3-Nitro-2-trifluoromethyl-2H-chromenes and products of their reduction. Synthesis and cytotoxicity evaluation

Article abstract of DOI:10.1007/s11172-014-0775-4

A series of 3-nitro-2-trifluoromethyl-2H-chromenes was synthesized by the reactions of salicylaldehyde derivatives with 3,3,3-trifluoro-1-nitroprop-1-ene. Further transformations of the synthesized chromenes gave hitherto unknown 3-amino-2-trifluoromethylchromanes promising as precursors for the synthesis of fused heterocyclic systems. Cytotoxicity assay first revealed the pronounced activity of several 2-nitro-2-trifluoromethyl-2H-chromenes against human tumor cell lines.

Full text of DOI:10.1007/s11172-014-0775-4

Russian Chemical Bulletin, International Edition, Vol. 63, No. 11, pp. 2551—2555, November, 2014
2551
3ꢀNitroꢀ2ꢀtrifluoromethylꢀ2Hꢀchromenes and products of their reduction.
Synthesis and cytotoxicity evaluation*
M. A. Baryshnikova,a A. Yu. Volkonskii,b D. V. Gusev,b N. O. Labodneva,c A. L. Sigan,b
N. G. Yakunina,a and N. D. Chkanikovb
aResearch Institute of Experimental Diagnostics and Tumor Therapy,
N. N. Blokhin Russian Cancer Research Center, Russian Academy of Sciences,
24 Kashirskoe shosse, 115478 Moscow, Russian Federation.
Fax: +7 (495) 324 6037. Eꢀmail: ma_ba@mail.ru
bA. N. Nesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences,
28 ul. Vavilova, 119991 Moscow, Russian Federation.
Fax: +7 (495) 135 5085. Eꢀmail: dgusev@ineos.ac.ru
cD. I. Mendeleev Russian University of Chemistry and Technology,
9 Miusskaya pl., 125047 Moscow, Russian Federation.
Fax: +7 (495) 135 5085. Eꢀmail: n.labodneva@gmail.com
A series of 3ꢀnitroꢀ2ꢀtrifluoromethylꢀ2Hꢀchromenes was synthesized by the reactions of
salicylaldehyde derivatives with 3,3,3ꢀtrifluoroꢀ1ꢀnitropropꢀ1ꢀene. Further transformations of
the synthesized chromenes gave hitherto unknown 3ꢀaminoꢀ2ꢀtrifluoromethylchromanes promꢀ
ising as precursors for the synthesis of fused heterocyclic systems. Cytotoxicity assay first revealed
the pronounced activity of several 2ꢀnitroꢀ2ꢀtrifluoromethylꢀ2Hꢀchromenes against human
tumor cell lines.
Key words: salicylaldehyde, 3,3,3ꢀtrifluoroꢀ1ꢀnitropropꢀ1ꢀene, condensation, 3ꢀnitroꢀ2ꢀ
trifluoromethylꢀ2Hꢀchromenes, 3ꢀaminoꢀ2ꢀtrifluoromethylchromanes, cytotoxicity.
Earlier, we by the reaction of a naturally occurring
phenolic aldehyde gossypol (2,2´ꢀdi(1,6,7ꢀtrihydroxyꢀ5ꢀ
isopropylꢀ3ꢀmethylꢀ8ꢀnaphthaldehyde) with 3,3,3ꢀtriꢀ
fluoroꢀ1ꢀnitropropꢀ1ꢀene have synthesized 6,6´ꢀdiisoproꢀ
pylꢀ8,8´ꢀdimethylꢀ2,2´ꢀdinitroꢀ3,3´ꢀbis(trifluoromethyl)ꢀ
3H,3´Hꢀ[9,9´]di[benzo(f)chromenyl]ꢀ5,10,5´,10´ꢀtetraol.
This compound is of interest since it comprises two fused
tricyclic systems with the chromene skeletons substituted
with two trifluoromethyl and two nitro groups. Biomediꢀ
cal evaluation of this compound shows extension of the
biological activity spectrum of the parent gossypol owing
to the appearance of pronounced antitumor and fungicidꢀ
al activities.1 In this case, new valuable properties are apꢀ
parently due to the presence of the trifluoromethylꢀsubstiꢀ
tuted chromene cores.
