Synthesis and anti-inflammatory activities of new cyanopyrane derivatives fused with steroidal nuclei

Article abstract of DOI:10.1002/ardp.200500209

Thirteen new heterocyclic derivatives containing a cyanopyrane ring fused to a steroidal moiety were conveniently synthesized and screened for their anti-inflammatory potencies comparable to that of the glucocorticoid prednisolone. Four compounds 5a, 5b,

Full text of DOI:10.1002/ardp.200500209

88
Arch. Pharm. Chem. Life Sci. 2006, 339, 88–95
Full Paper
Synthesis and Anti-inflammatory Activities of New
Cyanopyrane Derivatives Fused with Steroidal Nuclei
Abdel-Galil E. Amr1, Mohamed M. Abdulla2
1 Organic Chemistry Department, National Research Center, Cairo, Egypt
2 Research Unit, Egyptian Pharmacist Co., Cairo, Egypt
Thirteen new heterocyclic derivatives containing a cyanopyrane ring fused to a steroidal moiety
were conveniently synthesized and screened for their anti-inflammatory potencies comparable
to that of the glucocorticoid prednisolone. Four compounds 5a, 5b, 6b, and 8 exhibited superior
anti-inflammatory indices (in rats, protection against carrageenan induced edema and inhibi-
tion of plasma PGE). All the candidates were less toxic than the reference drug concerning LD50
values. Synthetic steroidal structures fused to a substituted cyanopyrane ring seem to be a pro-
mising approach in search for novel leads for potent anti-inflammatory agents.
Keywords: Cyanopyranes / Steroidal derivatives / Anti-inflammatory Agents /
Received: September 13, 2005; accepted: October 18, 2005
DOI 10.1002/ardp.200500209
Introduction
Natural steroids and their synthetic congeners were
extensively studied during the last decades [1, 2]. Sex hor-
Figure 1. Chemical structure of
prednisolone (I).
mones, cardiacs, diuretics, antibiotics, neuromusculars
are some representative examples. In particular, the ster-
oidal antiphlogistic activity, namely the anti-inflamma-
tory characteristics are still viewed as an active area of
the bioorganic medicinal chemistry literature. Regard-
ing their salt retention and gastric and peptic ulceration
the search for more potent synthetic agents represents a
challenging problem [2, 3].
In a previous work, we reported the synthesis of some
substituted pyrane and pyridine derivatives as analgesic,
anticonvulsant, anti-Parkinsonian, and antimicrobial
agents [4–8]. The androgenic, anabolic, and anti-inflam-
matory activities of many heterocyclic steroidal deriv-
atives were similarly reported [9]. On the other hand, cya-
nopyridones have promising antimicrobial [10, 11] and
anticancer [12–14] activities. Recently, we reported the
synthesis of some new chiral heterocyclic compounds as
anticancer and anti-inflammatory agents [15, 16].
In view of these reports and in continuation of our pre-
vious works in heterocyclic chemistry, we suggested and
newly synthesized thirteen cyanopyrane heterocyclics
fused with steroidal nuclei. The evaluation of the anti-
inflammatory potency of the synthesized compounds
was realized comparable to the synthetic corticosteroid
prednisolone (I, protection against carragenan induced
edema and inhibition of plasma prostaglandin E2). For
the structure of prednisolone see Figure 1.
Results and discussions
Correspondence: Abd El-Galil E. Amr, Applied Organic Chemistry De-
partment, National Research Center, Dokki, Cairo 12622, Egypt.
E-mail: aamr1963@yahoo.com
Chemistry
Arylmethylene of epiandrosterones 1a, b and the dehy-
droepiandrosterone 2 were prepared according to the lit-
Fax: +20 2 337-0931
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Arch. Pharm. Chem. Life Sci. 2006, 339, 88–95
Anti-inflammatory Activities of Cyanopyrane Derivatives
89
Scheme 1. Synthetic routes for com-
pounds 3–6.
erature [4, 17]. Condensation of 1a, b with ethyl cyanoace- aluminum isopropoxide resulted in the 3-oxo-analogue
tate in the presence of sodium ethoxide gave the corre- with delocalization of (D5-ene into (D4-ene derivative 10.
sponding cyanopyranes 3a, b, which were then reacted Modified Oppenauer oxidation [19] of 7 afforded the cor-
with p-toluene sulfonyl chloride (PTSCl) to afford the responding (D4,6-3-oxo-analogue 11 (Scheme 2).
