6-Substituted 1,4-Naphthoquinone Oxime Derivatives (III): Synthesis and Cytotoxic Evaluation

Article abstract of DOI:10.1134/S1070363218050316

As a continuous study, a set of 23 new 6-substituted 1,4-naphthoquinone oxime derivatives are synthesized and screened for their in vitro cytotoxic activity. Four of those oxime derivatives demonstrate more potent cytotoxic activity towards K562, HCT-15, and HCT-116 cell lines than a reference drug 5-Fu. In particular, compound 21g exhibits the strongest inhibitory activity against K562 cell lines with IC₅₀ values of 1.25 μM. According to flow cytometry data, compound 21g can arrest cell cycle at S phase and induce a strong apoptotic response in K562 cells. The preliminary structure-activity relationship study shows that the nature of substituents in positions 6 and 1' of 1,4-naphthoquinone derivatives significantly affect their cytotoxic activity.

Full text of DOI:10.1134/S1070363218050316

ISSN 1070-3632, Russian Journal of General Chemistry, 2018, Vol. 88, No. 5, pp. 1025–1035. © Pleiades Publishing, Ltd., 2018.
6-Substituted 1,4-Naphthoquinone Oxime Derivatives (III):
Synthesis and Cytotoxic Evaluation1
G. Huanga*, M. C. Liua, Q. Q. Menga, and S. S. Lia**
a School of Pharmacy, Shanghai Jiao Tong University, Shanghai, 200240 China
e-mail: *guanghuangmedchem@hotmail.com; **ssli@sjtu.edu.cn
Received April 1, 2018
Abstract—As a continuous study, a set of 23 new 6-substituted 1,4-naphthoquinone oxime derivatives are
synthesized and screened for their in vitro cytotoxic activity. Four of those oxime derivatives demonstrate more
potent cytotoxic activity towards K562, HCT-15, and HCT-116 cell lines than a reference drug 5-Fu. In
particular, compound 21g exhibits the strongest inhibitory activity against K562 cell lines with IC50 values of
1.25 μM. According to flow cytometry data, compound 21g can arrest cell cycle at S phase and induce a strong
apoptotic response in K562 cells. The preliminary structure-activity relationship study shows that the nature of
substituents in positions 6 and 1' of 1,4-naphthoquinone derivatives significantly affect their cytotoxic activity.
Keywords: 1,4-naphthoquinone, oxime derivatives, drug research, structure-activity relationships, cytotoxic
activity
DOI: 10.1134/S1070363218050316
INTRODUCTION
line [8, 9]. Therefore, incorporation of different
substituents into 1,4-naphthoquinone oxime scaffold
might result in the desirable biological activity.
Over the recent decades, 6-substituted 1,4-
naphthoquinone derivatives have attracted close
attention from medicinal chemists [1–4] due to a wide
variety of their biological activities. 1,4-Naphthoquinone
scaffold can generate reactive oxygen species (ROS)
and induce bioreductive alkylation, indiscriminately
leading to dearth of tumor and normal cells [5].
Encouraged by aforementioned studies and in
continuation of research on synthesis and cytotoxic
activity of 1,4-naphthoquinone oxime derivatives, we
introduced various substituents to 1,4-naphthoquinone
dioxime scaffold, modified the hydroxyl group of the
side chain and evaluated their in vitro cytotoxic
activity. The preliminary structure-activity relationship
was approached.
Our earlier study revealed that 6-substituted 5,8-
dimethoxy-1,4-naphthoquinone (DMNQ) derivatives
displayed stronger cytotoxicity than the corresponding
2-substituted DMNQs [6], but adverse effects
including hypotrichosis, weight reduction and much
bloody ascites were observed in mouse model.
Fortunately, our following study demonstrated that
oximation of the carbonyl groups afforded shikonin
oxime derivatives that exhibited more significant
cytotoxic activity against cancer cells along with lower
cytotoxicity towards normal cells than the lead
compound [7]. Mechanism of their strong inhibitory
effect was found to be not associated with bioreductive
alkylation and reactive oxygen species (ROS).
Recently, we have determined that some 6-substituted
1,4-naphthoquinone oxime derivatives demonstrated
high cytotoxic activity against a panel of cancer cell
RESULTS AND DISCUSSION
The general synthetic approaches to target
compounds are outlined in Schemes 1, 2, and 3. The
direct formylation of 1,4,5,8-tetramethoxynaphthalene
1 via the Vilsmeier-Haack reaction, followed by oxida-
tion of aldehyde 2 with Bu4NMnO4 yielded compound
3. Condensation of carboxylic acid 3 with alcohols
afforded compounds 4a–4c in excellent yields in the
presence of DCC and DMAP. Subsequent oxidation of
compounds 4a–4c with cerium(IV) ammonium nitrate
(CAN) gave corresponding 6-substituted 1,4-naphtho-
quinones 5a–5c [9]. Stirring of compounds 5a–5c at
50°C with hydroxylamine hydrochloride in the
presence of pyridine gave oximes 6a–6c. Following
hydrolysis of compound 6a with lithium hydroxide
1 The text was submitted by the authors in English.
1025

1026
HUANG et al.
Scheme 1.
OMe OMe
OMe OMe
POCl3, DMF
CH2Cl2
CHO
OMe OMe
OMe OMe
2
1
OMe OMe
OMe OMe
(1) Bu4NMnO4, pyridine
(2) NaHSO3
ROH, DCC, DMAP
CH2Cl2
COOR
COOH
OMe OMe
OMe OMe
OMe
4a−4c
3
HO
O
O
N
N
OMe
.
NH2OH HCl
CAN, EtOAc
H2O
EtOH, pyridine
COOR
COOR
OMe
OMe
6a−6c
HO
5a−5c
HO
N
OMe
.
