Synthesis and study of some new N-substituted imide derivatives as potential anticancer agents

Article abstract of DOI:10.1016/j.farmac.2005.01.011

A new series of N-substituted imide derivatives have been synthesized by treating phthalic anhydride, naphthalic anhydride and their substituted derivatives with 2-hydrazino-1-imidazoline hydrobromide, various para-substituted aryl amines, aminoglutethimide and 2,4-dinitrophenyl hydrazine. Compounds 9, 10, 12, 18, 19, 23, 24 and 34-36 have been selected and screened for antineoplastic activity by National Cancer Institute, Bethesda, USA. Some newer aminoglutethimide derivatives 37-39 have also been prepared in order to study the effect of N-substitution on its pharmacological profile for the treatment of carcinoma. These compounds (37-39) have exhibited weak inhibition of human placental aromatase as compared to aminoglutethimide.

Full text of DOI:10.1016/j.farmac.2005.01.011

Il Farmaco 60 (2005) 283–290
http://france.elsevier.com/direct/FARMAC/
Synthesis and study of some new N-substituted imide derivatives
as potential anticancer agents
Dharam Paul Jindal a, Vikas Bedi a, Birinder Jit a, Nalin Karkra a, Sheetal Guleria a,
Ranju Bansal a,*, Anja Palusczak b, Rolf W. Hartmann b
a University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh- 160014, India
b Pharmaceutical and Medicinal Chemistry, P.O. Box 15 11 50, D-66041, Saarland University, Saarbrucken, Germany
Received 25 November 2004; received in revised form 14 January 2005; accepted 27 January 2005
Abstract
A new series of N-substituted imide derivatives have been synthesized by treating phthalic anhydride, naphthalic anhydride and their
substituted derivatives with 2-hydrazino-1-imidazoline hydrobromide, various para-substituted aryl amines, aminoglutethimide and 2,4-
dinitrophenyl hydrazine. Compounds 9, 10, 12, 18, 19, 23, 24 and 34–36 have been selected and screened for antineoplastic activity by
National Cancer Institute, Bethesda, USA. Some newer aminoglutethimide derivatives 37–39 have also been prepared in order to study the
effect of N-substitution on its pharmacological profile for the treatment of carcinoma. These compounds (37–39) have exhibited weak inhi-
bition of human placental aromatase as compared to aminoglutethimide.
© 2005 Elsevier SAS. All rights reserved.
Keywords: Anticancer agents; aminoglutethimide; aromatase; 60-cell line; N-imide derivatives
1. Introduction
Another imide derivative, aminoglutethimide 4 (Chart 1)
is the pioneer drug of all nonsteroidal, reversible competitive
inhibitors of aromatase enzyme [18] and is used in the treat-
ment of mammary carcinoma. A number of modifications of
4 have been carried out with the hope of achieving better anti-
neoplastic agents. Aromatase is a cytochrome P450 enzyme,
which is responsible for the conversion of androgens to estro-
gens in the final step of the steroid biosynthetic pathway [19].
On the other hand, drugs that abolish trophic influences of
natural oestrogens have also been developed to treat breast
cancer. The antiestrogen, tamoxifen 5 is the gold standard
adjuvant treatment in primary breast cancer but has also been
linked with some adverse effects [20]. The alkylamino side
chain of tamoxifen plays an important role in its estrogen
modulating activity. Further, several studies have also revealed
an increased response of antitumor efficacy of tamoxifen in
combination with other cytotoxic chemotherapeutics and/or
aromatase inhibitors over tamoxifen alone [21,22]. In con-
tinuation with our earlier efforts on anticancer drug design
[23,24], it was thought worthwhile to synthesize some newer
N-substituted imide derivatives by incorporating the essen-
tial structural features of the above-mentioned potent cyto-
Intercalation to DNA by small organic molecules has been
extensively studied due to medicinal significance and still
occupies an important research area in the search for potent
antineoplastic agents [1]. DNA intercalating ability and anti-
tumor activity resides in a wide variety of chemical entities
such as anthraquinone derivatives [2,3], alkanesulfoanilides
[4], acridine [5,6], substituted phenazine-1-carboxamides [7],
methylindolo[3,2-b]quinolines [8], 6-phenylphenanthridine-
4-carboxamides [9], anthrapyrazoles [10], perimidines [11],
1,4-disubstituted anthracene derivatives [12] and
N-(aminoalkyl)imide derivatives etc. [13–15]. Of all, imide
derivatives have been reported as a potential class of antine-
oplastic agents with compounds like mitonafide 1, amona-
fide 2 and azonafide 3 in the market [14–16] (Chart 1). These
derivatives have also been explored as potential antifolate
thymidylate synthase inhibitors [17].
