Synthesis of 9-substituted derivatives of berberine as anti-HIV agents

Article abstract of DOI:10.1016/j.ejmech.2011.01.016

Naturally occurring protoberberine alkaloids, berberine and berberrubine along with 9-substituted derivatives of berberine were assessed for the anti-human immunodeficiency virus (HIV) activity. Berberine was found to be the most active compound with an EC₅₀ of 0.13 μM against HIV-1 NL4.3 virus in CEM-GFP cell lines. Berberrubine and two other compounds were found to be less active than berberine, at the same time they were less toxic than berberine. Enzyme based assay suggested that the anti-HIV activity of berberine and its analogs might be due to RTase inhibitory activity and some additional mechanisms.

Full text of DOI:10.1016/j.ejmech.2011.01.016

European Journal of Medicinal Chemistry 46 (2011) 1045e1049
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of 9-substituted derivatives of berberine as anti-HIV agents
,
,
,
a
a,
*
Hardik S. Bodiwala a 1, Sudeep Sabde b 1, Debashis Mitra b , Kamlesh Kumar Bhutani , Inder Pal Singh
**
a Department of Natural Products, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.A.S. Nagar, Punjab 160062, India
b National Center for Cell Science (NCCS), Pune University Campus, Ganeshkhind, Pune, Maharashtra 411007, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Naturally occurring protoberberine alkaloids, berberine and berberrubine along with 9-substituted
Received 22 July 2010
Received in revised form
6 January 2011
Accepted 12 January 2011
Available online 21 January 2011
derivatives of berberine were assessed for the anti-human immunodeficiency virus (HIV) activity.
Berberine was found to be the most active compound with an EC50 of 0.13 mM against HIV-1 NL4.3 virus
in CEM-GFP cell lines. Berberrubine and two other compounds were found to be less active than
berberine, at the same time they were less toxic than berberine. Enzyme based assay suggested that the
anti-HIV activity of berberine and its analogs might be due to RTase inhibitory activity and some addi-
tional mechanisms.
Keywords:
Berberine
Ó 2011 Elsevier Masson SAS. All rights reserved.
Berberrubine
Anti-HIV
Reverse transcriptase
1. Introduction
infection [19]. Berberine was found to be active against human
cytomegalo virus (HCMV). The anti-HCMV activity of 1 (IC50 0.68
mM)
Berberine (1) is a quaternary protoberberine alkaloid and is
a major constituent of many medicinal plants of families Papaver-
aceae, Berberidaceae, Fumariaceae, Menispermaceae, Ranuncula-
ceae, Rutaceae, Annonaceae etc. [1]. Berberine possesses various
biological activities likeantibacterial [2,3], antifungal[4], antimalarial
[5], antileishmanial [6], anticancer [7], antidiarrheal [8], cholesterol
lowering effect [9] and hypoglycemic effect [10]. A numberof 8, 9 and
13 substituted derivatives of berberine have been reported for
different activities. Various analogs using different chain lengths and
terminal amino groups have been synthesized to study the DNA-
binding affinity or as G-quadruplex stabilizing ligands [11e17].
Similar types of berberine derivatives have been reported as acetyl-
cholinesterase inhibitors [18].
was better than ganciclovir (IC50 0.91 M), a standard drug for the
m
treatment. The mechanism of action of 1 was proposed to be through
interventionwith intracellular events after virus penetration into the
host cells and before viral DNA synthesis [20]. Earlier, anti-HIV
reverse transcriptase (RT) activity of various protoberberine alka-
loids (Fig. 1) including berberine was reported. Columbamine (2)
iodide (IC50 58
rhizine (4) chloride (IC50 71
100 g/ml) were found to be moderately active, whereas compounds
that lacked quaternary nitrogen, e.g., tetrahydropalmatine and tet-
rahydroberberine, were inactive (25% inhibition at 200 g/ml)
mg/ml), coptisine (3) picrate (IC50 56
mg/ml), jatror-
mg/ml) and berberine (1) chloride (IC50
m
m
against HIV RT. The unavailability of more diversified analogs of
protoberberines prevented significant structure activity correlation
in this series of alkaloids [21]. There have been no further attempts to
study berberine derivatives as anti-HIV agents.
In continuation of our efforts to identify new anti-HIV lead
molecules based on various natural product scaffolds [22,23], we
synthesized various berberine-9-esters for evaluation of their anti-
HIV activity.
Recently, Zha et al. have reported that HIV-protease inhibitors like
amprenavir, atazanavir, lopinavir, andritonavirhave increased TNF-
and IL-6 expression in macrophages and thus caused stress. Ber-
berine inhibits the expression of TNF- and IL-6 in macrophages and
a
a
thus effectively reduces stress in mouse macrophages induced by
HIV-protease inhibitors. It was also concluded that berberine has the
potential application as a complimentary therapeutic agent for HIV
2. Chemistry
Structural modifications at C-9 of berberine can be envisaged
after converting the methoxy group to hydroxyl to obtain berber-
rubine. Various methods have been reported for the synthesis of
berberrubine (5) from berberine. Hong et al. reported that stirring
of 1 with urea at 200 ꢀC yielded berberrubine after purification by
* Corresponding author. Tel.: þ91 172 2214683x2144; fax: þ91 172 2214692.
** Corresponding author. Tel.: þ91 20 25708151.
E-mail addresses: dmitra@nccs.res.in (D. Mitra), ipsingh@niper.ac.in, ipsingh67@
yahoo.com (I.P. Singh).
1
Both the authors contributed equally to this paper.
