Potent and selective aminopyrimidine-based B-Raf inhibitors with favorable physicochemical and pharmacokinetic properties

Article abstract of DOI:10.1021/jm300016v

Recent clinical data provided proof-of-concept for selective B-Raf inhibitors in treatment of B-Raf^V600E mutant melanoma. Pyrazolopyridine-type B-Raf inhibitors previously described by the authors are potent and selective but exhibit low solubility requiring the use of amorphous dispersion-based formulation for achieving efficacious drug exposures. Through structure-based design, we discovered a new class of highly potent aminopyrimidine-based B-Raf inhibitors with improved solubility and pharmacokinetic profiles. The hinge binding moiety possesses a basic center imparting high solubility at gastric pH, addressing the dissolution limitation observed with our previous series. In our search for an optimal linker-hinge binding moiety system, amide-linked thieno[3,2-d]pyrimidine analogues 32 and 35 (G945), molecules with desirable physicochemical properties, emerged as lead compounds with strong efficacy in a B-Raf^V600E mutant mouse xenograft model. Synthesis, SAR, lead selection, and evaluation of key compounds in animal studies will be described.

Full text of DOI:10.1021/jm300016v

Article
pubs.acs.org/jmc
Potent and Selective Aminopyrimidine-Based B-Raf Inhibitors with
Favorable Physicochemical and Pharmacokinetic Properties
Simon Mathieu,† Stefan N. Gradl,† Li Ren,‡ Zhaoyang Wen,† Ignacio Aliagas,† Janet Gunzner-Toste,†
Wendy Lee,† Rebecca Pulk,† Guiling Zhao,† Bruno Alicke,† Jason W. Boggs,† Alex J. Buckmelter,‡
Edna F. Choo,† Victoria Dinkel,‡ Susan L. Gloor,‡ Stephen E. Gould,† Joshua D. Hansen,‡
Gregg Hastings,‡ Georgia Hatzivassiliou,† Ellen R. Laird,‡ David Moreno,‡ Yingqing Ran,†
Walter C. Voegtli,‡ Steve Wenglowsky,‡ Jonas Grina,‡ and Joachim Rudolph*,†
†Genentech, Inc., 1 DNA Way, South San Francisco, California 94080-4990, United States
‡Array BioPharma, 3200 Walnut Street, Boulder, Colorado 80301, United States
S
* Supporting Information
ABSTRACT: Recent clinical data provided proof-of-concept for selective B-Raf
inhibitors in treatment of B-RafV600E mutant melanoma. Pyrazolopyridine-type B-Raf
inhibitors previously described by the authors are potent and selective but exhibit low
solubility requiring the use of amorphous dispersion-based formulation for achieving
efficacious drug exposures. Through structure-based design, we discovered a new class
of highly potent aminopyrimidine-based B-Raf inhibitors with improved solubility and
pharmacokinetic profiles. The hinge binding moiety possesses a basic center imparting high solubility at gastric pH, addressing
the dissolution limitation observed with our previous series. In our search for an optimal linker-hinge binding moiety system,
amide-linked thieno[3,2-d]pyrimidine analogues 32 and 35 (G945), molecules with desirable physicochemical properties,
emerged as lead compounds with strong efficacy in a B-RafV600E mutant mouse xenograft model. Synthesis, SAR, lead selection,
and evaluation of key compounds in animal studies will be described.
Plexxikon,8 members of this series bind to the active
conformation of the kinase (DFG-in) and induce a character-
istic outward shift of the αC-helix. An essential structural factor
INTRODUCTION
The Raf enzyme family of protein kinases are members of the
mitogen activated protein kinase (MAPK) cascade, a key
■
pathway regulating cell proliferation, invasion, and survival.1
of this shift is a sulfonamide alkyl tail residue opening a
lipophilic pocket that accommodates said alkyl group, imparting
Through specific mutations, members of this pathway can
become constitutively active, leading to uncontrolled cell
growth. A frequent aberration in human cancers is a
substitution of a glutamic acid for valine at position 600 of
high kinase selectivity.8 Lead compounds 2 and 3 emerged as
potent inhibitors and exhibited robust efficacy in B-RafV600E
xenograft mouse models. However, the low solubility of 2 and 3
the Raf family member B-Raf (V600E).2 This mutation is
precluded the use of crystalline suspension formulations for
covering the dose ranges required for efficacy and toxicology
particularly prominent in melanoma, where it occurs with a
frequency of ∼60%.3 On the basis of epidemiology and
preclinical target validation, B-Raf has been recognized as an
excellent target opportunity for cancer treatment. In fact, recent
studies, and we resorted to amorphous spray-dried dispersion
formulation.7 Correspondingly, the sulfonamide-type clinical B-
Raf inhibitor vemurafenib (1) was reformulated as micro-
precipitated bulk powder (MBP) during phase 1 clinical studies
clinical data revealed a remarkable response of selective B-Raf
to achieve efficacious drug exposure levels.4,9 While amorphous
inhibitors, and vemurafenib (PLX4032/RG7204, Plexxikon/
dispersion formulations are increasingly being used for the
Roche, 1) recently received FDA approval for the treatment of
development of drugs with low solubility,10 traditional solid
dosage using crystalline drug remains the most straightforward
and preferred means of drug formulation. The study presented
in this account describes our efforts in identifying inhibitors
with improved physicochemical properties that would allow
dosage as crystalline suspension.
melanoma.4−6
Table 1 shows selected physicochemical data of compound 2.
The pKa of its most basic center at the hinge portion is low,
rendering this and related compounds largely unprotonated at
We recently reported the discovery of selective, orally
bioavailable, and efficacious inhibitors of B-RafV600E that are
based on a novel 3-methoxypyrazolopyridine hinge binding
template.7 Similarly to compounds previously reported by
Received: January 4, 2012
Published: February 15, 2012
© 2012 American Chemical Society
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Table 1. Measured Physicochemical Properties of
Compound 2
RESULTS AND DISCUSSION
■
Synthetic Chemistry. Representatives of the urea series
a
were synthesized as described in Schemes 1, 2, and 3. The
benzoic acid intermediate 67 was converted into the
corresponding amine 7 via a Curtius rearrangement reaction.
Coupling with carbamate 5 furnished the urea derivative 8,
which was subsequently reacted with ammonia (9a) or primary
amines 9b and 9c to form the target compounds 10−12. The
N-methyl urea analogue 15 was accessed via conversion of
benzoic acid 6 into its isocyanate derivative, coupling with an
appropriate methylaminopyrimidine 13, and further trans-
formation into the corresponding primary amine 15. The
pyrazolopyrimidine analogues 18 and 19 were obtained by a
similar procedure, however the isolatable phenyl carbamate 16
was used for the formation of the urea bond.
The chemistry used to synthesize 4-aminoquinazoline or 4-
aminothienopyrimidine core derivatives is described in
Schemes 4 and 5. For the 4-aminoquinazoline core (Scheme
4), 2-aminoisophtalic acid 20 was condensed with formamide
to afford benzoic acid intermediate 21, which was bis-
chlorinated and coupled with anilines 7a−7d. The resulting
chloroquinazolines 23a−d were then treated with ammonia to
obtain 24−27. For the synthesis of 4-aminothieno[3,2-
d]pyrimidine compounds (Scheme 5), precursor 28 was first
brominated at the 7-position, followed by a carbonylation in
methanol to generate the corresponding adduct 29. Con-
densation with phosphorus oxychloride, followed by hydrolysis
of the ester moiety, afforded acid 30. Similar chemistry as used
for scaffold A was performed to obtain 32−35.
Lead Optimization and Structure−Activity Relation-
ships. Following our design hypothesis explained in the
introduction, we embarked on the synthesis of urea linker
analogues, and, gratifyingly, prototype compounds, exemplified
by 10 and 19, were found to have higher potencies than their
“amide series” counterparts 36 and 2 (Figure 3). To validate
our design hypothesis, we attempted cocrystallization of
selected urea analogues in B-Raf and succeeded with compound
12 (Figure 2). As hypothesized by molecular modeling, the 4-
aminopyrimidine moiety forms similar hydrogen bond contacts
with the hinge residue Cys532 as the previous lead compound
2.7 The intramolecular hydrogen bond between the distal linker
urea −NH and the pyrimidine −N enforces a coplanar
arrangement of this moiety, and analogous to the cocrystal
structure of 2 in B-Raf, the hinge binding plane is oriented
forming an torsion angle of >60° relative to the central 2,6-
disubstituted phenyl ring (65.3°). Same as 2, the −NH and
−SO2 portions of the sulfonamide are interacting with Asp594
pKa (most pKa (most
mp
[°C]
thermodynamic solubility
b
a
logP
2.5
basic)
acidic)
[μg/mL], pH 1.2, 6.5, 7.4
0.7
7.5
226
3/4/9
a
b
Dissociation constant of the corresponding conjugated acid. Data
for highest melting crystalline polymorphs obtained in our hands.
gastric and intestinal tract pH values. In fact, compounds from
this series are weak acids as a consequence of a sulfonamide
−NH linked to an electron-deficient aromatic ring. Further-
more, melting points are typically high, indicating strong crystal
lattice forces.11 The combination of these factors likely
accounts for the low solubility.
Focus of our strategy on improving solubility was the
introduction of a basic center without adding significant
molecular weight. We preferred to maintain the sulfonamide
portion of the molecule due to its critical role in inferring kinase
selectivity and instead attempted to make key modifications at
the hinge binding portion of the molecule.
Two characteristic conformational features of the pyrazolo-
pyridine series are a >60° torsion angle of the amide moiety
relative to the 2,6-dihalo substituted central phenyl ring and a
near coplanar arrangement of the amide moiety with the hinge
binding heterocycle, a typical disposition of acylated anilines.12
The central phenyl ring and the hinge binding heterocycle are
in a near orthogonal spatial arrangement, and our goal was to
mimic this arrangement with an alternative linker−hinge binder
unit combination. This led us to the design of urea-linked
pyrimidine scaffolds that are geometrically held in place via an
intramolecular hydrogen bond (Figure 1). A similar pseudoring
approach has been employed by others to replace a bicyclic
core framework (e.g., pyrido[2,3-d]pyrimidin-7-one),13,14 but
to our knowledge, this approach had not been attempted to
mimic an acylated aniline moiety.
