Synthesis of novel N-(2-hydroxy-2-p-tolylethyl)-amide and N-(2-oxo-2-p-tolylethyl)-amide derivatives and their antidyslipidemic and antioxidant activity

Article abstract of DOI:10.1016/j.bmcl.2011.08.099

In continuation of our drug discovery program on metabolic diseases, we identified an alkaloidal amide, that is, Aegeline (V) from the plant Aegle marmelos leaves as a dual acting agent (antihyperlipidemic and antihyperglycemic). We therefore synthesized

Full text of DOI:10.1016/j.bmcl.2011.08.099

Bioorganic & Medicinal Chemistry Letters 21 (2011) 6393–6397
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of novel N-(2-hydroxy-2-p-tolylethyl)-amide
and N-(2-oxo-2-p-tolylethyl)-amide derivatives and their antidyslipidemic
and antioxidant activity q
T. Narender a, , K. Rajendar a, S. Sarkar a, V.K. Singh a, Upma Chaturvedi b, A.K. Khanna b, G. Bhatia b
⇑
a Medicinal and Process Chemistry Division, Central Drug Research Institute (CSIR), Lucknow 226 001, UP, India
b Biochemistry Division, Central Drug Research Institute (CSIR), Lucknow 226 001, UP, India
a r t i c l e i n f o
a b s t r a c t
Article history:
In continuation of our drug discovery program on metabolic diseases, we identified an alkaloidal amide,
that is, Aegeline (V) from the plant Aegle marmelos leaves as a dual acting agent (antihyperlipidemic and
antihyperglycemic). We therefore synthesized a series of alkaloidal amides [N-(2-hydroxy-2-p-tolyleth-
yl)-amides and N-(2-oxo-2-p-tolylethyl)-amide derivatives] related to Aegeline and screened for their
in vivo antihyperlipidemic activity in Triton induced hyperlipidemia model. The synthetic compounds
4, 17 and 20 showed equipotent activity to the natural product, that is, Aegeline (V). These compounds
also showed strong antioxidant activity, which support their antihyperlipidemic activity. Compound 12
showed better antihyperlipidemic and antioxidant profile than the natural product V.
Received 10 May 2011
Revised 12 August 2011
Accepted 24 August 2011
Available online 28 August 2011
Keywords:
Aegeline
Antidyslipidemic activity
Triton model
Ó 2011 Elsevier Ltd. All rights reserved.
Aegle marmelos
Phenylethanolamine derivatives
When carbohydrates are in low supply or their breakdown is
incomplete, fats become the preferred source of energy in diabetic
patients. As a result, the fatty acids are mobilized into the general
circulation leading to secondary triglyceridemia in which total ser-
um lipids in particular triglycerides as well as the levels of choles-
terol and phospholipids increase. This rise is proportional to the
severity of the diabetes. Uncontrolled diabetes is manifested by a
very high rise in triglycerides and fatty acid levels. An increase in
plasma lipids, particularly cholesterol, is a common feature of ath-
erosclerosis, a condition involving arterial damage, which may lead
to ischemic heart disease, myocardial infarction, and cerebrovascu-
lar accidents. These conditions are responsible for one-third of
deaths in industrialized nations.1
The discovery of new drugs from traditional medicine is not a
new phenomenon (Fig. 1). Christophe et al.2 discovered glucose in-
duced insulin secretion in vitro and ex vivo of 4-hydroxyisoleucine.
Furthermore, in type 2 diabetic rat model the compound was active
and partly corrected hyperglycemia and glucose tolerance. Recently
we have reported the lipid lowering activity of 4-hydroxyisoleucine
in high fat diet fed hamster model.3 Metformin (I) is currently used
as antidiabetic agent in the treatment of type 2 diabetes. Metformin
1 and its analogues4 were synthesized on the basis of a natural
product lead, that is, galegine (II).5 The synthetic cholesterol lower-
ing statins such as fluvastatin (III),6 cerivastatin7 were synthesized
on the basis of natural product lead, that is, mevastatin (IV).8 The
plant derived saponin derivative, pamaqueside (CP-148623),9 has
been reported for cholesterol absorption up to 35–40% and the fish
oils, which contain fatty acids such as eicosapentaenoic acid and
docosahexaenoic acids, have been reported for their lowering activ-
ity on triglycerides and cholesterol.10 The plant Aegle marmelos is
commonly known as ‘bael’ in India,11 belongs to the family of Ruta-
ceae, widely of diabetes mellitus. Our bioactivity-guided fraction-
ation and isolation work led to discovery of Aegeline (V) as an
antihyperglycemic and antidyslipidemic principle.12 Chatterjee
and Bose13 isolated the Aegeline (V) for the first time in 1952 and
its chemical structure and stereochemistry has been elucidated by
synthesis.14 Various methods for the synthesis of optically active
Aegeline (V) have also been appeared in the literature.15 As a part
of our drug discovery program on antidyslipidemic agents, we syn-
thesized several analogue compounds of the natural product V
(Aegeline) and studied their antidyslipidemic activity in Triton in-
duced hyperlipidemia model (Fig. 2).
