Structure-based design of isoindoline-1,3-diones and 2,3- dihydrophthalazine-1,4-diones as novel B-Raf inhibitors

Article abstract of DOI:10.1016/j.bmcl.2011.10.012

Structure-guided design led to the discovery of novel chemical scaffolds for B-Raf inhibitors. Both type I and type II kinase inhibitors have been explored and lead compounds with good potency and excellent selectivity have been identified.

Full text of DOI:10.1016/j.bmcl.2011.10.012

Bioorganic & Medicinal Chemistry Letters 21 (2011) 6941–6944
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure-based design of isoindoline-1,3-diones
and 2,3-dihydrophthalazine-1,4-diones as novel B-Raf inhibitors
Xiaolun Wang a,b, , Edward J. Salaski a, Dan M. Berger a, , Dennis Powell a, Yongbo Hu a,à
,
⇑
Donald Wojciechowicz a, Karen Collins a, Eileen Frommer a
a Pfizer Worldwide Research and Development, 401 N. Middletown Road, Pearl River, NY 10965, USA
b Pfizer Worldwide Research and Development, 200 Cambridgepark Drive, Cambridge, MA 02140, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Structure-guided design led to the discovery of novel chemical scaffolds for B-Raf inhibitors. Both type I
and type II kinase inhibitors have been explored and lead compounds with good potency and excellent
selectivity have been identified.
Received 7 September 2011
Revised 29 September 2011
Accepted 4 October 2011
Available online 12 October 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
B-Raf inhibitors
Structure-based drug design
Type I kinase inhibitors
Type II kinase inhibitors
The RAS-RAF-MEK-ERK signal transduction pathway plays an
important role in cell survival, growth, and proliferation.1 Mutant
B-Raf induces a constitutive activation of this pathway and is cru-
cial for the development of certain types of tumors.2 Therefore,
inhibition of B-Raf offers a viable means for treating cancers.3 In
previous reports, we disclosed the development of a series of
highly potent B-Raf inhibitors featuring a pyrazolopyrimidine core
and a pyridyl hinge binder.4,5 To further expand the chemical space
of B-Raf inhibitors, we underwent a campaign to design novel scaf-
folds utilizing available structure information.
HON
B-Raf KD = 0.16 nM
N
H
N
B-Raf IC50 = 22 nM
O
O
Cl
O
N
H
O
N
N
N
H
N
H
CF3
typeI
N
typeII
Sorafenib
SB-590885 (2FB8)
(1UWH)
Figure 1. B-Raf inhibitors. Hinge binder highlighted as blue, PDB code shown in
parentheses.
At the time we started our study both the X-ray structures of
type I and II kinase inhibitors with B-Raf had been published6
(Fig. 1). SB-5908857,8 is a tri-substituted imidazole binding to the
active conformation of B-Raf, while sorafenib2,9 is a di-substituted
urea interacting with the DFG-out inactive conformation of B-Raf.
Both structures employ a nitrogen containing heterocycle as the
hinge binder forming a hydrogen bond with hinge residue C532
which is also true for most of other B-Raf inhibitors.10 We envi-
sioned a different functional group as the hinge binder. As shown
in a recent review,11 amide groups are frequently seen in kinase
inhibitors and it could form two hydrogen bonds with the hinge
residue.12 Our design started with the imidazole B-Raf inhibitors8
where the pyridyl ring was replaced with an amide group and that
was positioned toward C532 residue by the construction of a fused
six-membered ring (Fig. 2). Dess–Martin oxidation of alcohol 4
provide aldehyde 5, which underwent a Fischer indolization with
4-hydrazinylbenzoic acid followed by amidation and then demeth-
ylation to afford 3 (Scheme 1). The IC50 of compound 3 against
B-Raf13 was greater than 10
lM, although the modeling study sug-
gested a favorable pose in the ATP binding pocket with the car-
bonyl group interacting with C532 of the active conformation
model14 (Fig. 3). We reasoned that there might be an energy pen-
alty to fix the conformation of the amide group. A literature search
revealed that imide groups can interact with the hinge residues
when placed in appropriate positions.15 Therefore, compound 11
with a tied imide group was proposed. The iodination of commer-
cially available 5-aminoisoindoline-1,3-dione (8) followed by a
intra-molecular Heck reaction gave tricycle compound 10.
⇑
Corresponding author. Tel.: +1 617 665 5212; fax: +1 617 665 5682.
E-mail address: xxw104@gmail.com (X. Wang).
Current address: OSI Pharmaceuticals1 Bioscience Park Drive, Farmingdale,
NY 11735, USA.
à
Current address: Millennium Pharmaceuticals, Inc., 40 Landsdowne Street,
Cambridge, MA 02139, USA.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.10.012

