Rapid development of piperidine carboxamides as potent and selective anaplastic lymphoma kinase inhibitors

Article abstract of DOI:10.1021/jm201565s

Piperidine carboxamide 1 was identified as a novel inhibitor of anaplastic lymphoma kinase (ALK enzyme assay IC₅₀ = 0.174 μM) during high throughput screening, with selectivity over the related kinase insulin-like growth factor-1 (IGF1R). The X-ray cocrystal structure of 1 with the ALK kinase domain revealed an unusual DFG-shifted conformation, allowing access to an extended hydrophobic pocket. Structure-activity relationship (SAR) studies were focused on the rapid parallel optimization of both the right- and left-hand side of the molecule, culminating in molecules with improved potency and selectivity over IGF1R.

Full text of DOI:10.1021/jm201565s

Article
pubs.acs.org/jmc
Rapid Development of Piperidine Carboxamides as Potent and
Selective Anaplastic Lymphoma Kinase Inhibitors
Marian C. Bryan,† Douglas A. Whittington,‡ Elizabeth M. Doherty,† James R. Falsey,† Alan C. Cheng,‡
Renee Emkey,‡ Rachael L. Brake,‡ and Richard T. Lewis*,‡
†Medicinal Chemistry Research Technologies, Amgen Inc., One Amgen Center Drive, Thousand Oaks, California 91320, United
States
‡Amgen Inc., 360 Binney Street, Cambridge Massachusetts 02142, United States
S
* Supporting Information
ABSTRACT: Piperidine carboxamide 1 was identified as a
novel inhibitor of anaplastic lymphoma kinase (ALK enzyme
assay IC50 = 0.174 μM) during high throughput screening,
with selectivity over the related kinase insulin-like growth
factor-1 (IGF1R). The X-ray cocrystal structure of 1 with the
ALK kinase domain revealed an unusual DFG-shifted
conformation, allowing access to an extended hydrophobic
pocket. Structure−activity relationship (SAR) studies were
focused on the rapid parallel optimization of both the right-
and left-hand side of the molecule, culminating in molecules
with improved potency and selectivity over IGF1R.
also exhibited moderate to excellent selectivity over the related
kinase family member insulin-like growth factor-1 (IGF1R, IC50
= 4.61 μM).12−14 Selectivity over IGF1R was of particular
interest due to its critical functions as a mediator of cell
proliferation, differentiation, and apoptosis, and broad
expression of this kinase in normal tissue.15
INTRODUCTION
■
Anaplastic lymphoma kinase (ALK) is a member of the insulin
receptor superfamily of tyrosine kinases.1 Although not widely
expressed in adult tissue, ALK is implicated in neuronal
development, differentiation, and basal dopaminergic signal-
ing.2,3 ALK is a putative oncogene and is expressed as part of
aberrant fusion proteins in a number of cancers including
anaplastic large-cell lymphomas (ALCL), inflammatory myofi-
broblastic tumors (IMT) and, more recently, a variety of solid
tumor types.1,4 Additionally, abnormal expression of full-length
ALK may play a role in oncogenesis given its aberrant
expression in a number of different tumor types including
diffuse large B-cell lymphoma, neuroblastoma, rhabdomyosar-
comas, glioblastomas, and esophageal squamous cell carcino-
mas.1,5−8 In these settings, ALK is believed to support
tumorigenesis through a variety of signaling mechanisms
leading to cell-cycle progression, survival, cell migration, and
cell shaping.9 Recently, the use of crizotinib for the targeted
inhibition of ALK has been shown effective in early phase
clinical trials against advanced non-small-cell lung cancers
carrying activated ALK kinase.10 Coupling these compelling
results with the knowledge that the protein is not widely
expressed in adult tissue presents ALK as an attractive oncology
target with a potentially large therapeutic window.
Determination of the cocrystal structure of 1 bound to the
ALK kinase domain (Figure 1) provided insights into possible
improvements in potency and selectivity (PDB ID 4DCE).16
The cocrystal structure of 1 with ALK shows a “DFG-shifted”
conformation, similar to a type 1 1/2 inhibitor conformation.17
When 1 is bound, Phe1271 of the DFG sequence, which
normally occupies a hydrophobic pocket in the apoprotein,18
shifts and forms a lid on top of the benzyl group, allowing for
an edge−face interaction with 1 and access to the extended
hydrophobic pocket flanked by Phe1174 and Ile1179 (Figure
1A,B). The carboxamide carbonyl hydrogen bonds to catalytic
Lys1150, which also hydrogen bonds to Glu1167. The amide
NH, in turn, accepts a hydrogen bond from the backbone
carbonyl of Gly1269, the residue preceding the DFG sequence.
