Synthesis and identification of novel berberine derivatives as potent inhibitors against TNF-α-induced nf-κb activation

Article abstract of DOI:10.3390/molecules22081257

Twenty-three new berberine (BBR) analogues defined on substituents of ring D were synthesized and evaluated for their activity for suppression of tumor necrosis factor (TNF)-α-induced nuclear factor (NF)-κB activation. Structure–activity relationship (SAR) analysis indicated that suitable tertiary/quaternary carbon substitutions at the 9-position or rigid fragment at position 10 might be beneficial for enhancing their anti-inflammatory potency. Among them, compounds 2d, 2e, 2i and 2j exhibited satisfactory inhibitory potency against NF-κB activation, with an inhibitory rate of around 90% (5 μM), much better than BBR. A preliminary mechanism study revealed that all of them could inhibit TNF-α-induced NF-κB activation via impairing IκB kinase (IKK) phosphorylation as well as cytokines interleukin (IL)-6 and IL-8 induced by TNF-α. Therefore, the results provided powerful information on further structural modifications and development of BBR derivatives into a new class of anti-inflammatory candidates for the treatment of inflammatory diseases.

Full text of DOI:10.3390/molecules22081257

molecules
Article
Synthesis and Identification of Novel Berberine
Derivatives as Potent Inhibitors against
TNF-α-Induced NF-κB Activation
Yan-Xiang Wang †, Lu Liu †, Qing-Xuan Zeng, Tian-Yun Fan, Jian-Dong Jiang, Hong-Bin Deng *
and Dan-Qing Song *
Institute of Medicinal Biotechnology, Chinese Academy of Medical Science and Peking Union Medical College,
Beijing 100050, China; wangyanxiang@imb.pumc.edu.cn (Y.-X.W.); liulu208@163.com (L.L.);
zqx50810793@163.com (Q.-X.Z.); fty1668@163.com (T.-Y.F.); jiang.jdong@163.com (J.-D.J.)
* Correspondence: hdeng@imb.pumc.edu.cn (H.-B.D.); songdanqingsdq@hotmail.com (D.-Q.S.);
Tel.: +86-10-6316-9876 (H.-B.D.); +86-10-6316-5268 (D.-Q.S.)
† These authors made equal contribution to this work.
Received: 27 June 2017; Accepted: 25 July 2017; Published: 27 July 2017
Abstract: Twenty-three new berberine (BBR) analogues defined on substituents of ring D were
synthesized and evaluated for their activity for suppression of tumor necrosis factor (TNF)-
nuclear factor (NF)- B activation. Structure–activity relationship (SAR) analysis indicated that
suitable tertiary/quaternary carbon substitutions at the 9-position or rigid fragment at position 10
might be beneficial for enhancing their anti-inflammatory potency. Among them, compounds 2d 2e
2i and 2j exhibited satisfactory inhibitory potency against NF- B activation, with an inhibitory rate
of around 90% (5 M), much better than BBR. A preliminary mechanism study revealed that all of
them could inhibit TNF- -induced NF- B activation via impairing I B kinase (IKK) phosphorylation
as well as cytokines interleukin (IL)-6 and IL-8 induced by TNF- . Therefore, the results provided
α-induced
κ
,
,
κ
µ
α
κ
κ
α
powerful information on further structural modifications and development of BBR derivatives into
a new class of anti-inflammatory candidates for the treatment of inflammatory diseases.
Keywords: berberine; anti-inflammatory; NF-κB; IκB kinase; structure-activity relationship
1. Introduction
Inflammation represents a response to tissue injury induced by a wide variety of stimuli, such as
pathogens, damaged cells, or irritants, and is a protective response involving immune cells, blood vessels,
and molecular mediators [
processes including arthritis, atherosclerosis, the metabolic syndrome, sepsis, and cancer. For most
of these conditions, no satisfactory treatment is available [ ]. Initial stages of inflammation involve
1–4]. As a consequence, diverse pathological conditions involve inflammatory
5,6
cytokine-mediated activation of the vascular endothelium leading to adhesion and transmigration
of leukocytes into the site of inflammation. Many of the pro-inflammatory processes elicited at the
endothelium and leukocytes are mediated by the transcription factor nuclear factor (NF)-
prevalent inducers of the NF- B signaling pathway are cytokines such as tumor necrosis factor (TNF)-
and interleukin (IL)-1, various mitogens, and bacterial components such as lipopolysaccharides (LPS).
NF- B is the name used for a family of homodimers and heterodimers, the NF- B dimers are
maintained in an inactive state in the cytoplasm bound to the inhibitor of B (I B) proteins, of which
the prototypical member is I B- ]. Upon a pro-inflammatory signal, such as binding of TNF- to
its membrane receptor, I B- B kinase (IKK). The IKK
becomes phosphorylated at Ser32/Ser36 by I
is a multi-subunit kinase complex, most typically composed of IKK-
κB. The most
κ
α
κ
κ
κ
κ
κ
α
[
7
α
κ
α
κ
α
and IKK-β and two molecules
of IKK
γ
/NF-kappa-B essential modulator (NEMO) [8]. The IKK-catalyzed phosphorylation triggers
Molecules 2017, 22, 1257; doi:10.3390/molecules22081257
www.mdpi.com/journal/molecules

Molecules 2017, 22, 1257
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degradation of I
it regulates gene expression [
inhibition of different mediators of the NF-
κ
B-
α
leading to the release of NF-
κ
B followed by its translocation to the nucleus where
9
]. As NF-
κ
B is a key regulator of many pro-inflammatory responses,
B signaling pathway, including IKK, has emerged as
κ
a promising approach for the development of anti-inflammatory candidates.
Natural isoquinoline alkaloid berberine (BBR, Figure 1), as a nonprescription anti-diarrhea drug,
has been extensively used in China for decades with a confirmed safety. Recently, several other
pharmacological and biological properties of BBR including anti-inflammatory and anti-carcinogenic
activities have been identified [10–16]. It was reported that BBR could suppress NF-κB activation
induced by various inflammatory agents or carcinogens with a mild potency [17]. In our study,
BBR’s moderate anti-inflammatory potency was further confirmed by an inhibitory rate of 36% at the
concentration of 5 µM. The unique isoquinoline skeleton of BBR provoked us to conduct structural
modification and optimization to enhance the anti-inflammatory effect. Therefore, in our present
study, taking BBR as the lead, a series of novel BBR analogues defined on the substituents of ring D
(Figure 1) was achieved to elucidate the structure-activity relationship (SAR) and develop a novel
class of anti-inflammatory agents. Specifically, novel ester, amide, and sulfonate BBR derivatives were
prepared and evaluated for their effect to inhibit TNF-
α-induced NF-κB activation, and the mechanism
exploration of the key compounds was carried out as well.
Figure 1. Chemical structures of berberine (BBR), and its structure modification strategy.
2. Results and Discussion
2.1. Chemistry
Firstly, taking commercial available BBR as the starting material, a demethylation reaction was
conducted to afford the key intermediate (Scheme 1) [18]. The esters 2a and sulfonates 3a were
obtained by esterification and sulfonation of compound with various acyl chloride and sulfonyl
chloride, respectively, using acetonitrile as solvent and triethylamine as the base. Compounds
and were prepared in the presence of corresponding amino compounds [19]. After acidification,
1
–k
–f
1
4
7
compound 6 was obtained taking pyridine as the base.
Scheme 1. Reagents and conditions: (a) 195 ◦C, 30–40 mmHg, 60 min; (b) R1COX/R1SO2Cl,
triethylamine, CH3CN, reflux; (c) 2,4-Dimethoxybenzylamine, 120 ◦C; (d) HCl, CH3OH; (e) R2COX,
◦
pyridine, CH2Cl2, reflux; (f) R-NH2, 120 C.

