Discovery of 3H-benzo[4,5]thieno[3,2-d]pyrimidin-4-ones as potent, highly selective, and orally bioavailable inhibitors of the human protooncogene proviral insertion site in moloney murine leukemia virus (PIM) kinases

Article abstract of DOI:10.1021/jm900943h

Pim-1, Pim-2, and Pim-3 are a family of serine/threonine kinases which have been found to be overexpressed in a variety of hematopoietic malignancies and solid tumors. Benzothienopyrimidinones were discovered as a novel class of Pim inhibitors that potent

Full text of DOI:10.1021/jm900943h

J. Med. Chem. 2009, 52, 6621–6636 6621
DOI: 10.1021/jm900943h
Discovery of 3H-Benzo[4,5]thieno[3,2-d]pyrimidin-4-ones as Potent, Highly Selective, and Orally
Bioavailable Inhibitors of the Human Protooncogene Proviral Insertion Site in Moloney Murine
Leukemia Virus (PIM) Kinases
Zhi-Fu Tao,* Lisa A. Hasvold, Joel D. Leverson, Edward K. Han, Ran Guan, Eric F. Johnson, Vincent S. Stoll,
Kent D. Stewart, Geoff Stamper, Nirupama Soni, Jennifer J. Bouska, Yan Luo, Thomas J. Sowin, Nan-Horng Lin,
Vincent S. Giranda, Saul H. Rosenberg, and Thomas D. Penning
Cancer Research, Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, Illinois 60064
Received June 26, 2009
Pim-1, Pim-2, and Pim-3 are a family of serine/threonine kinases which have been found to be
overexpressed in a variety of hematopoietic malignancies and solid tumors. Benzothienopyrimidinones
were discovered as a novel class of Pim inhibitors that potently inhibit all three Pim kinases with
subnanomolar to low single-digit nanomolar Ki values and exhibit excellent selectivity against a panel of
diverse kinases. Protein crystal structures of the bound Pim-1 complexes of benzothienopyrimidinones 3b
(PDB code 3JYA), 6e (PDB code 3JYO), and 12b (PDB code 3JXW) were determined and used to guide
SAR studies. Multiple compounds exhibited potent antiproliferative activity in K562 and MV4-11 cells
with submicromolar EC50 values. For example, compound 14j inhibited the growth of K562 cells with an
EC50 value of 1.7 μM and showed Ki values of 2, 3, and 0.5 nM against Pim-1, Pim-2, and Pim-3,
respectively. These novel Pim kinase inhibitors efficiently interrupted the phosphorylation of Bad in both
K562 and LnCaP-Bad cell lines, indicating that their potent biological activities are mechanism-based.
The pharmacokinetics of 14j was studied in CD-1 mice and shown to exhibit bioavailability of 76% after
oral dosing. ADME profiling of 14j suggested a long half-life in both human and mouse liver microsomes,
good permeability, modest protein binding, and no CYP inhibition below 20 μM concentration.
Introduction
overexpressed in human hepatoma cell lines and hepatocel-
lular carcinoma tissues and has been shown to promote
the growth of Ewing’s family tumor cell lines.20,21a,b More
recently, Pim-3 was found to be aberrantly expressed in
human colon and pancreatic cancers and to phosphorylate
Bad, thereby blocking Bad-mediated apoptosis.21c,d These
findings suggest that small molecule inhibitors of Pim kinases
may be of therapeutic value in the treatment of cancer.
The Pim kinases are a family of three serine/threonine
kinases, Pim-1, Pim-2, and Pim-3, which were first identified
in a series of retroviral insertional mutagenesis studies in
c-myc-induced murine lymphomas and named for the geno-
mic site, ProviralIntegration site of moloneyMurineleukemia
virus.1,2 The Pim kinases are favorable partners for myc-
induced tumorigenesis and can play functionally important
roles in this process.3-5 Pim-1/Pim-2/Pim-3 triple knock-
out mice are viable, but they are significantly smaller than
their wild-type counterparts, clearly due to reduced cell pro-
liferation.6 Pim kinases have been demonstrated to inhibit
apoptosis by inducing antiapoptotic Bcl-2 expression or
inactivating the pro-apoptotic protein Bada through phos-
phorylation.7-10 Pim-1 and Pim-2 have been found to be
overexpressed in solid tumors such as prostate cancer11-13
and a variety of human hematopoietic malignancies14-16 such
as leukemia and lymphoma. Pim kinases have been shown to
be involved in the growth, survival, perineural invasion, and
androgen independence of prostate cancer cells.17-19 Pim-3 is
Several classes of Pim inhibitors have been reported recently,
including ruthenium-containing organometallic complexes,22,23
bisindolylmaleimides,24 imidazo[1,2-b]pyridazines,25-27 pyri-
dones,28 flavonoids,29,30 benzoisoxazoles,31 and isoxazoloquino-
line-3,4(1H,9H)-diones.32 However, these molecules potently
inhibit only Pim-1, and the mechanism-based antitumor
activity of the majority of these reported Pim-1 inhibitors
remains to be described. Because all three Pim kinases have
been implicated in tumorigenesis, it may be necessary to
simultaneously target Pim-1, Pim-2, and Pim-3 to achieve
optimal cancer therapy efficacy.33-35 Pim triple knockout
mice are viable, suggesting that a favorable therapeutic win-
dow may be achieved for triple Pim-1, Pim-2, and Pim-3
inhibitors.6 Despite the high sequence conservation of the
active sites for Pim-1 and Pim-2, it has been reported that Pim-2
is intrinsically more difficult to target.25 During the pre-
paration of this article, 5-arylidene-2,4-thiazolidinediones36a
and cinnamic acids36b were reported to inhibit Pim-2. The
most potent Pim-2 inhibitors based on thiazolidinedione and
cinnamic acid have double-digit nanomolar IC50 values. The
mechanism-based in vitro antitumor activity of the latter
*To whom correspondence should be addressed. Phone: (847)-938-6772.
Fax: (847)-935-5165. E-mail: zhi-fu.tao@abbott.com.
a Abbreviations: APCI, atmospheric pressure chemical ionization; ATP,
adenosine-50-triphosphate; BAD, Bcl-2 antagonist of cell death; DMF,
N,N-dimethylformamide; DMSO, dimethyl sulfoxide; DTT, dithiothreitol;
EDTA, ethylenediaminetetraacetic acid; HEPES, N-(2-hydroxyethyl)-
piperazine-N0-2-ethanesulfonic acid; HTS, high-throughput screening;
Pim, proviral insertion site in moloney murine leukemia virus; SAR,
structure-activity relationship; THF, tetrahydrofuran.
r
2009 American Chemical Society
Published on Web 10/16/2009
pubs.acs.org/jmc

6622 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Tao et al.
Scheme 1a
a Reaction conditions: (a) 2-mercaptoacetate, aq NaOH, DMF, 0 °C, 95%; (b) formamide, 190 °C, 10 h, 13%; (c) HCl, 2-chloroacetonitrile, room
temperature, quantitative yield; (d) amine, DMF, room temperature; (e) 1,3-dihydroxylbenzene, K2CO3, DMF, 50 °C.
Scheme 2a
a Reaction conditions: (a) amines, biphenyl-2-yldi-tert-butylphosphine, Pd2(dba)3, NaOtBu, toluene, microwave 120 °C; (b) Pd(Ph3P)4, alkyne,
DMF, microwave 100 °C; (c) boronic acids, Pd(PPh3)2Cl2, microwave 160 °C, Na2CO3, DME/EtOH/H2O.
remains to be disclosed.36b To date, no potent Pim-3 inhibitor
has been reported. We herein report that tricyclic benzothie-
nopyrimidinones constitute a new class of Pim kinase inhibi-
tors. On the basis of a HTS hit, medicinal chemistry efforts led
to the identification of benzothienopyrimidinone-based Pim
inhibitors that not only inhibit all three Pim kinases at
subnanomolar to low single-digit nanomolar concentrations
but also exhibit excellent kinase selectivity. Interestingly, this
is the first time that a benzothienopyrimidinone scaffold has
been used for kinase inhibition, which is in contrast to the fact
that most other reported Pim inhibitors were developed based
on known kinase inhibitor templates.
further elaboration, being readily displaced by nucleophilic
amines and phenols. This simple alkylation expedited the
synthesis of amine derivatives 6 and ether analogue 7 and
thus greatly facilitated SAR studies.
Scheme 2 shows the synthesis of analogues derived from the
8-position of the phenyl ring. Compounds 8-11 were pre-
pared by using the same route as shown in Scheme 1. Suzuki
coupling of 8 with the corresponding boronic acid or boronic
ester readily provided compounds 12 and 14-16. Sonogashira
reaction between 8 and various alkynes afforded compounds
13. The amination of 8 was accomplished under Buchwald
conditions to provide 17. It is worth noting that all three Pd-
mediated couplings were performed under microwave heat-
ing, which greatly shortened the reaction times.
Chemistry
Displayed in Scheme 3 is the synthesis of analogues 18a and
18b derived from compound 14j. Advanced boronic esters 18c
and 18d were obtained from the commercially available
boronate 18e through direct alkylation in moderate yields.
Suzuki coupling under microwave conditions gave the desired
products 18a and 18b in low to moderate yields.
A general route for the synthesis of the 3H-benzo-
[4,5]thieno[3,2-d]pyrimidin-4-ones is shown in Scheme 1.37
Fluorobenzonitriles 1 were condensed with methyl 2-mer-
captoacetate and the resulting intermediates spontaneously
cyclized to benzothiophene 2 under basic conditions in ex-
cellent yield. Compound 3a was obtained through the con-
densation of 2 with formamide at high temperature. Com-
pound 3b was prepared by following a similar procedure used
for the synthesis of 3a. Compound 2 was reacted with
chloroacetonitrile in 4 N HCl in dioxane to quantitatively
provide the key intermediate 4, which readily underwent
cyclization under basic conditions to produce 5. The chloro-
methyl group at the 2-position of 5 was an excellent handle for
Results and Discussion
Initial Hit from High-Throughput Screening (HTS). Com-
pound 3b was identified as a HTS hit which showed Ki values of
63 and 160 nM against Pim-1 and Pim-2, respectively. However,
this compound also inhibited several other kinases (Table 5) such
as Clk4 (Ki = 90 nM) and Pbk (Ki = 316 nM) and did not show

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6623
Scheme 3a
a Reaction conditions: (a) K2CO3, DME, 20 h, 80 °C, 29-78%; (b) 8, Pd(PPh3)2Cl2, Na2CO3, DME/EtOH/H2O (7:2:3), microwave 150 °C, 10 min,
20-34%.
Figure 1. (a) X-ray crystal structure of compound 3b (orange carbons) bound to Pim-1 kinase. A hydrogen bond with Lys67 is indicated with a
black dotted line. (b,c) As in (a), with compounds 6e and 12b, respectively, with hydrogen bonds to Lys67 and Asn172/Asp186.
any cellular activity in multiple cell lines. The X-ray crystal-
lographic analysis of a 3b-Pim-1 complex indicated that the
compound binds to the ATP-binding pocket (Figure 1a). The
molecule is situated in the ATP-binding pocket in such a way
that the phenyl ring interacts with the proline-containing hinge
region, and the pyrimidone faces the pocket surrounded by
Lys67, Glu89, and Phe49. The 2-position of the pyrimidone ring
and the 8- and 9-positions of the phenyl ring are exposed to the
solvent-accessible region, indicating that these positions should
be able to accommodate a variety of substitutions to improve
potency and adjust physicochemical properties. Due to the ready
synthetic accessibility, we extensively investigated the SAR at the
2- and 8-positions based on 3a (vide infra).
Structure-Activity Relationship at the 2-Position of the
Pyrimidone Ring. A close examination of the X-ray structure
of the 3b-Pim-1 complex reveals that the 2-position of the
pyrimidone ring points toward the solvent-exposed region
through an area surrounded by Gly45, Phe49, Val52, Ile185,
Asp186, Asn172, etc. Table 1 shows the SAR results at the
2-position. Polar residues such as Asp186 and Asn172 pro-
vide opportunities for basic amines to make polar inter-
actions in this area. Therefore, we first introduced a series of
aliphatic amines at the 2-position (6a-6k). The methylene
linker between the amine nitrogen and the tricyclic core was
expected to enhance the hydrophobic interaction with the
surrounding Gly45, Phe49, Val52, and/or Ile185. Although
3a has slightly weaker affinity for Pim-1 and Pim-2 than the
initial HTS hit 3b, we chose to use 3a as the starting point for
this SAR exploration due to easier synthetic accessibility.
The introduction of a simple dimethylaminomethyl group
improved potency against Pim-1 and Pim-2 by 25- and
18-fold, respectively (6a vs 3a). However, larger substituents
such as piperidine (6b) and hexahydropyrimidine (6c) did not
improve the potency. The elaboration of the dimethylamino
group of 6a by removing a methyl group (6i) or lengthening
the methylene linker (6j, 6k) did not increase the potency
either. In contrast, the potency against Pim-2 was slightly
decreased by these modifications. To explore possible
H-bonding opportunities in this region, a hydroxyl group
was incorporated into the substituents to provide com-
pounds 6e-6h. Gratifyingly, the hydroxylpyrrolidyl ana-
logues (6e, 6f) fully maintained the affinity for both Pim-1
and Pim-2 relative to 6a, and the S configuration (6e) was
identified as the better of the two. Aromatic groups were next
introduced onto the 2-position (6l-6t). Substituents on the
aryl ring of this set of analogues allowed probing of poten-
tial protein interactions with the Gly-rich loop and/or the
side chains of Asp128/Glu171. Interestingly, the 30-hydroxyl-
phenyl analogue 6n showed subnamolar affinity to Pim-1,
which is a 397-fold increase relative to 3a. The Pim-2 inhibition

6624 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Table 1. SAR at the 2-Position of the Phenyl Ring
Tao et al.
of 6n was also improved 8-fold compared with 3a. Unfortu-
nately, other phenyl-containing analogues (6m, 6o-6t) were
significantly less potent than 6n. Extension of the linker
of 6n modestly improved the affinity for Pim-2 at the expense
of Pim-1 inhibition activity (6t vs 6n). Replacement of the
nitrogen linker with an oxygen considerably decreased the Pim-
1 inhibition activity and modestly reduced the Pim-2 affinity
(6n vs 7). Finally, it should be noted that all substitutions at the
2-position significantly enhanced the affinity for Pim-1 com-
pared with the parent compound 3a, while the potency against
Pim-2 varied significantly.
8-position points toward the solvent front and is situated in a
very hydrophobic area that is surrounded by Leu44, Ile104,
Val126, and Leu174. To enhance hydrophobic interactions
in this area, various lipophilic groups were installed at the
8-position (Table 2). Since the dimethylaminomethyl group
was identified as one of the best groups at the 2-position,
compound 9 was used as the starting point for the SAR
studies at the 8-position. Compared with compound 9, the
introduction of a simple halogen atom (6a, 8) or alkyl group
(10a, 10b, 11a, 11b) dramatically improved the affinity to
both Pim-1 and Pim-2. The vinyl analogue 12a is more
potent than its saturated counterpart 11a. The more hydro-
phobic cyclopropylvinyl group further increased the Pim-1
Structure-Activity Relationship at the 8-Position. The
X-ray structure of the 3b-Pim-1 complex indicates that the

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6625
Table 2. SAR at the 8-Position of the Pyrimidinone Ring
and Pim-2 inhibition potency (12b vs 12a). The acetylene
analogue 13a showed comparable activity to its alkene
counterpart 12a. However, the introduction of a polar
dimethylamino group (13b) onto the acetylene terminus
significantly decreased the Pim inhibition activity, consistent
with the hydrophobicity of this area. Extending the methyl-
ene linker pushed the polar amino group into the solvent-
exposed region and compound 13c thereby regained high
Pim inhibition activity. Compounds 14a-14q are phenyl-
substituted analogues. Again, simple phenyl substitution at
the 8-position considerably improved the Pim inhibition
activity (14a vs 9). Homologated analogue 14b was less
potent than 14a, but was still much more active than the
parent compound 9. The meta-substituted phenyl ana-
logues (14c-14f) maintained potency, and the 30-hydroxyl-
phenyl compound 14f stood out as the most active analogue.

