Synthesis and biological evaluation of novel 6,11-dihydro-5H-benzo[e]pyrimido- [5,4-b][1,4]diazepine derivatives as potential c-Met inhibitors

Article abstract of DOI:10.1016/j.ejmech.2017.08.060

Over expression of c-Met tyrosine kinase is known to promote tumorigenesis and metastasis, as well as to cause therapeutic resistance. Herein a series of novel 6,11-dihydro-5H-benzo[e]pyrimido[5,4-b][1,4]diazepine derivatives were designed, synthesised and evaluated for their c-Met kinase inhibition. Compounds 17e, 17f, 18a, and 18b were further examined for their anti-proliferative activities against four typical cancer cell lines (PC-3, Panc-1, HepG2, and Caki-1). The promising compound 17f was identified as a multi-target receptor tyrosine kinase inhibitor, which also displayed favourable pharmacokinetic properties in rats, had an acceptable safety profile in preclinical studies, and significant anti-tumour activity in the Caki-1 tumour xenograft model.

Full text of DOI:10.1016/j.ejmech.2017.08.060

Accepted Manuscript
Synthesis and biological evaluation of novel 6,11-dihydro-5H-benzo[e]pyrimido-
[5,4-b][1,4]diazepine derivatives as potential c-Met inhibitors
Daowei Huang, Lei Huang, Qingwei Zhang, Jianqi Li
PII:
S0223-5234(17)30672-4
10.1016/j.ejmech.2017.08.060
EJMECH 9704
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 14 April 2017
Revised Date: 26 August 2017
Accepted Date: 28 August 2017
Please cite this article as: D. Huang, L. Huang, Q. Zhang, J. Li, Synthesis and biological evaluation of
novel 6,11-dihydro-5H-benzo[e]pyrimido- [5,4-b][1,4]diazepine derivatives as potential c-Met inhibitors,
European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.08.060.
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ACCEPTED MANUSCRIPT
Graphical Abstract
A series of novel 6,11-dihydro-5H-benzo[e]pyrimido[5,4-b][1,4]diazepine
derivatives were designed, synthesised and evaluated for their c-Met kinase inhibition.
The promising compound 17f displayed favourable pharmacokinetic properties, an
acceptable safety profile, and significant anti-tumour activity in the Caki-1 tumour
xenograft model.

