucopyranosyl)-oxadiazole-
around a gas phase molecule is a key factor in determining its carboxamides with linkers 2–4. The results are shown in Table
ability to accept a proton; in this regard, for t2 (u ¼ 180ꢃ) the 1, with the most consistent results obtained using the Emodel
maximum ESP of the triazole NH nitrogen (62.55 kcal molꢂ1
)
scoring function which combines GlideScore, the non-bonded
and minimum ESP of the connected N (ꢂ50.84 kcal molꢂ1) is interaction energy and the excess internal energy of the gener-
consistent with H+ migration to form the most stable t1 ated ligand conformation. Non-bonded interactions in a
tautomer in the gas phase (Table 2). The intra-molecular NH scoring function are typically calculated as a sum of pair-wise
(amide) and triazole N (lone pair) contacts for the tautomers can interactions. Hence, a larger compound oen receives a more
only loosely be classied as hydrogen bonds under the current favorable score than a smaller compound. In order to overcome
IUPAC guidelines23 (donor angles are ꢀ103–105ꢃ, hence less this bias, a number of different normalization and scaling
than the preferred >110ꢃ), however, there is evidence of charge approaches have been proposed.29,30 Given that a size-depen-
transfer in the case of t1 and t2 tautomers where the magni- dent trend appeared to occur for our training set ligands, we
tudes of the potential at the N are less ꢀꢂ15 kcal molꢂ1. In any used the following equation to account for the different sizes of
case, favourable electrostatic stabilization occurs between these our aryl substituents (phenyl versus naphthyl):
atoms and the contacts are present in all free state conforma-
Emodelnorm ¼ Emodel/number of heavy atoms
(1)
tions. The conformational exibility and MESP of t2 compared
to the other tautomers become important factors when we
consider the binding at GPb.
Docking calculations were performed using Glide 5.8 in
extra-precision (XP) mode,18,26 a docking algorithm which has
proved effective in previous GPb catalytic site studies.15,27,28 The
predictive capability of Glide-XP in the current study was
measured by the ability of the algorithm to reproduce the trends
On initial inspection of Table 1, the predictive capability of
the docking does not appear obvious. For example, the score for
3b is clearly over-estimated. However, a reasonable correlation
R2 ¼ 0.67 between predicted (Emodelnorm) and experimental
(ln Ki) binding strengths was obtained. Further, the most
favorable aryl group for each of the linkers 2–4 was correctly
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Fig. 2 Molecular electrostatic potential (MESPs) can be used to provide insight into a number of hydrogen bonding phenomena. Shown are the
results of MESP calculations (M06-2X/6-31+G*) on a model of the isolated 5a ligand (Me groups instead of glucopyranose) in its different
tautomeric forms and most stable conformations (Table 2). The electrostatic potentials are mapped onto the electron density surface for each
molecule. Only the ESP range of ꢂ50 to 50 kcal molꢂ1 is used for a better visual demonstration of the tautomeric effects; actual minimum–
maximum ESP values were ꢂ55.4 to 63.7 kcal molꢂ1 obtained for the t2 tautomer. The minimum or maximum local electrostatic potential values
(kcal molꢂ1) for the relevant atoms are also shown.
predicted: 2-naphthyl (2b) in the case of 2, 1-naphthyl (3c) in the naphthyl substituents, linker 5 was predicted to give rise to
case of 3 (1-naphthyl) and phenyl (4a) in the case of 4. In terms more potent ligands.
