Isoflavonoids and other compounds from Psorothamnus arborescens with antiprotozoal activities

Article abstract of DOI:10.1021/np0502600

Bioactivity-guided fractionation of the root extract of Psorothamnus arborescens yielded the new isoflavone 5,7,3′,4′-tetrahydroxy- 2′-(3,3-dimethylallyl)isoflavone (1a) and the new 2-arylbenzofuran 2-(2′-hydroxy-4′,5′-methylenedioxyphenyl)-6-methoxybenzofuran- 3-carbaldehyde (2), together with seven known compounds, including three isoflavones, fremontin (3a), glycyrrhisoflavone (4a), and calycosin (5), two pterocarpans, maackiain (6) and 4-hydroxymaackiain (7), one triterpene, oleanolic acid (8), and one chalcone, isoliquiritigenin (9). In addition, the structure of the isoflavone fremontin was revised using spectroscopic and chemical methods and was assigned the new structure 3a. The isoflavone 1a and the chalcone 9 displayed leishmanicidal activity with IC₅₀ values of 13.0 and 20.7 μM, respectively, against Leishmania donovani axenic amastigotes. Calycosin (5) exhibited selective toxicity against Trypanosoma brucei brucei (IC₅₀ 12.7 μM) compared to L. donovani amastigotes and Vero cells (IC₅₀ 100 and 159 μM, respectively). These results prompted us to test a small group of structurally related isoflavones for their antitrypanosomal activities. Genistein and 7,3′,4′- trihydroxyisoflavone displayed promising activity (IC₅₀ values 4.2 and 7.1 μM, respectively) and selectivity (IC₅₀ versus Vero cells: 32.9 and 135 μM, respectively). These studies suggest that the isoflavone skeleton deserves further investigation as a template for novel antileishmanial and trypanocidal compounds.

Full text of DOI:10.1021/np0502600

J. Nat. Prod. 2006, 69, 43-49
43
Isoflavonoids and Other Compounds from Psorothamnus arborescens with Antiprotozoal
Activities
Manar M. Salem and Karl A. Werbovetz*
DiVision of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State UniVersity, Columbus, Ohio 43210
ReceiVed July 20, 2005
Bioactivity-guided fractionation of the root extract of Psorothamnus arborescens yielded the new isoflavone 5,7,3′,4′-
tetrahydroxy-2′-(3,3-dimethylallyl)isoflavone (1a) and the new 2-arylbenzofuran 2-(2′-hydroxy-4′,5′-methylenedioxy-
phenyl)-6-methoxybenzofuran-3-carbaldehyde (2), together with seven known compounds, including three isoflavones,
fremontin (3a), glycyrrhisoflavone (4a), and calycosin (5), two pterocarpans, maackiain (6) and 4-hydroxymaackiain
(7), one triterpene, oleanolic acid (8), and one chalcone, isoliquiritigenin (9). In addition, the structure of the isoflavone
fremontin was revised using spectroscopic and chemical methods and was assigned the new structure 3a. The isoflavone
1a and the chalcone 9 displayed leishmanicidal activity with IC50 values of 13.0 and 20.7 µM, respectively, against
Leishmania donoVani axenic amastigotes. Calycosin (5) exhibited selective toxicity against Trypanosoma brucei brucei
(IC50 12.7 µM) compared to L. donoVani amastigotes and Vero cells (IC50 100 and 159 µM, respectively). These results
prompted us to test a small group of structurally related isoflavones for their antitrypanosomal activities. Genistein and
7,3′,4′-trihydroxyisoflavone displayed promising activity (IC50 values 4.2 and 7.1 µM, respectively) and selectivity (IC50
versus Vero cells: 32.9 and 135 µM, respectively). These studies suggest that the isoflavone skeleton deserves further
investigation as a template for novel antileishmanial and trypanocidal compounds.
The genus Psorothamnus (name from the Greek psoros and
thamnus, meaning scurfy or scab shrub) belongs to the legume
family (Fabaceae). It was shown in our laboratory to be a rich source
of antiparasitic compounds.1 The genus includes nine species found
in the deserts of southwestern North America.2 Previously, we
isolated antileishmanial and trypanocidal compounds from P.
polydenius (S. Watson) Rydberg that displayed selectivity for these
organisms.1 The plant P. arborescens (Torrey ex A. Gray) Barneby
var. minutifolius (Parish) Barneby, Fabaceae-Papilionoideae2,3
[synonym Dalea fremontii Torrey ex A. Gray var. minutifolia
(Parish) L. Benson]4 is also known as Mojave dalea5 or indigo
bush.6 This plant is a small, thorny desert shrub up to 1 m in height
with deep indigo blue or violet flowers and is locally common
through the west-central and north Mojave Desert, California,
growing chiefly on granitic and volcanic bedrock.2,5 This species
has not been investigated before, and there are no previous reports
of any antiparasitic activity associated with this plant. The closely
related P. fremontii (D. fremontii), which occurs in California only
on the sedimentary ranges of the far eastern Mojave Desert,2 is
reported to have been used by the indigenous tribes to stop internal
hemorrhage and for the treatment of stomach problems.7
P. arborescens extracts exhibited significant activity against
Leishmania donoVani axenic amastigotes and bloodstream form
Trypanosoma brucei brucei. Bioassay-guided fractionation of the
ethanolic root extract from this plant resulted in the isolation of
several bioactive compounds. In this paper, we report on the
antiprotozoal activities of compounds 1a-9 and the structure
elucidation of compounds 1a and 2. The structure of fremontin, an
isoflavone previously isolated from P. fremontii,8 has also been
revised.
droxymaackiain (7),17,18 the triterpene oleanolic acid (8),10,19 and
the chalcone isoliquiritigenin (9).20,21 The structures of the known
compounds were determined by 1D and 2D NMR techniques and
confirmed by comparing the physical and spectroscopic/spectro-
metric data with those from the literature (NMR and MS).
Compound 1a isolated from P. arborescens had a 1H NMR
spectrum (see Table 1) showing a hydrogen-bonded OH group at
δ 13.00, a downfield singlet at δ 7.89, two o-coupled aromatic
protons at δ 6.56 and 6.76 (J ) 8.0 Hz), two m-coupled aromatic
protons at δ 6.29 and 6.43 (J ) 1.3 Hz), and a methylene group at
δ 3.30 displaying 1H-1H COSY correlation to a downfield methine
at δ 5.08, in addition to two methyls at δ 1.43 and 1.52. The 13C
and DEPT NMR spectra showed a carbonyl at δ 182.0, six
oxygenated carbons in the aromatic region at δ 165.0, 163.8, 159.2,
155.2, 145.6, and 144.2, and three methines at δ 124.4, 123.0, and
113.1, in addition to two relatively upfield methines at δ 99.8 and
94.5, one methylene at δ 27.3, two methyls at δ 25.6 and 17.6,
and five quaternary carbons at δ 130.8, 129.2, 125.3, 123.9, and
106.1. HRESIMS confirmed the deduced molecular formula
(measured [M + Na]+ 377.1000, calcd 377.1001 for C20H18NaO6).
The NMR and MS data indicated an isoflavone structure with a
5,7-dihydroxylated A-ring and a 2′,3′,4′-trisubstituted B-ring with
two hydroxyls and one isoprenyl group. This was supported by an
HMBC correlation between H-2 (δ 7.89) and C-1′, which is
correlated to H-5′ and H-1′′. H-6′ showed three-bond HMBC
correlations to C-3 and C-2′. H-1′′ and H-2′′ of the isoprenyl group
correlated to C-2′, thus establishing the position of the isoprenyl
group at C-2′. Further confirmation was provided via a selective
NOE experiment in which H-2 showed NOE correlations to H-6′,
H-1′′, and H-2′′. Assignment of ring B oxygenated carbons is based
on the HMBC correlation of H-1′′ to C-3′ and that of H-6′ to C-4′.
