Targeting KRAS Mutant Cancers via Combination Treatment: Discovery of a 5-Fluoro-4-(3 H)-quinazolinone Aryl Urea pan-RAF Kinase Inhibitor

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Targeting KRAS Mutant Cancers via Combination Treatment:

Discovery of a 5‑Fluoro-4-(3H)‑quinazolinone Aryl Urea pan-RAF

Kinase Inhibitor

Malcolm P. Huestis, Darlene Dela Cruz, Antonio G. DiPasquale, Matthew R. Durk, Charles Eigenbrot,

Paul Gibbons, Alberto Gobbi, Thomas L. Hunsaker, Hank La, Dennis H. Leung, Wendy Liu,

Shiva Malek, Mark Merchant, John G. Mo ﬀ at, Christine S. Muli, Christine J. Orr, Brendan T. Parr,

Frances Shanahan, Christopher J. Sneeringer, Weiru Wang, Ivana Yen, Jianping Yin, Michael Siu,*

and Joachim Rudolph *

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Supporting Information

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ABSTRACT: Optimization of a series of aryl urea RAF inhibitors led to the identiﬁcation of type II pan-RAF inhibitor GNE-0749

(7), which features a ﬂuoroquinazolinone hinge-binding motif. By minimizing reliance on common polar hinge contacts, this hinge

binder allows for a greater contribution of RAF-speciﬁc residue interactions, resulting in exquisite kinase selectivity. Strategic

substitution of ﬂuorine at the C5 position eﬃciently masked the adjacent polar NH functionality and increased solubility by

impeding a solid-state conformation associated with stronger crystal packing of the molecule. The resulting improvements in

permeability and solubility enabled oral dosing of 7. In vivo evaluation of 7 in combination with the MEK inhibitor cobimetinib

demonstrated synergistic pathway inhibition and signiﬁcant tumor growth inhibition in a KRAS mutant xenograft mouse model.

vemurafenib/cobimetinib in 2015,⁵and encorafenib/binimeti-

INTRODUCTION

■

nib in 2018.⁶

The RAS−ERK pathway is a central mitogen-activated protein

Despite the beneﬁts of type I BRAF inhibitors in targeting

kinase (MAPK) signal transduction cascade, and hyper-

BRAF^V600Emutant melanoma, inhibition of RAF kinases has

activation frequently promotes cancer. Given its importance,

not been successful in the context of KRAS mutant cancers.

this pathway has been the subject of intense drug discovery

RAS mutations are the most frequently observed oncogenic

eﬀorts, and a number of oral small-molecule drugs that target

mutations, found in almost 20% of all cancers, and eﬀective

RAF, MEK, and ERK have been approved over the past 10

years.¹The ﬁrst launched BRAF inhibitors, vemurafenib²and

dabrafenib,³approved in 2011 and 2013, respectively, were

designed to target BRAF^V600Emutant melanoma as single

agents (Figure 1). Despite initial promise, patients were found

to rapidly develop resistance, prompting the clinical explora-

tion of combined RAF and MEK inhibitor treatment, with the

goal of achieving more sustained inhibition of the MAPK

pathway. This led to the approval of the combination

treatment regimens of dabrafenib/trametinib in 2014,⁴

treatment of such cancers remains an area of signiﬁcant

medical need. Not only are KRAS mutant tumors refractory to

BRAF^V600Einhibitor treatment, but, even worse, the MAPK

Received: December 1, 2020

Published: March 29, 2021

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Figure 1. Marketed BRAF^V600Einhibitors and MEK inhibitor combination partners.

Figure 2. Recently disclosed pan-RAF inhibitors.

pathway was found to be “paradoxically” activated in such

contexts.⁷It is now understood that the inhibitor binding

mode is a crucial factor in determining the occurrence of

paradoxical activation. Vemurafenib and dabrafenib are

examples of type 1.5 binders, compounds that induce an

outward shift of the αC helix within the kinase domain of

BRAF.⁸These compounds are believed to bind to BRAF

monomers and robustly inhibit BRAF^V600E, which can signal in

the monomeric state.⁹However, in the context of KRAS

mutant tumors, type 1.5 binders promote formation of

nonsymmetrical RAF homo- and heterodimers, fueling the

pathway in such tumors and thus causing undesirable

activation.^1d,10

inhibitors were uniquely positioned to target KRAS mutant

cancers in combination with other MAPK pathway inhibitors.

The chemical structures of recently reported pan-RAF

inhibitors are shown in Figure 2. At the time of the study

reported here, the structures of naporafenib¹⁶(LXH254) and

belvarafenib¹⁷(GDC-5573) were not disclosed. Our interest

initially began with AZ628 but peaked with the report from Eli

Lilly and Deciphera describing LY3009120 (1), a pyrido[2,3-

d]pyrimidine aryl alkyl urea with potent in vivo activity in

multiple mouse xenograft tumor models. Following a favorable

pharmacological assessment of compound 1, the investigators

developed a formulation strategy to enable advancement into

phase 1 clinical trials.^11,14,18

Conversely, type II RAF inhibitors, compounds that

promote an outward shift of the DFG activation loop but

leave the αC helix in the “in position”, were found to have no

predisposition for causing paradoxical activation in KRAS

mutant/BRAF wild-type cells.¹⁰⁻¹²Examples of such com-

pounds are AZ628, a tool compound ﬁrst reported in 2007,¹³

and, later, LY3009120 (1).^11,14Although these compounds

promote rapid formation of RAF homo- or heterodimers, the

induced symmetrical dimer allows for full inhibitor occupancy

of both protomers, leading to robust inhibition of signaling.

Such compounds have the ability to inhibit not only

BRAF^V600Ebut also wild-type BRAF and CRAF and hence

are collectively referred to as RAF dimer inhibitors or pan-RAF

inhibitors. With no predisposition to induce RAF activation

and a clear rationale for targeting CRAF as a downstream

eﬀector of KRAS emerging at the time,¹⁵type II pan-RAF

RESULTS AND DISCUSSION

■

Motivated by the strong biological rationale for the

combination of pan-RAF and MEK inhibitors to target

KRAS mutant cancers,^7c,19we conﬁrmed in our laboratories

that literature pan-RAF inhibitors in combination with the

MEK inhibitor cobimetinib provided synergistic antiprolifer-

ative eﬀects in KRAS mutant A549 cells.^19bFrom there, we

initiated a medicinal chemistry campaign with the goal of

identifying a selective pan-RAF inhibitor with a pharmacoki-

netic proﬁle suitable for preclinical oral dosing.

Armed with the experience gained during our legacy

BRAF^V600Eprogram, we began with a strategy that sought to

use the dimethylbutyl phenyl urea of pan-RAF inhibitor

LY3009120 (1) in combination with a variety of diﬀerent

hinge binder motifs (Table 1) that we predicted to be

compatible based on docking studies. Investigation of various

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Table 1. Hinge Binder Optimization of RAF Inhibitors

Table 2. Physicochemical Properties and PK Data of Compounds 6 and 7

a

f

g

compound

mouse IV CL_b

mouse IV V_dss, t_1/2

mouse F (PO)

kinetic sol, mp

log D_7.4, TPSA, MDCK P_app(AB)

b

c

6

7

50 mL min⁻¹kg⁻¹

0.94 L kg⁻¹, 0.5 h

0% (MCT) , 2% (ASD)

<1 μM, 293 °C

4.47, 88 Å², 3.6 × 10⁻⁶cm s⁻¹

d

e

27 mL min⁻¹kg⁻¹

1.4 L kg⁻¹, 0.8 h

26% (PEG 400) , 60% (ASD)

3 μM, 215 °C

4.47, 88 Å², 13 × 10⁻⁶cm s⁻¹

a

b

c

d

e

f

IV: 1 mg kg⁻¹dose. 5 mg kg⁻¹dose. 25 mg kg⁻¹dose, hydroxypropyl methylcellulose acetate succinate. 5 mg kg⁻¹dose. 30 mg kg⁻¹dose. mp

= melting point; kinetic solubility in pH 7.4 PBS buﬀer. MDCK apical → basolateral P_app(apparent permeability).

g

hinge binders allowed for quick examination of kinase

selectivity SAR along this region of the active site.

Pharmacological evaluation included BRAF and CRAF

biochemical assays. To identify compounds with greater-

than-additive eﬀects when combined with a MEK inhibitor,

antiproliferative activity was compared in BRAF mutant A375

and KRAS mutant A549 cells along with A549 in the presence

of a sub-eﬃcacious concentration (100 nM) of cobimetinib

(Table 1).^19bCompounds with a signiﬁcant A549 potency shift

in the presence versus absence of cobimetinib cotreatment

were selected for a quantitative determination of synergistic

inhibition of A549 DNA synthesis using a full dose matrix

assay format (vide infra). Starting with the 3-methoxy-1H-

pyrazolo[3,4-b]pyridine benzamide hinge binder moiety

described in an earlier BRAF series (compound 2),²⁰we saw

similar potency but a loss in selectivity compared to compound

1. Inhibitors 3 and 4, which were based on a 4-amino-

pyrimidine hinge binder,²¹demonstrated biochemical potency

improvements. The cellular assays were found to be critical for

the triage of compounds since many compounds synthesized at

this stage of the program reached the lower limit of detection

in the biochemical assay. For reasons that are not immediately

apparent, compounds 3 and 4 showed greater selectivity in a

focused kinase panel²²(8/26 kinases >70% inhibition for 4) in

stark contrast to 4-aminoquinazoline 5, which was fairly

promiscuous (24/26 kinases >70% inhibition at 0.1 μM).

A conceivable strategy to increase kinase inhibitor selectivity

is to reduce reliance on common kinase hinge contacts.

Previously, it has been noted that compounds with a single

hinge H-bond contact tend to have higher selectivity, and

those with three hydrogen bonds have some of the lowest Gini

coeﬃcients.²³In this vein, we explored hinge binders with

lesser donor−acceptor−donor contacts and emphasized RAF-

speciﬁc residue interactions, particularly Trp424 (unique to

ARAF, BRAF, and CRAF) and Phe595 (DFG) interactions.

