Identification of a novel class of selective Tpl2 kinase inhibitors: 4-Alkylamino-[1,7]naphthyridine-3-carbonitriles

Article abstract of DOI:10.1016/j.bmc.2007.06.054

We have previously reported the discovery and initial SAR of the [1,7]naphthyridine-3-carbonitriles and quinoline-3-carbonitriles as Tumor Progression Loci-2 (Tpl2) kinase inhibitors. In this paper, we report new SAR efforts which have led to the identifi

Full text of DOI:10.1016/j.bmc.2007.06.054

Bioorganic & Medicinal Chemistry 15 (2007) 6425–6442
Identification of a novel class of selective Tpl2 kinase inhibitors:
4-Alkylamino-[1,7]naphthyridine-3-carbonitriles
Neelu Kaila,a,* Neal Green,a Huan-Qiu Li,a Yonghan Hu,a Kristin Janz,a
Lori Krim Gavrin,a Jennifer Thomason,a Steve Tam,a Dennis Powell,d John Cuozzo,e
J. Perry Hall,b Jean-Baptiste Telliez,b Sang Hsu,b Cheryl Nickerson-Nutter,b
Qin Wangc and Lih-Ling Linb
aChemical and Screening Sciences (CSS), Wyeth Research, 200 CambridgePark Drive, Cambridge, MA 02140, USA
bInflammation, Wyeth Research, 200 CambridgePark Drive, Cambridge, MA 02140, USA
cDrug Safety and Metabolism, Wyeth Research, 1 Burtt Road, Andover, MA 01810, USA
dCSS, Wyeth Research, Pearl River, NY, USA
ePraecis Pharmaceuticals, MA, USA
Received 1 May 2007; revised 20 June 2007; accepted 26 June 2007
Available online 4 July 2007
Abstract—We have previously reported the discovery and initial SAR of the [1,7]naphthyridine-3-carbonitriles and quinoline-3-car-
bonitriles as Tumor Progression Loci-2 (Tpl2) kinase inhibitors. In this paper, we report new SAR efforts which have led to the
identification of 4-alkylamino-[1,7]naphthyridine-3-carbonitriles. These compounds show good in vitro and in vivo activity against
Tpl2 and improved pharmacokinetic properties. In addition they are highly selective for Tpl2 kinase over other kinases, for example,
EGFR, MEK, MK2, and p38. Lead compound 4-cycloheptylamino-6-[(pyridin-3-ylmethyl)-amino]-[1,7]naphthyridine-3-carbonit-
rile (30) was efficacious in a rat model of LPS-induced TNF-a production.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
Several members of the mitogen-activated protein ki-
nase (MAP kinase) family, particularly the serine/threo-
nine kinase p38 (Fig. 1), have been investigated by
several companies. Four compounds have progressed
to the clinic,3 AMG-548, BIRB-796, SCIO-4694
(SCIO-323), and VX-702.4 Two of these, AMG-548
and BIRB-796, have failed to advance further due to
toxicity concerns.
Tumor Progression Loci-2 (Tpl2) kinase has been shown
to be involved in both production and signaling of TNF-
a.1 TNF-a is a pro-inflammatory cytokine and its role in
several inflammatory diseases, most notably rheumatoid
arthritis (RA), is well established.2 Thus, Tpl2 is an
attractive target for the treatment of RA, a chronic inca-
pacitating disease that currently affects more than five
million people worldwide. The use of kinase inhibitors
in the treatment of chronic diseases like RA has not
been clinically proven.3 Many compounds have failed
in phase 1 clinical trials due to adverse effects. The tox-
icity observed could be a result of poor selectivity
against other kinases.
LBP
LPS
CD14
TLR4
TNFR
MEKK
TPL2
MKK3/6
p38
MEK
ERK
MK2
AA
A
Keywords: Tpl2 kinase; [1,7]Naphthyridine-3-carbonitriles; Quinoline-
3-carbonitriles.
ATG
3’ARE
TNF
mRNA
*
Corresponding author. Tel.: +1 617 665 5626; fax: +1 617 665
5685; e-mail: nkaila@wyeth.com
Figure 1. Tpl2 is essential for TNF-a production and TNF-a signaling.
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.06.054

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N. Kaila et al. / Bioorg. Med. Chem. 15 (2007) 6425–6442
Tpl2 is a serine/threonine kinase in the MAP3K family
that is upstream of MEK in the ERK pathway
(Fig. 1).1 Tpl2 is the only known human kinase that
has a proline instead of a conserved glycine at the first
glycine in the GXGXXG motif of the ATP binding
loop.5a Tpl2 is also not inhibited by the pan kinase
inhibitor staurosporine.5b Therefore, we believe that
Tpl2 has some distinct structural features that will con-
fer selectivity against other kinases and circumvent un-
wanted side effects.
70% inhibition of TNF-a production in the LPS-mouse
model when dosed ip at 25 mg/kg. However, compound
2a has poor selectivity against epidermal growth factor
receptor (EGFR) tyrosine kinase. A pharmacokinetic
(PK) study of 2a revealed that it also showed poor bio-
availability (F) and very high total clearance (Cl) in rats
(F = 1%; Cl = 97 mL/min/kg). In this article, we report
the design and synthesis of carbonitriles that contain no-
vel headpieces and show selectivity against EGFR, im-
proved PK properties and activity in the LPS mouse
model.
In our previous work on Tpl26,7 we have reported the
[1,7]naphthyridine-3-carbonitriles (1) and quinoline-3-
carbonitriles (2) as potent, reversible, ATP-competitive
Tpl2 inhibitors with selectivity against certain kinases.
The naphthyridine carbonitriles 1 were identified via ‘di-
rected screening’ of our kinase library.6 Further SAR
evaluation of 1 indicated that, unlike other kinases,
the inhibitory activity of Tpl2 depended on substitution
at the C-6 position (tailpiece). In the literature, the C-6
position of naphthyridine-3-carbonitriles8 and 1,3-quin-
azolines9 has been shown to be useful for optimization
of solubility and permeability of kinase inhibitors, but
2. Chemistry
The synthesis of compounds with the general structure 1
was accomplished in a manner reported by Wissner
et al.10 As shown in Scheme 1 the key intermediate 7
was prepared in seven steps from commercially available
2-chloro-5-nitropyridine 3. Compound 7 and appropri-
ate headpiece amines were either refluxed in ethanol or
irradiated by microwave to give intermediates 8. The
6-fluoro groups of these intermediates were displaced
to have minimal effect on isolated kinase activity. How-
ever, several potent tailpieces were identified for class 1
compounds (1a–c). Further investigation showed that
the closely related quinoline-3-carbonitriles (2) had very
similar SAR to carbonitriles 1.7 A detailed study of the
C-6 substitution in carbonitriles 27 led to the identifica-
tion of a potent Tpl2 inhibitor, compound 2a, with
selectivity against several kinases (MEK, PKA, p38,
Src, CAMKII, MK2, PKC and S6). In vivo, 2a showed
with a variety of tailpiece amines using microwave irra-
diation or heating in pyridine to give final products 1
(Tables 1–3, 5, 6). Compound 21 (Table 1) was prepared
by addition–elimination reaction of intermediate 7 with
methyl-3-amino benzoate to give 8g, subsequent reac-
tion with 4-(2-aminoethyl)morpholine to afford 1d,
and finally hydrolysis of the methyl ester to give 21.
Compound 24 (Table 1) was synthesized in three steps
from intermediate 8j. Displacement of the fluoro group

N. Kaila et al. / Bioorg. Med. Chem. 15 (2007) 6425–6442
6427
Scheme 1. Synthesis of [1,7]naphthyridine-3-carbonitriles. Reagents and conditions: (a) i—KF, DMSO, 70 ꢁC, 18 h; ii—SnCl2–Æ2H2O, EtOAc; iii—
Boc2O, t-BuOH 40 ꢁC, 3 h, 53%, three steps; (b) i—n-BuLi, TMEDA, ether, ꢀ78 ꢁC; then CO2; ii—TMSCH2N2, CHCl3/MeOH, 51%, two steps; (c)
CH3CN, n-BuLi, THF, ꢀ78 ꢁC, 76%; (d) i—DMF–DMA, rt, 1 h; ii—oxalyl chloride, DMF, CH2Cl2, 59%, two steps; (e) RNH2, 2-ethoxyethanol or
EtOH, reflux; or RNH2, DME, 140 ꢁC microwave, 10 min; (f) R1NH2, THF or DMA or neat, 150–190 ꢁC microwave; or R1NH2, pyridine, 80 ꢁC.
with 4-(2-aminoethyl)morpholine generated 1e. The lat-
ter upon reduction with SnCl2 yielded 1f, which upon
treatment with methyl sulfonylchloride gave 24. Com-
pound 53 (Table 5) was prepared by N-methylation of
8p followed by reaction of intermediate 8u with (R)-1-
phenylethylamine. Compound 58 (Table 6) was
prepared by condensing intermediate 7 with N-Boc
piperidine to give 8r, subsequent reaction with pyridin-
3-ylmethylamine to give 1g, and removal of the Boc
group using HCl in dioxane to give 58.
33 (Table 3) started with commercially available para-
nitroaniline 9. The tailpiece was first installed by treating
9 with 4-(2-chloroethyl)morpholine, and the nitro com-
pound (10a) obtained was then reduced to 10b. The lat-
ter was alkylated by Michael addition–elimination to
give the 2-cyano-3-ethoxyacrylate 11b. Cyclization in
Dowtherm followed by chlorination gave 13b.
Nucleophilic displacement of chloride 13b with 4-phen-
oxyphenylamine yielded 33. Since this method was not
suitable for analoging at the 6 position it was not further
pursued. An alternate route using common intermediate
13a was used to synthesize all other such compounds.
Cyanoquinoline 13a7 was obtained in three steps from
Target compounds with the general structure 2 were
made according to Scheme 2.7 Synthesis of compound

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N. Kaila et al. / Bioorg. Med. Chem. 15 (2007) 6425–6442
Table 1. Tpl2 activity of 6-(2-morpholin-4-ylethylamino)-4-arylamino-
[1,7]napthyridine-3-carbonitriles
were not commercially available. Morpholin-4-ylacetal-
dehyde needed for the synthesis of 31 was prepared
in situ from its dimethyl acetal11 by heating with aque-
ous HCl overnight. Refluxing 2-picoline-N-oxide with
selenium dioxide in pyridine gave the 1-oxypyridine-2-
carbaldehyde12 required for the synthesis of 44 and 36.
3. Results and discussion
All compounds were studied for inhibition of isolated
Tpl2 enzyme via quantification of MEK phosphoryla-
tion in an ELISA format. For selected compounds,
EGFR selectivity was measured by looking at the inhi-
bition of EGFR kinase autophosphorylation in A431
cells overexpressing this enzyme, via quantification of
P-EGFR. Functional inhibition of TNF-a production
was measured using LPS-stimulated monocytes and
LPS-stimulated human blood. Compounds studied
in vivo were first assessed for selectivity against other ki-
nases (including p38a, CAMKII, PKC, PKA, MK2,
Src, S6K, IKKb, and Jnk-1).
Compound
X
Y
Tpl2 IC50 (lM)
1a
16
17
18
19
20
21
22
23
24
F
Cl
H
1.5
10.7
3.6
F
F
F
NHPh
NO2
H
H
7.6
7.0
H
I
7.1
H
COOH
CONH2
OPh
>10
>10
11.5
>10
H
H
H
NHSO2CH3
commercially available para-nitroaniline 9.8j Refluxing
intermediate 13a with various amines in ethanol or irra-
diation of these mixtures by microwave gave intermedi-
ates 14(a–d). The C-6 nitro group was reduced to give
intermediates 15(a–d). Tailpiece installation was carried
out by reductive amination of 15 with a variety of alde-
hydes to give 2 (Tables 3–5). Two of the aldehydes used
A brief SAR study of headpieces in the naphthyridine-3-
carbonitrile series (1)6 had revealed the 40-fluoro-30-
chloro aniline as the optimal arylamino substitution at
the 4-position. However, the effect of polar functionality
had not been explored. Thus, the headpiece moiety was
varied starting with the lead compound (1a) in this ser-
ies. As shown in Table 1, all these compounds showed
Table 2. Tpl2 activity and EGFR selectivity for 6-[(pyridine-3-ylmethyl)amino]-[1,7]naphthyridine-3-carbonitriles
Compound
Headpiece
Tpl2 IC50 (lM)
LPS-induced TNF-a inhibition
Monocyte IC50 (lM) Human blood IC50 (lM)
EGFR(A431) IC50 (lM)
1b
0.05
1.1
>20
20
<5
25
26
27
28
29
30
0.4
1.1
0.5
0.6
<5
>40
>40
>40
>40
>40
0.47
0.32
2.8
4.7
7.7
0.63
0.16
0.6
0.5
4.2
4.5

