Transformation of Azadiradione to Nimbocinol and 17β -Hydroxynimbocinol, and Structure-Pesticidal-Activity Relationship of Triterpenoids isolated from Azadirachta indica A. Juss. (Neem)

Article abstract of DOI:10.1002/hlca.200390277

During studies on the structure-pesticidal-activity relationship of Azadirachta indica constituents, azadiradione (1) was treated with methanolic K₂CO₃ to obtain 7-deacetyl derivative nimbocinol (2). Unexpectedly, 17β-hydroxynimbocinol (3), reported earlier as a natural product, was also formed. The structure-pesticidal-activity relationship of triterpenoids 1-16 against Anopheles stephensi Liston is described towards understanding the active functionalities responsible for the pesticidal activity of triterpenoids having apoeuphane(apotirucallane) and gedunin skeleta. The pesticidal activities of triterpenoids 2-8 are reported for the first time.

Full text of DOI:10.1002/hlca.200390277

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Helvetica Chimica Acta Vol. 86 (2003)
Transformation of Azadiradione to Nimbocinol and 17b-Hydroxynimbocinol,
and StructureÀPesticidal-Activity Relationshipof Triterpenoids isolated
from Azadirachta indica A. Juss. (Neem)
by Bina S. Siddiqui*a), Munawwer Rasheeda), Shaheen Faizia), Firdousa), S. Tariq Alia), Rajput M. Tariqb),
and Syed Naeem-ul-Hassan Naqvic)
a) HEJ Research Institute of Chemistry, International Center for Chemical Sciences, University of Karachi,
Karachi-75270, Pakistan (phone: 92-21-9243199; fax: ‡‡ 92-21-9243190, -91; e-mail: bina@khi.comsats.net.pk)
b) Department of Zoology, University of Karachi, Karachi-75270, Pakistan
c) Department of Pharmacology, Baqai Medical University, Toll Plaza, Karachi, Pakistan
During studies on the structureÀpesticidal-activity relationship of Azadirachta indica constituents,
azadiradione (1) was treated with methanolic K2CO3 to obtain 7-deacetyl derivative nimbocinol (2).
Unexpectedly, 17b-hydroxynimbocinol (3), reported earlier as a natural product, was also formed. The
structureÀpesticidal-activity relationship of triterpenoids 1 16 against Anopheles stephensi Liston is described
towards understanding the active functionalities responsible for the pesticidal activity of triterpenoids having
apoeuphane(apotirucallane) and gedunin skeleta. The pesticidal activities of triterpenoids 2 8 are reported for
the first time.
1. Introduction. Azadirachta indica A. Juss. (Syn. Melia indica Brandis; Melia
azadirachta Linn.), known commonly as neem (Urdu) and Indian lilac (English),
belongs to the Meliaceae family (order Rutales). Neem, a tropical to subtropical plant,
is native to the southern part of Asia, particularly Southeast Asia, including Pakistan. It
was introduced in Africa in the beginning of the last century and is now widely
cultivated in more than 30 countries, particularly along the Sahara×s southern fringe.
Over the last century or so, plantations of the tree have also been established in Fiji,
Mauritius, the Caribbean and many countries of Central and South America and the
USA. The tree, with an average lifespan of more than 200 years, starts fruiting after 3 to
5years and can produce up to 50 kg of fruit annually. The ripe, ellipsoidal drupe (fruit)
measures up to almost 2 cm in length, comprising sweet pulp and a seed covered in a
smooth yellow or yellowish-green coat [1].
Medicinal uses of various parts of neem are manifold and have been particularly
ascribed to its leaves, fruit, oil, and bark. Its different parts are highly reputed in
folklore and traditional medicine for the treatment of a variety of human ailments and
have been inherited as a tradition in the Indo-Pak culture [2][3]. Neem has been
studied for several decades for insecticidal and insect-repellant properties and the
chemical constituents responsible [1 10]. Since the report of azadirachtin, perhaps the
most outstanding potent, naturally occurring pesticide [11], scientists have begun to
undertake systematic chemical analyses to isolate and identify the biologically active
constituents, monitoring their entomological properties and have found that neem
botanicals can affect a broad spectrum of pests, including insects, nematodes, fungi,