This led us to synthesize 6-amino-4-(3H)-quinazolinone aryl

urea 6. We were pleased to see synergistic activity with

cobimetinib (A549 IC₅₀= 253 nM in combination with

cobimetinib) and improved kinase selectivity (5/26 kinases

>70% inhibition at 0.1 μM).²⁴

While excellent potency and kinase selectivity were achieved,

oral exposure proved to be challenging for this series of urea

analogues, presumably due to poor solubility (compounds 2−6

kinetic solubility <1 μM). A conventional crystalline

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Figure 3. Kinome tree for 7, tested against 222 kinases at a concentration of 0.1 μM. Red circles represent BRAF and CRAF.

suspension of 6 in aqueous MCT (0.5% methylcellulose with

0.2% Tween 80) resulted in no measurable oral absorption in

mouse (Table 2). The poor solubility of 6 likely results from a

combination of high lipophilicity (log D = 4.47) and strong

crystal lattice energy as evidenced by a high melting point (293

°C). A small-molecule X-ray crystal structure of 6 did indeed

show well-ordered crystal lattice interactions with a robust

intermolecular hydrogen-bond network (Figure S1). Interest-

ingly, we found the conformation adopted in the small-

molecule structure (torsional angle between the two ring

systems) to be signiﬁcantly diﬀerent from the expected

conformation in the kinase-bound state (vide infra). Not

even the use of enabling formulations such as PEG 400,

nanosuspension, or amorphous spray-dried dispersion (ASD)

formulations was able to provide remedy. We reasoned that the

high lipophilicity and crystal lattice energy presented a barrier

too diﬃcult to overcome and that the relatively low

permeability of the compound (P_app= 3.6 × 10⁻⁶cm s⁻¹in

MDCK cells) was likely a compounding factor.

Fortuitously, we found that the introduction of a ﬂuorine

substituent at the C5 position (Table 1, GNE-0749, 7)

provided improvements compared to compound 6 in cellular

potency (1.5×) and kinase selectivity (1/222 kinase >70%

inhibition at 0.1 μM, Table S3 and Figure 3). An X-ray crystal

structure of 7 bound in a mutant BRAF construct (Figure 4)

revealed the quinazolinone ring system interacting with

Cys532 backbone NH (3.1 Å) at the hinge of the protein in

addition to a polarized CH interaction with Cys532 CO (3.1

Å).²⁵With less discrete polarized hinge contacts, the exquisite

selectivity for RAF kinases may be due to van der Waals π-

stacking interactions of the heteroaryl hinge binder with

Trp531 in ARAF, BRAF, and CRAF.

The potency and selectivity gain associated with this single

ﬂuorine substitution were already desirable traits, but the most

impactful beneﬁts imparted by this modiﬁcation were property

improvements. Speciﬁcally, compound 7 demonstrated sig-

niﬁcantly increased permeability and a substantial decrease in

the melting temperature, associated with a gain in solubility

(Table 2). We reasoned that the presence of ﬂuorine masks the

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Figure 4. X-ray crystal structure of 7 bound in a mutant BRAF

construct (PDB ID: 7K0V).

Figure 6. Quantum mechanical (B3LYP-D3/6-311+g**) torsion scan

for model compounds representing 6 and 7. Torsion T₁was scanned

from −180 to 180° in steps of 30°. T₂was kept constant at −120°, the

angle found in the protein ligand crystal structure. The gray line

indicates the torsion angle of T₁found in the protein ligand crystal

structure, and the blue and orange lines denote the torsion angles of

T₁in the small-molecule X-ray structures of 6 and 7, respectively. T₂

in the small-molecule crystal structures is approximately −60°;

however, the change accounts for just 0.3 kcal mol⁻¹energy

diﬀerence.

adjacent polar NH functionality to improve permeability, but

the observed melting point decrease could not be easily

rationalized. We proceeded with determining the small-

molecule X-ray crystal structure of this compound (Figure

S2) and juxtaposed it with the small-molecule X-ray structure

of compound 6 (Figure S1). This comparison revealed

important diﬀerences. The ﬂuorine analogue 7 adopts a

rotational angle between the two rings that is similar to the

angle observed in its ligand protein cocrystal structure (Figure

5). Compound 6, on the other hand, adopts a conformation in

what is experimentally observed in the small-molecule X-ray

structure. A T₁of 180° is also the required conformation in the

kinase-bound state, and although other eﬀects might be

responsible for the 2-fold higher potency of 7 compared to 6,

this reduced conformational ﬂexibility of 7 and bias toward the

conformation required for kinase binding might be a

contributing factor to this potency gain.²⁶

The observation that T₁in compound 6 adopts a torsion

angle of ∼0 instead 180°, despite energetic equivalency, can

likely be explained by crystal packing beneﬁts. Indeed, we

conclude from a comparison to the small-molecule X-ray

structure of compound 7, which is conﬁned to a T₁of ∼180°,

that the network of molecular interactions in their respective

small-molecule X-ray structures is stronger for compound 6

than for 7, consistent with its higher melting point and lower

solubility. A detailed qualitative comparison is provided in the

Supporting Information. Taken together, the absence of a

ﬂuorine atom at C5 in compound 6 oﬀers two conformational

energy minima, and the molecule selects in the solid state for

the conformation that is associated with a stronger crystal

lattice. The ﬂuorine atom in C5 of compound 7, on the other

hand, does not allow for adopting an equivalent solid-state

conformation to compound 6 and leaves, as the only option,

adoption of a conformation that aﬀords a weaker crystal lattice

and resembles the conformation required for kinase binding.

Gratifyingly, the improvements in permeability and solubility

aﬀorded measurable oral exposure of 7 in mice when using

enabling formulations (F = 26% using PEG 400 and F = 60%

using ASD), marking a signiﬁcant improvement over

compound 6.

Figure 5. Conformations of 6 (yellow) and 7 (orange) determined by

small-molecule X-ray crystallography and overlay with protein ligand

conformation of 7 (gray). The largest diﬀerence between the

conformations of 6 and 7 is an ∼170° rotation around T₁(indicated).

In the overlay, the urea side chain is hidden for clarity.

the small-molecule X-ray structure that does not match the

required conformation in the kinase-bound state, namely, the

quinazoline ring is rotated by ∼170° (Figure 5).

To explain these observations, we generated a conforma-

tional energy proﬁle for the rotation around the quinazolinone

N bond (Figure 6), using simpliﬁed analogues of 6 and 7, 6′

and 7′, respectively. Compound 6 in the small-molecule X-ray

structure adopts a T₁of ∼0°, one of the two low energy

regions for 6′. While both T₁∼0 and ∼180° are ground-state

energy minima, we hypothesize that T₁at ∼0° is associated

with crystal packing beneﬁts, as discussed later. For the ﬂuorine

counterpart 7′, T₁at 0°, despite being located at a local

minimum, is 4 kcal mol⁻¹above the ground state, suﬃcient to

disfavor this torsion angle. 7′ only has one ground-state

conformation at ∼180°, and adoption of this angle is indeed

We have shown in a previous study that the combination of

type II RAF inhibitors and MEK inhibitors is eﬃcacious in

targeting KRAS mutant tumors.^19bThis synergy results from

the increased dependence of KRAS mutant tumors on RAF

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Figure 7. Synergistic activity of 7 combined with cobimetinib. (A) Proliferation of A549 cells, determined by incorporation of 5-ethynyl-2′-

deoxyuridine (EdU) into newly synthesized DNA, is inhibited in a dose-dependent manner by each agent separately (green curves), and potency is

strongly enhanced by increasing concentrations of the second compound. (B) Isobologram plot of fractional EdU uptake inhibition at diﬀerent

dose combinations shows that the doses required to attain 50% inhibition (blue line) are signiﬁcantly lower than predicted by a simple additivity

model (red line). (C) Diﬀerence between the observed percent eﬀect and expected fractional eﬀect according to the Loewe additivity model

plotted for each dose combination.

Figure 8. (A) In vivo tumor growth inhibition study over 21 days using HCT116 tumor-bearing mice treated with cobimetinib (5 mg kg⁻¹PO,

QD), GNE-0749 (7) (10 mg kg⁻¹PO, BID), or a combination. (B) In vivo PK/PD study over 4 days using HCT116 tumor-bearing mice treated

with cobimetinib (5 mg kg⁻¹PO, QD), GNE-0749 (7) (10 mg kg⁻¹PO, BID), or a combination.

signaling in the presence of a MEK inhibitor. RAF dimer (type

II) inhibitors are able to robustly inhibit MAPK signaling,¹¹

inhibition in KRAS mutant cells. This approach results in a

eﬀectively abrogating negative feedback caused by MEK

chemical synthetic lethal eﬀect where at drug concentrations in

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a

Scheme 1. Synthesis of Common Intermediates for Hinge Binder Analysis

a

Conditions: (a) 10% Pd/C (50% w/w), H₂(1 atm), EtOH, 23 °C, 94%; (b) PhOCOCl (1.1 equiv), NaHCO₃(2 equiv), THF, 23−60 °C; then,

NEt₃(2 equiv), 3,3-dimethylbutylamine (1.1 equiv), toluene, 23−90 °C, 20% (two steps); (c) LiOH·H₂O (1.1 equiv), THF/H₂O (5:1), 80 °C,

97%; (d) PhOCOCl (1.1 equiv), NaHCO₃(2 equiv), THF, 0−23 °C; then, NEt₃(2 equiv), 3,3-dimethylbutylamine (1.2 equiv), toluene, 23 °C,

95% (two steps); (e) tert-butyl carbamate (2 × 1.2 equiv), Pd₂(dba)₃(2 × 0.025 equiv), Xantphos (2 × 0.06 equiv), Cs₂CO₃(3 equiv), THF, 90−

110 °C, 32%; (f) tert-butyl carbamate (1.2 equiv), X-Phos Pd G2 (0.05 equiv), X-Phos (0.10 equiv), Cs₂CO₃(1.4 equiv), 1,4-dioxane, 110 °C;

then, HCl in isopropanol, 23 °C, 45% (two steps).

a

Scheme 2. Synthesis of Compounds in Table 1

a

Conditions: (a) DMF (0.2 equiv), oxalyl chloride (3 equiv), THF, 23 °C; then, 3-methoxy-1H-pyrazolo[3,4-b]pyridin-5-amine (1.2 equiv), NEt₃

