Potent and highly selective benzimidazole inhibitors of PI3-kinase delta

Article abstract of DOI:10.1021/jm300717c

Inhibition of PI3Kδ is considered to be an attractive mechanism for the treatment of inflammatory diseases and leukocyte malignancies. Using a structure-based design approach, we have identified a series of potent and selective benzimidazole-based inhibitors of PI3Kδ. These inhibitors do not occupy the selectivity pocket between Trp760 and Met752 that is induced by other families of PI3Kδ inhibitors. Instead, the selectivity of the compounds for inhibition of PI3Kδ relative to other PI3K isoforms appears to be due primarily to the strong interactions these inhibitors are able to make with Trp760 in the PI3Kδ binding pocket. The pharmacokinetic properties and the ability of compound 5 to inhibit the function of B-cells in vivo are described.

Full text of DOI:10.1021/jm300717c

Article
pubs.acs.org/jmc
Potent and Highly Selective Benzimidazole Inhibitors of PI3-Kinase
Delta
Jeremy M. Murray,*,† Zachary K. Sweeney,*,† Bryan K. Chan,† Mercedesz Balazs,† Erin Bradley,†
Georgette Castanedo,† Christine Chabot,† David Chantry,† Michael Flagella,† David M. Goldstein,§
Rama Kondru,§ John Lesnick,† Jun Li,† Matthew C. Lucas,§ Jim Nonomiya,† Jodie Pang,† Stephen Price,‡
Laurent Salphati,† Brian Safina,† Pascal P. A. Savy,‡ Eileen M. Seward,‡ Mark Ultsch,†
and Daniel P. Sutherlin†
†Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, United States
‡Argenta Discovery, 8/9 Spire Green Centre, Flex Meadow, Harlow CM19 5TR, United Kingdom
§Roche Research Center, 340 Kingsland Street, Nutley, New Jersey 07110, United States
S
* Supporting Information
ABSTRACT: Inhibition of PI3Kδ is considered to be an attractive mechanism for the treatment of inflammatory diseases and
leukocyte malignancies. Using a structure-based design approach, we have identified a series of potent and selective
benzimidazole-based inhibitors of PI3Kδ. These inhibitors do not occupy the selectivity pocket between Trp760 and Met752 that
is induced by other families of PI3Kδ inhibitors. Instead, the selectivity of the compounds for inhibition of PI3Kδ relative to
other PI3K isoforms appears to be due primarily to the strong interactions these inhibitors are able to make with Trp760 in the
PI3Kδ binding pocket. The pharmacokinetic properties and the ability of compound 5 to inhibit the function of B-cells in vivo
are described.
apparently essential role that these enzymes play in embryonic
development suggest that pan-PI3K inhibitors might have
limited tolerability in the treatment of PI3Kδ driven disease. As
such, there have been efforts directed toward the discovery of
selective inhibitors of PI3Kδ.11
The p110 subunits of several PI3K isoforms have been
structurally characterized.12−14 Williams, Shokat, and co-
workers described the ATP-binding site of this subunit as
including a hinge pocket, an affinity pocket, and a hydrophobic
region that lies below a nonconserved rim of the active site
(Figure 2A). Most selective PI3Kδ inhibitors occupy a
selectivity pocket between Trp760 and Met752 that is formed
by the concerted movement of these residues.13,15 However, a
modestly potent and selective PI3Kδ inhibitor that interacts
with residues near the nonconserved rim but does not induce
the selectivity pocket has also been described.12 Our group has
INTRODUCTION
■
Phosphoinositide 3-kinase delta (PI3Kδ) participates in the
activation of leukocytes by catalyzing the production of the
cellular second messenger phosphatidylinositol-3,4,5-trisphos-
phate (PIP3). Mice that express a form of PI3Kδ that is
catalytically inactive demonstrate deficient B-cell development
and reduced production of T-cell dependent antibodies.1,2
Inhibition of PI3Kδ is therefore considered to be an attractive
mechanism for the treatment of inflammatory diseases and
leukocyte malignancies.3−5
The class IA and class IB lipid kinases are structurally related
and include PI3Kα, PI3Kβ, PI3Kδ, and PI3Kγ.6 Several small
molecules that inhibit the kinase activity of all members of the
PI3K family, including 4-(2-(1H-indazol-4-yl)-6-((4-
(methylsulfonyl)piperazin-1-yl)methyl)thieno[3,2-d]pyrimidin-
4-yl)morpholine (GDC-0941)7 (1, Figure 1), are currently in
clinical trials for the treatment of cancer.8,9 While these
molecules have demonstrated efficacy in animal models,10 the
broad tissue distribution of PI3Kα and PI3Kβ and the
Received: May 22, 2012
Published: August 9, 2012
© 2012 American Chemical Society
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Figure 1. Structures of 1 (GDC-0941) and benzimidazole 2.
approaches the nonconserved rim (Figure 2A). In the affinity
pocket, the indazole group makes hydrogen bond interactions
with Asp787 from the kα3-helix and Tyr813 from kβ6. The
poor δ/γ selectivity of this compound can be attributed to the
broad distribution of the hydrophobic and electrostatic
intermolecular interactions that are conserved across the
various p110 isoforms.
In our previously reported work, benzimidazole 2 (Figure 1)
was discovered to be a reasonably selective inhibitor of PI3Kδ
(PI3Kα/PI3Kδ = 29).16 Although 2 had modest in vitro
permeability (MDCK A/B = 3 × 10−6 cm/s), it appeared to be
a promising candidate for further optimization. In particular, we
were encouraged that the benzimidazole inhibitors generally
demonstrated good potency in cellular assays and had low
potential for time-dependent inactivation of cytochrome p450
enzymes. The goal of the research described in this report was
to identify benzimidazole-based inhibitors of PI3Kδ with
improved selectivity against other PI3K isoforms as well as
improved in vitro and in vivo pharmacokinetic properties.17
We first examined the effect of the central bicyclic ring on
PI3Kδ and PI3Kα potency utilizing structure−activity relation-
ships developed from earlier studies. In particular, we
hypothesized that modification of the central heterocycle
would allow us to adjust the interaction of the piperidine
region of the inhibitors with Trp760 and residues located on
the nonconserved rim of the binding pocket. This would allow
us to specifically target the space created by the Thr750 side
chain in p110δ, which is electronically and structurally distinct
from the residues found in p110α, β, and γ (Arg770, Lys777,
and Lys802, respectively). Although Trp760 is conserved across
the different isoforms, the residues that interact with Trp760
are not. For example, the hydrophobic surface of Trp760 is
likely to be less accessible in PI3Kα because in this isoform, the
corresponding Trp residue (Trp780) is engaged in π-stacking
or cation-π interactions with Arg770. Additionally, in PI3Kα,
there is a hydrogen bond interaction between the indole ring of
Trp780 and Glu789 on the kβ5 strand that might limit the
mobility of the tryptophan ring.13 Taken together, these
interactions are likely to limit the access of small molecule
inhibitors to the hydrophobic face of the tryptophan ring in
PI3Kα. We were therefore particularly intrigued by the
prospect of improving the ability of our inhibitors to selectively
inhibit PI3Kδ by optimizing interactions with Trp760.