Molecules 2017, 22, 1257
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Secondly, compounds 16a and 16b were synthesized through a seven-step process (Scheme 2),
using commercially available 2,3-dihydroxybenzaldehyde ( ) as the starting materials with the
methods reported previously [20 21]. Condensation was conducted between 2-methoxy-3-hydroxy
benzaldehyde ( ) and homopiperonylamine (10) after selective methylation of . In the intermolecular
8
,
9
8
cyclization of the intermediate 12, the skeleton formed according to Pictet-Spengler cyclization and
Friedel-Crafts alkylation rule in one step, and, subsequently, the key intermediate 13 was obtained
with an ideal yield of 53%. After acidification, alkalization, and esterification, the final products 16a
and 16b were purified via flash column chromatography using methanol/dichloromethane as the
gradient eluent.
◦
Scheme 2. Reagents and conditions: (a) CH3I, room temperature (r.t.); (b) 100 C, 8 h; (c) NaBH4,
◦
methanol, reflux, 5 h; (d) glyoxal, formic acid, CuSO4, HCl, 100 C, 5 h; (e) methanol/H2O, CaO, r.t.,
2 h; (f) ethanol, HCl, r.t., 0.5 h; (g) RCOX, triethylamine, CH3CN, reflux.
2.2. Biology
2.2.1. SAR for Suppressing TNF-α-induced NF-κB Activation
All the newly synthesized compounds were examined in 293T cells for their anti-inflammatory
activities. Given the key role of NF-
their anti-inflammatory abilities by using TNF-
Structures of the analogues and their inhibitory rates on TNF-
activity are shown in Table 1.
κ
B signaling in inflammation, we investigated these analogues for
-induced NF- B-responsive promoter reporter assays.
-induced NF- B-responsive promoter
α
κ
α
κ
SAR analysis was first focused on the influence of substitutions on position 9 of ring D,
by which eleven new ester derivatives (2a ) were prepared and tested taking PS1145 as the
–
k
positive control [22]. Different kinds of cyclic carboxylic acid were introduced on 9-hydroxyl by
which cyclopropanecarboxylate (2a), cyclopentanecarboxylate (2b), 2-cyclopentylacetate (2c), and
1-methylcyclohexane-1-carboxylate (2d) derivatives were examined for their abilities to inhibit NF-
activation. An inspiring potency with the inhibitory rate of 96% was given by compound 2d while
compounds 2a only gave comparable inhibitory rates (34–56%) to BBR (36%). Then, two bridged-ring
derivatives (2e
effect on NF-
κB
–
c
,
f) were created; norbornane substituted compound 2e exhibited an improved inhibitory
κ
B activation with the rate of 83%, while the activity of chlorinated adamantane
compound 2f was only comparable to that of BBR. Finally, three alkyl ring-opening analogues
bearing tertiary carbon and quaternary carbon substitutions (2g ) were prepared and tested, and
–
i
they gave the improved activities with inhibitory rates of 81–96%. Based on the results above, it was
speculated that the introduction of an ester group bearing a suitable tertiary/quaternary carbon
substitution at the 9-position was beneficial for the activity. Moreover, the activity was retained when
a benzene or unsaturated heterocycle ring (2j,k) was inserted between the ester bond and quaternary
carbon substituent.

Molecules 2017, 22, 1257
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Table 1. Structures and inhibition effect of target compounds on tumor necrosis factor (TNF)-induced
nuclear factor (NF)-κB activities.
Inhibitory
Rate%
Inhibitory
Rate%
Inhibitory
Rate%
No.
R
No.
2i
R
No.
3f
R
2a
56
96
48
2b
2c
34
52
2j
88
72
4
5
43
52
2k
H
2d
96
3a
65
6
43
2e
2f
83
39
3b
3c
86
85
7
37
96
16a
2g
2h
81
92
3d
3e
87
82
16b
84
BBR
-
-
36
73
PS1145
Meanwhile, 6 sulfonate derivatives (3a
Compounds 3a , possessing substituted benzenesulfonate, were tested, and they could suppress
NF- B expression by 65–87%. It seemed that trifluoromethyl was a more favorable substitution than
a nitro group for the ability to inhibit TNF- -induced NF-
B activation. Then, 100-camphorsulfonyl
analogues 3e with different chiral configurations were generated, and apparently, compound 3e
–f) were designed to explore further SAR on position 9.
–
d
κ
α
κ
,f
with a D-configuration showed obviously higher activity than its entantiomer 3f, which indicated the
possible effect of chiral configuration.
Then, converting the sulfonyl linker to an amine or amide linker, and the generated
compounds 4–7 did not show improved activities compared to the lead BBR, and the result might hint
that amine and amide were not suitable to be applied as linkers for the inhibitory activity.
Next, the SAR study was conducted for the substituents on the 10-position of ring D. Introducing
adamantate at position 10 in BBR, compounds 16a and 16b gave satisfactory potencies with inhibitory
rates of 96% and 84%, respectively, which indicated that rigid structure on position 10 might be also
favorable for the ability to inhibit TNF-α-induced NF-κB activation.

Molecules 2017, 22, 1257
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Based on the preliminary SAR analysis, the IC50 of representative compounds 2d
inhibition of TNF- -induced NF-
the novel BBR derivatives on cell viability, the cytotoxic effects of representative compounds 2d
and 2j on 293T cells were determined by MTT assay. Our results revealed that 2d 2e 2i and 2j failed
to affect cell viability for 24 h at concentrations up to 20 M (Figure 2). These data demonstrated that
,
2e,
2i and 2j on
α
κ
B activation were tested, as listed in Table 2. To evaluate the effect of
2e 2i
,
,
,
,
µ
2d, 2e, 2i and 2j within 20 µM has little cytotoxic effects on 293T cells.
Table 2. The IC50 of compounds 2d, 2e, 2i and 2j on inhibition of TNF-α-induced NF-κB activation.
Compound
2d
2e
2i
2j
PS1145
IC50 (ìM)
1.01 ± 0.19
3.91 ± 1.04
0.55 ± 0.43
10.13 ± 1.40
2.493 ± 0.23
Figure 2. Cytotoxic effects of compounds 2d
, 2e, 2i and 2j on 293T cells. Following pretreatment with
compounds 2d 2e 2i and 2j at the indicated concentrations for 24 h, the cell viability of 293T cells were
,
,
determined by MTT assay.
2.2.2. Preliminary Mechanism Study
Nine key compounds (2d
investigate the preliminary mechanism of NF-
role in TNF- -induced NF- B activation, the experiment was carried out to verify if the 9 compounds
suppressed NF- B activation through the IKK pathway. The translocation of NF- B to the nucleus
is preceded by the phosphorylation, ubiquitination, and proteolytic degradation of I 23].
To determine whether inhibition of TNF- -induced NF- B activation was due to inhibition of I
degradation through IKK, we pretreated 293T cells with our representative compounds and then
exposed them to TNF- for 2 h. We then examined the cells for IKK phosphorylation with antibodies
specific for phospho-IKKα (Ser180), phosphor-IKKβ (Ser181), and IκBα degradation by Western blot.
As shown in Figure 3, TNF- induced IKK phosphorylation continued to increase at 120 min
but had no effect on compounds 2d 2e 2h 2j and 16a-pretreated cells, this was not the result of
the variation of IKK expression, as the total amount of IKK protein remained unchanged during the
–
j,
3b and 16a) with different types of structure were selected to
κ
B inactivation. Considering that IKK plays a critical
α
κ
κ
κ
κBα [
α
κ
κBα
α
α
α/β
,
,
, 2i,

Molecules 2017, 22, 1257
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treatment (Figure 3). In addition, compounds 2d
,
2e
,
2i and 2j delayed TNF-
α
-induced degradation of
I
κ
B
α
(Figure 3). Moreover, 2d
expression of cytokines including IL-6 and IL-8, as depicted in Figure 4. These results demonstrated
that compounds 2d 2e 2i and 2j inhibited both TNF- -induced IKK phosphorylation and I
, 2e, 2i, 2j, as well as the negative control 2f inhibited TNF-α-induced
,
,
α
κBα
degradation, indicating these compounds had great potential as anti-inflammatory agents. IKK serves
as a key node in inflammatory signaling, inhibition of which may represent a novel therapeutic
target for inflammatory diseases such as rheumatoid arthritis and atherosclerosis. Identification of
compounds 2d, 2e, 2i and 2j as novel IKK inhibitors will allow us to better define the potential of using
BBR derivatives for the treatment of inflammatory diseases.