(2 equiv), 55 °C, 48%; (b) Hunig’s base (2 equiv), 10% aq NaOH/DMF (2:1), 23 °C, 72%; (c) 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-

yl)pyridin-2-amine (1 equiv), X-Phos Pd G2 (0.05 equiv), X-Phos (0.05 equiv), K₃PO₄·H₂O (3 equiv), THF/H₂O (10:1), 80 °C, 80%; (d) 9a (1

equiv), 10a (1.4 equiv), Pd₂(dba)₃(0.02 equiv), Xantphos (0.06 equiv), Cs₂CO₃(1.4 equiv), 1,4-dioxane, 80 °C, 61%; (e) methylamine

hydrochloride (3.8 equiv), K₂CO₃(2 equiv), DMSO, 90 °C, 92%; (f) diphenyl carbonate (1.15 equiv), DMAP (1.2 equiv), DMF, 100 °C, 48%;

(g) 9c (1 equiv), DMAP (1.1 equiv), DMF, 90 °C, 14%; (h) TFA/CH₂Cl₂(1:1), 23 °C; then, 4-((2,4-dimethoxybenzyl)amino)quinazoline-8-

carboxylic acid (1 equiv), HATU (2 equiv), NEt₃(7 equiv), DMF, 23 °C, 54% (two steps); (i) TFA, 70 °C, 75%; (j) tert-butyl carbamate (1.5

equiv), Pd₂(dba)₃(0.025 equiv), Xantphos (0.05 equiv), Cs₂CO₃(2 equiv), THF, 80 °C, 95%; (k) TFA 23 °C; then, 9a (1 equiv), BrettPhos Pd

G1 (0.05 equiv), BrettPhos (0.05 equiv), NaOtBu (4 equiv), 1,4-dioxane, 100 °C, 80% (two steps); (l) 4.0 M HCl in 1,4-dioxane/(EtO)₃CH

(1:6), 110 °C, 69%; (m) tert-butyl carbamate (1.2 equiv), Pd₂(dba)₃(0.02 equiv), Xantphos (0.06 equiv), Cs₂CO₃(1.2 equiv), toluene, 90 °C,

86%; (n) 4.0 M HCl in 1,4-dioxane (12 equiv), EtOAc, 23 °C, 88%; (o) 9a (1 equiv), Pd(OAc)₂(0.05 equiv), Xantphos (0.05 equiv), Cs₂CO₃(3

equiv), 3 Å molecular sieves, 1,4-dioxane, 100 °C, 34%.

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CHEMISTRY²⁸

which neither inhibitor has single-agent activity, the combina-

tion prevents tumor growth.

■

The synthesis of LY3009120 (1) was accomplished as

previously described.^14aThe preparation of common inter-

mediates used in the synthesis of compounds 2−7 are outlined

in Scheme 1. Thus, hydrogenative reduction of methyl 4-

ﬂuoro-2-methyl-5-nitrobenzoate aﬀorded aniline 8a (94%

yield), which was elaborated to urea 8b by way of a one-pot

operation involving formation of phenyl carbamate and

displacement with 3,3-dimethylbutylamine (20% over two

steps). Saponiﬁcation of 8b produced benzoic acid 8c in 97%

yield.

Similarly, interception of the phenyl carbamate derived from

5-bromo-2-ﬂuoro-4-methylaniline and phenyl chloroformate

with 3,3-dimethylbutylamine resulted in 9a as a key

intermediate (Scheme 1). Under standard palladium-catalyzed

conditions, the aryl bromide urea 9a could then be converted

to aniline derivatives 9b and 9c.²⁹

The syntheses of RAF inhibitors 2−7 are shown in Scheme

2. Intermediate acid 8c was converted to an acid chloride

followed by treatment with 3-methoxy-1H-pyrazolo[3,4-b]-

pyridin-5-amine to aﬀord trione 8d (44% over two steps). The

trione motif could then be unwound to compound 2 using the

conditions of Ryabukhin (72% yield).³⁰The Suzuki−Miyaura

cross-coupling of 3-(Bpin)pyridin-2-amine with 6-chloro-N-

methylpyrimidin-4-amine aﬀorded 10a (80% yield).³¹Amino-

pyridine 10a could then be fastened to intermediate 9a

through a Buchwald−Hartwig reaction under the conditions of

Yin, aﬀording 3 in 61% yield.³²For the synthesis of diurea 4,

tert-butyl N-tert-butoxycarbonyl-N-(6-chloropyrimidin-4-yl)-

carbamate was subjected to nucleophilic aromatic substitution

with methylamine followed by formation of the phenyl

carbamate 11b (44% over two steps). Addition of aniline 9c

to 11b aﬀorded 4 in 14% yield. Acidic rupture of the tert-

butoxycarbamate in 9b aﬀorded the free aryl amine, which was

coupled to a protected aminoquinazoline using HATU (12a,

54% over two steps). Deprotection of the dimethoxybenzyl

group using triﬂuoroacetic acid aﬀorded 5 in 75% yield.

The preparation of 6 began from 6-bromo-3-methylquina-

zolin-4(3H)-one. Buchwald−Hartwig amination aﬀorded 13a

(95%), which was deprotected and subjected to amination

with aryl bromide 9a using BrettPhos (6, 80% over two

steps).³³For the synthesis of GNE-0749 (7), amino

benzamide 14b (prepared in two steps from 2-amino-6-

ﬂuorobenzoic acid) was cyclized to a quinazolinone 14c and

then elaborated to aminoquinazolinone 14e (52% over three

steps). Gram-scale amination of 9a with 14e aﬀorded 7 in 34%

yield.

To conﬁrm that the observed enhancement of the

antiproliferative eﬀect of compound 7 in combination with

cobimetinib met quantitative criteria for synergy, A549

(KRAS^G12Smutant) cells were treated with a combinatorial

matrix of doses of the two compounds (Figure 7). A robust

leftward shift in dose−response for each compound in the

presence of increasing dose of the second compound was

observed. When analyzed using the isobologram approach

(Figure 7B) and by calculating the excess activity over that

predicted using the Loewe additivity model (Figure 7C), a

strong and reproducible deviation from the additive model was

observed.²⁷A similar degree of synergy was observed with a

second KRAS mutant cell line, HCT116 (Figure S3), whereas

no greater-than-additive eﬀect was observed for a BRAF

mutant cell line (A375). In contrast, the combination of

vemurafenib (type 1.5 RAF inhibitor) and cobimetinib did not

demonstrate any synergistic eﬀect (Figure S4).

These encouraging cellular data motivated us to investigate

the combinatorial eﬀects of both compounds in vivo using the

KRAS^G12Dmutant HCT116 xenograft tumor model (Figure

8A). In this study, GNE-0749 (10 mg kg⁻¹, PO, BID) or

cobimetinib (5 mg kg⁻¹, PO, QD) showed, as expected,

minimal single-agent activity, whereas the combination

resulted in signiﬁcant antitumor eﬃcacy (111% TGI). While

there was apparent tolerated body weight loss with single-agent

7 (averaging ∼10% body weight loss), combination with

cobimetinib did not appear to exacerbate this eﬀect, and all

animals tolerated treatment to the end of the study (21 days).

Dedicated pharmacokinetic/pharmacodynamic (PK/PD)

studies also demonstrated improved pathway engagement

with the combination of 7 and cobimetinib at 2 and 8 h

following 4 days of treatment. Whereas single-agent

cobimetinib showed minimal activity with modest inhibition

of pERK at 2 and 8 h, single-agent 7 resulted in increased

stabilization of CRAF phosphorylation with a corresponding

transient decrease in downstream signaling at the level of

pMEK, pERK, and pRSK (Figure 8B). In contrast, the

combination of 7 and cobimetinib resulted in increased levels

of pCRAF, relative to 7 alone, and improved and sustained

reduction of pERK and pRSK through 8 h post-dose (Figure

8B).

CONCLUSIONS

■

In summary, we have reported here the discovery of pan-RAF

inhibitors that abrogate paradoxical activation based on a type

II kinase binding mode. Limiting hinge binder interactions led

to improved kinome selectivity (compound 6), and the

subsequent introduction of a ﬂuorine atom aﬀorded GNE-

0749 (7) as a potent and selective pan-RAF inhibitor with

properties that allowed for oral dosing. Cellular studies in two

KRAS mutant models revealed strong synergistic activity of

this compound in combination with the MEK inhibitor

cobimetinib. This in vitro synergy was recapitulated in vivo,

and oral treatment of a combination of 7 with cobimetinib in

the KRAS mutant HCT116 tumor-bearing mice demonstrated

synergistic pharmacodynamic eﬀects and substantial tumor

growth inhibition. The data provide preclinical proof-of-

concept for the combination of type II RAF inhibitors with

MEK inhibitors as an approach for targeting KRAS mutant

cancers.

EXPERIMENTAL SECTION

■

General Methods. Enzymatic Assays Measuring RAF Kinase

Activity. Compounds are evaluated for potency against BRAF (416-

766aa, Sigma B4062) and CRAF (Y340D Y341D, Life Technologies

PV3805), using a DELFIA assay by PerkinElmer. Phosphorylated

MAP2K1 (inactive, Carna 07-141-10-3000) is directly measured by

detection of Eu-anti-p-MEK1/2 (Ser 217/221) (PerkinElmer

TRF0213). Assay buﬀer (1×) shared by both enzymes includes the

following: 50 mM HEPES (pH 7.5), 10 mM MgCl₂, 0.01% Brij 35, 2

mM TCEP, and 0.05% bovine gamma-globulins. Inactive MAP2K1 is

50 nM [ﬁnal] in the assay. The assays are run at 10× ATP K_mfor both

enzymes, which translates to 500 μM [ATP] for CRAF and 50 μM

[ATP] for BRAF. The enzyme concentration for both CRAF and

BRAF is 1 nM [ﬁnal]. Endpoint assays are run for 90 min at room

temperature, with 30 min preincubations of the compound and

enzyme. Assays are quenched and transferred to glutathione-coated

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plates for the capture of the GST-MAP2K1, and time-resolved

ﬂuorescence of europium-ab is measured following the DELFIA assay

protocol. Potency is determined from the IC₅₀value and converted to

K_ivia the Cheng−Prusoﬀ equation.

Cell Lines. All cell lines and compounds were obtained from the

Genentech in-house repositories. Cell lines were maintained in RPMI-

1640 media and supplemented with 10% heat-inactivated FBS

(HyClone, SH3007003HI), 1× GlutaMAX (Gibco, 35050-061),

and 1× Pen Strep (Gibco, 15140-122). The cells were maintained

in a humidity-controlled environment (37 °C, 5% CO₂; Forma

Scientiﬁc II). All cell lines were utilized before passage 20 and treated

in an exponential growth phase at 50−75% conﬂuence.