Figure 2. (A) Schematic of p110δ ATP-binding site illustrating
important areas for inhibitor interactions. The figure is based on the
crystal structure of p110δ in complex with GDC-0941 (1) (PDB ID
2WXP). GDC-0941 is colored green and shown as ball and sticks. The
different regions are highlighted by labels of the corresponding colors.
(B) Compound 1 (GDC-0941) bound to p110δ (PDB ID 2WXP,
green) with an overlay of 1 bound to p110γ (PDB ID 1E8Y, violet).
The nonconserved residues close to the inhibitor binding site are
labeled.
recently reported that highly selective PI3Kδ inhibitors can be
obtained by appropriate modification of heterocycles that
occupy the affinity pocket of the PI3K enzymes.16
The pan-PI3K inhibitor 1 has a very similar binding mode in
crystal structures of this inhibitor bound to p110δ and p110γ
(Figure 2B).6 The oxygen atom of the morpholine ring forms a
hydrogen bond in the hinge region, while the sulfonylpiper-
azine adopts a dihedral angle of 60.5° relative to the plane
defined by the thienopyrimidine ring and occupies a region that
All compounds were tested in a biochemical assay that
measured the inhibition of phosphatidylinositol-3,4,5-trisphos-
phate (PIP3) production by recombinant PI3K isoforms and in
a cellular assay that monitored inhibition of phosphorylation of
AKT in a Ri-1 cell line.18 For this study, we chose to
incorporate a piperidinyl-propan-2-ol group in place of the
dimethylpiperidinamine group contained in 2, as we reasoned
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that replacement of the basic dimethylamino group should
reduce inhibitor promiscuity and improve measured perme-
ability.19 This substitution did not sufficiently influence
inhibitor potency or PI3Kα/PI3Kδ selectivity (i.e., 2 vs 3).
As shown in Table 1, most of the benzimidazole compounds
(Table 2). Ab initio calculations20 suggested that bulky groups
at the C(2)-position of the benzimidazole would strongly
disfavor coplanarity of the aromatic heterocycles.
Table 2. Inhibition of PI3Kδ Activity by Analogues 9−15
Table 1. Inhibition of PI3Kδ Activity by Analogues 3−8
a
Inhibition of phosphatidylinositol-3,4,5-trisphosphate (PIP3) pro-
duction by recombinant PI3Kδ (nM).
a
Inhibition of phosphatidylinositol-3,4,5-trisphosphate (PIP3) pro-
duction by recombinant PI3Kδ (nM).
In general, branched substituents at the C(2) position of the
benzimidazole were accommodated by both PI3Kα and PI3Kδ
isoforms (Table 2). Incorporation of larger groups, such as the
morpholine ring found in 15, reduced inhibition of PI3Kδ.
Generally, a small variation in isoform selectivity was observed
for inhibitors in this series, although some compounds with
excellent selectivity and activity in the cellular assay were
identified (e.g., ether 10).
initially prepared are potent inhibitors of PI3Kδ and feature
some selectivity against the α-isoform. Heterocycles that
contain a nitrogen atom instead of a sulfur atom in the 5-
position of the pyrimidine ring are generally more selective and
more potent inhibitors of PI3Kδ. Compound 8, in which the
position of the methylene linker is more dramatically shifted
relative to lead compound 2 was significantly less effective than
the other analogues. These observations are consistent with the
hypothesis that movement of the piperidine group closer to
Trp760 might increase selectivity for inhibition of PI3Kδ.
Purines 4 and 5 are particularly interesting analogues, as these
inhibitors maintain good potency in the cellular assay. Most
encouragingly, additional profiling of 5 revealed that this
compound is essentially inactive against PI3Kγ (IC50 > 10 μM,
γ/δ > 5000) and only a weak inhibitor of PI3Kβ (IC50 = 547
nM, β/δ = 228).
Further analysis of the calculated ground state conformation
of 5 indicated that this inhibitor may adopt a low-energy
conformation in which the purine and benzimidazole rings are
coplanar. In order to determine if restriction of the torsional
angle between these heterocycles would influence inhibitor
selectivity or improve the physical properties of these
compounds, we prepared a series of analogues with bulky
groups attached to the C(2) position of the benzimidazole ring
Structural characterization of 14 bound to p110γ21 showed
that there is a significant (32°) torsional angle between the
benzimidazole and purine rings in the bound conformation
(Figure 3). The benzimidazole ring engages in hydrogen-
bonding interactions with Lys833, while the dimethyl amino
group is twisted relative to the plane of the benzimidazole. One
of the methyl groups of the dimethylamine unit occupies a
small hydrophobic pocket formed by Pro810 and Lys833.
The piperidine ring of 14 engages in the targeted
hydrophobic contacts with Trp812 (Trp760 in p110δ). The
observed location of the piperidine ring of 14 is strikingly
different from that found for the analogous sulfonylpiperazine
group of 1 in cocrystal structures with p110γ (14, dihedral
angle =157°, Figure 3; 1, dihedral angle = 60°, Figure 2A) The
sulfonyl oxygen atoms of 1 contact the NH3 of Lys802 and the
backbone amide of Ala805. The isopropanol unit of 14 is likely
to be unable to interact productively with these residues and
instead shifts to form weak hydrophobic interactions with
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Table 3. Inhibition of PI3Kδ Activity by Benzimidazole
Analogues 5 and 16−20
Figure 3. Co-crystal structure of 14 and p110γ (PDB-ID 4GB9). The
dihedral angle between the plane formed by the purine ring and the
piperidine ring is 157°. The hydrogen bond interactions between
Val882 in the hinge and Lys833 of the affinity pocket are represented
as black dotted lines.
Trp812. The resulting suboptimal fit is likely to be partly
responsible for the modest potency of 14 against PI3Kγ (IC50 =
540 nM). As anticipated, substitution of the 4-(propan-2-ol)-
piperidine subunit of 5 with the 4-methanesulfonylpiperazine
unit of 1 (5 to 16, Table 3) resulted in a significant increase in
activity versus PI3Kα and PI3Kγ. These structure−activity
relationships highlight the crucial role of the amine subunit in
determining PI3K isoform selectivity for this series of
inhibitors.
Further optimization focused on replacement of the 4-
(propan-2-ol)-piperidine subunit of 5 with alternative sterically
demanding amines. Compounds containing a hydrophobic
group attached to the piperazine ring (18, Table 3), bulky
amine groups (19), and azetidine compounds (20) were also
potent, selective PI3K-δ inhibitors. A docking model of 1922
(Figure 4) suggests that several methylene groups of the
bicycloheptane unit form favorable packing interactions with
the indole ring of Trp760. In summary, the structure−activity
relationship of these inhibitors provide further evidence that
high PI3K isoform selectivity can be achieved for structurally
diverse PI3Kδ inhibitors that do not occupy the selectivity
pocket formed by the movement of Met752 and Trp760. These
inhibitors instead exploit residue differences close to Trp760
that influence the ability to make favorable interactions with
this residue in PI3Kδ but not in the other PI3K isoforms.12,13
Benzimidazoles 5 and 20 are generally representative of this
series of inhibitors and were selected for further profiling.
