4-Anilino-7-alkenylquinoline-3-carbonitriles as potent MEK1 kinase inhibitors

Article abstract of DOI:10.1016/j.bmc.2008.09.009

A series of substituted 7-alkenyl 4[3-chloro-4-(1-methyl-1H-imidazol-2-ylsulfanyl)]anilino-3-quinolinecarbonitrile analogs were synthesized and evaluated as MEK1 kinase inhibitors. The synthetic details, structure-activity relationships, biological activity, and selected oral exposure studies of these analogs are described. From these studies, compound 5m was chosen as a strong candidate for further evaluation. The selectivity of 5m was ascertained against a panel of 17 kinases, where activity was observed against EGFR, Src, Lyn, and IR kinases. Western blot studies in WM-266 cells demonstrated that 5m inhibited phosphorylation of ERK, while additional kinase pathways tested showed no inhibition at up to 10 μM of 5m. PK studies, as well as a xenograft and in vivo biomarker studies are described for 5m.

Full text of DOI:10.1016/j.bmc.2008.09.009

Bioorganic & Medicinal Chemistry 16 (2008) 9202–9211
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
4-Anilino-7-alkenylquinoline-3-carbonitriles as potent MEK1 kinase inhibitors
a
a
a
a
b
Dan M. Berger a, , Minu Dutia , Dennis Powell , Middleton B. Floyd , Nancy Torres , Robert Mallon ,
*
Donald Wojciechowicz b, Steven Kim b, Larry Feldberg b, Karen Collins b, Inder Chaudhary c
a Chemical & Screening Sciences, Wyeth Research, 401 N. Middletown Road, Pearl River, NY 10965, USA
b Oncology, Wyeth Research, 401 N. Middletown Road, Pearl River, NY 10965, USA
c Drug Safety and Metabolism, Wyeth Research, 401 N. Middletown Road, Pearl River, NY 10965, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of substituted 7-alkenyl 4[3-chloro-4-(1-methyl-1H-imidazol-2-ylsulfanyl)]anilino-3-quinoline-
carbonitrile analogs were synthesized and evaluated as MEK1 kinase inhibitors. The synthetic details,
structure–activity relationships, biological activity, and selected oral exposure studies of these analogs
are described. From these studies, compound 5m was chosen as a strong candidate for further evaluation.
The selectivity of 5m was ascertained against a panel of 17 kinases, where activity was observed against
EGFR, Src, Lyn, and IR kinases. Western blot studies in WM-266 cells demonstrated that 5m inhibited
Received 4 August 2008
Revised 27 August 2008
Accepted 5 September 2008
Available online 9 September 2008
Keywords:
Anticancer
MEK1
Kinase inhibitor
3-Quinolinecarbonitriles
phosphorylation of ERK, while additional kinase pathways tested showed no inhibition at up to 10 lM
of 5m. PK studies, as well as a xenograft and in vivo biomarker studies are described for 5m.
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
(AZD6244) is also currently being evaluated in clinical trials as a
potential anticancer agent.6a,7
The Ras-MAPK pathway is an essential signaling cascade that
controls cellular growth, proliferation, and survival. Activation of
this pathway under the influence of growth factor receptors or hor-
mones results in Ras being induced into an active GTP-bound state,
which in turn stimulates the serine/threonine kinase Raf. Raf
kinase phosphorylates and activates mitogen-activated protein
kinase kinases (MEK) 1 and 2, which phosphorylates mitogen-acti-
vated protein kinase (MAPK). The activated MAPK proteins translo-
cate to the nucleus, where they phosphorylate downstream targets
that result in growth and proliferation.1 Inappropriate Ras activa-
tion is associated with ꢀ30% of all human cancers, making both
Ras and its downstream effectors attractive targets for pharmaceu-
tical intervention.2a,b Additionally, B-Raf mutations have been
associated with certain tumor types, particularly melanoma and
colorectal cancer.3 It has been observed that tumor formation can
be associated with either Ras or Raf mutations, but not both.4 As
signaling by either mutation proceeds via the downstream target
MEK kinase, several groups have focused their attention on discov-
ering MEK inhibitors. Thus, researchers at Pfizer identified CI-1040
as a potent MEK1 kinase inhibitor with oral activity in vivo against
selected tumor lines.5 This compound entered human clinical tri-
als, but was subsequently replaced by analog PD0325901, which
demonstrated significantly improved bioavailability over CI-
High-throughput screening identified 6,7-dimethoxyquinoline-
3-carbonitriles as potent MEK1 kinase inhibitors.8,9 Of particular
interest was a series of 4-[3-chloro-4-(1-methyl-1H-imidazol-2-
ylsulfanyl)anilinoquinoline-3-carbonitriles I (Chart 1) which were
highly potent inhibitors in cellular proliferation assays. These com-
pounds were shown to be ATP competitive inhibitors of MEK1 ki-
nase,9 in contrast to the allosteric, non-ATP competitive MEK
inhibitors CI-1040, PD0325901, and AZD6244. Of these com-
pounds, II was identified as a potent MEK1 inhibitor (IC50: 3 nM),
with exceptional activity (IC50: 7 nM) against LoVo cells (a K-Ras
mutant human colon tumor line), as well as other selected cell
lines. Western blot studies revealed that this compound strongly
inhibited phosphorylation of ERK in LoVo cells, although additional
off-target activity was observed to cause inhibition of MEK phos-
phorylation as well. While II displayed potent in vitro activity as
well as in vivo activity following ip dosing, it was poorly soluble
in aqueous media and undetectable in plasma following oral dos-
ing. As a result, it was not orally active in vivo. Similarly, additional
C-6, C-7 dialkoxy substituted analogs10 were found to have poor
oral bioavailability. Other quinoline-3-carbonitrile analogs investi-
gated include the C-7 aryl11 analogs and C-7 amino12 substituted
series. A C-7 (4-diethylamino)piperidine substituted analog was
found to have oral activity against BXPC3 xenografts when dosed
orally at 25 and 50 mg/kg twice daily.12 However, further evalua-
tion of this compound revealed low oral bioavailability in rats (2%),
as well as highly variable blood levels following oral dosing. Thus,
further efforts were undertaken to find MEK1 kinase inhibitors
1040.6a,b
A MEK1 inhibitor discovered at Array Biopharma
* Corresponding author. Tel.: +1 845 602 3435; fax: +1 845 602 5561.
E-mail address: bergerd@wyeth.com (D.M. Berger).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.09.009

D. M. Berger et al. / Bioorg. Med. Chem. 16 (2008) 9202–9211
9203
Cl
Cl
S
N
S
N
N
N
HN
HN
N
R1O
O
CN
3
4
6
7
O
O
CN
R2
N
R3
N
N
n
I
II
N
n = 1-3
R1 = alkyl
NR2R3 = dialkylamines,
heterocyclic amines
Cl
S
N
N
HN
N
R
CN
R1
N
R = H, OMe
NR1R2 = heterocyclic amines,
dialkylamines
R2
n
III
Chart 1.
with improved oral bioavailability. The present work describes the
synthesis and detailed SAR of the C-7 alkenyl substituted quino-
line-3-carbonitriles. Structurally, they are described by the general
formula III shown in Chart 1.
Pure trans-substituted compounds 5l and m were obtained by
the methodology shown in Scheme 3. Hydroboration of alkynes
7a and b in the presence of Schwartz reagent provided the
intermediate boronates 8a and b with excellent trans-selectivity.17
Suzuki coupling of 8a and b with 4a in a mixture of toluene/EtOH/
H2O provided 5l and m in high yield and purity following silica gel
chromatography (the cis-isomer being 60.5% as determined by
analytical HPLC).
