Synthesis and biological evaluation of N-aryl-1,4-dihydropyridines as novel antidyslipidemic and antioxidant agents

European Journal of Medicinal Chemistry 45 (2010) 501–509

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European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech

Original article

Synthesis and biological evaluation of N-aryl-1,4-dihydropyridines as novel antidyslipidemic and antioxidant agents Atul Kumar a, *, Ram Awatar Maurya a, Siddharth Sharma a, Mukesh Kumar a, Gitika Bhatia b a b

Medicinal and Process Chemistry Division, Central Drug Research Institute, Lucknow, UP 226001, India Biochemistry Division, Central Drug Research Institute, Lucknow – 226001, India

a r t i c l e i n f o

a b s t r a c t

Article history: Received 26 May 2009 Received in revised form 16 August 2009 Accepted 22 October 2009 Available online 31 October 2009

N-aryl-1,4-dihydropyridines 2a–n were synthesized via iodine catalyzed three-component reaction of cinnamaldehydes, anilines and 2-keto esters in methanol. The synthesized compounds were screened for their antidyslipidemic and antioxidant activity in vivo and in vitro. Compounds 2a, 2g, and 2l have exhibited promising lipid and TG lowering activity, whereas compounds 2m and 2n have showed potent antioxidant activity. Ó 2009 Elsevier Masson SAS. All rights reserved.

Keywords: 1,4-Dihydropyridines Antidyslipidemic Antioxidant

1. Introduction Atherosclerosis and related diseases represent the prevalent cause of morbidity and mortality in industrialized countries [1–4]. Elevated level of plasma concentration of cholesterol, especially low density lipoprotein (LDL) and triglyceride along with free radical oxidative stress are recognized as leading cause in the development of atherosclerosis and coronary heart disease [5–10]. There is now an increasing amount of experimental and clinical evidences which shows the involvement of oxidative modifications of low density lipoproteins (LDL) in the pathogenesis of atherosclerosis. In most cases, oxidative damage takes place in the low density lipoprotein (LDL) of plasma by the hydroxyl radicals generated by the metal ions present in the serum due to the alterations in their oxidation states [11,12]. The drugs, currently being used in the treatment of dyslipidemia [13–19], act by lowering cholesterol or by lowering triglyceride levels in plasma. The commonly used antidyslipidemic statins have several side effects like myositis, arthralgias, gastrointestinal upset and elevated liver function tests. Therefore, there is need to discover potentially better antidyslipidemic agents with minimum side effects. The involvement of hydroxyl free radicals has been shown to be a major contributing factor for the peroxidative damage to

* Corresponding author. Tel.: þ91 522 2612411; fax: þ91 522 2623405. E-mail address: [email protected] (A. Kumar). 0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.ejmech.2009.10.036

lipoproteins present in the blood, which are responsible for the initiation and progression of atherosclerosis [20]. Hyperlipidemia may also induce other abnormalities like oxidation of free fatty acids, leading to the formation of ketone bodies as well as masking liver and muscles resistance to insulin which initiates the progress of diabetes in patients [21]. Furthermore, in hyperglycemic patients, several non-enzymatic glycosylation occurs accompanied by glucose oxidation catalyzed by Cu2þ and Fe2þ resulting in the  formation of O 2 and OH radicals which further accelerates the risk of cardiac diseases in dyslipidemic patients [22]. Therefore, besides cholesterol lowering property, a hypolipidemic agent incorporating antioxidant activity will be able to protect endothelial and myocardial function and could serve as a better antiatherosclerotic agent. Hantzsch 1,4-dihydropyridines (1,4-DHPs) 1 are an important class of biologically active heterocycles. The 1,4-DHPs 1 have been shown to possess diverse range biological activities like antihypertension [23], antitubercular, antioxidant [24,25], antiviral [26], and anticancer activity [27]. Many members of this series (1,4DHPs) such as nifedipine, nicardipine, amlodipine, and felodipine are clinically approved as drugs for the treatment of hypertension and cardiovascular diseases. Cerivastatin (Fig. 1) was a synthetic member of the class of statins, used to lower cholesterol and prevent cardiovascular disease. It was withdrawn from the market in 2001 because of the high rate of serious side effects [28]. Although Hantzsch 1,4-DHPs 1 have been extensively studied, the corresponding N-aryl, 5 or 5,6-unsubstituted 1,4-DHPs 2 (Fig. 1) are

502

A. Kumar et al. / European Journal of Medicinal Chemistry 45 (2010) 501–509

F R2

R

R3

O

R1

R4

N H

F

OH OH O

R1

R

N R2 2

1

OH OH

O

OH

HO

OH

N

N Demethylated cerivastatin

Cerivastatin

NO 2

Cl Cl COOEt MeOOC

MeOOC

O

Cl COOEt O

N H

N H Felodipine

NO 2 MeOOC

O

COOMe MeOOC N H

Nicardipine

Nifedipine

Amlodipine

O

N H

NH2

CH2 Ph N

Fig. 1. Bioactive dihydropyridines and pyridines.

R

R

R1 R2

NH2 R2

R4 CHO

3

R5 4

I2

R6

+

+

R6

O

R3

R1

O

N R3

Methanol O

rt, 1 hr R4

5

R5 2

Scheme 1. Synthesis of N–aryl–1, 4–dihydropyridine.

not well explored [29–31]. In our ongoing research towards the exploration of biologically important heterocycles [32–34], recently we also reported organocatalyzed N-aryl-1,4-dihydropyridines via a simple multi-component reaction for the synthesis of same class of compound [34]. we report here molecular iodine catalyzed synthesis and antidyslipidemic as well as antioxidant activity of 1,4-DHPs of type 2.

