UNC1062, a new and potent Mer inhibitor

Article abstract of DOI:10.1016/j.ejmech.2013.03.035

Abnormal activation of Mer kinase has been implicated in the oncogenesis of many human cancers including acute lymphoblastic and myeloid leukemia, non-small cell lung cancer, and glioblastoma. We have discovered a new family of small molecule Mer inhibitors, pyrazolopyrimidine sulfonamides, that potently inhibit the kinase activity of Mer. Importantly, these compounds do not demonstrate significant hERG activity in the PatchXpress assay. Through structure-activity relationship studies, 35 (UNC1062) was identified as a potent (IC₅₀ = 1.1 nM) and selective Mer inhibitor. When applied to live tumor cells, UNC1062 inhibited Mer phosphorylation and colony formation in soft agar. Given the potential of Mer as a therapeutic target, UNC1062 is a promising candidate for further drug development.

Full text of DOI:10.1016/j.ejmech.2013.03.035

European Journal of Medicinal Chemistry 65 (2013) 83e93
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
UNC1062, a new and potent Mer inhibitor
,
,
Jing Liu a 1, Weihe Zhang a 1, Michael A. Stashko a, Deborah DeRyckere d,
Christopher T. Cummings d, Debra Hunter c, Chao Yang a 2, Chatura N. Jayakody a,
,
Nancy Cheng a, Catherine Simpson a, Jacqueline Norris-Drouin a, Susan Sather d,
Dmitri Kireev a, William P. Janzen a c, H. Shelton Earp b c, Douglas K. Graham d,
,
,
Stephen V. Frye a c, Xiaodong Wang a
,
,
*
a Center for Integrative Chemical Biology and Drug Discovery, Division of Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy,
University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
b Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
c Lineberger Comprehensive Cancer Center, Department of Medicine, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill,
NC 27599, USA
d Department of Pediatrics, School of Medicine, University of Colorado Denver, Anschutz Medical Campus, Aurora, CO 80045, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Abnormal activation of Mer kinase has been implicated in the oncogenesis of many human cancers
including acute lymphoblastic and myeloid leukemia, non-small cell lung cancer, and glioblastoma. We
have discovered a new family of small molecule Mer inhibitors, pyrazolopyrimidine sulfonamides, that
potently inhibit the kinase activity of Mer. Importantly, these compounds do not demonstrate significant
hERG activity in the PatchXpress assay. Through structureeactivity relationship studies, 35 (UNC1062)
was identified as a potent (IC50 ¼ 1.1 nM) and selective Mer inhibitor. When applied to live tumor cells,
UNC1062 inhibited Mer phosphorylation and colony formation in soft agar. Given the potential of Mer as
a therapeutic target, UNC1062 is a promising candidate for further drug development.
Received 4 February 2013
Received in revised form
11 March 2013
Accepted 21 March 2013
Available online 2 April 2013
Keywords:
Mer inhibitors
Pyrazolopyrimidines
Sulfonamides
Ó 2013 Elsevier Masson SAS. All rights reserved.
Leukemia
Non-small cell lung cancer
Glioblastoma
1. Introduction
samples but not in normal bone marrow myeloid cells [5]. Finally,
Mer is frequently overexpressed in solid tumors, including non-
Mer belongs to the TAM (Tyro3, Axl, and Mer) family of receptor
tyrosine kinases (RTKs) [1]. Under normal physiological conditions,
Mer kinase promotes cell proliferation and survival, platelet ag-
gregation, macrophage clearance of apoptotic cells, and cytokine
release [2]. Abnormal activation of Mer has been implicated in the
oncogenesis of many human cancers and is often associated with
poor prognostic indicators [2,3]. For example, Mer is ectopically
expressed in both B- and T-cell acute lymphoblastic leukemias
(ALL) [4] but not in normal mouse and human T- and B-lympho-
cytes at any stage of development. Similarly, Mer is expressed in the
majority of acute myeloid leukemia (AML) cell lines and patient
small cell lung cancer (NSCLC) cell lines [6] and patient samples,
even though normal human bronchial epithelial (NHBE) cells and
lung tissue adjacent to tumors do not express Mer protein [6].
Similar findings have been reported in other solid tumors,
including glioblastoma (GBM) [3b,7] and metastatic melanoma [8].
This tumor specific expression pattern may confer a large thera-
peutic window e at least in these tumor types. Expression of a Mer
transgene in hematopoietic cells leads to development of leukemia
and/or lymphoma in mice [9]. In addition, when Mer is inhibited by
sh-RNA knockdown in leukemia [4c], NSCLC [6], or GBM cells [7a],
they are more susceptible to apoptotic death and exhibit reduced
colony formation in soft agar. Moreover, in orthotropic ALL and
AML xenograft mouse models, onset and progression of disease
were delayed and survival was significantly increased in mice
transplanted with Mer knockdown leukemia cell lines relative to
mice transplanted with wild-type cell lines [4c,5]. Similarly, in an
* Corresponding author. Tel.: þ1 919 843 8456; fax: þ1 919 843 8465.
E-mail address: xiaodonw@unc.edu (X. Wang).
1
These authors contributed equally.
Present address: 195 Blackhorse Ln, North Brunswick, NJ 08902, USA.
2
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.03.035

84
J. Liu et al. / European Journal of Medicinal Chemistry 65 (2013) 83e93
NSCLC xenograft mouse model growth of tumors is markedly
slowed by Mer knockdown. In addition, the combination of stan-
dard cytotoxic chemotherapies with shRNA-mediated Mer knock-
down results in synergistic tumor cell killing. Taken together, these
data demonstrate important roles of Mer for oncogenesis and anti-
apoptotic activity in multiple tumor types. Therefore, Mer provides
a novel therapeutic target for the treatment of ALL, AML, NSCLC,
and other Mer-related diseases. Consequently, Mer inhibitors are
expected to both mediate direct anti-tumor, proapoptotic effects
and augment chemosensitivity in combination with standard
therapies.
Few small molecule Mer inhibitors have been reported [10],
among them compound 1 (UNC569) (Fig. 1) is the most promising
one demonstrating inhibition of Mer at sub-nanomolar concen-
trations [11]. However, UNC569 has undesirable activity toward the
human ether-a-go-go-related gene potassium channel (hERG) in
Scheme 1. The synthetic routes for sulfonamide analogs.
the conventional PatchXpress assay (EC50 ¼ 1.7
mM) [12]. Inhibition
in which phosphorylated and unphosphorylated substrate peptides
were separated and analyzed using a LabChip EZ Reader. For
comparison, inhibition of other members in the TAM RTK family,
Tyro3 and Axl, was also measured. As shown in Table 1, when the
of hERG could lead to long QT syndrome and sudden death and is
the reason for the withdrawal of several FDA-approved drugs [13].
In an effort to decrease the hERG activity of UNC569 through analog
synthesis, we discovered that sulfonamide 2 (Fig. 1) had similar
activity against Mer kinase with better physical properties.
Consequently, compound 2 is a better starting point than UNC569
to develop potent Mer inhibitors as potential therapeutics. Herein
we report the structureeactivity relationship (SAR) of a series of
compounds related to 2 on inhibiting the TAM family kinases and
the discovery of potent Mer inhibitors with minimal hERG activity
and excellent cellular activity.
Table 1
Preliminary SAR at the R2 position.
2. Synthesis
The synthetic routes to sulfonamide analogs (Scheme 1) are
similar to what we have previously developed for the synthesis of
analogs of UNC569 [11]. Briefly, 3-chlorobenzoperoxoic acid (m-
CPBA) oxidation of the known compound 3 [14] followed by the
displacement of the methyl sulfinyl/sulfonyl group by amines
provided the intermediate 4, which was subsequently alkylated at
the N1 position of the pyrazole ring to yield 5. Suzuki coupling
between 5 and a boronic acid provided the final product 6.
Compound
R2
IC50 (nM)a
Mer
Axl
Tyro3
27
2
7
8
3.5
18
59
140
120
130
200
3. Results and discussion
35
Most hERG inhibitors contain a basic amine group and we thus
speculate that the hERG activity of 1 may be due to the presence of a
primary amine (NH2) at the R2 position (Table 1). Consequently, we
first synthesized a focused library of compounds with different R2
groups while keeping R1 fixed as N-methylbenzenesulfonamide.
Inhibition of Mer kinase activity by analogs was tested at the Km for
ATP using a microfluidic capillary electrophoresis (MCE) assay [15]
9
50
190
82
10
11
2.4
4.4
74
70
47
44
12
13
14
250
130
23
2400
5000
450
860
730
280
15
16
47
54
620
360
810
430
17
100
26000
720
a
Values are the mean of two or more independent assays performed at the
Fig. 1. The structures and enzymatic activities of 1 and 2.
ATP Km.

J. Liu et al. / European Journal of Medicinal Chemistry 65 (2013) 83e93
85
NH2 group at the R2 position (2) was replaced by a hydroxyl (OH)
Table 2
SAR study of R1.
group (7), the activity of the analog 7 was decreased 5-fold.
Increasing the length of the linker between the pyrazole ring and
the cyclohexyl group also decreased the activity of compounds (8, 9
vs 2). In contrast, when the length of the linker was shortened and
R2 was replaced with a trans-4-hydroxycyclohexyl group, the cor-
responding analog 10 had enhanced activity compared to the
parent 7. More specifically, the IC50 value was changed from 18 nM
for 7 to 2.4 nM for 10, suggesting that the conformational rigidity
and the size of R2 was important for Mer inhibition. Consistent with
this notion, analogs 12 and 13, which featured open chain versions
of cyclohexanol at the R2 position, had decreased Mer inhibition.