6626 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Tao et al.
Table 3. Ki Values for Analogues Combining Optimal Substituents at
the 2- and 8-Positions
Table 4. Ki Values for Pim-1, Pim-2, and Pim-3 for Select Compounds
Pim-1 inhibition Pim-2 inhibition Pim-3 inhibition
(Ki, nM)
compounds
(Ki, nM)
(Ki, nM)
12b
14a
14d
14h
14i
1
4
2
8
0.2
0.3
0.7
0.9
1
3
13
11
21
3
5
5
14j
2
0.5
4
19a
20a
20b
20c
21
8
60
3
5
<0.1
0.3
0.4
43
3
10
2
0.7
43
41
Chemical structure of compound 21.
unfavorable contact between the second nitrogen and one of the
hydrophobic amino acid residues in the extended hinge. Finally,
selected saturated heterocycles were assessed (17a, 17b), and it
was found that the pyrrolidine ring is much more favorable at
the 8-position than the piperidine ring (17a vs 17b).
Compounds Combining the Optimal 2- and 8-Position
Substituents. Table 3 shows compounds that combine some
of the best substituents identified for the 2- and 8-positions.
Compounds 19a, 19b, and 19c all have the 30-hydroxyl-
phenylaminomethyl group at the 2-position and all showed
low single-digit nanomolar potency against Pim-1 but much
weaker affinity to Pim-2. The 30-hydroxypyrrolidinyl group
at the 2-position was then combined with various groups at
the 8-position to provide compounds 20a-20e. All of these
compounds potently inhibit both Pim-1 and Pim-2. The
results in Table 3 suggest that selective inhibitors against
Pim-1 and dual inhibitors against both Pim-1 and Pim-2
can be readily obtained by fine-tuning the substitution at the
2-position, which will provide very useful tool compounds
for elucidating the biological functions of Pim kinases.
Pim-3 Inhibition Activity. Pim-3 has been implicated in
both human hematopoietic and malignant solid tumors.20,21
However, among the family of three serine/threonine Pim
kinases, Pim-3 has received the least attention and no potent
Pim-3 kinase inhibitors have been reported in the literature.
We therefore set up a Pim-3 kinase enzymatic assay and
tested a set of compounds from the tricyclic benzothieno-
pyrimidinone series. As shown in Table 4, these compounds
exhibited very high affinity for Pim-3. Notably, these com-
pounds were usually more potent against Pim-3 than the other
two Pim family kinases. The only exception was compound 21,
which was equipotent against all three Pim kinases.
An ortho-methyl-substitution negatively impacted activity
(14g vs 14a). Compounds 14h-14n contain a para-substi-
tuted phenyl ring and showed relatively flat SAR. Molecular
modeling indicated that the para substituent was situated in
the solvent front, consistent with the flat SAR at this posi-
tion. Compounds 14j and 14k showed superior activity for
both Pim-1 and Pim-2, likely due to the solvation of the
hydroxyl and amino groups at the para position. Further
elaboration of the hydroxyl group of 14j with solubilizing
groups (18a and 18b) fully retained the Pim inhibition
activity and could potentially improve the physicochemical
properties. The addition of a fluorine atom ortho to the
hydroxyl group had little effect on inhibition of either Pim-1
or Pim-2 (14m vs 14j). The 30,50-disubstituted analogues fully
maintained the inhibition activity to Pim-1 but lost signifi-
cant Pim-2 activity (14p vs 14c, 14q vs 14d). Analogues
containing a five-membered aromatic ring (15a-15c) exhib-
ited single-digit nanomolar Ki values for both Pim-1 and
Pim-2. A methyl substituent on the thiophene ring slightly
decreased activity (15d vs 15a). The introduction of a nitrogen
atom into the phenyl ring (16a) resulted in an increase of the
affinity for both Pim-1 and Pim-2 (16a vs 14a). However, the
introduction of an additional nitrogen atom caused significant
loss of activity (16b vs 14a, 16b vs 16a), possibly due to an
X-ray Crystallographic Analysis and Molecular Modeling
of the Inhibitor-Pim 1 Complexes. In order to guide the SAR
studies and/or better understand the SAR results and bind-
ing mode, the X-ray crystallographic analysis of three com-
pounds (3b (PDB code 3JYA), 6e (PDB code 3JYO), and
12b (PDB code 3JXW)) complexed with Pim-1 have been
performed (Figure 1a-c).

Article
The group of three Pim kinases is unique within the
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6627
Table 5. Kinase Selectivity of Pim Kinase Inhibitors
protein kinase family because of the presence of a proline
residue (Pro123 in Pim-1) in the hinge, eliminating a key
hydrogen-bond-donating hinge interaction to ATP and
canonical kinase inhibitors.24,38 This leaves the backbone
carbonyl of Glu121 as the sole hydrogen-bond-accepting
partner available to ATP or inhibitors of Pim-1 kinase. There
is also an additional proline (Pro 125) and a valine (Val 126)
in the extended hinge, which provides a larger hydrophobic
pocket for binding inhibitors. As shown in Figure 1a, the
crystal structure of the screening hit, 3b, exploits this novel
hinge arrangement by placing a ring with two chlorine atoms
nestled into the hydrophobic patch created by Pro123, the
hydrophobic side chain of Arg122, Ile104 for one chlorine
atom and the second more solvent-exposed in the direction
of Val126. 3b does not make any hydrogen bonds directly
with the hinge. The sole hydrogen bond made by 3b directly
to the protein is between the inhibitor carbonyl and Lys67.
The remainder of the inhibitor is sandwiched in the binding
site in van der Waals contacts between Ile104, Ile185, and
Leu174 on the bottom and the glycine-rich loop on the top.
The binding mode of 6e (Figure 1b) has only a single
chlorine atom pointed toward the extended hinge and has a
slightly altered binding mode from 3b. This shifts the single
chlorine atom from the corresponding position observed in
3b such that the remaining chlorine is in van der Waals
contact with the hydrophobic portion of Arg122, Pro123,
Val126, and Leu174. The carbonyl and the 3-N of the
pyrimidone ring of 6e form bidentate H-bonds with Lys67.
The core of 6e slightly shifts in upon removal of one chlorine
relative to 3b. Extension of a basic amine off the core
demonstrates a significant improvement in potency, and this
is most likely a result of electrostatic interaction with
Asp186, although there is no formal hydrogen bonding
observed. Interestingly, there is not a significant potency
gain by adding a tertiary alcohol, which is observed in good
hydrogen bonding distance to Asn172. This may be because
this hydrogen bond is fairly solvent-exposed, thereby limit-
ing its value.
The binding mode of 12b (Figure 1c) is essentially identical
to the monochloro compound, 6e; however, 12b is signifi-
cantly more potent than 6e. The binding modes of 12b and 6e
are preserved relative to 3b because the cyclopropyl exten-
sion on 12b mimics and extends on the chlorine interactions
of 6e. The cyclopropyl extension contributes to a highly
potent molecule for Pim-1 because it increases the van der
Waals interactions with the extended hinge residues, Val126
in particular, as well as the hydrophobic portion of Arg122
and displacing water from the binding site. 12b makes the
same carbonyl and 3-N bidentate hydrogen bonds to Lys62
as 6e. 12b also extends a tertiary amine forming a hydrogen
bond with Asp186, which has been demonstrated to signifi-
cantly improve affinity. Combining all of these features,
exploitation of the hydrophobic extended hinge, hydrogen
bonding to Lys62, and electrostatic interaction with Asp186,
results in the most potent analogue in this series for Pim-1.
In Tables 1 and 2 are shown the potencies for both Pim-1
and Pim-2 inhibition. In general, the compounds are more
potent against Pim-1, but there are several analogues where
there is equivalent activity. Active site differences within the
Pim family are located within the hinge region at residues
Glu124 and Val126 of Pim-1, which are Leu, Ala in Pim-2
and Glu, Ala in Pim-3, respectively. The electrostatic differ-
ence between Glu and Leu at position 124 would influence
kinase
3b (Ki, μM)a
20c (Ki, μM)
Pim-1
Pim-2
Pim-3
BTK
0.1301
0.1642
0.0110
0.6365
>1
0.0007
0.0021
0.0004
>1
CDK2
CLK4
EMK
IGF1R
IRAK4
MSK1
Map4k4
P70S6K
PBK
>1
>1
0.0896
>1
>1
>1
0.9909
>1
>1
>1
0.7285
0.8124
0.7734
0.3161
>1
>1
>1
>1
>1
PKA
PRKX
Syk
Src
0.5847
0.3514
>1
>1
>1
>1
>1
TrkC
0.8283
a The inhibition constant (Ki) values are calculated from the Cheng-
Prusoff equation, Ki = IC50/(1 þ ([ATP]/Km).
desolvation during inhibitor binding, and the change between
Val and Ala at 126 could influence steric and van der Waals
contacts near the 8-position of the analogues. There are no
residue differences in the Pim family in the immediate protein
region of the 2-position of the analogues, so it is less clear why
there is potency variation from probing that region.
Kinase Selectivity Profiles of Representative Pim Inhibi-
tors. Two Pim kinase inhibitors (3b, 20c) were tested for
selectivity against other kinases. Compound 20c is among
the most potent compounds in the enzymatic assays against
all three Pim kinases. Both compounds displayed remark-
able selectivity against a panel of diverse kinases (Table 5).
The initial HTS hit 3b showed weak affinity for several other
kinases. As discussed above, the crystal structure of 3b
provides a rationale for the Pim activity by showing that
the chlorophenyl group occupies a hydrophobic region near
the hinge provided by a proline residue (Pro123 in Pim-1).
This hydrophobic interaction would not be present in non-
Pim kinases because they do not have proline at this position
and possess a H-bond donor from the backbone amide;the
common kinase “hinge” pattern described for many other
kinases. Therefore, a polarity clash with the chlorophenyl
group of 3b would make the observed binding mode in Pim-1
unfavored in other kinases. Alternatively, it is conceivable
that simple, unsubstituted benzothienopyrimidinones, such
as 3b, could flip in the active site and present a canonical H-
bond donor-acceptor interaction with the hinge using the
pyrimidinone ring system, thus permitting weak activity on
other kinases. However, the highly optimized analogues
reported here with large substituents at positions 2 and 8 of
the ring system would make this flipped binding mode less
likely with concomitant enhanced kinome selectivity. The
selectivity of lead compound 20c was much improved com-
pared with 3b, exhibiting no inhibitory activity toward the
other panel kinases even at the highest concentration tested
(10 μM). With over 500 kinases in total, it has been very
challenging for kinase-targeting drug discovery programs to
achieve high selectivity.39 Thus, the high kinase selectivity of
the lead compound 20c is truly impressive.
Cell-Based Assays of Select Pim Inhibitors. Select com-
pounds with potent Pim-1 enzymatic activity in the enzymatic
assay were further characterized for their antiproliferative

6628 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Tao et al.
activity in K562 cells, a human leukemia cell line that over-
expresses the Pim kinases. As shown in Table 6, multiple
compounds potently killed these cancer cells and have sub-
micromolar to low single-digit micromolar EC50 values. The
K562 cell line has been reported to be resistant to Pim-1
selective inhibitors,25 suggesting that Pim-2 inhibition may
contribute significantly to the excellent cytotoxity of these
compounds against this cell line. The lack of a perfect
correlation between Pim enzymatic inhibition potency and
cellular antiproliferative activity may be due to physico-
chemical properties such as variation in cellular penetration
(for examples, please see ref 40), but our data did show a
general trend that compounds with weak Pim-2 inhibition
activity exhibit no antiproliferative activity in the K562 cell
lines (e.g., 14i and 20d). These compounds were also eval-
uated in MV4-11, another human leukemia cell line, and all
showed potent antiproliferative activity with EC50 values in
the submicromolar to low single-digit micromolar range.
To further establish that the cytotoxicity of the benzothie-
nopyrimidinone-based Pim inhibitors truly resulted from the
inhibition of Pim kinases, we examined the phosphorylation
of the pro-apoptotic protein Bad by Pim kinases in the
presence or absence of Pim inhibitors in representative
leukemia and prostate cancer cell lines. Bad, a downstream
target of the Pim kinases, has been demonstrated to be
directly phosphorylated at Ser112 and Ser136 by Pim-1,
Pim-2, and Pim-3. It was found that these Pim inhibitors
efficiently and dose-dependently inhibited the phosphoryla-
tion of Bad at Ser112 in K562 cells (Figure 2 and Table 7). In
addition, the inhibition of Bad phosphorylation by two Pim
inhibitors was tested in LnCaP prostate carcinoma cells
engineered to express high levels of Bad (LnCaP-Bad).41
Compounds 14m and 20e both inhibited Bad phosphoryla-
tion with submicromolar EC50 values. These results strongly
indicate that the benzothienopyrimidinone-based Pim inhi-
bitors exert their cytotoxic effects through the inhibition of
Pim kinases in cancer cells.
Pharmacokinetics of a Representative Pim Inhibitor. Since
compound 14j exhibited good overall activity in both enzy-
matic and cell-based assays, it was further characterized by
evaluation of pharmacokinetics (PK). The compound was
dosed into CD-1 mice with IV and oral administration.
Compound 14j is highly bioavailable and exhibited a bio-
availability of 76% with oral dosing (Table 8). This com-
pound showed a half-life of 2.1 h with IV dosing. Compound
14j was further profiled for its ADME properties (Table 9).
The compound was found to have good permeability, mod-
est protein binding, a long half-life in both human and mouse
liver microsomes, and no CYP inhibition below 20 μM
concentration.
Table 6. Anti-proliferative Activities of Select Pim Inhibitors in K562
and MV4-11 Cell Lines
compounds
14f 14i 14j 14m 15b 18a 18b 20b 20c 20d
K562 (EC50, μM)
0.3 >30 1.7 6.1 0.7 4.7 5.9 0.4 1.4 >30
MV4-11 (EC50, μM) 1.4 4.1 2.4 3.2 1.9 0.8 1.5 5.6 1.3 2.7
Conclusions
We have discovered benzothienopyrimidinones as a novel
class of Pim inhibitors. These new Pim inhibitors potently
inhibit all three Pim kinases with Ki values ranging from
subnanomolar to low single-digit nanomolar and exhibit
excellent kinase selectivity against a panel of diverse kinases.
More importantly, multiple compounds exhibited potent
antiproliferative activity in K562 and MV4-11 cells with
submicromolar EC50 values. The crystallographic structures
of compounds 3b, 6e, and 12b guided the SAR studies and will
continue to play an important role in the further elaboration
of this class of Pim inhibitors. These novel Pim inhibitors
efficiently interrupted the phosphorylation of Bad in both
K562 and LnCaP-Bad cell lines, supporting that their potent
biological activities are mechanism-based. PK studies of a
Figure 2. Compound 14j inhibits the phosphorylation of Bad at
serine-112. K562 cells were treated for 4 h with increasing concen-
trations of compound 14j. Cell lysates were then analyzed by
immunoblotting using an antibody directed against phospho-Bad
serine-112.
Table 7. EC50 Values for the Inhibition of the Phosphorylation of Bad
at Ser112 by Pim Inhibitors in the K562 and LnCaP-Bad Cell Lines
compounds
14f
14j
14m
20c
20e
K562 (EC50, μM)
LnCaP-Bad (EC50, μM)
0.5
NTa
0.5
NT
0.35
0.6
0.4
NT
0.15
0.7
a Not tested.
Table 8. Pharmacokinetic Properties of Compound 14j
IVa,b
POa,c
AUC (μmol h/L)
Cl (L/h/kg)
7.80
Vd (L/kg)
t1/2 (h)
AUC (μmol h/L)
Cmax (μM)
Tmax (h)
F (%)
3
3
14j
1.10
23.5
2.10
2.80
0.52
0.3
76
a Three animals were used for each dosing group, and all values presented here were the averages of three animals. b The IV dosage was 3 mg/kg. c The
oral dosage was 10 mg/kg.
Table 9. ADME Data for Compound 14j
mouse liver
microsomes
(t1/2, min)
human liver
microsomes
(t1/2, min)
CYP
(2C9, 2D6, 3A4)
(IC50, μM)
Pampa
(pH 7.4, %
transported)
protein binding in
10% human plasma
(% bound)
protein binding
in 100% human
plasma (% bound)
48
>60
>20
17.4
68
92

