Synthesis and structure-activity relationships of 1,2,3,4-tetrahydropyrido[2,3-b]pyrazines as potent and selective inhibitors of the anaplastic lymphoma kinase

Article abstract of DOI:10.1016/j.bmc.2010.04.087

Dysregulation of the anaplastic lymphoma kinase (ALK) is implicated in a variety of cancers. A series of tetrahydropyrido[2,3-b]pyrazines was constructed as ring-constrained analogs of a known aminopyridine kinase scaffold. Chemistry was developed to rapidly elaborate the SAR, structural elements impacting ALK inhibitory activity were exploited, and kinase selective analogs were identified that inhibit ALK with IC₅₀ values ～10 nM (enzyme) and ～150 nM (cell).

Full text of DOI:10.1016/j.bmc.2010.04.087

Bioorganic & Medicinal Chemistry 18 (2010) 4351–4362
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and structure–activity relationships of
1,2,3,4-tetrahydropyrido[2,3-b]pyrazines as potent and selective
inhibitors of the anaplastic lymphoma kinase
a
a
a
a
Karen L. Milkiewicz a, , Linda R. Weinberg , Mark S. Albom , Thelma S. Angeles , Mangeng Cheng ,
*
Arup K. Ghose a, Renee C. Roemmele b, Jay P. Theroff a, Ted L. Underiner a, Craig A. Zificsak a, Bruce D. Dorsey a
a Cephalon, Inc. 145 Brandywine Parkway, West Chester, PA 19380-4245, USA
b CPRD Cephalon Inc. 383 Phoenixville Pike, Malvern, PA 19355, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Dysregulation of the anaplastic lymphoma kinase (ALK) is implicated in a variety of cancers. A series of
tetrahydropyrido[2,3-b]pyrazines was constructed as ring-constrained analogs of a known aminopyri-
dine kinase scaffold. Chemistry was developed to rapidly elaborate the SAR, structural elements impact-
ing ALK inhibitory activity were exploited, and kinase selective analogs were identified that inhibit ALK
with IC50 values ꢀ10 nM (enzyme) and ꢀ150 nM (cell).
Received 19 March 2010
Revised 23 April 2010
Accepted 25 April 2010
Available online 29 April 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Anaplastic lymphoma kinase
Kinase inhibitors
1. Introduction
ALK and its putative ligand, pleiotrophin, are also overexpressed
in human glioblastomas.13 In mouse studies, depletion of ALK de-
Anaplastic lymphoma kinase (ALK) is a cell membrane-span-
ning receptor tyrosine kinase which belongs to the insulin receptor
subfamily. Normally, ALK has a restricted distribution in mamma-
lian cells; the most abundant expression of ALK occurs in the neo-
natal brain, suggesting a possible role for ALK in early brain
development.1
creased glioblastoma tumor growth and prolonged survival.14,15
In another investigative study, heritable mutations of ALK were
recognized as the main cause of familial neuroblastoma in chil-
dren, and the same mutations were implicated in non-inherited
neuroblastomas.16 Recently, a significant report implicated that
6–7% of non-small-cell lung cancers contain a novel translocation
where the echinoderm microtubule-associated protein-like 4 gene
(EML4) is fused to ALK.17 Studies have shown that an ALK inhibitor
may be a clinically effective treatment for non-small-cell lung can-
cer patients who express this translocation.18 Selective inhibition
of ALK activity has verified this as a tractable target for novel ther-
apies for several human cancers.19
ALK is implicated in the progression of several tumors. It was
initially identified as a result of its involvement in anaplastic
large-cell lymphomas, a subtype of non-Hodgkin lymphomas. Sixty
percent of anaplastic large cell lymphomas (ALCL) contain a chro-
mosomal mutation that generates a fusion protein comprised of
nucleophosmin (NPM) and the intracellular domain of ALK.2,3 This
mutant protein, NPM-ALK, possesses a constitutively active tyro-
sine kinase domain responsible for its oncogenic effect through
the activation of downstream effectors.1,3–5 The aberrant expres-
sion of constitutively active ALK is directly implicated in the path-
ogenesis of ALCL and inhibition of ALK can markedly impair the
growth of ALK+ lymphoma cells.6–11 In addition to ALCL, the consti-
tutively activated chimeric ALK has also been implicated in
approximately 60% of inflammatory myofibroblastic tumors
(IMTs), a slow-growing sarcoma that primarily affects children
and young adults.12
ALK inhibitors such as pyrazolo[3,4-c]isoquinoline A,20 5-aryl-
pyridone-carboxamide B,21 thiazole C,22 diaminopyrimidine D23
and 2-aminopyridines24–26 E (PF-2341066, Fig. 1) have been
reported for the treatment of both hematopoietic and non-hemato-
poietic malignancies. The latter compound is an orally bioavailable,
dual c-Met/ALK inhibitor with a reported ALK cellular IC50 value of
24 nM and is currently in phase 3 clinical trials targeting ALK.27
Constraining the conformational flexibility of ligands can facili-
tate binding by decreasing the entropic cost required to adopt a
preferred binding orientation. For ATP-competitive kinase inhibitor
scaffolds, this is particularly desirable, as the ATP binding pocket
typically favors a planar hinge-binding motif with distinct spacial
requirements for additional electrostatic interactions.28 Such a
strategy was employed to interrogate the structural requirements
* Corresponding author. Tel.: +1 610 738 6823; fax: +1 610 344 0065.
E-mail address: kmilkiew@cephalon.com (K.L. Milkiewicz).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.04.087

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K. L. Milkiewicz et al. / Bioorg. Med. Chem. 18 (2010) 4351–4362
H
H
N
O
O
N
N
H
N
O
O
N
N
O
OH
N
MeO
B
A
O
OCF3
N
N
N
H
S
H
Cl
C
H
N
N
N
NH2
O
N
N
O
Cl
HN
Cl
N
N
HN
F
N
N
iPrSO2
Cl
D
E
Figure 1. Reported ALK inhibitors.
of ALK inhibition after constraining the 2-aminopyridine scaffold
into a fused ring system (e.g., a tetrahydropyridopyrazine scaffold,
Fig. 2). This paper describes the synthesis of two distinct
constrained aminopyridine scaffolds, the 1,2,3,4-tetrahydropyri-
do[2,3-b]pyrazines (THPPs) and the 1,2,3,4-tetrahydro-[1,8]naph-
thyridines (THNTs) and the structure–activity relationships with
regards to the pendant aryl groups (Ar1 and Ar2) and the nature
of the linker (A-L) (Fig. 2).
R
N
I
I
O
NO2
Cl
a, b
d
N
N
H
N
1
2 R = H
c
3a-g R = CH2Ar2
Ar2
e
Ar2
R2
N
2. Chemistry
Ar1
N
O
N
N
H
The THPP/oxo-THPP scaffold was prepared as outlined in
Scheme 1. Coupling of pyridine 130 with glycine ethyl ester affor-
ded an aminopyridine which was subsequently reduced and cy-
N
N
H
4a-g R2 = I
e
5a-o R2 = Ar1
6a-c
pyridopyrazinone
Scheme 1. Synthesis of the THPP/oxo-THPP analogs and 6. Reagents and
conditions: (a) glycine ethyl ester HCl, EtOH, TEA, 80 °C; (b) SnCl2ꢁ2H2O, EtOH,
80 °C; (c) NaH, Ar2CH2Br, DMF; (d) DIBAL, CH2Cl2, ꢂ78 °C to rt; (e) aryl boronate,
K2CO3, PdCl2(PPh3)2, THF/water, 70 °C.
clized in one pot to afford oxo-THPP 2. Compound
2 was
pyridopyrazine
selectively alkylated with substituted benzyl bromides to form
3a–g which were subsequently subjected to a Suzuki–Miyaura
coupling to afford analogs 6a–c. Alternatively, the alkylated oxo-
THPP analogs 3a–g were reduced to the THPPs with DIBAL prior
to Suzuki–Miyaura coupling which in turn afforded compounds
5a–o. Similarly, THPPs with amide and sulfonamide linkers were
synthesized from oxo-THPP 2 in three steps as outlined in Scheme
2.
The synthesis of the phenoxy linked THNT scaffold is outlined in
Scheme 3. Reduction of 7-bromo-2,3-dihydro-1H-[1,8]naphthyri-
din-4-one31 10 with sodium borohydride afforded alcohol 11
which was then converted to regioisomer 12 by a debromina-
tion–bromination procedure; thus, metal-halogen exchange fol-
5
H
N
H
N
I
I
O
I
b
a
N
N
N
H
N
N
H
2
8
7
Ar2
Ar2
L
L
Ar1
N
N
c
9a: L = SO2
9b: L = COCH2
N
H
N
N
H
"hinge"
Met
Glu
1184
1182
Scheme 2. Synthesis of sulfonamide and amide linked THPPs. Reagents and notes:
(a) DIBAL, CH2Cl2, ꢂ78 °C to rt; (b) Ar2SO2Cl or Ar2COCl, pyridine; (c) Ar1B(OH)2,
K2CO3, PdCl2(PPh3)2 THF/water, 70 °C.
NH2
O
N
H
N
N
Ar1
Ar1
lowed by a MeOH quench gave non-brominated intermediate
that was brominated at C6 with NBS to afford 12. Suzuki coupling
followed by the ether forming Mitsunobu reaction afforded the de-
sired phenoxy THNT analogs 14.
Scheme 4 outlines the synthesis of the benzyl THNT scaffold 19.
Ketone 10 was converted to vinyl iodide 15 by means of the Takai
reaction,32 and was subsequently converted to 16 by Suzuki
A
L
Y
Ar2
Ar2
R
solvent
hydrophobic
region
2-Aminopyridine
Constrained Aminopyridine
Figure 2. Strategy to constrain the aminopyridine scaffold. A–L = N–CH2: THPP/
oxo-THPP; A–L = CH–CH2: benzyl THNT; A–L = CH–O: phenoxy THNT.

