Anti-inflammatory activities of selected synthetic homoisoflavanones

Article abstract of DOI:10.1080/14786419.2011.565004

Four homoisoflavanones of the 3-benzylidene-4-chromanone type, some of which were previously isolated from Caesalpinia pulcherrima, were synthesised to determine their anti-inflammatory activity and cytotoxicity. A range of four different homoisoflavanones (compounds 4a-4d) were synthesised from the corresponding substituted phenols.¹H-and ¹³C-NMR data together with high-resolution mass spectroscopy data were employed to elucidate the structures. Anti-inflammatory activity was determined in mice with acute croton oil-induced auricular dermatitis. Invitro cytotoxicity was tested against a Chinese hamster ovarian cell line using the 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazoliumbromide (MTT) assay. Compound 4a exhibited a tendency to inhibit oedema in a dose-dependent manner after 3 and 6 h of treatment. Compounds 4b-4d also inhibited oedema, although a clear dose-response relationship was not observed. Compounds 4a-4c were found to be less cytotoxic than compound 4d. Compound 4b was the least cytotoxic. Compounds 4a-4d exhibited anti-inflammatory activity and varying levels of cytotoxicity.

Full text of DOI:10.1080/14786419.2011.565004

This article was downloaded by: [New York University]
On: 13 February 2015, At: 05:49
Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered
office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Natural Product Research: Formerly
Natural Product Letters
Publication details, including instructions for authors and
subscription information:
http://www.tandfonline.com/loi/gnpl20
Anti-inflammatory activities of selected
synthetic homoisoflavanones
Mahidansha M. Shaikh a b , Hendrik G. Kruger a , Johannes
Bodenstein b , Peter Smith c & Karen du Toit b
a School of Chemistry , University of KwaZulu-Natal , Durban
4000 , South Africa
b School of Pharmacy and Pharmacology , University of KwaZulu-
Natal , Durban 4000 , South Africa
c Division of Pharmacology , University of Cape Town ,
Rondebosch , South Africa
Published online: 27 Sep 2011.
To cite this article: Mahidansha M. Shaikh , Hendrik G. Kruger , Johannes Bodenstein , Peter Smith
& Karen du Toit (2012) Anti-inflammatory activities of selected synthetic homoisoflavanones,
Natural Product Research: Formerly Natural Product Letters, 26:16, 1473-1482, DOI:
10.1080/14786419.2011.565004
To link to this article: http://dx.doi.org/10.1080/14786419.2011.565004
PLEASE SCROLL DOWN FOR ARTICLE
Taylor & Francis makes every effort to ensure the accuracy of all the information (the
“Content”) contained in the publications on our platform. However, Taylor & Francis,
our agents, and our licensors make no representations or warranties whatsoever as to
the accuracy, completeness, or suitability for any purpose of the Content. Any opinions
and views expressed in this publication are the opinions and views of the authors,
and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content
should not be relied upon and should be independently verified with primary sources
of information. Taylor and Francis shall not be liable for any losses, actions, claims,
proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or
howsoever caused arising directly or indirectly in connection with, in relation to or arising
out of the use of the Content.
This article may be used for research, teaching, and private study purposes. Any
substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing,
systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &

Conditions of access and use can be found at http://www.tandfonline.com/page/terms-
and-conditions

Natural Product Research
Vol. 26, No. 16, August 2012, 1473–1482
Anti-inflammatory activities of selected synthetic homoisoflavanones
Mahidansha M. Shaikhab, Hendrik G. Krugera, Johannes Bodensteinb,
Peter Smithc and Karen du Toitb
*
aSchool of Chemistry, University of KwaZulu-Natal, Durban 4000, South Africa;
bSchool of Pharmacy and Pharmacology, University of KwaZulu-Natal, Durban 4000,
South Africa; Division of Pharmacology, University of Cape Town, Rondebosch,
c
South Africa
(Received 29 July 2010; final version received 17 February 2011)
Four homoisoflavanones of the 3-benzylidene-4-chromanone type, some of
which were previously isolated from Caesalpinia pulcherrima, were synthe-
sised to determine their anti-inflammatory activity and cytotoxicity.
A range of four different homoisoflavanones (compounds 4a–4d) were
synthesised from the corresponding substituted phenols.1H- and 13C-NMR
data together with high-resolution mass spectroscopy data were employed
to elucidate the structures. Anti-inflammatory activity was determined in
mice with acute croton oil-induced auricular dermatitis. In vitro cytotox-
icity was tested against a Chinese hamster ovarian cell line using the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay.
Compound 4a exhibited a tendency to inhibit oedema in a dose-dependent
manner after 3 and 6 h of treatment. Compounds 4b–4d also inhibited
oedema, although a clear dose-response relationship was not observed.
Compounds 4a–4c were found to be less cytotoxic than compound 4d.
Compound 4b was the least cytotoxic. Compounds 4a–4d exhibited
anti-inflammatory activity and varying levels of cytotoxicity.
Keywords: homoisoflavanones; Caesalpinia pulcherrima; synthesis;
anti-inflammatory; cytotoxicity
1. Introduction
Inflammation represents a series of homeostatic events that form part of a
mechanism for human survival against pathogens and tissue injury. In general,
non-steroidal anti-inflammatory drugs inhibit cyclooxygenase enzymes and are
useful for the treatment of acute inflammation. However, their ability to inhibit the
progression of inflammation in chronic cases is controversial. These compounds also
possess several unwanted effects including intestinal ulceration, inhibition of platelet
function and alterations in renal functions (Jackson Roberts II & Morrow, 2001).
Therefore, needs for effective and safe anti-inflammatory drugs exist and new lead
compounds from natural sources are investigated. Plants are an established natural
source of lead compounds (Rao, Fang, & Tzeng, 2005).
*Corresponding author. Email: karent@ukzn.ac.za
ISSN 1478–6419 print/ISSN 1478–6427 online
ß 2012 Taylor & Francis
http://dx.doi.org/10.1080/14786419.2011.565004
http://www.tandfonline.com

