Cytotoxic, antioxidant activities and structure activity relationship of some newly synthesized terpenoidal oxaliplatin analogs

Article abstract of DOI:10.1016/j.ejmech.2008.05.024

The terpenoidal oxaliplatin derivatives (6) and (12) were newly synthesized using 2β,3α-dihydroxy-11-oxo-18β-olean-12-ene-30-oic acid (1) and 2α,2β-dihydroxy-18β-ursan-12-ene-28-oic acid (7) as starting materials. The synthesized compounds were evaluated for their cytotoxicity and antioxidant activities and were compared to Oxaliplatin and vitamin C as positive controls. Some of the compounds exhibited better cytotoxicity and antioxidant activities than the reference controls. The detailed synthesis, spectroscopic data, toxicity (LD₅₀) and pharmacological screening for the synthesized compounds were reported.

Full text of DOI:10.1016/j.ejmech.2008.05.024

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 44 (2009) 901e907
http://www.elsevier.com/locate/ejmech
Original article
Cytotoxic, antioxidant activities and structure activity relationship
of some newly synthesized terpenoidal oxaliplatin analogs
a,*
a
Abd El-Galil E. Amr , Korany A. Ali , Mohamed M. Abdalla
b
a
Applied Organic Chemistry Department, National Research Centre, El Tahrir Street, Dokki, Cairo, Egypt
b
Research Unit, Hi-Care Pharmaceutical Co., Cairo, Egypt
Received 24 April 2008; received in revised form 22 May 2008; accepted 22 May 2008
Available online 7 July 2008
Abstract
The terpenoidal oxaliplatin derivatives (6) and (12) were newly synthesized using 2b,3a-dihydroxy-11-oxo-18b-olean-12-ene-30-oic acid (1)
and 2a,2b-dihydroxy-18b-ursan-12-ene-28-oic acid (7) as starting materials. The synthesized compounds were evaluated for their cytotoxicity
Ò
and antioxidant activities and were compared to Oxaliplatin and vitamin C as positive controls. Some of the compounds exhibited better cy-
totoxicity and antioxidant activities than the reference controls. The detailed synthesis, spectroscopic data, toxicity (LD50) and pharmacological
screening for the synthesized compounds were reported.
Ó 2008 Published by Elsevier Masson SAS.
Keywords: Terpenoid diol; Oxaliplatin derivatives; Cytotoxic; Antioxidant activity
1
. Introduction
biological evaluation against colorectal carcinoma in compar-
ison to Oxaliplatin as the reference drug. Also the antioxidant
activities of the newly synthesized compound were estimated
and compared to that of vitamin C as positive control. The
chemical structures of glycyrrhetinic acid and Oxaliplatin
are given in Fig. 1.
The 2-benzylidene derivatives of glycyrrhetinic acid and
the C-30 esters and amides of glycyrrhetinic acid were pre-
pared [1,2]. The benzylidene derivatives showed potent anti-
ulcer activities, while both the ester and amide derivatives
showed potent analgesic activities [1,2]. Further chemical al-
terations on glycyrrhetinic acid and oleanolic acid lead to
the synthesis of their 3-menthyl carbonyl derivatives that ex-
hibited potent anti-ulcer activities [3], also synthesis of the
ureides derivatives of both glycyrrhetinic acids and oleanolic
acid of moderate anti-inflammatory potency has been reported
2. Results and discussion
2.1. Chemistry
2b,3a-Dihydroxy-11-oxo-18b-olean-12-ene-30-oic acid (1)
and 2a,2b-dihydroxy-18b-ursan-12-ene-28-oic acid (7) were
synthesized and isolated according to the literature [5e7]
and were used as starting materials. The carboxylic acid deriv-
atives 1 and 7 were esterified with diazomethane in chloroform
to give the corresponding methyl ester derivatives 2 and 8 this
reaction was completed according to the method given by
Dean et al. [13] for the protection of the carboxylic group.
The methyl esters 2 and 8 were reacted with sodium azide
in the presence of methane sulfonyl chloride and triethylamine
to give the corresponding diazide derivatives 3 and 9 accord-
ing to the literature method [14e17]. The terpenoidal diamine
[
4]. El-Gamal et al. [5,6] described the synthesis of 2b,3a-gly-
col derivatives of glycyrrhetic acid but they didnot investigate
its anticancer activity. Recently Chao-Mei et al. [7] described
the isolation of 2a-hydroxyl-ursolic acid (7) from the ethyl ac-
etate extract of the peel of apples (Malus pumila Mill). In view
of these reports and in continuation of our previous works in
chemistry of natural products [8e12], we have synthesized
some new compounds containing a terpenoid ring system for
*
Corresponding author. Fax: þ202 33370 931.
E-mail address: aamr1963@yahoo.com (A.E.-GalilE. Amr).
0223-5234/$ - see front matter Ó 2008 Published by Elsevier Masson SAS.
doi:10.1016/j.ejmech.2008.05.024

902
A.E-G.E. Amr et al. / European Journal of Medicinal Chemistry 44 (2009) 901e907
COOH
sodium hydroxide, gave the final oxalate complexes 6 and
2 in good yields Schemes 1 and 2.