Results and Discussion
2HꢀBenzopyranes (chromenes) are a class of naturally
occurring compounds found in a wide variety of plants.
Chromene core is the essential parts and the structural
determinant responsible for high biological activity of comꢀ
plex natural products (flavonoids, anthocyanins, etc.).2
Chromeneꢀbased synthetic compounds bearing various
substituents and functional groups have contribute subꢀ
stantially to the development of highly efficient theraꢀ
peutics possessing anticoagulant, antiꢀoxidant, antitumor,
antiviral, and HIV integrase inhibitory activities.3—6 The
high reactivity and structural diversity of chromenes
promote the development of novel synthetic approaches
towards these compounds and identifying the areas of their
practical applications.
Over the past several decades, the introduction of the
fluorine atoms into organic molecules has been used as an
efficient strategy to extend therapeutic efficacy and pharꢀ
macological activity of parent compounds.7 Until recentꢀ
ly, only few methods to access fluorinated or polyfluoroꢀ
alkylated chromenes have been developed. These multiꢀ
step syntheses require poorly available reagents and proꢀ
ceed with unsatisfactory yields.8—10 An convenient apꢀ
The aim of the present work is the synthesis of a library
of 2ꢀtrifluoromethylꢀsubstituted 3ꢀnitroꢀ2Hꢀchromenes
and 3ꢀaminochromanes and in vitro evaluation of their
biological activity.
* Dedicated to Academician of the Russian Academy of Sciences
Yu. N. Bubnov on the occasion of his 80th birthday and to the
60th anniversary of A. N. Nesmeyanov Institute of Organoꢀ
element Compounds of the Russian Academy of Sciences.
Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 11, pp. 2551—2555, November, 2014.
1066ꢀ5285/14/6311ꢀ2551 © 2014 Springer Science+Business Media, Inc.

2552
Russ.Chem.Bull., Int.Ed., Vol. 63, No. 11, November, 2014
Baryshnikova et al.
Scheme 1
Compound 1—3
R1
H
H
H
H
R2
H
H
Br
H
R3
H
H
H
H
R4
OMe
H
Br
Me
Compound 1—3
f
g
h
i
R1
H
H
H
H
R2
Cl
NO2
Me
R3
H
H
H
H
R4
H
H
H
H
a
b
c
d
OMe
proach for the synthesis of 3ꢀnitroꢀ2ꢀtrifluoromethylꢀ2Hꢀ
chromenes was first suggested by Korotaev and coꢀworkꢀ
ers.11 This approach involves catalytic condensation of
salicylic aldehydes with 3,3,3ꢀtrifluoroꢀ1ꢀnitropropꢀ1ꢀene
via nucleophilic addition of the hydroxy groups at the
activated C=C bond followed by the ring closure to give
the corresponding 2Hꢀchromene derivatives. Later, the
general character of the suggested approach was demonꢀ
strated by the reaction of various nitroalkenes with salicylꢀ
aldehyde derivatives and ketimines of oꢀhydroxyacetoꢀ
phenones.12,13
We also employed this synthetic pathway to obtain
a wide variety of novel trifluoromethylated chromenes for
the detailed study (Scheme 1). The reactions were carried
out either in dichloromethane (method A) or in propaneꢀ
2ꢀol (method B). To a mixture of salicylaldehyde derivaꢀ
tives 1a—i and 3,3,3ꢀtrifuoroꢀ1ꢀnitropropꢀ1ꢀene, a soluꢀ
tion of triethylamine used as a catalyst was added dropꢀ
wise over a period of 1 h at 5—10 C. When propanꢀ2ꢀol
was used as the solvent (5—10 C, then 20 C, reaction
time of 24 h), precipitation of the crystalline reaction prodꢀ
ucts, 3ꢀnitroꢀ2ꢀtrifuoromethylꢀ2Hꢀchromenes bearing the
corresponding substituents in the aromatic ring, was obꢀ
served in the most cases. Only in the cases of 5ꢀmethoxyꢀ
(1a) and 5ꢀmethylsalicylaldehydes (1d), the reactions carꢀ
ried out by method A require 40 h to be completed (TLC
data), which is due apparently to the electronꢀwithdrawꢀ
ing effect of the substituents decreasing the electrophiliciꢀ
ty of the carbonyl group of the starting compounds. It
should be noted that in these cases, no precipitation
occurred and the products were isolated after certain workꢀ
up of the reaction mixtures. The elemental analyses data
1
and H and 19F NMR spectral characteristics for comꢀ
pounds 2a—i are given in Tables 1 and 2, respectively.
