Structural modification of sanguinarine and chelerythrine and their in vitro acaricidal activity against Psoroptes cuniculi

Article abstract of DOI:10.1248/cpb.c12-00618

Sanguinarine (1) and chelerythrine (2) are two quaternary benzo[c]phenanthridine alkaloids (QBAs). Eighteen derivatives of 1 and 2 were synthesized by modification of C=N⁺ bond and evaluated for their in vitro acaricidal activity against Psoroptes cuniculi , a mange mite. A new method was developed to prepare 6-alkoxy dihydro derivatives of 1 and 2 (1a-e, 2a-e). Among all the compounds, only 6-alkoxy dihydrosanguinarines (1a-e) showed significant acaricidal activity at 5.0 mg/mL and 1a possessed the strongest activity (50% lethal concentrations (LC₅₀)=339.70±0.75 mg/L, 50% lethal time ( LT₅₀)=6.53±0.04 h), comparable with a standard drug ivermectin (LC₅₀=168.19±11.79 mg/L, LT ₅₀=16.54±0.11 h). The iminium moiety in 1 and 2 was proven to be the determinant for their acaricidal properties. 6-Alkoxy dihydro derivatives (1a-e, 2a-e) were prodrugs of 1 and 2. Compared with 7,8-dimethoxy groups, 7,8-methylenedioxy group was able to significantly improve the bioactivity. The present results suggested that QBAs are promising candidates or lead compounds for the development of new isoquinoline acaricidal agents.

Full text of DOI:10.1248/cpb.c12-00618

1508
Regular Article
Chem. Pharm. Bull. 60(12) 1508–1513 (2012)
Vol. 60, No. 12
Structural Modification of Sanguinarine and Chelerythrine and Their in
Vitro Acaricidal Activity against Psoroptes cuniculi
,a,b
Fang Miao,a Xin-Juan Yang,a,b Yan-Ni Ma,b Feng Zheng,b Xiao-Ping Song,c and Le Zhou*
b
a College of Life Science, Northwest A&F University; College of Science, Northwest A&F University; and
c Department of Veterinary Medicine, Northwest A&F University; Yangling, Shaanxi 712100, China.
Received July 16, 2012; accepted September 4, 2012; advance publication released online September 21, 2012
Sanguinarine (1) and chelerythrine (2) are two quaternary benzo[c]phenanthridine alkaloids (QBAs).
Eighteen derivatives of 1 and 2 were synthesized by modification of C=N+ bond and evaluated for their in
vitro acaricidal activity against Psoroptes cuniculi, a mange mite. A new method was developed to prepare
6-alkoxy dihydro derivatives of 1 and 2 (1a–e, 2a–e). Among all the compounds, only 6-alkoxy dihydrosan-
guinarines (1a–e) showed significant acaricidal activity at 5.0mg/mL and 1a possessed the strongest activity
(50% lethal concentrations (LC50)=339.70ꢀ0.75mg/L, 50% lethal time (LT50)=6.53ꢀ0.04h), comparable with
a standard drug ivermectin (LC50=168.19ꢀ11.79mg/L, LT50=16.54ꢀ0.11h). The iminium moiety in 1 and 2
was proven to be the determinant for their acaricidal properties. 6-Alkoxy dihydro derivatives (1a–e, 2a–e)
were prodrugs of 1 and 2. Compared with 7,8-dimethoxy groups, 7,8-methylenedioxy group was able to sig-
nificantly improve the bioactivity. The present results suggested that QBAs are promising candidates or lead
compounds for the development of new isoquinoline acaricidal agents.
Key words sanguinarine; chelerythrine; acaricidal activity; Psoroptes cuniculi; benzo[c]phenanthridine alka-
loid
Sanguinarine (1) and chelerythrine (2) (Fig. 1) belong to northwest of China. This plant is rich in compounds 1 and
quaternary benzo[c]phenanthridine alkaloids (QBAs) and 2 and has been used as Chinese traditional medicine for the
widely present in a number of plant species of the Fumaria- treatment of some skin diseases caused by pathogenic fungi or
ceae, Papaveraceae, and Rutaceae families.1) QBAs have given parasite, such as rosacea, scabies, brothers tinea, psoriasis and
rise to a lot of attention because of their extensive bioactivities so on. Our preliminary research revealed that the methanol
including antitumor,2–4) antimicrobial,5,6) anti-inflammatory,7) extract contained 4.75% 1 and 7.38% 2 of the plant possessed
antiviral8) including anti-human immunodeficiency virus significant in vitro acaricidal activity against P. cuniculi. This
(HIV),9) anti-platelet aggregation,10) anti-angiogenesis11) and result suggested that 1 and 2 as the main compounds of the
anti-acetylcholinesterase.12) Recently, QBAs were also found extract may have acaricidal activity. The purpose of the pres-
to have antiparasitic actions against Trichodina sp.,13) Dac- ent study is to examine the acaricidal activity in vitro of 1, 2
tylogyrus intermedius14) and malaria.15) However, the acari- and their derivatives against P. cuniculi and understand their
cidal activity of QBAs was not reported until now.
structure–activity relationship.
