Synthesis and antioxidant activities of berberine 9-: O -benzoic acid derivatives

Article abstract of DOI:10.1039/d1ra01339d

Although berberine (BBR) shows antioxidant activity, its activity is limited. We synthesized 9-O-benzoic acid berberine derivatives, and their antioxidant activities were screened via ABTS, DPPH, HOSC and FRAP assays. The para-position was modified with halogen elements on the benzoic acid ring, which led to an enhanced antioxidant activity and the substituent on the ortho-position was found to be better than the meta-position. Compounds 8p, 8c, 8d, 8i, 8j, 8l, and especially 8p showed significantly higher antioxidant activities, which could be attributed to the electronic donating groups. All the berberine derivatives possessed proper lipophilicities. In conclusion, compound 8p is a promising antioxidant candidate with remarkable elevated antioxidant activity and moderate lipophilicity.

Full text of DOI:10.1039/d1ra01339d

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Synthesis and antioxidant activities of berberine 9-
O-benzoic acid derivatives†
Cite this: RSC Adv., 2021, 11, 17611
a
Yanfei Liu,
Shuo Long,a Shanshan Zhang,a Yifu Tan,a Ting Wang,b Yuwei Wu,b
Ting Jiang,a Xiaoqin Liu,a Dongming Peng
and Zhenbao Liu
c
b
*
Although berberine (BBR) shows antioxidant activity, its activity is limited. We synthesized 9-O-benzoic acid
berberine derivatives, and their antioxidant activities were screened via ABTS, DPPH, HOSC and FRAP assays.
The para-position was modified with halogen elements on the benzoic acid ring, which led to an enhanced
antioxidant activity and the substituent on the ortho-position was found to be better than the meta-
position. Compounds 8p, 8c, 8d, 8i, 8j, 8l, and especially 8p showed significantly higher antioxidant
activities, which could be attributed to the electronic donating groups. All the berberine derivatives
possessed proper lipophilicities. In conclusion, compound 8p is a promising antioxidant candidate with
remarkable elevated antioxidant activity and moderate lipophilicity.
Received 18th February 2021
Accepted 17th April 2021
DOI: 10.1039/d1ra01339d
rsc.li/rsc-advances
antioxidant activity. At the same time, 8-oxocoptisine,16 indole,17
piperazine18 and benzothiazole19 derivatives of berberine have also
1. Introduction
been developed in recent years, while the C-9 position modication
of benzoic acid was seldom reported. In our previous work, we
designed and synthesized a series of berberine derivatives, and
demonstrated that the introduction of an aromatic nucleus on the C-
9 position of berberine had important inuences on hypoglycemic20
and anti-inammatory activities,21 which were closely related with
oxidative stress. Because antioxidant activation is closely related to
the electric potential distribution,22 introducing electron-donating
groups on the aromatic nucleus is a frequent approach for
achieving improved antioxidant activity. Therefore, we synthesized
a series of berberine-aromatic derivatives with electron-donating
groups, such as methoxy, methyl and acetoxyl groups (compounds
8a–8p), modied on the C-9 position. We further screened their
antioxidant activities by ABTS, DPPH, HOSC and FRAP assays, and
comprehensively analyzed the results via color grading proles.
Berberine (BBR), a natural isoquinoline alkaloid isolated from
Coptis chinensis and Hydrastis canadensis,1 has proved to possess
activity for various diseases, such as inammation,2 diabetes,3
insulin resistance, Alzheimer's disease, acute endotoxemia,4
and maintenance of the gut mucosal barrier function5 with
intestinal transport ability.6 It has been conrmed that the
inhibitory effect of BBR on oxidative stress was observed both in
cells and animal models, which were associated with oxidative
stress.7 In addition, the excessive accumulation of ROS is
attributed to the imbalance between the antioxidant defense
systems and production of reactive oxygen species (ROS),
including superoxide anion (O2ꢀc), hydroxyl radical (OHc), and
hydrogen peroxide (H2O2), leading to oxidative stress, which is
a key factor of metabolic disorder, acute respiratory distress
syndrome,8 idiopathic pulmonary brosis,9 neurodegenerative
disorders, insulin resistance, acceleration of the aging
process,10–12 impairing of the DNA repair systems13 and
inducing apoptosis of the pancreatic islet b-cells.14
2. Results and discussion
Although accumulating evidence has shown the usefulness of
BBR in reducing oxidative stress,15 its antioxidant activity is not
effective enough compared with other synthetic antioxidant agents,
which play a dominant role in clinical use. BBR derivates, containing
phenolic hydroxy groups, have been reported with enhanced
2.1. Synthesis
Compounds in this study were prepared according to Scheme 1.
Compound 2 containing an acetoxyl group was obtained by
alkylation of the phenolic hydroxyl group of compound 1 with
acetic anhydride in the presence of phosphoric acid at 90 ꢁC for
5–10 min. Compound 3 was synthesized from compound 1,
containing the phenolic hydroxyl group by treatment with
dimethyl sulfate in sodium hydroxide solution for 4.5 h
according to the literature.23 The leading compound (berberine,
6) was demethylated on the 9-O position, following a previously
described method.24 In simple terms, it was heated at 190 ꢁC in
a dry oven under vacuum (20–30 mmHg) for 0.5–1 h to obtain
a dark red solid, and puried by recrystallization from methyl
aDepartment of Pharmaceutical Engineering, College of Chemistry and Chemical
Engineering, Central South University, Changsha, 410083, China
bDepartment of Pharmaceutics, Xiangya School of Pharmaceutical Sciences, Central
South University, Changsha, 410013, China. E-mail: zhenbaoliu@csu.edu.cn
cDepartment of Medicinal Chemistry, School of Pharmacy, Hunan University of
Chinese Medicine, Changsha, 410208, China
† Electronic supplementary information (ESI) available. See DOI:
10.1039/d1ra01339d
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Scheme 1 Synthetic procedure and chemical structures of berberine derivatives. Reagents and conditions: (a) acetic anhydride, H3PO4, room
temperature; (b) dimethyl sulfate, NaOH, 95–100 ꢁC, 2 h; (c) SOCl2, pyridine, 75 ꢁC, 1–2 h; (d) vacuum (20–30 mmHg), 190 ꢁC, 0.5–1 h; (e)
pyridine, acetonitrile, room temperature, 2–4 h.
alcohol to obtain berberrubine (7). The benzoic acid derivatives
(2, 3, 4) were treated by thionyl chloride, then condensed with
berberrubine 7 in the presence of pyridine to obtain compound
(8). The compounds were puried by recrystallization
with methanol. Structures of all of the synthesized berberine
derivatives were explicitly characterized by IR, 1H NMR,
and HRMS.