Drug Combination Assays: Click-iT EdU Cell Proliferation

Imaging Assay of A549 Cell Line, Combination Studies with

Cobimetinib. A volume of 50 μL was dispensed in 384-well plates at

2000 cells per well, and compounds were dispensed directly to wells

via the Echo acoustic dispenser. Cells were grown in the presence of

compound for 24 h.

Pulse Cells. Plates were dispensed with 20 μM EdU 647, 5 μL of

volume dispensed to 50 μL cells in media, and incubated for 30 min

in 37 °C and 5% CO₂. All proceeding steps were performed at room

temperature on the bench.

Fix Cells. An equal volume of 4% paraformaldehyde and 2% ﬁnal

was dispensed to each well and incubated for 15 min. Cells were

washed for two cycles using PBS, with ﬁnal aspiration.

Permeabilize Cells. Triton X-100 at 0.5% in PBS was dispensed to

each well at 50 μL for 20 min. Cells were washed for two cycles using

PBS, with ﬁnal aspiration. At 25 μL per well, a solution of 1× Alexa

Fluor 647, 4 mM CuSO₄, and 2 mg mL⁻¹sodium ascorbate in TBS

was dispensed to the cells and incubated for 30 min. Cells were

washed for six cycles using PBS, with ﬁnal aspiration.

Stain Cells. A solution of 3.2 μM Hoechst 33342 in PBS was added

to the cells at 25 μL per well and incubated in the dark for 30 min.

Cells were washed for two cycles with ﬁnal aspiration. A PerkinElmer

PHENIX was used to image cellular DNA and cytoplasm (Alexa Fluor

647 multiplexed with Hoechst stain) and calculate cell number.

Columbus software was used for analysis and visualization. Genedata

was used to calculate potencies and visualize the synergies of the

double titrations of experimental compounds against cobimetinib.²⁷

Cloning, Expression, Puriﬁcation, and Crystallization of BRAF

Mutant. cDNA-encoding BRAF residue R444-K723 with H539K was

generated in the background of 16 mutations to improve expression

(I543A, I544S, I551K, Q562R, L588N, K630S, F667E, Y673S,

A688R, L706S, Q709R, S713E, L716E, S720E, P722S, and K723G)

with an N-terminal histidine tag that was generated by gene synthesis

for bacterial expression. Expression was auto-induced at 16 °C.

Escherichia coli cells were lysed in 25 mM Tris (pH 8.0), 150 mM

NaCl, 1 mM TCEP, 5% glycerol, and 10 μM GNE-0749 with protease

inhibitor tablets (Roche). The cell suspension was homogenized and

passed through a microﬂuidizer twice. The lysate was clariﬁed by

centrifugation at 8000 rpm for 30 min. The supernatant was loaded

onto a 4 mL Ni-NTA column (Qiagen) and then washed with 25 mM

Tris (pH 8.0), 150 mM NaCl, 1 mM TCEP, 5% glycerol, and 15 mM

imidazole. Protein was eluted with 0.3 M imidazole in the same buﬀer.

After veriﬁcation by SDS-PAGE, the protein was concentrated to 2

mL and loaded onto a HiLoad 16/60 Superdex 200 column (GE

Healthcare) pre-equilibrated with 25 mM HEPES, 150 mM NaCl, 1

mM TCEP, and 5% glycerol. The peak corresponding to monomeric

protein was pooled, diluted 3-fold with 25 mM HEPES, 5% glycerol,

and 1 mM TCEP, and directly loaded onto a 5 mL HiTrap SP HP

column (GE Healthcare). The protein was eluted with a 0−500 mM

NaCl gradient in 25 mM HEPES, 1 mM TCEP, and 5% glycerol. The

eluted protein was pooled and concentrated to 5 mg mL⁻¹.

Data Collection and Structure Determination of BRAF/GNE-

0749. The diﬀraction data of BRAF/GNE-0749 were collected using

monochromatic X-rays at the Advanced Light Source (ALS) beamline

5.0.2 using a PILATUS3 6M detector. The rotation method was

applied to a single crystal for each of the complete data set. The

crystals were kept at cryogenic temperature throughout the data

collection process. Data reduction was performed using the program

XDS³⁴and the CCP4 program suites.³⁵Data reduction statistics are

shown in Table S5.

The structures were phased by molecular replacement (MR) using

program PHASER.³⁶A previously published crystal structure of BRAF

(PDB code: 3C4C) was used as the MR search models. Manual

rebuilding was performed with graphics program Coot.³⁷The

structures were further reﬁned iteratively using program REFMAC5³⁸

and PHENIX³⁹using maximum likelihood target functions,

anisotropic individual B-factor reﬁnement and TLS reﬁnement, to

achieve ﬁnal statistics shown in Table S5.

Animal Studies. All individuals participating in animal care and use

are required to undergo training by the institution’s veterinary staﬀ.

Any procedures, including handling, dosing, and sample collection,

mandate training and validation of proﬁciency under the direction of

the veterinary staﬀ prior to performing procedures in experimental in

vivo studies. All animals were dosed and monitored according to

guidelines from the Institutional Animal Care and Use Committee

(IACUC) on study protocols approved by Genentech’s Laboratory

Animal Resource Committee at Genentech, Inc.

Xenograft Tumor Studies. All xenograft studies were done as

previously described.⁴⁰Brieﬂy, the HCT116 cells were grown in

normal growth media (RPMI 1640 with ^L-glutamine and 10% FCS),

harvested, and implanted subcutaneously into the right ﬂank of female

NCR nude mice (6−8 weeks old) obtained from Taconic (Cam-

bridge City, IN) weighing an average of 24−26 g. The mice were

housed at Genentech in standard rodent micro-isolator cages and

were acclimated to study conditions at least 3 days before tumor cell

implantation. Only animals that appeared to be healthy, free of

obvious abnormalities, and harbored tumors without signs of

ulceration were used for each study. Tumor volumes were determined

using digital calipers (Fred V. Fowler Company, Inc.) using the

formula (L × W × W)/2. Tumor growth inhibition (%TGI) was

calculated as the percentage of the area under the ﬁtted curve (AUC)

for the respective dose group per day in relation to the vehicle such

that %TGI = 100 × [1 − (AUC_treatment/day)/(AUC_vehicle/day)].

Curve ﬁtting was applied to log₂-transformed individual tumor

volume data using a linear mixed-eﬀect model using the R package

nlme, version 3.1−97 in R v2.12.0. Mice were weighed twice a week

using a standard scale and checked daily for signs of morbidity as

detailed above. Animals were euthanized within 4 h if deemed

moribund or if tumor volumes exceeded 1500 mm³.

Chemistry. All metal catalysts, ligands, and bases were purchased

from MilliporeSigma or Strem Chemicals, Inc. and stored in a

desiccator (weighing to air). Anhydrous solvents were purchased from

MilliporeSigma. Thin-layer chromatography was performed on EMD

TLC silica gel 60 F₂₅₄aluminum-backed plates and visualized with UV

light. Flash chromatographic puriﬁcations were performed with

Teledyne ICSO RediSep Rf Gold silica cartridges on a Teledyne

ISCO Combiﬂash Rf. Preparative HPLC puriﬁcation was conducted

on C18 (Gemini NX-C18 50 × 30 mm, 5 μm packing, 110 Å particle

size) with mixtures of acetonitrile and either 0.1% aq NH₄OH or

formic acid. Nuclear magnetic resonance data were acquired on a

Bruker Avance III HD 400 Ascend with a Z-GRD Prodigy BBO 5 mm

cryoprobe. Chemical shift values are reported relative to internal

standards and operating frequencies shown in parentheses: ¹H

(400.33 MHz), trimethylsilane = 0.00 ppm; ¹³C{¹H} (100.67

MHz), trimethylsilane = 0.00 ppm; and ¹⁹F (376.52 MHz),

trichloroﬂuoromethane = 0.00 ppm. In cases of uncertain assign-

ments, structural conﬁrmation was secured through 2D NMR

experiments. Reactions were monitored by HPLC/MS analysis on a

Shimadzu LC-30AD with a Waters 2.1 mm × 30 mm, 1.7 μm BEH

C18 column, UV detection at 254 nm, and dual ESI/APCI to a

Shimadzu LCMS-2020 single quadrupole mass analyzer. The method

The BRAF mutant GNE-0749 complex was crystallized by vapor

diﬀusion. Hanging drops were set up by mixing 1 μL of protein and 1

μL of well solution (18% PEG 3350, 0.2 M Na nitrate, and 0.1 M bis-

Tris propane (pH 6.5)) and incubated at 19 °C. Crystals grew after 2

days. Crystals were washed and transferred to a cryoprotectant

solution of 25% glycerol, 18% PEG 3350, 0.2 M Na nitrate, and 0.1 M

bis-Tris propane (pH 6.5) prior to ﬂash cooling in liquid nitrogen.

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was conducted at a ﬂow rate of 0.7 mL min⁻¹whereby mobile phase

A was 0.1% formic acid in water and mobile phase B was acetonitrile.

The method began at 2% B, ramping linearly to 98% B over 2 min.

The gradient was held at 98% B for 0.2 min, then ramped down to 2%

B over 0.1 min, and held at 2% B for 0.1 min. The purity of ﬁnal

compounds was veriﬁed by LCMS to be >95% in all cases using either

of the following methods: (1) 10 min LCMS method: experiments

were performed on an Agilent 1290 UHPLC coupled with an Agilent

MSD (6140) mass spectrometer using ESI as the ionization source.

The LC separation was done on a Phenomenex XB-C18, 1.7 μm, 50

× 2.1 mm column at a ﬂow rate of 0.4 mL min⁻¹. Mobile phase A was

water with 0.1% formic acid, and mobile phase B was acetonitrile with

0.1% formic acid. The gradient started at 2% B, ended at 98% B over 7

min, and held at 98% B for 1.5 min following equilibration for 1.5

min. LC column temperature was 40 °C. UV absorbance was

collected at 220 and 254 nm, and mass spectrometry full scan was

applied to all experiments. (2) 30 min LCMS method: experiments

were performed on an Agilent 1290 HPLC coupled with an Agilent

MSD (6140) mass spectrometer using ESI as the ionization source.