LiOH H2O
MeOH, H2O
COOH
N
OMe
HO
7
R = Me, Et, i-Pr.
monohydrate provided the target compound 7 in
relatively high yield. Reduction of aldehyde 2 by
NaBH4 afforded compound 10, which was further
converted into compounds 13a–13c via acylation. The
target compounds 15a–15c were synthesized in two
steps from esters 13a–13c by oxidation using CAN
and condensation with hydroxylamine hydrochloride.
In a similar manner, the target compound 12 was
synthesized by either alkaline hydrolysis of ester 15a
or direct oximation of compound 11 with hydroxyl-
amine hydrochloride.
title compounds 18a–18g and 21a–21g were then
synthesized following the method described above for
compounds 15a–15c.
The newly synthesized compounds were evaluated
for their in vitro cytotoxic activity against leukemia
(K562), human colorectal cancer (HCT-15 and HCT-
116) and human skin fibroblast (HSF) cell lines (see
the table). The drug 5-fluorouracil (5-Fu) was used as
the positive control.
According to the accumulated cytotoxicity data,
some 6-substituted 1,4-naphthoquinone oximes ex-
hibited moderated to good cytotoxicity towards K562,
HCT-15, and HCT-116 cells. All oximes derivatives
showed no obvious toxicity (IC50 >100 μM) against
The Grignard addition of alkyl magnesium bromide
to compound 2, followed by oxidative demethylation
with CAN gave 1,4-naphthoquinones 17a–17g. The
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 88 No. 5 2018

6-SUBSTITUTED 1,4-NAPHTHOQUINONE OXIME DERIVATIVES (III)
1027
Scheme 2.
HO
O
OMe
N
N
OMe
.
NH2OH HCl
CAN, EtOAc
H2O
1
EtOH, pyridine
O
OMe
OMe
HO
N
8
9
HO
N
HO
HO
OMe
OMe
(1) NaOH, MeOH
CH2OH(2) 2 M HCl
CH2OCOR
N
OMe
N
OMe
HO
15a−15c
12
.
NH2OH HCl
EtOH, pyridine
OMe OMe
O
OMe
OMe OMe
CAN, EtOAc
H2O
RCOOH, DCC
DMAP, CH2Cl2
NaBH4
EtOH
2
CH2OCOR
CH2OCOR
CH2OH
OMe OMe
O
OMe
OMe OMe
10
13a−13c
14a−14c
HO
N
O
O
OMe
OMe
.
NH2OH HCl
CAN, EtOAc
H2O
EtOH, pyridine
CH2OH
CH2OH
OMe
N
OMe
HO
11
12
R = Me, Et, i-Pr.
HSF. Cytotoxic activity of oxime derivatives was
largely dependent upon the substituents R. For
example, replacement of H atom on the 6-position of
1,4-naphthoquinone oxime scaffold (compound 9)
with carboxyl and hydroxymethyl group gave rise to
compounds 7 and 12, which caused the minor increase
in cytotoxic activity. Compounds 6a–6c bearing the
ester moiety displayed more potent cytotoxicity than
compound 12, and compound 6c demonstrated the
highest cytotoxic activity among these four products.
This tendency was also observed among compounds
15a–15c. Thus, steric hindrance of substituents R could
have influence on cytotoxic activity cytotoxic activity.
Among compounds 18a–18g with saturated alkyl
alcohol, compound 18g exhibited the highest in-
hibitory effects against K562, HCT-15, and HCT-116
cells with IC50 of 4.24, 8.17, and 9.41 μM, respec-
tively. Acylation of compounds 18a–18g afforded
products 21a–21g, which were characterized by sig-
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 88 No. 5 2018

1028
HUANG et al.
Scheme 3.
HO
HO
N
N
OMe
R
OMe OH
18a−18g
.
(1) 10% NaOH, H2O
NH2OH HCl
HO
O
O
OMe
N
OMe
(2) 2 M HCl
EtOH, pyridine
CAN, EtOAc
H2O
.
R
R
NH2OH HCl
EtOH, pyridine
OMe OH
17a−17g
N
OMe OAc
21a−21g
HO
O
OMe
OMe OMe
OMe OMe
Mg, RBr
(CH3CO)2O, DMAP
CAN, EtOAc
H2O
2
THF
R
R
R
O
OMe OAc
OMe OMe OAc
OMe OMe OH
19a−19g
16a−16g
20a−20g
R = Me, Et, Pr, i-Pr, n-butyl, i-butyl, i-pentyl.
nificant increase in cytotoxicity. These results were in
agreement with our earlier reports [6, 7] that
introduction of acyl groups to the 1'-OH of shikonin to
was an important factor for cytotoxic effects. The
compound 21g exhibited the strongest cytotoxic
activity towards K562, HCT-15, and HCT-116 cells,
which supported the influence of steric effects of
substituents R upon anticancer potency.
treated with different concentrations of 21g (0, 0.5, 1.0
or 2.0 μM) for 24 h and analyzed for cells changes in
apoptotic markers using flow cytometer. The percentage
of apoptotic cells was significantly increased after
treatment with enhancing concentrations of 21g
(Figs. 2a, 2b).
EXPERIMENTAL
The cell cycle analysis of compound 21g was
carried out using flow cytometry (Figs. 1a, 1b). The
percentage of cells in the S phase at indicated
concentrations (0.5, 1.0, or 2.0 μM) were 59.33%,
61.73% and 64.95%, respectively (55.19% for the
control), while the proportion of cells at G2/M phase
decreased significantly by treatment of 21g (p < 0.05,
p < 0.01 or p < 0.005) and the rate of cells at G1 phase
had no significant increase. These results suggested
that 21g-induced S phase of cell cycle arrest might be one
of the plausible mechanisms of cellular cytotoxicity.
