Selective class i phosphoinositide 3-kinase inhibitors: Optimization of a series of pyridyltriazines leading to the identification of a clinical candidate, AMG 511

Article abstract of DOI:10.1021/jm300846z

The phosphoinositide 3-kinase family catalyzes the phosphorylation of phosphatidylinositol-4,5-diphosphate to phosphatidylinositol-3,4,5-triphosphate, a secondary messenger which plays a critical role in important cellular functions such as metabolism, cell growth, and cell survival. Our efforts to identify potent, efficacious, and orally available phosphatidylinositol 3-kinase (PI3K) inhibitors as potential cancer therapeutics have resulted in the discovery of 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 (1). In this paper, we describe the optimization of compound 1, which led to the design and synthesis of pyridyltriazine 31, a potent pan inhibitor of class I PI3Ks with a superior pharmacokinetic profile. Compound 31 was shown to potently block the targeted PI3K pathway in a mouse liver pharmacodynamic model and inhibit tumor growth in a U87 malignant glioma glioblastoma xenograft model. On the basis of its excellent in vivo efficacy and pharmacokinetic profile, compound 31 was selected for further evaluation as a clinical candidate and was designated AMG 511.

Full text of DOI:10.1021/jm300846z

Article
pubs.acs.org/jmc
Selective Class I Phosphoinositide 3‑Kinase Inhibitors: Optimization
of a Series of Pyridyltriazines Leading to the Identification of a
Clinical Candidate, AMG 511
Mark H. Norman,*,† Kristin L. Andrews,‡ Yunxin Y. Bo,† Shon K. Booker,† Sean Caenepeel,⊥
Victor J. Cee,† Noel D. D’Angelo,† Daniel J. Freeman,⊥ Bradley J. Herberich,† Fang-Tsao Hong,†
Claire L. M. Jackson,† Jian Jiang,§ Brian A. Lanman,† Longbin Liu,† John D. McCarter,∥ Erin L. Mullady,○
Nobuko Nishimura,† Liping H. Pettus,† Anthony B. Reed,† Tisha San Miguel,∥ Adrian L. Smith,†
Markian M. Stec,† Seifu Tadesse,† Andrew Tasker,† Divesh Aidasani,§ Xiaochun Zhu,§ Raju Subramanian,§
Nuria A. Tamayo,† Ling Wang,⊥ Douglas A. Whittington,∇ Bin Wu,† Tian Wu,# Ryan P. Wurz,†
Kevin Yang,† Leeanne Zalameda,∥ Nancy Zhang,⊥ and Paul E. Hughes⊥
†Department of Medicinal Chemistry, ‡Department of Molecular Structure, §Department of Pharmacokinetics and Drug Metabolism,
⊥
#
∥Department of High-Throughput Screening/Molecular Pharmacology, Department of Oncology Research, and Department of
Pharmaceutics, Amgen Inc., One Amgen Center Drive, Thousand Oaks, California 91320, United States
∇Department of Molecular Structure and ○Department of High-Throughput Screening/Molecular Pharmacology, Amgen Inc., 360
Binney Street, Cambridge, Massachusetts 02142, United States
S
* Supporting Information
ABSTRACT: The phosphoinositide 3-kinase family catalyzes the phosphorylation of phosphatidylinositol-4,5-diphosphate to
phosphatidylinositol-3,4,5-triphosphate, a secondary messenger which plays a critical role in important cellular functions such as
metabolism, cell growth, and cell survival. Our efforts to identify potent, efficacious, and orally available phosphatidylinositol 3-
kinase (PI3K) inhibitors as potential cancer therapeutics have resulted in the discovery of 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 (1). In this paper, we describe the
optimization of compound 1, which led to the design and synthesis of pyridyltriazine 31, a potent pan inhibitor of class I PI3Ks
with a superior pharmacokinetic profile. Compound 31 was shown to potently block the targeted PI3K pathway in a mouse liver
pharmacodynamic model and inhibit tumor growth in a U87 malignant glioma glioblastoma xenograft model. On the basis of its
excellent in vivo efficacy and pharmacokinetic profile, compound 31 was selected for further evaluation as a clinical candidate and
was designated AMG 511.
regulatory subunit, whereas the catalytic, p110α, p110β, and
p110δ, subunits are the products of PIK3CA, PIK3CB, and
PIK3CD genes, respectively. All cell types express PI3Kα and
PI3Kβ, in contrast to PI3Kδ and PI3Kγ, which are
predominantly expressed in leukocytes.
PI3K-dependent signaling is frequently activated in human
cancers through a variety of genetic and epigenetic alterations,
including activating somatic mutations in PIK3CA, the gene
INTRODUCTION
■
The phosphatidylinositol 3-kinase (PI3K) signaling network
has a central role in several cellular processes critical to the
initiation and progression of cancer, including growth, survival,
and metabolism. Class I PI3Ks, separated from other classes of
PI3Ks by structure and function, are the most clearly implicated
in cancer.1 Class I PI3Ks are subdivided into class IA (PI3Kα, β,
and δ) and class IB (PI3Kγ) isoforms. Class IA PI3Ks are
heterodimers composed of a p85 regulatory subunit and an
isoform-specific catalytic p110 subunit, which define their
individual identity. The PIK3R1 gene encodes the p85
Received: June 15, 2012
Published: August 16, 2012
© 2012 American Chemical Society
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Figure 1. Lead compound 1 and major oxidative metabolites of compound 1.
encoding the catalytic subunit of PI3Kα, and the loss of
function of the lipid phosphatase PTEN (a negative regulator of
PI3K).2,3 For example, oncogenic mutations in PIK3CA are a
frequent event in many types of human tumors, notably in
breast and colon cancer; and amplification of PIK3CA has also
been observed in a significant number of ovarian and non-
small-cell lung tumors. In addition, loss-of-function mutations
of PTEN commonly occur in prostate cancers and
glioblastomas.4 Consequently, significant efforts have been
applied to discover and develop PI3K inhibitors as potential
cancer therapeutics,5 and several compounds have entered
clinical trials in recent years (e.g., SF1126,6 PX-866,7 BEZ235,8
BGT226,9 GDC-0941,10 XL765,11 and GSK-61512).
We recently reported the identification of a highly selective
series of pyridyltriazine-based inhibitors of the class I PI3Ks,
exemplified by 1 (Figure 1), in our efforts to identify novel
inhibitors of this pathway.13,14 Pyridyltriazine 1 showed potent
activities in both the biochemical (Ki values of 12, 5, 2, and 4
nM for PI3Kα, PI3Kβ, PI3Kδ, and PI3Kγ, respectively) and
cellular (U87 MG IC50 = 16 nM) assays. In addition,
compound 1 was active in vivo as determined by the inhibition
of AKT (Ser473) phosphorylation in a mouse liver
pharmacodynamic assay (EC50 = 137 ng/mL). It was also
shown to inhibit tumor growth in a U87 MG glioblastoma
tumor xenograft model in mice with an ED50 of 6.0 mg/kg
(AUC0−24h = 7.6 μM·h). Although pyridyltriazine 1 proved to
be a potent, selective, and efficacious PI3K inhibitor that
represented a promising novel lead, it had rapid iv clearance in
both rat and mouse (CL = 1.7 and 2.5 L/h/kg, respectively;
approximately 50% of liver blood flow in both species) and a
short mean residence time (MRT = 1.6 h for both rat and
mouse). Therefore, this compound was not suitable for further
advancement, and we sought to improve the pharmacokinetic
profile of this series to enhance the in vivo efficacy.
As an initial step toward improving the overall pharmaco-
kinetic profile of these PI3K inhibitors, we examined
compound 1 in more detail. A single intravenous dose of
compound 1 was administered to bile-duct-cannulated rats, and
the concentration of the parent drug was quantified in the
excreta (urine, bile, and feces) collected over 24 h postdose.15
The analysis revealed that less than 2.5% of the administered
dose of compound 1 was excreted as intact parent compound,
which led us to infer that metabolism was the dominant
clearance pathway. Therefore, metabolite identification studies
were carried out by incubating compound 1 with rat and
human liver microsomes or hepatocytes.16 LC−MS metabolite
profiling indicated that the majority of observed metabolites
resulted from oxidative metabolism. Two prominent metabolic
pathways were identified: one arising from O-demethylation,
giving pyridone 2, and another arising from oxidation around
the benzylic piperazine region, giving compound 3 (although
the exact site of oxidation was not ascertained). With this
information in hand, we prepared a series of analogues designed
to block the sites of metabolism of compound 1, focusing
initially on modifying the methoxypyridine to reduce O-
demethylation and, subsequently, on examining substitutions
around the benzylic piperazine region of the molecule. These
investigations culminated in the identification of the clinical
candidate AMG 511 (compound 31).
CHEMISTRY
■
The synthetic methods outlined below are organized by one of
three general target classes: analogues wherein the methox-
ypyridine is modified (Scheme 1), benzylic or piperazine
monomethyl-substituted (or trifluoromethyl-substituted) deriv-
atives (Scheme 2), and benzylic or piperazine dimethyl-
substituted analogues (Scheme 3).
The syntheses of analogues of compound 1 wherein the
methoxypyridine moiety was modified are shown in Scheme 1.
These derivatives were prepared from one of two known
intermediates, tert-butyl 4-((5-(4-(bis(4-methoxybenzyl)-
amino)-6-methyl-1,3,5-triazin-2-yl)-6-fluoropyridin-3-yl)-
methyl)piperazine-1-carboxylate (4) or 2-(2-fluoro-5-((4-
(methylsulfonyl)piperazin-1-yl)methyl)pyridin-3-yl)-4-methyl-
6-(methylthio)-1,3,5-triazine (22).14 For example, the reaction
of fluoropyridine 4 with 6-methoxypyridin-3-amine, 2-methox-
ypyrimidin-5-amine, or 5-fluoro-6-methoxypyridin-3-amine
provided intermediates 5−7, respectively. TFA-mediated Boc
removal of 5−7 followed by sulfonylation of the resulting
piperazines with methanesulfonyl chloride gave the correspond-
ing methylsulfonamides 9−11. Alternatively, fluoropyridine 4
was converted to methylsulfonamide 8, which was subsequently
treated with 6-chloropyridin-3-amine, 6-methylpyridin-3-amine,
or 5-fluoropyridin-3-amine to give pyridine−triazine inter-
mediates 12−14. The final products (1 and 15−19) were
obtained by removal of the bis(p-methoxybenzyl) (bis-PMB)
protecting groups of 9−14. Compound 1 was also demethy-
lated by treatment with iodotrimethylsilane to give the
pyridone metabolite 2. The final two compounds illustrated
in Scheme 1 (20 and 21) were derived from thiomethyl
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a
Scheme 1
a
Reagents and conditions: (a) 6-methoxypyridin-3-amine, 2-methoxypyrimidin-5-amine, or 5-fluoro-6-methoxypyridin-3-amine, LiHMDS, THF, 0
°C; (b) TFA, CH2Cl2, rt; (c) MsCl, TEA, CH2Cl2, 0 °C; (d) TFA, TfOH, Δ; (e) 6-chloropyridin-3-amine, 6-methylpyridin-3-amine, or 5-
fluoropyridin-3-amine, THF, NaHMDS, or LiHMDS, 0 °C to rt; (f) TFA, CH2Cl2, 0 °C to rt; (g) 6-(trifluoromethyl)pyridin-3-amine or 5-
methoxypyridin-3-amine, LiHMDS, THF, 0 °C; (h) 2 M NH3 in i-PrOH, sealed tube, 90 °C; (i) (TMS)I, CHCl3, 70 °C.
intermediate 22. Deprotection of the Boc group followed by
conversion of the resulting free amine to the corresponding
sulfonamide yielded 23. Treatment of triazine 23 with 6-
(trifluoromethyl)pyridin-3-amine or 5-methoxypyridin-3-amine
provided intermediates 24 and 25, which were heated with
ammonia in a sealed tube to give the requisite trifluoromethyl
and methoxy derivatives 20 and 21, respectively.
were prepared similarly to the corresponding methyl derivatives
by treatment with trifluoroacetic acid and subsequent
purification by chiral preparative SFC.
The two enantiomers containing monomethyl substitutions
on the piperazine ring (42 and 43) were derived from a
common intermediate, 38,14 where the key steps involved the
addition of a potassium trifluoroborate intermediate by the
method reported by Molander et al.17 (Scheme 2). The
potassium trifluoroborate reagents, prepared by the treatment
of potassium (bromomethyl)trifluoroborate and (R)- or (S)-
tert-butyl 3-methylpiperazine-1-carboxylate amine, were
coupled with chloropyridine intermediates (38 or 44) under
palladium-catalyzed cross-coupling conditions. The synthesis of
each enantiomer differed only in the sequence of the reaction
steps performed. For the R-enantiomer (42), the trifluor-
oborate reagent was added first, followed by methylsulfonamide
formation and addition of the 5-fluoro-6-methoxypyridin-3-
amine group, whereas with the S-enantiomer (43), the 5-fluoro-
6-methoxypyridin-3-amine was introduced initially followed by
the addition of the trifluoroborate reagent and subsequent
elaboration to the desired sulfonamide.
The syntheses of the derivatives that contain two methyl
groups α to the basic piperazine nitrogen (56−59) are outlined
in Scheme 3. The addition of methyl Grignard to 6-
fluoronicotinaldehyde (47) followed by treatment of the
resulting alcohol with thionyl bromide provided bromide 49.
Fluoropyridine 50 was prepared by the displacement of the
bromine in 49 with (R)-tert-butyl 3-methylpiperazine-1-
carboxylate in good yield. Deprotonation of 50 at the position
ortho to the fluoro group with n-butyllithium followed by
The preparation of derivatives containing a monosubstitution
on either the benzylic position or the piperazine ring is outlined
in Scheme 2. Both the methyl- and trifluoromethyl-substituted
benzylic derivatives 30−33 were prepared from a common
intermediate, fluoropyridine 26.14Introduction of the 5-fluoro-
6-methoxypyridin-3-amine group to 26 followed by hydrolysis
of the acetal group provided aldehyde 27. Addition of
methylmagnesium bromide to aldehyde 27 and subsequent
conversion of the resulting alcohol to the mesylate gave
intermediate 29. Displacement of the mesyl group with 1-
(methylsulfonyl)piperazine followed by removal of the PMB
protecting groups gave the racemic product, which was
separated and purified by chiral preparative supercritical fluid
chromatography (SFC) to provide both 30 and 31 in >98% ee.
The corresponding trifluoromethyl derivatives were prepared
by hydrolysis of acetal 26 followed by treatment of the resulting
aldehyde with trimethyl(trifluoromethyl)silane. Subsequent
addition of 5-fluoro-6-methoxypyridin-3-amine yielded alcohol
34. Intermediate 36 was prepared by conversion of 34 to the
corresponding chloride 35 followed by displacement with tert-
butyl 1-piperazinecarboxylate. Deprotection of 36 followed by
reaction with methanesulfonyl chloride provided methylsulfo-
namide 37. The final trifluoromethyl derivatives (32 and 33)
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a
Scheme 2
a
Reagents and conditions: (a) 5-fluoro-6-methoxypyridin-3-amine, LiHMDS, THF, 0 °C; (b) HCl, MeOH, 0 °C to rt; (c) MeMgBr, THF, 0 °C; (d)
MsCl, TEA, CH2Cl2, rt or 0 °C; (e) 1-(methylsulfonyl)piperazine, TEA, CH2Cl2; (f) TFA, 75 °C, or TFA, TfOH, 70−80 °C; (g) (TMS)CF3, THF,
0 °C; (h) tert-butyl 1-piperazinecarboxylate, CH3CN, TEA; (i) TFA, CH2Cl2, rt; (j) Pd(OAc)2, X-Phos, Cs2CO3, THF, H2O, microwave, 85−140
°C; (k) chiral separation by preparative SFC on a Chiralcel OJ-H column.
quenching with triisopropyl borate gave the corresponding
boronic acid 51. Suzuki−Miyaura coupling of 51 with 4-chloro-
N,N-bis(4-methoxybenzyl)-6-methyl-1,3,5-triazin-2-amine and
subsequent addition of 5-fluoro-6-methoxypyridin-3-amine
gave compound 53 as a mixture of two diastereomers epimeric
at the benzylic position. Deprotection of the Boc group
followed by methylsulfonamide formation with methanesulfon-
yl chloride provided the penultimate intermediates 54 and 55,
which were separable by column chromatography on silica gel.
Acid deprotection of 54 and 55 provided the first two desired
diastereomers, 56 and 57.
An alternative route was employed to prepare the final two
diastereomers, 58 and 59. Addition of 5-fluoro-6-methoxypyr-
idin-3-amine to fluoropyridine 38 followed by the Stille
coupling with tributyl(1-ethoxyvinyl)stannane provided ketone
60 in good yield (Scheme 3). Mesylate 62 was obtained by
reduction of 60 with sodium borohydride and subsequent
treatment with methanesulfonyl chloride. Reaction of mesylate
62 with (R)-tert-butyl 3-methylpiperazine-1-carboxylate pro-
vided 63 as a mixture of two diastereomers epimeric at the
benzylic position. Compound 63 was converted to the
methylsulfonamide derivative by the methods described
previously, and the resulting two diastereomers were separated
by column chromatography before deprotection to yield the
aminotriazines 58 and 59.