Nevertheless, QBAs have the common drawback of low
compatibility with physiological conditions. The highly polar
iminium moiety easily reacts with biological reducing agents
Results and Discussion
Chemistry Compounds 1a–i and 2a–i in Fig. 2 were
such as nicotinamide adenine dinucleotide (NADH), or non- synthesized by using 1 and 2 as the starting material, respec-
target nucleophiles in biological fluids to become the cor- tively. Compounds 1g–i and 2g–i were prepared according to
responding neutral nonactive phenanthridine derivatives.16–20) the methods previously reported by us.6) Compounds 1a–e
Therefore, during the past ten years, a great deal of work and 2a–e were respectively synthesized by reaction of 1h or
had focused on improving the chemical stability of QBAs by 2h with the corresponding alcohol (methanol, ethanol, n-pro-
structural modification.16,21)
panol, iso-propanol or n-butanol) in the presence of 20mol%
Psoroptes cuniculi is an important veterinary ectoparasite of CuCl2·2H2O under oxygen. Compounds 1 or 2 reacted with
in rabbits, goats, horses, sheep and so on.22) It can cause potassium cyanide in water solution to provide 1f or 2f.
intense pruritus, reduction of weight gain or even death of
In our previous research, 1a, 1b, 2a and 2b were respective-
animals.23,24) Therapy and control of both human scabies and ly synthesized by the reaction of 1 or 2 with sodium methylate
animal mange are based mainly on the use of effective drugs or ethylate. In addition, the reaction of 1 or 2 with the cor-
and chemicals. However, many of the chemical acaracides responding alcohol in the presence of triethylamine might also
have limitations such as resistance,25,26) toxicity27,28) and envi- give 1a–e or 2a–e. However, the methods above only gave
ronmental damage.27,28) Ivermectin is increasingly being used
to treat human scabies and animal mange but often treatment
failures, recrudescence and reinfection can occur.29) These
problem have lead to research efforts to discover new effective
acaricides derived from some active natural products.
Macleaya microcarpa (MAXIM.) FEDDE (the family Papav-
eraceae) is a perennial herb and widely distributed in the
The authors declare no conflict of interest.
Fig. 1. Structures of Sanguinarine (1) and Chelerythrine (2)
© 2012 The Pharmaceutical Society of Japan
*To whom correspondence should be addressed. e-mail: zhoulechem@yahoo.com.cn

December 2012
1509
Fig. 2. Derivatives of Sanguinarine (1) and Chelerythrine (2) (1a–i, 2a–i)
Table 1. Acaricidal Activity of Compounds 1, 2, 1a–i and 2a–i against
P. cuniculi at the Concentration of 5.0mg/mL at 24h
lower yields and lower purity of the products. In the present
research, we report a new method for preparation of 1a–e
and 2a–e, i.e., a CuCl2-catalyzed aerobic oxidative coupling
reaction of dihydrosanguinarine (1h) or dihydrochelerythrine
(2h) with aliphatic alcohols. Compared with the two methods
mentioned above, the present method has some obvious ad-
vantages such as nearly quantitative yields, simple work-up
operation and easily obtaining high purity products.
Mortality (%)
(mean S.D.)
Mortality (%)
(mean S.D.)a)
Compd. No.
Compd. No.