2.3. Antioxidant activities evaluation
2.3.1. ABTS assay. As shown in Fig. 1, BBR derivatives
showed diverse abilities in scavenging ABTS radicals, and most
of their abilities were greater than that of BBR, which may have
resulted from the enhancement of lipophilicity via esterication
Table 1 Log P values of berberine and berberine derivatives
2.2. Log P
Compounds
Log P
Compounds
Log P
The results are summarized in Table 1. Generally, moderate
lipophilicity (log P ¼ 0–3) is benet to the druggability of drug
candidates. As we expected, the lipophilicities of the synthe-
sized compounds were increased compared with that of
berberine. It is noteworthy that the values of log P of the
berberine derivatives were in a range from 1.23 to 2.13. Usually,
the compounds with log P of between 0–3 have good gastroin-
testinal absorption, indicating the synthesized compounds met
the requirements for oral administration.25 The different log P
may also lead to diverse antioxidant activities.
BBR
8a
8b
8c
8d
8e
8f
8g
8h
ꢀ0.18
1.23
1.29
1.71
1.83
1.31
1.66
1.24
1.67
8i
8j
1.46
1.41
1.56
1.56
1.24
1.27
2.06
2.13
8k
8l
8m
8n
8o
8p
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Fig. 1 ABTS radical scavenging capacities (A–C) and IC50 (D) of berberine derivatives.
process and the incorporation of aromatic acid. Compared with capacities, while 8b, 8k, 8m, and 8o showed slightly higher
BBR, compounds 8h, 8i, 8j and 8n, 8p, with the IC50 values antioxidant capacities, and compounds 8a and 8e exhibited
ranging from 1.06 ꢂ 0.15 to 2.94 ꢂ 0.32 mg mLꢀ1, exhibited lower antioxidant capacities.
superior efficacy in radicals scavenging (3–8-fold). Among them,
The ABTS assay is an electron-transfer-based assay that measures
compounds 8c, 8d, 8f, and 8g had moderate antioxidant the capacity of an antioxidant in reducing the oxidants. Among
Fig. 2 The DPPH radical scavenging capacities (A–C) and the IC50 (D) of BBR and BBR derivatives.
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these compounds, there is a correlation between the structure and > 8c and 8j > 8d, indicated that the acetoxyl group (–OCOCH3)
radical-scavenging capacities. Compound 8a showed the lowest substitution contributed superior antioxidant activity compared to
capacity, and this may be attributed to the introduction of electron- that of the methoxy group (–OCH3) substitution. We also found
donating groups, which was accordance with the research con- that when the benzene rings contain halogens, the antioxidant
ducted by Bhupendra Mistry.17 To investigate the relationship activities of the compounds will be signicantly improved, as
between different substituents and scavenging capacities, shown by the activities of 8c, 8d, 8h, 8i and 8j compared to that of
compound 8a was used as a basic compound. It was found that the 8b. The R2 position substituent contributed more to the activity
compounds possessing an aromatic nucleus showed enhanced than the R4 position, as conrmed by the activities of 8b > 8e, and
antioxidant capacities, which could be attributed to the oxygen- 8g > 8m. The substituent acetoxyl (–OCOH3) can provide better
containing moieties. The position of the substituent is also an antioxidant activity compared with methoxyl (–OCH3), as indicated
inuence factor. Different positions of the same substituents by the activities of 8e < 8m, 8c < 8i, and 8d < 8j.
exhibited different capacities, especially at the para-position. The
2.3.2. DPPH assay. As shown in Fig. 2, 8h, 8j, 8l have similar
activation of radical scavenging involves increased stabilization of activities with BBR. BBR and its derivatives possessed scavenging
radicals and other electron decient intermediates, which are capacities for DPPH radicals. The activities of compounds 8c, 8d, 8i,
formed during oxidation.26 Therefore, the introduced electron- 8p, and especially, 8c, 8d, 8p were better than that of BBR at the
donating groups were benecial to the reducing of the antioxidant concentration of 1.2 mg mLꢀ1, and the radical scavenging ratio of 8c,
activity, which may be attributed to the electronic effect contributed 8d, 8p reached 80%, 85%, and 97% (Fig. 2B), respectively, while BBR
to the free radical reaction on benzene.
is only 50%. Compound 8p was 6 times compared toBBR. In contrast,
Compounds 8g and 8f showed enhanced antioxidant capac- compounds 8a, 8b, 8e, 8f, 8g, 8m, 8n, 8o, and especially 8a, 8e
ities. The benzoic acid with acetoxyl substitution derivatives exhibited lower scavenging activities than BBR. The replacement of
(compounds 8g, 8i, 8j) were more efficient than their methoxy hydrogen on the aromatic ring by a halogen atom or methyl group
substitutes (compounds 8b, 8c, 8d). With regard to the aromatic donated enhanced activities (compounds 8c and 8d > 8b, compounds
substitution, the order of effectiveness was acetoxy group 8i and 8j > 8g). Aside from the aromatic substitution, the position of
(compound 8g) z ethoxy group (compound 8f) > methoxy group the substitution has signicant inuences on the activities. Para-
(compound 8b) > hydrogen group (compound 8a). The activity of 8i methoxy- (compound 8e) and para-acetoxyl- (compound 8m) benzoic
Fig. 3 The hydroxyl radical scavenging capacities (A–C) and the IC50 (D) of BBR and BBR derivatives.
