Total synthesis, antiprotozoal and cytotoxicity activities of rhuschalcone VI and analogs

Article abstract of DOI:10.1016/j.bmc.2010.02.055

The total synthesis of a potent antiplasmodial natural bichalcone, rhuschalcone VI, is described starting from simple and available resorcinol and 4-hydroxybenzaldehyde. Key steps include the solvent-free Aldol syntheses of chalcones, and the successful application of the Suzuki-Miyaura coupling reaction in the synthesis of bichalcones. The present work constitutes a general method for the rapid syntheses of a number of bichalcones related to rhuschalcone VI. Some of the bichalcones showed moderate antiprotozoal activities against Bodo caudatus, a preliminary screening system for antitrypanosomal activities, most of them with little or no cytotoxicity.

Full text of DOI:10.1016/j.bmc.2010.02.055

Bioorganic & Medicinal Chemistry 18 (2010) 2464–2473
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Total synthesis, antiprotozoal and cytotoxicity activities of rhuschalcone VI
and analogs
Shetonde O. Mihigo a, Wendimagegn Mammo b, Merhatibeb Bezabih a, Kerstin Andrae-Marobela c,
Berhanu M. Abegaz a,
*
a Department of Chemistry, University of Botswana, P. Bag 00704, Gaborone, Botswana
b Department of Chemistry, Addis-Ababa University, PO Box 1176, Addis-Ababa, Ethiopia
c Department of Biological Sciences, University of Botswana, P. Bag 00704, Gaborone, Botswana
a r t i c l e i n f o
a b s t r a c t
Article history:
The total synthesis of a potent antiplasmodial natural bichalcone, rhuschalcone VI, is described starting
from simple and available resorcinol and 4-hydroxybenzaldehyde. Key steps include the solvent-free
Aldol syntheses of chalcones, and the successful application of the Suzuki–Miyaura coupling reaction
in the synthesis of bichalcones. The present work constitutes a general method for the rapid syntheses
of a number of bichalcones related to rhuschalcone VI. Some of the bichalcones showed moderate anti-
protozoal activities against Bodo caudatus, a preliminary screening system for antitrypanosomal activi-
ties, most of them with little or no cytotoxicity.
Received 7 December 2009
Revised 23 February 2010
Accepted 24 February 2010
Available online 1 March 2010
Keywords:
Suzuki–Miyaura reaction
Bichalcone
Ó 2010 Elsevier Ltd. All rights reserved.
Rhuschalcone
Antiprotozoal activities
Bodo caudatus
OH O
1'"'
1. Introduction
β'
1''
3'"'
A'
B'
OH O
A
α'
4''
Rhus pyroides Burch. (Anacardiaceae) is a shrub to a medium-
sized tree widely distributed in the eastern part of Botswana and
South Africa, and is used against epilepsy in traditional medicine.1,2
R. pyroides is an exceptionally rich source of bioactive bichalcones
with novel structural characteristics.1 These bichalcones exhibit
potent antiplasmodial (unpublished results) and moderate anti-
proliferative properties against various strains and cancer cell lines
in standard assays.1 The Rhus bichalcones are basically of two
structural types: the bi-aryl ether type and the bi-aryl type; the lat-
ter having been reported from Rhus only. These compounds pos-
sess unique molecular structures and hence pose as worthwhile
targets for a detailed assessment of their biological properties.
However, their extreme scarcity renders any rational effort to eval-
uate their anti-malarial activities difficult if one relies on extrac-
tion from the producing Rhus species only. In an attempt to
circumvent this challenge, we have developed an efficient total
synthesis of rhuschalcone VI (1) using palladium-catalyzed Suzu-
ki–Miyaura cross-coupling reaction. Moreover, from purely chem-
ical perspective, this endeavor provided an excellent opportunity
to achieve the first use of Suzuki–Miyaura reaction in the synthesis
of bichalcones.
6'"'
HO
HO
OH
OH
4'
B
2
6'
1'
α
A
B
HO
OH
5
1
β
O
OH
2
1
Rhuschalcone VI (1) is a naturally occurring bichalcone com-
posed of two molecules of isoliquiritigenin (2) joined via a bi-aryl
linkage between the C-50 of the A-ring of one and the C-3 of the B-
ring of a second molecule of isoliquiritigenin. The natural material
was reported for the first time by Mdee et al.1 from the root bark of
R. pyroides and was shown to have a strong antiplasmodial activity
and a moderate antiproliferative activity against two colorectal
cancer cells.1 However, the quantities obtained from natural
sources were limited. As far as we are aware, rhuschalcone VI (1)
is the first and unique example of a natural dimer in which two
chalcones are linked by a C–C bond; and no group has reported
the total synthesis of C–C AB bichalcones. It is remarkable that
whereas (a) the total synthesis of the bi-aryl ether-type bichal-
cones (rhuschalcones and verbenachalcone) have been re-
ported,1,3,4 by employing a novel application of the Ullmann
synthesis and through anodic oxidation, respectively, and (b) a
* Corresponding author. Tel.: +267 355 2497; fax: +267 355 2836/4578.
E-mail address: abegazb@mopipi.ub.bw (B.M. Abegaz).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.02.055

S. O. Mihigo et al. / Bioorg. Med. Chem. 18 (2010) 2464–2473
2465
number of flavonoids have been employed in Suzuki reactions,5 the
use of chalcones and the synthesis of any bi-aryl type rhus bichal-
cones has not yet been reported. In order to provide ready access to
sufficient quantities of material for more complete biological stud-
ies, as well as a general route for the preparation of rhuschalcone
VI and its structural analogs, a total synthesis of rhuschalcone VI
is described herein. Most of the compounds prepared in the course
of this work were evaluated for their antiprotozoal activities using
a free-living, non-pathogenic protozoa Bodo caudatus (Bodonidae)
as a model. B. caudatus as well as the pathogenic human parasites
Trypanosoma cruzi and Trypanosoma brucei (Trypanosomatidae) fall
under the same order of Kinetoplastida.6
chalcones described later) under solvent free conditions, confirm-
ing reports by others that this methodology is superior to the syn-
thesis in solution, faster, cleaner and eco-friendly.14,15 Thus,
solvent-free aldol condensation, by grinding the mixture of equi-
molar quantities of 3b with anisaldehyde (4a) and NaOH, in a por-
celain mortar, gave bromochalcone 5 in 90% yield. Also, 3-
bromoanisaldehyde (4b) was subjected to solvent-free aldol con-
densation with 3a to give bromochalcone
6 in 97% yield
(Scheme 2).
