Strategies to mitigate the bioactivation of 2-anilino-7-aryl-pyrrolo[2,1-f ][1,2,4]triazines: Identification of orally bioavailable, efficacious ALK inhibitors

Article abstract of DOI:10.1021/jm2010767

Chemical strategies to mitigate cytochrome P450-mediated bioactivation of novel 2,7-disubstituted pyrrolo[2,1-f][1,2,4]triazine ALK inhibitors are described along with synthesis and biological activity. Piperidine-derived analogues showing minimal microsomal reactive metabolite formation were discovered. Potent, selective, and metabolically stable ALK inhibitors from this class were identified, and an orally bioavailable compound (32) with antitumor efficacy in ALK-driven xenografts in mouse models was extensively characterized.

Full text of DOI:10.1021/jm2010767

Article
pubs.acs.org/jmc
Strategies to Mitigate the Bioactivation of 2-Anilino-7-Aryl-
Pyrrolo[2,1-f ][1,2,4]triazines: Identification of Orally Bioavailable,
Efficacious ALK Inhibitors
Eugen F. Mesaros,* Tho V. Thieu, Gregory J. Wells, Craig A. Zificsak, Jason C. Wagner, Henry J. Breslin,
Rabindranath Tripathy, James L. Diebold, Robert J. McHugh, Ashley T. Wohler, Matthew R. Quail,
Weihua Wan, Lihui Lu, Zeqi Huang, Mark S. Albom, Thelma S. Angeles, Kevin J. Wells-Knecht,
Lisa D. Aimone, Mangeng Cheng, Mark A. Ator, Gregory R. Ott, and Bruce D. Dorsey
Worldwide Discovery Research, Cephalon, Inc., 145 Brandywine Parkway, West Chester, Pennsylvania 19380, United States
S
* Supporting Information
ABSTRACT: Chemical strategies to mitigate cytochrome P450-mediated bio-
activation of novel 2,7-disubstituted pyrrolo[2,1-f ][1,2,4]triazine ALK inhibitors are
described along with synthesis and biological activity. Piperidine-derived analogues
showing minimal microsomal reactive metabolite formation were discovered. Potent,
selective, and metabolically stable ALK inhibitors from this class were identified, and
an orally bioavailable compound (32) with antitumor efficacy in ALK-driven
xenografts in mouse models was extensively characterized.
INTRODUCTION
■
A set of chimeric oncoproteins that contain the catalytic
subunit of anaplastic lymphoma kinase (ALK)1 have been
identified in a number of clearly defined cancers,2 including
anaplastic large cell lymphoma3 (NPM-ALK), non-small cell
lung cancer4 (EML4-ALK), and inflammatory myofibroblastic
tumors5 (TPM3-ALK). Aberrant expression of full-length ALK
has been implicated in glioblastoma.6a Furthermore, somatic
and germline mutations of ALK have been shown to have a
causative role in neuroblastoma.6b ALK is a member of the insulin
receptor (IR) tyrosine kinase family, and full-length ALK
expression is restricted to the central and peripheral nervous
systems. The function of ALK has not been completely delineated,
although it is believed to be important in the physiological
development and function of the nervous system.2b On the basis
Figure 1. Recently published ALK inhibitors.
of disease pathologies associated with dysregulated ALK catalytic
activity, a number of ATP-competitive kinase inhibitors have been
macromolecules. Even though generalizations that associate reac-
tive metabolites with toxicities cannot be unequivocally made, bio-
activation is perceived as a potential risk.14 In this context, the
remarkable magnitude of bioactivation in this subset of pyrrolo-
triazines was quite unexpected by comparison to the much lower
levels listed in literature for inhibitors with similar structural
motifs.13,14 Nevertheless, the postulated mechanism for bio-
activation suggested that further structural modifications to the
susceptible fragment may attenuate this phenomenon.14
Herein, we describe a three-pronged strategy to mitigate
bioactivation/glutathione addition, including the installation of
electron withdrawing substituents to the site of oxidation,
shifting the position of the saturated heterocycle on the aniline
reported as potential therapies that act by disrupting the signal
transduction cascade of constitutively active ALK.7 The structures
of clinical level inhibitors PF-2341066 (1)8a−d and CH5424802
(2)8e have been published (Figure 1).
Our group has reported ALK inhibitors from diverse
structural classes including indolocarbazoles,9 tetrahydropyrido-
[2,3-b]pyrazines,10 uniquely substituted 2,4-diaminopyrimi-
dines11 (3, Figure 1), and most recently, 2-anilino-7-aryl-
pyrrolo[2,1-f ][1,2,4]triazines (4, Figure 1).12 Though orally
efficacious in ALK-dependent tumor xenografts in mouse,
preclinical testing of 4 and related analogues in bioactivation/
glutathione (GSH) trapping experiments in liver microsomes
identified extensive NADPH-mediated oxidation of the p-dianiline
moiety to a reactive quinone diimine.13 Reactive metabolites of
certain drugs have been linked to idiosyncratic adverse drug
reactions due to their ability to covalently conjugate endogenous
Received: August 10, 2011
Published: December 5, 2011
© 2011 American Chemical Society
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a
Scheme 1. Synthesis of Piperazine Derivatives 11−15
a
Reagents and conditions (for R1−5 definitions, see Table 1): (a) (i) for X = F (inhibitors 11 and 13), N-Boc-piperazine, K2CO3, DMF, 60 °C, 60−
90%, for X = Br (inhibitor 12), N-Boc-piperazine, Pd2(dba)3, XantPhos, Cs2CO3, 1,4-dioxane, 100 °C, 66%; (ii) TFA, DCM, rt, 80−97%; (b) (S)-2-
methyl-oxirane, MeOH, 42−82%; (c) H2, Pd/C, MeOH, 87−97%; (d) 9a (R1 = N(Me)SO2Me), DIPEA, 1-methoxy-2-propanol, microwave,
200 °C, 8%; (e) (i) 9b, (F3CSO2)2NPh, DIPEA, DMF, 0 °C → rt; (ii) 8 or 10, 85 °C, 30−47%.
Table 1. Enzyme and Cellular ALK Inhibition Data with Percent Glutathione Adduct for Compounds 4 and 11−15
a
IC50, (nM)
b
compd
R1
R2
R3
R4
R5
ALK enzyme
10
56 16
14
91 19
28
187 64
ALK cell
60
% GSH adducts
4
−N(Me)SO2Me
−N(Me)SO2Me
−N(Me)SO2Me
−N(Me)SO2Me
−N(Me)SO2Me
−OMe
H
H
F
A
A
A
A
H
H
H
F
−OMe
−OMe
−OMe
F
2
61
12
13
0.8
11
12
13
14
15
H
H
H
H
3
200
F
c
B
B
−OMe
−OMe
8
3.9
c
2.3
a
IC50 values are reported as the average of at least two separate determinations; standard deviations are indicated where at least three determinations
b
c
were made. Relative to the tested compound. See also ref 13. Due to O-demethylation and iminoquinone formation/trapping.
ring, as well as modifying the heterocycle to remove the
p-nitrogen of the ring. These approaches have led to the identi-
fication of an inhibitor that is basically devoid of the bio-
activation phenomenon and is also potent in enzymatic and
cellular assays, orally bioavailable, and displays antitumor
activity in ALK-driven subcutaneous tumor xenografts in mice.
Inhibitors 22−40 (cf. also Tables 2−4) were prepared
according to the general sequence depicted in Scheme 2. Suzuki
coupling of commercially available boronic ester 16 and
chloride 17 afforded 1,2,3,6-tetrahydropyridine derivative 18.
Nitrogen deprotection with trifluoroacetic acid, followed by
alkylation of the resulting amine, provided the corresponding
nitrophenyl-1,2,3,6-tetrahydropyridine intermediates.