2. Chemistry
The target compounds all possessed the 3-chloro-4-(1-methyl-
1H-imidazol-2-ylsulfanyl)aniline headpiece, as this provided the
optimal mixture of MEK1 enzyme and cellular activity for the 3-
quinolinecarbonitriles.9 Additionally, the final products were syn-
thesized with water-solubilizing amines attached via the alkene
linker at C-7, based on the SAR provided by the previously synthe-
sized MEK1 inhibitors.9–12 Scheme 1 outlines the method by which
we initially synthesized the desired target compounds, with instal-
lation of the different amines being the last step of the reaction
sequence. Thus, the reaction of propyn-1-ol, butyn-1-ol, and pen-
tyn-1ol with tributyltin hydride respectively provided 2a13 and
3a,14 2b, and 3b, as separable mixtures. Following chromatographic
separation of the isomers on silica gel, alcohols 2a and b were re-
acted with quinoline-3-carbonitrile intermediates 4a and b15 under
Stille coupling conditions16 to provide 5a–c. Allylic alcohol 5a was
converted to target compounds 5d–f via reaction with acetic anhy-
dride, followed by a palladium (II) acetate-mediated coupling with
the requisite amines. The alcohol residues of 5b and c were tosylat-
ed, then displaced by the corresponding amines to provide 5g–h, k,
and p. While this reaction sequence provided several analogs, one
issue was that the intermediate alcohols 5a–c were highly insoluble
in a variety of solvents, with the C-6 methoxy substituted 5c being
particularly insoluble. In addition, the target compounds in some
cases required more than one chromatographic separation to pro-
vide P98:2 trans/cis product ratios.
3. Biology
As previously determined for the 6,7-dialkoxy substituted 4-[3-
chloro-4-(1-methyl-1H-imidazol-2-ylsulfanyl)anilinoquinoline-3-
carbonitriles,9 the C-7 alkenyl substituted quinoline-3-carbonitr-
iles described here were confirmed to be MEK1 inhibitors. The
in vitro inhibitory activity of compounds II and 5a–r is detailed
in Table 1. Despite having low solubility (aqueous solubility:
1 lg/mL at pH 7.4), alcohols 5a–c had potent activity in both the
enzyme and cellular assays, demonstrating that the water-solubi-
lizing amines were not necessary for in vitro activity. Overall, the
in vitro potency of 5a and b was similar to the C-6 hydrogen substi-
tuted analogs 5d–o, which possess amine water-solubilizing
groups. While the C-7 substituted allylic amine analogs 5d–f ap-
peared to be slightly less potent than the comparably substituted
homoallylic amines, there was little variability in the activity of
the different amine substituted compounds. The importance of
the trans-geometry for the C-7 alkene was demonstrated by deter-
mining and comparing the IC50s of selected compounds for which
sufficient quantities of the cis-isomers were isolated. For example,
while 5j had a Raf/MEK IC50 of 20 nM, the cis-isomer was far less
potent with a Raf/MEK IC50 of 435 nM.
The most obvious improvement in activity was provided by the
C-6 methoxy substituent. Thus, homoallylic alcohol 5c, and amino-
substituted analogs 5p–r were the most potent MEK1 kinase inhib-
itors, with comparable activity to II. In cellular assays, however,
mixed results were observed for the C-6 methoxy substituted ana-
logs. While all of these analogs showed good potency against LoVo
cells, it proved to be challenging to obtain consistent antiprolifer-
ative IC50s when using the BXPC3 cells. Thus, for example, while
compounds 5p and r appeared to have some activity at low nano-
molar concentrations in several runs, the dose–response curves did
not reach 50% inhibition, and are therefore reported as having
In order to circumvent the solubility issues, another set of ana-
logs was prepared by the sequence outlined in Scheme 2. Interme-
diates 2b and c were first tosylated, and then reacted with amines
to provide amino intermediates 6a–e. Following the purification
by silica gel chromatography, the amino-substituted vinyl tin
intermediates 6a–e were reacted with 4a and b by Stille coupling
to provide target compounds 5i, j, n, o, q, and r. This methodology
proved useful in readily providing the target compounds, although
some were again found to require careful chromatography to pro-
vide P98:2 trans/cis product ratios.

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D. M. Berger et al. / Bioorg. Med. Chem. 16 (2008) 9202–9211
HO
Sn(Bu)3
HO
a,b
+
HO
Sn(Bu)3
n
n
n
3a: n = 1
3b: n = 2
1a: n = 1
2a: n = 1
2b: n = 2
1b: n = 2
Cl
Cl
S
S
N
N
N
N
HN
N
HN
N
c
R
CN
R
X
CN
HO
2a,b
n
5a: n = 1, R = H
5b: n = 2, R = H
5c: n = 2, R = OMe
4a: R = H, X = Br
4b: R = OMe, X = OTf
Cl
S
N
N
HN
N
d,e
R
CN
R1
N
or f,g
R2
n
5d: n = 1, R = H, NR1R2 = pyrrolidine
5e: n = 1, R = H, NR1R2 = N-methylpiperazine
5f: n = 1, R = H, NR1R2 = N-ethylpiperazine
5g: n = 2, R = H, NR1R2 = diethylamine
5h: n = 2, R = H, NR1R2 = dimethylamine
5k: n = 2, R = H, NR1R2 = piperidine
5p: n = 2, R = OMe, NR1R2 = diethylamine
Scheme 1. Preparation of 5a–h, k, and p. Reagents and conditions: (a) Bu3SnH, AIBN, 100 °C; (b) separation of isomers by silica gel chromatography (hexane/EtOAc);
(c) Pd(PPh3)4, PPh3, NMP, 100 °C; (d) (CH3CO)2O, HOAc, 50 °C; (e) R1R2NH, Pd(PPh3)4, NMP, 25 °C; (f) TosCl, 2,6-lutidine, 45 °C; (g) R1R2NH, THF, 60 °C.
>1000 nM activity. The same phenomenon was observed for analog
5n, which has a water-solubilizing diethylamine group attached at
C-7 via a trans-pent-1-enyl linker. It is possible that the low aque-
ous solubility of these analogs may be a contributing factor to
these results. The aqueous solubilities of 5n, p, and r were mea-
chosen for more extensive evaluation. As shown in Table 3, 5m
showed good selectivity versus a number of kinases, but it did have
significant activity against IR, EGFR, Src, and Lyn. These results
indicate that the biological effects of 5m on tumor cells may be
augmented by the inhibition of kinases in addition to MEK1. Of
these results, the potent activity against IR kinase was of most con-
cern, given the role it plays in the modulation of glucose.20
The cellular activity profile of compound 5m was determined
against a panel of additional cell lines. In addition to the LoVo and
BXPC3 activity as shown in Table 1, 5m was found to inhibit
H358 (lung), WM266-4, A375 (melanoma) cell lines with IC50s of
50, 25, and 26 nM, respectively. In contrast, 5m was a weak inhib-
itor of CaCo-2 cells (IC50: 1400 nM). The cellular profile was there-
fore as anticipated for a MEK1 kinase inhibitor, having significantly
more potent activity against K-ras mutant (LoVo, H358) and B-Raf
mutant (WM266-4, A375) cell lines than ras wild-type cells
(Caco-2). The cellular inhibition of MEK1 by 5m was measured in
sured to be 3, 3, and 2
to lower molecular weight analogs such as 5d and g (aqueous sol-
ubilities 20 and 28 g/mL, respectively, at pH 7.4).
lg/mL, respectively, at pH 7.4, in contrast
l
As it had been demonstrated that the majority of the synthe-
sized analogs had similar in vitro potency, our main focus was to
find a compound that would have the optimal physical character-
istics to provide improved oral bioavailability. Thus, a number of
analogs were tested in preliminary exposure studies, for which
blood levels were measured at 2 and 4 h following a single oral
dose of 50 mg/kg in a nude mouse model. Representative data
are shown in Table 2. Overall, the allylic amine substituted analogs
such as 5d and i had poor blood levels following a single oral dose
of 50 mg/kg. The homoallylic amines 5j, l, and m had significantly
higher blood levels, with 5m providing the best exposure at both
time points. Despite its poor solubility in aqueous methocel/
Tween, C-6 methoxy substituted 5q had measurable plasma levels
following oral dosing, although these were lower than the corre-
spondingly substituted 6-H substituted analog, 5l.
the WM266-4 cell line (Fig. 1). With an IC50 of 0.225 lM for the
inhibition of phospho-ERK, 5m did inhibit MEK1 in these cells, al-
beit at concentrations higher than that required to inhibit cellular
proliferation. Since 5m had shown good activity against additional
kinases, further studies were performed to determine whether it
would inhibit the associated pathways in the WM266-4 cells. Thus,
cell lysates have been tested for inhibition of phosphorylation of
EGFR, IR, IGFR, Src, SAP, JNK, P38, and overall phospho-tyrosine
With a potent in vitro activity profile, and good exposure levels
following oral administration to nude mice, compound 5m was

D. M. Berger et al. / Bioorg. Med. Chem. 16 (2008) 9202–9211
9205
R1
N
Sn(Bu)3
Sn(Bu)3
HO
a,b
R2
n
n
6a: n = 2, NR1R2 = pyrrolidine
2b: n = 2
2c: n = 3
6b: n = 2, NR1R2 = morpholine
6c: n = 2, NR1R2 = N-methylpiperazine
6d: n = 3, NR1R2 = diethylamine
6e: n = 3, NR1R2 = N-methylpiperazine
Cl
Cl
S
N
S
N
N
HN
N
HN
N
R
CN
R1
N
c
R
X
CN
6a-e
R2
N
n
4a: R = H, X = Br
4b: R = OMe, X = OTf
5i: n = 2, R = H, NR1R2 = morpholine
5j: n = 2, R = H, NR1R2 = pyrrolidine
5n: n = 3, R = H, NR1R2 = diethylamine
5o: n = 3, R = H, NR1R2 = N-methylpiperazine
5q: n = 2, R = OMe, NR1R2 = N-methylpiperazine
5r: n = 3, R = OMe, NR1R2 = N-methylpiperazine
Scheme 2. Preparation of 5i, j, n, o, q, and r. Reagents and conditions: (a) TosCl, 2,6-lutidine, 25 °C; (b) R1R2NH, THF, 25 °C, then 45 °C; (c) Pd(PPh3)4, PPh3, NMP, 100 °C.