* Corresponding author. Tel.: +91 172 2534116; fax: +91 172 2541142.
E-mail address: ranju29in@yahoo.co.in (R. Bansal).
0014-827X/$ - see front matter © 2005 Elsevier SAS. All rights reserved.
doi:10.1016/j.farmac.2005.01.011

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D.P. Jindal et al. / Il Farmaco 60 (2005) 283–290
Chart 1
.
Structures of mitonafide (1), amonafide (2), azonafide (3), aminoglutethimide (4) and tamoxifen (5).
toxic drugs in order to obtain synergistic effects, which we
report herein.
aniline derivatives 13–16 (0.45 g, 2.50 mmol) in pyridine
(40 ml) was refluxed for 6 h. The residue obtained after
removal of solvent was washed with distilled water, dried and
crystallized from methanol to afford 17–20 and 22–24, respec-
tively. Condensation of 11 (0.5 g, 2.06 mmol) with 2,4-
dinitrophenylhydrazine 25 (0.41 g) afforded compound 26.
2. Experimental Procedures
2.1. Chemistry
2.1.3. General procedure for the synthesis of compounds
30–32
Melting points (melting point apparatus MP I, Veego,
Mumbai, India) reported are uncorrected. H-NMR spectra
1
were recorded on Bruker AC-300F, 300 MHz NMR instru-
ment (Bruker, Fällanden, Switzerland) using tetramethylsi-
lane (TMS) as the internal standard (chemical shifts in d,
ppm). IR spectra were recorded on Perkin-Elmer 882 spec-
trophotometer model (Perkin-Elmer, Beaconsfield, Bucking-
hamshire, England) and were obtained with potassium bro-
mide pellets (vmax in cm–1). The purity of the compounds was
established by thin layer chromatography (TLC) and by
elemental analyses (C, H, N). Elemental analyses were per-
formed on a Perkin-Elmer 2400 (Perkin-Elmer, Norwalk, CT,
USA) and agreed with theoretical values to within 0.3%.
Anhydrous sodium sulphate was used as drying agent. Plates
for TLC were prepared with silica gel G using ethyl acetate.
Iodine vapors were used to develop the plates. The complete
NMR data of the compounds 18–20,23,24,30–32,35,36–
38,39 was in agreement with the proposed structures.
A solution of required amine in dichloromethane (10 ml)
was added drop-wise to a solution of 29 (0.25 g, 0.657 mmol)
in dichloromethane (100 ml). The reaction mixture was stirred
for 4 h at 5–8 °C. The reaction mixture was treated with
sodium carbonate solution, water and solvent removed under
reduced pressure to obtain a residue, which was crystallized
from solvent ether to afford 30–32, respectively.
2.1.4. General procedure for the synthesis of compounds
34–36
A solution of 33 (0.6 g, 2.58 mmol) and required anhy-
dride (6, 7, 11) in dry pyridine (10 ml) was refluxed for 1 h.
Solvent was removed under vacuum and water was added to
the obtained residue. The solid product obtained was filtered,
washed with water, dried and crystallized from methanol to
afford 34–36, respectively.
2.1.1. General procedure for the synthesis of compounds
9, 10, 12
2.1.5. General procedure for the synthesis
of hydrochlorides of compounds 37–39
A solution of 2-hydrazino-1-imidazoline hydrobromide 8
(0.25 g, 1.48 mmol) in absolute ethanol (25 ml) was added to
a solution of appropriate anhydride (6, 7, 11) in absolute etha-
nol. The resultant reaction mixture was stirred at 80 °C for
1 h. For compound 12, solution was stirred at room tempera-
ture for 3 h in the presence of 0.2 ml triethylamine. Solvent
was removed under vacuum and residue was dried in desic-
cator overnight. The dried residue was refluxed in toluene for
3 h. The solid material obtained after removal of solvent was
washed with ether, 5% solution of sodium bicarbonate, water,
and finally dried and crystallized from methanol to give 9, 10
and 12.
