Hydrogen-bonded rotamers of 2′,4′,6′-trihydroxy-3′-formyldihydrochalcone, an intermediate in the synthesis of a dihydrochalcone from Leptospermum recurvum

Article abstract of DOI:10.1016/S0040-4020(03)00940-2

Synthesis of 2′,4′,6′-trihydroxy-3′-methyldihydrochalcone, isolated as a natural product for the first time (ex Leptospermum recurvum), proceeds through 2′,4′,6′-trihydroxy-3′-formyldihydrochalcone. Two stable rotamers of this formyl derivative have been

Full text of DOI:10.1016/S0040-4020(03)00940-2

Tetrahedron 59 (2003) 6113–6120
Hydrogen-bonded rotamers of 20,40,60-trihydroxy-30-
formyldihydrochalcone, an intermediate in the synthesis of a
dihydrochalcone from Leptospermum recurvum
Kamarul’Ain Mustafa,a Henrik G. Kjaergaard,a Nigel B. Perryb and Rex T. Weaversa
,
*
aDepartment of Chemistry, University of Otago, P.O. Box 56, Dunedin 9001, New Zealand
bDepartment of Chemistry, Plant Extracts Research Unit, New Zealand Institute for Crop and Food Research Limited, University of Otago,
P.O. Box 56, Dunedin, New Zealand
Received 9 April 2003; revised 20 May 2003; accepted 12 June 2003
Abstract—Synthesis of 20,40,60-0trihydroxy-30-methyldihydrochalcone, isolated as a natural product for the first time (ex Leptospermum
recurvum), proceeds through 2 ,40,60-trihydroxy-30-formyldihydrochalcone. Two stable rotamers of this formyl derivative have been
identified and the problems associated with NMR assignments of this type of compound have been attributed to conformational exchange.
Rotamer ratios from molecular modelling and ab initio calculations agree well with those obtained from low temperature NMR studies. There
is also excellent correlation between experimental NMR chemical shifts for the hydrogen-bonded hydroxyl protons with those derived from
ab initio calculations. This formyl dihydrochalcone showed promising bioactivity in antiviral and antimicrobial assays.
q 2003 Elsevier Ltd. All rights reserved.
1. Introduction
by molecular mechanics and ab initio studies, that elucidate
the conformational exchange responsible for these
anomalies.
As an extension of our studies on the bioactive components
of the New Zealand manuka, Leptospermum scoparium,1,2
we have screened Leptospermum species for a range of
biological activities. This study revealed that the ethanol
extract of the foliage of Leptospermum recurvum Hook. f.
(family Myrtaceae) possessed antiviral activity. This plant,
endemic to Mt Kinabalu in the States of Sabah, Malaysia
has been reported to contain the polyphenols, cyanidin,
quercetin, ellagic acid, delphinidin and myricetin,3 but we
could find no reports on medicinal properties. However,
L. recurvum is almost identical (apart from leaf size) to
L. flavescens3 which has been used traditionally in Malaysia
to stimulate appetite and relieve stomach disorders and
menstrual discomfort.4
2. Results and discussion
The dried foliage of L. recurvum was extracted with ethanol
and, after repeated column chromatography and preparative
TLC on silica gel, yielded 20,40,60-trihydroxy-30-methyl-
dihydrochalcone 15 and an inseparable mixture of 2,5-
dihydroxy-6-methyl-7-methoxyflavanone (3) and its isomer
2,5-dihydroxy-8-methyl-7-methoxyflavanone (4).6 Further
components proved difficult to separate from the major
component, the mixture of hydroxyflavanones 3 and 4. This
mixture was dehydrated to convert 3 and 4 to the flavones 5
and 6. Chromatography yielded 5-hydroxy-6-methyl-7-
methoxyflavo0ne0 (5),7 5-hydroxy-8-methyl-7-methoxy-
flavone (6),8 2 ,6 -dihydroxy-30-methyl-40-methoxydihydro-
In this paper we describe the isolation of flavonoid
components of L. recurvum foliage, including the dihydro-
chalcone (1). Synthesis of 1 was subsequently achieved via
the formyl derivative (2). NMR studies of 2 produced
spectra with some broad peaks and some notable absences
of signals. Searching the literature likewise revealed
incomplete sets of NMR data. We here detail studies of
the solution chemistry of 2, by low temperature NMR and
Keywords: Myrtaceae; conformational exchange; molecular modelling; ab
initio calculation; NMR.
chalcone
(7),9
and
5-hydroxy-6-methyl-7-
methoxyflavanone (8).8 HPLC studies revealed that com-
pounds 1, 3, 4, 5, 7 and 8 were present in the total plant
*
Corresponding author. Tel.: þ64-3479-7925; fax: þ64-3479-7906;
e-mail: rweavers@alkali.otago.ac.nz
0040–4020/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0040-4020(03)00940-2

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K.’A. Mustafa et al. / Tetrahedron 59 (2003) 6113–6120
Scheme 1. Synthesis of dihydrochalcone 1.
extract. Although a significant peak was observed for
flavanone 5, the level of the isomeric flavanone 6 was below
detection in the extract.
cytotoxic. Further studies on the activity of 1 were
hampered by lack of sample and the unavailability of
further plant material. Synthesis of 1 was conducted by
following an established three-step method10 involving
acylation of phloroglucinol, followed by formylation and
reduction of the formyl group (Scheme 1).
