Synthesis of organic nitrates of luteolin as a novel class of potent aldose reductase inhibitors

Article abstract of DOI:10.1016/j.bmc.2013.04.066

Aldose reductase (AR) plays an important role in the design of drugs that prevent and treat diabetic complications. Aldose reductase inhibitors (ARIs) have received significant attentions as potent therapeutic drugs. Based on combination principles, three series of luteolin derivatives were synthesised and evaluated for their AR inhibitory activity and nitric oxide (NO)-releasing capacity in vitro. Eighteen compounds were found to be potent ARIs with IC ₅₀ values ranging from (0.099 ± 0.008) μM to (2.833 ± 0.102) μM. O⁷-Nitrooxyethyl-O^3′,O ^4′-ethylidene luteolin (La1) showed the most potent AR inhibitory activity [IC₅₀ = (0.099 ± 0.008) μM]. All organic nitrate derivatives released low concentrations of NO in the presence of l-cysteine. Structure-activity relationship studies suggested that introduction of an NO donor, protection of the catechol structure, and the ether chain of a 2-carbon spacer as a coupling chain on the luteolin scaffold all help increase the AR inhibitory activity of the resulting compound. This class of NO-donor luteolin derivatives as efficient ARIs offer a new concept for the development and design of new drug for preventive and therapeutic drugs for diabetic complications.

Full text of DOI:10.1016/j.bmc.2013.04.066

Bioorganic & Medicinal Chemistry 21 (2013) 4301–4310
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of organic nitrates of luteolin as a novel class of potent
aldose reductase inhibitors
a,
Qi-Qin Wang a, Ning Cheng a, Xiao-Wei Zheng a, Sheng-Ming Peng a,b, , Xiao-Qing Zou
⇑
⇑
a Key Laboratory of Environmentally Friendly Chemistry and Applications of Ministry of Education, College of Chemistry, Xiangtan University, Xiangtan 411105, China
b Postdoctoral Programme of Chemical Engineering & Technology, Xiangtan University, Xiangtan 411105, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Aldose reductase (AR) plays an important role in the design of drugs that prevent and treat diabetic com-
plications. Aldose reductase inhibitors (ARIs) have received significant attentions as potent therapeutic
drugs. Based on combination principles, three series of luteolin derivatives were synthesised and evalu-
ated for their AR inhibitory activity and nitric oxide (NO)-releasing capacity in vitro. Eighteen compounds
Received 19 March 2013
Revised 23 April 2013
Accepted 24 April 2013
Available online 3 May 2013
were found to be potent ARIs with IC50 values ranging from (0.099 0.008)
l
M to (2.833 0.102)
Nitrooxyethyl-O3 ,O4 -ethylidene luteolin (La1) showed the most potent AR inhibitory activity
[IC50 = (0.099 0.008) M]. All organic nitrate derivatives released low concentrations of NO in the pres-
ence of -cysteine. Structure–activity relationship studies suggested that introduction of an NO donor,
l
M. O7-
0
0
Keywords:
l
Diabetic complications
Organic nitrates
Luteolin
L
protection of the catechol structure, and the ether chain of a 2-carbon spacer as a coupling chain on
the luteolin scaffold all help increase the AR inhibitory activity of the resulting compound. This class
of NO-donor luteolin derivatives as efficient ARIs offer a new concept for the development and design
of new drug for preventive and therapeutic drugs for diabetic complications.
Aldose reductase inhibitors
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
ings, AR has become an attractive molecular target for novel drug
design.
Diabetes mellitus (DM) is a chronic, incurable metabolic disor-
der defined by the dysregulation of glucose homeostasis manifest-
ing as hyperglycaemia, abnormalities in lipid and protein
metabolism, and the development of both acute and long-term
complications.1 According to International Diabetes Federation
studies, approximately 366 million people worldwide were diag-
nosed with diabetes in 2011, and this number is expected to rise
to 522 million by 2030.2 DM is a leading cause of morbidity and
mortality in the world, particularly from complications such as
macrovascular complications, neuropathy, nephropathy, retinopa-
thy, and cataractogenesis.3,4 Increasing evidences suggest that
aldose reductase may provide a common biochemical link in the
pathogenesis of numerous diabetic complications and that the
hyperactivity of the polyol metabolic pathway catalysed by AR in
individuals with high blood glucose levels contributes to the
progression of diabetic complications.5
ARIs have received attentions as potential therapeutic drugs for
the prevention and treatment of diabetic complications.6,7 Over the
last three decades, many compounds with different structures
have been reported as ARIs, including alrestatin, tolrestat,
epalrestat, zopolrestat, zenarestat, ponalrestat, lidorestat,
naphtho[1,2-d]isothiazole derivatives, sorbinil, fidarestat, and rani-
restat.8 However, except for epalrestat, none of these compounds
are currently marketed. Many of the clinically evaluated ARIs have
proven to be inadequate as drug candidates because of their toxic
side effects or poor efficacy.8 Therefore, scientists are exerting
much effort into the development of novel ARIs with fewer side ef-
fects and excellent efficacy. Interest in flavonoids has steadily in-
creased because of their effectiveness, mild side effects, and
relatively low costs.9–11 A thorough survey of the related literature
revealed that flavonoids can modulate the activity of enzymes
(such as AR), affect the behaviour of many cell systems, and pro-
duce beneficial effects in the body.12
AR is an aldo–keto reductase that catalyses the nicotinamide
adenine dinucleotide phosphate (NADPH)-dependent reduction of
glucose to sorbitol in the first step of the polyol pathway. Sorbitol
is subsequently oxidised to fructose by sorbitol dehydrogenase
with concomitant reduction of NAD+ (Fig. 1).6 Based on these find-
⇑
Corresponding authors. Tel.: +86 073158293574.
E-mail address: pengshengming@21cn.com (S.-M. Peng).
Figure 1. Polyol pathway of glucose metabolism.
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.04.066

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Q.-Q. Wang et al. / Bioorg. Med. Chem. 21 (2013) 4301–4310
Luteolin
(2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4H-chro-
The synthetic route for compounds Lc1, Lc3, and Lc5 were sum-
marized in Scheme 3. Luteolin was heated with dic-
hlorodiphenylmethane in diphenyl ether at 175 °C for 30 min
yielded compound 6.25 Compounds 7a–c were synthesised accord-
ing to the method for 2a–c. Intermediates 7a–c were then reacted
with AgNO3 producing compounds 8a–c. Subsequent cleavage of
the diphenylmethyl group of 8a–c with a mixture of acetic acid
and water (4:1) gave the corresponding nitrate derivatives Lc1,
Lc3, and Lc5.26
men-4-one), a polyphenolic compound available in food products
of plant origin, belongs to the flavone subclass of flavonoids and
usually appears in its glycosylated form in celery, green pepper,
perilla leaf, and camomile tea.13 Preclinical studies have shown
that this flavone possesses a variety of pharmacological activities,
including anti-diabetic, by reducing glucose levels, antioxidant,
anti-inflammatory, and anticancer activities.14 Previous reports
have established that luteolin shows significant inhibitory activity
(IC50 = 0.6
l
M) against AR.15–17 Therefore, luteolin, as the scaffold
Luteolin was treated with 0.5 equiv bromoalkane and anhy-
drous K2CO3 to producing compounds Lc2, Lc4, and Lc6, respec-
tively (Scheme 4).
of ARIs, has considerable potential for the treatment of diabetic
complications.
NO as a gaseous signalling molecule participates in a plethora of
physiological processes, such as regulation of blood pressure,
3. Results and discussion
platelet
aggregation,
neurotransmission,
and
immune
responses.18,19 Considering the difficulty of performing meaningful
biological studies on NO gas, its progenitors (NO donors) are typi-
cally utilised in studies that investigate such diverse effects.18 Pre-
vious observations have shown that NO donors inhibit AR activity
and sorbitol accumulation in erythrocytes.20–22 Several studies
have demonstrated that inactivation of AR occurs by modification
of a hyper-reactive cysteine residue (Cys298) on the active site of
AR by thiol-modifying reagents, NO donors, and nitrosothiols.22,23
Furthermore, the vascular complications of diabetes are closely
associated with a decrease in NO generation. Thus, NO donors
could supply adequate amounts of NO to prevent AR activity and
diabetic complications.
