Computer-aided discovery of phenylpyrazole based amides as potent S6K1 inhibitors

Article abstract of DOI:10.1039/c9md00537d

Ribosomal protein S6 kinase beta-1 (S6K1) is an attractive therapeutic target. In this study, computational analysis of five thiophene urea-based S6K1 inhibitors was performed. Molecular docking showed that the five compounds formed hydrogen bonds with residues Glu173 and Leu175 of S6K1 and hydrophobic interactions with residues Val105, Leu97 and Met225, and these interactions were key elements for the inhibitory potency of the compounds. Binding free energy (?G_bind) decomposition analysis showed that Leu97, Glu173, Val 105, Leu175, Leu97 and Met225 contribute the most to ?G_bind. Based on the computer results, phenylpyrazole based amides (D1-D3) were designed and synthesized. Biological evaluation revealed thatD2exhibited 15.9 nM S6K1 inhibition, medium microsomal stability and desirable bioavailability.

Full text of DOI:10.1039/c9md00537d

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RESEARCH ARTICLE
Computer-aided discovery of phenylpyrazole
based amides as potent S6K1 inhibitors†
Cite this: DOI: 10.1039/c9md00537d
a
Yuxing Sun,a Lianhua Zhao,a Jinpeng Pana and Yangbo Fengbc
*
Yan Yin,
Ribosomal protein S6 kinase beta-1 (S6K1) is an attractive therapeutic target. In this study, computational
analysis of five thiophene urea-based S6K1 inhibitors was performed. Molecular docking showed that the
five compounds formed hydrogen bonds with residues Glu173 and Leu175 of S6K1 and hydrophobic
interactions with residues Val105, Leu97 and Met225, and these interactions were key elements for the
inhibitory potency of the compounds. Binding free energy (ΔGbind) decomposition analysis showed that
Leu97, Glu173, Val 105, Leu175, Leu97 and Met225 contribute the most to ΔGbind. Based on the computer
results, phenylpyrazole based amides (D1–D3) were designed and synthesized. Biological evaluation
revealed that D2 exhibited 15.9 nM S6K1 inhibition, medium microsomal stability and desirable
bioavailability.
Received 15th November 2019,
Accepted 4th April 2020
DOI: 10.1039/c9md00537d
rsc.li/medchem
computational work or X-ray cocrystal structures to support
Kablaoui's hypothesis. Thus, in this study, molecular docking
Introduction
Ribosomal protein S6 kinase beta-1 (S6K1), also known as p70
S6 kinase-1, is a serine/threonine kinase that contains two
non-identical kinase catalytic domains and phosphorylates
several proteins. S6K1 activity is suggested to be closely
associated with diverse cellular processes, including protein
synthesis,1 mRNA processing,2 glucose homeostasis,3 and cell
growth and apoptosis.4 Thus, S6K1 is an attractive therapeutic
target to develop drugs that treat aging-related diseases,5 type
2 diabetes6 and cancer.7 Although ATP-competitive small
molecular S6K1 inhibitors exist,8,9 there remains an urgent
need to identify new potent S6K1 inhibitors because of
increasing pharmacological interest.
was used to predict the binding modes of Kablaoui's S6K1
inhibitors, and molecular dynamics (MD) simulations of
Compd. 5 followed by calculation of the binding free energy
(ΔGbind
) with the molecular mechanics-generalized-Born/
surface area (MM/GBSA) method were used to gain insight
into the origin of the inhibitory activity at the amino acid
residue level. Three fluorine-containing S6K1 inhibitors
(D1–D3) were then designed according to the computational
results, and the chemical synthesis and an in vitro bioassay
test were performed to validate the molecular models.
Finally, the pharmacokinetic properties and metabolism
stabilities of D1 and D2 were investigated.
The group of Kablaoui has developed a series of potent
and selective thiophene urea-templated S6K1 inhibitors
(Compd. 1–5; Fig. 1). They divided where these compounds
interact with S6K1 into three regions, i.e., the hinge region,
hydrophobic pocket and solvent exposed region, and
provided a possible pharmacophore model.9 Computational
Results and discussion
As shown in Fig. 1, the replacement of the phenol with a
5-amino indazole increased the inhibitory activity (IC50 = 34
nM for Compd. 3 vs. 4 μM for Compd. 2). The IC50 of Compd.