We synthesized such urea-linked pyrimidine derivatives and
found them indeed to have comparable activities to
corresponding representatives of the first generation amide
series; cocrystal structure analysis revealed a binding mode as
hypothesized. Chemical stability problems prompted us to
abandon the urea series and led us to the development of the
related “reverse amide” linked 4-aminoquinazoline and 4-
aminothienopyrimidine series, providing superior overall
profiles. The following account will describe the development
of these series and the synthesis and biological evaluation of
selected compounds.
Figure 1. Structure models to illustrate the rescaffolding approach applied to the pyrazolopyridine series containing an amide linker unit (left).
Rescaffolding using a urea linker constrained through an intramolecular hydrogen bond (right) maintains the near coplanarity of the linker−hinge
binding plane and a near 90° torsion angle between this moiety and the connecting aryl unit. The red dotted line represents an intramolecular
hydrogen bond.
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a
Scheme 1. Synthesis of Aminopyrimidine Substituted Urea Analogues 10−12
a
Reagents and conditions: (a) phenylchloroformate, Cs2CO3, THF, 60 °C, 20 h, 37%; (b) diphenylphosphonic azide, NEt3, THF, 80 °C, 55%; (c) 5,
DCE, 90 °C, 75%; (d) 9a, 9b, or 9c, THF or DCE, 60−80 °C, 35−70%.
a
Scheme 2. Synthesis of Aminopyrimidine Substituted Methylurea Analogue 15
a
Reagents and conditions: (a) diphenylphosphonic azide, NEt3, THF, 80 °C, then 13, 25%; (b) 7 M NH3 in MeOH, rt, 40%.
a
Scheme 3. Synthesis of Pyrazolopyrimidine Substituted Urea Analogues 18 and 19
a
Reagents and conditions: (a) diphenylphosphonic azide, NEt3, dioxane, rt, then phenol, 100 °C, 5%; (b) 17a or 17b, DMSO, 80 °C, 5 h, 30−45%.
and Phe595-Gly596, respectively, and the propyl tail moiety
fills a narrow lipophilic pocket.
the replacement of the urea linker with a more stable amide-
linked 4-aminoquinazoline core. To our delight, prototype
“reverse amide” 24 displayed excellent enzyme and cellular
potencies without any chemical stability issues at low pH.16
Our premise was the installation of a basic center to address
the low solubility exhibited by the previous series, represented
here by compounds 2 and 3. As shown in Table 2, the
aminoquinazoline moiety of 24 provides a basic center with a
considerably higher pKa (4.45, measured data)17 compared to
the parent compound 2 (pKa = 0.7, measured data);
consequently, the solubility of 24 at pH 1.2 was substantially
increased, with a value of >1000 μg/mL (the solubility at pH
6.5 and 7.4 remained low at 1 μg/mL). As demonstrated later,
this translated into benefits in in vivo studies.
Upon repeated cell assay testing of one of the most potent
compounds in the urea series, 19, we noticed a continuous loss
of activity. Dedicated stability testings indeed revealed chemical
degradation that was particularly rapid under acidic conditions.
We believe that the intramolecular hydrogen bond is the prime
reason for the observed instability; indeed related carbamoyl
systems have been utilized as readily cleavable protecting
groups of nucleobases.15 This prompted us to abandon this
series.16
We then explored structural alternatives that maintain the
geometrical arrangement including the intramolecular hydro-
gen bond at the hinge binding region. One of our attempts was
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a
Scheme 4. Synthesis of 4-Aminoquinazoline analogues 24−27
a
Reagents and conditions: (a) formamide, formamidine acetate, 170 °C, 78%; (b) SOCl2, DMF, 77%; (c) 7a−d, pyridine, MgSO4, CHCl3, 22−76%;
(d) 2 M NH3/i-PrOH, 105 °C (microwave), 68−80%.
a
Scheme 5. Synthesis of 4-Aminothieno[3,2-d]pyrimidine Compounds 32−35
a
Reagents and conditions: (a) Br2, AcOH, 100 °C, 60%; (b) PdCl2(dppf)·DCM, Et3N, MeOH, CO (300 psi), 120 °C, 80%; (c) POCl3, reflux, 96%;
(d) LiOH, THF/H2O, rt, 81%; (e) oxalyl chloride, DMF (cat.), THF; (f) 22a−d, THF, rt, 61−100% for 2 steps; (g) 2 M NH3/i-PrOH, 95 °C
(thermal) or 110 °C (microwave), 61−89%.
Encouraged by the improved properties and potency of this
new scaffold, we explored alternative ring systems. In these
studies, 4-aminothieno[3,2-d]pyrimidine analogues, as repre-
sented by 32, emerged as promising leads with comparable
features to the aminoquinazolines. Compound 32 achieved
potencies of 3 and 15 nM, respectively, in the biochemical and
cellular assay (Table 3), and we succeeded in obtaining a
cocrystal structure of this compound with B-Raf (Figure 4).
Similar to the aminopyrimidine hydrogen bond contacts
observed in the B-Raf cocrystal structure of the urea analogue
12, the 4-aminothieno[3,2-d]pyrimidine moiety binds to the
hinge residue Cys532. Furthermore, this cocrystal structure
shared all the hallmark features observed in the cocrystal
structure of 12: (a) intramolecular hydrogen bond between the
linker amide −NH and the pyrimidine −N enforcing a coplanar
arrangement of this moiety, (b) hinge binding plane oriented in
a near orthogonal fashion relative to the central 2,6-
disubstituted phenyl ring (torsion angle of 82°), (c) −NH
and −SO2 portions of the sulfonamide interacting with Asp594
and Phe595-Gly596, respectively, (d) propyl tail moiety filling a
narrow lipophilic pocket. In alignment with kinase selectivity
profiles of other sulfonamide-type B-Raf inhibitors, 32
displayed high selectivity when tested against a large kinase
panel, showing >100-fold selectivity for 268/273 tested
kinases.18
Further SAR studies focused on the modification of the
central ring and tail group, and selected data are shown in Table
3. Varying the halogen groups at the −2 and −6 positions on
both scaffolds had subtle effects on enzyme and cellular
potencies. Interestingly, compounds with X = Cl were
consistently more potent than analogues with X = F in the
biochemical assay but showed a noticeably larger shift from
biochemical to cellular assay than the latter. We do not know if
this is due to differences in cell permeability or other reasons.
Placement of a fluorine at the end of the propyl tail group, as
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of the series described here would circumvent this limitation.
All compounds shown in Table 4 were dosed as crystalline
suspensions, and to our delight, oral bioavailability was
consistently found to be high. As an example, compound 24
was also dosed in mice in solution and the resulting exposure
was nearly matched by the exposure from dosing as crystalline
suspension (AUC of 331 vs 290 μM·h). Among the two
scaffolds investigated, we found the thieno[3,2-d]pyrimidine
compounds to exhibit higher oral exposure than their
quinazoline counterparts (Table 4). We account this to both
the lower clearance and better thermodynamic solubility at
neutral pH observed for the former series.
Extensive profiling of the compounds described above led us
to select 35 (G945), a thieno[3,2-d]pyrimidine analogue, for
advanced studies. 35 displays high overall kinase selectivity
when tested in a large panel,18 selectivity for inhibition of B-
RafV600E vs WT B-Raf and WT C-Raf, and strong antiprolifer-
ative efficacy in B-RafV600E mutant cell lines (Table 5).
35 showed no significant CYP inhibition (CYP2C9 IC50
=
9 μM, IC50 of all other major CYPs > 25 μM) and no time-
dependent CYP inhibition potential. Furthermore, it did not
exhibit any significant activity in a broad receptor panel or
hERG channel activity and was Ames-negative.
Figure 2. X-ray crystal structure of 12 (in green) in complex with B-
RafV600E. The cleft surface is rendered in violet, and select residues are
depicted in white. Hydrogen bonding interactions are illustrated with
yellow dashed lines.
In vivo pharmacokinetic studies with compound 35 in
rodents revealed extremely low IV clearance, very low volume
of distribution, and excellent oral exposure after dosing as
crystalline suspension (Table 6). Exposure increase was near
dose-proportional in rat between 5 and 25 mg/kg and slightly
less than proportional in mouse between 1 and 50 mg/kg. We
do not believe that enterohepatic recirculation can explain the
high rodent bioavailability, as clearance of compound 35 in
both mice and rats was very low (less than 1% of hepatic blood
flow), and metabolite levels will be low in turn. This is also
supported by in vitro metabolite identification studies using rat
and mouse hepatocytes which showed only minimal glucur-
onide metabolite formation.
The strong in vitro and in vivo profile of 35 led us to proceed
to in vivo efficacy studies using the Colo205 xenograft mouse
model. For benchmarking purposes, we dosed compound 2 in
the same study. 35 was found to be both significantly more
potent and efficacious in this model (Figure 5). We believe that
this large difference results from both the higher biochemical
activity and superior exposure of 35 compared to compound 2.
shown in a recent publication by us,19 led to an increase in
enzyme and cellular potencies in both scaffolds.
Profiling of Advanced Compounds. The favorable in
vitro profiles prompted us to evaluate these compounds in vivo
and selected in vivo as well as physicochemical data are shown
in Table 4. Following IV dosing, all compounds exhibited very
low clearance (CL) and volume of distribution (Vss). Similar to
previously reported B-Raf inhibitors with related chemotypes,7
solubility at neutral pH for these and other compounds from
this series is low. We believe that this is resulting from strong
crystal lattice force, as suggested by high melting points.
However, a dramatic difference to the previous series is the
universally observed high solubility at pH 1.2, presumably
resulting from the presence of the pyrimidine portion with
increased pKa. Previous B-Raf inhibitors with related chemo-
types consistently produced low exposures when dosed as
crystalline suspensions, likely as a result of a dissolution
limitation.20 Our hope was that the high solubility at gastric pH
Figure 3. Conceptual path from the “amide” to the “reverse amide” series.
