Improvement in oral bioavailability of 2,4-diaminopyrimidine c-Met inhibitors by incorporation of a 3-amidobenzazepin-2-one group

Article abstract of DOI:10.1016/j.bmc.2011.09.006

The hepatocyte growth factor (HGF)-c-Met signaling axis is involved in the mediation of many biological activities, including angiogenesis, proliferation, cell survival, cell motility, and morphogenesis. Dysregulation of c-Met signaling (e.g., overexpress

Full text of DOI:10.1016/j.bmc.2011.09.006

Bioorganic & Medicinal Chemistry 19 (2011) 6274–6284
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Improvement in oral bioavailability of 2,4-diaminopyrimidine c-Met
inhibitors by incorporation of a 3-amidobenzazepin-2-one group
⇑
Karen L. Milkiewicz , Lisa D. Aimone, Mark S. Albom, Thelma S. Angeles, Hong Chang,
Jennifer V. Grobelny, Jean Husten, Christine LoSardo, Sheila Miknyoczki, Seetha Murthy,
Damaris Rolon-Steele, Ted L. Underiner, Linda R. Weinberg, Candace S. Worrell, Kelli S. Zeigler,
Bruce D. Dorsey
Worldwide Discovery Research, Cephalon, Inc. 145 Brandywine Parkway, West Chester, PA 19380-4245, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 5 July 2011
Revised 30 August 2011
Accepted 6 September 2011
Available online 13 September 2011
The hepatocyte growth factor (HGF)-c-Met signaling axis is involved in the mediation of many biological
activities, including angiogenesis, proliferation, cell survival, cell motility, and morphogenesis. Dysregula-
tion of c-Met signaling (e.g., overexpression or increased activation) is associated with the proliferation and
metastasis of a wide range of tumor types, including breast, liver, lung, colorectal, gastric, bladder, and pros-
tate, among others. Inhibiting the HGF-c-Met pathway is predicted to lead to anti-tumor effects in many
cancers. Elaboration of the SAR around a series of 2,4-diaminopyrimidines led to a number of c-Met inhib-
itors in which pharmaceutical properties were modulated by substituents appended on the C2-benzazepi-
none ring. In particular, certain-3-amidobenzazepin-2-one analogs had improved oral bioavailability and
were evaluated in PK/PD and efficacy models. Lead compounds demonstrated tumor stasis with partial
regressions when evaluated in a GTL-16 tumor xenograft mouse model.
Keywords:
c-Met
Kinase inhibitors
Oral bioavailability
PK-PD
Ó 2011 Elsevier Ltd. All rights reserved.
In vivo efficacy
Under normal physiological conditions, during embryonic
development, and hepatic and cardiac injury repair, the c-Met
receptor tyrosine kinase and its high affinity ligand hepatocyte
growth factor (HGF, also known as scatter factor) mediate a variety
of cellular processes including proliferation, invasion, and angio-
genesis.1–4 In tumor cells, the activation of c-Met triggers a series
of signaling cascades that results in cell growth, invasion, prolifer-
ation, and protection from apoptosis.5,6 The over-expression of
HGF and c-Met is associated with more aggressive tumors and poor
patient prognosis.7 Tumor biopsies of many solid tumors indicate
expression of c-Met and HGF, and c-Met signaling has been noted
in a wide range of malignancies, including breast, liver, lung, colo-
rectal, gastric, bladder, prostate, head and neck, renal, pancreatic,
sarcomas, thyroid, melanoma, and hematopoietic malignancies.5,8,9
Activating mutations of c-Met have been identified in patients with
hereditary forms of cancer, which directly implicates c-Met activa-
tion in human tumorogenesis.10,11 Inhibition of c-Met kinase activ-
ity by small molecules would likely have broad therapeutic utility
due to the prevalence of Met amplification/overexpression and
mutations in a variety of human malignancies.12 Many reports
have indicated that the inhibition of c-Met kinase activity blocks
tumor cell growth and invasion in both in vitro and in vivo sys-
tems.13–17 There are many c-Met inhibitors in various stages of
development.18,19 Initial clinical results indicate that targeting
the c-Met-HGF axis is tolerated and can lead to efficacious results
either as single agent therapy or when combined with other tyro-
sine kinase inhibitors.20,21
As part of a c-Met discovery program, 2,4-diaminopyrimidines
containing a C4-aminobenzamide group and a C2-aminobenzazepi-
none group (1–3, Table 1) that demonstrated potent c-Met inhibi-
tion and selectivity over a panel of other kinases were identified.22
Analogs were evaluated for their inhibitory effects on the kinase
activity of recombinant c-Met enzyme and on c-Met autophospho-
rylation in over-expressing human gastric carcinoma (GTL-16) cells.
Counter-screening against the insulin receptor (IR) was also per-
formed.23 To assess kinase selectivity, select compounds were tested
in the Ambit Bioscience KINOMEscanTM 24
as S(90) values which reflect the fraction of kinases inhibited >90%
when screened at 1 M compound across a panel of >250 kinases.
;
these results are reported
l
The SAR of representative compounds from this series is re-
ported in Table 1. Although these compounds demonstrated potent
c-Met inhibitory activity in in vitro cellular assays, they suffered
from poor systemic exposure and low oral bioavailability in rats.
As described previously,22 optimal compounds in this series all
contained the 4-fluorobenzamide group as a means of balancing
potency and kinase selectivity. Efforts to replace this fluorobenz-
amide moiety led to compounds with decreased potency and/or
selectivity. Consequently, efforts to improve the pharmacokinetic
(PK) properties of the series focused on optimizing the C2-benzaz-
epinone portion of these analogs. Computer modeling studies
suggested that the 2-benzazepinone occupied a solvent exposed
⇑
Corresponding author. Tel.: +1 610 738 6823; fax: +1 610 344 0065.
E-mail address: kmilkiew@cephalon.com (K.L. Milkiewicz).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.09.006

K. L. Milkiewicz et al. / Bioorg. Med. Chem. 19 (2011) 6274–6284
6275
Table 1
SAR of benzazepinone analogs 1–3
O
O
Y
Y
N
Cl
H
N
N
4
2
O
HO
N
F
NH
O
N
N
H
O
R
N
H
O
Figure 2. Benazepril, a known orally bioavailable aminobenzazepinone.
a
a,b
compd
Y
R
c-Met enzyme IC50
(nM)
c-Met cell IC50
(nM)
F
c (%)
1
2
3
H
Me
H
H
30
54
99
10
44
8
1
0
subjected to the Staudinger reduction37 using polystyrene-triphen-
ylphosphine in wet THF to afford aminobenzazepinone 7 in good
yield. Attempts to directly displace the iodide of 5 with primary
and secondary amines led predominantly to elimination rather than
substitution products. After protecting benzazepinone 7 as its triflu-
oroacetamide 8a, reduction of the nitro group with H2/Pd afforded
aniline 9a. Utilizing previously reported chemistry,38 this aniline
was coupled to chloropyrimidine intermediate 10. The resulting dia-
minopyrimidine–trifluoroacetamide 11 was saponified to afford
amino-functionalized scaffold 12 (Scheme 1).
The SAR of diaminopyrimidine analogs 2, 11 and 12 is summa-
rized in Table 2 and shows that all three analogs possessed compa-
rable c-Met GTL-16 cellular inhibitory activities. Although c-Met
inhibition remained constant regardless of the benzazepinone sub-
stituent, amine analog 12 demonstrated more potent IR inhibition
and lower kinase selectivity (i.e., larger S(90) value) than parent 2.
On the other hand, amide 11 showed greater selectivity over IR and
demonstrated better kinase selectivity than either parent 2 or
amine 12. As predicted, amine 12 did achieve higher solubility than
either parent 2 or trifluoroacetamide 11. Amine 12 had a measured
Me Et 10
a
IC50 values are average of at least two separate determinations, see Experi-
mental Section for assay conditions.
b
GTL-16 cells.
c
IV dosing 1 mg/kg, 1 mL/kg administered in 3% DMSO 30% Solutol, 67% PBS, oral
dosing 5 mg/kg, 5 mL/kg administered in PEG400 in rat.
region, and thus might be amenable to modification with minimal
impact on c-Met kinase activity (Fig. 1). Additionally, metabolic ID
studies suggested that the C2-benzazepinone portion of the mole-
cule was a target for oxidation, so functionalizing this portion of
the molecule had a potential to decrease this metabolic liability.