cyclohexene (Ring A) derivatives 4a, b, respectively. Oxi-
dation of compounds 3a, b with Moffat oxidizing agents Pharmacological Screening
afforded the corresponding 3-oxo-analogue derivatives Anti-inflammatory potency
5a, b. Compounds 5a, b were oxidized with dichlorodi- Initially the acute toxicity of the compounds was assayed
cyanoquinone (DDQ) [18] to the corresponding 3-oxo- determining their LD50. Interestingly, all the compounds,
diene derivatives, 6a, b (Scheme 1).
except for 8, were less toxic than the reference drug
The dehydroepiandrosterone 2 was similarly reacted (Table 1). Then the newly synthesized compounds were
with ethyl cyanoacetate in the presence of sodium ethox- pharmacologically screened for their anti-inflammatory
ide to yield the corresponding cyanopyrane 7, which potency using male albino rats (Tables 2 and 3).
then was reacted with p-toluene sulfonylchloride to
afford the corresponding cyclohexene derivative 8. Mof- Purpose and rational
fat oxidation of compound 7 gave the 3-oxo analogue 9 For the determination of the antiphlogistic potency of
without affecting (D5-ene. Oppenauer oxidation of 7 with the synthesized compounds, two standard tests were rea-
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A.-G. E. Amr et al.
Arch. Pharm. Chem. Life Sci. 2006, 339, 88–95
Scheme 2. Synthetic routes for
compounds 7–11.
Table 1. Acute toxicity (LD50) of the synthesized compounds.
lized at 25 and 50 mg/kg body weight of the rats, namely
the protection against carragenan-induced edema
according to Winter et al. [20] and the inhibition of
plasma PGE2. The later is known as a good confirming
indicator for the carragenan-induced rat paw edema [21].
Regarding the protection against carragenan-induced
edema, four compounds, namely 5a, 6b, 8a, and 5b, were
found more potent (l120%) than prednisolone in a des-
cending order (Figure 2). For these compounds, a similar
but not identical activity profile was realized for the inhi-
bition of plasma PGE2, however, with an inversed order
for 8 and 5b.
Compound No
LD50 [mg/kg]
9
3a
5a
11b
12b
10
6b
6a
5b
3b
11
10
4b
4a
4.186
3.813
3.111
3.111
3.068
2.868
2.751
2.684
2.681
2.514
2.280
2.191
1.880
1.711
1.618
1.252
It is noteworthy that, regardless of the substitution on
the cyanopyrane ring, Moffat oxidation of the synthe-
sized structures was associated with a notable increase in
the anti-inflammatory activity. This was exemplified by
Prednisolone
8
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Arch. Pharm. Chem. Life Sci. 2006, 339, 88–95
Anti-inflammatory Activities of Cyanopyrane Derivatives
91
Table 2. Anti-inflammatory potencies of the synthesized com-
pounds (protection against carragenan-induced edema).
Table 3. Anti-inflammatory potencies of the synthesized com-
pounds (inhibition of plasma PGE2).
Compound No
Dose
[mg/kg]
Protection against
carragenan-in-
Compound No
Dose
[mg/kg]
Inhibition of plas-
ma PGE2 [%]
duced edema [%]
4b
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
–
4a
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
–
26.88
–
28.55
–
31.66
–
42.18
–
49.78
40.90
45.00
44.60
58.60
48.16
79.77
71.18
83.65
77.00
91.00
81.16
93.18
83.63
95.65
95.32
97.18
95.85
96.18
34.19
–
39.19
–
39.38
–
54.12
–
56.28
47.50
51.60
58.90
61.80
65.80
88.16
77.18
88.14
81.00
93.00
85.62
98.12
86.55
98.14
98.88
99.41
98.91
99.11
4a
3a
3a
4b
3b
10
10
3b
6a
6a
11
11
7
7
9
9
Prednisolone
Prednisolone
8
5b
8
5b
6b
5a
6b
5a
Figure 2. Structures of compounds with potent
anti-inflammatory activities.
compound 7 oxidized to yield 9 and more pronounced in significantly potent candidates 5, comparable to the
the case of compounds 3a, b upon oxidation to 5a, b, reference drug prednisolone (Figure 3). Further oxidation
respectively. In the latter case, the almost inactive (DDQ) of 5b to 6b was also accompanied with an
(regarding anti-inflammatory) compounds 3 afforded the enhanced activity.