1474
M.M. Shaikh et al.
Figure 1. The anti-inflammatory activity of synthetic homoisoflavanones.
Notes: Compounds 4a–4d (represented by A–D) were tested in a mouse model of acute croton
oil-induced auricular contact dermatitis. Oedema was measured after treatment with 0.05, 0.1
and 0.5 mg homoisoflavanone in acetone for 3 and 6 h. Treatment with 0.05 mg diclofenac
(‘Diclo’) was included as a positive control. Data are the mean ꢀ standard error of the mean of
duplicate measurements from four independent experiments. Statistical comparisons of
activity were by one-way ANOVA followed by Bonferroni’s post-test for multiple compar-
y
isons; *p50.05; ***p50.001 vs. croton oil only control; p50.05 vs. 6 h treatment.
Many homoisoflavanones have been isolated from different plants which are
used for medicinal purposes across the world (Du Toit, Drewes, & Bodenstein, 2010;
Pohl, Crouch, & Mulholland, 2000). These homoisoflavanone-containing plants are
mainly used for conditions that can be ascribed to pain, inflammation, microbial
infections, digestive disorders and endocrine dysfunctions (Du Toit et al., 2010).
Phytochemical investigations of Caesalpinia pulcherrima (L.) Sw. (Fabaceae), an
Indian medicinal plant, have resulted in the isolation of homoisoflavanones of
the 3-benzylidene-4-chromanone type with anti-inflammatory activity (Figure 1)
(Maheswara, Siddaiah, & Rao, 2006). This plant has been reported to be useful
in treating tridosha, fever, ulcers, asthma, tumours, vata and skin diseases and it is
also used as an abortifacient and emmenagogue (Maheswara et al., 2006; Watt &
Breyer-Brandwijk, 1962).

