Bioorganic & Medicinal Chemistry 22 (2014) 5824–5830
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Synthesis and evaluation of novel triazoles and mannich bases functionalized 1,4-dihydropyridine as angiotensin converting enzyme (ACE) inhibitors Ravindra M. Kumbhare a,⇑, Umesh B. Kosurkar a, Pankaj K. Bagul b,c, Abhinav Kanwal b,c, K. Appalanaidu a, Tulshiram L. Dadmal a, Sanjay Kumar Banerjee b,c,⇑ a
Fluoroorganic Division, CSIR-Indian Institute of Chemical Technology, Hyderabad 500607, India Medicinal Chemistry and Pharmacology Division, CSIR-Indian Institute of Chemical Technology, Hyderabad 500607, India c Present Address: Drug Discovery Research Center (DDRC), Translational Health Science and Technology Institute (THSTI), Gurgaon 122016, India b
a r t i c l e
i n f o
Article history: Received 28 July 2014 Revised 9 September 2014 Accepted 11 September 2014 Available online 19 September 2014 Keywords: Angiotensin converting enzyme Inhibitors 1,4-Dihydropyridine Triazole Mannich bases
a b s t r a c t A series of novel diethyl 2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate embedded triazole and mannich bases were synthesized, and evaluated for their angiotensin converting enzyme (ACE) inhibitory activity. Screening of above synthesized compounds for ACE inhibition showed that triazoles functionalized compounds have better ACE inhibitory activity compared to that of mannich bases analogues. Among all triazoles we found 6h, 6i and 6j to have good ACE inhibition activity with IC50 values 0.713 lM, 0.409 lM and 0.653 lM, respectively. Among mannich bases series compounds, only 7c resulted as most active ACE inhibitor with IC50 value of 0.928 lM. Ó 2014 Published by Elsevier Ltd.
1. Introduction Hypertension is the leading cause of cardiovascular disease affecting one in three adults worldwide.1 It is the primary cause of stroke, coronary artery disease and sudden cardiac death. Many factors are believed to be involved in the pathogenesis of hypertension, its progression and complications. Renin angiotensin aldosterone system (RAAS) plays an important role in controlling blood volume and hypertension.2,3 Angiotensin converting enzyme (ACE) is a key enzyme regulating the rate limiting step in the RAAS pathway. ACE converts angiotensin I to angiotensin II which is the main active component for hypertension. Angiotensin II causes vasoconstriction along with increased blood volume and water retention.4 To inhibit AngII mediated hypertension and associated complication, several drugs have been synthesized and tested as ACE inhibitors. Captopril was the first peptide-based ACE inhibitor which developed in 1975 and was considered a breakthrough. However, several side effects ⇑ ⇑Corresponding authors. Present Address: Drug Discovery Research Center (DDRC), Translational Health Science and Technology Institute (THSTI), Gurgaon 122016, India. Tel.: +91 11 26741358 (S.K.B.), tel.: +91 40 27191776; fax: +91 40 27193185 (R.M.K.). E-mail addresses: [email protected] (R.M. Kumbhare), [email protected]. in (S.K. Banerjee). http://dx.doi.org/10.1016/j.bmc.2014.09.027 0968-0896/Ó 2014 Published by Elsevier Ltd.
like skin rash, loss of taste and dry cough are reported with captopril.5 All those side effects of captopril are thought to be because of the presence of mercapto group which provides strong chelating action.6 After captopril, research has been aimed in finding potent, selective ACE inhibitors that would not contain a mercapto (SH) functional group and would have a weaker chelating function. Enalapril, lisinopril, ramipril and benzapril are the newly synthesized peptide based molecules available in market. However, all of them still carry the same set of side effects with less severity.7 So there is a need to synthesis non-peptide based analogue for ACE inhibition with fewer side effects. A survey of literature revealed that 1,4-dihydropyridine derivatives have received much attention during recent years due to its versatile biological activities such as antidepressant,8 antihypertensive,9 antithrombotic,10 anticonvulsant,11 cardiotonic,12 antibacterial,13 antiHIV,14 and calcium channel blockers.15 However, little attention has been directed towards the BACE-1 blocking activity of these compounds.16 DHP can be converted to the quaternary amines leading to their retention in the brain.17 Another characteristic feature of DHP scaffold is the possibility of structural modifications via introducing various chemical substituents in different positions of the DHP ring and hence providing remarkable changes in pharmacological profile.18 Dihydropyridine drugs, such
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N O N O
O O
O
O O O
O
O
Darodipine
N H Lacidipine
1
2
N H
O 2N
O O
N
O O
O
N
N H Manidipine 3
Figure 1. Drugs containing 1,4-dihydropyridine framework.
as nifedipine, nicardipine, amlodipine and other drugs must be used with caution in patients with coronary heart disease because of the increased mortality observed in patients treated with these drugs. One of the most discussed undesirable effects is the reflex tachycardia produced by dihydropyridines. Darodipine19 1 (Fig. 1) are dihydropyridine derivatives calcium antagonists that easily pass into the brain, showing high affinity for cerebral L-type voltage-sensitive calcium channel (VSCC). One of the most vascular selective of the dihydropyridines derivatives Lacidipine, 2 is a calcium channel blocker developed for oral administration for use in mild to moderate hypertension,20 and is widely used in therapy since the early 1990s. It exhibits anti-atherosclerotic and antioxidant effects.21 Manidipine 3 is a lipophilic and highly vasoselective agents22 and has long lasting activity for the treatments of hypertension. Manidipine is a dihydropyridine (DHP) calcium channel antagonist, which effectively reduced blood pressure in patients which mild to moderate hypertension and effectively maintains reduced blood pressure levels throughout the dosing periods of 24 h. Hence in continuous of our efforts for the structural modification on heterocyclic moiety23 and thus to improve their bio-
Compound
R
6a
5Br
6b
5Br
6c
R1
logical activity a new series of triazole and mannich base functionalized 1,4-dihydropyridine have been synthesized. 2. Results and discussion 2.1. Synthesis Substituted salicylaldehyde is the starting material in the synthesis of 1,4-dihydropyridine derivatives (6 and 7). It was propargylated with propargyl bromide to yield 4 in excellent yield. Aldehyde group from 4 reacted with ethylacetoacetate and ammonium acetate under reflux condition in ethanol solvent to give 1,4-dihydropyridine 5 in very good yield. Now, to obtain the triazole compounds (6a–j) by click reaction, 5 was reacted with various aliphatic and aryl azides in dry THF using 5 mol % CuI to provide 1,4-disubstituted triazole products. All the compounds obtained in very good yields (78–85%) were confirmed by IR, 1H and mass (ESI and HRMS) spectral data. Also, mannich bases (7a–k) of compound 5 were synthesized by reported method24 and products confirmed by IR, 1H and mass (ESI and HR-MS) spectral data (See Schemes 1-3).
Compound
NN CF3
S
3,5-dibromo
R 2N
R
7a
5Br
7b
5Br
O
HN
HN
N N OH
7c
3,5-dibromo HN
7d
3,5-dibromo
7e
3,5-dibromo HN
N
7f
3,5-dibromo HN
N
N
COOH 6d
6e 6f
3,5-dibromo
N
OCF3 F
SCF3
3,5-dibromo
N
3,5-dibromo O
6g
HN
3,5-dibromo
OH
3 OCH 3
6i
3 OCH 3
6j
3 OCH 3
3 OCH 3
HN
7h
3 OCH 3
HN
O
7i
3 OCH 3
HN
N
7j
3 OCH 3
7k
3 OCH 3
F
N
6h
7g
S
NN S
CF3 C 8 F17
HN
HN
N
COOC2H5
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R.M. Kumbhare et al. / Bioorg. Med. Chem. 22 (2014) 5824–5830 CHO
Br
R
CHO
K 2 CO 3 , Acetone
R
Reflux, 2 h
OH
O 4
R = 5 Br, 3,5-dibr omo, 3O CH 3
Scheme 1.
C2 H 5 OOC O O
CHO
Ethanol
R O
H 3C
O C2H5
NH 4 COOCH3
NH
R
Reflux,3h
O
COO2CH5
5
Scheme 2.
