In vitro cytotoxicity of hydrazones, pyrazoles, pyrazolo-pyrimidines, and pyrazolo-pyridine synthesized from 6-substituted 3-formylchromones

Article abstract of DOI:10.1515/znb-2014-0236

Pyrazoles 4a-f, hydrazones 5a-c and 6a-c, pyrazolo[1,5-a]pyrimidines 7a, b, and pyrazolo[3,4-b]pyridine 8 were prepared in good yields (80-95%) from the reaction of 6-substituted (H, Me, F) 3-formylchromones 1a-c with N-substituted hydrazines 2a-c and ami

Full text of DOI:10.1515/znb-2014-0236

Z. Naturforsch. 2015; 70(5)b: 305–310
Elier Galarraga*, Neudo Urdaneta, Keily J. Gutierrez and Julio C. Herrera
In vitro cytotoxicity of hydrazones, pyrazoles,
pyrazolo-pyrimidines, and pyrazolo-pyridine
synthesized from 6-substituted
3-formylchromones
Abstract: Pyrazoles 4a–f, hydrazones 5a–c and 6a–c,
Among the functionalized chromones, 3-formylchr-
pyrazolo[1,5-a]pyrimidines 7a, b, and pyrazolo[3,4-b]pyri- omone is a highly reactive compound, and is used as a
dine 8 were prepared in good yields (80–95 %) from the starting material in many reactions due to the presence
reaction of 6-substituted (H, Me, F) 3-formylchromones of three electron-deficient centers at C-2, C-4, and the
1a–c with N-substituted hydrazines 2a–c and aminopyra- C-3 formyl group. Reaction of the -CHO group with nitro-
zole 3. The cytotoxicity of the synthesized compounds was gen nucleophiles, such as hydrazine and aminopyrazole
assessed through the brine shrimp lethality assay. IC50 val- derivatives, has led to the formation of a variety of mol-
ues were between 80 and 300 μm. Fluorine substitution ecules that have been studied in detail for being of interest
decreased IC50 values.
to drug discovery [13–21].
π-Electron-rich compounds like chromone-3-car-
Keywords: brine shrimp lethality assay; hydrazones; boxyaldehydes react with aromatic primary hydrazines
pyrazolo[1,5-a]pyrimidines; pyrazolo[3,4-b]pyridines.
(1:1 molar ratio) mainly at the formyl group by a straight
forward 1,2-addition to form the corresponding hydrazone
[22–26]. On prolonged heating, a pyrazole-type structure
is produced by a 1,4-addition reaction accompanied by
pyrone ring-opening followed by recyclization and proton
transfer. Meanwhile, the reaction of 3-formylchromone
with equimolar quantities of aminopyrazole derivatives
affords only pyrazolo[1,5-a]pyrimidines, which is formed
by the abovementioned cyclization process of an imine
intermediate [27, 28].
The brine shrimp lethality assay (BSLA) is a prelimi-
nary standard bioassay, which is based on the use of a
simple zoologic organism, and is used to detect toxicity of
substances. BSLA is an easily performed and cost-effective
test. It showed positive correlations with specific cytotoxic
assays employing KB [29], 9KB [30], and 9PS [31] cancer
cell lines and it was also suitable in predicting trypano-
cidal [32] and pesticidal [33] activities.
The synthesis of nitrogenated derivatives of 3-for-
mylchromone is an important chemical issue; it may also
contribute to the identification of new compounds with
biological activity. In this work, we present the synthesis
and characterization of new pyrazoles 4d–f, hydrazones
5b, 5c, 6a–c and pyrazolo[3,4-b]pyridine 8, along with the
known pyrazoles 4a–c, hydrazone 5a and pyrazolo[1,5-a]
pyrimidines 7a and 7b. We also report on the in vitro tox-
icity of the obtained compounds against brine shrimps
(Artemia salina).
DOI 10.1515/znb-2014-0236
Received October 1, 2014; accepted February 5, 2015
1 Introduction
Benzopyrone group-based compounds, such as chr-
omones, are widely recognized as an important class of
biological active substances from both natural and syn-
thetic origins [1–3]. Numerous studies show their wide
range of activities, such as antioxidant [4], antimicrobial
[5, 6], antiviral [7, 8] and antitumor [9, 10]. The chromone
nucleus is also found within the chemical structure of
flavonoids, an important group of naturally occurring
substances that are of current interest because of their
cytotoxic activity [11, 12].
*Corresponding author: Elier Galarraga, Departamento de Química,
Edificio de Química y Procesos, Universidad Simón Bolívar (USB),
Apartado 89000, Caracas-1080A, Venezuela,
e-mail: eliergalarraga@usb.ve
Neudo Urdaneta, Keily J. Gutierrez and Julio C. Herrera:
Departamento de Química, Edificio de Química y Procesos,
Universidad Simón Bolívar (USB), Apartado 89000, Caracas-1080A,
Venezuela
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306ꢀ ꢀE. Galarraga et al.: Cytotoxicity of compounds synthesized from 6-substituted 3-formylchromones
ring. Compounds 5b and 5c had typical hydrazone IR
bands at 3273–2379 (NH), 3060–3064 (Nꢀ=ꢀCH), and 1516–
2 Results and discussion
1
1518 (Cꢀ=ꢀN) cm–1. H NMR resonances of 5b were observed
2.1 Synthesis
at δꢀ=ꢀ6.76 (dd, J ꢀ=ꢀ 2.2/8.4 Hz, 4′-H), 7.18 (d, J ꢀ=ꢀ 8.4 Hz, 3′-
H), and 7.50 (d, J ꢀ=ꢀ 2.2 Hz, 6′-H) ppm corresponding to
the 2,5-dichlorophenyl residue, while its 6-methyl group
appeared at 2.47 ppm. The presence of the hydrazone
moiety was established from signals at 8.12 (s, CHꢀ=ꢀN) and
8.60 (s, NH) ppm.