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6629
representative compound (14j) suggested that these Pim in-
hibitors are highly bioavailable. To the best of our knowledge,
these are the first examples of Pim inhibitors that simulta-
neously inhibit all three Pim kinases. They not only have great
potential to be developed as medically useful agents but also
have the potential to serve as powerful tools in the study
of Pim-involved biological processes in cancer biology and
immunology.42
(s, 2 H), 7.54 (dd, J = 8.82, 2.03 Hz, 1 H), 7.89 (d, J = 8.82 Hz,
1 H), 8.31 (d, J = 2.03 Hz, 1 H).
8-Chloro[1]benzothieno[3,2-d]pyrimidin-4(3H)-one (3a). 2 (242
mg, 1.0 mmol) was treated with formamide (8 mL, 0.2 mol) and
heated at 190 °C for 6 h. The reaction mixture was cooled to
room temperature and diluted with DMSO (2 mL). The result-
ing mixture was partitioned between ethyl acetate and water.
The organic layer was washed with water (3 ꢀ 50 mL) and brine,
dried (MgSO4), filtered, and concentrated. The concentrate was
triturated with MeOH and filtered to collect the solid. The solid
was further purified by reverse-phase preparative HPLC to
provide the title compound (12.8 mg, 5% yield): MS (ESI) m/z
Experimental Section
General Information. All reactions were carried out under N2
atmosphere unless otherwise specified. Reagents and solvents
were obtained from commercial suppliers and were used without
further purification. 1H NMR spectra were obtained on a
Varian UNITY or Inova (500 MHz), Varian UNITY (400
MHz), or Varian UNITY plus or Mercury (300 MHz) instru-
ment. Chemical shifts are reported as δ values (ppm) downfield
relative to TMS as an internal standard with multiplicities
reported in the usual manner. Mass spectral analyses were
performed on a Finnigan SSQ7000 GC/MS mass spectrometer
using different techniques, including electrospray ionization
(ESI), and atmospheric pressure chemical ionization (APCI),
as specified for individual compounds. Exact mass measure-
ments were performed on a Finnigan FTMS Newstar T70 mass
spectrometer. The compound is determined to be “consistent”
with the chemical formula if the exact mass measurement is
within 5.0 ppm relative mass error (RME) of the exact mono-
isotopic mass. Elemental analyses were performed by Quanti-
tative Technologies, Inc., Whitehouse, NJ. Column chromato-
graphy was carried out on a Horizon Pioneer system (Biotage,
Inc.).
1
237 (M þ H)þ; H NMR (300 MHz, DMSO-d6) δ ppm 7.72
(dd, J = 8.65, 2.20 Hz, 1 H), 8.19-8.26 (m, 2 H), 8.37 (s, 1 H),
12.92 (s, 1 H).
5-Chloro-3-[(2-chloroacetimidoyl)amino]benzo[b]thiophene-
2-carboxylic acid methyl ester (4). A suspension of 2 (6.2 g,
25.7 mmol) in 4 N hydrochloric acid in dioxane (70 mL) was
treated with 2-chloroacetonitrile (3.18 mL, 50.32 mmol) at room
temperature for 3 h. The white solid was collected by filtration
and dried to give the desired product as the HCl salt quantita-
tively. The material was used directly in the next step without
further purification: MS (APCI) m/z 317 (M þ H)þ.
8-Chloro-2-dimethylaminomethyl-3H-benzo[4,5]thieno[3,2-d]-
pyrimidin-4-one (6a). A mixture of 4 (30 mg, 0.076 mmol) and
2 M dimethylamine in methanol (2 mL) was stirred at room
temperature overnight and concentrated. The residue was puri-
fied by reverse-phase preparative HPLC to provide the title
compound in 56% yield: LCMS (APCI) m/z 294 (M þ H)þ;
1H NMR (400 MHz, DMSO-d6) δ ppm 3.04 (s, 6 H), 4.50 (s,
2 H), 7.73-7.78 (m, 1 H), 8.27 (d, J = 8.59 Hz, 1 H), 8.37 (d, J =
1.53 Hz, 1 H), 9.91 (br s, 1 H), 13.26 (br s, 1 H).
Compounds 6b-6t were prepared by using a similar proce-
dure described for the synthesis of 6a.
8-Chloro-2-(piperidin-1-ylmethyl)[1]benzothieno[3,2-d]pyrimidin-
Preparative reverse-phase HPLC was performed on an auto-
mated Gilson HPLC system, using a SymmetryPrep Shield
RP18 prep cartridge, 250 mm ꢀ 21.20 mm i.d., 10 μm, and a
flow rate of 15 mL/min; λ = 214, 245 nm; mobile phase A, 0.1%
TFA in H2O; mobile phase B, CH3CN; linear gradient 0-70%
of B in 40 min.
1
4(3H)-one (6b): LCMS (APCI) m/z 334 (M þ H)þ; H NMR
(500 MHz, methanol-d4) δ ppm 1.63-1.90 (m, 2 H), 1.89-2.10
(m, 4 H), 3.16-3.39 (m, 4 H), 4.51 (s, 2 H), 7.66 (dd, J = 8.54,
2.14 Hz, 1 H), 8.05 (d, J = 8.85 Hz, 1 H), 8.34 (d, J = 2.14 Hz,
1 H).
Analytical LCMS with two different solvent systems
(Methods A and B) indicated that the purity of all compounds
was not less than 95%. Analytical LCMS was performed on a
Finnigan Navigator mass spectrometer and Agilent 1100 HPLC
system running Xcalibur 1.2 and Open-Access 1.3 software. The
mass spectrometer was operated under positive APCI ionization
conditions. The HPLC system comprised an Agilent Quatern-
ary pump, degasser, column compartment, autosampler, and
diode-array detector, with a Sedere Sedex 75 evaporative light-
scattering detector. The column used was a Phenomenex Luna
8-Chloro-2-(tetrahydropyrimidin-1(2H)-ylmethyl)[1]benzothieno-
[3,2-d]pyrimidin-4(3H)-one (6c): LCMS (APCI) m/z 335 (M þ
1
H)þ; H NMR (500 MHz, DMSO-d6) δ ppm 1.80-2.01 (m,
2 H), 2.66-2.85 (m, 2 H), 3.07-3.15 (m, 2 H), 3.22-3.30 (m,
2 H), 4.36 (s, 2 H), 7.77 (dd, J = 8.70, 2.29 Hz, 1 H), 8.28 (d, J =
8.85 Hz, 1 H), 8.32 (d, J = 2.14 Hz, 1 H), 8.53 (s, 1 H), 8.75 (s,
1 H), 9.40 (s, 1 H), 13.24 (s, 1 H).
8-Chloro-2-(morpholin-4-ylmethyl)[1]benzothieno[3,2-d]pyrimidin-
1
4(3H)-one (6d): LCMS (APCI) m/z 336 (M þ H)þ; H NMR
˚
Combi-HTS C8(2) 5 μm 100 A (2.1 mm ꢀ 30 mm). TFA Method
(500 MHz, methanol-d4) δ ppm 3.37-3.50 (m, 4 H), 4.00 (t, J =
4.58 Hz, 4 H), 4.40 (s, 2 H), 7.66 (dd, J = 8.85, 2.14 Hz, 1 H), 8.05
(d, J = 8.54 Hz, 1 H), 8.35 (d, J = 2.14 Hz, 1 H).
(Method A): A gradient of 10-100% acetonitrile (solvent 1) and
0.1% trifluoroacetic acid in water (solvent 2) was used, at a flow
rate of 2 mL/min (0-0.1 min 10% solvent 1, 0.1-2.6 min
10-100% solvent 1, 2.6-2.9 min 100-10% solvent 1, 2.9-
3.0 min 100-10% solvent 1). Ammonium Method (Method B):
A gradient of 10-100% acetonitrile (solvent 1) and 10 mM
NH4OAc in water (solvent 2) was used, at a flow rate of 1.5
mL/min (0-0.1 min 10% solvent 1, 0.1-3.1 min 10-100%
solvent 1, 3.1-3.9 min 100-10% solvent 1, 3.9-4.0 min
100-10% solvent 1).
8-Chloro-2-{[(3S)-3-hydroxypyrrolidin-1-yl]methyl}[1]benzo-
thieno[3,2-d]pyrimidin-4(3H)-one (6e): LCMS (APCI) m/z 336
(M þ H)þ; 1H NMR (400 MHz, methanol-d4) δ ppm 2.13-2.28
(m, 1 H), 2.35 (br s, 1 H), 3.23-3.37 (m, 3 H), 3.81 (br s, 2 H),
4.64-4.69 (m, 1 H), 4.71 (s, 2 H), 7.65 (dd, J = 8.90, 2.15 Hz,
1 H), 8.04 (d, J = 8.29 Hz, 1 H), 8.38 (d, J = 2.15 Hz, 1 H); 1H
NMR (500 MHz, pyridine-d5) δ ppm 2.03-2.15 (m, 1 H),
2.18-2.33 (m, 1 H), 3.00-3.12 (m, 1 H), 3.24-3.33 (m, 2 H),
3.33-3.42 (m, 1 H), 4.27-4.46 (m, 2 H), 4.64-4.79 (m, 1 H),
7.57 (dd, J = 8.85, 2.14 Hz, 1 H), 7.95 (d, J = 8.85 Hz, 1 H), 8.46
(d, J = 2.14 Hz, 1 H).
8-Chloro-2-{[(3R)-3-hydroxypyrrolidin-1-yl]methyl}[1]benzo-
thieno[3,2-d]pyrimidin-4(3H)-one (6f): LCMS (APCI) m/z 336
(M þ H)þ; 1H NMR (500 MHz, pyridine-d5) δ ppm 2.01-2.12
(m, 1 H), 2.16-2.29 (m, 1 H), 2.92-3.04 (m, 1 H), 3.20-3.27
(m, 2 H), 3.27-3.35 (m, 1 H), 4.15-4.41 (m, 1 H), 4.56-4.83 (m,
1 H), 7.57 (dd, J = 2.14 Hz, 1 H), 7.95 (dd, J = 8.85, 2.14 Hz,
1 H), 8.47 (d, J = 2.14 Hz, 1 H).
3-Amino-5-chlorobenzo[b]thiophene-2-carboxylic acid methyl
ester (2). To a cold solution of 5-chloro-2-fluorobenzonitrile
(10.5 g, 67.5 mmol) in DMF at 0 °C was dropwise added methyl
2-mercaptoacetate (6.45 mL, 70.88 mmol). The reaction mixture
was stirred at 0 °C for 30 min, and then 5 M NaOH aqueous
solution (20.25 mL) was added dropwise. After stirring at 0 °C
for 3 h, the reaction mixture was quenched with ice-water. The
resulting precipitate was collected by filtration and dried to give
15.4 g of white solid in 95% yield: MS (APCI) m/z 242 (M þ
H)þ; 1H NMR (300 MHz, DMSO-d6) δ ppm 3.79 (s, 3 H), 7.18

