Synthesis, structure-activity relationship and in vitro anti-mycobacterial evaluation of 13-n-octylberberine derivatives

Article abstract of DOI:10.1016/j.ejmech.2012.03.012

Twenty-eight new 13-n-octylberberine derivatives were synthesized and evaluated for their activities against drug-susceptible Mycobacterium tuberculosis (M. tuberculosis) strain H₃₇Rv. Among these compounds, compound 16e was the most effective anti-tubercular agent with a MIC value of 0.125 μg/mL. Importantly, compound 16e exhibited more potent effect against rifampicin (RIF)- and isoniazid (INH)-resistant M. tuberculosis strains than both RIF and INH, suggesting a new mechanism of action. Therefore, it has been selected as a drug candidate for further investigation, or as a chemical probe for identifying protein target and studying tuberculosis biology. We consider 13-n-octylberberine analogs to be a promising novel class of antituberculars against multi-drug-resistant (MDR) strains of M. tuberculosis.

Full text of DOI:10.1016/j.ejmech.2012.03.012

European Journal of Medicinal Chemistry 52 (2012) 151e158
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, structureeactivity relationship and in vitro anti-mycobacterial
evaluation of 13-n-octylberberine derivatives
Yan-Xin Liu 1, Chun-Ling Xiao 1, Yan-Xiang Wang, Ying-Hong Li, Yan-Hui Yang, Yang-Biao Li,
**
*
Chong-Wen Bi, Li-Mei Gao, Jian-Dong Jiang , Dan-Qing Song
Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Twenty-eight new 13-n-octylberberine derivatives were synthesized and evaluated for their activities
Received 17 January 2012
Received in revised form
28 February 2012
Accepted 6 March 2012
Available online 21 March 2012
against drug-susceptible Mycobacterium tuberculosis (M. tuberculosis) strain H37Rv. Among these
compounds, compound 16e was the most effective anti-tubercular agent with a MIC value of 0.125 mg/
mL. Importantly, compound 16e exhibited more potent effect against rifampicin (RIF)- and isoniazid
(INH)-resistant M. tuberculosis strains than both RIF and INH, suggesting a new mechanism of action.
Therefore, it has been selected as a drug candidate for further investigation, or as a chemical probe for
identifying protein target and studying tuberculosis biology. We consider 13-n-octylberberine analogs to
Keywords:
be
a promising novel class of antituberculars against multi-drug-resistant (MDR) strains of
13-n-Octylberberine
Antitubercular
Structureeactivity relationship
MIC
M. tuberculosis.
Ó 2012 Elsevier Masson SAS. All rights reserved.
Drug-resistance
1. Introduction
new mechanism of action, or without cross-resistance with current
anti-mycobacterial drugs [7b].
Tuberculosis (TB) is more prevalent in the world today than at
any other time in human history. It is anticipated that there will be
about 9.8 million new TB cases this year, more than in any other
year in history [1,2]. Five percent of all TB cases are now estimated
to be multi-drug-resistant TB (MDR-TB), which is resistant to at
least rifampin (RIF) and isoniazid (INH), the two most important
first-line drugs used currently in clinic [3a,3b]. The overall inci-
dence of TB in HIV positive patients is 50 times that of the rate for
HIV negative individuals [4ꢀ6]. In recent years, the emergence of
MDR-TB and extensively-drug-resistant TB (XDR-TB) becomes one
of the biggest challenges in the treatment of this disease. Although
some TB drug candidates are being currently evaluated in clinical
trials, there have been no approved new chemical entities for
treatment of TB in the past 40 years [7a,7b]. Therefore, there is an
urgent need to discover the novel scaffold of anti-TB candiates with
In search for new chemical classes of anti-mycobacterial agents,
the compound libraries constructed in our laboratories were
screened against drug-susceptible Mycobacterium tuberculosis
(M. tuberculosis) strain H37Rv with RIF and INH as reference drugs
[8]. 13-n-Octylberberine (1, Fig. 1), identified from the library of
berberines, appeared to show a moderate anti-tubercular activity
with a MIC of 2.0
mg/mL (Table 1). In particular, compound 1
demonstrated a mild antimycobaterial activity (Table 2) against
RIF- and INH-resistant strains isolated from patients in China
(MIC ¼ 4.0
mg/mL), suggesting a new mechanism of action [9].
Therefore, the new chemical entity and ideal actitity with no cross
resistance of hit 1 provoked our strong interest to explore the
structureeactivity relationship (SAR) so as to discover candidates
with novel mode of action for treatment of TB, especially MDR-TB.
Among the 13-substituted berberine analogs, compounds 2ꢀ9
(Fig. 1) showed no anti-mycobacterial activities against
M. tuberculosis H37Rv in comparison with 1. The primary SAR
analysis revealed that n-octyl side-chain at the 13-position of 1
might be essential for the bactericidal activity. Therefore, in the
present study we retained the n-octyl group at position 13, and
focused our SAR study on the variation of substituents at positions
2-, 3- and/or 9-position(s) with 1 as the lead. On the basis of this
* Corresponding author. Tel./fax: þ86 10 6316 5268.
** Corresponding author. Tel.: þ86 10 8316 0005; fax: þ86 10 6301 7302.
E-mail addresses: jiang.jdong@163.com (J.-D. Jiang), songdanqingsdq@hotmail.
com (D.-Q. Song).
1
These authors made equal contributions to the work.
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.03.012

152
Y.-X. Liu et al. / European Journal of Medicinal Chemistry 52 (2012) 151e158
Table 2
4
In vitro anti-tubercular activities of the key compounds against MDR strains of
O
O
M. tuberculosisa (MIC: g/mLb)
m
6
Cl-
D
A
B
N+
C
Compd 44 83
164
431
926
H37Rv
1
4
4
0.5
0.25
64
4
4
4
1.0
0.5
> 64
4
4
4
4
ND
4
0.5
0.125
> 32
32
2.0
0.5
0.5
0.125
0.0625
0.0625
2
14g
16d
16e
RIF
ND
0.25
0.5
> 64
64
1
1.0
0.125
> 64
4
13
OCH3
OCH3
n-CnH2n+1
9
INH
a
MDR strains were isolated from patients with tuberculosis in China.
MIC: minimum inhibitory concentration.
10
b
compound 1:
compounds 2−9: n=0-7
n=8
2. Chemistry
Twenty-eight 13-n-octylberberine analogs were synthesized
with commercially available berberine (2) as the starting material
as described in Scheme 1, which includes three synthetic methods
(Route A, B and C). The intermediate 10 was obtained via a selective
reduction reaction, in which NaBH4 was used as a reducing agent
and methanol as the solvent [10]. Then, the intermediate 10 reacted
with n-octyl aldehyde in the solvent mixture of EtOH (80%) and
HOAc, and then acidified with 2 N HCl to yield the desired
compound 1 in a good yield [10,11].
Fig. 1. Chemical structures of compounds 1ꢀ9.
strategy, 28 new 13-n-octylberberine analogs were designed,
synthesized and evaluated for their in vitro anti-tubercular
activities against drug-susceptible and drug-resistant stains of
M. tuberculosis.
Table 1
Compound 1 was heated at 195ꢀ210 ꢁC for 10ꢀ15 min under
vacuum (30ꢀ40 mmHg) to afford the black oil [12], which was
recrystallized with ethanol/concentrated HCl (95:5) to afford 11. The
desired products 12aꢀd (Route A) were obtained through nucleo-
philic substitutionof 11 with different RX(X ¼Cl, Br, I)respectivelyin
yields of 36e40%, in which K2CO3/KOH was used as the base and
DMF as the solvent [13ꢀ15]. The products 12eꢀh were prepared by
esterification of 11 with the different acyl chloride, using pyridine as
the base and acetonitrile as the solvent with yields of 21%e47%
[16ꢀ17]. The key intermidiate 13 (Route B) was synthesized by
demethyl reaction of compound 1 with phloroglucinol in H2SO4
(60%) at 90ꢀ95 ꢁC [18]. In the thirdsynthetic route (Route C), another
key intermediate 15 was gotten through demethyl reaction of 1 with
AlCl3 (4.4 eq) in CH2Cl2 [19ꢀ21]. The final compounds in 12, 14 and
16 series were purified by flash chromatography using CH2Cl2/
CH3OH (95: 5) as the gradient eluent.
Structures and anti-mycobacterial activities of aimed compounds against M. tuber-
culosis strain H37Rv.