5
a
C2H5OOC
b
C2H5OOC
NH
R
NH
R O
COOC2H5
O
and histidyl-leucine (HL). The extent HA released is directly proportional to the ACE activity. In this screening method, the released hippuric acid from the substrate hippurryl-histyl-leucine (HHL) was mixed with pyridine and benzene sulfonyl chloride. The resulting yellow color (410 nm) is directly proportional to the released hippuric acid and thus with the ACE activity. All the new compounds 6a–j and 7a–k tested in the desired concentrations did not show any significant absorbance at 410 nm under control conditions. Being simple, sensitive and rapid nature, the colorimetric method is used for evaluating ACE inhibitory activity of new analogues 6a–j and 7a–k. Lisonipril, a known ACE inhibitor drug has been used as standard for comparison with the inhibitory activity of synthesized new analogues. The evaluation results of all 21 new compounds for ACE inhibitory activity are presented in Figure 2. The experiments carried out for test compounds at five different concentrations (100 nm to 2 lM) revealed that 6h, 6i, 6j and 7c showed dose dependent inhibition of ACE activity (Fig. 3). The mechanism for obtaining different ACE inhibition activity for different compounds need to be explored further. However, the increased chelating activity of those active compounds with zinc ions of the enzyme cannot be ruled out. The IC50 value of these active compounds are given in Table 1.The results obtained here will be explored further study the binding nature and to generate lead molecules for drug discovery. The toxicity evaluation of four most potent ACE inhibitors 6h, 6i, 6j and 7c was assessed by (3-5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay10 against A549 (adeno
COOC2H5
O 6a-j
N N N
HN R1
7a-k
NR2
Scheme 3. Reagents and conditions: (a) R1NHCOCH2N3, dry THF, CuI, 24 h, rt; (b) Neutral Al2O3, CuI, THF, HCHO, HNR2, 12 h, rt.
2.2. Pharmacology The in vitro angiotensin converting enzyme (ACE) inhibitory activity of new compounds 6a–j and 7a–k were measured using recent colorimetric high-throughput screening method developed by Jimsheena and Gowda.25 Most of the antihypertensive peptides have been characterized by the rabbit lung ACE inhibitor assay, based on the hydrolysis of the synthetic peptide hippurylhistidylleucine (HHL). HHL is hydrolyzed by ACE to hippuric acid (HA)
Figure 3. Dose-dependent angiotensin converting enzyme (ACE) inhibition activity of test compounds and standard compound.
Figure 2. In vitro angiotensin converting enzyme (ACE) inhibition of new analogues and standard drug.
R.M. Kumbhare et al. / Bioorg. Med. Chem. 22 (2014) 5824–5830 Table 1 IC50 values of active compounds selected from preliminary screening S. No.
Compound code
IC50 (lM)
1 2 3 4 5
6h 6i 6j 7c Std.
0.713 0.409 0.653 0.928 0.281
Table 2 MTT assay inhibitory activities (IC50) in lM S. No.
Compound code
A549-cells
HEK-cells
1 2 3 4 5
6h 6i 6j 7c Doxorubicin
>1000 >1000 >1000 >1000 2.6
>1000 >1000 >1000 >1000 0.69
carcinomic human alveolar basal epithelial) and HEK 293 (normal human embryonic kidney) cell lines. The inhibitory activities (IC50) in lM are summarized in Table 2. The compounds 6h, 6i, 6j and 7c exhibited their 50% inhibitory concentrations (IC50) >1000 lM and are considered nontoxic.
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than 200 mesh was used for column chromatography. Appropriate names for all the new compounds were given with the help of Chem Bio Office 2012. 4.1. General procedure for the preparation of compound (4) Salicylaldehyde (1 mmol) was dissolved in acetone (10 mL), K2CO3 (1.3 mmol) was added. The propargyl bromide (1 mmol) was slowly added drop wise to the above mixture over a period of 15 min at room temperature. Reaction mixture was refluxed for 2 h. After completion of reaction, solvent was removed under reduced pressure. The residue was washed with n-hexane repeatedly to remove excess propargyl bromide, treated with distilled water to remove excess potassium carbonate and solid product separated was filtered and dried to get product 4. 4.2. General procedure for the preparation of compound (5) An appropriate propargylated salicylaldehyde (10 mmol), ethyl acetoacetate (20 mmol) and ammonium acetate (22 mmol) were dissolved in ethanol and refluxed for 1 h. After completion of the reaction, reaction mixture was cooled to room temperature and product was extracted with ethyl acetate, washed with water and brine solution. Column chromatography was performed using silica gel (60–120 mesh), eluted with ethyl acetate and hexane (20%) to get product 5.
3. Conclusion 4.3. General procedure for the preparation of compound (6) In conclusion, we have described an efficient synthesis and evaluation of ACE inhibition activity of novel 1,4-dihydropyridine derivatives functionalized with triazoles and mannich bases. Compounds 6h, 6i, 6j and 7c exhibit promising ACE inhibition property comparable to Lisinopril drug. Cytotoxicity study of these compounds showed that all four compounds are nontoxic as assessed by measuring IC50 value in A549 and HEK cell lines, respectively. Screening all the new compounds using in vitro ACE inhibition assay resulted triazole as more potent ACE inhibitors than the respective mannich base analogues by thousand folds. Among the series of triazole 6a–j, three compounds 6h, 6i, 6j were resulted as most active ACE inhibitors with IC50 values of 0.713 lM, 0.409 lM and 0.653 lM, respectively. Compared with ACE inhibitory activity (IC50) of various triazole, mannich base 7c showed relatively higher IC50 value (0.928 lM). The results reveal that binding of heteroaryl triazole at ACE binding pocket is more pronounced through zinc ion chelation than the triazole of natural origin. The information generated here could be of use to generate lead molecules for drug discovery. 4. Experimental section Melting points were measured with a Fischer–Johns melting point apparatus and are uncorrected. IR spectra were recorded as neat liquids or KBr pellets and absorptions are reported in cm 1. 1 H NMR spectra were recorded on 300 (Bruker) and 500 MHz (Varian) spectrometers in appropriate solvents using TMS as internal standard or the solvent signals as secondary standards and the chemical shifts are shown in d scales. Coupling constants J are expressed in Hertz. High-resolution mass spectra were obtained by using ESI-QTOF mass spectrometry. Reagents and all solvents were analytically pure and were used without further purification. All the experiments were monitored by analytical thin layer chromatography (TLC) performed on silica gel GF254 pre-coated plates. After elution, plate was visualized under UV illumination at 254 nm for UV active materials. Further visualization was achieved by staining with PMA and charring on a hot plate. Silica gel finer
To a solution of dipolarophile 5 (1 mmol) in dry THF (5 mL), CuI (5 mol %) was added. Then aryl/aliphatic azide (1 mmol) was added slowly at room temperature under nitrogen atmosphere and continued stirring for 24 h. Solvent was removed under reduced pressure, the residue was diluted with water and extracted with ethyl acetate. The combined organic layers were washed with water, brine, dried over Na2SO4, filtered and concentrated under reduced pressure. This was further purified by column chromatography using (ethyl acetate/n-hexane, 40:60) as eluent to obtained the pure product 6. 4.3.1. Diethyl4-(5-bromo-2-((1-(2-(cyclohexylamino)-2oxoethyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-2,6-dimethyl1,4-dihydropyridine-3,5-dicarboxylate 6a Pale yellow solid, mp = 84 °C, IR (KBr) mmax: 3350, 2933, 1688, 1217, 1038 cm 1; 1H NMR (DMSO, 500 MHz): d = 1.07 (t, 6H, J = 6.9 Hz, CO2CH2Me), 1.13–1.44 (m, 5H), 1.54–1.83 (m, 5H), 2.21 (s, 6H, Me), 3.68 (s, 1H), 3.83–4.09 (m, 4H), 4.86 (s, 1H, pyridine CH), 4.99 (s, 2H, NCH2), 5.16 (s, 2H, OCH2), 6.83–6.89 (m, 1H, ArH), 7.16–7.23 (m, 1H, Ar-H), 7.58 (s, 1H, Ar-H), 7.77 (s, 1H, pyridine NH), 7.92 (s, 1H, CONH), 8.31 (s, 1H, triazole H). MS (ESI) m/z 644 [M+H]+; HR-MS (ESI) calcd for C30H38O6N5Br [M+H]+: 644.2078. Found: 644.2082. 4.3.2. Diethyl 4-(5-bromo-2-((1-(2-oxo-2-(5-(trifluoromethyl)1,3,4-thiadiazol-2-ylamino)ethyl)-1H-1,2,3-triazol-4-yl)meth oxy)phenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5dicarboxylate 6b White solid, mp = 160 °C, IR (KBr) mmax: 3349, 2935, 1690, 1216, 1039 cm 1; 1H NMR (DMSO, 300 MHz): d = 1.11 (t, 6H, J = 6.9 Hz, CO2CH2Me), 2.28 (s, 6H, Me), 3.88–4.15 (m, 4H), 4.89 (s, 1H, pyridine CH), 5.09 (s, 2H, OCH2), 5.28 (s, 2H, NCH2), 6.86–6.93 (m, 1H, Ar-H), 7.18–7.26 (m, 1H, Ar-H), 7.58–7.63 (m, 1H, Ar-H), 7.81 (s, 1H, pyridine NH), 7.98 (s, 1H, CONH), 8.39 (s, 1H, triazole H). MS (ESI) m/z 715 [M+H]+; HR-MS (ESI) calcd for C26H27O6N8BrF3S [M+H]+: 714.0826. Found: 714.0784.
