Discovery and SAR of 5-(3-Chlorophenylamino)benzo[ c ][2,6]naphthyridine-8- carboxylic Acid (CX-4945), the first clinical stage inhibitor of protein kinase CK2 for the Treatment of Cancer

Article abstract of DOI:10.1021/jm101251q

Herein we chronicle the discovery of CX-4945 (25n), a first-in-class, orally bioavailable ATP-competitive inhibitor of protein kinase CK2 in clinical trials for cancer. CK2 has long been considered a prime cancer drug target because of the roles of deregulated and overexpressed CK2 in cancer-promoting prosurvival and antiapoptotic pathways. These biological properties as well as the suitability of CK2s small ATP binding site for the design of selective inhibitors, led us to fashion novel therapeutic agents for cancer. The optimization leading to 25n (K_i = 0.38 nM) was guided by molecular modeling, suggesting a strong binding of 25n resulting from a combination of hydrophobic interactions, an ionic bridge with Lys68, and hydrogen bonding with the hinge region. 25n was found to be highly selective, orally bioavailable across species (20-51%) and efficacious in xenograft models. The discovery of 25n will allow the therapeutic targeting of CK2 in humans for the first time.

Full text of DOI:10.1021/jm101251q

J. Med. Chem. 2011, 54, 635–654 635
DOI: 10.1021/jm101251q
Discovery and SAR of 5-(3-Chlorophenylamino)benzo[c][2,6]naphthyridine-8-carboxylic Acid
(CX-4945), the First Clinical Stage Inhibitor of Protein Kinase CK2 for the Treatment of Cancer
Fabrice Pierre,* Peter C. Chua, Sean E. O’Brien, Adam Siddiqui-Jain, Pauline Bourbon, Mustapha Haddach, Jerome Michaux,
Johnny Nagasawa, Michael K. Schwaebe, Eric Stefan, Anne Vialettes, Jeffrey P. Whitten, Ta Kung Chen, Levan Darjania,
Ryan Stansfield, Kenna Anderes, Josh Bliesath, Denis Drygin, Caroline Ho, May Omori, Chris Proffitt, Nicole Streiner,
Katy Trent, William G. Rice, and David M. Ryckman
Cylene Pharmaceuticals, 5820 Nancy Ridge Drive, Suite 200, San Diego, California 92121, United States
Received September 27, 2010
Herein we chronicle the discovery of CX-4945 (25n), a first-in-class, orally bioavailable ATP-compet-
itive inhibitor of protein kinase CK2 in clinical trials for cancer. CK2 has long been considered a prime
cancer drug target because of the roles of deregulated and overexpressed CK2 in cancer-promoting
prosurvival and antiapoptotic pathways. These biological properties as well as the suitability of CK2’s
small ATP binding site for the design of selective inhibitors, led us to fashion novel therapeutic agents for
cancer. The optimization leading to 25n (Ki = 0.38 nM) was guided by molecular modeling, suggesting
a strong binding of 25n resulting from a combination of hydrophobic interactions, an ionic bridge
with Lys68, and hydrogen bonding with the hinge region. 25n was found to be highly selective, orally
bioavailable across species (20-51%) and efficacious in xenograft models. The discovery of 25n will
allow the therapeutic targeting of CK2 in humans for the first time.
Introduction
kinases, where genetic alterations lead to dysregulation of
their pathways, no CK2 mutations have been found to date,
yet its high expression and activity have been associated with
disease states.5,7 Overexpression of CK2 has been well docu-
mented in a number of cancers, including head and neck,8
breast,9 colorectal,10 renal,11 lung,12 leukemias,13 and pro-
state,14,15 and has also been linked to poor prognosis and
disease progression.5,16
These properties have defined CK2 as a prototypical “non-
oncogene target”,17 i.e., a nonmutated gene not directly
transformative but which is overexpressed to maintain the
cancer phenotype and protect cells from the increased cellular
stress resulting from oncogenic activation.18 The high depen-
dency of cancer cells on nononcogenes for survival has made
them an attractive yet underexploited new class of therapeutic
targets for the treatment of cancer.
Among the possible strategies to modulate CK2, antisense
nucleotides directed toward CK2R have scientifically vali-
dated CK2 as a target by inducing apoptosis in vivo in a xeno-
graft model of prostate cancer.19,20 A more practical therapy
would involve the oral administration of a small molecule
kinase inhibitor. The ATP binding site of CK2 is smaller than
most of the other protein kinases due tothe presence of unique
bulky residues,7 allowing for the design of very selective low
molecular weight ATP-competitive inhibitors.21,22
Many ATP-competitive inhibitors of CK2 have already
been reported in the literature,7,23-33 but none of them have
reached human clinical trials. Representative structural classes
of CK2 inhibitory reagents are shown in Figure 1. The chlo-
rinated nucleoside 1 (DRB) was one of the earliest described
inhibitors of CK2 with an IC50 of 15 μM.34 Flavonoids such
as emodin 2 displayed greater potency (IC50 =2 μM)35 but
generally lacked specificity. One of the most studied structural
Protein kinase CK2 is a pleiotropic, highly conserved
serine/threonine protein kinase ubiquitously distributed ac-
ross the compartments of multiple cell types.1-3 The protein is
comprised of a heterotetrameric complexcontaining two cata-
lytic isoforms (CK2Ror CK2R0) and two regulatory βsubunits
in various combinations. Abundant evidence supports a multi-
functional role for CK2 in a number of cellular events con-
tributing to the cancer phenotype. CK2 regulates many antia-
poptotic4 and pro-proliferative signaling cascades, including
PI3K/Akta and Wnt signaling cascades, NF-κB transcription,
and the DNA damage response (reviewed in refs 1 and 5).
Additionally, CK2 has been characterized as a regulator of
angiogenesis.6
CK2 is constitutively active and does not require phosphor-
ylation by other kinases for activation. Unlike other signaling
*To whom correspondence should be addressed. Phone: þ1 858 875
5113. Fax: 858-875-5101. E-mail: fpierre@cylenepharma.com.
a Abbreviations: PI3K, phosphatidylinositol-3-kinase; Akt, v-akt murine
thymoma viral oncogene homologue; NF-κB, nuclear factor κ light-chain
enhancer of activated B cells; TBB, 4,5,6,7-tetrabromobenzotriazole; IQA,
2-(5-oxo-5,6-dihydroindolo[1,2-a]quinazolin-7-yl)acetic acid; PTEN, phos-
phatase and tensin homologue; NaHMDS, sodium bis(trimethylsilyl)-
amide; PARP, poly(ADP-ribose) polymerase; NADþ, nicotinamide ade-
nine dinucleotide; PDK1, 3-phosphoinositide dependent protein kinase;
mTOR, mammalian target of rapamycin; FLT3, fms-like tyrosine kinase
receptor-3; PIM1, Proviral insertion site in Moloney murine leukemia 1;
CDK1, cyclin-dependent kinase 1; NMP, N-methyl-2-pyrrolidone; THF,
tetrahydrofuran; HOBt, hydroxybenzotriazole hydrate; DIEA, N,N-dii-
sopropylethylamine; EDCI, 1-ethyl-3-(3-dimethylaminopropyl) carbodi-
imide hydrochloride; PdCl2(dppf), 1,10-bis(diphenylphosphino)ferrocene
palladium(II) chloride complexed with dichloromethane; ES, electro-
spray; EtOAc, ethyl acetate; TFA, trifluoroacetic acid; LCMS, liquid
chromatography mass spectrometry; GCMS, gas chromatography mass
spectrometry.
r
2010 American Chemical Society
Published on Web 12/21/2010
pubs.acs.org/jmc

636 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2
Pierre et al.
classes of inhibitors include polyhalogenated-heteroaryls
such as tetrabromo-benzimidazole 3 (TBB),36-38 which has
higher selectivity and potency than many of the other known
agents.21 These compounds have been widely used as molec-
ular probes to elucidate the functional role of CK2. Although
3 has been reported to be well tolerated in mice,7 the poly-
halogenated aromatic nature of this molecule and its analo-
gues raises serious concerns about its potential long-term
toxicity in humans.39
discovery of 25n is of considerable importance as it will allow
for the evaluation of an entirely novel therapeutic strategy for
cancer.
Chemistry
Compounds 11a-b were efficiently synthesized (Scheme 1)
via a tandem Suzuki coupling/intramolecular amide forma-
tion between methyl 2-bromo-3-thiophene carboxylate 9 and
the commercially available boronic acids10a-b.46 Functional
group interconversions afforded several analogues 11c-e
bearing various substituents on R2.
The pharmaceutical industry’s effort to develop CK2 in-
hibitors has been met with limited success. Vangrevelinghe et
al. led the way by discovering the potent and selective CK2
inhibitor 6 (IQA) (Figure 2) (IC50 = 80 nM) by utilizing high
throughput docking methodologies.40 This molecule demon-
strated inhibition of CK2 in cells, and the structural basis for
its inhibition has been resolved by X-ray crystallography.22
Compound 6 was reported to decompose slowly in aqueous
media,33 precluding its use as a drug. A novel series of pyrazolo-
[1,5-a][1,3,5]triazines exemplified by analogue 4 (Figure 1)
emerged as a potent class of CK2 inhibitors, with the Ki values
of several analogues reported to be below 1 nM.41,42 These
molecules demonstrated cellular antiproliferative activity in
the submicromolar range. However, no further data have
been published in the literature and the current status of these
noteworthy molecules remains undisclosed. A recent publica-
tion described the discovery and cellular characterization of a
novel molecule 5, which induced antiproliferative responses in
cell culture, by inhibiting the phosphorylation of the CK2
substrate PTEN at Ser 370.43 Additionally, a recent review by
Prudent et al.44,45 discussed an emerging class of inhibitors
targeting structural elements located outside of the active site,
providing exciting opportunities for the development of allos-
teric inhibitors.
Lactam 11a was alkylated with 3-bromo-1-propanol to af-
ford a mixture of the N- and O-alkyl analogues 12a and 12b.
After separation by chromatography on silica gel followed by
ester hydrolysis, compounds 13a and 13b were isolated. Their
respective structures were assigned on the basis of their proton
NMR spectra. The loss of the carbonyl function in the
O-alkylated isomer 13b resulted in an upfield shift of the nearest
thiophene proton, from 7.77 ppm in 13a to 7.54 ppm in 13b.
Lactams 11a-b were alternatively reacted with phosphorus
oxychloride (POCl3) to afford chloroquinolines 14a-b. Inter-
mediate 14a was further derivatized into various substituted
4-aminothieno[3,2-c]quinoline-7-carboxylic acids 17a-p in
two steps. The first step involved nucleophilic displacement of
the chlorine of 14a with various substituted amines. The second
step was performed by hydrolysis of the ester function in basic
conditions. A tetrazole was introduced on R2 by reacting nitrile
16 with sodium azide. Alternatively, a nonacidic triazole was
also prepared in one pot starting from carboxamide 18, ob-
tained by oxidation of the corresponding cyano 16.
The chemistry described in Scheme 2 extended our previous
chemistry to various six-membered ring systems. Various ortho-
halogeno esters 22a, 22e-g, and 22i were prepared from their
commercially available carboxylic acids 21a, 21e-g, and 21i
using standard conditions. Under Suzuki conditions, ortho-
halogeno esters 22a and 22d-i reacted with boronic acids
10a-c to form lactams 23a-i in one step. These materials
were converted to various chloroquinolines 24a-i using phos-
phorus oxychloride.
Herein, we wish to report the discovery of the first-in-class
orally available ATP-competitive inhibitor of CK2 25n, cur-
rently in clinical trials for the treatment of cancer. The
Compound 24a was transformed (Scheme 3) into various
substituted benzo[c][2,6]naphthyridine-8-carboxylate 25a-t
in two steps by reaction with the desired amines followed by
ester hydrolysis. Functional group interconversions similar to
Scheme 1 were used to prepare 3-chloroanilines 26a-g bear-
ing various substitutions on R2. Nucleophilic displacement of
the chlorine by anilines in 24d-i (Scheme 4) and ester hydro-
lysis provided carboxylates 27d-e, 27h-i, 28d-g, and 28i.
The chemistry described in Schemes 5 and 6 allowed varia-
tions on the position and nature of functional groups on the
lower C-ring. Many ortho-substituted halogeno-anilines were
prepared from commercially available chemicals (Scheme 5).
The reaction of methyl 3-amino-2-methylbenzoate 29 with
iodination reagent NIS produced a mixture of isomers, the
Figure 1. Representative structures of known ATP-competitive
inhibitors of CK2.
Figure 2. Structure of CK2 inhibitor 6,40 PARP inhibitor 7,49 and lead molecule 11c.

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2 637
Scheme 1a
a Reagents and conditions: (a) (COCl)2, CH2Cl2, rt, then MeOH; (b) NaOAc, PdCl2(dppf), DMF, 120 °C; (c) K2CO3, 3-bromo-1-propanol, DMF,
100 °C; (d) NH4OH, 100 °C; (e) LiOH, MeOH, THF, H2O, rt; (f) LiAlH4, dioxane, 100 °C; (g) POCl3, (i-Pr)2NEt, toluene, reflux; (h) HNR3R4 (for
structuresofHNR3R4, seeTables1and 2), NMP, microwave120°C;(i) NaN3, NH4Cl, DMF, 120 °C;(j) H2O2, NaOH, NMP, 50 °C;(k) (MeO)2CHNMe2,
80 °C then AcOH, NH2NH2, 115 °C.
Scheme 2a
a Reagents and conditions: (a) (COCl)2, CH2Cl2, rt, then R1OH or H2SO4 (cat.), R1OH, reflux; (b) NaOAc, PdCl2(dppf), DMF, 120 °C or Cs2CO3,
PdCl2 (dppf), dioxane, 120 °C; (c) POCl3 reflux.
Scheme 3a
a Reagents and conditions: (a) R3NH2 (for structure of R3NH2, see Table 4) or 3-chloroaniline, NMP, microwave 120-160 °C or conventionnal
heating at 80 °C; (b) NaOH, H2O, EtOH, 50-80 °C; (c) LiAlH4, THF, -40 °C to rt; (d) HOBt, EDCI, (i-Pr)2NEt, NH4Cl, NMP, 70 °C; (e) NaN3,
NH4Cl, DMF, 120 °C; (f) (MeO)2CHNMe2, 80 °C then AcOH, NH2NH2, 80 °C.
major (86%) component of the mixture being methyl 3-amino-
6-iodo-2-methylbenzoate 30b and the minor (14%) component
being the desired methyl 3-amino-4-iodo-2-methylbenzoate
30a. The desired minor product was isolated pure by flash
chromatography on silica gel. The structure of this intermedi-
ate was unambiguously confirmed by its subsequent reactivity

638 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2
Pierre et al.
leading to the synthesis of lactam 39a (Scheme 6). The same
chemistry tested on the major isomer 30b led to a biaryl inter-
mediate that failed to provide any lactam in the presence of
NaHMDS. Methyl 5-amino-4-iodo-2-methylbenzoate 36
was the major isomer resulting from the iodination of methyl
5-amino-2-methylbenzoate 35 and could be easily isolated by
chromatography. Compound 37 was commercially available,
and the bromo aniline 32 was prepared in one step from the
acid 31.
Scheme 4a
Ortho-substituted halogeno-anilines 30a, 32, and 36-37
were coupled with boronic acid 3847,48 to yield 3-(o-amino-
phenyl)-pyridines intermediates (not shown) which was cy-
clized into 39a-d in the presence of NaHMDS. Finally,
conversions to molecules 41a-d were carried out via chloro-
quinolines 40a-d using the chemistry described above.
Results and Discussion
Structure-Based Design and Structure-Activity Relation-
ship (SAR). During our initial structure-based design efforts
to create a pharmaceutically suitable CK2 inhibitor, we noticed
an obvious similarity between the CK2 inhibitor 6 (Figure 2)22,40
and the poly(ADP-ribose) polymerase (PARP) inhibitor 7
developed by the same group and described elsewhere.49
Examination of our own collection of PARP inhibitors
as a potential source of novel CK2 inhibitors led us to
a Reagents and conditions: (a) H2N-Ph-R5, NMP, microwave 120-160 °C
or conventionnal heating at 80 °C; (b) NaOH, H2O, EtOH, 50-80 °C.
Scheme 5a
a Reagents and conditions: (a) NIS, AcOH, rt, minor isomer isolated by chromatography; (b) H2SO4 (cat.), MeOH, reflux; (c) H2, Pd/C, MeOH;
(d) NIS, AcOH, rt, major isomer isolated by chromatography.
Scheme 6a
a Reagents and conditions: (a) Cs2CO3, PdCl2 (dppf), dioxane, 120 °C, workup; (b) NaHMDS, THF, rt; (c) POCl3 reflux, (d) 3-chloroaniline, NMP,
microwave 120 °C; (e) NaOH, H2O, EtOH, 50-80 °C.