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Table 2. Potencies (Biochemical and Cellular) and Selected Measured Properties of Compounds Shown in Figure 2 and
Selected Compounds from the Urea Series
a,
b
b
,
c
de
,
g
series
amides
compd
B-RafV600E IC50 [nM]
pERK IC50 [nM]
measured pKa
solubility at pH 1.2 [μg/mL]
36
2
917
5
2400
19
nd
3
0.7
ureas
10
11
12
15
18
19
142
36
438
168
3400
240
88
nd
900
900
nd
7
64
83
5.6
1.6
f
1.8
4.5
49
reverse amides
24
2
10
>1000
a
b
c
d
Biochemical assay. Averages of at least three measurements. Cellular phosphorylation assay using B-RafV600E mutant Malme-3 M cell line. pKa
e
f
determination was performed at Sirius using the D-PAS technique. Dissociation constants of the corresponding conjugated acids. High variability
among different measurements with EC50 values ranging from 2.6 nM to 1.4 μM (n = 14). Thermodynamic solubility.
g
Table 3. Enzymatic and Cellular Potencies of 4-Aminoquinazoline and 4-Aminothieno[3,2-d]pyrimidine Scaffolds
ab
,
bc
,
compd
scaffold (A/B)
X
X′
R
B-RafV600E IC50 (nM)
pERK IC50 (nM)
24
25
26
27
32
33
34
35
A
A
A
A
B
B
B
B
F
F
H
H
H
F
1.8
9.5
9.5
7.7
2.5
15
Cl
F
F
0.59
0.54
0.11
3.2
Cl
F
Cl
F
F
H
H
H
F
Cl
F
F
0.9
19
Cl
1.1
15
Cl
F
0.18
4.6
a
b
c
Biochemical assay. Averages of at least three measurements. Cellular phosphorylation assay using B-RafV600E mutant Malme-3 M cell line.
It is especially noteworthy that the efficacy profile of compound
35 was achieved via dosing as crystalline suspension, while
compound 2 required formulation as amorphous dispersion.
Even with the help of an amorphous dispersion formulation,
compound 2 did not achieve >25% bioavailability at efficacious
doses in mice,7 which is in stark contrast to the high
bioavailability observed with crystalline suspensions of
compound 35.
CONCLUSION
■
In conclusion, we discovered a potent and selective B-Raf
inhibitor series that displays superior physicochemical charac-
teristics compared to other sulfonamide-type B-Raf inhibitors
previously reported by us. The key design step that led to the
identification of this series was the replacement of the amide
linked pyrazolopyridine hinge binding motif by a urea-linked
aminopyrimidine system that is constrained by an intra-
molecular hydrogen bond and provides equivalent planarity
of the hinge binding system compared to the previous series
studies therein. Further development of this idea led to the
identification of 4-aminoquinazoline and 4-aminothieno[3,2-
d]pyrimidine linked through an amide moiety. pKa of these
Figure 4. X-ray crystal structure of 32 (in green) in complex with B-
RafWT. The cleft surface is rendered in violet, and select residues are
depicted in white. Hydrogen-bonding interactions are illustrated with
yellow dashed lines.
systems is increased by several orders of magnitude affording
very good solubility at gastric pH and resulting in high
exposures after dosing as crystalline suspension, a key benefit
over sulfonamide-type B-Raf inhibitors previously reported by
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Table 4. Mouse Pharmacokinetic Profiles (IV, PO) and Selected Measured Physicochemical Data of Compounds 24, 32, and 35
a
a
b
c
d
CL
Vss
30 mg/kg PO AUC
mouse PPB
(%)
thermodynamic solubility pH
mp
(°C) logP
pKa (most
compd [mL/min/kg] [L/kg]
[μM·h]
30 mg/kg PO %F
1.2/6.5[μg/mL]
basic)
24
32
35
2.5
0.4
0.2
0.2
290
874
71
100
98.5
98.4
97.0
>1000/1
>1000/5
700/10
209
189
191
2.9
2.7
3.6
4.5
3.6
3.6
1.0
0.79
1680
>100
a
b
IV dose: 2.5 mg/kg. Compounds 24 and 32 dosed in 0.5% methylcellulose/0.2% Tween 80/99.3% water, compound 35 dosed in 20% HPBCD
c
d
vehicle (20% hydroxypropyl-β-cyclodextrin/80% water). Free-base form of highest melting crystalline polymorph found in our hands. Dissociation
constants of the corresponding conjugated acids.
Table 5. Biochemical Potencies of Compound 35 against B-
RafV600E and Wild-Type Raf Isoforms and Antiproliferative
Activities in Various Cell Lines
Adjusted Inhibitor IC50 at 1 mM ATP [nM]
B-RafV600E
WT B-Raf
WT C-Raf
2
a
22 (11-fold)
a
73 (152-fold)
Cellular Proliferation EC50 [nM]
b
A375-X1 (B-RafV600E
)
2
b
Colo205 (B-RafV600E
)
5
b
HT29 (B-RafV600E, EGFRhigh
RKO (B-RafV600E, EGFRhigh, PTENnull
)
24
2310
b
)
b
H226 (wt)
>10000
a
b
Selectivity of B-RafV600E vs shown wild-type Raf isoforms. Cell line
genotype.
the authors. Compound 35 emerged as a highly potent lead
compound with favorable overall properties and revealed both
remarkable potency and efficacy in the B-RafV600E mutant
Colo205 mouse xenograft in vivo model.
We did not detect adverse safety signals for compounds 32
and 35 in mouse efficacy studies, however, observations made
in higher species precluded the advancement of these
compounds, and we will report these data in due course.
Figure 5. Dose-ranging tumor growth inhibition studies in
subcutaneous Colo205V600E mouse xenograft model (qd × 21 d, po).
was performed on an ISCO system having prepacked silica gel
columns or on a Biotage model SP1 purification system running SPX
software with prepacked silica gel columns and UV detection at 220
and 254 nm. LC-MS experiments were performed on an Agilent 1100
HPLC coupled with an Agilent MSD mass spectrometer using ESI as
ionization source, employing two different methods (A and B).
Solvent A was water with 0.05% TFA and solvent B acetonitrile with
0.05% TFA. Method A: An Agilent ZORBAX SB-C18 30 mm × 2.1
mm column was used with a 0.4 mL/min flow rate. The gradient steps
consisted of 3−97% B over 7 min, holding 97% B for 1.5 min, followed
by equilibration for 1.5 min. Method B: An Agilent ZORBAX SB-C18
100 mm × 3.0 mm column was used with a 0.7 mL/min flow rate. The
gradient steps consisted of 2−98% solvent B over 25.5 min, holding
98% B for 2.5 min, followed by equilibration for 1.5 min. In both
methods, peaks were detected by UV absorbance at 220 and 254 nm,
and MS full scan was applied to all experiments. Melting points were
recorded on an electrothermal melting point apparatus, model 9100.
EXPERIMENTAL SECTION
■
Chemical Syntheses. The reactions set forth below were
conducted generally under a positive pressure of nitrogen or argon
or with a drying tube in anhydrous solvents, and the reaction flasks
were typically fitted with rubber septa for the introduction of
substrates and reagents via syringe or cannula. Glassware was oven-
dried and/or heat dried. All reagents and solvents were used without
further purification unless otherwise stated. Reactions were monitored
by either analytical TLC or analytical HPLC. Analytical TLC was
performed using glass plates precoated with silica gel (manufacturer:
EMD, Silica Gel 60 F254, 250 μm). Flash column chromatography
a
Table 6. PK Profile of Compound 35 in Rodents
dose [mg/kg]
mouse
rat
1 mg/kg IV
5 mg/kg PO
25 mg/kg PO
2.5 mg/kg IV
1 mg/kg PO
10 mg/kg PO
30 mg/kg PO
50 mg/kg PO
CL [mL/min/kg]
0.19
0.14
0.79
0.12
Vss [L/kg]
Cmax (PO) [μM]
AUC (PO) [μM·h]
%F
44.5
651
68
201
2970
62
7.93
84
73
162
1680
>100
249
3249
>100
867
>100
>100
a
Dosed as crystalline suspension, 20% HPBCD vehicle (20% hydroxypropyl-β-cyclodextrin/80% water).
2875
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1H NMR spectra were recorded on a Varian Mercury (400 MHz)
chloro-2-fluoro-3-(N-(propylsulfonyl)propylsulfonamido)benzoate
(5.5 g, 72%) as an oil that slowly solidified upon standing. H NMR
(CDCl3, 400 MHz) δ 7.28−7.45 (m, 7H), 5.42 (s, 2H), 3.58−3.66 (m,
2H), 3.43−3.52 (m, 2H), 1.08 (t, J = 8 Hz, 6H).
1
NMR spectrometer or on a Bruker AV III (400 or 500 MHz)
spectrometer. Chemical shifts are expressed in parts per million (ppm,
δ scale) using tetramethylsilane as the reference standard. When peak
multiplicities are reported, the following abbreviations are used: s
(singlet), d (doublet), t (triplet), q (quartet), m (multiplet), dd
(doublet of doublet), dt (doublet of triplet), br (broad). Coupling
constants are reported in hertz (Hz).
Step C: Benzyl 6-chloro-2-fluoro-3-(N-(propylsulfonyl)-
propylsulfonamido) benzoate (5.4 g, 10.98 mmol) was dissolved in
THF (100 mL) and 1 M aqueous KOH (100 mL). This mixture was
heated at reflux for 16 h and then allowed to cool to room
temperature. The mixture was then acidified to a pH of 2 with 2 M
aqueous HCl and extracted with EtOAc (2×). The extracts were
washed with water, dried over sodium sulfate, and concentrated to a
solid that was triturated with hexanes/ether to give 6-chloro-2-fluoro-
The purity of each compound that was synthesized and tested for
biological activity was ≥95% via HPLC analysis.
Phenyl 6-Chloropyrimidin-4-ylcarbamate (5). A 5 mL reaction
vial was charged with 6-chloropyrimidin-4-amine (4, 1043 mg, 8.05
mmol), phenylchloroformate (2.02 mL, 16.1 mmol), and cesium
carbonate (5246 mg, 16.1 mmol) in THF. The reaction vessel was
sealed and the mixture heated to 60 °C for 20 h. The solvent was
removed to afford phenyl 6-chloropyrimidin-4-ylcarbamate as yellow
solid (738 mg, 37%), which was used in the next step without further
purification. ESI-MS: m/z 250.1 (M + 1).
1
3-(propylsulfonamido)benzoic acid (2.2 g, 68%) as a solid. H NMR
(DMSO-d6, 400 MHz) δ 9.93 (s, 1H), 7.49 (t, J = 8 Hz, 1H), 7.38 (dd,
J = 8 Hz, 2 Hz, 1H), 3.11−3.16 (m, 2H), 1.68−1.78 (m, 2H), 0.97 (t, J
= 8 Hz, 3H).
