Highly potent and selective 3-N-methylquinazoline-4(3H)-one based inhibitors of B-RafV600E kinase

Article abstract of DOI:10.1016/j.bmcl.2014.03.007

Herein we describe the design of a novel series of ATP competitive B-Raf inhibitors via structure-based methods. These 3-N-methylquinazoline-4(3H)-one based inhibitors exhibit both excellent cellular potency and striking B-Raf selectivity. Optimization led to the identification of compound 16, a potent, selective and orally available agent with excellent pharmacokinetic properties and robust tumor growth inhibition in xenograft studies. Our work also demonstrates that by replacing an aryl amide with an aryl sulfonamide, a multikinase inhibitor such as AZ-628, can be converted to a selective B-Raf inhibitor, a finding that should have broad application in kinase drug discovery.

Full text of DOI:10.1016/j.bmcl.2014.03.007

Bioorganic & Medicinal Chemistry Letters 24 (2014) 1923–1927
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Highly potent and selective 3-N-methylquinazoline-4(3H)-one based
inhibitors of B-RafV600E kinase
a
a
a
a
Steve Wenglowsky a, Li Ren a, , Jonas Grina , Joshua D. Hansen , Ellen R. Laird , David Moreno ,
Victoria Dinkel a, Susan L. Gloor a, Gregg Hastings a, Sumeet Rana a, Kevin Rasor a, Hillary L. Sturgis a,
Walter C. Voegtli a, Guy Vigers a, Brandon Willis a, Simon Mathieu b, Joachim Rudolph b
⇑
a Array BioPharma, Inc., 3200 Walnut Street, Boulder, CO 80301, United States
b Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080-4990, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Herein we describe the design of a novel series of ATP competitive B-Raf inhibitors via structure-based
methods. These 3-N-methylquinazoline-4(3H)-one based inhibitors exhibit both excellent cellular
potency and striking B-Raf selectivity. Optimization led to the identification of compound 16, a potent,
selective and orally available agent with excellent pharmacokinetic properties and robust tumor growth
inhibition in xenograft studies. Our work also demonstrates that by replacing an aryl amide with an aryl
sulfonamide, a multikinase inhibitor such as AZ-628, can be converted to a selective B-Raf inhibitor, a
finding that should have broad application in kinase drug discovery.
Received 10 February 2014
Revised 28 February 2014
Accepted 3 March 2014
Available online 13 March 2014
Keywords:
B-Raf inhibitors
Type IIB inhibitor
Kinase drug discovery
Melonoma
Ó 2014 Elsevier Ltd. All rights reserved.
The Ras/Raf/MEK/ERK (MAPK) signaling pathway transduces
signals from cell surface receptors to the nucleus leading to cellular
proliferation, differentiation and survival.1 Raf kinases act down-
stream of RAS and are responsible for MEK and ERK activation.
BRAF gene mutations may lead to MAPK pathway amplification
via constitutive activation of B-Raf. Mutated B-Raf is present in
approximately 7% of all cancers,2 and is most frequently associated
with melanoma.3,4 The most common (>90%) mutation in B-Raf is a
glutamic acid for valine substitution at residue 600 (V600E),2
which leads to constitutive kinase activity 500-fold greater than
wild-type B-Raf,5 and correlates with increased malignancy and
decreased response to chemotherapy.6 A number of drug candi-
dates targeting the B-RafV600E mutation have been described, and
recently two novel and selective B-Raf inhibitors vemurafenib7
and dabrafenib8 have shown impressive clinical results in the
treatment of metastatic melonoma, thus validating B-RafV600E as
a cancer target.
and selectivity. Although the amide linker functioned efficiently as
a spacer and a conformational control, cleavage of the amide bond
leading to the formation of potentially toxic aniline metabolites
was observed in vivo for compound 1. To address this potential lia-
bility, we initiated a program to identify alternative linkers.10 An-
other approach was to rigidify and constrain the amide linker,
exemplified by our recently disclosed discovery of a series of po-
tent and selective inhibitors of B-RafV600E derived from the
pyridopyrimidin-7-one template (compound 2).11 In parallel to
this effort, we embarked on de novo design to identify new scaf-
folds. Our strategy was to retain the aryl sulfonamide moiety,
and evaluate linker/hinge-binder combinations that could make a
critical contact to the main chain-NH of Cys532 (Fig. 1).