6942
X. Wang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6941–6944
C532
O
O
I
a
b
N
H
N
HN
HN
O
NH2
K483
NH2
O
O
O
H
8
9
N
NH3
O
E501
H
O
OMe
c
O
OH
H
OH
O
HN
O
O
N
H2N
HN
N
HN
N
H
N
R
H
B-Raf
IC50 = 3.47 µM
O
R
O
10
11
2
1
Scheme 2. Reagents and conditions: (a) 2 equiv NIS, DMF, 45 °C, 50%; (b) 5, Pd,
DABCO, 30%; (c) BBr3, CH2Cl2, rt, 50%.
Figure 2. The design of B-Raf inhibitors.
Buchwald coupling reaction17 with two equivalents of 3-methoxy-
aniline afforded intermediate 14. Subsequential deprotection with
tetra-n-butylammonium fluoride and boron tribromide gave com-
pound 15. Compound 17 was synthesized in a similar route via
selective stepwise Buchwald coupling reactions (Scheme 3). Both
isoindoline-1,3-diones 15 and 2,3-dihydrophthalazine-1,4-dione
17 were more potent than tricyclic analog 11 supporting the
hypothesis that a bicyclic scaffold affords a more potent B-Raf
inhibitor. This data warranted optimization of the bicyclic scaffold.
The model of 15 with B-Raf (Fig. 4) suggested there are favorable
hydrogen bond interactions between the imide group and the
hinge region. The isoindoline-1,3-dione core sits in a hydrophobic
pocket surrounded by A481, V471, L514, and F583. One phenol
group interacts with K483 and E501 while the other phenol group
forms two hydrogen bonds with D594 and N581; this may explain
fivefold increases in potency compared to analog 17.
Our efforts for the further exploration of novel hinge binders led
to the discovery 2,3-dihydrophthalazine-1,4-dione 18 and 19,
which were prepared by the reaction of compound 15 and 17 with
hydrazine (Scheme 4). Interestingly, both compounds showed
comparable activity to the isoindoline-1,3-dione analogs and a
model of compound 19 with B-Raf suggests a binding mode very
similar to that of 15 (Fig. 5). A substructure search of diacylhydraz-
ide (CONHNHCO) motif against the RCSB protein data bank18 did
OMe
OH
O
b
HOOC
a
N
H
OMe
OMe
6
5
4
OH
OMe
d
c
H2NOC
H2NOC
N
H
3
N
H
B-Raf
IC50 > 10 µM
7
Scheme 1. Reagents and conditions: (a) Dess–Martin periodinane, CH2Cl2, rt; (b) 4-
hydrazinylbenzoic acid, HOAc, 130 °C, 45% two steps; (c). CDI, then NH4OH, 50%;
(d). BBr3, CH2Cl2, rt, 50%.
O
O
Cl
Cl
Cl
Cl
b
a
HN
TIPSN
O
O
12
13
O
OH
O
O
NH
NH
NH
c, d
TIPSN
HN
15
NH
O
O
14
O
B-Raf
OH
OH
IC50 = 0.338 µM
Figure 3. Model of compound 3a with B-Raf.
13
e
O
O
O
Deprotection with boron tribromide afforded the desired product
11 (Scheme 2). To our delight, compound 11 was active against
B-Raf with an IC50 of 3.47 lM.
To further optimize this series, we wondered if the flat tricyclic
core could be replaced with a more flexible system. To explore
other favorable interactions and improve physical properties,16
two bicyclic compounds 15 and 17 were designed and synthesized.
The preparation of compound 15 started with 5,6-dichloroisoindo-
line-1,3-dione (12). The silyl protection of 12 followed by a
NH
NH
NH
Cl
b, c, d
HN
TIPSN
17
O
B-Raf
IC50 = 1.68 µM
O
16
Scheme 3. Reagents and conditions: (a) TIPSCl, Et3N, rt, 74%; (b) ArNH2, cat. XPhos,
cat. Pd2(dba)3, K3PO4, toluene, reflux, 50–60%; (c) TBAF, rt; (d) BBr3, CH2Cl2, rt, 40–60%
two steps; (e) Ar0NH2, cat. t-BuXPhos, cat. Pd2(dba)3, K3PO4, toluene, reflux, 37%.