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2 639
Figure 3. Crystal structure of 6 bound to maize CK2R (PDB ID 1MO1).22 (A) 3D representation. Binding of 6 was largely determined by van
der Waals contacts with several hydrophobic residues (in green) of the ATP-binding pocket (mainly Val45, Val53, Ile66, Val95, Phe113, Val116,
Met163, and Ile174). The lactam -CONH- moiety of 6 was not directly involved in binding to the protein. (B) Schematic 2D view. The
carboxylate, essential for activity, formed hydrogen bonds with Lys68, the backbone nitrogen of Asp175, and one water molecule.
Table 1. CK2 and PARP Inhibitory Activity of Thieno[3,2-c]quinoline Analogues 11c-e, 13a-b, and 17a
4-oxo-4,5-dihydrothieno[3,2-c]quinoline-7-carboxylic acid
11c (Figure 2) because of its relatively close similarity with 6
and 7 and, in particular, because of its carboxylic acid group
at a position roughly overlaying with that of 6. Compound
11c was tested in an enzymatic assay using recombinant CK2
along with eight other molecules from our library. Molecule
11c was found to inhibit CK2 activity with an IC50 of 2.1 μM,
quickly providing us with a unique lead amenable to further
optimization.
The inhibitory activity of compounds bearing various
substituents on R2, 11c, 11e, and 11d (Table 1), suggested
that modification of the carboxylic group on R2 offered little
potential for increasing activity against CK2, a result con-
sistent with the published SAR of 6.40 Replacement of the
acid by a carboxamide in 11e or an alcohol in 11d suppressed
CK2 activity while improving inhibition of PARP.
examination of the published X-ray structure of 6 cocrys-
tallized with CK222 (Figure 3) revealed that the lactam
CONH motif of 6 did not form a H-bond with the CK2
protein. This led us to hypothesize that a chemical modifica-
tion of the same lactam motif in 11c could generate molecules
with decreased PARP interaction and potentially provide
improved selectivity for CK2. Compound 11a (Scheme 1)
was therefore subjected to chemical modifications to allow
the preparation of analogues in which the lactam had been
structurally modified.
The data summarized in Table 1 (compounds 11c, 13a-b,
and 17a) confirmed our hypothesis. O or N-substitution of
the lactam decreased inhibition of PARP while preserving
significant inhibition of CK2. Interestingly, substituted
4-aminothieno[3,2-c]quinoline-7-carboxylate 17a displayed grea-
ter activity against CK2. The straightforward access to 17a
from the activated intermediate 14a encouraged us to prepare
more amino-substituted analogues 17b-p, whose enzymatic
activities are summarized in Table 2. Shorter hydroxy alkyl-
substituted analogues 17b showed moderate activity against
CK2. Replacement of the hydroxy residue by a basic amine in
Most PARP inhibitors are designed to compete with the
cofactor NADþ, and therefore these generally display an es-
sential aryl/heteroaryl substituted amide motif in their struc-
ture (highlighted in red in Figure 2), which strongly interacts
with the PARP protein through three H-bonds.49,50 A close

640 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2
Pierre et al.
Table 2. CK2 Inhibitory Activity of Various C-4 and C-7 Substituted
Thieno[3,2-c]quinoline Analogues
Table 3. Effect of the A Ring on CK2 Inhibitory Activity
CK2
IC50, μM
compd
-NR3R4
-NH-(CH2)2OH
-NH-(CH2)2NMe2
pyrrolidino
R2
17b
17c
17d
17e
17f
17g
15
CO2H
CO2H
CO2H
CO2H
CO2H
CO2H
CO2Me
CN
1.26
0.102
1.78
-NH-phenyl
0.092
1.07
-NMe-phenyl
-NH-(2-Me-phenyl)
-NH-phenyl
-NH-phenyl
-NH-phenyl
0.970
>2.5
>2.5
>1
16
18
CONH2
C-(1H-tetrazol-5-yl)
C-(4H-1,2,4-triazol-3-yl) >1
19a
20
-NH-phenyl
-NH-phenyl
0.096
17h
17i
17j
-NH-(CH2)2Ph
-NH-(4-F-phenyl)
-NH-(3-F-phenyl)
CO2H
CO2H
CO2H
CO2H
CO2H
CO2H
CO2H
CO2H
0.516
0.219
0.068
0.178
0.032
0.077
0.028
0.395
0.129
0.129
0.075
17k -NH-(4-Cl-phenyl)
17l -NH-(3-Cl-phenyl)
17m -NH-(3-MeO-phenyl)
17n
17o
17p
19b
19c
-NH-(3-acetylenyl-phenyl)
-NH-(3-(PhO)-phenyl)
-NH-(3-(CONHMe)-phenyl) CO2H
-NH-(3-Cl-phenyl)
-NH-(3-F-phenyl)
C-(1H-tetrazol-5-yl)
C-(1H-tetrazol-5-yl)
17c resulted in a 10-fold increase of inhibition. A similar boost
was achieved with phenyl substituted compound 17e, leading us
into the potency range of 6.22,40 Introduction of a methyl group
onto the aniline nitrogen (17f) or in the ortho position of the
phenyl ring (17g) induced a notable loss of activity. This may be
explained by a decreased planarity of the inhibitors, resulting in
less favorable contact interactions within the small CK2 ATP
binding site. This observation was also noticed on tertiary
amine 17d.
27e, 25a, 27d, and 27i, Table 3), hypothesizing that we might
pick up favorable H-bond interactions with the hinge
region. This approach proved to be successful and resulted
in the discovery of compound 25a, which displayed a
dramatic boost in activity (>150-fold) over 27h and a
15-fold increase in activity over thieno[3,2-c]quinoline-
7-carboxylate 17e.
Various analogues lacking a carboxylate at C-7 (com-
pounds 15, 16, 18, and 20) were inactive, revealing again the
importance of an acidic function at that position. Tetrazole
19a retained a similar activity, while its nonacidic triazole 20
counterpart was inactive, in accordance with the previous
observation. Bulky moieties such as in 17h and 17o decreased
activity, suggesting that C-4 substituents were interacting with
a relatively small hydrophobic pocket. A number of substitu-
ted aniline analogues, bearing an acid (17i-p) or a tetrazole
(19b-c) at C-7, were prepared (Table 2). Anilines substituted
at the meta position by small hydrophobic groups showed the
greatest inhibition of CK2, leading to the most potent analo-
gues 3-chloroaniline 17l (IC50 = 0.032 μM) and 3-acetyleny-
laniline 17n (IC50 = 0.028 μM).
To improve potency, we considered replacing the thio-
phene ring with a six-membered ring, equivalent to the struc-
tural feature found in 6 (Figure 2). For that purpose, our
chemistry was successfully extended to ethyl ortho-bromo
benzoate 22h (Scheme 2 and Scheme 4). CK2 assay on the
resulting 27h (Table 3) revealed a loss of activity when replac-
ing the thiophene by a phenyl ring. On the basis of that ob-
servation, we began to explore analogues bearing nitrogen
atoms embedded at various places in the A ring (compounds
Encouraged by these results and guided by the thieno[3,2-c]-
quinoline-7-carboxylates SAR (Table 2), we prepared a
focused collection of C-5 substituted analogues of 25a whose
activities are shown in Table 4 (compounds 25b-t). All
compounds were generally more potent inhibitors of CK2,
and their IC50 values were 10- to 170-fold lower than their
thiophene counterparts.
Compounds bearing small substituents on C-5 (25d) and
various substituted aliphatic amino motifs (25b-c and
25e-f) achieved good activity, with IC50 values ranging from
8 to 27 nM. Remarkably, this position of the molecule ap-
peared to tolerate large extended phenyl substituted aliphatic
amino groups (25g-i); the phenethylamino substituted ana-
logue 25h being the most potent of the series. Derivatives of
anilines substituted by small hydrophobic groups in the meta
position (25j-l, 25n, and 25p-q) again demonstrated the
greatest inhibition of CK2, 3-chloroaniline 25n being the
most potent analogue of this novel class of CK2 inhibitors
with an IC50 of 1 nM. Large substituents (25r, 25s) or certain
polar groups (25t) decreased the activity of the inhibitors by
an order of magnitude. The 3-chloroaniline motif was con-
served in subsequent molecules to complete the structure-
activity relationship studies.

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2 641
Table 4. CK2 Inhibitory Activity of C-5 Substituted Benzo[c]-
[2,6]naphthyridine-8-carboxylate 25a-t
subjected to in situ minimization using the CHARMm force-
field as implemented in Discovery Studio 2.5.1 from Ac-
celrys, Inc., following manual placement of the compound.
The model oriented the compounds within the ATP binding
pocket of the kinase (the PDB structure 1JWH of human
CK2R was used as the structural template51). Figure 4 shows
the predicted binding of 25n to CK2R. The binding model,
which was continually revised to be consistent with informa-
tion derived from recently published X-ray structures and
the most recent in-house SAR data, was principally used to
prioritize synthesis and rationalize CK2 inhibition data.
The structural similarity of these compounds to 6 sug-
gested that the carboxylate group might bind in a similar
orientation to 6.22 The SAR confirmed the importance of
the carboxylate group for binding, so we predicted that the
carboxylate of 25n made analogous ionic interactions with
Lys68.
The mainly hydrophobic residues in the ATP binding site
of CK2R create a small, flat envelope into which ATP and
any inhibitor can bind, as outlined by previous works.22,51-55
The model suggests that the flat tricyclic cores could make
large areas of hydrophobic contacts with binding pocket
residues. The flat, hydrophobic nature of the tricyclic core is
likely to contribute strongly to the high degree of CK2 inhibi-
tion observed. The ring-A pyridine nitrogen of 25n (Figure 4) is
perfectly positioned to form a H-bond with the NH of Val116
in the hinge region, analogous to the interactions seen for
ATP analogues. The presence of a hydrogen-bond acceptor
in the preferred orientation can explain the increased activity
of pyridine analogues. It became obvious from this model
that displacement of the nitrogen or the carboxylate from
their optimal positions resulted in a geometrically unfavor-
able configuration, translated experimentally by a major loss
of activity in the CK2 assay (compounds 26c, 41d, 41b, 28i,
and 28d-f in Table 5). It is unclear why phenyl analogue 27h
is significantly less potent than thiophene 17e (Table 3). We
can speculate that it may be related to a greater affinity that
sulfur-bearing thiophenes (and hence a reorientation to
maximize the binding interaction) have for the sulfur group
at Met163.
CK2
IC50, μM
compd
R3
25a
25b
25c
25d
25e
25f
25g
25h
25i
-phenyl
-(CH2)2NMe2
-cyclopentyl
-OMe
-cyclopropyl
0.006
0.025
0.027
0.008
0.016
0.011
0.009
0.003
0.016
0.004
0.004
0.005
0.008
0.001
0.007
0.003
0.004
0.069
0.019
0.043
-(CH2)2O-i-Pr
-(CH2)phenyl
-(CH2)2phenyl
-(CH2)3phenyl
-(3-MeO-phenyl)
-(3-Cl, 4-F-phenyl)
-(3-F-phenyl)
-(2-Cl-phenyl)
-(3-Cl-phenyl)
-(4-Cl-phenyl)
-(3-acetylenyl-phenyl)
-(3-CN-phenyl)
-(4-(PhO)-phenyl)
-(3-(PhO)-phenyl)
-(3-(SO2NH2)-phenyl)
25j
25k
25l
25m
25n
25o
25p
25q
25r
25s
25t
The SAR of various 3-chloroanilines in Table 5 highlights
the role of the A-ring and the C-ring substituents. Com-
pounds 25n, 26a, and 26c-g confirmed the essential role of
the carboxylate on R2 for potent inhibition. Suppressing the
carboxylate (26c) or replacing it by a small nonacidic group
(26g-26a, 26d-e) resulted in over 400-fold loss of activity.
Acidic tetrazole 26f retained activity but at a somewhat lower
level.
Methyl groups adjacent to the carboxylate had a position
dependent effect. On R1 (41a), a methyl group maintained
activity at a potent level, while on R3 (41c), it suppressed
activity, suggesting an unfavorable interaction with the pro-
tein at that position. Molecules 41d and 41b, in which the
carboxylate was moved to adjacent carbons, were much less
active, showing that its position on R2 was necessary for
potent inhibition.
Molecules 28d-f and 28i confirmed the optimal position
of the ring-A nitrogen atom on A3, implying a discrete
interaction of the nitrogen with the protein. The potent acti-
vity of pyrimidine analogue 28g further strengthened this ob-
servation; showing that an additional nitrogen atom within
the ring did not negatively affect activity as far as a nitrogen
atom was present in A3 for H-binding.
The three components of the interaction of 25n, large
surface hydrophobic interaction, ionic bridge with the Lys68
region, and H-bond with the hinge region, account for the very
potent activity of the benzo[c][2,6]naphthyridine-8-carboxy-
late series. The 3-chloroaniline moiety has proved to maximize
potency on both scaffolds, and the chloro group is likely to fit
well within a small hydrophobic region, possibly in the glycine
rich loop. Interestingly, the 3D conformation of the anilino
moiety of our molecules overlay well with anilino substituents
of some pyrazolo[1,5-a][1,3,5]triazines inhibitors, previously
cocrystallized with CK2 and described by Nie et al.41
Finally, our model explains the loss of activity of R3-
methyl substituted analogue 41c (Table 5) by suggesting that
the methyl group would clash with the gatekeeper residue
phe113.
Binding Model. Compound 25n was confirmed to be a
potent, ATP-competitive inhibitor of CK2 with a Ki = 0.38 (
0.02 nM against the recombinant human holoenzyme
(RRββ). Drawing on analysis of critical interactions and
structural overlay with the numerous previously published
and publically available structural studies,22,51-55 we were
able to build a binding model that was consistent with the
SAR generated by the present CK2 program. No de novo
docking or scoring was performed. Each molecule was
Kinase Selectivity Profile. Compound 25n was screened at
0.5 μM against a panel of 238 kinases (Millipore) to assess its
selectivity profile. At 0.5 μM, 25n showed no major inhibi-
tion of the vast majority of tested kinases, including all
isoforms of PI3K, Akt, PDK1, and mTOR. IC50 values were
measured against the seven kinases for which it exhibited
greater than 90% inhibition at 0.5 μM (Table 6). As antici-
pated, the molecule displayed an excellent selectivity pro-
file, which is even more remarkable when considering the

642 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2
Pierre et al.
Table 5. CK2 Inhibitory Activity of Several Chloroanilines Derivatives
low molecular weight (349.8 g/mol) of the molecule. In
addition, 25n was evaluated at a concentration of 10 μM
in relevant cell-based assays for FLT3, PIM1, and CDK1
and was found to be functionally inactive against these
kinases.56
dephosphorylation of the Akt substrate p21(T145). Com-
pound 25n induced apoptosis in multiple cancer cell lines
as measured by concentration-dependent induction of cas-
pase 3/7 activity. Normal cells failed to show a detectable
change in caspase 3/7 activation within the concentration
range tested, indicating that cancer cells might be more
vulnerable to 25n than normal cells.56
Pharmacokinetics and Safety. The pharmacokinetic prop-
erties of 25n were evaluated in multiple species including
mouse, rat, and dog (Table 8). The drug exhibited low to
moderate clearance and a wide range of volume of distribu-
tion across species. Terminal half-life ranged between 5 and
12 h across species, and oral bioavailability was moderate in
all species ranging between 20 and 51%. Furthermore, 25n
showed minimal inhibition of five of the principal cyto-
chrome P450 isoforms (CYP1A2, CYP2C9, CY2C19,
CYP2D6, and CYP3A4).58 Also, there was no significant
inhibition of the hERG channel by this compound (<10%
inhibition at 0.1, 1.0, and 10.0 μM) in the patch clamp
assay,59 and the molecule was considered nonmutagenic
based on the Ames assay and also deemed nongenotoxic in
the mouse micronucleus test.
Cellular Characterization. Several of the most potent CK2
inhibitors were tested for their inhibition of the proliferation
of various cancer cell lines (Table 7). The molecules were
active against a broad range of cell types, including breast,
lung, and prostate cancers in which CK2 has been previously
shown to be overexpressed. The cellular biology of the potent
inhibitor 25n was further investigated. Compound 25n was
found to potently inhibit endogenous intracellular CK2 acti-
vity with an IC50 of 0.1 μM in Jurkat cells (Figure 5). The
differences between IC50 values of 25n in the kinase enzyme
assay relative to the cell proliferation assay may be due to
multiple factors. However, the greatest contributing factor is
likely that the ATP concentration in cells (1-10 mM) is far
higher than the ATP concentration (15 μM) used in molec-
ular screening assays. Thus, one would expect that higher
concentration of ATP-competitive 25n is needed to reach the
same level of CK2 inhibition in cells.
Additional data demonstrating a CK2-inhibition based
antiproliferative and antiangiogenic effect of 25n will be fully
discussed elsewhere.56 Briefly, in various cell lines, CK2
inhibition was confirmed by measuring the phosphorylation
level of the CK2 specific phosphorylation site on Akt(S129).
25n induced dephosphorylation of Akt(S129)57 and a rapid
Efficacy in Tumor Xenograft Studies. Compound 25n was
selected for in vivo efficacy in established human prostate
PC3 xenograft model in athymic mice (Figure 6). Mice
bearing subcutaneous PC3 tumors were treated with 25n
(25 mg/kg, 50 mg/kg, and 75 mg/kg, po, bid). 25n demon-
strated tumor growth inhibition (TGI = 19%, 40%, and

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2 643
Figure 4. Binding model of 25n (in green) in the ATP-pocket of CK2.
Table 6. IC50 Determination for 25n against Several Kinases
kinase
CK2R
CK2R0
DAPK3
17
FLT3
35
TBK1
35
CLK3
41
HIPK3
45
PIM1
46
CDK1/cyclinB
56
IC50, nM
1
1
Table 7. Cell Antiproliferative Activity of Derivatives for Selected Benzo[c][2,6]naphthyridine-8-carboxylate Analogues
cell viability IC50, μM
prostate
pancreatic
MIA
PC3 LNCaP BxPC3 PaCa-2 A549 H1299 Jurkat K-562 MCF7 Hs578T MB-231
lung
leukemia
breast
colorectal melanoma
HCT-
CK2 IC50
μM
,
MDA-
compd
116
A375
25n
25p
25h
25q
25k
25j
0.001
0.003
0.003
0.004
0.004
0.004
0.005
2.1 4.7
16.8 17.1
23.6 8.6
26.4 15.5
4.9 4.6
4.4
3.7
1.1
3.9
5.2
3.0
30.2
47.0
2.4
3.7
2.5
2.8
5.3
3.3
8.9
1.4
13.1
4.0
6.4
11.8
2.2
3.9
7.4
10.8
13.2
ND
21.0
11.1
22.0
2.7
ND
14.7
41.0
8.2
16.5
35.6
3.3
ND
ND
ND
3.2
3.6 >10
7.7 3.6
ND
ND
>50
6.8
ND
16.7
ND
9.2
5.0
28.7
6.1
>10
3.1
10.7
8.3
6.0
9.5
7.6
17.4 ND
9.2 ND
8.5
6.0
6.5 5.9 ND
3.2 >10 ND
12.9
5.9
ND
ND
ND
ND
25l
86%, respectively) compared to vehicle treated control,
and a dose responsive efficacy was observed (Figure 6).
Last, 25n was well tolerated in mice as assessed by minimal
changes in body weight during the course of treatment
compared to vehicle control. Other efficacy studies in
various models further confirmed the tolerability of the
drug and will be reported elsewhere.56
Conclusion
We have discovered a novel family of ATP-competitive
inhibitors of CK2 using both structure- and chemistry-based
optimization. This work culminated in the discovery of 25n, a
potent and selective inhibitor of CK2 that is efficacious in
animal models of cancer. The SAR information acquired

