Novel inhibitors of B-RAF based on a disubstituted pyrazine scaffold. Generation of a nanomolar lead

Article abstract of DOI:10.1021/jm050983g

B-RAF, a serine/threonine kinase, plays an important role in the development of certain classes of cancer, especially melanoma. As a result of high-throughput screening of a 23,000 compound library, 2-(3,4,5- trimethoxyphenylamino)-6-(3-acetamidophenyl)pyrazine, 1, was identified as a low micromolar (IC₅₀ = 3.5 μM) B-RAF inhibitor. This compound was chosen as the starting point of a program aimed at producing potent inhibitors of B-RAF. We have synthesized a series of 40 novel compounds, which involved extensive modifications to the 2-(3,4,5-trimethoxyphenylamino) moiety (ring A) of 1. Their biological profiles against isolated B-RAF and mutated B-RAF in a cellular assay have been determined. These efforts led to the identification of two compounds exhibiting activities lower than 800 nM against B-RAF.

Full text of DOI:10.1021/jm050983g

J. Med. Chem. 2006, 49, 407-416
407
Novel Inhibitors of B-RAF Based on a Disubstituted Pyrazine Scaffold. Generation of a
Nanomolar Lead
Ion Niculescu-Duvaz, Esteban Roman, Steven R. Whittaker, Frank Friedlos, Ruth Kirk, Ian J. Scanlon, Lawrence C. Davies,
Dan Niculescu-Duvaz, Richard Marais, and Caroline J. Springer*
Cancer Research UK Centre for Cancer Therapeutics at the Institute of Cancer Research, 15 Cotswold Road,
Sutton, Surrey SM2 5NG, United Kingdom
ReceiVed October 3, 2005
B-RAF, a serine/threonine kinase, plays an important role in the development of certain classes of cancer,
especially melanoma. As a result of high-throughput screening of a 23,000 compound library, 2-(3,4,5-
trimethoxyphenylamino)-6-(3-acetamidophenyl)pyrazine, 1, was identified as a low micromolar (IC50
)
3.5 µM) B-RAF inhibitor. This compound was chosen as the starting point of a program aimed at producing
potent inhibitors of B-RAF. We have synthesized a series of 40 novel compounds, which involved extensive
modifications to the 2-(3,4,5-trimethoxyphenylamino) moiety (ring A) of 1. Their biological profiles against
isolated B-RAF and mutated B-RAF in a cellular assay have been determined. These efforts led to the
identification of two compounds exhibiting activities lower than 800 nM against B-RAF.
Introduction
The protein kinase B-RAF forms part of a conserved signal
transduction pathway that regulates cellular responses to extra-
cellular signals.1,2 Under normal circumstances, B-RAF is
activated downstream of receptors in the cell membrane in a
RAS small G-protein dependent manner. It then phosphorylates
and activates the protein kinase MEK, which in turn activates
a third protein kinase called ERK. ERK phosphorylates tran-
scription factors such as ELK-1, regulating gene expression and
controlling how cells respond to extracellular signals.
B-RAF is mutated in approximately 7% of human cancers,3,4
especially melanoma (50-70%), ovarian (∼35%), thyroid
(∼30%) and colorectal (∼10%) cancers. The most common
mutation (∼90%) is a glutamic acid for valine substitution at
position 600 (V600E). The kinase activity of V600EB-RAF is
elevated 500-fold, providing cancer cells with both proliferation
and survival signals and allowing them to grow as tumors in
model systems.5 These data demonstrate that B-RAF is impor-
tant both in tumor induction and maintenance and that it is an
important and exciting new therapeutic target for several human
cancers. Novel anticancer drugs that inhibit the activity of this
kinase are therefore urgently sought.
High-throughput screening of a collection of potential inhibi-
tors resulted in the identification of a novel lead compound,
pyrazine 1. This compound exhibited low micromolar activity
against B-RAF and in a cellular assay in vitro. For this reason,
along with its synthetic tractability, compound 1 was chosen
as the initial point for structure optimization.
Rationale of the Design. RAF family proteins (A, B and C)
are serine-threonine kinases.6 A number of inhibitors, mainly
targeting C-RAF (Raf-1) such as benzylidene oxindoles,7 ZM
3363728 and sorafenib,9-12 have been developed. Recently
isoquinolines were reported as B-RAF inhibitors.13,14 The most
intensely studied, sorafenib, currently in phase III clinical trials,
proved to be active against B-RAF in cellular assays. However,
sorafenib, as a single agent, failed to show therapeutic activity
in the treatment of malignant melanoma, despite positive results
in renal carcinoma.15 This is probably because it also inhibits a
Figure 1. Structure of the hit 1.
number of other kinases (such as VEGFR, PDGFR),9 which
are also overexpressed in renal carcinoma. The reason for the
failure in malignant melanoma may be attributed to its inability
to reach a concentration in melanoma cells sufficient to inhibit
B-RAF. Therefore, more potent and more selective inhibitors
of B-RAF, able to produce clinical results in melanoma, are
needed.
The pyrazine hit described in this paper has a chemical
structure different from sorafenib and the other known RAF
inhibitors listed above. Therefore, it represents a unique starting
point for the development of B-RAF inhibitors.
Chemistry. Three domains can be described in compound 1
(Figure 1): a pyrazine central core containing an amino
functionality (denoted ring B), a trimethoxyphenyl moiety (ring
A) and an acetamidophenyl group (ring C). Our first objective
was the replacement of the trimethoxyphenyl group in order to
evaluate its importance in binding to B-RAF and to improve
the overall potency against the protein.
Compound 1 and 39 analogues (1, 6-44) modified on ring
A were synthesized in two steps starting from 2,6-dichloro-
pyrazine 2 (Scheme 1). A Suzuki coupling16,17 with 3-acet-
amidophenyl boronic acid in the presence of PdCl2-dppf:DCM
complex as catalyst and sodium carbonate as base gave
intermediate 3.
Intermediate 3 was treated with a number of commercially
available aromatic and heterocyclic amines (using liquid phase
parallel synthesis), via palladium amination conditions,18,19 to
generate a collection of analogues of the hit (compounds 6-30)
(Scheme 1). For the amination reactions, inert atmospheres (e.g.,
argon or nitrogen) and anhydrous degassed aprotic solvents
(toluene, dioxane) were used. The reactions were typically
performed at a temperature range of 80-110 °C.
Potassium tert-butoxide, sodium tert-butoxide or cesium
carbonate gave good results as bases, with reaction times within
* To whom correspondence should be addressed. Phone: +44 2087224214.
Fax: +44 2087224046. E-mail: caroline.springer@icr.ac.uk.
10.1021/jm050983g CCC: $33.50 © 2006 American Chemical Society
Published on Web 11/24/2005

408 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1
Niculescu-DuVaz et al.
Scheme 1. Synthesis of 2-N-Substituted-6-(3-acetamidophenyl)pyrazinesa
a Reagents and conditions: (a) 3-Acetamidophenyl boronic acid, PdCl2-dppf:DCM complex 1:1, 1 M Na2CO3, DME, 90 °C; (b) suitable arylamine,
Pd2dba3, imidazolium ligands, KOtBu, dioxane, conventional or microwave heating, 100 °C; (c) methylphenylamine, Pd2dba3, BINAP, NaOtBu, toluene, 90
°C; (d) suitable benzylamine, Et3N, DMF, 80 °C.
Scheme 2. Strategy for the Synthesis of Compound 10a
the range of 3-24 h under conventional heating. Alternatively,
microwave heating was employed to reduce reaction times (15-
30 min).
Tris(dibenzylideneacetone) dipalladium(0) (Pd2dba3) and pal-
ladacycles were used as palladium catalysts. Several ligands
were used, such as 1,3-bis(diphenylphosphino)propane (dppp),
rac-2,2′-bis(diphenylphosphino)-1,1′-binaphthyl (BINAP), 2(di-
cyclohexyl)phoshino-2′(dimethylamino) biphenyl, 1,3-bis(2,6-
diisopropylphenyl)-4,5-dihydro imidazolium tetrafluoroborate,
1,3-bis(2,4,6-trimethyl phenyl)-4,5-dihydroimidazolium tetra-
fluoroborate), and also 2-dicyclohexylphosphino-2′-(N,N-di-
methylamino)biphenyl.
For the synthesis of the benzylic amines, a different strategy
was used. Common intermediate 3 failed to react with benzylic
amines, probably because the chloropyrazine ring is rendered
less reactive upon introduction of the acetamido-phenyl group.
In contrast, the reaction of 2,6-dichloropyrazine with benzylic
amines in the presence of triethylamine proceeds with good yield
to afford intermediates 4a-p. These were subsequently treated
with 3-acetamidoboronic acid under Suzuki reaction conditions
to give the final targets 31-44 (route II, Scheme 1). The Suzuki
coupling was optimized in order to use liquid phase parallel
a Reagents and conditions: (a) KOtBu, K2CO3, DMF, 90 °C; (b) Pd2dba3,
KOtBu, imidazolium ligand, dioxane, 100 °C, microwave heating; (c) TFA,
r.t.
synthesis. We found this strategy to be more convenient for
in WM266.4 human melanoma cells expressing mutant B-RAF
the benzylic amines because of the ease of purification of the
with sulforhodamine-B (IC50SRB). The data are summarized
intermediates, since the reactions did not involve the use of a
in Table 1.
palladium catalyst or ligands and the yields were relatively high
(70-80%).
Compound 10 was synthesized via a protection-deprotection
strategy (Scheme 2). 3-Aminophenol 10a was reacted with
Our goal was to improve the potency of 1 by exploring the
SAR of ring A. First, we focused on investigating the role of
the methoxy groups (see Table 1). The removal of all of the
methoxy groups led to only a 2-fold decrease in potency
commercially available bromide 10b in the presence of potas-
(compound 6) for B-RAF.
sium tert-butoxide to afford amine 10c. Potassium carbonate
Of the three possible mono-methoxy isomers (8, 9, 11) only
was added to decrease the reaction time. The oxy-ethylamino
the 3-position appears particularly interesting since the corre-
chain was introduced as the protected t-Boc version by using
sponding isomer retains activity whereas the 4- and 2-methoxy
this method in order to prevent interference in the next step.
isomers are less active. Keeping two methoxy groups (in
Amine 10c was reacted under the amination conditions described
positions 3,4 and 3,5) (compounds 12 and 13) led to at least
equipotent compounds against B-RAF compared to 1. Other
functional groups were also introduced into the 3-position.
previously and subsequent cleavage of the t-Boc group with
trifluoroacetic acid (TFA) gave the target product 10.
Structure-Activity Relationships. The biological activities
of the compounds were determined using two assays: the
determination of the IC50 against the V600EB-RAF enzyme in
vitro (IC50B-RAF) and the determination of the growth inhibition
Neither the Cl nor the OH substituents (compounds 15, 16) led
to an improvement against B-RAF. However, bulky substituents
including solubilizing side chain (oxyethylamino as in com-
pound 10) or isoxazole (compound 19) were well tolerated by

Inhibitors of B-RAF
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1 409
Table 1. Analogues of Hit 1 by Modification of the Substituentsa
substituents designed at improving solubility were introduced
into the 4-position (compounds 20, 21), the potency was
maintained in the B-RAF assay, indicating that these modifica-
tions were tolerated.
Since the naked phenyl seemed a promising scaffold (com-
pound 6), several fused rings were also synthesized (compounds
22-28). Fluorine substitution of the fused ring proved detri-
mental (compounds 25-27) whereas the unsubstituted 5, 6,
7-membered rings (compounds 22-24) maintained the potency
but did not improve it. However, introduction of a fused lactone
(compound 28) produced a derivative with improved activity
against B-RAF (IC50 ) 0.74 nM). Replacement of the phenyl
ring (ring A) by different heterocycles resulted in compounds
29 and 30, the pyridine derivative 29, exhibited good potency
against the enzyme (IC50 ) 0.95 µM).
The role of the -NH- linker between rings A and B was
also explored. Introduction of an extra methyl onto the nitrogen
led to a tertiary amine, which turned out to be completely
inactive (compound 7). This finding suggests the -NH- linker
is involved in a crucial interaction with the protein.
Extra methylene groups [(CH2)n, n ) 1-3] between the amino
group and ring A were also introduced (compounds 31-44),
as well as some heterocycles. One extra methylene group is
well tolerated in the presence of different heterocyles (32, 33).
In general, however, introduction of extra methylenes is
detrimental, and the drop in potency parallels the number of
methylenes added (see 36-38).
