One-pot three-component synthesis of novel heterocyclic steroids as a central antioxidant and anti-inflammatory agents

Article abstract of DOI:10.1016/j.steroids.2012.09.001

Oxidative stress and inflammation have been implicated in several neurodegenerative and developmental brain disorders. The present work was devoted to the design and synthesis of novel steroid derivatives bearing promising heterocyclic moiety that would act to reduce neuro-inflammation and oxidative stress in brain. The novel heterocyclic steroids were synthesized and their chemical structures were confirmed by studying their analytical and spectral data. The tested compounds were assayed in the model of neuro-inflammation produced in rats by cerebral lipopolysacharide injection. The intracerebral administration of bacterial endotoxin resulted in cerebral inflammatory state evidenced by increased malondialdehyde (MDA), decreased reduced glutathione (GSH) level, increased nitric oxide as well as increased acetylcholinesterase (AChE) activity in the brain. Compounds 6, 10, 8b and 13a markedly increased reduced glutathione. Malondialadehyde and nitric oxide levels were reduced to normal values after treatment with all tested compounds. AChE activity was normalized by compound 8b and reduced to below normal values by compounds 10 and 14a. These results are exciting in that these agents might be useful candidates in treatment of cerebral inflammation.

Full text of DOI:10.1016/j.steroids.2012.09.001

Steroids 77 (2012) 1469–1476
Contents lists available at SciVerse ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
One-pot three-component synthesis of novel heterocyclic steroids as a
central antioxidant and anti-inflammatory agents
Nadia R. Mohamed a,b, Mervat M. Abdelhalim c, Yasser A. Khadrawy d, Gamal A. Elmegeed c,
,
⇑
Omar M.E. Abdel-Salam e
a Photochemistry Department, National Research Centre, Dokki, Cairo, Egypt
b Chemistry Department, Elaflaj Science and Humanity Studies College, Salman bin Abdul Aziz University, KSA
c Hormones Department, National Research Centre, Dokki, Cairo, Egypt
d Physiology Department, National Research Centre, Dokki, Cairo, Egypt
e Toxicology and Narcotics Department, National Research Centre, Dokki, Cairo, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 2 July 2012
Received in revised form 5 September 2012
Accepted 7 September 2012
Available online 20 September 2012
Oxidative stress and inflammation have been implicated in several neurodegenerative and developmen-
tal brain disorders. The present work was devoted to the design and synthesis of novel steroid derivatives
bearing promising heterocyclic moiety that would act to reduce neuro-inflammation and oxidative stress
in brain. The novel heterocyclic steroids were synthesized and their chemical structures were confirmed
by studying their analytical and spectral data. The tested compounds were assayed in the model of neuro-
inflammation produced in rats by cerebral lipopolysacharide injection. The intracerebral administration
of bacterial endotoxin resulted in cerebral inflammatory state evidenced by increased malondialdehyde
(MDA), decreased reduced glutathione (GSH) level, increased nitric oxide as well as increased acetylcho-
linesterase (AChE) activity in the brain. Compounds 6, 10, 8b and 13a markedly increased reduced glu-
tathione. Malondialadehyde and nitric oxide levels were reduced to normal values after treatment
with all tested compounds. AChE activity was normalized by compound 8b and reduced to below normal
values by compounds 10 and 14a. These results are exciting in that these agents might be useful candi-
dates in treatment of cerebral inflammation.
Keywords:
Anti-neuroinflammatory
Antioxidant
Pyridine
Pyridopyrimidine
Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction
pathogenesis of Alzheimer’s disease, the most common form of
dementia involves increased production and deposition of amyloid
Multi component reactions (MCRs) have emerged an efficient
method in synthetic organic chemistry, forcing the facile formation
of several new bonds in one-pot reactions. In the past decade, re-
search in academia and industry has increasingly emphasized the
use of MCRs as well as domino reactions sequences for a broad
range of products [1–3]. This is return to their convergence, pro-
ductivity, ease of execution and generally high yields of products
[4]. Pyridines generate a wide spread interest due to diverse phar-
macological activities [5,6]. Pyrimidines owing to their natural
occurrence in human in human genetic material are especially
important as anti-inflammatory agents [7,8]. Thus fused pyrido-
pyrimidines are associated with numerous biological activities
[9,10]. This is responsible for the abiding interest in this class of
compounds.
b-peptide in the brain [11]. Increased levels of lipid peroxidation
products have been detected in brains of patients with Alzheimer’s
disease [12]. The main molecular mechanisms leading to neuronal
cell death in Parkinson’s disease are thought to be oxidative stress
and mitochondrial dysfunction and the abnormal accumulation
and processing of mutant or damaged proteins due to gene-
environmental interactions [13]. Inflammation appears to be an
important contributor to neuronal injury [14]. Multiple sclerosis
the most common chronic inflammatory demyelinating disease is
also associated with increased generation of reactive oxygen spe-
cies [15]. Therefore, there is an immense need of findings new
drugs that would decrease brain oxidative stress and alleviate
neuroinflammation and/or neurodegenerative process. In light of
the above discoveries and in continuation to our previous work
[16–18], the present study investigate the use the MCRs technique
for synthesizing pyridines and pyridopyrimidines on steroidal
frame work. To our knowledge and according the literature survey,
this is the first use of MCRs on steroids. The compounds under
study were tested against oxidative stress and neuro-inflammation
caused by cerebral injection of lipopolysacharide endotoxin. The
Oxidative stress and inflammation have been implicated in sev-
eral neurodegenerative and developmental brain disorders. The
⇑
Corresponding author. Address: Hormones Department, National Research
Centre, 12622 Dokki, Cairo, Egypt. Tel.: +20 2 35682070; fax: +20 2 33370931.
E-mail address: gamalae@hotmail.com (G.A. Elmegeed).
0039-128X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.steroids.2012.09.001

1470
N.R. Mohamed et al. / Steroids 77 (2012) 1469–1476
administration of this bacterial wall endotoxin into the rat brain
induced secretion of cytokines, causes brain inflammation and
neuronal damage [19].
(C@C). 1H NMR (DMSO-d6, ppm): d = 0.76 (s, 3H, CH3-19), 1.15 (s,
3H, CH3-18), 1.50 (m, 1H, C5- H), 2.52 (s, 1H, OH, D2O-exchange-
able), 3.19 (m, 1H, C3- H), 4.57 (s, 1H, C-4 pyrane), 6.65 (s, 2H,
a
a
NH2), 7.22–7.54 (m, 5H, aromatic-H). 13C NMR (CDCl3, ppm):
d = 31.18 (C-1), 30.32, (C-2), 70.95 (C-3), 34.53 (C-4), 39.25 (C-5),
27.14 (C-6), 27.80 (C-7), 34.71 (C-8), 50.36 (C-9), 44.00 (C-10),
22.34 (C-11), 35.78 (C-12), 38.80 (C-13), 48.41 (C-14), 27.04 (C-
15), 110.92 (C-16), 149.30 (C-17), 19.52 (C-18), 20.22 (C-19),
43.07, 58.31, 159.34 (C-pyrane), 117.54 (CN), 129.32, 128.72,
142.41, 125.50 (C-phenyl). M.S (EI): m/z (%): 444 (M+, 56), 248
(23), 196 (74), 156 (29), 77 (100). Calc. for C29H37N2O2 (444.608):
C, 78.34; H, 8.16; N, 6.30; found: C, 78.56; H, 7.87; N, 6.17%.