Concurrent catalytic hydrogenation of the olefin and the
nitro group afforded precursors 19. Final union of anilines 19
and pyrrolotriazin-2-ols 20a12,15 under the same reaction
conditions as described earlier generated inhibitors 22, 23,
25, 27, 28, and 31−40. Aniline 21 was obtained by catalytic
hydrogenation of 18 and was reacted with pyrrolotriazin-2-ols
9b (with both R1 = N(Me)SO2Me and R1 = OMe). Deprotec-
tion of the amine afforded inhibitors such as N−H piperidine
24 (Table 2). Amide coupling reactions followed by Boc-amine
deprotection secured amino acid derivatives 29 and 30.
Reduction of Boc-amine 21 with LiAlH4 gave the correspond-
ing N-methyl piperidine intermediate which was converted to
inhibitor 26 (Table 3).
CHEMISTRY
■
We prepared the novel compounds discussed herein according
to synthetic procedures adapted from our previously reported
work.12,15 The synthesis of inhibitors 11−15 is outlined in
Scheme 1 (cf. also Table 1). Commercially available aromatic
halides 5 were reacted with N-Boc-piperazine, followed by
deprotection to generate amines 6, which were next alkylated
with (S)-2-methyl-oxirane. Nitro groups in 7 were reduced to
give anilines 8. Inhibitor 11 was prepared by reacting sulfoxide
9a (R1 = N(Me)SO2Me)12 with the appropriate aniline 8.
Superior yields were obtained for compounds 12−15 via 2-step
one-pot transformations: conversion of pyrrolotriazin-2-ols
9b12 to the corresponding trifluoromethanesulfonates 9c, followed
by in situ aromatic nucleophilic displacements with anilines 8
or 10.
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a
Scheme 2. Synthesis of Piperidine Derivatives 22−40
a
Reagents and conditions (for R and R1,7,8 definitions, see Tables 2−4): (a) Pd(PPh3)4, KHCO3, water/1,4-dioxane, 80 °C, 97%; (b) TFA, DCM, rt,
95%; (c) for inhibitors 22, 23, 25, 27, 28, epoxide, MeOH, 42−80%; for inhibitors 31−40, BrCH2CONHMe or ICH2CONH2, Cs2CO3, MeCN,
75 °C, 71−80%; (d) H2, Pd/C, MeOH, > 90%; (e) (i) 20a, (F3CSO2)2NPh, DIPEA, DMF, 0 °C → rt; (ii) aniline (e.g., 19 or 21), 85 °C, 13−58%;
(f) LiAlH4, THF, reflux, 92%; (g) N-Boc-aminoacid, EDCI, HOBt, DIPEA, DMF 50−60%.
Table 2. Enzyme and Cellular ALK Inhibition Data with Half-Lives (t1/2) in Liver Microsomes for Compounds 22−25
a
IC50 values are reported as the average of at least two separate determinations; standard deviations are indicated where at least three determinations
b
were made. M = mouse; R = rat; Mo = monkey; H = human.
One documented strategy for decreasing metabolic oxidation of
aromatics is to lower their electron density.16 Toward this end,
small electron withdrawing groups were installed on the C2-
aniline ring (Table 1) to arrive at analogues 11−13. Indeed,
monofluorinated derivatives 11 and 12 showed an 80%
reduction in adduct formation compared to 4, although the
levels of GSH conjugation were still above the threshold
considered high risk.13 Fluorination was also accompanied by
some erosion of activity in ALK enzyme and cell-based primary
assays. Enzyme activity of 12 was acceptable (IC50 = 14 nM),
RESULTS AND DISCUSSION
■
The level of covalent glutathione adduct formed by a xenobiotic
that has been incubated with liver microsomes and GSH in the
presence of NADPH was deemed a reasonable stratification
tool to assess the risk associated with reactive metabolite-
related idiosyncratic toxicity.13 We set our goal to synthesize
analogues with minimal/no in vitro GSH conjugation (reported
as % GSH-adduct relative to the tested compound), consistent
with literature reports on bioactivation of marketed drugs.13,14
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Table 3. Enzyme and Cellular ALK Inhibition Data with IR Inhibition, Kinome Selectivity, and iv Half-Life (t1/2), Clearance
(CL), and Oral Bioavailability (F %) in Rat for Compounds 26−37
a
IC50 values are reported as the average of at least two separate determinations; standard deviations are indicated where at least three determinations
b
were made. Kinase selectivity was determined using the Ambit Bioscience KINOMEscan technology, and is expressed as S(90), the fraction of
kinases inhibited >90% when screened at 1 μM across a panel of 256 kinases. See ref 19. For the complete list of PK parameters in rat, see the
c
Supporting Information.
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Table 4. Enzyme and Cellular ALK Inhibition Data with Half-Lives in Liver Microsomes and Oral Bioavailability in Rat for
Compounds 37−40
a
b
IC50 (nM)
ALK enzyme
liver microsome t1/2 (min)
c
compd
R
ALK cell
80
M
R
Mo
H
rat F %
37
38
39
40
H
15
11
6
4
1
2
29
<5
<5
<5
<5
6
<5
<5
18
37
OH
Me
Cl
<5
>40
>40
70
70
31
24
41
4.7 0.7
>40
15
a
IC50 values are reported as the average of at least two separate determinations; standard deviations are indicated where at least three determinations
b
c
were made. M = mouse; R = rat; Mo = monkey; H = human. For the complete list of PK parameters in rat, see the Supporting Information.
whereas the inhibitory activity against phosphorylation of
NPM-ALK in the cell-based assay was modest (IC50 = 200 nM).
Difluorinated compound 13 was sufficiently electron-deficient to
be resistant to metabolic activation, however, replacing the
electron-donating o-methoxy group with a fluorine atom con-
tributed to further weakening of ALK inhibition.
to generate analogue 25. This modification maintained enzyme
and cellular ALK activity and, interestingly, restored liver
microsome stability. However, the improved stability in rat liver
microsomes (t1/2 > 40 min; Table 2) did not correlate with the
iv parameters from the pharmacokinetic studies: short half-life
(t1/2 = 0.4 h) and moderately fast clearance (CL = 38 mL/
min/kg). Similar stability profile was observed for 25 in the rat S9
fraction in vitro assay (t1/2 > 40 min), suggesting that other and
possibly nonhepatic clearance mechanisms might play a role in
vivo. Exposure after oral dosing in rat was also low (e.g., F < 1%;
AUC0−∞ = 11 ng·h/mL, with a 5 mg/kg single oral dose; see also
the Supporting Information).
Alternatively, shifting the piperazine from the para- to meta-
position of the aniline ring (with respect to C2-NH, cf. Table 1)
would negate p-diiminoquinone formation, although it would
open the possibility of CYP-mediated hydroxylation at the (now)
unsubstituted p-position (R3 = H) and the potential to form a
p-iminoquinone. Interestingly, CYP-mediated formation of an
iminoquinone was observed, albeit mass spectrometric analysis of
the GSH-adduct demonstrated that the product originated from
demethylation of the o-methoxy unit (R5) rather than
hydroxylation para to C2-NH. The levels of covalent adducts
were greatly minimized for 14 and 15 (Table 1) relative to the
p-piperazine congeners; however, the potency of these analogues
was outside of the desired range. Combined with previous
structure−activity relationships (SAR),11,12 the above studies
suggested that the saturated para-heterocycle and ortho-methoxy
group substitutions were critical drivers of potency. This led us
to consider an alternative approach to mitigate bioactivation to
the p-diiminoquinones, namely replacing the piperazine with a
piperidine (cf. Table 2). In theory, p-CH-anilines would still be
capable of bioactivation to quinonemethide imine species;
however, the oxidation potential relative to dianilines was
expected to be dramatically diminished.