With bromochalcones 5 and 6 at hand the preparation of the
key intermediates (i.e., chalconylboronate esters, e.g., 7) for the
coupling reactions, was to be undertaken. The synthesis of boro-
nate ester 7 was envisaged via bromine-lithium exchange with
n-BuLi, followed by a reaction with 2-isopropoxy-4,4,5,5-tetra-
methyl-1,3,2-dioxaborolane in THF. The feasibility of this approach
was tested by reacting bromochalcone 8 (itself prepared by the al-
dol condensation of p-bromoacetophenone and anisaldehyde) with
n-BuLi and the dioxoborolane mentioned above. However, the ex-
pected boronate ester was not obtained; instead the 1,2- and 1,4-
2. Results and discussion
Among the various methodologies available to accomplish C–C
bond formation, we chose the Suzuki–Miyaura reaction, one of the
most popular and powerful methods for the coupling of aryl–aryl
moieties. This methodology has gained prominence7,8 and found
many applications7,9,10 both in research laboratories and for
large-scale industrial processes due to its compatibility with a vari-
ety of functional groups, the stability and the commercial availabil-
ity of a wide-range of organoboron starting materials, and the ease
of working up the reaction mixtures.7,11 The retrosynthetic analy-
sis of rhuschalcone VI (1) is shown in Scheme 1. Because phenols
are very sensitive to autoxidation even under slightly basic condi-
tions12 we decided to protect the phenolic hydroxyl groups as their
methyl ethers. This would lead to a permethylated bichalcone as
the penultimate target (not shown in Scheme 1), which would re-
quire complete demethylation to give bichaclone 1. We anticipated
that incomplete demethylation during the last step of the synthesis
may result in analogs of the targeted bichalcone (1), which may
provide a diversity of the natural product for biological evaluation.
The syntheses of bromochalcones 5 and 6 are shown in
Scheme 2. Thus, protection of the hydroxyl groups of resorcinol
(3) as methyl ethers and Friedel–Crafts acylation afforded com-
pound 3a, which was subsequently brominated with molecular
bromine to give 3b in 99% yield. The condensation of equimolar
quantities of 3a and 4b in the presence of NaOH, in methanol
according to a reported procedure,13 furnished 6 in 75% yield. Sub-
stantial improvement in yields (90–97%) were obtained by running
the aldol condensation (to synthesize 5, 6 as well as many other
addition products (9a and 9b) of n-BuLi to the
a,b-unsaturated car-
bonyl group of the chalcone were isolated.
R
O
Br
OMe
Br
OMe
8
R = O
9b R = OH,
n
-Bu
9a
Attempts to protect the carbonyl groups of the chalcones, using
ethylene glycol, 1,2-propanediol, 1,3-propanediol or 2,3-butane-
diol and p-TsOH (or H2NSO3H) in benzene (or toluene) were not
successful. Failure to convert the bromochalcones to the corre-
sponding boronate esters forced us to look for an alternative strat-
egy. At this point it was decided to form the boronate ester at an
early stage of the ketal of bromoacetophenone 10 to give 11 and
then perform the Suzuki–Miyaura coupling with bromochalcone
6. This would then allow the synthesis of various bichalcones by al-
dol condensation of the acetophenone derivative 13 with variously
substituted benzaldehydes. This approach was found successful
and the details are presented in Scheme 3. The synthesis com-
O
H
Br
MeO
O
Br
H
+
MeO
OMe
4b
OH
4
O
OH
O
O
OMe
OH
MeO
OH
OH
HO
HO
OH
3
+
OMe
3a
OH
O
H
OMeO
1
OMe
OMe
O
OMe
O
4a
OMe
MeO
+
B
Br
7
O
O
MeO
OMe
Br
O
MeO
3b
Scheme 1. Retrosynthetic analysis of rhuschalcone VI (1).

2466
S. O. Mihigo et al. / Bioorg. Med. Chem. 18 (2010) 2464–2473
OMeO
OMeO
(d)
MeO
MeO
OMe
Br
Br
5
3b
(c)
OMe
OMe
OH
(b)
(a)
O
OMe
Br
OH
OMe
3a
MeO
OMe
3
(g)
O
OMe
O
O
O
6
(e)
(f)
H
H
H
HO
HO
MeO
4
Br
Br
4b
Scheme 2. Syntheses of bromochalcones 5 and 6. Reagents, conditions and (yields): (a) K2CO3, Me2SO4, acetone, reflux, 4 h (94%); (b), CH3COCl, AlCl3, dichloromethane,
reflux, 30 min (60%); (c) Br2, AcOH, ꢀ20 °C, 43 min (99%); (d) 4a, NaOH, 23 min (90%); (e) Br2, methanol, ꢀ5 °C, 5 min (49%); (f) K2CO3, Me2SO4, acetone, reflux, 2.5 h (69%); (g)
NaOH, 10 min (97%).
OMe O
O
O
O
Br
B
Br
MeO
MeO
MeO
(a)
(c)
(b)
MeO
MeO
OMe
O
O
OMe O
OMeO
OMeO
11
3b
10
O
OMe
12
(d)
OMe O
R1
O
OMe O
(f)
MeO
MeO
R2
R3
R6
R5
(e)
MeO
MeO
OMe
OMe
OMe
O
OMe
O
R4
O
OMe
13
14
1 R1 = R2 = R3 = R4 = R5 = R6 = OH
15 R1 = R3 = R4 = R6 = OH, R2 = R5 = OCH3
16 R1 = R3 = R4 = R5 = R6 = OH, R2 = OCH3
Scheme 3. Synthesis of rhuschalcone VI (1) and bichalcones 15 and 16. Reagents, conditions and (yields): (a) 2,3-butanediol, H2NSO3H, toluene, reflux, 20 h (96%); (b) n-BuLi,
2-isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane, THF, ꢀ78 °C (92%); (c) 6, Pd(PPh3)4, toluene, reflux, 7 h (73%); (d) I2, acetone, reflux, 10 min (77%); (e) 3a, NaOH, rt,
8 min (98%); (f) BBr3, CH2Cl2, reflux, 3 h (52%).
menced with a smooth transformation of acetophenone 3b to ketal
10 using 2,3-butanediol/H2NSO3H in toluene.16 Metal-halogen ex-
change of 10 with n-BuLi followed by addition of 2-isopropoxy-
4,4,5,5-tetramethyl-1,3,2-dioxaborolane gave boronate ester 11
in 92% yield. Compound 11 was successfully coupled with brom-
ochalcone 6 under Suzuki–Miyaura conditions using tetrakis(tri-
phenylphosphine)Pd(0) as catalyst to give ketal 12 in 73% yield.
This was in turn deprotected by refluxing with 10 mol % of molec-
ular iodine in acetone17 to give methyl ketone 13 in 77% yield. Sol-
vent-free aldol condensation of compound 13 with 6.3 equiv of 4a
in presence of NaOH gave the hexamethoxy-bichalcone 14 in 98%
yield. Finally, bichalcone 14 was demethylated using BBr3 in
refluxing dichloromethane to give Rhuschalcone VI (1) in 52% yield
together with partially demethylated rhuschalcone VI derivatives
15 (18%) and 16 (6%).