R3
+Cl-
N
R2
R1
n-C8H17
OCH3
Compd
R1
R2
R3
MICa
(mg/mL)
1
11
OCH3
OH
OCH2O
OCH2O
OCH2O
OCH2O
OCH2O
OCH2O
OCH2O
OCH2O
OCH2O
OCH2O
2.0
4.0
4.0
2.0
8.0
4.0
4.0
4.0
16.0
8.0
12a
12b
12c
12d
12e
12f
12g
12h
13
14a
14b
14c
14d
14e
14f
14g
14h
14i
14j
14k
15
OCH2CH3
n-OC3H7
n-OC4H9
OCH2Ph
peCH3ePhCOO
peCH3OePhCOO
peCF3ePhSO3
peCNePhSO3
OCH3
3. Results and discussion
3.1. SAR analysis for anti-mycobacterial activity
Twenty-eight 13-substituted berberine derivatives were first
evaluated for their anti-tubercular activities against the multipli-
cation of drug-susceptible M. tuberculosis strain H37Rv with the
microplate alamar blue assay (MABA). RIF and INH were used as
reference drugs. Structures of 28 analogs and their anti-mycobac-
terial activities are shown in Table 1.
SAR study was first focused on the substituents at the 9-position
on the aromatic ring D of 1. Replacement methoxyl at the 9-position
with hydroxyl, ethoxyl, n-propoxy, n-butoxy, benzyloxy, benzoy-
loxy or benzenesulfonyl respectively, generated nine new analogs
(11, 12aꢀh). The result showed that compound 12b bearing an
ethoxyl afforded a mediated anti-tubercular activity at a MIC value
OH
OH
16.0
2.0
2.0
4.0
8.0
2.0
2.0
0.5
2.0
2.0
8.0
2.0
32.0
4.0
4.0
8.0
0.5
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OH
OH
OCH3
OH
OH
OH
OH
OCH3
OCH2CH3
n-OC3H7
n-OC4H9
OCH2Ph
OCH2CH3
n-OC3H7
n-OC4H9
OCH2Ph
OCH3
OCH2CH3
n-OC3H7
n-OC4H9
OCH2Ph
OCH2CH2O
OH
OH
16a
16b
16c
16d
16e
16f
INH
RIF
OCH2CH3
n-OC3H7
n-OC4H9
OCH2Ph
OCH2CH3
OCH2Ph
OH
OH
OH
OH
OCH2CH3
OCH2Ph
OCH2CH3
n-OC3H7
n-OC4H9
OCH2Ph
OCH2CH3
OCH2Ph
of 2.0
12a, 12cꢀh) showed decreased activities with MIC ranges between
4.0 and 16.0 g/mL, regardless of the size of the side-chains.
mg/mL, similar to that of the lead. The other compounds (11,
m
0.125
8.0
0.0625
0.0625
Next, SAR analysis was moved to the modifications at the 2- and
3-positions on the ring A. The oxacyclopentene was opened, the
resulting compound 13 bearing 2,3-dihydroxyl almost lost the
activity. A methoxyl, ethoxyl, n-propoxy, n-butoxy or benzyloxy
a
MIC: minimum inhibitory concentration.

Y.-X. Liu et al. / European Journal of Medicinal Chemistry 52 (2012) 151e158
153
O
O
O
O
O
O
Cl-
N+
Cl-
N
N+
b
a
OCH3
OCH3
OCH3
OCH3
OCH3
n-C8H17
OCH3
1
10
2
O
O
O
+ Cl-
+ Cl-
N
N
c
O
f or g
OH
R1
Route A
n-C8H17
n-C8H17
n-C8H17
n-C8H17
OCH3
OCH3
11
12a-h
R3
R2
HO
HO
Cl-
Cl-
N+
N+
f
d
Route B
OCH3
OCH3
n-C8H17
OCH3
OCH3
14a-k
13
R3
R2
Cl-
HO
HO
Cl-
N+
N+
f
e
Route C
R1
OH
n-C8H17
OCH3
OCH3
15
16a-f
Scheme 1. Reagents and conditions for the chemical synthesis: (a) NaBH4, NaOH; (b) octyl aldehyde, acetic acid, 80% ethanol; (c) 195 ꢁC, 30ꢀ40 mmHg, 10ꢀ15 min; (d) phlor-
oglucinol, 60% H2SO4, 90ꢀ95 ꢁC; (e) AlCl3 (4.4 eq), rt, CH2Cl2; (f) RX, K2CO3/KOH, 70 ꢁC; (g) RCOX, pyridine, CH3CN, reflux.
was attached at the 3-position of 13, respectively, with which five
new analogs (14aꢀe) were prepared and tested. Compounds 14aꢀb
and 14e afforded a similar anti-mycobacterial activity to the lead
3.2. Anti-resistance TB effect of the key compounds
As compounds 14g, 16d and 16e possessed excellent activity
against drug-susceptible M. tuberculosis strain H37Rv with MIC
with a MIC of 2.0 mg/mL. Then, introduction of a methoxyl, ethoxyl,
n-propoxy, n-butoxy, benzyloxy or ethylenedioxy at the positions 2
and 3 of 13 simultaneously, produced six compounds 14fꢀk. The
results showed that compounds 14f, 14hei and 14k demonstrated
values below 0.5 mg/mL, they were further examined the effect on
the MDR strains of M. tuberculosis. In this experiment,
M. tuberculosis strains 44, 83, 164, 431 and 926 isolated from
patients with tuberculosis in China, were resistant to both RIF and
INH. As described in Table 2, RIF and INH showed a decreased
activity against the drug-resistant stains partially or completely
a moderate activity at a MIC of 2.0
sessing 2, 3-diethoxyl exhibited an increased activity with a MIC
value of 0.5 g/mL, greater than that of the lead. It seemed that
mg/mL. Compound 14g pos-
m
introducting a substituent at the 2- and/or 3-position(s) might be
benefitable for the anti-mycobacterial activity.
with a MIC range between 4 and >64
16d and 16e inhibited the drug-resistant strains with a potency
(MIC ¼ 0.125e1.0 g/mL) similar to its effect on drug-susceptible
strain (MIC 0.125e0.5 g/mL). The results indicated that
compounds 16d and 16e were active for either drug-susceptible
M. tuberculosis or those resistant to RIF and INH, suggesting
a novel mechanism of action against TB.
mg/mL. However, compounds
In another variation, SAR study was carried out to explore the
substituents at positions 2, 3 and 9. The substituents including
a hydroxyl, ethoxyl, n-propoxy, n-butoxy or benzyloxy were added
at the 2-, 3- and 9-positions, respectively, by which seven new
analogs (15, 16aꢀf) were made and tested. Compounds 16dꢀe
m
¼
m
showed a potent activity at a MIC of 0.5 and 0.125 mg/mL, respec-
tively, in comparison with the lead. Especially, compound 16e
possessing tri-ethoxyl exhibited the strongest anti-tubercular
activity among the synthezied compounds, with 16-fold increase
over that of the lead. Thus, attachment of the suitable substituents
at the 2- 3- and 9-positions, especially an ethoxyl, might signifi-
cantly improve the activity against M. tuberculosis strain.
4. Conclusion
In conclusion, 28 new 13-n-octylberberines with different
substituents on the ring A and D were synthesized and evaluated
for their anti-mycobacterial activities against M. tuberculosis H37Rv
with 1 as the lead. SAR analysis revealed that (i) n-octyl group at

154
Y.-X. Liu et al. / European Journal of Medicinal Chemistry 52 (2012) 151e158
position 13 might be essential for the activity; (ii) introduction of
substituents at the 2-, 3- and/or 9-positions, especially an ethoxyl,
might significantly enhance the activity. Among the test
compounds, compound 16e exhibited a potential activity against
both drug-susceptible and MDR strains of M. tuberculosis, sug-
gesting a novel mode of action against TB. Thus, it has been selected
(1.14 g, 98%) as a dark red soild. mp: 122e124 ꢁC 1H NMR (CD3OD)
d:
0.85 (t, 3H, J ¼ 7.2 Hz), 1.23e1.41 (m, 10H), 1.82 (s, 2H), 3.04 (t, 2H,
J ¼ 6.0 Hz), 3.33 (t, 2H, J ¼ 8.4 Hz), 4.03 (s, 3H), 4.67 (t, 2H,
J ¼ 6.0 Hz), 6.06 (s, 2H), 6.96 (s, 1H), 7.23 (s, 1H), 7.79 (d, 1H,
J ¼ 9.2 Hz), 7.96 (d, 1H, J ¼ 9.2 Hz), 9.72 (s, 1H); 13C NMR (DMSO-d6)
d: 148.8, 146.5, 145.2, 144.6, 144.4, 134.8, 133.9, 133.6, 131.6, 124.6,
as
a
drug candidate for further investigation. In particular,
120.5, 117.2, 115.9, 109.1, 108.3, 102.0, 56.9, 56.7, 31.2, 30.2, 28.9,
28.6, 28.5, 28.4, 27.5, 22.0, 13.9. HRMS: calcd for C27H32NO4Cl
[MꢀCl]þ 434.2331, found 434.2340.
compound 16e might be used as a chemical probe for identifying
new protein targets, unraveling mechanism of action as well as
studing TB biology. We consider 13-n-octylberberines to be
a promising new class of anti-tubercular agents with an advantage
of inhibiting MDR strains of M. tuberculosis.