3.1. Measurement of nitric oxide
Griess assay is the most popular method for the analysis of NO
because of its low costs, simple execution, and straightforward
data analysis.27,28 The capacity of thiol-induced NO generation of
organic nitrates of luteolin was evaluated after incubation for 1 h
in the presence of L-cysteine. The effectiveness of the synthesised
compounds was determined with respect to sodium nitroprusside
(SNP) as an NO donor. These results are summarised in Table 1.
The percentages of released NO, which varied from
1.018 0.046% to 4.637 0.040%, were equivalent to those of or-
ganic nitrates of chrysin.24 However, the capacity of NO released
from SNP was substantially higher (10.42 1.80%) than organic ni-
trates of luteolin. These results should be evaluated based on the
actual additional amount of NO required by the body. The concen-
trations of NO required to mediate primarily protective effects are
extremely low (picomolar to nanomolar range).24 In the present
study, the release of adequate amounts of NO required to protect
the body were balanced with the concentration range demanded
for the sufficient activity of luteolin derivatives.
Our recent studies discovered that a derivative of chrysin I (Ta-
ble 1) exhibited in vitro inhibitory activities against AR
(IC50 = 0.290 0.009 lM) and advanced glycation end-product for-
mation.24 This derivative of I was even observed to increase the
glucose consumption of HepG2 cells.24 Therefore, to study the ef-
fect of variations in the lead compound in comparison with chrysin
on AR activity, luteolin derivatives were designed as analogues of
compound I. We postulated that NO donor hybrids that incorporat-
ing the active parts of luteolin may be more potent than any of the
initial compounds alone. In this study, we coupled NO donors (or-
ganic nitrates) to the 7-position of luteolin through a series of ester
or ether chains of different spacers (Fig. 2). The NO-releasing capac-
ities and AR inhibitory activities of the resulting derivatives were
evaluated in vitro. We believe that this class of NO-donor luteolin
compounds is worthy of further study as potential ARIs for inhib-
iting the polyol pathway and preventing the development of sec-
ondary diabetic complications.
3.2. Aldose reductase inhibitory activity of the target
compounds
All newly synthesised derivatives of luteolin were evaluated for
their potential inhibitory effect on AR isolated from bovine lenses
using quercetin as a reference drug. The assay was based on the
spectrophotometric monitoring of NADPH oxidation, which has
proven to be a reliable method, with DL-glyceraldehyde as the sub-
strate and NADPH as the cofactor.24,29 In Table 1, results of the cur-
rent study were compared with the results previously reported24
for I in a similar assay.
2. Chemistry
All derivatives including La1–6, Lb1–6, and Lc1–6 described in
this study have been obtained by synthesis starting from luteolin,
as shown in Schemes 1–4. The preparation of compounds La1–6
were outlined in Scheme 1. Treating luteolin with 1,2-dibromoeth-
ane at 70 °C for 30 min in anhydrous DMF catalyzed by anhydrous
K2CO3 yielded compound 1. Compounds 2a–c were prepared by
treating compound 1 with excessive amounts of the appropriate
dibromoalkane at reflux (rt) for 2–24 h in anhydrous acetone.13
These compounds were then reacted with AgNO3 producing prod-
ucts La1, La3, and La5, respectively.24 Compounds La2, La4, and
La6 were synthesised according to the method for 2a–c.
All of the luteolin derivatives exhibited moderate or significant
in vitro inhibitory activities on AR with IC50 values ranging from
(0.099 0.008) lM to (2.833 0.102) l
M. Compare with chrysin,24
the 7-hydroxyl and catechol moiety at the B ring of luteolin could
interacts with more AR binding site, therefore, luteolin
[(0.754 0.062)
tested compounds, La1 was the most active ARI, with an IC50 value
of (0.099 0.008) M. La1 was 7.5-fold more potent than luteolin
and 28.5-fold more active than quercetin [(2.850 0.040) M].
lM] exhibited the strong activity. Among the
l
l
These results indicate that replacement of the lead compound with
luteolin, as in compounds La3, La1, and Lc1, could improve AR
Compounds Lb1–6 were synthesised in four or five steps from
luteolin as shown in Scheme 2.
inhibitory activities. La3 [(0.127 0.011)
effective than compound I [(0.290 0.009)
l
M] was 2.3-fold more
M] under the same
l
Compound 1 was reacted with ethyl bromoacetate to afford
compound 3. Subsequent hydrolysis of this compound and reac-
tion with bromoalkane or dibromoalkane produced compounds
Lb2, Lb4, Lb6, and 5a–c. The intermediates 5a–c were then reacted
with AgNO3 producing products Lb1, Lb3, and Lb5, respectively.
conditions (Table 1).
Figure 3 shows the AR inhibitory potency of the newly synthes-
ised derivatives and a possible mechanism that explains the struc-
ture–activity relationships (SARs) described in the follow section.
Figure 3A shows that the AR inhibitory activity of compounds I,

Q.-Q. Wang et al. / Bioorg. Med. Chem. 21 (2013) 4301–4310
4303
Table 1
In vitro bovine lens AR inhibitory activities and NO-releasing properties of the target compounds
OH
OH
O
OH
OH
O
HO
O
R
O
O
R
O
O
OH
OH
O
OH
Luteolin
La, Lb
Lc
Compound
Structure
AR inhibitiona
%No releasedb
0.1% DMSO
La1
La2
La3
La4
R
IC50 M)
(l
NO3(CH2)2O
CH3CH2O
NO3(CH2)4O
CH3(CH2)3O
NO3(CH2)6O
CH3(CH2)5O
NO3(CH2)2COOCH2O
CH3CH2COOCH2O
NO3(CH2)4COOCH2O
CH3(CH2)3COOCH2O
NO3(CH2)6COOCH2O
CH3(CH2)5COOCH2O
NO3(CH2)2O
CH3CH2O
NO3(CH2)4O
CH3(CH2)3O
NO3(CH2)6O
0.099 0.008
1.188 0.097
0.127 0.011
1.251 0.009
0.445 0.036
1.711 0.125
0.445 0.026
0.506 0.037
0.491 0.028
0.523 0.045
1.302 0.086
2.833 0.102
0.168 0.007
0.604 0.046
0.472 0.025
0.769 0.067
0.491 0.032
1.810 0.130
2.111 0.020
2.813 0.050
1.328 0.090
1.018 0.046
1.351 0.035
1.140 0.035
1.070 0.035
2.345 0.020
4.637 0.040
La5
La6
Lb1
Lb2
Lb3
Lb4
Lb5
Lb6
Lc1
Lc2
Lc3
Lc4
Lc5
Lc6
CH3(CH2)5O
O2NOC4H8O
O
I24
0.290 0.009
0.754 0.062
1.430 0.040
OH
O
Luteolin
OH
HO
O
O
Quercetin24,c
2.850 0.040
OH
OH
Na+
O
Na+
N
N
OH2
N
N
SNPd
10.420 1.800
Fe-2
OH2
N
N
Each value represents the mean S.D. (n = 3).
a
The concentration required for a 50% inhibition of the decrease in the optical density of NADPH at 340 nm relative to 0.1% DMSO. IC50 values were calculated from the
dose inhibition curve.
b
Percent of nitric oxide released based on a theoretical maximum release of 1 mol of NO/mol of the target compounds.
Quercetin was used as positive control for AR inhibition test.
SNP (sodium nitroprusside) was used as positive control for NO releasing test.
c
d
La3, Lb3, and Lc3 could be related to their capacity to release NO as
well as their logP (lipophilicity).30 The logP of the compounds was
calculated using ChemBioDraw2010 software. Results showed that
the higher the values of both logP and NO-releasing capacity, the
higher the AR inhibitory activity of the compound is. Meanwhile,
remain organic nitrate derivatives also showed the trend. It was
found that the order of AR inhibition activity for compounds La1,
Lb1, and Lc1 was shown as La1 > Lc1 > Lb1 (Table 1) evidenced
by comparing the lipophilicity (La1 > Lc1 > Lb1) and the percent-
age (La1 > Lc1 > Lb1) of NO release of compounds. Effects of La5,
Lb5, and Lc5 on AR inhibition activity were in the rank order of
La5 > Lc5 > Lb5 (Table 1). Due to the lipophilicity and the percent-
age of NO release of Lb5 were lower than La5 and Lc5. Further-
more, La5 was more potent than Lc5, resulting from
comprehensive function of the lipophilicity and the percentage of
NO release.
