two -...
TRANSCRIPT
TWO NEW 5-DEO LATONES FROM ALBIZZIA ODORATISSIMA (MIMOSACEAE)
The genus Albizzia belongs to the family Mimosaceae consists of 50
,species of which 8 species occur in India1. A few plants belonging to this
genus are extensively in the treatment of hemicrania, piles, excessive
perspiration, bronchites, asthma, snake-bite, leprosy, ulcers, cough, diarrhea
and ophthalmia.
A systematic phytochemical investigation of the Albizzia odoratissina
now carried out has resulted in the isolation and characterisation of two new
5-deoxyflavones. Hence it is felt worthwhile t o review briefly the chemistry
of 5-deoxyflavones covering their distribution, classification, structure
determination by physical and chemical methods, and synthesis.
I. Distribution
Flavones with 5-deoxygenation occur abundantly in Faba~eaell-~~,
family followed by ~h~melaeaceae~ , Premulaceae6, CCyperaceae4' and
Myris t ica~eae~~~"~. The occurrence of 5-deoxyflavones in Primulaceae is
restricted to Primula genus only and have the special feature of less
oxygenation. The 5-deoxyflavones with high oxygenation are confirmed to
Mimosaceae52*54~.56'57. ~ ~ ~ ~ ~ ~ i t a c e a e ~ ~ and Convolvulaceae53.
The 5-deoxyflavones are also found scarcely in few members of
Bignoniaceae5, sapindaceaeg, ~ u n c a c e a e ~ ~ and Vochy~iaceae~~.
II. Classification
Naturally occurring 5-deoxyflavones can be conveniently classified into
four types based on the oxygenation pattern present in ring-B. They are:
i. 5-Deoxyflavones devoid of oxygenation in ring-B
ii. 5-Deoxyflavones having monooxygenation in ring-B
iii. 5-Deoxyflavones having dioxygenation in ring-B
iv. 5-Deoxyflavones having trioxygenation in ring-B
Table - 1: 5-Deoxyflavones devoid of oxygenation in ring-B
Compound Plant source Family Ref.
1. 6-Methoxyflavone (1) Pimeleu decora Thymelaeaceae 4
2. 7-Hydroxyflavone (2) Pirneleu simplex Thymelaeaceae 4
3. 7-Methoxyflavone (3) Pirneleu simplex Thymelaeaceae 4
4. 7,B-Dirnethoxyflavone (4) Godmania Bignoniaceae 5 aesculifolia
- -
From Table - 1 it is clear that excepting 7,8-dimethoxyflavone reported
from Godmania of Bignoniaceae all the 5-deoxyflavones devoid of oxygenation
in ring-B were reported from Pimelea of Thymelaeaceae.
R R, R, 1 OMe H H
2 H O H H
3 H OMe H 4 H OMe OMe
Table - 2: 5-Deoxyflavones having monooxygenation in ring-B
Compound
2'-Hydroxy- flavone (5)
Plant source Family Ref.
Pimelea simplex Thymelaeaceae Primula farina Primulaceae Dephnopsis Thymelae aceae selloniana
2'-Methoxy- flavone 16)
Pimelea simplex Thymelaeaceae Primula kewensis Primulaceae
3'-Hydroxy- flavone (7)
Pimelea decora Thymelaeaceae
3'-Methoxy- flavone (8)
P. decora Thymelaeaceae
4'-Hydroxy- flavone (9)
Sapind us saponaria Sapindaceae
4'-Methoxy- flavone (10)
S. saponaria Sapindaceae
6,3'-Dihydroxy- flavone (11)
Pimelea decora Thyrnelaeaceae
P. decora Thymelaeaceae 6,3'-Dimethoxy- flavone (12)
Prirnula macrophylla Primulaceae 7,2'-Dihydroxy- flavone (13)
Trifolium repens T . subterraneurn Medicago sativa Baptisia lecontei Glycyrrhiza pal lid i flora C, squamulosa 6. inflata Lespedeza nakaii Sophora viciifolia Castanospermum australe Pterocrapus
Fabaceae Fabaceae Fabaceae Fabaceae Fabaceae
7,4'-Dihydroxy- flavone (14)
Fabaceae Fabaceae Fabaceae Fabaceae Fabaceae
Fabaceae marsupium Vicia faba Fabaceae 26
Compound Plant source Family Ref.
