Discovery of an orally efficacious inhibitor of anaplastic lymphoma kinase

Article abstract of DOI:10.1021/jm201550q

Anaplastic lymphoma kinase (ALK) is a promising therapeutic target for the treatment of cancer, supported by considerable favorable preclinical and clinical activities over the past several years and culminating in the recent FDA approval of the ALK inhib

Full text of DOI:10.1021/jm201550q

Article
pubs.acs.org/jmc
Discovery of an Orally Efficacious Inhibitor of Anaplastic Lymphoma
Kinase
Diane E. Gingrich,* Joseph G. Lisko, Matthew A. Curry, Mangeng Cheng, Matthew Quail, Lihui Lu,
Weihua Wan, Mark S. Albom, Thelma S. Angeles, Lisa D. Aimone, R. Curtis Haltiwanger,
Kevin Wells-Knecht, Gregory R. Ott, Arup K. Ghose, Mark A. Ator, Bruce Ruggeri, and Bruce D. Dorsey
Worldwide Discovery Research, Cephalon, Inc., 145 Brandywine Parkway, West Chester, Pennsylvania 19380, United States
S
* Supporting Information
ABSTRACT: Anaplastic lymphoma kinase (ALK) is a pro-
mising therapeutic target for the treatment of cancer, supported
by considerable favorable preclinical and clinical activities over the
past several years and culminating in the recent FDA approval
of the ALK inhibitor crizotinib. Through a series of targeted
modifications on an ALK inhibitor diaminopyrimidine scaffold, our research group has driven improvements in ALK potency, kinase
selectivity, and overall pharmaceutical properties. Optimization of this scaffold has led to the identification of a potent and efficacious
inhibitor of ALK, 25b. A striking feature of 25b over previously described ALK inhibitors is its >600-fold selectivity over insulin
receptor (IR), a closely related kinase family member. Most importantly, 25b exhibited dose proportional escalation in rat compared
to compound 3 which suffered dose limiting absorption preventing further advancement. Compound 25b exhibited significant in vivo
antitumor efficacy when dosed orally in an ALK-positive ALCL tumor xenograft model in SCID mice, warranting further assessment
in advanced preclinical models.
amplification is functionally relevant in high-risk sporadic neuro-
blastoma.9 The dysregulated signaling resulting from these
rearrangements or mutations/gene amplification has led to a
state of “oncogenic addiction”, providing an opportunity for
intervention with small molecule inhibitors.10
INTRODUCTION
■
Despite progress made in the past decade, lung cancer con-
tinues to be the most frequent cause of cancer-related deaths
among men and women worldwide with greater than 200 000
new cases estimated this year in the United States alone.1 One
area of great promise in the treatment of lung and other can-
cers has been the advent of targeted kinase therapies.2 The
identification of genetic modifications resulting in tumor
development has provided an opportunity to target specific
kinases with the goal of providing more beneficial chemo-
therapeutic agents.3 One kinase that has garnered increasing
attention as a therapeutic target is anaplastic lymphoma kinase
(ALK) as illustrated by its growing pharmaceutical interest4 and
its role in non-small-cell lung cancer (NSCLC).5 ALK is a
receptor tyrosine kinase (RTK) member of the insulin receptor
(IR) superfamily, originally identified as part of the NPM-ALK
fusion gene in anaplastic large cell lymphoma (ALCL) with a
t(2;5) chromosomal translocation.6 While the physiological
role of ALK receptor has not been well-defined, involvement of
ALK in the oncogenesis of various human cancers has been well
documented and characterized. Besides NPM-ALK, various
other ALK fusion genes were subsequently detected in ALCLs,
inflammatory myofibroblastic tumor, diffuse large B-cell
lymphoma, systemic histiocytosis, and most notably, NSCLC,
resulting in the generation of oncogenic ALK fusion proteins
with constitutive phosphorylation/activation of ALK.7 The
constitutive kinase activity associated with ALK fusions appears
to play an essential role in proliferation and survival of human
cancer cells.8 Recently, it has also been reported that germ-
line mutations in ALK are the cause of most hereditary neuro-
blastoma cases, and ALK activation by mutation and/or gene
Crystal structures have been solved for the unphosphorylated
catalytic domain of ALK complexed with two ATP-competitive
inhibitors.11 This structural information can be parlayed into
the design of additional ALK inhibitors and also provides
insight into the basis for activation of ALK by the active site
mutations observed in neuroblastoma. Pfizer’s PF-02341066
(crizotinib, 1),12 a dual ALK/cMET inhibitor, was the first ALK
inhibitor advanced into clinical trials and more recently
received FDA approval for treatment of patients with locally
advanced or metastatic ALK-positive NSCLC.13,14 Other
pharmaceutical companies have also been active in the ALK
arena exploring an array of varied scaffolds. Novartis specifically
has published patent applications around a diaminopyrimidine
scaffold, as well as describing extensive pharmacology associated
with a lead compound NVP-TAE684 (2).15 In addition, several
groups have reported tyrosine kinase inhibitors which overcome
crizotinib resistance.16
Our research efforts have focused on developing potent
and selective inhibitors of ALK with modifications to the dia-
minopyrimidine scaffold, focusing on two major regions of the
molecule designated as the A- and C-rings (Figure 2).
Previously published research from our group identified a
bicyclo[2.2.1]hept-5-ene ring system of compound 3 (Figure 1)
Received: November 16, 2011
Published: May 7, 2012
© 2012 American Chemical Society
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Figure 1. Small molecule ALK inhibitors.
Figure 2. Optimization strategy and areas of modification on the diaminopyrimidine core.
as an alternative C-ring fragment impacting selectivity toward
IR.17 Our earlier report also highlighted that minor modi-
fications to the A-ring fragment can impact IR/ALK selectivity.
The combination of an A-ring methoxy selectivity determinant
combined with the [2.2.1] C-ring system provided improved
kinase selectivity observed with 3 versus other published
inhibitors. Selectivity toward IR is of particular interest because
of the involvement of IR in the regulation of glucose transport
and glycogen and fat biosynthesis.18,19 Avoiding potential com-
plications from this activity was a major objective in designing
second generation molecules with improved IR/ALK selectivity
over initial lead compound 3. While compound 3 provided a
balanced profile consisting of ALK potency and global kinase
selectivity, the modest IR/ALK selectivity led the team to
optimize the diaminopyrimidine series further in an effort to
improve selectivity toward IR (enzymatic IR/ALK IC50 ratio
of >100). In addition, compound 3 lacked the ability to be dose
escalated in preclinical toxicological species, precluding its further
development (Table 4).
Compound 4 (Figure 1) was also previously identified by our
group as an analogue with potent in vitro ALK activity but with a
potential hERG liability, as indicated by an in vitro IC50 of 3.9 μM
for hERG in a patch clamp assay (Supporting Information).
The 2,3,4,5-tetrahydro-1H-benzo[d]azepine A-ring motif of 4
resulted in potent ALK inhibition while providing a site for
functional group manipulation to balance potency with
favorable physicochemical properties. A variety of substituents
incorporated on the azepine nitrogen yielded changes in both
the enzyme and cell inhibition, depending on the nature of the
substituent. In an attempt to further modulate the potency and
selectivity differences observed with the azepine analogues, the
basic nitrogen was moved outside the seven-membered ring,
resulting in an aminobenzocycloheptene motif (Figure 2). The
resulting strategy was to identify suitable azepine or azepinone
A-ring replacements to combine with the previously established
bicyclo[2.2.1]hept-5-ene ring system, which would provide
potent ALK inhibition, improved selectivity against the IR receptor
and dose proportional increases in preclinical toxicology species.
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a
Scheme 1. Synthesis of 7-Aminosubstituted 1-Methoxy-6,7,8,9-tetrahydro-5H-benzocycloheptene Amine Fragments
a
Reagents and conditions: (a) NBS [2 equiv (X = H); 3 equiv (X = Br)], AIBN, CCl4, 80−99%; (b) (i) 3-oxopentanedioic acid diethyl ester, 0.6 M
sodium bicarbonate, tetra-n-butylammonium iodide, methylene chloride, 24 h; (ii) 1 M KOH, EtOH, reflux 3 h; 33% over two steps; (c) KNO3,
(CF3CO)2O, CH3CN, 53% (d) NaBH(OAc)3, amine, acetic acid, CH2Cl2, 44−70%; (e) H2, Pd/C wet, ethanol, 93%.
a
Scheme 2. Synthesis of 7-Aminosubstituted 3-Methoxy-6,7,8,9-tetrahydro-5H-benzocycloheptene Amine Fragments
a
Reagents and conditions: (a) (i) LiAlH4, THF, 0 °C, 65−97%; (ii) PBr3, CH2Cl2, 62−94%; (b) (i) 3-oxopentanedioic acid diethyl ester, 0.6 M
sodium bicarbonate, tetra-n-butylammonium iodide, CH2Cl2, 24 h; (ii) 1 M KOH, EtOH, reflux 3 h, 50% over two steps (c) KNO3, CH3CN, 53−
83%; (d) NaBH(OAc)3, amine, acetic acid, CH2Cl2, 65−90%; (e) H2, Pd/C wet, ethanol, 85−95%.
A focused medicinal chemistry effort evaluating modifications
to the aminobenzocycloheptene ring system was therefore
initiated.
without further purification and treated with potassium hydro-
xide in refluxing ethanol for 3 h to effect ester hydrolysis and
decarboxylation. Nitration of 8 was achieved with potassium
nitrate, trifluoroacetic anhydride in acetonitrile to give two nitra-
tion products, 10 and 12, in a 1.3:1 ratio, which were separable
with normal phase chromatography. Compounds 10 and 12 were
versatile intermediates that allowed the introduction of chemical
diversity through reductive amination chemistry. Compounds
possessing the 2-amino-1-methoxy-5,6,8,9-tetrahydro-7-aminoben-
zocycloheptene fragment, as illustrated by compound 10, provided
better ALK inhibition compared to the corresponding regiomeric
1-amino-4-methoxy-5,6,8,9-tetrahydro-7-aminobenzocyclohepte-
nane, 12. As a result, only reductive alkylation products with
2-amino-1-methoxy A-ring substitution were pursued. The
aminated products 13a−c were subjected to hydrogenation
conditions to provide the respective anilines in good yield.