The final analogue required for our investigation was the
benzylic gem-dimethyl analogue 69. This compound was
prepared from intermediate 38 as shown in Scheme 3. Addition
of 5-fluoro-6-methoxypyridin-3-amine followed by Suzuki−
Miyaura coupling of 38 with isopropenylboronic acid pinacol
ester provided propenyl intermediate 66. The benzylic amine
was introduced by the reaction of 66 with sodium azide in
trifluoroacetic acid to give azide 67, which was subsequently
reduced to the corresponding primary amine 68 by hydro-
genation over palladium on carbon. Bisalkylation of the
benzylic amine by treatment with ((methylsulfonyl)azanediyl)-
bis(ethane-1,2-diyl) dimethanesulfonate formed the piperazine
sulfonamide ring, and final deprotection of the PMB groups
provided the requisite gem-dimethyl derivative 69.
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a
Scheme 3
a
Reagents and conditions: (a) MeMgBr, THF, 0 °C; (b) SOBr2, CH2Cl2, 25 °C; (c) (R)-tert-butyl 3-methylpiperazine-1-carboxylate, K2CO3, KI,
CH3CN, 70 °C; (d) n-BuLi, B(O-i-Pr)3, THF; (e) 4-chloro-N,N-bis(4-methoxybenzyl)-6-methyl-1,3,5-triazin-2-amine,14 PdCl2(A-Phos)2, KOAc,
dioxane−H2O, 100 °C; (f) 5-fluoro-6-methoxypyridin-3-amine, LiHMDS, THF, 0 °C; (g) TFA, CH2Cl2, rt; (h) MsCl, TEA, CH2Cl2, 0 °C; (i)
diastereomeric separation; (j) TFA, TfOH, rt to 80 °C; (k) X-Phos, Pd(OAc)2, CsF, dioxane, tributyl(1-ethoxyvinyl)stannane, 110 °C; (l) NaBH4,
THF, 0 °C; (m) (S)-tert-butyl 3-methylpiperazine-1-carboxylate, 2,2,6,6-tetramethylpiperidine, CH3CN, reflux; (n) isopropenylboronic acid pinacol
ester, K2CO3, PdCl2(A-Phos)2, 4:1 dioxane/water, sealed tube, Δ; (o) NaN3, TFA, CHCl3, −15 °C to rt; (p) H2, Pd/C, THF, rt; (q)
((methylsulfonyl)azanediyl)bis(ethane-1,2-diyl) dimethanesulfonate, DIPEA, sealed vial, 130 °C.
crystal structure of compound 1 bound to the active site of the
PI3Kγ protein, which is highly homologous to the other class I
PI3K isoforms.14,20 The key interactions of compound 1 within
the active site of the enzyme are illustrated in Figure 2. In the
region of the active site referred to as the affinity pocket (Figure
2A), a hydrogen bond network exists between the pyridyl
nitrogen and a water molecule that is bridged between the
Asp841 carboxylate and the Tyr867 hydroxyl groups, as we
have observed in an earlier series of PI3K inhibitors.14,21 In
addition, the methyl of the methoxy group occupies a small
channel that projects toward Asp836, and the methoxy oxygen
atom forms a favorable interaction with Lys833. Since we had
established that the position of the pyridine nitrogen was
important for PI3K activity from our earlier SAR studies, we
retained this nitrogen and focused our modifications in the
latter two areas (i.e., exploring the pocket near Asp836 and
probing potential interactions with Lys833) as potential ways to
both increase potency and improve metabolic stability.
RESULTS AND DISCUSSION
■
The compounds described in this paper were inhibitors of all
class I PI3Ks, and the data, which were determined using
modified in vitro AlphaScreen assays, are reported in Tables 1
and 2.18 Cell-based activity was measured by the inhibition of
AKT (Ser473) phosphorylation in U87 MG glioblastoma cells.
In addition to the enzyme and cellular assays, the metabolic
stabilities of the compounds were evaluated in rat and human
liver microsomal (RLM and HLM) preparations for an initial
assessment of metabolism; these data are reported as intrinsic
clearance (CLint).19 These microsomal data were used to
prioritize potent compounds for additional pharmacokinetic
studies (vide infra).
In the initial phase of this investigation, we examined a small
set of analogues that contained replacements of the
methoxypyridine group in an attempt to increase potency and
reduce or eliminate the O-demethylation observed with
compound 1 (Table 1). To aid in the design of novel inhibitors
that could address this metabolic liability, we first analyzed the
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Figure 2. X-ray crystal structure of compound 1 bound to PI3Kγ determined at 2.95 Å resolution (PDB code 1E8Y). The gray surface indicates the
van der Waals surface area, and hydrogen bonds (or potential hydrogen bonds) are shown by dashed lines. (A) Affinity pocket region. (B) Ribose
pocket region. Note: Lys890 has been cut away in the surface view for clarity.
The first five compounds shown in Table 1 examined
substitutions on the 2-position of the pyridyl ring that project
into the Asp836 channel. A significant loss in PI3K activity was
observed when compound 1 was demethylated to give pyridone
2. Presumably, compound 2 exists predominantly in the form of
the pyridone tautomer (vs the hydroxypyridine tautomer), and
as such, the favorable interaction observed between the pyridyl
nitrogen of compound 1 and the bridged water molecule
between Asp841 and Tyr867 is disrupted, thereby resulting in
diminished activity. Replacement of the methoxy group of
compound 1 with small hydrophobic groups, such as chloro
(17) or methyl (18), was tolerated and gave only a slight loss in
activity on both the enzymatic and cellular levels; however, a
significant (∼10−15-fold) loss in potency was observed with
the larger trifluoromethyl substituent (compound 20). This
result is consistent with the narrow dimensions of the Asp836
channel observed in the crystal structure.
The next two compounds shown in Table 1 were designed to
alter the electron density in the ring (which we postulated may
affect the compound’s propensity for O-demethylation) as well
as to add a potential hydrogen bond with Lys833 (through
interactions with the 3-N pyrimidinyl nitrogen of compound 15
or with the 3-fluoro group of compound 16). The PI3K
activities of pyrimidine 15 were comparable to those of
compound 1; however, its cellular activity diminished by
approximately 4-fold. On the other hand, an increase in cellular
activity was obtained by the addition of a fluorine atom at the 3-
position of the pyridine ring (compound 16). Unfortunately,
the relative degrees of O-demethylation for compounds 1, 15,
and 16 could not be ascertained from the microsomal data,
since the intrinsic clearances of compounds 15 and 16 were
comparable to that of compound 1. However, when compound
16 was incubated in rat hepatocytes, metabolite profiling
revealed that oxidation on the methoxy group was significantly
reduced and that the dominant metabolite resulted from
oxidation on the core and not from demethylation of the
methoxy group. Although this was only a qualitative
observation, as a small amount of the demethylated metabolite
was still observed, the result was encouraging and suggested
that the added fluoro substituent could improve the overall
metabolic profile of the compound.
group was maintained (19) or by moving the methoxy group
from the 2-position to the 3-position (21). As seen in the
cocrystal structure of 1 with PI3Kγ (Figure 2A), the distance
between Lys833 and the 3-position of the pyridine ring was 4.3
Å. Modeling studies suggested that Lys833 could adopt
multiple conformations, including one where it was positioned
to favorably interact with the 3-position of the pyridine ring.
Taken together, the SAR results for the pyridine ring suggested
that interactions of the 2-methoxy group within the channel
were not critical for binding affinity, as a range of replacements
were tolerated.
Although the exact position of metabolism in compound 3
was not determined from our metabolism studies with
compound 1, oxidation on either side of the basic piperazine
nitrogen was probable, as benzylic C−H bonds and carbons
adjacent to a nitrogen atom are particularly prone to oxidation
by cytochrome P450 enzymes.22 In addition, the benzylic
position of compound 1 was also predicted to be the most
likely site of metabolism using MetaSite prediction software,
which was consistent with literature precedent.23,24 Therefore,
we examined the effect of methyl (and trifluoromethyl)
substitutions on the benzylic position and/or on the piperazine
ring in the second phase of our SAR investigations as a
potential means of blocking metabolism in this region of the
molecule. Figure 2B highlights the area of the enzyme being
explored with these derivatives and suggests that there would
be sufficient room to accommodate additional substituents in
this region. In addition, we postulated that we may also be able
to increase potency with these substituted analogues by gaining
additional hydrophobic interactions with the Thr887, Met953,
and Asp950 residues on the floor of the pocket (Figure 2B).
Since compound 16 had the best combination of cellular
potency and microsomal stability, the 5-fluoro-6-methoxypyr-
idin-3-amine moiety was retained in the next set of analogues
(Table 2). These derivatives were designed to systematically
examine the combinations of both monomethyl (30, 31, 42,
and 43) and dimethyl (56−59, and 69) substitutions on the
benzylic and piperazine positions. In general, all of the
substitutions were well tolerated within the PI3K enzymes, as
predicted by modeling studies. Both sets of monomethyl
derivatives (30 and 31; 42 and 43) showed a slight increase in
activities at the PI3Kα and PI3Kγ isoforms as compared to
compound 16. Although no significant stereochemical prefer-
Surprisingly, only a minimal loss in activity was observed by
the elimination of the 2-methoxy group while the 3-fluoro
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a
Table 1. Structure−Activity Relationships of Affinity Pocket Derivatives
a
The inhibition of PI3K activity was determined using a modified in vitro AlphaScreen assay with human p110α, p110β, p110δ, and p110γ enzymes.
b
The data represent an average of at least two determinations. Standard deviations are reported in the Supporting Information. Cellular IC50 values
were determined by an AKT (Ser473) phosphorylation assay using U87 MG glioblastoma cells. Single experimental values.19
c
ence was observed with regard to PI3K inhibition, the (R)-
methyl benzylic (31) and the (S)-methylpiperazine (43)
derivatives showed greater rat microsomal stabilities than
their corresponding enantiomers (30 and 42, respectively).
Increasing the size of the benzylic substituent to a
trifluoromethyl group (32 and 33) was detrimental to activity,
which was consistent with the steric constraints imposed by the
enzyme in this region of the binding site.
Combining methyl substitutions on both the benzylic and
piperazine positions provided the four diastereomers 56−59. In
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a
Table 2. Structure−Activity Relationships of Mono- and Disubstituted Derivatives
a
The inhibition of PI3K activity was determined using a modified in vitro AlphaScreen assay with human p110α, p110β, p110δ, and p110γ enzymes.
b
The data represent an average of at least two determinations. Standard deviations are reported in the Supporting Information. Cellular IC50 values
were determined by an AKT (Ser473) phosphorylation assay using U87 MG glioblastoma cells. Single experimental values.19
c
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Figure 3. X-ray cocrystal structure of compound 31 bound to PI3Kγ determined at 2.64 Å resolution (PDB code 4FLH). The gray surface indicates
the van der Waals surface area, and hydrogen bonds (or potential hydrogen bonds) are shown by dashed lines. (A, B) Side views highlighting the
linker and affinity pocket regions. (C, D) End views highlighting the ribose pocket region. Note: Lys890 has been cut away in the surface view for
clarity.
general, the bismethyl substitutions were also tolerated, with
the (S)-methylpiperazine diastereomers 58 and 59 being
preferred (with U87 MG IC50 values of 1−3 nM). As was
observed with the monomethyl derivatives, the stereochemis-
tries of the methyl groups had an impact on microsomal
stability within this series, with the R,S-isomer being more
stable (compound 58). The gem-dimethyl substitution on the
benzylic position was also tolerated and provided a derivative
(69) that exhibited excellent potencies in both the enzyme and
cellular assays.
All of the class I PI3K inhibitors prepared in this
investigation were highly selective over other members of the
closely related PIKK family of kinases, including the protein
kinase mTOR and the lipid kinases hVPS34 and PI4Ks
(biochemical IC50 values >10 μM). In addition, a selected set of
derivatives also showed excellent selectivity against several
protein kinases when evaluated in an Ambit KINOMEscan
selectivity screen.25,26 For example, compound 31 showed no
significant binding affinity toward 372 protein kinases when
tested at 1 μM. As noted previously,14 the high degree of
selectivity over protein kinases for this series of compounds
may be attributable, in large part, to the 2-methyl substituent
on the triazine hinge binder that occupies a small hydrophobic
pocket found in the PI3Ks (near Tyr867) which is generally
not present in protein kinases (excluding mTOR). The
additional structural features of the compounds optimized
from this investigation can be exemplified in the X-ray cocrystal
structure of compound 31 bound to PI3Kγ (Figure 3). As
observed previously for 1, the aminotriazine of compound 31
forms two hydrogen bonds to Val882 in the hinge region, and
the methoxypyridine nitrogen atom makes a hydrogen bond to
an ordered water molecule sitting between Tyr867 and Asp841
(Figure 3A,B). The methoxy oxygen atom and the additional 3-
fluoro substituent both form favorable interactions with Lys833
within the affinity pocket. The nitrogen in the pyridine central
core forms a hydrogen bond to a water molecule that is
associated with Asp964, an interaction that was not resolved in
the crystal structure with compound 1. Finally, the additional
methyl group on the benzylic position of compound 31
effectively fills the hydrophobic pocket formed by Thr887,
Ile963, and Asp950 residues on the floor of the enzyme, while
the methylsulfonamide oxygens engage in hydrogen bonds to
Lys802 and Ala805 (Figure 3C,D).
The SAR investigations described above enabled the
identification of several potent pan class 1 PI3K inhibitors
(<10 nM in the U87 MG cellular assay) that also showed good
metabolic stability in human and rat liver microsomes (CLint
<
100 μL/min/mg) (i.e., 16, 30, 31, 43, 58, and 69). This set of
compounds was evaluated further in pharmacokinetic experi-
ments, and the results of these studies are shown in Table 3.
Although there was not an exact correlation between in vitro rat
microsomal clearance values and the actual clearance observed
in vivo, generally, the rank order between the two measure-
ments held within this series. For example, compound 30,
which was the least stable in rat liver microsomes (CLint = 98
μL/min/mg), also demonstrated the highest in vivo clearance
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a
of AKT (Ser473) phosphorylation were determined 5 min
following the administration of HGF by a quantitative
electrochemiluminescence immunoassay. The results are
shown in Figure 4. Compound 31 significantly suppressed
PI3K signaling, as indicated by a dose-dependent decrease in
phosphorylated AKT (p-AKT) at Ser473 (Figure 4), and a
nonlinear regression analysis revealed a plasma EC50 of 228 ng/
mL. Inhibition of AKT phosphorylation directly correlated with
plasma concentrations in this study.
Given the potent pharmacodynamic effect of compound 31,
this compound was evaluated for its ability to inhibit tumor
growth in a mouse U87 MG glioblastoma xenograft model.28 In
this model, compound 31 was administered orally at 1, 3, or 10
mg/kg daily for 12 days (Figure 5). Treatment with compound
Table 3. Pharmacokinetic Profiles of Selected Compounds
b
c
iv
po
compd CL (L/kg/h) Vdss (L/kg) MRT (h) F (%) AUC (μM·h)
1
16
30
31
43
58
69
1.7
2.0
2.9
0.4
1.0
1.3
0.7
2.6
3.3
6.4
1.7
3.2
4.0
3.5
1.6
1.7
2.2
3.9
3.2
3.1
4.9
77
45
57
60
60
49
24
0.68
0.95
0.35
5.0
2.3
1.4
1.3
a
Pharmacokinetic parameters following administration in male
Sprague−Dawley rats: three animals per study. Dosed at 1 mg/kg
as a solution in DMSO. Dosed at 2 mg/kg as a solution or suspension
b
c
in 15% (hydroxypropyl)-β-cyclodextrin (HPβCD), 2% hydroxypropyl
methylcellulose (HPMC), and 1% Pluronic F68, pH 2.2, with
methanesulfonic acid (MSA).
in rats (CL = 2.9 L/kg/h), while the compound that was most
stable in microsomes (compound 31; CLint = 20 μL/min/mg)
showed the lowest clearance in vivo (CL = 0.4 L/kg/h). In
general, the compounds reported in Table 3 demonstrated
moderate to high volumes of distribution (Vdss ≈ 2−6 L/kg)
and moderate mean residence times (MRT ≈ 2−5 h). The
majority of the compounds also demonstrated good oral
bioavailabilities (F = 45−77%) (albeit with varying exposure
levels), with the exception of compound 69, which exhibited
oral bioavailability of only 24%. Overall, the benzylic (R)-
methyl analogue 31 had a superior pharmacokinetic profile with
low clearance (0.4 L/h/kg, 12% of liver blood flow), good oral
bioavailability (F = 60%), and a commensurate high oral
exposure (AUC = 5.0 μM·h).
With its excellent in vitro cellular potency and favorable
pharmacokinetic profile, pyridyltriazine 31 was evaluated
further in pharmacodynamic and xenograft studies. Initially,
compound 31 was tested in a mouse liver pharmacodynamic
model in which the inhibition of hepatocyte growth factor
(HGF)-induced AKT (Ser473) phosphorylation was meas-
ured.27 The compound was dosed orally at 3, 10, and 30 mg/
kg, and after 6 h the mice were administered HGF to activate
PI3K-dependent AKT phosphorylation in the liver. The levels
Figure 5. Effect of daily dosing of pyridine−triazine 31 in an
established U87 MG glioblastoma xenograft model grown in female
CD1 NU/NU mice. The arrow denotes the first day of dosing.
Statistical significance was evaluated by repeated measures analysis of
variance followed by Dunnett’s post hoc test.