Inhibitor 20 is soluble in aqueous solution (sol. at pH 6.5 = 338
μg/mL) and has good permeability in a standard MDCK assay
(Papp = 16 × 10−6 cm/s). Rat (90%) and human (91%) plasma
protein binding was moderate. There was no reversible or time-
dependent CYP inhibition associated with 20 in testing against
five CYP isoforms.23 In addition, very weak inhibition with (less
than 25% inhibition at 1 μM) was observed in testing against a
panel of 55 diverse kinases.24 Similar results were obtained for
compound 5. Pharmacokinetic profiling of compounds 5 and
20 in rat and dog indicated that 5 has moderate to low
a
Inhibition of phosphatidylinositol-3,4,5-trisphosphate (PIP3) pro-
duction by recombinant PI3Kδ (nM).
clearance in both species, while 20 is cleared more rapidly in
the rat (Table 4).
B-lymphocyte signaling plays a critical role in many
inflammatory diseases. We therefore chose to investigate if
our PI3Kδ-selective inhibitors could influence B-cell activation
in vivo. Activation of mouse spleen B-cells by anti-IgD
stimulates the expression of CD86 on the surface of B cells,
which can be quantified ex vivo by FACS analysis.25 To test if
PI3Kδ inhibitors might modulate the induced CD86
expression, compound 5 was orally administered to rats. After
30 min, anti-IgD was injected. Analysis of the percentage of B-
cells from the spleen that expressed CD86 after anti-IgD
treatment revealed that compound 5 could significantly inhibit
B-cell activation following oral doses as low as 1 mg/kg (Figure
5).
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a
Scheme 1
Figure 4. Model of 19 bound to p110δ. The possible hydrophobic
interaction between the bicycloheptane ring and Trp760 is represented
by the magenta dashed lines.
a
Reagents and conditions: (a) morpholine, DCM, 0 °C to rt, 1−24 h;
(b) n-BuLi, THF, −78 °C, 1 h then DMF, −78 °C, 30 min; (c) 4-(2-
propanol)-piperidine, NaHB(OAc)3, MS 4 Å, DCE, 20 h; (d) NaBH4,
MeOH, 0 °C, 1 h; (e) PBr3, DCM, 1 h; (f) 4-(2-propanol)-piperidine,
DIEA, DCM, 1−2 h; (g) 2-ethylbenzimidazole, Cs2CO3, Pd2(dba)3,
XPhos, 1,4-dioxane, 140 °C, microwave, 20−30 min; (h) 2-
ethylbenzimidazole, Cs2CO3, copper(I) thiophene-2-carboxylate,
NMP, 110 °C, 18 h; (i) PTSA, MeOH, 40 °C, 24 h.
Table 4. Pharmacokinetic Parameters for Inhibitors 5 and 20
in Rat and Dog
a
compd
species
Cl
Vdss (L/kg)
T1/2 (h)
F %
5
rat
19
5
4.1
2
1.5
6.4
2.6
3.6
92
dog
rat
>100
20
34
25
6.5
6.5
Compounds 9−11 and 13−15 were synthesized by coupling
of 36 and the corresponding C(2)-substituted benzimidazoles
(Scheme 3). Compound 12 was prepared through the
palladium-catalyzed coupling of 36 with phenylene-1,2-diamine
to provide intermediate 45. Treatment of 45 with refluxing
( )-lactic acid provided pure 12 following HPLC and SFC
purification.
dog
56
a
Reported as mL/min/kg.
This report describes the identification of a new family of
highly selective PI3Kδ inhibitors. Representative compounds
have good selectivity versus other PI3K isoforms, promising
physical properties, and anti-inflammatory properties in vivo.
The structural information disclosed supports the hypothesis
that PI3K-isoform selectivity can be achieved with inhibitors
that do not occupy the induced pocket between Trp760 and
Met752 in PI3Kδ. Improvements in the selectivity and
pharmacokinetic properties of this series of PI3K inhibitors
will be the subject of future reports.
Figure 5. Compound 5 inhibits the activation of B-cells, as measured
by CD86 expression, following anti-IgD stimulation. A: Saline. B:
MCT. C: anti-IgD + saline. D: anti-IgD + MCT.
Inhibitors 3−7 were synthesized according to the general
process as depicted in Scheme 1. Treatment of pyrimidines
21−25 with morpholine yielded tricyclic intermediates 26−
30.15 Deprotonation of tricycles 26−30 with n-butyllithium
followed by quenching with N,N-dimethylformamide afforded
the corresponding aldehydes 31−35. Reductive amination of
31−35 using 4-(2-propanol)-piperidine then generated com-
pounds 36−40. Alternatively, this transformation can be
achieved through a three-step sequence that includes reduction,
bromination and alkylation. The palladium-catalyzed coupling
of 2-chloropyrimidines 36−40 and 2-ethylbenzimidazole then
afforded compounds 3, 5−7, and 41. Treatment of 41 with acid
generated inhibitor 4. Analogous procedures were used for the
preparation of inhibitors 16−18 and 20. In a similar fashion,
the synthesis of inhibitor 19 was completed as outlined in
Scheme 2.
EXPERIMENTAL SECTION
■
All solvents and reagents were used as obtained. 1H NMR spectra were
recorded with a Bruker Avance DPX400 Spectrometer or a Varian
Inova 400 NMR spectrometer and referenced to tetramethylsilane.
Chemical shifts are expressed as δ units using tetramethylsilane as the
external standard (in NMR description, s, singlet; d, doublet; t, triplet;
q, quartet; m, multiplet; and br, broad peak). All coupling constants
(J) are reported in Hertz. Mass spectra were measured with an Agilent
quadrupole 6140 spectrometer using an ESI source coupled to an
Agilent 1200 HPLC system operating in reverse mode with an Agilent
SD-C18 (30 mm by 2.1 mm) with 1.8 μm sized particles. Analytical
purities of evaluated compounds were >95% unless stated otherwise.
The following analytic method was used, unless stated otherwise:
HPLC Agilent 1200, water with 0.05% TFA, acetonitrile with 0.05%
TFA, Agilent Zorbax SD-C18, 1.8 μM, 2.1 mm × 30 mm, 40 °C, 3−
95% B in 8.5 min, 95% in 2.5 min, 400 μL/min, 220 and 254 nm,
equipped with Agilent quadrupole 6140, ESI positive, and 110−800
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a
Scheme 2
a
Reagents and conditions: (a) 2-ethylbenzimidazole, Cs2CO3, Pd2(dba)3, XPhos, 1,4-dioxane, 140 °C, microwave, 20−30 min; (b) 44,
NaHB(OAc)3, MS 4 Å, DCE, 20 h; (c) TFA, DCM, 30 min; (d) oxetan-3-one, NaHB(OAc)3, MS 4 Å, DCE, 20 h.
a
The resulting precipitate was collected by filtration to afford 31 as a
light yellow solid (11 g, 99%).
Scheme 3
To a solution of 31 (0. 50 g, 2.0 mmol) in 1,2-dichloroethane (16
mL) was added 4-(propan-2-ol)-piperidine (0.254 g, 1.77 mmol),
micronized 4 Å molecular sieves (1.5 g), and N,N-diisopropylethyl-
amine (0.68 mL, 3.9 mmol). The resulting mixture was stirred at room
temperature for 2 h prior to the addition of sodium triacetoxybor-
ohydride (0.752 g, 3.55 mmol). The reaction was deemed complete
after stirring at room temperature for 20 h. The reaction mixture was
filtered and concentrated. The residue was purified by flash
chromatography (0−10% MeOH gradient in DCM) to give
intermediate 36 (1.56 g, 89.6%).