Our initial strategy to improve oral bioavailability of these 2,4-
diaminopyrimidine analogs involved appending amine functional-
ity onto the C2-benzazepinone group of analog 2. An amine substi-
tuent in this solvent exposed region could improve solubility of
the molecule and potentially lead to more orally bioavailable com-
pounds. Amino substituted benzazepinones are well known in the
literature, aiming at a multitude of pharmaceutical targets, includ-
ing PDK1 activators,25 Nav1.7 blockers,26 thrombin inhibitors,27 Fac-
tor Xa inhibitors,28 calcium channel blockers,29 cholecystokinin
receptor ligands,30 growth hormone secretagogues,31 gastrin recep-
tor antagonists,32 endothelin-converting enzyme inhibitors,33 and
perhaps most notably, angiotensin converting enzyme inhibitors,34
for example, benazepril. (Fig. 2)
solubility of >120
lg/mL, whereas the solubility of 2 and 11 were
21 g/mL and 8
l
lg/mL, respectively. In spite of the low solubility,
trifluoroacetamide 11 demonstrated greater oral exposure than
either 12 or 2 (Table 2). As a result of these unexpected data, a ser-
ies of amidobenzazepinone analogs was synthesized in attempts to
further improve the increased exposure seen with the trifluoroac-
etamide group. The goal with this series was to maintain c-Met cel-
lular activity (cell IC50 <50 nM) and achieve oral exposures that
would facilitate evaluation of analogs in PK/PD and anti-tumor effi-
cacy models. Another objective in the program was to increase
selectivity over IR (>50-fold), and improve kinase selectivity. An
S(90) of <0.20 was desirable in order to reduce the potential for
off-target liabilities.
The 3-aminobenzazepinones were prepared by a two-step iodin-
ation/amination sequence previously reported for other benzazepi-
nones35 and is illustrated in Scheme 1. Briefly, 436 was sequentially
treated with TMEDA, TMS-I, and then solid I2 to afford iodo-interme-
diate 5. After reaction with sodium azide, azido product 6 was then
Scheme 2 outlines the general synthesis of these amido-benzaz-
epinone analogs. Acylation of amine 7 with acid chlorides afforded
the corresponding amide intermediates 8b–f, which upon hydro-
genation yielded anilines 9b–f. Finally, acid-catalyzed coupling of
these anilines with aminopyrimidine 1036 led to the targeted dia-
minopyrimidines 13–17.
A range of acylating partners were selected to yield amide, car-
bamate and urea products (13–17) and these are listed in Table 2.
Initially, racemates were evaluated to determine which com-
pounds might warrant further investigation as single enantiomers.
As delineated in Table 2, all amide/urea/carbamate analogs (11,
13–17) demonstrated comparable c-Met cell and enzyme inhibi-
tory activity as parent 2, but showed greater selectivity over IR
and other kinases in the Ambit Bioscience KINOMEscan™ panel.
The PK properties (rat) of these functionalized, racemic benzaz-
epinones were evaluated and pertinent data is listed in Table 2. Ana-
logs 11, 15, 16, and 17 all had markedly higher oral AUC values than
initial lead compound 2, leading to bioavailabilities ranging from 9–
16% (as compared to 1%). It was therefore desirable to assess the
hinge region
M160
O
H
N
gate keeper residue
N
N
O
H
O
H
N
F159
O
L157
N
K110
Cl
H
N
N
NH3+
O
F
N
salt-bridge
O
solvent
COO-
D228
N
H
activation loop
Figure 1. Depiction of 2 in the c-Met ATP-binding pocket.

6276
K. L. Milkiewicz et al. / Bioorg. Med. Chem. 19 (2011) 6274–6284
TMSI
NaN3
I
TMEDA, I2
97%
DMF
76%
NO2
NO2
N
N
H
O
H
O
5
4
PS-PPh3
TFAA
H2N
N3
TEA, CH2Cl2
85%
NO2
Wet THF
76%
N
NO2
N
O
H
O
H
7
6
isopropanol
CSA, µwave, 120°C
F
F
F
F
F
O
O
H2
25%
F
N
H
N
H
Pd-C
NO2
90%
NH2
N
H
N
Cl
O
O
H
N
8a
9a
Cl
N
F
NH
O
F
F
O
NH
Cl
N
F
N
H
NaOH
10
N
N
F
NH
O
N
H
O
H
THF//H2O
97%
NH
11
Cl
N
H2N
N
N
F
NH
O
N
H
O
H
NH
12
Scheme 1. Appendage of exocyclic amine on benzazepinone and synthesis of diaminopyrimidine analogs.
properties of the individual enantiomers associated with these
racemates.
The R and S enantiomers of compound 16 were chosen for ad-
vanced studies due to the racemate’s superior PK properties over
other analogs (Table 2). The underlying reasons for the superior
oral exposure (rat) of R-16 over 2 are not clear (Table 2). Both ana-
logs share similarly low pH 7.4 solubilities (26 vs 21 lg/mL,
respectively), and comparably modest rat liver microsomal stabil-
Resolution of racemates 11, 15–17 was accomplished by super-
critical fluid chromatography of either the final targets or interme-
diate 7. In the latter case, both R-7 and S-7 were individually
carried on to R-12 and S-12, and ultimately converted to analogs
R- and S-11, 15–17, respectively (Scheme 3). To determine abso-
lute stereochemistry, a small molecule crystal structure of an acyl
analog of 12 was solved. This analog was synthesized from the first
eluting peak in the SFC separation of intermediate 7. This allowed
us to assign absolute stereochemistry to all compounds synthe-
sized from both the first and second eluting peaks from the sepa-
ration of 7. The enantiomeric purities of all final compounds
were then assessed by analytical chiral SFC.
Although the individual enantiomers 11, 15–17, had compara-
ble c-Met cellular and enzymatic inhibitory activities, there were
marked differences in kinase selectivity for R- and S-analogs (Table
3). In most cases, (compound 11 being the exception), the S-enan-
tiomer was more selective than the R-enantiomer. For c-Met ki-
nase, this region apparently has similar interactions with both
enantiomers of 15–17, whereas for other kinases, this region is
more sensitive to the enantiotopic disposition of the amide, urea,
or carbamate benzazepinone appendage. In general, these structur-
ally related analogs shared similar kinase inhibitory profiles, lar-
gely targeting the same kinases (see Supplementary data for a
comparison of enantiotopic profiles).
ity (t1/2 = 21 vs 15 min).
The PK profiles of the single enantiomers were found to be iden-
tical. Interestingly, the bioavailability and AUC for the free base of
racemate R/S-16 (as well as each enantiomer, R-16 and S-16) were
greater than those for the corresponding trifluoroacetate salt of
R/S-16 (Table 4).
Because both R and S-16 had reasonable oral bioavailability,
both compounds were taken forward to PK-PD studies. A five day
PK/PD study was conducted with R and S-16 (10 and 30 mg/kg
po, qd) to determine the plasma and tumor levels needed to inhibit
c-Met phosphorylation in GTL-16 tumor xenografts (Figs. 3 and 4).
Even though administration of either R-16 or S-16 led to com-
parable plasma and tumor exposures, dosing R-16 at 10 and
30 mg/kg, po, resulted in a greater PD response than dosing S-16
(63–95% vs 53–77% c-Met inhibition at the 24 h time point). Fur-
thermore, whereas c-Met inhibition remained relatively constant
from 2–24 h post dosing of R-16, c-Met inhibition was more vari-
able over the same period and less dose dependent upon treatment
with S-16. At this time, reasons for this behavior are not evident
and further studies are required to explain these observations.

K. L. Milkiewicz et al. / Bioorg. Med. Chem. 19 (2011) 6274–6284
6277
Table 2
SAR and PK properties of functionalized benzazepinones
Cl
N
R
N
N
F
NH
O
N
H
O
H
N
compd
Ra
c-Met enzyme IC50 (nM)
c-Met cell IC50 (nM)b
IR IC50
(nM)
S(90)c
IV AUC
Oral AUC
Vd
(L/kg)
CL
F (%)
(mg h/mL)d
(mg h/mL)e
(mL/min/kg)
2
H
54
13
17
14
22
27
10
16
12
11
18
22
153
473
71
370
3211
3111
0.29
0.23
0.36
0.22
0.16
0.14
1057
1311
787
825
654
46
0.5
1.2
4.6
3.0
2.0
1.3
16
16
38
32
33
20
1
9
11
12
13
14
15
CF3CONH–
NH2
Me2NCH2CONH–
MeCONH–
MeOCH2CONH–
606
NDf
61
48
497
NDf
1
2
12
846
O
16
17
18
28
16
20
1729
886
0.16
0.21
3425
1201
2631
987
0.8
1.0
6
15
16
N
N
H
MeOCONH–
15
a
b
c
d
e
f
Functionalized compounds 11, 13–17 were evaluated as mono-trifluoroacetic acid salts.