Furthermore, acid-catalyzed cyclization of the 2′-isoprenyl group
gave the dihydrobenzopyran derivative 1b, verifying the presence
of an OH group at the 3′ position. Further evidence against the
presence of an OH group in the 2′ position is the relatively
downfield position of the OH-5 signal (δ 13.0); a 2′-OH group in
an isoflavone structure would be expected to exert a significant
shielding effect on the 5-OH, resulting in a more upfield chemical
shift (δ 12.51-12.79).22,23 Thus compound 1a was identified as
the new 5,7,3′,4′-tetrahydroxy-2′-(3,3-dimethylallyl)isoflavone. The
isoflavanone arizonicanol D, isolated from the root of Sophora
Results and Discussion
Bioactivity-guided fractionation of the ethanolic extract of P.
arborescens yielded four isoflavones, the new 5,7,3′,4′-tetrahydroxy-
2′-(3,3-dimethylallyl)isoflavone (1a), fremontin (3a),8 glycyr-
rhisoflavone (4a),9 and calycosin (5),10-13 a new 2-arylbenzofuran,
2-(2′-hydroxy-4′,5′-methylenedioxyphenyl)-6-methoxybenzofuran-
3-carbaldehyde (2), the pterocarpans maackiain (6)14-17 and 4-hy-
* To whom correspondence should be addressed. Tel: (614) 292-5499.
Fax: (614) 292-2435. E-mail: werbovetz.1@osu.edu.
10.1021/np0502600 CCC: $33.50
© 2006 American Chemical Society and American Society of Pharmacognosy
Published on Web 12/22/2005

44 Journal of Natural Products, 2006, Vol. 69, No. 1
Salem and WerboVetz
Chart 1
Table 1. 1H and 13C NMR Data and HMBC Correlations for Compounds 1a and 1b in Acetone-d6
1a
1b
position
δC
δH (mult.; JHH, Hz)
HMBC
δC
δH (mult.; JHH, Hz)
HMBC
2
3
4
5
6
7
8
155.2
125.3
182.0
163.8
99.8
7.89 (s)
C-3, 4, 9, 10a,1′
155.5
124.9
181.5
163.9
99.9
8.02 (s)
C-3, 4, 9, 1′
6.29 (d; 1.3)
6.43 (d; 1.3)
C-4a, 5, 7, 8, 10
C-4a, 6, 7, 9, 10
6.30 (d; 1.3)
6.44 (d; 1.3)
C-4a, 5, 7, 8, 10
C-4a, 6, 7, 9, 10
165.0
94.5
165.1
94.6
9
159.2
106.1
123.9
129.2
144.2
145.6
113.1
123.0
27.3
159.3
106.0
122.9b
122.7b
142.4
147.5
112.7
122.8
21.3
33.3
75.2
26.8
26.8
10
1′
2′
3′
4′
5′
6′
1′′
2′′
3′′
4′′
5′′
5-OH
6.76 (d; 8.0)
6.56 (d; 8.0)
3.30 (d; 6.0)
5.08 (m)
C-1′, 2′a, 3′
6.70 (d; 8.0)
6.64 (d; 8.0)
2.66 (br t; 6.8)
1.78 (t; 6.8)
C-1′, 3′
C-3, 2′, 4′
C-1′, 2′, 3′, 2′′, 3′′
C-2′, 1′′, 3′′, 4′′, 5′′
C-3, 2′, 4′, 1′′a
C-1′, 2′, 3′, 2′′, 3′′
C-2′, 1′′, 4′′, 5′′
124.4
130.8
17.6
1.43 (s)
1.52 (s)
13.00 (s)
C-2′′, 3′′, 5′′
C-2′′, 3′′, 4′′
C-5, 6, 10
1.35 (s)
1.35 (s)
12.98 (s)
C-2′′, 3′′, 5′′
C-2′′, 3′′, 4′′
C-5, 6, 10
25.6
a Weak four-bond HMBC correlations. b Entries with the same superscript are interchangeable.
arizonica (Fabaceae), possesses a B-ring structure similar to that
The new 2-arylbenzofuran 2 exhibited a 1H NMR spectrum (see
Table 2) showing a downfield singlet at δ 10.00, a 1,2,4-
trisubstituted benzene ring with protons at δ 7.50 (d, J ) 8.4 Hz),
6.66 (dd, J ) 2.2 and 8.4 Hz), and 6.71 (J ) 2.2 Hz), two isolated
aromatic singlets at δ 7.52 and 7.16, a downfield methylene singlet
1
found in compound 1a, and their H and 13C NMR data are in
agreement.24 The structure of compound 1a is also closely related
to that of the isoflavone piscidone isolated from the root bark of
the Jamaican dogwood Piscidia erythrina (Fabaceae).23

IsoflaVonoids from Psorothamnus arborescens
Journal of Natural Products, 2006, Vol. 69, No. 1 45
Table 2. 1H and 13C NMR Data and HMBC Correlations for
Compound 2 in Acetone-d6
position
δC
δH (mult.; JHH
)
HMBC
2
3
4
5
6
7
8
9
1′
2′
3′
4′
5′
6′
163.5
118.3
133.5
108.9
159.9
100.5
162.5
109.8
119.5
150.4
94.1
147.8
146.7
100.9
187.7
56.1
7.50 (d; 8.4)
C-6, 8
6.66 (dd; 2.2,8.4)
C-6, 7, 9
Figure 1. Fremontin: literature and proposed structures.
6.71 (d; 2.2)
C-5, 6, 8, 9
1
The H and 13C NMR data (Table 3) as well as the physical
data for compound 3a closely matched those reported by Maniku-
mar et al.8 for the isoflavone fremontin (see Figure 1) isolated from
the related species P. fremontii. However, the 13C NMR data
showing ring B oxygenated carbons at relatively upfield positions
(δ 143.1 and 147.3) seemed more consistent with the OH groups
in ortho rather than meta positions to each other; oxygenated
carbons with no ortho/para oxygenation are expected to be more
downfield (δ 155-165).26 Furthermore, the OH-5 signal in acetone-
d6 occurred at δ 13.00, a more downfield value than that expected
for a 2′-hydroxylated isoflavone (δ 12.51-12.79),22,27 implying that
position 2′ is not hydroxylated. Tahara et al. suggested that the
structure of fremontin should be reexamined on the basis of the
OH-5 chemical shift22 and later proposed a possible B-ring structure
with two ortho-OH groups at positions 3′ and 4′ and the isoprenyl
group assigned to position 6′.28 Our HMBC data (see Table 3)
supported the proposed structure (Figure 1) where correlations of
H-2′ at δ 6.56 with C-3 and C-6′, and H-5′ at δ 7.02 with C-1′ and
-3′, were observed. To shed further light on the structure of this
compound, we performed selective NOE experiments to irradiate
the methyl group at δ 1.33, resulting in enhancement of the H-2
and the H-5′ singlets by 0.1 and 0.5%, respectively. Alternatively,
irradiating the H-2 singlet enhanced the signals of the nearby
isoprenyl methyl groups in the 6′ positions of ring B by 0.5 and
0.6%, while irradiating H-5′ strongly enhanced the isoprenyl
methyls by 9.1 and 3.1%. In addition, acetylation of fremontin gave
the tetraacetyl derivative 3b, whose 1H NMR signals for H-2′ and
H-5′ experienced a significant downfield shift of 0.39 and 0.37 ppm,
respectively, indicating that both protons are ortho to the acetylated