N. Kaila et al. / Bioorg. Med. Chem. 15 (2007) 6425–6442
6429
Table 3. Tpl2 activity of 4-arylamino-[1,7]naphthyridine-3-carbonitriles versus 4-arylaminoquinoline-3-carbonitriles
R1
R
X = N
Tpl2 IC50 (lM)
X = CH
Tpl2 IC50 (lM)
1a
1.5
31
1.0
32
1c
0.38
0.05
33
34
0.25
0.018
Table 4. Tpl2 activity of 4-cyclopentylaminoquinoline-3-carbonitrile and 4-tert-butylaminoquinoline-3-carbonitrile
Compound
R
Tpl2 IC50 (lM)
Compound
R1
Tpl2 IC50 (lM)
35
21
44
1.3
36
37
1.3
1.7
45
46
1.7
2.9
38
39
40
41
2.2
2.9
7.3
1.6
47
48
49
7.3
1.6
3
42
43
3
19.7

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N. Kaila et al. / Bioorg. Med. Chem. 15 (2007) 6425–6442
Table 5. SAR mapping of 4-alkylamino-[1,7]naphthyridine-3-carbonitriles
Compound
R1
R
X
N
Y
H
Tpl2 IC50 (lM)
30
0.16
50
27
51
52
53
54
55
56
57
C
H
2.6
0.32
2.4
N
C
H
H
N
N
N
N
N
N
H
0.92
>40
8
CH3
H
H
7.6
H
>40
5.2
H
diminished activity. This result is not unexpected since a
common feature seen in the ATP binding site for the
naphthyridine-3-carbonitriles8 and 1,3-quinazolines9 is
that the headpiece lies in a mostly lipophilic pocket bur-
ied deep within the active site with limited bulk toler-
ance. Next we looked at the cyclohexylamino
headpiece (Table 2). Here the more potent 3-pyridylm-
ethylamino group was used as a tailpiece. We found that
compound 26 was equipotent to 25. This result was a
pleasant surprise since an alkyl headpiece usually leads
to diminished activity in this core when applied to other
kinase targets. We expected that this unusal SAR would
grant selectivity over other kinases.
In the past, we had found that the [1,7]naphthyridine-3-
carbonitriles (1) and quinoline-3-carbonitriles (2) had
very similar SAR for Tpl2 inhibition. For example, in
Table 3, compounds 1a, 32, and 1c of the [1,7]naphthyri-
dine-3-carbonitrile series have similar Tpl2 activity to the
corresponding compounds 31, 33, and 34 in the quinoline-
3-carbonitriles series.7 Since the SAR tracks between the
two different cores, we made a library of quinoline carbo-
nitrile compounds combining the tert-butyl and cyclopen-
tylamino headpiece with our most potent tailpieces from
the naphthyridine carbonitrile series.6,7 (Table 4). Unfor-
tunately all compounds showed diminished activity.
These findings prompted us to do further SAR mapping
(Table 5). First, the quinoline analogs of naphthyridines
27 and 30 were evaluated. Both compounds (50 and 51)
showed 10-fold lower Tpl2 activity than their naphthyri-
dine analogs. It appears that N7 is important for activity
in the 4-alkylamino series. Next, the importance of the
4-NH group was tested. Methylating the C-4 nitrogen atom
in compound 52 abolished activity (53). One hypothesis
was that perhaps the 4-alkylamino analogs were binding
in a flipped mode compared to the 4-arylamino analogs,
with the key interaction made by the 3-cyano group
Further analoging of 26 and evaluation in other assays
showed that the 4-alkylamino substituted [1,7]naph-
thyridine-3-carbonitriles had moderate activity in the
Tpl2 enzymatic assay but improved activity in the
monocyte and human blood assays compared to the 4-
arylamino analogs (Table 2). Most importantly, these
compounds show excellent selectivity against other ki-
nases, particularly EGFR. In our earlier compounds,
lack of selectivity against EGFR had been a concern.

N. Kaila et al. / Bioorg. Med. Chem. 15 (2007) 6425–6442
6431
Scheme 2. Synthesis of quinoline-3-carbonitriles. Reagents and conditions: (a) 4-(2-chloroethyl)morpholine hydrochloride, 170 ꢁC, 14 h; (b) 10% Pd/
C, H2, rt, 24 h; (c) Cs2CO3, DMF, rt, 2 h; (d) Dowtherm A, 260 ꢁC, 4–5 h; (e) (COCl)2, DMF (cat.), DCE; or POCl3, reflux, 2-12 h; (f) RNH2, EtOH,
microwave or conventional heating; (g) SnCl2Æ2H2O, EtOH, microwave (or conventional heating) or Fe, NH4Cl, MeOH/H2O, heat; (h) R1CHO,
NaCNBH3, HOAc, EtOH.
replaced by one with the N7 atom. The 4-alkylamino
groups would then be behaving as the tailpieces. A few
compounds were made to test the hypothesis. The
6-(3-chloro-4-fluorophenylamino)-4-cyclohexylamino-
[1,7]naphthyridine-3-carbonitrile (54) and the 4-(3-
chloro-4-fluorophenylamino)-6-(alkylamino)-[1,7]naph-
thyridine-3-carbonitriles (55–57) did not show any
appreciable activity, indicating that our hypothesis was
probably incorrect.
evaluated for IV PK in rats (Table 7). Compound 30
showed good exposure and moderate clearance and
was taken forward for in vivo testing. It was examined
in the LPS-induced mouse model at an ip dose of
10 mg/kg. Compound 30 caused an 86% inhibition of
TNF-a production compared to vehicle in female
C57Bl/6 mice (Fig. 2). Upon screening for selectivity
over a panel of kinases in both in vitro and cellular as-
says (Table 8), it showed >200-fold selectivity against
MK2 and p38, two other kinases involved in TNF-a
production. It also had >200-fold selectivity against
MEK and other serine-threonine kinases. Table 8 also
shows a comparison of Tpl2 activity (in vitro and
in vivo), kinase selectivity profiles, and PK parameters
of lead candidate 30 versus our earlier lead 2a. Com-
pound 30 has an improved selectivity profile, lower
clearance, and improved exposure and bioavailability
in rats. In addition, 30 showed better in vivo activity
at 10 mg/kg (ip) compared to 2a at 25 mg/kg (ip) in
the LPS mouse model.
In an attempt to improve the activity of the alkylamino
series a few additional analogs were prepared (Table 6).
Insertion of heteroatoms into the alkyl headpiece was
not accepted (26 vs 58). Introduction of an alkynyl
group was tolerated (60). Although there was no signif-
icant improvement in activity, we were able to identify a
second compound (59) with comparable whole blood
activity to lead 30. This compound was twofold less po-
tent in the Tpl2 enzymatic and monocyte assay.
With this new alkylamino class of compounds we have
identified a series that is selective against EGFR and
demonstrates improved human blood activity
(Tables 2 and 6). Selected compounds were next
In summary, we have identified a novel class of selective
Tpl2 inhibitors, the 4-alkylamino-[1,7]naphthyridine-3-
carbonitriles. These compounds are potent in cellular

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N. Kaila et al. / Bioorg. Med. Chem. 15 (2007) 6425–6442
Table 6. Tpl2 activity and EGFR selectivity for 6-[(pyridine-3-ylmethyl)amino]-[1,7]naphthyridine-3-carbonitriles
Compound
Headpiece
Tpl2 IC50 (lM)
LPS-induced TNF-a inhibition
Monocyte IC50 (lM) Human blood IC50 (lM)
EGFR(A431) IC50 (lM)
58
59
60
>40
0.36
0.27
0.8
2
4.5
>40
NA
proved pharmacokinetic properties. On the basis of this
data the alkylamino-naphthyridine-carbonitriles have
been selected for further investigation.
Table 7. Rat PK parameters for potent 4-alkylamino naphthyridine
carbonitriles
Compound
Clearance
(mL/min/kg)
AUC iv/dose
(h kg ng/mL/mg)
29
27
30
50
28
30
328
732
4. Experimental
4.1. Chemistry
1041
Reactions were run using commercially available start-
ing materials and solvents, without further purification.
Proton NMR spectra were recorded at 400 MHz on a
Bruker AV-400 spectrometer using TMS (d 0.0) as
a reference. Combustion analyses were obtained using
a Perkin-Elmer Series II 2400 CHNS/O analyzer.
CHN analyses were carried out by Robertson-Microlit.
Where analyses are indicated by symbols of the ele-
ments, analytical results obtained for those elements
were within 0.4 of the theoretical values. Low resolu-
tion mass spectra were obtained using a Micromass
Platform Electrospray Ionization Quadrupole mass
spectrometer. High resolution mass spectra were
obtained using a Bruker APEXIII Fourier transform
ion cyclotron resonance (FT-ICR) mass spectrometer
equipped with an actively shielded 7 Tesla supercon-
ducting magnet (Magnex Scientific Ltd, UK) and an
external Bruker APOLLO electrospray ionization
6000
5000
4000
3000
2000
1000
0
71%
86%
2a
25 mg/kg
30
10 mg/kg
vehicle
Figure 2. Effect of compounds 2a (ip) and 30 (ip) on LPS/D-Gal-
induced TNF-a release.
and human blood assays indicating their functional abil-
ity to inhibit release of TNF-a. In addition, they show
good in vivo activity; a result, we believe, of their im-
Table 8. Kinase selectivity profile (IC50, lM), Tpl2 activity and PK parameters for compounds 30 and 2a
Compound
MEKa
p38a
Srca
CAMKIIa
MK2a
PKAa
PKCa
S6a
P-p38b
EGFRc
30
2a
>40
2.4
>40
30
38
NA
NA
NA
NA
NA
>40
5.84
>40
>10
>40
>10
NA
>20
0.03
2.11
Compound
Tpl2
IC50 (lM)
LPS-induced TNF-a inhibition
PK parameters in rat
Monocyte
IC50 (lM)
Human
blood IC50 (lM)
Clearance
(Cl/min/kg)
AUC iv/dose
(h kg ng/mL/mg)
Bioavailability (%)
30
2a
0.16
0.019
0.5
0.6
4.5
3.3
30
97
1041
171
14
1
NA, not active at 40 lM.
a Enzymatic assays.
b In human monocytes.
c In A431 cells.

