Biological investigation and structure-activity relationship studies on azadirone from Azadirachta indica A. Juss

Article abstract of DOI:10.1016/j.bmcl.2003.08.030

Azadirone 1, a limonoidal constituent of Azadirachta indica is found to possess potent cytotoxic activity against a panel of human cancer cell lines in our in vitro studies. In vitro screening of a number of semi-synthetic analogues of 1 revealed that the α,β-unsaturated enone moiety or its equivalent conjugated system in A-ring, C-7 acetyloxy/chloroacetyloxy or keto group in B-ring and the furan moiety are responsible for the activity of 1 and its analogues. Compound 1 and two of the semi-synthetic analogues 10 and 13 were found to possess good in vivo antitumor activity in modified hollow fiber animal models.

Full text of DOI:10.1016/j.bmcl.2003.08.030

Bioorganic & Medicinal Chemistry Letters 13 (2003) 4111–4115
Biological Investigation and Structure–Activity Relationship
Studies on Azadirone from Azadirachta indica A. Juss§
Srinivas Nanduri,a,* Siva Sanjeeva Rao Thunuguntla,a Vijay Kumar Nyavanandi,a
Sridevi Kasu,a P. Mahesh Kumar,a P. Sai Ram,a Sriram Rajagopal,b R. Ajaya Kumar,b
Dhanvanthri S. Deevi,b R. Rajagopalanb and A. Venkateswarlua
aDiscovery Chemistry, Dr. Reddy’s Laboratories Ltd., Discovery Research, Bollaram Road, Miyapur, Hyderabad 500 050, India
bDiscovery Biology, Dr. Reddy’s Laboratories Ltd., Discovery Research, Bollaram Road, Miyapur, Hyderabad 500 050, India
Received 4 June 2003; accepted 11 August 2003
Abstract—Azadirone 1, a limonoidal constituent of Azadirachta indica is found to possess potent cytotoxic activity against a panel
of human cancer cell lines in our in vitro studies. In vitro screening of a number of semi-synthetic analogues of 1 revealed that the
a,b-unsaturated enone moiety or its equivalent conjugated system in A-ring, C-7 acetyloxy/chloroacetyloxy or keto group in B-ring
and the furan moiety are responsible for the activity of 1 and its analogues. Compound 1 and two of the semi-synthetic analogues
10 and 13 were found to possess good in vivo antitumor activity in modified hollow fiber animal models.
# 2003 Elsevier Ltd. All rights reserved.
Introduction
Azadirone, a limonoidal triterpene was first isolated
from nim oil in 196728 and subsequently its structure
was elucidated.29 In the present study, 1 was isolated
from neem flowers for the first time. While there are no
reports in literature about the biological activity of 1,
the present study revealed 1 to be a potent cytotoxic
agent with good in vitro and in vivo activities. The
investigation was also extended to explore the structural
requirements for the activity of 1 and to synthesize
novel semi-synthetic analogues with improved activity.
The details of the study are presented in this communi-
cation (Fig. 1).
Limonoids, the triterpene derivatives present in Melia-
ceae and Rutaceae families have attracted much atten-
tion owing to their marked insect antifeedant,1 growth
regulatory2 properties and a variety of medicinal effects
including antibacterial, antifungal,3À5 antimalarial,6À9
antipyretic10,11 and antiinflammatory10,12 activities.
Anti-HIV properties of limonin and nomilin on infected
human mononuclear cells were recently reported.13 The
cytotoxic activity and structure–activity relationships of
various limonoids belonging to Meliaceae and Rutaceae
families against the murine P388 lymphocytic leukemia
cell lines were reviewed.14 Promising cytotoxic activity
of citrus limonoids against colon, breast and other can-
cers were reported.15À17 Antitumoral activity of Hap-
erforine A from Harrisonia perforate was described.18
Potent cytotoxic activity of a number of limonoids pre-
sent in Melia azedarach,19À22 Melia volkensii23À25 and
Melia toosendan26 were reported. Nimbolide and 28-
deoxonimbolide were identified as cytotoxic con-
stituents of Azadirachta indica leaves.27
Compound 1 was isolated from the methanolic extract
of dry neem flowers as a major constituent (0.05–0.1%
yield) and identified as azadirone based on extensive
spectral studies and comparison with published
As a part of our study to identify novel cytotoxic agents
of plant origin, the activity of azadirone 1 was studied.
