Aza-stilbenes as potent and selective c-RAF inhibitors

Article abstract of DOI:10.1016/j.bmcl.2006.07.063

The synthesis of several novel aza-stilbene derivatives was carried out. The compounds were tested for their c-RAF enzyme inhibition. Compound 27 possesses significant potency against c-RAF and demonstrates selectivity over other protein kinases. A hypoth

Full text of DOI:10.1016/j.bmcl.2006.07.063

Bioorganic & Medicinal Chemistry Letters 16 (2006) 5378–5383
Aza-stilbenes as potent and selective c-RAF inhibitors
Octerloney McDonald, Karen Lackey, Ronda Davis-Ward, Edgar Wood,
Vicente Samano, Patrick Maloney, Felix Deanda and Robert Hunter*
GlaxoSmithKline, Research Triangle Park, NC 27709, USA
Received 23 May 2006; revised 19 July 2006; accepted 21 July 2006
Available online 4 August 2006
Abstract—The synthesis of several novel aza-stilbene derivatives was carried out. The compounds were tested for their c-RAF
enzyme inhibition. Compound 27 possesses significant potency against c-RAF and demonstrates selectivity over other protein
kinases. A hypothesis for the binding mode, activity, and selectivity is proposed.
ꢀ
2006 Elsevier Ltd. All rights reserved.
The kinome has become a widely targeted class of sig-
naling proteins for drug intervention points due to the
multitude of cellular regulatory roles protein kinases
In our research, we sought to use these types of small
molecules as potential starting points in our effort to
find diverse series to inhibit the MAPK signaling path-
way since this may offer an ideal point to block cellular
1
play. At the basic level, all protein kinase enzymes bind
9
a molecule of ATP and peptide substrate in order to
propagate the signal cascade via transfer of the terminal
phosphate group to a tyrosine or serine/threonine. Early
research in the area targeted the protein substrate bind-
ing domain under the assumption that there was a great-
proliferation in cancer cells. Examples of c-RAF inhib-
itors currently in the literature include Sorfenib (BAY-
1
1,12
439006) and GW5074.
We began by performing a
substructure search of the GlaxoSmithKline compound
collection to select a few thousand compounds for
screening in a kinase enzyme cascade assay that included
2
,3
er probability for achieving selectivity.
Pioneering
1
0
work on piceatannol derivatives and diversely substitut-
ed tyrphostins showed a number of very important con-
c-RAF/MEK-1/ERK-2.
4
,5
cepts in the field of kinase inhibition. Peptide mimics
of the protein substrate proved difficult to make signifi-
cantly potent as compared with ATP binding kinase
6
activity inhibitors. However, small changes in substitu-
OH
tion patterns of non-peptide inhibitors of the protein
substrate phosphorylation, especially compound varia-
tions that would normally emerge from a medicinal
chemistry analog program, demonstrated interesting
mechanism of inhibition changes (Fig. 1). For example,
polyhydroxylated styrenes 1 and stilbenes (piceatannol)
CN
CN
OH
HO
HO
HO
HO
7
Piceatannol
Tyrphostin (1)
were inhibitors by virtue of their ability to compete with
5
peptide substrate in a kinase enzyme assay. When the
stilbene scaffold was substituted with halogens, as in 2,
the inhibition of kinase activity was competitive with
8
ATP, suggesting a different binding interaction.
N
Cl
CN
Cl
competative. vs ATP (2)
Keywords: Kinase; c-RAF; Aza-stilbene.
*
Corresponding author. Tel.: +1 919 483 5463; e-mail:
bob.n.hunter@gsk.com
Figure 1. Historical development of modulating kinase activity via
peptide substrate inhibition shifting to ATP competitive inhibition.
0
960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.07.063

