Synthesis and structure-activity relationships of a series of aporphine derivatives with antiarrhythmic activities and acute toxicity

Article abstract of DOI:10.3390/molecules21121555

Some aporphine alkaloids, such as crebanine, were found to present arrhythmic activity and also higher toxicity. A series of derivatives were synthesized by using three kinds of aporphine alkaloids (crebanine, isocorydine, and stephanine) as lead compound

Full text of DOI:10.3390/molecules21121555

molecules
Article
Synthesis and Structure-Activity Relationships of a
Series of Aporphine Derivatives with Antiarrhythmic
Activities and Acute Toxicity
Hui Wang, Xin Cheng, Shujun Kong, Zixian Yang, Hongmei Wang, Qiuyan Huang, Jingyu Li,
Cheng Chen and Yunshu Ma *
Department of Pharmaceutics Science, School of Chinese Materia Medica, Yunnan University of Traditional
Chinese Medicine, 1076#, Yuhua road, Chenggong, Kunming 650000, China; huiwang2016@sina.cn (H.W.);
chengxin920@126.com (X.C.); kongshujun1@126.com (S.K.); yangzixian2@126.com (Z.Y.);
15198906044@163.com (H.W.); 15912165160@163.com (Q.H.); lijingyu06@163.com (J.L.);
guoke10141@163.com (C.C.)
* Correspondence: yunshuma2@126.com; Tel./Fax: +86-871-6591-8230
Academic Editor: Maria Emília de Sousa
Received: 18 October 2016; Accepted: 7 November 2016; Published: 28 November 2016
Abstract: Some aporphine alkaloids, such as crebanine, were found to present arrhythmic
activity and also higher toxicity. A series of derivatives were synthesized by using three kinds
of aporphine alkaloids (crebanine, isocorydine, and stephanine) as lead compounds. Chemical
methods, including ring-opening reaction, bromination, methylation, acetylation, quaternization,
and dehydrogenation, were adopted. Nineteen target derivatives were evaluated for their
antiarrhythmic potential in the mouse model of ventricular fibrillation (VF), induced by CHCl3,
and five of the derivatives were investigated further in the rat model of arrhythmia, induced
by BaCl2. Meanwhile, preliminary structure-activity/toxicity relationship analyses were carried
out. Significantly, N-acetamidesecocrebanine (1d), three bromo-substituted products of crebanine
(2a, 2b, 2c), N-methylcrebanine (2d), and dehydrostephanine (4a) displayed antiarrhythmic effects
in the CHCl3-induced model. Among them, 7.5 mg/kg of 2b was able to significantly reduce
the incidence of VF induced by CHCl3 (p < 0.05), increase the number of rats that resumed sinus
rhythm from arrhythmia, induced by BaCl2 (p < 0.01), and the number of rats that maintained sinus
rhythm for more than 20 min (p < 0.01). Therefore, 2b showed remarkably higher antiarrhythmic
activity and a lower toxicity (LD50 = 59.62 mg/kg, mice), simultaneously, indicating that 2b could
be considered as a promising candidate in the treatment of arrhythmia. Structural-activity analysis
suggested that variationsin antiarrhythmic efficacy and toxicity of aporphines were related to the
C-1,C-2-methylenedioxy group on ring A, restricted ring B structural conformation, N-quaternization
of ring B, levoduction of 6a in ring C, and the 8-, 9-, 10-methoxy groups on ring D on the skeleton.
Keywords: aporphine derivatives; antiarrhythmia; 10,11-dibromocrebanine; 3-bromocrebanine;
crebanine; stephanine
1. Introduction
Although radio frequency ablation and other new techniques have been applied in clinical
therapy for the treatment of cardiac arrhythmia, drug therapy is still an important and essential
method of treatment. According to current methodology of the Vaughan Williams’ electrophysiological
classification, available anti-arrhythmic drugs are divided into four categories: Na+ channel blockers
(category I),
blockers (category IV) [
β
-receptor blockers (category II), K+ channel blockers (category III), and Ca+ channel
]. Unfortunately, many of the drugs that belong to these four categories have
1
also shown unavoidable side-effects, such as cardiac arrhythmia [2]. However, evidence shows that
Molecules 2016, 21, 1555; doi:10.3390/molecules21121555
www.mdpi.com/journal/molecules

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the new category III drugs, working through multiple ion (Ikr, Iks, Ito, INa) channels, are likely to
become the most prospective candidates for the treatment of arrhythmia, due to their higher efficiency,
as well as their lower risk of side-effects [3]. Therefore, searching for new components from natural
products and their derivatives that act on multiple ion channels seems to be one of the more important
approaches in the development of new antiarrhythmic drugs [4].
In the last 30 years, several aprophine-type compounds, such as isocorydine, L-dicentrine, and
crebanine, extracted from plants in the subgenus Tuberiphania Lo et M. Yang and the genus Stephania,
Menispermaceae, have been found possessing remarkable antiarrhythmic activities [5–7]. Based on
traditional medical records, some species from this subgenus have been used as medicinal herbs for
a long time in Southwest China, with medical functions such as pain-relief, heat-clearing, detoxing,
and activating blood circulation [8]. Studies have shown that almost all species of this subgenus
contain various types of isoquinoline alkaloids, including aprophines, morphinans, protoberberines,
etc., which display extensive physiological activities. They show activity on the K+, Ca2+, and Na+
channel, and some of them have been reported to be blockers of 5-hydroxytryptamine and dopamine
receptors [9]. Meanwhile, isoquinoline alkaloids, for example, berberine, l-tetrahydropalmatine, and
isocorydine, have been used in clinical treatment of cardiovascular and cranial disease in China.
The functions on multiple K+, Ca2+, and Na+ ion channels that these alkaloids exhibit coincides with
the current leading trend in the screening of new category III anti-arrhythmic drugs [10–12]. In our
previous observations, crebanine, an aprophine type alkaloid, showed a higher anti-arrhythmic effect
on experimental arrhythmia models compared with isocroydine and L-dicentrine [13]; however, it was
also found that the acute toxicity of crebanine was higher than that of isocroydine and L-dicentrine.
The effective arrhythmia dosage (2.5~5.0 mg/kg) of crebanine was close to its LD50 (9.38 mg/kg) in
mice [14], which implies that crebanine has a relatively narrow therapeutic window.
The present study is a further investigation regarding the structural modification and evaluation
of the arrhythmic activities of aprophine alkaloids. The authors have attempted to find new aprophine
derivatives with higher antiarrhythmia efficiencies and lower toxicities. A series of derivatives from
three kinds of aporphine (crebanine, isocroydine, and stephanine) were designed and synthesized by
connecting specific pharmacophores to enlarge the target distribution of antiarrhythmia. At the same
time, hydrophobic groups or lipophilic groups were introduced in order to improve the lipid-water
partition coefficient and membrane permeability. The antiarrhythmic effects of the derivatives were
preliminarily evaluated in a mouse model of ventricular fibrillation (VF), induced by CHCl3, and
further confirmed in a rat model of arrhythmia, induced by BaCl2. Six derivatives were obtained, and
are reported here for the first time, with antiarrhythmic effects and the structure-activity relationships
are discussed.
2. Results and Discussion
Nineteen products, including 13 derivatives of crebanine, four derivatives of isocorydine, and
two derivatives of stephanine, were synthesized, isolated, purified, and identified (Schemes 1–5), of
which 11 are reported for the first time: Secocrebanine (1a), N-cyclopropylmethylsecocrebanine
(
1b),
N-methylsulfonamidesecocrebanine (1e), 3,10,11-Tribromocrebanine (2a), 10,11-Dibromocrebanine
2b), 3-Bromocrebanine (2c), 11-Methoxy-N-methylisocorydine (5a), 11-Methoxyisocorydine (5b),
and 11-Ethoxyisocorydine (5c).
N-trifluoroacetamidesecocrebanine
(
1c),
N-acetamidesecocrebanine
(1d),
(

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Scheme 1. Synthesis of crebanine derivatives 1a, 1b, 1c, 1d, and 1e. Reagents and conditions:
(i) 1-chloroethyl chloroformate, K2CO3, 1,2-CH2ClCH2Cl, 85 ◦C, 6 h, MeOH, 37% HCl, refluxed for
2 h; (ii) cyclopropylmethyl bromide, K2CO3, MeCN, 80 ◦C, 4 h; (iii) trifluoroacetic anhydride, Et3N,
anhydrous CH2Cl2, 2 h, r.t.; (iv) acetic anhydride, 4-dimethylaminopyridine, Et3N, anhydous CH2Cl2,
r.t., 4 h; and (v) methanesulfonyl chloride, Et3N, anhydrous CH2Cl2, r.t., 2 h.
Scheme 2. Synthesis of crebanine analogues 2a, 2b, 2c, 2d, and 2e. Reagents and conditions:
(i) N-Bromosuccinimide, trifluoroacetic acid, r.t., 18 h; (ii) ether, CH3I, MeOH, r.t., 16 h; and
◦
(iii) AlBr3, anhydrous CH2Cl2, nitrobenzene, 5 C, argon, 18 h.
Scheme 3. Synthesis of crebanine and stephanine derivatives 3a, 3b, 4a and 4b. Reagents and
conditions: (i) 10% Pd/C, MeCN, N2, refluxed for 6 h; and (ii) NaCNBH3, EtOH, 2NHCl, r.t., 18 h.