5.4. General procedure for the synthesis of compounds 12aꢀd
(Route A)
5. Experimental section
To a stirred solution of 11 (200 mg, 0.43 mmol) and KOH
(78.4 mg,1.4 mmol) in DMF (10 mL), RX (1.0e1.5 eq) was added. The
reaction mixture was stirred at 70 ꢁC for 5ꢀ6 h. The solvent was
removed by evaporation, the residue was acidified by 2 N HCl, the
soild was collected by filtration and then purified by flash chro-
matography over silica gel using CH2Cl2/CH3OH (95:5) as the
gradient eluent, affording the title compounds 12aꢀd. Compounds
12aꢀd were gained following the same procedure using purchased
alkyl halide as material.
5.1. Chemistry
Melting point (mp) was obtained with CXM-300 melting point
apparatus and uncorrected. The 1H NMR spectra was performed on
a Varian Inova 400 MHz spectrometer (Varian, San Francisco, CA)
and 13C NMR on a Bruker Avance III 400 spectrometer in CD3OD or
DMSO-d6, with Me4Si as the internal standard. 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 CombiflashRf 200 (Teledyne,
Nebraska, USA), particle size 0.038 mm.
5.4.1. 2,3-Methylenedioxy-9-ethoxy-10-methoxy-13-n-octylproto-
berberine chloride (12a)
Compound 11 (200 mg, 0.43 mmol) was treated with ethyl
bromide (37.3
give the desired product 12a [8a,8c] as a brown solid, yield: 36%;
mp: 86e88 ꢁC; 1H NMR (400 MHz, CD3OD)
ml，0.5 mmol) according to the general procedure to
5.2. 2,3-Methylenedioxy-9,10-dimethoxy-13-n-octylprotoberberine
chloride (1)
d: 0.84 (t, 3H, J ¼ 7.2 Hz),
1.24e1.48 (m, 13H), 1.81 (t, 2H, J ¼ 7.6 Hz), 3.06 (t, 2H, J ¼ 6.0 Hz),
3.37 (t, 2H, J ¼ 8.0 Hz), 4.06 (s, 3H), 4.43 (q, 2H, J ¼ 6.8, 14.2 Hz), 4.74
(t, 2H, J ¼ 6.0 Hz), 6.07 (s, 2H), 6.98 (s, 1H), 7.24 (s, 1H), 8.08, 8.12
To a stirred solution of BBR (7.43 g, 20 mmol) and K2CO3 (8.30 g,
60 mmol) in methanol (250 mL), 5% NaOH (10 mL) solution con-
taining NaBH4 (0.80 g, 21 mmol) was added dropwise. The reaction
mixture was stirred at room temperature for 2 h and the precipi-
tated product was filtered, washed with water (20 mL) and 30%
ethanol (20 mL), and then recrystallized from absolute ethanol to
provide the intermediate 10 as a dark green solid.
(dd, 2H, J ¼ 9.6 Hz), 9.67 (s, 1H); 13C NMR (CD3OD)
d: 151.8, 151.3,
148.7, 145.3, 145.1, 137.7, 136.3, 135.1, 134.4, 127.0, 123.5, 122.1, 121.8,
110.4, 109.3, 103.7, 71.5, 58.9, 57.5, 32.9, 31.9, 30.5, 30.3 (2), 30.1,
29.3, 23.6, 15.9, 14.4. HRMS: calcd for C29H36NO4Cl [MꢀCl]þ
462.2644, found 462.2635.
To a stirred solution of the intermediate 10 (5.0 g, 15 mmol) in
80% ethanol (200 mL), n-octyl aldehyde (10 mL), HOAc (50 mL) was
added. The reaction mixture was heated to 85ꢀ95 ꢁC for 5 h. The
solvent was removed by evaporation to get the red oil. Diethyl ether
(200 mL) was added to this system and stirred at room temperature
for 2 h. After filtration, the diethyl ether layer was collected and
concentrated, and the residue was acidified by 2% HCl, stirred at
room temperature for 0.5 h. The solid was obtained by filtration to
afford the title compound 1 (4.6 g, 48%) as a yellow solid. mp:
5.4.2. 2,3-Methylenedioxy-9-n-propoxy-10-methoxy-13-n-octylpro-
toberberine chloride (12b)
Compound 11 (150 mg, 0.32 mmol) was treated with propyl
iodide (47
the desired product 12b as a brown solid, yield: 39%; mp: 95e97 ꢁC;
1H NMR (400 MHz, CD3OD)
ml, 0.48 mmol) according to the general procedure to give
d: 0.84 (t, 3H, J ¼ 6.8 Hz), 1.06 (t, 3H,
J ¼ 7.6 Hz), 1.24e1.26 (m, 10H), 1.41 (t, 2H, J ¼ 7.2 Hz), 1.79e1.92 (m,
2H), 3.06 (t, 2H, J ¼ 5.6 Hz), 3.37 (t, 2H, J ¼ 7.6 Hz), 4.06 (s, 3H), 4.31
(t, 2H, J ¼ 6.8 Hz), 4.74 (t, 2H, J ¼ 5.6 Hz), 6.07 (s, 2H), 6.98 (s, 1H),
7.24 (s, 1H), 8.08, 8.12 (dd, 2H, J ¼ 9.2 Hz), 9.63 (s, 1H); 13C NMR
111e113 ꢁC 1H NMR (CD3OD)
d
: 0.84 (t, 3H, J ¼ 6.8 Hz),1.24e1.28 (m,
8H), 1.39e1.41 (m, 2H), 1.79e1.81 (m, 2H), 3.06 (t, 2H, J ¼ 5.6 Hz),
3.37 (t, 2H, J ¼ 8.4 Hz), 4.07 (s, 3H), 4.15 (s, 3H), 4.73 (t, 2H,
J ¼ 5.6 Hz), 6.07 (s, 2H), 6.98 (s, 1H), 7.24 (s, 1H), 8.10 (d, 2H,
(DMSO-d6) d: 150.2, 149.0, 146.5, 144.1, 143.4, 135.7, 134.2, 134.0,
132.2, 125.8, 121.4, 121.2, 120.3, 109.1, 108.3, 102.1, 75.9, 57.1, 56.9,
31.2, 30.4, 28.9, 28.6, 28.5, 28.3, 27.4, 22.8, 22.0, 13.9, 10.2. HRMS:
calcd for C30H38NO4Cl [MꢀCl]þ 476.2801, found 476.2806.
J ¼ 9.6 Hz), 9.72 (s, 1H); 13C NMR (DMSO-d6)
d: 150.2, 149.0, 146.5,
144.3, 144.2, 135.7, 134.2, 134.0, 132.2, 125.9, 121.4, 121.2, 120.3,
109.1, 108.3, 102.1, 62.0, 57.0 (2), 31.2 (2), 30.4, 28.8, 28.5, 28.3, 27.3,
22.0, 13.9. HRMS: calcd for C28H34NO4Cl [MꢀCl]þ 448.2484, found
448.2506.