Cl
S
N
N
HN
N
R1
N
R1
N
CN
b
a
O
R2
B
O
R2
R1
N
R2
>99:1 trans/cis
7a: NR1R2 = N-methylpiperazine 8a: NR1R2 = N-methylpiperazine
7b: NR1R2 = N-ethylpiperazine 8b: NR1R2 = N-ethylpiperazine
5l: NR1R2 = N-methylpiperazine
5m: NR1R2 = N-ethylpiperazine
Scheme 3. Preparation of 5l and m. Reagents and conditions: (a) pinacolborane, Cp2ZrHCl, 25 °C; (b) 4a, Pd(PPh3)4, toluene/EtOH/aq K2CO3, reflux.
kinase inhibition. These studies showed that 5m did not inhibit
these pathways in WM266-4 cells at up to 10 M (data not shown),
half-life was 0.9 h, with moderate/high clearance (66 mL/min/kg).
The oral bioavailability was 17%, which was modest, but among
the highest observed for any 3-chloro-4-(1-methyl-1H-imidazol-
2-ylsulfanyl)aniline substituted quinoline-3-carbonitriles. A subse-
quent rat PK study provided similar results, with iv half-lives of 2.5
and 1.8 h, and somewhat improved volume of distribution of 5.7
and 7.6 L/kg in female and male rats, respectively. The oral bio-
availability of 5m was 23% and 13% in female and male rats,
respectively.
Given that 5m had modest plasma levels following oral dosing,
it was administered twice daily (bid) to nude mice implanted with
H358 xenografts at 25, 50, and 100 mg/kg orally (Fig. 2). At the
highest dose of 100 mg/kg bid, complete suppression of tumor
growth was observed. Statistically significant tumor growth inhibi-
tion was achieved at the 50 mg/kg bid dose, while the 25 mg/kg
dose had a marginal effect on inhibiting tumor growth. On removal
of the tumors at 2 h after the 100 mg/kg final dose was given, the
resulting lysates showed almost complete inhibition of phospho-
ERK (Fig. 3), consistent with the proposed mechanism of action
for 5m.
l
indicating that its activity against these additional kinases was not
contributing to the observed cellular antiproliferative activity.
In pharmaceutical profiling assays, 5m was found to be moder-
ately stable when exposed to male CD-1 mouse and rat microsomes,
with half-lives of 16.5 and 21 min, respectively. Permeability, as
measured in a PAMPA assay, was 0.45 Â 10À6 cm/s. Since compound
5m had shown the highest blood levels of the analogs evaluated in
the exposure studies following oral dosing, a full PK study was car-
ried out to determine its suitability for in vivo studies (Table 4).
Thus, 5m was dosed iv at 2 mg/kg in a pH 3 aqueous solution (it
was necessary to solubilize 5m at low pH, since the aqueous solu-
bility for this analog was a modest 4 lg/mL at pH 7.4) and at
50 mg/kg orally in female nude mice. Upon oral dosing, the com-
pound achieved a Cmax of 510 ng/mL, with the Tmax being achieved
at 1 h post-administration. The volume of distribution (Vss) was
moderate at 2.7 L/kg, and the half-life favorable at 4.7 h. As a sec-
ondary peak was observed with orally dosed 5m, this suggested
possible entero-hepatic recycling. Following an iv dose of 5m, the

9206
D. M. Berger et al. / Bioorg. Med. Chem. 16 (2008) 9202–9211
Table 1
Inhibition in the Raf/MEK1 coupled assay, and cellular activity for compounds II and 5a–r
Cl
S
N
N
HN
R
CN
X
N
n
a
a
a
Compound
R
n
X
Raf/MEK IC50 (nM)
LoVo IC50 (nm)
BXPC3 IC50 (nm)
II
—
H
H
OMe
H
H
H
H
H
H
H
H
H
H
H
H
—
1
2
2
1
1
1
2
2
2
2
2
2
2
3
3
2
2
3
—
OH
OH
OH
3
35
36
5
7
21
21
9
26
44
ND
25
40
40
42
73
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
Pyrrolidine
59
55
52
26
21
11
20
10
19
12
25
33
3
25
41
44
25
33
27
31
18
14b
34
37
20
8
N-Methylpiperazine
N-Ethylpiperazine
Diethylamine
Dimethylamine
Morpholine
32
39
51
Pyrrolidine
Piperidine
43
N-Methylpiperazine
N-Ethylpiperazine
Diethylamine
N-Methylpiperazine
Diethylamine
N-Methylpiperazine
N-Methylpiperazine
48b
48
>1000
41
OMe
OMe
OMe
>1000c
>1000c
>1000c
4
6
7
8
a
IC50 values reported represent the means of at least two separate determinations.
IC50 values are from a single determination.
Compounds highly insoluble, <50% inhibition was observed over a broad concentration range for this assay.
b
c
4. Summary and conclusion
Table 2
Plasma concentration of selected compounds following an oral dose of 50 mg/kg in
nude mice (average of three mice/measurement)
A series of C-7 alkenylquinoline-3-carbonitriles was evaluated
as MEK1 kinase inhibitors. For the initial synthetic pathway for this
series, our plan was to introduce the diverse amine water-solubi-
lizing groups in the last step. However, alternative pathways were
pursued due to the insolubility of several intermediates, as well as
significant quantities of the unwanted cis-isomer being present in
the product mixtures. Compounds of interest were subsequently
efficiently synthesized by utilizing vinylboronic acids and Suzuki
coupling chemistry under appropriate conditions to provide
essentially only the desired C-7 trans-alkenyl products.
A number of compounds were found to be potent MEK1 kinase
inhibitors in vitro, as was true for the previously explored series of
quinoline-3-carbonitriles possessing the C-4 [3-chloro-4-(1-
methyl-1H-imidazol-2-ylsulfanyl)]aniline substituent. However,
in contrast to the earlier series of analogs, these C-7 alkenyl substi-
tuted compounds showed significant plasma levels following oral
dosing. Of these, 5m was a clear lead candidate due to its good
in vitro potency profile, and highest plasma exposure levels ob-
served for the tested compounds.
Further biological evaluation of 5m revealed that it had
additional activity against kinases other than MEK1. Western blot
analyses carried out with 5m have so far revealed evidence of phos-
pho-ERK suppression, but no evidence of inhibition of other path-
ways that we have evaluated. Despite this observation, it is
certainly possible that additional kinase activities are contributing
to the potent cellular antiproliferative activity of 5m. Further studies
are underway to determine the activity of 5m against an expanded
set of kinases, and the biological relevance of any additional kinase
activities will be evaluated. At this time, we have established 5m
as a lead compound MEK1 inhibitor with oral activity against
H358 xenografts.
Compound
Plasma concentration
(2 h) ng/mL
Plasma concentration
(4 h) ng/mL
% CVa
(2 h, 4 h)
5d
5i
5j
5l
5q
5m
72b
95
319
368
267
494
11
22
82
85
76
132
43b, 69
55, 41
20, 31
33, 18
33, 17
53, 48
a
Coefficient of variation (CV) = standard deviation/mean  100.
