Highly potent and selective 3-N-methylquinazoline-4(3H)-one based inhibitors of B-RafV600E kinase
Steve Wenglowsky a, Li Ren a,⇑, Jonas Grina a, Joshua D. Hansen a, Ellen R. Laird a, David Moreno a, 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
aArray BioPharma, Inc., 3200 Walnut Street, Boulder, CO 80301, United States
bGenentech, Inc., 1 DNA Way, South San Francisco, CA 94080-4990, United States

a r t i c l e i n f o

Article history:
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
a b s t r a c t

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.
ti 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.
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

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 (>10ti) 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

⇑ Corresponding author. Tel.: +1 303 386 1448; fax: +1 303 386 1130.
E-mail address: [email protected] (L. Ren). http://dx.doi.org/10.1016/j.bmcl.2014.03.007
0960-894X/ti 2014 Elsevier Ltd. All rights reserved.
nanomolar inhibitor of B-RafV600E with a corresponding cellular IC50 of 44 nM.

H
N
N
4
HN
N3 OMe

F

O

F

1

OO
S
N
H

Ref. 11

HN

N

N

F

NO

F

2

OO
S
N
H

Table 1
B-Raf activity of compounds 3–12 F
Ar
N
H

6

2
1
R

O O
S
N
H

B-Raf V600E enzyme IC50 = 5 nM Malme-3M pERK IC 50 = 19 nM
B-Raf V600E enzyme IC 50 = 3 nM Malme-3M pERK IC 50 = 27 nM

Compd Ar
R1 B-RafV600E IC50a nM pERK IC50a nM

Ar

F

L

F
this work

O O
S
N
H

3

4

N
N

N

N

F 314

F 33

6251

118

Figure 1. Schematic illustration of the amide linker replacement strategy.
5
N
Cl 4 44

N
6 Cl 25 814
N
N
7N Cl 16 733
OMe
N
8N Cl 11 132
NHMe
N
9N Cl 100 878

10
NMe2
N
N
O

Cl 2

26

N

11

N
N

Cl 2 18

NO
N
12 Ph N Cl 17 581
O

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) 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 2ti 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/aC-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
a IC50 values reflect the average from at least two separate experiments.

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

N

N

O

F

N
H

10

Cl

OO
S
N
H

N

N

O

N
H

AZ-628

N
H

O

CN

N

N

O

N
H

AZ-628

N
H

O

CN

N

N

O

N
H

17

O O
S
N
H

B-Raf V600E enzyme IC50 = 2 nM Malme-3M pERK IC 50 = 26 nM
B-Raf V600E enzyme IC50 = 0.6 nM Malme-3M pERK IC50 = 27 nM
B-Raf V600E enzyme IC50 = 0.6 nM Malme-3M pERK IC50 = 27 nM
B-Raf V600E enzyme IC50 = 851 nM

Figure 3. Structures of 10 and AZ-628.

Figure 4. Structures of AZ-628 and 17.

Table 2
B-Raf activity of compounds 10, 13–17

Table 3
B-Raf activity of compounds 18–22

N
N

O
R2 N
H
6

2
R

1

O O
S
N
H

N
N

O
2
R

N R3
6

2 R1

O O
S
N
H

Compd
10
13
14
15
16
17

R1, R2 Cl, F
F, F F, Cl
CN, Cl CN, H H, Me
B-RafV600E IC50a nM 2
19
12
0.2
0.3
851
pERK IC50a nM 26
55
116
16
6 —b

Compd
18
19
20
21
22

R1, R2 F, F
F, Cl Cl, F F, F Cl, F

R3 Me
Me
Me
Et
Et

B-RafV600E IC50a nM 11
3
8
26
30

pERK IC50a nM 62
84
114
110
603

a IC50 values reflect the average from at least two separate experiments.
aIC50 values reflect the average from at least two separate experiments.
bNot determined.
Table 4

any substitution at the 2-position, showed very weak activity. These results are consistent with SAR from the amide9b and the
B-Raf activity of compounds 23–25
N

R2

6

O O

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

Compd
23
24
25

R2 Cl
F
H
N

O
O 2 NH S CN

B-RafV600E IC50 nMa 0.2
0.2
0.3

pERK IC50 nMa 15
3
7

of 17 was attributed to the different binding modes adopted by the sulfonamide versus the amide (Fig. 4).
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.
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
a IC50 values reflect the average from at least two separate experiments.

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] ti Km, ATP. No kinase showed >60% inhibition other than B-Raf and C-Raf.
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.

3500
3000
2500

Vehicle, 5 mL/kg 16, 30 mg/kg, QD

2000
1500
1000
500

65%

95%

99%

0
1 3 5 8

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.

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.
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.
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 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.

N

N

O

+
Br

F H2N

Cl

O O
S
N
H

N

N

O

F

N
H

Cl

O O
S
N
H

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 tiC.

N
N

O

28

+
OH
Cl

F

CN

29

F
a

N
N

O
Cl

O

30

CN

F
b

N
N

O
Cl

O
CN

23 – 25
O O
S
N
H

Scheme 2. Preparation of O-linked 3-N-methylquinazoline-4(3H)-ones. Reagents and conditions: (a) NaH, DMF; (b) n-PrSO2NH2, NaH, NMP, 40 ti C, 30 min; then 30, 120 ti C, 4 h.

Table 5
In vitro ADME and pharmacokinetic properties of 10 & 16
Compd PappABa Microsome clearanceb Observed clearancec Vdd AUCe %F
10 High 23 4.7 0.49 282 112

16 High 13 1.6
aLLC-PK1 cell permeability classification: low (<2 ti 10ti 6 cm/s), medium (2–8 ti 10ti 6 cm/s), high (>8 ti 10ti 6 cm/s).
bMouse microsome clearance (ml/min/kg).
cMouse IV PK at 2.5 mg/kg (ml/min/kg).
dL/kg.
eMouse PO PK at 30 mg/kg (lM h).
0.25 483 60

N
HN

H
N

F

O

F

O O S
N
H

Ref. 19

HN

N

H
N

F

O

F

N
H

O

CN

NOMe N OMe

N
1, type IIB DFG-in/αC-helix-out
selectvie B-Raf inhibitor

O

this work

N
31, type IIA DFG-out multikinase inhibitor

OO

N

O
N
H
N
H
CN
N

O
N
H CN
N
H
S

AZ-628, type IIA DFG-out
multikinase inhibitor
16, type IIB DFG-in/αC-helix-out selectvie B-Raf inhibitor

Figure 7. Conversion between type IIB selective B-Raf inhibitors and type IIA multikinase inhibitors.

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.

References and notes

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12.Inhibitor enzyme activity was determined utilizing full-length B-RafV600E. Inhibition of basal ERK phosphorylation in Malme-3M cells was used as the mechanistic cellular assay. For assay description see: Laird, E.; Lyssikatos, J.; Welch, M.; Grina, J.; Hansen, J.; Newhouse, B.; Olivero, A.; Topolav, G. WO 2006/084015 A2, 2006.
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18.See Supporting information for the list of 228 kinases.
19.Wenglowsky, S.; Moreno, D.; Laird, E. R.; Gloor, S. L.; Ren, L.; Risom, T.; Rudolph, J.; Sturgis, H. L.; Voegtli, W. C. Bioorg. Med. Chem. Lett. 2012, 22, 6237.