Table 1 Synthesis of N-substituted 1,4-DHPs.a

R6 O

2. Chemistry 1,4-DHPs 2 were synthesized via molecular iodine catalyzed three-component reaction of substituted cinnamaldehydes, anilines and 2-keto esters (Scheme 1). Although the reaction was sluggish in dichloromethane, chloroform, THF, DMF, high yields of products were obtained in methanol. Stirring a mixture of cinnamaldehyde (1 mmol), aniline (1 mmol), 2-keto ester (1 mmol), and molecular iodine (5 mol%) in methanol at room temperature was found to be an optimized reaction condition for the synthesis of 1,4DHPs 2. Using optimized reaction protocol, we synthesized a series of N-aryl-1,4-DHPs (Table 1). 3. Biological activity evaluation 3.1. Antidyslipidemic activity Lipid lowering and post heparin lipolytic activity: The antidyslipidemic activities of compounds 2a–n were evaluated in

R5

N

R

R3

R1 R2 2

R4

Entry

R

R1

R2

R3

R4

R5

R6

Product

Yield (%)b

1 2 3 4 5 6 7 8 9 10 11 12 13 14

H H H H H H H H H OCH3 H H H H

H H H H H NO2 H H H H H H H H

H H H H H H H CH3 CH3 H CH3 CH3 H H

H H H H H H H H H H H CH3 H H

H H H H H H H H H H Cl CH3 H H

H CH3 F Cl Br H H OCH3 H H H H H Cl

OC2H5 OC2H5 OC2H5 OC2H5 OC2H5 OC2H5 OCH3 OCH3 OCH3 OCH3 OCH3 OCH3 OtBu OtBu

2a 2b 2c 2d 2e 2f 2g 2h 2i 2j 2k 2l 2m 2n

90 88 89 85 88 90 89 88 92 87 91 90 81 80

a Reaction conditions: cinnamaldehyde (1 mmol), aniline (1 mmol), 2-keto ester (1 mmol), I2 (5 mol%), methanol, rt, 1 h. b Isolated yield.

-37.1%

-15.5%

-11.5%

503

-24.1%

-12.2%

-18.1%

-11.0%

-15.0%

-24.8%

-14.0%

-18.3%

-16.1%

-17.0%

300

-18.7%

350

-21.9%

Total cholesterol (mg/dl)

A. Kumar et al. / European Journal of Medicinal Chemistry 45 (2010) 501–509

250 200 150 100 50 Co nt ro l Tr it o n on ly Tr ito n + 2a Tr ito n + 2b Tr ito n + 2c Tr i to n + 2d Tr ito n + 2e Tr ito n + 2f Tr ito n + 2g Tr ito n + 2h Tr ito n + 2i Tr ito n +2 Tr j ito n + 2k Tr ito n + 2l Tr ito n +2 m Tr Tr ito ito n n + + 2n ge m fib ro zi l

0

Test samples Fig. 2. Total cholesterol lowering activity of compounds 2a–n and the standard drug gemfibrozil.

developed by administration of Triton WR-1339 (Sigma chemical co., St. Louis, USA) at a dose of 400 mg/kg body wt. intraperitoneally to animals of all groups except the control. Compounds 2a–n were macerated with gum acacia (0.2% w/v), suspended in water and fed simultaneously with triton at a dose of 100 mg/kg po to the animals of treated groups. Animals of the control and triton group without treatment with test compounds were given same amount of gum acacia suspension (vehicle). After 18 h of treatment 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 2500g) at 4  C for 10 min and the plasma was separated. Plasma was diluted with normal saline (ratio 1:3) and used for analysis of total cholesterol (TC), phospholipids (PL), triglycerides (Tg) by standard enzymatic procedures. Post heparin lipolytic activity (PHLA) was assayed (Wing and Robinson, 1968) using spectrophotometer and Beckmann auto-analyzer and standard kits purchased from Beckmann Coulter International, USA.

Table 2 Total cholesterol lowering activity of compounds 2a–n and the standard drug gemfibrozil. Entry

Compounds

Total cholesterol lowering activity (%)

S.Da

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Control Triton only Triton þ 2a Triton þ 2b Triton þ 2c Triton þ 2d Triton þ 2e Triton þ 2f Triton þ 2g Triton þ 2h Triton þ 2i Triton þ 2j Triton þ 2k Triton þ 2l Triton þ 2m Triton þ 2n Triton gemfibrozil

–

1.78 1.85 5.01 4.27 4.54 5.43 4.79 4.70 4.60 3.54 4.60 6.94 6.37 6.75 4.67 4.24 4.27

3.2. Antioxidant activity

Standard deviation.

200 150 100 50 0

Test samples

Fig. 3. Phospholipid lowering activity of compounds 2a–n and the gemfibrozil.

-39.1%

-13.5%

-12.8%

-23.9%

-13.4%

-18.7%

-11%

-5.7%

-12.5%

-16.5%

-15.3%

-23.7%

-28.5%

250

Co nt ro Tr l it o n on Tr ly it o n + 2a Tr it o n + 2b Tr it o n + 2c Tr it o n + 2d Tr it o n + 2e Tr it o n + Tr 2f it o n + 2g Tr it o n + 2h Tr it o n + 2i Tr it o n +2 Tr j it o n + 2k Tr it o n + Tr 2l it o n +2 m Tr Tr it o it o n n + + 2n ge m f ib ro zil

Phospholipid (mg/dl)

300

-15.3%

Antioxidant activity (generation of free radicals): Superoxide anions (O 2 ) were generated enzymatically by xanthine (160 mM), xanthine oxidase (0.04 U), and nitroblue tetrazolium (320 mM) in absence or presence of compounds 2a–n (100 mg/mL) in 100 mM phosphate buffer (pH 8.2). Fractions were sonicated well in phosphate buffer before use. The reaction mixtures were

a triton model [35]. Adult male Charles Foster rats (200  225 g) bred in the animal house of the institute were used for the lipid lowering activity. Rats were divided in control, triton induced, triton plus compounds and gemfibrozil (100 mg/kg) treated groups containing five rats in each. Hyperlipidemia was

-21.3%

a

3.50 21.9 18.7 17.0 16.0 18.3 14.0 24.8 15.0 11.0 18.1 12.2 24.1 11.5 15.5 37.1

504

A. Kumar et al. / European Journal of Medicinal Chemistry 45 (2010) 501–509

Table 3 Phospholipid lowering activity of compounds 2a–n and the gemfibrozil.