Mass spectrometry of compounds 2a—i revealed good
agreement between measured and calculated m/z values.
Table 1. Physicochemical characteristics and microanalysis data for compounds 2a—i
Comꢀ
pound
M.p/C
Yield
(%)
Found
Calculated
Molecular
formula
(%)
С
H
N
2a
2b
2c
2d
2e
2f
138—142
88—91
89
67
56
33
76
84
80
26
60
47.80
48.01
48.51
48.99
29.67
29.81
50.67
50.98
37.00
37.07
42.54
42.96
41.38
41.40
50.45
50.98
47.89
48.01
2.85
2.93
2.52
2.47
1.10
1.00
3.21
3.11
1.59
1.56
1.92
1.80
1.76
1.74
3.24
3.11
2.95
2.93
5.01
5.09
5.70
5.71
3.49
3.48
5.32
5.40
4.32
4.32
4.87
5.01
9.53
9.65
5.42
5.40
5.04
5.09
C11H8F3NO4
C10H6F3NO3
C10H4Br2F3NO3
C11H8F3NO3
C10H5BrF3NO3
C10H5ClF3NO3
C10H5F3N2O5
C11H8F3NO3
C11H8F3NO4
94—97
97—99
105—107
101—104
129—131
79—81
2g
2h
2i
84—85

3ꢀNitroꢀ2ꢀtrifluoromethylꢀ2Hꢀchromenes
Russ.Chem.Bull., Int.Ed., Vol. 63, No. 11, November, 2014 2553
Table 2. 1H and 19F NMR spectra (CDCl3) of compounds 2a—i
tivity for possible application as agrochemicals have been
described.14,15 However, no data on the synthesis of
3ꢀaminoꢀ2ꢀtrifluoromethylchromanes and their applicaꢀ
tions as valuable syntones have been published. To achieve
this goal, we performed exhaustive reduction of the double
bond and nitro group in compounds 2a—f,h,i with borane
in THF (see Scheme 1). The corresponding 3ꢀaminoꢀ2ꢀ
trifluoromethylchromanes 3a—f,h,i were isolated after cerꢀ
tain workꢀup. The elemental analysis data and NMR specꢀ
tral data for compounds 3a—f,h,i are summarized in
Tables 3 and 4, respectively.
Comꢀ
pound
NMR,  (J/Hz)
1H
19F
2a
2b
8.11 (s, 1 H, СH=); 7.02 (m, 3 H, R1,
R2, R3); 6.15 (m, 1 H, CHCF3); 3.92
(s, 1 H, OМe)
8.12 (s, 1 H, СН=); 7.45 (m, 1 H, R1);
7.37 (m, 1 H, R4); 7.08 (m, 2 H, R2, R3);
6.06 (m, 1 H, CHCF3)
8.02 (s, 1 H, CH=); 7.79 (s, 1 H, R1);
7.46 (s, 1 H, R3); 6.19 (m, 1 H, CHCF3)
8.17 (s, 1 H, CH=); 7.31 (m, 2 H, R1, R3); –0.37
7.05 (m, 1 H, R2); 6.19 (m, CHCF3);
2.34 (s, 3 H, Me)
–0.15
–0.16
–0.09
2c
2d
Note that in all described reactions, the product yields
do not exceed 50% regardless of the substrate nature. It
was found that all reaction mixtures obtained after isolaꢀ
tion of target 3ꢀaminoꢀ2ꢀtrifluoromethylchromanes conꢀ
tain 3ꢀhydroxylaminoꢀ2ꢀtrifluoromethylchromanes as the
main side products. In the case of compound 3c, readily
crystallized side product was isolated, to which a structure
of Nꢀ[6,8ꢀdibromoꢀ2ꢀ(trifluoromethyl)ꢀ3,4ꢀdihydroꢀ2Hꢀ
chromenꢀ3ꢀyl]hydroxylamine (4) was ascribed. Comꢀ
pound 4 is stable under reducing conditions (B2H6, THF)
2e
8.55 (s, 1 H, CH=); 7.91 (m, 1 H, R1);
7.68 (m, 1 H, R3); 7.14 (m, 1 H, R4);
6.63 (m, 1 H, CHCF3)
1.53
2f
8.05 (s, 1 H, CH=); 7.38 (m, 2 H, R3, R4); –1.14
7.04 (m, 1 H, R1); 6.08 (m, 1 H, CHCF3)
2g
2h
8.37 (s, 2 H, R1, R3); 8.20 (s, 1 H, CH=);
7.30 (s, 2 H, R4); 6.25 (m, 1 H, CHCF3)
8.08 (s, 1 H, CH=); 7.24 (m, 1 H, R1);
7.15 (m, 1 H, R3); 6.96 (m, 1 H, R4); 6.05
(m, 1 H, CHCF3); 2.33 (s, 1 H, Me)
–1.18
–0.09
1
even at prolong reflux. H and 19F NMR spectroscopy
revealed that formation of 3ꢀhydroxylaminoꢀsubstituted
chromanes is a general feature of these reactions and causes
the decrease in the target product yields.