1
3.3 2.9
100.0 0.0
73.3 2.9
71.7 2.9
73.3 2.9
71.7 2.9
6.7 2.9
2
2a
3.3 2.9
16.7 2.9
8.3 2.9
8.3 2.9
6.7 2.9
6.7 2.9
3.3 2.9
3.3 2.9
3.3 2.9
3.3 2.9
1.7 2.9
1a
1b
2b
1c
2c
1d
2d
The structures of all compounds were elucidated by spec-
troscopic analyses. Compounds 1 and 2 respectively revealed
1e
2e
1
1f
2f
singlet signals at δ 9.95 and 9.92ppm in H-NMR spectra due
to the corresponding HC=N+ proton (i.e. H-6), signals at δ
150.7 and 152.1ppm in 13C-NMR spectra due to the corre-
sponding C-6 and characteristic ion peaks at m/z 332 [M−Cl]+
and 348 [M−Cl]+ in electrospray ionization (ESI)-MS. With
respect to 1a–e and 2a–e, each compoumd showed a singlet
1g
1h
11.7 2.9
5.0 5.0
2g
2h
1i
6.7 2.9
2i
Ivermectin
100.0 0.0
Control
1
signal of H-6 in the region of δ 5.45–5.65ppm in H-NMR
spectum, one signal of C-6 in the region of δ 82–86ppm results above, the most effective compound 1a was further
13C-NMR spectrum and a pseudomolecular ion peak [M+Na]+ evaluated for its acaricidal toxicity. Ivermectin was used as
and a characteristic fragment ion peak [M−OR]+ in ESI-MS. a standard acaricide. The corrected mortality rates caused by
Compounds 1f and 2f respectively displayed singlet signals at 1a and ivermectin at different concentrations at 24h against
1
δ 5.94 and 5.93ppm in their H-NMR spectra due to the cor- P. cuniculi are shown in Fig. 3. Toxicity regression equations
responding H-6, and signals at δ 47.3 and 47.5ppm in their for concentration effect and the median lethal concentrations
13C-NMR spectra due to the corresponding C-6, and signals at (LC50) are given in Table 2. The mortality rates of 1a and
δ 118.0 and 118.5ppm due to the corresponding C≡N carbon. ivermectin increased as the concentration increased. Both 1a
In positive ESI-MS spectra, 1f and 2f showed a quasimolecu- and ivermectin showed significant linear correlation between
lar ion peak [M+H]+, a pseudomolecular ion peak [M+Na]+ the probability of mortality rate and log[concentration] value
and a characteristic fragment ion peak [M−CN]+.
(R2≥0.96). It was worth noting that there was a cross point
Pharmacology. Acaricidal Activity in Vitro The in at the concentration of ca. 0.7mg/mL between the curves
vitro acaricidal activity of compounds (1, 2, 1a–i and 2a–i) of 1a and ivermectin in Fig. 3, indicating that 1a was more
at the concentration of 5mg/mL are listed in Table 1. Among effective than ivermectin at more than ca. 0.7mg/mL. Be-
all the compounds, only the 6-alkoxy dihydrosanguinarines cause of the presence of the cross point, 1a showed a big-
(1a–e), also named sanguinarine pseudoalcoholates, displayed ger LC50 value (339.70 0.75mg/L) and a smaller LC90 value
significant acaricidal activity. Compared with 1a–e, all 2a–e (1243.90 36.09mg/L) than ivermectin (LC50=168.19 11.79,
only gave lower activity. Compound 1a showed the highest LC90=1693.63 126.18mg/L) (see Table 2).
activity with 100% of average mortality, which was the same
The corrected mortality rates of 1a and ivermectin at
as the standard acaricide ivermectin. Compared with the par- different times at 10mg/mL are showed in Fig. 4. Toxic-
ent compounds 1 and 2, all the pseudoalcoholates (1a–e or ity regression equations for post-treatment time effect and
2a–e) were able to remarkably enhance the activity. Among the median lethal time (LT50) are listed in Table 3. Similar
each group of the pseudoalcoholates (1a–e or 2a–e), 1a and to the case of the concentration effect, the mortality rates
2a gave the strongest activity while the other pseudoalcohol- caused by two compounds increased as the post-treatment
ates (1b–e or 2b–e) showed the nearly identical lower activity. time increased and showed significant linear correlations with
This result indicated that the activity improvement effect of logT value (R2≥0.99). From Fig. 4, it might be seen that the
6-methoxy group was far beyond that of the other 6-alkoxy acaricidal action of 1a was much faster than ivermectin. Both
group. Furthermore, comparison of the structures and activ- the LT50 and 90% lethal time (LT90) of 1a (LT50=6.53 0.04,
ity of 1a–e with that of 2a–e showed that 7,8-methylenedioxy LT90=7.93 0.11h) were significantly smaller than ivermectin
group was more benifical to the improvement of activity than (LT50=16.54 0.11, LT90=20.26 0.12h) (see Table 3).
7,8-dimethoxy group.
The higher activity of the pseudoalcoholates 1a–e and
Acaricidal Toxicity Based on the acaricidal screening 2a–e might be related with their hydrolyzable property, higher

1510
Vol. 60, No. 12
Fig. 3. Average Corrected Mortality of P. cuniculi for the Treatment of
1a and Ivermectin at Different Concentrations
Fig. 4. Average Corrected Mortality of P. cuniculi for the Treatment of
1a and Ivermectin (10mg/mL) at Various Time
lipophilicity and higher compatibility with physiological con-
ditions. 1a–e and 2a–e are structurally N,O-acetals and very Therefore, both 1a and 2a gave the best activity among the
easily converted to their corresponding iminium ion (1 or 2) corresponding pseudoalcoholates (1a–e or 2a–e).