The NMR spectra of an intermediate from this synthetic
work, 20,40,60-trihydroxy-30-formyldihydrochalcone 2,
showed interesting complexities. This dihydrochalcone
has been reported once as a natural product, isolated from
the twig and leaf extracts of the Peruvian plant Psidium
acutangulum (Myrtaceae).11 This report quoted some 13C
NMR data but gave no values for the hydroxylated carbons.
Sato et al. reported NMR data for the 50-methyl substituted
analogue, but also did not quote hydroxylated carbon
shifts.10 The reason for this became apparent when the 13C
NMR spectrum of0 2 was recorded at 258C; the peaks for
C-20, C-40, and C-6 were almost lost in the baseline (Fig. 1).
In the 1H NMR spectrum (Fig. 2) only one broad peak was
visible to represent all of the three OH groups.
The 40-methoxydihydrochalcone 7 was found to be antiviral
against Herpes-simplex virus, whereas its demethylated
analogue 1 showed no antiviral activity but was mildly
Figure 1. Temperature variance of 13C NMR spectra of dihydrochalcone 2 in d6-acetone.

K.’A. Mustafa et al. / Tetrahedron 59 (2003) 6113–6120
6115
Figure 2. Temperature variance of 1H NMR spectra of dihydrochalcone 2 in d6-acetone.
This was demonstrated by recording the NMR spectra at
lower temperatures. At 2808C two sets of peaks in the ratio
of 2:1 were clearly observable in slow exchange (Figs. 1 and
2). These corresponded to two of the three possible
hydrogen-bonded species. All signals could now be
accounted for.
Peaks observed were assignable to structures 2a (major) and
2b (minor) by way of 2D NMR spectroscopy at 2608C
(HSQC and CIGAR, Table 1). In the major form, C-1 (dC
206.0) had a two bond correlation in the CIGAR spectrum
via the H-bond12 to the intramolecular hydrogen-bonded
OH proton, resonating at dH 14.85. This O0H signal also
Figure 3. Possible formyl chalcone rotamers.
The presence of two carbonyl groups among the three OH
groups gives the possibility of three rotamers, each with two
strong intramolecular hydrogen-bonds (Fig. 3). Broadening
of NMR signals at 258C could result from being close to the
coalescence temperature for exchange between these forms.
had
a three-bond correlation to C-5 (dC 95.1)
establishing that this OH was located at C-60. The other
1
Table 1. H and 13C NMR data and observed CIGAR correlations for 2
Position
Major rotamer 2a
Minor rotamer 2b
13C
1H
CIGAR
13C
1H
CIGAR
1
2
3
205.3
47.0
30.7
142.2
129.2
126.7
103.5
170.4
104.3
167.5
95.1
173.8
–
–
3.27
2.87
–
7.30
7.20
–
205.9
46.9
30.3
142.3
129.2
126.7
103.6
172.2
104.6
169.5
94.6
169.0
–
–
3.27
2.87
–
7.30
7.20
–
1, 3
1, 2
–
3, 4, 7
5/6/8/9
–
–
–
–
10, 30 40, 60, 30-CHO
–
10, 20, 30, 30-CHO
Not observed
10, 100, 50, 60
2 , 3
1, 3
1, 2
–
3, 4, 7
5/6/8/9
–
–
–
–
10, 40, 30, 30-CHO
–
1, 10, 20, 30
30, 40, 50, 30-CHO
Not 0observed
30, 4 , 50
4
5/6/8/9
7
10
–
–
20
–
–
–
–
–
–
30
40
50
5.84
–
14.65
11.71
14.85
9.92
5.79
–
15.62
12.85
11.83
9.98
60
20-OH
40-OH
600-OH
3 -CHO
–
–
–
192.9
–
192.5
Recorded at 2808C in acetone-d6. 1H NMR determined at 500 MHz referenced to solvent (d 2.05) and 13C NMR at 125 MHz referenced to solvent (d 29.9).
CIGAR spectra were recorded at 2608C and correlations are from the proton signal to the signal of the numbered carbon.

6116
K.’A. Mustafa et al. / Tetrahedron 59 (2003) 6113–6120
calculations are more reliable than molecular mechanics and
allow prediction of hydrogen bond strengths and NMR
shifts.14 The ab initio calculations were performed with the
GAUSSIAN 94 program.15 The structures of rotamers 10a,
10b and 10c, were fully optimized with the HF/6-31G(d)
and B3LYP/6-31G(d) methods. Calculations gave 10a as
having the lowest energy and 10c the highest, and supported
the experimental ratio of 1.0:0.5:0.0 observed for 2a, 2b and
2c. The calculated relative energies and ratios of the
rotamers of 10 are compared with the experimental ratios
for 2 in Table 2.