Alternatively, 36 can be synthesized as follows: To a solution of 31
(15.0 g, 53.3 mmol) in MeOH (700 mL) was added sodium
borohydride (10.1 g, 266 mmol) at 0 °C. The reaction was then
allowed to warm to room temperature and stirred for 30 min. The
reaction mixture was reduced to approximately 1/3 of its volume
under vacuum. To the mixture was added 0.5 L of a 1:1 (v/v) mixture
of saturated sodium bicarbonate and water. The aqueous mixture was
extracted with EtOAc. The combined organic extracts were washed
with water and dried over MgSO4, filtered, and concentrated to a white
solid.
The solid was then dissolved in a mixture of 1,2-dichloroethane
(320 mL) and THF (250 mL) and cooled to 0 °C. To the cooled
mixture was slowly added PBr3 (8.7 mL, 93 mmol) to give a slurry.
After stirring at 0 °C for 1 h, the cold reaction mixture was filtered to
collect the solid. The solid was washed with 50 mL of a 1:1 (v/v)
mixture of water and saturated sodium bicarbonate and 150 mL of
water. The washed residue was then suspended in 80 mL of a 1:4
mixture of MeOH and water. The solid was collected by filtration and
air-dried to give 4-(8-(bromomethyl)-2-chloro-9-methyl-9H-purin-6-
yl)morpholine as a white solid (12.8 g).
A portion of the white solid (2.0 g) was then redissolved in DCM
(48 mL) and to the solution was added 4-(propan-2-ol)-piperidine
(0.827 g, 5.77 mmol) and N,N-diisopropylethylamine (1.5 mL, 8.7
mmol). The reaction was then stirred at room temperature for 18 h
prior to dilution with water. The aqueous mixture was extracted three
times with DCM. The combined extracts were washed with water,
dried over Na2SO4, filtered, and concentrated to give 36 (17 g, 78%).
To a microwave tube was added 36 (2.0 g, 4.89 mmol), 2-ethyl-1H-
benzo[d]imidazole (715 mg, 4.89 mmol), cesium carbonate (3.19 g,
9.78 mmol), tris(dibenzylideneacetone)dipalladium (305 mg, 0.333
mmol), XPhos (305 mg, 0.640 mmol), and DMF (37 mL). The
reaction was heated at 145 °C for 30 min in the microwave. The
reaction was then cooled to room temperature, diluted with EtOAc,
filtered through Celite, and concentrated. The crude product was then
purified by flash chromatography (0−10% MeOH gradient in DCM)
a
Reagents and conditions: (a) benzimidazole, Cs2CO3, Pd2(dba)3,
XPhos, DMF, 140 °C, microwave, 20−30 min; (b) benzimidazole,
NaOtBu, (tBu3P)2Pd, Pd(OAc)2, toluene, 140 °C, microwave, 30 min;
(c) 1,2-phenylenediamine, Pd2(dba)3, XPhos, DMF, 140 °C, micro-
wave, 30 min; (d) ( )-lactic acid, 125 °C.
amu. Microwave reactions were performed using a Biotage Initiator
Reactor.
Preparation of Compounds 26−30. Compounds 26−30 were
prepared according to a similar procedure previously described in ref
17 and references therein. Briefly, a mixture of the dichloropyrimidine
(21−25, 1.0 mmol) and morpholine (2.5 mmol) in MeOH (80 mL)
was stirred at 0 °C for 0.5 h. The mixture was then allowed to warm to
room temperature and stirred for 1.5 h. The solvent was evaporated.
The residue was redissolved in DCM and washed successively with 1
M HCl and brine, dried over Na2SO4, filtered, and concentrated to
give 26−30 (yield 96−99%).
Preparation of 2-(1-((2-(2-Ethyl-1H-benzo[d]imidazol-1-yl)-
9-methyl-6-morpholino-9H-purin-8-yl)methyl)piperidin-4-yl)-
propan-2-ol (5). To a solution of 26 (10.0 g, 39.4 mmol) and
N,N,N′N′-tetramethylethylenediamine (9.0 mL) in THF (200 mL) at
−78 °C was added a 2.5 M solution of n-butyllithium in THF (35 mL,
87.5 mmol). The solution was stirred at −40 °C for 30 min and then
recooled to −78 °C, whereupon DMF (8 mL) was added, and the
reaction was stirred for another hour. The reaction was quenched into
a cold 0.25 N HCl solution via 10 mL serological pipet aliquots. Ice
was added to keep the quenching solution temperature below 5 °C.
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followed by reverse phase HPLC to give 5 (1.8 g, 71%) as a white
solid. 1H NMR (400 MHz, DMSO-d6) δ 8.01 (m, 1H), 7.62 (m, 1H),
7.26 (m, 2H), 4.25 (s, br, 4H), 4.02 (s, 1H), 3.81 (s, 3H), 3.77 (m,
4H), 3.73 (s, 2H), 3.28 (m, 2H), 2.90 (d, J = 10.9, 2H), 1.99 (m, 2H),
1.66 (d, J = 10.8, 2H), 1.34 (t, J = 7.43, 3H), 1.19 (m, 3H), 1.02 (s,
6H). MS (ESI): m/z (M + H)+ 519. Analytical LC-MS on an Agilent
1200 using a 2.1 mm × 30 mm SD-C18 analytical column and H2O/
MeCN modified with 0.05% trifluoroacetic acid, running a linear
gradient from 3% MeCN to 95% MeCN, monitoring by a UV
wavelength of 254 nm and ESI+ TIC MS, showed 100% purity.
Preparation of 2-(1-((2-(2-Ethyl-1H-benzo[d]imidazol-1-yl)-
6-morpholino-9H-purin-8-yl)methyl)piperidin-4-yl)propan-2-
ol (4). Compound 41 was prepared from 22 according to a procedure
similar to that described for compound 5. Compound 41 was then
treated with equimolar of p-toluenesulfonic acid in methanol at 40 °C
for 24 h. The reaction was then concentrated, and 4 was isolated by
H2O/MeCN modified with 0.05% trifluoroacetic acid, running a linear
gradient from 3% MeCN to 95% MeCN, monitoring by a UV
wavelength of 254 nm and ESI+ TIC MS, showed 99% purity.