N. Kaila et al. / Bioorg. Med. Chem. 15 (2007) 6425–6442
6433
(ESI) source. The microwave procedures were carried
out with a Biotage microwave. Preparative HPLC
was run using a Waters reverse phase preperative
HPLC with Xterra C18 5 lM, 30 · 100 mm column.
The flow rate was 40 mL/min and mobile phase A
was water; mobile phase B was CH3CN; triethylamine
or formic acid was used as a modifier.
4.1.1.1. 4-(3-Chloro-4-fluorophenylamino)-6-(2-mor-
pholin-4-yl-ethylamino)-[1,7]naphthyridine-3-carbonitrile
(1a). Step 1 Method B. 4-(3-Chloro-4-fluorophenylami-
no)-6-fluoro-[1,7]naphthyridine-3-carbonitrile (8a). 1H
NMR (400 MHz, DMSO-d6) d 7.32–7.49 (m, 1H) 7.53
(t, J = 9.0 Hz, 1H) 7.72 (dd, J = 6.3, 2.3 Hz, 1H) 8.16
(s, 1H) 8.69 (s, 1H) 9.08 (s, 1H) 10.14 (s, 1H). Step 2
1
Method C. 44% yield. H NMR (400 MHz, CDCl3) d
4.1.1. General procedure for the synthesis of [1,7]naph-
thyridine-3-carbonitriles (1). Step 1 Method A. In a
250 mL round-bottomed flask fitted with a con-
2.5 (m, 4H) 2.6 (d, J = 11.9 Hz, 2H) 3.2 (m, 2H) 3.7
(m, 4H) 5.5 (t, J = 5.1 Hz, 1H) 6.2 (s, 1H) 6.9 (s, 1H)
7.1 (m, 1H) 7.2 (t, J = 8.6 Hz, 1H) 7.3 (m, 1H) 8.5 (s,
denser,
4-chloro-6-fluoro-[1,7]naphthyridine-3-carbo-
1H)
9.1
(d,
J = 0.5 Hz,
(C21H20ClFN6O + 0.9H2O) C, H, N.
1H).
Anal.
nitrile (7,10 1.25 g, 6.02 mmol) and the appropriate
aniline (1.32 mmol) were taken up in 100 mL of 2-
ethoxyethanol or ethanol and heated at reflux for
1–10 h, until t.l.c. analysis showed complete disap-
pearance of the 4-chloronaphthyridine. After cooling
to room temperature, the reaction mixture was par-
titioned between EtOAc and 5% Na2CO3. The aque-
ous layer was extracted twice more with EtOAc, and
the combined organic layers were washed three times
with brine, dried over anhydrous MgSO4, filtered,
and evaporated to give 4-substituted-6-fluoro-
[1,7]naphthyridine-3-carbonitriles which were consid-
ered of sufficient purity to be used directly in the
4.1.1.2. 4-(3-Chloro-4-fluorophenylamino)-6-(pyridin-
3-ylmethylamino)-[1,7]naphthyridine-3-carbonitrile (1b).
Intermediate 8a was treated with pyridin-3-ylmethyl-
amine following the procedure for Step 2 Method D.
1
24% yield. H NMR (400 MHz, DMSO-d6) d 4.58 (d,
J = 6.3 Hz, 2H) 7.10 (s, 1H) 7.26–7.39 (m, 2H) 7.46 (t,
J = 9.0Hz, 1H) 7.54 (t, J = 6.3 Hz, 1H) 7.59 (dd,
J = 6.6, 2.8 Hz, 1H) 7.75 (dt, J = 7.8, 1.6 Hz, 1H) 8.29
(s, 1H) 8.44 (dd, J = 4.7, 1.6 Hz, 1H) 8.60 (d,
J = 1.8 Hz, 1H) 8.87 (s, 1H) 9.67 (s, 1 H). Anal.
(C21H14ClFN6) C, H, N.
next step. Method B. In
a microwave vial, 4-
chloro-6-fluoro-[1,7]naphthyridine-3-carbonitrile (1.25 g,
6.02 mmol) and the appropriate aniline (6.6 mmol)
were taken up in DME. The vial was crimp-sealed
and heated in a microwave reactor at 140 ꢁC for
10 min. The contents of the vials were transferred
to a separatory funnel and worked up as described
above for Method A, giving 4-substituted-6-fluoro-
[1,7]naphthyridine-3-carbonitriles of sufficient purity
to be used directly in the next step. Step 2 Method
C. A solution of the appropriate amine (1.7 mmol)
and 6-fluoro-[1,7]naphthyridine-3-carbonitrile (1 mmol)
(from Step 1) in pyridine (3.3 mL) was heated to
80 ꢁC for 7 days. Pyridine was removed by evapora-
tion and crude products purified. Method D. The
6-fluoro-[1,7]naphthyridine-3-carbonitrile (1 mmol)
(from Step 1) was mixed with the appropriate amine
(20 mmol) in a microwave vial. The sealed vial was
heated in a microwave reactor at 190 ꢁC for 5–
10 min (until t.l.c. analysis showed complete con-
sumption of starting material). The contents of the
vial were transferred into a separatory funnel and
partitioned between 300 mL each EtOAc and brine,
and the aqueous layer extracted twice more with
EtOAc. The combined organic extracts were washed
with brine (3·), dried over anhydrous MgSO4,
filtered, and evaporated. Method E. The 6-fluoro-
[1,7]naphthyridine-3-carbonitrile (1 mmol) (from Step 1)
was taken up in a microwave vial in 5.6 mL THF
or DMA, with the appropriate amine (20 mmol).
The sealed vial was heated in a microwave reactor
at 150 ꢁC for 1 h, until t.l.c. analysis showed com-
plete disappearance of the starting material. Then
the THF was removed under reduced pressure. The
crude products from Step 2 were purified by recrys-
tallization, flash chromatography over silica gel or
preparative HPLC (water/acetonitrile, triethylamine
or formic acid modifier).
4.1.1.3. 4-[(3-Chloro-4-fluorophenyl)amino]-6-{[(1R)-
1-phenylethyl]amino}-[1,7]naphthyridine-3-carbonitrile
(1c). Intermediate 8a was treated with (1R)-1-phenyleth-
ylamine following the procedure for Step 2 Method E.
35% yield. 1H NMR (400 MHz, MeOD) d 1.51 (d,
J = 14.3 Hz, 3H), 3.46–3.61 (m, 1H), 4.34–4.56 (br s,
1H), 5.06 (br s, 1H), 6.73 (d, J = 15.9 Hz, 1H), 7.54–
7.69 (m, 5H), 7.73–7.85 (m, 4H), 7.90 (d, J = 15.9 Hz,
1H). HRMS (ESI+) calcd for C23H17ClFN5 (M+H)
418.1230, found 418.1231.
4.1.1.4. 4-(4-Fluorophenylamino)-6-(2-morpholinoeth-
ylamino)-[1,7]naphthyridine-3-carbonitrile (16). Step 1
Method
A.
4-(4-Fluorophenylamino)-6-fluoro-
[1,7]naphthyridine-3-carbonitrile (8b). 1H NMR
(400 MHz, DMSO-d6) d 7.27–7.36 (m, 2H) 7.41–7.49
(m, 2H) 8.19 (s, 1H) 8.63 (s, 1H) 9.05 (s, 1H) 10.09 (s,
1H). Step
2
Method E. 26% yield. 1H NMR
(400 MHz, DMSO-d6) d 2.45 (br s, 4H) 2.58 (br s, 2H)
3.38 (q, J = 6.4 Hz, 2H) 3.52–3.64 (m, 4H) 6.60 (t,
J = 6.2 Hz, 1H) 7.06 (s, 1H) 7.27 (t, J = 8.8 Hz, 2H)
7.33–7.43 (m, 2H) 8.22 (s, 1H) 8.82 (s, 1H) 9.60 (s,
1H). HRMS (ESI+) calcd for C21H22FN6O (M+H)
393.1834, found 393.1833.
4.1.1.5. 4-(3,4-Difluorophenylamino)-6-(2-morpholino-
ethylamino)-[1,7]naphthyridine-3-carbonitrile (17). Step 1
Method
B.
6-Fluoro-4-(3,4-difluorophenylamino)-
[1,7]naphthyridine-3-carbonitrile (8c). 1H NMR
(400 MHz, DMSO-d6) d 7.18–7.36 (m, 1H) 7.43–7.65
(m, 2H) 8.17 (s, 1H) 8.69 (s, 1H) 9.08 (s, 1H) 10.16 (s,
1
1H). Step 2 Method E. 24% yield. H NMR (400 MHz,
DMSO-d6) d 2.45 (br s, 4H) 2.57 (t, J = 6.4 Hz, 2H)
3.34–3.42 (m, 2H) 3.51–3.64 (m, 4H) 6.66 (t, J = 5.7 Hz,
1H) 7.02 (s, 1H) 7.13–7.24 (m, 1H) 7.43–7.58 (m, 2H)
8.28 (s, 1H) 8.85 (s, 1H) 9.68 (s, 1H). HRMS (ESI+) calcd
for C21H21F2N6O (M+H) 411.1740, found 411.1737.

6434
N. Kaila et al. / Bioorg. Med. Chem. 15 (2007) 6425–6442
4.1.1.6. 6-(2-Morpholinoethylamino)-4-(4-(phenylami-
no)phenylamino)-[1,7]naphthyridine-3-carbonitrile (18).
ethylamino)-[1,7]naphthyridine-4-ylamino)benzoic acid
(21) in 54% overall yield. H NMR (400 MHz, MeOD)
1
Step 1 Method A. 6-Fluoro-4-(4-(phenylamino)phenyl-
amino)-[1,7]naphthyridine-3-carbonitrile (8d). H NMR
d 3.13–3.23 (m, 6H) 3.72 (t, J = 6.1 Hz, 2H) 3.84–3.90
(m, 4H) 7.09 (s, 1H) 7.48–7.55 (m, 2H) 7.90–7.96 (m,
2H) 8.29 (s, 1H) 8.90 (s, 1H). HRMS (ESI+) calcd for
C22H22N6O3 (M+H), 419.1826, found 419.1821.
1
(400 MHz, DMSO-d6) d 6.86 (tt, J = 7.3, 1.0 Hz, 1H)
7.07–7.17 (m, 4H) 7.21–7.32 (m, 4H) 8.21 (s, 1H) 8.37
(s, 1H) 8.58 (s, 1H) 9.02 (s, 1H) 9.99 (s, 1H). Step 2
1
Method E. 13% yield. H NMR (400 MHz, DMSO-d6)
4.1.1.10. 3-(3-Cyano-6-(2-morpholinoethylamino)-[1,7]-
naphthyridine-4-ylamino)benzamide (22). Step
d 2.45 (br s, 4H) 2.58 (t, J = 6.4 Hz, 2H) 3.37 (q,
J = 6.7 Hz, 2H) 3.55–3.64 (m, 4H) 6.53 (t, J = 5.4 Hz,
1H) 6.85 (t, J = 7.2 Hz, 1H) 7.05–7.17 (m, 5H) 7.17–
7.31 (m, 4H) 8.17 (s, 1H) 8.31 (s, 1H) 8.79 (s, 1H) 9.53
(s, 1H). HRMS (ESI+) calcd for C27H28N7O (M+H)
466.2350, found 466.2350.
1
Method A. 3-(3-Cyano-6-fluoro-[1,7]naphthyridine-
4-ylamino)benzamide (8h). 1H NMR (400 MHz,
DMSO-d6) d 7.00–7.08 (m, 1H) 7.49–7.59 (m, 2H)
7.81–7.92 (m, 2H) 8.04 (s, 1H) 8.21 (s, 1H) 8.70 (s,
1H) 9.09 (s, 1H) 10.18 (s, 1H). Step 2 Method E.
37% yield. 1H NMR (400 MHz, DMSO-d6) d 2.38–
2.48 (m, 4H) 2.52–2.59 (m, 2H) 3.34–3.41 (m, 2H)
3.53–3.62 (m, 4H) 6.64 (s, 1H) 7.05 (s, 1H) 7.40–7.52
(m, 3H) 7.73–7.81 (m, 2H) 8.02 (s, 1H) 8.29 (s, 1H)
8.86 (s, 1H) 9.69 (s, 1H). HRMS (ESI+) calcd for
C22H23N7O2 (M+H), 418.1986, found 419.1980.
4.1.1.7.
6-(2-Morpholin-4-yl-ethylamino)-4-(4-nitro
phenylamino)-[1,7]naphthyridine-3-carbonitrile (19). Step
1 Method A gave crude 8e, which was taken to the next
step. Step 2 Method E. 18% yield. H NMR (400 MHz,
1
DMSO-d6) d 2.35–2.42 (m, J = 3.8 Hz, 4H) 2.46–2.50
(m, 2H) 3.34–3.39 (m, 2H) 3.52–3.57 (m, 4H) 6.78 (s,
1H) 6.89 (t, J = 5.6 Hz, 1H) 7.23 (d, J = 9.1 Hz, 2H)
8.18–8.23 (m, J = 9.7, 3.0, 2.7 Hz, 2H) 8.54 (s, 1H)
8.98 (s, 1H) 10.12 (s, 1H). HRMS calcd for
C21H21N7O3 (M+H), 420.1779, found 420.1777.
4.1.1.11. 6-(2-Morpholinoethylamino)-4-(3-phenoxy-
phenylamino)-[1,7]naphthyridine-3-carbonitrile (23). Step
1
[1,7]naphthyridine-3-carbonitrile (8i). NMR
Method A. 6-Fluoro-4-(3-phenoxyphenylamino)-
1H
(400 MHz, DMSO-d6) d 6.94–7.18 (m, 6H) 7.33–7.51
(m, 3H) 8.15 (s, 1H) 8.68 (s, 1H) 9.05 (s, 1H) 10.12 (s,
4.1.1.8. 4-(3-Iodophenylamino)-6-(2-morpholinoethyl-
amino)-[1,7]naphthyridine-3-carbonitrile (20). Step
1
1H). Step 2
Method E. 27% yield. 1H NMR
Method A. 6-Fluoro-4-(3-iodophenylamino)-[1,7]naph-
thyridine-3-carbonitrile (8f). 1H NMR (400 MHz,
DMSO-d6) d 7.25 (t, J = 8.0 Hz, 1H) 7.40 (ddd,
J = 8.1, 2.2, 0.9 Hz, 1H) 7.67 (dt, J = 7.8, 1.7 Hz, 1H)
7.75 (t, J = 1.8 Hz, 1H) 8.15 (s, 1H) 8.72 (s, 1H) 9.09
(400 MHz, DMSO-d6) d 2.44 (br s, 4H) 2.57 (t,
J = 6.3 Hz, 2H) 3.34–3.43 (m, 2H) 3.49–3.64 (m, 4H)
6.64 (t, J = 5.8 Hz, 1H) 6.90 (dd, J = 3.8, 1.5 Hz, 2H)
7.00 (s, 1H) 7.03–7.11 (m, 3H) 7.14 (t, J = 7.5 Hz, 1H)
7.33–7.47 (m, 3H) 8.28 (s, 1H) 8.84 (s, 1H) 9.64 (s,
1H). HRMS (ESI+) calcd for C27H27N6O2 (M+H)
467.2190, found 467.2188.
1
(s, 1H) 10.10 (s, 1H). Step 2 Method E. 17% yield. H
NMR (400 MHz, DMSO-d6) d 2.43 (s, 4H) 2.55 (t,
J = 6.4 Hz, 2H) 3.36 (q, J = 6.2 Hz, 2H) 3.53–3.61 (m,
4H) 6.66 (t, J = 5.4 Hz, 1H) 6.97 (s, 1H) 7.19 (t,
J = 7.8 Hz, 1H) 7.25–7.31 (m, 1H) 7.57 (d, J = 8.1 Hz,
1H) 7.62–7.65 (m, 1H) 8.31 (s, 1H) 8.86 (s, 1H) 9.60
(s, 1H). Anal. (C21H21IN6O) C, H, N.
4.1.1.12. N-(3-(3-Cyano-6-(2-morpholinoethylamino)-
[1,7]naphthyridine-4-ylamino)phenyl)methanesulfonamide
(24). Compound 7 was treated with 3-nitroaniline
following the procedure for Step 1 Method A to give
6-fluoro-4-(3-nitrophenylamino)-[1,7]naphthyridine-3-
carbonitrile (8j). 1H NMR (400 MHz, DMSO-d6) d 7.74
(t, J = 8.1 Hz, 1H) 7.80–7.85 (m, 1H) 8.12–8.16 (m, 1H)
8.17 (s, 1H) 8.21 (t, J = 2.0 Hz, 1H) 8.80 (s, 1H) 9.14 (s,
1H) 10.35 (s, 1H). The above compound was treated
4.1.1.9.
3-(3-Cyano-6-(2-morpholinoethylamino)-
[1,7]naphthyridine-4-ylamino)benzoic acid (21). Com-
pound 7 was treated with methyl 3-aminobenzoate
following the procedure for Step 1 Method A to give
methyl 3-(3-cyano-6-fluoro-[1,7]naphthyridine-4-ylami-
no)benzoate (8g). 1H NMR (400 MHz, DMSO-d6) d
3.88 (s, 3H) 7.62 (t, J = 7.7 Hz, 1H) 7.64–7.69 (m,
J = 8.5, 1.9, 1.5 Hz, 1H) 7.88–7.93 (m, 2H) 8.19 (s,
1H) 8.72 (s, 1H) 9.10 (s, 1H) 10.22 (s, 1H). Above
compound was treated with 4-(2-aminoethyl)morphol-
ine following the procedure for Step 2 Method E to
give methyl 3-(3-cyano-6-(2-morpholinoethylamino)-
[1,7]naphthyridine-4-ylamino)benzoate (1d). 1H NMR
(400 MHz, DMSO-d6) d 2.40–2.46 (m, 4H) 2.55 (t,
J = 6.4 Hz, 2H) 3.34–3.39 (m, 2H) 3.54–3.60 (m, 4H)
3.87 (s, 3H) 6.67 (t, J = 5.2 Hz, 1H) 7.01 (s, 1H) 7.56
(dt, J = 4.4, 1.2 Hz, 2H) 7.79–7.83 (m, 2H) 8.32 (s, 1H)
8.87 (s, 1H) 9.73 (s, 1H). To 1d (2 mmol) in tetrahydro-
furan (12 mL) were added methyl alcohol (4.5 mL) and
lithium hydroxide (1 N, 4.5 mL). After 12 h the solvents
were evaporated and the crude mixture was purified by
preparative HPLC to give 3-(3-cyano-6-(2-morpholino-
with
4-(2-aminoethyl)morpholine
following
the
procedure for Step 2 Method E to give 6-(2-morpholino-
ethylamino)-4-(3-nitrophenylamino)-[1,7]naphthyridine-
3-carbonitrile (1e). 19% yield. 1H NMR (400 MHz,
DMSO-d6) d 2.37–2.48 (m, 4H) 2.52–2.59 (m, 2H)
3.35–3.41 (m, 2H) 3.54–3.61 (m, 4H) 6.71–6.80 (m,
1H) 6.96 (s, 1H) 7.66–7.71 (m, 2H) 8.00–8.05 (m, 1H)
8.06–8.08 (m, 1H) 8.40 (s, 1H) 8.92 (s, 1H) 9.89 (s,
1H). A mixture of 1e (120 mg, 0.29 mmol) and
SnCl2.2H2O (348 mg, 1.54 mmol) in ethyl alcohol
(12 mL) was heated to reflux for 2.5 h. After cooling
to room temperature, water (10 mL) was added followed
by sodium bicarbonate (500 mg). The mixture was stir-
red at room temperature for 1 h. Ethyl acetate extrac-
tion followed by column chromatography (3–5%
methyl alcohol in dichloromethane) yielded 43 mg of
4-(3-aminophenylamino)-6-(2-morpholinoethylamino)-
[1,7]naphthyridine-3-carbonitrile, (1f) 39% yield. 1H