ACCEPTED MANUSCRIPT
Synthesis and biological evaluation of novel 6,11-dihydro-5H-benzo[e]pyrimido-
[5,4-b][1,4]diazepine derivatives as potential c-Met inhibitors
Daowei Huang,1,2 Lei Huang,1,2 Qingwei Zhang,1,2 Jianqi Li1,2,*
1Novel Technology Center of Pharmaceutical Chemistry, Shanghai Institute of
Pharmaceutical Industry, Shanghai, 201203, China;
2Shanghai Engineering Research Center of Pharmaceutical Process, Shanghai,
201203,China;
*Corresponding author: Tel.: +86-021-20572128. E-mail: lijianqb@126.com
Keywords c-Met; tyrosine kinase; diazepine derivatives; synthesis; antitumour
Abstract Over expression of c-Met tyrosine kinase is known to promote
tumorigenesis and metastasis, as well as to cause therapeutic resistance. Herein a
series of novel 6,11-dihydro-5H-benzo[e]pyrimido[5,4-b][1,4]diazepine derivatives
were designed, synthesised and evaluated for their c-Met kinase inhibition.
Compounds 17e, 17f, 18a, and 18b were further examined for their anti-proliferative
activities against four typical cancer cell lines (PC-3, Panc-1, HepG2, and Caki-1).
The promising compound 17f was identified as a multi-target receptor tyrosine kinase
inhibitor, which also displayed favourable pharmacokinetic properties in rats, had an
acceptable safety profile in preclinical studies, and significant anti-tumour activity in
the Caki-1 tumour xenograft model.
1. Introduction
Mesenchymal epithelial transition factor (c-Met), known as a hepatocyte growth
factor receptor, was discovered in 1984 as an oncogenic fusion protein, translocation
promoter region (TPR-MET) [1]. Physiologically, c-Met mediates a diverse array of
biological processes that lead to cell proliferation, migration, and invasion [2-5]. As
such, normal c-Met signalling plays an important role during embryogenesis, wound
healing, and liver regeneration in adulthood, however, the dysregulation of c-Met has
been associated with a wide range of solid tumours, including thyroid cancer, lung
cancer, gastric cancer, colorectal cancer, pancreatic cancer, prostate cancers, renal
cancer, etc. [6-7]. Accordingly, compelling evidence has linked c-Met over-activation
*Corresponding author. Tel: +86 21 20572128 (J.Q. Li)
E-mail address: lijianqb@126.com (J.Q. Li)
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to mediating intrinsic, or acquired, resistance to targeted therapies, and to the poor
prognosis in clinical studies [8]. As a result, c-Met has become an attractive target for
cancer therapy.
A variety of approaches have been used to target the c-Met signaling pathway
including antibodies [9,10], antagonists [11,12], and small-molecule kinase inhibitors
[13-17]. All reported small-molecule inhibitors have basically been categorised into
two classes based on their structures and binding modes in the c-Met kinase domain.
Type-I inhibitors (Crizotinib, JNJ-38877605, INCB-28060, MK-2461) bind in a
U-shaped conformation to the ATP-binding site at the entrance of the kinase pocket
and wrap around Met1211, while type-
inhibitors (Cabozantinib, Foretinib,
MGCD-265, Golvatinib) bind to c-Met with an extended conformation that stretches
from the ATP-binding site (hinge region) to the deep hydrophobic Ile1145 pocket near
the C-helix region. Among those representative compounds, shown in Figure 1,
Crizotinib and Cabozantinib were approved by the FDA in August, 2011 for the
treatment of non-small cell lung cancer (NSCLC) patients and in November, 2012 for
the treatment of patients with progressive metastatic medullary thyroid cancer,
respectively [18-19]. Furthermore, varieties of analogues have been reported recently
[20-29].
(Fig. 1. should be inserted here)
In general, type-I inhibitors showed high selectivity to c-Met kinase, however,
many projects involving these inhibitors in discovery, development or clinical were
stopped due to toxicity or unknown reasons. While type- inhibitors can inhibit other
kinases, display high potency and low toxicity, and endure drug-resistance.
Structurally, type- inhibitors are composed of three moieties (Figure 2): a
pharmacophore quinolone group (moiety A), a cyclopropyl-malonamide group
(moiety B), and a linking phenoxy group (moiety C). An early discovery found that
nitrogen in moiety A could form hydrogen bond with Met1160, so compounds could
bind to the kinase to inhibit its activity. In addition, the carbonyl oxygens in moiety B
engaged in hydrogen bonding with Lys1110 and Asp1222, respectively. The moiety C
is capable of participating in π-π interaction with Phe1223 to increase the affinity with
the c-Met kinase [30,31]. The binding mode of Cabozantinib with c-Met kinase is
shown in Figure 3. Generally, the modification of Cabozantinib mainly focused on
moiety A and moiety B, where moiety B was designed as a linear, or cyclic, five-atom
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chain bearing at least one amide bond between the two benzene groups, which is
known as “five-atom regulation” [32-35]. Additionally, the replacement of moiety A
by various N-containing heterocycles, such as substituted quinolone, thienopyridine,
pyrrolopyridine, aminopyridine, etc. has been investigated [36-39].
(Fig. 2. should be inserted here)
(Fig. 3. should be inserted here)
As shown in Figure 3, the ATP binding pocket is spacious enough to bind larger
groups, however, moiety A is reported as being limited to monocyclic, or bicyclic,
ring groups, and there are still no examples of the use of polycyclic ring to explore the
binding mode to the pocket residues. It is worth mentioning that several
6,11-dihydro-5H-benzo[e]pyrimido-[5,4-b][1,4]diazepine compounds, which have
pyridine nitrogen with potential to bind to the ATP binding pocket, have been reported
to exhibit excellent anti-tumour activities [40]. This inspired us to introduce tricyclic
groups, such as 6,11-dihydro-5H-benzo[e]pyrimido-[5,4-b][1,4]diazepine and its
derivatives, as a moiety A scaffold to investigate the requirements of the ATP binding
pocket. Therefore, twenty-two new compounds have been designed, synthesised and
evaluated (with regard to their biological activities) to study the structure-activity
relationships, and to develop novel c-Met inhibitors with good potential for use as
drug with low toxicity. The design strategy is shown in Figure 4.
(Fig. 4. should be inserted here)
The target compounds synthesised were evaluated for c-Met kinase inhibition,
and compounds 17e, 17f, 18a, and 18b were further tested for anti-proliferative
activities against four cancer cell lines (PC-3, Panc-1, HepG2, and Caki-1).
Furthermore, later research indicated that compound 17f exhibited favourable
pharmacokinetic properties, an acceptable safety profile in preliminary safety studies
(MTD and hERG), and potent activity in a c-Met-amplified (Caki-1) subcutaneous
tumour xenograft model.
2. Chemistry
A series of diazepine derivatives and their intermediates were synthesised
according to the pathways described in Schemes 1 to 6.
As depicted in Scheme 1, intermediate 9, obtained from the condensation of
commercially available cyclopropane-1,1-dicarboxylic acid with 4-fluoroaniline, was
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treated with (un) substituted 4-aminophenol to yield the key intermediate 10a-b. Then,
the substituted 5-nitro-4,6-dichloropyrimidine was reduced to yield 11a-c, which
reacted with 10a to give the desired intermediates 12a-c.
The target compounds 13a-c were synthesised by reaction of 12a-c with
substituted salicylic acid [41].
(Scheme 1. should be inserted here)
As shown in Scheme 2, the substituted 5-nitro-4,6-dichloropyrimidine, or
pyridine, reacted with substituted aniline, or phenol, to yield the intermediate 14a-i,
which reacted with 10a-b and was then reduced to yield 16a-i. Finally, the target
compounds 17a-i were prepared by cyclisation of 16a-i [42].
(Scheme 2. should be inserted here)
The target compounds 18a-d were prepared from starting material 17e-f and
17h-i under microwave conditions as outlined in Scheme 3.
(Scheme 3. should be inserted here)
Scheme 4 depicts the preparation of compounds 21a-b. Treatment of
commercially
available
4,6-dichloro-5-nitropyrimidine
with
methyl
6-amino-2,3-dimethoxybenzoate provided corresponding intermediate 19, which then
reacted with 10a-b to generate intermediates 20a-b. Intramolecular cyclisation of
20a-b furnished the target compounds 21a-b.
(Scheme 4. should be inserted here)
The general strategy to synthesise compounds 24a-b is outlined in Scheme 5
using the same method as was used to prepare 21a-b in Scheme 4, albeit modified so
that the inorganic base KF.2H2O was used instead of K2CO3 [43].
(Scheme 5. should be inserted here)
As shown in Scheme 6, compounds 25a-b were synthesised from 17e-f via
methylation.
(Scheme 6. should be inserted here)
The chemical structures of the target compounds were confirmed by MS, HRMS,
1H-NMR, and 13C-NMR spectroscopy.
3. Results and discussion
3.1. In vitro cytotoxicity and structure–activity relationships
The 22 prepared target compounds (13a-c, 17a-i, 18a-d, 21a-b, 24a-b, and
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25a-b) were evaluated for their in vitro inhibitory activity against c-Met kinase using
homogenous time-resolved fluorescence (HTRF) assay, and then the compounds with
potent c-Met enzyme activities were further tested against 4 c-Met-addicted cancer
cell lines including PC-3, Panc-1, HepG2, and Caki-1 by the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide
(MTT)
assay.
Cabozantinib was taken as a positive control. The results were expressed as
half-maximal inhibitory concentration (IC50) values and are presented in Table 1, as
mean values of experiments performed in triplicate.
As illustrated from Tables 1 to 4, six compounds (17d-g, and 18a-b) exhibited
promising activity against c-Met kinase with IC50 values ranging from 2322 nM to
24.4 nM, which suggested that the inhibition of c-Met kinase may be a main
mechanism underpinning the anti-tumour activity of the prepared compounds.
However, other compounds showed weak inhibitory activity with their values of IC50
being above 10 µM. Notably, compound 17f demonstrated the best activity with an
IC50 value of 24.4 nM, which is approximately equal to that of Cabozantinib (24.4 nM
versus 5.3 nM).
(Table 1. should be inserted here)
(Table 2. should be inserted here)
(Table 3. should be inserted here)
(Table 4. should be inserted here)
Four preferred compounds (17e-f, and 18a-b), mentioned above, were further
assayed for cancer cell activity (Table 5): all four compounds showed moderate, to
significant, anti-proliferative activities with IC50 values ranging from 0.748 to 7.125
µM, which is superior to Cabozantinib against all four tested cancer cells, which
suggested
that
the
introduction
of
6,11-dihydro-5H-benzo[e]pyrimido[5,4-b][1,4]diazepine group, as moiety A,
maintained the potent cytotoxic activity. In particular, the most prominent compound
(17f) displayed three to seven times more activity than that of Cabozantinib.
(Table 5. should be inserted here)
The biological activity data indicated that the tricyclic scaffold affected the
activity to a significant extent. For example, the introduction of an NH group into
position “Y” could make a better contribution to the inhibitory activity than an oxygen
(O) group (18a-b > 13c, 17e > 17g) (Table 1, 2), when “Z” was substituted by
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nitrogen, the activity increased (18a versus 18c, 18b versus 18d, 17e versus 17h, 17f
versus 17i) (Table 1, 2). The activities of compounds 17d (R1 = H) were significantly
higher than that of compounds 17b (R1 = CH3) (Table 1, 2). Besides, the inhibitory
activity increased significantly when groups R2 and R3 were substituted with a
methoxy group (17g > 17c, 17e > 17d) (Table 2), and this could be validated by the
subsequent binding mode analysis.
Compounds 17e-f with their CH2-NH group exhibited strong inhibitory activity
against c-Met kinase with IC50 values of 29.3 nM and 24.4 nM, respectively; however,
the activity declined in compounds 18a-b due to conformational restriction with the
CH=N group that affected the affinity to the kinase (Table 1, 2). In addition, the
inhibition against c-Met kinase vanished when NCH3 and CONH groups were
incorporated in 25a-b and 21a-b (Table 3, 4). In the end, modifications at position
“X” (whether fluorine substituted or not) could not affect the potency to any
significant extent, such as in compound 18a versus 18b, and 17e versus 17f (Table 1,
2).
3.2. Enzymatic selectivity assays
To examine whether, or not, 17f is a selective c-Met inhibitor, this compound
was screened against five other tyrosine kinases. Compared with its high potency
against c-Met, 17f also exhibited high inhibitory effects against VEGFR-2, EGFR,
RET, c-Kit and FLT-3. These results suggested that compound 17f is a promising
multi-target inhibitor of tyrosine kinase. The results are given in Table 6.
(Table 6. should be inserted here)
3.3. Acute toxicity test
To explore the safety profile of these 6,11-dihydro-5H-benzo[e]pyrimido-
[5,4-b][1,4]diazepine derivatives, the acute toxicity of 17f was measured in ICR mice
at a single dose of 420, 520, 620, 720, 820 and 920 mg/kg or vehicle control (n = 10)
by oral administration. Animals were monitored for 14 days. Treatment with 17f at
720 mg/kg killed 20 % of the mice, whereas the lower doses (420, 520, and 620
mg/kg) caused no abnormalities or animal deaths throughout the experiment. These
results suggest that administration of 17f at, or below, 620 mg/kg (PO) may be safe
for mice. The results are given in Table 7.
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(Table 7. should be inserted here)
3.4. Test of activity on hERG potassium currents
The activity of 17f on hERG potassium currents was evaluated, since activation
of hERG channels may induce cardiotoxicity. A compound solution was administrated,
from low- to high-concentrations, and each concentration was tested on at least three
cells. The IC50 value of 17f was greater than 40 µM, which demonstrated that
compound 17f did not activate hERG channels and had no cardiotoxicity. The results
are given in Table 8.
(Table 8. should be inserted here)
3.5. Pharmacokinetics study
The pharmacokinetics (PK) of 17f were measured in male SD rats. After
administration of 20 mg/kg (PO) 17f to SD rats, a Cmax of 1934.2 ng/mL was obtained
at 1.67 h. Compound 17f was well cleared (CLz/F = 2.93 L/h/kg) in rats. The
elimination half-life of 17f after administration was 2.36 h. Compound 17f was also
well distributed (Vz/F = 4.52 L/kg) and had a moderate oral bioavailability (F = 39 %)
in rats. The results are given in Table 9.
(Table 9. should be inserted here)
3.6. In vivo pharmacology
The in vivo activity of 17f was evaluated in a c-Met amplified (Caki-1)
subcutaneous tumour xenograft. Observations were made following oral
administration of 17f to the mouse model once daily for 21d: these demonstrated that
17f strongly inhibited tumour growth with inhibitory levels of 51.1 %, 58.87 %, and
64.54 % compared to untreated controls at respective dosages of 20, 40, and 80 mg/kg.
Moreover, 17f, at all dosage levels, was well tolerated by mice. The results are given
in Figure 6.
(Fig.5. should be inserted here)
4. Conclusions
In summary, 22 novel diazepine, oxazepines and pyridodiazepine derivatives
possessing 6,11-dihydro-5H-benzo[e]pyrimido-[5,4-b][1,4] diazepine derivatives as
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moiety A were designed, synthesised and evaluated to elucidate their c-Met kinase
inhibitory activities. Among these compounds, 17f exhibited excellent activity with an
IC50 value of 24 nM. Moreover, it displayed better anti-proliferative activities against
PC-3, HepG2, Caki-1, and Panc-1, and was five, to seven, times more active than
Cabozantinib. Preliminary SARs revealed that the C=N, N-CH3, and CONH groups
had a negative effect on c-Met kinase inhibitory activity compared with that of C-N
group, and the activity increased significantly when X, R2, and R3 were substituted
with fluorine and a methoxy group, respectively. Binding model analysis of 17f
suggested that the diazepine group could form two hydrogen-bonding interactions in
the ATP-binding site and another hydrogen-bonding interaction in moiety C, which
could be the reason for the aforementioned activity.
In addition, the primary PK profiles showed that compound 17f, with its
diazepine group, had favourable physico-chemical properties. There was no toxicity
observed in acute toxicity tests following administration in mice and in hERG
potassium channel testing. Meanwhile, 17f displayed significant anti-tumour activity
in a c-Met-amplified (Caki-1) subcutaneous tumor xenograft model. The additional
biological evaluation of 17f is currently in progress, and the novel scaffold that binds
to the ATP pocket in 17f provided a new starting point for further optimisation studies.
5. Experimental
5.1. Chemistry
Unless otherwise noted, all reagents and solvents employed were purchased
commercially and used as received. All reactions were conducted in microwave vials
or flasks containing Tefloncoated magnetic stirrer. Microwave irradiation experiments
were performed in a CEM-Discover@LabMate mono-mode microwave apparatus.
Reactions’ time and purity of the products were monitored by TLC on FLUKA
silica gel aluminum cards (0.2 mm thickness) with fluorescent indicator 254 nm.
Column chromatography was run on silica gel (200-300 mesh) from Qingdao Ocean
Chemicals (Qingdao, Shandong, China). All melting points were obtained on a
BüchiMelting Point B-540 apparatus (BüchiLabortechnik, Flawil, Switzerland) and
were uncorrected. Mass spectra (MS) were taken in ESI mode on Agilent 1100
1
LC-MS (Agilent, Palo Alto, CA, USA). H-NMR and 13C-NMR spectra were
generated on Bruker AM-400 spectrometers (Bruker Bioscience, Billerica, MA, USA)
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with TMS as an internal standard. All reagents were analytical reagent (AR) and
chemical pure (CP). Biological activities evaluation were performed at the Shanghai
Sudia Co., Ltd.
5.2. Preparation of 1-((4-fluorophenyl)carbamoyl)cyclopropane carboxylic acid (9)
To a solution of cyclopropane-1,1-dicarboxylic acid (13.01 g, 0.10 mol) in THF
(120 mL), trimethylamine (10.12 g, 0.10 mol) was added drop-wise under nitrogen
atmosphere at 0 . Then, SOCl2 (9.95 g, 0.10 mol) and a solution of 4-fluoroaniline
(12.22 g, 0.11 mol) in THF (60 mL) were added drop-wise at 0 , respectively. The
reaction solution was stirred in an ice bath for 2 h and monitored by thin-layer
chromatograph (TLC). After adjusting pH to 9.0 by 1 M NaOH, the solution was
acidified by 1 M HCl to pH 5.0, and the precipitate was filtered off, washed, dried in
vacuum, yielding 9 (14.71 g) as a white solid in 65.9% yield [44]. The production was
used for the next step without further purification. MS (ESI) m/z: 224.5 [M+H]+.
1H-NMR (400MHz, DMSO-d6) δ: 13.05 (s, 1H, COOH), 10.58 (s, 1H, CONH),
7.67~7.12 (m, 4H, Ar-H), 1.47 (s, 4H, CH2CH2).
5.3. General procedure for Preparation of N-(4-fluorophenyl)-N-(4-hydroxy-3-
substitutedphenyl)cyclopropane-1,1-dicarboxamide (10a-b)
To a solution of the intermediate 9 (1.0 g, 4.48 mmol) and substituted
aminophenol (5.38 mmol) in DMF (15 mL) was added EDCI (1.03 g, 5.38 mmol).
The solution was stirred at room temperature for 3 h. Then water (50 mL) was added
to precipitate white solid, adjusting pH to 4.0~5.0 by 1 M HCl. The white solid was
filtered off, washed and dried in vacuum to afford 10a-b [45].
5.3.1. N-(4-fluorophenyl)-N-(4-hydroxyphenyl)cyclopropane-1,1-dicarboxamide (10a)
1
Yield: 87.9%; MS (ESI) m/z: 315.4 [M+H]+. H-NMR (400MHz, DMSO-d6) δ:
10.17 (s, 1H, CONH), 9.73 (s, 1H, CONH), 9.23 (s, 1H, OH), 7.83~6.68 (m, 8H,
Ar-H), 1.48 (s, 4H, CH2CH2).
5.3.2.
N-(3-fluoro-4-hydroxyphenyl)-N-(4-fluorophenyl)cyclopropane-1,1-
dicarboxamide (10b)
1
Yield: 86.5%; MS (ESI) m/z: 332.9 [M+H]+. H-NMR (400MHz, DMSO-d6) δ:
10.21 (s, 1H, CONH), 9.84 (s, 1H, CONH), 9.32 (s, 1H, OH), 7.74~6.47 (m, 7H,
Ar-H), 1.42 (s, 4H, CH2CH2).
5.4. General procedure for Preparation of 4,6-dichloro-2-substituted-5-
nitropyrimidine (11a,b)
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10% Pd/C（0.05 g）was added to the solution of substituted pyrimidine (4.48
mmol) in ethyl acetate (EA, 20 mL) in Parr hydrogenater. Then, replace the air with
nitrogen three times, and react for 4 h at room temperature in normal pressure. Filter
the solution to remove Pd/C through Celite filter agent, the filtrate was concentrated
to yield the intermediates 11a,b [46].
5.4.1. 4,6-dichloro-2-methyl-5-nitropyrimidine (11a)
Pale solid; Yield: 84.0%; MS (ESI) m/z: 177.94 [M+H]+.
5.4.2. 4,6-dichloro-2-methyl-5-nitropyrimidine (11b)
Pale solid; Yield: 86.0%; MS (ESI) m/z: 163.87 [M+H]+.
5.5. General procedure for Preparation of N-(4-((5-amino-6-chloro-2-
substitutedpyrimidin-4-yl)oxy)phenyl)-N-(4-fluorophenyl)-cyclopropane-1,1-
dicarboxamide (12a,b)
To a solution of intermediates 11a,b (0.02 mol) and 10a (6.28 g, 0.02 mol) in
DMF (15 mL) was added K2CO3 (3.04 g, 0.022 mol). The mixture then was stirred at
60 ℃ for overnight. The reaction mixture was cooled to room temperature and ice
water was added, then viscous solid was precipitated, filtered off to obtain crude
product which was purified by stirring it with petroleum ether (40 mL) for 30 min to
yield the title compound 12a,b as a light white solid.
5.5.1. N-(4-((5-amino-6-chloro-2-methylpyrimidin-4-yl)oxy)phenyl)-N-(4-
fluorophenyl)cyclopropane-1,1-dicarboxamide (12a)
1
Pale white solid; Yield: 87.0%; MS (ESI) m/z: 456.08 [M+H]+. H-NMR
(400MHz, DMSO-d6) δppm: 10.24 (s, 1H, CONH), 10.12 (s, 1H, CONH), 8.11~7.07
(m, 8H, Ar-H), 5.74 (br, 2H, NH2), 1.43 (s, 4H, CH2CH2).
5.5.2. N-(4-((5-amino-6-chloropyrimidin-4-yl)oxy)phenyl)-N-(4-fluorophenyl)
cyclopropane-1,1-dicarboxamide (12b)
1
Pale white solid; Yield: 85.0%; MS (ESI) m/z: 442.13 [M+H]+. H-NMR
(400MHz, DMSO-d6) δppm: 10.20 (s, 1H, CONH), 10.14 (s, 1H, CONH), 8.06 (s, 1H,
pyrimidine-H), 8.01~7.13 (m, 8H, Ar-H), 5.81 (br, 2H, NH2), 2.21 (s, 3H, CH3), 1.46
(s, 4H, CH2CH2).
5.6. General procedure for Preparation of Compounds 13a-c
To a mixture of intermediates 12a-c (0.50 mmol), substituted salicylaldehyde
(0.50 mmol) and MgSO4 (0.18 g, 1.5 mmol) in DMF (15 mL) was added one drop
concentrated hydrochloric acid. The mixture then was stirred at 95 ℃for overnight.
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The reaction solution was cooled to room temperature and ice water was added, solid
was precipitated, filtered off to obtain crude product which was purified by silica gel
chromatography using PE/EA (3:1) to afford the title compounds 13a-c.
5.6.1. N-(4-fluorophenyl)-N-(4-((2-methylbenzo[f]pyrimido[4,5-b][1,4]oxazepin-4-yl)
oxyphenyl)cyclopropane-1,1-dicarboxamide (13a)
Yellow solid; Yield: 38.2%; m.p.: 183-185 ; MS (ESI) m/z: 524.18 [M+H]+,
1H-NMR (400MHz, DMSO-d6) δppm: 10.11 (s, 1H, CONH), 10.07 (s, 1H, CONH),
8.72 (s, 1H, CH=N), 7.86~7.13 (m, 12H, Ar-H), 2.41 (s, 3H, CH3), 1.46 (s, 4H,
CH2CH2). 13C NMR (100MHz, DMSO-d6): δ 15.39, 25.12, 30.29, 115.76, 117.11,
121.22, 121.58, 122.32, 125.29, 131.04, 133.67, 134.47, 136.33, 146.21, 154.79,
158.16, 160.05, 162.42, 163.67, 166.13, 168.18. HRMS Calcd for C29H22FN5O4
[M+H]+, 523.1716; found, 524.1825. Purity: > 95%.
5.6.2. N-(4-(benzo[f]pyrimido[4,5-b][1,4]oxazepin-4-yloxy)phenyl)-N-(4-fluorophen-
yl)cyclopropane-1,1-dicarboxamide (13b)
Yellow solid; Yield: 38.4%; m.p.: 170-172 ℃; MS (ESI) m/z: 510.16 [M+H]+,
1H-NMR (400MHz, DMSO-d6) δppm: 10.17 (s, 1H, CONH), 10.03 (s, 1H, CONH),
8.79 (s, 1H, CH=N), 8.36 (s, 1H, pyrimidine-H), 7.91~7.10 (m, 12H, Ar-H), 1.46 (s,
4H, CH2CH2). 13C NMR (100MHz, DMSO-d6): δ 15.42, 31.43, 114.99, 119.67,
121.29, 121.63, 122.40, 126.43, 131.05, 134.76, 135.14, 136.31, 148.25, 155.73,
157.11, 159.45, 162.85, 163.60, 165.90, 168.10. HRMS Calcd for C28H20FN5O4
[M+H]+, 509.1499; found, 510.1578. Purity: > 95%.
5.6.3. N-(4-((8,9-dimethoxybenzo[f]pyrimido[4,5-b][1,4]oxazepin-4-yl)oxy)phenyl)-N
-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (13c)
Yellow solid; Yield: 39.1%; m.p.: 196-199 ℃; MS (ESI) m/z: 570.15 [M+H]+, 1H
NMR (400MHz, DMSO-d6) δppm: 10.22 (s, 1H, CONH), 10.18 (s, 1H, CONH), 8.64
(s, 1H, CH=N), 8.46 (s, 1H, pyrimidine-H), 7.87~7.25 (m, 10H, Ar-H), 3.67 (s, 6H,
2×CH3), 1.43 (s, 4H, CH2CH2). 13C NMR (100MHz, DMSO-d6): δ 15.28, 30.69,
53.14, 113.97, 116.07, 121.19, 121.46, 123.08, 128.47, 130.61, 134.77, 135.82,
136.17, 144.49, 156.70, 157.72, 159.48, 160.59, 162.45, 165.96, 167.14. HRMS
Calcd for C30H24FN5O6 [M+H]+, 569.1728; found, 570.1532. Purity: > 95%.
5.7. General procedure for Preparation of the intermediates 14a-i
A mixture of substituted pyrimidine, or pyridine (1.0 mmol), substituted phenol,
or aniline (1.2 mmol) and K2CO3 (0.42 g，3.0 mmol) in DMF (3 mL) was heated at
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60 ℃ for 8 h. Upon cooling to room temperature, the reaction mixture was diluted
with EA (15 mL), washed by 10% NaOH solution (5 mL×2) and saturated bine (5
mL×3), and then dried by anhydrous Na2SO4, filtered and concentrated to yield the
intermediates 14a-i.
5.7.1. 4-chloro-2-methyl-5-nitro-6-phenoxypyrimidine (14a)
1
Light orange solid; Yield: 97.0%; MS (ESI) m/z: 266.01 [M+H]+, H NMR
(400MHz, DMSO-d6) δppm: 7.01~6.79 (m, 5H), 2.41 (s, 3H).
5.7.2. 6-chloro-N-(4-methoxyphenyl)-2-methyl-5-nitropyrimidin-4-amine (14b)
1
Light orange solid; Yield: 98.0%; MS (ESI) m/z: 295.07 [M+H]+, H NMR
(400MHz, DMSO-d6) δppm: 9.13 (s, 1H), 7.07~6.83 (m, 4H), 3.71 (d, J = 6.8 Hz, 3H),
2.44 (s, 3H).
5.7.3. 4-chloro-5-nitro-6-phenoxypyrimidine (14c)
1
Light orange solid; Yield: 96.5%; MS (ESI) m/z: 252.03 [M+H]+, H NMR
(400MHz, DMSO-d6) δppm: 8.01 (s, 1H), 7.01~6.79 (m, 5H).
5.7.4. 6-chloro-N-(4-methoxyphenyl)-5-nitropyrimidin-4-amine (14d)
1
Light orange solid; Yield: 97.6%; MS (ESI) m/z: 281.05 [M+H]+, H NMR
(400MHz, DMSO-d6) δppm: 9.17 (s, 1H), 8.04 (s, 1H), 7.11~6.78 (m, 4H), 3.65 (d, J
= 6.4 Hz, 3H).
5.7.5. 6-chloro-N-(3,4-dimethoxyphenyl)-5-nitropyrimidin-4-amine (14e,f)
1
Light orange solid; Yield: 98.2%; MS (ESI) m/z: 311.07 [M+H]+, H NMR
(400MHz, DMSO-d6) δppm: 9.14 (s, 1H), 7.97 (s, 1H), 7.15~6.83 (m, 3H), 3.74 (d, J
= 8.6 Hz, 6H).
5.7.6. 4-chloro-6-(3,4-dimethoxyphenoxy)-5-nitropyrimidine (14g)
1
Light orange solid; Yield: 95.4%; MS (ESI) m/z: 312.04 [M+H]+, H NMR
(400MHz, DMSO-d6) δppm: 9.06 (s, 1H), 7.22~6.91 (m, 3H), 3.57 (d, J = 8.0 Hz,
6H).
5.7.7.4-chloro-N-(3,4-dimethoxyphenyl)-3-nitropyridin-2-amine (14h-i)
1
Light orange solid; Yield: 95.4%; MS (ESI) m/z: 310.06 [M+H]+, H NMR
(400MHz, DMSO-d6) δppm: 9.11 (s, 1H), 7.99 (d, J = 4.0 Hz, 1H), 7.21 (d, J = 4.6 Hz,
1H), 7.14~6.87 (m, 3H), 3.76 (d, J = 8.4 Hz, 6H).
5.8. General procedure for Preparation of the intermediates 15a-i
A mixture of 14a-i (7.55 mmol), 10a-b (6.25 mmol) and K2CO3 (2.59 g, 18.75
mmol) in DMF (30 mL) was heated at 50 ℃ for 13 h. Upon cooling to room
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temperature, the reaction mixture was added water (100 mL), extracted by EA (15
mL×3). The organic phase was combined and concentrated to get the crude product,
which was purified by silica gel chromatography using a mixture of PE/EA (2:1~1:1)
to afford the intermediates 15a-i.
5.8.1. N-(4-fluorophenyl)-N-(4-((2-methyl-5-nitro-6-phenoxypyrimidin-4-yl)oxy)
phenyl)cyclopropane-1,1-dicarboxamide (15a)
1
Orange yellow solid; Yield: 72.8%; MS (ESI) m/z: 544.17 [M+H]+, H NMR
(400MHz, DMSO-d6) δppm: 10.15 (t, J = 12.0 Hz, 2H), 7.86~7.64 (m, 4H), 7.17~7.11
(m, 4H), 7.04~6.93 (m, 5H), 2.42 (s, 3H), 1.46 (s, 4H, CH2CH2).
5.8.2. N-(4-fluorophenyl)-N-(4-((6-((4-methoxyphenyl)amino)-2-methyl-5-
nitropyrimidin-4-yl)oxy)phenyl)cyclopropane-1,1-dicarboxamide (15b)
1
Orange yellow solid; Yield: 73.4%; MS (ESI) m/z: 573.20 [M+H]+, H NMR
(400MHz, DMSO-d6) δppm: 10.11 (t, J = 8.6 Hz, 2H), 7.94 (s, 1H), 7.76~7.68 (m,
4H), 7.25~7.17 (m, 4H), 7.01~6.89 (m, 4H), 2.43 (s, 3H), 1.46 (s, 4H, CH2CH2).
5.8.3. N-(4-fluorophenyl)-N-(4-((5-nitro-6-phenoxypyrimidin-4-yl)oxy)phenyl)
cyclopsropane-1,1-dicarboxamide (15c)
1
Orange yellow solid; Yield: 74.6%; MS (ESI) m/z: 530.16 [M+H]+, H NMR
(400MHz, DMSO-d6) δppm: 10.14 (t, J = 8.8 Hz, 2H), 8.03 (s, 1H), 7.79~7.64 (m,
4H), 7.27~7.14 (m, 4H), 7.01~6.89 (m, 5H), 1.46 (s, 4H, CH2CH2).
5.8.4. N-(4-fluorophenyl)-N-(4-((6-((4-methoxyphenyl)amino)-5-nitropyrimidin-4-yl)
oxy)phenyl)cyclopropane-1,1-dicarboxamide (15d)
1
Orange yellow solid; Yield: 72.5%; MS (ESI) m/z: 559.19 [M+H]+, H NMR
(400MHz, DMSO-d6) δppm: 10.12 (t, J = 6.8 Hz, 2H), 9.04 (s, 1H), 8.12 (s, 1H),
7.71~7.62 (m, 4H), 7.24~7.11 (m, 4H), 7.01~6.84 (m, 4H), 2.41 (s, 3H), 1.46 (s, 4H,
CH2CH2).
5.8.5. N-(4-((6-((3,4-dimethoxyphenyl)amino)-5-nitropyrimidin-4-yl)oxy)phenyl)-N-(4
-fluorophenyl)cyclopropane-1,1-dicarboxamide (15e)
1
Orange yellow solid; Yield: 75.1%; MS (ESI) m/z: 589.17 [M+H]+, H NMR
(400MHz, DMSO-d6) δppm: 10.14 (t, J = 8.4 Hz, 2H), 9.01 (s, 1H), 8.07 (s, 1H),
7.75~7.61 (m, 4H), 7.27~7.13 (m, 4H), 7.02~6.93 (m, 3H), 2.44 (s, 6H), 1.47 (s, 4H,
CH2CH2).
5.8.6. N-(4-((6-((3,4-dimethoxyphenyl)amino)-5-nitropyrimidin-4-yl)oxy)-3-
fluorophenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (15f)
13