644 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2
Pierre et al.
during the lead optimization of this new class of drugs has
been used to identify key structural elements necessary to
achieve potent CK2 inhibition. As a result, we have estab-
lished a binding model that rationalizes the multiple compo-
nents of the strong interaction of 25n with the ATP binding
pocket, providing valuable information for the design of
future classes of small molecule CK2 inhibitors.
While being beneficial for selectivity, the small size of the
cofactor site of CK2 also predisposes for the discovery of
hydrophobic inhibitors with poor drug-like properties. Re-
markably, 25n attained a favorable balance between potency
and pharmacokinetics, a challenging achievement that we
believe to be atypical with small molecule CK2 inhibitors.
The complexity of the regulation of CK2, its pleiotropy,
and the widedistribution of the proteinhas raised the question
of whether CK2 inhibitors can selectively kill cancer cells
while sparing normal cells. We have shown that 25n was
efficacious in xenograft models of cancer and remained very
well tolerated. Compound 25n (CX-4945) demonstrated an
acceptable pharmaceutical profile and is currently being
evaluated clinically as a first-in-class CK2 inhibitor for the
treatment of cancer.60
pler. Thin layer chromatography was performed Watman Partisil
˚
K6F60A silica gelplates (250 μm for analyticalor20cmꢀ 20cm,
1000 μm for preparative) with fluorescent indicator and visua-
lized with 254 nm UV light. Flash column chromatography was
˚
conducted either under medium pressure using Watman 60 A,
230-400 mesh ASTM bulk silica gel or on prepacked silica gel
cartridges (Biotage). Reverse phase HPLC purification was con-
ducted utilizing mass directed fraction collection employing a
Waters Micromass ZQ mass spectrometer with an electrospray
probe. Fraction collection was achieved with a Waters 2767 frac-
tion collector driven by a Waters 2525 binary pump. Fractions
were collected by mass, UV signal (Waters 2487 dual wave) or a
combination of both. The stationary phase is a Phenomenex
˚
Luna 5 μm C18 100 A Axia 21.2 mm ꢀ 50 mm column with a
mobile phase gradient of 25-95% CH3CN or MeOH in H2O
with 0.1% TFA. Compounds were generally isolated after pre-
parative HPLC by evaporation of the solvents using a Genevac
DD-4 evaporator with a fixed rotor. NMR spectra were recorded
in CDCl3, CD3OD, or DMSO-d6 on a Varian Mercury 400 with
an AutoX DB PFG probe and Protune accessory operating at
400 MHz for 1H and 100 MHz for 13C. Trimethyl silane (TMS)
was used as internal reference. Chemical shifts (δ) are reported in
parts per million (ppm) and coupling constants (J) in hertz (Hz).
The following abbreviations are used for multiplicities: s = sin-
glet, br = broad, d = doublet, t = triplet, m = multiplet, dd =
doublet of doublet, q = quadruplet, qi = quintuplet, sext = sex-
tuplet. Unless otherwise specified, all final compounds were
homogeneous with a purity of g95%, as determined by LCMS
analysis conducted on a Waters 2695 HPLC/MS system with
Micromass ZQ and 2487 UV detectors using a gradient of 10-
90% 0.05% TFA in ACN and 0.1% TFA in DI water.
Beyond cancer, CK2 plays multiple roles in inflammation
disorders, neurodegenerative diseases, and viral or parasitic
infections.5 The discovery of 25n and the human pharmacol-
ogy resulting from the modulation of CK2 may open the door
to the discovery of new treatments for diseases in which this
protein is dysregulated.
5-Bromopyrimidine-4-carboxylic acid 21g was prepared ac-
cording to a procedure previously described in a patent.61
4-(Diisopropylcarbamoyl)pyridin-3-ylboronic acid 38 was pre-
pared using a published procedure.47,48 Compounds 8, 10a-c,
21a, 21e-g, 21i, 22d, 22h, 29, 31, 33, 37, and the various amines
Experimental Section
Chemistry. General Methods. All solvents and reagents, un-
less otherwise stated, were reagent grade, commercially available,
and used without further purification. All experiments using
moisture sensitive reagents were carried out under a dry nitrogen
atmosphere. Microwave irradiation was carried out in a CEM
Discover series microwave reactor with the Explorer autosam-
Figure 6. In vivo growth inhibitory effect of 25n on human prostate
cancer PC3 xenografts in nude mice. Compound 25n dosed bid at
25 mg/kg (tumor growth inhibition TGI = 19%), 50 mg/kg (TGI =
40%), and 75 mg/kg (TGI = 86%).
Figure 5. Endogenous CK2 activity of 25n in Jurkat cells.
Table 8. Pharmacokinetic Parameters for Compound 25n
species (n = 3)
iv mg/kg
po mg/kg
Clsa L/h/kg
Vss L/kg
T1/2 Hr
po Cmax ng/mL
po AUC ng h/mL
%F
3
mouseb
rat
dog
5c
5.2c
1.9c
25c
10.5c
8.2d
2.5
0.1
0.7
18.1
2.0
5.0
11.6
8.3
311
7369
2178
2009
42343
5646
20
48
51
8.9
a Abbreviations: Cls, clearance; Vss, volume of distribution at steady state; T1/2, elimination half-life; Cmax, maximum concentration; AUC, area
under the curve; %F, bioavailability. b (n = 4). c The sodium salt of 25n was administered in a 25 mM sodium phosphate buffer. d Dogs received PO dose
via 25n sodium salt filled capsules.

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2 645
used to prepare analogues described in Tables 1-5 are commer-
cially available.
1H), 7.87 (d, J = 5.2, 1H), 7.92 (m, 2H), 8.11 (br s, 1H), 11.89 (br
s, 1H) ppm.
Methyl 2-Bromothiophene-3-carboxylate (9). 2-Bromo-3-
thiophene carboxylic acid 8 (1.0 equiv, 12.56 g, 60.66 mmol)
was suspended in CH2Cl2 (200 mL). Oxalyl chloride (1.1 equiv,
5.9 mL, 67.16 mmol) and 5 drops of DMF were added, inducing
formation of gas. The mixture was stirred overnight at room
temperature, and the volatiles were removed in vacuo. The re-
sulting solid was suspended in dry methanol (150 mL) and the
mixture heated to reflux. Evaporation of the solvents afforded
methyl 2-bromo-3-thiophene carboxylate 9 (13.16 g, 98%) as a
crude brown oil. LCMS (ES): >95% pure, m/z not detected. 1H
NMR (CDCl3, 400 MHz): δ 3.88 (s, 3H), 7.23 (d, J = 5.6, 1H),
7.56 (d, J = 5.6, 1H) ppm.
4-Oxo-4,5-dihydrothieno[3,2-c]quinoline-7-carbonitrile (11b).
In a microwave vial, methyl 2-bromo-3-thiophene carboxylate
9 (1.0 equiv, 250 mg, 1.13 mmol), 2-amino-3-cyanophenyl boronic
acid hydrochloric acid 10b (1.1 equiv, 250 mg, 1.24 mmol), sodium
acetate (3.0 equiv, 278 mg, 3.39 mmol), and PdCl2 (dppf) (0.007
equiv, 4.3 mg, 0.0082 mmol) were mixed together in anhydrous
DMF (2.5 mL). The mixture was heated in a microwave oven at
120 °C for 10 min. Water was added and the material extracted
with CH2Cl2. The organic extracts were washed with brine, dried
over Na2SO4, and the solvents removed in vacuo. The resulting
solid was sonicated in EtOAc, filtered, and dried to afford 4-oxo-
4,5-dihydrothieno[3,2-c]quinoline-7-carbonitrile 11b as a tan solid
1
(121 mg, 48%). LCMS (ES): 95% pure, m/z 227 [M þ H]þ. H
Methyl 4-Oxo-4,5-dihydrothieno[3,2-c]quinoline-7-carboxylate
(11a). In a microwave vial, methyl 2-bromo-3-thiophene car-
boxylate 9 (1.0 equiv, 260 mg, 1.18 mmol), 2-amino-4-(meth-
oxycarbonyl)phenylboronic acid hydrochloride 10a (1.1 equiv,
300 mg, 1.30 mmol), sodium acetate (3.0 equiv, 292 mg, 3.56 mmol),
and PdCl2 (dppf) (0.05 equiv, 31 mg, 0.059 mmol) were mixed
together in anhydrous DMF (2 mL). The mixture was heated in a
microwave oven at 120 °C for 10 min. Water was added and the
solid filtered and dried. The material was suspended in dichloro-
methane, filtered, and dried to afford methyl 4-oxo-4,5-dihydro-
thieno[3,2-c]quinoline-7-carboxylate 11a as a yellow solid (152 mg,
NMR (DMSO-d6, 400 MHz): δ 7.65 (m, 2H), 7.75 (br s, 1H), 7.97
(d, J = 5.2, 1H), 8.06 (d, J = 8.4, 1H), 12.03 (br s, 1H) ppm.
Methyl 4-(3-Hydroxypropoxy)thieno[3,2-c]quinoline-7-carbo-
xylate (12b) and Methyl 5-(3-Hydroxypropyl)-4-oxo-4,5-dihydro-
thieno[3,2-c]quinoline-7-carboxylate (12a). Methyl 4-oxo-4,5-
dihydrothieno[3,2-c]quinoline-7-carboxylate 11a (1.0 equiv, 18 mg,
0.069 mmol) was dissolved in anhydrous DMF (0.4 mL). K2CO3
(7.0 equiv, 70 mg, 0.506 mmol) and 3-bromo-1-propanol (16 equiv,
100 μL, 1.144 mmol) were added and the mixture stirred at 100 °C
for 1.5 h. After adding water, the mixture was extracted with
CH2Cl2. The combined extracts were dried over Na2SO4 and the
solvents removed in vacuo. Compounds 12a and 12b were
separated by preparative TLC on silica gel (eluted twice with
30% EtOAc in hexanes, then once with 50% EtOAc in hexanes).
The less polar compound was identified as methyl 4-(3-hydrox-
ypropoxy)thieno[3,2-c]quinoline-7-carboxylate 12b (12 mg).
TLC (50% EtOAc/hexanes) Rf = 0.5. LCMS (ES): 80% pure,
m/z 318 [M þ H]þ. The more polar compound was identified as
methyl 5-(3-hydroxypropyl)-4-oxo-4,5-dihydrothieno[3,2-c]quino-
line-7-carboxylate 12a (19 mg). TLC (50% EtOAc/hexanes) Rf =
0.2. LCMS (ES): 80% pure, m/z 318 [M þ H]þ. 1H NMR (CDCl3,
400 MHz): δ 2.08 (qi, J = 6.0, 2H), 3.61 (t, J = 5.2, 2H), 4.62 (t,
J = 6.0, 2H), 7.53 (d, J = 5.2, 1H), 7.77 (d, J = 5.2, 1H), 7.93 (d,
J = 8.0, 1H), 7.99 (dd, J = 1.2, J = 8.4, 1H), 8.26 (d, J = 0.8, 1H)
ppm. Structural assignement was based on NMR spectra of their
respective carboxylic acids. The two compounds wereusedfor the
following step without any further purification.
5-(3-Hydroxypropyl)-4-oxo-4,5-dihydrothieno[3,2-c]quinoline-
7-carboxylic Acid (13a). Methyl 5-(3-hydroxypropyl)-4-oxo-4,5-
dihydrothieno[3,2-c]quinoline-7-carboxylate 12a (1.0 equiv, 19 mg,
0.060 mmol) was dissolved in a 1:1:1 mixture of THF, methanol,
and water (0.5 mL). LiOH (40 mg) was added and the resulting
mixture stirred at room temperature for 1.5 h. Water, methanol,
and hydrochloric acid were added and the solution purified by
preparative HPLC. Genevac evaporation afforded 5-(3-hydro-
xypropyl)-4-oxo-4,5-dihydrothieno[3,2-c]quinoline-7-carboxylic
acid 13a as a white solid (4 mg, 22%). LCMS (ES): 95% pure, m/z
304 [M þ H]þ. 1H NMR (CDCl3/CD3OD, 9:1, 400 MHz): δ 2.08
(qi, J = 6.0, 2H), 3.61 (t, J = 5.2, 2H), 4.62 (t, J = 6.0, 2H), 7.53
(d, J = 5.2, 1H), 7.77 (d, J = 5.2, 1H), 7.93 (d, J = 8.0, 1H), 7.99
(dd, J = 1.2, J = 8.4, 1H), 8.26 (d, J = 0.8, 1H) ppm.
1
50%). LCMS (ES): 95% pure, m/z 260 [M þ H]þ. H NMR
(CDCl3, 400 MHz): δ 3. 99 (s, 3H), 7.54 (d, J = 5.2, 1H), 7.79 (d,
J = 4.8, 1H), 7.86 (d, J = 8.4, 1H), 7.91 (dd, J = 8.4, J = 1.6, 1H),
8.03 (d, J = 1.2, 1H) ppm.
4-Oxo-4,5-dihydrothieno[3,2-c]quinoline-7-carboxylic Acid (11c).
Methyl 4-oxo-4,5-dihydrothieno[3,2-c]quinoline-7-carboxylate 11a
(1.0 equiv, 618 mg, 2.38 mmol) was suspended in 10 mL of a mix-
ture of methanol, THF, and water (1:1:1, v:v:v). LiOH (2.0 equiv,
114 mg, 4.76 mmol) was added, and the mixture was stirred at room
temperature for 2 h. An additional amount of LiOH (114 mg) was
added, and the mixture was stirred for an hour. LiOH (50 mg) was
added and the mixture stirred for an additional 2 h. Water was
added and the solution filtered through a pad of Celite. The pad of
Celite was thoroughly washed with aqueous 1 N NaOH. The solu-
tion was acidified with 6 N aqueous hydrochloric acid to induce
precipitation of the expected material. Filtration and drying
afforded 11c as a yellow solid (562 mg, 96%). LCMS (ES):
95% pure, m/z 246 [M þ H]þ. 1H NMR (DMSO-d6, 400 MHz):
δ 7.61 (d, J = 5.2, 1H), 7.73 (dd, J = 1.6, J = 8.0, 1H), 7.88 (d,
J = 5.6, 1H), 7.92 (d, J = 8.4, 1H), 8.02 (d, J = 1.6, 1H), 11.92 (s,
1H), 13.21 (br s, 1H) ppm.
7-(Hydroxymethyl)thieno[3,2-c]quinolin-4(5H)-one (11d).
4-Oxo-4,5-dihydrothieno[3,2-c]quinoline-7-carboxylic acid 11c
(1.0 equiv, 38 mg, 0.155 mmol) was suspended in dioxane (1 mL).
LiAlH4 (7.0 equiv, 40 mg, 1.05 mmol) was added and the mixture
stirred at 100 °C for 45 min. Water was added, then methanol and
CH2Cl2. The solid salts were filtered off and washed with methanol
and CH2Cl2. After evaporation of the volatiles in vacuo, the materi-
al was dissolved in a mixture of NMP, methanol and water and
was purified by preparative HPLC. Genevac evaporation afforded
7-(hydroxymethyl)thieno[3,2-c]quinolin-4(5H)-one 11d as an off-
white solid (12 mg, 34%). LCMS (ES): 95% pure, m/z 232 [M þ
4-(3-Hydroxypropoxy)thieno[3,2-c]quinoline-7-carboxylic Acid
(13b). The title compound was prepared according to the proce-
dure used to prepare compound 13a starting from methyl 4-(3-
hydroxypropoxy)thieno[3,2-c]quinoline-7-carboxylate 12b. The
title compound 13b was isolated as a solid (3 mg, 26%). LCMS
(ES): 95% pure, m/z 304 [M þ H]þ. 1H NMR (CDCl3/CD3OD,
9:1, 400 MHz): δ 2.04 (qi, J = 6.0, 2H), 3.68 (t, J = 6.0, 2H), 4.69
(t, J = 6.4, 2H), 7.52 (d, J = 4.8, 1H), 7.54 (dd, J = 5.2, J = 1.6,
1H), 7.93 (d, J = 8.4, 1H), 7.97 (dd, J = 7.6, J = 0.8, 1H), 8.49 (d,
J = 0.8, 1H) ppm.
1
H]þ. H NMR (DMSO-d6, 400 MHz): δ 4.56 (s, 2H), 7.15 (d,
J = 7.6, 1H), 7.39 (br s, 1H), 7.55 (d, J = 5.2, 1H), 7.73 (d, J =
5.2, 1H), 7.76 (d, J = 8.0, 1H), 11.73 (s, 1H) ppm.
4-Oxo-4,5-dihydrothieno[3,2-c]quinoline-7-carboxamide (11e).
In a closed vessel, methyl 4-oxo-4,5-dihydrothieno[3,2-c]quino-
line-7-carboxylate 11a (44 mg, 0.170 mmol) was suspended in
concentrated aqueous ammonia (1 mL). The mixture was stirred
at 100 °C overnight. Aqueous 1N NaOH was added and the
mixture stirred at room temperature for 2 h. The solid was fil-
tered and dried to provide 4-oxo-4,5-dihydrothieno[3,2-c]quino-
line-7-carboxamide 11e as a brown solid (13 mg, 32%). LCMS
(ES): 95% pure, m/z 245 [M þ H]þ. 1H NMR (DMSO-d6, 400
MHz): δ 7.51 (br s, 1H), 7.62 (d, J = 4.8, 1H), 7.70 (d, J = 8.4,
4-Chlorothieno[3,2-c]quinoline-7-carboxylate (14a). Methyl
4-oxo-4,5-dihydrothieno[3,2-c]quinoline-7-carboxylate 11a (1.0
equiv, 1.50 g, 5.79 mmol) was suspended in dry toluene (15 mL).
Phosphorus oxychloride (1.2 equiv, 0.64 mL, 6.99 mmol) and

