Novel 2,3,4,5-tetrahydro-benzo[d]azepine derivatives of 2,4-diaminopyrimidine, selective and orally bioavailable ALK inhibitors with antitumor efficacy in ALCL mouse models

Article abstract of DOI:10.1016/j.bmcl.2010.10.115

The synthesis and biological evaluation of potent and selective anaplastic lymphoma kinase (ALK) inhibitors from a novel class of 2,4-diaminopyrimidines, incorporating 2,3,4,5-tetrahydro-benzo[d]azepine fragments, is described. An orally bioavailable anal

Full text of DOI:10.1016/j.bmcl.2010.10.115

Bioorganic & Medicinal Chemistry Letters 21 (2011) 463–466
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel 2,3,4,5-tetrahydro-benzo[d]azepine derivatives
of 2,4-diaminopyrimidine, selective and orally bioavailable ALK
inhibitors with antitumor efficacy in ALCL mouse models
⇑
Eugen F. Mesaros , Jason P. Burke, Jonathan D. Parrish, Benjamin J. Dugan, Andrew V. Anzalone,
Thelma S. Angeles, Mark S. Albom, Lisa D. Aimone, Matthew R. Quail, Weihua Wan, Lihui Lu, Zeqi Huang,
⇑
Mark A. Ator, Bruce A. Ruggeri, Mangeng Cheng, Gregory R. Ott , Bruce D. Dorsey
Worldwide Discovery Research, Cephalon, Inc., 145 Brandywine Parkway, West Chester, PA 19380, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and biological evaluation of potent and selective anaplastic lymphoma kinase (ALK)
inhibitors from a novel class of 2,4-diaminopyrimidines, incorporating 2,3,4,5-tetrahydro-benzo[d]aze-
pine fragments, is described. An orally bioavailable analogue (18) that displayed antitumor efficacy in
ALCL xenograft models in mice was identified and extensively profiled.
Received 21 September 2010
Revised 20 October 2010
Accepted 22 October 2010
Available online 27 October 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Kinase
ALK
ALCL
Diaminopyrimidine
Inhibitors
Anaplastic Lymphoma Kinase (ALK) is a member of the insulin
receptor tyrosine kinase family. ALK was initially identified in a chi-
meric protein (NPM–ALK) arising from a chromosomal translocation
between the ALK and nucleophosmin (NPM) genes.1 NPM–ALK is
constitutively active and plays an oncogenic role in 70–80% of all
anaplastic large cell lymphomas (ALCL).2 Two other ALK fusion mu-
tants of relevance in cancer pathogenesis were recently described,
tropomyosin 3 gene (TPM3–ALK) in inflammatory myofibroblastic
tumors (IMT)3 and echinoderm microtubule-associated protein-like
4 gene (EML4–ALK)4 in non-small cell lung cancers (NSCLC). Over-
expression of ALK was also observed in glioblastoma5 and neuro-
blastoma.6 Consequently, a number of ALK inhibitors in various
stages of development as therapies for ALK-dependent cancers have
been reported (Fig. 1), including PF-2341066 (1),7 NVP-TAE684 (2),8
and GSK1838705A (3).9
led inadvertently to 6 (Table 1), presumably arising from bis-addi-
tion of the requisite aniline to unreacted 2,4,5-trichloropyrimidine.
Interestingly, 6 demonstrated equivalent inhibition of ALK ki-
nase activity to 5, albeit with slightly lower (2.4-fold) cellular
Recent efforts from our laboratories10 towards ALK inhibitors
with therapeutic utility led to the discovery of a novel and potent
bridged bicyclic derivative (4, Fig. 1).2b,11 During the course of these
endeavors, we synthesized and profiled a variety of analogs related
to the reported kinase inhibitor NVP-TAE22612 (Table 1, 5), which
⇑
Corresponding authors. Tel.: +1 610 738 6168; fax: +1 610 738 6643 (E.F.M.);
tel.: +1 610 738 6861; fax: +1 610 738 6643 (G.R.O.).
E-mail addresses: emesaros@cephalon.com (E.F. Mesaros), gott@cephalon.com
(G.R. Ott).
Figure 1. Examples of reported ALK inhibitors.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.