O. McDonald et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5378–5383
5379
OH
the desired aza-stilbenes in good isolated yields, general-
1
7
ly 50 percent or better. Formation of the tetrazole was
accomplished with tributyl tin azide in toluene at re-
N
HO
OH
18
flux. Workup of the tributyl tin azide reaction consist-
ed of adding excess 4 M HCl in dioxane, dilution with
(
3)
N
(4)
OH
Et O, and collection of the solids by vacuum filtration.
2
Compound 10 was prepared in the same fashion from
methyl 5-bromo-nicotinate (Scheme 2).
p56lck
p56lck
IC50 1351 uM10
IC50 16.0 uM11
OH
O
HO
Compounds 11–14 were prepared from the ethyl ester by
stirring the desired stilbene with 33% methyl amine or
33% dimethyl amine in EtOH at room temperature
overnight. Compound 5 was prepared by the hydrolysis
of 10, and 15 was prepared by the esterification of 5-bro-
mo nicotinic acid and subsequent Stille and Heck reac-
(
5)
N
c-raf1/MEK/ERK
IC50 0.40 uM
Figure 2. Related stilbene derivatives that inhibit kinase activity.
1
9
tions as described above.
The enzymes used in the RAF/MEK/ERK Kinase Scin-
tillation Proximity Assay were prepared as previously
The literature examples 3 and 4 used for the basis of the
3,14
1
substructure search are shown in Figure 2.
A very
1
0
described. Standard compound screening assays were
performed in 96-well microtiter Optiplates (Packard
Instrument Co., Meriden, CT) where the final assay vol-
ume was 45 ll. Test compound (15 ll) dissolved in 6%
DMSO was added to the wells followed by substrate
solution (15 ll). The final assay concentrations of the re-
agents contained in the substrate solution were 10 lM
potent starting point emerged, also shown in Figure 2,
which formed the basis of a lead optimization program
targeting c-RAF. The synthesis, highlights of the
structure–activity relationships (SAR), and kinase selec-
tivity considerations will be described.
1
5
A facile synthetic route was used to produce diversely
substituted aza-stilbene analogs and is shown in
Scheme 1.
ATP, 10 mM MgCl , 2 lM peptide substrate, biotin-
2
3
3
AAATGPLSPGPFA, and 0.125 lCi [c- P]ATP/well,
0 mM Mops, pH 7.6. The reactions were initiated by
5
the addition of enzyme solution (15 ll). The final assay
concentrations of the enzymes were 10 nM c-RAF1,
The commercially available 5-bromo-nicotinonitrile,
methyl 5-bromo-nicotinate, and ethyl 5-bromo-nicotin-
ate served as good starting materials for the synthesis
of the desired compounds listed in Tables 1–4. In the
case of the nitrile and tetrazole containing 6–9, the 5-
bromo-nicotinonitrile was used in a Stille coupling with
tributylvinyl tin to generate the vinyl intermediate.
Subsequent reaction under Heck conditions with the
appropriately substituted bromo or iodo benzene gave
100 nM MEK1, and 300 nM ERK2. Positive control
wells contained 6% DMSO with no added compound.
Background control wells contained 50 mM EDTA.
The reaction was allowed to proceed for 120 min at
room temperature and was terminated by the addition
of 200 ll PBS containing 2.5 mg/ml streptavidin-coated
SPA beads (Amersham Biosciences, Piscataway, NJ),
1
6
50 lM ATP, 10 mM EDTA and 0.1% TX-100. The
microtiter plates were sealed and SPA beads were
allowed to settle for at least 6 h. The SPA signal was
measured using a Packard Topcount 96-well plate
scintillation counter (Packard Instrument Co.).
R3
R4
R1
R1
R1
Br
a
b
N
N
N
R2
To determine pIC50 values, data were normalized to
control values using the equation, 100 · (U1 À C2)/
(C1 À C2), where C1 is CPM in the absence of com-
pound, C2 is CPM in the presence of excess EDTA,
Scheme 1. Reagents and conditions: (a) tributylvinyl tin, LiCl, BHT,
Pd(PPh
3
)
2
Cl
2
2 3
, DMF, 70 ꢁC; (b) arylbromide(iodide), Pd dba , TEA,
P(o-tol)
3
, DMF, 95 ꢁC.
H
N
N
N
N
a
b
Br
N
N
O
N
O
O
H
N
N
Scheme 2. Reagents and conditions: (a) tributyl tin azide, toluene, reflux; (b) 33% dimethylamine, EtOH, rt.

