Synthesis and optimization of substituted furo[2,3-d]-pyrimidin-4-amines and 7H-pyrrolo[2,3-d]pyrimidin-4-amines as ACK1 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2012.08.020

Two classes of ACK1 inhibitors, 4,5,6-trisubstituted furo[2,3-d]pyrimidin4- amines and 4,5,6-trisubstituted 7H-pyrrolo[2,3-d]pyrimidin-4-amines, were discovered and evaluated as ACK1 inhibitors. Further structural refinement led to the identification of potent and selective dithiolane inhibitor 37.

Full text of DOI:10.1016/j.bmcl.2012.08.020

Bioorganic & Medicinal Chemistry Letters 22 (2012) 6212–6217
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Bioorganic & Medicinal Chemistry Letters
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Synthesis and optimization of substituted furo[2,3-d]-pyrimidin-4-amines and
7H-pyrrolo[2,3-d]pyrimidin-4-amines as ACK1 inhibitors
XianYun Jiao a, , David J. Kopecky a, , JinSong Liu b, JinQian Liu a, Juan C. Jaen a, Mario G. Cardozo b,
Rajiv Sharma a, Nigel Walker b, Holger Wesche c, Shyun Li c, Ellyn Farrelly d, Shou-Hua Xiao d,
Zhulun Wang b, Frank Kayser a
⇑
⇑
a Department of Medicinal Chemistry, Amgen Inc., 1120 Veterans Blvd., South San Francisco, CA 94080, USA
b Department of Molecular Structure & Characterization, Amgen Inc., 1120 Veterans Blvd., South San Francisco, CA 94080, USA
c Department of Oncology, Amgen Inc., 1120 Veterans Blvd., South San Francisco, CA 94080, USA
d Department of Lead Discovery, Amgen Inc., 1120 Veterans Blvd., South San Francisco, CA 94080, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Two classes of ACK1 inhibitors, 4,5,6-trisubstituted furo[2,3-d]pyrimidin4-amines and 4,5,6-trisubsti-
tuted 7H-pyrrolo[2,3-d]pyrimidin-4-amines, were discovered and evaluated as ACK1 inhibitors. Further
structural refinement led to the identification of potent and selective dithiolane inhibitor 37.
Ó 2012 Published by Elsevier Ltd.
Received 10 June 2012
Revised 25 July 2012
Accepted 1 August 2012
Available online 10 August 2012
Keywords:
ACK1
Cancer
Tyrosine kinase inhibitor
Furo[2,3-d]-pyrimidin-4-amine
7H-Pyrrolo[2,3-d]pyrimidin-4-amine
ACK1 (Activated Cdc42-associated tyrosine Kinase 1) is a non-
receptor tyrosine kinase that has attracted substantial interest as
a target for drug discovery research in recent years.1 Amplification
of the ACK1 gene in primary tumors has been reported to correlate
with poor prognosis.2 The expression of activated ACK1 in LNCaP
cells dramatically promotes prostate tumorigenesis.3a,b The Ack1
inhibitor inhibits androgen receptor transcription activity.3c Fur-
thermore, ACK1 is a mediator of EGF signals to Rho family GTP-
binding proteins through phosphorylation and activation of guan-
ine nucleotide exchange factors (GEFs) such as Dbl.4 These findings
suggest that ACK1 is a potential target for developing anti-cancer
therapeutics. The crystal structures of the human ACK1 kinase do-
mains in both the unphosphorylated and phosphorylated states
have been solved.5 In this Letter, the synthesis and SAR of two clas-
ses of ACK1 inhibitors are discussed.
Early structure-activity relationship (SAR) work was performed
upon this promising initial hit and revealed that appropriate N-
substitution could significantly enhance ACK1 inhibition levels. In-
deed, this effort led to the identification of potent (S)-N-(tetrahy-
drofuran-2-yl)methyl) furanopyrimidine
2 (ACK1 Ki = 0.01 lM,
Fig. 1). Subsequent SAR studies addressed the modification of other
regions of the lead structure as well as further refinement of the N-
substituent and are described below.
After the discovery of potent furanopyrimidine 26, we initially
sought to assess the viability of related core structures in order
to expand upon our available chemical space. We evaluated these
analogs in both ACK1 biochemical and cellular autophosphoryla-
tion assays.7 To this end, a number of new bicyclic structures were
A high-throughput small molecule ACK1 biochemical inhibition
screen was performed in-house and led to the identification of
1 lM inhibitor furanopyrimidine 1 (Fig. 1). Further binding studies
NH2
N
NH
N
early SAR
O
N
N
found compound 1 to be both ATP-competitive and reversible.
