Novel tricyclic azepine derivatives: Biological evaluation of pyrimido[4,5-b]-1,4-benzoxazepines, thiazepines, and diazepines as inhibitors of the epidermal growth factor receptor tyrosine kinase

Article abstract of DOI:10.1016/j.bmcl.2006.07.031

Novel tricyclic derivatives containing an oxazepine, thiazepine, or diazepine ring were studied for their EGFR tyrosine kinase inhibitory activity. While the oxazepines were in general more potent than thiazepines, the diazepines displayed somewhat differ

Full text of DOI:10.1016/j.bmcl.2006.07.031

Bioorganic & Medicinal Chemistry Letters 16 (2006) 5102–5106
Novel tricyclic azepine derivatives: Biological evaluation of
pyrimido[4,5-b]-1,4-benzoxazepines, thiazepines, and diazepines as
inhibitors of the epidermal growth factor receptor tyrosine kinase
Leon Smith, II,a,* Wai C. Wong,a Alexander S. Kiselyov,a, Sabina Burdzovic-Wizemann,a
Yunyu Mao,a Yongjiang Xu,a Matthew A. J. Duncton,a Ki Kim,a Evgueni L. Piatnitski,a
Jacqueline F. Doody,b Ying Wang,b Robin L. Rosler,b Daniel Milligan,a John Columbus,a
Chris Balagtas,a Sui Ping Lee,a Andrey Konovalova and Yaron R. Hadarib
aDepartment of Chemistry, ImClone Systems Incorporated, 710 Parkside Avenue, Suite 2, Brooklyn, NY 11226, USA
bDepartment of Protein Science, ImClone Systems Incorporated, 180 Varick Street, New York, NY 10014, USA
Received 1 June 2006; revised 8 July 2006; accepted 11 July 2006
Available online 2 August 2006
Abstract—Novel tricyclic derivatives containing an oxazepine, thiazepine, or diazepine ring were studied for their EGFR tyrosine
kinase inhibitory activity. While the oxazepines were in general more potent than thiazepines, the diazepines displayed somewhat
different structure–activity relationships. Moreover, the diazepines, in contrast to the oxazepines, showed appreciable inhibitory
activity against the KDR tyrosine kinase. Furthermore, both oxazepines and diazepines demonstrated significant ability to inhibit
autophosphorylation of EGFR in DiFi cells (generally, IC50 values in the single-digit micromolar to submicromolar range).
ꢀ 2006 Elsevier Ltd. All rights reserved.
Receptor tyrosine kinases (RTKs) are involved in cellu-
lar signal transduction pathways which influence cell
growth, differentiation, survival, and proliferation.1
Many human cancers are characterized by an up-regula-
tion of some of these RTKs.2 Consequently, they have
become targets of intense drug discovery efforts to iden-
tify novel anti-cancer agents.3 In particular, Erbitux, an
antibody which targets the epidermal growth factor
receptor (EGFR), has been approved for the treatment
of irinotecan-refractory colorectal cancer and squa-
mous-cell carcinoma of the head and neck.4 Iressa5
(1a) and Tarceva6 (1b), both of which belong to the same
chemical class of quinazolines, inhibit the EGFR kinase
and are being used to treat locally advanced, or meta-
static non-small cell lung cancer and certain types of
pancreatic cancer. We have reported a new class of
non-quinazoline oxazepine derivatives as inhibitors of
EGFR kinase, exemplified by structure (1c).7 It is inter-
esting that bromoanilino and dimethoxy moieties of (1c)
have also been described as desirable functionalities in
quinazoline-type EGFR kinase inhibitors,8 despite a sig-
nificant difference in the core ring systems between the
two structural classes (Fig. 1). In this report, we detail
structure–activity relationship results pertaining to the
azepine class of compounds, encompassing not only
oxazepines but also thiazepine and diazepine scaffolds.