of our key objective, to accurately predict the potential of linker
Despite their obvious importance, tautomeric states are still
5, we noted that the most favorable linker (from 2–4) for each oen not accurately considered in computer-aided drug design
aryl group was also correctly identied: linker 2 in the case of 2- efforts.31 The most favorable tautomeric state for binding of
naphthyl (Ki ¼ 30 mM), linker 3 for 1-naphthyl (Ki ¼ 33 mM), and ligands 5a–5c based on Emodel docking scores was t2 for both
for phenyl the linkers 3 and 4 were predicted to have similar 5a and 5c, and t1 for 5b (Table 3). The potential of using the
efficiency in line with kinetics (Ki's ¼ 104–136 mM). Encouraged lowest QM/MM complex energy for protein–ligand docking pose
by this, we then noted that for each of the phenyl, 1- and 2- selection has recently been highlighted, where QM/MM
Table 3 QM/MM gas and solution phase relative energies (kcal molꢂ1) for the optimized Glide docking poses of ligands 5a–5c considering the
different tautomeric forms as described in the text. The corresponding Emodel docking scores are also shown
QM/MM relative energies
b
Ligand
Tautomera
Emodel (Emodelnorm
)
u dihedral anglec (ꢃ)
Gas phase
Solution phase
5a
t1
t1
t2
t2
t3
t1
t2
t3
t1
t2
t2
t3
t3
ꢂ99.8 (ꢂ3.99)
ꢂ93.9 (ꢂ3.76)
ꢂ102.3 (ꢂ4.09)
ꢂ97.1 (ꢂ3.88)
ꢂ91.0 (ꢂ3.64)
ꢂ116.5 (ꢂ4.02)
ꢂ115.4 (ꢂ3.98)
ꢂ115.9 (ꢂ4.00)
ꢂ114.3 (ꢂ3.94)
ꢂ120.4 (ꢂ4.15)
ꢂ114.6 (ꢂ3.95)
ꢂ112.2 (ꢂ3.87)
ꢂ110.5 (ꢂ3.81)
34.3
178.0
19.7
16.6
7.7
3.8
18.1
19.6
0.0
10.7
13.3
15.8
0.0
8.9
16.5
1.3
ꢂ176.7
0.0
3.5
14.5
14.5
0.0
13.0
18.9
6.5
0.0
13.8
11.8
5b
5c
33.3
ꢂ10.1
3.2
16.2
43.3
ꢂ10.7
ꢂ0.5
ꢂ154.9
0.0
13.1
6.4
a
c.f. Table 2. b Normalized values as calculated using eqn (1). c Numbering scheme for dihedral angle u as shown in Table 2.
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energies were used to re-rank docking poses and compared with Asp283 backbone O (u ꢀ 50ꢃ). If u ꢀ 0ꢃ or u ꢀ ꢁ180ꢃ, although
their native crystallographic binding modes.32 Accordingly, to the 1,2,4-triazole NH forms no direct hydrogen bonds with GPb,
more accurately probe the binding of 5a–5c and its different there are favorable intra-molecular interactions from the car-
tautomers at GP, QM/MM optimizations with QSite 5.8 (ref. 18) boxamide NH with the lone pair of electrons on a triazole N
were performed on the Glide docking poses (Table 3). M06-2X/6- atom for both conformations, as mentioned earlier for the free
31+G* was used for the QM region (the ligands), while the GPb state ligand (Fig. 2). The smaller Ar group (phenyl) of 5a also
protein was described using MM with the OPLS-AA(2005) permits a ipped 1,2,4-triazole conformation (u ¼ ꢂ176.7ꢃ)
forceeld,33 with otherwise default options. Solution phase shown in Fig. 4, where the triazole NH is a little longer than
energies at the optimum geometries were then obtained, hydrogen bonding distance from the Glu88 sidechain carbox-
˚
employing a self-consistent reaction eld (SCRF) continuum ylate (distance ꢀ 3.3 A). Hence, 5a in its t2 tautomeric state
treatment for water solvation effects and which involved accu- exhibits considerable conformational exibility while bound at
rate numerical solution of the Poisson–Boltzmann (PBF) equa- GPb (favorable in terms of entropy). The 1,2,4-triazole has either
tion (M06-2X/6-31+G* + PBF).24,25 The results of the QM/MM intra- and intermolecular hydrogen bonding potential in these
calculations (Table 3) revealed the most stable free state solu- different conformations; while CH–p interactions from the
tion phase t2 tautomer of 5a–5c to also form the most favorable Asp283 CH to the ligand Ar (phenyl) group can further stabilize
complexes with GPb, in both gas and solution phases. From the the GPb–5a complex (Fig. 3). For the binding of ligand 5b
MESPs (Fig. 2) also, the t2 maximum and minimum atomic (Fig. 5), interactions are similar to that as described for 5a in
ESPs of the triazole NH nitrogen and amide CO oxygen (both Fig. 3, except the 2-naphthyl group extends deeper into the b-
conformations) are indicative of greater H-bond acceptor and cavity. However, the larger size of this Ar group limits somewhat
donor capabilities,34,35 respectively, for binding at GPb the conformational versatility (u dihedral angle) of the ligand,
compared to the other tautomers, although the value of ESP and it has much fewer poses compared to either 5a and 5c
maxima on molecular surfaces for predicting hydrogen bond (Table 3). For 5c, the orientation of the 1-naphthyl allows for
acidity has recently been questioned.36
greater conformational exibility in the GPb binding site
For the GPb–5a complex (Fig. 3), as well as forming the compared to its 2-naphthyl equivalent, and indeed the t3
standard hydrogen bonding interactions from the b-D-gluco- tautomer is able to adopt a pose (u ¼ ꢂ154.9ꢃ; Table 3) similar
pyranosyl hydroxyl groups and a hydrogen bond from the car- to that shown for 5a in Fig. 3, with a ip in the 1,2,4-triazole.