CHEMISTRY
■
Two differentially substituted methoxy A-ring fragments were
prepared to assess the impact of the methoxy group at the C1
versus C3 position of the A-ring on ALK potency and selec-
tivity for ALK versus IR. The initial synthesis of 1-methoxy-
6,7,8,9-tetrahydro-5H-benzocyclohepten-2-ylamines (14a−c)
outlined in Scheme 1 involved bromination of 1-methoxy-2,3-
dimethylbenzene with N-bromosuccinimide (NBS) in carbon
tetrachloride using either 2,2′-azo-bis-isobutyronitrile, benzoyl
peroxide, or ultraviolet light as the initiator to give initial inter-
mediate 6. Conversion of 6 to the seven-membered ring 8 was
accomplished using 3-oxopentanedioic acid diethyl ester in a
biphasic reaction of methylene chloride and 0.6 M aqueous
sodium bicarbonate with tetra-n-butylammonium iodide as a
phase transfer catalyst. The resultant product was carried on
A potential liability with the previously described synthetic
pathway was the poor selectivity observed under the nitration
conditions. To improve the overall yield of the desired nitration
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a
Scheme 3. A- and C-Ring Coupling Sequence
a
Reagents and conditions: (a) K2CO3, (1S,2S,3R,4R)-3-aminobicyclo[2.2.1]hept-5-ene-2-carboxylic acid amide, DMF; (b) HCl, 2-methoxyethanol,
substituted aminobenzocycloheptene A-ring fragment, 130 °C.
Table 1. ALK Inhibition Data for Select Diaminopyrimidine Analogues
a
b
compd
R
X
Y
ALK IC50, nM
Karpas-299 cell IC50, nM
IR/ALK enzyme IC50 ratio
S(90)
3
14
4
3
45
43
0.11
0.03
0.22
0.31
0.23
0.09
0.15
0.14
0.21
0.04
4
1
30
345
103
>333
46
23a
23b
24a
24b
25a
25b
26a
26b
27a
27b
28a
28b
MeO(CH2)2NH-
MeO(CH2)2NH-
MeO(CH2)2NH-
MeO(CH2)2NH-
morpholino-
OCH3
OCH3
H
H
H
21 11
65
9
1
2
30
OCH3
OCH3
H
4
40
H
11
3
65
67
OCH3
OCH3
H
35 11
120
20
>29
662
88
morpholino-
H
1.9 0.5
morpholino-
OCH3
OCH3
H
6
1
40
morpholino-
H
16
5
120
200
20
77
c
4-methylpiperazinyl-
4-methylpiperazinyl-
4-methylpiperazinyl-
4-methylpiperazinyl-
OCH3
OCH3
H
16.2 0.8
3.1 0.3
151
>320
>59
>67
NT
c
H
NT
OCH3
OCH3
17
15
5
6
150
150
0.15
0.03
H
a
Compounds designated “a” are the first eluting diastereomer and those designated “b” are the second eluting diastereomer from a reverse phase
b
c
preparative HPLC column. Average standard deviation of four or more determinations NT, not tested.
product, the C-6 position was temporarily blocked with bro-
mine as outlined in Scheme 1 (X = Br). Treatment of 5 with
3 equiv of NBS yielded the tribrominated product 7. Ring
annulation proceeded as previously described, and with the
position para to the methoxy group blocked, nitration occurred
ortho to the methoxy group to provide isomer 11 in good yield.
Desired reductive aminations then yielded nitro intermediates
13a−c, whose nitro groups were subsequently reduced to their
corresponding anilines under standard Pd/C hydrogenation
conditions, with concomitant reduction of the bromide to give
14a−c.
As illustrated in Scheme 2, the regiomeric methoxy A-ring,
3-methoxy-6,7,8,9-tetrahydro-5H-benzocycloheptene-2,7-dia-
mine, was prepared starting with 4-methoxyphthalic acid dimethyl
ester (15). Bromination using phosphorus tribromide in
methylene chloride provided 16. The 6/7-fused ring system was
formed in the same manner as previously outlined. Nitration of 17
yielded two nitrated products, 18 and 19, in roughly a 1:1.6 ratio,
respectively, which were separated on silica gel. Ketone 19 was
subjected to reductive alkylation conditions with a select number
of amines followed by hydrogenation to reduce the nitro group
to the corresponding anilines (20a−c). A small number of
analogues were prepared with the A-ring derived from com-
pound 18 to evaluate potential potency differences between the
A-ring methoxy regiomers. A 100-fold loss of ALK potency
was observed with analogues resulting from 18 compared to
regiomer 19. As a result, efforts were focused on elaborating com-
pound 19 with a diverse set of substituents.
The target analogues were assembled using our previously
published chemistry involving tandom nucleophilic aromatic
substitution reactions as shown in Scheme 3.15 The
(1S,2S,3R,4R)-3-aminobicyclo[2.2.1]hept-5-ene-2-carboxylic
acid amide fragment is installed first on the 2,4,5-trichloropyr-
imidine. The appropriately substituted aminobenzocyclohep-
tene ring fragment was subsequently incorporated to afford
the fully assembled diaminopyrimidine scaffold. Noteworthy,
analogues 23−28 were generated as diastereomeric pairs from
racemic anilines 14 and 20. Fortunately, the diasteromeric pairs
were easily separated via preparative reverse phase HPLC.
RESULTS
■
ALK enzyme and cellular inhibition data, as well as IR enzyme
inhibition data, were obtained for the above-described dia-
steriomeric pairs of targets as shown in Table 1.20 Several
trends were identified when evaluating the diastereomeric pairs
comprised of two methoxy regiomers (X or Y = OCH3). The
slower eluting 1-methoxy substituted diastereomer on reverse
phase HPLC chromatography, in general, appeared to be the
more active of the two diastereomers, exemplified when com-
paring 23a/b, 25a/b, and 27a/b. Each of the diastereomers,
24a/24b, 26a/26b, and 28a/28b, with 3-methoxy substitution
appeared comparable in terms of ALK enzyme and cellular
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potency (within 3-fold). All four analogues possessing the
flexible methoxyethylamino A-ring moiety displayed compara-
ble enzyme and cellular potency, regardless of the position of
the methoxy substituent. However, the isomers 25b and 27b,
possessing the more space constrained morpholino and
methylpiperazinyl fragments, were 5- to 10-fold more potent
ALK enzyme inhibitors compared to their match diasteriomers,
25a and 27a. This translated into increase cellular activity with
25b and 27b being the most potent in the Karpas-299 cell
assay. Another striking difference observed between methoxy
substituted analogues matched pairs was the difference in ALK
selectivity over IR (expressed as IR/ALK enzyme IC50 ratio).
The 1-methoxy substituted analogues had higher IR/ALK IC50
ratios compared to the ratios observed with the corresponding
3-methoxy substituted analogues. Of all of the compounds
evaluated, 25b displayed the highest IR/ALK enzyme IC50 ratio
of 662, a 15-fold improvement over 3. As a result, this com-
pound was profiled more extensively.
Figure 4. Binding mode of 25b with IR (brown ribbon), as derived by
Glide/XP docking program.
To help rationalize the observed improved selectivity for the
1-methoxy substituted analogues, both 25b and 3 were com-
putationally modeled in the 2XB7 (ALK) and the 3EKK (IRK)
X-ray structures as illustrated in Figure 3. Both analogues
this loop region in IR consistently folds back toward the ATP
binding pocket. With structures such as 25b, the loop had bad
steric overlap with Asn-1097 in the binding mode (Figure 4),
resulting in poor IR inhibition and thus providing a possible
hypothesis for the increase IR/ALK selectivity observed with
25b compared to 3.
Kinase selectivity for compounds in Table 1 was evaluated
using Ambit’s KINOMEScan panel of binding assays and
expressed as an S(90) value reflecting the fraction of kinases
inhibited of >90% when screened at 1 μM across a panel of
>353 kinases.23 The compounds displayed in Table 1 range in
S(90) values from 0.03 to 0.31 with 25b exemplifying broad
kinase specificity as measured by the S(90) value of 0.147. In
addition, 25b was evaluated in Millipore’s KinaseProfiler panel
of activity assays for 259 kinases, with IC50 values determined
for 15 kinases inhibited by ≥90% at 1 μM (Table 2). Except for
Table 2. Millipore KinaseProfiler IC50 Values for 25b
kinase
IC50, nM
Figure 3. Binding mode of 25b with ALK, as derived by Glide/XP
docking program.
ACK1 (h)
ARK5 (h)
BRK (h)
CHK2 (h)
FAK (h)
Fer (h)
41
26
73
29
6
3
4
7
docked into the active site with the expected bidentate binding
mode. This binding mode was consistent with several kinase
bound 2,4-dianilinopyrimidine structures available in the
Protein Data Bank.21 Similar binding modes were also observed
for IR; however, the docking scores generated computationally
when modeling these structures were considerably and con-
sistently higher (worse) for both ligands, in agreement with the
observed activity. Comparison of the active site showed several
critical residue differences between ALK and IR (Figure 4). The
gatekeeper residue (Leu) in ALK has been replaced by Met-
1076 in IR.22 Interaction of the chloro substituent on the
pyrimidine with the Leu-1196 in ALK provides a more favor-
able interaction compared to the more polarized Met-1076 in
IR. The hydrophobic residue Leu-1256 in ALK coming from
the β-sheet underneath the hinge region has been replaced by
Met-1076. IR is the first kinase where the DFG-out structure
was observed in the apo-form with the conformation of the
activation loop differing considerably in different structures. All
of these factors have affected the binding of a large ligand like
25b, since several other IR structures failed to dock such
ligands. However, the lack of activity of one of the stereo-
isomers of this type of ligand was resulting from the conforma-
tion difference in the loop region after the D-helix. Unlike ALK,
133 15
85
99 36
88
48 11
60
4
Fes (h)
Flt3 (h)
4
Flt4 (h)
GCK (h)
JNK1a1 (h)
Rsk1 (h)
Rsk2 (h)
Rsk3 (h)
Rsk4 (h)
9
112 17
61 10
13
7
4
1
19 11
Rsk2, -3, and -4 (IC50 values range of 7−19 nM), the IC50 for
any other kinase tested is at least 10-fold higher than that for
ALK (Table 2), supporting that 25b is a highly potent and
selective ALK inhibitor. Off-target kinase activities in cells could
provide a more relevant assessment of kinase selectivity of 25b.
In order to define the absolute stereochemistry of the
7-aminosubstituted 1-methoxy-6,7,8,9-tetrahydro-5H-benzocy-
cloheptene amine A-ring fragment, efforts to crystallize 25a or
25b were undertaken. A crystal of suitable quality was achieved
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Table 3. Pharmacokinetic Parameters of 25b in Mice, Sprague−Dawley Rat, Beagle Dog, and Cynomolgus Monkey
PK parameter
SCID mouse
CD-1 mouse
rat
dog
monkey
1.2 0.05
28
a
iv
t1/2 (h)
1.8
0.7
0.9 0.2
2.4 0.2
Cl (mL min−1 kg−1
Vd (L/kg)
)
6
29
17
2
40
1
4
0.8
1.8
1.3 0.2
8.2 0.5
409 16
2.9 0.3
615 89
170 51
4.7 1.3
1.2 0.05
954 209
AUC (ng·h/mL)
Cmax (ng/mL)
tmax (h)
3025
2753
1
584
880
0.25
2.3
989 127
b
c
po
599
3
427
1.3 0.3
6.2 1.6
4833 797
0.8
t1/2 (h)
1.8
3.3
AUC (ng·h/mL)
F (%)
21381
71
2993
51
2126
52
50
8
16
3
a
b
c
1 mg/kg dose. 10 mg/kg dose. n = 2.