5.4.3. 2,3-Methylenedioxy-9-n-butoxy-10-methoxy-13-n-octylpro-
toberberine chloride (12c)
Compound 11 (150 mg, 0.32 mmol) was treated with butyl
5.3. 2,3-Methylenedioxy-9-hydroxy-10-methoxy-13-n-octylprotob-
erberine chloride (11)
iodide (55
the desired product 12c as a brown solid, yield: 39%; mp:
114e116 ꢁC; 1H NMR (400 MHz, CD3OD)
ml, 0.48 mmol) according to the general procedure to give
d: 0.84 (t, 3H, J ¼ 6.8 Hz),
Compound 1 (1.2 g, 2.48 mmol) was heated at 195ꢀ210 ꢁC for
10ꢀ15 min under vacuum (30ꢀ40 mmHg) to afford the black oil,
which was acidified with ethanol/concentrated HCl (95:5). The
solvent was removed by evaporation, the residue was collected and
then purified by flash chromatography over silica gel using CH2Cl2/
CH3OH as the gradient eluent, affording the title compound 11
0.98 (t, 3H, J ¼ 7.6 Hz), 1.23e1.88 (m, 16H), 3.06 (t, 2H, J ¼ 5.6 Hz),
3.37 (t, 2H, J ¼ 8.0 Hz), 4.06 (s, 3H), 4.31 (t, 2H, J ¼ 6.4 Hz), 4.74 (t,
2H, J ¼ 5.6 Hz), 6.07 (s, 2H), 6.98 (s, 1H), 7.24 (s, 1H), 8.08, 8.11 (dd,
2H, J ¼ 9.2 Hz), 9.60 (s, 1H); 13C NMR (DMSO-d6)
d: 150.2, 149.0,
146.5, 144.1, 143.4, 135.7, 134.2, 134.0, 132.2, 125.8, 121.4, 121.2,
120.3, 109.1, 108.3, 102.1, 74.0, 57.1, 56.9, 31.5, 31.2, 30.3, 28.9, 28.6,

Y.-X. Liu et al. / European Journal of Medicinal Chemistry 52 (2012) 151e158
155
28.5, 28.3, 27.4, 22.0, 18.5, 13.9, 13.7. HRMS: calcd for C31H40NO4Cl
5.5.3. 2,3-Methylenedioxy-9-O-(4-trifluoromethylbenzenesulfonyl)-
10-methoxy-13-n- octylprotoberberine chloride (12g)
[MꢀCl]þ 490.2957, found 490.2969.
Compound 11 (110 mg, 0.23 mmol) was treated with anhydrous
5.4.4. 2,3-Methylenedioxy-9-benzyloxy-10-methoxy-13-n-octylpro-
toberberine chloride (12d)
pyridine (20 ml, 0.25 mmol) and 4-trifluoromethyl-benzenesulfonyl
chloride (0.24 g, 1.0 mmol), according to the general procedure to
Compound 11 (150 mg, 0.32 mmol) was treated with benzyl
give the desired product 12g as a brown solid, yield: 21%; mp:
bromide (42
give the desired product 12d as a brown solid, yield: 40%; mp:
102e103 ꢁC; 1H NMR (400 MHz, CD3OD)
m
l, 0.35 mmol) according to the general procedure to
103e104 ꢁC; 1H NMR (CD3OD)
d: 0.85 (t, 3H, J ¼ 6.8 Hz), 1.24e1.27
(m, 8H), 1.42 (t, 2H, J ¼ 6.8 Hz), 1.82 (t, 2H, J ¼ 6.8 Hz), 3.09 (t, 2H,
J ¼ 5.6 Hz), 3.42 (t, 2H, J ¼ 8.0 Hz), 3.67 (s, 3H), 4.83 (s, 2H), 6.09 (s,
2H), 7.02 (s, 1H), 7.29 (s, 1H), 7.67 (d, 2H, J ¼ 8.4 Hz), 7.92 (d, 2H,
J ¼ 8.0 Hz), 8.01, 8.17 (dd, 2H, J ¼ 8.4 Hz), 9.62 (s, 1H); 13C NMR
d: 0.84 (t, 3H, J ¼ 6.8 Hz),
1.12e1.40 (m, 10H), 1.78e1.80 (m, 2H), 3.00 (t, 2H, J ¼ 5.6 Hz), 3.36
(s, 2H), 4.11 (s, 3H), 4.61 (t, 2H, J ¼ 5.6 Hz), 5.40 (s, 2H), 6.06 (s, 2H),
6.96 (s, 2H), 7.21e7.29 (m, 4H), 7.42 (d, 1H, J ¼ 6.4 Hz), 8.11 (s, 2H),
(CD3OD) d: 153.0, 151.8, 150.3, 148.9, 143.9, 140.8, 139.2, 137.4, 135.3,
9.42 (s, 1H); 13C NMR (DMSO-d6)
d: 150.5, 149.0, 146.5, 144.2, 142.6,
134.9 (3), 127.7 (3), 126.4 (2), 126.1, 124.0, 121.5, 110.5, 109.4, 103.9,
59.4, 57.3, 33.0, 32.2, 30.7, 30.3 (2), 30.1, 29.0, 23.7, 14.4. HRMS:
calcd for C34H35F3NO6SCl [MꢀCl]þ 642.2137, found 642.2171.
136.4, 135.7, 134.2, 134.0, 132.1, 128.8 (2), 128.4, 128.3 (2), 125.7,
121.7, 121.6, 120.2, 109.1, 108.3, 102.1, 75.5, 57.1, 57.0, 31.2, 30.4, 28.8,
28.6 (2), 28.3, 27.4, 22.0, 13.9. HRMS: calcd for C34H38NO4Cl
[MꢀCl]þ 524.2801, found 524.2811.
5.5.4. 2,3-Methylenedioxy-9-O-(4-cyanobenzenesulfonyl)-10-
methoxy-13-n-octylprotoberberine chloride (12h)
5.5. General procedure for the synthesis of compounds 12eꢀh
Compound 11 (105 mg, 0.22 mmol) was treated with anhydrous
pyridine (20 ml, 0.25 mmol) and 4-cyano-benzenesulfonyl chloride
(Route A)
(0.20 g, 1.0 mmol), according to the general procedure to give the
To a solution of CH3CN (5 mL) was added 11 (105 mg,
0.22 mmol) at room temperature, then heated to form a dark-red
desired product 12h as a brown solid, yield: 40%; mp: 99e100 ꢁC;
1H NMR (CD3OD)
d
: 0.85 (t, 3H, J ¼ 6.8 Hz), 1.24e1.28 (m, 8H),
solution, to which was then added anhydrous pyridine (20
m
l)
1.41 (m, 2H), 1.81 (m, 2H), 3.09 (t, 2H, J ¼ 5.6 Hz), 3.42 (t, 2H,
J ¼ 8.0 Hz), 3.70 (s, 3H), 4.83 (s, 2H), 6.09 (s, 2H), 7.01 (s, 1H), 7.27 (s,
1H), 7.73 (d, 2H, J ¼ 8.0 Hz), 7.89 (d, 2H, J ¼ 8.4 Hz), 8.04, 8.14 (dd,
and acyl chloride or sulfuryl chloride. The resulting mixture was
refluxed at 75ꢀ80 ꢁC for 5ꢀ6 h and cooled to precipitate
completely. The crude product was filtered and purified by flash
chromatography over silica gel using CH2Cl2/CH3OH (95:5) as the
gradient eluent. Compounds 12eꢀh were gained following the
same procedure using purchased acyl chloride or sulfuryl chloride
as material.
2H, J ¼ 8.4 Hz), 9.62 (s, 1H); 13C NMR (DMSO-d6)
d: 152.6, 151.0,
149.4, 146.6, 142.5, 138.7, 137.2, 135.2, 134.2, 133.8, 132.0, 131.3,
129.3, 126.4, 125.2,121.6,120.0,118.6,117.6,111.0,109.2, 108.3, 102.2,
57.3, 56.8, 31.2, 30.6, 29.0, 28.5 (2), 28.3, 27.1, 22.0,13.9. HRMS: calcd
for C34H35N2O6ClS [MꢀCl]þ 599.2216, found 599.2237.