Natural Product Research
1475
Anti-inflammatory activities of the (E )- and (Z)-isomers of 7-hydroxy-
3-(40-methoxybenzylidene)chroman-4-one (bonducellin), previously isolated from
C. pulcherrima, were reported by Rao et al. (2005). Both isomers inhibited LPS/
IFN-ꢀ-induced NO production in mouse peritoneal macrophages with IC50
(concentration at 50% inhibition) values of 20 and 30 mM for the (Z)- and
(E )-isomer, respectively (Rao et al., 2005). Synthetic bonducellin and
(E )-7-methoxy-3-(40-methoxybenzylidene) chroman-4-one both previously isolated
from C. pulcherrima were also found to inhibit 5-lipoxygenase enzymes. Inhibitors of
these enzymes could be useful in treating asthma and other inflammatory disorders
(Siddaiah, Rao, Venkateswarlu, Krishnarajub, & Subbarajub, 2006).
In view of the anti-inflammatory properties displayed by the benzylidene-
4-chromanone compounds isolated from C. pulcherrima, this study aims to
synthesise two compounds previously isolated from the plant, including
(E )-7-methoxy-3-(40-methoxybenzylidene) chroman-4-one (Siddaiah et al., 2006) as
well as two structurally similar compounds of which one is novel. Furthermore, for
each compound, the anti-inflammatory activity in an acute croton oil-induced
auricular dermatitis mouse model as well as the cytotoxicity in a mammalian cell line
were determined.
2. Results and discussion
2.1. Chemistry
A range of four different homoisoflavanones were synthesised from the correspond-
ing substituted phenols. The synthesis of homoisoflavanones and its derivatives from
commercially available reagents was carried out using the general synthetic approach
shown in Scheme 1. The reaction of the substituted phenols (1a–1c) with
3-bromopropanoic acid using sodium hydride as base furnished substituted
3-phenyloxypropanoic acids (2a–2c) (Mahapatra, Jana, & Nanda, 2008). The
propanoic acids were cyclised using polyphosphoric acid (PPA) to give the
substituted chroman-4-ones (3a–3c) (Parham & Huestis, 1962). Base catalysed
condensation of substituted chroman-4-one with different substituted benzaldehydes
afforded the homoisoflavanones (4a–4d). The method was adapted from literature
(Evans & Lockhart, 1966; Mulvagh, Meegan, & Donnelly, 1979; Siddaiah et al.,
2006). The homoisoflavanone (4d) is novel whilst the other three homoisofla-
vanones (4a–4c) were previously reported (McPherson, Cordell, Soejarto, Pezzuto,
& Fong, 1983; Meegan & Dervilla, 1981; Siddaiah et al., 2006). The 1H-NMR
(NMR, nuclear magnetic resonance) data for homoisoflavanones (4a) and (4b)
coincides with the literature values, respectively (McPherson et al., 1983; Siddaiah
et al., 2006).
2.2. Reduction of croton oil-induced oedema
Four homoisoflavanones with different substitution patterns were synthesised and
tested for their anti-inflammatory activities. The ability of the homoisoflavanones to
inhibit acute croton oil-induced auricular contact dermatitis was assessed.
Compound 4a exhibited a tendency to inhibit oedema in a dose-dependent
manner after 3 and 6 h of treatment. Activity increased with increasing doses and

1476
M.M. Shaikh et al.
R1
R1
OH
R2
R2
O
Br
COOH
NaH,
COOH
PPA
DMF, rt, 35-40 %
80oC, 50 %
R3
R3
1
2
a) 1,R2=OMe, R3=H
b) 1,R3=H, R2=OMe
c) 1=H, R2, R3=OMe
R1
R1
R2
O
R2
O
R4
Substituted benzaldehyde
piperidine, 70-80oC,
56-76 %
R3
O
R3
O
4
3
4a R1,R2=OMe, R3=H, R4=OH
4b R1,R3=H, R2, R4=OMe
4c R1,R4=H R2, R3=OMe
4d R1=H, R2,R3=OMe, R4=OH
Scheme 1. Reagents and conditions for the chemical synthesis of substituted
homoisoflavanones.
optimal activity was observed at 0.5 mg. After 6 h of treatment, activity decreased for
all doses and was significant for 0.1 mg (3 h: 46.4 ꢀ 5.1%; 6 h: 29.0 ꢀ 5.0%).
Compounds 4b–4d also inhibited oedema, although a clear dose-response relation-
ship was not observed. For compounds 4b–4d, optimal activity was observed with
0.5, 0.1 and 0.05 mg, respectively, after 3 h of treatment. After 6 h of treatment, the
activities of compounds 4b and 4d, respectively, were significantly decreased at
0.5 mg (3 h: 54.2 ꢀ 5.6%; 6 h: 22.2 ꢀ 4.4%) and 0.05 mg (3 h: 49.2 ꢀ 8.3%; 6 h:
39.4 ꢀ 3.8%). However, compound 4c did not exhibit a significant decrease in
activity after 6 h at the doses tested.
Compounds 4a and 4d have similar substitution patterns. The presence of the
methoxy group at C-8 (4a) or at C-5 (4d) in ring A apparently has an insignificant
influence on the biological activity of these compounds. Compounds 4c and 4d have
the same substitution pattern in ring A, but 4c lacks the hydroxyl group at C-40 of
ring B. This compound exhibited higher activity than other compounds and activity
was maintained after 6 h. Overall, the compound with an unsubstituted B ring (4c)
seemed to show higher activity than compounds with substituted B rings (4a, 4b
and 4d).