OH group(s).11 Since those protons are in the para position to each
other, it follows that the only arrangement possible is that of the
structure 3b. Additional evidence came from the trimethylated
7.16 (s)
C-1′, 2′, 5′
7.52 (s)
10.00 (s)
3.86 (s)
6.08 (s)
C-2, 2′, 4′
C-3
C-6
3-CHO
6-OCH3
-OCH2O-
102.7
C-4′, 5′
at δ 6.08 indicating a methylenedioxy group, and a methoxy group
at δ 3.86. The 13C and DEPT NMR spectra showed a carbonyl
signal at δ 187.7, six oxygenated aromatic carbons at δ 163.5-
146.7, five aromatic methines and three quaternary carbons at δ
133.5-94.1, one methylenedioxy group at δ 102.7, and one
methoxy group at δ 56.1. The deduced molecular formula C17H12O6
was confirmed by HRESIMS (measured [M + Na]+ 335.0550,
calcd 335.0532). The carbonyl signal at δ 187.7 showed HSQC
correlation to the proton singlet at δ 10.00, thus indicating a
carbaldehyde group that was assigned to position 3 on the basis of
an HMBC correlation between the aldehydic proton and C-3. The
position of the methoxy group was established by an HMBC
correlation to C-6 and a NOESY correlation to H-5 and H-7. The
methylenedioxy protons showed HMBC correlations to the o-
oxygenated carbons of ring C (δ 146.7 and 147.8). The aromatic
singlet at δ 7.52 was assigned to H-6′ on the basis of its three-
bond HMBC correlations to C-2, while that at δ 7.16 to H-3′ on
the basis of three-bond correlations to C-1′ and 5′. Thus compound
2 was shown to be 2-(2′-hydroxy-4′,5′-methylenedioxyphenyl)-6-
methoxybenzofuran-3-carbaldehyde. The compound closest in
structure to 2 is cicerfuran isolated from the wild chickpea Cicer
bijugum.25
Table 3. 1H and 13C NMR Assignments and HMBC Correlations for Compounds 3a (acetone-d6) and 3c (CDCl3)
3a
δH (mult.; JHH,, Hz)
7.79 (s)
3c
δH (mult.; JHH,, Hz)
7.59 (s)
position
δC
HMBC
δC
HMBC
2
3
4
5
6
7
8
155.0
126.8
183.0
163.7
99.7
C-3, 4, 9, 1′
153.6
126.1
182.1
162.6
98.1
C-3, 4, 9, 1′
6.27 (d; 1.9)
6.39 (d; 1.9)
C-5, 7, 8, 10
C-6, 7, 9, 10
6.38 (d; 2.1)
6.40 (d; 2.1)
C-5, 7, 8, 10
C-6, 7, 9, 10
165.1
94.4
165.5
92.4
9
159.1
105.9
122.2
121.1
143.5
145.7
115.4
140.2
42.2
158.0
106.0
121.6
115.7
146.9
148.7
111.2
140.3
42.0
10
1′
2′
3′
4′
5′
6′
1′′
2′′
3′′
6.56 (s)
7.02 (s)
C-3, 3′,4′, 6′
6.54 (s)
7.06 (s)
C-3, 3′, 4′, 6′
C-1′, 3′, 4′, 1′′
C-1′, 3′, 4′, 6′, 1′′
150.0
109.4
6.01 (dd; 10.6, 17.6)
4.77 (dd; 1.2, 17.6)
4.64 (dd; 1.2, 10.6)
1.29 (s)
1.33 (s)
13.01 (s)
C-6′, 1′′, 4′′, 5′′
C-1′′, 2′′
C-1′′
C-6′, 1′′, 2′′, 5′′
C-6′, 1′′, 2′′, 4′′
C-5, 6, 10
148.5
109.8
6.02 (dd; 10.6, 17.5)
4.81 (d; 17.5)
4.73 (d; 10.6)
1.37 (s)
1.40 (s)
12.81 (s)
C-1′′, 4′′, 5′′
C-1′′, 2′′
C-1′′, 2′′
C-6′, 1′′, 2′′, 5′′
C-6′, 1′′, 2′′, 4′′
C-5, 6, 10
C-3′
4′′
5′′
5-OH
3′-OCH3
7′-OCH3
4′-OCH3
29.4
30.0
28.9
29.7
55.78a
55.84a
55.94a
3.83 (s)
3.88 (s)
C-7
3.92 (s)
C-4′
a Assignments are interchangeable.

46 Journal of Natural Products, 2006, Vol. 69, No. 1
Salem and WerboVetz
Table 4. Bioactivity of Compounds 1a-9 against Axenic L.
donoVani, T. b. brucei, and Two Mammalian Cell Lines
derivative 3c, prepared by reacting fremontin with dimethyl sulfate
in the presence of K2CO3, followed by separating the tri- (3c) and
tetramethylfremontin (3d) products. The OMe groups in 3c at δ
3.83, 3.88, and 3.92 were assigned at the 3′, 7, and 4′ positions,
respectively, due to HMBC correlations (see Table 3) with C-3′,
-7, and -4′, respectively. Irradiation of H-5′ at δ 7.06 resulted in
enhancement of the two isoprenyl methyl signals by 3.4 and 4.1%,
and only the 4′-OMe group at δ 3.92 by 6.5%, while irradiating
the H-2′ at δ 6.54 resulted in enhancing the 3′-OMe peak at δ 3.83
by 5.6% and the H-2 signal at δ 7.59 by 0.5%. On the other hand,
irradiating the H-2 proton at δ 7.59 resulted in enhancing the H-2′
signal at δ 6.54 (0.5%) and the isoprenyl group methyls at δ 1.37
and 1.40 by 0.5 and 0.7%.
IC50 (µM)a
L. donoVani axenic T. b. brucei
compound
1a
1b
2
3a
3b
3c
3d
4a
4b
4c
5
6
7
8
9
amastigotes
variant 221 Vero cells
PC-3 cells
13.0 ( 0.8
38.4 ( 6.2
>250
123 ( 4
42.7 ( 1.5
>250
86.0 ( 11.0
46.3 ( 2.5
68.3 ( 6.5
41.4 ( 2.7
100 ( 10
>250
12.1 ( 1.9 37.5 ( 10.2 12.7 ( 3.1
57.9 ( 2.5 NTb
107 ( 16
>250
>250
>250
75.3 ( 5.9 134 ( 27
67.8 ( 5.6 NT
32.3 ( 2.0 NT
41.7 ( 12.2 NT
78.5 ( 27.4
66.2 ( 28.5
146 ( 4
134 ( 30
33.0 ( 2.5 52.8 ( 11.3 62.1 ( 7.3
77.0 ( 3.4 NT
79.3 ( 9.6
7.8 ( 2.2 44.3 ( 7.3 30.9 ( 4.6
Glycyrrhisoflavone (4a), which is closely related in structure to
12.7 ( 0.7 159 ( 17
130 ( 13
3.7 ( 0.7
192 ( 29
>250
28.0 ( 10.0
211 ( 10
1
compound 1a, showed H and 13C NMR spectra consistent with
>250
3.3 ( 1.7
those reported in the literature.9 However, we assigned the following
signals differently due to the current availability of HMBC data.
The peak at δ 6.84 is assigned to H-6′ on the basis of its three-
bond correlation to C-3 and C-1′′, while the peak at δ 7.02 is
assigned to H-2′, as it shows correlation to C-3, -4′, and -6′. C-5′
is assigned to δ 128.8 on the basis of its correlation to H-2′′. Further
support for the structure was obtained in an NOE experiment where
the H-2 was irradiated, resulting in enhancement of the H-6′ and
to a lesser extent H-2′. The cyclized (4b) and methylated (4c)
derivatives were prepared for biological testing.
37.3 ( 1.0
>250
20.7 ( 2.3
NT
43.1 ( 8.3 >250
32.8 ( 2.0 65.6 ( 19.5 46.4 ( 7.8
NT
NT
NT
ansamitocin P3
pentamidine
podophyllo-
toxin
1.2 ( 0.3c
NT
NT
NT
NT
2.3 ( 0.4
NT
12.5 ( 1.1c
suramin
NT
0.19 ( 0.03 NT
NT
a Values represent the mean ( standard deviation of at least 3
independent experiments. b NT: not tested. c IC50 values in nM.