6630 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Tao et al.
8-Chloro-2-[(3-hydroxypiperidin-1-yl)methyl][1]benzothieno[3,2-d]-
pyrimidin-4(3H)-one (6g): LCMS (APCI) m/z 350 (M þ H)þ; 1H
NMR (500 MHz, DMSO-d6) δ ppm 1.18-2.26 (m, 5 H), 3.31
(s, 2 H), 3.97 (s, 2 H), 4.41 (s, 2 H), 5.55 (s, 1 H), 7.76 (dd, J =
8.70, 2.29 Hz, 1 H), 8.27 (d, J=8.54Hz, 1H), 8.31(d, J=1.83Hz,
1 H), 9.26-10.27 (m, 1 H), 13.20 (s, 1 H).
8-Chloro-2-((4-hydroxypiperidin-1-yl)methyl)[1]benzothieno[3,2-d]-
pyrimidin-4(3H)-one (6h): LCMS (APCI) m/z 350 (M þ H)þ; 1H
NMR (500 MHz, DMSO-d6) δ ppm 1.78 (br s, 2 H), 2.01 (br s,
2 H), 3.26 (br s, 2 H), 3.68 (br s, 2 H), 4.46 (br s, 2 H), 5.06 (br s,
1 H), 7.76 (dd, J = 8.70, 2.29 Hz, 1 H), 8.28 (d, J = 8.54 Hz, 1 H),
8.31 (d, J = 2.14 Hz, 1 H), 9.83 (br s, 1 H), 13.22 (br s, 1 H).
8-Chloro-2-[(methylamino)methyl][1]benzothieno[3,2-d]pyri-
midin-4(3H)-one (6i): LCMS (APCI) m/z 280 (M þ H)þ; 1H
NMR (500 MHz, DMSO-d6) δ ppm 2.79 (s, 3 H), 4.33 (s, 2 H),
7.76 (dd, J = 8.54, 2.14 Hz, 1 H), 8.27 (d, J = 8.54 Hz, 1 H), 8.35
(d, J = 2.14 Hz, 1 H), 9.29 (br s, 1 H).
2 H), 5.53 (t, J = 1.98 Hz, 1 H), 5.60 (d, J = 1.83 Hz, 2 H), 5.88 (s,
1 H), 7.72 (dd, J = 8.70, 2.29 Hz, 1 H), 8.22 (d, J = 8.85 Hz, 1 H),
8.26 (d, J = 2.14 Hz, 1 H), 8.82 (s, 2 H), 12.70 (s, 1 H).
8-Chloro-2-{[(3-hydroxy-2-methylphenyl)amino]methyl}[1]-
benzothieno[3,2-d]pyrimidin-4(3H)-one (6r): LCMS (APCI) m/z
372 (M þ H)þ; 1H NMR (500 MHz, DMSO-d6) δ ppm 2.04 (s,
3 H), 4.39 (d, J = 5.19 Hz, 2 H), 5.47 (s, 1 H), 6.05 (d, J = 7.93 Hz,
1 H), 6.18 (d, J = 7.93 Hz, 1 H), 6.73 (t, J = 8.09 Hz, 1 H), 7.72
(dd, J = 8.85, 2.14 Hz, 1 H), 8.20 (d, J = 2.14 Hz, 1 H), 8.22 (d,
J = 8.85 Hz, 1 H), 8.96 (s, 1 H), 12.71 (s, 1 H).
8-Chloro-2-{[(3-hydroxy-4-methylphenyl)amino]methyl}[1]benzo-
thieno[3,2-d]pyrimidin-4(3H)-one (6s): LCMS (APCI) m/z 372
(M þ H)þ; 1H NMR (500 MHz, DMSO-d6) δ ppm 1.94 (s, 3 H),
4.28 (s, 2 H), 5.82 (br s, 1 H), 6.07 (dd, J = 8.09, 2.29 Hz, 1 H),
6.17 (d, J = 2.14 Hz, 1 H), 6.75 (d, J = 7.93 Hz, 2 H), 7.71 (dd,
J = 8.54, 2.14 Hz, 1 H), 8.22 (d, J = 8.54 Hz, 1 H), 8.26 (d, J =
1.83 Hz, 1 H), 8.89 (s, 1 H), 12.70 (s, 1 H).
8-Chloro-2-[2-(dimethylamino)ethyl][1]benzothieno[3,2-d]pyri-
midin-4(3H)-one (6j): LCMS (APCI) m/z 308 (M þ H)þ; 1H
NMR (500 MHz, DMSO-d6) δ ppm 2.91 (d, J = 2.44 Hz, 6 H),
3.21 (t, J = 6.87 Hz, 2 H), 3.65 (d, J = 3.66 Hz, 2 H), 7.73 (dd,
J = 8.70, 2.29 Hz, 1 H), 8.24 (d, J = 8.85 Hz, 1 H), 8.30 (d, J =
2.14 Hz, 1 H), 9.28 (s, 1 H), 12.99 (s, 1 H).
8-Chloro-2-[3-(dimethylamino)propyl][1]benzothieno[3,2-d]-
pyrimidin-4(3H)-one (6k). A suspension of 2 (30 mg, 0.12 mmol)
in 4 N hydrochloric acid in dioxane (10 mL) was treated with
4-aminobutanenitrile (101 mg, 1.2 mmol) at ambient tempera-
ture for 3 days. The reaction mixture was concentrated, and the
residue was dissolved in DMF (1 mL). The resulting mixture was
treated with 2 M dimethylamine in methanol (5 mL) at 50 °C
overnight and concentrated. The residue was purified by HPLC
to give the desired product: LCMS (APCI) m/z 322 (M þ H)þ;
1H NMR (400 MHz, methanol-d4) δ ppm 2.29-2.41 (m, 2 H),
2.93 (t, J = 7.21 Hz, 2 H), 2.97 (s, 6 H), 3.29-3.36 (m, 2 H), 7.60
(dd, J = 8.59, 2.15 Hz, 1 H), 7.98 (d, J = 8.59 Hz, 1 H), 8.24 (d,
J = 2.15 Hz, 1 H).
2-(Anilinomethyl)-8-chloro[1]benzothieno[3,2-d]pyrimidin-4(3H)-
one (6l): LCMS (APCI) m/z 342 (M þ H)þ; 1H NMR (400 MHz,
DMSO-d6) δ ppm 4.36 (d, J = 5.52 Hz, 1 H), 6.11 (s, 1 H), 6.59
(t, J = 7.36 Hz, 1 H), 6.70 (d, J = 7.67 Hz, 2 H), 7.06-7.14 (m,
2 H), 7.70 (dd, J = 8.75, 2.30 Hz, 1 H), 8.21 (d, J = 9.21 Hz,
1 H), 8.24 (d, J = 2.15 Hz, 1 H), 12.79 (s, 1 H).
8-Chloro-2-{[(2-hydroxyphenyl)amino]methyl}[1]benzothieno-
[3,2-d]pyrimidin-4(3H)-one (6m): LCMS (APCI) m/z 358 (M þ
H)þ; 1H NMR (500 MHz, DMSO-d6) δ ppm 4.38 (s, 2 H), 5.57
(s, 1 H), 6.40-6.50 (m, 1 H), 6.53-6.65 (m, 2 H), 6.70 (dd, J =
7.63, 1.22 Hz, 1 H), 7.71 (dd, J = 8.70, 2.29 Hz, 1 H), 8.18 (d, J =
2.14 Hz, 1 H), 8.22 (d, J=8.54Hz, 1H), 9.43(s, 1H), 12.82(s, 1H).
8-Chloro-2-{[(3-hydroxyphenyl)amino]methyl}[1]benzothieno-
[3,2-d]pyrimidin-4(3H)-one (6n): LCMS (APCI) m/z 358 (M þ
H)þ; 1H NMR (500 MHz, DMSO-d6) δ ppm 4.31 (d, J = 5.80
Hz, 2 H), 5.99-6.05 (m, 2 H), 6.11 (t, J = 2.14 Hz, 1 H), 6.15 (dd,
J = 7.93, 1.53 Hz, 1 H), 6.87 (t, J = 7.93 Hz, 1 H), 7.71 (dd, J =
8.54, 2.14 Hz, 1 H), 8.22 (d, J = 8.85 Hz, 1 H), 8.26 (d, J = 2.14
Hz, 1 H), 9.01 (s, 1 H), 12.76 (s, 1 H).
8-Chloro-2-({[2-(3-hydroxyphenyl)ethyl]amino}methyl)[1]-
benzothieno[3,2-d]pyrimidin-4(3H)-one (6t): LCMS (APCI) m/z
386 (M þ H)þ; 1H NMR (400 MHz, DMSO-d6) δ ppm 1.26 (t,
J = 6.87 Hz, 2 H), 2.95-2.98 (m, 2 H), 4.37 (s, 2 H), 6.53 (br s,
1 H), 6.65-6.70 (m, 2 H), 6.72 (d, J = 7.93 Hz, 1 H), 7.15 (t, J =
8.09 Hz, 1 H), 7.77 (dd, J = 8.85, 2.14 Hz, 1 H), 8.28 (d, J = 8.85
Hz, 1 H), 8.32 (d, J = 2.14 Hz, 1 H), 9.27 (s, 1 H), 9.42 (s, 1 H).
8-Chloro-2-[(3-hydroxyphenoxy)methyl][1]benzothieno[3,2-d]-
pyrimidin-4(3H)-one (7). A mixture of 4 (30 mg, 0.076 mmol),
K2CO3 (52 mg, 0.38 mmol), and resorcinol (84 mg, 0.76 mmol)
in DMF (2 mL) was stirred at 50 °C for 20 h and concentrated.
The residue was purified by reverse-phase preparative HPLC to
provide the title compound in 56% yield: 1H NMR (400 MHz,
DMSO-d6) δ ppm 5.07 (s, 2 H), 6.41 (dd, J = 7.98, 2.15 Hz, 1 H),
6.48 (t, J = 2.30 Hz, 1 H), 6.49-6.54 (m, 1 H), 7.09 (t, J = 8.13
Hz, 1 H), 7.72 (dd, J = 8.75, 1.99 Hz, 1 H), 8.19 (d, J = 2.15 Hz,
1 H), 8.24 (d, J = 8.59 Hz, 1 H), 9.46 (s, 1 H), 13.11 (s, 1 H).
2-[(Dimethylamino)methyl]-8-bromo[1]benzothieno[3,2-d]pyri-
midin-4(3H)-one (8). The title compound was prepared using the
same synthetic sequences described for 6a: LCMS (APCI) m/z
338 (M þ H)þ; 1H NMR (500 MHz, methanol-d4) δ ppm 3.17
(s, 6 H), 4.56 (s, 2 H), 7.79 (dd, J = 8.70, 1.98 Hz, 1 H), 8.00 (d,
J = 8.54 Hz, 1 H), 8.56 (d, J = 1.83 Hz, 1 H).
2-[(Dimethylamino)methyl][1]benzothieno[3,2-d]pyrimidin-4(3H)-
one (9). The title compound was prepared using the same
synthetic sequences described for 6a: LCMS (APCI) m/z 260
(M þ H)þ; 1H NMR (400 MHz, DMSO-d6) δ ppm 3.03 (s, 6 H),
4.50 (s, 2 H), 7.66 (t, J = 7.02 Hz, 1 H), 7.69-7.75 (m, 1 H), 8.21
(d, J = 7.93 Hz, 1 H), 8.35 (d, J = 7.32 Hz, 1 H), 10.05 (s, 1 H),
13.17 (s, 1 H).
2-[(Dimethylamino)methyl]-8-methyl[1]benzothieno[3,2-d]pyri-
midin-4(3H)-one (10a). The title compound was prepared using
the same synthetic sequences described for 6a: LCMS (APCI)
m/z 274 (M þ H)þ; 1H NMR (500 MHz, DMSO-d6) δ ppm 2.52
(s, 3 H), 3.04 (s, 6 H), 4.50 (s, 2 H), 7.54 (dd, J = 8.54, 1.53 Hz,
1 H), 8.07 (d, J = 8.24 Hz, 1 H), 8.15 (s, 1 H), 10.19 (s, 1 H), 13.16
(s, 1 H).
2-[(Dimethylamino)methyl]-8-(trifluoromethyl)[1]benzothieno-
[3,2-d]pyrimidin-4(3H)-one (10b). The title compound was pre-
pared using the same synthetic sequences described for 6a:
LCMS (APCI) m/z 328 (M þ H)þ; 1H NMR (400 MHz,
DMSO-d6) δ ppm 2.99 (s, 6 H), 4.45 (s, 2 H), 8.03 (dd, J =
8.59, 1.84 Hz, 1 H), 8.49 (d, J = 8.29 Hz, 1 H), 8.63 (s, 1 H).
2-[(Dimethylamino)methyl]-8-ethyl[1]benzothieno[3,2-d]pyrimidin-
4(3H)-one (11a). A mixture of 12a (20 mg, 0.07 mmol) and 10%
Pd/C (20 mg) in methanol (8 mL) was stirred under hydrogen
atmosphere at room temperature overnight. The insoluble
material was filtered off through Celite, and the filtrate was
concentrated to give the desired product in quantitative yield:
LCMS (APCI) m/z 288 (M þ H)þ; 1H NMR (400 MHz, DMSO-
d6) δ ppm 1.29 (t, J = 7.52 Hz, 3 H), 2.83 (q, J = 7.67 Hz, 2 H),
3.01 (s, 6 H), 4.46 (s, 2 H), 7.58 (dd, J = 8.29, 1.84 Hz, 1 H), 8.09
(d, J = 8.29 Hz, 1 H), 8.16 (d, J = 1.23 Hz, 1 H).
8-Chloro-2-{[(4-hydroxyphenyl)amino]methyl}[1]benzothieno-
[3,2-d]pyrimidin-4(3H)-one (6o): LCMS (APCI) m/z 358 (M þ
H)þ; 1H NMR (500 MHz, DMSO-d6) δ ppm 4.31 (s, 2 H),
6.56-6.61 (m, 2 H), 6.61-6.69 (m, 2 H), 7.72 (dd, J = 8.54, 2.14
Hz, 1 H), 8.22 (d, J = 8.54 Hz, 1 H), 8.27 (d, J = 2.14 Hz, 1 H),
8.64 (br s, 1 H), 12.75 (br s, 1 H).
8-Chloro-2-((3-methoxyphenylamino)methyl)[1]benzothieno-
[3,2-d]pyrimidin-4(3H)-one (6p): LCMS (APCI) m/z 372 (M þ
H)þ; 1H NMR (500 MHz, DMSO-d6) δ ppm 3.66 (s, 1 H), 4.34
(d, J = 6.10 Hz, 2 H), 6.13-6.20 (m, 2 H), 6.28-6.31 (m, 2 H),
7.00 (t, J = 8.24 Hz, 1 H), 7.71 (dd, J = 8.54, 2.14 Hz, 1 H), 8.22
(d, J = 8.54 Hz, 1 H), 8.25 (d, J = 2.14 Hz, 1 H), 12.82 (s, 1 H).
8-Chloro-2-{[(3,5-dihydroxyphenyl)amino]methyl}[1]benzothieno-
[3,2-d]pyrimidin-4(3H)-one (6q): LCMS (APCI) m/z 374 (M þ
H)þ; 1H NMR (500 MHz, DMSO-d6) δ ppm 4.25 (d, J = 3.66 Hz,

Article
2-[(Dimethylamino)methyl]-8-isopropyl[1]benzothieno[3,2-d]pyri-
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6631
NMR (500 MHz, DMSO-d6) δ ppm 1.32-1.49 (m, 1 H), 1.55-
1.74 (m, 5 H), 1.75-1.89 (m, 4 H), 2.55 (t, J = 7.17 Hz, 2 H),
2.82-2.94 (m, 2 H), 3.03 (s, 6 H), 3.06-3.13 (m, 2 H), 3.40-3.62
(m, 2 H), 4.50 (s, 2 H), 7.67 (dd, J = 8.54, 1.83 Hz, 1 H), 8.20 (d,
J = 8.54 Hz, 1 H), 8.36 (d, J = 1.22 Hz, 1 H), 9.29 (s, 1 H), 10.11
(br s, 1 H), 13.24 (br s, 1 H).
Compounds 14a-14q, 15a-15d, and 16a-16b were prepared
under Suzuki coupling conditions as described for the synthesis
of 14j.
midin-4(3H)-one (11b). The title compound was prepared using
the same synthetic sequences described for 6a: LCMS (APCI)
m/z 302 (M þ H)þ; 1H NMR (400 MHz, DMSO-d6) δ ppm 0.94
(t, J = 7.32 Hz, 3 H), 1.65-1.74 (m, 2 H), 2.71-2.81 (m, 2 H),
3.03 (s, 6 H), 4.49 (s, 2 H), 7.57 (dd, J = 8.39, 1.68 Hz, 1 H), 8.10
(d, J = 8.24 Hz, 1 H), 8.14 (s, 1 H), 9.93 (s, 1 H), 13.14 (s, 1 H).
2-[(Dimethylamino)methyl]-8-vinyl[1]benzothieno[3,2-d]pyri-
midin-4(3H)-one (12a). The title compound was prepared using a
procedure similar to that described for the synthesis of 14j:
LCMS (APCI) m/z 285 (M þ H)þ; 1H NMR (400 MHz, DMSO-
d6) δ ppm 3.04 (s, 6 H), 4.49 (s, 2 H), 5.43 (d, J = 11.35 Hz, 1 H),
6.00 (d, J = 17.49 Hz, 1 H), 6.96 (dd, J = 17.49, 11.05 Hz, 1 H),
7.89 (dd, J = 8.59, 1.84 Hz, 1 H), 8.17 (d, J = 8.59 Hz, 1 H), 8.32
(s, 1 H), 10.01 (s, 1 H), 13.17 (s, 1 H).
2-[(Dimethylamino)methyl]-8-phenyl[1]benzothieno[3,2-d]pyri-
1
midin-4(3H)-one (14a): LCMS (APCI) m/z 336 (M þ H)þ; H
NMR (400 MHz, DMSO-d6) δ ppm 3.05 (s, 6 H), 4.51 (s, 2 H),
7.45 (t, J = 7.36 Hz, 1 H), 7.56 (t, J = 7.67 Hz, 2 H), 7.74-7.83
(m, 2 H), 8.02 (dd, J = 8.59, 1.84 Hz, 1 H), 8.30 (d, J = 8.90 Hz,
1 H), 8.57 (d, J = 1.53 Hz, 1 H), 9.89 (s, 1 H), 13.20 (s, 1 H).
2-[(Dimethylamino)methyl]-8-(1-phenylethyl)[1]benzothieno-
[3,2-d]pyrimidin-4(3H)-one (14b): LCMS (APCI) m/z 364 (M þ
H)þ; 1H NMR (500 MHz, DMSO-d6) δ ppm 1.70 (d, J = 7.02
Hz, 3 H), 3.02 (s, 6 H), 4.41 (q, J = 7.02 Hz, 1 H), 4.48 (s, 2 H),
7.15-7.23 (m, 1 H), 7.24-7.34 (m, 4 H), 7.60 (dd, J = 8.54, 1.83
Hz, 1 H), 8.10 (d, J = 8.54 Hz, 1 H), 8.23 (d, J = 1.53 Hz, 1 H),
10.04 (br s, 1 H), 13.15 (br s, 1 H).
8-[(E)-2-Cyclopropylvinyl]-2-[(dimethylamino)methyl][1]benzo-
thieno[3,2-d]pyrimidin-4(3H)-one (12b). The title compound was
prepared using a procedure similar to that described for the
1
synthesis of 14j: LCMS (APCI) m/z 326 (M þ H)þ; H NMR
(400 MHz, DMSO-d6) δ ppm 0.55-0.59 (m, 2 H), 0.82-0.87 (m,
2 H), 1.61-1.71 (m, 1 H), 3.04 (s, 6 H), 4.49 (s, 2 H), 6.02 (dd,
J = 15.80, 9.05 Hz, 1 H), 6.67 (d, J = 15.96 Hz, 1 H), 7.75 (dd,
J = 8.59, 1.84 Hz, 1 H), 8.09 (d, J = 8.29 Hz, 1 H), 8.19 (d, J =
1.53 Hz, 1 H), 10.09 (s, 1 H), 13.16 (s, 1 H).
2-[(Dimethylamino)methyl]-8-(3-fluorophenyl)[1]benzothieno-
[3,2-d]pyrimidin-4(3H)-one (14c): LCMS (APCI) m/z 354 (M þ
H)þ; 1H NMR (400 MHz, DMSO-d6) δ ppm 3.05 (s, 6 H), 4.51
(s, 2 H), 7.25-7.32 (m, 1 H), 7.57-7.66 (m, 3 H), 8.05 (dd, J =
8.44, 1.99 Hz, 1 H), 8.31 (d, J = 9.21 Hz, 1 H), 8.58 (d, J = 1.23
Hz, 1 H), 9.88 (s, 1 H), 13.22 (s, 1 H).
8-(3-Chlorophenyl)-2-[(dimethylamino)methyl][1]benzothieno-
[3,2-d]pyrimidin-4(3H)-one 14d): LCMS (APCI) m/z 370 (M þ
H)þ; 1H NMR (500 MHz, DMSO-d6) δ ppm 3.03 (s, 6 H), 4.50
(s, 2 H), 7.50-7.55 (m, 1 H), 7.59 (t, J = 7.93 Hz, 1 H), 7.76 (d,
J = 7.93 Hz, 1 H), 7.84 (t, J = 1.83 Hz, 1 H), 8.05 (dd, J = 8.54,
1.83 Hz, 1 H), 8.32 (d, J = 8.24 Hz, 1 H), 8.56 (d, J = 1.53 Hz,
1 H), 9.88 (br s, 1 H), 13.23 (br s, 1 H).
2-[(Dimethylamino)methyl]-8-[3-(trifluoromethoxy)phenyl][1]-
benzothieno[3,2-d]pyrimidin-4(3H)-one (14e): LCMS (APCI)
m/z 420 (M þ H)þ; 1H NMR (400 MHz, DMSO-d6) δ ppm 3.05
(s, 6 H), 4.51 (s, 2 H), 7.44-7.49 (m, 1 H), 7.70 (t, J = 7.98 Hz,
1 H), 7.75 (s, 1 H), 7.84 (d, J = 8.59 Hz, 1 H), 8.06 (dd, J = 8.44,
1.99 Hz, 1 H), 8.33 (d, J = 8.59 Hz, 1 H), 8.57 (d, J = 1.23 Hz,
1 H), 10.01 (s, 1 H), 13.21 (s, 1 H).
2-[(Dimethylamino)methyl]-8-ethynyl[1]benzothieno[3,2-d]pyri-
midin-4(3H)-one (13a). To a mixture of 8 (60 mg, 0.18 mmol),
ethynyltrimethylsilane (0.073 mL, 0.53 mmol), tetrakis(tri-
phenylphosphine)palladium(0) (30.7 mg, 0.03 mmol), and tri-
ethylamine (0.074 mL, 0.53 mmol) in N,N-dimethylformamide
(3 mL) was added copper(I) iodide (6.8 mg, 0.036 mmol), and
the mixture was heated at 100 °C for 600 s in a CEM microwave
synthesizer. After concentration, the residue was purified by
reverse-phase preparative HPLC to provide the TMS-pro-
tected 13a in 81% yield. This TMS-protected product (20 mg,
0.06 mmol) in methanol (1 mL) was treated with 5 M aqueous
NaOH (1 mL) for 10 min and concentrated. The residue was
purified by reverse-phase preparative HPLC to give the title
compound as the TFA salt in 91% yield: LCMS (APCI) m/z 284
(M þ H)þ; 1H NMR (400 MHz, DMSO-d6) δ ppm 3.03 (s, 6 H),
4.36 (s, 1 H), 4.50 (s, 2 H), 7.76 (dd, J = 8.44, 1.69 Hz, 1 H), 8.24
(d, J = 7.67 Hz, 1 H), 8.46 (d, J = 1.23 Hz, 1 H), 9.82 (br s, 1 H),
13.24 (br s, 1 H).
2-[(Dimethylamino)methyl]-8-[3-(dimethylamino)prop-1-ynyl][1]-
benzothieno[3,2-d]pyrimidin-4(3H)-one (13b). The title compound
was prepared using the same synthetic sequences as described for
2-[(Dimethylamino)methyl]-8-(3-hydroxyphenyl)[1]benzothieno-
[3,2-d]pyrimidin-4(3H)-one (14f): LCMS (APCI) m/z 352 (M þ
H)þ; 1H NMR (500 MHz, DMSO-d6) δ ppm 3.01 (s, 6 H), 4.50 (s,
2 H), 6.85 (dd, J = 7.93, 1.53 Hz, 1 H), 7.13 (t, J = 1.98 Hz, 1 H),
7.19 (d, J = 7.63 Hz, 1 H), 7.34 (t, J = 7.93 Hz, 1 H), 7.94 (dd, J =
8.54, 1.83 Hz, 1 H), 8.27 (d, J=8.54Hz, 1H), 8.52(d, J=1.53Hz,
1 H), 9.66 (br s, 1 H), 9.85 (br s, 1 H), 13.20 (br s, 1 H).
1
13a: LCMS (APCI) m/z 341 (M þ H)þ; H NMR (400 MHz,
DMSO-d6) δ ppm 2.91 (s, 6 H), 3.00 (s, 6 H), 4.38 (s, 2 H), 4.46
(s, 2 H), 7.82 (dd, J = 8.44, 1.69 Hz, 1 H), 8.29 (d, J = 8.59 Hz,
1 H), 8.46 (d, J = 1.53 Hz, 1 H).
8-(6-Chlorohex-1-ynyl)-2-[(dimethylamino)methyl][1]benzothieno-
[3,2-d]pyrimidin-4(3H)-one. To a mixture of 8 (60 mg, 0.18
mmol), 6-chlorohex-1-yne (0.064 mL, 0.53 mmol), Pd(PPh3)4
(30.7 mg, 0.03 mmol), and triethylamine (0.074 mL, 0.53 mmol)
in DMF (3 mL) was added CuI (6.8 mg, 0.036 mmol). The
resulting mixture was heated at 100 °C for 600 s in a CEM
microwave synthesizer and concentrated. The residue was puri-
fied by reverse-phase preparative HPLC to give the desired
product as the TFA salt in 65% yield: LCMS (APCI) m/z 374
(M þ H)þ; 1H NMR (400 MHz, DMSO-d6) δ ppm 1.62-1.79
(m, 2 H), 1.83-1.98 (m, 2 H), 2.54 (t, J = 7.21 Hz, 2 H), 3.03 (s,
6 H), 3.72 (t, J = 6.44 Hz, 2 H), 4.50 (s, 2 H), 7.67 (dd, J = 8.44,
1.69 Hz, 1 H), 8.18 (d, J = 8.29 Hz, 1 H), 8.37 (d, J = 1.23 Hz,
1 H), 9.82 (s, 1 H), 13.20 (s, 1 H).
8-(6-(Piperidin-1-yl)hex-1-ynyl)-2-[(dimethylamino)methyl][1]-
benzothieno[3,2-d]pyrimidin-4(3H)-one (13c). 8-(6-Chlorohex-1-
ynyl)-2-[(dimethylamino)methyl][1]benzothieno[3,2-d]pyrimidin-
4(3H)-one (10 mg, 0.021 mmol) in piperidine (1.5 mL) was heated
at 80 °C for 1 h. The reaction mixture was concentrated, and
the residue was purified by HPLC to give the desired product as
the TFA salt in 85% yield: LCMS (APCI) m/z 423 (M þ H)þ; 1H
2-[(Dimethylamino)methyl]-8-(2-methylphenyl)[1]benzothieno-
[3,2-d]pyrimidin-4(3H)-one (14g): LCMS (APCI) m/z 350 (M þ
H)þ; 1H NMR (400 MHz, DMSO-d6) δ ppm 2.26 (s, 3 H), 2.98
(s, 6 H), 4.46 (s, 1 H), 7.26-7.40 (m, 4 H), 7.69 (dd, J = 8.29, 2.15
Hz, 1 H), 8.25 (d, J = 8.29, 1 H), 8.26 (s, 1 H).
2-[(Dimethylamino)methyl]-8-(4-ethylphenyl)[1]benzothieno[3,2-d]-
pyrimidin-4(3H)-one (14h). LCMS (APCI) m/z 363 (M þ H)þ;
1H NMR (500 MHz, DMSO-d6) δ ppm 1.24 (t, J = 7.63 Hz,
3 H), 2.69 (q, J = 7.63 Hz, 2 H), 3.04 (s, 6 H), 4.51 (s, 2 H), 7.39
(d, J = 8.24 Hz, 2 H), 7.70 (d, J = 8.24 Hz, 2 H), 8.00 (dd, J =
8.54, 2.14 Hz, 1 H), 8.28 (d, J = 8.54 Hz, 1 H), 8.55 (d, J = 1.83
Hz, 1 H), 9.90 (br s, 1 H), 13.18 (br s, 1 H).
4-{2-[(Dimethylamino)methyl]-4-oxo-3,4-dihydro[1]benzothieno-
[3,2-d]pyrimidin-8-yl}benzonitrile (14i): LCMS (APCI) m/z 361
(M þ H)þ; 1H NMR (400 MHz, DMSO-d6) δ ppm 3.01 (s, 6 H),
4.47 (s, 2 H), 7.97-8.05 (m, 4 H), 8.09 (dd, J = 8.59, 1.84 Hz,
1 H), 8.36 (d, J = 8.59 Hz, 1 H), 8.61 (d, J = 1.23 Hz, 1 H).
8-(4-Hydroxylphenyl)-2-[(dimethylamino)methyl][1]benzothieno-
[3,2-d]pyrimidin-4(3H)-one (14j). To a mixture 8 (40 mg, 0.11
mmol), Pd(PPh3)2Cl2 (8.3 g, 0.012 mmol), and 4-hydroxylphenyl
boronic acid (19.9 mg, 0.14 mmol) in 2.5 mL of DME/EtOH/H2O