Since the dimethoxy substitution on the tricyclic phenyl
ring of (1c) appeared to be advantageous for activity,
our goal became to modify the aniline moiety, the
pyrimidine portion, and the seven-membered ring in
an attempt to enhance the kinase inhibitory activity of
the compounds. Select oxazepines were synthesized as
outlined in Scheme 1.9
Thus, 4,6-dichloro-5-aminopyrimidine (5) was coupled
with a diverse set of anilines (6) giving rise to diamino-
pyrimidine intermediates (7), which were reacted with
2,4,5-trihydroxybenzaldehyde to form the requisite tri-
cyclic core structure upon displacement of the chlorine
atom and concomitant imine formation.10 The remain-
Keywords: Epidermal growth factor receptor; Kinase inhibitor; Oxa-
zepine; Diazepine; Thiazepine.
*
Corresponding author. Tel.: +1 609 818 4121; fax: +1 609 818
3331; e-mail: leon.smith@bms.com
Present address: Chemical Diversity, 1155 Sorrento Valley Road,
Suite 5, San Diego, CA, 92121, USA.
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.07.031

L. Smith et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5102–5106
5103
F
Br
NH
N
Cl
NH
O
NH
N
H
N
O
O
N
O
O
N
N
N
O
O
O
N
O
1 b
1 a
O
1 c
Figure 1. IressaTM (1a), TarcevaTM (1 b), and benzoxazepine EGFR kinase inhibitor (1c).
R
Cl
NH
NH2
b
a
NH2
Cl
NH2
Cl
N
N
R
+
N
N
(6)
(7)
(5)
R
N
R
R
N
NH
N
NH
N
NH
N
H
d
c
N
N
N
N
OH
OH
O
O
O
O
O
O
(8)
O
(9)
(1a - 2h)
Scheme 1. Reagents and conditions: (a) DMF/120 ꢁC, or EtOH/H2O/HCl, 100 ꢁC, 59–72%; (b) 2,4,5-trihydroxybenzaldehyde, DMF, 100–120 ꢁC,
21–72%; (c) DMF, NaH or K2CO3, MeI or Me2SO4, 25–80 ꢁC, 3–93%; (d) NaBH4, THF, EtOH or MeOH, 40 ꢁC-reflux, 2–68%.
ing hydroxy moieties were then methylated and the
imine bond reduced to afford the desired target com-
pounds (1a–2k).
ylation level of poly-Glu-Ala-Tyr-biotin peptide in a
homogeneous time-resolved fluorescence (HTRF) at an
ATP concentration of 2.0 lM.13 To ensure quality
control, a literature EGFR tyrosine kinase inhibitor
(IressaTM, 1a) was included as an internal standard
(IC50 = 20 10 nM).14 Most of the compounds were
also assayed13 for their ability to inhibit the autophos-
phorylation level of EGFR in DiFi cells (1a,
IC50 = 55 15 nM). Results for enzymatic and cellular
assays were reported as an IC50 expressed in lM.
Some additional oxazepines, thiazepines, and diazepines
were prepared by a modified procedure described in
Scheme 2. In this more convergent approach, a dichloro-
pyrimidine (5 or 10) was reacted with 3,4-dimethoxyphe-
nol or thiophenol under basic condition, or with 3,4-
dimethoxyaniline under acidic condition, affording an
intermediate (11) which then underwent a cyclization
reaction11 in the presence of paraformaldehyde to form
an oxazepine, thiazepine or diazepine (12),12 respectively.
Subsequently, the remaining chloro group was displaced
with different anilines, amines, phenols, and thiophenols
to yield the target compounds.
Table 1 summarizes the biochemical and cell-based
inhibitory activity of oxazepines with various modifica-
tions. Substitution of the aniline portion of the oxaze-
pine core afforded modest results. For example,
replacing the bromine of the parent compound (1c) with
a methyl (2a) led to a dramatic drop in potency.