Helvetica Chimica Acta Vol. 86 (2003)
3343
bacteria, and even a few viruses. Unlike ordinary insecticides based on a single active
ingredient, neem botanicals contain several complex arrays of novel, active constit-
uents that collectively act in more than one way to produce abnormal insect
development and molting, hormonal disturbances, growth inhibition, antifertility,
antifeeding, and oviposition, but not as outright killers [1][4 6][9][10].
Chemical studies of neem×s active principles have led to the isolation of a host of
chemical constituents, characterization and structureÀactivity-relationship studies of
rearranged and modified azadirachtins against Spodoptera littoralis [12][13], synthetic
analogues of azadiradione (1) [14] and epoxyazadiradione (9) [15], and other
limonoids [16 18].
Anopheles stephensi Liston, a malarial-parasite-carrier mosquito was used in the
present study as it is the second-most-common anopheline in this region [19][20],
where malaria has been estimated to represent 2.3% of the overall global-disease
burden [21].
K2CO3 is ideal for selective deprotonation of acids and, in combination with various
solvents, has been used by investigators for the alkylation of phenols and 1,3-dicarbonyl
compounds, deamination of b-amino esters to the corresponding alcohols, and
epoxidation of amides with an a,b-aminoalcohol moiety, and to obtain intramolecular
Michael adducts [22]. Methanolic K2CO3 has been used for the detigloylation of
analogues obtained in oxidative studies on 11-acetoxy-22,23-dihydroazadirachtin [23]
and to convert allylic esters to the corresponding alcohols [24].
2. Results and Discussion. Systematic investigation of the ethanolic extract of the
fruit coat [25][26] following a pesticidal-activity-guided isolation, with monitoring of
An. stephensi Liston mortality, led to the isolation of five known triterpenoids,
azadirone (4) [27], deoxygedunin (5) [28], gedunin (6) [28], a-nimolactone (7) [29],
and b-nimolactone (8) [29], and the identification of various other natural products
[30]. The pesticidal activity (LC50) of these constituents determined against fourth-

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Helvetica Chimica Acta Vol. 86 (2003)
instar larvae of An. stephensi by the WHO method is compared with that of
azadiradione (1), epoxyazadiradione (9), desfuranoazadiradione (10), meliacinin (11),
azadironic acid (12), limocin A and B (13 and 14), isolated earlier from fruit coats [25],
and desfurano-6a-hydroxyazadiradione (15) and 22,23-dihydronimocinol (16) isolated
from leaves [31] towards gaining insight into the effect of structure on pesticidal
activity. The availability of these triterpenoids, each having the basic apoeuphane-
(apotirucallane) or gedunin skleton, provided us with an opportunity to understand the
active phores. To extend these studies to other analogues, 1 was hydrolyzed with
methanolic K2CO3, which afforded nimbocinol (2) [14][32] along with 17b-hydroxy-
nimbocinol (3), both of which are previously reported natural products [14][33].
A complete assignment of 1H and 13C nuclei of 3 has been made through
1
1
spectroscopic methods, including H- and 13C-NMR (BB and DEPT), J-resolved, H-
13C COSY, COSY-458, NOESY, HMQC, HMBC (Fig. 1), which has led to the
reassignment of some of the nuclei, including HÀC(5), Ha/bÀC(6), HÀC(9),
Ha/bÀC(11), Ha/bÀC(12), HÀC(21), HÀC(23), C(5), and C(9) (Table 1). The known
compounds have been identified through comparison of their physical and spectral data
with those reported in the literature [27 29][34][35].
Formation of 17b-Hydroxynimbocinol (3). The formation of 3 can be inferred on
the basis of the acidity of HÀC(17), which is also evident from the epimerization at
C(17) via enolization of 16-keto analogues in strong alkaline medium, e.g., in the
formation of 17-epinimbocinol [14]. Although methanolic K2CO3 is not strong enough
to cause complete enolization, it can readily oxidize the substrate carrying acidic
protons with O2 (analogous to 2-nitropropane, which autoxidizes in weakly but not
strongly basic solution). Auto-oxidation reactions are slow, and some of them seem to
proceed in the absence of the photosensitizer that would be required by classical theory
for the generation of singlet oxygen (1O2) [36][37]. Such free-radical reactions, called
electron-detachment- and -attachment reactions (ED/EA in the Scheme), are more-
favorable when the radical is formed at a tertiary C-atom, since the relative reactivity
for abstraction of H-atoms from tertiary aliphatic C-atoms is generally 4 6 times
higher than that from secondary aliphatic C-atoms. In the present case, electron
delocalization in the furan ring may stabilize the radical formed. Further, conforma-
tional strain contributes to retention of the a-orientation of furan [38][39].
StructureÀPesticidal-ActivityRelationships .
Neem constituents represent a
variety of structural diversity with varying order of biological activities. In the present
studies, their activities against An. stephensi were compared. The main triterpenoids
(tetranortriterpenoids) azadiradione (1), azadirone (4), and epoxyazadiradione (9),
e.g., possess a common ring A with 1-en-3-one system, an acetoxy substituent at C(7),
and a furan ring at C(17), but vary in ring D. The above compounds display LC50 values
of 15, 10, and 18 ppm, respectively (Fig. 2).
However, in gedunin (6), in which ring D is cleaved, the toxicity decreases to LC50
120 ppm. The same trend is observed in deoxygedunin (5; LC50 150 ppm), which also
has a cleaved ring D. The furan ring also contributes to the toxicity of neem compounds,
as could be inferred from the diminished toxicity of desfurano compounds relative to
azadiradione, like in a-nimolactone (7; LC50 60 ppm), b-nimolactone (8; LC50
45ppm), desfuranoazadiradione ( 10; LC50 37 ppm), and desfurano-6a-hydroxyazadi-
radione (15; LC50 43 ppm), as well as the 22,23-dihydronimocinol (16; LC50 60 ppm).

