Synthesis and Cytotoxic Activity of Fluorine-Containing 6,7-Dihydroindazolone and 6,7-Dihydrobenzisoxazolone Derivatives

Article abstract of DOI:10.1007/s11094-020-02258-z

The oximes of fluorine-containing 6,7-dihydroindazole-4,5-diones and 6,7-dihydrobenzisoxazole-4-5-diones, 5-[3-fluorobenzoyloxy)imino)]-6,6-dimethyl-3-(3-fluorophenyl)-6,7-dihydrobenz[d]]isoxazol-4(5H)-one, and 5,5-dimethyl-7-phenyl-9-(4-fluorophenyl)-3,5

Full text of DOI:10.1007/s11094-020-02258-z

DOI 10.1007/s11094-020-02258-z
Pharmaceutical Chemistry Journal, Vol. 54, No. 7, October, 2020 (Russian Original Vol. 54, No. 7, July, 2020)
SYNTHESIS AND CYTOTOXIC ACTIVITY OF FLUORINE-CONTAINING
6,7-DIHYDROINDAZOLONE AND 6,7-DIHYDROBENZISOXAZOLONE
DERIVATIVES
T. S. Khlebnikova,1,* Yu. A. Piven’,1 V. G. Zinovich,1
A. V. Baranovskii,1 É. B. Rusanov,2 O. V. Panibrat,1
S. É. Ogurtsova,1 and F. A. Lakhvich1
Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 54, No. 7, pp. 21 – 26, July, 2020.
Original article submitted March 5, 2020.
The oximes of fluorine-containing 6,7-dihydroindazole-4,5-diones and 6,7-dihydrobenzisoxazole-4-5-diones,
5-[3-fluorobenzoyloxy)imino)]-6,6-dimethyl-3-(3-fluorophenyl)-6,7-dihydrobenz[d]]isoxazol-4(5H)-one, and
5,5-dimethyl-7-phenyl-9-(4-fluorophenyl)-3,5,6,7-tetrahydro-2H-pyrazolo[4,3-f]quinoxaline were synthe-
sized. The cytotoxic activity of these compounds against cell lines MCF-7 (human breast carcinoma) and
HepG2 (human hepatocellular carcinoma) was determined. The fluorine-containing 6,7-dihydroindazolone
derivatives synthesized here included substances with marked antiproliferative activity, the mechanism of
which may be linked with induction of apoptosis due to impairment of the regular cell cycle, i.e., delayed of
tumor cells in the G2/M phase.
Keywords: 3-[fluoroalkyl(aryl)]-6,7-dihydroindazolone derivatives; 3-[fluoroalkyl(aryl)]-6,7-dihydrobenz-
isoxazolone derivatives, synthesis, cytotoxic activity.
Many heterocyclic compounds containing pyrazole and
isoxazole rings (both alone and condensed with other mono-
or polycyclic systems) are used as the base structures for the
design of numerous pharmacological agents [1, 2]. Deriva-
tives of indazole [3] and benzisoxazole [4] have a wide spec-
trum of biological activity, including antitumor, anti-HIV,
anti-inflammatory, analgesic, antipsychotic, and other activi-
ties. The main targets of the antitumor actions of indazole
and benzisoxazole derivatives are protein kinases, as well as
heat shock protein HSP90, which is involved in regulating
the cell cycle; this can lead to the induction of apoptosis
[5, 6]. An effective strategy for developing novel therapeutic
agents consists of selective introduction of fluorine atoms
and fluoroalkyl groups into biologically active molecules.
This is illustrated by the continuing increase in the number of
fluorine-containing drugs already approved for use and in
clinical trials [7]. Methods for synthesizing fluorine-contain-
ing heterocyclic structures as potential drugs are currently
under intense development [8, 9].