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4.3.3. 3-(2-(4-((2-(3,5-Bis(ethoxycarbonyl)-2,6-dimethyl-1,4dihydropyridin-4-yl)-4,6-dibromophenoxy)methyl)-1H-1,2,3triazol-1-yl)acetamido)benzoic acid 6c White solid, mp = 158 °C, IR (KBr) mmax: 3316, 2979, 1684, 1208, 1122 cm 1; 1H NMR (DMSO, 300 MHz): d = 1.08 (t, 6H, J = 6.9 Hz, CO2CH2Me), 2.25 (s, 6H, Me), 3.81–4.10 (m, 4H), 4.85 (s, 1H, pyridine CH), 5.11 (s, 2H, OCH2), 5.32 (s, 2H, NCH2), 6.91–6.98 (m, 4H, Ar-H), 7.22–7.29 (m, 2H, Ar-H), 7.79 (s, 1H, pyridine NH), 7.97 (s, 1H, CONH), 8.43 (s, 1H, Triazole H), 10.48 (s, 1H, COOH). MS (ESI) m/z 762 [M+H]+; HR-MS (ESI) calcd for C31H31Br2N5O8 [M+H]+: 761.9935. Found: 761.9915. 4.3.4. Diethyl4-(3,5-dibromo-2-((1-(2-oxo-2-(4-(trifluoromethoxy)benzylamino)ethyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate 6d Pale yellow solid, mp = 100 °C, IR (KBr) mmax: 3320, 2975, 1685, 1213, 1021 cm 1; 1H NMR (DMSO, 500 MHz): d = 1.08 (t, 6H, J = 6.9 Hz, CO2CH2Me), 2.19 (s, 6H, Me), 3.88–4.19 (m, 4H), 4.42 (d, 2H, J = 5.9 Hz, benzyl H), 4.81 (s, 1H, pyridine CH), 5.13 (s, 2H, OCH2), 5.23 (s, 2H, NCH2), 7.16–7.24 (m, 2H, Ar-H), 7.32–7.42 (m, 2H, Ar-H), 7.52 (s, 1H, pyridine NH), 7.72–7.80 (m, 2H, Ar-H), 8.05 (s, 1H, triazole H), 8.86 (t, 1H, J = 5.9 Hz, CONH). MS (ESI) m/ z 815 [M+H]+; HR-MS (ESI) calcd for C32H33O7N2Br2F4 [M+H]+: 814.0483. Found: 814.0525. 4.3.5. Diethyl4-(3,5-dibromo-2-((1-(2-oxo-2-(4-(trifluoromethyl thio)phenylamino)ethyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate 6e White solid, mp = 134 °C, IR (KBr) mmax: 3334, 2968, 1681, 1209, 1026 cm 1; 1H NMR (DMSO, 300 MHz): d = 1.24 (t, 6H, J = 6.9 Hz, CO2CH2Me), 2.18 (s, 6H, Me), 3.91–4.16 (m, 4H), 4.85 (s, 1H, pyridine CH) 5.17 (s, 2H, OCH2), 5.38 (s, 2H, NCH2), 7.49–7.79 (m, 6H, Ar-H), 7.85 (s, 1H, pyridine NH), 8.13 (s, 1H, triazole H), 10.66 (s, 1H, CONH). MS (ESI) m/z 818 [M+H] +; HR-MS (ESI) calcd for C31H30O7N4Br2F3S [M+H]+: 818.0284. Found: 818.0226. 4.3.6. Diethyl 4-(3,5-dibromo-2-((1-(2-(cyclopropylamino)-2oxoethyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-2,6-dimethyl1,4-dihydropyridine-3,5-dicarboxylate 6f Yellow solid, mp = 76 °C, IR (KBr) mmax: 3321, 2944, 1678, 1211, 1029 cm 1; 1H NMR (CDCl3, 500 MHz): d = 0.43–0.60 (m, 2H, cyclopropyl CH2), 0.70–0.91 (m, 2H, cyclopropyl CH2), 1.04 (t, 6H, J = 6.9 Hz, CO2CH2Me), 2.63 (s, 6H, Me), 2.72 (m, 1H, cyclopropyl CH), 3.88–4.21 (m, 4H), 4.88 (s, 1H, pyridine CH), 5.18 (s, 2H, OCH2), 5.40 (s, 2H, NCH2), 7.35–7.42 (m, 2H, Ar-H), 7.83 (s, 1H, pyridine NH), 8.11 (s, 1H, triazole H), 10.63 (s, 1H, CONH). MS (ESI) m/z 682 [M+H]+; HR-MS (ESI) calcd for C27H31Br2N5O6 [M+H]+: 681.0122. Found: 681.0132. 4.3.7. 3-(2-(4-((2-(3,5-Bis(ethoxycarbonyl)-2,6-dimethyl-1,4dihydropyridin-4-yl)-4,6-dibromo phenoxy)methyl)-1H-1,2,3triazol-1-yl)acetamido)propanoic acid 6g Yellow solid, mp = 78 °C, IR (KBr) mmax: 3332, 2932, 1727, 1234, 1105 cm 1; 1H NMR (DMSO, 500 MHz): d = 1.21 (t, 6H, J = 6.9 Hz, CO2CH2Me), 2.24 (s, 6H, Me), 2.42 (t, 2H, J = 7.2 Hz), 3.48–3.58 (m, 2H), 3.89–4.18 (m, 4H), 4.89 (s, 1H, pyridine CH), 5.20 (s, 2H, OCH2), 5.42 (s, 2H, NCH2CO), 7.52–7.68 (m, 2H, Ar-H), 7.89 (s, 1H, pyridine NH), 8.15 (s, 1H, triazole H), 10.58 (s, 1H, CONH), 13.20 (s, 1H, COOH). MS (ESI) m/z 714 [M+H]+; HR-MS (ESI) calcd for C27H31Br2N5O8 [M+H]+: 713.0154. Found: 713.0143.