Figure 3B shows that the AR inhibitory activity of the deriva-
tives may be attributed to the lipophilic B ring of the derivative
becoming deeply trapped in the lipophilic pocket of the AR active
site. Such entrapment releases NO, which could react with b-
mercaptoethanol to form RSNO (an analogue of S-nitrosoglutathi-
one) that combines with AR-SH (possibly Cys-298) to form a mixed
disulphide.23
3.3. Structure–activity relationship studies
Although the alkylate derivatives of luteolin (La2, La4, La6, Lb2,
Lb4, Lb6, Lc2, Lc4, and Lc6) showed reasonable IC50 values, they

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Q.-Q. Wang et al. / Bioorg. Med. Chem. 21 (2013) 4301–4310
Figure 2. Design concept of novel ARIs based on a luteolin scaffold.
Scheme 1. Preparation of derivatives La1–6. Reagents and conditions: (a) DMF, anhydrous K2CO3, BrCH2CH2Br, 70 °C; (b) acetone, anhydrous K2CO3, bromoalkane or
dibromoalkane, reflux; (c) CH3CN, THF, AgNO3, 75 °C.
were less effective at AR inhibition than organic nitrate derivatives
(Table 1). This phenomenon indicates that inactivation of AR is due
in part to the organic nitrates of luteolin releasing low concentra-
tions of NO. In a similar example, Srivastava et al. noted that NO
donors generate free NO against AR activity.22,23 Furthermore,
the r2 value of between NO-releasing capacity and IC50 of com-
pounds was 0.623 in the partial correlation analysis (Fig. 3). These
results indicate that NO could help increase the AR inhibitory
activity of the resulting compound.
Derivatives in which the B ring of the catechol structure was
protected by 1,2-dibromoethane showed appropriately enhanced
AR inhibitory activities compared with the corresponding 30,
40-unprotected derivatives. This finding was evidenced by compar-
ison of the biological activities of three pairs of derivatives, includ-
ing La1 and Lc1, La3 and Lc3, and La5 and Lc5 (Fig. 4). La1 was
1.7-fold more potent than Lc1. This result may be attributed to
the protection provided to the catechol structure, which increases
the lipophilicity of the La series. Except for Lc5, the percentages of
NO release by Lc1 and Lc3 were lower than those of the
corresponding La derivatives (Table 1).
the organic nitrates of La showed enhanced activities in contrast
to Lb. This finding was evidenced by comparison of the biological
activities of three pairs of derivatives, including La1 and Lb1, La3
and Lb3, and La5 and Lb5 (Fig. 5). The trends observed may be
due to the enhanced ability of the compounds of La to release
NO compared with the corresponding compounds in Lb as well
as their increased lipophilicity. Therefore, the AR inhibitory
activities of organic nitrate derivatives could be further increased
by insertion of an ether chain between the NO donor and luteolin,
but the insertion of ester chain shows slight improvement of
activities.
Figures 4 and 5 show that the length of the coupling chain is an
important factor in determining AR inhibitory activities. Shorter
compounds were more potent than longer ones in each series, as
deduced from these figures (La1 > La3 > La5, Lb1 > Lb3 > Lb5, and
Lc1 > Lc3 > Lc5). These trends suggest that elongation of the cou-
pling chain reduces the overall inhibition of AR. The ether chain
of the 2-carbon spacer as an optimal coupling chain may reason-
ably be assumed to perfectly fit the nitrooxy moiety at the 7-posi-
tion of compounds La1, Lb1, and Lc1 near Cys298 (present at the
active site of AR) to form RSNO, which would eventually form a
mixed disulphide between AR-SH and RSNO.
Further SAR studies on the importance of the coupling chain of
organic nitrates of La and Lb were conducted. We observed that

Q.-Q. Wang et al. / Bioorg. Med. Chem. 21 (2013) 4301–4310
4305
Scheme 2. Preparation of derivatives Lb1–6. Reagents and conditions: (a) DMF, anhydrous K2CO3, BrCH2CH2Br, 70 °C; (b) acetone, anhydrous K2CO3, bromo-acetate, rt; (c) (i)
KOH, THF, H2O, 70 °C, (ii) HCl; (d) acetone, anhydrous K2CO3, bromoalkane or dibromoalkane, rt; (e) CH3CN, THF, AgNO3, 75 °C.
Scheme 3. Preparation of derivatives Lc1, Lc3, and Lc5. Reagents and conditions: (a) Ph2CCl2, Ph2O, 175 °C, 30 min; (b) acetone, K2CO3, dibromoalkane, rt; (c) CH3CN, THF,
AgNO3, 75 °C; (d) AcOH/H2O (4:1), rt.
Scheme 4. Preparation of derivatives Lc2, Lc4, and Lc6. Reagents and conditions: (a) DMF, anhydrous K2CO3, bromoalkane, 70 °C.

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Q.-Q. Wang et al. / Bioorg. Med. Chem. 21 (2013) 4301–4310
Figure 3. (A) The AR inhibitory activity of compounds I, La3, Lb3, and Lc3 could be related to their logP and NO-releasing capacity. The correlation analysis of these two
factors and IC50 value should be belong to the partial correlation analysis. The partial correlation coefficient (r) of two variables was calculated using spss16.0 software. The r2
value of between NO-releasing capacity and IC50 of compounds was 0.623, however, the r2 value of between logP and IC50 of compounds was 0.767. (B) A possible mechanism
of regulation of AR activity by NO-donating derivatives of flavonoids in vitro.
In conclusion, we described the design, synthesis, evaluation,
and SARs of luteolin derivatives bearing nitrooxy alkyl or alkyl
groups at the 7-position of luteolin. All of the organic nitrates of
luteolin supplied low concentrations of NO to compensate NO
in vivo, which could prevent the development of diabetic compli-
cations. Alkyl compounds of luteolin exhibited moderate in vitro
AR inhibitory activities with IC50 values ranging from
(0.506 0.037)
olin showed more prominent AR inhibitory activities ranging from
(0.099 0.008) M to (1.302 0.086) M than alkylate derivatives.
lM to (2.833 0.102) lM. Organic nitrates of lute-
l
l
La1 was the most active derivative synthesised and exhibited a
28.5-fold gain in efficacy with respect to the positive reference
compound quercetin. We further demonstrated that inactivation
of AR may be related to the ability of the derivatives to release
NO as well as their lipophilicity. These results encourage us to con-
tinue our investigations on the design of more potent ARIs with
appropriate modifications based on La1. These hybrid ester NO-do-
nor prodrugs as favourable ARIs may provide a new way for
preventing and delaying diabetic complications.
Figure 4. Effect of organic nitrates of La and Lc on AR activity in vitro.
4. Experimental section
4.1. General
1H NMR spectra were obtained using a Bruker Avance 400
instrument with CDCl3, DMSO-d6. TMS was used as an internal
standard. Chemical shifts (d) are reported in ppm and coupling
constants (J) are expressed in Hz. Analysis (C, H, N) of the target
compounds was performed using an elementary Vario EL III ana-
lyser. Molecular masses were determined by matrix-assisted laser
desorption-ionisation time-of-flight mass spectrometry (MALDI-
TOF MS) using a Bruker Aupoflex-III mass spectrometer. Melting
points (mp) were determined on a Beijing Biotech X-4 micromelt-
ing point apparatus. Enzymatic reactions were monitored by a Bio-
Tek Synergy HT Multi-Mode microplate reader. All chemicals were
of reagent grade and commercially available. Luteolin (>98%) was
purchased from Shan Xi Huike Co., Ltd, Jiangsu, China, and used
without further purification. When not otherwise specified, anhy-
drous magnesium sulphate (MgSO4) was used as drying agent for
organic phases.
Figure 5. Effect of organic nitrates of La and Lb on AR activity in vitro.

Q.-Q. Wang et al. / Bioorg. Med. Chem. 21 (2013) 4301–4310
4307
4.2. Synthesis
4.2.2.4.
(La1)
O7-Nitrooxyethyl-O30,O40-ethylidene
luteolin
.