The NMR, UV, and IR data of maackiain (6) were in agreement
with the literature values.15-17 Despite the presence of two chiral
centers in the structure, only the 6aR,11aR and 6aS,11aS cis-
configurations are sterically possible.29 However, the specific
rotation data did not completely match those reported for either
the pure isomers or the racemate. The specific rotation is negative
([R]D24 ) -33.5, CHCl3), but the absolute value is much less than
that reported for the optically pure (-)-isomer ([R]D ) -251.7,
CHCl3).14 This suggests that the compound isolated is a mixture
of (-)-6aR,11aR-maackiain and the (+)-6aS,11aS-isomer with an
excess of the (-)-form. The same situation is true for the isolated
3,4-dihydroxy-8,9-methylenedioxypterocarpan (7), which is a 4-hy-
droxy analogue of maackiain (6). The specific rotation of compound
7 in CHCl3, [R]D24 ) +62.2, is less than that reported for the (+)-
isomer in the same solvent ([R]D ) +154). This, again, suggests
the presence of a mixture of (-)-6aR,11aR-4-hydroxymaackiain
and the (+)-6aS,11aS-isomer with an excess of the (+)-form.
The activities of the isolated compounds against L. donoVani
axenic amastigotes and T. b. brucei, as well as against two
mammalian cell lines (Vero cells and PC-3 prostate cancer cells),
are summarized in Table 4. Compounds 1a and 9 showed significant
antileishmanial and trypanocidal activities, displaying IC50 values
below 21 µM. However, they showed low selectivity (around 3-fold)
for the parasites over the mammalian Vero cells. The selectivity is
worse for pterocarpan 7, which shows higher toxicity toward Vero
cells than to the parasites. On the other hand, calycosin (5) displayed
selective toxicity (about 15-fold) toward T. b. brucei bloodstream
forms (IC50 12.7 µM) over Vero cells. Calycosin is reported to have
antiplasmodial,30,31 antileishmanial,32 and antigiardial effects.33 In
addition, calycosin was found to protect human umbilical vein
endothelial cells from hypoxia-induced impairment.34 Methylation
of the isoflavones 3a and 4a moderately improved the trypanocidal
but not the antileishmanial activity. Tetramethylglycyrrhisoflavone
(4c) was 3.7-fold more active toward trypanosomes than the parent
glycyrrhisoflavone (4a). Interestingly, compound 2 did not show
any antikinetoplastid activity in our assays. Related Fabaceae-
derived 2-arylbenzofurans were reported to possess antifungal25 and
antimalarial30 activities. The trypanocidal selectivity of calycosin
prompted us to assay a group of related natural isoflavones that
are commercially available (see Figure 2); the results are sum-
marized in Table 5. Genistein, a common dietary isoflavone
(especially in soy-based diets), was the most potent against
Figure 2. Structures of the additional compounds tested for
antiprotozoal activity.
Table 5. Bioactivity of Selected Isoflavones against Axenic L.
donoVani, T. b. brucei, and Vero Cells
IC50 (µM)
L. donoVani
T. b. brucei
compound
genistein
axenic amastigotes
variant 221
Vero cells
73.0 ( 5.6
89.9 ( 1.8
113 ( 6
NT
NT
NT
NT
NT
NT
4.2 ( 0.5
12.3 ( 1.0
94.2 ( 4.0
90.2 ( 14.1
20.2 ( 4.3
23.7 ( 5.0
>100
7.1 ( 1.6
11.9 ( 0.9
NT
32.9 ( 8.4
NTb
NT
NT
NT
NT
NT
135 ( 13
NT
biochanin A
7-hydroxyisoflavone
formononetin
prunetin
daidzein
7,4′-dimethoxyisoflavone
7,3′,4′-trihydroxyisoflavone
apigenin
pentamidine
suramin
1.5 ( 0.2
NT
NT
0.183 ( 0.025 NT
a Values represent the mean ( standard deviation of at least 3
independent experiments. b NT: not tested.
trypanosomes. This compound displayed an IC50 of 4.2 µM, making
it 3-fold more active than calycosin (IC50 of 12.7 µM) on a molar
basis. Past reports have shown that genistein inhibits trypanosomal
protein tyrosine kinase (PTK) activity.35 Later, it was shown that
genistein is an efficient inhibitor of trypanosomal protein synthesis
and phosphorylation (which is primarily serine and threonine
phosphorylation in these organisms), indicating that the antitrypano-
somal effect of this compound is not due to a specific effect on the
tyrosine kinase activity.36 Daidzein, a genistein analogue that is
known to be inactive against PTK,37 still displayed an inhibitory
effect (IC50 of 23.7 µM) against the parasites. Genistein was also
reported to show activity against both chloroquine-sensitive and
-resistant Plasmodium falciparum.31 7,3′,4′-Trihydroxyisoflavone

IsoflaVonoids from Psorothamnus arborescens
Journal of Natural Products, 2006, Vol. 69, No. 1 47
compound 4 (22.5 mg, tR 53 min), and crude compound 9 (20 mg, tR
8-12 min). The latter was purified by additional HPLC using a gradient
solvent of 40% to 60% B in A in 60 min to isolate 9.1 mg of 9 (tR )
48-54 min). F009-5 (500 mg) was chromatographed on a silica gel
column using a gradient solvent of 4% EtOAc-CHCl3 to 100% CHCl3
to 5% MeOH-CHCl3. Fractions were combined on the basis of their
TLC profile to give 150 mg of crude compound 1a, which was purified
by HPLC using a solvent gradient of 50% B in A increased to 60%
over 30 min then kept isocratic at 60% B, collecting fractions eluting
at tR 28-34 min to give 94 mg of pure compound 1a. F009-8 (917
mg) was subjected to silica gel chromatography eluting with a gradient
of 100% CH2Cl2 to 100% EtOAc. Additional silica gel column
chromatography of the fraction was performed eluting with 35% EtOAc
in CH2Cl2 (F009-8-3, 260 mg) using the isocratic solvent CHCl3-
MeOH-H2O (90:6:1, lower phase) followed by HPLC of subfraction
3 to obtain compound 5 (6 mg, isocratic 20% C in A, tR 32 min) and
subfraction 4 (50% B in A increased to 58% over 24 min, then kept
isocratic, collecting fractions eluting at tR 24-28 min) to give 42.6 mg
of compound 3a. A portion (95 mg) of F010 was purified using
preparative HPLC with an isocratic solvent system of 50% solvent B
in A to obtain 24 mg of compound 7 (tR 13.5 min) and 57 mg of
compound 6 (tR 29 min).
also showed a significant antitrypanosomal effect (IC50 of 7.1 µM),
while being virtually nontoxic to Vero cells. Apigenin, a flavone
with the same substitution pattern as that of genistein, was about
3-fold less active than genistein against T. b. brucei.
Our studies with compounds from P. arborescens and related
commercially available molecules revealed several isoflavonoids
with interesting antiprotozoal activities. These results indicate that
the isoflavone skeleton is worthy of further investigation as a
template for novel antitrypanosomal compounds.
Experimental Section
General Experimental Procedures. Melting points were measured
on a Thomas-Hoover capillary melting point apparatus and were
uncorrected. Optical rotations were determined on a Perkin-Elmer
polarimeter using a 100 mm glass microcell. UV-vis spectra were taken
in methanol using a SPECTRAmax PLUS spectrophotometer (Molec-
ular Devices, Sunnyvale, CA). IR spectra were obtained in KBr on a
Nicolet Prote´ge´ 460 FT-IR spectrophotometer. Nuclear magnetic
resonance (NMR) spectra were obtained on Bruker 300 and 400 MHz
spectrometers using the solvents CDCl3, CD3CN, methanol-d4, or
acetone-d6 (Aldrich) with TMS as the internal standard. 13C DEPT,
1H-1H COSY, DPFGSE-NOE (selective NOE),38 NOESY, HSQC, and
HMBC NMR spectra were obtained using standard Bruker pulse
sequences. All accurate mass experiments were performed on a
Micromass ESI-Tof II (Micromass, Wythenshawe, UK) mass spec-
trometer. Column chromatography was conducted using silica gel 60
(63-200 µm particle size) from EM Science or Sephadex LH-20 from
Amersham Biosciences. Silica gel 60 H (15-45 µm particle size) from
EM Science was used for vacuum-liquid chromatography (VLC).