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.01.016

1046
H.S. Bodiwala et al. / European Journal of Medicinal Chemistry 46 (2011) 1045e1049
therapeutic index (TI) of >60, almost 3.75 times than berberine. So,
attempts were made to introduce different substituents at C-9 of
berberine by preparing various esters.
Benzoyl derivative (6) was weakly active compared to 5.
Substitution at 40 in the benzoyl moiety by OMe (9) and SMe (10)
gave differences in activity. Compound 9 was active (w 44%
2
3
R1
R2
N
9
R3
10 R4
inhibition) at 0.02
substitution with five member hetero-aromatics with O and S as
hetero-atom (12 and 13), the case was reverse. Compound 12 with
furoyl moiety was weakly active at 18
compound 13 with thiophenoyl moiety was more active with an
EC50 value of 1.82 M and TI of 50. 2-Napthoyl derivative (7) was
moderately active whereas 1-napthoyl derivative (8) was inactive.
Compound 21 with bulky substitution like 1-adamantoyl showed
mM whereas 10 was inactive. In the case of
1: R1+ R2= OCH2O, R3= OCH3, R4= OCH3
2: R1= OH, R2= OCH3, R3= OCH3, R4= OCH3
mM concentration, while
3
: R1+ R2= OCH2O, R3+ R4= OCH2O
4: R1= OCH3, R2= OH, R3= OCH3, R4= OCH3
m
Fig. 1. Various protoberberine alkaloids.
more than 60% inhibition at 1 mM concentration. This suggested
that size and orientation of bulkiness at C-9 is important for anti-
HIV activity.
Electron withdrawing groups like F and CF3 on phenyl ring
(15e20) at C-9 of berberrubine were introduced to see their effect
on activity. 2,4-Difluoro substituted moiety (15) was the most
column chromatography using CHCl3: MeOH (4:1) [24]. Another,
method reported by Das and Srinivas involves microwave irradia-
tion [25]. The most efficient method was found to be vacuum
pyrolysis, which does not require any specialized instrument or
purification by column chromatography. It has been used widely for
the synthesis of 5 [5,26]. In our study (Scheme 1), compound 1 was
converted to 5 in 80e90% yield using vacuum pyrolysis. The next
step involved acylation of 5 in a polar aprotic solvent using various
acid chlorides. The desired final products were obtained in quan-
titative yield.
active with an EC50 value of 0.95 mM and TI of 60.15. Change in the
electronegativity at either position by CF3 reduced anti-HIV activity
(17 and 18). In addition, 3, 4 and 2, 6 di-substituted analogs were
also inactive.
Berberine showed 94% inhibiton of HIV-1 RT at a concentration
Different berberine-9-O-esters (6e21) were prepared using
different acid chlorides as shown in Fig. 2. All synthesized
compounds were identified by MS, IR, 1H and 13C NMR. All ester
derivatives showed a strong C]O stretching absorption band in the
region of 1730e1750 cmꢁ1 in IR spectra and a peak in the range of
of 20
inhibition of HIV-1 RT at the same concentration. The standard drug
Nevirapine showed 95.5% inhibition at 0.1 g/reaction concentra-
mg/reaction. All other screened compounds showed 99%
m
tion. The IC50 values showed that the most active compound (1)
was about 50 times less active than the standard drug. Berberine
and its derivatives showed low EC50 in cell based assay and high
IC50 in RT assay indicating that they act by additional mechanisms,
which remains to be clearly elucidated.
d
150e175 ppm in 13C NMR spectrum. All other signals of berberine
except eOCH3 at C-9 were observed in all synthesized compounds.
3. Biology
5. Conclusions
All synthesized compounds were tested for their cytotoxicity in
MTT assay before testing for cell based anti-HIV activity. Initially all
the synthesized derivatives were tested for anti-HIV activity at
highest non-toxic concentration. The results of cytotoxicity and
anti-HIV activity are shown in Table 1. Three out of the 17
compounds showed more than 70% inhibition of HIV replication in
CEM-GFP cells. These three compounds were analyzed further for
determination of EC50 and CC50 in order to look for a potential lead
molecule for further development. AZT was used as a positive
control.
In summary, a new series of 9-substituted berberine derivatives
has been synthesized. Naturally occurring berebrine (1) was found to
be the most active against HIV-1 NL4.3 in CEM-GFP cell line. Ber-
berrubine (5)a naturallyoccurringalkaloidwasfound tobelessactive
and less toxic than berberine (1). Two other compounds (13 and 15)
were also found to be less active and less toxic compared to berberine.
6. Experimental
In order to find mechanism of action, three active compounds
along with berberine were tested for HIV-1 RT inhibitory activity.
The results of anti-HIV RT activity are shown in Table 2.
6.1. General
1H and 13C NMR spectra were recorded on 400 MHz Bruker
FT-NMR (Avance II 400) spectrometer using tetramethylsilane as an
4. Results and discussion
internal standard and the chemical shifts are reported in d units.
Mass spectra were recorded on either CIMS (LtQ, Thermo, USA) or
MALDI MS (Bruker, USA). Merck silica gel 60 F254 plates were used
for TLC. Developed plates were visualized by UV light. All chemicals
were purchased from Sigma Aldrich. Solvents used for the chemical
synthesis purchased from commercial sources were of analytical
The parent compound berberine (1) was found to be active
against HIV-1 NL4.3 with an EC50 of 0.13
mM. Although berber-
rubine (5), a known natural product having free eOH group at C-9
was less active (EC50 of 2.8
mM) than 1, interestingly it had better
O
O
O
ii
i
Cl
Cl
Cl
N
N
N
O
O
O
OH
O
O
O
R
O
O
O
6 21
-
5
1
Scheme 1. Reagents and conditions: i. 190 ꢀC, 20e30 mm Hg, 2 h, ii. Acid chloride (RCOCl), CH3CN, RT, 30 min.