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Scheme 4. Synthesis of isocorydine derivatives 5a, 5b, 5c, and 5d. Reagents and conditions: (i)
Tetrabutylammonium bromide, K2CO3, anhydrous THF, CH3I, r.t., 8 h; (ii) N, N-diisopropylethylamine,
trimethylsilyldiazomethane, MeCN, MeOH, 15 h, r.t.; and (iii) Tetrabutylammonium bromide, K2CO3,
anhydrous THF, CH3CH2I, r.t., 8 h; and (iv) ether, CH3I, MeOH, r.t., 16 h.
Scheme 5. Synthesis of crebanine analogues 6c. Reagents and conditions: (
acid , methylbenzene, 120 C, 8 h; (ii) 5%Pd/C, MeOH, H2, 1 bar, 8 h; and (iii) K2CO3, anhydrous THF,
i
) 2,3-dimethoxy-benzoic
◦
CHI, r.t., 4 h.
The antiarrhythmic activities of the 19 products, respectively, were tested in the mouse model
of ventricular fibrillation (VF), induced by CHCl3. Six compounds, 1d
2d), and dehydrostephanine (4a), were found to possess significant antiarrhythmic effects (p < 0.05)
(Figure 1A–D). Furthermore, in the rat model of arrhythmia, induced by BaCl2, 2a 2b 2c 2d
and 4a demonstrated significant antiarrhythmia activity (Table 1). Lastly, in order to evaluate toxicity,
the LD50 of three active compounds, two bromo-substituted products of crebanine (2a 2b) and
, 2a, 2b, 2c, N-methylcrebanine
(
,
,
,
,
,
N-methylcrebanine (2d), were calculated using the sequential method in mice (Table 2) as 59.62, 65.15,
and 6.432 mg/kg, respectively.
Table 1. Antiarrhythmic activity of fiveaporphine derivatives (2a–d and 4a) in the model of arrhythmia,
induced by BaCl2 in anesthetized SD rats (iv) (n = 10).
Numbers of Rat
Maintaining
Numbers of Rat
Maintaining
Numbers of Rat
Maintaining
Dose
Numbers of Rat
Recovering
Recovery
Time (s)
Compounds
(mg/kg)
Time ≥ 3 min
Time ≥ 5 min
Time ≥ 20 min
NS
–
5
0
–
0
0
0
Lidocaine
7 **
8 **
7 **
9 **
7 **
8 **
8 **
8 **
10 **
22.2 ± 8.0
6 **
5 **
6 **
8 **
2
2
1
Crebanine
5
30.3 ± 31.8
35.0 ± 8.1
3
3
Stephanine
5
4 *
8 **
1
3
Isocorydine
10
15
7.5
7.5
3
736.4 ± 489.1
125.5 ± 145.9
113.5 ± 136.8
73.1 ± 58.2
131.9 ± 122.9
23.0 ± 11.0
4 *
0
3,10,11-Tribromocrebanine (2a)
10,11-Dibromocrebanine (2b)
3-Bromocrebanine (2c)
N-methylcrebanine (2d)
Dehydrostephanine (4a)
8 **
4 *
5 **
0
7 **
2
5 **
0
5 **
2
2
0
5
The chi-square test was used to estimate the significance between the numbers of rat recovering and the
numbers of rat maintaining time 3 min, 5 min, and 20 min in all comparisons. Significantly different from
≥
the control group, * p < 0.05, ** p < 0.01. Values of recovery time are expressed as mean
standard deviation.
±
SD. SD represents

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Figure 1.
(A–D) Antiarrthymic activity of 19 aporphine derivatives in a mouse VF model, induced
by CHCl3 (n = 10). Data represent the incidence percentage of VF in 10 experiments. Significantly
different from the control group, * p < 0.05, ** p < 0.01. 1d, 2a, 2b, 2c, 2d, and 4a possess significant
antiarrhythmic effects.
Table 2. Acute toxicity tests of three crebanine derivatives (2b, 2c, and 2d) in mice (n = 10).
Compounds
LD50 (mg/kg)
95% Confidence Intervals (CIs)
10,11-Dibromocrebanine (2b
3-Bromocrebanine (2c)
N-methylcrebanine (2d)
Crebanine [14]
)
59.62
65.15
6.43
62.23, 57.01
66.28, 64.02
7.53, 5.34
9.38
8.24, 10.53
2.1. Opening up of the N-C Bondon Ring B and N-Substitution and Antiarrhythmic Activity/Toxicity
Previously, we characterized some new type III anti-arrhythmic drugs, such as dronedarone [15],
most of which contained the N-substitution structure [3]. Therefore, we presumed that the
N-substitution in ring B of aporphine might be one of the key reasons for their anti-arrhythmic
activities. Five phenanthrene compounds were synthesized (Scheme 1) by opening up the N-C bond
(
(
1a) on ring B of crebanine, further connecting the N position with four kinds of lipophilic groups
1b 1e). The five obtained analogues were tested in the mouse model of VF, induced by CHCl3
–
(Figure 1A). The result showed that, except for compound 1d (10 mg/kg), which was able to return the
occurrence of VF to a normal sinus rhythm in 60% of tested mice (p < 0.01) (Figure 1A), the remaining
four ring B-opening derivatives (1a, 1b, 1c, and 1e) showed no significant effects on inhibiting rapid
VF in mice, even though the test dosages (10–40 mg/kg) were up to eight times higher than the dosage
of crebanine (5 mg/kg). Simultaneously, the tested mice also showed no obvious toxic symptoms.
These results indicate that antiarrhythmic efficiency and toxicity were both decreased when ring B of
crebanine was opened up. Therefore, it is speculated that a proper, closed ring B structure of aporphine
was necessary, as the flat structure of the restricted ring B structural conformation could embed into
myocardial cells more easily than the ring B-opening structure of phenanthrenes.

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2.2. The Lipophilicity/Hydrophilicity of Crebanine Derivatives and Antiarrhythmic Activity/Toxicity
Bromo-substituted derivatives often increase the lipophilicity of compounds. Bromine atoms
were first introduced into the structure of crebanine (Scheme 2) to explore the relationship between
lipophilicity and the antiarrhythmic activity/toxicity of aporphines. Compared with normal saline,
compounds 2a, 2b, and 2c, three kinds of crebanine bromoderivatives, exhibited a remarkable recovery
in mouse VF (p < 0.05) (Figure 1B). Notably, dibromide 2b of crebanine displayed better antiarrhythmic
activity than tribromide of crebanine 2a and monobromide of crebanine 2c (Table 1). After a 7.5 mg/kg
dosage of 2b was given, the number of animals that recovered, and the number of animals maintaining
sinus rhythm
≥
20 min was obviously higher than those of 2a (15 mg/kg) and 2c (7.5 mg/kg) in the rat
model of arrhythmia, induced by CHCl3 (Figure 1B). The results indicate that the different numbers
of Br replacements varied the lipid solubility of crebanine and changed the level of antiarrhythmic
efficiency. Furthermore, the LD50 of 2c (65.15 mg/kg) and 2b (59.62 mg/kg) in mice (Table 2) were
much higher than that of crebanine (LD50 9.38 mg/kg) [14], which showed that toxicity of brominated
crebanine was much lower than that of crebanine. This predicts that brominated crebanine may be a
prospective modified structure and would serve as a lead compound for antiarrhythmia.
On the other hand, N-methylation (quaternization) was introduced into the structure of
crebanine in order to determine how the improvement of hydrophilicity in aporphines impacted
the antiarrhythmic activity and toxicity. The quaternary ammonium of crebanine (2d, Scheme 2)
displayed a notable antiarrhythmic effect, even at a lower dosage of 3 mg/kg (Figure 1B), but the tested
mice showed symptoms such as polypnea, mydriasis, leaping, and tremors until death, which indicate
toxicity of the central nervous system (CNS) and the motor nervous system [16]. The LD50 of 2d was
calculated to be 6.432 mg/kg in mice (Table 2), much lower than that of crebanine (9.382 mg/kg),
which indicated that N-quaternizated aporphines could maintain anti-arrhythmic activity, but that the
toxicity was increased at the same time.
2.3. Ring D O-Demethylation and Antiarrhythmic Activity
The experiments were designed to remove the O-methyl groups in ring D of crebanine in
order to acquire more aporphine derivatives (Scheme 2). Table 3 shows all of reactions and their
derivatives [17–22]. Since ring B was unstable and easy to open using strong acids, it was difficult
to cleave the O-methyl groups in ring D when only strong acids (48%HBr/CH3COOH, 48%HBr,
and IH) were used [17–19]. Otherwise, when crebanine was treated with BBr3 in dichloromethane,
a molecular ion peak of the products was observed at m/z 300 [M + H]+, which implies that both the
O-methyl groups and methylenedioxy of crebanine had been removed simultaneously. Meanwhile,
the target product of the four phenolic hydroxyl groups was not acquired due to its high hydrophilicity
and instability. A 9-demethyl group derivative, namely stesakine (2e, Scheme 2) [20] in Table 3, was
obtained by utilizing the Lewis acids, BCl3, and AlBr3 [21,22], which indicated that the 9-methoxy
group in ring D of crebanine was less stable than the 8-methoxy group.
Table 3. O-Demethylation reaction of crebanine.
Entry
Solvent
Temperature (◦C)
Time (h)
Product
Yield (%)
1
2
3
4
5
6
7
48%HBr
48%HBr/CH3COOH
HI
BBr3/CH2Cl2
BCl3/CH2Cl2
AlBr3/CH2Cl2
AlBr3/PhNO2
120
130
100
25
25
25
3.5
4
2
–
1d
–
–
70
–
3
–
–
12
12
4
2d
2d
2d
10
10
20
40
“–” represents that no desired product was obtained.