Figure 3. BBR analogues inhibited TNF-
phosphorylation. Following pretreatment with BBR analogs 2d
were treated with TNF- (20 ng/mL) for 30 min. The phosphorylation of IKK
expression levels of IKK and I were measured by Western blot using corresponding antibodies.
GADPH = glyceraldehyde-3-phosphate dehydrogenase.
α
-induced NF-
κ
B activation by impairing I
3b and 16a (10 M) for 2 h, 293T cells
(Ser180/181) and
κB kinase (IKK)
–
j
,
µ
α
α/β
α
κBα
Figure 4. Effects of BBR analogues on TNF-
α
-induced cytokine of interleukin (IL)-6 and IL-8. Following
M) for 2 h, 293T cells were treated with TNF-
pretreatment with compounds 2d 2e 2f 2i and 2j (10
,
,
,
µ
α
(20 ng/mL) for 6 h, and then the medium was collected for ELISA analysis.
3. Materials and Methods
3.1. Apparatus, Materials, and Analysis Reagents
Antibodies against I
dehydrogenase (GADPH) were purchased from Cell Signaling Technology (Danvers, MA, USA). TNF-
was obtained from R&D Systems (Minneapolis, MN, USA). The p5 nuclear factor (NF)- B-luciferase
κBα, IKKα, phospho-IKKα/β (Ser180/181), and glyceraldehyde-3-phosphate
α
×
κ
reporter plasmid, the pRL-TK plasmid and Dual-luciferase reporter assay system were from Promega
(Madison, WI, USA).
Melting point (mp) was obtained with CXM-300 melting point apparatus and was uncorrected.
The 1H-NMR spectra was performed on a Varian Inova 500 or 600 MHz spectrometer (Varian, San
Francisco, CA, USA) and 13C-NMR on a Bruker Avance III 500 or 600 spectrometer in Dimethyl

Molecules 2017, 22, 1257
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sulfoxide (DMSO)-d6, or CDCl3, with Me4Si as the internal standard. Electrospray ionization (ESI)
high-resolution mass spectra (HRMS) were recorded on an Autospec UItima-TOF mass spectrometer
(Micromass UK Ltd., Manchester, UK). Flash chromatography was performed on a Combiflash Rf 200
(Teledyne, NE, USA), particle size 0.038 mm.
3.2. Synthesis
3.2.1. General Procedure for the Synthesis of 2a–k and 3a–f
BBR (3.71 g, 10 mmol) was heated at 195–210 ◦C for 10–15 min under vacuum (30–40 mmHg)
to afford the black oil, which was acidified with ethanol/concentrated HCl (95:5). The solvent was
removed by evaporation and the residue was collected and then purified by flash chromatography
over silica gel using CH2Cl2/CH3OH as the gradient eluent, giving the title compound
1
(2.85 g, 80%)
as an orange solid.
To a stirred solution of
1 (100 mg, 0.28 mmol) in anhydrous CH3CN, triethylamine (175 µL,
◦
1.26 mmol) was added and heated to 70 C. Then the RCOX/RSO2Cl (1.1–1.2 eq) was added and
stirred for 5–6 h. The mixture was cooled to precipitate completely, filtrated and washed with CH2Cl2
to afford target compounds 2a–k and 3a–f.
2,3-Methylenedioxy-9-((cyclopropanecarbonyl)oxy)-10-methoxy protoberberine chloride (2a). Compound
(100 mg, 0.28 mmol) was treated with cyclopropanecarbonyl chloride (28 L, 0.31 mmol) according to
the general procedure to give the desired product 2a as a yellow solid, yield: 34%; M.p.: 193–195 ◦C
(Dec.); 1H-NMR (500 MHz, DMSO-d6)
9.89 (s, 1H, CHarom), 9.08 (s, 1H, CHarom), 8.29 (d, J = 9.2 Hz,
1
µ
δ
1H, CHarom), 8.21 (d, J = 9.2 Hz, 1H, CHarom), 7.82 (s, 1H, CHarom), 7.11 (s, 1H, CHarom), 6.19 (s, 2H,
OCH2O), 4.98 (t, J = 6.4 Hz, 2H, CH2), 4.04 (s, 3H, OCH3), 3.22 (t, J = 6.4 Hz, 2H, CH2), 2.13 (tt, J = 7.8,
4.7 Hz, 1H, CH), 1.26–1.16 (m, 4H, 2
×
CH2); 13C-NMR (126 MHz, DMSO-d6)
δ 172.3, 151.0, 150.6, 148.3,
144.9, 138.7, 134.2, 133.5, 131.5, 127.3, 126.5, 121.8, 121.3, 121.0, 109.1, 106.2, 102.8, 57.9, 56.7, 26.8, 13.4,
10.4(2); HRMS: calcd. for C23H20NO5Cl [M − Cl]+ 390.1336, found 390.1342.
2,3-Methylenedioxy-9-((cyclopentanecarbonyl)oxy)-10-methoxy protoberberine chloride (2b). Compound
(100 mg, 0.28 mmol) was treated with cyclopentanecarbonyl chloride (38 L, 0.31 mmol) according to
the general procedure to give the desired product 2b as a yellow solid, yield: 32%; M.p.: 207–209 ◦C
(Dec.); 1H-NMR (500 MHz, DMSO-d6)
9.89 (s, 1H, CHarom), 9.07 (s, 1H, CHarom), 8.29 (d, J = 9.2 Hz,
1
µ
δ
1H, CHarom), 8.21 (d, J = 9.2 Hz, 1H, CHarom), 7.83 (s, 1H, CHarom), 7.11 (s, 1H, CHarom), 6.19 (s, 2H,
OCH2O), 4.96 (t, J = 6.3 Hz, 2H, CH2), 4.03 (s, 3H, OCH3), 3.47–3.38 (m, 1H, CH), 3.23 (t, J = 6.3 Hz, 2H,
CH2), 2.14–2.00 (m, 4H, 2
×
CH2), 1.79–1.64 (m, 4H, 2
×
CH2); 13C-NMR (126 MHz, DMSO-d6)
δ 174.1,
150.9, 150.6, 148.3, 145.1, 138.7, 134.5, 133.5, 131.5, 127.2, 126.5, 121.8, 121.2, 121.0, 109.1, 106.2, 102.8, 57.9,
56.0, 43.6, 30.1(2), 26.8, 26.1(2); HRMS: calcd. for C25H24NO5Cl [M − Cl]+ 418.1649, found 418.1653.
0
2,3-Methylenedioxy-9-(2 -cyclopentylacetoxy)-10-methoxy protoberberine chloride (2c). Compound
1
(100 mg,
0.28 mmol) was treated with 2-cyclopentylacetyl chloride (42
µ
L, 0.31 mmol) according to the general
◦
procedure to give the desired product 2c as a yellow solid, yield: 38%; M.p.: 186–188 C (Dec.);
1H-NMR (500 MHz, DMSO-d6)
δ 9.94 (s, 1H, CHarom), 9.06 (s, 1H, CHarom), 8.29 (d, J = 9.2 Hz, 1H,
CHarom), 8.21 (d, J = 9.2 Hz, 1H, CHarom), 7.82 (s, 1H, CHarom), 7.11 (s, 1H, CHarom), 6.19 (s, 2H,
OCH2O), 4.95 (t, J = 6.3 Hz, 2H, CH2), 4.03 (s, 3H, OCH3), 3.23 (t, J = 6.3 Hz, 2H, CH2), 2.88 (d,
J = 7.3 Hz, 2H, CH2), 2.41–2.30 (m, 1H, CH), 1.96–1.88 (m, 2H, CH2), 1.74–1.63 (m, 2H, CH2), 1.63–1.53
(m, 2H, CH2), 1.40–1.27 (m, 2H, CH2); 13C-NMR (126 MHz, CDCl3)
δ 171.5, 151.3, 151.0, 148.7, 147.3,
138.3, 136.3, 133.1, 131.0, 125.9, 125.5, 122.5, 120.2, 119.7, 108.9, 105.3, 102.4, 57.2, 55.3, 40.5, 36.7, 32.6(2),
27.8, 25.4(2); HRMS: calcd. for C26H26NO5Cl [M − Cl]+ 432.1805, found 432.1808.
