Structure-based design of potent and selective 3-phosphoinositide-dependent kinase-1 (PDK1) inhibitors

Article abstract of DOI:10.1021/jm101527u

Phosphoinositide-dependent protein kinase-1(PDK1) is a master regulator of the AGC family of kinases and an integral component of the PI3K/AKT/mTOR pathway. As this pathway is among the most commonly deregulated across all cancers, a selective inhibitor of PDK1 might have utility as an anticancer agent. Herein we describe our lead optimization of compound 1 toward highly potent and selective PDK1 inhibitors via a structure-based design strategy. The most potent and selective inhibitors demonstrated submicromolar activity as measured by inhibition of phosphorylation of PDK1 substrates as well as antiproliferative activity against a subset of AML cell lines. In addition, reduction of phosphorylation of PDK1 substrates was demonstrated in vivo in mice bearing OCl-AML2 xenografts. These observations demonstrate the utility of these molecules as tools to further delineate the biology of PDK1 and the potential pharmacological uses of a PDK1 inhibitor.

Full text of DOI:10.1021/jm101527u

ARTICLE
pubs.acs.org/jmc
Structure-Based Design of Potent and Selective 3-Phosphoinositide-
Dependent Kinase-1 (PDK1) Inhibitors†
Jesꢀus R. Medina,*,‡ Christopher J. Becker,‡ Charles W. Blackledge,‡ Celine Duquenne,‡ Yanhong Feng,‡
Seth W. Grant,‡ Dirk Heerding,‡ William H. Li,‡ William H. Miller,‡ Stuart P. Romeril,‡ Daryl Scherzer,‡
Arthur Shu,‡ Mark A. Bobko,‡ Antony R. Chadderton,‡ Melissa Dumble,‡ Christine M. Gardiner,‡
Seth Gilbert,‡ Qi Liu,‡ Sridhar K. Rabindran,‡ Valery Sudakin,‡ Hong Xiang,‡ Pat G. Brady,§
Nino Campobasso,§ Paris Ward,§ and Jeffrey M. Axten‡
‡Oncology Research and §Molecular Discovery Research, GlaxoSmithKline, Collegeville, Pennsylvania 19426, United States
ABSTRACT: Phosphoinositide-dependent protein kinase-1
(PDK1) is a master regulator of the AGC family of kinases
and an integral component of the PI3K/AKT/mTOR path-
way. As this pathway is among the most commonly deregu-
lated across all cancers, a selective inhibitor of PDK1 might
have utility as an anticancer agent. Herein we describe our
lead optimization of compound 1 toward highly potent and
selective PDK1 inhibitors via a structure-based design strat-
egy. The most potent and selective inhibitors demonstrated
submicromolar activity as measured by inhibition of phos-
phorylation of PDK1 substrates as well as antiproliferative
activity against a subset of AML cell lines. In addition,
reduction of phosphorylation of PDK1 substrates was demonstrated in vivo in mice bearing OCl-AML2 xenografts. These
observations demonstrate the utility of these molecules as tools to further delineate the biology of PDK1 and the potential
pharmacological uses of a PDK1 inhibitor.
’ INTRODUCTION
Phosphoinositide-dependent protein kinase-1 (PDK1), a
master regulator of the AGC kinase signal transduction, phos-
phorylates and activates at least 23 related AGC protein kinases
that are often constitutively activated in human cancers and
enable tumorigenesis.1-4 PDK1 is a direct downstream effector
of PI3K that positively regulates the AKT pathway,5-7 result-
ing in inhibition of apoptosis, promotion of cell division, and
Figure 1. Lead compound 1.
stimulation of glucose uptake and storage. Activation of PI3K,
mediated by the interaction of insulin and growth factors with
their receptors, results in the production of phosphatidylinositol
3,4,-diphosphate (PIP2) and phosphatidylinositol 3,4,5-triphos-
phate (PIP3), which colocalize AKT and PDK1 to the plasma
membrane through interaction with their pleckstrin homology
(PH) domains, allowing PDK1 to phosphorylate the activation
loop of AKT at Thr 308 and hence initiate the activation of AKT
in a PI3K-dependent manner.1-4 Furthermore, PDK1 is an acti-
vator of additional kinases involved in tumor progression such as
protein kinase C (PKC), serum-and glucocorticoid-induced protein
kinase (SGK), p70 ribosomal S6 kinase (S6K1), and p90 ribosomal
S6 kinase (RSK).2 These kinases lack a PH domain and are
therefore not dependent on colocalization with PDK1 at the plasma
membrane. For these kinases, phophorylation at the hydrophobic
motif by distinct upstream kinases enables PDK1 to recognize and
interact with these enzymes through its PIF-pocket, a small
phosphate binding groove located in the PDK1 catalytic domain.
This interaction facilitates the T-loop phosphorylation of these
substrates, activating them in a PI3K-independent manner.1-4
Because PDK1 is involved in several distinct signaling pathways
that are important for tumor progression, inhibitors of PDK1 might
be beneficial for the treatment of cancer.8-11 As such, several classes
of small-molecule PDK1 inhibitors have been reported.12,13 Herein,
we report our structure-based optimization of compound 1, a lead
generated from screening an in-house fragment library against
PDK1 (Figure 1).14
’ CHEMISTRY
Analogues substituted at the 2-position of the pyrimidine ring
were synthesized according to Scheme 1. Suzuki coupling of
Received: November 29, 2010
Published: February 22, 2011
r
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4-chloro-2-aminopyrimidine 2 with boronic acid 3 provided
intermediate 4, which was acetylated and then methylated to
give 5. Reaction with hydrazine monohydrate in ethanol at 95 °C
both removed the acetyl group and formed the indazole ring
to afford 8a. Compounds 8b and 8c were prepared from
intermediate 7, which was synthesized by the bis-pinacolato
diboron coupling of 6, followed by Suzuki coupling with 2,4-
dichloropyrimidine. Treatment of intermediate 7 with dimethy-
lamine or ethylamine followed by removal of the acetyl group
with acidic methanol afforded 8b and 8c, respectively. Com-
pound 8d was prepared by direct treatment of intermediate 7
with acidic methanol.
Scheme 1. Synthesis of Compounds of General Structure 8a
Two general synthetic routes were employed to obtain a
series of 6-substituted pyrimidine analogues 12 as described in
Scheme 3. Synthesis of R-Alkyl Substituted Cyclic Amines 17a
a Reagents and conditions: (a) Pd(PPh3)4, 1,4-dioxane, NaHCO3(aq),
95 °C; (b) (i) Ac2O, 80 °C, (ii) CH3I, Cs2CO3, DMF, rt; (c) (i)
a Reagents and conditions: (a) (i) benzaldehyde, toluene, reflux, (ii)
NaBH4, EtOH, 0 °C; (b) (i) chloroacetyl chloride, K2CO3, THF, H2O,
0 °C, (ii) NaOH(aq) (pH > 13), rt; (c) Red-Al, toluene, 0-60 °C; (d)
H2, Pd/C, HCl, CH3OH, rt; (e) (i) NaH, toluene, 0 °C, (ii) chloroacetyl
chloride, toluene, 90-110 °C; (f) LiAlH4, THF, 70 °C.
bis(pinacolato)diboron, KOAc, PdCl2(dppf) CH2Cl2, 1,4-dioxane,
3
100 °C, (ii) 2,4-dichloropyrimidine, PdCl2(dppf) CH2Cl2, 1,4-dioxane,
3
NaHCO3(aq), 100 °C; (d) for 8b and 8c, (i) amine, THF, 100 °C, (ii)
HCl, CH3OH, 60 °C; for 8d, HCl, CH3OH, 50 °C; (e) H2NNH2 H2O,
3
EtOH, 95 °C.
Scheme 2. Synthesis of Compounds 12a
a Reagents and conditions: (a) amine, (EtOH, CH3OH or THF), 50-100 °C; (b) 3, Pd(PPh3)4, 1,4-dioxane, NaHCO3(aq), 90-100 °C;
(c) H2NNH2 H2O, EtOH, 80-100 °C; (d) bis(pinacolato)diboron, KOAc, PdCl2(dppf) CH2Cl2, 1,4-dioxane, 100 °C; (e) (i) (9k, 9l, or 9m),
3
3
PdCl2(dppf) CH2Cl2, 1,4-dioxane, NaHCO3(aq), 100 °C, (ii) HCl, CH3OH, 60 °C; (f) (i) 3, Pd(PPh3)4, 1,4-dioxane, NaHCO3(aq), 95°C, (ii) PhB(OH)2,
3
Pd(PPh3)4, 1,4-dioxane, NaHCO3(aq), 95 °C.
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Scheme 4. Synthesis of Compounds of General Structure 21a
a Reagents and conditions: (a) amine, EDC, HOBt, N-methylmorpholine, rt; (b) TFA, CH2Cl2, rt.
Scheme 5. Synthesis of Bis-substituted Piperidine Analogues 28a
a Reagents and conditions: (a) (i) HCl, PtO2, CH3OH, (ii) CbzCl, DMAP, Et3N, CH2Cl2, 15-25 °C; (b) (i) 10% Pd/C, H2 (65 psi), EtOAc,
EtOH, rt, (ii) crystallization as the tartrate salt (L-(þ)-tartaric acid, IPA, water); (c) (i) LiOH H2O, THF, water, CH3OH, rt, (ii) aniline,
3
EDC, HOBt, H€unig’s base, DMF, rt, (iii) 10% Pd/C, H2, EtOAc, EtOH, rt; (d) (i) LiOH H2O, water, rt, (ii) 27, NaHCO3, 1,4-dioxane,
3
100 °C; (e) (i) amine, HATU, H€unig’s base, CH2Cl2, 0 °C, (ii) route 1 or 2; (f) (i) 1,4-dioxane, conc HCl, 100 °C, (ii) 9a, NaHCO3, 1,4-
dioxane, water, 120 °C, (iii) 3, Pd(PPh3)4, NaHCO3, 1,4-dioxane, water, 120 °C; (g) (i) cyclohexylamine, HATU, H€unig’s base, DMF, rt, (ii)
H2NNH2, EtOH, 110 °C.
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Scheme 6. Synthesis of Bis-substituted Morpholine Analogues 34a
a Reagents and conditions: (a) (i) (R)-(-)-epichlorohydrin, LiClO4, toluene, or 1,2-dichloroethane, rt, (ii) NaOCH3 in CH3OH or NaOEt in EtOH, rt;
(b) (i) HCl, EtOH, H2, 10% Pd/C (Degussa type), rt, (ii) for 30a (9a, H€unig’s base, CH3CN, 160 °C (MW)), for 30b (27, K2CO3, EtOH, reflux);
(c) (i) 10% Pd/C, H2, CH3OH, rt, (ii) Boc2O, H€unig’s base, THF, 40 °C, (iii) TEMPO, iodobenzene diacetate, CH2Cl2, 0 °C-rt; (d) (i) 3, 1,4-dioxane,
NaHCO3(aq), Pd(PPh3)4, 100 °C, (ii) for 31a (H5IO6/CrO3, wet CH3CN, 0-15 °C), for 31b (TEMPO, iodobenzene diacetate, CH2Cl2, water,
0 °C-rt); (e) (i) EDC, HOAt, amine, CH2Cl2, rt, (ii) HCl, dioxane, rt; (f) (i) aniline, EDC, HOBT, DMF, rt, (ii) H2NNH2 H2O, EtOH, 100 °C; (g)
3
(i) cyclohexylamine, HATU, H€unig’s base, DMF, rt, (ii) H2NNH2 H2O, 1,4-dioxane, 100 °C.
3
Scheme 7. Synthesis of Compounds 37a
a Reagents and conditions: (a) (i) LiOH H2O, THF, water, CH3OH, rt, (ii) DPPA, t-butyl alcohol, Et3N, 100 °C, (iii) Pd/C, H2(50 psi), EtOAc, rt;
3
(b) (i) conc HCl, 0 °C-rt, (ii) benzoyl chloride, NaHCO3, THF, water, 0 °C-rt, (iii) HCl, CH3OH, 65 °C.
Scheme 2. Route 1 involved the preparation of fluoronitrile
intermediate 10 prior to the final cyclization, leading to the desired
indazole analogues 12. 6-Substituted aminopyrimidine compounds
9 were either commercially available or prepared by reaction of
dichloropyrimidine 9a with the corresponding nucleophile. Suzuki
coupling of 9 with boronic acid 3 or the corresponding pinacol ester
followed by hydrazine cyclization afforded compounds 12b, 12e-j,
and 12l-m. Compound 12c was prepared by sequential Suzuki
coupling of dichloropyrimidine 9a with boronic acid 3 and then
phenyl boronic acid, followed by hydrazine cyclization. Alternatively
(route 2), intermediates 9k-m were coupled with indazole boronic
ester 11 under Suzuki conditions followed by acetyl group depro-
tection to provide the corresponding indazole analogues 12a, 12d,
and 12k.
Representative R-alkyl substituted cyclic amines were pre-
pared as described in Scheme 3. The R-methylmorpholine amine
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Scheme 8. Synthesis of Analogues 43a
a Reagents and conditions: (a) Boc2O, Et3N, CH3OH, rt; (b) (i) LiHMDS, THF, -78 °C, (ii) CH3I, -78 °C; (c) CaCl2, NaBH4, EtOH:THF (1:1),
0 °C; (d) MsCl, Et3N, CH2Cl2, 0 °C; (e) benzylamine, 70 °C; (f) (i) NaH, DMF, rt, (ii) CH3I, rt; (g) Pd/C (Degussa type), H2, EtOH, rt; (h) (i) 9a,
Et3N, EtOH, 100 °C, (ii) TFA, CH2Cl2, rt, (iii) 3,3-dimethylbutanoyl chloride, H€unig’s base, CH2Cl2, rt.
individual enantiomers 17a were prepared from the correspond-
ing optically pure 2-amino-1-propanols by reductive amination
with benzaldehyde followed by cyclization with chloroacetyl
chloride to provide the enantiomerically pure morpholinone
intermediates 14. Reduction with Red-Al provided the benzyl
protected morpholines 15, which upon debenzylation under
hydrogenolysis conditions afforded the desired R-methylmor-
pholines 17a. The R-ethyl and R-isopropyl morpholine amines
((R)-17b and (R)-17c, respectively) were prepared by direct
cyclization of the corresponding aminoalcohol with chloroacetyl
chloride to provide morpholinones 16a and 16b, respectively,
followed by reduction with LiAlH4. These amines were then used
to prepare analogues 18a-c (Table 3) following the synthetic
routes described in Scheme 2.
Compounds 21 were synthesized from their corresponding
amines 20 following either synthetic routes 1 or 2 described in
Scheme 2. Amines 20 were readily prepared from the Boc
protected 3-piperidine or 2-morpholineamine carboxylic acids
19 by coupling with the corresponding amines followed by Boc
deprotection (Scheme 4).
Several synthetic routes were used to prepare bis-substituted
analogues of general structure 28 (Scheme 5). Hydrogenation of
methyl 6-methylnicotinate using PtO2 as catalyst,15 followed by
Cbz protection of the resulting secondary amine, afforded a 3/1
ratio of cis- and trans-2,5-bis-substituted piperidine isomers 22,
which were separated by flash chromatography. Chiral prepara-
tive HPLC separation afforded individual optically pure cis
isomers (3S,6R)-22 and (3R,6S)-22.16 Compound 23 was
obtained after removal of the Cbz protecting group of (()-cis-22
and crystallization of the resulting secondary amine as the L-(þ)-
tartrate salt. Hydrolysis of esters (3S,6R)-22, (3R,6S)-22, and
(()-trans-22, and subsequent coupling of the resulting acid with
aniline followed by Cbz deprotection, provided the correspond-
ing amines 24. Compounds 28a were prepared from amines 24
following synthetic route 1 described in Scheme 2. Analogues
(3S,6R)-28b and (3S,6R)-28d were prepared from 23. Saponi-
fication of 23, followed by reaction of the resulting piperidine
acid derivative with 27, gave 25. Reaction of compound 25 with
the corresponding amines provided the amides, which were
converted to compounds (3S,6R)-28b and (3S,6R)-28d follow-
ing the synthetic routes described in Scheme 2. Compound
(3S,6R)-28c was prepared from intermediate 26, which was
prepared from simultaneous ester hydrolysis and Cbz deprotec-
tion of (3S,6R)-22 under acidic conditions, followed by reaction
of the resulting piperidine acid derivative with pyrimidine 9a and
in situ Suzuki cross-coupling with boronic acid 3. Reaction of
intermediate 26 with cyclohexylamine, followed by cyclization
with hydrazine, provided compound (3S,6R)-28c.
The syntheses of the morpholine analogues 34 are illustrated
in Scheme 6. Bis-substituted morpholines 29 were prepared
according to the reported protocol for similar compounds.17
Removal of the benzyl protecting group in 29a, followed by
reaction with dichloroaminopyrimidines 9a and 27, afforded
intermediates 30a and 30b, respectively. Suzuki coupling of 30
with boronic acid 3, followed by oxidation18 of the primary
alcohol to the carboxylic acid, provided intermediates 31. Reac-
tion of intermediates 31 with an amine, followed by hydrazine
cyclization, furnished the desired analogues 34a and 34c. Alter-
natively, replacement of the benzyl protecting group in 29b with
Boc, followed by oxidation of the primary alcohol, provided
carboxylic acid 32. Compound 32 was then converted to the
amines 33 by reaction with the corresponding amine followed by
deprotection of the Boc group. Final compounds 34b and 34d
were synthesized from amines 33 by following synthetic route 1
described in Scheme 2.
Compounds 37b and 37c were prepared from amines 36
and 35, respectively, following the general procedures
(Scheme 7). While amine 36 was commercially available,
amine 35 was prepared from compound 22 by ester hydro-
lysis, followed by Curtius rearrangement and subsequent
Cbz deprotection. Compound (()-37a was synthesized from
(()-37b.
A series of enantiomerically pure 5-methyl-3-aminopiperidine
compounds of general structure 43 was prepared according to
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Scheme 8. Boc protection of dimethyl D-glutamate, followed
by stereoselective R-methylation, provided compound 38.19 Re-
duction of the esters, followed by mesylation of the resulting diol
39 and subsequent treatment with neat benzylamine at 70 °C,
provided piperidine 40a with the desired regio- and stereochem-
istry. Methylation of the BocNH functionality on 40a provided
compound 40b. Debenzylation of compounds 40 afforded
amines 41, which were converted to compounds 43a-b by
following the general synthetic route 1. Additionally, 41b was
converted to 42 by reaction with 9a, Boc deprotection, and amide
formation, which was then elaborated to 43c.
’ RESULTS AND DISCUSSION
An X-ray crystal structure of compound 1 bound to PDK1
(Figure 2a) shows key hydrogen bonding interactions between
the aminoindazole and Ser160 and Ala162 in the hinge region.
Preliminary SAR at the 3-position of the indazole ring confirmed
the importance of the amino group as a hydrogen bond donor for
optimum binding (data not shown). In addition, the aminopyr-
imidine ring is engaged in a tight hydrogen bond network with
the protein, with the pyrimidine ring nitrogens acting as accep-
tors for the catalytic Lys111 and Thr222 at the floor of the
binding pocket. The amino group of the pyrimidine is a hydrogen
bond donor to the catalytic residue Glu130. In agreement with
the X-ray structural information, we previously demonstrated by
SAR the importance that each nitrogen functionality of the
aminopyrimidine contributes to PDK1 binding and inhibition
of kinase activity.14 Further inspection of the X-ray crystal
structure revealed a small lipophilic pocket in the region
occupied by the exocyclic amino group of the pyrimidine ring
and defined by amino acid residues Met134, Val143, and Leu159.
To further understand the SAR in this region, we varied
substituents at the 2-position of the pyrimidine ring (Table 1).
Consistent with the X-ray crystal structure, replacement of one
NH2 hydrogen with CH3 (8a) was tolerated, causing only a
minor reduction (0.4 log units) in enzyme potency relative to 1,
but increasing the size of the alkyl group to ethyl (8c) resulted in
a significant reduction in potency. In addition, substituting both
hydrogens with CH3 (8b) or replacing the pyrimidine NH2 with
a methoxy group (8d) resulted in a significant reduction in
potency, confirming the importance of a hydrogen bond donor at
this position for favorable contact with Glu 130. This excellent
agreement between the X-ray structural information and pre-
liminary SAR provided a basis for further structure-based opti-
mization of PDK1 activity.