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1
Orange yellow solid; Yield: 70.8%; MS (ESI) m/z: 607.14 [M+H]+, H NMR
(400MHz, DMSO-d6) δppm: 10.13 (t, J = 8.6 Hz, 2H), 9.02 (s, 1H), 8.04 (s, 1H),
7.72~7.67 (m, 3H), 7.25~7.18 (m, 4H), 7.08~6.97 (m, 3H), 2.41 (s, 6H), 1.44 (s, 4H,
CH2CH2).
5.8.7. N-(4-((6-(3,4-dimethoxyphenoxy)-5-nitropyrimidin-4-yl)oxy)phenyl)-N-(4-
fluorophenyl)cyclopropane-1,1-dicarboxamide (15g)
1
Orange yellow solid; Yield: 79.2%; MS (ESI) m/z: 590.11 [M+H]+, H NMR
(400MHz, DMSO-d6) δppm: 10.14 (t, J = 8.4 Hz, 2H), 9.04 (s, 1H), 7.78~7.66 (m,
3H), 7.27~7.13 (m, 4H), 7.01~6.84 (m, 3H), 2.43 (s, 6H), 1.46 (s, 4H, CH2CH2).
5.8.8. N-(4-((2-((3,4-dimethoxyphenyl)amino)-3-nitropyridin-4-yl)oxy)phenyl)-N-(4-
fluorophenyl)cyclopropane-1,1-dicarboxamide (15h)
1
Orange yellow solid; Yield: 61.4%; MS (ESI) m/z: 588.22 [M+H]+, H NMR
(400MHz, DMSO-d6) δppm: 10.14 (t, J = 8.0 Hz, 2H), 9.14 (s, 1H), 7.95 (d, J = 4.6
Hz, 1H), 7.74~7.62 (m, 4H), 7.42 (d, J = 4.4 Hz, 1H), 7.29~7.15 (m, 4H), 7.03~6.81
(m, 3H), 2.46 (s, 6H), 1.44 (s, 4H, CH2CH2).
5.8.9. N-(4-((2-((3,4-dimethoxyphenyl)amino)-3-nitropyridin-4-yl)oxy)-3-fluorophenyl
)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (15i)
1
Orange yellow solid; Yield: 62.5%; MS (ESI) m/z: 606.17 [M+H]+, H NMR
(400MHz, DMSO-d6) δppm: 10.15 (t, J = 8.2 Hz, 2H), 9.05 (s, 1H), 7.88 (d, J = 5.6
Hz, 1H), 7.71~7.57 (m, 3H), 7.40 (d, J = 4.8 Hz, 1H), 7.27~7.14 (m, 4H), 7.01~6.89
(m, 3H), 2.44 (s, 6H), 1.46 (s, 4H, CH2CH2).
5.9. General procedure for Preparation of the intermediates 16a-i
10% Pd/C (0.05 g) was added to the solution of 15a-i (1.84 mmol) in EA (20 mL)
in Parr hydrogenater. Then, replace the air with nitrogen three times, and react for
overnight at room temperature in normal pressure. Filter the solution to remove Pd/C
through Celite filter agent, the filtrate was concentrated to yield the intermediates
16a-i.
5.9.1. N-(4-((5-amino-2-methyl-6-phenoxypyrimidin-4-yl)oxy)phenyl)-N-(4-
fluoroph enyl)cyclopropane-1,1-dicarboxamide (16a)
1
Pale solid; Yield: 84.2%; MS (ESI) m/z: 514.19 [M+H]+, H NMR (400MHz,
DMSO-d6) δppm: 10.17 (t, J = 8.6 Hz, 2H), 7.82~7.69 (m, 4H), 7.34~7.21 (m, 4H),
7.08~6.97 (m, 5H), 5.83 (s, 2H), 2.41 (s, 3H), 1.44 (s, 4H, CH2CH2).
5.9.2. N-(4-((5-amino-6-((4-methoxyphenyl)amino)-2-methylpyrimidin-4-yl)oxy)
14