X. Wang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6941–6944
6943
not reveal any kinase X-ray crystal structures containing such a
hinge binder suggesting it is an uncommon binding element.
After we identified potent type I B-Raf inhibitor leads contain-
ing novel scaffolds by using structure-based drug design strategies,
we wondered if we could build a type II inhibitor using the same
chemical scaffolds that might be beneficial for better selectivity
and/or high biochemical efficiency.19,20 It has been known21 that
type I kinase inhibitors can be converted to type II kinase inhibitors
by the attachment of a lipophilic group at an appropriate position
presumably filling the pocket created by the movement of DFG
motif. Compound 20 was constructed by the replacement22 of
the phenol group with a benzamide group (Fig. 6). The amide
group was designed to form two hydrogen bonds with E501 and
D594, while the phenyl group could extend to the allosteric pocket
in DFG-out conformation (Fig. 7).23 The R2 group was also changed
Figure 4. Model of compound 15 with B-Raf (active conformation).
OH
OH
O
O
18 R1 = H
B-Raf IC50 = 3.77 µM
19 R1 = OH
B-Raf IC50 = 0.290 µM
R1
NH
NH
NH
NH
a
HN
HN
HN
O
O
R1
Scheme 4. Reagents and condition: (a) N2H4, rt, DMF, 80%.
Figure 7. Model of compound 20d with B-Raf (DFG-out conformation).
NHCOAr
NHCOAr
O
O
O
NH
Cl
NH
Cl
a, b
HN
HN
c
13
HN
O
20a-h
21a-f, h
Scheme 5. Reagents and conditions: (a) ArCOCl, pyridine, rt; (b) TBAF, rt, 50–80%
two steps; (c) N2H4 ꢀ80%.
Table 1
Type II B-Raf inhibitors
Figure 5. Model compound 19 with B-Raf (compound 15 shown as blue).
O
N
N
NH
tBu
R4
a-f
O
NH
Cl
g
X
E501
O
O
O
C532
H
N
H
N
X = NH
R4
B-Raf IC50
(l
M)
X = HNNH
R4
B-Raf IC50 (lM)
R3
O
O
20a
20b
20c
20d
20e
20f
Cl
Br
I
CF3
OCF3
CN
—
0.117
0.035
0.008
0.010
0.006
0.297
0.349
21a
21b
21c
21d
21e
21f
Cl
Br
I
CF3
OCF3
CN
—
0.035
0.019
0.008
0.017
0.018
0.296
0.293
O
NH
R2
HN
H
D594
N
O
O
20
Figure 6. Design of type II B-Raf inhibitors.
20g
21g