Molecular modeling studies suggested that a quinoline nitrogen
would be able to make the targeted hydrogen bond interaction
when connected to the aryl sulfonamide via an amine linker at
the 6-position (Fig. 2). Compound 3 was prepared to validate our
hypothesis and showed sub-micromolar activity in the enzyme as-
say (Table 1).12 Both the enzyme and cellular activity can be im-
proved dramatically (>10Â) when the quinoline hinge binding
template was replaced by quinazoline 4, an observation consistent
with findings in the pyridopyrimidin-7-one series.11 Potency was
further enhanced by substituting the 2-fluoro substituent on the
aryl sulfonamide with a chlorine. Compound 5 is now a single digit
nanomolar inhibitor of B-RafV600E with a corresponding cellular
IC50 of 44 nM.
Our lab recently reported the discovery of several novel series of
potent and selective inhibitors of B-RafV600E 9
, that featured an
amide linker connecting various hinge-binding templates to an
aryl sulfonamide (Fig. 1). The aryl sulfonamide has been shown
to be a key feature for engaging B-Raf in a DFG-in/aC-helix-out
conformation, which confers significant enhancements in potency
⇑
Corresponding author. Tel.: +1 303 386 1448; fax: +1 303 386 1130.
E-mail address: li.ren@arraybiopharma.com (L. Ren).
http://dx.doi.org/10.1016/j.bmcl.2014.03.007
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

1924
S. Wenglowsky et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1923–1927
Table 1
F
F
O
O
B-Raf activity of compounds 3–12
O
O
H
N
S
Ref. 11
S
N
N
6
F
N
N
H
H
O
O
S
F
O
F
HN
N
N
O
4
Ar
HN
N
H
N
2
N
H
R1
3
OMe
1
2
B-RafV600E enzyme IC50 = 5 nM
Malme-3M pERK IC50 = 19 nM
B-RafV600E enzyme IC50 = 3 nM
Malme-3M pERK IC50 = 27 nM
a
a
Compd
Ar
R1
F
B-RafV600E IC50 nM
pERK IC50 nM
N
this work
3
314
6251
118
44
F
N
N
O
O
4
5
6
F
33
4
Ar
S
N
N
L
N
H
F
Cl
Cl
Figure 1. Schematic illustration of the amide linker replacement strategy.
N
25
814
N
N
N
N
N
7
Cl
Cl
Cl
Cl
16
11
100
2
733
132
878
26
OMe
N
8
NHMe
N
9
NMe2
N
N
O
10
N
O
N
11
12
Cl
Cl
2
18
N
N
N
N
Ph
17
581
O
a
IC50 values reflect the average from at least two separate experiments.
Figure 2. Model of 3 (green) compared with the X-ray crystal structure of 1 (blue)
in complex with B-RafWT. The cleft surface is rendered in violet, and hydrogen-
bonding interactions are shown as dashed yellow lines. Sidechains of the hinge
residues are undisplayed for clarity. The propyl group resides in a pocket that is
enlarged by an outward shift of the
aC-helix. The DFG sequence (D594-G596)
and physiochemical properties in the pyrazolo[1,5-a]pyrimidine9e
and the imidazo[4,5-b]pyridine series.9d Indeed, the SAR trend mir-
rored our earlier findings. While a 5-membered heteroaryl such as
methylpyrazole 11 was able to maintain co-planarity with the qui-
nazoline-4(3H)-one hinge-binding template and make lipophilic
contacts with several residues that form the exit from the ATP
cleft11 (i.e., Ile463, Trp531, Ser535, Ser536) and remained active,
bulkier alkyl groups such as benzyl 12 resulted in significant po-
tency loss owing to an out-of-plane steric clash with the outer edge
of the cleft.