5.5.1. 2,3-Methylenedioxy-9-O-(4-methylbenzoyl)-10-methoxy-13-
neoctylprotoberberine chloride (12e)
5.6. 2,3-Dihydroxy-9,10-dimethoxy-13-n-octylprotoberberine
chloride (13)
Compound 11 (108 mg, 0.23 mmol) was treated with anhydrous
pyridine (20
m
l, 0.25 mmol) and 4-methylbenzoyl chloride (134 ul,
To a stirred solution of 60% H2SO4 (100 mL), phloroglucin (1.16 g,
7.14 mmol) was added portionwise to form a colorless solution,
then compound 1 (1.00 g, 2.07 mmol) was added portionwise and
the mixture was stirred at 90ꢀ95 ꢁC for 10ꢀ15 min. Then the
mixture was poured into saturated brine (20 mL) immediately with
violent stirring, and the resulting mixture was stirred at room
temperature for 2 h and cooled down to precipitate completely,
filtration, water washing, filter cake layer was adjusted to neutral
with NaOH (1 N), then adjusted to acidity with HCl (2 N), stirred,
filtration, collecting solid precipitation, recrystallization with
ethanol/concentrated HCl (95:5) to afford the title compound 13
(0.86 g, 88%) as a dark red soild. mp: 114.4e116.0 ꢁC; 1H NMR
1.0 mmol), according to the general procedure to give the desired
product 12e as a brown solid, yield: 47%; mp: 118e121 ꢁC; 1H NMR
(CD3OD)
d
: 0.85 (t, 3H, J ¼ 6.8 Hz), 1.25e1.31 (m, 8H), 1.44 (t, 2H,
J ¼ 6.8 Hz), 1.86 (m, 2H), 2.45 (s, 3H), 3.03 (t, 2H, J ¼ 5.6 Hz), 3.44 (t,
2H, J ¼ 8.0 Hz), 4.03 (s, 3H), 4.71 (t, 2H, J ¼ 5.6 Hz), 6.08 (s, 2H), 6.97
(s, 1H), 7.27 (s, 1H), 7.40 (d, 2H, J ¼ 8.0 Hz), 8.14 (d, 2H, J ¼ 8.0 Hz),
8.21, 8.41 (dd, 2H, J ¼ 9.6 Hz), 9.65 (s, 1H); 13C NMR (CD3OD)
d:
165.4, 152.3, 151.5, 148.8, 147.0, 144.0, 138.5, 137.1, 136.6, 135.2,
134.4, 131.8, 131.3, 130.7, 130.2, 126.7, 126.2, 125.9, 123.3, 121.7,
110.5, 109.3, 103.8, 59.0, 57.7, 33.0, 32.2, 30.6, 30.3 (2), 30.1, 29.1,
23.7, 21.8, 14.4. HRMS: calcd for C35H38NO5Cl [MꢀCl]þ 552.2750,
found 552.2743.
(DMSO-d6)
d
: 0.85 (t, 3H, J ¼ 6.8 Hz), 1.24e1.29 (m, 8H), 1.42e1.44
(m, 2H), 1.78 (m, 2H), 2.97 (t, 2H, J ¼ 5.6 Hz), 3.30 (t, 2H,
J ¼ 8.0 Hz), 4.07 (s, 6H), 4.75 (t, 2H, J ¼ 5.6 Hz), 6.85 (s, 1H), 7.18 (s,
1H), 8.13, 8.17 (dd, 2H, J ¼ 9.6 Hz), 9.83 (s, 1H); 13C NMR (DMSO-d6)
5.5.2. 2,3-Methylenedioxy-9-O-(4-methoxylbenzoyl)-10-methoxy-
13-n-octylprotoberberine chloride (12f)
Compound 11 (129 mg, 0.27 mmol) was treated with anhydrous
d: 149.8,148.0,144.2,144.1 (2),136.5,133.2,132.4,130.3,125.9,121.1,
pyridine (24
m
l, 0.30 mmol) and 4-methoxylbenzoyl chloride
121.0, 117.8, 116.5, 114.7, 62.0, 57.4, 57.0, 31.2, 30.6, 28.9, 28.8, 28.7,
28.5, 26.7, 22.0, 13.9. HRMS: calcd for C27H34NO4Cl [MꢀCl]þ
436.2488, found 436.2480.
(0.21 g，1.23 mmol), according to the general procedure to give
the desired product 12f as a brown solid, yield: 43%; mp:
118e120 ꢁC; 1H NMR (CD3OD)
d
: 0.85 (t, 3H, J ¼ 6.8 Hz), 1.25e1.29
(m, 8H), 1.44 (t, 2H, J ¼ 6.8 Hz), 1.86 (m, 2H), 3.03 (t, 2H, J ¼ 5.6 Hz),
3.44 (t, 2H, J ¼ 8.0 Hz), 3.89 (s, 3H), 4.03 (s, 3H), 4.72 (t, 2H,
J ¼ 5.6 Hz), 6.07 (s, 2H), 6.97 (s, 1H), 7.09 (d, 2H, J ¼ 8.8 Hz), 7.27 (s,
1H), 8.20 (m, 3H), 8.40 (d, 1H, J ¼ 9.6 Hz), 9.64 (s, 1H); 13C NMR
5.7. General procedure for the synthesis of compounds 14aꢀk
(Route B)
To a stirred solution of 13 (300 mg，0.62 mmol) and KOH
(139 mg，2.48 mmol) in DMF (10 mL), RX (1.0 or > 2.0 eq) was
added. The reaction mixture was stirred at 70 ꢁC for 5ꢀ6 h. The
solvent was removed by evaporation, the residue was acidified by
2 N HCl, the soild was collected by filtration and then purified by
flash chromatography over silica gel using CH2Cl2/CH3OH (95:5) as
(DMSO-d6) d: 164.2, 163.0, 150.2, 149.1, 146.6, 143.4, 136.4, 134.7,
134.4, 134.2, 132.7, 132.1, 131.3, 125.0, 124.7, 121.2, 120.2, 120.0,
114.4, 113.8, 109.2, 108.3, 102.1, 57.2, 57.0, 55.8, 31.2 (2), 30.5, 28.9,
28.6, 28.4, 27.2, 22.0, 13.9. HRMS: calcd for C35H38NO6Cl [MꢀCl]þ
568.2699, found 568.2685.

156
Y.-X. Liu et al. / European Journal of Medicinal Chemistry 52 (2012) 151e158
the gradient eluent, affording the title compounds 14aꢀk.
Compounds 14aꢀk were gained following the same procedure
using purchased alkyl halide as material.
113e115 ꢁC; 1H NMR (CD3OD)
d: 0.85 (t, 3H, J ¼ 6.8 Hz), 1.13e1.27
(m, 8H), 1.44e1.46 (m, 2H), 1.83 (m, 2H), 3.00 (s, 2H), 3.36 (t, 2H,
J ¼ 8.0 Hz), 4.06 (s, 3H), 4.14 (s, 3H), 4.71 (s, 2H), 5.26 (s, 2H), 7.06 (s,
2H), 7.28 (d,1H, J ¼ 9.2 Hz), 7.34 (m, 3H), 7.46 (d,1H, J ¼ 7.6 Hz), 8.09
5.7.1. 2-Hydroxy-3,9,10-trimethoxy-13-n-octylprotoberberine
chloride (14a)
(s, 2H), 9.68 (s, 1H); 13C NMR (DMSO-d6)
d: 150.0, 148.6, 145.5,
144.2, 144.1, 136.6, 136.0, 133.7, 132.3, 130.0, 128.4 (2), 127.9, 127.7
(2), 125.9, 121.2, 121.1, 119.4, 116.5, 112.7, 69.9, 62.0, 57.3, 56.9, 31.2
(2), 30.5, 28.9, 28.7, 28.4, 26.9, 22.0, 13.9. HRMS: calcd for
C34H40NO4Cl [MꢀCl]þ 526.2957, found 526.2942.