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Arch. Pharm. Chem. Life Sci. 2006, 339, 88–95
1.60–1.80 (m, 4H, 2CH2), 1.95 (m, 1H, CH), 2.20–2.40 (m, 2H,
CH2), 2.46 (s, 3H, Ph-CH3), 2.52 (m, 1H, CH), 3.10 (m, 1H, 5a-CH),
3.56 (m, 1H, 3a–CH), 7.28–7.41 (m, 4H, Ar-H), 10.45 (s, 1H, OH,
exchangeable with D2O). MS m/z (%): 457 (M+, 100), correspond-
ing to the molecular formula C30H35NO3 and also as base peak.
Synthesis of 2-oxo-3-cyano-6-substituted-androst-3-
ene[17,16-c]pyrane 4a and 4b
Figure 3. Anti-inflammatory activities of compounds tested.
A mixture of compounds 3a, b (5 mmol), p-toluene sulfonyl
chloride (0.4g, 5 mmol), and triethyl amine (1 mL) in dry ben-
zene (15 mL) was refluxed for 2 h. The solvent was evaporated
under reduced pressure. The obtained residue was solidified
with water and the solid formed was filtered off, washed with
water, and dried, then it was dissolved in dry benzene (15 mL)
and potassium tert-butoxide (25 mL, 0.5N, in DMSO) was added.
The reaction mixture was heated at 508C for 5 h, The formed
solid was filtered off and crystallized from the proper solvent to
give the corresponding oxidized cyanopyrane derivatives 4a, b,
respectively (Table 4).
Experimental
Chemistry
Melting points were determined on open glass capillaries using
an Electrothermal IA 9000 SERIES digital melting point appara-
tus (Electrothermal, Essex, U.K.) and are uncorrected. Elemental
analyses were performed with all final compounds on Elemen-
tar, Vario EL, Microanalytical Unit, National Research Centre,
Cairo Egypt and were found within l0.4% of the theoretical
values. Analytical data were obtained from the Microanalytical
Unit, Cairo University, Egypt. The IR spectra (KBr) were recorded
on a FT IR-8201 PC spectrophotometer (Shimadzu, Tokyo, Japan).
2-Oxo-3-cyano-6-(4-fluorophenyl)androst-3-ene-
[17,16-c]pyrane 4a
IR (KBr, cm– 1): 2235 (CN), 1735 (C=O). 1H-NMR (DMSO-d6) d: 0.77 (s,
3H, CH3, C-19), 0.83 (s, 3H, CH3, C-18), 0.88–0.95 (m, 1H, CH),
1.15–1.25 (m, 4H, 2CH2), 1.40–1.54 (m, 4H, 2CH2), 1.58–1.75 (m,
4H, 2CH2), 1.90 (m, 1H, CH), 2.25–2.40 (m, 2H, CH2), 2.56 (m, 1H,
CH), 3.25 (m, 1H, CH, C-5), 5.30 (d, 1H, CH-olefinic, C-3), 5.45 (d,
1H, CH-olefinic, C-4), 7.25-7.31 (m, 4H, Ar-H). MS m/z (%): 443 (M+,
45), corresponding to the molecular formula C29H30FNO2 and at
280 (100, base peak).
1
The H-NMR spectra were measured with Jeol FTGNM-EX 270,
270 MHz instrument (Jeol, Tokyo, Japan) in DMSO-d6 and the che-
mical shifts were recorded in (d, ppm) relative to TMS. The Mass
spectra were run at 70 eV with a Finnigan SSQ 7000 spectro-
meter (Thermo electron corporation, Madison, WI, USA) using EI
and the values of m/z are indicated in Dalton. TLC (Silica gel, alu-
minum sheets 60 F254, Merck, Darmstadt, Germany) followed the
reactions. The starting materials 1a, b and 2 were prepared
according to the published procedure [4, 17].