§DRL Publication No.: 297.
*Corresponding author. Tel.+91-40-2304-5439; fax:+91-40-2304-
5438; e-mail: nandurisrinivas@drreddys.com
Figure 1.
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.08.030

4112
S. Nanduri et al. / Bioorg. Med. Chem. Lett. 13 (2003) 4111–4115
Scheme 1. (i) DIBAL-H/benzene/rt/2 h; (ii) Ac2O/pyridine/DMAP/2 h; (iii) H2O2/2 N NaOH/MeOH/2 h; (iv) 10% alcoholic NaOH/24 h; (v)
LiAIH4/THF/reflux/24 h; (vi) H2NOHꢀ HCl/EtOH/pyridine/reflux/3 h; (vii) ClCH2CH2N(CH2CH3)2ꢀ HCl/K2CO3/acetone/reflux/8 h; (viii) 1-(2-
chloroethyl)pyrrolidineꢀ HCl/K2CO3/acetone/reflux/8 h.
Figure 2. C-7 derivatives of azadirone.
results.29 Semi-synthetic analogues of 1 were prepared
by modifying the a,b-unsaturated enone moiety in A-
ring (Scheme 1), C-7 acetyloxy group in B-ring (Scheme
2), and the furan moiety (Scheme 3). Compound 1 and
its analogues were evaluated for their in vitro cytotoxic
activity against eight cancer cells representing different
human cancers using SRB assay according to NCI pro-
tocol.30 In the preliminary screening, the percent growth
of the cancer cell lines at 10 mM concentration of the
compounds was measured (Table 1). Compounds
showing 50% or less growth in the preliminary screen-
ing were selected, subjected to dose response studies
with concentrations ranging from 100 to 0.1 mM and the
GI50’s were calculated (Table 2).

S. Nanduri et al. / Bioorg. Med. Chem. Lett. 13 (2003) 4111–4115
4113
Scheme 2. (i) EtOH/Aq. NaOH/reflux/2 h; (ii) PDC/DCM/8 h; (iii) Conditions given in Figure 2.
Scheme 3. (i) m-CPBA/NaOAc/DCW/rt/5 h.
In the above in vitro studies, 1 exhibited potent activity
against breast, melanoma and prostate cell lines having
GI50 values in sub micro molar concentrations. To
study the influence of a,b-unsaturated enone moiety in
A ring of 1, 3 (3-acetyloxy derivative of 1,), and 4
(1a,2a-epoxy derivative of 1) were synthesized (Scheme
1). Compound 3 was prepared by reducing 1 with
DIBAL-H to give 2, which was further acetylated to
obtain 3. Selective epoxidation of C-1,2 olefin bond
gave 4. Further, 2-alkoxy derivatives 6 and 8 were pre-
pared by refluxing 4 in methyl and ethyl alcohols in the
presence of alkali and acetylation of the resulting
2-alkoxy 7-deacetyl derivatives 5 and 7. C-3 oxime
derivative 11 and its corresponding ethers 12 and 13
were synthesized by usual methods.
11 moderate activity was observed in the preliminary
screening. However, activity was found to improve
considerably with the oxime ether derivatives 12 and 13.
These observations clearly show that a,b-unsaturated
enone moiety or its equivalent conjugated moieties in A
ring can confer substantial cytotoxic activity.
Compound 4 was further utilized as a key intermediate
in the synthesis of 9, a compound with 1,3-oxygena-
tion pattern in A ring. Compound 9 was obtained by
reductive opening of the epoxide in 4 with LiAlH4,
which on further acetylation gave 10, the correspond-
ing triacetyl derivative. Compound 10, exhibited good
in vitro cytotoxic activity against most of the cell
lines.