Derivative 22 validated this strategy, generating only 0.2%
GSH adduct, via demethylation and bioactivation to the
o-iminoquinone species with no formation of imino-quinone-
methide detected. This very low level of covalent adduct was
considered acceptable to pursue this strategy further.
Piperidines 22 and 23 (Table 2) were equally active in ALK
enzyme and cell assays to piperazine 4. Interestingly though,
they displayed suboptimal stability in liver microsomes.17 This
was unanticipated based on our previous experience with 4 and
ent-4,12 compounds with liver microsome t1/2 > 40 min across
all tested species (mouse, rat, monkey, and human).
Furthermore, N−H piperidine 24 also displayed poor micro-
some stability, suggesting that N-dealkylation at the piperidine
ring was not the major metabolic pathway.
Focus turned next to addressing pharmacokinetic properties
in rat by diversifying the piperidine N-substitution on analogues
with an o-methoxyphenyl group at C7 (cf. compounds 26−32
in Table 3). N-Methylpiperidine 26 was active in cells and
showed moderate selectivity against the insulin receptor (IR/
ALK IC50 ratio of 37)18 but at the expense of very low oral
exposure in rat. Kinome selectivity,19 expressed as the fraction
of kinases inhibited >90% when screened at 1 μM across a
panel of 256 kinases, S(90), was acceptable (≤0.10). Related
2-hydoxypropyl derivatives 27 and 28 showed good ALK
activity and acceptable kinase selectivity, including against IR.
Of note, diol 28 provided a 3-fold boost in cellular activity;
GSH adduct levels in microsomes were 0.5% and oral bioavailability
in rat improved to 15%. Amides 29 and 30 showed some erosion
in cellular activity but maintained comparable selectivity and oral
bioavailability. Notably, the iv half-lives in rat were progressively
longer going from compound 26 to 30 (Table 3) and were in good
correlation with the in vitro stability in both rat liver microsomes
and S9 fraction (t1/2 > 40 min for compounds 27−30). With the
exception of 27, the clearance was below the hepatic blood flow
(50 mL/min/kg12). 2-(N-Piperidine)-acetamide derivatives 31 and
32 were equipotent in the ALK cellular assay. Compound 32 was
significantly more stable in liver microsomes (t1/2 > 40 min in all
four species) than 31 (t1/2 < 12 min in monkey and human),
suggesting that the unsubstituted amide nitrogen is metabolically
more favorable. Acetamide 32 had very good ALK activity and
kinase selectivity (including against IR). The inhibitory activity
against NPM-ALK phosphorylation in cells was also favorable
(IC50 = 100 nM). In rat PK experiments, 32 showed a substantial
boost in iv half-life (3.4 h) and low clearance (12 mL/min/kg; only
24% of the hepatic blood flow), in excellent correlation with
the stability in rat liver microsomes (>40 min). The oral bio-
availability (Table 3) was also markedly improved. The propensity
On the basis of the knowledge accumulated with diamino-
pyrimidines11b and pyrrolotriazines,12 we replaced the o-N-
methyl(methylsulfonamide) group on C7-phenyl with o-methoxy
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of 32 for bioactivation was minimal (0.6% GSH adduct, from
O-demethylation). On the basis of the favorable profile, 32
advanced to in vivo studies (vide infra).
3 and 7 were activated in ALK-dependent cell lines while
remaining at basal levels in the ALK-independent ones (see
Supporting Information). Together with the data from the
primary ALK inhibition assays (Table 3), these experiments
suggest that the cytotoxity of 32 is driven by the inhibitory
activity against NPM-ALK phosphorylation.9
The pharmacokinetic parameters determined for 32 in
Sprague−Dawley (S−D) rats and Scid mice are shown in
Table 5. The compound was orally bioavailable and exhibited
Further SAR exploration of C7-aryl moieties with the
optimized acetamide piperidine was undertaken (cf. compounds
33−37 in Table 3). Para substitution on the C7 phenyl brought
to inhibitor 33 a tremendous increase in IR selectivity, paralleling
the corresponding selectivity determinant identified previously in
the diaminopyrimidine class.11b However, this improvement came
at the cost of lower cell activity. Replacement of o-methoxy group
in 32 with a nitrile generated analogue 34, which was 3-fold less
active. Regioisomeric 3-methoxy-pyridines 35 and 36 showed
dramatically different ALK inhibitory activities.
We postulate that the >100-fold lower activity of 35 (relative
to 36) may be due to electron lone pair repulsions between the
pyridyl and the core (N1) nitrogen atoms rendering the
dihedral about the C7-aryl bond much larger than required in
the active conformation.12 Similar to 22−24 (Table 2), sulfo-
namide 37 was active, but metabolically unstable: t1/2 < 5 min
in rat, monkey, and human liver microsomes. Both 36 and 37
showed acceptable levels of IR and kinome selectivity.
At this stage, we decided to reinvestigate N-methyl-
(methylsulfonamide) derivatives, focusing on the metabolic
fate of compound 37 in liver microsomes and anticipating that
we would be able to uncover the metabolic “hot-spot.” LC/MS-
based metabolite analyses were conducted for 37, indicating
oxidation to a compound of molecular weight (M + 16) to be
the major pathway in vitro. The fragmentation pattern in the
MS/MS spectrum narrowed the possible hydroxylation sites to
the pyrrolotriazine core, although the exact position could not
be unequivocally established. Suspecting that pyrrole oxidation
may have occurred, we prepared C5-hydroxy derivative 38
(Table 4), which showed identical LC retention time and MS/
MS spectrum to the (M + 16) metabolite of 37. Thus, blocking
the C5 position with either methyl (39) or chlorine (40) gave
substantially more stable analogues (Table 4). Analogues 39
and 40 displayed not only acceptable activity and selectivity
profiles (e.g., IR/ALK > 88 for both) but also high oral
bioavailability and plasma exposure levels in rat. Similar to 32,
in vitro metabolic stability of 39 and 40 correlated well with the
iv half-lives (t1/2 ≥ 2 h) and clearance (CL ≤ 4 mL/min/kg) in
rat. Less than 1% GSH conjugation was observed for 39 and 40
in liver microsomes, again through a O-demethylation/
oxidation route.
Table 5. Pharmacokinetic Parameters of 32 in S−D Rats and
in Scid Mice
a
PK parameters
S−D rat
Scid mouse
b
iv
dose (mg/kg)
1
1
6.0
7552
1.1
2
t1/2 (h)
3.4 0.2
1106 74
3.5 0.3
12 1.0
AUC0−t (ng·h/mL)
Vd (L/kg)
CL (mL/min/kg)
b
po
dose (mg/kg)
5
10
3473
2.0
Cmax (ng/mL)
tmax (h)
392 25
6.0 0.0
nd
t1/2 (h)
4.4
AUC0−t (ng·h/mL)
F%
1765 147
42534
56
43
3
a
Values are means of data from three animals; standard deviations are
b
indicated. Formulated as solutions in: 3% DMSO, 30% Solutol, 67%
PBS (i.v.); 100% PEG400 (p.o.).
high plasma exposures, long half-lives, and low clearance in
both species. Oral bioavailability in mice was 56%, comparable
to 43% in rat.
The results from the evaluation of 32 in single dose PK/PD
experiments in Scid mice bearing subcutaneous ALK-depend-
ent xenografts are shown in Figure 2. The pharmacodynamic
effect achieved with a 30 mg/kg oral dose in Sup-M2 xenograft
was 80−90% inhibition of NPM-ALK autophosphorylation
over 12 h postdose (Figure 2A). The levels of compound in
plasma reached 6−8 μM for up to 12 h with a ∼7-fold drop by
the 24 h time point (Figure 2B). The levels in tumors were
approximately 2-fold higher than in plasma at 12 h. These data
suggested that twice a day dosing in efficacy studies would be
appropriate.
