Potent and selective thiophene urea-templated inhibitors of S6K

Article abstract of DOI:10.1016/j.bmcl.2010.11.069

S6K1 (p70 S6 kinase-1) is thought to play a critical role in the development of obesity and insulin resistance, thus making it an attractive target in developing medicines for the treatment of these disorders. We describe a novel thiophene urea class of S6K inhibitors. The lead matter for the development of these inhibitors came from mining the literature for reports of weak off-target S6K activity. These optimized inhibitors exhibit good potency and excellent selectivity for S6K over a panel of 43 kinases.

Full text of DOI:10.1016/j.bmcl.2010.11.069

Bioorganic & Medicinal Chemistry Letters 21 (2011) 849–852
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Potent and selective thiophene urea-templated inhibitors of S6K
Ping Ye, Cyrille Kuhn , Miret Juan à, Rahul Sharma, Brendan Connolly, Gordon Alton §, Hu Liu,
⇑
Robert Stanton, Natasha M. Kablaoui
Cambridge South Laboratory, Pfizer Inc., 620 Memorial Drive, Cambridge, MA 02139, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
S6K1 (p70 S6 kinase-1) is thought to play a critical role in the development of obesity and insulin resis-
tance, thus making it an attractive target in developing medicines for the treatment of these disorders.
We describe a novel thiophene urea class of S6K inhibitors. The lead matter for the development of these
inhibitors came from mining the literature for reports of weak off-target S6K activity. These optimized
inhibitors exhibit good potency and excellent selectivity for S6K over a panel of 43 kinases.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 10 September 2010
Revised 16 November 2010
Accepted 17 November 2010
Available online 21 November 2010
Keywords:
p70 S6 kinase-1
Kinase
Inhibitor
Selectivity
S6K1
Thiophene urea
Activation of the mammalian target of rapamycin (mTOR) S6K1
signaling by nutrients has received broad attention due to its
implication in obesity.1 Morbidly obese patients are often affected
by increased insulin resistance, resulting in the development of
type II diabetes.2 Insulin resistance can be regulated by mTOR com-
plex1 activation of p70 S6 kinase-1 (S6K1).3,4 Recent studies have
shown that S6K1-deficient mice exhibit increased lipolysis and re-
duced adipose tissue mass. They also demonstrate that S6K1-
deficient mice are protected from diet- and age-induced obesity
making S6K1 an attractive target.4
S6K1 is a serine/threonine kinase that phosphorylates the ribo-
somal protein, S6. Insulin and nutrients activate S6K1 to regulate
protein synthesis. However, prolonged activation of S6K1 also
leads to the development of insulin resistance. Recent studies
revealed that insulin receptor substrate 1 (IRS1) S302, which is
proximal to the IRS1 phosphotyrosine-binding (PTB) domain and
contains an S6K1-recognition motif, is phosphorylated by
S6K1.5,6 Phosphorylation of S302 disrupts the ability of the PTB do-
main to interact with activated insulin receptor (IR), leading to de-
creased insulin signaling.5 This suggests that S6K1 may have a
critical function along with other signaling components in devel-
opment of obesity and insulin resistance, and may be an important
drug target in the treatment of patients suffering from these path-
ological disorders.
Despite increased reports suggesting S6K1 as a potential phar-
macological target, there are only limited classes of small molecule
inhibitors reported to date, such as Oltipraz and dithiolethione
derivatives,7–9 and LY303511 (Fig. 1).10 Our interest in pursuing
non classical binding inhibitors led us to the discovery of a series
of thiophene ureas as potent and selective S6K1 inhibitors (Fig. 2).
Our studies began with the synthesis of compound 1 (Table 1),
which was previously reported as a p38 inhibitor with an off-target
effect on S6K1.11 However, compound 1 showed no enzymatic
activity in an ADP hunter assay, a fluorescent intensity assay mon-
itoring the release of ADP product after phosphorylation of sub-
strate. When the nitrogen on heteroaryl-aryl ether was moved
from the 4 to the 3 position, compound 2, the IC50 dropped into
the measurable range at 9 lM. Activity was lost in conformational-
ly constrained analog 3. Replacement of the terminal pyridyl group
O
S
⇑
S
Corresponding author. Tel.: +1 617 551 3473.