2-Oxo-3-cyano-6-(4-methylphenyl)androst-3-ene-
[17,16-c]pyrane 4b
Synthesis of 2-oxo-3-cyano-substituted-
IR (KBr, cm– 1): 2226 (CN), 1732 (C=O). 1H-NMR (DMSO-d6) d: 0.76 (s,
3H, CH3, C-19), 0.81 (s, 3H, CH3, C-18), 0.90–0.95 (m, 1H, CH),
1.10–1.25 (m, 4H, 2CH2), 1.35–1.55 (m, 4H, 2CH2), 1.60–1.80 (m,
4H, 2CH2), 1.85 (m, 1H, CH), 2.20–2.42 (m, 2H, CH2), 2.48 (s, 3H,
Ph-CH3), 2.58 (m, 1H, CH), 3.30 (m, 1H, CH, C-5), 5.42 (d, 1H, CH-
olefinic, C-3), 5.48 (d, 1H, CH-olefinic, C-4), 7.18–7.23 (m, 4H, Ar-
H). MS m/z (%): 439 (M+, 25), corresponding to the molecular for-
mula C30H33NO2 and at 280 (100, base peak).
androsteno[17,16-c]pyran-3b-ol 3a and 3b
A mixture of 1 (10 mmol) and ethyl cyanoacetate (1.27 mL, 12
mmol) in 25 mL sodium ethoxide (920 mg sodium metal in 25
mL absolute ethanol) was refluxed for 7 h. The reaction mixture
was poured into ice water, the obtained solid was filtered off,
washed with water, dried, and crystallized from the proper sol-
vent to give the corresponding pyrano-steroidal derivatives 3a,
b, respectively (Table 4).
Synthesis of 2-oxo-3-cyano-6-(4-fluorophenyl)-
androstano[17,16-c]pyran-3-one 5a band 5b (Moffat
Oxidation)
2-Oxo-3-cyano-(4-fluorophenyl)androsteno[17,16-c]-
pyran-3b-ol 3a
IR (KBr, cm– 1): 3418 (OH), 2238 (CN), 1738 (C=O). 1H-NMR (DMSO-
d6) d: 0.81 (s, 3H, CH3, C-19), 0.83 (s, 3H, CH3, C-18), 0.95–1.00 (m,
1H, CH), 1.24–1.28 (m, 4H, 2CH2), 1.40–1.60 (m, 6H, 3CH2), 1.65-
1.85 (m, 4H, 2CH2), 1.98 (m, 1H, CH), 2.28–2.45 (m, 2H, CH2), 2.50
(m, 1H, CH), 3.00 (m, 1H, 5a-CH), 3.55 (m, 1H, 3a–CH), 7.23–7.50
(m, 4H, Ar-H), 10.55 (s, 1H, OH, exchangeable with D2O). MS m/z
(%): 461 (M+, 35), corresponding to the molecular formula
C29H32FNO3 and at 323 (100, base peak).
The appropriate compounds 3a, b (2 mmol) were dissolved in a
mixture of benzene (3 mL), dimethylsulphoxide (3 mL), pyridine
(0.16 mL), and trifluoroacetic acid (TFA, 0.08 mL). Dicyclohexyl-
carbodiimide (1.24 g, 6 mmol) was then added and the reaction
mixture was kept overnight at room temperature. Ether (50 mL)
was added followed by oxalic acid (0.54 g, 6 mmol) in methanol
(50 mL). After 30 minutes, water (50 mL) was added and the
obtained dicyclohexyl urea was removed by filtration. The fil-
trate was extracted with ether, washed with 5% sodium bicarbo-
nate, then with water. The ethereal solution was dried over
anhydrous sodium sulphate and evaporated under reduced pres-
sure. The formed residue was finally crystallized from the proper
solvent to give the corresponding oxo-compounds 5a, b, respec-
tively (Table 4).
2-Oxo-3-cyano-(4-methylphenyl)androsteno[17,16-c]-
pyran-3b-ol 3b
IR (KBr, cm-1): 3433 (OH), 2231 (CN), 1741 (C=O). 1H-NMR (DMSO-
d6) d: 0.78 (s, 3H, CH3, C-19), 0.82 (s, 3H, CH3, C-18), 0.90–0.95 (m,
1H, CH), 1.20–1.25 (m, 4H, 2CH2), 1.38–1.55 (m, 6H, 3CH2),
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Anti-inflammatory Activities of Cyanopyrane Derivatives
93
Table 4. Physico-chemical data of newly synthesized compounds.
Compound Ar
No
Yield
[%]
Mp.
