Design and synthesis of novel 2-methyl-4,5-substitutedbenzo[f]-3,3a,4,5- tetrahydro-pyrazolo[1,5-d][1,4]oxazepin-8(7H)-one derivatives as telomerase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2012.11.101

Eight novel 4,5-tetrahydropyrazolo[1,5-d][1,4]oxazepine derivatives have been synthesized and purified to be screened for anticancer activity. By a modified TRAP assay, some titled compounds were tested against telomerase, and compound 4a showed the most potent inhibitory activity with IC₅₀ value at 0.78 ± 0.22 μM. Western blot assays showed that compounds 4a and 4b could inhibit expression of Cyclin D1, TERT, phospho-AKT and PI3K/AKT pathway.

Full text of DOI:10.1016/j.bmcl.2012.11.101

Bioorganic & Medicinal Chemistry Letters 23 (2013) 720–723
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of novel 2-methyl-4,5-substitutedbenzo[f]-3,3a,4,5-tetra-
hydro-pyrazolo[1,5-d][1,4]oxazepin-8(7H)-one derivatives as telomerase
inhibitors
Xin-Hua Liu a,b, , Ying-Ming Jia c, Bao-An Song a, Zhi-xiang Pang b, Song Yang a,
⇑
⇑
a Key Laboratory of Green Pesticide and Agriculture Bioengineering, Ministry of Education, Guizhou University, Guiyang 550025, PR China
b School of Pharmacy, Anhui Medical University, Hefei 230032, PR China
c School of Chemistry and Chemical Engineering, Anhui University of Technology, Maanshan 243002, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Eight novel 4,5-tetrahydropyrazolo[1,5-d][1,4]oxazepine derivatives have been synthesized and purified
to be screened for anticancer activity. By a modified TRAP assay, some titled compounds were tested
against telomerase, and compound 4a showed the most potent inhibitory activity with IC50 value at
Received 11 September 2012
Revised 19 November 2012
Accepted 22 November 2012
Available online 3 December 2012
0.78 0.22
lM. Western blot assays showed that compounds 4a and 4b could inhibit expression of Cyclin
D1, TERT, phospho-AKT and PI3K/AKT pathway.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Synthesis
Hydropyrazolo-oxazepine
PI3K/AKT pathway
Antitumor agents
Telomerase represents one of the promising targets in drug dis-
covery.1–4 Human telomerase reverse transcriptase is a catalytic
enzyme that is required for cancer progression.5 Recent studies
indicate that the PI3K/AKT pathway is targeted by amplification,
mutation and translocation more frequently than any other path-
way in cancer patients.6
Many 3,4-dihydropyrazole-based derivatives have shown sev-
eral biological activities as seen in cannabinoid receptor 1 antago-
nist, monoamine oxidase inhibitors, and tumor necrosis
inhibitors,13 In our recent publications, several ary-dihydropyraz-
ole derivatives used as potent telomerase inhibitors were
described.14,15
Heterocyclic seven-membered ring constitute the core or a key
fragment of a number of bioactive compounds including isolated
from natural products.7–9 Among them, seven-membered hetero-
cycles with two heteroatoms in 1,4-distance are known to possess
manifold biological activity. In particular, [1,4]oxazepine units are
crucial moieties in several psychoactive pharmaceuticals (for
example, oxazepam), among a variety of physiologically active
1,4-oxazepines, the derivatives of their aryl-fused analogs repre-
sent a relatively little-explored group with interesting pharmaceu-
tical properties. They were described as effective protease
inhibitors, non-peptidergic GPCR inhibitors, integrin antagonists,
squalene synthase, and reverse transcriptase inhibitors.10–12
In an effort to design novel ary-dihydropyrazole systems as effi-
cient telomerase inhibitors, based on the protein structure of telo-
merase using LigandFit module within Discovery Studio 2.1, our
group has recently reported a novel docking model. When we
introduced the [1,4]oxazepine moiety in the above-mentioned
ary-dihydropyrazole skeleton, the novel moiety of dropyrazol-
[1,4]oxazepine with potentially higher activity against telomerase
was obtained.16 Recently, there have been only a few successful
hTERT inhibitors developed. BIBR1532 is one of the most promising
hTERT specific active site inhibitors.17 BIBR1532 is anon-nucleo-
tidic small molecule synthetic compound that inhibits telomerase
by non-competitively binding to the active site of hTERT.18,19 Since
there are only a very few systematic reports on the synthetic meth-
odology and evaluation of anticancer activity of this moiety, herein,
as a continuation of our research for finding novel, efficient telo-
merase inhibitors, we designed and synthesized a series of 2-
methyl-4,5-substitutedbenzo[f]-3,3a,4,5-tetrahydro-pyrazolo[1,5-
d][1,4]oxazepin-8(7H)-one derivatives.