Using a tetrahydropyran to replace the 4-hydroxycyclohexyl group
provided analogs that were less active (14, 15 and 16). Furthermore,
a cyclohexyl group alone at the R2 position resulted in a 28-fold
weaker inhibitor 17, which further confirmed our previous obser-
vation: a hydrogen bond between the polar substituent at the R2
position with the carbonyl of Arg727 of Mer protein was important
[11]. The configuration of the hydroxyl group was not critical, as the
cis-analog 11 had similar activity as trans-10.
Compound
R1
IC50 (nM)a
Mer
Axl
Tyro3
18
19
4.0
1.8
110
35
55
130
29
We also tested the activity of the synthetic analogs against Axl
and Tyro3 to monitor their selectivity for TAM family members and
develop a pan-TAM SAR. In general, these analogs showed modest
selectivity within the TAM family and are generally less potent
against Axl and Tyro3. In addition, to test our hypothesis regarding
on the cause of the hERG activity, compounds 2 and 10 were
characterized in the hERG binding assay. Indeed, 10 had no hERG
activity in the PatchXpress assay while 2 was active with an EC50 of
20
21
22
23
24
1.4
1.8
3.2
1.1
0.80
54
35
68
38
31
62
120
75
3.2 m
M. Removal of the primary amine at R2 position eliminated the
binding of our compounds to the hERG channel.
Next, the SAR at the R1 site was explored (Table 2). In general,
these analogs are very potent against Mer (at sub-nanomolar
concentrations) and have good selectivity for Mer over Axl (>16
fold) and Tyro3 (>9 fold). A reversed sulfonamide functional group
(18), sulfonic acid (19) and unsubstituted sulfonamide (20) at the R1
site were tolerated. When one of the acidic hydrogens in sulfon-
amide was substituted with a small alkyl group such as propyl (21)
or iso-propyl (22), the activity of the analogs remained the same. In
contrast, a cycloalkyl group such as cyclopropyl (23), cyclopentyl
(24) and cyclohexyl (25) at this position yielded a more active
analog. A tetrahydropyran (26) or 4-piperidinylmethyl (28) group
was also well tolerated at this position while a para-fluorophenyl
ring (27) was less suitable. Interestingly, 29, the precursor of 28
with a Boc protection group at piperidinyl nitrogen, was equally
potent, which could be explained by the fact that the R1 position
was exposed to solvents as observed in the X-ray structure of
UNC569 complexed with Mer [11]. To further confirm this expla-
nation, we introduced a large side chain at this position and found
that the corresponding derivative 30 was still active. Finally, when
the acidic hydrogen on the sulfonamide functional group was
replaced by a variety of alkyl groups, the activity and TAM RTK-
selectivity of the resulting analogs 31e36 were maintained [16].
Compared to UNC569, 35 (UNC1062) had improved selectivity
and activity against Mer relative to other TAM family RTKs (78-fold
selectivity over Axl and 54-fold over Tyro3). Morrison tight-binding
inhibition studies revealed that UNC1062 had a Ki of 0.33 nM
against Mer [17]. These outstanding inhibitory profiles of UNC1062
prompted us to further investigate its activity in intact cell systems.
The ability of UNC1062 to inhibit Mer auto-phosphorylation in
different cell lines was first tested. Briefly, human pre-B leukemia
697 cells were grown in suspension and treated with various
concentrations of UNC1062 for 1 h prior to addition of a phos-
phatase inhibitor to stabilize phospho-Mer. Mer protein was
immunoprecipitated from lysates and phospho-Mer and total Mer
proteins were detected by western blot. In this cell-based assay, the
67
25
26
27
1.1
1.2
9.7
350
69
45
28
6500
290
28
29
30
31
32
1.1
1.4
18
55
73
47
150
320
47
780
71
1.8
1.2
110
160
(continued on next page)

86
J. Liu et al. / European Journal of Medicinal Chemistry 65 (2013) 83e93
Table 2 (continued )
4. Conclusions
Compound
R1
IC50 (nM)a
In summary, we have identified and synthesized a new
family of small molecule Mer inhibitors, pyrazolopyrimidine
sulfonamides, which potently inhibit the kinase activity of Mer.
Importantly, this new generation of inhibitors do not show
significant hERG activity in the PatchXpress assay. Through
structureeactivity relationship studies, 35 (UNC1062) was
identified as both a potent (Ki ¼ 0.33 nM) and selective Mer
inhibitor. When applied to live cells, UNC1062 inhibited Mer
phosphorylation and colony formation in soft agar. These results
provide the first evidence of anti-tumor activity mediated by a
member of this novel class of inhibitors, thus further validate
Mer as a drug target for cancer.
Mer
Axl
Tyro3
27
33
34
35
1.0
42
0.65
46
85
22
60
1.1
1.4
36
23
76
5. Experimental
a
5.1. General
Values are the mean of two or more independent assays performed at the ATP
Km.
Microwave reaction was carried out using a Discover-S reactor
with a vertically-focused IR external temperature sensor and an
Explorer 72 autosampler. The dynamic mode was used to set up the
desired temperature and hold time with the following fixed pa-
rameters: Prestirring, 1 min; Pressure, 200 psi; Power, 200 W;
PowerMax, off; stirring, high. Flash chromatography was carried
out on pre-packed silica gel disposable columns. Analytical thin-
layer chromatography (TLC) was performed with silica gel 60
IC50 value for inhibition of phospho-Mer by UNC1062 was 6.4 nM
(Fig. 2). In comparison, the IC50 of UNC569 in the same assay was
141 nM [11], suggesting that UNC1062 was significantly more
potent than UNC569 both with purified Mer kinase and in intact
cells.
To determine if UNC1062 inhibits Mer phosphorylation in
adherent cells, similar studies were performed using a human,
adherent, pediatric rhabdoid brain tumor cell line (BT-12) and two
NSCLC cell lines (A549 and Colo699). Inhibition of Mer phosphor-
ylation was evident in cells when the concentration of UNC1062
was above 300 nM (Fig. 3A). Similarly, phosphorylation of Mer was
stably inhibited over 72 h of culture in A549 and Colo699 NSCLC
cells treated with 250 nM or 500 nM UNC1062 (Fig. 3B). Thus, while
solid tumor cells require significantly higher concentrations of
UNC1062 compared to non-adherent leukemia cells, possibly as a
result of decreased exposure across adherent cell membranes,
treatment with UNC1062 at sub-micromolar concentrations was
sufficient to inhibit Mer phosphorylation in adherent cell lines.
To determine if UNC1062 can mediate functional anti-tumor
effects, BT-12, A549, and Colo699 cells were cultured in soft agar
overlaid with medium containing UNC1062 or vehicle only. Treat-
F254, 0.25 mm pre-coated TLC plates. TLC plates were visualized
using UV254 or phosphomolybdic acid with charring. All 1H NMR
spectra were obtained with a 400 MHz spectrometer and 13C NMR
spectra were obtained with a 100 MHz spectrometer. Preparative
HPLC was performed with the UV detection at 220 or 254 nm. LCe
MS was performed with the UV detection at 220 nm, 254 nm, and
280 nm, and a single quadrupole mass spectrometer using elec-
trospray ionization (ESI) source. High-resolution (positive ion)
mass spectra (HRMS) were acquired using a LCMS-TOF mass
spectrometer.
5.2. Synthesis
5.2.1. 3-Bromo-N-butyl-1H-pyrazolo[3,4-d]pyrimidin-6-amine (4)
To a mixture of 3-bromo-6-(methylthio)-1H-pyrazolo[3,4-d]
pyrimidine (24.5 g, 100 mmol) in THF (100 mL) was added meta-
chloroperoxybenzoic acid (33.7 g, 77%, 150 mmol) in portions at
room temperature. The white mixture was stirred for 2 h and
transferred into a THF (50 mL) solution of n-butylamine (49.4 mL,
500 mmol) at 0 ꢀC. The resulting solution was heated at 60 ꢀC for
2.0 h. After removal of the solvent under reduced pressure, MeOH
was added and the mixture was filtered. The white solid was
ment with 1.0 mM UNC1062 was sufficient to completely abrogate
growth of BT-12 rhabdoid tumor colonies in soft agar (Fig. 4A).
Similarly, treatment with UNC1062 inhibited colony formation in
both A549 and Colo699 NSCLC cultures (Fig. 4B). Moreover, inhi-
bition of colony formation in long-term soft agar assays and inhi-
bition of Mer phosphorylation in short-term assays occurred at
similar concentrations of UNC1062 in these cell lines, consistent
with a causal relationship.
Fig. 2. UNC1062 inhibits accumulation of activated Mer protein in acute leukemia cells. 697 Cell cultures were treated with the indicated concentrations of UNC1062 for 1 h.
Pervanadate was added to cultures for 3 min to stabilize the phosphorylated form of Mer. Mer was immunoprecipitated from cell lysates and total Mer protein and Mer phos-
phoprotein were detected by immunoblot. A) Representative western blots. B) Relative levels of phospho-Mer and Mer proteins were determined. Mean values þ/ꢁ standard error
derived from 4 independent experiments are shown. IC50 ¼ 6.4 nM with a 95% confidence interval of 3.8e10.7 nM.