Table 4 Triglyceride lowering activity of compounds 2a–n and gemfibrozil.

Entry

Compounds

Phospholipid lowering activity (%)

S.Da

Entry

Compounds

Triglyceride lowering activity (%)

S.Da

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Control Triton only Triton þ 2a Triton þ 2b Triton þ 2c Triton þ 2d Triton þ 2e Triton þ 2f Triton þ 2g Triton þ 2h Triton þ 2i Triton þ 2j Triton þ 2k Triton þ 2l Triton þ 2m Triton þ 2n Triton gemfibrozil

–

2.40 4.91 4.79 5.22 5.81 3.85 11.15 6.00 3.98 3.14 2.60 3.80 4.85 4.64 6.33 3.74 6.42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Control Triton only Triton þ 2a Triton þ 2b Triton þ 2c Triton þ 2d Triton þ 2e Triton þ 2f Triton þ 2g Triton þ 2h Triton þ 2i Triton þ 2j Triton þ 2k Triton þ 2l Triton þ 2m Triton þ 2n Triton gemfibrozil

–

2.81 6.70 4.96 4.28 5.16 4.78 3.56 5.04 4.00 4.49 3.49 5.35 6.00 4.35 6.30 2.58 5.55

a

Standard deviation.

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 calculated spectrophotometrically. In another set of experiment effect of compounds on the generation of hydroxyl radical (OH) was studied by non-enzymatic reactants. Briefly, OH were generated in a non-enzymatic system comprising deoxy ribose (2.8 mM), FeSO4.7H2O (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 test compounds (100 mg/mL and 200 mg/mL) were incubated at 37  C for 90 min. The test compounds were also studied for their inhibitory action against microsomal lipid peroxidation in vitro by non-enzymatic inducer. Reference tubes and reagents blanks were also run simultaneously. Malondialdehyde (MDA) contents in both experimental and reference tubes were estimated spectrophotometrically by thiobarbituric acid as mentioned above. Alloprinol, Mannitol and a-tocopherol were used as standard drugs for superoxide, hydroxylations and microsomal lipid peroxidation.

-36.1%

-11.5%

-11.0%

-25.7%

-8.9%

-18.7%

-8.1%

-10.5%

-12.1%

-9.8%

-13.6%

-21.2%

200 150 100 50 0 Co nt ro Tr l ito n on ly Tr i to n + 2a Tr ito n + 2b Tr i to n + 2c Tr ito n + 2d Tr i to n + 2e Tr ito n + 2f Tr ito n + 2g Tr ito n + 2h Tr i to n + 2i Tr i to n +2 Tr j i to n + 2k Tr i to n + 2l Tr i to n +2 m Tr Tr ito ito n n + + 2n ge m fib ro zil

Triglycerides (mg/dl)

250

-25.3%

The total cholesterol (TC) of control groups were estimated as 91.51  5.62 mg/dl (Fig. 2, Table 2). Administration of triton

-20.7%

WR-1339 in rats induced marked hyperlipidemia as evidenced by 3.50 fold increase in the plasma levels of TC (320.60  8.64). The standard drug gemfibrozil decreased the TC level by 37.1% as compared to triton only group. The compounds 2a–n exhibited their TC lowering activity in 11.0%–24.8% range. Three most active compounds of the series were 2a (21.9%), 2g (24.8%) and 2l (24.1%). Treatment of rats with triton WR-1339 increased their plasma phospholipids (PL) by 3.12 folds (Fig. 3, Table 3). The triton only treated groups were compared with triton plus compound treated groups. The compounds 2a–n showed their PL lowering activities in 5.7%–28.5% range. In the series 2a (28.5%) was found to be the most active compound showing while gemfibrozil exhibited 39.1% PL lowering activity. Administration of triton in rats elevated their triglyceride (Tg) levels by 3.14 fold (Fig. 4, Table 4). Treatment of hyperlipidemic rats with compounds 2a–n reversed the plasma level of Tg with varying extents. Compound 2a (25.3%), 2g (18.7%), 2h (20.7%) and 2l (25.7%) showed potent Tg lowering activity, while other compounds showed mild lipid lowering activity as compared to triton. These data were compared with gemfibrozil, which showed a decrease in Tg levels by 36.1%. Triton induced rats caused inhibition of post heparin lipolytic activity (PHLA) [36,37] (32.6%) as compared to control. In PHLA, the treatment with compound partially reactivated these lipolytic

4. Results and discussion

300

3.14 25.3 21.2 13.6 9.8 12.1 10.5 18.7 20.7 8.1 18.7 8.9 25.7 11.0 11.5 36.1

Standard deviation.

-18.7%

a

3.12 28.5 23.7 15.3 16.5 12.5 5.7 21.3 15.3 11.0 18.7 13.4 23.9 12.8 13.5 39.1

Test samples

Fig. 4. Triglyceride lowering activity of compounds 2a–n and gemfibrozil.

+34.2%

505

+8.9%

+8.4%

+23.7%

+13.9%

+9.8%

+8.4%

+14.7%

+10.9%

+12.3%

+12.0%

14

+8.3%

16

+8.1%

+16.3%

18

+24.9%

20

12 10 8 6 4 2 0 Co nt ro Tr l it o n on ly Tr it o n + 2a Tr it o n + 2b Tr it o n + 2c Tr it o n + 2d Tr it o n + 2e Tr it o n + 2f Tr it o n + 2g Tr it o n + 2h Tr it o n + 2i Tr it o n +2 Tr j it o n + 2k Tr it o n + 2l Tr it o n +2 m Tr Tr it o it o n n + + 2n ge m f ib ro zil

PHLA (nmol free fatty acid formed/h/mL plasma)

A. Kumar et al. / European Journal of Medicinal Chemistry 45 (2010) 501–509

Test samples

Fig. 5. Post heparin lipolytic activity (PHLA) of compounds 2a–n and gemfibrozil.