Natural Product Research
1477
2.3. Cytotoxicity
Cytotoxicity is an important factor to consider when testing for any biological
activity. A relatively high value may reduce the therapeutic potential of the
compounds tested. The in vitro cytotoxicity of compounds 4a–4d was investigated
and the IC50 values were 214 ꢀ 52;4337; 153 ꢀ 29; 8 ꢀ 1.6 mM, respectively. The IC50
value for the emetine as control was 0.125 ꢀ 0.04 mM. Compounds 4a–4c were found
to be relatively less cytotoxic, whilst compound 4d showed some evidence of
cytotoxicity.
3. Experimental
3.1. Chemistry experimental
All reaction mixtures were magnetically stirred and monitored by thin-layer
chromatography (TLC) using Kieselgel 60 F254 from Merck (Darmstadt,
Germany).1H- and 13C-NMR spectra were recorded on a Bruker AVANCE III at
400 MHz with CDCl3 as internal reference. The value of the chemical shift (ꢁ) is
given in ppm and coupling constant (J) is reported in Hertz (Hz). Melting points
(m.p.) were recorded with a Mel-Temp m.p. apparatus in open capillaries and are
uncorrected. The high-resolution mass spectroscopy (HRMS) data were recorded on
a Waters Micromass Q-Tof Micro mass spectrometer with a lock spray source.
3.1.1. Preparation of substituted 3-phenoxypropanoic acid derivatives (2a–2c)
To a N,N-dimethylformamide (DMF) solution (10 mL), NaH (32.46 mmol) was
added at a temperature of 10–15ꢁC. Each substituted phenol (32.46 mmol) in DMF
(15 mL) was added to the reaction mixture and stirred at room temperature for 1 h.
A solution of 3-bromopropionic acid (38.96 mmol) in DMF (15 mL) was then added
dropwise at 0ꢁC and the reaction mixture was stirred for 12 h at room temperature.
It was then diluted with methanol (20 mL) and acidified with 10% HCl. The product
was extracted with ethyl acetate (3 ꢂ 50 mL). The combined ethyl acetate layer was
washed with water (1 ꢂ 50 mL), brine (1 ꢂ 50 mL) and then dried over magnesium
sulphate. The residue obtained after evaporation of the solvent was chromato-
graphed over a silica gel column, using a mixture of ethyl acetate/hexane (30 : 70) as
eluent to obtain the title products 2a–2c (yield, 35–40%).
3.1.2. 3-(2,3-Dimethoxyphenoxy) propanoic acid (2a)
1H-NMR (400 MHz, CDCl3) ꢁ: 2.88 (t, 2H, H-2), 3.81 (s, 3H, Ar–OMe-9), 3.84
(s, 3H, Ar–OMe-9), 4.29 (t, 2H, H-3), 6.59 (d, J ¼ 8.4 Hz, 2H, H-5 & H-7), 6.96 (dd,
J ¼ 8.3, 8.3 Hz, 1H, H-6).13C-NMR (100 MHz, CDCl3) ꢁ: 34.4 (C-2), 56.1 (8-OMe),
60.8 (9-OMe), 64.4 (C-3), 105.9 (C-7), 107.2 (C-5), 123.6 (C-6), 138.7 (C-9), 152.2
(C-4), 153.6 (C-8) and 176.8 (C-1). Mass m/z ¼ 227 [M þ 1]þ. Rf ¼ 0.23 for a mixture
of ethyl acetate/hexane (30 : 70) on silicagel.
3.1.3. 3-(3-Methoxyphenoxy) propanoic acid (2b)
1H-NMR (400 MHz, CDCl3) ꢁ: 2.84 (t, 2H, H-2), 3.78 (s, 3H, Ar–OMe-8), 4.23
(t, 2H, H-3), 6.46–6.53 (m, 3H, H-5, H-7, H-9) and 7.17 (t, 1H, H-6).