A mixture of aminoglutethimide 33 (0.2 g, 0.86 mmol)
and potassium carbonate (2.5 g) in dry acetone (50 ml) was
refluxed together for 4 h.Appropriate hydrochlorides of alky-
laminoethyl halides, potassium iodide (5 mg) and triethy-
lamine (four drops) were added to the refluxing solution and
the progress of the reaction was monitored by TLC. On
completion of the reaction, the reaction mixture was filtered
and the filtrate was removed under reduced pressure to obtain
an oily residue. The traces of moisture in the residue were
removed by repeatedly refluxing the residue with dry solvent
ether and recovering the solvent. The dried residue was then
refluxed with dry ether for about an hour and filtered. The
filtrate was dried to give an oily residue, which could not be
crystallized. Dry hydrochloride gas was passed through the
ethereal solution of the oily product to obtain the hygro-
scopic hydrochloride salts of 37–39.
2.1.2. General procedure for the synthesis of compounds
17–20, 22–24 and 26
A solution of suitable 3-substituted-1,8-naphthalic anhy-
dride 11 or 21 and appropriate 4-(2-dialkylaminoethoxy)-

D.P. Jindal et al. / Il Farmaco 60 (2005) 283–290
285
N-(4,5-Dihydro-1H-imidazol-2-ylamino)phthalimide (9)
From 6 (0.3 g, 2.025 mmol). Yield: 0.2 g, 43.47%.
m.p. > 300 °C. IR (KBr): 3413 (-NH), 1700 (C=O), 1660
(N-C=O) cm–1.
N-(4,5-Dihydro-1H-imidazol-2-ylamino)-5,6-dimethoxy-
phthalimide (10)
From 7 (0.25 g, 1.21 mmol). Yield: 0.2 g, 28.98%. m.p.
254–256 °C. IR (KBr): 3411 (-NH), 1704 (C=O), 1660
(N-C=O) cm–1.
IR (KBr): 3429 (N-H), 1701 (C=O), 1663 (N-C=O) cm–1.
1H-NMR: d 2.25 (s, 6H, -N(CH3)2), 2.67 (t, 2H, -CH2N<, J
= 6 Hz), 4.11 (t, 2H, -OCH2-, J = 6 Hz), 5.98 (s, 2H, -NH2,
disappeared on D2O exchange), 7.05 (d, 2H, Ar-H of phenyl
ring ortho to the six membered imide functionality, Jo = 9 Hz),
7.24 (d, 2H,Ar-H of phenyl ring ortho to the alkoxy function-
ality, Jo = 9 Hz), 7.32 (d, 1H, 4-CH, Jm = 2 Hz), 7.61 (t, 1H,
6-CH, Jo = 8 Hz), 7.98 (d, 1H, 2-CH, J = 2 Hz), 8.05 (m, 2H,
5-CH and 7-CH).
N-(4,5-Dihydro-1H-imidazol-2-ylamino)-1,8-naphtha-
limide (12)
N-[4-(2-Pyrrolidinoethoxy)phenyl]-3-amino -1, 8-naphtha-
limide (23)
From 11 (0.25 g, 1.028 mmol). Yield: 0.26 g, 78.78%.
m.p. > 350 °C. IR (KBr): 3404 (-NH), 1707 (C=O), 1660
(N-C=O) cm–1.
N-[4-(2-N,N-Dimethylaminoethoxy)phenyl]-3-nitro-1,8-
naphthalimide (17)
From 11 (0.5 g, 2.06 mmol) and 4-(2-N,N-dimethylamino-
ethoxy)aniline 13 (0.45 g, 2.50 mmol). Yield:0.3 g, 36.1%.
m.p. 226–228 °C. IR (KBr): 3060 (Ar C-H), 1705 (C=O),
1660 (N-C=O) cm–1. 1H-NMR: d 2.37 (s, 6H, -N(CH3)2), 2.79
(t, 2H, -CH2N<, J = 6 Hz), 4.14 (t, 2H, -OCH2-, J = 6 Hz),
7.10 (d, 2H, Ar-H of phenyl ring ortho to the six membered
imide functionality, Jo = 7 Hz), 7.20 (d, 2H, Ar-H of phenyl
ring ortho to the alkoxy functionality, Jo = 7 Hz), 7.98 (t, 1H,
6-CH, Jo = 8 Hz), 8.47 (d, 1H, 5-CH, Jo = 8 Hz), 8.81 (d, 1H,
7-CH, Jo = 7 Hz), 9.18 (d, 1H, 4-CH, Jm = 2 Hz), 9.32 (d, 1H,
2-CH, Jm = 2 Hz).