Figure 2. X-ray crystal structure of compound 1 bound to PDK1 (PDB
code 3NUN). (a) Hydrogen bond distances in Å. (b) Surface of G-loop
illustrating small hydrophobic pocket.
Table 1. SAR of the Hydrogen Bond Substitution at the
Pyrimidine 2-Position
Further examination of the crystal structure suggested that
substitution at the pyrimidine 6-position could fill the lipophilic
pocket under the G-loop (Figure 2b). Substitution with either
alkyl, aryl, alkoxy, thiolate, alkylamine, or arylamine was tolerated
and, in many instances, improved PDK1 potency (Table 2).
Additionally, many of these substituents conferred good-to-
excellent selectivity over other kinases such as Aurora A, Aurora
B, ALK5, and ROCK1. These kinases were chosen as represen-
tative specificity targets based on profiling of 1.14 Compound
12a, with a 6-methyl group, exhibited a 0.5 log increase in
potency and maintained ligand efficiency (LE)20,21 relative to
compound 1. Increasing the size of the alkyl substituent to i-Pr
(12b) retained potency relative to 12a with a concomitant
decrease in LE. The methoxy compound 12d showed similar
potency to 12b, whereas methylamine substitution (12e) in-
creased potency and LE. Interestingly, although dimethylamine
compd
R
PDK1 pIC50
LE
1
NH2
6.4
6.0
4.7
5.1
4.9
0.52
0.46
0.34
0.37
0.37
8a
8b
8c
8d
NHCH3
N(CH3)2
NHCH2CH3
OCH3
substitution (12j) did not result in an increase in potency relative
to 1, it significantly increased selectivity over other kinases such
as ALK5, Aurora A, Aurora B, and ROCK1. These observations
proved to be quite general, with monosubstituted amines
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Table 2. Selected SAR of 6-Substituted Pyrimidines
providing a higher level of potency and LE (12e-i) and tertiary
amines (12j-m) conferring a higher level of kinase selectivity.
The X-ray crystal structure of 12k bound to PDK1 (Figure 3a)
showed that the morpholine group was easily accommodated by
PDK1 and situated under the G-loop. Interestingly, the crystal
structure of the 6-ethylamino derivative 12f with PDK1
(Figure 3b) revealed that the ethyl group is oriented toward
the G-loop and is accommodated by a small lipophilic pocket. An
overlay of 12f and 12k (Figure 3c) suggested that an axial alkyl
group R to the amine on a cyclic amine could also fill this pocket.
This substitution had potential to provide the higher potency
observed for monoalkylamines as well as the high degree of
kinase selectivity associated with tertiary amine substitution. We
surmised that the axial conformation would be preferred based
on allylic strain principles.22
As predicted from the X-ray crystal structure overlay of 12f
and 12k, methyl substitution at the R-position of the morpholine
ring ((R)-18a) increased enzyme potency (∼0.5 log units)
and LE relative to unsubstituted morpholine 12k and also
maintained an excellent level of kinase selectivity (Table 3).
The R-methyl substituted piperidine analogue 18d exhibited
similar potency to (R)-18a. The predicted binding mode was
confirmed by a crystal structure of (R)-18a bound to PDK1
(Figure 4). Consistent with the structural data, the enantiomer
(S)-18a was less active than the unsubstituted morpholine
derivative 12k. Additionally, while ethyl substitution at the R-
position of the morpholine ring increased enzyme potency
(18b) relative to 12k, increasing the size further to isopropyl
(18c) had only a marginal effect.
with another lipophilic pocket along the G-loop could be
accessed through carboxamide substitution at the β-position of
either a morpholine or piperidine ring (Figure 5a). Both optically
pure N-phenylcarboxamide substituted morpholine (S)-21a
and piperidine (S)-21b analogues showed a significant in-
crease in potency (∼10-fold) relative to their unsubstituted
counterparts 12k and 12m, with good kinase selectivity for
PDK1 (Table 4). The binding mode was confirmed by a crystal
structure of (S)-21a bound to PDK1 (Figure 5b), wherein
interactions extending beyond the morpholine ring under the
G-loop are observed. The carboxamide offers good contacts
for H-bonding with a water molecule and serves as a linker for
the phenyl ring to extend into the lipophilic pocket. Consistent
with the binding mode, the corresponding enantiomers (R)-
21a and (R)-21b were less potent. Cyclohexylcarboxamide
substituted compound (S)-21c exhibited comparable potency
to (S)-21b. Cyclopentylcarboxamide (S)-21d showed a slight
reduction in potency, while compounds containing smaller
alkylcarboxamide substituents were significantly less active
(data not shown).
Examination of the X-ray crystal structures of (R)-18a and
(S)-21a revealed that the morpholine ring adopts the same
conformation with either the β-carboxamide or the R-alkyl
substituent. Therefore, we hypothesized that incorporating both
groups on a cis-disubstituted six-membered ring scaffold would
be beneficial. This requires that the carboxamide occupies an
equatorial position while the alkyl substituent occupies an axial
position, filling the lipophilic G-loop pockets. Indeed, the com-
bination of substituted carboxamides with alkyl substituents
provided an additive effect (Table 5). For instance, the bis-sub-
stituted piperidine (3S,6R)-28a and morpholine 34a analogues
Further examination of the X-ray crystal structure of 12k
bound to PDK1 suggested that additional favorable interactions
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dependent and independent pathways. PDK1-mediated phosphor-
ylation of Thr308-AKT (PI3K-dependent) and Ser221-RSK (PI3K-
independent) were evaluated as well as the PDK1-independent
phosphorylation state of Ser473-AKT. Consistent with potent and
selective inhibition of PDK1, compounds (3S,6R)-28a and 34a
exhibited submicromolar inhibition of the phosphorylation of both
Thr308-AKT and Ser221-RSK and did not affect the phosphor-
ylation of Ser473-AKT (IC50 > 29300 nM). Interestingly, methyl
substitution on the pyrimidine 2-amino group provided
compounds with increased cellular potency and kinase selecti-
vity ((3S,6R)-28b vs (3S,6R)-28a and (3S,6R)-28d (GSK-
2334470)23 vs (3S,6R)-28c). A similar trend was observed for
34b-d.
As part of our optimization process, we also investigated func-
tionalities at the β-position to the nitrogen of the six-membered
ring other than carboxamide. The activities of selected represen-
tatives are depicted in Table 6. The reverse amide (()-37a and
the carbamate (()-37b exhibited similar potency to phenylcar-
boxamide (S)-21b. Although the expected increase in potency
when adding the R-methyl group was observed ((()-cis-37c), it
was not as pronounced as in the carboxamide series. Surpris-
ingly, an X-ray crystal structure of (()-cis-37c with PDK1
(Figure 7) revealed that the opposite antipode occupied the
active site (compared to the carboxamide series). To accom-
modate this stereoisomer, the piperidine adopted the alternate
chair conformation, which places the methyl group away from
the pocket in the G-loop. Consistent with the X-ray results,
(3R,6S)-37c exhibited approximately 100-fold higher potency
relative to its enantiomer (3S,6R)-37c, although it showed only a
modest increase in potency (∼0.5 log units) relative to its
corresponding nonmethylated analogue (R)-37b.
Analysis of the X-ray crystal structure of (3R,6S)-37c in PDK1
suggested that moving the methyl group to the β-position of the
piperidine ring with a trans relationship to the carbamate func-
tionality could potentially fill the lipophilic pocket under the G-loop.
Thus a series of piperidine compounds of general structure 43
was investigated (Table 7). Indeed, compound 43a exhibited
high potency in the PDK1 enzyme assay as well as excellent
potency in the mechanistic cellular assays. The binding mode was
confirmed by an X-ray crystal structure of 43a bound to PDK1
(Figure 8). The β-methyl substituent points toward the lipophilic
pocket under the G-loop, and a hydrogen bond network invol-
ving the carbamate carbonyl group and both the catalytic lysine
111 and serine 94 is observed. The N-Me carbamate 43b
exhibited higher potency in the PDK1 enzyme assay (∼10-fold),
and the cellular mechanistic assay (∼5-fold), relative to the NH
carbamate 43a. In addition, replacement of the oxygen atom in
carbamate 43b with carbon afforded amide 43c, which also
exhibited a similar potency profile in the PDK1 enzyme and
mechanistic assays.
Selected compounds with potent mechanistic cellular activity
were tested for antiproliferative activity against a panel of leukemia
cell lines. We found that AML cell lines were more sensitive to
PDK1 inhibition (Table 8), which is consistent with published data
in which overexpression of PDK1 was found to be a common
feature of acute myeloid leukemia (45% of patients), promoting
monocyte colony formation and translocation of PKC.24 However,
we found that AML cell lines were not uniformly inhibited and only
a subset of AML cell lines was sensitive to growth inhibition by our
PDK1 inhibitors (data not shown).25
Figure 3. X-ray crystal structures and overlay of compounds 12k and
12f bound to PDK1.
showed an approximately 100-fold increase in potency relative to
the corresponding unsubstituted amines 12m and 12k, respec-
tively. The X-ray crystal structure of (3S,6R)-28a with PDK1
(Figure 6) confirmed the expected binding mode, where the
R-methyl substituent occupies a small pocket under the G-loop
and the phenyl carboxamide substituent extends beyond the
piperidine ring under the G-loop. Consistent with this binding
mode, the corresponding enantiomer (3R,6S)-28a and its race-
mic trans-stereoisomer (()-trans-28a showed >100-fold lower
potency.
Having achieved high enzyme potency and selectivity, we
evaluated several compounds in a mechanistic cellular assay in
PC-3 cells to determine the effect of inhibiting PDK1 on PI3-kinase
Compound (3S,6R)-28d was further evaluated through an in
vitro kinase selectivity panel of 285 protein and lipid kinases.26
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Table 3. SAR Results of r-Alkyl Substituted Cyclic Amines
Table 4. SAR of Carboxamide Substitution on Morpholine
and Piperidine Analogues
Figure 4. X-ray crystal structure of compound (R)-18a bound to
PDK1.
Figure 5. X-ray crystal structures of compounds 12k and (S)-21a
bound to PDK1.
Only 24 kinases were inhibited >50% in the presence of 10 μM
(3S,6R)-28d (Table 9). IC50 values available from our internal
kinase panel for a subset of kinases including members from the
AGC family are shown in Table 10. (3S,6R)-28d exhibited
>1000-fold selectivity against each of these kinases compared
to potency against PDK1.
The ability of (3S,6R)-28d to inhibit PDK1 signaling in
vivo was evaluated using SCID mice bearing OCI-AML2
xenografts. At its MTD dose (100 mg/kg, ip, single dose),
Cmax = 4510 ng/mL), (3S,6R)-28d exhibited 58% and 29%
reduction of AKTT308 phosphorylation at 3 and 6 h, respec-
tively, and 57% and 71% reduction of RSKS221 phosphoryla-
tion at 3 and 6 h, respectively (Figure 9). Consistent with the
in vitro findings, we observed no inhibition of AKTS473
phosphorylation.
which afforded high exposure (AUC = 31362 ng h/mL;
3
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Table 5. SAR of Bis-substituted Morpholine and Piperidine Analogues
a ND = not determined.
’ CONCLUSION
measured by inhibition of phosphorylation of PDK1 substrates
as well as antiproliferative activity against a subset of AML cell
lines. Experiments with compound (3S,6R)-28d did confirm the
ability of a PDK1 inhibitor to modulate downstream signaling in
vivo. However, preliminary results from an efficacy study (SCID
mice bearing OCI-AML2 tumors) indicated that compound
(3S,6R)-28d only modestly inhibits tumor growth inhibition in
vivo (data not shown). Taken together, these results advise that
further biology studies will be necessary to determine the utility
of a PDK1 inhibitor in the treatment of cancer either as a single
agent or in combination therapies. The discovery of potent and
highly selective PDK1 inhibitors, exemplified by (3S,6R)-28d,
permits further delineation of PDK1 mediated signal transduc-
tion and potential pharmacological uses of a PDK1 inhibitor.23,28
The optimization of compound 1 into highly potent and
selective PDK1 inhibitors was accomplished via a structure-based
design strategy targeting specific binding opportunities pre-
sented by the PDK1 G-loop to increase potency and selectivity.
Interestingly, the majority of kinase inhibitors do not exploit the
potential interactions presented by the G-loop, probably due to
its typical high flexibility, which could pose a challenge for
achieving high ligand binding affinity.27 However, X-ray crystal-
lography revealed distinct pockets in the PDK1 G-loop, which
enabled optimization of the morpholine and piperidine deriva-
tives through proper introduction of small alkyl groups on the
six-membered ring. The most potent and selective inhibitors
demonstrated submicromolar mechanistic cellular activity as
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Figure 6. X-ray crystal structure of compound (3S,6R)-28a bound
to PDK1.
Table 6. Selected SAR Data for Aminopiperidine Derivatives
of General Structure 37
Figure 7. X-ray crystal structure of compound (3R,6S)-37c bound to
PDK1.
0.1% TFA in each solvent). The products were detected by UV (254,
214, and 333 nm). All tested compounds were determined to be g95%
purity by LC-MS and analytical HPLC unless otherwise noted.
4-(2-Amino-4-pyrimidinyl)-2-fluorobenzonitrile (4). In a 25 mL sealed
tube under argon were combined 4-chloro-2-pyrimidinamine (100 mg,
0.772 mmol) and (4-cyano-3-fluorophenyl)boronic acid (127 mg, 0.772
mmol) in 1,4-dioxane (5 mL) and saturated aqueous NaHCO3 (1.25 mL).
This mixture was degassed for 10 min with argon. Pd(PPh3)4 (50 mg, 0.04
mmol) was then added, and the reaction sealed and heated at 95 °C in an oil
bath. After 3 h, the reaction was allowed to cool to room temperature and
then poured onto water (50 mL) and EtOAc (50 mL). The organic layer
was separated, dried (MgSO4), filtered through a pad of Celite503, and
concentrated to dryness. The resulting light-yellow solid was dissolved in
CHCl3 and purified by flash chromatography on SiO2 (eluent: 90/10/1
CHCl3/CH3OH/NH4OH) to afford the title compound (138 mg, 83%).
LC-MS (ES) m/z = 215 [M þ H]þ. 1H NMR (400 MHz, DMSO-d6):
δ 6.88 (s, 2H), 7.29 (d, J = 5.1 Hz, 1H), 8.04-8.22 (m, 3H), 8.43 (d, J =
5.1 Hz, 1H).
N-[4-(4-Cyano-3-fluorophenyl)-2-pyrimidinyl]-N-methylacetamide
(5). To 4-(2-amino-4-pyrimidinyl)-2-fluorobenzonitrile (230 mg, 1.07
mmol) was added acetic anhydride (10 mL), and the reaction mixture
was stirred overnight at 80 °C. LCMS analysis showed a ∼2:1 mixture of
mono- and diacetate products. The solvent was partially removed under
vacuum. Ethanol was added, and the solvents were evaporated. This
process was repeated 2 more times until most of the acetic anhydride and
acetic acid was removed. Methanol was added, and an insoluble white
solid was observed which was immediately filtered to afford N-[4-(4-
cyano-3-fluorophenyl)-2-pyrimidinyl]acetamide (145 mg, 53%) as a
white solid. LC-MS (ES) m/z = 279 [M þ Na]þ. 1H NMR (400 MHz,
DMSO-d6): δ 2.26 (s, 3H), 7.90 (d, J = 5.3 Hz, 1H), 8.12-8.19 (m, 1H),
8.22-8.28 (m, 1H), 8.28-8.34 (m, 1H), 8.83 (d, J = 5.1 Hz, 1H), 10.72
(s, 1H).
’ EXPERIMENTAL SECTION
Chemistry. General Methods. Unless otherwise noted, commer-
cially available materials were used without further purification. Air- or
moisture-sensitive reactions were carried out under a nitrogen or argon
atmosphere. Anhydrous solvents were obtained from Sigma-Adrich.
Flash chromatography was performed using silica gel under standard
techniques or using silica gel cartridges on an Analogix instrument.
NMR spectra were recorded on a Bruker 400 MHz spectrometer.
Chemical shifts (δ) are quoted in parts per million (ppm) relative to
an internal solvent referance. Coupling constants (J) are recorded in
hertz. LC-MS data were collected from a Sciex or Agilent instrument.
Reverse phase HPLC purifications were conducted on a Gilson HPLC
(monitoring at 215 and 254 nm) with a C18 column eluting with an
acetonitrile/water gradient with 0.1% TFA in each solvent unless
otherwise noted. Analytical HPLC data were generated by injecting
5 μL of very dilute sample solution in the appropriate solvent to a reverse
phase HPLC system run over 4 min (5-95% acetonitrile/water with
To N-[4-(4-cyano-3-fluorophenyl)-2-pyrimidinyl]acetamide (140 mg,
0.546 mmol) and Cs2CO3 (196 mg, 0.60 mmol) in DMF (5 mL) was
added iodomethane (0.038 mL, 0.60 mmol), and the reaction mixture
was stirred for 3 h at room temperature. Water was added (ca. 20 mL),
and a white precipitate was formed. The mixture was filtered, and the
solid was washed with more water. The solid was dissolved in EtOAc,
and the solution was dried (MgSO4), filtered, and concentrated to afford
the title compound (135 mg, 91%) as an off-white solid. LC-MS (ES)
1
m/z = 293 [M þ Na]þ. H NMR (400 MHz, DMSO-d6): δ 2.45 (s,
3H), 3.44 (s, 3H), 8.02 (d, J = 5.3 Hz, 1H), 8.15 (dd, J = 8.1, 6.8 Hz, 1H),
8.26 (dd, J = 8.1, 1.5 Hz, 1H), 8.33 (dd, J = 10.7, 1.4 Hz, 1H), 8.95 (d, J =
5.3 Hz, 1H).
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Table 7. SAR of 3-Methyl-5-aminopiperidine Analogues of General Structure 43
6-[2-(Methylamino)-4-pyrimidinyl]-1H-indazol-3-amine (8a). To
N-[4-(4-cyano-3-fluorophenyl)-2-pyrimidinyl]-N-methylacetamide
(135 mg, 0.500 mmol) in ethanol (8 mL) was added hydrazine mono-
hydrate (2 mL, 41 mmol), and the reaction mixture was stirred overnight
at 95 °C. The solution was poured onto water. A precipitate started to
form. The mixture was filtered, and the solid was washed with water
followed by Et2O. The residual solvent was removed under vacuum to
afford the title compound (85 mg, 71%) as a light-yellow solid. LC-MS
(ES) m/z = 241 [M þ H]þ. 1H NMR (400 MHz, DMSO-d6): δ 2.89 (d,
3H), 5.43 (s, 2H), 7.05-7.14 (m, 1H), 7.16 (d, J = 5.3 Hz, 1H), 7.62 (d,
J = 8.1 Hz, 1H), 7.77 (d, J = 8.3 Hz, 1H), 8.02 (br s, 1H), 8.34 (d, J =
4.8 Hz, 1H), 11.61 (s, 1H).
N-Acetyl-N-(1-acetyl-6-bromo-1H-indazol-3-yl)acetamide (6). In
a 100 mL flask under argon were combined 6-bromo-1H-indazol-3-amine
(2.47 g, 11.7 mmol), acetic anhydride (22 mL, 233 mmol), and DMAP
(0.071 g, 0.58 mmol), and the resulting mixture was heated at 120 °C for
5 h. The reaction was allowed to cool to room temperature, stirred over-
Figure 8. X-ray crystal structure of carbamate 43a bound to PDK1.
night, and then concentrated to dryness. The resulting residue was dry
loaded onto SiO2 using acetone and purified by flash chromatography
on SiO2 using EtOAc/Hex as the eluent to afford the title compound
(2.84 g, 68%) as a white solid. LC-MS (ES) m/z = 360, 362 [M þ Na]þ.
1H NMR (400 MHz, DMSO-d6): δ 2.31 (s, 6H), 2.70 (s, 3H), 7.67 (dd,
J = 8.6, 1.8 Hz, 1H), 7.83 (d, J = 8.3 Hz, 1H), 8.53 (d, J = 1.5 Hz, 1H).