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acid berberine derivatives showed lower antioxidant ability than their that of BBR (8.81 ꢂ 0.36 mM). We also found that having substit-
ortho-substitutes (compounds 8b and 8g), respectively. uent halogens at the R5 position was better than the methyl (–CH3)
2.3.3. HOSC assay. Most of the derivatives showed higher group due to the fact that 8c > 8b, 8d > 8b, 8i [ 8g, 8j [ 8g, 8i >
values than BBR (Fig. 3). The IC50 values range from 0.25 ꢂ 0.06 8l, 8j > 8l. The acetoxyl (–OCOCH3) substituent was better than the
to 73.70 ꢂ 0.11 mg mLꢀ1 compared with BBR (20.48 ꢂ 0.14 mg methoxy (–OCH3) substituent, as further conrmed by the activi-
mLꢀ1). Among them, compounds 8c, 8l, 8n and 8p exhibited ties of 8g > 8b, 8i > 8c, 8j > 8d, and 8m > 8e.
better antioxidant activities, while compounds 8e, 8m, and 8h
These results proved that there were regularities between the
showed poorer activities. This assay again showed that the para- ferric reducing power and the substituents on benzene. Compounds
substitution had a negative effect on the antioxidant activity introducing a benzene ring with an acetyl oxygen substituent
(compounds 8l > 8k, 8h < 8i, indicating that the R5 position was possessed potent ferric reducing capacities that were greater than
better than the R4 position for a substituent; compounds 8b > that modied with the methoxyl substituent. In addition, replacing
8e, 8g > 8m, indicating that the R2 position substituent was the hydrogen atom with oxygen-containing moieties on the ortho-
better than the R4 position substituent). Compound 8j > 8d, position of benzene donated reduced activities.
indicating that the acetoxyl (–OCOCH3) substituent was better
Based on the above experimental data, we speculated that
than the methoxy (–OCH3) substituent. Compound 8p exhibited the introduction of an electron-donating group on the benzene
signicantly enhanced activity, which was in accordance with ring could increase its electron cloud density, and the electric
other antioxidant assays.
effect was benecial to the free radical reaction of benzene,
2.3.4. FRAP assay. As shown in Fig. 4, these results showed thereby increasing the antioxidant activity to some degree.
that all the tested compounds exhibited a ferric reducing power. Furthermore, the effectiveness was acetoxy group > methoxy
Furthermore, most of the modied compounds, especially 8i, group, and this may be related to their electron-donating abil-
8j, 8p, exhibited signicantly higher activities than its natural ities. At the same time, we also found that the substitutes at the
precursor (BBR). When the concentration reached 187.5 mg mLꢀ1
,
ortho-position had a positive effect on the antioxidant activity,
the reducing capacities increased by 10, 10 and 40 times, respec- and the substitutions at the para-position hindered the anti-
tively, compared to BBR, while others, such as 8e, 8f, 8h and 8m, oxidant activity to some extent. Having a substituent at the
possessed a similar or slight decrease in the Fe(II) equivalent. ortho-position, and introduction of a halogen or a methyl group
Fig. 4D shows that at the same concentration of 187.5 mg mLꢀ1
,
at the meta-position can also enhance the antioxidant activity.
compound 8p possessed dramatically enhanced antioxidant Moreover, it was better to have multiple substituents on the
capacity (421.50 ꢂ 0.77 mM), which is nearly 50 times greater than benzene ring than to have only one substituent.
Fig. 4 (A–C) The Fe3+ reducing capacities of BBR and BBR derivatives. (D) The Fe3+ reducing capacities of BBR and BBR derivatives at
a concentration of 187.5 mM.
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Fig. 5 Comprehensive analysis of the antioxidant activities using the color grading profile.
antioxidation activity at 290 mm.29 g-Glutamylcysteine,
a precursor of glutathione, could enter cells to suppress LPS-
induced inammation, prevent ROS accumulation and GSH
depletion.30 Clinical trials have shown that oral administration
of g-glutamylcysteine increases the intracellular glutathione
levels above homeostasis.31 Ebselen protected peroxynitrite in
a dose-dependent manner, and its EC50 value is 8.0 mm.32 It is
a promising candidate for treatment of COVID-19 and other
respiratory virus infections.33 Ergothionine (EGT) inhibited
liposome oxidation by 67% and 100% at 20 mm and 100 mm,
respectively,34 which showed similar antioxidant activity to
coenzyme Q10. Polyphenols represent antioxidant natural
products from a variety of plant sources, and gallic acid has
been shown to be effective in scavenging free radicals. A DPPH
assay showed that gallic acid, which consumed free radicals
through a hydrogen-donating mechanism, was more effective
than vitamin E.35 Resveratrol and its derivatives play important
roles in scavenging H2O2 and inhibiting oxidative stress by
activating the antioxidation mechanism of Keap-1/Nrf2, as
shown in an asthma rat model, and have potential value as
functional food additives.36,37
In contrast, the lipophilic berberine was effective in inhibi-
tion against lipid peroxidation and in protecting rat hepatocytes
from oxidative stress.38 The scavenging activity of the hydroxyl
radical of berberine and its metabolite was 23% and 85% at
1 mM concentration, respectively. The hydroxy group at the C-9
position of berberine and its derivative played an important role
in the scavenging activity.39 Moreover, berberine protected mice
from LPS-induced oxidative damage and improved the survival
rate of mice.40 In addition, berberine has been shown to be able
to scavenge a variety of free radicals, which is may be related to
the involvement of berberine in the redox reaction.41 Experi-
ments using DPPH, ABTS radical scavenging, nitric oxide
scavenging, lipid peroxidation and other radical scavenging
methods compared berberine and vitamin C. It was found that
berberine showed antioxidant activity which was comparable to
vitamin C, and displayed a variety of mechanisms to achieve the
anti-oxidation effect, which proved the potential of berberine in
anti-oxidation therapy.41 In summary, the synthesis and explo-
ration of berberine-based derivatives has great potential and
good prospects in the treatment of reactive oxygen species-
related diseases, such as asthma and cytokine storms.