under an acidic condition30) even physiological acidic environ-
In conclusion, a series of C=N+-modified derivatives of
ment, such as the lysosome of cells.31) Moreover, unlike ionic 1 and 2 were synthesized and evaluated for their acaricidal
compounds 1 and 2, 1a–e and 2a–e are nonionic compounds activity in vitro against P. cuniculi. 6-Alkoxy dihydrosan-
and have higher lipophilicity. They are able to more easily guinarines (1a–e) were found to have significant acaricidal
penetrate target cell membranes than 1 and 2, and then ac- activity for the first time. Compared with ivermectin, the most
cumulate to high levels and convert back to the iminium ions effective compound 1a possessed a slightly bigger LC50 value
in the acidic environment of the lysosome. Unlike the higher and much smaller LT50 value. 6-Alkoxy dihydro derivatives
chemical activity of 1 and 2, 1a–e and 2a–e are inactive to of 1 and 2 were considered as prodrugs and the real active
biological reducing agents such as NADH and nucleophiles components were 1 and 2. The C=N+ double bond in 1 and 2
due to the absence of the iminium moiety. Based on the were the determinant for their acaricidal properties. The pres-
analysis above, we thought that the pseudoalcoholates might ent results suggested that quaternary benzo[c]phenanthridine
be prodrugs of 1 and 2 and the real active components should alkaloids are very good candidates or lead compounds for the
be 1 and 2. Although 1f–i and 2f–i have similar molecular development of new isoquinoline acaricidal agents.
structures to the pseudoalcoholates (1a–i, 2a–i), they cannot
convert back to 1 and 2 under physiological conditions. It may
be for this reason that 1f–i and 2f–i showed the lower or less
Experimental
Materials Ivermectin (≥91% 22,23-dihydroavermectin
activity. Obviously, the C=N+ double bond in 1 and 2 were B1 consisting of 95% avermectin B1a and 5% avermectin B1b)
the determinant for their acaricidal properties. To a certain was purchased from Sigma-Aldrich Trading Co., Ltd., Shang-
degree, the above conclusion was also supported by the fact hai, China. Other Chemicals used in the present study were
of the different activity of the pseudoalcoholates in the same purchased from J&K Chemical Ltd. (China) and used without
group (1a–e or 2a–e). Compared with the other 6-alkoxy further purification. Sanguinarine chloride (1) and chelery-
groups, 6-methoxy group has the smallest volume and the thrine chloride (2) were isolated by us from the entire plant of
weakest steric hindrance effect on the hydrolytic reaction. 1a Macleaya microcarpa (MAXIM) FEDDE according to the method
and 2a more easily converted back to the corresponding ion reported by us.6) Compound 1g–i, 2g–i were prepared accord-
forms (1, 2) than the other pseudoalcoholates (1b–e or 2b–e). ing to the methods previously reported by us using 1 and 2 as
Table 2. Toxicity Regression Equation for Concentration Effect and LC50 Values (mg/L) of 1a against P. cuniculi
Compd. No.
Toxicity regression equationa)
R2
LC50 S.D.
LC90 S.D.
1a
y=2.2344x−0.6541
y=1.2794x+2.1510
0.9677
0.9862
339.70 0.75
168.19 11.79
1243.90 36.09
1693.63 126.18
Ivermectin
a) y and x express the probability of mortality rate and log[concentration (mgL−1)], respectively.
Table 3. Toxicity Regression Equation for Time Effect and LT50 Values of 1a (10mg/mL) against P. cuniculi
Compd. No.
Toxicity regression equationa)
R2
LT50 S.D.
LT90 S.D.
1a
y=15.566x−7.6942
y=14.7330x−12.942
0.9970
0.9943
6.53 0.04
7.93 0.11
Ivermectin
16.54 0.11
20.26 0.12
a) y: the probability of mortality rate. x: logT (h).

December 2012
1511
1
starting materials.6)
yield 96%, H-, 13C-NMR, ESI-MS and mp data were agree-
Apparatus Melting points (mp) were determined on ment with that previously reported by us.6)
1
XT-4 micro-melting point apparatus and uncorrected. H- and
6-n-Propoxy Dihydrochelerythrine (2c): White granular
13C-NMR spectra were recorded with Bruker AVANCE III crystal (n-PrOH), yield 97%, mp 204–206°C, Rf 0.53 (petro-
1
operating at 500 and 125MHz, respectively and using TMS leum ether–ethyl acetate, v/v=1:1). H-NMR (CDCl3) δ: 7.77
as an internal standard. ESI-MS was measured on Trace mass (d, J=8.5Hz, 1H), 7.67 (s, 1H), 7.62 (d, J=8.6Hz, 1H), 7.46
spectrometer.
