Structure-based design of a novel series of potent, selective inhibitors of the class i phosphatidylinositol 3-kinases

Article abstract of DOI:10.1021/jm300184s

A highly selective series of inhibitors of the class I phosphatidylinositol 3-kinases (PI3Ks) has been designed and synthesized. Starting from the dual PI3K/mTOR inhibitor 5, a structure-based approach was used to improve potency and selectivity, resulting in the identification of 54 as a potent inhibitor of the class I PI3Ks with excellent selectivity over mTOR, related phosphatidylinositol kinases, and a broad panel of protein kinases. Compound 54 demonstrated a robust PD-PK relationship inhibiting the PI3K/Akt pathway in vivo in a mouse model, and it potently inhibited tumor growth in a U-87 MG xenograft model with an activated PI3K/Akt pathway.

Full text of DOI:10.1021/jm300184s

Article
pubs.acs.org/jmc
Structure-Based Design of a Novel Series of Potent, Selective
Inhibitors of the Class I Phosphatidylinositol 3-Kinases
,†
†
†
†
†
†
Adrian L. Smith,* Noel D. D’Angelo, Yunxin Y. Bo, Shon K. Booker, Victor J. Cee, Brad Herberich,
†
†
†
†
†
†
Fang-Tsao Hong, Claire L. M. Jackson, Brian A. Lanman, Longbin Liu, Nobuko Nishimura,
†
†
†
†
†
Liping H. Pettus, Anthony B. Reed, Seifu Tadesse, Nuria A. Tamayo, Ryan P. Wurz, Kevin Yang,
‡
§
⊥
⊥
Kristin L. Andrews, Douglas A. Whittington, John D. McCarter, Tisha San Miguel,
Leeanne Zalameda, Jian Jiang, Raju Subramanian, Erin L. Mullady, Sean Caenepeel,
Daniel J. Freeman, Ling Wang, Nancy Zhang, Tian Wu, Paul E. Hughes, and Mark H. Norman
⊥
∥
∥
∇
#
#
#
#
×
#
†
†
‡
∥
⊥
Departments of Medicinal Chemistry, Molecular Structure, Pharmacokinetics and Drug Metabolism, High-Throughput
#
×
Screening/Molecular Pharmacology, Oncology Research, and Pharmaceutics, Amgen Inc., One Amgen Center Drive, Thousand
Oaks, California 91320-1799, United States
§
∇
Departments of Molecular Structure and High-Throughput Screening/Molecular Pharmacology, Amgen Inc., 360 Binney Street,
Cambridge, Massachusetts 02142, United States
*
S Supporting Information
ABSTRACT: A highly selective series of inhibitors of the class I phosphatidylinositol 3-kinases (PI3Ks) has been designed and
synthesized. Starting from the dual PI3K/mTOR inhibitor 5, a structure-based approach was used to improve potency and
selectivity, resulting in the identification of 54 as a potent inhibitor of the class I PI3Ks with excellent selectivity over mTOR,
related phosphatidylinositol kinases, and a broad panel of protein kinases. Compound 54 demonstrated a robust PD−PK
relationship inhibiting the PI3K/Akt pathway in vivo in a mouse model, and it potently inhibited tumor growth in a U-87 MG
xenograft model with an activated PI3K/Akt pathway.
INTRODUCTION
Many of the functions of the class I PI3Ks relate to their
ability to activate protein kinase B (PKB, Akt) through
phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P3), with
downstream signaling stimulating cellular proliferation, differ-
■
The phosphatidylinositol 3-kinases (PI 3-kinases or PI3Ks) are
a family of lipid kinases involved in cellular functions such as
cell growth, proliferation, differentiation, motility, survival, and
intracellular trafficking. The PI3Ks are activated by receptor
tyrosine kinases and Ras and Rho family GTPases, and
subsequently, they phosphorylate the 3′-hydroxyl group of
phosphatidylinositol (PtdIns) and phosphoinositides, leading
to the activation of many intracellular signaling pathways. The
PI3K family is divided into three different classesclass I, class
II, and class IIIon the basis of primary structure, regulation,
and in vitro lipid substrate specificity. The class I PI3Ks have a
strong preference for phosphorylation of phosphatidylinositol-
2
entiation, and survival. PI3K signaling is constitutively
activated in many cancers by a variety of genetic alterations
that result in increased flux through the PI3K pathway.
Mutations in PIK3CA, the gene encoding p110α, are
particularly prominent in many cancers, including approx-
4
,5
imately 30% of breast, cervix, and endometrium tumors. In
addition, loss of function of the PI(3,4,5)P3 phosphatase
PTEN, a critical negative regulator of PI3K signaling, is also a
6
frequent event in many tumor types. PI3K signaling leads to
the phosphorylation of multiple substrates involved in survival
signaling by Akt. The mammalian target of rapamycin
4
,5-bisphosphate (PIP2). They are heterodimeric molecules
composed of a regulatory and a catalytic subunit and are further
divided between IA and IB subsets on the basis of sequence
similarity. The class IA PI3Ks (PI3Kα, PI3Kβ, and PI3Kδ) are
composed of heterodimers between a p110 catalytic subunit
(
mTOR), a downstream target of Akt, is a critical regulator
of PI3K/Akt signaling through positive and negative feedback
loops, and there is preclinical evidence that certain tumors with
(
p110α, p110β, and p110δ, respectively) and a p85 regulatory
subunit. The sole class IB subtype (PI3Kγ) is comprised of a
catalytic p110γ and a regulatory p101 subunit.
Received: February 9, 2012
Published: May 1, 2012
1
−3
©
2012 American Chemical Society
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deregulated PI3K/Akt signaling are particularly sensitive to
RESULTS AND DISCUSSION
■
7
−9
mTOR inhibition. Hence, it is clear that dysregulation of the
PI3K/Akt pathway contributes significantly to cellular trans-
formation and the development of cancer.
PI3K Inhibitor Design. The identification of the
benzimidazole triazine 5 (Figure 2) as a dual PI3K/mTOR
inhibitor has been described previously. As was noted in this
previous disclosure, the dual activity of 5 against the PI3Ks and
mTOR was a consequence of the high homology between the
PI3Ks and mTOR within the ATP binding site. Compound 5
was originally prepared during the course of an earlier program
targeting B-Raf, and it demonstrates potent inhibition of B-Raf
in addition to its PI3K/mTOR activities (
24
Upregulation of the PI3K/Akt pathway in many cancers and
its impact on tumor growth and survival has led to considerable
interest in several members of this pathway as possible
10−13
anticancer targets.
Close structural homology between
the p110 and mTOR kinase domains has permitted the design
12−21
of selective dual inhibitors of PI3K and mTOR,
offering
V600E
B-Raf IC50 = 3
the possibility of targeting cancers by inhibiting two critical
nodes in this pathway. In an attempt to balance efficacy with
clinical tolerability, recent efforts have also focused upon
nM; PI3Kα K = 350 nM; mTOR IC = 93 nM: see
i
50
Supporting Information for kinase selectivity data). Cocrystal
2
2,23
structures of PI3Kα with inhibitors had proven to be difficult to
deriving inhibitors with selectivity for either PI3K
mTOR.
or
Several compounds have recently entered
clinical development, including dual PI3K/mTOR inhibi-
8
,9,24−26
27,28
obtain at the outset of this work,
but a cocrystal structure of
inhibitor 5 with PI3Kγ (for which there is close sequence
homology with PI3Kα in the kinase domain) was readily
1
5,17,20
1
tors
and a more selective inhibitor of the class I PI3K
family (Figure 1). We report herein the structure-based
2
3
obtained (Figure 2). In this cocrystal structure, the amino-
triazine of compound 5 forms a bidentate hydrogen bond
interaction with Val882 in the hinge region of the kinase
domain of PI3Kγ, and the phenolic −OH forms a bridging
hydrogen bond between Asp841 and Tyr867 in a region of the
protein commonly referred to as either the affinity or selectivity
pocket. A similar phenolic substituent was reported for the lead
23
that ultimately gave rise to 4 (GDC-0941). The benzimida-
zole group itself serves as a central scaffold, with an
intramolecular hydrogen bond from the aminobenzimidazole
N−H to the adjacent triazine nitrogen atom locking the
conformation in a manner favorable for binding. The benzene
ring of the benzimidazole is orientated toward the ribose pocket
of the protein but does not substantially penetrate it.
The main liabilities of 5 as a starting point for a medicinal
chemistry program were the poor pharmacokinetic properties
24
associated with this scaffold. Not only was the phenolic
23
substituent susceptible to glucuronidation in vivo, but the
benzimidazole triazine scaffold itself was problematic due to
24
extensive metabolism of the benzimidazole. In contrast,
previous work from this group had demonstrated that it was
possible to obtain good in vivo properties with related
Figure 1. PI3K/mTOR dual inhibitors (1−3) and the class I selective
PI3K inhibitor 4.
29
pyridylpyrimidine and pyridylpurine scaffolds. This prompted
us to explore the general scaffold 6 (Figure 3), which contains
either a monocyclic or bicyclic hinge binder motif linked to a
central 2-aminopyridine core. We postulated that the central 6-
membered ring core would serve as a suitable template to
project the appropriate pharmacophoric moieties into each of
design of a new series of selective inhibitors of the class I PI3Ks
and its optimization to give a compound with potent antitumor
activity in a preclinical cancer model in vivo. A complementary
approach to a novel series of mTOR inhibitors from a similar
24
starting point has been described previously.
Figure 2. High throughput screening hit 5 and the cocrystal structure of 5 bound to the ATP binding site of PI3Kγ determined at 2.9 Å resolution.
5
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due to the presence of an additional amino acid in the hinge
region of the PI3Ks. This added substitution would give the
opportunity for significant selectivity over a broad range of
protein kinases, as was observed with the related series of
24
mTOR inhibitors.
The aminopyridine N−H of 6 would form an intramolecular
hydrogen bond with the adjacent hinge-binder nitrogen,
providing conformational rigidity to this bis-aryl combination
in a similar manner to that observed in 5 and in a conformation
that should favorably project the N-aryl substituent into the
affinity pocket. It was previously noted that the amino phenol
of 5 was likely to be subject to glucuronidation in vivo.
However, the methoxypyridine and indazole substituents of 3
1
7
(
PF-04691502) and 4 overlaid well with the phenol,
suggesting that it may be possible to replace the phenol
substituent in the affinity pocket.
Finally, molecular modeling suggested that substituents
projecting from the 5-position of the pyridine core into the
ribose pocket should be able to access similar interactions that
the respective alkoxycyclohexane and piperazine sulfonamide
substituents of 3 and 4 make in this region of the protein (the Y
substituent of generic structure 6 projecting toward Met804
and Ala805 of PI3Kγ). Some subtle residue differences exist
between the different PI3K isoforms and mTOR in the ribose
pocket region of these proteins, and we postulated that
substitution at the pyridine 5-position would offer the potential
to improve the potency, selectivity (over mTOR and between
PI3K isoforms), and pharmacokinetic properties of this new
series, as has been reported for these two clinical
Figure 3. Generic structure 6 showing potential interactions with
PI3Kγ.
the three key regions of the enzyme: the hinge region, the
affinity pocket, and the ribose pocket.
As illustrated in Figure 3, the monocyclic or bicyclic
heterocycle appended to the 3 position of the pyridine core
would provide the critical hydrogen bonding interactions with
the hinge region of the enzyme (Val882). In addition, the
cocrystal structure of compound 5 with PI3Kγ suggested that a
small lipophilic substituent X on the hinge-binding group of 6,
such as a methyl group, would favorably fill a small
hydrophobic pocket found in the PI3Ks (near Tyr867) which
is not generally present in protein kinases (excluding mTOR)
1
7,23
candidates.
Chemistry. The compounds highlighted in this report are
shown in Figure 4. Compounds 5, 7, and 8 have been
Figure 4. Structures of the scaffold 6 analogues described below.
5
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a
Scheme 1. Synthesis of Monocyclic Hinge-Binder Intermediates
a
Reagents and conditions: (a) MeMgBr, Et O; (b) NH , MeOH; (c) 4-methoxybenzyl chloride, NaH, DMF; (d) HI, CH Cl , H O; (e) NaSMe,
2
3
2
2
2
PhH; (f) i-PrMgCl, n-Bu SnCl, THF, −78 °C. *Intermediate 65d is commercially available.
3
a
Scheme 2. Main Routes for Coupling the Central Core with the Hinge-Binder and Affinity Pocket Substituents
a
Reagents and conditions: (a) Arylboronic acid + base, or arylstannane, Pd cat., Δ; (b) NaH, Boc O, DMF; (c) Method A: Ar-QH, base; Method B:
2
Ar-NH , HCl, 1,4-dioxane, H O, Δ; (d) L = N(PMB) : TFA, TfOH, Δ; (e) L = SMe: NH , 1,4-dioxane, H O, Δ; (f) L = N(Boc) : TFA, CH Cl .
2
2
2
3
2
2
2
2
2
4
previously described. The synthetic routes used to prepare
available 63a, 64b, 64c, 65d, and 65e. The chemistry is
exemplified by the versatility of 2,4,6-trichloro-1,3,5-triazine
(63a), where the differential reactivities of the three chlorine
atoms to successive substitution made it possible to selectively
replace the first chlorine atom with a methyl group (64a) and
compounds 9−62 are described below.
Scheme 1 summarizes the syntheses of key intermediates
used for the introduction of the monocylic hinge-binder
fragments onto the inhibitor scaffold from commercially
5
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the second with either an amino group (65a) or a thiomethyl
ether (68a). One of the more versatile intermediates was the
bis(4-methoxybenzyl)amino-protected derivative 66, which
incorporated two PMB protecting groups for the amino
substituent that facilitated subsequent chemistry steps. The
corresponding thiomethyl ether 68a provided an alternative
route for late-stage introduction of an amino substituent. The
third chloro substituent (e.g., on 66 or 68a) served as a suitable
coupling partner in palladium-catalyzed coupling reactions to
the central core in most cases, although the corresponding
iodides (e.g., 67) or stannanes (e.g., 70a,b; for reversed
coupling reactivity) were sometimes employed. The synthetic
routes involving the corresponding pyrimidine intermediates
the end (69a → 71h → 73h → 30) was also used, although
these strategies were generally less efficient than bis-PMB
protection of the amino group. The 5-chloro substituent on the
central core of 73j provided a convenient synthetic handle for
additional functionalization at this position to access the ribose
pocket, making 73j a versatile synthetic intermediate.
The synthesis of bicyclic pyridylpurine hinge-binder
analogues is outlined in Scheme 3. An efficient synthesis of
a
Scheme 3. Synthesis of Pyridylpurine Analogues
(
e.g., 65b or 68b) were very similar to those employed for the
triazines.
Scheme 2 summarizes the main strategies for coupling the
central core with the hinge-binder and affinity pocket
substituents. The syntheses of compounds 9−12 illustrate the
basic strategy used throughout this work, with a Pd-mediated
coupling between the chlorotriazine 65a and 2-fluoro-3-
pyridylboronic acid (to give 71a) being followed by
introduction of the affinity pocket substituent either through
a base-mediated S Ar displacement or an acid-catalyzed
N
displacement on the 2-fluoropyridine to give compounds 9−
1
2. Bis(di-tert-butyl(4-dimethylaminophenyl)phosphine)-
a
Reagents and conditions: (a) NH , H O, Δ; (b) (EtO) CH, Δ; (c)
3
2
3
dichloropalladium(II) [Pd(Amphos) Cl ] was the preferred
Pd catalyst with potassium acetate as base in either 1,4-
dioxane/water or ethanol/water as solvent for the Pd-mediated
Suzuki−Miyaura coupling reactions. The use of potassium
acetate as base helped minimize hydrolysis of the 2-
fluoropyridine under the reaction conditions compared with
stronger bases such as potassium or sodium carbonate. A
Suzuki−Miyaura cross-coupling reaction was used in most cases
throughout this work, although the pyrazines 71h,i were more
conveniently prepared from 2-fluoro-3-(tributylstannyl)-
2
2
3
,4-dihydro-2H-pyran, TsOH, CH Cl ; (d) 2-fluoropyridin-3-ylbor-
2 2
onic acid, Pd(Amphos) Cl , KOAc, EtOH, H O, Δ; (e) Ar-NH , HCl,
2
2
2
2
EtOH, H O, Δ; (f) 3-amino-6-methoxypyridine, LiHMDS, THF, 0
30
2
°
C; (g) HCl, H O, Δ. *Synthesis of 77b described in ref 29.
2
the intermediate 77a is shown starting from commercially
available 5-amino-4,6-dichloro-2-methylpyrimidine (74). Selec-
tive monoamination gave the diaminopyrimidine 75, which
furnished 6-chloro-2-methyl-9H-purine upon treatment with
triethyl orthoformate. Subsequent protection with a tetrahydro-
2H-pyran-2-yl (THP) protecting group gave purine 76, which
underwent a Suzuki−Miyaura coupling with 2-fluoro-3-
31
pyrazine using a Stille coupling. The S Ar reaction of amines
on 2-fluoropyridines using lithium bis(trimethylsilyl)amide
N
(
LiHMDS) as base in tetrahydrofuran (THF) was the most
convenient method for introducing many of the amino
substituents explored in the affinity pocket. The ether linkage
of 23 was introduced by phenolic displacement of the 2-
fluoropyridine using potassium carbonate in N,N-dimethylfor-
mamide (DMF). Some of the early compounds in this series,
such as 9−12, utilized an alternative acid-catalyzed coupling
using dilute hydrochloric acid in 1,4-dioxane at reflux to
facilitate introduction of the affinity pocket substituents onto
the 2-halopyridine central core.
A problem encountered in the syntheses of earlier
compounds such as 9−16 was the low isolated yields of the
desired products, partly due to their poor solubility and
competing deprotonation of the amino group when base-
mediated introduction of affinity pocket substituents was
attempted. The most successful strategy for overcoming this
was by masking the amino substituent with bis(4-methox-
ybenzyl) protecting groups (e.g., 66 → 71f → 73f → 24). This
significantly improved the solubility of intermediates and
resulted in much higher isolated yields. The PMB protecting
groups were readily removed at the end of the synthetic
sequence by heating in trifluoroacetic acid (TFA) containing a
small amount of trifluoromethanesulfonic acid (TfOH). Bis-
protection of the amino group with benzyloxycarbonyl (Boc)
groups (71m → 72m → 73m → 34) or masking as a
thiomethyl ether followed by displacement with ammonia at
29
pyridylboronic acid, giving 77a. Intermediates 77a and 77b
were converted to 17−19 and 21 by coupling with the relevant
amines to install the affinity pocket substituents followed by
removal of the THP protecting group under acidic conditions.
The 4-amino-1H-indazole affinity pocket substituent used for
compounds 10 and 14−17 was found to be chemically sensitive
and underwent extensive decomposition during the course of
the reactions used to prepare them, particularly under basic
conditions. It is likely that deprotonation at the 3-position of
the 1H-indazole under basic conditions gave rise to cleavage
and decomposition of the 1H-indazole. This problem could
be circumvented by THP-protection of 4-nitro-1H-indazole
(79) to give a readily separable mixture of 1H-indazole and 2H-
indazole products (80a,b) in approximately equal proportions,
which were reduced to the corresponding amines 81a,b
(Scheme 4). While the 1H-indazole derivative 81a was still
relatively sensitive to base, the 4-amino-2H-indazole 81b was
able to be coupled to the central core in moderate yield, as
illustrated in Scheme 5 with the pyridylpurine 77b and the
related 1H-pyrazolo[3,4-d]pyrimidine 84 (Scheme 6). The
overall yields for coupling and subsequent deprotection to
prepare 20 and 22 (27−37%) were noticeably improved
relative to the coupling yields for 10 and 14−17 (5−22%),
where the unprotected indazole was used.
32
5
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a
Scheme 4. Synthesis of Protected Indazole Intermediates
Scheme 7. Alternative Synthetic Routes Used To Access
a
Central-Core Analogues
a
Reagents and conditions: (a) 3,4-dihydro-2H-pyran, MP-TsOH,
EtOAc, Δ; (b) H , 10% Pd/C, EtOAc.
2
a
Scheme 5. Improved Synthesis of 1H-Indazole Analogues
a
Reagents and conditions: (a) 81b, LiHMDS, THF, 0 °C; (b) CSA,
CH Cl , MeOH, Δ.
2
2
Scheme 6. Synthesis of 1H-Pyrazolo[3,4-d]pyrimidine
a
Intermediate
a
Reagents and conditions: (a) 66, Pd(PPh ) , Na CO , DME, H O,
3
4
2
3
2
Δ; (b) 93, Cu(OAc) , i-Pr NEt, CH Cl ; (c) TFA, TfOH, Δ; (d) 70b,
2
2
2
2
Pd(PPh ) , toluene, Δ; (e) HCl, 1,4-dioxane, MeOH, Δ; (f) m-CPBA,
3
4
CH Cl then NH , 1,4-dioxane, Δ; (g) 70a, Pd(PPh ) , CuI, CsF,
2
2
3
3 4
THF, Δ; (h) NH , 1,4-dioxane, Δ.
3
enhanced sensitivity was presumably due to the amino
substituent on the central core participating in the opening of
the acetal group under acidic catalysis. The aldehydes 103a,b
were therefore readily obtained. Reduction and deprotection of
103a then provided 36.
a
Reagents and conditions: (a) LDA, N-methyl-N-(2-pyridyl)-
formamide, THF, −78 °C; then NH NH ; (b) 3,4-dihydro-2H-
2
2
pyran, MP-TsOH, EtOAc, Δ; (c) 2-fluoropyridin-3-ylboronic acid,
Pd(Amphos) Cl , KOAc, EtOH, H O, Δ.
2
2
2
The main methods used for late-stage introduction of a wide
range of ribose pocket substituents are illustrated in Scheme 9.
The chloropyridine 73j was an effective partner in a variety of
palladium-mediated coupling reactions. Alkyl substituents were
readily incorporated at the 5-pyridyl position using trifluor-
Scheme 7 illustrates the syntheses of some of the analogues
containing alternative central cores. The syntheses of 26, 28,
and 29 deviated from the previously described methodologies
33
34
by the use of a copper-mediated coupling of the boronic acid
3 with an amino-substituted central core to introduce the
oborate salts, amine substituents were introduced using
35
9
Buchwald−Hartwig aminations, and aryl substituents were
31
30
affinity pocket substituent. Stille reactions were used for
joining the central cores and hinge-binders in the syntheses of
introduced using Suzuki−Miyaura couplings. The aldehydes
103a,b were versatile intermediates for introduction of
aminomethyl substituents into the 5-pyridyl position via
reductive amination, with amine displacement on the
methanesulfonate 105 being an alternative route to these
aminomethyl analogues, as illustrated by the syntheses of
compounds 48 and 60.
2
8 and 29, reversing the polarity of the coupling relative to the
analogous couplings described so far. Additionally, the order of
attaching the affinity pocket and hinge-binding substituents to
the central core was switched during the synthesis of 29.
Scheme 8 outlines the synthesis of the key intermediates
1
03a and 105, which provided useful aldehyde and
During the later stages of this work, more efficient routes for
the synthesis of 5-(piperazin-1-ylmethyl)pyridine derivatives
were explored, particularly for the larger-scale synthesis of
compounds such as 54. The route shown in Scheme 10 was
found to be both versatile and efficient. Starting from
commercially available 2-fluoro-5-methylpyridine (109), ben-
zylic bromination followed by nucleophilic displacement with
tert-butyl piperazine-1-carboxylate gave 111. Selective ortho-
lithiation/boronic acid formation was possible to give 112, and
subsequent Suzuki−Miyaura cross-coupling reactions with 66
methanesulfonate functional handles for exploring the ribose
pocket. The thiomethyl ether derivative 103b was also used to a
lesser extent. Commercially available 6-fluoronicotinaldehyde
(97) underwent regioselective ortho-lithiation adjacent to the
fluoro-substituent after first protecting the aldehyde as an
acetal, allowing efficient formation of boronic acid 99. The
corresponding boronate ester 100 underwent Suzuki−Miyaura
coupling with triazines 66 and 68a, and the resulting products
(
101a,b) underwent a base-mediated S Ar displacement
N
reaction with 3-amino-6-methoxypyridine to give 102a,b.
Once the affinity pocket substituent was installed, the acetal
group became exceptionally sensitive to acid hydrolysis. This
or 68a (113a,b) followed by S Ar displacement with 3-amino-
N
6-methoxypyridine gave intermediates 114a,b. The Boc
protecting-group was readily removed with TFA in CH Cl
2
2
5
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a
Scheme 8. Synthesis of Key Intermediates for Exploring the Ribose Pocket
a
Reagents and conditions: (a) HOCH CH OH, TsOH, toluene, Δ; (b) LDA, (i-PrO) B, THF, −78 °C; (c) pinacol, MgSO , toluene; (d) 66 or
2
2
3
4
6
8a, Pd cat., base, 1,4-dioxane, H O, Δ; (e) 3-amino-6-methoxypyridine, LiHMDS, THF, 0 °C; (f) HCl, H O; (g) NaBH , CH Cl , MeOH, 0 °C;
2
2
4
2
2
(
h) MsCl, Et N, CH Cl , 0 °C; (i) TFA, TfOH, Δ.
3
2
2
a
Scheme 9. Alternative Methods for Introducing Ribose Pocket Substituents
a
− +
Reagents and conditions: (a) R-BF K or R R NH or R -B(OR) , Pd cat., solvent, Δ; (b) TFA, TfOH, Δ; (c) R R NH, NaBH (CN), CH Cl /
3
1
2
1
2
1
2
3
2
2
MeOH, or EtOH/AcOH; (d) NH , i-PrOH, Δ; (e) R R NH, Et N, CH Cl .
3
1
2
3
2
2
while leaving the PMB-protecting group of 114a intact, giving
15a,b. These could both be converted to the aminotriazine 50,
which was then converted to the carbamate 52. Compound
the methanesulfonamide 122 before final deprotection to 57.
The phenylsulfone 62 was efficiently prepared from 67.
Addition of the Grignard derived from 4-bromothioanisole to
the aldehyde of 67 gave the hydroxymethylene intermediate
1
1
1
15a was the more versatile of the two intermediates 115a and
15b and generally gave better overall yields of final products.
123. The thiomethyl ether substituent permitted a facile
The versatility of 115a is illustrated in Scheme 10 by its
conversion into 53−55 via intermediates 116−118.
Schemes 11 and 12 show the syntheses of compounds 57
and 62, respectively. The piperidinesulfonamide moiety of 57
was introduced by hydroboration of tert-butyl 4-methylenepi-
peridine-1-carboxylate (119), and coupling the resulting borane
to the chloride 73j to give 120, which was then converted to
reduction of the alcohol with triethylsilane in TFA by stabilizing
the benzylic cation intermediate in this reaction, resulting in
124. Ortho-lithiation was used to install a boronic acid group
on the pyridine (125), a Suzuki−Miyaura coupling with the
hinge-binder intermediate 66 gave 126, and then the
thiomethyl ether group was oxidized to the corresponding
5
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a
Scheme 10. Synthesis of 1-Piperazinylmethyl Ribose Pocket Analogues
a
Reagents and conditions: (a) NBS, Bz O , CCl , Δ; (b) tert-butyl piperazine-1-carboxylate, DMF; (c) LDA, (i-PrO) B, THF, −78 °C; (d) 66 or
2
2
4
3
6
8a, Pd cat., base, 1,4-dioxane, H O, Δ; (e) 3-amino-6-methoxypyridine, LiHMDS, THF, 0 °C; (f) TFA, CH Cl ; (g) L = N(PMB) : TFA, TfOH,
2
2
2
2
Δ; (h) L = SMe: NH , i-PrOH, Δ; (i) MeO(CO)Cl, Et N, CH Cl , 0 °C; (k) R-Cl, Et N, CH Cl .
3
3
2
2
3
2
2
a
Scheme 11. Synthesis of the Piperidine Sulfonamide 57
a
Reagents and conditions: (a) 119, 9-BBN, THF, Δ then 73j, Pd (dba) , X-Phos, Na CO , 1,4-dioxane, H O, Δ; (b) TFA, CH Cl ; (c) MsCl, Et N,
2
3
2
3
2
2
2
3
CH Cl , 0 °C; (d) TFA, TfOH, Δ.
2
2
sulfone 127. Installation of the affinity pocket substituent (128)
PI3Kγ, and PI3Kδ were measured in modified AlphaScreen
assays, mTOR kinase domain inhibitory activity was measured
in a LanthaScreen FRET assay, and cellular activity measuring
downstream inhibition of Akt phosphorylation at Ser473 was
measured in the U-87 MG human glioblastoma cell line using a
SureFire detection kit, all as previously described. The U-87
MG cell line was used, since it harbors a loss-of-function PTEN
mutation which results in an activated PI3K/Akt pathway. Data
from these assays is presented in Table 1 for compounds 5 and
followed by deprotection then gave 62.
