Biflavonoids as novel antituberculosis agents

Article abstract of DOI:10.1016/S0960-894X(01)00382-1

A series of naturally occurring and synthetic biflavonoids was evaluated for inhibitory activity against Mycobacterium tuberculosis H37Rv (Mtb). Compounds 6, 24, and 25 demonstrated 96, 95, and 87% inhibition, respectively, at a screening concentration of 12.5 μg/mL. The type of linkage and the presence of methoxy- and nitro-substituents in biflavonoids may contribute to the observed inhibitory activity. The results of this study represent the discovery of biflavonoids as a potential new class of antituberculosis agent.

Full text of DOI:10.1016/S0960-894X(01)00382-1

Bioorganic & Medicinal Chemistry Letters 11 (2001) 2101–2104
Biflavonoids as Novel Antituberculosis Agents
Yuh-Meei Lin,a,* Michael T. Flavin,a Constance S. Cassidy,a Aye Mara
and Fa-Ching Chenb
aMediChem Life Sciences, Woodridge, IL 60517, USA
bDepartment of Chemistry, National Taiwan University, Taipei, Taiwan
Received 6 March 2001; accepted 16 May 2001
Abstract—A series of naturally occurring and synthetic biflavonoids was evaluated for inhibitory activity against Mycobacterium
tuberculosis H37Rv (Mtb). Compounds 6, 24, and 25 demonstrated 96, 95, and 87% inhibition, respectively, at a screening con-
centration of 12.5 mg/mL. The type of linkage and the presence of methoxy- and nitro-substituents in biflavonoids may contribute
to the observed inhibitory activity. The results of this study represent the discovery of biflavonoids as a potential new class of
antituberculosis agent. # 2001 Elsevier Science Ltd. All rights reserved.
Tuberculosis (TB), an infection of Mycobacterium
tuberculosis (Mtb), is the leading cause of worldwide
death among infectious diseases.1,2 In turn, infectious
disease remains the leading cause of death in the world
today, greater than cardiovascular disease or cancer.3
Currently, one-third of the world’s population is infec-
ted with Mtb, a facultative intracellular bacillus.4 The
World Health Organization (WHO) estimates that
within the next 20 years, an estimated 1 billion people
will be newly infected, 200 million will become sick and
35 million will die.5 The recent resurgence of TB is
associated with the emergence of the HIV/AIDS epi-
demic6 and the rapid spread of multidrug resistant TB
strains. TB infection is a serious problem for hemodia-
lysis patients,7 and is also the leading cause of death in
women of childbearing age around the world.8
Biflavonoids are a series of naturally occurring com-
pounds that include flavone–flavone, flavanone–flavone
and flavanone–flavanone subunit linkages (Fig. 1).
More than 100 biflavonoids have been identified from
plants since the isolation of gingetin in 1929.11,12
A
variety of biological activities for biflavonoids have
been published, including antiinflammatory, anti-
microbial, antioxidant, and others.12,13 We have
recently reported the anti-HIV-1, anti-HBV, and anti-
viral activities of biflavonoids isolated from Rhus succe-
danea L. (Anacardiaceae) and Garcinia multiflora
Champ. (Guttiferae).12,14ꢀ16 In continuing our drug
discovery program, we present the screening results for a
series of naturally occurring and synthetic biflavonoids
against Mtb.
Symmetric biflavones with a carbon–carbon linkage
were produced by Ullmann condensation of mono-
halogenoflavonoids.17,18 Unsymmetrical biflavones (1–
5) were synthesized by Ullmann condensation of the
corresponding iodoflavones.19 Compound 6 (6–600 bia-
pigenin hexamethylether) was generated in four steps
from benzyl 4-iodo-3,5-dimethoxyphenylether (I)
(Scheme 1). 4,40-Dibenzyloxy-2,20,6,60-tetramethoxy-
biphenyl (II) was prepared from the Ullmann reaction
of compound I.20 The Hoesch reaction was performed
on compound II with CH3CN, ZnCl2 and HCl in dry
CHCl3/Et2O (1:1) to yield the key product, 4,40-dihy-
droxy-3,30-diacetyl-2,20,6,60-tetramethoxybihenyl (III).
Treatment with two mols of p-anisaldehyde in the pre-
sence of base afforded bichalcone IV. Oxidative cycliza-
tion of the bichalcone with SeO2 in dioxane followed by
Current treatment of TB requires a patient to take at
least two antibiotics in order to decrease the risk of drug
resistance. The leading combination therapy requires
isoniazid and rifampicin for an extended period of time.
This may be supplemented with pyrazinamide and
ethambutol when isoniazid resistance is suspected.
Recently, rifapentine,9 a derivative of rifamycin, was
approved by the FDA for the treatment of tubercu-
losis.10 The rapid spread of TB worldwide has intensi-
fied the need for more efficient drugs to combat this
disease.
*Corresponding author. Tel.: +1-630-783-4600; fax: +1-630-783-
4646; e-mail: ylin@medichem.com
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00382-1