K. L. Milkiewicz et al. / Bioorg. Med. Chem. 18 (2010) 4351–4362
4353
Table 1
OH
O
ALK enzyme potency of oxo-THPP versus THPP analogs
a
e
b,c
Br
N
N
H
Br
R
N
N
N
H
N
O
Ar2
10
11
Ar2
OH
N
O
Ar1
N
Y
N
H
N
N
H
N
N
H
12 R = Br
13 R = Ar1
14
phenoxyTHNT
d
Compound #
Ar2
Y
ALK enzyme IC50
a
5a
6a
H2
O
256
>10,000
Scheme 3. Synthesis of the phenoxy linked THNT analogs. Reagents and notes: (a)
NaBH4, MeOH; (b) n-BuLi, then MeOH; (c) NBS, CH2Cl2/AcOH; (d) Ar1B(OH)2, K2CO3,
PdCl2(PPh3)2, THF/water, 70 °C; (e) Ar2OH, DEAD, Ph3P, CH2Cl2, rt.
5b
6b
H2
O
27
278
Cl
Cl
F
R
Ar2
O
5c
6c
H2
O
15
145
R
c
a
F
Br
N
N
N
N
Br
N
N
H
H
H
a
IC50 values in nM reported as the average of at least two separate
10
15 R = I
17 R = H
18 R = Br
19 R = Ar1
d
b
determinations.
b
16 R = Ar2
benzylTHNT
the benzyl ring of the THPP influenced the potency of the series.
Compounds with 2,5-disubstitution and 2,3,6-trisubstitution had
the highest activity compared to the unsubstituted phenyl. Substit-
uents as large as a trifluoromethyl were tolerated in the 2 position
(5e), whereas a trifluoromethyl substituent was not tolerated in
the 5 position (5f).
Scheme 4. Synthesis of the benzyl linked THNT analogs. Reagents and notes: (a)
CrCl2, CHI3, THF; (b) Ar2B(OH)2, Na2CO3, Pd(PPh3)4, toluene/EtOH/water, 90 °C; (c)
Pd/C, H2, MeOH; (d) NBS, CH2Cl2, AcOH.
Keeping Ar2 = 2-trifluoromethyl-5-chlorobenzyl,
a series of
coupling with arylboronates. Subsequent hydrogenation reduced
both the alkene and the C7-Br to afford the core benzyl THNT 17.
Bromination of 17 with NBS and a second Suzuki coupling afforded
the desired benzyl THNT 19.
THPPs varying only in the C-7 substituent was prepared. The SAR
is summarized in Table 3. Whereas 4-phenylcarboxylic ester (5i)
or acid (5j) analogs showed weak ALK inhibitory activity, a variety
of 4-phenylcarboxamide (5k–m) and 2-aminopyridin-4-yl (5n–o)
analogs demonstrated comparatively higher inhibitory activity.
Phenylcarboxamides bearing a basic amine had as much as 10-fold
lower ALK IC50 values than those with non-ionizable functionality
(5m vs 5k, for example). On the other hand, modulating the basi-
city of the C7-piperazinylpyridine series, had less of an effect on
ALK IC50 values (5e and 5n vs 5o).
Compounds with IC50 values of less than 50 nM in the isolated
enzyme assay were tested for their ability to inhibit ALK phosphor-
ylation in Karpas-299 cells. Table 3 shows ALK cellular IC50 values
for selected compounds ranged from 150 to 450 nM.
The kinase selectivity of the more potent ALK inhibitors was as-
sessed using Ambit Bioscience’s KINOMEscan™ technology and is
expressed as an S(90) value; the fraction of kinases inhibited great-
3. Results
Four areas of the constrained aminopyridine scaffold were ex-
plored, including Ar1, Ar2, the core scaffold itself (varying A and
Y), and the linker (L) (Fig. 2). An initial series of THPPs (5) and
oxo-THPPs (6) were synthesized with Ar1 as a phenyl pyr-
rolomethylpyrrolidinyl amide. This initial Ar1 was chosen because
it was found to impart activity in the aminopyridine series.24 The
SAR for the target kinase, ALK, is summarized in Table 1.
As can be seen from the results in Table 1, ALK inhibitory activ-
ity was at least 10-fold weaker for oxo-THPP analogs (6, Y = O)
compared with THPP analogs (5, Y = H2). Also, ALK inhibition was
greater for analogs bearing halogenated rather than unsubstituted
benzyl groups (e.g., 5b–c, vs 5a and 6b–c vs 6a).
er than 90% when screened at 1 lM across a panel of 250–400 ki-
Another Ar1 group that proved active in the aminopyridine ser-
ies24 was methylpiperazinylpyridine, and a series of THPPs and
THNTs were prepared with this group held constant while varying
the nature of linker (L). The SAR summarized in Table 2 shows that
the nature of the linker (A-L) proved a critical determinant of
potency. Sulfonamide 9a (A-L = N–SO2, ALK IC50 = 465 nM) was
14-fold less active than benzyl amine 5d (ALK IC50 = 33 nM). Amide
analog 9b (A-L = N–COCH2) was devoid of ALK activity. In the THNT
series, both the phenoxy analog 14 (A-L = CH–O) and the benzyl
analog 19 (A-L = CH–CH2) had substantially lower potency than
the corresponding analogs in the THPP series (5h and 5d,
respectively).
nases.33 This panel includes representative kinases from all the
known kinase families. The THPP analogs demonstrated a high de-
gree of selectivity; even the least selective of them only had an
S(90) value of 0.051, indicating that at 1 lM, only 5% of the evalu-
ated kinases were inhibited greater than 90%. Percent inhibition
values against individual kinases are included in the Supplemen-
tary data.
The common kinases inhibited include ALK, ARK5, CSF-1R,
DCAMKL1-3, EPHB4, FAK, FLT3, JAK2, JAK3, KIT, LTK, MET, ROS1,
SNARK, and TNK2. The majority of these kinases are tyrosine ki-
nases. ALK shares a sequence homology with LTK and ROS, and
the activity against MET can be anticipated, as the aminopyridine
PF-2341066 is a known dual ALK/Met inhibitor. Because ALK be-
longs to the insulin receptor superfamily, inhibiting the insulin
receptor was a potential liability which could lead to dysregulation
As noted earlier, analogs with halogenated aryl rings had lower
ALK IC50 values than unsubstituted phenyl rings (e.g., 5b–c and 6b–
c vs 5a and 6a, Table 1). The nature of the halogenation pattern on

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K. L. Milkiewicz et al. / Bioorg. Med. Chem. 18 (2010) 4351–4362
Table 2
Influence of the aryl linker and substitution pattern on ALK enzyme potency
N
N
Ar2
L
N
A
N
N
R
Ar2
ALK enzyme IC50
33
S(90)b
0.012
a
Compound #
A
L
R
F
5d
N
CH2
H
F
Cl
5e
5f
N
N
N
N
CH2
CH2
CH2
CH2
H
H
H
H
10
0.022
NTc
CF3
CF3
Cl
506
11
Cl
5g
5h
0.020
0.025
Cl
F
11
F
Cl
F
9a
9b
14
19
20
N
SO2
COCH2
O
H
H
H
H
465
NT
NT
NT
NT
NT
F
F
F
N
>10,000
>10,000
1538
F
CH
CH
N
F
Cl
F
CH2
CH2
F
Cl
CH3
>3000
Cl
a
IC50 values in nM reported as the average of at least two separate determinations.
b
Kinase selectivity was determined using the Ambit Bioscience KINOMEscan™ technology, and is expressed as S(90), the fraction of kinases inhibited >90% when screened
M across a panel of 250–400 kinases.
NT = not tested.
at 1
l
c
of normal metabolic pathways. To confirm the lack of IR activity
seen in the Ambit scan, compounds with an ALK IC50 <100 nM were
evaluated and were found to be inactive (>3000 nM) against IR.
hydrophobic region on the bottom of the ATP pocket and the sub-
stituent Ar1 pointed out towards solvent exposed space.
The Glide XP score and Embrace
DE for ligand–protein complex
formation were computed for all ligands. It was possible to inter-
pret the SAR in the context of these docking poses. As depicted
in Figures 2 and 3, N4-H and N5 (in the THPP) make putative H-
bonds with residues in ALK (Glu-1182 and Met-1184). Consistent
with this model is the drop in inhibitory activity going from
N4-H analog 5g (ALK IC50 = 11) to N4-Me analog 20 (ALK IC50
>3000 nM Table 2).
Comparing compounds 5a–c versus 6a–c a major decrease in
activity is observed when a hydrophilic C = O is placed in a fairly
hydrophobic region (Fig. 4 arrow). The salt bridge Lys-35 is too
distant (ꢀ7 Å) to have any positive compensatory influence. The
3.1. Computational chemistry and discussion of results
Docked poses of 2-aminopyridine E and THPP analog 5d into
ALK homology models29 indicated that these related scaffolds
shared a high level of conformational overlap (Fig. 3). In general,
the predicted binding mode anchored the ligands into the ATP
binding pocket using the pyridine nitrogen to accept a hydrogen
bond from the gatekeeper+3 (gk+3) residue (Met-1184) and the
adjacent amino group to donate a hydrogen bond to the gk+1 res-
idue (Glu-1182). The pendant aryl group (Ar2) was directed into a

K. L. Milkiewicz et al. / Bioorg. Med. Chem. 18 (2010) 4351–4362
4355
Table 3
Influence of the 7-aryl substituent on ALK binding affinity
Cl
R
N
CF3
N
N
H
a
a
Compound #
R
ALK enzyme IC50
9
Karpas-299 cell IC50
S(90)
N
N
5e
200
0.022
NT
N
O
5i
5j
>3000
1302
NT
NT
O
O
0.023
HO
O
5k
5l
121
40
NT
0.008
0.019
N
O
N
450
O
O
N
5m
10
250
0.051
N
N
5n
5o
10
17
150
250
0.067
0.027
N
HN
N
N
N
O
IC50 values in nM reported as the average of at least two separate determinations.
a
halogens on the benzyl group enhanced binding by their ability to
interact with the salt bridge Lys34 as well as by the hydrophobic
interactions with residues like Leu-141 at the bottom of the ATP
binding site (Fig. 4).
4. Conclusion
Constraining the known aminopyridine kinase scaffold into a
THPP ring system proved a viable strategy to afford potent ALK
inhibitors. The nature of the linker (A-L, Fig. 2) was found to be a
critical determinant for activity, with the THPP scaffold providing
far greater inhibition than the oxo-THPP and THNT scaffolds. These
compounds also inhibited ALK phosphorylation in Karpas-299 cells
with IC50 values ranging from 100 to 450 nM. Additionally, the
constrained aminopyridine scaffold provided a means to afford po-
tent ALK inhibitors while maintaining selectivity over IR in partic-
ular and over other kinases in general. Computational modeling
was consistent with the empirical SAR and could be used to guide
further medicinal chemistry efforts.
Figure 3. The basic binding mode of the constrained 2-aminopyridine (5d), as
derived by Glide, along with the aligned ligand from the PDB file 2WGJ (PF-
2341066) (light pink). This binding mode was consistent with the empirical rules
for ligand binding mode prediction derived from the protein data bank kinase
structures. Here the amino-NH was bound to the gk+1 residue and the pyridine-N
was bound to the gk+3 residue. The gate keeper in the current model was Leu-81.