Step D: N-(3-Amino-4-chloro-2-fluorophenyl)propane-1-sulfona-
mide 7b was made using a similar procedure as described for N-(3-
1
N-(3-Amino-2,4-difluorophenyl)propane-1-sulfonamide
(7a). To a solution of 2,6-difluoro-3-(propylsulfonamido)benzoic acid1
(4.078 g, 14.6 mmol) in THF (60 mL) was added triethylamine (4.68
mL, 33.59 mmol) and diphenylphosphonic azide (3.73 mL, 16.8
mmol). The reaction mixture was stirred at room temperature for 3 h
and then warmed to 80 °C for 2 h. Water (10 mL) was added and the
mixture stirred at 80 °C for 15 h. The reaction mixture was diluted
with 300 mL of EtOAc, and the organic layer was washed with
saturated aq NaHCO3 solution and brine. The solvent was removed
under reduced pressure and the residue purified silica gel column
chromatography eluting with 30/70 EtOAc/hexane to obtain of N-(3-
amino-2,4-difluorophenyl)propane-1-sulfonamide (2.03 g, 55%). 1H
NMR (400 MHz, DMSO-d6) δ 9.32 (s, 1H), 6.90−6.80 (m, 1H), 6.51
(td, J = 8.7 Hz, 5.5 Hz, 1H), 5.28 (s, 2H), 3.05−2.96 (m, 2H), 1.82−
1.64 (m, 2H), 1.01−0.90 (m, 3H). ESI-MS: m/z 251.1 (M + 1).
N-(3-Amino-4-chloro-2-fluorophenyl)propane-1-sulfona-
mide (7b). Step A: A flame-dried flask equipped with a stir bar and
rubber septum was charged with 4-chloro-2-fluoroaniline (5.00 g,
34.35 mmol) and anhydrous THF (170 mL). This solution was cooled
to −78 °C, and n-BuLi (14.7 mL, 1.07 equiv of 2.5 M solution in
hexanes) was then added over a 15 min period. This mixture was
stirred at −78 °C for 20 min, and then a THF solution (25 mL) of 1,2-
bis(chlorodimethylsilyl)ethane (7.76 g, 1.05 equiv) was added slowly
(over a 10 min period) to the reaction mixture. The mixture was
stirred for 1 h, and then 2.5 M n-BuLi in hexanes (15.1 mL, 1.1 equiv)
was added slowly. After allowing the mixture to warm to room
temperature for 1 h, the mixture was cooled to −78 °C. A third
allotment of n-BuLi (15.66 mL, 1.14 equiv) was added slowly, and the
mixture was stirred at −78 °C for 75 min. Benzyl chloroformate (7.40
g, 1.2 equiv) was then added slowly, and the mixture was stirred at
−78 °C for 1 h. The cooling bath was then removed. The mixture was
allowed to warm for 30 min and then quenched with water (70 mL)
and concentrated HCl (25 mL). The mixture was allowed to continue
to warm to room temperature and then extracted with EtOAc. The
extracts were washed twice with a saturated NaHCO3 solution, once
with water, dried over sodium sulfate, and concentrated. The resulting
residue was purified via silica gel column chromatography (30% ethyl
acetate/hexane) to furnish benzyl 3-amino-6-chloro-2-fluorobenzoate
Amino-2,4-difluorophenyl)propane-1-sulfonamide 7a. H NMR (500
MHz, DMSO-d6) δ 9.54 (s, 1H), 7.02 (d, 1H), 6.58 (t, 1H), 5.50 (s,
2H), 3.09−2.95 (t, 2H), 1.81−1.64 (sx, 2H), 0.96 (t, 3H). ESI-MS: m/
z 267.1 (M + 1).
N-(3-Amino-2-chloro-4-fluorophenyl)propane-1-sulfona-
mide (7c). Step A: Into a 20 L 4-neck round flask was placed a
solution of 2-chloro-4-fluorobenzenamine (1300 g, 8.82 mol, 1.00
equiv, 99%) in toluene (10 L), 4-methylbenzenesulfonic acid (3.1 g,
17.84 mmol, 99%), and hexane-2,5-dione (1222.5 g, 10.62 mol, 1.20
equiv, 99%). The resulting solution was heated to reflux for 1 h in an
oil bath and cooled. The pH value of the solution was adjusted to 8
with sodium carbonate (1 mol/L). The resulting mixture was washed
with 1 × 5000 mL of water and concentrated under vacuum. The
crude product was purified by distillation and the fraction was
collected at 140 °C to afford 1-(2-chloro-4-fluorophenyl)-2,5-
dimethyl-1H-pyrrole (1700 g, 85%).
Step B: Into a 5000 mL 4-necked round-bottom flask purged and
maintained with an inert atmosphere of nitrogen was placed a solution
of 1-(2-chloro-4-fluorophenyl)-2,5-dimethyl-1H-pyrrole (390 g, 1.65
mol, 1.00 equiv, 95%) in THF (2000 mL). The reaction vessel was
cooled to −78 °C. To the above reaction vessel was added n-BuLi
(800 mL, 1.10 equiv, 2.5%) dropwise with stirring over 80 min and
methyl carbonochloridate (215.5 g, 2.27 mol, 1.20 equiv, 99%)
dropwise with stirring over 90 min. The reaction solution was further
stirred for 60 min at −78 °C and quenched by the addition of 1000
mL of NH4Cl/water. The resulting solution was extracted with 1500
mL of ethyl acetate. The organic layers were combined, washed with 1
× 1500 mL of water and 1 × 1500 mL of sodium chloride (aq), dried
over anhydrous magnesium sulfate, and concentrated under vacuum to
afford methyl 2-chloro-3-(2,5-dimethyl-1H-pyrrol-1-yl)-6-fluoroben-
zoate (crude, 566.7 g).
Step C: Into five 5000 mL 4-neck round-bottom flasks was placed a
solution of methyl 2-chloro-3-(2,5-dimethyl-1H-pyrrol-1-yl)-6-fluoro-
benzoate (1500 g, 5.05 mol, 1.00 equiv, 95%) in ethanol/H2O (7500/
2500 mL), NH2OH·HCl (5520 g, 79.20 mol, 15.00 equiv, 99%), and
triethylamine (2140 g, 20.98 mol, 4.00 equiv, 99%). The resulting
solution was heated at reflux for 18 h in an oil bath, cooled to room
temperature, concentrated, and extracted with 3 × 3000 mL of ethyl
acetate. The organic layers were combined, dried over anhydrous
sodium sulfate, and concentrated under vacuum. The residue was
purified using a silica gel column eluting with PE:EA (20:1−10:1) to
afford methyl 3-amino-2-chloro-6-fluorobenzoate (980 g, 95%).
Step D: Into four 5000 mL 4-neck round-bottom flasks was placed a
solution of methyl 3-amino-2-chloro-6-fluorobenzoate (980 g, 4.76
mol, 1.00 equiv, 99%) in dichloromethane (8000 mL). Triethylamine
(1454 g, 14.25 mol, 3.00 equiv, 99%) was added dropwise with stirring
at 0 °C over 80 min followed by the addition of propane-1-sulfonyl
chloride (1725 g, 11.94 mol, 2.50 equiv, 99%). The resulting solution
was stirred at room temperature for 2 h and diluted with 1000 mL of
water. The organic layer was washed with 1 × 1000 mL of hydrogen
chloride and 1 × 1000 mL of water, dried over sodium sulfate, and
1
(4.3 g, 45%) as an oil. H NMR (DMSO-d6, 400 MHz) δ 7.37−7.48
(m, 5H), 7.07 (dd, J = 8 Hz, 2 Hz, 1H), 6.87 (t, J = 8 Hz, 1H), 5.61
(br s, 2H), 5.40 (s, 2H).
Step B: Benzyl 3-amino-6-chloro-2-fluorobenzoate (4.3 g, 15.37
mmol) was dissolved in dry dichloromethane (270 mL). Triethyl-
amine (5.36 mL, 2.5 equiv) was added, and the mixture was cooled to
0 °C. Propane-1-sulfonyl chloride (3.63 mL, 32.3 mmol, 2.1 equiv)
was then added via a syringe, and a precipitate resulted. After the
addition was complete, the mixture was allowed to warm to room
temperature. The mixture was then diluted with dichloromethane (200
mL), washed with 2 M aqueous HCl (2 × 100 mL) and saturated
aqueous NaHCO3 solution, dried over sodium sulfate, and
concentrated. The resulting residue was purified via silica gel column
chromatography (40% ethyl acetate/hexane) to furnish benzyl 6-
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concentrated to afford methyl 2-chloro-6-fluoro-3-
(propylsulfonamido)benzoate as a brown solid (1500 g, 97%).
Step E: Into a 10000 mL 4-necked round-bottom flask was placed a
solution of methyl 2-chloro-6-fluoro-3-(propylsulfonamido)benzoate
(1500 g, 4.61 mol, 1.00 equiv, 95%) in THF/H2O (3000/3000 mL)
and potassium hydroxide (1000 g, 17.68 mol, 4.50 equiv, 99%). The
resulting solution was refluxed for 2 h, cooled to room temperature,
and extracted with 3 × 2000 mL of ethyl acetate. The aqueous layers
were combined, and the pH was adjusted to 2 with hydrogen chloride
(2 mol/L). The resulting solution was extracted with 2 × 3000 mL of
dichloromethane. The organic layers were combined, dried over
anhydrous sodium sulfate, and concentrated to afford 2-chloro-6-
mate (204 mg, 0.817 mmol) and N-(3-amino-2,4-difluorophenyl)-
propane-1-sulfonamide (225 mg, 0.899 mmol) were taken up in 1,2-
dichloroethane (3 mL, 41 mmol). The reaction mixture was heated at
90 °C for 15 h, cooled to room temperature, and concentrated under
reduced pressure. Purification via silica gel column chromatography
(eluent: ethylacetate/hexane 1:1) afforded N-(3-(3-(6-chloropyrimi-
din-4-yl)ureido)-2,4-difluorophenyl)propane-1-sulfonamide (250 mg,
75%). 1H NMR (400 MHz, DMSO-d6) δ 10.50−10.24 (s, 1H), 9.28−
9.12 (s, 1H), 8.73−8.64 (d, J = 1.0 Hz, 1H), 7.78−7.71 (d, J = 1.0 Hz,
1H), 7.38−7.28 (td, J = 8.8, 5.6 Hz, 1H), 7.20−7.07 (td, J = 9.3, 1.8
Hz, 1H), 6.45−6.50 (m, 1H), 3.06−2.99 (m, 2H), 1.79−1.68 (m, 2H),
1.01−0.92 (t, J = 7.4 Hz, 3H). ESI-MS: m/z 406.0 (M + 1).