The aim of the present work was to synthesize a group of
novel 6,7-dihydroindazolone and 6,7-dihydrobenzisoxazo-
lone derivatives, namely the oximes of fluorine-containing
6,7-dihydroindazole-4,5-diones and 6,7-dihydrobenzisoxa-
zole-4-5-diones, as well as 5,5-dimethyl-7-phenyl-9-(4-flu-
orophenyl)-3,5,6,7-tetrahydro-2H-pyrazolo[4,3-f]-quinoxa-
line and 5-[3-fluorobenzoyloxy)imino)]-6,6-dimethyl-3-(3-
fluorophenyl)-6,7-dihydrobenz[d]]isoxazol-4(5H)-one, and
to study their cytotoxic activities against cell lines MCF-7
(human breast carcinoma) and HepG2 (human hepatocellular
carcinoma).
The oximes of fluorine-containing 6,7-dihydroindazole-
4,5-diones and 6,7-dihydrobenzisoxazole-4,5-diones were
prepared using a synthesis approach based on 6,7-dihydroin-
dazolones and 6,7-dihydrobenzisoxazolones including oxi-
dation of these heterocycles on exposure to selenium dioxide
followed by oximation of the resulting dioxo derivatives
with hydroxylamine hydrochloride. Thus, boiling of indazo-
1
Institute of Bioorganic Chemistry, Belarus National Academy of Sciences,
5/2 Kuprevich Street, 22 – 141 Minsk, Belarus.
2
Institute of Organic Chemistry, Ukrainian National Academy of Sciences,
5 Murmanskaya Street, 02660 Kiev, Ukraine.
*
e-mail: khlebnicova@iboch.by
700
0091-150X/20/5407-0700 © 2020 Springer Science+Business Media, LLC

Synthesis and Cytotoxic Activity
701
X
N
X
X
N
R
NH2OH.HCl
Py
N
OH
O
N
SeO2
AcOH
N
N
IIIa - e
N
R
NH2(CH2)2NH2
O
R
O
N
O
N
Ia - e
IIa - e
N
N
IV
F
O
N
SeO2
AcOH
O
N
O
O
NH2OH.HCl
Py
O
3FC6H4COCl
Py
N
N
R
O
O
N
N
O
R
R
OH
O
O
O
F
VIa - c
VIIa - c
Va - c
VIII
F
I – III: R = CF , X = H (a); R = C F , X = H (b); R = 3F-C H , X = H (c); R = 4F-C H , X = H (d); R = 4F-C H , X = F (e); V-VII: R = CF
3
(a); 3F-C H (b); 4F-C H (c).
3
2 5
6
4
6
4
6
4
6
4
6 4
lones Ia – e in glacial acetic acid solution with a threefold
excess of selenium dioxide for 3 h and processing yielded
5,6-dihydroindazole-4,5-diones IIa – e, which without addi-
tional purification were treated with equimolar quantities of
hydroxylamine hydrochloride in pyridine for 20 h to produce
oximes IIIa – e with yields of 43 – 69%. Condensation of
6,7-dihydroindazole-4,5-dione IIc with an equivalent quan-
tity of ethylenediamine in ethanol with boiling for 3 h pro-
of oximes IIIa – e and VIIa – c contained signals from the
C4 carbonyl group carbon atom in the range
d
178.7 – 182.5 ppm and the C5 carbon atom bound to the
hydroxyimine group at d 152.3 – 153.8 ppm. Analysis of
two-dimensional NMR spectra for oxime IIIa showed that
oximation occurred at the C5 carbonyl group, as is also the
case for their nonfluorinated analogs [10, 11]. The position
of C5 was determined from the interaction between protons
at C7 and the methyl protons with this nucleus in the HMBC
spectrum.
duced
3,5,6,7-tetrahydro-2H-pyrazolo[4,3-f]quinoxaline
(IV) with a yield of 58%. The oximes of benzisoxa-
zole-4,5-diones VIIa – c were synthesized in conditions
analogous to those used for preparation of oximes IIIa – e,
though the duration of the oxidation reaction for
benzisoxazolones Va – c had to be increased to 8 h and the
subsequent oximation of the resulting dioxo derivatives
Va – c had to be increased to 72 h. The oximation reaction
was run with a four-fold excess of hydroxylamine hydrochlo-
ride. The yields of oximes VIIa – c were 59 – 62%.