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phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (16b)
1
Pale solid; Yield: 86.7%; MS (ESI) m/z: 543.24 [M+H]+, H NMR (400MHz,
DMSO-d6) δppm: 10.12 (t, J = 8.0 Hz, 2H), 7.95 (s, 1H), 7.77~7.64 (m, 4H),
7.26~7.14 (m, 4H), 7.04~6.84 (m, 4H), 5.81 (s, 2H), 2.41 (s, 3H), 1.43 (s, 4H,
CH2CH2).
5.9.3. N-(4-((5-amino-6-phenoxypyrimidin-4-yl)oxy)phenyl)-N-(4-fluorophenyl)
cyclopropane-1,1-dicarboxamide (16c)
1
Pale solid; Yield: 81.4%; MS (ESI) m/z: 500.14 [M+H]+, H NMR (400MHz,
DMSO-d6) δppm: 10.11 (t, J = 8.6 Hz, 2H), 8.01 (s, 1H), 7.74~7.61 (m, 4H),
7.23~7.17 (m, 4H), 7.02~6.81 (m, 5H), 5.86 (s, 2H), 1.44 (s, 4H, CH2CH2).
5.9.4. N-(4-((5-amino-6-((4-methoxyphenyl)amino)pyrimidin-4-yl)oxy)phenyl)-N-(4-
fluorophenyl)cyclopropane-1,1-dicarboxamide (16d)
1
Pale solid; Yield: 88.2%; MS (ESI) m/z: 529.20 [M+H]+, H NMR (400MHz,
DMSO-d6) δppm: 10.17 (t, J = 6.4 Hz, 2H), 9.07 (s, 1H), 8.19 (s, 1H), 7.78~7.69 (m,
4H), 7.23~7.15 (m, 4H), 7.02~6.87 (m, 4H), 5.80 (s, 2H), 2.43 (s, 3H), 1.44 (s, 4H,
CH2CH2).
5.9.5. N-(4-((5-amino-6-((3,4-dimethoxyphenyl)amino)pyrimidin-4-yl)oxy)phenyl)-N-
(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (16e)
1
Pale solid; Yield: 87.5%; MS (ESI) m/z: 559.20 [M+H]+, H NMR (400MHz,
DMSO-d6) δppm: 10.12 (t, J = 8.6 Hz, 2H), 9.03 (s, 1H), 8.05 (s, 1H), 7.74~7.62 (m,
4H), 7.28~7.19 (m, 4H), 7.06~6.87 (m, 3H), 5.74 (s, 2H), 2.46 (s, 6H), 1.43 (s, 4H,
CH2CH2).
5.9.6. N-(4-((5-amino-6-((3,4-dimethoxyphenyl)amino)pyrimidin-4-yl)oxy)-3-
fluorophenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (16f)
1
Pale solid; Yield: 87.9%; MS (ESI) m/z: 577.18 [M+H]+, H NMR (400MHz,
DMSO-d6) δppm: 10.11 (t, J = 8.4 Hz, 2H), 8.95 (s, 1H), 8.12 (s, 1H), 7.75~7.69 (m,
3H), 7.21~7.12 (m, 4H), 7.01~6.74 (m, 3H), 5.80 (s, 2H), 2.44 (s, 6H), 1.46 (s, 4H,
CH2CH2).
5.9.7. N-(4-((5-amino-6-(3,4-dimethoxyphenoxy)pyrimidin-4-yl)oxy)phenyl)-N-(4-
fluorophenyl)cyclopropane-1,1-dicarboxamide (16g)
1
Pale solid; Yield: 81.4%; MS (ESI) m/z: 560.17 [M+H]+, H NMR (400MHz,
DMSO-d6) δppm: 10.17 (t, J = 8.6 Hz, 2H), 9.01 (s, 1H), 7.71~7.67 (m, 3H),
7.29~7.15 (m, 4H), 7.07~6.86 (m, 3H), 5.86 (s, 2H), 2.47 (s, 6H), 1.42 (s, 4H,
15