Precoated TLC silica gel 60 F254 plates from EM Science were used
for thin-layer chromatography (0.25 and 2 mm layer thickness for
analytical and preparative TLC, respectively). Spots were visualized
using UV light or vanillin/sulfuric acid reagent. HPLC runs were carried
out using a System Gold model 127 pump equipped with a model 166
UV detector (Beckman) and 4.6 × 250 mm or 10 × 250 mm C18-A
Polaris columns (Varian) for analytical (flow rate ) 1 mL/min) or
semipreparative runs (flow rate 5 mL/min), respectively. The solvents
used were H2O, MeOH, and CH3CN, each with 0.1% HOAc, and are
designated as A, B, and C, respectively. Daidzein, 7,4′-dimethoxy-
isoflavone, 7,3′,4′-trihydroxyisoflavone, and apigenin were purchased
from Alfa Aesar and genistein from Indofine Chemical Company
(Somerville, NJ). Other compounds and reagents were obtained from
Sigma-Aldrich.
5,7,3′,4′-Tetrahydroxy-2′-(3,3-dimethylallyl)isoflavone (1a): greenish-
white residue; UV (MeOH) λmax (log ꢀ) 210 (4.53), 260 (4.42), 285 sh
(4.00) nm; IR νmax (KBr) 3446, 3223 (br OH), 3081, 2976, 2927, 1704,
1654, 1619, 1570, 1492, 1444, 1360, 1304, 1200, 1051 cm-1; 1H, 13
C
(acetone-d6, 300 and 75 MHz, respectively), and HMBC NMR data,
see Table 1; HRESIMS m/z 377.1000 [M + Na]+ (calcd for C20H18-
NaO6, 377.1001).
Acid-Catalyzed Cyclization of Compound 1a. Compound 1a (9.5
mg, 0.03 mmol) was stirred with 88% HCOOH (1 mL) at 80 °C
overnight. The product 1b (8.0 mg, 84% yield) was purified by HPLC
(50 to 100% solvent B in A in 50 min; tR ) 22 min; mp 247-249 °C;
1H, 13C (acetone-d6, 400 and 100 MHz, respectively), and HMBC NMR
data, see Table 1).
2-(2′-Hydroxy-4′,5′-methylenedioxyphenyl)-6-methoxybenzofuran-
3-carbaldehyde (2): white residue; UV (MeOH) λmax (log ꢀ) 247 (4.21),
295 (3.93), 346 (4.05) nm; IR νmax (KBr) 3281 (br OH), 2867, 1656,
1618, 1589,1506, 1461, 1314, 1202, 1141, 1034, 950, 893, 802 cm-1
;
1H, 13C (acetone-d6, 400 and 100 MHz, respectively), and HMBC NMR
data, see Table 2; HRESIMS m/z 335.0550 [M + Na]+ (calcd for
C17H12NaO6, 335.0532).
Fremontin (3a): greenish-white powder; UV (MeOH) λmax (log ꢀ)
259 (4.37), 292 (3.99) nm; IR νmax (film) 3366 (br OH), 1652, 1617,
1
1576, 1508, 1437, 1361, 1277, 1169 cm-1; H, 13C (acetone-d6, 400
and 100 MHz, respectively), and HMBC NMR data, see Table 3;
HRESIMS m/z 377.1027 [M + Na]+ (calcd for C20H18NaO6, 377.1001).
Acetylation of Fremontin. Fremontin (4 mg, 0.01 mmol) was
acetylated with acetic anhydride (0.3 mL) in pyridine (0.3 mL) by
stirring at room temperature overnight. Fremontin tetraacetate (3b, 3.5
mg, 59% yield) was purified by HPLC (gradient B in A, 70 to 100%
in 30 min; tR
Plant Material. P. arborescens var. minutifolius root was collected
on June 11, 2003, from E. Tulare Co., California, on the eastern slopes
of the southern Sierra Nevada in desert-chaparral transitional vegetation
by Dr. Richard Spjut (WBA 4841-13, SPJ-15357), World Botanical
Associates (Bakersfield, CA). Voucher specimens are deposited at the
Botanical Research Institute of Texas (BRIT), the United States National
Herbarium, the Smithsonian Institution (US), and the World Botanical
Associates (WBA).
) 7.5 min; mp 195-197 °C (lit.8 mp 195 °C); 1H NMR
(acetone-d6, 400 MHz) δ 8.00 (1H, s, H-2), 7.39 (1H, s, H-5′), 7.35
(1H, d, J ) 2.2 Hz, H-8), 6.97 (1H, d, J ) 2.2 Hz, H-6), 6.95 (1H, s,
H-2′), 5.99 (1H, dd, J ) 10.6, 17.5 Hz, H-2′′), 4.83 (1H, dd, J ) 1.1,
17.5 Hz, H-3′′a), 4.72 (1H, dd, J ) 1.1, 10.6 Hz, H-3′′b), 2.34, 2.29,
2.26, and 2.25 (3H each, s, COCH3), 1.38 and 1.33 (3H each, s, CH3-
4′′ and -5′′); 13C NMR (acetone-d6, 100 MHz) δ 175.9 (C, C-4), 169.3,
168.8, 168.7, and 168.5 (C each, COCH3), 158.6 (C, C-9), 155.2 (C,
C-7), 153.6 (CH, C-2), 151.6 (C, C-5), 148.7 (CH, C-2′′), 147.3 (C,
C-4′), 143.1 (C, C-3′), 141.0 (C, C-6′), 130.2 (C, C-1′), 128.9 (CH,
C-2′), 128.3 (C, C-3), 123.3 (CH, C-5′), 116.1 (C, C-10), 114.8 (CH,
C-6), 110.8 (CH2, C-3′′), 109.9 (CH, C-8), 42.8 (C, C-1′′), 29.9 and
29.5 (CH3 each, CH3-4′′ and -5′′), 21.0, 21.0, 20.53, and 20.45 (CH3
each, COCH3); HMBC correlations: H-2 to C-3, -4, -9, -1′, H-6 to
C-5, -7, -8, -10, H-8 to C-6, -7, -9, -10, H-2′ to C-3, -3′, -4′, -6′, H-5′
to C-1′, -3′, -6′, -1′′, H-2′′ to C-1′′, -4′′, -5′′, H-3′′ to C-1′′, -2′′, CH3-
4′′ to C-6′, -1′′, -2′′, -5′′, CH3-5′′ to C-6′, -1′′, -2′′, -4′′, COCH3 to
COCH3.