From 21 (0.5 g, 2.345 mmol) and 4-(2-pyrrolidino-
ethoxy)aniline 15 (0.5 g, 2.42 mmol).Yield: 0.50 g, 53.19%.
m.p. 174–176 °C. IR (KBr): 3377 (N-H), 1707 (C=O), 1667
(N-C=O) cm–1. 1H-NMR: d 1.77 (m, 4H, 3-CH2 and 4-CH2
-pyrrolidine), 2.61 (m, 4H, 2-CH2 and 5-CH2-pyrrolidine),
2.89 (t, 2H, -CH2N<, J = 6 Hz), 4.14 (t, 2H, -OCH2-, J = 6 Hz).
N-[4-(2-Piperidinoethoxy)phenyl]-3-amino-1,8-
naphthalimide (24)
From 21 (0.5 g, 2.345 mmol) and 4-(2-piperidino-
ethoxy)aniline 16 (0.6 g, 2.91 mmol).Yield: 0.48 g, 49.48%.
m.p. 92–94 °C. IR (KBr): 3349 (N-H), 1705 (C=O), 1664
(N-C=O) cm–1. 1H-NMR: d 1.43 (m, 2H, 4-CH2 -piperidine),
1.56 (m, 4H, 3-CH2 and 5-CH2 -piperidine), 2.51 (m, 4H,
2-CH2 and 6-CH2 -piperidine), 2.76 (t, 2H, -CH2N<, J
= 6 Hz), 4.14 (t, 2H, -OCH2, J = 6 Hz).
N-(2,4-Dinitro-1-anilino)-3-nitro-1,8-naphthalimide (26)
Yield: 0.45 g, 51.72%. m.p. 292–294 °C. IR (KBr): 3290
(N-H), 1705 (C=O), 1680 (N-C=O) cm–1. 1H-NMR: d 7.56
(d, 1H, 6-CH -dinitrophenylhydrazine ring, Jo = 9 Hz), 8.14
(t, 1H, 6-CH, Jo = 8 Hz), 8.21 (dd, 1H, 5-CH -dinitrophenyl-
hydrazine ring, Jo = 9 Hz, Jm = 2 Hz), 8.79 (d, 1H, 5-CH,
Jo = 8 Hz) 8.90 (d, 1H, 7-CH, Jo = 8 Hz), 9.01 (d, 1H, 3-CH
-dinitrophenylhydrazine ring, Jm = 2 Hz), 9.08 (d, 1H, 4-CH,
Jm = 2 Hz), 9.6 (d, 1H, 2-CH, Jm = 2 Hz), 10.62 (s, 1H, -NH,
disappeared on D2O exchange).
N-[4-(2-N,N-Diethylaminoethoxy)phenyl]-3-nitro-1,8-
naphthalimide (18)
From 11 (0.5 g, 2.06 mmol) and 4-(2-N,N-diethylamino-
ethoxy)aniline 14 (0.60 g, 2.88 mmol).Yield: 0.25 g, 27.7%.
m.p.196–198 °C. IR (KBr): 1705 (C=O), 1665 (N-C=O) cm–1.
1H-NMR: d 1.1 (t, 6H, -N(CH2CH3)2, J = 7 Hz), 2.66 (q, 4H,
-N(CH2CH3)2, J = 7 Hz), 2.93 (t, 2H, CH2N<, J = 6 Hz), 4.12
(t, 2H, -OCH2, J = 6 Hz).
N-[4-(2-Pyrrolidinoethoxy)phenyl]-3-nitro-1,8-
naphthalimide (19)
4-(3-Nitro-1,8-naphthalimido)benzoic acid (28)
A mixture of 11 (0.25 g, 1.03 mmol), p-aminobenzoic acid
27 (0.15 g, 1.09 mmol) and triethylamine (0.2 ml) in alde-
hyde free alcohol (150 ml) was stirred for 4 h. Excess ethanol
was removed and toluene (100 ml) was added to the residue
and refluxed for 4 h using Dean-Stark apparatus. Toluene was
removed under reduced pressure; distilled water was added
and allowed to stand overnight. The slurry was filtered,
washed thoroughly with distilled water, dried and crystal-
lized from methanol to afford 28 (0.30 g, 80.50%). Melting
point decompose at 280 °C. IR (KBr): 3100 (broad, -OH),
1710 (C=O), 1679 (N-C=O) cm–1. 1H-NMR: d 7.48 (d, 2H,
Ar-H of phenyl ring ortho to six membered imide function-
ality, Jo = 8.4 Hz), 8.07 (t, 1H, 6-CH), 8.13 (d, 2H, Ar-H of
phenyl ring ortho to carboxylic group, Jo = 7.8 Hz), 8.75 (m,
2H, 5-CH and 7-CH), 9.09 (d, 1H, 4-CH, Jm = 2.5 Hz), 9.48
(d, 1H, 2-CH, Jm = 2.2 Hz).