Plasma concentration of 5d was measured at 1 h post-dosing.
b
Table 3
Kinase selectivity profile of 5m
Kinase (ATP concentration)
IC50 (lM)
MEK1 (100
EGFR (10
IR (100
Src (100
Lyn (20
IGFR (100
KDR (1 M)
l
M)
M)
M)
M)
M)
M)
0.012
0.010
0.032
0.055
0.058
0.220
1.0
l
l
l
l
l
l
PDK1 (100
B-raf (100
IKK beta (2
l
l
l
M)
M)
M)
M)
PKC theta (6
PI3K alpha (25
AKT (20 M)
mTOR (100
Tpl2 (50 M)
MK2 (1 M)
3.10
8.80
9.17
20
27.4
>10
>30
>30
>40
>100
P70S6 (2
l
l
M)
M)
l
l
l
M)
l
l

D. M. Berger et al. / Bioorg. Med. Chem. 16 (2008) 9202–9211
9207
Concentration:
0
0.03
0.1
0.3
1
3
(μM)
pMAPK
MAPK
Dose (μM) % Inhibition
0
0.03
0.1
0.3
1
0
22
18
69
88
95
3
Figure 1. Inhibition of p-MAPK in WM266-4 cells treated with varying concentrations of 5m. Cells were incubated with compound for 2.5 h. Cell lysates were prepared and
analyzed as described in Section 5. Inhibition of p-MAPK was determined by densitometric measurement of autoradiograms, followed by normalizing these values by
calculating a p-MAPK/MAPK ratio. Ratios of treated samples were divided by the ratio determined for the control to derive the percent inhibition at that dose. By plotting
these values, the IC50 of p-MAPK inhibition was calculated be 0.225 lM.
Table 4
Pharmacokinetic properties of 5m in nude mouse and rat
Control
100 mg/kg 5m
Species
Female nude mouse
Female rat
Male rat
Route
Intravenous
Dose (mg/kg)
Cl (mL/min/kg)
Vss (L/kg)
T1/2 (h)
Route
Dose (mg/kg)
Cmax (ng/mL)
AUCINF (ng h/mL)
Oral bioavailability (% F)
2
1
1
pMAPK
MAPK
66.0
2.7
0.9
Oral
50
510
2170
17
35.7
5.7
2.5
70.3
7.6
1.8
10
10
61
204
13
298
988
23
Figure 3. p-MAPK suppression in H358 tumors following 21 days oral dosing with
5m at 100 mg/kg twice daily. Tumors excised from euthanized animals were stored
at À80 °C until assayed. Tumor lysates were prepared and analyzed as outlined in
Section 5.
Note. Cl, clearance; Vss, steady state volume of distribution; T1/2, half-life of com-
pound in plasma; Cmax, maximum observed plasma concentration; AUCINF, area
under the concentration curve extrapolated to infinity.
5. Experimental
500
450
400
350
300
250
200
5.1. General methods
Melting points were determined in open capillary tubes on a
Meltemp melting point apparatus and are uncorrected. 1H NMR
spectra were determined at 400 MHz on a DRX-400 spectrometer,
with chemical shifts (d) reported in parts per million referenced to
Me4Si. Electrospray (ES) mass spectra were recorded in positive
mode on a Micromass Platform or an LCT spectrometer. Electron
Impact (EI) mass spectra were obtained on a Finnigan MAT-90
spectrometer. All reagents and solvents obtained from commercial
suppliers were used without further purification. Reactions were
carried out under nitrogen as an inert atmosphere. Flash chroma-
150
100
50
control
tography was carried out with Baker 40 lM silica gel.
5m 25 mpk
5m 50 mpk
5m 100 mpk
5.2. Specific procedures
5.2.1. (E)-1-(tri-n-Butylstannyl)-1-buten-4-ol (2b)
0
T0
T7
T14
T21
A stirred mixture of 3-butyn-1-ol (3.50 g, 50 mmol) and AIBN
(0.25 g, 1.5 mmol), contained in large round-bottomed flask
equipped with a reflux condenser, was outgassed with N2 at
25 °C and treated with tri-n-butyltin hydride (20.2 mL, 75 mmol).
The mixture was slowly heated to 90 °C and the resulting vigorous
exothermic reaction was moderated by removal of the heating
bath and reflux of the butynol. After the initial reaction, the
days
Figure 2. Dose–response of 5m dosed orally twice daily (bid) versus H358 (lung)
xenografts implanted in nude mice. Each group (n = 15) was dosed twice daily with
compound in vehicle or vehicle alone (0.05% methocel/2% Tween). Tumor size at all
time points in treatment arms of 50 mg and 100 mg/kg were statistically different
than control tumors (p 6 0.05, Student’s t-test).

9208
D. M. Berger et al. / Bioorg. Med. Chem. 16 (2008) 9202–9211
solution was stirred at 100 °C for 18 h. The solution was cooled and
the crude product was subjected to chromatography on silica gel
with a 3–20% gradient of EtOAc in hexane. After elution of a small
amount of Z-isomer, the E-isomer was obtained as a colorless oil
(10.3 g, 57%). The NMR spectrum was identical to that reported.14
flash chromatography (silica gel, CH2Cl2/EtOAc/HOAc) to give
(2E)-3-[4-({3-chloro-4-[(1-methyl-1H-imidazol-2-yl)thio]pheny-
l}amino)-3-cyanoquinolin-7-yl]prop-2-enyl acetate as a light yel-
low solid, mp 181–193 °C (dec.) MS (ESI) m/z 490.1 [M+H]+; 1H
NMR (400 MHz, DMSO-d6) d 9.89 (s, 1H), 8.62 (s, 1H), 8.35 (d,
J = 9.3 Hz, 1H), 7.89 (br s, 2H), 7.55 (s, 1H), 7.46 (s, 1H), 7.18 (m,
1H), 7.17 (s, 1H), 6.89 (d, J = 6.9 Hz, 1H), 6.70 (m, 1H), 6.53 (d,
J = 6.9 Hz, 1H), 4.78 (d, J = 5.3 Hz, 2H), 3.61 (s, 3H), 2.10 (s, 3H).
Step 2: A stirred mixture of the crude acetate (2E)-3-[4-
({3-chloro-4-[(1-methyl-1H-imidazol-2-yl)thio]phenyl}amino)-3-
cyanoquinolin-7-yl]prop-2-enyl acetate (196 mg, 0.40 mmol), pyr-
5.2.2. 4-({3-Chloro-4-[(1-methyl-1H-imidazol-2-yl)thio]phenyl}-
amino)-7-[(1E)-3-hydroxyprop-1-enyl]quinoline-3-carbonitrile
(5a)
To 10 mL of N2-purged NMP was added Ph3P (280 mg,
1.08 mmol) and Pd(OAc)2 (61 mg, 0.27 mmol). The mixture was
warmed to 50 °C and stirred for 1 h. The resulting red solution of
palladium catalyst21 was cooled to 0 °C and treated successively
with 4a (1.26 g, 2.67 mmol) and a solution of stannane 2a13
(1.40 g, 4.0 mmol) in 3.4 mL of NMP. The mixture was warmed to
100 °C during 20 min, stirred for 1 h, cooled to 25 °C, and stirred
with H2O and 2:1 hexane/Et2O. The resulting tan solid was filtered
off, washed with H2O and 2:1 hexane/Et2O, and dried to give 1.18 g
(98% yield) of 5a. A portion of this material was digested in hot ace-
tone to provide a light tan solid, mp 220–240 °C (dec.); MS (ESI) m/
z 448.0 [M+H]+; 1H NMR (400 MHz, DMSO-d6) d 9.87 (s, 1H), 8.62
(s, 1H), 7.87 (s, 1H), 7.84 (s, 1H), 7.55 (s, 1H), 7.46 (d, J = 2.5 Hz,
1H), 7.19 (d, J = 2.5 Hz), 7.17 (s, 2H), 6.77 (m, 1H), 6.54 (d,
J = 8.7 Hz, 1H), 5.02 (t, J = 5.5 Hz, 1H), 4.20 (m, 2H), 3.61 (s, 3H). Ele-
mental Anal. Calcd for C23H18ClN5OSÁ0.8H2O: C, 59.75; H, 4.27; N,
15.15. Found: C, 60.01; H, 4.06; N, 14.05.22
rolidine (133 lL, 1.6 mmol), and 0.80 mL of NMP was treated with
Pd(Ph3P)4 (46 mg, 0.04 mmol) under N2 at 25 °C. After 1 h, the mix-
ture was stirred with 4:1 hexane/EtOAc and dilute NaHCO3. The
crude product was filtered off, washed with water and 4:1 hexane/
EtOAc, and dried. Purification by flash chromatography (silica gel,
25:25:2:1 CH2Cl2/EtOAc/MeOH/TEA) gave 5d as 106 mg (53%) of a
yellow solid, mp 182–190 °C; MS (ESI) m/z 501.1 [M+H]+; 1H NMR
(400 MHz, DMSO-d6) d 9.86 (s, 1H), 8.60 (s, 1H), 8.32 (d, J = 9.1 Hz,
1H), 7.88 (s, 1H), 7.86 (d, J = 9.1 Hz, 1H), 7.52 (d, J = 1.2 Hz, 1H),
7.44 (s, 1H), 7.16 (d, J = 1.2 Hz, 1H), 7.14 (m, 1H), 6.73 (m, 2H), 6.54
(d, J = 8.5 Hz, 1H), 3.61 (s, 3H), 3.37 (m, 2H), 2.61 (m, 2H), 1.74 (m,
4H). Elemental Anal. Calcd for C27H25ClN6SÁ0.3H2O: C, 64.03; H,
5.09; N, 16.59. Found: C, 64.06; H, 5.02; N, 16.54.
