Novel hinge binder improves activity and pharmacokinetic properties of BRAF inhibitors

Article abstract of DOI:10.1021/jm100383b

Mutated BRAF serine/threonine kinase is implicated in several types of cancer, with particularly high frequency in melanoma and colorectal carcinoma. We recently reported on the development of BRAF inhibitors based on a tripartite A?B?C system featuring an imidazo[4,5]pyridin-2-one group hinge binder. Here we present the design, synthesis, and optimization of a new series of inhibitors with a different A?B?C system that has been modified by the introduction of a range of novel hinge binders (A ring). The optimization of the hinge binding moiety has enabled the development of compounds with low nanomolar potencies in both BRAF inhibition and cellular assays. These compounds display optimal pharmacokinetic properties that warrant further in vivo investigations.

Full text of DOI:10.1021/jm100383b

J. Med. Chem. 2010, 53, 5639–5655 5639
DOI: 10.1021/jm100383b
Novel Hinge Binder Improves Activity and Pharmacokinetic Properties of BRAF Inhibitors
†
Alfonso Zambon, Delphine Menard, Bart M. J. M. Suijkerbuijk, Ion Niculescu-Duvaz, Steven Whittaker,
†
†
†
‡
ꢀ
Dan Niculescu-Duvaz,† Arnaud Nourry,† Lawrence Davies,† Helen A. Manne,† Filipa Lopes,† Natasha Preece,†
Douglas Hedley,† Lesley M. Ogilvie,† Ruth Kirk,‡ Richard Marais,‡ and Caroline J. Springer*,†
†The Institute of Cancer Research, Cancer Research UK Centre for Cancer Therapeutics, 15 Cotswold Road, Sutton, Surrey SM2 5NG,
United Kingdom, and ‡The Institute of Cancer Research, Cancer Research UK Centre for Cell and Molecular Biology, 237 Fulham Road,
London SW3 6JB, United Kingdom
Received March 26, 2010
Mutated BRAF serine/threonine kinase is implicated in several types of cancer, with particularly high
frequency in melanoma and colorectal carcinoma. We recently reported on the development of BRAF
inhibitors based on a tripartite A-B-C system featuring an imidazo[4,5]pyridin-2-one group hinge
binder. Here we present the design, synthesis, and optimization of a new series of inhibitors with a
different A-B-C system that has been modified by the introduction of a range of novel hinge binders
(A ring). The optimization of the hinge binding moiety has enabled the development of compounds with
low nanomolar potencies in both BRAF inhibition and cellular assays. These compounds display
optimal pharmacokinetic properties that warrant further in vivo investigations.
Introduction
disclosed), some of which are currently being evaluated in late
stage clinical trials.
BRAFa is a member of the RAF protein kinase family that
includes ARAF and CRAF (also known as RAF1). RAF
kinases are components of the mitogen-activated protein
kinase (MAPK) RAF-MEK-ERK signal transduction cas-
cade, a pathway that regulates several cell functions from pro-
liferation, and survival to differentiation and senescence. Acti-
vation of this pathway is a driver in several types of cancers,1
and large-scale genomic screens have detected mutations of
BRAF in malignant melanomas (50%), colorectal cancers
(15%), thyroid cancer (30%), and ovarian cancer (30%) and
at lower frequencies in several other cancer types.2,3 Over 100
mutations have been identified in BRAF, but the most com-
mon is a glutamic acid for valine substitution at position 600
(V600E). This single mutation accounts for over 90% of BRAF
mutations in cancer, confers 500-fold activation4 in melanoma,
and has been shown to stimulate cell proliferation and sur-
vival.4-6 Importantly, inhibition of BRAF blocks prolifera-
tion, induces apoptosis in vitro, and blocks tumor growth in
vivo,6,7 validating BRAF as a therapeutic target in human
cancer. Several drug development programs for the treatment
of mutant BRAF-driven malignancies have been initiated, with
compounds such as PLX40328 (structure not disclosed), N-(4-
chloro-3-(trifluoromethyl)phenyl)-1-methyl-5-(2-(5-(trifluoro-
methyl)-1H-imidazol-2-yl)pyridin-4-yloxy)-1H-benzo[d]imidazol-
2-amine (RAF265),9 (E)-5-(2-(4-(2-(dimethylamino)ethoxy)-
phenyl)-4-(pyridin-4-yl)-1H-imidazol-5-yl)-2,3-dihydro-1H-
inden-1-one oxime (SB590885),10 and XL28111 (structure not
We have developed a range of V600EBRAF inhibitors,12-15
targeting the inactive conformation of V600EBRAF. These
type II16 inhibitors use a common scaffold that comprises a
2,3-dihydroimidazo[4,5]pyridin-2-one moiety binding to the
hinge region of BRAF, a meta or para substituted aromatic
system occupying the lipophilic DFG-out pocket, a linker
interacting with the salt bridge formed between residues
Glu501 and Lys483, and a terminal aromatic group (phenyl
or pyrazole) that fills the allosteric pockets created by the
displacement of the DFG loop. For clarity, we designated the
hinge binder, the middle ring, and the terminal aromatic
group as rings A, B and C, respectively, and the salt bridge
binder as the BC linker.
The pyridoimidazolone moiety was envisaged as having
two functions: (1) to afford additional H-bond interactions
with the hinge backbone (one from the pyridine nitrogen
H-bond acceptor and one via the 3-NH donor of the imi-
dazolone) compared to compounds such as sorafenib, which
has a single H-bond from the pyridyl ring, and (2) to improve
the PK properties by reducing the number of rotable bonds.12
The structure and activities of sorafenib (the multikinase drug
designed to be a CRAF inhibitor) and relevant compounds 5a
and 5q were previously disclosed12 and are reported in Figure 1.
Interestingly, compound 5a from ref 12, an analogue of
sorafenib bearing the alternative pyridoimidazolone hinge
binder, has an activity comparable to that of sorafenib
(
V600EBRAF IC50 of 23 nM vs 43 nM). Furthermore, the
removal of the H-bond donor of the pyridoimidazolone
moiety by methylation on the 3 position (compound 5q of
ref 12) affects activity only marginally, decreasing it to the level
of sorafenib.
This behavior suggests that the H-bond formed between the
hinge region and NH group of the imidazolone ring is not
optimal. Indeed, docking of compound 5a from ref 12 into the
*To whom correspondence should be addressed. Phone: þ44 20 8722
4214. Fax: þ44 20 8722 4046. E-mail: caroline.springer@icr.ac.uk.
a Abbreviations: Boc, tert-butoxycarbonyl; BRAF, V-RAF murine
sarcoma viral oncogene homologue B1; ERK, extracellular regulated
kinase; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated
protein/extracellular regulated kinase; PK, pharmacokinetics; RAF, rapidly
growing fibrosarcoma; SAR, structure-activity relationship; THF, tetra-
hydrofuran.
r
2010 American Chemical Society
Published on Web 07/02/2010
pubs.acs.org/jmc

