Bis[3-(4′-substituted phenyl)prop-2-ene]disulfides as a new class of antihyperlipidemic compounds

Article abstract of DOI:10.1016/j.bmcl.2004.08.015

Novel derivatives of diallylsulfide, an active principle found in garlic, have been synthesized. The synthetic scheme, involving the Wittig reaction has been outlined. Among these derivatives, bis[3-(4′-nitrophenyl)prop-2-ene] disulfide has been found to be effective in lowering lipid levels and in inhibiting the activity of the enzyme HMG-CoA reductase, in Wistar rats. A series of bis[3-(4′-substituted phenyl)prop-2-ene]disulfides were prepared and their hypolipidemic activities were examined in hypercholesterolemic Wistar rats. Introduction of an electron withdrawing group to the phenyl ring in the parent compound led to the identification of compound 8 as a potent and efficacious hypolipidemic agent.

Full text of DOI:10.1016/j.bmcl.2004.08.015

Bioorganic & Medicinal Chemistry Letters 14 (2004) 5347–5350
Bis[3-(40-substituted phenyl)prop-2-ene]disulfides as a
new class of antihyperlipidemic compounds
Meenakshi Sharma, Manisha Tiwari and Ramesh Chandra*
Dr. B.R. Ambedkar Center for Biomedical Research, University of Delhi, Delhi 110 007, India
Received 1 June 2004; revised 5 August 2004; accepted 7 August 2004
Available online 8 September 2004
Abstract—A series of bis[3-(40-substituted phenyl)prop-2-ene]disulfides were prepared and their hypolipidemic activities were exam-
ined in hypercholesterolemic Wistar rats. Introduction of an electron withdrawing group to the phenyl ring in the parent compound
led to the identification of compound 8 as a potent and efficacious hypolipidemic agent.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
pounds diallyldisulfide (DADS) (2, Fig. 1), diallyltrisul-
fide (DATS) and dipropyldisulfide (DPDS), found in
garlic, have been shown to depress cholesterol synthesis
by 10–25% at low concentration (<0.5mmol/L). It has
been shown that incubation of rat hepatocyte culture
with diallydisulfide [a metabolite of allicin (3, Fig. 1)]
at a concentration of about 10lM blocked the forma-
tion of 7-dehydrocholesterol and reduced the produc-
tion of cholesterol.3
At present, the statins (1, Fig. 1) remain the drug of
choice for lowering cholesterol levels.1 However the sta-
tins have been known to be associated with undesirable
side effects including severe myopathy and statin associ-
ated memory loss.2 Consequently there emerges a need
for new therapeutic agents. The lipid-soluble com-
HO
O
The use of diallyldisulfide is restricted due to its extreme
volatility.4 Our objective was to synthesize substituted
derivatives of diallyldisulfide with greater stability and
greater hypolipidemic activity as compared to
diallyldisulfide.
O
O
H
O
H3C
H
CH3
H3C
2. Chemistry
1 Lovastatin
The compounds reported in Table 3 were synthesized by
oxidation of 2-mercaptoethanol (4) with Br2 in the pres-
ence of potassium bicarbonate as shown in Scheme 1 to
yield bis-(2-hydroxy ethyl)disulfide5 (5). The compound
5 was heated with 48% HBr along with concd H2SO4 in
a steam bath to yield bis-(2-bromoethyl)disulfide6 (6).
Compound 6 was refluxed with triphenylphosphine in
dry DMF7 to yield the phosphonium salt (7). The phos-
phonium salt (7) was refluxed with the appropriate alde-
hydes in the presence of lithium ethoxide to afford the
target compounds8,9 (8–12). Structural determination
of all synthesized compounds was carried out by spect-
roscopic analysis (1H NMR, IR and Mass) and CHN
analysis.9
S
S
CH
CH
CH
CH
CH
2
CH
2
2
2
2 Diallydisulphide
S(O)
3 Diallythiosulphinate (Allicin)
CH
CH
CH
CH
S
CH
2
CH
2
2
2
Figure 1.
Keywords: Diallyldisulphide; Hypercholesterolemia; HMG-CoA
reductase.
*
Corresponding author. Tel.: +91 11 27666272; fax: +91 11
27666248; e-mail: acbrdu@hotmail.com
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.08.015