Table 4. Dose Escalation Data Comparison for 3 and 25b
parameter
10 mg/kg
30 mg/kg
55 mg/kg
100 mg/kg
3
Cmax
925 192
1360 106
1450 115
1553 71
AUC0−12, ng·h/mL
6025 1608
11804 875
13826 1163
14011 562
25b
Cmax
349 40
1006 92
2253 73
3053 47
AUC0−12, ng·h/mL
2574 243
10895 1000
24110 1409
38187 2474
with 25a (Supporting Information). The solution of the X-ray
analysis confirmed the R-stereochemistry of 25a, thus con-
ferring S-stereochemistry at the morpholino carbon juncture for
compound 25b.
Compound 25b was advanced for in vivo evaluation based
on the ALK cell potency, IR selectivity, and overall kinase selec-
tivity. The pharmacokinetic properties of 25b were evaluated in
several species as highlighted in Table 3. Differences in expo-
sure of 25b were observed between the two mouse strains
SCID and CD-1, with lower clearance and volume of distribu-
tion terms and higher AUC obtained in SCID compared to
CD-1 mice. Oral exposure was demonstrated in all species with
bioavailability F ranging from 16% in cynomolgus monkeys to
71% in SCID mouse.
Compound 25b was evaluated in an oral dose escalation
study in rat at 10, 30, 55, and 100 mg/kg and is summarized in
Table 4. Dose proportional increases in both AUC and Cmax
were observed with 25b, displaying a ∼9-fold increase in Cmax
and ∼15-fold increase in AUC when comparing the 10 and
100 mg/kg doses. This is in contrast to the Cmax and AUC data
for 3 at 10 and 100 mg/kg, where there is only ∼2-fold increase
in Cmax and AUC despite the 10-fold increase in dose. This
comparative data between compounds 25b and 3 highlight the
improved PK exposure observed for 25b. Efforts to advance 3
preclinically were terminated and refocused on 25b.
A single oral dose of 25b at 30 mg/kg led to >90% inhibition
of NPM-ALK phosphorylation extending to 12 h postadminis-
tration in NPM-ALK positive ALCL Karpas-299 tumor xeno-
graft in SCID mouse (Figure 5A). The degree of NPM-ALK
phosphorylation inhibition was consistent with 25b levels in
tumor xenografts based on the calculated cellular activity of 25b
in murine plasma, suggesting that the target inhibition in tumor
xenografts was likely due to direct inhibitory effects exerted by
25b (Figure 5B).
Figure 5. Pharmacodynamics of 25b in Karpas-299 tumor xenografts
in mice.
activity was observed at both oral doses of 10 and 30 mg/kg,
b.i.d., with essentially complete tumor regressions achieved at the
30 mg/kg oral b.i.d. dose (Figure 6A). This is a significant
improvement over 3, which only induced tumor stasis of Karpas-
299 tumor xenografts when dosed up to 100 mg/kg, b.i.d.
(Supporting Information S3), consistent with the limited dose-
escalation of 3 observed in rat. Administration of 25b was well
tolerated with no overt toxicity and no significant body weight loss
at both dosing regimens. Dose-related levels of 25b were observed
in plasma and tumor lysates collected at 2 h after final dosing.
Observed levels of 25b in tumors were slightly less at the
10 mg/kg dose and 7-fold higher at the 30 mg/kg dose over the
A secondary in vivo study was measurement of the antitumor
efficacy of 25b, as assessed in a Karpas-299 tumor xenograft
model in SCID mouse. Compound 25b was administered to
SCID mice orally in PEG400 at doses of 10 and 30 mg/kg b.i.d.
for the 12-day duration of the study. Dose-dependent antitumor
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Dose-dependent antitumor activity was also observed in
other NPM-ALK-positive ALCL tumor xenografts and EML4-
ALK-positive NSCLC tumor xenografts in mice administered
with 25b orally, showing complete/near complete tumor regres-
sions following treatment at doses of 30 mg/kg b.i.d. or higher.24
In addition, 25b induced growth inhibition and cytotoxicity of
neuroblastoma cell lines harboring ALK activating mutants
(F1174L in NB-1643 cells and R1275Q in SH-SY5Y cells,
respectively) but not in the ones containing ALK WT without
gene amplification (SKNAS cells) (Figure 7), indicating that
25b is active against the two ALK activating mutants most
commonly observed in human neuroblastoma.
CONCLUSION
■
Optimization of the diaminopyrimidine chemotype has resulted
in the identification of a potent, selective, and orally bio-
available ALK inhibitor, 25b. This diaminopyrimidine analogue
possessed marked improved selectivity versus IR compared to
compound 3. Further optimization of the diaminopyrimidine
core by fixing the C-ring as the [2.2.1] fragment and re-
optimizing the A-ring fragment from an azepinone ring to a
substituted aminobenzocycloheptene ring has led to substantial
improvements in IR/ALK selectivity over those observed for 3
and improved hERG inhibition versus 4 (Supporting
Information). Molecular modeling suggests that the improve-
ment in IR/ALK selectivity observed for 25b may be due to
unfavorable interactions with the Met1139 located underneath
the hinge region. In addition to improved IR/ALK selectivity,
25b displayed dose proportional increases in rat exposure, pro-
viding a clear advantage over compound 3. Therefore, further
preclinical development of 3 was discontinued in favor of 25b.
Additional studies analyzing the impact of 25b and other
potent, selective, and orally bioavailable ALK inhibitors with
newly identified mutants will be necessary to provide improved
ALK inhibitors to treat NSCLC.25 Compound 25b demon-
strated potent inhibitory activity against ALK phosphorylation
both in vitro and in vivo and robust antitumor efficacy (tumor
regressions) in Karpas-299 tumor xenografts in mice.
Combining with its overall favorable pharmaceutical properties,
25b was identified as a preclinical development candidate and
was further examined in additional biological and pharmaceut-
ical assessments. Efficacy results from more advanced pre-
clinical models will be presented in subsequent publications.
Figure 6. Antitumor efficacy of 25b on Karpas-299 tumor xenografts
in mice.
EXPERIMENTAL SECTION
■
Chemistry. All reagents and anhydrous solvents were obtained
from commercial sources and used as received. 1H and 13C NMR were
obtained on a Bruker 400 MHz instrument in the solvent indicated
with tetramethylsilane as an internal standard. Coupling constants (J)
are in hertz (Hz). Analytical HPLC was run using a Zorbax RX-C8,
5 mm × 150 mm column, eluting with a mixture of acetonitrile and
water containing 0.1% trifluoroacetic acid with a gradient of 10−100%
over 6 min, monitoring at wavelengths 254, 290, and 215 nm. LCMS
analysis was either performed on a Waters Aquity Ultra Performance
LC with a 2.1 mm × 50 mm Waters Aquity UPLC BEH C18 1.7 μm
column or Agilent 1100 with a 2.1 mm × 30 mm 3.5 μm Eclipse
XDB-C8 column and a solvent system of 10−100% acetonitrile−water
with 0.1% formic acid. The mass spectrometry data were acquired on a
Micromass LC-ZQ 2000 quadrapole mass spectrometer (Waters
Aquity) or a Bruker Esquire 200 ion trap (Agilent 1100) instrument.
High resolution mass spectrometry was performed on a Waters Synapt
G2 Q-TOF mass spectrometer by positive ion electrospray using
leucine¬enkephalin as a lock-mass standard. Column chromatography
was typically performed using automation on an ISCO CombiFlash
Figure 7. Growth inhibition of 25b on neuroblastoma cell lines
harboring ALK activating mutations.
plasma adjusted ALK cell IC90 of 1500 nM (∼750 ng/mL), as
measured in the presence of murine plasma (Figure 6B), further
supporting the notion that sustained complete target inhibition
in tumor is required to achieve regression in NPM-ALK-positive
ALCL tumor xenografts.
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Companion using silica gel. Additional purification was accomplished
using a Gilson preparative HPLC system and reverse phase silica gel
and trilution LC software. Melting points were taken on a Mel-Temp
apparatus and are uncorrected. Purity of described compounds was
assessed by HPLC and shown to be >95% pure unless otherwise
noted.
Preparation of 1,2-Bis-bromomethyl-3-methoxybenzene
(6).26 1-Methoxy-2,3-dimethyl-benzene (40 g, 0.29 mol), N-bromo-
succinimide (104.6 g, 0.5877 mol), and 2,2′-azo-bis-isobutyronitrile
(2 g, 0.01 mol) were dissolved in carbon tetrachloride (800 mL). The
mixture was heated to reflux and was allowed to stir overnight. The
reaction mixture was cooled, and the solids were collected by filtration.
The filtrate was then washed with saturated sodium bicarbonate and
extracted with dichloromethane. Combined organic layers were dried
over sodium sulfate, filtered, and concentrated under vacuum to give
the desired product (86 g, 99% yield). 1H NMR (CDCl3) δ 7.23
(t, 1H, J = 8.05 Hz), 6.96 (d, 1H, J = 7.64 Hz), 6.86 (d, 1 H, J =
8.36 Hz), 4.77 (s, 2H), 4.61 (s, 2H), 3.89 (s, 3H).
Preparation of 1-Methoxy-5,6,8,9-tetrahydrobenzocyclo-
hepten-7-one (8).27 (i) To a 5 L flask was added tetra-n-
butylammonium iodide (30 g, 0.09 mol) in 0.6 M of sodium
bicarbonate in water (1250 mL) and methylene chloride (500 mL).
The reaction mixture was cooled to 0 °C, and a solution of 1,2-bis-
bromomethyl-3-methoxybenzene (43.0 g, 0.146 mol) and 3-
oxopentanedioic acid, diethyl ester (34.6 mL, 0.190 mol) in methylene
chloride (120 mL) was added slowly dropwise to the reaction
mixture via addition funnel. Following complete addition, the reaction
mixture was vigorously stirred at room temperature overnight. The
mixture was transferred in batches to a separatory funnel where the
layers were separated. The aqueous layer was washed with additional
methylene chloride, and the combined organic extracts were dried over
magnesium sulfate, filtered, and concentrated under vacuum to a dark
oil. The oil was triturated with diethyl ether, and the tetrabutylammo-
nium salts precipitated out of solution and were collected by filtration.
The filtrate was concentrated under vacuum to a dark oil which was
carried directly on to the next step without purification.
Preparation of 1-Bromo-2,3-bis-bromomethyl-4-methoxy-
benzene (7).28 To a stirred solution of 1-methoxy-2,3-dimethylben-
zene (1) (10.00 g, 0.073 mol) in carbon tetrachloride (100 mL, 1 mol)
was added N-bromosuccinimide (39.2 g, 0.22 mol) and benzoyl
peroxide (0.2 g, 0.7 mmol). The reaction mixture was heated to reflux
overnight. Additional NBS (1 equiv) and benzoyl peroxide (0.1 equiv)
were added, and the mixture continued to be heated at reflux. The
reaction mixture was cooled to room temperature and succinimide
removed by filtration. The filtrate was concentrated under vacuum to
1
give product. H NMR (DMSO-d6) δ 7.66 (d, 1H, J = 8.84 Hz), 1.06
(d, 1H, J = 8.84 Hz), 4.81 (s, 2H), 4.77 (s, 2H), 3.88 (s, 3H).