Helvetica Chimica Acta Vol. 86 (2003)
3345
Fig. 1. Spectroscopic data of 3. Top : Mass fragmentation pattern; middle: COSY-458 (bold lines), and NOE
correlations (arrows); bottom: HMBC (arrows) and HMQC (bold lines).
Oxidation of the furan ring to an acetal ring on the other hand, does not give rise to any
considerable alteration in activity (e.g., limocin A and B (13 and 14): LC50 19 ppm).
A further observation is that the presence of an a,b-unsaturated open-chain
carbonyl moiety or double bond (D20,22) in an open chain increases the activity

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Helvetica Chimica Acta Vol. 86 (2003)
Table 1. 1H-,13C-, and 1H,1H-COSY-458 Data (CDCl3) of 3. Literature data of 17b-hydroxyazadiradione
[34][35] included for comparison. Chemical shifts (d) in ppm, coupling constants (J) in Hz.
3
17b-Hydroxyazadiradione
d(H)
d(C) COSY-458
d(H)
d(C)
HÀC(1)
HÀC(2)
C(3)
7.11 (d, J ˆ 10.2)
5.83 (d, J ˆ 10.2)
157.5 5.83
125.7 7.11
204.7
7.16 (d, J ˆ 10.0)
5.89 (d, J ˆ 10.0)
157.15
125.82
204.11
44.07
C(4)
44.1
HaÀC(5)
HaÀC(6)
HbÀC(6)
HbÀC(7)
C(8)
2.42 ( J ˆ 13.0, 2.4)
44.4 1.99
25.5
25.5
71.7 1.83, 1.99, 2.42
46.8
36.9 1.80
40.2
15.6 1.58, 1.80
15.6 1.58, 1.80
22.2 1.80, 2.52, 1.58, 1.80, 2.03 2.12 1.55
22.2 1.80, 2.52, 1.58, 1.80, 2.03 2.12 1.55
50.4
2.20
38.81
22.39
1.83 (J ˆ 15.3, 2.7, 2.4)
1.99 (J ˆ 15.3, 13.0, 2.7)
4.16 (J ˆ 2.7, 2.7)
2.12 1.55
2.12 1.55
5.33
74.20
44.85
46.02
39.95
15.89
HaÀC(9)
2.35( J ˆ 12.1, 6.7)
2.24
C(10)
HaÀC(11) 2.03 (J ˆ 13.8, 9.6, 6.7, 3.4)
HbÀC(11) 1.80 (J ˆ 13.8, 12.1, 9.7, 3.2)
HaÀC(12) 1.58 (J ˆ 13.8, 9.7, 3.4)
HbÀC(12) 2.52 (J ˆ 13.8, 9.6, 3.2)
C(13)
C(14)
C(15)
C(16)
C(17)
2.12 1.55
2.12 1.55
23.50
50.36
193.42
120.33
205.84
80.67
195.4
120.2
206.2
80.7
5.87 (s)
5.78
Me(18)
Me(19)
C(20)
HÀC(21)
HÀC(22)
HÀC(23)
Me(28)
Me(29)
Me(30)
C(1')
0.93 (s)
1.19 (s)
30.2 2.52
19.1
1722.5
141.4 7.38, 6.37
109.57.38, 47.5
142.8 6.37, 7.54
21.4
0.96 (s)
1.23 (s)
30.84
19.08
122.5
6.39
7.54 (dd, J ˆ 1.84, 0.9)
6.37 (dd, J ˆ 1.69, 0.9)
7.38 (dd, J ˆ 1.84, 1.69)
1.08 (s)
1.13 (s)
1.27 (s)
7.58
142.62
109.63
7.42
141.42
21.22
27.01
25.16
169.69
20.96
1.09 (s)
1.07 (s)
1.35
27.1
25.5
Me(2')
17b-OH
1.94
3.43 (s)a)
a) Exchangeable with D2O.
significantly. This was exhibited by meliacinin (11; LC50 13 ppm, with CˆC in the side
chain at C(17)), azadironic acid (12; LC50 4.5ppm, with a,b-unsaturated carbonyl side
chain at C(17)), and its methyl derivative (12a; LC50 2.8 ppm).
Moreover, deacetylation at C(7) decreases the activity as noted in nimbocinol (7a-
deacetylazadiradione, 2; LC50 30 ppm). Hydroxylation at C(17), on the other hand
(7a-deacetyl-7a,17b-dihydroxyazadiradione, 3; LC50 15ppm), results in increased
activity, identical to that of 1 (Fig. 2). This relationship of the activities in compounds 1,
2, and 3 is analogous to earlier observations of pesticidal activity against Heliothis
virescens [33].
Replacement of the OH group by a MeO group at C(17) does not alter the activity
notably, as shown by 11 (17b-OMe) and 3 (17b-OH), and the same can be inferred from

Helvetica Chimica Acta Vol. 86 (2003)
3347
Scheme. Proposed ED/EA Pathwayfor Oxidation of 1 to 3 (D-ring)
15 and 16, which have an a-hydroxy substituent at C(6). These studies also reflect that
the right-hand part of the skeleton make an important contribution to the activity of
these limonoids, as alterations brought about in this part exhibit a notable change in the
order of activity.