Since a previous report on rhuschalcone VI (1) has shown that it
possesses interesting biological activities, unnatural rhuschalcone
VI analogs may show similar and/or other pharmacological activi-
ties, which would warrant additional investigation. Within this
context, and in order to demonstrate the applicability of our strat-
egy, we undertook and successfully completed the preparation of a
few such rhuschalcone VI analogues that would be of value in SAR
studies. These are 4,400,2000,4000-tetramethoxy-3,5000-bichalcone (17),
4,400,2000,4000-tetrahydroxy-3,5000-bichalcone (18), 4,5,400,2000,4000-penta-

S. O. Mihigo et al. / Bioorg. Med. Chem. 18 (2010) 2464–2473
2467
methoxy-3,5000-bichalcone
(19),
4,400,2000,4000-tetramethoxy-40-
for 15 and a methoxy singlet (3H) at d 3.89. The location of the two
methoxy signals in the bichalcone skeleton of 15 and one OMe
group in 16 were deduced from HMBC analyses of the correspond-
ing spectra. For compound 15 HMBC correlations were observed
between the methoxy signal and C-4000 at d 164.0, which in turn
showed correlation to H-3000 at d 6.63.
methyl-3,5000-bichalcone (20), and 4,5,20,40,400,2000,4000-heptameth-
oxy-3,5000-bichalcone (21). Scheme 4 outlines the syntheses of
bichalcones 17–21. Thus, bromochalcones 25–28 were synthesized
by solvent-free aldol condensation reactions between the appro-
priate aldehydes and acetophenone derivatives as shown in
Scheme 4. Coupling of these bromochalcones with 11 followed
by deprotection of the resulting chalconylketals, and finally aldol
condensation of the acetophenone derivatives with anisaldehyde
yielded compounds 17–21. Removal of protecting groups in 17
gave 18 in 22% yield (Scheme 4).
3. Antiprotozoal activity and cytotoxicity
The antiprotozoal activities of compounds generated in this
study, 1, 14–21 were evaluated using the free living protozoa B.
caudatus. This organism is a non-pathogenic member of the family
Bodonidae, which is related to the genera Leishmania and Trypano-
soma (Trypanosomatidae) by belonging to the same order of Kine-
toplastida.16 Leishmania and Trypanosoma include the pathogenic
human parasites such as T. cruzi and T. brucei.6 B. caudatus there-
fore can be considered as a preliminary screening system for anti-
The structure of the synthetic rhuschalcone VI was determined
mainly from 1H and 13C NMR spectral data that are similar to those
reported in the literature for the natural product. To further con-
firm the structure of the synthetic 1 an authentic natural product
was sought to make direct comparison possible. Such a sample
was not available and hence the natural compound was isolated
from the root barks of R. pyroides following the published proce-
dure.18 The 1H and 13C NMR spectroscopic data generated for the
natural product and the synthetic material were in complete
agreement (Table 1). Complete spectroscopic data (1D and 2D-
NMR data, HRMS) were generated for the rhuschalcone VI analogs
14, 17, 19, 20 and 21. 1H and 13C NMR data assignments were
achieved by analysis of HMBC and HMQC correlations as well as
by comparison of NMR data to that of rhuschalcone VI and the syn-
thetic intermediates. The permethylated compound 14 showed
signals for six methoxyl groups as expected. The partial demethyl-
ation products, 15 and 16, showed a 6H (2 ꢁ OMe) singlet at d 3.89
trypanosomal activity. Compound 29
a prenylated chalcone
(isobavachalcone) isolated from Dorstenia kameruniana19 which
has shown activity on other organisms20 was included in this study
for comparison purposes. As shown in Figure 1, compounds 1, 16,
29 and 18 induced the largest reduction in viability of protozoa
with an inhibition of 75–83% compared to controls after 24 h of
exposure to compounds at a concentration of 0.6 mg/mL. Inhibition
was higher after exposure of B. caudatus to compounds after 24 h
(Fig. 1 B) compared to a 4-h incubation with respective compounds
(Fig. 1, A). This most likely reflects the time period needed for up-
take and metabolism of compounds by the organism.
O
O
B
MeO
O
Br
Br
O
R2
MeO
R1
R3
MeO
R1
OMeO
NaOH
R3
+
11
O
(a)
O
R2
H
23 R2 = R3 = H
3a R2 = R3 = OMe
24 R2 = H, R3 = Me
25 R1 =R2 = R3 = H
4b R1 = H
26 R1 = OMe, R2 = R3 = H
27 R1 =R2 = H, R3 = Me
28 R1 =R2 = R3 = OMe
22 R = OMe
1
OMeO
O
OMeO
OMeO
(b)
(c)
MeO
MeO
MeO
OMe
R3
MeO
R3 MeO
R3
MeO
R1
R1
R1
O
R2
O
R2
O
R2
(d)
OH
O
18
17 R1 =R2 = R3 = H
19 R1 = OMe, R2 = R3 = H
20 R1 =R2 = H, R3 = Me
21 R1 =R2 = R3 = OMe
HO
HO
OH
O
HO
OH
18
OH
O
29
Scheme 4. Synthesis of bichalcones 17–21. Reagents and conditions: (a) Pd(PPh3)4, toluene, reflux; (b) I2, acetone, reflux; (c) anisaldehyde, NaOH, rt; (d) BBr3, CH2Cl2, reflux.

2468
S. O. Mihigo et al. / Bioorg. Med. Chem. 18 (2010) 2464–2473
Table 1
1H NMR (600 MHz) and 13C NMR (150 MHz) data for compounds 1, 14 and 17–21
Position
1 (acetone-d6)
dC
Synthetic Natural
14 (CDCl3)
dC
17 (CDCl3)
19 (CDCl3)
20 (CDCl3)
21 (CDCl3)
dH
dH
dH
dC
dH
dC
dH
dC
dH
dC
1
2
3
4
—
125.8
132.7
127.8
160.8
117.8
126.4
132.8
128.1
161.1
117.3
129.8
113.6
166.7a
102.9
164.8a
107.9
132.5
126.7
131.0
115.9
160.1
113.3
166.7b
103.7
166.2b
119.2
133.7
192.0
117.1
144.7
191.9
117.8
144.1
—
127.8
131.6
127.1
158.9
111.1
129.8
122.6
160.1
98.7
—
127.3
—
126.3
—
127.5
131.2
130.4
159.3
111.1
131.2
135.9
128.6
129.3
143.4
129.3
128.6
128.2
130.0
114.3
161.3
121.6
160.2a
95.3
—
7.15
—
—
—
7.16
—
—
130.9
124.2
132.0
149.1
152.7
111.4
122.4
160.2
98.7
7.89(2.1)
—
—
6.96(8.4)
7.71(8.4, 2.1) 129.5
—
—
6.37(2.3)
—
6.45(8.9, 2.4) 107.8
8.18(8.9)
—
7.74(8.6)
6.89(8.6)
—
—
—
6.39
—
—
8.20
—
7.84(15.2)
7.93(15.2)
—
7.92(15.4)
7.84(15.2)
7.53(1.6)
—
—
6.99(8.6)
7.58(8.5)
—
—
6.50(1.5)
—
6.57(8.6, 1.9) 105.0
7.74(8.7)
—
7.58(8.5)
6.94(8.6)
—