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Compound 2e showed weak activity at a maximum dissolvable dosage (2.5 mg/kg) in the mouse
VF model (Figure 1B), thus, the 9-methoxy group in ring D might be one of the functional groups
in aporphine alkaloids. Similarly, the 8- and/or 9- or 10-methoxylation in ring D of the aporphine
skeleton, such as crebanine, stephanine, and L-isocrydine, showed notable antiarrhythmic activity.
On the contrary, the removal of the 8- or 9- or 10-methyl groups of aporphine alkaloids in ring D may
reduce antiarrhythmic activity.
2.4. Racemization of Aporphines and Antiarrhythmic Activity
Two racemic compounds of crebanine and stephanne, namely (
±
) crebanine (3b) and ( )
±
stephanine (4b), were synthesized by reduction after dehydrogenation of 6a-H (Scheme 3) [23]. Both
compounds did not significantly manifest antiarrhythmic activity in the mouse model of VF induced by
CHCl3 (Figure 1C). The results indicated that the levoduction of the 6a configuration was a functional
group for maintaining antiarrhythmic activity.
2.5. 6a-H Dehydrogenated Derivatives of Aporphines and Antiarrhythmic Activity/Toxicity
Dehydrocrebanine (3a) was derived from dehydrogenating in 6a-H of crebanine and the formation
of a double bond in ring C of crebanine (Scheme 3). 3a showed a very high toxicity in animal tests.
In order to avoid animal poisoning and death, the test dosage had to be decreased to 1 mg/kg
(Figure 1C); however, antiarrhythmic activity was not obviously observed in the CHCl3-induced
VF model either. On the other hand, stephanine and its derivative of 6a-H dehydrogenation
(dehydrostephanine, 4a) (5 mg/kg), was able to notably confront VF, caused by CHCl3, in mice
(Figure 1C). In the rat model of arrhythmia induced by BaCl2, both stephanine and 4a increased the
number of animals that resumed normal sinus rhythm (p < 0.01), and significantly shortened the time
for recovery. However, 4a was unable to maintain a sinus rhythm, even after more than 3 min post
recovery (Table 1), and most of the tested animals died during the observation period (20 min), which
implies that the effective duration of 4a was much shorter and the toxicity was high. These findings
may be explained by the fact that dehydrogenation compounds increase the rigidity and conjugacy of
the skeleton due to ring C forming a double bond (3a, 4a), which facilitated the infusion of aporphine
molecules infusing myocardial cells, and consequently producing much faster and stronger toxic
effects relative to the prototype compounds.
2.6. 11-Alkylation and Quaternization of Isocorydine
Figure 1D and Table 1 show that isocorydine (10 mg/kg) displayed lower antiarrhythmic activity
tendencies compared to crebanine (5 mg/kg) and stephanine (5 mg/kg). The main difference in these
three compounds lies on the 1,2-dimethoxy group of ring A, which exists in crebanine and stephanine,
but not in isocorydine. This increase in antiarrhythmic activity may be related to the 1,2-dimethoxy
group structure, which widens the planar configuration of molecules.
All of the modified products of isocorydine, 5a, 5b, 5c, and 5d (Scheme 4) [24,25] were inactive in
the model of VF induced by CHCl3 (Figure 1D) at appropriate test dosages. Meanwhile, 5a and 5d
were both N-methylated as quaternary ammonium compounds, and presented such a high toxicity
that the test dosages had to be reduced to 1 mg/kg (5a) and 0.8 mg/kg (5d) (Table 1) to avoid animal
death. The reactions of toxicity appeared, much like those of N-methylcrebanine (2d), in the central
nervous system and the motor nervous system. The experiments also evidenced that the substitution
of the 11-methoxy group (5b) or 11-ethoxy group (5c) of isocorydine did not have any benefits for
antiarrhythmic activity.
2.7. Design of An Analogue of Crebanine
An analogue of crebanine, compound 6c, was designed and synthesized according to the
key group of crebanine (Scheme 5), which contained two basic sites, a tertiary amine group
and a dimethoxy-benzene group. However, animal tests showed that 6c could not significantly

Molecules 2016, 21, 1555
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inhibit the occurrence of VF induced in mice. Therefore, it is necessary to design a series of
dimethoxy-phenylamine derivatives in order to seek out the potential active compound.
3. Experimental Section
3.1. Synthesis
3.1.1. General Information
Isocorydine, crebanine, and stephanine were extracted from the plant, identified as Stephania
yunnanensis Lo by Professor Yunshu Ma, Yunnan University of Traditional Chinese Medicine, and as
reported previously [26,27]. Solvents and reagents used in syntheses were of analytical grade, were
purchased from commercial sources, and used without further purification.
All reactions were carried out under an atmosphere of argon, with magnetic stirring, in flame
dried or oven-dried glassware. Analytical thin layer chromatography was performed on 0.25-mm
silica gel GF 254 plates (Qingdao Haiyang Chemical Co., Ltd., Qingdao, China). Visualization was
accomplished with UV light and bismuth potassium iodide solution staining followed by heating.
1H-NMR spectra were recorded on a 400 MHz spectrometer (Amersham Pharmacia Biotech AB Inc.,
Tokyo, Japan) in CDCl3 or CD3OD at ambient temperature, using the solvent signal as an internal
standard. Data are reported as: (br/broad, s/singlet, d/doublet, t/triplet, q/quartet, m/multiplet;
integration; coupling constant(s) in hertz). 13C-NMR spectra were recorded on a 400 MHz spectrometer
(Amersham Pharmacia Biotech AB Inc., Tokyo, Japan) in CDCl3 at ambient temperature, using the
solvent signal as an internal standard. Electrospray ionization mass spectrometry was detected
with a Brukerama Zon SL (Bruker Daltonics Inc., Leipzig, Bremen, Germany); HR-MS was detected
with 6200 series TOF/6500 series (Agilent Technologies Inc., Santa Clara, UT, USA); rotation was
detected with a WZZ-three digital automatic polarimeter (Shanghai Shengguang Technology Co., Ltd.,
Shanghai, China).
3.1.2. Preparation of Secocrebanine (1a)
A mixture of crebanine (2 g, 5.9 mmol) and K2CO3 (1.7 g, 1.2 mmol) in 1,2-dichloroethane (20 mL)
was stirred and 1-chloroethyl chloroformate (1.0 g, 7.1 mmol) was added slowly. The mixture was
heated to 85 ◦C for 6 h, the 1,2-dichloroethane-insoluble material was filtered off, and the filtrate was
concentrated to give the residue, which was dissolved in MeOH (20 mL) and five drops of 37% HCl,
and refluxed for 2 h. The solution was concentrated and neutralized with saturated Na2CO3, and
the residue was extracted with CH2Cl2. The combined extracts were dried over anhydrous Na2SO4,
◦
filtered, and concentrated to get a crude product, which was crystallized from EtOAC at 4 C, yielding
1
compound 1a (1.2 g, 60%) as a pink-white solid. H-NMR (400 MHZ, CDCl3,
δ
; ppm) 8.83 (d, J = 9.2 HZ,
1H, 5-H), 7.93 (d, J = 9.6 HZ, 1H, 10-H), 7.87 (d, J = 9.6 HZ, 1H, 9-H), 7.29 (d, J = 8.8 HZ, 1H, 6-H), 7.10
(s, 1H, 2-H), 6.22 (s, 2H, -OCH2O-), 4.02 (s, 3H, 8-OCH3), 4.00 (s, 3H, 9-OCH3), 3.28–3.24 (t, J = 7.2 HZ,
2H, -CH2α), 2.97–2.93 (t, J = 7.2 HZ, 2H, -CH2β), 2.47 (s, 3H, -NCH3); 13C-NMR (100 MHZ, CDCl3,
δ;
ppm) 33.87 (t, -CH2β), 36.43 (q, N-CH3), 53.12 (t, -CH2α), 56.30 (q, 7-OCH3), 61.32 (q, 8-OCH3), 100.95
(t, -OCH2O-), 110.05 (d, C-2), 112.60 (d, C-6), 117.11 (s, C-8a), 118.38 (d, C-5), 123.26 (s, C-4a), 123.64 (d,
C-9), 123.79 (d, C-10), 125.19 (s, C-1a), 127.41 (s, C-5a), 130.88 (s, C-1), 141.76 (s, C-4), 143.23 (s, C-7),
144,98 (s, C-3), 149.96 (s, C-8), Positive ESI-MS m/z: 340.2 [M + H]+. HR-MS for C20H21NO4 [M + H]+;
calcd. 340.1543, found: 340.1542.
3.1.3. Preparation of N-Cyclopropylmethylsecocrebanine (1b)
Product 1a (200 mg, 0.59 mmol), K2CO3 (163 mg, 1.18 mmol), and cyclopropylmethyl bromide
(96 mg, 0.71 mmol) were added to MeCN (20 mL) and heated at 80 ◦C for 4 h. The reaction mixture
was filtered and concentrated to give a residue, which was purified by silica column chromatography
1
(CHCl3/MeOH 80:1), yielding compound 1b (180 mg, 78%) as a white solid. H-NMR (400 MHZ,