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K. L. Milkiewicz et al. / Bioorg. Med. Chem. 18 (2010) 4351–4362
5.3. ALK kinase assay
Compounds were tested for their ability to inhibit the kinase
activity of baculovirus-expressed ALK using a modification of the
ELISA protocol reported for trkA.36 Phosphorylation of the sub-
strate, phospholipase C-gamma (PLC-c) generated as a fusion pro-
tein with glutathione S-transferase (GST),37 was detected with a
europium-labeled anti-phosphotyrosine antibody and measured
by time-resolved fluorescence (TRF). Briefly, each 96-well plate
was coated with 100
C- ) in Tris-buffered saline (TBS). The assay mixture (total vol-
ume = 100 l/well) consisting of 20 mM HEPES (pH 7.2), 1 lM
ll/well of 10 lg/ml substrate (phospholipase
c
l
ATP (Km level), 5 mM MnCl2, 0.1% BSA, 2.5% DMSO, and various
concentrations of test compound was then added to the assay
plate. The reaction was initiated by adding enzyme (30 ng/ml
ALK) and was allowed to proceed at 37 °C for 15 min. Detection
of the phosphorylated product was performed by adding 100 ll/
well of Eu-N1 labeled PT66 antibody (Perkin Elmer # AD0041).
Incubation at 37 °C then proceeded for 1 h, followed by addition
of 100 ll enhancement solution (Wallac #1244-105). The plate
was gently agitated and after 30 min, the fluorescence of the
resulting solution was measured using the EnVision 2100 (or
2102) multilabel plate reader (Perkin Elmer).
Figure 4. Comparison of the binding mode of the THPP 5c (atom color) versus the
oxo-THPP 6c (pink). Gray surface represents nonpolar surface, red represents
negatively charged surface and blue represents positively charged surface. The oxo-
THPP C@O can be seen pointing into a hydrophobic region.
5. Experimental
Data analysis was performed using ActivityBase (IDBS, Guilford,
UK). IC50 values were calculated by plotting percent inhibition ver-
sus log10 of the concentration of compound and fitting to the non-
linear regression sigmoidal dose–response (variable slope)
equation in XLFit (IDBS, Guilford, UK).
All reagents and solvents were obtained from commercial
sources and used as received. 1H NMR spectra were obtained on
a Bruker Avance at 400 MHz in the solvent indicated with tetra-
methylsilane as an internal standard. Analytical HPLC was run
using a Zorbax RX-C8, 5 ꢃ 150 mm column eluting with a mixture
of acetonitrile and water containing 0.1% trifluoroacetic acid with a
gradient of 10–100%. LC–MS results were obtained on a Bruker Es-
quire 200 ion trap. Automated column chromatography was per-
formed on a CombiFlash Companion (ISCO, Inc.). Melting points
were taken on a Mel-Temp apparatus and are uncorrected.
5.4. Cellular ALK phosphorylation measured by
immunoblotting and ELISA assays
Immunoblotting of phospho-NPM-ALK and total NPM-ALK from
cell lysates was carried out according to the protocols provided by
the antibody suppliers. In brief, after treatment of compounds, cells
were lysed in Frak lysis buffer [10 mM Tris, pH 7.5, 1% Triton
X-100, 50 mM sodium chloride, 20 mM sodium fluoride, 2 mM
sodium pyrophosphate, 0.1% BSA, plus freshly prepared 1 mM acti-
vated sodium vanadate, 1 mM DTT, and 1 mM PMSF, protease
inhibitors cocktail III (1:100 dilution)]. After brief sonication, the
lysates were cleared by centrifugation, mixed with sample buffer
and subjected to SDS–PAGE. Following transfer to membranes,
the membranes were blotted with either rabbit phospho-NPM-
ALK(Y664) (Cat# 3341) or ALK antibody (Cat# 3342) from Cell
Signaling Technology (Beverly, MA), and then the HRP-conjugated
goat anti-rabbit antibodies (Santa Cruz, CA) after washed in TBS/
0.2% Tween-20. The protein bands were visualized with Enhanced
Chemiluminescence and quantitated with gel-pro analyzer.
To measure ALK tyrosine phosphorylation in cells with an ELISA
assay, fluoronunc plates (Cat# 437796, Nalge Nunc, Rochester, NY)
were pre-coated with goat anti-mouse IgG, and incubated with the
capture mouse ALK antibody (Cat# 35-4300, Zymed, Seattle, WA)
diluted 1:1000 in Superblock (Pierce, Rockford, IL). Following
blocking, cell lysates were added to plates and incubated overnight
at 4 °C. Plates were incubated with the detecting antibody, anti-
phospho-ALK (Y664) (Cell Signaling Technology), diluted at
1:2000, followed by incubation with goat anti-rabbit-IgG alkaline
phosphatase to amplify the detection. Wells were exposed to the
fluorogenic substrate 4 –MeUP and the signal quantified using a
CytoFluorÒ (series 4000) Fluorescence Multi-Well Plate Reader
(Applied Biosystems, Foster City, CA).
5.1. Homology modeling
A homology model of ALK was built from the kinase domain
(1116– 1392) of ALK_Human sequence (Swiss-Prot entry
Q9UM73). The Prime module of Schrodinger (www.schroding-
er.com) software was used for this purpose. A single DFG-in
homology model was built from insulin like growth factor receptor
(IGF1R) structures. Prime blast search identified IGF1R having the
highest sequence identity and homology with ALK. The sequence
alignment was provided in the Supplementary data. These obser-
vations were consistent with the finding of Gunby et. al.35
5.2. Docking study
The essential steps in the current docking experiment are sum-
marized below:
(i) Build 3D structures using LigPrep;
(ii) Calculate electrostatic potential fitted charge using 6-31G/
B3LYP in Jaguar;
(iii) 1000 steps mixed-torsional/low-mode conformational
search in MacroModel using the supplied atomic charges
and implicit water solvent model;
(iv) Glide/XP docking to keep top 10 binding poses;
(v) Selection of the binding mode using our knowledge based
approach;28
(vi) Embrace minimization of the ligand-protein complex.
5.5. 7-Iodo-3,4-dihydro-1H-pyrido[2,3-b]pyrazin-2-one (2)
Most of these modules are available in the Schrodinger molec-
ular modeling package.
2-Chloro-5-iodo-3-nitro-pyridine was dissolved in EtOH. Gly-
cine ethyl ester HCl was added (5.0 equiv) followed by TEA

K. L. Milkiewicz et al. / Bioorg. Med. Chem. 18 (2010) 4351–4362
4357
(5.0 equiv). The reaction mixture was heated to 80 °C for 4 h. The
5.6.5. 1-(5-Chloro-2-trifluoromethyl-benzyl)-7-iodo-3,4-
reaction mixture was concentrated to dryness and triturated
with H2O to obtain (5-iodo-3-nitro-pyridin-2-ylamino)-acetic
acid ethyl ester as a white solid in 82–94% yield. Mp 200 °C
(dec), LC–MS: m/z = 352 (M+H+), 1H NMR (CDCl3, 400 MHz) d
1.30 (t, J = 7.1 Hz, 3H), 4.25 (q, J = 7.1 Hz, 2H), 4.33 (d,
J = 5.6 Hz, 2 H), 8.44 (br s, 1H), 8.52 (d, J = 2.0 Hz, 1H), 8.69 (d,
J = 2.0 Hz, 1H). (5-Iodo-3-nitro-pyridin-2-ylamino)-acetic acid
ethyl ester was dissolved in EtOH. SnCl2ꢁ2H2O was added and
the reaction mixture was heated to 80 °C for 2 h. The resulting
dihydro-1H-pyrido[2,3-b]pyrazin-2-one (3e)
7-Iodo-3,4-dihydro-1H-pyrido[2,3-b]pyrazin-2-one 2 was re-
acted with 2-bromomethyl-4-chloro-1-trifluoromethyl-benzene
following the conditions in general procedure I to obtain 3e as a
crude insoluble solid which was taken forward to 1-(5-chloro-2-
trifluoromethyl-benzyl)-7-iodo-3,4-dihydro-1H-pyrido[2,3-b]pyr-
azine 4e without further purification.
5.6.6. 1-(2,5-Dichloro-benzyl)-7-iodo-3,4-dihydro-1H-
pyrido[2,3-b]pyrazin-2-one (3f)
precipitate was filtered and washed with EtOH to afford
2
as a rust colored solid in 59–77% yield. Mp 81–82 °C, LC–MS:
m/z = 276 (M+H+), 1H NMR (DMSO-d6, 400 MHz) d 3.94 (s, 2H),
6.94 (br s, 1H), 7.12 (d, J = 1.8 Hz, 1H), 7.74 (s, J = 1.8 Hz, 1H),
10.40 (br s, 1H).
7-Iodo-3,4-dihydro-1H-pyrido[2,3-b]pyrazin-2-one was reacted
with 2,5-dichlorobenzyl bromide following the conditions in gen-
eral procedure I to obtain 3f as a brown solid in 69% yield. Mp
>200 °C, LC–MS: m/z = 434 (M+H+); calcd for C14H10Cl2IN3O: 434
(M+H+), 1H NMR (DMSO-d6, 400 MHz) d 4.18 (s, 2H), 5.07 (s, 2H),
7.14 (d, J = 1.8 Hz, 1H), 7.15 (d, J = 2.3 Hz, 1H), 7.40 (dd, J = 8.6,
2.5 Hz, 1H), 7.56 (d, J = 8.6 Hz, 1H), 7.85 (d, J = 1.8 Hz, 1H).
5.6. General procedure I. N-Benzylation with NaH and benzyl
bromides to form 3a–g
5.6.7. 1-(2-Chloro-3,6-difluoro-benzyl)-7-iodo-3,4-dihydro-1H-
pyrido[2,3-b]pyrazin-2-one (3g)
7-Iodo-3,4-dihydro-1H-pyrido[2,3-b]pyrazin-2-one (2) was
suspended in anhydrous DMF and cooled to 0 °C. NaH (60% disper-
sion in mineral oil) (1 equiv) was added and a solution formed. The
respective benzyl bromide (1 equiv) was added and the resulting
reaction was stirred at rt for 1–4 h. The resulting product was puri-
fied by silica gel chromatography.
7-Iodo-3,4-dihydro-1H-pyrido[2,3-b]pyrazin-2-one was reacted
with 2 chloro, 3,6-difluorobenzyl bromide following the conditions
in general procedure I to afford 3g as an off-white solid in 63%
yield. Mp 237 °C, LC–MS: m/z = 436 (M+H+); calcd for C14H9ClF2I-
N3O: 436 (M+H+),1H NMR (DMSO-d6, 400 MHz) d 4.01 (s, 2H),
5.24 (s, 2H), 7.05 (d, J = 1.5 Hz, 1H), 7.23–7.32 (m, 1H), 7.41–7.48
(m, 1H), 7.79 (d, J = 1.5 Hz, 1H).
5.6.1. 1-Benzyl-7-iodo-3,4-dihydro-1H-pyrido[2,3-b]pyrazin-2-
one (3a)
7-Iodo-3,4-dihydro-1H-pyrido[2,3-b]pyrazin-2-one 2 was trea-
ted with benzyl bromide following the conditions in general proce-
dure I to obtain 3a as a light yellow solid in 38% yield. Mp 225–
226 °C, LC–MS: m/z = 366 (M+H+); calcd for C14H12IN3O: 366
(M+H+) 1H NMR (DMSO-d6, 400 MHz) d 4.14 (s, 2H), 5.10 (s, 2H),
7.13 (s, 1H), 7.22–7.29 (m, 4H), 7.31–7.38 (m, 2H), 7.78 (s, 1H).
5.7. General procedure II. Amide reduction with DIBAL to form
4a–g
Compound 3 was suspended in anhydrous CH2Cl2 and cooled to
0 °C. DIBAL (8.0 equiv) was added and the resulting solution was
allowed to warm to rt over 16 h. The reaction mixture was
quenched with methanol and a saturated solution of potassium so-
dium tartrate was added. The mixture was stirred at rt until the
layers separated (2 h), the layers were separated, and the organic
layer was dried over MgSO4, filtered, and concentrated. The prod-
uct was purified via silica gel chromatography.
5.6.2. 1-(2,6-Dichloro-benzyl)-7-iodo-3,4-dihydro-1H-
pyrido[2,3-b]pyrazin-2-one (3b)
7-Iodo-3,4-dihydro-1H-pyrido[2,3-b]pyrazin-2-one 2 was trea-
ted with 2,6-dichlorobenzyl bromide under the conditions of gen-
eral procedure I to afford 3b as an off-white solid. Mp >200 °C, LC–
MS: m/z = 434 (M+H+); calcd for C14H10Cl2IN3O: 434 (M+H+), 1H
NMR (DMSO-d6, 400 MHz) d 3.30 (s, 2H), 5.29 (s, 2H), 7.31 (d,
J = 8.6 Hz, 1H), 7.34 (d, J = 7.6 Hz, 1H), 7.45 (s, 1H), 7.47 (s, 1H),
7.76 (d, J = 1.0 Hz, 1H).
5.7.1. 1-Benzyl-7-iodo-1,2,3,4-tetrahydro-pyrido[2,3-b]pyrazine
(4a)
1-Benzyl-7-iodo-3,4-dihydro-1H-pyrido[2,3-b]pyrazin-2-one
(3a) was reduced via general procedure II to obtain 4a as a light
brown solid in 25% yield. Mp 147–148 °C, LC–MS: m/z = 352
(M+H+); calcd for C14H14IN3: 351 (M+H+),1H NMR (CDCl3,
400 MHz) d 3.09–3.32 (m, 2H), 3.51–3.54 (m, 2H), 4.36 (s, 2H), 6.84
(s, 1H), 7.23–7.30 (m, 1H), 7.31–7.39 (m, 4H), 7.58 (d, J = 1.5 Hz, 1H).
5.6.3. 1-(2,5-Difluoro-benzyl)-7-iodo-3,4-dihydro-1H-
pyrido[2,3-b]pyrazin-2-one (3c)
7-Iodo-3,4-dihydro-1H-pyrido[2,3-b]pyrazin-2-one 2 was re-
acted with 2,5-difluorobenzyl bromide following the conditions
in general procedure I to obtain 3c as a tan solid in 70% yield.
Mp >200 °C, LC–MS: m/z = 402 (M+H+), 1H NMR (DMSO-d6,
400 MHz) d 4.13 (s, 2H), 5.09 (s, 2H), 7.01 (m, 1H), 7.15 (m, 2H),
7.28 (m, 1H), 7.80 (s, 1H).
5.7.2. 1-(2,6-Dichloro-benzyl)-7-iodo-1,2,3,4-tetrahydro-
pyrido[2,3-b]pyrazine (4b)
1-(2,6-Dichloro-benzyl)-7-iodo-3,4-dihydro-1H-pyrido[2,3-b]-
pyrazin-2-one (3b) was reduced with general procedure II to afford
4b as a light yellow solid. Mp >200 °C, LC–MS: m/z = 420 (M+H+);
calcd for C14H12Cl2IN3: 420 (M+H+), 1H NMR (CDCl3, 400 MHz) d
3.03 (t, J = 4.8 Hz, 2H), 3.42 (m, 2H), 4.48 (s, 2H), 4.81 (s, 1H), 7.09
(s, 1H), 7.24 (m, 1H), 7.36 (d, J = 8.2 Hz, 2H), 7.63 (d, J = 1.6 Hz, 1H).
5.6.4. 1-(2-Chloro-5-trifluoromethyl-benzyl)-7-iodo-3,4-
dihydro-1H-pyrido[2,3-b]pyrazin-2-one (3d)
7-Iodo-3,4-dihydro-1H-pyrido[2,3-b]pyrazin-2-one 2 was re-
acted with 2-chloro-5-(trifluoromethyl) benzyl bromide following
the conditions in general procedure I to afford 3d as a white solid
in 39% yield. Mp 252–253 °C, LC–MS: m/z = 468 (M+H+); calcd for
C15H10ClF3IN3O: 468 (M+H+), 1H NMR (DMSO-d6, 400 MHz) d
4.16 (d, J = 1.2 Hz, 2H), 5.16 (s, 2H), 7.20 (s, 1H), 7.26 (d,
J = 1.3 Hz, 1H), 7.42 (s, 1H), 7.71 (dd, J = 8.3 Hz, 1.5 Hz, 1H), 7.77
(d, J = 8.6 Hz, 1H), 7.83 (d, J = 1.5 Hz, 1H).
5.7.3. 1-(2,5-Difluoro-benzyl)-7-iodo-1,2,3,4-tetrahydro-
pyrido[2,3-b]pyrazine (4c)
1-(2,5-Difluoro-benzyl)-7-iodo-3,4-dihydro-1H-pyrido[2,3-
b]pyrazin-2-one (3c) was reduced via general procedure II to afford
4c as a white solid in 42% yield. Mp 175–176 °C, LC–MS: m/z = 388
(M+H+); calcd for C14H12F2IN3: 388 (M+H+),1H NMR (DMSO-d6,
400 MHz) d 3.26–3.30 (m, 2H), 3.36–3.42 (m, 2H), 4.44 (s, 2H),