31 at 1 mg/kg QD resulted in significant inhibition of tumor
growth of approximately 70% compared to the vehicle control
group. Tumor stasis was observed in the cohort treated with 3
mg/kg, and tumor regression was observed in the 10 mg/kg
Figure 4. Effect of compound 31 in a mouse liver pharmacodynamic model measuring the inhibition of HGF-stimulated AKT (Ser473)
phosphorylation 6 h postdose. Asterisks denote p < 0.0001 compared with the vehicle/HGF group. Statistical significance was evaluated by
Dunnett’s method. Bars represent the average SD (n = 3). Tilted squares represent mean plasma concentrations.
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purified by preparative HPLC (Phenomenex Gemini C18, 100 × 30
mm, 5 μm) using a gradient of 5−40% CH3CN (0.1% TFA)/H2O
(0.1% TFA) over 20 min and then 100% CH3CN (0.1% TFA) for 3
min. The fractions containing product were concentrated under
vacuum, and the resulting solid was suspended in aqueous saturated
sodium bicarbonate, sonicated, and centrifuged. The supernatant
liquid was decanted, and the solid was washed with aqueous saturated
sodium bicarbonate (1×) and water (2×) and then dried under
vacuum to afford the title compound (0.46 g, 47%) as a yellow solid.
MS (ESI positive ion): m/z calcd for C20H25N9O3S 471, found 472 (M
+ H)+. 1H NMR (400 MHz, DMSO-d6): δ 11.41 (s, 1H), 11.36 (br s,
1H), 8.68 (d, J = 2.1 Hz, 1H), 8.17 (d, J = 2.1 Hz, 1H), 7.99 (br s,
1H), 7.83 (br s, 1H), 7.59−7.76 (m, 2H), 6.37 (d, J = 9.5 Hz, 1H),
3.47 (br s, 2H), 3.10 (t, J = 4.0 Hz, 4H), 2.86 (s, 3H), 2.47 (br s, 4H),
2.42 (s, 3H).
4 - ( 2 - ( ( 2 - M e t h ox y p y r im i d i n - 5 - y l ) a m i n o) - 5 - ( ( 4 -
(methylsulfonyl)piperazin-1-yl)methyl)pyridin-3-yl)-6-methyl-
1,3,5-triazin-2-amine (15). tert-Butyl 4-((5-(4-(Bis(4-
methoxybenzyl)amino)-6-methyl-1,3,5-triazin-2-yl)-6-((2-methoxy-
pyrimidin-5-yl)amino)pyridin-3-yl)methyl)piperazine-1-carboxylate
(6). A solution of tert-butyl 4-((5-(4-(bis(4-methoxybenzyl)amino)-6-
methyl-1,3,5-triazin-2-yl)-6-fluoropyridin-3-yl)methyl)piperazine-1-
carboxylate (4; 0.500 g, 0.78 mmol) and 2-methoxypyrimidin-5-amine
(0.117 g, 0.93 mmol) in THF (10 mL) was cooled to 0 °C and treated
dropwise via syringe with LiHMDS in THF (1.0 M, 2.33 mL, 2.33
mmol). The reaction mixture was stirred at 0 °C for 1 h and quenched
with water (10 mL). The reaction mixture was diluted with water (15
mL) and EtOAc (100 mL). The organic layer was separated, dried
over sodium sulfate, filtered, and concentrated in vacuo to give the title
compound (0.42 g, 72%). MS (ESI positive ion): m/z calcd for
C40H48N10O5 748, found 749 (M + H)+.
cohort. The ED50 of compound 31 was 0.6 mg/kg with an
AUC at EC50 of 3.6 μg·h/mL.
SUMMARY
■
In this investigation, we examined compounds designed to
block the proposed sites of metabolism of compound 1,
focusing initially on modifying the methoxypyridine to reduce
O-demethylation and then subsequently blocking metabolism
at the benzylic piperazine region of the molecule. In the first
phase of this investigation, we found that the 5-fluoro-6-
methoxypyridin-3-amine group in the affinity pocket provided
the best combination of both cellular potency and microsomal
stability (compound 16). Further SAR investigations revealed
that enhanced stability and improved potencies could be
obtained by adding a methyl group on the benzylic position.
Ultimately, this work led to identification of pyridyltriazine 31,
a potent pan inhibitor of class I PI3Ks with a superior
pharmacokinetic profile. Compound 31 was shown to potently
block the targeted PI3K pathway in a mouse liver
pharmacodynamic model with an EC50 of 228 ng/mL.
Furthermore, 31 potently inhibited tumor growth in a U87
MG glioblastoma xenograft model (ED50 = 0.6 mg/kg). On the
basis of its excellent pharmacokinetic properties and in vivo
efficacy, compound 31 was selected for further evaluation as a
clinical candidate for the treatment of cancer and was
designated AMG 511. A more detailed biological profile of
AMG 511 will be reported in due course.
N,N-Bis(4-methoxybenzyl)-4-(2-((2-methoxypyrimidin-5-yl)-
amino)-5-((4-(methylsulfonyl)piperazin-1-yl)methyl)pyridin-3-yl)-6-
methyl-1,3,5-triazin-2-amine (10). TFA (4 mL) was added to a
cooled (ice bath) solution of 6(0.413 g, 0.55 mmol) in CH2Cl2 (5
mL). The resulting mixture was stirred at room temperature for 1 h
and partially concentrated under vacuum. The residue was dissolved in
CH2Cl2 (5 mL), and triethylamine (0.38 mL, 2.76 mmol) and
methanesulfonyl chloride (0.13 mL, 1.65 mmol) were slowly added at
0 °C. The mixture was stirred at 0 °C for 1 h and concentrated under
vacuum. The crude product was partitioned between 1 M NaOH and
CH2Cl2 (20 mL each), and the separated aqueous layer was extracted
with CH2Cl2 (2 × 20 mL). The combined organic extracts were
washed with brine, dried over sodium sulfate, and concentrated to give
the crude product. The crude product was purified by silica gel
chromatography (0−3% 2 M NH3/MeOH in CH2Cl2) to give the title
compound (0.277 g, 69%) as a brown foam. MS (ESI positive ion): m/
z calcd for C36H42N10O5S 726.3, found 727.2 (M + H)+.
EXPERIMENTAL SECTION
■
General Procedures. Unless otherwise noted, all materials were
obtained from commercial suppliers and used without further
purification. Anhydrous solvents were obtained from Aldrich, Acros,
or EM Science and used directly. All reactions involving air- or
moisture-sensitive reagents were performed under a nitrogen or argon
atmosphere. Microwave-assisted reactions were conducted with either
an Initiator from Biotage, Uppsala, Sweden, or an Explorer from CEM,
Matthews, NC. Silica gel chromatography was performed using either
glass columns packed with silica gel (200−400 mesh, Aldrich
Chemical) or prepacked silica gel cartridges (Biotage or Redisep).
NMR spectra were determined with a Bruker 400 MHz spectrometer.
Chemical shifts were reported in parts per million (ppm, δ units). All
final compounds were purified to >95% purity as determined by LC−
MS obtained on an Agilent 1100 spectrometer using the following
methods: (A) Agilent SB-C18 column (50 × 3.0 mm, 2.5 μm) at 40
°C with a 1.5 mL/min flow rate using a 5−95% gradient of 0.1% TFA
in CH3CN in 0.1% TFA in water over 3.5 min; (B) Phenomenex
Gemini NX C18 column (50 × 3.0 mm, 3 μm) at 40 °C with a 1.5
mL/min flow rate using a 5−95% gradient of 0.1% formic acid in
CH3CN in 0.1% formic acid in water over 3.5 min. Low-resolution MS
data were obtained at the same time as the purity determination on the
LC−MS instrument using the ES ionization mode (positive).
Compound 1 and intermediates 4, 5, 9, 22, 26, and 38 were prepared
as previously described.14
5-((3-(4-Amino-6-methyl-1,3,5-triazin-2-yl)-5-((4-
(methylsulfonyl)piperazin-1-yl)methyl)pyridin-2-yl)amino)-
pyridin-2-ol (2). 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 (1; 1.00 g, 2.06 mmol), CHCl3 (16 mL), and
iodotrimethylsilane (0.88 mL, 6.18 mmol) were added to a resealable
vial. The reaction mixture was heated and stirred at 70 °C for 3 h, and
then an additional amount of iodotrimethylsilane (0.88 mL, 6.18
mmol) was added. After being stirred at 70 °C for a total of 19 h, the
solution was pipetted into a 1:1 saturated sodium bicarbonate/
Na2S2O3 aqueous solution (100 mL). The mixture was filtered, and
the filtercake was washed with water and dried under vacuum to give
the crude product as a dark-yellow solid. The crude material was
4-(2-((2-Methoxypyrimidin-5-yl)amino)-5-((4-(methylsulfonyl)-
piperazin-1-yl)methyl)pyridin-3-yl)-6-methyl-1,3,5-triazin-2-amine
(15). Two drops of trifluoromethanesulfonic acid were added to a
solution of 10 (0.301 g, 0.41 mmol) in TFA (0.6 mL) at room
temperature. The mixture was heated at 80 °C for 15 h. The reaction
mixture was allowed to cool to room temperature, and the solvent was
removed under vacuum. The crude product was first purified by silica
gel chromatography (0−3% MeOH in CH2Cl2). The isolated yellow
solid was dissolved in DMSO and subjected to a reversed-phase
preparative HPLC purification (Shimadzu using Phenomenex, Gemini
C18, 100 Å, 150 × 30 mm, 5 μm, 10−100% CH3CN/H2O with 0.1%
TFA). The fractions containing the product were combined, basified
with saturated aqueous sodium bicarbonate, and extracted with
CH2Cl2 (3 × 20 mL), and the combined organic layers were dried
over sodium sulfate, filtered, and concentrated in vacuo to give the title
compound (0.065 g, 32%) as a bright yellow solid. MS (ESI positive
1
ion): m/z calcd for C20H26N10O3S 486, found 487 (M + H)+. H
NMR (400 MHz, DMSO-d6): δ 11.78 (s, 1H), 9.03 (s, 2H), 8.73 (d, J
= 1.6 Hz, 1H), 8.23 (d, J = 1.8 Hz, 1H), 7.91 (br s, 1H), 7.75 (br s,
1H), 3.91 (s, 3H), 3.50 (s, 2H), 3.11 (br s, 4H), 2.86 (s, 3H), 2.46−
2.49 (m, 4H), 2.44 (s, 3H).
4-(2-((5-Fluoro-6-methoxypyridin-3-yl)amino)-5-((4-
(methylsulfonyl)piperazin-1-yl)methyl)pyridin-3-yl)-6-methyl-
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1,3,5-triazin-2-amine (16). tert-Butyl 4-((5-(4-(Bis(4-
methoxybenzyl)amino)-6-methyl-1,3,5-triazin-2-yl)-6-((5-fluoro-6-
methoxypyridin-3-yl)amino)pyridin-3-yl)methyl)piperazine-1-car-
boxylate (7). A solution of 4 (25 g, 38.8 mmol) and 5-fluoro-6-
methoxypyridin-3-amine (8.28 g, 58.3 mmol) in THF (400 mL)
contained in an oven-dried, 1 L, three-necked, round-bottomed flask
equipped with a nitrogen inlet was stirred at a temperature between
−5 and −10 °C and treated dropwise with LiHMDS in THF (1.0 M,
117 mL, 117 mmol). The dark pink solution was stirred between −5
and −10 °C for 1 h. The reaction was quenched with water (100 mL)
and saturated aqueous ammonium chloride (100 mL) and diluted with
EtOAc (350 mL). The separated aqueous layer was extracted with
EtOAc (2 × 300 mL), and the combined organic extracts were dried
over sodium sulfate, filtered, and concentrated in vacuo. The crude
product was purified by silica gel column chromatography (10−50%
EtOAc in hexanes) to give the title compound (26.8 g, 90%). MS (ESI
positive ion): m/z calcd for C41H48FN9O5 765, found 766 (M + H)+.
4-(2-((5-Fluoro-6-methoxypyridin-3-yl)amino)-5-((4-
(methylsulfonyl)piperazin-1-yl)methyl)pyridin-3-yl)-N,N-bis(4-me-
thoxybenzyl)-6-methyl-1,3,5-triazin-2-amine (11). A solution of 7
(25 g, 32.6 mmol) in CH2Cl2 (150 mL) and TFA (150 mL) was
stirred at room temperature for 1 h. The solution was concentrated
under vacuum, and the residue was dissolved in CH2Cl2 and carefully
neutralized with saturated aqueous sodium bicarbonate. The aqueous
layer was extracted with CH2Cl2, and the organic extracts were dried
over sodium sulfate, filtered, and concentrated in vacuo to give 4-(2-
((5-fluoro-6-methoxypyridin-3-yl)amino)-5-(piperazin-1-ylmethyl)-
pyridin-3-yl)-N,N-bis(4-methoxybenzyl)-6-methyl-1,3,5-triazin-2-
amine (20 g, 92%). The crude product was taken on to the next step
without purification. A 1 L, three-necked, round-bottomed flask
equipped with a thermometer was charged with 4-(2-((5-fluoro-6-
methoxypyridin-3-yl)amino)-5-(piperazin-1-ylmethyl)pyridin-3-yl)-
N,N-bis(4-methoxybenzyl)-6-methyl-1,3,5-triazin-2-amine (25.1 g,
37.7 mmol) and CH2Cl2 (500 mL). The suspension was stirred at
−15 °C and treated dropwise with triethylamine (52.4 mL, 0.38 mol).
The resulting solution was treated with methanesulfonyl chloride (8.92
mL, 113 mmol) and stirred at 0 °C for 1 h. The solvent was removed
under vacuum, and the crude product was washed with water (300
mL), suspended in water, stirred, and sonicated. The resulting
suspension was collected, washed with water, and dried to give the title
compound (26.5 g, 94%). MS (ESI positive ion): m/z calcd for
C37H42FN9O5S 743, found 744 (M + H)+.
The residue was dissolved in CH2Cl2 (5 mL), and TEA (0.40 mL, 2.9
mmol) was added. The mixture was cooled in an ice−water bath, and
methanesulfonyl chloride (0.100 mL, 1.28 mmol) was added dropwise.
The solution was stirred in an ice−water bath for 2 h and then
concentrated in vacuo. The crude product was purified by silica gel
column chromatography (0−6.25% 2-propanol with 10% NH4OH in
CHCl3) to give the title compound as a light yellow solid (0.223 g,
94%). MS (ESI positive ion): m/z calcd for C31H36FN7O4S 621, found
622 (M + H)+. 1H NMR (400 MHz, CDCl3): δ 8.46 (dd, J = 9.0, 2.1
Hz, 1H), 8.24 (d, J = 1.4 Hz, 1H), 7.23 (dd, J = 8.5, 6.9 Hz, 4H), 6.86
(t, J = 8.7 Hz, 4H), 4.81 (d, J = 2.0 Hz, 4H), 3.81 (d, J = 6.3 Hz, 6H),
3.60 (s, 2H), 3.23 (s, 4H), 2.74−2.80 (m, 3H), 2.56−2.61 (m, 4H),
2.55 (s, 3H).
4-(2-((6-Chloropyridin-3-yl)amino)-5-((4-(methylsulfonyl)-
piperazin-1-yl)methyl)pyridin-3-yl)-N,N-bis(4-methoxybenzyl)-6-
methyl-1,3,5-triazin-2-amine (12). A 10 mL vial was charged with 8
(0.110 g, 0.17 mmol), 6-chloropyridin-3-amine (0.046 g, 0.36 mmol),
and THF (1 mL). NaHMDS in THF (1.0 M, 0.61 mL, 0.61 mmol)
was added dropwise at 0 °C, and the dark red solution was stirred at
that temperature for 1 h. The reaction was quenched with saturated
aqueous ammonium chloride (0.2 mL) and partitioned between water
(20 mL) and EtOAc (20 mL). The aqueous phase was extracted with
EtOAc (20 mL). The combined organic extracts were washed with
brine (40 mL), dried over sodium sulfate, filtered, and concentrated in
vacuo. The crude product was purified by silica gel chromatography
(0−6.25% i-PrOH (with 10% NH4OH) in CHCl3) and then again by
silica gel chromatography (50−100% EtOAc in hexanes) to afford the
title compound (0.104 g, 81%) as a yellow solid. MS (ESI positive
ion): m/z calcd for C36H40ClN9O4S 729, found 730 (M + H)+.
4-(2-((6-Chloropyridin-3-yl)amino)-5-((4-(methylsulfonyl)-
piperazin-1-yl)methyl)pyridin-3-yl)-6-methyl-1,3,5-triazin-2-amine
(17). Trifluoromethanesulfonic acid (0.02 mL, 0.22 mmol) was added
to a solution of 12 (0.103 g, 0.14 mmol) in TFA (1 mL). The reaction
mixture was stirred at room temperature for 3.5 h. The solvent was
partially evaporated, and solid sodium carbonate was added followed
by saturated aqueous sodium bicarbonate. The resulting precipitate
was collected by filtration, and the crude product was purified by
preparative HPLC (Gilson using Phenomenex, Gemini C18, 100 × 30
mm, 5 μm, 1−90% CH3CN with 0.1% TFA in H2O with 0.1% TFA in
10 min). The fractions containing the desired compound were
combined and concentrated under vacuum, and the residue was
treated with saturated aqueous sodium bicarbonate. The resulting solid
was collected by filtration, washed with water, and dried in a vacuum
oven to yield the title compound (0.047 g, 68%) as a yellow solid. MS
(ESI positive ion): m/z calcd for C20H24ClN9O2S 489, found 490 (M
+ H)+. 1H NMR (400 MHz, DMSO-d6): δ 12.17 (s, 1H), 8.88 (d, J =
2.7 Hz, 1H), 8.75 (d, J = 2.1 Hz, 1H), 8.47 (dd, J = 8.7, 2.8 Hz, 1H),
8.30 (d, J = 2.1 Hz, 1H), 7.94 (br s, 1H), 7.79 (br s, 1H), 7.45 (d, J =
8.8 Hz, 1H), 3.52 (s, 2 H), 3.11 (br s, 4H), 2.86 (s, 3H), 2.45 (s, 3H).
Note: The four hydrogens on the piperazine ring were under the
DMSO signal and could not be assigned/integrated.