Acylation of the oxime of 6,7-dihydrobenzisoxazolone VIIb
with 3-fluorobenzoic acid chloranhydride in chloroform in
the presence of pyridine for 20 h produced 5-[(3-fluoro-
benzoyloxy)imino]-6,6-dimethyl-3-(3-fluorophenyl)-6,7-di-
hydrobenz[d]isoxazol-4(5H)-one (VIII) with a yield of 80%.
The structures of all compounds synthesized here were
Oximes IIIa – e and VIIa – c can exist in two configura-
tions – (Z) and (E). Structures were confirmed by x-ray anal-
1
confirmed by H, 13C, and 19F NMR spectroscopy and ele-
mental analysis. Along with signals from methyl and ethyl-
ene group protons and aromatic protons, the 1H NMR spectra
of the oximes of IIIa – e and VIIa – c contained signals
from the hydroxyl proton as a singlet at d 14.81 – 15.14 ppm
and d 14.13 – 14.45 ppm respectively, indicating the pres-
ence of strong intramolecular bonds. The 13C NMR spectra
Fig. 1. Molecular structure of oxime IIIc.

702
T. S. Khlebnikova et al.
toluene d 63 ppm (19F). Correlational spectra (HSQC, COSY,
HMBC, NOESY) were recorded and processed using stan-
dard software from Bruker Biospin. Crystals of oxime IIIc
were rhombic, spatial group Pba, a = 12.090(3),
b = 10.522(3), c = 28.570(7) Å, V = 3634.6(16) Å3. XRA of
a IIIc monocrystal was run on a Bruker Smart Apex II
ysis (XRA) of monocrystals of compound IIIc. Figure 1
shows the XRA of compound IIIc, confirming that this com-
pound is in the Z configuration.
EXPERIMENTAL CHEMICAL SECTION
1H, 13C, and 19F NMR spectra were obtained for solu-
diffractometer (lMoKa-radiation, q
25.05°). A total of
max
16,180 reflections were acquired, of which 3219 were inde-
pendent; structures were deciphered by a direct method and
refined by the least squares method and full-matrix
anisotropic approximation run in SHELXTL [12]. Final val-
ues for divergence factors R1(F) 0.0861, wR2(F2) 0.1758,
GOF 1.055 were from reflections with I > 2s(I). Melting
temperatures were measured on a Boetius apparatus. Ele-
mental analysis was run on a EuroVector EA3000 CHNS-O
tions in CDCl using a Bruker Biospin Avance 500 spec-
3
trometer with working frequencies of 500.13, 470.59, and
125.77 MHz for 1H, 19F, and 13C nuclei respectively. The in-
1
ternal standard for H NMR was the residual solvent signal
d 7.26 ppm (CDCl ) and the internal standard for 13C NMR
H
3
was the residual solvent signal d 77.16 ppm (CDCl ), and
C
3
the external standard was the signal from a,a,a-trifluoro-
TABLE 1. Spectral Characteristics of compounds IIIa – e, IV, VIIa – c and VIII
19F NMR spectrum,
1H NMR spectrum, d, ppm
