2,4-Diaminopyrimidine inhibitors of c-Met kinase bearing benzoxazepine anilines

Article abstract of DOI:10.1016/j.bmcl.2010.12.013

Elaboration of the SAR around a series of 2,4-diaminopyrimidines led to a number of c-Met inhibitors in which kinase selectivity was modulated by substituents appended on the C4-aminobenzamide ring and the nature of the C2-aminoaryl ring. Further lead opt

Full text of DOI:10.1016/j.bmcl.2010.12.013

Bioorganic & Medicinal Chemistry Letters 21 (2011) 660–663
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
2,4-Diaminopyrimidine inhibitors of c-Met kinase
bearing benzoxazepine anilines
⇑
Craig A. Zificsak , Jay P. Theroff, Lisa D. Aimone, Mark S. Albom, Thelma S. Angeles, Rebecca A. Brown,
Deborah Galinis, Jennifer V. Grobelny, Torsten Herbertz, Jean Husten, Laura S. Kocsis, Christine LoSardo,
Sheila J. Miknyoczki, Seetha Murthy, Damaris Rolon-Steele, Ted L. Underiner, Kevin J. Wells-Knecht,
Candace S. Worrell, Kelli S. Zeigler, 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:
Elaboration of the SAR around a series of 2,4-diaminopyrimidines led to a number of c-Met inhibitors in
which kinase selectivity was modulated by substituents appended on the C4-aminobenzamide ring and
the nature of the C2-aminoaryl ring. Further lead optimization of the C2-aminoaryl group led to ben-
zoxazepine analogs whose pharmaceutical properties were modulated by the nature of the substituent
on the benzoxazepine nitrogen. Tumor stasis (with partial regressions) were observed when an orally
bioavailable analog was evaluated in a GTL-16 tumor xenograft mouse model. Subsequent PK/PD studies
suggested that a metabolite contributed to the overall in vivo response.
Received 25 October 2010
Revised 29 November 2010
Accepted 2 December 2010
Available online 8 December 2010
Keywords:
Kinase
c-Met
Ó 2010 Elsevier Ltd. All rights reserved.
Diaminopyrimidine
Inhibitors
Aberrant expression of hepatocyte growth factor/scatter factor
(HGF/SF) and its endogenous receptor, c-Met, is associated with
the development and poor prognosis of a wide range of solid tu-
mors and implicated in the resistance to conventional therapies.1,2
Given the role of c-Met in mediating the growth, invasion, angio-
genesis and survival of multiple solid and hematological cancers,
and functioning as a ‘master switch in cancer progression’,3 the
HGF/c-Met axis is an attractive pharmacological target for oncol-
ogy drug discovery efforts.4
The Novartis FAK inhibitor (NVP-TAE 226, 1) has been reported
to possess sub-micromolar activity against c-Met.5 Initial screening
showed that C4-benzamide analogs provided c-Met activity.6 Fur-
thermore, removal of the methoxy substituent on the C2 aniline in
1 improved potency by 25-fold but at an expected cost in selectiv-
ity.7 Our first set of compounds focused on rescuing selectivity by
elaborating the C4-benzamide and utilizing a C2-benzo[d]azepine
aniline to mimic the basic amine of the morpholino-aniline
(Fig. 1). Analogs were evaluated in a c-Met enzymatic assay and
in a c-Met overexpressing human gastric carcinoma cell line
(GTL-16) assay and countered-screened against the insulin recep-
tor (IR).8,9 Select compounds were also evaluated for global kinase
selectivity using Ambit Bioscience KINOMEscanTM technology,10
expressed as an S(90) value which reflects the fraction of kinases
inhibited >90% when screened at 1
kinases.
lM across a panel of >250
Synthesis of the 2-chloro-4-anilino pyrimidine was achieved as
shown in Scheme 1. Isatoic anhydrides were reacted with methyl-
amine followed by addition into 2,4,5-trichloropyrimidine to afford
the dichloropyrimidines 4a–d. The corresponding C2 aniline was
prepared from benzo[d]azepine 5 by a straightforward three-step
sequence. Acid-catalyzed coupling afforded the target compounds
7a–d.
This initial set of analogs (7a–d) demonstrated potent inhibition
of c-Met activity (Table 1). Inhibition of the insulin receptor (IR)
was significant for 7a (X = H), but was strongly attenuated by
incorporation of a substituent at the 3-position of the benzamide.
Cellular activity suffered when chlorine (7b) and methyl (7c)
groups were incorporated, but fluorinated analog (7d) showed
excellent cellular activity (IC50 = 19 nM). The relative size of the
benzamide C3 substituent correlated with selectivity, with the
smallest, X = H, showing greater pan-kinase activity, and the larger,
X = Cl or Me, showing high selectivity. Based on its moderate
kinase selectivity and high potency in the cellular assay, the fluoro-
benzamide C4 aniline was selected for further optimization.
The C2-benzo[d]azepine lacked substitution in the benzylic
positions, and it was postulated that a C2 aniline with branching
at the 3- or 4-position could improve kinase selectivity. A substi-
tuted 1,5-benzoxazepine (such as 14 and 15) would furnish an
⇑
Corresponding author. Tel.: +1 610 738 6745; fax: +1 610 738 6643.
E-mail address: czificsa@cephalon.com (C.A. Zificsak).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.12.013