Abbreviations: PI3K, phosphatidylinositol 3-kinase; AKT, serine/threonine
Kinase; GPCR, G-protein coupled receptor; hTERT, human telomerase reverse
transcriptase subunit; BIBR1532, 2-[E)-3-naphtalen-2-yl-but-2-enylylamino]-ben-
zoic acid; MGC-803, The human gastric carcinoma cell line.
In order to further explore detailed molecular mechanism of the
synthesized compounds, inhibitive activities of hTERT and
⇑
Corresponding author. Tel.: +86 851 3161115; fax: +86 851 3620521.
E-mail addresses: xhliuhx@163.com (X.-H. Liu), yangsdqj@126.com (S. Yang).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.11.101

X.-H. Liu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 720–723
721
H3C
O
O
HO
(A)
CH3
N
H
(B)
R
R
R
N
H
OH
OH
1
2
O
H3C
HO
N
N
(D)
O
N
(C)
R
N
Br
(E)
O
R
4
3
Scheme 1. Synthesis of 2-methyl-substitutedbenzo-3,3a-dihydropyrazolo[1,5-d][1,4] oxazepin-8(7H)-one derivatives. Reagent and conditions: (A) CH3COCH3, NaOH,
C2H5OH, 20–30 °C, 10 h. (B) N2H4.H2O, CH3CH2OH, reflux, 2 h. (C) 2-Bromopropanoic acid, 4-nitrobenzenesulfonyl chloride, DMAP, reflux, 2 h. (D) NaHCO3, C2H5OH, reflux 3 h.
(E) 5% HCl. R = 7,9-2Br (4a); R = 7-Cl (4b); R = 9-OMe (4c); R = 7-CF3 (4d); R = 8-NO2 (4e); R = 8-Me (4f); R = 9-F (4g); R = H (4h).
Table 1
Inhibitory effects of slected compounds against telomerase
M) telomerasea
c
Compound
Structure
IC50
(l
O
N
N
N
O
O
4a
0.78 0.22
Br
Br
O
N
4b
4c
4d
1.71 0.51
10.82 1.32
3.14 1.07
Cl
O
N
N
N
N
O
O
OMe
O
F3C
O
N
N
O
4e
6.60 0.73
Figure 1. ORTEP drawing of compounds 4a and 4h.
NO2
phospho-AKT (p-AKT) proteins were evaluated, the results showed
title compounds 4a, 4b could inhibit the PI3K/AKT pathway.
(continued on next page)

722
X.-H. Liu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 720–723
V = 2052.42(9) nm3, T = 293(2) K, Z = 8, Dc = 1.400 g/cm3,
Table 1 (continued)
F(000) = 912, Reflections collected/unique = 5429/2020, Data/re-
straints/parameters = 2020/0/146, Goodness of fit on F2 = 1.080,
Fine, R1 = 0.0427, wR(F2) = 0.0900.