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CH2CH2).
5.9.8. N-(4-((3-amino-2-(3,4-dimethoxyphenoxy)pyridin-4-yl)oxy)phenyl)-N-(4-
fluorophenyl)cyclopropane-1,1-dicarboxamide (16h)
1
Pale solid; Yield: 81.4%; MS (ESI) m/z: 558.23 [M+H]+, H NMR (400MHz,
DMSO-d6) δppm: 10.12 (t, J = 7.6 Hz, 2H), 9.17 (s, 1H), 7.96 (d, J = 4.8 Hz, 1H),
7.71~7.67 (m, 4H), 7.46 (d, J = 4.8 Hz, 1H), 7.27~7.14 (m, 4H), 7.01~6.89 (m, 3H),
5.72 (s, 2H), 2.47 (s, 6H), 1.46 (s, 4H, CH2CH2).
5.9.9. N-(4-((3-amino-2-(3,4-dimethoxyphenoxy)pyridin-4-yl)oxy)-3-fluorophenyl)-N-
(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (16i)
1
Pale solid; Yield: 78.6%; MS (ESI) m/z: 576.14 [M+H]+, H NMR (400MHz,
DMSO-d6) δppm: 10.14 (t, J = 8.8 Hz, 2H), 9.11 (s, 1H), 7.90 (d, J = 5.6 Hz, 1H),
7.74~7.59 (m, 3H), 7.41 (d, J = 4.8 Hz, 1H), 7.26~7.17 (m, 4H), 7.04~6.81 (m, 3H),
5.77 (s, 2H), 2.46 (s, 6H), 1.44 (s, 4H, CH2CH2).
5.10. General procedure for Preparation of the title compounds 17a-i
A mixture of 16a-i (7.79 mmol), (HCHO)n (0.35 g, 11.69mmol), CF3COOH
(TFA, 0.80 g, 7.01 mmol) and MgSO4 (1.88 g, 15.58 mmol) in DCM (30 mL) was
heated at 40 ℃ for 12 h. Upon cooling to room temperature, the reaction mixture
was filtered off to remove MgSO4. The filtrate was concentrated to obtain crude
product which was purified by stirred with ethanol (20 mL) at 60 ℃for 1 h to give
the title compounds 17a-i.
5.10.1. N-(4-fluorophenyl)-N-(4-((2-methyl-5,6-dihydrobenzo[f]pyrimido[4,5-b][1,4]-
oxazepin-4-yl)oxy)phenyl)cyclopropane-1,1-dicarboxamide (17a)
1
Yield: 81.4%; m.p.: 121-123 ℃; MS (ESI) m/z: 526.19 [M+H]+, H NMR
(400MHz, DMSO-d6) δppm: 10.09 (s, 1H, CONH), 10.07 (s, 1H, CONH), 7.71~7.09
(m, 12H, Ar-H), 5.88 (br, 1H, CH2NH), 4.43 (s, 2H, CH2NH), 2.20 (s, 3H, CH3), 1.46
(s, 4H, CH2CH2). 13C NMR (100MHz, DMSO-d6): δ 15.71, 25.14, 32.06, 44.37,
113.70, 116.42, 117.68, 121.65, 122.54, 124.41, 126.89, 134.11, 146.95, 149.81,
151.94, 157.79, 161.62, 162.93, 165.84, 168.71. HRMS Calcd for C29H24FN5O4
[M+H]+, 525.1807; found, 526.1905. Purity: > 95%.
5.10.2. N-(4-fluorophenyl)-N-(4-((8-methoxy-2-methyl-6,11-dihydro-5H-benzo[e]
pyrimido[5,4-b][1,4]diazepin-4-yl)oxy)phenyl)cyclopropane-1,1-dicarboxamide (17b)
Yield: 77.2%; m.p.: 120-122 ℃; MS (ESI) m/z: 555.20 [M+H]+,1H NMR
(400MHz, DMSO-d6) δppm: 10.11 (s, 1H, CONH), 10.07 (s, 1H, CONH), 9.22 (s, 1H,
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Ar-NH), 7.71~6.69 (m, 11H, Ar-H), 5.36 (br, 1H, CH2NH), 4.19 (s, 2H, Ar-CH2), 3.71
(s, 3H, OCH3), 2.22 (s, 3H, pyrimidine-CH3), 1.46 (s, 4H, CH2CH2). 13C NMR
(100MHz, DMSO-d6): δ 15.84, 24.81, 31.09, 47.73, 55.84, 109.76, 112.06, 113.12,
116.72, 117.18, 119.46, 120.37, 122.59, 130.47, 131.23, 134.11, 134.44, 149.85,
152.97, 155.74, 157.36, 160.12, 162.34, 165.86, 168.47. HRMS Calcd for
C30H27FN6O4 [M+H]+, 554.2102; found, 555.2024. Purity: > 95%.
5.10.3. N-(4-((5,6-dihydrobenzo[f]pyrimido[4,5-b][1,4]oxazepin-4-yl)oxy)phenyl)-N-(
4-fluorophenyl)cyclopropane-1,1-dicarboxamide (17c)
1
Yield: 72.6%; m.p.: 117-119 ; MS (ESI) m/z: 512.16 [M+H]+, H NMR
(400MHz, DMSO-d6) δppm: 10.12 (s, 1H, CONH), 10.04 (s, 1H, CONH), 7.79 (s, 1H,
pyrimidine-H), 7.68~7.11 (m, 12H, Ar-H), 5.74 (br, 1H, CH2NH), 4.32 (s, 2H,
CH2NH), 1.41 (s, 4H, CH2CH2). 13C NMR (100MHz, DMSO-d6): δ 15.63, 31.01,
45.26, 114.28, 117.58, 119.02, 119.31, 120.62, 122.69, 125.07, 127.96, 134.41, 148.89,
150.84, 151.94, 158.27, 162.04, 164.25, 168.44. HRMS Calcd for C28H22FN5O4
[M+H]+, 511.1767; found, 512.1683. Purity: > 95%.
5.10.4. N-(4-fluorophenyl)-N-(4-((8-methoxy-6,11-dihydro-5H-benzo[e]pyrimido[5,4-
b][1,4]diazepin-4-yl)oxy)phenyl)cyclopropane-1,1-dicarboxamide (17d)
1
Yield: 84.7%; m.p.: 116-118 ; MS (ESI) m/z: 541.21 [M+H]+, H NMR
(400MHz, DMSO-d6) δppm: 10.09 (s, 1H, CONH), 10.07 (s, 1H, CONH), 9.22 (s, 1H,
Ar-NH), 7.87~6.71 (m, 12H, Ar-H), 5.52 (s, 1H, CH2NH), 4.20 (s, 2H, CH2NH), 3.71
(s, 3H, OCH3), 1.47 (s, 4H, CH2CH2). 13C NMR (100MHz, DMSO-d6): δ 15.14, 30.19,
46.33, 54.67, 107.54, 110.86, 113.42, 115.93, 117.78, 119.05, 120.49, 122.72, 130.39,
131.08, 133.01, 134.47, 149.18, 151.37, 153.14, 155.66, 160.32, 161.44, 165.46,
168.41. HRMS Calcd for C29H25FN6O4 [M+H]+, 540.1936; found, 541.2109. Purity: >
95%.
5.10.5. N-(4-((8,9-dimethoxy-6,11-dihydro-5H-benzo[e]pyrimido[5,4-b][1,4]diazepin-
4-yl)oxy)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (17e)
1
Yield: 88.2%; m.p.: 127-130 ; MS (ESI) m/z: 571.10 [M+H]+, H NMR
(400MHz, DMSO-d6) δppm: 10.08 (s, 1H, CONH), 10.07 (s, 1H, CONH), 9.11 (s, 1H,
NH), 7.69 (s, 1H, pyrimidine-H), 7.66~6.54 (m, 10H, Ar-H), 5.50 (s, 1H, CH2NH),
4.17 (s, 2H, CH2NH), 3.97 (s, 6H, 2×CH3), 1.47 (s, 4H, CH2CH2). 13C NMR
(100MHz, DMSO-d6): δ 15.46, 31.32, 49.73, 55.52, 56.02, 104.19, 113.08, 115.01,
117.61, 119.82, 121.49, 122.40, 134.94, 135.28, 142.51, 146.48, 148.14, 149.12,
17