J. Liu et al. / European Journal of Medicinal Chemistry 65 (2013) 83e93
87
Fig. 3. UNC1062 inhibits Mer activation in adherent tumor cell lines. A) BT-12 pediatric rhabdoid tumor cells were treated with UNC1062 or vehicle only for 1 h prior to the addition
of pervanadate for 15 min. Cell lysates were prepared, Mer protein was immunoprecipitated, and phospho-tyrosine and Mer protein were detected by western blot. B) The indicated
NSCLC cell lines were treated with UNC1062 or an equivalent volume of vehicle only for 72 h prior to treatment with pervanadate for 1.0 (Colo699) or 5.0 (A549) min. Whole cell
lysates were prepared, Mer protein was immunoprecipitated, and phosphorylated and total Mer proteins were detected by western blot. The results shown are representative of 3
independent experiments.
washed with MeOH (3ꢂ) and dried to afford the title compound 4
(23.6 g, 87%). 1H NMR (400 MHz, DMSO-d6)
8.59 (s, 1H), 7.63 (s,
5.2.3. 4-(1-((trans-4-Hydroxycyclohexyl)methyl)-6-(butylamino)-
1H-pyrazolo[3,4-d]pyrimidin-3-yl)-N-methylbenzenesulfonamide (7)
General procedure A: A mixture of 4 (0.054 g, 0.20 mmol), K2CO3
(0.11 g, 0.8 mmol), DMF (2.0 mL), and tert-butyl((trans-4-(chlor-
omethyl)cyclohexyl)oxy)dimethylsilane (0.066 g, 0.25 mmol) in a
10 mL microwave tube was heated under microwave irradiation at
150 ꢀC for 10 min. After the reaction mixture was cooled to room
temperature, (4-(N-methylsulfamoyl)phenyl)boronic acid (0.065 g,
0.30 mmol), Pd(PPh3)4 (0.023 g, 0.020 mmol), and H2O (1.0 mL)
were added sequentially. The mixture was stirred at room tem-
perature for 1.0 min and then heated under microwave irradiation
at 150 ꢀC for 15 min. After cooling to room temperature, the
mixture was quenched with H2O and extracted with EtOAc (3ꢂ).
The combined organic layers were dried (Na2SO4) and concen-
trated. The residue was purified by an ISCO silica gel column to
d
1H), 3.37e3.11 (m, 3H), 1.54e1.41 (m, 2H), 1.29 (dq, J ¼ 14.4, 7.3 Hz,
2H), 0.84 (t, J ¼ 7.3 Hz, 3H); 13C NMR (100 MHz, DMSO-d6)
d 162.0,
157.4, 153.1, 120.5, 107.1, 41.0, 31.0, 20.1, 14.2; MS m/z 270.1
[M þ H]þ.
5.2.2. 4-(1-((trans-4-Aminocyclohexyl)methyl)-6-(butylamino)-1H-
pyrazolo[3,4-d]pyrimidin-3-yl)-N-methylbenzenesulfonamide (2)
A mixture of 4 (0.054 g, 0.20 mmol), K2CO3 (0.11 g, 0.8 mmol),
DMF (2.0 mL), and tert-butyl (trans-4-(chloromethyl)cyclohexyl)
carbamate (0.062 g, 0.25 mmol) in a 10 mL microwave tube
was heated under microwave irradiation at 150 ꢀC for 10 min.
After cooling to room temperature, (4-(N-methylsulfamoyl)
phenyl)boronic acid (0.065 g, 0.30 mmol), Pd(PPh3)4 (0.023 g,
0.020 mmol), and H2O (1.0 mL) were added sequentially. The
resulting mixture was stirred at room temperature for 1.0 min
and then heated under microwave irradiation at 150 ꢀC for
15 min. After cooling to room temperature, the mixture was
quenched with H2O and extracted with EtOAc (3ꢂ). The com-
bined organic layer was dried (Na2SO4) and concentrated. The
residue was purified by an ISCO silica gel column to provide tert-
provide
4-(1-((trans-4-((tert-butyldimethylsilyl)oxy)cyclohexyl)
methyl)-6-(butylamino)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-N-
methylbenzenesulfonamide as a white solid. This white solid was
dissolved in THF (2.0 mL) and was added TBAF (1.0 M in THF, 1 mL)
at room temperature. The resulting solution was refluxed for 2.0 h
and was concentrated. The residue was purified by prep-HPLC to
provide the title compound 7 (0.040 g, 44% over 3 steps) as a white
butyl
(trans-4-((3-(4-(N-methylsulfamoyl)phenyl)-6-(butylla-
solid. 1H NMR (400 MHz, CDCl3)
d
9.30 (s, 1H), 8.20 (d, J ¼ 8.0 Hz,
mino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)cyclohexyl)
carbamate as a white solid. This white solid was dissolved in
CH2Cl2 (2.0 mL), then was added TFA (0.5 mL) at room tem-
perature. The resulting solution was stirred for 1.0 h and was
concentrated. The residue was purified by prep-HPLC to provide
the title compound 2 as a white solid (0.053 g, 48% over 3
2H), 8.01 (d, J ¼ 8.0 Hz, 2H), 4.26 (d, J ¼ 7.0 Hz, 2H), 3.58 (t,
J ¼ 7.2 Hz, 2H), 3.55e3.45 (m, 1H), 2.58 (s, 3H), 2.11e1.93 (m, 3H),
1.73 (td, J ¼ 14.9, 7.9 Hz, 4H), 1.49 (dq, J ¼ 14.8, 7.5 Hz, 2H), 1.32e1.14
(m, 4H), 1.02 (t, J ¼ 7.7Hz, 3H); LCeMS: >97% purity, tR ¼ 6.629 min;
MS m/z 473.3 [M þ 1]þ.
steps). 1H NMR (400 MHz, CDCl3)
d
9.17 (s, 1H), 8.20e8.14 (m,
5.2.4. 4-(1-(2-(trans-4-Hydroxycyclohexyl)ethyl)-6-(butylamino)-
1H-pyrazolo[3,4-d]pyrimidin-3-yl)-N-methylbenzenesulfonamide (8)
The title compound 8 (0.040 g, 42%) was prepared according
to general procedure A from tert-butyl((trans-4-(2-chloroethyl)
cyclohexyl)oxy)dimethylsilane (0.069 g, 0.25 mmol) as a white
2H), 8.01e7.95 (m, 2H), 4.26 (d, J ¼ 6.8 Hz, 2H), 3.52 (t, J ¼ 7.1 Hz,
2H), 3.13e3.00 (m, 1H), 2.81 (s, 3H), 2.15e2.01 (m, 3H), 1.86 (d,
J ¼ 12.0 Hz, 2H), 1.68 (dt, J ¼ 12.7, 7.4 Hz, 2H), 1.48 (dt, J ¼ 14.8,
7.3 Hz, 2H), 1.41e1.21 (m, 4H), 1.00 (t, J ¼ 7.4 Hz, 3H); LCeMS:
>97% purity, tR ¼ 4.379 min; MS m/z 472.3 [M þ 1]þ.
solid. 1H NMR (400 MHz, CDCl3)
d
8.79 (s, 1H), 7.92 (d, J ¼ 8.6 Hz,
Fig. 4. UNC1062 inhibits colony formation in solid tumor cell lines. A) BT-12 rhabdoid tumor cells were cultured in soft agar containing 1.0 mM UNC1062 or vehicle only and overlaid
with medium containing UNC1062 or vehicle only. Medium and UNC1062 were refreshed twice weekly. Colonies were stained and counted. Representative plates are shown. B)
A549 and Colo699 NSCLC cells were cultured in 0.35% soft agar overlaid with medium containing UNC1062 or vehicle only. Medium and UNC1062 were refreshed 3 times per week.
Colonies were stained and counted. Mean values ꢃ SEM derived from 3 independent experiments are shown. *Statistically significant results were determined using the student’s
paired t test (*p < 0.02, **p < 0.001 relative to vehicle only).

88
J. Liu et al. / European Journal of Medicinal Chemistry 65 (2013) 83e93
2H), 7.83 (d, J ¼ 8.6 Hz, 2H), 4.24 (t, J ¼ 7.0 Hz, 2H), 3.45e3.30
(m, 3H), 2.50 (s, 3H), 1.81 (t, J ¼ 11.5 Hz, 3H), 1.71 (dd, J ¼ 13.7,
6.8 Hz, 2H), 1.53 (dt, J ¼ 14.9, 7.3 Hz, 2H), 1.40e1.27 (m, 2H), 1.08
(dd, J ¼ 23.1, 12.5 Hz, 4H), 1.00e0.89 (m, 2H), 0.86 (t, J ¼ 7.9 Hz,
3H); LCeMS: >97% purity, tR ¼ 5.759 min; MS m/z 487.3
[M þ 1]þ.
NMR (400 MHz, CDCl3) d 8.86 (s, 1H), 8.00e7.92 (m, 2H), 7.91e7.83
(m, 2H), 4.42 (t, J ¼ 5.0 Hz, 2H), 4.01 (t, J ¼ 5.0 Hz, 2H), 3.38 (t,
J ¼ 7.1 Hz, 3H), 2.56 (s, 3H), 1.63e1.50 (m, 2H), 1.44e1.30 (m, 2H),
0.90 (t, J ¼ 7.3 Hz, 3H); LCeMS: >97% purity, tR ¼ 5.244 min; MS m/z
405.2 [M þ 1]þ.
5.2.8. 4-(1-(3-Hydroxypropyl)-6-(butylamino)-1H-pyrazolo[3,4-d]
pyrimidin-3-yl)-N-methylbenzenesulfonamide (13)
5.2.5. 4-(1-(3-(trans-4-Hydroxycyclohexyl)propyl)-6-(butylami
no)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-N-methylbenzenesulfona
mide (9)
The title compound 13 (0.045 g, 56%) was prepared according to
general procedure
C
from 3-chloropropan-1-ol (0.029 g,
8.88 (s,
The title compound 9 (0.055 g, 56%) was prepared according to
0.30 mmol) as a white solid. 1H NMR (400 MHz, CDCl3)
d
general procedure
cyclohexyl)oxy)dimethylsilane (0.069 g, 0.25 mmol) as a white
solid. 1H NMR (400 MHz, CDCl3)
9.03 (s, 1H), 8.17e8.09 (m, 2H),
7.99e7.92 (m, 2H), 4.32 (t, J ¼ 6.9 Hz, 2H), 3.48 (t, J ¼ 7.0 Hz, 2H),
3.42 (dt, J ¼ 10.8, 4.2 Hz, 1H), 2.58 (s, 3H), 2.02e1.84 (m, 4H), 1.76 (d,
J ¼ 11.6 Hz, 2H), 1.66 (dt, J ¼ 14.8, 7.3 Hz, 2H), 1.52e1.39 (m, 2H),
1.29e1.13 (m, 5H), 0.99 (t, J ¼ 7.4 Hz, 3H), 0.96e0.86 (m, 2H); LCe
MS: >97% purity, tR ¼ 5.856 min; MS m/z 501.3 [M þ 1]þ.