N-[6-(2-Chloro-4-pyrimidinyl)-1H-indazol-3-yl]acetamide (7). To
a mixture of N-acetyl-N-(1-acetyl-6-bromo-1H-indazol-3-yl)acetamide
(0.439 g, 1.299 mmol), N-(1-acetyl-6-bromo-1H-indazol-3-yl)acetamide
(0.063 g, 0.211 mmol), bis(pinacolato)diboron (0.384 g, 1.51 mmol),
and KOAc (0.445 g, 4.53 mmol) was added 1,4-dioxane (8 mL), and
nitrogen gas was bubbled through the mixture for 10 min. PdCl2-
[M þ H]þ. 1H NMR (400 MHz, DMSO-d6): δ 2.13 (s, 3H), 7.85 (d,
J = 8.8 Hz, 1H), 7.97 (d, J = 8.6 Hz, 1H), 8.25 (d, J = 5.3 Hz, 1H), 8.28-
8.36 (m, 1H), 8.84 (d, J = 5.3 Hz, 1H), 10.51 (s, 1H), 13.00 (br. s., 1H).
6-[2-(Dimethylamino)-4-pyrimidinyl]-1H-indazol-3-amine(8b). To
N-[6-(2-chloro-4-pyrimidinyl)-1H-indazol-3-yl]acetamide (20 mg, 0.070
mmol) was added dimethylamine (2.0 M in THF, 4 mL, 8.00 mmol), and
the reaction mixture was stirred overnight at 100 °C. The mixture was
poured into EtOAc and water, and the organic layer was separated,
washed with brine, dried (MgSO4), filtered, and concentrated. To the
resulting residue was added CH3OH (6 mL), followed by concentrated
HCl (0.3 mL, 3.60 mmol), and the reaction mixture was stirred for 3 h at
60 °C. The solution was concentrated and evaporated from toluene twice.
The resulting residue was triturated with Et2O to afford an HCl salt of the
desired product (20 mg, 88%) as a yellow solid. LC-MS (ES) m/z = 255
[M þ H]þ. 1H NMR (400 MHz, CD3OD): δ 3.39-3.56 (m, 6H), 7.68
(d, J = 6.8 Hz, 1H), 8.15 (s, 2H), 8.41-8.47 (m, 2H).
(dppf) CH2Cl2 (0.062 g, 0.076 mmol) was added, and the reaction
3
mixture was stirred for 2 h at 100 °C in a sealed tube. The reaction
mixture was cooled to room temperature and treated with 2,4-dichlor-
opyrimidine (0.225 g, 1.510 mmol) and saturated aqueous NaHCO3
(3 mL). Nitrogen gas was bubbled through the mixture for 10 min.
PdCl2(dppf) CH2Cl2 (0.062 g, 0.076 mmol) was added, and the
3
6-[2-(Ethylamino)-4-pyrimidinyl]-1H-indazol-3-amine (8c). The title
compound was prepared following synthetic procedures similar to the
preparation of 8b. LC-MS (ES) m/z= 255 [M þ H]þ.1HNMR(400MHz,
CD3OD): δ 1.40 (t, J = 7.2 Hz, 3H), 3.55-3.85 (m, 2H), 7.69 (d, J =
6.6 Hz, 1H), 8.09-8.23 (m, 2H), 8.43 (d, J = 6.6 Hz, 1H), 8.45 (s, 1H).
6-[2-(Methyloxy)-4-pyrimidinyl]-1H-indazol-3-amine (8d). To N-[6-
(2-chloro-4-pyrimidinyl)-1H-indazol-3-yl]acetamide (8.5 mg, 0.030 mmol)
reaction mixture was stirred overnight at 100 °C in a sealed tube. The
mixture was cooled to room temperature and poured into EtOAc and
water and then filtered. The filtrate was poured into a separatory funnel,
and the organic layer was separated, washed with brine, dried (MgSO4),
filtered, and concentrated. Flash chromatography on SiO2 (gradient:
CHCl3 to 90:10:1 CHCl3/CH3OH/NH4OH) afforded the title com-
pound (84 mg, 19%) as a yellow solid. LC-MS (ES) m/z = 288
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Table 8. Antiproliferative Activity of Selected PDK1 Inhibitors against a Panel of AML Cell Lines
cell IC50 (μM)
K562
OCI-AML2
OCI-AML3
HEL 92.1.7
F-36P
compd
(chronic myelogenous leukemia)
(acute myeloid leukemia)
(acute myeloid leukemia)
(erythroleukemia)
(acute myeloid leukemia)
(3S,6R)-28b
(3S,6R)-28d
34c
8
18
0.58
0.35
0.46
0.89
0.69
0.38
0.65
0.52
0.69
0.76
0.40
0.24
12
>30
19
0.46
0.28
0.39
0.46
0.82
0.19
>30
>30
24
34d
16
43a
18
43b
17
18
Table 9. Kinase Selectivity Data for (3S,6R)-28d Screened at
10 μMa
Table 10. Inhibitory Activity of (3S,6R)-28d against Other
Kinases
kinase
%RA
kinase
%RA
kinase
IC50 (nM)
SGK2(h)
PDK1(h)
PrKX(h)
Rsk4(h)
-2
0
Rsk2(h)
28
29
29
33
34
38
43
44
46
46
46
49
PDK1
2.5
>10000
>10000
>27542
39810
CHK2(h)
PKCζ(h)
AKT1
4
ALK5
12
13
14
16
17
21
22
23
26
BrSK1(h)
Rsk1(r)
ASK1
SGK(h)
Aurora A
Aurora B
EGFR
GSK3b
IKK1
Rsk3(h)
NLK(h)
3162
ARK5(h)
ROCK-II(r)
ZIPK(h)
PKBγ(h)
DRAK1(h)
ROCK-II(h)
Met(D1246H)(h)
BrSK2(h)
Aurora-B(h)
Ret(h)
>10000
>25118
>25118
25118
PI3Kγ
ROCK1
SYK
Rsk1(h)
7943
Met(Y1248H)(h)
>25118
>10000
a h = human; r = rat; RA = remaining activity.
VEGFR2
in CH3OH (4 mL) was added concentrated HCl (0.2 mL, 2.400 mmol),
and the reaction mixture was stirred overnight in a sealed tube at 50 °C. The
solution was poured into saturated aqueous NaHCO3 and EtOAc. The
organic layer was separated, washed with brine, dried (MgSO4), filtered, and
concentrated to afford the crude desired product (6.5 mg) as a yellow solid.
LC-MS (ES) m/z = 242 [M þ H]þ. 1H NMR (400 MHz, CD3OD): δ
4.13 (s, 3H), 7.65 (d, J = 5.3 Hz, 1H), 7.74-7.89 (m, 2H), 8.18 (s, 1H),
8.59 (d, J = 5.3 Hz, 1H).
water (0.5 mL) were added, and the solution was purified by reverse-
phase HPLC (CH3CN/H2O w/0.1%TFA). The fractions containing
the desired product were combined and concentrated until most of the
CH3CN was removed (∼half the original volume). The resulting
precipitate was filtered, washed with water, and dried under vacuum
to afford a TFA salt of the title compound (164 mg) as a white solid. LC-
1
MS (ES) m/z = 258 [M þ H]þ. H NMR (400 MHz, DMSO-d6):
δ 1.17 (t, J = 7.2 Hz, 3H), 3.37-3.50 (m, 2H), 6.38 (bs, 1H), 7.77
(bs, 1H), 7.94 (bs, 1H), 8.13-8.25 (m, 1H), 8.73 (bs, 1H).
6-Chloro-N4-ethyl-2,4-pyrimidinediamine (9d). In a 50 mL flask
under argon were combined 4,6-dichloro-2-pyrimidinamine (0.50 g,
3.05 mmol) and ethylamine (2.0 M in CH3OH, 15.24 mL, 30.5 mmol), and
the reaction mixture was stirred at 50 °C for 3 h. The mixture was cooled
to room temperature and concentrated. The resulting yellow solid was
dissolved in EtOAc, and the solution was washed with water. The organic
layer was dried (MgSO4), filtered, and concentrated to afford the title
compound (368 mg, 69%) as a yellow solid. LC-MS (ES) m/z = 173 [M þ
H]þ. 1H NMR (400 MHz, DMSO-d6): δ 1.08 (t, J = 7.2 Hz, 3H), 3.11-
3.30 (m, 2H), 5.70 (s, 1H), 6.37 (bs, 2H), 7.08 (bs, 1H).
4-[2-Amino-6-(ethylamino)-4-pyrimidinyl]-2-fluorobenzonitrile
(10d). In a 25 mL sealable tube under nitrogen were added (4-cyano-3-
fluorophenyl)boronic acid (0.29 g, 1.74 mmol) and 6-chloro-N4-ethyl-
2,4-pyrimidinediamine (0.30 g, 1.74 mmol), followed by 1,4-dioxane
(11.1 mL) and a saturated aqueous solution of NaHCO3 (2.8 mL). The
mixture was degassed with nitrogen for 10 min. Pd(Ph3P)4 (0.10 g,
0.087 mmol) was added, the vial was sealed, and the reaction mixture
was stirred for 16 h at 95 °C. The reaction was cooled to room tem-
perature, filtered, and concentrated. The resulting residue was parti-
tioned between EtOAc and water. The organic layer was separated, dried
(MgSO4), filtered, and concentrated. The resulting yellow oil was
dissolved in CH3CN (5 mL w/5 drops TFA). DMSO (3 drops) and
6-(3-Amino-1H-indazol-6-yl)-N4-ethyl-2,4-pyrimidinediamine (12f). To
a 25 mL flask under argon was added 4-[2-amino-6-(ethylamino)-
4-pyrimidinyl]-2-fluorobenzonitrile (0.164 g, 0.442 mmol), followed
by EtOH (3.5 mL). Hydrazine monohydrate (0.87 mL, 17.7 mmol) was
added, and the reaction mixture was stirred for 6 h at 80 °C. The reaction
was cooled to room temperature and concentrated to near dryness. Water
(3 mL) was added (orange oil was formed), followed by CH3CN (8 drops),
and the mixture was sonicated. A light-yellow precipitate formed. The
suspension was cooled on an ice bath and the solid was filtered, washed with
water, and dried under vacuum at 40 °C to afford the title compound (82
mg, 68%) as a light-yellow solid. LC-MS (ES) m/z = 270 [M þ H]þ. 1H
NMR (400 MHz, DMSO-d6): δ 1.13 (t, J = 7.2 Hz, 3H), 3.20-3.40 (m,
2H), 5.36 (s, 2H), 5.97 (s, 2H), 6.25 (s, 1H), 6.82 (bs, 1H), 7.41 (d, J = 8.3
Hz, 1H), 7.69 (d, J = 8.3 Hz, 1H), 7.84 (s, 1H), 11.50 (s, 1H).
Compounds 12b, 12e, 12g-j, and 12l-m were synthesized by follow-
ing synthetic procedures similar to the preparation of 12f (route 1).
6-[2-Amino-6-(1-methylethyl)-4-pyrimidinyl]-1H-indazol-3-amine
1
(12b). LC-MS (ES) m/z = 269 [M þ H]þ. H NMR (400 MHz,
DMSO-d6): δ 1.24 (d, J = 6.8 Hz, 6H), 2.83 (m, 1H), 5.42 (s, 2H), 6.54
(s, 2H), 7.07 (s, 1H), 7.60 (dd, J = 8.5, 1.4 Hz, 1H), 7.76 (d, J = 8.3 Hz,
1H), 7.99 (s, 1H), 11.59 (s, 1H).
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J = 8.6, 1.3 Hz, 1H), 7.64-7.72 (m, 2H), 7.77 (dd, J = 8.3, 0.8 Hz, 1H),
7.81-7.85 (m, 1H).
6-(3-Amino-1H-indazol-6-yl)-N4,N4-dimethyl-2,4-pyrimidinediamine
(12j). LC-MS (ES) m/z = 270 [M þ H]þ. 1H NMR (400 MHz, DM-
SO-d6): δ 3.07 (s, 6H), 5.37 (s, 2H), 6.02 (s, 2H), 6.44 (s, 1H), 7.56 (dd,
J = 8.3, 1.3 Hz, 1H), 7.71 (d, J = 8.6 Hz, 1H), 7.94 (s, 1H), 11.50 (s, 1H).
6-[2-Amino-6-(1-pyrrolidinyl)-4-pyrimidinyl]-1H-indazol-3-amine
1
(12l). LC-MS (ES) m/z = 296 [M þ H]þ. H NMR (400 MHz,
DMSO-d6): δ 1.93 (bs, 4H), 3.45 (bs, 4H), 5.37 (s, 2H), 6.00 (s, 2H),
6.27 (s, 1H), 7.54 (dd, J = 8.6, 1.3 Hz, 1H), 7.70 (d, J = 8.6 Hz, 1H), 7.92
(s, 1H), 11.49 (s, 1H).
6-[2-Amino-6-(1-piperidinyl)-4-pyrimidinyl]-1H-indazol-3-amine
1
(12m). LC-MS (ES) m/z = 310 [M þ H]þ. H NMR (400 MHz,
DMSO-d6): δ 1.53 (m, 4H), 1.64 (m, 2H), 3.47-3.79 (m, 4H), 5.37 (s,
2H), 6.03 (s, 2H), 6.58 (s, 1H), 7.57 (dd, J = 8.3, 1.3 Hz, 1H), 7.70 (d, J =
8.6 Hz, 1H), 7.94 (s, 1H), 11.49 (s, 1H).
4-(2-Amino-6-phenyl-4-pyrimidinyl)-2-fluorobenzonitrile (10a). In
a 25 mL sealable tube under argon were combined (4-cyano-3-fluorophenyl)-
boronic acid (0.50 g, 3.03 mmol), 4,6-dichloro-2-pyrimidinamine (0.497 g,
3.03 mmol), saturated aqueous NaHCO3 (4.04 mL), and 1,4-dioxane
(16.2 mL), and the mixture was degassed with argon for 5 min. Pd(Ph3P)4
(0.175 g, 0.152 mmol) was added, the vial was sealed, and the reaction
mixture was stirred for 16 h at 95 °C. The reaction was cooled to room
temperature, filtered through a plug of Celite503, and concentrated. The
resulting orange tar was partitioned between EtOAc and water. The
organic layer was separated, washed with brine, dried (MgSO4), filtered,
and concentrated. The resulting orange solid was sonicated in CHCl3
(50 mL) for 2 min. The mixture was filtrated, the solid was washed with
CHCl3, and the filtrate was concentrated. Flash chromatography on
SiO2 (40 g) of the resulting residue (gradient: CHCl3 to 5% EtOAc/
CHCl3) afforded intermediate 4-(2-amino-6-chloro-4-pyrimidinyl)-
2-fluorobenzonitrile (267 mg) as a yellow solid. LC-MS (ES) m/z = 249,
251 [M þ H]þ.
In a 25 mL sealable tube under argon were combined 4-(2-amino-6-
chloro-4-pyrimidinyl)-2-fluorobenzonitrile (0.196 g, 0.788 mmol) and
phenylboronic acid (0.096 g, 0.79 mmol) in 1,4-dioxane (5.0 mL)
and saturated aqueous NaHCO3 (1.3 mL), and the mixture was
degassed with argon for 5 min. Pd(Ph3P)4 (0.046 g, 0.04 mmol) was
added, the vial was sealed, and the reaction mixture was stirred overnight
at 95 °C. The reaction was cooled to room temperature and filtered,
and the filtrate was concentrated to dryness. The resulting solid was
partitioned between EtOAc and water. The organic layer was separated,
washed with brine, dried (MgSO4), filtered, and concentrated. Purifica-
tion of the resulting solids on reverse phase HPLC (CH3CN/H2O w/
0.1% TFA) afforded a TFA salt of the title compound (52 mg) as a
white solid. LC-MS (ES) m/z = 291 [M þ H]þ. 1H NMR (400 MHz,
DMSO-d6): δ 6.95 (s, 2H), 7.49-7.64 (m, 3H), 7.90 (s, 1H), 8.08-8.16
(m, 1H), 8.26 (dd, J = 8.1, 1.5 Hz, 2H), 8.30 (dd, J = 8.1, 1.5 Hz, 1H),
8.36 (dd, J = 11.1, 1.3 Hz, 1H).
6-(2-Amino-6-phenyl-4-pyrimidinyl)-1H-indazol-3-amine (12c). Into
a 25 mL sealable tube under argon were added 4-(2-amino-6-phenyl-
4-pyrimidinyl)-2-fluorobenzonitrile (0.052 g, 0.18 mmol) and EtOH
(5 mL). Hydrazine monohydrate (0.35 mL, 7.17 mmol) was added, the
vial was sealed, and the reaction mixture was stirred overnight at 95 °C.
The reaction was cooled to room temperature and concentrated. The
resulting solid was sonicated in a mixture of EtOH (1 mL) and water
(10 mL), filtered, washed with water, and dried under vacuum at 40 °C
to afford the title compound as a yellow solid (44 mg, 77%). LC-MS
(ES) m/z = 303 [M þ H]þ. 1H NMR (400 MHz, DMSO-d6): δ 5.43 (s,
2H), 6.74 (s, 2H), 7.46-7.59 (m, 3H), 7.69-7.84 (m, 3H), 8.12 (s,
1H), 8.23 (m, 2H), 11.63 (s, 1 H).
Figure 9. Effect of (3S,6R)-28d on the pharmacodynamic markers in
OCL-AML2 xenografts.
6-(3-Amino-1H-indazol-6-yl)-N4-methyl-2,4-pyrimidinediamine
1
(12e). LC-MS (ES) m/z = 256 [M þ H]þ. H NMR (400 MHz,
DMSO-d6): δ 2.81 (d, 3H), 5.37 (s, 2H), 6.00 (s, 2H), 6.25 (s, 1H), 6.80
(bs, 1H), 7.43 (d, J = 8.3 Hz, 1H), 7.69 (d, J = 8.6 Hz, 1H), 7.85 (s, 1H),
11.50 (s, 1H).
6-(3-Amino-1H-indazol-6-yl)-N4-(1-methylethyl)-2,4-pyrimidinedi-
amine (12g). LC-MS (ES) m/z = 284 [M þ H]þ. 1H NMR (400 MHz,
DMSO-d6): δ 1.15 (d, J = 6.6 Hz, 6H), 4.12 (bs, 1H), 5.36 (s, 2H), 5.94
(s, 2H), 6.24 (s, 1H), 6.71 (d, J = 7.6 Hz, 1H), 7.40 (d, J = 8.6 Hz, 1H),
7.69 (d, J = 8.3 Hz, 1H), 7.82 (s, 1H), 11.50 (bs, 1H).
6-(3-Amino-1H-indazol-6-yl)-N4-cyclopentyl-2,4-pyrimidinediamine
1
(12h). LC-MS (ES) m/z = 310 [M þ H]þ. H NMR (400 MHz,
DMSO-d6): δ 1.38-1.61 (m, 4H), 1.61-1.78 (m, 2H), 1.92 (m, 2H),
4.22 (bs, 1H), 5.37 (s, 2H), 5.97 (bs, 2H), 6.26 (s, 1H), 6.87 (bs, 1H),
7.40 (d, J = 8.6 Hz, 1H), 7.70 (d, J = 8.4 Hz, 1H), 7.82 (s, 1H), 11.51
(s, 1H).