Compound 13 (150 mg，0.32 mmol) was treated with methyl
iodide (20
give the desired product 14a as a brown solid, yield: 29%; mp:
88e90 ꢁC; 1H NMR (CD3OD)
ml，0.32 mmol), according to the general procedure to
d
: 0.85 (t, 3H, J ¼ 6.8 Hz), 1.24 (m, 8H),
1.43e1.45 (m, 2H), 1.83 (m, 2H), 3.06 (t, 2H, J ¼ 5.6 Hz), 3.37 (t, 2H,
J ¼ 8.0 Hz), 3.93 (s, 3H), 4.07 (s, 3H), 4.15 (s, 3H), 4.74 (s, 2H), 7.04 (s,
5.7.6. 2,3,9,10-Tetramethoxy-13-n-octylprotoberberine chloride (14f)
Compound 13 (106 mg，0.22 mmol) was treated with methyl
1H), 7.25 (s, 1H), 8.09 (s, 2H), 9.70 (s, 1H); 13C NMR (DMSO-d6)
d:
iodide (47
give the desired product 14f as a brown solid, yield: 31%; mp:
89e92 ꢁC; 1H NMR (CD3OD)
ml， 0.75 mmol), according to the general procedure to
150.0, 149.7, 145.4, 144.3, 144.2, 136.1, 133.6, 132.3, 130.0, 125.9,
121.2, 121.1, 119.1, 116.2, 111.3, 62.0, 57.3, 57.0, 55.8, 31.2 (2), 30.5,
28.9, 28.7, 28.4, 26.9, 22.0, 13.9. HRMS: calcd for C28H36NO4Cl
[MꢀCl]þ 450.26443, found 450.26432.
d
: 0.84 (t, 3H, J ¼ 6.8 Hz), 1.41e1.46 (m,
10H), 1.87 (t, 2H, J ¼ 7.6 Hz), 3.10 (t, 2H, J ¼ 6.0 Hz), 3.43 (t, 2H,
J ¼ 6.4 Hz), 3.87 (s, 3H), 3.90 (s, 3H), 4.07 (s, 3H), 4.16 (s, 3H), 4.74 (s,
2H), 7.08 (s, 1H), 7.34 (s, 1H), 8.10 (s, 2H), 9.73 (s, 1H); 13C NMR
5.7.2. 2-Hydroxy-3-ethoxy-9,10-dimethoxy-13-n-octylprotoberber-
ine chloride (14b)
(DMSO-d6) d: 150.7, 150.0, 147.3, 144.4, 144.2, 135.9, 133.7, 132.3,
132.2, 126.0, 121.2, 121.1, 119.0, 112.7, 111.2, 62.0, 57.2, 57.0 (2), 55.8,
31.2, 30.9, 29.1, 28.7, 28.6, 26.9, 22.1, 22.0, 13.9. HRMS: calcd for
C29H38NO4Cl [MꢀCl]þ 464.2801, found 464.2785.
Compound 13 (150 mg，0.32 mmol) was treated with ethyl
bromide (23.8
to give the desired product 14b as a brown solid, yield: 23%; mp:
92e93 ꢁC; 1H NMR (CD3OD)
ml，0.32 mmol), according to the general procedure
d
: 0.85 (t, 3H, J ¼ 7.2 Hz), 1.24e1.27 (m,
5.7.7. 2,3-Diethoxy-9,10-dimethoxy-13-n-octylprotoberberine
chloride (14g)
8H), 1.38e1.46 (m, 5H), 1.83 (m, 2H), 3.05 (t, 2H, J ¼ 5.6 Hz), 3.37 (t,
2H, J ¼ 8.0 Hz), 4.06 (s, 3H), 4.15 (s, 3H), 4.16e4.21 (m, 2H), 4.73 (t,
2H, J ¼ 5.6 Hz), 7.02 (s, 1H), 7.25 (s, 1H), 8.09 (s, 2H), 9.69 (s, 1H); 13C
Compound 13 (400 mg，0.83 mmol) was treated with ethyl
bromide (631
to give the desired product 14g as a brown solid, yield: 35%; mp:
85e86 ꢁC; 1H NMR (CD3OD)
ml，8.3 mmol), according to the general procedure
NMR (DMSO-d6) d: 150.0, 148.8, 145.3, 144.2 (2), 136.1, 133.6, 132.3,
130.2, 125.9, 121.2, 121.1, 119.0, 116.1, 112.0, 64.0, 62.0, 57.3, 57.0, 31.2
(2), 30.5, 28.8, 28.7, 28.4, 26.9, 22.0, 14.6, 13.9. HRMS: calcd for
C29H38NO4Cl [MꢀCl]þ 464.2801, found 464.2798.
d
: 0.85 (t, 3H, J ¼ 6.8 Hz), 1.24e1.29
(m, 8H), 1.38e1.47 (m, 8H), 1.84 (t, 2H, J ¼ 7.2 Hz), 3.08 (t, 2H,
J ¼ 5.6 Hz), 3.39 (t, 2H, J ¼ 8.0 Hz),4.06e4.18 (m, 4H), 4.07 (s, 3H),
4.15 (s, 3H), 4.74 (s, 2H), 7.06 (s, 1H), 7.34 (s, 1H), 8.10 (s, 2H), 9.72
5.7.3. 2-Hydroxy-3-n-propoxy-9,10-dimethoxy-13-n-octylprotober-
berine chloride (14c)
(s, 1H); 13C NMR (DMSO-d6)
d: 150.3, 150.0, 146.5, 144.4, 144.2,
135.9, 133.6, 132.4, 132.3, 126.0, 121.2, 121.1, 118.9, 114.6, 111.2, 64.4,
64.0, 62.0, 57.2, 57.0, 31.2, 30.9, 29.3, 29.1, 28.7, 28.6, 26.9, 22.0,
14.8, 14.6, 13.9. HRMS: calcd for C31H42NO4Cl [MꢀCl]þ 492.3114,
found 492.3113.
Compound 13 (150 mg，0.32 mmol) was treated with propyl
iodide (31.2
give the desired product 14c as a brown solid, yield: 29%; mp:
93e94 ꢁC; 1H NMR (DMSO-d6)
ml，0.32 mmol), according to the general procedure to
d
: 0.84 (t, 3H, J ¼ 6.8 Hz), 1.01 (t, 3H,
J ¼ 7.2 Hz), 1.22e1.27 (m, 10H), 1.40 (m, 2H), 1.74e1.81 (m, 4H), 3.02
(s, 2H), 4.03 (t, 2H, J ¼ 6.4 Hz), 4.07 (s, 6H), 4.78 (s, 2H), 7.06 (s, 1H),
7.26 (s, 1H), 8.14, 8.18 (dd, 2H, J ¼ 9.6 Hz), 9.87 (s, 1H); 13C NMR
5.7.8. 2,3-Di-n-propoxy-9,10-dimethoxy-13-n-octylprotoberberine
chloride (14h)
Compound 13 (150 mg，0.32 mmol) was treated with propyl
(CD3OD)
d
: 159.5, 158.6, 155.0, 153.7, 145.7, 143.2, 141.9, 139.7, 135.5,
iodide (100
give the desired product 14h as a brown solid, yield: 43%; mp:
67e69 ꢁC; 1H NMR (CD3OD)
ml，1.03 mmol), according to the general procedure to
134.5, 130.8, 130.7, 128.6, 125.8, 121.7, 79.4, 71.6, 66.5, 66.7, 40.8 (2),
40.0, 38.4, 38.3, 38.0, 36.5, 31.6, 31.5, 23.5, 20.0. HRMS: calcd for
C30H40NO4Cl [MꢀCl]þ 478.2957, found 478.2954.
d
: 0.90 (t, 3H, J ¼ 6.8 Hz), 1.06e1.14 (m,
10H), 1.30e1.34 (m, 5H), 1.52 (m, 2H), 1.81e1.93 (m, 5H), 3.13 (t, 2H,
J ¼ 5.6 Hz), 3.43 (t, 2H, J ¼ 8.0 Hz), 3.92e4.10 (m, 4H), 4.12 (s, 3H),
4.21 (s, 3H), 4.80 (t, 2H, J ¼ 5.2 Hz), 7.11 (s, 1H), 7.39 (s, 1H), 8.15 (s,
5.7.4. 2-Hydroxy-3-n-butoxy-9,10-dimethoxy-13-n-octylprotober-
berine chloride (14d)
2H), 9.77 (s, 1H); 13C NMR (CD3OD)
d: 153.2, 151.7, 149.1, 146.3,
Compound 13 (150 mg，0.32 mmol) was treated with butyl
145.2, 137.9, 135.8, 134.6, 133.9, 127.2, 123.1, 122.1, 120.6, 117.2, 113.5,
72.9, 71.7, 62.7, 59.2, 57.5, 33.0, 32.4, 30.9, 30.5, 30.4, 30.3, 28.8, 23.9,
23.7, 23.6, 14.4, 10.9, 10.8. HRMS: calcd for C33H46NO4Cl [MꢀCl]þ
520.3427, found 520.3433.