From 11 (0.5 g, 2.06 mmol) and 4-(2-pyrrolidinoethoxy)-
aniline 15 (0.5 g, 2.42 mmol). Yield: 0.415 g, 51.87%.
m.p.198–200 °C. IR (KBr): 1705 (C=O), 1660 (N-C=O) cm–1.
1H-NMR: d 1.83 (m, 4H, 3-CH2 and 4-CH2-pyrrolidine), 2.65
(m, 4H, 2-CH2 and 5-CH2-pyrrolidine), 2.95 (t, 2H, -CH2N<,
J = 6 Hz), 4.18 (t, 2H, -OCH2-, J = 6 Hz).
N-[4-(2-Piperidinoethoxy)phenyl]-3-nitro-1,
8-naphthalimide (20)
From 11 (0.5 g, 2.06 mmol) and 4-(2-(piperidinoethoxy)-
aniline 16 (0.6 g, 2.91 mmol). Yield: 0.43 g, 46.41%. m.p.
202–204 °C. IR (KBr): 1710 (C=O), 1660 (N-C=O) cm–1.1H-
NMR: d 1.48 (m, 2H, 4-CH2-piperidine), 1.65 (m, 4H, 3-CH2
and 5-CH2-piperidine), 2.53 (m, 4H, 2-CH2 and 6-CH2-
piperidine), 2.82 (t, 2H, -CH2N<, J = 6 Hz), 4.18 (t, 2H,
-OCH2-, J = 6 Hz).
N-[4-(2-N,N-Dimethylaminoethoxy)phenyl]-3-amino-1,
8-naphthalimide (22)
From 21 (0.5 g, 2.345 mmol) and 4-(2-N,N-dimethylamino-
ethoxy)aniline 13 (0.45 g, 2.50 mmol).Yield: 0.46 g, 52.27%.
4-(3-Nitro-1,8-naphthalimido)benzoyl chloride (29)
Compound 28 (0.25 g, 0.69 mmol) was refluxed in thionyl
chloride (3 ml) for 4 h under anhydrous conditions. Thionyl

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D.P. Jindal et al. / Il Farmaco 60 (2005) 283–290
chloride was removed under reduced pressure to afford a
brown colored solid 29 (0.25 g, 93.3%). IR (KBr): 1780
(C=O), 1745 (C=O stretch -aroyl chloride), 1670 (N-C=O)
cm–1.
4-(3-Nitro-1,8-naphthalimido)-N-(2-dimethylaminoethyl)-
benzamide (30)
From 2-dimethylaminoethyl chloride hydrochloride
(0.17 g, 1.18 mmol). IR (KBr): 3400, 3240 (N-H), 1710
(C=O), 1680 (N-C=O), 1120 (C-N stretch) cm–1. 1H-NMR: d
2.92 (s, 6H, -+N(CH3)2), 3.41 (m, 4H, -NH2), 3.33 (s, 4H,
5-CH2-piperidine-2,6-dione and -CH2-+N(CH3)2).
3-(4-Aminophenyl)-3-ethyl-1-(2-pyrrolidin-1-
ylethyl)piperidine-2,6-dione (39) hydrochloride
From 2-(N,N-dimethylamino)ethylamine (3 ml). Yield:
0.13 g, 45.8%. Melting point decompose at 290 °C. IR (KBr):
3310 (N-H), 1710 (C=O), 1670 (N-C=O), 1630 (amide C=O)
From 4-(2-chloroethyl)pyrrolidine hydrochloride (0.22 g,
1.29 mmol). IR (KBr): 3400, 3240 (N-H), 1710 (C=O), 1680
(N-C=O), 1120 (C-N stretch) cm–1. 1H-NMR: d 2.14 (m, 4H,
-CH2CH2- pyrrolidine), 3.21 (d, 4H, -+N(CH2)2 -pyrrolidine),
3.86 (m, 4H, -CH2-+N(CH2)2 -pyrrolidine and -NH2).
1
cm–1. H-NMR: d 2.32 (s, 6H, -N(CH3)2), 2.58 (t, 2H,
-CH2N(CH3)2, J = 6.2 Hz), 3.54 (q, 2H, -NHCH2-, J = 6.0 Hz).