6-(3-Amino-1H-indazol-6-yl)-N4-phenyl-2,4-pyrimidinediamine
(12i). LC-MS (ES) m/z = 318 [M þ H]þ. 1H NMR (400 MHz, CD3-
OD) δ 6.53 (s, 1H), 7.00-7.13 (m, 1H), 7.27-7.38 (m, 2H), 7.48 (dd,
6-[2-Amino-6-(4-morpholinyl)-4-pyrimidinyl]-1H-indazol-3-amine
(12k). In a 25 mL sealable tube under argon were combined N-acetyl-
N-(1-acetyl-6-bromo-1H-indazol-3-yl)acetamide (0.25 g, 0.74 mmol),
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bis(pinacolato)diboron (0.197 g, 0.776 mmol), and potassium acetate
(0.145 g, 1.48 mmol) in 1,4-dioxane (4.9 mL). The mixture was degassed
To a solution of (5R)-5-methyl-4-(phenylmethyl)-3-morpholinone
(11.7 g, 57 mmol) in toluene (140 mL) at 0 °C was added sodium bis
(2-methoxyethoxy)aluminumhydride (Red-Al, 35 mL, 3 mL/g of
morpholinone) slowly via addition funnel, and the reaction mixture was
stirred overnight at 60 °C. The reaction was cooled down to 0 °C and
quenched by dropwise addition of 1N aqueous NaOH (15 mL). The
resulting mixture was partitioned between Et2O (100 mL) and 1N
aqueous NaOH (100 mL). The organic layer was separated, and the
aqueous layer was further extracted with Et2O (50 mL). The combined
organic layers were dried (Na2SO4), filtered, and concentrated. The
resulting residue was azeotroped with CH3OH (50 mL) to afford the
title compound (10.69 g) as a colorless oil. LC-MS (ES) m/z = 192 [M
þ H]þ. 1H NMR (400 MHz, CDCl3): δ 1.11 (d, J = 6.1 Hz, 3H), 2.14-
2.32 (m, 1H), 2.45-2.56 (m, 1H), 2.61 (d, J = 11.9 Hz, 1H), 3.16 (d, J =
13.1 Hz, 1H), 3.26-3.42 (m, 1H), 3.57-3.66 (m, 1H), 3.68-3.79 (m,
2H), 4.08 (d, J = 13.1 Hz, 1H), 7.23-7.30 (m, 1H), 7.30-7.40 (m, 4H).
(3R)-3-Methylmorpholine ((R)-17a). To (3R)-3-methyl-4-(phenyl-
methyl)morpholine (10.7 g, 56 mmol) in CH3OH (110 mL) were
added 6N aqueous HCl (9.3 mL) and Pd/C (1.07 g, 10 wt %), and the
reaction mixture was stirred overnight at room temperature under a H2
atmosphere (balloon setup). The mixture was filtered through a glass
fiber filter, and the filter cake was washed with CH3OH. The combined
filtrate was concentrated and azeotroped with CH3OH (4 ꢀ 100 mL) to
afford the HCl salt of the title compound as a yellow oil that solidified
under high vacuum (7.91 g). 1H NMR (400 MHz, CD3OD): δ 3.95-
4.05 (m, 2H), 3.77 (m, 1H), 3.47-3.54 (m, 1H), 3.38-3.47 (m, 1H),
3.30-3.34 (m, 1H), 3.18-3.28 (m, 1H), 1.29 (d, J = 6.3 Hz, 3H).
6-{2-Amino-6-[(3R)-3-methyl-4-morpholinyl]-4-pyrimidinyl}-1H-
indazol-3-amine ((R)-18a). The title compound was prepared from
amine (R)-17a by following synthetic route 1. LC-MS (ES) m/z = 326
[M þ H]þ. 1H NMR (400 MHz, DMSO-d6): δ 1.19 (d, J = 6.8 Hz, 3H),
3.11 (m, 1H), 3.44 (m, 1H), 3.56-3.63 (m, 1H), 3.68-3.74 (m, 1H),
3.92 (dd, J = 11.1, 3.0 Hz, 1H), 4.05-4.14 (m, 1H), 4.49 (bs, 1H), 5.38
(s, 2H), 6.11 (s, 2H), 6.55 (s, 1H), 7.57 (dd, J = 8.6, 1.3 Hz, 1H), 7.71 (d,
J = 8.6 Hz, 1H), 7.95 (s, 1 H), 11.52 (s, 1H). Compound (S)-18a was
prepared similarly.
(5R)-5-Ethyl-3-morpholinone (16a). NaH (1.51 g, 37.9 mmol) in
dry toluene (10 mL) was cooled to 0 °C under nitrogen, and (2R)-2-
amino-1-butanol (1.5 g, 16.8 mmol) in toluene (5 mL) was added
dropwise. The mixture was stirred for 20 min, warming up to room
temperature, and chloroacetyl chloride (1.5 mL, 18.9 mmol) in toluene
(5 mL) was added dropwise. An exotherm was observed, so the mixture
was cooled in an ice bath during the addition. After the addition was
completed, the reaction mixture was heated to 110 °C overnight. The
mixture was cooled to room temperature, and 5 g of ammonium chloride
were added portionwise. The mixture was stirred for 20 min and filtered.
The filter cake was washed with toluene and discarded. The filtrate was
concentrated to give an orange oil that was dissolved in CH2Cl2 and
purified by SiO2 chromatography (gradient: CH2Cl2 to 90:10:1
CH2Cl2/CH3OH/NH4OH) to afford the title compound (1.2 g,
55%) as a yellow solid. 1H NMR (400 MHz, CDCl3): δ 0.96 (t,
J = 7.6 Hz, 3H), 1.52-1.63 (m, 2H), 3.40-3.51 (m, 2H), 3.85-3.94
(m, 1H), 4.07-4.20 (m, 2H), 7.55 (bs, 1H).
with argon for 5 min. PdCl2(dppf) CH2Cl2 (0.024 g, 0.03 mmol) was
3
added, the tube was sealed, and the reaction mixture was stirred for 5 h at
100 °C. The mixture was cooled to room temperature, the tube was unsealed,
and 4-chloro-6-(4-morpholinyl)-2-pyrimidinamine (0.175 g, 0.813 mmol),
NaHCO3 (0.25 g, 2.96 mmol), water (1.64 mL), and PdCl2(dppf) CH2Cl2
3
(0.024 g, 0.03 mmol) were added. The tube was resealed under argon, and
the reaction mixture was stirred overnight at 100 °C. The mixture was cooled
to room temperature, diluted with CH3CN (10 mL), filtered through a pad of
Celite503, and concentrated. The resulting brown solid was dissolved in 10
mL of solvent (30/70 CH3CN/H2O w/0.25 mL TFA), and the solution was
filtered through a 0.45 μ filter disk. The filtrate was purified by reverse-phase
HPLC (CH3CN/H2O w/0.1% TFA) to afford a TFA salt of the acetylated
Suzuki coupling intermediate (151 mg) as a white solid. LC-MS (ES) m/z =
354 [M þ H]þ. This intermediate was then dissolved in CH3OH (10 mL).
HCl (12 M, 0.69 mL, 8.3 mmol) was added, and the reaction mixture was
stirred at 60 °C for 4 h. The mixture was cooled to room temperature and
filtered. The solid was washed with hexanes to afford an HCl salt of the title
compound (75 mg) as a light-yellow solid. LC-MS (ES) m/z = 312 [M þ
H]þ. 1H NMR (400 MHz, DMSO-d6): δ 3.73 (m, 4H), 3.89 (m, 4H), 6.98
(s, 1H), 7.66 (d, J = 8.6 Hz, 1H), 8.08 (m, 2H).
Compounds 12a and 12d were synthesized following synthetic proce-
dures similar to the preparation of 12k (route 2).
6-(2-Amino-6-methyl-4-pyrimidinyl)-1H-indazol-3-amine (12a). HCl
salt. LC-MS (ES) m/z = 241 [M þ H]þ. 1H NMR (400 MHz, DMSO-d6):
δ 2.51 (s, 3H {assumed to be hidden beneath DMSO peak}), 7.59 (s, 1H),
7.81 (dd, J = 8.6, 1.3 Hz, 1H), 7.99 (d, J = 8.6 Hz, 1H), 8.21 (s, 1H),
12.72 (bs, 1H).
6-[2-Amino-6-(methyloxy)-4-pyrimidinyl]-1H-indazol-3-amine
1
(12d). LC-MS (ES) m/z = 257 [M þ H]þ. H NMR (400 MHz,
DMSO-d6): δ 3.86 (s, 3H), 5.40 (s, 2H), 6.58 (s, 1H), 6.64 (s, 2H),
7.54 (dd, J = 8.6, 1.0 Hz, 1H), 7.72 (d, J = 8.3 Hz, 1H), 7.95 (s, 1H),
11.57 (s, 1H).
(2R)-2-[(Phenylmethyl)amino]-1-propanol ((R)-13). To (2R)-2-amino-
1-propanol (4.5 g, 60 mmol) in toluene (120 mL) was added benzaldehyde
(636 mL). A Dean-Stark trap was placed on the flask, and the reaction
mixture was heated to reflux until no further water evolved. The reaction was
cooled down to room temperature and concentrated. The resulting residue
was dissolved in ethanol (120 mL) and treated with NaBH4 (5.67 g, 150
mmol) at 0 °C, followed by sufficient 4N HCl in dioxane to adjust the pH to 2.
The reaction mixture was stirred overnight at room temperature and then
concentrated in vacuo. The resulting residue was dissolved in 1N aq HCl
(200 mL) and washed with CH2Cl2 (2 ꢀ 100 mL). The aqueous phase was
then adjusted to pH > 13 with 6N aqueous NaOH and extracted with CH2Cl2
(2 ꢀ 150 mL). The combined organic phase was dried over anhydrous
Na2SO4, filtered, and concentrated to afford the title compound (9.44 g, 95%)
as a colorless oil, which solidified under high vacuum. LC-MS (ES) m/z= 166
[M þ H]þ. 1H NMR (400 MHz, CDCl3): δ 1.12 (d, J = 6.6 Hz, 3H), 2.07
(bs, 2H), 2.78-2.96 (m, 1H), 3.30 (dd, J = 10.6, 6.8 Hz, 1H), 3.63 (dd, J =
10.6, 4.0 Hz, 1H), 3.73-3.81 (m, 1H), 3.86-3.95 (m, 1H), 7.24-7.32 (m,
1H), 7.35 (m, 4H).
(3R)-3-Methyl-4-(phenylmethyl)morpholine ((R)-15). To (2R)-2-
[(phenylmethyl)amino]-1-propanol (8.43 g, 51 mmol) in THF (50 mL)
was added a solution of K2CO3 (21.15 g, 153 mmol) in water (50 mL).
To the resulting mixture at 0 °C was added slowly via syringe
chloroacetyl chloride (5.7 mL, 71.4 mmol) with vigorous stirring,
and the reaction mixture was stirred for 1 h at 0 °C. A 50% aqueous
NaOH solution was added to adjust the pH > 13, and the resulting
mixture was warmed up overnight to room temperature. The solution
was extracted with CH2Cl2 (2 ꢀ 200 mL), and the organic layer was
dried (Na2SO4), filtered, and concentrated to afford (5R)-5-methyl-
4-(phenylmethyl)-3-morpholinone ((R)-14) as a colorless oil. LC-MS
(ES) m/z = 206 [M þ H]þ.
(3R)-3-Ethylmorpholine ((R)-17b). A solution of 1 M LiAlH4 in
THF (7.7 mL, 7.7 mmol) was cooled in an ice bath under nitrogen. A
solution of (5R)-5-ethyl-3-morpholinone (500 mg, 3.87 mmol) in THF
(10 mL) was added dropwise, and the solution was heated to 70 °C for
16 h. After aproximately 2 h, a thick white precipitate had formed. The
reaction mixture was cooled down to room temperature and carefully
quenched with water (1 mL), 2 M NaOH (1 mL), and water (4 mL).
The resulting slurry was stirred at room temperature for 1 h and then
filtered through Celite. The filter cake was washed with EtOAc and
discarded. The filtrate was washed with brine, dried (MgSO4), and
filtered. HCl in ether (3.87 mL, 3.87 mmol) was added, producing a
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cloudy solution, and the solvent was evaporated to afford the HCl salt of
the title compound (251 mg, 56%) as an orange solid. 1H NMR (400
MHz, DMSO-d6): δ 0.88-0.97 (m, 3H), 1.46-1.72 (m, 2H), 2.91-
3.18 (m, 3H), 3.46 (dd, J = 12.3, 10.2 Hz, 1H), 3.71 (td, J = 11.8, 2.7 Hz,
1H), 3.82-3.97 (m, 2H), 9.68 (bs, 2H).
6-{2-Amino-6-[(3R)-3-ethyl-4-morpholinyl]-4-pyrimidinyl}-1H-
indazol-3-amine (18b). The title compound was prepared from amine
(R)-17b following synthetic route 1. LC-MS (ES) m/z = 340 [M þ H]þ.
1H NMR (400 MHz, DMSO-d6): δ 0.88 (t, J = 7.5 Hz, 3H), 1.63-1.80
(m, 2H), 3.09 (td, J = 12.8, 3.2 Hz, 1H), 3.37-3.46 (m, 2H), 3.51 (dd,
J = 11.4, 2.8 Hz, 1H), 3.80-3.91 (m, 2H), 4.30 (bs, 1H), 5.38 (s, 2H),
6.09 (bs, 2H), 6.56 (s, 1H), 7.56 (dd, J = 8.6, 1.0 Hz, 1H), 7.71 (d, J =
8.3 Hz, 1H), 7.94 (s, 1H), 11.52 (s, 1H).
6-{2-Amino-6-[(3R)-3-(1-methylethyl)-4-morpholinyl]-4-pyrimid-
inyl}-1H-indazol-3-amine (18c). The title compound was synthesized
following synthetic procedures similar to the preparation of 18b. LC-MS
(ES) m/z = 354 [M þ H]þ. 1H NMR (400 MHz, CD3OD): δ 0.88 (d,
J = 6.8 Hz, 3H), 1.10 (d, J = 6.6 Hz, 3H), 2.47-2.54 (m, 1H), 3.21-3.30
(m., 1H), 3.51-3.58 (m, 2H), 3.93 (dd, J = 11.2, 3.41 Hz, 1H), 4.10 (d,
J = 11.6 Hz, 1H), 6.46 (s, 1H), 7.47 (dd, J = 8.5, 1.4 Hz, 1H), 7.76 (d, J =
8.6 Hz, 1H), 7.79 (s, 1H).
6-{2-Amino-6-[(2R)-2-methyl-1-piperidinyl]-4-pyrimidinyl}-1H-in-
dazol-3-amine (18d). The title compound was prepared from com-
mercially available (2R)-2-methylpiperidine following synthetic route 1.
LC-MS (ES) m/z = 324 [M þ H]þ. 1H NMR (400 MHz, DMSO-d6): δ
1.14 (d, J = 6.8 Hz, 3H), 1.29-1.44 (m, 1H), 1.52-1.82 (m, 5H), 2.80-
2.98 (m, 1H), 4.35 (d, J = 12.6 Hz, 1H), 4.80 (bs, 1H), 5.38 (s, 2H), 6.02
(s, 2H), 6.53 (s, 1H), 7.56 (dd, J = 8.5, 1.1 Hz, 1H), 7.70 (d, J = 8.3 Hz,
1H), 7.93 (s, 1 H), 11.50 (s, 1H).
(3S)-N-Phenyl-3-piperidinecarboxamide ((S)-20b). To a mixture of
(3S)-1-{[(1,1-dimethylethyl)oxy]carbonyl}-3-piperidinecarboxylic acid
(500 mg, 2.181 mmol), HOBt (668 mg, 4.36 mmol), and EDC (502 mg,
2.62 mmol) in DMF (5 mL) was added N-methylmorpholine (1.20 mL,
10.90 mmol), and the solution was stirred for 10 min. Aniline (223 mg,
2.399 mmol) was added, and the reaction mixture was stirred overnight
at room temperature. The reaction was diluted with EtOAc (50 mL) and
then washed with water and brine. The organic layer was then dried
(Na2SO4), filtered, and concentrated to give a light-yellow oil. The oil
was purified on Biotage SP1 25 m column with a gradient of 0-40%
EtOAc in hexane over 14 column volumes. The residue was taken up in a
premixed 2:1 CH2Cl2:TFA solution (5 mL), and the resulting mixture
was stirred for 15 min and then concentrated to isolate the crude TFA
salt of the title compound. LC-MS (ES) m/z = 205 [M þ H]þ. 1H NMR
(400 MHz, DMSO-d6): δ 1.61-1.73 (m, 2H), 1.79-1.89 (m, 1H),
2.00-2.07 (m, 1H), 2.77-2.85 (m, 1H), 2.92 (d, J = 10.6 Hz, 1H), 3.08
(dd, J = 10.7, 5.9 Hz, 1H), 3.20 (d, J = 11.9 Hz, 1H), 3.33 (d, J = 10.9 Hz,
1H), 7.06 (t, J = 7.3 Hz, 1H), 7.31 (t, J = 8.0 Hz, 2H), 7.59 (d, J = 7.6 Hz,
2H), 8.61 (bs, 1H), 10.18 (s, 1H).
(dd, J = 8.6, 1.3 Hz, 1H), 7.69-7.74 (m, 3H), 7.97 (s, 1H), 9.84 (s, 1H),
11.53 (s, 1H).
The individual enantiomers (S)-21a and (R)-21a were separated by
preparative HPLC on a Chiralpak AD-H (250 mm ꢀ 20 mm) column:
5:95 = CH3OH:CH3CN (þ0.1% isopropylamine) at 20 mL/min;
23 °C; UV 254 nm.
(3S)-1-[2-Amino-6-(3-amino-1H-indazol-6-yl)-4-pyrimidinyl]-N-
cyclohexyl-3-piperidinecarboxamide ((S)-21c). Amine (S)-20c was
prepared according to the synthetic procedures described for (S)-20b,
and converted to (S)-21c following synthetic route 2. LC-MS (ES)
m/z = 435 [M þ H]þ. NMR spectrum observed a large water peak. 1H
NMR (400 MHz, DMSO-d6): δ 1.08-1.30 (m, 4H), 1.36-1.59 (m,
2H), 1.59-1.77 (m, 4H), 1.91 (d, J = 3.3 Hz, 1H), 3.09 (d, J = 11.9 Hz,
1H), 3.20 (d, J = 11.4 Hz, 1H), 4.38 (bs, 1H), 4.57-4.91 (m, 1H), 6.99
(d, J = 14.2 Hz, 1H), 7.57 (dd, J = 8.0, 4.7 Hz, 1H), 7.85 (d, J = 7.3 Hz,
1H), 7.94-8.01 (m, 2H), 12.62 (d, J = 1.0 Hz, 1H).
(3S)-1-[2-Amino-6-(3-amino-1H-indazol-6-yl)-4-pyrimidinyl]-N-cy-
clopentyl-3-piperidinecarboxamide ((S)-21d). Amine (S)-20d was
prepared according to the synthetic procedures described for (S)-20b
and converted to (S)-21d following synthetic route 2. LC-MS (ES) m/z =
1
421 [M þ H]þ. H NMR (400 MHz, DMSO-d6): δ 1.37 (m, 2H),
1.42-1.55 (m, 2H), 1.55-1.70 (m, 2H), 1.78 (m, 2H), 1.90 (m, 1H),
2.36 (m, 1H), 3.03-3.12 (m, 1H), 3.17 (m, 1H), 3.36-3.50 (m, 1H),
3.98 (m, 1H), 4.33 (m, 2H), 4.67 (d, J = 12.9 Hz, 1H), 4.86 (d, J =
11.4 Hz, 1H), 6.96-7.09 (m, 1H), 7.66 (m, 1H), 7.98 (d, J = 7.3 Hz,
1H), 8.02-8.10 (m, 2H), 12.83 (bs, 1H).
cis-3-Methyl 1-(Phenylmethyl)-6-methyl-1,3-piperidinedicarboxy-
late ((()-cis-22). A solution of methyl 6-methylnicotinate (50 g, 331
mmol, 1 equi.) in CH3OH (400 mL) and conc HCl (26 mL) was added
to a slurry of platinum(IV) oxide (2.0 g) in 50 mL of CH3OH/water (4/1)
in a Parr bottle. The mixture was hydrogenated at room temperature
under 60 psi of hydrogen gas for 4.5 h. The mixture was then filtered
through Celite. The filtrate was concentrated in vacuo, after chasing with
500 mL of toluene, to about 77 g of syrup. This residue was dissolved in
CH2Cl2 (500 mL) and chilled in an ice bath. To this stirred solution was
added DMAP (0.4 g, 3.31 mmol), followed by TEA (101 mL, 728 mmol,
2.2 equiv) portionwise. A suspension formed when TEA was added. This
mixture was chilled to 15 °C. To the resulting suspension was added
benzylchloroformate (52 mL, 364 mmol, 1.1 equiv) dropwise over a
25 min period such that the temperature of the mixture was kept at 15-
20 °C. After completion of benzylchloroformate addition, the mixture was
stirred chilled with an ice bath for another 30 min and then at ambient
temperature for 1 h. This mixture was washed with 300 mL of cold 1N HCl.