Structure−Activity Relationships. In vitro inhibitory
activities of compounds against recombinant PI3Kα, PI3Kβ,
14
a
Scheme 12. Synthesis of the Phenylsulfone 62
7
−31 highlighting basic structure−activity relationships (SARs)
around the hinge-binder, central core, and affinity pocket
regions of the scaffold 6. The data demonstrate a good
correlation between PI3K enzyme inhibition and cellular
activity, with minimal enzyme to cell shift being observed.
Additionally, compounds in this series had good cellular
permeability and generally little P-gp liability (see the
Supporting Information). Structure−activity relationship
(
SAR) discussions around PI3K enzymatic activity will focus
primarily upon PI3Kα, since mutations in this subtype are a
major contributor to many cancers. In vitro rat microsomal
stability data is also included in Table 1, providing an estimate
of the oxidative metabolism liability for the compounds to
guide selection of compounds for progession into rodent in
a
Reagents and conditions: (a) p-MeSC H MgBr, THF, −78 °C; (b)
6
4
TFA, Et SiH, CH Cl ; (c) LiTMP, (i-PrO) B, THF, −78 °C; (d) 66,
3
2
2
3
Pd(Amphos) Cl , KOAc, EtOH, H O, Δ; (e) m-CPBA, CH Cl ; (f) 3-
2
2
2
2
2
36
amino-6-methoxypyridine, LiHMDS, THF, 0 °C; (g) TFA, Δ.
vivo studies.
5
195
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a
Table 1. Structure−Activity Relationships: Hinge-Binder, Affinity Pocket, and Central Core
PI3K enzyme K (nM)
i
cmpd
α
β
γ
δ
mTOR enzyme IC50 (nM) U-87 MG cell pAkt IC50 (nM) rat microsomal CLint (μL/min/mg)
5
7
8
9
350
190
120
52
11
3
93
8
190
17
291
394
218
80
16
2
43
4
69
4
7
9
12
3
12
9
200
60
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
92
51
88
130
25
>10 000
7200
230
65
200
9
45
150
110
51
110
23
29
37
7800
360
28
35
51
56
360
300
62
150
110
91
370
>10 000
>10 000
>10 000
530
810
75
930
990
330
3600
12
810
>1000
>1000
340
71
3200
39
1300
28
>10 000
350
76
44
190
130
23
360
560
730
90
67
45
12
70
290
280
68
>10 000
8300
1700
>10 000
>10 000
4900
640
9700
>10 000
2300
>10 000
>10 000
>10 000
580
750
1100
450
>10 000
>10 000
2600
380
690
480
59
>10 000
5700
5000
>10 000
>10 000
6000
310
>10 000
5100
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
590
115
109
56
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
218
41
118
190
133
96
620
320
240
1300
120
610
910
81
62
110
41
520
740
90
300
120
130
58
130
72
a
Assays described in ref 14. Data represents an average of at least two determinations.
Starting with the moderately potent PI3K inhibitor 5,
kinases in a custom panel of 100 kinases (see Supporting
Information). This loss of activity against B-Raf was maintained
addition of a methyl group to the triazine hinge-binder in
order to occupy the previously mentioned small hydrophobic
for all compounds tested with a methyl substituent at this
24
V600E
pocket (compound 7) resulted in a 20-fold improvement in
PI3Kα inhibitory activity along with a similar increase in
cellular activity. In addition, this methyl substituent resulted in
extremely high kinase selectivity, as evidenced by the loss of B-
position on the hinge-binder (
B-Raf IC50 >1000 nM for
compounds 7−17, 19, 22−26, 29−31). Removal of the
cyanomethyl substituent from the aminotriazine (compound
24
8) gave an additional 10-fold improvement in PI3Kα activity
while retaining excellent broad protein kinase selectivity (see
Supporting Information). Little or no selectivity was observed
3
7
V600E
Raf activity
(
B-Raf IC : compound 5 = 3 nM;
50
compound 7 = 1200 nM) and broad selectivity over protein
5
196
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between the class I PI3K subtypes or over mTOR for 5, 7, and
amino-2-methylpyrimidines 13 and 14, substitution with a
carbon atom at this position only had a modest deleterious
effect on PI3K enzyme activity, although they were
approximately 4-fold weaker than the corresponding triazines
9 and 10 in the U-87 MG cellular assay. Fluoro substitution has
often been found to be a viable replacement for an aza
substituent, but in this case replacement of the triazine 1-aza
group with C−F severely impacted activity (compound 15; c.f.
14). Increasing the size of the 4-methyl substituent of the
hinge-binder to a trifluoromethyl group (compound 16)
resulted in a large loss of activity relative to that of 14,
indicating little room to push back further into the hydrophobic
pocket.
8. The promising levels of activity secured through these initial
changes to the hinge-binding region of the molecule therefore
allowed the SAR of the different regions of scaffold 6 to be
explored.
As a first step, the aminobenzimidazole central core was
replaced with the 2-aminopyridine core of 6 to give the
pyridyltriazine 9. This modification resulted in a modest loss of
activity against PI3Kα and in U-87 MG pAkt cellular activity
and, unexpectedly, caused a larger loss of activity against
mTOR, as noted in Table 1. The pyridyl substituent does not
penetrate as far back into the ribose pocket as the
benzimidazole, and it was presumed that the reduced
hydrophobic interactions in this region were responsible for
the observed reduction in activity. It was therefore hoped that it
would be possible to regain the modest loss of activity against
PI3Kα observed with this central core by suitable substitution
from the pyridine into the ribose pocket region (Figure 3:
structure 6, substituent Y). The data for 9 also demonstrated
that it was possible to engineer selectivity against mTOR into
the scaffold 6, although the source of the selectivity over
mTOR at this stage was not obvious from an examination of an
Expanding the size of the triazine hinge-binder to a larger
bicyclic group was also explored. The 2-methylpurine analogues
17−19 displayed similar levels of activity against the PI3Ks
compared with the triazines 10−12, with 19 being equipotent
to 12 in the U-87 MG cellular assay. There was, however, a
marked reduction in selectivity over mTOR. The importance of
the hinge-binder methyl group was confirmed at this stage with
the corresponding desmethyl purine analogues 20 and 21,
which both lost greater than 100-fold activity against the PI3Ks
compared with 17 and 19. Other bicyclic hinge-binders were
explored. For example, the 6-methyl-1H-pyrazolo[3,4-d]-
pyrimidine 22 was significantly less active compared with 17.
It was previously noted in the design of scaffold 6 that the
aminopyridine N−H should form an intramolecular hydrogen
bond with the adjacent hinge-binder aza group and lock the bis-
aryl group in a conformation favorable for binding to the PI3Ks.
Replacing the N−H with an O linker and a corresponding
pyrimidine hinge-binder has been an effective strategy for other
24
mTOR homology model derived from the PI3Kγ structure.
The improvement in rat microsomal stability going from 8 to 9
Table 1) was noteworthy, reflecting the previously noted
(
superior rat PK of the pyridyltriazine scaffold relative to the
benzimidazole trazine scaffold.
The next issue to address was finding a suitable replacement
for the phenol substituent with one more likely to have
favorable pharmacokinetic properties while maintaining PI3K
activity. With this goal in mind, the indazole, indole, and
methoxypyridine analogues 10, 11, and 12 were prepared. All
three compounds displayed similar levels of activity (Table 1),
with roughly a 10-fold drop in PI3K enzyme activity relative to
that of the phenol 9 being mitigated by a slightly smaller
kinase projects, particularly in improving physicochemical
39−42
properties such as solubility.
An unfavorable lone pair−
lone pair repulsion between the O linker and an adjacent
pyrimidine N should result in the hinge-binder rotating 180°
relative to the cases of 13−15 in its preferred binding
conformation, making 23 an appropriate test of this strategy.
The data for 23 suggested that incorporation of an O linker was
a highly disadvantageous strategy for the PI3Ks. Similarly, as
expected, methylation of the N−H (compound 24) severely
impacted activity, as did reversing the positions of the amino
and methyl substituents on the pyrimidine hinge-binder
(
approximately 4-fold) reduction in cellular activity. All three
compounds maintained good selectivity over mTOR, and 10
and 11 lacked activity against the class III PI3K hVPS34 (IC50
>9000 nM), giving the first indication of selectivity for this
scaffold against nonclass I PI3Ks. Compounds 10, 11, and 12
also displayed excellent kinase selectivity in a custom panel of
100 kinases (see Supporting Information). Rat microsomal
stabilities for the indazole 10 and especially the methoxypyr-
idine 12 were reasonable, with the indole 11 demonstrating the
least favorable stability. While compounds 10−12 were
significantly less active than the starting point (compound 8),
they had low molecular weights (MW = 318, 317, and 309,
respectively) and consequently high ligand efficiencies (LE =
(
compound 25). The 2-amino-4-methyl-1,3,5-triazine hinge-
binding motif was therefore focused upon in subsequent work
on this series.
Variations on the central pyridine core were next explored.
Replacing the pyridine with a phenyl ring (compound 26)
resulted in a 6-fold loss in PI3Kα activity relative to the case of
compound 12, and moving the pyridine nitrogen around the
ring (compounds 27 and 28) had a similar detrimental effect
on enzyme potency. Addition of a nitrogen atom to give a
pyrimidine central core (compound 29) had little effect on
PI3Kα enzyme activity but was deleterious to U-87 MG cellular
activity. The isomeric 2-pyrazinyltriazine 30 lost a little activity,
possibly due to an unfavorable lone pair−lone pair repulsion
between the pyrazine and triazine nitrogen atoms, while the
corresponding 2-pyrazinylpyrimidine 31 (where no such
interaction can exist) regained the lost enzyme activity. In
summary, the preliminary SAR efforts involving the hinge-
binder and central core variants suggested that the 2-
aminopyridine was an appropriate central core to build out
from into the ribose pocket in combination with the 2-amino-4-
38
0
.29, 0.28, and 0.30 for PI3Kα, respectively). The trade-off in
potency for the potential of improved in vivo properties was
therefore considered worthwhile. The favorable overall profile
of compounds containing the methoxypyridine affinity pocket
substituent relative to the corresponding indazole or indole
motifs led to the methoxypyridine being adopted during the
later stages of the work described herein.
Molecular modeling suggested that the 2-amino-4-methyl-
triazine hinge-binder formed favorable interactions with a hinge
Val residue of the PI3Ks through hydrogen bonds with the 3-
aza and 2-amino triazine substituents, while the 4-methyl group
formed a favorable hydrophobic interaction in a small pocket
flanked by a Tyr side chain, as indicated in the PI3Kγ model
with 6 (Figure 3). The triazine 1-aza group appeared to have
little interaction with the protein and, as can be seen with the 4-
5
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Article
a
Table 2. Structure−Activity Relationships: Ribose Pocket
PI3K enzyme K (nM)
i
cmpd
α
β
γ
δ
mTOR enzyme IC50 (nM)
U-87 MG cell (nM)pAkt IC50
rat microsomal CLint (μL/min/mg)
1
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
6
6
2
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
110
290
250
240
55
63
49
280
22
5
23
38
75
86
15
56
20
67
9
29
40
22
54
9
37
39
92
110
7
7800
>10 000
>10 000
>10 000
>10 000
2500
360
390
830
310
42
51
58
111
243
86
43
58
31
67
83
23
25
24
37
37
51
31
23
20
35
48
20
17
26
73
20
111
242
23
21
62
59
24
9
20
10
30
4
88
>10 000
>10 000
>10 000
>10 000
1400
150
150
83
99
4
3
2
2
47
39
8
7
21
8
16
7
88
89
6
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
3700
29
8
6
6
34
160
270
150
100
200
280
61
260
94
84
9
22
65
11
7
140
210
80
17
110
100
200
130
18
8
50
60
19
18
63
51
27
34
6
620
220
140
130
350
390
200
230
62
41
59
17
44
18
16
5
3
1400
140
16
4
2
4800
17
22
9
7
9
6
>10 000
>10 000
>10 000
>10 000
3600
23
5
9
3
46
15
61
5
8
4
54
33
16
73
59
3
46
5
21
3
65
38
13
48
2
30
11
7
13
15
1
>10 000
>10 000
>10 000
120
700
11
a
Assays described in ref 14. Data represents an average of at least two determinations.
methyltriazine hinge-binder and methoxypyridine affinity
pocket groups.
Metabolite ID studies with this scaffold had suggested that the
5-pyridyl position was a potential metabolic “soft spot”, but this
was not supported by the lack of improvement observed in
microsomal stability by substituting at this position (cf.
compounds 12 and 32). Deeper projection into the ribose
pocket with either a methoxy or hydroxymethyl group
(compounds 35, 36) gave a more sizable increase in enzyme
inhibitory activity, which translated into significant improve-
ments in cellular activity. Further extension into the ribose
pocket with compounds 37 and 38 resulted in a slight drop in
Table 2 shows the SAR obtained by substitution from the 5-
pyridyl position of compound 12, with the indicated
substituents Y and Y extending into the ribose pocket of
1
2
the PI3Ks. Compounds 32−62 all continued to demonstrate
good selectivity over mTOR. The introduction of small
substituents (Cl, Br, Me: compounds 32−34) into the ribose
pocket was well tolerated by the PI3Ks but had minimal effect
on enzyme and cellular activity relative to the case of 12.
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Journal of Medicinal Chemistry
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cellular activity relative to the case of 35, indicating that careful
exploration of substituents in the ribose pocket would be
necessary.
Substitution of the 5-pyridyl position with a range of
heterocyclic groups (for example, compounds 39−43) was
successful at improving activity, with 40, 42, and 43 inhibiting
the PI3K enzyme subtypes with K values <10 nM and
i
possessing cellular activities <50 nM. Selectivity over mTOR
was maintained with these compounds, and the intrinsic
clearances observed when incubated with rat liver microsomes
were low.
Modeling studies with the PI3Kγ crystal structure indicated
that a methylene spacer at the 5-pyridyl position would provide
a favorable trajectory for substitutents to access and obtain
favorable interactions in the ribose pocket, and considerable
effort was therefore put into exploring this substitution mode
(
compounds 44−62, Table 2). A range of aliphatic heterocycles
2
4−51), but they appeared to offer little improvement in
Figure 5. Cocrystal structure of 54 with PI3Kγ determined at 2.95 Å
resolution. Dashed lines indicate hydrogen bonds, and red spheres
represent ordered water molecules.
(
Y ) of varying ring size were well accommodated (compounds
4
inhibiting the PI3Ks relative to the case of 34. The piperazine
groups of compounds 50 and 51 were of particular interest,
however, since modeling in PI3Kγ suggested that substitution
from the distal piperazine nitrogen atom with hydrogen bond
acceptors capable of interacting with the side chain of Lys802
or the backbone N−H of Ala805 may give a boost in affinity by
picking up additional hydrogen bonding interactions with the
protein. The methyl carbamate 52 and N,N-dimethyl urea 53
provided only modest improvements in PI3K inhibition. By
contrast, the corresponding methyl sulfonamide-substituted
piperazine 54 resulted in a significant improvement in PI3K
inhibition that translated into a 15-fold improvement in cellular
activity relative to the cases of 50 and 51. In profiling
compound 54 more fully, it not only demonstrated extremely
high selectivity over a panel of protein kinases (minimal
inhibition at 1 μM against 98 protein kinases tested), but it also
demonstrated some selectivity against class II PI3Ks (PIK3C2α
IC50 >10 μM; PIK3C2β IC50 = 29 nM), excellent selectivity
against the class III PI3K VPS34 (IC50 >9 μM), excellent
selectivity against the PI4-kinases (PIK4α IC >10 μM; PIK4β
piperazine ring size (compounds 58 and 59) or incorporating
the SO group within the ring (the thiomorpholine 1,1-dioxide
2
60) appeared to offer no advantage. However, the methyl
phenylsulfone 62 was 4-fold more active at inhibiting the
PI3Kα enzyme and slightly more active in the cellular assay
compared with 54. The contribution of the methylsulfone
moiety to the activity of 62 was illustrated by comparison with
the case of analogue 61, which was 24-fold less active against
PI3Kα and 60-fold less active in the cellular assay.
In Vivo Studies. The rat pharmacokinetic (PK) properties
of some of the more potent analogues were evaluated in order
to select compounds to progress into in vivo pharmacodynamic
(PD) and xenograft efficacy studies. The key PK parameters for
compounds 54, 56, 57, and 62 are shown in Table 3. Rat iv
Table 3. Pharmacokinetic Parameters for Compounds 54,
56, 57, and 62
a
iv (1 mg/kg)
po
5
0
IC50 = 5.2 μM), and excellent selectivity against the PI3K-
related protein kinase DNA-PK (IC >10 μM), indicating that
MRT
(h)
CL
(L/(h·kg))
Vss
(L/kg)
AUC
(μM·h)
F
(%)
cmpd species
5
0
b
c
54
54
56
57
62
rat
1.6
1.6
1.3
1.0
1.2
1.7
2.5
2.5
2.8
1.8
2.6
4.1
2.9
2.7
0.68
77
5
4 was a highly selective inhibitor of the class I PI3Ks.
A cocrystal structure of 54 was obtained with PI3Kγ (Figure
). Compound 54 bound with two hydrogen bonds from the
d
d
mouse
rat
19.1
95
b
b
0.33
18
5
b
b
rat
0.04
3
aminotriazine ring to the hinge, with the triazine methyl
substituent oriented toward the Tyr867 side chain, and with the
b
b
rat
2.1
0.04
2
a
b
4
-methoxypyridine substituent projected into the affinity
Compound dosed iv as a DMSO solution. Compound dosed at 2
mg/kg po in 1% Pluronic F68/2% HPMC/15% HPBCD/82% H O/
pocket. The methoxypyridine nitrogen atom made a hydrogen
bond to an ordered water molecule sitting between Tyr867 and
Asp841, and the piperazine sulfonamide extended into the
ribose pocket with the sulfonamide oxygen atoms making
hydrogen bonds to the backbone N−H of Ala805 and the side
chain of Lys802. No specific interactions were observed
between the central pyridyl nitrogen atom and the protein.
Having identified the piperazine sulfonamide 54 as a potent
inhibitor of cellular Akt phosphorylation, modifications were
explored that attempted to maintain similar favorable
interactions with the protein. The N,N-dimethylsulfamide 55
was equipotent to 54 but displayed inferior rat microsomal
stability. The importance of the piperazine nitrogen atoms was
explored through the corresponding piperidines 56 and 57,
with both compounds showing an approximately 3-fold
reduction in cellular activity. Expanding or contracting the
2
c
pH 2.2 with MsOH. Compound dosed at 25 mg/kg po in 1%
Pluronic F68/2% HPMC/15% HPBCD/82% H O/pH 2.2 with
2
d
MsOH. Compound dosed at 25 mg/kg po in 1% Tween 80/2%
HPMC/97% H O/pH 2.2 with MsOH.
2
clearance was only moderate for both 54 and 62, and high for
56 and 57, despite the rat in vitro microsomal stabilities of
these compounds being generally good. The oral bioavailability
and plasma exposure of 54 were significantly better than those
obtained with 56, 57, or 62, and it had the lowest iv clearance
out of this group. Mouse PK was therefore obtained on 54
(Table 3), confirming that mouse PK parameters were
comparable to those in rat and that good oral exposure in
mouse could be achieved. In addition, plasma protein binding
(PPB) data for 54 indicated that there would be a high
5
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Journal of Medicinal Chemistry
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unbound fraction in plasma in vivo (PPB: mouse = 87.5%; rat =
by daily dosing with 54 at doses that achieved >60% inhibition
of PI3K for at least 8 h per day over 14 days, but maintaining
continuous robust inhibition of PI3K over 14 days may be
poorly tolerated.
8
2.6%; human = 82.7%).
The ability of 54 to inhibit HGF-stimulated PI3K signaling
was evaluated in a mouse liver PD assay. Compound 54 was
dosed at 25 and 75 mg/kg in CD1 nude mice. Human HGF
was injected iv at 3, 8, and 24 h postdose to induce PI3K-
dependent Akt phosphorylation in the liver, and pAkt(S473)
levels in the liver were then measured 5 min after HGF
administration. The levels of pAkt(S473) inhibition relative to
vehicle control are shown in Table 4, together with measured
CONCLUSIONS
■
A new series of highly selective inhibitors of the class I PI3Ks
has been designed and synthesized. The most potent
compounds have K values <10 nM against PI3Kα, PI3Kβ,
i
PI3Kγ, and PI3Kδ; have >1000-fold selectivity against mTOR;
and inhibit phosphorylation of Akt with IC values <50 nM in
Table 4. Liver PD Assay Data for Compound 54
50
the U-87 MG glioblastoma cell line containing an upregulated
PI3K/Akt pathway. Compound 54 demonstrated excellent
selectivity over related phosphatidylinositol kinases as well as a
broad panel of protein kinases. Compound 54 had good oral
exposure in mice, and this led to significant inhibition of PI3K/
Akt pathway signaling and antitumor efficacy in a mouse U-87
MG xenograft model. Further optimization of this series,
including substantial improvements in PK properties and in
vivo efficacy, will be reported in due course.
a
dose
2
b
5 mg/kg
75 mg/kg
b
time pAkt %Inh plasma conc (μM) pAkt %Inh plasma conc (μM)
3
8
h
h
96 ± 4
93 ± 7
11 ± 10
2.70
1.22
0.00
98 ± 2
99 ± 1
64 ± 32
7.36
2.80
0.54
24 h
a
Female CD1 nude mice were dosed po with 54 in 1% Pluronic F68/
% HPMC/15% HPBCD/82% H O/pH 2.2 with MsOH. Percent
b
2
2
inhibition of phosphorylation of Akt at Ser473 in liver determined by
comparison of pAkt(S473)/total Akt ratios between drug treated
groups and a 3 h vehicle-treated group (n = 3 per group).
EXPERIMENTAL SECTION
■
Chemistry. All reactions were run under N and stirred
2
using a PTFE-coated magnetic stirbar unless noted otherwise.
Reactions run at elevated temperature were performed using a
magnetic hot plate stirrer at the temperature indicated utilizing
an oil-bath, aluminum heating block, or aluminum beads for
heat transfer. Microwave reactions were run either in a Biotage
microwave reactor set to normal power at the indicated
temperature or in a CEM Discover microwave equipped with a
PowerMAX feature, and they were performed in sealed
microwave reaction vessels. All solvents and reagents obtained
from commercial sources were used without further purification
unless noted. Solutions were concentrated using a rotary
evaporator, and solids were collected by filtration using either a
plasma concentrations. The lower 25 mg/kg dose provided
near-complete target coverage for 8 h, but the relatively short
mean residence time of the compound resulted in loss of target
coverage by 24 h. By contrast, the higher 75 mg/kg dose was
able to maintain sufficient plasma concentrations for 24 h to
provide at least 64% target coverage over a 24 h period (plasma
free fraction concentration = 68 nM at 24 h). The ability of 54
to inhibit tumor growth in CD1 nude mice in an established U-
87 MG xenograft model was therefore evaluated (Figure 6).
̈
Buchner funnel or a sintered funnel. Residual solvent was
removed from all nonvolatile products and intermediates using
a vacuum manifold maintained at approximately 1 Torr. All
yields reported are isolated yields after removal of residual
solvents.
Analytical TLC was performed with EMD 0.25 mm silica gel
6
0 plates with a 254 nm fluorescent indicator. Plates were
developed in a covered chamber and visualized with ultraviolet
light. Silica gel column chromatography refers to column
chromatography as described by Still using EMD silica gel 60,
4
3
2
30−400 mesh, as the stationary phase, or by the use of an
automated medium pressure Teledyne ISCO purification
system using RediSep columns. The eluent solvent systems
and gradients are as noted.
H NMR spectra were obtained on a Bruker BioSpin GmbH
magnetic resonance spectrometer. H NMR spectra are
Figure 6. Daily dosing with 54 caused tumor growth inhibition in an
established U-87 MG glioblastoma xenograft model in female CD1
nude mice (n = 10 per group).
1
1
reported as chemical shifts in parts-per-million (ppm) relative
to an internal solvent reference. Peak multiplicity abbreviations
are as follows: s (singlet), br s (broad singlet), d (doublet), dd
(doublet of doublets), t (triplet), q (quartet), quin (quintet),
and m (multiplet).
Analytical HPLC and mass spectroscopy were conducted
using a reversed-phase Agilent 1100 Series HPLC-mass
spectrometer. Purities for final compounds were measured
using UV detection at 254 and 215 nm and are ≥95.0% unless
otherwise noted. A detailed description of the HPLC methods
Dosed orally at 3, 10, 25, and 75 mg/kg QD, compound 54
caused a dose-dependent inhibition of tumor growth with an
ED50 of 6.0 mg/kg (AUC0−24 h = 7.6 μM·h), and tumor stasis
was achieved at 25 mg/kg QD. No significant reduction in
body weight was observed over 14 days dosing up to 25 mg/kg
QD, but the highest 75 mg/kg QD dose resulted in 15% body
weight loss after dosing for 14 days. Figure 7 shows
corresponding U-87 MG tumor PD data from a single dose
study at 10 and 75 mg/kg. The data indicated that antitumor
efficacy could be achieved in this PTEN-null xenograft model
5
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Figure 7. U-87 MG tumor PD assay in female CD1 nude mice following a single dose with compound 54 at 10 mg/kg or 75 mg/kg PO in 1%
a
Pluronic F68/2% HPMC/15% HPBCD/82% H O/pH 2.2 with MsOH. Determined by comparison of pAkt(S473)/total Akt ratios between drug
2
treated groups and a 3 h vehicle-treated group (n = 3 per group). *p < 0.0006 (Dunnett’s method).
that were used for analyzing final compounds is included in the
Supporting Information.
filtration, washed with H O, and dried to give 65c (924 mg,
67%) as a white solid. LC−MS m/z: 163 (M + H) .
2
+
2,4-Dichloro-6-methyl-1,3,5-triazine (64a). A 3.0 M
4-Chloro-N,N-bis(4-methoxybenzyl)-6-methyl-1,3,5-
triazine-2-amine (66). A suspension of 65a (10.00 g, 69.2
mmol) in DMF (60 mL) at 0 °C was treated with NaH (60%
dispersion in mineral oil; 6.9 g, 160 mmol) portionwise. The
mixture was stirred for 30 min, and then 4-methoxybenzyl
chloride (2.1 mL, 15 mmol) was added dropwise. The mixture
was kept at 0 °C for 30 min, then at 22 °C for 3 h, and then
cooled to 0 °C, and the reaction was quenched with ice−water
(100 mL). The mixture was diluted with EtOAc (100 mL) and
separated, and the organic layer was dried (Na SO ), filtered,
solution of MeMgBr in Et O (10.0 mL, 30 mmol) was added
2
slowly to a white suspension of 2,4,6-trichloro-1,3,5-triazine
(
63a) (3.68 g, 20 mmol) in CH Cl (25 mL) at 0 °C, and the
2 2
resulting yellow suspension was allowed to warm to 22 °C and
was stirred for 3 h. The reaction was carefully quenched with
saturated aqueous NH Cl at 0 °C and then diluted with H O
4
2
and CH Cl (25 mL). The organic layer was separated, dried,
2
2
filtered, and concentrated to give 64a as a yellow solid (2.94 g,
1
9
0%), which was used without further purification. H NMR
2
4
(
400 MHz, CDCl ): δ 2.74 (s, 3H).
and concentrated. The residue was purified by silica gel column
3
4
-Chloro-6-methyl-1,3,5-triazin-2-amine (65a). A 2.0 M
chromatography (gradient: 0% → 8% EtOAc/hexanes) to give
66 (13.18 g, 50%) as a white solid. H NMR (400 MHz,
1
solution of NH in MeOH (36 mL, 72 mmol) was added
3
dropwise at 22 °C to a stirred suspension of 64a (2.94 g, 18.0
mmol) in toluene (20 mL) over 1.5 h [note: the reaction was
slightly exothermic]. The resulting mixture was stirred for an
additional 2.5 h, concentrated, and purified by silica gel column
chromatography (gradient: 0% → 10% MeOH/CH Cl ) to
CDCl ): δ 7.16 (t, J = 8.12 Hz, 4H), 6.95−6.81 (m, 4H), 4.74
3
(s, 2H), 4.69 (s, 2H), 3.81 (s, 6H), 2.45 (s, 3H).