4-(3-Nitro-1,8-naphthalimido)-N-(t-butyl)benzamide (31)
From t-butylamine (3 ml). Yield: 0.15 g, 54.7%. Melting
point decompose at 290 °C. IR (KBr): 3390 (N-H), 1710
(C=O), 1670 (N-C=O), 1640 (amide C=O) cm–1. 1H-NMR: d
1.48 (s, 9H, 3 × CH3).
4-(3-Nitro-1,8-naphthalimido)-N-pentylbenzamide (32)
From n-pentylamine (3 ml). Yield: 0.2 g, 70%. Melting
point decompose at 290 °C. IR (KBr): 3315 (N-H), 1710
(C=O), 1670 (N-C=O), 1630 (amide C=O) cm–1. 1H-NMR: d
0.93 (t, 3H, -CH3), 1.37 (m, 4H, -CH2CH2CH3), 1.64 (m,
2H, -NHCH2CH2-), 3.39 (q, 2H, -NHCH2).
2.2. Antineoplastic activity
2.2.1. 60-Cell line assay
The compounds (9, 10, 12, 18, 19, 23, 24 and 34–36)
selected by National Cancer Institute, Bethesda, USA have
been screened for their in vitro antitumor activity against the
cell panel consisting of 60-cell lines at a minimum of five
concentrations at 10-fold dilutions. A 48 h continuous expo-
sure protocol was used and a sulforhodamine B (SRB) pro-
tein assay was used to estimate cell viability or growth [25].
Two standard drugs, meaning that their activities against the
cell lines are well documented, are tested against each cell
line: NSC 19893 (5-Fluorouracil) and NSC 123127 (Adria-
mycin).
3-[4-(Phthalimido)phenyl]-3-ethyl-2,6-piperidinedione
(34)
From 6 (0.5 g). Yield: 0.8 g, 85.36%. Melting point 210–
212 °C. IR (KBr): 3192 (N-H), 1708 (C=O) cm–1. 1H-NMR:
d 0.90 (t, 3H, -CH3), 2.0 (m, 2H, -CH2CH3), 2.40 (m, 4H,
-CH2-CH2-piperidine-2,6-dione), 3.2 (s, 1H, NH, disap-
peared on D2O exchange), 7.56 (s, 4H, Ar), 8.00 (m, 4H, Ar).
3-[4-(4,5-Dimethoxyphthalimido)phenyl]-3-ethyl-2,6-
piperidinedione (35)
From 7 (0.44 g).Yield: 0.70 g, 76.96%. Melting point 282–
284 °C. IR (KBr): 3234 (N-H), 1710 (C=O) cm–1. 1H-NMR:
d 4.1 (s, 6H, -OCH3).
2.2.2. 3-Cell line assay
The compounds 37–39 have been selected and evaluated
by NCI for their antineoplastic activity using in vitro 3-cell
lines [MCF-7 (Breast), NCl-H460 (Lung) and SF-268 (CNS)]
one dose prescreen. In the current protocol, each cell line is
inoculated and preincubated on a microtiter plate. Test agents
are then added and the culture incubated for 48 h. End-point
determinations are made with alamar blue. Results for each
test agent are reported as the percent growth of the treated
cells when compared to the untreated control cells.
3-[4-(3-Nitro-1, 8-naphthalimido)phenyl]-3-ethyl-2,6-
piperidinedione (36)
From 11 (0.6 g, 2.46 mmol).Yield: 0.54 g, 48.2%. Melting
point 282–284 °C. IR (KBr): 3200 (N-H), 1685 (N-C=O),
1650 (amide C=O) cm–1. 1H-NMR: d 7.36 (d, 2H,Ar-H ortho
to piperidinedione ring, Jo = 8 Hz), 7.48 (d, 2H, Ar-H ortho
to imide functionality, Jo = 8 Hz).
2.3. Inhibition of aromatase in vitro
3-(4-Aminophenyl)-3-ethyl-1-(2-morpholin-4-
ylethyl)piperidine-2,6-dione (37) hydrochloride
From 4-(2-chloroethyl)morpholine hydrochloride (0.17 g,
0.86 mmol). IR (KBr): 3400, 3240 (N-H), 1710 (C=O), 1680
(N-C=O), 1120 (C-N stretch) cm–1. 1H-NMR: d 0.82 (t, 3H,
-CH2CH3), 1.99 (m, 2H, -CH2CH3), 2.39 (m, 2H, 4-CH2-
piperidine-2,6-dione), 2.71 (m, 2H, 5-CH2 piperidine-2,6-
dione) 3.16 (s, 2H, -+N(CH2)-morpholine), 3.34 (d, 2H,
N(CH2)2-morpholine) 3.68 (m, 2H, -OCH2 -morpholine), 3.97
(m, 2H, -OCH2-morpholine), 4.15 (m, 4H, -CH2- attached to
nitrogen of piperidine-2,6-dione ring and -CH2-+N-(CH2)2-),
7.33 (d, 2H, Jo = 8.34 Hz, Ar-H ortho to amino group), 7.53
(d, 2H, Jo = 8.44 Hz,Ar-H ortho to piperidine-2,6-dione ring),
11.6 (s, 1H, HCl).