The organic was concentrated in vacuo. The residue was partitioned
between toluene (400 mL), MTBE (200 mL), and water (250 mL). The
organic was washed with brine, dried over MgSO4, filtered, and concen-
trated in vacuo to give an oil (97 g) as the crude (NMR showed ∼3:1
cis/trans ratio of isomers). Silica gel column chromatography using
gradient elution of EtOAc in hexane gave 64.4 g (65% yield) of the title
compound. LC-MS (ES) m/z = 292 [M þ H]þ. 1H NMR (400 MHz,
CDCl3): δ 1.19 (d, J = 6.8 Hz, 3H), 1.51 (m, 1H), 1.65-1.84 (m, 2H),
1.90-2.00 (m, 1H), 2.44 (t, J = 11.2 Hz, 1H), 2.89-3.11 (m, 1H), 3.71
(s, 3H), 4.29 (bs, 1H), 4.51 (bs, 1H), 5.16 (s, 2H), 7.32-7.50 (m, 5H).
(()-trans-22. 1H NMR (400 MHz, CDCl3): δ 1.18 (d, J = 6.8 Hz,
3H), 1.37-1.42 (m, 1H), 1.78-1.91 (m, 2H), 2.04-2.10 (m, 1H), 2.61
(m, 1H), 3.13 (dd, J = 13.9, 4.3 Hz, 1H), 3.62 (s, 3H) 4.45 (m, 1H), 4.50
(d, J = 14.1 Hz, 1H), 5.14 (m, 2H), 7.25-7.40 (m, 5H).
(3S)-1-[2-Amino-6-(3-amino-1H-indazol-6-yl)-4-pyrimidinyl]-N-phenyl-
3-piperidinecarboxamide ((S)-21b). The title compound was prepared
from amine (S)-20b following synthetic route 1. LC-MS (ES) m/z = 429
1
[M þ H]þ. H NMR (400 MHz, DMSO-d6): δ 1.43-1.59 (m, 1H),
1.75-1.94 (m, 2H), 2.03-2.16 (m, 1H), 2.57-2.69 (m, 1H), 3.12-3.31
(m, 2H), 3.43-3.54 (m, 2H), 4.40-4.58 (m, 1H), 4.72-5.07 (m, 1H),
6.94-7.11 (m, 2H), 7.30 (m, 2H), 7.37-7.43 (m, 1H), 7.61 (d, J = 8.1 Hz,
2H), 7.75 (bs, 1H), 7.90 (d, 1H), 10.08 (d, J = 12.9 Hz, 1H), 12.10 (bs,
2H). Compound ((R)-21b) was prepared similarly.
4-[2-Amino-6-(3-amino-1H-indazol-6-yl)-4-pyrimidinyl]-N-phenyl-
2-morpholinecarboxamide ((()-21a). The title compound was synthe-
sized from (()-19a following synthetic procedures similar to the preparation
of ((S)-21b). LC-MS (ES) m/z = 431 [M þ H]þ. 1H NMR (400 MHz,
DMSO-d6): δ 2.99-3.16 (m, 2H), 3.70 (td, J = 11.4, 2.5 Hz, 1H), 4.09 (d,
J = 11.4 Hz, 1H), 4.15-4.29 (m, 2H), 4.65 (bs, 1H), 5.38 (s, 2H), 6.23 (bs,
2H), 6.68 (s, 1H), 7.09 (t, J = 7.45 Hz, 1H), 7.33 (t, J = 8.0 Hz, 2H), 7.60
(()-cis-22 was resolved by chiral stationary phase HPLC, super-
critical fluid chromatography (SFC) or by crystallization of a tartrate salt.
The following conditions were used for the HPLC analysis and
preparative resolution:
Analytical Separation Method. A ChiralPak AD-3 column (150 mm ꢀ
4.6 mm I.D.) was used eluting with a mixture of 15% ethanol and 85%
hexane containing 0.1% diethylamine at a flow rate of 1 mL/min.
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Preparative Separation Method. The separation was run in a Berger
Multi-Gram III preparative SFC instrument. The column used was a
ChiralPak IC (250 mm ꢀ 30 mm I.D.) eluting with a mixture of 75% SFC
þ14.5 (c 1.35, CHCl3). (3R,6S)-22. [R]23D = -13.1 (c 0.95, CHCl3).
vacuo. The resulting residue was extracted with EtOAc (2 ꢀ 300 mL)
and CHCl3 (2 ꢀ 300 mL), and the combined organic layers were dried
(MgSO4), filtered, and concentrated in vacuo to afford (3S,6R)-6-
methyl-1-{[(phenylmethyl)oxy]carbonyl}-3-piperidinecarboxylic acid
CO2 and 25% 2-propanol at a flow rate of 90 mL/min. (3S,6R)-22. [R]23
=
D
1
(17 g). LC-MS (ES) m/z = 278 [M þ H]þ. H NMR (400 MHz,
Resolution by Crystallization of the Tartrate Salt: Methyl (3S,6R)-6-
Methyl-3-piperidinecarboxylate L-(þ)-Tartaric Acid Salt (23). A solu-
tion of cis-3-methyl 1-(phenylmethyl)-6-methyl-1,3-piperidinedicarbox-
ylate (69 g, 237 mol) in EtOH (50 mL) and EtOAc (300 mL) was added
to a slurry of 10% Pd/C (3.7 g) in EtOAc (30 mL) and EtOH (10 mL)
under nitrogen in a Parr shaker bottle. The mixture was hydrogenated
under 65 psi at room temperature for 4 h. The mixture was filtered
through Celite and washed with EtOAc. The filtrate was concentrated
in vacuo to give 37 g of cis-3-methyl 1-(phenylmethyl)-6-methyl-1,3-
piperidinedicarboxylate as a liquid. LC-MS (ES) m/z = 158 [M þ H]þ.
1H NMR (400 MHz, CDCl3): δ 1.08 (d, J = 6.32 Hz, 3H), 1.15-1.33
(m, 1H), 1.53-1.64 (m, 1H), 1.64-1.78 (m, 1H), 2.13-2.25 (m, 1H),
2.31 (bs, 1H), 2.48-2.57 (m, 1H), 2.63-2.77 (m, 1H), 2.86 (dd, J =
13.1, 3.8 Hz, 1H), 3.39-3.51 (m, 1H), 3.74 (s, 3H).
CDCl3): δ 1.19 (d, J = 7.1 Hz, 3H), 1.57-1.91 (m, 3H), 1.94-2.05 (m,
1H), 2.48 (m, 1H), 3.02 (t, J = 12.8 Hz, 1H), 4.30 (bs, 1H), 4.53 (bs,
1H), 5.16 (s, 2H), 7.32-7.50 (m, 5H).
To a solution of (3S,6R)-6-methyl-1-{[(phenylmethyl)oxy]carbonyl}-
3-piperidinecarboxylic acid (17 g, 61.3 mmol) in DMF (400 mL) was
added aniline (8.56 g, 92 mmol), H€unig’s base (32.1 mL, 184 mmol),
HOBt (14.08 g, 92 mmol), and EDC (17.63 g, 92 mmol) at room tem-
perature. The reaction mixture was stirred overnight at room tempera-
ture then diluted with H2O (∼100 mL) and extracted with EtOAc (3 ꢀ
250 mL). The combined organic layers were washed with 1N HCl
(100 mL), H2O (3 ꢀ 100 mL), and brine (100 mL), and then dried
(MgSO4), filtered, and concentrated in vacuo to afford crude phenyl-
methyl (2R,5S)-2-methyl-5-[(phenylamino)carbonyl]-1-piperidinecar-
boxylate (21 g). LC-MS (ES) m/z = 353 [M þ H]þ.
A solution of phenylmethyl (2R,5S)-2-methyl-5-[(phenylamino)car-
bonyl]-1-piperidinecarboxylate (21 g, 59.6 mmol) in EtOAc (250 mL)
and EtOH (250 mL) was degassed. Pd/C (10%, 6.34 g, 5.96 mmol) was
added, and then H2 was bubbled through the mixture. The reaction
mixture was stirred at room temperature under H2 balloon overnight.
The mixture was filtered through a pad of Celite and concentrated in
vacuo to afford the crude title compound (12 g). LC-MS (ES) m/z = 219
[M þ H]þ. 1H NMR (400 MHz, CDCl3): δ 1.18 (d, J = 6.1 Hz, 3H),
1.36-1.51 (m, 1H), 1.55-1.65 (m, 1H), 1.68-1.83 (m, 1H), 2.10-
2.21 (m, 1H), 2.54-2.62 (m, 1H), 2.70-2.84 (m, 1H), 2.96 (dd, J =
12.0, 3.2 Hz, 1H), 3.40 (m, 1 H), 7.05-7.12 (m, 1H), 7.30-7.37 (m,
2H), 7.56-7.65 (m, 2H), 11.16 (bs, 1H).
A suspension of L-(þ)-tartaric acid (39 g, 260 mmol, 1.05 equiv) in
2-propanol (200 mL) and water (13 mL) was heated in a water bath
at 60 °C until all dissolved. To this hot stirred solution was added
cis-3-methyl 1-(phenylmethyl)-6-methyl-1,3-piperidinedicarboxylate (39 g,
248 mmol), followed by addition of 25 mL of 2-propanol rinse. The
resulting mixture was heated to 60 °C, resulting in a clear solution, and
then cooled to room temperature while the hot water bath was removed.
This hot solution was seeded with a sample of methyl (3S,6R)-6-methyl-
3-piperidinecarboxylate L-(þ)-tartaric acid salt that had a chiral purity of
98% ee and aged at ambient temperature (with the water bath removed)
for 20 min. The mixture turned into an oily texture with seeds still
present. To the mixture was added 5 mL of water and heated in the warm
water bath at 43 °C. The mixture became clear with the seeds still
present. The heating was stopped, and the mixture was stirred in the
warm water bath. After 20 min, the mixture gradually turned into a paste.
After another 10 min, the water bath was removed, and the mixture was
stirred at ambient temperature for another 1 h. The resulting paste was
filtered. The cake was washed with 50 mL of 2-propanol, giving 62 g of
wet solids. This cake was taken up in 150 mL of 2-propanol and 8 mL of
water and stirred as a slurry while being heated in a water bath to 60 °C
(internal temp 55 °C) for 5 min. The heating was turned off while the
mixture was still stirred in the warm water bath. After 30 min, the mixture
was filtered. The cake was washed with 100 mL of 2-propanol. Drying
under house vacuum at room temperature for 48 h gave 46.7 g of solids.
An analytical sample was derivatized to the corresponding N-Cbz derivative,
which was determined by chiral HPLC (methods used to analyze the
resolution of (()-cis-22 above) to have 85% ee. This material was taken up
in 2-propanol (420 mL) and water (38 mL) as a suspension. The mixture
was heated in a water bath to 65 °C, at which time the mixture became a
clear solution. The heating bath was removed. The mixture was seeded and
aged at ambient temperature for 20 h. The solids formed were filtered and
then washed with 100 mL of 2-propanol. The solids collected were dried
under house vacuum at room temperature for 24 h and then under vacuum
at room temperature for another 24 h to give 28.5 g of the title compound.
An analytical sample was converted to the N-Cbz derivative. The ee was
determined to be 97.7%. LC-MS (ES) m/z = 158 [M þ H]þ. 1H NMR
(400 MHz, DMSO-d6): δ 1.13 (d, J = 6.4 Hz, 3H), 1.24-1.43 (m, 1H),
1.59-1.79 (m, 2H), 1.93-2.06 (m, 1H), 2.77-2.88 (m, 1H), 2.98-3.14
(m, 2H), 3.38 (dd, J = 12.9, 2.8 Hz, 1H), 3.89 (s, 2H).
(3S,6R)-1-[2-Amino-6-(3-amino-1H-indazol-6-yl)-4-pyrimidinyl]-6-
methyl-N-phenyl-3-piperidinecarboxamide ((3S,6R)-28a). The title
compound was prepared from amine (3S,6R)-24 following synthetic
1
route 1. LC-MS (ES) m/z = 443 [M þ H]þ. H NMR (400 MHz,
DMSO-d6): δ 1.18 (d, J = 6.8 Hz, 3H), 1.75 (s, 2H), 1.83 (m, 1H), 1.92
(m, 1H), 5.37 (s, 2H), 6.10 (s, 2H), 6.64 (s, 1H), 7.03 (s, 1H), 7.32 (s,
2H), 7.29-7.34 (m, 2H), 7.64 (d, J = 7.3 Hz, 4H), 7.96 (s, 1H), 8.32 (s,
1H), 10.06 (s, 1H), 11.49 (s, 1H). Compound (3R,6S)-28a was prepared
similarly.
trans-1-[2-Amino-6-(3-amino-1H-indazol-6-yl)-4-pyrimidinyl]-6-
methyl-N-phenyl-3-piperidinecarboxamide ((()-trans-28a). The title
compound was synthesized following synthetic procedures similar to the
1
preparation of (3S,6R)-28a. LC-MS (ES) m/z = 443 [M þ H]þ. H
NMR (400 MHz, DMSO-d6): δ 1.21 (d, J = 6.6 Hz, 3H), 1.42-1.55 (m,
1H), 1.90-2.06 (m, 3H), 2.83 (bs, 1H), 3.35-3.41 (m, 1H), 4.61 (bs,
1H), 4.70 (d, J = 6.6 Hz, 1H), 5.37 (s, 2H), 6.07 (s, 2H), 6.55 (s, 1H),
6.90-7.06 (m, 1H), 7.23 (t, J = 8.0 Hz, 2H), 7.44-7.52(m, 1H), 7.58 (d,
J = 7.6 Hz, 2H), 7.67 (d, J = 8.3 Hz, 1H), 7.90 (s, 1H), 9.83 (s, 1H), 11.50
(s, 1H).
4,6-Dichloro-N-methyl-2-pyrimidinamine (27). Methylamine (2 M
solution in THF, 113 mL, 217 mmol, 2.05 equiv) was charged to a 1 L
three-neck flask fitted with a magnetic stirrer and a thermometer. The
mixture was chilled in an ice bath. To this stirred solution was added via
addition funnel a solution of 4,6-dichloro-2-(methylsulfonyl)pyrimidine
(25 g, 110 mmol) in EtOAc (250 mL) portionwise over a 25 min period.
The temp was between 5-10 °C. After completion of addition, the ice
bath was removed, and the mixture was stirred for 1 h at ambient
temperature. LC-MS showed complete conversion. The suspension was
filtered and washed with EtOAc. The filtrate was concentrated in vacuo.
The residue was partitioned between water (100 mL) and EtOAc
(450 mL). The organic layer was washed with brine, dried over MgSO4,
filtered, and concentrated in vacuo to give white solids, which were
triturated in 150 mL of CH2Cl2. These solids were collected by filtration
(3S,6R)-6-Methyl-N-phenyl-3-piperidinecarboxamide ((3S,6R)-24). To
a solution of 3-methyl 1-(phenylmethyl) (3S,6R)-6-methyl-1,3-piper-
idinedicarboxylate (15 g, 51.5 mmol) in THF/water/CH3OH (10/5/1,
480 mL) was added LiOH H2O (2.59 g, 61.8 mmol), and the reaction
3
mixture was stirred at room temperature for about 1 h. The mixture was
acidified with 1N HCl until the pH was ∼5 and then concentrated in
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Journal of Medicinal Chemistry
ARTICLE
and washed with cold CH2Cl2 (50 mL). Drying under house vacuum at
room temperature for 20 h and then high vacuum at room temperature
for 3 h gave the title compound (9.31 g) as a solid. LC-MS (ES) m/z =
179 [M þ H]þ. 1H NMR (400 MHz, CDCl3): δ 3.04 (s, 3H), 5.57 (bs,
1H), 6.63 (s, 1H).
(3S,6R)-1-[6-(3-Amino-1H-indazol-6-yl)-2-(methylamino)-4-pyrimi-
dinyl]-6-methyl-N-phenyl-3-piperidinecarboxamide ((3S,6R)-28b).
Methyl (3S,6R)-6-methyl-3-piperidinecarboxylate L-tartaric acid salt (4.0 g,
13.02 mmol) was dissolved in water (25 mL), to which was added
The title compound was prepared from (3S,6R)-1-[6-chloro-2-(methyl-
amino)-4-pyrimidinyl]-N-cyclohexyl-6-methyl-3-piperidinecarboxa-
mide following synthetic route 1. LC-MS (ES) m/z = 463 [M þ H]þ. 1H
NMR (400 MHz, CD3OD): δ 1.16-1.32 (m, 3H), 1.29 (d, J = 6.8 Hz,
3H), 1.34-1.45 (m, 2H), 1.65-1.68 (m, 1H), 1.76-1.81 (m, 5H),
1.85-1.92 (m, 2H), 1.97-2.05 (m, 1H), 2.35-2.42 (m, 1H), 2.97 (s,
3H), 3.11-3.15 (m, 1H), 3.64-3.70 (m, 1H), 4.45-4.65 (bs, 1H),
4.72-4.92 (bs, 1H), 6.45 (s, 1H), 7.52 (dd, J = 8.5, 1.1 Hz, 1H), 7.75 (d,
J = 8.3 Hz, 1H), 7.85 (s, 1H). 13C NMR (101 MHz, DMSO-d6): δ 14.6,
22.9, 24.6, 25.3, 27.9, 29.0, 32.5, 42.6, 47.2, 89.1, 107.7, 114.7, 116.6,
119.9, 136.7, 141.7, 149.2, 162.5, 163.0, 163.3, 172.3
(3S,6R)-1-[2-Amino-6-(4-cyano-3-fluorophenyl)-4-pyrimidinyl]-6-
methyl-3-piperidinecarboxylic acid (26). Into a 75 mL sealable tube
were added 3-methyl 1-(phenylmethyl) (3S,6R)-6-methyl-1,3-piperidi-
nedicarboxylate (10 g, 34.3 mmol), 1,4-dioxane (20 mL), and concen-
trated HCl (20 mL), and the mixture was stirred at 100 °C for 3 h. The
mixture was then cooled to room temperature. The solution was transferred
to a 500 mL round-bottom flask and concentrated to dryness. Tritura-
tion with Et2O and CH3CN afforded the HCl salt of (3S,6R)-6-methyl-
3-piperidinecarboxylic acid (6.17 g) as a colorless oil that turned into a
white solid after standing overnight. 1H NMR (400 MHz, CD3OD): δ
1.34 (d, J = 6.6 Hz, 3H), 1.46-1.63 (m, 1H), 1.83-2.03 (m, 2H), 2.13-
2.33 (m, 1H), 2.88-3.01 (m, 1H), 3.18 (dd, J = 13.1, 4.0 Hz, 1H), 3.22-
3.31 (m, 1H), 3.58-3.71 (m, 1H).
A mixture of 4,6-dichloro-2-pyrimidinamine (3.0 g, 18.3 mmol),
(3S,6R)-6-methyl-3-piperidinecarboxylic acid (3.98 g, 20.12 mmol),
and NaHCO3 (7.68 g, 91 mmol) in 1,4-dioxane (100 mL) and water
(50 mL) was stirred overnight at 117 °C in a sealed tube. The reaction
was allowed to cool to room temperature. LC-MS showed that most of
the 4,6-dichloro-2-pyrimidinamine had been consumed. Then 4-cyano-
3-fluorobenzeneboronic acid (3.32 g, 20.12 mmol) and Pd(Ph3P)4
(0.423 g, 0.366 mmol) were added, and the reaction mixture was stirred
for 24 h at 117 °C. The mixture was poured into water (300 mL) and
EtOAc (200 mL). The pH of the solution was 9, and the desired product
was in the aqueous layer. The aqueous layer was separated from the
organic layer, and then it was filtered. 6N HCl was added dropwise to the
filtrate to make the pH = 4. A precipitate formed. The precipitate was
filtered, washed with water, and dried to afford the crude title compound
(3.6 g) as a light-yellow solid. LC-MS (ES) m/z = 356 [M þ H]þ. 1H
NMR (400 MHz, DMSO-d6): δ 1.13 (d, J = 6.8 Hz, 3H), 1.56-1.94 (m,
5H), 2.26-2.39 (m, 1H), 2.86 (bs, 1H), 6.26 (s, 2H), 6.77 (s, 1H),
7.97-8.05 (m, 1H), 8.10-8.24 (m, 2H), 12.47 (bs, 1H).