8.08 (s, 1 H, CH=); 7.00 (m, 2 H, R3, R4);
6.85 (m, 1 H, R1); 6.04 (m, 1 H, CHCF3);
3.81 (s, 3 H, OMe)
0.04
Cytotoxicity assay of several selected compounds
(2a,b,d,e and 3e) was performed in vitro in Research Instiꢀ
tute of Experimental Diagnostics and Tumor Therapy of
N. N. Blokhin Russian Cancer Research Center of RAS.
Cytotoxicity of compounds 2a,b,d,e and 3e was examined
on human cell lines, namely, Jurkat (Tꢀlymphoblastic
leucosis), SKOVꢀ3 (ovarian carcinoma cells), HCTꢀ116
(colon cancer cells), A549 (lung adenocarcinoma), using
2i
To date, several examples of the involvement of 3ꢀnitroꢀ
2ꢀtrihalomethylꢀ2Hꢀchromenes in the synthesis of the
chromaneꢀbased compounds with potential biological acꢀ
Table 3. Physicochemical characteristics and microanalysis data for compounds 3a—f,h,i
Compoꢀ
und
Yield
(%)
Found
Calculated
Molecular
formula
Note
(%)
С
H
N
3a
3b
3c
3d
3e
3f
43
44
46
49
44
45
48
50
50.78
51.55
43.45
44.32
29.12
29.19
49.23
49.36
36.01
36.12
40.98
41.69
49.03
49.36
46.57
46.57
5.01
4.81
4.56
4.34
2.20
2.20
4.98
4.90
3.11
3.03
3.67
3.50
4.78
4.90
4.87
4.62
3.15
3.34
4.13
4.31
3.35
3.40
5.25
5.23
4.30
4.21
4.65
4.86
5.12
5.23
4.67
4.94
C11H12F3NO2•C7H8O3S
C10H10F3NO•C2H2O4•H2O
C10H8Br2F3NO•HCl
C11H12F3NO•HCl
Tosylate
Oxalate
Hydrochloride
Hydrochloride
Hydrochloride
Hydrochloride
Hydrochloride
Hydrochloride
C10H9BrF3NO•HCl
C10H9ClF3NO•HCl
C11H12F3NO•HCl
3h
3i
C11H12F3NO2•HCl

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Russ.Chem.Bull., Int.Ed., Vol. 63, No. 11, November, 2014
Baryshnikova et al.