(d, J=8.5Hz, 1H), 7.12 (s, 1H), 7.03 (d, J=8.6Hz, 1H), 6.05 (s,
Synthesis of 1a–e and 2a–e. General Procedure To 2H), 5.65 (s, 1H), 3.97 (s, 3H), 3.93 (s, 3H), 3.80–3.85 (m, 1H),
the solution of saguinarine chloride (1) or chelerythrine 3.57–3.62 (m, 1H), 2.73 (s, 3H), 1.42–1.61 (m, 2H), 0.76 (t,
chloride (2) (0.33mmol) in ca. 20mL of different alcohol J=7.4Hz, 3H); 13C-NMR (CDCl3) δ: 152.1, 147.8, 147.3, 146.6,
(methanol for 1a, 2a, ethanol for 1b, 2b, n-propanol for 1c, 138.7, 131.0, 126.8, 126.0, 125.0, 123.3, 122.7, 120.1, 119.0,
2c, iso-propanol for 1d, 2d or n-butanol for 1e, 2e) was added 112.8, 104.6, 101.0, 100.7, 84.7, 68.1, 55.9, 55.6, 40.6, 22.7, 10.7.
11mg (0.06mmol) of CuCl2·2H2O at room temperature. The ESI-MS m/z: 429.75 [M+Na]+, 348.27 [M−O(CH2)2CH3]+.
resulting solution was stirred at 80°C for 10h using an oil
6-iso-Propoxy Dihydrochelerythrine (2d): White needle
bath under an oxygen atmosphere. The reaction solution was crystal (iso-PrOH), yield 96%, mp 176–178°C, Rf 0.49 (petro-
1
passed through a pad of celite and washed with ca. 40mL leum ether–ethyl acetate, v/v=5:1). H-NMR (CDCl3) δ: 7.77
of the same alcohol. The combined filtrate was evaporated to (d, J=8.5Hz, 1H), 7.65 (s, 1H), 7.61 (d, J=8.6Hz, 1H), 7.46
dryness in high vacuum to give 1a–e and 2a–e as solids.
(d, J=8.5Hz, 1H), 7.12 (s, 1H), 7.02 (d, J=8.6Hz, 1H), 6.06
6-Methoxysanguinarine (1a): White crystal (MeOH), yield (s, 2H), 5.76 (s, 1H), 3.97 (s, 3H), 3.93 (s, 3H), 4.37–4.39 (m,
1
96%. H-, 13C-NMR, ESI-MS and mp data were in agreement 1H), 2.70 (s, 3H), 1.30 (d, J=6.0Hz, 1H), 0.87 (d, J=7.5Hz,
with that previously reported by us.6)
1H); 13C-NMR (CDCl3) δ: 152.2, 147.9, 147.3, 146.5, 138.8,
6-Ethoxysanguinarine (1b): White crystal (EtOH), yield 131.0, 126.8, 126.1, 125.1, 123.3, 122.8, 120.2, 119.1, 112.8,
1
97%. H-, 13C-NMR, ESI-MS and mp data were in agreement 104.6, 101.0, 100.7, 82.6, 66.9, 61.6, 56.0, 40.5, 23.6, 21.5.
with that previously reported by us.6)
ESI-MS m/z: 408.05 [M+H]+, 429.61 [M+Na]+, 348.14 [M−
6-n-Propoxy Dihydrosanguinarine (1c): White granular OCH(CH3)2]+.
solid (n-PrOH), yield 99%, mp 194–195°C, Rf 0.36 (petroleum
6-n-Butoxy Dihydrochelerythrine (1e): White needle crys-
ether–ethyl acetate, v/v=2:1). 1H-NMR (CDCl3) δ: 7.78 (d, tal (n-BuOH), yield 98%, mp 144–146°C, Rf 0.53 (petroleum
J=8.5Hz, 1H), 7.70 (1H, s, H-4), 7.49 (d, J=8.5Hz, 1H), 7.42 ether–ethyl acetate, v/v=5:1). 1H-NMR (CDCl3) δ: 7.77 (d,
(d, J=8.2Hz, 1H), 7.14 (s,1H), 6.95 (d, J=8.25Hz, 1H), 6.14 J=8.5Hz, 1H), 7.67 (s, 1H), 7.62 (d, J=8.5Hz, 1H), 7.46 (d,
(s, 2H), 6.08 (s, 2H), 5.49 (s, 1H), 3.83 (m, 1H), 3.63 (1H, m), J=8.5Hz, 1H), 7.12 (s, 1H), 7.03 (d, J=8.5Hz, 1H), 6.06 (s,
2.79 (s, 3H), 1.53 (m, 2H), 0.79 (t, J=7.5Hz, 3H); 13C-NMR 2H), 5.65 (s, 1H), 3.97 (s, 3H), 3.93 (s, 3H), 3.85–3.90 (m, 1H),
(CDCl3) δ: 148.0, 147.4, 147.3, 145.2, 138.6, 131.0, 126.9, 3.63–3.65 (m, 1H), 2.73 (s, 3H), 1.40–1.48 (m, 2H), 1.19–1.2
125.9, 123.6, 123.0, 120.3, 116.4, 113.5, 108.7, 104.6, 101.7, (m, 2H), 0.79 (t, J=7.5Hz, 3H); 13C-NMR (CDCl3) δ: 152.2,
101.0, 100.7, 84.4, 68.0, 40.9, 22.6, 10.6. ESI-MS m/z: 413.96 147.9, 147.3, 146.6, 138.7, 131.0, 126.8, 126.0, 125.0, 123.3,
[M+Na]+, 332.27 [M−O(CH2)2CH3]+.