5640 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Zambon et al.
Figure 1. Structural formulas and biological data of sorafenib and compounds 5a and 5q from ref 12.
Figure 2. Docking poses of compounds 21r from ref 13 (A) and 1a (B) on BRAF.
BRAF/sorafenib cocrystal structure (PDB code UWH)4 in-
dicates how, while the nitrogen atom of the pyridine ring
forms a strong H-bond with the backbone NH group of
Cys532, the geometry of the H-bond interaction between the
NH group of the imidazolone moiety and the backbone
carbonyl group of Cys532 is far from optimal, at a N-O
cycle moves the lactam moiety of the inhibitor closer to the
carbonyl group of Cys532, while maintaining the H-bond
between the pyridine ring and the amide moiety of Cys532
(Figure 2). Also, the pyrido[2,3-b]pyrazin-3(4H)-one group
presents a single H-bond donor versus the two H-bond donors
of the pyridoimidazolone moiety, a feature that is expected
to improve the PK properties and cell permeability of the
scaffold.
˚
distance of 4.0 A and an N-H-O angle of 140° (Figure 2A).
Thus, modifying the structure of the pyrimidazolone hinge
binder could allow the H-bond donor of the bicylic ring
moiety to form a more favorable interaction with the back-
bone, enhancing the inhibitory potency toward BRAF.
Furthermore, the terminal carbonyl group of the pyrimida-
zolone group points toward the solvent, and it can be used to
fine-tune the PK and solubility properties of the series.
By use of this rationale, alternative hinge binders were
explored in order to improve further the affinity for BRAF.
Thus, a series of ligands bearing a variety of alternative hinge
binders as ring A and a standard B-C system were docked
into the BRAF/sorafenib cocrystal structure (PDB code
UWH),4 and their binding modes were examined in compar-
ison to that of the corresponding pyridoimidazolone inhibi-
tor. A number of these compounds were then synthesized and
their activities and PK properties assessed. Here we describe
the synthesis of these target compounds and the ensuing
structure-activity relationship (SAR) studies.
The new analogues were synthesized according to the
synthetic routes outlined in Schemes 1-3. The target com-
pounds could be accessed either by forming the A-B ring
system first and then coupling the terminal group of the B ring
to introduce the linker/C ring group as in Scheme 1 and
Scheme 3 or, as in Scheme 2, by linking the C ring with the
system formed by the B ring and the nitro-amino-pyridine
moiety as a precursor of the A ring.12 The nitro group is then
reduced and the resulting diamino compound is condensed
with an appropriate dielectrophile to form the target bicylic A
ring. This second route, which requires a single step from the
common intermediate to the final product, is particularly
suitable for explorimg a range of different A ring systems in
a parallel fashion and has been used to access A ring systems
formed by a pyridine fused to five-membered heterocycles.
The synthesis of compounds 1a-ad and 2a-t, featuring a
substituted pyridopyrazinone unit, is outlined in Scheme 1.
Fused bicyclic systems are obtained by reaction of the already
reported diamino intermediates 6a-d12,13 with the appropri-
ate R-keto esters as electrophiles or with ethyl 2-ethoxy-2-
iminoacetate in the case of aminopyridopyrazinone compounds
1c, 1k, 1w, 1aa, 1ad and 2c, 2j, 2p, 2t. In the condensation
reaction both the pyridopyrazin-2(1H)-one and the pyridopyr-
azin-3(4H)-one isomers are obtained and are then separated
by chromatography. Removal of the Boc protecting group with
tetrabutylammonium fluoride in refluxing THF17 affords the
Design and Synthesis
Alternative bicyclic A rings formed by a pyridine fused with
a range of five- and six-membered rings bearing H-bond
donor and acceptor groups in various patterns were first
evaluated through docking on the BRAF/sorafenib cocrystal
structure. Among the structures considered, the pyrido[2,3-b]-
pyrazin-3(4H)-one group is of particular interest, as the exten-
sion of the second ring of the bicyclic system to a six-membered