1 with the pyridinylphenyl ether was lower than those of the
methods can predict affinity before
a compound is
synthesized and hence accelerate the drug discovery process
by reducing the number of iterations required and have often
provided novel structures. Recently, our research group
designed small bioactive organic molecules by using
combined
computational
studies.10
There
is
no
a School of Chemical and Environmental Engineering, Shanghai Institute of
Technology, 100 Hai Quan Rd., Shanghai, 201418, P. R. China.
E-mail: yinyan@sit.edu.cn
b Medicinal Chemistry, The Scripps Research Institute, 130 Scripps Way, Jupiter,
Florida 33458, USA
c Reaction Biology Corporation, Malvern, PA 19355, USA
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c9md00537d
Fig. 1 S6K1 inhibitors reported by Kablaoui's group.
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other compounds but still within a measurable range (IC50
=
9 μM). Substitutions at the 5-position of the thiophene ring
had clear effects on inhibition (IC50 = 223 nM for Compd. 4
with the methyl group vs. 15 nM for Compd. 5 with the
t-butyl group). Modifications to the amide region located at
the 3-position of the thiophene ring only gave a slight change
in activity (IC50 = 34 nM for Compd. 3 vs. 15 nM for Compd.
5). The following SAR explanation was provided by Kablaoui's
group:9 (a) the indazole moiety possibly interacts with the
hinge region of the S6K1 binding pocket; (b) the 5-position
substituent fits into a hydrophobic pocket of S6K1; (c) the
3-position lacks key interactions with the S6K1 binding
pocket and the 3-position of thiophene is a solvated region.
Molecular docking predicts the preferred orientation of a
ligand in
a protein–ligand complex. Knowledge of the
preferred orientation is used to predict the strength of the
association or binding affinity of the ligand–protein
complexes. Compd. 1–5 were docked into the binding site of
S6K1 (Fig. 2) to understand the pharmacophore models of
Kablaoui and guide the potent S6K1 inhibitor design.
In the S6K1–Compd. 1 complex (Fig. 2a and b), there was
one hydrogen bond (H-bond) between Leu175 and the
pyridine ring in the hinge binding region, seven hydrophobic
interactions (Leu97–phenyl ring, Leu97–pyridine ring,
Ala121–pyridine ring, Met225–pyridine ring, and Met225–
phenyl ring in the hinge binding region, and Val105–
thiophene and Val105–t-butyl group in the hydrophobic
region), and one unfavorable steric clash between the S atom
of Met225 and the O atom of the phenol. In the S6K1–
Compd. 2 complex (Fig. 2c and d), there were two H-bonds in
the hinge binding region (Leu175⋯OH and Met225⋯phenyl
ring) and six hydrophobic interactions (Leu97–phenyl ring,
Leu97–methyl group of the phenyl ring, Tyr174–methyl group
of the phenyl ring and Leu175–phenyl ring in the hinge
binding region and Val105–thiophene and Val105–t-butyl
group in the hydrophobic region). In the S6K1–Compd. 3
complex (Fig. 2e and f), there were two H-bonds in the hinge
binding region (Glu173–CO⋯H–N in the pyrazole ring and
Leu175–N–H⋯N in the pyrazole ring) and nine hydrophobic
interactions (Leu97–phenyl ring, Leu97–pyrazole ring,
Ala121–phenyl ring, Ala121–pyrazole ring, Leu175–phenyl
ring, Leu175–pyrazole ring and Met225–phenyl ring in the
hinge binding region and Val105–thiophene and Val105–t-
butyl group in the hydrophobic region). In the S6K1–Compd.
4 complex (Fig. 2g and h), there were three H-bonds (Glu173–
CO⋯H–N in the pyrazole ring and Leu175–N–H⋯N in the
pyrazole ring of the hinge binding region and Glu222–
CO⋯H–CHCH2NIJCH3)2 in the solvated region) and eight
hydrophobic interactions (Met225–phenyl ring, Leu97–phenyl
ring, Ala121–pyrazole ring, Leu175–phenyl ring, Leu17–
pyrazole ring and Met225–pyrazole ring in the hinge binding
region and Val105–thiophene and Val105–t-butyl group in the
Fig. 2 Docking results. (a) 3D view of the S6K1–Compd. 1 complex. (b)
2D view of the S6K1–Compd. 1 complex. (c) 3D view of the S6K1–
Compd. 2 complex. (d) 2D view of the S6K1–Compd. 2 complex. (e) 3D
view of the S6K1–Compd. 3 complex. (f) 2D view of the S6K1–Compd.