2.4. Comprehensive analysis of the antioxidant activity
using color grading prole
The results from different assays resulted in different results,
which may be attributed to the fact that the detection principles
were different, and thus led to differentiation. However, most of
the compounds showed similar results from the four assays.
From the results (a mixture color of the result color from four
assays) in Fig. 5, we can nd that compounds 8p, 8c, 8d, 8i, 8j,
and 8l showed signicant enhanced activities, and compounds
8b, 8g, 8h, 8n, and 8k showed slightly increased activities.
Compounds 8a, 8f, and 8o showed equal activities with BBR,
and compounds 8e, 8m showed lower activity.
2.5. The relation of the molecular electrostatic potentials
(MEPs) with the antioxidant activity
As shown in Fig. 6, the ESP distribution color at the position of
the berberine methylenedioxy group, which is the pharmaco-
phore of the antioxidant, was close to red, indicating that this
position has the trend of donating electrons. Compared with
BBR in Fig. 6A, compound 8p in Fig. 6B exhibited a more
extensive red area on the introduced groups at the C-9 and
acetoxyl groups. Thus, it can be considered that the trend of
donating electrons in compound 8p was more obvious
compared with berberine, which was in accordance with the
dramatically enhanced antioxidant activity.
N-Acetyl-L-cysteine, which can increase the GSH concentra-
tion in the lungs by supplementing L-cysteine with the GSH
precursor protein, has been shown to reduce ROS and reduce
airway inammation and AHR by regulating activation of NF-kb
and HIF-1a.27,28 N-Acetyllysine (Nal), which reduced the ROS
levels and inammatory responses, showed an IC50 value of the
Fig. 6 The MEPs distribution maps of (A) BBR and (B) compound 8p.
The molecular electrostatic potential distribution of the charge density
was presented by a color code from red to blue (from 0.05 to 0.143
hartree).
In this work, a series of 9-O-benzoic acid substituted
berberine derivatives were synthesized and their antioxidant
activities were evaluated by DPPH, ABTS, FRAP and HOSC
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ꢁ
assays. The antioxidant activities were not judged based on for 2 h at 95–100 C. The mixture was cooled to room temper-
a single test, but a comprehensive assessment by a series of ature, and 2 mL concentrated hydrochloric acid was added until
assays. According to the results of DPPH, ABTS, FRAP and the pH value changed to 1. The mixture became turbid, and the
HOSC, compounds 8p, 8c, 8d, 8i, 8j, and 8l showed superior product was extracted with diethyl ether. Removal of the solvent
antioxidant activity compared with BBR. In particular, from the extract gave 2-methoxybenzoic acid.
compound 8p can be used as a potential antioxidant, which had
3.3.3. Synthesis of compound 5. Taking benzoic acid as an
6 times, 8 times and 36 times greater radical scavenging example, a solution containing benzoic acid (3.7 g, 0.03 mol)
capacities in DPPH, ABTS and HOSC assays, respectively, and 48 and pyridine (3 d) in dry SOCl2 (3–10 mL) was heated at 75 ꢁC for
times greater ferric ion reducing antioxidant power compared 1–2 h under nitrogen atmosphere. Then, the reaction mixture
with that of BBR. This compound may be a potential drug for was concentrated under reduced pressure to give benzoyl
the treatment of the reactive oxygen species (ROS)-related chloride.
diseases, such as the treatment of cytokine storms which are
critical clinical conditions induced by the ROS in the patho- (16.8 g, 0.05 mmol) was heated at 190 C in a dry oven under
3.3.4. Synthesis of compound 7. Berberine chloride (6)
ꢁ
genesis of COVID-19.
vacuum (20–30 mmHg) for 0.5–1 h to obtain a dark red solid. It
was puried by recrystallizing from methyl alcohol to obtain
berberrubine (7).
3. Experimental
3.1. General experimental procedures
3.3.5. Synthesis of compound 8. Taking benzoic acid (3.7 g,
0.03 mol) as an example, benzoyl chloride was added to the
solution of berberrubine (7) (3.86 g, 0.012 mol) in 40 mL
acetonitrile and 4 mL pyridine, stirred for 2–4 h at room
temperature, and the reaction was monitored by thin-layer
chromatography (TLC). The crude product was recrystallized
twice from methyl alcohol to give the rened product.
3.3.5.1. Data for compound (8a). 73.8% yield; obtained as
a yellow solid; mp: 210–212 ꢁC; other characteristics are similar
with the literature we published before.20
3.3.5.2. Data for compound (8b). 68.2% yield; obtained as
a yellow solid; IR (KBr) nmax 3023, 2908, 1738, 1604, 1504, 1396,
1262, 1039, 750 cmꢀ1; 1H NMR (400 MHz, DMSO-d6): d 9.88 (s,
1H), 9.07 (s, 1H), 8.34 (d, J ¼ 9.3 Hz, 1H), 8.25 (d, J ¼ 9.2 Hz, 1H),
8.19 (dd, J ¼ 7.8, 1.8 Hz, 1H), 7.82 (d, J ¼ 11.9 Hz, 1H), 7.77 (ddd,
J ¼ 9.0, 7.4, 1.8 Hz, 1H), 7.33 (d, J ¼ 8.4 Hz, 1H), 7.20 (t, J ¼
7.6 Hz, 1H), 7.10 (s, 1H), 6.19 (s, 2H), 4.86–5.01 (m, 2H), 4.06 (d, J
¼ 13.6 Hz, 3H), 3.93 (s, 3H), 3.13–3.24 (m, 2H); HR-ESI-MS: m/z
456.1997 [M]+ (calcd for C27H22NO6, 456.1442).