4358
K. L. Milkiewicz et al. / Bioorg. Med. Chem. 18 (2010) 4351–4362
6.63 (br s, 1H), 6.77 (s, 1H), 7.05–7.10 (m, 1H), 7.15–7.19 (m, 1H),
7.20–7.27 (m, 1H), 7.41 (s, 1H).
(2.0 equiv), 1-hydroxybenzotriazole (1.7 equiv), and triethylamine
(1.4 equiv). After stirring at rt for 30 min, the respective amine
was added. The reaction mixture was stirred for 3–16 h. The result-
ing reaction mixture was concentrated, diluted with CH2Cl2,
washed with H2O, dried over MgSO4, and purified via silica gel
chromatography or RP preparative HPLC.
5.7.4. 1-(2-Chloro-5-trifluoromethyl-benzyl)-7-iodo-1,2,3,4-
tetrahydro-pyrido[2,3-b]pyrazine (4d)
1-(2-Chloro-5-trifluoromethyl-benzyl)-7-iodo-3,4-dihydro-1H-
pyrido[2,3-b]pyrazin-2-one (3d) was reduced with general proce-
dure VI to obtain 4d as a yellow solid in 28% yield. Mp 120–
125 °C, LC–MS: m/z = 454 (M+H+); calcd for C15H12ClF3IN3: 454
(M+H+), 1H NMR (CDCl3, 400 MHz) d 3.38 (t, J = 5.0 Hz, 2H), 3.55–
3.61 (m, 2H), 4.45 (s, 2H), 5.11 (s, 1H), 6.66 (s, 1H), 7.47 (s, 1H),
7.53 (dd, J = 8.3 Hz, 2H), 7.62 (d, J = 1.8 Hz, 1H).
5.9.1. [4-(1-Benzyl-1,2,3,4-tetrahydro-pyrido[2,3-b]pyrazin-7-
yl)-phenyl]-((S)-2-pyrrolidinylmethyl-pyrrolidin-1-yl)-
methanone—TFA salt (5a)
1-Benzyl-7-iodo-1,2,3,4-tetrahydro-pyrido[2,3-b]pyrazine (4a)
was reacted with 4-ethoxycarbonyl phenyl boronic acid following
general procedure IIIA to recover 4-(1-benzyl-1,2,3,4-tetrahydro-
pyrido[2,3-b]pyrazin-7-yl)-benzoic acid ethyl ester as a pale yel-
low solid in 42% yield. Mp 178–179 °C, LC–MS: m/z = 374
(M+H+); calcd for C23H23ClN3O2: 374 (M+H+), 1H NMR (CDCl3,
400 MHz) d 1.38 (t, J = 7.1 Hz, 3H), 3.40 (t, J = 5.1 Hz, 2H), 3.60–
3.63 (m, 2H), 4.35 (q, J = 7.1 Hz, 2H), 4.48 (s, 2H), 6.85 (s, 1H),
7.27–7.49 (m, 5H), 7.43 (d, J = 8.3 Hz, 2H), 7.73 (d, J = 2.0 Hz, 2H),
8.00 (d, J = 8.3 Hz, 2H). 4-(1-Benzyl-1,2,3,4-tetrahydro-pyrido[2,3-
b]pyrazin-7-yl)-benzoic acid ethyl ester was heated to 70 °C in a
1/1 mixture of THF and H2O with 5 equiv LiOHꢁH2O for 2–16 h.
The resulting solution was concentrated and neutralized with 1 N
HCl. The resulting precipitate was filtered to obtain 4-(1-benzyl-
5.7.5. 1-(5-Chloro-2-trifluoromethyl-benzyl)-7-iodo-1,2,3,4-
tetrahydro-pyrido[2,3-b]pyrazine (4e)
1-(2-Chloro-5-trifluoromethyl-benzyl)-7-iodo-3,4-dihydro-1H-
pyrido[2,3-b]pyrazin-2-one (3e) was reduced through general pro-
cedure II to afford 4e as an orange solid in 17% yield. Mp 189–
191 °C, LC–MS: m/z = 454 (M+H+); calcd for C15H12ClF3IN3: 454
(M+H+),1H NMR (DMSO-d6, 400 MHz) d 3.28–3.34 (m, 2H), 3.41–
3.46 (m, 2H), 4.53 (s, 2H), 6.52 (s, 1H), 6.75 (s, 1H), 7.44 (s, 2H),
7.59 (d, J = 8.4 Hz, 1H), 7.82 (d, J = 8.6 Hz, 1H).
5.7.6. 1-(2,5-Dichloro-benzyl)-7-iodo-1,2,3,4-tetrahydro-pyrido-
[2,3-b]pyrazine (4f)
1,2,3,4-tetrahydro-pyrido[2,3-b]pyrazin-7-yl)-benzoic acid as
a
1-(2,5-Dichloro-benzyl)-7-iodo-3,4-dihydro-1H-pyrido[2,3-b]-
pyrazin-2-one (3f) was reduced with general procedure II to obtain
4f as a brown solid in 85% yield. Mp 169–170 °C, LC–MS: m/z = 421
(M+H+); calcd for C14H12Cl2IN3: 421 (M+H+), 1H NMR (CDCl3,
400 MHz) d 3.40 (m, 3H), 3.59 (m, 2H), 4.39 (s, 2H), 4.85 (br s,
1H), 6.64 (s, 1H), 7.18 (d, J = 2.8 Hz, 1H), 7.22 (dd, J = 8.6, 2.8 Hz,
1H), 7.35 (d, J = 8.6 Hz, 1H), 7.63 (d, J = 1.5 Hz, 1H).
pale yellow solid in 90% yield. Mp >300 °C, LC–MS: m/z = 346
(M+H+); calcd for C21H19N3O2: 346 (M+H+), 1H NMR (DMSO-d6,
400 MHz) d 3.35–3.38 (m, 2H), 3.52–3.54 (m, 2H), 4.62 (s, 2H),
7.08 (s, 1H), 7.25–7.27 (m, 1H), 7.29–7.32 (m, 4H), 7.62 (d,
J = 8.4 Hz, 2H), 7.69 (d, J = 1.8 Hz, 1H), 7.92 (d, J = 8.4 Hz, 2H), 12.86
(br s, 1H). 4-(1-Benzyl-1,2,3,4-tetrahydro-pyrido[2,3-b]pyrazin-7-
yl)-benzoic acid was coupled with (S)-(+)-1-(2-pyrrolidinylmeth-
yl)pyrrolidine via general procedure IV. The reaction mixture was
purified via RP preparative HPLC to obtain 5a as a yellow foam in
43% yield. LC–MS: m/z = 482 (M+H+); calcd for C30H35N5O: 482
(M+H+), 1H NMR (CDCl3, 400 MHz) d 1.82–1.84 (m, 1H), 1.92–2.05
(m, 2H), 2.06–2.20 (m, 4H), 2.92–3.08 (m, 1H), 3.18–3.25 (m, 1H),
3.25–3.35 (m, 1H), 3.39–3.48 (m, 2H), 3.50–3.52 (m, 1H), 3.55–
3.63 (m, 1H), 3.65–3.71 (m, 1H), 3.72–3.77 (m, 2H), 3.82–3.94 (m,
1H), 4.01–4.11 (m, 1H), 4.48–4.59 (m, 3H), 6.92 (s, 1H), 7.26–7.42
(m, 8H), 7.56 (d, J = 8.1 Hz, 2H), 11.06 (br s, 1H).
5.7.7. 1-(2-Chloro-3,6-difluoro-benzyl)-7-iodo-1,2,3,4-tetrahy-
dro-pyrido[2,3-b]pyrazine (4g)
1-(2-Chloro-3,6-difluoro-benzyl)-7-iodo-3,4-dihydro-1H-pyr-
ido[2,3-b]pyrazin-2-one (3g) was reduced by general procedure II
to afford 4g as an off-white solid in 42% yield. Mp 156–157 °C,
LC–MS: m/z = 422 (M+H+); calcd for C14H11ClF2IN3: 422 (M+H+),
1H NMR (CDCl3, 400 MHz) d 3.12–3.20 (m, 2H), 3.42–3.50 (m,
2H), 4.41 (s, 2H), 4.89 (br s, 1H), 6.98–7.05 (m, 1H), 7.08 (s, 1H),
7.10–7.18 (m, 1H), 7.62 (d, J = 1.5 Hz, 1H).
5.9.2. {4-[1-(2,6-Dichloro-benzyl)-1,2,3,4-tetrahydro-
pyrido[2,3-b]pyrazin-7-yl]-phenyl}-(2-pyrrolidin-1-ylmethyl-
pyrrolidin-1-yl)-methanone (5b)
1-(2,6-Dichloro-benzyl)-7-iodo-1,2,3,4-tetrahydro-pyrido[2,3-
b]pyrazine (4b) was reacted with (S)-2-pyrrolidin-1-ylmethyl-pyr-
rolidine-1-carboxylic acid [4-(4,4,5,5-tetramethyl-[1,3,2]dioxa-
borolan-2-yl)-phenyl]-amide [obtained through amide coupling of
5.8. General procedure III. Suzuki coupling
General procedure IIIA: The starting iodo reactant was combined
with an aryl boronate (1.2–2.0 equiv), Pd(PPh3)4 (0.02–0.05 equiv),
and 2 M Na2CO3 (2.5 equiv) in a 6/1 mixture of toluene/EtOH. The
resulting reaction mixture was heated to 80 °C for 1–16 h. The
reaction mixture was then diluted with CH2Cl2, dried over MgSO4,
filtered, concentrated, and purified via silica gel chromatography.
General procedure IIIB: The starting iodo reactant, an aryl boro-
nate, (1.5–2.0 equiv), and PdCl2(PPh3)2 (0.1 equiv) were all dis-
solved in 6 mL THF. Potassium carbonate powder (5.0 equiv) was
dissolved in 6 mL of water in a separate flask. The potassium car-
bonate solution was added to the boronate solution, the mixture
was purged with nitrogen, and it was stirred at 70 °C for 1 h. The
reaction mixture was concentrated, and methylene chloride and
water were added. The aqueous layer was removed, and the organ-
ic layer was washed with saturated sodium bicarbonate solution,
followed by brine. It was dried with magnesium sulfate, filtered,
and then purified by silica gel column chromatography.
(S)-2-pyrrolidin-1-ylmethyl-pyrrolidine
4-(4,4,5,5-Tetramethyl-
[1,3,2]dioxaborolan-2-yl)-benzoic acid using standard amide cou-
pling conditions (EDCI, HOBT, DMF, DIEA, rt, 2 h)] following general
procedure IIIB to obtain 5b as a yellow solid in 22% yield. LC–MS:
m/z = 550 (M+H+); calcd for C30H33Cl2N5O: 550 (M+H+), 1H NMR
(CDCl3, 400 MHz) d 1.83 (m, 7H), 2.26 (m, 2H), 3.01 (m, 5H), 3.57 (m,
4H), 4.46 (br s, 1H), 4.61 (s, 2H), 5.17 (br s, 1H), 7.11 (d, J = 1.5 Hz,
1H), 7.25 (m, 1H), 7.37 (d, J = 8.1 Hz, 2H), 7.56 (m, 4H), 7.74 (s, 1H).
5.9.3. {4-[1-(2,5-Difluoro-benzyl)-1,2,3,4-tetrahydro-pyrido[2,3-
b]pyrazin-7-yl]-phenyl}-((S)-2-pyrrolidin-1-ylmethyl-
pyrrolidin-1-yl)-methanone (5c)
1-(2,5-Difluoro-benzyl)-7-iodo-1,2,3,4-tetrahydro-pyrido[2,3-
b]pyrazine (4c) (500 mg) was reacted with 4-ethoxycarbonyl
phenyl boronic acid following general procedure IIIA to obtain
4-[1-(2,5-difluoro-benzyl)-1,2,3,4-tetrahydro-pyrido[2,3-b]pyra-
zin-7-yl]-benzoic acid ethyl ester as an off-white solid in 63% yield.
5.9. General procedure IV. Amide coupling
The Aryl carboxylic acid was combined in anhydrous DMF with
N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide hydrochloride