6632 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Tao et al.
(7:2:3) was added 1 M Na2CO3 aqueous solution (0.2 mL). The
reaction mixture was heated for 600 s in a CEM microwave
synthesizer at 150 °C and concentrated. The residue was purified
by HPLC to give the desired product as the TFA salt (33 mg, 65%).
The TFA salt was converted into the HCl salt as follows: To a
solution of the TFA salt product in methanol was added a large
excess of 1 M HCl in ether. The resulting mixture was stirred at
room temperature for 4 h, and the white precipitate was collected
by filtration and dried at 60 °C for 3 days to give the title compound
1 H), 7.97 (dd, J = 8.54, 1.83 Hz, 1 H), 8.16 (d, J = 8.54 Hz,
1 H), 8.49 (d, J = 1.53 Hz, 1 H), 10.05 (br s, 1 H), 11.54 (s, 1 H),
13.14 (br s, 1 H).
2-[(Dimethylamino)methyl]-8-(3-furyl)[1]benzothieno[3,2-d]pyri-
1
midin-4(3H)-one (15c): LCMS (APCI) m/z 326 (M þ H)þ; H
NMR (500 MHz, DMSO-d6) δ ppm 3.06 (s, 6 H), 4.51 (s, 2 H),
7.06 (s, 1 H), 7.84 (t, J = 1.53 Hz, 1 H), 7.97 (dd, J = 8.90, 1.84
Hz, 1 H), 8.21 (d, J = 8.29 Hz, 1 H), 8.32 (s, 1 H), 8.45 (d, J =
1.84 Hz, 1 H), 10.27 (s, 1 H), 13.20 (s, 1 H).
1
as the HCl salt: LCMS (APCI) m/z 352 (M þ H)þ; H NMR
2-[(Dimethylamino)methyl]-8-(4-methylthien-3-yl)[1]benzothieno-
[3,2-d]pyrimidin-4(3H)-one (15d): LCMS (APCI) m/z 356 (M þ
H)þ; 1H NMR (500 MHz, DMSO-d6) δ ppm 2.28 (s, 3 H), 3.02
(s, 6 H), 4.50 (s, 2 H), 7.37 (d, J = 2.44 Hz, 1 H), 7.59 (d, J = 3.36
Hz, 1 H), 7.76 (dd, J = 8.24, 1.83 Hz, 1 H), 8.25 (d, J = 8.24 Hz,
1 H), 8.34 (d, J = 1.22 Hz, 1 H), 9.85 (s, 1 H), 13.20 (s, 1 H).
2-[(Dimethylamino)methyl]-8-pyridin-3-yl[1]benzothieno[3,2-d]-
pyrimidin-4(3H)-one (16a): LCMS (APCI) m/z 337 (M þ H)þ;
1H NMR (500 MHz, DMSO-d6) δ ppm 3.05 (s, 6 H), 4.52 (s,
2 H), 7.65 (dd, J = 7.93, 4.88 Hz, 1 H), 8.09 (dd, J = 8.54,
1.83 Hz, 1 H), 8.28 (d, J = 8.24 Hz, 1 H), 8.37 (d, J = 8.54 Hz,
1 H), 8.63 (d, J = 1.53 Hz, 1 H), 8.70 (dd, J = 4.88, 1.22 Hz,
1 H), 9.07 (d, J = 1.83 Hz, 1 H), 9.95 (br s, 1 H), 13.25 (br s, 1 H).
2-[(Dimethylamino)methyl]-8-pyrimidin-5-yl[1]benzothieno[3,2-d]-
pyrimidin-4(3H)-one (16b): LCMS (APCI) m/z 338 (M þ H)þ;
1H NMR (400 MHz, DMSO-d6) δ ppm 3.01 (s, 6 H), 4.45 (s,
2 H), 8.15 (dd, J = 8.44, 1.99 Hz, 1 H), 8.40 (d, J = 8.59 Hz, 1
H), 8.66 (d, J = 1.53 Hz, 1 H), 9.27 (s, 3 H).
(500 MHz, DMSO-d6) δ ppm 3.02 (s, 6 H), 4.52 (s, 2 H), 6.94 (d,
J = 8.54 Hz, 2 H), 7.63 (d, J = 8.54 Hz, 2 H), 7.93 (dd, J = 8.54,
1.83 Hz, 1 H), 8.21 (d, J = 8.54 Hz, 1 H), 8.52 (d, J = 1.53 Hz,
1 H), 9.73 (s, 1 H), 10.36 (br s, 1 H), 13.23 (br s, 1 H). Anal.
(C19H17N3O2S HCl 0.35H2O) C, H, N, Cl. HRMS (ESI-TOF)
3
3
calcd for C19H18N3O2S (M þ H)þ 352.1120; found 352.1122.
8-(4-Aminophenyl)-2-[(dimethylamino)methyl][1]benzothieno-
[3,2-d]pyrimidin-4(3H)-one (14k): LCMS (APCI) m/z 351 (M þ
H)þ; 1H NMR (400 MHz, DMSO-d6) δ ppm 3.04 (s, 6 H), 4.51 (s,
2 H), 6.86 (d, J = 8.59 Hz, 2 H), 7.57 (d, J = 8.59 Hz, 2 H), 7.92
(dd, J = 8.59, 2.15 Hz, 1 H), 8.19 (d, J = 8.59 Hz, 1 H), 8.47 (d,
J = 1.53 Hz, 1 H), 13.17 (s, 1 H).
2-[(Dimethylamino)methyl]-8-[4-(trifluoromethoxy)phenyl][1]-
benzothieno[3,2-d]pyrimidin-4(3H)-one (14l): LCMS (APCI) m/z
420 (M þ H)þ; 1H NMR (400 MHz, DMSO-d6) δ ppm 3.04 (s,
6 H), 4.50 (s, 2 H), 7.56 (d, J = 8.59 Hz, 2 H), 7.90 (d, J = 8.59 Hz,
2 H), 8.03 (dd, J = 8.44, 1.69 Hz, 1 H), 8.32 (d, J = 8.59 Hz, 1 H),
8.56 (d, J = 1.84 Hz, 1 H), 13.20 (s, 1 H).
2-[(Dimethylamino)methyl]-8-(3-fluoro-4-hydroxyphenyl)[1]benzo-
thieno[3,2-d]pyrimidin-4(3H)-one (14m): LCMS (APCI) m/z 370
(M þ H)þ; 1H NMR (500 MHz, DMSO-d6) δ ppm 3.05 (s, 6 H),
4.51 (s, 2 H), 7.12 (t, J = 8.70 Hz, 1 H), 7.45 (dd, J = 8.09, 1.98
Hz, 1 H), 7.59 (dd, J = 12.66, 2.29 Hz, 1 H), 7.94-8.00 (m, 1 H),
8.25 (d, J = 8.54 Hz, 1 H), 8.49 (d, J = 1.53 Hz, 1 H), 9.86(s, 1 H),
10.14 (s, 1 H), 13.20 (s, 1 H).
2-[(Dimethylamino)methyl]-8-[4-dimethylaminophenyl][1]benzo-
thieno[3,2-d]pyrimidin-4(3H)-one (14n): LCMS (APCI) m/z 379
(M þ H)þ; 1H NMR (500 MHz, DMSO-d6) δ ppm 2.98 (s, 6 H),
3.05 (s, 6 H), 4.52 (s, 2 H), 6.89 (d, J = 8.85 Hz, 2 H), 7.65 (d, J =
8.85 Hz, 2 H), 7.96 (dd, J = 8.54, 2.14 Hz, 1 H), 8.20 (d, J = 8.54
Hz, 1 H), 8.49 (d, J = 1.83 Hz, 1 H), 9.94 (s, 1 H), 13.18 (s, 1 H).
8-(4-Chloro-3-fluorophenyl)-2-[(dimethylamino)methyl][1]benzo-
thieno[3,2-d]pyrimidin-4(3H)-one (14o): LCMS (APCI) m/z 388
(M þ H)þ; 1H NMR (500 MHz, DMSO-d6) δ ppm 3.03 (s, 6 H),
4.49 (s, 2 H), 7.65-7.70 (m, 1 H), 7.73-7.81 (m, 1 H), 7.87 (dd,
J = 10.74, 2.15 Hz, 1 H), 8.06 (dd, J = 8.59, 2.15 Hz, 1 H), 8.32
(d, J = 9.21 Hz, 1 H), 8.58 (d, J = 1.23 Hz, 1 H).
8-(3-Chloro-5-fluorophenyl)-2-[(dimethylamino)methyl][1]benzo-
thieno[3,2-d]pyrimidin-4(3H)-one (14p): LCMS (APCI) m/z 388
(M þ H)þ; 1H NMR (500 MHz, DMSO-d6) δ ppm 3.03 (s, 6 H),
4.48 (s, 2 H), 7.48-7.58 (m, 1 H), 7.67 (d, J = 9.82 Hz, 1 H), 7.73
(s, 1 H), 8.08 (dd, J = 8.59, 1.53 Hz, 1 H), 8.33 (d, J = 8.59 Hz,
1 H), 8.57 (d, J = 1.53 Hz, 1 H).
8-(3,5-Dichlorophenyl)-2-[(dimethylamino)methyl][1]benzothieno-
[3,2-d]pyrimidin-4(3H)-one (14q): LCMS (APCI) m/z 404 (M þ
H)þ; 1H NMR (400 MHz, DMSO-d6) δ ppm 3.02 (s, 6 H), 4.48
(s, 2 H), 7.70 (t, J = 1.84 Hz, 1 H), 7.84 (d, J = 1.84 Hz, 2 H),
8.08 (dd, J = 8.59, 1.84 Hz, 1 H), 8.33 (d, J = 8.59 Hz, 1 H), 8.56
(d, J = 1.53 Hz, 1 H).
2-[(Dimethylamino)methyl]-8-thien-3-yl[1]benzothieno[3,2-d]-
pyrimidin-4(3H)-one (15a): LCMS (APCI) m/z 342 (M þ H)þ;
1H NMR (400 MHz, DMSO-d6) δ ppm 3.05 (s, 6 H), 7.66 (dd,
J = 5.06, 1.38 Hz, 1 H), 7.75 (dd, J = 5.06, 2.92 Hz, 1 H), 8.00
(dd, J = 2.92, 1.38 Hz, 1 H), 8.08 (dd, J = 8.44, 1.99 Hz, 1 H),
8.24 (d, J = 8.59 Hz, 1 H), 8.57 (d, J = 1.53 Hz, 1 H), 10.00 (s, 1
H), 13.16 (s, 1 H).
2-[(Dimethylamino)methyl]-8-pyrrolidin-1-yl[1]benzothieno[3,2-d]-
pyrimidin-4(3H)-one (17a). To a mixture of biphenyl-2-yldi-tert-
butylphosphine (5.3 mg, 0.018 mmol), Pd2(dba)3 (4.1 mg, 0.0044
mmol), and NaOt-Bu (26 mg, 0.27 mmol) in toluene (2.5 mL)
were added 8 (30 mg, 0.089 mmol) and pyrrolidine (0.015 mL,
0.18 mmol). The reaction mixture was purged with nitrogen,
heated at 120 °C for 20 min in a CEM microwave synthesizer,
and concentrated. The residue was purified by reverse-phase
preparative HPLC to give the desired product as the TFA salt
1
(25.6 mg, 52%): LCMS (APCI) m/z 329 (M þ H)þ; H NMR
(500 MHz, DMSO-d6) δ ppm 1.98-2.06 (m, 4 H), 3.02 (s, 6 H),
3.34 (t, J = 6.41 Hz, 4 H), 4.48 (s, 2 H), 7.06 (dd, J = 8.85, 2.44
Hz, 1 H), 7.32 (d, J = 2.44 Hz, 1 H), 7.91 (d, J = 8.85 Hz, 1 H),
10.05 (s, 1 H), 13.04 (s, 1 H).
2-[(Dimethylamino)methyl]-8-piperidin-1-yl[1]benzothieno[3,2-d]-
pyrimidin-4(3H)-one (17b). The title compound was prepared
using the same procedure as described for 17a: LCMS (APCI)
1
m/z 343 (M þ H)þ; H NMR (500 MHz, DMSO-d6) δ ppm
1.50-1.64 (m, 2 H), 1.64-1.81 (m, 4 H), 3.03 (s, 6 H), 3.19-3.36
(m, 4 H), 4.49 (s, 2 H), 7.49 (dd, J = 9.15, 2.44 Hz, 1 H), 7.77 (s,
1 H), 7.99 (d, J = 9.15 Hz, 1 H), 10.02 (br s, 1 H), 13.11 (br s, 1 H).
3-(4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)-
propane-1,2-diol (18c). 4-(4,4,5,5-Tetramethyl-1,3,2-dioxaboro-
lan-2-yl)phenol (220 mg, 1 mmol) in DME (5 mL) was treated
with 3-chloropropane-1,2-diol (166 mg, 1.5 mmol) and potassium
carbonate (691 mg, 5 mmol) and heated at 80 °C for 19 h.
Additional 3-chloropropane-1,2-diol (166 mg, 1.5 mmol) was
added, and heating was continued for 24 h. The reaction mixture
was cooled to room temperature, filtered through Celite, con-
centrated, and purified by column chromatography on silica gel
eluting with a solvent gradient of 0 to 1% MeOH in CH2Cl2 to
give 86 mg (29%) of the title compound: MS (DCI) m/z 312 (M þ
NH4)þ.
8-[4-(2,3-Dihydroxypropoxy)phenyl]-2-[(dimethylamino)methyl][1]-
benzothieno[3,2-d]pyrimidin-4(3H)-one (18a). A mixture of 8
(34 mg, 0.1 mmol), 18c (44 mg, 0.15 mmol), bis(triphenylphos-
phine)palladium(II) dichloride (7 mg, 0.01 mmol), and sodium
carbonate (2 M, 0.15 mL, 0.3 mmol) in a 7:2:3 mixture of DME/
EtOH/H2O (3 mL) was heated via microwave for 10 min at
150 °C, cooled to room temperature, filtered through a syringe
filter, concentrated, and purified by column chromatography on
silica gel eluting with 1:1 EtOAc/MeOH. The product was
further purified by reverse-phase preparative HPLC: 1H NMR
2-[(Dimethylamino)methyl]-8-(1H-pyrrol-2-yl)[1]benzothieno-
[3,2-d]pyrimidin-4(3H)-one (15b): LCMS (APCI) m/z 325 (M þ
H)þ; 1H NMR (500 MHz, DMSO-d6) δ ppm 3.06 (s, 6 H), 4.47
(s, 2 H), 6.08-6.27 (m, 1 H), 6.56-6.69 (m, 1 H), 6.86-7.01 (m,