122.7, 120.1, 119.0, 112.8, 104.6, 101.0, 100.7, 84.7, 66.2, 61.7,
6-iso-Propoxy Dihydrosanguinarine (1d): White granular 56.0, 40.6, 31.7, 19.4, 13.9. ESI-MS m/z: 443.88 [M+Na]+,
crystal (iso-PrOH), yield 98%, mp 228–230°C, Rf 0.53 (pe- 348.26 [M−O(CH2)3CH3]+.
troleum ether–ethyl acetate, v/v=5:1). 1H-NMR (CDCl3) δ:
Synthesis of 1f and 2f. General Procedure To the solu-
7.75 (d, J=7.0Hz, 1H), 7.64 (s, 1H), 7.46 (d, J=7.0Hz, 1H), tion of potassium cyanide (60mg, 0.92mmol) in ca. 20mL of
7.42 (d, J=6.5Hz, 1H), 7.11 (s, 1H), 6.95 (d, J=6.5Hz, 1H), water was added 1 or 2 (0.22mmol). The resulting solution
6.09 (s, 2H), 6.04 (s, 2H), 5.53 (s, 1H), 4.33 (brs, 1H), 2.72 was heated at 60°C for 30min using a water bath under stir-
(s, 3H), 1.27 (s, 3H), 0.91 (s, 3H); 13C-NMR (CDCl3) δ: 148.0, ring. White precipitate was filtered off, washed with a small
147.3, 147.3, 145.2, 138.7, 131.0, 127.0, 125.9, 123.6, 123.1, volume of water for several times and recrystallized in a
120.4, 116.4, 113.6, 108.6, 104.6, 101.7, 101.0, 100.6, 82.3, 66.9, mixed solution of chloroform and methanol to provide the de-
40.8, 23.5, 21.3. ESI-MS m/z: 413.92 [M+Na]+, 332.29 [M− sired compound 1f or 2f.
OCH(CH3)2]+.
6-Cyano Dihydrosaguinarine (1f): White prism crystal,
1
6-n-Butoxy Dihydrosanguinarine (1e): White solid (n- yield 83%, mp 242–243°C. H-NMR (DMSO-d6) δ: 7.86 (d,
BuOH), yield 99%, mp 178–179°C, Rf 0.52 (petroleum ether– J=8.5Hz, 1H), 7.67 (d, J=8.5Hz, 1H), 7.54 (d, J=8.2Hz,
ethyl acetate, v/v=5:1). 1H-NMR (CDCl3) δ: 7.75 (d, J=8.5Hz, 1H), 7.52 (s, 1H), 7.38 (s, 1H), 7.12 (d, J=8.2Hz, 1H), 6.26 (d,
1H), 7.66 (s, 1H), 7.46 (d, J=8.5Hz, 1H), 7.39 (d, J=8.0Hz, J=0.75Hz, 1H), 6.20 (d, J=0.75Hz, 1H), 6.17 (d, J=3.0Hz,
1H), 7.11 (s, 1H), 6.92 (d, J=8.0Hz, 1H), 6.11 (s, 2H), 6.06 2H), 5.94 (s, 1H), 2.60 (s, 3H); 13C-NMR (DMSO-d6) δ: 148.3,
(s, 2H), 5.45 (s, 1H), 3.85 (m, 1H), 3.62 (m, 1H), 2.75 (s, 3H), 147.6, 147.6, 144.6, 138.1, 130.7, 125.7, 125.0, 124.8, 122.5,
1.46 (m, 2H), 1.20 (m, 2H), 0.78 (t, J=7.0Hz, 3H); 13C-NMR 120.0, 118.0, 117.1, 109.3, 107.2, 104.3, 102.2, 101.4, 99.3, 47.3,
(CDCl3) δ: 148.0, 147.4, 147.3, 145.2, 138.5, 131.0, 126.9, 125.9, 40.8. ESI-MS m/z: 332.27 [M−CN]+, 359.25 [M+H]+, 380.82
123.6, 123.0, 120.3, 116.4, 113.5, 108.7, 104.6, 101.7, 101.0, [M+Na]+;
100.7, 84.4, 66.2, 40.9, 31.5, 19.3, 13.9. ESI-MS m/z: 406.08
6-Cyano Dihydrochelerythrine (2f): White prism crystal,
yield 89%, mp 244–246°C. H-NMR (DMSO-d6) δ: 7.88 (d,
1
[M+H]+, 427.96 [M+Na]+, 332.28 [M−O(CH2)3CH3]+.