Compound 2a (210 mg, 0.5 mmol), yellow powder,
4.2.1. Experimental procedure for the synthesis of O30,O40-
ethylidene luteolin (1)
yield: 67.5%, mp: 151–153 °C. 1H NMR (400 MHz, CDCl3) d: 12.88
0
0
(s, 1H, 5-OH), 7.62–7.59 (m, 2H, aromatic H2 ,6 ), 7.04–7.02 (d, 1H,
0
To a solution of luteolin (2.86 g, 10 mmol) in 50 mL of dry DMF
was added 1,2-dibromoethane (1.5 mL, 20 mmol) and anhydrous
potassium carbonate (0.7 g, 5 mmol), followed by heating at
70 °C for 30 min. To the reaction mixture was added ice water,
dropwise. The mixture was filtered, washed with water, dried un-
der reduced pressure. The residue was purified with a silica gel
column and was eluted with EtOAc/CH2Cl2 = 1:8 to afford 1, yellow
powder, yield 28.1%, mp: 307–309 °C. 1H NMR (400 MHz, DMSO-
d6, ppm) d: 12.90 (s, 1H, 5-OH), 10.87 (s, 1H, 7-OH), 7.60–7.57
J = 8 Hz, aromatic H5 ), 6.93 (s, 1H, aromatic H8), 6.86 (s, 1H, aro-
matic H3), 6.40 (s, 1H, aromatic H6), 4.91 (s, 2H, –CH2ONO2), 4.45
(s, 2H, –CH2–), 4.34–4.32 (d, 4H, J = 8 Hz,–CH2CH2–). MALDI-TOF:
m/z 424 ([M+Na]+). Anal. Calcd for C19H15NO9: C, 56.86; H, 3.77;
N, 3.49. Found: C, 56.53; H, 3.72; N, 3.30.
4.2.2.5. O7-Ethyl-O30,O40-ethylidene luteolin (La2).
Bromo-
ethane (0.75 mL, 10 mmol), yellow powder, yield: 69%, mp:
173–175 °C. 1H NMR (400 MHz, CDCl3) d: 7.41–7.37 (m, 2H, aro-
0
0
0
0
0
0
(m, 2H, PhH2 ,6 ), 7.04–7.02 (d, 1H, J = 8 Hz, PhH5 ), 6.87 (s, 1H,
PhH8), 6.51 (d, 1H, J = 1.7 Hz, PhH3), 6.20–6.19 (d, 1H, J = 4 Hz,
PhH6), 4.33–4.32 (d, 4H, J = 4 Hz, –CH2CH2–). MALDI-TOF: m/z
313 ([M+H]+).
matic H2 ,6 ), 6.98–6.96 (d, 1H, J = 8 Hz, aromatic H5 ), 6.54 (s, 1H,
aromatic H8), 6.45 (s, 1H, aromatic H3), 6.34 (s, 1H, aromatic H6),
4.33–4.32 (d, 4H, J = 4 Hz, –CH2CH2–), 4.13–4.08 (q, 2H, J = 6.7 Hz,
–CH2–), 1.47–1.43 (t, 3H, J = 8 Hz, –CH3). MALDI-TOF: m/z 341
([M+H]+).
4.2.2. General procedures for the synthesis of compounds La1–6
To a stirred solution of 1 (0.312 g, 1 mmol) in dry acetone
(20 mL), anhydrous potassium carbonate (0.14 g, 1 mmol) was
added. After 30 min, the appropriate bromo alkane or dibromo al-
kane (10 mmol) were added; the reaction mixture was stirred at
reflux until TLC evidenced complete consumption of starting mate-
rial (2–24 h). The solvent was removed under reduced pressure
(water pump) and the residue was washed with water
(3 ꢀ 20 mL), then dried. The residue was purified by column chro-
matography, eluting with CH2Cl2 to afford the desired derivatives
2a–c and La2, 4, 6.
4.2.2.6.
(La3).
O7-Nitrooxybutyl-O30,O40-ethylidene
2b (224 mg, 0.5 mmol), yellow powder, yield: 86%,
luteolin
mp: 150–153 °C. 1H NMR (400 MHz, CDCl3) d: 12.86 (s, 1H, 5-
0
0
OH), 7.62–7.59 (m, 2H, aromatic H2 ,6 ), 7.04–7.02 (d, 1H, J = 8 Hz,
aromatic H5 ), 6.91 (s, 1H, aromatic H8), 6.81 (s, 1H, aromatic H3),
6.36 (s, 1H, aromatic H6), 4.60 (s, 2H, –CH2ONO2), 4.34–4.32 (d,
4H, J = 8 Hz, –CH2CH2–), 4.14 (s, 2H, –CH2O–), 1.83 (s, 4H, –
CH2CH2–). MALDI-TOF: m/z 452 ([M+Na]+). Anal. Calcd for
0
C21H19NO9: C, 58.74; H, 4.46; N, 3.26. Found: C, 58.50; H, 4.08;
N, 3.14.
A
solution of the appropriate bromo derivatives 2a–c
(0.5 mmol) and AgNO3 (0.85 g, 5 mmol) in dry CH3CN (30 mL)
and THF (10 mL) was stirred at 75 °C. The reaction was allowed
to proceed for 2–8 h in the dark. Filtration was performed to re-
move any silver bromide precipitates. The filtrate was concen-
trated under reduced pressure and a yellow solid was produced.
This solid was washed with water until the runoff was clear. Solid
samples were dried and purified by a silica gel column, eluting
with CH2Cl2 to afford the final products La1, 3, 5.
4.2.2.7. O7-Butyl-O30,O40-ethylidene luteolin (La4).
Bromo-
butane (1.07 mL, 10 mmol), yellow powder, yield: 79%, mp: 154–
156 °C. 1H NMR (400 MHz, CDCl3) d: 7.41–7.37 (m, 2H, aromatic
0
0
0
H
2 ,6 ), 6.98–6.96 (d, 1H, J = 8 Hz, aromatic H5 ), 6.54 (s, 1H, aro-
matic H8), 6.45 (s, 1H, aromatic H3), 6.34 (s, 1H, aromatic H6),
4.33–4.32 (d, 4H, J = 4 Hz, –CH2CH2–), 4.04–4.01 (t, 2H, J = 6.2 Hz,
–CH2O–), 1.81–1.78 (t, 2H, J = 7 Hz, –CH2–), 1.53–1.47 (q, 2H,
J = 7.2 Hz, –CH2–), 1.01–0.97 (t, 3H, J = 7.2 Hz, –CH3). MALDI-TOF:
m/z 369 ([M+H]+).
4.2.2.1. O7-Bromethyl-O30,O40-ethylidene luteolin (2a).
1,2-
Dibromoethane (0.87 mL, 10 mmol), yellow powder, yield: 64%,
4.2.2.8.
(La5).
O7-Nitrooxyhexyl-O30,O40-ethylidene
2c (238 mg, 0.5 mmol), yellow powder, yield: 75%,
luteolin
mp: 218–220 °C. 1H NMR (400 MHz, CDCl3) d: 7.41–7.37 (m, 2H,
0
0
0
aromatic H2 ,6 ), 6.99–6.97 (d, 1H, J = 8 Hz, aromatic H5 ), 6.56 (s,
1H, aromatic H8), 6.48 (s, 1H, aromatic H3), 6.35 (s, 1H, aromatic
H6), 4.37–4.33 (m, 6H, CH2, –CH2CH2–), 3.68–3.65 (t, 2H, J = 6 Hz,
–CH2Br). MALDI-TOF: m/z 419 ([M+H]+).
mp: 156–158 °C. 1H NMR (400 MHz, CDCl3) d: 12.85 (s, 1H, 5-
0
0
OH), 7.62–7.59 (m, 2H, aromatic H2 ,6 ), 7.04–7.02 (d, 1H, J = 8 Hz,
0
aromatic H5 ), 6.90 (s, 1H, aromatic H8), 6.80 (s, 1H, aromatic H3),
6.35 (s, 1H, aromatic H6), 4.54–4.51 (t, 2H, J = 6.2 Hz, –CH2ONO2),
4.34–4.32 (d, 4H, J = 8 Hz, –CH2CH2–), 4.10–4.08 (t, 2H, J = 4 Hz, –
CH2O–), 1.75–1.68 (m, 4H, CH2, CH2), 1.43 (s, 4H, –CH2CH2–). MAL-
DI-TOF: m/z 480 ([M+Na]+). Anal. Calcd for C23H23NO9: C, 60.39; H,
5.07; N, 3.06. Found: C, 59.98; H, 4.98; N, 2.95, aromatic.