Proton signals of 5c were similar to those of 5b. The
presence of fluorine at position 6 was evidenced by the
multiplicity and chemical shift of the chromone protons at
δꢀ=ꢀ7.89 (dd, J ꢀ=ꢀ 3.1/8.2 Hz, 5-H), 7.42 (ddd, J ꢀ=ꢀ 3.1/7.6/9.2 Hz,
7-H), and 7.52 (dd, J ꢀ=ꢀ 9.2/4.4 Hz, 8-H) ppm. The isomeri-
zation of 5b and 5c to pyrazole structures was discarded
based on 13C NMR data, which showed the presence of
characteristic 1,4-benzopyrone carbonyl carbons between
δꢀ=ꢀ174.9–175.9 (C-4) ppm and vinylic CHꢀ=ꢀN resonances at
129.9–131.3 ppm.
The experimental procedure led to the synthesis of pyra-
zoles 4a–f, hydrazones 5a–c and 6a–c, pyrazolo[1,5-a]
pyrimidines 7a, 7b, and pyrazolo[3,4-b]pyridine 8. Deriva-
tives 4d–f, 5b, 5c, 6a–c, and 8 are newly synthesized
compounds. The products were obtained from the reac-
tion between equimolar quantities of 3-formylchromone
derivatives 1a–c, with the corresponding hydrazines 2a–c
or aromatic amine 3 in anhydrous THF. The pyrazolo[3,4-
b]pyridine 8 was obtained in two steps: the reaction of 1b
with 3 produced a precipitate, which was then refluxed
using AcOH as solvent in presence of I2 (Scheme 1). All
reactions were monitored by TLC and obtained in good
yields (80–95 %). The new compounds were fully charac-
terized using NMR, IR, and HRMS methods.
Acylated pyrazoles 4d–f showed IR bands at 3066–
3068 (Nꢀ=ꢀCH), 1755–1760 (O–Cꢀ=ꢀO), 1622–1640 (Cꢀ=ꢀO),
1583–1599 (Cꢀ=ꢀC), and 1504–1505 (Cꢀ=ꢀN) cm–1. Their 1H NMR
data exhibited the same characteristic signals as already
reported for pyrazoles 4a–c [34] and methyl resonances at
δꢀ=ꢀ2.11–2.14 ppm.
Hydrazones 6a–c displayed IR bands at approximately
3265–3287 (NH), 3114–3118 (Nꢀ=ꢀCH), 1515–1519 (Cꢀ=ꢀN) and
1
1584, 1334 (Nꢀ=ꢀO) cm–1. Distinctive H NMR signals for the
2,4-dinitrophenyl substituent were located at δ ꢀ=ꢀ 8.82–
8.85 (H-3′), 8.31–8.33 (H-5′) and 8.08–8.13 (H-6′) ppm,
while hydrazone-type protons were detected at 8.85–8.99
(CHꢀ=ꢀN) and 11.45–11.66 (NH) ppm. The methyl group
Hydrazones 5a–c and 6a–c showed signals in the IR
and NMR spectra, which were characteristic of a chromone signal of 6b was located at 2.47 ppm. Characteristic 13C
Scheme 1:ꢀReagents and conditions: (i) THF, reflux (1–2 h); (ii) Ac2O/H2SO4, reflux (5 h); (iii) AcOH/I2, reflux (3–4 h).
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E. Galarraga et al.: Cytotoxicity of compounds synthesized from 6-substituted 3-formylchromonesꢂ
ꢂ307
Table 1:ꢀBrine shrimp lethality assay of the synthesized
compounds.
Compound
IC50 (μm)
Compound
IC50 (μm)
4a
4b
4c
4d
4e
4f
261.8 34
202.2 39
154.6 49
296.5 15
319.4 19
185.8 24
161.2 26
170.0 28
5c
6a
6b
6c
7a
7b
8
98.1 14
95.3 26
113.2 16
83.0 16
101.3 11
94.7 14
110.4 37
5a
5b
Scheme 2:ꢀFormation of pyrazolo[3,4-b]pyridine 8.