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5641
Scheme 1. Synthesis of Pyridopyrazinone BRAF Inhibitors 1a-ad and 2a-ta
a Reagents and conditions: (a) dry EtOH. For R1 = H: ethyl glyoxylate (50% in toluene). For R1 = CH3: ethyl pyruvate. For R1 = NH2: ethyl
carboethoxyformimidate hydrochloride. For R1 = CF3: ethyl trifluoropyruvate. For R1 = OH: diethyl oxalate, microwave conditions (180 °C, 150W).
(b) 1 M TBAF in THF, reflux. (c) Isocyanates, dry DMSO, Ar atm, room temp.
Scheme 2. Synthesis of Compounds 3a-ha
a Reagents and conditions: (a) TFA (only for 11b); (b) isocyanate, dry THF, Ar; (c) H2, 10% Pd/C (d); for X = N isoamyl nitrite; for X = C-SH
carbon disulfide; for X = CH ethyl orthoformate; for X = C-NMe2 N-(dichloromethylene)-N-methylmethanaminium chloride; X = C-CH2NMe2,
X = 2-chloro-1,1,1-triethoxyethane, then dimethylamine.
common intermediates 9a-iand 10a-h, which are reacted with
the appropriate isocyanate to yield the final urea compounds
(Scheme 1). The regiochemistry of the two isomers 8a and 7a
chemical shifts of N4-H protons for isomers 7b-i and N1-H
protons for isomers 8b-h.
Compounds 3a-h, regioisomers 1c/2c and 1k/2j, and dike-
to compound 1q were synthesized according to Scheme 2; in
this case the starting materials are Boc-protected amines
11a-d. The Boc protecting group is removed as described
above, and the free amino moiety of the ring is reacted with
1-fluoro-4-trifluoromethylphenyl isocyanate or 3-tert-butyl-
1-phenyl-1H-pyrazole isocyanate to give ureas 13a-d. The
1
was assigned by NMR HMBC experiments by the H-13C
coupling between H3 and junction carbon C4a and between H2
and junction carbon C8a, respectively; furthermore, the struc-
ture of 8b was confirmed by X-ray crystallography (see Sup-
porting Information for X-ray data). Assignments of isomers
7b-i and 8b-h was made by analogy because of the characteristic