iodide (36.3
give the desired product 14d as a brown solid, yield: 28%; mp:
71e73 ꢁC; 1H NMR (CD3OD)
ml，0.32 mmol), according to the general procedure to
d
: 0.85 (t, 3H, J ¼ 6.8 Hz), 0.95 (t, 3H,
J ¼ 7.2 Hz),1.22e1.28 (m,10H),1.42e1.51 (m, 4H),1.73e1.79 (m, 4H),
3.03 (t, 2H, J ¼ 5.6 Hz), 3.33 (s, 2H), 4.08 (s, 6H), 4.78 (s, 2H), 7.09 (s,
1H), 7.23 (s, 1H), 8.17 (s, 2H), 9.31 (s, 1H), 9.85 (s, 1H); 13C NMR
5.7.9. 2,3-Di-n-butoxy-9,10-dimethoxy-13-n-octylprotoberberine
chloride (14i)
(CD3OD)
d: 149.8,148.9,145.2, 144.1,144.0, 136.0, 133.5,132.2, 130.0,
Compound 13 (150 mg，0.32 mmol) was treated with butyl
125.8, 121.1, 121.0, 118.8, 116.0, 111.9, 67.9, 61.9, 57.3, 56.9, 31.1, 30.5,
30.3, 28.7, 28.6, 28.4, 28.3, 26.8, 21.9,18.5,13.8,13.5. HRMS: calcd for
C31H42NO4Cl [MꢀCl]þ 492.3114, found 492.3117.
iodide (220
give the desired product 14i as a brown solid, yield: 44%; mp:
123e125 ꢁC; 1H NMR (CD3OD)
ml，1.94 mmol), according to the general procedure to
d: 0.85 (t, 3H, J ¼ 6.8 Hz), 0.94e0.99
(m, 6H), 1.24e1.29 (m, 8H), 1.46e1.55 (m, 6H), 1.73e1.88 (m, 6H),
3.08 (t, 2H, J ¼ 5.6 Hz), 3.37 (t, 2H, J ¼ 8.0 Hz), 4.02e4.11 (m, 4H),
4.07 (s, 3H), 4.15 (s, 3H), 4.74 (t, 2H, J ¼ 5.6 Hz), 7.06 (s, 1H), 7.34 (s,
5.7.5. 2-Hydroxy-3-benzyloxy-9,10-dimethoxy-13-n-octylprotober-
berine chloride (14e)
Compound 13 (200 mg，0.41 mmol) was treated with benzyl
1H), 8.10 (s, 2H), 9.72 (s, 1H); 13C NMR (CD3OD)
d: 153.3, 151.7, 149.1,
bromide (49.3
m
l，0.41 mmol), according to the general procedure
146.3, 145.2,138.0,135.9,134.6, 133.9,127.2,123.1,122.1, 120.6, 117.2,
113.5, 71.2, 70.0, 62.7, 59.2, 57.6, 33.1, 32.7, 32.4, 32.3, 30.9 (2), 30.6,
to give the desired product 14e as a brown solid, yield: 22%; mp:

Y.-X. Liu et al. / European Journal of Medicinal Chemistry 52 (2012) 151e158
157
30.5, 28.9, 23.7, 20.4, 20.3, 14.5, 14.2, 14.1. HRMS: calcd for
5.9.1. 2-Hydroxyl-3,9-diethoxy-10-methoxy-13-n-
octylprotoberberine chloride (16a)
Compound 15 (200 mg，0.44 mmol) was treated with ethyl
bromide (300 l，4.02 mmol), according to the general procedure
to give the desired product 16a as a brown solid, yield: 35%; mp:
90e92 ꢁC; 1H NMR (CD3OD)
C35H50NO4Cl [MꢀCl]þ 548.3740, found 548.3728.
5.7.10. 2,3-Dibenzyloxy-9,10-dimethoxy-13-n-octylprotoberberine
chloride (14j)
m
Compound 13 (150 mg，0.32 mmol) was treated with benzyl
d: 0.85 (t, 3H, J ¼ 7.2 Hz), 1.24e1.29 (m,
bromide (230
to give the desired product 14j as a brown solid, yield: 51%; mp:
71e73 ꢁC; 1H NMR (CD3OD)
ml，1.94 mmol), according to the general procedure
8H), 1.38e1.47 (m, 8H), 1.87 (m, 2H), 3.06 (t, 2H, J ¼ 5.6 Hz), 3.35 (t,
2H, J ¼ 8.0 Hz), 4.04 (s, 3H), 4.07e4.18 (m, 4H), 4.71 (t, 2H,
J ¼ 5.6 Hz), 7.05 (s, 1H), 7.33 (s, 1H), 7.82, 7.98 (dd, 2H, J ¼ 8.2 Hz),
d
: 0.80 (t, 3H, J ¼ 7.2 Hz), 1.12e1.28 (m,
12H), 1.72 (m, 2H), 3.06 (t, 2H, J ¼ 5.6 Hz), 4.06 (s, 3H), 4.14 (s, 3H),
9.74 (s, 1H); 13C NMR (DMSO-d6)
d: 150.1, 146.4, 145.0, 144.6, 144.2,
4.72 (t, 2H, J ¼ 5.6 Hz), 5.18 (s, 2H), 5.24 (s, 2H), 7.19 (s, 1H),
135.0, 133.0, 132.3, 131.8, 124.7, 119.1, 117.2, 115.7, 114.6, 114.2, 64.4,
64.0, 56.9 (2), 31.2, 30.7, 29.1, 28.7, 28.6, 27.0, 22.0, 15.3, 14.8, 14.6,
13.9. HRMS: calcd for C30H40NO4Cl [MꢀCl]þ 478.2957, found
478.2959.
7.27e7.48 (m, 11H), 8.08 (s, 2H), 9.70 (s, 1H); 13C NMR (CD3OD)
d:
153.0, 151.7, 148.6, 146.3, 145.2, 138.3, 138.0, 137.7, 136.2, 134.5,
134.3, 129.7 (3), 129.6, 129.3 (2), 128.7 (4), 127.2, 123.1, 122.2, 121.2,
118.7, 114.5, 73.1, 72.1, 62.7, 59.0, 57.6, 33.0 (2), 32.3, 30.5 (3), 28.9,
23.7, 14.5. HRMS: calcd for C41H46NO4Cl [MꢀCl]þ 616.3427, found
616.3474.
5.9.2. 2-Hydroxyl-3,9-di-n-propoxy-10-methoxy-13-n-octylprotober-
berine chloride (16b)
Compound 15 (200 mg，0.44 mmol) was treated with propyl
5.7.11. 2,3-Ethylenedioxy-9,10-dimethoxy ꢀ13-n-octylprotoberber-
ine chloride (14k)
iodide (293
give the desired product 16b as a yellow solid, yield: 45%; mp:
178e179 ꢁC; 1H NMR (CD3OD)
ml, 3.00 mmol), according to the general procedure to
Compound 13 (300 mg，0.62 mmol) was treated with 1,2-
d: 0.85 (t, 3H, J ¼ 7.2 Hz), 1.02e1.08
dibromoethane (267
procedure to give the desired product 14k as a brown solid, yield:
42%; mp: 110e112 ꢁC; 1H NMR (DMSO-d6)
ml，3.1 mmol), according to the general
(m, 6H), 1.24e1.29 (m, 8H), 1.43e1.47 (m, 2H), 1.81e1.92 (m, 6H),
3.05 (t, 2H, J ¼ 5.6 Hz), 3.37 (t, 2H, J ¼ 8.0 Hz), 4.05 (s, 3H), 4.08 (t,
2H, J ¼ 6.8 Hz), 4.32 (t, 2H, J ¼ 6.8 Hz), 4.75 (t, 2H, J ¼ 5.6 Hz), 7.02 (s,
d: 0.86 (t, 3H, J ¼ 6.8 Hz),
1.25e1.29 (m, 8H), 1.44e1.47 (m, 2H), 1.79 (m, 2H), 3.05 (t, 2H,
J ¼ 5.6 Hz), 3.30 (t, 2H, J ¼ 8.8 Hz), 4.08 (s, 6H), 4.32 (d, 2H,
J ¼ 4.8 Hz), 4.36 (d, 2H, J ¼ 4.8 Hz), 4.80 (s, 2H), 7.05 (s, 1H), 7.25 (s,
1H), 7.25 (s, 1H), 8.08 (s, 2H), 9.61 (s, 1H); 13C NMR (DMSO-d6)
d:
150.0, 149.0, 145.3, 144.0, 143.4, 136.1, 133.6, 132.4, 130.3, 125.9,
121.3, 121.0, 119.0, 116.2, 112.1, 75.8, 69.8, 57.5, 57.0, 31.2 (2), 30.5,
28.8, 28.7, 28.4, 26.9, 22.8, 22.0, 21.9,13.9,10.4,10.2. HRMS: calcd for
C32H44NO4Cl [MꢀCl]þ 506.3270, found 506.3266.