The synthesis of alkaloidal amide derivatives started with a
reaction between p-methyl phenacyl bromide (1) and NaN3 in
MeOH to provide p-methylphenacylazide (2). The phenacyl azide
(2) was reduced to give common synthon, p-methyl phenylacet-
amide (3) and the resultant p-methyl phenylacetamide was re-
acted with various acid/acid chlorides such as substituted
cinnamic acids, substituted benzoic acid, phenylacetic acids and
q
CDRI Communication No. 8125.
⇑
Corresponding author. Tel.: +91 522 2612411; fax: +91 522 2623405.
E-mail
addresses:
t_narendra@cdri.res.in,
tnarender@rediffmail.com
(T. Narender).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.08.099

6394
T. Narender et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6393–6397
NH NH
NH
N
N
H
NH 2
H2N
N
H
Galegine (II)
O
Metmorfin (I)
HO
COOH
OH
O
O
O
H
N
F
Fluvastatin (III)
Mevastatin (IV)
OH
H
N
O
Aegeline (V)
O
Figure 1. Naturally occurring and synthetic antidiabetic and antidyslipidemic agents.
O
O
O
a
NH3
Cl
Br
N3
b
3
2
1
c
OH
H
N
O
H
R
N
R
d
O
O
R
4 - 14
15 - 20
Figure 2. Synthesis of Aegeline analogues. Reagents: (a) NaN3, MeOH and H2O. (b) 10% Pd/C, concd HCl and dry MeOH. (c) acid, DEA, DIC, DCM and 0 °C to rt. (d) NaBH4 and
MeOH.
heterocyclic acids to provide the various amides 4–14 (Table 1) and
subsequent reduction of ketone with NaBH4 in MeOH at room tem-
perature resulted in the synthesis of p-methyl phenylethanolamine
derivatives 15–20 (Table 1). The double bond geometry has been
determined as trans for compounds 4–8 and 15–18 by their cou-
pling constant values in 1H NMR spectral data. The optical rotation
values of 15–20 indicated that these compounds appear to be mix-
ture of enantiomers.16
Male rats of Charles forest strain (100–150 g) were divided into
four groups [control, triton-induced, triton+compound (4–20), and
Gemfibrozil (100 mg/kg) treated] containing six rats in each group.
In this experiment of 18 h, hyperlipidemia was developed by
administration of triton WR-1339 (Sigma chemical company,
St. Louis, MO, USA) at a dose of 400 mg/kg. b.w. intraperitoneally
to animals of all the groups except the control. These derivatives
were macerated with gum acacia (0.2% w/v), suspended in water
and fed simultaneously with triton with a dose of 100 mg/kg po
to the animals of treated group and the diet being withdrawn.