1
fluoro-3-(propylsulfonamido)benzoic acid (517.5 g, 37%). H NMR
N-(3-(3-(6-Aminopyrimidin-4-yl)ureido)-2,4-difluorophenyl)
propane-1-sulfonamide (10). N-(3-(3-(6-Chloropyrimidin-4-yl)-
ureido)-2,4-difluorophenyl)propane-1-sulfonamide (8, 66 mg, 0.16
mmol) was added to a mixture of 7 M ammonia in methanol (2 mL).
The reaction stirred at 80 °C overnight. After removal of the solvent
under reduced pressure, the crude product was purified by reverse
phase HPLC, affording 20 mg (35%) of the title compound. HPLC
(400 MHz, CDCl3): δ 1.058−1.096 (m, J = 15.2 Hz, 3H), 1.856−
1.933 (m, 2H), 3.073−3.112 (m, 2H), 6.811 (1H, s), 7.156−7.199 (d,
J = 17.2 Hz, 1H), 7.827−7.863 (d, J = 14.4 Hz, 1H). ESI-MS: m/z
296.0 (M + 1).
Step F: N-(3-Amino-2-chloro-4-fluorophenyl)propane-1-sulfona-
mide 7c was made using a similar procedure as described for N-(3-
1
1
RT = 3.11 min (method A). ESI-MS: m/z 387.1 (M + 1). H NMR
amino-2,4-difluorophenyl)propane-1-sulfonamide 7a. H NMR (400
(400 MHz, DMSO-d6) 9.92 (s, 1H), 9.53 (s, 1H), 8.35 (s, 1H), 8.11
(s, 1H), 7.28 (dd, J = 14.7, 8.8, 1H), 7.08 (dd, J = 20.0, 9.8, 1H), 6.81
(s, 2H), 6.41 (s, 1H), 3.07−2.94 (m, 2H), 1.81−1.64 (m, 2H), 0.96 (t,
J = 7.4, 3H).
MHz, DMSO-d6) δ 9.20 (s, 1H), 7.28−6.99 (m, 1H), 6.63 (td, J = 8.7
Hz, 5.5 Hz, 1H), 5.45 (s, 2H), 3.07−2.99 (m, 2H), 1.88−1.69 (m,
2H), 1.03− 0.95 (m, 3H). ESI-MS: m/z 267.1 (M + 1).
N-(3-Amino-4-chloro-2-fluorophenyl)-3-fluoropropane-1-
sulfonamide (7d). Step A: Into a 5000 mL 4-necked round-bottom
flask was placed a solution of benzyl 3-amino-6-chloro-2-fluoroben-
zoate (200 g, 714.29 mmol, 1.00 equiv) (previously described in the
synthesis of 7b) in dichloromethane (2000 mL) and triethylamine
(216 g, 2.14 mol, 3.00 equiv) followed by the addition of a solution of
3-fluoropropane-1-sulfonyl chloride (227 g, 1.42 mol, 2.00 equiv) in
dichloromethane (300 mL) dropwise with stirring at about 8 °C over
60 min. After stirring at room temperature for 3 h, the resulting
mixture was washed with 500 mL of 5N HCl and 2 × 500 mL of water.
The organic layer was dried over anhydrous sodium sulfate and
concentrated under vacuum to afford benzyl 6-chloro-2-fluoro-3-(3-
fluoro-N-(3-fluoropropylsulfonyl)propylsulfonamido)benzoate (360 g,
91%) as a brown oil.
N-(2,4-Difluoro-3-(3-(6-(methylamino)pyrimidin-4-yl)-
ureido)phenyl)propane-1-sulfonamide (11). N-(3-(3-(6-Chloro-
pyrimidin-4-yl)ureido)-2,4-difluorophenyl)propane-1-sulfonamide (8,
30 mg, 0.07 mmol) was added to a solution of 2 M methylamine in
THF (2 mL), and the reaction mixture was stirred at 80 °C overnight.
The organic solvent was removed under reduced pressure and the
crude product purified by reverse phase HPLC affording 20 mg (70%)
of the title compound. HPLC RT = 3.20 min (method A). ESI-MS:
m/z 401.1 (M + 1). 1H NMR (400 MHz, DMSO-d6) 9.83 (br s, 2H),
9.48 (s, 1H), 8.18 (d, J = 6.8, 1H), 7.35−7.24 (m, 2H), 7.16 (t, J = 9.4,
1H), 6.45 (s, 1H), 3.14−2.98 (m, 2H), 2.72 (m, 3H), 1.87−1.63 (m,
2H), 0.99 (t, J = 7.4, 3H).
N-(2,4-Difluoro-3-(3-(6-(2-hydroxyethyl-amino)pyrimidin-4-
yl)ureido)phenyl)propane-1-sulfonamide (12). N-(3-(3-(6-
Chloropyrimidin-4-yl)ureido)-2,4-difluorophenyl)propane-1-sulfona-
mide (8, 30 mg, 0.07 mmol) and ethanolamine (40 μL, 0.7 mmol)
were dissolved in dichloroethane (0.6 mL). N,N-Diisopropyl-ethyl-
amine (100 μL, 0.7 mmol) was added followed by stirring at 60 °C
overnight. After removal of the solvent under reduced pressure, the
crude product was purified by silica gel chromatography eluting with
0−100% hexane/EtOAc to afford 15 mg (50%) of the title compound.
Step B: A solution of benzyl 6-chloro-2-fluoro-3-(3-fluoro-N-(3-
fluoropropylsulfonyl)propylsulfonamido)benzoate (360 g, 647.73
mmol, 1.00 equiv, 95%) in THF (1800 mL) and KOH (2M, 1680
mL) was stirred at 50 °C for 12 h. The resulting mixture was cooled
and concentrated under vacuum to remove most of THF. The residual
solution was washed with 3 × 500 mL of EtOAc. The aqueous layer
was adjusted to pH 2−3 with HCl (6M). The resulting solution was
extracted with 4 × 500 mL of ethyl acetate. The combined organic
layers were dried over anhydrous sodium sulfate and concentrated
u n d e r v a c u u m t o a f f o r d 6 - c h l o r o - 2 - f l u o r o - 3 - ( 3 -
1
HPLC RT = 3.10 min (method A). ESI-MS: m/z 431.1(M + 1). H
NMR (400 MHz, DMSO-d6) 9.83 (s, 1H), 9.49 (s, 1H), 8.19 (d, J =
15.6, 1H), 7.56−7.21 (m, 2H), 7.14 (t, J = 8.7, 1H), 6.52 (d, J = 24.6,
1H), 4.71 (m, 2H), 3.48 (m, 2H), 3.15−2.93 (m, 2H), 1.83−1.63 (m,
2H), 0.97 (t, J = 7.4, 3H).
1
fluoropropylsulfonamido)benzoic acid (190 g, 89%). H NMR (400
MHz, DMSO-d6) δ 9.65 (br s, 1H), 7.03 (m, 1H), 6.58 (m, 1H), 4.59
(m, 1H), 4.47 (m, 1H), 3.18 (m, 2H), 2.22−2.02 (m, 2H). ESI-MS:
m/z 312.1 [M-1].
6-Chloro-N-methylpyrimidin-4-amine (13). 4,6-Dichloropyri-
midine (978 mg, 6.56 mmol) was taken up in isopropyl alcohol (10
mL, 131 mmol) followed by cooling to 0−5 °C. A solution of 33%
methylamine in ethanol (1.768 mL, 13.2 mmol) was added, and the
reaction mixture was stirred for 15 h. The mixture was concentrated
under reduced pressure and suspended in water. The title compound
was obtained after filtration and drying in vacuo (772 mg, 82%). ESI-
Step C: Into a 3000 mL 3-necked round-bottom flask was placed a
solution of 6-chloro-2-fluoro-3-(3-fluoropropylsulfonamido)benzoic
acid (190 g, 574.8 mmol, 1.00 equiv, 95%) in N,N-dimethylformamide
(1500 mL) and triethylamine (184 g, 1.82 mol, 3.0 equiv) followed by
the addition of diphenylphosphoryl azide (250 g, 909.1 mmol, 1.50
equiv) dropwise with stirring at 5 °C over 10 min. After stirring at 5
°C for 2 h, water (500 mL) was added to the reaction mixture. The
resulting solution was stirred at 80 °C in an oil bath for an additional 2
h, cooled, and diluted with 2000 mL of EtOAc. The organic layer was
washed with 4 × 1000 mL of brine, dried over anhydrous sodium
sulfate, and concentrated under vacuum. The residue was purified via
silica gel column chromatography eluting with ethyl acetate/petroleum
ether (1:3) to afford N-(3-amino-4-chloro-2-fluorophenyl)-3-fluoro-
1
MS: m/z 144.1 (M + 1). H NMR (400 MHz, DMSO-d6) δ 8.27 (s,
1H), 7.65 (s, 1H), 6.50 (s, 1H), 2.99−2.67 (m, 3H).
N-(3-(3-(6-Chloropyrimidin-4-yl)-3-methylureido)-2,4-
difluorophenyl)propane-1-sulfonamide (14). 2,6-Difluoro-3-
(propylsulfonamido)benzoic acid (6, 1.05 g, 3.76 mmol) was taken
up in THF (20 mL, 0.2 mol), and triethylamine (1.2 mL, 8.64 mmol)
was added in. Then, diphenylphosphonic azide (930.9 μL, 4.32 mmol)
was added, and the mixture was stirred at room temperature for 2 h.
The mixture was then heated at 80 °C for 1 h, and 6-chloro-N-
methylpyrimidin-4-amine (674 mg, 4.70 mmol) was added. After
stirring at 80 °C overnight, the reaction mixture was diluted with
EtOAc (300 mL) and washed subsequently with saturated aqueous
Na2CO3 solution and brine (50 mL each). The organic phase was
1
propane-1-sulfonamide (76 g, 46%) as a white solid. H NMR (400
MHz, CDCl3) δ 7.04−7.06 (m, 1H), 6.91−6.87 (t, 1H), 6.39 (s, 1H),
4.62−4.59 (t, 1H), 4.40−4.57 (t, 1H), 4.15 (br s, 2H), 3.27−3.24 (t,
2H), 2.30−2.16 (m, 2H). ESI-MS: m/z 283.0 [M − 1].