(3S,6R)-1-[2-Amino-6-(3-amino-1H-indazol-6-yl)-4-pyrimidinyl]-
N-cyclohexyl-6-methyl-3-piperidinecarboxamide ((3S,6R)-28c). To
a solution of (3S,6R)-1-[2-amino-6-(4-cyano-3-fluorophenyl)-4-pyrimi-
dinyl]-6-methyl-3-piperidinecarboxylic acid (415 mg, 1.128 mmol) and
HATU (533 mg, 1.401 mmol) in DMF (3 mL) in a 10 mL round-bottom
flask was added H€unig’s base (0.408 mL, 2.34 mmol), and the resulting
mixture was stirred at room temperature for 15 min. Cyclohexylamine (0.16
mL, 1.401 mmol) was added, and the reaction mixture was stirred at room
temperature for 0.5 h. LC-MS showed the reaction was complete. The
reaction was poured into water, and EtOAc was added to extract the pro-
duct. The organic layer was concentrated, and the formed solid was recrys-
tallized from CH3CN to afford (3S,6R)-1-[2-amino-6-(4-cyano-3-fluoro-
phenyl)-4-pyrimidinyl]-N-cyclohexyl-6-methyl-3-piperidinecarboxamide
(245 mg) as a light-yellow solid. LC-MS (ES) m/z = 437 [M þ H]þ.
In a microwave vial, (3S,6R)-1-[2-amino-6-(4-cyano-3-fluorophenyl)-
4-pyrimidinyl]-N-cyclohexyl-6-methyl-3-piperidinecarboxamide (235 mg,
0.538 mmol), EtOH (5 mL), H€unig’s base (0.376 mL, 2.153 mmol), and
anhydrous hydrazine (0.101 mL, 3.23 mmol) were combined, and the
yellow suspension was heated overnight at 110 °C in an oil bath. When
the temperature of the reaction reached 100 °C all of the solids were
dissolved. After overnight, there was a yellow suspension as well as some
black-colored solid formed. LCMS showed mainly product. The black
LiOH H2O (1.80 g, 43.0 mmol, 3.3 equiv). The mixture was stirred at
3
room temperature for 20 h. LC-MS showed complete ester hydrolysis.
To this mixture was added NaHCO3 (4.81 mg, 57.3 mmol, 4.4 equiv),
4,6-dichloro-N-methyl-2-pyrimidinamine (2.32 g, 13.02 mmol, 1 equiv),
and 1,4-dioxane (25 mL). The reaction mixture was heated under reflux
at 100 °C for 24 h. The mixture was concentrated in vacuo. The resulting
residue was suspended in 40 mL of water, to which was added cold 2N
HCl until pH = 3. The resulting mixture was filtered, and the solids were
washed with water, dried under house vacuum at room temperature for
18 h, and then under vacuum over P2O5 at room temperature for 24 h to
give (3S,6R)-1-[6-chloro-2-(methylamino)-4-pyrimidinyl]-6-methyl-3-
piperidinecarboxylic acid (25) (3.23 g) as a solid. LC-MS (ES) m/z =
285 [M þ H]þ.
To (3S,6R)-1-[6-chloro-2-(methylamino)-4-pyrimidinyl]-6-methyl-
3-piperidinecarboxylic acid (880 mg, 3.09 mmol) in CH2Cl2 (15 mL)
at room temperature was added H€unig’s base (1.62 mL, 9.27 mmol,
3 equiv) and aniline (0.56 mL, 6.18 mmol, 2 equiv), and the resulting
mixture was chilled in an ice bath. To this stirred solution was added
HATU (1.29 g, 3.40 mmol, 1.1 equiv) in one portion. The resulting
suspension was stirred in the ice bath for 45 min. LC-MS showed
complete conversion. The mixture was filtered, and the filtrate was
concentrated in vacuo. Silica gel column chromatography with gra-
dient elution of 1% EtOAc in CHCl3 to 35% EtOAc in CHCl3 gave
(3S,6R)-1-[6-chloro-2-(methylamino)-4-pyrimidinyl]-6-methyl-N-phe-
nyl-3-piperidinecarboxamide (700 mg). LC-MS (ES) m/z = 360 [M þ
H]þ. 1H NMR (400 MHz, CD3OD): δ 1.27 (d, J = 6.8 Hz, 3H), 1.71-
1.95 (m, 3H), 2.03-2.14 (m, 1H), 2.49-2.62 (m, 1H), 2.87 (s, 3H),
3.08-3.23 (m, 1H), 6.11 (s, 1H), 7.06-7.16 (m, 1H), 7.26-7.38 (m,
2H), 7.52-7.62 (m, 2H).
The title compound was prepared from (3S,6R)-1-[6-chloro-2-(methyl-
amino)-4-pyrimidinyl]-6-methyl-N-phenyl-3-piperidinecarboxamide
following route 2. LC-MS (ES) m/z = 457 [M þ H]þ. 1H NMR (400
MHz, CD3OD): δ 1.33 (d, J = 6.8 Hz, 3H), 1.82-1.97 (m, 3H), 2.06-
2.16 (m, 1H), 2.57-2.65 (m, 1H), 2.98 (s, 3H), 3.15-3.25 (m, 1H),
6.51 (s, 1H), 7.12 (t, J = 7.5 Hz, 1H), 7.34 (t, J = 8.1 Hz, 2H), 7.54 (dd,
J = 8.5, 1.1 Hz, 1H), 7.60 (dd, J = 8.6, 1.3 Hz, 2H), 7.76 (d, J = 8.3 Hz,
1H), 7.87 (s, 1H).
(3S,6R)-1-[6-(3-Amino-1H-indazol-6-yl)-2-(methylamino)-4-pyrimi-
dinyl]-N-cyclohexyl-6-methyl-3-piperidinecarboxamide ((3S,6R)-28d).
To a suspension of (3S,6R)-1-[6-chloro-2-(methylamino)-4-pyrimidinyl]-
6-methyl-3-piperidinecarboxylic acid (3.05 g, 10.71 mmol) in CH2Cl2
(50 mL) at room temperature was added H€unig’s base (2.70 mL, 15.43
mmol, 1.3 equiv) and cyclohexylamine (1.60 mL, 14.2 mmol, 1.2 equiv),
and the resulting mixture was chilled in an ice bath. To this stirred
solution was added HATU (4.96 g, 13.1 mmol, 1.1 equiv) in one portion,
and the resulting suspension was stirred in the ice bath for 30 min. LC-
MS showed complete conversion. The mixture was diluted with CH2Cl2
(50 mL) and filtered through Celite. The filtrate was washed water (2 ꢀ
25 mL) and then brine. The organic layer was dried over Na2SO4,
filtered, and concentrated in vacuo. Silica gel column chromatography
using gradient elution of 1% EtOAc in CHCl3 to 50% EtOAc in
CHCl3 afforded (3S,6R)-1-[6-chloro-2-(methylamino)-4-pyrimidinyl]-
N-cyclohexyl-6-methyl-3-piperidinecarboxamide (4.26 g) as a foam. LC-
MS (ES) m/z = 366 [M þ H]þ.
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solid and the yellow solid were carefully separated due to the black solid
being heavier than yellow solid in the CH3OH solvent. The yellow solid
in CH3OH was filtered and washed with CH3OH to remove the color
and afford the title compound (157 mg) as a white solid. LC-MS (ES)
m/z = 449 [M þ H]þ. 1H NMR (400 MHz, DMSO-d6): δ 1.05-1.35 (m,
8H), 1.50-1.88 (m, 9H), 2.24 (s, 1H), 2.90 (s, 1H), 3.46-3.60 (m, 1H),
5.38 (s, 2H), 6.07 (s, 2H), 6.57 (s, 1H), 7.57 (d, J = 8.3 Hz, 1H), 7.70 (d, J =
8.3 Hz, 1H), 7.79 (d, J = 7.6 Hz, 1 H), 7.94 (s, 1H), 11.49 (s, 1H).
[(2S,5R)-5-Methyl-4-(phenylmethyl)-2-morpholinyl]methanol
(29a). To a stirred solution of (2R)-2-[(phenylmethyl)amino]-1-pro-
panol (1.65 g, 9.99 mmol) in toluene (50 mL) was added (R)-(-)-
epichlorohydrin (1.02 mL, 12.98 mmol), followed by lithium perchlo-
rate (1.062 g, 9.99 mmol) under nitrogen. After stirring 2 days, TLC (5%
methanol-dichloromethane) showed there was only a trace of starting
material and a major product. A solution of sodium methoxide (25 wt %
in CH3OH) (5.71 mL, 24.96 mmol) was then added and the mixture was
stirred for 3 days. Saturated aq NH4Cl (75 mL) was added, and the
product was extracted with EtOAc (3 ꢀ 75 mL). The combined organics
were washed with brine, dried (MgSO4), filtered, and evaporated to give
the crude product, which was purified by chromatography (Analogix RS-
120 silica cartridge) eluting with 20-50% EtOAc in hexanes to afford
the title compound (1.11 g) as a colorless oil. LC-MS (ES) m/z = 222
[M þ H]þ. 1H NMR (400 MHz, CDCl3): δ 1.12 (d, J = 6.6 Hz, 3H),
2.23-2.61 (m, 3H), 2.80 (m, 1H), 3.49-3.57 (m, 1H), 3.60-3.77 (m,
5H), 3.78-3.86 (m, 1H), 7.17-7.47 (m, 5H). [R]D = -5.2 (c = 1.55,
CH3OH, 23.5 °C).
[(2S,5R)-4-(2-Amino-6-chloro-4-pyrimidinyl)-5-methyl-2-morpho-
linyl]methanol (30a). To a stirred solution of [(2S,5R)-5-methyl-
4-(phenylmethyl)-2-morpholinyl]methanol (1.05 g, 4.74 mmol) in
ethanol (15 mL) was added concentrated hydrochloric acid (0.435 mL,
5.22 mmol). The mixture was purged with nitrogen to degas, and then
10% palladium on carbon (Degussa Type E101 NE/W, 50% wet, 150 mg,
0.070 mmol) was added and the mixture was purged with hydrogen and
then stirred under a balloon of H2. After stirring 4 h, TLC (50% EtOAc-
hexanes) showed no starting material and baseline product. The mixture
was degassed with nitrogen, filtered through Celite, and evaporated to
dryness to afford [(2S,5R)-5-methyl-2-morpholinyl]methanol (880 mg)
as the hydrochloride salt. 1H NMR (400 MHz, D2O): δ 1.34 (d, J = 7.1
Hz, 3H), 3.04-3.12 (m, 1H), 3.14-3.24 (m, 1H), 3.50-3.67 (m, 3H),
3.74-3.83 (m, 2H), 3.83-3.89 (m, 1H).
In a microwave vessel, H€unig’s base (0.59 mL, 3.35 mmol) was added
to [(2S,5R)-5-methyl-2-morpholinyl]methanol hydrochloride (208 mg,
1.12 mmol) in CH3CN (3 mL), and then 2-amino-4,6-dichloropyrimi-
dine (174 mg, 1.06 mmol) was added. The mixture was heated with stirring
in a microwave reactor at 160 °C for 1 h. HPLC indicated complete conver-
sion. The mixture was filtered through a 0.45 μm filter disk, evaporated, and
the product was purified further by chomatography (Analogix SF25-40 g
silica column) eluting with 2-5% CH3OH in CHCl3 to afford the title
compound (258 mg) as a white foam. LC-MS (ES) m/z = 260 [M þ H]þ.
1H NMR (400 MHz, CDCl3):δ1.28 (d, J= 6.8 Hz, 3H), 1.98 (bs, 1H), 2.99
(bs, 1H), 3.53-3.63 (m, 1H), 3.70 (bs, 1H), 3.75-3.82 (m, 2H), 3.83-3.89
(m, 1H), 4.87 (bs, 2H), 5.95 (s, 1H).
(2S,5R)-4-[2-Amino-6-(3-amino-1H-indazol-6-yl)-4-pyrimidinyl]-5-
methyl-N-phenyl-2-morpholinecarboxamide (34a). In a sealable vessel
was added (4-cyano-3-fluorophenyl)boronic acid (163 mg, 0.986 mmol),
[(2S,5R)-4-(2-amino-6-chloro-4-pyrimidinyl)-5-methyl-2-morpholi-
nyl]methanol (232 mg, 0.897 mmol) and 1,4-dioxane (5 mL), and
saturated aqueous NaHCO3 (2.5 mL). The mixture was purged with
nitrogen to degas, and then Pd(PPh3)4 (104 mg, 0.090 mmol) was added
and the vessel was sealed and heated at 100 °C overnight. HPLC and LC-
MS indicated the product had formed but some starting pyrimidine
remained. Another portion of (4-cyano-3-fluorophenyl)boronic acid
(50 mg, 0.303 mmol) was added, and the reaction was stirred at 120 °C
in a microwave for 30 min. The reaction was worked up by diluting with
toluene and water. The aqueous layer was extracted with toluene (2 ꢀ
5 mL) and the combined organics were washed with water and brine, dried
(MgSO4), filtered, and evaporated. The product was purified by silica gel
chromatography (Analogix SF25-40 g) eluting with 2-10% CH3OH in
CHCl3 to give 4-{2-amino-6-[(2S,5R)-2-(hydroxymethyl)-5-methyl-4-
morpholinyl]-4-pyrimidinyl}-2-fluorobenzonitrile (193 mg) as an off-white
foam. LC-MS (ES) m/z= 344 [M þ H]þ. 1H NMR (400 MHz, CDCl3):δ
1.32 (d, J = 6.8 Hz, 3H), 2.06 (d, J = 6.1 Hz, 1H), 3.05 (bs, 1H), 3.59-3.68
(m, 1H), 3.68-3.78 (m, 1H), 3.78-3.94 (m, 3H), 4.91 (bs, 2H), 6.31 (s,
1H), 7.70 (dd, J = 8.3, 6.6 Hz, 1H), 7.78-7.92 (m, 2H).
A stock solution of oxidant was prepared by dissolving H5IO6 (11.4 g,
50 mmol) and CrO3 (23 mg, 1.15 mol %) in wet CH3CN (0.75% water)
to a volume of 114 mL (takes about 2 h to dissolve).
In a 100 mL RB was placed 4-{2-amino-6-[(2S,5R)-2-(hydroxymethyl)-
5-methyl-4-morpholinyl]-4-pyrimidinyl}-2-fluorobenzonitrile (188 mg,
0.548 mmol) and wet CH3CN (0.75% water, 4.5 mL). The stirred
solution was cooled in an ice bath, and 4.37 mL of the above stock solution
was added very slowly dropwise (over 10 min). After 2 h, there was a small
amount of starting material observed by TLC (NaHCO3 added to an
aliquot, 5% CH3OH-CHCl3), so the reaction was placed in the refrigerator
overnight. At this time, there was no starting material by TLC. The reaction
was quenched by adding Na2HPO4 (0.5 g) in 5 mL of water. After stirring a
few minutes, the mixture was cloudy and the pH = 5. The product was
extracted with EtOAc (5 ꢀ 10 mL), and the combined organics were washed
with 5% NaHSO3, brine, dried (MgSO4), filtered, and evaporated to afford
the crude (2S,5R)-4-[2-amino-6-(4-cyano-3-fluorophenyl)-4-pyrimidinyl]-
5-methyl-2-morpholinecarboxylic acid (38a) (102 mg) as an off-white solid.
LC-MS (ES) m/z = 358 [M þ H]þ.
A 5 mL vial was charged with (2S,5R)-4-[2-amino-6-(4-cyano-3-
fluorophenyl)-4-pyrimidinyl]-5-methyl-2-morpholinecarboxylic acid (72 mg,
0.201 mmol), HOBt (29.9 mg, 0.222 mmol), and DMF (1 mL) under
nitrogen. The mixture was stirred and cooled in an ice bath, and then
EDC (42.5 mg, 0.222 mmol) was added. After stirring 10 min, aniline
(0.020 mL, 0.222 mmol) was added and the mixture was allowed to
warm to room temperature and stir. After 4 h, there was no change in
progress. The mixture was diluted with EtOAc and washed with water,
saturated NaHCO3, brine, and dried (MgSO4). After filtering, the filtrate
was concentrated, and the product was purified by silica gel chromatog-
raphy (Analogix SF-4 g) eluting with 20-50% EtOAc in CHCl3 to
afford (2S,5R)-4-[2-amino-6-(4-cyano-3-fluorophenyl)-4-pyrimidinyl]-
5-methyl-N-phenyl-2-morpholinecarboxamide (59 mg) as a light-orange
oil. LC-MS (ES) m/z = 433 [M þ H]þ.
(2S,5R)-4-[2-amino-6-(4-cyano-3-fluorophenyl)-4-pyrimidinyl]-5-
methyl-N-phenyl-2-morpholinecarboxamide (57 mg, 0.132 mmol) was
dissolved in ethanol (3 mL) with stirring in a 5 mL microwave vessel.
Hydrazine monohydrate (150 μL, 3.09 mmol) was added, and the
mixture was capped and heated at 100 °C in an oil bath for 24 h. The
mixture was partitioned between EtOAc and water, and the aqueous
layer was extracted with EtOAc. The combined organics were washed
with brine, dried (MgSO4), filtered, and evaporated. Purification by silica gel
chromatograpy (Analogix RS-4 g) eluting with 90:10:1 CHCl3:CH3OH:
conc aq NH4OH afforded the title compound (19 mg, 0.041 mmol,
30.8% yield) as a colorless film/foam. NMR showed a residual solvent
peak with singlets at 1.9 and 3.36, so the material was azeotroped
1
with CH3CN and then triturated to give a white powder. H NMR
(400 MHz, DMSO-d6): δ 1.24 (d, J = 6.3 Hz, 3H), 3.04 (bs, 1H),
3.82 (d, J = 9.8 Hz, 1H), 3.93 (d, J = 11.1 Hz, 1H), 4.07-4.23 (m, 1H),
4.53 (bs, 2H), 5.39 (bs, 2H), 6.22 (bs, 2H), 6.61 (s, 1H), 7.10 (t, J = 7.1
Hz, 1H), 7.34 (t, J = 7.7 Hz, 2H), 7.60 (d, J = 8.3 Hz, 1H), 7.69-7.78 (m,
3H), 7.96 (s, 1H), 9.87 (bs, 1H), 11.52 (s, 1H).
{(2S,5R)-4-[6-Chloro-2-(methylamino)-4-pyrimidinyl]-5-methyl-2-
morpholinyl}methanol (30b). The title compound was prepared simi-
larly to 30a but using pyrimidine 27. LC-MS (ES) m/z = 273, 275 [M þ
H]þ. 1H NMR (400 MHz, DMSO-d6): δ 1.15 (d, J = 6.8 Hz, 3H), 2.76
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(s, 3H), 2.85 (bs, 1H), 3.31-3.55 (m, 3H), 3.56-3.66 (m, 1H), 3.67-3.78
(m, 1H), 6.19 (bs, 1H).
aniline (0.088 mL, 0.964 mmol), HOAt (0.131 g, 0.964 mmol), and
EDC (0.222 g, 1.157 mmol), and the reaction mixture was stirred at room
temperature overnight. The reaction mixture was washed with water (2ꢀ),
and the organics were dried over Na2SO4 and concentrated to afford 1,1-
dimethylethyl (2S,5R)-5-ethyl-2-[(phenylamino)carbonyl]-4-morpholine-
carboxylate (0.247 g) as a brown oil. LC-MS (ES) m/z = 240 [M þ H]þ.
1,1-Dimethylethyl (2S,5R)-5-ethyl-2-[(phenylamino)carbonyl]-4-
morpholinecarboxylate (0.269 g, 0.804 mmol) was taken up in HCl in
1,4-dioxane (4 M, 0.024 mL, 0.804 mmol) and stirred at room tem-
perature overnight. The reaction was concentrated to yield an HCl salt of
(2S,5R)-5-ethyl-N-phenyl-2-morpholinecarboxamide (33a) (0.198 g)
as a brown oil. LC-MS (ES) m/z = 235 [M þ H]þ.