646 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2
Pierre et al.
DIEA (0.8 equiv, 0.81 mL, 4.65 mmol) were added and the mix-
ture vigorously stirred at 120 °C for 3 h under nitrogen atmo-
sphere. The mixture was hydrolyzed by addition of ice and water.
The compound was extracted with CH2Cl2 (4ꢀ). The combined
extracts were dried over Na2SO4 and the black solution filtered
through a pad of Celite. After evaporation of the volatiles in
vacuo, the resulting solid was triturated in a mixture of EtOAc
and hexanes. Filtration and drying provided methyl 4-chloro-
thieno[3,2-c]quinoline-7-carboxylate 14a as a yellow fluffy solid
pure form after dilution of the mixture, acidification by hydro-
chloric acid and filtration of the resulting precipitate. LCMS
(ES): >95% pure, m/z 303 [M þ H]þ. 1H NMR (DMSO-d6, 400
MHz): δ 1.89 (qi, J = 6.4, 2H), 3.56 (t, J = 6.4, 2H), 3.71 (q, J =
6.0, 2H), 7.86 (br s, 1H), 8.06 (br s, 3H), 8.40 (br s, 1H) ppm.
4-(2-Hydroxyethylamino)thieno[3,2-c]quinoline-7-carboxylic Acid
(17b). Yield, 21%. LCMS (ES): >95% pure, m/z 289 [M þ H]þ.
4-(2-(Dimethylamino)ethylamino)thieno[3,2-c]quinoline-7-car-
boxylic Acid, Trifluoroacetate Salt (17c). After preparative HPLC,
the material was isolated as a TFA salt. Yield, 57%. LCMS (ES):
>95% pure, m/z 316 [M þ H]þ, 271 [M þ H - NHMe2]þ.
4-(Pyrrolidin-1-yl)thieno[3,2-c]quinoline-7-carboxylic Acid (17d).
1
(1.14 g, 71%). LCMS (ES): 95% pure, m/z 278 [M þ H]þ. H
NMR (CDCl3, 400 MHz) δ 4.01 (s, 3H), 7.72 (d, J = 4.8, 1H),
7.74 (d, J = 5.2, 1H), 8.14 (d, J = 8.4, 1H), 8.25 (d, J = 8.4, 1H),
8.85 (d, J = 1.6, 1H) ppm.
1
Yield, 47%. LCMS (ES): >95% pure, m/z 299 [M þ H]þ. H
4-Chlorothieno[3,2-c]quinoline-7-carbonitrile (14b). The title
compound was prepared according to the procedure used to
prepare 14a starting from 4-chlorothieno[3,2-c]quinoline-7-car-
bonitrile 11b (1.00 g, 4.42 mmol). The title compound 14b was
isolated as a brown solid (833 mg, 77%). LCMS (ES): 95% pure,
m/z 245 [M þ H]þ. 1H NMR (DMSO-d6, 400 MHz): δ 7.80 (d,
J = 4.8, 1H), 8.04 (br d, J = 7.2, 1H), 8.27 (d, J = 5.2, 1H), 8.44
(d, J = 8.4, 1H), 8.63 (br s, 1H) ppm.
NMR (DMSO-d6, 400 MHz):δ 2.11 (br s, 4H), 3.95 (br s, 2H), 4.20
(br s, 2H), 7.95 (br s, 1H), 8.18 (br s, 3H), 8.82 (br s, 1H), 12.0 (br s,
1H) ppm.
4-(Methyl(phenyl)amino)thieno[3,2-c]quinoline-7-carboxylic Acid
(17f). Yield, 75%. LCMS (ES): >95% pure, m/z 335 [M þ H]þ. 1H
NMR (DMSO-d6, 400 MHz): δ 3.68 (s, 3H), 6.12 (d, J = 5, 1H),
7.38 (m, 3H), 7.49 (m, 2H), 7.73 (d, J = 5.2, 1H), 7.95 (d, J = 8.8,
1H), 8.15 (d, J = 8.8, 1H), 8.55 (br s, 1H) ppm.
Methyl 4-(Phenylamino)thieno[3,2-c]quinoline-7-carboxylate
(15). Methyl 4-chlorothieno[3,2-c]quinoline-7-carboxylate 14a
(1.0 equiv, 60 mg, 0.216 mmol) was mixed with aniline (10.1
equiv, 0.2 mL, 2.19 mmol) and NMP (0.2 mL). The mixture was
reacted in a microwave oven at 120 °C for 10 min. Water was
added, and the product was extracted with dichloromethane.
The organic phase was dried over Na2SO4, and the solvent was
removed in vacuo. The residue was triturated with dichloro-
methane, EtOAc, and hexanes to give a solid. After filtration
and drying, the title compound 15 was isolated as a brown solid.
(56 mg, 81%). LCMS (ES): 95% pure, m/z 335 [M þ H]þ.
4-(Phenylamino)thieno[3,2-c]quinoline-7-carbonitrile (16).
4-Chlorothieno[3,2-c]quinoline-7-carbonitrile 14b (1.0 equiv, 130 mg,
0.533 mmol), aniline (0.2 mL), and NMP (0.2 mL) were mixed in
a microwave vessel. The mixture was heated in a microwave oven
at120 °C for 10 min. Water was added, andthe resulting solid was
filtered and dried. 4-(Phenylamino)thieno[3,2-c]quinoline-7-car-
bonitrile 16 was isolated as a gray solid (132 mg, 83%). LCMS
(ES): 95% pure, m/z 302 [M þ H]þ. 1H NMR (DMSO-d6, 400
MHz): δ 7.07 (t, J = 7.2, 1H), 7.38 (td, J = 7.6, J = 2.0, 2H), 7.66
(dd, J = 8.0, J = 1.6, 1H), 8.08(dd, J = 8.8, J = 1.2, 2H), 8.11 (d,
J = 4.8, 1H), 8.14 (d, J = 8.4, 1H), 8.19 (d, J = 1.6, 1H), 8.26 (d,
J = 5.2, 1H), 9.40 (s, 1H) ppm.
4-(Phenylamino)thieno[3,2-c]quinoline-7-carboxylic Acid (17e).
Methyl 4-(phenylamino)thieno[3,2-c]quinoline-7-carboxylate 15
(15 mg, 0.045 mmol) was reacted in a mixture of THF (0.5 mL),
methanol (0.5 mL), water (0.5 mL), and lithium hydroxide
(60 mg) at room temperature overnight. Water was added and
the mixture acidified by addition of hydrochloric acid. The vola-
tiles solvents were removed in vacuo, and the resulting solid was
filtered and dried. The title compound 17e was isolated as an off-
white solid (6 mg, 42%). LCMS (ES): >95% pure, m/z 321 [M þ
H]þ. 1H NMR (DMSO-d6, 400 MHz): δ 7.28 (br s, 1H), 7.51 (t,
J = 6.8, 2H), 7.87 (br s, 2H), 7.93 (d, J = 8.8, 1H), 8.16 (m, 2H),
8.35 (d, J = 5.2, 1H), 8.47 (br s, 1H) ppm.
4-(o-Tolylamino)thieno[3,2-c]quinoline-7-carboxylic Acid (17g).
1
Yield, 90%. LCMS (ES): >95% pure, m/z 335 [M þ H]þ. H
NMR (DMSO-d6, 400 MHz): δ 2.31 (s, 3H), 7.44 (br s, 2H), 7.50
(br s, 1H), 7.55 (m, 1H), 7.98 (br m, 1H), 8.24 (br m, 2H), 8.40
(br s, 1H), 8.56 (br s, 1H) ppm.
4-(Phenethylamino)thieno[3,2-c]quinoline-7-carboxylic Acid (17h).
1
Yield, 56%. LCMS (ES): >95% pure, m/z 349 [M þ H]þ. H
NMR (DMSO-d6, 400 MHz): δ3.12 (t, J = 7.6, 2H), 4.01 (br s, 2H)
7.23(t, J= 6.8, 1H), 7.33 (t, J= 7.6, 2H), 7.39 (d, J= 7.6, 2H), 7.98
(br m, 1H), 8.18 (br m, 2H), 8.34 (br s, 1H), 8.78 (br s, 1H), 10.22 (br
s, 1H), 12.83 (br s, 1H), 13.45 (br s, 1H) ppm.
4-(4-Fluorophenylamino)thieno[3,2-c]quinoline-7-carboxylic Acid
(17i). Yield, 75%. LCMS (ES): >95% pure, m/z 339 [M þ H]þ. 1H
NMR (DMSO-d6, 400 MHz): δ 7.38 (br t, J = 7.4, 2H), 7.95 (m,
3H), 8.18 (m, 2H), 8.36 (br d, J = 5.9, 1H), 8.48 (br s, 1H) ppm.
4-(3-Fluorophenylamino)thieno[3,2-c]quinoline-7-carboxylic Acid
(17j). Yield, 75%. LCMS (ES): >95% pure, m/z 339 [M þ H]þ. 1H
NMR (DMSO-d6, 400 MHz): δ 7.06 (br t, J = 8.0, 1H), 7.51 (q,
J =6, 1H), 7.77(brd, J= 9.6, 1H), 7.96 (dd, J= 8.0, J =1.6, 1H),
8.04 (br d, J= 12.8, 1H), 8.16 (s, 1H), 8.17 (d, J= 4.0, 1H), 8.47 (m,
2H) ppm.
4-(4-Chlorophenylamino)thieno[3,2-c]quinoline-7-carboxylic Acid
(17k). Yield, 100%. LCMS (ES): 95% pure, m/z 355 [M þ H]þ. 1H
NMR (DMSO-d6, 400 MHz): δ 7.50 (d, J = 8.0, 2H), 7.92 (d, J =
8.4, 1H), 8.13 (m, 3H), 8.28 (d, J = 5.6, 1H), 8.41(s, 1H), 9.80 (br s,
1H) ppm.
4-(3-Chlorophenylamino)thieno[3,2-c]quinoline-7-carboxylic Acid
(17l). Yield, 49%. LCMS (ES): >95% pure, m/z 355 [M þ H]þ. 1H
NMR (DMSO-d6, 400 MHz): δ 7.20 (br d, J = 8, 1H), 7.47 (t, J =
8, 1H), 7.93 (d, J = 8.4, 1H), 8.05 (d, J = 8, 1H), 8.14 (m, 2H), 8.32
(m, 2H), 8.40 (m, 1H), 9.95 (br s, 1H) ppm.
4-(3-Methoxyphenylamino)thieno[3,2-c]quinoline-7-carboxylic
Acid (17m). Yield, 87%. LCMS (ES): >95% pure, m/z 351 [M þ
H]þ. 1H NMR (DMSO-d6, 400 MHz): δ 3.83 (s, 3H), 6.88 (br s,
1H), 7.42 (m, 2H), 7.62 (br, 1H), 7.96 (br d, J = 9, 2H), 8.19 (br d,
J = 8, 2H), 8.38 (br d, J = 4.1, 1H), 8.50 (br s, 1H) ppm.
4-(3-Ethynylphenylamino)thieno[3,2-c]quinoline-7-carboxylic
Acid (17n). Yield, 59%. LCMS (ES): >95% pure, m/z 345 [M þ
H]þ. 1H NMR (DMSO-d6, 400 MHz): δ 4.23 (s, 1H), 7.26 (br s,
1H), 7.47(t, J = 7.6, 1H), 7.93 (d, J = 8.4, 1H), 8.13 (m, 3H), 8.22
(s, 1H), 8.31 (d, J = 5.2, 1H), 8.39 (s, 1H), 9.78 (br, 1H) ppm.
4-(3-Phenoxyphenylamino)thieno[3,2-c]quinoline-7-carboxylic
Acid (17o). Yield, 80%. LCMS (ES): 95% pure, m/z 413 [M þ
H]þ. 1H NMR (DMSO-d6, 400 MHz): δ 6.91 (br m, 1H), 7.19
(m, 3H), 7.46 (m, 3H), 7.64 (br d, J = 8, 1H), 7.82 (br s, 1H), 7.93
(d, J = 8.8, 1H), 8.13 (d, J = 6, 2H), 8.36 (br s, 1H), 8.48 (br d,
J = 4.4, 1H) ppm.
General Procedure for the Synthesis of Compounds 17a-d and
17f-p. Synthesis of 4-(3-Hydroxypropylamino)thieno[3,2-c]qui-
noline-7-carboxylic Acid (17a). Methyl 4-chlorothieno[3,2-c]-
quinoline-7-carboxylate 14a (7 mg, 0.025 mmol) was mixed with
3-amino-1-propanol (0.1 mL) and NMP (0.1 mL). The mixture
was reacted in a microwave oven at 100 °C for 5 min. The mix-
ture was diluted with NMP and purified by preparative HPLC.
After evaporation of the solvents in vacuo, the resulting ester
was reacted with lithium hydroxide (40 mg, 1.67 mmol) in a 1:1:1
mixture of THF/methanol/water (0.5 mL) at room temperature
for 2 h. The resulting mixture was diluted and the compound
purified by preparative HPLC. After evaporation of the solvents
in vacuo, the title compound 17a was isolated as a white solid
(4.6 mg, 61%). Alternatively, materials could be isolated in a
4-(3-(Methylcarbamoyl)phenylamino)thieno[3,2-c]quinoline-7-car-
boxylic Acid (17p). Yield, 49%. LCMS (ES): 95% pure, m/z 378