11. 7-Hydroxy-4'- Bauhinia manca Caesalpiniaceae 27
rnethoxyflavone
12. 7-Methoxy-4'- Bauhinia manca Caesalpiniaceae 27
hydroxyflavone Trifol ium hybridum Fabaceae 28
(16)
13. 7,4'-Dimethoxy- Virola venosa Myristicaceae 29
flavone (17) V. michelli Myris ticaceae 30
14. 8,2'-Dihydroxy- Primula pulverulenta Primulaceae 31
flavone (18)
15. 7,8,4'-Trihydroxy- Acacia nigrescens Mimosaceae 32
flavone (19)
From Table-2 it is evident that majority of 5-deoxyflavones with mono
oxygenation in ring-I3 are found in Pimelea of Thymelaeaceae, Primula of
Primulaceae, Glycerrhiza of Fabaceae and Virola of Myristicaceae. The
occurrence of compounds 5-10 constitute the rare report of flavones without
any substitution in ring-A. The isolation of compounds 11 and 12 with
monooxygenation at 6-position is of very rare occurrence. The occurrence of
2-oxygenated flavones with 5-deoxygenation (13 and 18) in Nature is also
very rare.
5 O H H H
6 OMe H H
7 H O H H
8 H OMe H
9 H H O H
R, 11 OH OH
1 2 OMe OMe
10 H H OMe
R R, R2 14 OH H OH
15 OH H OMe
16 OMe H OH
17 OMe H OMe
19 OH OH OH
Table - 3: 5-Deoxyflavones having dioxygenation in ring-B
Compound
1. 2',5'-Dihydroxy- flavone (20)
2. 2',5'-Dihydroxy- flavone 5'-acetate (21)
3. 3',4'-Dihydroxy- flavone (22)
4. 6,2',3'-Trirnethoxy- flavone (23)
5. 7,3',4'-Trihydroxy- flavone (24)
Plant source Familv Ref.
Primula spp. Primulaceae 33
P. pulverulenta Primulaceae 33
Primula spp. Primulaceae 34,35
Pimelea decora Thymelaeaceae 4
Trifolium repense Fabaceae Baptisia lecontei Fabaceae Sophora uiciifoliu Fabaceae Vicia faba Fabaceae Trifblium Fabaceae subterraneum Medicago sativa Fabaceae Juncus trifidus Juncaceae Luzula purpurea Juncaceae Salvertia Vochysiaceae convallariodora Vochysia Vochysiaceae cinnamonea V. tucanorurn Vochysiaceae Carex Cyperaceae longebrachiata Schoenus spp. Cyperaceae Uicinia spp. C yperace ae Medicago arborea Fabaceae Medicago strasseri Fabaceae Cyperus ref ixus Cyperaceae C. distans Cyperaceae C. laevigatus Cyperaceae C. betchei Cyperaceae C. lhotskyanus Cyperaceae C. rigidellus Cyperaceae Pinnati betchei Cyperaceae P, rigidellus Cyperaceae Albizzia julibrisson Mimosaceae
Compound Plant source Family Ref.
6. 7,3'-Dihydroxy-4'- Sophoraviciifolia Fabaceae 23 methoxyflavone Acacia farnesiana Mimosaceae 44 (Farnisin) (25)
7. 7,4'-Dihydroxy-3'- Salvertia Vochysiaceae 39 rnethoxyflavone convallariodora (Geraldone) (26) Vochysia Vochysiaceae 39
cinnamonea Trifol i um Fabaceae 45 subterraneum
8. 7,4'-Dimethoxy-3'- Virola venosa Myristicaceae 29 hydroxyflavone V. rnichelli Myristicaceae 30 (Tithonine) (27) Tithonia Compositaceae 46
tubaeformis Tithonia spp. Compositaceae 47 Virola surinamensis Myristicaceae 48
9. 7,3',4'-Trimethoxy- Virola venosa Myristicaceae 29 flavone (28) V. michelli Myristicaceae 30
Umtixa listerana Fabaceae 49
10. 7-Methoxy-3',4'- Virola uenosa Myristicaceae 29 methylenedioxy- flavone (29)
11. 6,7,3',4'- Abrus precatorius Fabaceae 50 Tetrahydroxy- flavone (30)
12. 6,4'-Dimethoxy-7,3'- A. precatorius Fabaceae 50 dihydroxyflavone (Abrectorin) (31)
13. 6,7,8,3',4'- SaturejcL montana Cyperaceae 51 Pentamethoxy- flavone (32)
From Table - 3 it is clear that majority of flavones with dioxygenation
in ring-B possess high degree of oxygenation at 3/:4' followed by 2':5'
positions.