Extraction and Isolation. The dried and powdered root material
(0.6 kg) was percolated with 2.9 L of 95% EtOH. The resulting gummy
extract (38 g) was partitioned between H2O and CH2Cl2. The organic
layer was then partitioned between 90% MeOH (F001, 12.0 g) and
hexane (F002, 2.0 g). The aqueous fraction was further partitioned
between EtOAc (F003, 0.5 g) and H2O (F004, 10.4 g). The activity
was concentrated in F001 (IC50 ) 16.5 µg/mL in the axenic amastigote
assay), which was chromatographed over Sephadex LH-20 eluting with
MeOH. The fractions were combined according to their TLC profile
into seven pools (F006-F012). The bioactivity was found to be highest
in fractions F008, F009, and F010. F008 (0.86 g) was chromatographed
on a silica gel column eluting with a gradient of 20% EtOAc-hexane
to 100% EtOAc. The fraction eluted with 25% EtOAc-hexane was
purified using preparative HPLC with an isocratic solvent system of
90% B in A, λmax 210 nm, to collect 23 mg of compound 8 (tR 11
min). F009 (9.4 g) was subjected to silica gel chromatography using a
solvent gradient from 30% EtOAc-hexane to 100% EtOAc to 25%
MeOH-EtOAc to obtain nine fractions, F009-1 to F009-9. F009-4 (750
mg) was subjected to Sephadex LH-20 chromatography followed by
HPLC using a gradient from 50% to 60% B in A over 30 min then
isocratic 60% B in A to yield compound 2 (3.5 mg, tR 37 min),
Methylation of Fremontin. Fremontin (20 mg, 0.06 mmol) in
anhydrous acetone (9 mL) was refluxed for 2 h with excess K2CO3
(125 mg) and Me2SO4 (0.1 mL, 1.06 mmol) with molecular sieves (4
Å). PTLC (2% MeOH in CHCl3) of the products yielded trimethyl-
fremontin (3c, 10.5 mg, 47% yield), which was recrystallized from
EtOAc-hexane as grayish needles (Rf 0.47; mp 163-164 °C; 1H, 13
C

48 Journal of Natural Products, 2006, Vol. 69, No. 1
Salem and WerboVetz
(CDCl3, 400 and 100 MHz, respectively), and HMBC NMR data, see
Table 3) and tetramethylfremontin (3d, 11.0 mg, 47% yield, Rf 0.23,
yellow residue): 1H NMR (acetone-d6, 400 MHz) δ 7.62 (1H, s, H-2),
7.05 (1H, s, H-5′), 6.59 (1H, s, H-2′), 6.56 (1H, d, J ) 2.3 Hz, H-8),
6.48 (1H, d, J ) 2.3 Hz, H-6), 6.05 (1H, dd, J ) 10.6, 17.6 Hz, H-2′′),
4.82 (1H, dd, J ) 1.2, 17.6 Hz, H-3′′a), 4.68 (1H, dd, J ) 1.2, 10.6
Hz, H-3′′b), 3.93 (3H, s, 7-OCH3), 3.86 (3H, s, 5-OCH3), 3.84 (3H, s,
4′-OCH3), 3.75 (3H, s, 3′-OCH3), 1.38 (3H, s, CH3-4′′), 1.33 (3H, s,
CH3-5′′); 13C NMR (acetone-d6,100 MHz) δ 176.0 (C, C-4), 164.8 (C,
C-7), 162.3 (C, C-5), 160.8 (C, C-9), 151.6 (CH, C-2), 150.2 (CH,
C-2′′), 149.5 (C, C-4′), 147.9 (C, C-3′), 140.8 (C, C-6′), 129.7 (C, C-3),
125.1 (C, C-1′), 118.1 (CH, C-2′), 112.7 (C, C-5′), 110.5 (C, C-10),
109.5 (CH2, C-3′′), 96.7 (CH, C-6), 93.6 (CH, C-8), 56.47, 56.25, 56.21,
and 56.09 (CH3 each, OCH3-5, 7, 3′, and 4′), 42.8 (C, C-1′′), 29.7 (CH3,
CH3-4′′), 28.9 (CH3, CH3-5′′); HMBC correlations: H-2 to C-3, -4,
-9, -1′, H-6 to C-5, -7, -8, -10, H-8 to C-6, -7, -9, -10, H-2′ to C-3, -4′,
-6′, H-5′ to C-1′, -3′, -1′′, H-2′′ to C-1′′, -4′′, -5′′, H-3′′ to C-1′′, -2′′,
CH3-4′′ to C-6′, -1′′, -2′′, -5′′, CH3-5′′ to C-6′, -1′′, -2′′, -4′′, OCH3-3′
to C-3′, OCH3-4′ to C-4′, OCH3-5 to C-5, OCH3-7 to C-7.
Glycyrrhisoflavone (4a): brownish-white residue; UV (MeOH) λmax
(log ꢀ) 261 (4.48), 292 sh (4.13) nm; IR νmax (film) 3373 (br OH),
1651, 1622, 1575, 1504, 1442, 1367, 1307, 1284, 1178, 1051, 838
cm-1;1H NMR (acetone-d6, 400 MHz) δ 13.07 (1H, br s, 5-OH), 8.09
(1H, s, H-2), 7.02 (1H, d, J ) 1.2 Hz, H-2′), 6.84 (1H, d, J ) 1.2 Hz,
H-6′), 6.40 (1H, br s, H-8), 6.28 (1H, br s, H-6), 5.37 (1H, m, H-2′′),
3.37 (2H, d, J ) 7.3 Hz, H-1′′), 1.73 (3H, s, CH3-4′′), 1.70 (3H, s,
CH3-5′′); 13C NMR (acetone-d6,100 MHz) δ 181.6 (C, C-4), 165.1 (C,
C-7), 163.9 (C, C-5), 159.0 (C, C-9), 154.2 (CH, C-2), 144.9 (C, C-4′),
144.2 (C, C-3′), 132.3 (C, C-3′′), 128.8 (C, C-5′), 124.3 (C, C-3), 123.7
(CH, C-2′′), 122.7 (C, C-1′), 122.1 (CH, C-6′), 114.7 (CH, C-2′), 106.0
(C, C-10), 99.8 (CH, C-6), 94.4 (CH, C-8), 29.0 (CH2, C-1′′), 25.9
(CH3, CH3-4′′), 17.8 (CH3, CH3-5′′); HMBC correlations: H-2 to C-3,
-4, -9, -1′, H-6 to C-5, -7, -8, -10, H-8 to C-6, -7, -9, -10, H-2′ to C-3,
-3′, -4′, -6′, H-6′ to C-3, -2′, -4′, -1′′, H-1′′ to C-4′, -5′, -6′, -2′′, -3′′,
H-2′′ to C-5′, 1′′, -4′′, -5′′, CH3-4′′ to C-2′′, -3′′, -5′′, CH3-5′′ to C-2′′,
-3′′, -4′′; HRESIMS m/z 377.1012 [M + Na]+ (calcd for C20H18NaO6,
377.1001).
Acid-Catalyzed Cyclization of Glycyrrhisoflavone. Glycyrrhisofla-
vone (4.0 mg, 0.01 mmol) in 1 mL of HCOOH (88%) was stirred at
90 °C for 5 h. The reaction mixture was partitioned between H2O (2
mL) and EtOAc (3 × 2 mL). After drying the organic layer, the product
4b (2.5 mg, 63% yield) was purified by HPLC (50 to 100% B in A in
50 min; tR ) 25 min): 1H NMR (acetone-d6, 400 MHz) δ 13.07 (1H,
br s, 5-OH), 8.13 (1H, s, H-2), 6.93 (1H, d, J ) 2.1 Hz, H-2′), 6.84
(1H, d, J ) 2.1 Hz, H-6′), 6.41 (1H, d, J ) 1.9 Hz, H-8), 6.28 (1H, d,
J ) 1.9 Hz, H-6), 2.81 (2H, t, J ) 6.7 Hz, H-1′′), 1.86 (2H, t, J ) 6.7
Hz, H-2′′), 1.35 (6H, s, CH3-4′′ and -5′′); 13C NMR (acetone-d6,100
MHz) δ 181.5 (C, C-4), 165.8 (C, C-7), 163.6 (C, C-5), 159.1 (C, C-9),
154.2 (CH, C-2), 146.6 (C, C-3′), 142.7 (C, C-4′), 124.1a (C, C-3),
123.2a (C, C-1′), 122.4 (CH, C-6′), 121.9 (C, C-5′), 114.3 (CH, C-2′),
105.8 (C, C-10), 100.0 (CH, C-6), 94.6 (CH, C-8), 75.7 (C, C-3′′),
33.5 (CH2, C-2′′), 27.0 and 27.0 (CH3 each, CH3-4′′ and -5′′), 22.9
(CH2, C-1′′) (assignments with the same superscript may be inter-
changed); HMBC correlations, H-2 to C-3, -4, -9, -1′, H-6 to C-5, -7,
-8, -10, H-8 to C-6, -7, -9, -10, H-2′ to C-3, -3′, -4′, -6′, H-6′ to C-3,
-2′, -4′, -1′′, H-1′′ to C-4′, -5′, -6′, -2′′, -3′′, H-2′′ to C-5′, 1′′, -3′′, -4′′,
-5′′, CH3-4′′ and -5′′ to C-2′′, -3′′.