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2 647
[M þ H]þ. 1H NMR (DMSO-d6, 400MHz):δ2.83(d, J = 4, 3H),
7.58 (br t, 1H), 7.71 (br s, 1H), 7.95 (d, J = 8, 1H), 8.17 (m, 2H),
8.33 (br s, 1H), 8.46 (br m, 2H), 8.55 (br s,1H) ppm.
J = 7.2, 2H), 7.57 (d, J = 5.2, 1H), 8.57 (d, J = 4.8, 1H), 8.04
(s, 1H) ppm.
Methyl 5-Bromopyrimidine-4-carboxylate (22g). Crude 5-bro-
mopyrimidine-4-carboxylic acid 21g (prepared according to the
procedure described in literature61) (1.0 equiv, 6.14 g, 30.2 mmol)
was suspended in CH2Cl2 (100 mL). Oxalylchloride (1.1 equiv,
2.9 mL, 33.0 mmol) was added followed by 2 drops of DMF. The
mixture was stirred at room temperature overnight, and the
volatiles were removed in vacuo. The residue was dissolved in
methanol (50 mL) and heated. After evaporation of methanol in
vacuo, the compound was dissolved in CH2Cl2 and poured on a
prepacked silica gel column. The material was eluted using 20%
of ethyl acetate in hexanes. Evaporation of the solvent provided
the title compound 22g as a light-orange crystalline solid (2.54 g,
39%). LCMS (ES): 95% pure, m/z 217 [M]þ, 219 [M þ 2]þ. 1H
NMR (CDCl3, 400 MHz) δ 4.04 (s, 3H), 9.02 (s, 1H), 9.21 (s, 1H)
ppm. 13C NMR (CDCl3, 100 MHz) δ 53.73, 118.31, 155.49,
156.78, 161.52, 163.85 ppm.
4-(Phenylamino)thieno[3,2-c]quinoline-7-carboxamide (18).
4-(Phenylamino)thieno[3,2-c]quinoline-7-carbonitrile 16 (34 mg,
0.113 mmol) was dissolved in NMP (0.3 mL). Aqueous H2O2
(30%, 0.2 mL) was added, followed by addition of 6N NaOH
(50μL). The mixture was stirred at 50 °C for 2 h. An extra amount
of 30% aqueous H2O2 (0.3 mL) and 6N NaOH (100 μL)
were added to reach 70% conversion in 30 min. Waterwas added
and the solid filtered and dried. The material was further
reacted under the same conditions in order to achieve a complete
transformation. 4-(Phenylamino)thieno[3,2-c]quinoline-7-car-
boxamide 18 was isolated as a solid (30 mg, 83%). LCMS (ES):
95% pure, m/z 320 [M þ H]þ.
N-Phenyl-7-(4H-1,2,4-triazol-3-yl)thieno[3,2-c]quinolin-4-amine
(20). 4-(Phenylamino)thieno[3,2-c]quinoline-7-carboxamide 18
(28 mg, 0.088 mmol) was suspended in N,N-dimethylformamide
dimethylacetal and the mixture stirred at 80 °C under nitrogen
atmosphere for 2 h. The volatiles were removed in vacuo. Acetic
acid (0.5 mL) and anhydrous hydrazine (0.1 mL) were added, and
the mixture was stirred at 115 °C for 1 h. Water and brine were
added and the solid was filtered. The material was purified by pre-
parative HPLC. Genevac evaporation and trituration in EtOAc/
hexanes afforded the title compound 20 as an off-white solid (9 mg,
Methyl 3-Bromopicolinate (22i). 3-Bromopicolinic acid 21i
(1.0 equiv, 5.67 g, 28.08 mmol) was reacted with oxalyl chloride
(1.2 equiv, 3.0 mL, 34.39 mmol) in dichloromethane (75 mL).
DMF (0.2 mL) was added, and the mixture was stirred for 2 h.
The mixture was cooled to 0 °C, and methanol (10 mL) was
added. The mixture was diluted with dichloromethane (200 mL).
The organic phase was washed with a saturated solution of sodium
bicarbonate (2 ꢀ 100 mL) and brine (100 mL). The solution was
dried over Na2SO4 and the volatiles removed invacuotogivemethyl
3-bromopicolinate 22i as a brownish oil (5.82 g, 96%). 1H NMR
(CDCl3, 400 MHz) δ 4.00 (s, 3H), 7.30 (dd, J = 8.1, J = 4.0, 1H),
8.00 (dd, J=8.1, J= 1.3, 1H), 8.61 (dd, J=4.0, J= 1.3, 1H) ppm.
Methyl 2-Bromonicotinate (22e). 2-Bromopicolinic acid 21e
(1.0 equiv, 2.51 g, 12.45 mmol) was reacted with oxalyl chloride
(1.3 equiv, 1.4 mL, 15.81 mmol) in dichloromethane (40 mL).
DMF (0.1 mL) was added, and the mixture was stirred for 2 h.
An additional amount of oxalyl chloride (1.4 mL) was added
and the reaction stirred for an additional hour. The mixture was
cooled to 0 °C, and methanol (5 mL) was added. The mixture
was diluted with dichloromethane (200 mL). The organic phase
was washed with a saturated solution of sodium bicarbonate
(2 ꢀ 100 mL) and brine (100 mL). The solution was dried over
Na2SO4 and the volatiles removed in vacuo to give methyl 2-bro-
1
30%). LCMS (ES): 94% pure, m/z 344 [M þ H]þ. H NMR
(CDCl3/CD3OD, 9:1, 400 MHz) δ 6.57 (d, J = 4.8, 1H),
7.42-7.44 (m, 3H), 7.47-7.52 (m, 3H), 8.10 (d, J = 8.4, 1H),
8.14 (s, 1H), 8.30 (d, J = 7.2, 1H), 8.39 (s, 1H) ppm.
General Procedure for the Synthesis of Tetrazole Analogues
19a-c. N-Phenyl-7-(1H-tetrazol-5-yl)thieno[3,2-c]quinolin-4-amine
(19a). 4-Chlorothieno[3,2-c]quinoline-7-carbonitrile 14b (1.0 equiv,
23 mg, 0.094 mmol), aniline (0.1 mL), and NMP (0.1 mL) were
mixed in a microwave vial. The mixture was heated in a microwave
oven at 120 °C for 10 min. Water was added and the resulting inter-
mediate 4-(phenylamino)thieno[3,2-c]quinoline-7-carbonitrile
16 was filtered and dried. This crude material was mixed in a vial
with DMF (0.5 mL), NH4Cl (50 mg), and NaN3 (50 mg). The
mixture was stirred at 120 °C for 3 h. After addition of water and
filtration, N-phenyl-7-(1H-tetrazol-5-yl)thieno[3,2-c]quinolin-4-
amine 19a was isolated as a beige solid (13 mg, 41%). LCMS
(ES): >95% pure, m/z 345 [M þ H]þ, 317 [M þ H - N2]þ. 1H
NMR (DMSO-d6, 400 MHz): δ 7.07 (t, J = 7.2, 1H), 7.40 (t, J =
7.6, 2H), 8.00 (dd, J = 1.6, J = 8.4, 1H), 8.04 (d, J = 5.2, 1H),
8.10 (dd, J = 1.2, J = 8.8, 2H), 8.19 (d, J = 8.0, 1H), 8.25 (d, J =
5.6, 1H), 8.43 (d, J = 1.6, 1H), 9.34 (s, 1H) ppm.
1
mopicolinate 22e as an oil (1.97 g, 73%). H NMR (CDCl3, 400
MHz) δ 3.95 (s, 3H), 7.32 (dd, J = 7.7, J = 5.1, 1H), 8.17 (dd, J =
7.7, J = 2.2, 1H), 8.60 (dd, J = 5.1, J = 2.0, 1H) ppm.
Ethyl 3-Chloropyrazine-2-carboxylate (22f). 3-Chloropyra-
zine-2-carboxylic acid 21f (1.0 equiv, 2.08 g, 13.11 mmol) was
reacted with oxalyl chloride (1.6 equiv, 1.8 mL, 20.63 mmol) in
dichloromethane (75 mL). DMF (0.4 mL) was added, and the
mixture was stirred at room temperature overnight. The mixture
was concentrated and dissolved in dichloromethane (50 mL).
Ethanol (5 mL) was added at 0 °C, and the mixture was stirred
for one hour at room temperature. The mixture was diluted with
dichloromethane (200 mL). The organic phase was washed with
a saturated solution of sodium bicarbonate (2 ꢀ 100 mL) and
brine (100 mL). The solution was dried over Na2SO4 and the
volatiles removed in vacuo. The solution was concentrated and
filtered through a plug of silica gel. Evaporation of the solvents
gave the title compound 22f as an oil (1.65 g, 67%). One spot
TLC. LCMS (ES): >80% pure, m/z 187 [M þ H]þ. The material
was used for next step without any further purification.
Methyl 5-Oxo-5,6-dihydrobenzo[c][2,6]naphthyridine-8-car-
boxylate (23a). Ethyl 3-bromoisonicotinate 22a (1.15 g, 5.0
mmol), 2-amino-4-methoxycarbonyl-phenylboronic acid hydro-
chloride 10a (1.04 g, 4.5 mmol), sodium acetate (1.64 g,
20 mmol), PdCl2 (dppf) (182 mg, 0.25 mmol), and dimethylfor-
mamide (7.5 mL) were combined in a flask. The flask was eva-
cuated and filled with nitrogen twice and heated to 125 °C with
stirring for 12 h or until LCMS indicated the absence of any
starting material. The mixture was cooled to room temperature
and water (100 mL) was added to form a brown precipitate. The
N-(3-Chlorophenyl)-7-(1H-tetrazol-5-yl)thieno[3,2-c]quinolin-
4-amine (19b). Yield, 13%. LCMS (ES):>95% pure, m/z 379
[M þ H]þ, 351 [M þ H - N2]þ.
N-(3-Fluorophenyl)-7-(1H-tetrazol-5-yl)thieno[3,2-c]quinolin-
4-amine (19c). Yield, 47%. LCMS (ES): > 95% pure, m/z 363
[M þ H]þ, 335 [M þ H - N2]þ. 1H NMR (DMSO-d6, 400 MHz):
δ 6.89 (td, J = 8.4, J = 2.4, 1H), 7.43 (q, J = 7.9, 1H), 7.85 (dd,
J = 1.2, J = 8.0, 1H), 8.06-8.09 (m, 2H), 8.23 (d, J = 8.0, 1H),
8.27 (d, J = 5.6, 1H), 8.33 (dt, J = 2.0, J = 12.4, 1H), 8.51 (d,
J = 1.2, 1H), 9.53 (s, 1H) ppm.
Ethyl 3-Bromoisonicotinate (22a). 3-Bromoisonicotinic acid
21a (3.0 g, 14.9 mmol) in ethanol (100 mL) was treated with
concentrated sulfuric acid (5 mL). The mixture was brought to
reflux, at which time everything went into solution. After 12 h
at reflux, LCMS indicated that the reaction was complete. The
reaction mixture was cooled to room temperature and con-
centrated on a rotary evaporator to a third of its original
volume. The mixture was then diluted with 250 mL of ethyl
acetate and washed twice with saturated aqueous sodium
bicarbonate. Concentration on a rotary evaporator yielded
ethyl 3-bromoisonicotinate 22a (3.25 g, 95%) as a yellowish oil
which was sufficiently pure for subsequent chemical trans-
formations. LCMS (ES): >95% pure, m/z 230 [M þ H]þ.
1H NMR (CDCl3, 400 MHz) δ 1.37 (t, J = 8.0, 3H), 4.39 (q,

648 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2
Pierre et al.
precipitate was filtered to yield 637 mg (56%) of methyl 5-oxo-
5,6-dihydrobenzo[c][2,6]naphthyridine-8-carboxylate 23a. LCMS
Methyl 5-Oxo-5,6-dihydrobenzo[c][2,7]naphthyridine-8-car-
boxylate (23d). Yield, 90%. LCMS (ES): >95% pure, m/z 255
[M þ H]þ. 1H NMR (DMSO-d6, 400 MHz): δ 3.91 (s, 3H), 7.80
(d, J = 8.8, 1H), 7.99 (s, 1H), 8.45 (d, J = 5.6, 1H), 8.58 (d, J =
8.0, 1H), 8.96 (d, J = 5.6, 1H), 9.45 (br s, 1H), 12.07 (s, 1H) ppm.
Methyl 5-Oxo-5,6-dihydropyrazino[2,3-c]quinoline-8-carbox-
ylate (23f). Yield, 34%. LCMS (ES): >95% pure, m/z 256
1
(ES): 95% pure, m/z 255 [M þ H]þ. H NMR (DMSO-d6, 400
MHz): δ 3.87 (s, 3H), 7.69 (d, J = 8.0, 1H), 7.93 (s, 1H), 8.08 (d,
J = 5.2, 1H), 8.60 (d, J = 8.4, 1H), 8.79 (d, J = 5.2, 1H), 9.85 (s,
1H) ppm.
5-Oxo-5,6-dihydrobenzo[c][2,6]naphthyridine-8-carbonitrile (23b).
Sodium acetate (410 mg, 5 mmol) and PdCl2(dppf) (36 mg, 0.05
mmol) were added to a mixture of ethyl 3-bromoisonicotinate 22a
(230 mg, 1.0 mmol) and 2-amino-4-cyanophenylboronic acid
hydrochloric acid salt 10b (179 mg, 0.9 mmol). The mixture was
connected to an exit bubbler and heated to 120 °C for 18 h, at which
time LCMS analysis indicated that the reaction was done based on
the disappearance of starting material. After cooling to room tem-
perature, water was added and the dark solids were filtered and
washed with dichloromethane to give 5-oxo-5,6-dihydrobenzo[c]-
[2,6]naphthyridine-8-carbonitrile 23b (156 mg, 46%) as a gray
1
[M þ H]þ. H NMR (DMSO-d6, 400 MHz): δ 3.92 (s, 3H),
7.57 (d, J = 8.4, 1H), 8.01 (s, 1H), 8.65 (d, J = 8.4, 1H), 9.03 (d,
J = 2.0, 1H), 9.13 (d, J = 1.6, 1H), 12.05 (s, 1H) ppm.
Methyl 5-Oxo-5,6-dihydrobenzo[f][1,7]naphthyridine-8-car-
boxylate (23i). Yield, 29%. The crude solid was used for next
step without any further purification and characterization.
Methyl 2-Amino-3-bromobenzoate (32). Commercially avail-
able 2-amino-3-bromobenzoic acid 31 (1.00 g, 4.62 mmol) was
mixed with methanol (10 mL) and concentrated sulfuric acid
(1 mL). The mixture was stirred at reflux for 31 h. The solvent
were evaporated, and saturated aqueous sodium bicarbonate
was carefully added. The solid was extracted with CH2Cl2 (3ꢀ).
The combined extracts were dried over Na2SO4 and the solvents
removed in vacuo to afford methyl 2-amino-3-bromobenzoate
32 as a semicrystalline solid (976 mg, 91%). LCMS (ES): >85%
pure, m/z 230 [M þ H]þ. 1H NMR (CDCl3, 400 MHz): δ 3.88 (s,
3H), 6.34 (br s, 2H), 6.53 (t, J = 8.0, 1H), 7.57 (dd, J = 1.6, J =
7.6, 1H), 7.85 (dd, J = 1.6, J = 8.4, 1H) ppm.
Methyl 3-Amino-4-iodo-2-methylbenzoate (30a). Commer-
cially available methyl 3-amino-2-methylbenzoate 29 (1.0 equiv,
11.62 g, 70.4 mmol) was suspended in acetic acid (100 mL).
N-Iodosuccinimide (1.03 equiv, 16.32 g, 72.0 mmol) was added
in several portions. The reaction mixture was stirred at room
temperature for 30 min. The solvent was evaporated and the
residue diluted with EtOAc (150 mL). The organic phase was
washed with a solution of potassium carbonate (100 mL), water
(3 ꢀ 40 mL), and brine (3 ꢀ 40 mL). After drying over Na2SO4,
the solvent was removed by rotary evaporation. The reaction
produced a mixture of compounds, the major (86%) component
of the mixture being methyl 3-amino-6-iodo-2-methylbenzoate
30b (Rf = 0.25, SiO2, 25% ethyl acetate in hexanes, GCMS
m/z = 291) and the minor (14%) component being the desired
methyl 3-amino-4-iodo-2-methylbenzoate 30a (Rf = 0.5, SiO2,
25% ethyl acetate in hexanes, GCMS m/z = 291). The desired
minor product was isolated pure by flash chromatography on
silica gel (EtOAc 15% to 25% gradient in hexanes) to afford the
title compound 30a as a yellow solid (1.23 g, 6%). GCMS:
>95% pure, m/z 291. 1H NMR (CDCl3, 400 MHz): δ 2.08 (s,
3H), 3.60 (br s, 2H), 3.94 (s, 3H), 6.45 (d, J = 8.8, 1H), 7.39 (d,
J = 8.0, 1H) ppm.
1
solid. LCMS (ES): >90% pure, m/z 222 [M þ H]þ. HNMR
(400 MHz, DMSO-d6): δ 12.2 (1H, s), 9.96 (1H, s), 8.90 (1H, d, J =
5.1), 8.77 (1H, d, J = 8.2), 8.13 (1H, d, J = 5.1), 7.73 (1H, dd J =
8.2, J = 1.6), 7.70 (1H, d, J = 1.6) ppm.
Benzo[c][2,6]naphthyridin-5(6H)-one (23c). Ethyl 3-bromoi-
sonicotinate 22a (1.0 equiv, 1.76 g, 7.65 mmol), 2-aminophenyl-
boronic acid hydrochloride 10c (1.0 equiv, 1.33 g, 7.67 mmol),
and cesium carbonate (2.0 equiv, 4.99 g, 15.31 mmol) were sus-
pended in dioxane (15 mL). The mixture was degassed by bub-
bling nitrogen for 10 min. PdCl2 (dppf) (0.05 equiv, 280 mg, 0.383
mmol) was added, and the mixture was stirred at reflux for 2 h.
The resulting solid was filtered, washed with methanol, water,
and methanol, and dried. Benzo[c][2,6]naphthyridin-5(6H)-one
23c was isolated as an off-white solid (823 mg, 55%). LCMS(ES):
95% pure, m/z 197 [M þ H]þ.
Methyl 6-Oxo-5,6-dihydrophenanthridine-3-carboxylate (23h).
Ethyl 2-bromobenzoate 22h (1.3 equiv, 300 mg, 1.3 mmol) was
combined with 2-amino-4-methoxycarbonyl-phenylboronic acid
hydrochloride 10a (1.0 equiv, 195 mg, 1.0 mmol), sodium acetate
(3.9 equiv, 226 mg, 3.9 mmol), PdCl2 (dppf) (0.025 equiv, 25 mg,
0.025 mmol), and dimethylformamide (2.5 mL). The resulting
mixture was reacted in a microwave oven at 120 °C for 10 min.
Water was added, and the material was extracted with EtOAc
(2ꢀ). The organic phase was dried over Na2SO4 and filtered
through a plug of silica gel. Trituration with dichloromethane
and hexanes provided methyl 6-oxo-5,6-dihydrophenanthridine-
3-carboxylate 23h as a solid (125 mg, 49%). The crude solid was
used for next step without any further characterization and
purification.
General Procedure for the Synthesis of Compounds 23d-g and
23i. Synthesis of Methyl 5-Oxo-5,6-dihydropyrimido[4,5-c]-
quinoline-8-carboxylate (23g). Methyl 5-bromopyrimidine-4-
carboxylate 22g (1.0 equiv, 10.0 g, 46.08 mmol) was mixed in a
flask with 2-amino-4-methoxycarbonyl-phenylboronic acid hy-
drochloride 10a (1.1 equiv, 11.70 g, 50.56 mmol), cesium carbo-
nate (3.0 equiv, 45.0 g, 138.11 mmol), and PdCl2 (dppf) (0.015
equiv, 505 mg, 0.69 mmol) in anhydrous dioxane (200 mL). The
reaction mixture was degassed by bubbling nitrogen through the
suspension of reagents for about 10 min. The reaction was
stirred under reflux for 3 h, at which time LCMS monitoring
indicated the presence of starting materials. The conversion was
completed after adding an extra amount of palladium catalyst
(505 mg) and water (200 μL) and further reacting at reflux
overnight. After adding water and then brine, the precipitate
was filtered and washed with water (200 mL) and methanol (200
mL). The solid was stirred in ethyl acetate for 2 h, filtered, and
dried. The title compound 23g was isolated as a tan solid (9.39 g,
Methyl 2-Methyl-5-nitrobenzoate (34). Commercially avail-
able 2-methyl-5-nitrobenzoic acid 33 (24 g, 132.5 mmol) was
dissolved in a mixture of methanol (240 mL) and concentrated
sulfuric acid (8 mL). The mixture was stirred at reflux overnight.
Upon cooling, the ester crystallized out. The material was
isolated by filtration to afford methyl 2-methyl-5-nitrobenzoate
34 as a white solid (19.0 g, 74%). A second crop of material (4.92
g, 19%) was isolated upon concentration and addition of water
to the mother liquor. The crude solid was used for next step
without any further characterization and purification.
Methyl 5-Amino-2-methylbenzoate (35). Methyl 2-methyl-5-
nitrobenzoate 34 (5.06 g, 25.95 mmol) was suspended in metha-
nol (100 mL). The mixture was degassed by bubbling nitrogen
for 15 min. Pd/C (10% wet Degussa type E101 NE/WW, 260
mg) was added and the mixture stirred under hydrogen atmo-
sphere (balloon) overnight. The suspension was filtered and the
solvents evaporated to afford the title compound 35 as an
orange oil (4.18 g, 97%). 1H NMR (400 MHz, CDCl3): δ 2.45
(s, 3H), 3.45 (br s, 2H), 3.85 (s, 3H), 6.74 (dd, J = 7.4, J = 2.3,
1H), 7.02 (d, J = 7.4, 1H), 7.25 (d, J = 2.5, 1H) ppm.
1
80%). LCMS (ES): 95% pure, m/z 256 [M þ H]þ. H NMR
(DMSO-d6, 400 MHz): δ 3.79 (s, 3H), 7.81 (d, J = 8.0, 1H), 8.68
(d, J = 8.8, 1H), 9.49 (s, 1H), 10.19 (s, 1H), 12.37 (s, 1H) ppm.
Methyl 5-Oxo-5,6-dihydrobenzo[c][2,6]naphthyridine-8-car-
boxylate (23e). Yield, 21%. LCMS (ES): >95% pure, m/z 255
[M þ H]þ.
Methyl 5-Amino-4-iodo-2-methylbenzoate (36). Methyl
5-amino-2-methylbenzoate 35 (1.0 equiv, 3.75 g, 22.7 mmol)
was dissolved in acetic acid (70 mL). N-Iodosuccinimide