5348
M. Sharma et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5347–5350
a
OH
OH
SH
OH
S
(CH )
2 2
S
(CH )
2
(CH )
2 2
2
5
4
b
c
+
-
+
-
Br
S
S
(CH )
2 2
Br
(CH )
S
(CH )
2 2
BrPh P
S
(CH )
2
2
2
PPh Br
3
3
2
6
7
R
d-h
Compound 8
Compound 9
NO2
NH2
OCH3
CH CH
CH
CH
S
S
CH
2
R
CH
2
R
Compound 10
8-12
Compound 11
Compound 12
H
COOH
Scheme 1. Reagents and conditions: (a) Br2, KHCO3, DCM (33.8% from 4): (b) HBr, H2SO4 (55% from 5); (c) PPh3, DMF, reflux (71.7% from 6);
(d) lithium ethoxide, p-nitro benzaldehyde (60.6% from 7); (e) lithium ethoxide, p-amino benzaldehyde (67.0% from 7); (f) lithium ethoxide,
p-methoxy benzaldehyde (77.8% from 7); (g) lithium ethoxide, benzaldehyde (69.3% from 7); (h) lithium ethoxide, p-carboxy benzaldehyde (49.2%
from 7).
3. Animal treatment
by Brooke Bond Lipton India Ltd. Control hyperchol-
esterolemic groupanimals were fed with 5% cholesterol
in their diet for one week (group2) ( Table 1). The thio-
sulfinate fraction of garlic and lovastatin (20mg/kgbwt)
were taken as two positive control groups and supple-
mented with 5% cholesterol in their diet (groups 3, 4).
The animals in groups 5, 6, 7, 8 and 9 were fed 5% cho-
lesterol containing diet supplemented with synthesized
compounds (8–12) (20mg/kgbwt). The experiment was
repeated three times. Lovastatin and test compounds
were orally administered (suspension in water) to Wistar
rats.
Adult male rats of Wistar strain weighing 150–200g
were used in the investigation. The rats were selected
at random from the stock colony maintained in the ani-
mal house facility, Dr. B.R. Ambedkar Center for Bio-
medical Research, University of Delhi. Animals were
maintained in an air-conditioned room. The room was
maintained at 25 2°C with natural daytime and no
light after 1900h, until morning. Lovastatin was pro-
cured from Ranbaxy Labs India.
The rats were divided into nine groups of six animals
each. The control groupwas treated with saline with a
normal diet. Gold Mohur rat feed was used supplied
The food intake of the animals and weight gain taking
place in the animals is elaborated in Table 1.
Table 1. Food intake and body weight gain in rats fed the respective diets for 7days (values are mean SE of six rats in each group)
Day 1
Food intake (g)
Day 2
Day 3
Day 4
Day 5
Day 6
Day 7
Group1
Group2
Group3
Group4
Group5
Group6
Group7
Group8
Group9
10.25 1.25
11.00 0.95
11.05 0.75
10.95 1.24
12.85 1.34
13.05 0.5
10.50 0.5
11.94 1.5
11.00 1.0
10.50 0.55
10.05 1.0
11.95 0.35
10.45 1.0
12.00 1.25
12.95 0.25
10.85 0.5
13.56 2.0
10.55 0.95
10.00 1.0
11.50 2.0
12.50 2.5
10.40 1.5
12.75 1.0
12.35 0.75
10.00 0.85
12.45 0.53
10.35 0.80
10.30 0.74
12.10 0.45
12.00 1.5
12.90 2.0
12.00 1.5
12.30 2.0
11.05 0.95
12.80 2.56
11.00 0.95
11.05 1.0
11.50 2.0
11.20 2.0
13.05 1.0
10.05 2.0
9.85 0.85
11.25 2.0
10.35 1.5
12.05 3.0
10.75 0.5
11.20 3.0
11.25 1.0
13.00 2.0
9.95 1.5
10.65 3.0
9.75 1.5
12.90 2.0
12.45 1.5
10.05 1.0
9.85 1.5
11.06 3.0
13.75 2.0
10.94 2.0
10.55 0.5
9.25 0.5
12.40 1.0
9.55 2.0
Body weight (g)
Group1
Group2
175 5.25
170 4.85
160 10.05
165 5.5
180 4.5
175 5.65
185 4.5
180 8.5
190 6.5
175 3.5
170 5.5
160 10.05
166 4.5
180 4.6
176 3.65
186 4.4
180 8.6
191 5.5
177 2.5
172 4.5
161 5.05
166 7.5
182 2.6
177 1.5
187 3.5
182 5.6
191 2.5
177 5.5
172 8.5
162 4.05
167 5.5
182 6.5
177 4.5
187 9.5
183 6.5
192 2.5
178 5.5
173 8.5
162 5.05
167 5.5
183 5.5
177 4.5
187 9.5
183 6.5
192 2.5
178 5.5
174 4.5
163 5.05
168 5.5
185 5.5
178 4.5
188 4.5
184 5.5
193 2.5
179 5.5
176 4.5
163 9.05
168 7.5
185 3.5
178 2.5
188 1.5
185 5.5
194 2.5
Group3
Group4
Group5
Group6
Group7
Group8
Group9