Figure 4. Selected CIGAR correlations of the major and minor rotamers of
2.
major hydrogen-bonded OH peak at dH 14.65 did not
correlate to C-50, but did correlate to the C-30 aldehyde
carbon signal (dC 192.9). These results established the major
rotamer as 2a (Fig. 4).
To calculate NMR chemical shifts, structures 10a and 10b
were optimized with the B3LYP/6-31G(d) method.14 This
led to the optimized geometries summarised in Table 3.
Calculations of NMR chemical shifts for both the hydrogen-
bonded species 10a and 10b were done with the HF/6-
31G(d) method at the B3LYP/6-31G(d) optimized geome-
try.15 The chemical shift values in Table 4 are corrected
with respect to calculated TMS values.
The identity of the minor form was established similarly.
The C-1 carbonyl carbon (dC 205.9) had a two-bond
correlation to the hydrogen-bonded OH proton signal at dH
15.62. This signal also had three-bond correlations to C-10
(dC 103.6) and C-30 (dC 104.6), showing that this OH was
located at C-20. The second hydrogen-bonded OH observed
for this minor form, resonating at dH 12.85 ppm, had three
bond correlations to C-50 (dC 94.6) and C-30, so it had to be
located at C-40. Thus the minor form was 2b (Fig. 4). Peaks
corresponding to the third form, 2c (Fig. 3) were not
observed in the temperature range 280 to 258C. This is the
first time that the identities of the rotamers of formyl
chalcones such as 2 have been established.
Hydrogen bond strengths were compared to the OH
bond length. The longer the OH bond the stronger the
hydrogen bonded interaction, and therefore the more
downfield the signal will be observed in the NMR spectrum.
The longest calcu0lated OH bond (Table 3) is that of Ob–Hd
(1.012 A), the 2 -OH, for rotamer 10b. The 1H NMR
˚
chemical shift of the proton of this hydroxyl group in 2b was
the most downfield, observed at d 15.6 ppm (calculated for
10b as d 14.6) (Table 4). The second longest calculated OH
The identity and assignment of the major and minor forms
were supported by both molecular mechanics and ab initio
energy calculations done on a simplified structure with a
CH3 group in place of the –CH2CH2C6H5 group, (10). An
extended labelling system has been used for 10 to identify
non-carbon atoms. Molecular mechanics calculations
(MMX force field13) and conformational searching pre-
dicted the two most stable rotamers for 10 as 10a (71%) and
10b (29%) at 2808C (Table 2). The 10c rotamer was
predicted to be undetectable by NMR (0.04%). These
rotamers are analogous to 2a, 2b and 2c, respectively
(Fig. 3).
0
e
h
˚
bond is that of O –H (1.010 A), the 6 -OH, for rotamer
10a. This corresponds to the second most downfield OH
proton signal for 2 at d 14.9 ppm (calculated for 10a as d
14.2). In general the calculated chemical shifts for the two
rotamers of compound 10 agree well with those found
experimentally for 2 (Table 4) except for those of the non-
intramolecular hydrogen bonded hydroxyl groups (the 40-
OH of rotamer a; and the 60-OH of rotamer b) where the
calculated shifts were much smaller than those observed.
Table 3. B3LYP/6-31G(d) optimized geometries of 10a and 10b
10a
10b
10a
10b
Bonded O–H
r(OeHh)
H-bonds
r(OgHd)
1.010
1.009
0.970
0.971
1.012
1.003
1.613
1.575
–
–
–
r(ObHd)
r(OaHh)
r(OdHf)
r(OaHd)
r(OgHf)
1.556
1.668
–
Bonded C–H
Bond angles
/C1OaHh
/C1OaHd
/C100OgHf
/C100OgHd
/C1’C1Oa
5
g
0
r(C H )
r(C2Ha)
r(C2Hb)
r(C2Hc)
r(C100He)
1.086
1.092
1.094
1.094
1.100
1.086
1.091
1.094
1.094
1.100
106.2
107.0
109.2
123.9
119.8
108.8
106.1
106.7
123.4
120.0
˚
Bond distances in A and angles in degrees.