Animals of control and triton group without treatment with com-
pounds 4–20 were given same amount of gum acacia suspension
(vehicle). After 18 h of treatment the animals were anaesthetized
with thiopentone solution (50 mg/kg b.w.) prepared in normal sal-
ine and then 1.0 ml blood was withdrawn from retro-orbital sinus
using glass capillary in EDTA coated Eppendorf tube (3.0 mg/ml
blood). The blood was centrifuged at 4 °C for 10 min and plasma
was separated. Plasma was diluted with normal saline (ratio of
1:3) and used for analysis of total cholesterol (TC), triglycerides
(TG) and phospholipids (PL) by standard enzymatic methods17,18
and post-heparin lipolytic activity (PHLA) were assayed using
spectrophotometer and Beckmann auto-analyzer and standard kits
purchased from Beckmann Coulter International, USA.19
The naturally occurring Aegeline (V) lowered the TC by 24%, TG
by 22% and PL by 23%, protein by 24% and also reactivated the post-
heparin lipolytic activity (PHLA) by 15% as compared to control at a
dose of 100 mg/kg.12,20 All the synthesized compounds were also
screened in the same model at a dose of 100 mg/kg. In the

T. Narender et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6393–6397
6395
Table 1
Antihyperlipidemic activity of keto-amide (4–14) and amino-alcohol (15–20) series compounds in Triton induced hyperlipidemia model at a dose of 100 mg/kg
S. No
Compound
Total cholesterola
Triglyceridea
Phospholipida
Proteinb
PHLAc
OH
H
N
V
À24⁄⁄⁄
À22⁄⁄
À23⁄⁄⁄
À24⁄⁄⁄
+14⁄
O
O
AEGELINE
OH
O
O
H
N
04
05
À22⁄⁄
À11⁄
À21⁄⁄
À09
À23⁄⁄⁄
À10⁄
20⁄⁄
+19⁄
+10⁄
O
H
N
À08 NS
O
OH
OH
O
H
À10⁄
À12⁄
À06
À10⁄
À13⁄
+13⁄
+06
N
06
07
À05
O
O
Cl
O
H
N
À12⁄
À12⁄
Cl
O
O
S
O
O
O
H
N
08
09
À07
À04
À09
À06
+07
O
O
H
N
O
O
À10⁄
À12⁄
À14⁄
À14⁄
+12⁄
O
O
H
N
10
11
12
À14⁄
À04
À11⁄
À09
À15⁄
À05
À19⁄
À09
+06
O
F
Cl
O
H
N
+08
NO2
N
O
O
O
H
N
À24⁄⁄⁄
À22⁄⁄
À24⁄⁄⁄
À21⁄⁄
+20⁄⁄
O
O
H
N
13
14
15
À07
À04
À07
À05
À02
À09
À07
À06
À09
À07
À04
À08
+08
+06
+07
N
N
O
H
N
O
O
OH
OH
H
N
(continued on next page)

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T. Narender et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6393–6397
Table 1 (continued)
S. No
Compound
Total cholesterola
Triglyceridea
Phospholipida
Proteinb
PHLAc
+05
OH
OH
OH
H
N
16
À08
À09
À07
À08
O
O
O
OH
OH
H
N
17
18
À24⁄⁄⁄
À16⁄
À05
À23⁄⁄⁄
À14⁄
À22⁄⁄
À13⁄
À07
À21⁄⁄
À15⁄
À11⁄
+21⁄⁄
+14⁄
+08
O
Cl
H
N
Cl
Cl
OH
OH
H
N
19
20
À11⁄
NO2
O
H
N
À23⁄⁄⁄
À33⁄⁄⁄
À25⁄⁄⁄
À31⁄⁄⁄
À22⁄⁄
À23⁄⁄⁄
À31⁄⁄⁄
+19⁄
N
O
Gemfibrozil (standard)
À36⁄⁄⁄
+23⁄⁄⁄
Units of keto-amide series compounds: a: mg/dl; b: g/dl; c: n mol of free fatty acids formed/h/ml of plasma; d: Triton treated group compared with control and Triton plus
drug treated group compared with Triton group: Values are mean SD of six rats, ⁄⁄⁄p <0.001; ⁄⁄p <0.01; ⁄p <0.05; values without star = Non significant.
Table 2
Antioxidant activity of Aegeline and synthetic compounds
Super oxide anions (O2À
)
Hydroxyl radicals (ÅOH)b
Microsomal lipid peroxidationb
a
S. No
Compound
Dose (lg/ml)
100
200
À19⁄
À18⁄
À12⁄
OH
H
N
À25⁄⁄⁄
À22⁄⁄
À31⁄⁄⁄
V
O
O
Aegeline
100
200
À17⁄
À17⁄
À22⁄⁄
OH
À31⁄⁄⁄
À29⁄⁄⁄
À29⁄⁄⁄
O
H
N
4
O
100
200
À24⁄⁄⁄
À31⁄⁄⁄
À19⁄
À19⁄
N
À33⁄⁄⁄
À29⁄⁄⁄
O
H
N
12
O
100
200
À21⁄⁄
À18⁄
À19⁄
OH
O
OH
OH
H
N
À32⁄⁄⁄
À27⁄⁄⁄
À23⁄⁄⁄
17
20
O
O
100
200
À17⁄
À17⁄
À22⁄⁄
H
N
À31⁄⁄⁄
À29⁄⁄⁄
À29⁄⁄⁄
N
Standards
200
À69⁄⁄⁄ (Allopurinol)
À45⁄⁄⁄ (Mannitol)
À63*** (
a-tocoferol)
Units: a: n mole formazone formed/minute. b: n mole MDA formed/h. Values are mean SD of six rats, ⁄⁄⁄p <0.001; ⁄⁄p <0.01; ⁄p <0.05.