1
The H NMR spectra were recorded on Bruker Avance III 500
MHz and Avance III 400 MHz NMR spectrometers from Bruker
(Karlsruhe, Germany), respectively, with tetramethylsilane
(TMS) as the internal standard. The IR spectra were measured
by a Nicolet 6700 FTIR spectrometer from Thermo Fisher
Scientic (Boston, MA, USA). Samples were ground with KBr
and compressed to pellets. The HRMS were measured by
a Water XEVO G2-XS QT high-resolution mass spectrometer
from Waters (Boston, MA, U.S.A.).
3.2. Material
Berberine chloride was purchased from Lion Biological Tech-
nology (Zhengzhou, China). Benzoic acid, salicylic acid and other
chemical raw materials were purchased from Bide Pharmatech,
Ltd. (Shanghai, China). 1,1-Diphenyl-2-trinitrophenylhydrazine
(DPPH), 2,20-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) dia-
mmonium salt (98%) (ABTS) and 2,4,6-tri-(2-pyridyl)-1,3,5-triazine
(TPTZ) were from Aladdin (Shanghai, China). Other commonly
used reagents were from Sinopharm Chemical Reagent Co., Ltd.
(Shanghai, China).
3.3.5.3. Data for compound (8c). 65.4% yield; obtained as
a yellow solid; IR (KBr) nmax 3009, 2846, 1748, 1614, 1504, 1395,
1274, 1207, 1103, 1035, 818, 764 cmꢀ1 1H NMR (400 MHz,
;
DMSO-d6) d 9.94 (s, 1H), 9.10 (s, 1H), 8.31–8.40 (m, 1H), 8.27 (d, J
¼ 9.2 Hz, 1H), 8.18 (d, J ¼ 2.8 Hz, 1H), 7.85 (d, J ¼ 3.1 Hz, 1H),
7.83 (d, J ¼ 2.8 Hz, 1H), 7.39 (d, J ¼ 9.1 Hz, 1H), 7.11 (s, 1H), 6.19
3.3. Synthesis of berberine derivatives
3.3.1. Synthesis of compound 2. Taking salicylic acid as (s, 2H), 4.84–5.02 (m, 2H), 4.06 (d, J ¼ 10.7 Hz, 3H), 3.94 (s, 3H),
example, a mixture of salicylic acid (2.0 g, 0.015 mol) and acetic 3.20 (dd, J ¼ 15.3, 9.0 Hz, 2H); HR-ESI-MS: m/z 490.1633 [M]+
anhydride (5 mL, 0.05 mol) was added to a ask, and phos- and m/z 492.1617 [M + 2]+ (calcd for C27H21ClNO6, 490.1052).
phoric acid (5–7 d) was dropped into the mixture under stirring.
Then, the mixture was stirred for 5–10 min at 90 ꢁC. Finally, the a yellow solid; IR (KBr) nmax 3404, 2904, 1750, 1613, 1505, 1394,
mixture was cooled to room temperature to obtain white 1276, 1207, 1102, 1034, 816, 621 cmꢀ1 1H NMR (500 MHz,
crystals.
DMSO-d6) d 9.93 (s, 1H), 9.07 (s, 1H), 8.34 (d, J ¼ 9.1 Hz, 1H),
3.3.5.4. Data for compound (8d). 63.7% yield; obtained as
;
3.3.2. Synthesis of compound 3. Taking salicylic acid as an 8.27 (d, J ¼ 5.0 Hz, 1H), 8.26 (d, J ¼ 9.2 Hz, 1H), 7.94 (d, J ¼
example, a solution of 8 g sodium hydroxide in 47 mL water was 8.9 Hz, 1H), 7.82 (d, J ¼ 2.7 Hz, 1H), 7.33 (d, J ¼ 9.1 Hz, 1H), 7.11
cooled to 0 ꢁC, and 0.1 mol salicylic acid was added under (d, J ¼ 7.4 Hz, 1H), 6.18 (d, J ¼ 9.6 Hz, 2H), 4.92 (s, 2H), 4.02–
stirring. 10 mL dimethyl sulfate was quickly added under cool 4.09 (m, 3H), 3.93 (s, 3H), 3.21 (d, J ¼ 6.1 Hz, 2H); HR-ESI-MS: m/
condition, and the mixture was heated to 35 C and stirred for z 534.1188 [M]+ and m/z 536.1167 [M + 2]+ (calcd for
ꢁ
ꢁ
20 min at 35 C. An additional 10 mL of dimethyl sulfate was
C
27H21BrNO6, 534.0547).
3.3.5.5. Data for compound (8e). 68.2% yield; obtained as
ꢁ
added. The mixture was stirred for 10 min at 45 C, heated to
95–100 C, and stirred for 2 h at 95–100 C. A solution of 3.9 g a yellow solid; other characteristics are similar with the litera-
NaOH in 13.3 mL water was added, and the mixture was stirred ture reported.42
ꢁ
ꢁ
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7.83 (d, J ¼ 16.3 Hz, 1H), 7.68 (dd, J ¼ 8.3, 1.9 Hz, 1H), 7.29 (t, J ¼
8.9 Hz, 1H), 7.12 (d, J ¼ 17.6 Hz, 1H), 6.21 (d, J ¼ 12.2 Hz, 2H),
4.87–5.02 (m, 2H), 4.04 (s, 3H), 3.21 (dd, J ¼ 13.7, 7.6 Hz, 2H),
2.47 (s, 3H), 2.21 (s, 3H); HR-ESI-MS: m/z 498.2138 [M]+ (calcd
for C29H24NO7, 498.1547).