1478
M.M. Shaikh et al.
13C-NMR (100 MHz, CDCl3) ꢁ: 34.3 (C-2), 55.2 (8-OMe), 63.0 (C-3), 101.1 (C-9),
106.6 (C-7), 106.8 (C-5), 129.9 (C-6), 159.6 (C-4), 160.8 (C-8) and 177.1 (C-1). Mass
m/z ¼ 197 [M þ 1]þ. Rf ¼ 0.25 for a mixture of ethyl acetate/hexane (30 : 70) on
silicagel.
3.1.4. 3-(3,5-Dimethoxyphenoxy) propanoic acid (2c)
1H-NMR (400 MHz, CDCl3) ꢁ: 2.83 (t, 2H, H-2), 3.76 (s, 6H, Ar–OMe-6,8), 4.20
(t, 2H, H-3) and 6.09 (s, 3H, H-5, H-7, H-9).13C-NMR (100 MHz, CDCl3) ꢁ: 34.2
(C-2), 55.3 (6-OMe and 8-OMe), 63.0 (C-3), 93.4 (C-7), 93.5 (C-5 and C-9), 160.2
(C-4), 161.5 (C-8) and 176.8 (C-1). Mass m/z ¼ 227 [M þ 1]þ. Rf ¼ 0.24 for a mixture
of ethyl acetate/hexane (30 : 70) on silicagel.
3.2. Preparation of substituted chroman-4-ones (3a–3c)
A mixture of substituted 3-phenoxypropanoic acid (10 mmol) and PPA (10 g) was
stirred whilst being heated to 85–90ꢁC for 2 h. The red coloured syrup thus obtained
was poured into crushed ice and extracted with diethyl ether (2 ꢂ 50 mL). The extract
was washed with 3 N NaOH (30 mL), water (50 mL) and dried over magnesium
sulphate. The residue obtained after evaporation of the solvent was chromato-
graphed through a silica gel column using an appropriate mixture of ethyl acetate/
hexane (30 : 70) as eluent to obtain the title product (3a–3c) (yield, 50–55%).
3.2.1. 7,8-Dimethoxychroman-4-one (3a)
1H-NMR (400 MHz, CDCl3) ꢁ: 2.74 (t, 2H, H-3), 3.83 (s, 3H, Ar–OMe-7), 3.88
(s, 3H, Ar–OMe-8), 4.54 (t, 2H, H-2), 6.60 (d, J ¼ 8.9, 1H, H-6), 7.64 (d, J ¼ 8.9 Hz,
1H, H-5).13C-NMR (100 MHz, CDCl3) ꢁ: 37.5 (C-3), 56.1 (7-OMe), 61.0 (8-OMe),
67.6 (C-2), 105.5 (C-6), 116.5 (C-4a), 123.1 (C-5), 136.7 (C-8), 155.7 (C-8a), 158.5
(C-7) and 190.7 (C-4). Mass m/z ¼ 209 [M þ 1]þ. Rf ¼ 0.55 for a mixture of ethyl
acetate/hexane (30 : 70) on silicagel.
3.2.2. 7-Methoxychroman-4-one (3b)
1H-NMR (400 MHz, CDCl3) ꢁ: 2.74 (t, 2H, H-3), 3.83 (s, 3H, Ar–OMe-7), 4.51
(t, 2H, H-2), 6.40 (d, J ¼ 2.4 Hz, 1H, H-8), 6.57 (dd, J ¼ 2.4, 8.9 Hz, 1H, H-6), 7.82
(d, J ¼ 8.8 Hz, 1H, H-5).13C-NMR (100 MHz, CDCl3) ꢁ: 37.4 (C-3), 55.6 (C-7-OMe),
67.3 (C-2), 100.7 (C-8), 109.8 (C-6), 115.2 (C-4a), 128.8 (C-5), 163.7 (C-8a), 165.9
(C-7) and 190.5 (C-4). Mass m/z ¼ 179 [M þ 1]þ. Rf ¼ 0.57 for a mixture of ethyl
acetate/hexane (30 : 70) on silicagel.
3.2.3. 5,7-Dimethoxychroman-4-one (3c)
1H-NMR (400 MHz, CDCl3) ꢁ: 2.71 (t, 2H, H-3), 3.81 (s, 3H, Ar–OMe-7), 3.87
(s, 3H, Ar–OMe-5), 4.43 (t, 2H, H-2) and 6.04 (s, 2H, H-6 & H-8).13C-NMR
(100 MHz, CDCl3) ꢁ: 38.7 (C-3), 55.5 (C-7-OMe), 56.0 (C-5-OMe), 66.7 (C-2), 92.8
(C-8), 93.3 (C-6), 106.3 (C-4a), 162.2 (C-5), 165.2 (C-8a), 165.7 (C-7) and 189.1 (C-4).