2. Experimental section
2.1. Synthetic methods, analytical and spectral data
Starting steroids, epi-androsterone, were purchased from Sigma
Company, USA. All solvents were anhydrated by distillation prior
to using. All melting points were measured using an Electrother-
mal apparatus and are uncorrected. The IR spectra were recorded
in (KBr discs) on a shimadzu FT-IR 8201 PC spectrometer and ex-
pressed in cmꢀ1. The 1H NMR and 13C NMR spectra were recorded
with Jeol instrument (Japan), at 270 and 125 MHz, respectively, in
DMSO-d6 or CDCl3 as solvent and chemical shifts were recorded in
ppm relative to TMS. The spin multiplicities were abbreviated by
2.1.2. 3b-Hydroxy-30,50,80-trihydro-50-phenyl-5
70]pyrido[20,30-d]pyrimidin-40-one (8a), 3b-hydroxy-30,50,80-trihydro-
20-methyl-50-phenyl-5 -androstan[16,17:6,70]pyrido[20,30-d]pyrimi-
din-40-one (8b), 3b-hydroxy-30,50,80-trihydro-20-(chloromethyl)-50-
phenyl-5
-androstan[16,17:6,70]pyrido[20,30-d]pyrimidin-40-one (8c)
a-androstan[16,17:6,
a
a
the letters: s-singlet, d-doublet, t-triplet, q-quartet and
m
2.1.2.1. General procedure. A suspension of compound 4 (0.44 g,
1 mmol) in formic acid, acetic acid or chloroacetic acid (5 ml)
was heated under reflux for 4–6 h until all starting materials had
disappeared as indicated by TLC. The reaction mixture was evapo-
rated under vacuum and the remaining residue was treated with
ice/water mixture, neutralized with KOH (10%). The obtained solid
product was collected by filtration, dried and crystallized from the
appropriate solvent.
(multiplet, more than quartet). Mass spectra were recorded on a
GCMS-QP 1000 ex spectra mass spectrometer operating at 70 eV.
Elemental analyses were carried by the Microanalytical Data Unit
at the National Research Centre, Giza, Egypt and the Microanalyti-
cal Data Unit at Cairo University, Giza, Egypt. The reactions were
monitored by thin layer chromatography (TLC) which was carried
out using Merck 60 F254 aluminum sheets and visualized by UV
light (254 nm). The mixtures were separated by preparative TLC
and gravity chromatography. All described compounds showed
the characteristic spectral data of cyclopentanoperhydrophenanth-
rene nuclei of androstane series were similar to those reported in
literature [20,21]. For the nomenclature of steroid derivatives, we
used the definitive rules for the nomenclature of steroids published
by the Joint Commission on the Biochemical Nomenclature (JCBN)
of IUPAC [22,23].
Compound 8a: Yellow crystals from absolute ethanol, mp 113–
115 °C, yield 68%, IR (KBr, cmꢀ1):
t = 3560–3442 (OH, 2NH), 3028
(CH-aromatic), 2928, 2865 (CH-aliphatic), 1680 (CO), 1596 (C@C).
1H NMR (DMSO-d6, ppm): d = 0.78 (s, 3H, CH3-19), 1.14 (s, 3H,
CH3-18), 1.46 (m, IH, C5-
aH), 2.52 (s, 1H, OH, D2O-exchangeable),
3.35 (m, 1H, C3- H), 4.53 (s, IH, C-5 pyridine), 7.06–7.18 (m, 5H, aro-
a
matic-H), 7.52 (s, IH, C-2 pyrimidine), 8.09, 8.85 (2s, 2H, 2NH, D2O-
exchangeable). 13C NMR (DMSO-d6, ppm): d = 32.29 (C-1), 32.52
(C-2), 71.85 (C-3), 37.43 (C-4), 38.50 (C-5), 27.11 (C-6),27.60 (C-7),
38.31 (C-8),52.56 (C-9), 45.00 (C-10), 22.34 (C-11), 34.30 (C-12),
39.80 (C-13), 50.61 (C-14), 28.18 (C-15), 111.34 (C-16),134.30
(C-17), 20.32 (C-18), 20.02 (C-19), 33.70 (C-5 pyridine), 100.07,
163.21, 150.74, 157.28 (C-pyrimidine), 129.29, 128.70, 142.30,
125.25 (C-phenyl). M.S (EI): m/z (%): 470 (M+-l, 67), 248 (12), 223
(29), 131 (67), 94 (100), 77 (58). Calc. for C30H37N3O2 (471.634): C,
76.40; H, 7.91; N, 8.91; found: C, 76.14; H, 8.16; N, 9.20%.
2.1.1. 20-Amino-3b-hydroxy-40H-40-phenyl-5 -androstan[16,17:50,60]
a
pyridin-30-carbonitrile (4), 20-amino-3b-hydroxy-40H-40-phenyl-5
androstan[16,17:50,60]pyran-30-carbonitrile (6)
a-
2.1.1.1. General procedure. A solution of epi-androsterone 1 (1.45 g,
5 mmol), benzaldehyde 2 (0.53 g, 5 mmol) and malononitrile 3
(0.33 g, 5 mmol) in ethanol (10 ml) containing ammonium acetate
(0.98 g, 2% excess) or piperidine (1 ml) was heated under reflux for
4–5 h until all starting materials had disappeared as indicated by
TLC. The oil product that formed in each case on removal of the sol-
vent in vacuum, was solidified by boiling in petroleum ether (60–
80 °C), collected by filtration, dried and crystallized from the
appropriate solvent.