Additional interactions were observed between hinge residue
Met1199 and N1 of the aminopyrimidine ring and the anilinic
NH (Figure 1A). Finally, the trimethoxyphenyl group sat in a
narrow groove sandwiched by Leu1122 and the hinge region.
Comparison of the cocrystal structure of ALK with 1 and a
crystal structure of a benzimidazole inhibitor-bound IGF1R19
shows residue differences in the region adjacent to the benzyl
ring (Leu1196 vs Met1049 and Ile1171 vs Met1024 in IGF1R
We identified piperidine carboxamide 1 (Scheme 1) from a
high-throughput screen of our proprietary sample collection
against a recombinant, truncated ALK enzyme construct.
Compound 1 inhibited the ALK construct with an IC50
=
0.174 μM and demonstrated comparable functional activity in a
whole cell assay monitoring ALK phosphorylation (IC50
=
Received: November 21, 2011
Published: January 20, 2012
0.384 μM in ALK-positive Karpas-299 cells).11 The compound
© 2012 American Chemical Society
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Scheme 1. Retrosynthetic Analysis of 1
Figure 1. (A) Co-crystal structure of 1 with ALK at 2.0 Å resolution highlighting key interactions with the DFG residues (shown in green). (B) Co-
crystal structure of 1 with ALK illustrating the extended hydrophobic pocket. (PDB ID 4DCE).
and ALK, respectively) as well as conformational differences in
the DFG region (Figure 2).20 In the IGF1R crystal structure,
benzyl ring of compound 1. In addition, Asp1123 is oriented
inward, where it impinges on the binding of compound 1.
Substitution of Ile1171 (ALK) with Met1024 (IGF1R) also
narrows the entrance to the pocket and modifies its shape.
On the basis of these observations, we synthesized a series of
analogues designed to probe the extended hydrophobic pocket
and the linker binder region to optimize nonbonded
interactions while maintaining or improving selectivity over
IGF1R.
CHEMISTRY
■
Retrosynthetic analysis of 1 (Scheme 1) reveals a lead amenable
to a modular, parallel-synthesis approach. Arrays of analogues
were prepared either via SNAr with chloropyrimidine
intermediate 2 (route A, Scheme 1) or by amide coupling to
carboxylic acid intermediate 3 (route B, Scheme 1).
Chloropyrimidine 2 was prepared from (S)-1-(tert-
butoxycarbonyl)piperidine-3-carboxylic acid (4) and p-tolylme-
thylamine (5) according to Scheme 2. HATU-mediated
coupling of 4 with 5 provided intermediate 6. Cleavage of
the Boc protecting group under acidic conditions followed by
reaction with 2,4-dichloropyrimidine afforded predominately
the desired 4-substituted key intermediate 2. The three steps
Figure 2. Overlay of cocrystal structures of compound 1 with ALK
(PDB ID 4DCE) and a structure of IGF1R bound to a benzimidazole
inhibitor (PDB ID 2OJ9). ALK is in brown, 1 is in orange, and IGF1R
is in green.
Phe1124 of the DFG region is flipped away from the pocket,
precluding a putative favorable stacking interaction with the
a
Scheme 2. Synthesis of Compounds 7a−l via Chloropyrimidine Intermediate 2
a
Reagents and conditions: (a) Et3N, HATU, CH2Cl2, 22 °C, 70%; (b) 1 M HCl, CH2Cl2, 22 °C, quant; (c) 2,4-dichloropyrimidine, Et3N, EtOH, 22
°C, 32%; (d) DMSO, 90 °C.
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a
Scheme 3. Synthesis of Compounds 11a−v via Carboxylic Acid Intermediate 3
a
Reagents and conditions: (a) 2,4-dichloropyrimidine, Et3N, EtOH, 0−22 °C, 88%; (b) DMSO, 90 °C, 88%; (c) LiOH, H2O, dioxane, 22 °C, quant;
(d) Et3N, HATU, DCE, 22 °C.
a
Scheme 4. Synthesis of R-Enantiomer of 1
a
Reagents and conditions: (a) 2,4-dichloropyrimidine, Et3N, EtOH, 0−22 °C; (b) 3,4,5-trimethoxyaniline, DMSO, 90 °C; (c) TFA, CH2Cl2, 22 °C;
(d) 5, HATU, Et3N, CH2Cl2, 22 °C.