Natural Product Research
1479
Mass m/z ¼ 209 [M þ 1]þ. Rf ¼ 0.54 for a mixture of ethyl acetate/hexane (30 : 70) on
silicagel.
3.3. Preparation of substituted homoisoflavanones
A mixture of substituted chroman-4-one (2.4 mmol), substituted benzaldehyde
(3.6 mmol) and piperidine (7–10 drops) was heated at 80ꢁC for 2–48 h. The reaction
was monitored by TLC, using 3 : 7 acetate/hexane as solvent system. The reaction
mixture was cooled, diluted with water (15 mL), acidified with 10% HCl and then
extracted with ethyl acetate (3 ꢂ 30 mL). The combined ethyl acetate layer was
washed with water (30 mL), brine (30 mL) and dried over magnesium sulphate. The
residue obtained after evaporation of the solvent was chromatographed with a silica
gel column using an appropriate mixture of ethyl acetate/hexane as eluent to obtain
the homoisoflavanones (yield, 58–86%).
3.3.1. (E)-7,8-dimethoxy-3-(40-hydroxybenzylidene)chroman-4-one (4a)
Yield 58%; m.p. 172–175ꢁC; yellow powder.
1H-NMR (400 MHz, CDCl3) ꢁ: 3.87 (s, 3H, Ar–OMe-7), 3.94 (s, 3H, Ar–OMe-8),
5.41 (d, J ¼ 1.52 Hz, 2H, H-2), 5.90 (broad s, 1H, OH), 6.70 (d, J ¼ 8.8 Hz, 1H, H-6),
6.93 (d, J ¼ 8.8 Hz, 2H, H-30,50), 7.22 (d, J ¼ 8.5 Hz, 2H, H-20,60), 7.80 (s, 1H, H-9),
7.81 (d, J ¼ 8.8 Hz, 1H, H-5).13C-NMR (100 MHz, CDCl3) ꢁ: 54.3 (C-7-OMe), 59.3
(C-8-OMe), 66.3 (C-2), 104.2 (C-6), 114.0 (C-4a), 115.3 (C-30,50), 122.2 (C-5), 125.2
(C-10), 126.8 (C-20,60), 130.3 (C-3), 134.8 (C-9), 135.4 (C-8), 153.0 (C-8a), 155.2 (C-4),
156.7 (C-7) and 179.7 (C-4). HRMS calculated for C18H17O5 [M þ H]þ 313.0998,
found 313.1071. Rf ¼ 0.51 on silicagel with ethyl acetate/hexane (30 : 70).
3.3.2. (E)-7-methoxy-3-(40-methoxybenzylidene)chroman-4-one (4b)
Yield 70%; mp 126–128ꢁC; pale yellow needles.
1H-NMR (400 MHz, CDCl3) ꢁ: 3.83 (s, 3H, Ar–OMe-7), 3.85 (s, 3H, Ar–OMe-40),
5.35 (d, J ¼ 1.8 Hz, 2H, H-2), 6.39 (d, J ¼ 2.4 Hz, 1H, H-8), 6.62 (dd, J ¼ 2.4, 8.8 Hz,
1H, H-6), 6.95 (d, J ¼ 8.8 Hz, 2H, H-30,50), 7.25 (d, J ¼ 8.8 Hz, 2H, H-20,60), 7.80 (s, 1H,
H-9) and 7.95 (d, J ¼ 8.8 Hz, 1H, H-5).13C-NMR (100 MHz, CDCl3) ꢁ: 55.3
(C-40-OMe), 55.6 (C-7-OMe), 68.0 (C-2), 100.7 (C-8), 110.3 (C-6), 114.2 (C-4a),
115.8 (C-30,50), 127.1 (C-10), 128.8 (C-5), 129.6 (C-20,60), 131.9 (C-3), 136.5 (C-9), 160.5
(C-40), 162.9 (C-8a), 165.8 (C-7) and 180.9 (C-4). HRMS calculated for C18H17O4
[M þ H]þ 297.1049, found 297.1121. Rf ¼ 0.62 on silicagel with ethyl acetate/hexane
(30 : 70).
3.3.3. (E)-5,7-dimethoxy-(3-benzylidene)chroman-4-one (4c)
Yield 76%; mp 100–103ꢁC; dark yellow powder.
1H-NMR (400 MHz, CDCl3) ꢁ: 3.82 (s, 3H, Ar–OMe-5), 3.91 (s, 3H, Ar–OMe-7),
5.2 (d, J ¼ 1.7 Hz, 2H, H-2), 6.12 (d, J ¼ 2.2 Hz, 1H, H-6), 6.06 (d, J ¼ 2.2 Hz, 1H
H-8), 7.27 (d, J ¼ 8.0 Hz, 2H, H-20,60), 7.27-7.37 (m, 3H, H-30, H-40, H-50) and 7.82
(s, 1H, H-9).13C-NMR (100 MHz, CDCl3) ꢁ: 67.4 (C-2), 131.9 (C-3), 179.5 (C-4),
107.2 (C-4a), 164.7 (C-5), 55.6 (C-5-OMe), 93.6 (C-6), 165.8 (C-7), 55.1 (C-7-OMe),
93.6 (C-8), 162.0 (C-8a), 134.8 (C-10), 129.9 (C-20,60) and 128.6 (C-40). HRMS
calculated for C18H17O4 [M þ H]þ 297.1049, found 297.1121. Rf ¼ 0.58 on silicagel
with ethyl acetate/hexane (30 : 70).