2,3-Methylenedioxy-9-((10-methylcyclohexane-10-carbonyl)oxy)-10-methoxy protoberberine chloride (2d).
Compound
1 (100 mg, 0.28 mmol) was treated with 1-methyl-1-cyclohexanecarboxylic acid (48 mg,
0.34 mmol) according to the general procedure to give the desired product 2d as a yellow solid, yield:
◦
1
39%; M.p.: 215–217 C (Dec.); H-NMR (500 MHz, DMSO-d6)
δ 9.42 (s, 1H, CHarom), 9.09 (s, 1H,

Molecules 2017, 22, 1257
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CHarom), 8.30 (d, J = 9.2 Hz, 1H, CHarom), 8.22 (d, J = 9.2 Hz, 1H, CHarom), 7.84 (s, 1H, CHarom), 7.12 (s,
1H, CHarom), 6.19 (s, 2H, OCH2O), 4.96 (t, J = 6.2 Hz, 2H, CH2), 4.03 (s, 3H, OCH3), 3.21 (t, J = 6.2 Hz, 2H,
CH2), 2.24–2.15 (m, 2H, CH2), 1.70–1.34 (m, 11H, 4
δ 175.1, 150.8, 150.6, 148.4, 144.4, 138.7, 134.4, 133.7, 131.6, 127.3, 126.6, 121.5, 121.4, 121.0, 109.1, 106.2,
102.8, 57.8, 56.5, 44.0, 35.6(2), 26.8, 26.3, 25.9, 23.1(2); HRMS: calcd. for C27H28NO5Cl [M
446.1962, found 446.1962.
×
CH2 and CH3); 13C-NMR (126 MHz, DMSO-d6)
−
Cl]+
2,3-Methylenedioxy-90-(2-(bicyclo[2.2.1]heptan-2-yl)acetoxy)-10-methoxy protoberberine chloride
Compound (100 mg, 0.28 mmol) was treated with 2-norbornaneacetic acid (49 L, 0.34 mmol)
(2e).
1
µ
according to the general procedure to give the desired product 2e as a yellow solid, yield: 24%; M.p.:
189–191 ◦C (Dec.); 1H-NMR (500 MHz, DMSO-d6) δ 10.01–9.94 (m, 1H, CHarom), 9.07 (s, 1H, CHarom),
8.29 (d, J = 9.2 Hz, 1H, CHarom), 8.21 (d, J = 9.2 Hz, 1H, CHarom), 7.82 (s, 1H, CHarom), 7.11 (s, 1H,
CHarom), 6.19 (s, 2H, OCH2O), 4.96 (t, J = 6.3 Hz, 2H, CH2), 4.03 (s, 3H, OCH3), 3.23 (t, J = 6.3 Hz,
2H, CH2), 2.83 (dd, J = 16.0, 7.5 Hz, 1H, CH2), 2.72 (dd, J = 16.0, 7.5 Hz, 1H, CH2), 2.29–2.24 (m, 1H,
CH), 2.18–2.14 (m, 1H, CH2), 2.05–1.97 (m, 1H, CH2), 1.62–1.44 (m, 4H, 2
×
CH2), 1.29–1.12 (m, 4H,
2
×
CH2); 13C-NMR (126 MHz, DMSO-d6)
δ
170.5, 151.0, 150.6, 148.3, 145.1, 138.7, 134.2, 133.5, 131.5,
127.3, 126.5, 121.8, 121.2, 121.0, 109.1, 106.2, 102.8, 57.8, 55.9, 41.2, 40.8, 38.6, 38.0, 36.9, 35.5, 30.1, 28.9,
26.8; HRMS: calcd. for C28H28NO5Cl [M − Cl]+ 458.1962, found 458.1963.
2,3-Methylenedioxy-9-((30-chloroadamantane-10-carbonyl)oxy)-10-methoxy protoberberine chloride (2f).
Compound
1 (100 mg, 0.28 mmol) was treated with 3-chloroadamantane-1-carboxylic acid (73 mg,
0.34 mmol) according to the general procedure to give the desired product 2f as a brown solid, yield:
◦
1
33%; M.p.: 206–208 C (Dec.); H-NMR (500 MHz, DMSO-d6)
δ 9.63 (s, 1H, CHarom), 9.08 (s, 1H,
CHarom), 8.29 (d, J = 9.2 Hz, 1H, CHarom), 8.22 (d, J = 9.2 Hz, 1H, CHarom), 7.83 (s, 1H, CHarom), 7.12 (s,
1H, CHarom), 6.19 (s, 2H, OCH2O), 4.99 (t, J = 6.3 Hz, 2H, CH2), 4.03 (s, 3H, OCH3), 3.23 (t, J = 6.3 Hz,
2H, CH2), 2.56 (s, 2H CH2), 2.38–2.31 (m, 2H, CH2), 2.21–2.17 (m, 4H, 2
×
CH2), 2.17–2.08 (m, 4H,
2
×
CH2), 1.79–1.67 (m, 2H, 2 173.2, 150.6, 150.6, 148.4, 144.6,
×
CH); 13C-NMR (126 MHz, DMSO-d6)
δ
138.7, 134.3, 133.6, 131.56, 127.4, 126.5, 121.5, 121.4, 121.0, 109.1, 106.2, 102.8, 69.1, 58.0, 56.2, 48.0, 46.7(2),
45.4, 37.2(2), 34.3, 31.2(2), 26.8; HRMS: calcd. for C30H29NO5Cl2 [M − Cl]+ 518.1728, found 518.1733.
2,3-Methylenedioxy-9-pivaloyloxy-10-methoxy protoberberine chloride (2g). Compound
0.28 mmol) was treated with pivaloyl chloride (38 L, 0.31 mmol) according to the general procedure
to give the desired product 2g as a yellow solid, yield: 32%; M.p.: 205–207 C (Dec.); H-NMR
(500 MHz, DMSO-d6) 9.58 (s, 1H, CHarom), 9.09 (s, 1H, CHarom), 8.29 (d, J = 9.2 Hz, 1H, CHarom), 8.22
1 (100 mg,
µ
◦
1
δ
(d, J = 9.2 Hz, 1H, CHarom), 7.83 (s, 1H, CHarom), 7.12 (s, 1H, CHarom), 6.19 (s, 2H, OCH2O), 4.99 (t,
J = 6.3 Hz, 2H, CH2), 4.03 (s, 3H, OCH3), 3.22 (t, J = 6.3 Hz, 2H, CH2), 1.47 (s, 9H,(CH3)3); 13C-NMR
(151 MHz, CD3OD)
δ 175.7, 150.9, 150.9, 148.5, 143.4, 138.8, 134.7, 133.7, 130.6, 126.4, 125.5, 121.7,
120.7, 120.3, 107.9, 105.2, 102.3, 56.3, 56.1, 39.3, 26.6, 26.2(3); HRMS: calcd. for C24H24NO5Cl [M
406.1649, found 406.1653.
−
Cl]+
2,3-Methylenedioxy-9-((30,30-dimethylbutanoyl)oxy)-10-methoxy protoberberine chloride (2h). Compound
(100 mg, 0.28 mmol) was treated with 3,3-dimethylbutyryl chloride (43 L, 0.31 mmol) according to
the general procedure to give the desired product 2h as a yellow solid, yield: 27%; M.p.: 212–214 ◦C
(Dec.); 1H-NMR (500 MHz, DMSO-d6)
9.96 (s, 1H, CHarom), 9.07 (s, 1H, CHarom), 8.30 (d, J = 9.2 Hz, 1H,
1
µ
δ
CHarom), 8.22 (d, J = 9.2 Hz, 1H, CHarom), 7.83 (s, 1H, CHarom), 7.11 (s, 1H, CHarom), 6.19 (s, 2H, OCH2O),
4.96 (t, J = 6.3 Hz, 2H, CH2), 4.04 (s, 3H, OCH3), 3.23 (t, J = 6.3 Hz, 2H, CH2), 2.75 (s, 2H, CH2), 1.18
(s, 9H, (CH3)3); 13C-NMR (126 MHz, DMSO-d6)
δ 169.5, 151.0, 150.6, 148.3, 145.1, 138.7, 134.1, 133.6, 131.5,
127.3, 126.5, 121.8, 121.2, 121.0, 109.1, 106.2, 102.8, 57.7, 55.9, 47.6, 31.4, 30.0(3), 26.8; HRMS: calcd. for
C25H26NO5Cl [M − Cl]+ 420.1805, found 420.1813.