K. L. Milkiewicz et al. / Bioorg. Med. Chem. 18 (2010) 4351–4362
4359
1H), 7.46–7.54 (m, 4H), 7.63 (d, J = 1.8 Hz, 1H), 8.18 (d, J = 2.3 Hz,
1H).
Mp 173–174 °C, LC–MS: m/z = 410 (M+H+); calcd for C23H21F2N3O2:
410 (M+H+), 1H NMR (CDCl3, 400 MHz) d 1.39 (t, J = 7.1 Hz, 3H),
3.45 (t, J = 5.0 Hz, 2H), 3.61–3.66 (m, 2H), 4.37 (q, J = 7.1 Hz, 2H),
4.49 (s, 2H), 5.32 (br s, 1H), 6.78 (d, J = 1.5 Hz, 1H), 6.89–6.96 (m,
1H), 6.99–7.08 (m, 2H), 7.45 (d, J = 8.4 Hz, 2H), 7.76 (d, J = 1.8 Hz,
2H), 8.02 (d, J = 8.6 Hz, 2H). 4-[1-(2,5-Difluoro-benzyl)-1,2,3,4-tet-
rahydro-pyrido[2,3-b]pyrazin-7-yl]-benzoic acid ethyl ester was
heated to 70 °C in a 1/1 mixture of THF and H2O with 5 equiv
LiOHꢁH2O for 2–16 h. The resulting solution was concentrated
and neutralized with 1 N HCl. The resulting precipitate was filtered
to obtain 4-[1-(2,5-difluoro-benzyl)-1,2,3,4-tetrahydro-pyrido-
[2,3-b]pyrazin-7-yl]-benzoic acid as a beige solid in 91% yield.
Mp >300 °C, LC–MS: m/z = 382 (M+H+); calcd for C21H17F2N3O2:
382 (M+H+), 1H NMR (DMSO-d6, 400 MHz) d 3.28–3.33 (m, 2H),
3.40–3.49 (m, 2H), 4.58 (s, 2H), 6.58 (s, 1H), 6.77 (s, 1H), 7.10–
7.20 (m, 2H), 7.26–7.34 (m, 1H), 7.59 (d, J = 8.3 Hz, 2H), 7.73 (s,
1H), 7.89 (d, J = 8.3 Hz, 2H), 12.92 (br s, 1H). 4-[1-(2,5-Difluoro-
benzyl)-1,2,3,4-tetrahydro-pyrido[2,3-b]pyrazin-7-yl]-benzoic acid
was reacted with(S)-(+)-1-(2-pyrrolidinylmethyl)pyrrolidine follow-
ing general procedure IV to obtain 5c as a pale yellow solid in 53%
yield. Mp 143–144 °C, LC–MS: m/z = 518 (M+H+); calcd for
C30H33F2N5O: 518 (M+H+), 1H NMR (CDCl3, 400 MHz) d 1.48–1.58
(m, 1H), 1.87–2.15 (m, 4H), 2.17–2.22 (m, 3H), 2.55–2.64 (m, 4H),
2.83–2.89 (m, 1H), 3.12–3.23 (m, 1H), 3.42–3.50 (m, 2H), 3.60–
3.64 (m, 2H), 4.40–4.49 (m, 3H), 5.07 (br s, 1H), 6.76 (s, 1H), 6.86–
7.09 (m, 3H), 7.41 (d, J = 8.1 Hz, 2H), 7.45–7.52 (m, 2H), 7.73 (s, 1H).
5.9.7. 1-(2,5-Dichloro-benzyl)-7-[6-(4-methyl-piperazin-1-yl)-
pyridin-3-yl]-1,2,3,4-tetrahydro-pyrido[2,3-b]pyrazine (5g)
1-(2,5-Dichloro-benzyl)-7-iodo-1,2,3,4-tetrahydro-pyrido[2,3-
b]pyrazine (4f) was coupled to 1-methyl-4-[5-(4,4,5,5-tetra-
methyl-[1,3,2]dioxaborolan-2-yl)-pyridin-2-yl]-piperazine follow-
ing general procedure IIB to afford 5g as a brown solid in 50%
yield. LC–MS: m/z = 469 (M+H+); calcd for C24H26Cl2N6: 469
(M+H+), 1H NMR (CDCl3, 400 MHz)
d 2.33 (s, 3H), 2.51 (t,
J = 5.1 Hz, 4H), 3.48 (m, 2H), 3.55 (t, J = 5.1 Hz, 4H), 3.64 (t,
J = 4.7 Hz, 2H), 4.46 (s, 2H), 5.20 (br s, 1H), 6.52 (d, J = 1.5 Hz,
1H), 6.65 (d, J = 8.8 Hz, 1H), 7.19 (dd, J = 8.5, 2.4 Hz, 1H), 7.26 (d,
J = 2.3 Hz, 1H), 7.33 (d, J = 8.3 Hz, 1H), 7.52 (dd, J = 8.6, 2.5 Hz,
1H), 7.62 (d, J = 1.5 Hz, 1H), 8.20 (d, J = 2.5 Hz, 1H).
5.9.8. 1-(2-Chloro-3,6-difluoro-benzyl)-7-[6-(4-methyl-
piperazin-1-yl)-pyridin-3-yl]-1,2,3,4-tetrahydro-pyrido[2,3-
b]pyrazine (5h)
1-(2-Chloro-3,6-difluoro-benzyl)-7-iodo-1,2,3,4-tetrahydro-
pyrido[2,3-b]pyrazine (4g) was coupled to 1-methyl-4-[5-(4,4,5,5-
tetramethyl-[1,3,2]dioxaborolan-2-yl)-pyridin-2-yl]-piperazine
through general procedure IIB to afford 5h as a light brown solid in
16% yield. Mp = 219–220 °C, LC–MS: m/z = 471 (M+H+); calcd for
C24H25ClF2N6: 471 (M+H+), 1H NMR (CDCl3, 400 MHz) d 2.36 (s,
3H), 2.54 (t, J = 5.1 Hz, 4H), 3.27 (t, J = 4.8 Hz, 2H), 3.51 (t,
J = 4.7 Hz, 2H), 3.59 (t, J = 5.1 Hz, 4H), 4.50 (d, J = 1.0 Hz, 2H), 5.01
(br s, 1H), 6.70 (d, J = 8.8 Hz, 1H), 7.03 (m, 2H), 7.11 (m, 1H), 7.61
(dd, J = 8.8, 2.5 Hz, 1H), 7.64 (d, J = 1.8 Hz, 1H), 8.35 (d, J = 2.3 Hz,
1H).
5.9.4. 1-(2,5-Difluoro-benzyl)-7-[6-(4-methyl-piperazin-1-yl)-
pyridin-3-yl]-1,2,3,4-tetrahydro-pyrido[2,3-b]pyrazine (5d)
1-(2,5-Difluoro-benzyl)-7-iodo-1,2,3,4-tetrahydro-pyrido[2,3-
b]pyrazine (4c) was reacted with 1-methyl-4-[5-(4,4,5,5-tetra-
methyl-[1,3,2]dioxaborolan-2-yl)-pyridin-2-yl]-piperazine follow-
ing general procedure IIIA to afford 5d as a beige solid in 53%
yield. Mp 192–193 °C, LC–MS: m/z = 437 (M+H+); calcd for
C24H26F2N6: 437 (M+H+), 1H NMR (CDCl3, 400 MHz) d 2.34 (s,
3H), 2.52–2.58 (m, 4H, 3.43–3.50 (m, 2H), 3.52–3.59 (m, 4H),
3.60–3.65 (m, 2H), 4.47 (s, 2H), 4.90 (s, 1H), 6.62 (m, 2H), 6.91–
7.09 (m, 3H), 7.52 (dd, J = 8.8 Hz, 2.5 Hz, 1H), 7.62 (d, J = 1.8 Hz,
1H), 8.2 (d, J = 2.5 Hz, 1H).
5.9.9. 4-{1-[5-Chloro-2-(trifluoromethyl)benzyl]-1,2,3,4-
tetrahydropyrido[2,3-b]pyrazin-7-yl}benzoic acid ethyl ester
(5i)
1-[5-Chloro-2-(trifluoromethyl)benzyl]-7-iodo-1,2,3,4-tetrahy-
dropyrido[2,3-b]pyrazine (7.0 g) was reacted with 4-ethoxycar-
bonyl phenyl boronic acid as in general procedure IIIA. 5i was
obtained as a yellow solid in 71% yield. Mp 154–155 °C, LC–MS:
m/z = 476 (M+H+); calcd for C24H21ClF3N3O2: 476 (M+H+), 1H
NMR (CDCl3, 400 MHz) d 1.40, (t, 3H), 3.49 (m, 2H), 3.71 (m, 2H),
4.35 (q, 2H), 4.61 (s, 2H), 5.72 (br s, 1H), 6.62 (s, 1H), 7.44 (m,
3H), 7.50 (s, 1H), 7.63 (d, 1H), 7.72 (s, 1H), 8.07 (d, 2H).
5.9.5. 1-(5-Chloro-2-trifluoromethyl-benzyl)-7-[6-(4-methyl-
piperazin-1-yl)-pyridin-3-yl]-1,2,3,4-tetrahydro-pyrido[2,3-
b]pyrazine (5e)
1-(5-Chloro-2-trifluoromethyl-benzyl)-7-iodo-1,2,3,4-tetrahy-
dro-pyrido[2,3-b]pyrazine (4e) was reacted with 1-methyl-4-[5-
(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-pyridin-2-yl]-piper-
azine following general procedure IIA to obtain 5e as a pale orange
solid in 28% yield. Mp 226–227 °C, LC–MS: m/z = 504 (M+H+); calcd
for C25H26ClF3N6: 504 (M+H+), 1H NMR (CDCl3, 400 MHz) d 2.34 (s,
3H), 2.51 (t, J = 5.0 Hz, 4H), 3.47–3.56 (m, 6H), 3.62–3.69 (m, 2H),
4.61 (s, 2H), 4.92 (br s, 1H), 6.49 (d, J = 1.0 Hz, 1H), 6.64 (d,
J = 8.8 Hz, 1H), 7.34 (d, J = 8.3 Hz, 1H), 7.52–7.58 (m, 2H), 7.61–
7.65 (m, 2H), 8.18 (d, J = 2.3 Hz, 1H).
5.9.10. 4-{1-[5-Chloro-2-(trifluoromethyl)benzyl]-1,2,3,4-
tetrahydropyrido[2,3-b]pyrazin-7-yl}benzoic acid (5j)
4-{1-[5-Chloro-2-(trifluoromethyl)benzyl]-1,2,3,4-tetrahydro-
pyrido[2,3-b]pyrazin-7-yl}benzoic acid ethyl ester (5i) (5.1 g) was
heated to 70 °C in a 1/1 mixture of THF and H2O with 5 equiv
LiOHꢁH2O for 2–16 h. The resulting solution was concentrated
and neutralized with 1 N HCl. The resulting precipitate was filtered
to obtain 5j as a light orange solid in 88% yield. LC–MS: m/z = 448
(M+H+); calcd for C22H17ClF3N3O2: 448 (M+H+), 1H NMR (DMSO-d6,
400 MHz) d 3.41 (m, 2H), 3.60 (m, 2H), 4.80 (s, 2H), 7.07 (s, 1H),
7.63 (m, 3H), 7.74 (s, 1H), 7.82 (d, 1H), 7.95 (m, 2H), 8.81 (s, 1H),
13.01 (br s, 1H).
5.9.6. 1-(2-Chloro-5-trifluoromethyl-benzyl)-7-[6-(4-methyl-
piperazin-1-yl)-pyridin-3-yl]-1,2,3,4-tetrahydro-pyrido[2,3-
b]pyrazine (5f)
5.9.11. {1-[(5-Chloro-2-trifluoromethyl)benzyl]-1,2,3,4-tetra-
hydro-pyrido[2,3-b]pyrazin-7-yl}piperidin-1-yl-methanone
(5k)
4-{1-[5-Chloro-2-(trifluoromethyl)benzyl]-1,2,3,4-tetrahydro-
pyrido[2,3-b]pyrazin-7-yl}benzoic acid (5j) (89 mg) was reacted
with piperidine as in general procedure IV to give 5k as an orange
solid in 64% yield. Mp 216–217 °C, LC–MS: m/z = 515 (M+H+); calcd
for C27H26ClF3N4O: 515 (M+H+), 1H NMR (CDCl3, 400 MHz) d 1.61
(m, 6H), 2.88 (s, 1H), 2.95 (s, 1H), 3.38 (s, 2H), 3.49 (m, 2H), 3.69
1-(2-Chloro-5-trifluoromethyl-benzyl)-7-iodo-1,2,3,4-tetrahy-
dro-pyrido[2,3-b]pyrazine (4d) was reacted with 1-methyl-4-[5-
(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-pyridin-2-yl]-piper-
azine following general procedure IIA to obtain 5f as a yellow film in
13% yield. LC–MS: m/z = 503 (M+H+); calcd for C25H26ClF3N6: 503
(M+H+), 1H NMR (CDCl3, 400 MHz) d 2.34 (s, 3H), 2.51 (t, J = 5.3 Hz,
4H), 3.28 (t, J = 5.3 Hz, 2H), 3.54 (t, J = 5.0 Hz, 4H), 3.66–3.72 (m,
2H), 4.53 (s, 2H), 4.89 (br s, 1H), 6.53 (s, 1H), 6.64 (d, J = 8.8 Hz,