ACCEPTED MANUSCRIPT
150.52, 156.63, 157.08, 159.46, 168.15. HRMS Calcd for C30H27FN6O5 [M+H]+,
570.2064; found, 571.1055. Purity: > 95%.
5.10.6. N-(4-((8,9-dimethoxy-6,11-dihydro-5H-benzo[e]pyrimido[5,4-b][1,4]diazepin-
4-yl)oxy)-3-fluorophenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (17f)
1
Yield: 86.3%; m.p.: 132-135 ; MS (ESI) m/z: 589.20 [M+H]+, H NMR
(400MHz, DMSO-d6) δppm: 10.27 (s, 1H, CONH), 10.03 (s, 1H, CONH), 9.14 (s, 1H,
NH), 7.87 (s, 1H, pyrimidine-H), 7.77~6.55 (m, 9H, Ar-H), 5.57 (s, 1H, CH2NH),
4.17 (s, 2H, CH2NH), 3.71 (s, 6H, 2×CH3), 1.41 (s, 4H, CH2CH2). 13C NMR
(100MHz, DMSO-d6): δ 15.33, 31.69, 49.67, 55.52, 56.01, 104.22, 108.54, 113.05,
114.99, 116.30, 116.81, 119.86, 122.42, 123.95, 134.68, 135.17, 135.51, 142.54,
146.31, 148.13, 150.55, 152.21, 154.63, 156.12, 157.07, 159.46, 168.05. HRMS
Calcd for C30H26F2N6O5 [M+H]+, 588.1924; found, 589.2036. Purity: > 95%.
5.10.7. N-(4-((8,9-dimethoxy-5,6-dihydrobenzo[f]pyrimido[4,5-b][1,4]oxazepin-4-yl)
oxy)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (17g)
1
Yield: 77.2%; m.p.: 114-117 ; MS (ESI) m/z: 572.11 [M+H]+, H NMR
(400MHz, DMSO-d6) δppm: 10.10 (s, 1H, CONH), 10.07 (s, 1H, CONH), 7.70 (s, 1H,
pyrimidine-H), 7.65~6.87 (m, 10H, Ar-H), 6.10 (s, 1H, CH2NH), 4.42 (s, 2H,
CH2NH), 3.78 (s, 6H, 2×CH3), 1.46 (s, 4H, CH2CH2). 13C NMR (100MHz,
DMSO-d6): δ 15.03, 30.12, 45.13, 52.64, 59.46, 102.15, 113.07, 114.87, 116.92,
119.87, 120.54, 122.47, 132.19, 137.23, 144.58, 146.02, 148.39, 149.82, 150.07,
154.43, 157.28, 159.67, 168.74. HRMS Calcd for C30H26FN5O6 [M+H]+, 571.1906;
found, 572.1163. Purity: > 95%.
5.10.8. N-(4-((8,9-dimethoxy-6,11-dihydro-5H-benzo[e]pyrido[3,2-b][1,4]diazepin-4-
yl)oxy)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (17h)
Yield: 79.4%; m.p.: 129-131 ; MS (ESI) m/z: 570.21 [M+H]+,1H NMR
(400MHz, DMSO-d6) δppm: 10.11 (s, 1H, CONH), 10.02 (s, 1H, CONH), 8.54 (s, 1H,
NH), 7.66~6.56 (m, 12H, Ar-H), 5.36 (s, 1H, CH2NH), 4.16 (s, 2H, CH2NH), 3.71 (d,
13
J = 12.0 Hz, 6H, 2×CH3), 1.46 (s, 4H, CH2CH2). C NMR (100MHz, DMSO-d6): δ
15.27, 31.83, 49.24, 55.61, 58.94, 101.47, 106.67, 113.22, 115.36, 117.69, 120.80,
121.41, 126.49, 134.96, 137.25, 144.51, 146.48, 148.38, 149.95, 153.57, 156.03,
157.48, 159.21, 168.15. HRMS Calcd for C31H28FN5O5 [M+H]+, 569.2116; found,
570.2139. Purity: > 95%.
5.10.9. N-(4-((8,9-dimethoxy-6,11-dihydro-5H-benzo[e]pyrido[3,2-b][1,4]diazepin-4-
18

ACCEPTED MANUSCRIPT
yl)oxy)-3-fluorophenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (17i)
1
Yield: 75.8%; m.p.: 131-134 ; MS (ESI) m/z: 588.14 [M+H]+, H NMR
(400MHz, DMSO-d6) δppm: 10.22 (s, 1H, CONH), 10.05 (s, 1H, CONH), 8.53 (s, 1H,
NH), 7.72~6.51 (m, 11H, Ar-H), 5.41 (s, 1H, CH2NH), 4.16 (d, J = 4.0 Hz, 2H,
CH2NH), 3.71 (d, J = 8.0 Hz, 6H, 2×CH3), 1.45 (s, 4H, CH2CH2). 13C NMR (100MHz,
DMSO-d6): δ 15.12, 31.49, 47.62, 54.53, 56.06, 101.36, 103.27, 107.55, 113.26,
114.79, 116.08, 116.64, 119.81, 122.72, 126.91, 134.61, 135.52, 138.17, 143.76,
146.39, 148.03, 150.51, 152.40, 154.93, 156.72, 157.77, 163.14, 168.43. HRMS
Calcd for C30H26F2N6O5 [M+H]+, 587.2067; found, 588.1493. Purity: > 95%.
5.11. General procedure for Preparation of the title compounds 18a-b
A dried vial was charged with compounds 17e-f (3.51 mmol) and Cs2CO3 (3.43 g,
10.53 mmol) in DMF (15 mL). The resulting suspension was irradiated at 100 for 1
h. Upon cooling to room temperature, the mixture was concentrated, and the residue
was purified by silica gel chromatography using PE/EA (1:1) to afford the compounds
18a-b.
5.11.1. N-(4-((8,9-dimethoxy-11H-benzo[e]pyrimido[5,4-b][1,4]diazepin-4-yl)oxy)
phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (18a)
White solid; Yield: 34.0%; m.p.: 127-129 ; MS (ESI) m/z: 569.12 [M+H]+, 1H
NMR (400MHz, DMSO-d6) δppm: 10.17 (s, 1H, CONH), 10.11 (s, 1H, CONH), 7.75
(s, 1H), 7.62 (t, J = 6.8 Hz, 2H), 7.52 (d, J = 8.0 Hz, 2H), 7.44 (d, J = 8.0 Hz, 1H),
7.37 (s, 1H), 7.12 (t, J = 12.0 Hz, 2H), 6.85 (d, J = 8.0 Hz, 1H), 6.71 (s, 1H), 6.03 (t, J
= 4.0 Hz, 2H), 3.73 (s, 3H, CH3), 3.68 (s, 3H, CH3), 1.46 (s, 4H, CH2CH2). 13C NMR
(100MHz, DMSO-d6): δ 15.24, 31.66, 54.60, 102.27, 110.58, 114.72, 115.47, 118.33,
121.69, 122.15, 122.97, 134.25, 143.78, 145.26, 146.69, 148.42, 150.43, 154.82,
157.64, 161.76, 166.04. HRMS Calcd for C30H26F2N6O5 [M+H]+, 568.1933; found,
569.1241. Purity: > 95%.
5.11.2. N-(4-((8,9-dimethoxy-11H-benzo[e]pyrimido[5,4-b][1,4]diazepin-4-yl)oxy)-3-
fluorophenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (18b)
White solid; Yield: 32.7%; m.p.: 136-138 ; MS (ESI) m/z: 587.22 [M+H]+, 1H
NMR (400MHz, DMSO-d6) δppm: 10.28 (s, 1H, CONH), 10.02 (s, 1H, CONH), 8.17
(s, 1H), 7.76 (t, J = 8.0 Hz, 2H), 7.63 (dd, J = 8.0 Hz, 12.0 Hz, 2H), 7.54 (s, 1H), 7.37
(d, J = 8.0 Hz, 1H), 7.23~7.12 (m, 4H), 6.70 (s, 1H), 6.44 (s, 1H), 6.70 (s, 1H), 3.69
(d, J = 8.0 Hz, 6H, OCH3), 1.45 (s, 4H, CH2CH2). 13C NMR (100MHz, DMSO-d6): δ
19

ACCEPTED MANUSCRIPT
15.62, 33.21, 55.25, 102.71, 104.56, 110.71, 112.30, 114.75, 115.02, 118.22, 120.36,
121.47, 122.39, 122.90, 133.55, 134.51, 137.93, 143.73, 145.02, 149.14, 150.39,
152.57, 156.41, 159.62, 162.47, 168.17. HRMS Calcd for C30H26F2N6O5 [M+H]+,
586.1820; found, 587.2216. Purity: > 95%.
5.12. General procedure for Preparation of the title compounds 18c-d
A dried vial was charged with compounds 17h-i (3.51 mmol) and Cs2CO3 (3.43 g,
10.53 mmol) in DMF (15 mL). The resulting suspension was irradiated at 100 for 1
h. Upon cooling to room temperature, the mixture was concentrated, then the residue
was purified by silica gel chromatography using PE/EA (3:1) to afford the compounds
18c-d.
5.12.1. N-(4-((8,9-dimethoxy-11H-benzo[e]pyrido[3,2-b][1,4]diazepin-4-yl)oxy)
phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (18c)
Brown solid; Yield: 27.0%; m.p.: 134-137 ; MS (ESI) m/z: 567.98 [M+H]+, 1H
NMR (400MHz, DMSO-d6) δppm: 10.11 (s, 1H, CONH), 10.01 (s, 1H, CONH), 7.73
(s, 1H), 7.64 (t, J = 8.0 Hz, 2H), 7.56 (d, J = 8.0 Hz, 2H), 7.48 (d, J = 8.0 Hz, 1H),
7.43 (s, 1H), 7.15 (t, J = 12.0 Hz, 2H), 6.94 (d, J = 8.0 Hz, 2H), 6.70 (s, 1H), 6.26 (t, J
= 4.0 Hz, 2H), 3.72 (s, 3H, CH3), 3.67 (s, 3H, CH3), 1.45 (s, 4H, CH2CH2). 13C NMR
(100MHz, DMSO-d6): δ 15.41, 31.28, 55.64, 103.22, 110.72, 114.99, 115.41, 118.02,
120.77, 121.62, 122.31, 122.95, 134.88, 143.90, 145.07, 146.11, 150.44, 152.69,
154.43, 159.66, 163.71, 168.07. HRMS Calcd for C30H26F2N6O5 [M+H]+, 587.2067;
found, 568.1989. Purity: > 95%.
5.12.2. N-(4-((8,9-dimethoxy-11H-benzo[e]pyrido[3,2-b][1,4]diazepin-4-yl)oxy)-3-
fluorophenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (18d)
Brown solid; Yield: 24.7%; m.p.: 139-142 ; MS (ESI) m/z: 585.94 [M+H]+, 1H
NMR (400MHz, DMSO-d6) δppm: 10.29 (s, 1H, CONH), 10.04 (s, 1H, CONH), 7.76
(s, 1H), 7.63 (t, J = 8.2 Hz, 2H), 7.54 (d, J = 8.2 Hz, 2H), 7.46 (d, J = 8.2 Hz, 1H),
7.41 (s, 1H), 7.18 (t, J = 10.0 Hz, 2H), 6.94 (s, 1H), 6.74 (s, 1H), 6.52 (d, J = 4.0 Hz,
2H), 3.76 (s, 3H, CH3), 3.68 (s, 3H, CH3), 1.46 (s, 4H, CH2CH2). 13C NMR (100MHz,
DMSO-d6): δ 15.37, 31.22, 54.63, 101.29, 103.20, 104.55, 105.32, 107.81, 111.76,
114.39, 115.26, 119.01, 120.31, 121.68, 122.39, 122.62, 134.82, 140.62, 143.91,
145.24, 146.17, 150.43, 153.67, 155.02, 159.61, 165.78, 167.22. HRMS Calcd for
C30H26F2N6O5 [M+H]+, 585.1861; found, 585.9426. Purity: > 95%.
5.13.
Preparation
of
methyl
20