H.S. Bodiwala et al. / European Journal of Medicinal Chemistry 46 (2011) 1045e1049
1047
O
Cl
N
O
O
O
R
O
R =
6
10
18
14
S
F3C
F
F
S
7
19
O
O
11
15
F
F
CF3
F
8
O
S
12
16
20
F
F3C
9
F
13
17
O
21
F
CF3
Fig. 2. Structures of synthesized 9-substituted berberine derivatives.
grade and were used without further purification. Concentration of
solutions after reactions involved the use of a rotatory evaporator
(Buchi, Switzerland) operating at a reduced pressure.
6.2.1.1. Berberine chloride (1). Yellow solid; IR (KBr) nmax 3405,
3049, 1598, 1567, 1506, 1480 cmꢁ1; 1H NMR (CD3OD)
9.76 (s, 1H),
d
8.66 (s, 1H), 8.09 (s, 1H), 7.99 (s, 1H), 7.62 (s, 1H), 6.95 (s, 1H), 6.09
(s, 2H), 4.93 (s, 2H), 4.20 (s, 3H), 4.09 (s, 3H), 3.30 (s, 1H), 3.25
(s, 1H); 13C NMR (CD3OD)
d 150.7, 150.6, 148.4, 145.0, 144.3, 138.2,
6.2. Chemistry
133.7, 130.4, 126.6, 123.1, 121.9, 120.4, 120.0, 108.0, 105.1, 102.2, 61.2,
56.2, 55.8, 26.8; MS: m/z 336 [M ꢁ Cl]þ.
6.2.1. Procedure for synthesis of berberrubine (5)
Commercially available berberine (1, 1 g) chloride was heated at
190 ꢀC in a vacuum oven under reduced pressure (20e30 mm Hg)
for 1e2 h to obtain berberrubine (5, 885 mg) in 90% yield.
6.2.1.2. Beberrubine chloride (5). Dark red solid; yield 80e90%; IR
(KBr) nmax 3365, 2901, 1634, 1614, 1568, 1543, 1505, 1471 cmꢁ1; 1H
NMR (CD3OD)
6.80 (s, 2H), 6.00 (s, 2H), 4.85 (s, 2H), 3.83 (s, 3H), 3.09 (s, 2H); 13C
NMR (CD3OD) 162.7, 149.6, 149.4, 148.0, 145.8, 134.0, 132.2, 129.2,
d 9.19 (s, 1H), 7.90 (s, 1H), 7.45 (s, 1H), 7.34 (s, 1H),
Table 1
d
Anti-HIV activity of 9-substituted berberine derivatives in CEM-GFP cell line.
122.5, 121.4, 120.1, 118.2, 107.7, 106.9, 104.3, 101.8, 55.2, 54.1, 27.5,
Code Conc. (
m
M)a % Inhibition (p24) EC50
(
m
M)b
CC50
2.09
169.2
ND
ND
ND
ND
ND
ND
ND
91.87
ND
57.14
ND
ND
ND
ND
ND
ND
(
m
M)c
TId
MS: m/z 323 [M þ 1-Cl]þ [5].
1
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
AZT
0.29
15.52
4.69
2.10
1.05
0.02
5.30
5.31
18.03
4.62
2.24
4.32
21.65
9.77
4.88
4.88
1.95
1.03
5
66.0
76.95
0.13
2.8
16.07
60.42
ND
ND
ND
ND
ND
ND
ND
50.47
ND
60.15
ND
ND
ND
ND
ND
ND
6.2.2. General procedure for synthesis of 6e21
37.2
58.82
0
44.26
0
45.63
22.62
73.13
0
71.65
0
23.36
0
0
0
ND
ND
ND
ND
ND
ND
ND
1.82
ND
0.95
ND
ND
ND
ND
ND
ND
To the solution of 5 in CH3CN various acid chlorides were added.
Reaction mixture was allowed to stir for 30 min at room tempera-
ture. Solvent was evaporated under vacuum and crude product was
washed with n-hexane/diethyl ether to yield 6e21 in 92e96% yield.
6.2.2.1. 9-O-(Benzoyl) berberrubine chloride (6). Yellowish brown
solid; yield 94%; IR (KBr) nmax 3406, 3014, 1745, 1609, 1507 cmꢁ1; 1H
NMR (CD3OD)
d
9.72 (s,1H), 8.84 (s,1H), 8.32 (d, 2H, J ¼ 6.8 Hz), 8.22
(d, 2H, J ¼ 8.8 Hz), 7.78e7.64 (m, 4H), 6.95 (s, 1H), 6.11 (s, 2H), 4.91
Table 2
HIV-1 reverse transcriptase activity of 9-substituted berberine derivatives.
64.23
Compound
m
g/reaction
% Inhibition
IC50 (mg/reaction)
89.75 ꢂ 7.07
1.05 ꢂ 0.07 24.06 ꢂ 0.63 22.91
1
5
13
15
20
20
20
20
94
99
99
99
2.1 ꢂ 0.5
6.1 ꢂ 0.1
3.8 ꢂ 0.2
3.5 ꢂ 0.1
0.041
a
Highest noncytotoxic concentration.
b
c
EC50 ¼ concentration of compound to achieve 50% inhibition of infected cells.
CC50 ¼ concentration of compound indicating 50% cytotoxicity in uninfected
cells.
Nevirapine
0.1
95.5
d
TI ¼ CC50/EC50; ND ¼ Not determined.