—
—
6.60
—
—
7.73
—
7.38(15.7)
7.68(15.4)
—
7.46(15.7)
7.71(15.5)
3.83
—
4.03
3.88
3.89
7.63(m)
—
—
131.3a 6.84
113.8
133.4
151.5
148.5
108.2
138.5
128.4
128.5
132.5
128.5
128.4
128.1
130.1
114.3
161.3
121.9
160.3a
95.4
7.62(m)
—
—
7.01(9.1)
7.62(m)
—
7.96(8.0)
7.31(8.0)
—
7.31(8.0)
7.96(8.0)
—
7.59(8.7)
6.95(8.6)
—
—
—
6.61
—
—
7.75
—
7.47(15.5) 119.9
7.83(15.5) 144.3
—
7.48(15.7) 125.0
7.72(15.7) 142.1
3.84
—
127.4
159.4
111.1
—
—
—
5
7.01(9.1)
7.63(m)
—
8.05(7.3)
7.51(m)
7.59(8.5)
7.51(m)
8.05(7.3)
—
7.59(8.5)
6.95(8.6)
—
—
—
6
130.5a 7.33
10
113.7
166.7a
102.9
164.8a
138.5
128.5
128.6
132.6
128.6
128.5
128.2
130.0
114.3
161.3
121.6
160.2b
95.3
—
20
7.96(7.4)
7.48(7.7)
7.55(7.3)
7.48(7.7)
7.96(7.4)
—
7.58(8.6)
6.92(8.6)
—
—
—
6.59
—
30
6.51(1.9)
—
6.58(8.6, 2.0) 105.1
7.73(8.6)
—
7.58(8.8)
6.93(8.7)
—
—
—
6.60
—
—
7.72
—
7.39(15.7)
7.63(15.7)
—
7.45(15.7)
7.70(15.8)
3.71
3.96
3.90
4.03
40
163.9
164.0
50
60
132.4
126.9
130.8
115.8
160.0
112.6
166.7b
104.2
166.4b
120.5
133.4
192.0
116.6
145.1
191.3
118.1
143.4
132.6
128.2
130.0
114.3
161.2
121.6
160.2a
95.3
132.7
128.2
130.0
114.3
161.3
121.5
160.2
95.2
161.0
119.8
133.7
190.9
126.5
142.3
190.3
124.9
142.2
60.9
100
200, 600
300, 500
400
1000
2000
3000
6.61
—
—
7.76
4000
161.2a
119.7
134.1
191.0
125.2
142.4
190.4
125.0
142.1
55.9
161.3b
119.7
134.1
190.6
160.8a
121.3
134.2
190.8
161.3a
119.7
134.1
190.1
5000
—
7.66
—
6000
C@O
a
b
—
7.47(15.6) 119.9
7.85(15.7) 144.8
—
7.48(15.8) 125.0
7.72(15.7) 142.1
3.85
—
7.33(15.6) 120.3
7.62(15.7) 144.5
—
7.45(15.9) 124.9
7.71(15.7) 142.4
3.94
4.03
—
C@O0
a0
190.4
190.5
190.4
b0
OMe-4
OMe-5
OMe-20
OMe-40
OMe-400
55.9
56.0
56.0
—
55.9
—
—
—
55.9
55.8
56.1
13.79a
56.1
—
—
—
—
55.5b
55.8b
55.4
—
—
—
3.87a
4.03a
3.90a
55.4c,d 3.86
55.4
56.0b
55.9b
3.90a,b
4.03a
3.87b
2.45
55.9a,b 3.87
55.4
OMe-2000 13.73a
OMe-4000
3.87
3.90
56.1c
56.0d
4.03a
3.84a
56.1a
55.4b
21.6
3.89a
3.90a
55.6a
55.8a
55.8b
CH3-40
Values with the same letter superscripts in the same column are interchangeable.
Figures in parentheses are J values in hertz.
Figure 1. Growth inhibition of Bodo caudatus. Protozoa were exposed for 4 h (A) and 24 h (B) to compounds 1, 14–21 and 29 at a concn of 0.6 mg/mL. G/S represents growth
control of protozoa in solvent (end concn 8% DMSO). (+) is positive control with CuSO4 in the same concn as compounds. Note 100% inhibition of protozoa growth with CuSO4,
while compounds 1, 16, 18, 29 and 18 showed growth inhibition in a time-dependent manner of 75–83%. Assays were performed in triplicates, bars represent mean SD.
However, it must be noted that none of the compounds were as
active as CuSO4, which served as positive control killing 100% of
the protozoa at the same concentration. LC50 concentrations were
determined for the four most active compounds. As is evident from
Figure 2, the most active compound (29) displayed LC50 concentra-
2.30
killed protozoa with lesser efficacy at LC50 concentrations of
20.59 g/mL (18), 53.32 g/mL (1) and 118.20 g/mL (16), respec-
tively. Compounds 1 and 29 induced significant cell death of BHK
cells (Fig. 3) after exposure for 48 h at a concentration of 100 g/
mL, which corresponded to a CC50 value of 97.59 g/mL and
lg/mL for CuSO4 (data not shown). The other compounds
l
l
l
l
tion of 4.36
lg/mL, which was comparable to LC50 concentration of
l

S. O. Mihigo et al. / Bioorg. Med. Chem. 18 (2010) 2464–2473
2469
Figure 2. Antiprotozoal activity of compounds 1, 29, 16 and 18. The lethal concn of compounds for 50% of Bodo caudatus cells (LC50) was determined by the MTT-viability
assay. Compound 29 was the most active with a LC50 concentration of 4.36 ( g/mL) (B), followed by compound 18 (LC50 = 20.59 g/mL, D), compound 1 (LC50 = 53.30 g/mL,
A) and compound 16 (LC50 = 118.20 g/mL, C), respectively. Data are presented as the mean SD of triplicate determinations.
l
l
l
l
Figure 3. Cytotoxicity of compounds with antiprotozoal activity. (A–D) BHK cells in exponential growth phase were exposed to compounds 1, 29, 16 and 18 in a concn range
of 0.0001–100 g/mL for 48 h. Cell survival was determined in comparison to untreated cells by the MTT assay. Note that compounds 1 and 29 induced significant cell death
at a concn of 100 g/mL corresponding to a CC50 value of 97.59 g/mL and 86.88 g/mL, respectively (A, B), while cell viability was maintained after exposure to compounds
16 and 18 at the same concn (C, D). Data are presented as means SD from triplicate determinations.
l
l
l
l
86.88
is approximately 20 times higher than its antiprotozoal concentra-
tion (LC50 = 4.36 g/mL), while antiprotozoal activity of compound
1 is in the same range as its cytotoxic concentration. Compounds
16 and 18 induced a small decrease in cell viability compared to
non-treated cells, but did not exhibit significant cytotoxicity up
lg/mL, respectively. The CC50 concentration of compound 29
4. Conclusions
l
The total synthesis of rhuschalcone VI (1) has been achieved in
12 steps starting from resorcinol and 4-hydroxybenzaldehyde. This
synthesis constitutes the first application of the Suzuki–Miyaura
reaction in the synthesis of C–C AB linked bichalcones. Conver-
gence of the strategy enables the syntheses of a wide-range of
to a concentration of 100 lg/mL.

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S. O. Mihigo et al. / Bioorg. Med. Chem. 18 (2010) 2464–2473
bichalcones bearing a C–C linkage by simple structural modifica-
tion of the starting acetophenone and benzaldehyde derivatives.
The first total syntheses of eight (14–21) rhuschalcone VI-type
bichalcones were achieved, indicating that the general methodol-
ogy developed here is of practical use in the syntheses of more
congeners carrying the same carbon-framework.