1
H
O
H
H2
N
2.2. Pharmacological screening
O
O
H
Pt
Initially, the acute toxicity of the compounds was assayed
via the determination of their LD . All compounds were in-
HO
O
O
N
H2
50
terestingly less toxic than the reference control (Table 1).
The 12 compounds 1e12 were further studied for their cyto-
toxic and antioxidant activities.
Glycyrrhetinic
Oxaliplatin
Fig. 1.
ligands 4 and 10 were isolated after catalytic hydrogenation of
diazide derivatives 3 and 9 over palladium charcoal (Pd/C) in
formic acid [18,19] (Schemes 1 and 2).
The diaminodichloroplatinum complexes 5 and 11 were
synthesized via the reaction of potassium tetrachloroplatinate
2.2.1. Cytotoxic activity
From Table 2, all the newly synthesized tested compounds
showed potent cytotoxic activities against HT-29 cell line (co-
lorectal carcinoma). The cytotoxic activities increase as the
doses increase, therefore the 150 mg/ml doses induced more
cell death than the 100 mg/ml doses and the latters induced
more cell death than 50 mg/ml doses. Also the cytotoxic activ-
ities increase by increasing the time of contact between cell
line and cytotoxic agents at any given dose level. So the cell
(
II) (K PtCl ) with diamine ligands 4 and 10. The reaction
2 4
of sodium oxalate with diaminodichloroplatinum complexes
and 11 in water in the presence of silver nitrate (AgNO3)
as a precipitating agent for chloride and in the presence of
5
COOH
COOCH3
O
O
CH N
2
2
HO
HO
HO
HO
CHCl3
1
2
CH SO Cl
TEA
3
2
NaN3
COOCH3
COOCH3
O
O
N3
H N
2
Pd/C
HCOOH
H N
N3
2
4
3
K PtCl4
2
COOCH3
O
H N
2
Cl
Cl
Pt
COOCH3
H N
2
5
O
COONa
O
O
NaOH
O
O
H N
2
COONa
AgNO3
Pt
H N
2
6
Scheme 1.

A.E-G.E. Amr et al. / European Journal of Medicinal Chemistry 44 (2009) 901e907
903
CH N
2
2
COOH
COOCH3
HO
HO
HO
HO
CHCl3
8
7
CH SO Cl
TEA
3
2
NaN3
COOCH3
N3
COOCH3
H N
2
Pd/C
HCOOH
H N
2
N3
10
9
K PtCl4
2
COOCH3
H N
2
Cl
Cl
Pt
H N
2
11
COOH
COOH
AgNO3
O
O
COOCH3
O
O
H N
NaOH
2
Pt
H N
2
12
Scheme 2.
death after 48 h is greater than that after 24 h and the cell
death after 24 h is higher than that after 12 h. The compounds
arranged in descending order of cytotoxic activity are 12, 6,
2.2.2. Antioxidant activities
The antioxidant activities of the newly synthesized com-
pounds were obtained by measuring the scavenging effects
of the synthesized compounds on the superoxide produced
by HX/XO system. We measure the scavenging activities of
the tested compounds at a dose level of 50 mg/ml.
From the results in Table 3, it was found that all compounds
showed antioxidant activities via inhibition of NBT reduction
in the following descending order 12, 6, 11, 5, 10, 4, 9, 3, 8
and 2.
1
1, 5, 10, 4, 9, 3, 8 and 2.
Table 1
Acute toxicity (LD50) of the synthesized compounds
Compound
LD50 (mg/kg)
1
2
3
4
5
6
7
8
9
656 ꢁ 0.62
793 ꢁ 0.78
898 ꢁ 0.78
893 ꢁ 0.88
864 ꢁ 0.89
871 ꢁ 0.85
816 ꢁ 0.88
818 ꢁ 0.82
825 ꢁ 0.86
836 ꢁ 0.85
846 ꢁ 0.89
822 ꢁ 0.99
223 ꢁ 0.28
2.2.3. Structureeactivity relationship (SAR)
ꢀ
The triterpenoid function is essential for the cytotoxic and
antioxidant activities, the ursane one induces higher activ-
ities than the olean one.
10
11
12
ꢀ The oxaloplatin moiety sharply increases the cytotoxic and
antioxidant activities.