GTL-16 cells.
Kinase selectivity was determined using the Ambit Bioscience KINOMEscanTM technology.
1 mg/kg, 1 mL/kg IV administered in 3% DMSO 30% Solutol, 67% PBS in rats.
5 mg/kg, 5 mL/kg oral administered in 100% PEG400 in rats.
ND = not determined—oral levels were too low to calculate.
O
R
RCOCl
H2
H2N
N
H
N+O
N+O
O
N
N
Pd-C
TEA, CH2Cl2
20-90%
O
H
O
H
O
7
8b-f
Cl
N
Cl
N
F
NH
O
O
O
Cl
R
N
N
R
N
H
N
H
N
N
F
NH
O
N
H
O
H
NH2
N
H
O
N
isopropanol
CSA, µwave, 120°C
9b-f
13-17
19-45%
85-97%
Scheme 2. Synthesis of amidobenzazepinones.
Based on the greater and more enduring c-Met inhibition demon-
strated by R- over S-16, the anti-tumor efficacy of R-16 was eval-
uated in a GTL-16 tumor xenograft, murine model.
ity over IR >50-fold, kinase selectivity S(90) <0.20 and oral expo-
sure that would facilitate evaluation of analogs in PK/PD and
anti-tumor efficacy models. In particular, analog R-16 had a c-
Met IC50 value of 16 nM in GTL-16 cells, 96-fold selectivity over
IR, and a kinase selectivity S(90) value of 0.19 (68/353 kinases were
Administration of R-16 at 10 and 30 mg/kg, po, bid and 100 mg/
kg po, qd resulted in 70% (p 60.001), 80% (p 60.001) and 90% (p
60.001) tumor growth inhibition, respectively. Initial tumor
growth stasis [d 3–5 (10 and 30 mg/kg) or d 3–10 (100 mg/kg)]
was followed by a short period of tumor growth followed by
growth stasis between d 12 and 15 (Fig. 5). A follow-up study in
this xenograft model confirmed this tumor growth inhibition
pattern (data not shown).
In conclusion, appending an exocyclic amide or urea functional-
ity onto a known benzazepinone 2,4-diaminopyrimidine c-Met
inhibitor led to compounds with improved selectivity and oral bio-
availability. Compounds with this functionality were identified
that met program criteria: c-Met cellular activity <50 nM, selectiv-
inhibited greater than 90% at 1 lM, see Supplementary data for a
complete list). Administration of R-16 at 10 and 30 mg/kg, po,
bid and 100 mg/kg po, qd, resulted in tumor growth inhibition, tu-
mor growth stasis, and partial regressions in the c-Met dependent
GTL-16 human gastric carcinoma xenograft model. Interestingly,
while c-Met in vitro data and the PK data were comparable for
the enantiomers of compound 16, R-16 demonstrated a stronger
PK/PD response than its enantiomer, S-16. Efforts are underway
to further evaluate how the nature of the amide functionality teth-
ered to the benzazepinone affects the in vivo properties of this dia-
minopyrimidine series.

6278
K. L. Milkiewicz et al. / Bioorg. Med. Chem. 19 (2011) 6274–6284
SFC
O
+
H2N
H2N
H2N
O
N+
O
+ O
N+
O
N
H
N
N
N
H
O
H
O
O
O
7
S-7
R-7
O
Cl
Cl
N
R
RCOCl
N
H2N
N
H
N
H
N
F
NH
O
N
N
N
NH
O
N
H
O
H
H
O
F
NH
NH
S-12 & R-12
S & R 13-17
Scheme 3. Synthesis of R- and S-12–17.
Table 3
SAR single enantiomer analogs R- and S-11, 15–17
O
Cl
R
N
N
H
N
N
F
NH
O
N
H
H
O
NH
S(90)b
a
Compd
R
c-Met enzyme IC50 (nM)
c-Met cell IC50 (nM)
IR IC50 (nM)
R/S-11
R-11
S-11
R/S-15
R-15
S-15
R/S-16
R-16
S-16
R/S-17
R-17
S-17
CF3–
CF3–
CF3–
MeOCH2–
MeOCH2–
MeOCH2–
Pyrrolidine–
Pyrrolidine–
Pyrrolidine–
MeO–
13
26
38
27
28
37
18
24
70
28
78
24
16
12
17
22
34
26
16
26
34
20
7
473
583
945
3111
1807
2999
1729
1418
2999
886
0.23
0.14
0.24
0.14
0.21
0.09
0.16
0.18
0.09
0.21
0.27
0.11
MeO–
MeO–
273
598
12
a
GTL-16 cells.
b
Kinase selectivity was determined using the Ambit Bioscience KINOMEscanTM technology.
product was performed by adding 100
lL/well of Eu-N1 labeled
1. Experimental
PT66 antibody (PerkinElmer #AD0041; diluted 1:5000 in TBS con-
taining 1% BSA). Incubation at 37 °C then proceeded for 1 h, fol-
1.1. In vitro assays
lowed by addition of 100 lL enhancement solution (PerkinElmer
#1244-105). The plate was gently agitated and after 5–10 min,
the fluorescence of the resulting solution was measured using
the PerkinElmer EnVisionÒ 2100 or 2102 multi-label plate reader.
Data analysis was performed using ActivityBase (IDBS, Guildford,
UK). IC50 values were calculated by plotting percent inhibition ver-
sus log10 of the concentration of compound and fitting to the non-
linear regression sigmoidal dose-response (variable slope)
equation in XLFit (IDBS, Guildford, UK).
The insulin receptor enzymatic screen was similar, with an as-
say mixture consisting of 20 mM HEPES (pH 7.2), 20 lM ATP,
5 mM MnCl2 and 0.1% BSA. Enzyme (20 ng/mL bIRCD) was added
and the reaction was allowed to proceed at room temperature
for 20 min. Detection of the phosphorylated product was per-
c-Met activity assay. Example compounds were tested for their
ability to inhibit the kinase activity of baculovirus-expressed c-
Met using a modification of the assay protocol reported for trkA.39
Phosphorylation of the substrate, phospholipase C-gamma (PLC-c)
generated as a fusion protein with glutathione S-transferase (GST)
as reported,40 was detected with a europium-labeled anti-phos-
photyrosine antibody and measured by time-resolved fluorescence
(TRF). Briefly, each 96-well plate was coated with 100
20 g/mL substrate (PLC- ) in Tris-buffered saline (TBS). The assay
mixture (total volume = 100 L/well) consisting of 50 mM HEPES
(pH 7.2), 50 mM NaCl, 1 M ATP (Km level), 4 mM MnCl2, 0.01% Tri-
lL/well of
l
c
l
l
tonX-100, and various concentrations of test compound (diluted in
DMSO; 2.5% DMSO final in assay) was then added to the assay
plate. The reaction was initiated by adding 10 ng/mL c-Met (cyto-
plasmic domain, in-house generated) and was allowed to proceed
at room temperature for 30 min. Detection of the phosphorylated
formed by adding 100 lL/well of Eu-N1 labeled PY100 antibody
(PerkinElmer #AD0160; diluted 1:10,000 in TBS-T containing
0.25% BSA). Incubation at 37 °C for 1 h was followed by addition

K. L. Milkiewicz et al. / Bioorg. Med. Chem. 19 (2011) 6274–6284
6279
Table 4
with deionized H2O (3ꢀ) followed by addition of FRAK lysis buffer
at 150 L/well. The plates were then mixed for about 5 min and
Comparison of the PK properties of the individual enantiomers of compound 16
l
placed in a ꢁ80 °C freezer. Analysis of the samples for phosphory-
lated c-Met was performed using high-binding Greiner-Bio-One
white assay plates (Greiner #655074) that were pre-coated with
purified goat-anti-rabbit antibody (‘Immunopure’; Pierce
#31210; diluted 1:400 in TBS) followed by rabbit anti-c-Met anti-
body (VWR #71-8000; diluted 1:700 in TBS containing 1% BSA).
The cell lysate plates were first thawed at 37 °C and spun down
at 3500 rpm for about 10–15 min. For the capture step, lysates
Compd
IV AUC Oral AUC Vd (L/kg) CL (mL/min/kg) F (%)
R-16
S-16
R/S-16
3460
3194
2331
5851
5548
4662
2631
0.6
0.6
0.8
0.8
5
5
8
6
34
35
40
15
R/S-16 (TFA salt) 3425
of 50 lL enhancement solution (Wallace #1244-105). Plates were
agitated for 10 min and the fluorescence was measured using the
PerkinElmer EnVisionÒ 2102 or 2104 multi-label plate reader.