extra hepatic tissues resulted an increase in the levels of circulatory lipids [38,39]. The Structure activity relationship revealed that compounds with methyl and ethyl esters are more active in comparison to tbutyl ester. Electron donating and electron withdrawing substitution on phenyl rings have not shown any significant effect on activity. The antioxidant activities of compounds 2a–n were evaluated by generating free radicals [superoxide ions (O 2 ), hydroxyl radicals (OH), microsomal lipid peroxidation] in vitro in the absence and presence of these compounds. The scavenging potential of compounds 2a–n and the standard drug alloprinol at 100 and 200 mg/mL against formation of superoxide ions (O 2 ) are shown in Fig. 6/Table 6. The compounds of the series 2a–n showed significant antioxidant activity. The Structure activity relationship revealed that compounds with tertiary butyl ester functionality (2m and 2n) were most active compounds of the series. Mannitol was taken as standard drug in order to compare the Hydroxyl (OH) radical scavenging potential of compounds 2a–n. Mannitol showed 31.1% and 44.5% hydroxyl radical scavenging activity at 100 and 200 mg/mL respectively. The compounds 2a–n exhibited 10.9%–26.9% activity at 100 mg/mL concentration and 22.6%–37.1% activity at 200 mg/mL concentrations. 2e, 2i, 2m and 2n were among the most active compounds of the series. The results of this study are shown in Fig. 7/Table 7.

Table 5 Post heparin lipolytic activity (PHLA) of compounds 2a–n and gemfibrozil. Entry

Compounds

Post heparin lipolytic activity (%)

S.Da

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Control Triton only Triton þ 2a Triton þ 2b Triton þ 2c Triton þ 2d Triton þ 2e Triton þ 2f Triton þ 2g Triton þ 2h Triton þ 2i Triton þ 2j Triton þ 2k Triton þ 2l Triton þ 2m Triton þ 2n Triton gemfibrozil

– 32.6 16.3 24.9 8.1 8.3 12.0 12.3 10.9 14.7 8.4 9.8 13.9 23.7 8.4 8.9 34.2

0.51 0.09 0.12 0.09 0.26 0.12 0.12 0.11 0.10 0.17 0.24 0.09 0.25 0.17 0.38 0.17 0.27

Standard deviation.

activities in plasma of hyperlipidemic rats. However, gemfibrozil causes the significant reversal of these enzymes level (Fig. 5, Table 5). Triton WR-1339 acts as surfactant, suppresses the action of lipase and blocks the uptake of lipoproteins from the circulation of

-77.5%

-66.4%

-24.5% -48.0%

-52.9%

-30.7%

-23.6% -32.2%

-21.3% -35.3%

-29.8% -44.8%

-21.1% -48.0%

-43.0%

-25.9%

200 microgram/mL

-20.6% -33.6%

-18.7% -28.4%

-19.1% -37.4%

-25.7% -42.5%

-22.9%

-35.8%

100 90 80 70 60 50 40 30 20 10 0

-21.5% -34.0%

Superoxide ions (nmol formazone formed/min)

100 microgram/mL

-19.8% -34.7%

a

Compounds Fig. 6. Superoxide ions

(O 2)

scavenging potential of compounds 2a–n and alloprinol.

A. Kumar et al. / European Journal of Medicinal Chemistry 45 (2010) 501–509

Table 6 Superoxide ions (O 2 ) scavenging potential of compounds 2a–n and alloprinol.

a b c d

6. Experimental 6.1. Materials and general Unless otherwise specified all the reagents and catalysts were purchased from Sigma–Aldrich and were used without further any purification. The common solvents were purchased from Ranbaxy. Organic solutions were concentrated under reduced pressure on a Bu¨chi rotary evaporator. Chromatographic purification of products was accomplished using flash chromatography on 230–400 mesh

5. Conclusion

-31.2%

40 30 20 10

Fig. 7. Hydroxyl radical scavenging potential of compounds 2a–n and mannitol.

2n an ni to l

Compounds

M

2g

2f

2e

2d

2c

2b

2a

0

-44.5%

50

-34.6%

-19.2%

-23.1%

-26.9% -23.0%

-26.9%

-35.7%

-31.1%

-11.5%

200 microgram/mL -14.8%

-20.8%

-10.9% -25.5%

60

-32.7%

-29.3%

-20.7%

-13.8%

100 microgram/mL

70

Co nt ro l

Generation of hydroxyl radicals (nmol MDA formed/h/mg protein)

In conclusion, we have synthesized N-aryl-1,4-DHPs via a molecular iodine catalyzed three-component reaction of

80

Standard deviation for 100 mg/ml. Standard deviation for 200 mg/ml. Dose of 100 mg/ml. Dose of 200 mg/ml.

cinnamaldehyde, anilines and 2-keto esters. All the synthesized compounds were screened for their lipid lowering, and antioxidant activities in vivo and in vitro. The compounds 2a, 2g, and 2l have shown promising antidyslipidemic activity whereas compound 2m and 2n have exhibited significant antioxidant activity. It is interesting to observe that ester group is playing important role in segregating antidyslipidemic and antioxidant activity. Compounds having methyl/ethyl ester groups are exhibiting promising antidyslipidemic activity whereas compounds with tertiary butyl ester functionality are showing potent antioxidant activity.

The microsomal lipid peroxidation scavenging activities of Naryl-1,4-DHPs 2a–n was studied. a-Tocopherol was taken as standard drug which showed 43.9% and 52.1% activity at 100 and 200 mg/mL concentrations respectively. The synthesized compounds 2a–n exhibited 14.7%–35.7% activity and 22.8%–38.4% activity at 100 and 200 mg/mL concentrations respectively. The results are shown in Fig. 8/Table 8.