Furthermore, introducing an ethynyl group (2b) resulted
in a reduction in enzymatic activity while maintaining
The EGFR tyrosine kinase inhibitory activity of the
compounds was determined by measuring the phosphor-
Cl
Cl
NH2
NH2
Cl
HT
O
O
N
b
N
a
+
O
Y
N
T
Y
N
O
(11)
(5) Y = H
(10) Y = Me
T = NH, O, S
R
R
X
N
H
X
N
d
Cl
N
H
N
N
N
T
c
N
N
N
X=T=O
O
O
O
Y
T
Y
Y
T
O
(2i - 4e)
(2p, 2q)
O
(12)
O
Scheme 2. Reagents and conditions: (a) K2CO3/DMF/60 ꢁC or EtOH/H2O/HCl/reflux, 24–84%; (b) CH2Cl2, trifluoroacetic acid, MgSO4, (CHO)n,
40 ꢁC, 18–76%; (c) Anilines/amines, HCl, 2-propanol or DMF, 94–150 ꢁC, or neat/120–150 ꢁC, or K2CO3/DMF/65–80 ꢁC for phenols and
thiophenols, 8–79%; (d) Cs2CO3, MeI, DMF, 70 ꢁC, 33–38%.

5104
L. Smith et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5102–5106
Table 1. Inhibition results for oxazepines9
Ar
Z
N
X
N
N
O
Y
O
O
Compound
Ar
X
Y
Z
Enzymatic
a
IC50 (lM)
DiFi cell
a
IC50 (lM)
1c
2a
2b
2c
2d
2e
2f
3-Br–Ph
3-Me–Ph
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
O
H
H
H
H
H
H
H
H
H
H
H
H
Me
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
Me
H
H
0.3
7.4
0.5
4.4
0.9
>100
7.6
0.7
4.6
3.9
>100
nt
3-Ethynyl–Ph
4-Br–Ph
4-F–Ph
3.2
8.7
7.1
1.2
3-Cl–4-F–Ph
3-Cl–2-F–Ph
5-Cl–2-F–Ph
2-Cl–4-F–Ph
6-Indazolyl
2-Naphthyl
6-Benzthiazolyl
3-Br–Ph
0.3
1.1
2g
2h
2i
3.4
1.2
2j
0.5
5.6
2.3
nt
2k
2l
>100
>100
0.7
nt
2m
2n
2o
2p
2q
2r
2s
3-Cl–2-F–Ph
3-Br–Ph
nt
1.8
6.1
nt
3-Cl–4-F–Ph
3-Br–Ph
O
1.2
4.6
O
3-Cl–4-F–Ph
3-Br–Ph
3-Cl–Ph
O
10.8
1.3
nt
S
1.2
2.4
S
1.0
nt, not tested; generally, enzymatic activity too low to test in cellular assay.
a Data are reported as means of n P 2 determinations.
Table 2. Inhibition results for thiazepines9
submicromolar cell-based potency. A substitution at the
para position (2c, 2d) did not confer any benefit as well.
Among the dihalo-substituted analogs (2e–2h), (2f) was
the most active analog. Interestingly, compound (2e),
with the same substitution as Iressa, was more potent
in the cellular assay than in the enzymatic assay, essen-
tially being equipotent to (1c). Of the bicyclic anilines
(2i–2k), only the naphthyl analog (2j) was comparable
to (1c) in activity. Compounds (2l) and (2m) clearly
showed that further substitution on the pyrimidine ring
was undesirable. We also evaluated the necessity of the
aniline NH- in inhibiting EGFR. While the O-linked
analog (2n) was comparable to the corresponding NH-
linked compound (1c), having twofold lower activity,
the S-linked compound (2r) was somewhat less potent.
Lastly, we established that the NH- of the seven-mem-
bered ring was necessary for good activity. When meth-
ylated there was a significant loss (>6.5-fold) in
inhibitory activity (2p, 2q).