Molecules 2016, 21, 1555
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CDCl3, δ; ppm) 8.81(d, J = 9.2 HZ, 1H, 5-H), 8.04 (d, J = 9.6 HZ, 1H, 10-H), 7.93 (d, J = 9.2 HZ, 1H, 9-H),
7.31 (d, J = 8.4 HZ, 1H, 6-H), 7.16 (s, 1H, 2-H), 6.23 (s, 2H, -OCH2O-), 4.02 (s, 3H, 8-OCH3), 3.99 (s, 3H,
9-OCH3), 3.84–3.78 (m, 1H, CH2α), 3.66–3.60 (m, 1H, -CH2β), 3.47–3.42 (m, 1H, -CH2β), 3.18–3.15 (m,
2H, N-CH2), 2.97 (s, 3H, -NCH3), 2.93–2.89 (m, 1H, CH2α), 1.30–1.25 (m, 1H, -CH), 0.85–0.82 (m, 2H,
-CH2), 0.50–0.47 (m, 1H, -CH2). .13C-NMR (100 MHZ, CDCl3,
δ; ppm) 100.62 (t, -OCH2O-), 52.82 (t,
-CH2β), 29.59 (t, CH2α), 29.26 (t, -CH2), 29.05 (t, -CH2), 61.00 (q, 8-OCH3), 56.02 (q, 8-OCH3), 43.97 (q,
N-CH3), 115.77 (d, C-5), 114.88 (d, C-6),62.40(d, -CH), 152.55 (s, C-8), 145.17 (s, C-3), 145.02 (s, C-7),
141.99 (s, C-4), 133.19 (s, C-1),130.69 (s, C-5a), 125.73(s, C-1a), 124.66 (s, C-4a), 114.88(s, C-8a). Positive
ESI-MS m/z: 394.2 [M + H]+. HR-MS for C24H27NO4 [M + H]+; calcd. 394.2013, found: 394.2026.
3.1.4. Preparation of N-Trifluoroacetamidesecocrebanine (1c)
A solution of 1a (200 mg, 0.59 mmol) and Et3N (72 mg, 0.71 mmol) in anhydrous CH2Cl2
(30 mL), trifluoroacetic anhydride (149 mg, 0.71 mmol) was slowly added under an ice-bath, then, the
solution was stirred for 2 h at room temperature. The mixture was diluted with water (20 mL) and
extracted with CH2Cl2 (3
×
10 mL). The combined extracts were dried over anhydrous Na2SO4, filtered
and concentrated to give a crude product, which was purified by silica gel column chromatography
(CH2Cl2/MeOH 100:1), and yielding compound 1c (210 mg, 82%) as a white solid. H-NMR (400 MHZ,
1
CDCl3, δ; ppm) 8.83 (d, J = 9.2 HZ, 1H, 5-H), 7.99 (d, J = 9.2 HZ, 1H, 10-H), 7.94 (d, J = 9.6 HZ, 1H, 9-H),
7.31 (d, J = 9.2 HZ, 1H, 6-H), 7.07 (s, 1H, 2-H), 6.24 (s, 1H, -OCH2O-), 6.23 (s, 1H, -OCH2O-), 4.03 (s,
3H, 8-OCH3), 4.00 (s, 3H, 9-OCH3), 3.73–3.69 (t, J = 7.4 HZ, 2H, -CH2α), 3.36–3.32 (t, J = 7.2 HZ, 2H,
-CH2β), 3.11 (s, 1H, -NCH3), 2.99 (s, 2H, -NCH3). 13C-NMR (100 MHZ, CDCl3,
δ; ppm) 30.67 (t, CH2α),
36.04 (q, N-CH3), 51.79 (t, -CH2β ), 56.32 (q, 7-OCH3), 61.33(q, 8-OCH3), 101.21 (t, -OCH2O-), 110.13 (d,
C-2), 112.91 (d, C-6), 117.15 (s, C-8a), 119.12 (s, C-4a), 119.28 (d, C-5), 122.08 (s, CF3), 123.77 (d, C-9),
123.81 (d, C-10), 125.39 (C-1a), 127.47 (C-5a), 128.83 (s, C-1), 142.27 (s, C-4), 143.32 (s, C-7), 145.08 (s,
C-3), 150.15 (s, C-8). Positive ESI-MS m/z: 458.2 [M + Na]+. HR-MS for C22H20F3NO5 [M + Na]+; calcd.
458.1186, found: 458.1188.
3.1.5. Preparation of N-Acetamidesecocrebanine (1d)
To a stirred solution of 1a (200 mg, 0.59 mmol), 4-dimethylaminopyridine (144 mg, 1.18 mmol),
and Et3N (72 mg, 0.71 mmol) in anhydrous CH2Cl2 (20 mL), acetic anhydride (72 mg, 0.71mmol) was
slowly added and stirred for 4 h at room temperature. The mixture was diluted with water (10 mL) and
extracted with CH2Cl2 (3
×
10mL). The combined extracts were dried over anhydrous Na2SO4, filtered,
and concentrated to get a crude product, which was purified by silica gel column chromatography
(CH2Cl2/MeOH 100:1), yielding compound 1d (180 mg, 80%) as a white solid. H-NMR (400 MHZ,
1
CDCl3, δ; ppm) 8.83 (d, J = 8.4 HZ, 1H, 5-H), 8.00 (d, J = 10.0 HZ, 1H, 10-H) 7.96 (d, J = 9.6 HZ, 1H,
9-H), 7.32 (d, J = 9.2 HZ, 1H, 6-H), 7.09 (s, 1H, 2-H), 6.23 (s, 1H, -OCH2O-), 6.22 (s, 1H, -OCH2O-), 4.03
(s, 6H, 8-OCH3), 4.00 (s, 6H, 9-OCH3), 3.67–3.61 (dd, J = 15.6 HZ, J = 8.0 HZ, 2H, -CH2α), 3.31–3.27
(t, J = 7.4 HZ, 2H, -CH2β), 3.00 (s, 1H, -NCH3), 2.81 (s, 2H, -NCH3), 2.09 (s, 2H, -NOCH3), 1.85 (s,
1H, -NOCH3). 13C-NMR (100 MHZ, CDCl3,
δ; ppm) 22.03 (q, -NOCH3), 32.57 (t, -CH2α), 37.39 (q,
N-CH3), 51.99 (t, -CH2β), 56.33 (q, 7-OCH3), 61.32 (q, 8-OCH3), 100.08 (t, -OCH2O-), 110.28 (d, C-2),
112.88 (d, C-6), 117.17 (s, C-8a), 119.10 (d, C-5),123.76 (d, C-9), 123.81 (d, C-10), 125.55 (s, C-4a), 127.50
(C-1a), 128.72 (C-5a), 130.31 (s, C-1), 142.30 (s, C-4), 143.28 (s, C-7), 145.14 (s, C-3), 150.11 (s, C-8), 170.69
(s, -C=O).Positive ESI-MS m/z: 404.2 [M + Na]+.HR-MS for C22H23NO5 [M + Na]+; calcd. 404.1468,
found: 404.1474.
3.1.6. Preparation of N-Methylsulfonamidesecocrebanine (1e)
To a mixture of 1a (200 mg, 0.59 mmol) and Et3N (72 mg, 0.71 mmol) in anhydrous CH2Cl2
(30 mL) under an ice-bath, methanesulfonyl chloride (81 mg, 0.71 mmol) was slowly added and stirred
for 2 h at room temperature. The mixture was diluted with water (20 mL) and extracted with CH2Cl2
(3
×
10 mL). The combined extracts were dried over anhydrous Na2SO4, filtered, and concentrated to