4.2.2.2. O7-Brombutyl-O30,O40-ethylidene luteolin (2b).
1,4-
Dibromobutane (1.22 mL, 10 mmol), yellow solid, yield: 52.8%, mp:
156–158 °C. 1H NMR (400 MHz, CDCl3) d: 7.41–7.37 (m, 2H, aro-
0
0
0
matic H2 ,6 ), 6.98–6.96 (d, 1H, J = 8 Hz, aromatic H5 ), 6.54 (s, 1H,
aromatic H8), 6.46 (s, 1H, aromatic H3), 6.34 (s, 1H, aromatic H6),
4.33–4.32 (d, 4H, J = 4 Hz, –CH2CH2–), 4.08–4.06 (t, 2H, J = 4 Hz, –
OCH2–), 3.51–3.48 (t, 2H, J = 6.1 Hz, –CH2Br), 2.09–1.99 (m, 4H, –
CH2CH2–). MALDI-TOF: m/z 447 ([M+H]+).
4.2.2.9. O7-Hexyl-O30,O40-ethylidene luteolin (La6).
Bro-
mohexane (1.42 mL, 10 mmol), yellow powder, yield: 68.7%, mp:
125–127 °C. 1H NMR (400 MHz, CDCl3) d: 7.40–7.36 (m, 2H, aro-
0
0
0
matic H2 ,6 ), 6.97–6.95 (d, 1H, J = 8 Hz, aromatic H5 ), 6.52 (s, 1H,
aromatic H8), 6.44 (s, 1H, aromatic H3), 6.33 (s, 1H, aromatic H6),
4.32–4.31 (d, 4H, J = 4 Hz, –CH2CH2–), 4.03–4.00 (t, 2H, J = 6.5 Hz,
–CH2O–), 1.82–1.78 (t, 2H, J = 8 Hz, –CH2–), 1.46–1.34 (m, 6H, –
CH2CH2CH2–), 0.91 (s, 3H, –CH3). MALDI-TOF: m/z 397 ([M+H]+).
4.2.2.3. O7-Bromhexyl-O30,O40-ethylidene luteolin (2c).
Dibromohexane (1.56 mL, 10 mmol), yellow powder, yield: 68%,
mp: 149–151 °C. 1H NMR (400 MHz, CDCl3) d: 7.41–7.38 (m, 2H,
aromatic H2 ,6 ), 6.98–6.96 (d, 1H, J = 8 Hz, aromatic H5 ), 6.54 (s,
1H, aromatic H8), 6.46 (s, 1H, aromatic H3), 6.34 (s, 1H, aromatic
H6), 4.33 (d, 4H, J = 2.4 Hz, –CH2CH2–), 4.04–4.02 (t, 2H, J = 4 Hz,
–OCH2–), 3.45–3.42 (t, 2H, J = 6.3 Hz, –CH2Br), 1.91–1.83 (m, 4H,
CH2, CH2), 1.52(s, 4H, –CH2CH2–). MALDI-TOF: m/z 475 ([M+H]+).
1,6-
0
0
0
4.2.3. General procedures for the synthesis of compounds Lb1–6
4.2.3.1. O7-(Ethoxycarbonyl)methyl-O30,O40-ethylidene luteolin
(3).
Anhydrous potassium carbonate (10 mmol, 1.38 g) was
added to a stirred solution of 1 (10 mmol, 3.12 g) in dry acetone

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Q.-Q. Wang et al. / Bioorg. Med. Chem. 21 (2013) 4301–4310
(150 mL), followed by refluxing until the solution became clear.
Ethyl bromoacetate (30 mmol, 3.5 mL) was then dribbled into the
mixture, and acetone (10 mL) was added. The solution was re-
fluxed for 4 h and vacuum filtered. The filter liquor was concen-
trated to obtain a yellow solid, which was subsequently washed
with petroleum, water. The solid was then dried and dissolved in
dichloromethane. Any undissolved solid were discarded. The solu-
tion was concentrated and dried to produce a yellow powder 3,
yield: 79.6%, mp: 133–135 °C. 1H NMR (400 MHz, CDCl3) d: 12.81
4.32 (d, 4H, J = 4 Hz, –CH2CH2–), 3.83 (s, 2H, –CH2OCO–), 1.34–
1.30 (t, 3H, J = 7.2 Hz, –CH3). MALDI-TOF: m/z 399 ([M+H]+).
Compound Lb4: Bromobutane (15 mmol, 1.61 mL), yield: 88.6%,
mp: 141–143 °C. 1H NMR (400 MHz, CDCl3) d: 12.81 (s, 1H, 5-OH),
0
0
7.41–7.37 (m, 2H, aromatic H2 ,6 ), 6.99–6.97 (d, 1H, J = 8 Hz, aro-
0
matic H5 ), 6.55 (s, 1H, aromatic H8), 6.48 (s, 1H, aromatic H3),
6.35 (s, 1H, aromatic H6), 4.69 (s, 2H, –CH2O–), 4.33 (s, 4H, –
CH2CH2–), 4.25–4.22 (t, 2H, J = 6.1 Hz, –CH2OCO–), 1.67–1.64 (t,
2H, J = 6.8 Hz, –CH2–), 1.40–1.35 (q, 2H, J = 7.1 Hz, –CH2–), 0.94–
0.91 (t, 3H, J = 7 Hz, –CH3). MALDI-TOF: m/z 427 ([M+H]+).
0
0
(s, 1H, 5-OH), 7.41–7.37 (m, 2H, aromatic H2 ,6 ), 6.99–6.97 (d, 1H,
0
J = 8 Hz, aromatic H5 ), 6.55 (s, 1H, aromatic H8), 6.48–6.47 (d,
Compound Lb6: Bromohexane (15 mmol, 2.13 mL), yield: 85.2%,
mp: 110–112 °C. 1H NMR (400 MHz, CDCl3) d: 12.80 (s, 1H, 5-OH),
7.41–7.38 (m, 2H, aromatic H2 ,6 ), 6.99–6.97 (d, 1H, J = 8 Hz, aro-
1H, J = 4 Hz, aromatic H3), 6.35 (s, 1H, aromatic H6), 4.70 (s, 2H, –
CH2O–), 4.33–4.32 (d, 4H, J = 4 Hz, –CH2CH2–), 3.83 (s, 2H, –CH2O-
CO–), 1.34–1.30 (t, 3H, J = 7.2 Hz, –CH3).
0
0
0
matic H5 ), 6.56 (s, 1H, aromatic H8), 6.48 (d, 1H, J = 1.6 Hz, aro-
matic H3), 6.35 (d, 1H, J = 1.5 Hz, aromatic H6), 4.69 (s, 2H, –
CH2O–), 4.34–4.33 (d, 4H, J = 4 Hz, –CH2CH2–), 4.24–4.21 (t, 2H,
J = 6.6 Hz, –CH2OCO–), 1.68–1.62 (m, 2H, –CH2–), 1.29–1.25 (m,
6H, –CH2CH2CH2–), 0.87–0.85 (d, 3H, J = 8 Hz, –CH3). MALDI-TOF:
m/z 458 ([M+H]+).
4.2.3.2. O7-Carboxymethyl-O30,O40-ethylidene luteolin (4).
The solution of 3 (5 mmol, 1.99 g) in THF (25 mL), H2O (100 mL) and
ethanol (200 mL) was stirred at 70 °C for 15 min, and then a solution
of potassium hydroxide (30 mmol, 1.68 g) in H2O was added dropwise.
The reaction mixture was stirred at 70 °C until TLC evidenced complete
consumption of starting material (8 h). The resulting homogeneous
solution was concentrated and dissolved in H2O. Any undissolved solid
were discarded. Then, the pH was adjusted to the desired acidity with
hydrochloric acid. The solution underwent vacuum filtration, as well as
water washing and drying to afford a yellow solid 4, yield: 52.7%, mp:
320–322 °C. 1H NMR (400 MHz, DMSO-d6, ppm) d: 12.87 (s, 1H, 5-OH),
4.2.3.4.O7-[(Nitrooxyl)ethoxycarbonyl]methyl-O30,O40-ethylidene
luteolin (Lb1, Lb3, Lb5).
Compounds Lb1, Lb3 and Lb5 were
synthesized according to the method for La1.