1480
M.M. Shaikh et al.
3.3.4. Novel (E)-5,7-dimethoxy-3-(40-hydroxybenzylidene)chroman-4-one (4d)
Yield 68%; mp 197–200ꢁC; yellow powder.
1H-NMR (400 MHz, CDCl3) ꢁ: 3.76 (s, 3H, Ar–OMe-7), 3.82 (s, 3H, Ar–OMe-5),
5.17 (d, J ¼ 1.4 Hz, 2H, H-2), 6.05 (d, J ¼ 2.2 Hz, 1H, H-6), 6.00 (d, J ¼ 2.2 Hz, 1H,
H-8), 7.11 (d, J ¼ 8.5 Hz, 2H, H-20,60), 6.85 (d, J ¼ 8.5 Hz, 2H, H-30,50), 7.70 (s, 1H,
H-9).13C-NMR (100 MHz, CDCl3) ꢁ: 66.5 (C-2), 128.0 (C-3), 178.8 (C-4), 106.1
(C-4a), 164.7 (C-5), 55.0 (OMe–C-5), 92.5 (C-6), 164.7 (C-7), 54.5 (OMe–C-7), 92.5
(C-8), 163.6 (C-8a), 126.0 (C-10), 130.9 (C-20,60), 114.7 (C-30,50), 155.9 (C-40) and
135.0 (C-9). HRMS calculated for C18H17O5 [M þ H]þ 313.0998, found 313.1071.
Rf ¼ 0.50 on silicagel with ethyl acetate/hexane (30 : 70).
3.4. Assessment of croton oil-induced oedema
The synthesised homoisoflavanones were assessed for their potential anti-inflam-
matory activity. Ethical approval (003/09/Animal) from the University of KwaZulu-
Natal Animal Ethics subcommittee was obtained prior to the investigation of acute
croton oil-induced auricular dermatitis in a mouse model. Guidelines by the
University of KwaZulu-Natal Animal Ethics Subcommittee and Biomedical
Resources Unit for the maintenance and treatment of laboratory animals were
followed.
Eight-week-old male Balb/c mice of approximately 30 g each were divided into
two groups for the 3 (eight mice) and 6 h (eight mice) treatment, respectively, for each
homoisoflavanone tested. In each group, two mice per condition were used,
representing duplicate measurements. Equal volumes of croton oil (25 mL) were
mixed with acetone (25 mL) as vehicle and applied (50 mL total volume; 1 h) onto the
inner surface of the right auricle of each mouse to induce oedema (Della Loggia, Del
Negro, Tubaro, Barone, & Parrilli, 1989). Acetone has not been documented to have
an anti-inflammatory effect by itself (Lee et al., 2009). Thereafter, the homoiso-
flavanones were dissolved in acetone and applied onto the right auricle (0.05, 0.1 and
0.5 mg in 50 mL; 3 or 6 h treatment) to assess the reduction in oedema. The non-
steroidal anti-inflammatory drug diclofenac (0.05 mg; 6 h treatment) was included as
a positive control.
Mice were euthanised after treatment for 3 and 6 h. From each mouse, left and
right auricle biopsy specimens were obtained with a 6 mm biopsy punch and then
weighed.
3.5. Cytotoxicity assay
In vitro cytotoxicity of the synthesised homoisoflavanones was tested against a
Chinese hamster ovarian (CHO-K1) cell line using the 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT) assay. This cell line was obtained from
American Type Culture Collection (ATCC, CCL-61).
The MTT assay is a colourimetric assay to determine cellular growth
and survival, and compares well with other available assays (Mosman,
1983; Rubinstein et al., 1990). The tetrazolium salt MTT was used to measure cell
viability.

Natural Product Research
1481
Homoisoflavanones were prepared in a 2 mg mLꢃ1 stock solution containing
10% v/v DMSO (DMSO, dimethyl sulphoxide). Emetine was used as the reference
drug at an initial concentration of 100 mg mLꢃ1 serially diluted in 10-fold to obtain
six concentrations, the lowest being 0.001 mg mLꢃ1. Homoisoflavanones were diluted
similarly. The DMSO solvent system had no measurable effect on cell viability
(data not shown).
3.6. Data analysis
Data are reported as the mean ꢀ standard error of the mean of three (cytotoxicity
assays) and at least four (anti-inflammatory assays) independent experiments with
triplicate and duplicate measurements, respectively. For the anti-inflammatory
assays, oedema was quantified by calculating the difference in weights of the right
and left auricular biopsy specimens. The value is expressed as a percentage of the
croton oil control. For the cytotoxicity assays, the 50% inhibitory concentration
(IC50) values were obtained from full dose-response curves using a non-linear
dose-response curve fitting analysis. GraphPad Prism version 5 (GraphPad Software,
San Diego, CA, USA) was used to analyse and present the data. Statistical
comparisons were made by one-way ANOVA followed by Bonferroni’s post-test for
multiple comparisons, or by Student’s two-tailed paired t-test to determine p-values.
A value of p50.05 was considered significant.
4. Conclusions
In conclusion, we were able to synthesise four structurally similar homoisoflava-
nones of which one was novel. Structures were assigned based on NMR and mass
spectrometric data. These compounds were tested for the first time for
anti-inflammatory properties in an animal model and all of them were found to be
active. It appeared as if the substitution pattern in ring A did not have a significant
effect on the activity, whilst an unsubstituted B ring increased activity. Compound 4c
lacks the hydroxyl group at C-40 of ring B and was the only compound that exhibited
a maintained activity after 6 h. Compounds 4a–4c were relatively less cytotoxic,
whilst compound 4d showed some degree of cytotoxicity, although this was 64-fold
lower than the cytotoxic control, emetine.
Acknowledgements
The authors acknowledge the University of KwaZulu-Natal Competitive Research Fund and
Rolexsi (Pty) Ltd for financial support, as well as Ms Sithabile Buthelezi and Mr Dennis
Ndwandwe for experimental assistance.
References
Della Loggia, R., Del Negro, P., Tubaro, A., Barone, G., & Parrilli, M. (1989).
Homoisoflavanones as anti-inflammatory principles. Planta Medica, 55, 587.