B
from tert-butyl((trans-4-(3-chloropropyl)
1H), 7.98 (d, J ¼ 8.6 Hz, 2H), 7.90 (d, J ¼ 8.6 Hz, 2H), 4.43 (t,
J ¼ 6.2 Hz, 2H), 3.50 (t, J ¼ 5.8 Hz, 2H), 3.40 (t, J ¼ 7.1 Hz, 2H), 2.59 (s,
3H), 2.10e2.00 (m, 2H), 1.60 (dt, J ¼ 14.9, 7.4 Hz, 2H), 1.40 (dq,
J ¼ 14.5, 7.3 Hz, 2H), 0.92 (t, J ¼ 7.3 Hz, 3H); LCeMS: >97% purity,
tR ¼ 5.336 min; MS m/z 419.2 [M þ 1]þ.
d
5.2.9. N-Methyl-4-(6-(butylamino)-1-(tetrahydro-2H-pyran-4-yl)-
1H-pyrazolo[3,4-d]pyrimidin-3-yl)benzenesulfonamide (14)
General procedure D: A mixture of 4 (0.054 g, 0.20 mmol),
K2CO3 (0.11 g, 0.8 mmol), DMF (2.0 mL), and 4-chlorotetrahydro-
2H-pyran (0.072 g, 0.60 mmol) in a 10 mL microwave tube was
heated under microwave irradiation at 200 ꢀC for 30 min. After
cooling to room temperature, (4-(N-methylsulfamoyl)phenyl)
boronic acid (0.065 g, 0.30 mmol), Pd(PPh3)4 (0.023 g,
0.020 mmol), and H2O (1.0 mL) were added sequentially. The
resulting mixture was stirred at room temperature for 1.0 min and
then heated under microwave irradiation at 150 ꢀC for 15 min.
After cooling to room temperature, the mixture was quenched
with H2O and extracted with EtOAc (3ꢂ). The combined organic
layers were dried (Na2SO4) and concentrated. The combined
organic layer was dried (Na2SO4) and concentrated under reduced
pressure. The residue was purified by an ISCO silica gel column to
provide the title compound 14 as a white solid (0.052 g, 61% over 2
5.2.6. 4-(1-(trans-4-Hydroxycyclohexyl)-6-(butylamino)-1H-
pyrazolo[3,4-d]pyrimidin-3-yl)-N-methylbenzenesulfonamide (10)
and 4-(1-(cis-4-hydroxycyclohexyl)-6-(butylamino)-1H-pyrazolo
[3,4-d]pyrimidin-3-yl)-N-methylbenzenesulfonamide (11)
General procedure B: A mixture of 4 (0.054 g, 0.20 mmol), K2CO3
(0.11 g, 0.8 mmol), DMF (2.0 mL), and 4-chlorocyclohexanol
(0.081 g, 0.6 mmol) in a 10 mL microwave tube was heated under
microwave irradiation at 200 ꢀC for 30 min. After cooling to room
temperature, (4-(N-methylsulfamoyl)phenyl)boronic acid (0.065 g,
0.30 mmol), Pd(PPh3)4 (0.023 g, 0.020 mmol), and H2O (1.0 mL)
were added sequentially. The resulting mixture was stirred at room
temperature for 1.0 min and then heated under microwave irradi-
ation at 150 ꢀC for 15 min. After cooling to room temperature, the
mixture was quenched with H2O and extracted with EtOAc (3ꢂ).
The combined organic layer was dried (Na2SO4) and concentrated.
The residue was purified by prep-HPLC to provide the title com-
pounds 10 (0.011 g, 13% over 3 steps) and 11 (0.020 g, 23% over 3
steps) as white solids. Compound 10: 1H NMR (400 MHz, CDCl3)
steps). 1H NMR (400 MHz, CDCl3)
d
8.96 (s, 1H), 8.07 (d, J ¼ 8.5 Hz,
2H), 7.97 (d, J ¼ 8.5 Hz, 2H), 5.33 (s, 1H), 4.92e4.79 (m, 1H), 4.32
(dd, J ¼ 10.8, 5.4 Hz, 1H), 4.17 (dd, J ¼ 11.7, 3.3 Hz, 2H), 3.64 (dd,
J ¼ 11.9, 10.2 Hz, 2H), 3.52 (dd, J ¼ 12.9, 6.9 Hz, 2H), 2.71 (d,
J ¼ 5.4 Hz, 3H), 2.48 (ddd, J ¼ 25.0, 12.5, 4.7 Hz, 2H), 1.97 (d,
J ¼ 10.8 Hz, 2H), 1.66 (dt, J ¼ 14.8, 7.2 Hz, 2H), 1.47 (dq, J ¼ 14.5,
7.3 Hz, 2H), 0.99 (t, J ¼ 7.3 Hz, 3H); LCeMS: 96% purity,
tR ¼ 5.689 min; MS m/z 445.2 [M þ 1]þ.
d
8.78 (s, 1H), 7.92 (d, J ¼ 8.4 Hz, 2H), 7.82 (d, J ¼ 8.3 Hz, 2H), 4.51 (s,
1H), 3.64 (s, 1H), 3.37 (t, J ¼ 7.0 Hz, 2H), 2.50 (s, 3H), 2.17e1.86 (m,
6H), 1.61e1.29 (m, 6H), 0.87 (t, J ¼ 7.3 Hz, 3H); LCeMS: >97% purity,
tR ¼ 5.402 min; HRMS (TOF, ESIþ) m/z: [M þ H]þ calculated for
C
22H31N6O3S, 459.2178; found 459.2155. Compound 11: 1H NMR
(400 MHz, CDCl3 þ CD3OD)
d
8.82 (s, 1H), 7.95 (dd, J ¼ 8.2, 6.2 Hz,
5.2.10. N-Methyl-4-(6-(butylamino)-1-((tetrahydro-2H-pyran-4-yl)
methyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)benzenesulfonamide (15)
The title compound 15 (0.062 g, 70% over 2 steps) was prepared
according to general procedure C from 4-(bromomethyl)tetrahy-
dro-2H-pyran (0.045 g, 0.25 mmol) as a white solid. 1H NMR
2H), 7.84 (dd, J ¼ 8.5, 1.8 Hz, 2H), 4.66e4.48 (m, 1H), 3.44e3.33 (m,
3H), 2.53 (s, 3H), 2.46e2.32 (m, 1H), 1.99e1.81 (m, 3H), 1.79e1.61
(m, 3H), 1.55 (dt, J ¼ 14.8, 7.4 Hz, 2H), 1.36 (td, J ¼ 14.8, 7.4 Hz, 3H),
0.88 (t, J ¼ 7.3 Hz, 3H); LCeMS: >97% purity, tR ¼ 5.590 min; MS m/z
459.2 [M þ 1]þ.
(400 MHz, CDCl3)
d 8.95 (s, 1H), 8.10e8.03 (m, 2H), 8.00e7.93 (m,
2H), 5.37 (br s, 1H), 4.41e4.30 (m, 1H), 4.24 (d, J ¼ 7.1 Hz, 2H), 4.03e
3.91 (m, 2H), 3.51 (dd, J ¼ 13.0, 6.9 Hz, 2H), 3.38 (td, J ¼ 11.5, 2.6 Hz,
2H), 2.71 (d, J ¼ 5.4 Hz, 3H), 2.40e2.24 (m, 1H), 1.71e1.61 (m, 2H),
1.60e1.56 (m, 1H), 1.54e1.38 (m, 5H), 0.99 (t, J ¼ 7.3 Hz, 3H); LCe
MS: >97% purity, tR ¼ 5.714 min; MS m/z 459.3 [M þ 1]þ.
5.2.7. 4-(1-(2-Hydroxyethyl)-6-(butylamino)-1H-pyrazolo[3,4-d]
pyrimidin-3-yl)-N-methylbenzenesulfonamide (12)
General procedure C: A mixture of 4 (0.054 g, 0.20 mmol), K2CO3
(0.11 g, 0.8 mmol), DMF (2.0 mL), and 2-bromoethanol (0.038 g,
0.30 mmol) in a 10 mL microwave tube was heated under micro-
wave irradiation at 150 ꢀC for 10 min. After cooling to room tem-
perature, (4-(N-methylsulfamoyl)phenyl)boronic acid (0.065 g,
0.30 mmol), Pd(PPh3)4 (0.023 g, 0.020 mmol), and H2O (1.0 mL)
were added sequentially. The resulting mixture was stirred at room
temperature for 1.0 min and then heated under microwave irradi-
ation at 150 ꢀC for 15 min. After cooling to room temperature, the
mixture was quenched with H2O and extracted with EtOAc (3ꢂ).