4-Methyl-6-(2-((6-methylpyridin-3-yl)amino)-5-((4-
(methylsulfonyl)piperazin-1-yl)methyl)pyridin-3-yl)-1,3,5-tria-
zin-2-amine (18). N,N-Bis(4-methoxybenzyl)-4-methyl-6-(2-((6-
methylpyridin-3-yl)amino)-5-((4-(methylsulfonyl)piperazin-1-yl)-
methyl)pyridin-3-yl)-1,3,5-triazin-2-amine (13). LiHMDS in THF
(1.0 M, 0.72 mL, 0.72 mmol) was added dropwise to a solution of 8
(0.149 g, 0.24 mmol) and 6-methylpyridin-3-amine (0.039 g, 0.36
mmol) in THF (5 mL) at 0 °C under an atmosphere of argon. The
solution was allowed to stir at 0 °C for 2 h, and then the reaction was
quenched by the addition of saturated aqueous ammonium chloride.
The reaction mixture was extracted with EtOAc (50 mL), dried over
magnesium sulfate, filtered, and concentrated in vacuo. The crude
product was purified by silica gel chromatography (1−15% MeOH in
CH2Cl2) to give the title compound (0.138 g, 81%) as a yellow
semisolid. MS (ESI positive ion): m/z calcd for C37H43N9O4S 709,
found 710 (M + H)+. 1H NMR (400 MHz, CDCl3): δ 11.92 (s, 1H),
8.71 (d, J = 2.5 Hz, 1H), 8.58 (d, J = 2.3 Hz, 1H), 8.23 (d, J = 2.3 Hz,
1H), 8.07 (dd, J = 8.3, 2.6 Hz, 1H), 7.17−7.24 (m, 4H), 7.07 (d, J =
8.4 Hz, 1H), 6.86 (dd, J = 12.4, 8.7 Hz, 4H), 4.84 (d, J = 5.3 Hz, 4H),
4-(2-((5-Fluoro-6-methoxypyridin-3-yl)amino)-5-((4-
(methylsulfonyl)piperazin-1-yl)methyl)pyridin-3-yl)-6-methyl-1,3,5-
triazin-2-amine (16). A three-necked 500 mL flask equipped with an
overhead stirrer, a thermocouple, and a nitrogen inlet was charged
with 11 (26.9 g, 36.2 mmol) and TFA (175 mL). Upon complete
dissolution, the solution was warmed to 45 °C, and trifluorometha-
nesulfonic acid (16.1 mL, 181 mmol) was added dropwise. The
reaction mixture was stirred for 1 h and then cooled to 20 °C. A
separate 2 L flask equipped with an overhead stirrer and thermocouple
was charged with 10% aqueous trisodium citric acid (500 mL) and
cooled to 0 °C. The reaction mixture was added dropwise to the
cooled aqueous solution. The product precipitated out of solution,
CH2Cl2 was added, and the slurry was stirred at 20 °C for 16 h. The
solid was collected by filtration, washed with copious water and
copious ethanol, and dried to give the title compound (15.06 g, 83%)
as a yellow solid. MS (ESI positive ion): m/z calcd for C21H26FN9O3S
1
503, found 504 (M + H)+. H NMR (400 MHz, DMSO-d6): δ 11.96
(s, 1H), 8.73 (d, J = 2.1 Hz, 1H), 8.42 (d, J = 2.1 Hz, 1H), 8.37 (dd, J
= 12.7, 2.1 Hz, 1H), 8.26 (d, J = 2.1 Hz, 1H), 7.93 (br s, 1H), 7.78 (br
s, 1H), 3.93 (s, 3H), 3.50 (s, 2H), 3.11 (br s, 4H), 2.87 (s, 3H), 2.45−
2.49 (m, 4H), 2.44 (s, 3H).
4-(2-((6-Chloropyridin-3-yl)amino)-5-((4-(methylsulfonyl)-
piperazin-1-yl)methyl)pyridin-3-yl)-6-methyl-1,3,5-triazin-2-
amine (17). 4-(2-Fluoro-5-((4-(methylsulfonyl)piperazin-1-yl)-
methyl)pyridin-3-yl)-N,N-bis(4-methoxybenzyl)-6-methyl-1,3,5-tria-
zin-2-amine (8). A 100 mL round-bottomed flask was charged with 4
(0.246 g, 0.383 mmol) and CH2Cl2 (3 mL). TFA (3.0 mL, 38.9
mmol) was added dropwise, and the reaction mixture was stirred at
room temperature for 45 min. The mixture was concentrated in vacuo.
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3.81 (s, 3H), 3.79 (s, 3H), 3.50 (s, 2H), 3.18 (d, J = 4.3 Hz, 4H), 2.70
(s, 3H), 2.59 (s, 3H), 2.55 (t, J = 4.6 Hz, 4H), 2.51 (s, 3H).
3-(4-Methyl-6-(methylthio)-1,3,5-triazin-2-yl)-5-((4-
(methylsulfonyl)piperazin-1-yl)methyl)-N-(6-(trifluoromethyl)-
pyridin-3-yl)pyridin-2-amine (24). A stirred solution of 23 (0.109 g,
0.26 mmol) and 6-(trifluoromethyl)pyridin-3-amine (0.064 g, 0.39
mmol) in THF (1.5 mL) at 0 °C was treated with LiHMDS in THF
(1.0 M, 0.79 mL, 0.79 mmol), and the mixture was stirred for 1 h. The
reaction mixture was diluted with water (10 mL) and EtOAc (15 mL).
The separated aqueous layer was extracted with EtOAc (2 × 10 mL),
and the combined organic extracts were washed with brine, dried over
sodium sulfate, and concentrated in vacuo to give the title compound
(0.098 g, 67%) as a yellow solid. MS (ESI positive ion): m/z calcd for
C22H25F3N8O2S2 554, found 555 (M + H)+.
4-Methyl-6-(2-((6-methylpyridin-3-yl)amino)-5-((4-
(methylsulfonyl)piperazin-1-yl)methyl)pyridin-3-yl)-1,3,5-triazin-2-
amine (18). 13 (0.138 g, 0.19 mmol) was treated with TFA (5 mL)
and heated at 80 °C for 16 h. The excess TFA was removed under
reduced pressure, and the crude residue was treated with ice and a
saturated aqueous solution of sodium bicarbonate. The resulting
yellow suspension was filtered through a medium-porosity sintered
glass frit and washed with water. The yellow solid was washed with a
small amount of i-PrOH, and the resulting yellow amorphous solid was
purified by silica gel chromatography (0−3% MeOH in CH2Cl2),
affording the title compound (0.051 g, 56%) as a yellow solid. MS (ESI
positive ion): m/z calcd for C21H27N9O2S 469, found 470 (M + H)+.
1H NMR (400 MHz, DMSO-d6): δ 11.96 (s, 1H), 8.83 (d, J = 2.7 Hz,
4-Methyl-6-(5-((4-(methylsulfonyl)piperazin-1-yl)methyl)-2-((6-
(trifluoromethyl)pyridin-3-yl)amino)pyridin-3-yl)-1,3,5-triazin-2-
amine (20). 24 (0.087 g, 0.16 mmol) and 2 M NH3 in i-PrOH (2 mL)
were added to a 10 mL reaction vial. The tube was sealed and heated
at 90 °C overnight. After cooling, the mixture was adsorbed onto silica
gel, and the material was purified by silica gel chromatography (0−
10% MeOH in CH2Cl2) to give the title compound (0.033 g, 40%) as
a yellow solid. MS (ESI positive ion): m/z calcd for C21H24F3N9O2S
1H), 8.73 (d, J = 2.3 Hz, 1H), 8.22−8.28 (m, 2H), 7.91 (br s, 1H),
7.77 (br s, 1H), 7.20 (d, J = 8.6 Hz, 1H), 3.50 (s, 2H), 3.33 (br s, 4H),
3.11 (br s, 4H), 2.87 (s, 3H), 2.45 (s, 3H), 2.43 (s, 3H).
4-(2-((5-Fluoropyridin-3-yl)amino)-5-((4-(methylsulfonyl)-
piperazin-1-yl)methyl)pyridin-3-yl)-6-methyl-1,3,5-triazin-2-
amine (19). 4-(2-((5-Fluoropyridin-3-yl)amino)-5-((4-
(methylsulfonyl)piperazin-1-yl)methyl)pyridin-3-yl)-N,N-bis(4-me-
thoxybenzyl)-6-methyl-1,3,5-triazin-2-amine (14). LiHMDS in THF
(1.0 M, 0.45 mL, 0.45 mmol) was added dropwise to a solution of 8
(0.080 g, 0.13 mmol) and 3-amino-5-fluoropyridine (0.029 g, 0.26
mmol) in THF (5 mL) at 0 °C under an atmosphere of argon. The
solution was allowed to stir at 0 °C for 20 min and quenched by the
addition of saturated aqueous ammonium chloride. The mixture was
extracted with EtOAc (2 × 15 mL), dried over magnesium sulfate,
filtered, and concentrated in vacuo. The crude product was purified by
silica gel chromatography (25−50% CH2Cl2 in 4% MeOH/EtOAc) to
give the title compound (0.072 g, 78%) as a yellow solid. MS (ESI
positive ion): m/z calcd for C36H40FN9O4S 713, found 714 (M + H)+.
4-(2-((5-Fluoropyridin-3-yl)amino)-5-((4-(methylsulfonyl)-
piperazin-1-yl)methyl)pyridin-3-yl)-6-methyl-1,3,5-triazin-2-amine
(19). A microwave vial was charged with 14 (0.070 g, 0.10 mmol) and
TFA (1.5 mL). The mixture was sealed under argon and heated in a
microwave reactor at 120 °C for 30 min. The solvent was removed
under vacuum, and the residue was purified by preparative HPLC
(Phenomenex, Gemini C18, 100 × 30 mm, 10 μm, 10−40% CH3CN/
H2O with 0.1% TFA). Product-containing fractions were concentrated
and treated with saturated aqueous sodium bicarbonate and CH2Cl2.
The aqueous layer was extracted with CH2Cl2 (3×). The combined
organic extracts were dried over sodium sulfate, filtered, and
concentrated in vacuo to give the title compound (0.027 g, 58%) as
a yellow solid. MS (ESI positive ion): m/z calcd for C20H24FN9O2S
1
523, found 524 (M + H)+. H NMR (400 MHz, DMSO-d6): δ 12.49
(s, 1H), 9.12 (d, J = 2.5 Hz, 1H), 8.79 (d, J = 2.1 Hz, 1H), 8.74 (dd, J
= 8.7, 2.0 Hz, 1H), 8.36 (d, J = 2.1 Hz, 1H), 7.97 (br s, 1H), 7.84 (d, J
= 8.6 Hz, 2H), 3.55 (s, 2H), 3.12 (br s, 4H), 2.87 (s, 3H), 2.48−2.50
(m, 4H), 2.47 (s, 3H).
4-(2-((5-Methoxypyridin-3-yl)amino)-5-((4-(methylsulfonyl)-
piperazin-1-yl)methyl)pyridin-3-yl)-6-methyl-1,3,5-triazin-2-
amine (21). A stirred solution of 23 (0.200 g, 0.48 mmol) and 5-
methoxypyridin-3-amine (0.090 g, 0.73 mmol) in DMF (5 mL) was
treated with LiHMDS in THF (1.0 M, 1.94 mL, 1.94 mmol) at 0 °C,
and the mixture was stirred for 1 h. The reaction mixture was diluted
with water (10 mL) and EtOAc (15 mL). The separated aqueous layer
was extracted with EtOAc (2 × 10 mL), and the combined organic
layers were washed with brine, dried over sodium sulfate, and
concentrated in vacuo to give the crude residue, which was heated with
2 M NH3 in i-PrOH (3 mL) in a sealed tube at 90 °C overnight. After
cooling, the mixture was adsorbed onto silica gel, and the material was
purified by silica gel chromatography (0−5% MeOH in CH2Cl2) to
give the title compound (0.044 g, 19%) as a yellow solid. MS (ESI
positive ion): m/z calcd for C21H27N9O3S 485, found 486 (M + H)+.
1H NMR (400 MHz, DMSO-d6): δ 12.13 (s, 1H), 8.74 (s, 1H), 8.55
(s, 1H), 8.31 (s, 1H), 8.19 (br s, 1H), 7.93 (br s, 2H), 7.78 (br s, 1H),
3.86 (s, 3H), 3.52 (s, 2H), 3.11 (br s, 4H), 2.87 (s, 3H), 2.46−2.50
(m, 4H), 2.46 (s, 3H).
4-(2-((5-Fluoro-6-methoxypyridin-3-yl)amino)-5-(1-(4-
(methylsulfonyl)piperazin-1-yl)ethyl)pyridin-3-yl)-6-methyl-
1,3,5-triazin-2-amines 30 and 31. 5-(4-(Bis(4-methoxybenzyl)-
amino)-6-methyl-1,3,5-triazin-2-yl)-6-((5-fluoro-6-methoxypyridin-
3-yl)amino)nicotinaldehyde (27). 4-(5-(1,3-Dioxolan-2-yl)-2-fluoro-
pyridin-3-yl)-N,N-bis(4-methoxybenzyl)-6-methyl-1,3,5-triazin-2-
amine (26; 1.69 g, 3.26 mmol) and 5-fluoro-6-methoxypyridin-3-
amine (0.477 g, 3.36 mmol) were dissolved in THF (28 mL), and the
reaction flask was cooled in an ice−water bath. The solution was
placed under a nitrogen atmosphere, and LiHMDS in THF (1.0 M, 9.5
mL, 9.5 mmol) was added. The reaction mixture was stirred for 35
min. Aqueous HCl (5 M, 4.5 mL) and MeOH (5.8 mL) were added,
and the reaction was allowed to warm to room temperature and
stirred. After 10 min, the reaction was treated with water (20 mL) and
extracted with CH2Cl2. The organic extracts were combined, dried
over sodium sulfate, filtered, and concentrated in vacuo. Purification by
silica gel chromatography (0−10% MeOH in CH2Cl2) gave the
product. This material was dissolved in THF (20 mL) and MeOH (4
mL), and aqueous HCl (5 M, 2 mL) was added. The reaction mixture
was stirred at room temperature for 25 min. The reaction was diluted
with water (20 mL) and CH2Cl2 (20 mL). The layers were separated,
and the aqueous phase was extracted with CH2Cl2 (10 mL). The
organic extracts were combined, dried over sodium sulfate, filtered,
concentrated, and dried under high vacuum overnight to give the title
compound (1.10 g, 57%). MS (ESI positive ion): m/z calcd for
C32H30FN7O4 595, found 596 (M + H)+. 1H NMR (400 MHz,
CDCl3): δ 12.58 (s, 1H), 9.92 (s, 1H), 9.25 (d, J = 2.3 Hz, 1H), 8.75
1
473, found 474 (M + H)+. H NMR (400 MHz, DMSO-d6): δ 12.33
(s, 1H), 8.82 (s, 1H), 8.77 (s, 1H), 8.56 (d, J = 12.1 Hz, 1H), 8.35 (s,
1H), 8.17 (d, J = 2.0 Hz, 1H), 7.97 (br s, 1H), 7.81 (br s, 1H), 3.54 (s,
2H), 3.12 (br s, 4H), 2.87 (s, 3H), 2.46 (br s, 7H).
4-Methyl-6-(5-((4-(methylsulfonyl)piperazin-1-yl)methyl)-2-
((6-(trifluoromethyl)pyridin-3-yl)amino)pyridin-3-yl)-1,3,5-tria-
zin-2-amine (20). 2-(2-Fluoro-5-((4-(methylsulfonyl)piperazin-1-
yl)methyl)pyridin-3-yl)-4-methyl-6-(methylthio)-1,3,5-triazine (23).
TFA (4 mL) was slowly added to a stirred solution of tert-butyl 4-
((6-fluoro-5-(4-methyl-6-(methylthio)-1,3,5-triazin-2-yl)pyridin-3-yl)-
methyl)piperazine-1-carboxylate (0.853 g, 1.96 mmol) in CH2Cl2 (5
mL) at 0 °C. The reaction mixture was stirred at room temperature for
1 h. The mixture was concentrated, and the sticky residue obtained
was dissolved in CH2Cl2 (10 mL) and cooled in an ice bath.
Triethylamine (1.37 mL, 9.82 mmol) followed by methanesulfonyl
chloride (0.46 mL, 5.89 mmol) was added to this solution. The
reaction mixture was stirred at 0 °C for 1 h, and the solvent was
removed under vacuum. The crude product was purified by silica gel
chromatography (0−10% MeOH in CH2Cl2). The product was
washed with i-PrOH to give the title compound (0.640 g, 79%) as a
white solid. MS (ESI positive ion): m/z calcd for C16H21FN6O2S2 412,
found 413 (M + H)+. 1H NMR (400 MHz, DMSO-d6): δ 8.58 (dd, J =
9.3, 2.2 Hz, 1H), 8.37 (s, 1H), 3.67 (s, 2H), 3.01−3.19 (m, 4H), 2.87
(s, 3H), 2.61 (s, 3H), 2.60 (s, 3H), 2.47−2.49 (m, 4H).