76.4 °C; 1H NMR (400 MHz, CDCl3): d 1.23 (3H, d, J = 6 Hz), 1.30
(3H, d, J = 6 Hz), 1.73 (3H, s), 3.52 (1H, m), 3.75 (1H, m), 3.89
(3H, s), 3.90 (3H, s), 6.51 (1H, s), 7.12 (1H, s); 13C NMR
(100 MHz, CDCl3): d 16.47, 16.79, 26.78, 56.24, 56.37, 78.20,
78.90, 97.79, 101.14, 106.72, 126.17, 130.71, 156.19, 157.43. HRMS
(TOF EI+) m/z: M+ calcd for C14H19BrO4 330.0467; found 330.0465.
5.4. 2-(2,4-Dimethoxy-5-(2,4,5-trimethyl-1,3-dioxolan-2-
yl)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (11)
5. Experimental
5.1. General
To a cooled (ꢀ78 °C) solution of ketal 10 (4.00 g, 12.08 mmol,
1 equiv) in THF (10 mL) under nitrogen atmosphere was added
1.6 M n-BuLi (8.31 mL, 0.85 g, 13.29 mmol, 1.1 equiv) and the mix-
ture was stirred for about 5 min. 2-Isopropoxy-4,4,5,5-tetra-
methyl-1,3,2-dioxaborolane (4.94 g, 26.58 mmol, 2.2 equiv) was
added quickly and the mixture was left to warm to room temper-
ature overnight. The resulting yellowish solution was poured onto
water (50 mL) and extracted with diethyl ether (5 ꢁ 20 mL). The
combined extract was dried (Na2SO4) and the solvent was removed
and the resulting solid was dried overnight in a vacuum oven
(55 °C) to afford boronate ester 11 (4.21 g, 92%) as a whitish solid.
Mp 134.9–137.6 °C; 1H NMR (400 MHz, CDCl3): d 1.24 (3H, d, J =
6 Hz), 1.31 (3H, d, J = 6 Hz), 1.34 (12H, m), 1.75 (3H, s), 3.53 (1H,
m), 3.79 (1H, m), 3.88 (3H, s), 3.91 (3H, s), 6.44 (1H, s), 7.90 (1H,
s); 13C NMR (100 MHz, CDCl3): d 16.63, 16.80, 24.74, 24.81,
2 ꢁ 24.92, 26.85, 55.68, 56.14, 77.95, 78.86, 2 ꢁ 83.08, 95.74,
107.46, 123.91, 135.22, 160.85, 165.91. HRMS (TOF EI+) m/z: M+
calcd for C20H31BO6 378.2214; found 378.2235.
Commercially available reagents and solvents were used with-
out further purification other than those detailed below. THF was
distilled from sodium–benzophenone under nitrogen immediately
before use. Analytical thin layer chromatography was carried out
using aluminum or glass-backed plates coated with Merck Kiesel-
gel 60 GF254. Developed plates were visualised under ultra-violet
light (254 nm) and/or sprayed with vanillin-sulfuric acid. Column
chromatography was conducted on columns of different sizes
using Silica Gel 60, particle size 0.040–0.063 mm (Merck). Fully
characterized compounds were chromatographically homoge-
neous. Melting points were determined using a Büchi mp B545
apparatus and are uncorrected. The ultraviolet and visible (UV–
vis) spectra were measured on a Shimadzu UV-2101PC UV–VIS
scanning spectrometer. Infrared (IR) spectra were measured on
Perkin Elmer System 2000 FT-IR spectrometer as KBr pellets.
NMR spectra were recorded on Bruker Avance 300, 400 or
600 MHz spectrometers. Low-resolution mass spectra were ob-
tained on Finnigan MAT LCQDECA instrument and high-resolution
mass spectra were obtained on GCT Premier Instrument.
5.5. Tetrakis(triphenylphosphine)Pd(0)
Dimethoxybenzene was prepared by methylation21 of resor-
cinol. 2,4-Dimethoxyacetophenone (3a) was prepared by Friedel–
Crafts acetylation of dimethoxybenzene using standard proce-
dures.22 5-Bromo-2,4-dimethoxyacetophenone (3b) and 50-Bro-
mo-20,40,4-trimethoxychalcone (5) were prepared as reported
previously.18 3-Bromo-4-methoxybenzaldehyde (4b) was prepared
by methylation of 3-bromo-4-hydroxybenzaldehyde, which was it-
self prepared by bromination23 of 4-hydorxybenzaldehyde.
Tetrakis(triphenylphosphine)palladium(0) was prepared fol-
lowing a modified literature24 procedure. Thus, triphenylphos-
phine (7.40 g, 28.24 mmol, 5 equiv) was added to a solution of
palladium chloride (1.00 g, 5.64 mmol, 1 equiv) in DMSO (70 mL)
under nitrogen atmosphere and the resulting mixture was refluxed
in an oil bath until complete solubilization (140 °C, 2.4 h). Then the
oil bath was removed and the solution was rapidly stirred for 5 min
after which hydrazine hydrate (1.2 mL) was slowly added over
1 min during which a vigorous reaction took place with evolution
of gas. The resulting dark solution was then cooled with a water
bath for about 5 min and left to crystallize at rt, filtered under
nitrogen atmosphere on a sintered-glass funnel and was washed
successively with absolute ethanol (2 ꢁ 15 mL) and diethyl ether
(2 ꢁ 15 mL), then dried to give tetrakis(triphenylphosphine)Pd(0)
as a yellow crystalline solid (6.39 g, 98%).
5.2. 3-Bromo-20,40,4-trimethoxychalcone (6)
A mixture of 4b (3.00 g, 13.95 mmol, 1 equiv), 3a (2.51 g,
13.94 mmol, 1 equiv), and NaOH (6 pellets) was ground in a porce-
lain mortar at room temperature. After 10 min, the mixture turned
to a yellow solid which was treated with water (60 mL) and filtered
to give bromochalcone 6 (5.12 g, 97%) as a yellow solid. Mp 152.1–
153.3 °C); IR (KBr)
m
max 2943, 1649, 1593, 1508, 1460, 1423, 1389,
5.6. (E)-1-(2,4-Dimethoxyphenyl)-3-(20,40,6-trimethoxy-50-
(2,4,5-trimethyl-1,3-dioxolan-2-yl)biphenyl-3-yl)prop-2-en-1-
one (12)
1323, 1271, 1215, 1166, 1020, 978, 816, 650 cmꢀ1
;
1H NMR
(300 MHz, CDCl3): d 3.90 (3H, s, OCH3), 3.94 (3H, s, OCH3), 3.96
(3H, s, OCH3), 6.52 (1H, d, J = 2.1 Hz, H-30), 6.59 (1H, dd, J = 2.1,
8.4 Hz, H-50), 6.93 (1H, d, J = 8.4 Hz, H-5), 7.41 (1H, d, J = 15.8 Hz,
A
mixture of 6 (1.50 g, 3.98 mmol, 1 equiv), 11 (1.50 g,
H ), 7.52 (1H, dd, J = 1.8, 8.4 Hz, H-6), 7.60 (1H, d, J = 15.8 Hz,
a
Hb), 7.77 (1H, d, J = 8.4 Hz, H-60), 7.84 (1H, d, J = 1.8 Hz, H2).