Methylation of Glycyrrhisoflavone. Glycyrrhisoflavone (4a, 9.0
mg, 0.03 mmol) was heated to reflux with excess K2CO3 (280 mg)
and excess Me2SO4 (0.5 mL, 5.3 mmol) in 9.0 mL of anhydrous acetone
under 4 Å molecular sieves for 5 h. The product 4c (6.2 mg, 59%
yield) was purified by HPLC (50 to 100% B in A in 50 min; tR 34
min): 1H NMR (CDCl3, 400 MHz) δ 7.77 (1H, s, H-2), 7.11 (1H, d,
J ) 1.9 Hz, H-2′), 6.81 (1H, d, J ) 1.9 Hz, H-6′), 6.45 (1H, d, J ) 2.2
Hz, H-8), 6.38 (1H, d, J ) 2.2 Hz, H-6), 5.28 (1H, m, H-2′′), 3.94
(3H, s, 5-OCH3), 3.89 (3H, s, 7-OCH3), 3.88 (3H, s, 3′-OCH3), 3.82
(3H, s, 4′-OCH3), 3.36 (2H, d, J ) 7.2 Hz, H-1′′), 1.72 (3H, s, CH3-
4′′), 1.71 (3H, s, CH3-5′′); 13C NMR (CDCl3,100 MHz) δ 175.3 (C,
C-4), 163.8 (C, C-7), 161.4 (C, C-5), 159.8 (C, C-9), 152.2 (C, C-3′),
150.4 (CH, C-2), 146.8 (C, C-4′), 135.2 (C, C-5′), 132.1 (C, C-3′′),
127.7 (C, C-3), 126.2 (C, C-1′), 123.0 (CH, C-2′′), 122.1 (CH, C-6′),
111.8 (CH, C-2′), 109.9 (C, C-10), 96.2 (CH, C-6), 92.5 (CH, C-8),
60.5, 56.4, 55.8, and 55.7 (CH3 each, 5-, 7-, 3′-, 4′-OCH3), 28.6 (CH2,
C-1′′), 25.8 (CH3, CH3-4′′), 17.8 (CH3, CH3-5′′); HMBC correlations,
H-2 to C-3, -4, -9, -1′, H-6 to C-5, -7, -8, -10, H-8 to C-6, -7, -9, -10,
H-2′ to C-3, -3′, -4′, -6′, H-6′ to C-3, -2′, -4′, -1′′, H-1′′ to C-4′, -5′,
-6′, -2′′, -3′′, H-2′′ to 1′′, -4′′, -5′′, CH3-4′′ to C-2′′, -3′′, -5′′, CH3-5′′
to C-2′′, -3′′, -4′′).
Calycosin (5): white residue; UV (MeOH) λmax (log ꢀ) 248 (4.44),
260 (sh 4.41), 290 (4.21) nm; IR νmax (film) 3205 (br OH), 1623, 1585,
1
1511, 1454, 1280, 1196, 1133, 1024, 852 cm-1; H, 13C, and HMBC
NMR data consistent with literature values;10-12 HRESIMS m/z
307.0578 [M + Na]+ (calcd for C16H12NaO5, 307.0582).
24
Maackiain (6): amorphous white powder; [R]D -33.5 (c 0.13,
CHCl3); UV (MeOH) λmax (log ꢀ) 213 (4.38), 286 (3.66), 310 (3.85)
nm; IR νmax (KBr) 3393 (br OH), 2927, 1624, 1474, 1456, 1347, 1320,
1186, 1144, 1122 cm-1; 1H, 13C, and HMBC NMR data consistent with
literature values;14-17 HRESIMS m/z 307.0603 [M + Na]+ (calcd for
C16H12NaO5, 307.0582).
3,4-Dihydroxy-8,9-methylenedioxypterocarpan (7): amorphous
24
white powder; [R]D +62.2 (c 0.188, CHCl3); UV (MeOH) λmax (log
ꢀ) 213 (4.66), 310 (3.85) nm; IR νmax (film) 3423 (br OH), 1625, 1476,
1
1459, 1338, 1206, 1144, 1052, 936 cm-1; H, 13C, and HMBC NMR
data consistent with literature values;17,18 HRESIMS m/z 323.0521 [M
+ Na]+ (calcd for C16H12NaO6, 323.0532).
Oleanolic acid (8): amorphous white powder; [R]D +72; UV
(MeOH) λmax < 220 nm; IR νmax (KBr) 3421 (br OH), 2944, 2874,
1697 (sh), 1458, 1387, 1210, 1167, 1029 1051 cm-1 1H, 13C, and
;
HMBC NMR data consistent with literature values;19,39 HRESIMS m/z
479.3501 [M + Na]+ (calcd for C30H48NaO3, 479.3501).
Isoliquiritigenin (9): yellow powder; UV (MeOH) λmax (log ꢀ) 242
sh (3.86), 298 sh (3.78), 371 (4.30) nm; IR νmax (KBr) 3357 (br OH),
1630, 1605, 1586, 1547, 1514, 1503, 1220, 1166, 1142;1H, 13C, and
HMBC NMR data consistent with literature values;20,21 HRESIMS m/z
279.0647 [M + Na]+ (calcd for C15H12NaO4, 279.0633).
Antileishmanial Assay Using Axenic Amastigotes. The antileish-
manial activity of the isolated compounds was tested in vitro against
L. donoVani amastigote-like parasites (WHO designation: MHOM/
SD/62/1S-CL2D) in a three-day assay using the tetrazolium dye-based
CellTiter reagent (Promega) as described previously.40-42
Antitrypanosomal Assay. Compounds were tested for their activity
against bloodstream-form T. b. brucei (MITat 1.2, variant 221)
axenically cultured in HMI-9 medium as described by Bodley et al.43
with minor modifications. Briefly, 100 µL of late log phase parasites
were incubated in 96-well plates (Costar) at an initial concentration of
105 cells/mL with or without test compounds at 37 °C in a humidified
5% CO2 atmosphere for 72 h. Ten microliters of a 20 mg/mL solution
of p-nitrophenyl phosphate (prepared in 1 M NaOAc, pH 5.5, 1%
TritonX-100) was then added to each well, and plates were reincubated
at 37 °C as before for 6-8 h. Optical densities were then measured at
405 nm using a SpectraMax Plus microplate reader (Molecular Devices).
IC50 values, the concentration of the compound that inhibited cell growth
by 50% compared to untreated control, were determined with the aid
of the software program SoftMax Pro (Molecular Devices). This
program uses the dose-response equation y ) ((a - d)/(1 + (x/c)b))
+ d, where x ) the drug concentration, y ) absorbance at 405 nm, a
) upper asymptote, b ) slope, c ) IC50, and d ) lower asymptote.
This assay has the advantage of not being subject to interference by
samples with a reducing potential (such as the o-catechols isolated and
assayed in this study), as it depends on measuring acid phosphatase
activity in surviving parasites.
Cytotoxicity Assay. Cytotoxicity was evaluated against two cell
lines, Vero cells and PC-3 prostate cells, obtained from the American
Type Culture Collection (ATCC, Rockville, MD) as described previ-
ously.2,44
Acknowledgment. M.S. was supported by a Jane Chen fellowship
and a Presidential fellowship at The Ohio State University. Additional
support was provided by The Ohio State University, College of
Pharmacy. We would like to thank Dr. J. Cassady for giving us the
opportunity to sample his library of plant extracts for screening. We
would like also to thank Dr. R. Spjut for the invaluable comments and
suggestions regarding the taxonomy of P. arborescens and Dr. C.
Cottrell for useful discussions and help with the NMR techniques.