4.3.8. Diethyl 4-(2-((1-(2-(6-fluorobenzo[d]thiazol-2-ylamino)2-oxoethyl)-1H-1,2,3-triazol-4-yl)methoxy)-3-methoxyphenyl)2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate 6h Pale green solid, mp = 100 °C, IR (KBr) mmax: 3334, 2936, 1701, 1231, 1078 cm 1; 1H NMR (DMSO, 500 MHz): d = 1.26 (t, 6H, J = 6.9 Hz, CO2CH2Me), 2.31 (s, 6H,Me), 3.28 (s, 3H, OCH3), 3.62–4.18 (m, 4H), 4.91 (s, 1H, pyridine CH), 5.22 (s, 2H, OCH2), 5.45 (s, 2H, NCH2), 6.57–7.47 (m, 3H, Ar-H), 7.51–7.83 (m, 3H, Ar-H), 8.0 (s, 1H, pyridine NH), 8.13 (s, 1H, triazole H), 10.24 (s, 1H, CONH). MS (ESI) m/z 665 [M+H]+; HR-MS (ESI) calcd for C32H34O7N6FS [M+H]+: 665.2188. Found: 665.2193. 4.3.9. Diethyl4-(3-methoxy-2-((1-(2-oxo-2-(5-(trifluoromethyl)1,3,4-thiadiazol-2-ylamino)ethyl)-1H-1,2,3-triazol-4-yl)meth oxy) phenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate 6i White solid, mp = 132 °C, IR (KBr) mmax: 3351, 2933, 1689, 1215, 1039 cm 1; 1H NMR (DMSO, 500 MHz): d = 1.14 (t, 6H, J = 6.9 Hz, CO2CH2Me), 2.15 (s, 6H, Me), 3.82 (s, 3H, OCH3), 3.89–4.13 (m, 4H), 5.0 (s, 1H, pyridine CH), 5.17 (s, 2H, OCH2), 5.53 (s, 2H, NCH2), 6.65–7.17 (m, 3H, Ar-H), 7.43 (s, 1H, pyridine NH), 8.03 (s, 1H, triazole H), 10.92 (s, 1H, CONH). MS (ESI) m/z 666 [M+H]+; HR-MS (ESI) calcd for C28H29O7N7F3S [M+H]+: 664.1795. Found: 664.1802. 4.3.10. Diethyl 4-(2-((1-(3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10heptadecafluorodecyl)-1H-1,2,3-triazol-4-yl)methoxy)-3methoxyphenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5dicarboxylate 6j Yellow viscous liquid, IR (neat) mmax: 3353, 2939, 1679, 1228, 1054 cm 1; 1H NMR (DMSO, 300 MHz): d = 1.15 (t, 6H, J = 6.9 Hz, CO2CH2Me), 2.26 (s, 6H, Me), 2.85 (t, 2H, J = 7.6 Hz, CF2CH2), 3.84 (s, 3H, OCH3), 3.94–4.26 (m, 4H), 4.72 (t, 2H, J = 7.6 Hz, NCH2), 5.08 (s, 1H, pyridine CH), 5.20 (s, 2H, OCH2), 6.66–7.12 (m, 3H, Ar-H), 7.27 (s, 1H, pyridine NH), 7.91 (s, 1H, triazole H). MS (ESI) m/z 903 [M+H]+; HR-MS (ESI) calcd for C33H30O6N4F17 [M+H]+: 901.1888. Found: 901.1871. 4.4. General procedure for the preparation of compound (7) To a solution of terminal acetylene (2 mmol) in THF was added secondary amine (2.1 mmol), 37% formaldehyde (2 mmol), CuI (0.2 mmol) and 3 g neutral alumina. This reaction mixture was stirred at room temperature for 12 h. The progress of reaction was monitored by TLC. Solvent was removed under reduced pressure and extracted with ethyl acetate, washed with water, brine and dried over Na2SO4. Product was purified by column chromatography using (ethyl acetate/n-hexane, 50:50) as eluent to afford the pure product 7. 4.4.1. Diethyl4-(5-bromo-2-(4-morpholinobut-2-ynyloxy) phenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate 7a Brown solid, mp = 73 °C, IR (KBr) mmax: 3324, 2935, 1692, 1213 cm 1; 1H NMR (CDCl3, 500 MHz): d = 1.31 (t, 6H, J = 6.9 Hz, CO2CH2Me), 2.39 (s, 6H, Me), 2.61 (m, 4H), 3.38 (s, 2H, NCH2), 3.82 (m, 4H, morpholine), 4.14 (m, 4H, morpholine), 4.73 (s, 2H, OCH2), 5.21 (s, 1H, pyridine CH), 5.94 (s, 1H, pyridine NH), 7.20–7.57 (m, 3H, Ar-H). MS (ESI) m/z 562 [M+H]+; HR-MS (ESI) calcd for C27H33O6N2Br [M+H]+: 561.1595. Found: 561.1578.
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4.4.2. Diethyl 4-(5-bromo-2-(4-(4-(pyridin-2-yl)piperazin-1yl)but-2-ynyloxy)phenyl)-2,6-dimethyl-1,4-dihydropyridine3,5-dicarboxylate 7b Brown solid, mp = 76 °C, IR(KBr) mmax: 3329, 2929, 1691, 1437, 1212 cm 1; 1H NMR (DMSO, 300 MHz): d = 1.21 (t, 6H, J = 6.9 Hz, CO2CH2Me), 2.29 (s, 6H, Me), 2.64 (m, 4H), 3.02 (s, 2H, NCH2), 3.55 (m, 4H, piperazine H), 4.04 (m, 4H, piperazine H), 4.62 (s, 2H, OCH2), 5.11 (s, 1H, pyridine CH), 5.94 (s, 1H, pyridine NH), 6.43–6.86 (m, 3H, Ar-H), 7.07–7.69 (m, 3H, Ar-H), 8.19 (m, 1H, Ar-H). MS (ESI) m/z 638 [M+H]+; HR-MS (ESI) calcd for C37H26O3N7 [M+H]+: 637.1989. Found: 637.2004. 4.4.3. Diethyl4-(3,5-dibromo-2-(4-(4-(2-hydroxyethyl)piperazin -1-yl)but-2-ynyloxy)phenyl)-2,6-dimethyl-1,4-dihydropyridine3,5-dicarboxylate 7c Brown solid, mp = 74 °C, IR (KBr) mmax: 3412, 3327, 2932, 1694, 1441, 1215 cm 1; 1H NMR (CDCl3, 500 MHz): d = 1.05 (t, 6H, J = 6.9 Hz, CO2CH2Me), 2.28 (s, 6H, Me), 2.62 (s, 8H, piperazine H), 2.69 (t, 2H, J = 5.9 Hz), 3.41 (s, 2H, NCH2), 3.48 (m, 2H), 3.66 (s, 1H), 4.10 (m, 4H), 4.36 (s, 2H, OCH2), 4.76 (s, 1H, pyridine CH), 5.13 (s, 1H, pyridine NH), 7.36 (d, 1H, J = 2.3 Hz, Ar-H), 7.50 (d, 1H, J = 2.5 Hz, Ar-H). MS (ESI) m/z 682 [M+H] +; HR-MS (ESI) calcd for C32H28O7N4Br [M+H]+: 682.1034. Found: 682.0975. 4.4.4. Diethyl4-(3,5-dibromo-2-(4-(4-methylpiperazin-1-yl)but2-ynyloxy)phenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5dicarboxylate 7d Brown solid, mp = 81 °C, IR (KBr) mmax: 3328, 2931, 1696, 1439, 1214 cm 1; 1H NMR (CDCl3, 300 MHz): d = 1.21 (t, 6H, J = 6.9 Hz, CO2CH2Me), 2.31 (s, 9H), 2.36 (s, 8H), 3.39 (s, 2H, NCH2), 4.05 (m, 4H), 4.59 (s, 2H, OCH2), 5.08 (s, 1H, pyridine CH), 6.98 (s, 1H, pyridine NH), 7.36 (d, 1H, J = 2.3 Hz, Ar-H), 7.50 (d, 1H, J = 2.5 Hz, Ar-H). MS (ESI) m/z 654 [M+H]+; HR-MS (ESI) calcd for C28H38O5N3 Br2 [M+H]+: 654.1173. Found: 654.1006. 4.4.5. Diethyl 4-(3,5-dibromo-2-(4-(4-(4-fluorophenyl)piperazin -1-yl)but-2-ynyloxy)phenyl)-2,6-dimethyl-1,4-dihydropyridine3,5-dicarboxylate 7e Brown solid, mp = 70 °C, IR (KBr) mmax: 3331, 2930, 1693, 1440, 1215 cm 1; 1H NMR (CDCl3, 300 MHz): d = 1.22 (t, 6H, J = 6.9 Hz, CO2CH2Me), 2.31 (s, 6H, Me), 2.60–2.82 (m, 4H), 2.99–3.26 (m, 4H), 3.41 (s, 2H, NCH2), 3.92–4.20 (m, 4H), 4.69 (s, 2H, OCH2), 5.11 (s, 1H, pyridine CH), 6.37 (s, 1H, pyridine NH), 6.75–7.08 (m, 4H, Ar-H), 7.34 (d, 1H, J = 2.3 Hz, Ar-H), 7.49 (d, 1H, J = 2.5 Hz, ArH). MS (ESI) m/z 734 [M+H]+; HR-MS (ESI) calcd for C33H37O6N2Br2F [M+H]+: 734.0997. Found: 734.1058. 