4360
K. L. Milkiewicz et al. / Bioorg. Med. Chem. 18 (2010) 4351–4362
(m, 2H), 4.62 (s, 2H), 5.26 (s, 1H), 6.60 (s, 1H), 7.37 (m, 5H), 7.51 (s,
1H), 7.64 (d, J = 8.3 Hz, 1H), 7.75 (d, J = 1.5 Hz, 1H).
(m, 2H), 3.27–3.29 (m, 2H), 5.78 (s, 1H), 6.37 (s, 1H), 6.78 (d,
J = 2.0 Hz, 1H), 7.33 (d, J = 2.0 Hz, 1H). 7-Iodo-1,2,3,4-tetrahydro-
pyrido[2,3-b]pyrazine (7) was dissolved in anhydrous pyridine.
2,5-Difluorobenzene sulfonyl chloride (1.0 equiv) was added and
the reaction mixture was stirred at rt for 16 h. The reaction mixture
was concentrated and the resulting residue was purified by silica
gel chromatography to afford 1-(2,5-difluoro-benzenesulfonyl)-7-
iodo-1,2,3,4-tetrahydro-pyrido[2,3-b]pyrazine (8) as a pale yellow
solid in 13% yield. Mp 187–188 °C, LC–MS: m/z = 438 (M+H+); calcd
for C13H10F2N3O2S: 438 (M+H+), 1H NMR (CDCl3, 400 MHz) d 3.29–
3.33 (m, 2H), 3.83 (t, J = 4.8 Hz, 2H), 5.08 (br s, 1H), 7.11–7.19 (m,
1H), 7.26–7.33 (m, 1H), 7.58–7.63 (m, 1H), 7.96 (d, J = 1.5 Hz, 1H),
8.03 (d, J = 1.8 Hz, 1H). 1-(2,5-Difluoro-benzenesulfonyl)-7-iodo-
1,2,3,4-tetrahydro-pyrido[2,3-b]pyrazine (8) was reacted with
1-methyl-4-[5-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-
pyridin-2-yl]-piperazine following general procedure IIIA to obtain
9a as a yellow foam in 35% yield. LC–MS: m/z = 487 (M+H+); calcd
for C23H26F2N6O2S: 487 (M+H+), 1H NMR (CDCl3, 400 MHz) d 2.34
(s, 3H), 2.54 (t, J = 5.3 Hz, 4H), 3.35–3.38 (m, 2H), 3.60 (t,
J = 5.0 Hz, 4H), 3.91 (t, J = 4.8 Hz, 2H), 5.05 (br s, 1H), 6.71 (d,
J = 8.8 Hz, 1H), 7.11–7.17 (m, 1H), 7.24–7.29 (m, 1H), 7.58–7.63
(m, 2H), 7.90 (d, J = 2.0 Hz, 1H), 8.05 (d, J = 2.3 Hz, 1H), 8.34 (d,
J = 2.6 Hz, 1H).
5.9.12. {1-[(5-Chloro-2-trifluoromethyl)benzyl]-1,2,3,4-tetra-
hydropyrido[2,3-b]pyrazin-7-yl}-(4-methoxy-piperidin-1-yl)-
methanone (5l)
4-{1-[5-Chloro-2-(trifluoromethyl)benzyl]-1,2,3,4-tetrahydro-
pyrido[2,3-b]pyrazin-7-yl}benzoic acid (5j) (80 mg) was reacted
with 4-methoxypiperidine HCl as in general procedure IV to give
5l as an orange solid in 58% yield. LC–MS: m/z = 545 (M+H+); calcd
for C28H28ClF3N4O2: 545 (M+H+), 1H NMR (CDCl3, 400 MHz) d 1.73
(m, 4H), 2.88 (s, 1H), 2.95 (s, 1H), 3.36 (s, 3H), 3.47 (m, 4H), 3.68
(m, 3H), 4.62 (s, 2H), 5.35 (br s, 1H), 6.59 (d, J = 1.3 Hz, 1H), 7.38
(m, 5H), 7.51 (s, 1H), 7.64 (d, J = 8.3 Hz, 1H), 7.75 (s, 1H).
5.9.13. {4-[1-(5-Chloro-2-trifluoromethyl-benzyl)-1,2,3,4-tetra-
hydro-pyrido[2,3-b]pyrazin-7-yl]-phenyl}-(4-pyrrolidin-1-yl-
piperidin-1-yl)-methanone (5m)
1-(5-Chloro-2-trifluoromethyl-benzyl)-7-iodo-1,2,3,4-tetrahy-
dro-pyrido[2,3-b]pyrazine (4e) was reacted with (4-pyrrolidin-1-
yl-piperidin-1-yl)-[4-(4,4,5,5-tetramethyl [1,3,2]dioxaborolan-2-
yl)-phenyl]-methanone following general procedure IIIA to obtain
5m as an orange foam in 19% yield. LC–MS: m/z = 585 (M+H+);
calcd for C31H33ClF3N5O: 585 (M+H+), 1H NMR (CDCl3, 400 MHz)
d 1.43–1.62 (m, 3H), 1.65–1.99 (m, 3H), 2.21–2.30 (m, 2H), 2.52–
2.61 (m, 6H), 2.87–3.08 (m, 3H), 3.47–3.51 (m, 2H), 3.65–3.71
(m, 2H), 4.62 (s, 2H), 5.02 (s, 1H), 6.59 (d, J = 1.5 Hz, 1H), 7.31–
7.39 (m, 5H), 7.50 (s, 1H), 7.65 (d, J = 8.4 Hz, 1H), 7.74 (s, 1H).
5.9.17. (2,5-Difluoro-phenyl)-{7-[4-((S)-2-pyrrolidin-1-
ylmethyl-pyrrolidine-1-carbonyl)-phenyl]-3,4-dihydro-2H-
pyrido[2,3-b]pyrazin-1-yl}-methanone (9b)
7-Iodo-1,2,3,4-tetrahydro-pyrido[2,3-b]pyrazine was dissolved
in anhydrous THF. Pyridine (1.0 equiv) was added, and the reaction
was cooled to 0 °C. 2,5-Difluorophenyl acetyl chloride (1.0 equiv)
was added, and a white precipitate formed. The reaction was stir-
red at 0 °C for 15 min, allowed to warm to rt, and stirred at rt for
16 h. The reaction was diluted with EtOAc, washed with NaHCO3,
dried over MgSO4, filtered, concentrated, and purified by silica
gel chromatography to afford (2,5-difluoro-phenyl)-(7-iodo-3,4-
dihydro-2H-pyrido[2,3-b]pyrazin-1-yl)-methanone as an off-white
solid in 14% yield. Mp 152–153 °C, LC–MS: m/z = 416 M+H+); calcd
for C15H12F2IN3O: 416 (M+H+), 1H NMR (CDCl3, 400 MHz) d 3.47–
3.54 (m, 2H), 3.80–3.87 (m, 4H), 5.44 (br s, 1H), 6.90–7.04
(m, 3H), 8.03 (s, 1H). 2-(2,5-Difluoro-phenyl)-1-(7-iodo-3,4-dihy-
dro-2H-pyrido[2,3-b]pyrazin-1-yl)-ethanone was reacted with 1-
methyl-4-[5-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-pyri-
din-2-yl]-piperazine following general procedure IIIA to afford
2-(2,5-difluoro-phenyl)-1-{7-[2-(4-methyl-piperazin-1-yl)-pyri-
din-4-yl]-3,4-dihydro-2H-pyrido[2,3-b]pyrazin-1-yl}-ethanone as
5.9.14. 1-(5-Chloro-2-trifluoromethyl-benzyl)-7-(2-piperazin-1-
yl-pyridin-4-yl)-1,2,3,4-tetrahydro-pyrido[2,3-b]pyrazine (5n)
1-(5-Chloro-2-trifluoromethyl-benzyl)-7-iodo-1,2,3,4-tetrahy-
dro-pyrido[2,3-b]pyrazine (4e) was reacted with 2-(piperazin-1-
yl)pyridine-4-boronic acid pinacol ester following general proce-
dure IIIB to obtain 5n as an off-white solid. Mp 189–190 °C, LC–
MS: m/z = 489; calcd for C24H24ClF3N6: 489 (M+H+), 1H NMR1H
NMR (CDCl3, 400 MHz) d 2.98 (m, 4H), 3.49 (6H, m), 3.71 (m,
2H), 4.61 (s, 2H), 5.29 (s, 1H), 5.34 (br s, 1H), 6.55 (m, 2H), 6.64
(dd, J = 5.2, 1.4 Hz, 1H), 7.36 (d, J = 8.1 Hz, 1H), 7.52 (s, 1H), 7.64
(d, J = 8.3 Hz, 1H), 7.77 (d, J = 2.0 Hz, 1H), 8.12 (d, J = 5.3 Hz, 1H).
5.9.15. 1-[4-(4-{1-[5-Chloro-2-(trifluoromethyl)benzyl]-1,2,3,4-
tetrahydro-pyrido[2,3-b]pyrazin-7-yl}pyridin-2-yl)piperazin-1-
yl]ethanone (5o)
To a solution of 1-[5-chloro-2-(trifluoromethyl)benzyl]-7-(2-
piperazin-1-yl-pyridin-4-yl)-1,2,3,4-tetrahydro-pyrido[2,3-b]pyra-
zine (5n) (63 mg) in anhydrous DMF (1 mL) was added acetyl
chloride (1.3 equiv) and triethylamine (1.1 equiv). The mixture
was stirred at room temperature under nitrogen overnight and
then concentrated. The residue was purified on normal phase silica
gel chromatography to give 5o as a yellow solid in 56% yield. Mp
241–242 °C, LC–MS: m/z = 531 (calcd for C26H26ClF3N6O: 531
(M+H+), 1H NMR (CDCl3, 400 MHz) d 2.15 (s, 3H), 3.47 (m, 2H),
3.52 (m, 2H), 3.57 (m, 2H), 3.62 (m, 2H), 3.73 (m, 4H), 4.61 (s,
2H), 5.25 (br s, 1H), 6.55 (m, 2H), 6.69 (d, J = 5.3 Hz, 1H), 7.37 (d,
J = 8.3 Hz, 1H), 7.52 (s, 1H), 7.65 (d, J = 8.3 Hz, 1H), 7.78 (br s,
1H), 8.12 (d, J = 5.3 Hz, 1H).
a
light yellow solid in 15% yield. Mp >300 °C, LC–MS: m/
z = 465.02 (M+H+); calcd for C25H26F2N6O: 465 (M+H+), 1H NMR
(CDCl3, 400 MHz) d 2.36 (s, 3H), 2.52–2.57 (m, 2H), 3.52–3.62 (m,
6H), 3.90–3.96 (m, 4H), 5.11 (br s, 1H), 6.70 (d, J = 8.8 Hz, 1H),
6.90–7.04 (m, 3H), 7.41–7.58 (m, 2H), 8.07 (s, 1H), 8.32 (s, 1H).
5.9.18. 7-Bromo-1,2,3,4-tetrahydro-[1,8]naphthyridin-4-ol (11)
7-Bromo-2,3-dihydro-1H-[1,8]naphthyridin-4-one (10) was
placed in methanol and sodium borohydride (1.84 equiv) was
added portionwise over 5 min. The reaction was stirred at rt for
15 min and then quenched with acetic acid. The reaction mixture
was concentrated under reduced pressure and the residue was ta-
ken up in toluene. Silica gel was added and the mixture was con-
centrated under reduced pressure. Purification by silica gel
chromatography using dry loading and a gradient of 0–80%
EtOAc/hex as the eluting solvent afforded 11 as a pale yellow solid
in 78% yield. Mp 130–131 °C, LC–MS: m/z = 231 (M+H+); calcd for
C8H9Br2N2O: 230 (M+H+), 1H NMR (MeOH-d4, 400 MHz) d 1.80–
1.94 (m, 2H), 3.33–3.48 (m, 2H), 4.66 (t, J = 4.4 Hz, 1H), 6.66 (d,
J = 7.6 Hz, 1H), 7.31 (d, J = 7.6 Hz, 1H).
5.9.16. 1-(2,5-Difluoro-benzenesulfonyl)-7-[6-(4-methyl-
piperazin-1-yl)-pyridin-3-yl]-1,2,3,4-tetrahydro-pyrido[2,3-
b]pyrazine (9a)
7-Iodo-3,4-dihydro-1H-pyrido[2,3-b]pyrazin-2-one was treated
with general procedure V using 4 equiv DIBAL-H to obtain 7-iodo-
1,2,3,4-tetrahydro-pyrido[2,3-b]pyrazine as a white solid in 31%
yield. Mp 126–128 °C, LC–MS: m/z = 262 (M+H+); calcd for
C7H8IN3: 262 (M+H+), 1H NMR (DMSO-d6, 400 MHz) d 3.13–3.17