Having established the 3-N-methylquinazoline-4(3H)-one as a
promising scaffold, the next stage of our optimization focused on
varying the substitutions at the 2 and 6-position on the central
phenyl ring. Selected examples are shown in Table 2. Among the
halogens evaluated, the 2-Cl, 6-F substitution pattern gave the
highest potencies both in biochemical and cellular assays (10 vs
13 and 14, 5 vs 4). The potency of 14 can be rescued by replacing
the 2-F with a CN group and compound 15 is a sub-nanomolar
inhibitor of B-RafV600E with a pERK IC50 of 16 nM. Removing the
6-Cl substituent did not adversely affect activity as 16 remained
similarly active, indicating that this position is less critical for po-
tency than the 2-position. Indeed, compound 17, an analog without
resides in its active (DFG-in) conformation. The model was generated by inspection
and analogy to the X-ray crystal structure of 1 using the Maestro suite: Schrodinger
Release 2009-1, Schrodinger LLC, New York, NY, 2009.
Keeping the 2-Cl substituent on the aryl sulfonamide constant
and moving the nitrogen to the 4-position gave compound 6, a
quinoxaline analog with reduced activity. Further modifying the
quinazoline template by substituting the 4-position with OMe,
NHMe and NMe2 (7–9) all led to potency loss, likely arising from
steric interference with the P-loop. However, 3-N-methylquinazo-
line-4(3H)-one 10, a regio-isomer of 7 (N- vs O-methylation),
showed a 2Â improvement in both biochemical and cellular po-
tency over 5. It is worth mentioning that AZ-628 (Fig. 3), a Type
II multikinase inhibitor with B-Raf activity contains the same hinge
binding motif.13 We anticipated, however, that the aryl sufonamide
tail would allow compound 10 to bind B-Raf in a type IIB fashion
(i.e.; the DFG-in/a
C-helix-out kinase conformation)14, typically
imparting excellent kinase selectivity (vide infra).
Comparing a model of 10 to the X-ray crystal structure of 1
suggests that the N-methyl group of 10 contacts residues at the
exit of the ATP cleft, an area that was explored to balance potency

S. Wenglowsky et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1923–1927
1925
N
O
F
N
O
N
O
N
O
O
O
O
O
O
O
N
S
N
N
N
S
N
H
N
N
H
N
H
CN
N
H
N
N
H
N
H
CN
H
H
Cl
10
AZ-628
AZ-628
17
B-Raf V600E enzyme IC50 = 851 nM
B-RafV600E enzyme IC50 = 2 nM
Malme-3M pERK IC50 = 26 nM
B-Raf V600E enzyme IC50 = 0.6 nM
Malme-3M pERK IC50 = 27 nM
B-RafV600E enzyme IC50 = 0.6 nM
Malme-3M pERK IC50 = 27 nM
Figure 3. Structures of 10 and AZ-628.
Figure 4. Structures of AZ-628 and 17.
Table 2
Table 3
B-Raf activity of compounds 10, 13–17
B-Raf activity of compounds 18–22
R2
N
6
R2
N
6
N
N
O
O
O
O
N
S
S
N
H
N
2
N
H
2
R1
H
O
R3 R1
O
R1, R2
B-RafV600E IC50 nM
pERK IC50 nM
a
a
Compd
R1, R2
R3
B-RafV600E IC50 nM
pERK IC50 nM
a
a
Compd
10
13
14
15
16
17
Cl, F
F, F
F, Cl
CN, Cl
CN, H
H, Me
2
26
55
116
16
6
18
19
20
21
22
F, F
Me
Me
Me
Et
11
3
8
26
30
62
84
114
110
603
19
12
0.2
0.3
851
F, Cl
Cl, F
F, F
Cl, F
Et
b
—
a
IC50 values reflect the average from at least two separate experiments.
a
IC50 values reflect the average from at least two separate experiments.
Not determined.
b
Table 4
B-Raf activity of compounds 23–25
any substitution at the 2-position, showed very weak activity.