O
O
1
2
ACK1 Ki = 1 µM
ACK1 Ki = 0.01 µM
⇑
Corresponding authors. Tel.: +1 650 244 2103; fax: +1 650 837 9369 (X.Y.J.);
tel.: +1 650 244 2146; fax: +1 650 837 9369 (D.J.K.).
E-mail addresses: xjiao@amgen.com (X. Jiao), dkopecky@amgen.com
(D.J. Kopecky).
Figure 1. Initial HTS hit 1 and subsequent derivative 2 resulting from amino group
substitution.
0960-894X/$ - see front matter Ó 2012 Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.bmcl.2012.08.020

X. Jiao et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6212–6217
6213
NH
N
NH
NH
N
O
O
O
N
N
N
N
N
O
S
N
2
3
4
µ
ACK1 Ki = 0.01
ACK1 Cell IC50 = 1.2
M
µ
ACK1 Ki >10 µM
ACK1 Ki = 7.3 µM
M
NH
O
NH
NH
N
O
O
N
N
N
O
N
N
N
N
H
5
6
7
ACK1 Ki = 1.8 µM
ACK1 Ki = 0.07 µM
ACK1 Ki = 0.006 µM
µ
µ
ACK1 Cell IC50 = 5.6
M
ACK1 Cell IC50 = 0.62
M
Figure 2. Various furanopyrimidine core changes that were tested. The biochemical and cellular potencies of pyrrolopyrimidine 7 were found to be similar to those of
furanopyrimidine 2.
Table 1
Table 1 (continued)
ACK1 inhibition for (S)-5,6-diphenyl-N-((tetrahydrofuran-2-yl)methyl)furo[2,3-
d]pyrimidin-4-amines and (S)-5,6-diphenyl-N-((tetrahydrofuran-2-yl)methyl)-7H-
pyrrolo[2,3-d]pyrimidin-4-amines with selected 6-aryl substituents R
Compound
X
R
ACKi Ki (
l (l
M)a ACKi Cell IC50 M)b
O
N
27
NH
0.006
0.02
NH
O
O
N
R
a
Values are means of at least two experiments.
Average of at least two IC50 values.
b
X
N
Compound
X
R
ACKi Ki (
l
M)a ACKi Cell IC50 M)b
(l
synthesized and evaluated (Fig. 2). Changes that exhibited little or
no ACK1 inhibition include thienopyrimidine 3, imidazopyrimidine
4, and imidazotriazine 5. Furanopyridine 6 proved to be a potent
2
7
8
9
10
11
12
13
14
15
16
O
NH
O
NH OMe
O
H
H
OMe
0.01
1.2
0.006
0.005
0.006
0.008
0.007
0.002
0.004
0.002
0.005
0.006
0.62
0.55
0.35
0.25
0.10
0.09
0.30
0.04
0.03
0.02
ACK1 inhibitor in vitro (ACK1 Ki = 0.07 lM, Fig. 2) but showed a
SO2Me
significant erosion in cellular activity relative to furanopyrimidine
2. Gratifyingly, it was discovered that pyrrolopyrimidine 7 was
roughly equipotent to furanopyridine 2 in both biochemical and
cellular assays, and thus represented a second core scaffold for fur-
ther SAR exploration.
NH SO2Me
NH SO2NMe2
O
SO2NHMe
NH SO2NHMe
O(CH2)2NMe2
O
NH O(CH2)2NMe2
A large number of substituents were introduced at the para-po-
sition of the 6-phenyl ring for both the furanopyrimidine 2 and
pyrrolopyrimidine 7 lead structures (Table 1). While methoxy
and methanesulfonyl groups were tolerated in both series (8–11),
significant improvements in both biochemical and cellular ACK1
inhibition were observed for more polar sulfonamides attached
to the pyrrolopyrimidine core structure (12 and 14, ACK1
O
O
17
O
0.004
0.16
O
N
18
19
NH
NH
0.006
0.006
0.08
0.06
O
O
O
N
N
N
Ki = 0.002 lM and ACK1 Cell IC50 <0.10 lM in both cases). Building
upon this finding, a series of polar 2-aminoethoxy groups was
examined in the hope of improving cellular activity (15–21). It
was found that a 2-dimethylaminoexthoxy moiety induced very
high levels of ACK1 inhibition in enzymatic and cellular assays
for both core structures (15 and 16, ACK1 Cell IC50 <0.03 lM in
both cases). Moreover, the attachment of a 2-pyrrolidinonylethoxy
group to the pyrrolopyrimidine scaffold was highly favorable to
cellular ACK1 inhibition (21, ACK1 Cell IC50 = 0.02 lM). In addition,
a number of amides were examined (22–27), and several were
shown to be quite potent in the pyrrolopyrimidine series (23, 25
20
O
0.004
0.004
0.23
O
O
O
N
21
NH
O
0.02
22
23
24
25
C(O)NMe2
0.01
0.18
0.02
2.05
0.04
NH C(O)NMe2
C(O)NHMe
0.002
0.013
0.003
O
NH C(O)NHMe
and 27, ACK1 Cell IC50 = 0.02–0.04 lM).