N
O
N
S
E-mail address: natasha.m.kablaoui@pfizer.com (N.M. Kablaoui).
Present address: Boehringer Ingelheim, 2100 Cunard Street, Laval, Québec,
Canada H7S 2G5.
NH
N
à
Present address: Ariad Pharmaceuticals, 26 Landsdowne Street, Cambridge, MA
02139, United States.
Oltipraz
LY 303511
§
Present address: Visionary Pharmaceuticals, 8019 Montara Ave, San Diego, CA
92126, United States.
Figure 1. S6K1 inhibitors.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.11.069

850
P. Ye et al. / Bioorg. Med. Chem. Lett. 21 (2011) 849–852
Table 2
H
O
S6K1 enzymatic activity of R2-modified thiophene ureas
N
H
N
H
N
N
H
O
N
N
H
H
N
O
S
N
N
N
H
N
S
O
N
R2
H
Figure 2. Selective S6K1 inhibitor.
Compound
R2
S6K1 IC50 (nM)
15
Table 1
9
S6K1 enzymatic activity of R1-modified thiophene ureas
O
S
10
11
29
62
O
O
N
H
H
N
N
R1
12
13
70
O
S
223
14
1900
OH
OH
Compound
1
R1
S6K1 IC50
>20
(lM)
15
196
N
16
379
O
O
O
O
O
N
N
2
3
4
9
pyl compound (10), suggesting that R2 substituents fit in a hydro-
phobic pocket of S6K.
Complete removal of the amide group at the thiophene 3-
position from compound 8 maintains activity as illustrated by
compound 17 (Table 3). This suggests that the R3 position lacks
key interactions with S6K binding pocket, and instead leads to
solvent or an open part of the binding pocket. Taking advantage
of possible solvent exposure of R3, we therefore explored the
tolerance of functional groups that can be used to modulate phys-
iochemical properties of the series. Table 3 shows the modifica-
tions and their respective solubility and permeability data. The
carboxylic acid (18) and primary aliphatic amines (19–21) retain
potencies in the 100 nanomolar range. Compounds 9, 18 and 21
have increased solubility, while compound 18 shows good perme-
ability. The tolerance of R3 substitution has steric limits. Secondary
cyclic amine (22) and benzyl amines (23–24) led to a decrease in
potency to 200 nanomolar range. Taken together, a possible bind-
ing model incorporating SAR of the R1, R2, and R3 positions is illus-
trated in Figure 3. Interactions of compound 9 with the hinge
region, a hydrophobic pocket and a solvated region are depicted.
While the central urea moiety has been shown to bind to p38 in
a DFG out configuration,14 the configuration for binding of this ser-
ies to S6K can only be fully revealed with an X-ray co-crystal
structure.
>20
>20
5
6
>20
>20
O
F
O
OH
7
8
4
N
0.034
N
H
with aryl and substituted aryl also resulted in a loss of activity (4–
6). These preliminary results suggested that a hydrogen bond
acceptor with a certain conformation is critical to the activity. Clip-
ping off the terminal ring displayed a modest gain in potency to
Several of the compounds were tested in a whole cell activity
assay measuring the phosphorylation of ribosomal protein S6. Of-
ten there can be a disconnect between the activity of a compound
in an enzymatic assay compared to a cellular assay. We found this
to be the case for this series of S6K inhibitors. For example, the cel-
1.4
lM (7). Replacement of the phenol with a 5-amino indazole re-
sulted in highly active compound 8 (0.034
l
M). The indazole moi-
lular IC50 of 19 was 0.685
lM, about 70-fold less active than enzy-
ety possibly interacts with the hinge region of the S6K1 binding
pocket. There are a number of publications which depict indazole
as a pharmacophore for kinase inhibitors.12,13
Thiophene 5-position substitution is critical to the activity. Ta-
ble 2 details these R2 substituents. The large tBu proved to be the
optimal substituent tested (9), followed closely by isopropyl (10)
then ethyl, cyclopropyl and methyl (11–13) in a clear size-depen-
dant trend (15, 29, 62, and 233 nM respectively). A phenyl substi-
tution in the R2 position (14) was not tolerated, dropping the
matic activity. Similarly, the cellular IC50 of 9 was 0.521
l
M, about
40-fold less active. This discrepancy between assay results can be
due to the differences between the interactions of the compound
with a purified recombinant enzyme compared to its interactions
with the endogenous enzyme; differences in ATP concentrations
in the assays can also contribute. In addition to the inherent differ-
ences between the enzymatic and cellular assays, poor cell perme-
ability could also account for the difference in measured inhibitory
activity.