NMR resonances for carbonyl and vinylic CHꢀ=ꢀN carbons 5c [IC50 ꢀ=ꢀ 83.0 16, 94.7 14, 95.3 26 and 98.1 14 μm
at δꢀ=ꢀ174.6–175.1 and 125.2–125.8 ppm, excluded a pyrazole respectively] among the more active ones. The observed
structure for 6a–c.
toxicities are slightly lower than the cytotoxicity of Podo-
When chromones were substituted with either H (1a) phyllotoxin, a well known bioactive compound [IC50 ꢀ=ꢀ
or F (1c) at C-6, the reaction with 3 produced the expected 5.8 μm] [31].
pyrazolo[1,5-a]pyrimidines 7a and 7c. However, the reac-
The reasons for this toxic effect are not clear. However,
tion product between 6-methyl substituted chromone 1b the presence of fluorine increased activity, as could be
with aromatic amine 3 yielded the pyrazolo[3,4-b]pyridine observed for compounds 4c, 5c, 6c, and 7b. Such results are
8 after two steps (see general procedures). IR bands for 8 consistent with the general observation, which states that
were present at 3400 (OH), 3203 (NH), 3036 (Nꢀ=ꢀCH), 1634 the presence of aromatic fluorine enhances the overall bio-
1
(Cꢀ=ꢀO), and 1479 (Cꢀ=ꢀN) cm–1. Its H NMR spectra revealed logical activity of organic compounds on a moderate scale
signals of a p-hydroxy-methylphenyl residue at δꢀ=ꢀ2.53 (s, [35, 36]. The presence of the hydroxyl group may also play a
5′-Me), 6.90 (d, J ꢀ=ꢀ 8.4 Hz, 3′-H), 7.22 (d, J ꢀ=ꢀ 2.2 Hz, 6′-H), role in the toxicity, as evidenced by the increase in the IC50
7.27 (dd, J ꢀ=ꢀ 2.2/8.4 Hz, 4′-H), and 10.09 (s, OH) ppm. The values for acylated derivatives 4d–f compared with 4a–c.
formation of the pyrazolo[1,5-a]pyrimidine structure was More specific cytotoxic studies that use tumor cell lines
discarded due to the absence of the characteristic C-3 reso- MCF-7 (breast) and PC3 (prostate) are currently underway.
nance between δꢀ=ꢀ93.8–96.9 ppm in the 13C NMR spectra.
The presence of 3-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl
signals at 2.26 (s, 3-Me), 8.52 (d, J ꢀ=ꢀ 1.8 Hz, 4-H), 8.78 (d,
3 Experimental section
J ꢀ=ꢀ 1.8 Hz, 6-H) and 13.60 (s, NH) ppm, confirmed the for-
mation of 8.
The formation of regioisomer 8 may arise from an 3.1 Chemistry
imine intermediary (Scheme 2) that undergoes 1,4-addi-
tion at C-2 by attack of C-4′ from the pyrazole instead of the Melting points were determined using a Krüss-Optronic (San
nitrogen atom N-2′. To the best of our knowledge, this is Diego, CA, USA) apparatus, and they were not corrected.
the first report regarding the formation of pyrazolo[3,4-b] 1H NMR, 13C NMR, and DEPT-135 spectra were recorded on a
pyridines by intramolecular attack of an sp2 carbon atom. JEOL Eclipse Plus 400 (400 MHz) (Peabody, MA, USA) and
Bruker Avance500* (500 MHz) (Billerica, MA, USA) spec-
trometers, using CDCl3 and [D6]DMSO as solvents. IR spectra
were obtained from KBr pellets with Shimadzu IR-408
(Columbia, MD, USA) equipment. The HRMS (70 eV) analy-
2.2 Brine shrimp lethality assay
The toxicity of all compounds was assayed against Artemia ses were conducted in a JEOL JMS-AX505WA (Peabody, MA,
salina. The dose-dependent assays were carried out USA) double focus mass spectrometer, using the electron
between 10 and 500 ppm using a negative control, Tween impact (EI) method. Analytical thin-layer chromatography
®
80 , at this concentration did not affect this bioassay. The was carried out on 0.25 mm layers of silica gel PF254 (Merck)
IC50 values of the compounds are presented in Table 1. The (Kenilworth, NJ, USA).
study showed that all tested compounds exhibited a toxic
Reagents 1a–c, 2a–c, and 3 were purchased from
effect to the brine shrimp, with derivatives 6c, 7b, 6a and Aldrich (Milwaukee, WI, USA) as ‘synthetic grade’ and used
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ꢀꢀꢀꢁ
308ꢀ ꢀE. Galarraga et al.: Cytotoxicity of compounds synthesized from 6-substituted 3-formylchromones
without further purification. Compounds 4a–c, 5a, 7a, and (C-5), 132.7 (C-5′), 135.8 (C-4′), 139.2 (C-1″), 142.5 (C-3), 145.9
7b were prepared as described in the general procedures. (C-2′), 169.5 (O–Cꢀ=ꢀO), 187.1 (Cꢀ=ꢀO) ppm. – HRMS: m/z ꢀ=ꢀ
Their physical constants and spectroscopic data were in 320.1116 (calcd. 320.1161 for C19H16N2O3).
agreement with those described in the literature [16, 28, 37].