The ring A 2,3-disubstituents play an important role in
regulating the cytotoxic and antioxidant activities. In
ꢀ
Oxaliplatin

904
A.E-G.E. Amr et al. / European Journal of Medicinal Chemistry 44 (2009) 901e907
Table 2
Cytotoxic effect on HT-29 at a concentration of 50, 100, 150 mg/ml after time
in hours
added with stirring at room temperature. The reaction mixture
was left overnight at the same temperature. The solvent was
evaporated under reduced pressure to dryness. The residue
was crystallized from methanol/chloroform to give the corre-
Compound
Cytotoxic effect at different concentrations
ꢂ
25
5
1
0 mg/ml
2 h 24 h
100 mg/ml
150 mg/ml
sponding methyl ester 2. Yield 96%, mp 106 C, [a] þ91
D
ꢃ1
1
48 h
12 h
24 h
48 h
12 h
24 h
(c 1, MeOH); IR (KBr, cm ): 1746, 1710 (2C]O);
H
NMR (DMSO-d ): d 0.78, 0.82 (2s, 6H, 2CH ), 0.86 (s, 3H,
2
3
4
5
6
8
9
19
29
40
50
65
22
33
44
52
70
20
21
31
43
52
70
29
35
47
54
75
23
33
36
45
55
73
35
40
50
57
77
33
39
42
48
58
75
40
44
56
60
80
40
41
46
50
63
77
44
48
60
65
83
40
42
52
58
65
79
50
55
62
70
86
44
40
55
65
73
81
53
61
70
75
88
50
52
60
70
76
88
58
66
75
78
89
50
6
3
CH ), 1.06 (s, 3H, CH ), 1.18 (s, 3H, CH ), 1.20e1.23 (m,
3
3
3
4H, 2CH ), 1.26 (s, 3H, CH ), 1.31 (s, 3H, CH ), 1.42e1.50
2
3
3
(m, 4H, 2CH ), 1.60e1.70 (m, 6H, 3CH ), 1.75 (m, 1H,
CH), 1.95 (m, 1H, CH), 2.41 (m, 1H, CH), 2.61 (m, 1H,
2
2
CH), 3.61 (m, 1H, CH), 3.75e3.78 (m, 2H, CH ), 4.42 (s,
2
1
1
1
0
1
2
3H, COOCH ), 4.50, 4.55 (2br s, 2H, 2OH, exchangeable
3
1
3
with D O), 5.78 (s, 1H, CH]C); C NMR (DMSO-d6):
2
d 39.11, 89.09, 91.12, 47.61, 55.12, 18.91, 37.0, 37.21,
Oxaliplatin
4
3
5.16, 40.13, 191.18, 128.61, 170.28, 48.55, 26.71, 23.41,
2.81, 53.17, 41.56, 43.31, 32.71, 31.27, 30.19, 18.91,
a descending order of activities, we found the oxaliplati-
nium, chloroplatinum, diamino, diazido and, lastly the
diol functional groups.
17.51, 23.27, 24.16, 30.81, 31.91, 108.7 (C1eC30), 62.17
þ
(OCH ); MS (EI): m/z 500 (25%) [M ]. Anal. Calcd for
3
C H O : C, 74.36; H, 9.66. Found: C, 74.30; H, 9.60.
31 48 5
3
1
.1.2. Synthesis of methyl 2b,3a-diazedo-11-oxo-18b-olean-
2-ene-30-oate (3)
3
. Experimental
To a mixture of ester (2) (20 mmol) and methane sulfonyl
3
.1. Chemistry
chloride (3.6 ml) in benzene (60 ml), triethylamine (1.8 ml)
was slowly added with stirring at room temperature for 1 h
and then filtered off. The filtrate was evaporated under reduced
pressure, the residue was dissolved in ethanol (75 ml) and then
sodium azide (2.4 g) was added. The reaction mixture was re-
fluxed for 4 h followed by periodic additions of water, stirred
at room temperature overnight, diluted with water and ex-
tracted with dichloromethane. The extracted part was washed
with water, dried over anhydrous calcium chloride, and evap-
orated under reduced pressure. The residue was suspended
with a solution of sodium hydroxide (32 g/100 ml water),
Melting points were determined on open glass capillaries
using a Electrothermal IA 9000 digital melting point apparatus
and are uncorrected. Elemental analyses were performed on
Elementar, Vario EL, Microanalytical Unit, Cairo University,
Egypt and were found within ꢁ0.4% of the theoretical values.
Infrared (IR) spectra were recorded on Carlzeise Spectropho-
tometer model ‘‘UR 10’’ spectrophotometer using the KBr
disc technique. The NMR spectra were recorded on Varian
Gemini 270 MHz spectrometer (DMSO-d ) and the chemical
6
shifts are given in d (ppm) downfield from tetramethylsilane
(TMS) as an internal standard. The mass spectra (MS) were
measured using a Finnigan SSQ 7000 mass spectrometer.
ꢂ
stirred at 45 C for 0.5 h, and extracted with dichloromethane.