potency to 1.9
lM. Both enantiomers of 1-ethanol substituted
The synthesis of the thiophene urea analogs such as 9 is de-
picted in Scheme 1. 2-Amino thiophene (27) was synthesized
compounds 15 and 16 are significantly less potent than the isopro-

P. Ye et al. / Bioorg. Med. Chem. Lett. 21 (2011) 849–852
851
Table 3
R3-modified thiophene ureas
S
O
O
NH2
R3
b
a
N
+
H
H
N
N
O
H
O
N
O
26
25
S
S
O
27
N
H
H
N
O
S
c
Compound
R3
Solubilityb
PAMPAc
(e-6 cm/s)
a
d
NH2
S6K1 IC50
(nM)
O
(
lg/mL)
O
28
17
18
H
COOH
19
52
7
3
17
Na
O
O
29
143
10
O
O
19
20
21
9
10
7
4
4
5
2
H
N
S
N
H
OH
H
N
H
O
N
e
N
NH
9
HN
N
O
O
O
O
O
HN
O
NH
S
O
10
41
N
N
18
H
OH
N
19
15
Scheme 1. Reagents and conditions: (a) sulfur, TEA, DMF, 50 °C to rt overnight
(68%); (b) NaOH/H2O, microwave, 150 °C, 30 min; (c) triphosgene, toluene, rt, 2 h
(83%); (d) 5-aminoindazole, TEA, ACN, 50 °C, 4 h or microwave, 100 °C, 30 min; (e)
N,N-dimethylethane-1,2-diamine, HATU, DIPEA, DMF, rt 1 h (81%).
N
H
171
N
H
22
23
24
207
201
191
n/a
n/a
n/a
n/a
n/a
n/a
Table 4
N
In vitro metabolic stability, CYPs, and dofetilide interaction
O
O
O
Compounds
9
17
18
19
20
21
a
HLM stability_T1/2 (min)
HHEP stability_T1/2 (min)
38
55
n/a
36
52
20
4
>120
n/a
6
55
0
>120
138
40
11
12
>120
367
45
14
0
>120
263
52
30
9
N
H
b
125
18
0
CYP1A2c (% inh)
CYP2C9 (% inh)
CYP2D6 (% inh)
CYP3A4 (% inh)
Dofetilided (% inh)
F
7
0
N
H
0
0
11
2
0
0
0
28
0
0
a
b
c
a
b
c
Enzymatic activity.
Kinetic solubility was measures at pH 6.5 in 50 mM phosphate buffer.
Permeability was measured across a synthetic lipid bilayer.
Compound stability in human liver microsomes at 1
Compound stability in human hepatocyte at 1 M. LC/MS/MS detection.
Compound drug–drug interaction at 3 M using clinical substrate probes in
lM. LC/MS/MS detection.
l
l
HLM. % Inhibition reported for CYP3A4, 2D6, 2C9, 1A2. LC/MS/MS detection.
d
Measured by the displacement of a known hERG channel inhibitor, dofetilide. %
Inhibition reported.
solvent exposed
region
N
hydrogen bonding
Human liver microsomal (HLM) lability assays are useful for deter-
mining the clearance of a compound due to Phase I (including cyto-
chrome P450) metabolism. The series shows moderate to good
stability in HLM. Hepatocytes are an ideal in vitro system to mon-
itor hepatic metabolism since these intact cells contain all the he-
patic enzymes found in in vivo. Human hepatocyte (HHEP) stability
assay of the series suggests that they have low metabolic liability.