The activity in the SRB test shows figures comparable to
sorafenib activity. However, no direct relationship was found
between the potency of B-RAF inhibition and the efficacy in
the SRB assay. The outstanding value reported for compound
22 (32 nM) in this test is believed to be an off-target effect.
Conclusions
High-throughput screening of a library of compounds resulted
in the identification of hit 1 with a novel structure, which served
as starting point for the design of new B-RAF inhibitors. This
has resulted in two compounds (19, 28) that both show an IC50
below 800 nM for B-RAF. Our lead compound 28 has a similar
IC50 for the SRB assay. The secondary amino group between
rings A and B seems to be essential for biological activity.
Additional methylene groups proved detrimental for the activity
except in some cases where different heterocycles were intro-
duced. Additionally, we found that the potency against B-RAF
was essentially maintained upon replacement of the trimethoxy-
phenyl unit by appropriate solubilizing groups. These findings
will be combined with efforts aimed at further optimization of
the rest of the molecule.
Experimental Section
All starting materials, reagents and solvents for reactions were
reagent grade and used as purchased. Chromatography solvents were
HPLC grade and were used without further purification. Reactions
were monitored by thin-layer chromatography (TLC) analysis using
Merck silica gel 60 F-254 thin layer plates. Flash column chro-
matography was carried out on Merck silica gel 60 (0.015-0.040
mm) or in disposable Isolute Flash Si and Si II silica gel columns.
Preparative TLC was performed on either Macherey-Nagel [809
023] precoated TLC plates SIL G-25 UV254 or Analtech [2015]
precoated preparative TLC plates, 2000 microns with UV254. LC-
MS analyses were performed on a Micromass LCT/Water’s Alliance
2795 HPLC system with a Discovery 5 µm, C18, 50 mm × 4.6
mm i.d. column from Supelco at a temperature of 22 °C using the
following solvent system: Solvent A: methanol; Solvent B: 0.1%
formic acid in water, at a flow rate of 1 mL/min. Gradient starting
with 10% A/90% B from 0 to 0.5 min then 10% A/90% B to 90%
a See Experimental Section for a description of the assay conditions.
b The H was replaced by a methyl group, which generated a tertiary amine.
c ND ) Not determined.
the protein. The 3-oxazole-phenyl-substituted analogue 19 was
the most potent compound against B-RAF in this series (IC50
) 0.79 µM). The 4-substitution resulted in inactive compounds
(compounds 17 and 18) against B-RAF. However, when bulky

410 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1
Niculescu-DuVaz et al.
A/10% B from 0.5 min to 6.5 min and continuing at 90% A/10%
B up to 10 min. From 10 to 10.5 min the gradient reverted back to
10% A/90% B and was held until 12 min. UV detection was at
254 nm and ionization was positive or negative ion electrospray.
The molecular weight scan range was 50-1000. Samples were
supplied as 1 mg/mL in DMSO or methanol with 3 µL injected on
a partial loop fill. NMR spectra were recorded in DMSO-d6 on a
Bruker DPX 250 operating at 250.13 MHz or on a Bruker advance
500 operating at 500.26 MHz. The signal of the deuterated solvent
was used as internal reference; the chemical shifts are expressed
in ppm (δ). Preparative HPLC purifications were performed on an
Axial Chromatospac Prep 10 (Jobin-Yvon) using Merck Kieselgel
60 (0.015-0.040). Microwave reactions were run on a CM
discovery unit operating at 200 W with simultaneous stirring. The
parallel synthesis in liquid phase were run on a Carousel (12 place
reaction station) from Radley Discovery Technology.
pure 1 (18.4%) was obtained. 1H NMR (DMSO-d6) δ 2.07 (s, 3H),
3.63 (s, 3H), 3.80 (s, 6H), 7.25 (s, 2H), 7.43 (t, 1H, J ) 7.9 Hz),
7.65 (d, 1H, J ) 8.1 Hz), 7.65 (d, 1H, J ) 7.8 Hz), 8.16 (s, 1H),
8.27 (s, 1H), 8.38 (s, 1H), 9.57 (s, 1H), 10.06 (s, 1H); 13C NMR
(DMSO-d6), δ: 23.91, 55.42, 60.09, 117.40, 121.34, 129.18, 129.83,
131.90, 133.64, 136.87, 137.30, 139.78, 147.70, 151.81, 152.81,
169.70; LC-MS, tR ) 7.17min; m/z: 395 (M + H)+ (C21H22N4O4);
HRMS: (M + H)+ calcd for C21H22N4O4 395.1719, found
395.1720.
2-(Phenylamino)-6-(3-acetamidophenyl)pyrazine (6). Method
C. 2-(3-Acetamidophenyl)-6-chloropyrazine 3 (100 mg, 0.404
mmol), aniline (54 mg, 0.581 mmol), potassium tert-butoxide (68
mg, 0.605 mmol), 1,3-bis(2,6-di-i-propylphenyl)-4,5-dihydroimi-
dazolium tetrafluoroborate (19 mg, 0.040 mmol) and Pd(0)2dba3
(18.5 mg, 0.020 mmol) in dry dioxane (2.5 mL) were added to a
round-bottomed flask in an Ar atmosphere were placed and stirred
at 95 °C and for 3.5 h. The solvent was evaporated under vacuum
to yield a solid that was submitted to column chromatography
2-(3-Acetamidophenyl)-6-chloropyrazine (3). Method A.
Amounts of 1.00 g (6.70 mmol) 2,6-dichloropyrazine, 450 mg (0.54
mmol) PdCl2.dppf:DCM 1:1, and 1.44 g (8.04 mmol) 3-acetami-
dophenyl boronic acid were dissolved in 100 mL dimethyl ethylene
glycol (DME). To this solution 14 mL aq. 1 M Na2CO3 solution
was added under stirring and the reaction mixture was heated at
90 °C (bath temperature) for 5 h. The reaction mixture was
evaporated to dryness, the residue taken up in 50 mL AcOEt and
filtered and the organic solution washed (2 × 30 mL brine), dried
(MgSO4) and evaporated to 20 mL. A pale yellow solid, 160 mg
was obtained. After filtration, the filtrate was submitted to prepara-
tive HPLC (AcOEt) and another fraction (749 mg) of 3 was
1
(AcOEt) to produce 32 mg (26%) of 6. H NMR (DMSO-d6) δ
2.11 (s, 3 H), 6.99 (t, 1H, J ) 7.4 Hz), 7.33-7.49 (m, 3 H), 7.62
(d, 1 H, J ) 7.9 Hz), 7.73 (d, 1 H, J ) 7.9 Hz), 7.87 (d, 1 H, J )
7.6 Hz), 8.19 (s, 1 H), 8.44 (s, 1 H), 8.46 (bs, 1 H), 9.60 (s, 1 H),
10.10 (s, 1 H); 13C NMR (DMSO-d6) δ: 24.11, 117.12, 118.21,
119.93, 121.04, 121.30, 128.91, 129.20, 129.96, 133.54, 137.05,
139.97, 140.66, 147.87, 151.46, 168.46; LC-MS: tR ) 7.11 min,
m/z 305.1 (M + H)+, >90% purity; HRMS m/z (M + H)+ calcd
for C18H17N4O: 305.1402, found 305.1401.
2-(Phenyl-N-methylamino)-6-(3-acetamidophenyl)pyrazine (7).
Method D. 3 (150 mg, 0.61 mmol), N-methylaniline (93 mg, 0.87
mmol), tris(dibenzylidene acetone)dipalladium (0) (Pd(0)2dba3) (28
mg, 0.030 mmol), 1,3-bis (2,6-di-i-propyl phenyl)-4,5-dihydro-
imidazolium tetrafluoro-borate (29 mg, 0.061 mmol), potassium
tert-butoxide (102 mg, 0.91 mmol) and dioxane (3.5 mL) were
added to a vessel suitable for microwave irradiation provided with
stirring and Ar atmosphere. The vessel was irradiated for 30 min
at 100 °C. The crude reaction mixture was passed through a very
short silica gel column (2-3 g silica) and washed with AcOEt.
After evaporation of the solvent, 117 mg (61%) of 7 was produced.
1H NMR (DMSO-d6) δ 2.08 (s, 3 H), 3.52 (s, 3 H), 7.32 (t, 1 H,
J ) 7.0 Hz), 7.38-7.54 (m, 5 H), 7.73 (d, 2 H, J ) 8.6 Hz), 7.83
(s, 1 H), 8.31 (bs, 1 H), 8.38 (s, 1 H), 10.09 (s, 1 H); 13C NMR
(DMSO-d6) δ: 24.03, 37.69, 117.13, 120.06, 121.13, 126.04,
126.24, 128.99, 129.14, 129.95, 130.46, 136.93, 139.85, 144.69,
147.98, 153.29, 168.46; LCMS: tR ) 6.87 min, m/z 319.0, (M +
H)+, >95% purity; HRMS: (M + H)+ calcd for C19H19N4O
319.1543, found 319.1559.
1
obtained. Total yield: 909 mg (54.7%). H NMR (DMSO-d6) δ
2.07 (s, 3H, CH3CO), 7.47 (t, 1H, J ) 7.9 Hz), 7.79-7.84 (2d,
2H), 8.28 (s, 1H), 8.28 (s, 1H), 8.76 (s, 1H), 10.17 (s, 1H); 13C
NMR (DMSO-d6) δ: 23.99, 117.26, 121.07, 121.53, 129.58, 134.67,
140.08, 140.19, 142.78, 147.81, 151.32, 168.55. LC-MS: tR
)
6.27min, m/z 248.0 (M + H)+, >98% purity; HRMS m/z (M +
H)+ calcd for C12H10ClN3O 248.0591, found 248.0592.
2-(3,4,5-Trimethoxyphenylamino)-6-chloropyrazine (4). Method
B. To 5.00 g (33.5 mmol) 2,6-dichloropyrazine dissolved in 200
mL dry toluene in an oven dried flask were added under stirring
0.42 g (0.46 mmol) Pd(0)2dba3, 0.83 g (1.34 mmol) 2,2′-bis-
(diphenylphosphino)-1,1′-binaphthyl (BINAP), 7.36 g (40.2 mmol)
3,4,5-trimethoxyaniline and 4.41 g (46.9 mmol) sodium tert-
butoxide. The reaction mixture was heated at 90 °C (bath temper-
ature) for 4 h under N2. After cooling, the toluene solution was
filtered. The solid was taken up in 100 mL acetone and filtered
through a 5 cm column of Kieselgel 60, and the resulting solution
was evaporated under vacuum. A portion of 2.53 g of 4 was
obtained. The toluene filtrate was evaporated to dryness and then
retaken in 70-80 mL boiling AcOEt. The solution was kept at 4
°C overnight, and 2.57 g of the solid title compound was collected
by filtration. The filtrate was reduced to 18-20 mL and submitted
to HPLC (AcOEt:cycloxane 2:1). A third fraction of 0.94 g of 4
was collected. The total yield was 6.04 g (61.1%). 1H NMR
(DMSO-d6) δ 3.63 (s, 3H), 3.77 (s, 6H), 7.02 (s, 2H), 7.96 (s, 1H),
8.14 (s, 1H), 9.79 (s, 1H); 13C NMR (DMSO-d6), δ: 56.04, 60.03,
128.16, 128.86, 132.73, 135.61, 136.80, 144.87, 149.89, 149.89,
151.54, 152.84; LC-MS, tR ) 7.43 min; m/z: 296 (M + H)+
(C13H13ClN3O3); HRMS: (M + H)+ calcd for C13H14ClN3O3
296.0802, found 296.0801.
2-(2-Methoxyphenylamino)-6-(3-acetamidophenyl)pyrazine (8).
Using method D with o-anisidine (107 mg, 0.87 mmol), 103 mg
1
(51%) of 8 was produced. H NMR (DMSO-d6) δ 2.09 (s, 3H),
3.89 (s, 3H), 7.01-7.08 (m, 3H), 7.42 (t, 1H, J ) 7.9 Hz), 7.61 (d,
1H, J ) 7.6 Hz), 7.70 (d, 1H, J ) 7.9 Hz), 8.40 (s, 1H), 8.41 (s,
1H), 8.42 (s, 1H), 8.46-8.48 (m, 1H), 8.76 (s, 1H), 10.11 (s, 1H);
13C NMR (DMSO-d6) δ: 24.14, 55.72, 110.93, 117.10, 119.60,
119.92, 120.70, 121.09, 122.29, 129.03, 129.23, 130.02, 134.15,
137.13, 139.95, 147.72, 149.09, 151.59, 168.49; LC-MS: tR ) 7.21
min, m/z 335.1 (M + H)+, (C19H18N4O2), >98% purity. HRMS
m/z (M + H)+ calcd for C19H19N4O2 335.1508, found 335.1516.