In hydrogen-bonded and conjugated structures, ab initio
Table 2. Relative energies (kcal mol21) and ratio of the rotamers of 10 calculated at 2808C
Rotamer
Expt. for 2
MMX
HF/6-31G(d)
Ratio
B3LYP/6-31G(d)
DE Ratio
Ratio
DE
Ratio
DE
a
b
c
1
0.5
0.0
0
0.35
2.92
1
0.40
5.0£1024
0
0.22
6.94
1
0.57
1.8£1028
0
0.61
5.06
1
0.24
2.0£1026

K.’A. Mustafa et al. / Tetrahedron 59 (2003) 6113–6120
6117
Table 4. Calculated 1H and 13C NMR chemical shifts for 10a and 10b
compared with experimental values for 2a and 2b
(12), which has three hydrogen bonds to break, are
consistent with slow exchange at room temperature.16
10a (calc.a)
2a (expt.b)
10b (calc.a)
2b (expt.b)
H-50
5.2
14.1
4.8
14.2
10.3
98.5
177.1
94.9
168.3
86.2
181.1
207.7
193.9
5.8
14.7
11.7
14.9
5.3
14.6
12.9
5.0
10.5
95.6
178.8
98.4
177.5
85.6
169.4
206.8
194.9
5.8
15.6
12.9
11.8
20-OH
40-OH
60-OH
30-CHO
C-10
9.9
10.0
103.5
170.4
104.3
167.5
95.1
173.8
205.3
192.9
103.6
172.2
104.6
169.5
94.6
169.0
205.9
192.5
C-20
C-30
C-40
C-50
C-60
C-1
Assays on synthetic 1 confirmed the biological activity of
the natural sample from L. recurvum, and showed
antimicrobial activity against Bacillus subtilis and Tricho-
phyton mentagrophytes at 60 mg/disk.
30-CHO
a
Calculated with the HF/6-31G(d)//B3LYP/6-31G(d) method. Calculated
as (s–sTMS), where for TMS, sH¼32.6 ppm and sC¼200.0, and s is the
GIAO Nuclear Magnetic Shielding Tensor.
Recorded at 2808C in acetone-d6. 1H NMR determined at 500 MHz
referenced to solvent (d 2.05) and 13C NMR at 125 MHz referenced to
solvent (d 29.9).
b
Compound 2 exhibited stronger cytotoxic, antiviral and
antimicrobial activities that will be described elsewhere in a
paper
on
dihydrochalcones.
structure–activity
relationships
of
As the NMR spectra of compound 2 were obtained in
deuterated acetone, some intermolecular hydrogen bonded
interaction between the free OH groups and the carbonyls of
the solvent would be anticipated. Such interaction would
account for the discrepancies between the observed and
calculated values.
3. Conclusions
Although dihydrochalcone 7, flavanones 3, 4 and 8 and
flavone 5 have all been reported from natural sources, this is
the first report of dihydrochalcone 1 as a natural product.
These flavonoids are unusual in that they have C-methylated
A-rings and non-oxygenated B-rings. Synthesis of 1 has
confirmed the cytotoxicity observed for the natural sample.
1H NMR spectra of methyl dihydrochalcones 1 and 7 each
showed only one strongly hydrogen-bonded signal (d 13.91
and 13.55, respectively) along with hydroxyl proton signals
at higher field. Two and three bond correlations in the
HMBC spectra showed tha0t the predominant rotamer in
each case is that where the 2 -hydroxyl group is involved in
the intra-molecular hydrogen bond (Fig. 5, rotamers 1a and
7b).
The complex NMR spectra of formyl dihydrochalcone 2
have been demonstrated to arise from conformational
exchange between hydrogen-bonded rotamers. Low tem-
perature NMR studies have shown clearly that two of the
three possible hydrogen-bonded forms 2a and 2b are present
at the lower exchange limit. The experimentally determined
ratio of rotamers for 2 matched well with ratios calculated
for the simplified structure 10 from both molecular
mechanics and ab initio methods, and NMR chemical shifts
calculated for the various forms of 10 corresponded well
with the experimentally determined frequencies for 2.
Conformational exchange between these rotamers at room
temperature explains the in0complete NMR data previously
reported for 211 and for its 5 -methyl analogue.10 Compound
10, modelled in this study, has been synthesized previously,
Figure 5. Possible methyl chalcone rotamers.
0
0
1
and the H NMR signals of H-5 , the 3 -CHO and 2-CH3
reported with no mention of broadening. No OH signals
were reported.17 We could find no report of the 13C NMR
spectrum of 10 which we would anticipate to show
broadening.
We propose that this preference may be dictated by
optimisation of intermolecular hydrogen bonding inter-
actions involving the free hydroxyl group(s) or by steric
interaction distorting the 20-hydroxyl out of the plane of ring
A. The symmetric dihydrochalcone (9)0 showed only one
broad signal (d 11.75) for the 20 and 6 -hydroxyl groups.
This is consistent with fast exchange between the two
equivalent hydrogen-bonded rotamers. This conformational
exchange should have a lower rotational barrier than that
experienced between the rotamers of the formyl derivative
2, as only one hydrogen bond needs to be broken. By
Simperler and Mikenda18 have carried out ab initio
calculations to derive the relative energies of diacyl
phloroglucinols using B3LYP/6-31G(d,p). Of the com-
pounds studied, the methyl derivative (11) bears closest
resemblance to 10. Rotamers 11a and 11b (Fig. 3) were
considered, with 11a calculated to be the more stable by
0.72 kcal mol21, similar to our results for 10 (Table 2).