4-Iodo-N,N-bis(4-methoxybenzyl)-6-methyl-1,3,5-tria-
zin-2-amine (67). A mixture of 66 (1.02 g, 2.65 mmol) and
67% aqueous HI (0.50 mL, 6.6 mmol) in CH Cl (10 mL) was
2
2
2
2
1
give 65a (1.88 g, 73%) as a yellow solid. H NMR (300 MHz,
stirred at 22 °C for 19 h. The reaction mixture was diluted with
saturated aqueous NaHCO3 (30 mL) and extracted with
EtOAc (2 × 40 mL). The organic extract was washed with
saturated brine (20 mL), dried (Na SO ), filtered, concen-
CD OD): δ 2.32 (s, 3H).
3
6-Chloro-2-methylpyrimidin-4-amine (65b). NH3 (2
M) in MeOH (6 mL, 12 mmol) was added to a stirred
2
4
suspension of 4,6-dichloro-2-methylpyrimidine (64b) (487 mg,
trated, and purified by silica gel column chromatography (50%
CH Cl /hexanes) to give crude 67 (826 mg, 65%) as a 3:1
2.99 mmol) in 1,4-dioxane (10 mL) at 22 °C. The reaction
2
2
vessel was sealed and stirred at 70 °C for 16 h. After cooling,
the reaction mixture was concentrated and the residue was
purified by silica gel column chromatography (gradient: 0% →
mixture of 67 and 66. This was used in subsequent reactions
without further purification. LC−MS m/z: 476 (M + H) .
2-Chloro-4-methyl-6-(methylthio)-1,3,5-triazine (68a).
MeSNa (490 mg, 7.0 mmol) was added portionwise at 0 °C to
a stirred cloudy solution of 64a (1.04 g, 6.3 mmol) in toluene
(10 mL) over 15 min. The pale-yellow mixture was stirred at 0
+
1
0% MeOH/CH Cl ) to give 65b (250 mg, 58%) as a white
2 2
+
solid. LC−MS m/z: 144 (M + H) .
-Chloro-5-fluoro-2-methylpyrimidin-4-amine (65c).
A mixture of 4,6-dichloro-5-fluoro-2-methylpyrimidine (64c)
6
°C for a further 1 h, and then H O (10 mL) was added. The
2
(
1.55 g, 8.60 mmol) in aqueous NH (∼28% w/v) (10 mL)
separated aqueous layer was extracted with EtOAc (2 × 20
mL), and the combined organic layers were washed with
saturated brine, dried (Na SO ), concentrated, and purified by
3
and MeOH (1 mL) was heated at 70 °C for 2 h (sealed tube).
After cooling, H O (10 mL) was added and the mixture was
2
2
4
stirred for 30 min. The resulting solid was collected by
silica gel column chromatography (gradient: 0% → 70%
5
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Article
CH Cl /hexanes) to give 68 (870 mg, 78%) as a white solid.
4-(2-Fluoropyridin-3-yl)-6-methyl-1,3,5-triazin-2-
amine (71a). A mixture of 65a (423 mg, 2.93 mmol), 2-
fluoropyridin-3-ylboronic acid (619 mg, 4.39 mmol), Pd-
2
2
1
H NMR (400 MHz, DMSO-d ): δ 2.55 (s, 3H), 2.51 (br s,
6
3
H).
-Chloro-2-methyl-6-(methylthio)pyrimidine (68b). A
suspension of 64b (11.50 g, 70.6 mmol) and MeSNa (5.93 g,
5 mmol) in toluene (100 mL) was stirred at 22 °C for 24 h.
4
(Amphos) Cl (91 mg, 146 μmol), and KOAc (862 mg, 8.78
2 2
mmol) in 1,4-dioxane (6 mL) was heated at 100 °C for 16 h.
The mixture was allowed to cool and passed through a short
plug of diatomaceous earth, washing with EtOAc (3 × 15 mL).
The filtrate was concentrated and purified by silica gel column
8
The reaction mixture was concentrated, and the residue was
partitioned between EtOAc (100 mL) and saturated brine (100
mL). The organic layer was separated, washed with saturated
brine (50 mL), and dried (Na SO ). The crude product was
chromatography (gradient: 0% → 3% MeOH/CH
Cl
71a (454 mg, 76%) as a pale-brown powder. H NMR (400
MHz, DMSO-d ): δ 8.48 (ddd, J = 9.91, 7.65, 2.01 Hz, 1H),
) to give
2
2
1
2
4
purified by recrystallization from hexanes to give 68b (5.50 g,
6
1
4
1
5%) as white crystals. H NMR (400 MHz, CDCl ): δ 7.01 (s,
8.39 (d, J = 5.02 Hz, 1H), 7.65 (br s, 2H), 7.51 (ddd, J = 7.15,
5.14, 1.76 Hz, 1H), 2.37 (s, 3H).
6-(2-Fluoropyridin-3-yl)-2-methylpyrimidin-4-amine
(71b). A mixture of 65b (256 mg, 1.78 mmol), 2-fluoropyridin-
3
H), 2.64 (s, 3H), 2.56 (s, 3H).
2-Iodo-4-methyl-6-(methylthio)-1,3,5-triazine (69a). A
mixture of 68a (2.11 g, 12.0 mmol) and 67% aqueous HI (2.3
mL, 30 mmol) in CH Cl (4 mL) was stirred at 22 °C for 3 h.
3-ylboronic acid (376 mg, 2.67 mmol), Pd(Amphos) Cl (55
2 2
2
2
The solid was collected by filtration and washed with CH Cl .
mg, 89 μmol), and KOAc (524 mg, 5.34 mmol) in 1,4-dioxane
(3 mL) was heated at 120 °C for 30 min in a microwave
reactor. The mixture was allowed to cool and passed through a
short plug of diatomaceous earth, washing with EtOAc (3 × 10
mL). The filtrate was concentrated and purified by silica gel
2
2
The solid was treated with saturated aqueous NaHCO (10
3
mL) and extracted with EtOAc (2 × 30 mL). The combined
organic extracts were washed with saturated brine (10 mL),
dried (Na SO ), filtered, concentrated, and purified by silica gel
2
4
column chromatography (50% CH Cl /hexanes) to give 69a
column chromatography (gradient: 0% → 5% MeOH/CH Cl )
2 2
2
2
1
(
2.10 g, 65%) as a yellow solid. H NMR (300 MHz, CDCl ): δ
to give 71b (304 mg, 84%) as a white solid. LC−MS m/z: 205
3
+
2
.52 (s, 3H), 2.50 (s, 3H).
-Iodo-2-methyl-6-(methylthio)pyrimidine (69b). A
solution of 68b (5.30 g, 30.3 mmol) and 67% aqueous HI
(M + H) .
4
5-Fluoro-6-(2-fluoropyridin-3-yl)-2-methylpyrimidine-
4-amine (71c). A mixture of 2-fluoropyridin-3-ylboronic acid
(
5.7 mL, 76 mmol) in CH Cl (20 mL) was stirred at 22 °C for
(666 mg, 4.73 mmol), 65c (509 mg, 3.15 mmol), Na
(835 mg, 7.89 mmol), and Pd(PPh (364 mg, 0.32 mmol) in
DME (6 mL) and H O (0.6 mL) was heated at 100 °C for 60
min in a microwave reactor. The mixture was diluted with H O
2
CO ·H O
2 3 2
2
2
2
0 h. The solid was collected by filtration and washed with
)
3 4
CH Cl . The solid was suspended in saturated aqueous
2
2
2
NaHCO (100 mL) and extracted with EtOAc (3 × 100
mL). The combined organic extracts were washed with
saturated brine, dried (Na SO ), and concentrated to give
3
(100 mL), and the precipitate was collected by filtration,
washing with CH Cl , to give 71c (418 mg, 60%) as an off-
2
4
2
2
1
1
6
9b (7.30 g, 90%) as a light-yellow solid. H NMR (400 MHz,
white powder. H NMR (400 MHz, DMSO-d ): δ 8.39 (d, J =
6
CDCl ): δ 7.43 (s, 1H), 2.62 (s, 3H), 2.52 (s, 3H).
4.02 Hz, 1H), 8.18 (t, J = 8.03 Hz, 1H), 7.53 (t, J = 5.27 Hz,
1H), 7.38 (br s, 2H), 2.37 (s, 3H).
3
2-Methyl-4-(methylthio)-6-(tributylstannyl)-1,3,5-tria-
zine (70a). A solution of 69a (2.67 g, 10.0 mmol) in THF (10
mL) at −78 °C was treated dropwise with a 2.0 M solution of i-
PrMgCl in THF (6.0 mL, 12.0 mmol). The reaction mixture
6-(2-Fluoropyridin-3-yl)-2-(trifluoromethyl)pyrimidin-
4-amine (71d). A mixture of 6-chloro-2-(trifluoromethyl)-
pyrimidin-4-amine (65d) (400 mg, 2.02 mmol), 2-fluoropyr-
idin-3-ylboronic acid (428 mg, 3.04 mmol), Pd(PPh ) Cl (63
was stirred at −78 °C for 30 min, and then Bu SnCl (2.7 mL,
3
3 2
2
1
0.0 mmol) was added dropwise. The reaction mixture was
mg, 101 μmol), and KOAc (596 mg, 6.08 mmol) in 1,4-dioxane
(3 mL) was heated at 120 °C for 30 min in a microwave
reactor. The mixture was passed through a short plug of
diatomaceous earth and washed with CH Cl (3 × 20 mL).
allowed to warm to 22 °C for 16 h. The reaction mixture was
diluted with H O (10 mL) and extracted with EtOAc (2 × 40
2
mL). The organic extract was washed with saturated brine (10
2
2
mL), dried (Na SO ), filtered, concentrated, and purified by
The filtrate was concentrated and purified by silica gel column
chromatography (gradient: 0% → 5% MeOH/CH Cl ) to give
71d as a white solid (180 mg, 34%). H NMR (400 MHz,
2
4
silica gel column chromatography (50% CH Cl /hexanes) to
2
2
2
2
1
1
give 70a (2.40 g, 56%) as a clear colorless oil. H NMR (400
MHz, CDCl ): δ 2.51 (s, 3H), 2.47 (s, 3H), 1.70−1.40 (m,
DMSO-d ): δ 8.57 (t, J = 8.80 Hz, 1H), 8.38 (d, J = 3.91 Hz,
3
6
6
H), 1.40−1.10 (m, 12H), 0.90 (t, 9H).
1H), 7.83 (br s, 2H), 7.63−7.46 (m, 1H), 7.15 (s, 1H).
4-(2-Fluoropyridin-3-yl)-6-methylpyrimidin-2-amine
(71e). A mixture of Pd(PPh ) (58 mg, 50 μmol), 2-amino-4-
2
-Methyl-4-(methylthio)-6-(tributylstannyl)pyrimidine
70b). A solution of 69b (1.63 g, 6.12 mmol) in 30 mL of THF
at −78 °C was treated with 2.0 M i-PrMgCl in THF (3.67 mL,
.35 mmol). After 10 min, Bu SnCl (1.65 mL, 6.12 mmol) was
(
3
4
chloro-6-methylpyrimidine (65e) (143 mg, 0.10 mmol), 2-
fluoropyridin-3-ylboronic acid (211 mg, 1.49 mmol), and CsF
(454 mg, 2.99 mmol) in 1,4-dioxane (2 mL) was heated at 150
°C for 30 min in a microwave reactor. The mixture was
concentrated and purified by silica gel column chromatography
(gradient: 33% → 50% EtOAc/hexanes) to give 71e (55 mg,
7
3
added and the mixture was stirred at −78 °C for 1 h. The
mixture was allowed to warm to 22 °C and stirred for 16 h. The
mixture was diluted with EtOAc (100 mL) and saturated
aqueous KF (100 mL). The layers were separated and the
aqueous layer was extracted with EtOAc (2 × 50 mL). The
combined organic layers were dried (Na SO ), filtered,
+
27%) as a white solid. LC−MS m/z: 205.1 (M + H) .
4-(2-Fluoropyridin-3-yl)-N,N-bis(4-methoxybenzyl)-6-
methyl-1,3,5-triazin-2-amine (71f). A mixture of 66 (1.57 g,
4.08 mmol), 2-fluoropyridin-3-ylboronic acid (632 mg, 4.48
mmol), Pd(Amphos) Cl (144 mg, 0.20 mmol), and KOAc
2
4
concentrated, and purified by silica gel column chromatography
gradient: 0% → 5% EtOAc/hexanes) to give 70b (1.39 g,
(
1
5
7
1
3%) as a clear colorless oil. H NMR (300 MHz, CDCl ): δ
3
2
2
.07 (s, 1H), 2.62 (s, 3H), 2.51 (s, 3H), 1.70−1.44 (m, 6H),
.42−1.21 (m, 6H), 1.21−0.98 (m, 6H), 0.91 (t, 9H).
(1.22 g, 12.4 mmol) in EtOH (15 mL) and H O (1.5 mL)
2
contained in a 20 mL microwave tube was degassed by
5
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Journal of Medicinal Chemistry
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1
bubbling Ar through it for 5 min. The mixture was heated in a
12%) as a colorless oil. H NMR (300 MHz, CDCl ): δ 8.68 (s,
3
microwave reactor at 100 °C for 20 min. The reaction mixture
1H), 8.32 (s, 1H), 7.67 (s, 1H), 2.79 (s, 3H), 2.63 (s, 3H).
4-(5-Chloro-2-fluoropyridin-3-yl)-N,N-bis(4-methoxy-
benzyl)-6-methyl-1,3,5-triazin-2-amine (71j). A mixture of
5-chloro-2-fluoropyridin-3-ylboronic acid (1.22 g, 6.96 mmol),
66 (2.30 g, 5.98 mmol), KOAc (1.20 g, 12.2 mmol), and
was partitioned between a 1:1 mixture of H O and saturated
2
brine (150 mL) and EtOAc (50 mL). The aqueous phase was
extracted with EtOAc (50 mL). The combined organic phases
were washed with saturated brine (50 mL). The organic phase
was dried (Na SO ), filtered, concentrated, and purified by
Pd(Amphos)
and H
Cl
O (2 mL) under Ar was heated at 95 °C. After 2 h, more
2
(150 mg, 0.21 mmol) in 1,4-dioxane (10 mL)
2
4
2
2
silica gel column chromatography (gradient: 10% → 50%
EtOAc/hexanes) to give 71f (1.65 g, 91%) as a pale-yellow oil.
5-chloro-2-fluoropyridin-3-ylboronic acid (600 mg) was added
1
and the mixture was heated for another 1 h. The mixture was
H NMR (400 MHz, CDCl ): δ 8.62−8.49 (m, 1H), 7.99−7.86
3
allowed to cool to 22 °C, saturated aqueous NH Cl (5 mL) was
(
6
m, 1H), 7.36−7.27 (m, 1H), 7.23 (dd, J = 8.31, 4.99 Hz, 4H),
4
added, and the mixture was partitioned between H O (5 mL)
.87 (t, J = 8.51 Hz, 4H), 3.81 (s, 3H), 4.81 (s, 4H), 3.80 (s,
2
and EtOAc (30 mL). The organic layer was dried (Na SO ),
3
H), 2.55 (s, 3H).
2
4
concentrated, and purified by silica gel column chromatography
4
-(3-Chloropyridin-4-yl)-N,N-bis(4-methoxybenzyl)-6-
(
8
gradient: 10% → 50% EtOAc/hexanes) to give 71j (2.30 g,
methyl-1,3,5-triazin-2-amine (71g). A mixture of 67 (306
mg, 0.64 mmol), 3-chloropyridine-4-boronic acid (101 mg, 0.64
mmol), dichloro 1,1′-bis(diphenylphosphino)ferrocene
palladium(II) (52 mg, 0.064 mmol), and Cs CO (251 mg,
1
0%) as a light-yellow solid. H NMR (400 MHz, CDCl ): δ
3
8
.53 (dd, J = 8.02, 2.74 Hz, 1H), 8.25 (d, J = 1.37 Hz, 1H), 7.21
(
d, J = 8.41 Hz, 4H), 6.86 (t, J = 8.22 Hz, 4H), 4.81 (d, J = 7.24
Hz, 4H), 3.80 (2s, 6H), 2.54 (s, 3H).
-(5-Bromo-2-fluoropyridin-3-yl)-6-methyl-1,3,5-tria-
2
3
0
.77 mmol) in 1,4-dioxane (6 mL) and H O (1 mL) was stirred
2
4
at 90 °C for 1 h. The mixture was diluted with H O (20 mL)
2
zin-2-amine (71k). A glass microwave reaction vessel was
charged with 5-bromo-2-fluoropyridin-3-ylboronic acid (760
mg, 3.46 mmol), 65a (500 mg, 3.46 mmol), Pd(PPh ) (200
and extracted with EtOAc (2 × 30 mL). The combined organic
extracts were washed with saturated brine (10 mL), dried
3
4
(
Na SO ), concentrated, and purified by silica gel column
2 4
mg, 0.17 mmol), Na CO ·H O (1.29 g, 10.4 mmol), 1,2-
2
3
2
chromatography (50% EtOAc/hexanes) to give 71g (129 mg,
1
dimethoxyethane (16 mL), and H
2
O (62 μL, 3.4 mmol) under
4
8
(
4%) as a glass. H NMR (300 MHz, CDCl ): δ 8.69 (s, 1H),
3
N . The reaction mixture was heated in a microwave reactor at
2
.58 (d, J = 4.97 Hz, 1H), 7.71 (d, J = 4.82 Hz, 1H), 7.26−7.10
m, 4H), 6.86 (t, J = 8.77 Hz, 4H), 4.80 (s, 4H), 3.81 (s, 3H),
1
00 °C for 60 min. The mixture was filtered through
diatomaceous earth, washing with EtOAc (100 mL). The
3
.80 (s, 3H), 2.56 (s, 3H).
-(3-Fluoropyrazin-2-yl)-4-methyl-6-(methylthio)-
,3,5-triazine (71h). A 1.6 M solution of n-BuLi in hexanes
filtrate was concentrated and purified by silica gel column
2
chromatography (gradient: 5% → 10% 2 M NH in MeOH/
3
1
CH Cl ) to give 71k (345 mg, 35%) as a brown solid. LC−MS
2
2
(
(
0.92 mL, 11 mmol) was added to 2,2,6,6-tetramethylpiperidine
2.0 mL, 12 mmol) in THF (50 mL) at −50 °C. Following the
+
m/z: 284.1/286.2 (M + H) .
4
-(2-Fluoro-5-methylpyridin-3-yl)-6-methyl-1,3,5-tria-
zin-2-amine (71m). A 2.5 M solution of n-BuLi in hexanes
9.6 mL, 24 mmol) was added to a mixture of i-Pr NH (3.4
addition, the mixture was stirred at 0 °C for 20 min and then
cooled to −100 °C. 2-Fluoropyrazine (980 mg, 10 mmol) in
(
2
THF (5 mL) was then added dropwise. After 5 min, Bu SnCl
3
mL, 24 mmol) in THF (5 mL) at 0 °C, and the resulting pale-
(
3.3 mL, 12 mmol) in THF (5 mL) was added dropwise and
yellow solution was stirred for 30 min and then cooled to −78
stirring was continued for 1 h. The reaction was quenched with
a solution of 35% aqueous HCl, EtOH, THF (1:4:5) and
allowed to warm to 22 °C. The reaction mixture was diluted
°
C. A suspension of 2-fluoro-5-methylpyridine (2.22 g, 20
mmol) in THF (5 mL) was slowly added. The resulting bright-
yellow solution was stirred at −78 °C for 1 h, treated with a
with saturated aqueous NaHCO (30 mL) and extracted with
3
solution of (i-PrO) B (6.9 mL, 30 mmol) in THF (10 mL), and
3
EtOAc (2 × 50 mL). The combined organic extracts were
then allowed to warm to 22 °C. The yellow suspension was
quenched with 1 M aqueous NaOH until basic (pH ∼10), and
the organic layer was separated. The aqueous layer was carefully
acidified with 6 M aqueous HCl until slightly acidic and then
was extracted with EtOAc (3 × 50 mL). The combined organic
layers were dried (Na SO ), filtered, and concentrated. The
washed with saturated brine (30 mL), dried (Na SO ),
2
4
concentrated, and purified by silica gel column chromatography
50% CH Cl /hexanes) to give 2-fluoro-3-(tributylstannyl)-
(
2
2
1
pyrazine (2.98 g, 77%) as a colorless oil. H NMR (300 MHz,
CDCl ): δ 8.63 (s, 1H), 8.02 (s, H), 1.68−1.46 (m, 6H), 1.42−
3
2
4
1.12 (m, 12H), 0.88 (t, J = 7.23 Hz, 9H).
resulting white solid was washed with Et O to give 2-fluoro-5-
2
A mixture of 69a (100 mg, 0.37 mmol), 2-fluoro-3-
methylpyridin-3-ylboronic acid (2.92 g, 94%) as a white solid.
+
(
(
tributylstannyl)pyrazine (145 mg, 0.37 mmol), and Pd(PPh3)4
43 mg, 0.037 mmol) in toluene (2 mL) was stirred at 110 °C
LC−MS m/z: 156 (M + H) .
1
,4-Dioxane (3 mL) was added to a mixture of Pd-
for 18 h. The mixture was allowed to cool, concentrated, and
(
Amphos) Cl (270 mg, 0.43 mmol), 2-fluoro-5-methylpyr-
2 2
purified by silica gel column chromatography (40% EtOAc/
idin-3-ylboronic acid (1.60 g, 10 mmol), 65a (1.24 g, 8.5
mmol), and KOAc (2.50 g, 26 mmol) in 1,4-dioxane (3 mL)
that was heated at 120 °C for 30 min in a microwave reactor.
The mixture was passed through a short plug of diatomaceous
earth, washing with CH Cl (3 × 20 mL). The filtrate was
1
hexanes) to give 71h (42 mg, 47%) as a colorless oil. H NMR
(
300 MHz, CDCl ): δ 8.73 (s, 1H), 8.42 (s, 1H), 2.73 (s, 3H),
3
2.65 (s, 3H).
4
-(3-Fluoropyrazin-2-yl)-2-methyl-6-(methylthio)-
2
2
pyrimidine (71i). A mixture of 69b (266 mg, 1.00 mmol), 2-
fluoro-3-(tributylstannyl)pyrazine (see: 71h) (387 mg, 1.00
mmol), and Pd(PPh ) (116 mg, 0.10 mmol) in toluene (3
mL) was heated in a microwave reactor at 140 °C for 40 min.
The mixture was concentrated and purified by silica gel column
chromatography (40% EtOAc/hexanes) to give 71i (28 mg,
concentrated and purified by silica gel column chromatography
(gradient: 0% → 5% MeOH/CH Cl ) to give 71m (760 mg,
2
2
+
3
4
41%) as a white solid. LC−MS m/z: 220 (M + H) .
4-(2-Chloro-5-methoxypyridin-3-yl)-6-methyl-1,3,5-
triazin-2-amine (71n). K CO (500 mg, 3.6 mmol) followed
2
3
by MeI (0.20 mL, 2.9 mmol) were added to a stirred mixture of
5
203
dx.doi.org/10.1021/jm300184s | J. Med. Chem. 2012, 55, 5188−5219

Journal of Medicinal Chemistry
-bromo-6-chloropyridin-3-ol (500 mg, 2.4 mmol) in DMF (3
Article
1
5
H NMR (400 MHz, DMSO-d ): δ 12.00 (s, 1H), 11.15 (br s,
6
mL). The mixture was heated at 45 °C for 4 h and then allowed
1H), 8.77 (dd, J = 7.78, 1.76 Hz, 1H), 8.38 (dd, J = 4.52, 1.51
Hz, 1H), 8.09 (d, J = 6.02 Hz, 1H), 7.81−7.53 (m, 2H), 7.35 (t,
J = 2.76 Hz, 1H), 7.19−6.99 (m, 2H), 6.90 (dd, J = 7.78, 4.77
Hz, 1H), 6.66 (br s, 1H), 2.55 (s, 3H).
to stand at 22 °C for 16 h. The mixture was diluted with H O
2
(
100 mL), and the precipitate was collected by filtration and
dried to give 3-bromo-2-chloro-5-methoxypyridine as a tan
1
solid. H NMR (400 MHz, CDCl ): δ 8.04 (s, 1H), 7.49 (d, J =
4-(2-(6-Methoxypyridin-3-ylamino)pyridin-3-yl)-6-
methyl-1,3,5-triazin-2-amine (12). A solution of 71a (50
mg, 0.24 mmol) and 3-amino-6-methoxypyridine (30 mg, 0.24
mmol) in 1,4-dioxane (1 mL) was treated with 2 M aqueous
HCl (0.12 mL, 0.24 mmol), and the mixture was heated at 150
°C for 16 h. After cooling, the reaction mixture was
concentrated and purified by silica gel column chromatography
(gradient: 0% → 5% MeOH/CH Cl ) to give 12 (60 mg, 80%)
3
1
.37 Hz, 1H), 3.86 (s, 3H).
A 2.5 M solution of n-BuLi in hexanes (3.7 mL, 9.3 mmol)
was added slowly to a stirred premixed solution of 3-bromo-2-
chloro-5-methoxypyridine (1.73 g, 7.8 mmol) and (i-PrO)3B
(
2.1 mL, 9.3 mmol) in THF (10 mL) at −78 °C. The resulting
dark mixture was stirred for 1 h and then allowed to warm up
to 22 °C over 1 h. The reaction was quenched with 1 M
aqueous NaOH (50 mL), and then EtOAc (50 mL) was added.
The separated aqueous layer was acidified with 5 M HCl to pH
2
2
1
as a yellow solid. H NMR (400 MHz, DMSO-d ): δ 11.74 (br
6
s, 1H), 8.77 (dd, J = 5.02, 2.51 Hz, 1H), 8.53 (br s, 1H), 8.29
(br s, 1H), 8.23−8.08 (m, 1H), 7.84 (br s, 1H), 7.71 (br s, 1H),
6.88 (ddd, J = 7.78, 4.27, 4.02 Hz, 1H), 6.82 (dd, J = 8.78, 4.27
Hz, 1H), 3.84 (d, J = 5.02 Hz, 3H), 2.42 (d, J = 4.02 Hz, 3H).
3-(3-(6-Amino-2-methylpyrimidin-4-yl)pyridin-2-
ylamino)phenol (13). A solution of 71b (121 mg, 0.59
mmol) and 3-aminophenol (78 mg, 0.71 mmol) in 1,4-dioxane
(1 mL) was treated with 2 M aqueous HCl (0.30 mL, 0.60
mmol), and the mixture was heated at 100 °C for 16 h. After
cooling, the reaction mixture was concentrated and purified by
silica gel column chromatography (gradient: 0% → 5% MeOH/
∼
5 and then extracted with EtOAc (2 × 50 mL). The
combined organic layers were washed with saturated brine,
dried (Na SO ), and concentrated to give 2-chloro-5-
2
4
methoxypyridin-3-ylboronic acid (1.03 g, 71%) as a brown
+
solid. LC−MS m/z: 188/190 (M + H) .
A stirred mixture of 2-chloro-5-methoxypyridin-3-ylboronic
acid (1.03 g, 5.48 mmol), 65a (720 mg, 4.98 mmol),
Na CO ·H O (1.32 g, 12.5 mmol), and Pd(PPh ) (576 mg,
2
3
2
3 4
0
.1 equiv) in 1,4-dioxane (10 mL) and H O (2.5 mL) under N2
2
was heated at 90 °C for 16 h. The mixture was concentrated
and purified by silica gel column chromatography (gradient: 0%
1
CH Cl ) to give 13 (19.7 mg, 11%) as a yellow solid. H NMR
2
2
→
5% MeOH/CH Cl ) to give 71n (644 mg, 51%) as a white
(400 MHz, DMSO-d ): δ 12.23 (s, 1H), 9.25 (d, J = 2.35 Hz,
2
2
6
1
solid. H NMR (400 MHz, DMSO-d ): δ 8.24 (d, J = 1.17 Hz,
1H), 8.35−8.18 (m, 1H), 8.02 (d, J = 7.63 Hz, 1H), 7.36 (br s,
1H), 7.16−6.95 (m, 4H), 6.93−6.84 (m, 1H), 6.74 (d, J = 1.76
Hz, 1H), 6.35 (dd, J = 6.65, 1.96 Hz, 1H), 2.62−2.51 (s, 3H).
N-(3-(6-Amino-2-methylpyrimidin-4-yl)pyridin-2-yl)-
1H-indazol-4-amine (14). A solution of 71b (136 mg, 0.67
mmol) and 1H-indazol-4-amine (106 mg, 0.81 mmol) in 1,4-
dioxane (1 mL) was treated with 2 M aqueous HCl (0.33 mL,
0.66 mmol), and the mixture was heated at 100 °C for 16 h.