The LC separation was done on an Agilent Zorbax Eclipse XDB-C18,

3.5 μm, 100 × 3.0 mm column at a ﬂow rate of 0.7 mL min⁻¹. Mobile

phase A was water with 0.1% formic acid, and mobile phase B was

acetonitrile with 0.1% formic acid. The gradient started at 2% B,

ended at 98% B over 25.5 min, and held at 98% B for 2.5 min

following equilibration for 1.5 min. LC column temperature was 40

°C. UV absorbance was collected at 220 and 254 nm, and mass

spectrometry full scan was applied to all experiments.

methylbenzoic acid (60.0 mg, 0.203 mmol) in anhydrous tetrahy-

drofuran (1.0 mL) was added anhydrous DMF (0.003 mL, 0.04

mmol) followed by oxalyl chloride (52.8 μL, 0.606 mmol). After

stirring for 5 min, the reaction mixture was concentrated to dryness

and redissolved in anhydrous tetrahydrofuran (1.0 mL). To the

solution was added 3-methoxy-1H-pyrazolo[3,4-b]pyridin-5-amine

(39.9 mg, 0.243 mmol) followed by triethylamine (56.4 μL, 0.405

mmol), and the mixture was heated at 55 °C for 66 h. The volatiles

were removed, and the residue was subjected to ﬂash column

chromatography (dichloromethane/methanol, 100:0 to 90:10) to

1

aﬀord the title compound as a white solid (48.7 mg, 48%). H NMR

(400 MHz, DMSO-d₆): δ 12.54 (s, 1H), 10.70 (s, 1H), 8.63 (d, J =

2.4 Hz, 1H), 8.46 (d, J = 2.4 Hz, 1H), 7.63 (d, J = 7.2 Hz, 1H), 7.49

(d, J = 10.9 Hz, 1H), 4.01 (s, 3H), 3.63−3.52 (m, 2H), 2.48 (s, 3H),

1.61−1.47 (m, 2H), 0.95 (s, 9H).

5-(3-(3,3-Dimethylbutyl)ureido)-4-ﬂuoro-N-(3-methoxy-1H-

pyrazolo[3,4-b]pyridin-5-yl)-2-methylbenzamide (2). To solid 5-(3-

(3,3-dimethylbutyl)-2,4,5-trioxoimidazolidin-1-yl)-4-ﬂuoro-N-(3-me-

thoxy-1H-pyrazolo[3,4-b]pyridin-5-yl)-2-methylbenzamide (18.2 mg,

0.0367 mmol) were added N,N-diisopropylethylamine (13 μL, 0.073

mmol), anhydrous N,N-dimethylformamide (70 μL), and aqueous

10% sodium hydroxide (0.18 mL), and the reaction mixture was

stirred for 2.5 h. The mixture was concentrated and puriﬁed by

preparative HPLC to aﬀord the title compound as a white solid (11.6

1

mg, 72% yield, >99% HPLC purity). H NMR (400 MHz, DMSO-

d₆): δ 12.50 (s, 1H), 10.46 (s, 1H), 8.65 (d, J = 2.2 Hz, 1H), 8.47 (d, J

= 2.5 Hz, 1H), 8.40−8.23 (m, 2H), 7.16 (d, J = 12.3 Hz, 1H), 6.52 (t,

J = 5.6 Hz, 1H), 4.01 (s, 3H), 3.17−3.07 (m, 2H), 2.32 (s, 3H),

1.43−1.25 (m, 2H), 0.90 (s, 9H).

Methyl 5-Amino-4-ﬂuoro-2-methylbenzoate (8a). Under a nitro-

gen atmosphere, palladium on carbon (4.99 g, 10 mol % [5 wt %, 50%

wet]) and methyl 4-ﬂuoro-2-methyl-5-nitrobenzoate (5.00 g, 23.5

mmol) were suspended in ethanol (78 mL). The atmosphere was

evacuated and replaced with hydrogen gas balloon, and the mixture

was stirred vigorously for 23 h. The mixture was ﬁltered through

Celite, rinsed with methanol, and then concentrated to a light purple

1-(5-Bromo-2-ﬂuoro-4-methylphenyl)-3-(3,3-dimethylbutyl)urea

(9a). To a mixture of 5-bromo-2-ﬂuoro-4-methylaniline (10.00 g,

49.01 mmol) and sodium bicarbonate (8.23 g, 98.0 mmol) in

anhydrous THF (196 mL) under nitrogen and at 0 °C was added

phenyl chloroformate (6.8 mL, 54 mmol). After stirring for 15 min,

the cooling bath was removed and the reaction mixture was allowed

to stir for 2.5 h. The mixture was ﬁltered, rinsed with anhydrous

toluene, and then concentrated. The residue was taken up in

anhydrous toluene (163 mL), and while stirring, triethylamine (13.7

mL, 98.0 mmol) and 3,3-dimethylbutylamine (7.9 mL, 59 mmol)

were added sequentially. After 3 h, the mixture was ﬁltered and the

solid product was rinsed with heptane and toluene to aﬀord the title

1

solid (4.03 g, 94%). H NMR (400 MHz, CDCl₃): δ 7.41 (d, J = 9.3

Hz, 1H), 6.86 (d, J = 11.9 Hz, 1H), 3.86 (s, 3H), 3.67 (br s, 2H), 2.48

(s, 3H).

Methyl 5-(3-(3,3-Dimethylbutyl)ureido)-4-ﬂuoro-2-methylben-

zoate (8b). To a suspension of methyl 5-amino-4-ﬂuoro-2-

methylbenzoate (3.76 g, 20.5 mmol) and sodium bicarbonate (3.45

g, 41.1 mmol) in anhydrous tetrahydrofuran (103 mL) under a

nitrogen atmosphere was added phenyl chloroformate (2.84 mL, 22.6

mmol), and the mixture was heated to 60 °C for 5 min. The mixture

was then cooled to room temperature and ﬁltered, and the solids were

rinsed with dichloromethane. The mother liquor was concentrated to

aﬀord the crude carbamate as a white solid (1.42 g, 4.67 mmol). The

solid was dissolved in anhydrous toluene (23 mL), and to this

solution was added triethylamine (1.30 mL, 9.34 mmol) followed by

3,3-dimethylbutylamine (0.692 mL, 5.14 mmol). The solution was

heated at 90 °C for 1.5 h and cooled to rt that aﬀorded a white solid,

which was ﬁltered and rinsed with heptane and toluene to aﬀord the

1

compound as a white solid (15.16 g, 95%). H NMR (400 MHz,

DMSO-d₆): δ 8.38 (d, J = 7.9 Hz, 1H), 8.32 (d, J = 2.2 Hz, 1H), 7.23

(d, J = 12.6 Hz, 1H), 6.52 (t, J = 5.5 Hz, 1H), 3.16−3.03 (m, 2H),

2.25 (s, 3H), 1.41−1.29 (m, 2H), 0.90 (s, 9H).

6-(2-Aminopyridin-3-yl)-N-methylpyrimidin-4-amine (10a). Into

a vial were weighed 6-chloro-N-methylpyrimidin-4-amine (310 mg,

2.16 mmol), 2-aminopyridine-3-boronic acid pinacol ester (485 mg,

2.16 mmol), chloro(2-dicyclohexylphosphino-2′,4′,6′-triisopropyl-

1,1′-biphenyl)[2-(2′-amino-1,1′-biphenyl)]palladium(II) (86.7 mg,

0.108 mmol), 2-dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl

(52.5 mg, 0.108 mmol), and potassium phosphate tribasic

monohydrate (1.54 g, 6.48 mmol). The vial was purged with nitrogen

gas, charged with degassed tetrahydrofuran (10.8 mL) and distilled

water (1.1 mL), and then sealed, and the reaction mixture was stirred

at 80 °C for 4 h. After cooling to rt, the mixture was concentrated to

dryness. The reaction residue thus obtained was puriﬁed by ﬂash

column chromatography (100:0 to 20:80 CH₂Cl₂/90:9:1 CH₂Cl₂/

MeOH/aq NH₄OH) to aﬀord the title compound as a white solid

1

title compound (1.29 g, 20% over two steps). H NMR (400 MHz,

DMSO-d₆): δ 8.68 (d, J = 8.6 Hz, 1H), 8.30 (d, J = 2.5 Hz, 1H), 7.17

(d, J = 12.3 Hz, 1H), 6.49 (t, J = 5.6 Hz, 1H), 3.80 (s, 3H), 3.14−3.05

(m, 2H), 2.44 (s, 3H), 1.40−1.29 (m, 2H), 0.91 (s, 9H).

5-(3-(3,3-Dimethylbutyl)ureido)-4-ﬂuoro-2-methylbenzoic Acid

(8c). A solution of methyl 5-(3-(3,3-dimethylbutyl)ureido)-4-ﬂuoro-

2-methylbenzoate (400 mg, 1.29 mmol) and lithium hydroxide

monohydrate (59.5 mg, 1.42 mmol) in tetrahydrofuran (6.4 mL) and

water (1.3 mL) was stirred at 80 °C for 4 h. Tetrahydrofuran was

carefully evaporated, and to the stirring aqueous solution was added

hydrogen chloride (2.0 mL, 8.0 mmol, [4.0 M in 1,4-dioxane]). The

solid precipitate was ﬁltered and rinsed with water to aﬀord the title

1

(346 mg, 80%). H NMR (400 MHz, DMSO-d₆): δ 8.49 (br s, 1H),

8.04 (dd, J = 4.7, 1.8 Hz, 1H), 7.85 (br s, 1H), 7.54−7.02 (m, 3H),

6.77 (d, J = 1.1 Hz, 1H), 6.64 (dd, J = 7.6, 5.0 Hz, 1H), 2.85 (d, J =

4.7 Hz, 3H).

1-(3,3-Dimethylbutyl)-3-(2-ﬂuoro-4-methyl-5-((3-(6-

(methylamino)pyrimidin-4-yl)pyridin-2-yl)amino)phenyl)urea (3).