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Helvetica Chimica Acta Vol. 86 (2003)
Fig. 2. Triterpenoids 1 16: structures and LC50 values for pesticidal activityagainst An. stephensi
¾
A synthetic pesticide of pyrethroid class, permethrin (Coopex EC 2.5%) was also
assayed for pesticidal activity in parallel. Its LC50 value against fourth instar larvae of
An. stephensi was 0.125ppm.

Helvetica Chimica Acta Vol. 86 (2003)
3349
Experimental Part
General. UV spectra: MeOH solns.; Hitachi U-3200 spectrophotometer; lmax in nm. FT-IR-ATR
(Attenuated Total Reflectance) Spectra: CHCl3 solns.; Bruker Vector-2000-FTIR spectrophotometer, with
.
Opus (Version 3.0) software; n in cmÀ1 1H-NMR Spectra: CDCl3 solns.; Bruker Avance-400 spectrometer at
400 MHz; 13C-NMR, DEPT, COSY-458, NOESY, J-resolved, HMQC, and HMBC experiments: Bruker Avance-
500 spectrometer at 500 MHz; d in ppm rel. to SiMe4, J in Hz. 1H-13C COSY Spectra: Bruker Aspect-300
instrument at 300 MHz. Spectral assignments made on the basis of combined spectroscopic data. LR-EI-MS and
HR-EI-MS; double-focussing Finnigan MAT-312 and Jeol JMS-HX-110 mass spectrometers, resp.; ion source at
2508 and 70 eV; m/z (rel. %). Elemental analyses: Carlo-Erba 1106 instrument.
Anal. TLC and prep. thin-layer chromatography (prep. TLC): Merck Kieselgel 60 PF254 coated on glass
plates; detection by UV illumination (254 and 366 nm) and by iodine spray. Vacuum liquid chromatography
(VLC): Merck Kieselgel 60. Flash column chromatography (FC): Eyela model EF-10 or Aldrich flash column
chromatograph; Merck 9385 silica gel, 0.040 0.063 mm. Prep. HPLC: Shimadzu LC-8A; UV SPD-6A detector
(at 254 nm); Shimpack PREP ODS column (No. 2025752, 250 mm  22 mm i.d.; Shimadzu, Kyoto, Japan);
mobile phase 50 90% aq. MeOH; flow rate 50 120 ml/min in steps.
Bioassay-Guided Extraction and Isolation. Plant material and bioassay-guided extraction and isolation of
compounds has been described for the thirteen VLC fractions −A× to −M× in a previous communication [25].
Fractions −A× to −C× were found to contain constituents other than triterpenoids [30]. The mother liquor of
−D× after crystallization of 9, on prep. TLC (silica gel, petroleum ether/AcOEt 70 :30, 254 nm) afforded
azadirone [27] (4; 26 mg). Fractions −E× afforded 1 and 9 in equal amounts. Fractions −F× (petroleum ether/
AcOEt 50 :50 (2) eluate) and −G× [petroleum ether/AcOEt, 40 :60 (2) eluate] were combined, dissolved in a
minimum quantity of AcOEt and poured into excess petroleum ether to precipitate limonoids. The clear
supernatant that separated on standing overnight was decanted, and the insoluble residue separated by prep.
TLC (silica gel, petroleum ether/AcOEt 60 :40) to afford 1, 9, and 5 [28]. Fractions −D× through −G×, showing one
or two major spots on anal. TLC were further purified to give compounds 1, 4, 5, and 9. The pesticidal activities
of 1 and 9 have been reported earlier [25], while those of 4 and 5 are reported in the present communication.
Fraction −L× (1.53 g), the most-active fraction, was subjected to FC to afford 16 fractions (LFC-1 LFC-16).
Three of the fractions eluted with a gradient from CHCl3/MeOH 99 :1 to CHCl3/MeOH 95.5 :4.5 (elution vol.,
1600 ml), i.e., fractions −LFC-6× (181.9 mg; LC50 23.5ppm) and −LFC-7× (41 mg; LC50 25.3 ppm), and −LFC-8×
(70.2 mg; LC50 19.2 ppm) were combined to give 293.1 mg, that was resubmitted to FC on an Aldrich
chromatograph (silica gel, CHCl3, CHCl3/MeOH, and MeOH in order of increasing polarity). The fractions
were finally combined on the basis of TLC to give 22 fractions, −LFC-601× to −LFC-622×.
Fraction −LFC-611× (27 mg), which was eluted with CHCl3/MeOH, 99 :1 (elution vol. 750 ml), on further purifi-
cation by prep. TLC, afforded pure gedunin [28] (6; 22 mg; LC50 120 ppm). Fraction −LFC-618× (38 mg), which eluted
with CHCl3/MeOH, 96 :4 (elution vol. 250 ml) and 95 :5 (elution vol. 500 ml) furnished pure a-nimolactone [29]
(7; 12 mg) and b-nimolactone [29] (8; 16 mg) on prep. TLC (silica gel, CHCl3 with a few drops of MeOH)].
17b-Hydroxynimbocinol (ˆ21,23-Epoxy-7a,17b-dihydroxy-24,25,26,27-tetranorapotirucalla-1,14,20,22-tet-
raene-3,16-dione; 3). Hydrolysis of 1: Azadiradione (1; 100 mg, 0.222 mmol) was dissolved in a minimum
amount of MeOH. To this soln., a super-sat. soln. of methanolic K2CO3 (10 ml, along with residual insoluble
alkali) [23][24] was added, and the mixture stirred vigorously at r.t. for 8 h. The reaction was monitored by anal.
TLC for the disappearance of 1, poured into cold sat. aq. NH4Cl (20 ml), and extracted with AcOEt (3Â). The
AcOEt layer was dried (anh. Na2SO4), evaporated, and purified by reverse-phase prep. HPLC (RP-18, MeOH/
H2O 30 :70) to furnish crystalline nimbocinol (deacetylazadiradione; 2, 60 mg) and 17b-hydroxynimbocinol (3,
11.4 mg) in a ratio of 5.2 :1.
Data of 3: Needle-like crystals (11.4 mg). M.p.: 2388 (dec.). ‰aŠ2D7 : ‡ 54.84 (c ˆ 0.248, MeOH). UV: 238. IR:
3600 3200 (OH), 1698, 1665(RCH ˆCHCOOR, RCHˆCHCOR), 1595 (CˆC), 1454, 1386 (Me2C), 1159,
1033 (CÀO), 870 (trisubstituted CˆC, furan). 1H- and 13C-NMR and COSY-458: see Table 1. HR-EI-MS:
‡
‡
‡
‡
‡
424.2251 (38, C26H32O5, M ), 409.1996 (28, C25H29O5 , [M À Me] ), 406.2117 (3.5, C26H30O4 , [M À H2O] ),
‡
‡
‡
‡
391.1927 (5, C25H27O4 , [M À Me À H2O] ), 329.2117 (11, C21H29O3 , d), 229.1154 (2, C15H17O2 ), 149.0978 (12,
‡
‡
‡
C10H13O , a), 137.0982 (12, C9H13O , b), 95.0150 (100, C5H3O2 , c) (Fig. 1). Anal. calc. for C26H32O5 (424): C 73.6,
H 7.6; found: C 71.8, H 8.6.
Pesticidal Activity: Rearing: The fourth-instar larvae of An. stephensi Liston (Orangi Town Wild Strain), a
vector of the malarial parasite, were collected directly from the natural environment especially established for
this research work. The size of the pond was 2.4 Â 1.2 m with a depth of 0.6 m. The pupae from the pond were
collected and kept in cages for hatching.

