J. Ren et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2525–2529
2527
Table 1
calculations, it was shown that the binding of triazolopyridine at
the ATP site of HSP90 is enthalpy favorable and the electrostatic
interaction between triazolopyridine group and HSP90 is less
important than the van der Waals interactions. As pointed by
numerous analyses that the enthalpy and entropy compensation
is a common phenomenon in protein–ligand interactions16 that
says favorable enthalpy interactions such as hydrogen bonding,
The fluorescence polarization assay of the identified active fragments and compounds
from initial optimization
R1
HO
R2
p–p stacking can countpoise by the reduction in entropy contribu-
tion due to restriction of the flexibility of the receptor. Therefore,
the actually improvement in binding free energy of compound 9
may be low than the enthalpy contribution since the triazolopyri-
dine group clearly hinders the mobility of its surrounding residues
of HSP90. Nevertheless, we thought the binding interaction of
triazolopyridine group is not the destructive factor accounting for
the low activity of compound 9.
OH
O
6-15
R2
a
Compd
R1
FP IC50 (lM)
N
N
N
Br
Br
8
8.8 2.1%@5
18.7 5.1%@5
0.63 0.08
l
Mb
Mb
N
N
N
N
N
N
N
Then we performed the solvation free energy calculations on
compound 9. By utilizing the density functional M06-2X at 6-
31+G(d) level combined with the SMD solvation model17 imple-
mented in Gaussian program, we found that compound 9 has a
low solvation free energy about ꢀ26.66 kcal/mol, which indicated
that when binding to the HSP90, the compound needs to overcome
a large desolvation effect to transfer from aqueous solution to the
protein binding site. The solvation free energy of the virtual com-
pound without the triazolopyridine group is ꢀ19.72 kcal/mol. Tak-
ing together, it means that adding the triazolopyridine group needs
to supply about 7 kcal/mol desolvation free energy. Therefore, in
the next round of optimization, we focused on the issue of the
hydrophilicity of the compounds, and tried to reduce the ligand
solvation free energy to improve the binding affinity to HSP90.
Based on the previous SAR of HSP90 inhibitors,18 the tetrahy-
droisoquinoline in compound 9 is not the optimal for binding.
Therefore, together with above mentioned rationale, several het-
ero-aromatic rings were used to substitute the [12,4]triazolo[4,3-
a]pyridine group, and produced compounds 10–15. Comparison
of compounds 10 to 8 or 11 to 9, the only difference occurs at
the triazolopyridine ring substituted with the indole group; the
binding activities from FP assay are dramatically improved. This
is consistent with the calculation that the solvation free energy
of compound 11 is ꢀ13.98 kcal/mol, much less than the value of
compound 9 (ꢀ26.66 kcal/mol). Similar trend can be deduced from
the inhibition activities of 13–15. Compounds 14 and 15 are about
3 and 7-fold less active than ligand 13 respectively, which rein-
forces the hydrophobic property of the fused bicyclic ring is critical
for the binding. From compounds 10–13 in Table 1, it was found
that compound 13 with isoindoline group at R2 is the most potent
inhibitor, consistent with the data reported in the previous struc-
ture–activity relationship about R2 group.12 Consequently, we
selected compound 13 to carry out the further optimization by
attaching various functional groups to the indole moiety.
The synthesis route of compounds in Table 2 is outlined in
Scheme 1 (see the Supporting material for details). Generally, the
synthesized compound 16 was hydrolyzed with lithium hydroxide
to give compound 17. Compound 18 was prepared by coupling the
key intermediate 17 with isoindoline using standard EDC/HOBt
coupling conditions. After coupling of 18 with different substituted
indoles,19 followed by de-protection of the benzyl protecting
groups, the final product 28–36 were obtained.
Inspecting the co-crystal structure, it was thought that the sub-
pocket accommodating the indole ring cannot permit a large sub-
stituent on the indole, especially at 6-, 7-position of indole ring.
Based on the in silico modeling, we prepared 9 compounds with
various substituents at three positions of indole. As listed in
Table 2, modification of the R1-position of indole with linear polar
groups (compounds 28–30) decreases the binding affinity consid-
erably, at least 30-fold less active than the parent compound 13,
especially the compound with acidic group (30). Due to the limited
N
N
9
l
N
10
11
12
13
N
0.13 0.01
N
N
N
S
0.33 0.02
0.038 0.006
N
N
N
14
15
0.13 0.01
0.25 0.03
N
N
N
N
a
All the inhibition ratios or IC50 values were obtained from triple measurements.
The compounds Geldanamycin and NVP-AUY922 were used as control, with IC50
values 74.0 7 nM and 8.0 0.2 nM, respectively.
b
The inhibition ratio measured with FP assay method at ligand concentration
5 lM.
methods to quantify two important components of the protein–
ligand binding free energy, namely the binding interaction energy
and solvation free energy, of ligand 9 with or without the triazolo-
pyridine group. The Glide docking program15 in Schrödinger soft-
ware package was used to calculate the protein–ligand binding
energy. Since we solved the crystal structure of ligand 9 bound
to the N-terminal of HSP90, the SCORE-ONLY mode was used to
obtain the binding interaction energy. From the calculation, it
was found that the non-covalent interaction energy between com-
pound 9 and the N-terminal of HSP90 is ꢀ68.78 kcal/mol, and the
large contribution stems from van der Waals interactions
(ꢀ56.83 kcal/mol). While for the virtual compound by deleting
the triazolopyridine group (see chemical structure 9v in Support-
ing materials) from ligand 9, its interaction energy is
ꢀ48.77 kcal/mol. The large difference of non-covalent interactions
between two compounds is rooted from the difference of their van
der Waals interactions (ligand 9 is ꢀ20.25 kcal/mol lower), while
the coulomb electrostatic interactions are almost the same (ligand
9: ꢀ11.95 kcal/mol; the virtual compound without the triazolo-
pyridine group: ꢀ12.18 kcal/mol). From these docking energy