22
R, OM e
20 011 Of1
21 OH OAc
M e 0
0
2 3
R R, K, 24 OH OH OH
25 01.1 OH OMe
26 OH OMe OH
27 OMe OH OMe
28 OMe OMe OMe
R R, R, R3 30 OH OH OH OH
31 OMe OH OH OMe
OMe
Table - 4: 5-Deoxyflavones having trioxygenation in ring-B
Compound
1. 7,2',4',5'-Tetramethoxy- flavone (33)
2. 7,3',4',5'-Tetrahydroxy- flavone (34)
3. 6,7,3',4',St- Pent ahydroxy- flavone (35)
4. 6,7,4'-Trihydroxy- 3',5'-dimethoxy- flavone (36)
5. 6,7-Dihydroxy-3',4',5'- trimethoxyflavone (Prosogerin E) (37)
6. 7-Hydroxy-6,3',4',5'- tetramethoxyflavone (Prosogerin D) (38)
7. 6,7,3',4',5'-Pentamethox - flavone (Prosogerin C) (39)
Plant source Family Ref.
Calliandra Mimosaceae 52 californica Evo Lvulus Convolvulaceae 53 nummularius Prosopis spicigera Mimosaceae 54
Artemisia giraldii Compositaceae 55
Prosopis spicigera Mimosaceae 56
P. spicigera Mimosaceae 54
P. spicigera Mimosaceae 57
From Table-4 it is clear that all the 5-deoxyflavones with
trioxygenation in ring-B have oxygenation at 2':4':5' or 3':4':5' only.
R R, 33 OMe H
34 E-I OMe
R R, R, R3 K4
35 OH OH OH OH OH
36 OH OEI OMe OH OMe
37 OH OH OMe OMe OMe
38 OMe OH OMe OMe OMe
39 OMe OMe OMe OMe OMe
IlI. Structure Determination
The various methods which are generally employed for the structure
determination of 5-deoxyflavones are:
i. Physical methods
ii. Chemical methods
iii. Synthesis
i, Physical methods
The physical methods widely employed in the identification and
structure elucidation of 5-deoxyflavones are:
a. Ultraviolet spectroscopy
b. IR spectroscopy
c. 'El-NMR spectroscopy
d. I3C-NMIt spectroscopy
e. Mass spectroscopy
a. Ultraviolet spectroscopy
UV spectroscopy, a major technique for the structure analysis of
flavonoids, widely is used to distinguish 5-deoxyflavones from 5-
hydroxyflavoness8. Flavones with bdeoxygenation usually appear as bright
yellow / bluish green fluorescent spots under W light. 5-Deoxyflavones
exhibit two absorption bonds in the region 340-350 and 240-280 nm in band
I and band 11, as in the case of &oxygenated flavones but intensity of band
I1 absorption maxima in 5-deoxyflavones in methanol are usually very weak
than those of 5-hydroxyflavones58.
b. IR speetroseopy
5-Deoxyflavones are readily distinguished from 5-oxygenated flavones
from their IR spectra", as they show carbonyl absorption band in the region
1620-1650 cm" as distinct from 5-hydroxyflavones which usually exhibit
carbonyl absorption in the region 1650-1660 crne1.
R spectroscopy
In the structure elucidation of 5-deoxflavones certain useful
information can be obtained by comparison of their proton NMR spectra with
&oxygenated flavones. The chemical shift of H-5 proton of 5-deoxyflavones is
strongly deshielded by the 4-keto group and appears between 7.8-8.1 ppm. In
5-deoxy-7-oxygenated flavones the signals for both H-6 and H-8 occur at lower
field than in the 5,7-dihydroxyflavonoids5g. Moreover in 5-deoxyflavones the
H-8 may absorb a t either lower or higher field than the H-6 proton as
evidenced from 5,7-dihydroxy flavones, in which H-6 proton always absorbs
at higher field than the H-8 proton60.
R speetroseopy
13 C-NMR spectroscopy provides an elegant method for structure
establishment of 5-deoxyflavones. Lack of oxygenation at C-5 position will
have marked influence on the carbonyl carbon (C-4) resonance due to the
absence of intramolecular hydrogen bonding with carbonyl carbon. An upfield
shift of about +5 ppm of the C-4 resonance is typical of 5-deoxygenated
flavones6I.
e. Mass spectroscopy
The fragmentation pattern of 5-deoxyflavones is similar to 5-
hydrox$avones. The molecular ion peak is generally the base peak with
other major peaks corresponding to [M-HI', [M-CO]", A,'", [A,-CO]" and B,"
fragments in Chart - 1.
CHART - 1
ii. Chemical methods
Alkaline degradation, widely employed for the structure determination
of 5-oxygenated flavones may be employed for structure determination of 5-
deoxyflavones also.
a. Alkaline degradation
When 5-deoxyflavones are subjected to alkaline degradation with 50%
potassium hydroxide in methanol under nitrogen atmosphere for 5-8 h afford
2'-hydroxyacetophenone and benzoic acid derivatives.