However, Simperler and Mikenda did not comment on
1
contrast, reported H and 13C NMR spectra of jensenone

6118
K.’A. Mustafa et al. / Tetrahedron 59 (2003) 6113–6120
rotamer 11c. Consideration of IR spectra did not enable
them to study the rotamer distribution experimentally. Our
study shows that employing variable temperature NMR
methods may offer a more fruitful approach.
isotropic magnetic shielding in tetramethylsilane (TMS)
minus that of the compound. The structure of TMS was fully
optimized with the B3LYP/6-31G(d) method. For both the
compounds and the TMS reference the GIAO shielding
tensors were calculated with the HF/6-31G(d) method on
the B3LYP/6-31G(d) optimized structure.24,25 All calcu-
lations were performed by using Gaussian94.15
4. Experimental
4.1. General
4.3. Plant material
L. recurvum was collected from Lincoln Landcare Research
Garden, New Zealand in November 1997. A voucher
specimen (voucher code 971103-13) is kept at the Plant
Extracts Research Unit, University of Otago, Dunedin, New
Zealand.
UV and IR spectra were recorded on a Jasco 7800 UV–Vis
spectrometer and a Perkin–Elmer 1600 FTIR instrument
respectively. Melting points were determined on a hot bench
Leica AG melting point apparatus first calibrated with
standard samples of known melting points. NMR spectra
were recorded on a Varian INOVA-500 spectrometer
1
operating at 500 MHz for H and 125 MHz for 13C NMR
4.4. Extraction and isolation
spectra were recorded on ca. 0.075 M solutions in d6-
acetone (referenced to solvent, d 2.05 for 1H and d 29.9 for
Foliage of L. recurvum (60 g) was dried, ground, extracted
(EtOH, 1£700 mL, 2£350 mL) and fractionated by RP
column chromatography (50 g, C-18) by gradient elution
from H2O through CH3CN to CHCl3. The fractions were
pooled into three groups: A, B and C based on their UV-
active spots on TLC silica (4:4:1, cyclohexane/EtOAc/
EtOH). The first group, A (78 mg) was subjected to flash
chromatography on silica gel (2 g), developed in steps from
cyclohexane to EtOAc. Fractions eluted with 4:1, cyclo-
hexane/EtOAc, gave an inseparable mixture of isomeric
hydroxyflavanones 3 and 4 (8 mg). Fractions eluted with 4:1
and 3:1, cyclohexane/EtOAc gave dihydrochalcone 1
(11 mg). Fraction B (63 mg) consisted of 3 and 4. Fraction
C (136 mg) containing 3, 4, 5, 7 and 8, was dehydrated as
follows.26
1
13C) or CDCl3 (referenced to solvent, d 7.25 for H and d
77.7 for 13C). Spectra were assigned with the aid of double-
quantum filtered COSY (1H–1H correlations), HSQC (one
bond 1H–13C correlations), and HMBC-CIGAR experi-
ments19 (two and three bond 1H–13C correlations).
Preparative TLC was carried out using Merck PLC-glass
plated silica gel 60 F254, 20 cm£20 cm (0.5 mm thickness).
HPLC equipment consisted of a Waters 717 autosampler,
600 pump and controller, and a 490E UV programmable
multi-wavelength detector controlled by Millennium soft-
ware. Samples were analyzed using an RP-18 analytical
column (Merck 100 RP-18, LichroCART 250£4 mm,
5 mm) fitted with a guard column (Merck 100 RP-18,
LichroCART 4£4 mm, 5 mm). Samples for analysis were
passed through ISOLUTE SPE Columns (100 mg, C-18 EC)
primed with MeCN (3£300 mL) prior to filtration and
filtered (0.45 mm) and 10 mL injected using the auto-
sampler. Silica gel 60, 200–400 Mesh, 40–63 mm (Merck)
was used in the column chromatography and octadecyl-
functionalised silica gel (C-18 Aldrich) was used for
reversed-phase (RP) chromatography. Biological assays
were performed as described previously.20
A solution of fraction C in AcOH (8 ml) containing six
drops of 96% H2SO4 was refluxed for 30 minutes. The
precipitate obtained by dilution with H2O was filtered off
and washed until neutral. It was then subjected to flash
chromatography on silica gel (13 g). Fractions eluted with
19:1 cyclohexane/EtOAc gave flavanone 8 (7 mg) and a
mixture of two components which, after preparative TLC
(toluene), gave flavones 5 (5 mg) and 6 (3 mg). Fractions
eluted with 17:1 cyclohexane/EtOAc gave dihydrochalcone
7.
HCl gas from Matheson Gas was dried by passing through
conc. H2SO4. Hydrocinnamonitrile (99% purity) from
AJAX Chemicals was used without further purification.
Phloroglucinol dihydrate (97% purity) from BDH was
dehydrated by heating under reflux in toluene using a
Dean–Stark apparatus.21 The residual material after solvent
removal was crystallized from EtOAc and cyclohexane.
Analytical RP HPLC showed that compounds 1, 3, 4, 5, 7
and 8, but not flavone 6, were present in the crude extract.