5.2.6. 4-({3-Chloro-4-[(1-methyl-1H-imidazol-2-yl)thio]phenyl}-
amino)-7-[(1E)-3-(4-methylpiperazin-1-yl)prop-1-enyl]quino-
line-3-carbonitrile (5e)
5.2.3. 4-({3-Chloro-4-[(1-methyl-1H-imidazol-2-yl)thio]phenyl}-
amino)-7-[(1E)-4-hydroxybut-1-enyl]quinoline-3-carbonitrile (5b)
Compound 5b was prepared by the procedure used for the
preparation of compound 5a using stannane 2b. Flash chromatog-
raphy (silica gel, 10:1 CH2Cl2/MeOH) gave a 77% yield of 5b as an
amber solid, mp 205–210 °C; MS (ESI) m/z 462.2 [M+H]+; 1H
NMR (400 MHz, DMSO-d6) d 9.86 (s, 1H), 8.60 (s, 1H), 8.31 (s,
1H), 7.82 (s, 1H), 7.80 (s, 1H), 7.55 (d, J = 1.3 Hz, 1H), 7.44 (s, 1H),
7.17 (d, J = 1.3 Hz, 1H), 6.66 (m, 1H), 6.53 (d, J = 8.5 Hz, 1H), 4.66
(t, J = 5.3 Hz, 1H), 3.61 (s, 3H), 3.58 (m, 2H), 2.42 (m, 2H). Elemental
Anal. Calcd for C24H20ClN5OSÁ0.9CH3OH: C, 60.93; H, 4.85; N, 14.27.
Found: C, 61.09; H, 4.46; N, 14.05.
Following the procedure described for 5d, allylic alcohol 5a was
acetylated and reacted with N-methylpiperazine to provide com-
pound 5e as a yellow solid in 27% yield, mp 209–212 (dec.); MS
(ESI) m/z 530.1 [M+H]+; 1H NMR (400 MHz, DMSO-d6) d 9.89 (s,
1H), 8.60 (s, 1H), 7.54 (s, 1H), 7.44 (s, 1H), 7.16 (br s, 2H), 6.76 (d,
1H, J = 8.0 Hz), 6.58–6.67 (br m, 3H), 6.55 (d, 1H, J = 10.0 Hz), 3.61
(s, 3H), 3.15 (d, J = 6.7 Hz, 2H), 2.50 (s, 3H), 2.44 (br s, 4H), 2.34 (br
s, 4H), 2.17 (s, 3H). Elemental Anal. Calcd for C28H28ClN7SÁ0.45CH3
OH: C, 62.76; H, 5.35; N, 18.00. Found: C, 62.61; H, 5.57; N, 17.64.
5.2.7. 4-({3-Chloro-4-[(1-methyl-1H-imidazol-2-yl)thio]phenyl}-
amino)-7-[(1E)-3-(4-ethylpiperazin-1-yl)prop-1-enyl]quinoline-
3-carbonitrile (5f)
5.2.4. 4-({3-Chloro-4-[(1-methyl-1H-imidazol-2-yl)thio]phenyl}-
amino)-7-[(1E)-4-hydroxybut-1-enyl]-6-methoxyquinoline-3-car-
bonitrile (5c)
Following the procedure described for 5d, allylic alcohol 5a was
acetylated and reacted with N-ethylpiperazine to provide com-
pound 5f as a yellow solid in 36% yield, mp 224–225; MS (ESI)
m/z 544.1 [M+H]+; 1H NMR spectrum (400 MHz, DMSO-d6) d 9.89
(s, 1H), 8.60 (s, 1H), 7.54 (s, 1H), 7.44 (s, 1H), 7.16 (br s, 2H), 6.76
(d, 1H, J = 8.0 Hz), 6.58–6.67 (br m, 3 H), 6.55 (d, 1H, J = 10.0 Hz),
3.61 (s, 3H), 3.15 (d, J = 6.7 Hz, 2H), 2.50 (br s, 2H), 2.44 (br s,
4H), 2.34 (d, J = 6.0 Hz, 4H), 0.99 (t, 3H). Elemental Anal. Calcd for
C29H30ClN7SÁ 0.75H2O: C, 62.46; H, 5.69; N, 17.58. Found: C,
62.14; H, 5.62; N, 17.40.
Compound 5c was prepared from triflate 4b and stannane 2b by
the procedure used for the preparation of 5b. Digestion of the
crude product with 10:1 CH2Cl2/MeOH gave a 35% yield of 5c as
a white solid, mp 273–278 °C; MS (ESI) m/z 492.0 [M+H]+; 1H
NMR (400 MHz, DMSO-d6) d 9.67 (s, 1H), 8.50 (s, 1H), 7.98 (s,
1H), 7.70 (s, 1H), 7.55 (d, J = 1.2 Hz, 1H), 7.44 (m, 1H), 7.16 (d,
J = 1.2 Hz, 1H), 6.79 (d, J = 16.2 Hz, 1H), 6.62 (m, 2H), 6.56 (d,
J = 8.6 Hz, 1H), 4.65 (t, J = 5.3 Hz), 1H), 3.94 (s, 3H), 3.60 (s, 3H),
3.94 (s, 3H), 3.56 (m, 2H), 2.42 (m, 2H). Elemental Anal. Calcd for
C25H22ClN5O2SÁ0.5CH3OH: C, 60.29; H, 4.76; N, 13.79. Found: C,
60.55; H, 4.44; N, 13.84.
5.2.8. 4-({3-Chloro-4-[(1-methyl-1H-imidazol-2-yl)thio]phenyl}-
amino)-7-[(1E)-4-(diethylamino)but-1-enyl]quinoline-3-carboni-
trile (5g)
5.2.5. 4-({3-Chloro-4-[(1-methyl-1H-imidazol-2-yl)thio]phenyl}-
amino)-7-[(1E)-3-pyrrolidin-1-ylprop-1-enyl]quinoline-3-carbo-
nitrile (5d)
Step 1: A mixture of 5b (0.77 g, 1.67 mmol), tosyl chloride
(1.59 g, 8.35 mmol), and 16.7 mL of 2,6-lutidine was stirred at
45 °C for 18 h and stirred at 25 °C with water (0.75 mL; 42 mmol)
for 1 h. The resulting suspension was partitioned with CH2Cl2 and
dilute NaHCO3. The organic layer was washed with water, dried,
and concentrated. The residue was stirred in 10:1 Et2O/acetone.
The tan solid was filtered off, washed with Et2O, and dried at
25 °C to give 0.84 g, mp 135–150 °C (dec.). The crude (2E)-3-[4-
({3-chloro-4-[(1-methyl-1H-imidazol-2-yl)thio]phenyl}amino)-3-
cyanoquinolin-7-yl]but-3-enyl 4-methylphenylsulfonate was used
without further purification.