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2 649
(1.03 equiv, 5.27 g, 23.4 mmol) was added in small portions over
60 min. The mixture was stirred at room temperature for 30 min.
Acetic acid was evaporated. The residue was diluted with ethyl
acetate (80 mL) and neutralized with saturated sodium carbo-
nate (80 mL). The organic layer was washed with 1 M sodium
thiosulfate (2 ꢀ 40 mL) and then water (2 ꢀ 40 mL) and brine
(2 ꢀ 40 mL). The material was purified by flash chromatography
on silica gel (gradient 10-30% ethyl acetate in hexanes) to
provide methyl 5-amino-4-iodo-2-methylbenzoate 36 as a yellow-
was obtained after drying in vacuo. LCMS (ES): 95% pure, m/z
273 [M þ H]þ. 1H NMR (CDCl3, 400 MHz): δ 4.03 (s, 3H), 8.25
(dd, J = 6.0, J = 1.2, 1H), 8.40 (dd, J = 8.4, J = 1.6, 1H), 8.73
(d, J = 8.8, 1H), 8.82 (d, J = 2.0, 1H), 9.03 (d, J = 5.6, 1H),
10.08 (s, 1H) ppm.
5-Chlorobenzo[c][2,6]naphthyridine-8-carbonitrile (24b). Yield,
77%. LCMS (ES): 95% pure, m/z 240 [M þ H]þ.
Methyl 6-Chlorophenanthridine-3-carboxylate (24h). Yield,
97%. The crude solid was used for next step without any further
characterization and purification.
1
orange solid (3.19 g, 49%). GCMS >95% pure, m/z 291. H
NMR (400 MHz, DMSO-d6): δ 2.30 (s, 3H), 3.78 (s, 3H), 5.27
(br s, 2H), 7.24 (s, 1H), 7.54 (s, 1H) ppm.
Methyl 5-Chlorobenzo[h][1,6]naphthyridine-8-carboxylate (24e).
Yield, 57%. LCMS (ES): >95% pure, m/z 273 [M þ H]þ.
Methyl 5-Chloropyrazino[2,3-c]quinoline-8-carboxylate (24f).
Yield, 97%. LCMS (ES): >90% pure, m/z 274 [M þ H]þ.
Methyl 5-Chlorobenzo[f][1,7]naphthyridine-8-carboxylate (24i).
Yield, 80%. LCMS (ES): >85% pure, m/z 273 [M þ H]þ. The
material was used for next step without any further purification.
Methyl 5-Chlorobenzo[c][2,7]naphthyridine-8-carboxylate (24d).
A solution of methyl 5-oxo-5,6-dihydrobenzo[c][2,7]naphthyridi-
ne-8-carboxylate 23d (650 mg, 2.56 mmol) in phosphorus oxy-
chloride (4.0 mL) was heated at 120 °C for 2.5 h. The reaction was
concentrated under reduced pressure and diluted with ACN (20
mL) and H2O (40 mL). The solution was neutralized with NaOH
(3N), and the resulting precipitate was collected by filtration to give
the title compound 24d (600 mg, 86%). LCMS (ES): >95% pure,
m/z 273 [M þ H]þ.
General Procedure for the Synthesis of Compounds 39a-d.
Synthesis of Methyl 5-Oxo-5,6-dihydrobenzo[c][2,6]naphthyr-
idine-7-carboxylate (39b). Methyl 2-amino-3-bromobenzoate
32 (1.0 equiv, 652 mg, 2.61 mmol) and 4-(diisopropylcarbam-
oyl)pyridin-3-ylboronic acid 38 (prepared according to a proce-
dure described in literature),47,48 (1.0 equiv, 600 mg, 2.61 mmol)
were combined with cesium carbonate (2.0 equiv, 1.699 g, 5.21
mmol) in dioxane containing 5% of water (6 mL). The mixture
was degassed by bubbling nitrogen for 10 min. PdCl2 (dppf) (0.05
equiv, 95 mg) was added and the reaction stirred at reflux for 2 h.
Dioxane was evaporated, water was added, and the material
extracted with CH2Cl2 (3ꢀ). The combined extracts were dried
over Na2SO4 and the solvents removed in vacuo. The material
was purified by flash chromatography on silica gel (eluent 0.5%
methanol in CH2Cl2) to afford the intermediate methyl 2-amino-
3-(4-(diisopropylcarbamoyl)pyridin-3-yl)benzoate as a greenish
foam. (LCMS (ES): >95% pure, m/z 356 [M þ H]þ). This
material was dissolved under nitrogen atmosphere in anhydrous
THF (1.5 mL). A NaHMDS solution (1.0 M in THF, 2.0 equiv,
1.4 mL, 1.4 mmol) was added dropwise from a syringe. The
resulting suspension was stirred at room temperature for 1 h. The
reaction was quenched by addition of a saturated aqueous
solution of ammonium chloride. The solid that formed was
filtered and dried. After trituration in methanol and filtration,
the title compound was isolated as a gray fluffy solid (93 mg, 14%
over two steps). LCMS (ES): >95% pure, m/z 255 [M þ H]þ. 1H
NMR (400 MHz, DMSO-d6): δ 3.97 (s, 3H), 7.44 (t, J = 7.6, 1H),
8.11 (dd, J = 4,8, J = 0.8, 1H), 8.20 (dd, J = 7.6, J = 1.2, 1H),
8.87 (dd, J = 5.2, 1H), 8.91 (d, J = 7.2, 1H), 9.94 (s, 1H), 11.77
(br s, 1H) ppm.
Methyl 5-Chloropyrimido[4,5-c]quinoline-8-carboxylate (24g).
In a vial, methyl 5-oxo-5,6-dihydropyrimido[4,5-c]quinoline-8-
carboxylate 23g (1.0 equiv, 151 mg, 0.59 mmol) was mixed in
toluene (1 mL) with DIEA (1.5 equiv, 155 μL, 0.89 mmol) and
phosphorus oxychloride (5 equiv, 270μL, 3.0mmol). The mixture
was stirred at 120 °C for 1 h and cooled down to room tem-
perature. After adding ice and water, the compound was ex-
tracted with CH2Cl2 (4ꢀ). The solution was filtered over Na2SO4
and filtered through a pad of Celite. After evaporation of the
volatiles, the material was triturated in a mixture of ethyl acetate
and hexanes, filtered, and dried to afford methyl 5-chloropy-
rimido[4,5-c]quinoline-8-carboxylate 24g as a light-brown fluffy
solid (115 mg, 71%). LCMS (ES): 95% pure, m/z 274 [M þ H]þ.
1H NMR (DMSO-d6, 400 MHz): δ 3.96 (s, 3H), 8.37 (dd, J = 1.6,
J = 8.4, 1H), 8.60 (d, J = 1.6, 1H), 9.15 (d, J = 8.8, 1H), 9.74 (s,
1H), 10.61 (s, 1H) ppm.
Methyl 7-Methyl-5-oxo-5,6-dihydrobenzo[c][2,6]naphthyridine-
8-carboxylate (39a). Yield, 52%. LCMS (ES): >95% pure, m/z
269 [M þ H]þ. 1H NMR (400 MHz, DMSO-d6): δ 2.60 (s, 3H),
3.88 (s, 3H), 7.60 (d, J = 7, 1H), 8.16 (d, J = 5, 1H), 8.56 (d,
J = 7, 1H), 8.88 (d, J = 5, 1H), 9.94 (s, 1H), 11.13 (br s, 1H) ppm.
Methyl 9-Methyl-5-oxo-5,6-dihydrobenzo[c][2,6]naphthyridine
-8-carboxylate (39c). Yield, 41%. LCMS (ES): 89% pure, m/z
269 [M þ H]þ. 1H NMR (400 MHz, DMSO-d6): δ 2.62 (s, 3H),
3.88 (s, 3H), 7.89 (s, 1H), 8.13 (d, J = 4.8, 1H), 8.58 (s, 1H), 8.87
(d, J = 5.2, 1H), 9.95 (s, 1H), 12.07 (br s, 1H) ppm.
5-Chlorobenzo[c][2,6]naphthyridine (24c). Benzo[c][2,6]naph-
thyridin-5(6H)-one 23c (1.0 equiv, 813 mg, 4.15 mmol) was mixed
with phosphorus oxychloride (5.0 equiv, 2 mL, 21.84 mmol) and
acetonitrile (10 mL). The mixture was stirred at reflux for 5 h. The
mixture was poured on ice and the resulting solid filtered and dried.
5-Chlorobenzo[c][2,6]naphthyridine 24c was isolated as a gray
solid (459 mg, 52%). LCMS (ES): 95% pure, m/z 215 [M þ H]þ.
General Procedure for the Synthesis of Compounds 40a-d.
Synthesis of Methyl 5-Chlorobenzo[c][2,6]naphthyridine-7-car-
boxylate (40b). Methyl 5-oxo-5,6-dihydrobenzo[c][2,6]naphth-
yridine-7-carboxylate 39b (1.0 equiv, 85 mg, 0.334 mmol) was
stirred in phosphorus oxychloride (2 mL) at 120 °C for 2 h. The
solvent was removed in vacuo. Ice and water were added. The
resulting solid was filtered and dried to afford the title com-
pound 40b as a solid (84 mg, 92%). LCMS (ES): >95% pure,
m/z 273 [M þ H]þ.
Methyl 5-Oxo-5,6-dihydrobenzo[c][2,6]naphthyridine-9-car-
boxylate (39d). Yield, 41%. LCMS (ES): 90% pure, m/z 255
[M þ H]þ.
General Procedure for the Synthesis of Compounds 24a-b,
24e-f, and 24h-i. Methyl 5-Chlorobenzo[c][2,6]naphthyridine-
8-carboxylate (24a). Methyl 5-oxo-5,6-dihydrobenzo[c][2,6]-
naphthyridine-8-carboxylate 23a (200 mg, 0.787 mmol) was
combined with phosphorus oxychloride (1 mL) and heated to
reflux. After 2 h, LCMS indicated the absence of any starting
material. The volatiles were removed under reduced pressure.
The residue was taken up in dichloromethane (50 mL) and
washed twice with saturated aqueous sodium bicarbonate. The
organic phase was dried over sodium sulfate and concentrated
on a rotary evaporator to give methyl 5-chlorobenzo[c][2,6]-
naphthyridine-8-carboxylate 24a (140 mg, 65%) as a grayish
solid. An alternate workup consisted in adding acetonitrile after
evaporation of phosphorus oxychloride. The material was pre-
cipitated by addition of ice and water. Reasonably pure material
Methyl 5-Chloro-7-methylbenzo[c][2,6]naphthyridine-8-car-
boxylate (40a). Yield, 85%. LCMS (ES): >95% pure, m/z 287
1
[M þ H]þ. H NMR (400 MHz, DMSO-d6): δ 2.95 (s, 3H),
3.94 (s, 3H), 8.14 (d, J = 7.8, 1H), 8.28 (d, J = 5.7, 1H), 8.98 (d,
J = 9, 1H), 9.08 (d, J = 6, 1H), 10.37 (s, 1H) ppm.
Methyl 5-Chloro-9-methylbenzo[c][2,6]naphthyridine-8-car-
boxylate (40c). Yield, 76%. LCMS (ES): >95% pure, m/z 287
1
[M þ H]þ. H NMR (400 MHz, DMSO-d6): δ 2.79 (s, 3H),
3.94 (s, 3H), 8.25 (d, J = 5, 1H), 8.45 (s, 1H), 9.02 (s, 1H), 9.07
(d, J = 5, 1H), 10.35 (s 1H) ppm.
Methyl 5-Chlorobenzo[c][2,6]naphthyridine-9-carboxylate (40d).
Yield, 50%. LCMS (ES): >95% pure, m/z 273 [M þ H]þ.