The structure of abrectorin isolated from Abrus precatorius49 was
established as 7,3'-dihydroxy-6,4'-dimethoxyflavone (31), based on the
formation of 2,4-dihydroxy-5-methoxyacetophenone (31a) and 3-hydroxy-4-
methoxybenzoic acid (31b) when abrectorin was treated with 50% potassium
hydroxide (Chart - 2).
CHART - 2
OH OMe
50% KOH HOqL ~ Q M ~
MeOH, N,, 8h M e 0 HOOC /
0 31a 31b
0 31
Alkaline degradation of Abrectorin
iii. Synthesis
5-Deoxyflavones are preferably synthesised by Baker-Venkataraman
rearrangement. In this rearrangement ortho-hydroxyacetophenone derivative
is condensed with a substituted benzoyl chloride in the presence of potassium
carbonate. The resulting ester on treatment with potassium hydroxide in
pyridine is converted into a diketone which was cyclised in glacial acetic acid
and anhydrous sodium acetate yielded the corresponding 5-deoxyflavone.
Bhardwaj et als2 have carried out the total synthesis of prosogerin D
(38) by condensing 4-benzyloxy-2-hydroxy-5-methoxyacetophenone (40) with
tri-0-methylgalloylchlorde (41) in the presence of dry pyridine to give an ester
(42) which undergoes isomerization in the presence of pyridine-potassium
hydroxide to give 4-benzyloxy-2-hydroxy-5,3',4',5'-tetramethoxy
dibenzoylmethane (43). Cyclization of this P-diketone with acetic acid-soidum
acetate yielded 7-benzyloxy-6,3',4',5'-tetramethoxflavone (44) which on
catalytic (PdC) debenzylation in acetic acid resulted prosogerin D (38)
(Chart - 3).
Present work
ALbizzia odoratissima Benth is a large tree widely distributed in the
tropical India and Sri Lanka'. In traditional medicine, the bark of this plant
is used as a remedy for leprosy, ulcers and the leaves for coughs2s3.
Earlier chemical investigation on A. odoratissima has resulted in the
isolation of triterpenic acids63 and saponins from seeds"$' and flavonoids from
h e a r t w o ~ d ~ ~ .
A systematic chemical examination of the root bark of A. odoratissima
has now been undertaken as this part of this species has not been examined
earlier.
Chemical investigation of the root bark of Aodoratissima
The shade dried and powdered root bark (2 Kg) ofA. odoratissima was
successively extracted with n-hexane, acetone and methanol. Further work
up of methanol extract didn't yield any crystalline principle.
Examination of Wexane Extract
The hexane extract on concentration yielded dark green syrupy mass
(20 g). It was column chromatographed over silica gel using hexane-ethyl
acetate step gradient. The hexane-ethyl acetate, 9:1, 8:2 eluates yielded two
yellow solids and were designated as AO-1 and AO-2, respectively.
AO-1 was obtained as yellow amorphous powder (20 mg) from
methanol, mp 252-254°C. It gave negative ferric chloride test and orange
colour with Mg-HCI. I t was bluish green flourescent under UV and UV/NH,.
AO-1 showed [M-tKl', [M+NaIt and [M+Hl+ ions at mlz 365.0547,
349.0788 and 327.0983, respectively in its positive ESIMS (Fig. 1)
corresponding to the molecular formula C,,K,,O,. This was corroborated by
the decoupled 13C-NMR DEPT spectrum (Fig. 2) which showed signals for all
the eighteen carbons of the molecule. The UV absorption maxima of AO-1
(Fig. 3) in MeOH (254, 325 nm) suggested AO-1 to be a flavoneG7. The
addition of aluminium chloride and sodium acetate caused no shifts in its UV
spectrum indicating the absence of free hydroxyl groups a t C-5 and C-7,
respectively. The IR spectrum (Fig. 4) showed a carbonyl absorption band at
1648 and a aromatic C=C band at 1620 crnm1, respectively.