90

29.3% 32.7% 25.5% 31.1% 35.7% 26.9% 23.0% 22.6% 33.1% 27.7% 23.0% 29.3% 37.1% 34.6% 44.5%

-37.1%

Alloprinol

13.8% 20.7% 10.9% 20.8% 14.8% 11.5% 26.9% 13.9% 14.1% 18.3% 12.0% 19.7% 23.1% 19.2% 31.2%

-19.7%

2n

2.50 3.16 1.80 1.47 3.59 1.47 1.70 1.68 1.67 1.84 1.30 1.45 2.59 1.75 0.96

-29.3%

2m

3.78 2.95 3.06 2.52 1.24 2.68 1.25 1.47 2.02 2.27 2.51 2.09 5.07 1.38 2.53 4.3

51.62 49.08 54.35 50.29 46.91 53.35 56.16 56.51 48.18 52.77 56.22 51.58 45.89 47.76 40.52

2m

2l

72.98 60.92 57.84 65.00 57.83 62.15 64.60 53.35 62.85 62.70 59.62 64.23 58.57 56.05 58.95 50.24

2l

2k

Control 2a 2b 2c 2d 2e 2f 2g 2h 2i 2j 2k 2l 2m 2n Mannitol

-12.0%

2j

Activityd

-23.0%

2i

Activityc

2k

2h

SDb

-18.3%

2g

– 21.5% 34.0% 22.9% 35.8% 25.7% 42.5% 19.1% 37.4% 18.7% 28.4% 20.6% 33.6% 25.9% 43.0% 19.8% 34.7% 21.1% 48.0% 29.8% 44.8% 21.3% 35.3% 23.6% 32.2% 30.7% 52.9% 24.5% 48.0% 66.4% 77.5%

SDa

-27.7%

2f

3.04 2.47 4.29 2.61 2.33 2.46 2.44 3.68 2.92 4.11 2.54 2.94 3.50 2.95 2.01 2.47 3.54 1.77 2.20 2.95 2.28 1.94 2.15 2.78 2.50 2.07 3.97 2.12 1.86 1.88 0.75

Generation of hydroxyl radicals (OH)

2j

2e

90.06 70.71 59.45 69.46 57.85 66.9 51.76 72.82 56.41 73.21 64.45 71.48 59.76 66.70 51.34 72.21 58.85 71.03 46.84 63.20 49.74 70.84 58.29 68.84 61.04 62.45 42.42 65.30 46.80 30.24 20.29

Test compound

-14.1%

2d

Activity

2i

2c

S.D

-13.9%

2b

100 200 100 200 100 200 100 200 100 200 100 200 100 200 100 200 100 200 100 200 100 200 100 200 100 200 100 200 100 200

Generation of superoxide ions (O 2)

-22.6%

Control 2a

Conc. of comp. (mg/mL)

2h

Test compound

Table 7 Hydroxyl radical scavenging potential of compounds 2a–n and mannitol.

-33.1%

506

A. Kumar et al. / European Journal of Medicinal Chemistry 45 (2010) 501–509

-43.9% -52.1%

-42.1%

-18.5% -26.6%

-35.7%

-22.7% -33.1%

-16.2% -22.8%

-17.6% -37.4%

-30.3%

-22.7%

-14.7% -28.9%

-21.5% -37.3%

-25.1%

-34.4%

-35.8%

60

-38.4%

70

-32.1%

80

-19.2%

200 microgram/mL -20.7%

-25.5%

90

-28.5%

-23.5%

-19.9%

100

-37.1%

50 40 30 20 10 0 2n To co ph er o

Compounds

a-

2m

2l

2k

2j

2i

2h

2g

2f

2e

2d

2c

2b

2a

Co n

tro

l

l

Microsomal lipid peroxidation (nmol MDA formed/h/mg protein)

100 microgram/mL

507

Fig. 8. Microsomal lipid peroxidation scavenging potential of compounds 2a–n and a-tocopherol.

silica gel. Reactions were monitored by thin-layer chromatography (TLC) on 0.25 mm silica gel plates visualized under UV light, iodine or KMnO4 staining. 1H and 13C NMR spectra were recorded on a Brucker DRX -300 Spectrometer. Chemical shifts (d) are given in ppm relative to TMS and coupling constants (J) in Hz. The purity of all the synthesized compounds is >98%. IR spectra were recorded on an FT IR spectrophotometer Shimadzu 8201 PC and are reported in terms of frequency of absorption (cm1). Mass spectra (ESI MS) were obtained by Micromass Quattro II instrument. 6.2. General procedure for the synthesis of N-substituted-1,4dihydropyridines Cinnamaldehyde (1 mmol), aniline (1 mmol), acetoacetate ester (1 mmol) and molecular iodine (5 mol %) were taken in 25 ml round bottom flask and stirred for 1 h. After completion the reaction mixture was diluted with water and extracted with ethyl acetate. The ethyl acetate layer was dried over anhydrous sodium sulphate and concentrated to give crude, which was purified by silica gel column chromatography. Table 8 Microsomal lipid peroxidation scavenging potential of compounds 2a–n and atocopherol. Test compound

Microsomal lipid peroxidationa

SDb

SDc

Control 2a 2b 2c 2d 2e 2f 2g 2h 2i 2j 2k 2l 2m 2n a-Tocopherol

86.03 68.91 65.83 61.55 68.38 68.19 69.50 67.51 73.37 66.46 70.87 72.07 66.49 55.32 70.15 48.24

4.27 2.78 1.68 1.59 1.67 2.43 1.82 1.31 9.38 1.75 1.48 1.68 1.61 2.24 1.79 2.56

0.83 2.37 1.23 1.28 0.98 1.34 2.16 1.23 1.86 1.17 1.92 1.29 1.36 3.23 1.56 0.52

a b c d e

54.11 52.95 55.08 56.44 58.38 64.39 53.90 61.12 53.87 59.94 66.32 57.59 49.83 63.12 41.21

Activityd (%)

Activitye (%)

19.9% 23.5% 28.5% 25.5% 20.7% 19.2% 21.5% 14.7% 22.7% 17.6% 16.2% 22.7% 35.7% 18.5% 43.9%

37.1% 38.4% 35.8% 34.4% 32.1% 25.1% 37.3% 28.9% 37.4% 30.3% 22.8% 33.1% 42.1% 26.6% 52.1%

Microsomal lipid peroxidation (nmol MDA formed/h/mg protein). Standard deviation for 100 mg/ml. Standard deviation for 200 mg/ml. Dose of 100 mg/ml. Dose of 200 mg/ml.