keto-amide series (4–14) compounds 4 and 12 showed good lipid
lowering activity similar to the natural products. Compound 4 low-
ered the TC by 22%, TG by 21% PL by 23%, Protein by 20% and PHLA
reactivation by 19%, where as 12 lowered the TC by 24%, TG by 22%,
PL by 24%, Protein by 21% and reactivation of PHLA by 20%. In the
hydroxyl amine series (15–20) compound 17 and 20 showed good
lipid lowering activity. Compound 17 lowered the TC by 24%, TG by
23% and PL by 22%, Protein by 21% and PHLA reactivation by 21%.
The amino-alcohol derivative with heterocyclic acid 20 lowered
the TC by 23%, TG by 25% and PL by 22%, Protein by 23% and PHLA
reactivation by 19%.
Recent studies have demonstrated that the generation of large
quantities of reactive oxygen species can cause activation of lipid
peroxidation, protein modification, which leads to cardiovascular
diseases (CVD).21 Therefore we have screened Aegeline (V) and
synthetic compounds (4–20) for their antioxidant activity (Table

T. Narender et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6393–6397
6397
2). Superoxide anions were generated enzymatically22 by xanthine
Supplementary data
(160 mM), xanthine oxidase (0.04 U) and nitroblue tetrazolium
(320 mM) in the absence or presence of compounds (100 mg/ml)
in 100 mM phosphate buffer (pH 8.2). Fractions were sonicated
well in phosphate buffer before use. The reaction mixtures were
incubated at 37 °C and after 30 min the reaction was stopped by
adding 0.5 ml glacial acetic acid. The amount of formazone formed
was measured at 560 nm on a spectrophotometer. Percentage inhi-
bition was calculated taking absorption coefficient of formazone as
7.2 Â 103 M/cm. In another set of experiment, an effect of com-
pounds on generation of hydroxyl radicals (OHÀ) was also studied
by non-enzymic reactants.23 Briefly OHÀ was generated in a non-
enzymic system comprised of deoxyribose (2.8 mM), FeSO4Á7H2O
(2 mM), sodium ascorbate (2.0 mM) and H2O2 (2.8 mM) in
50 mM KH2PO4 buffer, pH 7.4 to a final volume of 2.5 ml. The above
reaction mixtures in the absence or presence of compounds
(100 mg/ml) were incubated at 37 °C for 90 min. Reference sam-
ples and reagent blanks were also run simultaneously. Malondial-
dehyde (MDA) content in both experimental and reference
samples were estimated spectrophotometrically by thiobarbituric
acid method as mentioned above.18,24
Supplementary data (spectroscopic characterization of all new
compounds along with their IR, 1H, 13C spectra, Mass and biological
screening) associated with this article can be found, in the online
version, at doi:10.1016/j.bmcl.2011.08.099.
References and notes
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3. Narender, T.; Anju, P.; Shweta; Tanvir, K.; Rashmi, S.; Bhatia, G.; Chander, R.
Bioorg. Med. Chem. Lett. 2006, 16, 293.
4. Werner, E. A.; Bell, J. J. Chem. Soc. 1922, 221, 1790;; Shapiro, S. L.; Parrino, V. A.;
Freedman, L. J. Am. Chem. Soc. 1959, 81, 3728.
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Dyerberg, J. Acta Med. Scand. 1972, 192, 85; Bang, H. O.; Dyerberg, J.; Hjorne, N.
Acta Med. Scand. 1975, 200, 69.