3.2.3 (2-Acetoxy-5-fluorophenyl)(1-phenyl-1H-pyrazol-
3.2 General procedure for the synthesis
4-yl)methanone (4f)
of 4d–f
Pale yellow crystals. Yield: 91%. M.p. 102–104 °C. – IR
A portion of compound 4a–c (0.4 mmol) was dissolved
(KBr): vꢀ=ꢀ3068 (Nꢀ=ꢀCH), 1755 (O–Cꢀ=ꢀO), 1628 (Cꢀ=ꢀO), 1583
in an excess of Ac2O (4 mL), after which H2SO4 (18 m,
1
(Cꢀ=ꢀC), 1504 (Cꢀ=ꢀN). – H NMR (500 MHz, CDCl3): δꢀ=ꢀ2.13
0.1 mL) was added and refluxed for 5 h. Once finished,
(s, 3H, CH3 acetate), 7.14 (dd, J ꢀ=ꢀ 4.4/9.2 Hz, 1H, 3′-H), 7.28
15 mL of iced water was added and extracted with CH2Cl2
(ddd, Jꢀ=ꢀ2.9/7.6/9.2 Hz, 1H, 4′-H), 7.36 (d, J ꢀ=ꢀ 7.0 Hz, 1H, 4″-H),
(2 ꢀ×ꢀ 6 mL). The organic phase was washed with 15 %
7.45 (dd, J ꢀ=ꢀ 7.0/8.1 Hz, 2H, 3″/5″-H), 7.66 (dd, J ꢀ=ꢀ 2.9/8.8 Hz,
NaHCO3 (aq.) (4 ꢀ×ꢀ 15 mL), and dried over Na2SO4, after
1H, 6′-H), 7.69 (d, J ꢀ=ꢀ 8.1 Hz, 2H, 2″/6″-H), 8.05 (s, 1H, 3-H),
which the solvent was evaporated under reduced pressure.
8.30 (s, 1H, 5-H) ppm. – 13C NMR (125 MHz, CDCl3): δ ꢀ=ꢀ 20.6
The obtained solid was recrystallized from hexane-EtOAc
(CH3), 115.9/116.2 (C-3′), 118.6/118.9 (C-4′), 119.7 (C-2″/C-6″),
(1:1) solvent mixture.
124.2 (C-4), 125.0/125.1 (C-5′), 127.8 (C-4″), 129.6 (C-3″/C-5″),
130.8 (C-5), 133.6/133.7 (C-1′), 139.0 (C-1″), 142.4 (C-3), 143.9
(C-2′), 157.9/161.2 (C-6′), 169.3 (O–Cꢀ=ꢀO), 185.4 (Cꢀ=ꢀO) ppm.
3.2.1 (2-Acetoxy-phenyl)(1-phenyl-1H-pyrazol-4-yl)
– HRMS: m/z ꢀ=ꢀ 324.0988 (calcd. 324.0910 for C18H13FN2O3).
methanone (4d)
Colorless crystals. Yield: 85 %. M.p. 108–110 °C. – IR (KBr):
v ꢀ=ꢀ 3066 (Nꢀ=ꢀCH), 1756 (O–Cꢀ=ꢀO), 1622 (Cꢀ=ꢀO), 1599 (Cꢀ=ꢀC), 3.3 General procedure for the synthesis of
1
1504 (Cꢀ=ꢀN). – H NMR (500 MHz, CDCl3): δ ꢀ=ꢀ 2.14 (s, 3H,
4a–c, 5a–c, and 6a–c
CH3 acetate), 7.18 (d, J ꢀ=ꢀ 7.8 Hz,1H, 5′-H), 7.31 (d, J ꢀ=ꢀ 7.7 Hz,
1H, 3′-H), 7.33 (d, Jꢀ=ꢀ7.3 Hz,1H, 4″-H), 7.43 (dd, J ꢀ=ꢀ 7.3/8.2 Hz, 3-Formylchromone derivative 1a–c (1.0 mmol) was dis-
2H, 3″/5″-H), 7.53 (td, J ꢀ=ꢀ 1.5/7.7 Hz,1H, 4′-H), 7.67 (d, J ꢀ=ꢀ solved in hot anhydrous THF (4 mL) and a solution of
8.2 Hz, 2H, 2″/6″-H), 7.61 (dd, J ꢀ=ꢀ 1.5/7.7 Hz, 1H, 6′-H), 8.06 hydrazine 2a–c (1.0 mmol) in THF (2 mL) was added
(s, 1H, 3-H), 8.29 (s, 1H, 5-H) ppm. – 13C NMR (125 MHz, slowly. The mixture was refluxed for 1–2 h; once cooled the
CDCl3): δ ꢀ=ꢀ 20.9 (CH3), 119.8 (C-2″/C-6″), 123.6 (C-4), 124.8 solid was filtered, washed with water, and recrystallized
(C-1′), 125.9 (C-5′), 127.9 (C-3′), 129.6 (C-4″), 129.7 (C-3″/C-5″), from absolute EtOH.
131.0 (C-6′), 132.3 (C-5), 132.6 (C-4′), 139.3 (C-1″), 142.6 (C-3),
148.3 (C-2′), 169.5 (O–Cꢀ=ꢀO), 187.1 (Cꢀ=ꢀO) ppm. – HRMS:
m/z ꢀ=ꢀ 306.0998 (calcd. 306.1004 for C18H14N2O3).