The robust PD response observed in the single dose PK/PD
experiments correlated well with the inhibition of tumor growth
observed in the efficacy studies. In Sup-M2 xenografts (Figure 3)
oral dosing for 12 days at 30 mg/kg bid effected partial tumor
regression. Increasing the dose to 55 mg/kg bid resulted in
complete tumor regression following 12 days of dosing.
Importantly, no body weight loss or other overt toxicity was
noticed with either of these dosing regimens.
On the basis of the favorable in vitro profiles and pharmaco-
kinetic properties in rat (Tables 3 and 4), 32, 39, and 40 were
selected for in vivo PK/PD evaluation in tumor xenograft
models. Initially, single dose PK/PD studies monitoring ALK
signaling in Sup-M2 tumor xenografts in Scid mice along with
drug exposure in plasma/tumors were used to assess in vivo
activity. Oral dosing of 39 or 40 at 30 mg/kg, po exerted much
less than 75% inhibition of NPM-ALK phosphorylation over
12 h, demonstrating that significantly higher doses would be
required to achieve sustained target inhibition (see Supporting
Information).
Inhibitor 32, on the other hand, displayed strong inhibition
at 30 mg/kg, po (vide infra), which warranted further in vitro
and in vivo profiling. In cytotoxicity assays in cell cultures,
compound 32 demonstrated concentration-related growth
inhibition of ALK-dependent cell lines Sup-M2 and Karpas-
299, whereas it had a minimal effect on the viability of ALK-
independent cell lines K562 and Hut-102 (see Supporting
Information). Upon treatment with 32, pro-apoptotic caspases
CONCLUSIONS
■
In summary, we have described several chemical strategies to
ameliorate bioactivation of lead structures in the 2-anilino-7-
aryl-pyrrolo[2,1-f ][1,2,4]triazine class of ALK inhibitors.
Fluorination of the aniline ring substantially reduced metabolic
bioactivation of piperazine derivatives, although complete
mitigation was accompanied by loss of potency toward the
target enzyme. Moving the piperazine to the meta position of
the aniline ring also reduced bioactivation/conjugation; again,
target potency suffered. Ultimately, replacing the piperazine- for
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Figure 2. Results of PK/PD experiment in Sup-M2 xenograft in Scid mice: (A) inhibition of NPM-ALK autophosphorylation with a single dose of
32: 30 mg/kg po as a solution in PEG400; (B) compound concentration in plasma and tumor.
(0.1% formic acid/water):95% (acetonitrile/0.1% formic acid)), or a
Bruker Esquire 200 ion trap. 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. Automated column chromatography (SiO2) was performed
on CombiFlash Companion instruments (ISCO, Inc.). Melting points
were taken on a Mel-Temp apparatus and are uncorrected.
4-(3-Methoxy-4-nitro-phenyl)-3,6-dihydro-2H-pyridine-1-
carboxylic Acid tert-Butyl Ester (18). A mixture of 4-chloro-2-
methoxy-1-nitro-benzene (4.9 g, 25.9 mmol), 4-(4,4,5,5-tetramethyl-
[1,3,2]dioxaborolan-2-yl)-3,6-dihydro-2H-pyridine-1-carboxylic acid
tert-butyl ester (8.0 g, 25.9 mmol), tetrakis(triphenylphosphine)-
palladium(0) (1.5 g, 1.3 mmol), and a 2 M solution of potassium
bicarbonate in water (32.5 mL, 65.0 mmol) in 1,4-dioxane (75 mL)
was heated at 80 °C overnight. The reaction mixture was cooled to room
temperature and partitioned between dichloromethane (200 mL) and
water (200 mL). The layers were separated and the aqueous phase was
extracted with dichloromethane (100 mL). The combined organic
Figure 3. Antitumor efficacy of 32, dosed po as a solution in PEG400.
extracts were dried (MgSO4) and then filtered, and the solvent was
evaporated under reduced pressure. The product (8.5 g, 97% yield) was
piperidine-containing pyrrolo[2,1-f][1,2,4]triazines proved suc-
cessful. Interestingly, this replacement shifted metabolism to
the core pyrrolotriazine for a subset of inhibitors containing a
N-methyl(methylsulfonamide) moiety on the C7-aryl ring.
We identified a product of oxidation at C5 and were able to
block this position with a chlorine or methyl group to sub-
stantially improve in vitro stability while maintaining potency;
these analogues displayed acceptable oral bioavailability. A C7
o-methoxyphenyl analogue (32) had excellent ALK activity,
selectivity, and metabolic stability and displayed <1% in vitro
bioactivation/conjugation. Furthermore, 32 was orally bioavail-
able in two species and demonstrated dose-related antitumor
efficacy in ALK-driven tumor xenografts in mouse.
isolated by column chromatography (Silicagel, EtOAc/hexanes 25−
50%). 1H NMR (CDCl3) δ 7.87 (d, J = 8.6 Hz, 1H), 7.01 (m, 2H), 6.17
(br s, 1H), 4.12 (m, 2H), 3.98 (s, 3H), 3.66 (m, 2H), 2.53 (m, 2H), 1.50
(s, 9H).
2-[4-(4-Amino-3-methoxy-phenyl)-piperidin-1-yl]-acet-
amide (19; R7 = CH2C(O)NH2). 4-(3-Methoxy-4-nitro-phenyl)-3,6-
dihydro-2H-pyridine-1-carboxylic acid tert-butyl ester (3.0 g, 9.0 mmol)
was treated with trifluoroacetic acid (3.5 mL, 44.9 mmol) in methylene
chloride (20 mL) at room temperature for 2 h. Minimum amount of
saturated sodium carbonate was added to pH 8, and the mixture was
extracted with dichloromethane (3 × 100 mL). The combined extracts
were dried (MgSO4) and then filtered, and the solvent was evaporated
under reduced pressure to afford 4-(3-methoxy-4-nitro-phenyl)-1,2,3,6-
tetrahydro-pyridine (2.0 g, 95% yield), which was used in the next step
1
without further purification. H NMR (CDCl3) δ 7.96 (d, J = 8.4 Hz,
1H), 7.02 (m, 2H), 6.28 (m,1H), 3.98 (s, 3H), 3.598 (m, 2H), 3.13 (m,
2H), 2.46 (m, 2H), 1.76 (br s, 1H).
EXPERIMENTAL SECTION
■
Into a 1-neck round-bottom flask was added 4-(3-methoxy-4-nitro-
phenyl)-1,2,3,6-tetrahydro-pyridine (7.5 g, 32.0 mmol), iodoacetamide
(5.9 g, 32.0 mmol), and cesium carbonate (15.7 g, 48.0 mmol) in
acetonitrile (300 mL). The reaction was stirred overnight at reflux, and
then cooled, when the product precipitated. Water was added and the
product was collected by filtration, washed with water, and dried under
high vacuum. Trituration from dichloromethane afforded the final
product, 2-[4-(3-methoxy-4-nitro-phenyl)-3,6-dihydro-2H-pyridin-
General Methods. All commercial reagents and solvents were
used as received unless otherwise indicated. H and 13C NMR spectra
1
were recorded on a Bruker Avance spectrometer at 400 and 100 MHz,
respectively, in the solvent indicated, with tetramethylsilane as an
internal standard. To asses the purity of the final compounds,
analytical HPLC was run on a Zorbax RX-C8, 5 mm × 150 mm
column, eluting with a 10−100% gradient mixture of acetonitrile and
water containing 0.1% trifluoroacetic acid, over 5 min; purity, deter-
mined from the UV peak area, was ≥95%, unless indicated otherwise.