1048
H.S. Bodiwala et al. / European Journal of Medicinal Chemistry 46 (2011) 1045e1049
(s, 2H), 4.07 (s, 3H), 3.22 (s, 2H); 13C NMR (CD3OD)
d
163.9, 151.2,
6.2.2.8. 9-O-(2-Thiopheneoyl) berberrubine chloride (13). Yellowish
brown solid; yield 95%; IR (KBr) nmax 3403, 2925, 1734, 1609, 1507,
151.0, 148.6, 143.9, 138.9, 134.4, 134.2, 133.8, 130.7, 130.3, 128.6,
128.1, 126.8, 125.6, 122.0, 120.7, 108.0, 105.3, 102.3, 56.4, 56.0, 26.6;
MS: m/z 427 [M þ 1-Cl]þ [24].
1480 cmꢁ1; 1H NMR (CD3OD)
d 9.72 (s, 1H), 8.84 (s, 1H), 8.27 (d, 1H,
J ¼ 9.2 Hz), 8.22 (d, 1H, J ¼ 9.2 Hz), 8.16 (d, 1H, J ¼ 3.6 Hz), 8.01
(d, 1H, J ¼ 4.8 Hz), 7.69 (s, 1H), 7.34 (t, 1H, J ¼ 4.4 Hz), 6.95 (s, 1H),
6.11 (s, 2H), 4.91 (t, J ¼ 6.4 Hz), 4.07 (s, 3H), 3.22 (t, 2H, J ¼ 6.4 Hz);
6.2.2.2. 9-O-(2-Napthoyl) berberrubine chloride (7). Yellowish brown
solid; yield 96%; IR (KBr) nmax 3826, 3739, 3616, 1735, 1613,
13C NMR (CD3OD)
d 159.2, 151.3, 151.0, 148.6, 143.8, 139.0, 135.8,
1508 cmꢁ1; 1H NMR (CD3OD)
d
9.75 (s, 1H), 8.96 (s, 1H), 8.79 (s, 1H),
135.1, 133.8, 133.7, 130.9, 130.7, 128.3, 127.0, 125.6, 121.9120.7, 120.3,
8.26e8.18 (m, 3H), 8.06 (t, 2H, J ¼ 8.6 Hz), 8.00 (d, 2H, J ¼ 8.4 Hz),
7.70 (t, 1H, J ¼ 7.8 Hz), 7.66 (s, 1H), 7.62 (t, 1H, J ¼ 7.4 Hz), 6.92 (s, 1H),
6.09 (s, 2H), 4.89 (t, J ¼ 6.0 Hz), 4.05 (s, 3H), 3.20 (t, 2H, J ¼ 6.0 Hz);
108.0, 105.2, 102.3, 56.4, 56.0, 26.6; MS: m/z 433 [M þ 1-Cl]þ.
6.2.2.9. 9-O-(2-Thiopheneacetonyl) berberrubine chloride (14). Brown
solid; yield 94%; IR (KBr) nmax 3415, 1782, 1607, 1508 cmꢁ1; 1H NMR
13C NMR (CD3OD)
d 165.5, 152.6, 152.4, 150.0, 145.3, 140.2, 137.6,
135.8,135.1, 133.9,133.8,132.0, 130.6, 130.4,129.8, 128.3, 128.3,127.0,
126.6, 126.5, 123.3, 122.0, 121.6, 109.4, 106.7, 103.7, 57.9, 57.3, 28.0;
MS: m/z 477 [M þ 1-Cl]þ.
(CD3OD)
d
9.69 (s,1H), 8.78 (s,1H), 8.21 (d,1H, J ¼ 9.2 Hz), 8.15 (d,1H,
J ¼ 9.2 Hz), 7.66 (s,1H), 7.40 (d,1H, J ¼ 5.2 Hz), 7.18 (d,1H, J ¼ 6.3 Hz),
7.07 (t, 1H, J ¼ 3.4 Hz), 6.97 (s,1H), 6.11 (s, 2H), 4.89 (brs), 4.45 (s, 2H),
4.02 (s, 3H), 3.23 (t, 2H, J ¼ 6.4 Hz); 13C NMR (CD3OD)
d 168.1, 151.0,
6.2.2.3. 9-O-(1-Napthoyl) berberrubine chloride (8). Yellowish brown
solid; yield 92%; IR (KBr) nmax 3415, 3020, 1734, 1609, 1507 cmꢁ1; 1H
151.0, 148.5, 143.7, 138.5, 134.1, 133.6, 130.6, 127.4, 126.8, 126.6, 125.5,
125.2,121.6,120.5,120.2,108.0,105.2,102.3, 56.3, 56.0, 33.9, 26.6; MS:
m/z 447 [M þ 1-Cl]þ.
NMR (CD3OD)
d
9.79 (s, 1H), 9.00 (d, 1H, J ¼ 9.5 Hz), 8.86 (s, 1H), 8.75
(d, 1H, J ¼ 7.2 Hz), 8.32e8.27 (overlapped signal, 2H), 8.03 (d, 1H,
J ¼ 8.4 Hz), 7.72e7.52 (m, 4H), 6.95 (s, 1H), 6.12 (s, 2H), 4.90
(t, J ¼ 6.0 Hz), 4.07 (s, 3H), 3.22 (t, 2H, J ¼ 6.0 Hz); 13C NMR (CD3OD)
6.2.2.10. 9-O-(2,4-Diflourobenzoyl) berberrubine chloride (15). Yellow
solid; yield 93%; IR (KBr) nmax 3368, 3026, 1751, 1611, 1568,
d
168.1, 155.2, 154.9, 152.5, 148.0, 143.0, 138.9, 138.5, 138.0, 137.8,
1506 cmꢁ1; 1H NMR (CD3OD)
d 9.78 (s, 1H), 8.85 (s, 1H), 8.37 (dd, 1H,
136.0, 135.6, 134.6, 132.6, 132.0, 130.7, 130.3, 129.6, 129.0, 128.4,
128.0,126.0, 124.6,124.2,111.9, 109.2,106.2, 60.4, 59.9, 30.6; MS: m/z
477 [M þ 1-Cl]þ.