M. Sharma et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5347–5350
5349
All the test compounds were found to be soluble in or-
ganic solvents and insoluble in water. The compounds
were stable under ordinary conditions of use and
storage.
4. Biochemical assays
The animals were anaesthetized at the end of the exper-
imental period, after overnight starvation. Blood was
drawn from retroorbital sinus using capillary tubes, into
dried test tubes. The serum was separated for cholesterol
estimation. The animals were immediately dissected
to remove their tissues, which were washed in ice-cold
saline (0.85% NaCl). The extraneous material was re-
moved.10,11 Approximately 1g of tissue was kept for
estimation of lipids and the remaining was used for
other biochemical assays.12
Hepatic and serum cholesterol was determined using
the method of Zlatkis et al.13 Hepatic triglyceride
method was determined by the method of Van Han-
del and Zilversmit.14 HMG-CoA reductase activity
was determined by the method of Venugopala Rao
et al.15
Hepatotoxicity was evaluated by measuring alanine
transferase,16 aspartate transferase17 and alkaline phos-
phatase18 levels in serum. Nephrotoxicity was evaluated
by measuring blood urea nitrogen levels19 and creati-
nine20 in serum (Tables 3 and 4).
5. Results and discussion
In the present communication we describe the synthesis
of several derivatives of diallyldisulfide and an evalua-
tion of their antilipidemic activity12–14 in male Wistar
rats. The rats were fed 5% cholesterol in their diet for
one week (Table 1). The test compounds were simulta-
neously administered to the rats for 1week at a dose
of 20mg/kgbwt. No significant difference was found in
body weight gain and diet consumption in all the treated
groups as compared to control (Table 1). During the
course of this search it was found that, bis[3-(40-nitro-
phenyl)prop-2-ene]disulfide showed significant anti-
hyperlipidemic activity.
The derivatives with electron withdrawing substituents
(8, 12) on the phenyl ring were found to lower hepatic
cholesterol levels by 37% and 13%, respectively, and in-
hibit the activity of HMG-CoA reductase by 56% and
24%, respectively (Table 2). The derivatives with elec-
tron donating substituents (9 and 10) on the phenyl ring
showed weaker activity as evidenced by their reduced
ability to lower hepatic cholesterol levels and inhibit
HMG-CoA reductase. The derivative with no substitu-
ents on the phenyl ring showed low antihyperlipidemic
activity. A comparison of these percentages with those
evoked on treatment with lovastatin demonstrated a
potential use of bis-(4-nitrophenyl)disulfide in lowering
lipid levels.