ACCEPTED MANUSCRIPT
6-((6-chloro-5-nitropyrimidin-4-yl)amino)-2,3-dimethoxybenzoate (19)
A mixture of 4,6-dichloro-5-nitropyrimidine (3.0 g, 15.55 mmol), methyl
6-amino-2,3-dimethoxybenzoate (3.94 g, 18.66 mmol) and K2CO3 (6.44 g, 46.65
mmol) in DMF (20 mL) was heated at 60
for 8 h. Upon cooling to room
temperature, the reaction mixture was added water (100 mL), and filtered off to get
the crude product, which was purified by silica gel chromatography using PE/EA
(10:1) to afford the intermediate 19 as a yellow solid (4.98 g, 87%). MS (ESI) m/z:
1
368.94 [M+H]+, H NMR (400MHz, DMSO-d6) δppm: 10.91 (s, 1H), 8.57 (s, 1H),
7.72 (s, 1H), 7.43 (s, 1H), 3.82 (t, J = 8.0 Hz, 9H).
5.14. General procedure for Preparation of the intermediates 20a-b
A mixture of 19 (2.0 g, 5.43 mmol), 10a-b (4.53 mmol) and K2CO3 (1.88 g,
13.59 mmol) in DMF (15 mL) was heated at 50 for 13 h. Upon cooling to room
temperature, the reaction mixture was added water (100 mL), and extracted by EA (15
mL×3). The organic phase was combined and concentrated to get the crude product,
which was purified by silica gel chromatography using PE/EA (5:1) to afford the
intermediates 20a-b as a pale-yellow solid.
5.14.1. Methyl6-((6-(4-(1-((4-fluorophenyl)carbamoyl)cyclopropanecarboxamido)
phenoxy)-5-nitropyrimidin-4-yl)amino)-2,3-dimethoxybenzoate (20a)
1
Pale-yellow solid; Yield: 84.0%; MS (ESI) m/z: 647.11 [M+H]+, H NMR
(400MHz, DMSO-d6) δppm: 10.92 (s, 1H), 10.17 (t, J = 8.6 Hz, 2H), 8.59 (s, 1H),
7.77 (s, 1H), 7.44 (s, 1H), 7.29~7.12 (m, 4H), 7.02~6.84 (m, 4H), 3.88 (t, J = 8.6 Hz,
9H), 1.45 (s, 4H).
5.14.2. Methyl6-((6-(2-fluoro-4-(1-((4-fluorophenyl)carbamoyl)cyclopropane
carboxamido)phenoxy)-5-nitropyrimidin-4-yl)amino)-2,3-dimethoxybenzoate (20b)
1
Pale-yellow solid; Yield: 82.7%; MS (ESI) m/z: 665.19 [M+H]+, H NMR
(400MHz, DMSO-d6) δppm: 10.90 (s, 1H), 10.16 (t, J = 6.8 Hz, 2H), 8.54 (s, 1H),
7.71 (s, 1H), 7.42 (s, 1H), 7.19~7.11 (m, 4H), 6.97~6.81 (m, 4H), 3.83 (t, J = 6.8 Hz,
9H), 1.44 (s, 4H).
5.15. General procedure for Preparation of the title compounds (21a-b)
A mixture of 20a-b (4.64 mmol) and iron dust (1.30 g, 23.20 mmol) in AcOH
(40 mL) was heated at 110
for 12 h. Upon cooling to room temperature, the
reaction mixture was filtered off and the filtrate was concentrated to obtain the crude
product, which was purified by stirred with ethanol (20 mL) at 80 for 1 h giving the
21

ACCEPTED MANUSCRIPT
title compounds 21a-b.
5.15.1. N-(4-((8,9-dimethoxy-6-oxo-6,11-dihydro-5H-benzo[e]pyrimido[5,4-b][1,4]
diazepin-4-yl)oxy)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (21a)
Pale-white solid; Yield: 64.0%; m.p.: 252-254 ; MS (ESI) m/z: 585.19 [M+H]+,
1H NMR (400MHz, DMSO-d6) δppm: 10.10 (s, 1H, CONH), 10.04 (s, 1H, CONH),
8.85 (s, 1H, CONH), 7.65~7.55 (m, 5H), 7.20~7.05 (m, 5H), 5.65 (s, 2H), 3.97 (s, 3H,
13
CH3), 3.92 (s, 3H, CH3), 1.45 (s, 4H, CH2CH2). C NMR (100MHz, DMSO-d6): δ
15.21, 30.34, 55.87, 56.73, 102.61, 106.09, 108.34, 114.03, 117.47, 124.41, 129.02,
133.07, 136.11, 142.75, 144.93, 149.24, 151.82, 154.48, 156.26, 157.47, 162.63,
168.72. HRMS Calcd for C30H26F2N6O5 [M+H]+, 584.1867; found, 585.1936.
Purity: > 95%.
5.15.2. N-(4-((8,9-dimethoxy-6-oxo-6,11-dihydro-5H-benzo[e]pyrimido[5,4-b][1,4]
diazepin-4-yl)oxy)-3-fluorophenyl)-N-(4-fluorophenyl)cyclopropane-1,1-
dicarboxamide (21b)
Pale-white solid; Yield: 61.0%; m.p.: 257-259 ; MS (ESI) m/z: 602.98 [M+H]+,
1H NMR (400MHz, DMSO-d6) δppm: 10.24 (s, 1H, CONH), 10.02 (s, 1H, CONH),
8.80 (s, 1H, CONH), 7.77 (dd, J = 2.4 Hz, 13.2 Hz, 1H), 7.65~7.62 (m, 2H), 7.50 (s,
1H), 7.38 (dd, J = 1.2 Hz, 9.2 Hz, 1H), 7.22 (t, J = 8.8 Hz, 1H), 7.17~7.12 (m, 3H),
13
5.67 (s, 2H), 3.96 (s, 3H, CH3), 3.90 (s, 3H, CH3), 1.46 (s, 4H, CH2CH2). C NMR
(100MHz, DMSO-d6): δ 15.37, 31.66, 55.93, 56.06, 105.01, 106.70, 108.63, 108.89,
115.01, 116.41, 120.33, 122.43, 122.58, 127.86, 135.17, 136.39, 136.71, 141.02,
141.72, 144.89, 147.88, 151.83, 154.26, 156.35, 157.01, 159.48, 168.08. HRMS
Calcd for C30H26F2N6O5 [M+H]+, 602.1743; found, 602.9826. Purity: > 95%.
5.16. Preparation of methyl 6-((4-chloro-3-nitropyridin-2-yl)amino)-2,3-
dimethoxybenzoate (22)
A solution of 4,6-dichloro-5-nitropyridine (4.0 g, 0.02 mol), methyl
6-amino-2,3-dimethoxybenzoate (5.07 g, 0.024 mol) and KF.2H2O (5.65 g，0.06 mol)
in DMF (25 mL) was heated at 130 ℃for 9 h. Upon cooling to room temperature, the
reaction solution was added water (100 mL), and filtered off to get the crude product,
which was purified by silica gel chromatography using PE/EA (5:1) to afford the
1
intermediate 22 as a yellow solid (2.94 g, 40%). MS (ESI) m/z: 367.95 [M+H]+, H
NMR (400MHz, DMSO-d6) δppm: 10.91 (s, 1H), 8.57 (d, J = 6.4 Hz, 1H), 7.96 (d, J
= 8.6 Hz, 1H), 7.85 (s, 1H), 7.47 (s, 1H), 3.86 (t, J = 8.8 Hz, 9H).
22

ACCEPTED MANUSCRIPT
5.17. General procedure for Preparation of the intermediates 23a-b
A solution of 22 (3.0 g, 8.17 mmol), 10a-b (9.80 mmol) and KF.2H2O (2.31 g，
24.51 mmol) in DMF (20 mL) was heated at 140 ℃for 15 h. Upon cooling to room
temperature, water (100 mL) was added to the reaction solution. The precipitate was
collected by filtration, stirred with ethanol (20 mL) at 80 ℃for 1 h, and filtered to get
the crude product, which was purified by silica gel chromatography using PE/EA (3:2)
to afford the intermediates 23a-b as yellow solids.
5.17.1. Methyl6-((4-(4-(1-((4-fluorophenyl)carbamoyl)cyclopropanecarboxamido)
phenoxy)-3-nitropyridin-2-yl)amino)-2,3-dimethoxybenzoate (23a)
Yellow solid; Yield: 76.0%; MS (ESI) m/z: 646.20 [M+H]+, 1H NMR (400MHz,
DMSO-d6) δppm: 10.94 (s, 1H), 10.12 (t, J = 8.6 Hz, 2H), 8.56 (d, J = 6.8 Hz, 1H),
7.91 (d, J = 8.4 Hz, 1H), 7.82 (s, 1H), 7.62 (s, 1H), 7.27~7.14 (m, 4H), 7.07~6.82 (m,
4H), 3.84 (t, J = 8.8 Hz, 9H), 1.44 (s, 4H).
5.17.2. Methyl6-((4-(2-fluoro-4-(1-((4-fluorophenyl)carbamoyl)cyclopropanecarbox
amido)phenoxy)-3-nitropyridin-2-yl)amino)-2,3-dimethoxybenzoate (23b)
Yellow solid; Yield: 74.8%; MS (ESI) m/z: 664.14 [M+H]+, 1H NMR (400MHz,
DMSO-d6) δppm: 10.92 (s, 1H), 10.14 (t, J = 8.0 Hz, 2H), 8.51 (d, J = 6.4 Hz, 1H),
7.93 (d, J = 8.6 Hz, 1H), 7.79 (s, 1H), 7.67 (s, 1H), 7.21~7.12 (m, 3H), 7.06~6.87 (m,
4H), 3.85 (t, J = 6.8 Hz, 9H), 1.44 (s, 4H).
5.18. General procedure for Preparation of the title compounds (24a-b)
A mixture of 23a-b (3.10 mmol) and iron dust (0.87 g, 15.50 mmol) in AcOH
(35 mL) was heated at 110 ℃ for 12 h. Upon cooling to room temperature, the
reaction mixture was filtered off and the filtrate was concentrated to obtain the crude
product, which was purified by stirred with ethanol (20 mL) at 80 ℃for 1 h giving the
title compounds 24a-b as white solids.
5.18.1. N-(4-((8,9-dimethoxy-6-oxo-6,11-dihydro-5H-benzo[e]pyrido[3,2-b][1,4]
diazepin-4-yl)oxy)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (24a)
Yellow solid; Yield: 60.0%; m.p.: 241-244 ℃; MS (ESI) m/z: 583.91 [M+H]+, 1H
NMR (400MHz, DMSO-d6) δppm: 10.11 (s, 1H, CONH), 10.08 (s, 1H, CONH), 8.95
(s, 1H, CONH), 8.32 (s, 1H, NH), 7.64~7.55 (m, 5H, Ar-H), 7.22~6.88 (m, 5H, Ar-H),
6.67~6.62 (m, 2H, Ar-H), 3.80 (s, 3H, CH3), 3.72 (s, 3H, CH3), 1.46 (s, 4H, CH2CH2).
13C NMR (100MHz, DMSO-d6): δ 15.61, 32.17, 53.66, 58.06, 102.33, 104.58, 106.27,
108.64, 115.06, 120.40, 123.12, 126.55, 134.16, 136.19, 142.55, 146.39, 148.21,
23