Table 4. 1H and 19F NMR spectra (CDCl3) of compounds 3a—f,h,i
Table 5. In vitro cytotoxicity of compounds 2a,b,d,e and 3e
Comꢀ
pound
NMR,  (J/Hz)
Comꢀ Concentration
pound
Cell survival rate (%)
/mol L–1
1H
19F
Jurkat SKOVꢀ3 HCTꢀ116 A549
3a
8.34 (s, 3 H, NH3+); 7.35 (m, 4 H, Tos);
7.01—6.75 (m, 3 H, R1, R2, R3); 5.12
(m, 1 H, CHCF3); 4.19 (br.s, 1 H, CH2CH—);
3.78 (s, 3 H, OMe); 3.2 (m, 1 H, CH2CH—);
2.99 (m, 1 H, CH2CH—); 2.28 (s, 3 H, Me, Tos)
7.20—6.93 (m, 4 H, R1, R2, R3, R4); 4.97
(m, 1 H, CHCF3); 3.98 (m, 4 H, CH2CH—,
NH3+); 3.25 (m, 1 H, CН2CH—); 2.86
(m, 1 H, CH2CH—)
7.55 (s, 1 H, R3); 7.16 (s, 1 H, R1); 4.36
(m, 1 H, CHCF3); 3.70 (br.s, 1 H,
4.70
2a
1•10–7
1•10–6
1•10–5
1•10–4
1•10–7
1•10–6
1•10–5
1•10–4
1•10–7
1•10–6
1•10–5
1•10–4
1•10–7
1•10–6
1•10–5
1•10–4
1•10–7
1•10–6
1•10–5
1•10–4
87
79
33
10
88
84
54
11
73
71
57
19
95
84
50
19
89
87
87
72
194
101
176
117
100
104
101
142
183
116
188
131
195
186
198
123
193
195
193
182
198
102
173
119
193
188
186
111
185
194
194
112
187
182
186
110
196
195
105
180
191
195
190
119
198
191
193
115
186
181
176
117
185
186
180
114
101
100
102
183
2b
2d
2e
3e
3b
5.16
3.66
4.43
3c*
3d
CH2CH—); 3.16 (m, 1 H, CH2CH—); 2.75
(m, 1 H, CH2CH—); 1.32 (s, 2 H, NH2)
8.68 (s, 3 H, NH3+); 7.03—6.86 (m, 3 H, R1,
R2, R3); 5.12 (m, 1 H, CHCF3); 4.16
(br.s, 1 H, CH2CH—); 3.48 (br.s, 1 H,
CH2CH—); 3.27 (m, 1 H, CH2CH—);
2.18 (s, 3 H, Me)
8.78 (s, 3 H, NH3+); 7.46—7.37 (m, 2 H, R2,
R3); 6.98 (m, 1 H, R1); 5.21 (m, 1 H, CHCF3);
4.19 (br.s, 1 H, CH2CH—); 3.53—3.23
(m, 2 H, CH2CH—, CH2CH—)
8.78 (s, 3 H, NH3+); 7.29 (m, 2 H, R3, R4);
7.03 (m, 1 H, R1); 5.22 (m, 1 H, CHCF3);
4.19 (br.s, 1 H, CH2CH—); 3.29 (m, 1H,
CH2CH—); 3.29 (m, 1 H, CH2CH—)
8.76 (s, 3 H, NH3+); 7.03—6.86 (m, 3 H, R1,
R3, R4); 5.12 (m, 1 H, CHCF3); 4.11
(br.s, 1 H, CH2—СH); 3.28 (m, 2 H,
CH2—CH, CH2—CH); 2.23 (s, 3 H, Me)
3e
3f
4.77
4.77
4.78
4.79
In summary, the obtained results show a good promise
for further studies of physiological activities of this class of
compounds.
3h
3i
Experimental
1
H and 19F NMR spectra were run on Bruker AMXꢀ400 and
Bruker AMXꢀ300 instruments with working frequencies of 400.13
and 376.50 MHz, respectively, at 20 C. The chemical shifts are
given in the  scale relative to the residual solvent signal (1H)
and CF3COOH (19F, an external standard). Mass spectra (EI,
70 eV) were obtained with a Kratos MSꢀ890 instrument. The startꢀ
ing compounds 1a—i are commercially available. 3,3,3ꢀTrifluoꢀ
roꢀ1ꢀnitropropꢀ1ꢀene was synthesized by the known procedure.16
8ꢀMethoxyꢀ3ꢀnitroꢀ2ꢀtrifluoromethylꢀ2Hꢀchromene (2a). A.