Percent inhibitions for CYP3A4, 2D6, 2C9, and 1A2 are reported in
Table 4 as an early indication of possible drug–drug interactions.
The series shows no or low risk of inhibition against 3A4 and
2D6. Compounds 17, 19, 20, and 21 showed moderate risk against
1A2, while 17, 18, and 21 showed moderate risk against 2C9.
Assessment for QT interval elongation measured by displacement
of a known hERG potassium ion channel inhibitor, dofetilide,
showed no risk.19
Selectivity was examined against a panel of 43 kinases (Fig. 4).
The series was highly selective for inhibition of S6K1. For example,
the heat map of percent inhibition of compound 18 against other
kinases was cold and clean, only JAK3, CLK1, and CHEK2 showed
low levels of inhibition.
In summary, a novel series of small molecule S6K1 inhibitors
with good potency, good physiochemical properties, and excellent
selectivity profile was reported.
O
NH
NH
H
H
O
N
N
HN
O
HN
S
O
N
NH
hydrophobic pocket
hinge region
Figure 3. Possible pharmacophore model.
through the Gewald reaction.15,16 Saponification of 27 gave the
corresponding carboxylate acid salt (28). Thiaisatoic anhydride
(29) was prepared as reported.17 Ring opening of 29 by 5-aminoin-
dazole gave the carboxylic analog 18. The de-carboxylation
product 17 (Table 3) was isolated when reaction temperature in-
creased to 150 °C under microwave conditions. Carboxylic product
18 coupled with the amine to give final product 9. Compound 9
was isolated by column chromatography. All compounds gave sat-
isfactory spectral and analytical data.18
Several compounds were selected for in vitro pharmacokinetics
and toxicity evaluation, the results of which are reported in Table 4.

852
P. Ye et al. / Bioorg. Med. Chem. Lett. 21 (2011) 849–852
Figure 4. Selectivity profile of compound 18 against 43 kinases.
18. Selected experimental and analytical information: all 1H NMR data were
Acknowledgments
recorded by a 400 MHz Bruker NMR spectrometer. DMSO-d6 was used as NMR
solvent. All compounds >95% purity by HPLC (UV, 254 nm) and 1H NMR.
The authors thank Scott Wildman, Simon Low, Darcy Kohls, and
Yuan-hua Ding for useful discussions, Deborah Moshinsky for run-
ning the kinase selectivity panel, Bridget Reaney and Evan Walters
for analytical support.
Compound
27:
methyl
2-amino-5-tert-butylthiophene-3-carboxylate:
triethylamine (225 mg, 2.5 mmol) was added to a vigorously stirred mixture
of cyanoacetic acid methyl ester (500 mg, 5 mmol), sulfur powder (162 mg,
5 mmol) and DMF (10 mL) at 50 °C. 3,3-Dimethylbutyraldehyde (505 mg,
5 mmol) in 4 mL of DMF was added dropwise to this suspension, while
maintaining the temperature at 50 °C. When the addition was complete, the
reaction mixture was allowed to cool to rt and stirred overnight. The reaction
mixture was partitioned in water and ether. The ether layer was washed with
water (15 mL Â 4), dried over anhydrous sodium sulfate, and concentrated
under reduced pressure to afford a yellow solid, 68% yield. m/z ES+ = 214.08,
calcd 214.08; 1H NMR d ppm 7.15 (s, 2H), 6.48 (s, 1H), 3.69 (s, 3H), 1.25 (s, 9H).
References and notes
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2. Jürgens, H. S.; Neschen, S.; Ortmann, S.; Scherneck, S.; Schmolz, K.; Schüler, G.;
Schmidt, S.; Blüher, M.; Klaus, S.; Perez-Tilve, D.; Tschöp, M. H.; Schürmann, A.;
Joost, H. G. Diabetologia 2007, 50, 1481.
3. Nobukuni, T.; Kozma, S.; Thomas, G. Curr. Opin. Cell Biol. 2007, 19, 135.
4. Um, S. H.; Frigerio, F.; Watanabe, M.; Picard, F.; Joaquin, M.; Sticker, M.;
Fumagalli, S.; Allegrini, P. R.; Kozma, S. C.; Auwerx, J.; Thomas, G. Nature
(London, UK) 2004, 431, 200.