5642 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Zambon et al.
Scheme 3. Synthesis of Meta-Substituted Pyridopyrazinone BRAF Inhibitors 4a-na
a Reagents and conditions: (a) dry EtOH. For R1 = H: ethyl glyoxylate (50% in toluene). For R1 = CH3: ethyl pyruvate. (b) 1 M TBAF in THF,
reflux. (c) Isocyanates, dry DMSO, Ar atm, room temp.
nitro group of the pyridine ring is then reduced by catalytic
hydrogenation to diamino intermediates 14a-d, and conden-
sation with the corresponding electrophiles forms the bicyclic
A ring system.
compounds inhibit cell growth through inhibition of the ERK
pathway, thus suggesting that the observed activity is not due to
off-target effects on proliferation but to BRAF inhibition.13,15
The focus of the current work was the identification of
effective novel A ring as hinge binders. The SAR analysis was
then extended to probe the effects on activity of structural
diversity on the B-C ring system, including the change from
para to meta substitution pattern on the B ring. Finally, the
PK properties of some of the most effective and representative
compounds are presented.
Selection of A Ring Fragments. The influence of the
structure of ring A on the activity of the compounds in the
assays was initially evaluated on compounds bearing a
3-fluoro substituted B-ring connected through a urea linker
to a 2-fluoro-5-(trifluoromethyl)phenyl ring C. The bench-
mark compound for this study is 21r from ref 13, in which the
same B/C ring system is connected to a pyridoimidazolone A
ring. The results are summarized in Table 1.
A clear observation from Table 1 is that pyridopyrazin-
3(4H)-one compounds 1a-d have both enzymatic and cel-
lular values that are improved or comparable to those of our
benchmark compound. The presence of an amino group at
the 2 position of the pyrazinone ring leads to a slight decrease
in enzymatic activity (compare 1c and 1a), while the presence
of a lipophilic methyl group in the same position leads to a
2-fold improvement in cellular activity (compare 1b and 1a).
More strikingly, pyridopyrazin-2(1H)-one compounds are
noticeably less active than their pyridopyrazin-3(4H)-one
counterparts on both the enzymatic and cellular assays. The
difference in enzymatic activity is more marked between
pyrazinone and methyl pyrazinone compounds (compare
1a,b and 2a,b) than between aminopyrazinone compounds
(compare 1c with 2c). Compound 1d, with two lactam
groups, has the same level of activity as pyridopyrazin-
3(4H)-one compounds 1a-c. With the exception of com-
pound 3e, which exhibits a 100-fold decrease in activity with
respect to the parent compound 21r from ref 13, compounds
with five-membered heterocycles fused with the pyridine ring
show only a slight decrease in both the enzymatic and cellular
potencies and generally exhibit an activity that is between
those of the related pyridopyrazin-3(4H)-one and pyrido-
pyrazin-2(1H)-one compounds.
The synthesis of meta compounds 4a-n was carried out
according to Scheme 3, following synthetic routes we recently
presented for analogous compounds.14 Thus, compounds
4a-m were obtained by condensation of diamino compound
15a14 with the appropriate electrophiles as described above,
followed by deprotection of the NHBoc group and reaction
with isocyanate or acyl chloride to give the corresponding
ureas or amides. Compound 4n, bearing a reverse amide
linker, was obtained using 3-methoxy benzoate 15b as starting
material. The pyridopyrazin-2(1H)-one moiety is then intro-
duced as described above, and the coupling with aminophenyl-
pyrazole was carried out in the presence of trimethyl-
aluminum18 as coupling agent.
Importantly, pyridopyrazinones are more amenable to
chemical manipulation with respect to the pyridoimidazole
moiety previously presented. In particular, the endocyclic
double bond of final compounds 1l and 2d can be hydro-
genated by sodium borohydride (compounds 5a,b) directly,
while the carbonyl group of the pyrazinone of intermediate 7b
can be converted to the corresponding chloropyrazine by a
formal chloro dehydroxylation withN-chlorosuccinimideand
PPh3 in 1,4-dioxane at reflux.19 The resulting chloropyrazine
21 can be synthetically elaborated through nucleophilic aro-
matic substitution by reaction with amines, allowing the
introduction of solubilizing groups on the pyridopyrazinone
scaffold, which followed by deprotection and coupling with
the appropriate isocyanate gives compounds 5c-k (Scheme 4).