Into a vial were weighed 1-(5-bromo-2-ﬂuoro-4-methylphenyl)-3-

(3,3-dimethylbutyl)urea (80.0 mg, 0.242 mmol), tris-

(dibenzylideneacetone)dipalladium(0) (5.5 mg, 0.0060 mmol), 4,5-

bis(diphenylphosphino)-9,9-dimethylxanthene (8.4 mg, 0.014 mmol),

6-(2-aminopyridin-3-yl)-N-methylpyrimidin-4-amine (48.6 mg, 0.242

1

compound as a white solid (371.8 mg, 97%). H NMR (400 MHz,

DMSO-d₆): δ 8.58 (d, J = 8.8 Hz, 1H), 8.15 (d, J = 2.6 Hz, 1H), 7.07

(d, J = 12.2 Hz, 1H), 6.43 (t, J = 5.5 Hz, 1H), 3.12−3.08 (m, 2H),

2.43 (s, 3H), 1.40−1.32 (m, 2H), 0.91 (s, 9H).

5-(3-(3,3-Dimethylbutyl)-2,4,5-trioxoimidazolidin-1-yl)-4-ﬂuoro-

N-(3-methoxy-1H-pyrazolo[3,4-b]pyridin-5-yl)-2-methylbenzamide

(8d). To a solution of 5-(3-(3,3-dimethylbutyl)ureido)-4-ﬂuoro-2-

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mmol), and cesium carbonate (110 mg, 0.338 mmol). Under a stream

of nitrogen gas, the vessel was charged with anhydrous 1,4-dioxane

(1.2 mL) and the vial was sealed. The reaction mixture was stirred at

80 °C for 66 h. After cooling to rt, the mixture was ﬁltered through

Celite and rinsed with dichloromethane and methanol. The mother

liquor was concentrated, and the residue was puriﬁed by preparative

HPLC to aﬀord the title compound as a yellow solid (66.0 mg, 61%

yield, >99% HPLC purity). ¹H NMR (400 MHz, DMSO-d₆): δ 11.21

(br s, 1H), 8.67−8.48 (m, 2H), 8.16 (dd, J = 4.8, 1.8 Hz, 1H), 8.04

(d, J = 2.3 Hz, 1H), 8.00 (br s, 1H), 7.53 (br s, 1H), 7.03 (d, J = 11.9

Hz, 1H), 6.92 (d, J = 1.4 Hz, 1H), 6.84 (dd, J = 7.6, 5.0 Hz, 1H), 6.38

(t, J = 5.6 Hz, 1H), 3.13−3.00 (m, 2H), 2.88 (d, J = 4.6 Hz, 3H), 2.21

(s, 3H), 1.41−1.25 (m, 2H), 0.90 (s, 9H).

1-(5-Amino-2-ﬂuoro-4-methylphenyl)-3-(3,3-dimethylbutyl)urea

hydrochloride (9c). Into a pressure ﬂask were weighed 1-(5-bromo-2-

ﬂuoro-4-methylphenyl)-3-(3,3-dimethylbutyl)urea (2.00 g, 6.04

mmol), chloro(2-dicyclohexylphosphino-2′,4′,6′-triisopropyl-1,1′-

biphenyl)[2-(2′-amino-1,1′-biphenyl)]palladium(II) (225 mg, 0.302

mmol), 2-dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl (294

mg, 0.604 mmol), tert-butyl carbamate (866 mg, 7.25 mmol), and

cesium carbonate (2.75 g, 8.45 mmol). Under a stream of nitrogen

gas, the vessel was charged with anhydrous 1,4-dioxane (60 mL) and

the pressure ﬂask was sealed. The reaction mixture was stirred at 110

°C for 18 h. After cooling to rt, the mixture was ﬁltered through Celite

and rinsed with dichloromethane. The mother liquor was

concentrated, and the residue was dissolved in a solution of

hydrochloric acid in isopropanol (30 mL, molarity unknown), stirred

for 20 h, and then ﬁltered. The solid was rinsed with isopropyl acetate

to aﬀord the title compound as a white solid (831 mg, 45% over two

steps). ¹H NMR (400 MHz, DMSO-d₆): δ 8.23 (br s, 1H), 8.11 (d, J

= 7.6 Hz, 1H), 7.07 (d, J = 11.9 Hz, 1H), 6.53 (br s, 1H), 3.14−3.06

(m, 2H), 2.19 (s, 3H), 1.39−1.32 (m, 2H), 0.91 (s, 9H).

tert-Butyl (6-(Methylamino)pyrimidin-4-yl)carbamate (11a). To

a mixture of methylamine hydrochloride (3.13 g, 46.4 mmol) and tert-

butyl N-tert-butoxycarbonyl-N-(6-chloropyrimidin-4-yl)carbamate

(4.00 g, 12.1 mmol) in dimethyl sulfoxide (50 mL) was added

potassium carbonate (3.35 g, 24.3 mmol) at room temperature. The

reaction mixture was stirred at 90 °C for 10 h, then cooled to rt, and

concentrated under reduced pressure. The residue was puriﬁed by

ﬂash column chromatography (100:0 to 50:50 petroleum ether/

EtOAc) to aﬀord the title compound as a white solid (2.50 g, 92%).

LCMS m/z: 225.1 (M + H⁺).

Phenyl (6-Aminopyrimidin-4-yl)(methyl)carbamate (11b). To a

mixture of tert-butyl (6-(methylamino)pyrimidin-4-yl)carbamate (650

mg, 2.90 mmol) and 4-dimethylaminopyridine (425 mg, 3.48 mmol)

in N,N-dimethylformamide (5 mL) was added diphenyl carbonate

(714 mg, 3.33 mmol) dropwise at room temperature. The resulting

mixture was then stirred at 100 °C for 16 h. Thereafter, the reaction

mixture was cooled to rt, diluted with water, and extracted with ethyl

acetate. The organic layer was washed with water 2× and brine and

dried over anhydrous sodium sulfate. Following concentration, the

residue thus obtained was puriﬁed by ﬂash chromatography (100:0 to

34:66 petroleum ether/EtOAc) to aﬀord the title compound as a

white solid (340 mg, 48%). LCMS m/z: 244.8 (M + H⁺).

1-(5-bromo-2-ﬂuoro-4-methylphenyl)-3-(3,3-dimethylbutyl)urea

(2.00 g, 6.04 mmol), tris(dibenzylideneacetone)dipalladium(0) (138

mg, 0.151 mmol), 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene

(210 mg, 0.362 mmol), tert-butyl carbamate (8.66 g, 7.25 mmol), and

cesium carbonate (5.90 g, 18.1 mmol). Under a stream of nitrogen

gas, the vessel was charged with anhydrous tetrahydrofuran (30 mL)

and the pressure ﬂask was sealed. The reaction mixture was stirred at

90 °C for 18 h at which time HPLC indicated only minimal of

conversion to product. Additional tris(dibenzylideneacetone)-

dipalladium(0) (138 mg, 0.151 mmol), 4,5-bis(diphenylphosphino)-

9,9-dimethylxanthene (210 mg, 0.362 mmol), and tert-butyl

carbamate (8.66 g, 7.25 mmol) were added, and the reaction was

continued at 110 °C for a further 24 h. After cooling to rt, the mixture

was ﬁltered through Celite and rinsed with CH₂Cl₂. The mother

liquor was concentrated, and the residue was puriﬁed by ﬂash

chromatography (100:0 to 50:50 heptane/EtOAc) to aﬀord the title

1

compound as a white solid (717 mg, 32%). H NMR (400 MHz,

CDCl₃): δ 7.98 (d, J = 7.8 Hz, 1H), 6.83 (d, J = 11.2 Hz, 1H), 6.33

(br s, 1H), 6.14 (br s, 1H), 4.97−4.78 (m, 1H), 3.31−3.20 (m, 2H),

2.17 (s, 3H), 1.51 (s, 9H), 1.47−1.41 (m, 2H), 0.93 (s, 9H).

4-((2,4-Dimethoxybenzyl)amino)-N-(5-(3-(3,3-dimethylbutyl)-

ureido)-4-ﬂuoro-2-methylphenyl)quinazoline-8-carboxamide

(12a). A solution of tert-butyl (5-(3-(3,3-dimethylbutyl)ureido)-4-

ﬂuoro-2-methylphenyl)carbamate (99.0 mg, 0.269 mmol) in dichloro-

methane (1.3 mL) and triﬂuoroacetic acid (1.3 mL) was stirred for 45

min and then concentrated to dryness. Onto the solid residue were

weighed HATU (209.0 mg, 0.539 mmol) and 4-((2,4-

dimethoxybenzyl)amino)quinazoline-8-carboxylic acid (91.4 mg,

0.269 mmol), and then anhydrous N,N-dimethylformamide (1.3

mL) and triethylamine (0.263 mL, 1.89 mmol) were added. The

reaction mixture was stirred for 18 h, then diluted with dichloro-

methane, and washed with 10% aqueous potassium carbonate and

then brine. The organics were concentrated and puriﬁed by ﬂash

chromatography (dichloromethane/methanol, 100:0 to 95:5) to

aﬀord the title compound as a yellow solid (86.3 mg, 54%). ¹H

NMR (400 MHz, CDCl₃): δ 13.45 (s, 1H), 8.87 (dd, J = 7.5, 1.5 Hz,

1H), 8.69 (s, 1H), 8.45 (d, J = 8.0 Hz, 1H), 7.85 (dd, J = 8.1, 1.6 Hz,

1H), 7.58 (dd, J = 8.1 Hz, 1H), 7.32 (d, J = 8.4 Hz, 1H), 6.94 (d, J =

11.0 Hz, 1H), 6.59−6.40 (m, 3H), 6.29 (br s, 1H), 4.99 (t, J = 5.5 Hz,

1H), 4.82 (d, J = 5.4 Hz, 2H), 3.91 (s, 3H), 3.81 (s, 3H), 3.33−3.14

(m, 2H), 2.41 (s, 3H), 1.51−1.42 (m, 2H), 0.93 (s, 9H).