These results are consistent with SAR from the amide9b and the
pyridopyrimidin-7-one series11 owing to the expectation that the
aryl sufonamide occupies similar spaces. Possible explanations
for the importance of the 2-position include: effects on torsion an-
gle, hydrophobic contact with protein, and/or a beneficial effect on
the pKa of the sulfonamide motif.
Given the structural similarities between AZ-628 and 17 (same
hinge binder, linker and phenyl spacer), the poor B-RafV600E activity
of 17 was attributed to the different binding modes adopted by the
sulfonamide versus the amide (Fig. 4).
R2
O
6
N
N
O
O
S
N
2
H
O
CN
Compd
R2
B-RafV600E IC50 nMa
pERK IC50 nMa
23
24
25
Cl
F
H
0.2
0.2
0.3
15
3
7
a
IC50 values reflect the average from at least two separate experiments.
The effect of modifying the linker was also investigated as mod-
eling studies suggested the –NH linker was not making any specific
interaction with the protein. It was reasoned that permeability and
absorption could be improved by removing the unnecessary
hydrogen bond donor. Alkylation of the linker nitrogen was exam-
ined and selected examples are shown in Table 3. For the 2-F ana-
logs, methylation was well tolerated and no loss in activity was
observed (18 vs 13 and 19 vs 14). The SAR was different for the
2-Cl series, where the –NMe linker led to a modest drop in potency
(20 vs 10). The loss in potency likely arises from the larger Cl atom
imparting an unfavorable dihedral angle between the two rings.
Consistent with our hypothesis, larger alkyl group on the linker
nitrogen also resulted in less active compounds (21 vs 18 and 22
vs 20).
The oxygen linker was also briefly evaluated and selected exam-
ples are shown in Table 4. Modeling evaluation predicted a slight
shift of the template, suggesting that compounds should be simi-
larly active. Synthetic accessibility led to the choice of the 2-CN
series to test our hypothesis. IC50 values were comparable to the
NH-linked counterparts (23 vs 15, 25 vs 16), providing some of
the most potent compounds within the series.
others.16 Meanwhile, an oxygen atom of the sulfonamide forms
H-bonds to the backbone NH of Phe595 and G596 of the DFG
sequence.
The N-linked 3-N-methylquinazoline-4(3H)-ones were prepared
according to Scheme 1.17 6-Bromo-3-N-methylquinazoline-4(3H)-
one 26 was used in a Buchwald coupling reaction with N-(3-ami-
no-2-chloro-4-fluorophenyl)-1-cyclopropylmethanesulfonamide
27 to furnish compounds 10–22.
The
O-linked
3-N-methylquinazoline-4(3H)-ones
were
prepared according to Scheme 2.17 6-Hydroxyl-3-N-methylquinaz-
oline-4(3H)-one 28 was coupled with 3-chloro-2,6-difluorobenzo-
nitrile 29 in the presence of NaH to form the biaryl ether 30.
Displacing the 3-F with propyl sulfonamide under basic conditions
gave compounds 23–25.
General kinase selectivity for this series of compounds was ex-
pected to be excellent because of the uncommon DFG-in/aC-helix-
out binding mode that is induced by the sulfonamide tail. Selected
analogs such as 10 and 16 were screened in a large kinase panel at
a concentration of 1 lM. For example, the inhibitory activity of 16
was assessed against a panel of 228 kinases18 from across the hu-
man kinome at [ATP] ꢀ Km, ATP. No kinase showed >60% inhibition
other than B-Raf and C-Raf.
An X-ray crystal structure of B-RafWT in complex with 18 con-
firmed our anticipated binding mode and is depicted in Figure 5.15
The quinazoline nitrogen makes a key H-bond to the NH of Cys532
at the hinge. The nitrogen atom of the sulfonamide moiety is with-
in H-bond distance to the main-chain NH group of Asp594. Such an
interaction suggests that the nitrogen atom is deprotonated, an
observation consistant with previous reports from us11 as well as
In vitro ADME properties of 10 and 16 were also determined
(Table 5). Both compounds were highly permeable and intrinsically
stable in mouse microsomes (Table 5). The in vivo profiles for both
of them showed low clearance, high oral exposure and excellent
bioavailability.