Results and Discussion
Enzymatic and cellular potencies of the target compounds
were evaluated using three previously described in vitro
assays.12,13 In brief, the enzymatic assay measures the IC50
of purified V600EBRAF (IC50, V600EBRAF) in vitro, whereas
the other two cellular assays assess the inhibition IC50 of the
formation of phosphoERK and the proliferation GI50. We
previously discussed how the concordance between pERK
IC50 and SRB GI50 values, which is observed throughout
the series, provides evidence for the hypothesis that our

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Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5643
Scheme 4. Derivatization of Pyridopyrazinone Compoundsa
a Reagents and conditions: (a) NaBH4, dry EtOH; (b) N-chlorosuccinimide, PPh3, dry 1,4-dioxane, Ar, reflux; (c) X = methylpiperazine:
N-methylpiperazine, dry CH2Cl2, Ar, room temp. X = dimethylamino: Me2NH (2 M in THF), dry THF, 0 °C to room temp. X = morpholino:
morpholine, dry CH2Cl2, Ar, room temp. X = methylamino: CH3NH2 (2 M in THF), dry THF, room temp; (d) 1 M TBAF in THF, reflux; (e)
Isocyanates, dry DMSO, Ar atm, room temp.
This behavior is consistent with our postulated mode of
binding, as higher affinity for the BRAF enzyme is to be
expected from compounds bearing an H-bond donor group
at N4, next to the pyridine nitrogen, which allows for the
formation of a second H-bond with the Cys532 backbone
carbonyl group. This explains the difference in activity
between compounds 1a-d, which can form a second H-bond
with the hinge through the lactam moiety, and compounds
2a-c, which are unable to form the H-bond through the
endocyclic imine group in position 3. Compounds 3a-d
show intermediate activities, since, as discussed above, the
geometry of the five-membered ring leads to the formation of
a less favorable H-bond with the backbone.
This model is further validated by the chemical modifica-
tion of some pyridopyrazinone compounds. When the endo-
cyclic double bond of pyridopyrazin-2(1H)-one compound
2d is reduced, the enzymatic activity of the resulting com-
pound 5a is improved from 1.29 μM to 54 nM (compare 2d
and 5a in Table 2). Conversely, when the same reaction is
carried out on pyridopyrazin-3(4H)-one compound 1l to
give reduced compound 5b, the two inhibitors show very
close activities (compare 1l and 5b in Table 2).
The SAR observation proposed above is also valid for the
compounds in Table 2; inhibitors with substituted or unsub-
stituted pyridopyrazin-3(4H)-one A rings are consistently
more active than their pyridopyrazin-2(1H)-one counter-
parts on both enzymatic and cellular assays, generally one
order of magnitude or greater (e.g., compare 1e with 2d, 1n
with 2k, 1p with 2l, and 1v with 2o). As observed for the
pyridoimidazolone-based compounds,15 the introduction of
3-tert-butyl-1-phenyl-1H-pyrazole or a 3-tert-butyl-1-p-to-
lyl-1H-pyrazole group as C-ring leads to an increase of
potency in the cell-based pERK and the SRB cell prolifera-
tion assays.
The combination of some of the new A ring moieties with
the most active B-C ring fragments that had been previously
reported led to a series of compounds with cellular activity at
the single-digit nanomolar level, for example, compounds
1s-u (Table 2), a marked improvement with respect to the
most effective inhibitors previously reported. A similar im-
provement in cellular activity is not observed for compounds
3f-g, which feature a dimethylaminoimidazole and a benzo-
triazole group fused to the pyridine ring, respectively, and to
a lesser extent for compound 3h, featuring an imidazolethiol
moiety. A possible explanation for the discrepancy between
this behavior and the other A-rings investigated can be
ascribed to the polarity of the benzotriazole and dimethyl-
aminoimidazole groups, as polar moieties are known to
impede the transport of compounds through the cell mem-
brane. As already observed, all other factors being equal, the
potency of the inhibitors increases in the order phenyl <
naphthyl ∼ 3-F-phenyl < 3-MeS-phenyl.13,15
Variation of the B-C Fragment. After identifying suitable
new A-ring fragments of the inhibitors, we focused on
connecting the new A rings to a variety of B-C ring frag-
ments similar to those reported in previous studies (see
Table 2 for synthesized compounds).12-15 Compounds 2ad
and 2z from ref 15, the most active inhibitors from the
previous series and their activities, are reported in Figure 3
for comparison.