The aqueous fraction was acidified with concentrated hydro-
chloric acid and extracted with dichloromethane, dried over
anhydrous calcium chloride, and then evaporated under re-
duced pressure. The residue was solidified with ethanol, fil-
tered off, dried and crystallized from ethanol to give the
corresponding diazide derivatives (3). Yield 81%, mp
3.1.1. Synthesis of methyl 2b,3a-dihydroxy-11-oxo-18b-
olean-12-ene-30-oate (2)
To a solution of acid derivative 1 (2.1 mmol) in dry chloro-
form (25 ml), ethereal solution of diazomethane (25 ml) was
ꢂ
25
ꢃ1
3
(
21 C, [a]D þ67 (c 1, MeOH); IR (KBr, cm ): 2231
1
N^N), 1742, 1715 (2C]O); H NMR (DMSO-d ): d 0.79,
Table 3
The % scavenging effect of compounds on superoxide at a dose 50 mg/ml
6
0.82 (2s, 6H, 2CH ), 0.88 (s, 3H, CH ), 1.10 (s, 3H, CH ),
3 3 3
1.18 (s, 3H, CH ), 1.20e1.24 (m, 4H, 2CH ), 1.25 (s, 3H,
CH ), 1.30 (s, 3H, CH ), 1.43e1.51 (m, 4H, 2CH ), 1.60e
Compound
% Scavenging at a 50 mg/ml
Relative potency
to vitamin C
3
2
3
3
2
1.68 (m, 6H, 3CH ), 1.76 (m, 1H, CH), 1.92 (m, 1H, CH),
2.43 (m, 1H, CH), 2.57 (m, 1H, CH), 3.66 (m, 1H, CH),
2
3
4
5
6
8
9
65.12
68.12
70.12
75.12
86.66
67.98
69.14
74.15
80.14
91.24
45.00
1.447
1.513
1.558
1.669
1.925
1.510
1.536
1.647
1.780
2.027
1.000
2
3.80e3.82 (m, 2H, CH ), 4.52 (s, 3H, COOCH ), 5.82 (s,
3
2
13
H, CH]C); C NMR (DMSO-d ): d 39.11, 48.71, 47.6,
7.71, 55.11, 18.91, 37.0, 37.19, 45.5, 40.16, 191.18,
28.41, 170.16, 48.33, 26.51, 23.4, 32.8, 55.16, 41.41,
1
3
1
6
10
11
12
43.31, 32.7, 31.17, 30.0, 18.9, 17.5, 23.21, 24.16, 30.83,
31.9, 168.7 (C1eC30), 62.16 (OCH ); MS (EI): m/z 550
3
þ
(
12%) [M ]. Anal. Calcd for C H N O : C, 67.61; H,
3
1 46 6 3
Vitamin C
8.42; N, 15.26. Found: C, 67.55; H, 8.36; N, 15.20.

A.E-G.E. Amr et al. / European Journal of Medicinal Chemistry 44 (2009) 901e907
905
3
.1.3. Synthesis of methyl 2b,3a-diamino-11-oxo-18b-
mixture was stirred for a period of one day at room temperature.
The precipitated silver chloride was filtered off, and then a mix-
ture of oxalic acid (240 mg, 2.70 mmol) in NaOH solution
(5.4 ml) was added with stirring. The reaction mixture was
stirred overnight at room temperature, the precipitate was fil-
tered off and dried under reduced pressure over P O to give
olean-12-ene-30-oate (4)
A mixture of the azide derivative 3 (10 mmol) and formic
acid (20 mmol) in ethanol (50 ml) was warmed, and then pal-
ladium (10% on carbon) was added slowly until evolution of
nitrogen gas. The mixture was filtered though Celite and the ex-
cess of ethanol was removed under reduced pressure. The res-
idue was dissolved in water, neutralized with sodium carbonate
2
5
the corresponding oxalo platinum complex (6). Yield 56%, mp
ꢃ1
ꢂ
25
328 C dec., [a]D þ71 (c 1, MeOH); IR (KBr, cm ): 3341
1
(
1 N) to pH w7, the product was extracted with methylene
(NH ), 1748, 1723, 1712 (4C]O); H NMR (DMSO-d ):
2
6
chloride, and dried over calcium chloride anhydrous. The sol-
vent was evaporated under reduced pressure to dryness to af-
ford the crude diamino terpenoidal derivative (4). Yield 69%,
d 0.79, 0.83 (2s, 6H, CH ), 0.88 (s, 3H, CH ), 1.02 (s, 3H,
3 3
CH ), 1.19 (s, 3H, CH ), 1.21e1.26 (m, 4H, 2CH ), 1.32 (s,
3
3
2
3H, CH ), 1.34 (s, 3H, CH ), 1.38e1.48 (m, 4H, 2CH ),
3
3
2
ꢂ
25
ꢃ1
mp 216 C, [a] þ128 (c 1, MeOH); IR (KBr, cm ):
1.54e1.61 (m, 6H, 3CH ), 1.79 (m, 1H, CH), 1.88 (m, 1H,
2
D
1
338e3324 (2NH ), 1747, 1712 (2C]O); H NMR (DMSO-
3
CH), 2.41 (m, 1H, CH), 2.48 (m, 1H, CH), 2.61 (m, 1H, CH),
3.11, 3.31 (2br s, 4H, 2NH , exchangeable with D O), 3.82
2
d ): d 0.78, 0.82 (2s, 6H, 2CH ), 0.84 (s, 3H, CH ), 1.04 (s,
6
3
3
2
2
3
H, CH ), 1.17 (s, 3H, CH ), 1.19e1.23 (m, 4H, 2CH ), 1.27
2
(m, 2H, CH ), 4.48 (s, 3H, COOCH ), 5.86 (s, 1H, CH]C);
2 3
C NMR (DMSO-d ): d 39.61, 48.91, 48.12, 37.91, 55.75,
6
3
3
1
3
(
s, 3H, CH ), 1.31 (s, 3H, CH ), 1.41e1.51 (m, 4H, 2CH ),
3
3
2
1
.58e1.67 (m, 6H, 3CH ), 1.78 (m, 1H, CH), 1.96 (m, 1H,
2
18.20, 37.13, 37.51, 45.73, 40.27, 191.18, 128.58, 171.28,
48.63, 26.41, 23.55, 32.88, 55.91, 41.38, 43.19, 32.71, 31.68,
30.15, 18.73, 17.39, 23.41, 24.72, 30.87, 31.26, 168.89 (C1e
CH), 2.40 (m, 1H, CH), 2.61 (m, 1H, CH), 3.18, 3.25 (2br s,
H, 2NH , exchangeable with D O), 3.84e3.86 (m, 2H,
CH ), 3.71 (m, 1H, CH), 4.34 (s, 3H, COOCH ), 5.76 (s, 1H,
4
2
2
C30), 62.13 (OCH ), 172.85, 173.60 (2CO); MS (EI): m/z 781
3
2
3
1
3
þ
CH]C); C NMR (DMSO-d ): d 34.41, 48.71, 47.91,
6
(8%) [M ]. Anal. Calcd for C H N O Pt: C, 50.70; H, 6.45;
33 50 2 7
3
1
4
3
7.91, 55.81, 18.83, 37.11, 37.39, 45.51, 40.17, 191.28,
28.53, 171.16, 48.56, 26.41, 23.39, 32.83, 55.56, 41.39,
3.21, 32.67, 31.61, 30.19, 18.79, 17.43, 23.19, 24.26, 30.93,
N, 3.58. Found: C, 50.65; H, 6.42; N, 3.52.