3 complex. (g) 3D view of the S6K1–Compd. 4 complex. (h) 2D view of
the S6K1–Compd. 4 complex. (i) 3D view of the S6K1–Compd.
5
complex. (j) 2D view of the S6K1–Compd. 5 complex. In the 3D view,
carbon atoms of the ligand and protein are shown in yellow and white,
respectively. In the 2D view, conventional H-bonds, carbon H-bonds,
and pi–donor H-bonds are shown as green dotted lines. Pi–sulfur, alkyl,
pi–alkyl and pi–sigma interactions (hydrophobic interactions) are
shown as pink dotted lines, except for the pi–sigma interactions, which
are colored purple.
hydrophobic region). In the S6K1–Compd.
5 complex
(Fig. 2i and j), there were three H-bonds (Glu173–CO⋯H–N
in the pyrazole ring and Leu175–N–H⋯N in pyrazole ring in
hinge binding region and Glu222–CO⋯H–CHCH2NIJCH3)2
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Table 1 The binding free energies for the S6K1–Compd. 5 complexa
in the solvated region) and ten hydrophobic interactions
(Leu97–pyrazole ring, Leu97–phenyl ring, Ala121–phenyl ring,
Ala121–pyrazole ring, Leu175–pyrazole ring, Met225–phenyl
ring and Met225–pyrazole ring in the hinge binding region,
Val105–thiophene and Val105–t-butyl group in the
hydrophobic region, and Leu172–t-butyl group in the solvated
region).
ΔGvdw
ΔGSA
ΔGele
ΔGGB
ΔGbind
Ave.
S.D.
−54.60
−6.08
−26.74
43.31
2.03
−44.12
1.19
0.12
2.10
1.32
a
All energies are in kcal mol−1
.
There were several key binding elements that contributed
to the high potency of Compd. 3–5. Fig. 2f, h, and j indicate
that the pyrazole headgroup binds to the hinge region of
S6K1 through H-bonds. The first H-bond is between the NH
of Leu175 and the N of the pyrazole ring, and the second
H-bond is between the N of Glu173 and the NH of the
pyrazole ring. The second H-bond probably accounts for the
higher inhibitory activity of Compd. 3–5 when compared with
that of Compd. 1 and Compd. 2, because this H-bond is
absent in these two compounds (Fig. 2b and d). Importantly,
there is a hydrophobic pocket under the P-loop in these
enzyme–ligand complexes, and the substitution groups at the
5-position of thiophene in Compd. 3–5 were buried in this
pocket with the phenyl headgroup and thiophene ring in
close proximity to this pocket. Thus, hydrophobic
interactions with the hydrophobic surface of this pocket are
postulated to be dominant factors that contribute to the
higher potencies of Compd. 3> and Compd. 5 vs. Compd. 4
(Fig. 2e and i vs. g).
contributions (ΔGele + ΔGGB) were less important than non-
polar contributions (ΔGvdw + ΔGSA) to the S6K1 inhibitory
activity of Compd. 5, and van der Waals interactions were
crucial to the binding free energy of the inhibitor.
The total ΔGbind of the S6K1–Compd. 5 complex was
decomposed using the MM/GBSA method to yield the
individual energy contributions.11 The contributions of the
residues are presented in Fig. 2j and are −2.9527 kcal mol−1,
−2.1242 kcal mol−1, −2.1029 kcal mol−1, −1.8915 kcal mol−1,
−1.8517 kcal mol−1, −1.3419 kcal mol−1, −0.8753 kcal mol−1
and −0.2025 kcal mol−1 for Glu173, Val105, Leu175, Leu97,
Met225, Glu222, Ala121 and Leu172, respectively (Fig. 4). We
concluded that Leu97, Glu173, Leu175 and Met225 located in
the hinge region and Val105 located in the hydrophobic
pocket have important energy contributions (more than −1.85
kcal mol−1). Additionally, Glu222, Ala121 and Leu172 showed
positive contributions to the binding.