The 'H-NMR spectrum of AO-1 (Fig. 5) showed two rnethoxyl singlets at 6
3.97 and 4.0 I, a two-proton singlet at 6 6.05 assigned to a methylenedioxy group
and a sharp one-proton singlet at 6 6.61 ascribed to H-3. The EIMS of AO-1
(Fig. 6) showed two retro-Diels Alder fragments (Chart-4) at mlz 181 [A,+H]'
and 146 [B,]+ indicating the presence of two rnethoxyl groups in ring-A and a
methylenedioxy group in ring-B, respectively. The 'H-NMR spectrum of AO-1
further showed two ortho-coupled aromatic doublets at 6 7.91 and 7.01 and were
assigned to H-5 and H-6, respectively as the former showed correlations
with C-6, C-7, C-4, and C-8a, and the latter with C-4a, C-5, C-7 and C-8 in
its HMBC spectrum (Fig. 7). It also displayed three aromatic proton signals
at 6 7.50 (IH, dd, J = 8.2, 1.6 HZ), 7.36 (lH, d, J = 1.6 Hz) and 6.91 (IH, d,
J = 8.2 Hz) assigned to H-6', H-2' and H-5' respectively, characteristic of 3',
4'-dioxygenated f l a v ~ n e ~ ~ . This fixes the attachment of a rnethylenedioxy
group at 6 6.05 to 3' and 4' positions, further evidenced by the 3J correlation
of the methylene protons with (2-3' (6 148.4) and C-4' (6 150.4). Of the two
rnethoxyl groups in ring-A, the one at 6 3.97 was placed at C-8 as it resonated
at 61.6 ppm in its 13C-NM~ spectrum which is characteristic of a di-ortho-
substituted methoxyl group6'. This fixes the placement of another methoxyl
group at (6 4.01) to C-7, further supported by its NOESY spectrum (Fig. 8).
The NMR spectral assignments for AO-1 were further confirmed by HSQC
(Fig. 9 and Table - 51, HMBC (Fig. 7 and Table - 6), 'H-'H COSY (Fig. 10 and
Table - 7) studies.
Significant HMBC (-+-) and
NOESY (4-------b) correlations for 45
Thus from the foregoing spectral studies the structure of AO-1 was
characterised as 7,8-dimethoxy-3',4'-methylenedioxyflavon (45).
Table - 5: COSY lJcH (HSQC) Date of AO-1
Proton chemical shift (6)
7.91 (H-5)
7.50 (H-6')
7.36 (H-2')
7.01 (H-6)
6.91 (H-5')
6.61 (H-3)
6.05 (0CE120)
4.011 (OMe-7)
3.97 (OMe-8)
Correlated carbon chemical shift (6) Assignment
Table - 6: HMBC (2-3J,,) Correlations of AO-1
Proton
H-5
H-6'
H-2'
H-6
H-5'
H-3
OCH,O
OMe-7
OMe-8
Chemical shift (6)
7.91
7.50
7.36
7.01
6.91
6.61
6.05
4.01
3.97
Correlated carbon(s)
C-6, C-7, C-4, C-8a
C-2, C-2', C-5', C-4'
C-l', C-2, C-3', C-4'
(3-5, C-4a, C-7, C-8
6-l', C-4', C-3'
C-2, C-l', 6-4, C-4a
C-3', C-4'
C-7
C-8
Fig. 1 : ESI Mass Spectrum of AO-1
200 250 300 350 400
Wave length (nm)
Fig. 3 : UV Spectrum (MeOH) of AO-1
Table - 7: 'H-lH COSY Data of AO-1
Chemical shift of coupled protons Type of coupling Assignment
7.91 and 7.01
7.50 and 7.36
7.50 and 6.91
ortho
meta
ortho
H-5 and H-6
H-6' and H-2'
H-6' and H-5'
AO-2 was obtained as yellow amorphous solid (16 mg) from methanol,
mp. 128-130°C. It gave negative ferric chloride test and pale pink colour with
Mg-HC1. It was bluish green flourescent under W and WNH, .
AO-2 showed [M+H]+ ion peak at mlz 313.1000 in its positive ESIMS
(Fig. l l ) , consistent with the molecular formula C,,H,,O,, and was
corroborated by decoupled 13C-NMR DEFT spectrum (Fig. 12), which showed
signals for all the eighteen carbons of the molecule. The W absorption
maxima of 2 in MeOH at 238 and 339 nm (Fig. 13) is typical of a flavone".