1926
S. Wenglowsky et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1923–1927
3500
Vehicle, 5 mL/kg
16, 30 mg/kg, QD
3000
2500
2000
1500
1000
500
99%
65%
95%
0
1
3
5
8
Day of study
Figure 6. Tumor growth inhibition of 16 in LOX xenograft.
In summary, we have discovered a series of 3-N-methylquinaz-
oline-4(3H)-one based B-Raf inhibitors with excellent potency and
selectivity profiles, without the liability of releasing potentially
toxic aniline metabolite associated with an earlier scaffold. Optimi-
zation led to the identification of compound 16, a potent, selective
and orally available B-Raf inhibitor with excellent pharmacokinetic
properties and robust tumor growth inhibition in xenograft
studies.
Figure 5. X-ray crystal structure of 18 in complex with B-RafWT. The protein is
rendered white, and 18 is colored green. The surface and hydrogen-bonding
interactions are as described in Figure 2, and the observed interactions are
consistent with those depicted for compound 3.
Additionally, our current work demonstrated that a type IIA
multikinase inhibitor such as AZ-628 can be converted to a selec-
tive type IIB inhibitor by replacing an aryl amide with an aryl sul-
fonamide functionality (Fig. 7). Previously, we have also shown
that the process can be reversed to rationally design a DFG-out
Compound 16 was further advanced to a tumor growth inhibi-
tion (TGI) study in nude mice with established LOX (B-RafV600E
)
xenografts, at a daily dose of 30 mg/kg QD from day 1 to day 4. Tu-
mor volume was measured on day 1, 3, 5 and 8 (Fig. 6). After 4 days
of dosing, a 95% TGI response was registered on day 5. More impor-
tantly, prolonged inhibition was observed even after dosing was
stopped. For example, 99% TGI was reported on day 8. These find-
ings suggest that compound 16 is a highly efficacious B-Raf
inhibitor.
multikinase inhibitor 31 from a DFG-in/aC-helix-out selective B-
Raf inhibitor such as 1.19 Although these findings were discovered
in the context of B-Raf, application to other kinase targets should
be considered in the future.
F
N
O
N
O
F
O
O
O
O
+
S
N
N
S
H2N
N
Br
N
H
N
H
H
Cl
Cl
26
27
10 - 22
Scheme 1. Preparation of N-linked 3-N-methylquinazoline-4(3H)-ones. Reagents and conditions: Pd2(dba)3, BINAP, NaOt-Bu, PhMe, 100 °C.
Cl
N
O
N
O
Cl
N
O
Cl
a
b
O
O
+
N
N
N
S
F
F
OH
O
F
O
N
H
CN
29
CN
CN
28
23 - 25
30
Scheme 2. Preparation of O-linked 3-N-methylquinazoline-4(3H)-ones. Reagents and conditions: (a) NaH, DMF; (b) n-PrSO2NH2, NaH, NMP, 40 °C, 30 min; then 30, 120 °C,
4 h.
Table 5
In vitro ADME and pharmacokinetic properties of 10 & 16
Compd
PappABa
Microsome clearanceb
Observed clearancec
Vdd
AUCe
%F
10
16
High
High
23
13
4.7
1.6
0.49
0.25
282
483
112
60
a
b
c
LLC-PK1 cell permeability classification: low (<2 Â 10À6 cm/s), medium (2–8 Â 10À6 cm/s), high (>8 Â 10À6 cm/s).
Mouse microsome clearance (ml/min/kg).
Mouse IV PK at 2.5 mg/kg (ml/min/kg).
L/kg.
d
e
Mouse PO PK at 30 mg/kg (lM h).