5644 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Zambon et al.
Table 1. Comparison of the Potencies of Inhibitors with Different A-Ringsa
a Where errors are lower than 10-3 μM, the error values are reported as 0.000.

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Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5645
Table 2. Comparison of the Potencies of Inhibitors with Different Combinations of A-, B-, and C-Rings

5646 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Table 2. Continued
Zambon et al.

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Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5647
Table 2. Continued

5648 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Table 2. Continued
Zambon et al.

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Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5649
Table 2. Continued
Introduction of Water-Soluble Groups onto A Ring Frag-
ments. We describe above how the reactivity of the pyrido-
pyrazinone fragment allows the introduction of solubilizing
groups on the A ring through its conversion to a pyrido-
chloropyrazine followed by a nucleophilic aromatic substi-
tution. The activities of the resulting compounds are sum-
marized in Table 4. Predictably, given the removal of an
H-bond donor from the hinge binder, the activities of these
compounds are markedly lower than those of their pyrido-
pyrazin-3(4H)-one counterparts (e.g., compare 5c, 5e, 5g
with 1a and compare 5i with 1l) and in line with those of pyri-
dopyrazin-2(1H)-one compounds (e.g., compare 5c, 5e, 5g
with 2a and compare 5d, 5f, 5h with 2h).
Figure 3. Structural formulas and biological data of compounds
2ad and 2z from ref 15.
Kinase and Cellular Selectivity. We previously reported
how elaboration on the B-ring confers a remarkable selec-
tivity profile, particularly in the case of compounds with
meta-substituted B-rings.13,14 To evaluate the effect of the
new hinge binders on selectivity, compounds 1p and 4e,
representing both the para and the meta scaffold, were
submitted to the MRC National Centre for Protein Kinase
Profiling20 for the assessment of their activity at a single con-
centration of 1 μM in a panel of ∼80 protein kinases (see
Table 1 in Supporting Information for full results). As shown
in Figure 4, the compounds inhibit only a few kinases in the
panel, with SRC, LCK, and p38 being common hits between
the two panels, thus confirming the selectivity of this series.
Selectivity of the series versus WTBRAF can be evaluated
by comparing the in-house IC50 versus V600EBRAF of com-
pounds 1a and 4e with the corresponding WTBRAF results
obtained by Invitrogen. Compound 1a has a WTBRAF IC50
of 83 nM and a V600EBRAF IC50 of 4 nM, and 4e has a
WTBRAF IC50 of 87 nM and a V600EBRAF IC50 of 918 nM.
The differential correlates well with the ratio between
Extension to the Meta-Scaffold. We recently reported
the synthesis of BRAF inhibitors in which the aromatic
middle ring connects to the hinge binder and the allosteric
pocket binder in a meta substitution pattern.14 Compounds
with this scaffold show low-nanomolar level of inhibition
on enzymatic assays and show inhibition on cellular assays
in the high nanomolar range. Introduction of the new
A-rings on the meta scaffold has led to the synthesis of
compounds 4a-n, whose activity is summarized in Table 3,
which also includes the relevant counterparts from ref 14. In
this case, the activity of pyridopyrazin-3(4H)-one com-
pounds is generally comparable with those of the parent
compounds. The noteworthy exception is of the cellular
activity of pyrazole compounds bearing an urea linker,
whose potency is enhanced by the presence of the pyrido-
pyrazin-3(4H)-one fragment, leading to compounds with
activities in the 100 nM range on pERK and on SRB assays
(compare 4c, 4e, and 4g with compounds 27 and 26 from
ref 14).