The addition of aluminium chloride and sodium acetate didn't cause any shift
in its UV spectrum indicating the absence of free hydroxyl groups at C-5 and
(2-7, respectively. The IR spectrum (Fig. 14) showed two strong absorption
bands a t 1640 and 1600 ern-', due to carbonyl and aromatic C=C bonds,
The 'H-NMR spectrum of AO-2 (Fig. 15) displayed three methoxyl singlets
at 6 3.83, 3.86 and 3-87 and a sharp one-proton singlet at F 7.03 characteristic
of H-3 of 2'-oxygenated flavone70. The EIMS of AO-2 (Fig. 16) showed the
[W' ion peak at mlz 312 and two retro-Diels Alder fragments (Chart-5) at
mlz 151 [A,+H]+ and 162 [BJ' consistent with the presence of one methoxyl
group in ring-A and two methoxyl groups in ring-B, respectively. The 'H-NMR
spectrum of AO-2, further revealed two aromatic ABX spin coupled systems,
at 6 8.07 (IH, d, J = 8.8 Hz), 6.90 (1H, dd, J = 8.8, 2.3 Hz) and 6.85 (lH, d,
J = 2.3 Hz) assigned to H-5, H-6 and H-8, and 6 7.82 ( lH, d, J = 8.1 Hz), 6.57
(IH, dd, J = 8.1, 2.3 Hz) and 6.50 (lH, d, J = 2.3 Hz) assigned to H-6', H-5'
and H-3', confirming the presence of mono-substitution in ring-A and
disubstitution in ring-B. The three methoxyl groups at 6 3.83, 3.86 and 3.87
were placed at C-4', C-2' and C-7 positions as these methoxyls protons showed
correlations with these carbons at 163.0, 159.4, 163.8 ppm respectively in its
HMBC spectrum (Fig. 17). The placement of methoxyl groups were further
supported by the NOE studies (Fig. 18). The NMR spectral assignments were
further confirmed by HSQC (Fig. 19 and Table - 8), HMBC (Fig. 17 and Table
- 9) and *H-'H COSY (Fig. 20 and Table - 10) studies.
Significant HMBC (+) and
NOESY (4------+) correlations for 46
Thus from the foregoing spectral studies compound AO-2 was
established as 7,2',4'-trimethoxyflavone (46). The isolation of AO-2 constitutes
the first report of the occurrence of a 2'-oxygenated flavone from Albizia
genus.
Table - 8: 'H-"C-COSY (HSQC) Data of AO-2
Proton chemical shift Correlated carbon (6) chemical shift (6)
8.07 (H-5) 126.8
7.82 (H-6') 130.2
7.03 (H-3) 111.1
6.90 (H-6) 113.9
6.85 (H-8) 100.2
6.57 (H-5') a
105.2
6.50 (H-3') 98.8
3.87 (OMe-7) 55.7
3.86 (OMe-2') 55.6
3.83 (OMe-4') 55.7
Assignment
Table - 9: HMBC (2-3~c,) Correlations of AO-2
Proton
H-5
H-6'
H-3
H-6
H-8
H-5'
H-3'
OMe-7
OMe-2'
OMe-4'
Chemical shift (6)
8.07
7.82
7.03
6.90
6.85
6.57
6.50
3.87
3.86
3.83
Correlated carbon(s)
C-4, C-6, C-7, C-8a
C-2, C-2', C-4'
C-l', C-2, 6-4, C-4a
C-4a, C-7, C-8
C-4a, (3-6, C-7, C-8a
C-4', C-3', C-1'
C-l', C-2', C-4', (3-5'
C-7
C-2'
C-4'
IZE'BLI Wdd
200 250 300 350 400
Wave length (nm)
Fig. 13 : UV Spectrum (MeOH) of AO-2
I-" -. .-
1 3 0
' ' I " ' ' r *
0
v,
' . . . , . . ,
. , , , , , , , , , , , , , , , , . , , I . . . I , ,
Table - 10: 'I3-lE-I COSY Data of AO-2
chemical shift of coupled protons ( 6 ) Type of coupling Assignment
8.07 and 6.90 ortho H-5 and H-6
6.90 and 6.85 meta H-6 and H-8
7.82 and 6.57 H-6' and H-5'
6.57 and 6.50 meta H-5' and H-3'
Examination of Acetone Extract
The acetone extract ofA. odoratissima was concentrated under reduced
pressure to give dark brown gummy mass (25 g). It was segregated into
n-hexane soluble (10 g) and insoluble fraction (15 g). The hexane soluble
fraction on further workup didn't yield any crystalline principle.
The hexane insoluble fraction (15 g) on purification over a silica gel
column using hexane-ethyl acetate (7:3) as eluent furnished yellow solid, and
crystallized from methanol to give yellow needle and it was designated as
AO-3.
(Tithonine, 27)
AO-3 was crystallized from methanol as yellow needles (20 mg) mp
190-192°C. I t gave dark green colour with alcoholic ferric chloride and orange-
red colour with Mg-HC1. It was analysed for C,,H,,O, which is consistant
with the molecular ion at mlz 298 in its EI mass spectrum (Fig. 21) and
was corroborated by the presence of seventeen carbon signals in its 13C NMR
spectrum (Fig- 22). Its W absorption maxima (Fig. 23) at 235 and 338 nm
closely resembled that of a 5-deoxyflavone derivative6?. Addition of sodium
acetate didn't cause any change in band I1 absorption maximum indicating
the absence of a free hydroxyl a t (2-7. The IR spectrum (Fig. 24) showed two
strong absorption bands at 3278 and 1643 cm-' due to hydroxyl and
conjugated carbonyl functions, respectively.