4.4.1. 20,40,60-Trihydroxy-30-methyldihydrochalcone 1.5
Pale yellow oil. UV (MeOH) lmax (log 1): 322 (3.62), 290
(4.23); IR (film) nmax cm21: 3353 broad, 3015, 2974, 1620
broad, 1523, 1426, 1215. 1H NMR (acetone-d6): 13.81 (1H,
s, 20-OH), 9.68 (1H, br. s, 60-OH), 9.08 (1H, s, 40-OH), 7.27
(2H, m, H-5/H-9), 7.17 (3H, m, H-6/H-7/H-8), 6.07 (1H, s,
H-50), 3.39 (2H, t, J¼8 Hz, H-2), 2.98 (2H, t, J¼8 Hz, H-3),
1.96 (3H, s, 30-Me). 13C NMR (acetone-d6): 205.2 (C-1),
165.1 (C-20), 162.8 (C-40), 160.4 (C-60), 142.8 (C-4), 1290.1
(C-5/C-9), 126.9 (C-6/C-7/C-8), 104.9 (C-10), 103.3 (C-3 ),
94.8 (C-50), 46.2 (C-2), 31.3 (C-3), 8.0 (30-Me).
4.2. Computational methods
Molecular mechanics calculations were performed with
PCModel version 7.13 Conformational searches used the
MMX force field, with the mixed Monte Carlo coordinate
movements/bond rotations strategy22 for the generation of
initial structures. The default cut-off criteria were employed.
The structures of rotamers 10a, 10b and 10c, were fully
optimized with the HF/6-31G(d) and B3LYP/6-31G(d)
methods. NMR shielding tensors were calculated with
the gauge-including atomic orbitals (GIAO) method.23 The
theoretical 1H and 13C chemical shifts were calculated as the
4.4.2. 2,5-Dihydroxy-6-methyl-7-methoxyflavanone 3
and its isomer, 2,5-dihydroxy-8-methyl-7-methoxyflava-
none 4.6 Yellowish oil. [a]D21¼þ24.2, c 0.1, CHCl3 (lit.,6

K.’A. Mustafa et al. / Tetrahedron 59 (2003) 6113–6120
6119
21
21
546 nm
þ20.7, c 0.105, CHCl3); [a]
¼22.84, [a]
¼
17 Hz, H-3), 2.83 (1H, dd, J¼3, 17 Hz, H-3), 2.01 (3H, s,
6-Me). 13C NMR (CDCl3): 195.8 (C-4), 165.8 (C-7), 161.1
(C-5), 160.4 (C-9), 138.5 (C-10), 128.9 (C-30/C-40/C-50),
126.1 (C-20/C-60), 106.1 (C-6), 102.8 (C-10), 90.7 (C-8),
79.4 (C-2), 55.8 (7-OMe), 43.5 (C-3), 6.8 (6-Me).
577 nm
21
405 nm
21
435 nm
227.4, [a]
¼230.4, [a]
¼2300.4 (all c 0.1,
CHCl3). UV (MeOH) lmax nm (log 1): 289 (4.38), 335
(3.80) (shoulder). IR (film) nmax cm21: 3500–3100, 2953,
1638–1600, 1448, 1297, 1215, 1125. EIMS m/z 300 (Mþ
1
for C17H16O5), 282, 105 (100), 77. H NMR (CDCl3): 3/4;
11.92/11.970(2£10H, s, 5-OH), 7.67 (4H, m, H-20/H-60), 7.44
(4H, m, H-3 /H-5 ), 7.41 (2H, m, H-40), 6.12/6.11 (2£1H, s,
H-8/H-6), 3.84/3.85 (2£3H, s, 7-OMe), 3.03/3.02 (2£2H, s,
H-3), 2.00/2.04 (2£3H, s, 6-Me/8-Me); 13C NMR (CDCl3):
3/4, 194.2/194.7 (C-4), 165.7/166.1 (C-7), 160.2/1620.0
(C-5), 157.7/155.5 (C-9), 1401.9/142.2 (C-10), 129.2 (C-4 ),
128.8 (C-30/50), 125.0 (C-2 /60), 106.8/92.6 (C-6), 102.3
(C-10), 101.1/101.6 (C-2), 91.6/105.7 (C-8), 55.8/55.9
(7-OMe), 48.4/48.2 (C-3), 6.9/7.8 (6-Me/8-Me).