3.3.1 3-[(2,5-Dichlorophenyl)hydrazonomethyl]-
6-methyl-chromen-4-one (5b)
3.2.2 (2-Acetoxy-5-methylphenyl)(1-phenyl-1H-pyrazol-
4-yl)methanone (4e)
Yellow solid. Yield: 87%. M.p. 221–222 °C. – IR (KBr): v ꢀ=ꢀ
3273 (NH), 3060 (Nꢀ=ꢀCH), 1649 (Cꢀ=ꢀO), 1620 (Cꢀ=ꢀC), 1518
(Cꢀ=ꢀN). – 1H NMR (400 MHz, CDCl3): δ ꢀ=ꢀ 2.47 (s, 3H, CH3),
Pale yellow crystals. Yield: 87%. M.p. 99–101 °C.– IR (KBr): 6.76 (dd, J ꢀ=ꢀ 2.2/8.4 Hz, 1H, 4′-H), 7.18 (d, J ꢀ=ꢀ 8.4 Hz, 1H,
v ꢀ=ꢀ 3065 (Nꢀ=ꢀCH), 1760 (O–Cꢀ=ꢀO), 1640 (Cꢀ=ꢀO), 1598 (Cꢀ=ꢀC), 3′-H), 7.40 (d, J ꢀ=ꢀ 8.8 Hz, 1H, 8-H), 7.48 (d, J ꢀ=ꢀ 8.8 Hz, 1H,
1
1505 (Cꢀ=ꢀN). – H NMR (500 MHz, CDCl3): δ ꢀ=ꢀ 2.11 (s, 3H, 7-H), 7.50 (d, J ꢀ=ꢀ 2.2 Hz, 1H, 6′-H), 8.05 (bs, 1H, 5-H), 8.12
CH3 acetate), 2.34 (s, 3H, CH3), 7.06 (d, J ꢀ=ꢀ 8.8 Hz, 1H, 3′-H), (s, 1H, CHꢀ=ꢀN), 8.14 (s, 1H, 2-H), 8.60 (s, 1H, NH) ppm.
7.33 (dd, J ꢀ=ꢀ 0.0/8.8 Hz, 1H, 4′-H), 7.40 (d, J ꢀ=ꢀ 7.7 Hz, 1H, 4″-
–
13C NMR (100 MHz, CDCl3): δ ꢀ=ꢀ 21.0 (CH3), 113.8 (C-6′),
H), 7.45 (dd, J ꢀ=ꢀ 7.7/8.1 Hz, 2H, 3″/5″-H), 7,67 (d, Jꢀ=ꢀ2.0 Hz, 115.2 (C-3), 118.2 (C-8), 118.9 (C-2′), 119.9 (C-4′), 123.6 (C-10),
1H, 6′-H), 7,69 (d, J ꢀ=ꢀ 8.1 Hz, 2H, 2″/6″-H), 8.06 (s, 1H, 3-H), 125.3 (C-5), 129.9 (CHꢀ=ꢀN), 132.1 (C-3′), 133.8 (C-5′), 135.2
8.29 (s, 1H, 5-H) ppm. – 13C NMR (125 MHz, CDCl3): δ ꢀ=ꢀ 20.7 (C-7), 135.8 (C-6), 141.1 (C-1′), 152.6 (C-2), 154.6 (C-9), 175.9
(CH3), 20.8 (CH3), 119.7 (C-2″/C-6″), 123.1 (C-1′), 124.8 (C-4), (C-4) ppm. – HRMS: m/z ꢀ=ꢀ 346.0288 (calcd. 346.0276 for
127.7 (C-3′), 129.7 (C-3″/C-5″), 129.8 (C-4″), 130.8 (C-6′), 132.2 C17H12Cl2N2O2).
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ꢂ309
3.3.2 3-[(2,5-Dichlorophenyl)hydrazonomethyl]-
6-fluoro-chromen-4-one (5c)
3.3.5 3-[(2,4-Dinitrophenyl)hydrazonomethyl]-
6-fluor-chromen-4-one (6c)
Yellow solid. Yield: 85%. M.p. 211–213 °C. – IR (KBr): v ꢀ=ꢀ Orange solid. Yield: 83%. M.p. 261–262 °C. IR (KBr): v ꢀ=ꢀ
3279 (NH), 3064 (Nꢀ=ꢀCH), 1646 (Cꢀ=ꢀO), 1620 (Cꢀ=ꢀC), 1516 3287 (NH), 3118 (Nꢀ=ꢀCH), 1640 (Cꢀ=ꢀO), 1605 (Cꢀ=ꢀC), 1519
1
(Cꢀ=ꢀN). – H NMR (400 MHz, CDCl3): δ ꢀ=ꢀ 6.77 (dd, J ꢀ=ꢀ (Cꢀ=ꢀN), 1584, 1340 (Nꢀ=ꢀO). – 1H NMR (400 MHz, [D6]DMSO):
2.5/8.5 Hz, 1H, 4′-H), 7.18 (d, J ꢀ=ꢀ 8.5 Hz, 1H, 3′-H), 7.42 (ddd, δ ꢀ=ꢀ 7.72 (m, 1H, 7-H), 7.81 (m, 1H, 8-H), 7.83 (bs, 1H, 5-H),
Jꢀ=ꢀ3.1/7.6/9.2 Hz, 1H, 7-H), 7.50 (d, J ꢀ=ꢀ 2.5 Hz, 1H, 6′-H), 7.52 8.12 (d, J ꢀ=ꢀ 9.5 Hz, 1H, 6′-H), 8.33 (dd, J ꢀ=ꢀ 2.5/9.5 Hz 1H,
(dd, J ꢀ=ꢀ 4.4/9.2 Hz, 1H, 8-H), 7.89 (dd, J ꢀ=ꢀ 3.1/8.2 Hz 1H, 5′-H), 8.74 (s, 1H, 2-H), 8,85 (d, J ꢀ=ꢀ 2.5 Hz, 1H, 3′-H), 8.99