6-Methoxychelerythrine (2a): Yellow–white prism (MeOH), J=8.6Hz, 1H), 7.76 (d, J=8.7Hz, 1H), 7.67 (d, J=8.6Hz, 1H),
1
yield 95%, H-, 13C-NMR, ESI-MS and mp data were agree- 7.52 (s, 1H), 7.38 (s, 1H), 7.27 (d, J=8.7Hz, 1H), 6.18 (s, 1H),
ment with that previously reported by us.6)
6.17 (s, 1H), 5.93 (s, 1H), 3.92 (s, 3H), 3.90 (s, 3H), 2.58 (s,
6-Ethoxychelerythrine (2b): Yellow–white prism (EtOH), 3H); 13C-NMR (DMSO-d6) δ: 152.0, 148.1, 147.5, 145.3, 138.0,

1512
Vol. 60, No. 12
130.6, 125.5, 124.7, 123.7, 122.3, 119.7, 119.7, 119.4, 118.5, described above. The mites in each well were observed under
113.9, 104.2, 99.2, 101.2, 60.7, 55.7, 47.5, 40.6. ESI-MS m/z: a stereomicroscope every 0.5h or 1.0h and the percentage
375.29 [M+H]+, 396.78 [M+Na]+.
mortality and corrected percentage mortality for each well
Pharmacology. Screening of Acaricidal Activity in Vitro in each set time was calculated. The tested compound was
In vitro acaricidal activity of 1, 2 and their derivatives (1a–2i) performed in three test groups and each group consisted of
were performed according to the literature method with slight triplicate. The corrected percentage mortality of each group
modification.32) Psoroptes cuniculi adult mites of both sexes at each set time was expressed as means S.D. Probit value
isolated from naturally infected rabbits were used as tested of the corrected percentage mortality for each post-treatment
mites. The scabs and the cerumen, collected from the infected time and the corresponding log[post-treatment time (h)] value
ears, were observed by means of a stereoscopic microscope were used to establish toxicity regression equation for time ef-
to isolate adult mites of both sexes. Mites were placed in 24- fect. LT50 was calculated from it.
well flat-bottomed cell culture plates (20 adult mites per each
well). All tested compounds and the standard drug ivermectin
Acknowledgements This project was supported by the
were tested at the concentration of 5mg/mL in 10% dimethyl “National Natural Science Foundation” of China (NNSF; Nos.
sulfoxide (DMSO) and 10% Tween-80 in normal saline. Half 31172365, 30771454, 31000865).
milliliter of each tested solution was directly added to each
well. Three replicates were made for each concentration. As
untreated control, the same solution except for the tested com-
pound was used while ivermectin in the same solvent repre-
sented the treated control.
All the plates were placed in separate humidity chambers
in saturated humidity conditions at 22°C. After 24h each
plate was observed under a stereomicroscope for 5min. When
the persistent immobile mites were stimulated with a needle,
lack of reaction was considered as the indication of death.
Mortality rates were calculated as the following formula and
expressed as means S.D.
References
1) Krane B. D., Fagbule M. O., Shamma M., Gözler B., J. Nat. Prod.,
47, 1–43 (1984).
2) Ahsan H., Reagan-Shaw S., Breur J., Ahmad N., Cancer Lett., 249,
198–208 (2007).
3) Jang B. C., Park J. G., Song D. K., Baek W. K., Yoo S. K., Jung K.
H., Park G. Y., Lee T. Y., Suh S. I., Toxicol. In Vitro, 23, 281–287
(2009).
4) Vrba J., Doležel P., Víčar J., Ulrichová J., Toxicol. In Vitro, 23,
580–588 (2009).
5) Meng F. Y., Zuo G. Y., Hao X. Y., Wang G. C., Xiao H. T., Zhang J.
Q., Xu G. L., J. Ethnopharmacol., 125, 494–496 (2009).
6) Miao F., Yang X. J., Zhou L., Hu H. J., Zheng F., Ding X. D., Sun
D. M., Zhou C. D., Sun W., Nat. Prod. Res., 25, 863–875 (2011).
7) Lenfeld J., Kroutil M., Marsálek E., Slavík J., Preininger V.,
Simánek V., Planta Med., 43, 161–165 (1981).
8) Colombo M. L., Bosisio E., Pharmacol. Res., 33, 127–134 (1996).
9) Kerry M. A., Duval O., Waigh R. D., Mackay S. P., J. Pharm. Phar-
macol., 50, 1307–1315 (1998).
number of death mites
number of the tested mites
mortality (%)=
×100
Acaricidal Toxicity Assay 6-Methoxy dihydrosanguina-
rine (1a) with the strongest acaricidal activity was used for
acaricidal toxicity determination including toxicity regression
equation for concentration effect and time effect, LC50 and
LT50. Ivermectin was used as the treated control.