4-Amino-N-(5-(3-(3,3-dimethylbutyl)ureido)-4-ﬂuoro-2-

methylphenyl)quinazoline-8-carboxamide (5). A solution of 4-

((2,4-dimethoxybenzyl)amino)-N-(5-(3-(3,3-dimethylbutyl)ureido)-

4-ﬂuoro-2-methylphenyl)quinazoline-8-carboxamide (41.0 mg,

0.0696 mmol) in triﬂuoroacetic acid (0.7 mL) was stirred at 70 °C

for 3 h and then concentrated to dryness. The residue was puriﬁed by

preparative HPLC to aﬀord the title compound as a white solid (22.9

1

mg, 75% yield, >99% HPLC purity). H NMR (400 MHz, DMSO-

d₆): δ 13.43 (s, 1H), 8.93 (d, J = 8.5 Hz, 1H), 8.70 (dd, J = 7.5, 1.9

Hz, 1H), 8.60 (s, 1H), 8.48 (dd, J = 8.1, 1.5 Hz, 1H), 8.30 (br s, 2H),

8.12 (d, J = 2.3 Hz, 1H), 7.68 (dd, J = 7.9 Hz, 1H), 7.12 (d, J = 12.0

Hz, 1H), 6.43 (t, J = 5.6 Hz, 1H), 3.16−3.05 (m, 2H), 2.37 (s, 3H),

1.43−1.32 (m, 2H), 0.92 (s, 9H).

1-(6-Aminopyrimidin-4-yl)-3-(5-(3-(3,3-dimethylbutyl)ureido)-4-

ﬂuoro-2-methylphenyl)-1-methylurea (4). To a mixture of 1-(5-

amino-2-ﬂuoro-4-methylphenyl)-3-(3,3-dimethylbutyl)urea (220 mg,

0.82 mmol) and phenyl (6-aminopyrimidin-4-yl)(methyl)carbamate

(200 mg, 0.82 mmol) in N,N-dimethylformamide (5 mL) was added

4-dimethylaminopyridine (110 mg, 0.90 mmol) at rt. The resulting

mixture was stirred at 90 °C for 16 h, cooled to rt, and concentrated

to dryness. The residue thus obtained was puriﬁed by preparative

HPLC to aﬀord the title compound as a white solid (50 mg, 14%

tert-Butyl (3-Methyl-4-oxo-3,4-dihydroquinazolin-6-yl)-

carbamate (13a). Into a pressure ﬂask were weighed 6-bromo-3-

methyl-quinazolin-4-one (4.456 g, 18.64 mmol), tris-

(dibenzylideneacetone)dipalladium(0) (427 mg, 0.466 mmol), 4,5-

bis(diphenylphosphino)-9,9-dimethylxanthene (539 mg, 0.932

mmol), tert-butyl carbamate (3.34 g, 28.0 mmol), and cesium

carbonate (12.15 g, 37.28 mmol). Under a stream of nitrogen gas, the

vessel was charged with anhydrous tetrahydrofuran (93 mL) and the

pressure ﬂask was sealed. The reaction mixture was stirred at 80 °C

for 66 h. After cooling to rt, the mixture was ﬁltered through Celite

and rinsed with dichloromethane. The mother liquor was

concentrated nearly to dryness and then ﬁltered. The solid thus

collected was rinsed with heptane and a small amount of isopropyl

acetate to aﬀord the title compound as a white solid (4.877 g, 95%).

¹H NMR (400 MHz, DMSO-d₆): δ 9.75 (s, 1H), 8.35 (d, J = 2.5 Hz,

1

yield, 97% HPLC purity). H NMR (400 MHz, DMSO-d₆): δ 9.46

(br s, 2H), 8.52 (d, J = 8.4 Hz, 1H), 8.19 (s, 1H), 8.09 (s, 1H), 7.27

(br s, 1H), 7.04 (d, J = 11.6 Hz, 1H), 6.43−6.40 (m, 1H), 3.36 (s,

3H), 3.11−3.06 (m, 2H), 2.18 (s, 3H), 1.36−1.32 (m, 2H), 0.90 (s,

9H).

tert-Butyl (5-(3-(3,3-Dimethylbutyl)ureido)-4-ﬂuoro-2-

methylphenyl)carbamate (9b). Into a pressure ﬂask were weighed

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1H), 8.25 (s, 1H), 7.79 (dd, J = 8.9, 2.5 Hz, 1H), 7.59 (d, J = 8.9 Hz,

1H), 3.48 (s, 3H), 1.50 (s, 9H).

pressure, and the residue was puriﬁed by column chromatography on

silica gel to aﬀord the title compound (48.9 g, 86%). H NMR (400

1

1-(3,3-Dimethylbutyl)-3-(2-ﬂuoro-4-methyl-5-((3-methyl-4-oxo-

3,4-dihydroquinazolin-6-yl)amino)phenyl)urea (6). A solution of

tert-butyl (3-methyl-4-oxo-3,4-dihydroquinazolin-6-yl)carbamate (722

mg, 2.63 mmol) in triﬂuoroacetic acid (8.8 mL) was stirred for 1 h

and then concentrated to a white solid. Onto this solid were weighed

1-(5-bromo-2-ﬂuoro-4-methylphenyl)-3-(3,3-dimethylbutyl)urea

(869 mg, 2.63 mmol), chloro[2-(dicyclohexylphosphino)-3,6-dime-

thoxy-2′,4′,6′-triisopropyl-1,1′-biphenyl][2-(2-aminoethyl)phenyl]-

palladium(II) methyl-tert-butyl ether adduct (107 mg, 0.131 mmol),

2-(dicyclohexylphosphino)-3,6-dimethoxy-2′,4′,6′-triisopropyl-1,1′-

biphenyl (74.2 mg, 0.131 mmol), and sodium tert-butoxide (1.01 g,

10.5 mmol). Under a stream of nitrogen gas, the vessel was charged

with anhydrous 1,4-dioxane (26 mL) and the pressure ﬂask was

sealed. The reaction mixture was stirred at 100 °C for 19 h before

cooling to rt. After concentration, the crude residue was puriﬁed by

ﬂash chromatography (100:0 to 90:10 CH₂Cl₂/MeOH). The

fractions containing the product were concentrated to dryness and

then triturated with dichloromethane. After ﬁltration, the white solid

was washed with isopropyl acetate to aﬀord the title compound as a

MHz, DMSO-d₆): δ 9.24 (s, 1H), 8.31 (s, 1H), 8.01 (t, J = 8.4 Hz,

1H), 7.44 (dd, J = 1.2, 8.8 Hz, 1H), 3.44 (s, 3H), 1.48 (s, 9H).

6-Amino-5-ﬂuoro-3-methylquinazolin-4(3H)-one (14e). To a

solution of tert-butyl 5-ﬂuoro-3-methyl-4-oxo-3,4-dihydroquinazolin-

6-ylcarbamate (100 g, 0.340 mol) in ethyl acetate (1000 mL) was

added hydrogen chloride (1000 mL, 4 mol, [4.0 M in ethyl acetate]).

The reaction mixture was stirred for 12 h and then ﬁltered. The solid

was collected, suspended in water, and neutralized with aqueous

sodium bicarbonate. The resulting solid was collected by ﬁltration and

1

dried to aﬀord the title compound (57.6 g, 88%). H NMR (400

MHz, DMSO-d₆): δ 8.06 (s, 1H), 7.24−7.27 (m, 2H), 5.54 (br s,

2H), 3.41 (s, 3H).

1-(3,3-Dimethylbutyl)-3-(2-ﬂuoro-5-((5-ﬂuoro-3-methyl-4-oxo-

3,4-dihydroquinazolin-6-yl)amino)-4-methylphenyl)urea (GNE-

0749, 7). Into a pressure ﬂask were weighed 6-amino-5-ﬂuoro-3-

methylquinazolin-4(3H)-one (0.583 g, 3.02 mmol), palladium(II)

acetate (34.0 mg, 0.151 mmol), 4,5-bis(diphenylphosphino)-9,9-

dimethylxanthene (87.3 mg, 0.151 mmol), 1-(5-bromo-2-ﬂuoro-4-

methylphenyl)-3-(3,3-dimethylbutyl)urea (1.00 g, 3.02 mmol),

cesium carbonate (2.95 g, 9.07 mmol), and 3 Å molecular sieves

(2.50 g). Under a stream of nitrogen gas, the vessel was charged with

anhydrous 1,4-dioxane (15 mL) and the pressure ﬂask was sealed.

The reaction mixture was stirred at 100 °C for 19 h. After cooling to

rt, the mixture was ﬁltered and rinsed with dichloromethane and

methanol. The mother liquor was concentrated, and the residue was

puriﬁed by ﬂash chromatography (100:0 to 90:10 CH₂Cl₂/MeOH).

This sequence was repeated with double the amount of reactants and

without molecular sieves, and the two runs were combined for ﬁnal

puriﬁcation via preparative HPLC to aﬀord the title compound as a

white solid (1.369 g, 34% yield, >99% HPLC purity). mp 215 °C. ¹H

NMR (400 MHz, DMSO-d₆): δ 8.17−8.13 (m, 2H), 7.89 (d, J = 7.9

Hz, 1H), 7.51 (d, J = 2.0 Hz, 1H), 7.31 (d, J = 8.7 Hz, 1H), 7.14−

7.00 (m, 2H), 6.44 (t, J = 5.5 Hz, 1H), 3.44 (s, 3H), 3.11−2.98 (m,

2H), 2.09 (s, 3H), 1.38−1.24 (m, 2H), 0.88 (s, 9H). ¹³C{¹H} NMR

(101 MHz, DMSO-d₆): δ 157.7 (d, J_C−F= 3.0 Hz), 154.6, 148.2 (d,

1

white solid (889 mg, 80% over two steps, >99% HPLC purity). H

NMR (400 MHz, DMSO-d₆): δ 8.17 (d, J = 2.1 Hz, 1H), 8.11 (s,

1H), 8.03 (d, J = 8.0 Hz, 1H), 7.90 (br s, 1H), 7.50 (d, J = 8.8 Hz,

1H), 7.27 (dd, J = 8.8, 2.7 Hz, 1H), 7.21 (d, J = 2.6 Hz, 1H), 7.11 (d,

J = 12.2 Hz, 1H), 6.46 (t, J = 5.5 Hz, 1H), 3.44 (s, 3H), 3.08−3.01

(m, 2H), 2.09 (s, 3H), 1.38−1.27 (m, 2H), 0.88 (s, 9H).

6-Amino-3-bromo-2-ﬂuorobenzoic Acid Hydrobromide (14a).

To a solution of 2-amino-6-ﬂuorobenzoic acid (100 g, 0.640 mol) in

methanol (1500 mL) at −78 °C was added bromine (103 g, 0.640

mol), and the reaction mixture was stirred for 2 h. The reaction was

quenched with ice−water (800 mL) and aqueous sodium thiosulfate

(670 mL), and the precipitate was collected by ﬁltration to aﬀord a

brown solid. The brown solid was slurried in ethyl acetate (500 mL)

1

and then ﬁltered to aﬀord the title compound (89 g, 44%). H NMR

(400 MHz, DMSO-d₆): δ 7.40 (dd, J = 7.6, 9.2 Hz, 1H), 6.56 (dd, J =

1.2, 9.2 Hz, 1H).