The 'H NMR spectrum (Fig. 25) of AO-3 showed a downfield signal at
6 9.44 assignable to a phenolic hydroxyl group. It also showed signals for two
methoxyl groups (6 3.84 and 3.88) and six aromatic protons (6 7.89, 7.51,
7.43, 7.2 1, 7.03 and 7.01). Six aromatic protons appearing as two sets of ABX
type s i w a l s a t 6 7.51 (IH, dd, J = 8.9, 1.5 Hz), 7.43 (IH, d, s, H-2', J = 1.5
Hz), and 7.03 (lH, d, J = 8.9 Hz); 7.89 (lH, d, J = 8.9 Hz), 7.01 (IH, d, J =
1.5 Hz) and 7.21 ( lH, d, J = 1.5 Hz) were assigned to H-6', H-2', H-5' and H-
5, H-6, H-8, respectively. A sharp one proton singlet at 6 6.70 was assigned
to H-3 proton of flavone. Two retro-Diels Alder fragments at mlz 150 and
148, in its EI mass spectrum (Fig. 26) indicated the presence of a rnethoxyl
group in ring-A, and a hydroxyl and a methoxyl group in ring-B, respectively.
The methoxyl groups at 6 3.88 and 3.84 were placed at C-7 and C-4'
positions, based on the absence of bathochromic shift of the band-I1 and band-
I W absorption maxima of AO-3 with sodium acetate and sodium methoxide,
respectively.
Thus the structure of AO-3 was elucidated as 7,4'-dimethoxy-3'-
hydroxyflavone (tithonine) (27) by comparison of its spectral data with
literature v a l ~ e s ~ ~ > ~ ~ ' ~ ~ ~ ~ ~ .
LLB'L L06 ' L
EXPERIMENTAL
The root bark ofA. odoratissima used in the present investigation was
collected from Tirupati, Andhra Pradesh, India.
The dried and ground root bark of A. odoratissima (2 Kg) was
successively extracted with n-hexane (3 x 51), Me, CO (3 x 51) and MeOH (3
x 51). The MeOH extract on further workup didn't yield any crystalline
principle.
Examination of Hexane Extract
The hexane extract on concentration under reduced pressure gave a
dark green syrupy mass (20 g). It was found to be a mixture 'of two
components on preliminary TLC examination and was subjected to column
chromatography over silica gel (200 g) using hexane-ethyl acetate as eluent.
A total of 25 fractions of 50 ml each were collected by slow elution and the
column fractions were monitored by TLC (silica gel) using hexane-ethyl
acetate 9:1, 8:2 as solvent systems and alcoholic ferric chloride as the
detecting agent (Table - 11).
Fraction No. Eluent Nature of the Eluate
1-5 Hexane Fatty material (negligible)
6- 11 Hexane-EtOAc (9: 1) Yellow solid (25 mg)
12-15 Hexane-Et OAc (9: 1) No residue
16-20 Hexane-EtOAc (8:2) Yellow solid (20 rng)
21-25 Hexane-EtOAc (1: 1) No residue
The yellow solid (25 mg) obtained from fractions 6-11 on crystallization
from methanol afforded yellow amorphous powder (20 mg), rnp 252-254OC. It
was designated as AO-1.
Fractions 16-20 on concentration yielded yellow solid (20 mg) which on
crystallization from MeOH gave yellow amorphous powder (16 mg), mp 128-
130°C. It was designated as AO-2.
AO-1 was crystallized from MeOH as yellow amorphous solid (20 mg);
mp 252-254'6. It gave negative ferric chloride test and orange colour with
Mg-HCl. It was bluish green flourescent under UV and UV/NH,.
UV: h max (MeOH) (log E) 254 (4.231, 325 (4.14) nm.
IR: v max (KBr) 1648 (>C=O), 1620, 1596, 1544,1490 cm-l.
R: (400 MHz, CDCl,) 6 7.91 (lH, d, J = 8.9 Hz, H-5), 7.50 (IH, dd, J
= 8.9, 1.6 HZ, H-60, 7.36 (lH, d, J = 1.6 HZ, H-29, 7.01 (lH, d, J = 8.9 HZ, H-
6), 6.91 (lH, d, J = 8.2 HZ, H-5'), 6.61 (lH, S, H-3), 6.05 (2H, S, -0-CH,-0-),
4.01 (3H, s, OMe-7), 3.97 (3H, s, OMe-8).
R: (75 MHz, CDC1,) 6 178.0 (C-4), 162.6 (C-2), 156.6 (C-7), 150.5 (C-
8a), 150.4 ((2-47, 148.4 (C-3'), 136.9 (C-8), 125.8 (C-1'1, 121.4 (C-6'), 120.9 (C-
51, 118.6 (C-4a), 109.8 (C-6), 108.8 (G-5'), 106.3 (C-2'1, 105.9 (C-3), 101.9
(OCH,O), 61.6 (OMe-8), 56.4 (OMe-7).