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(d, J = 2.1 Hz, 1H), 8.03−7.97 (m, 2H), 7.21 (dd, J = 15.1, 8.4 Hz,
4H), 6.87 (dd, J = 11.1, 8.6 Hz, 4H), 4.89 (s, 2H), 4.85 (s, 2H), 4.04
(s, 3H), 3.82 (s, 3H), 3.79 (s, 3H), 2.61 (s, 3H).
8.31 (t, J = 2.2 Hz, 1H), 8.27 (d, J = 2.3 Hz, 1H), 8.05 (d, J = 2.1 Hz,
1H), 5.42 (br s, 2H), 4.04 (s, 3H), 3.56 (q, J = 6.6 Hz, 1H), 3.24 (t, J =
4.7 Hz, 4H), 2.78 (s, 3H), 2.54−2.68 (m, 7H), 1.44 (t, J = 6.8 Hz,
3H).
1-(5-(4-(Bis(4-methoxybenzyl)amino)-6-methyl-1,3,5-triazin-2-
yl)-6-((5-fluoro-6-methoxypyridin-3-yl)amino)pyridin-3-yl)ethanol
(28). 27 (1.054 g, 1.77 mmol) was suspended in THF (16 mL), and
the reaction flask was cooled to 0 °C. A solution of methylmagnesium
bromide in diethyl ether (3.0 M, 1.80 mL, 5.40 mmol) was added via
syringe, and the reaction was stirred at 0 °C for 25 min. The reaction
was treated with saturated aqueous ammonium chloride (3 mL,
dropwise at first as gas evolution was observed) and water (20 mL).
The reaction was diluted with EtOAc, and the layers were separated.
The aqueous phase was extracted with EtOAc, and the organic extracts
were combined, dried over sodium sulfate, filtered, concentrated, and
purified by silica gel chromatography (1−5% MeOH in CH2Cl2) to
give the title compound (0.894 g, 83%). MS (ESI positive ion): m/z
The racemic mixture of 4-(2-((5-fluoro-6-methoxypyridin-3-yl)-
amino)-5-(1-(4-(methylsulfonyl)piperazin-1-yl)ethyl)pyridin-3-yl)-6-
methyl-1,3,5-triazin-2-amine was separated using chiral preparative
SFC on a Chiralcel OJ-H column (250 × 20 mm) using 78:22
supercritical CO2/MeOH (1% DEA). The two separate peaks were
collected, concentrated, and dried under high vacuum to afford the
two enantiomers (30 and 31). The absolute stereochemistries of the
two enantiomers were assigned on the basis of the X-ray crystal
structure of compound 31 cocrystallized with PI3Kγ (see Figure 3).
(R)-4-(2-((5-Fluoro-6-methoxypyridin-3-yl)amino)-5-(1-(4-
(methylsulfonyl)piperazin-1-yl)ethyl)pyridin-3-yl)-6-methyl-1,3,5-tria-
zin-2-amine (30) was the second eluting peak and was isolated as a
yellow powder. MS (ESI positive ion): m/z calcd for C22H28FN9O3S
1
calcd for C33H34FN7O4 611, found 612 (M + H)+. H NMR (400
1
517, found 518 (M + H)+. H NMR (400 MHz, CDCl3): δ 11.93 (s,
MHz, CDCl3): δ 11.86 (s, 1H), 8.83 (d, J = 2.3 Hz, 1H), 8.32 (d, J =
2.3 Hz, 1H), 8.01 (dd, J = 12.1, 2.1 Hz, 1H), 7.96 (d, J = 2.3 Hz, 1H),
7.21 (dd, J = 10.8, 8.6 Hz, 4H), 6.87 (dd, J = 12.1, 8.8 Hz, 4H), 4.81−
4.94 (m, 5H), 4.02 (s, 3H), 3.82 (s, 3H), 3.79 (s, 3H), 2.59 (s, 3H),
1.73 (d, J = 3.7 Hz, 1H), 1.53 (d, J = 6.5 Hz, 3H).
1H), 8.71 (d, J = 2.3 Hz, 1H), 8.26−8.32 (m, 2H), 8.05 (d, J = 2.1 Hz,
1H), 5.41 (br s, 2H), 4.04 (s, 3H), 3.56 (q, J = 6.4 Hz, 1H), 3.24 (t, J =
4.5 Hz, 4H), 2.78 (s, 3H), 2.53−2.68 (m, 4H), 2.59 (s, 3H), 1.44 (d, J
= 6.6 Hz, 3H).
1-(5-(4-(Bis(4-methoxybenzyl)amino)-6-methyl-1,3,5-triazin-2-
yl)-6-((5-fluoro-6-methoxypyridin-3-yl)amino)pyridin-3-yl)ethyl
Methanesulfonate (29). Triethylamine (0.90 mL, 6.5 mmol) and
methanesulfonyl chloride (0.41 mL, 5.3 mmol) were added to a
solution of 28 (0.867 g, 1.42 mmol) in CH2Cl2 (22 mL) at 0 °C under
a nitrogen atmosphere. The reaction mixture was stirred at 0 °C for 20
min, diluted with CH2Cl2 (125 mL), and treated with water (25 mL).
The layers were separated, and the aqueous phase was extracted with
CH2Cl2. The organic extracts were combined, dried over sodium
sulfate, filtered, and concentrated in vacuo to give the title compound,
which was used without further purification.
4-(2-((5-Fluoro-6-methoxypyridin-3-yl)amino)-5-(1-(4-
(methylsulfonyl)piperazin-1-yl)ethyl)pyridin-3-yl)-N,N-bis(4-me-
thoxybenzyl)-6-methyl-1,3,5-triazin-2-amine. The crude product
from the previous step was dissolved in CH2Cl2 (15 mL) and treated
with triethylamine (0.90 mL, 6.5 mmol) and 1-(methylsulfonyl)-
piperazine (0.754 g, 4.59 mmol). The reaction mixture was stirred
under nitrogen at room temperature overnight and treated with water
(20 mL) and CH2Cl2 (10 mL). The layers were separated, and the
aqueous phase was extracted with CH2Cl2. The organic extracts were
combined, dried over sodium sulfate, filtered, and concentrated, and
the crude material was purified by silica gel chromatography (2%
MeOH in CH2Cl2 and then a gradient of 3.5−10% 2 M NH3/MeOH
in CH2Cl2) to give the title compound (0.457 g, 42%). MS (ESI
positive ion): m/z calcd for C38H44FN9O5S 757, found 758 (M + H)+.
1H NMR (400 MHz, CDCl3): δ 11.87 (s, 1H), 8.72 (d, J = 2.3 Hz,
(S)-4-(2-((5-Fluoro-6-methoxypyridin-3-yl)amino)-5-(1-(4-
(methylsulfonyl)piperazin-1-yl)ethyl)pyridin-3-yl)-6-methyl-1,3,5-tria-
zin-2-amine (31) was the first eluting peak and was isolated as a yellow
powder. MS (ESI positive ion): m/z calcd for C22H28FN9O3S 517,
found 518 (M + H)+. 1H NMR (400 MHz, CDCl3): δ 11.93 (s, 1H),
8.71 (d, J = 2.3 Hz, 1H), 8.26−8.32 (m, 2H), 8.05 (d, J = 2.3 Hz, 1H),
5.40 (br s, 2H), 4.03 (s, 3H), 3.56 (q, J = 7.0 Hz, 1H), 3.24 (t, J = 4.9
Hz, 4H), 2.78 (s, 3H), 2.53−2.68 (m, 4H), 2.59 (s, 3H), 1.44 (d, J =
6.6 Hz, 3H).
4-(2-((5-Fluoro-6-methoxypyridin-3-yl)amino)-5-(2,2,2-tri-
fluoro-1-(4-(methylsulfonyl)piperazin-1-yl)ethyl)pyridin-3-yl)-
6-methyl-1,3,5-triazin-2-amines 32 and 33. 1-(5-(4-(Bis(4-
methoxybenzyl)amino)-6-methyl-1,3,5-triazin-2-yl)-6-fluoropyridin-
3-yl)-2,2,2-trifluoroethanol. To a stirred solution of 5-(4-(bis(4-
methoxybenzyl)amino)-6-methyl-1,3,5-triazin-2-yl)-6-fluoronicotinal-
dehyde (3.78 g, 7.99 mmol) in THF (50 mL) was added
trimethyl(trifluoromethyl)silane (1.70 g, 11.98 mmol) followed by
CsF (0.243 g, 1.60 mmol) at 0 °C, and the mixture was stirred for 10
min before being allowed to warm to room temperature followed by
stirring for 3.5 h. The reaction was quenched with 1 M HCl and stirred
for another 30 min, and then EtOAc (10 mL) was added. The
separated aqueous layer was extracted with EtOAc (2 × 20 mL), and
the combined organic extracts were washed with brine, dried over
sodium sulfate, and concentrated in vacuo to give the title compound
as a brown foam, which was used without further purification.
1-(5-(4-(Bis(4-methoxybenzyl)amino)-6-methyl-1,3,5-triazin-2-
yl)-6-((5-fluoro-6-methoxypyridin-3-yl)amino)pyridin-3-yl)-2,2,2-tri-
fluoroethanol (34). LiHMDS in THF (1 M, 42.7 mL, 42.7 mmol) was
added dropwise to a stirred mixture of 5-fluoro-6-methoxypyridin-3-
amine (2.43 g, 17.07 mmol) and 1-(5-(4-(bis(4-methoxybenzyl)-
amino)-6-methyl-1,3,5-triazin-2-yl)-6-fluoropyridin-3-yl)-2,2,2-trifluor-
oethanol (4.64 g, 8.54 mmol) in THF (50 mL) at −10 °C. The
mixture was stirred for 30 min. The reaction was quenched with water
(25 mL) and saturated aqueous ammonium chloride (25 mL) and
diluted with EtOAc (25 mL). The separated aqueous layer was
extracted with EtOAc (2 × 50 mL), and the combined organic extracts
were washed with brine, dried over sodium sulfate, and concentrated
in vacuo to give the crude product. Purification by silica gel column
chromatography (0−50% EtOAc in hexanes) gave the title compound
(5.14 g, 91%) as a brown foam.
1H), 8.25 (d, J = 2.3 Hz, 1H), 8.07 (d, J = 12.3, 2.1 Hz, 1H), 7.97 (d, J
= 2.3 Hz, 1H), 7.22 (t, J = 8.5 Hz, 4H), 6.82−6.91 (m, 4H), 4.76−4.93
(m, 4H), 4.02 (s, 3H), 3.82 (s, 3H), 3.79 (s, 3H), 3.55 (q, J = 6.8 Hz,
1H), 3.16 (t, J = 4.6 Hz, 4H), 2.69 (s, 3H), 2.60 (s, 3H), 2.49−2.58
(m, 4H), 1.38 (d, J = 6.6 Hz, 3H).
4-(2-((5-Fluoro-6-methoxypyridin-3-yl)amino)-5-(1-(4-
(methylsulfonyl)piperazin-1-yl)ethyl)pyridin-3-yl)-6-methyl-1,3,5-tri-
azin-2-amines 30 and 31. 4-(2-((5-Fluoro-6-methoxypyridin-3-
yl)amino)-5-(1-(4-(methylsulfonyl)piperazin-1-yl)ethyl)pyridin-3-yl)-
N,N-bis(4-methoxybenzyl)-6-methyl-1,3,5-triazin-2-amine (0.457 g,
0.61 mmol) was dissolved in TFA (9.5 mL) and heated at 75 °C
for 16 h. The reaction was allowed to cool to room temperature and
concentrated under vacuum. The crude product was suspended in
CH2Cl2 (40 mL) and treated with saturated aqueous sodium
bicarbonate and NaOH (5 M) to raise the pH of the aqueous phase
to ∼8. The layers were separated, and the aqueous phase was extracted
with CH2Cl2 and CH2Cl2/MeOH (10:1). The organic extracts were
combined, dried over sodium sulfate, filtered, and concentrated in
vacuum. The crude product was purified by silica gel column
chromatography (3−20% 2 M NH3/MeOH in CH2Cl2) to give the
racemic compound (0.195 g, 62%) as a yellow solid. MS (ESI positive
4-(5-(1-Chloro-2,2,2-trifluoroethyl)-2-((5-fluoro-6-methoxypyri-
din-3-yl)amino)pyridin-3-yl)-N,N-bis(4-methoxybenzyl)-6-methyl-
1,3,5-triazin-2-amine (35). Triethylamine (0.54 mL, 3.87 mmol) and
methanesulfonyl chloride (0.24 mL, 3.09 mmol) were added to a
solution of 34 (1.03 g, 1.55 mmol) in CH2Cl2 (20 mL) at 0 °C, and
the mixture was stirred for 3 h. The reaction mixture was allowed to
warm to room temperature and stirred for an additional 2 h. The
reaction was quenched with saturated aqueous ammonium chloride
and water. The aqueous layer was extracted with CH2Cl2 (2 × 50 mL).
1
ion): m/z calcd for C22H28FN9O3S 517, found 518 (M + H)+. H
NMR (400 MHz, CDCl3): δ 11.93 (s, 1H), 8.71 (d, J = 2.5 Hz, 1H),
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The combined organic extracts were dried over sodium sulfate,
concentrated, and dried under vacuum to give the crude product,
which was used without further purification. MS (ESI positive ion): m/
z calcd for C33H30ClF4N7O3 683, found 684 (M + H)+.
J = 9.4 Hz, 1H), 3.95 (s, 3H), 3.13 (t, J = 4.6 Hz, 4H), 2.86 (s, 3H),
2.69−2.78 (m, 2H), 2.58−2.69 (m, 2H), 2.45 (s, 3H).
(R)-4-(2-((5-Fluoro-6-methoxypyridin-3-yl)amino)-5-((2-
methyl-4-(methylsulfonyl)piperazin-1-yl)methyl)pyridin-3-yl)-
6-methyl-1,3,5-triazin-2-amine (42). Potassium (R)-((4-(tert-Bu-
toxycarbonyl)-2-methylpiperazin-1-yl)methyl)trifluoroborate. A
mixture of potassium (bromomethyl)trifluoroborate (1.20 g, 5.38
mmol) and (R)-tert-butyl 3-methylpiperazine-1-carboxylate (1.12 g,
5.65 mmol) in THF (7 mL) was heated at 80 °C under nitrogen for 24
h. The mixture was allowed to cool to room temperature and
concentrated under vacuum. The residue was dissolved in acetone
(125 mL) and treated with potassium carbonate (1 equiv). The
suspension was stirred at room temperature for 30 min and then
filtered through a short plug of diatomaceous earth. The filter cake was
washed with additional acetone, and the combined organic extracts
were concentrated to give the crude product as a colorless foam (1.51
g, 88%). The pure material was obtained as a beige powder by adding
acetone/diethyl ether (2:1) to the foam and then slowly adding
hexanes while sonicating the mixture. 19F NMR (377 MHz, acetone-
d6): δ −141.36.
tert-Butyl 4-(1-(5-(4-(Bis(4-methoxybenzyl)amino)-6-methyl-
1,3,5-triazin-2-yl)-6-((5-fluoro-6-methoxypyridin-3-yl)amino)-
pyridin-3-yl)-2,2,2-trifluoroethyl)piperazine-1-carboxylate (36). A
solution of 35 (1.059 g, 1.55 mmol) in CH3CN (10 mL) was treated
with tert-butyl 1-piperazinecarboxylate (0.577 g, 3.10 mmol) and
triethylamine (1.08 mL, 7.74 mmol), and the mixture was heated at
100 °C for 3 h. The reaction mixture was concentrated, and the crude
product was purified by silica gel column chromatography (0−5%
MeOH/2 M NH3 in CH2Cl2) to give the title compound (1.24 g,
96%) as a brown foam. MS (ESI positive ion): m/z calcd for
C42H47F4N9O5 833, found 834 (M + H)+.
4-(2-((5-Fluoro-6-methoxypyridin-3-yl)amino)-5-(2,2,2-trifluoro-
1-(4-(methylsulfonyl)piperazin-1-yl)ethyl)pyridin-3-yl)-N,N-bis(4-
methoxybenzyl)-6-methyl-1,3,5-triazin-2-amine (37). TFA (3 mL)
was added to a solution of 36 (1.24 g, 1.49 mmol) in CH2Cl2 (5 mL),
and the reaction mixture was stirred at room temperature for 1 h. The
mixture was concentrated under vacuum to remove as much TFA as
possible. The sticky residue was dissolved in a solution of CH2Cl2 (5
mL), and triethylamine (1.04 mL, 7.44 mmol) and methanesulfonyl
chloride (0.35 mL, 4.46 mmol) were slowly added at 0 °C. The
mixture was stirred for 1 h. The crude product was partitioned
between 1 M NaOH (20 mL) and CH2Cl2 (20 mL). The separated
aqueous layer was extracted with CH2Cl2 (2 × 20 mL), and the
combined organic layers were washed with brine, dried over sodium
sulfate, and concentrated in vacuo to give the title compound (1.20 g,
99%) as a dark foam.