10) Tsai I. L., Wun M. F., Teng C. M., Ishikawa T., Chen I. S., Phyto-
chemistry, 48, 1377–1382 (1998).
The tested compound was completely dissolved in 0.2mL
DMSO, and then 0.2mL of Tween-80 and 1.6mL of normal
11) Eun J. P., Koh G. Y., Biochem. Biophys. Res. Commun., 317, 618–
624 (2004).
saline were added and completely mixed to prepare a series of 12) Cho K. M., Yoo I. D., Kim W. G., Biol. Pharm. Bull., 29, 2317–2320
(2006).
concentrations of 1.0, 0.8, 0.6, 0.4, 0.2, 0.1mg/mL of 1a and
13) Yao J. Y., Li X. L., Shen J. Y., Pan X. Y., Hao G. J., Xu Y., Ying W.
L., Ru H. S., Liu X. L., Aquaculture, 318, 235–238 (2011).
14) Wang G. X., Zhou Z., Jiang D. X., Han J., Wang J. F., Zhao L. W.,
Li J., Vet. Parasitol., 171, 305–313 (2010).
15) Nyangulu J. M., Hargreaves S. L., Sharples S. L., Mackay S. P.,
a series of concentrations of 1.5, 0.5, 0.2, 0.1, 0.05, 0.02mg/
mL of ivermectin. Acaricidal activity of each solution was as-
sayed at 24h according to the method described in “Screening
of Acaricidal Activity in Vitro.” Untreated control mites were
treated with the same solution except for the tested compound.
Each of the tested concentration was performed in three test
Waigh R. D., Duval O., Mberu E. K., Watkins W. M., Bioorg. Med.
Chem. Lett., 15, 2007–2010 (2005).
groups and each test group consisted of triplicate. The cor-
rected mortality of each group was calculated as the following
formula and expressed as means S.D.
16) Nakanishi T., Masuda A., Suwa M., Akiyama Y., Hoshino-Abe N.,
Suzuki M., Bioorg. Med. Chem. Lett., 10, 2321–2323 (2000).
17) Eldin S., Jencks W. P., J. Am. Chem. Soc., 117, 4851–4857 (1995).
18) Parenty A. D. C., Smith L. V., Pickering A. L., Long D. L., Cronin
L., J. Org. Chem., 69, 5934–5946 (2004).
corrected mortality (%)
% test mortality  % control mortality
19) Shimizu M., Itou H., Miura M., J. Am. Chem. Soc., 127, 3296–3297
(2005).
20) Yoshida J., Suga S., Suzuki S., Kinomura N., Yamamoto A., Fuji-
wara K., J. Am. Chem. Soc., 121, 9546–9549 (1999).
21) Parenty A. D. C., Smith L. V., Guthrie K. M., Long D. L., Plumb J.,
Brown R., Cronin L., J. Med. Chem., 48, 4504–4506 (2005).
22) Bates P. G., Vet. Parasitol., 83, 201–217 (1999).
23) Flatt R. E., Wiemers J., Lab. Anim. Sci., 26, 758–761 (1976).
24) Yeatts J. W., Vet. Rec., 134, 359–360 (1994).
=
×100
1.00%  % control mortality
Probit value of the corrected mortality at the test concentra-
tions and the corresponding log[concentration (mg/L)] value
were used to establish toxicity regression equation for concen-
tration effect by the linear least-square fitting method. LC50
value was calculated from the equation.
The test solution containing 10mg/mL 1a or ivermectin in
the same solvent was prepared and used to determine LT50
value, respectively. The determination of acaricidal activ-
ity of each solution was carried out according to the method
25) Synge B. A., Bates P. G., Clark A. M., Stephen F. B., Vet. Rec., 137,
51 (1995).
26) Clark A. M., Stephen F. B., Cawley G. D., Bellworthy S. J., Groves

December 2012
B. A., Vet. Rec., 139, 451 (1996).
1513
155–184 (2002).
27) Halley B. A., VandenHeuvel W. J. A., Wislocki P. G., Vet. Parasi- 31) Walterová D., Ulrichová J., Válka I., Vicar J., Vavrecková C., Tá-
tol., 48, 109–125 (1993).
borská E., Harjrader R. J., Meyer D. L., Cerná H., Simánek V., Acta
Univ. Palacki. Olomuc. Fac. Med., 139, 7–16 (1995).
32) Fichi G., Flamini G., Giovanelli F., Otranto D., Perrucci S., Exp.
Parasitol., 115, 168–172 (2007).
28) O’Brien D. J., Vet. Parasitol., 83, 177–185 (1999).
29) Currie B. J., Harumal P., McKinnon M., Walton S. F., Clin. Infect.
Dis., 39, e8–e12 (2004).
30) Dostál J., Slavík J., Studies in Natural Products Chemistry, 27,