13C NMR spectrum, d, ppm
Compound
d, ppm
IIIa
IIIb
IIIc
1.36 (s, 6H, 2CH3), 3.04 (s, 2H, CH2), 7.46 – 7.51 27.5, 36.9, 40.4, 115.8, 120.1 (q, JC-F 269 Hz), 124.4, 130.0,
(m, 2H, Harom), 7.53 – 7.62 (m, 3H, Harom), 14.81 (s, 137.1, 142.1 (q, JC-F 40 Hz), 150.4, 153.0, 178.7
1H, OH)
–63.31 (3F)
1.35 (s, 6H, 2CH3), 3.05 (s, 2H, CH2), 7.47 – 7.52 27.4, 36.9, 40.2, 110.3 (t.q., JC-F 253, 40 Hz), 117.0, 118.8 (qt,
–82.84 (3F),
–111.39 (2F)
(m, 2H, Harom), 7.53 – 7.62 (m, 3H, Harom), 14.85 (s,
1H, OH)
JC-F 287, 37 Hz), 124.4, 130.0, 137.1, 141.2 (t, JC-F 31 Hz),
150.4, 153.0, 179.0
1.37 (s, 6H, 2CH3), 3.05 (s, 2H, CH2), 7.11 – 7.18
(m, 1H, Harom), 7.39 – 7.48 (m, 1H, Harom),
7.49–7.62 (m, 5H, Harom), 7.85 – 7.92 (m, 1H,
27.5, 37.1, 40.1, 115.9, 116.1, 116.6 (d, JC-F 21 Hz), 124.4,
124.8, 129.4, 129.8, 129.9, 133.1 (d, JC-F 9 Hz), 137.8, 150.1,
152.4, 153.8, 162.8 (d, JC-F 245 Hz), 180.5
–113.00 (1F)
H
arom), 7.92 – 7.98 (m, 1H, Harom), 15.14 (s, 1H, OH)
1.37 (s, 6H, 2CH3), 3.04 (s, 2H, CH2), 7.10 – 7.19 27.5, 37.1, 40.2, 115.4 (d, JC-F 22 Hz), 115.8, 124.4, 127.2,
(m, 2H, Harom), 7.47 – 7.61 (m, 5H, Harom), 129.3, 129.8, 131.1 (d, JC-F 9 Hz), 137.8, 150.0, 152.7, 153.8,
8.09 – 8.16 (m, 1H, Harom), 15.13 (s, 1H, OH) 163.8 (d, JC-F 250 Hz), 180.6
1.37 (s, 6H, 2CH3), 3.00 (s, 2H, CH2), 7.12 – 7.18 27.6, 37.0, 40.2, 115.5 (d, JC-F 22 Hz), 115.8, 116.9 (d, JC-F
IIId
IIIe
–111.19 (1F)
–110.77 (1F),
–111.02 (1F)
(m, 2H, Harom), 7.23 – 7.30 (m, 2H, Harom),
7.48–7.55 (m, 2H, Harom), 8.07 – 8.14 (m, 2H,
arom), 15.10 (s, 1H, OH)
23 Hz), 126.4 (d, JC-F 9 Hz), 127.0, 131.1 (d, JC-F 9 Hz), 133.9,
150.1, 152.8, 153.7, 162.7 (d, JC-F 250 Hz), 163.8 (d, JC-F
250 Hz), 180.5
H
IV
1.26 (s, 6H, 2CH3), 2.98 (s, 2H, CH2), 3.52 (s, 4H, 26.4, 37.1, 40.0, 44.5, 45.3, 114.4, 114.9 (d, JC-F 21 Hz), 124.2,
2CH2), 7.10 (t, 2H, Harom, J 8.6 Hz), 7.40 – 7.48 (m, 128.3, 128.8, 129.6, 131.5 (d, JC-F 8 Hz), 138.7, 143.7, 150.2,
1H, Harom), 7.48 – 7.60 (m, 4H, Harom), 8.18 (dd, J 150.5, 163.2 (d, JC-F 248 Hz), 165.9
8.6 Hz, 5.5, 2H, Harom
-113.22 (1F)
)
VIIa
VIIb
1.43 (s, 6H, 2CH3), 3.19 (s, 2H, CH2), 14.13 (s, 1H, 27.7, 37.0, 40.1, 112.8, 118.8 (q, JC-F 272 Hz), 152.0 (q, JC-F
OH) 41 Hz), 152.3, 177.6, 182.5
-63.53 (3F)
-111.77 (1F)
1.43 (s, 6H, 2CH3), 3.16 (s, 2H, CH2), 7.21 – 7.27 27.7, 37.2, 40.0, 113.7, 116.5 (d, JC-F 24.0 Hz), 118.3 (d, JC-F
(m, 1H, Harom), 7.48 (td, JC-F 8.0 Hz, 5.8, 1H, Harom), 21.0 Hz), 125.1, 128.4 (d, JC-F 8 Hz), 130.4 (d, JC-F 8 Hz),
7.80 (dt, JC-F 9.7 Hz, 2.1, 1H, Harom), 7.85 (dt, JC-F 153.1, 159.8, 162.7 (d, JC-F 247 Hz), 180.1, 182.2