(R)-tert-Butyl 4-((5-(4-(Bis(4-methoxybenzyl)amino)-6-methyl-
1,3,5-triazin-2-yl)-6-fluoropyridin-3-yl)methyl)-3-methylpiperazine-
1-carboxylate (39). 4-(5-Chloro-2-fluoropyridin-3-yl)-N,N-bis(4-me-
thoxybenzyl)-6-methyl-1,3,5-triazin-2-amine14 (38; 1.00 g, 2.08
mmol), 2-(dicyclohexylphosphino)-2′,4′,6′,-triisopropyl-1,1′-biphenyl
(0.100 g, 0.21 mmol), cesium carbonate (2.04 g, 6.25 mmol),
potassium (R)-((4-(tert-butoxycarbonyl)-2-methylpiperazin-1-yl)-
methyl)trifluoroborate (0.701 g, 2.19 mmol), and palladium(II)
acetate (0.023 g, 0.104 mmol) were added to a 20 mL microwave
tube, and the tube was purged with argon. The solids were then
treated with THF (10 mL) and water (1 mL) and heated under
microwave irradiation at 85 °C for 16 h. The mixture was filtered
through a short plug of diatomaceous earth, washed with EtOAc (3×),
and concentrated. The crude product was purified by silica gel
chromatography (0−5% MeOH in CH2Cl2) to obtain the title
compound (1.01 g, 74%) as a yellow foam. MS (ESI positive ion): m/z
calcd for C36H44FN7O4 657, found 658 (M + H)+.
(R)-4-(2-Fluoro-5-((2-methyl-4-(methylsulfonyl)piperazin-1-yl)-
methyl)pyridin-3-yl)-N,N-bis(4-methoxybenzyl)-6-methyl-1,3,5-tria-
zin-2-amine (40). TFA (4 mL) was added to a cooled solution of 39
(1.01 g, 1.53 mmol) in CH2Cl2 (5 mL), and the mixture was stirred at
room temperature for 1 h. The mixture was concentrated, and the
sticky residue was dissolved in CH2Cl2 (10 mL), to which
triethylamine (2.14 mL, 15.35 mmol) and methanesulfonyl chloride
(0.60 mL, 7.68 mmol) were slowly added at 0 °C. The mixture was
stirred at 0 °C for 1 h and concentrated, and the crude product was
partitioned between 1 M NaOH and CH2Cl2 (20 mL each). The
separated aqueous layer was extracted with CH2Cl2 (2 × 20 mL), and
the combined organic extracts were washed with brine, dried over
sodium sulfate, and concentrated in vacuo to give the crude residue.
Purification by silica gel column chromatography (10−70% EtOAc in
hexanes) gave the title compound (0.478 g, 49%) as a pale yellow
foam. MS (ESI positive ion): m/z calcd for C32H38FN7O4S 635, found
636 (M + H)+.
4-(2-((5-Fluoro-6-methoxypyridin-3-yl)amino)-5-(2,2,2-trifluoro-
1-(4-(methylsulfonyl)piperazin-1-yl)ethyl)pyridin-3-yl)-6-methyl-
1,3,5-triazin-2-amines 32 and 33. Trifluoromethanesulfonic acid (0.5
mL) was added to a solution of 37 (1.20, 1.48 mmol) in TFA (5 mL),
and the reaction mixture was heated at 70 °C for 2 h. The reaction was
allowed to cool to room temperature and concentrated under vacuum.
The crude product was purified by silica gel column chromatography
(0−5% 2 M NH3/MeOH in CH2Cl2), followed by preparative TLC
(EtOAc/CH2Cl2, 3:2) to give the racemic compound (0.841 g, 100%)
as a yellow solid. MS (ESI positive ion): m/z calcd for C22H25F4N9O3S
1
571, found 572 (M + H)+. H NMR (400 MHz, DMSO-d6): δ 11.96
(s, 1H), 8.84 (d, J = 2.1 Hz, 1H), 8.40 (d, J, = 2.3 Hz, 1H), 8.29−8.39
(m, 2H), 8.03 (br s, 1H), 7.88 (br s, 1H), 4.86 (q, J = 9.4 Hz, 1H),
3.95 (s, 3H), 3.13 (t, J = 4.6 Hz, 4H), 2.86 (s, 3H), 2.69−2.78 (m,
2H), 2.58−2.69 (m, 2H), 2.45 (s, 3H).
The racemic mixture of 4-(2-((5-fluoro-6-methoxypyridin-3-yl)-
amino)-5-(2,2,2-trifluoro-1-(4-(methylsulfonyl)piperazin-1-yl)ethyl)-
pyridin-3-yl)-6-methyl-1,3,5-triazin-2-amine was separated using chiral
preparative SFC on a Chiralcel OJ-H column (250 × 20 mm) using
78:22 supercritical CO2/MeOH (1% DEA). The two separate peaks
were collected, concentrated, and dried under high vacuum to afford
the two enantiomers (32 and 33). The absolute stereochemistries
were assigned on the basis of eluting peak order and by analogy to
compounds 30 and 31.
(R)-4-(2-((5-Fluoro-6-methoxypyridin-3-yl)amino)-5-((2-methyl-
4-(methylsulfonyl)piperazin-1-yl)methyl)pyridin-3-yl)-N,N-bis(4-me-
thoxybenzyl)-6-methyl-1,3,5-triazin-2-amine (41). LiHMDS in THF
(1 M, 2.26 mL, 2.26 mmol) was added dropwise to a stirred mixture of
5-fluoro-6-methoxypyridin-3-amine (0.118 g, 0.83 mmol) and 40
(0.478 g, 0.75 mmol) in THF (10 mL) at −10 °C, and the mixture was
stirred for 30 min. The reaction was quenched with water and
saturated aqueous ammonium chloride (25 mL each) and diluted with
EtOAc (25 mL). The separated aqueous layer was extracted with
EtOAc (2 × 50 mL), and the combined organic extracts were washed
with brine, dried over sodium sulfate, and concentrated to give the
crude product. Purification by silica gel column chromatography (0−
1% MeOH in CH2Cl2) followed by washing with i-PrOH gave the title
compound (0.377 g, 66%).
(R)-4-(2-((5-Fluoro-6-methoxypyridin-3-yl)amino)-5-(2,2,2-tri-
fluoro-1-(4-(methylsulfonyl)piperazin-1-yl)ethyl)pyridin-3-yl)-6-meth-
yl-1,3,5-triazin-2-amine (32) was the second eluting peak and was
isolated as a yellow powder. MS (ESI positive ion): m/z calcd for
1
C22H25F4N9O3S 571, found 572 (M + H)+. H NMR (400 MHz,
DMSO-d6): δ 12.01(s, 1H), 8.85 (d, J = 2.0 Hz, 1H), 8.40 (d, J = 2.3
Hz, 1H), 8.29−8.39 (m, 2H), 7.93 (br s, 1H), 7.79 (br s, 1H), 4.84 (q,
J = 9.6 Hz, 1H), 3.94 (s, 3H), 3.12 (t, J = 4.7 Hz, 4H), 2.86 (s, 3H),
2.69−2.78 (m, 2H), 2.58−2.69 (m, 2H), 2.45 (s, 3H).
(S)-4-(2-((5-Fluoro-6-methoxypyridin-3-yl)amino)-5-(2,2,2-tri-
fluoro-1-(4-(methylsulfonyl)piperazin-1-yl)ethyl)pyridin-3-yl)-6-meth-
yl-1,3,5-triazin-2-amine (33) was the first eluting peak and was isolated
as a yellow powder. MS (ESI positive ion): m/z calcd for
1
C22H25F4N9O3S 571, found 572 (M + H)+. H NMR (400 MHz,
(R)-4-(2-((5-Fluoro-6-methoxypyridin-3-yl)amino)-5-((2-methyl-
4-(methylsulfonyl)piperazin-1-yl)methyl)pyridin-3-yl)-6-methyl-
1,3,5-triazin-2-amine (42). 41 (0.377 g, 0.48 mmol), TFA (2 mL),
DMSO-d6): δ 11.96 (s, 1H), 8.84 (d, J = 2.1 Hz, 1H), 8.40 (d, J = 2.3
Hz, 1H), 8.29−8.39 (m, 2H), 8.03 (br s, 1H), 7.88 (br s, 1H), 4.86 (q,
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and trifluoromethanesulfonic acid (0.22 mL) were added to a 25 mL
round-bottomed flask. The reaction mixture was stirred at 70 °C for 1
h and allowed to cool to room temperature. The volatiles were
removed under vacuum, the residual sticky oil was cooled to 0 °C, and
ice (∼100 g) was added followed by 1 M NaOH until basic. The
resulting yellow precipitate was filtered and purified by silica gel
column chromatography (0−5% MeOH with 2 M NH3 in CH2Cl2) to
give the title compound (0.125 g, 48%) as a yellow solid. MS (ESI
positive ion): m/z calcd for C22H28FN9O3S 517, found 518 (M + H).
1H NMR (400 MHz, DMSO-d6): δ 11.95 (s, 1H), 8.71 (d, J = 2.3 Hz,
1H), 8.41 (d, J = 2.1 Hz, 1H), 8.33−8.40 (m, 1H), 8.27 (d, J = 2.1 Hz,
1H), 7.90 (br s, 1H), 7.76 (br s, 1H), 3.93 (s, 3H), 3.91 (s, 1H), 3.13−
3.30 (m, 3H), 2.87−2.95 (m, 1H) 2.85 (s, 3H), 2.53−2.79 (m, 3H),
2.44 (s, 3H), 2.14−2.28 (m, 1H), 1.16 (d, J = 6.3 Hz, 3H).
(S)-4-(2-((5-Fluoro-6-methoxypyridin-3-yl)amino)-5-((2-
methyl-4-(methylsulfonyl)piperazin-1-yl)methyl)pyridin-3-yl)-
6-methyl-1,3,5-triazin-2-amine (43). Potassium (S)-((4-(tert-Bu-
toxycarbonyl)-2-methylpiperazin-1-yl)methyl)trifluoroborate. A
mixture of potassium (bromomethyl)trifluoroborate (1.20 g, 5.38
mmol) and (S)-tert-butyl 3-methylpiperazine-1-carboxylate (1.13 g,
5.65 mmol) in THF (7 mL) was heated at 80 °C under nitrogen for 24
h. The mixture was allowed to cool to room temperature, and the
solvent was removed under vacuum. The residue was redissolved in
acetone (125 mL) and treated with potassium carbonate (1 equiv).
The suspension was stirred at room temperature for 30 min and then
filtered through a short plug of diatomaceous earth. The filter cake was
washed with acetone, and the combined organic phases were
concentrated to give the crude product as a colorless foam (1.51 g,
88%). The pure material was obtained as a beige powder by adding
acetone/diethyl ether (2:1) to the foam and then slowly adding
hexanes while sonicating the mixture. 19F NMR (377 MHz, acetone-
d6): δ −141.28.
4-(5-Chloro-2-((5-fluoro-6-methoxypyridin-3-yl)amino)pyridin-3-
yl)-N,N-bis(4-methoxybenzyl)-6-methyl-1,3,5-triazin-2-amine (44).
A stirred mixture of 3814 (2.53 g, 5.28 mmol) and 5-fluoro-6-
methoxypyridin-3-amine (0.834 g, 5.87 mmol) in THF (50 mL) was
treated dropwise with LiHMDS in THF (1 M, 16.74 mL, 16.74 mmol)
at −15 °C and stirred for 40 min. The reaction was quenched with
water and saturated ammonium chloride (25 mL each) and diluted
with EtOAc (25 mL). The separated aqueous layer was extracted with
EtOAc (2 × 50 mL), and the combined organic extracts were washed
with brine, dried over sodium sulfate, and concentrated. The residue
was purified by silica gel chromatography (0−1% MeOH in CH2Cl2)
followed by washing with i-PrOH to give the title compound (2.07 g,
88%). MS (ESI positive ion): m/z calcd for C31H29ClFN7O3 601,
found 602 (M + H). 1H NMR (400 MHz, CDCl3): δ 8.77 (d, J = 2.5
Hz, 1H), 8.23 (d, J = 2.5 Hz, 1H), 7.95 (d, J = 2.0 Hz, 1H), 7.90 (d, J
= 12.1 Hz, 1H), 7.18 (dd, J = 17.4, 8.4 Hz, 4H), 6.86 (t, J = 8.1 Hz,
4H), 4.86 (s, 2H), 4.82 (s, 2H), 4.01 (s, 3H); 3.81 (s, 3H), 3.79 (s,
3H), 2.60 (s, 3H).
stirred at room temperature for 30 min. The solvent was removed
under vacuum, and the brown oil obtained was redissolved in CH2Cl2
(5 mL). The mixture was cooled to −15 °C, and triethylamine (0.42
mL, 2.94 mmol) and methanesulfonyl chloride (0.14 mL, 1.77 mmol)
were added sequentially. The reaction mixture was stirred at −15 °C
for 1 h. The reaction was quenched with water and saturated aqueous
sodium bicarbonate (10 mL each) and diluted with EtOAc (10 mL).
The separated aqueous layer was extracted with EtOAc (2 × 10 mL),
and the combined organic extracts were washed with brine, dried over
sodium sulfate, and concentrated. The crude product was used without
further purification.
(S)-4-(2-((5-Fluoro-6-methoxypyridin-3-yl)amino)-5-((2-methyl-4-
(methylsulfonyl)piperazin-1-yl)methyl)pyridin-3-yl)-6-methyl-1,3,5-
triazin-2-amine (43). 46 (0.219 g, 0.29 mmol), TFA (2 mL), and
trifluoromethanesulfonic acid (0.1 mL) were added to a 25 mL round-
bottomed flask. The reaction mixture was stirred at 80 °C for 16 h and
allowed to cool to room temperature. The volatiles were removed
under vacuum, and the crude product was purified by silica gel column
chromatography [0 −5% MeOH (with NH3 in CH2Cl2)] to give the
title compound (0.098 g, 53%) as a yellow solid. MS (ESI positive
ion): m/z calcd for C22H28FN9O3S 517, found 518 (M + H). 1H NMR
(400 MHz, DMSO-d6, mixture of rotamers): δ 12.06 (d, J = 5.1 Hz,
1H), 9.44−9.97 (m, 1H), 8.76−9.09 (m, 1H), 8.21−8.54 (m, 2H),
7.97 (br s, 1H), 7.87 (br s, 1H), 4.08−4.96 (m, 2H), 3.95 (s, 3H),
3.61−3.87 (m, 2H), 2.79−3.36 (m, 8H), 2.45 (s, 3H), 1.34−1.65 (m,
3H).
4-(2-((5-Fluoro-6-methoxypyridin-3-yl)amino)-5-((R)-1-((R)-
2-methyl-4-(methylsulfonyl)piperazin-1-yl)ethyl)pyridin-3-yl)-
6-methyl-1,3,5-triazin-2-amine (56). 1-(6-Fluoropyridin-3-yl)-
ethanol (48). A clear solution (prepared by filtration of a slightly
cloudy suspension) of 6-fluoronicotinaldehyde (47; 9.88 g, 79 mmol)
in THF (100 mL) was added dropwise via an addition funnel to a
solution of methylmagnesium bromide in diethyl ether (3 M, 31.6 mL,
95 mmol) in THF (280 mL) at −6 °C. The addition was completed
over about 20 min, and the reaction temperature was kept below −5
°C during the addition. MeOH (10 mL) was added dropwise, followed
by saturated aqueous ammonium chloride (300 mL) and a sufficient
amount of water to dissolve the precipitate. EtOAc (200 mL) was then
added, and the organic layer was separated. The aqueous layer was
extracted with CH2Cl2 (2 × 150 mL), and the combined organic
extracts were dried over sodium sulfate, filtered, and concentrated in
vacuo to provide the title compound (10.55 g, 95%) as a light yellow
oil. 1H NMR (400 MHz, CDCl3): δ 8.18 (d, J = 2.0 Hz, 1H), 7.85 (td,
J = 8.1, 2.5 Hz, 1H), 6.92 (dd, J = 8.5, 2.8 Hz, 1H), 4.98 (q, J = 6.5 Hz,
1H), 2.21 (br s, 1H), 1.53 (d, J = 6.5 Hz, 3H).
5-(1-Bromoethyl)-2-fluoropyridine (49). Thionyl bromide (11.60
mL, 149 mmol) was added dropwise over a 15 min period to a
solution of 48 (10.55 g, 74.7 mmol) in CH2Cl2 (300 mL) at 25 °C,
and the resulting orange solution was stirred at 25 °C for 3 h. The
excess of thionyl bromide was carefully quenched with water (150 mL)
at 0 °C with vigorous stirring, and NaOH (5 M, 100 mL) was then
carefully added at 0 °C (over ∼10 min). The pH was adjusted to ∼9
by the addition of saturated aqueous sodium bicarbonate. The
resulting mixture was vigorously stirred at 0 °C for 5 min and
partitioned between CH2Cl2 (200 mL) and aqueous sodium
bicarbonate (300 mL of saturated sodium bicarbonate and 300 mL
of water). The organic layer was separated, and the aqueous layer was
extracted with CH2Cl2 (300 mL). The combined organic extracts were
sequentially washed with saturated aqueous sodium bicarbonate (500
mL) and brine (500 mL), dried over sodium sulfate, filtered, and
concentrated in vacuo to provide the title compound (14.58 g, 96%) as
a yellow oil. This material was used without further purification.