Preparation of 1-Bromo-4-methoxy-5,6,8,9-tetrahydroben-
zocyclohepten-7-one (9). (i)To a stirred suspension of tetra-n-
butylammonium iodide (1.2 g, 0.0032 mol) in 0.6 M of sodium
bicarbonate in water (43 mL) and methylene chloride (5 mL,
0.08 mol) was added a solution of 3-oxopentanedioic acid, diethyl
ester (1.3 mL, 0.0070 mol) and 1-bromo-2,3-bis-bromomethyl-4-
methoxybenzene (2.01 g, 0.00539 mol) in methylene chloride (5 mL)
dropwise. The biphasic reaction mixture was stirred vigorously
overnight, during which time the mixture changed color from pale
yellow to orange. The reaction mixture was quenched with ammonium
chloride and extracted with methylene chloride (3 × 30 mL). The
organic extracts were dried over magnesium sulfate, filtered, and
concentrated under vacuum. The diastereomeric mixture was taken on
without purification to the next step.
(ii) A stirred solution of 1-bromo-4-methoxy-7-oxo-6,7,8,9-tetrahy-
dro-5H-benzocycloheptene-6,8-dicarboxylic acid diethyl ester (102 g,
0.247 mol) in ethanol (2000 mL) and 1 M aqueous potassium hydro-
xide (1300 mL) was heated to reflux for 2 h. The reaction mixture was
cooled to room temperature. HCl (1 N) was added to quench the
reaction and the product extracted with methylene chloride, washed
with water and brine, dried over magnesium sulfate, filtered, and con-
centrated under vacuum to give a light orange-tan solid. The material
was purified by ISCO chromatography using ethyl acetate−hexane
(25−50%) to give the desired product as a pale orange-tan solid
(58 g, 87% yield). 1H NMR (CDCl3) δ 7.44 (d, 1H, J = 8.84 Hz), 6.70
(d, 1H, J = 9.09 Hz), 3.82 (s, 3H), 3.18 (m, 2H), 3.08 (m, 2H), 2.57
(m, 4H).
Preparation of 4-Bromo-1-methoxy-2-nitro-5,6,8,9-tetrahy-
drobenzocyclohepten-7-one (11). To a stirred solution of
1-bromo-4-methoxy-5,6,8,9-tetrahydrobenzocyclohepten-7-one (20.0 g,
0.074 mol) in sulfuric acid (200 mL) at 0 °C under an atmosphere of
nitrogen was added potassium nitrate (7.89 g, 0.078 mol) in portions.
After being stirred for 15 min, the reaction mixture was poured over
ice−water. A gummy solid formed which was dissolved in methylene
chloride, transferred to a separatory funnel and the water layer re-
moved. The organic phase was washed with saturated aqueous bicar-
bonate and water, dried over magnesium sulfate, filtered, and
concentrated under vacuum to an orange solid. Product was purified
by ISCO chromatography on silica gel (350 g column) using ethyl
acetate−hexane (10−100%). Fractions corresponding to product were
combined and concentrated under vacuum to give product as an orange
solid (18 g, 77% yield). 1H NMR (CDCl3) δ 8.04 (s, 1H), 3.90 (s, 3H),
3.25 (m, 2H), 3.15 (m, 2H), 2.64 (m, 4H).
(ii) A stirred solution of 1-methoxy-7-oxo-6,7,8,9-tetrahydro-5H-
benzocycloheptene-6,8-dicarboxylic acid diethyl ester (80 g, 0.2 mol)
in ethanol (1500 mL, 26 mol) and aqueous potassium hydroxide
solution (1 M, 950 mL) was heated to reflux for 3 h. After 3 h, the
mixture was cooled to room temperature and carefully quenched
with 1 N HCl. The reaction mixture was concentrated under vacuum
to remove ethanol and then extracted with methylene chloride. The
organic layer was dried over magnesium sulfate, filtered, and concen-
trated under vacuum to provide a dark brown-black oil. Purification
was accomplished using ISCO chromatography on silica gel with hexane−
ethyl acetate as the eluant. Fractions corresponding to product were
combined and concentrated under vacuum, during which time pro-
1
duct crystallized as a yellow solid. H NMR (CDCl3) δ 7.16 (t, 1H, J =
7.58 Hz), 6.83 (dd, 2H), 3.83 (s, 3H), 3.01 (m, 2H), 2.91 (m, 2H), 2.59
(m, 4H).
Preparation of 1-Methoxy-2-nitro-5,6,8,9-tetrahydrobenzo-
cyclohepten-7-one (10). A flask containing 1-methoxy-5,6,8,9-
tetrahydrobenzocyclohepten-7-one (5.76 g, 30.3 mmol) in acetonitrile
(142.3 mL) was cooled to 0 °C, and the mixture was stirred for 5 min.
Potassium nitrate (3.06 g, 30.3 mmol) was added in one portion, and
the mixture was stirred for 5−10 min at 0 °C and then warmed to
room temperature. Changes in color from light yellow to a deeper
orange were observed over 2 h at room temperature. After 2 h, the
reaction mixture was poured into saturated sodium bicarbonate. Solid
bicarbonate was also added to adjust the pH between 4.5 and 6.0. The
reaction mixture was extracted with methylene chloride (3 × 100 mL).
The organic extracts were combined and dried over magnesium
sulfate, filtered, and concentrated under vacuum to give the product as
an orange waxy solid (7.55 g). Purification was accomplished on silica
gel using ISCO chromatography and ethyl acetate−hexanes (0−50%)
as the eluant (2.10 g, 29.5%). Additional material was isolated by
Preparation of ((4-Bromo-1-methoxy-2-nitro-6,7,8,9-tetra-
hydro-5H-benzocyclohepten-7-yl)-(2-methoxyethyl)amine
(13a). To a stirred solution of 4-bromo-1-methoxy-2-nitro-5,6,8,9-
tetrahydrobenzocyclohepten-7-one (2.2 g, 7 mmol) in methylene
chloride (150 mL) was added 2-methoxyethylamine (6.1 mL,
70.4 mmol), powdered 4 Å molecular sieves, and acetic acid (4 mL,
70.4 mmol). The reaction mixture was heated to reflux overnight and
then cooled to room temperature. Sodium triacetoxyborohydride (4.5 g,
21 mmol) was added in one portion, and the mixture was stirred for 2 h.
The reaction solids were removed by filtration, and the filtrate was
neutralized with 33% aqueous sodium hydroxide, adjusting the pH
between 8 and 10. The product was extracted with methylene chloride
(3 × 30 mL). The organic extract was dried over magnesium sulfate,
filtered, and concentrated under vacuum. Purification was accom-
plished on silica gel using methanol−methylene chloride (10%)
1
subjecting the filtrate to chromatography. H NMR (CDCl3) δ 7.71
(d, 1H, J = 7.92 Hz), 7.11 (d, 1H, J = 8.29 Hz), 3.90 (s, 3H), 3.05 (m,
2H), 2.99 (m, 2H), 2.63 (m, 4H).
1
as the eluant to give the product as a red oil. H NMR (CDCl3)
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δ 7.93 (s, 1H), 3.85 (s, 3H), 3.51 (t, 2H, J = 6.3 Hz), 3.39 (m, 2H),
3.37 (s, 3H), 2.83 (m, 4H), 2.62 (t, 1H, J = 12 Hz), 2.08 (m, 2H), 1.33
(m, 2H).
Preparation of 4-(4-Bromo-1-methoxy-2-nitro-6,7,8,9-tetra-
hydro-5H-benzocyclohepten-7-yl)morpholine (13b). The prod-
uct was isolated as a brown oil (17.8 g, 79% yield). 1H NMR (CDCl3)
δ 7.92 (s, 1H), 3.86 (s, 3H), 3.70 (t, 4H, J = 4.5 Hz), 3.43 (m, 1H),
2.90 (t, 2H, J = 4.5 Hz), 2.68 (m, 2H), 2.55 (t, 2H, J = 4.5 Hz), 2.47
(m, 2H), 2.13 (m, 2H), 1.42 (q, 2H, J = 10 Hz).
Preparation of 1-(4-Bromo-1-methoxy-2-nitro-6,7,8,9-tetra-
hydro-5H-benzocyclohepten-7-yl)-4-methylpiperazine (13c).
The product was isolated as a brown oil (1.87 g, 72% yield). 1H
NMR (CDCl3) δ 7.96 (s, 1H), 3.96 (s, 3H), 3.72 (t, 4H, J = 4.5 Hz),
3.43 (m, 1H), 2.90 (t, 2H, J = 4.5 Hz), 2.68 (m, 2H), 2.55 (m, 2H),
2.47 (m, 2H), 2.26 (s, 3H), 2.06 (m, 2H), 1.74 (m, 2H).
Preparation of (2-Methoxyethyl)-(2-nitro-6,7,8,9-tetrahy-
dro-5H-benzocyclohepten-7-yl)amine (14a). 4-Bromo-1-me-
thoxy-2-nitro-6,7,8,9-tetrahydro-5H-benzocyclohepten-7-yl)-(2-
methoxyethyl)amine (2.4 g, 6.4 mmol) was dissolved in ethanol
(50 mL) in a 500 mL Parr flask. Palladium on carbon, 10% (90:10
carbon black/palladium, 250 mg, 19 mmol), was added to the flask and
the material subjected to hydrogen at 50 psi for 16 h. The catalyst was
removed by filtration through Celite and the resulting filtrate con-
centrated under vacuum to provide the desired product as a red oil
(HBr salt). 1H NMR (CDCl3) δ 6.68 (d, 1H, J = 8.1 Hz), 6.52 (d, 1H,
J = 8.1 Hz), 3.77 (m, 4H), 3.68 (s, 3H), 3.40 (s, 3H), 3.07 (m, 4H),
2.75 (m, 1H), 2.39 (m, 2H), 1.55 (m, 2H).
in water (300 mL) and methylene chloride (130 mL, 2.1 mol) was
added a solution of 1,2-bis-bromomethyl-4-methoxybenzene (16.00 g,
0.054 mol) and 3-oxopentanedioic acid, diethyl ester (14.31 g,
0.071 mol) in methylene chloride (40 mL, 0.6 mol). The solution was
stirred vigorously at room temperature for ∼20 h. Saturated
ammonium chloride solution was added to the reaction mixture.
The product was extracted with ethyl acetate (3 × 100 mL). The ethyl
acetate extracts were washed with water and brine, then dried over
magnesium sulfate, filtered, and concentrated under vacuum to give a
yellow oil. The oil was triturated with ether, and a solid precipitated
out of solution and was removed by filtration (tetrabutylammonium
salts). The filtrate was concentrated to an oil (20.0 g, 100%) that was
carried on to the next step without purification.