Reagents and solvents were obtained from
commercial suppliers. Column chromatography was
conducted on silica gel (100–200 mesh) purchased
1
from Qingdao Ocean Chemical Factory. H NMR
spectra were measured on a Varian MERCURY
spectrometer using CDCl3 or DMSO-d6 as solvents
and TMS as the internal reference. HRMS spectra
were recorded on a Shimadzu LC-MS-2010EV mass
spectrometer.
1,4,5,8-Tetramethoxy-2-naphthaldehyde (2) [7].
A solution of 1,4,5,8-tetramethoxynaphthalene (1)
(38.0 g, 0.15 mol) in CH2Cl2 (250 mL) was added to a
Annexin V/PI staining was used for measuring the
effect of 21g on K562 cells apoptosis. Cells were
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 88 No. 5 2018

6-SUBSTITUTED 1,4-NAPHTHOQUINONE OXIME DERIVATIVES (III)
1029
In vitro cytotoxic activity of 1,4-naphthoquinone oxime derivatives against K562, HCT-15, HCT-116, and HSF cell lines
HO
N
OMe
R
N
OMe
HO
IC50a on cell lines, μM
K562 HCT-15 HCT-116 HSF
IC50a on cell lines, μM
K562 HCT-15 HCT-116 HSF
Comp.
no.
Comp.
no.
R
O
R
1'
6a
35.7
33.2
40.4
>100 18d
22.8
30.3
28.5
>100
O
OH
1'
O
6b
6c
7
26.5
22.4
49.2
84.3
29.6
25.8
53.4
90.8
35.1
28.7
65.9
>100
>100 18e
>100 18f
>100 18g
>100 21a
9.43
7.21
4.24
33.2
20.6
15.2
8.17
41.8
15.4
13.9
9.41
39.5
>100
>100
>100
>100
O
OH
1'
O
O
O
OH
1'
OH
OH
1'
9
H
OAc
1'
OH
12
67.1
20.3
73.9
26.6
82.3
29.2
>100 21b
>100 21c
30.7
19.8
29.6
20.3
26.7
17.8
>100
>100
OAc
1'
O
15a
O
OAc
O
O
1'
15b
16.5
22.4
27.8
>100 21d
10.7
13.4
19.5
>100
O
O
OAc
1'
15c
18a
18b
18c
12.4
67.4
52.6
34.2
17.9
73.5
60.4
45.7
21.6
82.1
65.8
39.6
>100 21e
>100 21f
>100 21g
>100 5-Fu
5.78
2.23
1.25
15.6
8.06
2.79
1.63
8.24
10.8
3.91
2.47
12.5
>100
>100
>100
>100
OAc
1'
1'
OAc
OH
1'
1'
OH
1'
OAc
̲
OH
a IC50 values were calculated from three independent experiments.
mixture of POCl3 (570 mL, 0.76 mol) with N,N-
dimethylformamide (59 mL, 0.77 mol). The reaction
mixture was refluxed for 12 h, then quenched with ice
water and extracted with CH2Cl2. The organic phases
were combined, washed with brine, dried, and
concentrated. The crude product was recrystallized
from hexane to give compound 2 (38.8 g, yield 92%)
1
as a yellow solid. H NMR spectrum, δ, ppm: 3.91 s
(3H, ArOCH3), 3.92 s (3H, ArOCH3), 3.98 s (3H,
ArOCH3), 3.99 s (3H, ArOCH3), 6.93 d (J = 7.4 Hz,
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 88 No. 5 2018

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HUANG et al.
(a)
0 μM
0.5 μM
100
80
100
80
60
60
40
40
20
0
20
0
0
50
100 150
200 250
0
50
100 150
200 250
Channels P1-A
Channels P1-A
1.0 μM
2.0 μM
100
80
100
80
60
40
60
40
20
0
20
0
0
50
100
150
200 250
0
50
100
150
200 250
Channels P1-A
Channels P1-A
(b)
S
100
80
G2/M
60
40
20
G1
0
μM
Fig. 1. Compound 21g induced cell cycle arrest of K562 cells. (a) Diagram of the cell cycle for each phase based on flow
cytometry. (b) The rate of cells in G1, S, and G2/M phases. The data were analyzed by Student’s t-test, *p < 0.05, **p < 0.01,
***p < 0.005.
1H, ArH), 7.01 d (J = 7.8 Hz, 1H, ArH), 7.16 s (1H,
ArH), 10.56 s (1H, ArCHO).
permanganate (3.62 g, 10.0 mmol) in portion. The
mixture was stirred at room temperature for 18 h.
Saturated sodium bisulfite solution was added drop-
wise to the reaction mixture and a yellow solid was
precipitated. The filtrate was acidified with 2 M
hydrochloric acid and then extracted with dichloro-
1,4,5,8-Tetramethoxy-2-naphthoic acid (3) [10].
To a stirred solution of compound 3 (1.38 g, 10.0 mmol)
in pyridine (30 mL) was added tetrabutylammonium
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6-SUBSTITUTED 1,4-NAPHTHOQUINONE OXIME DERIVATIVES (III)
1031
(a)
104
104
Q1
Q2
Q1
Q2
0.021%
2.22%
0.041%
3.03%
103
102
103
102
0 μM
0.5 μM
(b)
100
Q4
93.2%
Q3
4.59%
Q4
91.5%
Q3
5.46%
101
101
80
60
40
100
104
100
104
100 101 102 103 104
100 101 102 103 104
Q1
Q2
59.5%
Q1
Q2
8.32%
PI
0.232%
1.43%
20
0
103
102
103
102
1.0 μM
2.0 μM
0
0.5
1.0
2.0
μM
Q4
16.1%
Q4
78.9%
101
100
101
100
Q3
22.9%
Q3
12.5%
100 101 102 103 104
100 101 102 103 104
Annexin V-FITC
Fig. 2. Compound 21g induced apoptosis of K562 cells. (a) K562 cells apoptosis was detected by Annexin V/PI on flow cytometry.