(3R)-tert-Butyl 4-(1-(6-Fluoropyridin-3-yl)ethyl)-3-methylpipera-
zine-1-carboxylate (50). 49 (3.90 g, 19.11 mmol), CH3CN (75
mL), (R)-tert-butyl 3-methylpiperazine-1-carboxylate (4.02 g, 20.07
mmol), potassium carbonate (3.16 g, 22.94 mmol), and potassium
iodide (0.634 g, 3.82 mmol) were added to a 100 mL round-bottomed
flask. The reaction mixture was stirred at 70 °C for 16 h. The reaction
mixture was diluted with water (50 mL) and CH2Cl2 (50 mL), and the
organic layer was separated. The separated aqueous layer was extracted
(S)-tert-Butyl 4-((5-(4-(Bis(4-methoxybenzyl)amino)-6-methyl-
1,3,5-triazin-2-yl)-6-((5-fluoro-6-methoxypyridin-3-yl)amino)-
pyridin-3-yl)methyl)-3-methylpiperazine-1-carboxylate (45). 44
(0.197 g, 0.33 mmol), 2-(dicyclohexylphosphino)-2′,4′,6′,-triisoprop-
yl-1,1′-biphenyl (0.016 g, 0.03 mmol), cesium carbonate (0.32 g, 0.98
mmol), potassium (S)-((4-(tert-butoxycarbonyl)-2-methylpiperazin-1-
yl)methyl)trifluoroborate (0.126 g, 0.39 mmol), and palladium acetate
(4 mg, 0.02 mmol) were added to a 10 mL reaction vial. The vial was
sealed and purged with argon and then treated with THF (1 mL) and
water (0.1 mL). The mixture was heated in a microwave reactor at 140
°C for 30 min. After cooling, the mixture was filtered through a short
plug of diatomaceous earth, washed with EtOAc (3×), and
concentrated in vacuo. The crude product was purified by silica gel
column chromatography (0−5% MeOH in CH2Cl2) to give the title
compound (0.230 g, 90%) as a yellow syrup. MS (ESI positive ion):
m/z calcd for C42H50FN9O5 779, found 780 (M + H)+.
(S)-tert-Butyl 4-((5-(4-(Bis(4-methoxybenzyl)amino)-6-methyl-
1,3,5-triazin-2-yl)-6-((5-fluoro-6-methoxypyridin-3-yl)amino)-
pyridin-3-yl)methyl)-3-methylpiperazine-1-carboxylate (46). 45
(0.230 g, 0.29 mmol), CH2Cl2 (3 mL), and TFA (3 mL) were
added to a 25 mL round-bottomed flask. The reaction mixture was
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with CH2Cl2 (3 × 50 mL), and the combined organic extracts were
washed with brine, dried over sodium sulfate, and concentrated. The
residue was purified by silica gel column chromatography (0−50%
EtOAc in hexanes) to provide the title compound (3.05 g, 49%).
(5-(1-((R)-4-(tert-Butoxycarbonyl)-2-methylpiperazin-1-yl)ethyl)-
2-fluoropyridin-3-yl)boronic Acid (51). 50 (3.0 g, 9.28 mmol) and
THF (100 mL) were added to a 150 mL round-bottomed flask. The
reaction mixture was cooled to −78 °C and treated dropwise with n-
butyllithium in hexanes (2.5 M, 4.08 mL, 10.20 mmol). The solution
was stirred at −78 °C for 1 h and treated in one portion with
triisopropyl borate (2.55 mL, 11.13 mmol). The mixture was stirred at
−78 °C for 1 h, 1 M NaOH (30 mL) was added, and the solution was
stirred at room temperature for 30 min. The aqueous layer was
separated, and the organic layer was extracted with additional 1 M
NaOH (30 mL). The combined aqueous layers were carefully acidified
with 5 M HCl (12 mL) to a final pH of ∼4−5. The reaction mixture
was extracted with EtOAc (3 × 100 mL), and the combined organic
extracts were dried over sodium sulfate, filtered, and concentrated
under vacuum to give the title compound (2.5 g, 73%). This material
was used without further purification.
(3R)-tert-Butyl 4-(1-(5-(4-(Bis(4-methoxybenzyl)amino)-6-methyl-
1,3,5-triazin-2-yl)-6-fluoropyridin-3-yl)ethyl)-3-methylpiperazine-1-
carboxylate (52). A glass microwave reaction vessel was charged with
51 (1.40 g, 3.81 mmol), 4-chloro-N,N-bis(4-methoxybenzyl)-6-
methyl-1,3,5-triazin-2-amine (1.46 g, 3.81 mmol), 1,4-dioxane (10
mL), potassium acetate (1.12 g, 11.44 mmol), water (1 mL), and
Pd(A-Phos)Cl2 (0.27 g, 0.38 mmol). The solution was stirred and
heated in a microwave reactor at 120 °C for 20 min. The reaction
mixture was diluted with water (20 mL) and CH2Cl2 (25 mL). The
organic layer was separated, and the aqueous layer was extracted with
CH2Cl2 (3 × 50 mL). The combined organic extracts were dried over
sodium sulfate, filtered, and concentrated in vacuum. The crude
product was purified by silica gel chromatography (0−50% EtOAc in
hexanes) to provide the title compound (1.26 g, 49%).
(3R)-tert-Butyl 4-(1-(5-(4-(Bis(4-methoxybenzyl)amino)-6-methyl-
1,3,5-triazin-2-yl)-6-((5-fluoro-6-methoxypyridin-3-yl)amino)-
pyridin-3-yl)ethyl)-3-methylpiperazine-1-carboxylate (53). 52 (1.0 g,
1.49 mmol), 5-fluoro-6-methoxypyridin-3-amine (0.317 g, 2.23 mmol),
and THF (20 mL) were added to a 250 mL round-bottomed flask.
The solution was cooled to −40 °C and treated dropwise via an
addition funnel with LiHMDS in THF (1 M, 4.47 mL, 4.47 mmol).
The solution was stirred at −40 °C for 30 min, quenched with water
(20 mL) and saturated aqueous ammonium chloride (20 mL), and
diluted with EtOAc (20 mL). The organic layer was separated, and the
aqueous layer was extracted with EtOAc (2 × 20 mL). The combined
organic extracts were washed with brine (20 mL), dried over sodium
sulfate, and concentrated. The crude product was purified by silica gel
chromatography (40 g, 5−50% EtOAc in hexanes) to provide (R)-tert-
butyl 4-((R)-1-(5-(4-(bis(4-methoxybenzyl)amino)-6-methyl-1,3,5-tri-
azin-2-yl)-6-((5-fluoro-6-methoxypyridin-3-yl)amino)pyridin-3-yl)-
ethyl)-3-methylpiperazine-1-carboxylate (0.45 g, 38%) and (R)-tert-
butyl 4-((S)-1-(5-(4-(bis(4-methoxybenzyl)amino)-6-methyl-1,3,5-tri-
azin-2-yl)-6-((5-fluoro-6-methoxypyridin-3-yl)amino)pyridin-3-yl)-
ethyl)-3-methylpiperazine-1-carboxylate (0.35 g, 30%).
removed under vacuum. The crude product was washed with water
(30 mL), stirred, and sonicated. The resulting solid was collected,
washed with water, and dried in a vacuum oven to give the title
compound (0.150 g, 77%).
4-(2-((5-Fluoro-6-methoxypyridin-3-yl)amino)-5-((R)-1-((R)-2-
methyl-4-(methylsulfonyl)piperazin-1-yl)ethyl)pyridin-3-yl)-6-meth-
yl-1,3,5-triazin-2-amine (56). 54 (0.130 g, 0.168 mmol), TFA (5 mL),
and trifluoromethanesulfonic acid (0.2 mL) were added to a 50 mL
round-bottomed flask. The solution was stirred at 70 °C for 30 min.
The volatiles were removed under vacuum, and the residue was cooled
in an ice bath and neutralized with 1 M NaOH. The mixture was
stirred at room temperature for 16 h, and the precipitate obtained was
filtered and washed with water. Purification by preparative TLC using
5% MeOH in CH2Cl2 gave the title compound (0.047 g, 52%). MS
(ESI positive ion): m/z calcd for C23H30FN9O3S 531, found 532 (M +
1
H). H NMR (400 MHz, DMSO-d6): δ 11.93 (s, 1H) 8.70−8.74 (m,
1H), 8.37−8.43 (m, 2H), 8.31−8.34 (m, 1H), 7.88−7.92 (m, 1H),
7.74−7.78 (m, 1H), 4.00 (d, J = 6.8 Hz, 1H), 3.94 (s, 3H), 3.01−3.18
(m, 3H), 2.84 (s, 5H), 2.45−2.55 (m, 5H), 1.38 (d, J = 6.6 Hz, 3H),
1.07 (d, J = 6.3 Hz, 3H).
4-(2-((5-Fluoro-6-methoxypyridin-3-yl)amino)-5-((S)-1-((R)-
2-methyl-4-(methylsulfonyl)piperazin-1-yl)ethyl)pyridin-3-yl)-
6-methyl-1,3,5-triazin-2-amine (57). 4-(2-((5-Fluoro-6-methoxy-
pyridin-3-yl)amino)-5-((S)-1-((R)-2-methyl-4-(methylsulfonyl)-
piperazin-1-yl)ethyl)pyridin-3-yl)-N,N-bis(4-methoxybenzyl)-6-
methyl-1,3,5-triazin-2-amine (55). (R)-tert-Butyl 4-((S)-1-(5-(4-(bis-
(4-methoxybenzyl)amino)-6-methyl-1,3,5-triazin-2-yl)-6-((5-fluoro-6-
methoxypyridin-3-yl)amino)pyridin-3-yl)ethyl)-3-methylpiperazine-1-
carboxylate (0.150 g, 0.19 mmol), CH2Cl2 (5 mL), and TFA (5 mL)
were added to a 100 mL round-bottomed flask. The reaction mixture
was stirred at room temperature for 1 h, and the solvent was removed
under vacuum. The residue was dissolved in CH2Cl2 (10 mL) and
slowly washed with a saturated aqueous solution of sodium
bicarbonate. The organic phase was separated, dried over sodium
sulfate, filtered, and concentrated. The residue was dried in a vacuum
oven, dissolved in CH2Cl2 (5 mL), and cooled to −50 °C.
Triethylamine (0.27 mL, 0.19 mmol) and methanesulfonyl chloride
(0.08 mL, 0.19 mmol) were added, the solution was stirred at −50 °C
for 30 min, and the solvent was removed under vacuum. The crude
product was washed with water (30 mL), stirred, and sonicated. The
resulting solid was collected, washed with water, and dried in a vacuum
oven to give the title compound (0.100 g, 69%), which was used
without further purification.
4-(2-((5-Fluoro-6-methoxypyridin-3-yl)amino)-5-((S)-1-((R)-2-
methyl-4-(methylsulfonyl)piperazin-1-yl)ethyl)pyridin-3-yl)-6-meth-
yl-1,3,5-triazin-2-amine (57). 55 (0.095 g, 0.12 mmol), TFA (5 mL),
and trifluoromethanesulfonic acid (1 mL) were added to a 50 mL
round-bottomed flask. The solution was stirred at 70 °C for 30 min.
The volatiles were removed under vacuum, and the residue was cooled
in an ice bath and neutralized with 1 M NaOH. The mixture was
stirred at room temperature for 16 h, and the resulting precipitate was
filtered and washed with water. Purification by preparative TLC using
5% MeOH in CH2Cl2 gave the title compound (0.031 g, 47%). MS
(ESI positive ion): m/z calcd for C23H30FN9O3S 531, found 532 (M +
1
H). H NMR (400 MHz, DMSO-d6): δ 11.95 (br s, 1H), 8.75 (br s,
4-(2-((5-Fluoro-6-methoxypyridin-3-yl)amino)-5-((R)-1-((R)-2-
methyl-4-(methylsulfonyl)piperazin-1-yl)ethyl)pyridin-3-yl)-N,N-bis-
(4-methoxybenzyl)-6-methyl-1,3,5-triazin-2-amine (54). (R)-tert-
Butyl 4-((R)-1-(5-(4-(bis(4-methoxybenzyl)amino)-6-methyl-1,3,5-tri-
azin-2-yl)-6-((5-fluoro-6-methoxypyridin-3-yl)amino)pyridin-3-yl)-
ethyl)-3-methylpiperazine-1-carboxylate (0.200 g, 0.25 mmol), CH2Cl2
(5 mL), and TFA (5 mL) were added to a 100 mL round-bottomed
flask. The reaction mixture was stirred at room temperature for 1 h,
and the solvent was removed under vacuum. The residue was dissolved
in CH2Cl2 (10 mL) and slowly washed with a saturated aqueous
sodium bicarbonate solution. The organic phase was separated, dried
over sodium sulfate, filtered, concentrated, and dried in a vacuum
oven. The residue was dissolved in CH2Cl2 (5 mL) and cooled to −50
°C. To the reaction mixture were added triethylamine (0.35 mL, 2.52
mmol) and methanesulfonyl chloride (0.10 mL, 1.26 mmol). The
solution was stirred at −50 °C for 30 min, and the solvent was
1H), 8.32−8.49 (m, 3H), 7.90 (br s, 1H), 7.76 (br s, 1H), 4.05 (d, J =
5.7 Hz, 1H), 3.94 (s, 3H), 3.25 (br s, 2H), 3.04 (br s, 2H), 2.85 (br s,
5H), 2.45 (s, 4H), 2.32 (d, J = 7.2 Hz, 1H), 1.38 (d, J = 6.6 Hz, 3H),
1.16 (d, J = 5.7 Hz, 3H).
4-(2-((5-Fluoro-6-methoxypyridin-3-yl)amino)-5-((R)-1-((S)-
2-methyl-4-(methylsulfonyl)piperazin-1-yl)ethyl)pyridin-3-yl)-
6-methyl-1,3,5-triazin-2-amine (58). 1-(5-(4-(Bis(4-
methoxybenzyl)amino)-6-methyl-1,3,5-triazin-2-yl)-6-((5-fluoro-6-
methoxypyridin-3-yl)amino)pyridin-3-yl)ethanone (60). A stirred
mixture of 38 (0.602 g, 1.00 mmol), X-Phos-Pd(OAc)2 remilled mix
(1:1, 0.070 g, 0.10 mmol), and cesium fluoride (0.532 g, 3.50 mmol)
in 1,4-dioxane (5 mL) was treated with tributyl(1-ethoxyvinyl)-
stannane (0.507 mL, 1.50 mmol) under nitrogen. The mixture was
sealed and heated at 110 °C for 16 h, allowed to cool to room
temperature, and filtered through a short plug of diatomaceous earth.
The filter cake was washed with EtOAc (3 × 20 mL) and
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concentrated. The crude product was purified by silica gel
chromatography (0−5% MeOH in CH2Cl2) to give a mixture of the
desired product and a small amount of dechlorinated product. This
mixture was subjected to flash column chromatographic purification
(30% EtOAc in hexanes) to give the title compound (0.482 g, 79%) as
a light yellow solid. MS (ESI positive ion): m/z calcd for
C33H32FN7O4 609, found 610 (M + H)+.
1-(5-(4-(Bis(4-methoxybenzyl)amino)-6-methyl-1,3,5-triazin-2-
yl)-6-((5-fluoro-6-methoxypyridin-3-yl)amino)pyridin-3-yl)ethanol
(61). A stirred solution of 60 (0.135 g, 0.22 mmol) in THF (5 mL)
was treated with sodium borohydride (0.042 g, 1.11 mmol) at 0 °C,
and the mixture was stirred for 10 min before being allowed to warm
to room temperature for 1 h. The suspension was quenched with 1 M
NaOH, and the separated aqueous layer was extracted with EtOAc (2
× 10 mL). The combined organic extracts were washed with brine,
dried over sodium sulfate, and concentrated in vacuo to give the title
compound, which was used directly in the next step without further
purification.
MS (ESI positive ion): m/z calcd for C23H30FN9O3S 531, found 532
(M + H). 1H NMR (400 MHz, DMSO-d6): δ 11.93 (s, 1H), 8.72 (d, J
= 2.4 Hz, 1H), 8.35−8.45 (m, 3H), 7.89 (br s, 1H), 7.76 (br s, 1H),
3.96−4.08 (m, 1H), 3.93 (s, 3H), 3.07−3.15 (m, 2H), 3.05 (dd, J =
10.9, 2.8 Hz, 1H), 2.73−2.90 (m, 5H), 2.51−2.55 (m, 2H), 2.44 (s,
3H), 1.37 (d, J = 6.8 Hz, 3H), 1.07 (d, J = 6.1 Hz, 3H).
4-(2-((5-Fluoro-6-methoxypyridin-3-yl)amino)-5-((S)-1-((S)-2-
methyl-4-(methylsulfonyl)piperazin-1-yl)ethyl)pyridin-3-yl)-6-
methyl-1,3,5-triazin-2-amine (59). A solution of 65 (0.101 g, 0.13
mmol) in CH2Cl2 (3 mL) was treated with TFA (3 mL), and the
mixture was stirred at room temperature for 30 min before being
concentrated to give a brown oil. This was redissolved in CH2Cl2 (3
mL) and treated with triethylamine (0.09 mL, 0.64 mmol) followed by
slow addition of methanesulfonyl chloride (0.03 mL, 0.38 mmol) at
−15 °C. The mixture was stirred for 1 h and then quenched with
saturated aqueous sodium bicarbonate and water. The separated
aqueous layer was extracted with CH2Cl2 (2×), and the combined
organic extracts were dried over sodium sulfate and concentrated. The
crude product was treated with TFA (2 mL) and heated at 80 °C
overnight. The resulting mixture was allowed to cool to room
temperature and concentrated, and the crude product was purified by
silica gel chromatography (0−5% MeOH in CH2Cl2) to give the title
compound (0.029 g, 43%) as a yellow solid. MS (ESI positive ion): m/
1-(5-(4-(Bis(4-methoxybenzyl)amino)-6-methyl-1,3,5-triazin-2-
yl)-6-((5-fluoro-6-methoxypyridin-3-yl)amino)pyridin-3-yl)ethyl
methanesulfonate (62). A solution of 61 (0.436 g, 0.71 mmol) in
CH2Cl2 (10 mL) was treated with triethylamine (0.30 mL, 2.14 mmol)
and methanesulfonyl chloride (0.14 mL, 1.78 mmol). The mixture was
stirred for 2 h and then quenched with water (10 mL). The aqueous
layer was extracted with CH2Cl2 (2 × 5 mL), and the combined
organic extracts were washed with 1 M HCl, saturated aqueous sodium
bicarbonate, and brine and dried over sodium sulfate. Removal of the
solvent under vacuum gave the title compound as a yellow residue,
which was used directly in the next step without further purification.