Preparation of 2-(1-((9-Methyl-6-morpholino-2-(2-(tetrahy-
drofuran-2-yl)-1H-benzo[d]imidazol-1-yl)-9H-purin-8-yl)-
methyl)piperidin-4-yl)propan-2-ol (9). To a microwave tube was
charged 36 (65 mg, 0.16 mmol), 2-(tetrahydrofuran-2-yl)-1H-
benzo[d]imidazole (61 mg, 0.323 mmol), palladium diacetate (5.4
mg, 0.024 mmol), bis(tritert-butylphosphine)palladium (12 mg, 0.024
mmol) and sodium tert-butoxide (31 mg, 0.32 mmol). The tube was
flushed with nitrogen for 5 min prior to the addition of toluene (2.6
mL). The reaction was then sealed and heated to 145 °C for 30 min in
a microwave. The reaction mixture was diluted with MeOH and
loaded onto a Biotage Isolute SCX-2 column. The column was washed
with MeOH followed by elution of the desired product by the addition
of a 2 M ammonia solution in methanol. After concentration, the
crude product was purified by reverse phase HPLC to give the title
1
reverse phase HPLC purification. Yield = 38%. H NMR (400 MHz,
1
DMSO-d6) δ 7.96 (m, 1H), 7.62 (m, 1H), 7.23 (m, 2H), 4.24 (s, br,
4H), 4.02 (s, 1H), 3.77 (m, 4H), 3.64 (s, 2H), 3.25 (m, 2H), 2.91 (d, J
= 11.1, 2H), 1.00 (t, J = 10.9, 2H), 1.65 (d, J = 11.7, 2H), 1.30 (t, J =
11.1, 3H), 1.12−1.30 (m, 3H), 1.02 (s, 6H). MS (ESI): m/z (M + H)+
505. Analytical LC-MS on an Agilent 1200 using a 2.1 mm × 30 mm
SD-C18 analytical column and H2O/MeCN modified with 0.05%
trifluoroacetic acid, running a linear gradient from 5% MeCN to 100%
MeCN, monitoring by a UV wavelength of 254 nm and ESI+ TIC MS,
showed 100% purity.
compound (35 mg, 39%). H NMR (400 MHz, DMSO-d6) δ 7.99
(dd, J = 6.99, J = 1.75, 1H), 7.70 (dd, J = 6.79, J = 1.76, 1H), 7.29 (m,
2H), 5.88 (m, 1H), 4.25 (s, br, 4H), 4.02 (s, 1H), 3.80−3.85 (m, 4H),
3.77 (m, 4H), 3.73 (s, 2H), 2.91 (d, J = 11.4, 2H), 2.53−2.59 (m, 1H),
2.26−2.33 (m, 1H), 1.90−2.09 (m, 4H), 1.66 (d, J = 11.1, 2H), 1.14−
1.26 (m, 3H), 1.02 (s, 6H). MS (ESI): m/z (M + H)+ 561. Analytical
LC-MS on an Agilent 1200 using a 2.1 mm × 30 mm SD-C18
analytical column and H2O/MeCN modified with 0.05% trifluoro-
acetic acid, running a linear gradient from 3% MeCN to 95% MeCN,
monitoring by a UV wavelength of 254 nm and ESI+ TIC MS, showed
100% purity.
Preparation of 2-(1-((2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-
4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)piperidin-4-
yl)propan-2-ol (5). Compound 3 was prepared from 23 according to
a procedure similar to that described for compound 5. Yield = 42%. 1H
NMR (400 MHz, DMSO-d6) δ 8.00 (m, 1H), 7.65 (m, 1H), 7.42 (s,
1H), 7.25 (m, 2H), 4.07 (s, 1H), 3.97 (m, 4H), 3.83 (s, 2H), 3.81 (m,
4H), 3.27 (m, 2H), 2.99 (d, J = 10.9, 2H), 1.99 (t, J = 11.3, 2H), 1.67
(d, J = 11.3, 2H), 1.32 (t, J = 7.43, 3H), 1.11−1.29 (m, 3H), 1.04 (s,
6H). MS (ESI): m/z (M + H)+ 521. Analytical LC-MS on an Agilent
1200 using a 2.1 mm × 30 mm SD-C18 analytical column and H2O/
MeCN modified with 0.05% trifluoroacetic acid, running a linear
gradient from 5% MeCN to 100% MeCN, monitoring by a UV
wavelength of 254 nm and ESI+ TIC MS, showed 99.8% purity.
Preparation of 2-{1-[5-(2-Ethylbenzoimidazol-1-yl)-7-mor-
pholin-4-yl-thiazolo[4,5-d]pyrimidin-2-ylmethyl]piperidin-4-
yl}propan-2-ol (6). Compound 6 was prepared from 24 according to
a procedure similar to that described for compound 3. Yield = 37%. 1H
NMR (400 MHz, CDCl3): δ 8.11 (1H, m), 7.77−7.70 (1H, m), 7.23
(2H, m), 4.01−3.99 (6H, m), 3.87 (5H, m), 3.42−3.39 (2H, m), 3.10
(2H, m), 2.27 (2H, m), 1.79 (2H, m), 1.50 (2H, m), 1.46−1.40 (3H,
m), 1.32 (1H, m), 1.20 (6H, s). MS (ESI): m/z (M + H)+ 522.
Analytical LC-MS on an Agilent 1200 using a 2.1 mm × 30 mm SD-
C18 analytical column and H2O/MeCN modified with 0.05%
trifluoroacetic acid, running a linear gradient from 5% MeCN to
100% MeCN, monitoring by a UV wavelength of 254 nm and ESI+
TIC MS, showed 97.3% purity.
Preparation of 2-(1-((2-(2-(1-Methoxyethyl)-1H-benzo[d]-
imidazol-1-yl)-9-methyl-6-morpholino-9H-purin-8-yl)methyl)-
piperidin-4-yl)propan-2-ol (10). To a microwave tube was charged
36 (0.15 g, 0.37 mmol), 2-(1-methoxyethyl)-1H-benzo[d]imidazole
(0.097 g, 0.55 mmol), tris(dibenzylideneacetone)dipalladium(0) (23
mg, 0.025 mmol), XPhos (23 mg, 0.048 mmol), cesium carbonate
(0.24 g, 0.73 mmol), and DMF (3 mL). The reaction was then sealed
and heated to 145 °C for 20 min in a microwave. The reaction was
then concentrated, and the crude product was purified by reverse
1
phase HPLC to give the title compound (160 mg, 29.7%). H NMR
(400 MHz, DMSO-d6) δ 7.95 (m, 1H), 7.70 (m, 1H), 7.28 (m, 2H),
5.42 (m, 1H), 4.28 (s, br, 4H), 4.00 (s, 1H), 3.82 (s, 3H), 3.76 (m,
4H), 3.74 (s, 2H), 3.09 (s, 3H), 2.97 (m, 2H), 2.00 (m, 2H), 1.67 (m,
4H), 1.19 (m, 2H), 1.02 (s, 6H), 0.92 (m, 1H). MS (ESI): m/z (M +
H)+ 549. Analytical LC-MS on an Agilent 1200 using a 2.1 mm × 30
mm SD-C18 analytical column and H2O/MeCN modified with 0.05%
trifluoroacetic acid, running a linear gradient from 3% MeCN to 95%
MeCN, monitoring by a UV wavelength of 254 nm and ESI+ TIC MS,
showed 100% purity.