(b) The proportion of apoptotic (Annexin V positive) cells increased dependently with 21g treatment concentration. The data were
analyzed by Student’s t-test, **p < 0.01, ***p < 0.005.
methane, washed with brine, dried, and concentrated.
The crude product was purified by flash column
chromatography to afford compound 3 (1.07 g, yield
To a stirred solution of carboxylic acid (0.60 mmol)
and alcohol (0.50 mmol) in anhydrous dichloromethane
(5.0 mL) were added DCC (124 mg, 0.60 mmol) and
DMAP (31 mg, 0.25 mmol). After stirring overnight at
room temperature, petroleum ether was added to the
reaction mixture at 4°C to facilitate precipitation. The
solution was filtered and concentrated. The residue
was purified by flash chromatography to afford 4a–4c
and 13a–13c as colorless oils.
1
73%) as a light-yellow solid. H NMR spectrum, δ,
ppm: 3.92 s (3H, ArOCH3), 3.94 s (3H, ArOCH3), 3.98
s (3H, ArOCH3), 4.00 s (3H, ArOCH3), 6.99 s (2H,
ArH), 7.48 s (1H, ArH), 11.95 s (1H, ArCOOH).
2-Hydroxymethyl-1,4,5,8-tetramethoxynaph-
thalene (10). To a stirred solution of compound 2
(2.76 g, 10.0 mmol) in absolute ethanol (100 mL) was
added sodium borohydride (3.03 g, 40.0 mmol) in
portion. The mixture was heated at reflux overnight
and then cooled. The ethanol was removed under
reduced pressure, the residue was diluted with water
and extracted with dichloromethane, washed with
brine, dried and concentrated. The crude product was
purified by flash column chromatography to afford
compound 10 (2.60 g, yield 94%) as light-yellow solid.
1H NMR spectrum, δ, ppm: 2.40 s (1H, OH), 3.78 s
(3H, ArOCH3), 3.89 s (3H, ArOCH3), 3.92 s (3H,
ArOCH3), 3.93 s (3H, ArOCH3), 6.80 s (2H, ArH),
6.90 s (1H, ArH).
Synthesis of 2-substituted 1,4,5,8-tetramethoxy-
naphthalenes (16a–16g) (general procedure). 1-Bromo-
alkane (14.1 mmol) was added dropwise at 50°C to a
stirred suspension of magnesium powder (0.34 g,
14.3 mmol) and a catalytic amount of iodine in
anhydrous THF (10.0 mL). After stirring at 50°C for
2 h, a solution of aldehyde 2 (0.78 g, 2.82 mmol) in
dry THF (4.0 mL) was added to the solution of the
prepared alkylmagnesium bromide. The mixture was
stirred overnight at room temperature and then
quenched with saturated NH4Cl solution. The mixture
was extracted with dichloromethane, washed with
brine, dried, and concentrated. The residue was puri-
fied by flash column chromatography to afford 16a–
16g as colorless oils.
Synthesis of 2-substituted 1,4,5,8-tetramethoxy-
naphthalenes (4a–4c and 13a–13c) (general procedure).
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HUANG et al.
General procedures for 2-substituted 1,4,5,8-
3.65 s (3H, COOCH3), 3.82 s (3H, ArOCH3), 5.11–
5.22 m [1H, COOCH(CH3)2], 7.27 s (1H, ArH), 7.40 s
(2H, QuinH), 12.18 s (1H, =N–OH), 12.26 s (1H, =N–
OH). HRMS (ESI), m/z: 335.1240 [M + H]+.
C16H19N2O6. Calculated: 335.1243.
tetramethoxy-naphthalenes (19a–19g). To a stirred
solution of an alcohol 16a–16g (2.0 mmol) in acetic
anhydride (20 mL) was added 4-dimethylamino-
pyridine (0.4 mmol). The reaction mixture was stirred
at room temperature for 12 h and then quenched with
methanol. The mixture was extracted with dichloro-
methane, washed with brine, dried, and concentrated to
afford 19a–19g as yellow oils, that were used directly
in the next step without purification.
(1E,4E)-5,8-Dimethoxy-1,4-naphthoquinone di-
1
oxime (9). Yield 81%. H NMR spectrum, δ, ppm:
3.76 s (6H, 2ArOCH3), 7.13 s (1H, ArH), 7.37 s (2H,
QuinH), 12.18 s (2H, =N–OH). HRMS (ESI), m/z:
249.0874 [M + H]+. C12H13N2O4. Calculated: 249.0875.
General procedure for 6-substituted 1,4-naphtho-
quinones (5a–5c, 8, 11, 14a–4c, 17a–17g, and 20a–20g).
A solution of CAN (1.42 g, 2.6 mmol) in H2O
(2.5 mL) was added dropwise to a solution of 2-
substituted 1,4,5,8-tetramethoxynathalene (1.0 mmol)
in EtOAc (10.0 mL) at 0°C. In 8 min, the reaction
mixture was extracted with EtOAc, washed with brine,
dried, and concentrated. The residue was purified by
flash column chromatography to afford 6-substituted
1,4-naphthoquinone as a yellow oil.
(1E,4E)-6-Acetoxymethyl-5,8-dimethoxy-1,4-
naphthoquinone dioxime (15a). Yield 84%. 1H NMR
spectrum, δ, ppm: 2.11 s (3H, COCH3), 3.62 s (3H,
ArOCH3), 3.80 s (3H, ArOCH3), 5.15 s (2H, ArCH2O),
7.18 s (1H, ArH), 7.39 s (2H, QuinH), 12.10 s (2H,
=N–OH); HRMS (ESI), m/z: 321.1087 [M + H]+.