Compound Lb1: Compound 5a (239 mg, 0.5 mmol), AgNO3
(0.85 g, 5 mmol), yield: 66.1%, mp: 210–212 °C. 1H NMR
(400 MHz, CDCl3) d: 12.83 (s, 1H, 5-OH), 7.41–7.38 (m, 2H, aro-
0
0
0
0
0
7.64–7.60 (m, 2H, aromatic H2 ,6 ), 7.04–7.02 (d, 1H, J = 8 Hz, aromatic
matic H2 ,6 ), 6.99–6.97 (d, 1H, J = 8 Hz, aromatic H5 ), 6.56 (s, 1H,
aromatic H8), 6.48 (s, 1H, aromatic H3), 6.35 (s, 1H, aromatic H6),
4.75 (s, 2H, –CH2O–), 4.71 (s, 2H, –CH2ONO2), 4.33(d, 2H,
J = 3.9 Hz, –CH2OCO–). MALDI-TOF: m/z 460 ([M+H]+). Anal. Calcd
for C21H17NO11: C, 54.91; H, 3.73; N, 3.05. Found: C, 54.53; H,
3.58; N, 2.93.
0
H5 ), 6.94 (s, 1H, aromatic H8), 6.80 (s, 1H, aromatic H3), 6.36 (s, 1H, aro-
matic H6), 4.77 (s, 2H, –CH2O–), 4.34–4.32 (d, 4H, J = 8 Hz, –CH2CH2–).
4.2.3.3. O7-[(Bromoethoxyl)carbonyl]methyl-O30,O40-ethylidene
luteolin (5a–c, Lb2, Lb4 and Lb6).
Triethylamine (18 mmol,
2.55 mL) was added to a solution of 4 (3 mmol, 1.11 g) in DMSO
(5 mL) and acetone (100 mL). The reaction mixture was then re-
fluxed for 30 min. 1,2-Dibromoethane (15 mmol, 1.31 mL) was
dribbled into the solution, followed by refluxing for 24 h and filtra-
tion to remove any precipitates that may have formed. The filter li-
quor was concentrated. The residue was added ice water to
produce a yellow solid. The solid was filtered and dried to obtain
desired compound 5a, yield: 73.2%, mp: 193–195 °C. 1H NMR
(400 MHz, CDCl3) d: 12.81 (s, 1H, 5-OH), 7.41–7.37 (m, 2H, aro-
Compound Lb3: Compound 5b (253 mg, 0.5 mmol), AgNO3
(0.85 g, 5 mmol), yield: 53.3%, mp: 143–145 °C. 1H NMR
(400 MHz, CDCl3) d: 12.83 (s, 1H, 5-OH), 7.41–7.38 (m, 2H, aro-
0
0
0
matic H2 ,6 ), 6.99–6.97 (d, 1H, J = 8 Hz, aromatic H5 ), 6.56 (s, 1H,
aromatic H8), 6.48 (s, 1H, aromatic H3), 6.34 (s, 1H, aromatic H6),
4.71 (s, 2H, –CH2O–), 4.46 (s, 2H, –CH2ONO2), 4.34–4.33 (d, 4H,
J = 4 Hz, –CH2CH2–), 4.27 (s, 2H, –CH2OCO–), 1.80 (s, 4H, –
CH2CH2–). MALDI-TOF: m/z 488 ([M+H]+). Anal. Calcd for
C23H21NO11: C, 56.68; H, 4.34; N, 2.87. Found: C, 56.52; H, 4.18;
N, 2.73.
0
0
0
matic H2 ,6 ), 6.98–6.96 (d, 1H, J = 8 Hz, aromatic H5 ), 6.55 (s, 1H,
aromatic H8), 6.49 (s, 1H, aromatic H3), 6.36 (s, 1H, aromatic H6),
4.75 (s, 2H, –CH2O–), 4.56–4.53 (t, 2H, J = 5.6 Hz, –CH2–), 4.33–
4.32 (d, 4H, J = 4 Hz, –CH2CH2–), 3.56–3.54 (t, 2H, J = 8 Hz, –CH2Br).
Compounds 5b–c, Lb2, Lb4 and Lb6 were synthesized according
to the method for 5a.
Compound Lb5: Compound 5c (267 mg, 0.5 mmol), AgNO3
(0.85 g, 5 mmol), yield: 68.1%, mp: 138–140 °C. 1H NMR
(400 MHz, CDCl3) d: 12.82 (s, 1H, 5-OH), 7.41–7.37 (m, 2H, aro-
0
0
0
matic H2 ,6 ), 6.99–6.97 (d, 1H, J = 8 Hz, aromatic H5 ), 6.56 (s, 1H,
aromatic H8), 6.48 (s, 1H, aromatic H3), 6.34 (s, 1H, aromatic H6),
4.70 (s, 2H, –CH2O–), 4.43–4.40 (t, 2H, J = 6.4 Hz, –CH2ONO2),
4.33 (d, 4H, J = 3.6 Hz, –CH2CH2–), 4.25–4.21 (t, 2H, J = 6.3 Hz, –
CH2OCO–), 1.72–1.67 (q, 4H, J = 6.5 Hz, CH2, CH2), 1.40 (s, 4H, –
CH2CH2–). MALDI-TOF: m/z 516 ([M+H]+). Anal. Calcd for
Compound 5b: 1,4-Dibromobutane (15 mmol, 1.83 mL), yield:
78.7%, mp: 167–169 °C. 1H NMR (400 MHz, CDCl3) d: 12.81 (s,
0
0
1H, 5-OH), 7.41–7.37 (m, 2H, aromatic H2 ,6 ), 6.99–6.97 (d, 1H,
0
J = 8 Hz, aromatic H5 ), 6.55 (s, 1H, aromatic H8), 6.48 (s, 1H, aro-
matic H3), 6.34 (s, 1H, aromatic H6), 4.70 (s, 2H, –CH2O–), 4.33–
4.25 (m, 6H, –CH2CH2–, –CH2OCO–), 3.42–3.39 (t, 2H, J = 6 Hz, –
CH2Br), 1.91–1.84 (m, 4H, –CH2CH2–).
C25H25NO11: C, 58.25; H, 4.89; N, 2.72. Found: C, 57.96; H, 4.68;
N, 2.53.
Compound 5c: 1,6-Dibromohexane (15 mmol, 2.34 mL), yield:
4.2.4. General procedures for the synthesis of compounds Lc1–6
and 8a–c
73.6%, mp: 155–157 °C. 1H NMR (400 MHz, CDCl3) d: 12.81 (s,
1H, 5-OH), 7.41–7.40 (m, 2H, aromatic H2 ,6 ), 6.99–6.97 (d, 1H,
4.2.4.1. O30,O40-Diphenylmethane luteolin (6).
Dichloro-
0
0
0
J = 8 Hz, aromatic H5 ), 6.56 (s, 1H, aromatic H8), 6.48 (s, 1H, aro-
diphenylmethane (2 mL, 10.42 mmol) was added to a stirred mix-
ture of luteolin (2 g, 6.99 mmol) in diphenyl ether (20 mL) and the
reaction mixture was heated at 175 °C for 30 min.
After cooled to 60 °C, the dark solution was poured into petro-
leum (100 mL), the precipitation was filtered. The filtrate was con-
centrated and purified by column chromatography (eluent,
petroleum ether/EtOAc = 6:1 to 2:1) to give 6, respectively. Yield:
50.7%, yellow solid, mp: 139–141 °C. 1H NMR (400 MHz, DMSO-
d6, ppm) d: 12.86 (s, 1H, 5-OH), 10.86 (s, 1H, 7-OH), 7.78 (s, 1H,
matic H3), 6.35 (s, 1H, aromatic H6), 4.70 (s, 2H, –CH2O–), 4.33–
4.22 (m, 6H, CH2CH2, –CH2OCO–), 3.40–3.36 (t, 2H, J = 6.4 Hz, –
CH2Br), 1.85–1.67 (m, 4H, CH2, CH2), 1.45–1.36 (m, 4H, –CH2CH2–).