C. A. Zificsak et al. / Bioorg. Med. Chem. Lett. 21 (2011) 660–663
661
Figure 1. Novartis FAK inhibitor and initial targets 2.
Table 1
Benzazepine analog SAR
Analog
X
c-Met enzyme
IC50 (nM)
c-Met cell
IC50 (nM)
IR enzyme
IC50 (nM)
Ambit
S(90)
7a
7b
7c
7d
H
41
24
26
8.0
220
550
1400
19
20
>10,000
>3000
230
0.58
0.06
0.06
0.35
Cl
Me
F
treatment with alkoxide generated benzoxazepine carbamates
(10a–e, 11, respectively, Scheme 2). Simple hydrogenation fur-
nished the carbamate capped anilines. Hydrolysis of the ethyl car-
bamates, (10b, 11) yielded intermediates that ultimately provided
N-alkyl (12f–g, 13b) or N-acyl (12h–k, 13c) benzoxazepine anilines
in 2–4 steps.
Acid-catalyzed coupling of the dichloropyrimidine 4d with the 5-
substituted 8- or 7-aminobenzoxazepine furnished targets 17a–k or
18a–c, respectively (Scheme 3).12 Table 2 summarizes the c-Met
inhibitory data (enzyme and cell), as well as the selectivity data
[IR and the kinome (Ambit S(90) values at 1 lM)] for these
benzoxazepines. The aliphatic-capped analogs (17f, 17g) were
the most potent in the cellular assay, but gave unacceptably low
oral exposure in rat PK (5 mg/kg PO AUC0–1 <100 ng h/mL). All of
the carbamate-capped analogs (17a–e) had similar cellular activity
(28–53 nM) and kinase selectivity [S(90) = 0.14–0.17]. Carbamate
17e, featuring an aliphatic amine, had the least amount of activity
againstIR, butalso displayedlow oral exposure in rat PK. Analog 17b,
however, furnished acceptable rat PK (1 mg/kg IV, 5 mg/kg PO;
IVt1/2 = 1.2 h; CL = 15.3 mL/min/kg; IV AUC0–1 = 1224 ng h/mL; PO
Scheme 1. Reagents: (a) MeNH2, THF; (b) 2,4,5-trichloropyrimidine, DIEA, NMP,
100 °C; (c) nitric acid; (d) bromoethyl methyl ether, K2CO3; (e) H2, Pd/C; (f) 4a–d,
camphorsulfonic acid, 2-BuOH, reflux.
aniline nitrogen handle to facilitate SAR elaboration in order to test
this hypothesis. Following a known procedure,11 alkylation of 6- or
5-nitrobenzoxazolidone (8 or 9) followed by a ring-expanding
Scheme 2. Reagents: (a) 1-bromo-3-chloropropane, K2CO3, acetone; (b) NaOR1, NMP; (c) H2, Pd/C; (d) NaOH; (e) CH3CHO, NaBH(OAc)3; (f) ClCH2COCl, Et3N, DMAP;
(g) pyrrolidine; (h) TFAA or R2COCl, pyr, DMAP; (i) BH3ꢀTHF; See Table 2 for R group descriptions in analogs 12a–e, 12h–k, 13a, 13c.