2-(3-Methoxyphenylamino)-6-(3-acetamidophenyl)pyrazine (9).
Method D with m-anisidine (107 mg, 0.87 mmol) gave 24 mg
(11.8%) of 9. 1H NMR (DMSO-d6) δ 2.08 (s, 3 H), 3.77 (s, 3 H),
6.56 (d, 1 H, J ) 7.4 Hz), 7.25 (t, 1 H, J ) 8.1 Hz), 7.38-7.47
(m, 2 H), 7.56 (bs, 1 H), 7.65 (d, 1 H, J ) 7.8 Hz), 7.73 (d, 1 H,
J ) 9.5 Hz), 8.20 (s, 1 H), 8.33 (bs, 1 H), 8.42 (s, 1 H), 9.61 (s,
1 H), 10.09 (s, 1 H); 13C NMR (DMSO- d6) δ 24.02, 54.78, 103.29,
107.48, 110.59, 117.26, 120.13, 121.21, 129.22, 129.65, 130.09,
133.66, 137.15, 139.88, 141.91, 147.98, 151.46, 159.67, 168.43;
LC-MS: tR ) 7.17 min, m/z 335.1 (M + H)+, >98% purity; Anal.
(C19H18N4O2); C, H, N. (calcd C, 68.25; H, 5.43; N, 16.76. Found:
C, 67.81; H, 5.49; N, 16.47). HRMS m/z (M + H)+ calcd for
C19H19N4O2 335.1508, found 335.1498.
2-(3,4,5-Trimethoxyphenylamino)-6-(3-acetamidophenyl)pyra-
zine (1). The catalyst was prepared in an oven dried flask by adding
with stirring under an Ar atmosphere 40 mg (0.09 mmol) dppb
and 30 mg (0.077 mmol) PdCl2(benzonitrile)2 to 10 mL dry,
degassed toluene. After 30 min, 225 mg (0.76 mmol) 2-(3,4,5-
trimethoxyphenylamino)-6-chloropyrazine (4) 161 mg (0.9 mmol)
3-acetamido-boronic acid, 1.60 mL EtOH and 1.5 mL aq. 1 M Na2-
CO3 solution in 10 mL toluene were added, and the reaction mixture
was heated at 90 °C (bath temperature) for 8 h. After cooling,
AcOEt (50 mL) was added to the reaction, and the mixture was
washed with brine (2 × 100 mL), dried (MgSO4), and evaporated
to dryness. An amount of 165 mg of a solid was obtained and
purified by preparative HPLC (AcOEt:EtOH 9:1). Finally, 55 mg

Inhibitors of B-RAF
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1 411
tert-Butyl 2-(3-Aminophenoxy)ethylcarbamate (10c). 3-
Aminophenol 10a (600 mg, 5.48 mmol), K2CO3 (380 mg, 2.76
mmol), potassium tert-butoxide (648 mg, 5.76 mmol) and dry DMF
(8 mL) were added to a round-bottomed flask provided with Ar
atmosphere. The reaction mixture was stirred for 10 min, and a
solution of 2-(Boc-amino) ethyl bromide (1.23 g, 5.50 mmol) in 6
mL of dry DMF was added dropwise and the temperature increased
to 90 °C for 3 h. To this mixture was added a solution of sodium
hydroxide (3.5 g) in water (100 mL). This mixture was extracted
with AcOEt (3 × 40 mL). The organic layers were combined, dried
(MgSO4), filtered and evaporated under vacuum to produce a crude
mixture that was chromatographed using a mixture of AcOEt: DCM
NMR (DMSO-d6) δ 23.91, 54.92, 94.33, 96.21, 117.21, 117.42,
118.10, 120.32, 121.28, 121.35, 129.20, 130.22, 133.71, 137.24,
139.80, 140.70, 142.43, 148.07, 151.47, 160.60, 168.38; LC-MS,
tR ) 7.35 min; m/z: 365.3 (M + H)+ (C20H20N4O3); HRMS: (M
+ H)+ calcd for C20H21N4O3, 365.1637, found 365.1614.
2-(3,4-Dimethoxyphenylamino)-6-(3-acetamidophenyl)pyra-
zine (13). Using the same method B and the workup described for
compound 12, from 180 mg (0.73 mmol) 3 and 134 mg (0.88 mmol)
3,4-dimethoxyaniline (reaction time: 6 h), 72 mg (27.1%) of pure
13 was obtained after preparative HPLC (AcOEt) purification of
the whole batch. 1H NMR (DMSO-d6) δ 2.07 (s, 3H), 3.73 (s, 3H),
3.77 (s, 3H), 6.95 (d, 1H, J ) 8.7 Hz), 7.26 (q, 1H, Ja ) 8.6 Hz,
Jb ) 2.3 Hz), 7.42 (t, 1H, J ) 7.9 Hz), 7.62 (s, 1H), 7.64 (d, 1H),
7.72 (d, 1H, J ) 7.6 Hz), 8.13 (s, 1H), 8.30 (s, 1H), 8.35 (s, 1H),
9.45 (s, 1H), 10.07 (s, 1H); 13C NMR (DMSO-d6) δ 24.00, 55.07,
55.84, 103.51, 110.03, 112.63, 117.62, 120.09, 121.20, 129.16,
129.37, 133.38, 134.39, 137.28, 139.82, 143.58, 147.87, 148.71,
151.60, 168.44; LC-MS: tR ) 5.85 min; m/z: 365.3 (M + H)+
(C20H20N4O3); HRMS: (M + H)+ calcd for C20H21N4O3, 365.1614,
found 365.1602.
1
(15:85) to furnish 713 mg (49%) of 10c. H NMR (DMSO-d6) δ
1.38 (s, 9H), 3.24 (c, 2 H, J ) 5.8 Hz), 3.33 (s, 1 H), 3.82 (t, 2 H,
J ) 5.9 Hz), 5.02 (s, 2 H), 6.06 (d, 1 H, J ) 9.1 Hz), 6.12-6.16
(m, 2 H), 6.88 (t, 1 H, J ) 7.7 Hz), 6.95 (bt, 1 H, J ) 5.2 Hz). 13
C
NMR (DMSO-d6) δ: 28.19, 65.81, 77.70, 99.98, 101.90, 106.91,
129.49, 149.93, 155.64, 159.37. LC-MS: tR ) 4.16 min, >77%
purity. HRMS m/z (M + Na)+ calcd for C13H20N2O3Na 275.1372,
found 275.1378.
2-[3-(tert-Butylcarbamoyl-N-ethyloxy)phenylamino]-6-(3-
acetamidophenyl)pyrazine (10d). Method D with tert-butyl 2-(3-
aminophenoxy)ethylcarbamate 10c (190 mg, 0.753 mmol) was
applied with heating for 20 min. The reaction mixture was filtered
and washed with AcOEt (45 mL). The liquid was dried under
vacuum and the residue chromatographed using a mixture of
2-(2,4-Dimethoxyphenylamino)-6-(3-acetamidophenyl)pyra-
zine (14). Using method B, 3 (60 mg, 0.24 mmol), 1,3-dimethoxya-
niline (54 mg, 0.35 mmol), Pd(0)2dba3 (9 mg, 0.01 mmol), BINAP
(50 mg, 0.04 mmol) and sodium tert-butoxide (33 mg, 0.34 mmol)
in degassed toluene (5 mL) were reacted at 85 °C for 16 h. The
solvent was evaporated under vacuum and the resulting solid was
washed with 20 mL hot AcOEt and filtered. The volume was
reduced to 2 mL and chromatographed on silica gel (AcOEt) to
furnish 61 mg of a mixture of two products. Preparative TLC
(AcOEt) and crystallization in chloroform afforded 12 mg (14%)
of 14. 1H NMR (DMSO-d6) δ 2.08 (s, 3 H), 3.77 (s, 3 H), 3.85 (s,
3 H), 6.59 (dd, 1 H, Ja ) 8.9 Hz, Jb ) 2.7 Hz), 6.68 (d, 1 H, J )
2.6 Hz), 7.40 (t, 1 H, J ) 7.9 Hz), 7.65 (t, 2 H, J ) 9.3 Hz), 8.08
(d, 1 H, J ) 8.8 Hz), 8.19 (s, 1 H), 8.29 (bs, 1 H), 8.32 (s, 1 H),
8.55 (s, 1 H), 10.07 (s, 1 H); 13C NMR (DMSO-d6) δ 24.07, 55.29,
55.73, 99.03, 104.39, 117.11, 119.88, 121.09, 121.83, 122.25,
129.11, 129.26, 133.01, 137.23, 139.82, 147.89, 151.45, 152.12,
155.83, 168.40; LC-MS: tR ) 7.10 min, m/z 365.1 (M + H)+,
>99% purity; HRMS: (M + H)+ calcd for C20H21N4O3 365.1605,
found 365.1614.
2-(3-Chlorophenylamino)-6-(3-acetamidophenyl)pyrazine (15).
Using method A with 300 mg (1.25 mmol) 2-(3-chlorophen-
ylamino)-6-chloropyrazine, 4b, and 268 mg (1.5 mmol) 3-aceta-
midoboronic acid (reaction time: 16 h), 78 mg (28.1%) pure title
15 were obtained after purification by preparative HPLC (AcOEt).
1H NMR (DMSO-d6) δ 2.09 (s, 3H), 7.03 (d, 1H, J ) 7.12 Hz),
7.39 (t, 1H, J ) 6.4 Hz), 7.45 (t, 1H, J ) 7.9 Hz), 7.63 (d, 1H, J
) 7.9 Hz), 7.73 (d, 1H, J ) 7.7 Hz), 7.89 (m, 2H), 8.20 (s, 1H),
8.48 (s, 1H), 9.81 (s, 1H), 10.09 (s, 1H); 13C NMR (DMSO-d6) δ
20.73, 24.02, 55.29, 110.87, 116.92, 119.82, 120.94, 123.11, 123.62,
127.67, 129.02, 130.26, 132.04, 137.30, 139.76, 140.32, 143.31,
147.87, 153.84, 168.42; LC-MS: tR ) 6.43 min, m/z 309.1 (M +
H)+ C17H16N4O2; HRMS: (M + H)+ calcd for C17H16N4O4
309.1352, found 309.1376.
1
AcOEt: DCM (1:1) to furnish 120 mg (43%) of 10d. H NMR
(DMSO-d6) δ 1.37 (s, 9 H), 2.09 (s, 3 H), 3.32 (m, 2 H), 3.98 (m,
2 H), 6.56 (dd, 1 H, Ja ) 8.0 Hz, Jb ) 2.4 Hz), 7.01 (bt, 1 H), 7.25
(t, 1 H, J ) 8.1 Hz), 7.36 (bd, 1 H, J ) 8.4 Hz), 7.45 (t, 1 H, J )
7.9 Hz), 7.59 (bs, 1 H), 7.66 (bd, 1 H, J ) 9.2 Hz), 7.74 (bd, 1 H,
J ) 8.0 Hz), 8.19 (s, 1 H), 8.34 (bs, 1 H), 8.42 (s, 1 H), 9.60 (s,
1 H), 10.09 (s, 1 H). 13C NMR (DMSO-d6) δ 23.99, 28.18, 66.23,
77.77, 103.95, 107.82, 110.74, 117.11, 120.07, 121.24, 129.24,
129.66, 130.08, 133.66, 137.10, 139.90, 141.88, 147.96, 151.44,
155.65, 158.87, 168.53. LC-MS: tR ) 7.67 min, m/z 364.2 (M -
Boc + H)+ (C20H21N5O2), >98% purity. HRMS m/z (M + H)+
calcd for C25H30N5O4 464.2298, found 464.2284.
2-[3-(2-Aminoethoxy)phenylamino]-6-(3-acetamidophenyl)-
pyrazine (10). Compound 10b (60 mg, 0.129 mmol) was depro-
tected by stirring with trifluoroacetic acid (TFA) (2.5 mL) for 3 h
at room temperature. The TFA was evaporated under vacuum, and
340 µL triethylamine was added. The residue was chromatographed
using a gradient of AcOEt:EtOH 85:15 to 50:50 to give 15 mg of
1
10. Yield: 32%. H NMR (250 MHz, DMSO-d6) δ 2.09 (s, 3 H),
2.90 (t, 2 H, J ) 5.5 Hz), 3.96 (t, 2 H, J ) 5.7 Hz), 6.56 (d, 1 H,
J ) 8.0 Hz), 7.24 (t, 1 H, J ) 8.0 Hz), 7.34 (d, 1 H, J ) 6.8 Hz),
7.44 (t, 1 H, J ) 7.9 Hz), 7.64 (bs, 1 H), 7.66 (d, 1 H, J ) 9.0 Hz),
7.73 (d, 1 H, J ) 8.0 Hz), 8.20 (s, 1 H), 8.34 (bs, 1 H), 8.42 (s, 1
H), 9.64 (s, 1 H), 10.16 (s, 1 H). 13C NMR (DMSO-d6) δ 23.98,
40.82, 69.56, 103.92, 107.96, 110.61, 117.24, 120.19, 121.26,
129.22, 129.61, 130.08, 133.68, 137.15, 139.90, 141.89, 147.98,
151.47, 159.08, 168.51. LC-MS: tR ) 4.46 min, m/z 364.2 (M +
H)+ (C21H20N5O2).