ACCEPTED MANUSCRIPT
151.25, 155.47, 156.19, 160.22, 162.37, 167.53. HRMS Calcd for C30H26F2N6O5
[M+H]+, 583.1975; found, 583.9168. Purity: > 95%.
5.18.2. N-(4-((8,9-dimethoxy-6-oxo-6,11-dihydro-5H-benzo[e]pyrido[3,2-b][1,4]
diazepin-4-yl)oxy)-3-fluorophenyl)-N-(4-fluorophenyl)cyclopropane-1,1-
dicarboxamide (24b)
Yellow solid; Yield: 64.0%; m.p.: 243-245 ; MS (ESI) m/z: 602.19 [M+H]+, 1H
NMR (400MHz, DMSO-d6) δppm: 10.16 (s, 1H, CONH), 10.02 (s, 1H, CONH), 8.91
(s, 1H, CONH), 8.54 (s, 1H, NH), 7.63~7.49 (m, 4H, Ar-H), 7.20~6.78 (m, 5H, Ar-H),
6.62~6.57 (m, 2H, Ar-H), 3.86 (s, 3H, CH3), 3.74 (s, 3H, CH3), 1.45 (s, 4H, CH2CH2).
13C NMR (100MHz, DMSO-d6): δ 15.22, 30.63, 54.91, 56.74, 102.61, 104.16, 106.73,
108.66, 109.34, 112.07, 116.48, 120.19, 122.52, 124.35, 127.82, 133.11, 136.26,
136.88, 141.01, 142.79, 144.89, 147.52, 150.82, 154.64, 156.40, 157.36, 159.42,
167.04. HRMS Calcd for C30H26F2N6O5 [M+H]+, 601.1821; found, 602.1907.
Purity: > 95%.
5.19. General procedure for Preparation of the title compounds (25a-b)
17e-f (1.0 mmol) was added drop-wise to HCOOH (2.5 mL) at 10-15 , and
then 37% aq. HCHO (0.25 mL) was added. The solution was heated at 90 for 2 h.
Upon cooling to room temperature, the reaction solution was added to 20 mL
saturated aq. NaHCO3, and extracted by EA (15 mL×3). The organic phase was
combined and concentrated to get the crude product, which was purified by silica gel
chromatography using PE/EA (1:1 to 2:3, to 1:2) to afford the target compounds
25a-b as white solids.
5.19.1. N-(4-((8,9-dimethoxy-5-methyl-6,11-dihydro-5H-benzo[e]pyrimido[5,4-b]-[1,
4]diazepin-4-yl)oxy)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide
(25a)
White solid; Yield: 40.0%; m.p.: 247-249 ; MS (ESI) m/z: 585.13 [M+H]+, 1H
NMR (400MHz, DMSO-d6) δppm: 10.09 (s, 1H, CONH), 10.07 (s, 1H, CONH), 9.29
(s, 1H, NH), 7.87~6.83 (m, 11H, Ar-H), 3.98 (s, 2H, CH2), 3.72 (d, J = 4.8 Hz, 6H,
13
CH3), 2.55 (s, 3H, NCH3),1.46 (s, 4H, CH2CH2). C NMR (100MHz, DMSO-d6): δ
15.72, 31.39, 54.48, 55.11, 56.83, 104.09, 112.01, 114.32, 116.17, 119.82, 121.40,
123.34, 131.07, 135.33, 137.29, 147.31, 151.44, 153.45, 157.36, 159.13, 164.07,
167.01. HRMS Calcd for C30H26F2N6O5 [M+H]+, 584.2214; found, 585.1316.
Purity: > 95%.
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5.19.2. N-(4-((8,9-dimethoxy-5-methyl-6,11-dihydro-5H-benzo[e]pyrimido[5,4-b]-[1,
4]diazepin-4-yl)oxy)-3-fluorophenyl)-N-(4-fluorophenyl)cyclopropane-1,1-
dicarboxamide (25b)
White solid; Yield: 44.0%; m.p.: 251-253 ; MS (ESI) m/z: 603.21 [M+H]+, 1H
NMR (400MHz, DMSO-d6) δppm: 10.27 (s, 1H, CONH), 10.04 (s, 1H, CONH), 9.36
(s, 1H, NH), 7.86~6.84 (m, 10H, Ar-H), 4.00 (s, 2H, CH2), 3.72 (d, J = 6.0 Hz, 6H,
13
CH3), 2.56 (s, 3H, NCH3),1.46 (s, 4H, CH2CH2). C NMR (100MHz, DMSO-d6): δ
15.35, 31.68, 55.50, 55.89, 56.35, 104.45, 108.51, 113.94, 114.99, 116.27, 116.30,
119.93, 122.43, 123.85, 134.57, 135.17, 135.61, 137.08, 148.35, 151.42, 152.13,
154.55, 157.08, 158.57, 159.47, 162.91, 168.04. HRMS Calcd for C30H26F2N6O5
[M+H]+, 602.2089; found, 603.2164. Purity: > 95%.
5.20. c-Met kinase assay
The effects of indicated compounds on the activities of c-Met kinase determined
using enzyme-linked immunosorbent (ELISAs) with purified recombinant proteins
[47]. Briefly, 20 µg/mL poly (Glu, Tyr)4:1 (Sigma, St. Louis, MO, USA) was
pre-coated in 96-well plates as a substrate. A 50-µL aliquot of 10 µg/mL ATP solution
diluted in kinase reaction buffer (50 mmol/L HEPES [pH 7.4], 50 mmol/L MgCl2, 0.5
mmol/L MnCl2, 0.2 mmol/L Na3VO4, and 1 mmol/L DTT) was added to each well; 1
µL of various concentrations of indicated compound diluted in 1% DMSO (v/v)
(sigma) were then added to each reaction well. DMSO (1%, v/v) was used as the
negative control. The kinase reaction was initiated by the addition of purified c-Met
tyrosine kinase proteins diluted in 49 µL of kinase reaction buffer. After incubation for
60 min at 37 , the plate was washed three times with phosphate-buffered saline
(PBS) containing 0.1% Tween 20 (T-PBS). Anti-phosphotyrosine (PY99) antibody
(100µL; 1:500, diluted in 5 mg/mL BSA T-PBS) was then added. After a 30-min
incubation at 37 , the plate was washed three times, and 100 µL horseradish
peroxidase-conjugated goat anti-mouse IgG (1:2000, diluted in 5 mg/mL BSA T-PBS)
was added. The plate was then incubated at 37 ℃ for 30 min and washed 3 times. A
100-µL aliquot of
a
solution containing 0.03% H2O2 and
2
mg/mL
o-phenylenediamine in 0.1 mol/L citrate buffer (pH 5.5) was added. The reaction was
terminated by the addition of 50 µL of 2 mol/L H2SO4 as the color changed, and the
plate was analyzed using a multi-well spectrophotometer (SpectraMA 190, Molecular
Devices, Sunnyvale, CA, USA) at 490 nm. The inhibition rate (%) was calculated
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using the following equation: [1-(A490/A490 control)] × 100%. The IC50 values
were calculated from the inhibition curves in two separate experiments.
5.21. Cell Proliferation Assay
Cells were essded in 96-well tissue culture plates. On the next day, the cells were
exposed to various concentrations of compounds and further cultured for 72 h. Cell
proliferation was then determined using sulforhodamine B (SRB, Sigma, St. Louis,
MO, USA). The IC50 values were calculated by concentration-response curve fitting
using the four-parameter method [47].
5.22. Pharmacokinetic study
Male SD rats (SLRC laboratory Animal Inc., Shanghai, China) were used. For
intravenous administration, prepared dosing solution was injected via the femoral vein.
The rats were fasted overnight before drug administration and until 6 h after dosing.
For the PO experiment, rats (24 in each group) were given a single dose of 20 mg/kg,
and heparinized samples of blood were collected at 5, 15, 30 min, 1, 2, 4, 8, and 24 h
postdose. For the iv experiment, rats (24 in each group) were given a single 4 mg/kg
dose, and blood samples were collected at 5, 15, 30 min, 1, 2, and 4 h postdose.
Plasma was harvested after centrifugation and stored frozen at -40 until analyzed.
The concentrations of compounds in plasma were determined by LC/MS/MS
(Shimadzu LC-30AD). The results are shown as the maximum plasma concentration
(Cmax), the time to reach peak plasma concentration (Tmax), terminal half-life (T1/2),
and the area under the plasma concentration-time curve from zero to time infinity
(AUC0-inf) [48].
5.23. Acute toxicity test
Male and female KM mice (18-22 g) were purchased from SLRC Laboratory
Animal Inc., Shanghai, China. Mice were randomly divided into six groups with ten
mice each (five males, five females). Mice were orally given 17f with a single dose
420, 520, 620, 720, 820 and 920 mg/kg, or vehicle conrol, respectively. The mouse
death was monitored daily, and recored up to 14 days after treatment. All animals
were euthanized and necropsied for gross lesion examination for possible damage to
the heart, liver, and kidneys [49].
5.24. Compound activity on hERG
Whole-cell recordings were performed using automated Qpatch (Sophion). Cells
were voltage clamped at a holding potential of -80 mV. The hERG current was
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activated by depolarizing at +20 mV for 5 sec, after which the voltage was taken back
to -50 mV for 5 sec to remove the inactivation and observe the deactivating hERG tail
current. The voltage stimulation was applied per 15 sec. Compounds solutions were
administrated from low to high concentration, 2 min for each concentration and 10
µM cisapride was applied at the end of perfusion of compound solution. Each
concentration was tested [50].
on at least 3 cells.
5.25. In vivo antitumor activity assay
Female nude mice (4-6 weeks old) were housed and maintained under
specific-pathogen free conditions. Animal procedures were performed according to
institutional ethical guidelines of animal care [51]. The Caki-1 cells at a density of 5 ×
106 in 200 µL were injected s.c. into the right flank of nude mice and then allowed to
grow to 700-800 mm3, defined as a well-developed tumor. After that, the
well-developed tumors were cut into 1 mm3 fragments and transplanted s.c. into the
right flank of nude mice using a trocar. When the mean tumour volume reached
120-130 mm3, the mice were randomly assigned into vehicle and treatment groups (n
= 6 in treated group, n = 12 in vehicle group). Vehicle groups were given vehicle
alone, and treatment groups received 17f as indicated doses via intraperitoneal
injection once daily for 3 weeks in Caki-1 model. The sizes of the tumours were
measured twice per week using microcaliper. The tumour volume (V) was calculated
as follows: V = [length (mm) × width2 (mm2)]/2. Percent (%) inhibition values (TGI)
were measured on the final day of study for drugtreated compared with
vehicle-treated mice and were calculated as 100% × (1- ((treatedfinal day – treatedday
0)/controlfinal day – controlday 0))).
Acknowledgments
This work was financially supported by the National Natural Science Foundation
of China (No.81273373), and Science and Technology Commission of Shanghai
Municipality (No. 12431901101, 14YF1412800).
SUPPLEMENTARY MATERIAL
MS spectra, 1H NMR, 13C NMR and HRMS spectra of compounds 17e, 17f and
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25b; Means of tumor weight during experiment; Means of body weight of rats during
experiment.
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