4.4.6. Diethyl 4-(3,5-dibromo-2-(4-(4-(pyridin-2-yl)piperazin-1yl)but-2-ynyloxy)phenyl)-2,6-dimethyl-1,4-dihydropyridine3,5-dicarboxylate 7f Yellow solid, mp = 75 °C, IR (KBr) mmax: 3329, 2928, 1692, 1437, 1211 cm 1; 1H NMR (CDCl3, 300 MHz): d = 1.22 (t, 6H, J = 6.9 Hz, CO2CH2Me), 2.28 (s, 6H, Me), 2.55–2.76 (m, 4H, piperazine H), 3.40 (s, 2H, NCH2), 3.48–3.62 (m, 4H, piperazine H), 3.93–4.17 (m, 4H), 4.65 (s, 2H, OCH2), 5.09 (s, 1H, pyridine CH), 6.23 (s, 1H, pyridine NH), 6.54–6.75 (m, 3H, Ar-H), 7.34 (d, 1H, J = 2.3 Hz, ArH), 7.49 (d, 1H, J = 2.5 Hz, Ar-H), 8.15–8.22 (m, 1H, Ar-H). MS (ESI) m/z 717 [M+H]+; HR-MS (ESI) calcd for C33H39O6N2Br2 [M+H]+: 717.1169. Found: 717.1118. 4.4.7. Diethyl 4-(3-methoxy-2-(4-(pyrrolidin-1-yl)but-2-ynyloxy) phenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate 7g Brown solid, mp = 122 °C, IR (KBr) mmax: 3334, 2938, 1693, 1210 cm 1; 1H NMR (CDCl3, 300 MHz): d = 1.19 (t, 6H, J = 6.9 Hz, CO2CH2Me), 1.62–1.93 (m, 4H), 2.30 (s, 6H, Me), 2.46–2.74 (m, 4H), 3.41 (s, 2H, NCH2), 3.79 (s, 3H, OCH3), 3.93–4.13 (m, 4H),
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4.60 (s, 2H, OCH2), 5.09 (s, 1H, pyridine CH), 6.49 (s, 1H, pyridine NH), 6.70 (dd, 1H, J = 2.5 Hz, Ar-H), 6.83–6.95 (m, 2H, Ar-H). MS (ESI) m/z 497 [M+H]+; HR-MS (ESI) calcd for C28H37O6N2 [M+H]+: 497.2646. Found: 497.2628. 4.4.8. Diethyl 4-(3-methoxy-2-(4-morpholinobut-2-ynyloxy) phenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate 7h Yellow solid, mp = 80 °C, IR (KBr) mmax: 3324, 2934, 1692, 1215 cm 1; 1H NMR (CDCl3, 300 MHz): d = 1.20 (t, 6H, J = 6.9 Hz, CO2CH2Me), 2.29 (s, 6H, Me), 2.50 (m, 4H), 3.30 (s, 2H, NCH2), 3.72 (t, 4H, J = 4.5 Hz), 3.81 (s, 3H, OCH3), 3.98–4.10 (m, 4H), 4.65 (s, 2H, OCH2), 5.12 (s, 1H, pyridine CH), 6.17 (s, 1H, pyridine NH), 6.70 (dd, 1H, J = 2.5 Hz, Ar-H), 6.86–6.93 (m, 2H, Ar-H). MS (ESI) m/z 513 [M+H]+; HR-MS (ESI) calcd for C28H37O7N2 [M+H]+: 513.2595. Found: 513.2586. 4.4.9. Diethyl 4-(3-methoxy-2-(4-(4-methylpiperazin-1-yl)but2-ynyloxy)phenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate 7i Yellow solid, mp = 118 °C, IR (KBr) mmax: 3328, 2930, 1695, 1439, 1214 cm 1; 1H NMR (CDCl3, 300 MHz): d = 1.29 (t, 6H, J = 6.9 Hz, CO2CH2Me), 2.29 (s, 9H), 2.37 (s, 8H), 3.37 (s, 2H, NCH2), 3.80 (s, 3H, OCH3), 3.96–4.08 (m, 4H), 4.57 (s, 2H, OCH2), 5.08 (s, 1H, pyridine CH), 6.49 (s, 1H, pyridine NH), 6.70 (dd, 1H, J = 2.5 Hz, Ar-H), 6.83–6.95 (m, 2H, Ar-H). MS (ESI) m/z 526 [M+H]+; HR-MS (ESI) calcd for C29H40O6N3 [M+H]+: 526.2912. Found: 526.2904. 4.4.10. Diethyl 4-(3-methoxy-2-(4-(4-phenylpiperazin-1-yl)but2-ynyloxy)phenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate 7j Brown solid, mp = 82 °C, IR (KBr) mmax: 3335, 2937, 1692, 1438, 1216 cm 1; 1H NMR (CDCl3, 300 MHz): d = 1.19 (t, 6H, J = 6.9 Hz, CO2CH2Me), 2.28 (s, 6H, Me), 2.45–2.87 (m, 4H), 2.95–3.30 (m, 4H), 3.38 (s, 2H, NCH2), 3.79 (s, 3H, OCH3), 3.94–4.12 (m, 4H), 4.65 (s, 2H, OCH2), 5.12 (s, 1H, pyridine CH), 6.32 (s, 1H, pyridine NH), 6.65–7.05 (m, 5H, Ar-H), 7.18–7.40 (m, 3H, Ar-H). MS (ESI) m/z 588 [M+H]+; HR-MS (ESI) calcd for C34H42O6N3 [M+H]+: 588.3068. Found: 588.3069. 4.4.11. Diethyl 4-(2-(4-(4-(ethoxycarbonyl)piperidin-1-yl)but-2ynyloxy)-3-methoxyphenyl)-2,6-dimethyl-1,4-dihydropyridine3,5-dicarboxylate 7k Brown solid, mp = 84 °C, IR (KBr) mmax: 3339, 2936, 1693, 1436, 1218 cm 1; 1H NMR (CDCl3, 300 MHz): d = 1.19 (t, 6H, J = 6.9 Hz, CO2CH2Me), 1.26 (t, 3H, J = 7.6 Hz), 1.63–2.01 (m, 4H), 2.30 (s, 6H, Me), 2.34–2.51 (m, 1H), 2.59–2.97 (m, 4H), 3.30 (s, 2H, NCH2), 3.80 (s, 3H, OCH3), 4.03 (q, 2H, J = 6.8 Hz), 4.08–4.22 (m, 4H), 4.62 (s, 2H, OCH2), 5.10 (s, 1H, pyridine CH), 6.36 (s, 1H, pyridine NH), 6.70 (dd, 1H, J = 3.0 Hz, Ar-H), 6.85–6.93 (m, 2H, Ar-H). MS (ESI) m/z 583 [M+H]+; HR-MS (ESI) calcd for C32H43O8N2 [M+H]+: 583.3014. Found: 583.3017. 5. ACE inhibition assay ACE inhibition assay was performed using the method described by Jimsheena and Gowda.25 Rabbit lung acetone powder (Sigma Aldrich, USA) was used as a source of ACE enzyme. 1 gm of rabbit lung acetone powder was incubated with 10 mL of 0.05 M sodium borate buffer pH 8.2 containing 0.3 M NaCl and 0.5% Triton X-100 at 4 °C for 24 h followed by centrifugation at 4 °C, 12,000 rpm for 30 min. The supernatant was then collected and stored in aliquots and was used as a source of ACE enzyme. ACE activity was assayed by monitoring the release of Hippuric acid (HA) from the hydrolysis of hippuryl-histidyl-leucine (HHL) (Sigma–Aldrich, USA). ACE solution was preincubated with test
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and standard drug solution for 10 min at 37 °C. The enzyme reaction was started by adding 0.05 M sodium borate buffer (pH 8.2) containing 0.3 M NaCl and 5 mM substrate (HHL) followed by incubation at 37 °C for 30 min. The reaction was arrested by the addition of 100 lL of 0.1 M HCl. After stopping the reaction, 200 lL of pyridine (SD Fine chemical, India) was added followed by 100 lL of benzyl sulfonyl chloride (BSC) (SD Fine chemical, India). The solution was mixed by inversion for 1 min and cooled on ice. The yellow color developed was measured at 410 nm. The decreased concentration of HA in the test reaction compared with the control reaction was expressed as percentage inhibition and calculated from the equation: Inhibition% = 100 [T/C] 100, where T = absorbance of test reaction and C = absorbance of control reaction. The therapeutic drug Lisinopril was used as reference ACE inhibitor. 