2.3.1. Preparation of Aromatase
The enzyme was obtained from the microsomal fraction
of freshly delivered human term placental tissue according to
the procedure of Thompson and Siiteri [26]. The isolated
microsomes were suspended in the minimum volume of phos-
phate buffer (0.05 M, pH 7.4) and stored at –30 °C as
described [26]. No loss of activity was observed within
4 months.
2.3.2. Inhibition of aromatase
This assay was performed similar to described methods
[27,28] monitoring enzyme activity by measuring the 3H2O
formed from [1b,2b-3H] testosterone during aromatization.
Each incubation tube contained 0.225 µCi of (1b,2b-3H) tes-
3-(4-Aminophenyl)-1-(dimethylaminoethyl)-3-
ethylpiperidine-2,6-dione (38) hydrochloride

D.P. Jindal et al. / Il Farmaco 60 (2005) 283–290
287
tosterone, 5 µM unlabeled testosterone, 2 mM NADPH,
20 mM glucose-6-phosphate, 1EU glucose-6-phosphate dehy-
drogenase, and inhibitor (0–250 µM) in phosphate buffer
(0.05 M, pH 7.4). The test compounds had been dissolved in
ethanol and diluted with buffer. The final ethanol concentra-
tion of control and inhibitor incubation was 2%. Each tube
was preincubated for 5 min at 30 °C in a shaking water bath.
Microsomal protein (0.5 mg) was added to start the reaction.
The total volume for each incubation was 0.5 ml. The reac-
tion was terminated by withdrawing 100 µl aliquots at 0, 7,
14 and 21 min and pipetting them into 200 µl of a cold 1 mM
HgCl2 solution. After addition of 200 µl of an aqueous
dextran-coated charcoal (DCC) suspension (2%), the vials
were shaken for 20 min and centrifuged at 1500 g for 5 min
to separate the charcoal-adsorbed steroids. Aliquots of the
supernatant were assayed for 3H2O by counting in a scintil-
lation mixture in a Beckman liquid scintillation spectrometer
(LS 8000).
naphthalic anhydride 21 were condensed with appropriate
4-(2-dialkylaminoethoxy)aniline derivatives 13–16 in reflux-
ing pyridine to afford 17–20 and 22–24, respectively (Scheme
2). Similarly, 2,4-dinitrophenylhydrazone derivative 26 was
also prepared by treating 11 with 2,4-dinitrophenylhydrazine
25 (Scheme 2). Spectral studies and elemental data were in
agreement with the proposed structures.
Treatment of 11 with p-aminobenzoic acid (27) in pres-
ence of triethylamine in aldehyde free alcohol yielded 28,
1
which exhibited H-NMR signals at d 9.09 (d, 4-CH) and
9.48 ppm (d, 2-CH). Characteristic bands for six membered
imides appeared at 1710 and 1679 cm–1 in IR spectrum.
Refluxing 28 with thionyl chloride yielded 29, which showed
peak at 1745 cm–1 in IR spectrum characteristic for aroyl chlo-
rides. Compound 29 was treated with different amines in
dichloromethane at 5–8 °C to obtain compounds 30–32,
respectively (Scheme 3).
It was also thought worthwhile to condense anhydrides 6,
7 and 11 with aminoglutethimide 33, an established aro-
matase inhibitor, to have synergistic effects. This was achieved
by refluxing these anhydrides and 33 in pyridine to yield
phthalic anhydride derivatives 34, 35 and naphthalic anhy-
dride derivative 36, respectively (Scheme 4).A triplet of -CH3
group appeared at d 0.90 whereas two protons of -CH2 of
substituted ethyl group coupled separately with -CH3 and
4-CH2 of piperidinedione ring (long range coupling) to give
two multiplets in close proximity at d 2.0 (merged) for 34,
1.96 (merged) for 35 and 1.95 and 2.07 ppm for 36.