We previously developed potent and highly selective Rho
kinase inhibitors with a pyrazole-phenyl scaffold and realized
that hydrophobic benzodioxane and chroman groups bound
within the hydrophobic pocket.12,13 The optimization of
Compd. 5 started by replacing the central thiazole ring with a
benzodioxane group or a chroman group while at the same
time the indazole headgroup was lengthened to a pyrazole
phenyl group. Since fluorine substituents affect nearly all
adsorption, distribution, metabolism and excretion
properties of a lead compound, fluorine substituents have
become widespread and important drug components. After
focusing previously on the design of bioactive fluorine-
containing compounds,14,15 we estimated that F substituents
should contribute to enzyme inhibition and were introduced
in this work. Three phenylpyrazole-based amides (D1–D3)
with fluorine in the middle phenyl ring were designed as
S6K1 inhibitors (Scheme 1). As expected, D1–D3 interacted
with Glu173 and Leu175 through H-bonds and with Val 105,
Leu97, Met225 and Ala121 through hydrophobic interactions
(Fig. 5). Molecular docking results indicated that the F
Compd. 5 exhibited the highest inhibitory activity among
Compd. 1–5. Thus, Compd. 5 was used in 10 ns molecular
dynamics (MD) simulations to validate the accuracy of the
docking and calculate ΔGbind
. The root mean square
deviation (RMSD) plot revealed that Compd. 5 reached
equilibrium after 2 ns (RMSD = 3.35 0.48 Å; Fig. 3a), and
the initial and final structures were similar (Fig. 3b), which
indicated that the binding pocket and the conformation of
the ligand were stable and the docking results were reliable.
After the MD simulations, we extracted the 7–8 ns period
of the Compd. 5–S6K1 complex to calculate the ΔGbind by
using the MM/GBSA method.11 The individual energy terms
including the van der Waals contribution (ΔGvdw),
electrostatic contribution (ΔGele), polar contribution to
solvation free energy (ΔGGB) and non-polar contribution to
solvation free energy (ΔGSA) were also calculated. ΔGvdw, ΔGSA
,
ΔGele, ΔGGB, and ΔGbind were −54.60 kcal mol−1, −6.08 kcal
mol−1, −26.74 kcal mol−1, 43.31 kcal mol−1 and −44.12 kcal
mol−1, respectively (Table 1). We concluded that polar
Fig. 3 MD simulation results. (a) Plot of RMSD versus time. (b) S6K1–
Compd. 5 complex. The initial and final structures are shown in red
and green, respectively.
Fig. 4 Energy contributions of key residues.
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of cytochrome P450 isoforms 1A2/2C9/2D6/3A4 at 10 μM were
42/63/40/38 for D1 and 12/84/61/43 for D2, which indicated
that 2C9 was the main CYP isoform causing the metabolism
instabilities.
Experimental
Chemistry
All reagents and solvents were from commercially available
sources and used without further purification unless
otherwise stated. Thin layer chromatography (TLC) analysis
was performed with
chromatography was carried out on silica gel (200–300 mesh).
NMR spectra were recorded with Bruker 500 MHz
a precoated silica gel. Column
Scheme 1 Synthesis of D1–D3.
a
spectrometer. The line positions of multiplets were given in
ppm (δ), and the coupling constants ( J) were given in hertz.
Elemental analysis was performed on a Thermo Fisher
elemental analyzer. HRMS was performed with Solaril X70
FT-MS apparatus.
substituent at the third position of the middle phenyl ring
formed a H-bond with Met225 (Fig. 5b, d, and f), and a
H-bond interaction at the 2-position with an F substituent
would decrease the activity of the inhibitor by distorting the
inhibitor and forcing the benzodioxane ring away from the
hydrophobic pocket (Fig. 5e).
Encouraged by the docking results for inhibitors D1–D3,
the synthesis of these newly designed compounds was carried
out (Scheme 1). D1 and D2 were readily synthesized by a
short sequence that began with amide condensation of
commercially available aniline and a chroman carboxylic acid
to yield amide A. The pyrazole was then incorporated through
standard palladium-catalyzed Suzuki coupling to give the
desired products D1 and D2. Similarly, a short sequence
involving sequential Suzuki coupling between the
4-pyrazoleboronic acid pinacol ester and 4-bromo-2-fluoro-1-
nitrobenzene, Pd/C catalyzed nitro reduction and the amide
condensation reaction gave the inhibitor D3.
D1–D3 were then shipped to the Reaction Biology
Corporation in the USA for biological activity testing. D1–D3
exhibited similar inhibitory activity against S6K1 to Compd.