UV detection was set at 254 and 290 nm wavelengths. LC/MS data
were recorded on either of the following instruments: a Waters Aquity
Ultra Performance LC coupled with Micromass LC-ZQ 2000
quadrupole mass spectrometer (2.1 mm × 50 mm Waters Aquity
UPLC BEH C18 1.7 μm column, target column temperature 45 °C,
run time 2 min, flow rate 0.600 mL/min, and solvent mixture of 5%
1
1-yl]-acetamide (7.4 g, 80% yield). H NMR (CDCl3) δ 7.87 (d, J =
8.3 Hz, 1H), 7.03 (m, 3H), 6.21 (m, 1H), 5.46 (br s, 1H), 3.99 (s,
3H), 3.31 (m, 2H), 3.17 (s, 2H), 2.85 (m, 2H), 2.60 (m, 2H).
To a solution of 2-[4-(3-methoxy-4-nitro-phenyl)-3,6-dihydro-2H-
pyridin-1-yl]-acetamide (7.4 g, 25.5 mmol) in methanol (350 mL) was
added 10% Pd/C (2.71 g). The mixture was shaken in a Parr apparatus
under an atmosphere of hydrogen (50 PSI) for 2.5 h. Filtration
121
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Journal of Medicinal Chemistry
Article
through Celite and evaporation of the solvent provided crude 19 (6.4
g, 95% yield), which was used without further purification. H NMR
(CDCl3) δ 7.14 (br s, 1H), 6.65 (m, 3H), 5.39 (br s, 1H), 3.85
(s, 3H), 3.70 (br s, 2H), 3.03 (s, 2H), 2.98 (m, 2H), 2.42 (m, 1H),
2.28 (m, 2H), 1.85 (m, 2H), 1.73 (m, 2H).
1H), 6.83 (d, J = 3.7 Hz, 1H), 7.76 (d, J = 8.6 Hz, 1H), 6.42 (d, J = 8.6
Hz, 1H), 3.85 (s, 3H), 3.82 (s, 3H), 2.85 (br s, 4H), 2.41 (br s, 4H),
2.34 (s, 3H). LC/MS (ESI+) m/z 445.1 (M + H)+.
1
N-[2-(2-{4-[1-(2-Hydroxy-ethyl)-piperidin-4-yl]-2-methoxy-
henylamino}-pyrrolo[2,1-f][1,2,4]triazin-7-yl)-phenyl]-N-meth-
yl-methanesulfonamide (22). 1H NMR (CDCl3) δ 8.72 (s, 1H),
8.07 (d, J = 8.4 Hz, 1H), 7.95 (m, 1H), 7.53 (m, 3H), 7.45 (s, 1H),
7.03 (d, J = 4.8 Hz, 1H), 6.86 (d, J = 4.8 Hz, 1H), 6.73 (d, J = 1.6 Hz,
1H), 6.64 (dd, J = 8.4, 1.6 Hz, 1H), 3.90 (s, 3H), 3.65 (m, 2H), 3.12
(s, 3H), 3.05 (m, 2H), 2.67 (s, 3H), 2.59 (m, 2H), 2.47 (m, 1H), 2.19
(m, 3H), 1.78 (m, 4H). LC/MS (ESI+) m/z 551.1 (M + H)+; mp 99−
111 °C.
N-[2-(2-{4-[1-((R)-2-Hydroxy-propyl)-piperidin-4-yl]-2-
methoxy-phenylamino}-pyrrolo[2,1-f][1,2,4]triazin-7-yl)-phenyl]-
N-methyl-methanesulfonamide (23). 1H NMR (CDCl3) δ 8.72
(s, 1H), 8.07 (d, J = 8.4 Hz, 1H), 7.95 (d, J = 3.9 Hz, 1H), 7.53 (br s,
3H), 7.45 (s, 1H), 7.02 (d, J = 4.0 Hz, 1H), 6.86 (d, J = 3.9 Hz, 1H),
6.72 (s, 1H), 6.64 (d, J = 8.3 Hz, 1H), 3.90 (s, 3H), 3.89 (m, 1H), 3.15
(m, 1H), 3.12 (s, 3H), 2.95 (m, 1H), 2.67 (s, 3H), 2.37 (m, 4H), 2.05
(m, 1H), 1.79 (m, 4H), 1.26 (br s, 1H), 1.16 (d, J = 7.0 Hz, 3H). LC/
MS (ESI+) m/z 565.2 (M + H)+; mp 91−103 °C; HPLC purity 90%.
N-{2-[2-(2-Methoxy-4-piperidin-4-yl-phenylamino)-pyrrolo-
[2,1-f][1,2,4]triazin-7-yl]-phenyl}-N-methyl-methanesulfona-
mide (24). 1H NMR (CDCl3) δ 8.72 (s, 1H), 8.08 (d, J = 8.4 Hz,
1H), 7.95 (m, 1H), 7.53 (m, 3H), 7.45 (s, 1H), 7.03 (d, J = 4.7 Hz,
1H), 6.86 (d, J = 4.7 Hz, 1H), 6.74 (s, 1H), 6.65 (d, J = 8.0 Hz, 1H),
3.89 (s, 3H), 3.23 (m, 2H), 3.12 (s, 3H), 2.76 (m, 2H), 2.67 (s, 3H),
2.57 (m, 1H), 2.30 (br s, 1H), 1.84 (m, 2H), 1.70 (m, 2H). LC/MS
(ESI+) m/z 507.0 (M + H)+; HPLC purity 92%.
General Procedure for the Synthesis of Inhibitors 12−15
and 22−40: 2-(4-{3-Methoxy-4-[7-(2-methoxy-phenyl)-pyrrolo-
[2,1-f ][1,2,4]triazin-2-ylamino]-phenyl}-piperidin-1-yl)-acet-
amide (32). 7-(2-Methoxy-phenyl)-pyrrolo[2,1-f ][1,2,4]triazin-2-ol
(0.50 g, 2.1 mmol) was dissolved in N,N-dimethylformamide (40 mL)
at 0 °C, and N,N-diisopropylethylamine (1.1 mL, 6.2 mmol) was added.
The reaction stirred for 30 min, then N-phenylbis(trifluoromethane-
sulphonimide) (0.81 g, 2.3 mmol) was added and the reaction was
allowed to warm to room temperature. After complete conversion to the
corresponding trifluoromethanesulfonate intermediate (approximately
1 h; monitored by HPLC), 2-[4-(4-amino-3-methoxy-phenyl)-piperidin-
1-yl]-acetamide (0.68 g, 2.6 mmol) was added and the reaction was
heated at 95 °C overnight. N,N-Dimethylformamide was evaporated
under reduced pressure, and the residue was partitioned between water
(100 mL) and ethyl acetate (200 mL). The aqueous layer was further
extracted with ethyl acetate (200 mL). The combined organic extracts
were dried (MgSO4), filtered, and the solvent evaporated under reduced
pressure. The pure product (485 mg, 48% yield) was isolated by column
chromatography (Silicagel, 0−10% MeOH/DCM), followed by Et2O
1
trituration to afford 32. H NMR (CDCl3) δ 8.69 (s, 1H), 8.30 (d, J =
8.7 Hz, 1H), 8.00 (dd, J = 7.6, 1.5 Hz, 1H), 7.44 (m, 2H), 7.13 (m, 2H),
7.07 (d, J = 8.7 Hz, 1H), 7.03 (d, J = 4.6 Hz, 1H), 6.84 (d, J = 4.6 Hz,
1H), 6.72 (m, 2H), 5.42 (br s, 1H), 3.91 (s, 3H), 3.85 (s, 3H), 3.04 (s,
2H), 3.00 (m, 2H), 2.48 (m, 1H), 2.29 (m, 2H), 1.85 (m, 2H), 1.77 (m,
2H). 13C NMR (CDCl3) δ 173.7, 157.2, 152.5, 151.2, 147.5, 138.9,
131.1, 129.6, 127.9, 127.7, 121.1, 120.1, 119.4, 118.7, 117.8, 115.3, 111.0,
108.3, 105.1, 62.0, 55.7, 55.6, 54.9, 41.7, 33.9. LC/MS (ESI+) m/z 487.3
(M + H)+; mp: 88−95 °C; high resolution mass spectrum (ESI) m/z
487.2457 [(M + H)+ calcd for C27H30N6O3: 487.2458].