N-(3-(3-(6-Chloropyrimidin-4-yl)ureido)-2,4-difluorophenyl)-
propane-1-sulfonamide (8). Phenyl 6-chloropyrimidin-4-ylcarba-
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Journal of Medicinal Chemistry
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concentrated under reduced pressure and the residue purified via silica
gel chromatography (eluent: 0−50% ethylacetate in hexane) to obtain
365 mg (25%) of the title compound. ESI-MS: m/z 420.1 (M + 1).
N-(3-(3-(6-Aminopyrimidin-4-yl)-3-methylureido)-2,4-
difluorophenyl)propane-1-sulfonamide (15). N-(3-(3-(6-Chlor-
opyrimidin-4-yl)-3-methylureido)-2,4-difluorophenyl)propane-1-sulfo-
namide (14, 26 mg, 0.062 mmol) was added to a 7 M solution of
ammonia in methanol (1 mL, 6.7 mmol). The reaction mixture was
stirred at room temperature overnight, concentrated under reduced
pressure, and purified via reverse phase HPLC to obtain 15 mg (40%)
of the title compound. HPLC RT = 3.37 min (method A). ESI-MS:
(dd, J = 14.6, 8.8, 1H), 7.20 (t, J = 9.5, 1H), 3.16−3.01 (m, 2H), 1.84−
1.67 (m, 2H), 0.98 (t, J = 7.4, 3H).
4-Hydroxyquinazoline-8-carboxylic Acid (21). A 10 L reactor
was charged with 2-aminoisophthalic acid 20 (600 g, 3.3 mol) and
formamidine acetate (1035 g, 9.9 mol, 3.0 equiv). After stirring for 25
min, formamide (132 mL, 3.3 mol) was added. The mixture was
heated at 170 °C with a sand bath and continuously stirred with a
heavy duty overhead mechanical stirrer for 5 h. HPLC analysis
indicated no presence of 2-aminoisophthalic acid. The temperature
was lowered to 80 °C. Water (5 L) was slowly added to the reactor.
The resulting suspension was heated under reflux for 1 h. The reaction
mixture was then cooled to room temperature and filtered. The filter
cake was washed with water (2 × 2 L) and MeOH (2 × 2 L). The
filter cake was dried in an oven over 40 °C for 17 h. The first crop of
the final product was obtained (384 g). To the previously obtained
filtrate, concentrated HCl was added and the pH adjusted to 0.2. The
mixture was filtered, and the filter cake washed with water (500 mL).
Drying in the oven at 40 °C for 16 h yielded a second crop of the
product which was combined with the first crop. Both product crops
were combined to afford 4-hydroxyquinazoline-8-carboxylic acid (500
g, 87%). 1H NMR (400 MHz, DMSO-d6) δ 8.51 (s, 1H), 8.45 (dd, J =
7.6, 1.6 Hz, 1H), 8.35 (dd, J = 7.9, 1.6 Hz, 1H), 7.68 (t, J = 7.8 Hz,
1H).
1
m/z 401.1 (M + 1). H NMR (400 MHz, DMSO-d6) 12.10 (s, 1H),
9.80 (s, 1H), 8.22 (s, 1H), 7.27 (dd, J = 14.6, 8.8, 1H), 7.07 (t, J = 9.2,
1H), 6.99 (s, 2H), 6.14 (s, 1H), 3.31 (s, 3H), 3.06−2.88 (m, 2H),
1.83−1.60 (m, 2H), 0.96 (t, J = 7.4, 3H).
P h e n y l 2 , 6 - D i f l u o r o - 3 - ( p r o p y l s u l f o n a m i d o ) -
phenylcarbamate (16). 2,6-Difluoro-3-(propylsulfonamido)benzoic
acid7 (4 g, 14 mmol) was dissolved in 1,4-dioxane (100 mL), and
triethylamine (2.2 mL, 16 mmol) and diphenylphosphonic azide (3.4
mL, 16 mmol) were added. The reaction mixture was stirred at room
temperature for 3 h and then added dropwise to a solution of phenol
(15 g, 160 mmol) in 1,4-dioxane (100 mL) at 100 °C. The mixture
was stirred at 100 °C for 3 h and then cooled to room temperature.
Silica was added, and the mixture was concentrated. Purification via
flash chromatography (gradient elution, solvent: 0−30% ethyl acetate
4-Chloroquinazoline-8-carbonyl Chloride (22). 4-Hydroxyqui-
nazoline-8-carboxylic acid 21 (2.50 g, 13.1 mmol) was suspended in
thionyl chloride (40 mL), and DMF (0.20 mL, 2.63 mmol) was added.
The reaction mixture was heated at reflux for 2 h, and the remaining
undissolved solid was filtered off. The filtrate was concentrated in
vacuo and the residue redissolved in chloroform and reconcentrated in
vacuo. The same process was repeated twice with toluene. The
obtained solid was triturated with heptane and filtered to afford 4-
1
in heptanes) yielded the title compound (2.9 g, 55%). H NMR (400
MHz, DMSO-d6) δ 9.85 (s, 1H), 9.68 (s, 1H), 7.49−7.30 (m, 3H),
7.30−7.11 (m, 4H), 3.11−3.03 (m, 2H), 1.83−1.61 (m, 2H), 0.96 (t, J
= 7.4, 3H).
3-Methoxy-1H-pyrazolo[3,4-d]pyrimidin-4-amine (17b). To a
mixture of tetracyanoethylene (300 g, 2.34 mol) and urea (47.7 g, 0.79
mol) at rt was added methanol (1 L). The reaction mixture was heated
at 35 °C for 20 min, cooled to room temperature, and diluted with
diethyl ether (4 L). The mixture was cooled at −78 °C for 3 h, filtered,
washed with cold ether (400 mL), and dried in vacuo to give 2-
(dimethoxymethylene)malononitrile (200 g, 62%) as a white solid,
which was used directly in the next step.
1
chloroquinazoline-8-carbonyl chloride (2.30 g, 77%). H NMR (400
MHz, DMSO-d6) δ 8.56 (s, 1H), 8.48 (dd, J = 7.6, 1.6 Hz, 1H), 8.39
(dd, J = 7.9, 1.6 Hz, 1H), 7.71 (t, J = 7.8 Hz, 1H). m/z (ESI-MS) (M
− (2 Cl) + (2 -OMe) + 1) = 219.2.
4-Amino-quinazoline-8-carboxylic Acid [2,6-Difluoro-3-(pro-
pane-1-sulfonylamino)-phenyl]-amide (24). Step A: To a
solution of N-(3-amino-2,4-difluorophenyl)propane-1-sulfonamide 7a
(170 mg, 0.679 mmol) in chloroform (3 mL) was added magnesium
sulfate (150 mg) and pyridine (0.16 mL, 2.04 mmol). A suspension of
4-chloroquinazoline-8-carbonyl chloride (0.20 g, 0.88 mmol) in
chloroform (4 mL) was then added at room temperature. The
reaction mixture was heated at 60 °C for 1 h, and the magnesium
sulfate was removed by filtration. The filtrate was diluted with
dichloromethane and washed with a saturated solution of NaHCO3.
The aqueous layer was extracted twice with dichloromethane and the
combined organic layers dried with sodium sulfate, filtered, and
concentrated in vacuo. The crude product was purified by flash
chromatography eluting with 10−20% EtOAc/DCM to afford 4-
chloro-N-(2,6-difluoro-3-(propylsulfonamido)phenyl)quinazoline-8-
carboxamide 23a (145 mg, 48%). Step B: In a microwave vessel, 4-
chloro-N-(2,6-difluoro-3-(propylsulfonamido)phenyl)quinazoline-8-
carboxamide 23a (0.08 g, 0.18 mmol) was dissolved in a 2 M ammonia
solution in isopropyl alcohol (4 mL) and heated in a microwave
reactor at 105 °C for 15 min. The reaction mixture was concentrated
in vacuo and the crude product then purified by SFC (or reverse phase
HPLC) to afford the title compound (55 mg, 71%) as a solid. HPLC
To a mixture of 2-(dimethoxymethylene)malononitrile (200 g, 1.45
mol) in H2O (2.7 L) at rt was added hydrazine monohydrate (78 mL,
1.63 mol). The reaction mixture was stirred for 14 h, filtered, washed
with H2O (400 mL), and dried in vacuo to afford 5-amino-3-methoxy-
1
1H-pyrazole-4-carbonitrile (140 g, 70%) as a light-yellow solid. H
NMR (400 MHz, DMSO-d6) δ 11.04 (bs, 1 H), 6.35 (bs, 2 H), 3.76
(s, 3 H).
A mixture of 5-amino-3-methoxy-1H-pyrazole-4-carbonitrile (140 g,
1.01 mol) and formamidine acetate (140 g, 1.34 mol) was heated at
145 °C for 1 h, cooled to rt, and diluted with H2O (1.2 L). The
mixture was vigorously stirred for 2 h and filtered. The solid was
washed with methanol (400 mL) and dried in vacuo to afford 3-
methoxy-1H-pyrazolo[3,4-d]pyrimidin-4-amine (105 g, 64%) as a
brown solid. 1H NMR (400 MHz, DMSO-d6) δ 12.31 (bs, 1 H), 8.07
(s, 1 H), 7.48 (bs, 1 H), 6.63 (bs, 1H), 3.94 (s, 3 H).
N-(2,4-Difluoro-3-(3-(3-methoxy-1H-pyrazolo[3,4-d]-
pyrimidin-4-yl)ureido)phenyl)propane-1-sulfonamide (19).