1H), 8.19 (s, 2H), 9.88 (s, 1H); 13C NMR (DMSO-d6)
d: 150.2, 145.2,
144.5, 144.2, 142.1, 135.4, 134.1, 132.1, 132.0, 125.9, 121.3, 121.2, 119.8,
118.0, 116.2, 64.6, 64.1, 62.0, 57.3, 57.0, 31.2, 30.4, 28.9, 28.6, 28.5,
28.3, 26.6, 22.0, 13.9. HRMS: calcd for C29H36NO4Cl [MꢀCl]þ
462.2644, found 462.2632.
5.9.3. 2-Hydroxyl-3,9-di-n-butoxy-10-methoxy-13-n-octylprotober-
berine chloride (16c)
Compound 15 (165 mg，0.36 mmol) was treated with butyl
5.8. 2,3,9-Trihydroxyl-10-methoxy-13-n-octylprotoberberine
chloride (15)
iodide (340
give the desired product 16c as a yellow solid, yield: 5%; mp:
151e153 ꢁC; 1H NMR (CD3OD)
ml，3.0 mmol), according to the general procedure to
d: 0.85 (t, 3H, J ¼ 7.2 Hz), 0.94e1.00
To a stirred solution of 1 (2.0 g，4.14 mmol) in CH2Cl2 (100 mL),
AlCl3 (2.43 g，18.2 mmol) was added. The reaction mixture was
stirred at room temperature for 3 days. The solvent was removed by
evaporation, and the residue was acidified by 4 N HCl, and then was
redissolved in MeOH. The solvent was removed by evaporation, and
the residue was purified by flash chromatography over silica gel
using CH2Cl2/CH3OH as the gradient eluent, affording the title
compound 15 (1.5 g，80%) as a dark brown soild. mp: 128e130 ꢁC;
(m, 6H), 1.24e1.29 (m, 8H), 1.45e1.57 (m, 6H), 1.78e1.88 (m, 6H),
3.05 (t, 2H, J ¼ 5.6 Hz), 3.37 (t, 2H, J ¼ 8.0 Hz), 4.06 (s, 3H), 4.08e4.14
(m, 2H), 4.34e4.38 (m, 2H), 4.74 (t, 2H, J ¼ 5.6 Hz), 7.06 (s, 1H), 7.34
(s, 1H), 8.08 (s, 2H), 9.60 (s, 1H); 13C NMR (DMSO-d6)
d: 149.8, 148.0,
144.2, 143.9, 143.3, 136.4, 133.2, 132.5, 130.3, 125.8, 121.2, 120.9,
117.8, 116.5, 114.7, 74.0, 57.5, 57.0 (2), 35.8, 31.5, 31.3, 30.7, 30.6, 28.9
(2), 28.7, 28.5, 26.7, 18.7, 18.5, 14.0, 13.7 (2). HRMS: calcd for
C34H48NO4Cl [MꢀCl]þ 534.3588, found 534.3568.
1H NMR (DMSO-d6)
d
: 0.85 (t, 3H, J ¼ 6.8 Hz),1.25e1.29 (m, 8H),1.43
(m, 2H), 1.79 (m, 2H), 2.94 (t, 2H, J ¼ 5.6 Hz), 3.27 (t, 2H, J ¼ 7.2 Hz),
4.03 (s, 3H), 4.71 (s, 2H), 6.84(s, 1H), 7.17 (s, 1H), 7.19, 8.05 (dd, 2H,
J ¼ 9.2 Hz), 9.32 (s, 1H), 9.84 (s, 1H), 9.89 (s, 1H), 11.20 (s, 1H); 13C
5.9.4. 2-Hydroxy-3,9-dibenzyloxy-10-methoxy-13-n-octylprotober-
berine chloride (16d)
Compound 15 (150 mg，0.33 mmol) was treated with benzyl
NMR (DMSO-d6)
d: 148.6, 145.5, 145.0, 144.9 (2), 136.3, 133.3, 132.7,
bromide (356
give the desired product 16d as a yellow solid, yield: 15%; mp:
171e173 ꢁC; 1H NMR (CD3OD)
ml，3.0 mmol), according to the general procedure to
130.9, 125.4, 118.6, 117.8, 117.4, 116.3, 115.5, 57.8, 57.6, 32.0 (2), 31.0,
29.6, 29.5, 29.2, 27.6, 22.8, 14.7. HRMS: calcd for C26H32NO4Cl
[MꢀCl]þ 422.2331, found 422.2321.
d
: 0.85 (t, 3H, J ¼ 6.8 Hz), 1.24e1.29
(m, 8H), 1.41e1.43 (m, 2H), 1.80e1.82 (m, 2H), 2.95 (t, 2H,
J ¼ 5.6 Hz), 3.33 (t, 2H, J ¼ 8.0 Hz), 4.10 (s, 3H), 4.58 (t, 2H,
J ¼ 5.6 Hz), 5.25 (s, 2H), 5.39 (s, 2H), 7.04 (s, 1H), 7.24e7.47 (m, 11H),
5.9. General procedure for the synthesis of compounds 16aꢀf
(Route C)
8.10 (s, 2H), 9.38 (s, 1H); 13C NMR (DMSO-d6)
d: 150.3, 148.7, 145.6,
144.2, 142.5, 136.7, 136.4, 135.9, 133.7, 132.3, 129.9, 128.8, 128.4 (3),
128.3 (3), 127.9, 127.8, 127.4, 125.7, 121.5, 121.4, 119.4, 116.5, 112.7,
75.4, 69.9, 57.5, 57.0, 31.2 (2), 30.5, 28.9, 28.7, 28.4, 26.9, 22.0, 13.9.
HRMS: calcd for C40H44NO4Cl [MꢀCl]þ 602.3270, found 602.3252.
To a stirred solution of 15 (300 mg，0.66 mmol) and KOH
(140 mg，2.50 mmol) in DMF (10 mL), RX (>2.0 eq) was added. The
reaction mixture was stirred at 70 ꢁC for 5ꢀ6 h. The solvent was
removed by evaporation, the residue was acidified by 2 N HCl, the
soild was collected by filtration and then purified by flash chro-
matography over silica gel using CH2Cl2/CH3OH (95:5) as the
gradient eluent, affording the title compounds 16aꢀf. Compounds
16aꢀf were gained following the same procedure using purchased
alkyl halide as material.
5.9.5. 2,3,9-Triethoxy-10-methoxy-13-n-octylprotoberberine
chloride (16e)
Compound 15 (300 mg，0.66 mmol) was treated with ethyl
bromide (485
ml，6.5 mmol), according to the general procedure to
give the desired product 16e as a brown solid, yield: 39%; mp:

158
Y.-X. Liu et al. / European Journal of Medicinal Chemistry 52 (2012) 151e158
71e73 ꢁC; 1H NMR (CD3OD)
d
: 0.85 (t, 3H, J ¼ 7.2 Hz), 1.24e1.29 (m,
Appendix A. Supplementary material
9H), 1.38e1.47 (m, 10H), 1.86 (m, 2H), 3.08 (t, 2H, J ¼ 5.6 Hz), 3.39 (t,
2H, J ¼ 8.0 Hz), 4.06 (s, 3H), 4.07e4.18 (m, 4H), 4.41e4.46 (m, 2H),
4.76 (t, 2H, J ¼ 5.6 Hz), 7.06 (s, 1H), 7.34 (s, 1H), 8.09 (s, 2H), 9.67 (s,
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.ejmech.2012.03.012.
1H); 13C NMR (DMSO-d6)
d: 150.2, 150.1, 146.4, 144.3, 143.1, 135.8,
133.5, 132.3, 132.2, 125.8, 121.5, 121.0, 118.8, 114.5, 112.1, 70.0, 64.4,
64.0, 57.2, 56.9, 31.2, 30.8, 29.2, 29.1, 28.7, 28.6, 26.9, 22.0, 15.3, 14.7,
14.5, 13.9. HRMS: calcd for C32H44NO4Cl [MꢀCl]þ 506.3270, found
506.3258.
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octylberberine analogs were examined for their activities against
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m
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This work was supported by the National Natural Science Fun-
dation for Young Scientists (81102312) and the National S&T Major
Special Project on Major New Drug Innovation (2012ZX0
9301002ꢀ001ꢀ017).