3. Results
3.1. Chemistry
Respective imidazoline derivatives 9, 10 and 12 were pre-
pared by treating ethanolic solutions of 2-hydrazino-1-
imidazoline hydrobromide 8 with phthalic anhydride (6),
dimethoxyphthalic anhydride 7 and 3-nitro-1,8-naphthalic
anhydride 11 and subsequent reflux in toluene (Scheme 1).
3-Nitro-1,8-naphthalic anhydride 11 and 3-amino-1,8-
N-Substituted aminoglutethimide derivatives were pre-
pared by treating 33 with various hydrochlorides of alkylami-
Scheme 1. Synthetic procedures of compounds 9, 10 and 12. Reagents and
conditions: (i) absolute alcohol, 80 °C, 1 h and (ii) absolute alcohol, triethy-
lamine, room temperature.
Scheme 2
. Synthetic procedures of compounds 17–20, 22–24 and 26. Rea-
gents and conditions: (i) pyridine, reflux.

288
D.P. Jindal et al. / Il Farmaco 60 (2005) 283–290
Cancer Institute, based in general, on the basis of degree of
novelty of the structure and computer modeling techniques,
were assayed in vitro against a panel consisting of 60 human
tumor cell lines, derived from nine cancers types (leukemia,
lung, colon, CNS, melanoma, ovarian, renal, prostate and
breast cancers), using five concentrations at 10-fold dilu-
tions, the highest being 10–4 M. Mean log dose response
parameters such as GI50 (drug concentration resulting in a
50% reduction in the net protein increase), TGI (drug con-
centration of total growth inhibition) and LC50 (concentra-
tion of drug resulting in a 50% reduction in the measured
protein at the end of the drug treatment as compared to that at
the beginning) are summarized in Table 1.
The aminoglutethimide derivatives 37–39 were tested using
a one dose (10–4 M) primary anticancer in vitro assay against
tumor in the three-cell line panel consisting of MCF7 (breast),
NCI-H460 (lung) and SF-268 (CNS) and the data are shown
in Table 2. Compounds, which reduce the growth of any one
of the cell lines to approximately 32% or less, are passed on
for evaluation in the full panel of 60-cell lines over a 5-log
dose range.
Scheme 3. Synthetic procedures of compounds 27–32. Reagents and condi-
tions: (i) triethylamine, aldehyde free alcohol; (ii) thionyl chloride and (iii)
dichloromethane, 5–8 °C, respective amines.
noethyl chlorides in acetone under anhydrous conditions to
afford 37–39 as oily residues, respectively (Scheme 4). Since
the oily residues could not be crystallized, the compounds
were characterized as their hydrochloride salts.
3.2. Antineoplastic activity
3.3. Aromatase inhibitory activity
The compounds 9, 10, 12, 18, 19, 23, 24 and 34–36
The aromatase inhibitory activity of aminoglutethimide
selected by Drug Synthesis and Chemistry Branch, National
derivatives 37–39 was determined in vitro using human pla-
Scheme 4. Synthetic procedures of compounds 34–39. Reagents and conditions: (i) pyridine, reflux and (ii) respective hydrochlorides of alkylaminoethyl
chlorides, potassium carbonate, potassium iodide, triethylamine, dry acetone.
Table 1
Mean log dose–response parameters such as GI50, TGI and LC50 in the 60-cell line screening
Compound
NSC No.
664226
664227
645467
679263
679264
682467
682468
663902
663901
679226
Mean log10 GI50 (Molar) Mean log10 TGI (Molar) Mean log10 LC50 (Molar)
9 (DPJ-231)
10 (DPJ-236)
12 (DPJ-229)
18 (DPJ-519)
19 (DPJ-525)
23 (DPJ-544)
24 (DPJ-545)
34 (DPJ-329)
35 (DPJ-327)
36 (DPJ-534)
–4.00
–4.00
–4.00
–5.12
–4.76
–4.77
–4.80
–5.00
–5.00
–4.80
–4.00
–4.00
–4.00
–4.57
–4.28
–4.44
–4.49
–5.00
–5.00
–4.26
–4.00
–4.00
–4.00
–4.18
–4.05
–4.15
–4.21
–5.00
–5.00
–4.05

D.P. Jindal et al. / Il Farmaco 60 (2005) 283–290
289
Table 2
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Acknowledgements
Thanks are due to University Grants Commission, New
Delhi, India for financial support and Panjab University, India
for providing necessary facilities. We also thank Dr. V.L.
Narayanan, NCI, Bethesda, USA for carrying out the antine-
oplastic activity.
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