5. Harrington, L. S.; Findlay, G. M.; Gray, A.; Tolkacheva, T.; Wigfield, S.; Rebholz,
H.; Barnett, J.; Leslie, N. R.; Cheng, S.; Shepherd, P. R.; Gout, I.; Downes, C. P.;
Lamb, R. F. J. Cell Biol. 2004, 166, 213.
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Cancer Ther. 2009, 8, 2791.
10. Kristof, A. S.; Pacheco-Rodriguez, G.; Schremmer, B.; Moss, J. J. Pharmacol. Exp.
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11. Moore, W. R.; Springman, E.; Michelotti, E. PCT Int. Appl. WO062984 A2,
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Compound
29:
6-tert-butyl-1H-thieno[2,3-d][1,3]oxazine-2,4-dione:
compound 27 (63 mg, 0.30 mmol), 2-aminothiophene and 0.6 mL of 1 M
NaOH aqueous solution were combined in a conical bottom microwave vial.
The slurry was irradiated under microwave at 150 °C for 30 min. Triphosgene
(44 mg, 0.15 mmol) in 1 mL toluene was added slowly with vigorous stirring.
The resulting mixture was allowed to stir at rt for 2 h. The precipitate was
filtered and washed successively with water, hexane and ether, then dried in
vacuo to give compound 29 with 83% yield. m/z ES+ = 226.09, calcd 226.05; 1
NMR d ppm 12.60 (s, 1H), 6.92 (s, 1H), 1.23 (s, 9H).
H
Compound
carboxylic acid: compound 29 (125 mg, 0.56 mmol), 5-aminoindazole
(74 mg, 0.56 mmol) and 4.5 mL of acetonitrile were added to 5 mL
microwave vial After the subsequent addition of neat triethylamine (154 L,
1.11 mmol), the resulting mixture was irradiated under microwave at 100 °C
for 30 min. The solvent was removed in vacuo. Flash column chromatography
(0–10% MeOH in dichloromathane, linear gradient over 30 min) provided
compound 18 as a off-white solid. m/z ES+ = 359.32, calcd 359.11; 1H NMR d
ppm 12.94 (br s, 1H), 10.05 (s, 1H), 8.00 (s, 1H), 7.99 (d, J = 1.20 Hz, 1H), 7.48 (d,
J = 8.84 Hz, 1H), 7.36 (dd, J = 8.84, 1.60 Hz, 1H), 6.77 (s, 1H), 1.32 (s, 9H). Urea
NH absent.
Compound 9: 1-(5-tert-butyl-3-((2-(dimethylamino)ethyl)carbamoyl)thiophen-
2-yl)-3-(1H-indazol-5-yl)urea: compound 18 (1.79 g, 5 mmol), N,N-dimethyl-
ethane-1,2-diamine (0.44 g, 5 mmol), and 40 mL of DMF were added to a round
bottom flask and stirred until completely dissolved. Diisopropylethylamine
(776 mg, 6 mmol) was added, followed by HATU (2.09 g, 5.5 mmol). The
resulting solution was stirred at rt for 1 h. The solvent was removed in vacuo.
Flash column chromatography (NH4OH/MeOH/DCM, 0.5:5:94.5) gave
compound 9 with 81% yield. m/z ES+ = 429.29, calcd 429.20; 1H NMR d ppm
12.95 (s, 1H), 11.35 (s, 1H), 10.06 (s, 1H), 8.14 (t, J = 5.84 Hz, 1H), 7.99 (s, 2H),
7.47 (d, J = 8.84 Hz, 1H), 7.36 (dd, J = 8.84, 1.64 Hz, 1H), 7.11 (s, 1H), 3.28–3.41
(m, 2H), 2.40 (t, J = 6.95 Hz, 2H), 2.19 (s, 6H), 1.34 (s, 9H).
18:
2-(3-(1H-indazol-5-yl)ureido)-5-tert-butylthiophene-3-
a
l
14. Pargellis, C.; Tong, L.; Churchill, L.; Cirillo, P. F.; Gilmore, T.; Graham, A. G.;
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