464
E. F. Mesaros et al. / Bioorg. Med. Chem. Lett. 21 (2011) 463–466
Table 1
Biological data for compounds 5–7
a
Compds
A
B
IC50 (nM)
ALK enzyme
ALK cell
IR enzyme
25
5
6
5
4
50
120
175
1860
7
34
>10,000
a
IC50 values are reported as the average of at least two separate determinations.
Table 2
Biological data for compounds 14–18
R1
IC50 (nM)
a
Compds
ALK enzyme
ALK cell
IR enzyme
14
15
16
17
18
MeO(CH2)2–
CHF2CH2–
MeS(O)2CH2CH2–
MeNHC(O)CH2–
(Me)2NC(O)CH2–
7
58
10
6
150
—
100
100
30
2694
>10,000
>3000
>3000
1380
4
a
IC50 values are reported as the average of at least two separate determinations.
Scheme 1. Reagents and conditions: (a) (i) KNO3, TFAA, MeCN, 0 °C?rt; (ii)
separation of regioisomers; (iii) 20–40% aq NaOH, MeCN, rt; (b) (i) R1-X, K2CO3,
MeCN, 75 °C; (ii) H2, Pd/C, MeOH; (c) K2CO3, DMF or DIPEA, THF, rt to 50 °C; (d) HCl,
MeO(CH2)2OH, 100 °C. See Tables 2 and 3 for R1–3 definitions.
secured intermediates 13. Finally, a second SNAr reaction between
10 and 13 provided the desired target molecules 14–24.
Gratifyingly, enzyme activity of 14 (Table 2) was improved rel-
ative to 7 and comparable to 6 (Table 1). IR selectivity was main-
tained, though cellular activity still required improvement. We
focused first on analogues diversely substituted on the benzaze-
pine nitrogen and observed that subtle changes to the nature of
R1 groups impacted both enzyme and cell activity.
Though likely not the only contributing factor, the basicity of
this nitrogen appeared to be of particular importance. For example,
fluorinated derivative 15 lost eightfold in enzyme activity relative
to the more basic amine 14. N-Substituents in 16 and 17 restored
ALK activity for these compounds and brought moderate improve-
ments in cellular activity. Amide 18 was the most promising lead
(Table 2), active against ALK with good cell activity (cell/enzyme
IC50 ratio of 7–8) and highly selective against IR (IR/ALK IC50 ratio
of 345). These results prompted further exploration of the SAR with
respect to the R2,3 substituents (Table 3).
Examples 19 and 20 comprise truncated versions of 18, which
define the selectivity determinants on the aromatic ring B. Com-
pound 19 with a hydrogen as R2 resulted in a 12-fold loss in ALK
activity relative to 18. Furthermore, hydrogen in place of morpho-
line for R3 (20) led to a significant loss in IR selectivity (IR/ALK IC50
ratio of 16) as well as broader kinome selectivity (S(90) 0.12),17
showcasing the importance of a substituent on the 4-position of
the B ring as a key selectivity determinant. The morpholine
activity. More importantly however, 6 exhibited a tremendous in-
crease in selectivity against the highly homologous insulin recep-
tor (IR). Selectivity over IR is desirable since recent reports of
concomitant inhibition of IR with small molecule kinase inhibitors
have shown compound-dependent fluctuation in glucose homeo-
stasis in preclinical animal models.9,13
Capitalizing on this serendipitous finding, we incorporated mor-
pholine-phenyl fragment B (Table 1) and benzazepinone A (cf. 4 in
Fig. 1),2b,11 providing novel compound 7, though this resulted in a
drop-off in both ALK enzymatic activity as well as cellular potency.
IR selectivity was maintained. At this juncture we sought to improve
the potency of these inhibitors while maintaining this high degree of
IR selectivity. In an effort to provide synthetically tractable function-
ality with a handle to modulate both potency and physiochemical
properties, the 2,3,4,5-tetrahydro-1H-benzo[d]azepine motif was
incorporated into this pharmacophore.
The synthesis of these analogues is outlined in Scheme 1.14 Ben-
zazepine 8 was prepared from inexpensive starting materials
employing reported procedures.15 Nitration of 8 in presence of
trifluoroacetic anhydride16 afforded an intermediate trifluoroaceta-
mide, which was hydrolyzed to the corresponding amine 9. N-Alky-
lations followed by reduction of the nitro group generated anilines
10. Reaction of 2,4,5-trichloropyrimidine (11) with anilines 12