Table 1. Hinge Region Modifications
a
IC50 (μM) ( SD)
compd
R
ALK
IGF1R
1
3,4,5-OMePh
Me
0.174 ( 0.012)
4.61 ( 0.75)
c
c
7a
7b
7c
7d
7e
7f
>25
>25
b
c
Ph
2.32 ( 0.13)
>25
c
c
Bn
>25
>25
c
3-ClPh
2.01 ( 0.45)
>25
c
c
4-ClPh
6.30
>25
3-OMePh
4-OMePh
3-EtPh
1.02 ( 0.09)
3.10 ( 2.06)
0.912 ( 0.167)
9.022
c
7g
7h
7i
>25
c
>25
c
c
4-EtPh
>25
>25
c
7j
3,4-OMePh
3,4-benzodioxane
3,5-OMePh
1.73 ( 1.31)
1.48 ( 0.48)
>25
c
7k
7l
>25
b
b
0.325 ( 0.173)
2.93 ( 1.20)
a
b
c
All values are the average of n ≥ 3 standard deviation unless indicated otherwise. n = 2 standard deviation. n = 1.
proceeded without epimerization, as determined by chiral
supercritical fluid chromatography (SFC).21 Arrays of ana-
logues were then rapidly assembled via SNAr reaction of 2 with
various primary amines under thermal conditions. Final
compounds were then obtained after high throughput mass-
triggered preparative HPLC purification.22
following high throughput mass-triggered preparative HPLC
purification.22
In addition, the enantiomer of compound 1 (14) was
generated from (R)-(−)-nipecotic acid ethyl ester (12) via
carboxylic acid 13 using the method described for 3 (Scheme
4).
The synthesis of intermediate 3 is outlined in Scheme 3.
Reaction of 2,4-dichloropyrimidine with ethyl-(S)-nipecotate
(8) following the procedure for intermediate 2 afforded
pyrimidine 9. Coupling with 3,4,5-trimethoxyaniline under
thermal conditions gave aniline 10, which was saponified with
LiOH to generate carboxylic acid 3. HATU-mediated coupling
of 3 with various amines proceeded with minimal racemization
of the chiral center.23 Final compounds were obtained as before
RESULTS AND DISCUSSION
■
Analogues were assayed for inhibition of ALK enzyme activity
as well as selectivity over IGF1R.24 ALK functional activity was
measured using a time-resolved fluorescence resonance energy
transfer (TR-FRET) assay which measured the phosphoryla-
tion of a peptide substrate.11 FRET assays monitoring
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Table 2. Amide Bond Modifications
a
IC50 (μM) ( SD)
compd
X
Y
stereocenter
ALK
IGF1R
1
CO
CO
NH
NH
S
0.174 ( 0.01)
4.12 ( 0.43)
4.61 ( 0.75)
>25
14
R
a
All values are the average of n ≥ 3 standard deviation.
Table 3. Right-Hand Side Modifications
a
IC50 (μM) ( SD)
compd
R
ALK
IGF1R
1
−CH2-(4-MePh)
−Ph
−CH2-Ph
−(CH2)2-Ph
−(CH2)3-Ph
−CH2-CO-Ph
−CH(S-Me)-Ph
−CH(R-Me)-Ph
0.174 ( 0.012)
0.833 ( 0.063)
0.364 ( 0.073)
0.358 ( 0.415)
6.71 ( 1.07)
4.61 ( 0.75)
c
11a
11b
11c
11d
11e
11f
11g
6.59
b
6.12 ( 0.75)
b
>25
b
6.39 ( 1.17)
2.94 ( 0.83)
4.10
c
c
2.60
10.54
c
c
2.01
>25
a
b
c
All values are the average of n ≥ 3 standard deviation unless indicated otherwise. n = 2. n = 1.
Table 4. Right-Hand Side Substituent Effects
a
IC50 (μM) ( SD)
compd
R
ALK
IGF1R
1
4-Me
0.174 ( 0.012)
0.341 ( 0.019)
0.083 ( 0.018)
4.61 ( 0.75)
b
11h
11i
11j
11k
11l
2-Me
8.32 ( 2.62)
0.722 ( 0.046)
0.116 ( 0.015)
0.798 ( 0.088)
b
b
b
3-Me
b
3-OCF3
4-OCF3
2-Cl
0.016 ( 0.001)
0.010 ( 0.002)
c
c
0.587
7.08
b
b
11m
11n
11o
11p
11q
11r
11s
11t
11u
3-Cl
0.080 ( 0.040)
0.095 ( 0.012)
0.070 ( 0.014)
2.93 ( 0.59)
0.364 ( 0.050)
b
4-Cl
1.87 ( 0.69)
3-NO2
4-NO2
3-CO2Me
4-CO2Me
3-Ph
0.503 ( 0.062)
b
>25
b
b
0.060 ( 0.016)
1.70 ( 0.08)
8.89 ( 4.29)
b
>25
c
0.019 ( 0.01)
0.138 ( 0.026)
0.192 ( 0.054)
4.23
b
4-Ph
>25
b
3-OPh
2.77 ( 0.48)
a
b
c
All values are the average of n ≥ 3 standard deviation unless indicated otherwise. n = 2. n = 1.
phosphorylation of a peptide substrate were also used for the
counter-screening assay for IGF1R.