3.98 mmol, 1 equiv), and Pd(PPh3)4 (5 mol %, relative to 6) in tolu-
ene (15 mL) was refluxed under nitrogen atmosphere for 10 min. A
20% aqueous solution of tetraethylammonium hydroxide
(12.25 mL, 2.45 g, 16.71 mmol, 4.2 equiv) was added and the
resulting mixture was refluxed following the progress of reaction
by TLC. After 5 h, the mixture was cooled to room temperature
and water (10 mL) was added and the mixture extracted with
diethyl ether (4 ꢁ 20 mL) and dried (Na2SO4). The solvent was re-
moved and the residue was chromatographed over silica gel
(petroleum ether/ethyl acetate 2:3–1:1). The resulting solid was
dried overnight in a vacuum oven to afford ketal 12 (1.59 g, 73%).
1H NMR (400 MHz, CDCl3): d 1.25 (3H, d, J = 6 Hz), 1.32 (d, J =
6 Hz), 1.83 (3H, s), 3.60 (1H, m), 3.78 (1H, m), 3.81 (3H, s), 3.82
(3H, s), 3.85 (3H, s), 3.88 (3H, s), 3.96 (3H, s), 6.50 (1H, d, J =
5.3. 2-(5-Bromo-2,4-dimethoxyphenyl)-2,4,5-trimethyl-1,3-
dioxolane (10)
In a round-bottomed flask equipped with a Dean-Stark trap and
a reflux condenser, compound 3b (13.87 g, 53.55 mmol, 1 equiv)
was treated with 2,3-butanediol (6.26 g, 69.55 mmol, 1.3 equiv)
and H2NSO3H (0.52 g, 5.36 mmol, 10 mol % relative to the diol) in
refluxing toluene (50 mL) until the reaction was complete (TLC
monitoring, 20 h). The mixture was filtered and the residue was
washed with diethyl ether (30 mL). After evaporation of the sol-
vent, the resulting solid was dried overnight in a vacuum oven
(55 °C) to give ketal 10 as pink solid (17.02 g, 96%). Mp 73.7–

S. O. Mihigo et al. / Bioorg. Med. Chem. 18 (2010) 2464–2473
2471
2.0 Hz), 6.56 (1H, dd, J = 2.0, 8.4 Hz), 6.62 (1H, s), 6.99 (1H, d, J =
8.8 Hz), 7.41 (1H, d, J = 15.6 Hz), 7.52 (1H, s), 7.54 (1H, d, J =
2.0 Hz), 7.58 (1H, dd, J = 2.0, 8.4 Hz), 7.71 (1H, d, J = 15.6 Hz),
7.74 (1H, d, J = 8.4 Hz); 13C NMR (100 MHz, CDCl3): d 16.54,
16.83, 27.00, 55.52, 55.77, 55.77, 55.87, 56.01, 78.15, 78.84,
97.67, 98.63, 105.10, 107.37, 111.20, 117.77, 122.53, 124.04,
124.99, 127.69, 128.13, 128.81, 129.11, 132.08, 132.65, 142. 51,
157.54, 157.69, 159.01, 160.22, 163.93, 190.84.
5.10. 4,20,400,2000-Tetrahydroxy-40,4000-dimethoxy-3,5000-bichalcone
(15)
Yellow solid; IR (KBr)
1367, 1276, 1209, 1155, 1130 cmꢀ1; UV (MeOH) kmax (log
(4.38), 262 (4.03) nm, (MeOH + NaOMe) kmax (log ) 433 (4.44),
279 (3.99) nm, (MeOH + NaOAc) kmax (log ) 374 (4.36), 306
(4.02) nm, (MeOH + AlCl3) kmax (log ) 373 (4.36), 288 (3.95) nm,
(MeOH + AlCl3 + HCl) kmax (log
) 377 (4.32) nm; 1H NMR (ace-
m
max 3423, 2926, 1628, 1555, 1502, 1450,
e) 375
e
e
e
e
5.7. (E)-3-(50-Acetyl-20,40,6-trimethoxybiphenyl-3-yl)-1-(2,4-
dimethoxyphenyl)prop-2-en-1-one (13)
tone-d6, 300 MHz) d 13.89 (1H, s), 13.73 (1H, s), 8.20 (1H, d, J =
9 Hz, H-60), 8.15 (1H, s; H-6000), 7.91–7.89 (4H, overlapping multi-
plet; H-a
, H-a0, H-b, H-b0), 7.78–7.74 (4H, overlapping multiplet;
Ketal 12 (3.73 g, 6.80 mmol) was treated with molecular iodine
(0.150 g, 10 mol %) in refluxing acetone (30 mL) for 10 min.17 After
cooling to room temperature, acetone was distilled off and the res-
idue suspended in CH2Cl2 (30 mL) and washed successively with
5% aq Na2S2O3 (20 mL), water (2 ꢁ 20 mL) and brine (20 mL). The
resulting solution was dried (MgSO4), filtered and the solvent
evaporated in vacuo to give chalconylacetophenone 13 (2.51 g,
77%) as a dark solid. 1H NMR (400 MHz, CDCl3): d 2.63 (3H, s),
3.82 (3H, s), 3.87 (3H, s), 3.88 (3H, s), 3.89 (3H, s), 4.01 (3H, s),
6.50 (1H, d, J = 2.4 Hz), 6.56 (1H, s), 6.57 (1H, dd, J = 2.4, 8.8 Hz),
6.98 (1H, d, J = 8.8 Hz), 7.37 (1H, d, J = 15.6 Hz), 7.49 (1H, d, J =
2.0 Hz), 7.58 (1H, dd, J = 2.0, 8.4 Hz), 7.67 (1H, d, J = 15.6 Hz),
7.73 (1H, d, J = 8.4 Hz, H), 7.82 (1H, s); 13C NMR (100 MHz, CDCl3):
d 31.99, 55.55, 55.67, 55.81, 55.81, 55.90, 94.82, 98.66, 104.99,
111.02, 119.57, 120.11, 122.54, 125.14, 127.02, 127.81, 129.75,
131.52, 132.63, 133.93, 142.33, 158.90, 160.19, 160.99, 161.81,
163.85, 190.94, 197.55. HRMS (TOF EI+) m/z: M+ calcd for
C28H28O7 476.1835; found 476.1870.
H-2, H-6, H-200, H-600), 7.05 (1H, d, J = 9 Hz, H-5), 6.90 (2H, d, J =
9 Hz, H-300, H-500), 6.63 (1H, s, H-3000), 6.52–6.47 (2H, overlapping
multiplet, H-50, H-30), 3.89 (6H, s, –OMe-40, –OMe-4000).
5.11. 4,20,40,400,2000-Pentahydroxy-4000-methoxy-3,5000-bichalcone
(16)
Yellow solid; IR (KBr)
1275, 1209, 1157 cmꢀ1; UV (MeOH) kmax (log
(4.45) nm, (MeOH + NaOMe) kmax (log ) 433 (4.78), 293 (4.26)
nm, (MeOH + NaOAc) kmax (log ) 379 (4.70), 377 (4.65) nm, (MeO-
H + AlCl3) kmax (log 375 (4.68), 273 (4.19) nm, (MeO-
H + AlCl3 + HCl) kmax (log
) 377 (4.65) nm; 1H NMR (acetone-d6,
300 MHz) d 13.88 (1H, s), 13.66 (1H, s), 8.15–8.13 (2H, overlapping
multiplet; H-6000, H-60), 7.91–7.86 (4H, overlapping multiplet; H-
mmax 3424, 2924, 1631, 1555, 1502, 1366,
e) 374 (4.70), 241
e
e
e
)
e
a
,
H-a0, H-b, H-b0), 7.76–7.74 (4H, overlapping multiplet; H-2, H-6, H-
200, H-600), 7.05 (1H, d, J = 6 Hz, H-5), 6.90 (2H, d, J = 9 Hz, H-300, H-
500), 6.63 (1H, s, H-3000), 6.45 (1H, dd, J = 3 Hz, 9 Hz, H-50), 6.38 (1H,
dd, J = 3 Hz, 9 Hz, H-30), 3.89 (3H, s, –OMe-4000).