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X. Wang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6941–6944
Figure 8. Selectivity of compounds 20d and 21d.
6. The only known B-Raf crystal structures at that time were 1UWH, 1UWJ with
sorafenib and 2FB8 with SB-590885. Updated to 8/2011, there are 18 X-ray
crystal structures of B-Raf with small ligands in RCSB Protein Data Bank
(1UWH, 1UWJ, 2FB8, 3C4C, 3C4D, 3D4Q, 3IDP, 3II5, 3OG7, 3PPJ, 3PPK, 3PRF,
3PRI, 3PSB, 3PSD, 3Q4C, 3Q96, 3SKC).
7. King, A. J.; Patrick, D. R.; Batorsky, R. S.; Ho, M. L.; Do, H. T.; Zhang, S. Y.; Kumar,
R.; Rusnak, D. W.; Takle, A. K.; Wilson, D. M.; Hugger, E.; Wang, L.; Karreth, F.;
Lougheed, J. C.; Lee, J.; Chau, D.; Stout, T. J.; May, E. W.; Rominger, C. M.;
Schaber, M. D.; Luo, L.; Lakdawala, A. S.; Adams, J. L.; Contractor, R. G.; Smalley,
K. S. M.; Herlyn, M.; Morrissey, M. M.; Tuveson, D. A.; Huang, P. S. Cancer Res.
2006, 66, 11100–11105.
8. Takle, A. K.; Brown, M. J. B.; Davies, S.; Dean, D. K.; Francis, G.; Gaiba, A.; Hird, A.
W.; King, F. D.; Lovell, P. J.; Naylor, A.; Reith, A. D.; Steadman, J. G.; Wilson, D. M.
Bioorg. Med. Chem. Lett. 2006, 16, 378–381.
9. Wilhelm, S. M.; Carter, C.; Tang, L.; Wilkie, D.; McNabola, A.; Rong, H.; Chen, C.;
Zhang, X.; Vincent, P.; McHugh, M.; Cao, Y.; Shujath, J.; Gawlak, S.; Eveleigh, D.;
Rowley, B.; Liu, L.; Adnane, L.; Lynch, M.; Auclair, D.; Taylor, I.; Gedrich, R.;
Voznesensky, A.; Riedl, B.; Post, L. E.; Bollag, G.; Trail, P. A. Cancer Res. 2004, 64,
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10. Wong, K. K. Recent Patents Anticancer Drug Discov. 2009, 4, 28–35.
11. Ghose, A. K.; Herbertz, T.; Pippin, D. A.; Salvino, J. M.; Mallamo, J. P. J. Med.
Chem. 2008, 51, 5149–5171.
12. Very recently a series of B-Raf inhibitors with an amide group as the hinged
binder was published in this journal. Packard, G. K.; Papa, P.; Riggs, J. R.;
Erdman, P.; Tehrani, L.; Robinson, D.; Harris, R.; Shevlin, G.; Perrin-Ninkovic, S.;
Hilgraf, R.; McCarrick, M. A.; Tran, T.; Fleming, Y.; Bai, A.; Richardson, S.; Katz,
J.; Tang, Y.; Leisten, J.; Moghaddam, M.; Cathers, B.; Zhu, D.; Sakata, S. Bioorg.
Med. Chem. Lett. 2011. doi:10.1016/j.bmcl.2011.03.006.
13. The B-Raf enzymatic assay protocol has been described in the following
reference. Gopalsamy, A.; Ciszewski, G.; Hu, Y.; Lee, F.; Feldberg, L.; Frommer,
E.; Kim, S.; Collins, K.; Wojciechowicz, D.; Mallon, R. Bioorg. Med. Chem. Lett.
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from a phenyl to a chlorine atom because the modeling study
suggested that a large R2 may have an unfavorable steric interac-
tion with the transposed activation loop.24 Isoindoline-1,3-diones
20a–g were prepared from intermediate 13 by acylation and then
deprotection (Scheme 5). Further reaction with hydrazine provided
2,3-dihydrophthalazine-1,4-diones 21a–g (Scheme 5).
Both the isoindoline-1,3-dione analogs and 2,3-dihydrophthal-
azine-1,4-dione analogs exhibited good activity against B-Raf
(Table 1). The potency increased from meta-chloro, bromo, to iodo
analogs (20a–c/21a–c) indicated large meta lipophilic substituents
on the phenyl ring were preferred to fill the hydrophobic pocket
formed by seven non-polar amino acid residues (Fig. 7). The
meta-trifluoromethylphenyl group has been documented9,25 as a
good hydrophobic group to fill the DFG-out pocket and analogs
(20d, 21d, 20e, and 21e) with such a group or meta-trifluorometh-
oxylphenyl indeed showed low nanomolar IC50s. As we expected,
compounds with a polar substitute (20g, 21g) were less potent.
Interestingly a tert-butyl pyrazole group, which was optimal for a
p38 type II inhibitor,26 did not give better potency.
The selectivity profile of compound 20d and 21d was assessed
against a panel of 22 protein kinases (Fig. 8). Both of the
compounds exhibited excellent selectivity against all of tested
kinases including p38alpha, PKCbeta, and VEGFR2, which were
inhibited by our previous pyrazolopyrimidine series5 and the well
known type II Raf inhibitor sorafenib.9
14. A model built from 2FB8 (PDB) was used.
In summary, both type I and type II B-Raf inhibitor leads with
novel chemical scaffolds and hinge binders have been developed
with the aid of structure-based drug design technology. Com-
pounds with low nanomolecular activity have been identified
and they are suitable for further optimization.
15. Zhao, B.; Bower, M. J.; McDevitt, P. J.; Zhao, H.; Davis, S. T.; Johanson, K. O.;
Green, S. M.; Concha, N. O.; Zhou, B.-B. S. J. Biol. Chem 2002, 277, 46609–46615.
16.
A recent analysis suggested that disruption of molecular planarity has a
positive impact on the solubility. Ishikawa, M.; Hashimoto, Y. J. Med. Chem.
2011, 54, 1539–1554.
17. Hennessy, E. J.; Buchwald, S. L. J. Org. Chem. 2005, 70, 7371–7375.
18. RCSB Protein Data Bank website: www.pdb.org.
19. Backes, A.; Zech, B.; Felber, B.; Klebl, B.; Müller, G. Expert Opin. Drug Discov.
2008, 3, 1427–1449.
Acknowledgments
20. Alton, G. R.; Lunney, E. A. Expert Opin. Drug Discov. 2008, 3, 595–605.
21. Liu, Y.; Gray, N. S. Nat. Chem. Biol. 2006, 2, 358–364.
22. Wolin, R. L.; Bembenek, S. D.; Wei, J.; Crawford, S.; Lundeen, K.; Brunmark, A.;
Karlsson, L.;Edwards,J. P.; Blevitt, J. M. Bioorg. Med.Chem.Lett.2008, 18, 2825–2829.
23. A model built from 1UWH (PDB) was used.
We thank Drs. Tarek Mansour, Robert Abraham, John Ellingboe,
Ariamala Gopalsamy, John Mathias, Katherine Lee and Joseph
Strohbach for their support of this work.
24. To test this hypothesis
a close analog to compound 20d with a meta-
methoxyphenyl replacing chlorine atom was prepared and it was eight fold
less potent than 20d.
References and notes
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