6. Cytotoxicity assay in cell lines A549-cells (Human Lung Adenocarcinoma Epithelial Cell Line) and HEK293 cells (Human Embryonic Kidney Cell Line) were purchased from National Centre for Cell Science (NCCS), Pune. Both cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Sigma, USA) with 10% heat-inactivated fetal bovine serum (FBS, Lonza), 100 U/mL penicillin, 100 U/mL streptomycin, and 2 mM L-glutamine at 37 °C in a humidified atmosphere of 5% CO2. Cells were passaged every 2–3 days to maintain exponential growth. 7. In vitro cytotoxicity assay The cytotoxicity of the different compounds (Table 1) was studied by means of a colorimetric micro culture assay using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide.10 1 104 cells/well were seeded in 100 lL DMEM, supplemented with 10% FBS in each well of 96-well micro culture plates and incubated for 24 h at 37 °C in a CO2 incubator. The desired concentrations (10 lM–1000 lM) of the compounds were made and added to the wells with respective vehicle control. After 24 h of incubation, 10 lL MTT (5 mg/mL) was added to each well and the plates were further incubated for 4 h. Then the supernatant from each well was carefully removed, formazan crystals were dissolved in 100 lL of DMSO, and absorbance was recorded at 540-nm wavelength. Cell survival rate was determined by comparing the absorbance value of treated cells with that in the control cells. 50% effective concentration (EC50) values were decided by probit analysis. Acknowledgments We are thankful to the Director of IICT for providing facilities. U.B.K., P.K.B., A.K. and K. A. thank for Council of Scientific and Industrial research (CSIR) and T.L.D. thank for UGC, New Delhi,
for the award of a fellowship and R.M.K thanks S.E.R.C., Department of Science & Technology, Government of India for financial assistance under the Fast Track Scheme for young scientists (SR/FTP/CS-031/2010). SKB is thankful to Department of Biotechnology (DBT) for providing Ramalingaswami Fellowship. References and notes 1. 2. 3. 4. 5. 6. 7. 8.
9.
10. 11.
12. 13. 14. 15.
16.
17. 18. 19. 20.
21.
22. 23.
24. 25.
Bhuyan, B. J.; Mugesh, G. Org. Biomol. Chem. 2011, 9, 5185. Hall, J. E. Clin. Cardiol. 1991, 114, 6. Hall, J. E.; Coleman, T. G.; Guyton, A. C. J. Am. Geriatr. Soc. 1989, 37, 801. Parish, R. C.; Miller, L. J. Drug Safety 1992, 7, 14. Sweitzer, Nancy. K. Circulation 2003, 108, 16. Migdalof, B. H.; Antonaccio, M. J.; McKinstry, D. N.; Singhvi, S. M.; Lan, S. J.; Egli, P.; Kripalani, K. J. Drug Metab. Rev. 1984, 15, 841. Izzo, J. L.; Weir, M. R. J. Clin. Hypertens. (Greenwich) 2011, 13, 667. (a) Hassan, H. A.; Abdel Aziz, M.; Abuo Rahma Gel, D; Farag, H. H. Bioorg. Med. Chem. 2009, 15, 1681; (b) Czyrak, A.; Mogilnicka, E.; Maj, J. Neuropharmacology 1989, 28, 229. (a) Gaudio, A. C.; Korolkovas, A.; Takahata, Y. J. Pharm. Sci. 1994, 83, 1110; (b) Schleifer, K. J. J. Med. Chem. 1999, 42, 2204; (c) Visentin, S.; Amiel, P.; Frittero, R.; Boschi, D.; Roussel, C.; Giusta, L. J. Med. Chem. 1999, 42, 1422. Carlos Sunkel, E.; Fau de Casa Juana, Miguel; Cillero, Francisco Javier; Priego, Jaime G.; Pilar Ortega, M. J. Med. Chem. 1988, 31, 1886. (a) Surendra Kumar, R.; Idhayadhulla, A.; Jamal Abdul Nasser, A.; Kavimani, S.; Indumathy, S. Indian J. Pharm. Sci. 2010, 72, 719; (b) Subudhi, B. B.; Panda, P. K.; Swain, S. P.; Sarangi, P. Acta Pol. Pharm. 2009, 66, 147. Beyer, T.; Gansohr, N.; Gjörstrup, P.; Ravens, U. Naunyn Schmiedebergs Arch Pharmacol. 1986, 334, 488. Refat, Hala M.; Fadda, A. A. Eur. J. Med. Chem. 2013, 70, 419. Chu, C. K.; Bhadti, V. S.; Doshi, K. J.; Etse, J. T.; Gallo, J. M.; Boudinot, F. D.; Schinazi, R. F. J. Med. Chem. 1990, 33, 2188. (a) Miri, R.; Javidnia, K.; Mirkhani, H.; Kazemi, F.; Hemmateenejad, B.; Edraki, N.; Mehdipour, A. R. DARU 2008, 16, 263; (b) Kumar, R. S.; Idhayadhulla, A.; Abdul Nasser, A. J.; Selvin, J. Eur. J. Med. Chem. 2011, 46, 804. (a) Choi, S. J.; Cho, J. H.; Im, I.; Lee, S. D.; Jang, J. Y.; Oh, Y. M.; Jung, Y. K.; Jeon, E. S.; Kim, Y. C. Eur. J. Med. Chem. 2010, 45, 2578; (b) Al-Nadaf, A.; Abu Sheikha, G.; Taha, M. O. Bioorg. Med. Chem. 2010, 1, 9. (a) Bodor, N.; Buchwald, P. Adv. Drug Deliver. Rev. 1999, 36, 229; (b) Bodor, N.; Buchwald, P. Drug Discovery Today 2002, 7, 766. Wang, P.; Song, L.; Yi, H.; Zhang, M.; Zhu, S.; Deng, H.; Shao, M. Tetrahedron Lett. 2010, 51, 3975. Matucci, R.; Ottaviani, M. F.; Barbieri, M.; Cerbai, E.; Mugelli, A. Br. J. Pharmacol. 1997, 122, 1353. (a) van Amsterdam, F. T.; Roveri, A.; Maiorino, M.; Ratti, E.; Ursini, F. Free Radic. Biol. Med. 1992, 12, 183; (b) Wang, Fan; Chou, Ann; Segatori, Laura Chem. Biol. 2011, 18, 766. Cominacini, Luciano; Fratta Pasini, Anna; Garbin, Ulisse; Pastorino, Antonio M.; Davoli, Anna; Nava, Cristina; Campagnola, Mario; Rossato, Paolo; Lo Cascio, V. Biochem. Biophys. Res. Commun. 2003, 302, 679; (b) Tirzitis, Gunars; Tirzite, Dace; Hyvonen Czech, Zhanna J. Food Sci. 2000, 19, 81. Wirtz, Susanne; Herzig Br, Stefan J. Pharmacol. 2004, 142, 275. (a) Ravindra Kumbhare, M.; Janaki Ramaiah, M.; Dadmal, Tulshiram; Pushpavalli, S. N. C. V. L.; Mukhopadhyay, Debasmita; Divya, B.; Anjana Devi, T.; Kosurkar, Umesh; Pal Bhadra, Manika Eur. J. Med. Chem. 2011, 46, 4258; (b) Ravindra Kumbhare, M.; Kosurkar, Umesh B.; Janaki Ramaiah, M.; Dadmal, Tulshiram L.; Pushpavalli, S. N. C. V. L.; Pal Bhadra, Manika Bioorg. Med. Chem. Lett. 2012, 22, 5424; (c) Kantevari, Srinivas; Addla, Dinesh; Bagul, Pankaj K.; Sridhar, Balasubramanian; Banerjee, Sanjay K. Bioorg. Med. Chem. 2011, 19, 4772. Sharifi, Ali; Farhangian, Hossein; Mohsenzadeh, Farshid; Naimi-Jamal, M. R. Monatsh. Chem. 2002, 133, 199. (a) Jimsheena, V. K.; Gowda, L. R. Anal. Chem 2009, 81, 9388; (b) Jimsheena, V. K.; Gowda, L. R. Peptides 2010, 31, 1165.