O
Table 2 summarizes the results from a broad survey of C-5 mod-
ifications of the pyrrolopyrimidine scaffold. ACK1 inhibition was
diminished substantially when the C-5 phenyl moiety was re-
placed with a cyclopropyl group (30), while substituents with a
N
26
O
0.006
0.19
O

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X. Jiao et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6212–6217
Table 2
Table 3
ACK1 inhibition for (S)-6-ethylphenyoxy-N-((tetrahydrofuran-2-yl)methyl)-7H-pyr-
rolo[2,3-d]pyrimidin-4-amine with selected 5-position substituents R1 and 6-eth-
oxyphenyl substituents R2
ACK1 inhibition for 6-ethylphenyloxy-5-phenyl-7H-pyrrolo[2,3-d]pyrimidin-4-
amines and 6-ethylphenoxy-5-phenyl furo[2,3-d]pyrimidin-4-amines with selected
N-substituents R1 and 6-ethoxyphenyl substituents R2
NH
N
R1
R2
R1
O
NH
R2
N
O
N
N
O
H
X
N
Compound
R1
R2
ACKi Ki
ACKi Cell
(l
M)a
IC50
(l
M)b
Compound
X
R1
R2
ACKi Ki
M)a
ACKi Cell
(l
IC50 (l
M)b
16
19
Ph
Ph
NMe2
0.006
0.006
0.02
N
0.06
0.50
15
16
O
NMe2
NMe2
0.005
0.006
0.03
O
28
NMe2
0.01
NH
0.02
O
S
29
30
NMe2
NMe2
N
0.02
1.98
0.24
35
36
NH
O
NMe2
0.0003
0.0002
0.005
0.01
n.d.
S
S
N
31
32
0.04
0.01
0.39
0.16
NMe2
S
S
N
N
37
38
39
40
41
NH
O
0.0003
0.008
0.013
0.069
0.004
0.01
3.00
2.00
7.16
0.55
OMe
S
N
N
NMe2
NMe2
NMe2
NMe2
33
34
0.02
0.16
0.04
S (rac.)
O
O
0.004
O
F
O
a
Values are means of at least two experiments.
Average of at least two IC50 values.
S
S
b
O
S
OH
similar ring size to phenyl were tolerated (28–29). This result may
be due to an increase in unfavorable steric interactions between
the C-5 and C-6 groups in the case of R1 = cyclopropyl since early
co-crystallographic studies suggested that the C-5 and C-6 aryl
substituents are orthogonally disposed, where the C-6 aryl ring
adopts a coplanar orientation relative to the pyrrolopyrimidine
core.8 Various electron-rich and electron-poor six-membered aryl
and pyridyl groups in this position were also investigated (31–
34), and one compound of this series, ortho-fluorophenyl analog
34, proved to be a potent ACK1 inhibitor.
42
43
44
45
46
NH
NH
NH
NH
NH
NMe2
NMe2
NMe2
NMe2
NMe2
0.03
0.02
0.04
0.05
0.05
0.41
0.42
0.46
0.78
1.30
(rac.)
OMe
(rac.)