4.4.7. 20,40,60-Trihydroxydihydrochalcone 9.10 Into a
mixture of dry phloroglucinol (2.30 g, 0.0183 mol), hydro-
cinnamonitrile (3.88 g, 0.0230 mol), and ZnCl2 (0.91 g,
0.00670 mol) in dry Et2O (46.0 mL) was passed dry HCl gas
under vigorous stirring by mechanical stirrer with cooling in
an ice–MeOH bath (210 to 2208C) for 3 h. The reaction
mixture was allowed to stand overnight in a freezer, and
again dry HCl gas was passed through it for 3 h. After
further standing in a freezer, the reaction mixture was
decanted and the residual brown viscous syrup was
hydrolysed by refluxing in H2O (69.0 mL) for 2 h. The
yellow solid was collected by filtration. A further crop of
crystals was obtained as yellow needles by concentration of
the filtrate to give a total mass of 3.32 g (71%) of 20,40,60-
trihydroxydihydrochalcone 9. Mp 1448C (lit.,10 142–
1438C). UV (MeOH) lmax (log 1): 325 (3.68), 287
(4.43) nm. IR (neat) nmax 3293, 3016, 1631, 1605, 1518,
1452 cm21; ESI (2ve) m/z 2570[M2H]; 1H NMR (acetone-
d6): 11.75 (2H, br. s, 20-OH/6 -OH), 9.27 (1H, s, 40-OH),
7.27 (4H, m, H-5/H-6/H-8/H-9), 7.18 (1H, m, H-7), 5.94
(2H, s, H-30/H-50), 3.39 (2H, t, J¼8 Hz, H-2), 2.97 (2H, t,
J¼80Hz, H-3); 13C NMR (acetone-d6): 203.3 (C-1), 164.0
(C-4 ), 163.6 (C-20/C-60), 141.2 (C-4), 127.7 (C-5/C-6/
C-8/C-9), 125.2 (C-7), 103.1 (C-10), 94.1 (C-30/C-50), 44.4
(C-2), 29.6 (C-3).
4.4.3. 5-Hydroxy-6-methyl-7-methoxyflavone 5.7 Yellow-
ish gum. UV (MeOH) lmax (log 1): 314 (4.00), 273 (4.28),
249 (4.04). IR (film) nmax cm21: 3440 broad, 3030, 2930,
1652, 1615, 1588, 1492, 1456, 1349, 1216, 1139. 1H NMR
(CDCl3): 12.73 (1H, br. s, 5-OH), 7.89 (2H, m, H-20/H-60),
7.54 (1H, m, H-40), 7.51 (2H, m, H-30/H-50), 6.68 (1H, s,
H-3), 6.50 (1H, s, H-8), 3.93 (3H, s, 7-OMe), 2.11 (3H, s,
6-Me). 13C NMR (CDCl3): 182.5 (C-4), 163.6 (C-2/C-7),
158.5 (C-5), 155.05 (C-9), 131.5 (C-10/C-40), 129.1 (C-30/
C-50), 126.3 (C-2 /C-60), 109.3 (C-6), 106.0 (C-3), 105.5
(C-10), 89.5 (C-8), 55.8 (7-OMe), 7.4 (6-Me).
4.4.4. 5-Hydroxy-8-methyl-7-methoxyflavone 6.8 Pale
yellow oil. UV (MeOH) lmax (log 1): 340 (3.70), 335
(3.70), 274 (4.40). IR (film) nmax cm21: 3456 broad, 3015,
2964, 1651, 1610, 1523, 1476, 1420, 1220. 1H0 NMR
(CDCl3): 12.76 (1H, br. s, 5-OH), 7.91 (2H, m, H-2 /H-60),
7.54 (1H, m, H-40), 7.52 (2H, m, H-30/H-50), 6.66 (1H, s,
H-3) 6.41 (1H, s, H-6), 3.91 (3H, s, 7-OMe), 2.30 (3H, s,
8-Me). 13C NMR (CDCl3): 183.1 (C-4), 163.8 (C-2), 163.3
(C-7), 1600.3 (C-5), 154.6 (C-9), 131.8 (C-10/C-40), 129.1
(C-30/C-5 ), 126.3 (C-20/C-60), 105.3 (C-10), 105.1 (C-3),
104.1 (C-8), 95.3 (C-6), 56.0 (7-OMe), 7.7 (8-Me).
4.4.8. 20,40,60-Trihydroxy-30-formyldihydrochalcone 210.
Into a vigorously stirred solution of 20,40,60-trihydroxy-
chalcone (0.20 g, 0.000780 mol), Zn(CN)2 (0.18 g,
0.00155 mol) and AlCl3 (0.21 g, 0.00155 mol) in dry Et2O
(4.0 mL) was passed dry HCl gas with cooling in an ice-
MeOH bath (210 to 2208C) for 3 h. The reaction mixture
was allowed to stand overnight in a freezer. This operation
was repeated once more. After decantation, the residual dark
yellow viscous syrup was heated under reflux in H2O
(4.0 mL) for 1 h. After filtration, the residue was dissolved
in EtOAc (4.0 mL). The EtOAc soln was washed with H2O
(3£4 mL), and brine (3£4 mL), dried over Na2SO4, and
then concentrated using a rotary evaporator to give a reddish
brown syrup which was then purified by passing through a
silica column (Davisil silica, cyclohexane/EtOAc; 3:1) to
give 2 as pinkish powder (0.14 g, 61%). Mp 1588C (lit.,10
157–1588C). IR (film) nmax: 3149, 3026, 2975, 2923, 1626
(broad), 1430, 1315, 1215, 758 cm21; UV (MeOH) lmax
(log 1): 325 (3.73), 273 (4.52) nm; ESI (2ve) m/z: 285
[M2H]; found: C, 67.2; H, 5.0%; calcd for C16H14O5: C,
4.4.5. 20,60-Dihydroxy-30-methyl-40-methoxydihydro-
chalcone 7.9 Pale yellow oil. UV (MeOH) lmax (log 1):
324 (3.55), 288 (4.29). IR (film) nmax cm21: 3292, 3026,
2974, 1625, 1600, 1517, 1426, 1215. 1H NMR (acetone-d6):
13.45 (1H, s, 20-OH), 9.98 (1H, s, 60-OH), 7.27 (2H, m,
H-5/H-9), 7.17 (3H, m, H-6/H-7/H-8), 6.14 (1H, s, H-50),
3.81 (3H, s, 40-OMe), 3.41 (2H, t, J¼8 Hz, H-2), 2.98 (2H, t,
J¼8 Hz, H-3), 1.92 (3H, s, 30-Me). 13C NMR (acetone-d6):
205.8 (C-1), 170.5 (C-20), 164.6 (C-40), 161.1 (C-60), 1420.8
(C-4), 129.02 (C-5/C-9),0126.6 (C-06/C-7/C-8), 105.3 (C-1 ),
104.4 (C-3 ), 91.0 (C-5 ), 55.7 (4 -OMe), 46.5 (C-2), 31.3
(C-3), 7.2 (30-Me).