boxamide carbonyl O with the Leu136 backbone NH, through
rotation around the dihedral angle u the 1,2,4-triazole in its
favored solution phase t2 tautomeric form has the potential to
Synthesis
hydrogen bond with the Asp339 carboxylate on one side of the b-
cavity (water-bridged), and on the other side directly with the
Based on the computational results, synthesis of the ligands
was considered worthwhile. Several methods have been
Fig. 3 The lowest solution phase energy pose of ligand 5a (tautomer t2) bound at GPb with a dihedral angle (u ¼ 19.7ꢃ; Table 3), as calculated
using QM/MM and described in the text.
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Fig. 4 The flipped 1,2,4-triazole binding conformation of tautomer t2 of 5a at GPb (dihedral angle u ¼ ꢂ176.7ꢃ; Table 3), as calculated using QM/
MM and described in the text. The tautomeric t2 state allows for favorable intra-molecular hydrogen bond contacts between the carboxamide
NH and a 1,2,4-triazole N when u is close to 0ꢃ (Fig. 2) or flipped (above). In the flipped conformation, the 1,2,4-triazole NH is close to hydrogen
bonding distance with the Glu88 carboxylate.
published for the preparation of 3,5-disubstituted 1,2,4-tri- the desired triazoles under thermal conditions,37–39 the
azoles.37 While acyl-amidrazones (prepared from amides, cycloaddition of nitriles with nitrile-imines or nitrile-imi-
thioamides, nitriles, or amidines) could be transformed into nium ions gave 2,3,5-trisubstituted 1,2,4-triazoles in
Fig. 5 The lowest solution phase energy pose of ligand 5b (tautomer t2) bound at GPb (dihedral angle u ¼ ꢂ10.1ꢃ; Table 3), calculated using QM/
MM as described in the text.
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Scheme 1 Synthesis of the predicted molecules 5a and 5b.
moderate to good yields.40–43 The reaction of 5-substituted shown in Scheme 1. The imidoyl chlorides 10a,b prepared from
tetrazoles with imidoyl chlorides (easily obtained from the N-benzyl-amides 9a,b as described earlier,13 were reacted with
corresponding amides by SOCl2) gave 3,4,5-trisubstituted tetrazole 6 (used without any purication) to give the corre-
1,2,4-triazoles in good yields.44,45
sponding 4-benzyl-N-(2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl)-
This latter method was extensively applied for the prepara- 5-aryl-1,2,4-triazole-3-carboxamides 11a,b in moderate yields. In
tion of 3-(C-glycosyl)-5-substituted-1,2,4-triazoles in our group.13 the case of the 1-naphthyl derivative 5c, the expected formation
The predicted target compounds of this study (5a,b) have been of 11c from 10c was unsuccessful, no transformation was
prepared using this methodology starting from tetrazole 6 16 as
Table 4 Results of ADMET property predictions for the different tautomers of the N-(b-D-glucopyranosyl)-1,2,4-triazolecarboxamides inhibitors
(5a–5c) studied in this worka
Jorgensen's rule of three and
Lipinski's rule of ve and violations (V)b
HBDg HBAh log P(o/w)
violations (V)b
Caco-2i
2 c
˚
Inhibitor/ Mr [Da]
log S
PSA [A ]
2
d
tautomer (<500)
(#5)
(#10) (<5)
V
[nm sꢂ1] (>22) (>ꢂ5.7)
NMPj (<7)
V
(<140 A ) log Khsa
log BBe
TSWf
˚
5a
t1
t2
t3
350.3
350.3
350.3
6
6
6
10
10
10
ꢂ1.6
ꢂ1.1
ꢂ1.4
1
1
1
19.2*
19.2*
18.9*
ꢂ2.7
ꢂ2.8
ꢂ2.8
5
5
5
1
1
1
173.3
173.3
173.4
ꢂ1.02
ꢂ0.93
ꢂ0.97
ꢂ2.8
ꢂ2.8
ꢂ2.8
—
—
—
5b
t1
t2
t3
400.4
400.4
400.4
6
6
6
10
10
10
ꢂ0.8
ꢂ0.3
ꢂ0.6
1
1
1
19.2*
19.2*
19.0*
ꢂ3.4
ꢂ3.6
ꢂ3.6
5
5
5
1
1
1
173.3
173.2
173.4
ꢂ0.85
ꢂ0.72
ꢂ0.80
ꢂ2.9
ꢂ2.9
ꢂ2.9
—
—
—
5c
t1
t2
400.4