After cooling, the reaction mixture was concentrated and
purified by silica gel column chromatography (gradient: 0% →
5% MeOH/CH Cl ) to give 14 (32.9 mg, 16%) as a yellow
6
1
2
H), 7.68 (br s, 2H), 7.65 (d, J = 1.17 Hz, 1H), 3.87 (s, 3H),
.36 (s, 3H).
3
-(3-(4-Amino-6-methyl-1,3,5-triazin-2-yl)pyridin-2-
ylamino)phenol (9). A solution of 71a (133 mg, 0.65 mmol)
and 3-aminophenol (85 mg, 0.78 mmol) in 1,4-dioxane (1 mL)
was treated with 2 M aqueous HCl (0.33 mL, 0.66 mmol), and
the mixture was heated at 100 °C for 16 h. After cooling, the
reaction mixture was concentrated and purified by silica gel
column chromatography (gradient: 0% → 5% MeOH/CH Cl )
2
2
1
to give 9 (66 mg, 34%) as a yellow solid. H NMR (400 MHz,
2
2
1
DMSO-d ): δ 11.97 (s, 1H), 9.27 (s, 1H), 8.76 (dd, J = 7.78,
solid. H NMR (400 MHz, DMSO-d ): δ 13.09 (br s, 1H),
6
6
1
2
.76 Hz, 1H), 8.35 (dd, J = 4.77, 1.76 Hz, 1H), 7.92−7.64 (m,
12.29 (s, 1H), 8.35 (d, J = 4.52 Hz, 1H), 8.22−7.89 (m, 3H),
7.29 (t, J = 7.78 Hz, 1H), 7.20−6.86 (m, 4H), 6.79 (s, 1H),
2.62 (s, 3H).
H), 7.51 (s, 1H), 7.28−6.99 (m, 2H), 6.89 (dd, J = 7.53, 4.52
Hz, 1H), 6.40 (d, J = 7.03 Hz, 1H), 2.45 (s, 3H).
N-(3-(4-Amino-6-methyl-1,3,5-triazin-2-yl)pyridin-2-
yl)-1H-indazol-4-amine (10). A solution of 71a (300 mg,
.15 mmol) and 1H-indazol-4-amine (292 mg, 2.19 mmol) in
,4-dioxane (5 mL) was treated with 2 M aqueous HCl (0.73
N-(3-(6-Amino-5-fluoro-2-methylpyrimidin-4-yl)-
pyridin-2-yl)-1H-indazol-4-amine (15). Aqueous HCl (2 M,
0.45 mL, 0.90 mmol) was added to a stirred mixture of 71c
(400 mg, 1.80 mmol) and 1H-indazol-4-amine (360 mg, 2.70
mmol) in 1,4-dioxane (3 mL), and the reaction vessel was
heated at 150 °C for 40 min in a microwave reactor. After
cooling, the mixture was concentrated and purified by silica gel
column chromatography (gradient: 0% → 10% MeOH/
0
1
mL, 1.46 mmol), and the reaction vessel was heated at 150 °C
for 40 min in a microwave reactor. After cooling, the reaction
mixture was concentrated and purified by silica gel column
chromatography (gradient: 0% → 10% MeOH/CH Cl ) to
2
2
1
1
give 10 (21.5 mg, 4.6%) as a yellow powder. H NMR (400
CH Cl ) to give 15 (134 mg, 22%) as a yellow powder. H
2
2
MHz, DMSO-d ): δ 13.10 (s, 1H), 12.26 (s, 1H), 8.80 (d, J =
NMR (400 MHz, DMSO-d ): δ 13.07 (br s, 1H), 10.87 (s,
6
6
7
=
.82 Hz, 1H), 8.42 (d, J = 4.50 Hz, 1H), 8.19 (s, 1H), 8.13 (d, J
7.43 Hz, 1H), 7.75 (br s, 2H), 7.31 (t, J = 8.02 Hz, 1H), 7.16
1H), 8.34 (d, J = 3.72 Hz, 1H), 8.18−7.82 (m, 3H), 7.47 (br s,
2H), 7.36−7.19 (m, 1H), 7.11 (d, J = 8.22 Hz, 1H), 7.05−6.75
(m, 1H), 2.56 (s, 3H).
(
d, J = 8.41 Hz, 1H), 7.07−6.89 (m, 1H), 2.56 (s, 3H).
N-(3-(4-Amino-6-methyl-1,3,5-triazin-2-yl)pyridin-2-
N-(3-(6-Amino-2-(trifluoromethyl)pyrimidin-4-yl)-
pyridin-2-yl)-1H-indazol-4-amine (16). A mixture of 71d
(157 mg, 0.61 mmol) and 1H-indol-4-amine (162 mg, 1.22
mmol) in 1,4-dioxane (2 mL) and 2 M aqueous HCl (0.30 mL,
0.60 mmol) was heated at 100 °C for 16 h. After cooling, the
reaction mixture was concentrated and purified by silica gel
column chromatography (gradient: 0% → 5% MeOH/CH Cl )
yl)-1H-indol-4-amine (11). Aqueous HCl (2 M, 0.42 mL,
0
0
.83 mmol) was added to a stirred mixture of 71a (171 mg,
.83 mmol) and 1H-indol-4-amine (132 mg, 1.00 mmol) in 1,4-
dioxane (4 mL), and the mixture was heated at 100 °C for 16 h.
After cooling, the reaction mixture was concentrated and
purified by silica gel column chromatography (gradient: 0% →
2
2
1
5
% MeOH/CH Cl ) to give 11 (58 mg, 22%) as a yellow solid.
to give 16 as a yellow solid (14.4 mg, 6.4%). H NMR (400
2
2
5
204
dx.doi.org/10.1021/jm300184s | J. Med. Chem. 2012, 55, 5188−5219

Journal of Medicinal Chemistry
Article
MHz, DMSO-d ): δ 13.06 (br s, 1H), 10.82 (s, 1H), 8.36 (d, J
mL). The organic extract was washed with saturated brine (5
6
=
(
7
4.52 Hz, 1H), 8.11 (d, J = 7.53 Hz, 1H), 7.99 (s, 1H), 7.89
d, J = 7.53 Hz, 1H), 7.81 (br s, 2H), 7.29 (t, J = 7.53 Hz, 1H),
.14 (d, J = 8.03 Hz, 1H), 7.07 (s, 1H), 7.05−6.95 (m, 1H).
-(2-(1H-Indol-4-yloxy)pyridin-3-yl)-6-methylpyrimi-
mL) and dried (Na SO ). The solution was filtered,
2
4
concentrated, and purified by silica gel column chromatography
1
(20% THF/CH Cl ) to give 73g (9 mg, 24%). H NMR (300
2
2
4
MHz, CDCl ): δ 10.32 (s, 1H), 8.68−8.49 (m, 1H), 8.33 (s,
3
din-2-amine (23). A stirred mixture of 71e (57 mg, 0.28
mmol), 4-hydroxyindole (74 mg, 0.56 mmol), and K CO (116
mg, 0.84 mmol) in DMF (2 mL) was heated at 180 °C for 30
min in a microwave reactor. The mixture was allowed to cool,
concentrated, and purified by silica gel column chromatography
1H), 8.24 (d, J = 5.12 Hz, 2H), 8.03 (d, J = 5.12 Hz, 2H), 7.95
(d, J = 1.61 Hz, 1H), 7.38 (dd, J = 8.70, 2.41 Hz, 2H), 7.21 (d, J
= 8.33 Hz, 2H), 7.14 (d, J = 8.33 Hz, 2H), 6.98 (s, 1H), 6.87
(d, J = 8.33 Hz, 2H), 6.79 (d, J = 8.33 Hz, 2H), 6.72 (d, J =
8.77 Hz, 1H), 4.86 (s, 2H), 4.79 (s, 2H), 3.81 (s, 3H), 3.76 (s,
3H), 2.56 (s, 3H).
2
3
(
gradient: 0% → 10% MeOH/CH Cl ) to give 23 (19.5 mg,
2 2
1
2
1
4
1
2
2%) as a gray powder. H NMR (400 MHz, DMSO-d ): δ
A solution of 73g (7 mg, 0.013 mmol) and TfOH (3.4 μL,
0.04 mmol) in TFA (0.1 mL) was stirred at 22 °C for 2 h. The
reaction mixture was diluted with saturated aqueous NaHCO3
(5 mL) and extracted with EtOAc (2 × 20 mL). The combined
organic extracts were washed with saturated brine (5 mL) and
dried (Na SO ). The solution was filtered, concentrated, and
6
1.24 (br s, 1H), 8.37 (dd, J = 7.78, 1.76 Hz, 1H), 8.10 (dd, J =
.77, 1.76 Hz, 1H), 7.48−7.16 (m, 4H), 7.08 (t, J = 7.78 Hz,
H), 6.77 (d, J = 7.53 Hz, 1H), 6.63 (s, 2H), 6.03 (br s, 1H),
.26 (s, 3H).
4
-(2-((6-Methoxypyridin-3-yl)(methyl)amino)pyridin-
2
4
3
-yl)-6-methyl-1,3,5-triazin-2-amine Trifluoroacetate
purified by silica gel column chromatography (EtOAc) to give
27 (3.2 mg, 81%) as a yellow film. H NMR (300 MHz,
1
(
24). A solution of 6-methoxy-N-methylpyridin-3-amine (123
mg, 0.89 mmol) and 71f (263 mg, 0.59 mmol) in THF (5 mL)
was cooled to 0 °C in an ice−water bath and treated dropwise
with 1.0 M LiHMDS in THF (1.8 mL, 1.8 mmol). The solution
was stirred at 0 °C for 4 h. The reaction mixture was quenched
CDCl ): δ 10.38 (s, 1H), 8.38 (s, 1H), 8.24 (d, J = 5.12 Hz,
3
1H), 8.13 (s, 1H), 8.07 (d, J = 4.97 Hz, 1H), 7.58 (dd, J = 8.77,
2.05 Hz, 1H), 6.81 (d, J = 8.77 Hz, 1H), 5.41 (s, 2H), 3.96 (s,
3H), 2.54 (s, 3H).
4-(3-(6-Methoxypyridin-3-ylamino)pyrazin-2-yl)-6-
methyl-1,3,5-triazin-2-amine (30). A mixture of 71h (61
mg, 0.26 mmol), 3-amino-6-methoxypyridine (38 μL, 0.31
with a saturated solution of NH Cl at 0 °C and extracted with
4
EtOAc (30 mL), dried (MgSO ), filtered, and concentrated to
4
+
give 73f (415 mg). LC−MS m/z: 564.0 (M + H) .
A solution of 73f (94.6 mg, 0.168 mmol) in TFA (8 mL) was
heated at 80 °C for 48 h. The mixture was concentrated to
about 50% of the original volume under a stream of Ar. The
remaining mixture was pipetted carefully into a saturated
mmol), CuI (5 mg, 0.03 mmol), and i-Pr NEt (89 μL, 0.51
2
mmol) in 1,4-dioxane (1 mL) was heated at 100 °C for 24 h.
The mixture was concentrated and purified by silica gel column
chromatography (20% EtOAc/CH Cl ) to give N-(6-methox-
2
2
aqueous solution of NaHCO , which had been cooled in an
ypyridin-3-yl)-3-(4-methyl-6-(methylthio)-1,3,5-triazin-2-yl)-
pyrazin-2-amine (73h) (67 mg, 76%) as a yellow solid. H
3
1
ice−water bath. The resulting precipitate was removed by
filtration and washed with H O. The aqueous layer (containing
NMR (300 MHz, CDCl ): δ 11.34 (s, 1H), 8.36 (d, J = 1.90
2
3
the product) was lyophilized to give a yellow solid. This was
purified by silica gel column chromatography (EtOAc) followed
by reversed-phase preparative HPLC (Phenomenex C18
column: 150 mm × 30 mm; 5 μm; 10% → 90% MeCN/
Hz, 1H), 8.26 (s, 2H), 8.00 (dd, J = 8.99, 2.56 Hz, 1H), 6.80 (d,
J = 8.77 Hz, 1H), 3.96 (s, 3H), 2.77 (s, 3H), 2.68 (s, 3H).
A mixture of 73h (44 mg, 0.129 mmol), concentrated
aqueous NH (1 mL), and 1,4-dioxane (1 mL) was heated at
3
H O; 0.1% TFA additive) to give 24 (20 mg, 26%) as a yellow
100 °C for 16 h and then concentrated and purified by silica gel
2
1
film. H NMR (400 MHz, CD OD): δ 8.44 (dd, J = 5.1, 1.8 Hz,
column chromatography (10% MeOH/EtOAc) to give 30 (34
3
1
1H), 7.83 (dd, J = 7.5, 1.9 Hz, 1H), 7.79 (d, J = 2.7 Hz, 1H),
7.42 (dd, J = 8.9, 2.8 Hz, 1H), 7.07 (dd, J = 7.5, 5.0 Hz, 1H),
6.67 (d, J = 8.8 Hz, 1H), 3.84 (s, 3H), 3.50 (s, 3H), 2.34 (s,
3H).
mg, 85%) as a yellow solid. H NMR (300 MHz, CDCl ): δ
3
11.83 (s, 1H), 8.37 (d, J = 2.05 Hz, 1H), 8.24 (s, 1H), 8.20 (s,
1H), 8.04 (dd, J = 8.92, 2.48 Hz, 1H), 6.80 (d, J = 8.77 Hz,
1H), 5.71 (s, 2H), 3.95 (s, 3H), 2.62 (s, 3H).
3
-(3-(2-Amino-6-methylpyrimidin-4-yl)pyridin-2-
6-(3-(6-Methoxypyridin-3-ylamino)pyrazin-2-yl)-2-
methylpyrimidin-4-amine (31). A mixture of 71i (21 mg,
0.09 mmol), 3-amino-6-methoxypyridine (22 μL, 0.18 mmol),
ylamino)phenol (25). A mixture of 71e (50 mg, 0.25 mmol)
and 3-aminophenol (32 mg, 0.29 mmol) in 1,4-dioxane (1 mL)
was treated with 2 M aqueous HCl (0.12 mL, 0.24 mmol) and
heated at 100 °C for 16 h. The reaction mixture was
concentrated and purified by silica gel column chromatography
CuI (2 mg, 9 μmol), and i-Pr NEt (23 mg, 0.18 mmol) in 1,4-
2
dioxane (1 mL) was heated at 100 °C for 24 h. The reaction
mixture was diluted with H O (5 mL) and extracted with
2
(
gradient: 0% → 5% MeOH/CH Cl ) to give 25 (3.0 mg, 4%)
EtOAc (2 × 20 mL). The combined organic extracts were
2
2
1
as a yellow solid. H NMR (400 MHz, CD OD): δ 8.30−8.19
washed with saturated brine (5 mL) and dried (Na SO ). The
3
2
4
(
m, 1H), 8.17 (d, J = 7.53 Hz, 1H), 7.38 (d, J = 2.01 Hz, 1H),
solution was filtered, concentrated, and purified by silica gel
column chromatography (30% EtOAc/hexanes) to give N-(6-
methoxypyridin-3-yl)-3-(2-methyl-6-(methylthio)pyrimidin-4-
7
1
.19−6.97 (m, 4H), 6.92−6.72 (m, 1H), 6.47 (d, J = 8.03 Hz,
H), 2.41 (s, 3H).
1
4
-(3-(6-Methoxypyridin-3-ylamino)pyridin-4-yl)-6-
yl)pyrazin-2-amine (73i) (14 mg, 46%). H NMR (300 MHz,
methyl-1,3,5-triazin-2-amine (27). A mixture of 71g (32
mg, 0.069 mmol), 3-amino-6-methoxypyridine (17 μL, 0.14
mmol), chloro[2-(dicyclohexylphosphino)-3,6-dimethoxy-2′-4′-
CDCl ): δ 12.20 (s, 1H), 8.42 (d, J = 2.05 Hz, 1H), 8.22 (s,
3
1H), 8.18 (s, 1H), 8.09 (dd, J = 8.92, 2.63 Hz, 1H), 8.02 (d, J =
1.90 Hz, 1H), 6.79 (d, J = 8.77 Hz, 1H), 3.95 (s, 3H), 2.78 (s,
3H), 2.63 (s, 3H).
6
′-tri-iso-propyl-1,1′-biphenyl][2-(2-aminoethyl)phenyl]-
palladium(II) (BrettPhos precatalyst, 2 mg), and t-BuONa (17
mg, 0.17 mmol) in 1,4-dioxane (1 mL) was heated at 100 °C
for 16 h. The reaction mixture was diluted with saturated
A solution of 73i (11 mg, 0.032 mmol) and in 1,4-dioxane (1
mL) was treated with m-CPBA (11 mg, 0.065 mmol) and
stirred at 22 °C for 1 h. The mixture was then treated with
aqueous NH Cl (10 mL) and extracted with EtOAc (2 × 20
concentrated aqueous NH (0.5 mL, 13 mmol) and 1,4-dioxane
4
3
5
205
dx.doi.org/10.1021/jm300184s | J. Med. Chem. 2012, 55, 5188−5219

Journal of Medicinal Chemistry
Article
(
1 mL). The reaction mixture was heated at 100 °C for 2 h.
mL) at 22 °C. The mixture was stirred for 1 h and then diluted
The mixture was concentrated and purified by silica gel column
with saturated aqueous NH Cl (5 mL) and H O (5 mL), and
4
2
chromatography (80% EtOAc/hexanes) to give 31 (8.2 mg,
extracted with EtOAc (3 × 10 mL). The combined organic
1
8
1
1
2
3%). H NMR (300 MHz, CDCl ): δ 12.46 (s, 1H), 8.42 (s,
layers were washed with saturated brine, dried (Na SO ), and
3
2
4
H), 8.15 (s, 1H), 8.10 (dd, J = 8.77, 2.48 Hz, 1H), 7.98 (d, J =
.32 Hz, 1H), 7.47 (s, 1H), 6.78 (d, J = 8.62 Hz, 1H), 4.95 (s,
H), 3.95 (s, 3H), 2.64 (s, 3H).
concentrated to give crude 73m. This was dissolved in CH Cl
2 2
(2 mL) and treated with TFA (2 mL). The mixture was stirred
for 1 h. The reaction mixture was concentrated, H O (10 mL)
2
4
-(5-Chloro-2-(6-methoxypyridin-3-ylamino)pyridin-
was added, and the mixture was extracted with CH Cl (3 × 10
2
2
3
2
-yl)-N,N-bis(4-methoxybenzyl)-6-methyl-1,3,5-triazin-
-amine (73j). A solution of 3-amino-6-methoxypyridine (1.41
mL). The combined organic layers were washed with saturated
brine, dried (Na SO ), concentrated, and purified by silica gel
2
4
g, 11.3 mmol) and 71j (3.62 g, 7.54 mmol) in THF (15 mL)
was cooled to 0 °C and treated dropwise with 1.0 M LiHMDS
in THF (22.6 mL, 22.6 mmol). The resulting reaction mixture
was stirred for 30 min and then quenched with a saturated
column chromatography (gradient: 0% → 5% MeOH/CH Cl )
followed by another silica gel column chromatography
2 2
purification (gradient: 0% → 80% EtOAc/hexanes) to give
1
34 (11 mg, 13%) as a yellow solid. H NMR (400 MHz,
aqueous solution of NH Cl (50 mL). The mixture was
DMSO-d ): δ 11.53 (br s, 1H), 8.66 (br s, 1H), 8.35 (br s, 1H),
4
6
extracted with EtOAc (50 mL), dried (MgSO ), filtered,
concentrated, and purified by silica gel column chromatography
8.16 (br s, 1H), 8.09 (d, J = 10.37 Hz, 1H), 6.79 (d, J = 9.19
Hz, 1H), 5.38 (br s, 2H), 3.95 (s, 3H), 2.56 (s, 3H), 2.29 (s,
3H).
4
(
8
gradient: 20% → 100% EtOAc/hexanes) to give 73j (3.77 g,
6%) as a bright-yellow crystalline solid. H NMR (400 MHz,
1
4-(5-Methoxy-2-(6-methoxypyridin-3-ylamino)-
pyridin-3-yl)-6-methyl-1,3,5-triazin-2-amine (35). 1,4-Di-
oxane (0.1 mL) and 2 M aqueous HCl (0.22 mL, 0.44 mmol)
were added to a mixture of 71n (110 mg, 0.44 mmol) and 3-
amino-6-methoxypyridine (81 mg, 0.66 mmol), and the mixture
was heated at 140 °C for 60 min in a microwave reactor. The
resulting mixture was concentrated and purified by silica gel
column chromatography (gradient: 0% → 5% MeOH/
CH Cl ). The product was washed with methanol (3 × 5
CDCl ): δ 11.64 (s, 1H), 8.73 (d, J = 2.74 Hz, 1H), 8.25 (d, J =
3
2.54 Hz, 1H), 8.19 (d, J = 2.74 Hz, 1H), 7.83 (dd, J = 9.00, 2.74
Hz, 1H), 7.19 (dd, J = 16.43, 8.61 Hz, 4H), 6.92−6.78 (m, 4H),
6
3
.70 (d, J = 8.80 Hz, 1H), 4.86 (s, 2H), 4.81 (s, 2H), 3.92 (s,
H), 3.80 (d, J = 9.19 Hz, 6H), 2.57 (s, 3H).
4
-(5-Chloro-2-(6-methoxypyridin-3-ylamino)pyridin-
3
-yl)-6-methyl-1,3,5-triazin-2-amine (32). A solution of 73j
(
1
110 mg, 0.19 mmol) in TFA (10 mL) was heated at 80 °C for
2
2
6 h. The mixture was concentrated to a slurry, diluted with
mL) and recrystallized from MeOH to give 35 (21 mg, 14%) as
1
saturated aqueous NaHCO (5 mL), and filtered. The solid was
a yellow-green powder. H NMR (400 MHz, DMSO-d ): δ
3
6
washed with H O, MeOH, and 25% EtOAc/hexanes to give 32
as a brown solid (65 mg, 100%). H NMR (400 MHz, DMSO-
d6): δ 11.69 (br s, 1H), 8.72 (br s, 1H), 8.50 (d, J = 3.33 Hz,
11.47 (s, 1H), 8.52 (d, J = 2.74 Hz, 1H), 8.39 (d, J = 3.33 Hz,
1H), 8.18−8.13 (m, 1H), 8.12 (d, J = 3.13 Hz, 1H), 7.86 (br s,
1H), 7.73 (br s, 1H), 6.79 (d, J = 8.80 Hz, 1H), 3.83 (s, 3H),
3.82 (s, 3H), 2.44 (s, 3H).
6-Chloro-2-methyl-9-(tetrahydro-2H-pyran-2-yl)-9H-
purine (76). A mixture of 2-methyl-4,6-dichloro-5-amino-
2
1
1
H), 8.31 (br s, 1H), 8.09 (br s, 1H), 7.94 (br s, 1H), 7.80 (br
s, 1H), 6.84 (br s, 2H), 3.85 (br s, 3H), 2.43 (br s, 3H).
-(5-Bromo-2-(4-methoxyphenylamino)pyridine-3-
yl)-6-methyl-1,3,5-triazin-2-amine (33). A mixture of 71k
645 mg, 2.27 mmol) and 3-amino-6-methoxypyridine (282
4
pyrimidine (74) (1.05 g) and aqueous NH (∼25% w/v) (3
3
(
mL) was heated in a microwave reactor at 120 °C for 25 min.
This procedure was performed nine times to process a total of
9.94 g (55.8 mmol) of 74. The reaction mixtures were
combined and concentrated, and the residue (75; 8.89 g) was
suspended in triethyl orthoformate (100 mL, 600 mmol) and
heated at 100 °C for 75 min. The mixture was concentrated
and treated with hexanes (100 mL), and the resulting solid was
collected by filtration (9.45 g). The solid was suspended in
CH Cl (100 mL), and TsOH (12% in acetic acid, 0.90 mL, 5.6
mg, 2.27 mmol) in THF (5 mL) was cooled in an ice−water
bath and treated dropwise with 1.0 M LiHMDS in THF (13.6
mL, 13.6 mmol). The mixture was stirred for 30 min and then
quenched with H O (60 mL). The mixture was extracted with
2
CH Cl (3 × 50 mL), and the combined organic extracts were
2
2
dried (MgSO ), concentrated, and purified by silica gel column
4
chromatography (5% 2 M NH in MeOH/CH Cl ) to give 33
3
2
2
1
(
450 mg, 51%) as a yellow solid. H NMR (400 MHz, DMSO-
2
2
d6): δ 11.70 (d, J = 1.17 Hz, 1H), 8.84 (br s, 1H), 8.49 (d, J =
mmol) and 3,4-dihydro-2H-pyran (6.6 mL, 73 mmol) were
added. The mixture was heated at 50 °C for 30 min and then
stirred at 22 °C for 16 h. The reaction mixture was diluted with
4
7
.30 Hz, 1H), 8.37 (br s, 1H), 8.07 (dd, J = 4.21, 1.86 Hz, 1H),
.81 (br s, 1H), 6.84 (d, J = 2.35 Hz, 1H), 3.84 (br s, 3H), 2.50
(
d, J = 0.98 Hz, 3H).
-(2-(6-Methoxypyridin-3-ylamino)-5-methylpyridin-
-yl)-6-methyl-1,3,5-triazin-2-amine (34). NaH (60%
CH Cl2 (100 mL) and washed with saturated aqueous
2
4
NaHCO (75 mL). The organic extract was dried (Na SO )
3
2
4
1
3
and concentrated to give 76 (13.73 g, 97% over 3 steps). H
dispersion in mineral oil) (410 mg, 10 mmol) was carefully
NMR (400 MHz, CDCl ): δ 8.26 (s, 1H), 5.78 (d, J = 10.56
3
added to a solution of 71m (905 mg, 4.1 mmol) and Boc O
Hz, 1H), 4.19 (d, J = 11.93 Hz, 1H), 3.84−3.76 (m, 1H), 2.80
2
(
2.70 g, 12 mmol) in DMF (3.0 mL), and the mixture was
(s, 3H), 2.20−1.96 (m, 3H), 1.89−1.64 (m, 3H).
stirred at 22 °C for 16 h. The solution was quenched with ice
6-(2-Fluoropyridin-3-yl)-2-methyl-9-(tetrahydro-2H-
pyran-2-yl)-9H-purine (77a). A mixture of 76 (532 mg, 2.10
mmol), 2-fluoropyridin-3-ylboronic acid (596 mg, 4.23 mmol),
KOAc (629 mg, 6.41 mmol), and Pd(Amphos) Cl (37 mg, 53
and diluted with H O (50 mL), and the resulting precipitate
2
was collected by filtration to give di-tert-butyl 4-(2-fluoro-5-
methylpyridin-3-yl)-6-methyl-1,3,5-triazin-2-yl-dicarbamate
2
2
(
72m) (1.13 g, 65%) as a yellow solid. LC−MS m/z: 420 (M +
μmol) under N was suspended in EtOH (5 mL) and H O (1
2
2
+
H) .
mL), degassed, and heated at reflux for 2 h. The mixture was
poured into saturated aqueous NaHCO3 (100 mL) and
extracted with EtOAc (3 × 100 mL). The combined EtOAc
1.0 M LiHMDS in THF (0.80 mL, 0.80 mmol) was added
dropwise to a stirred mixture of 72m (112 mg, 0.27 mmol) and
3
-amino-6-methoxypyridine (50 mg, 0.40 mmol) in THF (3
extracts were dried (MgSO ), concentrated, and purified by
4
5
206
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Article
silica gel column chromatography (EtOAc) to give 77a (519
N-(6-Methoxypyridin-3-yl)-3-(2-methyl-9H-purin-6-
yl)pyridin-2-amine (19). A solution of 78a (95 mg, 0.23
mmol) in 2 M aqueous HCl (2 mL, 4 mmol) was heated briefly
at 100 °C in an oil bath, and then the heater was turned off and
the mixture was allowed to slowly cool to 22 °C. The solution
was concentrated and purified by strong cation-exchange
mg, 79%) as a pale-yellow oil which crystallized to give a white
1
solid upon trituration with Et O. H NMR (400 MHz, DMSO-
2
d ): δ 8.79 (s, 1H), 8.53−8.46 (m, 1H), 8.46−8.43 (m, 1H),
6
7.62−7.55 (m, 1H), 5.85−5.78 (m, 1H), 4.08−4.00 (m, 1H),
3.80−3.70 (m, 1H), 2.78 (s, 3H), 2.40−2.26 (m, 1H), 2.06−
1.95 (m, 2H), 1.87−1.72 (m, 1H), 1.67−1.56 (m, 2H).