Ar
X
H
N
N
O
Y
N
S
O
Compound
Ar
X
Y
Enzymatic
a
IC50 (lM)
3a
3b
3c
3d
3e
3f
3-Br–Ph
NH
NH
NH
NH
NH
NMe
NMe
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
O
H
5.9
8.2
3-Cl–4-F–Ph
3-Cl–2-F–Ph
6-Indazolyl
H
H
62.9
14.1
>100
>100
27.0
30.7
>100
45.9
>100
>100
17.0
14.5
15.2
24.6
16.2
21.7
19.3
1.6
H
6-Benzthiazolyl
3-Br–Ph
3-Cl–Ph
H
H
3g
3h
3i
H
3-Br–Ph
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
3-Br–4-Me–Ph
3-Cl–4-F–Ph
3-Cl–2-F–Ph
4-Cl–3-Me–Ph
2-Naphthyl
5-Benzimidazolyl
6-Benzthiazolyl
6-Indazolyl
5-Indazolyl
3-Br–Ph
3j
3k
3l
Table 2 lists the thiazepines prepared and tested for
enzymatic inhibitory activity. Compound (3a), in
comparison to (1c), was at least 10-fold less active. Sim-
ilarly, other analogs (3b–3e) were less potent than their
counterparts in the oxazepine series (2e, 2f, 2i, and 2k,
respectively). Steric hindrance of the sulfur atom over
oxygen is a possible explanation for decrease in activity
seen in thiazepines. Substituting the bridge nitrogen with
a methyl group (3f, 3g) decreased activity. As was the
case with the oxazepines, adding a methyl substituent
3m
3n
3o
3p
3q
3r
3s
3t
3-Cl–2-F–Ph
3-Br–Ph
3-Cl–Ph
O
H
S
H
3u
S
H
2.9
a Data are reported as means of n P 2 determinations.

L. Smith et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5102–5106
5105
Table 3. Inhibition results for diazepines9
As seen in our earlier report, some compounds in this
study display little correlation between enzymatic
activity and cellular activity. Poor solubility of such
compounds is a possible explanation for these discrep-
ancies. In summary, we have identified and studied the
structure–activity relationships of a novel series of
R
A
NH
N
H
N
N
non-quinazoline compounds containing
a tricyclic
O
oxazepine, thiazepine or diazepine ring system. While
the oxazepines were in general more potent EGFR
kinase inhibitors than thiazepines, the diazepines
showed somewhat different SAR, and were moderately
potent inhibitors of the KDR kinase as well. Further-
more, both oxazepines and diazepines demonstrated
significant ability to inhibit cell-based phosphorylation
in DiFi cells (generally, IC50 values in the single-digit
micromolar to submicromolar range). Mono- and
multi-kinase targeting and orally efficacious analogs of
these compound series have been obtained and will be
reported in due course.
N
H
O
Compound
R
A
Enzymatic DiFi cell
a
IC50 (lM) IC50a (lM)
4a
4b
4c
4d
4e
3-Br —
3-Cl–4-F CH2
1.5
0.3
0.8
0.2
nt
1.6
3-MeO
CH2
CH2
11.7
0.9
H
H
(R)-MeCH 0.15
0.5
nt, not tested; enzymatic activity too low to be tested in cellular assay.
a Data are reported as means of n P 2 determinations.
Acknowledgments
to the pyrimidine ring led to poorly active compounds
(3h–3q). Replacing the bridge nitrogen with an oxygen
(3r, 3s) also significantly decreased activity. Having a
sulfur bridge as in (3t) and (3u) actually maintained
some of the potency relative to (3a), (2r) and (2s).
Because of their overall moderate enzymatic potencies,
only a handful of thiazepines were assayed for their
cellular potency in DiFi cells. Analogs that were tested
showed weak activity (IC50 P 17 lM).
We are grateful to Dr. Peter Bohlen and Dr. Marc
Labelle for their strong support.
References and notes
1. Traxler, P. Exp. Opin. Ther. Patents 1998, 8, 1599.
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Table
3 summarizes the activity profiles of the
diazepines. Compound (4a) showed reduced activity
compared to the oxazepine counterpart (1c). However,
when the nitrogen bridge was extended by one methy-
lene unit (A = CH2), the activity was less sensitive to
the substituent variation on the phenyl ring such that
even a methoxy group (4c) or lack of a substituent
(4d) led to enzymatic potencies comparable to that of
(1c). Indeed, an additional methyl moiety on the bridge
(4e) did not adversely affect potency at all. When
assayed in the DiFi cells, (4e) emerged as the most
potent compound among the diazepines, and equipotent
to (1c).
6. Herbst, R. S.; Johnson, D. H.; Mininberg, E.; Carbone, D.
P.; Henderson, T.; Kim, E. S.; Blumenschein, G., Jr.; Lee,
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7. (a) Smith, L. M., II.; Hadari, Y. PCT. Int. Appl.