The mixture of phenyl 2,6-difluoro-3-(propylsulfonamido)-
phenylcarbamate (638 mg, 1.72 mmol) and 3-methoxy-1H-pyrazolo-
[3,4-d]pyrimidin-4-amine (313 mg, 1.89 mmol) was suspended in
dimethyl sulfoxide (2 mL). The reaction mixture was stirred at 80 °C
for 5 h. Purification by HPLC afforded 540 mg (30%) of the title
compound. HPLC RT = 4.17 min (method A). ESI-MS: m/z 442.0
1
RT = 7.90 min (method B). ESI-MS: m/z 422.1 (M + 1). H NMR
(400 MHz, DMSO-d6) δ 13.25 (s, 1H), 9.67 (s, 1H), 8.65 (d, J = 6.6
Hz, 1H), 8.57 (s, 1H), 8.53 (d, J = 8.1, 1H), 8.33 (s, 2H), 7.68 (t, J =
7.8 Hz, 1H), 7.37 (dd, J = 14.4, 8.6 Hz, 1H), 7.22 (t, J = 8.6 Hz, 1H),
3.14−3.01 (m, 2H), 1.82−1.69 (m, 2H), 0.98 (t, J = 7.4 Hz, 3H).
4-Amino-quinazoline-8-carboxylic Acid [6-Chloro-2-fluoro-
3-(propane-1-sulfonylamino)-phenyl]-amide (25). Step A: 4-
Chloro-N-(6-chloro-2-fluoro-3-(propylsulfonamido)phenyl)-
quinazoline-8-carboxamide 23b was made using a similar procedure as
described for 4-chloro-N-(2,6-difluoro-3-(propylsulfonamido)phenyl)-
quinazoline-8-carboxamide 23a. Step B: 4-Amino-quinazoline-8-
carboxylic acid [6-chloro-2-fluoro-3-(propane-1-sulfonylamino)-phe-
nyl]-amide 25 was made using a similar procedure as described for 4-
1
(M + 1). H NMR (400 MHz, DMSO-d6) 13.09 (s, 1H), 11.10 (s,
1H), 9.81 (s, 1H), 8.52 (d, J = 22.0, 2H), 7.35 (dd, J = 14.1, 8.3, 1H),
7.17 (t, J = 9.1, 1H), 4.05 (s, 3H), 3.05 (m, 2H), 1.86−1.65 (m, 2H),
0.97 (t, J = 7.4, 3H).
N-(3-(3-1H-Pyrazolo[3,4-d]pyrimidin-4-ylureido)-2,4-
difluorophenyl)propane-1-sulfonamide (18). The title com-
pound was synthesized using an analogous procedure as described
for compound 19. HPLC RT = 3.63 min (method A). ESI-MS: m/z
412.0 (M + 1). 1H NMR (400 MHz, DMSO-d6) 13.99 (s, 1H), 11.13
(s, 1H), 11.03 (s, 1H), 9.68 (s, 1H), 8.59 (s, 1H), 8.54 (s, 1H), 7.37
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amino-quinazoline-8-carboxylic acid [2,6-difluoro-3-(propane-1-sulfo-
nylamino)-phenyl]-amide 24. HPLC RT = 9.07 min (method B). ESI-
MS: m/z 438.0 (M + 1). 1H NMR (400 MHz, DMSO-d6) δ 13.47 (s,
1H), 9.90 (s, 1H), 8.65 (dd, J = 7.5, 1.4 Hz, 1H), 8.58 (s, 1H), 8.53
(dd, J = 8.3, 1.4 Hz, 1H), 8.43 (s, 1H), 8.26 (s, 1H), 7.69 (t, J = 7.9
Hz, 1H), 7.47−7.36 (m, 2H), 3.16−3.08 (m, 2H), 1.75 (sx, J = 7.4 Hz,
2H), 0.97 (t, J = 7.4 Hz, 3H).
d]pyrimidine-7-carboxylate (2.00 g, 8.75 mmol) in THF (60 mL) and
water (20 mL) was added lithium hydroxide monohydrate (0.59 g,
14.0 mmol). The reaction mixture was stirred at room temperature for
2 h, after which the volatiles were concentrated in vacuo. Water was
added and a solid was obtain after filtration, which was rinsed with
water and dried on a lyophilizer to afford 4-chlorothieno[3,2-
1
d]pyrimidine-7-carboxylic acid (1.56 g, 81%). H NMR (500 MHz,
4-Amino-quinazoline-8-carboxylic Acid [2-Chloro-6-fluoro-
3-(propane-1-sulfonylamino)-phenyl]-amide (26). Step A: 4-
Chloro-N-(2-chloro-6-fluoro-3-(propylsulfonamido)phenyl)-
quinazoline-8-carboxamide 23c was made using a similar procedure as
described for 4-chloro-N-(2,6-difluoro-3-(propylsulfonamido)phenyl)-
quinazoline-8-carboxamide 23a. Step B: 4-Amino-quinazoline-8-
carboxylic acid [2-chloro-6-fluoro-3-(propane-1-sulfonylamino)-phe-
nyl]-amide 26 was made using a similar procedure as described for 4-
amino-quinazoline-8-carboxylic acid [2,6-difluoro-3-(propane-1-sulfo-
nylamino)-phenyl]-amide 24. HPLC RT = 8.00 min (method B). ESI-
MS: m/z 438.0 (M + 1). 1H NMR (400 MHz, DMSO-d6) δ 13.25 (s,
1H), 9.68 (s, 1H), 8.69−8.61 (m, 1H), 8.57 (s, 1H), 8.56−8.49 (m,
1H), 8.32 (s, 2H), 7.68 (t, J = 7.8 Hz, 1H), 7.37 (td, J = 8.8, 5.7 Hz,
1H), 7.22 (t, J = 8.7 Hz, 1H), 3.15−3.03 (m, 2H), 1.84−1.68 (m, 2H),
1.04 (d, J = 6.1 Hz, 1H), 1.02−0.93 (m, 3H).
4-Amino-quinazoline-8-carboxylic Acid [6-Chloro-2-fluoro-
3-(3-fluoro-propane-1-sulfonylamino)-phenyl]-amide (27).
S t e p A : 4 - C h l o r o - N - ( 6 - c h l o r o - 2 - f l u o r o - 3 - ( 3 -
fluoropropylsulfonamido)phenyl)quinazoline-8-carboxamide 23d was
made using a similar procedure as described for 4-chloro-N-(2,6-
difluoro-3-(propylsulfonamido)phenyl)quinazoline-8-carboxamide
23a. Step B: 4-Amino-quinazoline-8-carboxylic acid [6-chloro-2-fluoro-
3-(3-fluoro-propane-1-sulfonylamino)-phenyl]-amide 27 was made
using a similar procedure as described for 4-amino-quinazoline-8-
carboxylic acid [2,6-difluoro-3-(propane-1-sulfonylamino)-phenyl]-
amide 24. HPLC RT = 3.40 min (method A). ESI-MS: m/z 456.1,
458.1 (M + 1). 1H NMR (400 MHz, DMSO-d6) δ 13.46 (s, 1H), 9.98
(s, 1H), 8.68−8.62 (m, 1H), 8.58 (s, 1H), 8.55−8.50 (m, 1H), 8.30
(br s, 2H), 7.68 (t, J = 7.8 Hz, 1H), 7.47−7.37 (m, 2H), 4.60 (t, J = 6.0
Hz, 1H), 4.48 (t, J = 6.0 Hz. 1H), 3.27−3.21 (m, 2H), 2.19−2.04 (m,
2H).
DMSO-d6) δ 8.98 (s, 1H), 8.69 (s, 1H).
4-Amino-thieno[3,2-d]pyrimidine-7-carboxylic Acid [6-
Chloro-2-fluoro-3-(propane-1-sulfonylamino)-phenyl]-amide
(33). Step A: To a solution of 4-chlorothieno[3,2-d]pyrimidine-7-
carboxylic acid 30 (0.93 g, 4.31 mmol) in THF (45 mL) at 0 °C was
added oxalyl chloride (730 μL, 8.63 mmol) followed by DMF (14.5
μL, 0.19 mmol). The reaction mixture was stirred at room temperature
for 75 min and then concentrated in vacuo to give crude 4-
chlorothieno[3,2-d]pyrimidine-7-carbonyl chloride, which was used
directly in the next step. Step B: Crude 4-chlorothieno[3,2-
d]pyrimidine-7-carbonyl chloride was dissolved in THF (40 mL),
and N-(3-amino-4-chloro-2-fluorophenyl)propane-1-sulfonamide 7b
(1.00 g, 3.75 mmol) was added. The reaction mixture was stirred at
55 °C for 90 min, cooled to room temperature, and diluted with
dichloromethane and a saturated aqueous solution of NaHCO3. The
layers were separated and the aqueous layer extracted with
dichloromethane (2×). The combined organic layers were dried
over sodium sulfate, filtered, and concentrated in vacuo. The crude
product was purified by flash chromatography to afford 4-chloro-N-(6-
chloro-2-fluoro-3-(propylsulfonamido)phenyl)thieno[3,2-d]-
1
pyrimidine-7-carboxamide 31b (1.65 g, 95%). H NMR (500 MHz,
DMSO-d6) δ 10.72 (s, 1H), 9.88 (s, 1H), 9.36 (s, 1H), 9.26 (s, 1H),
7.47−7.42 (m, 2H), 3.19−3.07 (m, 2H), 1.79−1.71 (m, 2H), 0.98 (t, J
= 7.4 Hz, 3H). ESI-MS: m/z 463.0, 465.0 (M + 1). Step C: A sealed
tube was charged with 4-chloro-N-(6-chloro-2-fluoro-3-
(propylsulfonamido)phenyl)thieno[3,2-d]pyrimidine-7-carboxamide
31b (1.65 g, 3.56 mmol), and a 2 M ammonia solution in isopropyl
alcohol (27 mL) was added. The reaction mixture was heated at 95 °C
for 24 h and then concentrated in vacuo. The crude product was
triturated with 2% i-PrOH/water solution (crude could also be
purified by reverse phase HPLC) to afford the title compound (1.41 g,
89%). HPLC RT = 9.69 min (method B). ESI-MS: m/z 444.0 (M +
Methyl 4-Hydroxythieno[3,2-d]pyrimidine-7-carboxylate
(29). Step A: To a solution of 3H-thieno[3,2-d]pyrimid-4-one 28
(25 g, 164 mmol) in acetic acid (200 mL) was added bromine (26
mL) dropwise. The reaction mixture was heated at 100 °C for 8 h. The
resulting suspension was cooled to room temperature, poured into
water, and neutralized with solid sodium bicarbonate. The solid
product was collected by vacuum filtration to yield 7-bromo-3H-
1
1). H NMR (400 MHz, DMSO-d6) δ 11.47 (s, 1H), 9.90 (s, 1H),
8.98 (s, 1H), 8.55 (s, 1H), 7.97 (s, 2H), 7.49−7.36 (m, 2H), 3.18−
3.07 (m, 2H), 1.81−1.67 (m, 2H), 0.97 (t, J = 7.4 Hz, 3H).