In the preliminary screening, 3 and 4 exhibited loss of
activity indicating that the a,b-unsaturated enone moi-
ety plays an important role for the activity of 1. Com-
pounds 2-methoxy and 2-ethoxy derivatives 6 and 8,
respectively, showed moderate activity in the pre-
liminary assay. In further evaluation, while 6 exhibited
loss of activity against all the cell lines, 8 showed good
activity against breast, CNS and colon cell lines. When
the enone moiety was converted to its C-3 oxime as in
Modifications carried out in B ring (Scheme 2) include
hydrolysis of C-7 acetyloxy moiety leading to the cor-
responding hydroxy derivative 14, which on further
oxidation gave 7-keto derivative (15). Several C-7 ester
and ether derivatives (16–25) of 14 were synthesized and
are listed in Figure 2.
The screening results indicated that 14 exhibited good
activity against breast, CNS, melanoma and prostate

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S. Nanduri et al. / Bioorg. Med. Chem. Lett. 13 (2003) 4111–4115
Table 1. In vitro cytotoxic activities of azadirone derivatives (preliminary screening)
Cytotoxicity (% growth at 10À5 M concentration)
Compd
Breast
CNS
Colon
Lung
H522
Melanoma
M14
Ovarian
SKOV3
Prostate
DU145
Renal
A498
MCF-7/ADR
U251
SW620
1
2
3
4
À18
76
78
59.3
58
À4
100
46
100
63
29
79.6
59
100
35
29
97
86
69.6
82
À6
100
76
100
67
30
100
19.3
100
38
À6
100
74
63.1
47
23
88.2
69
42.7
50
6
7
8
17
51
54
70
36
65
57
52
67
40
67
61
84
35
52
33
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
38
55
28
22
36
30
66
54
29
57
57
55
66
62
À24
À60
8
44
26
À13.9
100
43
69
75
48
40
54
69
63
53
51
À22
42
87
À7
À35
64
43
100
49
61
90
84
86
85
48
100
14
À19
À5
5
47
23
38
35
56
46
20
7
5
À24
À51
100
3
81
60
51
49
61
86
62
67
64
12
26
15.8
7
61
86
39
59
95
41
39
58
32
46
7
76
77
67
68
69
81
25
63
76
13
24.8
À16
46
27
65
9
45
59
67
43
74
48
5
33
100
À8
70.3
63
52
40
15
40
41
77
53
55
À2
71
22
44.3
74
À3
5
73.3
À13
100
71.3
41.6
Table 2. In vitro cytotoxic activities of azadirone derivatives
Cytotoxicity (GI50 mM)a
Compd
Breast
CNS
Colon
Lung
H522
Melanoma
Ovarian
SKOV3
Prostate
DU145
Renal
A498
MCF-7/ADR
U251
SW620
M14
1
6
8
10
12
13
14
15
23
25
0.06
95
8
4
6
3
5
3
60
3
2.5
20
5
30
4
4
3.5
6
7
20
5
3
4
6
40
30
40
3
4
0.08
40
20
5
4
5
5
35
40
9
3
4
0.05
35
50
4
7
6
9.5
6
20
5
2
40
40
15
8
3
>100
3
4
>100
25
15
9
>100
7.5
65
5
>100
4
0.6
20
5
50
6
20
5
12
4
4
aCytotoxicity GI50 values are the concentrations corresponding to 50% growth inhibition.
cell lines with GI50’s in micro molar concentrations and
showed loss of activity against other cell lines. However,
the corresponding 7-keto derivative 15 showed good
activity against all the cell lines. The biological eval-
uation of C-7 ester and ether derivatives of 14 showed
the following interesting pattern. Replacement of the
acetyl moiety with ethyl substituent (16) led to loss of
activity indicating that an ester linkage at C-7 is essen-
tial. Changing of the acetyl to trifluoroacetyl (17) resul-
ted in loss of activity. Moderate activity was observed
with propionyl moiety (18) which decreased with butyryl
(19) and isobutyryl (20) derivatives. More bulky groups
like cinnamoyl, glycinyl, and carbamoyl derivatives
(21–24) also showed loss of activity. Interestingly, activ-
ity was found to be retained with C-7 chloroacetyloxy
derivative (25). The above results clearly indicate that a
keto group or an ester linkage at C-7 can result in good
cytotoxic activity and bulkier esters reduce the activity.