Supporting Information Available: The 1H and 13C NMR spectra
of compounds 1a and 2 and the NMR data of compounds 5-9. This
material is available free of charge via the Internet at http://
pubs.acs.org.

IsoflaVonoids from Psorothamnus arborescens
Journal of Natural Products, 2006, Vol. 69, No. 1 49
(23) Tahara, S.; Moriyama, M.; Ingham, J. L.; Mizutani, J. Phytochemistry
1992, 31, 679-682.
(24) Tanaka, T.; Ohyama, M.; Iinuma, M.; Shirataki, Y.; Komatsu, M.;
Burandt, C. L. Phytochemistry 1998, 48, 1187-1193.
(25) Stevenson, P.; Veitch, N. Phytochemistry 1998, 48, 947-951.
(26) Markham, K. R. Techniques of FlaVonoid Identification; Academic
Press: New York, 1982; pp 72-93.
References and Notes
(1) Salem, M. M.; Werbovetz, K. A. J. Nat. Prod. 2005, 68, 108-111.
(2) Barneby, R. C. Daleae Imagines: An Illustrated ReVision of Erra-
zuriza Philippi, Psorothamnus Rydberg, Marina Leibmann, and Dalea
Lucanus emend. Barneby, Including All Species of Leguminosae Tribe
Amorpheae BorissoVa EVer Referred to Dalea; New York Botanical
Garden: Bronx, NY, 1977; pp 31-38.
(3) Jepson, W. L.; Hickman, J. C. The Jepson Manual: Higher Plants
of California; University of California Press: Berkeley, CA, 1993;
pp 642-643.
(4) Munz, P. A.; Keck, D. D. A California Flora; University of California
Press: Berkeley, CA, 1973; pp 852-854.
(5) Cronquist, A. Intermountain Flora; Vascular Plants of the Inter-
mountain West, U.S.A; Published for the New York Botanical Garden
by Hafner Pub. Co.: New York, 1972; pp 29-32.
(6) DeDecker, M. Flora of the Northern MojaVe Desert, California;
California Native Plant Society: Berkeley, CA, 1984; p 61.
(7) Train, P.; Archer, W. A.; Henrichs, J. R. Medicinal Uses of Plants
by Indian Tribes of NeVada; Quarterman Publications: Lawrence,
MA, 1982; pp 42-44.
(8) Manikumar, G.; Gaetano, K.; Wani, M. C.; Taylor, H.; Hughes, T.
J.; Warner, J.; McGivney, R.; Wall, M. E. J. Nat. Prod. 1989, 52,
769-773.
(9) Hatano, T.; Kagawa, H.; Yasuhara, T.; Okuda, T. Chem. Pharm. Bull.
1988, 36, 2090-2097.
(27) Tahara, S.; Moriyama, M.; Ingham, J. L.; Mizutani, J. Phytochemistry
1991, 30, 2769-2775.
(28) Tahara, S. Kagaku to Seibutsu 1991, 29, 493-502.
(29) Harborne, J. B. The FlaVonoids: AdVances in Research Since 1986,
1st ed.; Chapman & Hall: London, 1994; pp 166-180.
(30) Kraft, C.; Jenett-Siems, K.; Siems, K.; Solis, P. N.; Gupta, M. P.;
Bienzle, U.; Eich, E. Phytochemistry 2001, 58, 769-774.
(31) Kraft, C.; Jenett-Siems, K.; Siems, K.; Gupta, M. P.; Bienzle, U.;
Eich, E. J. Ethnopharmacol. 2000, 73, 131-135.
(32) Araujo, C. A. C.; Alegrio, L. V.; Leon, L. L. Phytochemistry 1998,
49, 751-754.
(33) ElSohly, H. N.; Joshi, A. S.; Nimrod, A. C. Planta Med. 1999, 65,
490.
(34) Fan, Y.; Wu, D.-Z.; Gong, Y.-Q.; Zhou, J.-Y.; Hu, Z. B. Eur. J.
Pharmacol. 2003, 481, 33-40.
(35) Wheeler-Alm, E.; Shapiro, S. Z. J. Protozool. 1992, 39, 413-416.
(36) Gale, M., Jr.; Carter, V.; Parsons, M. J. Cell Sci. 1994, 107, 1825-
1832.
(37) Akiyama, T.; Ishida, J.; Nakagawa, S.; Ogawara, H.; Watanabe, S.;
Itoh, N.; Shibuya, M.; Fukami, Y. J. Biol. Chem. 1987, 262, 5592-
5595.
(38) Braun, S.; Kalinowski, H.-O.; Berger, S. 150 and More Basic NMR
Experiments: A Practical Course, 2nd expanded ed.; VCH: Wein-
heim, 1998; pp 460-463.
(39) Zhang, Z.; Koike, K.; Jia, Z.; Nikaido, T.; Guo, D.; Zheng, J. J. Nat.
Prod. 1999, 62, 740-745.
(40) Havens, C. G.; Bryant, N.; Asher, L.; Lamoreaux, L.; Perfetto, S.;
Brendle, J. J.; Werbovetz, K. A. Mol. Biochem. Parasitol. 2000, 110,
223-236.
(41) Werbovetz, K. A.; Brendle, J. J.; Sackett, D. L. Mol. Biochem.
Parasitol. 1999, 98, 53-65.
(42) Werbovetz, K. A.; Sackett, D. L.; Delfin, D.; Bhattacharya, G.; Salem,
M.; Obrzut, T.; Rattendi, D.; Bacchi, C. Mol. Pharmacol. 2003, 64,
1325-1333.
(43) Bodley, A. L.; McGarry, M. W.; Shapiro, T. A. J. Infect. Dis. 1995,
172, 1157-1159.
(10) Kamnaing, P.; Fanso Free, S. N. Y.; Nkengfack, A. E.; Folefoc, G.;
Fomum, Z. T. Phytochemistry 1999, 51, 829-832.
(11) Markham, K. R.; Mabry, T. J.; Swift, T. W., III. Phytochemistry
1968, 7, 803-808.
(12) Parthasarathy, M. R.; Puri, R. N.; Seshadri, T. R. Indian J. Chem.
1969, 7, 118-120.
(13) Kobayashi, M.; Noguchi, H.; Sankawa, U. Chem. Pharm. Bull. 1985,
33, 3811-3816.
(14) Suginome, H. Experientia 1962, 18, 161-163.
(15) Shibata, S.; Nishikawa, Y. Chem. Pharm. Bull. 1963, 11, 167-177.
(16) To˜ke´s, A. L.; Litkei, G.; Gula´csi, K.; Antus, S.; Baitz-Ga´cs, E.;
Sza´ntay, C.; Darko´, L. L. Tetrahedron 1999, 55, 9283-9296.
(17) Chaudhuri, S. K.; Huang, L.; Fullas, F.; Brown, D. M.; Wani, M.
C.; Wall, M. E.; Tucker, J. C.; Beecher, C. W. W.; Kinghorn, A. D.
J. Nat. Prod. 1995, 58, 1966-1969.
(18) Cook, J. T.; Ollis, W. D.; Sutherland, I. O.; Gottlieb, O. R.
Phytochemistry 1978, 17, 1419-1422.
(19) Seebacher, W.; Simic, N.; Weis, R.; Saf, R.; Kunert, O. Magn. Reson.
Chem. 2003, 41, 636-638.
(20) Markham, K. R.; Ternai, B. Tetrahedron 1976, 32, 2607-2612.
(21) Saitoh, T.; Noguchi, H.; Shibata, S. Chem. Pharm. Bull. 1978, 26,
144-147.
(44) Cottrell, D. M.; Capers, J.; Salem, M. M.; DeLuca-Fradley, K.; Croft,
S. L.; Werbovetz, K. A. Bioorg. Med. Chem. 2004, 12, 2815-2824.
(22) Tahara, S.; Ingham, J. L.; Hanawa, F.; Mizutani, J. Phytochemistry
1991, 30, 1683-1689.
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