C15H17N2O6. Calculated: 321.1087.
(1E,4E)-6-Propionyloxymethyl-5,8-dimethoxy-
1
1,4-naphthoquinone dioxime (15b). Yield 86%. H
NMR spectrum, δ, ppm: 1.07 t (J = 6.8 Hz, 3H,
COCH2CH3), 2.42 q (J = 7.8 Hz, 2H, COCH2CH3),
3.63 s (3H, ArOCH3), 3.79 s (3H, ArOCH3), 5.17 s
(2H, ArCH2O), 7.17 s (1H, ArH), 7.39 s (2H, QuinH),
12.10 s (2H, =N–OH). HRMS (ESI), m/z: 335.1242
[M + H]+. C16H19N2O6. Calculated: 335.1243.
General procedure for the target compounds
(6a–6c, 9, 12, 15a–15c, 18a–18g, and 21a–21g). A
mixture of a 6-substituted 1,4-naphthoquinone derivative
(0.50 mmol) with hydroxylamine hydrochloride (0.24 g,
3.5 mmol) and pyridine (0.28 g, 3.5 mmol) in
anhydrous ethanol (12 mL) was stirred at 50°C for
24 h under nitrogen atmosphere. After cooling down,
the reaction mixture was concentrated and then poured
into ice-water. The resulting precipitate was filtered off
and recrystallized from ethanol to give the target
compounds 6a–6c, 9, 12, and 15a–15c as yellow solids.
(1E,4E)-6-(2-Methyl-1-oxopropoxy)methyl-5,8-
dimethoxy-1,4-naphthoquinone dioxime (15c). Yield
1
85%. H NMR spectrum, δ, ppm: 1.15 d (J = 6.8 Hz,
6H, COCH(CH3)2), 2.61–2.72 m [1H, OCOCH(CH3)2],
3.64 s (3H, ArOCH3), 3.79 s (3H, ArOCH3), 5.19 s
(2H, ArCH2O), 7.15 s (1H, ArH), 7.40 s (2H, QuinH),
12.11 s (2H, =N–OH). HRMS (ESI), m/z: 349.1898
[M + H]+. C17H21N2O6. Calculated: 349.1900.
(1E,4E)-6-Methoxyformyl-5,8-dimethoxy-1,4-
1
naphthoquinone dioxime (6a). Yield 82%. H NMR
spectrum, δ, ppm: 3.66 s (3H, COOCH3), 3.82 s (3H,
ArOCH3), 3.88 s (3H, ArOCH3), 7.33 s (1H, ArH),
7.40 s (2H, QuinH), 12.16 s (1H, =N–OH), 12.23 s
(1H, =N–OH). HRMS (ESI), m/z: 307.0928 [M + H]+.
C14H15N2O6. Calculated: 307.0930.
(1E,4E)-6-Hydroxymethyl-5,8-dimethoxy-1,4-
1
naphthoquinone dioxime (12). Yield 84%. H NMR
spectrum, δ, ppm: 3.55 s (3H, ArOCH3), 3.76 s (3H,
ArOCH3), 4.59 d (J = 5.6 Hz, 2H, ArCH2OH), 5.22 t
(J = 5.6 Hz, 1H, ArCH2OH), 7.22 s (1H, ArH), 7.38 s
(2H, QuinH), 11.95 s (2H, =N–OH). HRMS (ESI),
m/z: 279.0979 [M + H]+. C13H15N2O5. Calculated:
279.0981.
(1E,4E)-6-Ethoxyformyl-5,8-dimethoxy-1,4-
1
naphthoquinone dioxime (6b). Yield 84%. H NMR
spectrum, δ, ppm: 1.34 t (J = 7.2 Hz, 3H, CH2CH3),
3.66 s (3H, COOCH3), 3.82 s (3H, ArOCH3), 4.34 q
(J = 7.2 Hz, 2H, COOCH2CH3), 7.31 s (1H, ArH), 7.40
s (2H, QuinH), 12.19 s (1H, =N–OH), 12.27 s (1H,
=N–OH). HRMS (ESI), m/z: 321.1086 [M + H]+.
C15H17N2O6. Calculated: 321.1087.
Alkaline hydrolysis of ester 15a. A mixture of com-
pound 15a (0.15 g, 0.47 mmol) with NaOH (56.4 mg,
1.41 mmol) and methanol (20 mL) was stirred at room
temperature for 1 h. After completion of the process,
the reaction mixture was concentrated and acidified
with 2 M hydrochloric acid. The yellow solid was
precipitated and filtered off. The crude product was
(1E,4E)-6-Isopropoxyformyl-5,8-dimethoxy-1,4-
naphthoquinone dioxime (6c). Yield 88%. 1H NMR spec-
trum, δ, ppm: 1.35 d [J = 6.2 Hz, 6H, COOCH(CH3)2],
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6-SUBSTITUTED 1,4-NAPHTHOQUINONE OXIME DERIVATIVES (III)
1033
recrystallized from petroleum ether/ ethyl acetate to
give compound 12 (0.11 g, yield 85%) as a yellow solid.
6.6 Hz, 6H, CHCH3), 1.32–1.38 m [1H, CH2CH(CH3)2],
1.52–1.59 m [1H, 1/2(ArCHCH2)], 1.77–1.86 m [1H,
1/2(ArCHCH2)], 3.61 s (3H, ArOCH3), 3.80 s (3H,
ArOCH3), 5.02 d.d (J = 8.8, 4.2 Hz, 1H, ArCHCH2),
5.13 d (J = 4.0 Hz, 1H, OH), 7.25 s (1H, ArH), 7.39 s
(2H, QuinH), 11.98 s (2H, =N–OH). HRMS (ESI),
m/z: 335.1605 [M + H]+. C17H23N2O5. Calculated:
335.1607.