OH
A wide range of aliphatic substituents R1 was tolerated on the
pyrimidine ring C-4 amino group (Table 3). In general, four and
five-membered rings with a methylene linker were well tolerated,
while the larger six-membered ring derivatives exhibited reduced
ACK1 inhibition. While a (S)-tetrahydrofuranylmethyl group was
reasonable starting point for both core structures (15–16), further
SAR identified the 1,3-dithiolanylmethyl group as the most potent
amino substituent discovered to date (35–37). Indeed, dithiolane
35 in particular is highly potent in the biochemical assay
(Ki = 0.3 nM) as well as the cellular assay (IC50 = 5 nM). The exqui-
site potency of 35 is consistent with molecular modeling results;
while the C3-methylene of the (S)-tetrahydrofuranylmethyl group
was postulated to sterically clash with the L259 residue of ACK1, a
dithiolane moiety was predicted to avoid this unfavorable
interaction.
OMe
OMe
a
Values are means of at least two experiments.
Average of at least two IC50 values.
b
in rat hepatocytes relative to dimethylamine 35, unfortunately
both 35 and 37 displayed high iv clearances in Sprague–Dawley
rats (Table 4). Subsequent in vitro rat liver S9 metabolite identifi-
cation studies of 35 indicated that extensive oxidation of both
the dithiolane ring and the dimethylamino group occurred. In
terms of kinase selectivity, compound 37 was reasonably selective
(Ki = 0.3 nM) for ACK1 relative to the following related kinases: LCK
Since pyrrolopyrimidine dithiolanes 35 and 37 both displayed
particularly excellent levels of ACK1 inhibition, these analogs were
viewed as potential candidates for further investigation in tumor
xenograft experiments. While in vitro metabolic studies indicated
that pyrrolidine 37 was predicted to be significantly more stable

X. Jiao et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6212–6217
6215
Table 4
R2
NH
N
NH2
NH2
N
R1
R1
Sprague–Dawley rat pharmacokinetic parameters and predicted rat hepatocyte
I
clearances of pyrrolopyrimidine 35 and 37 (1 mg/kg iv dose)a
N
N
a
b, c
N
SiEt3
SiEt3
Compound
Cl (L/h⁄kg)
Vss (L/kg)
MRT (h)
Predicted Cl in
rat hepatocytes
N
H
48
N
H
N
NH2
(l
L/min⁄10À6 cells)
47
49
35
37
7.5
6.8
26.6
15.6
3.6
2.3
2.5
0.16
d, e
a
Three animals were used per study.
R2
NH
N
R1
N
R3
N
H
E206
T205
7, 9 11-12 14, 16
,
,
,
L207
18-19 21, 23 25,
, ,
27 28 34 42 46
,
-
,
-
A208
Scheme 1. Reagents and conditions: (a) 1-R1-2-(triethylsilyl)acetylene (2.5 equiv),
Pd(dppf)Cl2 (0.1 equiv), Na2CO3 (2 equiv), LiCl, DMF, 95 °C; (b) R2CO2H, EDC, HOBT,
DMAP (cat), DMF; (c) LiAlH4, THF, 0–50 °C, 2 h; (d) NIS, DMF, rt to 45 °C; (e)
R3B(OH)2 (1.5 equiv), Pd(dppf)Cl2 (0.1 equiv), 2 M aq Na2CO3 (2.5 equiv), LiCl
(3 equiv), 1:1 toluene/EtOH, 80 °C.
G269
P209
to account for the high levels of biochemical and cellular inhibition
observed for 35.9 The structure also shows that the 2-dim-
ethylaminoexthoxy group is exposed to solvent.
The formation of indole-type derivatives by the intramolecular
palladium-catalyzed Heck reaction has reported.10 Figure 4 shows
our general route used to access the 4-aminopyrrolopyrimidine
core structure from 4,6-diamino-5-iodopyrimidine (47)11 via intra-
molecular Heck chemistry. Indeed, the coupling of an appropri-
ately-substituted 2-triethylsilylalkyne and organopalladium
complex 47a (generated by an oxidative addition of compound
D134
Figure 3. X-ray co-crystal structure of compound 35 bound to human ACK1 kinase
domain at 2.5 Å resolution. RCSB file name (4EWH).