3.1.6. Synthesis of methyl-2a,3b-dihydroxy-18b-ursan-12-
ene-28-oate (8)
1.91, 169.12 (C1eC30), 62.63 (OCH ); MS (EI): m/z 498
3
þ
(5%) [M ]. Anal. Calcd for C H N O : C, 74.65; H, 10.10;
N, 5.62. Found: C, 74.60; H, 10.05; N, 5.56.
The same procedure for the preparation of compound 2 was
used with compound 7 as the starting material. Yield 71%, mp
3
1 50 2 3
ꢂ
25
ꢃ1
1
83 C, [a]D þ22 (c 1, MeOH); IR (KBr, cm ): 3340e3335
1
(2OH), 1742, 1705 (2C]O); H NMR (DMSO-d ): d 0.82,
3
1
.1.4. Synthesis of dichloro (methyl 2b,3a-diamino-11-oxo-
8b-olean-12-ene-30-oate) platinum (5)
6
0.86 (2s, 6H, 2CH ), 0.91 (s, 3H, CH ), 1.05 (m, 1H, CH),
3
3
A solution of 4 (7.22 mmol) and potassium tetrachloroplati-
1.11 (s, 3H, CH ), 1.18e1.24 (m, 4H, 2CH ), 1.28 (s, 3H,
3 2
nate(II) (3.00 g, 7.23 mmol) in 160 ml of water was stirred at
room temperature. A yellow solid was formed, which was fil-
tered off and dried under reduced pressure over P O to give
CH ), 1.36 (s, 3H, CH ), 1.41 (s, 3H, CH ), 1.43e1.55 (m,
3 3 3
4H, 2CH ), 1.60e1.71 (m, 4H, 2CH ), 1.75 (m, 1H, CH),
2
2
1.81 (m, 1H, CH), 1.95 (m, 1H, CH), 2.24e2.28 (m, 4H,
2CH ), 2.41 (m, 1H, CH), 3.78 (m, 1H, CH), 3.92 (m, 1H,
2
5
the corresponding dichloro compound (5). Yield 66%, mp
25
2
ꢂ
ꢃ1
2
(
71 C dec., [a] þ39 (c 1, MeOH); IR (KBr, cm ): 3341
CH), 4.48 (s, 3H, COOCH ), 4.50, 4.55 (2br s, 2H, 2OH, ex-
3
D
1
NH ), 1741, 1710 (2C]O); H NMR (DMSO-d ): d 0.79,
13
changeable with D O), 5.60 (m, 1H, CH]C); C NMR
2
6
2
0
1
.82 (2s, 6H, 2CH ), 0.86 (s, 3H, CH ), 1.06 (s, 3H, CH ),
3
(DMSO-d ): d 39.12, 89.19, 91.16, 47.18, 55.71, 18.33,
3
3
6
.19 (s, 3H, CH ), 1.22e1.25 (m, 4H, 2CH ), 1.28 (s, 3H,
3
37.16, 37.41, 45.19, 40.41, 40.41, 39.16, 128.36, 136.31,
48.61, 26.73, 23.61, 32.71, 53.76, 41.12, 44.17, 32.71, 32.16,
31.12, 19.83, 17.56, 23.67, 24.18, 169.17, 31.56, 30.18 (C1e
2
CH ), 1.32 (s, 3H, CH ), 1.41e1.50 (m, 4H, 2CH ), 1.58e
3
3
2
1
2
2
.62 (m, 6H, 3CH ), 1.81 (m, 1H, CH), 1.92 (m, 1H, CH),
2
þ
.39 (m, 1H, CH), 2.58 (m, 1H, CH), 3.14, 3.28 (2br s, 4H,
NH , exchangeable with D O), 3.68 (m, 1H, CH), 3.78e3.80
C30), 62.63 (OCH ); MS (EI): m/z 486 (5%) [M ]. Anal. Calcd
3
for C H O : C, 76.50; H, 10.35. Found: C 76.44; H, 10.28.