2-(3-Hydroxyphenylamino)-6-(3-acetamidophenyl)pyrazine (16).
Method B with 3-hydroxyaniline (95 mg, 0.87 mmol) afforded 158
mg (80%) of 16. 1H NMR (DMSO-d6) δ 2.11 (s, 3H), 6.41 (d, 1H,
J ) 7.9 Hz), 7.13 (t, 1H, J ) 7.9 Hz), 7.27 (d, 1H, J ) 7.9 Hz),
7.40 (s, 1H), 7.44 (t, 1H, J ) 7.9 Hz), 7.59 (d, 1H, J ) 7.6 Hz),
7.76 (d, 1H, J ) 7.2 Hz), 8.18 (s, 1H), 8.42 (s, 1H), 8.51 (s, 1H),
9.29 (s, 1H), 9.45 (s, 1H), 10.15 (s, 1H); 13C NMR (DMSO-d6) δ
24.08, 105.15, 108.73, 109.33, 117.36, 119.97, 121.22, 129.21,
129.59, 129.77, 133.60, 137.02, 139.74, 141.71, 147.80, 151.52,
157.66, 168.84; LC-MS: tR ) 6.58 min; m/z 321.0 (M + H)+,
>94% purity; HRMS: (M + H)+ calcd for C18H17N4O2: 321.1330,
found 321.1352.
2-[4-(Trifluoromethoxy)phenylamino]-6-(3-acetamidophenyl)-
pyrazine (17). Method B was followed starting from 4-(trifluoro-
methoxy)aniline (51 mg, 0.29 mmol). Purification by preparative
TLC (AcOEt) furnished 19 mg (21%) of 17. 1H NMR (DMSO-d6)
δ 2.10 (s, 3 H), 7.34-7.47 (m, 3 H), 7.60 (d, 1 H, J ) 7.9 Hz),
2-(3,5-Dimethoxyphenylamino)-6-(3-acetamidophenyl)pyra-
zine (12). Using method B, 180 mg (0.73 mmol) 2-(3-acetami-
dophenyl)-6-chloropyrazine (3), dissolved in 5 mL dry toluene in
an oven dried flask, was reacted under N2 with 24 mg (0.03 mmol)
Pd(0)2dba3, 75 mg (0.12 mmol) BINAP, 134 mg (0.88 mmol) 3,4,5-
trimethoxyaniline and 98 mg (1.02 mmol) sodium tert-butoxide at
90 °C (bath temperature) for 5 h. The toluene solution was filtered
and evaporated under vacuum. The solid was taken up in 40 mL
AcOEt, washed (2 × 40 mL water) and dried (MgSO4). The catalyst
was filtered and the mixture extracted with 10 mL of acetone. The
organic solutions were pooled, evaporated to 10 mL volume and
left at 4 °C overnight to produce 160 mg of 12. The filtrate,
submitted to HPLC (AcOEt) gave an additional 40 mg of 12. The
1
total yield was 170 mg (63.9%). H NMR (DMSO-d6) δ 2.07 (s,
3H), 3.75 (s, 6H), 6.15 (s, 1H), 7.12 (s, 2H), 7.44 (t, 1H, J ) 7.8
Hz), 7.67 (d, 1H, J ) 7.9 Hz), 7.73 (d, 1H, J ) 7.6 Hz), 8.19 (s,
1H), 8.24 (s, 1H), 8.39 (s, 1H), 9.64 (s, 1H), 10.03 (s, 1H); 13C

412 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1
Niculescu-DuVaz et al.
7.74 (d, 1 H, J ) 7.8 Hz), 8.00 (d, 2 H, J ) 9.1 Hz), 8.21 (s, 1 H),
8.51 (bs, 2 H), 9.82 (s, 1 H), 10.10 (s, 1 H); 13C NMR (DMSO-d6)
δ 24.09, 117.01, 119.09, 119.87, 120.94, 121.68, 129.15, 130.40,
133.52, 136.73, 139.92, 141.96, 141.99, 147.66, 151.00, 168.45;
LCMS: tR ) 8.06 min; m/z 389.3 (M + H)+, (C19H15F3N4O2),
>96% purity; HRMS: (M + H)+ calcd for C18H16N4O2: 361.1295,
found 361.1301.
2-(3-Chloro-4-fluorophenylamino)-6-(3-acetamidophenyl)-
pyrazine (18). Method B was followed starting from 3-chloro-4-
fluoroaniline (42 mg, 0.29 mmol). Purification by preparative TLC
(AcOEt) produced 4 mg (5%) of 18. 1H NMR (DMSO-d6) δ 2.09
(s, 3 H), 7.38 (d, 1 H, J ) 9.5 Hz), 7.46 (d, 1 H, J ) 7.9 Hz), 7.61
(d, 1 H, J ) 7.9 Hz), 7.72 (d, 1 H, J ) 7.5 Hz), 7.86-7.91 (m, 1
H), 8.00-8-04 (m, 1 H), 8.18 (s, 1 H), 8.40 (bs, 1 H), 8.48 (s, 1
H), 9.80 (s, 1 H), 10.08 (s, 1 H); LCMS: tR ) 6.79 min; m/z 357.2
(M + H)+, (C18H14ClFN4O), >55% purity.
2-[3-(Oxazol-5-yl)phenylamino]-6-(3-acetamidophenyl)pyra-
zine (19). Method B was followed starting from 3-(1,3-oxazol-5-
yl)aniline (56 mg, 0.35 mmol), Preparative TLC (AcOEt) afforded
23 mg (26%) of 19. 1H NMR (DMSO-d6) δ 2.08 (s, 3 H), 7.36 (d,
1 H, J ) 8.0 Hz), 7.48 (t, 2 H, J ) 8.3 Hz), 7.61 (s, 1 H), 7.65 (d,
1 H, J ) 8.4 Hz), 7.82 (d, 2 H, J ) 7.3 Hz), 8.23 (s, 1 H), 8.39
(bs, 2 H), 8.46 (s, 1 H), 8.47 (s, 1 H), 9.82 (s, 1 H), 10.09 (s, 1 H);
13C NMR (DMSO-d6) δ 24.0, 113.4, 117.1, 117.3, 118.3, 120.3,
121.3, 121.8, 127.9, 129.3, 129.8, 130.3, 133.7, 137.0, 139.9, 141.4,
147.9, 150.8, 151.3, 151.7, 168.5; LC-MS: tR ) 7.23 min, m/z
372.1 (M + H)+, >96% purity (C21H17N5O2); HRMS: (M + H)+
calcd for C21H18N5O2: 372.1461, found 372.1436.
2-[4-(Morpholinosulfonyl)phenylamino]-6-(3-acetamido-
phenyl)pyrazine (20). Method B was followed starting from
4-(morpholinosulfonyl)aniline (85 mg, 0.35 mmol). Column chro-
matography (AcOEt) afforded 34 mg (31%) of 20. 1H NMR
(DMSO-d6) δ 2.11 (s, 3 H), 2.86-2.89 (m, 4 H), 3.62-3.65 (m, 4
H), 7.45 (t, 1 H, J ) 7.8 Hz), 7.54 (d, 1 H, J ) 8.1 Hz), 7.73-7.80
(m, 3 H), 8.14 (d, 2 H, J ) 8.9 Hz), 8.28 (s, 1 H), 8.57 (bs, 1 H),
8.61 (s, 1 H), 10.13 (s, 1 H), 10.18 (s, 1 H); 13C NMR (DMSO-d6)
δ 24.19, 45.90, 65.28, 117.09, 117.50, 120.06, 121.09, 125.43,
129.22, 129.33, 131.54, 134.02, 136.56, 140.07; LC-MS: tR ) 6.74
min; m/z 454.2 (M + H)+, >98% purity; HRMS: (M + H)+ calcd
for C22H24N5O4S: 454.1549, found 454.1524.
7.25-7.38 (m, 1 H), 7.43 (t, 1 H, J ) 8.0 Hz), 7.68 (t, 1 H, J )
7.8 Hz), 8.11 (s, 1 H), 8.28 (s, 1 H), 8.34 (s, 1 H), 9.38 (s, 1 H),
10.08 (s, 1 H); 13C NMR (DMSO-d6) δ 24.64, 64.29, 64.51, 110.82,
115.00, 117.69, 118.20, 121.10, 122.61, 129.56, 130.37, 131.03,
132.75, 137.34, 138.58, 140.38, 143.82, 149.32, 121.10, 122.61,
129.56, 130.37, 131.03, 132.75, 137.34, 138.58, 140.38, 143.82,
149.32, 151.99, 168.59; LC-MS: tR ) 6.96 min; m/z 363.1 (M +
H)+ (C20H18N4O3) >95% purity; HRMS: (M + H)+ calcd for
C20H19N4O3: 363.1457, found 363.1466.
2-(3,4-Dihydro-2H-benzo[b][1,4]dioxepin-7-ylamino)-6-(3-
acetamidophenyl)pyrazine (24). Method B was followed starting
from 3,4-dihydro-7-amino-2H-1,5-benzo[b][1,4]dioxepin (58 mg,
0.35 mmol). Purification by preparative TLC (AcOEt) produced
33 mg (37%) of 24. 1H NMR (DMSO-d6) δ 2.09 (bs, 5 H), 4.00-
4.13 (m, 4 H), 6.98 (d, 1 H, J ) 8.7 Hz), 7.33 (d, 1 H, J ) 2.7
Hz), 7.43 (t, 1 H, J ) 7.9 Hz), 7.54 (dd, 1 H, Ja ) 8.7 Hz, Jb ) 2.6
Hz), 7.62 (bd, 1 H, J ) 6.8 Hz), 7.70 (bd, 1 H, J ) 7.9 Hz), 8.12
(s, 1 H), 8.35 (bs, 1 H), 8.38 (s, 1 H), 9.47 (s, 1 H), 10.08 (s, 1 H);
13C NMR (DMSO-d6) δ 24.06, 31.99, 70.61, 111.53, 113.48,
117.21, 120.01, 121.08, 121.69, 129.21, 129.75, 133.29, 136.24,
137.10, 139.89, 145.73, 147.92, 151.17, 151.40, 168.42; LC-MS:
tR ) 7.02 min; m/z 377.2 (M + H)+, (C21H20N4O3), >98% purity;
HRMS: (M + H)+ calcd for C21H21N4O3: 377.1601, found
377.1614.
2-(2,2,3,3-Tetrafluoro-2,3-dihydrobenzo[b][1,4]dioxin-6-yl-
amino)-6-(3-acetamidophenyl)pyrazine (25). Method B was fol-
lowed starting from 2,2,3,3-tetrafluoro-6-amino-1,4-benzodioxine
(78 mg, 0.35 mmol). Crystallization from AcOEt gave 11 mg (10%)
of 25. 1H NMR (DMSO-d6) δ 2.08 (s, 3 H), 7.44 (d, 1 H, J ) 9.1
Hz), 7.47 (d, 1 H, J ) 7.8 Hz), 7.59 (bd, 1 H, J ) 7.6 Hz), 7.704-
7.759 (m, 2 H), 7.96 (d, 1 H, J ) 2.4 Hz), 8.21 (s, 1 H), 8.45 (bs,
1 H), 8.51 (s, 1 H), 9.95 (s, 1 H), 10.10 (s, 1 H); 13C NMR (DMSO-
d6) δ 24.00, 106.13, 115.92, 117.34, 118.01, 120.13, 121.08, 129.31,
130.04, 130.98, 133.62, 136.16, 136.79, 139.03, 139.99, 147.90,
150.84, 168.40; LC-MS: LC-MS: tR ) 8.38 min; m/z 435.1 (M +
H)+, (C20H14F4N4O3), >99% purity; HRMS: (M + H)+ calcd for
C20H15F4N4O3: 435.1080, found 435.1027.