Compound Lb2: Bromoethane (15 mmol, 1.13 mL), yield: 79.6%,
mp: 133–135 °C. 1H NMR (400 MHz, CDCl3) d: 12.81 (s, 1H, 5-OH),
7.41–7.37 (m, 2H, aromatic H2 ,6 ), 6.99–6.97 (d, 1H, J = 8 Hz, aro-
matic H5 ), 6.55 (s, 1H, aromatic H8), 6.48–6.47 (d, 1H, J = 4 Hz, aro-
matic H3), 6.35 (s, 1H, aromatic H6), 4.70 (s, 2H, –CH2O–), 4.33–
0
0
0

Q.-Q. Wang et al. / Bioorg. Med. Chem. 21 (2013) 4301–4310
4309
0
0
0
0
aromatic H2 ), 7.72–7.70 (d, 1H, J = 8 Hz, aromatic H6 ), 7.56–7.46
OH), 7.58–7.57 (m, 4H, aromatic H2 ,6 , aromatic H, aromatic H),
7.47–7.39 (m, 8H, diphenylmethylene H), 7.00–6.98 (d, 1H,
(m, 10H, diphenylmethylene H), 7.24–7.22 (d, 1H, J = 8 Hz, aro-
0
0
matic H5 ), 6.88 (s, 1H, aromatic H8), 6.52 (s, 1H, aromatic H3),
J = 8 Hz, aromatic H5 ), 6.52 (s, 1H, aromatic H8), 6.44 (s, 1H, aro-
6.20 (s, 1H, aromatic H6).
matic H3), 6.33 (s, 1H, aromatic H6), 4.48–4.45 (t, 2H, J = 6.1 Hz, –
CH2ONO2), 4.02 (s, 2H, –CH2O–), 1.83–1.77 (m, 4H, CH2, CH2),
1.51 (s, 4H, –CH2CH2–).
4.2.4.2 O7-Bromethyl-O30,O40-diphenylmethane luteolin (7a–c).
To a stirred solution of 6 (1.8 g, 4 mmol) in acetone (150 mL),
was added anhydrous potassium carbonate (0.6 g, 4.4 mmol), the
reaction mixture was stirred at reflux for 30 min. 1,2-Dibromoeth-
ane (12 mmol, 1.1 mL) was then added dropwise for 10 min. The
mixture was stirred at reflux for 24 h, the precipitation was fil-
tered. The filter liquor was concentrated under reduced pressure
to obtain a yellow solid. The solid was washed with water
(3 ꢀ 20 mL), dried and purified by column chromatography (elu-
ent, CH2Cl2) to give 7a, respectively. Yield: 70.3%, yellow solid,
mp: 127–129 °C. 1H NMR (400 MHz, CDCl3) d: 12.78 (s, 1H, 5-
4.2.4.4. O7-Nitrooxyethyl luteolin (Lc1, Lc3, Lc5).
Com-
pounds 8a–c (1 mmol) was added to a mixture of acetic acid/water
(80:20, 50 mL). The solution was refluxed for 4–5 h. Then EtOAc
(50 mL) and water (50 mL) were added. The organic layer was
washed with a NaHCO3 saturated aqueous solution (3 ꢀ 30 ml)
and dried over anhydrous MgSO4. After the solution was concen-
trated, the residue was purified by recrystallization from CH2Cl2
to afford Lc1, Lc3, Lc5, respectively.
Compound Lc1: Compound 8a (539.5 mg, 1 mmol), yield: 45.5%,
yellow solid, mp: 228–230 °C. 1H NMR (400 MHz, DMSO-d6, ppm)
d: 13.00 (s, 1H, 5-OH), 10.02 (s, 1H, 40-OH), 9.39 (s, 1H, 30-OH),
0
0
OH), 7.58–7.57 (m, 4H, aromatic H2 ,6 , aromatic H, aromatic H),
7.47–7.39 (m, 8H, diphenylmethylene H), 7.00–6.98 (d, 1H,
0
0
0
J = 8 Hz, aromatic H5 ), 6.53 (s, 1H, aromatic H8), 6.47 (s, 1H, aro-
7.46–7.43 (m, 2H, aromatic H2 ,6 ), 6.91–6.89 (d, 1H, J = 8 Hz, aro-
0
matic H3), 6.35 (s, 1H, aromatic H6), 4.36–4.33 (t, 2H, J = 6 Hz, –
CH2O–), 3.67–3.64 (t, 2H, J = 6 Hz, –CH2Br).
Compounds 7b and 7c were synthesized according to the meth-
od for 7a.
matic H5 ), 6.79–6.75 (d, 2H, J = 13.3 Hz, aromatic H8, aromatic
H3), 6.41 (s, 1H, aromatic H6), 4.91 (s, 2H, –CH2ONO2), 4.46 (s,
2H, –CH2O–). MALDI-TOF: m/z 376 ([M+H]+). Anal. Calcd for
C17H13NO9: C, 54.41; H, 3.49; N, 3.73. Found: C, 54.12; H, 3.28;
N, 3.51.
Compound 7b: 1,4-Dibromobutane (12 mmol, 1.5 mL), yield:
69.8%, yellow solid, mp: 150–152 °C. 1H NMR (400 MHz, CDCl3)
Compound Lc3: Compound 8b (567.5 mg, 1 mmol), yield:
43.7%, yellow solid, mp: 224–226 °C. 1H NMR (400 MHz, DMSO-
d6, ppm) d: 12.98 (s, 1H, 5-OH), 10.00 (s, 1H, 40-OH), 9.39 (s, 1H,
0
0
d: 12.74 (s, 1H, 5-OH), 7.58–7.57 (m, 4H, aromatic H2 ,6 , aromatic
H, aromatic H), 7.46–7.39 (m, 8H, diphenylmethylene H), 7.00–
30-OH), 7.43 (s, 2H, aromatic H2 ,6 ), 6.90–6.89 (d, 1H, J = 6.4 Hz, aro-
0
0
0
6.98 (d, 1H, J = 8 Hz, aromatic H5 ), 6.52 (s, 1H, aromatic H8), 6.44
0
(s, 1H, aromatic H3), 6.33 (s, 1H, aromatic H6), 4.08–4.05 (t, 2H,
J = 5.2 Hz, –CH2O–), 3.51–3.48 (t, 2H, J = 6.1 Hz, –CH2Br), 2.09–
1.98 (m, 4H, –CH2CH2–).
matic H5 ), 6.73 (s, 2H, ArH8, aromatic H3), 6.37 (s, 1H, aromatic H6),
4.60 (s, 2H, –CH2ONO2), 4.15 (s, 2H, –CH2O–), 1.83 (s, 4H, –
CH2CH2–). MALDI-TOF: m/z 488 ([M+H]+). Anal. Calcd for
Compound 7c: 1,6-Dibromohexane (12 mmol, 1.9 mL), yield:
C
19H17NO9: C, 56.58; H, 4.25; N, 3.47. Found: C, 56.29; H, 4.02;
83.1%, yellow solid, mp: 110–112 °C. 1H NMR (400 MHz, CDCl3)
N, 3.12.
0
0
d: 12.74 (s, 1H, 5-OH), 7.58–7.57 (m, 4H, aromatic H2 ,6 , aromatic
Compound Lc5: Compound 8c (595.6 mg, 1 mmol), yield: 54.5%,
H, aromatic H), 7.47–7.39 (m, 8H, diphenylmethylene H), 7.00–
yellow solid, mp: 203–205 °C. 1H NMR (400 MHz, DMSO-d6, ppm)
d: 12.96 (s, 1H, 5-OH), 9.98 (s, 1H, 40-OH), 9.37 (s, 1H, 30-OH), 7.45–
0
6.98 (d, 1H, J = 8 Hz, aromatic H5 ), 6.52 (s, 1H, aromatic H8), 6.44
0
0
(s, 1H, aromatic H3), 6.34 (s, 1H, aromatic H6), 4.04–4.01 (t, 2H,
J = 5.6 Hz, –CH2O–), 3.45–3.42 (t, 2H, J = 6.4 Hz, –CH2Br), 1.91–
1.83 (m, 4H, CH2, CH2), 1.52 (s, 4H, –CH2CH2–).
7.43 (m, 2H, aromatic H2 ,6 ), 6.90–6.88 (d, 1H, J = 8 Hz, aromatic
0
H5 ), 6.72 (s, 2H, aromatic H8, aromatic H3), 6.35 (s, 1H, aromatic
H6), 4.54–4.51 (t, 2H, J = 6.4 Hz, –CH2ONO2), 4.11–4.08 (t, 2H,
J = 6 Hz, –CH2O–), 1.75–1.68 (m, 4H, CH2, CH2), 1.43 (s, 4H, –
CH2CH2–). MALDI-TOF: m/z 432 ([M+H]+). Anal. Calcd for
4.2.4.3. O7-Nitrooxyethyl-O30,O40-diphenylmethane luteolin
(8a–c).