5650 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Zambon et al.
Table 3. Comparison of the Potencies of Inhibitors with Different A-Rings and a Meta Substitution Pattern

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Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5651
Table 3. Continued
the Km(ATP)app values of WTBRAF and V600EBRAF,21 sug-
gesting similar affinities (Ki) of the series for the mutant and
wild-type form of the enzyme.
H-bond group. Compounds with a naphthyl B-ring and
phenyl C-ring have poor F and peak concentrations
(compounds 1y, 1z), while compound 3h has similar PK
parameters to its pyridoimidazolone analogue 2z from ref 15.
It was observed15 that phenyl C rings have a detrimental
effect on oral bioavavilability; indeed, compound 1v, which
bears both a naphthyl B ring and a phenyl C ring, has
particularly poor PK parameters and no appreciable half-
life. Meta compound 4e, with a tolylpyrazole C ring, shows an
improved F compared with its phenylpyrazole analogue 4c.
Besides compound 4e, many of the compounds assessed
exhibit concentrations well above their GI50 values (some
over 100 000-fold) for extended periods following oral ad-
ministration. In particular, compound 1t shows a high oral
bioavailability along with a long half-life, maintaining a
concentration greater than the GI50 for over 24 h, and
represents an improvement in PK properties compared to
the inhibitors previously reported.15 This, combined with the
potency of the compound, suggests potential for therapeutic
efficacy in vivo in human tumor xenograft models.
The selectivity of compound 4e toward BRAF mutant cell
lines was also assessed by comparing its SRB GI50 values on
the WM266.4 mutant BRAF melanoma cell line and the
WM1361 (BRAF wild type/NRAS mutant) and the D24
(BRAF/NRAS wild type) cell lines. The results are presented
in Figure 5 and show that 4e is 60- and 130-fold selective
toward the WM1361 and D24 cell line, respectively, support-
ing evidence that our proposed mechanism of action is
through the inhibition of oncogenic BRAF.
PK Properties. The oral bioavailability (F), half-life (t1/2),
and maximum concentrations (Cmax) of some of the inhibi-
tors were determined in vivo. The results are presented in
Table 5.14
It is worth noting that diketo compound 1m shows lower F
and peak concentration Cmax with respect to its unsubsti-
tuted analogue. These poor PK parameters could be ex-
plained by the presence on the A ring of an additional

5652 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Zambon et al.
Table 4. Comparison of the Potencies of Inhibitors Featuring Solubilizing Groups on the A-Ring

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Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5653
Conclusions
yloxy)naphthalen-1-ylcarbamate,13 tert-butyl 4-(2-amino-3-
nitropyridin-4-yloxy)-2-fluorophenylcarbamate,13 tert-butyl 4-(2-
amino-3-nitropyridin-4-yloxy)-2-(methylthio)phenylcarbamate,13
tert-butyl 3-(3-oxo-3,4-dihydropyrido[2,3-b]pyrazin-8-yloxy)-
phenylcarbamate,14 and 3-tert-butyl-1-phenyl-1H-pyrazol-5-amine14
were synthesized according to described procedures. Chromatogra-
phy solvents were HPLC grade and were used without further
purification. Reactions were monitored by thin layer chromatogra-
phy (TLC) analysis using Merck silica gel 60 F-254 thin layer plates.
Flash column chromatography 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 μm with UV254. LCMS
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 22 °C using the following solvent systems:
solvent A, methanol; solvent B, 0.1% formic acid in water at a flow
rate of 1 mL/min. Gradient started with 10% A/90% B from 0 to
0.5 min and then from 10% A/90% B to 90% A/10% B from 0.5 to
6.5 min and continued at 90% A/10% B to 10 min. From 10 to
10.5 min the gradient reverted back to 10% A/90% B where the
concentrations remained until 12 min. UV detection was at 254 nm,
and ionization was positive or negative ion electrospray. The mole-
cular 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
MHz or a Bruker Advance 500 MHz spectrometer. Chemical shifts
(δ) are given in ppm and are referenced to residual, not fully
deuterated solvent signal (i.e., DMSO-d5). Coupling constants (J)
are given in Hz. The purities of the final compounds described here
were determined by HPLC as described above and are 95% or higher
unless stated otherwise.
Potential improved mutant BRAF inhibitors bearing alter-
native hinge binders (A-rings) have been synthesized. This
study identified 2-substituted pyridopyrazin-3(4H)-one groups
as optimized A rings, which we postulate form enhanced
interactions with the hinge region. The combination of this
new scaffold with substituted phenyl B rings and 3-tert-butyl-1-
aryl-1H-pyrazoles C rings leads to inhibitors exhibiting single-
digit nanomolar potencies on both enzymatic and cellular
assays, with an improvement compared to the most potent
compounds reported thus far. One of the new compounds
shows an oral bioavailability of over 70% in mouse and main-
tains a concentration above the GI50 for 24 h after administra-
tion, which suggests the potential to exhibit therapeutic efficacy
in vivo in human tumor xenograft models.
Experimental Section
Materials and Methods. Starting materials, reagents, and
solvents for reactions were reagent grade and used as purchased.
tert-butyl 4-(2,3-diaminopyridin-4-yloxy)-2-fluorophenylcar-
bamate,13 tert-butyl 4-(2,3-diaminopyridin-4-yloxy)phenylcar-
bamate,12 tert-butyl 4-(2,3-diaminopyridin-4-yloxy)-2-(methyl-
thio)phenylcarbamate,13 tert-butyl 4-(2,3-diaminopyridin-4-
Docking. Inhibitors were docked on BRAF (PDB code
UWH) using GOLD, version 3.1.1.22 In order to prepare the
receptor for docking, the crystal structure was protonated using
Table 5. Pharmacokinetic Data for Selected Compounds, Based on
Plasma Levels after a 10 mg/kg Single Dose po
compda
F, %
t1/2, hb
Cmax, nMc
1g
1l
17
24
1
6.3 (24)
5.9 (24)
5.4 (>18)
7.9 (24)
(6)
31853 (0.5, 510)
33640 (3, 2240)
5536 (3, 70)
1m
1t
71
4
101199 (3, 101200)
316 (3, 9)
1v
1y
3h
4c
4e
7
1.7 (11)
3.3 (24)
1.4 (6)
14165 (0.25, 130)
11652 (3, 76)
13
6
29543 (0.5, 170)
26989 (0.5, 142)
27
4.0 (24)
a 10 mg/kg single dose po in mouse. b In parentheses the number
of hours the concentration of a compound is above its GI50 after oral
administration. c In parentheses the tmax [h] and the ratio between the
plasma concentration of the compound and its GI50 at tmax after oral
administration.
Figure 4. Kinase selectivity of compounds 1p (A) and 4e (B) tested
at 1 μM against a panel of ∼80 kinases at the MRC National Centre
for Protein Kinase Profiling. The kinases inhibited by >70% are
presented: (C) 1p and (D) 4e.
Figure 5. Antiproliferative effect of 4e in WM266.4 (BRAF mutant), WM1361 (NRAS mutant), and D24 (BRAF/NRAS wildtype) as
determined by SRB assay. The selectivity index is calculated by expressing the GI50 values relative to the GI50 value for WM266.4 cells.