Compound 8b: Brown crystals from absolute ethanol, mp 178–
180 °C, yield 73%, IR (KBr, cmꢀ1):
t = 3558–3440 (OH, 2NH), 3027
(CH-aromatic), 2930, 2864 (CH-aliphatic), 1687 (CO), 1595
(C = C). 1H NMR (DMSO-d6, ppm): d = 0.78 (s, 3H, CH3-19), 1.17
(s, 3H, CH3-18), 1.39 (m, 1H, C5-
a H), 2.32 (s, 1H, OH, D2O-
Compound 4: Yellow crystals from absolute ethanol, mp 103–
exchangeable), 3.20 (m, 1 H, C3- H), 4.43 (s, 1 H, C-5 pyridine),
a
105 °C, yield 85%, IR (KBr, cmꢀ1):
t
= 3654–3469 (OH, NH2, NH),
7.08–7.22 (m, 5H, aromatic-H), 8.29, 8.93 (2s, 2H, 2NH, D2O-
exchangeable). 13C NMR (DMSO-d6, ppm): d = 32.12 (C-1), 32.42,
(C-2), 71.25 (C-3), 37.40 (C-4), 38.65 (C-5), 27.00 (C-6), 27.16
(C-7), 38.30 (C-8), 52.46 (C-9), 44.28 (C-10), 22.34 (C-11), 34.30
(C-12), 40.28 (C-13), 50.76 (C-14), 27.28 (C-15), 111.04 (C-16),
133.70 (C-17), 20.52 (C-18), 21.02 (C-19), 33.75 (C-5 pyridine),
100.16, 163.20, 150.74, 157.34 (C-pyrimidine), 129.29, 128.70,
142.30, 125.53 (C-phenyl). M.S (EI): m/z (%): 485 (M+, 34), 248
(84), 238 (34), 131 (100), 108 (24), 77 (65). Calc. for C31H39N3O2
(485.304): C, 76.67; H, 8.09; N, 8.65; found: C, 76.84; H, 8.36; N,
8.42%.
3037 (CH-aromatic), 2930, 2859 (CH-aliphatic), 2224 (CN), 1583
(C@C). 1H NMR (DMSO-d6, ppm): d = 0.81 (s, 3H, CH3-19), 1.12 (s,
3H, CH3-18), 1.53 (m, 1H, C5-aH), 2.02 (s, 1H, OH, D2O-exchange-
able), 3.24 (m, 1H, C3- H), 4.50 (s, 1H, C-4 pyridine), 6.82 (s, 2H,
a
NH2), 7.24–7.49 (m, 5H, aromatic-H), 8.09 (s, 1H, NH, D2O-
exchangeable). 13C NMR (DMSO-d6, ppm): d = 31.29 (C-1), 30.32,
(C-2), 72.85 (C-3), 34.43 (C-4), 38.50 (C-5), 27.11 (C-6), 27.86
(C-7), 35.31 (C-8), 50.56 (C-9), 44.00 (C-10), 21.34 (C-11), 34.3
(C-12), 38.80 (C-13), 47.61 (C-14), 120.18 (C-15), 111.64 (C-16),
134.30 (C-17), 19.32 (C-18), 20.02 (C-19), 44.67, 57.81, 162.31
(C-pyridine), 117.54 (CN), 129.19, 128.76, 142.31, 125.51 (C-phe-
nyl). M.S (EI): m/z (%): 443 (M+ꢁ, 33), 376 (87), 260 (16), 193 (46),
77 (100). Calc. for C29H37N3O (443.624): C, 78.51; H, 8.41; N,
9.47; found: C, 78.20; H, 7.24; N, 9.21%.
Compound 8c: Yellowish brown powder from 1,4-dioxane, m.p.
192–193 °C, yield 77%, IR (KBr, Cmꢀ1):
t = 3567–3446 (OH, 2NH),
3032 (CH-aromatic), 2945, 2860 (CH-aliphatic), 1693 (CO), 1607
(C = C). 1H NMR (DMSO-d6, ppm): d = 0.83 (s, 3H, CH3-19), 1.23
Compound 6: Pale brown crystals from absolute ethanol, mp
(s, 3H, CH3-18), 1.40 (m, 1H, C5-
exchangeable), 3.18 (m, 1H, C3- H), 3.47 (s, 2H, CH2Cl), 4.40 (s,
1H, C-5 pyridine), 7.06–7.25 (m, 5H, aromatic-H), 8.34, 8.98 (2s,
a H), 2.27 (s, 1 H, OH, D2O-
193–195 °C, yield 76%, IR (KBr, cmꢀ1):
t
= 3563–3354 (OH, NH2),
a
3032 (CH-aromatic), 2942, 2878 (CH-aliphatic), 2222 (CN), 1613

N.R. Mohamed et al. / Steroids 77 (2012) 1469–1476
1471
2H, 2NH, D2O-exchangeable). 13C NMR (DMSO-d6, ppm): d = 32.52
(C-1), 32.40, (C-2), 71.30 (C-3), 37.40 (C-4), 37.35 (C-5), 27.30 (C-6),
27.76 (C-7), 38.53 (C-8), 52.42 (C-9), 45.08 (C-10), 22.31 (C-11),
35.70 (C-12), 45.29 (C-13), 50.46 (C-14), 28.18 (C-15), 111.24 (C-
16), 134.20 (C-17), 20.72 (C-18), 21.12 (C-19), 33.75 (C-5 pyridine),
100.12, 162.20, 164.34, 157.30 (C-pyrimidine), 49.74 (CH2-Cl),
129.03, 128.74, 142.30, 125.43 (C-phenyl). M.S (El): m/z (%): 520
(M+, 33), 272 (17), 247 (54), 141 (100), 91 (24). Calc. for C31H38
N3O2Cl (520.105): C, 71.59; H, 7.36; N, 8.08; found: C, 71.82; H,
7.13; N, 8.29%.
(C-16), 134.20 (C-17), 20.70 (C-18), 21.32 (C-19), 38.37 (C-5 pyri-
dine), 90.32, 152.24, 181.43, 196.05 (C-pyrimidine), 129.13,
128.27, 141.13, 125.43 (C-phenyl). M.S (EI): m/z (%): 519 (M+,
32), 248 (64), 271 (26), 141 (100), 131 (76), 77 (45). Calc. for
C30H37N30S2 (519.764): C, 69.32; H, 7.18; N, 8.08; S, 12.34; found:
C, 69.50; H, 7.01; N, 7.79; S, 12.56%.
2.1.5. N-(3b-hydroxy-30-cyano-10,40-dihydro-40-phenyl-5a-androstan
[16,17:50,60]pyridine-20-yl)-N,N-dimethylformamidine (11)
To a solution of compound 4 (0.88 g, 2 mmol) in acetonitrile
(10 mL), dimethylformamid-dimethylacetal (0.24 g, 2 mmol) was
added dropwise with stirring. After complete addition, the reaction
mixture was heated in a water bath at 70 °C for 2 h. After cooling at
room temperature, the reaction mixture poured over ice/ water
mixture and the resulted semisolid was subjected to extraction
with chloroform (2 ꢂ 30 mL). The organic layer was dried over
anhydrous magnesium sulfate and then filtered. The oil product
that formed on removal of the solvent in vacuum, was solidified
by boiling in petroleum ether (60–80 °C), collected by filtration
and crystallized from absolute ethanol to afford pale yellow pow-
der of compound 11, m.p. 187–189 °C, yield 80%, IR (KBr, cmꢀ1):
2.1.3. 3b-Hydroxy-30,50,80-trihydro-20-methyl-50-phenyl-5a-androstan
[16,17:60,70]pyrido[20,30-d]pyrimidin-40-one (8b), 3b-Hydroxy-30,50,80-
trihydro-20,50-phenyl-5a-androstan[16,17:60,70]pyrido[20,30-d]pyrimi-
din-40-one (9)
2.1.3.1. General procedure. To a solution of compound 4 (0.44 g,
1 mmol) in dimethyl formamide (DMF) (20 mL), equimolar amount
of acetyl chloride (0.078 g, 1 mmol) or benzoyl chloride (0.14 g,
1 mmol) was added dropwise with stirring. After complete addi-
tion, the reaction mixture was refluxed for 6–8 h until all the start-
ing materials had disappeared as indicated by TLC. The reaction
mixture, then left to cool at room temperature, poured over
crushed ice and left in a refrigerator at 4 °C overnight. The formed
solid product, in each case, was collected by filtration and crystal-
lized from the appropriate solvent.