5-H), 8.07 (s, 1H, CHꢀ=ꢀN), 8.14 (s, 1H, 2-H), 8.60 (s, 1H, NH) (s, 1H, CHꢀ=ꢀN), 11,66 (s, 1H, NH) ppm. – 13C NMR (100 MHz,
ppm. – 13C NMR (100 MHz, CDCl3): δ ꢀ=ꢀ 110.2 (C-5), 110.5 [D6]DMSO): δ ꢀ=ꢀ 110.3 (C-5), 110.6 (C-8), 117.7 (C-6′), 118.4
(C-8), 114.1 (C-6′), 119.2 (C-2′), 119.9 (C-4′) 122.1 (C-7), 123.2 (C-3′), 122.1 (C-10), 123.2 (C-7), 123.4 (C-5′), 125.3 (CHꢀ=ꢀN),
(C-3), 125.2 (C-10), 131.3 (CHꢀ=ꢀN), 133.4 (C-3′), 134.2 (C-5′), 129.9 (C-3), 130.4 (C-2′), 137.9 (C-4′), 142.1 (C-1′), 152.8 (C-9),
142.9 (C-1′), 154.6 (C-2), 158.5/160.9 (C-6), 174.9 (C-4) ppm. – 156.5/161.2 (C-6), 158.7 (C-2), 174.6 (C-4) ppm. – HRMS:
HRMS: m/z ꢀ=ꢀ 350.0030 (calcd. 350.0025 for C16H9Cl2FN2O2). m/z ꢀ=ꢀ 372.0599 (calcd. 372.0506 for C16H9FN4O6).
3.3.3 3-[(2,4-Dinitrophenyl)hydrazonomethyl]-
chromen-4-one (6a)
3.4 General procedure for the synthesis of
7a and 7b
Orange solid. Yield: 90%. M.p. 294–295 °C. – IR (KBr): 3-Formylchromone derivative 1a or 1c (1.0 mmol) was
v ꢀ=ꢀ 3265 (NH), 3114 (Nꢀ=ꢀCH), 1644 (Cꢀ=ꢀO), 1605 (Cꢀ=ꢀC), 1519 mixed with aminopyrazole 3 (1.0 mmol) and refluxed in
(Cꢀ=ꢀN), 1580, 1334 (Nꢀ=ꢀO). – 1H NMR (400 MHz, [D6]DMSO): 10 mL of anhydrous THF for 1 h. After cooling, the solid
δ ꢀ=ꢀ 7.56 (dd, J ꢀ=ꢀ 7.3/8.1 Hz, 1H, 6-H), 7.72 (d, J ꢀ=ꢀ 8.4 Hz, 1H, was filtered, washed repeatedly with hot THF, and recrys-
8-H), 7.86 (dd, J ꢀ=ꢀ 7.3/8.4 Hz, 1H, 7-H), 8.13 (d, J ꢀ=ꢀ 9.5 Hz, tallized from EtOH to produce TLC pure compounds.
1H, 6′-H), 8.16 (d, J ꢀ=ꢀ 8.1 Hz, 1H, 5-H), 8.31 (dd, J ꢀ=ꢀ 2.2/9.5
Hz, 1H, 5′-H), 8.75 (s, 1H, 2-H), 8.85 (d, J ꢀ=ꢀ 2.2 Hz, 1H, 3′-H),
8.97 (s, 1H, CHꢀ=ꢀN), 11.65 (s, 1H, NH) ppm. – 13C NMR (100 3.5 General procedure for the synthesis of 8
MHz, [D6]DMSO): δ ꢀ=ꢀ 117.6 (C-6′), 118.1 (C-3′), 119.1 (C-8),
123.3 (C-5′), 121.5 (C-10), 124.2 (C-5), 125.8 (CHꢀ=ꢀN), 129.9 Equimolar quantities of 1b and 3 (1.0 mmol) were refluxed
(C-3), 130.5 (C-2′), 135.1 (C-7), 126.6 (C-6), 135.8 (C-4′), 142.4 in anhydrous THF for 1–2 h, until the formation of a pre-
(C-1′), 156.0 (C-9), 159.5 (C-2), 175.1 (C-4) ppm. – HRMS: cipitate. Once separated from the solution, this precipi-
m/z ꢀ=ꢀ 354.0634 (calcd. 354.0600 for C16H10N4O6).
tate was dissolved in AcOH (7 mL) in the presence of I2
(1.0 mmol) and then refluxed for a period of 3–4 h. Upon
completion of the TLC reaction, the mixture was poured
into crushed ice and treated with NaHCO3 and Na2SO3. The
solid was filtered, washed with cold water, and dried.