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Y.-M. Lin et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2101–2104
Figure 1. Structures of flavonoids and biflavonoids.
(pigment B, 13), with intermediate solubility in ethanol,
was precipitated and removed by filtration. The filtrate
separated from pigment B was evaporated to yield pig-
ment C, a brown solid.21 Pigment A was purified by
silica gel column chromatography with toluene/pyri-
dine/formic acid (36:10:2) as the eluting solvent to yield
hinokiflavone (11) and robustaflavone (12), successively.
Alternatively, robustaflavone hexaacetate was obtained
by the reaction of pigment A with acetic anhydride/
pyridine and recrystallization from a mixture of meth-
ylene chloride/ethyl acetate (ꢁ9:1). Hydrolysis of this
compound with 2M NaOH produced robustaflavone
(12). Repeated recrystallization of pigment B from
MeOH afforded amentoflavone (13). Pigment C was
purified by silica gel (toluene/ethyl acetate, 4:1–1:9)
and polyamide (water/methanol 9:1–1:9) column
chromatography, providing agathisflavone (14), rhus-
flavanone (15), and succedaneaflavanone (16).21
Scheme 1. (i) Ullmann reaction; (ii) CH3CN, ZnCl2 and HCl in
CHCl3/Et2O (1:1); (iii) p-anisaldehyde/alkali; (iv) SeO2/dioxane; (v)
H3PO4/EtOH.
Biflavonoids 17–23 were isolated from the heartwood of
G. multiflora.22 Briefly, the dried heartwood shavings
were extracted with hot methanol. The methanol extract
was treated with toluene and ethyl acetate, successively.
The ethyl acetate-soluble fraction was purified by both
silica gel chromatography (toluene/ethyl acetate, 7:3–
1:9) and polyamide chromatography (methanol/water,
1:9–9:1).
purification by preparative TLC yielded 6,600-biapigenin
hexamethylether (6). Cyclization of bichalcone (IV) by
refluxing with alcoholic H3PO4 under acidic conditions
for 3 weeks afforded 6,600-Binarigenin hexamethylether
(10).
Naturally occurring biflavonoids were isolated from
seed kernels of R. succedanea and G. multiflora.21,22
Briefly, seed kernels were defatted with hexane followed
by extraction with 95% ethanol and concentration of
the extract solution. Hinokiflavone (11) and Robusta-
flavone (12) were precipitated as pigment A during the
early stages of concentration due to their low solubilities.
The solution was filtered to remove pigment A and the
filtrate was further concentrated. Amentoflavone
Volkensiflavone hexamethylether (24) and GB-1a hexa-
methylether (25) were prepared by methylation of volk-
ensiflavone (17) and GB-1a (19) with dimethylsulfate in
dry acetone and potassium carbonate.22 Rhusflavanone
hexaacetate (26), succedaneaflavanone hexaacetate
(27), morelloflavone heptaacetate (28), and spicataside
acetate (29) were prepared by acetylation of com-