7.7 Hz, 1.3, 1H, Harom), 14.45 (s, 1H, OH)
VIIc
VIII
1.43 (s, 6H, 2CH3), 3.15 (s, 2H, CH2), 7.16 – 7.22 27.7, 37.3, 39.9, 113.7, 116.0 (d, JC-F 22 Hz), 122.6, 131.6 (d,
(m, 2H, Harom), 8.04 – 8.09 (m, 2H, Harom), 14.45 (s, C-F 9 Hz), 153.1, 159.9, 164.7 (d, JC-F 252 Hz), 180.2, 182.1
1H, OH)
-108.28 (1F)
J
1.55 (s, 6H, 2CH3), 3.26 (s, 2H, CH2), 7.20 – 7.28 25.7, 38.6, 41.4, 115.3, 116.2 (d, JC-F 24 Hz), 117.0 (d, JC-F
-111.71 - (-111.85)
(m), -111.48 -
(m, 1H, Harom), 7.30 (td, J 8.3 Hz, 2.7, 1H, Harom),
7.41 – 7.57 (m, 2H, Harom), 7.76 (dt, J 9.3 Hz, 2.0,
1H, Harom), 7.83 – 7.91 (m, 2H, Harom), 7.91 – 8.00
24 Hz), 118.3 (d, JC-F 21 Hz), 121.0 (d, JC-F 21 Hz), 124.9 (d,
C-F 3 Hz), 125.9 (d, JC-F 3 Hz), 128.6 (d, JC-F 9 Hz), 130.4 (d,
C-F 9 Hz), 130.5 (d, JC-F 2 Hz), 130.5 (d, JC-F 3 Hz), 159.4,
162.4, 162.7 (d, JC-F 248 Hz), 162.8 (d, JC-F 247 Hz), 164.8,
176.2, 180.6
J
(-111.65) (m)
J
(m, 1H, Harom
)

Synthesis and Cytotoxic Activity
703
analyzer. Elemental analysis data were consistent with calcu-
lated values. Column chromatography (CC) was run on silica
gel eluted with a mixture of ethyl acetate and petroleum
ether. 6,7-Dihyroindazolones Ia – e [13, 14] and 6,7-dihyd-
robenzisoxazolones Va – c [15, 16] were prepared by stan-
dard methods.
roform was removed in a rotary evaporator. The residue was
dissolved in 10 ml pyridine and 0.70 g (10.0 mmol) of
hydroxylamine hydrochloride was added and mixed at room
temperature for 72 h. The reaction mix was poured into a
mixture of 120 ml of 18% HCl and 40 g of ice and extracted
with chloroform (5 ´ 30 ml). The combined organic layers
were dried over Na SO . Solvent was removed in a rotary
Oximes of 3-[fluoroalkyl(aryl)]-6,7-dihydroindazole-
4,5-diones (IIIa – e). Selenium dioxide (0.27 g, 2.4 mmol)
was added to a solution of 0.8 mmol of 6,7-dihydroindazo-
lone Ia – e in 25 ml of glacial acetic acid and the solution
was boiled for 3 h. Acetic acid was removed on a rotary
evaporator. Chloroform (40 ml) was added to the residue and
filtered and the chloroform was removed in a rotary evapora-
tor. The residue was dissolved in 4 ml of pyridine and 0.06 g
(0.8 mmol) of hydroxylamine hydrochloride was added with
mixing for 20 h at room temperature. The reaction mix was
poured into a mixture of 60 ml of 18% HCl and 20 g of ice
and extracted with chloroform (5 ´ 20 ml). The combined or-
ganic layers were dried over Na SO . Solvent was removed
2
4
evaporator and CC was used to extract oximes VIIa – c as
colorless crystals.