(1E,4E)-6-(1-Hydroxyethyl)-1,4-naphthoquinone
dioxime (18a). Yield 84%. 1H NMR spectrum, δ, ppm:
1.34 d (J = 6.0 Hz, 3H, ArCHCH3), 3.60 s (3H,
ArOCH3), 3.80 s (3H, ArOCH3), 5.05–5.15 m [1H,
ArCH(OH)CH3], 5.20 d (J = 4.0 Hz, 1H, OH), 7.27 s
(1H, ArH), 7.38 s (2H, QuinH), 11.98 s (2H, =N–OH).
HRMS (ESI), m/z: 293.1134 [M + H]+. C14H17N2O5.
Calculated: 293.1137.
(1E,4E)-6-(1-Hydroxy-4-methylpentyl)-1,4-
naphthoquinone dioxime (18g). Yield 82%. 1H NMR
spectrum, δ, ppm: 0.86 d [J = 6.4 Hz, 6H, CH(CH3)2],
1.17–1.26 m [1H, 1/2(CHCH2CH2)], 1.29–1.38 m (1H,
(1E,4E)-6-(1-Hydroxypropyl)-1,4-naphthoquinone
1
dioxime (18b). Yield 86%. H NMR spectrum, δ,
CH(CH3)2), 1.49–1.65
m
[3H, ArCHCH2 and
ppm: 0.88 t (J = 7.2 Hz, 3H, CH2CH3), 1.54–1.70 m
(2H, CHCH2CH3), 3.58 s (3H, ArOCH3), 3.77 s (3H,
ArOCH3), 4.85 t (J = 7.0 Hz, 1H, ArCHCH2), 5.13 d
(J = 4.0 Hz, 1H, OH), 7.27 s (1H, ArH), 7.36 s (2H,
QuinH), 11.95 s (2H, =N–OH). HRMS (ESI), m/z:
307.1293 [M + H]+. C15H19N2O5. Calculated: 307.1294.
1/2(CHCH2CH2)], 3.59 s (3H, ArOCH3), 3.78 s (3H,
ArOCH3), 4.89 d.d (J = 9.4, 4.8 Hz, 1H, ArCHCH2),
5.13 d (J = 4.8 Hz, 1H, OH), 7.23 s (1H, ArH), 7.38 s
(2H, QuinH), 11.98 s (2H, =N–OH). HRMS (ESI),
m/z: 349.1760 [M + H]+. C18H25N2O5. Calculated: 349.1763.
(1E,4E)-6-(1-Acetoxyethyl)-1,4-naphthoquinone
dioxime (21a). Yield 83%. 1H NMR spectrum, δ, ppm:
1.50 d (J = 5.0 Hz, 3H, CHCH3), 2.10 s (3H, COCH3),
3.65 s (3H, ArOCH3), 3.82 s (3H, ArOCH3), 6.10–6.16
m (1H, ArCHCH3), 7.09 s (1H, ArH), 7.39 s (2H,
QuinH), 12.10 s (2H, =N–OH). HRMS (ESI), m/z:
335.1243 [M + H]+. C16H19N2O6. Calculated: 335.1243.
(1E,4E)-6-(1-Hydroxybutyl)-1,4-naphthoquinone
dioxime (18c). Yield 84%. 1H NMR spectrum, δ, ppm:
0.90 t (J = 7.2 Hz, 3H, CH2CH3), 1.25–1.47 m (2H,
CH2CH2CH3), 1.53–1.72 m (2H, CHCH2), 3.60 s (3H,
ArOCH3), 3.79 s (3H, ArOCH3), 4.95 t (J = 7.0 Hz,
1H, ArCHCH2), 5.14 d (J = 4.0 Hz, 1H, OH), 7.24 s
(1H, ArH), 7.38 s (2H, QuinH), 11.97 s (2H, =N–OH).
HRMS (ESI), m/z: 321.1449 [M + H]+. C16H21N2O5.
Calculated: 321.1450.
(1E,4E)-6-(1-Acetoxypropyl)-1,4-naphthoquinone
1
dioxime (21b). Yield 81%. H NMR spectrum, δ,
ppm: 0.88 t (J = 7.4 Hz, 3H, CH2CH3), 1.70–1.82 m
(2H, CHCH2CH3), 2.10 s (3H, COCH3), 3.65s (3H,
ArOCH3), 3.81 s (3H, ArOCH3), 6.03 t (J = 6.4 Hz,
1H, ArCHCH2), 7.02 s (1H, ArH), 7.39 s (2H, QuinH),
12.11 s (2H, =N–OH). HRMS (ESI), m/z: 349.1397
[M + H]+. C17H21N2O6. Calculated: 349.1400.
(1E,4E)-6-(1-Hydroxy-2-methylpropyl)-1,4-
naphthoquinone dioxime (18d). Yield 86%. 1H NMR
spectrum, δ, ppm: 0.86 d (J = 5.4 Hz, 3H, CHCH3),
0.87 d (J = 5.4 Hz, 3H, CHCH3), 1.83–1.92 m (1H,
ArCHCH(CH3)2), 3.59 s (3H, ArOCH3), 3.78 s (3H,
ArOCH3), 4.67 d (J = 4.8 Hz, 1H, ArCHCH), 5.13 d
(J = 4.0 Hz, 1H, OH), 7.19 s (1H, ArH), 7.39 s (2H,
QuinH), 12.00 s (2H, =N–OH). HRMS (ESI), m/z:
321.1450 [M + H]+. C16H21N2O5. Calculated: 321.1450.