400.4
400.4
130–
725
6
6
6
0–6
10
10
10
2–20
ꢂ0.8
ꢂ0.4
ꢂ0.7
ꢂ2.0–
6.5
1
1
1
—
18.2*
18.8*
18.3*
<25 poor
>500 great
ꢂ3.4
ꢂ3.6
ꢂ3.5
ꢂ6.5–
0.5
5
5
5
1–8
1
1
1
—
174.7
174.2
174.6
7–200
ꢂ0.85
ꢂ0.72
ꢂ0.80
ꢂ1.5–
1.5
ꢂ2.9
ꢂ2.9
ꢂ2.9
ꢂ3.0–
1.2
—
—
—
—
t3
Rangek
a
ADMET data calculated using Qikprop 3.5;18 predicted properties outside the range for 95% of known drugs indicated with an asterisk (*). b Rules
as listed in the columns, with any violations of the rules highlighted in italics. c PSA represents the van der Waals (polar) surface areas of N and O
2
d
e
atoms; recommended PSA < 140 A according to Veber et al.54 log Khsa: predicted binding to human serum albumin. log BB: predicted blood-
˚
f
g
h
brain barrier coefficient. Toxicity structural warnings from FAF-Drugs2.48 Number of hydrogen bond donors. Number of hydrogen bond
acceptors. i Caco-2 cell permeability. j Number of primary metabolites. Range for 95% of known drugs.18
k
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detected and the desired compound has not been synthesized
yet using any other synthetic methods.
The protecting groups were cleaved in a two-steps procedure.
The N-benzyl group of 11a,b was removed rst by catalytic
hydrogenation in excellent yields (94 and 85%). Subsequently,
Experimental
Computational details (additional)
Free ligand calculations. To determine the important (low
energy) tautomeric forms/conformations of the model ligands 5
(Table 2), 1000 steps of the Monte Carlo Multiple Minima
(MCMM) method were performed using MacroModel 9.9,18 the
OPLS-AA(2005) forceeld33 and GB/SA continuum model for
H2O solvation effects.55 The conformations were then optimized
using DFT (M06-2X/6-31+G*) with Jaguar 8.0 18 and frequency
calculations used to characterize the stationary points as true
minima, as well as for calculation of the gas-phase Gibbs free
energies at 298.15 K. For the solution phase QM calculations
(M06-2X/cc-pVTZ++)22 at these geometries, a SCRF continuum
treatment of solvation with the PBF equation was used.24,25
Protein preparation. The initial setup of the GPb receptor for
calculations was performed using Schrodinger's “Protein Prep-
aration Wizard”18 starting from the GPb–1b co-crystallised
complex. Water molecules were deleted, bond orders assigned,
and hydrogen atoms added, with protonation states for basic
and acidic residues based on residue pKa values at normal pH
(7.0). Subsequent optimization of hydroxyl groups, histidine
protonation states and C/N atom “ips”, and side-chain O/N
atom ips of Asn and Gln was based on optimizing hydrogen
bonding patterns. The phosphate in pyridoxal-phosphate (PLP)
was assigned in monoanionic form. Finally, an “Impref” mini-
mization of the GPb complex was performed using the OPLS-
AA(2005) force eld33 to remove steric clashes and bad contacts
´
O-deacetylation was effected by the Zemplen method to give
fully deprotected N-(b-D-glucopyranosyl)-5-aryl-1,2,4-triazole-3-
carboxamides 5a,b.
Enzyme kinetics
The inhibition constants (Ki) against rabbit muscle glycogen
phosphorylase b (RMGPb) of compounds 5a,b were determined
according to the protocol described earlier.46 The results are
summarized in Table 1 together with the Ki's of 1,2,4- and 1,3,4-
oxadiazole-carboxamide type inhibitors. As the data show, the
1,2,4-triazole analogues 5a (Ki ¼ 1 mM) and 5b (Ki ¼ 9.2 mM) are
more potent inhibitors than the oxadiazole analogues, consis-
tent with our computational predictions for favorable binding
at GPb.