6.2.1. 2-Methyl-1,4-diphenyl-1,4-dihydro-pyridine-3-carboxylic acid ethyl ester (2a) Physical state: Oily. ESI MS (m/z) ¼ 320 (M þ H). IR (Neat, cm1): 1691, 1568, 1221. 1H NMR (CDCl3, 200 MHz) d ¼ 1.19 (t, J ¼ 7.1 Hz, 3H), 2.21 (s, 3H), 4.08 (q, J ¼ 7.1 Hz, 2H), 4.75 (d, 1H, J ¼ 5.5 Hz, CH), 5.07 (dd, J ¼ 5.5 & 7.6 Hz, 1H), 6.23 (d, J ¼ 7.6 Hz, 1H), 7.24–7.47 (m, 10H). 13C NMR (CDCl3, 50 MHz) d ¼ 14.7, 19.2, 40.6, 59.9, 102.1, 107.9, 126.6, 127.8, 127.9, 128.0, 128.8, 129.9, 130.0, 144.1, 148.4, 148.9, 169.2. Elemental analysis calculated for C21H21NO2: C, 78.97; H, 6.63; N, 4.39. Found: C, 78.82; H, 6.50; N, 4.27. 6.2.2. 2-Methyl-4-phenyl-1-(4-methylphenyl)-1,4-dihydropyridine-3-carboxylic acid ethyl ester (2b) Physical state: Oily. ESI MS (m/z) ¼ 334 (M þ H). IR (Neat, cm1): 1691, 1568, 1222. 1H NMR (CDCl3, 200 MHz) d ¼ 1.19 (t, J ¼ 7.1 Hz, 3H), 2.21 (s, 3H), 2.43 (s, 3H), 4.08 (q, J ¼ 7.1 Hz, 2H), 4.75 (d, J ¼ 5.4 Hz, 1H), 5.05 (dd, J ¼ 5.4 & 7.6 Hz, 1H), 6.19 (d, J ¼ 7.6 Hz,1H), 7.14 (d, J ¼ 8.2 Hz, 2H), 7.25–7.44 (m, 7H). 13C NMR (CDCl3, 50 MHz) d ¼ 14.6, 19.1, 23.1, 40.7, 59.8, 101.7, 107.7, 126.6, 127.8, 128.0, 128.7, 130.0, 130.6, 137.7, 141.5,148.7, 149.1,169.3. Elemental analysis calculated for C22H23NO2: C, 79.25; H, 6.95; N, 4.20. Found: C, 79.12; H, 6.80; N, 4.27. 6.2.3. 1-(4-Fluorophenyl)-2-methyl-4-phenyl-1,4-dihydropyridine-3-carboxylic acid ethyl ester (2c) Physical state: Oily. ESI MS (m/z) ¼ 338 (M þ H). IR (Neat, cm1): 1693, 1569, 1216. 1H NMR (CDCl3, 200 MHz) d ¼ 1.19 (t, J ¼ 7.1 Hz, 3H), 2.20 (s, 3H), 4.09 (q, J ¼ 7.1 Hz, 2H), 4.75 (d, J ¼ 5.4 Hz, 1H), 5.06 (dd, J ¼ 5.4, 7.6 Hz, 1H), 6.14 (d, J ¼ 7.6 Hz, 1H), 7.11–7.20 (m, 5H), 7.24–7.33 (m, 4H). 13C NMR (CDCl3, 50 MHz) d ¼ 14.7, 19.0, 40.6, 59.9, 102.3, 108.0, 116.9 (d, J ¼ 22.6 Hz), 126.6, 128.0, 128.8, 129.8 (d, J ¼ 8.2 Hz), 129.9, 140.1 (d, J ¼ 3.8 Hz), 148.2, 148.8, 161.9 (d, J ¼ 247.2 Hz), 169.1. Elemental analysis calculated for C21H20FNO2: C, 74.76; H, 5.97; N, 4.15. Found: C, 74.61; H, 5.82; N, 4.07. 6.2.4. 1-(4-Chlorophenyl)-2-methyl-4-phenyl-1,4-dihydropyridine-3-carboxylic acid ethyl ester (2d) Physical state: Oily. ESI MS (m/z) ¼ 355 (M þ H). IR (Neat, cm1): 1693, 1568, 1222. 1H NMR (CDCl3, 200 MHz) d ¼ 1.18 (t, J ¼ 7.1 Hz, 3H), 2.19 (s, 3H), 4.07 (q, J ¼ 7.1 Hz, 2H), 4.73 (d, J ¼ 5.4 Hz, 1H), 5.07 (dd, J ¼ 5.4, & 7.6 Hz, 1H), 6.16 (d, J ¼ 7.6 Hz, 1H), 7.16–7.44 (m, 9H). 13 C NMR (CDCl3, 50 MHz) d ¼ 14.6, 19.1, 40.6, 59.9, 102.9, 108.2, 126.7, 127.9, 128.8, 129.2, 129.5, 130.2, 133.4, 142.6, 147.7, 148.6, 169.1. Elemental analysis calculated for C21H20ClNO2: C, 71.28; H, 5.70; N, 3.96. Found: C, 71.11; H, 5.85; N, 3.87.