GTL-16 cells (human gastric carcinoma cell line) were grown to
about 85% confluency by seeding cells at 40,000 cells/well in 96-
well, collagen 1-treated plates (Becton Dickinson #356407) a day
before the experiment. Cells were washed 3X with TBS followed
by addition of 100 lL/well of serum-free DMEM with 0.05% BSA
(control). Test compounds were added and the plates were incu-
bated for 2 h at 37 °C. Cells were washed at the end of incubation
were transferred to the assay plates (100 lL/well) and incubated
overnight at 4 °C. Plates were then washed 10ꢀ with deionized
H2O followed by a 2-hour incubation step with Eu-N1 labeled
PY100 antibody (PerkinElmer #AD0160; diluted 1:2000 in TBS
containing 1% BSA). After washing the plates 10ꢀ with water,
150 lL/well of enhancement solution (Wallace #1244-105) was
added and the plates were read on the PerkinElmer EnVisionÒ
2102 or 2104 multi-label plate reader.
R-16 (10 mg/kg po, qdX5d)
R-16 (30 mg/kg po, qdX5d)
10000
1000
100
IC50=26 nM
(15 ng/mL)
10
1
Plasma (ng/mL)
Tumor (ng/g)
100
80
60
*
40
***
***
***
20
**
***
***
***
0
R-16 (10 mg/kg)
R-16 (30 mg/kg)
*p≤0.05, **p≤0.01, ***p≤0.001-vehicle as compared to R-16
Figure 3. R-16 PK/PD study.

6280
K. L. Milkiewicz et al. / Bioorg. Med. Chem. 19 (2011) 6274–6284
100000
10000
1000
100
S-16 (10 mg/kg po qdX5d)
S-16 (30 mg/kg po qdX5d)
IC50=34 nM
(20 ng/mL)
10
1
Time (h)
Plasma (ng/mL)
Tumor (ng/g)
100
80
60
40
20
0
**
**
***
**
S-16(10 mg/kg)
S-16 (30 mg/kg)
**p≤0.01, ***p≤0.001-vehicle as compared to S-16
Figure 4. S-16 PK/PD study.
1.2. Pharmacokinetic screening
frozen on dry ice and stored at approximately ꢁ20 °C pending
analysis.
Three adult male Sprague–Dawley rats were used in each treat-
ment group. The rats were fasted overnight prior to oral dose
administration. Intravenous administration was via the lateral tail
vein and oral doses were administered by gavage. The compound
was administered IV in a vehicle of 3% DMSO: 30% Solutol: 67%
phosphate buffered saline. Oral dosing was in PEG400.
Plasma was prepared for high performance liquid chromatogra-
phy (HPLC)/mass spectrometric analysis according to standard
protocol following protein precipitation with acetonitrile contain-
ing an internal standard. The plasma samples were then analyzed
for both test compounds and alprenolol (internal standard) via
HPLC coupled with tandem mass spectrometry.
For blood collection, each rat (unanaesthetized) was placed in a
clear PlexiglasÒ restraining tube, and blood samples (approxi-
mately 0.25 mL) were drawn from a lateral tail vein into heparin-
ized collection tubes at predetermined sampling times (0.083,
0.25, 0.5, 1, 2, 4, and 6 h post dose). No pre-dose samples were ob-
tained. The exception to this procedure was the last sampling time
in which the animals were sacrificed by decapitation and trunk
blood was obtained rather than blood via a tail vein. The blood
samples were placed on wet ice until centrifuged to separate plas-
ma. The plasma fraction was transferred into clean dry tubes,
The plasma concentration data for all rats were entered into Ex-
cel spreadsheets in preparation for pharmacokinetic analysis. Phar-
macokinetic parameters for bortezomib were estimated for each
rat by non-compartmental analysis (Gibaldi M, Perrier D. Pharma-
cokinetics, 2nd edition, Marcel Dekker, New York, Chapter 11,
1982) of the plasma concentration versus time data using WinNon-
lin software (Professional Version 4.1, Pharsight Corporation, Palo
Alto, CA, 1997).
The maximum plasma concentration (Cmax) was the highest ob-
served plasma concentration after an oral dose; tmax was the corre-

K. L. Milkiewicz et al. / Bioorg. Med. Chem. 19 (2011) 6274–6284
6281
Vehicle (PEG 400 po, bid)
R-16 (10 mg/kg po, bid)
R-16 (30 mg/kg po, bid)
R-16(100 mg/kg po, qd)
duplicate. The level of c-Met phosphorylation was normalizedfor to-
tal c-Met content.
1250
1050
850
For anti-tumor efficacy studies, mice were administered R-16
over 3 concentrations for a two week period. Mice were weighed
and tumor volumes determined every 2–3 days. Mice were moni-
tored for signs of morbidity (behavior and body weight loss). At
the termination of the study, tumors and plasma samples were ta-
ken for PK analysis.
All reagents and solvents were obtained from commercial
sources and used as received. 1H NMR spectra were obtained on a
Bruker Avance at 400 MHz in the solvent indicated with tetrameth-
ylsilaneas an internalstandard. AnalyticalHPLC was run using a Zor-
650
450
bax Eclipse XDB-C8 3.5
l
m 4.6 ꢀ 75 mm column eluting with a
250
0
2
4
6
8
10
12
14
16
mixture of acetonitrile and water containing 0.1% trifluoroacetic
acid with a 5 min gradient of 10–100%. LCMS results were obtained
on either of two instruments. A Waters Acquity Ultra Performance
Days
LC with a Waters Aquity UPLC BEH C18 1.7
was paired with a Micromass-ZQ 2000 quadrupole mass spectrom-
eter with electrospray ionization. Alternatively, an Agilent 1100 ser-
ies HPLC with a Zorbax Eclipse XDB-C8 3.5
was paired with a Bruker Esquire 3000 mass spectrometer with elec-
trospray ionization. Preparative HPLC was performed on a Gilson
l
m 2.1 ꢀ 50 mm column
Vehicle vs.
Vehicle vs.
Vehicle vs.
Day of Dosing R-16-10 mg/kg bid R-16-30 mg/kg bid R-16-100 mg/kg qd
1
3
5
ns
ns
ns
ns
ns
p<0.05
p<0.05
p<0.001
p<0.0001
p<0.0001
p<0.0001
l
m 2.1 ꢀ 30 mm column
p<0.05
p<0.01
p<0.05
p<0.05
p<0.001
p<0.05
p<0.01
p<0.01
p<0.001
p<0.001
8
10
12
15
HPLC using a Phenomenex Gemini NX 5
column with UV detection. Supercritical Fluid Chromatography
m),
l
m C18, 21.2 ꢀ 100 mm
was performed on a AS-H Chiralpak column (10 ꢀ 250 mm, 5
l
with a flow rate of 6 ml/min (35% MeOH, 0.1% DEA) at a pressure
of 120 bar and a temperature of 35 °C. Automated column chroma-
tography was performed on a CombiFlash Companion (Teledyne
Isco Inc.). Melting points were taken on a Mel-Temp apparatus and
are uncorrected.
Figure 5. Anti-tumor efficacy of R-16.
sponding time when Cmax was observed. The terminal rate constant
for elimination from plasma (b) was estimated by linear regression
of the terminal portion of the semi-logarithmic plasma concentra-
tion versus time curve. The apparent terminal half-life (t1/2) was
calculated as 0.693 divided by b. The area under the plasma con-
centration versus time curve from time zero to the time of the last
measurable concentration (AUC0–t) after a single dose was deter-
mined by the linear trapezoidal rule. The area from zero to infinity
(AUC0–1) was calculated as the sum of AUC0–t and the area extrap-
olated from the last measurable concentration to infinity (Clast/b).
Concentrations pre-dose were all assumed to be zero for the pur-
pose of calculation of the AUC. Oral bioavailability was determined
by dividing the dose normalized oral AUC0–1 by the AUC0–1 from
IV dosing and multiplying by 100 to express the ratio as a percent.
Racemic samples were separated into the individual enantio-
mers using supercritical fluid chromatography on a chiralcel OJ-H
column (250 ꢀ 4.6 mm, 5
lm) with 40% methanol. Spectral data
for the single enantiomers were identical to that of the racemate.