N. Kaila et al. / Bioorg. Med. Chem. 15 (2007) 6425–6442
6435
NMR (400 MHz, DMSO-d6) 2.40–2.48 (m, 4H) 2.53–
2.60 (m, 2H) 3.34–3.40 (m, 2H) 3.54–3.63 (m, 4H)
5.18–5.27 (m, 2H) 6.39–6.47 (m, 3H) 6.57 (s, 1H) 7.01–
7.08 (m, 2H) 8.22 (s, 1H) 8.81 (s, 1H) 9.43 (s, 1H). Com-
pound 1f (111 mg, 0.29 mmol) was suspended in methy-
lene chloride (10 mL). Triethylamine (44 lL, 0.32 mmol)
was added and the mixture was cooled to 0 ꢁC. Meth-
ylsulfonyl chloride (24 lL, 0.31 mmol) was added and
the mixture was stirred at room temperature for 12 h.
Another 44 lL triethylamine and 48 lL methylsulfonyl
chloride were added and the reaction mixture was stirred
for 12 h. Work-up and preparative HPLC yielded
45.5 mg of product 24 (34% yield). 1H NMR
(400 MHz, DMSO-d6) d 2.39–2.47 (m, 4H) 2.56 (t,
J = 6.8 Hz, 2H) 2.99–3.03 (m, 3H) 3.27–3.42 (m, 2H)
3.55–3.61 (m, 4H) 6.63 (t, J = 6.1 Hz, 1H) 7.00–7.04
(m, 1H) 7.04 (s, 1H) 7.08 (d, J = 8.6 Hz, 1H) 7.12–7.16
(m, 1H) 7.36 (t, J = 8.3 Hz, 1H) 8.26 (s, 1H) 8.84 (s,
1H) 9.69 (s, 2H). HRMS (ESI+) calcd for C22H25N7O3S
(M+H), 468.1812, found 468.1810.
Method B. 4-Cyclopropylamino-6-fluoro-[1,7]naph-
thyridine-3-carbonitrile (8n). 1H NMR (400 MHz,
DMSO-d6) d 0.89–1.01 (m, 4H) 3.09–3.18 (m, J = 6.2,
3.9 Hz, 1H) 8.11 (s, 1H) 8.54 (s, 1H) 8.65 (s, 1H) 8.95
(s, 1H). Step 2 Method D. 22% yield. 1H NMR
(400 MHz, DMSO-d6) d 0.80–0.89 (m, J = 3.5, 3.5 Hz,
1H) 0.95 (dd, J = 6.8, 1.8 Hz, 2H) 4.54 (d, J = 6.3 Hz,
2H) 7.02–7.08 (m, 1H) 7.26–7.40 (m, 2H) 7.76 (dd,
J = 7.8, 1.5 Hz, 1H) 8.15 (d, J = 2.3 Hz, 1H) 8.23 (s,
1H) 8.43 (dd, J = 4.8, 1.5 Hz, 1H) 8.60 (d, J = 2.0 Hz,
1H) 8.73 (s, 1H). HRMS (ESI+) calcd for C18H16N6
(M+H), 317.1509, found 317.1504.
4.1.1.17. 4-tert-Butylamino-6-[(pyridin-3-ylmethyl)-
amino]-[1,7]naphthyridine-3-carbonitrile (29). Step
1
Method B. 4-tert-Butylamino-6-fluoro-[1,7]naphthyri-
1
dine-3-carbonitrile (8o). H NMR (400 MHz, DMSO-
d6) d 1.62 (s, 9H) 7.26 (s, 1H) 8.17 (s, 1H) 8.66 (s, 1H)
8.96 (s, 1H). Step 2 Method D. 37% yield. H NMR
1
(400 MHz, DMSO-d6) d 1.42–1.62 (m, 9H) 4.58 (d,
J = 5.8 Hz, 2H) 6.58 (s, 1H) 6.89 (s, 1H) 7.25–7.38 (dd,
J = 7.7, 4.9 Hz, 1H) 7.45–7.53 (m, 1H) 7.71–7.81 (m,
1H) 8.15 (s, 1H) 8.26 (s, 1H) 8.43 (d, J = 4.8 Hz, 1H)
8.62 (s, 1H). HRMS (ESI+) calcd for C19H20N6
(M+H), 333.1822, found 333.1824.
4.1.1.13. 4-Phenylamino-6-[(pyridin-3-ylmethyl)-ami-
no]-[1,7]naphthyridine-3-carbonitrile (25). Step 1 Method
B. 6-Fluoro-4-phenylamino-[1,7]naphthyridine-3-carbo-
nitrile (8k). 1H NMR (400 MHz, DMSO-d6) d 7.25–
7.33 (m, 3H) 7.42 (t, J = 7.7 Hz, 2H) 8.13 (s, 1H) 8.52
(s, 1H) 8.96 (s, 1H) 10.15 (s, 1H). Step 2 Method D.
4.1.1.18. 4-Cycloheptylamino-6-[(pyridin-3-ylmethyl)-
1
38% yield. H NMR (400 MHz, DMSO-d6) d 4.56 (d,
amino]-[1,7]naphthyridine-3-carbonitrile (30). Step
Method B. 4-Cycloheptylamino-6-fluoro-[1,7]naphthyri-
1
J = 6.3 Hz, 2H) 7.10–7.16 (m, 1H) 7.22–7.30 (m, 3H)
7.33 (dd, J = 7.6, 4.8 Hz, 1H) 7.41 (t, J = 7.8 Hz, 2H)
7.44–7.54 (m, 1H) 7.75 (d, J = 7.8 Hz, 1H) 8.25 (s, 1H)
8.39–8.48 (m, 1H) 8.59 (s, 1H) 8.85 (s, 1H) 9.62 (s,
1H). Anal. (C21H16N6 + 0.8 H2O) C, H, N.
1
dine-3-carbonitrile (8p). H NMR (400 MHz, DMSO-
d6) d 1.11–1.58 (m, 6H) 1.60–1.87 (m, 4H) 2.03–2.12
(m, 2H) 4.29 (s, 1H) 7.84–7.96 (m, 1H) 8.26 (s, 1H)
8.57 (s, 1H) 8.92 (s, 1H). Step 2 Method D. 33% yield.
1H NMR (400 MHz, DMSO-d6) d 1.46–1.84 (m, 10H)
1.95–2.11 (m, 2H) 4.38–4.52 (m, 1H) 4.58 (d,
J = 6.6 Hz, 2H) 7.11 (d, 1H) 7.22–7.45 (m, 3H) 7.77
(d, J = 7.8 Hz, 1H) 8.17 (s, 1H) 8.43 (dd, J = 4.7,
1.4 Hz, 1H) 8.61 (s, 1H) 8.71 (s, 1H). Anal.
(C22H24N6 + 2.0 H2O) C, H, N.
4.1.1.14. 4-Cyclohexylamino-6-[(pyridin-3-ylmethyl)-
amino]-[1,7]naphthyridine-3-carbonitrile (26). Step
Method B. 4-Cyclohexylamino-6-fluoro-[1,7]naphthyri-
1
1
dine-3-carbonitrile (8l). H NMR (400 MHz, DMSO-
d6) d 1.48–1.84 (m, 8H) 2.01–2.11 (m, 2H) 4.53 (d,
J = 5.1 Hz, 1H) 7.86 (d, J = 8.8 Hz, 1H) 8.28 (s, 1H)
8.57 (s, 1H) 8.92 (s, 1H). Step 2 Method D. 63% yield.
1H NMR (400 MHz, DMSO-d6) d 1.05–1.88 (m, 8H)
2.05 (s, 2H) 4.23 (s, 1H) 4.48–4.68 (m, 2H) 7.07–7.13
(m, 1H) 7.26–7.44 (m, 3H) 7.77 (d, J = 7.8 Hz, 1H)
8.17 (s, 1H) 8.43 (d, J = 6.1 Hz, 1H) 8.61 (s, 1H) 8.71
(s, 1H). Anal. (C21H22N6 + 1.8H2O) C, H, N.
4.1.1.19. 6-[(2-Morpholin-4-ylethyl)amino]-4-[(4-phen-
oxyphenyl)amino]-[1,7]naphthyridine-3-carbonitrile (32).
Step 1 Method A. 6-Fluoro-4-(4-phenoxyphenylami-
no)-[1,7]naphthyridine-3-carbonitrile (8q). 1H NMR
(400 MHz, DMSO-d6) d 7.1 (m, 2H) 7.1 (m, 3H) 7.4
(m, 4H) 8.2 (s, 1H) 8.6 (s, 1H) 9.1 (s, 1H) 10.1 (m,
1
1H) Step 2 Method C. 44% yield. H NMR (400 MHz,
4.1.1.15. 4-Cyclopentylamino-6-[(pyridin-3-ylmethyl)-
amino]-[1,7]naphthyridine-3-carbonitrile (27). Step 1
Method B. 4-Cyclopentylamino-6-fluoro-[1,7]naphthyri-
DMSO-d6) d 2.5 (m, 4H) 2.6 (t, J = 6.8 Hz, 2H) 3.4
(m, 2H) 3.6 (m, 4H) 6.6 (t, J = 5.6 Hz, 1H) 7.0 (m,
2H) 7.1 (m, 4H) 7.4 (m, 3H) 8.2 (s, 1H) 8.3 (s, 1H) 8.8
(s, 1H) 9.6 (s, 1H). HRMS (ESI+) calcd for
C27H26N6O2 (M+H), 467.2190, found 467.2196.
1
dine-3-carbonitrile (8m). H NMR (400 MHz, DMSO-
d6) d 1.59–1.66 (m, 2H) 1.74–1.88 (m, 4H) 2.03–2.12 (m,
2H) 4.69–4.74 (m, 1H) 7.88 (d, J = 7.3 Hz, 1H) 8.30 (s,
1H) 8.59 (s, 1H) 8.93 (s, 1H). Step 2 Method D. 50% yield.
1H NMR (400 MHz, DMSO-d6) d 1.71–1.84 (m, 2H)
2.02–2.12 (m, 1H) 3.32 (s, 6H) 4.58 (d, J = 6.3 Hz, 2H)
7.06–7.14 (m, 1H) 7.28–7.36 (m, 2H) 7.41 (d, J = 7.6 Hz,
1H) 7.77 (dd, J = 7.8, 1.8 Hz, 1H) 8.19 (s, 1H) 8.43 (dd,
J = 4.7, 1.6 Hz, 1H) 8.61 (d, J = 2.0 Hz, 1H) 8.72 (s,
1H). Anal. (C20H20N6 + 1.2H2O) C, H, N.
4.1.1.20. 4-Cycloheptylamino-6-(R)-1-phenylethylami-
no-[1,7]naphthyridine-3-carbonitrile (52). Intermediate 8p
was treated with (R)-1-phenylethylamine following the
1
procedure for Step 2 Method D. 7% yield. H NMR
(400 MHz, DMSO-d6) d 1.41 (d, J = 6.8 Hz, 3H)
1.46–1.89 (m, 12H) 4.32–4.52 (m, 1H) 4.89–5.10 (m,
1H) 7.10–7.21 (dd, J = 7.3, 7.3 Hz, 1H) 7.22–7.44
(m, 5H) 7.85 (s, 1H) 8.18 (s, 1H) 8.38 (s, 2H). HRMS
(ESI+) calcd for C24H27N5 (M+H), 386.2339, found
386.2335.
4.1.1.16. 4-Cyclopropylamino-6-[(pyridin-3-ylmethyl)-
amino]-[1,7]naphthyridine-3-carbonitrile (28). Step
1