c
Compound
Structure
IC50 (l
M) telomerasea
O
The molecular structures of compounds 4a, 4h were shown in
Figure 1. Crystallographic data (excluding structure factors) for
the structure had been deposited with the Cambridge Crystallo-
graphic Data Center as supplementary publication No. CCDC-
887779, 899791.23
N
N
O
4f
21.90 2.07
Some purified title compounds were assayed for telomerase
inhibition,25 using a MGC-803 cell extract, also included the activ-
ity of reference compound ethidium bromide.26 The results were
summarized in Table 1. The results suggested some compounds
showed strong inhibitory activity with telomerase. Especially
compounds 4a, 4b and 4g with IC50 values of 0.78 0.22,
O
O
N
N
N
N
O
O
4g
2.11 0.87
F
1.71 0.51 and 2.11 0.87 lM, respectively, which were even
better than that of the ethidium bromide. From the data pre-
sented in Table 1, it can be concluded that halophenyl structure
show higher inhibitory activity than any other structure, this
pointed out the direction for us to further optimize the structures
of 2-substituted-4,5-substitutedbenzo[f]-3,3a,4,5-tetrahydro-pyr-
azolo[1,5-d][1,4]oxazepin-8(7H)-one derivatives as potential telo-
merase inhibitors.
4h
14.80 1.01
2.39 0.12
In order to examine whether some titled compounds change the
expression of TERT, we performed western blot assays.27 As shown
in Figure 2, compared with adjacent control group, TERT protein
was expressed at lower level in MGC-803 cells, which were treated
with compounds 4a, 4b, 4c, 4d, 4f and 4g, respectively. The result
suggested that telomerase activity was significantly down-regu-
lated within 48 h when the cells exposed to compounds 4a, 4b.
In summary, based on finding novel, efficient telomerase inhib-
itors, we designed and synthesized some novel 2-methyl-4,5-
substitutedbenzo[f]-3,3a,4,5-tetrahydro-pyrazolo[1,5-d][1,4]oxa-
zepin-8(7H)-one derivatives as potential telomerase inhibitors,
followed by chemical synthesis and biological evaluated for them.
Among them, 4a and 4h were determined by X-ray. In order to
examine whether some titled compounds change the expression
of TERT, western blot assays showed that compounds 4a and 4b
could inhibit the expression of Cyclin D1, TERT, phospho-AKT
and the PI3K/AKT pathway. These results are of help in the rational
design of more efficient telomerase inhibitors for chemoprevention
and chemotherapy in further.
Ethidium
bromideb
a
Telomerase supercoiling activity.
b
Ethidium bromide is reported as a control. The inhibition constant of ethidium
toward telomerase has been reported previously.
1 l
M = 10À6 mol/L.
c
The synthesis of compound 1 (Scheme 1) started from substi-
tuted salicylaldehyde and catalyzed by NaOH at 20–30 °C was
added acetone. Compound 2 was prepared according to a previ-
ously published report.20 Using catalysts 4-nitrobenzenesulfonyl-
chloride and DMAP, proved to be an efficient alternative for the
synthesis of title compound 3. The general synthetic procedure
process and spectral data of compound 4 can be found in the sup-
porting information.21
The structures of compounds 4a, 4h were determined by X-ray
crystallography. Crystal data of 4a: Colorless crystals, yield, 70%;
mp 177–178 °C; C12 H10 Br2 N2 O2, M = 374.04, Monoclinic,
space group C2/c; a = 37.69(3), b = 9.820(9), c = 14.002(12) (Å);
Acknowledgments
a
= 90, b = 99.851(9),
c
= 90 (°), V = 5107(8) nm3, T = 293(2) K,
Z = 16, Dc = 1.946 g/cm3, F(000) = 2912, Reflections collected/un-
The authors wish to thank the National Natural Science Founda-
tion of China (No. 21172048, 21272008), China Postdoctoral Sci-
ence Foundation funded project (2012M511948) and the
Research Fundation of Doctor, Anhui Medical University
(XJ201120).
ique = 12579/4565,
Data/restraints/parameters = 4565/36/327,
Goodness of fit on F2 = 1.010, Fine, R1 = 0.0976, wR(F2) = 0.2461.