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Helvetica Chimica Acta Vol. 86 (2003)
PreliminaryScreening Procedure: Ten young fourth-instar larvae of An. stephensi were collected in 5ml of
tap water and transferred to a 100 ml glass beaker containing 45ml of distilled water. The fractions were tested
at 28 Æ 18 at five final concentrations. Controls were similarly prepared. Each concentration and control was run
as a duplicate set, and mortality was recorded after 24 h. See Table 2.
Table 2. LC50 Values Determined for Fractions from A. indica Fruit Coats and Seeds against Fourth-Instar
Larvae of An. stephensi
Fraction No.
LC50
Fraction
LC50
[ppm] No.
[ppm]
Main fractions:
1
RB-b (Fresh fruit-coat extract)a)
RB-a (Fresh seed extract)a)
290
784
6
7
PE −S× (Petroleum ether
soluble fraction of RB-b −N×)
PE −I× (Petroleum ether
159
154
106
15
2
3
4
insoluble fraction of RB-b −N×)
AcOEt phase of FFC (RB-b) after 1658
−ES× (Ether soluble
liquidÀliquid partitioning
fraction of PE −S×)
−EI× (Ether insoluble
fraction of PE −I×)c)
RB-b −A×b) (Acidic fraction
of AcOEt phase)
144
43
9
5RB-b −N× (Neutral fraction
of AcOEt phase)
VLC-fractions from −ES×
10
11
12
13
14
−A×
−B×
−C×
−D× −G×
− H×
100
200
150
15−I×
16
17
107
−J×
−K×
−L×
136
212
66
d
)
18
21519
−M×
182
a) The LC50 value determined with fourth-instar larvae of Aedes aegypti was found to be 319 ppm for RB-b and
446 ppm for RB-a. b) This extract, when tested after 6 months, gave a LC50 value of 300 ppm. c) Consisted
exclusively of azadiradione (2). d) These fractions were mixtures of 1, 4, 5, and 9, and were, therefore, not
assayed as such, although the activity of pure compounds was determined (see Fig. 2).
MortalityTesting: The WHO method [40] was modified for this application. A batch of ten fourth-instar
larvae were released in a 100-ml beaker containing 50 ml filtered tap water. The conc. selected in the preliminary
screening of each compound were tested at 28 Æ 18. A group of seven beakers was set up, five for the different
concentrations of pesticide and one each for positive and negative controls. Each experiment was repeated five
times. Experiments in which the mortality in the negative control was found to be more than 10% were
discarded. The mortality was recorded after 24 h and readings were subjected to Abbot×s formula [41].
Calculation of LC50: The lethal concentrations (LC50) were calculated by means of the PROBIT analysis
[42]. Both RB-b and RB-a were also tested for toxicity against Aedes aegypti L., and LC50 were found to be 319
and 446 ppm, resp. The detailed statistical data is presented in Table 3.