N-[2-(2-{3-Fluoro-4-[4-((S)-2-hydroxy-propyl)-piperazin-1-
yl]-2-methoxy-phenylamino}-pyrrolo[2,1-f][1,2,4]triazin-7-yl)-
phenyl]-N-methylmethanesulfonamide, Trifluoroacetic Acid
Salt (11). 1H NMR (DMSO-d6) δ 9.48 (br s, 1H), 8.97 (s, 1H),
8.14 (s, 1H), 7.96 (m, 1H), 7.62 (m, 2H), 7.53 (m, 2H), 6.99 (dd, J =
15.0; 5.0 Hz, 2H), 6.70 (t, J = 8.0 Hz, 1H), 4.12 (m, 1H), 3.83 (m,
3H), 3.76 (m, 2H), 3.58 (m, 2H), 3.20 (m, 4H), 3.07 (s, 3H), 3.09 (s,
2H), 2.88 (s, 3H), 1.14 (d, J = 6.0 Hz, 3H). LC/MS (ESI+) m/z 584.0
(M + H)+; HPLC purity 92%.
N-[2-(2-{5-Fluoro-4-[4-((S)-2-hydroxy-propyl)-piperazin-1-
yl]-2-methoxy-phenylamino}-pyrrolo[2,1-f][1,2,4]triazin-7-yl)-
phenyl]-N-methylmethanesulfonamide (12). 1H NMR (DMSO-
d6) δ 8.96 (s, 1H), 7.98 (d, J = 8.0 Hz, 1H), 7.84 (d, J = 15.0 Hz, 1H),
7.66 (m, 2H), 7.54 (m, 2H), 7.00 (dd, J = 17.0; 5.0 Hz, 2H), 6.68 (d,
J = 8.0 Hz, 1H), 4.30 (m, 1H), 3.85 (s, 3H), 3.79 (m, 1H), 3.07 (s, 3H),
2.99 (m, 4H), 2.90 (s, 3H), 2.57 (m, 4H), 2.27 (m, 2H), 1.07 (d, J =
6.0 Hz, 3H); LC/MS (ESI+) m/z 584 (M + H); mp 116−120 °C.
N-[2-(2-{2,5-Difluoro-4-[4-((S)-2-hydroxy-propyl)-piperazin-
1-yl]-phenylamino}-pyrrolo[2,1-f][1,2,4]triazin-7-yl)-phenyl]-N-
methyl-methanesulfonamide, Trifluoroacetic Acid Salt
(13). 1H NMR (DMSO-d6) δ 9.54 (br, 1H), 8.99 (s, 1H), 8.88 (s,
1H), 7.96 (d, J = 8.0 Hz, 1H), 7.76 (m, 1H), 7.64 (d, J = 8.0 Hz, 1H),
7.49 (m, 2H), 7.06 (m, 1H), 7.00 (dd, J = 15.0; 5.0 Hz, 2H), 4.12 (m,
1H), 3.58 (d, J = 12.0 Hz, 2H), 3.44 (t, J = 12.0 Hz, 2H), 3.22 (m, 4H),
3.09 (s, 6H), 2.89 (s, 3H), 1.15 (d, J = 6.0 Hz, 3H); LC/MS (ESI+)
m/z 572 (M + H).
N-(2-{2-[2-Methoxy-5-(4-methyl-piperazin-1-yl)-phenylami-
no]-pyrrolo[2,1-f][1,2,4]triazin-7-yl}-phenyl)-N-methyl-metha-
nesulfonamide (14). 1H NMR (CDCl3) δ 8.75 (s, 1H), 7.28 (s,
1H), 7.71 (m, 1H), 7.50 (m, 4H), 6.88 (br s, 2H), 6.78 (d, J = 8.6 Hz,
1H), 6.45 (d, J = 8.8 Hz, 1H), 3.87 (s, 3H), 3.16 (s, 3H), 2.75 (br s,
4H), 2.52 (s, 3H), 2.40 (br s, 4H), 2.35 (s, 3H). LC/MS (ESI+) m/z
522.0 (M + H)+.
(R)-1-(4-{3-Methoxy-4-[7-(2-methoxy-phenyl)-pyrrolo[2,1-f]-
][1,2,4]triazin-2-ylamino]-phenyl}-piperidin-1-yl)-propan-2-ol
(25). 1H NMR (CDCl3) δ 8.69 (s, 1H), 8.29 (d, J = 8.1 Hz, 1H), 7.99
(d, J = 7.7 Hz, 1H), 7.43 (m, 2H), 7.14 (m, 1H), 7.07 (d, J = 8.4 Hz,
1H), 7.02 (d, J = 4.4 Hz, 1H), 6.83 (d, J = 4.4 Hz, 1H), 6.73 (s, 1H),
6.71 (d, J = 8.1 Hz, 1H), 3.90 (s, 3H), 3.89 (m, 1H), 3.84 (s, 3H), 3.15
(m, 1H), 2.94 (m, 1H), 2.35 (m, 4H), 2.06 (m, 1H), 1.79 (m, 4H), 1.30
(br s, 1H), 1.16 (d, J = 5.8 Hz, 3H). LC/MS (ESI+) m/z 488.2 (M +
H)+; mp 67−74 °C. HPLC purity 94%.
[2-Methoxy-4-(1-methyl-piperidin-4-yl)-phenyl]-[7-(2-methoxy-
phenyl)-pyrrolo[2,1-f][1,2,4]triazin-2-yl]-amine (26). 1H NMR
(CDCl3) δ 8.69 (s, 1H), 8.29 (d, J = 8.8 Hz, 1H), 7.98 (dd, J = 8.0; 1.7
Hz, 1H), 7.42 (m, 2H), 7.12 (m, 1H), 7.07 (d, J = 8.4 Hz, 1H), 7.01
(d, J = 5.6 Hz, 1H), 6.83 (d, J = 5.6 Hz, 1H), 6.74 (s, 1H), 6.73
(m, 1H), 3.88 (s, 3H), 3.84 (s, 3H), 3.07 (m, 2H), 2.45 (m, 1H), 2.41
(s, 3H), 2.15 (m, 2H), 1.87 (m, 4H). LC/MS (ESI+) m/z 444.22
(M + H)+; mp 71−84 °C.
(
)-1-Fluoro-3-(4-{3-methoxy-4-[7-(2-methoxy-phenyl)-
pyrrolo[2,1-f][1,2,4]triazin-2-ylamino]-phenyl}-piperidin-1-yl)-
propan-2-ol (27). 1H NMR (CDCl3) δ 8.69 (s, 1H), 8.29 (d, J = 8.8
Hz, 1H), 8.00 (dd, J = 7.6 Hz; 1.5 Hz, 1H), 7.44 (m, 2H), 7.14 (m, 1H),
7.08 (d, J = 8.3 Hz, 1H), 7.02 (d, J = 4.6 Hz, 1H), 6.83 (d, J = 4.6 Hz,
1H), 6.73 (s, 1H), 6.72 (d, J = 7.6 Hz, 1H), 4.44 (m, 2H), 3.95 (m, 1H),
3.91 (s, 3H), 3.85 (s, 3H), 3.10 (m, 1H), 2.95 (m, 1H), 2.47 (m, 4H),
2.10 (m, 1H), 1.80 (m, 4H), 1.59 (br s, 1 OH, and water peak). LC/MS
(ESI+) m/z 506.0 (M + H)+; mp 73−79 °C; HPLC purity 94%.