2-(2,2-Difluorobenzo[d][1,3]dioxol-5-ylamino)-6-(3-acetami-
dophenyl)pyrazine (26). Method B was followed starting from
5-amino-2,2-difluoro-1,3-benzodioxine (61 mg, 0.35 mmol). Crys-
tallization from DCM and cyclohexane (4:1) provided 6 mg (6%)
of 26. 1H NMR (DMSO-d6) δ 2.09 (s, 3 H), 7.37 (d, 1 H, J ) 8.8
Hz), 7.44 (t, 7.9 Hz), 7.57-7.65 (m, 2 H), 7.70 (d, 1 H, J ) 7.7
Hz), 7.94 (d, 1 H, J ) 2.1 Hz), 8.20 (s, 1 H), 8.40 (bs, 1 H), 8.45
(s, 1 H), 9.88 (s, 1 H), 10.12 (s, 1 H); 13C NMR (DMSO-d6) δ
24.00, 110.06, 113.48, 117.09, 119.91, 121.00, 129.20, 130.44,
133.49, 136.83, 136.95, 137.51, 139.91, 142.76, 147.81, 151.05,
168.38; LC-MS: tR ) 8.03 min; m/z 385.1 (M + H)+ (C19H14-
F2N4O3), >98% purity; HRMS: (M + H)+ calcd for C19H15-
F2N4O3: 385.1112, found 385.1073.
2-(4-Morpholinophenylamino)-6-(3-acetamidophenyl)pyra-
zine (21). Method B was followed starting from 4-morpholinoa-
niline (155 mg, 0.87 mmol). Crystallyzation from AcOEt produced
1
70 mg (30%) of 21. H NMR (DMSO-d6) δ 2.09 (s, 3 H), 3.03-
3.07 (bt, 4 H), 3.72-3.76 (bt, 4 H), 6.97 (d, 2 H, J ) 9.0 Hz), 7.42
(t, 1 H, J ) 7.9 Hz), 7.65-7.68 (m, 2 H), 7.70 (d, 2 H, J ) 8.9
Hz), 8.11 (s, 1 H), 8.33 (bs, 2 H), 9.36 (s, 1 H), 10.09 (s, 1 H); 13
C
NMR (DMSO-d6) δ 24.11, 49.24, 66.16, 116.01, 117.08, 119.61,
119.89, 121.08, 129.16, 133.03, 133.15, 137.23, 139.87, 146.10,
147.95, 151.70, 168.45; LC-MS: LC-MS: tR ) 6.42 min; m/z 390.2
(M + H)+, >88% purity; HRMS: (M + H)+ calcd for C22H24-
N5O2: 390.1923, found 390.1930.
2-(2,2,3,3-Tetrafluoro-2,3-dihydrobenzo[b][1,4]dioxin-5-ylami-
no)pyrazin-2-yl)phenyl)acetamide (27). Method B was followed
starting from 2,2,3,3-tetrafluoro-5-amino-1,4-benzodioxin (78 mg,
0.35 mmol). The solvent was evaporated in vacuo, the resulting
crude was washed with 30 mL DCM and filtered. The insoluble
solid was washed with 30 mL of hot AcOEt and filtered. The AcOEt
2-(3,4-Methylenedioxyphenylamino)-6-(3-acetamidophenyl)-
pyrazine (22). Using method B starting from 200 mg (0.33 mmol)
3 and 138 mg (1.0 mmol) 3,4-methylendioxyaniline, 84 mg (29.0%)
pure 22 was obtained after preparative HPLC (AcOEt) purification
1
1
of the whole batch. Reaction time: 18 h. H NMR (DMSO-d6) δ
was evaporated under vacuum to produce 39 mg (37%) of 27. H
2.08 (s, 3H), 5.99 (s, 2H), 6.91 (d, 1H, J ) 8.4 Hz), 7.32 (q, 1H,
Ja ) 8.4 Hz, Jb ) 2.0 Hz), 7.40 (d, 1H, J ) 1.8 Hz), 7.43 (t, 1H,
J ) 7.9 Hz), 7.65 (d, 1H, J ) 8.9 Hz), 7.68 (d, 1H, J ) 7.8 Hz),
8.12 (s, 1H), 8.30 (s, 1H), 8.35 (s, 1H), 9.42 (s, 1H), 10.09 (s, 1H);
13C NMR (DMSO-d6) δ 24.07, 100.62, 100,74, 108.27, 111.15,
117.07, 119.92, 121.09, 129.22, 129.61, 133.29, 135.25, 137.14,
139.87, 141.75, 147.19, 147.95, 151.53, 168.44; LC-MS, tR ) 7.07
min; m/z: 349.1 (M + H)+ (C19H16N4O3); HRMS: (M + H)+ calcd
for C19H17N4O3: 349.1301, found 349.1308.
NMR (DMSO-d6) δ 2.08 (s, 3 H), 7.11 (d, 1 H, J ) 7.8 Hz), 7.34-
7.44 (m, 2 H), 7.60 (d, 1 H, J ) 7.2 Hz), 7.70 (d, 1 H, J ) 7.3
Hz), 8.34 (d, 1 H, J ) 7.4 Hz), 8.42 (bs, 1 H), 8.45 (s, 1 H), 8.57
(s, 1 H), 9.41 (s, 1 H), 10.07 (s, 1 H); 13C NMR (DMSO-d6) δ
24.09, 110.20, 117.01, 117.41, 120.01, 120.98, 125.83, 127.29,
129.13, 129.79, 131.57, 133.89, 139.49, 136.72, 139.88, 147.68,
150.70, 168.39; LC-MS: tR ) 8.17 min; m/z 435.1 (M + H)+,
(C20H13F4N4O3), >95% purity; HRMS: (M + H)+ calcd for
C20H14F4N4O3: 435.1013, found 435.1013.
2-(2,3-Dihydrobenzo[b][1,4]dioxin-6-ylamino)-6-(3-acetami-
dophenyl)pyrazine (23). Method B was followed starting from
6-amino-3,4-benzodioxane (45 mg, 0.29 mmol). Column chroma-
tography (AcOEt) furnished 40 mg (46%) of 23. 1H NMR (DMSO-
d6) δ 2.09 (s, 3 H), 4.20-4.24 (m, 4 H), 6.85 (d, 1 H, J ) 8.7 Hz),
2-(3-Oxo-1,3-dihydroisobenzofuran-5-ylamino)-6-(3-acetami-
dophenyl)pyrazine (28). Method B was followed starting from
6-amino-1,3-dihydroisobenzofuran-1-one (52 mg, 0.35 mmol). The
solvent was evaporated in vacuo and the resulting solid washed
with 30 mL DCM and filtered. The insoluble solid was washed

Inhibitors of B-RAF
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1 413
with acetone (60 mL), the acetone evaporated and the resulting
solid crystallized from DMF:DCM; 1:10 to give 23 mg (26%) of
28. H NMR (DMSO-d6) δ 2.09 (s, 3 H), 5.39 (s, 2 H), 7.45 (t, 1
2H, J ) 5.7 Hz), 5.99 (s, 2H), 6.81-6.93 (m, 3H), 7.71 (s, 1H),
7.91 (s, 1H), 7.96 (t, 1H); 13C NMR (DMSO-d6) δ 43.61, 100.83,
108.07, 108.08, 120.89, 128.38, 132.38, 143.08, 145.87, 146.23,
154.43; LC-MS: tR ) 7.24 min, m/z: 278.05 (M + H)+, (C12H12-
ClN3O2); HRMS: (M + H)+ calcd for C14H14ClN4O2 305.0805,
found 305.0812.
2-[(5-Methyl-3-isoxazolyl)methylamino]-6-chloropyrazine (4f).
Using method E from 140 mg (1.25 mmol) 5-methyl-3-isoxazolyl-
methylamine and 202 mg (1.38 mmol) 2,6-dichloropyrazine, 164
mg (58.4%) of 4f was obtained after purification on an Isolute
column (AcOEt:cyclohexane 2:1). 1H NMR (DMSO-d6) δ 2.38 (s,
3H), 4.48 (d, 2H, J ) 5.9 Hz), 6.17 (s, 1H), 7.78 (s, 1H), 7.96 (s,
1H), 8.07 (t, 1H); 13C NMR (DMSO-d6) δ 11.73, 30.64, 101.15,
128.86, 131.26, 145.79, 154.28, 161.67, 169.55; LC-MS, tR ) 5.59
min, m/z: 224.9 (M + H)+, (C9H9ClN4O); HRMS: (M + H)+ calcd
for C9H10ClN4O 225.0543, found 225.0556.
2-[(1,5-Dimethyl-1H-pyrazol-3-yl)methylamino]-6-chloro-
pyrazine (4g). Using method E from 140 mg (1.12 mmol) (1,5-
dimethyl-1H-pyrazol-3-yl)methylamine and 180 mg (1.23 mmol)
2,6-dichloropyrazine, 105 mg (61.2%) of 4g was obtained after
purification on an Isolute column (AcOEt). Mp 122-3 °C; 1H NMR
(DMSO-d6) δ 2.21 (s, 3H), 3.67 (s, 3H), 4.29 (d, 2H, J ) 5.6 Hz),
5.96 (s, 1H), 7.70 (s, 1H), 7.84 (t, 1H, J ) 5.2 Hz), 7.92 (s, 1H);
LC-MS, tR ) 5.72 min, m/z: 237.9 (M + H)+, (C10H13ClN5).
2-(2-Pyridylmethylamino)-6-chloropyrazine (4h). Using method
E from 140 mg (1.30 mmol) 2-pyridylmethylamine and 210 mg
(1.43 mmol) 2,6-dichloropyrazine, 53 mg (48.7%) of 4h was
obtained after purification on an Isolute column (AcOEt:cyclo-
hexane 2:1). Mp 107-8.5 °C; 1H NMR (DMSO-d6) δ 4.57 (d, 2H,
J ) 5.89), 7.26-7.38 (m, 2H), 7.74 (s, 1H), 7.73-7.80 (m, 1H),
8.02 (s, 1H), 8.13 (s, 1H), 8.54 (d, 1H, J ) 4.51); 13C NMR
(DMSO-d6) δ 45.56, 121.29, 122.25, 128.43, 128.40, 131.37,
136.76, 145.84, 154.57, 157.97; LC-MS, tR ) 2.69 min, m/z: 220.9
(M + H)+ (C10H19ClN4). HRMS: (M + H)+ calcd for C10H10ClN4
221.0594, found 221.0600.
2-(Benzylamino)-6-chloropyrazine (4i). Using method E with
100 mg (0.93 mmol) benzylamine and 146 mg (1.00 mmol) 2,6-
dichloropyrazine, 144 mg (70.5%) of 4i was obtained after
purification on an Isolute Si-II column (AcOEt:cyclohexane 2:1).
1H NMR (DMSO-d6) δ 4.47 (d, 2H, J ) 5.9 Hz), 7.23-7.37 (m,
5H), 7.72 (s, 1H), 7.94 (s, 1H), 8.05 (t, 1H, J ) 5.6 Hz); 13C NMR
(DMSO-d6) δ 43.78, 126.88, 127.95, 128.36, 128.40, 131.16,
138.80, 145.89, 154.55; LC-MS: tR ) 7.38 min, m/z: 219.95 (M
+ H)+, (C11H10ClN3). HRMS: (M + H)+ calcd for C11H11ClN3
220.0642, found 220.0661.
2-(2-Phenylethylamino)-6-chloropyrazine (4j). Using method
E with 400 mg (3.30 mmol) 2-phenylethylamine and 533 mg (3.63
mmol) 2,6-dichloropyrazine, 586 mg (53.4%) of 4j was ob-
tained after purification on an Isolute Si-II column, 20 g
(AcOEt:cyclohexane 2:1). 1H NMR (DMSO-d6) δ 2.85 (t, 2H, J )
7.6 Hz), 3.48 (q, 2H), 7.18-7.34 (m, 5H), 7.64 (m, 1H), 7.68 (s,
1H), 7.88 (s, 1H); 13C NMR (DMSO-d6) δ 34.55 41.88, 126.28,
127.25, 128.30, 128.67, 131.14, 143.09, 145.97, 154.65; LC-MS:
tR ) 7.64 min, m/z: 233.1 (M + H)+, (C12H11ClN3); HRMS: (M
+ H)+ calcd for C12H12ClN3 234.0798, found 234.0791.