47 with a palladium (0) species) gives the p-alkyne-r-organo-pal-
(Ki = 138 nM), JAK3 (Ki = 6.5 nM), KDR (Ki = 380 nM), and TIE2
(Ki = 200 nM).
ladium (0) complex 47b, which transforms into carbopalladium
adduct 47c. Intermediate 47c subsequently forms a nitrogen-con-
taining palladacycle 47d via iodide displacement from the palla-
dium by one of the pendant nitrogen nucleophiles. The
palladacycle 47d then converts to the 6-triethylsilyl-4-aminopyr-
rolopyrimidine product 47e by reductive elimination.12 This meth-
od is highly regioselective to form 5-subsituted-6-(triethylsilyl)-
7H-pyrrolo[2,3-d]pyrimidin-4-amine of general structure 47e due
to coordination effects in the carbo-palladation step.10,13 Our opti-
mized protocol is as follows: 4,6-diamino-5-iodopyrimidine is
treated with a triethylsilylalkyne (2.5 equiv), sodium carbonate
(2.0 equiv), LiCl (1 equiv) and 10 mol % of Pd(dppf)Cl2 in DMF and
then heated at 95 °C overnight. The yields of the reaction ranged
from good to excellent.
The X-ray co-crystal structure of dithiolane 35 bound to the hu-
man ACK1 catalytic kinase domain was obtained at 2.5 Å resolution
(Fig. 3). Compound 35 is anchored into the ATP-binding site of the
ACK1 protein via two key protein-ligand hinge interactions: the
pyrrolopyrimidine NH acts as a hydrogen bond donor to the car-
bonyl oxygen of A208, while the pyrimidine N-1 accepts a hydro-
gen bond from the backbone amide NH of L207 (shown in purple
dashed lines). The rest of the compound makes numerous van
der Waals interactions with the protein, including two close van
der Waals contacts between the sulfur atoms of the dithiolane
moiety and the carbonyl oxygen atoms of D134 and G269 (shown
in grey dashed lines). These dithiolane interactions are postulated
R
NH2
NH2
R
NH2
N
NH2
N
I
I
PdI
Pd
SiEt3
I
N
N
N
N
N
SiEt3
Pd
N
NH2
N
NH2
NH2
NH2
47
47a
47b
47c
NH2
NH2
N
R
R
SiEt3
N
SiEt3
Pd
N
N
N
H
H
47e
47d
Figure 4. Proposed intermediates in the intramolecular palladium-catalyzed formation of 4-aminopyrrolopyrimidines.

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X. Jiao et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6212–6217
Ph
OH
N
NC
H2N
OMe
Ph
Br
a
b
N
OMe
Ph
O
OMe
O
O
50
51
52
c, d
R1
NH
Cl
Ph
Ph
N
N
O
e
OH
OH
O
O
N
N
f
53
54
R1
NH
N
Ph
R2
N
O
15, 17, 20, 36, 38-40
Scheme 2. Reagents and conditions: (a) malononitrile, Et3N; (b) HCO2H, reflux; (c) POCl3, 105 °C; (d) BBr3, DCM; (e) R1CH2NH2, n-BuOH; (f) Cs2CO3, R2Br, DMF.
MeO
MeO
S
NH
N
NH
N
S
a
N
N
n=1,
R
R
X
X
35-37, 41
55 (X = O, NH)
Scheme 3. Reagents and conditions: (a) 1,2-ethanedithiol or 1,3-propanedithiol, p-TsOH, toluene, reflux.
Our general route to ACK1 pyrrolopyrimidine inhibitors is
References and notes
shown in Scheme 1. After palladium-catalyzed pyrrolopyrimidine
formation as described above, N-monoalkylation of the 4-amino
group of amine 48 was achieved by stepwise acylation and reduc-
tion to give the N-alkylated product 49. Subsequent transforma-
tion of the C-6 triethylsilyl group of 49 into the corresponding
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iodide followed by
a Suzuki coupling with an appropriate
arylboronic acid provided the pyrrolopyrimidine analogs listed
in Tables 1–3.
Many of the furanopyrimidine analogs with alkoxy substituents
attached to the 4-position of the 6-phenyl ring that are listed in Ta-
ble 1 and Table 3 were prepared by the sequence shown in
Scheme 2. Specifically, condensation of acetophenone 5014 with
malononitrile afforded furan 51 which was further transformed
into furanopyrimidine 52 by exposure to refluxing formic acid.
Subsequent chlorination, demethylation and N-alkylation with an
appropriate amine provided amine 54, which was then O-alkylated
with an appropriate alkyl bromide in the presence of cesium car-
bonate to generate the target molecules. Note that 1,3-dithiolane
derivatives 35–37 and 41 were generated from 1,1-dimethylacetal
intermediate 55 by treatment with either 1,2-ethanedithiol or 1,3-
propanedithiol and p-toluenesulfonic acid in refluxing toluene
6a,c
(Scheme 3).