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6633
(300 MHz, DMSO-d6) δ ppm 2.95 (s, 6 H), 3.48 (d, J = 5.43 Hz,
2 H), 3.83 (dq, J = 5.26, 5.03 Hz, 1 H), 3.94 (m, 1 H), 4.08 (dd,
J = 9.83, 4.41 Hz, 1 H), 4.40 (s, 2 H), 4.69 (s, 1 H), 4.97 (s, 1 H),
7.11 (m, 2 H), 7.72 (m, 2 H), 7.97 (dd, J = 8.65, 1.86 Hz, 1 H),
8.24 (d, J = 8.48 Hz, 1 H), 8.50 (d, J = 1.70 Hz, 1 H).
1H NMR (500 MHz, DMSO-d6) δ ppm 4.33 (d, J = 4.88 Hz,
2 H), 6.03 (dd, J = 7.48, 1.98 Hz, 2 H), 6.13 (t, J = 2.14 Hz, 1 H),
6.16 (d, J = 7.93 Hz, 1 H), 6.87 (t, J = 8.09 Hz, 1 H), 7.72 (d, J =
2.14 Hz, 2 H), 8.03 - 8.11 (m, 2 H), 8.19 (d, J = 8.24 Hz, 1 H), 8.53
(d, J = 1.53 Hz, 1 H), 9.02 (s, 1 H), 12.68 (s, 1 H).
2-(3-(4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)-
propyl)isoindoline-1,3-dione (18d). 4-(4,4,5,5-Tetramethyl-1,3,2-
dioxaborolan-2-yl)phenol (220 mg, 1 mmol) in DME (5 mL) was
treated with 2-(3-bromopropyl)isoindoline-1,3-dione (402 mg,
1.5 mmol) and potassium carbonate (691 mg, 5 mmol) and
heated at 80 °C for 19 h. Additional 2-(3-bromopropyl)isoindo-
line-1,3-dione (305 mg, 1.14 mmol) was added, and heating was
continued for 9 h. The reaction mixture was cooled to room
temperature, filtered through Celite, concentrated, and purified
by column chromatography on silica gel eluting with a solvent
gradient of 0 to 1% MeOH in CH2Cl2: MS (ESI) m/z 408 (M þ
H)þ; 1H NMR (500 MHz, DMSO-d6) δ ppm 2.05 (m, 2 H), 2.97
(s, 6 H), 3.00 (m, 2 H), 4.14 (t, J = 6.10 Hz, 2 H), 4.43 (s, 2 H),
7.11 (m, 2 H), 7.75 (m, 2 H), 7.82 (s, 2 H), 7.98 (dd, J = 8.54, 1.83
Hz, 1 H), 8.25 (d, J = 8.54 Hz, 1 H), 8.50 (d, J = 1.22 Hz, 1 H),
13.15 (s, 1 H).
8-[4-(3-Aminopropoxy)phenyl]-2-[(dimethylamino)methyl][1]benzo-
thieno[3,2-d]pyrimidin-4(3H)-one (18b). A mixture of 8 (34 mg,
0.1 mmol), 18d (61 mg, 0.15 mmol), bis(triphenylphosphine)-
palladium(II) dichloride (7 mg, 0.01 mmol), and sodium carbo-
nate (2 M, 0.15 mL, 0.3 mmol) in a 7:2:3 mixture of DME/
EtOH/H2O (3 mL) was heated via microwave for 10 min at
150 °C, cooled to room temperature, filtered through a syringe
filter, concentrated, and purified by column chromatography on
silica gel eluting with 0-50% MeOH in EtOAc. The product
was further purified by reverse-phase preparative HPLC: MS
(ESI) m/z 409 (M þ H)þ; 1H NMR (500 MHz, DMSO-d6) δ ppm
2.05 (m, 2 H), 2.79 (s, 6 H), 3.00 (m, 2 H), 4.14 (t, J = 6.10 Hz,
2 H), 4.43 (s, 2 H), 7.11 (m, 2 H), 7.75 (m, 2 H), 7.82 (s, 2 H), 7.98
(dd, J = 8.54, 1.83 Hz, 1 H), 8.25 (d, J = 8.54 Hz, 1 H), 8.50 (d,
J = 1.22 Hz, 1 H), 13.14 (s, 1 H).
Methyl 3-amino-5-bromobenzo[b]thiophene-2-carboxylate. To
a cold solution of 5-bromo-2-fluorobenzonitrile (13.5 g, 67.5
mmol) in DMF at 0 °C was added dropwise methyl 2-mercap-
toacetate (6.45 mL, 70.88 mmol). The reaction mixture was
stirred at 0 °C for 30 min, and then 5 M NaOH aqueous solution
(20.25 mL) was added dropwise. After stirring at 0 °C for 3 h, the
reaction mixture was quenched with ice-water. The resulting
precipitate was collected by filtration and dried to give 18.5 g of
white solid in 96% yield: LCMS (APCI) m/z 287 (M þ H)þ.
5-Bromo-3-[(2-chloroacetimidoyl)amino]benzo[b]thiophene-2-
carboxylic acid methyl ester. A suspension of methyl 3-amino-
5-bromobenzo[b]thiophene-2-carboxylate (7.2 g, 25.16 mmol)
in 4 N hydrochloric acid in dioxane (70 mL) was treated with
2-chloroacetonitrile (3.18 mL, 50.32 mmol) at room tempera-
ture for 3 h. The white solid was collected by filtration and dried
to give the desired product as the HCl salt quantitatively: LCMS
(APCI) m/z 362 (M þ H)þ.
2-{[(3-Hydroxyphenyl)amino]methyl}-8-phenyl[1]benzothieno-
[3,2-d]pyrimidin-4(3H)-one (19c). The title compound was pre-
pared under similar Suzuki coupling conditions as those described
1
for the synthesis of 14j: LCMS (APCI) m/z 400 (M þ H)þ; H
NMR (500 MHz, DMSO-d6) δ ppm 4.33 (d, J = 6.10 Hz, 2 H),
5.99-6.08 (m, 2 H), 6.13 (t, J = 2.14 Hz, 1 H), 6.14-6.21 (m, 1 H),
6.86 (t, J = 7.93 Hz, 1 H), 7.44 (t, J = 7.32 Hz, 1 H), 7.54 (t, J =
7.63 Hz, 2 H), 7.81 (d, J = 7.32 Hz, 2 H), 7.99 (dd, J = 8.54, 1.83
Hz, 1 H), 8.25 (d, J = 8.54 Hz, 1 H), 8.48 (d, J = 1.83 Hz, 1 H),
9.00 (s, 1 H), 12.70 (s, 1 H).
8-Bromo-2-{[(3S)-3-hydroxypyrrolidin-1-yl]methyl}[1]benzo-
thieno[3,2-d]pyrimidin-4(3H)-one. The title compound was pre-
pared using a procedure similar to that described for 6a: LCMS
1
(APCI) m/z 380 (M þ H)þ; H NMR (500 MHz, DMSO-d6)
δ ppm 1.56-1.66 (m, 1 H), 1.97-2.09 (m, 1 H), 2.51-2.61 (m,
2 H), 2.75-2.87 (m, 2 H), 3.67-3.78 (m, 2 H), 4.09-4.28 (m,
1 H), 7.81 (dd, J = 8.54, 2.14 Hz, 1 H), 8.15 (d, J = 8.85 Hz,
1 H), 8.31 (d, J = 2.14 Hz, 1 H).
2-{[(3S)-3-Hydroxypyrrolidin-1-yl]methyl}-8-thien-3-yl[1]benzo-
thieno[3,2-d]pyrimidin-4(3H)-one (20a). The title compound was
prepared under similar Suzuki coupling conditions as those
described for the synthesis of 14j: LCMS (APCI) m/z 384
(M þ H)þ; 1H NMR (500 MHz, DMSO-d6) δ ppm 2.00 (br s,
1 H), 2.04-2.37 (m, 1 H), 2.62-3.11 (m, 2 H), 3.69-4.10 (m, J =
73.54 Hz, 2 H), 4.51 (br s, 1 H), 4.63 (d, J = 13.43 Hz, 2 H), 5.55
(br s, 1 H), 7.66-7.71 (m, 1 H), 7.75 (dd, J = 5.03, 2.90 Hz, 1 H),
8.03 (dd, J = 2.90, 1.37 Hz, 1 H), 8.08 (dd, J = 8.54, 1.83 Hz,
1 H), 8.24 (d, J = 8.54 Hz, 1 H), 8.56 (s, 1 H), 10.47 (br s, 1 H),
13.15 (br s, 1 H).
2-{[(3S)-3-Hydroxypyrrolidin-1-yl]methyl}-8-phenyl[1]benzo-
thieno[3,2-d]pyrimidin-4(3H)-one (20b). The title compound was
prepared under similar Suzuki coupling conditions as those
described for the synthesis of 14j: LCMS (APCI) m/z 378 (M þ
1
H)þ; H NMR (500 MHz, pyridine-d5) δ ppm 1.98-2.10 (m,
1 H), 2.16-2.28 (m, 1 H), 2.87-2.98 (m, 1 H), 3.13-3.22 (m,
2 H), 3.22-3.30 (m, 1 H), 4.08-4.35 (m, 2 H), 4.58-4.75 (m,
1 H), 7.41 (t, J = 7.32 Hz, 1 H), 7.51 (t, J = 7.63 Hz, 2 H), 7.77
(d, J = 7.32 Hz, 2 H), 7.90 (dd, J = 8.39, 1.98 Hz, 1 H), 8.07 (d,
J = 8.24 Hz, 1 H), 8.79 (d, J = 1.53 Hz, 1 H).
8-(4-Hydroxyphenyl)-2-{[(3S)-3-hydroxypyrrolidin-1-yl]methyl}-
[1]benzothieno[3,2-d]pyrimidin-4(3H)-one (20c). The title com-
pound was prepared under similar Suzuki coupling conditions
as those described for the synthesis of 14j: LCMS (APCI) m/z
1
394 (M þ H)þ; H NMR (500 MHz, DMSO-d6) δ ppm 1.97
(br s, 1 H), 2.08-2.34 (m, 1 H), 3.34 (br s, 2 H), 3.87 (br s, 2 H),
4.49 (s, 1 H), 4.60 (br s, 2 H), 5.52 (s, 1 H), 6.93 (d, J = 8.85 Hz,
2 H), 7.61 (d, J = 8.54 Hz, 2 H), 7.93 (dd, J = 8.54, 1.83 Hz,
1 H), 8.22 (d, J = 8.54 Hz, 1 H), 8.45 (s, 1 H), 9.68 (s, 1 H),
10.17-10.72 (br s, 1 H), 13.12 (s, 1 H).
2-{[(3-Hydroxyphenyl)amino]methyl}-8-thien-3-yl[1]benzothieno-
[3,2-d]pyrimidin-4(3H)-one (19a). A mixture of 5-bromo-3-[(2-
chloroacetimidoyl)amino]benzo[b]thiophene-2-carboxylic acid
methyl ester (30 mg, 0.076 mmol) and 3-aminophenol (41 mg,
0.38 mmol) in DMF (2 mL) was stirred at room tempera-
ture overnight and concentrated. The residue was purified by
reverse-phase preparative HPLC to provide the desired product
as the TFA salt in 56% yield: LCMS (APCI) m/z 404 (M þ H)þ;
1H NMR (500 MHz, DMSO-d6) δ ppm 4.31 (d, J = 5.80 Hz,
2 H), 6.00-6.05 (m, 2 H), 6.11 (t, J = 2.14 Hz, 1 H), 6.15 (dd,
J = 7.93, 1.53 Hz, 1 H), 6.87 (t, J = 7.93 Hz, 1 H), 7.82 (dd, J =
8.70, 1.98 Hz, 1 H), 8.15 (d, J = 8.85 Hz, 1 H), 8.40 (d, J = 1.83
Hz, 1 H), 9.02 (s, 1 H), 12.76 (br s, 1 H).
4-(2-{[(3S)-3-Hydroxypyrrolidin-1-yl]methyl}-4-oxo-3,4-dihydro-
[1]benzothieno[3,2-d]pyrimidin-8-yl)benzonitrile (20d). The title
compound was prepared under similar Suzuki coupling condi-
tions as those described for the synthesis of 14j: LCMS (APCI)
1
m/z 403 (M þ H)þ; H NMR (500 MHz, pyridine-d5) δ ppm
2.00-2.09 (m, 1 H), 2.16-2.27 (m, 1 H), 2.83-2.92 (m, 1 H),
3.10-3.19 (m, 2 H), 3.20-3.27 (m, 1 H), 4.08-4.30 (m, 2 H),
4.56-4.79 (m, 1 H), 7.58 (s, 2 H), 7.88 (dd, J = 8.39, 1.98 Hz,
1 H), 8.13 (d, J = 8.24 Hz, 1 H), 8.78 (d, J = 1.22 Hz, 1 H).
8-[(E)-2-Cyclopropylvinyl]-2-{[(3S)-3-hydroxypyrrolidin-1-yl]-
methyl}[1]benzothieno[3,2-d]pyrimidin-4(3H)-one (20e). The title
compound was prepared under similar Suzuki coupling condi-
tions as those described for the synthesis of 14j: LCMS (APCI)
2-{[(3-Hydroxyphenyl)amino]methyl}-8-thien-3-yl[1]benzothieno-
[3,2-d]pyrimidin-4(3H)-one (19b). The title compound was pre-
pared under similar Suzuki coupling conditions as those described
for the synthesis of 14j: LCMS (APCI) m/z 406 (M þ H)þ;
1
m/z 368 (M þ H)þ; H NMR (400 MHz, DMSO-d6) δ ppm
0.53-0.62 (m, 2 H), 0.78-0.89 (m, 2 H), 1.56-1.74 (m, 1 H),
1.91-2.36 (m, 2 H), 3.76-4.04 (m, 2 H), 4.57 (d, J = 46.95 Hz,