5.8. (E)-1-(2,4-Dimethoxyphenyl)-3-(20,40,6-trimethoxy-50-((E)-
3-(4-methoxyphenyl)acryloyl)biphenyl-3-yl)prop-2-en-1-one
(14)
5.12. Synthesis of rhuschalcone VI (1) analogs
5.12.1. General procedure
A mixture of chalconylacetophenone 13 (0.12 g, 0.25 mmol,
1 equiv), anisaldehyde (0.21 g, 1.58 mmol, 6.3 equiv), and NaOH
(1 pellet) was ground in a porcelain mortar at room temperature.
After 8 min, a yellowish solid was formed which was treated with
water (100 mL) and filtered to give 14 (0.146 g, 98%) as a dark-yel-
low solid. Mp 184.9–187.1 °C; IR (KBr) mmax 2916, 1649, 1597,
A solution of boronate ester 11, the appropriate chalcone (25–
28), and Pd(PPh3)4 (5 mol %, relative to 11) in toluene (20 mL)
was refluxed under nitrogen atmosphere for 10 min. A 20% aque-
ous solution of tetraethylammonium hydroxide (4.2 equiv) was
added and the resulting mixture refluxed following the progress
of reaction by TLC (6.5 h for 17 and 19; 5 and 7 h for 20 and 21,
respectively), the mixture was cooled to room temperature and
water (10 mL) was added and the mixture extracted with diethyl
ether (4 ꢁ 20 mL) and dried (MgSO4). The solvent was removed
and the crude product passed through a column of silica gel (hex-
ane/ethyl acetate 3.5:1.5), and the purified solid product was dried
overnight in a vacuum oven to afford coupled intermediates 17a,
19a, 20a and 21a in 86%, 80%, 95% and 79%, respectively. One
equivalent of each of these intermediates (1.32 g of 17a; 1.20 g of
19a; 0.37 g of 20a and 1.12 g of 21a) was refluxed with I2
(10 mol %) in acetone (20 mL) for 12 min (5 min for 20a). After
evaporation of solvent, the resulting residue was dissolved in
dichloromethane (40 mL) and washed successively with 5% aq
Na2S2O3 (3 ꢁ 20 mL), water (2 ꢁ 20 mL), brine (30 mL) and dried
over anhydrous MgSO4. After evaporation of solvent, the crude
product was purified over silica gel column chromatography
(petroleum ether/ethyl acetate 3.5:1.5) to afford the deketalized
ketones 17b (900 mg, 80%), 19b (588 mg, 57%), 20b (118 mg,
37%) and 21b (302 mg, 79%). One equivalent of each of 17b
(100 mg, 1 equiv), 19b (50 mg, 1 equiv), 20b (110 mg, 1 equiv)
and 21b (50 mg, 1 equiv) were mixed with anisaldehyde (5, 6, 1,
8 equiv, respectively), and NaOH (1 pellet). The mixtures were
ground in a porcelain mortar at room temperature. After 8, 6, 13,
10 min, a yellowish solid was formed, treated with water and fil-
tered. The resulting solid was purified using prep TLC (petroleum
ether/ethyl acetate 4:2).
1502, 1458, 1423, 1254, 1202, 1159, 1024, 986, 823 cmꢀ1 1H
;
and 13C NMR, see Table 1. HRMS (TOF EI+) m/z: M+ calcd for
C36H34O8 594.2254; found 594.2256.
5.9. Rhuschalcone VI (1)
To a solution of 14 (0.050 g, 0.08 mmol, 1 equiv) in CH2Cl2
(5 mL), 1 M BBr3 in CH2Cl2 (1.5 mL, 0.380 g, 1.52 mmol, 6 equiv)
was added. The resulting mixture turned pink and was heated un-
der reflux, for 3 h. After cooling to room temperature, and the mix-
ture was quenched by slow addition of methanol, evaporated in
vacuo and the resulting orange solid was dissolved in KOH (2.5%
in water). The resulting yellowish solution was acidified to pH 6
by drop wise addition of 3 M HCl and a precipitate formed. This
was filtered, dried, and purified by preparative TLC (0.25 mm silica
gel plates) using chloroform/methanol (46:04) as eluent to afford
rhuschalcone VI 1 (22 mg, 52%, Rf = 0.20), 15 (8 mg, 18%, Rf
=
0.27), and 16 (3 mg, 6%, Rf = 0.35). Rhuschalcone VI (1): yellow so-
lid; Mp 192–198 °C; IR (KBr) mmax 3429, 2924, 1626, 1555, 1502,
1362, 1219, 1171 cmꢀ1; UV (MeOH) kmax (log
e
) 370 (4.54), 275
) 414 (4.56), 305 (4.32)
) 378 (4.68), 304 (4.52) nm, (MeO-
374 (4.76), 236 (4.50) nm, (MeO-
e
) 375 (4.72) nm. 1H and 13C NMR see
(4.25) nm, (MeOH + NaOMe) kmax (log
nm, (MeOH + NaOAc) kmax (log
H + AlCl3) kmax (log
e
e
e)
H + AlCl3 + HCl) kmax (log
Table 1.

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S. O. Mihigo et al. / Bioorg. Med. Chem. 18 (2010) 2464–2473
5.12.2. 4,400,2000,4000-Tetramethoxy-3,5000-bichalcone (17)
5.14. Protozoa and mammalian cells
The crude 17 obtained following the general procedure above
was purified using prep TLC (petroleum ether/ethyl acetate 4:2)
to afford compound 17 (104 mg, 81%): yellow solid; Mp 155.8–
157.1 °C; IR (KBr) mmax 2934, 1655, 1593, 1499, 1280, 1252, 1205,
B. caudatus protozoa cultures were a generous gift of the Global
Institute for Bioexploration (Gibex, Rutgers University, New Bruns-
wick, NJ, USA). Protozoa were maintained at 30 °C in cereal grass
medium (Wards, Rochester, NY, USA) diluted 1:1 with sterile
1144, 1020, 822 cmꢀ1; UV (CHCl3) kmax (log ) 342 (4.40) nm; 1H
e
and 13C NMR see Table 1. ; HRMS (TOF EI+) m/z: M+ calcd for
water, supplemented with a liquid E. coli culture (strain DH5
a
C34H30O6 534.2043; found 534.2065.
was donated by G. Joseph, Gibex, Rutgers University, New Bruns-
wick, NJ, USA) grown in LB medium (Sigma) and subcultured every
7–12 days. Baby hamster kidney (BHK) cells were kindly donated
by Botswana Vaccine Institute. BHK cells were grown in DMEM/
F10 1:1 (Sigma) supplemented with 10% fetal calf serum (Highveld
Biological, South Africa) and antibiotics penicillin and streptomy-
cin (Sigma) in a 37 °C/5% CO2 incubator.