2,3-Methylenedioxy-9-((20-propylpentanoyl)oxy)-10-methoxy protoberberine chloride (2i). Compound
1
(100 mg, 0.28 mmol) was treated with 2-propylpentanoyl chloride (58
to the general procedure to give the desired product 2i as a yellow solid, yield: 24%; M.p.: 199–201 C
µL, 0.34 mmol) according
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Molecules 2017, 22, 1257
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(Dec.); 1H-NMR (500 MHz, DMSO-d6)
δ
9.68 (s, 1H, CHarom), 9.10 (s, 1H, CHarom), 8.30 (d, J = 9.2 Hz,
1H, CHarom), 8.23 (d, J = 9.2 Hz, 1H, CHarom), 7.83 (s, 1H, CHarom), 7.11 (s, 1H, CHarom), 6.19 (s, 2H,
OCH2O), 4.95 (t, J = 6.3 Hz, 2H, CH2), 4.02 (s, 3H, OCH3), 3.23 (t, J = 6.3 Hz, 2H, CH2), 2.95 (p, J = 6.6 Hz,
1H, CH), 1.88–1.79 (m, 2H, CH2), 1.72–1.63 (m, 2H, CH2), 1.56–1.42 (m, 4H, 2
×
CH2), 0.99 (t, J = 7.3 Hz,
6H, 2 173.4, 150.9, 150.6, 148.4, 144.6, 138.7, 134.1, 133.6,
×
CH3); 13C-NMR (126 MHz, DMSO-d6)
δ
131.5, 127.3, 126.5, 121.6, 121.3, 121.0, 109.1, 106.2, 102.8, 57.8, 56.2, 44.4, 33.6(2), 26.8, 20.3(2), 14.7(2);
HRMS: calcd. for C27H30NO5Cl [M − Cl]+ 448.2118, found 448.2128.
2,3-Methylenedioxy-9-((p-tert-butylbenzoyl)oxy)-10-methoxy protoberberine chloride (2j). Compound
(100 mg, 0.28 mmol) was treated with 4-tert-butylbenzoyl chloride (61 L, 0.31 mmol) according
to the general procedure to give the desired product 2j as a brown solid, yield: 28%; M.p.: 205–207 ◦C
(Dec.); 1H-NMR (500 MHz, DMSO-d6)
10.00 (s, 1H, CHarom), 9.11 (s, 1H, CHarom), 8.35 (d, J = 9.2 Hz,
1H, , CHarom), 8.27 (d, J = 9.2 Hz, 1H, CHarom), 8.20 (d, J = 8.5 Hz, 2H, 2 CHarom), 7.85 (s, 1H, CHarom),
7.72 (d, J = 8.5 Hz, 2H, 2 CHarom), 7.10 (s, 1H, CHarom), 6.19 (s, 2H, OCH2O), 4.91 (t, J = 6.3 Hz,
2H, CH2), 4.03 (s, 3H, OCH3), 3.20 (t, J = 6.3 Hz, 2H, CH2), 1.38 (s, 9H, (CH3)3); 13C-NMR (126 MHz,
DMSO-d6) 164.0, 158.6, 151.1, 150.6, 148.4, 145.1, 138.8, 134.3, 133.6, 131.5(2), 131.1, 127.5, 126.6(2),
1
µ
δ
×
×
δ
126.5, 125.9, 122.0, 121.3, 121.0, 109.1, 106.2, 102.8, 57.9, 55.8, 35.8, 31.5(3), 26.8; HRMS: calcd. for
C30H28NO5Cl [M − Cl]+ 482.1962, found 482.1962.
2,3-Methylenedioxy-9-((30-tert-butyl-10-methyl-1H-pyrazole-50-carbonyl)oxy)-10-methoxy protoberberine
chloride (2k). Compound
carbonyl chloride (59 L, 0.34 mmol) according to the general procedure to give the desired product
2k as a yellow solid, yield: 42%; M.p.: 207–209 C (Dec.); 1H-NMR (500 MHz, DMSO-d6)
1 (100 mg, 0.28 mmol) was treated with 5-tert-butyl-2-methylpyrazole-3-
µ
◦
δ
10.03 (s, 1H,
CHarom), 9.12 (s, 1H, CHarom), 8.36 (d, J = 9.2 Hz, 1H, CHarom), 8.29 (d, J = 9.2 Hz, 1H, CHarom), 7.84 (s,
1H, CHarom), 7.15 (s, 1H, CHpyrazole), 7.11 (s, 1H, CHarom), 6.19 (s, 2H, OCH2O), 4.92 (t, J = 6.4 Hz,
2H, CH2), 4.13 (s, 3H, NCH3), 4.06 (s, 3H, OCH3), 3.22 (t, J = 6.4 Hz, 2H, CH2), 1.34 (s, 9H, (CH3)3);
13C-NMR (126 MHz, DMSO-d6)
δ 160.5, 157.2, 151.1, 150.7, 148.4, 145.1, 138.9, 133.6, 133.1, 131.5, 131.2,
127.9, 126.5, 121.8, 121.3, 121.0, 109.6, 109.1, 106.2, 102.8, 58.0, 55.9, 40.0, 32.5, 31.0(3), 26.8; HRMS: calcd.
for C28H28N3O5Cl [M − Cl]+ 486.2023, found 486.2027.
2,3-Methylenedioxy-9-((p-nitrophenylsulfonyl)oxy)-10-methoxy protoberberine chloride (3a). Compound
1
(100 mg, 0.28 mmol) was treated with 4-nitrobenzenesulfonyl chloride (75 mg, 0.34 mmol) according to
the general procedure to give the desired product 3a as an orange solid, yield: 31%; M.p.: 146–148 ◦C
(Dec.); 1H-NMR (500 MHz, DMSO-d6)
δ
9.69 (s, 1H, CHarom), 9.14 (s, 1H, CHarom), 8.52 (d, J = 8.6 Hz,
CHarom), 8.30 (d, J = 9.3 Hz, 1H, CHarom), 8.27 (d, J = 8.6 Hz, 2H, 2 CHarom), 8.22 (d, J = 9.3 Hz,
1H, CHarom), 7.83 (s, 1H, CHarom), 7.13 (s, 1H, CHarom), 6.20 (s, 2H, OCH2O), 4.98 (t, J = 6.3 Hz, 2H,
CH2), 3.69 (s, 3H, OCH3), 3.22 (t, J = 6.3 Hz, 2H, CH2); 13C-NMR (126 MHz, DMSO-d6)
151.8, 151.8,
2H, 2
×
×
δ
150.7, 148.2, 144.2, 140.4, 139.3, 133.9, 131.6, 131.1, 130.8(2), 129.3, 126.5, 125.4(2), 122.1, 121.5, 120.6, 108.9,
106.1, 102.7, 57.3, 56.1, 26.6; HRMS: calcd. for C25H19N2O8SCl [M − Cl]+ 507.0856, found 507.0860.
2,3-Methylenedioxy-9-((p-trifluoromethylphenyl sulfonyl)oxy)-10-methoxy protoberberine chloride (3b).
Compound
(83 mg, 0.34 mmol) according to the general procedure to give the desired product 3b as a yellow solid,
yield: 41%; M.p.: 190–191 ◦C (Dec.); 1H-NMR (500 MHz, DMSO-d6)
9.70 (s, 1H, CHarom), 9.17 (s, 1H,
CHarom), 8.30 (d, J = 9.2 Hz, 1H, CHarom), 8.24–8.20 (m, 4H, 4 CHarom), 8.15 (d, J = 8.5 Hz, 2H, 2
CHarom), 7.84 (s, 1H, CHarom), 7.13 (s, 1H, CHarom), 6.20 (s, 2H, OCH2O), 4.98 (t, J = 6.3 Hz, 2H, CH2),
3.23 (t, J = 6.3 Hz, 2H, CH2); 13C-NMR (126 MHz, DMSO-d6)
152.0, 150.9, 148.4, 144.5, 139.5, 139.2,
1 (100 mg, 0.28 mmol) was treated with 4-(trifluoromethyl)benzene-1-sulfonyl chloride
δ
×
×
δ
135.3, 134.1, 131.7, 131.3, 130.4(2), 129.5, 127.6(2), 126.6, 123.8, 122.3, 121.7, 120.8, 109.1, 106.3, 102.9,
57.4, 56.3, 26.8; HRMS: calcd. for C26H19F3NO6SCl [M − Cl]+ 530.0879, found 530.0884.