5380
O. McDonald et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5378–5383
Table 1. c-RAF/MEK/ERK inhibition and SAR for compounds 5–15
Table 4. c-RAF/MEK/ERK inhibition data SAR for compounds
8–32
2
R4
R1
H
R4
N
N
N
N
N
1
4
Compound
R
R
c-RAF/MEK/ERK
inhibition pIC50
N
4
Compound
R
c-RAF/MEK/ERK inhibition pIC50
5
6
7
8
9
COOH
CN
CN
OH
OH
H
6.4 (0.15), n = 4
6.1 (0.25), n = 4
6.0 (0.28), n = 10
8.4 (0.35), n = 10
7.9 (0.3), n = 5
6.2 (0.26), n = 3
5.1, n = 1
28
29
30
31
32
2-Furan
6.2 (0.28), n = 3
2-Thiophene 6.5 (0.32), n = 3
2-Pyridyl 7.0 (0.63), n = 4
3-Thiophene 6.0, n = 1
3-Pyridyl 6.2 (0.25), n = 2
Tetrazole
Tetrazole
COOMe
CON(Me)
OH
H
10
11
12
13
14
15
OH
OH
OH
H
2
Standard deviation is given in parentheses. n values are as shown
n = replicates).
CONHMe
CONHMe
6.6 (0.15), n = 4
6.6 (0.52), n = 4
6.8 (0.23), n = 4
5.4, n = 1
(
CONH
t-Butyl ester
2
OH
OH
and U is CPM in the presence of compound. Com-
pounds were serially diluted 3-fold and added to the
reaction mixture to produce an 11-point dose–response
curve ranging from 0.0001 to 10 lM. IC50 values were
estimated using the equation Y = Vmax · (1 À (X/
Standard deviation is given in parentheses. n values are as shown
n = replicates).
(
(
IC50 + X))) where Y is rate of product formation, V
max
Table 2. c-RAF/MEK/ERK inhibition data SAR for compounds
6–20
is the rate of the reaction in the absence of an inhibitor,
and X is the inhibitor concentration. The IC50 value was
1
R3
then converted to a pIC50.
H
O
N
Based on initial observations of the SAR, the aza-stil-
bene core with the ortho di-methyl groups was retained
to achieve potent c-RAF inhibition. The data in Table 1
H
N
R
2
1
2
3
show that residues in the R position which contain a
Compound
R
R
c-RAF/MEK/ERK
inhibition pIC50
hydrogen-bond acceptor/donor such as the acid, amide,
or tetrazole are generally more potent in the c-RAF/
MEK/ERK enzyme assay. Removal of the hydroxyl
13
CH
CH
Cl
3
2
CH
3
6.6 (0.52), n = 4
4.6, n = 1
16
CH
3
CH
Cl
H
2
CH
3
4
group at R had very little effect on the enzyme potency.
17
18
19
20
6.6 (0.46), n = 4
5.5, n = 1
5.7, n = 1
CH
Cl
3
H
Cl
Structure–activity relationships generated via the initial
screening set suggested di-substitution ortho to the stil-
bene was important for enzyme activity. Maintaining a
hydrogen in the 4-position of the pendant phenyl ring
and the N-methyl amide on the pyridyl ring, Table 2,
we synthesized a set of stilbenes to probe the SAR in this
region. The diethyl substitution 16 was 100-fold less ac-
tive than the parent, indicating a size restriction.
Replacement of the methyl groups by chlorines, 17,
resulted in a compound that was equipotent with 13 in
the enzyme assay. Removal of one of the methyl or chlo-
ro groups, 18 and 19, proved to be 10-fold less active
than the parent 13. Interestingly, when only one of
methyl groups was replaced with chlorine, as in 20, the
activity was increased by about 3-fold.
CH
3
7.1 (0.56), n = 4
Standard deviation is given in parentheses. n values are as shown
n = replicates).
(
Table 3. c-RAF/MEK/ERK inhibition data SAR for compounds
1–27
2
H
O
N
R1
O
N
1
Compound
R
c-RAF/MEK/ERK
inhibition pIC50
Placement of a N-methyl carbamate in the 4-position of
the pendant phenyl ring, 21–27 in Table 3, was accom-
plished by reaction of the compounds where R is OH
21
CN
CON(Me)
7.4 (0.1), n = 3
5.1, n = 1
4
22
2
23
24
25
26
27
CONHMe
CONH
8.0 (0.22), n = 4
8.1 (0.18), n = 4
7.5 (0.53), n = 4
7.0 (0.33), n = 3
8.2 (0.09) n = 3
in Table 1 with methylisocyanate in DMF. Incorpora-
tion of the carbamate increased enzyme potency by a
minimum of 15-fold in 21 and a maximum of 60-fold
2
COOMe
t-Butyl ester
COOH
23
in 27.
Standard deviation is given in parentheses. n values are as shown
n = replicates).
Consideration of kinase selectivity is important due
to the similarities of the ATP-binding pockets of
(