4h: Colorless crystals, yield, 87%; mp 168–169 °C; C12 H12 N2 O2,
M = 216.24, Orthorhombic, space group Pbca; a = 7.9121(2),
b = 14.6270(4), c = 17.7344(5) (Å);
a
= 90, b = 90,
c
= 90 (°),
DMSO Control
LY
4g
4f
4c
4b
4a
TERT
Cyclin D1
p-Akt
Akt
β-actin
Figure 2. Western blot analyses of MGC-803 cells lysates was performed for Cyclin D1, TERT, phospho-AKT and total AKT after 48 h of incubation. b-actin served as loading
control. LY294002 (25 M) is the classic inhibitor of PI3 K/AKT pathway and DMSO was employed as negative control.
l

X.-H. Liu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 720–723
723
4g: Colorless crystals, yield, 74%; mp 175–177 °C; 1H NMR (400 MHz, CDCl3) d
(ppm): 2.17 (s, 3H, –CH3), 3.35 (dd, 1H, J1 = 4.0 Hz, J2 = 8.0 Hz, pyrazole 4-Ha),
3.65 (dd, 1H, J1 = 8.0 Hz, J2 = 16.0 Hz, pyrazole 4-Hb), 4.12 (d, 1H, J = 16.0 Hz,
oxazepane 2-Ha), 5.05 (d, 1H, J = 16.0 Hz, oxazepane 2-Hb), 5.72 (dd, 1H,
J1 = 8.0 Hz, J2 = 12.0 Hz, pyrazole 5-H), 6.89–7.01 (m, 3H). Anal. Calcd for
References and notes
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Qi, X. B. Bioorg. Med. Chem. Lett. 2010, 20, 5705.
16. Liu, X. H.; Li, J.; Shi, J. B.; Song, B. A.; Qi, X. B. Eur. J. Med. Chem. 2012, 51, 294.
17. Damm, K.; Hemmann, U.; Garin-Chesa, P. EMBO J. 2001, 20, 6958.
18. Pascolo, E.; Wenz, C.; Lingner, J.; Hauel, N.; Priepke, H.; Kauffmann, I. J. Biol.
Chem. 2002, 277, 15566.
19. El-Daly, H.; Kull, M.; Zimmermann, S.; Pantic, M.; Waller, C. F.; Martens, U. M.
Blood 2005, 105, 1742.
C
12H11FN2O2: C, 61.53; H, 4.73; N, 11.96. Found: C, 61.25; H, 4.99; N, 12.10.
4h. Colorless crystals, yield, 87%; mp 168–169 °C; 1H NMR (400 MHz, CDCl3) d
(ppm): 2.05 (s, 3H, –CH3), 3.32 (dd, 1H, J1 = 4.0 Hz, J2 = 8.0 Hz, pyrazole 4-Ha),
3.60 (dd, 1H, J1 = 8.0 Hz, J2 = 16.0 Hz, pyrazole 4-Hb), 4.17 (d, 1H, J = 16.0 Hz,
oxazepane 2-Ha), 5.00 (d, 1H, J = 16.0 Hz, oxazepane 2-Hb), 5.63 (dd, 1H,
J1 = 8.0 Hz, J2 = 12.0 Hz, pyrazole 5-H), 6.84-7.00 (m, 4H). Anal. Calcd for:
C12H12N2O2: C, 66.65; H, 5.59; N, 12.96. Found: C, 66.70; H, 5.47; N, 13.18.
22. The reactions were monitored by thin layer chromatography (TLC) on Merck
pre-coated silica GF254 plates. Melting points (uncorrected) were determined
on a XT4MP apparatus (Taike Corp., Beijing, China). 1H NMR spectra were
collected on PX400 spectrometer at room temperature with TMS and solvent
signals allotted as internal standards. Chemical shifts are reported in ppm (d).
Elemental analyses were performed on a CHN–O-Rapid instrument, and were
within 0.4% of the theoretical values.