Helvetica Chimica Acta Vol. 86 (2003)
3351
Table 3. Statistical Analysis of Pesticidal Activity
Entry
Dose [ppm]
Mean mortality
Dose
[ppm]
Mean mortality
Dose
[ppm]
Mean mortality
[% Æ SD]a)
[% Æ SD]a)
[% Æ SD]a)
Main fractions:
RB-b
RB-a
400.0
EtOAc phase of FFC
1
2
3
4
5
100.0
200.0
300.0
400.0
500.0
20 Æ 7
22 Æ 4.48
Æ 5.48
80.0
120.0
160.0
200.0
240.0
20 Æ 7.07
34 Æ 5.48
42 Æ 4.48
66 Æ 5.48
82 Æ 4.48
34 Æ 5600.0
52 Æ 4
36
8
800.0
1000.0
1200.0
54 Æ 5.48
72 Æ 4.48
96 Æ 4.48
82 Æ 4
98 Æ 4
RB-b −A×
RB-b −N×
PE −S×
1
2
3
4
5
125.0
150.0
175.0
200.0 0.0
225.0
36 Æ 520.0
Æ 4
Æ 4
80.0
120.0
160.0
200.0
22 Æ 4
36 Æ 5
44 Æ 5
72 Æ 4
Æ 4
54 Æ 530.0
72 Æ 4
28
40.0
52 Æ 8
Æ 4
6284 Æ 55
92 Æ 4
60.0
ES
76 Æ 5240.0
88
PE −I×
EI
1
2
3
4
5
80.0 .0
120.0
160.0
200.0
240.0
4424 Æ 575
36 Æ 5
Æ 510.0
54 Æ 5
34
68
Æ 5
50 Æ 7
Æ 4
125.0
15.0
.0
.0
5666Æ 5175
7746Æ 5225
98 Æ 4
Æ 520.0
8Æ4525
.0
275.0
Æ 5
88 Æ 4
30.0
92 Æ 4
VLC-Fractions from −ES×:
Fraction −A×
Fraction −B×
110.2
Fraction −C×
1
2
3
4
5
55.5
74.0
92.548
111.0
8 Æ 4
26 Æ 5147.0
Æ 4
2 Æ 4
Æ 4
92.2
123.0
153.7
14 Æ 5
22 Æ 4
52 Æ 8
Æ 5
8
183.7
6220.55
257.2
36 Æ 5
7Æ65184.5
82 Æ 8
78 Æ 4
Æ 4
229.592
215.2
94 Æ 5
Fraction − H×
Fraction −I×
50.0
Fraction −J×
1
2
3
4
5
50.0
100.0
8 Æ 4
28 Æ 4
Æ 4
50.0
100.0
150.0
200.0
250.0
24 Æ 5
36 Æ 5
56 Æ 5
62 Æ 4
88 Æ 4
14 Æ 5100.0
36 7Æ2515
44 Æ 5200.0
66 9Æ6525
48
88
150.0
200.0
250.0
0.0
0.0
Æ 4
Æ 4
Æ 4
Fraction −K×
Fraction −L×
25.0
Fraction −M×
1
2
3
4
5
50.0
100.0 0.0
150.0
8 Æ 4
20 Æ 7
Æ 5100.0
Æ 4
50.0
150.0
250.0
22 Æ 4
1444Æ 55
34 Æ 5100.0
46 7Æ6515
62 Æ 4
34
5
Æ 5
42 Æ 4
Æ 5
62
200.0
250.0
0.0
Æ65200.0
96 Æ 4
200.0
76 Æ 5
Purified triterpenoids and permethrin
1
2
3
1
2
3
4
5
10.0
15.0
20.0
25.0
30.0
34 Æ 510.0
50 Æ 7
22
76
Æ 4
10.0
15.0
20.0
36 Æ 5
50 Æ 7
72 Æ 4
Æ 4
20.0
30.0
.0
36 Æ 5
68 Æ 4
50 Æ 7
84 Æ 540.0
92 Æ 4
88Æ 525
94 Æ 530.0
50.0
98
Æ 4