Pharmacokinetic predictions
The early evaluation of the pharmacokinetic properties of
lead compounds in drug design efforts is desirable due to the
potential for failure in late stage clinical trials. Accordingly,
the absorption, distribution, metabolism and excretion
(ADME) properties of 5a–5c were predicted using the QikProp
3.5 program in normal mode.18 Toxicity is the leading cause
of drug attrition in clinical trials, together with lack of effi-
cacy.47 The FAF-Drugs2 server was used to extract any
potential toxicity structural warnings.48 Results are shown in
Table 4.
An orally active drug should have no more than one
violation of Lipinski's ‘rule of ve’,49 while for Jorgensen's
‘rule of three’50,51 more drug-like molecules have fewer viola-
tions. The N-(b-D-glucopyranosyl)-1,2,4-triazolecarboxamides
5a–5c had only one violation of each. The Lipinski violation is
due to too many hydrogen bond donors (HBDs), 6 instead of
#5 in each case, but this is still within the range for 95% of
known drugs and consistent with some recent ‘beyond rule of
ve’ studies.52,53 In terms of Jorgensen's rules, the Caco-2 cell
permeability (>22 nm sꢂ1) is violated for the ligands (ꢀ19 nm
sꢂ1), where previously we noted that the sensitive balance
between adequate lipophilicity and solubility may need
attention in lead optimization of heterocyclic derivatives
conjugated to glucose.16 Meanwhile, the log Khsa's (degree of
human serum albumin affecting bioavailability) is ꢀꢂ0.9 to
ꢂ0.7 and within the range for 95% of known drugs (ꢂ1.5 to
˚
but with heavy atoms constrained to within 0.3 A (RMSD) of
their crystallographic positions.
Docking details. For the Glide 5.8 docking calculations in
extra-precision (XP) mode, the shape and properties of the GPb
catalytic binding site were rst mapped onto grids with
˚
dimensions of ꢀ26.7 ꢄ 26.7 ꢄ 26.7 A centered on the native co-
˚
crystallized ligand (1b). Core constraints (1 A) on the six glucose
ring atoms + the ligands' amide moieties to retain them close to
the native ligand crystallographic positions were applied. Post-
docking minimization of the ligand poses was performed (with
strain correction) with a maximum of 5 poses per ligand saved.
Poses were considered conformationally distinct for RMSDs
˚
(heavy atoms) > 0.5 A.
Synthesis
The general procedures employed for the synthesis are
described in the ESI,† together with the NMR data.
GP inhibition assay
1.5), while the log BB (blood-brain barrier coefficients) values Glycogen phosphorylase b was prepared from rabbit skeletal
(ꢂ2.9 to ꢂ2.8) are also within the desirable range (ꢂ3.0 to 1.2). muscle according to the method of Fischer and Krebs56 using 2-
Importantly, there were no toxicity structural warnings for the mercaptoethanol instead of L-cysteine, and recrystallized at
ligands from FAF-Drugs2. Overall, the pharmacokinetic least three times before use. The kinetic studies with glycogen
results are similar with the results for the N-(b-D-glucopyr- phosphorylase were performed as described previously.46
anosyl)-oxadiazole-carboxamides
previously
reported.16 Kinetic data for the inhibition of rabbit skeletal muscle
However, in terms of pharmacodynamics the 5a,b ligands glycogen phosphorylase by monosaccharide compounds were
proved via kinetics experiments to be much more potent collected using different concentrations of a-D-glucose-1-phos-
inhibitors of GP.
phate (4, 6, 8, 10, 12 and 14 mM) and constant concentrations of
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glycogen (1% w/v) and AMP (1 mM). The enzymatic activities
were presented in the form of double-reciprocal plots (Line-
weaver–Burk) applying a nonlinear data-analysis programme.
The inhibitor constants (Ki) were determined by Dixon plots, by
replotting the slopes from the Lineweaver–Burk plots against
the inhibitor concentrations. The means of standard errors for
all calculated kinetic parameters averaged to less than 10%.3,57
IC50 values were determined in the presence of 4 mM glucose 1-
phosphate, 1 mM AMP, 1% glycogen, and varying concentra-
tions of an inhibitor.
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