1.3.1. 3-Iodo-5,5-dimethyl-8-nitro-1,3,4,5-tetrahydro-1-
benzazepin-2-one (5)
5,5-Dimethyl-8-nitro-1,3,4,5-tetrahydro-benzo[b]azepin-2-one
(4) (4.00 g, 17 mmol) in Methylene chloride (54 mL) was cooled to
0 °C and treated with N,N,N0,N0-tetramethylethylenediamine
(7.73 mL, 51.2 mmol) then treated dropwise with iodotrimethylsi-
lane (7.29 mL, 51.2 mmol). The mixture was stirred at 0 °C for
60 min after which solid Iodine (6.50 g, 25.6 mmol) was added in
one portion and the mixture stirred at 0 °C for 60 min. The reaction
was quenched with 10% Na2S2O3 and filtered to recover a beige so-
lid (2.63 g) The organic layer was diluted with EtOAc and washed
with Na2S2O3. The organic layer was dried over MgSO4, filtered,
and concentrated to a brown solid (1.80 g). The two solids were
combined (5.43 g, 88%) LCMS 361 (M+H). The resulting solid was
directly taken forward to the next step without purification.
1.3. Pharmacodynamic and anti-tumor efficacy studies
Compounds meeting the proposed selectivity, pharmacokinetic
properties, and bioactivity in the previously mentioned assays
underwent an initial scale-up for in vivo evaluation of anti-tumor
activity. For the pharmacodynamic and anti-tumor efficacy studies,
female athymic nude mice were injected with 1 ꢀ 106 GTL-16 hu-
man gastric carcinoma cells. Upon xenografts reaching a mean vol-
ume of 140 mm3 mice were randomized into the appropriate
treatment groups (n = 3 PK/PD study or n = 10 anti-tumor study).
For the PK/PD studies mice were dosed for 5 d and were sacrificed
at 2, 6, 10, and 24 h. Tumor lysates were analyzed for the inhibition
of c-Met autophosphorylation or for determination of tumor com-
pound levels by mass spectrometry along with the corresponding
plasma and tissue samples. A commercially available ELISA (Bio-
source, Camarillo, CA) was used to evaluate the effects of c-Met
inhibitors on c-Met phosphorylation. Tumors were homogenized
in lysis buffer then incubated in a 96 well plate coated with anti-c-
Met for 2 h. Plates were washed and then incubated with a phos-
pho-c-Met antibody (Tyr1230/34/35) or total c-Met antibody for
1 h. Plates were washed and incubated with a anti-rabbit IgG-HRP
antibody for 30 min. Tetramethylbenzidine was added to the wells
and phosphorylation was quantitated by reading the absorbance
on a microplate reader at 450 nm. Each sample was conducted in
1.3.2. 3-Azido-5,5-dimethyl-8-nitro-1,3,4,5-tetrahydro-1-
benzazepin-2-one (6)
3-Iodo-5,5-dimethyl-8-nitro-1,3,4,5-tetrahydro-1-benzazepin-
2-one (5) (5.37 g, 14.9 mmol), sodium azide (6.49 g, 100 mmol), and
N,N-dimethylformamide (100 mL) were stirred at rt for 2 h. The
reaction mixture was diluted with H2O and the resulting beige ppt
was filtered and taken to the next step without purification.
1.3.3. 3-Amino-5,5-dimethyl-8-nitro-1,3,4,5-tetrahydro-1
benzazepin-2-one (7)
3-Azido-5,5-dimethyl-8-nitro-1,3,4,5-tetrahydro-1-benzazepin
-2-one (6) (3.10 g, 10.3 mmol), triphenylphosphine resin (1.0 mmol/
g loading; 12 g, 11.55 mmol), tetrahydrofuran (120 mL) and water
(0.9 mL) were combined and stirred at rt overnight. The reaction
mixture was filtered and the resin was washed with MeOH. The

6282
K. L. Milkiewicz et al. / Bioorg. Med. Chem. 19 (2011) 6274–6284
resulting solution was concentrated under reduced pressure and the
resulting brown solid was triturated with CH2Cl2 to a beige solid.
(HPLC purity >98%) 1H NMR (DMSO-d6, 400 MHz) d 10.33 (br s,
1H), 8.00 (dd, J = 8.6 Hz, 1.7 Hz, 1H), 7.81 (d, J = 1.7 Hz, 1H), 7.66
(d, J = 8.6 Hz, 1H), 4.81 (dd, J = 7.5 Hz, 7.5 Hz, 1H), 2.71–2.66 (m,
1H), 2.61–2.55 (m, 1H), 1.39 (s, 3H), 1.35 (s, 3 H).
solid (66 mg, 97%). LCMS 498.22 (M+H), HPLC purity 94%, 1H
NMR (CDCl3, 400 MHz) d 8.90 (br s, 1H), 8.05 (s, 1H), 7.93 (br s,
1H), 7.63 (br s, 1H), 7.44–7.39 (m, 2H), 7.35–7.31 (m, 2H), 7.28–
7.26 (m, 2H), 7.20 (d, J = 7.5 Hz, 1H), 6.90 (d, J = 8.2 Hz, 1H), 6.71
(br s, 1H), 3.40–3.37 (m, 1H), 2.93 (d, J = 4.7 Hz, 3H), 2.31–2.27
(m, 1H), 1.99–1.96 (m, 2H), 1.88–1.85 (m, 1H), 1.38 (s, 3H), 1.28
(s, 3H).
1.3.4. N-(5,5-Dimethyl-8-nitro-2-oxo-2,3,4,5-tetrahydro-1H-1-
benzazepin-3-yl)-2,2,2-trifluoro-acetamide (8a)
1.3.8. 2-{5-Chloro-2-[3-(2-dimethylamino-acetylamino)-5, 5-
dimethyl-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepin-8-ylami
no]-pyrimidin-4-ylamino}-3-fluoro-N-methyl-benzamide (13)
2-Dimethylamino-N-(5,5-dimethyl-8-nitro-2-oxo-2,3,4,5-tetra-
hydro-1H-1-benzazepin-3-yl)-acetamide (8b), dimethylamino-
acetic acid (0.124 g, 1.20 mmol), EDCI (0.461 g, 2.41 mmol), HOBT
(0.276 g, 2.05 mmol) and DMF(1.00 mL, 12.9 mmol) were stirred
at rt for 30 min, followed by the addition of TEA (0.235 mL,
1.68 mmol) and 3-amino-5,5-dimethyl-8-nitro-1,3,4,5-tetrahy-
dro-1-benzazepin-2-one 7 (0.300 g, 1.20 mmol). The reaction mix-
ture was stirred at rt for 3 h, diluted with ether and washed with
water. The organic layer was dried over MgSO4, filtered, and con-
centrated under reduced pressure. Title compound was isolated
as a white solid (80 mg, 20%).
Into a 1-neck round-bottom flask was added 3-amino-5,5-di-
methyl-8-nitro-1,3,4,5-tetrahydro-1-benzazepin-2-one (7) (4.10 g,
16.4 mmol), methylene chloride (200 mL), pyridine (3.99 mL,
49.3 mmol), and trifluoroacetic anhydride (3.48 mL, 24.7 mmol).
The reaction mixture was stirred at rt for 1 h. HPLC indicated com-
plete conversion. The reaction mixture was washed with 10% citric
acid to remove pyridine and the organic layer was dried over
MgSO4, filtered, and concentrated under reduced pressure. Product
isolated as an off-white solid. 1H NMR (DMSO-d6, 400 MHz) d 10.38
(br s, 1H), 9.69 (d, J = 7.88 Hz, 1H), 8.01 (dd, J = 8.66 Hz, 2.47 Hz,
1H), 7.87 (d, J = 2.47 Hz, 1H), 7.71 (d, J = 8.66 Hs, 1H), 4.35–4.28
(m, 1H), 2.30–2.27 (m, 2H), 1.47 (s, 3H), 1.35 (s, 3H).
1.3.5. N-(8-Amino-5,5-dimethyl-2-oxo-2,3,4,5-tetrahydro-1H-1-
benzazepin-3-yl)-2,2,2-trifluoro-acetamide (9a)
1.3.9. N-(8-Amino-5,5-dimethyl-2-oxo-2,3,4,5-tetrahydro-1H-1-
benzazepin-3-yl)-2-dimethylamino-acetamide (9b)
N-(5,5-Dimethyl-8-nitro-2-oxo-2,3,4,5-tetrahydro-1H-1-benza
zepin-3-yl)-2,2,2-trifluoro-acetamide (8a) (2.80 g, 8.1 mol), 10%
palladium on carbon (50% Wet), 0.546 g, 0.25 mmol), and Metha-
nol (7 mL) were combined in a Parr flask. The reaction mixture
was shaken under an atmosphere of hydrogen (40 psi) for
90 min. The resulting mixture was filtered through Celite and the
filtrate was concentrated to afford desired product as a white foam.