In conclusion, two novel series of furo[2,3-d]pyrimidin4-amines
and 7H-pyrrolo[2,3-d]pyrimidin-4-amines which exhibit potent
in vitro inhibitor activity against ACK1 have been identified and
evaluated. 1,3-Dithiolane-substituted pyrrolopyrimidine 37 dis-
plays excellent ACK1 cellular inhibition, good kinase selectivity,
and a suitable in vitro metabolic profile. Unfortunately, the phar-
macokinetic profile of 37 was poor and prevented this inhibitor
from being further evaluated in tumor xenograft studies.
7. (a) Xiao, S.-H.; Farrelly, E.; Anzola, J.; Crawford, D.; Jiao, X.-Y.; Liu, J.-Q.; Ayres,
M.; Li, S.; Huang, L.; Sharma, R.; Kayser, F.; Wesche, H.; Young, S. W. Anal.

X. Jiao et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6212–6217
6217
Biochem. 2007, 367, 179–189 (b) ACK1 enzymatic assay Ki determination: The
assay utilizes a protein expressed in baculovirus infected Hi-5 cells (a fusion of
an N-terminal (His)6 Tag with amino acids 117 to 489 of ACK1) purified by
affinity chromatography on a Ni-NTA column. The substrate for the reaction is
ACK1 itself (autophosphorylation) and poly-Glutamic acid-Tyrosine (PGT (4:1),
Sigma catalog #PO275). The PGT is coated to Nunc 96 well plates at 80 mg/mL
overnight at 4 °C. The morning after coating, the plates are washed twice, and
80 mL reaction buffer (10 mM Hepes, pH 7.6; 20 mM MgCl2; 75 mM NaCl,
0.125% TWEEN20 (polyoxyethylene sorbitan monolaurate); 1 mM DTT) with
lines are seeded in 96 well tissue culture plates (BD Falcon) at a density of 2 to
4 Â 104 in DMEM/F12 (C8) or DMEM (HeLa) with 0.125% FCS in the presence of
ACK1 inhibitors (final DMSO concentration is 0.5%, all tissue culture media are
from Cellgro). After 20 to 24 h incubation at 37 °C and 5% CO2, cell viability is
determined using the Cytotox One kit (Promega) according to the
manufacturer’s instructions.
8. Unpublished work.
9. Iwaoka, M.; Takemoto, S.; Tomoda, S. J. Am. Chem. Soc. 2002, 124, 10613.
10. Cacchi, S.; Fabrizi, G. Chem. Rev. 2005, 105, 2873. And references therein.
11. Lee, C.-H.; Jiang, M.; Cowart, M.; Gfesser, G.; Perner, R.; Kim, K.-H.; Gu, Y.-G.;
Williams, M.; Jarvis, M. F.; Kowaluk, E. A.; Stewart, A. O.; Bhagwat, S. S. J. Med.
Chem. 2001, 44, 2133.
5
l
M ATP are added to each well. Test compounds are added in 10
lL DMSO,
and the reaction is started by addition of 10 L kinase in assay buffer. The
l
reaction proceeds 2 h at room temperature. Next, the plates are washed four
times, and the level of tyrosine phosphorylation in a given well is quantified by
standard ELISA assay utilizing a phosphotyrosine antibody (PY20, Pierce). (c)
ACK1 cell based assay IC50 determination: The assay is based on the
dependence of certain transformed cell lines (e.g. C8 cells, a Ras and E1A
transformed fibroblast line) on ACK1 for survival under low serum conditions,
whereas other cell lines (e.g. HeLa) do not. This dependency was confirmed
utilizing ACK1 specific siRNAs. For this assay, test (C8) and control (HeLa) cell
12. (a) Larock, R. C.; Yum, E. K.; Refvik, M. D. J. Org. Chem. 1998, 63, 7652; (b)
Larock, R. C.; Yum, E. K. J. Am. Chem. Soc. 1991, 113, 6689.
13. (a) Arcadi, A.; Cacchi, S.; Marinelli, F. Tetrahedron Lett. 1986, 27, 6397; (b) Kang,
S. K.; Park, S. S.; Kim, S. S.; Choi, J. K.; Yum, E. K. Tetrahedron Lett. 1999, 40, 4379.
14. Shridhar, D. R.; Ram, B.; Sarma, C. R.; Saxena, N. K. Indian J. Chem., Sect B: Org.
Chem. Med. Chem. 1982, 21, 860. Made by bromination of 1-(4-
methoxyphenyl)-2-phenylethanone.