S. Wenglowsky et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1923–1927
1927
F
F
O
O
O
H
N
Ref. 19
H
N
S
N
N
N
N
H
CN
H
O
F
O
F
HN
HN
N
N
OMe
OMe
1,
31,
type IIB DFG-in/αC-helix-out
selectvie B-Raf inhibitor
type IIA DFG-out
multikinase inhibitor
N
O
N
O
this work
O
O
O
N
N
S
N
H
N
H
CN
N
H
N
H
CN
16,
AZ-628,
type IIB DFG-in/αC-helix-out
selectvie B-Raf inhibitor
type IIA DFG-out
multikinase inhibitor
Figure 7. Conversion between type IIB selective B-Raf inhibitors and type IIA multikinase inhibitors.
9. (a) Wenglowsky, S.; Ren, L.; Ahrendt, K. A.; Laird, E. R.; Aliagas, I.; Alicke, B.;
Buckmelter, A. J.; Choo, E. F.; Dinkel, V.; Feng, B.; Gloor, S. L.; Gould, S. E.; Gross,
S.; Gunzner-Toste, J.; Hansen, J. D.; Hatzivassiliou, G.; Liu, B.; Malesky, K.;
Mathieu, S.; Newhouse, B.; Raddatz, N. J.; Ran, Y.; Rana, S.; Randolph, N.; Risom,
T.; Rudolph, J.; Savage, S.; Selby, L. T.; Shrag, M.; Song, K.; Sturgis, H. L.; Voegtli,
W. C.; Wen, Z.; Willis, B. S.; Woessner, R. D.; Wu, W.-I.; Young, W. B.; Grina, J.
ACS Med. Chem. Lett. 2011, 2, 342; (b) Wenglowsky, S.; Ahrendt, K. A.;
Buckmelter, A. J.; Feng, B.; Gloor, S. L.; Gradl, S.; Grina, J.; Hansen, J. D.; Laird, E.
R.; Lunghofer, P.; Mathieu, S.; Moreno, D.; Newhouse, B.; Ren, L.; Risom, T.;
Rudolph, J.; Seo, J.; Sturgis, H. L.; Voegtli, W. C.; Wen, Z. Bioorg. Med. Chem. Lett.
2011, 21, 5533; (c) Wenglowsky, S.; Moreno, D.; Rudolph, J.; Ran, Y.; Ahrendt, K.
A.; Arrigo, A.; Colsen, B.; Gloor, S. L.; Hasting, G. Bioorg. Med. Chem. Lett. 2012,
22, 912; (d) Newhouse, B. J.; Wenglowsky, S.; Grina, J.; Laird, E. R.; Voegtli, W.
C.; Ren, L.; Ahrendt, K.; Buckmelter, A. J.; Gloor, S. L.; Klopfenstein, N.; Rudolph,
J.; Wen, Z.; Li, X.; Feng, B. Bioorg. Med. Chem. Lett. 2013, 23, 5896; (e) Ren, L.;
Laird, E.; Buckmelter, A. J.; Dinkel, V.; Gloor, S. L.; Grina, J.; Newhouse, B.; Rasor,
K.; Hastings, G.; Gradl, S. N.; Rudolph, J. Bioorg. Med. Chem. Lett. 2012, 22, 1165.
10. Mathieu, S.; Gradl, S.; Ren, L.; Wen, Z.; Aliagas, I.; Gunzner-Toste, J.; Pulk, R.;
Zhao, G.; Alicke, B.; Boggs, J.; Buckmelter, A.; Choo, E.; Dinkel, V.; Gloor, S.;
Gould, S.; Hansen, J.; Hastings, G.; Hatzivassiliou, G.; Laird, E.; Moreno, D.; Ran,
Y.; Voegtli, W.; Wenglowsky, S.; Grina, J.; Rudolph, J. J. Med. Chem. 2012, 55,
2869.
Acknowledgment
The authors thank Susan Rhodes and Jennifer Otten for perme-
ability determinations.
Supplementary data
Supplementary data associated with this article can be found, in
the
online
version,
at
http://dx.doi.org/10.1016/j.bmcl.
2014.03.007.
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18. See Supporting information for the list of 228 kinases.
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