Structure−activity relationships specific to the hinge-binding
region of the molecule are shown in Table 1, where
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Table 5. Comparison of 1, 11q, and 11s in Enzyme and Cellular Assays
a
IC50 (μM) ( SD)
compd
R
cLogP
ALK
Karpas
cell shift
1
4-Me
3-Ph
4.24
5.63
4.77
4.46
3.71
3.20
0.174 ( 0.012)
0.019 ( 0.011)
0.016 ( 0.001)
0.080 ( 0.040)
0.060 ( 0.016)
0.384 ( 0.126)
0.224 ( 0.122)
0.059 ( 0.021)
2.2
11.8
3.7
11s
11j
11m
11q
11v
b
3-OCF3
c
3-Cl
0.226
2.8
b
b
3-CO2Me
3-morpholine
0.061 ( 0.032)
1.0
b
0.031 ( 0.002)
0.028 ( 0.022)
0.9
a
b
c
All values are the average of n ≥ 3 standard deviation unless indicated otherwise. n = 2. n = 1.
modifications generally resulted in a loss of potency.
Replacement of the trimethoxyphenyl group with Me (7a)
resulted in loss of detectible potency. An unsubstituted phenyl
ring (7b) maintained measurable potency (IC50 = 2.32 μM),
although extending this group by one methylene unit (7c) was
not tolerated. C-3 substitutions proved more potent than C-4
(7d vs 7e, 7f vs 7g, 7h vs 7i), with 3-ethyl 7h being the most
potent (IC50 = 0.912 μM). Disubstituted phenyl analogues such
as 3,4-dimethoxyphenyl 7j or 3,4-benzodioxane 7k gave an
order of magnitude loss in potency relative to 1 but were still
active. 3,5-Dimethoxy analogue 7l alone gave comparable
potency (IC50 = 0.325 μM) to 1, most likely by mimicking the
minimal interactions the meta-position methoxy groups make
with the protein in the crystal structure. This also highlights the
insignificance of the methoxy group at C-4, which projects into
the solvent in the crystal structure. None of these modifications
significantly impacted IGF1R selectivity.
Modification of the amide portion of piperidinyl carboxamide
1 proved detrimental to potency (Table 2). The R-enantiomer
of compound 1 (14) was active, but less potent in the
enzymatic assay (IC50 = 4.12 μM) compared with the S-
enantiomer. This finding is consistent with the interactions
noted in the cocrystal structure where the carbonyl oxygen is
hydrogen bonded to catalytic Lys1150 and the amide NH
forms a hydrogen bond with the backbone carbonyl of Gly1269
(Figure 1A).27
Structure−activity relationships specific to the hydrophobic
pocket are seen in Table 3. The spacing between the piperidine
carboxamide and the pendant aryl ring as well as the
substitution of the alkyl linker between these two moieties
proved important for both ALK enzyme potency and
selectivity. A direct linkage was tolerated (11a, IC50 = 0.833
μM), although a one- or two-carbon tether length was preferred
(11b, IC50 = 0.364 μM, and 11c, IC50 = 0.358 μM,
respectively). Further elongation of the tether (n = 3) gave
an 18-fold loss in activity (11d vs 11c). Substitution of the
linker with either a carbonyl or methyl also negatively impacted
potency (11e vs 11c; 11f,11g vs 11b).
The effect of alternative phenyl substitution patterns on ALK
potency is shown in Table 4. In general, substitution at the C-3
and C-4 positions was preferred over C-2 substitution. The C-2
methylated analogue 11h was equipotent to the unsubstituted
phenyl analogue 11b (IC50 = 0.341 μM vs IC50 = 0.364 μM).
Methylation at C-3 (11i, IC50 = 0.083 μM) gave a 4-fold
increase in potency relative to 1, although a reduction in IGF1R
selectivity was observed (8-fold for 11i vs 17-fold for 11b).
Introduction of a trifluoromethoxy group at C-3 or C-4
improved potency (11j IC50 = 0.016 μM and 11k IC50 = 0.010
μM, respectively). As with 11i, 11j showed a decrease in
selectivity over IGF1R. C-3 or C-4 halo-derivatives showed
improved potency relative to C-2 (11l, 11m, and 11n),
although C-3 analogue 11m also exhibited increased activity for
IGF1R. Strongly electron withdrawing groups such as nitro or
methyl carboxylate maintained potency when appended to C3
(11o IC50 = 0.070 μM and 11q IC50 = 0.060 μM, respectively)
and decreased potency at C4 (11p IC50 = 2.93 μM and 11r
IC50 = 1.70 μM, respectively).