K. L. Milkiewicz et al. / Bioorg. Med. Chem. 18 (2010) 4351–4362
4361
5.9.19. 6-Bromo-1,2,3,4-tetrahydro-[1,8]naphthyridin-4-ol (12)
7-Bromo-1,2,3,4-tetrahydro-[1,8]naphthyridin-4-ol (11) was
dissolved in anhydrous THF and cooled to ꢂ78 °C. A solution of
n-butyllithium in THF was added (5–7 equiv) and the reaction
was warmed slowly to room temperature. After 2–5 h, methanol
was added and the contents were concentrated to dryness. The res-
idue was dissolved in DCM/AcOH (1:1) and NBS (1.2 equiv) was
added. After 45 min, the mixture was concentrated to dryness onto
silica gel, and purified by silica gel chromatography to afford 12 as
yellow solids in 25–65% yield. Mp 125–128 °C, LC–MS: m/z = 229
(M+H+); calcd for C8H9Br2N2O: 230 (M+H+), 1H NMR (DMSO-d6,
400 MHz) d 1.72 (m, 2H), 3.25 (m, 2H), 4.56 (m, 1H), 5.33 (d,
J = 5.0 Hz, 1H), 6.8 (br s, 1H), 7.47 (d, J = 2.5 Hz, 1H), 7.87 (d,
J = 2.5 Hz, 1H).
5.9.23. 7-Bromo-4-[1-(2,5-difluoro-phenyl)-methylidene]-1,2,3,
4-tetrahydro [1,8]naphthyridine (16)
7-Bromo-4-[1-iodo-methylidene]-1,2,3,4-tetrahydro-[1,8]
naphthyridine, 2,5-difluorophenylboronic acid (15) (1.2 equiv),
and Pd(PPh3)4 (0.075 equiv) were placed in 4:1 toluene/EtOH and
2 N Na2CO3 (10.0 equiv) was added. The reaction was heated at
85 °C for 5 h and then celite was added to the reaction mixture
which was then concentrated under reduced pressure. The dry
powder was then loaded onto a silica gel column and a gradient
of 0–20% EtOAc/hex was used as the eluting solvent to obtain 16
as a yellow solid in 70% yield. Mp 178–179 °C, LC–MS: m/z = 337
(M+H+); calcd for C15H11BrF2N2: 338 (M+H+), 1H NMR (CDCl3,
400 MHz) d 2.62 (t, J = 5.8 Hz, 2H), 3.55–3.62 (m, 2H), 5.25 (br s,
1H), 6.26 (s, 1H), 6.45 (d, J = 7.9 Hz, 1H), 6.85–6.95 (m, 1H), 6.96–
7.12 (m, 3H).
5.9.20. 6-[6-(4-Methyl-piperazin-1-yl)-pyridin-3-yl]-1,2,3,4-
tetrahydro-[1,8]naphthyridin-4-ol (13)
5.9.24. 4-(2,5-Difluoro-benzyl)-1,2,3,4-tetrahydro-[1,8]naph-
thyridine (17)
6-Bromo-1,2,3,4-tetrahydro-[1,8]naphthyridin-4-ol (12) was
reacted with 1-methyl-4-[5-(4,4,5,5-tetramethyl-[1,3,2] dioxa-
borolan-2-yl)-pyridin-2-yl]-piperazine following general proce-
dure IIIA. The product was purified by silica gel chromatography
using a gradient of 0–10% (5% NH4OH in MeOH)/CH2Cl2 as the elut-
ing solvent to obtain 13 as brown solids in 22% yield. Mp 124–
128 °C, LC–MS: m/z = 326 (M+H+); calcd for C18H23N5O: 326
(M+H+), 1H NMR (CDCl3, 400 MHz) d 2.00 (m, 1H), 2.08 (s, 1H),
2.62 (t, J = 5.0 Hz, 4H), 3.49 (m, 2H), 3.58 (m, 2H), 3.63 (t,
J = 5.0 Hz, 4H), 4.85 (t, J = 4.0 Hz, 1H), 6.68 (s, 1H), 6.71 (s, 1H),
7.59 (dd, J = 2.6, 8.8 Hz, 1H), 7.66 (d, J = 2.2 Hz, 1H), 8.01 (d,
J = 2.3 Hz, 1H), 8.32 (d, J = 2.5 Hz, 1H).
7-Bromo-4-[1-(2,5-difluoro-phenyl)-methylidene]-1,2,3,4-tet-
rahydro-[1,8]naphthyridine (16) was dissolved in methanol and
Pd/C was added. The reaction was hydrogenated at atmospheric
pressure for 1.5 h. The reaction mixture was then filtered through
celite and concentrated under reduced pressure to obtain 17 as a
clear colorless oil in quantitative yield. LC–MS: m/z = 261
(M+H+); calcd for C15H11F2N2: 261 (M+H+), 1H NMR (MeOH-d4,
400 MHz) d 1.68–1.86 (m, 2H), 2.80 (dd, J = 9.0 Hz, 13.3 Hz, 1H),
2.96 (dd, J = 10.0 Hz, 13.3 Hz, 1H), 3.02–3.12 (m, 1H), 3.34–3.41
(m, 1H), 3.46–3.55 (m, 1H), 6.44 (dd, J = 5.2 Hz, 7.2 Hz, 1H), 6.93–
7.03 (m, 2H), 7.03–7.11 (m, 2H), 7.72 (dd, J = 1.0 Hz, 5.0 Hz, 1H).
5.9.25. 6-Bromo-4-(2,5-difluoro-benzyl)-1,2,3,4-tetrahydro-
[1,8] naphthyridine (18)
5.9.21. 4-(2-Chloro-3,6-difluoro-phenoxy)-6-[6-(4-methyl-
piperazin-1-yl)-pyridin-3-yl]-1,2,3,4-tetrahydro-
4-(2,5-Difluoro-benzyl)-1,2,3,4-tetrahydro-[1,8] naphthyridine
(17) was dissolved in a 10:1 mixture of CH2Cl2/AcOH and NBS
(1.2 equiv) was added. The reaction was stirred at rt for 1 h. and
was then concentrated under reduced pressure. The residue was
purified by silica gel chromatography using a gradient of 0–60%
EtOAc/hex as the eluting solvent to obtain 18 as a clear, colorless
oil in 30% yield. LC–MS: m/z = 339 (M+H+); calcd for C15H11BrF2N2:
338 (M+H+), 1H NMR (CDCl3, 400 MHz) d 1.66–1.75 (m, 1H), 1.75–
1.86 (m, 1H), 2.73 (dd, J = 9.6 and 13.4 Hz, 1H), 2.95 (dd, J = 6.0 and
13.4 Hz, 1H), 2.99–3.08 (m, 1H), 3.34–3.43 (m, 1H), 3.45–3.55 (m,
1H), 5.22 (br s, 1H), 6.80–6.86 (m, 1H), 6.88–6.96 (m, 1H), 6.98–
7.05 (m, 1H), 7.16 (d, J = 1.9 Hz, 1H), 7.91 (d, J = 1.9 Hz, 1H).
[1,8]naphthyridine (14)
6-[6-(4-Methyl-piperazin-1-yl)-pyridin-3-yl]-1,2,3,4-tetrahy-
dro-[1,8]naphthyridin-4-ol (13) (1 equiv), 2-chloro-3,6-difluoro-
phenol (1–3.0 equiv), and triphenylphosphine (1–3.0 equiv) were
placed in anhydrous THF and DIAD (3.0 equiv) was added. The
reaction mixture was stirred at rt for 15 min and then concentrated
under reduced pressure. The resulting crude product was purified
by silica gel chromatography using a gradient of 0–10% MeOH/
CH2Cl2 as the eluting solvent to obtain 14 as a yellow foam in
54% yield. LC–MS: m/z = 472 (M+H+); calcd for C24H24ClF2N5O:
472 (M+H+), 1H NMR (DMSO-d6, 400 MHz) d 1.89 (m, 1H), 2.21
(s, 3H), 2.24 (m, 1H), 2.39 (t, J = 4.8 Hz, 4H), 3.40 (m, 1H), 3.47 (t,
J = 4.8 Hz, 4H), 3.63 (m, 1H), 6.82 (d, J = 8.8 Hz, 1H), 7.03 (d,
J = 2.3 Hz, 1H), 7.17 (d, J = 2.3 Hz, 1H)7.25 (m, 1H), 7.39 (m, 1H),
7.52 (dd, J = 2.5, 8.8 Hz, 1H), 8.08 (d, J = 2.5 Hz, 1H), 8.19 (d,
J = 2.5 Hz, 1H).
5.9.26. 4-(2,5-Difluoro-benzyl)-6-[6-(4-methyl-piperazin-1-yl)-
pyridin-3-yl]-1,2,3,4-tetrahydro-[1,8] naphthyridine (19)
6-Bromo-4-(2,5-difluoro-benzyl)-1,2,3,4-tetrahydro-[1,8]
naphthyridine (18) (1 equiv), 1-methyl-4-[5-(4,4,5,5-tetramethyl-
[1,3,2]dioxaborolan-2-yl)-pyridin-2-yl]-piperazine (1.4 equiv), and
Pd(PPh3)4 (0.1 equiv) were placed in a 4:1 mixture of toluene/EtOH
and 2 N Na2CO3 (4.0 equiv) was added. The reaction was heated at
90 °C for 6 h. The reaction mixture was then concentrated under
reduced pressure and the residue was taken up in CH2Cl2 and
washed with water. The organic layer was dried over Na2SO4, fil-
tered, and concentrated under reduced pressure. Purification by
silica gel chromotograhpy using a gradient of 0–20% MeOH/CH2Cl2
as the eluting solvent afforded 19 as a yellow foam in 26% yield.
LC–MS: m/z = 436 (M+H+); calcd for C25H27F2O5: 436 (M+H+), 1H
NMR (CDCl3, 400 MHz) d 1.7–2.0 (m, 2H), 2.37 (s, 3H), 2.55–2.60
(m, 4H), 2.81 (dd, J = 9.1 and 13.3 Hz, 1H), 2.98 (dd, J = 6.4 and
13.3 Hz, 1H), 3.08–3.18 (m, 1H), 3.40–3.50 (m, 1H), 3.53–3.70 (m,
5H), 5.18 (br s, 1H), 6.68 (d, J = 8.8 Hz, 1H), 6.81–6.87 (m, 1H),
6.89–6.96 (m, 1H), 6.98–7.06 (m, 1H), 7.16 (d, J = 1 Hz, 1H), 7.50
(dd, J = 2.5 and 8.8 Hz, 1H), 8.05 (d, J = 1 Hz, 1H), 8.24 (d,
J = 2.5 Hz, 1H).
5.9.22. 7-Bromo-4-[1-iodo-methylidene]-1,2,3,4-tetrahydro-
[1,8] naphthyridine (15)
7-Bromo-2,3-dihydro-1H-[1,8]naphthyridin-4-one (10)32 was
suspended in anhydrous THF and a solution of CrCl2 (8.35 equiv)
and CHI3 (2.13 equiv) in anhydrous THF was added. The reaction
was stirred at rt for 16 h and was then diluted with methylene
chloride and washed with saturated NaHCO3. The aqueous phase
was extracted twice with methylene chloride and the combined
organic layers were washed with brine and then dried over Na2SO4.
The solution was filtered and concentrated under reduced pres-
sure. Purification by silica gel chromatography using a gradient
of 0–30% EtOAc/hex as the eluting solvent to obtain 15 as a yellow
solid in 27% yield. Alkene geometry determined by NOE. LC–MS:
m/z = 352 (M+H+); calcd for C9H8BrIN2: 352 (M+H+), 1H NMR (CDCl3,
400 MHz) d 2.69 (t, J = 6.0 Hz, 2H), 3.43–3.51 (m, 2H), 5.25 (br s, 1H),
6.23 (s, 1H), 6.74 (d, J = 8.0 Hz, 1H), 8.19 (d, J = 8.0 Hz, 1H).