A solution of the appropriate bromo derivatives 7a–c
C21H21NO9: C, 58.47; H, 4.91; N, 3.25. Found: C, 58.16; H, 4.69;
(2 mmol) and AgNO3 (3 g, 20 mmol) in dry CH3CN (60 mL) and
THF (20 mL) was stirred at 75 °C. The reaction was allowed to pro-
ceed for 8–12 h in the dark. Filtration was performed to remove
any silver bromide precipitates. The filtrate was concentrated un-
der reduced pressure and a yellow solid was produced. This solid
was washed with water until the runoff was clear. Solid samples
were dried and purified by column chromatography (eluent, petro-
leum ether/EtOAc = 8:1 to CH2Cl2) to afford 8a–c, respectively.
Compound 8a: Compound 7a (1.12 g, 2 mmol), yield: 73.9%,
yellow solid, mp: 98–100 °C. 1H NMR (400 MHz, CDCl3) d: 12.81
N, 2.97.
4.2.4.5. O7-Ethyl luteolin (Lc2, Lc4, Lc6).
Bromoethane
(0.38 mL, 5 mmol) was added to a stirred solution of luteolin
(2.86 g, 10 mmol) and anhydrous potassium carbonate (0.7 g,
5 mmol) in dry DMF (50 mL) and the reaction mixture was heated
at 70 °C for 3 h.The mixture was added ice water, dropwise. The
precipitation was filtered, washed with water, dried under reduced
pressure. The residue was purified by column chromatography
(eluent, CH2Cl2/EtOAc = 20:3 to 2:1) to afford Lc2, respectively.
Yield: 7.85%, yellow solid, mp: 230–232 °C. 1H NMR (400 MHz,
DMSO-d6, ppm) d: 12.97 (s, 1H, 5-OH), 9.98 (s, 1H, 40-OH), 9.39
0
0
(s, 1H, 5-OH), 7.58 (s, 4H, aromatic H2 ,6 , aromatic H, aromatic
H), 7.47–7.40 (m, 8H, diphenylmethylene H), 7.01–6.99 (d, 1H,
J = 8 Hz, aromatic H5 ), 6.54 (s, 1H, aromatic H8), 6.46 (s, 1H, aro-
(s, 1H, 30-OH), 7.45–7.43 (m, 2H, aromatic H2 ,6 ), 6.90–6.88 (d,
0
0
0
0
matic H3), 6.34 (s, 1H, aromatic H6), 4.85 (s, 2H, –CH2ONO2), 4.31
(s, 2H, –CH2O–).
1H, J = 8 Hz, aromatic H5 ), 6.73–6.70 (d, 2H, J = 10.1 Hz, aromatic
H8, aromatic H3), 6.35 (s, 1H, aromatic H6), 4.18–4.13 (q, 2H,
J = 6.7 Hz, –CH2O–), 1.37–1.34 (t, 3H, J = 6.8 Hz, –CH3). MALDI-
TOF: m/z 315 ([M+H]+).
Compounds Lc4 and Lc6 were synthesized according to the
method for Lc2.
Compound 8b: Compound 7b (1.17 g, 2 mmol), yield: 71.9%,
yellow solid, mp: 129–131 °C. 1H NMR (400 MHz, CDCl3) d: 12.77
0
0
(s, 1H, 5-OH), 7.58 (s, 4H, aromatic H2 ,6 , aromatic H, aromatic
H), 7.47–7.40 (m, 8H, diphenylmethylene H), 7.00–6.99 (d, 1H,
0
J = 7.2 Hz, aromatic H5 ), 6.53 (s, 1H, aromatic H8), 6.44 (s, 1H, aro-
Compound Lc4: Luteolin (2.86 g, 10 mmol), bromobutane
(0.54 mL, 5 mmol), yield: 7.63%, yellow solid, mp: 218–220 °C. 1H
NMR (400 MHz, DMSO-d6, ppm) d: 12.96 (s, 1H, 5-OH), 9.98 (s,
matic H3), 6.33 (s, 1H, aromatic H6), 4.55 (s, 2H, –CH2ONO2), 4.07
(s, 2H, –CH2O–), 1.83 (s, 4H, –CH2CH2–).
Compound 8c: 7c (1.23 g, 2 mmol), yield: 76.3%, yellow solid,
1H, 40-OH), 9.37 (s, 1H, 30-OH), 7.46–7.43 (m, 2H, aromatic H2 ,6 ),
0
0
mp: 153–155 °C. 1H NMR (400 MHz, CDCl3) d: 12.75 (s, 1H, 5-
6.90–6.88 (d, 1H, J = 8 Hz, aromatic H5 ), 6.73 (s, 2H, aromatic H8,
0

4310
Q.-Q. Wang et al. / Bioorg. Med. Chem. 21 (2013) 4301–4310
aromatic H3), 6.35 (s, 1H, aromatic H6), 4.11–4.08 (t, 2H, J = 5.9 Hz,
–CH2O–), 1.74–1.70 (t, 2H, J = 6.7 Hz, –CH2–), 1.47–1.41 (q, 2H,
J = 7.2 Hz, –CH2–), 0.96–0.92 (t, 3H, J = 7.2 Hz, –CH3). MALDI-TOF:
m/z 343 ([M+H]+).
Compound Lc6: Luteolin (2.86 g, 10 mmol), bromohexane
(0.71 mL, 5 mmol), yield: 8.57%, yellow solid, mp: 243–245 °C.
1H NMR (400 MHz, DMSO-d6, ppm) d: 12.96 (s, 1H, 5-OH), 9.99
(s, 1H, 40-OH), 9.37 (s, 1H, 30-OH), 7.46–7.43 (m, 2H, aromatic
Acknowledgments
This work was financially supported by the Foundation of Re-
search Fund for the Doctoral Program of Higher Education of China
(20114301120004), Hunan Provincial Natural Science Foundation
of China (12JJ6081) and the Dr.’s Start-up Foundation of Xiangtan
University (06KZ|KZ08035).
0
0
0
H
2 ,6 ), 6.90–6.88 (d, 1H, J = 8 Hz, aromatic H5 ), 6.72 (s, 2H, aromatic
Supplementary data
H8, aromatic H3), 6.35 (s, 1H, aromatic H6), 4.09–4.07 (d, 2H,
J = 5.8 Hz, –CH2O–), 1.73 (s, 2H, –CH2–), 1.41–1.31 (m, 6H, –
CH2CH2CH2–), 0.88 (s, 3H, –CH3). MALDI-TOF: m/z 371 ([M+H]+).
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.04.066.
4.3. Biological activity
References and notes
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M L-
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Calf eyes were obtained from a local abattoir soon after slaugh-
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5 M PBS and dialysed overnight in the same solution. The dialysed
material was used for enzymatic assay and stored at ꢁ70 °C.
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4.3.3. Enzymatic inhibition24,32,33
Test enzyme and inhibitory activities of the target compounds
were determined spectrophotometrically by monitoring changes
in absorbance at 340 nm. Such changes are due to the oxidation
of NADPH catalysed by AR.
Determination of the AR inhibitory activities of the newly syn-
thesised compounds was conducted using an optimised volume of
200
tions of the compounds (20
(pH 6.2) containing b-mercaptoethanol (20
l
L of enzyme (750
l
g/mL protein) and different concentra-
L, 0.1–10 mol/L) in 50 mM PBS
L, 5 mM), NADPH
L, 0.4 M), and glyceraldehyde
L, 2.5 mM). The reaction was initiated by addition of glyceral-
l
l
l
(20
(20
lL, 0.24 mM), Li2SO4 (40 l
l
29. Pegklidou, K.; Koukoulitsa, C.; Nicolaou, I.; Demopoulos, V. J. Bioorg. Med. Chem.
2010, 18, 2107.
dehyde, and the decrease in optical density of NADPH at 340 nm
was recorded for 10 min. The IC50 values of the compounds were
calculated using Sigmaplot software and expressed as the
mean S.D. of triplicate experiments. The flavonoid quercetin
was used as a reference during the AR assay.
30. Maccari, R.; Ciurleo, R.; Giglio, M.; Cappiello, M.; Moschini, R.; Corso, A. D.;
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4.3.4. Statistical analysis
Data were shown as mean S.D. Differences between individual
groups were analyzed by using ANOVA followed by Dunett’s test. A
difference with a P value of <0.05 was considered to be significant.