Step 1: A solution of 5a (1.43 g, 3.2 mmol) and acetic anhydride
(24 mL, 254 mmol) in 24 mL of HOAc was stirred at 50 °C for 19 h
and concentrated to dryness. The residue was stirred in dilute NaH-
CO3 and 1:1 hexane/Et2O. The resulting solid was filtered, washed
with water and 1:1 hexane/Et2O, and dried to give 1.64 g (100%) of
(2E)-3-[4-({3-chloro-4-[(1-methyl-1H-imidazol-2-yl)thio]phenyl}
amino)-3-cyanoquinolin-7-yl]prop-2-enyl acetate as an amber so-
lid, sufficiently pure for the next step. A portion was subjected to

D. M. Berger et al. / Bioorg. Med. Chem. 16 (2008) 9202–9211
9209
Step 2: To a stirred suspension of crude (2E)-3-[4-({3-chloro-4-
[(1-methyl-1H-imidazol-2-yl)thio]phenyl}amino)-3-cyanoquino-
lin-7-yl]but-3-enyl 4-methylphenylsulfonate (185 mg, 0.30 mmol)
in 5.0 mL of THF was added diethylamine (2.5 mL, 24 mmol). The
resulting solution was stirred at 60 °C for 20 h. The mixture was
concentrated to remove THF, and the residue was partitioned be-
tween 20:1 CH2Cl2/MeOH and dilute NaHCO3. The organic layer
was washed with water, dried, and concentrated. Flash chromatog-
raphy (silica gel, EtOAc/MeOH/TEA) gave a 30% yield of 5g as an
off-white solid, mp 230–235 °C; MS (ESI) m/z 517.1 [M+H]+; 1H
NMR (400 MHz, DMSO-d6) d 9.85 s, 1Y, 8.59 s, 1H), 8.30 (d,
J = 8.8 Hz, 1H), 7.81 (s, 1H), 7.78 (d, J = 8.8 Hz, 1H), 7.54 (d,
J = 1.1 Hz, 1H), 7.43 (s, 1H), 7.16 (d, J = 1.1 Hz, 1H), 7.15 (m, 1H),
6.67 (m, 2H), 6.54 (d, J = 8.6 Hz, 1H), 3.61 (s, 3H), 2.56 (m, 2H),
2.37 (q, J = 6.8 Hz, 4H), 0.98 (t, J = 6.8 Hz, 6H). Elemental Anal. Calcd
for C28H29ClN6S: C, 65.04; H, 5.65; N, 16.25. Found: C, 64.74; H,
5.42; N, 16.06.
(s, 1H), 7.80 (d, J = 8.6 Hz, 1H), 7.55 (d, J = 1.1 Hz, 1H), 7.44 (s,
1H), 7.17 (d, J = 1.1 z, 1H), 7.16 (m, 1H), 6.66 (m, 2H), 6.53 (d,
J = 8.6 Hz, 1H), 3.61 (s, 3H), 3.58 (m, 4H), 2.43 (m, 8H). Elemental
Anal. Calcd for C28H27ClN6OS: C, 63.33; H, 5.12; N, 15.82. Found:
C, 63.13; H, 4.89; N, 15.47.
5.2.12. 4-({3-Chloro-4-[(1-methyl-1H-imidazol-2-yl)thio]phenyl}-
amino)-7-[(1E)-4-pyrrolidin-1-ylbut-1-enyl]quinoline-3-carbonitrile
(5j)
Following the procedure described for 5i, 6a was reacted with
4a to provide compound 5j as an off-white solid in 40% yield, mp
185–191 °C; MS (ESI) m/z 515.1 [M+H]+; 1H NMR (400 MHz,
DMSO-d6)
d 9.87 (s, 1H), 8.31 (d, J = 8.6 Hz, 1H), 7.81 (d,
J = 8.6 Hz, 1H), 7.55 (d, J = 1.1 Hz, 1H), 7.44 (s, 1H), 7.17 (d,
J = 1.1 Hz, 1H), 7.16 (m, 1H), 6.66 (m, 2H), 6.53 (d, J = 8.6 Hz, 1H),
3.61 (s, 3H), 2.55 (m, 4H), 2.45 (m, 4H), 1.71 (m, 4H). Elemental
Anal. Calcd for C28H27ClN6SÁ0.25H2O: C, 64.73; H, 5.33; N, 16.17.
Found: C, 64.67; H, 5.14; N, 16.07.
5.2.9. 4-({3-Chloro-4-[(1-methyl-1H-imidazol-2-yl)thio]phenyl}-
amino)-7-[(1E)-4-(dimethylamino)but-1-enyl]quinoline-3-carbo-
nitrile (5h)
5.2.13. 4-({3-Chloro-4-[(1-methyl-1H-imidazol-2-yl)thio]phenyl}-
amino)-7-[(1E)-4-piperidin-1-ylbut-1-enyl]quinoline-3-carboni-
trile (5k)
Following the procedure described for 5g, allylic alcohol 5b was
tosylated and reacted with dimethylamine to provide compound
5h as an off-white solid in 32% yield, mp 191–198 °C; MS (ESI)
m/z 489.1 [M+H]+; 1H NMR (400 MHz, DMSO-d6) d 9.87 s, 1H),
8.59 (s, 1H), 8.30 (d, J = 9.3 Hz, 1H), 7.81 (s, 1H), 7.80 (d,
J = 9.3 Hz, 1H), 7.55 (d, J = 1.1 Hz, 1H), 7.44 (s, 1H), 7.17 (d,
J = 1.1 Hz, 1H), 7.16 (m, 1H), 6.66 (m, 2H), 6.53 (d, J = 8.4 Hz, 1H),
3.61 (s, 3H), 2.40 (m, 4H), 2.18 (s, 6H). Elemental Anal. Calcd for
C26H25ClN6SÁ0.25CH3CO2C2H5: C, 63.45; H, 5.32; N, 16.44. Found:
C, 63.68; H, 5.09; N, 16.28.
Following the procedure described for 5g, allylic alcohol 5b was
tosylated and reacted with piperidine to provide compound 5k as
an off-white solid in 48% yield, mp 205–210 °C; MS (ESI) m/z 529.1
[M+H]+; 1H NMR (400 MHz, DMSO-d6) d 9.86 (s, 1H), 8.60 (s, 1H),
8.30 (d, J = 9.1 Hz, 1H), 7.81 (s, 1H), 7.80 (d, J = 9.1 Hz, 1H), 7.55
d, J = 1.1 Hz, 1H), 7.44 (s, 1H), 7.17 (d, J = 1.1 Hz, 1H), 7.16 (m,
1H), 6.66 (m, 2H), 6.53 (d, J = 8.6 Hz, 1H), 3.61 (s, 3H), 2.43 (m,
2H), 2.38 (m, 4H), 1.50 (m, 4H), 1.38 (m, 4H). Elemental Anal. Calcd
for C29H29ClN6S: C, 65.83; H, 5.52; N, 15.88. Found: C, 65.59; H,
5.13; N, 15.48.
5.2.10. Preparation of 6a–e. Representative procedure:1-methyl-
4-[(3E)-4-(tri-n-butylstannyl)but-3-enyl]piperazine (6c)
5.2.14. 4-({3-Chloro-4-[(1-methyl-1H-imidazol-2-yl)thio]phenyl}-
amino)-7-[(1E)-4-(4-methylpiperazin-1-yl)but-1-enyl]quinoline-3-
carbonitrile (5l)
Step 1: To a stirred solution of (E)-1-(tri-n-butylstannyl)-1-bu-
ten-4-ol (5.42 g, 15 mmol) in 30 mL of 2,6-lutidine was added tosyl
chloride (8.58 g, 45 mmol) while maintaining at 25 °C. After 20 h,
the excess tosyl chloride was decomposed by the addition of
30 mL of water and 5 mL of pyridine while cooling to maintain at
25 °C. The resulting mixture was portioned with CH2Cl2 and dilute
NaHCO3. The organic layer was washed with water and dried over
MgSO4. Following concentration at <35 °C, finally at 0.5 mmHg,
crude (3E)-4-(tri-n-butylstannyl)but-3-enyl 4-methylbenzenesulf-
onate was obtained as an oil.
Step 2: A solution of the crude (3E)-4-(tri-n-butylstannyl)but-3-
enyl 4-methylbenzenesulfonate (1.55 g, 3.0 mmol), 1-methylpiper-
azine (1.33 mL, 12 mmol), and 3.0 mL of THF was stirred for 24 h at
25 °C and subsequentlyheated to45 °C for2 h. Thesolutionwas con-
centrated to dryness under vacuum, and the residue was partitioned
with 1:1 hexane/Et2O and dilute NaHCO3. The organic layer was
washed with water, dried, and concentrated at <30 °C to give 6c as
an amber oil which was used in the next step without purification.