Appending a less electron-withdrawing, hydrophobic phenyl
ring to the C3 position (11s) increased potency for ALK and
improved selectivity over IGF1R (ALK IC50 = 0.019 μM,
IGF1R IC50 = 4.23 μM) to give the most selective analogue
identified in these studies. C4-substituted phenyl 11t also
maintained potency relative to methyl 1 (IC50 = 0.138 μM vs
IC50 = 0.174 μM) without negatively impacting selectivity.
Introduction of an ether spacer to 11s (11u) led to a 10-fold
loss in ALK potency and decreased IGF1R selectivity relative to
11s.
The functional activity of piperidine carboxamide 1 and
potent analogues 11j, 11m, 11q, and 11s was subsequently
measured in an ALK-positive Karpas-299 whole cell assay
(Table 5). An apparent correlation between cell shift and the
calculated logP (cLogP) was observed, such that as cLogP
increased (11q < 1 < 11m < 11j < 11s), the cell shift also
increased. These findings suggest that reducing clogP will lead
to decreased cell shifts. To test this hypothesis, morpholine
analogue 11v was generated using the method described in
Scheme 3. Gratifyingly, the resulting compound proved highly
potent in the ALK enzyme and cellular assays (ALK IC50
=
0.031 μM, cell IC50 = 0.028 μM). To evaluate morpholine 11v
for kinase selectivity, the compound was analyzed in the IGF1R
assay as well as tested at 1 μM against a panel of 99 kinases
using the Ambit Biosciences KINOMEscan platform.25 Gratify-
ingly, morpholine 11v was >200-fold selective over IGF1R
(IC50 = 7.39 μM) and showed no effect on IGF1R in the
kinome panel. In addition, only 4 kinases, namely ALK, KIT,
TRKA, and FLT3, have <30% percent-of-control (POC) for
the compound. Compound 1 also showed activity toward these
kinases as well as PAK2 and MAP2K5, with a POC <30%
highlighting the improved selectivity of 11v.26
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MHz, CDCl3) δ ppm 7.08−7.20 (m, 4 H), 6.13 (br s, 1 H), 4.38 (d, J
= 5.26 Hz, 2 H), 3.92 (d, J = 13.30 Hz, 1 H), 3.62−3.83 (m, 1 H), 3.21
(dd, J = 13.01, 9.65 Hz, 1 H), 3.00 (dd, J = 10.40 Hz, 1 H), 2.33 (s, 3
H), 2.20−2.30 (m, 1 H), 1.80−1.95 (m, 2 H), 1.58−1.71 (m, 1 H),
1.43−1.52 (m, 1 H), 1.42 (s, 9 H). MS (ESI, positive ion) m/z: 355.2
[M + Na] 355.2.
CONCLUSIONS
■
In this investigation, a modular, parallel-synthesis approach was
used to swiftly examine the hinge-binding region and
hydrophobic pocket occupied by compound 1, a selective
ALK inhibitor. This approach allowed for rapid SAR develop-
ment across different sections of the molecule concurrently and
greatly increased the speed with which interesting and potent
compounds were discovered. The SAR of this series of ALK
inhibitors demonstrated that modification of the hinge-binding
region fails to improve potency or selectivity. These findings are
reasonable given that this part of the molecule is in a solvent-
exposed region with minimal contacts to the protein outside of
the linker binder. However, changes to the section of the
molecule occupying the extended hydrophobic pocket created
by an unusual DFG-shifted conformation were effective.
Furthermore, we found that cellular potency correlated with
the cLogP of the molecule and used these findings to generate
morpholine 11v, which was highly potent in both the ALK
enzyme and cellular assays as well as selective over IGF1R and a
broad range of kinases in a kinase scan. Tesaro, Inc. signed an
agreement with Amgen granting Tesaro exclusive worldwide
rights for the development, manufacture, commercialization,
and distribution of small-molecule inhibitors of ALK. This work
has established a path forward for the generation of more
potent and selective ALK inhibitors currently underway.