3352
Helvetica Chimica Acta Vol. 86 (2003)
Table 3 (cont.)
Entry
Dose [ppm]
Mean mortality
Dose
[ppm]
Mean mortality
Dose
[ppm]
Mean mortality
[% Æ SD]a)
[% Æ SD]a)
[% Æ SD]a)
4
5
6
1
2
3
4
5
2.514 0.0
5.0
7.536
10.0
12.568
14Æ 55
28 Æ 4
Æ 5
Æ 540.0
34 Æ 580.0
50 Æ 7
8
Æ 4
Æ 4
100.0
150.0
200.0
250.0
28
120.0
160.0
200.0
50 Æ 7
72 Æ 4
92 Æ 4
50 Æ 7
Æ 4
68 Æ 4
88 Æ 4
7
8
9
1
2
3
4
5
20.0
40.0
60.0
80.0
20 Æ 7
15.0
.0
45.0
60.0
75.0
22 Æ 4
13.0
14 Æ 5
Æ 5
36 Æ 530.0
50 Æ 7
34
34
3Æ4515
50 Æ 7
17.0
36 Æ 5
Æ 7
72 Æ 4
84 Æ4519.0
96 Æ 4
5
100.0
98 Æ 4
21.0
12
72 Æ 4
10
11
1
2
3
4
5
18.520
27.7536
37.0
46.2566 .0
55.5
Æ 7
9.0
22 Æ 4
2.2
8 Æ 4
Æ 5
Æ 511.0
50 Æ 7
14Æ 52.75
50 Æ 7
13.0
3.30
3.8536
28 Æ 4
Æ 5
72Æ 515
82 Æ 8
Æ 4
17.0
94 Æ 54.40
48
Æ 4
12a
13
14
1
2
3
4
5
1.4
1.758
2.1
2.4528
2.8
2 Æ 4
Æ 4
13.0
15.0
.0
19.0
21.0
8 Æ 4
11.0
13.0
2 Æ 4
8 Æ 4
Æ 4
20 Æ 7
14 Æ 517.0
Æ 4
34
Æ 52125
50 Æ 7
17.0
5
36 Æ 5
Æ 7
50 Æ 7
66 Æ 5109.0
15
16
Permethrin
0.0312
1
2
3
4
5
10.0
20.0
30.0
40.0
50.0
8 Æ 4
20.0
22 Æ 4
22 Æ 4
Æ 5
24 Æ 540.0
360Æ 560.0
44 Æ 580.0
56 Æ 5100.0
34
5
76
36Æ 50.0625
Æ 7
0.1250
52 Æ 8
Æ 5
66Æ 50.1875
94
00
77Æ 50.25
Æ 4
a) Standard deviation (the value for both the positive and negative controls was 0 Æ 0).
REFERENCES
[1] N. D. Vietmeyer, −Neem A Tree for Solving Global Problems×, National Academy Press, Washington,
D.C., 1992, and refs. cited therein.
[2] K. M. Nadkarni, −Indian Materia Medica×, revised by A. K. Nadkarni, Popular Prakashan Ltd., Bombay,
1976, Vol. 1.
[3] Department of Ayurveda, −Ayurveda Pharmacopoeia×, Colombo, Sri Lanka, 1979, Vol. 1, 256; Vol. 2, 68.
[4] −The Neem Tree: Azadirachta indica A. Juss. and other Meliaceous Plants×, Ed. H. Schmutterer, VCH,
Weinheim, 1995, and refs. cited therein.
[5] W. Kraus, M. Bokel, M. Schwinger, B. Vogler, R. Soellner, D. Wendisch, R. Steffens, U. Wachendorff, in
−Phytochemistry and Agriculture×, Ed. T. In van Beek, H. Breteler, Oxford University Press, Oxford, 1993.
[6] H. Rembold, in −Insecticides of Plant Origin×, Ed. J. T. Arnason, B. J. R. Philogene, M. Morand, ACS
Symposium Series 387, Washington, D.C., 1989.
[7] K. Chatterjee, Indian Med. Gaz. 1919, 54, 171.