(1E,4E)-6-(1-Acetoxybutyl)-1,4-naphthoquinone
dioxime (21c). Yield 84%. 1H NMR spectrum, δ, ppm:
0.92 t (J = 7.4 Hz, 3H, CH2CH3), 1.25–1.44 m (2H,
CH2CH2CH3), 1.66–1.76 m [1H, 1/2(ArCHCH2)], 1.80–
1.91 m [1H, 1/2(ArCHCH2)], 2.11 s (3H, COCH3),
3.68 s (3H, ArOCH3), 3.82 s (3H, ArOCH3), 6.08 t (J =
7.0 Hz, 1H, ArCHCH2), 7.03 s (1H, ArH), 7.38 s (2H,
QuinH), 12.07 s (2H, =N–OH). HRMS (ESI), m/z:
363.1555 [M + H]+. C18H23N2O6. Calculated: 363.1556.
(1E,4E)-6-(1-Hydroxypentyl)-1,4-naphthoquinone
dioxime (18e). Yield 78%. 1H NMR spectrum, δ, ppm:
0.87 t (J = 7.0 Hz, 3H, CH2CH3), 1.24–1.46 m (4H,
CH2CH2), 1.55–1.65 m (2H, CHCH2CH2), 3.62 s (3H,
ArOCH3), 3.81 s (3H, ArOCH3), 4.92 d.d (J = 11.2,
6.0 Hz, 1H, ArCHCH2), 5.13 d (J = 4.0 Hz, 1H, OH),
7.23 s (1H, ArH), 7.38 s (2H, QuinH), 11.97 s (2H,
=N–OH). HRMS (ESI), m/z: 335.1603 [M + H]+.
C17H23N2O5. Calculated: 335.1607.
(1E,4E)-6-(1-Acetoxy-2-methylpropyl)-1,4-naphtho-
quinone dioxime (21d). Yield 86%. 1H NMR spectrum,
δ, ppm: 0.82 d (J = 5.2 Hz, 3H, CHCH3), 0.97 d (J =
5.2 Hz, 3H, CHCH3), 2.00–2.07 m [1H, CH(CH3)2],
2.11 s (3H, COCH3), 3.66 s (3H, ArOCH3), 3.80 s (3H,
ArOCH3), 6.07 t (J = 7.0 Hz, 1H, ArCHCH2), 6.98 s
(1H, ArH), 7.39 s (2H, QuinH), 12.09 s (2H, =N–OH).
(1E,4E)-6-(1-Hydroxy-3-methylbutyl)-1,4-naphtho-
quinone dioxime (18f). Yield 79%. 1H NMR spectrum,
δ, ppm: 0.92 d (J = 6.6 Hz, 3H, CHCH3), 0.96 d (J =
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 88 No. 5 2018

1034
HUANG et al.
HRMS (ESI), m/z: 363.1551 [M + H]+. C18H23N2O6.
Calculated: 363.1556.
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]
dye reduction assay. Exponentially growing cells were
seeded in 96-well microplates (100 μL/well at a
density of 3.5×104 cell/mL for the adherent cells and
3×104 cells/mL for the suspension cells) and allowed
to grow for 24 h prior to the exposure to the studied
compounds. Stock solutions of the tested compounds
were freshly prepared in DMSO and then diluted with
growth medium. At the final dilutions the solvent
concentration never exceeded 0.5%. Cells were
exposed to the tested agents for 72 h, whereby for each
concentration a set of six separate wells were used.
After the exposure period MTT solution (5 mg/mL in
PBS) aliquots were added to each well. The plates
were further incubated for 4 h at 37oC and formazan
crystals formed were dissolved by adding 110 μL of
5% HCOOH in 2-propanol. Absorption of the samples
was measured by Multiskan MK3 microplate reader
(Thermo Scientific, USA) at 570 nm. Concentration of
each compound was examined in triplicate, and IC50
values were calculated by linear regression analysis
using IBM SPSS statistics software (version 21.0).
(1E,4E)-6-(1-Acetoxypentyl)-1,4-naphthoquinone
dioxime (21e). Yield 87%. 1H NMR spectrum, δ, ppm:
0.87 t (J = 6.8 Hz, 3H, CH2CH3), 1.24–1.36 m (4H,
CH2CH2), 1.70–1.78 m [1H, 1/2(ArCHCH2)], 1.81–
1.89 m [1H, 1/2(ArCHCH2)], 2.10 s (3H, COCH3),
3.65 s (3H, ArOCH3), 3.80 s (3H, ArOCH3), 6.15 t (J =
7.0 Hz, 1H, ArCHCH2), 7.03 s (1H, ArH), 7.37 s (2H,
QuinH), 12.07 s (2H, =N–OH). HRMS (ESI), m/z:
377.1713 [M + H]+. C19H25N2O6. Calculated: 377.1713.
(1E,4E)-6-(1-Acetoxy-3-methylbutyl)-1,4-naphtho-
quinone dioxime (21f). Yield 87%. 1H NMR spectrum,
δ, ppm: 0.94 d [J = 5.4 Hz, 6H, CH(CH3)2], 1.40–1.54
m
[1H, CH2CH(CH3)2], 1.59–1.67
m
[1H,
1/2(ArCHCH2)], 1.80–1.88 m [1H, 1/2(ArCHCH2)],
2.11 s (3H, COCH3), 3.67 s (3H, ArOCH3), 3.81 s (3H,
ArOCH3), 6.15 t (J = 7.0 Hz, 1H, ArCHCH2), 7.03 s
(1H, ArH), 7.39 s (2H, QuinH), 12.09 s (2H, =N–OH).
HRMS (ESI), m/z: 377.1710 [M + H]+. C19H25N2O6.
Calculated: 377.1713.