[8C]
[a]D25
(c 1, MeOH)
Color and Solvent
for crystallization
Mol. Formulaa)
(Mol. wt)
3a
3b
4a
4b
5a
5b
6a
6b
7
8
9
10
11
C6H4-p-F
C6H4-p-CH3
C6H4-p-F
C6H4-p-CH3
C6H4-p-F
C6H4-p-CH3
C6H4-p-F
C6H4-p-CH3
–
–
–
–
–
72
65
55
70
72
75
58
77
72
62
56
68
82
271
+ 88
+ 76
+ 98
+ 84
+ 88
+ 96
+ 83
+ 66
+ 96
+ 69
+ 88
+ 95
+ 69
Yellow MeOH
Pale yellow AcOEt
White EtOH
Orange MeOH
Yellow MeOH
Brown MeOH
White MeOH
Pale yellow MeOH
Orange MeOH-AcOEt (2:1) C29H30ClNO3 (476.01)
White MeOH
Yellow MeOH
C29H32FNO3 (461.57)
C30H35NO3 (457.61)
C29H30FNO2 (443.56)
C30H33NO2 (439.59)
C29H30FNO3 (459.56)
C30H33NO3 (455.59)
C29H26FNO3 (455.52)
C30H29NO3 (451.56)
283
A320
310
239
166
234
209
189
119
261
130
243
C29H28ClNO2 (457.99)
C29H28ClNO3 (473.99)
C29H28ClNO3 (473.99)
C29H26ClNO3 (471.98)
Brown MeOH
Yellow MeOH-AcOEt (2:1)
a)
Confirmed by elemental analysis showing values within (0.4% of the theoretical values unless otherwise stated.
1.40–1.60 (m, 2H, CH2), 1.65–1.70 (m, 2H, CH2), 1.75 (m, 1H, CH),
1.85–2.00 (m, 2H, CH2), 2.30–2.45 (m, 1H, CH), 5.75 (d, 1H, CH-
olefinic, C-2), 5.87 (s, 1H, CH-olefinic, C-4), 6.91 (d, 1H, CH-olefi-
nic, C-1), 7.18–7.29 (m, 4H, Ar-H). MS m/z (%): 455 (M+, 45), corre-
sponding to the molecular formula C29H26FNO3 and at 292 (100,
base peak).
2-Oxo-3-cyano-6-(4-fluorophenyl)androstano[17,16-
c]pyran-3-one 5a
IR (KBr, cm– 1): 2248 (CN), 1735 (C=O).1H-NMR (DMSO-d6) d: 0.77 (s,
3H, CH3, C-19), 0.88 (s, 3H, CH3, C-18), 1.05–1.15 (m, 1H, CH),
1.22–1.28 (m, 4H, 2CH2), 1.40–1.61 (m, 6H, 3CH2), 1.64–1.90 (m,
4H, 2CH2), 1.98 (m, 1H, CH), 2.25–2.38 (m, 2H, CH2), 2.52 (m, 1H,
CH), 3.05 (m, 1H, 5a-CH), 7.28-7.39 (m, 4H, Ar-H). MS m/z (%): 459
(M+, 100, base peak), corresponding to the molecular formula
C29H30FNO3.
2-Oxo-3-cyano-6-(4-methylphenyl)androst-4,6-
diene[17,16-c]pyran-3-one 6b
IR (KBr, cm– 1): 2241 (CN), 1778–1767 (C=O, enone), 1728 (C=O,
ketone). 1H-NMR (DMSO-d6) d: 0.77 (s, 3H, CH3, C-19), 0.81 (s, 3H,
CH3, C-18), 1.15–1.20 (m, 1H, CH), 1.24–1.32 (m, 4H, 2CH2),
1.36–1.58 (m, 2H, CH2), 1.60–1.70 (m, 2H, CH2), 1.76 (m, 1H, CH),
1.90–2.05 (m, 2H, CH2), 2.32 (s, 3H, Ph-CH3), 2.35–2.45 (m, 1H,
CH), 5.71 (d, 1H, CH-olefinic, C-2), 5.91 (s, 1H, CH-olefinic, C-4),
6.89 (d, 1H, CH-olefinic, C-1), 7.21–7.31 (m, 4H, Ar-H). MS m/z (%):
451 (M+, 100, base peak), corresponding to the molecular for-
mula C30H29NO3.
2-Oxo-3-cyano-6-(4-methylphenyl)androstano[17,16-
c]pyran-3-one 5b
IR (KBr, cm-1): 2231 (CN), 1738 (C=O). 1H-NMR (DMSO-d6) d: 0.78 (s,
3H, CH3, C-19), 0.88 (s, 3H, CH3, C-18), 1.05–1.10 (m, 1H, CH),
1.24–1.30 (m, 4H, 2CH2), 1.42–1.60 (m, 6H, 3CH2), 1.62–1.92 (m,
4H, 2CH2), 2.10 (m, 1H, CH), 2.34 (s, 3H, Ph-CH3), 2.38–2.40 (m,
2H, CH2), 2.50 (m, 1H, CH), 3.15 (m, 1H, 5a-CH), 7.21–7.38 (m, 4H,
Ar-H). MS m/z (%): 455 (M+, 16), corresponding to the molecular
formula C30H33NO3 and at 296 (100, base peak).