To a mixture of 7a (1.52 g, 10.0 mmol) and 4,4,5,5-tetramethyl-
[1,3,2]dioxaborolane (2.98 g, 17.2 mmol) was added bis(cyclopen-
tadienyl)-zirconium chloride hydride (0.13 g, 0.05 mmol). The
resulting mixture was stirred at room temperature for 24 h to pro-
vide (E)-1-methyl-4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)but-3-enyl)piperazine (8a), which was subsequently dissolved
in toluene/ethanol/water (100 mL/10 mL/10 mL). Compound 4a
(2.36 g, 5.0 mmol), K2CO3 (2.1 g, 15.2 mmol), and Pd(PPh3)4
(0.46 g, 0.4 mmol) were added. The reaction mixture was heated
at 85 °C for 5 h and allowed to cool to room temperature. To this
mixture was added 100 mL of a saturated NaHCO3 solution. The
resulting solid was collected by filtration, washed with water,
EtOAc, and dried under vacuum. The crude solid was purified by
silica gel flash column chromatography (gradient 95:5–4:1
CH2Cl2/MeOH) to give 1.70 g (63%) of 5l as an off-white solid, mp
142–143 °C; MS (ESl) m/z 544.2 [M+H]+; 1H NMR (400 MHz,
DMSO-d6) d 9.86 (s, 1H), 8.59 (s, 1H), 8.30 (d, J = 8.7 Hz, 1H), 7.80
(s, 1H), 7.79 (d, J = 8.7 Hz, 1H), 7.54 (d, J = 1.1 Hz, 1H), 7.44 (s,
1H), 7.16 (d, J = 1.1 Hz, 1H), 7.15 (m, 1H), 6.64 (m, 2H), 6.54 (d,
J = 8.6 Hz, 1H), 3.61 (s, 3H), 2.37 (m, 12H), 2.15 (s, 3H). HPLC anal-
ysis (CH3CN/H2O/TFA reverse phase) showed the trans/cis ratio to
be 98.5:0.5. Elemental Anal. Calcd for C29H30ClN7SÁ0.2H2O: C,
63.59; H, 5.59; N, 17.90. Found: C, 63.46; H, 5.57; N, 17.84.
5.2.11. 4-({3-Chloro-4-[(1-methyl-1H-imidazol-2-yl)thio]phenyl}
amino)-7-[(1E)-4-morpholin-4-ylbut-1-enyl]quinoline-3-carbo-
nitrile (5i)
To a N2-purged mixture of 4a (188 mg, 0.40 mmol), 6b (0.24 g,
0.56 mmol), and 2.0 mL of NMP was added Pd(Ph3P)4 (46 mg,
0.04 mmol). The mixture was warmed to 100 °C, and the resulting
solution was stirred for 1 h, cooled, and partitioned with CH2Cl2
and water. The organic layer was dried and concentrated to give
a residue that was stirred in 10:1 Et2O/hexane. The resulting solid
was filtered off and subjected to flash chromatography (silica gel,
5:1 EtOAc/MeOH) to give 134 mg (63%) of 5i as a light yellow solid,
mp 232–238 °C; MS (ESI) m/z 531.1 [M+H]+; 1H NMR (400 MHz,
DMSO-d6) d 9.86 (s, 1H), 8.60 (s, 1H), 8.31 (d, J = 8.6 Hz, 1H), 7.81
5.2.15. 4-({3-Chloro-4-[(1-methyl-1H-imidazol-2-yl)thio]phenyl}-
amino)-7-[(1E)-4-(4-ethylpiperazin-1-yl)but-1-enyl]quinoline-
3-carbonitrile (5m)
Following the procedure described for 5l, 7b was converted 8b,
and reacted with 4a to provide compound 5m as a yellow solid in
73% yield, mp 194–204 °C; MS (ESI) m/z 558.1 [M+H]+; 1H NMR

9210
D. M. Berger et al. / Bioorg. Med. Chem. 16 (2008) 9202–9211
(400 MHz, DMSO-d6) d 9.84 (s, 1H), 8.60 (s, 1H), 8.30 (d, J = 8.8 Hz,
1H), 7.79 (m, 2H), 7.54 (s, 1H), 7.43 (s, 1H), 7.16 (s, 2H), 6.65 (m,
2H), 6.54 (d, J = 8.8 Hz, 1H), 3.61 (s, 3H), 2.46–2.29 (m, 14H), 0.98
(t, J = 6.8 and 7.2 Hz, 3H). Elemental Anal. Calcd for
C30H32ClN7SÁ0.3H2O: C, 63.94; H, 5.83; N, 17.40. Found: C, 64.01;
H, 5.67; N, 17.14.
215–221 (dec.) °C; MS (ESI) m/z 588.1 [M+H]+; 1H NMR
(400 MHz, DMSO-d6) d 9.66 (s, 1H), 8.50 (s, 1H), 7.98 (s, 1H), 7.69
(s, 1H), 7.54 (d, J = 1.2 Hz, 1H), 7.44 (s, 1H), 7.17 (m, 1H), 7.16 (d,
J = 1.2 Hz, 1H), 6.75 (d, J = 15.9 Hz, 1H), 6.63 (dt, J = 15.9 and
6.7 Hz, 1H), 6.57 (d, J = 8.6 Hz, 1H), 3.94 (s, 3H), 3.60 (s, 3H), 2.29
(m, 12H), 2.16 (s, 3H), 1.62 (m, 2H). Elemental Anal. Calcd for
C31H34ClN7OSÁ0.5CH3OH: C, 62.62; H, 6.01; N, 16.23. Found: C,
62.64; H, 5.71; N, 16.14.
5.2.16. 4-({3-Chloro-4-[(1-methyl-1H-imidazol-2-yl)thio]phenyl}-
amino)-7-[(1E)-5-(diethylamino)pent-1-enyl]quinoline-3-carbo-
nitrile (5n)
5.3. Biological evaluation
Following the procedure described for 5i, 6d was reacted with
4a to provide compound 5n as an off-white solid in 49% yield,
mp 220–225 °C; MS (ESI) m/z 531.1 [M+H]+; 1H NMR (400 MHz,
DMSO-d6) d 9.86 (s, 1H), 8.59 (s, 1H), 8.30 (d, J = 8.8 Hz, 1H), 7.81
(s, 1H), 7.80 (d, J = 8.8 Hz, 1H), 7.54 (d, J = 1.1 Hz, 1H), 7.42 (d,
J = 1.8 Hz, 1H), 7.16 (d, J = 1.1 Hz, 1H), 7.14 (d, J = 1.8 Hz, 1H),
6.66 (m, 2H), 6.54 (d, J = 8.6 Hz, 1H), 3.61 (s, 3H), 2.47 (q,
J = 7.3 Hz, 4H), 2.43 (m, 2H), 2.26 (m, 2H), 1.59 (m, 2H), 0.95 (t,
J = 7.3 Hz, 6H). Elemental Anal. Calcd for C29H31ClN6S: C, 65.58;
H, 5.88; N, 15.82. Found: C, 65.59; H, 5.91; N, 15.79.
5.3.1. Enzyme studies
MEK1 enzyme activities were determined by the previously de-
scribed high throughput, non-radioactive Raf/MEK1/MAPK ELISA
assay.18 Additional in-house protein kinase assays were performed
by immunologically based ELISA or DELFIA protocols using the
appropriate phospho-specific antibodies.
5.3.2. Cellular studies
Cellular proliferation assays19 (Sulfo-rhodamine B staining)
were carried out using LoVo, CaCo-2 (colon), BXPC3 (pancreatic)
and WM266-4 and A375 (melanoma) human tumor cell lines.
5.2.17. 4-({3-Chloro-4-[(1-methyl-1H-imidazol-2-yl)thio]phenyl}-
amino)-7-[(1E)-5-(4-methylpiperazin-1-yl)pent-1-enyl]quinoline-
3-carbonitrile (5o)
5.3.3. Phosphorylation studies
Following the procedure described for 5i, 6e was reacted with 4a
to provide compound 5o as an off-white solid in 64% yield, mp 219–
227 (dec.) °C; MS (ESI) m/z 558.1 [M+H]+; 1H NMR (400 MHz, DMSO-
d6) d 9.85 (s, 1H), 8.58 (s, 1H), 8.30 (d, J = 8.8 Hz, 1H), 7.81 (s, 1H), 7.80
(d, J = 8.8 Hz, 1H), 7.54 (d, J = 1.1 Hz, 1H), 7.42 (m, 1H), 7.16 (d,
J = 1.1 Hz, 1H), 7.14 (m, 1H), 6.65 (m, 2H), 6.54 (d, J = 8.8 Hz, 1H),
3.61 (s, 3H), 2.51 (m, 6H), 2.31 (m, 6H), 2.14 (s, 3H), 1.62 (m, 2H). Ele-
mental Anal. Calcd for C30H32ClN7S: C, 64.56; H, 5.78; N, 17.57.