To a solution of 3ꢀmethoxysalicylaldehyde 1a (0.25 g, 1.61 mmol)
and 3,3,3ꢀtrifluoroꢀ1ꢀnitropropꢀ1ꢀene (0.23 g, 1.63 mmol) in
dichloromethane (1.5 mL), a solution of triethylamine (16 mg,
0.16 mmol) in dichloromethane (1.5 mL) was added dropwise
over a period of 1 h at 5 C. The obtained solution was stirred for
40 h, washed with 1% aqueous HCl and water (315 mL). The
aqueous layer was extracted with dichloromethane (210 mL),
the organic layer was separated, dried with sodium sulfate, and
the solvent was removed in vacuo. The dark brown residue was
recrystallized from hexane, the crystals were washed with hexꢀ
ane and dried. Yield 87%. Compounds 2b—i were synthesized
similarly. Physicochemical characteristics and spectral data for
compounds 2a—i are given in Tables 1 and 2.
8.73 (s, 3 H, NH3+); 6.94—6.78 (m, 3 H, R1,
R3, R4); 5.1 (s, 1 H, CHCF3); 4.15
(br.s, 1 H, CH2CH—); 3.70 (s, 3 H, OMe);
3.25 (m, 2 H, CH2CH—, CH2CH—)
* Data for a free base obtained from hydrochloride.
MTT colorimetric assay. Cancer cell cultures were obꢀ
tained from the culture collection of N. N. Blokhin Russian
Cancer Research.
The MMT assay is the best known method for the
cytotoxicity evaluation. MTT assay involves the reduction
of yellow waterꢀsoluble 3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀ
diphenyltetrazolium bromide (MTT) by mitochondrial and
cytoplasmic dehydrogenases of metabolically active cells
to give waterꢀinsoluble purple formazan crystals. The
amount of produced formazan directly correlates to the
number of metabolically active cells and their viability and
can be quantified using spectrophotometry.
B. To a solution of 3ꢀmethoxysalicylaldehyde 1a (4.56 g,
30 mmol) and 3,3,3ꢀtrifluoroꢀ1ꢀnitropropene (4.2 g, 30 mmol)
in propanꢀ2ꢀol (15 mL), a solution of triethylamine (0.3 g,
3 mmol) in propanꢀ2ꢀol (15 mL) was added dropwise over a period
The results of cytotoxicity evaluation are given in
Table 5. Compounds 2a,b,d,e exhibit the most pronounced
cytotoxic effect against the Jurkat cell line.

3ꢀNitroꢀ2ꢀtrifluoromethylꢀ2Hꢀchromenes
Russ.Chem.Bull., Int.Ed., Vol. 63, No. 11, November, 2014 2555
of 1 h at 5 C. The reaction mixture was stirred for 24 h at ~20 C
and then cooled to 5 C. The precipitate formed was collected by
filtration, washed with cold propanꢀ2ꢀol, and dried in vacuo. No
further purification of the product was required. Yield 89%. Comꢀ
pounds 2b—i were synthesized similarly.
The test compounds were examined at concentrations of
1•10–7, 1•10–6, 1•10–5, and 1•10–4 mol L–1 in triplicate. For negꢀ
ative control, 2 L of DMSO and 18 L of complete RPMIꢀ1640
medium per well were added. After 72 h, 20 L of a MTT soluꢀ
tion (stock concentration 5 mg mL–1, final concentration
1 mg mL–1) was introduced into each well and the plates were
incubated at 37 C for 4 h under a 5% CO2 environment.
After formazan formation, the medium was removed. The
precipitate was dissolved by adding 150 L of DMSO and the
plates were incubated at 37 C for 5—7 min followed by gentle
shaking on an orbital shaker. Then, the absorbance were meaꢀ
sured at  = 530 nm with a photometric immunoassay analyzer
AIFPꢀ01 Uniplan (Pikon Ltd., Russia). The absorbance value is
directly proportional to the number of viable cells.