t
= 3487–3395 (OH, NH), 3028 (CH-aromatic), 2978, 2856 (CH-ali-
phatic), 2225 (CN), 1576 (C = C). 1H NMR (DMSO-d6, ppm): d = 0.95
(s, 3H, CH3-19), 1.19 (s, 3H, CH3-18), 1.43 (m, IH, C5- H), 2.25
(s, 1H, OH, D2O-exchangeable), 2.47 (s, 6H, NMe2), 3.24 (m, 1H,
C3- H), 4.40 (s, 1H, C-5 pyridine), 7.07–7.18 (m, 5H, aromatic-H),
a
Data of compound 8b from this method is a fingerprint to that
of the preceding method.
a
7.85 (s, 1H, enamine CH), 8.63 (s, 1H, NH, D2O-exchangeable).
M.S (EI): m/z (%): 497 (M+-1, 43), 248 (14), 250 (36), 181 (100),
77 (65), 71 (28). Calc. for C32H42N4O (498.702): C, 77.07; H, 8.49;
N, 11.23; found: C, 76.78; H, 8.71; N, 11.39%.
Compound 9: Pale brown crystals from 1,4-dioxane, m.p. 213–
214 °C, yield 74%, IR (KBr, cmꢀ1):
t = 3546–3475 (OH, 2NH), 3030
(CH-aromatic), 2986, 2845 (CH-aliphatic), 1697 (CO), 1587 (C@C).
1H NMR (CDCl3, ppm): d = 0.87 (s, 3H, CH3-19), 1.19 (s, 3H, CH3-
18), 1.42 (m, IH, C5-
a
H), 2.07 (s, 1H, OH, D2O-exchangeable), 3.27
2.1.6. N-(3b-hydroxy-30-cyano-10,40-dihydro-40-phenyl-5
[16,17:50,60]pyridin-20-yl)ethoxymethanamine (13a), N-(3b-hydroxy-
30-cyano-40H-40-phenyl-5 -androstan [16,17:50,60]pyran-20-yl)
ethoxymethanamine (13b)
a-androstan
(m, 1H, C3- H), 4.47 (s, 1H, C-5 pyridine), 7.14-7.35 (m, 10H, aro-
a
matic-H), 8.54, 9.20 (2s, 2H, 2NH, D2O-exchangeable). 13C NMR
(CDCl3, ppm): d = 32.42 (C-l), 32.57, (C-2), 71.34 (C-3), 37.92 (C-
4), 37.05 (C-5), 27.43 (C-6), 28.06 (C-7), 38.50 (C-8), 52.56 (C-9),
45.28 (C-10), 23.01 (C-11), 35.72 (C-12), 45.29 (C-13), 50.74 (C-
14), 28.68 (C-15), 111.04 (C-16), 134.20 (C-17), 20.52 (C-18),
20.72 (C-19), 33.70 (C-5 pyridine), 100.12, 162.20, 161.30, 157.35
(C-pyrimidine), 129.03, 128.24, 142.30, 125.43, 126.43, 128.98,
128.71, 130.04 (C-phenyl). M.S (EI): m/z (%): 547 (M+, 38), 299
(27), 248 (34), 131 (100), 170 (24), 77 (46). Calc. for C36H41N3O2
(547.730): C, 78.94; H, 7.54; N, 7.67; found: C, 78.72; H, 7.73; N,
7.39%.
a
A mixture of compound 4 (0.88 g, 2 mmol) or compound 6
(0.88 g, 2 mmol) and triethylorthoformate (0.30 g, 2 mmol) in ace-
tic anhydride (10 mL) was heated under reflux for 5–7 h. The reac-
tion was controlled by TLC, after cooling at room temperature, the
reaction mixture poured over an ice/ water mixture and the re-
sulted semisolid was subjected to extraction with chloroform
(2 ꢂ 30 mL). The organic layer was dried over magnesium sulfate
and then filtered. The oil product that formed on removal of the
solvent in vacuum was solidified by boiling in petroleum ether
(60–80 °C), collected and crystallized from the appropriate solvent.
Compound 13a: Brown powder from 1,4-dioxane, m.p. 117–
2.1.4. 3b-Hydroxy-10,30,50,80-tetrahydro-50-phenyl-5
a-androstan
[16,17:60,70]pyrido[20,30d]pyrimidin-20,40-dithione (10)
119 °C, yield 82%, lR (KBr, cmꢀ1):
t = 3482–3395 (OH, NH), 3030
To a solution of compound 4 (0.44 g, 1 mmol) in 15 mL of etha-
nol/KOH (10%), excess of carbon disulphide was added. The reac-
tion mixture was heated under reflux for 4 h until all the starting
materials had disappeared as indicated by TLC. The reaction sol-
vent was evaporated under vacuum and the remaining residue
was treated with ice/water mixture, neutralized with dilute hydro-
chloric acid. The obtained solid product was collected by filtration,
dried and crystallized from the absolute ethanol to afford dark yel-
low crystals of compound 10. m.p. 220–222 °C, yield 72%, IR (KBr,
(CH-aromatic), 2984, 2867 (CH-aliphatic), 2225 (CN), 1593 (C@C).