J

_C−F= 237.4 Hz), 147.5 (d, J_C−F= 259.6 Hz), 145.6, 141.0, 135.6 (d,

_C−F= 3.0 Hz), 132.6 (d, J_C−F= 10.8 Hz), 126.3 (d, J_C−F= 11.4 Hz),

6-Amino-3-bromo-2-ﬂuoro-N-methylbenzamide (14b). To a

solution of 6-amino-3-bromo-2-ﬂuorobenzoic acid hydrobromide

(80.6 g, 0.260 mol) and methylamine hydrochloride (86.3 g, 1.28

mol) in N,N-dimethylformamide (300 mL) were added triethylamine

(258 g, 2.55 mol) and HATU (194 g, 0.510 mol). The reaction

mixture was stirred overnight, then cooled to 0 °C, and quenched

with saturated aqueous ammonium chloride (500 mL). The resulting

mixture was extracted with dichloromethane (500 mL × 3), and the

combined organic layers were washed with water (500 mL × 3), dried

over anhydrous sodium sulfate, and concentrated under reduced

pressure. The residue was puriﬁed by column chromatography on

silica gel (petroleum ether/EtOAc = 4:1) to aﬀord the title compound

125.4 (d, J_C−F= 7.2 Hz), 122.9 (d, J_C−F= 4.0 Hz), 122.2 (d, J_C−F

=

5.5 Hz), 116.3 (d, J_C−F= 18.5 Hz), 115.4, 111.3 (d, J_C−F= 2.8 Hz),

43.3, 35.5, 33.2, 29.4, 29.2, 16.9. ¹⁹F NMR (377 MHz, DMSO-d₆): δ

−130.8 (s, 1F), −136.1 (s, 1F). HRMS m/z: [M + H]⁺calcd for

C₂₃H₂₇F₂N₅O₂, 444.2206; found, 444.2212.

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c02085.

■

sı

1

as a white solid (36.5 g, 58%). H NMR (400 MHz, DMSO-d₆): δ

Molecular formula strings of compounds 1−14 (CSV)

Crystallographic data of compound 6 (CIF)

Crystallographic data of compound 7 (CIF)

8.31 (d, J = 3.6 Hz, 1H), 7.30 (dd, J = 8.0, 8.8 Hz, 1H), 6.49 (dd, J =

1.2, 8.8 Hz, 1H), 5.95 (br s, 2H), 2.74 (d, J = 4.8 Hz, 3H).

6-Bromo-5-ﬂuoro-3-methylquinazolin-4(3H)-one (14c). To a

mixture of 6-amino-3-bromo-2-ﬂuoro-N-methylbenzamide (75 g,

0.30 mol) in triethyl orthoformate (450 mL) was added hydrogen

chloride (75 mL, 300 mmol, [4.0 M in 1,4-dioxane]). The reaction

mixture was stirred at 110 °C for 3 h and then cooled to rt. The

precipitate was collected by ﬁltration and dried to aﬀord the title

compound (54 g, 69%). ¹H NMR (400 MHz, CDCl₃): δ 8.05 (s, 1H),

7.87 (dd, J = 6.8, 8.8 Hz, 1H), 7.42 (dd, J = 1.6, 8.8 Hz, 1H), 3.58 (s,

3H).

tert-Butyl 5-Fluoro-3-methyl-4-oxo-3,4-dihydroquinazolin-6-yl-

carbamate (14d). To a nitrogen-sparged suspension of 6-bromo-5-

ﬂuoro-3-methylquinazolin-4(3H)-one (50 g, 0.19 mol) and tert-butyl

carbamate (27.3 g, 0.230 mol) in toluene (1000 mL) were added

tris(dibenzylideneacetone)dipalladium(0)-chloroform adduct (4.03 g,

3.90 mmol), 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (6.75

g, 11.7 mmol), and cesium carbonate (76.1 g, 0.230 mol). The

reaction mixture was stirred at 90 °C overnight before being cooled to

rt and ﬁltered. The mother liquor was concentrated under reduced

Detailed synthetic chemistry schemes, NMR and HPLC

spectra for compound 7, small-molecule X-ray crystal

structures and statistics for compounds 6 and 7, detailed

kinase selectivity panel data for compounds 1−7,

crystallographic statistics for protein cocrystal structure

of 7, data on synergistic eﬀects of GNE-0749 +

cobimetinib and nonsynergistic eﬀects of vemurafenib

+ cobimetinib in KRAS mutant cell line, HCT116

(PDF)

AUTHOR INFORMATION

Corresponding Authors

■

Michael Siu − Discovery Chemistry, Genentech, Inc., South

San Francisco, California 94080, United States;

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J. Med. Chem. 2021, 64, 3940−3955

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Article

Notes

orcid.org/0000-0002-2822-6584; Phone: +1-650-467-

7764; Email: siu.michael@gene.com

The authors declare the following competing ﬁnancial

interest(s): All authors are employees/former employees of

Genentech, Inc. and may hold stock in Roche Holding AG. All

studies were funded by Genentech, Inc.

Coordinates and structure factors for the BRAF domain

complex are available in the PDB with accession code 7K0V

(7). The authors will release the atomic coordinates and

experimental data upon article publication.

Joachim Rudolph − Discovery Chemistry, Genentech, Inc.,

South San Francisco, California 94080, United States;

Phone: +1-650-467-8867; Email: rudolph.joachim@

gene.com

Authors

Malcolm P. Huestis − Discovery Chemistry, Genentech, Inc.,

South San Francisco, California 94080, United States;

orcid.org/0000-0002-6038-3838

Darlene Dela Cruz − Translational Oncology, Genentech, Inc.,

South San Francisco, California 94080, United States

Antonio G. DiPasquale − Small Molecule Pharmaceutical

Sciences, Genentech, Inc., South San Francisco, California

94080, United States

ACKNOWLEDGMENTS

■

M.P.H. thanks Kim Huard for support and encouragement.

Kinase selectivity data courtesy of SelectScreen service from

Life Technologies, a Thermo Fisher company, Madison, WI,

USA. We thank the staﬀ at beamline 5.0.2 of the Advanced

Light Source for assistance during data collection and Xiaolin

Zhang (Genentech, Inc.) for BA analysis.

Matthew R. Durk − Drug Metabolism and Pharmacokinetics,

Genentech, Inc., South San Francisco, California 94080,

United States

ABBREVIATIONS USED

■

ASD, amorphous spray-dried dispersion; CL, clearance;

DELFIA, dissociation-enhanced lanthanide ﬂuorescence im-

munoassay; DFG, Asp−Phe−Gly; ERK, extracellular signal-

regulated kinase; F, bioavailability; HCT, human colorectal

cancer cells; IV, intravenous; KRAS, Kirsten rat sarcoma; log

D, log of distribution coeﬃcient; MDCK, Madin−Darby

canine kidney cell; MEK, mitogen-activated protein kinase

kinase; MAPK, mitogen-activated protein kinase; MCT,

methylcellulose-tween; mp, melting point; MS, mass spec-

trometry; Ni-NTA, nickel-nitrilotriacetic acid; P_app, perme-

ability coeﬃcient; PBS, phosphate-buﬀered saline; PEG,

polyethylene glycol; PK, pharmacokinetic; PKPD, pharmaco-

kinetic/pharmacodynamic; PO, per os (oral); RAF, rapidly

accelerated ﬁbrosarcoma; SAR, structure−activity relationship;

TCEP, tris(2-carboxyethyl)phosphine; TGI, tumor growth

inhibition; TPSA, topological polar surface area; V_dss, volume

of distribution

Charles Eigenbrot − Structural Biology, Genentech, Inc.,

South San Francisco, California 94080, United States

Paul Gibbons − Discovery Chemistry, Genentech, Inc., South

San Francisco, California 94080, United States

Alberto Gobbi − Discovery Chemistry, Genentech, Inc., South

San Francisco, California 94080, United States

Thomas L. Hunsaker − Translational Oncology, Genentech,

Inc., South San Francisco, California 94080, United States

Hank La − Drug Metabolism and Pharmacokinetics,

Genentech, Inc., South San Francisco, California 94080,

United States

Dennis H. Leung − Small Molecule Pharmaceutical Sciences,

Genentech, Inc., South San Francisco, California 94080,

United States

Wendy Liu − Discovery Chemistry, Genentech, Inc., South San

Francisco, California 94080, United States

Shiva Malek − Molecular Oncology, Genentech, Inc., South

San Francisco, California 94080, United States

Mark Merchant − Translational Oncology, Genentech, Inc.,

South San Francisco, California 94080, United States

John G. Moﬀat − Biochemical and Cellular Pharmacology,

Genentech, Inc., South San Francisco, California 94080,

United States

Christine S. Muli − Small Molecule Pharmaceutical Sciences,

Genentech, Inc., South San Francisco, California 94080,

United States

Christine J. Orr − Translational Oncology, Genentech, Inc.,

South San Francisco, California 94080, United States

Brendan T. Parr − Discovery Chemistry, Genentech, Inc.,

South San Francisco, California 94080, United States

Frances Shanahan − Molecular Oncology, Genentech, Inc.,

South San Francisco, California 94080, United States

Christopher J. Sneeringer − Biochemical and Cellular

Pharmacology, Genentech, Inc., South San Francisco,

California 94080, United States

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Weiru Wang − Structural Biology, Genentech, Inc., South San

Francisco, California 94080, United States

Ivana Yen − Molecular Oncology, Genentech, Inc., South San

Francisco, California 94080, United States

Jianping Yin − Structural Biology, Genentech, Inc., South San

Francisco, California 94080, United States

Complete contact information is available at:

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J. Med. Chem. 2021, 64, 3940−3955

Article Doi

Targeting KRAS Mutant Cancers via Combination Treatment: Discovery of a 5-Fluoro-4-(3 H)-quinazolinone Aryl Urea pan-RAF Kinase Inhibitor

DOI: 10.1021/acs.jmedchem.0c02085

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Full text of DOI:10.1021/acs.jmedchem.0c02085

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