5654 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Zambon et al.
the Protonate3D tool of MOE, and the ligand and water
molecules were then removed. The active site was defined using
Method 4: Introduction of the Linker/C Ring. A solution of
appropriate regioisomer (1 mmol) in dry DMSO (1 mL) under
Ar was treated with the isocyanate (1 mmol), and the solution
was stirred at room temperature. After 3 h, the solution was
diluted with H2O and the precipitate was isolated by filtration.
1-(2-Fluoro-4-(3-oxo-3,4-dihydropyrido[3,2-b]pyrazin-8-yloxy)-
phenyl)-3-(2-fluoro-5-(trifluoromethyl)phenyl)urea 1a. Method 4
was used with 4-(4-amino-3-fluorophenyloxy)pyridoprazine-
3(4H)-one 9b and 1-fluoro-2-isocyanato-4-(trifluoromethyl)-
benzene (yield = 80%). 1H NMR δ: 6.67 (d, 1H, HPy, J = 5.5),
7.08 (m, 1H, Harom), 7.34 (m, 1H, Harom), 7.40 (m, 1H, Harom),
7.51 (m, 1H, Harom), 8.17 (s, 1H, Harom), 8.23 (t, 1H, Harom, J =
8.1), 8.38 (d, 1H, HPy, J = 5.5), 8.64 (m, 1H, Harom), 9.20 (s, 1H,
NH), 9.35 (s, 1H, NH), 12.91 (br s, 1H, NHAr). 19F NMR δ:
-60.8, -124.0, 125.2. LCMS: m/z 478 (M þ H, 100, 5.04 min).
HRMS (3.38 min): m/z calcd for C21H13F5N5O3 ([M þ H]þ)
478.0933; found 478.0935.
1-(3-tert-Butyl-1-p-tolyl-1H-pyrazol-5-yl)-3-(2-(methylthio)-
4-(3-oxo-3,4-dihydropyrido[2,3-b]pyrazin-8-yloxy)phenyl)urea 1t.
Method 4 was used with 8-(4-amino-3-(methylthio)phenoxy)-
pyrido[3,2-b]pyrazin-3(4H)-one 9d and 3-tert-butyl-5-isocyanato-
1-p-tolyl-1H-pyrazole. The title compound (5 mg, 8%) was
obtained as a white powder after purification on silica gel
(eluent DCM/EtOAc, 1/1, Rf = 0.57). 1H NMR δ: 1.31 (s, 9H,
tBu), 2.23 (s, 3H, CH3), 2.31 (s, 3H, SCH3), 6.30 (s, 1H), 6.36 (s,
1H), 6.49 (d, 1H, HPy, J = 5.8), 7.02 (dd, 1H, Harom, J = 8.9, J =
2.7), 7.19 (m, 4H, Harom), 7.31 (d, 1H, Harom, J = 8.3), 7.81 (s, 1H,
NH or CH), 8.16 (d, 1H, Harom, J = 8.9), 8.26 (s, 1H, NH or CH),
8.30 (d, 1H, HPy, J = 5.8), 11.37 (s, 1H, NH). LCMS: m/z 556
(M þ H, 100). HRMS: m/z calcd for C29H29N7O3S ([M þ H]þ)
556.2125; found 556.2124.
˚
a radius of 10 A from the backbone oxygen atom of Asp594 of
the ATP binding pocket. Partial charges of the ligands were
derived using the Charge-2 CORINA 3D package in TSAR 3.3
and their geometries optimized using the COSMIC module of
TSAR. Ten docking solutions were generated per docking run
with GOLD, and the best three were stored for analysis.
X-ray Crystallographic Analysis. Crystallographic data for
compound 8b have been deposited with the Cambridge Crystallo-
graphic Data Center as Supplementary Publication. Copies
of the data can be obtained, free of charge, on application to
CCDC, 12 Union Road, Cambridge CH21EZ, U.K. (fax, þ44
1223 336 033; e-mail, deposit@ccdc.cam.ac.uk), CCDC deposi-
tion number 778231. Figure 1 (in Supporting Information)
shows an ORTEP-3 diagram of 8b.
Biological Assays. V600EBRAF Kinase Assay and SRB IC50
for BRAF Inhibitors. These assays have been described by
Niculescu-Duvaz et al.12
pERK Kinase Assay. This assay has been described by
ꢀ
13
Menard et al.
Pharmacokinetics. Female BALB/cAnNCrl mice at least 6
weeks of age were used for the PK analyses except for the
intravenous administration of 1m, which was carried out with
female Crl:CD1-Foxn1nu mice bearing V600E mutant BRAF
WM266.4 tumor xenografts. The mice were dosed intravenously
(2 mg/kg, equivalent to ∼4 μmol/kg, 10 mL/kg, in DMSO/
Tween-20/water 10:1:89 v/v) or orally by gavage. Samples were
taken at seven or eight time-points between 5 min and 18 or 24 h
for the intravenous route and at six or eight time-points between
15 min and 18 or 24 h for the oral route. Three mice were used
per time-point per route. They were placed under halothane or
isoflurane anesthesia, and blood for plasma preparation was
taken by terminal cardiac puncture into heparinized syringes.
Plasma samples were snap frozen in liquid nitrogen and then
stored at -70 °C prior to analysis. All procedures involving
animals were performed in accordance with national Home
Office regulations under the Animals (Scientific Procedures)
Act 1986 and within guidelines set out by the Institute’s Animal
Ethics Committee and the United Kingdom Coordinating
Committee for Cancer Research’s ad hoc Committee on the
Welfare of Animals in Experimental Neoplasia.23
Acknowledgment. We thank Cancer Research UK (Grants
C309/A8274 and C107/A10433), the Wellcome Trust, the
Instituteof Cancer Research, andthe Isleof ManAnti-Cancer
Association for funding. We thank Dr. Peter N. Horton and
Dr. Michael B. Hursthouse of the EPSRCNational Crystallo-
graphy Service (University of Southampton, U.K.) for collec-
tion of diffraction data and structure determination of com-
pound 8b. We are grateful to Neus Cantarino for providing
technical assistance, Meirion Richards, Dr. Maggie Liu, and
Dr. Amin Mirza for analytical chemistry support and to
Professors Julian Blagg and Paul Workman for suggestions,
support, and helpful comments. This work was carried out as
part of a research collaboration between the Institute of
Cancer Research, The Wellcome Trust and Cancer Research
U.K. Please note that all authors who are, or have been,
employed by The Institute of Cancer Research are subject to a
‘Rewards to Inventors Scheme’ that may reward contributors
to a program that is subsequently licensed.
Method 2: Synthesis of the 2-Substituted Pyrido[2,3-b]pyrazin-
3(4H)-one and 3-Substituted Pyrido[2,3-b]pyrazin-2(1H)-one Re-
gioisomers. To a solution of the appropriate tert-butyl 4-(2,3-
diaminopyridin-4-yloxy-2-phenylcarbamate in dry EtOH were
ꢀ
˚
added consecutively molecular sieves (3 A) and ethyl glyoxylate
(3.6 mL of a 50% solution in toluene, 1.7 equiv). The solution
was stirred at room temperature until the starting material was
consumed (monitored by TLC). Then the mixture was evaporated
to dryness and subjected to column chromatography (elution with
CH2Cl2 to EtOAC/CH2Cl2 (1/2, v/v toward 1/1, v/v).
tert-Butyl 4-(3-Oxo-3,4-dihydropyrido[2,3-b]pyrazin-8-yloxy)-
phenylcarbamate 7a and tert-Butyl 4-(2-Oxo-1,2-dihydropy-
rido[2,3-b]pyrazin-8-yloxy)phenylcarbamate 8a. Method 2 was
used overnight with tert-butyl 4-(2,3-diaminopyridin-4-yloxy)-
phenylcarbamate12 (0,86 g, 2.71 mmol) to provide 7a (0.200 g,
21% yield) and 8a (0.430 g, 45% yield).
Supporting Information Available: Experimental details of the
synthesis and analytical characterization of all compounds;
X-ray diffraction data for compound 8b; selectivity profile for
compounds 1p and 4e. This material is available free of charge
via the Internet at http://pubs.acs.org.
tert-Butyl 4-(3-Oxo-3,4-dihydropyrido[2,3-b]pyrazin-8-yloxy)-
1
t
phenylcarbamate 7a. H NMR δ (in CDCl3): 1.54 (s, 9H, Bu),
6.55 (d, 1H, HPy, J=5.5), 6.67(bs, 1H, Harom), 7.14(d, 2H, Harom
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