The combined organic layers were dried (Na2SO4) and concen-
trated. The residue was purified by an ISCO silica gel column to
provide the title compound 12 (0.044 g, 57%) as a white solid. 1H
5.2.11. N-Methyl-4-(6-(butylamino)-1-(2-(tetrahydro-2H-pyran-4-
yl)ethyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)benzenesulfonamide(16)
The title compound 16 (0.058 g, 63% over 2 steps) was pre-
pared according to general procedure C from 4-(2-bromoethyl)
tetrahydro-2H-pyran (0.048 g, 0.25 mmol) as a white solid. 1H
NMR (400 MHz, CDCl3) d 8.95 (s, 1H), 8.10e8.03 (m, 2H), 7.99e7.93
(m, 2H), 5.38 (br s, 1H), 4.47e4.33 (m, 3H), 3.95 (dd, J ¼ 11.5,
3.5 Hz, 2H), 3.49 (dd, J ¼ 13.1, 6.8 Hz, 2H), 3.33 (td, J ¼ 11.7, 1.8 Hz,
2H), 2.71 (d, J ¼ 5.4 Hz, 3H), 1.97e1.86 (m, 2H), 1.76 (d, J ¼ 12.7 Hz,
2H), 1.65 (dt, J ¼ 12.7, 7.4 Hz, 2H), 1.55e1.30 (m, 5H), 0.98

J. Liu et al. / European Journal of Medicinal Chemistry 65 (2013) 83e93
89
(t, J ¼ 7.3 Hz, 3H); LCeMS: 97% purity, tR ¼ 5.835 min; MS m/z
473.3 [M þ 1]þ.
a white solid. 1H NMR (400 MHz, CD3OD þ CDCl3)
d
8.74 (s, 1H),
7.87e7.78 (m, 4H), 4.46e4.43 (m,1H), 3.59e3.55 (m,1H), 3.33e3.28
(m, 2H), 2.03e1.93 (m, 4H), 1.90e1.85 (m, 2H), 1.50e1.45 (m, 2H),
1.39e1.33 (m, 2H), 1.30e1.25 (m, 2H), 1.08e1.06 (m, 3H), 0.80 (t,
J ¼ 7.4 Hz, 3H); LCeMS: 97% purity, tR ¼ 5.147 min; MS m/z 445.2
[M þ 1]þ.
5.2.12. 4-(1-Cyclohexyl-6-(butylamino)-1H-pyrazolo[3,4-d]
pyrimidin-3-yl)-N-methylbenzene sulfonamide (17)
The title compound 17 (0.035 g, 40% over 2 steps) was pre-
pared according to general procedure D from chlorocyclohexane
(0.071 g, 0.60 mmol) as a white solid. 1H NMR (400 MHz, CDCl3)
5.2.17. trans-4-(6-(Butylamino)-1-(4-hydroxycyclohexyl)-1H-
pyrazolo[3,4-d]pyrimidin-3-yl)-N-propylbenzenesulfonamide (21)
The title compound 21 (0.019 g, 13%) was prepared according to
general procedure B from (4-(N-propylsulfamoyl)phenyl)boronic
acid (0.109 g, 0.45 mmol) as a white solid. 1H NMR (400 MHz, CDCl3)
d
8.94 (s, 1H), 8.10e8.03 (m, 2H), 7.99e7.92 (m, 2H), 5.30 (br s, 1H),
4.68e4.56 (m, 1H), 4.33 (q, J ¼ 5.4 Hz, 1H), 3.52 (dd, J ¼ 12.9,
7.0 Hz, 2H), 2.70 (d, J ¼ 5.4 Hz, 3H), 2.16e1.89 (m, 6H), 1.77 (d,
J ¼ 12.7 Hz, 1H), 1.66 (dt, J ¼ 14.8, 7.3 Hz, 2H), 1.54e1.28 (m, 5H),
0.99 (t, J ¼ 7.3 Hz, 3H); LCeMS: >97% purity, tR ¼ 6.358 min; MS
m/z 443.2 [M þ 1]þ.
d
8.83 (s, 1H), 7.98 (s, 4H), 4.68e4.62 (m, 1H), 4.42 (t,, J ¼ 6.16 Hz,
1H), 3.87e3.81 (m, 1H), 3.57e3.51 (m, 2H), 2.98 (dd, J ¼ 6.95,
13.46 Hz, 2H), 2.25e2.16 (m, 4H), 2.10e2.07 (m, 2H), 1.71 (dt,
J ¼ 7.43, 14.83 Hz, 2H), 1.60e1.42 (m, 6H), 1.00 (t, J ¼ 7.36 Hz, 3H),
0.89 (t, J ¼ 7.40 Hz, 3H); LCeMS: >97% purity, tR ¼ 6.217 min; MS m/
z 487.3 [M þ 1]þ.
5.2.13. trans-4-(3-Bromo-6-(butylamino)-1H-pyrazolo[3,4-d]
pyrimidin-1-yl)cyclohexanol
To
a
solution of
4
(2.70 g, 10 mmol) and cis-4-
chlorocyclohexanol (4.04 g, 30 mmol) in DMF (50 mL) was added
K2CO3 (5.52 g, 40 mmol). The reaction mixture was heated at 150 ꢀC
for overnight, quenched with water at room temperature and
extracted with EtOAc (3ꢂ). The combined organic layer was dried
(Na2SO4) and concentrated. The residue was purified by an ISCO
silica gel column to afford the title compound (1.83 g, 52%) as a
5.2.18. trans-4-(6-(Butylamino)-1-(4-hydroxycyclohexyl)-1H-
pyrazolo[3,4-d]pyrimidin-3-yl)-N-isopropylbenzenesulfonamide (22)
The title compound 22 (0.016 g, 13%) was prepared according to
general procedure
boronic acid (0.095 g, 0.39 mmol) as a white solid. 1H NMR
(400 MHz, CDCl3)
B
from (4-(N-isopropylsulfamoyl)phenyl)
white solid. 1H NMR (400 MHz, CD3OD)
d
8.49 (s,1H), 4.59e4.41 (m,
d
8.91 (s, 1H), 8.00 (dd, J ¼ 8.62, 20.77 Hz, 4H),
1H), 3.69e3.56 (m, 1H), 3.42 (t, J ¼ 7.1 Hz, 2H), 2.13e2.00 (m, 4H),
4.69e4.61 (m, 1H), 4.26 (d, J ¼ 7.61 Hz, 1H), 3.88e3.80 (m, 1H),
3.57e3.48 (m, 2H), 2.27e2.18 (m, 4H), 2.07 (d, J ¼ 10.85 Hz, 2H),
1.71e1.53 (m, 6H), 1.47 (dd, J ¼ 7.36, 14.90 Hz, 2H), 1.11 (d,
J ¼ 6.52 Hz, 6H), 0.99 (t, J ¼ 7.34 Hz, 3H); LCeMS: >97% purity,
tR ¼ 5.826 min; MS m/z 487.3 [M þ 1]þ.
1.98e1.89 (m, 2H), 1.66e1.56 (m, 2H), 1.52e1.36 (m, 4H), 0.96 (t,
J ¼ 7.4 Hz, 3H); 13C NMR (100 MHz, DMSO-d6)
d 161.6, 155.0, 153.4,
119.1, 110.3, 107.7, 68.3, 55.5, 34.6, 30.9, 29.8, 20.0, 14.1; MS m/z
369.1 [M þ H]þ.
5.2.14. trans-N-(4-(6-(Butylamino)-1-(4-hydroxycyclohexyl)-1H-
pyrazolo[3,4-d]pyrimidin-3-yl)phenyl)methanesulfonamide (18)
The title compound 18 (0.008 g, 18%) was prepared according to
general procedure B from (4-(methylsulfonamido)phenyl)boronic
acid (0.033 g, 0.15 mmol) as a white solid. 1H NMR (400 MHz, CDCl3)
5.2.19. 4-(6-(Butylamino)-1-(trans-4-hydroxycyclohexyl)-1H-pyra-
zolo[3,4-d]pyrimidin-3-yl)-N-cyclopropylbenzenesulfonamide (23)
The title compound 23 (0.003 g, 4%) was prepared according to
general procedure
boronic acid (0.090 g, 0.375 mmol) as a white solid. 1H NMR
(400 MHz, CD3OD).
E from (4-(N-cyclopropylsulfamoyl)phenyl)
d
8.76 (s, 1H), 7.76e7.72 (m, 2H), 7.28e7.24 (m, 2H), 4.56e4.48 (m,
d
9.11 (s, 1H), 8.17 (d, J ¼ 8.6 Hz, 2H), 8.00 (d,
1H), 3.71e3.61 (m, 1H), 3.42e3.39 (m, 1H), 3.29e3.27 (m, 2H), 2.92
(s, 3H), 2.14e2.02 (m, 4H), 1.97e1.90 (m, 2H), 1.61e1.53 (m, 2H),
1.47e1.32 (m, 6H), 0.90 (t, J ¼ 7.36 Hz, 3H); LCeMS: >97% purity,
tR ¼ 5.538 min; MS m/z 459.3 [M þ 1]þ.
J ¼ 8.6 Hz, 2H), 4.73e4.63 (m, 1H), 3.79e3.67 (m, 1H), 3.51 (t,
J ¼ 7.1 Hz, 2H), 2.30e2.01 (m, 7H), 1.69 (td, J ¼ 14.7, 7.4 Hz, 2H),
1.61e1.42 (m, 4H), 1.01 (t, J ¼ 7.4 Hz, 3H), 0.62e0.47 (m, 4H); LCe
MS: 95% purity, tR ¼ 5.957 min; MS m/z 485.0 [M þ 1]þ.