J ¼ 8.4, 14.8 Hz), 8.29 (d, 1H, J ¼ 9.2 Hz), 8.24 (d, 1H, J ¼ 9.2 Hz), 7.70
(s,1H), 7.31e7.23 (m, 2H), 6.94 (s,1H), 6.11 (s, 2H), 4.92 (t, J ¼ 6.0 Hz),
4.09 (s, 3H), 3.23 (t, 2H, J ¼ 6.0 Hz); 13C NMR (CD3OD)
d 162.0, 152.5,
152.4, 150.0, 145.3, 140.4, 136.3, 136.2, 135.2, 135.1, 132.1, 128.4, 127.0,
123.2, 122.1, 121.7, 113.4, 113.2, 109.4, 107.0, 106.7, 106.4, 103.8, 57.9,
57.3, 28.0; MS: m/z 463 [M þ 1-Cl]þ.
6.2.2.4. 9-O-(4-Methoxybenzoyl) berberrubinechloride(9). Yellowish
brown solid; yield 96%; IR (KBr) nmax 3343, 1734, 1605, 1508 cmꢁ1
;
1H NMR (CD3OD)
d 9.68 (s, 1H), 8.81 (s, 1H), 8.26e8.18 (m, 4H), 7.68
(s, 1H), 7.14 (d, 2H, J ¼ 8.4 Hz), 6.90 (s, 1H), 6.10 (s, 2H), 4.90
6.2.2.11. 9-O-(3,4-Diflourobenzoyl) berberrubine chloride (16).
Yellowish brown solid; yield 94%; IR (KBr) nmax 3410, 3022, 2924,
(t, J ¼ 6.0 Hz), 4.05 (s, 3H), 3.94 (s, 3H), 3.21 (t, 2H, J ¼ 6.0 Hz); 13C
NMR (CD3OD)
d
164.9, 163.7, 151.2, 150.9, 148.6, 143.9, 138.8, 134.5,
1752, 1611, 1507 cmꢁ1; 1H NMR (CD3OD)
d 9.78 (s, 1H), 8.85 (s, 1H),
133.8, 132.5, 131.4, 130.6, 126.6, 125.6, 122.0, 120.6, 120.3, 120.0,
113.9, 113.2, 108.0, 105.2, 102.3, 56.4, 55.9, 54.9, 26.6; MS: m/z 457
[M þ 1-Cl]þ.
8.31e8.18 (m, 4H), 7.70 (s, 1H), 7.57 (m, 1H), 6.96 (s, 1H), 6.12 (s, 2H),
4.92 (t, J ¼ 6.4 Hz), 4.07 (s, 3H), 3.23 (t, 2H, J ¼ 6.4 Hz); 13C NMR
(CD3OD)
d 165.9, 155.0, 155.0, 152.5, 147.8, 143.0, 137.9, 137.7, 134.6,
131.9, 131.0, 129.5, 125.7, 124.6, 124.2, 123.5, 123.3, 121.9, 121.7, 111.9,
6.2.2.5. 9-O-(4-Methylthiobenzoyl) berberrubine chloride (10).
Yellowish brown solid; yield 92%; IR (KBr) nmax 3416, 1733, 1608,
109.2, 106.3, 60.4, 59.8, 30.6; MS: m/z 463 [M þ 1-Cl]þ.
1593, 1506 cmꢁ1
;
1H NMR (CD3OD)
d
9.72 (s, 1H), 8.84 (s, 1H),
6.2.2.12. 9-O-(2-Flouro-4-triflouromethylbenzoyl) berberrubine chlo-
ride (17). Brownish yellow solid; yield 96%; IR (KBr) nmax 3425,
8.29e8.22 (m, 4H), 7.70 (s, 1H), 7.47 (d, 2H, J ¼ 6.8 Hz), 6.96 (s, 1H),
6.12 (s, 2H), 4.90 (brs), 4.07 (s, 3H), 3.23 (t, 2H, J ¼ 6.4 Hz), 2.61
2925, 1756, 1609, 1507 cmꢁ1 1H NMR (CD3OD)
; d 9.83 (s, 1H), 8.85
(s, 3H); 13C NMR (CD3OD)
d
163.7, 151.2, 151.0, 148.6, 148.3, 143.9,
(s, 1H), 8.49 (t, 1H, J ¼ 7.2 Hz), 8.33 (d, 1H, J ¼ 12.8 Hz), 8.27 (d, 1H,
J ¼ 12.0 Hz), 7.79 (d, 2H, J ¼ 10.0 Hz), 7.69 (s, 1H), 6.96 (s, 1H), 6.11
(s, 2H), 4.86 (brs), 4.08 (s, 3H), 3.17 (brs, 2H); 13C NMR (CD3OD)
138.8,134.4,133.7,130.6,130.5,126.7,125.5,124.7,124.5,123.7,122.0,
120.6, 120.3, 108.0, 105.2, 102.3, 56.3, 55.9, 26.6, 13.1; MS: m/z 473
[M þ 1-Cl]þ.
d
165.7, 162.2, 161.7, 152.8, 150.4, 145.7, 140.9, 138.9, 135.7, 135.6,
135.3, 132.5, 129.1, 127.4, 126.4, 123.5, 123.0, 122.5, 122.0, 116.6,
6.2.2.6. 9-O-(Piperonyl)berberrubinechloride(11). Yellow solid; yield
116.3, 109.8, 107.1, 104.2, 58.3, 57.8, 28.5; MS: 513 [M þ 1-Cl]þ.