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M. Sharma et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5347–5350
Table 3. Effect of single and five doses of synthesized compounds on liver marker enzymes
Single dose
Five doses
Alanine
transferase
(GPT) IU
Aspartate
transferase
(GOT) IU
Alklaline
phosphatase
KA Units
Alanine
transferase
(GPT) IU
Aspartate
transferase
(GOT) IU
Alklaline
phosphatase
KA Units
Normal range
Control
18–30
22
46–81
14–32
18–30
22
46–81
48.5 2.5
14–32
45.59 5.8
3
41
51
6
1
30.2 18.2
29.5 12.5
40.6 5.2
45.6 9.0
34.0 0.5
31.0 0.3
4
15.5 0.50
Compound 8
Compound 9
Compound 10
Compound 11
Compound 12
21 3.5
26.5 1.5
26.5 0.5
17.5 0.5
25 1.0
49
37
1
7
48
8
51.5 2.50
21
23
27
2
2
1
46.9 6.7
25.2 9.4
33.6 12.5
32.8 2.5
49
51
4
6
29.5 4.5
39
56
3
4
53 3.5
28 2.5
Table 4. Effect of single and five doses of synthesized compounds on kidney marker enzymes
Single dose
Five doses
Blood urea nitrogen mg/dL
Creatinine mg/dL
BUN mg/dL
Creatinine mg/dL
Normal range
Control
5–29
0.20–0.80
0.715 0.075
0.4 0.06
5–29
0.20–0.80
0.79 0.04
0.8 0.04
10.45 1.41
7.65 0.15
12.96 0.07
7.43 0.65
12.39 1.05
10.8 1.89
6.48 0.015
14.64 1.23
8.48 0.59
7.37 0.31
17.4 0.45
Compound 8
Compound 9
Compound 10
Compound 11
Compound 12
0.55 0.28
0.595 0.145
0.58 0.06
0.61 0.15
0.84 0.175
0.495 0.06
0.57 0.05
0.70 0.03
20.23
2
7. Wittig, G.; Geissler, G. Ann. 1958, 10, 619.
8. Maercker, A. Org. Rea. J. 1965, 14, 270.
9. Satisfactory analytical data were obtained for all the
target compounds. Example, compound 10: mp155–
157°C; 1H NMR (300MHz, CDCl3): 1.63 (s, 4H); 3.91 (s,
6H); 6.90 (m, 2H); 7.27 (d, 2H, J = 3.4Hz); 7.33–7.87 (m,
8H); (traces of aldehydic impurity evident at d 9.91 and d
3.83); MS (FAB): m/z 358 (M+), 357 (Mꢀ1)+: Elemental
analysis % Calcd: C, 67.03; H, 6.14. Found: C, 66.51; H,
5.80.
10. Hogeboom, G. H. In Methods in Enzymology; Colowick,
S. P., Kaplar, N. O., Eds.; Academic: New York, 1955;
Vol. 1, p16.
11. Umbeit, W. W.; Burries, R. H.; Stalifier, S. F. Manometric
Techniques: A Manual Describing Methods Applicable to
the Study of Tissue Metabolism. Burgess Publishing:
Minneapolis, 1957; Vol. 10.
6. Conclusion
Results shows that certain novel derivatives of diallyldi-
sulfide such as bis[3(4-nitrophenyl)prop-2-ene]disulfide
are effective in lowering cholesterol levels and would
be potentially beneficial in treating hyperchol-
esterolemia.
Acknowledgements
The authors wish to acknowledge the financial support
received from the Department of Science and Technol-
ogy, India and the facilities provided by the Dr. B.R.
Ambedkar Center for Biomedical Research, University
of Delhi, Delhi 110007.
12. Bligh, E. G.; Dyer, W. J. Can. J. Biochem. Physiol. 1959,
37, 911.
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