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Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc
Synthesis and evaluation of novel triazoles and mannich bases functionalized 1,4-dihydropyridine as angiotensin converting enzyme (ACE) inhibitors Ravindra M. Kumbhare a,⇑, Umesh B. Kosurkar a, Pankaj K. Bagul b,c, Abhinav Kanwal b,c, K. Appalanaidu a, Tulshiram L. Dadmal a, Sanjay Kumar Banerjee b,c,⇑ a
Fluoroorganic Division, CSIR-Indian Institute of Chemical Technology, Hyderabad 500607, India Medicinal Chemistry and Pharmacology Division, CSIR-Indian Institute of Chemical Technology, Hyderabad 500607, India c Present Address: Drug Discovery Research Center (DDRC), Translational Health Science and Technology Institute (THSTI), Gurgaon 122016, India b
a r t i c l e
i n f o
Article history: Received 28 July 2014 Revised 9 September 2014 Accepted 11 September 2014 Available online 19 September 2014 Keywords: Angiotensin converting enzyme Inhibitors 1,4-Dihydropyridine Triazole Mannich bases
a b s t r a c t A series of novel diethyl 2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate embedded triazole and mannich bases were synthesized, and evaluated for their angiotensin converting enzyme (ACE) inhibitory activity. Screening of above synthesized compounds for ACE inhibition showed that triazoles functionalized compounds have better ACE inhibitory activity compared to that of mannich bases analogues. Among all triazoles we found 6h, 6i and 6j to have good ACE inhibition activity with IC50 values 0.713 lM, 0.409 lM and 0.653 lM, respectively. Among mannich bases series compounds, only 7c resulted as most active ACE inhibitor with IC50 value of 0.928 lM. Ó 2014 Published by Elsevier Ltd.
1. Introduction Hypertension is the leading cause of cardiovascular disease affecting one in three adults worldwide.1 It is the primary cause of stroke, coronary artery disease and sudden cardiac death. Many factors are believed to be involved in the pathogenesis of hypertension, its progression and complications. Renin angiotensin aldosterone system (RAAS) plays an important role in controlling blood volume and hypertension.2,3 Angiotensin converting enzyme (ACE) is a key enzyme regulating the rate limiting step in the RAAS pathway. ACE converts angiotensin I to angiotensin II which is the main active component for hypertension. Angiotensin II causes vasoconstriction along with increased blood volume and water retention.4 To inhibit AngII mediated hypertension and associated complication, several drugs have been synthesized and tested as ACE inhibitors. Captopril was the first peptide-based ACE inhibitor which developed in 1975 and was considered a breakthrough. However, several side effects ⇑ ⇑Corresponding authors. Present Address: Drug Discovery Research Center (DDRC), Translational Health Science and Technology Institute (THSTI), Gurgaon 122016, India. Tel.: +91 11 26741358 (S.K.B.), tel.: +91 40 27191776; fax: +91 40 27193185 (R.M.K.). E-mail addresses: [email protected] (R.M. Kumbhare), [email protected]. in (S.K. Banerjee). http://dx.doi.org/10.1016/j.bmc.2014.09.027 0968-0896/Ó 2014 Published by Elsevier Ltd.
like skin rash, loss of taste and dry cough are reported with captopril.5 All those side effects of captopril are thought to be because of the presence of mercapto group which provides strong chelating action.6 After captopril, research has been aimed in finding potent, selective ACE inhibitors that would not contain a mercapto (SH) functional group and would have a weaker chelating function. Enalapril, lisinopril, ramipril and benzapril are the newly synthesized peptide based molecules available in market. However, all of them still carry the same set of side effects with less severity.7 So there is a need to synthesis non-peptide based analogue for ACE inhibition with fewer side effects. A survey of literature revealed that 1,4-dihydropyridine derivatives have received much attention during recent years due to its versatile biological activities such as antidepressant,8 antihypertensive,9 antithrombotic,10 anticonvulsant,11 cardiotonic,12 antibacterial,13 antiHIV,14 and calcium channel blockers.15 However, little attention has been directed towards the BACE-1 blocking activity of these compounds.16 DHP can be converted to the quaternary amines leading to their retention in the brain.17 Another characteristic feature of DHP scaffold is the possibility of structural modifications via introducing various chemical substituents in different positions of the DHP ring and hence providing remarkable changes in pharmacological profile.18 Dihydropyridine drugs, such
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N O N O
O O
O
O O O
O
O
Darodipine
N H Lacidipine
1
2
N H
O 2N
O O
N
O O
O
N
N H Manidipine 3
Figure 1. Drugs containing 1,4-dihydropyridine framework.
as nifedipine, nicardipine, amlodipine and other drugs must be used with caution in patients with coronary heart disease because of the increased mortality observed in patients treated with these drugs. One of the most discussed undesirable effects is the reflex tachycardia produced by dihydropyridines. Darodipine19 1 (Fig. 1) are dihydropyridine derivatives calcium antagonists that easily pass into the brain, showing high affinity for cerebral L-type voltage-sensitive calcium channel (VSCC). One of the most vascular selective of the dihydropyridines derivatives Lacidipine, 2 is a calcium channel blocker developed for oral administration for use in mild to moderate hypertension,20 and is widely used in therapy since the early 1990s. It exhibits anti-atherosclerotic and antioxidant effects.21 Manidipine 3 is a lipophilic and highly vasoselective agents22 and has long lasting activity for the treatments of hypertension. Manidipine is a dihydropyridine (DHP) calcium channel antagonist, which effectively reduced blood pressure in patients which mild to moderate hypertension and effectively maintains reduced blood pressure levels throughout the dosing periods of 24 h. Hence in continuous of our efforts for the structural modification on heterocyclic moiety23 and thus to improve their bio-
Compound
R
6a
5Br
6b
5Br
6c
R1
logical activity a new series of triazole and mannich base functionalized 1,4-dihydropyridine have been synthesized. 2. Results and discussion 2.1. Synthesis Substituted salicylaldehyde is the starting material in the synthesis of 1,4-dihydropyridine derivatives (6 and 7). It was propargylated with propargyl bromide to yield 4 in excellent yield. Aldehyde group from 4 reacted with ethylacetoacetate and ammonium acetate under reflux condition in ethanol solvent to give 1,4-dihydropyridine 5 in very good yield. Now, to obtain the triazole compounds (6a–j) by click reaction, 5 was reacted with various aliphatic and aryl azides in dry THF using 5 mol % CuI to provide 1,4-disubstituted triazole products. All the compounds obtained in very good yields (78–85%) were confirmed by IR, 1H and mass (ESI and HRMS) spectral data. Also, mannich bases (7a–k) of compound 5 were synthesized by reported method24 and products confirmed by IR, 1H and mass (ESI and HR-MS) spectral data (See Schemes 1-3).
Compound
NN CF3
S
3,5-dibromo
R 2N
R
7a
5Br
7b
5Br
O
HN
HN
N N OH
7c
3,5-dibromo HN
7d
3,5-dibromo
7e
3,5-dibromo HN
N
7f
3,5-dibromo HN
N
N
COOH 6d
6e 6f
3,5-dibromo
N
OCF3 F
SCF3
3,5-dibromo
N
3,5-dibromo O
6g
HN
3,5-dibromo
OH
3 OCH 3
6i
3 OCH 3
6j
3 OCH 3
3 OCH 3
HN
7h
3 OCH 3
HN
O
7i
3 OCH 3
HN
N
7j
3 OCH 3
7k
3 OCH 3
F
N
6h
7g
S
NN S
CF3 C 8 F17
HN
HN
N
COOC2H5
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R.M. Kumbhare et al. / Bioorg. Med. Chem. 22 (2014) 5824–5830 CHO
Br
R
CHO
K 2 CO 3 , Acetone
R
Reflux, 2 h
OH
O 4
R = 5 Br, 3,5-dibr omo, 3O CH 3
Scheme 1.