(ii) 2-Methoxy-7-oxo-6,7,8,9-tetrahydro-5H-benzocycloheptene-6,
8-dicarboxylic acid diethyl ester (18.2 g, 0.0544 mol) was dissolved
in ethanol, and the solution was treated with potassium hydroxide
(24.4 g, 0.435 mol) in water (140 g, 7.6 mol). The mixture was then
refluxed until HPLC showed consumption of starting material (∼5 h).
The mixture was then acidified with 1 N HCl, and the product was
extracted with dichloromethane. Organic extracts were dried over
sodium sulfate, filtered, and concentrated under vacuum. The crude
mixture was filtered through a plug of silica, rinsing with dichloromethane
before purification. The filtrate was concentrated under vacuum and
purified by ISCO flash column chromatography (hexane−ethyl acetate).
Combined fractions were reduced under vacuum to afford 2-methoxy-
1
5,6,8,9-tetrahydrobenzocyclohepten-7-one (6.0 g, 58% yield). H NMR
(CDCl3) δ 7.14 (d, 1H, J = 8.8 Hz), 6.79 (s, 1H), 6.75 (d, 1H, J =
8.8 Hz), 3.81 (s, 3H), 2.86 (m, 4H), 2.56 (m, 4H).
Preparation of 1-Methoxy-7-morpholin-4-yl-6,7,8,9-tetrahy-
dro-5H-benzocyclohepten-2-ylamine (14b). The product was
isolated as an HBr salt (11 g, 79% yield). 1H NMR (CDCl3) δ 6.72 (d,
1H, J = 7.83 Hz), 6.55 (d, 1H, J = 7.83 Hz), 4.15 (broad s, 4H), 3.70
(s, 3H), 3.47 (m, 1H), 3.35 (broad s, 1H), 3.10 (broad s, 4H), 2.59−
2.86 (m, 4H), 2.37 (m, 1H), 1.48 (m, 3H).
Preparation of 2-Methoxy-1-nitro-5,6,8,9-tetrahydrobenzo-
cyclohepten-7-one (18) and 2-Methoxy-3-nitro-5,6,8,9-tetra-
hydrobenzocyclohepten-7-one (19). 2-Methoxy-5,6,8,9-tetrahy-
drobenzocyclohepten-7-one (6.00 g, 0.0315 mol) was dissolved in
acetonitrile (280 mL) and was added to a mixture of trifluoroacetic
anhydride (13.4 mL, 0.095 mol) in acetonitrile (60 mL) at 0 °C.
Potassium nitrate (3.19 g, 0.032 mol) was then added and the mixture
allowed to warm to room temperature. Following complete con-
sumption of starting material, the mixture was poured over saturated
sodium bicarbonate, and organics were extracted with ethyl acetate−
dichloromethane. Combined organics were dried over sodium sulfate,
filtered, and reduced under vacuum. The crude mixture was purified by
ISCO flash column chromatography using hexane−ethyl acetate (0−
50%). Combined fractions were reduced under vacuum to afford
2-methoxy-1-nitro-5,6,8,9-tetrahydrobenzocyclohepten-7-one (1.80 g,
25% yield) (1H NMR (CDCl3) δ 7.30 (d, 1H, J = 8.6 Hz), 6.90 (d,
1H, J = 8.6 Hz), 3.89 (s, 3H), 2.93 (m, 2H), 2.77 (m, 2H), 2.60−2.65
(m, 4H)) and 3.62 g (49%) of 2-methoxy-3-nitro-5,6,8,9-tetrahy-
drobenzocyclohepten-7-one (1H NMR (CDCl3) δ 7.78 (s, 1H), 6.96
(s, 1H), 3.97 (s, 3H), 2.94 (m, 4H), 2.64 (m, 4H)).
Preparation of 3-Methoxy-N*7*-(2-methoxyethyl)-6,7,8,9-
tetrahydro-5H-benzocycloheptene-2,7-diamine (20a). (i) To a
stirred solution of 2-methoxy-3-nitro-5,6,8,9-tetrahydrobenzocyclo-
hepten-7-one (0.81 g, 0.0034 mol) in 1,2-dichloroethane (12 mL)
under nitrogen at room temperature were added 2-methoxyethylamine
(0,31 g, 0.004 mol) and acetic acid (0.24 mL, 0.004 mol). The reaction
mixture was stirred at room temperature for 3 h. The mixture was then
cooled to 0 °C, and sodium triacetoxyborohydride (2.2 g, 0.01 mol)
was added to the reaction mixture in one portion. The reaction
mixture was stirred at 0 °C for 30 min and then warmed to room
temperature for 4 h. The reaction mixture was quenched with water
and pH adjusted to 8−10 with 10 N NaOH solution. The mixture was
extracted with methylene chloride, dried over magnesium sulfate, and
concentrated under vacuum. Purification was accomplished on silica
gel using ISCO chromatography with methylene chloride−methanol
with ammonium hydroxide. The product was isolated as a brown film.
1H NMR (CDCl3) δ 8.42 (s, 1H), 8.16 (s, 1H), 3.87 (s, 3H), 3.67 (m, 2H),
Preparation of 1-Methoxy-7-(4-methylpiperazin-1-yl)-
6,7,8,9-tetrahydro-5H-benzocyclohepten-2-ylamine (14c). The
1
product was isolated as a dark brown oil HBr salt (1.5 g, 89%). H
NMR (CDCl3) δ 6.91 (d, 1H, J = 8.84 Hz), 6.49 (s, 1H), 6.44 (d, 1H,
J = 8.84 Hz), 3.63 (m, 1H), 2.54−2.75 (m, 6H), 2.31−2.45 (m, 5H),
2.31 (s, 3H), 2.05 (m, 2H), 1.61 (m, 1H), 1.37 (m, 2H).
Preparation of 1,2-Bis-bromomethyl-4-methoxybenzene
(16).29 (i) To a stirred suspension of lithium tetrahydroaluminate
(16.6 g, 0.44 mol) in tetrahydrofuran (300 mL) at 0 °C under nitrogen
was added dropwise a solution of 4-methoxyphthalic acid dimethyl
ester (24.46 g, 0.11 mol) in tetrahydrofuran (100 mL). The mixture
was stirred at 0 °C for 1 h and then warmed to room temperature
overnight. The mixture was recooled at 0 °C and the reaction quen-
ched with addition of water (125 mL) carefully dropwise, followed by
1 N NaOH (100 mL) and additional water (125 mL). Evolution of gas
was observed upon initial quenching with water. Aluminum salts
precipitated out of solution and were removed by filtration following
complete quenching of the reaction mixture. The filtrate was diluted
with ethyl acetate, washed with water, dried over magnesium sulfate,
filtered, and concentrated under vacuum to provide (2-hydroxymethyl-
4-methoxyphenyl)methanol as a colorless oil (17.8 g, 97% yield).
1H NMR (CDCl3) δ 7.24 (d, 1H, J = 8.2 Hz), 6.91 (s, 1H), 6.80
(d, 1H, J = 8.2 Hz), 4.63 (d, 4H, J = 6.3 Hz), 3.8 (s, 3H), 3.44 (broad
s, 1H), 3.16 (broad s, 1H).
(ii) (2-Hydroxymethyl-4-methoxyphenyl)methanol (17.8 g, 0.11 mol)
was dissolved in chloroform (200 mL, 2 mol) and treated with
phosphorus tribromide (60.2 g, 0.222 mol) dropwise over 6 h. After
being stirred overnight at room temperature, the mixture was cooled
at 0 °C and treated with water (50 mL). The reaction mixture was
poured over saturated sodium bicarbonate and extracted with
dichloromethane. Combined organics were dried over sodium sulfate,
filtered, and concentrated under vacuum to give the product (16.0 g,
51% yield) which was used without further purification. 1H NMR
(CDCl3) δ 7.29 (d, 1H, J = 8.4 Hz), 6.9 (s, 1H), 6.83 (d, 1H, J = 8.4
Hz), 4.66 (s, 2H), 4.62 (s, 2H), 3.82 (s, 3H).
3.38 (m, 5H), 3.22 (m, 2H), 2.62−2.88 (m, 5H), 2.36 (m, 2H).
(ii) 2-Methoxyethyl-(2-methoxy-3-nitro-6,7,8,9-tetrahydro-5H-ben-
zocyclohepten-7-yl)amine (2.50 g, 0.0085 mol) in ethanol (45 mL)
was added to a Parr vessel containing palladium on carbon, 10%
(90:10 carbon black/palladium, 0.5 g, 0.038 mol), under nitrogen. The
Preparation of 2-Methoxy-5,6,8,9-tetrahydrobenzocyclo-
hepten-7-one (17). (i) To a stirred solution of tetra-n-butyl-
ammonium iodide (12.1 g, 0.033 mol) in 0.6 M of sodium bicarbonate
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vessel was placed on a Parr shaker and subjected to hydrogen at 50 psi
[2.2.1]hept-5-ene-2-carboxamide was isolated as a white solid (276
mg, 56% yield). H NMR (DMSO-d6) δ 8.61 (d, 1H, J = 7.32 Hz),
1
for 2 h. Catalyst was removed by filtration and the product used
1
without purification. H NMR (CDCl3) δ 7.67 (s, 1H), 6.83 (s, 1H),
8.20 (s, 1H), 7.86 (s, 1H), 7.32 (s, 1H), 6.33 (br s, 1H), 6.30 (br s,
1H), 3.99 (m, 1H), 2.88 (s, 1H), 2.75 (s, 1H), 2.04 (d, 1H, J = 8.84
Hz), 1.40 (d, 1H, J = 8.84 Hz); LCMS (m/e) 299, 300 (M + H).
General Procedure for Coupling Reactions: Preparation of
(1S,2S,3R,4R)-3-[5-Chloro-2-((S)-1-methoxy-7-morpholin-4-yl-
6,7,8,9-tetrahydro-5H-benzocyclohepten-2-ylamino)-
pyrimidin-4-ylamino]bicyclo[2.2.1]hept-5-ene-2-carboxylic
Acid Amide (23a) and (1S,2S,3R,4R)-3-[5-Chloro-2-((R)-1-me-
thoxy-7-morpholin-4-yl-6,7,8,9-tetrahydro-5H-benzocyclo-
hepten-2-ylamino)pyrimidin-4-ylamino]bicyclo[2.2.1]hept-5-
ene-2-carboxylic Acid Amide (23b). To a microwave vial
containing methoxy-7-morphoin-4-yl-6,7,8,9-tetrahydro-5H-benzocy-
clohepten-2-ylamine (56 mg, 0.2 mmol) and (1S,2S,3R,4R)-3-(2,5-
dichloropyrimidin-4-ylamino)bicycle[2.2.1]hept-5-ene-2-carboxylic
acid amide (50 mg, 0.2 mmol) in isopropyl alcohol (4 mL) was added
4 M HCl in 1,4-dixoane (40 μL) dropwise. The reaction mixture was
subjected to microwaves at 140 °C for 50 min. The mixture was
concentrated under vacuum to remove solvent and purified by reverse
phase preparative HPLC with acetonitrile−water containing 0.1% TFA
(5−50% over 17 min) as the eluant. Two diastereomers were isolated,
redissolved in methanol (3 mL), and treated separately with MP-
carbonate (solid support). The MP-carbonate beads were removed by
filtration and the respective filtrates concentrated under vacuum.