(S)-1-(2-Chloropyrimidin-4-yl)-N-(4-methylbenzyl)-
piperidine-3-carboxamide (2). To a solution of 6 (506 mg) in
CH2Cl2 (15 mL) was added 1 M HCl in diethyl ether (8 mL). The
reaction was maintained at 23 °C for 20 h while stirring. The reaction
was concentrated to dryness and taken on to the next step without
further purification. To a solution of the intermediate (409 mg) in
EtOH (3 mL) was added 2,4-dichloropyrimidine (227 mg) and Et3N
(423 μL). The resulting solution was stirred at 23 °C for 20 h. The
reaction was then concentrated and the residue was adsorbed onto a
plug of silica gel and purified by chromatography through a 50 g silica
gel column, eluting with 0.5−5% MeOH in CH2Cl2, to provide 2 (32%
yield) as a white solid. 1H NMR (300 MHz, CDCl3) δ ppm 1.49−1.63
(m, 1 H) 1.74−1.85 (m, 1 H) 1.93−2.09 (m, 2 H) 2.29−2.43 (m, 4
H) 3.11−3.22 (m, 1 H) 3.57 (m, 1 H) 4.03 (m, 1 H) 4.18 (m, 1 H)
4.40 (m, 2 H) 6.40 (d, J = 6.28 Hz, 1 H) 7.14 (s, 4 H) 8.00 (d, J = 6.14
Hz, 1 H). ESI-MS [M + H] 345.0. 8.00 (d, J = 6.14 Hz, 1 H), 7.14 (s,
4 H), 6.40 (d, J = 6.28 Hz, 1 H), 4.40 (m, 2 H), 4.18 (m, 1 H), 4.03
(m, 1 H), 3.57 (m, 1 H), 3.11−3.22 (m, 1 H), 2.29−2.43 (m, 4 H),
1.93−2.09 (m, 2 H), 1.74−1.85 (m, 1 H), 1.49−1.63 (m, 1 H). MS
(ESI, positive ion) m/z: 345.0 [M + H].
(S)-N-(4-Methylbenzyl)-1-(2-((3,4,5-trimethoxyphenyl)-
amino)-4-pyrimidinyl)-3-piperidinecarboxamide (1). Intermedi-
ate 2 (86 mg) and 3,4,5-trimethoxyaniline (46 mg) were dissolved in
DMSO (1 mL) and heated at 80 °C for 20 h. The reaction was cooled
to 23 °C, diluted with water, and extracted with CH2Cl2 (2×). The
combined organic layers were dried over Na2SO4, filtered, and
concentrated. The crude material was absorbed onto a plug of silica gel
and purified by chromatography through silica gel (10 g), eluting with
0.5− 5% MeOH in CH2Cl2, to provide 1 (65% yield) as a light-purple
solid. 1H NMR (400 MHz, CDCl3) δ 7.95 (d, J = 6.06 Hz, 1H), 7.02−
7.17 (m, 5H), 6.83 (s, 2H), 6.36 (br s, 1H), 6.03 (d, J = 6.06 Hz, 1H),
4.26−4.43 (m, 2H), 4.22 (d, J = 12.32 Hz, 1H), 4.01 (br s, 1H), 3.74−
3.88 (m, 9H), 3.56 (dd, J = 9.29, 13.20 Hz, 1H), 3.14 (t, J = 10.47 Hz,
1H), 2.24−2.41 (m, 4H), 1.91−2.05 (m, 2H), 1.72 (dd, J = 4.30, 9.19
Hz, 1H), 1.46−1.61 (m, 1H). HRMS: calcd for (C27H33N5O4)H+,
492.25978; found, 492.26004.
Preparation of 7a−l. General Procedure A. A mixture of the
amine (435 μmol) and 2 (290 μmol) in DMSO (600 μL) was heated
at 90 °C for 16 h. The mixture was then cooled to 23 °C, filtered
through a course frit, and purified by mass-triggered preparative HPLC
(Phenomenex Gemini-NX C18 110A column (100 mm × 21 mm, 5
μ), 44 mL/min flow rate, 5−95% [0.1% TFA in acetonitrile] in [0.1%
TFA in water] over 10 min, mass spectral data were acquired from 100
to 850 amu in electrospray positive mode using MS, Waters SQ; UV,
Waters 2487 or Waters PD) to provide the desired products as their
TFA salts.
(S)-Ethyl 1-(2-Chloropyrimidin-4-yl)piperidine-3-carboxylate
(9). To a solution of (S)-(+)-nipecotic acid ethyl ester (1 g) in EtOH
(8 mL) was added Et3N (885 μL), and the reaction was stirred at 23
°C. After 18 h, the reaction was concentrated and purified by
chromatography through a 50 g silica gel column, eluting with 10−
90% ethyl acetate in hexanes, to provide the desired product (88%
yield) as a clear oil. MS (ESI, positive ion) m/z: 270.0 [M + H].
(S)-Ethyl 1-(2-(3,4,5-Trimethoxyphenylamino)-pyrimidin-4-
yl)piperidine-3-carboxylate (10). To a solution of 9 (9.5 g) in
DMSO (70.1 mL) was added 3,4,5-trimethoxyaniline (6.4 g). The
resulting solution was heated to 90 °C while stirring. After 3 d, the
reaction was cooled to 23 °C and diluted with CH2Cl2. The organic
phase was washed with water (3×). The combined aqueous layers
were then extracted with CH2Cl2 (1×). The combined organic layers
were then dried over sodium sulfate, filtered, and concentrated to give
a residue. The residue was adsorbed onto a plug of silica gel and
purified by chromatography through a 100 g silica gel column, eluting
with 10−100% ethyl acetate in hexanes to provide 10 (88% yield) as
EXPERIMENTAL DETAILS
■
General. All reagents and solvents were obtained from commercial
suppliers and used without further purification. Silica gel chromatog-
raphy was performed using prepacked silica gel cartridges (Biotage).