3.3.5.6. Data for compound (8f). 60.8% yield; obtained as
a yellow solid; IR (KBr) nmax 3029, 2893, 1748, 1606, 1508, 1397,
1280, 1219, 1105, 1040, 759 cmꢀ1; 1H NMR (400 MHz, DMSO-d6)
d 9.88 (s, 1H), 9.08 (s, 1H), 8.34 (d, J ¼ 9.3 Hz, 1H), 8.22–8.30 (m,
1H), 8.15–8.21 (m, 1H), 7.83 (d, J ¼ 2.7, 1H), 7.73 (ddd, J ¼ 8.9,
7.4, 1.8 Hz, 1H), 7.31 (d, J ¼ 8.3 Hz, 1H), 7.18 (t, J ¼ 7.5 Hz, 1H),
7.10 (s, 1H), 6.19 (d, J ¼ 4.0 Hz, 2H), 4.87–5.00 (m, 2H), 4.20 (q, J
¼ 6.9 Hz, 2H), 4.05 (s, 3H), 3.18–3.25 (m, 2H), 3.17 (s, 2H), 1.36
(t, J ¼ 6.9 Hz, 3H); HR-ESI-MS: m/z 470.2158 [M]+ (calcd for
3.3.5.13. Data for compound (8m). 58.3% yield; obtained as
a yellow solid; IR (KBr) nmax 3308, 2986, 1749, 1603, 1514, 1367,
1278, 1208, 1036, 779 cmꢀ1 1H NMR (500 MHz, DMSO-d6)
;
d 10.02 (s, 1H), 9.07 (d, J ¼ 8.5 Hz, 1H), 8.36 (d, J ¼ 9.3 Hz, 1H),
8.30–8.34 (m, 2H), 8.27 (d, J ¼ 9.2 Hz, 1H), 7.83 (d, J ¼ 2.6 Hz,
1H), 7.43–7.52 (m, 2H), 7.10 (s, 1H), 6.19 (d, J ¼ 5.4 Hz, 2H),
4.86–4.96 (m, 2H), 4.03 (s, 3H), 3.18–3.24 (m, 2H), 2.36 (s, 3H);
HR-ESI-MS: m/z 484.0838 [M]+ (calcd for C28H22NO7,
484.1391).
C
28H24NO6, 470.1598).
3.3.5.7. Data for compound (8g). 61.2% yield; obtained as
a yellow solid; other characteristics are similar with the litera-
ture we published before.21
3.3.5.8. Data for compound (8h). 53.8% yield; obtained as
a yellow solid; IR (KBr) nmax 3426, 2953, 1763, 1614, 1506,
1396, 1278, 1226, 1125, 1068, 1039 cmꢀ1; 1H NMR (500 MHz,
DMSO-d6) d 9.97 (s, 1H), 9.10 (s, 1H), 8.41 (d, J ¼ 8.4 Hz, 1H),
8.35 (d, J ¼ 9.3 Hz, 1H), 8.26–8.30 (m, 1H), 7.81–7.87 (m, 1H),
7.70–7.75 (m, 1H), 7.68 (dd, J ¼ 8.1, 1.9 Hz, 1H), 7.11 (s, 1H),
6.19 (s, 2H), 4.86–4.97 (m, 2H), 4.06 (d, J ¼ 20.3 Hz, 3H), 3.21
(dd, J ¼ 14.3, 8.1 Hz, 2H), 2.24 (s, 3H); HR-ESI-MS: m/z
3.3.5.14. Data for compound (8n). 49.8% yield; obtained as
a yellow solid; IR (KBr) nmax 3419, 2980, 2943, 2906, 1740, 1603,
1
1504, 1399, 1373, 1334, 1279, 1231, 1122, 1034 cmꢀ1; H NMR
(500 MHz, DMSO-d6): d 9.97 (s, 1H), 9.09 (s, 1H), 8.33 (d, J ¼
9.3 Hz, 1H), 8.26–8.31 (m, 1H), 7.83 (s, 1H), 7.54 (s, 2H), 7.11 (s,
1H), 6.18 (s, 2H), 4.86–4.98 (m, 2H), 4.00–4.09 (m, 3H), 3.93 (d, J
¼ 8.0 Hz, 6H), 3.83 (s, 3H), 3.17–3.25 (m, 2H); HR-ESI-MS: m/z
516.2278 [M]+ (calcd for C29H26NO8, 516.1653).
518.0922 [M]+ and m/z 520.0892 [M
+
2]+ (calcd for
3.3.5.15. Data for compound (8o). 40.6% yield; obtained as
a yellow solid; IR (KBr) nmax 3403, 2890, 1768, 1608, 1505, 1395,
C
28H21ClNO7, 518.1001).
3.3.5.9. Data for compound (8i). 51.5% yield; obtained as
a yellow solid; IR (KBr) nmax 3385, 3019, 2842, 1738, 1604, 1504,
1395, 1262, 1214, 1135, 1103, 1037, 930, 758 cmꢀ1 1H NMR
1
1370, 1324, 1277, 1221, 1106, 1060 cmꢀ1; H NMR (500 MHz,
DMSO-d6): d 9.97 (s, 1H), 9.09 (s, 1H), 8.35 (d, J ¼ 9.3 Hz, 1H),
8.27 (d, J ¼ 9.2 Hz, 1H), 7.84 (s, 1H), 7.50 (s, 2H), 7.11 (s, 1H),
6.20 (s, 2H), 4.86–4.99 (m, 2H), 4.07–4.31 (m, 6H), 4.04 (s, 3H),
3.14–3.26 (m, 2H), 1.40 (t, J ¼ 6.9 Hz, 6H), 1.25–1.35 (m, 3H);
HR-ESI-MS: m/z 558.2906 [M]+ (calcd for C32H32NO8, 558.2122).