6634 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Tao et al.
3 H), 5.40 (s, 1 H), 6.03 (dd, J = 15.65, 8.90 Hz, 1 H), 6.68
(d, J = 15.65 Hz, 1 H), 7.76 (dd, J = 8.59, 1.84 Hz, 1 H),
8.08 (d, J = 8.29 Hz, 1 H), 8.18 (s, 1 H), 10.41 (s, 1 H), 13.04
(s, 1 H).
per manufacturer’s instructions. Analysis was performed using
an fmax fluorescence microplate reader (Molecular Devices,
Sunnyvale, CA), set at the excitation wavelength of 544 nm and
emission wavelength of 595 nm. Data were analyzed using SOFT-
max PRO software provided by the manufacturer.
8-Bromo-2-((phenethylamino)methyl)[1]benzothieno[3,2-d]pyri-
midin-4(3H)-one (21). The title compound was prepared using
the same procedure as described for the synthesis of 19a: H
Effect of PIM inhibitors on the Bad Phosphorylation. Cells
were treated with Pim inhibitors for 2 h and then scraped into
cold PBS, pelleted, and then resuspended in 100-200 μL ice-
cold insect cell lysis buffer supplemented with protease inhibi-
tors. Cells were lysed by sonication, and the debris was cleared in
a microcentrifuge. Forty micrograms of lysate was loaded in
each well of 10% Tris-glycine polyacrylamide minigels for
SDS-PAGE analysis. Proteins were transferred to PVDF
membranes, blocked for 1 h in TBS-T plus 5% (w/v) powdered
blotting grade milk, and then probed overnight at 4 °C with
primary antibody (P-Bad antibody, S112 antibody from Cell
Signaling Technology) at a 1:2000 dilution in blocking solution.
Blots were developed using enhanced chemiluminescence (ECL
Plus) reagents from Amersham Biosciences. P-Bad bands were
scanned using a GS-800 densitometer to calculate the EC50
values.
1
NMR (400 MHz, DMSO-d6) δ ppm 3.03-3.12 (m, 2 H),
3.40-3.47 (m, 2 H), 4.39 (s, 2 H), 7.27-7.34 (m, 3 H), 7.38
(t, J = 7.48 Hz, 2 H), 7.87 (dd, J = 8.70, 1.98 Hz, 1 H), 8.21 (d,
J = 8.54 Hz, 1 H), 8.47 (d, J = 2.14 Hz, 1 H).
Pim Kinase Assays. Kinase assays were conducted as follows
with final concentrations as listed. In 384-well v-bottom poly-
propylene plates, 10 μL of compound (2% DMSO) was mixed
with 20 μL of Pim-1 (50 pM), Pim-2 (500 pM), or Pim-3 (300 pM)
and peptide substrate (biotin-C6linker-VRRLRRLTAREAA)
(2 μM), followed by immediate initiation with 20 μL of λ-[33P]-
ATP (5 μM, 2mCi/μmol) using a reaction buffer comprising
25 mM HEPES, pH 7.5, 0.5 mM DTT, 10 mM MgCl2, 100 μM
Na3VO4, and 0.075 mg/mL Triton X-100. Reactions were quen-
ched after 1 h by the addition of 50 μL stop buffer (50 mM
EDTA, 2 M NaCl). Eighty microliters of the stopped reactions
were transferred to 384-well streptavidin-coated plates (Flash-
Plate Plus, Perkin-Elmer), incubated 30 min at room temperature
and washed three times with 0.05% Tween-20/PBS using an
ELX-405 automated plate washer (BioTek) and counted on a
TopCount Scintillation Plate Reader (Packard).
Selectivity Kinase Assays. Kinases were assayed using either
radiometric or homogeneous time-resolved fluorescence
(HTRF) in vitro kinase methods with the choice of format to
provide maximal reagent efficiency. Radiometric assays were
conducted exactly as described for the Pim in vitro assay except
for the enzyme concentration and substrate used, which were
custom to each kinase. Substrates were either chosen from those
described in literature/vendor protocols or identified through
screening of a 720 peptide Jerini kinase substrate set (Jerinin
AG). HTRF assays were conducted using kinase-specific kinase
concentration, ATP concentrations approximately 5-fold the
respective ATP Km, 0.5 μM biotinylated peptide substrate, 1 h
reaction time, followed by addition of stop/detection buffer
(30 mM EDTA, 1 μg/mL streptavidin-APC (Prozyme), 50 ng/
mL europium cryptate-conjugated phospho-specific mono-
clonal antibodiy (CisBio), 30 mM HEPES, pH 7.5, 120 mM
KF, 0.005% Tween-20, 0.05% BSA). The antibody was PT66-K
europium cryptate for tyrosine kinases or that from the Ser/Thr
KinEase assay kit for Ser/Thr kinases. Quenched reactions were
allowed to stand at room temperature for 1 h and then read in a
time-resolved fluorescence detector (Envision, Perkin-Elmer) at
615 and 665 nm simultaneously. The ratio between the signal of
615 and 665 nm was used in the calculation of the IC50. Serine
kinases were assayed in 25 mM HEPES, pH 7.5, 0.5 mM DTT,
10 mM MgCl2, 100 μM Na3VO4, 0.075 mg/mL Triton X-100,
and 2% DMSO. Tyrosine kinases were assayed in 50 mM
HEPES, pH 7.5, 10 mM MgCl2, 2 mM MnCl2, 0.1% BSA,
1 mM DTT, and 2% DMSO. Kinase-specific reagents (phos-
photidyl serine, diacyl glycerol, calcium chloride, calmodulin,
cGMP) were employed only where required.
X-ray Crystal Structure. Pim-1 ([Pim-1 (29-313)]-VD-6His)
was expressed in Escherichia coli, purified, and crystallized using
an adapted version of the method described by Kumar et al.43 In
brief, the protein was purified by nickel affinity, followed by ion
exchange and finally size-exclusion chromatography. Protein
ligand complexes were prepared by adding compound dissolved
in DMSO (dimethyl sulfoxide) to the protein (18 mg/mL) to
provide a final compound concentration of 1 mM. Crystal-
lization plates (hanging drop vapor diffusion) were set using an
equal volume of protein (18 mg/mL) and reservoir solution
(0.4-0.9 M sodium acetate, 0.1 M imidazole pH 6.5) with the
plate being incubated at 4 °C. Crystals were cryo-cooled using
30% glycerol. Data were collected at APS, IMCA CAT BM-17,
and the data were processed using HKL200044 and refined using
Refmac.45-47 Crystals of 3b diffracted to 2.1 A, with space
˚
group P65, a = b = 96.8, c = 80.8, and refined to an Rwork
23.3% and Rfree = 25.7%. Crystals of 6e diffracted to 2.40 A,
with space group P65, a = b = 98.3, c = 80.6, and refined to an
=
˚
R
work = 22.8% and Rfree = 26.2%. Crystals of 12b diffracted to
˚
2.8 A, with space group P65, a = b = 96.8, c = 80.8, and refined
to an Rwork = 21.6% and Rfree = 26.6%.
Acknowledgment. The authors thank Dr. Andrew Souers
for critical reading of the manuscript, and Dr. Steven Elmore
for support. The authors also wish to thank Dr. Michael
Cohen for providing LaCaP-BAD cell line, the Department of
Structural Chemistry for measuring NMR and MS spectra,
the High-Through-Put Purification Group for analytical
LC-MS, the Protein Biochemistry Group for the purification
of Pim-1 protein used for X-ray crystallographic analysis, and
the HT-ADME Group for the ADME profile of compound
14j. The X-ray diffraction data were collected at beamline
17-BM in the facilities of the Industrial Macromolecular
Crystallography Association Collaborative Access Team
(IMCA-CAT) at the Advanced Photon Source. These facili-
ties are supported by the companies of the Industrial
Macromolecular Crystallography Association through a con-
tract with Illinois Institute of Technology (IIT), executed
through IIT’s Center for Synchrotron Radiation Research
and Instrumentation. Use of the Advanced Photon Source
was supported by the U.S. Department of Energy, Basic
Energy Sciences, Office of Science, under Contract No.
W-31-109-Eng-38.
Cell Viability Assays. K562 cells (ATCC, Manassas, VA) were
cultured in Iscove’s medium plus 10% fetal bovine serum
(Invitrogen, Carlsbad, CA) and maintained at 37 °C (5% CO2)
in a humidified incubator. MV4-11 cells (ATCC, Manassas, VA)
were cultured under the same conditions in RPMI þ 10% fetal
bovine serum (Invitrogen, Carlsbad, CA). Cells were seeded into
96-well tissue culture plates at 50 000 cells per well (100 μL medium
per well), and compounds were then added at 2ꢀ concentrations in
100 μL medium to give final concentrations ranging from 0.01 to
30 μM (half-log dilutions). The cells were incubated in the presence
of compounds for 48 h at 37 °C (5% CO2) and then analyzed
by adding 20 μL alamarBlue reagent (Invitrogen, Carlsbad, CA)
to each well and incubating until the reaction was complete, as
Note Added after ASAP Publication. This paper was
published ASAP on October 16, 2009 with typographical errors.
The revised version was published on October 20, 2009.