Synthesis of 2-oxo-3-cyano-6-(4-chlorophenyl)androst-5-
Synthesis of 2-oxo-3-cyano-6-substituted-androst-4,6-
ene[17,16-c]-pyran-3b-ol 7
The compound was prepared by the method given for com-
pounds 3, using compound 2 as starting material (Table 4). IR
diene[17,16-c]pyran-3-one 6a and 6b
Anhydrous hydrogen chloride gas was bubbled into a solution of
compound 5a, b (6 mmol) and dichlorodicyanoquinone (DDQ)
(1.8 g, mmol) in dioxane (40 mL). The mixture was kept for 30
minutes at room temperature. The precipitated hydroquinone
was then removed by filtration. The filtrate was extracted with
ether. The ethereal solution was washed with 1% sodium hydro-
xide solution, then water and dried over anhydrous sodium sul-
fate. The solvent was evaporated under reduced pressure; the
residue formed was crystallized from methanol to give the corre-
sponding cyclohexyldiene derivatives 6a, b, respectively (Table
4).
1
(KBr, cm– 1): 3518–3416 (OH), 2248 (CN), 1723 (C=O). H-NMR
(DMSO-d6) d: 0.76 (s, 3H, CH3, C-19), 0.81 (s, 3H, CH3, C-18), 0.90–
1.00 (m, 1H, CH), 1.25–1.30 (m, 4H, 2CH2), 1.45–1.60 (m, 6H,
3CH2), 1.66–1.80 (m, 2H, CH2), 1.95 (m, 1H, CH), 2.30–2.50 (m,
2H, CH2), 2.60 (m, 1H, CH), 3.65 (m, 1H, 3a–CH), 5.70 (m, 1H, CH-
olefinic, C-6), 7.33–7.41 (m, 4H, Ar-H), 10.45 (s, 1H, OH, exchange-
able with D2O). MS m/z (%): 476 (M+, 10), corresponding to the
molecular formula C29H30ClNO3, at 478 (M+ +2, 3) and at 279
(100, base peak).
Synthesis of 2-oxo-3-cyano-6-(4-chlorophenyl)androst-
3,5-diene[17,16-c]-pyran 8
The compound was prepared by the method given for com-
pounds 4 using compound 7 as starting material (Table 4). IR
(KBr, cm– 1): 2257 (CN), 1723 (C=O). 1H-NMR (DMSO-d6) d: 0.74 (s,
3H, CH3, C-19), 0.79 (s, 3H, CH3, C-18), 0.88–0.95 (m, 1H, CH),
2-Oxo-3-cyano-6-(4-fluorophenyl)androst-4,6-
diene[17,16-c]pyran-3-one 6a
IR (KBr, cm– 1): 2235 (CN), 1775–1765 (C=O, enone), 1723 (C=O,
ketone). 1H-NMR (DMSO-d6) d: 0.79 (s, 3H, CH3, C-19), 0.84 (s, 3H,
CH3, C-18), 1.10–1.15 (m, 1H, CH), 1.25–1.30 (m, 4H, 2CH2),
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A.-G. E. Amr et al.
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1.15–1.25 (m, 4H, 2CH2), 1.40–1.54 (m, 4H, 2CH2), 1.58–1.75 (m,
2H, CH2), 1.90 (m, 1H, CH), 2.25–2.40 (m, 2H, CH2), 2.56 (m, 1H,
CH), 5.25 (m, 1H, CH-olefinic, C-3), 5.50 (m, 1H, CH-olefinic, C-4),
5.65 (m, 1H, CH-olefinic, C-6), 7.31–7.48 (m, 4H, Ar-H). MS m/z
(%): 458 (M+, 32), corresponding to the molecular formula
C29H28ClNO2, 460 (M++2, 10) and 277 (100, base peak).