1H NMR spectra were obtained on either a Bruker UltraShield 300
MHz or Bruker DRX 400 (400 MHz) spectrometer and reported as
ppm downfield from the deuterated solvent. All tested compounds
were purified to >95% purity at 215 and 254 nm as determined by
HPLC. HPLC analysis was obtained on an Agilent 1100, using one of
the following two methods: [A] Agilent SB-C18 column (50 mm ×
3.0 mm, 2.5 μ) at 40 °C with a 1.5 mL/min flow rate using a gradient
of 5−95% [0.1% TFA in acetonitrile] in [0.1% TFA in water] over 3.5
min; [B] Agilent Zorbax SB-C18 (50 mm × 3.0 mm, 3.5 μ) at 40 °C
with a 1.5 mL/min flow rate using a gradient of 5−95% [0.1% TFA in
acetonitrile] in [0.1% TFA in water] over 3.5 min. Enantiomeric excess
was obtained by SFC using a Chiralpak AD-H column (4.6 mm × 15
cm, 5 um particle size) with carbon dioxide (gradient A) and methanol
with 0.2% diethylamine (gradient B) as the mobile phase. A gradient of
5% B to 60% B for a run time of 7 min (40 °C, 4.0 mL/min, back
pressure at 100 bar). The analysis software used were MassWare v.
4.01, MassLynx version 4.0 SP1 and Agilent LC/MSD Chemstation
Rev.B.03.01. Exact mass (HRMS) measurements were performed on a
Fourier-transform ion cyclotron resonance (FT-ICR) mass spectrom-
eter operating at 7 T (Bruker Daltonics, Billerica MA). Ions were
generated by electrospray ionization (positive mode). The instrument
was externally calibrated with a PEG300/600 solution using the
standard Francel equation. The calculated mass error for each calibrant
ion was less than 1.0 ppm from the measured value. For each spectra
512 k data points were collected using a 1.25 MHz sweep width of
detection. The time domain data were not processed prior to
performing a magnitude mode Fourier transform.
(S)-tert-Butyl 3-(4-Methylbenzylcarbamoyl)-piperidine-1-
carboxylate (6). To a solution of (S)-1-(tert-butoxycarbonyl)-
piperidine-3-carboxylic acid (Beta Pharma Inc., 500 mg) and Et3N
(912 μL) in CH2Cl2 (11 mL) was added HATU (912 mg), followed
by 4-methylbenzylamine (305 μL). The resulting solution was stirred
at 23 °C for 48 h. The reaction was concentrated, and the residue was
adsorbed onto a plug of silica gel and purified by chromatography
through a 50 g silica gel column, eluting with 0.5−5% MeOH in
1
CH2Cl2, to provide 6 (70% yield) as a white foam. H NMR (300
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dx.doi.org/10.1021/jm201565s | J. Med. Chem. 2012, 55, 1698−1705

Journal of Medicinal Chemistry
Article
1
an off-white foam. H NMR (300 MHz, CDCl3) δ 7.98 (d, J = 5.99
MEscan results, and crystal structure information. This material
Hz, 1H), 7.12 (s, 1H), 6.89 (s, 2H), 6.07 (d, J = 6.14 Hz, 1H), 4.38 (d,
J = 12.86 Hz, 1H), 4.15 (q, J = 7.11 Hz, 3H), 3.78−3.90 (m, 10H),
3.28 (dd, J = 10.01, 13.08 Hz, 1H), 3.13 (t, J = 10.82 Hz, 1H), 2.47−
2.59 (m, 1H), 2.11 (d, J = 8.92 Hz, 1H), 1.71−1.88 (m, 3H), 1.48−
1.65 (m, 1H), 1.25 (t, J = 7.09 Hz, 3H). MS (ESI, positive ion) m/z:
417.1 [M + H].
is available free of charge via the Internet at http://pubs.acs.org.
Accession Codes
The cocrystal structure of ALK + 1 has been deposited in the
RCSB (PDB ID 4DCE).
(S)-1-(2-(3,4,5-Trimethoxyphenylamino)-pyrimidin-4-yl)-
piperidine-3-carboxylic Acid (3). To a solution of 10 (1073 mg) in
dioxane (9 mL) was added lithium hydroxide monohydrate (162 mg)
in water (9 mL). The resulting reaction was stirred at 23 °C. After 1 h,
the reaction was diluted with CH2Cl2 and washed with 1 N
hydrochloric acid. The organic layer was extracted with aqueous
sodium hydroxide. The aqueous layer was then acidified with 5 N
hydrochloric acid to give a precipitate. The desired product was
collected by suction filtration and air-dried to give 3 (86% yield) as a
AUTHOR INFORMATION
■
Corresponding Author
*Phone: 617-444-5232. Fax: 617-577-9511. E-mail: richard.
lewis@amgen.com.