N-(3-(2-Methyl-9H-purin-6-yl)pyridin-2-yl)-1H-inda-
44
chromatography (gradient: MeOH → 2 M NH /MeOH)
3
1
to give 19 (72 mg, 95%) as an orange solid. H NMR (400
zol-4-amine (17). A mixture of 77a (320 mg, 1.02 mmol) and
H-indazol-4-amine (187 mg, 1.41 mmol) was suspended in
EtOH (10 mL), and 5 M aqueous HCl (0.25 mL, 1.25 mmol)
MHz, DMSO-d ): δ 13.60 (br s, 1H), 12.68 (s, 1H), 9.80 (dd, J
6
1
= 7.82, 1.96 Hz, 1H), 8.60 (s, 1H), 8.54 (d, J = 2.69 Hz, 1H),
8.31 (dd, J = 4.65, 1.96 Hz, 1H), 8.20 (dd, J = 8.92, 2.81 Hz,
1H), 7.00 (dd, J = 7.95, 4.77 Hz, 1H), 6.85 (d, J = 8.80 Hz,
1H), 3.85 (s, 3H), 2.86 (s, 3H).
was added. The mixture was heated at 100 °C for 75 min,
cooled to 22 °C, and treated with 2 M NH in MeOH (5 mL).
3
The reaction mixture was concentrated and purified by
reversed-phase preparative HPLC (Phenomenex C18 column:
6-Methoxy-N-(3-(9-(tetrahydro-2H-pyran-2-yl)-9H-
purin-6-yl)pyridin-2-yl)pyridin-3-amine (78b). A solution
of 77b (197 mg, 0.66 mmol) and 3-amino-6-methoxypyridine
(102 mg, 0.82 mmol) in THF (2 mL) was cooled in an ice−
water bath and treated with 1.0 M LiHMDS in THF (3.0 mL,
3.0 mmol). The mixture was stirred for 1 h and then quenched
1
50 mm × 30 mm; 5 μm; 10% → 90% MeCN/H O; 0.1% TFA
2
additive). The resulting product was dissolved in CH Cl ,
2
2
filtered through a short silica gel plug, washing with 2 M NH3
1
in MeOH, and concentrated to give 17 (33.9 mg, 10%). H
NMR (400 MHz, DMSO-d ): δ 13.12 (s, 1H), 12.72 (s, 1H),
with H O (0.1 mL). The mixture was extracted into EtOAc
6
2
9.78 (s, 1H), 8.62 (s, 1H), 8.41 (dd, J = 4.50 Hz, 1.76 Hz, 1H),
8.27 (s, 1H), 8.10 (d, J = 7.43 Hz, 1H), 7.33 (t, J = 8.02 Hz,
1H), 7.17 (d, J = 8.22 Hz, 1H), 7.09 (dd, J = 7.83 Hz, 4.69 Hz,
1H), 2.94 (s, 3H).
from saturated aqueous NaHCO , concentrated, and purified
by silica gel column chromatography (50% EtOAc/hexane;
3
yellow band from column) to give 78b (190 mg, 72%) as a
1
yellow crystalline solid. H NMR (400 MHz, CDCl ): δ 12.06
3
N-(3-(2-Methyl-9H-purin-6-yl)pyridin-2-yl)-1H-indol-4-
(s, 1H), 9.68 (dd, J = 7.82, 1.76 Hz, 1H), 8.99 (s, 1H), 8.44 (d,
J = 2.54 Hz, 1H), 8.28 (dd, J = 4.69, 1.76 Hz, 1H), 8.34 (s, 1H),
8.05 (dd, J = 8.90, 2.64 Hz, 1H), 6.88 (dd, J = 7.82, 4.69 Hz,
1H), 6.77 (d, J = 8.80 Hz, 1H), 5.86 (dd, J = 10.37, 2.35 Hz,
1H), 4.25−4.16 (m, 1H), 3.95 (s, 3H), 3.82 (s, 1H), 2.24−1.98
(m, 3H), 1.89−1.61 (m, 3H).
N-(6-Methoxypyridin-3-yl)-3-(9H-purin-6-yl)pyridin-2-
amine (21). A solution of 78b (190 mg, 0.47 mmol) in 2 M
aqueous HCl (2 mL, 4 mmol) was heated for 5 min at 100 °C
and then allowed to slowly cool to 22 °C and stand for 16 h.
amine (18). A mixture of 77a (270 mg, 0.86 mmol) and 4-
aminoindole (159 mg, 1.21 mmol) in EtOH (5 mL) was
treated with 5 M aqueous HCl (0.21 mL, 1.1 mmol) and
heated at 100 °C for 3 h. The mixture was allowed to cool and
was then diluted with 2 M NH in MeOH (4 mL) and allowed
3
to stand for 16 h. The mixture was concentrated, treated with
DMF (5 mL), and filtered. The solid was washed with CH Cl ,
2
2
and the filtrate was concentrated and purified by reversed-phase
preparative HPLC (Phenomenex C18 column: 150 mm × 30
mm; 5 μm; 10% → 90% MeCN/H O; 0.1% TFA additive).
The solution was neutralized with aqueous NH , and the
2
3
The resulting product was dissolved in CH Cl , filtered through
precipitated product was collected by filtration, washing with a
2
2
a short silica gel plug, washing with 2 M NH in MeOH, and
concentrated to give 18 (13.7 mg, 5%). H NMR (400 MHz,
small volume of H O and dried under vacuum. 21 (120 mg,
80%) was obtained as an orange solid. H NMR (400 MHz,
3
2
1
1
DMSO-d ): δ 13.60 (s, 1H), 12.44 (s, 1H), 11.16 (s, 1H), 9.77
DMSO-d ): δ 13.80 (br s, 1H), 12.33 (br s, 1H), 9.73 (br s,
6
6
(
d, J = 7.04 Hz, 1H), 8.61 (s, 1H), 8.37 (dd, J = 4.69 Hz, 1.76
1H), 9.10 (s, 1H), 8.70 (s, 1H), 8.58−8.50 (m, 1H), 8.34−8.25
(m, 1H), 8.20−8.08 (m, 1H), 7.07−6.95 (m, 1H), 6.90−6.79
(m, 1H), 3.85 (s, 3H).
Hz, 1H), 8.05 (dd, J = 6.85 Hz, 1.56 Hz, 1H), 7.37 (t, J = 2.64
Hz, 1H), 7.11−7.04 (m, 2H), 7.01 (dd, J = 7.82 Hz, 4.69 Hz,
1
H), 6.72 (s, 1H), 2.92 (s, 3H).
4-Nitro-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole
(80a) and 4-Nitro-2-(tetrahydro-2H-pyran-2-yl)-2H-inda-
zole (80b). A suspension of 4-nitro-1H-indazole (4.07 g, 25
mmol) in EtOAc (50 mL) was treated with 3,4-dihydro-2H-
pyran (6.8 mL, 75 mmol) and MP-TsOH resin (Biotage) (380
mg, 4.3 mmol/g, 0.06 equiv) and heated at reflux for 2 h. The
mixture was filtered, concentrated, and purified by silica gel
column chromatography (gradient: 5% EtOAc/hexane →10%
EtOAc/10% CH Cl /hexane) to give 80a (3.04 g, 49%) as a
6
-Methoxy-N-(3-(2-methyl-9-(tetrahydro-2H-pyran-2-
yl)-9H-purin-6-yl)pyridin-2-yl)pyridin-3-amine (78a). A
solution of 77a (190 mg, 0.61 mmol) and 3-amino-6-
methoxypyridine (94 mg, 0.76 mmol) in THF (2 mL) was
cooled in an ice−water bath and treated with 1.0 M LiHMDS
in THF (3.0 mL, 3.0 mmol). The mixture was stirred for 1 h
and then quenched with H O (0.1 mL). The mixture was
extracted into EtOAc (3 × 50 mL) from saturated aqueous
2
2
2
NaHCO (50 mL), concentrated, and purified by silica gel
column chromatography (gradient: 25% → 50% EtOAc/
pale-yellow crystalline solid (recrystallized from EtOAc/
hexane) followed by 80b (2.34 g, 38%) as a pale-yellow oil.
Structural assignments were confirmed by NOESY (N−CH−O
3
hexane; yellow band from column) to give 78a (96.3 mg,
1
3
8%) as a yellow crystalline solid. H NMR (400 MHz, DMSO-
to aromatic protons).
1
d6): δ 12.50 (s, 1H), 9.72 (dd, J = 7.82, 1.71 Hz, 1H), 8.87 (s,
80a: H NMR (400 MHz, DMSO-d ): δ 8.57 (s, 1H), 8.32
6
1
1
H), 8.53 (d, J = 2.69 Hz, 1H), 8.32 (dd, J = 4.65, 1.96 Hz,
H), 8.18 (dd, J = 8.93, 2.81 Hz, 1H), 7.01 (dd, J = 7.82, 4.65
(d, J = 8.53 Hz, 1H), 8.21 (d, J = 7.53 Hz, 1H), 7.69 (t, J = 8.03
Hz, 1H), 6.04 (dd, J = 9.54, 2.01 Hz, 1H), 3.93−3.84 (m, 1H),
3.83−3.74 (m, 1H), 2.46−2.36 (m, 1H), 2.12−1.98 (m, 2H),
Hz, 1H), 6.85 (d, J = 9.05 Hz, 1H), 5.89−5.80 (m, 1H), 4.12−
3.97 (m, 1H), 3.85 (s, 3H), 3.82−3.67 (m, 1H), 2.89 (s, 3H),
2.38−2.27 (m, 1H), 2.12−1.93 (m, 2H), 1.88−1.72 (m, 1H),
1.70−1.55 (m, 2H).
1.84−1.70 (m, 1H), 1.67−1.56 (m, 2H).
1
80b: H NMR (400 MHz, DMSO-d ): δ 8.92 (s, 1H), 8.26−
6
8.22 (m, 2H), 7.54 (t, J = 8.03 Hz, 1H), 5.93 (dd, J = 9.54, 2.51
5
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Journal of Medicinal Chemistry
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Hz, 1H), 4.01 (m, 1H), 3.83−3.71 (m, 1H), 2.30−2.18 (m,
1H), 6.02−5.91 (m, 1H), 3.99−3.91 (m, 1H), 3.77−3.67 (m,
1H), 2.72 (s, 3H), 2.48−2.36 (m, 1H), 2.08−1.96 (m, 1H),
1.95−1.87 (m, 1H), 1.84−1.32 (m, 3H).
4-(2-Fluoropyridin-3-yl)-6-methyl-1-(tetrahydro-2H-
pyran-2-yl)-1H-pyrazolo[3,4-d]pyrimidine (84). A mixture
of 83 (108 mg, 0.43 mmol), 2-fluoropyridin-3-ylboronic acid
(120 mg, 0.85 mmol), and KOAc (105 mg, 1.07 mmol) in
1
H), 2.15−1.34 (m, 5H).
1
-(Tetrahydro-2H-pyran-2-yl)-1H-indazol-4-amine
(
81a). A solution of 80a (1.05 g, 4.25 mmol) in EtOAc (100
mL) was treated with 10% Pd/C (60 mg) and stirred under an
atmosphere of H for 22 h. The reaction was filtered and
2
concentrated. The residue was triturated with Et O to give 81a
2
1
(
892 mg, 97%) as an off-white solid. H NMR (400 MHz,
EtOH (1.25 mL) and H O (0.25 mL) was placed under
2
DMSO-d ): δ 8.11 (s, 1H), 7.03 (t, J = 7.82 Hz, 1H), 6.74 (d, J
vacuum for 5 min and then flushed with nitrogen for 5 min and
6
=
8.22 Hz, 1H), 6.18 (d, J = 7.43 Hz, 1H), 5.78 (s, 2H), 5.67−
treated with Pd(Amphos) Cl2 (8.0 mg, 11 μmol). The
suspension was heated at 80 °C for 60 min. The reaction
mixture was poured into EtOAc (100 mL) and saturated
2
5
2
1
.59 (m, 1H), 3.92−3.82 (m, 1H), 3.74−3.63 (m, 1H), 2.45−
.30 (m, 1H), 2.08−1.96 (m, 1H), 1.94−1.84 (m, 1H), 1.80−
.65 (m, 1H), 1.55 (br s, 2H).
aqueous NaHCO (100 mL) and extracted. The organic layer
3
2
-(Tetrahydro-2H-pyran-2-yl)-2H-indazol-4-amine
was dried (MgSO ), filtered, concentrated, and purified by silica
gel column chromatography (gradient: 25% → 50% EtOAc/
4
(
81b). A solution of 80b (2.34 g, 9.45 mmol) was dissolved in
EtOAc (100 mL) and treated with 10% Pd/C (100 mg). The
hexanes) to give 84 (105 mg, 79%) as a pale-yellow oil which
slowly crystallized upon standing. H NMR (400 MHz,
1
resulting suspension was stirred under an atmosphere of H for
2
1
6 h and then was filtered, concentrated, and purified by silica
DMSO-d ): δ 8.57−8.46 (m, 2H), 8.41 (d, J = 3.51 Hz, 1H),
6
gel column chromatography (50% EtOAc/hexane) to give 81b
7.64 (s, 1H), 6.04 (d, J = 8.03 Hz, 1H), 4.02−3.92 (m, 1H),
3.79−3.68 (m, 1H), 2.82 (s, 3H), 2.48−2.41 (m, 1H), 2.10−
2.00 (m, 1H), 1.99−1.88 (m, 1H), 1.87−1.72 (m, 1H), 1.59 (d,
J = 3.51 Hz, 2H).
1
(
584 mg, 29%) as an orange foam. H NMR (400 MHz,
DMSO-d ): δ 8.47 (s, 1H), 6.92 (t, J = 7.82 Hz, 1H), 6.72 (d, J
6
=
3
2
8.61 Hz, 1H), 5.99 (d, J = 7.04 Hz, 1H), 5.69−5.54 (m, 3H),
.98 (d, J = 11.74 Hz, 1H), 3.78−3.63 (m, 1H), 2.13−2.02 (m,
H), 2.00−1.87 (m, 1H), 1.80−1.65 (m, 1H), 1.59 (br s, 2H).
2-(Tetrahydro-2H-pyran-2-yl)-N-(3-(9-(tetrahydro-2H-
pyran-2-yl)-9H-purin-6-yl)pyridin-2-yl)-2H-indazol-4-
amine (85a). A solution of 81b (37.4 mg, 0.17 mmol) and 77b
(51.5 mg, 0.17 mmol) in THF (1 mL) was cooled in an ice−
water bath and treated dropwise with 1.0 M LiHMDS in THF
(0.55 mL, 0.55 mmol). The mixture was stirred for 60 min and
4-Chloro-6-methyl-1H-pyrazolo[3,4-d]pyrimidine (82).
LDA was prepared by dropwise addition of 2.5 M n-BuLi in
hexanes (14.7 mL, 36.8 mmol) to i-Pr NH (5.42 mL, 38.4
2
mmol) in THF (40 mL) cooled in an ice−water bath. The
LDA solution was cooled to −78 °C, and a solution of 4,6-
dichloro-2-methylpyrimidine (64b) (5.45 g, 33.4 mmol) in
THF (50 mL) was added dropwise over 1 h. A solution of N-
methyl-N-(2-pyridyl)formamide (4.8 mL, 40 mmol) in THF
then quenched with H O (50 μL). The mixture was extracted
2
with EtOAc (100 mL) from saturated aqueous NaHCO (100
3
mL), dried (MgSO ), concentrated, and purified by silica gel
4
column chromatography (50% EtOAc/hexane) to give 85a
(
20 mL) was added dropwise to the solution at −78 °C over 20
(36.6 mg, 43%) as a yellow oil which crystallized upon
1
min. The resulting solution was stirred for 30 min and then
quenched with a solution of acetic acid (2.1 mL, 37 mmol) in
THF (20 mL) added dropwise at −78 °C over 10 min. The
solution was stirred at −78 °C for 30 min.
The resulting solution of 4,6-dichloro-2-methylpyrimidine-5-
carbaldehyde was treated dropwise with anhydrous hydrazine
standing. H NMR (400 MHz, CDCl ): δ 12.75 (br s, 1H),
3
9.77 (d, J = 7.82 Hz, 1H), 9.12 (s, 1H), 8.44 (d, J = 3.52 Hz,
1H), 8.38 (s, 2H), 8.06 (d, J = 7.04 Hz, 1H), 7.41 (d, 1H), 7.35
(d, J = 7.63 Hz, 1H), 7.03−6.92 (m, 1H), 5.88 (d, 1H), 5.73 (d,
1H), 4.30−4.13 (m, 2H), 3.84 (br s, 2H), 2.35−2.17 (m, 3H),
2.16−2.00 (m, 3H), 1.79 (br s, 6H).
(
1.1 mL, 35 mmol) at −78 °C. The mixture was stirred for 15
N-(3-(9H-Purin-6-yl)pyridin-2-yl)-1H-indazol-4-amine
(± )-10-Camphorsulfonate (20). A solution of 85a (34.8 mg,
70 μmol) in CH Cl /MeOH (4 mL; 1:1) was treated with CSA
min, and then the cooling bath was removed and the mixture
stirred at 22 °C for 1 h. The mixture was concentrated and
partitioned between H O (110 mL) and EtOAc (110 mL). The
organic layer was washed with saturated aqueous NaHCO3
(
2
2
(38 mg, 2.2 equiv), and the mixture was stirred at 40 °C for 3 h.
The volume of the reaction mixture was reduced by ∼50%
under a stream of N to remove CH Cl , and the solution was
2
100 mL), separated, dried (MgSO ), treated with activated
4
2
2
2
charcoal, and filtered through a plug of silica gel, washing with
EtOAc. The filtrate was concentrated and purified by silica gel
column chromatography (gradient: 5% acetone/CH Cl →
triturated with Et O, resulting in the formation of a precipitate
2
that was collected by filtration, washed with Et O, and dried
2
1
under vacuum to give 20 (33.7 mg, 86%) as a brown solid. H
2
2
2
5% EtOAc/hexane). The residue was suspended in CH Cl (3
NMR (400 MHz, DMSO-d ): δ 13.17 (br s, 1H), 9.89 (d, 1H),
2
2
6
mL) and cooled in a freezer for 16 h (−20 °C), and the solid
9.33 (s, 1H), 8.76 (s, 1H), 8.46−8.41 (m, 1H), 8.31 (s, 1H),
8.24 (d, 1H), 7.35 (t, 1H), 7.21 (d, 1H), 7.14 (t, 1H), 2.88 (d,
1H), 2.73−2.63 (m, 1H), 2.42−2.34 (d, 1H), 2.29−2.18 (m,
1H), 1.97−1.90 (m, 1H), 1.90−1.74 (m, 2H), 1.27 (m, 2H),
1.05 (s, 3H), 0.75 (s, 3H).
was collected by filtration to give 82 (330 mg, 5.9%) as a tan
1
solid. H NMR (400 MHz, DMSO-d ): δ 14.25 (br s, 1H), 8.35
6
(
s, 1H), 2.68 (s, 3H).
4
-Chloro-6-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-
pyrazolo[3,4-d]pyrimidine (83). A suspension of 82 (325
mg, 1.93 mmol) in EtOAc (3 mL) was treated with 3,4-
dihydro-2H-pyran (0.53 mL, 5.8 mmol) and MP-TsOH resin
N-(3-(6-Methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-
pyrazolo[3,4-d]pyrimidin-4-yl)pyridin-2-yl)-2-(tetrahy-
dro-2H-pyran-2-yl)-2H-indazol-4-amine (85b). A solution
of 81b (80 mg, 0.37 mmol) and 84 (112 mg, 0.36 mmol) in
THF (1 mL) was stirred in an ice−water bath and treated
dropwise with a 1.0 M solution of LiHMDS in THF (1.1 mL,
1.1 mmol). The mixture was stirred for 45 min and then
(Biotage) (72 mg, 4.3 mmol/g, 0.15 equiv), and the resulting
suspension was heated at 90 °C for 3 h. The solution was
filtered, washed with EtOAc, and concentrated to give a pale-
yellow oil. Purification by silica gel column chromatography
(
gradient: 5% → 20% EtOAc/hexanes) gave 83 (482 mg, 99%)
quenched with H O (0.1 mL). The mixture was extracted into
2
1
as a colorless oil. H NMR (400 MHz, DMSO-d ): δ 8.44 (s,
EtOAc (100 mL) from saturated aqueous NaHCO (100 mL),
6
3
5
208
dx.doi.org/10.1021/jm300184s | J. Med. Chem. 2012, 55, 5188−5219

Journal of Medicinal Chemistry
Article
dried (MgSO ), concentrated, and purified by silica gel column
bromopyridin-3-ylcarbamate (89) (481 mg, 1.76 mmol) and
4
chromatography (gradient: 25% → 30% EtOAc/hexanes) to
give 85b (56.9 mg, 31%) as a yellow oil. H NMR (400 MHz,
Pd(PPh ) (170 mg, 0.15 mmol) under N was treated with a
3
4
2
1
solution of 70b (630 mg, 1.47 mmol) in toluene (7 mL). The
mixture was heated at 120 °C for 16 h, concentrated, and
purified by silica gel column chromatography (gradient: 0% →
CDCl ): δ 11.97 (br s, 1H), 8.53−8.43 (m, 1H), 8.42−8.30 (m,
3
3
1
H), 7.96 (br s, 1H), 7.48−7.38 (m, 1H), 7.34 (d, J = 7.43 Hz,
H), 7.03−6.94 (m, 1H), 6.16 (d, J = 10.17 Hz, 1H), 5.71 (d, J
25% EtOAc/hexanes) to give 90 (462 mg, 95%) as a white
1
=
9.00 Hz, 1H), 4.14 (d, J = 9.98 Hz, 2H), 3.95−3.73 (m, 2H),
solid. H NMR (400 MHz, CDCl ): δ 12.61 (s, 1H), 8.82−8.76
3
3.04 (s, 3H), 2.76−2.58 (m, 1H), 2.39−2.26 (m, 1H), 2.25−
(m, 1H), 8.30 (dd, J = 4.30, 1.56 Hz, 1H), 8.21 (s, 1H), 7.33
(dd, J = 8.61, 4.30 Hz, 1H), 2.73 (s, 3H), 2.62 (s, 3H), 1.56 (s,
9H).
1.94 (m, 4H), 1.93−1.59 (m, 6H).
N-(3-(6-Methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-
pyridin-2-yl)-1H-indazol-4-amine (22). A solution of 85b
2-(2-Methyl-6-(methylthio)pyrimidin-4-yl)pyridin-3-
amine (91). A mixture of 90 (462 mg, 1.39 mmol), 4.0 M HCl
in 1,4-dioxane (7.0 mL, 28 mmol), and MeOH (8 mL) was
heated at 60 °C for 16 h. The reaction mixture was
concentrated, and the yellow residue was partitioned between
CH Cl (100 mL) and saturated aqueous NaHCO (100 mL).
(
57 mg, 0.11 mmol) in CH Cl (1 mL) and MeOH (2 mL)
2
2
was treated with CSA (114 mg, 4.4 equiv) and stirred at 60 °C
for 1 h. The mixture was concentrated and purified by strong
cation-exchange chromatography (gradient: MeOH → 2 M
44
NH /MeOH) to give 22 (35 mg, 87%) as a brown solid. The
3
2
2
3
1
H NMR of the free base thus obtained gave very broad signals,
The organic layer was dried (Na SO ), filtered, and
2 4
1
1
and so ∼2 mg of 22 was converted to the HCl salt for better H
concentrated to give 91 (380 mg, 100%) as a yellow oil. H
NMR (400 MHz, CDCl ): δ 8.13 (s, 1H), 8.06 (dd, J = 4.30,
NMR analysis by dissolving 22 in MeOH (1 mL) containing 2
3
drops of 2 M aqueous HCl followed by concentration to
1.57 Hz, 1H), 7.14 (dd, J = 8.22, 4.30 Hz, 1H), 7.04 (dd, J =
8.31, 1.47 Hz, 1H), 6.48 (br s, 1H), 2.70 (s, 3H), 2.61 (s, 3H).
6-Methoxy-N-(2-(2-methyl-6-(methylthio)pyrimidin-4-
yl)pyridin-3-yl)pyridin-3-amine (92). A mixture of 91 (380
mg, 1.64 mmol), 6-methoxypyridin-3-ylboronic acid (751 mg,
1
dryness. HCl salt: H NMR (400 MHz, DMSO-d ): δ 14.18 (br
6
s, 1H), 11.38 (s, 1H), 10.43 (s, 1H), 9.76 (s, 1H), 9.42 (d, 1H),
9
7
.04 (s, 1H), 8.64 (d, 1H), 8.46 (s, 1H), 7.98−7.85 (m, 2H),
.69 (d, J = 9.54 Hz, 1H), 2.14 (s, 3H).
4
-(2-Aminophenyl)-N,N-bis(4-methoxybenzyl)-6-
4.91 mmol), Cu(OAc)
2
(446 mg, 2.45 mmol), and i-Pr
NEt
2
methyl-1,3,5-triazin-2-amine (87). A mixture of 2-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (86) (292 mg, 1.33
mmol), Pd(PPh ) (59 mg, 0.05 mmol), 66 (395 mg, 1.03
(1.1 mL, 6.5 mmol) in CH
2
Cl (10 mL) was stirred for 72 h at
2
22 °C. The mixture was filtered through diatomaceous earth,
concentrated, and purified by silica gel column chromatography
3
4
mmol), Na CO ·H O (218 mg, 2.05 mmol), DME (5 mL), and
2
(gradient: 5% → 50% EtOAc/hexane) to give 92 (76 mg, 14%)
2
3
2
1
H O (1.5 mL) was purged with Ar and then heated at 90 °C for
as a yellow solid. H NMR (400 MHz, CDCl
): δ 11.23 (s,
3
2
h. The reaction mixture was diluted with H O (10 mL) and
1H), 8.22 (s, 1H), 8.15−8.06 (m, 2H), 7.52 (dd, J = 8.71, 2.84
Hz, 1H), 7.32 (dd, J = 8.51, 1.47 Hz, 1H), 7.13 (dd, J = 8.51,
4.21 Hz, 1H), 6.80 (d, J = 8.61 Hz, 1H), 3.97 (s, 3H), 2.70 (s,
3H), 2.62 (s, 3H).
2
extracted with EtOAc (20 mL), and the extract was dried
Na SO ), filtered, concentrated, and purified by silica gel
(
2
4
column chromatography (gradient: 0% → 1% MeOH/CH Cl )
2
2
1
to give 87 (244 mg, 54%) as a light-yellow oil. H NMR (400
6-(3-(6-Methoxypyridin-3-ylamino)pyridin-2-yl)-2-
methylpyrimidin-4-amine (28). A solution of 92 (76 mg,
0.22 mmol) in CH Cl (6 mL) was treated with m-CPBA (103
MHz, DMSO-d ): δ 8.25 (dd, J = 8.02, 1.57 Hz, 1H), 7.28−
6
7
1
.06 (m, 8H), 6.89 (t, J = 8.12 Hz, 4H), 6.72 (d, J = 7.63 Hz,
H), 5.47 (s, 1H), 4.78 (s, 2H), 4.74 (s, 2H), 3.73 (s, 3H), 3.72
2
2
mg, 0.45 mmol) at 22 °C and stirred for 90 min. The reaction
(
s, 3H), 2.44 (s, 3H).
mixture was partitioned between CH Cl2 (50 mL) and
2
N,N-Bis(4-methoxybenzyl)-4-(2-(6-methoxypyridin-3-
ylamino)phenyl)-6-methyl-1,3,5-triazin-2-amine (88). A
saturated aqueous NaHCO (50 mL). The organic layer was
3
dried (Na SO ), filtered, and concentrated to give crude 6-
2
4
mixture of 87 (540 mg, 1.22 mmol), Cu(OAc) (330 mg, 1.83
mmol), 6-methoxypyridin-3-ylboronic acid (561 mg, 3.67
methoxy-N-(2-(2-methyl-6-(methylsulfonyl)pyrimidin-4-yl)-
pyridin-3-yl)pyridin-3-amine (113 mg) as an orange oil. LC−
2
+
mmol), and i-Pr NEt (0.85 mL, 4.89 mmol) in CH Cl (15
MS m/z: 372.0 (M + H) .
2
2
2
mL) was stirred at 22 °C for 16 h, filtered through a pad of
NH gas was bubbled through a solution of crude 6-methoxy-
3
diatomaceous earth, concentrated, and purified by silica gel
N-(2-(2-methyl-6-(methylsulfonyl)pyrimidin-4-yl)pyridin-3-
yl)pyridin-3-amine (83 mg, 0.22 mmol) in 1,4-dioxane (7 mL)
for 5 min, and then the vessel was heated at 100 °C for 18 h.