3–5 (IC50 = 56.7 nmol L−1 for D1, 15.9 nmol L−1 for D2, and
179 nmol L−1 for D3), which indicated that the information
obtained from the computational efforts successfully guided
the design of potent S6K1 inhibitors.
The pharmacokinetic (PK) properties of D1 and D2 in
Sprague-Dawley rats were studied (Table 2). D1 demonstrated
28 mL min−1 kg−1 clearance (Cl), 0.7 L kg−1 volume of
distribution (Vd), 1.6 μM h AUC, 0.67 nM oral Cmax and 25%
bioavailability (%F), whereas D2 demonstrated 29 mL min−1
kg−1 Cl, 0.8 L kg−1 Vd, 1.5 μM h AUC, 0.60 nM oral Cmax and
23% bioavailability.
Synthesis of D1–D3
4-Bromo-3,5-difluoroaniline (1 mmol) was added to a stirred
solution of a carboxylic acid derivative (1 mmol), HATU (1
mmol), and DIEA (3 mmol) in DMF at room temperature.
After the amide coupling was finished, detected by TLC,
DMF was removed under reduced pressure, and the
obtained residue was extracted with EtOAc (3 × 20 mL). The
combined organic phases were then washed with saturated
brine (3 × 5 mL), dried over Na2SO4, and evaporated in a
rotavap to give intermediate A, which was used without
further purification.
PdijPIJPh)3]4 (0.15 mmol) was added to a degassed mixture
of 4-pyrazoleboronic acid pinacol ester (1.2 mmol), K2CO3 (3
mmol), and intermediate A (1 mmol) in dioxane/H2O (2 mL,
4 : 1 by volume) under nitrogen protection. This solution was
stirred at 95 °C until the complete disappearance of A. Then,
the reaction mixture was concentrated under reduce
pressure, extracted with EtOAc (3 × 20 mL), washed with
brine (3
× 5 mL), dried over anhydrous Na2SO4, and
evaporated in a rotavap to give a residue. Finally, the
obtained residue was purified through flash column
chromatography to give the targeted inhibitors D1 and D2.
N - ( 3 , 5 - D i f l u o r o - 4 - ( 1 H - p y r a z o l - 4 - y l ) p h e n y l ) - 6 -
methoxychromane-3-carboxamide (D1): (58% yield over two
steps). 1H NMR (DMSO-d6, 500 MHz) δ: 12.61 (br, 1H), 8.21
(br, 1H), 8.13–8.10 (m, 2H), 7.74–7.68 (m, 2H), 6.74–6.67 (m,
3H), 4.42–4.39 (m, 1H), 4.09–4.04 (m, 1H), 3.69 (s, 3H), 3.09–
2.96 (m, 3H). 13C NMR (DMSO-d6, 125 MHz) δ: 159.95,
154.26, 152.11, 143.55, 142.78, 130.36, 130.05, 128.11, 119.77,
118.71, 114.53, 113.57, 112.22, 111.33, 58.75, 55.18, 52.19,
12.57. Anal. calcd for C20H17F2N3O3: C, 62.33; H, 4.45; N,
10.90. Found: C, 62.32; H, 4.46; N, 10.91. HRMS calcd for
C20H18F2N3O3 [M + H+]: 386.1316, found 386.1314.
Metabolism stabilities in liver microsomes of D1 and D2
were examined (Table 3). Neither D1 nor D2 showed high
microsome stability. Half-lives (t1/2) in humans were 23 and
37 min for D1 and D2, respectively, whereas t1/2 values in rats
were 17 and 15 min for D1 and D2, respectively.
To understand the reason for the short t1/2 values of D1
and D2, P450 inhibition of four major isoforms was evaluated
using a cocktail inhibition assay. Percent inhibition (% inh.)
N-(3,5-Difluoro-4-(1H-pyrazol-4-yl)phenyl)chromane-3-
carboxamide (D2): (54% yield over two steps). 1H-NMR
(DMSO-d6, 500 MHz) δ 8.46–8.44 (m, 1H), 8.10–8.09 (m, 2H),
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Fig. 5 Docking results of D1–D3. (a) 3D view of the S6K1–D1 complex. (b) 2D view of the S6K1–D1 complex. (c) 3D view of the S6K1–D2 complex.