Found: C, 64.27; H, 5.67; N, 17.54.
WM266-4 human melanoma cells were grown in DMEM with
10% FBS. For phosphoblot analysis, cells were grown in 6-well
plates and when semi-confluent, exposed to compound for 2.5 h.
Cells were solubilized at 4 °C in cell lysis buffer (150 mM NaCl,
10 mM Tris, pH 7.4, 1% (w/v) Triton X-100, 0.5% (w/v) sodium
deoxycholate, 0.1% (w/v) SDS with protease inhibitors: 1 mM
EDTA, 7.5 TIU/mL aprotinin, 10 lg/mL pepstatin A, 30 lM leupep-
tin and a 1:100 dilution of phosphatase inhibitor cocktails I (P-
2850) and II (P-5726) (Sigma Chemical Co., St. Louis, MO)). Lysate
was clarified by centrifugation at 17,000g for 10 min. Total lysate
protein was determined by the BCA protein assay (Pierce Biochem-
5.2.18. 4-({3-Chloro-4-[(1-methyl-1H-imidazol-2-yl)thio]phenyl}-
amino)-7-[(1E)-4-(diethylamino)but-1-enyl]-6-methoxyquinoline-
3-carbonitrile (5p)
ical). Equal amounts of protein lysate (30 lg) were separated on
SDS–PAGE gels (4–20% gradient) and transferred to nitrocellulose.
After blocking non-specific binding sites on the nitrocellulose blot
with 3% (w/v) BSA in TBS-T (Tris-buffered saline with 0.1% Tween
20), blots were incubated with anti-MAPK (diluted 1:1000) or anti-
phosphoMAPK (diluted 1:2000) (Sigma Chemical Co., St. Louis,
MO) for 1.5 h followed by washing in TBS-T. The blots were then
incubated with appropriate secondary antibody conjugated to
horse radish peroxidase (Sigma Chemical Co, St. Louis, MO) The
blots were again washed with TBS-T followed by exposure to ECL
and exposure to autoradiographic film.
Following the procedure described for 5g, allylic alcohol 5c was
tosylated and reacted with diethylamine to provide compound 5p
as a yellow solid in 54% yield, mp 194–202 °C; MS (ESI) m/z 547.2
[M+H]+; 1H NMR (400 MHz, DMSO-d6) d 9.70 (s, 1H), 8.50 (s, 1H),
7.96 (s, 1H), 7.71 (s, 1H), 7.54 (d, J = 1.2 Hz, 1H), 7.44 (s, 1H), 7.16
(d, J = 1.2 Hz, 1H), 7.15 (m, 1H), 6.79 (d, J = 15.9 Hz, 1H), 6.61 (dt,
J = 15.9 and 7.0 Hz, 1H), 6.56 (d, J = 8.8 Hz), 1H), 5.30 (s, 1H), 3.94
(s, 3H), 3.60 (s, 3H), 3.36 (q, J = 7.0 Hz, 4H), 2.57 (m 3H), 0.98 (t,
J = 7.0 Hz, 6H). Elemental Anal. Calcd for C29H31ClN6OSÁ1.25H2O:
C, 61.15; H, 5.93; N, 14.75. Found: C, 61.44; H, 6.02; N, 14.41.
5.3.4. In vivo studies
5.2.19.4-({3-Chloro-4-[(1-methyl-1H-imidazol-2-yl)thio]phenyl}-
amino)-6-methoxy-7-[(1E)-4-(4-methylpiperazin-1-yl)but-1-en-
yl]quinoline-3-carbonitrile (5q)
All animal studies were performed in accordance with internal
IACUC standards. Human non-small cells lung cancer cells (Amer-
ican Type Culture Collection, Rockville, Maryland # CRL-5807)
were grown in RPMI (Gibco/BRL, Gaithersburg, MD) supplemented
with 10% FBS (Gemini Bio-Products Inc., Calabasas, CA). Athymic
nu/nu female mice (Charles River, Wilmington, MA) were injected
SC in the flank area with 10 Â 106 cells plus Matrigel (BD Biosci-
ences, Billerica, MA). When tumors attained a mass of between
200 and 250 mg, the mice were randomized into treatment groups
(day zero), 10 animals per group. Animals were administered com-
pound orally BID on days 1 through 20 post-staging (day zero) with
either 25, 50, or 100 mg/kg/dose of compound prepared in 0.5%
methocel/2% Tween 80 as the vehicle control. Tumor mass was
determined every 7 days [(length  width2)/2] for 21 days post-
staging. Relative tumor growth (mean tumor mass on days 7, 14,
and 21 divided by the mean tumor mass on day zero) was deter-
mined for each treatment group. Statistical analysis of the vehicle
control tumor group versus the treated groups at each time point
Following the procedure described for 5i, 6c was reacted with
4b to provide compound 5q as an off-white solid in 50% yield,
mp 223–230 (dec.) °C; MS (ESI) m/z 574.2 [M+H]+; 1H NMR
(400 MHz, DMSO-d6) d 9.67 (s, 1H), 8.50 (s, 1H), 7.96 (s, 1H), 7.70
(s, 1H), 7.54 (d, J = 1.2 Hz, 1H), 7.44 (s, 1H), 7.17 (m, 1H), 7.16 (d,
J = 1.2 Hz, 1H), 6.79 (d, J = 15.4 Hz, 1H), 6.59 (dt, J = 15.4 and
6.0 Hz, 1H), 6.56 (d, J = 8.9 Hz, 1H), 3.94 (s, 3H), 3.60 (s, 3H), 2.38
(m, 12H), 2.16 (s, 3H). Elemental Anal. Calcd for C30H32ClN7OS: C,
62.76; H, 5.62; N, 17.08. Found: C, 62.44; H, 5.54; N, 16.97.
5.2.20.4-({3-Chloro-4-[(1-methyl-1H-imidazol-2-yl)thio]phenyl}-
amino)-6-methoxy-7-[(1E)-5-(4-methylpiperazin-1-yl)pent-1-en-
yl]quinoline-3-carbonitrile (5r)
Following the procedure described for 5i, 6e was reacted with
4b to provide compound 5r as a yellow solid in 50% yield, mp

D. M. Berger et al. / Bioorg. Med. Chem. 16 (2008) 9202–9211
9211
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5.3.5. Exposure studies and pharmacokinetics
Compounds for oral (po) administration were made as a suspen-
sion in 0.5% methylcellulose and 1% Tween 80. For iv administration,
the compounds were dissolved in 0.5% methylcellulose and 1%
Tween 80, with the pH lowered to 3 by aqueous HCl. Mice were man-
ually randomized and treated with a single dose of 50 mg/kg by po
gavage. After drug dosing, three mice per treatment group were sac-
rificed at 0.25, 0.5, 1, 4, 8, and 24 h by cardiac stick exsanguinations
under isoflurane anesthesia, and plasma samples were collected.
Analysis of plasma was carried out using liquid chromatography/
mass spectrometry analysis. In brief, 50-lL plasma samples were ex-
tracted with 0.2 mL acetonitrile. The organic layer was collected fol-
lowing extraction, evaporated to dryness, and reconstituted in
0.05 mL of acetonitrile/water (1:1, v/v). Samples were analyzed with
an API-3000 triple quadrupole mass spectrometer (PE Sciex, Foster
City, CA). Analysis of data for the calculation of pharmacokinetic
parameters was carried out using noncompartmental analysis with
WinNonlin v. 4.1 (Pharsight Corporation, Mountain View, CA).
Acknowledgments
The authors acknowledge the Wyeth Analytical Chemistry
Department for providing spectral data and elemental analyses,
and the Chemical Technology group for the pharmaceutical profil-
ing data. Additionally, we thank Dr. Tarek Mansour for his support.
18. Mallon, R.; Feldberg, L. R.; Kim, S. C.; Collins, K.; Wojciechowicz, D.; Hollander,
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References and notes
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