The reaction mixture was concentrated and purified by silica gel
column chromatography (gradient: 10% → 60% EtOAc/
column chromatography (gradient: 0% → 2% 2 M NH in
3
MeOH/CH Cl ), to give 88 (384 mg, 57%) as a dark-yellow
2
2
+
oil. LC−MS m/z: 548.8 (M + H) .
-(2-(6-Methoxypyridin-3-ylamino)phenyl)-6-methyl-
,3,5-triazin-2-amine (26). A solution of 88 (384 mg, 0.70
4
1
hexanes). The product was further purified by trituration with
1
mmol) in TFA (3 mL) was treated with 3 drops of TfOH and
heated at 80 °C for 3 h. The solvent was removed in vacuo, and
the residue was purified by reversed-phase preparative HPLC
Et O to give 28 (22 mg, 22%) as a yellow solid. H NMR (400
2
MHz, CDCl ): δ 11.38 (br s, 1H), 8.12 (dd, J = 2.54, 0.39 Hz,
3
1H), 8.06 (dd, J = 4.30, 1.37 Hz, 1H), 7.52 (dd, J = 8.80, 2.74
Hz, 1H), 7.47 (s, 1H), 7.36−7.29 (m, 1H), 7.11 (dd, J = 8.41,
4.30 Hz, 1H), 6.79 (d, J = 8.80 Hz, 1H), 4.94 (br s, 2H), 3.96
(s, 3H), 2.57 (s, 3H).
5-Bromo-N-(6-methoxypyridin-3-yl)pyrimidin-4-
amine (95). A mixture of 5-bromopyrimidin-4-amine (94)
(344 mg, 1.98 mmol), 6-methoxypyridin-3-ylboronic acid (93)
(
Phenomenex C18 column: 150 mm × 30 mm; 5 μm; 10% →
9
2
1
2
7
8
0% MeCN/H O; 0.1% TFA additive) to give 26 (47 mg,
2
1
2%) as a yellow solid. H NMR (400 MHz, DMSO-d ): δ
6
0.92 (s, 1H), 8.44 (dd, J = 8.12, 1.66 Hz, 1H), 8.12 (d, J =
.74 Hz, 1H), 7.68 (dd, J = 8.80, 2.74 Hz, 2H), 7.53 (br s, 1H),
.28 (ddd, J = 8.46, 6.99, 1.57 Hz, 1H), 6.88 (dd, J = 12.81,
.51 Hz, 2H), 6.82−6.69 (m, 1H), 3.86 (s, 3H), 2.38 (s, 3H).
tert-Butyl 2-(2-Methyl-6-(methylthio)pyrimidin-4-yl)-
(907 mg, 5.93 mmol), i-Pr NEt (1.4 mL, 7.9 mmol), and
2
anhydrous Cu(OAc) (539 mg, 2.97 mmol) in CH Cl (2 mL)
2
2
2
pyridin-3-ylcarbamate (90). A mixture of tert-butyl 2-
was stirred at 22 °C for 16 h. The solid was filtered off and
5
209
dx.doi.org/10.1021/jm300184s | J. Med. Chem. 2012, 55, 5188−5219

Journal of Medicinal Chemistry
Article
washed with CH Cl . The filtrate was concentrated and purified
stirred under N at 22 °C for 72 h. The suspension was filtered,
2
2
2
by silica gel column chromatography (50% EtOAc/hexanes) to
and the solid was washed with EtOAc. The filtrate was washed
with saturated brine (2 × 200 mL) and dried (Na SO ),
1
give 95 (36 mg, 6.5%). H NMR (300 MHz, CDCl ): δ 8.55 (s,
3
2
4
1
1
H), 8.46 (s, 1H), 8.28 (s, 1H), 7.85 (dd, J = 8.77, 2.48 Hz,
H), 6.98 (s, 1H), 6.80 (d, J = 8.92 Hz, 1H), 3.95 (s, 3H).
N-(6-Methoxypyridin-3-yl)-5-(4-methyl-6-(methyl-
filtered, and concentrated to give 100 (19.19 g, 100%) as a
1
light-yellow powder. H NMR (400 MHz, CDCl ): δ 8.39 (d, J
3
= 2.54 Hz, 1H), 8.28 (dd, J = 8.02 Hz, 2.54 Hz, 1H), 5.84 (s,
thio)-1,3,5-triazin-2-yl)pyrimidin-4-amine (96). A mixture
of 5-bromo-N-(6-methoxypyridin-3-yl)pyrimidin-4-amine (95)
1H), 4.17−4.04 (m, 4H), 1.37 (s, 12H).
4-(5-(1,3-Dioxolan-2-yl)-2-fluoropyridin-3-yl)-N,N-bis-
(4-methoxybenzyl)-6-methyl-1,3,5-triazin-2-amine
(101a). A mixture of 66 (2.10 g, 5.46 mmol), 100 (1.90 g, 6.44
mmol), Pd(Amphos) Cl (201 mg, 0.28 mmol), and KOAc
(
0
23 mg, 0.08 mmol), 70a (35.2 mg, 0.08 mmol), CuI (15 mg,
.08 mmol), CsF (206 mg, 0.82 mmol), and Pd(PPh ) (9.5
3
4
mg, 8.2 μmol) in THF (1 mL) was heated at 140 °C in a
microwave reactor for 30 min. The reaction mixture was diluted
2
2
(1.51 g, 15.4 mmol) was suspended in H O (4 mL) and 1,4-
2
with H O (10 mL) and extracted with EtOAc (2 × 30 mL).
dioxane (20 mL), and N was bubbled through the suspension
2
2
The combined organic extracts were washed with saturated
for 30 s. The mixture was heated at 100 °C for 4 h, cooled to 22
brine (5 mL), dried (Na SO ), filtered, concentrated, and
°C, treated with H O (40 mL), and extracted with EtOAc (3 ×
2
4
2
purified by silica gel column chromatography (60% EtOAc/
100 mL). The combined organic extracts were dried (Na SO ),
2
4
1
hexanes) to give 96 (15 mg, 54%). H NMR (300 MHz,
filtered through a diatomaceous earth, concentrated, and
purified by silica gel column chromatography (gradient: 0%
CDCl ): δ 11.58 (s, 1H), 9.65 (s, 1H), 8.74 (s, 1H), 8.35 (d, J =
3
1
2
.19 Hz, 1H), 8.07 (dd, J = 8.84, 2.56 Hz, 1H), 6.82 (d, J = 8.92
Hz, 1H), 3.96 (s, 3H), 2.67 (s, 3H), 2.65 (s, 3H).
-(4-(6-Methoxypyridin-3-ylamino)pyrimidin-5-yl)-6-
methyl-1,3,5-triazin-2-amine (29). A mixture of 96 (14 mg,
.04 mmol), 28% aqueous NH (0.5 mL, 12.8 mmol), and 1,4-
→ 2% MeOH/CH Cl ) to give 101a (3.18 g). H NMR (400
2
2
MHz, CDCl ): δ 8.65 (dd, J = 8.90 Hz, 2.45 Hz, 1H), 8.42 (d, J
3
4
= 1.37 Hz, 1H), 7.23 (dd, J = 8.41 Hz, 5.87 Hz, 4H), 6.87 (dd, J
= 10.27 Hz, 8.71 Hz, 4H), 5.93 (s, 1H), 4.83 (s, 2H), 4.81 (s,
2H), 4.17−4.04 (m, 4H), 3.82 (s, 3H), 3.80 (s, 3H), 2.55 (s,
3H).
0
3
dioxane (1 mL) was heated at reflux for 1 h. The solid formed
was collected by filtration and washed with EtOAc (2 mL) to
2-(5-(1,3-Dioxolan-2-yl)-2-fluoropyridin-3-yl)-4-meth-
yl-6-(methylthio)-1,3,5-triazine (101b). A suspension of
100 (715 mg, 3.53 mmol), 68a (682 mg, 3.88 mmol),
Pd(PPh ) (408 mg, 0.35 mmol), and Na CO ·H O (935 mg,
1
give 29 (10 mg, 79%) as a yellow solid. H NMR (300 MHz,
DMSO-d ): δ 11.78 (s, 1H), 9.37 (s, 1H), 8.65 (s, 1H), 8.48 (s,
6
1
(
H), 8.10 (d, J = 8.33 Hz, 1H), 7.94 (s, 1H), 7.79 (s, 1H), 6.88
d, J = 8.77 Hz, 1H), 3.87 (s, 3H), 2.43 (s, 3H).
-(1,3-Dioxolan-2-yl)-2-fluoro-3-(4,4,5,5-tetramethyl-
,3,2-dioxaborolan-2-yl)pyridine (100). 6-Fluoronicotinal-
3
4
2
3
2
8.82 mmol) in 1,4-dioxane (16 mL) and H O (3.3 mL) was
2
5
heated at 90 °C for 2 h. The mixture was filtered and washed
with EtOAc (2 × 20 mL), and the combined organic phases
were concentrated and purified by silica gel column
chromatography (gradient: 0% → 30% EtOAc/hexanes) to
give 101b (526 mg, 48%) as a white solid. LC−MS m/z: 309
1
dehyde (97) (21.96 g, 176 mmol) was suspended in toluene
340 mL), and ethylene glycol (10.4 mL, 186 mmol) and
(
TsOH (15% in acetic acid, 1.10 mL) were added. The mixture
was heated at 120 °C for 45 min, cooled to 22 °C, diluted with
saturated aqueous NaHCO (50 mL) and H O (150 mL), and
+
(M + H) .
5-(4-(Bis(4-methoxybenzyl)amino)-6-methyl-1,3,5-tri-
azin-2-yl)-6-(6-methoxypyridin-3-ylamino)-
nicotinaldehyde (103a). A mixture of 101a (3.25 g, 6.28
mmol) and 3-amino-6-methoxypyridine (858 mg, 6.91 mmol)
in THF (63 mL) was cooled to 0 °C and treated dropwise with
1.0 M LiHMDS in THF (19 mL, 19 mmol). The mixture was
stirred at 0 °C for 2 h, and then 5 M aqueous HCl (10 mL, 50
mmol) was added and the mixture was stirred for 20 min. The
reaction mixture was diluted with CH Cl (200 mL) and
3
2
extracted with EtOAc (2 × 150 mL). The combined organic
layers were dried (Na SO ), filtered, concentrated, and purified
2
4
by silica gel column chromatography (gradient: 0% → 2.5%
MeOH/CH Cl ) to give 5-(1,3-dioxolan-2-yl)-2-fluoropyridine
2
2
(
98) (19.06 g, 64%) as a pale-yellow liquid. LC−MS m/z: 170
+
(
M + H) .
Compound 98 (18.80 g, 111 mmol) was dissolved in THF
(
2
300 mL) and cooled in a dry ice−acetone bath under N . A
2
2
2
.0 M solution of LDA in heptane/THF/ethylbenzene (89 mL,
washed with H O (2 × 100 mL), and the organic layer was
2
178 mmol) was added via syringe over 20 min. After 75 min, (i-
dried (Na SO ), filtered, and concentrated to give 103a (3.25 g,
2
4
1
PrO) B (40.8 mL, 178 mmol) was added via syringe over 5
90%) as a yellow solid. H NMR (400 MHz, DMSO-d ): δ
3
6
min, and then the reaction was allowed to slowly warm to 22
12.09 (s, 1H), 9.90 (s, 1H), 9.14 (d, J = 2.35 Hz, 1H), 8.77 (d, J
= 2.35 Hz, 1H), 8.31 (d, J = 2.74 Hz, 1H), 7.87 (dd, J = 8.80,
2.74 Hz, 1H), 7.28 (d, J = 8.61 Hz, 2H), 7.21 (d, J = 8.80 Hz,
2H), 6.94−6.87 (m, 2H), 6.87−6.78 (m, 3H), 4.83 (d, J = 7.24
Hz, 4H), 3.85 (s, 3H), 3.74 (s, 3H), 3.69 (s, 3H), 2.58 (s, 3H).
5-(1,3-Dioxolan-2-yl)-N-(6-methoxypyridin-3-yl)-3-(4-
methyl-6-(methylthio)-1,3,5-triazin-2-yl)pyridin-2-amine
(102b). A mixture of 101b (425 mg, 1.38 mmol) and 3-amino-
6-methoxypyridine (257 mg, 2.09 mmol) in THF (3 mL) was
cooled to 0 °C and treated dropwise with 1.0 M LiHMDS in
THF (4.1 mL, 4.1 mmol) and then stirred for 1 h. The reaction
°
C. After 4.5 h, the reaction mixture was treated with 1 M
aqueous NaOH (300 mL) and stirred for 15 min. The layers
were separated, and the organic layer was discarded. The stirred
aqueous phase was treated with concentrated aqueous HCl to
lower the pH to ∼5 and was then extracted with 10:1 CH Cl /
2
2
MeOH (3 × 250 mL). The aqueous phase was treated with 5
M aqueous HCl during the extractions to maintain the pH at
∼
5−6, and saturated brine was also added to the aqueous phase
to aid extraction. The combined organic extracts were
concentrated to give 5-(1,3-dioxolan-2-yl)-2-fluoropyridin-3-
ylboronic acid (99) (14.56 g, 62%). LC−MS m/z: 214 (M +
mixture was diluted with saturated aqueous NH Cl (10 mL)
4
+
H) .
and extracted with EtOAc (3 × 10 mL). The combined organic
extracts were washed with saturated brine (10 mL), dried
(Na SO ), concentrated, and purified by silica gel column
Compound 99 (14.56 g, 68.4 mmol) was suspended in
toluene (300 mL), and anhydrous MgSO (41.20 g, 342 mmol)
4
2
4
and pinacol (8.27 g, 70.0 mmol) were added. The reaction was
chromatography (gradient: 0% → 50% EtOAc/hexanes) to give
5
210
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Journal of Medicinal Chemistry
02b (35 mg, 6%) as a yellow solid. LC−MS m/z: 413 (M +
Article
1
H) .
The title compound was synthesized from 106a (37 mg, 0.06
mmol), following an analogous deprotection procedure to that
+
6
-(6-Methoxypyridin-3-ylamino)-5-(4-methyl-6-
described for compound 36, and isolated (5.7 mg, 26%) as a
1
(
methylthio)-1,3,5-triazin-2-yl)nicotinaldehyde (103b). A
yellow solid. H NMR (400 MHz, CDCl ): δ 11.73 (br s, 1H),
3
solution of 102b (35 mg, 0.09 mmol) in THF (3 mL) was
treated with 2 M aqueous HCl (1.5 mL, 3.0 mmol), and the
mixture was stirred at 22 °C for 2 h. A yellow precipitate
gradually formed. The THF was removed in vacuo, and the
precipitate was collected and dried to give 103b (27 mg, 86%)
8.85 (d, J = 2.35 Hz, 1H), 8.37 (d, J = 2.35 Hz, 1H), 8.28 (d, J
= 2.35 Hz, 1H), 8.11 (dd, J = 8.90, 2.45 Hz, 1H), 6.80 (d, J =
8.80 Hz, 1H), 5.41 (br s, 2H), 4.42 (s, 2H), 3.96 (s, 3H), 3.39
(s, 3H), 2.57 (s, 3H).
4-(2-(6-Methoxypyridin-3-ylamino)-5-morpholinopyr-
idin-3-yl)-6-methyl-1,3,5-triazin-2-amine (39). A mixture
+
as a yellow solid. LC−MS m/z: 369 (M + H) .
of 73j (100 mg, 0.17 mmol), Pd(OAc) (5.8 mg, 0.03 mmol),
(
5-(4-(Bis(4-methoxybenzyl)amino)-6-methyl-1,3,5-tri-
2
X-Phos (49 mg, 0.10 mmol), Cs CO (167 mg, 0.51 mmol),
azin-2-yl)-6-(6-methoxypyridin-3-ylamino)pyridin-3-yl)-
methanol (104). NaBH (103 mg, 2.72 mmol) was added to a
suspension of 103a (523 mg, 0.91 mmol) in MeOH (5 mL)
and CH Cl (5 mL) at 0 °C. The resulting solution was stirred
at 0 °C for 5 min and then allowed to warm to 22 °C and
stirred for 45 min. Saturated aqueous NH Cl (10 mL) and H O
(
CH Cl (2 × 50 mL). The combined organic extracts were
dried (Na SO ), filtered, and concentrated to give 104 (505
2
3
and morpholine (0.10 mL, 1.2 mmol) in toluene (2 mL) was
stirred under Ar at 160 °C for 24 h. The mixture was allowed to
cool to 22 °C and purified by silica gel column chromatography
4
2
2
(
gradient: 0% → 90% EtOAc/hexanes) to give N,N-bis(4-
methoxybenzyl)-4-(2-(6-methoxypyridin-3-ylamino)-5-mor-
4
2
pholinopyridin-3-yl)-6-methyl-1,3,5-triazin-2-amine (106b)
20 mL) were added, and the mixture was extracted with
1
(
95.6 mg, 88%) as an orange solid. H NMR (400 MHz,
2
2
CDCl ): δ 11.32 (br s, 1H), 8.44 (d, J = 2.93 Hz, 1H), 8.25 (d,
3
2
4
J = 2.54 Hz, 1H), 8.06 (d, J = 2.93 Hz, 1H), 7.86 (dd, J = 8.80,
2.74 Hz, 1H), 7.21 (d, J = 8.41 Hz, 2H), 7.17 (d, J = 8.61 Hz,
mg, 96%) as a yellow-orange solid. LC−MS m/z: 580 (M +
H) .
+
2
H), 6.87 (d, J = 8.41 Hz, 2H), 6.83 (d, J = 8.61 Hz, 2H), 6.68
(
5-(4-(Bis(4-methoxybenzyl)amino)-6-methyl-1,3,5-tri-
(
d, J = 8.80 Hz, 1H), 4.87 (s, 2H), 4.80 (s, 2H), 3.92 (s, 3H),
azin-2-yl)-6-(6-methoxypyridin-3-ylamino)pyridin-3-yl)-
methyl Methanesulfonate (105). A suspension of 104 (1.00
g, 1.73 mmol) in CH Cl (25 mL) was stirred at 0 °C and
treated with Et N (1.1 mL, 7.9 mmol) followed by MsCl (0.50
mL, 6.4 mmol). The resulting solution was stirred for 30 min,
diluted with CH Cl (10 mL) and H O (10 mL), and extracted
with CH Cl (2 × 50 mL). The combined organic extracts were
dried (Na SO ), filtered, and concentrated to give 105 (935
mg, 82%). LC−MS m/z: 658 (M + H) .
3
4
.87−3.83 (m, 4H), 3.81 (s, 3H), 3.78 (s, 3H), 3.06−3.00 (m,
H), 2.57 (s, 3H).
2
2
The title compound was synthesized from 106b (110 mg,
.17 mmol) following an analogous deprotection procedure to
3
0
that described for compound 36 and isolated as an orange solid
2
2
2
1
(
10.3 mg, 15%). H NMR (400 MHz, CDCl ): δ 11.39 (br s,
3
2
2
1
H), 8.49 (d, J = 3.13 Hz, 1H), 8.34 (d, J = 2.54 Hz, 1H), 8.10
2
4
(
1
3
d, J = 2.74 Hz, 1H), 8.10−8.07 (m, 1H), 6.76 (d, J = 8.80 Hz,
+
H), 5.38 (br s, 2H), 3.94 (s, 3H), 3.86−3.92 (m, 4H), 3.16−
(
5-(4-Amino-6-methyl-1,3,5-triazin-2-yl)-6-(6-methox-
.09 (m, 4H), 2.56 (s, 3H).
ypyridin-3-ylamino)pyridin-3-yl)methanol (36). A solu-
tion of 104 (101 mg, 0.17 mmol) in TFA (3.5 mL) was treated
with TfOH (70 μL) and stirred at 75 °C for 1 h. The mixture
was allowed to cool to 22 °C and then concentrated. 1.0 M
Aqueous NaOH (2.0 mL) followed by MeOH (1 mL) were
added, and the mixture was stirred at 22 °C for 5 min. The
MeOH was removed in vacuo, and the resulting solid was
collected by filtration, washing with H O (20 mL) and then
Et O (6 mL). Purification by silica gel column chromatography
(
7
4
-(5-(3,6-Dihydro-2H-pyran-4-yl)-2-(6-methoxypyri-
din-3-ylamino)pyridin-3-yl)-6-methyl-1,3,5-triazin-2-
amine (40). A mixture of 2-(3,6-dihydro-2H-pyran-4-yl)-
4
7
,4,5,5-tetramethyl-1,3,2-dioxaborolane (64 mg, 0.31 mmol),
3j (162 mg, 0.28 mmol), Pd dba (10 mg, 0.01 mmol), and X-
2
3
Phos (11 mg, 0.02 mmol) in 1,4-dioxane (3 mL) was purged
with Ar and treated with 1.0 M aqueous Na CO ·H O (0.69
2
3
2
2
mL, 0.69 mmol). The mixture was heated at 140 °C in a
microwave reactor for 30 min and then treated with 1 M
aqueous NaOH (30 mL) and extracted with EtOAc (30 mL).
The organic extract was washed with saturated brine, dried
2
gradient: 0% → 10% MeOH/CH Cl ) gave 36 (41.8 mg,
1%) as a yellow solid. H NMR (400 MHz, CD OD): δ 8.94
2 2
1
3
(
2
d, J = 2.54 Hz, 1H), 8.48 (d, J = 2.35 Hz, 1H), 8.25 (d, J =
.54 Hz, 1H), 8.07 (dd, J = 8.80, 2.74 Hz, 1H), 6.85 (d, J = 9.00
Hz, 1H), 4.60 (s, 2H), 3.94 (s, 3H), 3.38 (s, 2H), 2.52 (s, 3H).
-(5-(Methoxymethyl)-2-(6-methoxypyridin-3-
ylamino)pyridin-3-yl)-6-methyl-1,3,5-triazin-2-amine
37). A mixture of 73j (109 mg, 0.19 mmol), potassium
trifluoro(methoxymethyl)borate (34 mg, 0.22 mmol), X-Phos
8.9 mg, 0.019 mmol), Pd(OAc) (2.1 mg, 9.3 μmol), and
(
MgSO ), filtered, and concentrated to give 4-(5-(3,6-dihydro-
4
2
H-pyran-4-yl)-2-(6-methoxypyridin-3-ylamino)pyridin-3-yl)-
N,N-bis(4-methoxybenzyl)-6-methyl-1,3,5-triazin-2-amine
4
(
106c) (175 mg) as a bright-yellow crystalline solid.
The title compound was synthesized from 106c (175 mg)
(
following an analogous deprotection procedure to that
described for compound 36 as a bright-yellow amorphous
1
(
2
solid (38 mg, 35%). H NMR (400 MHz, DMSO-d ): δ 11.76
6
Cs CO (182 mg, 0.56 mmol) in 1,4-dioxane (1.2 mL) and
H O (0.12 mL) under N was heated in a microwave reactor at
1
plug of diatomaceous earth, washing with EtOAc, and the
filtrate was concentrated and purified by silica gel column
chromatography (gradient: 0% → 50% EtOAc/CH Cl ) to give
N,N-bis(4-methoxybenzyl)-4-(5-(methoxymethyl)-2-((6-me-
thoxypyridin-3-yl)amino)pyridin-3-yl)-6-methyl-1,3,5-triazin-2-
2
3
(s, 1H), 8.83 (d, J = 2.5 Hz, 1H), 8.54 (d, J = 2.7 Hz, 1H), 8.44
(d, J = 2.5 Hz, 1H), 8.17 (dd, J = 8.8, 2.7 Hz, 1H), 7.87 (br s,
1H), 7.73 (br s, 1H), 6.83 (d, J = 8.8 Hz, 1H), 6.22 (br s, 1H),
4.24 (d, J = 2.5 Hz, 2H), 3.87−3.82 (m, 5H), 2.47 (m, 2H),
2.44 (s, 3H).
4-(2-(6-Methoxypyridin-3-ylamino)-5-(4-methylpiper-
azin-1-yl)pyridin-3-yl)-6-methyl-1,3,5-triazin-2-amine
(41). The title compound (17.2 mg, 49%) was synthesized as
an orange solid following a similar two step procedure to that
described for 39 using 73j (50 mg, 0.09 mmol) and 1-
2
2
40 °C for 30 min. The mixture was filtered through a short
2
2
amine (106a) (37 mg, 33%) as a yellow solid. LC−MS m/z:
5
+
94 (M + H) .
5
211
dx.doi.org/10.1021/jm300184s | J. Med. Chem. 2012, 55, 5188−5219

Journal of Medicinal Chemistry
Article
1
methylpiperazine (20 μL, 0.18 mmol). H NMR (400 MHz,
ybenzyl)-6-methyl-1,3,5-triazin-2-amine (106h) (110 mg, 63%)
as a viscous yellow oil. LC−MS m/z: 640 (M + H) .
+
CDCl ): δ 11.41 (br s, 1H), 8.51 (d, J = 2.93 Hz, 1H), 8.34 (d,
J = 2.54 Hz, 1H), 8.11 (d, J = 2.93 Hz, 1H), 8.11−8.07 (m,
H), 6.76 (d, J = 8.80 Hz, 1H), 5.34 (br s, 2H), 3.93 (s, 3H),
3
A solution of 106h (110 mg, 0.17 mmol) in TFA (265 μL,
3.44 mmol) was treated with TfOH (15 μL, 0.17 mmol), and
the mixture was heated at 80 °C for 16 h. The mixture was
concentrated and purified by silica gel column chromatography
(gradient: 0% → 3% 2 M NH in MeOH/CH Cl ) to give 61
1
3
.22 (br s, 4H), 2.71 (br s, 4H), 2.56 (s, 3H), 2.45 (br s, 3H).
5
-(4-Amino-6-methyl-1,3,5-triazin-2-yl)-N-(6-methox-
ypyridin-3-yl)-6′-methyl-3,3′-bipyridin-6-amine (42).
The title compound was synthesized as a yellow crystalline
solid (57 mg, 56%) following a similar two step procedure to
that described for 40 using 73j (144 mg, 0.25 mmol) and 6-
methylpyridin-3-ylboronic acid (40.5 mg, 0.30 mmol).
NMR (400 MHz, DMSO-d ): δ 11.85 (s, 1H), 9.03 (d, J = 2.7
Hz, 1H), 8.77 (d, J = 2.2 Hz, 1H), 8.67 (d, J = 2.5 Hz, 1H),
.56 (d, J = 2.7 Hz, 1H), 8.20 (dd, J = 8.8, 2.7 Hz, 1H), 7.99
3
2
2
1
(15 mg, 22%) as a yellow solid. H NMR (400 MHz, DMSO-
d6): δ 11.68 (s, 1H), 8.61 (d, J = 2.15 Hz, 1H), 8.53 (d, J = 2.54
Hz, 1H), 8.23 (d, J = 2.15 Hz, 1H), 8.16 (dd, J = 8.80, 2.74 Hz,
1H), 7.83 (br s, 1H), 7.69 (br s, 1H), 7.11−7.45 (m, 5H), 6.81
(d, J = 8.80 Hz, 1H), 3.94 (s, 2H), 3.84 (s, 3H), 2.41 (s, 3H).
4-(5-((2-Methoxyethylamino)methyl)-2-(6-methoxy-
pyridin-3-ylamino)pyridin-3-yl)-6-methyl-1,3,5-triazin-2-
amine (38). A solution of 103a (240 mg, 0.42 mmol) in
CH Cl (2 mL) and MeOH (2 mL) was treated with 2-
1
H
6
8
(
(
(
dd, J = 8.1, 2.2 Hz, 1H), 7.91 (br s, 1H), 7.77 (br s, 1H), 7.38
d, J = 8.2 Hz, 1H), 6.85 (d, J = 8.8 Hz, 1H), 3.86 (s, 3H), 2.52
s, 3H), 2.46 (s, 3H).