As 26, a mixture of hydroxy butenolide derivatives of 1,
showed loss of activity (Table 1), it is inferred that the
furan moiety in 1 is also very important for the cyto-
toxic activity of 1 (Scheme 1).
In summary, the above studies resulted in 10, 12, 13, 15,
and 25 with good in vitro anticancer activity. Of these,
10 and 13 along with azadirone 1, which exhibited good
cytotoxic activity and possess unique structural features
were evaluated for their in vivo potential in Swiss
Albino Mice using modified hollow fiber assay.31,32 Poly
vinylidine fluoride hollow fibers containing different
types of human cancer cell lines were implanted intra-
peritoneally (ip) and subcutaneously (sc) into the mice.
The compounds were administered by IP route at two
different doses for 4 days. The doses were determined
based on the MTDvalues of the compounds. The per-
centage growth inhibition of cancer cells determined in

S. Nanduri et al. / Bioorg. Med. Chem. Lett. 13 (2003) 4111–4115
4115
Table 3. In vivo hollow fiber assay results of azadirone derivatives
9. Rochanankij, S.; Thebtaranonth, Y.; Yenjai, Ch.; Yutha-
vong, Y. Southeast Asian J. Trop. Med. Public Health 1985,
16, 16.
10. Okpanyi, S. N.; Ezeukwu, G. C. Planta Med. 1981, 41, 34.
11. Khattak, S. G.; Gilani, S. N.; Ikram, M. J. Ethnopharma-
col. 1985, 14, 45.
12. Fujiwara, T.; Sugishita, E.; Takeda, T.; Ogihara, Y.; Shi-
mizu, M.; Nomura, T.; Tomita, Y. Chem. Pharm. Bull. 1984,
32, 385.
13. Mengoni, F.; Lichtner, M.; Russo, G.; Matroianni, C. M.;
Vullo, V.; Battinelli, L.; Mazzanti, G.; Saija, A. Abstracts of
Papers: 40th ICAAC (Interscience Conference on Anti-
microbial Agents and Chemotherapy), Toronto, Ontario,
Canada, Sept. 17–20, 2000: abstract no. 1846.
14. Pettit, G. R.; Barton, D. H. R.; Herald, C. L.; Polonsky,
J.; Schmidt, J. M.; Connolly, D. J. J. Nat. Prod. 1983, 46, 379.
15. Tanaka, T.; Maeda, M.; Kohno, H.; Murakami, M.;
Kagami, S.; Miyake, M.; Wada, K. Carcinogenesis 2001, 22, 193.
16. Guthrie, N.; Morley, K.; Hasegawa, S.; Manners, G. D.;
Vandenberg, T. ACS Symp. Ser. 2000, 758, 164.
17. Miller, E. G.; Taylor, S. E.; Berry, C. W.; Zimmerman,
J. A.; Hasegawa, S. ACS Symp. Ser. 2000, 758, 132.
18. Potier, P. J.; Khuong-Huu.; Francoise, C. C.; Khuong-
Huu, Q. PCT Int. Appl. WO 2001030776 A1, Chem. Abstr.
2001, 134, 344565.
Compd
Dose
(mg/kg)
sc
Score
ip
Score
Total
score
1
75
150
75
150
50
12
24
6
24
8
14
24
12
24
14
24
36
48
18
48
22
46
10
13
100
22
both the compartments was an index of the compound’s
anticancer potency. Compounds inhibiting 50% or
more growth of cancer cells compared to the vehicle
treated control cells were considered active and a score
of 2 was assigned at the dose tested. Each compound
was tested against 12 human cancer cell lines. The
results obtained are summarized in Table 3. The results
indicated that the compounds are moderately active at
low doses and fairly active at higher doses.
In conclusion, we have identified azadirone 1, present in
neem flowers, as a potent cytotoxic agent with good in
vitro and in vivo activity. Semi-synthetic studies per-
formed and cytotoxic activity screening of the derived
analogues resulted in 10 and 13 with good activity. The
studies also revealed that the a,b-unsaturated enone
moiety or its equivalent conjugated system of A-ring,
C-7 acetyloxy/chloroacetyloxy or keto group of B-ring
and the furan moiety are the structural requirements for
the potent activity of 1 and its analogues.