LCMS 316.15 (M+H), H NMR (CDCl3) d 8.22, (s, 1H), 7.59 d,
J = 6.1 Hz, 1H), 7.21 (d, J =8.5 Hz, 1H), 6.56 (dd, J = 1.0 Hz, 8.5 Hz,
1H), 6.31 (s, 1H), 4.58–4.52 (m, 1H), 3.75 (s, 2H), 2.64–2.59 (m,
1H), 1.92 (t, J = 12.1 Hz, 1H), 1.44 (s, 3H), 1.35 (s, 3H).
Title compound was prepared from 2-(2,5-dichloro-pyrimidin-
4-ylamino)-3-fluoro-N-methyl-ben zamide (10) in an analogous
manner to 11. Title compound was isolated as a white solid.
(55 mg, 45%). LCMS 585.30 (M+H), 1H NMR (DMSO-d6, 400 MHz)
d 9.67 (br s, 2H), 9.42 (s, 1H), 9.35 (s, 1H), 8.86 (d, J = 7.8 Hz, 1H),
8.50 (d, J = 4.9 Hz, 1H), 8.19 (s, 1H), 7.49–7.46 (m, 2H), 7.36–7.31
(m, 2H), 7.21 (s, 1H), 7.11 (d, J = 8.7 Hz, 1H), 3.93 (s, 2H), 2.78 (s,
6H), 2.74 (d, J = 4.1 Hz, 6H), 2.22–2.18 (m, 1H), 1.94–1.79 (m,
2H), 1.35 (s, 3H), 1.25 (s, 3H).
1.3.11. N-(5,5-Dimethyl-8-nitro-2-oxo-2,3,4,5-tetrahydro-1H-1-
benzazepin-3-yl)-acetamide (8c)
1.3.6. 2-{5-Chloro-2-[5,5-dimethyl-2-oxo-3-(2,2,2-trifluoro-
acetylamino)-2,3,4,5-tetrahydro-1H-1-benzazepin-8-ylamino]-
pyrimidin-4-ylamino}-3-fluoro-N-methyl-benzamide (11)
Into a 10 ml sealed tube was added N-(8-amino-5,5-dimethyl-2-
oxo-2,3,4,5-tetrahydro-1H-1-benzazepin-3-yl)-2,2,2-trifluoro-
acetamide (9a) (72 mg, 0.228 mmol), 2-(2,5-dichloro-pyrimidin-4
-ylamino)-3-fluoro-N-methyl-benzamide (10) (72 mg, 0.228 mmol),
10-camphorsulfonic acid (5.3 mg, 0.023 mmol) and isopropyl alcohol
(2 mL). The reaction was heated under microwave radiation
(300 watts) at 120 °C for 30 min. The title compound was isolated
as a white solid after reverse phase HPLC and lyophilization (73 mg,
25%). HPLC purity 99%, LCMS 594.24 (M+H), 1H NMR (DMSO-d6,
400 MHz) d 9.73 (s, 1H), 9.60 (d, J = 7.8 Hz, 1H), 9.43 (s, 1H), 9.37 (s,
1H), 8.50 (d, J = 4.5 Hz, 1H), 8.19 (s, 1H), 7.48–7.44 (m, 2H), 7.35–
7.30 (m, 2H), 7.18 (s, 1H), 7.10 (d, J = 8.9 Hz, 1H), 4.27–4.20 (m, 1H),
2.73 (d, J = 4.2 Hz, 3H), 2.18–2.12 (m, 2H), 1.35 (s, 3H), 1.26 (s, 3H).
Title compound was prepared from 3-amino-5,5-dimethyl-8-
nitro-1,3,4,5-tetrahydro-1-benzazepin-2-one (7) and acetic anhy-
dride in an analogous manner to 8a. Title compound was isolated
as a white solid. (420 mg, 90%), HPLC 99% purity, H NMR (DMSO-
d6) 10.21 (s, 1H), 8.14 (d, J = 7.8 Hz, 1H), 7.99 (d, J = 8.6 Hz, 1H),
7.84 (s, 1H), 7.69 (d, J = 8.7 Hz, 1H), 4.28–4.21 (m, 1H), 2.23–2.17
(m, 1H), 2.02–1.95 (m, 1H), 1.81 (s, 3H), 1.45 (s, 3H), 1.32 (s, 3H).
1.3.12. N-(8-Amino-5,5-dimethyl-2-oxo-2,3,4,5-tetrahydro-1H-
1-benzazepin-3-yl)-acetamide (9c)
Title compound was prepared from N-(5,5-dimethyl-8-nitro-2-
oxo-2,3,4,5-tetrahydro-1H-1-benzazepin-3-yl)-acetamide in an
analogous manner to 9a. (260 mg, 97%). HPLC purity = 90%, LCMS
(262 (M+H). 1H NMR (DMSO-d6, 400 MHz) d 9.59 (s, 1H), 7.98 (d,
J = 8.0 Hz, 1H), 7.01 (d, J = 8.4 Hz, 1H), 6.38 (d, J = 8.3 Hz, 1H), 6.25
(s, 1H), 5.32 (br s, 2H), 4.26–4.22 (m, 1H), 2.06–2.00 (m, 1H), 1.80–
1.77 (m, 1H), 1.77 (s, 3H), 1.29 (s, 3H), 1.21 (s, 3H).
1.3.7. 2-[2-(3-Amino-5,5-dimethyl-2-oxo-2,3,4,5-tetrahydro-1H-
1-benzazepin-8-ylamino)-5-chloro-pyrimidin-4-ylamino]-3-flu
oro-N-methyl-benzamide (12)
1.3.13. 2-[2-(3-Acetylamino-5,5-dimethyl-2-oxo-2,3,4,5-tetrahy
dro-1H-1-benzazepin-8-ylamino)-5-chloro-pyrimidin-4-ylamin
o]-3-fluoro-N-methyl-benzamide (14)
2-{5-Chloro-2-[5,5-dimethyl-2-oxo-3-(2,2,2-trifluoro-acetyl-
amino)-2,3,4,5-tetrahydro-1H-1-benzazepin-8-ylamino]-pyrim-
idin-4-ylamino}-3-fluoro-N-methyl-benzamide (11) (80 mg, 0.13
mmol) was dissolved in tetrahydrofuran (12 mL). 1.00 M of sodium
hydroxide in water (2.4 mL) was added, and the reaction mixture
was stirred at room temperature for 6 h, at which point HPLC indi-
cated complete consumption of starting material. The reaction
mixture was diluted with H2O and extracted with CH2Cl2. The or-
ganic layer was dried over magnesium sulfate, filtered, and con-
centrated under reduced pressure to afford product as a white
Title compound was prepared from N-(5,5-dimethyl-8-nitro-2-
oxo-2,3,4,5-tetrahydro-1H-1-benzazepin-3-yl)-acetamide and 2-
(2,5-dichloro-pyrimidin-4-ylamino)-3-fluoro-N-methyl-benzam-
ide (10) in an analogous manner to 11. (62 mg, 43%). HPLC pur-
ity = 99%, LCMS 524 (M+H), 1H NMR (DMSO-d6, 400 MHz) d 9.53
(s, 1H), 9.39 (s, 1H), 9.35 (s, 1H), 8.51 (d, J = 4.6 Hz, 1H), 8.19 (s,
1H), 8.04 (d, J = 8.0 Hz, 1H), 7.49–7.45 (m, 2H), 7.33–7.26 (m,
2H), 7.15 (s, 1H), 7.08 (d, J = 9.0 Hz, 1H), 4.22–4.17 (m, 1H), 2.73

K. L. Milkiewicz et al. / Bioorg. Med. Chem. 19 (2011) 6274–6284
6283
(d, J = 4.4 Hz, 3H), 2.08–2.06 (m, 1H), 1.87–1.84 (m, 1H), 1.84 (s,
3H), 1.32 (s, 3H), 1.23 (s, 3H).
zazepin-3-yl)-amide (9e) and 2-(2,5-dichloro-pyrimidin-4-ylami-
no)-3-fluoro-N-N-methyl-benzamide (10) in an analogous
manner to 11. Title compound was isolated as a white solid
(29 mg, 22%), HPLC purity = 94%, LCMS 597 (M+H), 1H NMR
(DMSO-d6, 400 MHz) d 9.47 (s, 1H), 9.40 (s, 1H), 9.33 (s, 1H), 8.54
(d, 1H), 8.19 (s, 1H), 7.52–7.42 (m, 2H), 7.38–7.25 (m, 2H), 7.15–
7.07 (m, 2H), 5.92 (m, 1H), 4.09 (m, 1H), 3.22–3.18 (m, 4H), 2.73
(d, J = 3.5 Hz, 3H), 1.81–1.77 (m, 4H), 1.32 (s, 3H), 1.23 (s, 3H).