Y.-M. Lin et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2101–2104
2103
pounds 15, 16, 18, and 21, with acetic anhydride and
pyridine.22,23
linkage, were completely devoid of activity. These com-
pounds contain polar substituents such as hydroxy- (17
and 18), acetoxy- (28 and 29) or glucosyl- (20). Methyl-
ation of 17 to provide hexamethylether 24 led to a sig-
nificant increase in inhibitory activity of 95% against
Mtb.
The compounds were screened for TB inhibition against
Mtb H38Rv in BACTEC 12B medium using the BAC-
TEC 460 radiometric system.24 Biflavones composed of
two flavone units0w00 ith linkages of 8–2000 (1), 8–3000 (2 and
13), 7–3000 (3), 6–2 (4 and 5), 6–60 (6), 3–30 (7), 6–80 (14),
6–3000 (12), 3–O–4000 (8), and 6–O–4000 (11) (Fig. 1) were
screened against Mtb and the results are summarized in
Table 1. Among these biflavones, biflavone 6, which is
constructed with two molecules of apigenin trimethyl
ether in a 6–600 linkage, exhibited highest inhibitory
activity (96%) at a concentration of 12.5 mg/mL. The
MIC value was estimated to be greater than 12.5 mg/
mL. It is notable that biflavones 5 and 7, which consist
of the same flavone units as 6, but with different lin-
kages (6–2000 and 3–300), were completely devoid of
activity. This result suggests that the 6–600 linkage is
required for the inhibitory activity of biflavone. Bifla-
vone 8, which possesses a nitro-group and a linkage of
3–O–4000, exhibited moderate activity with 61% inhibi-
tion against Mtb. In the absence of methoxy- or nitro-
substituents, biflavones 1–4 were inactive (1 and 3) or
weakly (4) to moderately active (2). In addition, com-
pounds with hydroxy substituents (11–14) were completely
inactive against Mtb. These results indicated that meth-
oxy- and nitro-groups and a linkage of C6–C60 or C3–O–
C4000 might contribute to the biflavone anti-TB activity.
All biflavanones with
a
3–800 linkage possessing
hydroxy- (19 and 20), acetoxy- (26 and 27) or glucosyl-
(22 and 23) groups were inactive against Mtb. However,
compound 25, the hexamethylether of GB-1a (19),
demonstrated an inhibitory activity of 87% against Mtb
growth (Table 3). These results indicate that methoxy
groups may play a role in the increased activity of a
flavanone–flavone or a biflavanone with a 3–800 linkage.
Other biflavanones such as 6–800 (15) and 6–600 (16)
binaringenins and their hexaacetates 26 and 27 were
inactive. Compounds 9 and 10 with 3–300 and 6–600 lin-
kages, respectively, were inactive, despite the presence
of methoxy substituents. Both methoxy groups and a 3–
800 linkage appear to be important for the anti-TB
activity of biflavanones.
It has been reported that a C-8 methoxy group
increased the bacteriostatic activities of fluoro-
quinolones and quinolones against moderately resistant,
clinical isolates of Mtb,25,26 and that introducing nitro-
or methoxy-substituents to 3-hydrazono-1H-2-indoli-
nones increased their anti-TB activities.27 These results
coincide with our observations that methoxyl- and
nitro-groups are requirements for the anti-TB activity of
biflavonoids. Factors besides the lipophilic character of
a compound may therefore affect inhibitory activity
against Mtb.
The inhibitory activities of biflavonoids that are com-
posed of flavanone–flavone subunits are listed in
Table 2. Biflavonoids 17, 18, 21, 28, and 29, with a 3–800
Table 1. Antituberculosis activities of biflavones
The above results represent the discovery of biflavo-
noids as a new class of anti-TB agents. A 6–600 linkage
for biflavone hexamethylether, a 3–800 linkage for flava-
none–flavone compound and biflavanone hexamethyl-
ether, and increased lipophilic properties are required
for inhibitory activity. The mechanistic and pharmaco-
logical properties and the determination of structure–
activity relationships of biflavonoids are critical for the
development of new anti-TB drugs.
Compound
number
Compound name
% Inhibition at
12.5 mg/mL
1
2
3
4
8-2000-Biflavone
8-3000-Biflavone
0
49
0
9
1
96
0
61
0
0
0
7-3000-Biflavone
6-2000-Biflavone
5
6
7
8
11
12
13
14
6-2000-Biapigenin hexamethylether
6-600-Biapigenin hexamethylethera
3-300-Biapigenin hexamethylether
30-Nitro-3-O-40-biflavone
Hinokiflavone (6-O-4000-biapigenin)
Robustaflavone (6-3000-biapigenin)
Amentoflavone (8-3000-biapigenin)
Agathisflavone (6-800-biapigenin)
Table 3. Antituberculosis activities of biflavanones
0
Compound
number
Compound name
% Inhibition at
12.5 mg/mL
aMIC>12.5 mg/mL.
9
3-300-Bi-(7-methoxyflavanone)
6-600-Binaringenin hexamethylether
Rhusflavanone (6-800 binarigenin)
Succedaneaflavanone (6-600-binaringenin)
GB-1a (3-800 binaringenin)
0
2
0
10
15
16
19
20
22
23
25
Table 2. Antituberculosis activities of flavanone–flavones
0
NDa
0
0
0
Compound
number
Compound name
% Inhibition at
12.5 mg/mL
GB-2a (3-800 naringenyleriodictyol)
GB-1a glucoside
GB-2a glucoside
17
18
21
24
28
29
Volkensiflavone (3-800-naringenylapigenin)
Morelloflavone (3-800-naringenylluteolin)
Spicataside (volkensiflavone-7-glucoside)
Volkensiflavone hexamethylethera
Morelloflavone heptaacetate
0
0
0
95
0
0
GB-1a hexamethyletherb
(3-800-Binarigenin hexamethylether)
Rhusflavanone hexaacetate
Succedaneaflavanone hexaacetate
87
0
0
26
27
Spicataside acetate
aND, not determined.
bMIC>12.5 mg/mL.
aMIC=12.5 mg/mL.

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Y.-M. Lin et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2101–2104
11. Geiger, H.; Quinn, C. In The Flavonoids Advances in
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The authors thank Advanced Life Sciences (Woodridge,
IL, USA) for funding. We thank Dr. Robert C. Rey-
nolds, Southern Research Institute, Tuberculosis Anti-
microbial Acquisition and Coordinating Facility
(TAACF), National Institute of Allergy and Infectious
Diseases, for administrative arrangement of the activity
tests. Antimycobacterial data were provided by the
Tuberculosis Antimicrobial Acquisition and Coordinat-
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ment contract with the US National Institute of Allergy
and Infectious Diseases.
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