Notes
The authors declare the following competing financial
interest(s): The Authors are employees of Amgen Inc.
1
white solid. H NMR (300 MHz, DMSO-d6) δ ppm 10.49 (s, 1 H),
7.95 (d, J = 7.60 Hz, 1 H), 6.86 (s, 2 H), 6.69 (d, J = 7.60 Hz, 1 H),
3.98−4.21 (m, 1 H), 3.57−3.77 (m, 12 H), 2.53−2.64 (m, 1 H), 1.93−
2.07 (m, 1 H), 1.65−1.86 (m, 2 H), 1.47−1.65 (m, 1 H). MS (ESI,
positive ion) m/z: 389.1 [M + H].
ACKNOWLEDGMENTS
■
We thank Hao Chen and Linda Epstein for expression and
purification of recombinant ALK enzyme, Violeta Yu for helpful
discussion and analysis, John Eschelbach and Mqhele Ncube for
preparative HPLC purification, and Kyung-Hyun Gahm for
chiral SFC and Paul Schnier for HRMS.
Preparation of 11a−v. General Procedure B. To a solution of 3
(50 mg) in ClCH2CH2Cl (1.5 mL) in a 24-well plate (Thomson
Instrument Company) was added Et3N (16 μL). To this mixture was
added HATU (67 mg) in ClCH2CH2Cl (1.0 mL) followed by the
amine (353 μmol). The reaction plate was then sealed with a
pierceable plate seal (Thomson Instruments) and shaken at 256 rpm
on a variable speed mini vortex shaker (Eberbach Corporation) for 24
h. The reaction was diluted with MeOH (2 mL) and filtered. The
filtrate was concentrated, and the reaction mixture was purified by
mass-triggered preparative HPLC (Phenomenex Gemini-NX C18
110A column (100 mm × 21 mm, 5 μ), 44 mL/min flow rate, 5% to
95% [0.1% TFA in acetonitrile] in [0.1% TFA in water] over 10 min,
mass spectral data were acquired from 100 to 850 amu in electrospray
positive mode using MS, Waters SQ, UV, Waters 2487 or Waters PD)
to provide the desired products as their TFA salts.
ABBREVIATIONS USED
■
ALK, anaplastic lymphoma kinase; ALCL, anaplastic large-cell
lymphoma; DCC, N,N′-dicyclohexylcarbodiimide; DCE, di-
chloroethane; DFG, Asp-Phe-Gly sequence in ATP binding
site; FRET, fluorescence resonance energy transfer; HATU, 2-
(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hex-
afluorophosphate; IGF1R, insulin-like growth factor-1 receptor;
IMT, inflammatory myofibroblastic tumors; KIT, tyrosine−
protein kinase (also known as CD117 or mast/stem cell growth
factor receptor); PDB ID, protein data bank identity number;
POC, percent of control; SAR, structure−activity relationship;
SFC, supercritical fluid chromatography; TFA, trifluoroacetic
acid; TR-FRET, time-resolved fluorescence resonance energy
transfer
(S)-Methyl 3-((1-(2-(3,4,5-Trimethoxy-phenylamino)-
pyrimidin-4-yl)piperidine-3-carboxamido)methyl)benzoate
1
(11q). H NMR (300 MHz, CDCl3) δ ppm 7.81−7.92 (m, 3 H),
7.29−7.40 (m, 2 H), 6.92−7.19 (m, 1 H), 6.69−6.84 (m, 3 H), 6.06
(d, J = 6.28 Hz, 1 H), 4.29−4.51 (m, 2 H), 4.13 (dd, J = 13.59, 2.48
Hz, 1 H), 3.68−3.96 (m, 15 H), 2.39−2.52 (m, 1 H), 2.03−2.15 (m, 1
H), 1.89−2.03 (m, 1 H), 1.65−1.80 (m, 1 H), 1.49−1.65 (m, 1 H);
HRMS: calcd for (C28H33N5O6)H+, 536.24958; found, HRMS [M +
H] 536.24978.
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ASSOCIATED CONTENT
■
S
* Supporting Information
Synthesis of 7a−l, 11a−p, 11s−t, and 14, analytical data for
final compounds, biological assay protocols, complete KINO-
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a single enantiomer.
(24) A minimum significant ratio of 1.73 was calculated for the
enzyme assays and 2.9 for the cellular assay.
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(21) Supercritical fluid chromatography performed using Chiralpak
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DEA) as additive in supercritical CO2. Total flow was 4 mL/min.
Column temperature was kept at 40 °C and outlet pressure was
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