3ꢀAminoꢀ8ꢀmethoxyꢀ2ꢀtrifluoromethylchromane (3a). A threeꢀ
necked flask (100 mL) equipped with a thermometer, a pressure
equalizing dropping funnel, and a condenser was charged with
1 M solution of B2H6 in THF (10 mL, 10 mmol) followed by
cooling to 5 C. Then, a solution of compound 2a (0.6 g,
2.5 mmol) in THF (10 mL) was added over a period of 2 h with
stirring. After 1 h, NaBH4 (50 mg) was added, the cooling was
removed, and the reaction mixture was refluxed for 8 h. After
cooling down, the mixture was poured onto ice (200 g) with
stirring. The obtained mixture was acidified with 1 M HCl to pH
2 and then heated at 70 C for 2 h with stirring. After cooling to
20 C, the mixture was extracted with diethyl ether (325 mL).
The aqueous layer was separated, basified with 10% aqueous
NaOH, and extracted with diethyl ether. Combined organics
were dried with sodium sulfate and the solvent was removed
in vacuo. The residue was dissolved in MeCN (20 mL) and treatꢀ
ed with a solution of pꢀtoluenesulfonic acid (0.45 g, 5 mmol) in
MeOH (5 mL). Slow removal of the solvents resulted in tosylꢀ
ate 3a, colorless crystals.
Cell survival rate was calculated by the following equation:
Cell survival rate (%) = (Dexp/Dcontrol)•100%,
where Dexp is an absorbance of the experimental wells, Dcontrol is
an absorbance of the negative control wells.
References
1. Pat. EA 015364; Byul. Izobret. [Invention Bull.], 2011, No. 3;
http://www.eapo.org/ru/patents/reestr/.
Oxalate 3b was obtained similarly with the use of oxalic acid.
Hydrochlorides 3c—f,h,i were obtained as follows: gaseous
HCl was bubbled through a solution of the corresponding neutral
compound in anhydrous diethyl ether, the crystals of thus obtained
hydrochloride were washed with diethyl ether and dried. Yields
and spectral data for compounds 3a—f,h,i are given in Tables 3 and 4.
Nꢀ[6,8ꢀDibromoꢀ2ꢀ(trifluoromethyl)ꢀ3,4ꢀdihydroꢀ2Hꢀ
chromenꢀ3ꢀyl]hydroxylamine (4) was isolated from acidified
aqueous layer obtained in the synthesis of 2c by extraction with
diethyl ether. Removal of the solvent, purification of the residue by
column chromatography, and subsequent recrystallization from
methanol afforded compound 4 in 70% yield (based on the amount
of the residue obtained by extraction), m.p. 142—144 C. Found (%):
C, 30.85; H, 2.08; N, 3,44. C10H8Br2F3NO2. Calculated (%):
C, 30.72; H, 2.06; N, 3.58. MS (ESI), m/z (Irel (%)): 391 (42).
1H NMR (DMSOꢀd6), : 3.03 (d, 2 H, CH2, J = 3.0 Hz); 3.60
(s, 1 H, CHNHOH); 5.05 (m, 1 H, CHCF3); 5.71 (d, 1 H, NH,
J = 6.7 Hz); 7.41 (s, 1 H, Ar); 7.50 (s, 1 H, Ar); 7.6 (d, 1 H, OH,
J = 2.0 Hz). 19F NMR (DMSOꢀd6), : 6.13 (s, 3 F, CF3).
Cell culturing technique. Cells were cultured in RPMIꢀ1640
medium supplemented with 10% fetal calf serum, 10 mmol L–1
HEPES (Sigma, USA), 2 mmol L–1 Lꢀglutamine (Sigma, USA),
40 ng mL–1 gentamicin (ICN, USA), 0.1% amino acid solution,
and 0.1% vitamin solution (PanEco, Russia) at 37 C under a 5%
CO2 environment. In order to maintain log phase growth, the
cells were passaged every 3—4 days. The primary adherent cells
were detached from polystyrene culture vessels with Versene
and washed with serumꢀfree RPMIꢀ1640 medium.
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MTT assay. The SKOVꢀ3, HCTꢀ116, and A549 cell were
planted in clearꢀbottom 96ꢀwell plate at a density of 4•103 cells
per mL, and the Jurkat cells, at a density of 15•103 cells per mL
in complete RPMIꢀ1640 culture medium (180 L, Costar, USA).
For cytotoxicity evaluation, 20 L of a solution containing 2 L
of a stock solution of the test compound in DMSO and 18 L
of complete RPMIꢀ1640 medium were added in each well and
the plates were incubated for 72 h at 37 C under a 5% CO2
environment.
16.
S. Iwata, Y. Ishiguro, M. Utsugi, K. Mitsuhashi, K. Tanaka,
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Received June 5, 2014;
in revised form June 25, 2014