31 50 4
2
2
(
m, 2H, CH ), 4.51 (s, 3H, COOCH ), 5.84 (s, 1H, CH]C);
2 3
C NMR (DMSO-d ): d 39.58, 48.83, 48.11, 37.83, 55.73,
6
1
3
3.1.7. Synthesis of methyl-2a,3b-diazido-18b-olean-12-ene-
28-oate (9)
1
4
3
8.91, 37.12, 37.41, 45.61, 40.17, 191.28, 128.61, 171.17,
8.53, 26.36, 23.41, 32.91, 55.73, 41.41, 43.21, 32.67, 31.61,
0.15, 18.73, 17.41, 23.24, 24.16, 30.91, 31.16, 169.17 (C1e
The same procedure for the preparation of compound 3 was
used with compound 8 as the starting material. Yield 79%, mp
þ
ꢂ
25
ꢃ1
C30), 62.63 (OCH ); MS (EI): m/z 764 (15%) [M ]. Anal. Calcd
3
for C H Cl N O Pt: C, 48.69; H, 6.59; Cl, 9.27; N, 3.66.
3
152 C, [a]D þ117 (c 1, MeOH); IR (KBr, cm ): 2231
1
(N^N), 1738, 1712 (2C]O); H NMR (DMSO-d ): d 0.84,
1
50
2
2
3
6
Found: C, 48.62; H, 6.55; Cl, 9.22; N, 3.60.
0.89 (2s, 6H, 2CH ), 0.92 (s, 3H, CH ), 1.03 (m, 1H, CH),
3
3
1.13 (s, 3H, CH ), 1.17e1.25 (m, 4H, 2CH ), 1.26 (s, 3H,
3 2
3
.1.5. Synthesis of (methyl 2b,3a-diamino-11-oxo-18b-
CH ), 1.37 (s, 3H, CH ), 1.41 (s, 3H, CH ), 1.48e1.55 (m,
3 3 3
olean-12-ene-30-oate)oxalo platinum (6)
4H, 2CH ), 1.60e1.75 (m, 4H, 2CH ), 1.78 (m, 1H, CH),
2 2
To a suspension of 5 (2.84 mmol) in water (60 ml), silver ni-
trate (920 mg, 5.40 mmol) was added in one portion. The
1.81 (m, 1H, CH), 1.96 (m, 1H, CH), 2.25e2.30 (m, 4H,
2CH ), 2.40 (m, 1H, CH), 3.77 (m, 1H, CH), 3.91 (m, 1H,
2

906
A.E-G.E. Amr et al. / European Journal of Medicinal Chemistry 44 (2009) 901e907
1
CH), 4.46 (s, 3H, COOCH ), 5.60 (m, 1H, CH]C); C NMR
3
ꢂ
25
mp 347 C dec., [a]D þ116 (c 1, MeOH); IR (KBr, cm ):
ꢃ1
3
1
3356e3326 (NH ), 1740, 1728, 1712 (3C]O); H NMR
(
DMSO-d ): d 39.12, 48.63, 47.51, 47.18, 55.83, 18.41, 37.66,
6
2
3
2
1
6
7.93, 45.29, 40.41, 39.17, 128.46, 136.51, 48.66, 26.81,
3.57, 32.61, 53.17, 41.12, 44.17, 32.61, 32.17, 31.16,
9.81, 17.56, 23.77, 24.19, 169.17, 31.56, 30.19 (C1eC30),
(DMSO-d ): d 0.85, 0.89 (2s, 6H, 2CH ), 0.94 (s, 3H, CH ),
1.05 (m, 1H, CH), 1.17 (s, 3H, CH ), 1.20e1.24 (m, 4H,
3
6
3
3
2CH ), 1.30 (s, 3H, CH ), 1.36 (s, 3H, CH ), 1.42 (s, 3H,
2
3
3
þ
2.61 (OCH ); MS (EI): m/z 536 (2%) [M ]. Anal. Calcd
CH ), 1.49e1.54 (m, 4H, 2CH ), 1.59e1.64 (m, 4H, 2CH ),
3 2 2
3
for C H N O : C, 69.37; H, 9.01; N, 15.66. Found: C,
3
1.679 (m, 1H, CH), 1.85 (m, 1H, CH), 1.97 (m, 1H, CH),
2.24e2.30 (m, 4H, 2CH ), 2.38 (m, 1H, CH), 3.31, 3.47
1 48 6 2
6
9.32; H, 8.95; N, 15.60.
2
(2br s, 4H, 2NH , exchangeable with D O), 3.88 (m, 1H,
2 2
3
.1.8. Synthesis of methyl-2a,3b-diamino-18b-ursan-12-
CH), 3.97 (m, 1H, CH), 4.51 (s, 3H, COOCH ), 5.67 (m,
3
1
3
ene-28-oate (10)
1H, CH]C); C NMR (DMSO-d ): d 39.18, 48.56, 47.66,
6
The same procedure for the preparation of compound 4 was
used with compound 9 as the starting material. Yield 56%, mp
47.14, 55.46, 18.69, 37.66, 37.00, 45.17, 40.30, 39.16,
128.41, 136.71, 48.17, 26.78, 23.49, 32.61, 33.56, 41.13,
44.13, 35.60, 32.17, 31.17, 19.71, 17.51, 23.61, 24.13,
ꢂ
25
ꢃ1
2
75 C, [a] þ167 (c 1, MeOH); IR (KBr, cm ): 3363e3362
D
1
(
2NH ), 1743, 1714 (2C]O); H NMR (DMSO-d ): d 0.80,
169.22, 31.48, 30.15 (C1eC30), 63.11 (OCH ), 172.9, 173.2
2
6
3
þ
0
.87 (2s, 6H, CH ), 0.90 (s, 3H, CH ), 1.05 (m, 1H, CH),
3
(2CO, oxalate); MS (EI): m/z 767 (22%) [M ]. Anal. Calcd
for C H N O Pt: C, 51.62; H, 6.83; N, 3.65. Found: C,
51.56; H, 6.78; N, 3.60.