650 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2
Pierre et al.
Methyl 5-(3-Chlorophenylamino)benzo[c][2,6]naphthyridine-
8-carboxylate (26a). Methyl 5-chlorobenzo[c][2,6]naphthyri-
dine-8-carboxylate 24a (1.0 equiv, 529 mg, 1.94 mmol) was
combined with 3-chloroaniline (1.5 equiv, 370 mg, 2.92 mmol)
in NMP (4 mL) in a flask, and the mixture was heated to 80 °C
for 2 h. The mixture was dissolved in CH2Cl2, washed with
saturated aqueous sodium bicarbonate, and dried over Na2SO4.
The material was purified by flash chromatography (SiO2, 1:1 to
9:1 gradient of EtOAc/hexanes) to obtain the title compound
The final carboxylic acid materials could be isolated by filtra-
tion as pure solids after adding water and neutralizing the
solution with an aqueous solution of hydrochloric acid or could
be isolated by filtration as sodium carboxylate salts when
precipitation occurred during the reaction. An alternate isola-
tion method consisted in diluting the reaction mixture with a
mixture of acetonitrile, water, and trifluoroacetic acid. The
resulting solutions were submitted to preparative HPLC, pro-
viding the pure carboxylic acids after Genevac evaporation.
Compounds bearing a basic moiety were isolated as trifluoro-
acetate salts.
1
26a. LCMS (ES): >95% pure, m/z 364 [M þ H]þ. H NMR
(DMSO-d6, 400 MHz): δ 3.91 (s, 3H), 7.16 (dd, J = 1.2, J = 8.0,
1H), 7.44 (t, J = 8.0, 1H), 7.94 (dd, J = 8.4, J = 2.0, 1H), 8.06
(dd, J = 8.4, J = 1.2, 1H), 8.20-8.22 (m, 2H), 8.82-8.86 (m,
2H), 9.04 (d, J = 5.6, 1H), 10.05 (br s, 1H), 10.21 (s, 1H) ppm.
N-(3-Chlorophenyl)benzo[c][2,6]naphthyridin-5-amine (26c).
5-Chlorobenzo[c][2,6]naphthyridine 24c (1.0 equiv, 20 mg,
0.093 mmol) was mixed with 3-chloroaniline (15 equiv, 150 μL,
1.42 mmol) and NMP (0.8 mL). The mixture was stirred at 60 °C
overnight. Water was added and the material extracted with
dichloromethane. The organic phase was extracted with water
(2ꢀ) and the solvents evaporated. The material was purified by
preparative TLC (SiO2, 5% methanol in dichloromethane). The
title compound 26c was isolated as a solid (19 mg, 68%). LCMS
(ES): >95% pure, m/z 306 [M þ H]þ. 1H NMR (DMSO-d6, 400
MHz): δ 7.12(brd, J = 8.4, 1H), 7.42 (t, J = 8.0, 1H), 7.52 (t, J =
7.2, 1H), 7.70 (t, J = 7.2, 1H), 7.77 (d, J = 7.2, 1H), 8.07 (d, J =
8.0, 1H), 8.34 (s, 1H), 8.58 (d, J = 5.2, 1H), 8.79 (d, J = 8.0, 1H),
8.97 (br s, 1H), 9.62 (s, 1H), 10.18 (br s, 1H) ppm.
(5-(3-Chlorophenylamino)benzo[c][2,6]naphthyridin-8-yl)methanol
(26d). Methyl 5-(3-chlorophenylamino)benzo[c][2,6]naphthyridine-
8-carboxylate 26a (50 mg, 0.137 mmol) was dissolved in THF
(4 mL) and the solution cooled to -40 °C. A solution of LiAlH4
(1.0 M in diethylether, 270 μL, 0.27 mmol) was added, and the
reaction mixture was allowed to warm to room temperature. After
aqueous workup, the product was purified by flash chromatogra-
phy on silica gel (ethyl acetate/hexanes gradient from 1:1 to 9:1).
The title compound 26d was isolated as a solid (46 mg, 100%).
LCMS (ES): >95% pure, m/z 336 [M þ H]þ. 1H NMR (DMSO-
d6, 400 MHz): δ 4.72 (s, 2H), 7.16 (dd, J = 8, J = 1.2, 1H),
7.43-7.49 (m, 2H), 7.75 (s, 1H), 8.05 (br d, J = 8.4, 1H), 8.32 (s,
1H), 8.69 (d, J = 5.6, 1H), 8.74 (d, J = 8.8, 1H), 8.96 (d, J = 6.0,
1H), 9.82 (br s, 1H), 10.18 (s, 1H) ppm.
5-(Phenylamino)benzo[h][1,6]naphthyridine-8-carboxylic Acid
(27e). Yield, 88%. LCMS (ES): >95% pure, m/z 316 [M þ H]þ.
1HNMR (400 MHz, DMSO-d6): δ 7.10 (t, J = 7.2, 1H), 7.43 (t,
J = 7.6, 2H), 7.89 (dd, J = 4.0, J = 8.0, 1H), 7.95 (dd, J = 2,
J = 8.4, 1H), 8.10 (d, J = 1.6, 2H), 8.25 (d, J = 1.6, 1H), 8.90 (d,
J = 8.4, 1H), 9.14 (dd, J = 1.6, J = 8.4, 1H), 9.21 (dd, J = 1.2,
J = 4.4, 1H), 9.54 (s, 1H), 13.16 (s, 1H) ppm.
5-(Phenylamino)benzo[c][2,6]naphthyridine-8-carboxylic Acid,
Trifluoroacetate Salt (25a). Yield, 23%. LCMS (ES): >95%
pure, m/z 316 [M þ H]þ. 1HNMR (400 MHz, CD3OD):δ 10.17
(1H, s), 9.67 (1H, br), 8.99 (1H, d, J = 5.9), 8.83 (1H, d, J = 8.6),
8.62 (1H, d, J = 5.9), 8.24 (1H, d, J = 1.6), 8.04 (1H, s), 8.02 (1H,
s), 7.93 (1H, dd, J = 8.2, J = 1.6), 7.43 (1H, d, J = 7.4), 7.41 (1H,
d, J = 7.4), 7.10 (1H, m) ppm.
5-(Phenylamino)benzo[c][2,7]naphthyridine-8-carboxylic Acid
(27d). Yield, 72%. LCMS (ES): 82% pure, m/z 316 [M þ H]þ.
1H NMR (400 MHz, DMSO-d6): δ 7.10 (t, J = 7.6, 1H), 7.43 (t,
J = 7.6, 2H), 7.93 (dd, J = 8.4, J = 1.6, 1H), 8.09 (d, J = 7.6,
2H), 8.22 (d, J = 1.6, 1H), 8.67 (d, J = 6.0, 1H), 8.71 (d, J = 8.4,
1H), 8.99 (d, J = 5.6, 1H), 9.69 (s, 1H), 10.01 (s, 1H), 13.20 (br s,
1H) ppm.
Sodium 5-(Phenylamino)benzo[f][1,7]naphthyridine-8-carbox-
ylate (27i). Yield, 10%. LCMS (ES): >95% pure, m/z 316 [M þ
H]þ. 1H NMR (400 MHz, DMSO-d6): δ 7.05 (t, J = 6.8, 1H),
7.42 (t, J = 8, 2H), 7.95 (m, 2H), 8.26 (s, 1H), 8.32 (d, J = 8.0,
2H), 8.46 (d, J = 8.4, 1H), 9.02 (d, J = 4.4, 1H), 9.17 (d, J = 8.4,
1H), 9.73 (s, 1H) ppm.
2-(8-Carboxybenzo[c][2,6]naphthyridin-5-ylamino)-N,N-di-
methylethanaminium Trifluoroacetate (25b). Yield, 45%. LCMS
(ES): >95% pure, m/z 311 [M þ H]þ, 266 [M þ H - NHMe2]þ.
1H NMR (400 MHz, DMSO-d6): δ 2.90 (s, 6H), 3.47 (br t, 2H),
3.95 (br s, 2H), 7.88 (dd, J = 8.4, J = 2.0, 1H), 8.24 (m, 2H), 8.41
(br s, 1H), 8.79 (d, J = 8.8, 1H), 8.94 (d, J = 5.2, 1H), 9.49 (br,
1H), 10.12 (s, 1H) ppm.
5-(Cyclopentylamino)benzo[c][2,6]naphthyridine-8-carboxylic
Acid (25c). Yield, 39%. LCMS (ES): 94% pure, m/z 308 [M þ
H]þ. 1H NMR (400 MHz, DMSO-d6): δ 1.62-1.77 (m, 6H), 2.12
(m, 2H), 4.64 (sext, J = 6.8, 1H), 7.70 (br, 1H), 7.84 (m, 1H),
8.16 (br s, 1H), 8.42 (br s, 1H), 8.74 (d, J = 8.0, 1H), 8.89 (m,
1H), 10.07 (s, 1H), 13.0 (br s, 1H) ppm.
5-(Methoxyamino)benzo[c][2,6]naphthyridine-8-carboxylic
Acid (25d). Yield, 26%. LCMS (ES): 94% pure, m/z 270 [M þ
H]þ. 1H NMR (400 MHz, DMSO-d6): δ 3.92 (s, 3H), 7.55 (d,
J = 8.0, 1H), 7.83 (d, J = 5.2, 1H), 8.09 (s, 1H), 8.35 (d, J =
8.4, 1H), 8.58 (d, J = 5.6, 1H), 9.56 (s, 1H), 10.30 (s, 1H),
12.24 (br s, 1H) ppm.
General Procedure for the Synthesis of Compounds 25a-t,
27d-e, 27h-i, 28d-g, 28i, and 41a-d. 6-(Phenylamino)phenan-
thridine-3-carboxylic Acid (27h). Methyl 6-chlorophenanthri-
dine-3-carboxylate 24h (1.0 equiv, 53 mg, 0.20 mmol) was
combined with aniline (3 equiv, 57 mg, 0.59 mmol) and
N-methyl pyrrolidinone (0.2 mL) in a microwave tube, and the
mixture was heated to 120 °C for 10 min, at which time LCMS
indicated that the reaction was complete as indicated by the
absence of any starting material. 6N NaOH (0.5 mL) was added,
and the mixture was stirred at 50 °C for 30 min. The mixture was
neutralized with 1N hydrochloric acid. The material was fil-
tered. Purification by preparative HPLC and Genevac evapora-
tion afforded the title compound 27h as a solid (28 mg, 45%).
1
LCMS (ES): >95% pure, m/z 315 [M þ H]þ. H NMR (400
MHz, CD3OD): δ 7.56-7.70 (m, 5H), 7.99 (t, J = 8.4, 1H), 8.17
(td, J = 8, J = 1.6, 1H), 8.19 (br t, J = 6.8, 1H), 8.49 (d, J = 1.6,
1H), 8.70 (d, J = 8.2, 1H), 8.73 (d, J = 7.2, 1H), 8.86 (d, J = 8,
1H) ppm.
5-(Cyclopropylamino)benzo[c][2,6]naphthyridine-8-carboxylic
Acid Trifluoroacetate Salt (25e). Yield, 49%. LCMS (ES): 85%
pure, m/z 280 [M þ H]þ.
5-(2-Isopropoxyethylamino)benzo[c][2,6]naphthyridine-8-car-
boxylic Acid (25f). Yield, 30%. LCMS (ES): 91% pure, m/z 326
[M þ H]þ. 1H NMR (400 MHz, DMSO-d6): δ 1.10 (d, J = 6.0,
6H), 3.61-3.76 (m, 5H), 7.84 (d, J = 8.5, 1H), 8.06 (br, 1H), 8.16
(br s, 1H), 8.30 (d, J = 6.4, 1H), 8.75 (d, J = 8.5, 1H), 8.90 (d,
J = 6.4, 1H), 10.08 (s, 1H), 13.10 (br s, 1H) ppm.
The following compounds were prepared using a similar two-
step procedure. The nucleophilic substitutions between various
chloro compounds 24a, 24d-i, and 40a-d and various amines
was achieved in NMP at temperatures ranging from 80 to 120 °C
using microwave (10-15 min) or conventional heating (2 h). The
ester hydrolyses were carried out in one pot by adding aqueous
(3N or 6N) NaOH to the precedent mixture and heating the
solution at 50-80 °C for up to 2 h. Alternatively, the hydrolysis
reaction could be accomplished after isolation of the ester by
precipitation in water and purification by flash chromatography.
5-(Benzylamino)benzo[c][2,6]naphthyridine-8-carboxylic Acid
Trifluoroacetate Salt (25g). Yield, 69%. LCMS (ES): >95%
pure, m/z 330 [M þ H]þ. 1H NMR (400 MHz, DMSO-d6): δ 4.87
(s, 2H), 7.23 (t, J = 6.8, 1H), 7.33 (m, 2H), 7.47 (m, 2H), 7.83 (d,

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2 651
J = 8.4, 1H), 8.13 (s, 1H), 8.37 (d, J = 4.4, 1H), 8.73 (d, J = 8.0,
1H), 8.91 (J = 4.8, 1H), 10.08 (s, 1H), 12.20 (br s, 1H) ppm.
5-(Phenethylamino)benzo[c][2,6]naphthyridine-8-carboxylic Acid
Trifluoroacetate Salt (25h). Yield, 33%. LCMS (ES): >95% pure,
m/z 344 [M þ H]þ. 1H NMR (400 MHz, CD3OD): δ 3.21 (t, J =
6.8, 2H), 4.11 (t, J = 7.2, 2H), 7.21 (m, 1H), 7.31 (m, 2H), 7.37 (m,
2H), 8.20 (dd, J = 8, J = 1.2, 1H), 8.39 (br d, J = 6, 1H), 8.58 (d,
J = 1.6, 1H), 8.80 (d, J = 8.4, 1H), 9.02 (d, J = 6, 1H), 10.10 (s,
1H) ppm.
5-(3-Phenylpropylamino)benzo[c][2,6]naphthyridine-8-carboxylic
Acid Trifluoroacetate Salt (25i). Yield, 50%. LCMS (ES): >95%
pure, m/z 358 [M þ H]þ. 1HNMR (400 MHz, DMSO-d6): δ 2.09
(qi, J=7.2, 2H), 2.77(t,J=7.2, 2H), 3.72(brq, J=4.8, 2H), 7.18
(m, 1H), 7.28(m, 4H), 7.91 (d, J=7.6, 1H), 8.40(brs, 1H), 8.44(d,
J = 4.0, 1H), 8.78 (d, J = 8.4, 1H), 8.97 (d, J = 5.2, 1H), 10.09 (s,
1H) ppm.
5-(3-Methoxyphenylamino)benzo[c][2,6]naphthyridine-8-carbo-
xylic Acid Trifluoroacetate Salt (25j). Yield, 48%. LCMS (ES):
>95% pure, m/z 346 [M þ H]þ. 1HNMR (400 MHz, CD3OD): δ
3.89 (s, 3H), 7.05 (d, J = 8, 1H), 7.28 (d, J = 7.6, 1H), 7.38 (br s,
1H), 7.52 (t, J =8, 1H), 8.20 (d, J = 8.8, 1H), 8.51(s, 1H), 8.56 (d,
J = 5.6, 1H), 8.82 (d, J = 8.4, 1H), 9.05 (d, J = 5.6, 1H), 10.16 (s,
1H) ppm.
5-(3-Chloro-4-fluorophenylamino)benzo[c][2,6]naphthyridine-
8-carboxylic Acid Trifluoroacetate Salt (25k). Yield, 40%.
LCMS (ES): 95% pure, m/z 368 [M þ H]þ. 1H NMR (400
MHz, CD3OD): δ 7.34 (t, J = 9.2, 1H), 7.84-7.88 (m, 1H),
8.11 (dd, J = 8.0, J = 1.2, 1H), 8.20 (dd, J = 6.8, J = 2.8, 1H),
8.42 (d, J = 1.6, 1H), 8.48 (d, J = 5.6, 1H), 8.75 (d, J = 8.0, 1H),
8.94 (d, J = 6.0, 1H), 10.11 (s, 1H) ppm.
5-(3-Sulfamoylphenylamino)benzo[c][2,6]naphthyridine-8-car-
boxylic Acid (25t). Yield, 26%. LCMS (ES): >95% pure, m/z
395 [M þ H]þ. 1H NMR (400 MHz, DMSO-d6): δ 7.43 (s, 2H),
7.56 (d, J = 8.0, 1H), 7.64 (t, J = 7.6, 1H), 7.99 (dd, J = 8.4, J =
1.6, 1H), 8.32 (d, J = 1.6, 1H), 8.40 (d, J = 8.0, 1H), 8.68 (m,
2H), 8.88 (d, J = 8.4, 1H), 9.02 (d, J = 5.6, 1H), 9.89 (s, 1H),
10.20 (s, 1H), 13.20 (br s, 1H) ppm.
Sodium 5-(3-Chlorophenylamino)-7-methylbenzo[c][2,6]napht-
hyridine-8-carboxylate (41a). Yield, 46%. LCMS (ES): >95%
pure, m/z 364 [M þ H]þ. 1H NMR (400 MHz, DMSO-d6): δ 2.76
(s, 3H), 7.08 (dd, J = 8, J = 2, 1H), 7.41 (t, J = 8.4, 1H), 7.47 (d,
J = 8.4, 1H), 8.00 (dd, J = 8.4, J = 2, 1H), 8.45 (d, J = 8.4, 1H),
8.55 (d, J = 5.6, 1H), 8.61 (d, J = 2, 1H), 8.86 (d, J = 2, 1H),
9.66 (s, 1H), 10.08 (s, 1H) ppm.
5-(3-Chlorophenylamino)-9-methylbenzo[c][2,6]naphthyridine-
8-carboxylic Acid (41c). Yield, 16%. LCMS (ES): >95% pure,
m/z 364 [M þ H]þ. 1H NMR (400 MHz, DMSO-d6): δ 2.73 (s,
3H), 7.15 (d, J = 8.0, 1H), 7.45 (t, J = 8.0, 1H), 8.08 (d, J = 6,
1H), 8.20 (s, 1H), 8.29 (br s, 1H), 8.65 (m, 1H), 8.73 (s, 1H), 9.01
(d, J = 6.0, 1H), 9.74 (br s, 1H), 10.22 (br s, 1H) ppm.
5-(3-Chlorophenylamino)benzo[c][2,6]naphthyridine-9-carbo-
xylic Acid (41d). Yield, 21%. LCMS (ES): 91% pure, m/z 350
[M þ H]þ. 1H NMR (400 MHz, DMSO-d6): δ 7.19 (d, J = 8.4,
1H), 7.47 (t, J = 8.0, 1H), 7.83 (d, J = 8.8, 1H), 8.09 (d, J = 8.4,
1H), 8.21 (d, J = 8.8, 1H), 8.34 (br s, 1H), 8.69 (d, J = 5.6, 1H),
9.03 (br d, J = 5.6, 1H), 9.29 (s, 1H), 9.90 (br s, 1H), 10.23 (br s,
1H) ppm.
5-(3-Chlorophenylamino)benzo[c][2,6]naphthyridine-7-carboxylic
Acid (41b). Yield, 9%. LCMS (ES): 95% pure, m/z 350 [M þ H]þ.
5-(3-Chlorophenylamino)benzo[f][1,7]naphthyridine-8-carboxylic
Acid (28i). Yield, 10%. LCMS (ES): >95% pure, m/z 350 [M þ
H]þ. 1H NMR (400 MHz, DMSO-d6): δ 7.12 (dd, J = 7, J = 2,
1H), 7.43 (t, J = 7, 1H), 7.97 (dd, J = 7, J = 2, 1H), 8.05 (dd,
J = 7, J = 4, 1H), 8.22 (dd, J = 7, J = 2, 1H), 8.32 (d, J = 1.6,
1H), 8.62 (t, J = 2, 1H), 8.72 (d, J = 7, 1H), 9.12 (dd, J = 5, J =
2, 1H), 9.28 (d, J = 7, 1H), 10.12 (s, 1H), 12.25 (br s, 1H) ppm.
5-(3-Chlorophenylamino)benzo[c][2,7]naphthyridine-8-carboxylic
Acid (28d). Yield, 40%. LCMS (ES): 89% pure, m/z 350 [M þ H]þ.
1H NMR (400 MHz, DMSO-d6):δ7.15 (dd, J=8, J=2, 1H), 7.45
(t,J=8,1H),7.96(d,J=8,1H),8.08(d,J=8,1H),8.25(d,J=2,
1H), 8.31 (t, J = 2, 1H), 8.70 (d, J = 5.6, 1H), 8.74 (d, J = 8.4, 1H),
9.01 (d, J = 6, 1H), 9.82 (s, 1H), 10.00 (s, 1H), 13.3 (br s, 1H) ppm.
5-(3-Chlorophenylamino)benzo[h][1,6]naphthyridine-8-carboxylic
Acid (28e). Yield, 18%. LCMS (ES): 95% pure, m/z 350 [M þ H]þ.
1H NMR (400 MHz, DMSO-d6): δ 7.11 (d, J = 7.2, 1H),7.42 (t,
J = 7.6, 1H), 7.84 (br m, 1H), 7.95 (d, J = 8, 1H), 8.05 (d, J = 7.6,
1H), 8.22 (s, 1H), 8.30 (s, 1H), 8.86 (d, J = 8.4, 1H), 9.06 (d, J = 8,
1H), 9.16 (s, 1H), 9.61 (s, 1H), 13.18 (br s, 1H) ppm.
5-(3-Chlorophenylamino)pyrazino[2,3-c]quinoline-8-carboxylic
Acid (28f). Yield, 83%. LCMS (ES): >95% pure, m/z 351 [M þ
H]þ. 1HNMR (400 MHz, DMSO-d6): δ 7.14 (dd, J = 8.4, J =
1.6, 1H), 7.44 (t, J = 8.4, 1H), 8.02 (dd, J = 8.4, J = 1.6, 1H),
8.26 (br d, J = 7.6, 1H), 8.32 (br s, 1H), 8.56 (m, 1H), 8.84 (d, J =
8.4, 1H), 9.17 (d, J = 2.4, 1H), 9.33 (d, J = 2.0, 1H), 10.16 (s,
1H), 13.3 (br s, 1H) ppm.
5-(3-Chlorophenylamino)pyrimido[4,5-c]quinoline-8-carboxylic
Acid (28g). Yield, 83%. LCMS (ES): >95% pure, m/z 351 [M þ
H]þ. 1H NMR (400 MHz, DMSO-d6): δ 7.14 (dd, J = 7.6, J =
2.0, 1H), 7.44 (t, J = 8.0, 1H), 8.02 (dd, J = 8.4, J = 1.6, 1H),
8.26 (d, J = 8.0, 1H), 8.32 (d, J = 1.6, 1H), 8.56 (t, J = 2.4, 1H),
8.86 (d, J = 8.4, 1H), 9.66 (s, 1H), 10.18 (s, 1H), 10.44 (s, 1H),
13.33 (br s, 1H) ppm.
N-(3-Chlorophenyl)-8-(1H-tetrazol-5-yl)benzo[c][2,6]naphthy-
ridin-5-amine Trifluoroacetate Salt (26f). A mixture of the
5-chlorobenzo[c][2,6]naphthyridine-8-carbonitrile 24b (25 mg,
0.104 mmol), 3-chloroaniline (50 mg, 0.394 mmol), and NMP
(0.2 mL) was heated to 100 °C in a microwave reactor for 20 min.
LCMS indicated the absence of starting material. Sodium azide
(100 mg, 1.53 mmol), ammonium chloride (100 mg, 1.89 mmol),
and DMF (0.2 mL) were added to the crude mixture. The
5-(3-Fluorophenylamino)benzo[c][2,6]naphthyridine-8-carboxylic
Acid Trifluoroacetate Salt (25l). Yield, 22%. LCMS (ES): >95%
pure, m/z 334 [M þ H]þ. 1H NMR (400 MHz, DMSO-d6): δ 6.90
(td, J = 8.4, J = 2.8, 1H), 7.43 (q, J = 8.4, 1H), 7.87 (d, J = 8.4,
1H), 7.96 (dd, J = 8.4, J = 2.0, 1H), 8.14 (br d, J = 12, 1H), 8.60
(d, J = 5.6, 1H), 8.86 (d, J = 8.4, 1H), 9.00 (d, J = 6,0, 1H), 9.72
(br s, 1H), 10.18 (s, 1H) ppm.
5-(2-Chlorophenylamino)benzo[c][2,6]naphthyridine-8-carboxylic
Acid (25m). Yield, 22%. LCMS (ES): 94% pure, m/z350 [M þ H]þ.
Sodium 5-(3-Chlorophenylamino)benzo[c][2,6]naphthyridine-
8-carboxylate (25n). Yield, 86%. LCMS (ES): >95% pure,
m/z 350 [M þ H]þ. 1H NMR (400 MHz, DMSO-d6): δ 7.10 (dd,
J = 7.6, J = 2.0, 1H), 7.43 (t, J = 8.0, 1H), 8.04 (dd, J = 8.4,
J = 2.0, 1H), 8.13 (dd, J = 8.0, J = 1.6, 1H), 8.27 (d, J = 1.6,
1H), 8.46 (t, J = 2.0, 1H), 8.65 (d, J = 7.6, 1H), 8.67 (d, J = 8.4,
1H), 8.90 (d, J = 6.0, 1H), 9.75 (s, 1H), 10.2 (s, 1H) ppm. 13
C
NMR (100 MHz, DMSO-d6) δ 116.0, 116.43, 118.64, 119.19,
119.61, 120.70, 121.53, 125.67, 127.0, 127.56, 129.96, 132.74,
142.12, 142.44, 143.18, 146.38, 147.37, 149.3, 169.25 ppm.
5-(4-Chlorophenylamino)benzo[c][2,6]naphthyridine-8-carboxylic
Acid Trifluoroacetate Salt (25o). Yield, 62%. LCMS (ES): 95%
pure, m/z 350 [M þ H]þ. 1H NMR (400 MHz, DMSO-d6): δ 7.48
(d, J =8.0, 2H), 7.96 (d, J =7.0, 1H), 8.17(d, J =6.4, 2H), 8.27(s,
1H), 8.61 (d, J = 5.9, 1H), 8.86 (d, J = 8.8, 1H), 9.00 (d, J = 5.9,
1H), 9.69 (s, 1H), 10.19 (s, 1H), 13.20 (br s, 1H) ppm.
5-(3-Ethynylphenylamino)benzo[c][2,6]naphthyridine-8-carboxylic
Acid (25p). Yield, 61%. LCMS (ES): 95% pure, m/z 340 [M þ H]þ.
1H NMR (400 MHz, DMSO-d6): δ 4.21 (s, 1H), 7.21 (d, J = 7.6,
1H), 7.98 (dd, J = 8.4, J = 2.0, 1H), 8.22 (br d, J = 8, 1H), 8.26 (br
s, 1H), 8.28 (br s, 1H), 8.63 (d, J=6, 1H), 8.86(d, J= 8.8, 1H), 9.00
(d, J = 6, 1H), 9.66 (s, 1H), 10.19 (s, 1H) ppm.
5-(3-Cyanophenylamino)benzo[c][2,6]naphthyridine-8-carboxylic
Acid Trifluoroacetate Salt (25q). Yield, 30%. LCMS (ES): 95%
pure, m/z 341 [M þ H]þ.
5-(4-Phenoxyphenylamino)benzo[c][2,6]naphthyridine-8-carbo-
xylic Acid (25r). Yield, 54%. LCMS (ES): 95% pure, m/z 408
[M þ H]þ.
5-(3-Phenoxyphenylamino)benzo[c][2,6]naphthyridine-8-carbo-
xylic Acid (25s). Yield, 70%. LCMS (ES): 90% pure, m/z 408
[M þ H]þ.