Cell cycle analysis. K562 cells were plated in 6-well
plates and then treated with different concentrations of
21g for 24 h. After treatment, trypsinized adherent
cells were harvested (1×106 mL) and washed with cold
PBS. Subsequently, cells were centrifuged and fixed in
70% ethanol at 4°C overnight, that were resuspended
in PBS containing 100 μL of RNase A and 400 μL of
PI (Cell Cycle and Apoptosis Analysis Kit, Beyotime,
China). Cellular DNA content for cell cycle
distribution assay was detected by a flow cytometry
(FACS Calibur, BD Biosciences) and results were
analyzed using Modfit software.
(1E,4E)-6-(1-Acetoxy-4-methylpentyl)-1,4-naphtho-
quinone dioxime (21g). Yield 79%. 1H NMR spectrum,
δ, ppm: 0.86 d [J = 5.4 Hz, 6H, CH(CH3)2], 1.20–1.34
m (2H, CH2), 1.50–1.58 m [1H, CH2CH(CH3)2], 1.72–
1.78 m [1H, 1/2(ArCHCH2)], 1.82–1.87 m [1H,
1/2(ArCHCH2)], 2.10 s (3H, COCH3), 3.64 s (3H,
ArOCH3), 3.80 s (3H, ArOCH3), 6.02 t (J = 7.8 Hz,
1H, ArCHCH2), 7.02 s (1H, ArH), 7.38 s (2H, QuinH),
12.09 s (2H, =N–OH). HRMS (ESI), m/z: 391.1864
[M + H]+. C20H27N2O6. Calculated: 391.1869.
Synthesis of (1E,4E)-6-carboxyl-5,8-dimethoxy-
1,4-naphthoquinone dioxime (7). A mixture of com-
pound 6a (0.16 g, 0.52 mmol) with lithium hydroxide
monohydrate (0.13 g, 3.25 mmol), methanol (20 mL)
and water (5 mL) was stirred at room temperature for
12 h. After completion, the reaction mixture was con-
centrated and acidified with 2 M hydrochloric acid.
The precipitated red solid was filtered off and
recrystallized from petroleum ether/ ethyl acetate to
give compound 7 (0.14 g, yield 92%) as light red solid.
1H NMR spectrum, δ, ppm: 3.65 s (3H, ArOCH3), 3.80
s (3H, ArOCH3), 7.30 s (1H, ArH), 7.38 s (2H,
QuinH), 12.55 s (3H, =N–OH + COOH). HRMS
(ESI), m/z: 293.0772 [M + H]+. C13H13N2O6.
Calculated: 293.0774.
Apoptosis analysis. K562 cells were incubated in
6-well plates overnight. Cells were then treated with
vehicle and different concentrations of 21g for 24 h.
After incubation, cells were collected and washed
twice with PBS. Next, the cells were resuspended in
195 μL of Annexin V−FITC binding buffer. Sub-
sequently, 5 μL of binding buffer and 10 μL of PI were
added and incubated in darkness for 15 min at room
temperature. Percentage of apoptotic cells was deter-
mined by flow cytometry.
CONCLUSIONS
A total of 23 new 1,4-naphthoquinone oxime
derivatives were synthesized and evaluated for their in
vitro cytotoxicity against three cancer cell lines and
Cytotoxic activity assay [11]. Cytotoxicity of the
compounds was assessed using the MTT [3-(4,5-
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 88 No. 5 2018

6-SUBSTITUTED 1,4-NAPHTHOQUINONE OXIME DERIVATIVES (III)
doi 10.1021/jm00348a023
1035
one normal cell line. Cell-based assays demonstrated
that all oxime derivatives exhibited high selectivity
between cancer and normal cells. Some oxime deriva-
tives displayed moderate to high cytotoxic activity
towards cancer cells. Compound 21g was determined
to be the most cytotoxic towards K562, HCT-15, and
HCT-16 cell lines with IC50 values of 1.25, 1.63, and
2.47 μM. Compound 21g induced cell cycle arrest at S
phase and a strong apoptotic response in K562 cells.
The preliminary structure-activity relationship studies
revealed that the nature of substituents at positions 6
and 1' of 1,4-naphthoquinone played a vital role in
cytotoxicity of the products.
3. Gordaliza, M., Corral, J.M.M., Castro, M.A., Ma-
hiques, M.M., Gralvalos, M.D.G., and Feliciano, A.S.,
Bioorg. Med. Chem. Lett., 1996, vol. 6, p. 1859. doi
10.1016/0960-894X(96)00326-5
4. Song, G.Y., Kim, Y., Zheng, X.G., You, Y.J., Cho, H.,
Chung, J.H., Sok, D.E., and Ahn, B.Z., Eur. J. Med.
Chem., 2000, vol. 35, p. 291. doi 10.1016/S0223-5234
(00)00129-X
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doi 10.1016/0009-2797(91)90029-7
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Li, S.S., Eur. J. Med. Chem., 2011, vol. 46, p. 3420. doi
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ACKNOWLEDGMENTS
7. Huang, G., Zhao, H.R., Meng, Q.Q., Zhang, Q.J.,
Dong, J.Y., Zhu, B.Q., and Li, S.S., Eur. J. Med. Chem.,
2018, vol. 143, p. 166. doi 10.1016/j.ejmech.2017.11.031
This research was supported by National Natural
Science Foundation of China (nos. 81373274 and
81673281), Shanghai Science and Technology Innova-
tion (no. 17431902000) and Ph.D. Programs
Foundation of Ministry of Education China
(no. 20120073110068).
8. Huang, G., Zhao, H.R., Zhou, W., Dong, J.Y.,
Zhang, Q.J., Meng, Q.Q., Zhu, B.Q., and Li, S.S.,
Monatsh. Chem., 2017, vol. 148, p. 1011. doi 10.1007/
s00706-016-1899-z
9. Huang, G., Zhao, H.R., and Li, S.S., Russ. J. Gen.
Chem., 2017, vol. 87, p. 2979. doi 10.1134/
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