20055009384, Chem. Abstr. 2005, 142, 172179; (b) Smith,
L., II; Piatnitski, E. L.; Kiselyov, A. S.; Ouyang, X.; Chen,
X.; Burdzovic-Wizemann, S.; Xu, Y.; Wang, Y.; Rosler,
R. L.; Patel, S. N.; Chiang, H. H.; Milligan, D. L.;
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Since KDR (or VEGFR-2), another receptor tyrosine
kinase, has long been within our area of interest,15
some of the test compounds were also assayed for
their enzymatic inhibitory activity against the KDR
kinase.16 While the oxazepines in general showed very
weak KDR activity (IC50 > 10 lM), diazepines
(4b–4e) displayed appreciable potency with IC50 in
the range of 2–5 lM. It is perceivable that the diaze-
pine nitrogen between two aromatic rings, and the
adjacent pyrimidine nitrogen together form hydrogen
bonding interactions at the active site, analogous to
that proposed for AEE788 which is a pyrrolo[2,3-
d]pyrimidine reported to be an inhibitor of both
EGFR and KDR.17 This is in contrast to the oxaze-
pines and thiazepines that lack a potential hydrogen
bond donating group present in the diazepine
scaffold.

5106
L. Smith et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5102–5106
9. All structures described in this report are consistent with
their H NMR and LC/MS data.
The anti-EGFR antibodies were prepared in a buffer made
with Na2CO3 (0.2 M, 16 ml) and NaHCO3 (0.2 M, 34 ml)
and the pH was adjusted to 9.6. Prior to adding cell lysates
to the wells, the plates were washed three times with
PBS + 0.1% Tween 20 and blocked by 3% BSA in PBS
(200 ll for 1 h incubation). Eighty microliters of cell
lysates was transferred to the coated wells and incubated
for 1 h at 4 ꢁC. After incubation, the plates were washed
three times with PBS + 0.1% Tween 20. To detect auto-
phosphorylated EGFR (tyrosine residues), 100 ll of anti-
phosphotyrosine antibodies (RC20:HRP, Transduction
Laboratories) was added per well (final concentration
0.5 lg/ml in PBS) and incubated for 1 h. The plates were
then washed six times with PBS + 0.1% Tween 20.
1
10. Levkovskaya, L. G.; Sazonov, N. V.; Grineva, N. A.;
Mameeva, I. E.; Serochkina, L. A.; Safonova, T. S. Chem.
Heterocycl. Compd. (English Translation) 1985, 21, 100.
11. (a) Duncton, M. A. J.; Smith, L. M., II; Burdzovic-
Wizeman, S.; Burns, A.; Liu, H.; Mao, Y.; Wong, W. C.;
Kiselyov, A. S. J. Org. Chem. 2005, 70, 9629; (b) Aversa,
M. C.; Bonaccorsi, P.; Giannetto, P. J. Heterocycl. Chem.
1989, 26, 1383; (c) Aversa, M. C.; Giannetto, P. J. Chem.
Res., Synop. 1984, 200.
12. Structurally similar diazepines have recently been reported:
Yang, J.; Che, X.; Dang, W.; Wei, Z.; Gao, S.; Bai, X. Org.
Lett. 2005, 7, 1541.
13. Enzymatic assay conditions: EGFR tyrosine kinase inhi-
bition was determined by measuring the phosphorylation
level of poly-Glu-Ala-Tyr-biotin (pGAT-biotin) peptide in
a homogeneous time-resolved fluorescence (HTRF) assay.
Into a black 96-well Costar plate was added 2 ll/well of
25· compound in DMSO (final compound concentration
in the 50-ll kinase reaction was typically 1 nM to 10 lM).