(S)-3-(4-{3-Methoxy-4-[7-(2-methoxy-phenyl)-pyrrolo[2,1-f]-
[1,2,4]triazin-2-ylamino]-phenyl}-piperidin-1-yl)-propane-1,2-
diol (28). 1H NMR (CDCl3) δ 8.69 (s, 1H), 8.29 (d, J = 8.0 Hz, 1H),
8.99 (dd, J = 8.0, 1.6 Hz, 1H), 7.44 (m, 2H), 7.13 (m, 1H), 7.07 (d, J =
8.0 Hz, 1H), 7.02 (d, J = 4.7 Hz, 1H), 6.83 (d, J = 4.7 Hz, 1H), 6.72 (s,
1H), 6.71 (d, J = 8.0 Hz, 1H), 3.90 (s, 3H), 3.86 (m, 1H), 3.85 (s,
3H), 3.77 (m, 1H), 3.55 (m, 1H), 3.22 (d, J = 10.6 Hz, 1H), 3.08 (d,
J = 10.6 Hz, 1H), 2.77 (br s, 2H), 2.69 (m, 1H), 2.48 (m, 3H), 2.20
(m, 1H), 1.85 (m, 4H). LC/MS (ESI+) m/z 504.19 (M + H)+; mp
66−80 °C.
2-Amino-1-(4-{3-methoxy-4-[7-(2-methoxy-phenyl)-pyrrolo-
[2,1-f][1,2,4]triazin-2-ylamino]-phenyl}-piperidin-1-yl)-2-meth-
yl-propan-1-one (29). 1H NMR (CDCl3) δ 8.69 (s, 1H), 8.30 (d,
J = 8.8 Hz, 1H), 7.99 (dd, J = 7.6; 1.6 Hz, 1H), 7.44 (m, 2H), 7.13 (m,
1H), 7.08 (d, J = 8.4 Hz, 1H), 7.02 (d, J = 4.7 Hz, 1H), 6.83 (d, J = 4.7
Hz, 1H), 6.70 (m, 2H), 4.84 (m, 2H), 3.89 (s, 3H), 3.84 (s, 3H), 2.88
2-Methoxy-5-(4-methyl-piperazin-1-yl)-phenyl]-[7-(2-me-
thoxy-phenyl)-pyrrolo[2,1-f][1,2,4]triazin-2-yl]-amine (15). 1H
NMR (CDCl3) δ 8.69 (s, 1H), 7.95 (br s, 1H), 7.84 (d, J = 7.4 Hz,
1H), 7.52 (s, 1H), 7.40 (m, 1H), 7.08 (m, 2H), 6.93 (d, J = 3.7 Hz,
122
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(m, 2H), 2.72 (m, 1H), 1.90 (m, 2H), 1.63 (m, 4H), 1.45 (s, 6H). LC/
(m, 1H), 2.06−2.23 (m, 2H), 1.60−1.87 (m, 4H). LC/MS (ESI+)
m/z 580.15 (M + H)+.
MS (ESI+) m/z 515.0 (M + H)+; mp 81−89 °C.
2-[4-(4-{7-[2-(Methanesulfonyl-methyl-amino)-phenyl]-5-
methylpyrrolo[2,1-f][1,2,4]triazin-2-ylamino}-3-methoxy-phe-
nyl)-piperidin-1-yl]-acetamide (39). 1H NMR (DMSO-d6) δ 8.95
(s, 1H), 7.93−7.98 (m, 1H), 7.88 (d, J = 8.2 Hz, 1H), 7.61−7.67 (m,
1H), 7.57 (s, 1H), 7.51−7.56 (m, 2H), 7.21 (s, 1H), 7.14 (s, 1H), 6.89
(s, 1H), 6.81 (s, 1H), 6.68 (d J = 8.5 Hz, 1H), 3.84 (s, 3H), 3.05 (s,
3H), 2.85−2.95 (m, 7H), 2.35−2.45 (m, 4H), 2.08−2.28 (m, 2H),
1.64−1.81 (m, 4H). LC/MS (ESI+) m/z 578.16 (M + H)+.
2-[4-(4-{5-Chloro-7-[2-(methanesulfonyl-methyl-amino)-
phenyl]-pyrrolo[2,1-f][1,2,4]triazin-2-ylamino}-3-methoxy-
phenyl)-piperidin-1-yl]-acetamide (40). 1H NMR (DMSO-d6) δ
9.48 (br s, 1H), 8.98 (s, 1H), 7.95 (m, 3H), 7.79 (d, J = 8.1 Hz, 1H),
7.72 (s, 1H), 7.67 (d, J = 7.2 Hz, 1H), 7.55 (m, 2H), 7.01 (s, 1H), 6.88
(s, 1H), 6.68 (d, J = 8.2 Hz, 1H), 3.92 (s, 2H), 3.84 (s, 3H), 3.55 (m,
2H), 3.15 (m, 2H), 3.09 (s, 3H), 2.91 (s, 3H), 2.76 (m, 1H), 1.99 (m,
4H). LC/MS (ESI+) m/z 598.0 (M + H)+; HPLC purity 90%.
In Vitro Assays. ALK enzyme inhibition assay, cell-based NPM-
ALK tyrosine phosphorylation assay, cytotoxicity assay, and caspase
3/7 activation assay were performed as described in ref 9. Metabolic
stability assays in liver microsomes were performed as described in ref
17; metabolic stability assays in rat S9 fraction were performed in a
similar manner. In vitro microsome incubations/GSH-trapping
experiments were performed as described in ref 13.
(
)-Azetidin-2-yl-(4-{3-methoxy-4-[7-(2-methoxy-phenyl)-
pyrrolo[2,1-f][1,2,4]triazin-2-ylamino]-phenyl}-piperidin-1-yl)-
methanone (30). 1H NMR (CDCl3) δ 8.69 (s, 1H), 8.30 (m, 1H),
7.98 (m, 1H), 7.44 (m, 2H), 7.13 (m, 1H), 7.07 (d, J = 8.3 Hz, 1H),
7.03 (m, 1H), 6.84 (d, J = 4.6 Hz, 1H), 6.67 (m, 2H), 4.80 (m, 1H),
4.39 (m, 1H), 3.90 (s, 3H), 3.89* (s, 3H, *amide rotamer), 3.84 (s,
3H), 3.68 (m, 1H), 3.56 (m, 1H), 3.45 (m, 1H), 3.07 (m, 1H), 2.88
(m, 1H), 2.70 (m, 2H), 2.32 (m, 2H), 1.91 (m, 2H), 1.63 (m, 2H).
LC/MS (ESI+) m/z 513.0 (M + H)+; mp 103−112 °C.
2-(4-{3-Methoxy-4-[7-(2-methoxy-phenyl)-pyrrolo[2,1-f]-
][1,2,4]triazin-2-ylamino]-phenyl}-piperidin-1-yl)-N-methyl-
acetamide (31). 1H NMR (CDCl3) δ 8.69 (s, 1H), 8.30 (d, J = 8.7
Hz, 1H), 8.00 (d, J = 6.3 Hz, 1H), 7.45 (m, 2H), 7.24 (m, 1H), 7.14
(m, 1H), 7.05 (d, J = 13.0 Hz, 1H), 7.03 (m, 1H), 6.84 (d, J = 4.7 Hz,
1H), 6.73 (m, 2H), 3.91 (s, 3H), 3. 85 (s, 3H), 3.04 (s, 2H), 2.96 (m,
2H), 2.87 (d, J = 5.0 Hz, 3H), 2.47 (m, 1H), 2.29 (m, 2H), 1.73 (m,
4H). LC/MS (ESI+) m/z 501.21 (M + H)+; HPLC purity 91%.