2
2
methoxyethanamine (54 μL, 0.62 mmol) and NaBH(OAc)3
(264 mg, 1.25 mmol). The mixture was stirred for 2 h,
concentrated, diluted with CH Cl (100 mL), washed with
4
-(2-(6-Methoxypyridin-3-ylamino)-5-(1-methyl-1H-
pyrazol-4-yl)pyridin-3-yl)-6-methyl-1,3,5-triazin-2-
amine (43). The title compound was synthesized as an orange
crystalline solid (72 mg, 83%) following a similar two step
procedure to that described for 40 using 73j (97 mg, 0.17
2
2
saturated aqueous NaHCO (2 × 100 mL), dried (Na SO ),
3
2
4
and concentrated to give N,N-bis(4-methoxybenzyl)-4-(5-(((2-
methoxyethyl)amino)methyl)-2-((6-methoxypyridin-3-yl)-
amino)pyridin-3-yl)-6-methyl-1,3,5-triazin-2-amine (107a),
which was used without further purification.
mmol) and 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-
1
2
-yl)-1H-pyrazole (42 mg, 0.20 mmol). H NMR (400 MHz,
DMSO-d ): δ 11.69 (s, 1H), 8.87 (d, J = 2.5 Hz, 1H), 8.55 (t, J
The 107a thus obtained was deprotected using the procedure
6
=
(
3
3.1 Hz, 2H), 8.18 (dd, J = 8.8, 2.7 Hz, 1H), 8.12 (s, 1H), 7.90
s, 1H), 7.82 (s, 1H), 7.77 (s, 1H), 6.84 (d, J = 8.8 Hz, 1H),
.88 (s, 3H), 3.85 (s, 3H), 2.46 (s, 3H).
-(2-(6-Methoxypyridin-3-ylamino)-5-((4-methylpi-
perazin-1-yl)methyl)pyridin-3-yl)-6-methyl-1,3,5-triazin-
-amine (51). A mixture of 73j (160 mg, 0.27 mmol), X-Phos
13 mg, 0.03 mmol), Cs CO (268 mg, 0.82 mmol), potassium
described for 36 to give 38 (37 mg, 22%) as a yellow crystalline
1
solid. H NMR (400 MHz, CDCl ): δ 11.63 (s, 1H), 8.78 (d, J
3
= 2.54 Hz, 1H), 8.35 (d, J = 2.54 Hz, 1H), 8.24 (d, J = 2.54 Hz,
1H), 8.12 (dd, J = 8.80, 2.74 Hz, 1H), 6.77 (d, J = 8.80 Hz,
1H), 5.63 (s, 2H), 3.94 (s, 3H), 3.78 (s, 2H), 3.57−3.52 (m,
2H), 3.36 (s, 3H), 2.86−2.80 (m, 2H), 2.55 (s, 3H).
4
2
(
4-(5-(Azetidin-1-ylmethyl)-2-(6-methoxypyridin-3-
ylamino)pyridin-3-yl)-6-methyl-1,3,5-triazin-2-amine
(44). The title compound was synthesized as a yellow
crystalline solid (11 mg, 7%) following the two step procedure
described for 38 using 103a (228 mg, 0.40 mmol) and
2
3
1
-methyl-4-trifluoroboratomethylpiperazine (66 mg, 0.30
mmol), and Pd(OAc) (3.1 mg, 0.014 mmol) in THF (1
mL) and H O (0.1 mL) was purged with Ar and heated in a
microwave reactor at 140 °C for 30 min. The mixture was
filtered through a short plug of diatomaceous earth, washing
with EtOAc, and the filtrate was concentrated and purified by
silica gel column chromatography (gradient: 0% → 5% MeOH/
CH Cl ) to give N,N-bis(4-methoxybenzyl)-4-(2-(6-methox-
2
2
1
azetidine hydrochloride (48 mg, 0.51 mmol). H NMR (400
MHz, CDCl ): δ 11.75 (s, 1H), 8.90 (d, J = 2.35 Hz, 1H), 8.37
3
(d, J = 2.74 Hz, 1H), 8.17 (d, J = 2.35 Hz, 1H), 8.08 (dd, J =
8.90, 2.64 Hz, 1H), 6.77 (d, J = 8.80 Hz, 1H), 3.94 (s, 3H),
3.76 (s, 2H), 3.53 (t, J = 7.34 Hz, 4H), 2.53 (s, 3H), 2.28 (quin,
J = 7.38 Hz, 2H).
4-(2-(6-Methoxypyridin-3-ylamino)-5-(pyrrolidin-1-
ylmethyl)pyridin-3-yl)-6-methyl-1,3,5-triazin-2-amine
(45). The title compound was synthesized as a yellow
crystalline solid (81 mg, 46%) following the two step procedure
2
2
ypyridin-3-ylamino)-5-((4-methylpiperazin-1-yl)methyl)-
pyridin-3-yl)-6-methyl-1,3,5-triazin-2-amine (106g) (161 mg,
+
8
9%) as a viscous yellow oil. LC−MS m/z: 662 (M + H) .
The title compound was synthesized from 106g (175 mg,
.28 mmol) as a yellow solid (98 mg, 96%) following an
0
analogous deprotection procedure to that described for
compound 36. H NMR (400 MHz, DMSO-d ): δ 11.74 (s,
1
described for 38 using 103a (257 mg, 0.45 mmol) and
6
1
1
8
=
H), 8.69 (d, J = 2.15 Hz, 1H), 8.54 (d, J = 2.74 Hz, 1H),
.33−8.00 (m, 2H), 7.86 (br s, 1H), 7.71 (br s, 1H), 6.82 (d, J
pyrrolidine (48 mg, 0.67 mmol). H NMR (400 MHz, CDCl ):
3
δ 11.64 (s, 1H), 8.71 (d, J = 2.35 Hz, 1H), 8.36 (d, J = 2.54 Hz,
1H), 8.20 (d, J = 2.35 Hz, 1H), 8.13 (dd, J = 8.80, 2.74 Hz,
1H), 6.76 (d, J = 8.80 Hz, 1H), 6.33 (br s, 2H), 3.94 (s, 3H),
3.58 (s, 2H), 2.57 (br s, 4H), 2.54 (s, 3H), 1.90−1.80 (m, 4H).
4-(2-(6-Methoxypyridin-3-ylamino)-5-(piperidin-1-
ylmethyl)pyridin-3-yl)-6-methyl-1,3,5-triazin-2-amine
(46). The title compound was synthesized as a yellow
crystalline solid (52 mg, 28%) following the two step procedure
8.80 Hz, 1H), 3.84 (s, 3H), 3.81−3.49 (m, 4H), 3.40 (s, 2H),
2
.44 (s, 3H), 2.35 (m, 4H), 2.14 (s, 3H).
-(5-Benzyl-2-(6-methoxypyridin-3-ylamino)pyridin-
-yl)-6-methyl-1,3,5-triazin-2-amine (61). A mixture of 2-
4
3
benzyl-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (90 mg, 0.41
mmol), 73j (160 mg, 0.27 mmol), Pd dba (12 mg, 0.014
mmol), X-Phos (13 mg, 0.027 mmol), and Na CO ·H O (73
mg, 0.69 mmol) in 1,4-dioxane (2.5 mL) and H O (0.25 mL)
2
3
2
3
2
described for 38 using 103a (264 mg, 0.46 mmol) and
2
1
was purged with Ar and heated in a microwave reactor at 140
piperidine (68 μL, 0.69 mmol). H NMR (400 MHz, CDCl ):
3
°
C for 30 min. The mixture was filtered through a short plug of
δ 11.64 (s, 1H), 8.72 (d, J = 2.35 Hz, 1H), 8.35 (d, J = 2.54 Hz,
1H), 8.21 (d, J = 2.35 Hz, 1H), 8.14 (dd, J = 8.90, 2.84 Hz,
1H), 6.77 (d, J = 9.00 Hz, 1H), 5.56 (br s, 2H), 3.94 (s, 3H),
3.45 (s, 2H), 2.56 (s, 3H), 2.41 (br s, 3H), 1.60 (dt, J = 10.95,
5.48 Hz, 5H), 1.46−1.39 (m, 2H).
diatomaceous earth, washing with EtOAc. The filtrate was
concentrated and purified by silica gel column chromatography
(
(
gradient: 0% → 30% EtOAc/hexanes) to give 4-(5-benzyl-2-
6-methoxypyridin-3-ylamino)pyridin-3-yl)-N,N-bis(4-methox-
5
212
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Journal of Medicinal Chemistry
- ( 2 - ( 6 - M e t h o x y p y r i d i n - 3 - y l a m i n o ) - 5 -
Article
4
(± )-4-(2-(6-Methoxypyridin-3-ylamino)-5-((3-
(methylsulfonyl)pyrrolidin-1-yl)methyl)pyridin-3-yl)-6-
methyl-1,3,5-triazin-2-amine (59). The title compound was
synthesized as a yellow crystalline solid (78 mg, 42%) following
the two step procedure described for 38 using 103a (230 mg,
(
morpholinomethyl)pyridin-3-yl)-6-methyl-1,3,5-triazin-
-amine (47). Morpholine (18 μL, 0.20 mmol) and AcOH (4
μL, 0.07 mmol) were added to a suspension of 103b (25 mg,
.07 mmol) in EtOH (3 mL), the mixture was stirred at 22 °C
2
0
for 1 h, and then NaBH (CN) (4.3 mg, 0.07 mmol) was added.
0.40 mmol) and 3-(methylsulfonyl)pyrrolidine (89 mg, 0.60
3
1
The mixture was stirred for 16 h, diluted with H O (10 mL),
mmol). H NMR (400 MHz, CDCl ): δ 11.63 (s, 1H), 8.73 (d,
2
3
and extracted with EtOAc (3 × 10 mL). The combined organic
J = 2.35 Hz, 1H), 8.36 (d, J = 2.54 Hz, 1H), 8.21 (d, J = 2.15
Hz, 1H), 8.10 (dd, J = 8.80, 2.74 Hz, 1H), 6.78 (d, J = 8.80 Hz,
1H), 5.55 (br s, 2H), 3.94 (s, 3H), 3.68−3.56 (m, 3H), 3.05−
2.99 (m, 1H), 2.95−2.88 (m, 1H), 2.87 (s, 3H), 2.84−2.76 (m,
1H), 2.70−2.61 (m, 1H), 2.56 (s, 3H), 2.35−2.25 (m, 2H).
1-((5-(4-Amino-6-methyl-1,3,5-triazin-2-yl)-6-(6-me-
thoxypyridin-3-ylamino)pyridin-3-yl)methyl)piperidin-
4-ol (48). A solution of 105 (510 mg, 0.78 mmol) in CH Cl
layers were washed with saturated brine, dried (Na SO ),
2
4
filtered, concentrated, and purified by silica gel column
chromatography (gradient: 0% → 10% MeOH/CH Cl ) to
2
2
give N-(6-methoxypyridin-3-yl)-3-(4-methyl-6-(methylthio)-
,3,5-triazin-2-yl)-5-(morpholinomethyl)pyridin-2-amine
1
(
+
107e) (19 mg, 64%) as a yellow solid. LC−MS m/z: 440 (M
+
H) .
2
2
A mixture of 107e (19 mg, 0.04 mmol) in 2.0 M NH /i-
(25 mL) was treated with 4-hydroxypiperidine (254 mg, 2.51
3
PrOH (3 mL) was heated at 90 °C for 24 h. The reaction
mixture was concentrated and purified by silica gel column
chromatography (gradient: 0% → 5% MeOH/CH Cl ) to give
mmol) and Et N (0.49 mL, 3.5 mmol). The resulting
3
suspension was stirred for 16 h, diluted with H O (20 mL),
2
and extracted with CH Cl (3 × 25 mL). The combined
2
2
2
2
1
47 (6.0 mg, 34%) as a yellow solid. H NMR (400 MHz,
organic extracts were dried (Na SO ), concentrated, and
2 4
CDCl , with one drop of DMSO-d ): δ 11.69 (br s, 1H), 8.76
purified by silica gel column chromatography (gradient: 5%
→ 20% 2 M NH in MeOH/CH Cl ) to give 1-((5-(4-(bis(4-
methoxybenzyl)amino)-6-methyl-1,3,5-triazin-2-yl)-6-(6-me-
thoxypyridin-3-ylamino)pyridin-3-yl)methyl)piperidin-4-ol
(108a) (385 mg, 75%) as a yellow gum.
3
6
(
br s, 1H), 8.39 (br s, 1H), 8.23 (br s, 1H), 8.12 (br s, 1H),
.77 (d, J = 9.00 Hz, 1H), 5.92 (br s, 2H), 3.93 (br s, 3H), 3.49
br s, 2H), 2.55 (br s, 3H), 2.50 (br s, 4H), 2.23 (br s, 4H).
R)-(1-((5-(4-Amino-6-methyl-1,3,5-triazin-2-yl)-6-(6-
3
2
2
6
(
(
methoxypyridin-3-ylamino)pyridin-3-yl)methyl)-
pyrrolidin-3-yl)methanol (49). The title compound was
synthesized as a pale-yellow solid (65 mg, 28%) following the
two step procedure described for 38 using 103a (310 mg, 0.54
A solution of 108a (310 mg, 468 mmol) in TFA (1 mL) and
TfOH (0.2 mL) was stirred at 90 °C for 1 h. The solution was
allowed to cool, concentrated, and diluted with 2 M aqueous
NaOH (10 mL). The mixture was stirred for 10 min and then
extracted with CH Cl (3 × 25 mL). The combined organic
mmol) and (R)-pyrrolidin-3-yl-methanol (0.16 mL, 1.6 mmol).
2
2
1
H NMR (400 MHz, DMSO-d ): δ 11.83 (s, 1H), 10.12−10.01
extracts were dried, concentrated, and purified by silica gel
column chromatography (gradient: 5% → 20% 2 M NH in
MeOH/CH Cl ) to give 48 (120 mg, 61%) as a yellow solid.
H NMR (400 MHz, DMSO-d ): δ 11.75 (s, 1H), 8.68 (d, J =
6
(
dd, J = 5.28, 2.35 Hz, 1H), 8.91 (dd, J = 5.28, 2.35 Hz, 1H),
3
8.54 (dd, J = 2.54, 0.39 Hz, 1H), 8.42 (dd, J = 3.72, 2.35 Hz,
1H), 8.15 (dd, J = 8.90, 2.84 Hz, 1H), 7.93 (d, J = 1.17 Hz,
1H), 7.82 (d, J = 0.78 Hz, 1H), 6.85 (d, J = 9.00 Hz, 1H), 4.38
2
2
1
6
0.78 Hz, 1H), 8.54 (d, J = 2.74 Hz, 1H), 8.18 (d, J = 2.74 Hz,
1H), 8.16 (t, J = 2.35 Hz, 1H), 7.87 (d, J = 1.37 Hz, 1H), 7.71
(br s, 1H), 6.83−6.80 (d, J = 9.00 Hz, 1H), 4.54 (d, J = 4.11
Hz, 1H), 3.84 (s, 3H), 3.38 (br s, 3H), 2.67 (dd, J = 4.60, 2.64
Hz, 2H), 2.44 (s, 3H), 1.99 (m, 2H), 1.69 (d, J = 3.33 Hz, 2H),
1.38 (d, J = 10.76 Hz, 2H).
(
3
1
dd, J = 6.85, 0.39 Hz, 2H), 3.85 (s, 3H), 3.51−3.31 (m, 4H),
.29−3.07 (m, 2H), 2.86−2.55 (m, 1H), 2.44 (s, 3H), 2.14−
.92 (m, 1H), 1.82−1.61 (m, 1H).
4
-(2-(6-Methoxypyridin-3-ylamino)-5-((4-
(
methylsulfonyl)piperidin-1-yl)methyl)pyridin-3-yl)-6-
methyl-1,3,5-triazin-2-amine (56). The title compound was
synthesized as a yellow crystalline solid (87 mg, 52%) following
the two step procedure described for 38 using 103a (200 mg,
4-((5-(4-Amino-6-methyl-1,3,5-triazin-2-yl)-6-((6-me-
thoxypyridin-3-yl)amino)pyridin-3-yl)methyl)-
thiomorpholine 1,1-Dioxide (60). A mixture of 105 (126
mg, 0.19 mmol), thiomorpholine 1,1-dioxide (78 mg, 0.58
0
.35 mmol) and 4-(methylsulfonyl)piperidine (85 mg, 0.52
1
mmol). H NMR (400 MHz, CDCl ): δ 11.67 (s, 1H), 8.71 (d,
mmol), and Et N (0.11 mL, 0.77 mmol) in THF (4 mL) was
3
3
J = 2.15 Hz, 1H), 8.36 (d, J = 2.54 Hz, 1H), 8.18 (d, J = 2.35
Hz, 1H), 8.11 (dd, J = 9.00, 2.74 Hz, 1H), 6.77 (d, J = 8.80 Hz,
stirred at 80 °C for 4 h. The reaction mixture was diluted with
H O (25 mL) and extracted with EtOAc (3 × 25 mL), and the
2
1
H), 5.76 (s, 2H), 3.94 (s, 3H), 3.49 (s, 2H), 3.10 (d, J = 11.35
Hz, 2H), 2.86−2.80 (m, 4H), 2.55 (s, 3H), 2.18−2.00 (m, 4H),
.00−1.84 (m, 3H).
-(2-(6-Methoxypyridin-3-ylamino)-5-((4-(methylsul-
fonyl)-1,4-diazepan-1-yl)methyl)pyridin-3-yl)-6-methyl-
,3,5-triazin-2-amine (58). The title compound was
combined organic layers were dried (Na SO ), filtered,
2
4
concentrated, and purified by silica gel column chromatography
(gradient: 25% → 100% EtOAc/hexanes) to give 4-((5-(4-
(bis(4-methoxybenzyl)amino)-6-methyl-1,3,5-triazin-2-yl)-6-
((6-methoxypyridin-3-yl)amino)pyridin-3-yl)methyl)-
thiomorpholine 1,1-dioxide (108b) (80 mg, 60%) as a yellow
oil.
2
4
1
synthesized as a yellow crystalline solid (25 mg, 12%) following
the two step procedure described for 38 using 103a (242 mg,
Compound 108b (80 mg, 0.115 mmol) was deprotected
0
.42 mmol) and 1-(methylsulfonyl)-1,4-diazepane hydrochlor-
using the procedure described for 36 to give 60 (35 mg, 57%)
1
1
ide (135 mg, 0.63 mmol). H NMR (400 MHz, CD OD): δ
as a yellow crystalline solid. H NMR (400 MHz, DMSO-d ): δ
3
6
8
=
8
3
2
.90 (d, J = 2.35 Hz, 1H), 8.45 (d, J = 2.74 Hz, 1H), 8.22 (d, J
2.35 Hz, 1H), 8.02 (dd, J = 8.80, 2.74 Hz, 1H), 6.80 (d, J =
.80 Hz, 1H), 3.97 (s, 2H), 3.91 (s, 3H), 3.59−3.51 (m, 2H),
.47 (t, J = 6.26 Hz, 2H), 3.14−3.03 (m, 4H), 2.91 (s, 3H),
.09−2.00 (m, 2H).
11.76 (s, 1H), 8.75−8.67 (m, 1H), 8.54 (dd, J = 2.25, 0.49 Hz,
1H), 8.28−8.21 (m, 1H), 8.21−8.12 (m, 1H), 7.93−7.83 (m,
1H), 7.74 (br s, 1H), 6.82 (d, J = 8.80 Hz, 1H), 3.84 (s, 3H),
3.64 (s, 2H), 3.15−3.08 (m, 4H), 2.94−2.86 (m, 4H), 2.44 (s,
3H).
5
213
dx.doi.org/10.1021/jm300184s | J. Med. Chem. 2012, 55, 5188−5219

Journal of Medicinal Chemistry
-((4-(tert-Butoxycarbonyl)piperazin-1-yl)methyl)-2-
Article
5
Pd(Ph P) (175 mg, 0.15 mmol). The suspension was heated
at 80 °C for 16 h and filtered through a short plug of solid
Na SO , washing with EtOAc, and the filtrate was concentrated.
3
4
fluoropyridin-3-ylboronic acid (112). Benzoyl peroxide
(
1.57 g, 6.48 mmol) and NBS (23.2 g, 130 mmol) were added
2
4
to a stirred solution of 2-fluoro-5-methylpyridine (14.4 g, 130
The residue was purified by silica gel column chromatography
mmol) in CCl (125 mL), and the suspension was heated at
reflux for 2 h. After cooling, the solution was filtered,
concentrated, and purified by silica gel column chromatography
(gradient: 0% → 30% EtOAc/hexanes) to give 113b (968 mg,
4
1
74%) as a white solid. H NMR (400 MHz, CDCl ): δ 8.57 (d,
3
J = 9.00 Hz, 1H), 8.30 (s, 1H), 3.58 (s, 2H), 3.44 (br s, 4H),
2.66 (s, 3H), 2.64 (s, 3H), 2.43 (d, J = 4.50 Hz, 4H), 1.46 (s,
9H).
(
gradient: 0% → 10% EtOAc/hexanes) to give 5-(bromo-
methyl)-2-fluoropyridine (110) (15.10 g, 61%) as a yellow
+
solid. LC−MS m/z: 191 (M + H) .
N,N-Bis(4-methoxybenzyl)-4-(2-(6-methoxypyridin-3-
ylamino)-5-(piperazin-1-ylmethyl)pyridin-3-yl)-6-meth-
yl-1,3,5-triazin-2-amine (115a). A 1.0 M solution of
LiHMDS in THF (46.6 mL, 46.6 mmol) was added dropwise
to a solution of 113a (10.0 g, 15.5 mmol) and 3-amino-6-
methoxypyridine (2.9 mL, 23 mmol) in THF (150 mL) at 0
°C. The mixture was stirred for 1 h and then quenched with
H O (200 mL), diluted with saturated aqueous NH Cl (200
tert-Butyl piperazine-1-carboxylate (17.8 g, 95 mmol) was
added to a stirred solution of 110 (15.1 g, 80 mmol) in DMF
(
120 mL) at 0 °C, and the suspension was stirred at room
temperature for 16 h. The reaction was quenched with cold
H O (50 mL), the resulting suspension was stirred for 30 min,
2
and the resulting solid was collected by filtration, washing with
cold H O (50 mL). The off-white precipitate was dried under
2
2
4
vacuum to give tert-butyl 4-((6-fluoropyridin-3-yl)methyl)-
mL), and extracted with CH Cl (1 × 400 mL; 3 × 200 mL).
2 2
piperazine-1-carboxylate (111) (19.8 g, 84%) as a white solid.
The combined organic extracts were washed with saturated
1
H NMR (400 MHz, DMSO-d ): δ 8.13 (s, 1H), 7.90 (dt, J =
brine (500 mL) and then dried (Na SO ), filtered, concen-
6
2
4
8
2
.02, 1.37 Hz, 1H), 7.15 (dd, J = 8.31, 2.05 Hz, 1H), 3.51 (s,
H), 3.30 (br s, 4H), 2.40−2.10 (m, 4H), 1.39 (s, 9H).
trated, and purified by silica gel column chromatography
(gradient: 5% → 50% EtOAc/hexanes) to give tert-butyl 4-((5-
(4-(bis(4-methoxybenzyl)amino)-6-methyl-1,3,5-triazin-2-yl)-
6-(6-methoxypyridin-3-ylamino)pyridin-3-yl)methyl)-
piperazine-1-carboxylate (114a) (9.50 g, 82%). LC−MS m/z:
2.5 M n-BuLi in hexanes (36 mL, 90 mmol) was added
dropwise to a solution of i-Pr NH (12.8 mL, 90 mmol) in THF
2
(
150 mL, 75 mmol) at −40 °C, and the pale-yellow solution of
+
LDA was stirred for 1 h and then cooled to −78 °C. A solution
of 111 (22.2 g, 75 mmol) in THF (100 mL) was added
dropwise via cannula into the LDA solution over 30 min. The
brown mixture was stirred at −78 °C for 1.5 h, and then a
748 (M + H) .
A solution of 114a (57.0 g, 76 mmol) in CH
and TFA (200 mL) was stirred for 2 h and concentrated, and
the residue was dissolved in CH Cl (400 mL) and neutralized
by slow addition of saturated aqueous NaHCO . The aqueous
layer was extracted with CH Cl (2 × 200 mL), and the
combined organic extracts were dried (Na SO ), filtered, and
Cl (200 mL)
2
2
2
2
solution of (i-PrO) B (26 mL, 113 mmol) in THF (50 mL)
3
3
was added slowly. The resulting mixture was stirred at −78 °C
for 30 min, and then the cooling bath was removed. After the
reaction mixture had warmed up to 22 °C, the yellow mixture
was quenched with 1.0 M aqueous NaOH (50 mL) and stirred
for an additional 30 min. The separated aqueous layer was
carefully treated with 5 M aqueous HCl until acidic (pH ∼4−
2
2
2
4
concentrated to give 115a (45.6 g, 92%). This material was
+
used without further purification. LC−MS m/z: 648 (M + H) .
4-(2-((6-Methoxypyridin-3-yl)amino)-5-(piperazin-1-
ylmethyl)pyridin-3-yl)-6-methyl-1,3,5-triazin-2-amine
(50). Method A. A 1.0 M solution of LiHMDS in THF (1.5
mL, 1.5 mmol) was added to a solution of 113b (220 mg, 0.51
mmol) and 3-amino-6-methoxypyridine (94 mg, 0.76 mmol) in
THF (3 mL) at 0 °C, and the mixture was stirred for 1 h. The
5
), and the resulting cloudy mixture was diluted with EtOAc
(
150 mL). The separated aqueous layer was extracted with
EtOAc (2 × 150 mL), and the combined organic phases were
dried (Na SO ), filtered, and concentrated to give 112 (21.9 g,
2
4
+
86%) as a pale-yellow solid. LC−MS m/z: 340 (M + H) .
reaction mixture was diluted with saturated aqueous NH Cl (10
4
tert-Butyl 4-((5-(4-(Bis(4-methoxybenzyl)amino)-6-
mL), H O (10 mL), and EtOAc (10 mL). The separated
2
methyl-1,3,5-triazin-2-yl)-6-fluoropyridin-3-yl)methyl)-
piperazine-1-carboxylate (113a). A mixture of 66 (27.0 g,
aqueous layer was extracted with EtOAc (2 × 10 mL), and the
combined organic layers were washed with saturated brine,
dried (Na SO ), concentrated, and purified by silica gel column
70 mmol), 112 (28.6 g, 84 mmol), KOAc (17.2 g, 175 mmol),
2
4
and Pd(Amphos) Cl (2.59 g, 3.65 mmol) in 1,4-dioxane (300
chromatography (gradient: 0% → 50% EtOAc/hexanes) to give
tert-butyl 4-((6-(6-methoxypyridin-3-ylamino)-5-(4-methyl-6-
(methylthio)-1,3,5-triazin-2-yl)pyridin-3-yl)methyl)piperazine-
2
2
mL) and H O (60 mL) was stirred at 100 °C for 16 h under
2
N . The mixture was concentrated to remove most of the 1,4-
2
1
dioxane and then partioned between H O (250 mL) and
1-carboxylate (114b) (133 mg, 49%) as a yellow solid. H
2
EtOAc (500 mL). The organic layer was washed with H O
NMR (400 MHz, CDCl ): δ 11.42 (s, 1H), 8.81 (br s, 1H),
2
3
(
250 mL), dried (Na SO ), filtered, concentrated, and purified
8.36 (d, J = 2.74 Hz, 1H), 8.27 (br s, 1H), 8.10 (dd, J = 8.71,
2.64 Hz, 1H), 6.79 (d, J = 8.80 Hz, 1H), 3.95 (s, 3H), 3.50 (s,
2H), 3.43 (br s, 4H), 2.67 (s, 3H), 2.66 (br s, 3H), 2.42 (br s,
4H), 1.45 (s, 9H).