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N. Kaila et al. / Bioorg. Med. Chem. 15 (2007) 6425–6442
4.1.1.21. 4-(Cycloheptylmethylamino)-6-(R)-1-phenyl
ethylamino-[1,7]naphthyridine-3-carbonitrile (53). To a
following the procedure for Step 2 Method D. 19%
yield. H NMR (400 MHz, DMSO-d6) d 1.27–2.03 (m,
1
solution
of
4-(cycloheptylmethylamino)-6-fluoro-
12H) 3.66–3.83 (m, 1H) 6.72–6.87 (m, 2H) 7.27–7.38
(m, 1H) 7.45 (t, J = 9.1 Hz, 1H) 7.52–7.62 (m, 1H)
8.26 (s, 1H) 8.83 (s, 1H) 9.61 (s, 1H). Anal.
(C22H21ClFN5 + 0.6CH3OH) C, H, N.
[1,7]naphthyridine-3-carbonitrile (8p, 0.2 g, 0.7 mmol)
in DMF (3 mL) was added NaH (60% in mineral oil,
40 mg, 0.8 mmol) at room temperature. After stirring
for 10 min 0.2 mL of MeI was added. The reaction mix-
ture was stirred for 4 h, then quenched with water. The
aqueous solution was filtered to give 4-(cycloheptylm-
ethylamino)-6-fluoro-[1,7]naphthyridine-3-carbonitrile
(8u) as a beige solid (150 mg, 71% yield). 1H NMR
(400 MHz, DMSO-d6) d 1.50–1.84 (m, 12H) 3.32 (s,
3H) 7.69 (d, J = 2.0 Hz, 1H) 8.14 (s, 1H) 8.47 (s, 1H).
Intermediate 8u was treated with (R)-1-phenylethyl-
amine following the procedure for Step 2 Method D
4.1.1.26. 4-(Piperidin-4-ylamino)-6-[(pyridin-3-ylmeth-
yl)-amino]-[1,7]naphthyridine-3-carbonitrile (58). Compound
7 was treated with 4-aminopiperidine-1-carboxylic acid
tert-butyl ester following the procedure for Step 1 Method
B to give 4-(3-cyano-6-fluoro-[1,7]naphthyridin-4-yla-
mino)piperidine-1-carboxylic acid tert-butyl ester (8r).
Crude 8r was treated with pyridin-3-ylmethylamine
following the procedure for Step 2 Method D to give 4-
{3-cyano-6-[(pyridin-3-ylmethyl)-amino]-[1,7]naphthyridine-
4-ylamino}-piperidine-1-carboxylic acid tert-butyl ester
(1g). Crude 1g (0.285 mg, 0.621 mmol) from above was
treated with 4 M HCl in dioxane (21 mL) at 0 ꢁC. The
reaction mixture was stirred overnight at 0 ꢁC and then
purified using preparative HPLC to give compound 58.
1
to give compound 53. 10% yield. H NMR (400 MHz,
DMSO-d6) d 1.37–1.85 (m, 12H) 1.84–2.17 (m, 2H)
3.32 (s, 3H) 4.31–4.49 (m, J = 4.0 Hz, 1H) 4.96–5.08
(m, J = 7.8 Hz, 1H) 6.89–6.99 (m, 1H) 7.10–7.32 (m,
5H) 7.47 (d, J = 7.3 Hz, 2H) 8.13 (s, 1H) 8.66 (s, 1H).
HRMS (ESI+) calcd for C25H29N5 (M+H), 400.2496,
found 400.2499.
1
10% yield. H NMR (400 MHz, DMSO-d6) d 1.61–1.86
(m, 2H) 2.03 (d, J = 13.1 Hz, 2H) 2.71 (t, J = 12.4 Hz,
2H) 3.18 (d, J = 12.4 Hz, 3H) 4.33 (s, 1H) 4.58 (d,
J = 6.3 Hz, 2H) 7.13–7.20 (m, 1H) 7.26–7.39 (m, 2H)
7.53 (d, J = 7.3 Hz, 1H) 7.77 (d, J = 7.8 Hz, 1H) 8.19 (s,
1H) 8.29 (s, 1H) 8.42 (d, J = 4.6 Hz, 1H) 8.61 (s, 1H)
8.73 (s, 1H). HRMS (ESI+) calcd for C20H21N7 (M+H),
360.1931, found 360.1925.
4.1.1.22. 6-(3-Chloro-4-fluorophenylamino)-4-(cyclo-
pentylamino)-[1,7]naphthyridine-3-carbonitrile (54).
A
mixture of 3-chloro-4-fluorobenzeneamine (550 mg,
3.78 mmol), 4-(cyclopentylamino)-6-fluoro-[1,7]naph-
thyridine-3-carbonitrile (8m, 28 mg, 0.1 mmol), and ce-
sium carbonate (506 mg, 1.55 mmol, 15.5 equiv) in
DMF (4 mL) was heated to 200 ꢁC in a microwave tube
for 1 h using a microwave reactor. Preparative HPLC
purification yielded 15 mg product (36% yield). 1H
NMR (400 MHz, DMSO-d6) d 1.52–1.68 (m, 2H)
1.70–1.91 (m, 4H) 1.99–2.16 (m, 2H) 4.58–4.76 (m,
1H) 7.33 (t, J = 9.1 Hz, 1H) 7.45–7.56 (m, 1H) 7.76 (s,
1H) 7.80 (d, J = 7.1 Hz, 1H) 7.87–7.97 (m, 1H) 8.32 (s,
1H) 8.90 (s, 1H) 9.64 (s, 1H). HRMS (ESI+) calcd for
C20H17ClFN5 (M+H), 382.1229, found 382.1237.
4.1.1.27. 4-(1,1-Dimethylpropylamino)-6-[(pyridin-3-
ylmethyl)amino]-[1,7]naphthyridine-3-carbonitrile (59).
Step 1 Method B. 4-(1,1-Dimethylpropylamino)-6-flu-
oro-[1,7]naphthyridine-3-carbonitrile (8s). 1H NMR
(400 MHz, DMSO-d6) d 0.90 (t, J = 7.5 Hz, 3H) 1.56
(s, 6H) 2.02 (q, J = 7.3 Hz, 2H) 7.12 (s, 1H) 8.18 (s,
1H) 8.65 (s, 1H) 8.97 (s, 1H). Step 2 Method D. The
reaction mixture was heated at 140 ꢁC for 10 min. 19%
yield. 1H NMR (400 MHz, DMSO-d6) d 0.82 (t,
J = 7.5 Hz, 3H) 1.47 (s, 6H) 1.79–2.02 (m, 2H) 4.59 (d,
J = 5.8 Hz, 2H) 6.44 (s, 1H) 6.90 (s, 1H) 7.32 (dd,
J = 8.1, 4.8 Hz, 1H) 7.49 (t, J = 6.4 Hz, 1H) 7.76 (d,
J = 7.8 Hz, 1H) 8.25 (s, 1H) 8.43 (d, J = 3.5 Hz, 1H)
8.61 (d, J = 1.3 Hz, 1H) 8.77 (s, 1H). Anal.
(C20H22N6 + 1.3H2O) C, H, N.
4.1.1.23. 4-(3-Chloro-4-fluorophenylamino)-6-cyclo-
pentylamino-[1,7]naphthyridine-3-carbonitrile (55). Step
1 Method A. 4-(3-Chloro-4-fluorophenylamino)-6-flu-
oro-[1,7]naphthyridine-3-carbonitrile (8a). Step 2 Meth-
od D. 41% yield. 1H NMR (400 MHz, DMSO-d6) d
1.43–1.63 (m, 4H) 1.65–1.78 (m, 2H) 1.91–2.07 (m,
J = 9.4 Hz, 2H) 3.84–4.02 (m, 1H) 6.81–6.98 (m, 2H)
7.30–7.40 (m, 1H) 7.41–7.53 (m, 1H) 7.56–7.65 (m,
1H) 8.26 (s, 1H) 8.83 (s, 1H) 9.63 (s, 1H). Anal.
(C20H17ClFN5 + 1.1 H2O) C, H, N.
4.1.1.28. 4-(1,1-Dimethylprop-2-ynylamino)-6-[(pyri-
din-3-ylmethyl)-amino]-[1,7]naphthyridine-3-carbonitrile
(60). Step 1 Method B. The reaction mixture was
heated at 140 ꢁC for thirty minutes to give 8t. Crude
8t was taken forward to the next step. Step 2 Method
D. The reaction mixture was heated at 140 ꢁC for ten
minutes. 10% yield. 1H NMR (400 MHz, DMSO-d6)
d 1.80 (s, 6H) 3.50–3.61 (m, 1H) 4.57 (d, J = 6.6 Hz,
2H) 6.95 (d, J = 14.4 Hz, 2H) 7.29–7.37 (m, 1H)
7.52 (t, J = 6.4 Hz, 1H) 7.77 (dd, J = 7.8, 2.0 Hz, 1H)
8.31 (s, 1H) 8.38–8.45 (dd, J = 4.7, 1.6 Hz, 1H) 8.62
(s, 1H) 8.80 (s, 1H). Anal. (C20H18N6 + 0.4H2O) C, H,
N.
4.1.1.24. 6-tert-Butylamino-4-(3-chloro-4-fluorophe-
nylamino)-[1,7]naphthyridine-3-carbonitrile (56). Inter-
mediate 8a was treated with tert-butylamine following
the procedure for Step 2 Method E. 21% yield. 1H
NMR (400 MHz, DMSO-d6) d 1.32–1.51 (m, 9H)
6.40–6.48 (m, 1H) 6.94 (s, 1H) 7.28 (s, 1H) 7.37–7.59
(m, 2H) 8.27 (s, 1H) 8.85 (s, 1H) 9.67 (s, 1H). HRMS
(ESI+) calcd for C19H17ClFN5 (M+H), 370.1229, found
370.1229.
4.1.1.25. 4-(3-Chloro-4-fluoro-phenylamino)-6-cyclo-
heptylamino-[1,7]naphthyridine-3-carbonitrile (57). Inter-
mediate 8a was treated with cycloheptylamine
4.1.2. General procedure for the synthesis of quinoline-3-
carbonitriles (2). Step 1 Method F. In a microwave vial,
4-chloro-6-nitro-quinoline-3-carbonitrile (13a,8j 0.4 g,