1H NMR (CDCl3, ppm): d = 1.04 (s, 3H, CH3-19), 1.15 (t, 3H, ester-
CH3), 1.25 (s, 3H, CH3-18), 1.40 (m, 1H, C5-
a H), 2.08 (s, 1H, OH,
D2O-exchangeable), 3.28 (m, 1H, C3- H), 3.87 (s, 2H, ester-CH2),
a
4.78 (s, 1H, C-5 pyridine), 7.23–7.37 (m, 5H, aromatic-H), 7.58 (s,
1H, N@CH), 8.73 (s, 1H, NH, D2O-exchangeable). M.S (EI): m/z
(%): 499 (M+, 56), 248 (34), 179 (100), 77 (65), 72 (35). Calc. for
C32H41N3O2 (499.687): C, 76.92; H, 8.27; N, 8.41; found: C, 77.12;
H, 8.02; N, 8.29%.
cmꢀ1):
t
= 3538–3454 (OH, 3NH), 3032 (CH-aromatic), 2986, 2845
Compound 13b: Pale brown powder from methanol, m.p. 139–
(CH-aliphatic), 1589 (C@C), 1199, 1193 (2C@S). 1H NMR (DMSO-d6,
ppm): d = 0.98 (s, 3H, CH3-19), 1.23 (s, 3H, CH3-18), 1.40 (m, 1H,
141 °C, yield 77%, IR (KBr, cmꢀ1):
t = 3432 (OH), 3042 (CH-aro-
matic), 2979, 2854 (CH-aliphatic), 2225 (CN), 1590 (C@C). 1H
NMR (DMSO-d6, ppm): d = 1.02 (s, 3H, CH3-19), 1.17 (t, 3H, ester-
C5-
a H), 2.21 (s, 1H, OH, D2O-exchangeable), 3.12 (m, 1H, C3-a
H), 4.43 (s, 1H, C-5 pyridine), 7.06–7.15 (m, 5H, aromatic-H),
8.63 (s, 1H, NH, D2O-exchangeable), 9.34 (s, 2H, 2NH,
D2O-exchangeable). 13C NMR (DMSO-d6, ppm): d = 32.54 (C-1),
32.70, (C-2), 70.74 (C-3), 38.02 (C-4), 37.15 (C-5), 27.40 (C-6),
28.06 (C-7), 38.50 (C-8), 52.40 (C-9), 44.85 (C-10), 22.43 (C-11),
35.70 (C-12), 46.20 (C-13), 50.72 (C-14), 28.38 (C-15), 111.24
CH3), 1.20 (s, 3H, CH3-18), 1.40 (m, 1H, C5-
aH), 2.27 (s, 1H, OH,
D2O-exchangeable), 3.35 (m, 1H, C3- H), 4.04 (s, 2H, ester-CH2),
a
4.73 (s, 1H, C-5 pyrane), 7.20–7.27 (m, 5H, aromatic-H), 7.53 (s,
1H, N@CH), 8.73 (s, 1H, NH, D2O-exchangeable). 13C NMR (CDCl3,
ppm): d = 37.02 (C-l), 31.07, (C-2), 71.13 (C-3), 37.92 (C-4), 43.25
(C-5), 27.63 (C-6), 31.16 (C-7), 34.50 (C-8), 51.06 (C-9), 38.28

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N.R. Mohamed et al. / Steroids 77 (2012) 1469–1476
(C-10), 21.21 (C-11), 34.72 (C-12), 44.59 (C-13), 50.04 (C-14), 27.88
(C-15), 110.34 (C-16), 149.87 (C-17), 18.02 (C-18), 19.70 (C-19),
44.12, 76.20, 170.30 (C-pyrane), 117.23 (CN), 156.09 (CHOEt),
63.00 (CH2-ester), 15.78 (CH3-ester), 127.03, 128.25, 142.13,
125.53 (C-phenyl). M.S (EI): m/z (%): 500 (M+, 46), 248 (24), 180
(100), 77 (45), 72 (17). Calc. for C32H40N2O3 (500.672): C, 76.77;
H, 8.05; N, 5.60; found: C, 76.89; H, 8.22; N, 5.39%.
2.2.2. Surgical procedures
At the beginning of the experiment, the number of rats was 50.
Rats were anaesthetized with sodium pentobarbital (40 mg/kg,
i.p.). After shaving the hair from the front-occipital area, antisepsis
was performed with 2% iodine solution. A hole of 0.5 cm was made
using orthodontic roof motor and number 2 drill to the right of the
bregma until the dura matter was exposed. With the use of a Ham-
ilton syringe fitted with a 30-gauge needle, lipopolysaccharide
(5 lg in a volume of 2 ll PBS) was injected in the cisterna pontis
2.1.7. 30-Amino-3b-hydroxy-40-imino-50-phenyl-5
a-androstan
(basal), an enlargement of the subarachnoid space on the ventral
surface of the pons [24,25]. A group of rats (n = 6) was subjected
to the same surgical procedure but injected with saline (0.9%)
and served as negative control. The dura matter was left open
and the skin together with the remainder of the subcutaneous tis-
sue was sutured with a nylon thread 4.0.
[16,17:60,70]-50H-pyrido[20,30-d]pyrimidine (14a), 30-amino-3b-
hydroxy-40-imino-50-phenyl-5 -androstan[16,17:60,70]-50H-pyrano
a
[20,30-d]pyrimidine (14b)
To a mixture of compound 13a (0.49 g, 1 mmol) or 13b (0.50 g,
1 mmol) and hydrazine hydrate (0.05 g, 1 mmol) in 25 mL absolute
ethanol, a catalytic amount of triethyl amine was added (0.5 mL).
The reaction mixture was heated to 80 °C with stirring and main-
tained at this temperature for 4–6 h with continued stirring. The
reaction was monitored by TLC, concentrated under vacuum and
the remaining residue was treated with ice/water mixture
(50 mL) and neutralized with dilute hydrochloric acid. The formed
solid product, in each case, was filtered off, dried and crystallized
from absolute ethanol.
2.2.3. Experimental design
Starting from the 2nd day of lipopolysaccharide injection, rats
were randomly divided into six groups (n = 6 each). One of these
groups was treated daily with saline orally and served as positive
control. Each of the remaining lipopolysaccharide-injected groups
was treated daily with one of compounds 6, 10, 8b, 13a or 14a dis-
solved in 2% Tween 80 and injected subcutaneously in a dose of
50 mg kgꢀ1 b.wt. for three consecutive days. Four hours after the
last injection the rats were sacrificed and the cortex was dissected
out on ice-cold Petri-dish. The samples were weighed and kept fro-
zen at ꢀ20 °C until analysis.
Compound 14a: Orange powder, m.p. 172–174 °C, yield 76%, IR
(KBr, cmꢀ1):
t
= 3480–3375 (OH, NH, NH2), 3042 (CH-aromatic),
2988, 2863 (CH-aliphatic), 1603 (C = C). 1H NMR (CDCI3, ppm):
d = 1.06 (s, 3H, CH3-19), 1.17 (s, 3H, CH3-18), 1.42 (m, 1H, C5- H),
2.87 (s, 1H, OH, D2O-exchangeable), 3.28 (m, 1H, C3- H), 4.76 (s,
a
a
1H, C-5 pyridine), 6.52 (s, 2H, NH2), 7.23-7.33 (m, 5H, aromatic-
H), 7.56 (s, 1H, C-2 pyrimidine), 8.58, 8.73 (2s, 2H, 2NH, D2Oex-
changeable). l3C NMR (CDCl3, ppm): d = 37.50 (C-l), 31.17, (C-2),
71.00 (C-3), 37.02 (C-4), 42.95 (C-5), 27.32 (C-6), 31.24 (C-7),
34.50 (C-8), 53.06 (C-9), 36.28 (C-10), 21.32 (C-11), 35.72 (C-12),
45.79 (C-13), 50.24 (C-14), 29.08 (C-15), 111.04 (C-16), 134.87
(C-17), 16.52 (C-18), 19.72 (C-19), 29.31 (C-5 pyridine), 151.12,
90.20, 158.09, 143.56 (C-pyrimidine), 142.45, 127.13, 128.25,
125.73 (C-phenyl). M.S (EI): m/z (%): 485 (M+, 26), 248 (19), 128
(100), 77 (65). Calc. for C30H39N5O (485.664): C, 74.19; H, 8.09;
N, 14.42; found: C, 73.95; H, 8.30; N, 14.23%.