O. McDonald et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5378–5383
5381
8
7
6
5
4
.5
9
5
24 27
8
9
30
8
8
7
.5
8
.5
7
.5
7
.5
6
*
*
6.5
6
.5
5
*
*
*
*
*
5.5
5
.5
4
4
.5
4
**
*
*
*
Figure 3. (*) < Value at maximum concentration tested (10 lM) also
10
inactive on JNK3 and P38. RME = RAF-MEK-ERK,
(mouse), LCK (mouse).
FYN
Figure 4. (*) < Value at maximum concentration tested (10 lM) also
10
inactive on JNK3 and P38. RMK = RAF-MEK-ERK,
(mouse), LCK (mouse).
FYN
protein kinases. As shown in Figure 3, the incorpora-
tion of the carbamate group yielded potent com-
pounds, and it resulted in compounds that were
quite selective over other representative kinases in
the screening panel.
Unfortunately, the combination of both the tetrazole
and the carbamate functionalities in the same mole-
cule was not explored due to the instability of the
carbamate (ex vivo studies in both mouse and human
plasma demonstrated the carbamate exhibited rapid
2
4
biphasic degradation to the parent phenol). We in-
stead focused on the preparation of compounds
which contained the tetrazole and potential replace-
ments for the carbamate. To do this, we prepared
the triflate of 6 and used it in a second Stille reac-
20
tion. Listed in Table 4 are examples of the com-
pounds prepared.
Replacement of the carbamate with a 2-pyridyl ring, 30,
resulted in only a modest loss of c-RAF activity as com-
pared to 8 and 9. However, this modification introduces
some kinase selectivity challenges because it demon-
strates activity on the SRC kinase family (FYN, LCK,
LYN, and SRC).
Figure 5. Structural model of c-RAF (carbon atoms in gray) in
complex with compound 5 (carbon atoms in green). H-bond interac-
tions are designated by thin yellow lines.
strated a change in activity at all, where substitution
of the hydroxyl with a hydrogen atom resulted in a
modest 3-fold decrease in c-RAF inhibitory activity.
To possibly explain why tetrazole led to an increase
in potency, models of 5 and 8 in complex with
c-RAF were constructed.
In efforts to explain the observed c-RAF SAR and selec-
tivity in terms of protein–ligand binding, a homology
model of c-RAF was constructed from available
1
1
b-RAF crystallographic data. The sequence identity
across their kinase domains was roughly 80% with no
residue differences in their ATP-binding sites. The
2
1
c-RAF model was constructed using MVP, and then
2
geometry optimized in Insight’s Discover module.
Illustrated in Figure 5 is a docking model of 5 in com-
plex with c-RAF. Note that the inhibitor’s pyridyl ring
binds to the kinase at the hinge region with its nitrogen
atom forming an H-bond interaction with the backbone
NH of Cys424. The 4-hydroxy-2,6-dimethylphenyl moi-
ety is positioned deep into the ATP-binding pocket
toward the aC helix and is surrounded by mostly hydro-
phobic residues including Val363, Ala373, Leu397,
Leu406, Ile419, and Phe487. One face of the phenyl ring
makes van der Waals contact with the alkyl portion of
Lys375’s side chain, while the other interacts with the
2
Designating compound 5 as the parent, substitution of
the carboxylate with tetrazole resulted in a 100-fold in-
crease in c-RAF inhibitory activity, Table 1. From Fig-
ures 3 and 4, note that this substitution also led to an
increase in potency of 1.30–1.60 log units against
FYN, LCK, and SRC. Such dramatic changes in activ-
ity were not observed from the two functional groups
4
used at the R position. Indeed, only 8 and 9 demon-