3.3.5.16. Data for compound (8p). 48.0% yield; obtained as
a yellow solid; IR (KBr) nmax 3403, 2890, 1768, 1608, 1505, 1395,
;
(500 MHz, DMSO-d6) d 9.99 (s, 1H), 9.07 (s, 1H), 8.39 (d, J ¼
2.6 Hz, 1H), 8.34 (d, J ¼ 9.3 Hz, 1H), 8.27 (d, J ¼ 9.2 Hz, 1H),
7.98 (dd, J ¼ 8.7, 2.7 Hz, 1H), 7.83 (s, 1H), 7.51 (d, J ¼ 8.7 Hz,
1H), 7.11 (s, 1H), 6.18 (d, J ¼ 11.2 Hz, 2H), 4.86–4.96 (m, 2H),
3.99–4.12 (m, 3H), 3.19–3.25 (m, 2H), 2.20 (s, 3H); HR-ESI-MS:
m/z 518.1614 [M]+ and m/z 520.1600 [M + 2]+ (calcd for
1
1370, 1324, 1277, 1221, 1160, 1060 cmꢀ1; H NMR (500 MHz,
C
28H21ClNO7, 518.1001).
DMSO-d6): d 10.07 (s, 1H), 9.08 (s, 1H), 8.36 (d, J ¼ 9.4 Hz, 1H),
8.27–8.30 (m, 1H), 8.12 (s, 2H), 7.84 (s, 1H), 7.10 (d, J ¼ 2.8 Hz,
1H), 6.19 (s, 2H), 4.87–4.92 (m, 2H), 4.05 (d, J ¼ 3.5 Hz, 3H),
3.19–3.23 (m, 2H), 2.40 (s, 3H), 2.35 (s, 6H); HR-ESI-MS: m/z:
600.3245 [M]+ (calcd for C32H26NO11, 600.1500).
3.3.5.10. Data for compound (8j). 53.7% yield; obtained as
a yellow solid; IR (KBr) nmax 3338, 3016, 1752, 1616, 1506, 1480,
1366, 1273, 1203, 1100, 1031, 920, 761 cmꢀ1; 1H NMR (500 MHz,
DMSO-d6) d 9.99 (s, 1H), 9.07 (s, 1H), 8.39 (d, J ¼ 2.6 Hz, 1H),
8.34 (d, J ¼ 9.3 Hz, 1H), 8.27 (d, J ¼ 9.2 Hz, 1H), 7.98 (dd, J ¼ 8.7,
2.7 Hz, 1H), 7.83 (s, 1H), 7.51 (d, J ¼ 8.7 Hz, 1H), 7.11 (s, 1H),
6.19 (s, 2H), 4.83–5.01 (m, 2H), 4.04 (s, 3H), 3.17–3.27 (m, 2H),
2.25 (s, 3H); HR-ESI-MS: 562.1165 m/z [M]+ and m/z 564.1152 [M
+ 2]+ (calcd for C28H21BrNO7, 562.0496 m/z).
3.3.5.11. Data for compound (8k). 59.1% yield; obtained as
a yellow solid; IR (KBr) nmax 3302, 3026, 2906, 1745, 1615, 1504,
1436, 1366, 1340, 1204, 1039, 822, 777 cmꢀ1; 1H NMR (500 MHz,
DMSO-d6) d 9.91 (s, 1H), 9.11 (s, 1H), 8.34 (d, J ¼ 9.3 Hz, 1H),
8.29 (t, J ¼ 5.4 Hz, 1H), 8.27 (d, J ¼ 9.2 Hz, 1H), 7.84 (s, 1H), 7.41
(d, J ¼ 8.0 Hz, 1H), 7.25 (s, 1H), 7.10 (s, 1H), 6.19 (s, 2H), 4.87–
4.99 (m, 2H), 4.03 (s, 3H), 3.21 (dd, J ¼ 13.2, 7.0 Hz, 2H), 2.48 (s,
3H), 2.22 (s, 3H); HR-ESI-MS: m/z 498.2134 [M]+ (calcd for
3.4. The calculation of the partition coefficient (log P)
The partition coefficient, abbreviated as P, is dened as
a particular distribution ratio of molecules in oil and water, and
is useful in predicting the absorption of drugs within the body.
Hence, the log P is a criterion used in decision-making by
medicinal chemists in pre-clinical drug discovery. To investi-
gate the druggability of berberine derivatives, log P of BBR and
its derivatives were calculated using ALOGPS 2.1 soware.
3.5. Antioxidant activity assays
The antioxidant activities of berberine derivatives 8a–8p were
measured by FRAP, ABTS, DPPH and HOSC assays. IC50 was
calculated by means of SPSS Statistics 21 soware. A signicant
difference is considered at the level of p < 0.05.
3.5.1. ABTS assay. The ABTS assay is a widely applied
method for measuring the radical-scavenging ability of antiox-
idants. ABTS can be oxidized to green ABTSc+ (maximum
C
29H24NO7, 498.1547).
3.3.5.12. Data for compound (8l). 54.3% yield; obtained as
a yellow solid; IR (KBr) nmax 3340, 3033, 2904, 1744, 1618, 1504,
1440, 1366, 1340, 1275, 1186, 1033, 928, 822, 573 cmꢀ1; 1H NMR
(500 MHz, DMSO-d6) d 9.93 (d, J ¼ 9.9 Hz, 1H), 9.11 (s, 1H), 8.33
(t, J ¼ 11.6 Hz, 1H), 8.23–8.31 (m, 1H), 8.21 (t, J ¼ 6.9 Hz, 1H),
17618 | RSC Adv., 2021, 11, 17611–17621
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absorbance at 734 nm) under the action of an oxidant, and the radical scavenging activity (HRSA%) was calculated. The equa-
generation of ABTSc+ can be inhibited by an antioxidant. The tion is shown below:
assay was carried out according to a previously reported
Ax ꢀ Ax0
method.43 Generally, the ABTSc+ aqueous mother solution was
prepared by mixing the ABTS solution and oxidant, and stored
in the dark at room temperature for 12–16 h. Aliquots con-
taining 10 mL of different concentrations of sample in DMSO
were added to 200 mL ABTSc+ solution (A734 nm ¼ 0.700 ꢂ 0.02)
(Ax) by dilution of the ABTSc+ aqueous mother solution with
ethanol (20% of water). Methanol was set as the blank (A0), and
different concentrations of assays were set as the control (Ax0).