2.2.4. Neurochemical analysis
Lipid peroxidation was assayed by measuring the level of mal-
ondialdehyde (MDA) in the cortex. MDA was determined by mea-
suring thiobarbituric acid reactive species using the method of
Ruiz-Larrea et al. [26] in which the thiobarbituric acid reactive sub-
stances react with thiobarbituric acid to produce a red colored
complex having absorbance peak at 532 nm. The assay of reduced
glutathione (GSH) levels was performed spectrophotometrically
using the method of Ellman’s [27]. It depends on the reduction of
5,50-dithiobis-2-nitrobenzoic acid with glutathione to produce a
yellow color whose absorbance is measured at 405 nm. The assay
of nitric oxide was carried out using Biodiagnostic kit No. NO 25
33 (Biodiagnostic Co., Egypt). This method is based on the spectro-
photometric method of Montgomery and Dymock [28] which de-
pends on the measurement of endogenous nitrite concentration
as an indicator of nitric oxide production. The procedure used for
the determination of acetylcholinesterase activity (AChE) in the
cortex was a modification of the method of Ellman et al. [29] as de-
scribed by Gorun et al. [30]. The principle of the method is the
measurement of the thiocholine produced as acetylthiocholine is
hydrolyzed. The color was read immediately at 412 nm.
Compound 14b: Yellow powder, m.p. 186–188 CC, yield 78%, IR
(KBr, cmꢀ1):
t
= 3482–3373 (OH, NH, NH2), 3038 (CH-aromatic),
2987, 2863 (CH-aliphatic), 1608 (C@C). 1H NMR (CDCl3, ppm):
d = 1.02 (s, 3H, CH3-19), 1.13 (s, 3H, CH3-18), 1.42 (m, 1H, C5- H),
2.85 (s, 1H, OH, D2O-exchangeable), 3.34 (m, 1H, C3- H), 4.75 (s,
a
a
1H, C-5 pyrane), 6.67 (s, 2H, NH2), 7.26–7.35 (m, 5H, aromatic-
H), 7.60 (s, 1H, C-2 pyrimidine), 8.96 (s, 1H, NH, D2O-exchange-
able). l3C NMR (CDCl3, ppm): d = 37.25 (C-l), 31.07, (C-2), 71.08
(C-3), 37.12 (C-4), 42.46 (C-5), 27.13 (C-6), 31.25 (C-7), 34.62 (C-
8), 53.06 (C-9), 36.25 (C-l0), 21.22 (C-11), 35.72 (C-12), 45.82 (C-
13), 50.34 (C-14), 30.28 (C-15), 111.34 (C-16), 149.85 (C-17),
18.02 (C-18), 20.32 (C-19), 28.45 (C-5 pyrane), 159.30, 90.20,
158.04, 143.56 (C-pyrimidine), 142.35 127.23, 128.15, 125.70 (C-
phenyl). M.S (EI): m/z (%): 486 (M+, 54), 272 (39), 214 (65), 84
(100), 77 (43). Calc. for C30H38N4O2 (486.648): C, 74.04; H, 7.87;
N, 11.51; found: C, 73.78; H, 8.12; N, 11.28%.
3. Results and discussion
3.1. Chemistry
Heterocyclic steroids serve as platform for developing pharma-
ceutical agents for various applications. We have attempted a
straightforward synthesis of these compounds using MCRs. Thus,
2.2. Biological assay
2.2.1. Experimental animals
A mixture of epi-androsterone (3b-hydroxy-5a-androstan-3-one)
The experimental animals used in the present study were adult
Wistar albino rats weighing 130–150 g. The animals were obtained
from the animal house of the National Research Center, Egypt. They
were maintained on stock diet and kept under fixed appropriate
conditions of housing and handling. All experiments were carried
out in accordance with the research protocols established by the
animal care committee of the National Research Center - Egypt.
1, benzaldhyde 2 and malononitrile 3 was heated in absolute eth-
anol containing ammonium acetate under reflux to afford the cor-
responding aminoandrostano-1,4-dihydropyridine derivative
4
(Scheme 1). The reaction proceeded via the formation of the ben-
zylidene adduct at first followed by the addition of malononitrile
and cyclization by loss of water. It was explained previously
that the formed adduct may undergo aromatization via loss of

N.R. Mohamed et al. / Steroids 77 (2012) 1469–1476
1473
O
H
CN
CH3COONH4
H2C
PhCHO +
+
H
H
CN
HO
2
3
NH2
1
H
HN
CN
EtOH
Pip
H
Ph
4
H
NH2
O
CN
H
X
Ph
H
6
NH2
N
CN
Ph
H
5
Scheme 1.
hydrogen to produce structure 5 [31]. The 1H NMR spectrum of the
synthesized adduct revealed the presence of hydrogen proton at C-
4 of the 1,4-dihydropyridine ring at d = 4.50 ppm and D2O-exange-
able NH proton at d = 8.09 ppm. This excluded structure 5 and sup-
ported the formation of the adduct 4. Carrying out the previous
reaction in presence of piperidine instead of ammonium acetate
led to the formation of the aminoandrostanodihydropyrane deriv-
ative 6 (Scheme 1). The 1H NMR spectrum of product 6 showed the
disappearance of the characteristic signal of NH group of the pyri-
dine ring. All the microanalytical and spectroscopic data were in
accordance with the suggested structure 6.
Concerning the stereochemistry at position 4 of compounds 4
and 6, the reaction conditions produced only one isomer [6]. Also,
thin layer chromatography examination of the reaction mixture re-
vealed one product. Identification of 1,4-dihydropyridine or dihyd-
ropyran isomers is beyond the scope of this study.
and the other spectroscopic data were in accordance with the
structure of 10 (Scheme 3). Treatments of 4 with dimethylformam-
ide-dimethylacetal in acetonitrile yielded the open enamine ad-
duct, aminoandrostanopyridine-N,N-dimethylformamidine 11.
The 1H NMR spectrum of compound 11 revealed, beside the disap-
pearance of the characteristic signal of the amino group, the pres-
ence of singlet signal at d = 2.47 (6H) for NMe2 group and singlet at
d = 7.85 (1H) for the enamine CH (c.f. Experimental section).