662
C. A. Zificsak et al. / Bioorg. Med. Chem. Lett. 21 (2011) 660–663
Table 2
Benzoxazepine analog SAR
Analog
R
c-Met enzyme IC50 (nM)
c-Met cell IC50 (nM)
IR enzyme IC50 (nM)
Ambit S(90)
17a
17b
17c
17d
17e
17f
17g
17h
17i
CO2Me
CO2Et
CO2-i-Pr
CO2(CH2)2OMe
CO2(CH2)3NMe2
Et
(CH2)2-pyrrolidine
H
32
25
47
81
45
14
21
42
28
44
13
140
120
17
46
36
53
50
28
18
22
57
30
19
51
440
41
49
550
1000
820
800
1400
370
300
560
500
2300
930
NT
0.16
0.17
0.14
0.14
0.16
0.22
0.25
0.21
0.17
0.12
0.15
NT
Ac
17j
(CO)CH2-pyrrolidine
(CO)CH2OMe
CO2Et
Et
Ac
17k
18a
18b
18c
490
1200
0.21
0.20
Scheme 3. Reagents: (a) 12a–k, camphorsulfonic acid, 2-BuOH, reflux; (b) 13a–c,
camphorsulfonic acid, 2-BuOH, reflux.
Figure 3. Inhibition of c-Met phosphorylation by 17b and 17h on day 5 of dosing,
1 h post dose.
samples taken at day 5 showed low levels of 17b. For example, at a
3 mg/kg dose, tumor levels of 17b barely exceeded its cellular
c-Met IC50 (35 nM or 18 ng/mL). A subsequent in vitro metabolite
ID study (i.e., incubation of 17b with mouse liver microsomes) re-
vealed that 17h was a primary metabolite (carbamate hydrolysis).
Since 17h had comparable c-Met cellular activity as 17b, it was
possible that the observed in vivo efficacy could have been driven,
to varying degrees, by these two agents.
A five-day PK/PD experiment was conducted to simultaneously
assess levels of 17b and 17h in plasma and tumor samples after dos-
ing GTL-16 tumor xenograft-bearing nude mice with 17b or 17h at
100 mg/kg (po, qd, Figs. 3 and 4). The PD response for each analog
was similar, with 17h exhibiting slightly greater knockdown of c-
Met phosphorylation over the 24 h period following the last dose
(Fig. 3). Whether dosed as 17h or 17b, similar tumor levels of 17h
were observed and these remained above 200 ng/g over 10 h
(Fig. 4b). Although plasma levels of 17b quickly fell below its cellular
IC50 value after 2 h (Fig. 4), tumor levels remained constant from 2 to
10 h (125 ng/g). Thus the original tumor growth inhibition (Fig. 2)
might not solely be attributed to 17b, and presumably metabolites
such as 17h contributed to the overall biological response. The lack
of a durable PD effect at a relatively high dose (100 mg/kg) coupled
with generally poor rat PK properties of 17h, such as high clearance
(CL = 34 mL/min/kg) and a short half-life (IV t1/2 = 0.3 h), reduced
interest in its further development.
Figure 2. Effect of 17b on GTL-16 tumor xenograft.
AUC0–1 = 913 ng h/mL; %F = 15%) and was selected for in vivo effi-
cacy experiments. Amide capped analogs (17i–k) fared poorly in
rat PK studies. In the 7-aminobenzoxazepine series, carbamate 18a
was significantly less active in the cellular assay and 18b and 18c
displayed poor oral exposure in rat PK.
Oral administration of 17b at 3, 10 or 30 mg/kg (po, qd) in a 15-
day, GTL-16 xenograft tumor model (Fig. 2) showed tumor stasis at
the higher doses, with partial regressions in 40% and 50% of the test
animals, respectively. At 3 mg/kg, 60% tumor growth inhibition
was achieved. All dosing regimens were well tolerated, with no sig-
nificant reduction in body weight. Paradoxically, tumor and plasma
In conclusion, potent inhibitors of c-Met were prepared con-
taining a benzoxazepine aniline at C2 of the 2,4-diaminopyrimi-
dine scaffold; selectivity was modulated by incorporation of
fluorine on the C4 aniline. Analog 17b was orally bioavailable

C. A. Zificsak et al. / Bioorg. Med. Chem. Lett. 21 (2011) 660–663
663
(rat) and demonstrated in vivo tumor stasis in a GTL-16 tumor
xenograft mouse model. Oral administration of 17b in mice led
to formation of an equipotent metabolite (17h), which contributed
to overall tumor growth inhibition.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.12.013.
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Figure 4. PK data for 17b and 17h on day 5 of dosing, 1 h post dose.