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give a crude product, which was purified by silica gel column chromatography (CH2Cl2/MeOH 100:1),
1
yielding 1e (210 mg, 82%) as a white solid. H-NMR (400 MHZ, CDCl3,
δ; ppm) 8.83 (d, J = 9.2 HZ,
1H, 5-H), 7.98 (d, J = 9.6 HZ, 1H, 10-H), 7.85 (d, J = 9.6 HZ, 1H, 9-H), 7.31(d, J = 9.2 HZ, 1H, 6-H),
7.11 (s, 1H, 2-H), 6.23 (s, 2H, -OCH2O-), 4.03 (s, 3H, 8-OCH3), 4.00 (s, 3H, 9-OCH3), 3.46–3.42 (m, 2H,
-CH2α), 3.37–3.34 (m, 2H, -CH2β), 2.92 (s, 3H, -NCH3), 2.74 (s, 3H, -NSO2CH3). 13C-NMR (100 MHZ,
CDCl3, δ; ppm) 33.26 (t, -CH2α), 35.38 (t, -NSO2CH3), 51.56 (t, -CH2β), 56.31 (q, 7-OCH3), 61.34 (q,
8-OCH3), 101.10 (t, -OCH2O-), 110.30 (d, C-2), 112.76 (d, C-6),117.13 (s, C-8a), 119.06 (d, C-5),122.72 (d,
C-9),123.77 (d, C-10),125.21 (s, C-4a),127.40 (C-1a), 128.88 (s, C-1), 142.20 (s, C-4),143.29 (s, C-7), 145.07
(s, C-3),150.07 (s, C-8). Positive ESI-MS m/z: 440.2 [M + Na]+. HR-MS for C21H23NO6S [M + Na]+;
calcd. 440.1138, found: 440.1140.
3.1.7. Preparation of 3,10,11-Tribromocrebanine (2a), 10,11-Dibromocrebanine (2b) and
3-Bromocrebanine (2c)
Crebanine (200 mg, 0.59 mmol) and N-Bromosuccinimide (213.6 mg, 1.2 mmol) were added
to trifluoroacetic acid (5 mL), the mixture was stirred for 18hat room temperature, diluted with
water (10 mL), and neutralized with saturated NaHCO3 and extracted with CH2Cl2 (3
The combined CH2Cl2 solution was brined with water, dried over anhydrous Na2SO4, filtered, and
concentrated to give a crude product, which was purified by silica gel column chromatography
×
10 mL).
(CH2Cl2/MeOH 200:1
and compound 2c (80 mg, 33%).
Compound 2a, pale yellow solid. H-NMR (400 MHZ, CDCl3,
→
100:1), yielding compound 2a (52 mg, 27%), compound 2b (50 mg, 17%),
1
δ; ppm) 6.16 (1H, s, 9, -OCH2O-),
6.06 (1H, s, -OCH2O-), 3.93 (3H, s, 8-OCH3), 3.87 (3H, s, 9-OCH3), 3.54 (1H, d, J = 12.8 HZ, 6a-H),
3.16–3.09 (m, 1H, 7-Ha), 2.99–2.91 (m, 2H, 5-Ha, 7-Hb), 2.77–2.72 (m, 1H, 5-Ha), 2.60 (s, 3H, -NCH3),
2.53–2.50 (m, 1H, 4-Ha), 2.15–2.02 (m, 1H, 4-Hb). 13C-NMR (100 MHZ, CDCl3,
δ; ppm), 29.29 (t, C-7),
29.33 (t, C-4), 52.78 (t, C-5), 44.02 (t, -NCH3), 60.82 (q, 9-OCH3), 61.17 (q, 8-OCH3), 62.18 (d, C-6a),
100.77 (t, -OCH2O-),102.96 (s, C-3), 115.29 (s, C-10), 118.63 (s, C-11), 125.78 (s, C-7a), 129.83 (s, C-1b),
130.73 (s, C-3a), 132.67 (s, C-11a), 142.26 (s, C-1), 145.28 (s, C-8), 149.72 (s, C-2), 150.88 (s, C-9a). Positive
ESI-MS m/z: 579.2 [M + 3]+. HR-MS for C20H18Br3NO4 [M + H]+; calcd.573.8859, found:573.8853.
1
Compound 2b, white solid. H-NMR (400 MHZ, CDCl3,
δ; ppm) 7.13 (s, 1H, 3-H), 6.14 (s, 1H,
-OCH2O-), 6.05 (s, 1H, -OCH2O-), 3.89 (s, 3H, 8-OCH3), 3.79 (s, 3H, 9-OCH3), 3.61–3.57 (m, 1H, 6a-H),
3.11–3.07 (m, 1H, 7-Ha), 2.97–2.87 (m, 2H, 5-Ha, 7-Hb), 2.80–2.66 (m, 1H, 5-Hb), 2.56 (s, 3H, -NCH3),
2.52–2.47 (m, 1H, 4-Ha), 2.11–2.04 (t, J = 13.6 HZ, 1H, 4-Hb). 13C-NMR (100 MHZ, CDCl3,
δ; ppm)
100.62 (t, -OCH2O-), 52.82 (t, C-5), 29.26 (t, C-4), 29.05 (t, C-7), 62.40 (d, C-6a), 115.95 (d, C-3), 152,55 (s,
C-9), 145.17 (s, C-2), 145.09 (s, C-8), 141.99 (s, C-1), 133.19 (s, C-11a), 130.69 (s, C-3a), 125.73 (s, C-7a),
124.66 (s, C-1a). Positive ESI-MS m/z: 498.0 [M + H]+. HR-MS for C20H19Br2NO4 [M + H]+; calcd.
495.9754, found: 495.9759.
1
Compound 2c, white solid. Positive ESI-MS m/z: 418 [M]+, 420.0 [M + 2]+. H-NMR (400 MHZ,
CDCl3, δ; ppm) 7.76 (d, J = 8.8 HZ, 1H, 11-H), 6.90 (d, J = 8.8 HZ, 1H, 10-H), 6.22 (s, 1H, -OCH2O-),
6.07(s, 1H, -OCH2O-), 3.91 (s, 3H, 8-OCH3), 3.84 (s, 3H, 9-OCH3), 3.82–3.80 (m, 1H, 6a-H), 3.09-3.04 (m,
2H, 5-Ha, 7-Hb), 2.90–2.82 (m, 2H, 5-Ha, 7-Hb), 2.17 (s, 3H, -NCH3), 1.95–1.87 (m, 1H, 4-Ha), 1.79–1.72
(m, 1H, 4-Hb).
3.1.8. Preparation of N-Methylcrebanine (2d)
A mixture of crebanine (200 mg, 0.59 mmol), ether (109 mg, 1.48 mmol) and CH3I (184.5 mg,
1.3 mmol) in MeOH (10 mL) was stirred for 16 h at room temperature. The reaction mixture was
concentrated and extracted with CHCl3 (3
Na2SO4, filtered, and concentrated to give a crude product, which was purified by silica gel column
×
10 mL). The combined extracts were dried over anhydrous
chromatography (CH2Cl2/MeOH 30:1), yielding compound 2d (180 mg, 86%) as a white solid.
1H-NMR (400 MHZ, CDCl3,
δ; ppm) 7.70 (d, J = 8.8 Hz, 1H, 11-H), 7.05 (d, J = 8.8 Hz, 1H, 10-H),
6.78 (s, 1H, 3-H), 6.18 (s, 1H, -OCH2O-), 5.98 (s, 1H, -OCH2O-), 4.74–4.70 (m, 1H, 6a-H), 3.80 (s, 3H,