Furthermore, the interaction of either compound 4 or 6 with
triethylorthoformate in acetic anhydride solution yielded the acy-
clic structures 13a and 13b, respectively. The expected cyclized
adducts 12a, b were excluded dependant on the spectroscopic
data. The I.R spectrum of compound 13a as example showed
the presence of the nitrile group at
sence of the carbonyl group of the pyrrole ring in 12a structure.
Treatment of compounds 13a, with hydrazine hydrate in
t
= 2225 cmꢀ1 and the ab-
b
The androstanopyridopyrimidinone derivatives 8a–c were
formed via boiling compound 4 in different organic acids like for-
mic, acetic and chloroacetic acids 7a–c (Scheme 2). It is believed
that the nitrile group was converted into amide via hydrolysis fol-
lowed by cyclization. Dimorth rearrangement took place to furnish
the final fused pyridopyrimidinone adduct [32,33]. On the other
hand interaction of compound 4 with acetyl chloride or benzoyl
chloride in the presence of triethylamine yielded the correspond-
ing fused candidates androstanopyridopyrimidinone derivatives
8b and 9, respectively (Scheme 2).
ethanol absolute under reflux led to the formation of the fused
aminoandrostanopyridopyrimidine adducts 14a, b respectively
(Scheme 4).
3.2. Bioassay
The in vivo potent anti-neuroinflammatory and anti-oxidative
stress in brain of the novel synthesized heterocyclic steroids, com-
pounds 6, 10, 8b, 13a and 14a was investigated. The tested com-
pounds were assayed in the model of neuro-inflammation
produced in rats by cerebral lipopolysacharide injection. The intra-
cerebral administration of bacterial endotoxin resulted in cerebral
inflammatory state evidenced by increased lipid peroxidation
(malondialdehyde; MDA), decreased reduced glutathione (GSH)
Aminoandrostano-1,4-dihydropyridine 4 was allowed to react
with excess of carbon disulfide in alcoholic-potassium hydroxide
solution (10%) to form the androstanopyridopyrimidine-dithione
adduct 10. The microanalysis supported the presence of sulfur

1474
N.R. Mohamed et al. / Steroids 77 (2012) 1469–1476
R
N
NH
HN
+
4
RCOOH
O
7a-c
Ph
H
R'COCl
-HCl
8a-c
7,8a, R=H
b
R'
, R=CH3
c, R=CH2Cl
N
NH
HN
O
Ph
H
8b, R'=CH3
9, R'=Ph
Scheme 2.
S
HN
NH
S
HN
CS2
4
EtOH /KOH
Ph
A
M
D
H
-
F
10
M
D
H
N
NMe2
HN
CN
Ph
H
11
Scheme 3.
level, increased nitric oxide as well as increased acetylcholinester-
ase (AChE) activity in the brain (Table 1). The test compounds were
administered subcutaneously to rats treated with intracerebral
lipopolysaccahride. Compounds 6, 10, 8b and 13a markedly in-
creased GSH that reached normal values with compounds 8b, 10
and 13a and was even raised above control value in rats treated
with compound 6. MDA was reduced to normal values after treat-
ment with all tested compounds, almost normalized nitric oxide
levels while marked inhibition of nitric oxide was seen after treat-
ment with compound 6. AChE activity, a marker of inflammation,
was normalized by compound 8b and reduced to below normal
values by compounds 10 and 14a. AChE activity was markedly re-
duced by compound 6. These results are exciting in that, these
compounds might be useful candidates in treatment of cerebral
inflammation.
Directed by the structure of tested compounds it is probably
that these compounds have a dual mechanism for their anti-neur-
oinflammatory and anti-oxidative activities. Further studies should
be made to establish the mechanism of action with the possibility
to formulate potent anti-cerebral inflammatory agents.

N.R. Mohamed et al. / Steroids 77 (2012) 1469–1476
1475
H
OEt
N
X
CN
NH2NH2
4 or 6
+
CH(OEt)3
Ph
H
X
13a,b
OEt
O
N
N
NH2
N
X
X
NH
Ph
Ph
H
H
12a,b
14a,b
12-14a,
X=NH
b, X=O
Scheme 4.
Table 1
Effect of test compounds on reduced glutathione (GSH), malondialdehyde (MDA), nitric oxide (NO) and acetylcholinesterase activity (AChE) in the brain of rats subjected to
intracerebral lipopolysaccharide (LPS) injection.
GSH (mmol/g fresh tissues)
MDA (nmol/g fresh tissue)
No (
l
mol/g)
AChE (l mol SH/g/min)
Vehicle
LPS
0.583 0.082
0.149 0.033*
0.606 0.127
0.193 0.045*
0.455 0.051
0.439 0.053
0.818 0.064
18.476 2.277
23.736 1.468
15.937 1.22
18.824 0.895
18.427 0.895
21.194 0.487
19.216 1.742
0.083 0.007
0.141 0.019*
0.073 0.004
0.102 0.008
0.123 0.014
0.072 0.007
0.077 0.0 II
4.729 0.671
6.830 0.713
4.487 0.561
3.456 0.288
6.145 0.944
2.176 0.252*
1.319 0.401*
LPS + 8b
LPS + 14a
LPS + 13a
LPS + 10
LPS + 6
*
Indicate significant difference compared vehicle-treated control values at p < 0.05.
[2] Heravi MM, Zakeri M, Pooremamy S, Oskooie HA. Clean and efficient synthesis
of polyhydroquinoline derivatives under solvent-free conditions catalyzed by
morpholine. Synth Commun 2011;41:113–20.
[3] Nemati F, Bigdeli MA, Mahdavinia GH, Kiani H. 2,4,6-Trichloro[1,3,5]triazine
(TCT)-catalyzed one-pot Mannich-type reaction: three component synthesis of
b-amino carbonyl compounds. Green Chem Lett Rev 2010;3(2):89–92.
[4] Quiroga J, Cruz S, Insuasty B, Abonía R, Nogueras M, Cobo J. Three-component
synthesis of hexahydropyridopyrimidine–spirocyclohexanetriones induced by
microwave. Tetrahedron Lett 2006;47(1):27–30.
[5] Kidwai M, Thakur R, Rastogi S. Ecofriendly synthesis of substituted pyridine
and pyrido[2,3-d]pyrimidine derivatives. Russ Chem Bull 2005;54(6):1523–6.
[6] Mohamed NR, Khaireldin NY, Fahmy AF, El-Sayed AA. Facile synthesis of fused
nitrogen containing heterocycles as anticancer agents. Der Pharma Chemica
2010;2(1):400–17.