2,3-Methylenedioxy-9-((m-trifluoromethylphenylsulfonyl)oxy)-10-methoxy protoberberine chloride (3c).
Compound
1 (100 mg, 0.28 mmol) was treated with 3-(trifluoromethyl)benzenesulfonyl chloride
(55 L, 0.34 mmol) according to the general procedure to give the desired product 3c as an orange
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Molecules 2017, 22, 1257
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solid, yield: 37%; M.p.: 184–186 ◦C (Dec.); 1H-NMR (500 MHz, DMSO-d6)
δ
9.71 (s, 1H, CHarom), 9.18
(s, 1H, CHarom), 8.31–8.26 (m, 2H, 2
1H, CHarom), 8.07–8.03 (m, 1H, CHarom), 7.99–7.94 (m, 1H, CHarom), 7.13 (s, 1H, CHarom), 6.20 (s, 2H,
OCH2O), 5.04 (t, J = 6.4 Hz, 2H, CH2), 3.46 (s, 3H, OCH3), 3.24 (t, J = 6.4 Hz, 2H, CH2); 13C-NMR
×
CHarom), 8.17 (d, J = 9.4 Hz, 1H, CHarom), 8.13 (t, J = 7.7 Hz,
(126 MHz, DMSO-d6)
δ 151.5, 150.7, 148.2, 144.2, 139.3, 136.4, 134.0, 133.8, 133.9, 133.0, 131.6, 131.4,
129.7, 129.3, 127.8, 126.3, 122.9, 122.4, 121.6, 120.6, 108.9, 106.1, 102.7, 57.2, 56.2, 26.6; HRMS: calcd. for
C26H19F3NO6SCl [M − Cl]+ 530.0879, found 530.0883.
2,3-Methylenedioxy-9-((o-trifluoromethylphenylsulfonyl)oxy)-10-methoxy protoberberine chloride
Compound (100 mg, 0.28 mmol) was treated with 2-(trifluoromethyl)benzenesulfonyl chloride
(52 L, 0.34 mmol) according to the general procedure to give the desired product 3d as an orange
solid, yield: 38%; M.p.: 183–185 ◦C (Dec.); 1H-NMR (500 MHz, DMSO-d6)
9.73 (s, 1H, CHarom), 9.17
(s, 1H, CHarom), 8.36–8.29 (m, 3H, 3 CHarom), 8.24 (d, J = 1.8 Hz, 1H, CHarom), 8.23 (d, J = 9.4 Hz,
(3d).
1
µ
δ
×
1H, CHarom), 8.01 (t, J = 7.9 Hz, 1H, CHarom), 7.84 (s, 1H, CHarom), 7.13 (s, 1H, CHarom), 6.20 (s, 2H,
OCH2O), 4.98 (t, J = 6.3 Hz, 2H, CH2), 3.66 (s, 3H, OCH3), 3.23 (t, J = 6.3 Hz, 2H, CH2); 13C-NMR
(126 MHz, DMSO-d6)
δ 151.8, 150.7, 148.2, 144.4, 139.2, 136.3, 134.0, 133.2, 132.8, 132.0, 131.5, 131.1,
130.7, 129.3, 126.3, 125.6, 123.6, 122.2, 121.5, 120.6, 108.9, 106.1, 102.7, 57.3, 56.1, 26.6; HRMS: calcd. for
C26H19F3NO6SCl [M − Cl]+ 530.0879, found 530.0880.
2,3-Methylenedioxy-9-((D-(+)-100-camphorsulfonyl)oxy)-10-methoxy
protoberberine
chloride
(3e).
Compound
1 (100 mg, 0.28 mmol) was treated with D(+)-10-camphorsulfonyl chloride (85 mg,
0.34mmol) according◦to the general procedure to give the desired product 3e as a yellow solid, yield:
1
20%; M.p.: 193–195 C (Dec.); H-NMR (500 MHz, DMSO-d6)
δ
9.77 (s, 1H, CHarom), 9.14 (s, 1H,
CHarom), 8.37 (d, J = 9.2 Hz, 1H, CHarom), 8.29 (d, J = 9.2 Hz, 1H, CHarom), 7.84 (s, 1H, CHarom), 7.12
(s, 1H, CHarom), 6.20 (s, 2H, OCH2O), 5.01 (t, J = 6.4 Hz, 2H, CH2), 4.20 (d, J = 15.0 Hz, 1H ,COCH2),
4.13 (s, 3H, OCH2), 3.98 (d, J = 15.0 Hz, 1H, COCH2), 3.23 (t, J = 6.4 Hz, 2H, CH2), 2.48–2.40 (m, 1H,
CH2), 2.39–2.30 (m, 1H, CH2), 2.14 (t, J = 4.5 Hz, 1H, CH), 2.07–1.98 (m, 2H, CH2), 1.73–1.64 (m, 1H,
CH2), 1.53–1.44 (m, 1H, CH2), 1.10 (s, 3H, CH3), 0.91 (s, 3H, CH3); 13C-NMR (126 MHz, DMSO-d6)
δ
214.2, 152.3, 150.8, 148.4, 144.8, 139.1, 134.0, 132.1, 131.7, 128.6, 127.1, 122.5, 121.5, 120.8, 109.1, 106.2,
102.8, 58.5, 58.1, 56.4, 50.6, 48.8, 42.9, 42.6, 26.9, 26.8, 25.9, 20.0, 19.9; HRMS: calcd. for C29H30NO7SCl
[M − Cl]+ 536.1737, found 536.1745.
0
2,3-Methylenedioxy-9-((L-(
−
)-10 -camphorsulfonyl)oxy)-10-methoxy protoberberine chloride (3f). Compound
1
(100 mg, 0.28 mmol) was treated with L(
to the general procedure to give the desired product 3f as a yellow solid, yield: 21%; M.p.: 201–203 ◦C
(Dec.); 1H-NMR (500 MHz, DMSO-d6)
9.76 (s, 1H, CHarom), 9.13 (s, 1H, CHarom), 8.37 (d, J = 9.2 Hz,
−
)-10-camphorsulfonyl chloride (85 mg, 0.34 mmol) according
δ
1H, CHarom), 8.29 (d, J = 9.2 Hz, 1H, CHarom), 7.84 (s, 1H, CHarom), 7.12 (s, 1H, CHarom), 6.20 (s, 2H,
OCH2O), 5.01 (t, J = 6.4 Hz, 2H, CH2), 4.20 (d, J = 15.0 Hz, 1H, COCH2), 3.97 (d, J = 15.0 Hz, 1H, COCH2),
3.23 (t, J = 6.4 Hz, 2H, CH2), 2.47–2.40 (m, 1H, CH2), 2.38–2.31 (m, 1H, CH2), 2.14 (t, J = 4.5 Hz, 1H,
CH), 2.06–1.98 (m, 2H, CH2), 1.73–1.64 (m, 1H, CH2), 1.53–1.46 (m, 1H, CH2), 1.10 (s, 3H), 0.91 (s, 3H);
13C-NMR (126 MHz, DMSO-d6)
δ 214.2, 152.3, 150.8, 148.4, 144.8, 139.3, 134.0, 132.1, 131.7, 128.6, 127.9,
122.5, 121.5, 120.8, 109.1, 106.2, 102.8, 58.5, 58.1, 56.4, 50.6, 48.8, 42.9, 42.6, 26.9, 26.8, 25.9, 20.0, 19.9;
HRMS: calcd. for C29H30NO7SCl [M − Cl]+ 536.1737, found 536.1742.