(1S,2S,3R,4R)-3-[5-Chloro-2-((R)-1-methoxy-7-morpholin-4-
yl-6,7,8,9-tetrahydro-5H-benzocyclohepten-2-ylamino)-
pyrimidin-4-ylamino]bicyclo[2.2.1]hept-5-ene-2-carboxylic
Acid Amide (25a). The product was isolated as tan solid (35.6 mg,
3.94 (s, 3H), 3.53 (t, 1H, J = 14 Hz), 3.37 (s, 6H), 2.83−2.92 (m, 5H),
2.71 (m, 2H), 2.12 (m, 2H), 1.39 (m, 2H).
Preparation of 3-Methoxy-7-morpholin-4-yl-6,7,8,9-tetrahy-
dro-5H-benzocyclohepten-2-ylamine (20b). (i) 2-Methoxy-3-
nitro-5,6,8,9-tetrahydrobenzocyclohepten-7-one (4.94 g, 0.021 mol)
in methylene chloride (100 mL) was treated with morpholine (18.30 g,
0.21 mol) followed by acetic acid (12.61 g, 0.21 mol). Powdered 4 Å
molecular sieves (2 mass equiv) was added and the mixture heated
to reflux for 4 h. The solution was then cooled to room temperature,
and sodium triacetoxyborohydride (8.90 g, 0.042 mol) was added.
The reaction mixture was poured over saturated sodium bicarbonate,
and organics were extracted with ethyl acetate−dichloromethane.
Combined organics were dried over sodium sulfate, filtered, and
concentrated under vacuum. The crude mixture was purified by ISCO
flash column chromatography (methylene chloride−methanol). Com-
bined fractions were reduced under vacuum to afford 4-(2-methoxy-3-
nitro-6,7,8,9-tetrahydro-5H-benzocyclohepten-7-yl)morpholine (5.4 g,
84% yield).
(ii) 4-(2-Methoxy-3-nitro-6,7,8,9-tetrahydro-5H-benzocyclohepten-
7-yl)morpholine (5.40 g, 0.018 mol) was dissolved in ethanol
(100 mL) and the reaction mixture carefully added to 10% palladium
on carbon (0.75 g) under nitrogen in a Parr vessel. The mixture was
then placed on a Parr shaker until uptake of hydrogen had ceased
(5 h). Catalyst was removed by filtration and the filtrate concentrated
under vacuum to afford 3-methoxy-7-morpholin-4-yl-6,7,8,9-tetrahy-
dro-5H-benzocyclohepten-2-ylamine (4.1 g, 84% yield). 1H NMR
(DMSO-d6) δ 6.56 (s, 1H), 6.40 (s, 1H), 4.39 (m, 2H), 3.17 (s, 3H),
3.53 (m, 4H), 3.34 (m, 2H), 2.62 (m, 1H), 2.44 (m, 4H), 1.91 (m,
2H), 1.24 (q, 2H, J = 12 Hz).
Preparation of 3-Methoxy-7-(4-methyl-piperazin-1-yl)-
6,7,8,9-tetrahydro-5H-benzocyclohepten-2-ylamine (20c). (i)
2-Methoxy-3-nitro-5,6,8,9-tetrahydrobenzocyclohepten-7-one (1.5 g,
6.38 mmol) and 1-methylpiperazine (6.4 g, 63.8 mmol) were dissolved
in methylene chloride (150 mL) and treated with acetic acid (3.8 g,
63.8 mmol). The mixture was then heated to reflux for 4 h. When
the mixture cooled to room temperature, sodium triacetoxyborohy-
dride (2.7 g, 12.8 mmol) was added in one portion. The reaction
mixture was stirred at room temperature overnight. The reaction
mixture was poured over saturated aqueous sodium bicarbonate
solution and extracted with dichloromethane. The organic phase was
dried over sodium sulfate, filtered, and concentrated under vacuum.
The crude reaction mixture was purified on silica gel using ISCO
chromatography with hexane−ethyl acetate as the eluant. Fractions
corresponding to product were dried to provide the desired product
which was carried on without purification.
1
33% yield). H NMR (CDCl3) δ 8.02 (d, 1H, J = 8.3 Hz), 7.81 (s,
1H), 6.88 (d, 1H, J = 8.3 Hz), 6.31 (m, 1H), 6.25 (m, 1H), 5.85 (s,
1H), 5.55 (s, 1H), 4.19 (t, 1H, J = 7.3 Hz), 3.01 (s, 4H), 3.72 (s, 3H),
3.49 (m, 2H), 3.07−3.15 (m, 4H), 2.91 (m, 2H), 2.76 (m, 1H), 2.38−
2.47 (m, 3H), 2.18 (d, 1H, J = 9.6 Hz), 1.61 (d, 1H, J = 9.3 Hz), 1.48
(m, 2H). 13C (NMR) δ 176.01, 157.99, 156.38, 152.82, 146.59,
139.08, 137.47, 136.43, 134.74, 130.79, 123.79, 117.48, 103.77, 66.65,
65.74, 61.01, 52.12, 48.66, 47.58, 46.95, 43.55, 43.43, 31.64, 29.53,
29.36, 22.82. LCMS (m/e) 539 (M + H). HRMS: calcd for
C28H35ClN6O3 (M + H)+ 539.2537, found 539.2542.
(1S,2S,3R,4R)-3-[5-Chloro-2-((S)-1-methoxy-7-morpholin-4-
yl-6,7,8,9-tetrahydro-5H-benzocyclohepten-2-ylamino)-
pyrimidin-4-ylamino]bicyclo[2.2.1]hept-5-ene-2-carboxylic
Acid Amide (25b). The product was isolated as an off-white solid
1
(42.8 mg, 39% yield). H NMR (CDCl3) δ 8.16 (d, 1H, J = 7.5 Hz),
7.86 (s, 2H), 7.41 (broad s, 1H), 6.88 (d, 1H, J = 7.5 Hz), 6.31 (s,
2H), 5.81 (s, 1H), 5.62 (s, 1H), 4.28 (m, 1H), 3.97 (s, 4H), 3.73 (s,
3H), 3.39−3.54 (m, 2H), 3.09 (m, 4H), 2.90 (m, 2H), 2.75 (m, 1H),
2.36−2.5 (m, 4H), 2.20 (d, 1H, J = 9 Hz), 1.61 (d, 1H, J = 9 Hz), 1.48
(m, 2H), 1.26 (m, 1H). 13C NMR (DMSO-d6) δ 175.99, 157.98,
156.36, 152.83, 146.71, 139.09, 137.57, 136.42, 134.80, 130.76, 123.77,
117.54, 103.72, 67.04, 66.71, 61.00, 52.09, 48.67, 47.56, 46.92, 43.53,
43.42, 31.66, 29.56, 29.43, 22.85. LCMS (m/e) 539 (M + H). HRMS:
calcd for C28H35ClN6O3 (M + H)+ 539.2537, found 539.2533.
(1S,2S,3R,4R)-3-{5-Chloro-2-[1-methoxy-7-(2-methoxy-ethyl-
amino)-6,7,8,9-tetrahydro-5H-benzocyclohepten-2-ylamino]-
pyrimidin-4-ylamino}bicyclo[2.2.1]hept-5-ene-2-carboxylic
Acid Amide (23a). The product was isolated as the beige foam
(ii) 1-(2-Methoxy-3-nitro-6,7,8,9-tetrahydro-5H-benzocyclohepten-
7-yl)-4-methylpiperazine (1.89 g 5.92 mmol) was dissolved in ethanol
(40 mL). Palladium on carbon, 10% (90:10 carbon black/palladium,
0.5 g, 37.5 mmol), was added to the reaction mixture, and the con-
tents were placed under hydrogen (50 psi) for 2 h. The catalyst was
removed by filtration through Celite and the filtrate concentrated
1
under vacuum to give product. H NMR (DMSO-d6) δ 6.56 (s, 1H),
1
3.7 (s, 3H), 3.48 (m, 4H), 3.34 (m, 4H), 2.6 (m, 1H), 2.45 (m, 2H),
2.27 (m, 2H), 1.91 (m, 2H), 1.24 (q, 2H, J = 12 Hz).
(58.2 mg, 27% yield). H NMR (CDCl3) δ 8.17 (d, J = 8.3 Hz, 1H),
7.88 (s, 1H), 7.39 (s, 1H), 6.94 (m, 1H), 6.86 (d, J = 8.4 Hz, 1H), 6.37
(m, 1H), 6.31 (m, 1H), 5.61 (br s, 1H), 5.36 (br s, 1H), 4.37 (m, 1H),
3.72 (s, 3H), 3.51 (m, 2H), 3.26 (s, 3H), 3.23 (m, 1H), 3.06 (s, 1H),
2.94 (s, 1H), 2.85−2.70 (m, 6H), 2.49 (d, J = 8.4 Hz, 2H), 2.24 (d, J =
8.9 Hz, 1H), 2.09 (m, 2H), 1.63 (m, 2H), 1.26 (m, 4H). 13C NMR
(DMSO-d6): δ 175.99, 157.99, 156.37, 152.83, 146.62, 139.08, 137.73,
136.44, 134.94, 130.68, 123.72, 117.43, 103.71, 71.91, 60.96, 57.85,
52.09, 47.57, 46.91, 45.74, 43.54, 43.43, 34.09, 33.55, 30.74, 21.85.
HPLC purity: >90%. LCMS (m/e) 527 (M + H). HRMS: calcd for
C27H35ClN6O3 (M + H)+ 527.2537, found 539.2538.