2
4
by silica gel column chromatography (gradient: 10% → 50%
EtOAc/hexanes) to give 113a (37.3 g, 83%). H NMR (400
MHz, CDCl ): δ 8.47 (dt, J = 9.05, 1.34 Hz, 1H), 8.24 (d, J =
1
3
1
.37 Hz, 1H), 7.22 (dd, J = 7.82, 7.24 Hz, 4H), 6.86 (t, J = 8.90
Hz, 4H), 4.81 (d, J = 4.30 Hz, 4H), 3.80 (d, J = 7.43 Hz, 6H),
.41 (dd, J = 5.18, 4.79 Hz, 4H), 2.54 (s, 4H), 2.40 (d, J = 5.09
TFA (2 mL) was added to a stirred mixture of 114b (66 mg,
0.12 mmol) in CH Cl (2 mL), and the mixture was stirred at
2
2
3
22 °C for 1 h. The reaction mixture was concentrated, diluted
Hz, 4H), 1.45 (s, 9H).
with saturated aqueous NaHCO (20 mL each), and extracted
3
tert-Butyl 4-((6-Fluoro-5-(4-methyl-6-(methylthio)-
,3,5-triazin-2-yl)pyridin-3-yl)methyl)piperazine-1-car-
with CH Cl (3 × 10 mL). The combined organic layers were
2
2
1
washed with saturated brine, dried (Na SO ), and concentrated
2 4
boxylate (113b). 1,4-Dioxane (15 mL) and H O (3 mL) were
added to a mixture of 68a (560 mg, 3.19 mmol), 112 (1.03 g,
to give 115b. This material was treated with a 2.0 M solution of
2
NH in i-PrOH (2 mL, 4 mmol), and the reaction vessel was
3
3
.04 mmol), Na CO ·H O (805 mg, 7.59 mmol), and
sealed and heated at 90 °C for 16 h. The mixture was
2
3
2
5
214
dx.doi.org/10.1021/jm300184s | J. Med. Chem. 2012, 55, 5188−5219

Journal of Medicinal Chemistry
Article
concentrated and purified by silica gel column chromatography
combined solids were purified by silica gel column chromatog-
raphy (gradient: 0% → 10% MeOH/CH Cl ), and the resulting
(
gradient: 0% → 10% MeOH/CH Cl ), followed by washing
2 2
2
2
the isolated solid with Et O and EtOAc, to give 50 (2.5 mg,
product was suspended in i-PrOH (20 mL) and sonicated for
35 min. The solid was collected by filtration and dried to give
54 (1.41 g, 78%) as a yellow solid. H NMR (400 MHz,
2
1
1
1
1
3%) as a yellow solid. H NMR (400 MHz, DMSO-d ): δ
1.74 (br s, 1H), 8.69 (br s, 1H), 8.55 (br s, 1H), 8.19 (br s,
H), 8.16 (br s, 1H), 7.87 (br s, 1H), 7.72 (br s, 1H), 6.82 (d, J
6
1
DMSO-d ): δ 11.76 (d, J = 1.96 Hz, 1H), 8.70 (d, J = 1.76 Hz,
6
=
=
8.41 Hz, 1H), 4.21−3.94 (m, 2H), 3.84 (br s, 3H), 3.42 (d, J
1H), 8.54 (br s, 1H), 8.19 (b, 2H), 7.87 (br s, 1H), 7.73 (d, J =
3.13 Hz, 1H), 6.83 (br s, 1H), 3.84 (br s, 3H), 3.48 (d, J = 2.15
Hz, 2H), 3.33 (d, J = 2.15 Hz, 3H), 3.10 (dd, J = 4.60, 2.25 Hz,
4H), 2.87 (d, J = 2.15 Hz, 3H), 2.44 (d, J = 0.78 Hz, 3H).
4-((5-(4-Amino-6-methyl-1,3,5-triazin-2-yl)-6-((6-me-
thoxypyridin-3-yl)amino)pyridin-3-yl)methyl)-N,N-dime-
thylpiperazine-1-sulfonamide (55). A mixture of 115a (520
mg, 0.80 mmol) and Et N (1.1 mL, 8.0 mmol) in CH Cl (50
13.50 Hz, 4H), 2.84 (br s, 4H), 2.45−2.40 (m, 3H), 2.32 (br
s, 1H).
4
-(2-((6-Methoxypyridin-3-yl)amino)-5-(piperazin-1-
ylmethyl)pyridin-3-yl)-6-methyl-1,3,5-triazin-2-amine
(
(
50). Method B. The title compound was alternatively prepared
250 mg, 79%) from 115a (500 mg, 0.77 mmol) following the
deprotection procedure described for 36.
3
2
2
Methyl 4-((5-(4-Amino-6-methyl-1,3,5-triazin-2-yl)-6-
mL) was stirred at 0 °C and treated dropwise with
dimethylsulfamoyl chloride (0.26 mL, 2.4 mmol). The mixture
was stirred for 2 h, concentrated, and purified by silica gel
column chromatography (gradient: 5% → 50% EtOAc/
hexanes) to give 4-((5-(4-(bis(4-methoxybenzyl)amino)-6-
methyl-1,3,5-triazin-2-yl)-6-(6-methoxypyridin-3-ylamino)-
pyridin-3-yl)methyl)-N,N-dimethylpiperazine-1-sulfonamide
(
6-methoxypyridin-3-ylamino)pyridin-3-yl)methyl)-
piperazine-1-carboxylate (52). A solution of 50 (500 mg,
.23 mmol) and Et N (0.51 mL, 3.7 mmol) in CH Cl (5 mL)
1
3
2
2
was cooled to 0 °C and treated with methyl chloroformate
(
0.19 mL, 2.45 mmol), and the suspension was stirred at 0 °C
for 1 h. The mixture was concentrated and purified by silica gel
+
column chromatography (gradient: 2% → 10% 2 M NH in
(118) (250 mg, 41%). LC−MS m/z: 755 (M + H) .
Compound 118 (210 mg, 0.28 mmol) was deprotected
3
MeOH/CH Cl ) to give 52 (160 mg, 28%) as a yellow solid.
2
2
1
H NMR (400 MHz, DMSO-d ): δ 11.75 (s, 1H), 8.70 (d, J =
following the procedure described for 36 to give 55 (110 mg,
6
1
1
.96 Hz, 1H), 8.54 (d, J = 2.54 Hz, 1H), 8.18 (s, 1H), 8.16 (d, J
77%) as a yellow solid. H NMR (400 MHz, DMSO-d
): δ
6
=
2.74 Hz, 1H), 7.87 (s, 1H), 7.71 (s, 1H), 6.82 (d, J = 8.80 Hz,
11.75 (s, 1H), 8.70 (d, J = 2.15 Hz, 1H), 8.55 (d, J = 0.39 Hz,
1H), 8.19 (d, J = 2.54 Hz, 2H), 8.17 (dd, J = 8.90, 2.84 Hz,
1H), 7.86 (d, J = 0.98 Hz, 1H), 7.71 (d, J = 1.76 Hz, 1H), 6.82
(d, J = 8.80 Hz, 1H), 3.84 (s, 3H), 3.47 (s, 2H), 3.31 (s, 2H),
3.16 (d, J = 4.89 Hz, 3H), 2.75 (s, 6H), 2.47 (s, 2H), 2.44 (s,
3H).
1
4
H), 3.84 (s, 3H), 3.58 (s, 3H), 3.44 (s, 2H), 3.39−3.33 (m,
H), 2.44 (s, 3H), 2.38−2.30 (m, 4H).
4
-((5-(4-Amino-6-methyl-1,3,5-triazin-2-yl)-6-((6-me-
thoxypyridin-3-yl)amino)pyridin-3-yl)methyl)-N,N-dime-
thylpiperazine-1-carboxamide (53). A solution of 115a
(
320 mg, 0.50 mmol) in CH Cl (25 mL) was treated with
3
0.14 mL, 1.5 mmol). The solution was stirred at 22 °C for 16
4-(2-((6-Methoxypyridin-3-yl)amino)-5-((1-
(methylsulfonyl)piperidin-4-yl)methyl)pyridin-3-yl)-6-
methyl-1,3,5-triazin-2-amine (57). tert-Butyl 4-methylene-
piperidine-1-carboxylate (119) (117 mg, 0.59 mmol) was
treated with a 0.5 M solution of 9-BBN in THF (1.18 mL, 0.59
mmol), and the mixture was heated at reflux for 4 h. The
resulting solution was transferred into a stirred mixture of 73j
(288 mg, 0.49 mmol), Pd (dba) (23 mg, 0.03 mmol), X-Phos
2
2
Et N (0.69 mL, 5.0 mmol) and dimethylcarbamyl chloride
(
h and then concentrated to give 116 as a yellow oil, which was
used directly without further purification.
The crude 116 was deprotected following the procedure
1
described for 36 to give 53 (315 mg) as a green solid. H NMR
(
8
0
J = 9.00 Hz, 1H), 3.84 (s, 3H), 3.43 (br s, 2H), 3.09 (dd, J =
4
4
400 MHz, DMSO-d ): δ 11.75 (s, 1H), 8.72−8.67 (m, 1H),
6
2
3
.56−8.50 (m, 1H), 8.20−8.16 (m, 1H), 8.15 (dd, J = 2.25,
.49 Hz, 1H), 7.88−7.83 (m, 1H), 7.73−7.66 (m, 1H), 6.81 (d,
(24 mg, 0.05 mmol), and Na CO ·H O (131 mg, 1.23 mmol)
2 3 2
in 1,4-dioxane (1 mL) and H O (0.25 mL). The mixture was
2
heated in a microwave reactor at 140 °C for 30 min. The
resulting mixture was filtered through a short plug of
diatomaceous earth, washing with EtOAc (3 × 10 mL). The
filtrate was concentrated and purified by silica gel column
chromatography (gradient: 0% → 30% EtOAc/hexanes) to give
tert-butyl 4-((5-(4-(bis(4-methoxybenzyl)amino)-6-methyl-
1,3,5-triazin-2-yl)-6-(6-methoxypyridin-3-ylamino)pyridin-3-
yl)methyl)piperidine-1-carboxylate (120) (261 mg, 71% yield)
.89, 4.30 Hz, 4H), 2.71 (s, 6H), 2.43 (s, 3H), 2.40−2.26 (m,
H).
4
-(2-((6-Methoxypyridin-3-yl)amino)-5-((4-
(
methylsulfonyl)piperazin-1-yl)methyl)pyridin-3-yl)-6-
methyl-1,3,5-triazin-2-amine (54). A solution of 115a (3.20
g, 4.94 mmol) and Et N (6.9 mL, 49 mmol) in CH Cl (65
mL) was stirred at −15 to −10 °C and treated dropwise with
MsCl (1.2 mL, 15 mmol). The resulting solution was stirred at
3
2
2
+
as a viscous yellow oil. LC−MS m/z: 747 (M + H) .
−
10 °C for 1 h. The solution was concentrated and purified by
TFA (4 mL, 52 mmol) was slowly added to a solution of 120
silica gel column chromatography (gradient: 0% → 10% 2 M
NH in MeOH/CH Cl ) to give N,N-bis(4-methoxybenzyl)-4-
(261 mg, 0.35 mmol) in CH Cl (5 mL), and the mixture was
2
2
stirred at 22 °C for 1 h. The mixture was concentrated to
remove as much TFA as possible. The sticky residue (121) was
dissolved in CH Cl (10 mL), and Et N (0.49 mL, 3.5 mmol)
3
2
2
(
2-(6-methoxypyridin-3-ylamino)-5-((4-(methylsulfonyl)-
piperazin-1-yl)methyl)pyridin-3-yl)-6-methyl-1,3,5-triazin-2-
2
2
3
amine (117) (3.01 g, 84%) as light-yellow solid.
followed by MsCl (82 μL, 1.05 mmol) were slowly added at 0
°C. The mixture was stirred for 1 h and then concentrated. The
crude product was partitioned between 1 M aqueous NaOH
(20 mL) and CH Cl (20 mL), the layers were separated, and
A solution of 117 (2.70 g, 3.72 mmol) in TFA (2 mL, 27
mmol) and TfOH (50 μL, 3.7 mmol) was stirred at 80 °C for 4
h. The dark solution was concentrated to a slurry and
2
2
neutralized with saturated aqueous NaHCO . The resulting
the aqueous layer was extracted with CH Cl (2 × 20 mL). The
3
2 2
precipitate was collected by filtration and washed with H O (50
combined organic layers were washed with saturated brine (20
2
mL). The aqueous washings were extracted with CH Cl (3 ×
mL), dried (Na SO ), filtered, concentrated, and purified by
silica gel column chromatography (gradient: 10% → 50%
2
2
2
4
5
0 mL), dried (Na SO ), filtered, and concentrated. The
2
4
5
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dx.doi.org/10.1021/jm300184s | J. Med. Chem. 2012, 55, 5188−5219

Journal of Medicinal Chemistry
Article
EtOAc/hexanes) to give N,N-bis(4-methoxybenzyl)-4-(2-(6-
methoxypyridin-3-ylamino)-5-((1-(methylsulfonyl)piperidin-4-
°C before being quenched with H O (50 mL) and stirred for
2
15 min. The layers were separated, and the organic phase was
extracted with 1 M aqueous NaOH (3 × 50 mL). The organic
phase was discarded, and the combined aqueous phases were
treated with concentrated HCl to lower the pH to 5. Then, the
aqueous phase was extracted with 10:1 CH Cl /MeOH (3 × 50
yl)methyl)pyridin-3-yl)-6-methyl-1,3,5-triazin-2-amine (122)
+
(
192 mg, 76%) as a yellow film. LC−MS m/z: 725 (M + H) .
Compound 57 (88 mg, 69%) was prepared as a yellow solid
from 122 (192 mg, 0.26 mmol) following the deprotection
2
2
1
procedure described for 36. H NMR (400 MHz, DMSO-d ): δ
mL). The organic extracts were combined and concentrated to
6
1
1
1
3
2
1
1.66 (s, 1H), 8.60 (d, J = 2.54 Hz, 1H), 8.54 (d, J = 2.54 Hz,
H), 8.17 (dd, J = 8.90, 2.64 Hz, 1H), 8.14 (d, J = 2.15 Hz,
H), 7.84 (br s, 1H), 7.71 (br s, 1H), 6.81 (d, J = 8.80 Hz, 1H),
.84 (s, 3H), 3.62−3.45 (m, 2H), 2.82 (s, 3H), 2.72−2.61 (m,
H), 2.43 (s, 3H), 1.68−1.67 (m, 2H), 1.64−1.46 (m, 1H),
.32−1.15 (m, 2H).
give 2-fluoro-5-(4-(methylthio)benzyl)pyridin-3-ylboronic acid
(125) (1.89 g, 71%) as a waxy solid. H NMR (400 MHz,
1
DMSO-d ): δ 8.40 (br s, 2H), 8.14−8.11 (m, 1H), 7.90−7.85
6
1
9
(m, 1H), 7.20 (s, 4H), 3.91 (s, 2H), 2.44 (s, 3H). F NMR
(377 MHz, DMSO-d ): δ −64.21 (s, 1F).
6
A mixture of 66 (2.37 g, 6.15 mmol), 125 (1.89 g, 6.83
4
- ( 2 - ( 6 - M e t h o x y p y r i d i n - 3 - y l a m i n o ) - 5 - ( 4 -
mmol), Pd(Amphos) Cl (224 mg, 0.32 mmol), and KOAc
2
2
(
methylsulfonyl)benzyl)pyridin-3-yl)-6-methyl-1,3,5-tri-
(2.72 g, 27.7 mmol) was suspended in EtOH (30 mL) and
H O (7.5 mL). N was bubbled through the suspension for
azin-2-amine (62). Magnesium turnings (214 mg, 8.79 mmol)
in a minimal amount of THF were treated with 1,2-
dibromoethane (50 μL, cat.), and the mixture was allowed to
stand until effervescence was observed (1 min). A solution of 4-
bromothioanisole (1.71 g, 8.39 mmol) in THF (20 mL) was
added dropwise, and the mixture was stirred for 2 h,
occasionally heating to reflux with a heat gun, to give a cloudy
pale-yellow solution. The resulting Grignard solution was
added dropwise over 10 min to a solution of 6-fluoronicoti-
naldehyde (67) (1.00 g, 7.99 mmol) in THF (10 mL) cooled in
a dry ice−acetone bath. The mixture was stirred at −78 °C for
2
2
about 20 s, and the mixture was then heated at 88 °C for 2.75 h.
The reaction was allowed to cool to 22 °C, treated with H O
2
(90 mL), and extracted with CH Cl (3 × 100 mL). The
2
2
combined organic extracts were dried (Na SO ), filtered,
2
4
concentrated, and purified by silica gel column chromatography
(gradient: 80% CH Cl /hexanes → CH Cl → 3.5% MeOH/
2
2
2
2
CH Cl ) to give 4-(2-fluoro-5-(4-(methylthio)benzyl)pyridin-
2
2
3-yl)-N,N-bis(4-methoxybenzyl)-6-methyl-1,3,5-triazin-2-amine
1
(126) (2.84 g, 80%). H NMR (400 MHz, CDCl ): δ 8.32 (dd,
3
J = 9.00 Hz, 2.35 Hz, 1H), 8.14 (dd, J = 1.56 Hz, 1H), 7.24−
7.18 (m, 6H), 7.13−7.09 (m, 2H), 6.90−6.83 (m, 4H), 4.81 (s,
2H), 4.78 (s, 2H), 3.99 (s, 2H), 3.82 (s, 3H), 3.80 (s, 3H), 2.53
(s, 3H), 2.46 (s, 3H).
3
0 min, and then the reaction was quenched by the dropwise
addition of 2 M aqueous HCl (9 mL, 18 mmol). The cooling
bath was removed, and the mixture was allowed to warm to
ambient temperature. The mixture was extracted into EtOAc (3
A solution of 126 (625 mg, 1.07 mmol) in CH Cl (11 mL)
2
2
×
200 mL) from H O (500 mL), dried (MgSO ), and
was cooled in an ice−water bath and treated with a solution of
2
4
concentrated to give (6-fluoropyridin-3-yl)(4-(methylthio)-
m-CPBA (557 mg, 3.23 mmol) in CH Cl (17.5 mL). The
2
2
1
phenyl)methanol (123) (1.86 g, 93%) as a colorless oil. H
NMR (400 MHz, DMSO-d ): δ 8.23 (s, 1H), 7.87 (t, J = 8.22
reaction was allowed to warm to 22 °C and stirred for 35 min.
The mixture was treated with saturated aqueous NaHCO (25
6
3
Hz, 1H), 7.32 (d, J = 8.02 Hz, 2H), 7.22 (d, J = 7.82 Hz, 2H),
mL) and saturated aqueous Na S O (6 mL) and stirried for an
2
2
3
7
3
.11 (d, J = 8.41 Hz, 1H), 6.11 (br s, 1H), 5.78 (s, 1H), 2.44 (s,
H).
additional 50 min. The layers were separated, and the aqueous
phase was extracted with CH Cl (3 × 25 mL). The combined
2
2
A solution of 123 (1.86 g, 7.96 mmol) in CH Cl (3 mL)
organic layers were dried (Na SO ), filtered, concentrated, and
2
2
2 4
was treated with TFA (2.6 mL, 34 mmol), resulting in a green
purified by silica gel column chromatography (gradient: 0% →
2% MeOH/CH Cl ) to give 4-(2-fluoro-5-(4-(methylsulfonyl)-
solution. The mixture was stirred for 5 min, and then Et SiH
3
2
2
(
3.3 mL, 21 mmol) was added dropwise. The green color
benzyl)pyridin-3-yl)-N,N-bis(4-methoxybenzyl)-6-methyl-
1,3,5-triazin-2-amine (127) (540 mg, 82%). H NMR (400
1
dissipated rapidly to give a straw-colored solution, and a brief
exotherm was observed. The mixture was stirred for 30 min and
then extracted into CH Cl (3 × 100 mL) from saturated
MHz, CDCl ): δ 8.34 (dd, J = 8.90 Hz, 2.45 Hz, 1H), 8.16 (d, J
3
= 1.76 Hz, 1H), 7.89 (d, J = 8.41 Hz, 2H), 7.40 (d, J = 8.22 Hz,
2H), 7.21 (d, J = 8.61 Hz, 4H), 6.90−6.82 (m, 4H), 4.82 (s,
2H), 4.79 (s, 2H), 4.13 (s, 2H), 3.82 (s, 3H), 3.80 (s, 3H), 3.03
(s, 3H), 2.54 (s, 3H).
2
2
aqueous NaHCO (100 mL). The organic extracts were dried
3
(
(
(
MgSO ) and purified by silica gel column chromatography
4
gradient: 5% → 7.5% EtOAc/hexanes) to give 2-fluoro-5-(4-
methylthio)benzyl)pyridine (124) (1.67 g, 89%) as a colorless
A solution of 127 (503 mg, 0.82 mmol) and 3-amino-6-
methoxypyridine (112 mg, 0.91 mmol) in THF (8 mL) was
cooled to 0 °C, treated dropwise with a 1.0 M solution of
LiHMDS in THF (2.5 mL, 2.5 mmol), and stirred for 40 min.
The mixture was treated with ice−water (0.60 mL), diluted
with CH Cl (100 mL), dried (Na SO ), filtered, concentrated,
1
oil. H NMR (400 MHz, DMSO-d ): δ 8.15 (s, 1H), 7.81 (t, J
=
2
(
6
8.22 Hz, 1H), 7.20 (s, 4H), 7.10 (d, J = 8.41 Hz, 1H), 3.94 (s,
1
9
H), 2.44 (s, 3H). F NMR (376 MHz, DMSO-d ): δ −72.37
6
s, 1F).
A solution of LiTMP was generated by dropwise addition of
2
2
2
4
a 1.6 M solution of n-BuLi in hexanes (7.4 mL, 11.8 mmol) to a
solution of 2,2,6,6-tetramethylpiperidine (2.1 mL, 12 mmol) in
THF (24 mL) cooled in an ice−water bath. The resulting
yellow solution was stirred for 15 min. A solution of 124 (2.26
and purified by silica gel column chromatography (gradient: 0%
→ 2% MeOH/CH Cl ) to give N,N-bis(4-methoxybenzyl)-4-
(2-(6-methoxypyridin-3-ylamino)-5-(4-(methylsulfonyl)-
2
2
benzyl)pyridin-3-yl)-6-methyl-1,3,5-triazin-2-amine (128) (412
1
g, 9.67 mmol) and (i-PrO) B (4.5 mL, 20 mmol) in THF (30
mg, 70%). H NMR (400 MHz, CDCl ): δ 11.58 (s, 1H), 8.61
3
3
mL) was cooled in a dry ice−acetone bath and treated dropwise
via cannula with the above LiTMP solution over 15 min to give
a yellow/brown solution. The solution was stirred at −78 °C
for 1 h, and then it was slowly allowed to warm up to 22 °C
over 1.5 h. The solution was stirred for an additional 1 h at 22
(d, J = 2.35 Hz, 1H), 8.26 (d, J = 2.54 Hz, 1H), 8.13 (d, J =
2.35 Hz, 1H), 7.87 (dd, J = 8.90 Hz, 2.64 Hz, 1H), 7.81 (d, J =
8.22 Hz, 2H), 7.37 (d, J = 8.22 Hz, 2H), 7.21 (d, J = 8.61 Hz,
2H), 7.15 (d, J = 8.61 Hz, 2H), 6.87 (d, J = 8.61 Hz, 2H), 6.82
(d, J = 8.41 Hz, 2H), 6.70 (d, J = 8.80 Hz, 1H), 4.85 (s, 2H),
5
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Journal of Medicinal Chemistry
Article
4
3
.77 (s, 2H), 4.00 (s, 2H), 3.93 (s, 3H), 3.82 (s, 3H), 3.80 (s,
H), 2.98 (s, 3H), 2.57 (s, 3H).
A suspension of 128 (412 mg, 0.57 mmol) in TFA (6 mL, 78
for microsomal stability data; the Amgen PKDM in vivo group
for PK experiments; and Peter Yakowec and Jin Tang for
expression and purification of PI3Kγ.
mmol) was heated at 75 °C for 16 h. The mixture was
concentrated and diluted first with saturated aqueous NaHCO3
and then with 5 M aqueous NaOH to raise the pH to ∼8−9.
The suspension was filtered, and the solid was washed with
ABBREVIATIONS USED
■
Amphos, di-tert-butyl(4-dimethylaminophenyl)phosphine; m-
CPBA, 3-chloroperoxybenzoic acid; CSA, (±)-camphorsulfonic
acid; DMSO, methyl sulfoxide; HGF, hepatocyte growth factor;
HPLC, high performance liquid chromatography; LC−MS,
liquid chromatography−mass spectrometry; LDA, lithium
diisopropylamide; LiHMDS, lithium bis(trimethylsilyl)amide;
MP-TsOH, macroporous polystyrene-supported p-toluenesul-
fonic acid; mTOR, mammalian target of rapamycin; NBS, N-
bromosuccinimide; PI3K, phosphatidylinositol 3-kinase; PTEN,
phosphatase and tensin homologue; PTFE, polytetrafluoro-
ethylene; TFA, trifluoroacetic acid; TfOH, trifluoromethane-
sulfonic acid; THP, tetrahydro-2H-pyranyl; TLC, thin layer
chromatography; TsOH, p-toluenesulfonic acid; X-Phos,
dicyclohexyl(2′,4′,6′-triisopropylbiphenyl-2-yl)phosphine
H O and purified by silica gel column chromatography
2
(
gradient: 3% → 5% MeOH/CH Cl ). The resulting solid
2 2
was suspended in MeOH (5 mL), filtered, and dried to give 62
1
(
126 mg, 46%) as a yellow powder. H NMR (400 MHz,
CDCl ): δ 11.64 (s, 1H), 8.65 (d, J = 2.15 Hz, 1H), 8.36 (d, J =
3
2
2
2
3
.35 Hz, 1H), 8.17 (d, J = 1.56 Hz, 1H), 8.11 (dd, J = 8.90 Hz,
.25 Hz, 1H), 7.88 (d, J = 8.22 Hz, 2H), 7.42 (d, J = 7.82 Hz,
H), 6.78 (d, J = 8.80 Hz, 1H), 5.36 (br s, 2H), 4.04 (s, 2H),
.95 (s, 3H), 3.04 (s, 3H), 2.56 (s, 3H).
In Vitro Assays. Enzyme assays for PI3Kα, PI3Kβ, PI3Kγ,
PI3Kδ, mTOR, hVPS34, and B-Raf, as well as the U-87 MG
pAKT-inhibition cellular assay were performed as previously
1
4,37
described.
In Vivo Assays. A mouse liver PD assay and a mouse U-87
MG tumor xenograft efficacy study were performed in CD1
nude mice (Charles Rivers Laboratories) with compound 54 at
the indicated doses and analyzed following the procedures
previously described.
Crystallography: Determination of p110γ Crystal
REFERENCES
■
(
1) Engelman, J. A.; Luo, J.; Cantley, L. C. The evolution of
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14
(2) Cantley, L. C. The phosphoinositide 3-kinase pathway. Science
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kinases. Annu. Rev. Biochem. 1998, 67, 481−507.
45
purified, and crystallized according to published procedures.
(
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2
2
0 h at room temperature in cryo solutions (mother liquor plus
0% glycerol) containing 1 mM compound (5% DMSO).
1
(
Crystals were then flash frozen in liquid nitrogen prior to data
collection. Diffraction data for p110γ + 54 was collected at the
Advanced Photon Source, beamline 22-BM using λ = 1.0000 Å
and a MAR 225 mm CCD detector. Data were reduced using
46
the HKL software suite, and the structures were solved by
molecular replacement using apo human p110γ as a search
model (PDB ID 1E8Y). Structures were refined using
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(
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data for compounds 5, 7, 8, 10−12, and 54; and X-ray cocrystal
data for compound 54 with PI3Kγ. This material is available
free of charge via the Internet at http://pubs.acs.org.
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(
1
(
AUTHOR INFORMATION
Corresponding Author
Phone: 805-447-4662. E-mail: adrians@amgen.com.
Gualberto, A. Development of inhibitors of the IGF-IR/PI3K/Akt/
■
*
mTOR pathway. Rev. Recent Clin. Trials 2010, 5, 189−208.
(14) D’Angelo, N. D.; Kim, T.-S.; Andrews, K.; Booker, S. K.;
Caenepeel, S.; Chen, K.; Freeman, D.; Jiang, J.; McCarter, J. D.; San
Miguel, T.; Mullady, E. L.; Schrag, M.; Subramanian, R.; Tang, J.;
Wahl, R. C.; Wang, L.; Whittington, D. A.; Wu, T.; Xi, N.; Xu, Y.;
Yakowec, P.; Zalameda, L. P.; Zhang, N.; Hughes, P.; Norman, M. H.
Discovery and Optimization of a Series of Benzothiazole Phosphoi-
nositide 3-Kinase (PI3K)/Mammalian Target of Rapamycin (mTOR)
Dual Inhibitors. J. Med. Chem. 2011, 54, 1789−1811.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors thank their colleagues Paul Brennan, Mqhele
Ncube, and Hui-Ling Wang for the design and synthesis of
compound 5 as a B-Raf inhibitor; Laurie Schenkel for the
syntheses of compounds 7 and 8; Josette Carnahan and Carol
Babij for providing B-Raf enzyme assay data; Bernd Bruenner
(15) Knight, S. D.; Adams, N. D.; Burgess, J. L.; Chaudhari, A. M.;
Darcy, M. G.; Donatelli, C. A.; Luengo, J. I.; Newlander, K. A.; Parrish,
C. A.; Ridgers, L. H.; Sarpong, M. A.; Schmidt, S. J.; Van Aller, G. S.;
Carson, J. D.; Diamond, M. A.; Elkins, P. A.; Gardiner, C. M.; Garver,
5
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dx.doi.org/10.1021/jm300184s | J. Med. Chem. 2012, 55, 5188−5219