(3S)-tert-Butyl 4-(1-(5-(4-(Bis(4-methoxybenzyl)amino)-6-methyl-
1,3,5-triazin-2-yl)-6-((5-fluoro-6-methoxypyridin-3-yl)amino)-
pyridin-3-yl)ethyl)-3-methylpiperazine-1-carboxylate (63). A stirred
solution of crude 62 (0.492 g, 0.71 mmol) in CH3CN (10 mL) was
treated with 2,2,6,6-tetramethylpiperidine (0.18 mL, 1.07 mmol) and
(S)-tert-butyl 3-methylpiperazine-1-carboxylate (0.186 g, 0.93 mmol).
The mixture was heated at reflux overnight. The reaction mixture was
allowed to cool to room temperature, and the resulting suspension was
diluted with water and CH2Cl2 (20 mL each). The separated aqueous
layer was extracted with CH2Cl2 (2 × 20 mL), and the combined
organic extracts were washed with brine, dried over sodium sulfate,
and concentrated under vacuum. The residue was purified by silica gel
column chromatography (0−15% EtOAc in CH2Cl2 followed by 5%
MeOH in CH2Cl2) to give the two separated diasteromers (S)-tert-
butyl 4-((S)-1-(5-(4-(bis(4-methoxybenzyl)amino)-6-methyl-1,3,5-tri-
azin-2-yl)-6-((5-fluoro-6-methoxypyridin-3-yl)amino)pyridin-3-yl)-
ethyl)-3-methylpiperazine-1-carboxylate (65) (first eluting diaster-
eomer; 0.101 g, 18%) followed by (S)-tert-butyl 4-((R)-1-(5-(4-
(bis(4-methoxybenzyl)amino)-6-methyl-1,3,5-triazin-2-yl)-6-((5-fluo-
ro-6-methoxypyridin-3-yl)amino)pyridin-3-yl)ethyl)-3-methylpipera-
zine-1-carboxylate (64) (second eluting diastereomer; 0.101 g, 18%).
The absolute stereochemistries at the diastereomeric centers of the
two diastereomers were assigned by the cocrystal structure of
compound 58 (derived from the second eluting distereomer) in
complex with PI3Kγ at 2.9 Å resolution.
4-(2-((5-Fluoro-6-methoxypyridin-3-yl)amino)-5-((R)-1-((S)-2-
methyl-4-(methylsulfonyl)piperazin-1-yl)ethyl)pyridin-3-yl)-6-meth-
yl-1,3,5-triazin-2-amine (58). A solution of 64 (0.101 g, 0.13 mmol)
in CH2Cl2 (3 mL) was treated with TFA (3 mL), and the mixture was
stirred at room temperature for 30 min. The solution was concentrated
under vacuum to give a brown oil. This residue was redissolved in
CH2Cl2 (3 mL) and treated with triethylamine (0.09 mL, 0.64 mmol)
followed by the slow addition of methanesulfonyl chloride (0.03 mL,
0.38 mmol) at −15 °C. The reaction mixture was stirred for 1 h and
then quenched with saturated aqueous sodium bicarbonate and water.
The separated aqueous layer was extracted with CH2Cl2 (2×), and the
combined organic extracts were dried over sodium sulfate and
concentrated. The crude product was treated with TFA (2 mL) and
heated at 80 °C overnight. The resulting mixture was allowed to cool
to room temperature and concentrated, and the resulting residue was
purified by silica gel column chromatography (0−5% MeOH in
CH2Cl2) to give the title compound (0.029 g, 43%) as a yellow solid.
1
z calcd for C23H30FN9O3S 531, found 532 (M + H). H NMR (400
MHz, DMSO-d6): δ 11.95 (s, 1H), 8.74 (d, J = 2.5 Hz, 1H), 8.27−8.46
(m, 2H), 7.90 (br s, 1H), 7.77 (br s, 1H), 3.99−4.09 (m, 1H), 3.93 (s,
3H), 3.20−3.30 (m, 1H), 2.94−3.11 (m, 2H), 2.84 (s, 3H), 2.75−2.84
(m, 2H), 2.45 (s, 3H), 2.40 −2.45(m, 1H), 2.29−2.35 (m, 1H), 1.28
(d, J = 6.6 Hz, 3H), 1.15 (d, J = 6.3 Hz, 3H).
4-(2-((5-Fluoro-6-methoxypyridin-3-yl)amino)-5-(2-(4-
(methylsulfonyl)piperazin-1-yl)propan-2-yl)pyridin-3-yl)-6-
methyl-1,3,5-triazin-2-amine (69). 4-(2-((5-Fluoro-6-methoxypyr-
idin-3-yl)amino)-5-(prop-1-en-2-yl)pyridin-3-yl)-N,N-bis(4-methoxy-
benzyl)-6-methyl-1,3,5-triazin-2-amine (66). A mixture of isoprope-
nylboronic acid pinacol ester (0.214 mL, 1.14 mmol), potassium
carbonate (0.316 g, 2.28 mmol), bis(4-(di-tert-butylphosphino)-N,N-
dimethylbenzenamine)palladium dichloride (0.040 g, 0.057 mmol),
and 44 (0.344 g, 0.571 mmol) in 4:1 1,4-dioxane/water (3.75 mL) was
sealed in a vial and heated at 80 °C for 24 h. The reaction mixture was
allowed to cool to room temperature and partitioned between water
and CH2Cl2. The aqueous layer was extracted with CH2Cl2 (3×), and
the combined organic layers were dried over anhydrous sodium
sulfate, filtered, and concentrated in vacuo. The material was purified
by silica gel chromatography (0−30% EtOAc/hexane) to afford the
title compound (0.282 g, 81%) as an orange solid. MS (ESI positive
ion): m/z calcd for C34H34FN7O3 607, found 608 (M + H). 1H NMR
(400 MHz, CDCl3): δ 11.92 (br s, 1H), 8.94 (d, J = 2.5 Hz, 1H), 8.46
(d, J = 2.7 Hz, 1H), 8.05 (dt, J = 12.4, 1.1 Hz, 1H), 7.97 (d, J = 2.3 Hz,
1H), 7.18−7.26 (m, 4H), 6.82−6.91 (m, 4H), 5.34 (s, 1H), 5.02 (t, J =
1.5 Hz, 1H), 4.89 (s, 2H), 4.83 (s, 2H), 4.02 (s, 3H), 3.82 (s, 3H),
3.79 (s, 3H), 2.60 (s, 3H), 2.14 (s, 3H).
4-(5-(2-Azidopropan-2-yl)-2-((5-fluoro-6-methoxypyridin-3-yl)-
amino)pyridin-3-yl)-N,N-bis(4-methoxybenzyl)-6-methyl-1,3,5-tria-
zin-2-amine (67). A slurry of sodium azide (0.151 g, 2.32 mmol) and
66 (0.282 g, 0.464 mmol) in CHCl3 (1 mL) at −15 °C (MeOH/ice)
was treated with a solution of TFA (0.36 mL, 4.64 mmol) in CHCl3 (1
mL) via a syringe over 10 min. The reaction was sealed in a vial and
heated at 60 °C. After 2 h, the temperature was increased to 80 °C.
After 2 h, an additional 2.5 equiv of sodium azide and 5 equiv of TFA
were added. The reaction was sealed and heated at 80 °C for 20 min.
The reaction was allowed to cool to room temperature and then
placed in a freezer over the weekend. The reaction was allowed to
warm to ambient temperature and then heated at 80 °C for 1 h. The
reaction mixture was treated with ice and 10 N NaOH until basic, and
the solution was partitioned between water and CH2Cl2. The aqueous
layer was extracted with CH2Cl2 (3×), and the combined organic
extracts were dried over anhydrous sodium sulfate, filtered, and
concentrated in vacuo to give the title compound (0.280 g, 93%) as an
orange oil. MS (ESI positive ion): m/z calcd for C34H35FN10O3 650,
found 651 (M + H). 1H NMR (400 MHz, CDCl3): δ 11.89 (br s, 1H),
8.88−8.93 (m, 1H), 8.38−8.43 (m, 1H), 8.03 (dt, J = 12.1, 1.2 Hz,
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1H), 7.90−7.98 (m, 1H), 7.23 (t, J = 8.6 Hz, 4H), 6.82−6.92 (m, 4H),
4.91 (s, 2H), 4.82 (s, 2H), 4.02 (s, 3H), 3.82 (s, 3H), 3.79 (s, 3H),
2.60 (s, 3H), 1.64 (s, 6H).
Accession Codes
The cocrystal structure of PI3Kγ + compound 31 has been
deposited in the Protein Data Bank with PDB code 4FLH.
4-(5-(2-Aminopropan-2-yl)-2-((5-fluoro-6-methoxypyridin-3-yl)-
amino)pyridin-3-yl)-N,N-bis(4-methoxybenzyl)-6-methyl-1,3,5-tria-
zin-2-amine (68). A mixture of 67 (0.280 g, 0.430 mmol) and
palladium on 10% carbon with 50% water (0.366 g) was flushed with
nitrogen and treated with THF (7.5 mL). The atmosphere was
replaced with hydrogen from a balloon. The reaction was stirred
rapidly for 1.5 h and flushed with nitrogen. The solution was filtered
through diatomaceous earth, which was then rinsed with CH2Cl2/
MeOH. The filtrate was concentrated in vacuo to give the title
compound (0.272 g, 100%), which was used without further
purification. MS (ESI positive ion): m/z calcd for C34H37FN8O3
AUTHOR INFORMATION
Corresponding Author
*Phone: (805) 447-1552. Fax: (805) 480-3015. E-mail:
markn@amgen.com.
Notes
■
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
1
624, found 625 (M + H). H NMR (400 MHz, CDCl3): δ 11.79 (s,
We thank Randy Hungate, Rick Kendall, Terry Rosen, and
Glenn Begley for their support of this research program.
Thanks also go to Paul Andrews for his assistance with the in
vitro cellular assays, Ronya Primack for running the rat PK
assay, and Mike Hayashi for performing the hepatocyte
incubations. In addition, we are grateful to Jin Tang and
Peter Yakowec for expression and purification of the PI3Kγ
enzyme and to Wes Barnhart, Sam Thomas, Fang Xia, Zheng
Hua, and Kyung Gahm for their support in compound
purification.
1H), 8.94 (d, J = 2.7 Hz, 1H), 8.51 (d, J = 2.7 Hz, 1H), 8.00−8.10 (m,
1H), 7.93 (d, J = 2.3 Hz, 1H), 7.13−7.25 (m, 4H), 6.80−6.93 (m,
4H), 4.89 (s, 3H), 4.82 (s, 2H), 4.00 (s, 3H), 3.81 (s, 3H), 3.77 (s,
3H), 2.58 (s, 2H), 1.55 (s, 6H).
4-(2-((5-Fluoro-6-methoxypyridin-3-yl)amino)-5-(2-(4-
(methylsulfonyl)piperazin-1-yl)propan-2-yl)pyridin-3-yl)-6-methyl-
1,3,5-triazin-2-amine (69). A mixture of 68 (0.272 g, 0.435 mmol)
and ((methylsulfonyl)azanediyl)bis(ethane-1,2-diyl) dimethanesulfo-
nate (0.222 g, 0.653 mmol) in DIPEA (3 mL) was added to a 50 mL
round-bottomed flask. The flask was fitted with a water-cooled reflux
condenser and placed in a 130 °C oil bath. After 2.5 h, the reaction
mixture was allowed to cool to room temperature and partitioned
between saturated aqueous sodium bicarbonate and CH2Cl2. The
aqueous layer was extracted with CH2Cl2 (3×), and the combined
organic layers were dried over anhydrous sodium sulfate, filtered, and
concentrated in vacuo. The crude material was purified by silica gel
chromatography (0−100% EtOAc/hexane) to give 4-(2-((5-fluoro-6-
methoxypyridin-3-yl)amino)-5-(2-(4-(methylsulfonyl)piperazin-1-yl)-
propan-2-yl)pyridin-3-yl)-N,N-bis(4-methoxybenzyl)-6-methyl-1,3,5-
triazin-2-amine (0.180 g, 54%) as an orange oil. MS (ESI positive ion):
m/z calcd for C39H46FN9O5S 771, found 772 (M + H). 1H NMR (400
MHz, CDCl3): δ 11.87 (br s, 1H), 8.91 (dd, J = 2.2, 0.4 Hz, 1H), 8.47
(dd, J = 2.3, 0.4 Hz, 1H), 8.11 (dt, J = 12.5, 1.2 Hz, 1H), 7.91−8.01
(m, 1H), 7.18−7.25 (m, 4H), 6.77−6.94 (m, 4H), 4.77−4.89 (m, 4H),
4.02 (s, 3H), 3.82 (s, 3H), 3.79 (s, 3H), 3.10−3.16 (m, 4H), 2.56−
2.65 (m, 10H), 1.37 (s, 6H).
A solution of 4-(2-((5-fluoro-6-methoxypyridin-3-yl)amino)-5-(2-
(4-(methylsulfonyl)piperazin-1-yl)propan-2-yl)pyridin-3-yl)-N,N-bis-
(4-methoxybenzyl)-6-methyl-1,3,5-triazin-2-amine (0.180 g, 0.233
mmol) in TFA (2 mL) was treated with trifluoromethanesulfonic
acid (0.104 mL, 1.17 mmol). The reaction was sealed and heated to 80
°C for 30 min. The reaction was cooled with ice and treated with ice
and 10 N NaOH until basic. The solution was partitioned between
water and CH2Cl2. The aqueous layer was extracted with CH2Cl2
(3×), and the combined organic layers were dried over anhydrous
sodium sulfate, filtered, and concentrated in vacuo. The material was
treated with CH2Cl2 and purified by silica gel chromatography (0−
10% MeOH in CH2Cl2). The product-containing fractions were
concentrated, and the residue was sonicated in diethyl ether (2 mL),
filtered, and rinsed with diethyl ether (1 mL). The resulting solid was
dried in vacuo to give the title compound (0.067 g, 54%) as a yellow
solid. MS (ESI positive ion): m/z calcd for C23H30FN9O3S 531, found
532 (M + H). 1H NMR (400 MHz, DMSO-d6): δ 11.91 (s, 1H), 8.86
(d, J = 2.5 Hz, 1H), 8.51 (d, J = 2.5 Hz, 1H), 8.37−8.45 (m, 2H), 7.88
(br s, 1H), 7.74 (br s, 1H), 3.93 (s, 3H), 3.09 (m, 4H), 2.86 (s, 3H),
2.51−2.58 (m, 4H), 2.44 (s, 3H), 1.37 (s, 6H).
ABBREVIATIONS USED
■
Ac, acetyl; AKT, serine−threonine kinase Akt; A-Phos, di-tert-
butyl(4-(dimethylamino)phenyl)phosphine; DIPEA, diisopro-
pylethylamine; DEA, diethylamine; DMF, N,N-dimethylforma-
mide; DMSO, dimethyl sulfoxide; EC, effective concentration;
ED, effective dose; HGF, hepatocyte growth factor; HLM,
human liver microsome; HPβCD, (hydroxypropyl)-β-cyclo-
dextrin; HPMC, hydroxypropyl methylcellulose; iv, intra-
venous; LiHMDS, lithium hexamethyldisilazide; mTOR,
mammalian target of rapamycin; MG, malignant glioma;
MSA, methanesulfonic acid; NADPH, nicotinamide adenine
dinucleotide phosphate; PD, pharmacodynamic; PIP2, phos-
phatidylinositol-4,5-bisphosphate; PIP3, phosphatidylinositol-
3,4,5-triphosphate; PI3K, phosphoinositide 3-kinase; PK,
pharmacokinetic; po, per os (orally); PTEN, phosphatase and
tensin homologue gene; RLM, rat liver microsome; rt, room
temperature; SAR, structure−activity relationship; SFC, super-
critical fluid chromatography; TEA, triethylamine; TFA,
trifluoroacetic acid; THF, tetrahydrofuran; X-Phos, 2-(dicyclo-
hexylphosphino)-2′,4′,6′-triisopropyl-1,1′-biphenyl
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ASSOCIATED CONTENT
■
S
* Supporting Information
Biological assays, in vivo study protocols, selectivity data,
MetaSite metabolite predictions for compound 1, standard
deviation information, X-ray experimental procedures, and X-
ray crystallographic data. This material is available free of
charge via the Internet at http://pubs.acs.org.
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collected at 0−4, 4−8, and 8−24 h postdose. The parent drug
concentrations were determined in each excreta from each collection
period using an LC−MS/MS assay.
(16) Compound 1 (1 μM) was incubated in rat and human liver
microsomes in potassium phosphate buffer (66.6 μM, pH 7.4) with
NADPH (1 mM) and MgCl2 (3 mM) at 37 °C for 1 h. Hepatocytes
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