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8-OCH3), 3.76–3.73 (m, 1H, 7-Ha), 3.72 (s, 3H, 9-OCH3), 3.64–3.55 (m, 2H, 7-Hb,5-Ha), 3.33 (s, 3H,
-NCH3), 3.18–3.09 (m, 1H, 5-Hb), 2.97 (s, 3H, -NCH3), 2.94–2.89 (m, 1H, 4-Ha), 2.70–2.63 (t, J = 14.2 Hz,
1H, 4Hb). Positive ESI-MS m/z: 354.2 [M]+.
3.1.9. Preparation of Stesakine (2e)
Crebanine (200 mg, 0.59 mmol) in 2mL of anhydrous CH2Cl2 was slowly added into a suspension
of AlBr3 (320 mg, 1.2 mmol) in nitrobenzene (5 mL) at 5 ◦C under argon. The solution was stirred for
18 h, diluted with water (20 mL) and neutralized with saturated NaHCO3. The mixture was extracted
with CH2Cl2 (3
×
10 mL), and the combined CH2Cl2 solution was brined with water, dried with
anhydrous Na2SO4, filtered, and concentrated to give a crude product, which was purified by silica
gel column chromatography (CH2Cl2/MeOH 50:1), yieldingcompound 2e (56 mg, 29%) as a colorless
1
oil. H-NMR (400 MHZ, CDCl3,
δ
; ppm) 7.63 (d, J = 8.8 HZ, 1H, 11-H), 6.83 (d, J = 8.8 HZ, 1H, 10-H),
6.53 (s, 1H, 3-H), 6.06 (s, 1H, -OCH2O-), 5.92 (s, 1H, -OCH2O-), 5.77 (s, 1H, 9-OH), 3.93 (s, 3H, 8-CH3),
3.69–3.64 (dd, J = 16.0 HZ, J = 4.0 HZ, 1H, 6a-H), 3.19–3.03 (m, 3H, 7-H, 5-Ha), 2.65–2.61 (m, 1H, 5-Hb),
2.60 (s, 3H, -NCH3), 2.56–2.50 (m, 1H, 4-Ha), 2.32–2.25 (t, J = 10.4 HZ, 1H, 4-Hb), 13C-NMR (100 MHZ,
CDCl3, δ; ppm) 142.2 (s, C-1), 116.7 (s, C-1a), 126.5 (s, C-1b), 146.6 (s, C-2), 106.7 (d, C-3), 126.7 (s, C-3a),
26.3 (t, C-4), 56 (t, C-5), 61.9 (d, 6a), 29.2 (t, C-7), 124.8 (s, C-7a), 156.4 (s, C-8), 145.8 (s, C-9), 108.5 (d,
t-10), 118.7 (d, C-11), 126.6 (s, C-11a), 100.1 (t, -CH2O-), 44.0 (q, -NCH3), 56.0 (q, 8-OCH3). Positive
ESI-MS m/z: 326 [M + H]+.
3.1.10. Preparation of Dehydrocrebanine (3a)
Pd/C (10%, 210 mg) was added to a suspension of crebanine (200 mg, 0.59 mmol) in MeCN
(20 mL), and the solution was refluxed for 6 h under N2. The mixture reaction solution was filtered and
1
the filtrate was evaporated to give compound 3a (181 mg, 91%) as a yellowish-green solid. H-NMR
(400 MHZ, CDCl3, δ; ppm) 8.66 (d, J = 8.8 Hz, 1H, 11-H), 7.03 (d, J = 9.2 Hz, 1H, 10-H), 6.89 (s, 2H, 3-H,
7-H), 6.19 (s, 2H, -OCH2O-), 3.99 (s, 3H, 8-OCH3), 3.97 (s, 3H, 9-OCH3), 3.34–3.37 (t, J = 5.8 Hz, 2H,
5-H), 3.24–3.31 (t, J = 5.8 Hz, 2H, 4-H), 3.13 (s, 3H, -NCH3). Positive ESI-MS m/z: 348.2 [M + H]+.
3.1.11. Preparation of (±) Crebanine (3b)
A mixture of 3a (150 mg, 0.44 mmol) and NaCNBH3 (36 mg, 0.57 mmol) in absolute EtOH was
stirred, and a mixture of EtOH and 2NHCl was added until the pH approached 3.0 (within 4 h).
Then, the solution was continuously stirred for 18 h at room temperature. After evaporation of the
reaction mixture, the pH was adjusted to 8 with saturated Na2CO3 and the mixture solution was
extracted with CH2Cl2 (3
×
10 mL). The combined extracts were dried over anhydrous Na2SO4, filtered,
and concentrated to get a crude product, which was purified by silica gel column chromatography
(CH2Cl2/MeOH 50:1), yielding compound 3b (120 mg, 80%) as a yellow oil. [α]2D5: 0◦ (C 0.2, MeOH).
1H-NMR (400 MHZ, CDCl3,
δ; ppm) 7.81 (d, J = 8.8 Hz, 1H, 11-H), 6.88 (d, J = 8.8 Hz, 1H, 10-H), 6.53 (s,
1H, 3-H), 6.07 (s, 1H, -OCH3O-), 5.90 (s, 1H, -OCH3O-), 3.90 (s, 3H, 8-OCH3), 3.81 (s, 3H, 9-OCH3),
3.70–3.65 (dd, J = 14.8 Hz, J = 4.4 Hz, 1H, 6a-H), 3.18–3.04 (m, 3H, 7-H, 5-Ha), 2.66–2.61 (m, 1H, 5-Hb),
2.59 (s, 3H, -NCH3), 2.56–2.49 (m, 1H, 4-Ha), 2.33–2.26 (t, J = 14.2 Hz, 1H, 4-Hb). Positive ESI-MS m/z:
340.2 [M + H]+.
3.1.12. Preparation of Dehydrostephanine (4a)
In a similar manner used in the preparation of 3a, stephanine (200 mg, 0.65 mmol) was subjected
to a dehydrogenation reaction to give compound 4a (162 mg, 81%) as a yellowish-green solid. 1H-NMR
(400 MHZ, CDCl3, δ; ppm) 8.56 (d, J = 8.4 HZ, 1H, 11-H), 8.29 (d, J = 8.0 HZ, 10-H), 7.08 (s, 1H, 9-H),
6.96 (s, H, 3-H), 6.93 (s, H, 7-H), 6.21 (s, 2H, -OCH2O-), 4.02 (s, 3H, 8-OCH3), 3.40–3.37 (t, J = 5.6 HZ,
2H, 5-CH2), 3.27–3.24 (m, 2H, 5-CH2), 3.12 (s, 3H). Positive ESI-MS m/z: 308.2 [M + H]+.