The absorbance was read every 20 s at 30 ꢁC in 6 min by ELISA.
The radical scavenging activity in percentage (RSA%) was
calculated according to Nanjappa et al.,44 with some modica-
tions as described in the equation below:
HOSA% ¼ 1 ꢀ
ꢃ 100%
A0
3.5.4. Ferric reducing antioxidant power (FRAP) assay. The
principle of FRAP is that antioxidants can restore ferric-
tripyridyltriazine (TPTZ-Fe(III)) in acidic conditions, and
generate the stable ferrous form (TPTZ-Fe(II)), which has the
maximum absorbance wavelength at 593 nm, and then the
antioxidant potential of the compounds was estimated accord-
ing to the absorbance. The FRAP assay was carried out in
accordance with the previous literature.49 Briey, 10 mM TPTZ
was dissolved in 40 mM hydrochloric acid (HCl), 20 mM
FeCl3$6H2O, 0.3 M acetate buffer to a nal pH of 3.6, and
different concentrations of the sample and FeSO4$6H2O (1.25,
2.5, 5, 25, 100, 150, 200, 400, 500 mM) were prepared. 1.9 mL
FRAP reagent containing TPTZ, FeCl3$6H2O and acetate buffer
Ax ꢀ Ax0
RSA% ¼ 1 ꢀ
ꢃ 100%
A0
3.5.2. DPPH assay. Radical scavenging efficiencies of the according to the proportion of 10 : 1 : 1 with 100 mL sample
compounds were evaluated by DPPH analysis. DPPH, a kind of were mixed. Before reading the absorbance at 595 nm by ELISA,
stable radical, with absorption at 517 nm, can be paired with the mixture was shaken and reacted at 37 ꢁC for 30 min. Iron(II)
a free radical scavenger, generating a pale yellow non-radical sulfate solution was used for calibration. The FRAP values are
form (DPPH-H), which has no absorption at 517 nm. The expressed as concentrations of iron(II). All analyses were done in
DPPH radical solution was prepared by dissolving 1 mg of triplicate.
DPPH in 20 mL of methanol and stored in the dark. 1.5 mL
DPPH and 1 mL different concentrations of the sample dis-
solved in DMSO were mixed (Ax), shaken and reacted in the
dark for 30 min. The mixture containing 1.5 mL DPPH and
1 mL DMSO was used as a negative control (A0), the referrals
anti-oxidant (ascorbic acid) in methanol and DPPH radical
solution were set as positive controls. Furthermore, mixture of
1.5 mL methanol and 1 mL sample was measured as back-
ground signal (Ax0). All measurements were carried out in
triplicate. The decrease of the absorbance of the compound
mixture at 517 nm represented the capability of the samples.
The radical scavenging activity in percentage (RSA%) was
determined according to Mensor et al.,45,46 with some modi-
cations as described in the equation below:
3.6. Comprehensive analysis of the antioxidant activity
using a color grading prole
To comprehensively understand the antioxidant activities of
these BBR derivatives, we compared their activities with BBR
by comprehensive analysis of the antioxidant activities from
ABTS, DPPH, HOSC, and FRAP assays. The compounds with
superior activities were labeled with red color, the compounds
with inferior activities were labeled with green color, and the
compounds with equal activities to BBR were labeled with
white color. The darkness of the color indicated the degree of
superiority and inferiority. The nal results are presented
using the mixed color of the four colors that resulted from
ABTS, DPPH, HOSC and FRAP assays to indicate the antioxi-
dant activities.
Ax ꢀ Ax0
RSA% ¼ 1 ꢀ
ꢃ 100%
A0
3.7. The molecular electric potentials (MEPs)
3.5.3. HOSC assay. The hydroxyl radical ($OH) scavenging
effects of the synthesized compounds were examined. Genera-
tion of $OH was carried out through the Fenton reaction by
mixing H2O2 and Fe(II) with salicylic acid.47,48 The reaction
system contained 1 mL of 9 mM FeSO4 and 1 mL of 9 mM sal-
icylic acid dissolved in ethanol and 1 mL of various concen-
trations of samples. The reaction system was freshly prepared
and reacted at 37 ꢁC for 30 min prior to the detection of
absorbance at 510 nm (Ax). Two controls were applied for this
test: deionized water was set as a negative control (blank) (A0).
To avoid the disturbance of color, the reaction system without
the addition of H2O2 was set as the control (Ax0). Each value is
presented as the average of three determinations. The hydroxyl
The molecular electric potentials (MEPs) of berberine and
compound 8p were analyzed. The optimized structures are
characterized as minima by means of vibrational analysis using
ChemBio3D Ultra 14.0. Density functional theory (DFT/B3LYP)
at the 3-21G (d,p) was used to calculate the MEPs, and the
calculation was performed using the Gaussian 09W program
package with default convergence. The MEPs of compounds on
the electron density distribution were plotted with GaussView,
and were presented by a color code ranging from 0.05 to 0.143
hartree.
Conflicts of interest
There are no conicts to declare.
© 2021 The Author(s). Published by the Royal Society of Chemistry
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19 B. M. Mistry, H. S. Shin, Y. S. Keum, M. Pandurangan,
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Acknowledgements
This work was nancially supported by the Huxiang Young
Talent Support Program of Hunan Province (2018RS3005), the
Innovation-Driven Project of Central South University
(2020CX048), the Changsha Science and Technology Plan
Project (kq2005001, kq2004086), the National Natural Science
Foundation of China (81301258), the Natural Science Founda-
tion of Hunan Province (2019JJ60071, 2020JJ4680), the Shen-
ghua Yuying Project of Central South University, the
Postgraduate Innovation Project of Central South University
(2020zzts819, 2020zzts408 and 2020zzts409), and the Open-End
Fund for the Valuable and Precision Instruments of Central
South University.
25 C. S. Liu, Y. R. Zheng, Y. F. Zhang and X. Y. Long, Fitoterapia,
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