Preparation of 2-(1-((2-(2-Cyclopropyl-1H-benzo[d]-
imidazol-1-yl)-9-methyl-6-morpholino-9H-purin-8-yl)methyl)-
piperidin-4-yl)propan-2-ol (11). To a microwave tube was charged
36 (0.415 g, 1.01 mmol), 2-cyclopropyl-1H-benzo[d]imidazole (0.200
g, 1.26 mmol), tris(dibenzylideneacetone)dipalladium(0) (28 mg,
0.030 mmol), XPhos (29 mg, 0.061 mmol), cesium carbonate (0.661
g, 2.0 mmol) and DMF (5 mL). The reaction was then heated to 140
°C for 30 min in a microwave. The reaction mixture was diluted with
MeOH and loaded onto a Biotage Isolute SCX-2 column. The column
was washed with MeOH followed by elution of the desired product by
the addition of a 2 M ammonia solution in methanol. After
concentration, the crude product was purified by reverse phase
Preparation of 2-{1-[5-(2-Ethylbenzoimidazol-1-yl)-7-mor-
pholin-4-yl-thiazolo[5,4-d]pyrimidin-2-ylmethyl]piperidin-4-
yl}propan-2-ol (7). Compound 40 was first prepared from 25
according to a procedure similar to that described for compound 3. A
mixture of 40 (100 mg, 0.24 mmol), 2-ethyl-1H-benzimidazole (42
mg, 0.29 mmol), copper(I) thiophene-2-carboxylate (9 mg, 0.048
mmol), and cesium carbonate (119 mg, 0.36 mmol) in NMP (0.5 mL)
was then stirred at 110 °C for 18 h. The reaction mixture was diluted
with MeOH and loaded onto an Isolute SCX-2 cartridge (5 g). The
cartridge was washed with MeOH, and the desired product was eluted
with 2 M NH3 in MeOH. The solvents were removed, and the residue
was subjected to flash chromatography (0−20% MeOH in EtOAc)
followed by reverse phase HPLC to give the title compound as a beige
1
HPLC to give the title compound (160 mg, 29.7%). H NMR (400
MHz, DMSO-d6) δ 7.95 (m, 1H), 7.56 (m, 1H), 7.21 (m, 2H), 4.26
(s, br, 4H), 4.02 (s, 1H), 3.82 (s, 3H), 3.77 (m, 4H), 3.74 (s, 2H), 2.89
(m, 3H), 2.00 (t, J = 10.7, 2H), 1.66 (d, J = 10.4, 2H), 1.05−1.30 (m,
7H), 1.02 (s, 6H). MS (ESI): m/z (M + H)+ 531. Analytical LC-MS
on an Agilent 1200 using a 2.1 mm × 30 mm SD-C18 analytical
column and H2O/MeCN modified with 0.05% trifluoroacetic acid,
running a linear gradient from 3% MeCN to 95% MeCN, monitoring
by a UV wavelength of 254 nm and ESI+ TIC MS, showed 96% purity.
Preparation of 2-(1-((2-(2-(1-Hydroxyethyl)-1H-benzo[d]-
imidazol-1-yl)-9-methyl-6-morpholino-9H-purin-8-yl)methyl)-
piperidin-4-yl)propan-2-ol (12). To a microwave tube was charged
1
solid (45 mg, 36%). H NMR (400 MHz, CDCl3): δ 8.03−7.98 (m,
1H); 7.77−7.73 (m, 1H); 7.32−7.24 (m, 2H); 4.41 (m, 4H); 3.90−
3.85 (m, 6H); 3.35 (q, J = 7.5, 2H); 3.11 (m, 2H); 2.33−2.12 (m,
2H); 1.79 (m, 2H); 1.57 (m, 2H); 1.44 (t, J = 7.5, 3H); 1.34 (m, 1H);
1.22 (s, 6H). MS (ESI): m/z (M + H)+ 522. Analytical LC-MS on an
Agilent 1200 using a 2.1 mm × 30 mm SD-C18 analytical column and
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36 (2.2 g, 5.42 mmol), phenylene-1,2-diamine (0.73 g, 6.78 mmol),
tris(dibenzylideneacetone)dipalladium(0) (0.39 g, 0.43 mmol), XPhos
(0.41 g, 0.87 mmol), cesium carbonate (3.54 g, 10.9 mmol), and DMF
(42 mL). The reaction was then sealed and heated to 140 °C for 40
min in a microwave. The reaction mixture was allowed to cool to room
temperature and filtered. The filtrate was then concentrated to give
compound 45 as a purple solid (3.2 g). This crude material was used
in the following step without further purification.
DMSO-d6) δ 8.01 (m, 1H), 7.63 (m, 1H), 7.24 (m, 2H), 4.26 (s,
br, 4H), 3.86 (m, 7H), 3.77 (s, 2H), 3.26 (m, 2H), 3.25 (br, s, 4H),
2.76 (s, 3H), 2.60 (m, 4H), 1.45 (m, 3H). MS (ESI): m/z (M + H)+
540. Analytical LC-MS on an Agilent 1200 using a 2.1 mm × 30 mm
SD-C18 analytical column and H2O/MeCN modified with 0.05%
trifluoroacetic acid, running a linear gradient from 3% MeCN to 95%
MeCN, monitoring by a UV wavelength of 254 nm and ESI+ TIC MS,
showed 100% purity.
Crude 45 (0.53 g) was combined with ( )-lactic acid (4 mL) and
heated to 125 °C in a sealed vessel for 3.5 h. The reaction mixture was
cooled to room temperature and concentrated. The crude product was
purified by reverse phase HPLC. The enantiomers were separated by
Preparation of 2-(4-((2-(2-Ethyl-1H-benzo[d]imidazol-1-yl)-
9-methyl-6-morpholino-9H-purin-8-yl)methyl)piperazin-1-yl)-
2-methylpropanamide (17). Compound 17 was prepared according
to a procedure similar to that described for compound 3, using 2-
1
1
methyl-2-(piperazin-1-yl)propanamide. Yield = 42%. H NMR (400
chiral SFC to give 12 (71 mg, 12% from crude 45). H NMR (400
MHz, DMSO-d6) δ 8.03 (m, 1H), 7.63 (m, 1H), 7.26 (m, 2H), 7.07
(s, 1H), 6.98 (s, 1H), 4.26 (s, 4H), 3.82 (s, 3H), 3.78 (m, 6H), 3.27
(m, 2H), 2.43 (s, br, 4H), 1.34 (t, J = 7.44, 3H), 1.06 (s, 6H). MS
(ESI): m/z (M + H)+ 547. Analytical LC-MS on an Agilent 1200 using
a 2.1 mm × 30 mm SD-C18 analytical column and H2O/MeCN
modified with 0.05% trifluoroacetic acid, running a linear gradient
from 3% MeCN to 95% MeCN, monitoring by a UV wavelength of
254 nm and ESI+ TIC MS, showed 100% purity.
Preparation of 4-(2-(2-Ethyl-1H-benzo[d]imidazol-1-yl)-9-
methyl-8-((4-(methylsulfonyl)piperazin-1-yl)methyl)-9H-purin-
6-yl)morpholine (18). Compound 18 was prepared according to a
procedure similar to that described for compound 3, using 1-tert-
butylpiperazine. Yield = 64%. 1H NMR (400 MHz, CDCl3) δ 8.01 (m,
1H), 7.75 (m, 1H), 7.25 (m, 2H), 4.40 (s, br, 4H), 3.85 (m, 7H), 3.77
(s, 2H), 3.40 (m, 2H), 2.60 (s, 8H), 1.40 (m, 3H), 1.02 (s, 9H). MS
(ESI): m/z (M + H)+ 518. Analytical LC-MS on an Agilent 1200 using
a 2.1 mm × 30 mm SD-C18 analytical column and H2O/MeCN
modified with 0.05% trifluoroacetic acid, running a linear gradient
from 3% MeCN to 95% MeCN, monitoring by a UV wavelength of
254 nm and ESI+ TIC MS, showed 100% purity.