E. F. Mesaros et al. / Bioorg. Med. Chem. Lett. 21 (2011) 463–466
465
Table 3
Biological data for compounds 18–24
R2
R3
IC50 (nM)
S(90)
a
b
Compds
ALK enzyme
ALK cell
IR enzyme
1380
18
19
MeO–
H
4
30
—
0.03
—
49
504
20
21
22
23
24
MeO–
MeO–
MeO–
MeO–
MeO–
H
4
6
4
7
5
65
65
40
65
40
62
573
351
233
189
0.12
0.11
0.09
0.06
0.11
–C(O)NH2
–O(CH2)2OMe
–CN
–OMe
a
IC50 values are reported as the average of at least two separate determinations.
b
Kinase selectivity was determined using the Ambit Bioscience KINOMEscan™ technology, and is expressed as S(90), the fraction of kinases inhibited >90% when screened
M across a panel of 256 kinases.
at 1
l
Table 4
Pharmacokinetic parameters of 18 in Sprague–Dawley rats and in CD-1 mice
PK parameters
S–D rata
CD-1 mouse
iv
Doseb (mg/kg)
t1/2 (h)
1
1
0.8 0.05
1019 91
1.1 0.1
16 1.5
0.7
830
1.2
20
AUC0–t (ngÁh/mL)
Vd (L/kg)
CL (mL/min/kg)
po
Doseb (mg/kg)
Cmax (ng/mL)
tmax (h)
5
10
1192
0.5
306 53
3.3 0.7
n.d.
t1/2 (h)
3.1
AUC0–t (ngÁh/mL)
1395 281
3895
64
F (%)
30
6
a
Values are means of data from three animals.
Formulated as solutions in: 3% DMSO, 30% Solutol, 67% PBS (iv); 100% PEG400
b
(po).
replacements in 21–24 afford lower molecular weight and some-
what increased polarity, while maintaining acceptable levels of
ALK activity and IR selectivity. Noteworthy, the IR activity is re-
lated to the size of these functional groups.
Compared to 18, 21–24 were 2–3 times less selective against
the tested kinome panel (S(90) 0.06–0.11) and showed suboptimal
rat PK data (oral bioavailability F in the 0–12% range, low expo-
sures and short iv half-lives). Inhibitor 18 had acceptable pharma-
cokinetics in both rat and mouse (Table 4) for in vivo evaluation.
In cell proliferation assays, 18 demonstrated concentration
dependent growth inhibition in ALK-positive ALCL cell lines Kar-
pas-299 (calculated IC50 ꢀ 160 nM) and Sup-M2 (calculated
IC50 ꢀ 80 nM), while it had a minimal effect on ALK-negative
K562 cells up to 1000 nM tested.
Based upon the potency against ALK in enzyme and cellular as-
says (inhibition of phosphorylation and proliferation) combined
with the promising oral PK, 18 was evaluated in a single-dose
PK/PD experiment (30 mg/kg, po) in mice bearing ALK-positive tu-
mor xenografts. 18 provided sustained inhibition of NPM–ALK
autophosphorylation (75% inhibition for up to 12 h post-dose).
Figure 2. Antitumor efficacy of 18, dosed po as a solution in PEG400.
obtained (Fig. 2). 18 exhibited tumor regressions at 30 mg/kg and
55 mg/kg b.i.d., po doses, at which it was well tolerated. No overt
toxicity or body weight loss was observed.
Further profiling for potential off-target liabilities demonstrated
that 18 was selective with respect to a panel of 64 receptors; inhi-
bition levels at 10
10
M).18 In a functional patch clamp assay to assess activity
against the hERG ion channel, 18 displayed an IC50 = 3.9
M.19
Against the major CYP isoforms, minimal inhibition for CYP1A2
and 2D6 was seen (IC50 >20 M), however, inhibition of the 2C9,
2C19, and 3A4 isoforms was 1.2, 0.8, 2.0 M, respectively.
lM were <50%, except for 5HT-1A (67% at
l
l
l
l
In summary, the synthesis and biological characterization of no-
vel benzazepine-derived 2,4-diaminopyrimidines were described.
The research efforts outlined above resulted in identification of po-
tent and highly selective (including against IR) ALK inhibitors. Or-
ally bioavailable analogues (exemplified by the tool compound 18)
from this series demonstrated in vivo efficacy in ALCL tumor
models.
Plasma concentrations of 3–5 lM were achieved out to 12 h with
comparable levels observed in tumors. Mouse plasma protein
binding of 97.2% rendered unbound concentrations of 18 of 90–
130 nM, approximately 3–4-fold above the cellular IC50 of 30 nM.
Consistent with the PK/PD response, dose-dependent antitumor
efficacy in the Sup-M2 tumor xenograft model in SCID mice was

466
E. F. Mesaros et al. / Bioorg. Med. Chem. Lett. 21 (2011) 463–466
10. For recent work on ALK inhibitors from a different chemotype see: Milkiewicz,
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