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6.2.5. 1-(4-Bromophenyl)-2-methyl-4-phenyl-1,4-dihydropyridine-3-carboxylic acid ethyl ester (2e) Physical state: Oily. ESI MS (m/z) ¼ 399 (M þ H). IR (Neat, cm1): 1691, 1565, 1221. 1H NMR (CDCl3, 200 MHz) d ¼ 1.18 (t, J ¼ 7.1 Hz, 3H), 2.20 (s, 3H), 4.08 (q, J ¼ 7.1 Hz, 2H), 4.74 (d, J ¼ 5.5 Hz, 1H), 5.07 (dd, J ¼ 5.5 & 7.6 Hz, 1H), 6.17(d, J ¼ 7.6 Hz, 1H), 7.12 (d, J ¼ 8.1 Hz, 2H), 7.23–7.38 (m, 5H), 7.58 (d, J ¼ 8.1 Hz, 2H). 13C NMR (CDCl3, 50 MHz) d ¼ 4.7, 19.2, 40.6, 60.1, 103.0, 108.3, 121.3, 126.7, 128.0, 128.8, 29.5, 129.6, 135.2, 143.1, 147.6, 148.6, 169.0. Elemental analysis calculated for C21H20BrNO2: C, 63.33; H, 5.06; N, 3.52. Found: C, 63.22; H, 5.05; N, 3.37. 6.2.6. 2-Methyl-4-(2-nitrophenyl)-1-phenyl-1,4-dihydro-pyridine3-carboxylic acid ethyl ester (2f) Physical state: Oily. ESI MS (m/z) ¼ 365 (M þ H). IR (Neat, cm1): 1694, 1568, 1524, 1355, 1222. 1H NMR (CDCl3, 200 MHz) d ¼ 0.95 (t, J ¼ 7.1 Hz, 3H), 2.24 (s, 3H), 3.90 (q, J ¼ 7.1 Hz, 2H), 5.21– 5.25 (m, 2H), 6.14–6.16 (m, 1H), 7.22–7.50 (m, 6H), 7.62–7.65 (m, 1H), 7.75 (dd, J ¼ 8.1 & 1.5 Hz, 2H). 13C NMR (CDCl3, 50 MHz) d ¼ 14.3, 19.0, 36.6, 59.9, 100.6, 106.6, 123.6, 127.1, 128.0, 128.1, 130.1, 130.4, 131.6, 133.6, 143.7, 143.8, 148.2, 150.3, 168.3. Elemental analysis calculated for C21H20N2O4: C, 69.22; H, 5.53; N, 7.69. Found: C, 69.17; H, 5.40; N, 7.54. 6.2.7. 2-Methyl-1,4-diphenyl-1,4-dihydro-pyridine-3-carboxylic acid methyl ester (2g) Physical state: Oily. ESI MS (m/z) ¼ 306 (M þ H). IR (Neat, cm1): 3062, 2943, 1688, 1570, 1225. 1H NMR (CDCl3, 200 MHz) d ¼ 2.07 (s, 3H), 3.50 (s, 3H), 4.58 (d, J ¼ 5.6 Hz, 1H), 4.61 (dd, J ¼ 5.6 Hz & 7.6 Hz, 1H), 6.08 (d, J ¼ 7.8 Hz, 1H), 7.07–7.37 (m, 10H). 13C NMR (CDCl3, 50 MHz) d ¼ 19.1, 40.4, 51.1, 101.8, 107.8, 126.5, 126.9, 127.4, 127.7, 127.9, 128.1, 128.2, 128.9, 129.0, 129.5, 129.9, 144.0, 148.6, 148.7, 169.6. Elemental analysis calculated for C20H19NO2: C, 78.66; H, 6.27; N, 4.59. Found: C, 78.60; H, 6.22; N, 4.48. 6.2.8. 1-(4-Methoxy-phenyl)-2,5-dimethyl-4-phenyl-1,4-dihydropyridine-3-carboxylic acid methyl ester (2h) Physical state: Oily. ESI MS (m/z) ¼ 350 (M þ H). IR (Neat, cm1): 2947, 1690, 1567, 1508, 1439. 1H NMR (CDCl3, 300 MHz) d ¼ 1.59 (s, 3H), 2.18 (s, 3H), 3.61 (s, 3H), 3.84 (s, 3H), 4.51 (s, 1H), 6.00 (d, J ¼ 1.3 Hz, 1H), 6.94–6.99 (m, 2H), 7.13–7.42 (m, 7H). 13C NMR (CDCl3, 75 MHz) d ¼ 12.8, 12.9, 44.0, 48.8, 54.2, 98.6, 113.4, 114.1, 124.4, 124.5, 124.9, 126.4, 126.6, 126.7, 126.8, 127.5, 127.6, 127.7, 135.4, 145.6, 146.8, 157.3, 168.0. Elemental analysis calculated for C22H23NO3: C, 75.62; H, 6.63; N, 4.01. Found: C, 76.54; H, 6.57; N, 3.98. 6.2.9. 2,5-Dimethyl-1,4-diphenyl-1,4-dihydro-pyridine-3carboxylicacid methyl ester (2i) Physical state: Oily. ESI MS (m/z) ¼ 320 (M þ H). IR (Neat, cm1): 2945, 1690, 1567, 1493, 1382, 1226. 1H NMR (CDCl3, 300 MHz) d ¼ 1.48 (s, 3H), 2.06 (s, 3H), 3.50 (s, 3H), 4.39 (s, 1H), 5.96 (d, J ¼ 1.3 Hz, 1H), 7.09–7.37 (m, 10H). 13C NMR (CDCl3, 50 MHz) d ¼ 17.3, 17.4, 44.5, 50.5, 99.7, 114.7, 124.4, 125.1, 126.1, 126.5, 126.7, 127.1, 128.5, 142.7, 145.6, 146.5, 168.2. Elemental analysis calculated for C21H21NO2: C, 78.97; H, 6.63; N, 4.39. Found: C, 78.82; H, 6.58; N, 4.31. 6.2.10. 4-(4-Methoxy-phenyl)-2-methyl-1-phenyl-1,4-dihydropyridine-3-carboxylic acid methyl ester (2j) Physical state: Oily. ESI MS (m/z) ¼ 336 (M þ H). IR (Neat, cm1): 2946, 1692, 1595, 1236. 1H NMR (CDCl3, 200 MHz) d ¼ 2.15 (s, 3H), 3.45 (s, 3H), 3.83 (s, 3H), 4.96–5.05 (m, 2H), 5.91 (d, J ¼ 7.4 Hz, 1H), 6.77–6.86 (m, 2H), 7.07–7.31(m, 7H). 13C NMR (CDCl3, 50 MHz) d ¼ 19.0, 33.9, 51.0, 55.7, 107.6, 110.7, 121.2, 121.4, 127.2, 127.5, 128.0, 128.3, 129.5, 129.8, 156.0, 168.0. Elemental