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3.1.13. Preparation of (±) Stephanine (4b)
In a similar manner used for the preparation of 3b
,
4a (150 mg, 0.49 mmol) was subjected to a
racemization reaction to give compound 4b (125 mg, 83%) as a yellowish-green solid. [α]2D5: 0◦ (C 0.2,
MeOH). 1H-NMR (400 MHZ, CDCl3,
δ; ppm) 7.70 (d, J = 8.0 Hz, 1H, 11-H), 7.31–7.26 (t, J = 8.0 Hz, 1H,
10-H), 6.85 (d, J = 8.0 Hz, 1H, 9-H), 6.58 (s, 1H, 3-H), 6.10 (s, 1H, -OCH2O-), 5.95 (s, 1H, -OCH2O-),
3.86 (s, 1H, 8-OCH3), 3.78–3.74 (dd, J = 14.8 Hz, J = 3.6 Hz, 1H, 6a-H), 3.40–3.34 (m, 3H, 7-H, 5a-H),
2.90–2.83 (m, 1H, 5a-H), 2.80 (s, 3H, -NCH3), 2.77–2.72 (m, 1H, 4a-H), 2.58–2.51 (t, J = 14.4 Hz, 1H,
4b-H). Positive ESI-MS m/z: 310.2 [M + H]+.
3.1.14. Preparation of 11-Methoxy-N-Methylisocorydine (5a)
To a mixture of isocorydine (200 mg, 0.59 mmol), Tetrabutylammonium bromide (16 mg,
0.03 mmol), and K2CO3 (163 mg, 1.18 mmol) in anhydrous THF (10 mL) CH3I (185 mg, 1.30 mmol)
was successively added over a 10-min period and stirred for 8 h at room temperature. The reaction
mixture was condensed and extracted with CHCl3. The combined extracts were dried over anhydrous
Na2SO4, filtered, and concentrated to give a crude product, which was purified by silica gel column
chromatography (CH2Cl2/MeOH 50:1), yielding compound 5a (154 mg, 71%) as a white solid.
1H-NMR (400 MHZ, CDCl3,
δ; ppm) 6.95 (d, J = 8.0 HZ, 1H, 8-H), 6.84 (d, J = 8.4 HZ, 1H, 9-H),
6.66 (s, 1H, 3-H), 3.88 (d, J = 4.0 HZ, 6H, 1-OCH3,2-OCH3), 3.87 (s, 3H,11-OCH3), 3.72 (s, 3H, 10-OCH3),
3.17–3.10 (m, 1H, 6a-H), 3.04–2.99 (m, 2H, 7-Ha, 5-Ha), 2.87 (d, J = 12.4 HZ, 1H, 7-Hb), 2.69 (d,
J = 16.8 HZ, 1H, 5-Hb), 2.53–2.48 (m, 4H, -NCH3, 4-Ha), 2.40–2.33 (t, J = 13.0 HZ, 1H, 4-Hb).13C-NMR
(100 MHZ, CDCl3, δ; ppm) 29.73 (t, C-4), 23.03 (t, C-7), 42.94 (q, N-CH3), 53.07 (q, N-CH3), 55.83 (q,
10-OCH3), 55.97(q, 2-OCH3), 60.00 (t, C-5), 60.21 (q, 1-OCH3), 60.40 (q, 11-OCH3), 111.46 (d, C-3),
112.72 (d, C-9), 121.63 (s, C-11a). 122.36 (d, C-8), 123.98 (s, C-1a), 123.96 (s, C-7a), 126.18 (s, C-1b), 146.35
(s, C-1), 147.04 (s, C-10), 152.20 (s, C-11), 152.54 (s, C-20). Positive ESI-MS m/z: 370.2 [M + H]+. HR-MS
for C22H28NO4 [M + H]+; calcd. 370.2013, found: 370.2026.
3.1.15. Preparation of 11-Methoxyisocorydine (5b)
A solution of isocorydine (200 mg, 0.59 mmol) in MeCN (5 mL) and MeOH (5 mL) was treated
with N,N-diisopropylethylamine (91 mg, 0.71 mmol) and trimethylsilyldiazomethane (0.36 mL, 2M in
hexane), the mixture was stirred at room temperature for 15 h, condensed, and extracted with CHCl3.
The combined extracts were dried over anhydrous Na2SO4, filtered, and concentrated to give a crude
product, which was purified by silica gel column chromatography (CHCl3–MeOH = 80:1), yielding
1
compound 5b (51 mg, 24%) as a colorless oil. H-NMR (400 MHZ, CDCl3,
δ; ppm) 6.95 (d, J = 8.0 HZ,
1H, 8-H), 6.84 (d, J = 8.4 HZ, 1H, 9-H), 6.66 (s, 1H, 3-H), 3.88 (d, J = 4.0 HZ, 6H, 1-OCH3,2-OCH3), 3.87
(s, 3H,11-OCH3), 3.72 (s, 3H, 10-OCH3), 3.17–3.10 (m, 1H, 6a-H), 3.04–2.99 (m, 2H, 7-Ha, 5-Ha), 2.87 (d,
J = 12.4 HZ, 1H, 7-Hb), 2.69 (d, J = 16.8 HZ, 1H, 5-Hb), 2.53–2.48 (m, 4H, -NCH3, 4-Ha), 2.40–2.33 (t,
J = 13.0 HZ, 1H, 4-Hb). Positive ESI-MS m/z: 356.2 [M + H]+. HR-MS for C21H25NO4 [M + H]+; calcd.
356.1856, found: 356.1864.
3.1.16. Preparation of 11-Ethoxyisocorydine (5c)
A mixture of isocorydine (200 mg, 0.59 mmol), Tetrabutylammonium bromide (16 mg, 0.03 mmol),
and K2CO3 (163 mg, 1.18 mmol) in anhydrous THF (10 mL) was successively added CH3CH2I
(203 mg, 1.30 mmol) over a 10-min period and stirred for 8 h at room temperature. The reaction
mixture was condensed and extracted with CHCl3. The combined extracts were dried over anhydrous
Na2SO4, filtered, and concentrated to get a crude product, which was purified by silica gel column
1
chromatography (CH2Cl2/MeOH 50:1), yielding compound 5c (80 mg, 23%) as a colorless oil. H-NMR
(400 MHZ, CDCl3, δ; ppm) 6.95 (d, J = 8.0 HZ, 1H, 8-H), 6.83 (d, J = 8.0 HZ,1H, 9-H), 6.66 (s, 1H, 3-H),
3.87 (d, J = 2.8 HZ, 6H, 1-OCH3, 2-OCH3), 3.84–3.79 (m, 1H, 6a-H), 3.66 (s, 3H, 10-OCH3), 3.19–3.14
(m, 1H, 7-Ha), 3.05–2.98 (m, 2H,11-CH2), 2.87–2.83 (m, 1H, 7-Hb), 2.71–2.67 (m 1H, 5-Ha), 2.54 (s, 3H,

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-NCH3), 2.50–2.47 (m, 4-Ha), 2.40–2.34 (t, 1H, J = 12.8 HZ, 4-Hb), 1.14–1.10 (t, J = 7.0 HZ, 3H, 11-CH3).
13C-NMR (100 MHZ, CDCl3,
; ppm) 15.79 (q, 11-CH2CH3), 25.87 (t, C-7), 33.34 (t, C-4). 56.21 (q,
δ
10-OCH3), 60.96 (q, 1-OCH3), 61.14 (q, 2-OCH3), 68.89 (t, 11-CH2CH3), 111.41 (d, C-3), 112.72 (d, C-9),
122.04 (d, C-8), 124.00 (C-7a), 125.01 (s, C-1a), 127.00 (s, C-1b), 146 .89 (s, C-1), 147.04 (s, C-10), 152.82 (s,
C-11), 153.42 (s, C-2). Positive ESI-MS m/z: 370.2 [M + H]+. HR-MS for C22H27NO4 [M + H]+; calcd.
370.2013, found: 370.2022.
3.1.17. Preparation of N-Methylisocorydine (5d)
In a similar manner used in the preparation of 2d, isocorydine (200 mg, 0.59 mmol) was subjected
to a N-methylation reaction to give compound 5d (183 mg, 88% yield) as a white solid. 1H-NMR
(400 MHZ, CDCl3, δ; ppm) 8.60 (s, 1H, 11-OH), 7.01 (d, J = 8.0 HZ, 1H, 8-H), 6.89 (d, J = 8.0 Hz, 2H,
9-H), 6.86 (s, 1H, 3-H), 4.77–4.73 (dd, J = 12.0 HZ, J = 5.2 HZ, 1H, 6a-H), 4.24 (d, J = 13.2, 1H, 5a-H),
3.96 (s, 3H, 1-OCH3), 3.89 (s, 3H, 2-OCH3), 3.76 (s, 3H, 10-OCH3), 3.74 (s, 3H, -NCH3), 3.65–3.58 (m,
2H, 7a-H), 3.54–3.41 (m, 2H, 5b-H, 7a-H), 3.33 (s, 3H, -NCH3), 3.11–3.07 (m, 1H, 4-Ha), 2.88–2.81 (t,
J = 12.0 HZ, 1H, 4-Hb). Positive ESI-MS m/z: 356.2 [M]+.
3.1.18. Preparation of (Z)-N-2,3-Dimethoxybenzylidene-3,4-Dimethoxy-2-Phenyl-Ethanamine (6a)
A mixture of 2-(3,4-dimethoxy-phenyl)-ethylamine (1 g, 5.5 mmol) and 2,3-dimethoxy-benzoic
acid (1.1 g, 6.1 mmol) in methylbenzene (50 mL) was heated to 120 ◦C for 8 h. The reaction mixture
was condensed and extracted with EtOAC. The combined extracts were washed with water three
times, dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was drawn off
by a vacuum pump over 4 h to give compound 6a (1.78 g, 92% Purity, 91% yield) as a brown-red
oil. 1H-NMR (400 MHZ, CDCl3,
δ; ppm) 7.54 (d, J = 7.6 HZ 1H, N-CH2CH2Ph-2H), 7.09–7.05 (t,
J = 8.0 HZ, 1H, N-CH2Ph-4H), 6.96 (d, J = 8.0 HZ, 1H, N-CH2CH2Ph-5H), 6.76 (d, J = 12.0 HZ,
3H, N-CH2CH2Ph-6H, N-CH2Ph-3H, N-CH2Ph-5H), 3.90–3.88 (m, 1H, N-CH2β), 3.87–3.76 (m, 12H,
4×-OCH3), 2.98–2.94 (t, J = 7.0 HZ, 2H, N-CH2α). Positive ESI-MS m/z: 330.2 [M + H]+.
3.1.19. Preparation of N-2,3-Dimethoxybenzyl-3,4-Dimethoxy-2-Phenylethanamine (6b)
Amixture of 6a (1.5 g, 4.6 mmol) and Pd/C(5%, 150 mg) in MeOH (50 mL) was stirred under a
hydrogen atmosphere (1 bar) for 8 h. The MeOH-insoluble material was filtered off and the filtrate
was evaporated to give a crude product, which was purified by silica gel column chromatography
1
(CH2Cl2/petroleum ether 1:1), yielding compound 6b (1.3 g, 87%) as a yellow oil. H-NMR (400 MHZ,
CDCl3,
δ; ppm), 7.01–6.97 (t, J = 8.0 HZ, 1H, N-CH2Ph-4H), 6.83 (d, J = 6.4 HZ, 2H, N-CH2CH2Ph-5H,
N-CH2CH2Ph-6H), 6.78 (d, J = 8.0 HZ, 1H, N-CH2CH2Ph-5H), 6.73 (d, 2H, J = 11.6, N-CH2Ph-3H,
N-CH2Ph-5H), 3.84–3.77 (m, 12H, 4 -OCH3), 2.87–2.83 (t, J = 7.0 HZ, 2H, N-CH2α), 2.78–2.74 (t,
×
J = 6.8 HZ, 2H, N-CH2β), 1.89 (s, 2H, N-CH2). Positive ESI-MS m/z: 310.2 [M + H]+.
3.1.20. Preparation of N-Methyl-N-2,3-Dimethoxybenzyl-3,4-Dimethoxy-2-Phenyl-Ethanamine (6c)
To a mixture of 6b (1.2 g, 3.6 mmol) and K2CO3 (1.0 g, 7.2 mmol) in anhydrous THF (20 mL),CHI
(610.6 mg, 4.3 mmol) was slowly added under an ice-bath over a 10-min period. The solution was
stirred for 4 h at room temperature, condensed, and extracted with EtOAC. The combined extracts
were dried over anhydrous Na2SO4 and filtered, and the filtrate was concentrated to give a crude
product, which was purified by silica gel column chromatography (CH2Cl2/petroleum ether 10:1),
1
yielding compound 6c (950 mg, 76% yield) as a colorless oil. H-NMR (400 MHZ, CDCl3,
δ; ppm)
7.06–7.00 (dd, 2H, J = 9.6 HZ, J = 4.8 HZ, N-CH2CH2Ph-5H, N-CH2CH2Ph-6H), 6.86 (t, 1H, J = 4.2 HZ,
N-CH2Ph-4H), 6.78 (d, 1H, J = 8.4, N-CH2CH2Ph-2H), 6.73 (d, 2H, J = 7.2, N-CH2Ph-5H, N-CH2Ph-3H),
3.86–3.82 (m, 12H, 4
×
-OCH3), 3.74 (s, 2H, N-CH2), 2.87 (t, 2H, J = 7.2, N-CH2α), 2.77–2.71 (m, 2H,
N-CH2β), 2.37 (s, 3H). Positive ESI-MS m/z: 346.2 [M + H]+.