23. Crystallographic studies: X-ray single-crystal diffraction data for compounds 4a,
4h (a sample of size 0.26 Â 0.23 Â 0.22 and 0.31 Â 0.27 Â 0.21 mm3) was
collected on a Bruker SMART APEX CCD diffractometer at 296(2) K using MoK
a
radiation (k = 0.71073 Å) by the
x scan mode. The program SAINT was used for
integration of the diffraction profiles. The structure was solved by direct
methods using the SHELXS program of the SHELXTL package and refined by
full-matrix least-squares methods with SHELXL.24 The corrections for LP
factors were applied. All non-hydrogen atoms of compounds 4a, 4h were
refined with anisotropic thermal parameters. All hydrogen atoms were
generated theoretically onto the parent atoms and refined isotropically with
fixed thermal factors.
24. Sheldrick, G. M. SHELXTL-97, Program for Crystal Structure Solution and
Refinement; University of Göttingen: Göttingen, Germany, 1997.
25. Kim, N. W.; Piatyszek, M. A.; Prowse, K. R.; Harley, C. B.; West, M. D.; Ho, P. L.;
Coviello, G. M.; Wright, W. E.; Weinrich, S. L.; Shay, J. W. Science 2011, 1994, 266.
26. Telomerase activity assay: Compounds 4 were tested in a search for small
molecule inhibitors of telomerase activity by using the TRAP-PCR-ELISA assay.
In detail, the MGC-803 cells were firstly maintained in DMEM medium (GIBCO,
New York, USA) supplemented with 10% fetal bovineserum (GIBCO, New York,
20. Liu, X. H.; Zhu, J.; Zhou, A. N.; Song, B. A.; Zhu, H. L.; Bai, L. S.; Bhadury, P. S.;
Pan, C. X. Bioorg. Med. Chem. Lett. 2009, 17, 1207.
USA), streptomycin (0.1 mg/mL) and penicillin (100 IU/mL) at 37 °C in
a
21. General synthetic procedure process for compound 4: To a C2H5OH (20 ml)
solution of NaHCO3 (10 mmol) was added 2-bromo-1-(5-(2-hydroxy-
substitutedphenyl)-3-methyl-4,5-dihydropyrazol-1-yl)ethanone 3 (10 mmol),
then the reaction mixture was refluxed for 3 h. The mixture was allowed to
cool to room temperature and 30 ml water was added, adjusted pH to 7 with
5% HCl solution, allowed to stand at 0 °C over night. The product was collected
by filtration and the crude residue was purified by chromatography on SiO2
(acetone/petroleum, v:v = 2:1) to give title compound 4 (Scheme 1) as colorless
solids.22
humidified atmosphere containing 5% CO2. After trypsinization, 5 Â 104
cultured cells in logarithmic growth were seeded into T25 flasks (Corning,
New York, USA) and cultured to allow to adherence. The cells were then
incubated with Staurosporine (Santa Cruz, Santa Cruz, USA) and the drugs with
a series of concentration as 60, 20, 6.67, 2.22, 0.74, 0.25 and 0.082 l g/mL,
respectively. After 24 h treatment, the cells were harvested by cell scraper
orderly following by washed once with PBS. The cells were lysed in 150 lL
RIPA cell lysis buffer (Santa Cruz, Santa Cruz, USA), and incubated on ice for
30 min. The cellular supernatants were obtained via centrifugation at 12,000 g
for 20 min at 4 °C and stored at À80 °C. The TRAP-PCR-ELISA assay was
4a: Colorless crystals, yield, 70%; mp 177–178 °C; 1H NMR (400 MHz, CDCl3) d
(ppm): 2.06 (s, 3H, –CH3), 3.32 (dd, 1H, J1 = 4.0 Hz, J2 = 8.0 Hz, pyrazole 4-Ha),
3.61 (dd, 1H, J1 = 8.0 Hz, J2 = 16.0 Hz, pyrazole 4-Hb), 4.29 (d, 1H, J = 16.0 Hz,
oxazepane 2-Ha), 5.00 (d, 1H, J = 16.0 Hz, oxazepane 2-Hb), 5.84 (dd, 1H,
J1 = 8.0 Hz, J2 = 12.0 Hz, pyrazole 5-H), 7.67 (d, 1H, J = 1.6 Hz), 7.93 (d, 1H,
J = 2.4 Hz). Anal. Calcd for C12H10Br2N2O2: C, 38.53; H, 2.69; N, 7.49. Found: C,
38.19; H, 3.00; N, 7.25.