(2S,5R)-4-[6-(3-amino-1H-indazol-6-yl)-2-(methylamino)-4-pyrimi-
dinyl]-N-cyclohexyl-5-methyl-2-morpholinecarboxamide (34c). The
title compound was prepared from 30b following similar synthetic
procedures to the synthesis of 34a. LC-MS (ES) m/z = 465 [M þ
H]þ. 1H NMR (400 MHz, DMSO-d6): δ 1.04-1.16 (m, 1H), 1.19 (d,
J = 6.6 Hz, 3H), 1.23-1.37 (m, 4H), 1.52-1.63 (m, 1H), 1.63-1.79 (m,
4H), 2.76-2.99 (m, 4H), 3.55-3.68 (m, 1H), 3.74 (dd, J = 11.4, 2.8 Hz,
1H), 3.86 (d, J = 11.1 Hz, 1H), 3.91 (dd, J = 11.4, 2.8 Hz, 1H), 4.20-
4.83 (bs, 2H), 5.39 (s, 2H), 6.50-6.71 (m, 2H), 7.56-7.69 (m, 2H),
7.72 (d, J = 8.3 Hz, 1H), 7.99 (s, 1H), 11.50 (s, 1H).
[(2S,5R)-5-Ethyl-4-(phenylmethyl)-2-morpholinyl]methanol (29b). To
(R)-(-)-2-amino-1-butanol (5 g, 56.1 mmol) in CH3OH (120 mL) was
added benzaldehyde (6.24 mL, 61.7 mmol), and the reaction mixture
was stirred under nitrogen for 15 min. The mixture was then cooled in an
ice bath, and sodium borohydride (2.33 g, 61.7 mmol) was added
portionwise. The mixture was stirred in the ice bath for 1.5 h. NaOH
(6 N, 25 mL) was added, and the mixture was concentrated. The resulting
residue was taken up in 100 mL of H2O and extracted with Et2O (2ꢀ).
The organics were combined, dried over Na2SO4, and concentrated to
afford (2R)-2-[(phenylmethyl)amino]-1-butanol (10.92 g) as a fluffy
white solid. LC-MS (ES) m/z = 180 [M þ H]þ.
The title compound was prepared from amine 33a following route 1.
LC-MS (ES) m/z = 473 [M þ H]þ. 1H NMR (400 MHz, DMSO-d6): δ
0.92 (t, 3H), 1.74-1.86 (m, 2H), 3.34 (s, 3H), 3.71-3.74 (m, 1H),
3.74-3.77 (m, 1H), 4.03 (bs, 1H), 4.06 (bs, 1H), 4.10-4.16 (m, 1H),
4.64-4.85 (m, 1H), 5.39 (bs, 2H), 6.60-6.67 (m, 2H), 7.07-7.13 (m,
1H), 7.31-7.36 (m, 2H), 7.60-7.64 (m, 1H), 7.70-7.74 (m, 3H), 8.00
(s, 1H), 9.87 (bs, 1H), 11.50 (bs, 1H).
(2S,5R)-4-[6-(3-Amino-1H-indazol-6-yl)-2-(methylamino)-4-pyrimi-
dinyl]-N-cyclohexyl-5-ethyl-2-morpholinecarboxamide (34d). The
title compound was synthesized following synthetic procedures
similar to the preparation of 34b. TFA salt. LC-MS (ES) m/z = 479
[M þ H]þ. 1H NMR (400 MHz, DMSO-d6): δ 0.85-0.92 (m, 3H),
1.07-1.15 (m, 2H), 1.20-1.35 (m, 4H), 1.52-1.61 (m, 2H), 1.65-
1.80 (m, 4H), 2.81-2.88 (m, 5H), 3.56-3.63 (m, 1H), 3.63-3.69
(m, 1H), 3.86-3.92 (m, 1H), 3.93-3.99 (m, 1H), 4.57-4.83 (m,
2H), 5.38 (bs, 1H), 6.53-6.64 (m, 2H), 7.58-7.67 (m, 2H), 7.70-
7.74 (m, 1H), 7.97 (bs, 1H), 11.49 (bs, 1H).
1,1-Dimethylethyl {1-[2-Amino-6-(3-amino-1H-indazol-6-yl)-4-pyrimi-
dinyl]-3-piperidinyl}carbamate ((()-37b). The title compound was
prepared from amine 36 following synthetic route 1. LC-MS (ES) m/z =
425 [M þ H]þ. 1H NMR (400 MHz, DMSO-d6): δ 1.38 (s, 9H), 1.43
(m, 2H), 1.66-1.79 (m, 1H), 1.80-1.95 (m, 1H), 2.78-2.93 (m, 1H),
2.95-3.10 (m, 1H), 3.29-3.40 (m, 1H), 4.11 (d, J = 12.6 Hz, 1H), 4.26
(bs, 1H), 5.38 (s, 2H), 6.08 (bs, 2H), 6.57 (s, 1H), 6.93 (d, J = 7.3 Hz,
1H), 7.55 (d, J = 8.3 Hz, 1H), 7.70 (d, J = 8.3 Hz, 1H), 7.93 (s, 1H),
11.51 (s, 1H).
To a solution of (2R)-2-[(phenylmethyl)amino]-1-butanol (10.06 g,
56.1 mmol) in 1,2-dichloroethane (DCE, 250 mL) was added (R)-(-)-
epichlorohydrin (6.75 g, 72.9 mmol), followed by lithium perchlorate
(5.97 g, 56.1 mmol) under nitrogen. The reaction was stirred for 2 days
at room temperature, then sodium ethoxide (21 wt % in ethanol, 52.4
mL, 140 mmol) was added and the reaction continued to stir for 3 days.
Saturated NH4Cl was added, and the product was extracted with EtOAc.
The organic layers were combined, dried over Na2SO4, and concen-
trated to afford the title compound (16.2 g) as a colorless oil. LC-MS
(ES) m/z = 236 [M þ H]þ. 1H NMR (400 MHz, DMSO-d6): δ 0.80 (t,
J = 7.5 Hz, 3H), 1.44-1.71 (m, 2H), 2.26-2.45 (m, 3H), 3.19-3.50 (m,
4H), 3.49-3.80 (m, 3H), 4.60 (bs, 1H), 7.19-7.27 (m, 1H), 7.28-7.37
(m, 4H).
(2S,5R)-4-[6-(3-Amino-1H-indazol-6-yl)-2-(methylamino)-4-pyrimi-
dinyl]-5-ethyl-N-phenyl-2-morpholinecarboxamide (34b). [(2S,5R)-
5-Ethyl-4-(phenylmethyl)-2-morpholinyl]methanol (0.5 g, 2.125 mmol)
was dissolved in CH3OH (20 mL) and placed under a nitrogen atmosphere.
Palladium on carbon (10 wt %, 0.023 g, 0.212 mmol) was added, and the
flask was flushed with nitrogen and evacuated (3ꢀ). Then the reaction was
placed under an atmosphere of hydrogen (balloon) and stirred at room
temperature overnight. The reaction mixture was filtered through Celite and
concentrated to afford [(2S,5R)-5-ethyl-2-morpholinyl]methanol (0.309 g)
as a colorless oil. LC-MS (ES) m/z = 146 [M þ H]þ.
Each pure enantiomer of 37b was prepared following the same synthetic
route as described for ((()-37b), starting from the corresponding pure
enantiomer of 36.
N-{1-[2-Amino-6-(3-amino-1H-indazol-6-yl)-4-pyrimidinyl]-3-pi-
peridinyl}benzamide ((()-37a). 1,1-Dimethylethyl {1-[2-amino-
6-(3-amino-1H-indazol-6-yl)-4-pyrimidinyl]-3-piperidinyl}carbamate
(865 mg, 2.04 mmol) was added portionwise to ice-cooled concen-
trated hydrochloric acid (8 mL, 96 mmol) with stirring. A solid yellow
mass formed. The ice bath was removed, and the reaction was allowed
to warm to room temperature with stirring. HPLC indicated com-
plete conversion. The mixture was diluted with ice-water (20 mL),
and the solution was concentrated under reduced pressure. The
residue was dissolved in ethanol (15 mL) and evaporated, followed by
suspending in CH3CN (15 mL) and evaporating. The residue was
then triturated with 2-propanol (10 mL) and filtered, washed with
2-propanol followed by hexanes, and dried under vacuum to afford
6-[2-amino-6-(3-amino-1-piperidinyl)-4-pyrimidinyl]-1H-indazol-3-amine
trihydrochloride (761 mg) as a yellow powder. A 140 mg portion was
suspended in CH3OH (2 mL) and water (1 mL) and was brought to
pH 10 with 1 N NaOH. The solution was filtered and purified by
Gilson automated reverse phase HPLC (Gemini Phenomenex C18
5 μ, 100 mm ꢀ 300 mm), eluting with 5-90% CH3CN in water þ
0.1%NH4OH). Pure fractions were combined, evaporated, and dried
to afford the free base (72 mg) as a white solid. LC-MS (ES) m/z =
325 [M þ H]þ. 1H NMR (400 MHz, DMSO-d6): δ 1.15-1.29 (m,
1H), 1.31-1.46 (m, 1H), 1.51 (bs, 2H), 1.62-1.75 (m, 1H), 1.87 (d,
To [(2S,5R)-5-ethyl-2-morpholinyl]methanol (0.309 g, 2.125 mmol)
in THF (10 mL) were added Boc2O (0.493 mL, 2.125 mmol) and
H€unig’s base (0.371 mL, 2.125 mmol), and the reaction mixture was
heated to 40 °C and stirred overnight. The reaction was then cooled to
room temperature and concentrated to afford 1,1-dimethylethyl
(2S,5R)-5-ethyl-2-(hydroxymethyl)-4-morpholinecarboxylate (0.488 g).
LC-MS (ES) m/z = 246 [M þ H]þ.
To a vigorously stirred solution of 1,1-dimethylethyl (2S,5R)-5-ethyl-
2-(hydroxymethyl)-4-morpholinecarboxylate (2.52 g, 10.27 mmol) in
CH2Cl2 (50 mL) that was cooled to 0 °C were added TEMPO (0.321 g,
2.054 mmol) and (diacetoxyiodo)benzene (7.28 g, 22.60 mmol). The
ice bath was removed, and the reaction was allowed to warm to room tem-
perature and stirred overnight. The reaction was quenched with CH3OH
and then concentrated to afford crude (2S,5R)-4-{[(1,1-dimethylethyl)-
oxy]carbonyl}-5-ethyl-2-morpholinecarboxylic acid (32) (0.711 g) as a
yellow oil. LC-MS (ES) m/z = 260 [M þ H]þ.
To (2S,5R)-4-{[(1,1-dimethylethyl)oxy]carbonyl}-5-ethyl-2-morpho-
linecarboxylic acid (0.250 g, 0.964 mmol) in CH2Cl2 (10 mL) was added
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Journal of Medicinal Chemistry
ARTICLE
J = 10.6 Hz, 1H), 2.53-2.68 (m, 2H), 2.83 (t, J = 12.1 Hz, 1H), 4.30
(m, 2H), 5.38 (s, 2H), 6.03 (bs, 2H), 6.58 (s, 1H), 7.56 (d, J = 8.6 Hz,
1H), 7.70 (d, J = 8.3 Hz, 1H), 7.94 (s, 1H), 11.52 (s, 1H).
1-dimethylethyl)oxy]carbonyl}amino)-2-methyl-1-piperidinecarbox-
ylate (88.28 g) as a thick clear syrup. LC-MS (ES) m/z = 349 [M þ
H]þ. 1H NMR (400 MHz, CD3OD): δ 1.17 (d, J = 7.1 Hz, 3H), 1.45
(s, 9H), 1.56-1.66 (m, 2H), 1.73-1.78 (m, 2H), 2.66 (t, J = 12.0 Hz,
1H), 4.41-4.44 (m, 1H), 5.14 (s, 2H), 7.31-7.39 (m, 5H).
Pd/C (6.30 g) was added to a 2 L Parr bottle, followed by addition of
EtOAc (50 mL) under nitrogen. The mixture was stirred as a slurry,
followed by addition of a solution of phenylmethyl (2S,5R)-5-({[(1,1-
dimethylethyl)oxy]carbonyl}amino)-2-methyl-1-piperidinecarboxylate
(58 g, 166 mmol) in EtOAc (330 mL). The mixture was shaken under 50
psi of hydrogen at room temperature for 4 h. LC-MS showed complete
conversion. The mixture was filtered through Celite and rinsed with
EtOAc. The filtrate was concentrated in vacuo to give the title com-
pound (35.47 g) as an oil. LC-MS (ES) m/z = 215 [M þ H]þ. 1H NMR
(400 MHz, CD3OD): δ 1.13 (d, J = 6.3Hz, 3H), 1.34-1.40 (m, 1H), 1.47
(s, 9H), 1.56-1.62 (m, 1H), 1.66-1.75 (m, 1H), 1.79-1.86 (m, 1H),
2.71-2.78 (m, 1H), 2.87 (m, 1H),) 2.98-3.05 (m, 1H), 3.61 (bs, 1H).
1,1-Dimethylethyl {(3R,6S)-1-[2-Amino-6-(3-amino-1H-indazol-
6-yl)-4-pyrimidinyl]-6-methyl-3-piperidinyl}carbamate ((3R,6S)-
37c). The title compound was prepared from (3R,6S)-35 following
synthetic route 1. LC-MS (ES) m/z = 439 [M þ H]þ. 1H NMR (400
MHz, CD3OD): δ 1.25 (d, J = 7.1 Hz, 3H), 1.48 (s, 9H), 1.64-1.77
(m, 2H), 1.78-1.87 (m, 2H), 2.73 (t, J = 12.1 Hz, 1H), 3.41-3.48
(m, 1H), 4.50-4.60 (bs, 1H), 4.75-4.85 (bs, 1H), 6.52 (s, 1H), 7.48 (d,
J = 8.3 Hz, 1H), 7.77 (d, J = 8.3 Hz, 1H), 7.80 (s, 1H).
Sodium bicarbonate (145 mg, 1.73 mmol) was added to a solution of
6-[2-amino-6-(3-amino-1-piperidinyl)-4-pyrimidinyl]-1H-indazol-3-amine
trihydrochloride (150 mg, 0.346 mmol) in water (1.5 mL) with stirring.
Tetrahydrofuran (1.5 mL) was added, the mixture was cooled in an ice
bath, and benzoyl chloride (0.044 mL, 0.380 mmol) was added dropwise
with stirring. The mixture was then warmed to room temperature and stirred
for 2 h. LC-MS showed starting material, product, and bis-benzoylation.
Additional portions of benzoyl chloride (0.040 mL, 0.345 mmol) and
NaHCO3 (145 mg, 1.73 mmol) were added, and the mixture was stirred
for 1 h. HPLC showed complete conversion to multibenzoylated product.
The reaction was diluted with EtOAc, washed with water, dried (Na2SO4),
filtered, and evaporated. The crude residue was suspended in CH3OH
(8 mL), concentrated aqueous HCl (1 mL, 12 mmol) was added, and the
mixture was stirred for 3 days at 65 °C. HPLC showed conversion to the
desired product. The mixture was concentrated to ca. 2 mL and then
diluted with CH3CN (ca. 8 mL) and heated. Additional CH3CN was
added to the hot solution until turbid. The mixture was allowed to cool
slowly to room temperature with strring over 2 h. The precipitate was
collected by filtration and washed with CH3CN, then Et2O, and finally
hexanes. Drying afforded the title compound (156 mg) dihydrochloride
as an off-white solid. A 100 mg portion was purified further by reverse
phase HPLC (Gemini Phenomenex C18 5 μ, 100 mm ꢀ 300 mm),
eluting with 5-90% CH3CN in water þ 0.1%NH4OH) to give the title
compound (55 mg) as a white solid. LC-MS (ES) m/z = 429 [M þ H]þ.
1H NMR (400 MHz, DMSO-d6): δ 1.43-1.60 (m, 1H), 1.60-1.74 (m,
1H), 1.76-1.86 (m, 1H), 1.92-2.03 (m, 1H), 2.87-3.00 (m, 2H),
3.83-3.95 (m, 1H), 4.27-4.39 (m, 1H), 4.49 (bs, 1H), 5.38 (s, 2H),
6.12 (s, 2H), 6.64 (s, 1H), 7.46 (t, J = 7.3 Hz, 2H), 7.50-7.54 (m, 1H),
7.54-7.61 (m, 1H), 7.71 (d, J = 8.6 Hz, 1H), 7.87 (d, J = 7.1 Hz, 2H),
7.94 (s, 1H), 8.39 (d, J = 7.6 Hz, 1H), 11.52 (s, 1H).
1,1-Dimethylethyl [(3R,6S)-6-Methyl-3-piperidinyl]carbamate ((3R,6S)-
35). (3R,6S)-6-Methyl-1-{[(phenylmethyl)oxy]carbonyl}-3-piperi-
dinecarboxylic acid (prepared as described in the experimental procedure
for (3S,6R)-28b, 85 g, 307 mmol) in a 2 L RB flask was azeotroped with
toluene (3 ꢀ 200 mL). The residue (an oil) was dissolved in 500 mL
of t-butanol (anhydrous grade). To this solution was added triethy-
lamine (64.1 mL, 460 mmol, 1.5 equiv) at room temperature in one
portion. Diphenyl azidophosphate (110 g, 398 mmol, 1.3 equiv) was
added via an addition funnel at room temperature portionwise into
this stirred mixture. Addition took 20 min. The resulting mixture
(a light-yellow clear solution) was stirred at room temperature for
30 min, followed by heating in an oil bath to 100 °C under reflux for
20 h. LC-MS showed complete conversion. The mixture was con-
centrated in vacuo to remove as much t-butanol as possible. The
residue was diluted with EtOAc (1 L), which was washed with 200 mL
of cold 2N HCl, followed by 200 mL of 2.5 N NaOH. The NaOH
portion was salted with NaCl and back-extracted with EtOAc (400 mL). All
the organic portions were combined, washed with brine, dried over
MgSO4, filtered, and concentrated in vacuo. The residue was taken
up in hexane (500 mL) and EtOAc (20 mL). A waxy paste developed.
The mixture was chilled in the refrigerator for 20 h, resulting in a
two-phase mixture. The top layer was decanted off and gave 42 g as
an oil after concentration in vacuo, which would undergo further
silica gel column purification. The waxy bottom component was
taken up in CHCl3 (300 mL) and EtOAc (30 mL). The mixture was
stirred for 5 min and filtered. The filtrate was concentrated in vacuo
to give an oil (150 g), which would undergo further silica gel column
purification. The waxy solids (30 g) collected were identified as
(PhO)2POOH and were discarded. Silica gel column chromatogra-
phy of the above two oils on multiple runs using gradient elution of
1-50% EtOAc in CHCl3 gave phenylmethyl (2S,5R)-5-({[(1,
Compounds (()-cis-37c and (3S,6R)-37c were prepared from (()-
cis-35 and (3S,6R)-35, respectively, following the synthetic procedures
described for (2S,5R)-37c.
Dimethyl (4R)-N-{[(1,1-Dimethylethyl)oxy]carbonyl}-4-methyl-D-
glutamate (38). In a 20 mL RB flask was dissolved dimethyl D-glutamate
(25 g, 118 mmol) in CH3OH (150 mL). Triethylamine (36.2 mL,
260 mmol) was added, followed by Boc2O (33.5 g, 154 mmol), and the
reaction mixture was stirred at room temperature for 18 h. The reaction
was concentrated and then dissolved in 200 mL of CH2Cl2. The resulting
organic solution was washed with 1N HCl (2 ꢀ 50 mL), followed by
brine, dried over Na2SO4, filtered, and concentrated. Purification was
done on a 350 g Biotage SNAP column with gradient of 0-35% EtOAc in
hexane over 35 min to isolate dimethyl N-{[(1,1-dimethylethyl)oxy]-
1
carbonyl}-D-glutamate (30.7 g) as a clear oil. H NMR (400 MHz,
DMSO-d6): δ 1.38 (s, 9H), 1.79 (ddd, J = 14.5, 8.8, 6.1 Hz, 1H), 1.94 (t, J =
13.4 Hz, 1H), 2.33-2.43 (m, 2H), 3.59 (s, 3H), 3.62 (s, 3H), 4.00 (dd, J =
9.4, 7.6 Hz, 1H), 7.29 (d, J = 7.8 Hz, 1H).
Dimethyl N-{[(1,1-dimethylethyl)oxy]carbonyl}-D-glutamate (4 g,
14.53 mmol) in THF (40 mL) was placed in a 250 mL RB flask, and it
was cooled to -78 °C. LiHMDS (1.0 M in THF, 30.5 mL, 30.5 mmol)
was added dropwise, and the mixture was stirred at -78 °C for 0.5 h.