3
1
CH ), 1.35 (s, 3H, CH ), 1.42 (s, 3H, CH ), 1.44e1.52 (m,
.12 (s, 3H, CH ), 1.19e1.23 (m, 4H, 2CH ), 1.31 (s, 3H,
3
2
33 52 2 6
3
3
3
4
1
2
H, 2CH ), 1.58e1.65 (m, 4H, 2CH ), 1.78 (m, 1H, CH),
2 2
.86 (m, 1H, CH), 1.99 (m, 1H, CH), 2.22e2.28 (m, 4H,
CH ), 2.45 (m, 1H, CH), 3.21, 3.41 (2br s, 4H, 2NH , ex-
3.2. Biological methodology
2
2
changeable with D O), 3.79 (m, 1H, CH), 3.97 (m, 1H, CH),
2
1
.48 (s, 3H, COOCH ), 5.62 (m, 1H, CH]C); C NMR
DMSO-d ): d 39.12, 48.61, 47.78, 47.16, 55.51, 18.63,
6
3
4
(
3.2.1. Determination of acute toxicity (LD50)
The LD50 were determined by injection of increasing doses
of the tested compounds to adult male albino rats, and then the
dose causing 50% animals’ death was calculated according to
Austen et al. [20].
3
3
2
3
7.66, 37.51, 45.20, 40.39, 39.31, 128.46, 136.70, 48.53,
6.81, 23.60, 32.63, 53.66, 41.13, 44.13, 32.65, 32.17,
1.15, 19.71, 17.55, 23.66, 24.17, 169.16, 31.55, 30.18
þ
(C1eC30), 62.61 (OCH ); MS (EI): m/z 484 (6%) [M ].
Anal. Calcd for C H N O : C, 76.81; H, 10.81; N, 5.78.
3
3
1 52 2 2
Found: C, 76.76; H, 10.78; N, 5.72.
.1.9. Synthesis of dichloro (methyl-2a,3b-diamino-18b-
3.2.2. Determination of the cytotoxicity in HT-29 cells
3
3.2.2.1. Cell cultures. HT-29 cells were obtained from the Ko-
rean cell line Bank (KCLB, Seoul, Korea) and cultured in
ursan-12-ene-28-oate) platinum (11)
The same procedure for the preparation of compound 5 was
used with compound 10 as the starting material. Yield 49%,
DMEM supplemented 10% FBS, 100 mg/ml penicillin and
ꢂ
100 mg/ml streptomycin at 37 C under 5% CO atmosphere.
2
ꢂ
25
ꢃ1
6
ꢃ1
mp 211 C dec., [a] þ36 (c 1, MeOH); IR (KBr, cm ):
Cells (10 ml ) were grown in 35 mm culture dishes and in
96-well plates (Becton Dickinson Lab ware). The final vol-
umes of culture media were 2 ml for the 35 mm culture dish
and 100 ml for each well on the 96-well plate.
D
1
348e3330 (NH ), 1742, 1711 (2C]O); H NMR (DMSO-
3
2
d ): d 0.84, 0.88 (2s, 6H, 2CH ), 0.93 (s, 3H, CH ), 1.04 (m,
6
3
3
1
H, CH), 1.14 (s, 3H, CH ), 1.16e1.22 (m, 4H, 2CH ), 1.31
3 2
(
s, 3H, CH ), 1.37 (s, 3H, CH ), 1.40 (s, 3H, CH ), 1.47e
3 3 3
1
1
.53 (m, 4H, 2CH ), 1.58e1.63 (m, 4H, 2CH ), 1.76 (m,
2
H, CH), 1.84 (m, 1H, CH), 1.94 (m, 1H, CH), 2.26e2.32
3.2.2.2. Neutral red assay. All experiments were performed
2
6
ꢃ1
on the cultured HT-29 cells. Briefly, cells (10 ml ) were
exposed to 100 mU/ml GO in culture media containing
0.5% D-glucose without fetal bovine serum and then incu-
bated in the presence of various concentrations of heterocy-
clic compounds of thiazoldinone derivatives. After 2 days
of growth, a neutral red uptake assay was performed accord-
ing to the method of Wadsworth and Koop (1999) [21]. Cells
(
m, 4H, 2CH ), 2.41 (m, 1H, CH), 3.28, 3.38 (2br s, 4H,
2
2
NH , exchangeable with D O), 3.81 (m, 1H, CH), 3.94 (m,
2 2
1
3
1
NMR (DMSO-d ): d 39.17, 48.60, 47.76, 47.14, 55.48,
1
4
3
3
H, CH), 4.50 (s, 3H, COOCH ), 5.61 (m, 1H, CH]C);
C
3
6
8.59, 37.56, 37.60, 45.19, 40.30, 39.20, 128.40, 136.69,
8.15, 26.79, 23.51, 32.59, 33.60, 41.11, 44.10, 32.59,
2.11, 31.16, 19.69, 17.48, 23.57, 24.11, 169.11, 31.48,
6
ꢃ1
(10 ml ) were cultured in a 96-well plate for 12 and 24 h.