2-(3-Phenylpropylamino)-6-chloropyrazine (4k). Using method
E with 100 mg (0.74 mmol) 3-phenylpropylamine and 117 mg (0.80
mmol) 2,6-dichloropyrazine, 136 mg (74.3%) of 4k was ob-
tained after purification on an Isolute Si-II column, 10 g
(AcOEt:cyclohexane 2:1). 1H NMR (DMSO-d6) δ: 1.78-1.91 (m,
2H), 2.67 (t, 2H, J ) 7.3 Hz), 3.24 (q, 2H), 7.14-7.33 (m, 5H),
7.61 (t, 1H, J ) 5.2 Hz), 7.65 (s, 1H), 7.88 (s, 1H); 13C NMR
(DMSO-d6) δ 30.07, 30.67, 32.45, 125.74, 127.95, 128.26, 128.40,
131.11, 141.56, 145.97, 154.78; LC-MS: tR ) 7.96 min, m/z:
247.97 (M + H)+, (C13H14ClN3). HRMS: (M + H)+ calcd for
C13H15ClN3 248.0955, found 248.0941.
1
H, J ) 8.0 Hz), 7.62-7.69 (m, 2 H), 7.76 (d, 1 H, J ) 7.9 Hz),
8.23 (s, 1 H), 8.24-8.29 (m, 2 H), 8.39 (bs, 1 H), 8.50 (s, 1 H),
9.99 (s, 1 H), 10.06 (s, 1 H); 13C NMR (DMSO-d6) δ 24.05, 69.78,
112.78, 117.31, 120.16, 121.13, 123.36, 124.58, 125.59, 129.29,
130.73, 133.63, 136.83, 139.71, 139.94, 141.47, 147.88, 151.05,
168.47, 170.75; LC-MS: tR ) 6.73 min; m/z 361.1 (M + H)+
(C18H15N4O2), 100% purity; HRMS: (M + H)+ calcd for
C18H16N4O2: 361.1295, found 361.1301.
2-(2,6-Dichloropyridin-4-ylamino)-6-(3-acetamidophenyl)-
pyrazine (29). Method B was followed starting from 4-amino-2,6-
dichloropyridine (57 mg, 0.35 mmol). Preparative TLC (AcOEt)
1
afforded 7 mg (8%) of 29. H NMR (DMSO-d6) δ 2.08 (s, 3 H),
7.48 (t, 1 H, J ) 7.9 Hz), 7.65 (d, 1 H, J ) 8.0 Hz), 7.71 (d, 1 H,
J ) 7.6 Hz), 7.85 (s, 2 H), 8.30 (s, 1 H), 8.32 (bs, 1 H), 8.66 (s,
1 H), 10.10 (s, 1 H), 10.55 (s, 1 H); 13C NMR (DMSO-d6) δ 23.9,
110.3, 117.47, 120.6, 121.2, 129.4, 133.4, 134.2, 136.3, 140.0,
148.3, 149.5, 149.7, 151.4, 168.3; LC-MS: tR ) 6.47 min; m/z
374.1 (M + H)+, (C17H13Cl2N5O), >80% purity.
2-(4-tert-Butylthiazol-2-ylamino)-6-(3-acetamidophenyl)pyra-
zine (30). Method B was followed starting from 4-(tert-butyl)-1,3-
thiazolyl-2-amine (55 mg, 0.35 mmol). Column chromatography
on silica gel (AcOEt: DCM, 2:1) furnished 19 mg (21%) of 30.
1H NMR (DMSO-d6) δ 1.30 (s, 9 H), 2.10 (s, 3 H), 6.69 (s, 1 H),
7.47 (t, 1 H, J ) 7.9 Hz), 7.69 (d, 1 H, J ) 8.0 Hz), 7.89 (d, 1 H,
J ) 7.8 Hz), 8.38 (s, 1 H), 8.45 (bs, 1 H), 8.58 (s, 1 H), 10.11 (s,
1 H), 11.78 (bs, 1 H); 13C NMR (DMSO-d6) δ 24.05, 29.80, 34.12,
103.13, 117.42, 120.48, 121.54, 129.30, 131.54, 133.28, 136.40,
139.92, 147.95, 158.12, 160.43, 168.46; LC-MS: tR ) 7.10 min;
m/z 368.2 (M + H)+, (C19H21N5OS), >50% purity.
2-(3-Chlorophenylamino)-6-chloropyrazine (4b). The title
compound was prepared by method B using 3-chloroaniline (520
mg, 3.5 mmol). The workup was different: to the reaction mixture
50 mL was added Et2O, and the mixture was extracted with 100
mL H2O. The aqueous layer was extracted again with 50 mL Et2O
and the organic solution pooled, dried (MgSO4) and evaporated to
dryness. A solid (0.852 g) was obtained and purified by preparative
HPLC (AcOEt:cycloxane 1:2). 0.419 g (50.0%) of 4b was collected.
1H NMR (DMSO-d6) δ 7.02 (m, 1H, J ) 7.9 Hz), 7.36 (t, 1H, J )
8.1 Hz), 7.51 (m, 1H, J ) 8.3 Hz), 7.83 (t, 1H, J ) 2.0 Hz), 8.03
(s, 1H), 8.18 (s, 1H), 10.03 (s, 1H); LC-MS, tR ) 8.41 min; m/z:
296 (M - H)-, (C10H7Cl2N3).
2-(4-Methoxyanilin)-6-chloropyrazine (4c). Using method B
with 4-methoxyaniline (500 mg, 3.35 mmol) (reaction time: 5 h),
4c was obtained (265 mg, 33.7%), after purification by preparative
1
HPLC (cyclohehane:AcOEt 1:1). H NMR (DMSO-d6) δ 3.73 (s,
3H), 6.94 (d, 2H, J ) 9.0 Hz), 7.51 (d, 2H), 7.89 (s, 1H), 8.08 (s,
1H), 9.65 (s, 1H).
2-(2,3-Dehydro-1,4-benzodioxin-6-methylamino)-6-chloro-
pyrazine (4d). Method E. Amounts of 100 mg (0.61 mmol) 2,3-
dehydro-1,4-benzodioxin-6-methylamine, 96 mg (0.15 mmol) 2,6-
dichloropyrazine and 170 mL triethylamine were stirred in 10 mL
DMF for 18 h at 80 °C. After cooling, the reaction mixture was
diluted with 25 mL H2O and extracted with 25 mL AcOEt. The
organic layer was washed (2 × 25 mL brine), dried, (MgSO4) and
evaporated to 2-3 mL. The resulting solution was purified on an
Isolute flash Si-II column (10 g Si) (AcOEt:cyclohexane 2:1), from
which 133 mg (78.6%) of 4d were obtained, as a thick oil. 1H NMR
(DMSO-d6) δ: 4.22 (s, 2H), 4.33 (d, 2H, J ) 5.3 Hz), 6.81 (s,
1H), 6.83 (d, 2H), 7.71 (s, 1H), 7.91 (s, 1H), 7.96 (t, 1H); 13C
NMR (DMSO-d6) δ 43.19, 63.95, 116.20, 116.87, 120.37, 128.14,
131.73, 142.40, 143,13, 145.87, 154.44; LC-MS, tR ) 7.15 min;
m/z: 278.05 (M + H)+, (C13H12ClN3O2); HRMS: (M + H)+ calcd
for C13H12ClN3O2: 319.0962, found 319.0965.
2-(1,3-Benzodioxol-5-ylmethylamino)-6-chloropyrazine (4e).
Using method E from 140 mg (0.92 mmol) 1,3-benzodioxol-5-
ylmethylamine and 148 mg (1.01 mmol) 2,6-dichloropyrazine, 248
mg (72.6%) of 4e was obtained after purification on an Isolute
column (AcOEt:cyclohexane 2:1). 1H NMR (DMSO-d6) δ 4.37 (d,
2-(2-Phenoxyethylamino)-6-chloropyrazine (4l). Using method
E with 400 mg (2.90 mmol) 2-phenoxyethylamine and 468 mg (3.19
mmol) 2,6-dichloropyrazine, 467 mg (64.7%) of 4l was obtained
after purification by chromatography on an Isolute Si-II column
1
10 g (AcOEt:cyclohexane 2:1). H NMR (DMSO-d6) δ 3.64 (q,

414 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1
Niculescu-DuVaz et al.
2H), 4.12 (t, 2H, J ) 5.6 Hz), 6.94-7.00 (m, 3H), 7.26-7.33 (m,
2H), 7.72 (s, 1H), 7.82 (t, 1H, J ) 5.5 Hz), 7.88 (s, 1H); 13C NMR
(DMSO-d6) δ 65.65, 114.47, 120.68, 128.12, 129.47, 131.43,
143.09, 145.85, 154.69, 158.31; LC-MS: tR ) 7.52 min; m/z:
247.97 (M + H)+, (C12H12ClN3O). HRMS: (M + H)+ calcd for
C12H13ClN3O 250.0747, found 250.0742.
a twelve-tube carousel. After purification on an Isolute column,
Flash Si-II, 10 g (AcOEt), 89 mg (65.7%) of 31 was obtained. 1H
NMR (DMSO-d6) δ 2.08 (3, 3H), 4.22 (s, 2H), 4.86 (d, 2H, J )
5.8 Hz), 6.80 (d, 2H, J ) 7.7 Hz), 6.89 (d, 2H, J ) 7.6 Hz), 6.91
(s, 1H), 7.39 (t, 1H), 7.59 (s, 1H), 7.66-7.68 (m, 2H), 7.91 (s,
1H), 8.19 (s, 1H), 8.24 (s, 1H), 10.05 (s, 1H); 13C NMR (DMSO-
d6) δ 24.02, 43.03, 59.72, 63.95, 116.31, 116.79, 120.60, 127.66,
129.00, 131.86, 132.83, 137.33, 139.72, 142.28, 143.12, 147.97,
153.89, 168.38; LC-MS: tR ) 6.84 min; m/z: 377.1 (M + H)+,
(C21H20N4O3). HRMS: (M + H)+ calcd for C21H21N4O3 377.1614,
found 377.1586.
2-[2-(4-Methoxyphenoxy)ethylamino]-6-chloropyrazine (4m).
Using method E with 140 mg (0.84 mmol) 2-(4-methoxyphenoxy)-
ethylamine and 135 mg (0.92 mmol) 2,6-dichloropyrazine, 176 mg
(75.2%) of 4m was obtained after purification on an Isolute Si-II
1
column 10 g (AcOEt:cyclohexane 2:1). H NMR (DMSO-d6) δ
3.60 (q, 2H, J ) 5.5 Hz), 3.70 (s, 3H), 4.06 (t, 2H, J ) 5.6 Hz),
6.86 (d, 2H), 6.92 (m, 2H), 7.72 (s, 1H), 7.80 (t, 1H, J ) 5.3 Hz),
7.96 (s, 1H); 13C NMR (DMSO-d6) δ 55.33, 65.65, 114.60, 115.67,
128.08, 131.44, 143.09, 145.85, 151.80, 154.69; LC-MS: tR ) 6.80
min; m/z: 279.9 (M + H)+, (C13H14ClN3O2); HRMS; (M + H)+
calcd for C15H18ClN4O2 321.1118, found 321.1122.
2-(3,4-Methylenedioxyphenylaminomethyl)-6-(3-acetamido-
phenyl)pyrazine (30). Using method A, 100 mg (0.38 mmol)
2-(3,4-methylenedioxyphenylamino methyl)-6-chloropyrazine (4e)
were reacted with 102 mg (0.58 mmol) 3-acetamido phenyl boronic
acid (reaction time: 4 h), and after purification on a Isolute column,
Flash SiII, 10 g (AcOEt), 70.0 mg (50.8%) of 30 was obtained. 1H
NMR (DMSO-d6) δ 2.08 (s, 3H), 4.76 (d, 2H, J ) 5.8 Hz), 5.97
(s, 2H), 6.86 (d, 1H, J ) 7.9 Hz), 6.93 (d, 1H, J ) 8.0 Hz), 7.93
(t, 1H, J ) 7.9 Hz), 7.61 (t, 1H, J ) 5.8 Hz), 7.78 (d, 2H, J ) 7.8
Hz), 7.92 (s, 1H), 8.20 (s, 1H), 8.26 (s, 1H), 10.05 (s, 1H), 13C
NMR (DMSO-d6) δ 24.02, 43.41, 100.74, 108.03, 108.25, 117.01,
119.81, 120.92, 127.71, 129.01, 131.88, 133.68, 137.30, 139.72,
146.06, 147.20, 147.94, 153.83, 168.39; LC-MS: tR ) 6.92 min;
m/z: 363.1 (M + H)+, (C20H18N4O3); HRMS; (M + H)+ calcd for
C20H19N4O3 363.1457, found 363.1464.