(Z)-5-(Hydroxyimino)-6,6-dimethyl-3-(trifluorome-
thyl)-6,7-dihydrobenz[d]isoxazol-4(5Í)-one (VIIa). The
yield was 61%. T was 158 – 161°C. C H F N O .
m
10
9
3
2
3
(Z)-5-(Hydroxyimino)-6,6-dimethyl-3-(3-fluorophe-
nyl)-6,7-dihydrobenz[d]isoxazol-4(5Í)-one (VIIb). The
yield was 62%. T was 138 – 140°C. C H FN O .
m
15 13
2
3
(Z)-5-(Hydroxyimino)-6,6-dimethyl-3-(4-fluorophe-
nyl)-6,7-dihydrobenzî[d]isoxazol-4(5Í)-one (VIIc). The
yield was 59%. T was 145 – 148°C. C H FN O .
m
15 13
2
3
(Z)-6,6-Dimethyl-5-[(3-fluorobenzoyloxy)imino]-3-(3-
fluorophenyl)-6,7-dihydrobenz[d]isoxazol-4(5H)-one
(VIII). Pyridine (0.1 ml) was added to a solution of 0.05 g
(0.17 mmol) of oxime VIIb in 15 ml of chloroform, fol-
lowed by 0.036 g (0.26 mmol) of 3-fluorobenzoic acid
chloranhydride. The reaction was mixed for 20 h, washed
with 3% HCl (2 ´ 5 ml), and dried over Na SO . Compound
2
4
on a rotary evaporator and CC was used to extract oximes
IIIa – e as colorless crystals.
(Z)-5-(Hydroxyimino)-6,6-dimethyl-1-phenyl-3-triflu-
oromethyl-1,5,6,7-tetrahydro-4H-indazol-4-one
(IIIa).
The yield was 55%. T was 107 – 110°C. C H F N O .
m
16 14
3
3
2
(Z)-5-(Hydroxyimino)-6,6-dimethyl-1-phenyl-3-per-
fluoroethyl-1,5,6,7-tetrahydro-4H-indazol-4-one (IIIb).
The yield was 69%. T was 111 – 114°C. C H F N O .
2
4
VIII was extracted from the residue by CC as colorless crys-
tals. The yield was 0.057 g (80%). T was 152 – 155°C.
m
m
17 14
5
3
2
C H F N O .
(Z)-5-(Hydroxyimino)-6,6-dimethyl-1-phenyl-3-(3-flu-
orophenyl)-1,5,6,7-tetrahydro-4H-indazol-4-one (IIIc).
The yield was 43%. T was 145 – 148°C. C H FN O .
22 16
2
2
4
EXPERIMENTAL BIOLOGICAL SECTION
m
21 18
3
2
(Z)-5-(Hydroxyimino)-6,6-dimethyl-1-phenyl-3-(4-flu-
orophenyl)-1,5,6,7-tetrahydro-4H-indazol-4-one (IIId).
The yield was 48%. T was 159 – 162°C. C H FN O .
Studies of the cytotoxicity of 6,7-dihydroindazolone de-
rivatives IIIa – e and 6,7,-dihydrobenzisoxazolone deriva-
tives VIIa – c, VIII were performed using the MTT test [17].
MCF-7 (human breast carcinoma) and HepG2 (human
hepatocellular carcinoma) cells obtained from the Russian
Collection of Cell Cultures (Institute of Cytology, Russian
Academy of Sciences, St. Petersburg) were cultured in
DMEM and MEM nutrient media respectively. Media were
supplemented with 10% fetal bovine serum and a mixture of
m
21 18
3
2
(Z)-5-(Hydroxyimino)-6,6-dimethyl-1,3-bis(4-fluoro-
phenyl)-1,5,6,7-tetrahydro-4H-indazol-4-one (IIIe). The
yield was 66%. T was 147 – 149°C. C H F N O .