1
67.1; H, 4.9%. H and 13C NMR (Table 1).
4.4.6. 5-Hydroxy-6-methyl-7-methoxyflavanone 8.8
Yellowish gum. [a]2D1.7¼þ34.7, c 0.01, MeOH (lit.,27
22
4.4.9. 20,40,60-Trihydroxy-30-methyldihydrochalcone 1.5
A solution of 2 (0.023 g, 0.08 mmol) in MeOH (0.80 mL)
was heated under reflux with Zn–Hg amalgam (0.46 g),
conc. HCl (0.4 mL) and H2O (0.1 mL) for 15 min. After
decantation to remove the Zn–Hg amalgam, H2O (1.0 mL)
was added to the supernatant solution. The mixture was
extracted with EtOAc (3£4 mL) and the combined extracts
were washed with H2O (3£10 mL) and brine (1£10 mL)
and dried over anhydrous Na2SO4. The solvent was
22
577 nm
22
22
546 nm
þ45, c 0.02, MeOH); [a]
¼2184.0, [a]
¼
2545.3, [a] ¼23298 (all c 0.01,
MeOH). UV (MeOH) lmax (log 1): 290 (4.30), 326 (3.54)
(shoulder). IR (film) nmax cm21: 3015, 2925–2950, 1638,
¼22802, [a]
435 nm
405 nm
1
1580, 1449, 1293, 1205, 1156, 1129. H NMR (CDCl3):
12.05 (1H, s, 5-OH), 7.45 (2H, m, H-20/H-60), 7.42 (3H, m,
H-30/H-40/H-50), 6.09 (1H, s, H-8), 5.41 (1H, dd, J¼3,
13 Hz, H-2), 3.83 (3H, s, 7-OMe), 3.07 (1H, dd, J¼13,

6120
K.’A. Mustafa et al. / Tetrahedron 59 (2003) 6113–6120
P. A.; Swithenbank, C.; Lidert, Z. Phytochemistry 1991, 30,
1131–1132.
removed by rotary evaporation to give 1 as a yellow oil
(0.019 g, 87%). A sample for spectral studies and bioassays
was further purified by prep. TLC (cyclohexane/EtOAc;
1:1), yield 73%. UV (MeOH) lmax (log 1): 325 (3.73), 273
12. Borisov, E. V.; Zhang, W.; Bolvig, S.; Hansen, P. E. Magn.
Reson. Chem. 1998, 36, S104–S110.
13. Serena Software, PC Model V 7.0; Molecular Modeling
Software: Bloomington, Indiana, 1999.
(4.52) nm; ESI (2ve) m/z: 271 [M2H]; IR (neat) nmax
1
:
3394, 3015, 2923, 1718, 1626 cm21. H and 13C NMR (as
above).
14. Foresman, J. B.; Frisch, A.; 2nd ed. Exploring Chemistry with
Electronic Structure Methods; Gaussian Inc: Pittsburgh, 1996.
15. Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Gill, P. M. W.;
Johnson, B. G.; Robb, M. A.; Cheeseman, J. R.; Keith, T.;
Petersson, G. A.; Montgomery, J. A.; Raghavachari, K.;
Al-Laham, M. A.; Zakrzewski, V. G.; Ortiz, J. V.; Foresman,
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Acknowledgements
Thanks to W. Harris and J. W. van Klink for plant material,
M. Thomas and W. Redmond for assistance with NMR
experiments, B. Clark for MS analysis; G. Ellis for
biological assays, and M. Dick for microanalysis. This
research was supported in part by the New Zealand
Foundation for Research, Science and Technology and by
the New Zealand Official Development Assistance
(NZODA), Ministry of Foreign Affairs and Trade (PhD
scholarship).
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