N. Kaila et al. / Bioorg. Med. Chem. 15 (2007) 6425–6442
6437
1.71 mmol) and the appropriate amine (2.05 mmol) were
taken up in 2 mL EtOH. The vial was crimp-sealed and
heated in a microwave reactor at 150 ꢁC for 45 min. The
solvent was removed and the residue was partitioned be-
tween ether and H2O. This gave a suspension, which was
filtered, washed with H2O, and dried on a high vacuum
pump overnight to give a solid, which was used in the
next step without purification.
(s, 1H). Step 2 Method I was used to obtain 6-amino-
4-(3-chloro-4-fluorophenylamino)quinoline-3-carbonit-
rile (15a) in 90% yield. H NMR (400 MHz, DMSO-d6)
1
d 5.78 (s, 2H) 7.12–7.19 (m, 2H) 7.25 (dd, J = 8.8,
2.3 Hz, 1H) 7.34–7.42 (m, 2H) 7.70 (d, J = 9.1 Hz, 1H)
8.34 (s, 1H) 9.36 (s, 1H). 4-(2,2-Dimethoxyethyl)mor-
11
pholine
was heated with 37% aqueous HCl (0.6 mL
for 0.5 mmol) overnight at 70 ꢁC. This reaction mixture
was treated with intermediate 15a following the proce-
dure for Step 3 Method J. 60% yield. 1H NMR
(400 MHz, MeOD) d 2.49 (m, 2H), 2.51–2.53 (m, 2H),
3.18–3.30 (m, 4H), 3.49–3.52 (m, 2H), 3.57 (m, 2H),
6.88 (s, 1H), 7.02–7.23 (m, 3H), 7.25–7.33 (m, 1H),
7.58 (d, J = 9.1 Hz, 1H), 8.18 (s, 1H). HRMS (ESI+)
calcd for C22H21ClFN5O (M+H), 426.1491; found
426.1497.
Method G. 4-Chloro-6-nitroquinoline-3-carbonitrile
(13a,8j 2.33 g, 10 mmol) and the appropriate amine
(1.74 g, 12 mmol) were suspended in ethanol (60 mL)
and heated at reflux for 3 h or until t.l.c. analysis showed
that the reaction was complete. After cooling, the sol-
vent was removed in vacuo and the residue was stirred
with ether/saturated aqueous NaHCO3 (100 mL/
75 mL) for 2.5 h to triturate. The solid was collected
by suction filtration and used in the next step.
4.1.2.2. 4-(3-Chloro-4-fluorophenylamino)-6-[(pyridin-
3-ylmethyl)amino]quinoline-3-carbonitrile (34). Interme-
diate 15a was treated with pyridine-3-carbaldehyde
following the procedure for Step 3 Method J. 95% yield.
1H NMR (400 MHz, DMSO-d6) d 6.35 (dd, J = 3.3,
0.8 Hz, 1H) 6.39 (dd, J = 3.3, 1.8 Hz, 1H) 6.79 (t,
J = 5.6 Hz, 1H) 7.21–7.27 (m, 3H) 7.35 (dd, J = 9.1,
2.5 Hz, 1H) 7.43 (t, J = 9.0 Hz, 1H) 7.48 (dd, J = 6.7,
2.7 Hz, 1H) 7.60 (dd, J = 1.8, 0.8 Hz, 1H) 7.70 (d,
J = 9.1 Hz, 1H) 8.33 (s, 1H). HRMS (ESI+) calcd for
C21H14ClFN4O (M+H) 393.0913, found 393.0917.
Step 2 Method H. In a microwave vial, 4-amino-6-nitro-
quinoline-3-carbonitrile (14, 0.97 mmol) and SnCl2Æ2-
H2O (1.09 g, 4.83 mmol) were taken up in 2 mL
EtOH. The vial was sealed and heated in a microwave
reactor at 110 ꢁC for 10 min, until LC/MS analysis
showed complete disappearance of the nitroquinoline.
The reaction mixture was poured into ice water, neutral-
ized with saturated NaHCO3, and extracted with EtOAc
(2· 150 mL). The combined organic layers were washed
with brine, dried over anhydrous Na2SO4, filtered, and
evaporated. These products were used in the next step
without purification.
4.1.2.3. 6-Benzylamino-4-cyclopentylaminoquinoline-
3-carbonitrile (35). Step 1 Method F was used to synthe-
size 4-cyclopentylamino-6-nitroquinoline-3-carbonitrile
1
Method I. 4-Amino-6-nitroquinoline-3-carbonitrile (14,
7.29 mmol) was suspended in ethanol (85 mL), then
SnCl2Æ2H2O (8.3 g, 36.5 mmol) was added and the reac-
tion mixture heated at reflux for 2.5 h or until complete
by t.l.c. The reaction mixture was diluted with 100 mL
of water, and then solid NaHCO3 was added until the
pH was basic (ꢁ11). The solution was extracted with
chloroform, washed with brine, treated with activated
charcoal, dried over MgSO4, and solvent evaporated.
These products were used in the next step without
purification.
(14b) in 66% yield. H NMR (400 MHz, DMSO-d6) d
1.59–1.68 (m, 2H) 1.76–1.93 (m, 4H) 2.05–2.15 (m,
2H) 4.72–4.80 (m, 1H) 7.97 (d, J = 9.1 Hz, 1H) 8.40 (s,
1H) 8.46 (dd, J = 9.1, 2.3 Hz, 1H) 8.66 (s, 1H) 9.56 (d,
J = 2.3 Hz, 1H). Step 2 Method H was used to obtain
6-amino-4-cyclopentylaminoquinoline-3-carbonitrile
1
(15b) in 50% yield. H NMR (400 MHz, DMSO-d6) d
1.60 (s, 2H) 1.77 (d, J = 3.0 Hz, 4H) 2.01–2.11 (m, 2H)
4.64 (d, J = 5.8 Hz, 1H) 5.47 (s, 2H) 7.00–7.06 (m,
J = 7.6 Hz, 1H) 7.11–7.16 (m, 1H) 7.25 (d, J = 1.8 Hz,
1H) 7.54 (d, J = 8.6 Hz, 1H) 8.15 (s, 1H). Step 3 Method
1
J gave product 35 in 90% yield. H NMR (400 MHz,
Step 3 Method J. In a 50 mL round-bottomed flask, the
6-aminoquinoline-3-carbonitrile (15, 0.29 mmol) was ta-
ken up in 3 mL EtOH, and the appropriate aldehyde
(0.37 mmol) was added. Acetic acid was added to bring
the pH of the solution to 4, and the mixture stirred for
15 min. NaCNBH3 (12.5 mg, 0.20 mmol) was then
added, and the reaction mixture allowed to stir from 4
to 24 h at 25–30 ꢁC. Ethanol was then removed under
reduced pressure and the crude product was purified
by preparative HPLC.
DMSO-d6) d 1.57–1.67 (m, 2H) 1.73–1.83 (m, 4H)
2.02–2.11 (m, 2H) 4.42–4.47 (m, J = 5.8 Hz, 2H) 4.62–
4.70 (m, 1H) 6.72 (t, J = 5.7 Hz, 1H) 7.05–7.13 (m,
2H) 7.22–7.28 (m, 2H) 7.33 (t, J = 7.5 Hz, 2H) 7.44 (d,
J = 7.3 Hz, 2H) 7.55 (d, J = 8.8 Hz, 1H) 8.21 (s, 1H).
HRMS (ESI+) calcd for C18H20N6 (M+H), 321.1822;
found 321.1822.
4.1.2.4.
4-Cyclopentylamino-6-[(1-oxypyridin-2-yl-
methyl)amino]quinoline-3-carbonitrile (36). Intermediate
15b was treated with 1-oxypyridine-2-carbaldehyde12
following the procedure for Step 3 Method J. 28% yield.
1H NMR (400 MHz, DMSO-d6) d 1.59 (s, 2H) 1.69–1.78
(m, 4H) 2.00–2.08 (m, 2H) 4.59–4.68 (d, J = 6.6 Hz, 2H)
6.76 (t, J = 6.8 Hz, 1H) 7.03 (d, J = 7.8 Hz, 1H) 7.20–
7.43 (m, 5H) 7.59 (d, J = 8.8 Hz, 1H) 8.19 (d,
J = 7.3 Hz, 2H) 8.32 (d, J = 6.3 Hz, 1H). HRMS
(ESI+) calcd for C21H21N5O (M+H), 360.1819; found
360.1819.
4.1.2.1. 4-[(3-Chloro-4-fluorophenyl)amino]-6-[(2-mor-
pholin-4-ylethyl)amino]quinoline-3-carbonitrile (31). Step
1 Method G was used to synthesize 4-(3-chloro-4-flu-
oro-phenylamino)-6-nitroquinoline-3-carbonitrile (14a)
1
in 80% yield. H NMR (400 MHz, DMSO-d6) d 7.28–
7.35 (m, 1H) 7.47 (t, J = 9.0 Hz, 1H) 7.56 (dd, J = 6.7,
2.7 Hz, 1H) 7.99 (d, J = 9.1 Hz, 1H) 8.49 (dd, J = 9.4,
2.5 Hz, 1H) 8.64 (s, 1H) 9.47 (d, J = 2.3 Hz, 1H) 10.72