(d) 2D view of the S6K1–D2 complex. (e) 3D view of the S6K1–D3 complex. (f) 2D view of the S6K1–D3 complex. In the 3D view, carbon atoms in
the ligand and protein are shown in yellow and white, respectively. In the 2D view, conventional hydrogen bonds and carbon H-bonds are shown
as green dotted lines. Pi–sulfur, alkyl, pi–alkyl, pi–anion, pi–pi stacked and pi–sigma interactions are hydrophobic interactions. Pi–sulfur and pi–anion
interactions are shown as yellow dotted lines, alkyl and pi–alkyl interactions are shown as pink dotted lines, and pi–sigma and pi–pi stacking are
shown as purple dotted lines.
7.90–7.87 (m, 1H), 7.20–7.18 (m, 1H), 7.13–7.09 (m, 1H),
6.91–6.88 (m, 1H), 6.87–6.80 (m, 1H), 4.60–4.57 (m, 1H),
4.37–4.33 (m, 1H), 3.93–3.38 (m, 1H), 3.32–3.21 (m, 2H). 13C
NMR (DMSO-d6, 125 MHz) δ: 159.34, 154.39, 152.13, 146.36,
142.72, 138.65, 130.14, 130.06, 128.01, 118.83, 116.20, 115.98,
114.55, 111.24, 58.70, 52.33, 12.57. Anal. calcd for
C19H15F2N3O2: C, 64.22; H, 4.26; N, 11.83. Found: C, 64.20; H,
4.25; N, 11.81. HRMS calcd for C19H16F2N3O2 [M + H+]:
356.1211, found 356.1214.
Following the procedure of D1 and D2 from intermediate
A, intermediate B was obtained as a white solid from
4-bromo-2-fluoro-1-nitrobenzene. Then,
a
mixture of
intermediate B (1 mmol), 5% Pd/C, and H2 in CH3OH (10
mL) was stirred at room temperature until the complete
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Table 2 Rat pharmacological data for D1 and D2a
Compd.
D1
28
0.7 0.2
0.47 0.1
1.6 0.3
D2
Cl (mL min−1 kg−1
)
5
29
6
Vd (L K−1
)
0.8 0.2
0.5 0.1
1.5 0.2
0.6 0.04
t1/2 (h)
AUC (μM h)
Cmax (μM)
F (%)
0.67 0.03
25
5
23
5
a
Data were the means of three determinations.
Fig. 6 Compounds' IC50 values against S6K1.
Table 3 Metabolism stabilities of D1 and D2a
% inh. at 10 μM
t1/2 (min)
with 3-fold serial dilution starting at 20 μM. Reactions were
carried out at 5 μM ATP for S6K1 (Fig. 6).
Compd.
1A2/2C9/2D6/3A4
Human
Rat
Pharmacokinetics.16
Pharmacokinetic studies were
D1
D2
42/63/40/38
12/84/61/43
23
37
17
15
conducted on Sprague Dawley rats. The compound was
formulated in a generic formulation at 1 mg mL−1 and dosed
at 1 mg kg−1 intravenously into the femoral vein or 2 mg kg−1
by oral gavage. Blood was obtained at t = 5 min, 15 min, 30
min, 1 h, 2 h, 4 h, 6 h, and 8 h. Blood was collected in EDTA
containing tubes and plasma was generated by standard
centrifugation methods. All procedures and handling were
according to the standard operating procedures approved by
the IACUC at Scripps Florida. All animal procedures were
performed in accordance with the Guidelines for Care and
Use of Laboratory Animals of The Scripps Research Institute
at Scripps Florida and approved by the Animal Ethics
Committee of The Scripps Research Institute.
In order to assess in vivo pharmacokinetic parameters, an
LC-MS/MS bioanalytical method was developed where 25 μl
of plasma was treated with 125 μl of acetonitrile containing
an internal standard in a Millipore Multiscreen Solvinert 0.45
micron low binding PTFE hydrophilic filter plate
(#MSRLN0450) and allowed to shake at room temperature for
five minutes. The plate was then centrifuged for 5 minutes at
4000 rpm in a tabletop centrifuge and the filtrate was
collected in a polypropylene capture plate. The filtrate (10 μl)
is injected into an Agilent 1200 HPLC equipped with a
Thermo Betasil C18 HPLC column 5 μ (50 × 2.1 mm) #70105-
052130. Mobile phase A was water with 0.1% formic acid.
Mobile phase B was acetonitrile with 0.1% formic acid. The
flow rate was 375 μl min−1 using a gradient of 90% A/10% B
from 0–0.5 min, ramped to 5% A/95% B at 2 min, held at 5%
A/95% B until 3.0 min, ramped to 90% A/10% B at 4 min,
and held at 90% A/10% B until 7 min.
a
Data were the means of three experiments with errors within 30%
of the mean.