2-[(5-Methyl-3-isoxazolyl)methylamino]-6-(3-acetamido-
phenyl)pyrazine (33). Using method A, 100 mg (0.44 mmol) 2-[(5-
methyl-3-isoxazolyl)methylamino]-6-chloropyrazine (4f) and 120.0
mg (0.67 mmol) 3-acetamidophenyl boronic acid, (reaction time:
4 h) wrer reacted, and after purification on an Isolute column, Flash
SiII, 10 g (AcOEt), 54.0 mg (34%) of 33 resulted. 1H NMR
(DMSO-d6) δ 2.08 (s, 3H), 2.36 (s, 3H), 4.62 (d, 2H, J ) 5.9 Hz),
6.20 (s, 1H), 7.40 (t, 1H, J ) 7.9 Hz), 7.61 (t, 1H, J ) 5.8 Hz),
7.67-7.70 (m, 3H), 7.96 (s, 1H), 8.23 (s, 1H), 8.25 (s, 1H), 10.05
(s, 1H), 13C NMR (DMSO-d6) δ 11.73, 24.02, 36.99, 101.32,
117.00, 119.90, 121.10, 128.25, 129.06, 131.97, 137.17, 139.73,
147.99, 153.63, 162.40, 168.40, 169.40; LC-MS: tR ) 5.98 min;
m/z: 323.1 M + , (C17H17N5O2); HRMS; (M + H)+, calcd for
C17H18N5O2 324.1461, found 324.1486.
2-(3,4,5-Trimethoxybenzylamino)-6-chloropyrazine (4n). Us-
ing method E with 400 mg (2.03 mmol) 2-phenylethylamine and
327 mg (2.23 mmol) 2,6-dichloropyrazine, 434 mg (69.2%) of 4n
was obtained after purification on an Isolute Si-II column, 20 g
1
(AcOEt:cyclohexane 2:1). H NMR (DMSO-d6) δ 3.64 (s, 6H),
3.77 (s, 3H), 4.38 (d, 2H, J ) 5.7 Hz), 6.71 (s, 2H), 7.73 (s, 1H),
7.73 (m, 2H); 13C NMR (DMSO-d6) δ 44.22, 55.82, 59.95, 105.15,
128.26, 131.28, 134.26, 136.57, 145.84, 152.83, 154.48;
LC-MS: tR ) 6.83 min; m/z: 309.93 (M + H)+, (C16H16ClN3O3);
HRMS; (M + H)+ calcd for C16H17ClN3O3 310.0958, found
310.0948.
3-(3-Fluorobenzylamino)-6-chloropyrazine (4o). In an oven
dried flask, 50 mg (034 mmol) 3,6-dichloropyrazine, 50 mg (0.40
mmol) 3-fluoro-benzylamine, 48 mg (0.50 mmol) sodium tert-
butoxide and 3.20 mg (0.007 mmol) palladacycle 220 were
suspended in 2 mL dry toluene under stirring and N2. The reaction
mixture was heated for 2 h at 90 °C (bath temperature). The solvent
was evaporated under vacuum, the residue taken up in 10 mL
AcOEt, washed (2 × 10 mL brine), dried (MgSO4) and evaporated
to dryness and 40 mg of 4o, as a brown oil, was obtained.
Using method D with 50 mg (0.34 mmol) of 2,6-dichloropyrazine
and 50 mg (0.40 mmol) 3-fluorobenzylamine and, after purification
on an Isolute flash Si column, 10 g (AcOEt) 80 mg (62.2%) of 4o,
1
as a thick oil, was obtained. H NMR (DMSO-d6) δ 4.49 (d, 2H,
2-[(1,5-Dimethyl-1H-pyrazol-3-yl)methylamino]-6-(3-acetami-
dophenyl)pyrazine (34). Using method A, 100 mg (0.42 mmol)
2-[(1,5-dimethyl-1H-pyrazol-3-yl)methylamino]-6-chloropyrazine
(4g) and 113.0 mg (0.63 mmol) 3-acetamido phenyl boronic acid
(reaction time: 4 h) gave, after purification on an Isolute column,
Flash SiII, 10 g (AcOEt), 47.0 mg (34.6%) of 34. 1H NMR (DMSO-
d6) δ: 2.08 (s, 3H), 2.20 (s, 3H), 3.68 (s, 1), 4.46 (d, 2H, J ) 5.6
Hz), 6.00 (s, 1H), 7.36-7.42 (m, 2H), 7.68-7.73 (m, 2H), 7.93 (s,
1H), 8.17 (s, 1H), 8.23 (s, 1H), 10.05 (s, 1H), 13C NMR (DMSO-
d6) δ 10.63, 24.01, 35.06, 37.78, 103.78, 117.03, 119.79, 121.03,
127.56, 129.00, 131.94, 137.40, 138.87, 139.71, 147.90, 153.96,
168.39; LC-MS: tR ) 6.01 min; m/z: 337.1 (M + H)+, (C18H20N6O);
HRMS; (M + H)+, calcd for C18H20N6O 337.1777, found 337.1763.
2-(2-Pyridylmethylamino)-6-(3-acetamidophenyl)pyrazine (35).
Using method A, 100 mg (0.42 mmol) 2-(2-pyridylmethylamino)-
6-chloropyrazine (4h) and 122.0 mg (0.68 mmol) 3-acetamido
phenyl boronic acid (reaction time: 4 h) gave after purification on
an Isolute column, Flash SiII, 10 g (AcOEt), 117.0 mg (77.4%) of
35. 1H NMR (DMSO-d6) δ 2.07 (s, 3H), 4.46 (d, 2H, J ) 5.9 Hz),
7.24-7.29 (m, 1H), 7.37 (t, 1H, J ) 7.9 Hz), 7.45 (d, 1H), 7.59-
7.79 (m, 4H), 8.02 (s, 1H), 8.19 (s, 1H), 8.20 (s, 1H), 8.55 (d, 1H),
10.05 (s, 1H), 13C NMR (DMSO-d6) δ 24.01, 57.72, 119.83, 121.05,
121.45, 122.10, 127.92, 128.99, 132.00, 136.68, 137.26, 139.69,
148.00, 153.91, 158.96, 168.38; LC-MS: tR ) 3.61 min; m/z: 320.1
(M + H)+, (C18H17N5O); HRMS; (M + H)+, calcd for C18H18N5O
320.1511, found 320.1520.
J ) 5.9 Hz), 7.05-7.19 (m, 3H), 7.35-7.44 (m, 1H), 7.74 (s, 1H),
7.96 (s, 1H), 8.09 (t, 1H, J ) 5.5 Hz); 13C NMR (DMSO-d6) δ
43.25, 113.79, 123.36, 128.53, 130.34, 131.21, 141.92, 145.84,
152.43, 161.26, 163.19; LC-MS: tR ) 7.36 min; m/z: 238 (M +
H)+, (C11H9ClFN3). HRMS: (M + H)+ calcd for C11H10ClFN3
238.0547, found 238.0533.
2-(3-Furylmethylamino)-6-chloropyrazine (4p). Using method
B, with 100 mg (0.67 mmol) 2,6-dichloropyrazine and 80 mg (0.82
mmol) 3-furylmethylamine and after purification by preparative
HPLC (AcOEt:cyclohexane 2:1), 4p was obtained as thick oil
crystallizing on storage: 128 mg (50.5%). 1H NMR (DMSO-d6) δ
4.28 (d, 2H, J ) 5.6 Hz), 7.31 (s, 1H), 7.62-767 (m, 2H), 7.73 (s,
1H), 7.83 (t, 1H, J ) 5.2 Hz), 7.92 (s, 1H); LC-MS, tR ) 6.45
min; m/z: 210 (M + H)+, (C9H8ClFN3O).
2-(3-Chloro-4-fluorobenzylamino)-6-chloropyrazine (4q). Us-
ing method B with 100 mg (0.67 mmol) 2,6-dichloropyrazine and
159 mg (1.00 mmol) 3-chloro-4-fluorobenzylamine and purification
on Isolute column, 10 g (AcOEt) 4q was obtained as a thick oil,
1
138 mg (76%). H NMR (DMSO-d6) δ 4.49 (d, 2H, J ) 5.9 Hz),
7.05-7.19 (m, 3H), 7.35-7.44 (m, 1H), 7.74 (s, 1H), 7.96 (s, 1H),
8.09 (t, 1H, J ) 5.5 Hz); 13C NMR (DMSO-d6) δ 42.66, 116.85,
128.09, 128.63, 129.47, 131.25, 136.88, 145.81, 154.30, 155.27,
157.22; LC-MS: tR ) 7.94 min; m/z: 238 (M + H)+, (C11H8-
ClFN3). HRMS: (M + H)+ calcd for C11H9Cl2FN3 272.0158, found
272.0155.
2-(2,3-Dehydro-1,4-benzodioxin-6-methylamino)-6-(3′-aceta-
midophenyl)pyrazine (31). Using method A, 100 mg (0.36 mmol)
2-(2,3-dehydro-1,4-benzodioxin-6-methylamino)-6-chloro-
pyrazine, 4d, and 97 mg (0.54 mmol, 1.5 equiv) 3-acetamidophenyl
boronic acid were reacted at 80 °C (bath temperature) for 20 h in
2-(Benzylamino)-6-(3-acetamidophenyl)pyrazine (36). Using
method A with 100 mg (0.46 mmol) 2-benzylamino-6-chloro-
pyrazine (4i) and 123 mg (0.68 mmol) 3-acetamidophenyl boronic
acid, 116 mg (79.2%) of 36 were obtained after purification on a
1
Isolute Si-II column, 10 g (AcOEt). H NMR (DMSO-d6) δ 2.08

Inhibitors of B-RAF
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1 415
(s, 3H), 4.62 (d, 2H, J ) 5.8 Hz), 7.21-7.46 (m, 7H), 7.64-7.72
2-(3-Fluorophenylmethylamino)-6-(3′-acetamidophenyl)pyra-
zine (42). Using method A and starting from 80 mg (0.33 mmol)
2-(3-fluorophenylmethyl amino)-6-chloropyrazine (4o) and 62 mg
(0.38 mmol) 3-fluorobenzylamine, 26 mg (23.6%) pure 42 was
obtained after purification by preparative HPLC (AcOEt:EtOH 95:
5). Reaction time: 18 h. 1H NMR (DMSO-d6) δ 2.07 (s, 3H), 4.63
(d, 2H, J ) 5.9 Hz), 7.06-7.50 (m, 5H), 7.60-7.70 (m, 2H), 7.74
(t, 1H, J ) 5.7 Hz), 7.95 (s, 1H), 8.22 (s, 1H), 8.25 (s, 1H), 10.04
(s, 1H); 13C NMR (DMSO-d6) δ 24.01, 43.14, 113.44, 114.14,
117.03, 119.86, 121.00, 123.63, 127.95, 129.00, 130.25, 131.90,
137.22, 143.06, 147.96, 153.79, 161.26, 168.37; LC-MS: tR ) 7.07
min; m/z 337.1 (M + H)+, (C19H17FN4O); HRMS; (M + H)+, calcd
for C19H18FN4O 337.1465, found 337.1456.
(m, 2H), 7.94(s, 1H), 8.20 (s, 1H), 8.25 (s, 1H), 10.04 (s, 1H); 13
C
NMR (DMSO-d6) δ 24.02, 43.61, 117.01, 119.81, 121.08, 126.80,
127.70, 128.30, 131.89, 137.32, 139.72, 148.00, 153.95, 168.38;
LC-MS: tR ) 7.00 min; m/z 319.1 (M + H)+, (C19H18N4O); HRMS;
(M + H)+, calcd for C19H19N4O 319.1559, found 319.1582.
2-(Phenylethylamino)-6-(3-acetamidophenyl)pyrazine (37).
Using method A with 100 mg (0.43 mmol) 2-(phenylethylamino)-
6-chloropyrazine (4j) and 115 mg (0.64 mmol) 3-acetamidophenyl
boronic acid, 96 mg (67.6%) of 37 was obtained, after purification
on an Isolute Si-II column, 10 g (AcOEt). 1H NMR (DMSO-d6) δ:
2.08 (s, 3H), 2.93 (t, 2H, J ) 7.6 Hz) 3.52 (q, 2H), 7.17-7.44 (m,
7H), 7.69 (d, 2H), 7.89 (s, 1H), 8.17 (s, 1H), 8.25 (s, 1H), 10.05
(s, 1H); 13C NMR (DMSO-d6) δ 14.06, 34.87, 41.94, 117.12,
119.82, 121.05, 126.03, 127.45, 128.30, 128.80, 128.99, 131.95,
137.53, 139.69, 139.71, 148.15, 154.13, 168.37; LC-MS: tR ) 7.38
min; m/z 332.1 M + , (C20H20N4O); HRMS; (M + H)+, calcd for
C20H21N4O 333.1715, found 333.1736.