analysis calculated for C21H21NO3: C, 75.20; H, 6.31; N, 4.18. Found: C, 75.02; H, 6.18; N, 4.11. 6.2.11. 1-(3-Chloro-phenyl)-2,5-dimethyl-4-phenyl-1,4-dihydropyridine-3-carboxylic acid methyl ester (2k) Physical state: Oily. ESI MS (m/z) ¼ 354 (M þ H). IR (Neat, cm1): 2943, 1694, 1577, 1478, 1227. 1H NMR (CDCl3, 200 MHz) d ¼ 1.56 (s, 3H), 2.15 (s, 3H), 3.59 (s, 3H), 4.46 (s, 1H), 6.03 (d, J ¼ 1.3 Hz, 1H), 7.08–7.37 (m, 9H). 13C NMR (CDCl3, 50 MHz) d ¼ 18.7, 18.9, 45.9, 51.1, 102.3, 116.6, 125.4, 126.1, 126.7, 127.8, 128.1, 128.6, 130.8, 135.4, 145.3, 146.7, 147.1169.4. Elemental analysis calculated for C21H20ClNO2: C, 71.28; H, 5.70; N, 3.96. Found: C, 71.12; H, 5.60; N, 3.87. 6.2.12. 1-(2,3-Dimethyl-phenyl)-2,5-dimethyl-4-phenyl-1,4dihydro-pyridine-3-carboxylic acid methyl ester (2l) The compound was isolated as 1.13:1 rotamer mixture (2la major rotamer, 2lb minor rotamer). Physical state: Oily. ESI MS (m/ z) ¼ 348 (M þ H). IR (Neat, cm1): 2938, 1690, 1566, 1385, 1231. 1H NMR (CDCl3, 300 MHz) d ¼ 1.58 (s, 3H), 2.09 (s, 3H), 2.19 and 2.25 (2s, 2la and 2lb, 3H, CH3), 2.36 (s, 3H), 3.60 and 3.61 (2s, 2la and 2lb, 3H, OCH3), 4.52 and 4.61 (2s, 2la and 2lb, 1H, CH), 5.78 and 5.87 (2d, J ¼ 1.0 Hz, 2la and 2lb, 1H, CH), 7.00–7.57 (m, 8H). 13C NMR (CDCl3, 75 MHz) d ¼ 12.8, 12.9, 16.1, 16.3, 17.2, 17.3, 19.1, 19.2, 44.3, 44.5, 49.2, 97.3, 97.4, 98.7, 113.6, 114.2, 123.3, 123.9, 124.7, 124.8, 124.9, 125.1, 125.2, 125.3, 126.5, 126.7, 126.8, 127.4, 128.1, 128.2, 128.3, 133.8, 133.9, 134.0, 137.1, 137.5, 137.6, 141.1, 141.5, 145.6, 145.9, 146.9, 147.0, 148.5, 168.1, 168.2. Elemental analysis calculated for C23H25NO2: C, 79.51; H, 7.25; N, 4.03. Found: C, 79.42; H, 7.16; N, 3.97. 6.2.13. 2-Methyl-1,4-diphenyl-1,4-dihydro-pyridine-3-carboxylic acid tert-butyl ester (2m) Physical state: Oily. ESI MS (m/z) ¼ 348 (M þ H). IR (Neat, cm1): 2956, 1690, 1571, 1235. 1H NMR (CDCl3, 200 MHz) d ¼ 1.34 (s, 9H), 2.16 (s, 3H), 4.71 (d, J ¼ 5.1 Hz, 1H), 5.00 (dd, J ¼ 5.1 & 7.7 Hz, 1H), 6.16 (d, J ¼ 7.7 Hz, 1H), 7.22–7.47 (m, 10H). 13C NMR (CDCl3, 50 MHz,) d ¼ 19.1, 28.6, 41.3, 79.6, 103.7, 107.6, 126.5, 127.6, 128.0, 128.7, 129.7, 129.9, 144.2, 147.2, 149.2, 168.0. Elemental analysis calculated for C23H25NO2: C, 79.51; H, 7.25; N, 4.03. Found: C, 79.40; H, 7.12; N, 3.90. 6.2.14. 1-(4-Chlorophenyl)-2-methyl-4-phenyl-1,4-dihydropyridine-3-carboxylic acid tert-butyl ester (2n) Physical state: Oily. ESI MS (m/z) ¼ 382 & 384 (M þ H). IR (Neat, cm1): 2966, 1691, 1570, 1236. 1H NMR (CDCl3, 200 MHz) d ¼ 1.33 (s, 9H), 2.14 (s, 3H), 4.69 (d, J ¼ 5.1 Hz, 1H), 4.99 (dd, J ¼ 5.1 & 7.7 Hz, 1H), 6.10 (d, J ¼ 7.7 Hz, 1H), 7.14–7.41 (m, 9H). 13C NMR (CDCl3, 50 MHz) d ¼ 19.1, 28.6, 41.3, 79.7, 104.5, 108.0, 126.6, 127.9, 128.8, 129.2, 129.3, 130.1, 133.2, 142.8, 146.5, 148.9, 168.6. Elemental analysis calculated for C23H24ClNO2: C, 72.34; H, 6.33; N, 3.67. Found: C, 72.41; H, 6.23; N, 3.51. Acknowledgments Acknowledgments: R. A. Maurya, S. Sharma and M. Kumar are thankful to CSIR, New Delhi for financial support. References [1] [2] [3] [4]

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