ESIMS: m/z (positive ion mode) [M+Hl+, 327.0983 (calc. for C,,H,,O,:
327.3015).
Finally AO-1 was characterized as 7,8-dimethoxy-3',4'-
methylenedioxyflavone (45).
AO-2 was crystallized from MeOH as yellow amorphous solid (16 mg),
mp 128-130°C. It gave negative ferric chloride and pale pink colour with Mg-
HC1. I t was bluish green flourescent under UV and UVINH,.
UV: h max (MeOH) (log E) 238 (4.18), 339 (4.03) nm.
XR: v max (KBr) 1640 (>C=O), 1600, 1509, 1440, 1376 crnml.
R: (400 MHz, CDC1,) 6 8.07 (IH, d, J = 8.8 HZ, H-5), 7.82 (lH, d, J =
8.7 HZ, H-6'), 7.03 (lH, S, H-3), 6.90 (lH, dd, J = 8.8, 2.3 HZ, H-6), 6.85 (IH,
d, J = 2.3 Hz, H-8), 6.57 (lH, dd, J = 8.7, 2.3 HZ, H-5'1, 6.50 (lH, d, J = 2 . 3
Hz, H-3'), 3.87 (3H, s, OMe-7), 3.86 (3H, s, OMe-27, 3.83 (3H, s, OMe-4').
R: (75 MHz, CDCI,) G 178.3 (C-4), 163.8 (C-7), 163.0 (C-4'), 160.4 (C-
21, 159.4 (C-2'), 158.0 (C-8a), 130.2 (C-6'), 126.8 (C-5), 117.6 (C-4a), 113.9 (C-
6), 113.5 (C-1'), 111.1 (C-3), 105.2 (C-5'), 100.2 (C-8), 98.8 (C-3% 55.7 (OMe-71,
55.6 (OMe-2'), 55.5 (OMe-4').
ESIMS: (positive ion mode) mlz [M+H]+ 313.1000 (calc. for C,,H,,O,:
313.3239).
Finally, AO-2 was characterized as 7,2',4'-trimethoxyflavone (46).
Examination of Acetone Extract
The acetone extract was concentrated to yield a dark brown gummy
mass (25 g). It was defatted with n-hexane. The residue left behind (15 g) was
column chromatographed over silica gel using step gradient of hexane and
ethyl acetate. A total of 20 fractions were collected and the column fractions
were monitored by TLC (silica gel) using hexane-EtOAc (7:3) as the solvent
system and alcoholic ferric chloride as detecting agent. The column
chromatographic details are shown in Table - 12.
TABLE - 12
Fraction No. Eluent Nature of Eluate
1-5 Hexane Fatty material
6-10 Hexane-E t OAc (8: 2) No residue
11-15 Hexane-EtOAc (7:3) Colourless solid (30 mg)
16-20 EtOAc No residue
The colourless solid (30 mg) obtained by evaporation of hexane-EtOAc
(7:3) eluates on crystallization from MeOH furnished colourless needles (25
mg) and was designated as AO-3.
A 0 -3
(Tithonine, 27)
AO-3 was crystallized from MeOH as colourless needles (25 mg), rnp
190-192°C. It gave dark green colour with alcoholic ferric chloride, orange
colour with Mg-HC1.
W: hmax (hTeOH) (log E): 235 (4.411, 314 sh, 338 (4.20) nm.
IR: v m a . ( D r ) 3278 (OH), 2972,2840,1643 (>C=O), 1602,1511,1440,1379
cm-l.
1: (300 MHz, DMSO-d,) 8 9.44 (IH, S, OH-3'), 7.89 (IH, d, J = 8.9 Hz,
H-5), 7.51 ( lH, dd, J = 8.9, 1.5 HZ, H-67, 7.43 (lH, d, J = 1.5 Hz, H-Z'), 7.21
(IH, d, J = 1.5 HZ, H-8), 7.03 (lH, d, J = 8.9 HZ, H-5'), 7.01 (lH, dd, J = 8.9,
1.5 Hz, H-6), 6.70 (IH, s, H-3), 3.88 (3H, s, OMe-7), 3.84 (3H, s, OMe-4').
EIMS: mlz (rel. int. %) 298 (loo), 283 (5), 270 (4), 255 ( l l ) , 165 (lo), 151
(32).
Finally the identity of AO-3 as 7,4'-dimethoxy-3'-hydroxyflavone
(tithonine) (27) was confirmed by comparing with literature values of
tithonine.
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