Following the various treatments, the medium was removed
and the cells were incubated in 100 ml of new medium con-
0.11 (C1eC30), 62.11 (OCH ); MS (EI): m/z 750 (14%)
3
þ
[M ]. Anal. Calcd for C H Cl N O Pt: C, 49.60; H, 6.98;
Cl, 9.44; N, 3.73. Found: C, 49.55; H, 6.92; Cl, 9.40; N, 3.68.
3
1
52
2 2 2
ꢂ
taining 10 mg/ml neutral red for 90 min at 37 C. After neu-
tral red treatment, the medium was removed and the wells
were washed three times with 100 ml PBS. One hundred mi-
croliters of 50% ethanol containing 50 mM sodium citrate
(pH 4.2) was added into each well on the plate. After
20 min, the absorbance was measured at 510 nm using
3
.1.10. Synthesis of (methyl-2a,3b-diamino-18b-ursan-12-
ene-28-oate) oxalo platinum (12)
The same procedure for the preparation of compound 6 was
used with compound 11 as the starting material. Yield 53%,

A.E-G.E. Amr et al. / European Journal of Medicinal Chemistry 44 (2009) 901e907
907
a Spectra Count TM (Packard Instrument Co., Downers
Grove, USA) ELISA reader.
[2] M.H.A. El-Gamal, H.M. Safwat, S.R. Benawy, M.M. Abdulla, Bull. Fac.
Pharm. Cairo Univ. 38 (2000) 191e196.
[
3] M.H.A. El-Gamal, H.M. Safwat, M.M. Hussein, M.M. Abdulla, Bull.
Fac. Pharm. Cairo Univ. 40 (2002) 41e46.
3
.2.3. Determination of antioxidant activity
Superoxide radicals were generated by xanthine/xanthine
[
4] M.M. Abdulla, Mans. Sci. Bull. (A Chem) 29 (2000) 101e124.
5] M.H.A. El-Gamal, B.A.H. El-Tawil, M.B.E. Fayez, Tetrahedron 30
(1974) 4083e4087.
[
oxidase (XO) and measured by the Nitroblue Tetrazolium
NBT) reduction method. A test sample was mixed in
a 100 mM phosphate buffer solution (pH w7.0) containing
[
6] M.H.A. El-Gamal, B.A.H. El-Tawil, M.B.E. Fayez, J. Pharmaceutical,
Science 62 (1973) 1557e1558.
[7] M. Chao-Mei, Q.C. Shao, R.C. Jing, Q.W. Rui, F. Tu, H. PengMasao,
(
ꢃ2
ꢂ
XO (1.65 ꢄ 10 units/ml) and NBT (133 mM) at 25 C in
D. Mohsen, Eur. J. Med. Chem. 40 (2005) 582e589.
8] A.E. Amr, M.M. Abdalla, Bioorg. Med. Chem. 14 (2006) 4341e4352.
96-well flat-bottomed microassay plates. The measurement
was started by adding xanthine (164 mM). Production of super-
[
[9] A.E. Amr, M.M. Abdalla, Arch. Pharm. Chem. Life Sci. 339 (2006) 88e95.
10] A.E. Amr, N.A. Abdel-Latif, M.M. Abdalla, Acta Pharm. 56 (2006)
203e218.
[
[
[
oxide radical was followed spectrophotometerically at 560 nm
ꢂ
at 25 C for 10 min. The superoxide scavenging activity was
calculated according to the following formula:
11] A.E. Amr, N.A. Abdel-Latif, M.M. Abdalla, Bioorg. Med. Chem. 14
(
12] N.A. Abd El-Latif, M.A. Abdulla, A.E. Amr, Acta Pharm. 58 (2008) 43e59.
2006) 373e384.
Superoxide scavenging activityð%Þ
Absorbancecontrol ꢃ Absorbancesample
[13] P.D.G. Dean, T.G. Halsall, M.W. Whitehouse, J. Pharm. Pharmacol. 19
1967) 533e544.
[14] G. Swift, D.J. Swern, Org. Chem. 32 (1967) 511e517.
15] A. Scheurer, P. Mosset, R.W. Saalfrank, Tetrahedron: Asymmetry 8
1997) 1243e1251.
[16] A.G. Schultz, J.P. Dittami, S.O. Myong, C.K. Sha, J. Am. Chem. Soc.
05 (1983) 3273e3276.
(
¼
ꢄ 100
Absorbancecontrol
[
(
where Absorbancecontrol and Absorbancesample represent the in-
creased absorbance in the absence and presence of samples,
respectively.
1
[
[
17] E.F.V. Scriven, K. Turnbull, Chem. Rev. 88 (1988) 297e368.
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9
[
20] R.F. Austen, W.E. Broklehurst, J. Exp. Med. 113 (1961) 521e525.
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[1] M.H.A. El-Gamal, H.M. Safwat, S.R. Benawy, M.M. Abdulla, Bull. Fac.
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[