1
conversion of intermediate B detected by H NMR. Then, the
reaction mixture was filtered and concentrated to give
aniline C.
COCl2 (1.5 mmol) was added to
a mixture of 2,3-
dihydrobenzoij1,4]dioxine-2-carboxylic acid (1.1 mmol) in dry
CH2Cl2 (5 mL) at 0 °C. Then, DMF (3–5 drops) was added to
the mixture, and then the reaction mixture was stirred at
room temperature for another 30 minutes and concentrated
to dryness with a rotavap to give acid chloride as a white
solid. To a solution of intermediate C (1 mmol) in dry CH2Cl2
(2 mL), acid chloride in dry CH2Cl2 (2 mL) and Et3N (3 mmol)
were added with nitrogen protection at 0 °C. After complete
conversion of C detected by TLC, the reaction was quenched
with saturated NaHCO3 (2 mL) and extracted with EtOAc (3 ×
20 mL). The combined organic phases were washed with
brine (3 × 5 mL) again, dried over Na2SO4, concentrated
under reduced pressure, and purified through flash column
chromatography to give the targeted inhibitor D3.
N-(2-Fluoro-4-(1H-pyrazol-4-yl)phenyl)-2,3-dihydrobenzoijb]-
ij1,4]dioxine-2-carboxamide (D3): (50% yield over three steps).
1H-NMR (DMSO-d6, 500 MHz) δ 11.54 (br, NH, 1H), 8.43–8.40
(m, 2H), 8.04–8.03 (m, 1H), 7.90–7.89 (m, 1H), 7.61–7.59 (m,
1H), 7.13–7.11 (m, 1H), 6.88–6.87 (m, 2H), 6.63–6.62 (m, 1H),
5.12–5.11 (m, 1H), 4.46–4.42 (m, 1H), 4.34–4.31 (m, 1H). 13C
NMR (DMSO-d6, 125 MHz) δ: 159.31, 158.76, 158.43, 154.89,
153.27, 144.01, 143.53, 141.72, 129.36, 128.57, 126.43, 121.74,
119.22, 117.87, 112.77, 112.05, 62.66, 54.94. Anal. calcd for
C18H14FN3O3: C, 63.71; H, 4.16; N, 12.38. Found: C, 63.73; H,
An API Sciex 4000 equipped with a turbo ion spray source
was used for all analytical measurements. MRM methods
were carried out in positive ion mode. Peak areas of the
analyte ions were measured against the peak areas of the
internal standard. Data were fitted using WinNonLin using
an IV bolus model.
4.15; N, 12.40. HRMS calcd for C18H15FN3O3 [M
H+]:340.1097, found 340.1096.
+
S6K1 assay. S6K1 assay was carried out at the Reaction
Biology Corporation and followed the protocols described on
its web site. Compounds were tested in 10-dose IC50 mode
with 2.5-fold serial dilution starting at 10 μM. Control
compound staurosporine was tested in 10-dose IC50 mode
P450 inhibition.16 P450 inhibition of four major isoforms
were evaluated using a cocktail inhibition assay, where the
metabolism of specific marker substrates (CYP1A2,
phenacetin demethylation to acetaminophen; CYP2C9,
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tolbutamide hydroxylation to hydroxytolbutamide; CYP2D6,
bufuralol hydroxylation to 4′-hydroxybufuralol; CYP3A4,
midazolam hydroxylation to 1′-hydroxymidazolam) in the
presence or absence of 10 μM probe compound was
evaluated. The concentration of each marker substrate was
approximately its Km. The conditions were similar to those
described by Tesino and Patonay, except that 2C19 was not
evaluated as we found that the stock solution of the 2C19
probe substrate, omeprazole, had poor stability. Specific
inhibitors for each isoform were included in each run to
validate the system.