The natural products V exhibited moderate antioxidant activity
at 200 lg/ml concentration. The compounds 4, 12, 17 and 20,
which are active in hyperlipidemia studies exhibited better antiox-
idant activity than the natural products at the same concentration
(Table 2).
11. Kirtikar, B. D.; Basu, K. R. Indian Med. Plants Part I 1993.
12. Narender, T.; Shweta, S.; Tiwari, P.; Papi Reddy, K.; Khaliq, T.; Prathipati, P.;
Puri, A.; Srivastava, A. K.; Chander, R.; Agarwal, S. C.; Raj, K. Bioorg. Med. Chem.
Lett. 2007, 17, 1808.
13. Chatterjee, A.; Bose, S. J. Ind. Chem. Soc. 1952, 29, 425.
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15. Brown, R. F. C.; Jackson, W. R.; McCarthy, T. D. Tetrahedron: Asymmetry 1993, 4,
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16. Please see the Supplementary data for procedures for the synthesis of
compounds, spectral data, stereochemistry, optical rotation and melting
points of synthetic compounds.
17. Deeg, R.; Ziegehorn, J. Clin. Chem. 1983, 29, 1798; Buccolo, G.; David, H. Clin.
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18. Data were analyzed using Student’s t-test. The hyperlipidemic groups were
compared with control drug treated groups. Similarly the generations of
oxygen free radicals with different derivatives were compared with that of
their formation without compounds. P <0.05 was considered to be significant.
19. Wing, D. R.; Robinson, D. F. Biochem. J. 1968, 109, 841.
20. We previously reported Aegeline’s (V) antihyperlipidemic activity in hamster
model (Bioorg. Med. Chem. Lett. 2007, 17, 1808). In hamster model Aegeline (V)
was fed from day 4 to day 10 (7 days: 7 Â 50 mg = 350 mg/kg body weight and
7 Â 30 mg = 210 mg/kg body weight) and the cumulative lipid profile was
analyzed after that (Day 10). In current manuscript the antihyperlipidemic
activity of Aegeline (V) was studied in triton model. In triton model the
Aegeline (V) and synthetic analogues 4–20 were fed for only one day (100 mg/
kg body weight) and the lipid profile was analyzed after 18 h (on the same day)
of oral administration of compound tested.
21. Kaliora, A. C.; Dedoussis, G. V. Z.; Schmidt, H. Atherosclerosis 2006, 18, 1;
Wattanapitayakul, S. K.; Bauer, J. A. Pharmacol. Ther. 2001, 89, 187; Griendling,
K. K.; Ushio-Fukai, M. J. Lab. Clin. Med. 1998, 132, 9; Hirio, S.; Harada, H.; Nishi,
H. Biochem. Biophys. Res. Commun. 1999, 261, 332.
In conclusion a series of alkaloidal amides (4–20) related to nat-
ural product Aegeline (V) have been synthesized in our laboratory
and screened for their antihyperlipidemic and antioxidant activity.
Some of the compounds of this series (4, 17 and 20) are equipotent
to the natural product V in antihyperlipidemia studies and better
antioxidants than the natural product V. Compound 12 showed
better antihyperlipidemic and antioxidant profile than the natural
product V. Our preliminary structure activity relationship data
indicated that p-methylphenylacetamide derivatives (4 and 12)
are as potent as p-methyl phenylethanolamine derivatives (17
and 20). It appears that stereochemistry is not so important in
the activity, however further studies are required to confirm the
importance of stereochemistry. Further work is in progress to syn-
thesize large number of compounds to confirm the structure activ-
ity relationship and also to evaluate the antihyperglycemic activity
of the compounds presented in this manuscript to develop dual
acting synthetic agent..
Acknowledgments
Authors are thankful to Dr. T. K. Chakrobarthy, Director CDRI for
constant encouragement for the program on the synthesis of natu-
ral products analogues of biological importance, SAIF for spectral
data and CSIR, New Delhi for financial support, Ms. Deepika for
technical support.
22. Bindoli, A.; Valente, M.; Cavallini, L. Pharmacol. Res. Commun. 1985, 17, 831.
23. Halliwell, B.; Gutteridge, J. M. C.; Arouma, O. Anal. Biochem. 1987, 165, 215.
24. Okhawa, H.; Ohishi, N.; Yagi, K. Anal. Biochem. 1978, 95, 351.