Preparation of (1S,2S,3R,4R)-3-(2,5-Dichloropyrimidin-4-
ylamino)bicyclo[2.2.1]hept-5-ene-2-carboxylic Acid Amide
(22). A mixture of (2-exo,3-exo)3-aminobicyclo[2.2.1]hept-5-ene-2-
carboxamide (250 mg, 1.64 mmol), 2,4,5-trichloropyrimidine (19)
(366 mg, 1.99 mmol), and sodium bicarbonate (280 mg, 3.33 mmol)
in methanol (4 mL) and water (2 mL) was stirred at room tem-
perature for 69 h. The mixture was diluted with water (10 mL) and
extracted with ethyl acetate (2 × 25 mL). The combined organic
extracts were washed successively with water and brine. After drying
over magnesium sulfate, the reaction mixture was filtered and con-
centrated under vacuum. The product was triturated with diethyl
ether, filtered, and washed with additional diethyl ether. The pro-
duct (2-exo,3-exo)-3-(2,5-dichloropyrimidin-4-ylamino)bicyclo
(1S,2S,3R,4R)-3-{5-Chloro-2-[1-methoxy-7-(2-methoxyethy-
lamino)-6,7,8,9-tetrahydro-5H-benzocyclohepten-2-ylamino]-
pyrimidin-4-ylamino}bicyclo[2.2.1]hept-5-ene-2-carboxylic
Acid Amide (23b). The product was isolated as a beige foam (45.27 mg,
4589
dx.doi.org/10.1021/jm201550q | J. Med. Chem. 2012, 55, 4580−4593

Journal of Medicinal Chemistry
Article
1
23% yield). H NMR (CDCl3) δ 8.17 (d, J = 8.3 Hz, 1H), 7.88 (s,
(21.7 mg, 7% yield). 1H NMR (CDCl3) δ 11.74 (s, 1H), 9.13 (d, 1H,
J = 8.08 Hz), 7.71 (s, 1H), 7.62 (d, 1H, J = 8.34), 6.90 (d, 1H, J =
8.34 Hz), 6.30 (m, 1H), 6.10 (m, 1H), 5.91 (s, 1H), 5.53 (s, 1H),
4.00 (t, 1H, J = 7.32 Hz), 3.72 (s, 3H), 3.42−3.58 (m, 8H), 3.08 (s,
1H), 2.90 (m, 1H), 2.88 (s, 1H), 2.83 (s, 3H), 2.75 (m, 2H), 2.35−
2.45 (m, 6H), 2.10 (d, 1H, J = 10 Hz), 1.59 (d, 1H, J = 8.33 Hz), 1.49
(m, 2H). 13C NMR (DMSO-d6) δ 176.42, 156.88, 154.09, 147.86,
139.46, 138.5, 136.11, 134.46, 129.48, 124.28, 119.39, 118.2, 115.5,
104.27, 67.42, 61.46, 52.96, 51.18, 47.73, 47.56, 45.19, 43.58, 42.77,
42.33, 30.86, 27.95, 22.22. LCMS (m/e) 552 (M + H). LCMS (m/e)
539. HRMS: calcd for C29H38ClN7O2 (M + H)+ 552.2854, found
552.2866.
(1S,2S,3R,4R)-3-{5-Chloro-2-[1-methoxy-7-(4-methylpipera-
zin-1-yl)-6,7,8,9-tetrahydro-5H-benzocyclohepten-2-ylamino]-
pyrimidin-4-ylamino}bicyclo[2.2.1]hept-5-ene-2-carboxylic
Acid Amide (27b). The product was was isolated as an off-white solid
(12.6 mg, 4% yield). 1H NMR (CDCl3) δ 10.98 (s, 1H), 9.26 (d, 1H,
J = 7.07 Hz), 7.68 (s, 1H), 7.43 (d, 1H, J = 8.09 Hz), 6.92 (d, 1H, J =
8.08 Hz), 6.58 (s, 1H), 6.20 (m, 1H), 5.91 (m, 1H), 5.52 (s, 1H), 3.80
(m, 1H), 3.70 (s, 3H), 3.39−3.62 (m, *H), 3.03 (s, 1H), 2.93 (m,
2H), 2.85 (s, 3H), 2.77 (m, 2H), 2.38 (m, 5H), 1.53 (d, 1H, J = 9.34
Hz), 1.45 (d, 1H, J = 12.4 Hz), 1.27 (m, 2H). LCMS (m/e) 552 (M +
H). HRMS: calcd for C29H38ClN7O2 (M + H)+ 552.2854, found
552.2866.
1H), 7.39 (s, 1H), 6.94 (m, 1H), 6.86 (d, J = 8.4 Hz, 1H), 6.37 (m,
1H), 6.31 (m, 1H), 5.61 (br s, 1H), 5.36 (br s, 1H), 4.37 (m, 1H),
3.72 (s, 3H), 3.51 (m, 2H), 3.26 (s, 3H), 3.23 (m, 1H), 3.06 (s, 1H),
2.94 (s, 1H), 2.85 − 2.70 (m, 6H), 2.49 (d, J = 8.4 Hz, 2H), 2.24 (d,
J = 8.9 Hz, 1H), 2.09 (m, 2H), 1.63 (m, 2H), 1.26 (m, 4H). 13C NMR
(DMSO-d6) δ 176.00, 157.97, 156.35, 152.83, 146.65, 139.09, 137.73,
136.43, 134.95, 130.69, 123.73, 117.4, 103.69, 71.90, 60.96, 57.85,
52.08, 47.56, 46.91, 45.74, 43.53, 43.41, 34.10, 33.57, 30.73, 28.91,
21.99. LCMS (m/e) 527 (M + H). HRMS: calcd for C27H35ClN6O3
(M + H)+ 527.2537, found 539.2531.
(1S,2S,3R,4R)-3-{5-Chloro-2-[3-methoxy-7-(2-methoxyethy-
lamino)-6,7,8,9-tetrahydro-5H-benzocyclohepten-2-ylamino]-
pyrimidin-4-ylamino}bicyclo[2.2.1]hept-5-ene-2-carboxylic
Acid Amide (24a). The product was isolated as a white trifluoroacetic
1
acid salt (20.5 mg, 20% yield). H NMR (CDCl3) δ 10.63 (broad s,
1H), 9.38 (broad s, 1H), 8.99 (d, 1H, J = 8.08 Hz), 8.45 (broad s, 1H),
7.63 (s, 1H), 7.22 (s, 1H), 6.71 (s, 1H), 6.53 (s, 1H), 6.29 (s, 1H), 5.94
(s, 1H), 5.47 (s, 1H), 3.83 (s, 4H), 3.72 (m, 2H), 3.41 (s, 3H), 3.35 (m,
1H), 3.25 (m, 2H), 3.01 (s, 1H), 2.69−2.85 (m, 5H), 2.54 (d, 1H, J =
7.58 Hz), 2.38 (m, 2H), 2.06 (d, 1H, J = 9.09 Hz), 1.63 (m, 3H), 1.52
(d, 1H, J = 9.09 Hz). 13C NMR (DMSO-d6) δ 174.84, 157.80, 156.39,
152.85, 146.29, 139.19, 136.39, 136.09, 133.79, 126.06, 120.17, 111.60,
103.57, 73.78, 71.79, 59.90, 57.91, 57.85, 55.73, 51.82, 47.62, 46.72,
45.68, 43.77, 34.27, 34.00,31.05. LCMS (m/e) 527 (M + H). HRMS:
calcd for C27H35ClN6O3 (M + H)+ 527.2537, found 527.2554.
(1S,2S,3R,4R)-3-{5-Chloro-2-[3-methoxy-7-(4-methylpipera-
zin-1-yl)-6,7,8,9-tetrahydro-5H-benzocyclohepten-2-ylamino]-
pyrimidin-4-ylamino}bicyclo[2.2.1]hept-5-ene-2-carboxylic
Acid Amide (28a). The product was was isolated as a light brown
powder (19.5 mg, 12% yield). 1H NMR (400 MHz, DMSO-d6) δ 8.12
(s, 1H), 7.89 (s, 1H), 7.78 (m, 1H), 7.38 (s, 1H), 6.96 (s, 1H), 6.35
(m, 1H), 6.14 (m, 1H), 4.02 (m, 2H), 3.82 (s, 3H), 3.50 (m, 4H), 3.30
(m, 3H), 3.14 (m, 2H), 2.78 (s, 3H), 2.71−2.92 (m, 6H), 2.16 (m,
2H), 2.05 (m, 2H), 1.43 (m, 4H). LCMS (m/e) 552 (M + H). HRMS:
calcd for C29H38ClN7O2 (M + H)+ 552.2854, found 552.285.
(1S,2S,3R,4R)-3-{5-Chloro-2-[3-methoxy-7-(4-methylpipera-
zin-1-yl)-6,7,8,9-tetrahydro-5H-benzocyclohepten-2-ylamino]-
pyrimidin-4-ylamino}bicyclo[2.2.1]hept-5-ene-2-carboxylic
Acid Amide (28b). The product was isolated as a light brown powder
(1S,2S,3R,4R)-3-{5-Chloro-2-[3-methoxy-7-(2-methoxyethy-
lamino)-6,7,8,9-tetrahydro-5H-benzocyclohepten-2-ylamino]-
pyrimidin-4-ylamino}bicyclo[2.2.1]hept-5-ene-2-carboxylic
Acid Amide (24b). The product was isolated as a white
1
trifluoroacetic acid salt (18.9 mg, 18% yield). H NMR (CDCl3) δ
10.23 (broad s, 1H), 9.37 (broad s, 1H), 8.82 (s, 1H), 8.54 (s, 1H),
7.67 (s, 1H), 7.52 (s, 1H), 6.80 (s, 1H), 6.68 (s, 1H), 6.35 (s, 1H),
6.12 (s, 1H), 5.54 (s, 1H), 4.18 (m, 1H), 3.84 (s, 3H), 3.71 (m, 2H),
3.40 (m, 4H), 3.23 (m, 2H), 3.05 (s,1H), 2.63−2.83 (m, 4H), 2.36 (m,
2H), 2.17 (d, 2H, J = 8.59 Hz), 1.60 (m, 4H). 13C NMR (DMSO-d6) δ
175.83, 157.83, 156.38, 152.83, 146.41, 139.17, 136.43, 136.16, 133.77,
126.00, 120.42, 111.60, 103.60, 73.78, 71.63, 59.89, 57.85, 55.70,
51.78, 47.63, 46.69, 45.63, 43.81, 43.75, 34.00, 33.75, 30.94. LCMS
(m/e) 527 (M + H). HRMS: calcd for C27H35ClN6O3 (M + H)+
527.2537, found 527.2536.
1
(23.8 mg, 14% yield). H NMR (DMSO-d6) δ 8.12 (s, 1H), 7.89 (s,
1H), 7.78 (m, 1H), 7.38 (s, 1H), 6.96 (s, 1H), 6.35 (m, 1H), 6.14 (m,
1H), 4.02 (m, 2H), 3.82 (s, 3H), 3.50 (m, 4H), 3.30 (m, 3H), 3.14 (m,
2H), 2.78 (s, 3H), 2.71−2.92 (m, 6H), 2.16 (m, 2H), 2.05 (m, 2H),
1.43 (m, 4H). HPLC purity: >86%. LCMS (m/e) 552 (M + H).
HRMS: calcd for C29H38ClN7O2 (M + H)+ 552.2854, found 552.2861.