1.3.14. N-(5,5-Dimethyl-8-nitro-2-oxo-2,3,4,5-tetrahydro-1H-1-
benzazepin-3-yl)-2-methoxy-acetamide (8d)
Title compound was prepared from 3-amino-5,5-dimethyl-8-ni-
tro-1,3,4,5-tetrahydro-1-benzazepin-2-one (7) and methoxyacetyl
chloride in an analogous manner to 8a. (338 mg, 66%). HPLC pur-
ity = 99%, LCMS 250 (M+H), 1H NMR (DMSO-d6, 400 MHz) d 10.33
(s, 1H), 8.00 (dd, J = 8.8 Hz, 2.0 Hz, 1H), 7.85 (d, J = 2.0 Hz, 1H), 7.82
(d, J = 7.8 Hz, 1H), 7.71 (d, J = 8.8 Hz, 1H), 4.30–4.27 (m, 1H), 3.80
(s, 2H), 3.31 (s, 3H), 2.27–2.24 (M, 1H), 2.10 (t, J = 13.4 Hz, 1H),
1.45 (s, 3H), 1.32 (s, 3H).
1.3.20. (5,5-Dimethyl-8-nitro-2-oxo-2,3,4,5-tetrahydro-1H-1-
benzazepin-3-yl)-carbamic acid methyl ester (8f)
Title compound was prepared from 3-amino-5,5-dimethyl-8-
nitro-1,3,4,5-tetrahydro-1-benzazepin-2-one (7) and methyl chlo-
roformate in an analogous manner to 8a. Title compound is an
off white solid. (94 mg, 38%) LCMS 308 (M+H).
1.3.15. N-(8-Amino-5,5-dimethyl-2-oxo-2,3,4,5-tetrahydro-1H-
1-benzazepin-3-yl)-2-methoxy-acetamide (9d)
Title compound was prepared from N-(5,5-dimethyl-8-nitro-2-
oxo-2,3,4,5-tetrahydro-1H-1-benzazepin-3-yl)-2-methoxy-acet-
amide (8d) in an analogous manner to 9a. (white foam, 170 mg,
HPLC purity = 99%) LCMS 292 (M+H), 1H NMR (CDCl3, 400 MHz) d
7.42 (d, J = 7.3 Hz, 1H), 7.30 (s, 1H), 7.20 (d, J = 8.4 Hz, 1H), 6.38
(s, 1H), 4.66–4.59 (m, 1H), 3.93–3.87 (m, 2H), 3.69 (br s, 2H),
3.43 (s, 3H), 2.56–2.51 (m, 1H), 1.91–1.85 (m, 1H), 1.42 (s, 3H),
1.35 (s, 3H),
1.3.21. (8-Amino-5,5-dimethyl-2-oxo-2,3,4,5-tetrahydro-1H-1-
benzazepin-3-yl)-carbamic acid methyl ester (9f)
Title compound was prepared from (5,5-dimethyl-8-nitro-2-
oxo-2,3,4,5-tetrahydro-1H-1-benzazepin-3-yl)-carbamic
acid
methyl ester in an analogous manner to 9a. Title compound was
isolated as a yellow solid (80 mg, 94%), LCMS 278 (M+H).
1.3.22. {8-[5-Chloro-4-(2-fluoro-6-methylcarbamoyl-phenylami
no)-pyrimidin-2-ylamino]-5,5-dimethyl-2-oxo-2,3,4,5-tetrahy
dro-1H-1-benzazepin-3-yl}-carbamic acid methyl ester (17)
Title compound was prepared from (8-amino-5,5-dimethyl-2-
oxo-2,3,4,5-tetrahydro-1H-1-benzazepin-3-yl)-carbamic acid met
hyl ester (9f) and 2-(2,5-dichloro-pyrimidin-4-ylamino)-3-fluoro-
N-methyl-benzamide (10) in an analogous manner to 11. Title com-
pound was isolated as a pale yellow solid (30 mg, 19%) LCMS 558
(M+H), H NMR (DMSO-d6) 9.51 (s, 1H), 9.39 (s, 1H), 9.34 (s, 1H),
8.51 (d, 1H), 8.19 (s, 1H), 7.49–7.46 (m, 2H), 7.39–7.25 (m, 3H),
7.15 (s, 1H), 7.09 (m, 1H), 3.93–3.90 (m, 1H), 3.50 (s, 3H), 2.74 (d,
J = 3.8 Hz, 3H), 2.09–2.06 (m, 1H), 1.95–1.89 (m, 1H), 1.32 (s, 3H),
1.22 (s, 3H).
1.3.16. 2-{5-Chloro-2-[3-(2-methoxy-acetylamino)-5,5-dimeth
yl-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepin-8-ylamino]-pyri
midin-4-ylamino}-3-fluoro-N-methyl-benzamide (15)
Title compound was prepared from N-(8-amino-5,5-dimethyl-
2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepin-3-yl)-2-methoxy-acet-
amide and 2-(2,5-dichloro-pyrimidin-4-ylamino)-3-fluoro-N-
methyl-benzamide (10) in an analogous manner to 11. Title com-
pound was isolated as a white solid (40 mg, 38%) HPLC pur-
ity = 99%, LCMS = 572 (M+H), 1H NMR (DMSO-d6, 400 MHz) d
9.72 (s, 1H), 9.40 (s, 1H), 9.34 (s, 1H), 8.51 (m, 1H), 8.18 (s, 1H),
7.70 (d, J = 7.8 Hz, 1H), 7.49–7.44 (m, 2H), 7.31–7.27 (m, 2H),
7.16 (s, 1H), 7.10 (d, 1H), 4.23–4.21 (m, 1H), 3.82 (s, 2H), 3.33 (s,
3H), 2.73 (d, J = 4.8 Hz, 3H), 2.22–2.18 (m, 1H), 1.92–1.88 (m,
1H), 1.33 (s, 3H), 1.24 (s, 3H).
Acknowledgments
1.3.17. Pyrrolidine-1-carboxylic acid (5,5-dimethyl-8-nitro-2-
oxo-2,3,4,5-tetrahydro-1H-1-benzazepin-3-yl)-amide (8e)
Title compound was prepared from 3-amino-5,5-dimethyl-8-
nitro-1,3,4,5-tetrahydro-1-benzazepin-2-one (7) and pyrrolidine-
1-carbonyl chloride in an analogous manner to 8a. Title compound
was isolated as a pale yellow solid (256 mg, 92%). LCMS 347 (M+H).
HPLC purity 99%.
The authors would like to thank Mark Olsen and Emily Kord-
witz for chiral separation of the racemates, and Rebecca Brown
for bioanalysis.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.09.006.
1.3.18. Pyrrolidine-1-carboxylic acid (8-amino-5,5-dimethyl-2-
oxo-2,3,4,5-tetrahydro-1H-1-benzazepin-3-yl)-amide (9e)
Title compoundwas prepared frompyrrolidine-1-carboxylic acid
(5,5-dimethyl-8-nitro-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepin-
3-yl)-amide (8e) in an analogous manner to 9a. Title compound was
isolated as a white solid, (140 mg, 85% yield). LCMS 317 (M+H), 1H
NMR (CDCl3, 400 MHz) d :7.52 (s, 1H), 7.17 (d, J = 8.3 Hz, 1H), 6.50
(d, J = 8.3 Hz, 1H), 6.25 (s, 1H), 5.27 (d, J = 6.6 Hz, 1H), 4.54–4.51
(m, 1H), 3.70 (s, 2H), 3.36–3.34 (m, 4H), 2.55–2.50 (m, 1H), 1.91–
1.87 (m, 6H), 1.42 (s, 3H), 1.32 (s, 3H).
References and notes
1. Nakamure, T.; Mizuno, S.; Matsumoto, K.; Sawa, Y.; Matsuda, H.; Nakamura, T.
J. Clin. Invest. 2000, 106, 1511.
2. Comoglio, P. M.; Boccaccio, C. Semin. Cancer Biol. 2001, 11, 153.
3. Boccoccio, C.; Comoglio, P. M. Nat. Rev. Cancer 2006, 6, 637.
4. Huh, C. G.; Factor, V. M.; Sanchez, A.; Uchida, K.; Conner, E. A.; Thorgeirsson, S.
S. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 4477.
5. Birchmeier, C.; Birchmeier, W.; Gherardi, E.; VandeWoude, G. F. Nat. Rev. Mol.
Cell Biol. 2003, 4, 915.