3.3.4 3-[(2,4-Dinitrophenyl)hydrazonomethyl]-
6-methyl-chromen-4-one (6b)
Orange solid. Yield: 87%. M.p. 271–272 °C. – IR (KBr): v ꢀ=ꢀ
3270 (NH), 3118 (Nꢀ=ꢀCH), 1644 (Cꢀ=ꢀO), 1595 (Cꢀ=ꢀC), 1515 3.5.1 (2-Hydroxy-5-methylphenyl)(3-methyl-
(Cꢀ=ꢀN), 1584, 1334 (Nꢀ=ꢀO). – 1H NMR (400 MHz, [D6]DMSO):
δ ꢀ=ꢀ 2.47 (s, 3H, CH3), 7.56 (d, J ꢀ=ꢀ 8.4 Hz, 1H, 8-H), 7.65 (d,
1H-pyrazolo[3,4-b]pyridin-5-yl)methanone (8)
J ꢀ=ꢀ 8.4 Hz, 1H, 7-H), 7,95 (bs, 1H, 5-H), 8.08 (d, J ꢀ=ꢀ 9.5 Hz, Pale yellow solid. Yield: 80%. M.p. 198–200 °C. – IR (KBr):
1H, 6′-H), 8.32 (dd, J ꢀ=ꢀ 2.5/9.5 Hz 1H, 5′-H), 8.67 (s, 1H, 2-H), v ꢀ=ꢀ 3400 (OH), 3203 (NH), 3036 (Nꢀ=ꢀCH), 1634 (Cꢀ=ꢀO), 1597
1
8.82 (bs, 1H, 3′-H), 8.85 (s, 1H, CHꢀ=ꢀN), 11.45 (s, 1H, NH) (Cꢀ=ꢀC), 1479 (Cꢀ=ꢀN). – H NMR (400 MHz, [D6]DMSO): δ ꢀ=ꢀ
ppm. – 13C NMR (100 MHz, [D6]DMSO): δ ꢀ=ꢀ 20.8 (CH3), 118.1 2.26 (s, 3H, CH3 pyrazolopyridine), 2.53 (s, 3H, CH3 2-
(C-6′), 118.8 (C-3′), 117.6 (C-8), 121.4 (C-10), 123.1 (C-5′), 125.4 hydroxy-5- methylphenyl), 6.90 (d, J ꢀ=ꢀ 8.4 Hz, 1H, 3′-H),
(C-5), 125.2 (CHꢀ=ꢀN), 129.8 (C-3), 129.9 (C-2′), 135.7 (C-7), 136.0 7.22 (d, Jꢀ=ꢀ2.2 Hz, 1H, 6′-H), 7.27 (dd, J ꢀ=ꢀ 2.2/8.4 Hz, 1H, 4′-
(C-6), 138.5 (C-4′), 142.6 (C-1′), 154.7 (C-9), 157.8 (C-2), 174.9 H), 8.52 (d, J ꢀ=ꢀ 1.8 Hz, 1H, 4-H), 8.78 (d, J ꢀ=ꢀ 1.8 Hz, 1H, 6-H),
(C-4) ppm. – HRMS: m/z ꢀ=ꢀ 368.0757 (calcd. 368.0757 for 10.09 (s, 1H, OH), 13.60 (s, 1H, NH) ppm. – 13C NMR (100
C17H12N4O6).
MHz, [D6]DMSO): δ ꢀ=ꢀ 12.7 (CH3), 20.5 (CH3), 113.9 (C-5), 117.3
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ꢀꢀꢀꢁ
310ꢀ ꢀE. Galarraga et al.: Cytotoxicity of compounds synthesized from 6-substituted 3-formylchromones
[11] W. Ren, Z. Qiao, H. Wang, L. Zhu, L. Zhang, Med. Res. Rev.
2003, 23, 519.
(C-3′), 125.2 (C-1′), 126.9 (C-9), 128.6 (C-4), 131.0 (C-4′), 132.3
(C-6′), 134.6 (C-5′), 143.9 (C-3), 150.8 (C-6), 153.8 (C-8), 155.1
(C-2′), 196.1 (Cꢀ=ꢀO) ppm. – HRMS: m/z ꢀ=ꢀ 267.1044 (calcd.
267.1008 for C15H13N3O2).
[12] N. Li, J.-H. Liu, J. Zhang, B.-Y. Yu, J. Agric. Food Chem. 2008, 56,
3876.
[13] D. W. Engers, A. Y. Frist, C. W. Lindsley, C. C. Hong, C. R. Hop-
kins, Bioorg. Med. Chem. Lett. 2013, 23, 3248.
[14] A. Kamal, J. R. Tamboli, V. L. Nayak, S. F. Adil, M. V. P. S. Vishnu-
vardhan, S. Ramakrishna, Bioorg. Med. Chem. Lett. 2013, 23,
3208.
3.6 BSLA
[15] Z. Baráth, R. Radics, G. Spengler, I. Ocsovszki, M. Kawase, N.
Motohashi, Y. Shirataki, A. Shah, J. Molnár, In vivo 2006, 20,
645.
[16] A. Lazarenkow, J. Nawrot-Modranka, E. Brzezinka, U. Kra-
jewska, M. Rózalski, Med. Chem. Res. 2012, 21, 1861.
[17] P. Foltínová, M. Lácová, D. Loos, Il Farmaco 2000, 55, 21.
[18] H. M. El-Shaaer, P. Foltínová, M. Lácová, J. Chovancová, H.
Stankovicova, Il Farmaco 1998, 53, 224.
[19] B.-d. Wang, Z.-Y. Yang, T.-r. Li, Bioorg. Med. Chem. 2006, 14,
6012.
The assay was performed as described previously by Meyer
[31] with some minor modifications. Brine shrimp eggs
®
(Gulf Breeze ) were hatched in artificial sea water pre-
®
pared with commercial salt mixture (Instant Ocean ), and
then illuminated and oxygenated with an aquarium pump.