6438
N. Kaila et al. / Bioorg. Med. Chem. 15 (2007) 6425–6442
4.1.2.5. 6-(2-Cyanobenzylamino)-4-cyclopentylamino-
quinoline-3-carbonitrile (37). Intermediate 15b was trea-
ole-2-carbaldehyde following the procedure for Step 3
Method J. 35% yield. 1H NMR (400 MHz, DMSO-d6) d
1.60–1.66 (m, 2H) 1.74–1.84 (m, 4H) 2.08–2.16 (m, 2H)
3.76 (s, 3H) 4.68 (d, J = 5.1 Hz, 3H) 6.65 (t, J = 5.2 Hz,
1H) 6.98 (d, J = 7.8 Hz, 1H) 7.14 (d, J = 2.3 Hz, 1H)
7.22 (dd, J = 9.2, 2.2 Hz, 1H) 7.27 (d, J = 1.8 Hz, 1H)
7.55 (d, J = 1.8 Hz, 1H) 7.61 (d, J = 9.1 Hz, 1H) 8.23 (s,
1H) 8.30 (s, 1H). HRMS (ESI+) calcd for C20H22N6
(M+H), 347.1979; found 347.1974.
ted with 2-cyanobenzaldehyde following the procedure
for Step 3 Method J. 55% yield. H NMR (400 MHz,
1
DMSO-d6) d 1.55–1.65 (m, 2H) 1.66–1.82 (m, 4H)
1.98–2.10 (m, 2H) 4.58–4.68 (m, 3H) 6.69–6.76 (dd,
J = 5.8, 5.8 Hz, 1H) 6.97 (d, J = 7.8 Hz, 1H) 7.11 (d,
J = 2.0 Hz, 1H) 7.24 (dd, J = 9.1, 2.5 Hz, 1H) 7.45–
7.52 (m, 1H) 7.56–7.70 (m, 3H) 7.87 (d, J = 7.8 Hz,
1H) 8.19 (s, 1H). Anal. (C23H21N5 + 1.0 H2O) C, H, N.
4.1.2.11. 4-Cyclopentylamino-6-(2-morpholin-4-yleth-
ylamino)quinoline-3-carbonitrile (43). 4-(2,2-Dimethoxy-
4.1.2.6. 6-(3-Cyanobenzylamino)-4-cyclopentylamino-
quinoline-3-carbonitrile (38). Intermediate 15b was trea-
ted with 3-cyanobenzaldehyde following the procedure
11
ethyl)morpholine was heated with 37% aqueous HCl
(0.6 mL for 0.5 mmol) overnight at 70 ꢁC. This reaction
mixture was treated with intermediate 15b following the
procedure for Step 3 Method J. 22% yield. H NMR
1
for Step 3 Method J. 95% yield. H NMR (400 MHz,
1
DMSO-d6) d 1.54–1.64 (m, 2H) 1.68–1.79 (m, 4H) 2.00–
2.11 (m, 2H) 4.49–4.54 (m, J = 6.1 Hz, 2H) 4.59–4.66
(m, 1H) 6.79–6.86 (dd, J = 5.8, 5.8 Hz, 1H) 6.92 (d,
J = 6.3 Hz, 1H) 7.03 (d, J = 2.0 Hz, 1H) 7.21 (dd, J =
9.1, 2.3 Hz, 1H) 7.51–7.59 (m, 2H) 7.74 (dd, J = 18.8,
8.0 Hz, 2H) 7.90 (s, 1H) 8.17 (s, 1H). HRMS (ESI+) calcd
for C23H21N5 (M+H), 368.1870; found 368.1866.
(400 MHz, DMSO-d6) d 1.59–1.65 (m, 2H) 1.75–1.83
(m, 4H) 2.05–2.14 (m, 2H) 2.43–2.48 (m, J = 8.1 Hz,
4H) 2.55–2.60 (m, 2H) 3.27–3.34 (m, 4H) 3.57–3.64
(m, 4H) 4.64–4.70 (m, 1H) 5.87 (t, J = 4.2 Hz, 1H)
7.03–7.09 (m, 2H) 7.21 (dd, J = 9.1, 2.3 Hz, 1H) 7.54
(d, J = 8.8 Hz, 1H) 8.16 (s, 1H). HRMS (ESI+) calcd
for C21H27N5O (M+H), 366.2288; found 366.2291.
4.1.2.7. 4-Cyclopentylamino-6-(3-methanesulfonyl-
benzylamino)quinoline-3-carbonitrile (39). Intermediate
15b was treated with 3-methanesulfonylbenzaldehyde
following the procedure for Step 3 Method J. 40% yield.
1H NMR (400 MHz, DMSO-d6) d 1.55–1.63 (m, 2H)
1.69–1.81 (m, 4H) 2.01–2.11 (m, 2H) 3.18 (s, 3H) 4.58
(d, J = 5.8 Hz, 2H) 4.60–4.66 (m, 1H) 6.83 (t,
J = 6.1 Hz, 1H) 6.96 (d, J = 7.3 Hz, 1H) 7.10 (d,
J = 2.3 Hz, 1H) 7.23 (dd, J = 8.8, 2.3 Hz, 1H) 7.54–
7.64 (m, 2H) 7.77–7.84 (m, 2H) 8.00 (t, J = 1.6 Hz,
1H) 8.17 (s, 1H). Anal. (C23H24N4O2S + 1.3H2O) C,
H, N.
4.1.2.12. 4-(tert-Butylamino)-6-{[(1-oxidopyridin-2-yl)-
methyl]amino}quinoline-3-carbonitrile (44). Step
1
Method F was used to synthesize 4-cyclopentylamino-
6-nitroquinoline-3-carbonitrile (14c). The crude product
was taken to the next step. Step 2. To a solution of 14c
(800 mg, 2.95 mmol) in methanol (5 mL) and water
(2 mL) were added iron powder (935 mg, 16.7 mmol)
and ammonium chloride (1.47 g, 27.7 mmol). The mix-
ture was stirred vigorously and heated to 75 ꢁC for
4 h. After allowing the mixture to cool to rt, EtOAc
(25 mL) and saturated aqueous NaHCO3 (10 mL) were
added. The organic layer was dried over MgSO4 and
concentrated to afford 6-amino-4-(tert-butylami-
no)quinoline-3-carbonitrile (15c) as a tan solid in quan-
titative yield. The product was used without further
4.1.2.8. 4-[(3-Cyano-4-cyclopentylaminoquinolin-6-yl-
amino)methyl]benzenesulfonamide (40). Intermediate
15b was treated with 4-formyl-benzenesulfonamide fol-
lowing the procedure for Step 3 Method J. 86% yield.
1H NMR (400 MHz, DMSO-d6) d 1.61 (t, J = 4.8 Hz,
2H) 1.71–1.81 (m, 4H) 2.01–2.11 (m, 2H) 4.53 (d,
J = 6.1 Hz, 2H) 4.61–4.67 (m, 1H) 6.79 (t, J = 5.9 Hz,
1H) 6.98 (d, J = 7.8 Hz, 1H) 7.09 (d, J = 2.5 Hz, 1H)
7.21 (dd, J = 9.1, 2.3 Hz, 1H) 7.29 (s, 2H) 7.56 (d, J =
9.1 Hz, 1H) 7.61 (d, J = 8.3 Hz, 2H) 7.75–7.80 (m, 2H)
8.17 (s, 1H). Anal. (C22H23N5O2S + 1.0 CH3OH) C, H, N.
1
purification. H NMR (400 MHz, DMSO-d6) d 1.52 (s,
9H) 5.67 (br s, 2H), 5.99 (br s, 1H), 7.12 (s, 1H), 7.69
(d, J = 9.1 Hz, 1H) 8.28 (s, 1H). Intermediate 15c was
treated with 1-oxypyridine-2-carbaldehyde12 following
the procedure for Step 3 Method J, giving product 44
1
in 60% yield. H NMR (400 MHz, MeOD) d 1.61 (s,
9H), 3.15–3.26 (m, 2H), 7.57 (s, 1H), 7.74–7.96 (m,
4H), 8.11 (d, J = 9.1 Hz, 1H), 8.77 (d, J = 9.1 Hz, 1H)
8.99 (s, 1H). HRMS (ESI+) calcd for C20H21N5O
(M+H), 348.1819; found 348.1821.
4.1.2.9. 4-Cyclopentylamino-6-[(2H-pyrazol-3-ylmeth-
yl)amino]quinoline-3-carbonitrile (41). Intermediate 15b
was treated with 2H-pyrazole-3-carbaldehyde following
the procedure for Step 3 Method J. 28% yield. 1H
NMR (400 MHz, DMSO-d6) d 1.62 (s, 2H) 1.79 (s,
4H) 2.05–2.14 (m, 2H) 4.40 (d, J = 5.3 Hz, 2H) 4.64–
4.72 (m, 1H) 6.27 (d, J = 1.8 Hz, 1H) 6.38 (t,
J = 5.3 Hz, 1H) 7.03 (d, J = 7.8 Hz, 1H) 7.16 (s, 1H)
7.26 (dd, J = 9.0, 2.15 Hz, 1H) 7.55 (d, J = 8.8 Hz, 1H)
7.60 (s, 1H) 8.17 (s, 1H) 8.20 (s, 1H). HRMS (ESI+)
calcd for C19H20N6 (M+H), 333.1822; found 333.1818.
4.1.2.13. 4-(tert-Butylamino)-6-[(3-cyanobenzyl)-
amino]quinoline-3-carbonitrile (45). Intermediate 15c
was treated with 3-cyanobenzaldehyde following the
1
procedure for Step 3 Method J. 65% yield. H NMR
(400 MHz, MeOD) d 1.48 (s, 9H), 3.32–3.49 (m, 2H),
4.58 (s, 1H), 6.74 (d, J = 2.3 Hz, 1H), 7.29 (dd, J = 9.1,
2.3 Hz, 1H), 7.45 (m, 1H), 7.55–7.78 (m, 4H), 8.28 (s,
1H). HRMS (ESI+) calcd for C22H21N5 (M+H),
356.1870; found 356.1872.
4.1.2.10.
dazol-2-ylmethyl)amino]quinoline-3-carbonitrile
Intermediate 15b was treated with 1-methyl-1H-imidaz-
4-Cyclopentylamino-6-[(1-methyl-1H-imi-
4.1.2.14. 4-(tert-Butylamino)-6-{[3-(methylsulfonyl)-
benzyl]amino}quinoline-3-carbonitrile (46). Intermediate
15c was treated with 3-methanesulfonylbenzaldehyde
(42).

N. Kaila et al. / Bioorg. Med. Chem. 15 (2007) 6425–6442
6439
following the procedure for Step 3 Method J. 42% yield.
1H NMR (400 MHz, MeOD) d 1.61 (s, 9H), 3.03 (s,
3H), 3.16–3.24 (m, 2H), 4.58 (s, 1H) 6.90 (d, J = 2.3 Hz,
1H) 7.37 (dd, J = 9.1, 2.27 Hz, 1H) 7.52 (t, J = 7.7 Hz,
1H), 7.59 (d, J = 9.1 Hz, 1H) 7.68 (d, J = 8.3 Hz, 1H),
7.76 (dd, J = 7.8, 2.0 Hz, 1H), 7.90 (s, 1H) 8.55 (s, 1H).
HRMS (ESI+) calcd for C22H24N4O2S (M+H),
409.1693, found 409.1692.
6.98 (d, J = 8.3 Hz, 1H) 7.11 (s, 1H) 7.22 (dd, J = 9.1,
2.3 Hz, 1H) 7.36 (dd, J = 7.5, 5.2 Hz, 1H) 7.56 (d,
J = 9.1 Hz, 1H) 7.82 (d, J = 7.8 Hz, 1H) 8.17 (s, 1H)
8.46 (d, J = 3.0 Hz, 1H) 8.67 (s, 1H). HRMS (ESI+)
calcd for C21H21N5 (M+H), 344.1870; found 344.1872.
4.1.3. 6-[(2-Morpholin-4-ylethyl)amino]-4-[(4-phenoxy-
phenyl)amino]quinoline-3-carbonitrile (33). To a 250 mL
Erlenmeyer flask equipped with a large stir bar were
added 4-nitroaniline (27.6 g, 0.2 mol) and 4-(2-chloro-
ethyl)morpholine hydrochloride (18.6 g, 0.1 mol). The
mixture was heated at a sand bath temperature of
170 ꢁC for 14 h. After allowing the reaction mixture to
cool to 60 ꢁC, EtOAc (100 mL) was added. The thick
mixture was partitioned between EtOAc (600 mL) and
water (600 mL). The aqueous phase was extracted with
EtOAc (3· 200 mL), then adjusted to pH 9 with 1 N
NaOH and further extracted with dichloromethane (3·
200 mL). The organic phase was washed with brine
and dried over Na2SO4 to provide 10.1 g (40%) of N-
(2-morpholinoethyl)-4-nitroaniline (10a) isolated as a
yellow solid. The material was used without further
4.1.2.15. 4-({[4-(tert-Butylamino)-3-cyanoquinolin-6-
yl]amino}methyl)benzenesulfonamide (47). Intermediate
15c was treated with 4-formylbenzenesulfonamide fol-
lowing the procedure for Step 3 Method J. 55% yield.
1H NMR (400 MHz, MeOD) d 1.40 (s, 9H), 3.22–3.34
(m, 2H), 4.56 (s, 1H) 6.92 (s, 1H), 7.43–7.49 (m, 1H),
7.72–7.79 (m, 2H), 7.82 (d, J = 9.1 Hz, 1H), 8.02 (br s,
2H), 8.44 (s, 1H). HRMS (ESI+) calcd for
C21H23N5O2S (M+H), 410.1645; found 410.1640.
4.1.2.16. 4-(tert-Butylamino)-6-[(1H-pyrazol-5-ylm-
ethyl)amino]quinoline-3-carbonitrile (48). Intermediate
15c was treated with 2H-pyrazole-3-carbaldehyde fol-
lowing the procedure for Step 3 Method J. 70% yield.
1H NMR (400 MHz, MeOD) d 1.68 (s, 9H), 3.46 (s,
2H), 4.57 (s, 1H), 6.42 (s, 1H), 7.07 (s, 1H), 7.38 (dd,
J = 9.1, 2.3, 1H), 7.71 (s, 1H), 7.79 (d, J = 9.1 Hz, 1H),
8.41 (s, 1H), 8.59 (s, 1H). HRMS (ESI+) calcd for
C18H20N6 (M+H), 321.1822, found 321.1822.
1
purification. H NMR (400 MHz, DMSO-d6) d 2.29–
2.58 (m, 4H), 3.15–3.40 (m, 4H), 3.48–3.70 (m, 4H),
6.58–6.76 (m, 2H), 7.18 (t, J = 5.3 Hz, 1H), 7.99 (d,
J = 9.4 Hz, 2H).
To a solution of 10a (8.5 g, 33.7 mmol) in methanol
(200 mL) was added a slurry of 10% Pd/C (1.2 g) in
EtOAc (100 mL). Hydrogen gas was bubbled through
the solution for 1 min. The mixture was then stirred at
rt under an atmosphere of hydrogen gas for 24 h, at
which time N2 gas was bubbled into the mixture for
15 min. The mixture was filtered through a pad of Celite
and the filtrate concentrated to give 7.4 g (99%) of N1-
(2-morpholinoethyl)benzene-1,4-diamine (10b) as a red
syrup. The material was used without further purifica-
4.1.2.17.
dazol-2-yl)methyl]amino}quinoline-3-carbonitrile
4-(tert-Butylamino)-6-{[(1-methyl-1H-imi-
(49).
Intermediate 15c was treated with 1-methyl-1H-imidaz-
ole-2-carbaldehyde following the procedure for Step 3
1
Method J. 47% yield. H NMR (400 MHz, MeOD) d
1.58 (s, 9H), 3.26 (s, 2H), 3.85 (s, 3H), 7.20 (s, 1H),
7.32 (s, 1H), 7.38 (dd, J = 9.1, 2.3, 1H), 7.47 (s, 1H),
7.69 (d, J = 9.1 Hz, 1H), 8.67 (s, 1H). HRMS (ESI+)
calcd for C19H22N6 (M+H), 335.1979; found 335.1980.
1
tion. H NMR (400 MHz, CD2Cl2-d2) d 2.26–2.41 (m,
2H), 2.43–2.56 (m, 2H), 2.89–3.04 (m, 4H), 3.54–3.63
(m, 4H), 6.33–6.44 (m, 2H), 6.44–6.53 (m, 2H)
4.1.2.18. 4-(Cycloheptylamino)-6-[(pyridin-3-ylmethyl)-
amino]quinoline-3-carbonitrile (50). Step 1 Method F
was used to synthesize 4-cycloheptylamino-6-nitroquin-
oline-3-carbonitrile (14d) in 70% crude yield. Step 2
Method H was used to prepare 6-amino-4-cycloheptyla-
minoquinoline-3-carbonitrile (15d, 83% crude yield).
Step 3 Method J gave compound 50 in 69.5% yield.
1H NMR (400 MHz, DMSO-d6) d 1.37–1.78 (m, 10H)
1.89–2.05 (m, 2H) 4.31–4.39 (m, 1H) 4.41 (d,
J = 5.8 Hz, 2H) 6.65 (t, J = 6.0 Hz, 1H) 6.88 (d,
J = 8.3 Hz, 1H) 7.05 (d, J = 2.3 Hz, 1H) 7.15 (dd,
J = 9.0, 2.4 Hz, 1H) 7.29 (dd, J = 7.3, 4.3 Hz, 1H) 7.49
(d, J = 9.1 Hz, 1H) 7.71–7.80 (m, 1H) 8.10 (d,
J = 6.3 Hz, 1H) 8.39 (dd, J = 4.8, 1.8 Hz, 1H) 8.60 (d,
J = 1.8 Hz, 1H). HRMS (ESI+) calcd for C23H25N5
(M+H) 372.2182, found 372.2186.
To a solution of 10b (5.0 g, 22 mmol) in toluene
(100 mL) was added ethyl 2-cyano-3-ethoxyacrylate
(3.8 g, 22 mmol). The solution was heated at 110 ꢁC
for 8 h, allowed to cool to rt, and stirred overnight. Hex-
anes (800 mL) were added with vigorous stirring. The
resulting suspension was heated to 60 ꢁC and EtOAc
was added slowly until a copious precipitate formed.
After allowing to cool to rt the solid was isolated by fil-
tration to give 4.17 g of 2-cyano-3-(4-(2-morpholinoeth-
ylamino)phenylamino)acrylic acid ethyl ester (11b) as a
yellow powder. After standing for 2 h, the mother liquor
was filtered to give 3.47 g of a yellow solid for a com-
bined yield of 7.54 g (99%), which was used directly in
the next step. Dowtherm A (300 mL) was bubbled with
Ar gas for 1h while being heated to 100 ꢁC. The acrylate
11b (3.76 g, 10.9 mmol) was added in three portions and
the mixture was heated to 259 ꢁC under a stream of Ar.
After gently refluxing for 6h, the reaction mixture was
allowed to cool below 50 ꢁC and then poured into
1500 mL of hexanes. Filtration provided 4-hydroxy-6-
(2-morpholin-4-yl-ethylamino)quinoline-3-carbonitrile
(12b) as a tan powder (1.15 g, 37%). 1H NMR
4.1.2.19. 4-Cyclopentylamino-6-[(pyridin-3-ylmethyl)-
amino]quinoline-3-carbonitrile (51). Intermediate 15b
was treated with pyridine-3-carbaldehyde following the
1
procedure for Step 3 Method J. 20% yield. H NMR
(400 MHz, DMSO-d6) d 1.58–1.66 (m, 2H) 1.73–1.81
(m, 4H) 2.01–2.11 (m, 2H) 4.48 (d, J = 5.8 Hz, 2H)
4.61–4.69 (m, 1H) 6.50 (s, 1H) 6.72 (t, J = 6.1 Hz, 1H)