652 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2
Pierre et al.
mixture was heated for 18 h at 125 °C, at which time LCMS
analysis indicated the absence of any starting material. The mix-
ture was diluted with water and purified by preparative HPLC to
give the title compound 26f (12 mg, 24%). LCMS (ES): >95%
20ꢀ PARP buffer diluted with high-purity water) and 15 μL of
diluted PARP-HSA enzyme (diluted in 1ꢀ PARP buffer, 0.1
unit per well) to 25 μL of PARP1 cocktail (prepared from 10ꢀ
PARP cocktail and 10ꢀ activated DNA, both 2.5 μL per well
and 20 μL per well of 1ꢀ PARP buffer). The reactions were in-
cubated at ambient temperature for 60 min, and then the liquid
was removed. After washing the wells four times with PBS
(200 μL), 50 μL of STREP-HRP (horseradish peroxidase)
solution (diluted 500-fold in 1ꢀ Strep-Diluent) was added,
and the reactions were allowed to incubate for 30 min at ambient
temperature. The liquid was removed and, after washing the
wells four times with PBS (200 μL), 50 μL each of PeroxyGlo A
and B (chemiluminescent horseradish peroxidase substrates)
were added and the resulting chemiluminescence quantified on
the SpectraMax M5 plate reader.
Kinase Selectivity Panel. The 238 kinase selectivity panel was
conducted using the Kinase Profiler service offered by Millipore
(Billerica, MA), which utilizes a radiometric filter-binding as-
say. The % inhibition of each kinase was estimated using 0.5 μM
of 25n at ATP concentrations equivalent to the Km value for
ATP for each respective human recombinant kinase. The deter-
mination of IC50 values was conducted at similar concentrations
of ATP (equivalent to the Km for ATP) using nine concentra-
tions of 25n over a range of 0.0001-1 μM.
1
pure, m/z 374 [M þ H]þ, 346 [M þ H - N2]þ. H NMR (400
MHz, DMSO-d6): δ 7.18 (dd, J=7.5, J=1.6, 1H), 7.47 (t, J=8,
1H), 8.08 (d, J = 7.5, 1H), 8.15 (dd, J = 8.4, J = 1.6, 1H), 8.31
(br s, 1H), 8.43 (d, J = 1.6, 1H), 8.68 (d, J = 6, 1H), 9.00 (d, J =
8.8, 1H), 9.03 (d, J = 6, 1H), 9.83 (br s, 1H), 10.24 (s, 1H) ppm.
5-(3-Chlorophenylamino)benzo[c][2,6]naphthyridine-8-carbox-
amide (26e). Sodium 5-(3-chlorophenylamino)benzo[c][2,6]naph-
thyridine-8-carboxylate 25n (1.0 equiv, 58 mg, 0.16 mmol) was
reacted with HOBt (2.0 equiv, 42 mg, 0.31 mmol), ammonium
chloride (7.7 equiv, 65 mg, 1.21 mmol), DIEA (4.0 equiv, 109 μL,
0.63 mmol), and EDCI (2.0 equiv, 60 mg, 0.31 mmol) in NMP
(0.5 mL) at 70 °C for 30 min. Water was added, and the resulting
precipitate was filtered and washed with water. The crude material
was suspended in a mixture of EtOAc and hexanes, filtered, and
dried. The title compound 26e was isolated as a tan solid (27 mg,
1
50%). LCMS (ES): >95% pure, m/z 349 [M þ H]þ. H NMR
(400 MHz, DMSO-d6): δ 7.15 (br d, J = 9.2, 1H), 7.45 (t, J = 8.4,
1H), 7.54 (br s, 1H), 7.97 (dd, J = 1.6, J = 8.4, 1H), 8.11 (dd, J =
1.2, J = 8, 1H), 8.279 (br s, 1H), 8.284 (s, 1H), 8.31 (t, J = 2, 1H),
8.59 (d, J = 6, 1H), 8.85 (d, J = 8.4, 1H), 8.99 (d, J = 6, 1H), 9.67
(s, 1H), 10.20 (s, 1H) ppm.
Cell Viability Assay. Various cell lines were seeded at a density
of 3000 cells per well 24 h prior to treatment, in appropriate
media, and then treated with indicated concentrations of CK2
inhibitors. Suspension cells were seeded and treated on the
same day. Following 4 days of incubation, 20 μL of Alamar
Blue (10% of volume/well) was added and the cells were further
incubated at 37 °C for 4-5 h. Fluorescence with excitation
wavelength at 530-560 nm and emission wavelength at 590 nm
was measured.
N-(3-Chlorophenyl)-8-(4H-1,2,4-triazol-3-yl)benzo[c][2,6]-
naphthyridin-5-amine (26g). 5-(3-Chlorophenylamino)benzo-
[c][2,6]naphthyridine-8-carboxamide 26e (1.0 equiv, 36 mg) was
stirred in N,N-dimethylformamide dimethyl acetal (2 mL) at
80 °C for 4 h. The volatiles were removed in vacuo. Acetic acid
(0.5 mL) and hydrazine hydrate (0.1 mL) were added. The
mixture was stirred at 80 °C for 1 h. Water was added and the
solid filtered and dried. After trituration in a mixture of CH2Cl2
and hexanes, the title compound 26g was isolated as a solid
(22 mg, 67%). LCMS (ES): 95% pure, m/z373 [M þ H]þ.1HNMR
(400 MHz, DMSO-d6): δ 7.15 (br d, J = 7.4, 1H), 7.46 (t, J = 8.4,
1H), 8.10(d, J= 7.5, 1H), 8.15 (d, J=7.5, 1H), 8.36(s, 1H), 8.39(s,
1H), 8.59 (d, J = 5.6, 1H), 8.89 (d, J = 5.6, 1H), 8.97 (d, J = 5.6,
1H), 9.68 (s, 1H), 10.18 (s, 1H) ppm.
CK2 Kinase Assay. Test compounds in aqueous solution were
added at a volume of 10 μL to a reaction mixture comprising
10 μL of assay dilution buffer (ADB; 20 mM MOPS, pH 7.2,
25 mM β-glycerolphosphate, 5 mM EGTA, 1 mM sodium ortho-
vanadate, and 1 mM dithiothreitol), 10 μL of substrate peptide
(RRRDDDSDDD, dissolved in ADB at a concentration of 1 mM),
10 μL of recombinant human CK2 (RRββ-holoenzyme, 25 ng dis-
solved in ADB; Millipore). Reactions were initiated by the addition
of 10 μL of ATP solution (90% 75 mM MgCl2, 75 μM ATP (final
ATP concentration = 15 μM) dissolved in ADB; 10% [γ-33P]ATP
(stock 1 mCi/100 μL; 3000 Ci/mmol (Perkin-Elmer) and maintained
for 10 min at 30 °C. The reactions were quenched with 100 μL of
0.75% phosphoric acid and then transferred to and filtered through
a phosphocellulose filter plate (Millipore). After washing each well
five times with 0.75% phosphoric acid, the plate was dried under
vacuum for 5 min and, following the addition of 15 μL of scintilla-
tion fluid to each well, the residual radioactivity was measured using
a Wallac luminescence counter. The IC50 values were derived from
eight concentrations of test inhibitors.
Endogenous CK2 Assay. The human leukemia Jurkat T-cell
line were cultured in RPMI 1640 (Cambrex) supplemented with
10% fetal calf serum and 50 ng/mL gentamycin. Before treatment,
cells were washed, resuspended at a density of ∼106 cells/mL in
medium containing 1% fetal calf serum, and incubated in the
presence of 25n at 0.01, 0.1, and 1.0 μM for 2 h. Cells were
recovered by centrifugation, lysed using a hypotonic buffer
(20 mMTris/HCl pH 7.4; 2 mM EDTA; 5 mM EGTA; 10 mM
mercaptoethanol; 10 mM NaF; 1 μM okadaic acid; 10% v/v
glycerol; 0.05% NP-40; 1% protease inhibitor cocktail) and
protein from the cleared lysate was diluted to 1 μg per μL in
assay dilution buffer (ADB; 20 mM MOPS, pH 7.2, 25 mM
β-glycerolphosphate, 5 mM EGTA, 1 mM sodium orthovana-
date, and 1 mM dithiothreitol). To 20 μL of diluted protein was
added 10 μL of substrate peptide (RRRDDDSDDD, dissolved
in ADB at a concentration of 1 mM) and 10 μL of PKA inhibitor
cocktail (Upstate/Millipore). Reactions were initiated by the
addition of 10 μL of ATP solution (90% 75 mM MgCl2, 500 μL
ATP dissolved in ADB; 10% [γ-32P]ATP: stock 1 mCi/100 μL;
3000 Ci/mmol (Perkin-Elmer) and maintained for 15 min at
32 °C. The reactions were quenched with 100 μL of 0.75% phos-
phoric acid and then transferred to and filtered through a
phosphocellulose filter plate (Millipore). After washing each
well 5 times with 0.75% phosphoric acid, the residual radio-
activity was measured using a Wallac luminescence counter.
In Vivo Pharmacokinetics. The pharmacokinetic profile of
25n was investigated in overnight fasted ICR mice, SD rats, and
beagle dogs following a single dose given intravenously (iv) and
orally (po). The compound was a sodium salt formulated in
phosphate buffer for the mice and rats and as a sodium salt dry
powder filled capsule for the dogs. Eight mice (four for each leg),
six rats (three for each leg), and six dogs (three for each leg) were
treated with 25n. Blood samples of each mouse were collected at
0.08, 0.25, 0.50, 1, 2, 4, 6, 8, and 24 h following iv dosing and at
0.25, 0.50, 1, 2, 4, 6, 8, and 24 h following oral dosing. Blood
samples of each rat were collected at 0.08, 0.25, 0.50, 1, 2, 4, 6, 8,
12, 24, 36, and 48 h following iv dosing and at 0.25, 0.50, 1, 2, 4,
25n Ki Measurement. The Ki value (inhibition constant) for
25n against recombinant human CK2 (RRββ-holoenzyme) was
determined by plotting the IC50 values of 25n determined in the
presence of various concentrations of ATP against the concen-
tration of ATP (performed at Millipore). The Ki value is equi-
valent to the Y-intercept according to the Cheng-Prussoff
equation62 (Ki = IC50/(1 þ [ATP]/Km), where Ki is the inhibition
constant and Km is the Michaelis constant.
PARP1 Assay. PARP assays were conducted using a chemi-
luminescent PARP1 assay kit (Trevigen). Briefly, reactions were
performed in histone-coated strip wells, by adding 10 μL of test
compound dissolved in 1ꢀ PARP buffer (prepared by mixing

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2 653
6, 8, 12, 24, 36, and 48 h following oral dosing. Blood samples of
each dog were collected at 0.08, 0.25, 0.50, 1, 2, 4, 6, 8, 12, and
24 h following iv dosing and at 0.25, 0.50, 1, 2, 4, 6, 8, 12, and 24 h
following oral dosing. Samples were centrifuged at 8000 rpm for
5 min and the plasma collected and stored at -80 °C until
analysis. Samples were analyzed by LC-MS/MS technique. The
pharmacokinetic parameters were derived by noncompartmen-
tal analysis.
In Vivo Antitumor Efficacy in PC3 Xenograft. Tumors were
initiated by subcutaneous injection of PC3 tumor cell suspen-
sions into the right hind flank region of each mouse. When
tumors reached a designated volume of 150-200 mm3 mice were
randomized and divided into groups of 10 mice per group. The
sodium salt of 25n was administered as a solution in 25 mM
NaH2PO4 buffer, by oral gavage twice daily at 25 mg/kg,
50 mg/kg and 75 mg/kg. Tumor volumes and body weights were
measured twice weekly. The length and width of the tumor were
measured with calipers and the volume calculated using the
following formula: tumor volume = (length ꢀ width2)/2. Per-
cent tumor growth inhibition (TGI) values were calculated on
the final day of the study for 25n-treated compared to vehicle-
treated mice and were calculated as 100 ꢀ {1 - [(TreatedFinal
Day - TreatedDay 1)/(ControlFinal Day - ControlDay 1)]}.
The significance of the differences between the treated versus
vehicle groups were determined using one-way ANOVA
(Graphpad Prism).
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Acknowledgment. We thank Dr. John K.C. Lim for re-
viewing the manuscript and Cosmin Borsan for assistance in
recording NMR spectra.
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