5.12.3. 4,400,2000,4000-Tetrahydroxy-3,5000-bichalcone (18)
Yellow solid; IR (KBr) mmax 3429, 2924, 1631, 1555, 1502, 1450,
1371, 1280, 1209, 1163, 1030, 825 cmꢀ1; 1H NMR (300 MHz, CDCl3)
d 13.71 (1H, s), 8.19–8.14 (3H, overlapping multiplet; H-30, H-50, H-
6000), 7.89–7.80 (5H, overlapping multiplet; H-b , H-b0, H-2, H-200, H-
600), 7.75–7.70 (3H, overlapping multiplet; H-20, H-40, H-60), 7.55–
7.51 (2H, overlapping multiplet, H-a
, H-a0), 7.00 (1H, d, J = 9 Hz, H-
5.15. B. caudatus viability assay
5), 6.94 (3H, overlapping multiplet, H-6, H-300, H-500).
The growth inhibition assay was performed as described by
5.12.4. 4,5,400,2000,4000-Pentamethoxy-3,5000-bichalcone (19)
Joseph25 with some modifications. 100
l
L of a fresh subculture
containing 10,000 protozoa were transferred into wells of a
flat-bottomed 96-well plate and 10 of mixture of
5000 units/mL penicillin, streptomycin and 100 g/mL kanamy-
The crude 19 obtained following the general procedure above
was purified using prep TLC (petroleum ether/ethyl acetate
6.3:3.7) to afford compound 19 (49.8 mg, 79%): yellow solid; Mp
76.5–78.3 °C; IR (KBr) mmax 2934, 1653, 1597, 1502, 1454, 1258,
l
L
a
l
cin were added. B. caudatus were then exposed to different com-
pounds for 4 h or 24 h at a final concentration of 0.6 mg/mL.
CuSO4 served as positive control at the same concentration. Fi-
1202, 1163, 1016, 827 cmꢀ1 1H- and 13C NMR see Table 1. HRMS
;
(TOF EI+) m/z: M+ calcd for C35H32O7 564.2148; found 564.2170.
nally, 5 lL of a 5 mg/mL stock solution of 3-(4,6-dimethylthia-
zole-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma) was
added. After 2 h incubation, supernatant was carefully removed
from each well and 100 lL DMSO was added to dissolve MTT
crystals. Absorbance at 570 nm indicative of viability of protozoa
was measured with a multiwell-plate reader (Tecan Sunrise,
Waesi Pharmaceuticals, Gaborone, Botswana). Viability assays
were performed in triplicates.
5.12.5. 4,400,2000,4000-Tetramethoxy-40-methyl-3,5000-bichalcone
(20)
The crude 20 obtained following the general procedure above
was purified using prep TLC (petroleum ether/ethyl acetate
5.3:4.5) to afford compound 20 (25 mg, 18%): yellow solid; Mp
112.4–114.1 °C; IR (KBr) mmax 2934, 1653, 1597, 1454, 1253,
1202, 1024, 814 cmꢀ1 1H and 13C NMR See Table 1. HRMS (TOF
;
EI+) m/z: M+ calcd for C35H32O6 548.2199; found 548.2213.
5.16. Determination of LC50 concentrations and cytotoxicity
5.12.6. 4,5,20,40,400,2000,4000-Heptamethoxy-3,5000-bichalcone (21)
The crude 21 obtained following the general procedure above
was purified using prep TLC (petroleum ether/ethyl acetate 3:2)
to afford compound 21 (34 mg, 54%): yellow solid; Mp 113.1–
114.9 °C; IR (KBr) mmax 2924, 1649, 1597, 1460, 1414, 1261, 1205,
Lethal concentration of compounds for 50% of B. caudatus (LC50
)
and general cytotoxicity of compounds (CC50) were determined by
the MTT viability assay as described by Mosmann26 with some
modifications. Briefly, BHK cells or B. caudatus protozoa were ad-
1167, 1115, 1020, 825 cmꢀ1 1H and 13C NMR d see Table 1.
;
justed to 1 ꢁ 105/mL in their respective growth media. 100
lL of
BHK cell suspension was transferred to wells of a 96-well plate
5.13. Isolation of natural of rhuschalcone VI (1) from R.
pyroides
and left to adhere overnight. 100
directly after adding 20 L of a mixture of 5000 units/mL penicillin,
streptomycin and 100 g/mL kanamycin into wells. Cells or proto-
lL of B. caudatus cells were used
l
l
The air-dried and powdered R. pyroides (root bark, 1.6 kg) was
extracted exhaustively by cold percolation with a mixture of meth-
ylene chloride–methanol (1:1) followed by 100% methanol. The
combined extracts (167.02 g), obtained by evaporation under re-
duced pressure was suspended in a mixture of water–methanol
(1:1; 800 mL) and extracted with methylene chloride
(4 ꢁ 300 mL) to give, after concentration in vacuo, 16.32 g of a
methylene chloride soluble extract. This extract was adsorbed on
25 g of silica gel and chromatographed through a column packed
with 200 g silica gel in chloroform. The column was eluted using
the following solvent systems, and 19 fractions of ca. 500 mL each
were collected: (a) neat chloroform: fractions 1–3, (b) chloroform–
methanol (96:04): fractions 4–11, (c) chloroform–methanol
(93:07): fractions 12–14, (d) chloroform–methanol (90:10): frac-
tions 15–17, and (e) chloroform–methanol (96:04): fractions 18–
19. TLC monitoring using synthetic rhuschalcone VI (1) as refer-
ence showed that fractions 7 (0.375 g), and 8–11 (1.045 g) con-
tained the sought after substance (rhuschalcone VI (1)), which
were independently subjected to chromatographic separations on
Sephadex LH-20 followed by preparative TLC (chloroform–metha-
nol 46:04) to give 1 (24 mg and 79 mg, respectively).
zoa were exposed for 48 h to serial dilutions of compounds with
the respective growth media to an end concentration ranging from
0.0001 to 1000 lg/mL in a 37 °C/5% CO2 incubator. 10 lL of a MTT
(Sigma) stock solution of 5 mg/mL was added to each well and the
plate was incubated for further 4 h. Supernatant was removed
carefully and MTT crystals were dissolved by adding 100 lL DMSO.
Absorbance was measured at 570 nm using a microplate reader
(Tecan Sunrise, Waesi Pharmaceuticals, Gaborone, Botswana). As-
says were performed in triplicates and best-fit curves were gener-
ated using GraphPad Prism 4.0 software (GraphPad Software, La
Jolla, CA, USA).
Acknowledgements
S.O.M. is gratefully indebted to DAAD-NAPRECA for a Ph.D. fel-
lowship. Financial support from IPICS (Uppsala University, Swe-
den) to NABSA is gratefully acknowledged. We would like to
thank Gili Joseph at the Global Institute of Bioexploration (Gibex,
USA) who developed and provided the anti-protozoa assay
protocols.

S. O. Mihigo et al. / Bioorg. Med. Chem. 18 (2010) 2464–2473
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