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6635
References
transcriptional target of divergent EWS/ETS oncoproteins. Mol.
Cell. Biol. 2003, 23, 3897–3908. (b) Fuji, C.; Nakamoto, Y.; Lu, P.;
Tsuneyama, K.; Popivanova, B. K.; Kaneko, S.; Mukaida, N. Aberrant
expression of serine/threonine kinase Pim-3 in hepatocellular carcino-
ma development and its role in the proliferation of human hepatoma cell
lines. Int. J. Cancer 2005, 114, 209–218. (c) Li, Y.-Y.; Popivanova, B.
K.; Nagai, Y.; Ishikura, H.; Fujii, C.; Mukaida, N. Pim-3, a proto-
oncogene with serine/threonine kinase activity, is aberrantly expressed
in human pancreatic cancer and phosphorylates Bad to block Bad-
mediated apoptosis in human pancreatic cancer cell lines. Cancer Res.
2006, 66, 6741–6747. (d) Popivanova, B. K.; Li, Y.-Y.; Zheng, H.;
Omura, K.; Fujii, C.; Tsuneyama, K.; Mukaida, N. Proto-oncogene,
Pim-3 with serine/threonine kinase activity, is aberrantly expressed in
human colon cancer cells and can prevent Bad-mediated apoptosis.
Cancer Sci. 2007, 98, 321–328.
(1) Cuypers, H. T.; Selten, G.; Quint, W.; Zijlstra, M.; Maandag, E. R.;
Boelens, W.; van Wezenbeek, P.; Melief, C.; Berns, A. Murine
leukemia virus-induced T-cell lymphomagenesis: Integration of pro-
viruses in a distinct chromosomal region. Cell 1984, 37, 141–150.
(2) Mikkers, H.; Allen, J.; Knipscheer, P.; Romeyn, L.; Hart, A.; Vink,
E.; Berns, A. High-throughput retroviral tagging to identify com-
ponents of specific signaling pathways in cancer. Nat. Genet. 2000,
32, 153–159.
(3) van Lohuizen, M.; Verbeek, S.; Krimpenfort, P.; Domen, J.; Saris,
C.; Radaszkiewicz, T.; Berns, A. Predisposition to lymphomagen-
esis in pim-1 transgenic mice: Cooperation with c-myc and N-myc
in murine leukemia virus-induced tumors. Cell 1989, 56, 673–682.
(4) van Lohuizen, M.; Verbeek, S.; Scheijen, B.; Wientjens, E.; van der
Gulden, H.; Berns, A. Identification of cooperating oncogenes in
Eμ-myc transgenic mice by provirus tagging. Cell 1991, 65, 737–
752.
(5) van der Lugt, N. M. T.; Domen, J.; Verhoeven, E.; Linders, K.; van
der Gulden, H.; Allen, J.; Berns, A. Proviral tagging in Eμ-myc
transgenic mice lacking the Pim-1 proto-oncogene leads to com-
pensatory activation of Pim-2. EMBO J. 1995, 14, 2536–2544.
(6) Mikkers, H.; Nawijn, M.; Allen, J.; Brouwers, C.; Verhoeven, E.;
Jonkers, J.; Berns, A. Mice deficient for all Pim kinases display
reduced body size and impaired responses to hematopoietic growth
factors. Mol. Cell. Biol. 2004, 24, 6104–6115.
(7) Lilly, M.; Sandholm, J.; Cooper, J. J.; Koskinen, P. J.; Kraft, A.
The Pim-1 serine kinase prolongs survival and inhibits apoptosis-
related mitochondrial dysfunction in part through a bcl-2-depen-
dent pathway. Oncogene 1999, 18, 4022–4031.
(8) Fox, C. J.; Hammerman, P. S.; Cinalli, R. M.; Master, S. R.;
Chodosh, L. A.; Thompson, C. B. The serine/threonine kinase
Pim-2 is a transcriptionally regulated apoptotic inhibitor. Genes
Dev. 2003, 17, 1841–1854.
(9) Yan, B.; Zemskova, M.; Holder, S.; Chin, V.; Kraft, A.; Koskinen,
P. J.; Lilly, M. The Pim-2 kinase phosphorylates Bad on serine 112
and reverses Bad-induced cell death. J. Biol. Chem. 2003, 278,
45358–45367.
(10) Macdonald, A.; Campbell, D. G.; Toth, R.; McLauchlan, H.;
Hastie, C. J.; Arthur, J. S. Pim kinases phosphorylate multiple
sites on Bad and promote 14-3-3 binding and dissociation from
Bcl-XL. BMC Cell Biol. 2006, 7, 1.
(11) Dhanasekaran, S. M.; Barrette, T. R.; Ghosh, D.; Shah, R.;
Varambally, S.; Kurachi, K.; Pienta, K. J.; Rubin, M. A.; Chin-
naiyan, A. M. Delineation of prognostic biomarkers in prostate
cancer. Nature 2001, 412, 822–826.
(22) Debreczeni, J. E.; Bullock, A. N.; Atilla, G. E.; Williams, D. S.;
Bregman, H.; Knapp, S.; Meggers, E. Ruthenium half-sandwich
complexes bound to protein kinase pim1. Angew. Chem., Int. Ed.
2006, 45, 1580–1585.
(23) Bregman, H.; Meggers, E. Ruthenium half-sandwich complexes as
protein kinase inhibitors: an N-succinimidyl ester for rapid deriva-
tizations of the cyclopentadienyl moiety. Org. Lett. 2006, 8, 5465–
5468.
(24) Bullock, A. N.; Debreczeni, J. E.; Fedorov, O. Y.; Nelson, A.;
Marsden, B. D.; Knapp, S. Structural basis of inhibitor specificity
of the human protooncogene proviral insertion site in moloney
murine leukemia virus (Pim-1) kinase. J. Med. Chem. 2005, 48,
7604–7614.
(25) Pogacic, V.; Bullock, A. N.; Fedorov, O.; Filippakopoulos, P.;
Gasser, C.; Biondi, A.; Meyer-Monard, S.; Knapp, S.; Schwaller, J.
Structural analysis identifies imidazo[1,2-b]pyridazines as Pim
kinase inhibitors with in vitro antileukemic activity. Cancer Res.
2007, 67, 6916–6924.
(26) Bearss, D.; Liu, X.-H.; Grand, C.; Gourley, E.; Lamb, J.; Lloyd,
M.; Vankayalapati, H. Discovery and characterization of a small
molecule inhibitor for Pim-1 kinase. Abstract 414, American
Society of Hematology National Meeting, 2006.
(27) (a) Lamb, J.; Gourley, E.; Liu, X.-H.; Grand, C.; Vankayalapati,
H.; Warner, S.; Bearss, D. A small molecule inhibitor of Pim-1
kinase with activity in both hematological and solid tumor malig-
nancies. Abstract 985, AACR-NCI-EOTRC International Con-
ference on Molecular Targets and Cancer Therapeutics, San
Francisco, CA, 2007. (b) Bearss, D. J.; Liu, X.-H.; Vankayalapati,
H.; Xu, Y. Imidazo[1,2-b]pyridazine and pyrazolo[1,5-a]pyrimidine
derivatives and their use as protein kinase inhibitors. WO
2008058126, 2005.
(12) Valdman, A.; Fang, X.; Pang, S. T.; Ekman, P.; Egevad, L. Pim-1
expression in prostatic intraepithelial neoplasia and human pros-
tate cancer. Prostate 2004, 60, 367–371.
(13) Xu, Y.; Zhang, T.; Tang, H.; Zhang, S.; Liu, M.; Ren, D.; Niu, Y.
Overexpression of Pim-1 is a potential biomarker in prostate
carcinoma. J. Surg. Oncol. 2005, 92, 326–330.
(14) Kim, K. T.; Baird, K.; Ahn, J. Y.; Meltzer, P.; Lilly, M.; Levis, M.;
Small, D. Pim-1 is up-regulated by constitutively activated FLT3
and plays a role in FLT3-mediated cell survival. Blood 2005, 105,
1759–1767.
(15) Pasqualucci, L.; Neumeister, P.; Goosens, T.; Nanjangud, G.;
Chaganti, R. S.; Kuppers, R.; Dalla-Favera, R. Hypermutation
of multiple proto-oncogenes in B-cell diffuse large-cell lymphomas.
Nature 2001, 412, 341–346.
(28) Cheney, I. W.; Yan, S.; Appleby, T.; Walker, H.; Vo, T.; Yao, N.;
Hamatake, R.; Hong, Z.; Wu, J. Z. Identification and structure-
activity relationships of substituted pyridones as inhibitors of Pim1
kinase. Bioorg. Med. Chem. Lett. 2007, 17, 1679–1683.
(29) Holder, S.; Lilly, M.; Brown, M. L. Comparative molecular field
analysis of flavonoid inhibitors of the Pim-1 kinase. Bioorg. Med.
Chem. 2007, 15, 6463–6473.
(30) Holder, S.; Zemskova, M.; Zhang, C.; Tabrizizad, M.; Bremer, R.;
Neidigh, J. W.; Lilly, M. B. Characterization of a potent and
selective small-molecule inhibitor of the Pim1 kinase. Mol. Cancer
Ther. 2007, 6, 163–172.
(31) (a) Pierce, A. C.; Jacobs, M.; Stuver-Moody, C. Docking study
yields four novel inhibitors of the protooncogene Pim-1 kinase.
J. Med. Chem. 2008, 51, 1972–1975.
(32) Tong, Y.; Stewart, K. D.; Thomas, S.; Przytulinska, M.; Johnson,
E. F.; Klinghofer, V.; Leverson, J.; McCall, O.; Soni, N. B.; Luo,
Y.; Lin, N. -H.; Sowin, T. J.; Giranda, V. L.; Penning, T, D.
Isoxazolo[3,4-b]quinoline-3,4(1H,9H)-diones as unique, potent
and selective inhibitors for Pim-1 and Pim-2 kinases: Chemistry,
biological activities, and molecular modeling. Bioorg. Med. Chem.
Lett. 2008, 18, 5206–5208.
(33) Hammerman, P. S.; Fox, C. J.; Birnbaum, M. J.; Thompson, C. B.
Pim and Akt oncogenes are independent regulators of hemato-
poietic cell growth and survival. Blood 2005, 105, 4477–4483.
(34) Mizuki, M.; Schwable, J.; Steur, C.; Choudhary, C.; Agrawal, S.;
(16) Gaidano, G.; Pasqualucci, L.; Capello, D.; Berra, E.; Deambrogi,
C.; Rossi, D.; Larocca, L. M.; Gloghini, A.; Carbone, A.;
Dalla-Favera, R. Abberant somatic hypermutation in multiple
subtypes of AIDS-associated non-Hodgkin lymphoma. Blood
2003, 102, 1833–1841.
(17) Chen, W. W.; Chan, D. C.; Donald, C.; Lilly, M. B.; Kraft, A. S.
Pim family kinases enhance tumor growth of prostate cancer cells.
Mol. Cancer Res. 2003, 3, 443–451.
(18) Dai, J. M.; Zhang, S. Q.; Zhang, W.; Lin, R. X.; Ji, Z. Z.; Wang,
S. Q. Antisense oligodeoxynucleotides targeting the serine/threo-
nine kinase Pim-2 inhibited proliferation of DU-145 cells. Acta
Pharmacol. Sinica 2005, 26, 364–368.
(19) Dai, H.; Li, R.; Wheeler, T.; Diaz de Vivar, A.; Frolov, A.; Tahir,
S.; Agoulnik, I.; Thompson, T.; Rowley, D.; Ayala, G. Pim-2
upregulation: biological implications associated with disease pro-
gression and perineural invasion in prostate cancer. Prostate 2005,
65, 276–286.
€
Sargin, B.; Steffen, B.; Matsumura, I.; Kanakura, Y.; Bohmer,
F. D.; Muller-Tidow, C.; Berdel, W. E.; Serve, H. Suppression of
€
myeloid transcription factors and induction of STAT response
genes by AML-specific Flt3 mutations. Blood 2003, 101, 3164–
3173.
(35) Adam, M.; Pogacic, V.; Bendit, M.; Chappuis, R.; Nawijn, M. C.;
Duyster, J.; Fox, C. J.; Thompson, C. J.; Cools, J.; Schwaller, J.
Targeting Pim kinases impairs survival of hematopoietic cells
transformed by kinase inhibitor-sensitive and kinase inhibitor-
resistant forms of Fms-like tyrosine kinase 3 and BCR/ABL.
Cancer Res. 2006, 66, 3828–835.
(20) For a review, see: Li, X.; Zhu, S; Liu, L.; Huang, Y. Molecular
characterization and biological effects of the Pim-3 gene. Yixue
Fenzi Shengwuxue Zazhi 2008, 5, 146–148.
(21) (a) Deneen, B.; Welford, S. M.; Ho, T.; Hernandez, F.; Kurland,
I.; Denny, C. T. Pim3 proto-oncogene kinase is a common

6636 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Tao et al.
(36) (a) Xia, Z.; Knaak, C.; Ma, J.; Beharry, Z. M.; McInnes, C.; Wang,
W.; Kraft, A. S.; Smith, C. D. Synthesis and evaluation of novel
inhibitors of Pim-1 and Pim-2 protein kinases. J. Med. Chem. 2009,
52, 74–86. (b) Qian, K.; Wang, L.; Cywin, C. L.; Farmer, B. T.; Hickey,
E.; Homon, C.; Jakes, S.; Kashem, M. A.; Lee, G.; Leonard, S.; Li, J.;
Magboo, R.; Wang, M.; Pack, E.; Peng, C.; Prokopowicz, A.; Welzel,
M.; Wolak, J.; Morwick, T. Hit to lead account of the discovery of a
new class of inhibitors of Pim kinases and crystallographic studies
revealing an unusual kinase binding mode. J. Med. Chem. 2009, 52,
1814–1827.
Fesik, S. W.; Rosenberg, S.; Elmore, S. W. Studies leading to potent,
dual inhibitors of Bcl-2 and Bcl-xL. J. Med. Chem. 2007, 50, 641–662.
(d) Zhu, G. D.; Gandhi, V. B.; Gong, J.; Thomas, S.; Woods, K. W.;
Song, X.; Li, T.; Diebold, R. B.; Luo, Y.; Liu, X.; Guan, R.; Klinghofer,
V.; Johnson, E. F.; Bouska, J.; Olson, A.; Marsh, K. C.; Stoll, V. S.;
Mamo, M.; Polakowski, J.; Campbell, T. J.; Martin, R. L.; Gintant,
G. A.; Penning, T. D.; Li, Q.; Rosenberg, S. H.; Giranda, V. L. Syntheses
of potent, selective, and orally bioavailable indazole-pyridine series of
protein kinase B/Akt inhibitors with reduced hypotension. J. Med.
Chem. 2007, 50, 2990–3003. (e) Park, C.-M; Bruncko, M.; Adickes, J.;
Bauch, J.; Ding, H.; Kunzer, A.; Marsh, K. C.; Nimmer, P.; Shoemaker,
A. R.; Song, X.; Tahir, S. K.; Tse, C.; Wang, X.; Wendt, M. D.; Yang, X.;
Zhang, H.; Fesik, S. W.; Rosenberg, S. H.; Elmore, S. W. Discovery of
an orally bioavailable small molecule inhibitor of prosurvival B-cell
lymphoma 2 proteins. J. Med. Chem. 2008, 51, 6902–6915.
(41) Taghiyev, A. F.; Guseva, N. V.; Harada, H.; Knudson, C. M.;
Rokhlin, O. W.; Cohen, M. B. Overexpression of Bad potentiates
sensitivity to tumor necrosis factor-related apoptosis-inducing
ligand treatment in the prostatic carcinoma cell line LNCaP. Mol
Cancer Res. 2003, 1, 500–507.
(42) Pim kinases have emerged as a potential target in immunology:
(a) Aho, T. L. T.; Lund, R. J.; Ylikoski, E. K.; Matikainen, S.;
Lahesmaa, R.; Koskinen, P. J. Expression of human pim family
genes is selectively up-regulated by cytokines promoting T helper
type 1, but not T helper type 2, cell differentiation. Immunology
2005, 116, 82–88. (b) Li, J.; Peet, G. W.; Balzarano, D.; Li, X.; Massa,
P.; Barton, R. W.; Marcu, K. B. Novel NEMO/IκB kinase and NF-κB
target genes at the pre-B to immature B cell transition. J. Biol. Chem.
2001, 276, 18579–18590. (c) Rainio, E.; Sandholm, J.; Koskinen, P. J.
Cutting edge: transcriptional activity of NFATc1 is enhanced by the
Pim-1 kinase. J. Immunol. 2002, 168, 1524–1527. (d) Zhu, N.;
Ramirez, L. M.; Lee, R. L.; Magnuson, N. S.; Bishop, G. A.; Gold,
M. R. CD40 signaling in B cells regulates the expression of the Pim-1
kinase via the NF-κB pathway. J. Immunol. 2002, 168, 744–754.
(e) Fox, C. J.; Hammerman, P. S.; Thompson, C. B. The Pim kinases
control rapamycin-resistant Tcell survival and activation. J. Exp. Med.
2005, 201, 259–266.
(37) (a) Robba, M.; Touzot, P.; Riquelme, R. M. Synthese de benzo(1)-
thieno[2,3 d]pyrimidines et de benzo(1)thieno[3,2-d]pyrimidines.
Tetrahedron Lett. 1972, 44, 4549–4551. (b) Robba, M.; Touzot, P.; El-
Kashef, H. [1]Benzothienopyrimidines. I. Etude de la 3H-benzothieno-
[3,2-d]pyrimidone-4. J. Heterocycl. Chem. 1980, 17, 923–928.
(c) Bridges, A. J.; Zhou, H. Synthetic methodology for the preparation
of benzo[b]thieno[3,2-d]pyrimidines substituted with electron donating
substituents on the benzene ring. J. Heterocycl. Chem. 1997, 34, 1134–
1139. (d) Showalter, H. D. H.; Bridges, A. J.; Zhou, H.; Sercel, A. D.;
McMichael, A.; Fry, D. W. Tyrosine kinase inhibitors. 16. 6,5,6-
Tricyclic benzothieno[3,2-d]pyrimidines and pyrimido[5,4-b]- and
-[4,5-b]indoles as potent inhibitors of the epidermal growth factor
receptor tyrosine kinase. J. Med. Chem. 1999, 42, 1814–1827.
(38) (a) Qian, K. C.; Wang, L.; Hickey, E. R.; Studts, J.; Barringer, K.;
Peng, C.; Kronkaitis, A.; Li, J.; White, A.; Mische, S.; Farmer, B.
Structural basis of constitutive activity and a unique nucleotide
binding mode of human Pim-1 kinase. J. Biol. Chem. 2005, 280,
6130–6137. (b) Jacobs, M. D.; Black, J.; Futer, O.; Swenson, L.; Hare,
B.; Fleming, M.; Saxena, K. Pim-1 ligand-bound structures reveal the
mechanism of serine/threonine kinase inhibition by LY294002. J. Biol.
Chem. 2005, 280, 13728–13734. (c) Kumar, A.; Mandiyan, V.; Suzuki,
Y.; Zhang, C.; Rice, J.; Tsai, J.; Artis, D. R.; Ibrahim, P.; Bremer, R.
Crystal structures of proto-oncogene kinase Pim1: A target of aberrant
somatic hypermutations in diffuse large cell lymphoma. J. Mol. Biol.
2005, 348, 183–193. (d) Bullock, A. N.; Debreczeni, J. E.; Amos, A. L.;
Knapp, S.; Turk, B. E. Structure and substrate specificity of the Pim-1
kinase. J. Biol. Chem. 2005, 280, 41675–41682.
(39) Knight, Z. A.; Shokat, K. M. Features of selective kinase inhibi-
(43) Kumar, A.; Mandiyan, V.; Suzuki, Y.; Zhang, C.; Rice, J.; Tsai, J.;
Artis, D. R.; Ibrahim, P.; Bremer, R. Crystal structures of proto-
oncogene kinase Pim-1: A target of aberrant somatic hypermuta-
tions in diffuse large cell lymphoma. J. Mol. Biol. 2005, 348, 183–
193.
(44) Otwinowski, Z.; Minor, W. In Methods in Enzymology, Macro-
molecular Crystallography, Part A; Carter, C. W., Jr., Sweet, R. M.,
Eds.; Academic Press: New York, 1997; Vol. 276, pp 307-326.
(45) Murshudov, G.; Vagin, A.; Dodson, E. Application of maximum
likelihood refinement in the refinement of protein structures. Proc.
Daresbury Study Weekend 1996.
(46) Murshudov, G. N.; Vagin, A.; Dodson, E. J. Refinement of
macromolecular structures by the maximum-likelihood method.
Acta Crystallogr., Sect. D 1997, D53, 240–255.
(47) Pannu, N. J.; Murshudov, G. N.; Dodson, E. J.; Read, R. J.
Incorporation of prior phase information strengthens maximum-
likelihood structure refinement. Acta Crystallogr., Sect. D 1998,
D54, 1285–1294.
tors. Chem. Biol. 2005, 12, 621–637.
(40) (a) Tao, Z.-F.; Wang, L.; Stewart, K. D.; Chen, Z.; Gu, W.; Bui,
M.; Merta, P.; Zhang, H.; Kovar, P.; Johnson, E.; Park, C.; Judge,
R.; Rosenberg, S.; Sowin, T.; Lin, N.-H. Structure-based design,
synthesis, and biological evaluation of potent and selective macro-
cyclic checkpoint kinase 1 inhibitors. J. Med. Chem. 2007, 50, 1514–
1527. (b) Wendt, M. D.; Shen, W.; Kunzer, A.; McClellan, W. J.;
Bruncko, M.; Oost, T. K.; Ding, H.; Joseph, M. K.; Zhang, H.; Nimmer,
P. M.; Ng, S.-C.; Shoemaker, A. R.; Petros, A. M.; Oleksijew, A.;
Marsh, K.; Bauch, J.; Oltersdorf, T.; Belli, B. A.; Martineau, D.; Fesik,
S. W.; Rosenberg, S. H.; Elmore, S. W. Discovery and structure-
activity relationship of antagonists of B-cell lymphoma 2 family
proteins with chemopotentiation activity in vitro and in vivo. J. Med.
Chem. 2006, 49, 1165–1181. (c) Bruncko, M.; Oost, T. K.; Belli, B. A.;
Ding, H.; Joseph, M. K; Kunzer, A.; Martineau, D.; McClellan, W. J.;
Mitten, M.; Ng, S.; Nimmer, P. M.; Oltersdorf, T.; Park, C.-M.; Petros,
A.; Shoemaker, A. R.; Song, X.; Wang, X.; Wendt, M. D.; Zhang, C.;