5.2.15. 4-(1-(trans-4-Hydroxycyclohexyl)-6-(butylamino)-1H-
pyrazolo[3,4-d]pyrimidin-3-yl)benzenesulfonic acid (19)
5.2.20. trans-4-(6-(Butylamino)-1-(4-hydroxycyclohexyl)-1H-
pyrazolo[3,4-d]pyrimidin-3-yl)-N-cyclopentylbenzenesulfonamide (24)
The title compound 24 (0.015 g, 10%) was prepared according to
General procedure E: A mixture of trans-4-(3-bromo-6-(buty-
lamino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)cyclohexanol (0.055 g,
0.15 mmol), K3PO4 (0.063 g, 0.30 mmol), 4-boronobenzenesulfonic
acid (0.045 g, 0.225 mmol), Pd(PPh3)4 (0.017 g, 0.015 mmol),
dioxane (2.0 mL) and H2O (0.5 mL) in a 10 mL microwave tube was
heated under microwave irradiation at 150 ꢀC for 10 min. After
cooling to room temperature, the mixture was quenched with H2O
and extracted with EtOAc (3ꢂ). The combined organic layer was
dried (Na2SO4) and concentrated. The residue was filtered through
a plug of Celite and then purified by prep-HPLC to afford the title
compound 19 (0.027 g, 30%) as a white solid. 1H NMR (400 MHz,
general procedure
boronic acid (0.121 g, 0.45 mmol) as a white solid. 1H NMR
(400 MHz, CDCl3)
B from (4-(N-cyclopentylsulfamoyl)phenyl)
d
8.82 (s, 1H), 7.99 (q, J ¼ 8.67 Hz, 4H), 4.68e4.64
(m, 1H), 4.44 (d, J ¼ 7.34 Hz, 1H), 3.86e3.82 (m, 1H), 3.67 (dd,
J ¼ 6.82, 13.53 Hz, 1H), 3.55e3.50 (m, 2H), 2.22e2.16 (m, 4H), 2.10e
2.07 (m, 2H), 1.84 (dd, J ¼ 5.70, 12.36 Hz, 2H), 1.71 (dd, J ¼ 7.63,
14.79 Hz, 2H), 1.63e1.57 (m, 4H), 1.54e1.48 (m, 4H), 1.40e1.35 (m,
2H), 1.00 (t, J ¼ 7.35 Hz, 3H); LCeMS: 95% purity, tR ¼ 6.186 min; MS
m/z 513.3 [M þ 1]þ.
CD3OD)
d
9.19 (s, 1H), 8.05e7.93 (m, 4H), 4.77e4.61 (m, 1H), 3.73 (tt,
5.2.21. trans-4-(6-(Butylamino)-1-(4-hydroxycyclohexyl)-1H-
pyrazolo[3,4-d]pyrimidin-3-yl)-N-cyclohexylbenzenesulfonamide (25)
The title compound 25 (0.015 g, 11%) was prepared according to
J ¼ 10.7, 4.1 Hz, 1H), 3.57 (t, J ¼ 7.1 Hz, 2H), 2.31e2.02 (m, 6H), 1.77e
1.67 (m, 2H), 1.61e1.44 (m, 4H), 1.03 (t, J ¼ 7.4 Hz, 3H); LCeMS:
>97% purity, tR ¼ 4.890 min; MS m/z 444.2 [M ꢁ 1]ꢁ.
general procedure
boronic acid (0.110 g, 0.39 mmol) as a white solid. 1H NMR
(400 MHz, CDCl3)
B from (4-(N-cyclohexylsulfamoyl)phenyl)
5.2.16. trans-4-(6-(Butylamino)-1-(4-hydroxycyclohexyl)-1H-
pyrazolo[3,4-d]pyrimidin-3-yl)benzenesulfonamide (20)
The title compound 20 (0.064 g, 71%) was prepared according
to general procedure
dioxaborolan-2-yl)benzenesulfonamide (0.086 g, 0.306 mmol) as
d
8.95 (s, 1H), 8.04 (d, J ¼ 8.61 Hz, 2H), 7.97 (d,
J ¼ 8.59 Hz, 2H), 5.36 (br s, 1H), 4.67e4.64 (m, 1H), 4.34 (d,
J ¼ 7.55 Hz, 1H), 3.87e3.80 (m, 1H), 3.52 (dd, J ¼ 7.03, 12.98 Hz, 2H),
3.24e3.15 (m, 1H), 2.28e2.18 (m, 4H), 2.09e2.06 (m, 2H), 1.82e1.79
(m, 2H), 1.67e1.56 (m, 6H), 1.52e1.46 (m, 4H), 1.27e1.14 (m, 6H),
E
from 4-(4,4,5,5-tetramethyl-1,3,2-

90
J. Liu et al. / European Journal of Medicinal Chemistry 65 (2013) 83e93
1.00 (t, J ¼ 7.35 Hz, 3H); LCeMS: >97% purity, tR ¼ 6.171 min; MS m/
z 527.0 [M þ 1]þ.
2.0 equiv) as a white solid. 1H NMR (400 MHz, CD3OD)
d 9.17 (s, 1H),
8.95 (s, 1H), 8.06 (d, J ¼ 8.54 Hz, 2H), 7.96 (d, J ¼ 8.33 Hz, 2H), 7.34e
7.31 (m, 2H), 6.90e6.86 (m, 2H), 4.68e4.62 (m, 1H), 3.75e3.70 (m,
1H), 3.50 (t, J ¼ 7.90 Hz, 2H), 2.24e2.13 (m, 4H), 2.06e2.03 (m, 2H),
1.71e1.64 (m, 2H), 1.58e1.38 (m, 6H), 1.10 (dd, J ¼ 4.67, 7.89 Hz, 2H),
1.00 (t, J ¼ 7.36 Hz, 3H); LCeMS: 96% purity, tR ¼ 6.051 min; MS m/z
622.3 [M þ 1]þ.
5.2.22. trans-4-(6-(Butylamino)-1-(4-hydroxycyclohexyl)-1H-pyraz-
olo[3,4-d]pyrimidin-3-yl)-N-(tetrahydro-2H-pyran-4-yl)benzenesulf-
onamide (26)
The title compound 26 (0.016 g, 10%) was prepared according to
general procedure B from (4-(N-(tetrahydro-2H-pyran-4-yl)sulfa-
moyl)phenyl)boronic acid (0.128 g, 0.45 mmol) as a white solid. 1H
5.2.27. 4-(6-(Butylamino)-1-(trans-4-hydroxycyclohexyl)-1H-
pyrazolo[3,4-d]pyrimidin-3-yl)-N,N-dimethylbenzenesulfonamide (31)
The title compound 31 (0.052 g, 74%) was prepared according to
NMR (400 MHz, CDCl3)
d 9.44 (br s, 1H), 8.92 (s, 1H), 8.00 (q,
J ¼ 8.67 Hz, 4H), 4.69e4.61 (m, 2H), 3.90e3.84 (m, 4H), 3.55 (t,
J ¼ 7.04 Hz, 2H), 3.43e3.33 (m, 2H), 2.21e2.07 (m, 6H), 1.79e1.68
(m, 4H), 1.60e1.44 (m, 6H), 1.00 (t, J ¼ 7.36 Hz, 3H); LCeMS: 95%
purity, tR ¼ 5.747 min; MS m/z 529.3 [M þ 1]þ.
general procedure
boronic acid (0.052 g, 0.225 mmol) as a white solid. 1H NMR
(400 MHz, CDCl3)
E from (4-(N,N-dimethylsulfamoyl)phenyl)
d
8.94 (s, 1H), 8.07 (d, J ¼ 8.4 Hz, 2H), 7.88 (d,
J ¼ 8.3 Hz, 2H), 5.34 (br s,1H), 4.73e4.59 (m,1H), 3.90e3.76 (m,1H),
3.52 (dd, J ¼ 13.0, 6.8 Hz, 2H), 2.75 (s, 6H), 2.29e2.15 (m, 4H), 2.13e
2.02 (m, 2H), 1.66 (dt, J ¼ 14.9, 7.3 Hz, 2H), 1.59e1.52 (t, J ¼ 10.2 Hz,
3H), 1.47 (dd, J ¼ 15.0, 7.4 Hz, 2H), 1.00 (t, J ¼ 7.3 Hz, 3H); LCeMS:
97% purity, tR ¼ 6.011 min; MS m/z 473.3 [M þ 1]þ.
5.2.23. trans-4-(6-(Butylamino)-1-(4-hydroxycyclohexyl)-1H-pyraz-
olo[3,4-d]pyrimidin-3-yl)-N-(4-fluorophenyl)benzenesulfonamide (27)
The title compound 27 (0.009 g, 6%) was prepared according to
general procedure B from 4-(N-(4-fluorophenyl)sulfamoyl)phenyl)
boronic acid (0.133 g, 0.45 mmol) as a white solid. 1H NMR
(400 MHz, CDCl3)
d
9.38 (s, 1H), 8.82 (s, 1H), 7.94e7.89 (m, 2H),
5.2.28. N-(tert-Butyl)-4-(6-(butylamino)-1-(trans-4-hydroxycyclo-
hexyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-N-methylbenzenesulfon-
amide (32)
The title compound 32 (0.058 g, 76%) was prepared according to
general procedure E from ((4-(N-(tert-butyl)-N-methylsulfamoyl)
phenyl)boronic acid (0.061 g, 0.225 mmol) as a white solid. 1H NMR
7.85e7.81 (m, 2H), 7.09e7.05 (m, 2H), 6.98e6.94 (m, 2H), 6.57 (s,
1H), 4.67e4.61 (m, 1H), 3.98e3.80 (m, 1H), 3.57e3.51 (m, 2H),
2.22e2.08 (m, 6H), 1.71 (dt, J ¼ 7.29, 14.74 Hz, 2H), 1.62e1.57 (m,
2H),1.45 (dt, J ¼ 7.37,14.45 Hz, 2H), 0.99 (t, J ¼ 7.38 Hz, 3H); LCeMS:
>97% purity, tR ¼ 6.198 min; MS m/z 539.3 [M þ 1]þ.