5382
O. McDonald et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5378–5383
side chain of Thr421. The ligand’s hydroxyl is posi-
tioned toward Glu393 forming an H-bond interaction.
At the opposite end of the molecule, the carboxylate is
positioned at the entrance of the ATP-binding site near
the protein–solvent interface where it makes van der
Waals contact with three hydrophobic residues, Ile355,
Phe475, and Phe487.
From Figure 6, note that the binding mode for 8 is nearly
identical to that of 5, with the difference being the tet-
razole. However, given that tetrazole is significantly
more lipophilic than the carboxylate, it is reasonable
to conclude that 8 will have a more favorable hydropho-
bic interaction with Ile355, Phe475, and Phe487. Not
only does the tetrazole interact more favorably with
these residues, but it also makes van der Waals contact
with a fourth hydrophobic residue, Trp423. Given these
observations, it is apparent why the tetrazole analogues
Figure 7. Structural model of c-RAF (carbon atoms in gray) in
complex with compound 27 (carbon atoms in green). H-bond
interactions are designated by thin yellow lines.
8 and 9 are significantly more potent than the other
aza-stilbene derivatives from Table 1.
Recall that substitution of the carboxylate with tetrazole
also led to an increase in potency of 1.30–1.60 log units
against FYN, LCK, and SRC. From our sequence analy-
ses and superposition of LCK and SRC X-ray structures
onto c-RAF, it was discovered that the four c-RAF
hydrophobic residues predicted to interact with 8 corre-
sponded to hydrophobic residues in FYN, LCK, and
SRC, suggesting that the increase in potency for these
other kinases was related to favorable hydrophobic inter-
actions. In FYN, these residues are Leu277, Tyr344,
Leu397, and Phe409. For LCK and SRC, the residues
are Leu238, Tyr305, Leu353, Phe370 and Leu282,
Tyr349, Leu402, Phe414, respectively.
Illustrated in Figure 7 is a docking model of compound
27 in complex with c-RAF. The inhibitor’s binding
mode is similar to that already described for compounds
5 and 8. However, compound 27 forms one additional
H-bond with c-RAF. Similar to 5 and 8, this inhibitor
forms an H-bond interaction with the side chain of
Glu393 through the carbamate NH. The additional
H-bond interaction is formed between the inhibitor’s
carbonyl and the backbone NH of Asp486. We hypoth-
esize that the increase in enzyme potency for 21–27 may
result in part from this additional H-bond interaction
with c-RAF. Note that the residue which immediately
precedes Asp486 is Gly485. Based on our docking mod-
el, it is evident that formation of the H-bond interaction
between the inhibitor’s carbonyl and Asp486 is not ste-
rically hindered by this glycine. However, if this residue
were mutated to a larger amino acid, even one the size of
alanine, the increase in side-chain volume may perhaps
be sufficient to hinder the formation of this H-bond.
This in turn may affect the formation of the H-bond be-
tween the carbamate NH and the glutamate (which is
present in all seven kinases). In FYN, LCK, LYN,
SRC, and TIE2, the residue which corresponds to
Gly485 in c-Raf is an alanine. For VEGFR2, that resi-
due is a cysteine. We hypothesize that the observed
c-Raf selectivity may result in part over the ability of
these non-glycine residues to sterically hinder formation
of the specified H-bond interaction.
4
Replacement of the hydroxyl at the R position with
N-methyl carbamate resulted in significant increases in
c-RAF inhibitory activities, Table 3. Consider as an
example 27, which had an increase in c-RAF inhibitory
activity comparable to that seen with 8. Moreover and
perhaps more importantly, 27 proved to be selective
for c-RAF (pIC50 = 8.16) over the other kinases
(
pIC50 < 5.0) in the screening panel, Figure 3.
From a Piceatannol and Tyrphostin substructure search
of the GSK compound collection, an aza-stilbene series
was identified (5) for a c-RAF lead optimization effort
(IC50 value 0.4 lM). The most potent c-RAF compound
was 3,5-dimethyl-4-{(E)-2-[5-(1H-tetrazol-5-yl)-3-pyrid-
inyl]ethenyl}phenol (8, IC50 value 0.004 lM), and it also
inhibited SRC family kinases (e.g., IC50 value for FYN
0
.040 lM). 4-{(E)-2-[5-(aminocarbonyl)-3-pyridinyl]eth-
enyl}-3,5-dimethylphenyl methylcarbamate (25) and
-[(E)-2-(2,6-dimethyl-4-{[(methylamino)carbonyl]oxy}-
5
phenyl)ethenyl]-3-pyridinecarboxylic acid (27) were the
most potent and selective c-RAF inhibitors synthesized.
SAR was established for enhancing c-RAF potency and
Figure 6. Structural model of c-RAF (carbon atoms in gray) in
complex with compound 8 (carbon atoms in green). H-bond interac-
tions are designated by thin yellow lines.

O. McDonald et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5378–5383
5383
selectivity, and can be explained using a homology
model of c-RAF.
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23. Analytical data for 27, 1H NMR (300 MHz, DMSO-d ) d
6
ppm 2.3 (s, 6H) 2.6 (d, J = 4.6 Hz, 3H) 6.8 (m, 3H) 7.5 (d,
J = 16.6 Hz, 1H) 7.6 (q, J = 4.7 Hz, 1H) 8.5 (t, J = 1.9 Hz,
1H) 9.0 (d, J = 1.9 Hz, 1H) 9.0 (d, J = 2.1 Hz, 1H), LCMS
1
À
m/z: 325 [MÀH] .
24. Data unpublished.