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3.2. Pharmacological Evaluation
3.2.1. Materials
Verapamil hydrochloride (Shanghai Harvest Pharmaceutical Co., Ltd., Shanghai, China), lidocaine
hydrochloride (Shanghai ZhaohuiPharmaceutical Co., Ltd., Shanghai, China), isocorydine, crebanine,
and stephanine were used as positive control groups. Normal saline (NS) (Kunming Nanjiang
Pharmaceutical Co., Ltd., Kunming, China) was used as negative control group. Other chemicals
used—chloral hydrate, barium chloride, chloroform, sodium hydroxide, and hydrochloric acid were
of analytical grade.
All aporphine alkaloid derivatives were used for the tested drug groups, and were synthesized,
purified, and determined according to Sections 3.1.2–3.1.20 in this paper. The compounds were
dissolved in 10% hydrochloric acid solution and were adjusted to pH5–7 with 40% sodium hydroxide.
3.2.2. Animals
The experiments were carried out on Kunming mice (28–35 g) and SD rats (180–210 g), which
were provided by Hunan SJA Laboratory Animal Co., Ltd. (SYXK (Dian) K 2011-0011, Changsha,
China). Half of the animals were m◦ale and half were female, and they were housed in a controlled
environment (temperature 22
±
1 C; 60%
±
10% humidity; in a 12/12 h light-dark cycle) with
free access to standard pellet artificial diet and tap water. All animal experiments performed were
in compliance with the Animal Experimental Care and Ethic Committee of Yunnan University of
Traditional Chinese Medicine.
Determination of the dosage for tested drugs: The animals were first given 5 mg/kg of tested
drugs, based on the effective dose of crebanine in mice and rat [7]. The dosage was reduced or
increased according to the exhibited symptoms of poisoning, or death, and the antiarrhythmic effect
on the animals.
3.2.3. Statistical Analyses
Data are expressed as the means
using the Chi-square test. Differences were considered significant when p < 0.05. LD50 was determined
±
SD and incidence ratio. Statistical significance was calculated
using a related formulate of up-and-down procedure (UDP) [28].
3.2.4. Antiarrhythmic Activity Test of Aporphine Derivatives in a Mouse Model of Ventricular
Fibrillation Induced by CHCl3
Mice were randomly divided into a negative control group (NS), 4 positive control groups
(verapamil hydrochloride, crebanine, iscorydine, and stephanine), and 19 tested groups for aporphine
derivatives (Figure 1). Tested drugs were injected though the caudal vein at a chosen dose. Five
minutes later, each mouse was placed in a 500-mL inverted beaker, which contained a cotton ball
that had been soaked with 1mL of chloroform, and then were removed immediately after respiratory
arrest. The chest of the asphyxiated mouse was opened in order to observe the condition of ventricular
fibrillation (VF) [7,29]. The occurrence ratios of VF in mice are listed in Figure 1.
3.2.5. Anti-Arrhythmic Activity of Five Kinds of Aporphine Derivatives in a Rat Model of Arrhythmia
Induced by BaCl2
Rats were randomly divided into 10 groups: an NS group, 4 groups of positive control (lidocaine,
crebanine, iscorydine and stephanine) groups, and 5 tested groups of active derivatives (2a–d and
4a) (Table 1). First, rats were anesthetized with 10% choral hydrate (ip, 300 mg/kg), fixed on their
backs and connected to a physiological signal acquisition system in order to record normal II lead
electrocardiogram (ECG). Then, a barium chloride solution was injected into the caudal vein of each rat
(4 mg/kg, at a volume of 1 mL/kg) to trigger arrhythmia. Three minutes later, rats were injected with
different treatments though the lingual vein at a certain doses. Then, ECG was recorded. The number

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of rats that recovered to a normal sinus rhythm, the duration needed to recover, and number of
rats that maintained normal rhythm for more than 3 min, 5 min, and 20 min, in each group, were
calculated [7,29] (Table 1).
3.2.6. Acute Toxicity Test in Mice
Mice were injected with the test compounds via the caudal vein at a certain dose. The Dm
(100% death) and Dn (0% death) of the compounds were found in preliminary tests; then, 5–6 dose
groups (n = 10) of the compounds were set up, according to the requirements of the up-and-down
procedure (UDP) method, death number was recorded and LD50 was determined [28].
4. Conclusions
In this study, derivatives of three kinds of aporphine alkaloids, crebanine, isocorydine, and
stephanine, were designed and synthesized through the reactions of ring-opening, N-substitution,
demethylation, methylation, acetylation, quaternization, and dehydrogenation. The use of NBS for the
bromination reaction was successfully applied to the synthesis of the three kinds of bromocrebanines.
Among these derivatives, compound 2b, dibromocrebanine, proved to be the most promising
candidate, showing maximum potency against arrhythmia and minimum toxicity compared to the
other compounds prepared in this family. Derivatives 2a, 2c, 2d, and 4a also displayed impressive
potency. Finally, derivative 1d exhibited a lower, though still significant, activity.
The relationship of the structure–antiarrhythmic activities of aporphines are summarized as:
(1) C-1,C-2-methylenedioxy group on ring A of aporphines increases the planar configuration of
molecules and plays an important role in both activity and toxicity; (2) a closed structure of the B
ring is essential for both antiarrhythmic efficacy and toxicity; additionally, N-quaternization of ring B
could increase toxicity; (3) levoduction of 6a in ring C is an active group; and (4) 8-, 9-, and 10-methoxy
groups on ring D is an important functional groups. Further explorations are underway to evaluate
the biological activities and the ion channel mechanisms of these aporphine derivatives.
Acknowledgments: The National Natural Science Foundation of China (81260651) and the Science Foundation of
Education Department of Yunnan Province (2015J102) are gratefully acknowledged for their financial support.
We acknowledge members of Minglong Yuan’s lab, Yunnan Minzu University, for chemical modifications.
We also thank. Qing Zhao, Hongping He and Aixue Zuo for chemical structure analyses, and Jia Chen for
language assistance.
Author Contributions: Yunshu Ma and Hui Wang conceived and designed the experiments; Hui Wang and
Shujun Kong performed the experiments; Hui Wang, Zixian Yang and Hongmei Wang analyzed the data;
Qiuyan Huang, Jingyu Li and Cheng Chen contributed reagents and materials; Yunshu Ma, Hui Wang and
Xin Cheng wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: The compounds 1a, 2a, 2b, 1e, 2d and 6b are avilable, other compounds are not provided
from us.
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2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).