performed using
a telomerase detection kit (Roche, Basel, Switzerland)
according to the manufacturer’s protocol. In brief, 2
mixed with 48 L TRAP reaction mixtures. PCR was then initiated at 94 °C,
120 s for predenaturation and performed using 35 cycles each consisting of
94 °C for 30 s, 50 °C for 30 s, 72 °C for 90 s. Then 20 L of PCR products were
lL of cell extracts were
l
l
hybridized to a digoxigenin (DIG)-labeled telomeric repeat specific detection
probe. And the PCR products were immobilized via the biotin-labeled primer to
a streptavidin-coated microtiter plate subsequently. The immobilized DNA
fragment were detected with a peroxidase-conjugated anti-DIG antibody and
visualized following addition of the stop regent. The microtitre plate was
assessed on TECAN Infinite M200 microplate reader (Mannedorf, Switzerland)
at a wavelength of 490 nm, and the final value were presented as mean SD.
27. Western blotting: Mouse anti-TERT monoclonal antibody and rabbit anti-Cyclin
D1 monoclonal antibody were purchased from Abcam (Cambridge, UK) and
vector (Switzerland), respectively. AKT and phospho-AKT antibodies were
purchased from Cell Signaling (Beverly, MA, USA). Secondary antibodies for
goat anti-rabbit immunoglobulin (Ig) G horse radish peroxidase (HRP), goat
anti-mouse IgG HRP was purchased from Santa Cruz Biotechnology (California,
USA). b-actin antibody was obtained from Santa Cruz Biotechnology
(California, USA). Dimethyl sulfoxide (DMSO) was purchased from Sigma Inc.
(St. Louis, MO, USA). Human MGC-803 cells were lysed with RIPA lysis buffer
(Beyotime, China). Whole extracts were prepared, and protein concentration
was detected using a BCA protein assay kit (Beyotime, China). Total protein (30
or 50 mg) from samples were separated by SDS–PAGE and blotted onto a PVDF
membrane (Millipore Corp, Billerica, MA, USA). After blockade of nonspecific
protein binding, nitrocellulose blots were incubated for 1 h with primary
antibodies diluted in TBS/Tween-20 (0.075%) containing 3% Marvel. Mouse
monoclonal antibody recognizing TERT (Abcam, UK) was used 1:500 as was
anti-b-actin (Santa Cruz, USA), rabbit monoclonal antibody recognizing Cyclin
D1 (vector, Switzerland) was used 1:500, rabbit monoclonal anti phospho-AKT
(Cell Signaling, USA) was diluted 1:500 as was anti-AKT (Cell Signaling, USA).
Horseradish peroxidase conjugated anti-mouse and anti-rabbit antibodies
were used as secondary antibodies correspondingly. After extensive washing in
TBS/Tween-20, the blots were processed with distilled water for detection of
antigen using the enhanced chemiluminescence system. Proteins were
visualized with ECL-chemiluminescent kit (ECL-plus, Thermo Scientific).
4b: Colorless crystals, yield, 60%; mp 182–183 °C; 1H NMR (400 MHz, CDCl3) d
(ppm): 2.00 (s, 3H, –CH3), 3.37 (dd, 1H, J1 = 4.0 Hz, J2 = 8.0 Hz, pyrazole 4-Ha),
3.69 (dd, 1H, J1 = 8.0 Hz, J2 = 16.0 Hz, pyrazole 4-Hb), 4.21 (d, 1H, J = 16.0 Hz,
oxazepane 2-Ha), 5.08 (d, 1H, J = 16.0 Hz, oxazepane 2-Hb), 5.77 (dd, 1H,
J1 = 8.0 Hz, J2 = 12.0 Hz, pyrazole 5-H), 7.21–7.40 (m, 3H, ArH‘s). Anal. Calcd for
C
12H11ClN2O2: C, 57.49; H, 4.42; N, 11.17. Found: C, 57.65; H, 4.71; N, 11.03.