Anti-inflammatory activity
Carrageenan-induced edema (rats paw test)
Groups of adult male albino rats (150–180 g), each of eight ani-
mals were orally dosed with tested compounds at a dose level of
25–50 mg/kg one hour before carrageenan challenge. All ani-
mals were obtained from the Animal House Colony, Research
Institute of Ophthalmology, Giza, Egypt. Foot paw edema was
induced by subplantar injection of 0.05 mL of 1% suspension of
carrageenan in saline into the plantar tissue of one hind paw.
An equal volume of saline was injected to the other hand paw
and served as control. Four hours after drug administration the
animals were decapitated, blood was collected, and the paws
were rapidly excised. The average weight of edema was exam-
ined for the treated as well as the control group and the percen-
tage inhibition of weight of edema was also evaluated. Predniso-
lone (5 mg/kg) was employed as standard reference against
which the tested compounds were compared.
Synthesis of 2-oxo-3-cyano-6-(4-chlorophenyl)androst-5-
ene[17,16-c]-pyran-3-one 9
The compound was prepared by the method given for com-
pounds 5 using compound 7 as starting material (Table 4). IR
(KBr, cm– 1): 2231 (CN), 1731 (C=O). 1H-NMR (DMSO-d6) d: 0.74 (s,
3H, CH3, C-19), 0.79 (s, 3H, CH3, C-18), 0.95–1.00 (m, 1H, CH),
1.24–1.30 (m, 4H, 2CH2), 1.42–1.60 (m, 6H, 3CH2), 1.65–1.92 (m,
2H, CH2), 2.05 (m, 1H, CH), 2.32–2.42 (m, 2H, CH2), 2.48 (m, 1H,
CH), 5.58 (m, 1H, CH-olefinic, C-6), 7.18– 7.33 (m, 4H, Ar-H). MS
m/z (%): 474 (M+, 19), corresponding to the molecular formula
C29H28ClNO3, 476 (M++2, 6) and at 229 (100, base peak).
Estimation of plasma prostaglandin E2 (PGE2)
Heparinized blood samples were collected from rats (n = 8),
plasma was separated by centrifugation at 12,000 g for 2 min at
408C, immediately frozen, and stored at 208C until use. The
design correlate EIA prostaglandin E2 (PGE2) kit (Aldrich, Stein-
heim, Germany) is a competitive immuno assay for the quantita-
tive determination of PGE2 in biological fluids. The kit uses a
monoclonal antibody to PGE2 to bind, in a competitive manner,
the PGE2 in the sample after a simultaneous incubation at room
temperature. The excess reagents were washed away and the
substrate was added, after a short incubation time the enzyme
reaction was stopped, and the yellow color generated was read
on a microplate reader DYNATech, MR 5000 at 405 nm (Dyna-
tech Industries Inc., McLean, VA, USA). The intensity of the
bound yellow color is inversely proportional to the concentra-
tion of PGE2 in either standard or samples.
Synthesis of 2-oxo-3-cyano-6-(4-chlorophenyl)androst-4-
ene[17,16-c]-pyran-3-one (10) (Oppenauer Oxidation)
To a solution of compound 7 (6.9 mmol) in a mixture of cyclo-
hexanone (50 mL)/dry benzene (45 mL), freshly distilled alumi-
num isopropoxide (2 g, 9.7 mmol) in benzene (5 mL) was added.
The reaction mixture was refluxed for 16 h. The reaction mix-
ture was treated dropwise with water (4 mL) and the precipitated
aluminum salt was collected by filtration. The filtrate was evapo-
rated under reduced pressure and the obtained residue was crys-
tallized from the proper solvent to give the corresponding oxi-
dized derivative 10 (Table 4). IR (KBr, cm– 1): 2234 (CN), 1768
1
(C=O, enone), 1731 (C=O, ketone). H-NMR (DMSO-d6) d: 0.73 (s,
3H, CH3, C-19), 0.82 (s, 3H, CH3, C-18), 1.00–1.10 (m, 1H, CH),
1.20–1.30 (m, 4H, 2CH2), 1.45–1.55 (m, 4H, 2CH2), 1.60–1.75 (m,
4H, 2CH2), 1.80 (m, 1H, CH), 1.88–2.05 (m, 2H, CH2), 2.35–2.40
(m, 1H, CH), 5.71 (s, 1H, CH-olefinic, C-4), 7.23–7.38 (m, 4H, Ar-H).
MS m/z (%): 474 (M+, 100, base peak), corresponding to the mole-
cular formula C29H28ClNO3 and at 476 (M++2, 35).
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