2-(4-{4-[7-(2,4-Dimethoxy-phenyl)-pyrrolo[2,1-f][1,2,4]-
][1,2,4]triazin-2-ylamino]-3-methoxy-phenyl}-piperidin-1-yl)-
acetamide (33). 1H NMR (MeOH-d4) δ 8.78 (br s, 1H), 8.22 (d, J =
4.83 Hz, 1H), 7.77 (d, J = 4.87 Hz, 1H), 6.98 (s, 2H), 6.89 (s, 1H),
6.64−6.78 (m, 3H), 3.97 (s, 2H), 3.91 (s, 3H), 3.87 (s, 3H), 3.78
(s, 3H), 3.66−3.74 (m, 2H), 3.15−3.28 (m, 2H), 2.87 (m, 1H), 2.03−
2.15 (m, 4H). LC/MS (ESI+) m/z 517.0 (M + H)+; mp 119−121 °C;
HPLC purity 93%.
In Vivo Assays. Rat and mouse pharmacokinetic assays, in vivo
biochemical efficacy (PK/PD) assays, and antitumor efficacy studies
were performed as described in refs 11a and 12.
2-(4-{4-[7-(2-Cyano-phenyl)-pyrrolo[2,1-f][1,2,4]triazin-2-yla-
mino]-3-methoxy-phenyl}-piperidin-1-yl)-acetamide, Tri-
fluoroacetic Acid Salt (34). 1H NMR (DMSO-d6) δ 9.51 (b s,
1H), 9.08 (s, 1H), 8.09 (d, J = 7.80 Hz, 1H), 8.05 (d, J = 7.80 Hz, 1H),
7.98 (s, 1H), 7.94 (d, J = 8.09 Hz, 1H), 7.88 (m, 2H), 7.72 (s, 1H),
7.65 (m, 1H), 7.17 (m, 1H), 7.04 (m, 1H), 6.90 (s, 1H), 6.71 (d, J =
8.20 Hz, 1H), 3.93 (s, 2H), 3.88 (s, 3H), 3.55 (d, J = 11.64 Hz, 2H),
3.15 (m, 2H), 2.67 (m, 1H), 2.01 (m, 4H). LC/MS (ESI+) m/z
482.17 (M + H)+.
ASSOCIATED CONTENT
■
S
* Supporting Information
Additional biological data for compounds 25−30, 32, 39, and
40 and 1H NMR and 13C NMR spectra for compound 32. This
material is available free of charge via the Internet at http://
pubs.acs.org.
2-(4-{3-Methoxy-4-[7-(3-methoxy-pyridin-2-yl)-pyrrolo[2,1-
f][1,2,4]triazin-2-ylamino]-phenyl}-piperidin-1-yl)-acetamide,
trifluoroacetic acid salt (35). 1H NMR (DMSO-d6) δ 9.48 (br s,
1H), 9.02 (s, 1H), 8.37 (d, J = 4.4 Hz, 1H), 8.14 (d, J = 8.2 Hz, 1H),
7.97 (s, 1H), 7.71 (m, 3H), 7.54 (dd, J = 8.4; 4.6 Hz, 1H), 7.07 (d, J = 4.6
Hz, 1H), 6.98 (d, J = 4.6 Hz, 1H), 6.88 (s, 1H), 6.71 (d, J = 8.4 Hz, 1H),
3.93 (s, 2H), 3.88 (s, 3H), 3.83 (s, 3H), 3.55 (m, 2H), 3.15 (m, 2H), 2.75
(m, 1H), 1.99 (m, 4H). LC/MS (ESI+) m/z 488.0 (M + H)+; HPLC
purity 90%.
2-(4-{3-Methoxy-4-[7-(3-methoxy-pyridin-4-yl)-pyrrolo[2,1-
f][1,2,4]triazin-2-ylamino]-phenyl}-piperidin-1-yl)-acetamide
(36). 1H NMR (CDCl3) δ 8.75 (s, 1H), 8.48 (s, 1H), 8.43 (d, J = 4.0
Hz, 1H), 8.27 (d, J = 8.2 Hz, 1H), 8.21 (d, J = 4.0 Hz, 1H), 7.49 (s,
1H), 7.20 (d, J = 4.0 Hz, 1H), 7.19 (br s, 1H), 6.84 (d, J = 3.8 Hz,
1H), 6.79 (d, J = 8.3 Hz, 1H), 6.76 (s, 1H), 5.56 (br s, 1H), 3.99 (s,
3H), 3.92 (s, 3H), 3.05 (s, 2H), 3.02 (m, 2H), 2.49 (m, 1H), 2.31 (m,
2H), 1.79 (m, 4H). LC/MS (ESI+) m/z 488.0 (M + H)+; HPLC
purity 92%.
2-[4-(4-{7-[2-(Methanesulfonyl-methyl-amino)-phenyl]-
pyrrolo[2,1-f][1,2,4]triazin-2-ylamino}-3-methoxy-phenyl)-pi-
peridin-1-yl]-acetamide (37). 1H NMR (CDCl3) δ 8.73 (s, 1H),
8.08 (d, J = 8.3 Hz, 1H), 7.95 (m, 1H), 7.53 (m, 3H), 7.46 (s, 1H),
7.12 (br s, 1H), 7.02 (d, J = 4.7 Hz, 1H), 6.86 (d, J = 4.7 Hz, 1H), 6.72
(d, J = 1.3 Hz, 1H), 6.64 (dd, J = 8.3, 1.3 Hz, 1H), 5.53 (br s, 1H),
3.90 (s, 3H), 3.13 (s, 3H), 3.04 (s, 2H), 2.99 (m, 2H), 2.66 (s, 3H),
2.46 (m, 1H), 2.29 (m, 2H), 1.85 (m, 2H), 1.75 (m, 2H). LC/MS
(ESI+) m/z 564.17 (M + H)+; mp 103−112 °C; HPLC purity 94%.
2-[4-(4-{5-Hydroxy-7-[2-(methanesulfonyl-methyl-amino)-
phenyl]-pyrrolo[2,1-f][1,2,4]triazin-2-ylamino}-3-methoxy-
phenyl)-piperidin-1-yl]-acetamide (38). 1H NMR (DMSO-d6) δ
9.94 (s, 1H), 8.74 (s, 1H), 7.94−8.02 (m, 1H), 7.88 (d, J = 8.3 Hz,
1H), 7.59−7.67 (m, 1H), 7.48−7.56 (m, 2H), 7.40 (s, 1H), 7.21 (br s,
1H), 7.13 (br s, 1H), 6.85 (s, 1H), 6.67 (d, J = 8.3 Hz, 1H), 6.35
(s, 1H), 3.83 (s, 3H), 3.05 (s, 3H), 2.77−2.99 (m, 7H), 2.32−2.45
AUTHOR INFORMATION
■
Corresponding Author
*Phone: 610-738-6168. Fax: 610-738-6643. E-mail: emesaros@
cephalon.com.
ACKNOWLEDGMENTS
■
We thank Dr. Renee Roemmele and James Haley for assistance
in multigram scale preparation of intermediates.
ABBREVIATIONS USED
■
ALK, anaplastic lymphoma kinase; ATP, adenosine triphos-
phate; AUC, area under the curve; bid, bis in die (twice a day);
Cl, intrinsic clearance; Cmax, maximum concentration; CYP,
cytochrome P450; EML4, echinoderm microtubule-associated
protein-like 4; F(%), fraction absorbed (oral bioavailability);
GSH, glutathione; HPLC, high-pressure/performance liquid
chromatography; IR, insulin receptor; iv, intravenous; LC,
liquid chromatography; MS, mass spectrometry; NMR, nuclear
magnetic resonance; NPM, nucleophosmin; PEG400, poly-
ethyleneglycol-400; PK/PD, pharmacokinetic/pharmacody-
namic; po, per os (oral); S(90), selectivity score 90% inhibition;
SAR, structure−activity relationship; t1/2, half-life; tmax, time of
maximum concentration; TPM3, tropomyosin α-3 chain; Vd,
volume of distribution
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