95%; IR (KBr) nmax 3434, 2925, 1734, 1610, 1507 cmꢁ1 1H NMR
;
(CD3OD)
d
9.70 (s,1H), 8.84 (s,1H), 8.27 (d,1H, J ¼ 9.2 Hz), 8.23 (d,1H,
6.2.2.13. 9-O-(4-Flouro-2-triflouromethylbenzoyl) berberrubine chlo-
ride (18). Yellowish brown solid; yield 95%; IR (KBr) nmax 3423,
J ¼ 9.2 Hz), 7.94 (d,1H, J ¼ 8.0 Hz), 7.70 (s, 2H), 7.06 (d,1H, J ¼ 8.4 Hz),
6.96 (s, 1H), 6.15 (s, 2H), 6.12 (s, 2H), 4.89 (t, J ¼ 6.4 Hz), 4.07 (s, 3H),
3027, 1768, 1611, 1506 cmꢁ1 1H NMR (CD3OD)
; d 9.72 (s, 1H), 8.86
3.22 (t, 2H, J ¼ 6.0 Hz); 13C NMR (CD3OD)
d
164.7, 154.6, 152.7, 152.4,
(s, 1H), 8.62 (t, 1H, J ¼ 6.8 Hz), 8.31 (d, 1H, J ¼ 9.2 Hz), 8.26 (d, 1H,
J ¼ 9.2 Hz), 7.80 (d, 1H, J ¼ 9.2 Hz), 7.70 (s, 2H), 6.97 (s, 1H), 6.12
(s, 2H), 4.94 (t, J ¼ 6.4 Hz), 4.08 (s, 3H), 3.25 (t, 2H, J ¼ 6.0 Hz); 13C
150.0, 149.7, 145.3, 140.3, 135.9, 135.2, 132.1, 128.1, 128.1, 127.0, 123.4,
123.1, 122.0, 121.7, 111.0, 109.4, 109.4, 106.7, 103.9, 103.7, 57.8, 57.3,
28.0; MS: m/z 471 [M þ 1-Cl]þ.
NMR (CD3OD)
d 168.0, 164.6, 162.7, 152.5, 150.1, 144.9, 140.6, 137.0,
136.8, 135.3, 135.0, 133.9, 132.2, 128.7, 127.1, 125.7, 123.1, 122.2, 121.7,
120.6, 120.4, 117.0, 116.9, 116.6, 116.5, 109.5, 106.8, 103.8, 57.9, 57.5,
28.1; MS: m/z 513 [M þ 1-Cl]þ.
6.2.2.7. 9-O-(2-Furoyl) berberrubine chloride (12). Yellow solid;
yield 93%; IR (KBr) nmax 3414, 3022, 1750, 1621, 1609, 1506 cmꢁ1; 1H
NMR (CD3OD)
d
9.73 (s, 1H), 8.83 (s,1H), 8.27 (d,1H, J ¼ 8.8 Hz), 8.22
(d,1H, J ¼ 9.2 Hz), 7.97 (s,1H), 7.69 (s,1H), 7.66 (s,1H, J ¼ 3.2 Hz), 6.96
(s, 1H), 6.80 (s, 1H), 6.11 (s, 2H), 4.91 (t, J ¼ 5.6 Hz), 4.08 (s, 3H), 3.23
6.2.2.14. 9-O-(4-Flouro-3-triflouromethylbenzoyl) berberrubine chlo-
ride (19). Yellowish brown solid; yield 93%; IR (KBr) nmax 3405,
(t, 2H, J ¼ 6.0 Hz); 13C NMR (CD3OD)
d
151.1,151.0,148.6,148.6,143.8,
3026, 1757, 1709, 1623, 1609, 1506 cmꢁ1 1H NMR (CD3OD)
; d 9.82
142.8,138.5,133.7,133.4,130.7,127.0,125.6,121.9,120.9,120.6,120.3,
(s,1H), 8.86 (s, 1H), 8.61 (m, 2H), 8.32e8.27 (m, 2H), 7.70 (s,1H), 7.67
(t, 1H, J ¼ 10.0 Hz), 6.96 (s, 1H), 6.11 (s, 2H), 4.91 (t, J ¼ 6.0 Hz), 4.08
112.4, 108.0, 105.3, 102.3, 56.4, 56.0, 26.6; MS: m/z 417 [M þ 1-Cl]þ.

H.S. Bodiwala et al. / European Journal of Medicinal Chemistry 46 (2011) 1045e1049
1049
(s, 3H), 3.23 (t, 2H, J ¼ 5.6 Hz); 13C NMR (CD3OD)
d 166.7, 163.6,
[2] K. Iwasa, M. Kamigauchi, M. Ueki, M. Taniguchi, Antibacterial activity and
structure-activity relationships of berberine analogs, Eur. J. Med. Chem. 31
(1996) 469e478.
[3] S. Samosorn, B. Tanwirat, N. Muhamad, G. Casadei, D. Tomkiewicz, K. Lewis,
A. Suksamrarn, T. Prammananan, K.C. Gornall, J.L. Beck, J.B. Bremner, Anti-
bacterial activity of berberine-NorA pump inhibitor hybrids with a methylene
ether linking group, Bioorg. Med. Chem. 17 (2009) 3866e3872.
[4] K.D. Park, J.H. Lee, S.H. Kim, T.H. Kang, J.S. Moon, S.U. Kim, Synthesis of
13-(substituted benzyl) berberine and berberrubine derivatives as antifungal
agents, Bioorg. Med. Chem. Lett. 16 (2006) 3913e3916.