(400 MHz, CDCl3)
d 8.93 (s, 1H), 8.07e7.97 (m, 2H), 7.96e7.89 (m,
5.2.24. trans-tert-Butyl 4-((4-(6-(butylamino)-1-(4-hydroxycyclohe-
xyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)phenylsulfonamido)methyl)
piperidine-1-carboxylate (29)
The title compound 29 (0.011 g, 9%) was prepared according to
general procedure B from (4-(N-((1-(tert-butoxycarbonyl)piper-
2H), 5.40 (br s, 1H), 4.73e4.58 (m, 1H), 3.91e3.76 (m, 1H), 3.52 (dd,
J ¼ 12.9, 6.9 Hz, 2H), 3.01 (s, 3H), 2.30e2.14 (m, 4H), 2.07 (d,
J ¼ 10.8 Hz, 2H), 1.68e1.44 (m, 7H), 1.38 (d, J ¼ 4.8 Hz, 9H), 0.99 (t,
J ¼ 7.3 Hz, 3H); LCeMS: 96% purity, tR ¼ 6.328 min; MS m/z 515.3
[M þ 1]þ.
idin-4-yl)methyl)sulfamoyl)phenyl)boronic
0.30 mmol, 1.5 equiv) as a white solid. 1H NMR (400 MHz,
CDCl3 þ CD3OD) 8.78 (s, 1H), 7.90 (d, J ¼ 8.44 Hz, 2H), 7.80 (d,
acid
(0.119
g,
5.2.29. trans-4-(3-(4-(Azetidin-1-ylsulfonyl)phenyl)-6-(butylami-
no)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)cyclohexanol (33)
The title compound 33 (0.054 g, 74%) was prepared according to
general procedure E from (4-(azetidin-1-ylsulfonyl)phenyl)boronic
acid (0.054 g, 0.225 mmol) as a white solid. 1H NMR (400 MHz,
d
J ¼ 7.34 Hz, 2H), 4.46e4.43 (m, 1H), 3.91 (d, J ¼ 12.95 Hz, 2H), 3.63e
3.61 (m, 1H), 2.36 (t, J ¼ 7.08 Hz, 2H), 3.22 (dt, J ¼ 1.61, 3.24 Hz, 3H),
2.65 (d, J ¼ 6.62 Hz, 2H), 2.54e2.51 (m, 2H), 2.08e2.00 (m, 4H),
1.93e1.90 (m, 2H), 1.58e1.46 (m, 4H), 1.31 (s, 12H), 0.97e0.91 (m,
2H), 0.89 (t, J ¼ 7.35 Hz, 3H); LCeMS: 96% purity, tR ¼ 6.305 min; MS
m/z 642.4 [M þ 1]þ.
CDCl3)
d
8.96 (s, 1H), 8.11 (d, J ¼ 8.3 Hz, 2H), 7.95 (d, J ¼ 8.2 Hz, 2H),
5.33 (s, 1H), 4.75e4.57 (m, 1H), 3.92e3.74 (m, 5H), 3.53 (dd, J ¼ 13.1,
6.7 Hz, 2H), 2.32e2.16 (m, 4H), 2.15e2.03 (m, 4H), 1.66 (dt, J ¼ 14.8,
7.2 Hz, 2H), 1.55e1.50 (m, 3H), 1.45 (dd, J ¼ 14.6, 7.3 Hz, 2H), 1.00 (t,
J ¼ 7.3 Hz, 3H); LCeMS: >97% purity, tR ¼ 5.992 min; MS m/z 485.3
[M þ 1]þ.
5.2.25. trans-4-(6-(Butylamino)-1-(4-hydroxycyclohexyl)-1H-pyra
zolo[3,4-d]pyrimidin-3-yl)-N-(piperidin-4-ylmethyl)benzenesulfona-
mide (28)
A mixture of 29 (20 mg, 0.03 mmol) and TFA (2.3 mL) in
dichloromethane (4.7 mL) was stirred at room temperature for
2.0 h, then diluted with dichloromethane (20 mL) and washed with
a saturated aqueous NaHCO3 solution, water and brine. The organic
layer was dried (Na2SO4) and concentrated to afford the title
compound 28 (12.7 mg, 75%) as a white solid. 1H NMR (400 MHz,
5.2.30. trans-4-(6-(Butylamino)-3-(4-(pyrrolidin-1-ylsulfonyl)
phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)cyclohexanol (34)
The title compound 34 (0.020 g, 27%) was prepared according to
general procedure
boronic acid (0.096 g, 0.375 mmol) as a white solid. 1H NMR
(400 MHz, CD3OD)
E from (4-(pyrrolidin-1-ylsulfonyl)phenyl)
d
9.20 (s, 1H), 8.20 (d, J ¼ 8.5 Hz, 2H), 7.98 (d,
CDCl3þCD3OD)
d
8.25 (s, 1H), 7.23 (d, J ¼ 8.45 Hz, 2H), 7.14 (d,
J ¼ 8.5 Hz, 2H), 4.75e4.64 (m, 1H), 3.78e3.67 (m, 1H), 3.55 (t,
J ¼ 7.1 Hz, 2H), 3.32e3.23 (m, 4H), 2.30e2.02 (m, 6H), 1.85e1.64 (m,
6H), 1.62e1.42 (m, 4H), 1.02 (t, J ¼ 7.4 Hz, 3H); LCeMS: >97% purity,
tR ¼ 6.100 min; MS m/z 499.0 [M þ 1]þ.
J ¼ 8.20 Hz, 2H), 3.85e3.80 (m, 1H), 2.96e2.91 (m, 1H), 2.72 (t,
J ¼ 7.15 Hz, 2H), 2.54e2.52 (m, 3H), 2.07e1.99 (m, 4H),1.41e1.32 (m,
4H), 1.26e1.23 (m, 2H), 1.12 (d, J ¼ 13.63 Hz, 2H), 0.98e0.94 (m, 1H),
0.91e0.84 (m, 2H), 0.79e0.75 (m, 2H), 0.67e0.56 (m, 4H), 0.17 (t,
J ¼ 7.35 Hz, 3H); LCeMS: >97% purity, tR ¼ 4.975 min; MS m/z 542.3
[M þ 1]þ.
5.2.31. trans-4-(6-(Butylamino)-3-(4-(morpholinosulfonyl)phenyl)-
1H-pyrazolo[3,4-d]pyrimidin-1-yl)cyclohexanol (35, UNC1062)
The title compound 35 (0.063 g, 82%) was prepared according
5.2.26. trans-1-(4-(6-(Butylamino)-1-(4-hydroxycyclohexyl)-1H-
pyrazolo[3,4-d]pyrimidin-3-yl)phenylsulfonamido)-N-(4-fluorophe-
nyl)cyclopropanecarboxamide (30)
to general procedure
dioxaborolan-2-yl)phenyl)sulfonyl)morpholine (0.079 g, 0.225
mmol) as a white solid. 1H NMR (400 MHz, CD3OD).
9.20 (s, 1H),
E from 4-((4-(4,4,5,5-tetramethyl-1,3,2-
d
The title compound 30 (0.009 g, 7%) was prepared according to
general procedure B from (4-(N-(1-((4-fluorophenyl)carbamoyl)
cyclopropyl)sulfamoyl)phenyl)boronic acid (0.160 g, 0.42 mmol,
8.26e8.20 (m, 2H), 7.95e7.89 (m, 2H), 4.75e4.64 (m, 1H), 3.79e
3.68 (m, 1H), 3.73 (dd, J ¼ 10.2, 5.7 Hz, 4H), 3.55 (t, J ¼ 7.1 Hz, 2H),
3.08e2.97 (m, 4H), 2.36e2.01 (m, 6H), 1.76e1.65 (m, 2H), 1.62e1.42

J. Liu et al. / European Journal of Medicinal Chemistry 65 (2013) 83e93
91
(m, 4H), 1.02 (t, J ¼ 7.4 Hz, 3H); 13C NMR (100 MHz, DMSO-d6)
from previous titration studies (data not shown) [17b] (Y ¼ velocity,
X ¼ inhibitor concentration).
d
160.6,155.2,154.0,140.6,136.9,133.7,128.3,127.0,105.1, 68.0, 65.3,
55.0, 45.9, 40.3, 34.2, 30.6, 29.4, 19.6, 13.7; LCeMS: >97% purity,
tR ¼ 5.657 min; HRMS (TOF, ESIþ) m/z: [M þ H]þ calculated for
For UNC1062, the Morrison Ki was 0.33 ꢃ 0.083 nM (n ¼ 3, error
reported as S.D.).
C
25H35N6O4S, 515.2440; found 515.2421.
5.5. hERG binding assay
5.2.32. trans-(1-((4-(6-(Butylamino)-1-(4-hydroxycyclohexyl)-1H-
pyrazolo[3,4-d]pyrimidin-3-yl)phenyl)sulfonyl)piperidin-4-yl)(morp-
holino)methanone (36)
Assay buffer: Hank’s balanced salt solution, 20 mM HEPES,
2.5 mM probenecid, pH 7.4, membrane potential sensitive dye
(Molecular Devices).