4c: Colorless crystals, yield, 71%; mp 166–167 °C; 1H NMR (400 MHz, CDCl3) d
(ppm): 2.11 (s, 3H, –CH3), 3.42 (dd, 1H, J1 = 4.0 Hz, J2 = 8.0 Hz, pyrazole 4-Ha),
3.75 (dd, 1H, J1 = 8.0 Hz, J2 = 16.0 Hz, pyrazole 4-Hb), 3.83 (s, 3H, –OCH3), 4.27
(d, 1H, J = 16.0 Hz, oxazepane 2-Ha), 5.02 (d, 1H, J = 16.0 Hz, oxazepane 2-Hb),
5.64 (dd, 1H, J1 = 8.0 Hz, J2 = 12.0 Hz, pyrazole 5-H), 6.88–7.19 (m, 3H). Anal.
Calcd for C13H14N2O3: C, 63.40; H, 5.73; N, 11.38. Found: C, 63.18; H, 6.02; N,
11.21.
4d: Colorless crystals, yield, 60%; mp 170–172 °C; 1H NMR (400 MHz, CDCl3) d
(ppm): 2.02 (s, 3H, –CH3), 3.33 (dd, 1H, J1 = 4.0 Hz, J2 = 8.0 Hz, pyrazole 4-Ha),
3.65 (dd, 1H, J1 = 8.0 Hz, J2 = 16.0 Hz, pyrazole 4-Hb), 4.15 (d, 1H, J = 16.0 Hz,
oxazepane 2-Ha), 4.97 (d, 1H, J = 16.0 Hz, oxazepane 2-Hb), 5.65 (dd, 1H,
J1 = 8.0 Hz, J2 = 12.0 Hz, pyrazole 5-H), 7.08–7.35 (m, 3H). Anal. Calcd for
C
13H11F3N2O2: C, 54.93; H, 3.90; N, 9.86. Found: C, 55.15; H, 4.04; N, 10.10.
4e: Colorless crystals, yield, 81%; mp 182–183 °C; 1H NMR (400 MHz, CDCl3) d
(ppm): 2.11 (s, 3H, –CH3), 3.39 (dd, 1H, J1 = 4.0 Hz, J2 = 8.0 Hz, pyrazole 4-Ha),
3.71 (dd, 1H, J1 = 8.0 Hz, J2 = 16.0 Hz, pyrazole 4-Hb), 4.19 (d, 1H, J = 16.0 Hz,
oxazepane 2-Ha), 5.04 (d, 1H, J = 16.0 Hz, oxazepane 2-Hb), 5.77 (dd, 1H,
J1 = 8.0 Hz, J2 = 12.0 Hz, pyrazole 5-H), 7.45–7.94 (m, 3H). Anal. Calcd for
C
12H11N3O4: C, 55.17; H, 4.24; N, 16.09. Found: C, 55.42; H, 4.53; N, 15.87.
4f: Colorless crystals, yield, 72%; mp 164–165 °C; 1H NMR (400 MHz, CDCl3) d
(ppm): 2.11 (s, 3H, –CH3), 2.49 (s, 3H, –CH3), 3.30 (dd, 1H, J1 = 4.0 Hz, J2 = 8.0 Hz,
pyrazole 4-Ha), 3.62 (dd, 1H, J1 = 8.0 Hz, J2 = 16.0 Hz, pyrazole 4-Hb), 4.17 (d, 1H,
J = 16.0 Hz, oxazepane 2-Ha), 5.00 (d, 1H, J = 16.0 Hz, oxazepane 2-Hb), 5.63 (dd,
1H, J1 = 8.0 Hz, J2 = 12.0 Hz, pyrazole 5-H), 6.75–7.19 (m, 3H). Anal. Calcd for
C13H14N2O2: C, 67.81; H, 6.13; N, 12.17. Found: C, 68.03; H, 5.87; N, 12.45.