Preparation of 2-(2-Ethyl-benzoimidazol-1-yl)-9-methyl-6-
morpholin-4-yl-8-((1S,4S)-5-oxetan-3-yl-2,5-diazabicyclo-
[2.2.1]hept-2-ylmethyl)-9H-purine (19). To a pressure tube was
charged 31 (5.64 g, 20.0 mmol), 2-ethyl-1H-benzimdazole (3.21 g,
22.0 mmol), tris(dibenzylideneacetone)dipalladium(0) (0.458 g, 0.5
mmol), XPhos (0.954 g, 2.0 mmol), cesium carbonate (9.78 g, 30.0
mmol), and dioxane (80. mL). The reaction was degassed for 5 min,
sealed, and heated at reflux for 18 h. The reaction mixture was then
filtered through a pad of Celite while hot, and the pad was washed
with hot dioxane. The combined filtrate was concentrated to give a
residue which was triturated with Et2O (100 mL), filtered, and dried
under vacuum to give 42 (4.5 g, 58%)
A mixture of 42 (200 mg, 0.51 mmol), 44 (122 mg, 0.62 mmol),
and molecular sieves (4 Å, powdered, 520 mg) in DCE (10 mL) was
stirred at ambient temperature for 3 h. Sodium triacetoxyborohydride
(162 mg, 0.76 mmol) was added and the mixture stirred for 17 h, then
loaded onto an Isolute SCX-2 cartridge (25 g). The cartridge was then
washed with methanol, and the desired product was subsequently
eluted using 2 M NH3 in MeOH. The eluant was collected and
concentrated in vacuo. The resultant residue was purified by flash
chromatography (0−15% MeOH in EtOAc) to give 43 as a white solid
(294 mg, quant.).
MHz, DMSO-d6) δ 7.98 (m, 1H), 7.70 (m, 1H), 7.28 (m, 2H), 5.60
(m, 1H), 5.40 (d, J = 6.35, 1H), 4.24 (s, br, 4H), 4.05 (s, 1H), 3.81 (s,
3H), 3.78 (m, 4H), 3.73 (s, 2H), 2.90 (d, J = 10.9, 2H), 1.99 (t, J =
10.7, 2H), 1.66 (d, J = 10.4, 2H), 1.61 (d, J = 6.49, 3H), 1.21 (m, 3H),
1.02 (s, 6H). MS (ESI): m/z (M + H)+ 535. Analytical LC-MS on an
Agilent 1200 using a 2.1 mm × 30 mm SD-C18 analytical column and
H2O/MeCN modified with 0.05% trifluoroacetic acid, running a linear
gradient from 3% MeCN to 95% MeCN, monitoring by a UV
wavelength of 254 nm and ESI+ TIC MS, showed 100% purity.
Preparation of 2-(1-((2-(2-Isopropyl-1H-benzo[d]imidazol-1-
yl)-9-methyl-6-morpholino-9H-purin-8-yl)methyl)piperidin-4-
yl)propan-2-ol (13). Compound 13 was prepared from 36 according
to a coupling procedure similar to that described for compound 11,
using 2-isopropyl-1H-benzo[d]imidazole. Yield = 26%. 1H NMR (400
MHz, DMSO-d6) δ 7.89 (m, 1H), 7.64 (m, 1H), 7.23 (m, 2H), 4.26
(s, br, 4H), 4.00 (s, 1H), 3.92 (m, 1H), 3.81 (s, 3H), 3.77 (m, 4H),
3.73 (s, 2H), 2.91 (d, J = 11.0, 2H), 2.00 (t, J = 11.3, 2H), 1.66 (d, J =
12.1, 2H), 1.35 (d, J = 6.8, 6H), 1.21 (m, 3H), 1.02 (s, 6H). MS (ESI):
m/z (M + H)+ 533. Analytical LC-MS on an Agilent 1200 using a 2.1
mm × 30 mm SD-C18 analytical column and H2O/MeCN modified
with 0.05% trifluoroacetic acid, running a linear gradient from 3%
MeCN to 95% MeCN, monitoring by a UV wavelength of 254 nm and
ESI+ TIC MS, showed 100% purity.
Preparation of 2-(1-((2-(2-(Dimethylamino)-1H-benzo[d]-
imidazol-1-yl)-9-methyl-6-morpholino-9H-purin-8-yl)methyl)-
piperidin-4-yl)propan-2-ol (14). Compound 14 was prepared from
36 according to a coupling procedure similar to that described for
compound 9, using N,N-dimethyl-1H-benzo[d]imidazol-2-amine.
1
Yield = 38.1%. H NMR (400 MHz, DMSO-d6) δ 7.53 (d, J = 7.76,
1H), 7.30 (d, J = 7.74, 1H), 7.07 (t, J = 7.62, 1H), 6.94 (t, J = 7.62,
1H), 4.21 (s, br, 4H), 4.00 (s, 1H), 3.80 (s, 3H), 3.75 (m, 4H), 3.72 (s,
2H), 2.90 (s, 6H), 1.99 (t, J = 11.2, 2H), 1.66 (d, J = 11.8, 2H), 1.21
(m, 3H), 1.02 (s, 6H). MS (ESI): m/z (M + H)+ 534. Analytical LC-
MS on an Agilent 1200 using a 2.1 mm × 30 mm SD-C18 analytical
column and H2O/MeCN modified with 0.05% trifluoroacetic acid,
running a linear gradient from 3% MeCN to 95% MeCN, monitoring
by a UV wavelength of 254 nm and ESI+ TIC MS, showed 100%
purity.
Preparation of 2-(1-((9-Methyl-6-morpholino-2-(2-morpho-
lino-1H-benzo[d]imidazol-1-yl)-9H-purin-8-yl)methyl)-
piperidin-4-yl)propan-2-ol (15). Compound 15 was prepared from
36 according to a coupling procedure similar to that described for
compound 9, using 4-(1H-benzo[d]imidazol-2-yl)morpholine. Yield =
60%. 1H NMR (400 MHz, DMSO-d6) δ 7.66 (d, J = 7.6, 1H), 7.39 (d,
J = 7.6, 1H,), 7.12 (dd, J = 11.4, J = 3.77, 1H), 7.03 (m, 1H), 4.27 (s,
br, 4H), 4.02 (s, 1H), 3.82 (s, 3H), 3.76 (m, 4H), 3.72 (s, 2H), 3.64
(m, 4H), 3.23 (m, 4H), 2.90 (d, J = 11.1, 2H), 1.99 (t, J = 10.9, 2H,),
1.66 (d, J = 10.8, 2H), 1.23 (m, 3H), 1.02 (s, 6H). MS (ESI): m/z (M
+ H)+ 576. Analytical LC-MS on an Agilent 1200 using a 2.1 mm × 30
mm SD-C18 analytical column and H2O/MeCN modified with 0.05%
trifluoroacetic acid, running a linear gradient from 3% MeCN to 95%
MeCN, monitoring by a UV wavelength of 254 nm and ESI+ TIC MS,
showed 100% purity.