After an incubation period of 48 h at 27 °C, 10 shrimps
were transferred with a Pasteur pipette to three sample
vials for each of the three doses (500, 100, 10 μg mL–1), for
a total of nine vials per sample. The compound samples
(10 mg) were dissolved in CHCl3 (5 mL). Aliquotes of testing
solutions (1250, 250, or 25 μL for the 500, 100 and 10 ppm
doses, respectively) were placed on vials (5 mL) and the
solvent was evaporated. The residue was redissolved in
[20] Q. Wang, Z.-Y. Yang, G.-F. Qi, D.-D. Qin, Eur. J. Med. Chem.
2009, 44, 2425.
[21] K. M. Khan, N. Ambreen, S. Hussain, S. Perveen, M. I. Choud-
hary, Bioorg. Med. Chem. 2009, 17, 2983.
[22] A. O. Fitton, J. R. Frost, H. Suschitzky, Tetrahedron Lett. 1975,
25, 2099.
®
10 μL of Tween 80 , after which artificial sea water (5 mL)
[23] A. O. Fitton, J. R. Frost, H. Suschitzky, J. Chem. Soc., Perkin
Trans. 1 1979, 1691.
[24] C. K. Ghosh, J. Heterocycl. Chem. 1983, 20, 1437.
[25] G. Sabitha, Aldrichimica Acta 1996, 29, 15.
[26] A. S. Plaskon, O. O. Grygorenko, S. V. Ryabukhin, Tetrahedron
2012, 68, 2743.
was added. Survivors were counted and the percent deaths
at each dose were determined. Control samples were
included and assayed simultaneously. IC50 values were cal-
culated from 24 h counts using the probit analysis [38].
[27] J. Quiroga, D. Mejía, B. Insuasty, R. Abonía, M. Nogueras, A.
Sanchez, J. Cobo, J. N. Low, J. Heterocycl. Chem. 2002, 39, 51.
[28] J. R. Zimmerman, B. J. Myers, S. Bouhall, A. McCarthy, O. John-
tony, M. Manpadi, Tetrahedron Lett. 2014, 55, 936.
[29] P. N. Solis, C. W. Wrigth, M. M. Anderson, M. P. Gupta, J. D. Phil-
lipson, Planta Medica 1993, 59, 250.
References
[1] A. M. Edwards, J. B. L. Howell, Clin. Exp. Allergy 2000, 30, 756.
[2] L. H. Hurley, Nat. Rev. Cancer 2002, 2, 188.
[3] A. K. Mukherjee, S. Basu, N. Sarkar, A. C. Ghosh, Curr. Med.
Chem. 2001, 8, 1467.
[4] W. Samee, P. Nunthanavanit, J. Unwitayatorn, Int. J. Mol. Sci.
2008, 9, 235.
[5] S. M. Hasan, M. M. Alam, A. Husain, S. Khanna, M. Akhtar, M.
S. Zaman, Eur. J. Med. Chem. 2009, 44, 4896.
[6] R. Amin, B. Krammer, N. Abdel-Kader, T. Verwanger, A. El-
Ansary, Eur. J. Med. Chem. 2010, 45, 372.
[7] J. Ungwitayatorn, W. Samee, J. Pimthon, J. Mol. Struct. 2004,
689, 99.
[30] J. E. Anderson, C. M. Goetz, J. M. McLaughlin, Phytochem. Anal.
1991, 2, 107.
[31] B. N. Meyer, N. R. Ferrigni, J. E. Putnam, L. B. Jacobsen, D. E.
Nichols, J. L. McLaughlin, Planta Med. 1982, 45, 31.
[32] C. L. Zani, P. P. Chavez, R. Queiroz, A. B. de Oliveira, J. E. Car-
doso, A. M. Anjos, T. S. Grandi, Phytomedicine 1995, 2, 47.
[33] L. P. Santos Pimenta, G. B. Pinto, J. A. Takahashi, L. G. F. e
Silva, M. A. D. Boaventura, Phytomedicine 2003, 10, 209.
[34] G. Sabitha, R. S. Babu, J. S. Jadav, Synth. Commun. 1998, 28,
4571.
[35] A. A. Gakh, M. N. Burnett, J. Fluorine Chem. 2011, 132, 88.
[36] J.-P. Bégué, D. Bonnet-Delpont in Fluorine and Health, Elsevier,
2008, chapter 13, p. 553.
[8] P. Nunthanavanit, N. G. Anthony, B. F. Johnston, S. P. Mackay, J.
Ungwitayatorn, Arch. Pharm. Chem. Life Sci. 2008, 341, 357.
[9] W. Huang, M.-Z. Liu, Y. Li, Y. Tan, G.-F. Yang, Bioorg. Med. Chem. [37] G. J. Reddy, D. Latha, C. Thirupathaiah, K. S. Rao, Heterocycl.
2007, 15, 5191.
Commun. 2003, 9, 391.
[38] D. Finney, Probit Analysis, 3rd Edition, Cambridge University
Press, Cambridge, 1971, chapter 3, p. 21.
[10] W. Huang, Y. Ding, Y. Miao, M.-Z. Liu, Y. Li, G.-F. Yang, Eur. J.
Med. Chem. 2009, 44, 3687.
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