2-(3-Phenylpropylamino)-6-(3-acetamidophenyl)pyrazine (38).
Using method A with 100 mg (0.40 mmol) 2-(3-phenylpropyl-
amino)-6-chloropyrazine (4k) and 107 mg (0.60 mmol) 3-aceta-
midophenyl boronic acid, 124 mg (89.5%) of 38 was obtained after
purification on an Isolute Si-II column (AcOEt). 1H NMR (DMSO-
d6) δ 1.85-1.98 (m, 2H), 2.72 (t, 2H, J ) 7.3 Hz), 3.40 (q, 2H),
7.14-7.42 (m, 7H), 7.66 (dd, 2H), 7.89 (s, 1H), 8.15 (s, 1H), 8.22
(s, 1H), 10.05 (s, 1H); 13C NMR (DMSO-d6) δ 24.02, 30.57, 32.66,
117.03, 119.78, 121.04, 125.86, 127.25, 128.24, 128.31, 128.97,
131.86, 137.48, 139.68, 141.82, 148.08, 154.08, 168.37; LC-MS:
tR ) 7.67 min; m/z 347.1 (M + H)+, (C21H22N4O); HRMS; (M +
H)+, calcd for C21H23N4O 347.1845, found 347.1847.
2-Phenoxyethylamino-6-(3-acetamidophenyl)pyrazine (39).
Using method A, 100 mg (0.40 mmol) 2-phenoxyethylamino-6-
chloropyrazine (4l) and 107.0 mg (0.60 mmol) 3-acetamidophenyl
boronic acid were reacted, and after purification on an Isolute
column, Flash Si-II (AcOEt), 112.0 mg (80.3%) of 39 was obtained.
1H NMR (DMSO-d6) δ 3.79 (dd, 2H, J ) 5.5 Hz), 4.20 (t, 2H),
3.40 (q, 2H), 6.90-7.01 (m, 3H), 7.25-7.43 (m, 4H), 7.69 (t, 2H,
J ) 7.5 Hz), 7.97 (s, 1H), 8.19 (s, 1H), 8.22 (s, 1H), 10.06 (s, 1H);
13C NMR (DMSO-d6) δ 24.03, 65.98, 114.48, 117.08, 119.79,
120.60, 121.08, 127.73, 129.48, 132.15, 137.43, 139.73, 148.09,
154.10, 158.42, 168.40; LC-MS: tR ) 7.29 min; m/z 349.1 (M +
H)+, (C20H20N4O2); HRMS; (M + H)+, calcd for C20H21N4O2
349.1665, found 349.1673.
2-(3-Furylmethylamino)-6-(3-acetamidophenyl)pyrazine (43).
Using method A with 160 mg (0.76 mmol) 2-(3-furylmethylamino)-
6-chloropyrazine (4p) and 168 mg (0.87 mmol) 3-acetamidophenyl
boronic acid, 114 mg (48.7%) pure 43 was obtained after purifica-
tion by preparative HPLC (AcOEt:EtOH 95:5). Reaction time: 18
1
h. H NMR (DMSO-d6) δ 2.08 (s, 3H), 4.43 (d, 2H, J ) 5.8 Hz),
6.53 (s, 1H), 7.30-7.50 (m, 2H), 7.52-7.75 (d, 4H), 7.95 (s, 1H),
8.22 (s, 1H), 8.30 (s, 1H), 10.04 (s, 1H); 13C NMR (DMSO-d6) δ
20.73, 24.02, 110.87, 116.92, 119.82, 120.94, 123.11, 127.67,
129.02, 130.26, 132.04, 137.30, 139.76, 140.32, 143.31, 147.87,
153.84, 168.42; LC-MS: tR ) 6.43 min; m/z 309.1 (M + H)+,
(C17H16N4O2); HRMS; (M + H)+, calcd for C17H17N4O2 309.1352,
found 309.1376.
2-(3-Chloro-4-fluorobenzylamino)-6-(3-acetamidophenyl)-
pyrazine (44). Using method A with 100 mg (0.37 mmol) 2-(3-
chloro-4-fluorobenzylamino)-6-chloropyrazine (4q) and 76 mg (0.42
mmol) 3-acetamidophenyl boronic acid, 40 mg (29.2%) pure 44
was obtained after purification by preparative HPLC (AcOEt:EtOH
1
95:5). Reaction time: 18 h. H NMR (DMSO-d6) δ 2.08 (s, 3H),
4.58 (d, 2H, J ) 5.8 Hz), 7.30-7.55 (m, 3H), 7.70 (d, 3H), 7.78
(s, 1H), 7.95 (s, 1H), 8.22 (s, 1H), 8.28 (s, 1H), 10.04 (s, 1H); 13
C
NMR (DMSO-d6) δ 24.08, 100.62, 100.74, 111.15, 117.08, 119.92,
121.09, 129.22, 129.61, 133.28, 135.06, 137.15, 139.88, 141.76,
147.20, 147.96, 151.93, 168.45; LC-MS: tR ) 7.56 min; m/z 371.1
(M + H)+, (C19H16ClFN4O); HRMS; (M + H)+, calcd for C19H17-
ClFN4O 371.1075, found 371.1061.
V600EB-RAF Kinase Assay. Full-length rabbit MEK1 protein was
expressed with a GST tag at the N-terminus and a C-terminal
histidine tag in Escherichia coli JM109 bacteria and purified by
nickel-agarose affinity chromatography. V600EB-RAF was generated
by infection of SF9 insect cells cultured in SF-900 II medium
(Invitrogen, Paisley, Scotland) with a baculovirus containing full-
length human B-RAF with an N-terminal histidine tag. Assay buffer
was 20 mM MOPS, pH 7.2, containing 5 mM EGTA, 10 mM
MgCl2, 0.1% â-ME and 25 mM â-glycerophosphate. All incuba-
tions were at room temperature with shaking. Amounts of 1 µg
GST-MEK1, 0.07 µL insect cell V600EB-RAF lysate and 0.5 µL
inhibitor at the required concentrations (0.001 to 100 µM final
concentration) were added to the wells of a glutathione-coated plate,
and the plate was preincubated for 10 min. ATP in assay buffer
(10 µL), to give a final concentration of 100 µM, was added to
each well, and the plates were incubated for 45 min. The plates
were then washed 3× with 200 µL 0.1% tween20/water. Primary
antibody (rabbit anti-phospho MEK1/2 diluted 1/2000, Cell Signal-
ing Technologies, Hitchin, UK) and Eu-labeled anti-rabbit second-
ary antibody (diluted 1/1000, Perkin-Elmer, Turku, Finland) were
preincubated for 30 min and 50 µL added to the washed plates,
which were incubated for a further hour. The plates were washed
as before, and 100 µL DELFIA enhancement solution (Perkin-
Elmer, Turku, Finland) was added. The plates were sealed and
incubated for 30 min and europium counts measured on a Victor 2
reader (Perkin-Elmer, Turku, Finland).
2-(4-Methoxyphenyloxyethylamino)-6-(3-acetamidophenyl)-
pyrazine (40). Using method A with 228 mg (0.97 mmol) 2-(4-
methoxyphenyloxyethylamino)-6-chloropyrazine (4m) and 147 mg
(0.82 mmol) 3-acetamidophenyl boronic acid, 33 mg (10.2%) pure
40 was obtained, after purification on an Isolute SiII column, 10 g
1
(AcOEt). Reaction time: 24 h. H NMR (DMSO-d6) δ: 2.09 (s,
3H), 3.75 (s, 3H), 7.27 (s, 2H), 6.96 (d, 2H, J ) 9.0 Hz), 7.42 (t,
1H, J ) 7.9 Hz), 7.63 (d, 2H, J ) 7.7 Hz), 7.71 (d, 2H), 7.75 (d,
2H), 8.11 (s, 1H), 8.23 (d, 2H), 8.36 (s, 1H), 9.41 (s, 1H), 10.09
(s, 1H); 13C NMR (DMSO-d6) δ 24.03, 55.30, 66.63, 114.58,
115.46, 117.06, 119.77, 121.08, 127.69, 129.01, 132.16, 137.43,
139.73, 148.08, 152.42, 153.40, 154.11, 161.33, 168.40; LC-MS:
tR ) 7.13 min; m/z 379.1 (M + H)+, (C21H22N4O3); HRMS; (M +
H)+, calcd for C21H23N4O3 379.1770, found 379.1757.
2-(3,4,5-Trimethoxybenzylamino)-6-(3-acetamidophenyl)pyra-
zine (41). Using method A with 100 mg (0.32 mmol) 2-(3,4,5-
trimethoxybenzylamino)-6-chloropyrazine (4n) and 87 mg (0.49
mmol) 3-acetamidophenyl boronic acid, 114 mg (87.2%) of 41 was
obtained, after purification on an Isolute Si-II column, 10 g (AcOEt).
1H NMR (DMSO-d6) δ 2.07 (s, 3H), 3.62 (s, 6H), 3.74 (s, 3H),
4.53 (d, 2H, J ) 4.9 Hz), 6.79 (s, 2H), 7.39 (t, 1H, J ) 7.9 Hz),
7.62-7.72 (m, 3H), 7.94 (s, 1H), 8.23 (s, 1H), 8.33 (s, 1H), 10.06
(s, 1H); 13C NMR (DMSO-d6) δ 23.99, 55.71, 59.91, 105.29,
116.95, 119.80, 120.94, 127.65, 128.99, 132.01, 135.44, 136.33,
137.24, 139.74, 147.77, 152.74, 153.85, 168.39; LC-MS: tR ) 6.51
min; m/z 408.1 (M + H)+, (C22H24N4O4); HRMS; (M + H)+, calcd
for C22H25N4O4 409.1876, found 409.1874.
SRB IC50 for B-RAF Inhibitors. WM266.4 melanoma cells
were cultured in DMEM/10% fetal bovine serum, at 37 °C, in 5%
CO2 water saturated atmosphere. Cell suspensions (10 000/mL)
were prepared and 100 µL/well dispensed into 96-well plates giving
1000 cells/well. The plates were returned to the incubator for 24 h
to allow the cells to reattach. The compounds were initially prepared

416 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1
Niculescu-DuVaz et al.
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N. et al. Paradoxical activation of Raf by a novel Raf inhibitor. Chem.
Biol. 1999, 6, 559-568.
(9) Wilhelm, S. M.; Carter, C.; Tang, l. y.; Wilkie, D.; McNabola, A. et
al. BAY 43-9006 exhibits broad spectrum oral antitumor activity
and targets the RAF/MEK/ERK pathway and receptor tyrosine
kinases involved in tumor progression and angiogenesis. Cancer Res.
2004, 64, 7099-7109.
(10) Bollag, G.; Freeman, S.; Lyons, G. F.; Post, L. E. Raf pathway
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at 20 mM in DMSO. Aliquots (200 µL) were diluted into 20 mL
culture medium giving 200 µM, and 10 serial dilutions of 3×
prepared. Aliquots (100 µL) of each dilution were added to the
wells, giving doses ranging from 100 µM to 0.005 µM. After a
further 6 days growth, the cells are fixed in trichloroacteic acid
(10%) and stained with sulforhodamine-B (0.1%). After rinsing,
the bound stain was taken into solution and the absorbance at 540
nm determined. The percent control growth was plotted against
the logarithm of the drug concentration and analyzed by nonlinear
regression to a four-parameter logistic equation (Graphpad Prism,
Graphpad Software Inc., San Diego, CA). The IC50 generated by
this procedure is the concentration of the drug that produces a
percentage control A540 midway between the saturation and zero-
effect plateaus.
(12) Lee, J. T.; McCubrey, J. A. BAY 43-9006 Bayer/Onix. Curr. Opin.
InVestig. Drugs 2003, 4, 757-763.
(13) Batt, D. B.; Bold, G.; Kim, S.; Ramsey, T. M.; Sabio, M. L. Use of
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(14) Fink, C. A.; Perez, L. B.; Ramsey, T. M.; Yusuf, N.; Versace, R. W.
et al. 1,4-Disubstituted isoquinoline deriVatiVes as RAF-kinase
inhibitors useful for the treatment of proliferatiVe diseases; Novartis
AG.: Switzerland, 2005; p 144.
Acknowledgment. This work is supported by Cancer
Research UK (CUK) grant numbers: C309/A2187 and C107/
A3096, the Wellcome Trust and The Institute of Cancer
Research.
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