m
21 17
2
3
2
5,5-Dimethyl-7-phenyl-9-(4-fluorophenyl)-3,5,6,7-tet-
rahydro-2H-pyrazolo[4,3-f]quinoxaline (IV). Selenium di-
oxide (0.17 g, 1.5 mmol) was added to a solution of 0.17 g
(0.5 mmol) of indazolone Id in 15 ml of glacial acetic acid
and the mixture was boiled for 3 h. Acetic acid was removed
in a rotary evaporator. Chloroform (40 ml) was added to the
residue and filtered; the chloroform was evaporated in a ro-
tary evaporator. The residue was dissolved in 15 ml of etha-
nol and 0.03 ml (0.5 mmol) of ethylenediamine was added
and the mixture was boiled for 3 h. Solvent was removed in a
rotary evaporator and CC was used to extract 0.104 g (58%)
of compound IV from the residue in the form of colorless
crystals. T was 158 – 161°C. C H FN .
TABLE 2. Effects of Compounds IIIb – e on Cell Cycle Phases of
HepG2 Tumor Cells
Cell cycle phase, %
Compound
Apoptosis, %
G1
S
G2/M
Control
63.07 ± 4.56 13.30 ± 3.47 23.88 ± 3.40 1.56 ± 0.38
36.55 ± 3.25 2.54 ± 0.72 60.91 ± 3.97 32.63 ± 2.26
37.44 ± 6.99 12.57 ± 3.52 49.99 ± 3.47 19.50 ± 3.25
38.01 ± 3.95 6.63 ± 4.57 55.36 ± 0.62 13.97 ± 4.19
31.00 ± 0.63 21.25 ± 3.99 46.92 ± 3.90 19.59 ± 3.14
40.68 ± 2.39 19.65 ± 2.35 39.69 ± 4.74 15.44 ± 4.23
m
23 21
4
Cisplatin
IIIb
Oximes of 3-[fluoroalkyl(aryl)]-6,7-dihydrobenzisoxa-
zole-4,5-diones (VIIa – c). Selenium dioxide (0.83 g,
7.5 mmol) was added to a solution of 2.5 mmol of isoxa-
zolone Va – c in 45 ml of glacial acetic acid and boiled for
8 h. Acetic acid was removed in a rotary evaporator. Chloro-
form (80 ml) was added to the residue and filtered; the chlo-
IIIc
IIId
IIIe

704
T. S. Khlebnikova et al.
140
120
100
80
140
120
100
80
IIIa
IIIb
IIIc
IIId
IIIe
IV
VIIa
VIIb
VIIc
60
60
VIII
40
40
20
20
0
0
10
30
50
70
90
110
10
30
50
70
90
110
Compounds, mM
Compounds, mM
HepG2
HepG2
140
120
100
80
160
140
120
100
80
IIIa
IIIb
IIIc
IIId
IIIe
IV
VIIa
VIIb
VIIc
VIII
60
60
40
40
20
20
0
0
10
30
50
70
90
110
10
30
50
70
90
110
Compounds, mM
Compounds, mM
Fig. 2. Viability of MCF-7 and HepG2 cells after treatment with solutions of compounds IIIa – e, IV, VIIa – c, and VIII.
medium containing test compounds at the IC determined in
penicillin (100 U/ml), streptomycin (100 mg/ml), and
amphotericin B (25 mg/ml) and incubation was at 37°C in a
50
the MTT test. After 48 h, cells were removed from plates and
fixed with cold 70% ethanol at -20°C. Cells were then
humid atmosphere containing 5% CO . Cells of both lines
2
stained with propidium iodide (50 mg/ml) + RNase
A
were seeded into 96-well plates at a concentration of
7000 cells/well and were incubated for 24 h. Test substances
were added to concentrations of 0.1, 0.5, 1, 10, 50, and
100 mM. The concentration of stock solutions of test com-
pounds was 20 mM. Solutions were added to the final con-
centrations in incubation medium. Controls were supple-
mented with 0.5% solvent. After 72 h, each well was supple-
mented with 20 ml MTT (5 mg/ml). After 3 h exposure at
(1 mg/ml) in phosphate buffer for 30 min in the dark at room
temperature. The reference agent was cisplatin, with IC
50
17 mM. Experiments were performed in three repeats. Statis-
tical significance was p < 0.05.