Helvetica Chimica Acta Vol. 86 (2003)
[8] S. Siddiqui, Curr. Sci. 1942, 11, 278.
3353
[9] −Azadirachta indica A. Juss.; Proceedings of the Fifth International Neem Conference×, Gatton, Australia,
February 4 9, 1996, Ed. R. P. Singh, R. C. Saxena, Science Publishers, Enfield, 1999.
[10] −Practice Oriented Results on Use and Production of Neem Ingredients and Pheromones VIII:
Proceedings of 8th Workshop×, Hohensholms, Germany, February 16 18, 1998, Ed. H. Kleeberg,
C. P. W. Zebitz, Trifolio-M GmbH, Lahnau, 1998.
[11] J. H. Butterworth, E. D. Morgan, J. Chem. Soc. (D), Chem. Commun. 1968, 23.
[12] S. V. Ley, J. C. Anderson, W. M. Blaney, E. D. Morgan, R. N. Sheppard, M. S. J. Simmond, A. M. Z. Slawin,
S. C. Smith, D. J. Williams, A. Wood, Tetrahedron 1991, 47, 9231.
[13] S. V. Ley, J. C. Anderson, W. M. Blaney, P. S. Jones, Z. Lidert, E. D. Morgan, N. G. Robinson, D.
Santafianios, M. S. J. Simmond, P. L. Toogood, Tetrahedron 1989, 45, 5175.
[14] S. Siddiqui, B. S. Siddiqui, Ghiasuddin, S. Faizi, Pak. J. Sci. Ind. Res. 1991, 34, 1.
[15] S. Siddiqui, B. S. Siddiqui, Ghiasuddin, S. Faizi, Pak. J. Sci. Ind. Res. 1990, 33, 359.
[16] A. P. Jarvis, S. Johnson, E. D. Morgan, M. Simmonds, S. J. Blaney, M. Wally, J. Chem. Ecol. 1997, 23, 2841.
[17] S. R. Rojatkar, D. D. Sawaikar, B. R. Patewad, S. Gurunath, P. P. Pujar, B. A. Nagasampagi, Nat. Prod. Lett.
1998, 11, 181.
[18] S. Siddiqui, S. Faizi, T. Mahmood, B. S. Siddiqui, J. Chem. Soc., Perkin Trans. 1, 1986, 1021.
[19] M. Suleman, K. Khan, S. Khan, Pak. J. Zool. 1993, 25, 321.
[20] F. P. Amerasinghe, M. Mukhtar, N. Herrel, J. Med. Entomol. 2002, 39, 28.
[21] 13th Programme Report of WHO/TDR, 1995 96, p. 40.
[22] −Encyclopedia of Reagents for Organic Synthesis×, Vol. 6, Ed. L. A. Paquette, John Wiley & Sons Ltd.,
Chichester, 1995, 4203.
[23] S. V. Ley, P. J. Lovell, A. M. Z. Slavin, S. C. Smith, D. J. Williams, A. Wood, Tetrahedron 1993, 49, 1675.
[24] S. F. Martin, C. L. Campbell, Tetrahedron Lett. 1987, 28, 503.
[25] B. S. Siddiqui, M. Rasheed, Ghiasuddin, S. Faizi, S. N. H. Naqvi, R. M. Tariq, Tetrahedron 2000, 56, 3547.
[26] B. S. Siddiqui, M. Rasheed, Helv. Chim. Acta 2001, 84, 1962.
[27] D. Lavie, E. C. Levy, M. K. Jain, Tetrahedron 1971, 27, 3927.
[28] J. W. Powell, J. Chem. Soc. C 1966, 1794.
[29] S. Siddiqui, B. S. Siddiqui, Ghiasuddin, S. Faizi, Phytochemistry 1992, 31, 4275.
[30] B. S. Siddiqui, M. Rasheed, T. Gulzar, Firdous, S. N. H. Naqvi, R. M. Tariq, Z. Naturforsch. 2003, in press.
[31] B. S. Siddiqui, F. Afshan, S. Faizi, R. M. Tariq, S. N. H. Naqvi, J. Nat. Prod. 2002, 65, 1216.
[32] S. Siddiqui, S. Faizi, B. S. Siddiqui, Z. Naturforsch. 1986, 41B, 922.
[33] S. M. Lee, J. I. Olsen, M. P. Schweizer, J. A. Klocke, Phytochemistry 1988, 27, 2773.
[34] W. Kraus, R. Cramer, Tetrahedron Lett. 1978, 2395.
[35] S. Siddiqui, S. Fuchs, J. Lucke, W. Voelter, Tetrahedron Lett. 1978, 611.
[36] L-K. Sy, G. D. Brown, Tetrahedron 2002, 58, 897.
[37] L-K. Sy, G. D. Brown, Tetrahedron 2002, 58, 909.
[38] J. Fossey, D. Lefort, J. Sorba, −Free Radicals in Organic Chemistry×, John Wiley & Sons, Chichester, 1995.
[39] −Reactive Intermediates in Organic Chemistry: Free Radicals×, Ed. J. K. Kochi, John Wiley & Sons, New
York, 1973.
[40] 17th Report of WHO Expert Committee on Insecticides, World Health Organization Techn. Rep. Ser.
No. 443, 1970.
[41] W. S. Abbot, J. Econ. Ent. 1925, 18, 265.
[42] M. Raymond, G. Proto, D. Ratsira, PROBIT Analysis of Mortality Assays Displaying Quantal Response.
Ver. 3.3, License L93019, Praxeme, 34680, St. Georges d×Orques, 1993.
Received June 6, 2003