Ligand preparation. The ligand preparation was
performed using Sybyl-2.0. 3D structures of all compounds
were constructed using the Sketch Molecule module and were
minimized using the Tripos force field by the Powell gradient
algorithm with Gasteiger–Hückel charges. The maximum
iterations for the minimization were set to 10 000. The
minimization was terminated when an energy gradient
convergence criterion of 0.005 kcal mol−1 Å−1 was reached.
Molecular docking.10 Molecular docking was performed
using the Surflex-Dock module in Sybyl (Tripos, USA). The
crystal structure of human S6K1 for molecular docking was
downloaded from the RCSB Protein Data Bank (http:/www.
rcsb.org/pdb/home/home.do, PDB:3A60). The protein was
prepared by using the biopolymer module implemented. All
water molecules were removed, hydrogen atoms were added,
the end residues were repaired, and energy minimization was
performed on the protein. Docking calculations were
performed through protomol generation using the ligand,
the protein was fixed while the ligand was flexible, and the
default parameters were used as described in the Sybyl
manual unless otherwise specified. Subsequently, the ligand
was docked into the ATP-binding site of S6K1 by an empirical
scoring function. By default, each ligand generated 20
conformations, and the scores were forecasted based on the
strength of the receptor–ligand interactions. The best docked
conformation was selected for further studies.
ns production run was carried out by the NPT ensemble at
300 K and 1.013 × 105 kPa. RMSD calculations of the
equilibrated system and cluster analysis were carried out for
selected compounds.
At length, a production run of 10 ns under conditions of
constant pressure and temperature was carried out, and the
MD trajectories of all systems were taken to calculate the
binding free energy with the MM/GBSA method. To increase
the accuracy of the binding free energy calculations, all
energy components were calculated using 50 snapshots
extracted from 7 to 8 ns based on the RMSD results. Finally,
MM/GBSA free energy decomposition was run using SANDER
in MMPBSA.py to obtain the quantitative information about
the interactions between the inhibitors and the residues.
Conclusions
Based on five thiophene urea-based S6K1 inhibitors, molecular
docking demonstrated that H-bonding interactions to residues
Glu173 and Leu175 and hydrophobic interactions to residues
Val105, Leu97 and Met225 were key elements for S6K1 inhibitory
activities. 10 ns MD simulations supported the docking results.
Binding free energy decomposition also indicated that Glu173,
Val105, Leu175, Leu97 and Met225 contributed significantly to
the binding free energy. S6K1 inhibitors D1–D3 were then
designed according to the computational results. The inhibitory
activities of D1–D3 obtained after synthesis and biological
evaluation by S6K1 assay were as good as the lead compounds
Compd. 3–5, which indicated that the molecular models were
used effectively to design highly potent S6K1 inhibitors. Only
medium bioavailabilities and microsome stabilities were
observed. Future efforts with these compounds will focus on
biological evaluations in cell lines and improving their
metabolism stabilities.
Conflicts of interest
There are no conflicts to declare.
MD simulations.10,11 MD simulations were performed
using AmberTools 14.0 package with the ff99SB force field
for the kinase and the general AMBER force field (gaff) for
the ligands. MD simulations followed the procedures of
preparation, minimization, heating, density, balance, and
production. In the beginning, the complex system was
simulated in a TIP3P water box environment. In this process,
the SHAKE algorithm was applied to restrain the stretching
vibrations of all bonds involving hydrogen atoms, and the
entire system suffered from energy minimization to eliminate
possible space collisions with two steps: (1) the atom position
of all solute species was restrained by a force of 100 kcal
mol−1 Å−2. The water molecules and ligands were minimized
by 500 steps of the steepest descent method followed by 500
steps of the conjugated gradient method; (2) the entire
system was energy optimized by 5000 steps of the steepest
descent method followed by 5000 steps of the conjugated
gradient method. Then, the system was heated from 0 to 300
K, followed by equilibration at 300 K over 50 ps. Finally, a 10
Acknowledgements
The authors acknowledge financial support from the National
Natural Science Foundation of China (Grant No. 21502117),
the Shanghai Gaofeng & Gaoyuan Project for University
Academic Program Development, and the Shanghai Key
Laboratory of Chemical Assessment and Sustainability
(19DZ2271500). We are also grateful to Prof. Guanjun Wang
for his support. Experimental help for Tables 2 and 3 from
Lin Li and modification–polish of the resubmitted
manuscript from the Edanz Group (https://en-author-services.
edanzgroup.com/) are also greatly appreciated.
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