4362
K. L. Milkiewicz et al. / Bioorg. Med. Chem. 18 (2010) 4351–4362
13. Stoica, G. E.; Kuo, A.; Aigner, A.; Sunitha, I.; Souttou, B.; Malerczyk, C.; Caughey,
D. J.; Wen, D.; Karavanov, A.; Riegel, A. T.; Wellstein, A. J. Biol. Chem. 2001, 276,
16772.
14. Powers, C.; Aigner, A.; Stoica, G. E.; McDonnell, K.; Wellstein, A. J. Biol. Chem.
2002, 277, 14153.
5.9.27. 1-(2,5-Dichloro-benzyl)-4-methyl-7-[6-(4-methyl-pip-
erazin-1-yl)-pyridin-3-yl]-1,2,3,4-tetrahydro-pyrido[2,3-b]-
pyrazine (20)
1-(2,5-Dichloro-benzyl)-7-iodo-1,2,3,4-tetrahydro-pyrido[2,3-
b]pyrazine (4f) was dissolved in anhydrous DMF. NaH (1.6 equiv)
was added followed by iodomethane (1.4 equiv). The reaction
mixture was stirred at rt for 48 h. Concentration of the reaction
mixture followed by column chromatography afforded 1-(2,5-di-
chloro-benzyl)-7-iodo-4-methyl-1,2,3,4-tetrahydro-pyrido[2,3-
b]pyrazine as a yellow solid in 56% yield. LC–MS: m/z = 434
(M+H+); calcd for C15H14Cl2IN3: 434 (M+H+), 1H NMR (CDCl3,
400 MHz) d 3.09 (s, 3H), 3.42 (m, 2H), 3.48 (m, 2H), 4.36 (s, 2H),
6.54 (1H, d, J = 1.8 Hz), 7.17 (1H, d, J = 2.3 Hz), 7.21 (1H, dd,
J = 8.6, 2.5 Hz), 7.34 (1H, d, J = 4.3 Hz), 7.72 (1H, d, J = 1.8 Hz). 1-
(2,5-Dichloro-benzyl)-7-iodo-4-methyl-1,2,3,4-tetrahydro-pyrido
[2,3-b]pyrazine was coupled to to 1-methyl-4-[5-(4,4,5,5-tetra-
methyl-[1,3,2]dioxaborolan-2-yl)-pyridin-2-yl]-piperazine through
general procedure IIIB to afford 20 as a brown solid in 43% yield.
LC–MS: m/z = 483 (M+H+); calcd for C25H28Cl2N6: 483 (M+H+), 1H
NMR (CDCl3, 400 MHz) d 2.33 (s, 3H), 2.52 (t, J = 5.1 Hz, 4H), 3.16
(s, 3H), 3.53 (m, 8H), 4.45 (s, 2H), 6.45 (d, J = 1.8 Hz, 1H), 6.65 (d,
J = 8.8 Hz, 1H), 7.18 (dd, J = 8.5, 4.8 Hz, 1H), 7.25 (d, J = 2.3 Hz,
1H), 7.32 (d, J = 8.3 Hz, 1H), 7.52 (dd, J = 8.6, 2.5 Hz, 1H), 7.75 (d,
J = 1.8 Hz, 1H), 8.22 (d, J = 2.5 Hz, 1H).
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.04.087.
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