3.3. Conclusion
In this study we have attempted a straightforward synthesis of
novel pyrido- and pyrido-pyrimidine steroids that would act to re-
duce neuro-inflammation and oxidative stress in brain, using mul-
ti-component reactions. The tested compounds were assayed in
the model of neuro-inflammation produced in rats by cerebral
lipopolysacharide injection. We investigated also the pharmaceuti-
cal importance of incorporating heterocylic moiety to the steroid
nucleus to form new effective hybrid molecules. Compounds 6,
10, 8b, 13a and 14a showed anti-neuroinflammatory and anti-
oxidant activities with various intensities. Results suggest tested
compounds are useful candidates in treatment of cerebral
inflammation.
[7] Amir M, Javed SA, Kumar H. Pyrimidine as antiinflammatory agent: a review.
Indian J Pharm Sci 2007;69(3):337–43.
[8] Yejella RP, Atla SR. A study of anti-inflammatory and analgesic activity of new
2,4,6-trisubstituted pyrimidines. Chem Pharm Bull (Tokyo) 2011;59(9):
1079–82.
[9] Gyires K, Fürst S, Miklya I, Budavari I, Knoll J. Analysis of the analgesic and anti-
inflammatory effects of rimazolium, a pyrido-pyrimidine derivative, compared
with that of prostaglandin synthesis inhibitors and morphine. Drugs Exp Clin
Res 1985;11(8):493–500.
References
[10] Huron DR, Gorre ME, Kraker AJ, Sawyers CL, Rosen N, Moasser MM. A novel
pyridopyrimidine inhibitor of abl kinase is a picomolar inhibitor of Bcr-abl-
driven K562 cells and is effective against STI571-resistant Bcr-abl mutants.
Clin Cancer Res 2003;9(4):1267–73.
[1] Yu L, Chen B, Huang X. Multicomponent reactions of allenes, diaryl diselenides,
and nucleophiles in the presence of iodosobenzene diacetate: direct synthesis
of 3-functionalized-2-arylselenyl substituted allyl derivatives. Tetrahedron
Lett 2007;48(6):925–7.

1476
N.R. Mohamed et al. / Steroids 77 (2012) 1469–1476
[11] Lovell MA, Ehmann WD, Mattson MP, Markesbery WR. Elevated 4-
hydroxynonenal in ventricular fluid in Alzheimer’s disease. Neurobiol Aging
1997;18:457–61.
[23] IUPAC. Joint Commission on Biochemical Nomenclature (JCBN). The
nomenclature of steroids, Recommendations 1989. Eur
1989;186:429–58.
J
Biochem
[12] Reitz C, Tang M, Luchsinger J, Mayeux R. Relation of plasma lipids to Alzheimer
disease and vascular dementia. Arch Neurol 2004;61(5):705–14.
[13] Gandhi S, Wood NW. Molecular pathogenesis of Parkinson’s disease. Hum Mol
Genet 2005;14:2749–55.
[14] Nagatsu T, Sawada M. Inflammatory process in Parkinson’s disease: role for
cytokines. Curr Pharm Des 2005;11:999–1016.
[24] Kim W, Mohney RP, Wilson B, Jeohn G, Liu B, Hong J. Regional difference in
susceptibility to lipopolysaccharide-induced neurotoxicity in the rat brain:
Role of microglia. J Neurosci 2000;20(16):6309–16.
[25] Cai Z, Fan LW, Lin S, Pang Y, Rhodes PG. Intranasal administration of insulin-
like growth factor-1 protects against lipopolysaccharide-induced injury in the
developing rat brain. Neuroscience 2011;194:195–207.
[15] Carlson NG, Rose JW. Antioxidants in multiple sclerosis: do they have a role in
therapy? CNS Drugs 2006;20(6):433–41.
[16] Mohareb RM, Elmegeed GA, Baiuomy AR, Eskander EF, William MG. Evaluation
of anti-inflammatory, anti-nociceptive and anti-ulcerogenic, activities of novel
synthesized thiazolyl and pyrrolyl steroids. Arch Pharm Chem Life Sci
2011;344:595–604.
[26] Ruiz-Larrea MB, Leal AM, Liza M, Lacort M, de Groot H. Antioxidant effect of
estradiol and 2-hydroxyestradiol on iron-induced lipid peroxidation of rat
liver microsome. Steriods 1994;59:383–8.
[27] Ellman GL. Tissue sulfhydryl groups. Arch Biochem Biophys 1959;82:70–7.
[28] Montgomery HAC, Dymock JF. Determination of nitric oxide. Analyst
1961;86:414–7.
[17] Mohareb RM, Elmegeed GA, Abdel-Salam OME, Doss SH, William MG.
[29] Ellman GL, Courtney KD, Andres Jr V, Feather-Stone RM. A new and rapid
colorimetric determination of acetylcholinesterase activity. Biochem
Pharmacol 1961;7:88–95.
[30] Gorun V, Proinov I, Baltescu V, Balaban G, Barzu O. Modified Ellman procedure
for assay of cholinesterases in crude enzymatic preparation. Anal Biochem
1978;86:324–6.
Synthesis of modified steroids as
a novel class of non-ulcerogenic, anti-
inflammatory and anti-nociceptive agents. Steroids 2011;76:1190–203.
[18] Mohareb RM, Ahmed HH, Elmegeed GA, Abd-Elhalim MM, Shafic RW.
Development of new indole-derived neuroprotective agents. Bioorg Med
Chem 2011;19:2966–74.
[19] Koprich JB, Reske-Nielsen C, Mithal P, et al. Neuroinflammation mediated by
IL-lb increases susceptibility of dopamine neurons to degeneration in
[31] Hassan KM, El-Dean AMK, Youssef MSK, Atta FM, Abbady MS. Some reactions
of 3-amino-2-carboethoxy-4,6-dimethylthieno[2,3-b]-pyridine: Synthesis of
some new thienopyridopyrimidines. Phosphorus Sulfur Silicon Relat Elem
1990;47:283–9.
an animal model of Parkinson’s disease.
8–20.
J Neuroinflammation 2008;5:
[20] Gacs-Baitz E, Minuti L, Taticchi A. Synthesis and complete 1H and 13C NMR
analysis of some 4-androsten-3- one derivatives. Synop 1996;27:324–5.
[21] Frenkel M, Marsh KN. TRC Data Series: Spectral Data for Steroids. Texas,
TX: Thermodynamics Research Center; 1994. 386pp.
[22] IUPAC. Joint Commission on Biochemical Nomenclature (JCBN). Nomenclature
of steroids. Pure Appl Chem, 1989;61:1783–1822.
[32] Shishoo CJ, Devani MB, Ullas GV, Ananthan S, Bhadti VS. Studies on the
synthesis of 2-(2-arylvinyl)thieno(2,3-d)pyrimidines and 5-(2-arylvinyl)
triazolothieno(3,2-e)pyrimidines. J Heterocyc Chem 1985;22:825–30.
[33] Abdelrazek FM. Heterocyclic synthesis with nitriles: synthesis of some new
thiophene and thieno[2,3-d]pyrimidin derivatives iv. Phosphorus Sulfur
Silicon Relat Elem 1996;119:271–7.