[5] K. Iwasa, H.-S. Kim, Y. Wataya, D.-U. Lee, Antimalarial activity and structure-
activity relationships of protoberberine alkaloids, Eur. J. Med. Chem. 33 (1998)
65e69.
[6] J.L. Vennerstrom, J.K. Lovelace, V.B. Waits, W.L. Hanson, D.L. Klayman, Berberine
derivatives as antileishmanial drugs, Antimicrob. Agents Chemother. 34 (1990)
918e921.
[7] S. Letasiová, S. Jantová, L. Cipák, M. Múcková, Berberine-antiproliferative
activity in vitro and induction of apoptosis/necrosis of the U937 and B16 cells,
Cancer Lett. 239 (2006) 254e262.
[8] K. Yamamoto, H. Takase, K. Abe, Y. Saito, A. Suzuki, Pharmacological studies on
antidiarrheal effects of a preparation containing berberine and geranii herba,
Nippon Yakurigaku Zasshi 101 (1993) 169.
[9] P. Yang, D.Q. Song, Y.H. Li, W.J. Kong, Y.X. Wang, L.M. Gao, S.Y. Liu, R.Q. Cao,
J.D. Jiang, Synthesis and structure-activity relationships of berberine
analogues as a novel class of low-density-lipoprotein receptor up-regulators,
Bioorg. Med. Chem. Lett. 18 (2008) 4675e4677.
[10] L.-Q. Tang, W. Wei, L.-M. Chen, S. Liu, Effects of berberine on diabetes induced
by alloxan and a high-fat/high-cholesterol diet in rats, J. Ethnopharmacol. 108
(2006) 109e115.
163.2, 152.9, 150.4, 145.8, 140.8, 139.0, 138.9, 137.8, 137.7, 135.5,
132.4, 131.5, 129.0, 127.3, 127.0, 123.5122.4, 122.1, 119.9, 119.6, 118.7,
109.8, 107.0, 104.1, 58.3, 57.7 28.4; MS: m/z 513 [M þ 1-Cl]þ.
6.2.2.15. 9-O-(2-Flouro-6-triflouromethylbenzoyl) berberrubine chlo-
ride (20). Yellowish brown solid; yield 95%; IR (KBr) nmax 3406,
3026, 1768, 1612, 1507 cmꢁ1 1H NMR (CD3OD)
; d 9.72 (s, 1H), 8.86
(s, 1H), 8.62 (t, 1H, J ¼ 7.6 Hz), 8.31 (d, 1H, J ¼ 9.2 Hz), 8.26 (d, 1H,
J ¼ 9.2 Hz), 7.81 (d, 1H, J ¼ 8.8 Hz), 7.70 (s, 2H), 6.97 (s, 1H), 6.11
(s, 2H), 4.94 (t, J ¼ 6.0 Hz), 4.07 (s, 3H), 3.25 (t, 2H, J ¼ 6.0 Hz); 13C
NMR (CD3OD)
d 152.9, 145.3, 137.2, 129.1, 127.4, 122.6, 109.8, 107.1,
104.1, 58.2, 57.8, 28.4; MS: m/z 513 [M þ 1-Cl]þ.
6.2.2.16. 9-O-(1-Adamantoyl) berberrubine chloride (21). Yellowish
brown solid; yield 96%; IR (KBr) nmax 3414, 2906, 2850, 1750, 1609,
1507 cmꢁ1; 1H NMR (CD3OD)
d 9.38 (s, 1H), 8.80 (s, 1H), 8.20 (d, 1H,
J ¼ 9.2 Hz), 8.15 (d, 1H, J ¼ 9.2 Hz), 7.67 (s, 1H), 6.96 (s, 1H), 6.11
(s, 2H), 4.94 (t, J ¼ 6.4 Hz), 4.05 (s, 3H), 3.22 (t, 2H, J ¼ 6.0 Hz),
2.25e1.88 (m, 15H, adamantyl protons); 13C NMR (CD3OD)
d 174.7,
151.0,150.8, 148.5,143.4, 138.7, 134.7, 133.7,130.7, 126.4, 125.5, 121.7,
120.7, 120.3, 108.0, 105.2, 102.3, 56.2, 56.2, 41.5, 38.7, 38.6, 36.1, 35.9,
28.0, 26.7; MS: m/z 485 [M þ 1-Cl]þ.
[11] W.H. Chen, J.Y. Pang, Y. Qin, Q. Peng, Z. Cai, Z.H. Jiang, Synthesis of linked
berberine dimers and their remarkably enhanced DNA-binding affinities,
Bioorg. Med. Chem. Lett. 15 (2005) 2689e2692.
6.3. Biology
[12] J.Y. Pang, Y. Qin, W.H. Chen, G.A. Luo, Z.H. Jiang, Synthesis and DNA-binding
affinitiesofmonomodifiedberberines, Bioorg. Med.Chem. 13 (2005)5835e5840.
[13] M. Franceschin, L. Rossetti, A. D’Ambrosio, S. Schirripa, A. Bianco, G. Ortaggi,
M. Savino, C. Schultes, S. Neidle, Natural and synthetic G-quadruplex inter-
active berberine derivatives, Bioorg. Med. Chem. Lett. 16 (2006) 1707e1711.
[14] Y.H. Long, L.P. Bai, Y. Qin, J.Y. Pang, W.H. Chen, Z. Cai, Z.L. Xu, Z.H. Jiang, Spacer
length and attaching position-dependent binding of synthesized proto-
berberine dimers to double-stranded DNA, Bioorg. Med. Chem. 14 (2006)
4670e4676.