1482
M.M. Shaikh et al.
Du Toit, K., Drewes, S.E., & Bodenstein, J. (2010). The chemical structures, plant origins,
ethnobotany and biological activities of homoisoflavanones. Natural Product Research,
24, 412–445.
Evans, D.L., & Lockhart, I.M. (1966). Reaction of aromatic aldehydes and nitroso
compounds with 4-chromanones. Journal of the Chemical Society (C) Organic,
711–712.
Jackson Roberts II, L., & Morrow, J. (2001). Analgesic-antipyretic and anti-inflammatory
agents and drugs employed in the treatment of gout. In J. Hardman
&
L. Limbird (Eds.), The pharmacological basis of therapeutics (pp. 687–731).
New York, NY: McGraw-Hill.
Lee, D., Choo, B., Yoon, T., Cheon, M., Lee, H., Lee, A., & Kim, H. (2009).
Anti-inflammatory effects of Asparagus cochinchinensis extract in acute and chronic
cutaneous inflammation. Journal of Ethnopharmacology, 121, 28–34.
Mahapatra, T., Jana, N., & Nanda, S. (2008). Chemoenzymatic synthesis and resolution of
compounds containing a quaternary stereocenters adjacent to a carbonyl group.
Tetrahedron: Asymmetry, 19, 1224–1232.
Maheswara, M., Siddaiah, V., & Rao, C.V. (2006). Two new homoisoflavanoids from
Caesalpinia pulcherrima. Chemical and Pharmaceutical Bulletin, 54, 1193–1195.
McPherson, D.D., Cordell, G.A., Soejarto, D.D., Pezzuto, J.M., & Fong, H.H.S. (1983).
Peltogynoids and homoisoflavonoids from Caesalpinia pulcherrima. Phytochemistry, 22,
2835–2838.
Meegan, M.J.D., & Dervilla, M.X. (1981). Dihydrochalcones from 3-benzylidinechroman-
4-ones. Journal of Chemical Research, Synopses 1, 4.
Mosmann, T. (1983). Rapid colorimetric assay for cellular growth and survival: application to
proliferation and cytotoxicity assays. Journal of Immunological Methods, 65, 55–63.
Mulvagh, D., Meegan, M.J.,
&
Donnelly, D.M. (1979). Rearrangements of
3-benzylidenechroman-4-ones. Journal of Chemical Research Synopsis, 4, 137.
Parham, W.E., & Huestis, L.D. (1962). The Reaction of dichlorocarbene with 2,3-chromene
and 3,4-chromene. Journal of the American Chemical Society, 84, 813–816.
Pohl, T., Crouch, N., & Mulholland, D. (2000). Southern African Hyacinthaceae: chemistry,
bioactivity and ethnobotany. Current Organic Chemistry, 4, 1287–1324.
Rao, Y.K., Fang, S.F., & Tzeng, Y. (2005). Anti-inflammatory activities of flavonoids isolated
from Caesalpinia pulcherrima. Journal of Ethnopharmacology, 100, 249–253.
Rubinstein, L.V., Shoemaker, R.H., Paull, K.D., Simon, R.M., Tosini, S., Skehan, P., &
Boyd, M.R. (1990). Comparison of in vitro anticancer-drug-screening data generated
with a tetrazolium assay against a diverse panel of human tumor cell lines. Journal of the
National Cancer Institute, 82, 1113–1118.
Siddaiah, V., Rao, C.V., Venkateswarlu, S., Krishnarajub, A.V., & Subbarajub, G.V. (2006).
Synthesis, stereochemical assignments, and biological activities of homoisoFavonoids.
Bioorganic and Medicinal Chemistry, 14, 2545–2551.
Watt, J., & Breyer-Brandwijk, M. (1962). The medicinal and poisonous plants of southern and
eastern Africa. Edinburgh: Livingston Ltd.