3.2.2. Synthesis of 2,3-Methylenedioxy-9-(o,p-dimethoxybenzyl amino)-10-methoxy Protoberberine
Chloride (4)
To a stirred solution of BBR (7.4 g, 20 mmol) in 2,4-dimethoxybenzylamine (15 mL, 78 mmol) at
120 ◦C for 6–8 h. The mixture was cooled to room temperature and washed with acetone (3
×
50 mL)
to remove the remaining amine. The reside was purified by flash chromatography over silica gel using
CH2Cl2/CH3OH (96.5:3.5) as the gradient eluent to afford red solid
, yield: 37%; M.p.: 239–240 ◦C
(Dec.); 1H-NMR (500 MHz, DMSO-d6)
9.98 (s, 1H, CHarom), 8.73 (s, 1H, CHarom), 7.88 (d, J = 8.8, 1H,
CHarom), 7.77 (s, 1H, CHarom), 7.52 (d, J = 8.8 Hz, 1H, CHarom), 7.14 (d, J = 8.3 Hz, 1H, CHarom), 7.09
4
δ

Molecules 2017, 22, 1257
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(s, 1H, CHarom), 6.52 (d, J = 2.2 Hz, 1H, CHarom), 6.44 (t, J = 6.5 Hz, 1H, NH), 6.42–6.39 (m, 1H, CHarom),
6.17 (s, 2H, OCH2O), 4.80 (t, J = 6.2 Hz, 2H ,NCH2), 4.66 (d, J = 6.3 Hz, 2H, CH2), 3.87 (s, 3H, OCH3),
3.77 (s, 3H, OCH3), 3.71 (s, 3H, OCH3), 3.21 (t, J = 6.3 Hz, 2H, CH2); 13C-NMR (126 MHz, DMSO-d6)
δ
160.5, 158.5, 150.1, 148.2, 148.1, 147.1, 137.5, 136.3, 133.5, 130.9, 130.3, 124.8, 121.2, 120.5, 120.3, 118.0,
117.7, 109.1, 105.9, 104.8, 102.6, 98.9, 57.5, 56.0, 55.8, 55.6, 47.0, 27.3; HRMS: calcd. for C28H27N2O5Cl
[M − Cl]+ 471.1914, found 471.1919.
3.2.3. Synthesis of 2,3-Methylenedioxy-9-amino-10-methoxy Protoberberine Chloride (5)
Compound 4 (3 g, 6.4 mmol) was dissolved in CH3OH, and hydrochloric acid 3 mL was added.
The mixture was stir◦red for 5–6 h, filtered, and washed with 80% ethanol to afford red solid
5, yield:
1
80%; M.p.: 212–214 C (Dec.); H-NMR (500 MHz, DMSO-d6)
δ
10.19 (s, 1H, CHarom), 8.64 (s, 1H,
CHarom), 7.84 (d, J = 8.6 Hz, 1H, CHarom), 7.76 (s, 1H, CHarom), 7.32 (d, J = 8.6 Hz, 1H, CHarom),
7.08 (s, 1H, CHarom), 6.89 (s, 2H, NH2), 6.16 (s, 2H, OCH2O), 4.70 (t, J = 6.3 Hz, 2H, CH2), 3.98 (s, 3H,
OCH3), 3.20 (t, J = 6.3 Hz, 2H, CH2); 13C-NMR (126 MHz, DMSO-d6)
δ 149.8, 148.0, 147.0, 143.9, 138.1,
135.4, 132.1, 130.4, 123.0, 121.2, 119.8, 113.7, 113.3, 108.9, 105.6, 102.4, 56.9, 55.1, 27.2; HRMS: calcd. for
C19H17N2O3Cl [M − Cl]+ 321.1233, found 321.1235.
3.2.4. Synthesis of 2,3-Methylenedioxy-9-(20-propylpentanamido)-10-methoxy Protoberberine
Chloride (6)
To a stirred solution of
CH2Cl2 (5 mL), the 2-propylpentanoyl chloride (143
h. The solvent was removed by evaporation and purified by flash chromatography over silica gel using
CH2Cl2/CH3OH (95:5) as the gradient eluent to give yellow solid
, yield: 34%; M.p.: 249–251 ◦C
(Dec.); 1H-NMR (600 MHz, DMSO-d6)
10.04 (s, 1H, CHarom), 9.26 (s, 1H, CHarom), 8.99 (s, 1H, CHarom),
5
(100 mg, 0.28 mmol) and pyridine (100
µL, 1.24 mmol) in anhydrous
µL, 0.84 mmol) was added and refluxed for 10–12
6
δ
8.25–8.13 (m, 2H, CHarom), 7.79 (s, 1H, CHarom), 7.09 (s, 1H, CHarom), 6.15 (s, 2H, CHarom), 4.87 (t,
J = 6.4 Hz, 2H, OCH2O), 3.99 (s, 3H, OCH3), 3.19 (t, J = 6.4 Hz, 2H, CH2), 2.67 (s, 1H, CH), 1.70–1.59 (m,
2H, CH2), 1.48–1.36 (m, 6H, 3
176.0, 154.8, 150.3, 148.1, 146.1, 137.8, 133.7, 131.0, 127.8, 125.8, 124.2, 122.3, 121.2, 120.8, 108.9, 106.0,
102.5, 57.4, 56.4, 45.6, 34.9(2), 26.8, 20.6(2), 14.6(2); HRMS: calcd. for C27H31N2O4Cl [M
found 447.2284.
×
CH2), 0.99 – 0.90 (m, 6H, 2
×
CH3); 13C-NMR (151 MHz, DMSO-d6)
δ
−
Cl]+ 447.2278,
3.2.5. Synthesis of 2,3-Methylenedioxy-9-(20-ethylhexyl amino)-10-methoxy Protoberberine
Chloride (7)
To a stirred solution of BBR (2.5 g, 6.7 mmol) in 2,4-dimethoxybenzylamine (5 mL, 26 mmol) at
120 ◦C for 6–8 h. The mixture was cooled to room temperature and washed with acetone (3
to remove the remaining amine. The reside was purified by flash chromatography over silica gel using
×
20 mL)
◦
CH2Cl2/CH3OH (95:5) as the gradient eluent to afford red solid
7
, yield: 41%; M.p.: 219–211 C (Dec.);
1H-NMR (500 MHz, DMSO-d6)
δ
10.29 (s, 1H, CHarom), 8.69 (s, 1H, CHarom), 7.88 (d, J = 8.7 Hz, 1H,
CHarom), 7.76 (s, 1H, CHarom), 7.44 (d, J = 8.7 Hz, 1H, CHarom), 7.07 (s, 1H, CHarom), 6.54 (t, J = 6.0 Hz,
1H, NH), 6.16 (s, 2H, OCH2O), 4.82 (t, J = 6.3 Hz, 2H, CH2), 3.95 (s, 3H, OCH3), 3.50 (t, J = 6.4 Hz,
2H, NCH2), 3.20 (t, J = 6.3 Hz, 2H, CH2), 1.70–1.62 (m, 1H, CH), 1.45–1.15 (m, 7H, 2
×
CH2 and CH3),
0.89–0.80 (m, 7H, 2 150.1, 148.2, 147.0, 146.9, 138.1,
×
CH2 and CH3); 13C-NMR (126 MHz, DMSO-d6)
δ
136.1, 133.6, 130.8, 124.9, 121.2, 120.2, 117.2, 116.2, 109.0, 105.8, 102.6, 57.4, 55.2, 51.0, 40.4, 30.9, 28.9,
27.3, 24.2, 23.2, 14.6, 11.3; HRMS: calcd. for C27H33N2O3Cl [M − Cl]+ 433.2485, found 433.2489.
3.2.6. Synthesis of 2,3-Methylenedioxy-9-methoxy-10-(adamantane-10-carbonyl)oxy Protoberberine
Chloride (16a)
Compound 15 was obtained according to the reported procedure as an orange solid [18].