[15] Y. Qin, J.Y. Pang, W.H. Chen, Z. Cai, Z.H. Jiang, Synthesis, DNA-binding affinities,
and binding mode of berberine dimers, Bioorg. Med. Chem. 14 (2006) 25e32.
[16] W.J. Zhang, T.M. Ou, Y.J. Lu, Y.Y. Huang, W.B. Wu, Z.S. Huang, J.L. Zhou, K.Y. Wong,
L.Q. Gu, 9-Substituted berberine derivatives as G-quadruplex stabilizing ligands
in telomeric DNA, Bioorg. Med. Chem. 15 (2007) 5493e5501.
[17] Y. Ma, T.M. Ou, J.Q. Hou, Y.J. Lu, J.H. Tan, L.Q. Gu, Z.S. Huang, 9-N-Substituted
berberine derivatives: stabilization of G-quadruplex DNA and down-regula-
tion of oncogene c-myc, Bioorg. Med. Chem. 16 (2008) 7582e7591.
[18] L. Huang, A. Shi, F. He, X. Li, Synthesis, biological evaluation, and molecular
modeling of berberine derivatives as potent acetylcholinesterase inhibitors,
Bioorg. Med. Chem. 18 (2010) 1244e1251.
[19] W. Zha, G. Liang, J. Xiao, E.J. Studer, P.B. Hylemon, W.M. Pandak Jr., G. Wang,
X. Li, H. Zhou, Berberine inhibits HIV protease inhibitor-induced inflammatory
response by modulating ER stress signaling pathways in murine macrophages,
PLoS One 5 (2010) e9069.
[20] K. Hayashi, K. Minoda, Y. Nagaoka, T. Hayashi, S. Uesato, Antiviral activity of
berberine and related compounds against human cytomegalovirus, Bioorg.
Med. Chem. Lett. 17 (2007) 1562e1564.
[21] T.B. Ng, B. Huang, W.P. Fong, H.W. Yeung, Anti-human immunodeficiency
virus (anti-HIV) natural products with special emphasis on HIV reverse
transcriptase inhibitors, Life Sci. 61 (1997) 933e949.
[22] N. Ahmed, K.G. Brahmbhatt, S. Sabde, D. Mitra, I.P. Singh, K.K. Bhutani,
Synthesis and anti-HIV activity of alkylated quinoline 2,4-diols, Bioorg. Med.
Chem. 18 (2010) 2872e2879.
[23] S.K. Chauthe, S.B. Bharate, S. Sabde, D. Mitra, K.K. Bhutani, I.P. Singh, Biomi-
metic synthesis and anti-HIV activity of dimeric phloroglucinols, Bioorg. Med.
Chem. 18 (2010) 2029e2036.
6.3.1. Cell cytotoxicity assay using MTT
Cytotoxicity of potential candidates was assayed using MTT
based Cell proliferation assay kit (Roche) in the CEM-GFP cell line
according to the manufacturer’s protocol. Briefly, 2 ꢃ 104 cells/well
were seeded in 96-well plate; samples were then added into the
wells at different concentrations keeping untreated and vehicle
treated wells as controls. After 48 h incubation, 10
mL of MTT
reagent (5 mg/ml) was added in to the wells to allow the reaction.
Formazan crystals produced during the reaction were solubilized
and color development was read at 540 nm.
6.3.2. Anti-HIV screening in CEM-GFP cells
Human CD4þ T cell line, CEM-GFP cells were infected with HIV-
1 NL4.3 virus at a multiplicity of infection (MOI) of 0.05 using the
standard protocol previously published from the lab [27,28]. The
infection was monitored by GFP visualization under the micro-
scope. The cells were incubated with samples for up to 7 days post
infection. Virus production was assayed in the culture supernatant
on day-7 post infection by p24 antigen capture ELISA (Perkin Elmer
Life Science, USA). AZT was used as a positive control.
6.3.3. HIV-1 reverse transcriptase inhibition assay
A colorimetric assay was performed to analyze the HIV-1 RT
inhibitory activity of the synthesized compounds using HIV-1 RT
assay kit (Roche, Germany) according to manufacturer’s protocol.
Nevirapine was used as a positive control.
[24] S.W. Hong, S.H. Kim, J.A. Jeun, S.J. Lee, S.U. Kim, J.H. Kim, Antimicrobial activity
of 9-O-acyl- and 9-O-benzoyl substituted berberrubines, Planta Med. 66
(2000) 361e363.
Acknowledgment
[25] B. Das, K. Srinivas, Conversion of berberine into berberrubine by selective
demethylation under microwave irradiation, Synth Commun. 32 (2002)
3027e3029.
[26] J.H. Kim, S.J. Lee, S.H. Kim, New derivatives of berberrubine ant their salts for
antibiotics and antifungal agents. WO 03/051875, 2003.
[27] M. Kumar, D. Mitra, Heat shock protein 40 is necessary for human immuno-
deficiency virus-1 Nef-mediated enhancement of viral gene expression and
replication, J. Biol. Chem. 280 (2005) 40041e40050.
Authors are thankful to Department of Biotechnology, Govern-
ment of India for the financial support to this project (grant no. BT/
PR7020/Med/14/930/2005 dt. 23/05/2006).
References
[28] J.S. Ladha, M.K. Tripathy, D. Mitra, Mitochondrial complex
I activity is
impaired during HIV-1-induced T-cell apoptosis, Cell Death Differ. 12 (2005)
[1] L. Grycova, J. Dostal, R. Marek, Quaternary protoberberine alkaloids, Phyto-
1417e1428.
chemistry 68 (2007) 150e175.