To screen for drugs that block or open HERG Kþ channels, we
employ a fluorescence-based membrane potential assay (Molecular
Devices). HEK293 cells stably expressing recombinant human
HERG (provided by Dr. J. Overholt, Case Western Reserve University,
Cleveland, OH; cells originally from Drs. A. Brown and E. Ficker,
The title compound 36 (0.065 g, 66%) was prepared according to
general procedure E from (4-((4-(morpholine-4-carbonyl)piper-
idin-1-yl)sulfonyl)phenyl)boronic acid (0.091 g, 0.24 mmol) as a
white solid. 1H NMR (400 MHz, CD3OD þ CDCl3)
d 9.21 (s,1H), 8.37e
8.28 (m, 2H), 8.24e8.16 (m, 2H), 5.04e4.94 (m, 1H), 4.60e4.38 (m,
2H), 4.23e4.07 (m, 3H), 3.99e3.94 (m, 3H), 3.93e3.82 (m, 4H),
3.78e3.68 (m, 2H), 2.91e2.82 (m, 2H), 2.76 (ddd, J ¼ 10.4, 7.2,
3.9 Hz, 1H), 2.61e2.46 (m, 4H), 2.46e2.36 (m, 2H), 2.30e2.17 (m,
2H), 2.15e2.06 (m, 2H), 2.06e1.98 (m, 2H), 1.98e1.86 (m, 2H), 1.85e
1.74 (m, 2H), 1.33 (t, J ¼ 7.4 Hz, 3H); LCeMS: 96% purity,
tR ¼ 5.468 min; MS m/z 626.3 [M þ 1]þ.
MetroHealth Medical Center, Cleveland, OH) are seeded in poly-
lysine-coated 96-well plates (45,000 cells/100 L DMEM supple-
mented with 10% fetal bovine serum/well) one day prior to assay.
The next day, the medium is removed and replaced with 30 L/well
L-
m
m
of assay buffer containing membrane potential dye (Molecular
Devices) (the lyophilized dye is reconstituted with 15 mL of assay
buffer). After a 15 min incubation at 37 ꢀC, 30
lutions of terfenadine (a known HERG blocker used as a reference
m
L/well of 2ꢂ di-
5.3. Microfluidic capillary electrophoresis (MCE) assay
compound) or test compound (final assay concentration ranging
Activity assays were performed in a 384 well, polypropylene
from 0.1 nM to 10
assayed in triplicate). Baseline fluorescence (excitation 530 nM,
emission 565 nM) is measured over 15 min, then 140 L of depo-
mM) are added to the cells (each concentration
microplate with a final volume of 50
containing 10 mM MgCl2, 1.0 mM DTT, 0.01% Triton X-100, 0.1%
bovine serum albumin (BSA), using 1.0 M fluorescent substrate
(Table 3) and ATP at Km for each enzyme (Table 3). All reactions
were terminated by addition of 20 L of 70 mM EDTA. Phosphor-
mL in 50 mM Hepes, pH 7.4
m
m
larization solution (143 mM KCl in distilled water) containing test
or reference compound (1ꢂ) is added to the cells and fluorescence
is recorded for 3 min. Raw fluorescence data are analyzed by
GraphPad Prism 4.0.
m
ylated and unphosphorylated substrate peptides (Table 3) were
separated following a 180 min incubation on a LabChip EZ Reader
equipped with a 12-sipper chip in separation buffer supplemented
with 1 ꢂ CR-8 and analyzed using EZ Reader software.
5.6. Cell based assays for Mer kinase inhibition
697, BT-12, A549, or Colo699 cells were cultured in the presence
of UNC1062 or vehicle only for 1.0 h (697, BT-12) or 72 h (A549,
Colo699). Pervanadate solution was prepared fresh by combining
20 mM sodium orthovanadate in 0.9ꢂ PBS in a 1:1 ratio with 0.3%
(w/w) hydrogen peroxide in PBS for 15e20 min at room tempera-
5.4. Morrison Ki measurement
Inhibition of Mer kinase by UNC1062 was measured in the MCE
assay using the Morrison tight-binding method [17b]. Reactions
were performed in a 384 well, polypropylene microplate in a final
ture. Cultures were treated with 120
mM pervanadate prior to
volume of 80
1.0 mM DTT, 0.01% Triton X-100, 0.1% bovine serum albumin (BSA),
and 5.0 M ATP. Compounds were tested in 20 dilution (1.5 fold)
dose curves spanning a concentration range of 60e0.027 nM,
2.0 nM Mer,1.0 M peptide substrate, and 5.0 M ATP, final. To 40
of enzyme (4 nM Mer), 8.0 L of compound in 10% DMSO was added
and allowed to incubate for 10 min followed by addition of 32 L of
mL in 50 mM Hepes, pH 7.4 containing 10 mM MgCl2,
collection for preparation of whole cell lysates, immunoprecipita-
tion of Mer, and analysis by western blot.
m
697, A549, and Colo699 cells were treated with pervanadate for
3.0, 5.0, and 1.0 min respectively. Cell lysates were prepared in
50 mM HEPES pH 7.5, 150 mM NaCl, 10 mM EDTA, 10% glycerol, and
1% Triton X-100, supplemented with protease inhibitors (Roche
Molecular Biochemicals, #11836153001). Mer protein was immu-
noprecipitated with a monoclonal anti-Mer antibody (R&D Sys-
tems, #MAB8912) and Protein G agarose beads (InVitrogen).
Phospho-Mer was detected by western blot using a polyclonal
anti-phospho-Mer antibody raised against a peptide derived from
the tri-phosphorylated activation loop of Mer. Nitrocellulose
membranes were stripped and total Mer protein was detected us-
ing a second anti-Mer antibody (Epitomics Inc., #1633-1). For 697
cells, relative phosphorylated and total Mer protein levels were
determined by densitometry and IC50 values were determined by
non-linear regression.
m
m
mL
m
m
substrate mix (containing 2.5
ATP).
mM peptide substrate and 12.5 mM
Kinetic reads were taken from 7.0 to 209 min, 12 reads total, on
an EZ Reader, using upstream voltage ¼ ꢁ2250 V, downstream
voltage ¼ ꢁ500 V and pressure ¼ ꢁ1.0 psi (sip time 0.2 s, buffer
30 s). The steady-state velocity was analyzed by linear regression of
the peptide as percent-conversion/min, and a plot of nM compound
(UNC1062) vs. velocity was fit to the Morison equation using
GraphPad Prism 5.0, Y ¼ Vo*ð1 ꢁ ððððEt þ X þ ðKi*ð1 þ ðS=KmÞÞÞÞꢁ
ðððEt þ X þ ðK*ð1 þ ðS=KmÞÞÞÞ2Þ ꢁ 4*Et*XÞ0:5ÞÞ=ð2*EtÞÞÞ. The total
i
enzyme concentration (Et) was fixed to 0.855 nM as determined
Confluent BT-12 cultures were treated with pervanadate for
15 min. Cell lysates were prepared in 20 mM HEPES pH 7.5, 500 mM
NaCl, 5.0 mM EDTA, 10% glycerol, and 1% Triton X-100, supple-
Table 3
Assay conditions for MCE assays currently used.
mented with protease inhibitors (10
phenylmethylsulfonyl fluoride, and 20
phatase inhibitors (50 mM sodium fluoride and 1.0 mM sodium
orthovanadate) and Mer protein was immunoprecipitated using a
custom polyclonal rabbit anti-Mer antisera raised against a peptide
m
g/mL leupeptin, 10
mg/mL
Kinase
Peptide substrate
Kinase (nM)
ATP (uM)
mg/ml aprotinin) and phos-
Mer
Axl
Tyro
5-FAM-EFPIYDFLPAKKK-CONH2
5-FAM-KKKKEEIYFFF-CONH2
5-FAM-EFPIYDFLPAKKK-CONH2
2.0
120
10
5.0
65
21

92
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containing 0.5ꢂ RPMI medium, 7.5% FBS, and the indicated concen-
trations of UNC1062 or DMSO vehicle only and overlaid with 0.5 mL
of 1ꢂ RPMI medium containing 15% FBS and UNC1062 or DMSO
vehicle only. Medium and UNC1062 or vehicle were refreshed 2
times per week. Colonies were stained with thiazolyl blue tetrazo-
lium bromide (Sigma Aldrich, #M5655) and counted after 3 weeks.
A549 or Colo699 cells (15,000 cells) were cultured in 1.5 mL of
0.35% soft agar containing 1ꢂ RPMI medium and 10% FBS and
overlaid with 2.0 mL of 1ꢂ RPMI medium containing 10% FBS and
the indicated concentrations of UNC1062 or DMSO vehicle only.
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[8] (a) J. Schlegel, M.J. Sambade, S. Sather, S.J. Moschos, A.-C. Tan, A. Winges,
D. DeRyckere, C.C. Carson, D.G. Trembath, J.J. Tentler, S.G. Eckhardt, P.-F. Kuan,
R.L. Hamilton, L.M. Duncan, C.R. Miller, N. Nikolaishvili-Feinberg, B.R. Midkiff,
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HERG data was generously provided by the National Institute of
Mental Health’s Psychoactive Drug Screening Program, Contract #
HHSN-271-2008-025C(NIMH PDSP). The NIMH PDSP is directed by
Bryan L. Roth MD, PhD at the University of North Carolina at Chapel
Hill and Project Officer Jamie Driscol at NIMH, Bethesda MD, USA.
We also thank Yingqiu Zhou for her help with MCE assays. This
work was supported by the University of North Carolina Cancer
Research Fund and Federal Funds from the National Cancer
Institute, National Institute of Health, under Contract No.
HHSN261200800001E. The content of this publication does not
necessarily reflect the views or policies of the Department of Health
and Human Services, nor does mention of trade names, commercial
products, or organizations imply endorsement by the U.S.
Government.
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Supplementary data
(c) S. Howard, V. Berdini, J.A. Boulstridge, M.G. Carr, D.M. Cross, J. Curry,
L.A. Devine, T.R. Early, L. Fazal, A.L. Gill, M. Heathcote, S. Maman,
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Supplementary data associated with this article can be found in
the online version, at http://dx.doi.org/10.1016/j.ejmech.2013.03.
035. These data include MOL files and InChiKeys of the most
important compounds described in this article.
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