Compound 15 (100 mg, 0.28 mmol) was treated with 1-adamantanecarbonyl chloride (61 mg,
0.31 mmol) according to the general procedure to give the desired product 16a as a brown solid,

Molecules 2017, 22, 1257
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yield: 27%; M.p.: 144–146 ◦C (Dec.); 1H-NMR (500 MHz, DMSO-d6)
δ
10.03 (s, 1H, CHarom), 9.07 (s,
1H, CHarom), 7.98 (d, J = 9.1 Hz, 1H, CHarom), 7.96 (d, J = 9.1 Hz, 1H, CHarom), 7.86 (s, 1H, CHarom),
7.12 (s, 1H, CHarom), 6.20 (s, 2H, OCH2O), 4.97 (t, J = 6.3 Hz, 2H, CH2), 4.10 (s, 3H, OCH3), 3.23 (t,
J = 6.3 Hz, 2H, CH2), 2.14–2.07 (m, 9H, 3
×
CH and 3
×
CH2), 1.80–1.74 (m, 6H, 3
×
CH2); 13C-NMR
(126 MHz, DMSO-d6) 175.5, 151.0, 148.9, 148.4, 146.9, 141.6, 140.5, 138.0, 135.4, 132.0, 123.8, 122.2, 121.0,
δ
120.8, 109.1, 106.4, 102.9, 63.8, 55.8, 41.3, 38.8(3), 36.4(3), 27.9(3), 26.8; HRMS: calcd. for C30H30NO5Cl
[M − Cl]+ 484.2118, found 484.2122.
3.2.7. Synthesis of 2,3-Methylenedioxy-9-methoxy-10-(20-(adamantan-1-yl)acetoxy) Protoberberine
Chloride (16b)
Compound 15 (100 mg, 0.28 mmol) was treated with 2-(1-adamantyl)acetyl chloride (66 mg,
0.31 mmol) according to the general procedure to give the desired product 16b as an orange solid,
yield: 28%; M.p.: 141–143 ◦C (Dec.); 1H-NMR (500 MHz, CDCl3)
δ 10.85 (s, 1H, CHarom), 8.32 (s, 1H,
CHarom), 7.78 (d, J = 8.8 Hz, 1H, CHarom), 7.74 (d, J = 8.8 Hz, 1H, CHarom), 7.39 (s, 1H, CHarom), 6.85 (s,
1H, CHarom), 6.12 (s, 2H, OCH2O), 5.42 (t, J = 6.2 Hz, 2H, CH2), 4.38 (s, 3H, OCH3), 3.32 (d, J = 6.2 Hz,
2H, COCH2), 2.45 (s, 2H, CH2), 2.09–2.04 (m, 3H, 3
×
CH), 1.79 (d, J = 2.8 Hz, 6H, 3
×
CH2), 1.78–1.70
(m, 6H, 3
×
CH2); 13C-NMR (126 MHz, CDCl3)
δ 169.4 151.4, 150.0, 148.7, 148.3, 141.6, 140.2, 137.7,
135.3, 131.5, 122.8, 122.1, 120.0, 119.5, 108.9, 105.5, 102.5, 64.2, 55.9, 48.5, 42.5(3), 36.8(3), 33.6, 28.7(3),
27.7; HRMS: calcd. for C31H32NO5Cl [M − Cl]+ 498.2275, found 498.2275.
3.3. Biology Assay
3.3.1. Cell Culture and NF-κB Luciferase Reporter Gene Assay
The 293T cell line was purchased from the American Type Culture Collection (ATCC, VA, USA).
Cells were cultured in Dulbecco’s Modified Eagle’s medium (DMEM) (Gibico, NY, USA) supplemented
with 10% fetal bovine serum (Hyclone, UT, USA), 100 U/mL penicillin, and 100
sulfate and incubated at 37 ◦C in a humidified atmosphere with 5% CO2.
µg/mL streptomycin
The 293T cells were co-transfected with pNF-
using the Vigofect transfection reagent (Vigorous Biotechnology, Beijing, China) as instructed by the
manufacturers [23]. After 24 h of transfection, cells were pretreated with 5 M of BBR analogues for 2 h
and then stimulated with TNF- (20 ng/mL) for 6 h. Following TNF- treatment, the cells were lysed,
κB-Lucreporter plasmid plus the pRL-TK plasmid
µ
α
α
and the luciferase activity was determined using the Dual-luciferase reporter assay system (Promega,
Madison, CA, USA) according to the manufacture0s protocols. The luciferase activity values were
normalized to the expression of the Renilla luciferase and presented as the percentages of luciferase
activity measured without TNF-α or BBR analogues treatment.
3.3.2. Western Blot
Western blot was performed as described previously. Briefly, cells were washed with
phosphate-buffered saline (PBS) and were lysed in lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM
NaCl, 10 mM
NaF, 1 mM Na3VO4, 0.5% Triton X-100, and 1 mM dithiothreitol (DTT)) supplemented with protease
inhibitors (1 mM phenylmethylsulfonyl fluoride (PMSF), 5 g/mL leupeptin, 5 g/mL pepstatin A,
and 5 g/mL aprotinin). Proteins were separated by SDS-PAGE and were electrically transferred
β-glycerophosphate, 5 mM egtazic acid (EGTA), 1 mM sodium pyrophosphate, 5 mM
µ
µ
µ
to a polyvinylidene difluoride membrane. The membranes were blocked for one hour at room
temperature in TBS (150 mM NaCl, 20 Mm Tris, pH 7.5, 0.05% Tween-20) containing 5% milk and
probed with specific first antibodies for one hour at room temperature. Blots were then incubated with
horseradish peroxidase (HRP)-conjugated anti-rabbit (7074, Cell Signaling) or anti-mouse (7076, Cell
Signaling) secondary antibody. Immunoreactive proteins were visualized using the ECL detection
system (Bio-Rad, Hercules, CA, USA).

Molecules 2017, 22, 1257
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3.3.3. Determination of Cytokines Production
The 293T cells cultured in 24-well plates were pretreated with compounds 2d
,
2e
,
2i and 2j
for 2 h and then stimulated with TNF-α (20 ng/mL) for 6 h. Levels of IL-6 and IL-8 in the culture
media were quantified using ELISA kits (R&D Systems Inc., MN, USA) in accordance with the
manufacturer’s instructions.
3.3.4. Cell Survival Assay
The cell survival was evaluated by MTT assay. Briefly, Cell suspensions (100
at concentration of 50% confluence were seeded into the 96-well plates, and then were treated with
various concentrations of BBR derivate. After 24 h of incubation, 10 L of the MTT (1 mg/mL)
solution was added into each plate and incubated for 2 h at 37 ◦C, 5% CO2. Subsequently, the culture
supernatant was replaced with 100 L DMSO to dissolve the formazan crystal made from succinic
µL) of 293T cells
µ
µ
dehydrogenase in the mitochondria and its substrate MTT. The optical density (OD) at 550 and 630 nm
were measured using a microplate reader. The net absorbance (OD630–OD550) indicates the enzymatic
activity of mitochondria and provides information on cell viability.
4. Conclusions
To conclude, 23 new BBR analogues defined on substituents of the ring D were synthesized and
evaluated for their effect to inhibit TNF-
tertiary or quaternary carbon substitutions on position 9 or rigid fragment on position 10 might be
beneficial for the activity. Among them, compounds 2d 2e 2i and 2j exhibited satisfactory potency
with inhibitory rates of over 90% at the concentration of 5 M compared with that of BBR. A preliminary
mechanism study revealed that all of them could inhibit TNF- -induced NF- B activation via impairing
IKK phosphorylation as well as TNF- induced expression of cytokines including IL-6, and IL-8. Our
current study supports the potential role of compounds 2d 2e 2i and 2j in the prevention and
α-induced NF-κB activation. SAR analysis indicated that
,
,
µ
α
κ
α
,
,
treatment of inflammatory diseases, and they have been selected for the further investigation.
Acknowledgments: This work was supported by the National Natural Science Foundation of China (81321004
and 81473248) and CAMS initiative for innovative medicine 2016-12M-1-011.
Author Contributions: Y.-X.W. performed part of synthetic experiments and wrote the paper, L.L. performed the
biological assay, Q.-X.Z. and T.-Y.F. performed part of synthetic experiments, J.-D.J., H.-B.D. and D.-Q.S. conceived
and designed the experiments.
Conflicts of Interest: The authors declare no conflict of interest.
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2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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