19. Takeya, K.; Qiao, Z.; Hirobe, C.; Itokawa, H. Bioorg.
Med. Chem. 1996, 4, 1355.
20. Takeya, K.; Qiao, Z.; Hirobe, C.; Itokowa, H. Phyto-
chemistry 1996, 42, 709.
21. Itokawa, H.; Qiao, Z.; Hirobe, C.; Takeya, K. Chem.
Pharm. Bull. 1995, 43, 1171.
22. Ahn, J.; Choi, S.; Lee, C. Phytochemistry 1994, 36, 1493.
23. Rogers, L. L.; Zeng, L.; Kozlowski, J. F.; Shimada, H.;
Alali, F. Q.; Johnson, H. A.; McLaughlin, J. L. J. Nat. Prod.
1998, 61, 64.
24. Rogers, L. L.; Zeng, L.; McLaughlin, J. L. Tetrahedron
Lett. 1998, 39, 4623.
References and Notes
25. Zeng, L.; Gu, Z.; Fang, X.; Fanwick, P. E.; Chang, C.;
Smith, D. L.; McLaughlin, J. L. Tetrahedron 1995, 51, 2477.
26. Kigodi, P. G. K.; Blasko, G.; Thebtaranonth, Y.; Pezzuto,
J. M.; Cordell, G. A. J. Nat. Prod. 1989, 52, 1246.
27. Phillip, G. K. K.; Gabor, B.; Yodhathai, T.; John, M. P.;
Geoffrey, A. C. J. Nat. Prod. 1989, 52, 1246.
1. Champagne, D. E.; Koul, O.; Isman, M. B.; Scudder,
G. G. E.; Towers, G. H. N. Phytochemistry 1992, 31, 377.
2. Naqvi, S. N. H.; Ahmed, S. O.; Mohmmad, F. A. Pak. J.
Pharm. Sci. 1991, 4, 71.
3. Siddiqui, S.; Siddiqui, B. S.; Faizi, S.; Mahmood, T. J. Nat.
Prod. 1988, 51, 30.
4. Ara, I.; Siddiqui, B. S.; Faizi, S.; Siddiqui, S. J. Nat. Prod.
1988, 51, 1054.
5. Obaseki, A. O.; Jegede-fadusin, H. A. Fitoterapia 1986,
LVII, 247.
6. Khalid, S. A.; Farouk, A.; Geary, T. G.; Jenesen, J. B. J.
Ethnopharmacol. 1986, 15, 201.
28. Lavie, D.; Jain, M. K. Chem. Commun. 1967, 267.
29. Lavie, D.; Levy, E. C.; Jain, M. K. Tetrahedron 1971, 27,
3927.
30. Monks, A.; Scudiero, D.; Skehan, P.; Shoemaker, R.;
Paull, K.; Vistica, D.; Hose, C.; Langley, J.; Cronise, P.;
Vaigro-Wolff, A. J. Nat. Cancer Inst. 1991, 83, 757.
31. Sriram, R.; Sunil Kumar, K. B.; Dhanvanthri, S. D.;
Shravan, K. R.; Krishna, N. S.; Sony, P.; Ajay Kumar, R.;
Seshagiri Rao, C.; Rajagopalan, R. Abstracts of Papers; Clin-
ical Cancer Research, 2001; abstract no. #164.
7. Khalid, S. A.; Duddeck, H.; Gonzales-Sierra, M. J. Nat.
Prod. 1989, 52, 922.
8. MacKinnon, S.; Durst, T.; Arnason, J. T.; Angerhofer, C.;
Pezzuto, J.; Sanchez-Vindas, P. E.; Poveda, L. J.; Gbeassor,
M. J. Nat. Prod. 1997, 60, 336.
32. Hollingshead, M. G.; Alley, M. C.; Camalier, R. F.;
Abbott, B. J.; Mayo, J. G.; Malspeis, L.; Grever, M. R. Life
Sciences 1995, 57, 131.