Results from the MTT test are shown in Fig. 2. Among
the study compounds, only benzisoxazolone oxime VIIa had
no cytotoxic effect in relation to both lines. The activity of
the other compounds was dose-dependent: low concentra-
tions (0.1 – 1 mM) stimulated tumor cell growth, while high
concentrations (>10 mM) inhibited growth. Indazolone deriv-
atives IIIb – e, IV decreased tumor cell growth in both lines
at a concentration of 50 mM by ?50%, while a concentration
of 100 mM decreased the growth of HepG2 cells by 80% and
that of MCF-7 by 64%. Isoxazolone derivatives VIIb, c and
VIII at 50 mM inhibited the growth of cells of both lines less
effectively: by only 30 – 40%. Overall, human liver carci-
noma cells HepG2 were more sensitive to the actions of the
study compounds.
37°C and 5% CO , granules of reduced product (formazan)
2
were dissolved in 200 ml of DMSO and the optical density of
the solution was measured at 570 nm on an AIF-M/340 plate
reader. Percent viability = OD experimental wells/OD con-
trol wells ´100%, where OD is optical density.
Drug concentrations inhibiting cell viability by 50%
(IC ) were determined graphically using the dose curve in
50
Excel. All experiments were run in three repeats. Statistical
significance was at p < 0.05. Analysis of the effects of syn-
thetic compounds on the distribution of HepG2 cells in the
cell cycle and the types of cell death (apoptosis or necrosis)
induced was performed using a Beckman Coulter FC500
flow cytometer in the FL-3 channel. Levels of apoptosis
were determined using the “subG1” peak. Analyses were run
using Kaluza software (Beckman Coulter).
The effects of indazolone derivatives IIIb – e most ac-
tive against HepG2 cells on the distribution of HepG2 cells
by cell cycle phase and the type of cell death (apoptosis of
the necrosis) induced by compounds were studied (Table 2;
HepG2 cells were seeded at a rate of 300,000 cells/Petri
dish (6 cm2). After 24 h, culture medium was replaced with
Figs. 3 and 4). The IC values of these compounds were in
50
the range 40 – 60 mM. Compounds IIIb – e terminated the

Synthesis and Cytotoxic Activity
705
b
a
c
f
d
e
Fig. 3. Distributions of HepG2 cells in the cell cycle after exposure to test compounds: a) controls; b) IIIb; c) IIIc; d) IIId; e) IIIe; f) cisplatin.
cell cycle in the G2/M phase, increasing the proportion of
cells in this phase twofold from the level in controls and de-
creasing the proportion in G1. They had virtually no effect
on the S phase (except compound IIIc). The result of termi-
nation of the cell cycle was an increase in the level of
apoptosis from 2% in controls to 13 – 19% with study com-
pounds. Cisplatin increased the level of apoptosis to 33%.
The mechanism of the cytotoxic action of study compounds
in relation to tumor cells appears to be associated with induc-
tion of cell death by the mechanism of apoptosis due to im-
pairment to the regular cell cycle, i.e., delay of tumor cells in
the G2/M phase.
Thus, among the fluorine-containing 6,7-dihydroindazo-
lone derivatives synthesized here, substances with marked
antiproliferative activity were found, which is evidence of
the value of continuing studies of this series of compounds.
b
a
c
d
f
e
Fig. 4. Level of apoptosis of HepG2 cells after exposure to test compounds: a) controls; b) IIIb; c) IIIc; d) IIId; e) IIIe; f) cisplatin.

706
T. S. Khlebnikova et al.
G. A. Selivanova, E. V. Panteleeva, et al., Rus. Chem. Rev.,
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