6. Liu, X.; Yao, W.; Newton, R. C.; Scherle, P. A. Expert Opin. Investig. Drugs 2008,
17, 997.
7. Boccaccio, C.; Comoglio, P. M. Nat. Rev. Cancer 2006, 6, 637.
8. Trusolino, L.; Comoglio, P. M. Nat. Rev. Cancer 2002, 2, 289.
9. Peruzzi, B.; Bottaor, D. P. Clin. Cancer Res. 2006, 12, 3657.
10. Schmidt, L.; Duh, F. M.; Chen, F., et al Nat. Genet. 1997, 16, 68.
11. Jeffers, M.; Schmidt, L.; Nakaigawa, N., et al Proc. Natl. Acad. Sci. U.S.A. 1997, 94,
11445.
1.3.19. Pyrrolidine-1-carboxylic acid {8-[5-chloro-4-(2-fluoro-6-
methylcarbamoyl-phenylamino)-pyrimidin-2-ylamino]-5,5-di
methyl-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepin-3-yl}-amide
(16)
Title compound was prepared from pyrrolidine-1-carboxylic
acid (8-amino-5,5-dimethyl-2-oxo-2,3,4,5-tetrahydro-1H-1-ben-
12. Peruzzi, B.; Bottaro, D. P. Clin. Cancer Res. 2006, 12, 3657.

6284
K. L. Milkiewicz et al. / Bioorg. Med. Chem. 19 (2011) 6274–6284
13. Wang, X.; Le, P.; Liang, C.; Chan, J.; Kiewlich, D.; Miller, T.; Harris, D.; Sun, L.;
Rice, A.; Vasile, S.; Black, R. A.; Howlett, A. R.; Patel, N.; McMahon, G.; Lipson, K.
E. Mol. Cancer Ther. 2003, 2, 1085.
14. Sattler, M.; Pride, Y. B.; Ma, P.; Gramlich, J. L.; Chu, S. C.; Quinnan, L. A.;
Shirazian, S.; Liang, C.; Podar, K.; Christensen, J. M.; Salgia, R. Cancer Res. 2003,
63, 5462.
15. Christensen, J. G.; Schreck, R.; Burrows, J.; Kuruganti, P.; Chan, E.; Le, P.; Chen,
J.; Wang, X.; Ruslim, L.; Blake, R.; Lipson, K. E.; Ramphal, J.; Do, S.; Cui, J. J.;
Cherrington, J. M.; Mendel, D. B. Cancer Res. 2003, 63, 7345.
16. Hov, H.; Holt, R. U.; Ro, T. B.; Fagerli, U.-M.; Hjorth-Hansen, H.; Baykov, V.;
Christensen, J. G.; Waage, A.; Sundan, A.; Borset, M. Clin. Cancer Res. 2004, 10,
6686.
17. Zou, H. Y.; Li, Q.; Lee, J. H.; Arango, M. E.; McDonnell, S. R.; Yamazaki, S.;
Koudriakova, T. B.; Alton, G.; Cui, J. J.; Kung, P.-P.; Nambu, M. D.; Los, G.;
Bender, S. L.; Mroczkowski, B.; Christensen, J. G. Cancer Res. 2007, 67, 4408.
18. Underiner, T. L.; Herbertz, T.; Miknyoczki, S. J. Anti-Cancer Agents Med. Chem.
2010, 10, 7.
25. Wei, L.; Gao, X.; Warne, R.; Hao, X.; Bussiere, D.; Gu, X.; Uno, T.; Liu, Y. Bioorg.
Med. Chem. Lett. 2010, 20, 3891.
26. Hoyt, S. B.; London, C.; Ok, H.; Gonzalez, E.; Duffy, J. L.; Abbadie, C.; Dean, B.;
Felix, J. P.; Garcia, M. L.; Jochnowitz, N., et al Bioorg. Med. Chem. Lett. 2007, 17,
6172.
27. Ho, J. Z.; Gibson, T. S.; Semple, J. E. Bioorg. Med. Chem. Lett. 2002, 12, 743.
28. Tamura, S. Y.; Goldman, E. A.; Bergum, P. W.; Semple, J. E. Bioorg. Med. Chem.
Lett. 1999, 9, 2573.
29. Kimball, S. D.; Floyd, D. M.; Das, J.; Hunt, J. T.; Krapcho, J.; Rovnyak, G.; Duff, K.
J.; Lee, V. G.; Moquin, R. V. J. Med. Chem. 1992, 35, 780.
30. Lowe, J. A., III; Hageman, D. L.; Drozda, S. E.; McLean, S.; Bryce, D. K.; Crawford,
R. T.; Zorn, S.; Morrone, J.; Bordner, J. J. Med. Chem. 1994, 37, 3789.
31. Schoen, W. R.; Pisano, J. M.; Prendergast, K.; Wyvratt, M. J., Jr.; Fisher, M. H.;
Cheng, K.; Chan, W. W.-S.; Butler, B.; Smith, R. G.; Ball, R. G. J. Med. Chem. 1994,
37, 897.
32. Kawanishi, Y.; Ishihara, S.; Tsushima, T.; Seno, K.; Hagishita, S.; Ishikawa, M.;
Ishihara, Y. Bioorg. Med. Chem. 1997, 5, 1411.
19. Porter, J. Expert Opin. Ther. Pat. 2010, 20, 159.
33. Wallace, E. M.; Moliterni, J. A.; Moskal, M. A.; Neubert, A. D.; Marcopulos, N.;
Stamford, L. B.; Trapani, A. J.; Savage, P.; Chou, M.; Jeng, A. Y. J. Med. Chem. 1998,
41, 1513.
34. Watthey, J. W.; Stanton, J. L.; Desai, M.; Babiarz, J. E.; Finn, B. M. J. Med. Chem.
1985, 28, 1511.
20. Stabile, L. P.; Rothstein, M. E.; Keohavong, P.; Lenzner, D.; Land, S. R.; Gaither-
Davis, A. L.; Kim, K. J.; Kaminski, N.; Siegfried, J. M. Cancers 2010, 2, 2153.
21. Zhang, Y.; Staal, B.; Essenburg, C.; Su, Y.; Kang, L.; West, R.; Kaufman, D.;
DeKoning, T.; Eagleson, B.; Buchanan, S. G.; Vande Woude, G. F. Cancer Res.
2010, 70, 6880.
35. Zheng, N.; Armstrong, J. D.; Eng, K. K.; Keller, J.; Liu, T.; Lynch, R. J.; Hartner, F.
22. Weinberg, L. R.; Albom, M. S.; Angeles, T. S.; Husten, J.; Lisko, J. G.; McHugh, R.
J.; Milkiewicz, K. L.; Murthy, S.; Ott, G. R.; Theroff, J. P.; Tripathy, R.; Underiner,
T. L.; Zificsak, C. A.; Dorsey, B. D. Bioorg. Med. Chem. Lett. 2011, 21, 164.
23. As a key regulator of glucose metabolism, selectivity against IR was desired to
be >10-fold in the enzymatic assays.
24. Fabian, M. A.; Biggs, W. H., III; Treiber, D. K.; Atteridge, C. R.; Azimioara, M. D.;
Benedetti, M. G.; Carter, T. A.; Ciceri, P.; Edeen, P. T.; Floyd, M.; Ford, J. M.;
Galvin, M.; Gerlach, J. L.; Grotzfeld, R. M.; Herrgard, S.; Insko, D. E.; Insko, M. A.;
Lai, A. G.; Lelias, J.-M.; Mehta, S. A.; Milanov, Z. V.; Velasco, A. M.; Wodicka, L.
M.; Patel, H. K.; Zarrinkar, P. P.; Lockhart, D. J. Nat. Biotechnol. 2005, 23, 329.
W.; Volante, R. P. Tetrahedron: Asymmetry 2003, 14, 3435–3446.
22
36. Synthesis described in Ref.
(Weinberg, et al.)
37. Staudinger, H.; Meyer, J. Helv. Chim. Acta 1919, 2, 635.
22
38. Synthesis described in Ref.
(Weinberg, et al.)
39. Angeles, T. S.; Steffler, C.; Bartlett, B. A.; Hudkins, R. L.; Stephens, R. M.; Kaplan,
D. R.; Dionne, C. A. Anal. Biochem. 1996, 236, 49.
40. Rotin, D.; Margolis, B.; Mohammadi, M.; Daly, R. J.; Daum, G.; Li, N.; Fischer, E.
H.; Burgess, W. H.; Ullrich, A.; Schlessinger, J. EMBO J. 1992, 11, 559.