Next, 38 ll of reaction buffer (25 mM Hepes, pH 7.5,
5 mM MgCl2, 5 mM MnCl2, and 2 mM DTT) containing
1.5 pmol pGAT-biotin and 1–2 ng EGFR enzyme was
added to each well. After 5–10 min preincubation, the
kinase reaction was initiated by the addition of 10 ll of
10 lM ATP in reaction buffer, after which the plate was
incubated at room temperature for 45 min. The reaction
was stopped by the addition of 50 ll KF buffer (50 mM
Hepes, pH 7.5, 0.5 M KF) containing 100 mM EDTA and
0.23 lg/ml PY20K (Eu-cryptate labeled anti-phosphoty-
rosine antibody, CIS Biointernational). After 30 min,
100 ll of 5 nM SV-XL (modified-APC-labeled Streptavi-
din, CIS Biointernational) in KF buffer was added, and
after an additional 2-h incubation at room temperature,
the plate was read in a RUBYstar HTRF Reader.
14. Barker, A. J.; Gibson, K. H.; Grundy, W.; Godfrey, A. A.;
Barlow, J. J.; Healy, M. P.; Woodburn, J. R.; Ashton, S.
E.; Curry, B. J.; Scarlett, L.; Henthorn, L.; Richards, L.
Bioorg. Med. Chem. Lett. 2001, 11, 1911.
15. (a) Piatnitski, E. L.; Duncton, M. A. J.; Kiselyov, A. S.;
Katoch-Rouse, R.; Sherman, D.; Milligan, D.; Balagtas,
C.; Wong, W. C.; Kawakami, J.; Doody, J. F. Bioorg.
Med. Chem. Lett. 2005, 15, 4696; (b) Duncton, M. A. J.;
Piatnitski, E. L.; Katoch-Rouse, R.; Smith, L. M., II;
Kiselyov, A. S.; Milligan, D. L.; Balagtas, C.; Wong, W.
C.; Doody, J. F. Bioorg. Med. Chem. Lett. 2006, 16, 1579.
16. VEGFR tyrosine kinase inhibition is determined by
measuring the phosphorylation level of poly-Glu-Ala-
Tyr-biotin (pGAT-biotin) peptide in a homogeneous time-
resolved fluorescence (HTRF) assay. Into a black 96-well
Costar plate is added 2 ll/well of 25· compound in 100%
DMSO (final concentration in the 50 ll kinase reaction is
typically 1 nM to 10 lM). Next, 38 ll of reaction buffer
(25 mM Hepes, pH 7.5, 5 mM MgCl2, 5 mM MnCl2, 2
mM DTT, and 1 mg/ml BSA) containing 0.5 mmol
p-GAT-biotin and 3–4 ng KDR enzyme is added to each
well. After 5–10 min preincubation, the kinase reaction is
initiated by the addition of 10 lM ATP in the reaction
buffer, after which the plate is incubated at room
temperature for 45 min. The reaction is stopped by
addition of 50 ll KF buffer (50 mM Hepes, pH 7.5,
0.5 M KF, and 1 mg/ml BSA) containing 100 mM EDTA
and 0.36 lg/ml PY20K (Eu-cryptate labeled anti-phos-
photyrosine antibody, CIS Bio International) in KF buffer
is added, and after 2 h incubation at room temperature,
the plate is read in a RUBY Star HTRF reader.
EGFR cellular phosphorylation assay: DiFi cells were
plated in 96-well Costar plates (2.5 · 105 cells per well) and
incubated for 4 h. The cells were then starved in serum-
free medium overnight. The next morning test compounds
were added to individual wells. Following a 2 h incuba-
tion, cells were lysed in 100 ll of lysis buffer (150 mM
NaCl, 50 mM Hepes, 0.5% Triton X-100, 10 mM NaPPi,
50 mM NaF, 1 mM Na3VO4, and protease inhibitors, pH
7.5), and rocked for 1 h at 4 ꢁC. The autophosphorylation
level of EGFR was then analyzed by ELISA using anti-
phosphotyrosine antibodies. ELISA: 96-well ELISA plates
were coated with 100 ll/well of 1 lg/ml anti-EGFR
antibodies (Erbitux), and incubated overnight at 4 ꢁC.
17. Caravatti, G.; Bold, G.; Bruggen, J.; Furet, P.; Lane, H.;
Mestan, J.; Meyer, T.; Tang, C.; Wartmann, M.; Wood,
J.; Zimmerlin, A.; Traxler, P. AACR-NCI-EORTC Meet-
ing, Boston, MA, November 17–21, 2003, Poster #A118.