6440
N. Kaila et al. / Bioorg. Med. Chem. 15 (2007) 6425–6442
(400 MHz, DMSO-d6) d 2.33–2.59 (m, 4H), 3.13–3.27
(m, 4H), 3.51–3.65 (m, 4H), 6.06 (t, J = 5.3 Hz, 1H),
7.08 (d, J = 2.8 Hz, 1H), 7.17 (dd, J = 8.8, 2.8 Hz, 1H),
7.41 (d, J = 9.1 Hz, 1H), 8.47 (s, 1H).
MA). The detection of test articles was performed on
a PESCIEX API-3000 triple quadrupole mass spectrom-
eter (Applied Biosystems, Concord, Ontario, L4K4V8)
using TurboIon Spray source. Plasma standard curves
were generated by plotting peak area ratio of test article
and internal standard against nominal concentrations.
A solution of the quinoline 12b (1.0 g, 3.3 mmol) in
phosphorus oxychloride (50 mL) was heated at 120 ꢁC
for 12 h. The reaction mixture was concentrated under
reduced pressure and the resulting residue was stirred
in dichloromethane (200 mL) and cooled to 0 ꢁC. Water
(100 mL) was added followed by solid Na2CO3, added
slowly until the aqueous phase reached pH 9. The or-
ganic layer was dried over Na2SO4 and concentrated
to provide 4-chloro-6-(2-morpholinoethylamino)quino-
line-3-carbonitrile (13a) (800 mg, 69%) as a light yellow
The pharmacokinetic parameters were determined using
WinNonlin (version 4.1, Pharsight, Mountain View,
CA). Calculations were performed using the non-com-
partmental analysis approach. The estimation of area
under the plasma concentration versus time curve
(AUC) was based upon the log trapezoidal rule. The ter-
minal rate constant (k) was derived from the slope of the
terminal log-linear phase of plasma concentrations–time
curves. The apparent terminal half-life (t1/2) was calcu-
lated as 0.693/k. No statistical analysis other than
descriptive statistics was conducted.
1
powder. H NMR (400 MHz, DMSO-d6) d 2.37–2.54
(m, 4H), 2.55–2.67 (m, 4H), 3.33 (s, 4H), 3.61 (s, 4H),
6.82 (d, 1H), 6.87 (d, J = 2.5 Hz, 1H), 7.52 (dd,
J = 9.2, 2.7 Hz, 1H), 7.87 (d, J = 9.1 Hz, 1H), 8.74 (s,
1H).
4.3. Biology
To a solution of 13b (135 mg, 0.39 mmol) in 2-ethoxy-
ethanol (5 mL) was added 4-phenoxyaniline (57 mg,
0.39 mmol). The mixture was heated at 135 ꢁC for
36 h. After allowing to cool to rt the reaction mixture
was diluted with ether and hexane (1:1, 20 mL), stirred
for 4 h, and filtered. The solid was washed with ether
and dried on a vacuum pump to give 33 (106 mg,
4.3.1. Tpl2 enzymatic assay. Tpl2/Cot activity was di-
rectly assayed using GST-MEK1 as a substrate. The
phosphorylation on serine residues 217 and 221 of
GST-MEK1 was detected by an ELISA. Briefly,
0.4 nM Tpl2 was incubated with 35 nM GST-MEK1
in a kinase reaction buffer of pH 7.2 containing
20 mM Mops, 50 lM ATP, 20 mM MgCl2, 1 mM
DTT, 25 mM b-glycerophosphate, 5 mM EGTA, and
1 mM sodium orthovanadate for 1 h at 30 ꢁC. Com-
pounds solubilized in 100% DMSO were pre-diluted in
assay buffer so that the final concentration of DMSO
in the reaction was 1%. The kinase reaction was carried
out in 100 ll volume on 96-well plates. The kinase reac-
tion was then stopped by the addition of 100 mM
EDTA. The entire reaction mixture was then transferred
to the detection plate, a 96-well Immunosorb plate that
had been pre-coated with anti-GST antibody (Amer-
sham). After 1 h incubation at room temperature, the
detection plate was washed four times with TBST
(TBS + 0.05% Tween 20) and then incubated for an-
other hour at room temperature with anti-phospho-
MEK1 antibody (Cell Signaling) 1:1000 in 10 mM Mops
7.5, 150 mM NaCl, 0.05% Tween 20, 0.1% gelatin,
0.02% NaN3, and 1% BSA. The detection plate was
washed again and incubated for 30 min with DELFIA
Europium (Eu) labeled goat anti-rabbit IgG (Perkin-El-
mer), 1:4000 in the same buffer used for the primary
incubation. After a final wash, Eu detection solution
was added to each well and the Eu signal was measured
in a Wallac Victor Multilabel Counter. Data were im-
ported into Excel and IC50 calculations were performed
using the Xlfit (IDBS) software package.
1
55%) as an orange solid. H NMR (400 MHz, DMSO-
d6) d 3.01–3.55 (m, 6H), 3.65–4.19 (m, 6H), 7.03–7.08
(m, 1H), 7.09–7.20 (m, 4H), 7.34–7.50 (m, 5H), 7.55
(m, 1H), 7.82 (d, J = 9.1 Hz, 1H), 8.65 (s, 1H). HRMS
(ESI+) calcd for C28H27N5O2 (M+H), 466.2238; found
466.2234.
4.2. Pharmacokinetic studies
The animals used in the pharmacokinetic (PK) studies
were male adult Sprague–Dawley rats (Charles River
Laboratories, Wilmington, MA). All the PK studies
were performed at Wyeth Research Laboratories
(Andover, MA) under the supervision of the Institu-
tional Animal Care and Use Committee. The dose for-
mulation for intravenous administration was 50%
DMSO/50% polyethylene glycol 200 (v/v, 1 mL/kg).
The oral dose formulation was an aqueous suspension
containing 2% Polysorbate 80 (a.k.a. Tween 80), 0.5%
methylcellulose, and 0.06% lactic acid. Blood samples
of approximately 0.25 mL were collected into K2 EDTA
coated sampling tubes at 0.083, 0.25, 0.5, 1, 2, 4, 7, and
24 h post dose administration. Plasma samples were har-
vested and stored at ꢀ80 ꢁC until analysis.
In a 0.5 mL 96-well plate, 50 ll of plasma sample was
precipitated with 100 ll acetonitrile containing 500 ng/
mL internal standard. The internal standard is a com-
pound with a similar chemical structure as that of the
test article. The samples were vortexed and centrifuged
at 5700 rpm for 10 min. Supernatants were subjected
to LC-MS/MS analysis. HPLC separation was per-
formed on a Perkin-Elmer Series 200 HPLC system
(Perkin-Elmer, Norwalk, CT) using an XTerra MS
C18 column (2.1 · 20 mm, 2.5 lm; Waters, Milford,
4.3.2. Inhibition of TNF-a in primary human monocytes.
Human blood buffy coat (containing 1 mM EDTA) was
incubated with RosetteSep human monocyte enrich-
ment antibody cocktail (Stem Cell Technologies
#15068). This mixture was then diluted with an equal
volume of PBS/2% FBS/1 mM EDTA, layered onto an
equal volume of Histopaque-1077 (Sigma #H8889),
and centrifuged for 20 min at 1500 rpm. The monocytes
were then recovered from the interface, diluted, and

N. Kaila et al. / Bioorg. Med. Chem. 15 (2007) 6425–6442
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Chem. 2001, 44, 2719–2734; (b) Smaill, J. B.; Showalter,
H. D. H.; Zhou, H.; Bridges, A. J.; McNamara, D. J.;
Fry, D. W.; Nelson, J. M.; Sherwood, V.; Vincent, P.
W.; Roberts, B. J.; Elliott, W. L.; Denny, W. A. J. Med.
Chem. 2001, 44, 429–440; (c) Hennequin, L. F.; Stokes,
reisolated on a Histopaque-1077 cushion. After this sec-
ond round of purification, the monocytes were washed
twice in PBS/2% FBS/1 mM EDTA, resuspended in
RPMI/0.5% FBS to 2 · 106 cells/mL, plated at 800,000
cells per well in 48-well formats, and incubated at
37 ꢁC/5% CO2. Thirty minutes prior to LPS stimulation,
compounds (in 100% DMSO) were added. LPS was then
added to 10 ng/mL and the cultures allowed to incubate
for an additional ꢁ3 h. Media supernatants were then
harvested and analyzed for TNF-a by standard ELISA
or electrochemiluminescence on a Sector6000 reader
(Meso Scale Discovery).
4.3.3. Inhibition of TNF-a in human blood. Blood was
drawn from healthy male volunteers into heparin tubes.
At 37 ꢁC, the blood was diluted 1–5 with pre-warmed
RPMI/3% FCS, and then dispensed into 96-well-format-
ted 200 lL tube strips and capped. Thirty minutes prior
to LPS stimulation compounds (in 100% DMSO) were
added. Final DMSO concentrations equaled 1.0%.
LPS was then added to 10 ng/mL and the cultures are al-
lowed to incubate for an additional ꢁ3 h. Samples were
centrifuged in a strip rotor at 6500 rpm, and the plasma
supernatants were analyzed by standard ELISA or elect-
rochemiluminescence on a Sector6000 reader (Meso
Scale Discovery).
4.3.4. LPS-induced acute TNF-a production in mice.
LPS/D-Gal-induced acute TNF-a production in mouse
sera: female C57Bl/6 mice, 8–10 weeks of age, were
obtained from the Jackson Laboratory. The animals
were fed food and water ad libitum and all procedures
were approved by the Institutional Animal Care and
Use Committee. Compound at 10 or 25 mg/kg, or
vehicle, was administered to the mice by the intraperi-
toneal (ip) route. One hour after the compound, LPS
plus D-galactosamine in PBS was administered ip. Fi-
nal LPS and D-gal concentrations in each animal were
2 and 160 ng/kg, respectively. The mice were eutha-
nized with carbon dioxide 1.5 hour after the LPS/D-
Gal injection and bled by cardiac puncture. TNF-a
levels were measured in the serum samples using a
TNF-a ELISA.
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