4-Amino-thieno[3,2-d]pyrimidine-7-carboxylic Acid [2,6-Di-
fluoro-3-(propane-1-sulfonylamino)-phenyl]-amide (32). Step
A: 4-Chloro-N-(2,6-difluoro-3-(propylsulfonamido)phenyl)thieno-
[3,2-d]pyrimidine-7-carboxamide 31a was made using a similar
procedure as described for 4-chloro-N-(6-chloro-2-fluoro-3-
(propylsulfonamido)phenyl)thieno[3,2-d]pyrimidine-7-carboxamide
31b. Step B: 4-Amino-thieno[3,2-d]pyrimidine-7-carboxylic acid [2,6-
difluoro-3-(propane-1-sulfonylamino)-phenyl]-amide 32 was made
using a similar procedure as described for 4-amino-thieno[3,2-
d]pyrimidine-7-carboxylic acid [6-chloro-2-fluoro-3-(propane-1-sulfo-
nylamino)-phenyl]-amide 33. HPLC RT = 8.72 min (method B). ESI-
MS: m/z 428.0 (M + 1). 1H NMR (400 MHz, DMSO-d6) δ 11.49 (s,
1H), 9.62 (s, 1H), 8.98 (s, 1H), 8.56 (s, 1H), 7.97 (s, 2H), 7.47 (dd, J
= 9.1, 5.3 Hz, 1H), 7.39 (t, J = 9.1 Hz, 1H), 3.18−3.09 (m, 2H), 1.77
(dd, J = 15.2, 7.5 Hz, 2H), 0.99 (t, J = 7.4 Hz, 3H).
1
thieno[3,2-d]pyrimid-4-one (21.4 g, 60%) as a solid. H NMR (500
MHz, DMSO-d6) δ 12.75 (s, 1H), 8.38 (s, 1H), 8.27 (s, 1H). Step B:
7-Bromo-3H-thieno[3,2-d]pyrimid-4-one (10.0 g, 40.7 mmol), [1,1′-
bis-(diphenyl-phosphino)ferrocene]dichloropalladium(II) complex
with dichloromethane (1:1) (830.5 mg, 1.017 mmol), triethylamine
(28.35 mL, 203.4 mmol), and methanol (80 mL) were combined in an
autoclave fitted with a large stir bar. The mixture was purged with
nitrogen for 5 min. The vessel was placed under an atmosphere of
carbon monoxide (300 psi) and heated to 120 °C for 3 h. The vessel
was cooled to room temperature, and the reaction mixture was filtered.
The collected solids were washed with methanol (250 mL). The solids
were air-dried to give the title compound (6.8 g, 80%). 1H NMR (400
MHz, DMSO-d6) δ 12.74 (s, 1H), 8.90 (s, 1H), 8.27 (s, 1H), 3.89 (s,
3H).
4-Chlorothieno[3,2-d]pyrimidine-7-carboxylic Acid (30). Step
A: Methyl 4-hydroxythieno[3,2-d]pyrimidine-7-carboxylate 29 (6.8 g,
31 mmol) was dissolved in phosphoryl chloride (100 mL, 1000 mmol)
and heated to reflux for 2 h. The mixture was stirred at room
temperature overnight. The phosphoryl chloride was distilled off, and
the solids were neutralized with ice and sodium bicarbonate. The
resulting suspension was filtered to give a solid, which was triturated
with anhydrous ether. The resulting suspension was filtered to yield
methyl 4-chlorothieno[3,2-d]pyrimidine-7-carboxylate as a solid (6.76
g, 96%). 1H NMR (400 MHz, DMSO-d6) δ 9.30 (s, 1H), 9.17 (s, 1H),
3.91 (s, 3H). Step B: To a solution of methyl 4-chlorothieno[3,2-
4-Amino-thieno[3,2-d]pyrimidine-7-carboxylic Acid [2-
Chloro-6-fluoro-3-(propane-1-sulfonylamino)-phenyl]-amide
(34). Step A: 4-Chloro-N-(2-chloro-6-fluoro-3-(propylsulfonamido)-
phenyl)thieno[3,2-d]pyrimidine-7-carboxamide 31c was made using a
similar procedure as described for 4-chloro-N-(6-chloro-2-fluoro-3-
(propylsulfonamido)phenyl)thieno[3,2-d]pyrimidine-7-carboxamide
31b. Step B: 4-Amino-thieno[3,2-d]pyrimidine-7-carboxylic acid [2-
chloro-6-fluoro-3-(propane-1-sulfonylamino)-phenyl]-amide 34 was
made using a similar procedure as described for 4-amino-thieno[3,2-
d]pyrimidine-7-carboxylic acid [6-chloro-2-fluoro-3-(propane-1-sulfo-
nylamino)-phenyl]-amide 33. HPLC RT = 9.06 min (method B). ESI-
MS: m/z 444.0 (M + 1). 1H NMR (400 MHz, DMSO-d6) δ 11.49 (s,
1H), 9.62 (s, 1H), 8.98 (s, 1H), 8.56 (s, 1H), 7.97 (s, 2H), 7.47 (dd, J
2879
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Journal of Medicinal Chemistry
Article
= 9.1, 5.3 Hz, 1H), 7.39 (t, J = 9.1 Hz, 1H), 3.18−3.09 (m, 2H), 1.77
(dd, J = 15.2, 7.5 Hz, 2H), 0.99 (t, J = 7.4 Hz, 3H).
(dibenzylideneacetone)dipalladium(0); PEG, polyethylene gly-
col; Ph, phenyl; PIPES, piperazine-N,N′-bis(2-ethanesulfonic
acid); PK, pharmacokinetics; PO, oral administration; PPB,
plasma protein binding; qd, dosing once daily; rmsd, root-
mean-square deviation; rt, room temperature; RT, retention
time; SAR, structure−activity relationship; TCEP, tris(2-
carboxyethyl)phosphine; TGI, tumor growth inhibition; THF,
tetrahydrofuran; Vss, steady-state volume of distribution; TLC,
thin-layer chromatography; UPLC, ultraperformance liquid
chromatography; WT, wild-type
4-Amino-thieno[3,2-d]pyrimidine-7-carboxylic Acid [6-
Chloro-2-fluoro-3-(3-fluoro-propane-1-sulfonylamino)-phe-
nyl]-amide (35). Step A: 4-Chloro-N-(6-chloro-2-fluoro-3-(3-
fluoropropylsulfonamido)phenyl)thieno[3,2-d]pyrimidine-7-carboxa-
mide 31d was made using a similar procedure as described for 4-
chloro-N-(6-chloro-2-fluoro-3-(propylsulfonamido)phenyl)thieno[3,2-
d]pyrimidine-7-carboxamide 31b. Step B: 4-Amino-thieno[3,2-d]-
pyrimidine-7-carboxylic acid [6-chloro-2-fluoro-3-(3-fluoro-propane-
1-sulfonylamino)-phenyl]-amide 35 was made using a similar
procedure as described for 4-Amino-thieno[3,2-d]pyrimidine-7-
carboxylic acid [6-chloro-2-fluoro-3-(propane-1-sulfonylamino)-phe-
nyl]-amide 33. HPLC RT = 9.06 min (method B). ESI-MS: m/z
462.1, 464.1 (M + 1). 1H NMR (400 MHz, DMSO-d6) δ 11.49 (br s,
1H), 10.03 (br s, 1H), 8.97 (s, 1H), 8.55 (s, 1H), 7.96 (br s, 2H), 7.43
(m, 2H), 4.61 (m, 1H), 4.49 (m, 1H), 3.25 (m, 2H), 2.17−2.04 (m,
2H).
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ASSOCIATED CONTENT
* Supporting Information
■
S
Kinase activity profiles of compounds 32 and 35. Biological
assay and solubility assay information. Chemical stability data
for 19 and 24. Protein expression and purification of B-Raf and
B-RafV600E protein constructs for crystallography. Crystal
structure determination of B-Raf complex with compound 12
and 32, respectively. Colo205 xenograft efficacy study details
for compound 35 and 2. This material is available free of charge
via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: 650 467 8867. E-mail: rudolph.joachim@gene.com.
■
Notes
The authors declare no competing financial interest.
(6) Another selective B-Raf inhibitor in advanced clinical studies is
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ACKNOWLEDGMENTS
We thank Laura Afflerbaugh and Jane Kuo for their drug
formulation support.
■
ABBREVIATIONS USED
■
Ac, acetyl; ACN, acetonitrile; ADME, absorption, distribution,
metabolism, and excretion; aq, aqueous; AUC, area under
curve; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-
propanesulfonate; CL, clearance; Cmax, maximal concentration;
CYP, cytochrome P-450; d, day; DCE, dichloroethane; DCM,
dichloromethane; DFG, sequence of the three amino acids
aspartic acid-phenylalanine-glycine; DMF, N,N-dimethylforma-
mide; DMSO, dimethylsulfoxide; D-PAS, dip probe absorption
spectroscopy; EC50, half-maximal effective concentration; ED50,
half-maximal effective dose; ED90, dose associated with 90% of
the maximum effectiveness; ESI, electrospray ionization; %F,
oral bioavailability; equiv, equivalent; FDA, Food and Drug
Administration; mp, melting point; HBSS, Hank’s balanced salt
solution; hERG, human ether-a-go-go related gene; HFBA,
heptafluorobutyric acid; HPLC, high performance liquid
chromatography; HPMCAS, hydroxypropylmethylcellulose ac-
etate succinate; IC50, half-maximal inhibitory concentration; h,
hour; IPA, isopropyl alcohol; IV, intravenous administration;
LC, liquid chromatography; MAPK, mitogen activated protein
kinase; MS, mass spectrometry; MW, microwave; MW,
molecular weight; PdCl2 (dppf)·DCM, 1,1′-bis-
(diphenylphosphino)ferrocene-palladium(II)dichloride di-
c h l o r o m e t h a n e c o m p l e x ; P d 2 ( d b a ) 3 , t r i s -
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Journal of Medicinal Chemistry
Article
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(17) pKa determination was performed at Sirius using the D-PAS
technique.
(18) Testing against a large kinase panel was performed at Invitrogen.
The data are shown in the Supporting Information.
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