TFA (3 mL) was added to a solution of 43 (294 mg, 0.51 mmol) in
DCM (10 mL) and the mixture stirred at ambient temperature for 30
min, then loaded onto an Isolute SCX-2 cartridge (25 g). The
cartridge was then washed with methanol, and the desired product was
subsequently eluted using 2 M NH3 in MeOH. The eluant was
collected and concentrated in vacuo. The resultant residue was purified
by flash chromatography (0−15% MeOH in DCM) to afford 8-
[(1S,4S)-1-(2,5-diazabicyclo[2.2.1]hept-2-yl)methyl]-2-(2-ethylben-
zoimidazol-1-yl)-9-methyl-6-morpholin-4-yl-9H-purine as a white solid
(153 mg, 63%).
A solution of of 8-[(1S,4S)-1-(2,5-diazabicyclo[2.2.1]hept-2-yl)-
methyl]-2-(2-ethyl-benzoimidazol-1-yl)-9-methyl-6-morpholin-4-yl-
9H-purine (250 mg, 0.53 mmol), oxetan-3-one (40 mg, 0.31 mmol),
and molecular sieves (4 Å, powdered, 200 mg) in DCE (7 mL) was
stirred at ambient temperature for 4 h. Sodium triacetoxyborohydride
Preparation of 4-(2-(2-Ethyl-1H-benzo[d]imidazol-1-yl)-9-
methyl-8-((4-(methylsulfonyl)piperazin-1-yl)methyl)-9H-purin-
6-yl)morpholine (16). Compound 16 was prepared according to a
procedure similar to that described for compound 3, using 1-
(methylsulfonyl)piperazine. Yield = 77%. 1H NMR (400 MHz,
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(148 mg, 0.70 mmol) was added and the mixture stirred for 16 h, then
loaded onto an Isolute SCX-2 cartridge (10 g). The cartridge was then
washed with methanol, and the desired product was subsequently
eluted using 2 M NH3 in MeOH. The eluant was collected and
concentrated in vacuo. The resultant residue was purified by flash
chromatography (0−10% MeOH in DCM) to afford 19 as a white
#K151CAD, Gaithersburg, MD). IC50 values were calculated from
the fit of the dose−response curves to a 4-parameter equation.
Inhibition of CD86 Production in Mice Following Anti-IgD
Injection with Compound 5. BALB/c mice (4/group) were separated
into eight groups. Control groups were treated with saline (po),
vehicle (po), and 150 μg/mouse anti-IgD IV + saline (po), or 150 μg/
mouse anti-IgD IV + MCT. Compound treated animals were first
treated with compound (oral gavage) and, after 0.5 h, injected with
150 μg anti-IgD. Mice were euthanized 6 h after treatment with anti-
IgD. Blood was analyzed for B cell numbers and expression of CD86
by FACS.
1
solid (125 mg, 45%). H NMR (400 MHz, CDCl3,) δ 8.02 (m, 1H);
7.76 (m, 1H); 7.26 (m, 2H); 4.72−4.65 (m, 4H); 4.31 (m, 4H); 4.01−
3.86 (m, 10H); 3.35 (m, 4H); 2.99 (m, 1H); 2.82 (m, 1H); 2.76 (m,
2H); 1.79 (m, 2H), 1.45 (t, J = 7.5, 3H). MS (ESI): m/z (M + H)+
530. Analytical LC-MS on an Agilent 1200 using a 2.1 mm × 30 mm
SD-C18 analytical column and H2O/MeCN modified with 0.05%
trifluoroacetic acid, running a linear gradient from 3% MeCN to 95%
MeCN, monitoring by a UV wavelength of 254 nm and ESI+ TIC MS,
showed 100% purity.
ASSOCIATED CONTENT
* Supporting Information
■
S
Details of the cocrystal structure of compound 14 with PI3Kγ.
This material is available free of charge via the Internet at
http://pubs.acs.org.
Preparation of 4-(1-((2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-
9-methyl-6-morpholino-9H-purin-8-yl)methyl)azetidin-3-yl)-
morpholine (20). Compound 20 was prepared according to a
procedure similar to that described for compound 3, using 4-azetidin-
1
AUTHOR INFORMATION
Corresponding Author
3-ylmorpholine. Yield = 11%. H NMR (400 MHz, CDCl3): δ 8.02−
■
7.98 (m, 1H); 7.77−7.73 (m, 1H); 7.27−7.23 (m, 2H); 4.35 (m, 4H);
3.89−3.82 (m, 6H); 3.83 (s, 3H); 3.72 (m, 4H); 3.56 (m, 2H); 3.34
(q, J = 7.5, 2H); 3.15−3.03 (m, 3H); 2.34 (m, 4H) and 1.44 (t, J = 7.5,
3H). MS (ESI): m/z (M + H)+ 518. Analytical LC-MS on an Agilent
1200 using a 2.1 mm × 30 mm SD-C18 analytical column and H2O/
MeCN modified with 0.05% trifluoroacetic acid, running a linear
gradient from 3% MeCN to 95% MeCN, monitoring by a UV
wavelength of 254 nm and ESI+ TIC MS, showed 100% purity.
Characterization of Biochemical and Cellular Activity in Vitro.
Assays were performed as described previously.17 Briefly, the
enzymatic activity of the Class I PI3K isoforms was measured using
a fluorescence polarization assay that monitors formation of the
product 3,4,5-inositoltriphosphate molecule as it competes with
fluorescently labeled PIP3 for binding to the GRP-1 pleckstrin
homology domain protein. An increase in phosphatidyl inositide-3-
phosphate product results in a decrease in fluorescence polarization
signal as the labeled fluorophore is displaced from the GRP-1 protein
binding site. Class I PI3K isoforms were purchased from Millipore or
were expressed and purified as heterodimeric recombinant proteins.
Tetramethylrhodamine-labeled PIP3 (TAMRA-PIP3), di-C8-PIP2,
and PIP3 detection reagents were purchased from Echelon
Biosciences. PI3K isoforms were assayed under initial rate conditions
in the presence of 10 mM Tris (pH 7.5), 25 μMATP, 9.75 μMPIP,
2,5%glycerol, 4 mMMgCl2, 50 mMNaCl, 0.05% (v/v) Chaps, 1 mM
dithiothreitol, and 2% (v/v) DMSO at the following concentrations
for each isoform: PI3Kα,β at 60 ng/mL; PI3Kγ at 8 ng/mL; and
PI3Kδ at 45 ng/mL. After assaying for 30 min at 25 °C, reactions were
terminated with a final concentration of 9 mM EDTA, 4.5 nM
TAMRAPIP3, and 4.2 μg/mL GRP-1 detector protein before reading
fluorescence polarization on an Envision plate reader. IC50 values were
calculated from the fit of the dose−response curves to a 4-parameter
equation.
*E-mail: murray.jeremy@gene.com (J.M.M.); zachary.
sweeney@novartis.com (Z.K.S.).
Notes
The authors declare no competing financial interest.
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