Anaplastic Lymphoma Kinase (ALK) Assay. Compounds were
tested for their ability to inhibit the kinase activity of baculovirus-
expressed human ALK cytoplasmic domain using a modification of
the assay protocol reported for trkA.30 Phosphorylation of the sub-
strate, phospholipase C-γ (PLC-γ) generated as a fusion protein with
glutathione S-transferase (GST),31 was detected with a europium-
labeled antiphosphotyrosine antibody and measured by time-resolved
fluorescence (TRF). Briefly, each 96-well plate was coated with
100 μL/well of 10 μg/mL substrate solution (recombinant GST-
PLCγ) in Tris-buffered saline (TBS). The ALK assay mixture (total
volume of 100 μL/well) consisting of 20 mM HEPES (pH 7.2), 1 μM
ATP, 5 mM MnCl2, 0.1% BSA, and test compound (diluted in DMSO,
2.5% DMSO final in assay) was then added to the assay plate. Enzyme
(50 ng/mL ALK) was added, and the reaction was allowed to proceed
at 37 °C for 15 min. Detection of the phosphorylated product was
performed by adding 100 μL/well of Eu-N1 labeled PT66 antibody
diluted 1:5000 in 0.25% BSA in TBS containing 0.1% Tween-20
(TBS-T). Incubation at 37 °C then proceeded for 1 h, followed by
addition of 100 μL of enhancement solution. The plate was gently
agitated, and after 30 min, the fluorescence of the resulting solution
was measured using the PerkinElmer EnVision 2102 (or 2104)
multilabel plate reader (PerkinElmer, Waltham, MA). Data analysis
was performed using ActivityBase (IDBS, Guilford, U.K.). IC50 values
were calculated by plotting percent inhibition versus log10 of the
concentration of compound and fitting to the nonlinear regression
sigmoidal dose−response (variable slope) equation in XLFit (IDBS,
Guilford, U.K.).
(1S,2S,3R,4R)-3-[5-Chloro-2-(3-methoxy-7-morpholin-4-yl-
6,7,8,9-tetrahydro-5H-benzocyclohepten-2-ylamino)-
pyrimidin-4-ylamino]bicyclo[2.2.1]hept-5-ene-2-carboxylic
Acid Amide (26a). The product was isolated as an off-white powder
1
(439 mg, 26% yield). H NMR (DMSO-d6) δ 9.60 (m, 1H), 8.12 (s,
1H), 7.90 (s, 1H), 7.79 (m, 1H), 7.39 (s, 1H), 6.98 (s, 1H), 6.36 (m,
1H), 6.16 (m, 1H), 4.00 (m, 3H), 3.83 (s, 3H), 3.30 (m, 5H), 2.74 −
2.90 (m, 6H), 2.39 (m, 3H), 1.94 (d, 1H, J = 4.80 Hz), 1.44 (m, 3H),
1.28 (m, 1H), 1.04 (s, 1H), 0.74 (s, 1H). 13C NMR (DMS0-d6) δ
175.84, 157.82, 156.38, 152.86, 146.39, 139.18, 136.39, 135.99, 133.67,
126.12, 120.30, 111.62, 103.57, 67.29, 66.71, 55.74, 51.80, 48.60, 47.63,
46.72, 43.76, 32.08, 32.03, 29.97, 29.66, 15.06. LCMS (m/e) 539.
HRMS: calcd for C28H35ClN6O3 (M + H)+ 539.2537, found 539.2534.
(1S,2S,3R,4R)-3-[5-Chloro-2-(3-methoxy-7-morpholin-4-yl-
6,7,8,9-tetrahydro-5H-benzocyclohepten-2-ylamino)-
pyrimidin-4-ylamino]bicyclo[2.2.1]hept-5-ene-2-carboxylic
Acid Amide (26b). The product was was isolated as a single
1
diastereomer as an off-white solid (422 mg, 25% yield). H NMR
(DMSO-d6) δ 9.56 (m, 1H), 8.05 (s, 1H), 7.81 (m, 2H), 7.35 (s, 1H),
6.89 (s, 1H), 6.35 (m, 1H), 6.16 (m, 1H), 4.04 (m, 4H), 3.83 (s, 3H),
3.71 (m, 4H), 2.90 (m, 3H), 2.76 (m, 5H), 2.32 (m, 3H), 2.13 (m,
1H), 1.41 (m, 4H). 13C NMR (DMSO-d6) δ 175.83, 157.85, 156.39,
152.82, 146.52, 139.12, 136.46, 136.07, 133.68, 126.08, 120.54, 111.63,
103.64, 67.09, 66.71, 63.37, 55.73, 51.80, 48.66, 47.63, 46.70, 43.81,
31.94, 30.05, 29.87, 29.75. LCMS (m/e) 539. HRMS: calcd for
C28H35ClN6O3 (M + H)+ 539.2537, found 539.2560.
(1S,2S,3R,4R)-3-{5-Chloro-2-[1-methoxy-7-(4-methylpipera-
zin-1-yl)-6,7,8,9-tetrahydro-5H-benzocyclohepten-2-ylamino]-
pyrimidin-4-ylamino}bicyclo[2.2.1]hept-5-ene-2-carboxylic
Acid Amide (27a). The product was was isolated as an off-white solid
4590
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Journal of Medicinal Chemistry
Article
Insulin Receptor (IR) Kinase Assay. The IR kinase assay was
performed using the TRF assay as described above for ALK. The
nucleotide substrate ATP was used at 20 μM, and recombinant human
baculovirus-expressed IR cytoplasmic domain was added to the assay
at a final concentration of 20 ng/mL. In place of Eu-N1 labeled PT66
antibody, Eu-N1 labeled PY100 antibody (diluted 1:10000 in antibody
dilution buffer) was utilized for detection.
Cellular NPM-ALK Phosphorylation Assay. The cellular NPM-
ALK tyrosine phosphorylation assay was carried out as described
previously.19 To calculate the cellular IC90 values in murine plasma, the
Karpas-299 cells were incubated with different concentrations of CEP-
28122 in murine plasma for about 2 h, and the cells were then pro-
cessed for NPM-ALK phosphorylation assay as described.20
were monitored. When the tumor xenograft volumes reached
approximately 600 mm3, the tumor-bearing mice were randomized
into different treatment groups (8−10 mice/group) and were
administered orally with either vehicle (PEG-400) or compound
20b formulated in vehicle at indicated doses and with indicated dosing
frequency, with 100 μL per dosing volume. The length (L) and width
(W) of each tumor was measured with a vernier caliper, and the mouse
body weight was determined every 2−3 days. The tumor volumes
were then calculated with the formula 0.5236LW(L + W)/2. Statistical
analyses of tumor volumes and mouse body weight were carried out
using the Mann−Whitney rank sum test. Plasma and tumor samples
were obtained at 2 h after final dose, and the compound levels in
plasma and tumor lysates were measured by LCMS/MS.
Docking Study. The computational work was done using
Schrodinger/Maestro molecular modeling package (Maestro, version
9.1.107, Schrodinger, LLC, New York, NY). The essential steps in the
current docking experiment included preparation of representative
ALK (PDB code 2XB7) and IRK (PDB code 3EKK) structures from
the Protein Data Bank using Maestro protein preparation workflow.
These structures had similar ligands and were expected to give the best
docking results. The procedure involved (1) grid generation around
the ligand, (2) preparation of ligands (25b and 3) using the LigPrep
module, (3) use of Glide/XP docking to keep the top 10 binding poses
for each compound, and (4) selection of the binding mode using our
knowledge based approach.32
X-ray Crystallography. The X-ray intensity data were measured
on a Bruker CCD area detector system (Cu Kα, 2631 frames, 0.5° in
ω for 30 s): orthorhombic, P212121, a = 5.1493(6) Å, b = 18.930(2) Å,
c = 27.837(3) Å, Z = 4, C28H35ClN6O3. Solved and refined using
SHELXTL (Bruker, version 6.1), the final anisotropic full-matrix least-
squares refinement on F2 (4110 reflections, 345 variables) converged
at R1 = 9.25% for the observed data and wR2 = 24.22% for all data.
The absolute stereochemistry can be assigned at a 99.5% reliability
level with a Flack parameter of 0.08(5) for the stereochemistry as
shown and 0.91(5) for the inverted model. The expected value is 0
for the correct absolute configuration and 1 for the inverted stereo-
chemistry. The Hamilton R-factor ratio is 0.234/0.220 = 1.054,
indicating greater than 99.5% reliability in the determination of the
difference between the two models.
Pharmacokinetics. Adult female SCID mice, male CD-1 mice,
male Sprague−Dawley rat (Charles River, Kingston, NY), male beagle
dogs (Cephalon, Inc., Maisons Alfort, France), and male cynomolgus
monkeys (Covance Laboratories, Alice, TX) were used in the experi-
ments. All animal usage was approved by the Cephalon IACUC. Mice
were dosed via the lateral tail vein for iv administration (3% DMSO,
30% Solutol, 67% phosphate buffered saline) or via oral gavage (100%
PEG400) using a dose volume of 100 μL. Trunk blood was collected
from three mice per time point over a 6 h period. Rats were also dosed
via the lateral tail vein for iv administration (1 mL/kg, vehicle as
described above) or via oral gavage (5 mL/kg in 40% HPβCD). Serial
blood samples were collected from the lateral tail vein into heparinized
collection tubes over a 6 h period. In dogs and primate iv admi-
nistration (0.5 mL/kg in 40% HPβCD) was via the saphenous vein.
Dogs received a 1 mL/kg oral dose via gavage, while monkeys were
dosed via nasogastric tube (2 mL/kg). Serial blood samples were
collected from the femoral vein into heparinized collection tubes over
a 24 h period. All animals were fasted overnight prior to oral
administration. The plasma was separated by centrifugation, and the
sample was prepared for analysis by protein precipitation with ace-
tonitrile. The plasma samples were analyzed for drug and internal
standard via LCMS/MS protocol. The pharmacokinetic parameters
were calculated by a noncompartmental method using WinNonlin
software (Professional, version 4.1, Pharsight Corporation, Palo Alto,
CA, 1997). Parameters were calculated using plasma concentration
time data for individual animals (rat, dog, and monkey, n = 3), and
these data are presented as the mean SEM. Mouse data are pre-
sented as the composite mean.
ASSOCIATED CONTENT
■
S
* Supporting Information
Structure of compound 25a, hERG inhibition data for select
inhibitors, and antitumor efficacy of compound 3. This material
is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: degingrich12@gmail.com.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors gratefully acknowledge the scientific contributions
of James Haley and Renee Roemmele for the preparation of key
intermediates. Special thanks are given to Damaris Rolon-
Steele, Kelli Zeigler, Christine LoSardo, and Rebecca Brown for
their efforts with the pharmacokinetic data generation and
analysis. The authors also thank Edward Hellriegel and Mehran
Yazdanian for their contributions regarding the dog and
monkey pharmacokinetic studies, Dr. Yael Mosse of Children
Hospital of Philadelphia, PA, for kindly providing the
neuroblastoma cell lines (SKNAS, SH-SY5Y, and NB-1643),
and Henry Breslin for scientific discussion contributions.
Pharmacodynamics. Scid mice bearing Karpas-299 sc tumor
xenografts were administered 25b at 30 mg/kg, po. P-NPM-ALK and
total NPM-ALK in tumor samples were detected by immunoblotting,
and relative NPM-ALK phosphorylation was calculated as described
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