Methyl iodide (1.817 mL, 29.1 mmol) was then added as rapidly as
possible, and the solution was stirred at -78 °C for 4.5 h. The reaction
was then quenched with 1N HCl solution (40 mL) and extracted with
EtOAc (3 ꢀ 50 mL). The organics were combined and washed with
saturated NaHCO3 followed by brine, then dried over Na2SO4 and
concentrated to give a red-amber colored oil. The oil was purified on a
Biotage SNAP 50 g column with a gradient of 0 to 35% EtOAc in hexane
over 35 min to give the title compound (1.37 g) as a clear oil. 1H NMR
(400 MHz, DMSO-d6): δ 1.08 (d, J = 7.1 Hz, 3H), 1.38 (s, 9H), 1.71
(ddd, J = 13.6, 8.3, 5.1 Hz, 1H), 1.94 (ddd, J = 13.9, 10.1, 6.1 Hz, 1H),
2.43-2.49 (m, 1H), 3.60 (s, 3H), 3.62 (s, 3H), 4.01 (ddd, J = 9.9, 8.4, 5.2
Hz, 1H), 7.29 (d, J = 8.3 Hz, 1H).
1,1-Dimethylethyl [(1R,3R)-4-hydroxy-1-(hydroxymethyl)-3-methyl-
butyl]carbamate (39). To a solution of dimethyl (4R)-N-{[(1,1-di-
methylethyl)oxy]carbonyl}-4-methyl-D-glutamate (1.79 g, 6.19 mmol)
in ethanol (15 mL) and THF (15 mL) was added CaCl2 (2.83 g, 24.8
mmol), and the resulting mixture was cooled in an ice bath. Sodium
borohydride (1.873 g, 49.5 mmol) was added portionwise, and the white
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Journal of Medicinal Chemistry
ARTICLE
cloudy mixture was stirred for 0.5 h in an ice bath. The reaction was then
allowed to stir overnight at room temperature. A 10% Na2CO3 solution
was added to quench the reaction, but the solution was too thick and an
additional 20 mL of water was added. The white slurry was extracted
with EtOAc (3 ꢀ 60 mL). The organics were combined and washed with
saturated NaCl solution, dried over Na2SO4, filtered, and concentrated
to afford the crude title compound (1.56 g) as a clear oil. 1H NMR (400
MHz, DMSO-d6): δ 0.82 (d, J = 6.6 Hz, 3H), 1.18 (t, J = 7.1 Hz, 1H),
1.31 (d, J = 3.0 Hz, 1H), 1.37 (s, 9H), 1.49-1.60 (m, 1H), 3.09-3.33
(m, 4H), 3.45 (td, J = 7.0, 5.1 Hz, 2H), 4.37 (dt, J = 9.2, 5.2 Hz, 1H), 4.56
(t, J = 5.7 Hz, 1H).
1,1-Dimethylethyl [(3R,5R)-5-Methyl-1-(phenylmethyl)-3-piperidinyl]-
carbamate (40a). 1,1-Dimethylethyl [(1R,3R)-4-hydroxy-1-(hydroxy-
methyl)-3-methylbutyl]carbamate (1.56 g, 6.69 mmol) was dissolved in
CH2Cl2 (30 mL) in a 250 mL RB flask capped with a rubber septa. The
reaction flask was charged with N2 and cooled in an ice bath. Triethy-
lamine (3.73 mL, 26.7 mmol) was added, followed by dropwise addition
of methanesulfonyl chloride (1.563 mL, 20.06 mmol). After the addi-
tion, the reaction was allowed to stir at 0 °C for 1 h. The reaction was
then diluted with CH2Cl2 (30 mL) and washed with saturated NaHCO3
(30 mL) then water, dried with Na2SO4, filtered, and concentrated to
afford the crude (2R,4R)-2-({[(1,1-dimethylethyl)oxy]carbonyl}amino)-
4-methyl-5-[(methylsulfonyl)oxy]pentyl methanesulfonate (2.61 g).
LC-MS (ES) m/z = 390 [M þ H]þ.
0.966 mmol), and the resulting mixture was vigorously stirred for 30 min.
CH3I (0.044 mL, 0.708 mmol) was added, and the reaction mixture was
stirred for 3 h at room temperature. Saturated aqueous NaHCO3 was added
carefully (ca. 5 mL, initial vigorous bubbling), and the resulting mixture was
poured onto water and EtOAc. The organic layer was separated, washed with
brine, dried (MgSO4), filtered, and concentrated. Flash chromatography on
SiO2 (0-30% EtOAc in hexane gradient) afforded the title compound
(162 mg) as a colorless oil. LC-MS (ES) m/z = 319 [M þ H]þ. 1H NMR
(400 MHz, CDCl3): δ 1.10 (d, J = 7.1 Hz, 3H), 1.39-1.54 (m, 10H), 1.64-
1.75 (m, 1H), 2.01 (dt, J = 7.0, 3.7 Hz, 1H), 2.09-2.24 (m, 2H), 2.32 (bs,
1H), 2.68 (dd, J= 10.5, 3.9 Hz, 1H), 2.85 (s, 3H), 3.32-3.60 (m, 2H), 7.21-
7.28 (m, 1H), 7.29-7.36 (m, 4H).
1,1-Dimethylethyl {(3R,5R)-1-[2-Amino-6-(3-amino-1H-indazol-6-
yl)-4-pyrimidinyl]-5-methyl-3-piperidinyl}methylcarbamate (43b). A
solution of 1,1-dimethylethyl methyl[(3R,5R)-5-methyl-1-(phenylmethyl)-
3-piperidinyl]carbamate (160 mg, 0.502 mmol) in ethanol (6 mL) was
degassed with N2 for 10 min. Pd/C 10% (Degussa type, 54 mg) was
added, and the resulting mixture was stirred for 3 days at room
temperature under a hydrogen atmosphere (balloon setup). The mixture
was degassed with N2 and filtered through an Acrodisk, rinsing with
ethanol (ca. 15 mL). The filtrate was concentrated under vacuum to
afford crude 1,1-dimethylethyl methyl[(3R,5R)-5-methyl-3-piperidi-
nyl]carbamate (41b). LC-MS (ES) m/z = 229 [M þ H]þ. The residue
(41b) was taken up into 1,4-dioxane (6 mL), and to that solution were
added 4,6-dichloro-2-pyrimidinamine (79 mg, 0.477 mmol) and satd aq
NaHCO3 (3 mL). The mixture was stirred overnight at 100 °C in a
sealed tube and then allowed to cool to room temperature. (4-Cyano-3-
fluorophenyl)boronic acid (124 mg, 0.754 mmol) was added, and N2 gas
was bubbled through the mixture for 10 min. Pd(PPh3)4 (29.0 mg, 0.025
mmol) was added, the vessel was sealed, and the reaction mixture was
stirred for 4 h at 100 °C. The mixture was then poured into water and
EtOAc. The organic layer was separated, and the aqueous layer was
further extracted with EtOAc. The combined organic layers were washed
with brine, dried (MgSO4), filtered, and concentrated. Flash chroma-
tography on SiO2 (0-70% EtOAc in hexane gradient) afforded 1,1-
dimethylethyl {(3R,5R)-1-[2-amino-6-(4-cyano-3-fluorophenyl)-4-pyri-
midinyl]-5-methyl-3-piperidinyl}methylcarbamate (170 mg) as a thick
oil. LC-MS (ES) m/z = 441 [M þ H]þ.
Into a 10 mL sealable vial was added (2R,4R)-2-({[(1,1-dimethyl-
ethyl)oxy]carbonyl}amino)-4-methyl-5-[(methylsulfonyl)oxy]pentyl
methanesulfonate (1.05 g, 2.70 mmol), followed by benzylamine (5.88 mL,
53.9 mmol), and the reaction was capped and heated at 70 °C for 24 h.
The reaction was allowed to cool, and then it was transferred into a 1N
NaOH solution (20 mL). The resulting oily mixture was then extracted
with hexane (3 ꢀ 20 mL). The combined extracts were washed with
saturated NaCl solution, dried over MgSO4, filtered, and concentrated.
The residue was purified by flash chromatography on a 20 g Biotage
SNAP column with gradient elution of 0-20% EtOAc in hexane over
20 min to afford the title compound (452 mg) as a clear oil. LC-MS
(ES) m/z = 305 [M þ H]þ. 1H NMR (400 MHz, DMSO-d6): δ 0.85-
0.92 (m, 3H), 1.22-1.28 (m, 1H), 1.37 (s, 9H), 1.50 (m, 1H), 1.90 (bs,
2H), 2.29 (bs, 2H), 2.35-2.44 (m, 1H), 3.43 (m, 2H), 3.64 (bs, 1H),
6.44 (d, J = 6.8 Hz, 1H), 7.24 (m, 1H), 7.31 (m, 4H).
1,1-Dimethylethyl [(3R,5R)-5-Methyl-3-piperidinyl]carbamate
(41a). Into a Parr shaker jar was added 10% Pd/C (316 mg, 0.148 mmol,
Degussa type), followed by a solution of 1,1-dimethylethyl [(3R,5R)-5-
methyl-1-(phenylmethyl)-3-piperidinyl]carbamate (452 mg, 1.485 mmol)
in ethanol (20 mL). The Parr shaker jar was then placed into the shaker,
flushed with N2, and then pressurized with hydrogen to 30 psi. The reaction
was shaken at room temperature overnight. The reaction was filtered and
concentrated to give the crude title compound (310 mg). LC-MS (ES)
m/z = 215 [M þ H]þ. 1H NMR (400 MHz, DMSO-d6): δ 0.79 (d, J = 6.6
Hz, 3H), 1.16-1.27 (m, 1H), 1.39 (s, 9H), 1.65 (m, 2H), 2.11 (dd, J= 12.4,
8.6 Hz, 1H), 2.60 (m, 2H), 2.71 (dd, J = 12.1, 3.0 Hz, 1H), 3.27 (d, J =
7.1 Hz, 1H), 6.66 (m, 1H).
1,1-Dimethylethyl{(3R,5R)-1-[2-amino-6-(3-amino-1H-indazol-6-yl)-
4-pyrimidinyl]-5-methyl-3-piperidinyl}carbamate (43a). The title
compound was prepared from amine 41a following synthetic route 1.
LC-MS (ES) m/z = 439 [M þ H]þ. 1H NMR (400 MHz, DMSO-d6):
δ 0.88 (d, J = 6.8 Hz, 3H), 1.29 (s, 9H), 1.37-1.52 (m, 2H), 1.68 (m,
1H), 1.93-2.07 (m, 1H), 2.83 (bs, 1H), 3.38 (bs, 1H), 3.63 (bs, 1H),
3.89 (m, 1H), 5.37 (s, 2H), 6.04 (bs, 2H), 6.49 (s, 1H), 6.82 (d, J = 6.3
Hz, 1H), 7.50 (d, J = 8.6 Hz, 1H), 7.69 (d, J = 8.3 Hz, 1H),
7.89 (s, 1H), 11.49 (s, 1H).
1,1-Dimethylethyl Methyl[(3R,5R)-5-methyl-1-(phenylmethyl)-3-pi-
peridinyl]carbamate (40b). To 1,1-dimethylethyl [(3R,5R)-5-methyl-
1-(phenylmethyl)-3-piperidinyl]carbamate (196 mg, 0.644 mmol) in
DMF (3 mL) was added NaH (60% dispersion in mineral oil, 38.6 mg,
To 1,1-dimethylethyl {(3R,5R)-1-[2-amino-6-(4-cyano-3-fluorophenyl)-
4-pyrimidinyl]-5-methyl-3-piperidinyl}methylcarbamate (170 mg, 0.386
mmol) in ethanol (6 mL) was added hydrazine monohydrate (0.70
mL, 14.3 mmol), and the reaction mixture was stirred overnight at
100 °C in a sealed tube. The mixture was poured into water (ca. 150
mL), and a white precipitate formed. It was filtered, and the solid was air-
dried for 2 h. The resulting white solid was dried under vacuum at 45 °C
for 1 h to afford the title compound (110 mg). LC-MS (ES) m/z = 453
1
[M þ H]þ. H NMR (400 MHz, DMSO-d6): δ 0.92-1.02 (m, 3H),
1.41 (s, 9H), 1.46-1.55 (m, 1H), 1.85-1.99 (m, 1H), 2.07-2.19 (m,
1H), 2.76 (s, 3H), 3.02-3.14 (m, 1H), 3.12-3.23 (m, 1H), 3.90-4.15
(m, 2H), 4.35 (m, 1H), 5.38 (s, 2H), 6.07 (s, 2H), 6.60 (s, 1H), 7.57 (dd,
J = 8.6, 1.3 Hz, 1H), 7.70 (d, J = 8.6 Hz, 1H), 7.95 (s, 1H), 11.50 (s, 1H).
N-{(3R,5R)-1-[2-Amino-6-(3-amino-1H-indazol-6-yl)-4-pyrimidinyl]-
5-methyl-3-piperidinyl}-N,3,3-trimethylbutanamide (43c). In a 20 mL
sealable vial was added 1,1-dimethylethyl methyl[(3R,5R)-5-methyl-3-piperi-
dinyl]carbamate (41b, 443 mg, 1.94 mmol), 4,6-dichloro-2-pyrimidinamine
(477 mg, 2.91 mmol), triethylamine (0.541 mL, 3.88 mmol), and ethanol
(10 mL). The vial was sealed, and the reaction mixture was heated overnight
at 100 °C. The reaction was concentrated, and the resulting residue was
purified by flash chromatography on SiO2 (gradient: 0-55% EtOAc in
hexane) to afford 1,1-dimethylethyl [(3R,5R)-1-(2-amino-6-chloro-4-
pyrimidinyl)-5-methyl-3-piperidinyl]methylcarbamate (678 mg). LC-MS
1
(ES) m/z = 356, 358 [M þ H]þ. H NMR (400 MHz, DMSO-d6): δ
0.91 (d, J = 7.1 Hz, 3H), 1.41 (s, 9H), 1.44-1.54 (m, 1H), 1.90 (td,
J = 11.9, 4.8 Hz, 1H), 2.04-2.17 (m, 1H), 2.72 (s, 3H), 3.01 (t, J = 11.5 Hz,
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1H), 3.10 (dd, J = 13.3, 2.9 Hz, 1H), 3.79 (bs, 1H), 4.03 (m, 1H), 6.08 (s,
1H), 6.47 (bs, 2H).
(S473) signals were normalized to total AKT, while phospho-AKT
(T308) and phospho-RSK signals were analyzed without normaliza-
tion. All values were expressed as percent of the DMSO-treated
controls. IC50s were determined from inhibition curves using XLfit4
software (IDBS, Guildford, UK).
Pharmacodynamic Studies. Female SCID mice bearing sub-
cutaneous OCI-AML2 tumor xenografts of ∼500-600 mm3 in size
were treated with vehicle (1% DMSO, 20% PEG400, pH 4.2) or PDK1
inhibitor (100 mg/kg) by intraperitoneal administration. Three and six
hours later, 10 μg of hIGF-I (via the tail vein) were administered, and
animals were euthanized after 10 min for collection of tumor tissue and
blood. Frozen tumors samples were homogenized in lysis buffer and
analyzed for phosphor- and total AKT and RSK by ELISA, as described
above. Concentration of compound in the blood and tumor samples was
also analyzed by LC/MS/MS.
To 1,1-dimethylethyl [(3R,5R)-1-(2-amino-6-chloro-4-pyrimidinyl)-
5-methyl-3-piperidinyl]methylcarbamate (75 mg, 0.211 mmol) in CH2-
Cl2 (2 mL) was added TFA (2 mL), and the reaction was allowed to sit at
room temperature for 2 h. Toluene (∼15 mL) was added, and the
resulting mixture was evaporated under vacuum. To the resulting residue
were added CH2Cl2 (2 mL) and H€unig’s base (0.18 mL, 1.05 mmol),
followed by 3,3-dimethylbutanoyl chloride (0.03 mL, 0.21 mmol), and
the resulting mixture was stirred overnight at room temperature.
Saturated aqueous NaHCO3 (ca. 20 mL) was added, and the resulting
mixture was extracted with EtOAc (ca. 50 mL). The organic layer was
separated, washed with brine, dried (MgSO4), filtered, and concentrated
to afford crude N-[(3R,5R)-1-(2-amino-6-chloro-4-pyrimidinyl)-5-methyl-
3-piperidinyl]-N,3,3-trimethylbutanamide (42, 80 mg). LC-MS (ES)
m/z = 354 [M þ H]þ.
The title compound was prepared from compound 42 following
synthetic route 1 as a mixture of rotamers. LC-MS (ES) m/z = 451 [M þ
H]þ. 1H NMR (400 MHz, DMSO-d6 þ 1 drop D2O): δ 0.98 (m, 12H),
1.41 and 1.52 (m, 1H), 1.87-2.41 (m, 4H), 2.76 and 2.90 (s, 3H),
2.99-3.25 (m, 2H), 3.84-3.99 (m, 1H), 4.01-4.26 (m, 1H), 4.39-
4.66 (m, 1H), 6.57 and 6.62 (s, 1H), 7.50-7.60 (m, 1H), 7.70 (m, 1H),
7.92 (s, 1H).
Accession Codes
†PDB codes: 12f, 3QCQ; 12k, 3QCS; (R)-18a, 3QCX; (S)-21a,
3QCY; (3S,6R)-28a, 3QD0; (3R,6S)-37c, 3QD3; 43a, 3QD4.
’ AUTHOR INFORMATION
Biochemical Characterization of PDK1 Inhibitors and
Crystallographic Studies. pIC50 = -log10 (IC50), where the IC50
is the molar concentration of compound required to inhibit the kinase
activity by 50%. Experimental details describing (1) the in vitro PDK1
inhibition assays for pIC50 determination and (2) PDK1 crystallography
(Table 11) are described in a previous publication.14
Corresponding Author
*Phone: 610-917-5889. Fax: 610-917-4206. E-mail: jesus.r.
medina@gsk.com.
’ ABBREVIATIONS USED
PDK1, phosphoinositide-dependent protein kinase-1; PI3K,
phosphatidylinositol 3-kinase; AKT, protein kinase B; PIP2,
phosphatidylinositol 3,4,-diphosphate; PIP3, phosphatidylinosi-
tol 3,4,5-triphosphate; PKC, protein kinase C; SGK, serum- and
glucocorticoid-induced protein kinase; S6K1, p70 ribosomal S6
kinase; RSK, p90 ribosomal S6 kinase; ALK5, TGF-β type I
receptor; ROCK1, Rho-associated protein kinase-1; LE, ligand
efficiency; G-loop, glycine rich loop; MTD, maximum tolerated
dose; AUC, area under the curve; AML, acute myeloid leukemia
Phospho-AKT (S473, T308, Total AKT) and Phospho-RSK
ELISA. PC3 cells (ATCC, Manassas, VA) were plated in 96-well flat
bottom plates (Corning, Lowell, MA) at a density of 15000 cells/well in
RPMI 1640 medium supplemented with 10% FBS. Cells were incubated
at 37 °C, 5% CO2, for 18-20 h. Compounds (dissolved in 100% DMSO)
were diluted in an 11-point 3-fold dilution in DMSO. Compound dilu-
tion stocks were further diluted in RPMI 1640 with 10% FBS and added
to each cell well. DMSO without compound was used in control wells.
Final concentration of DMSO in each well was 0.15%. After 6 h at 37 °C,
cells were washed with cold PBS (without calcium or magnesium) and
lysed in lysis buffer (Meso Scale Discovery, Gaithersburg, MD) supple-
mented with 1 protease inhibitor tablet/10 mL (Roche, Indianapolis,
IN), 10 mM NaF, and 200 μL/10 mL Sigma phosphatase inhibitors
1 and 2 (Sigma Aldrich, St Louis, MO) for 30 min at 4 °C. All washes
were performed on a Bio Tek ELx405 plate washer (Bio Tek Instru-
ments, Winooski, VT).
’ REFERENCES
(1) Pearce, L. R.; Komander, D.; Alessi, D. R. The nuts and bolts of
AGC protein kinases. Nature Rev. Mol. Cell. Biol. 2010, 11, 9–22.
(2) Mora, A.; Komander, D.; Van Aalten, D. M.; Alessi, D. R. PDK1,
the master regulator of AGC kinase signal transduction. Semin. Cell. Dev.
Biol. 2004, 15, 161–170.
ELISA plates (Meso Scale Discovery; AKT Duplex, cat. N41100B-1;
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