C2 H 5 OOC O O
CHO
Ethanol
R O
H 3C
O C2H5
NH 4 COOCH3
NH
R
Reflux,3h
O
COO2CH5
5
Scheme 2.
5
a
C2H5OOC
b
C2H5OOC
NH
R
NH
R O
COOC2H5
O
and histidyl-leucine (HL). The extent HA released is directly proportional to the ACE activity. In this screening method, the released hippuric acid from the substrate hippurryl-histyl-leucine (HHL) was mixed with pyridine and benzene sulfonyl chloride. The resulting yellow color (410 nm) is directly proportional to the released hippuric acid and thus with the ACE activity. All the new compounds 6a–j and 7a–k tested in the desired concentrations did not show any significant absorbance at 410 nm under control conditions. Being simple, sensitive and rapid nature, the colorimetric method is used for evaluating ACE inhibitory activity of new analogues 6a–j and 7a–k. Lisonipril, a known ACE inhibitor drug has been used as standard for comparison with the inhibitory activity of synthesized new analogues. The evaluation results of all 21 new compounds for ACE inhibitory activity are presented in Figure 2. The experiments carried out for test compounds at five different concentrations (100 nm to 2 lM) revealed that 6h, 6i, 6j and 7c showed dose dependent inhibition of ACE activity (Fig. 3). The mechanism for obtaining different ACE inhibition activity for different compounds need to be explored further. However, the increased chelating activity of those active compounds with zinc ions of the enzyme cannot be ruled out. The IC50 value of these active compounds are given in Table 1.The results obtained here will be explored further study the binding nature and to generate lead molecules for drug discovery. The toxicity evaluation of four most potent ACE inhibitors 6h, 6i, 6j and 7c was assessed by (3-5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay10 against A549 (adeno
COOC2H5
O 6a-j
N N N
HN R1
7a-k
NR2
Scheme 3. Reagents and conditions: (a) R1NHCOCH2N3, dry THF, CuI, 24 h, rt; (b) Neutral Al2O3, CuI, THF, HCHO, HNR2, 12 h, rt.
2.2. Pharmacology The in vitro angiotensin converting enzyme (ACE) inhibitory activity of new compounds 6a–j and 7a–k were measured using recent colorimetric high-throughput screening method developed by Jimsheena and Gowda.25 Most of the antihypertensive peptides have been characterized by the rabbit lung ACE inhibitor assay, based on the hydrolysis of the synthetic peptide hippurylhistidylleucine (HHL). HHL is hydrolyzed by ACE to hippuric acid (HA)
Figure 3. Dose-dependent angiotensin converting enzyme (ACE) inhibition activity of test compounds and standard compound.
Figure 2. In vitro angiotensin converting enzyme (ACE) inhibition of new analogues and standard drug.
R.M. Kumbhare et al. / Bioorg. Med. Chem. 22 (2014) 5824–5830 Table 1 IC50 values of active compounds selected from preliminary screening S. No.
Compound code
IC50 (lM)
1 2 3 4 5
6h 6i 6j 7c Std.
0.713 0.409 0.653 0.928 0.281
Table 2 MTT assay inhibitory activities (IC50) in lM S. No.
Compound code
A549-cells
HEK-cells
1 2 3 4 5
6h 6i 6j 7c Doxorubicin
>1000 >1000 >1000 >1000 2.6
>1000 >1000 >1000 >1000 0.69
carcinomic human alveolar basal epithelial) and HEK 293 (normal human embryonic kidney) cell lines. The inhibitory activities (IC50) in lM are summarized in Table 2. The compounds 6h, 6i, 6j and 7c exhibited their 50% inhibitory concentrations (IC50) >1000 lM and are considered nontoxic.
5827
than 200 mesh was used for column chromatography. Appropriate names for all the new compounds were given with the help of Chem Bio Office 2012. 4.1. General procedure for the preparation of compound (4) Salicylaldehyde (1 mmol) was dissolved in acetone (10 mL), K2CO3 (1.3 mmol) was added. The propargyl bromide (1 mmol) was slowly added drop wise to the above mixture over a period of 15 min at room temperature. Reaction mixture was refluxed for 2 h. After completion of reaction, solvent was removed under reduced pressure. The residue was washed with n-hexane repeatedly to remove excess propargyl bromide, treated with distilled water to remove excess potassium carbonate and solid product separated was filtered and dried to get product 4. 4.2. General procedure for the preparation of compound (5) An appropriate propargylated salicylaldehyde (10 mmol), ethyl acetoacetate (20 mmol) and ammonium acetate (22 mmol) were dissolved in ethanol and refluxed for 1 h. After completion of the reaction, reaction mixture was cooled to room temperature and product was extracted with ethyl acetate, washed with water and brine solution. Column chromatography was performed using silica gel (60–120 mesh), eluted with ethyl acetate and hexane (20%) to get product 5.
3. Conclusion 4.3. General procedure for the preparation of compound (6) In conclusion, we have described an efficient synthesis and evaluation of ACE inhibition activity of novel 1,4-dihydropyridine derivatives functionalized with triazoles and mannich bases. Compounds 6h, 6i, 6j and 7c exhibit promising ACE inhibition property comparable to Lisinopril drug. Cytotoxicity study of these compounds showed that all four compounds are nontoxic as assessed by measuring IC50 value in A549 and HEK cell lines, respectively. Screening all the new compounds using in vitro ACE inhibition assay resulted triazole as more potent ACE inhibitors than the respective mannich base analogues by thousand folds. Among the series of triazole 6a–j, three compounds 6h, 6i, 6j were resulted as most active ACE inhibitors with IC50 values of 0.713 lM, 0.409 lM and 0.653 lM, respectively. Compared with ACE inhibitory activity (IC50) of various triazole, mannich base 7c showed relatively higher IC50 value (0.928 lM). The results reveal that binding of heteroaryl triazole at ACE binding pocket is more pronounced through zinc ion chelation than the triazole of natural origin. The information generated here could be of use to generate lead molecules for drug discovery. 4. Experimental section Melting points were measured with a Fischer–Johns melting point apparatus and are uncorrected. IR spectra were recorded as neat liquids or KBr pellets and absorptions are reported in cm 1. 1 H NMR spectra were recorded on 300 (Bruker) and 500 MHz (Varian) spectrometers in appropriate solvents using TMS as internal standard or the solvent signals as secondary standards and the chemical shifts are shown in d scales. Coupling constants J are expressed in Hertz. High-resolution mass spectra were obtained by using ESI-QTOF mass spectrometry. Reagents and all solvents were analytically pure and were used without further purification. All the experiments were monitored by analytical thin layer chromatography (TLC) performed on silica gel GF254 pre-coated plates. After elution, plate was visualized under UV illumination at 254 nm for UV active materials. Further visualization was achieved by staining with PMA and charring on a hot plate. Silica gel finer
To a solution of dipolarophile 5 (1 mmol) in dry THF (5 mL), CuI (5 mol %) was added. Then aryl/aliphatic azide (1 mmol) was added slowly at room temperature under nitrogen atmosphere and continued stirring for 24 h. Solvent was removed under reduced pressure, the residue was diluted with water and extracted with ethyl acetate. The combined organic layers were washed with water, brine, dried over Na2SO4, filtered and concentrated under reduced pressure. This was further purified by column chromatography using (ethyl acetate/n-hexane, 40:60) as eluent to obtained the pure product 6. 4.3.1. Diethyl4-(5-bromo-2-((1-(2-(cyclohexylamino)-2oxoethyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-2,6-dimethyl1,4-dihydropyridine-3,5-dicarboxylate 6a Pale yellow solid, mp = 84 °C, IR (KBr) mmax: 3350, 2933, 1688, 1217, 1038 cm 1; 1H NMR (DMSO, 500 MHz): d = 1.07 (t, 6H, J = 6.9 Hz, CO2CH2Me), 1.13–1.44 (m, 5H), 1.54–1.83 (m, 5H), 2.21 (s, 6H, Me), 3.68 (s, 1H), 3.83–4.09 (m, 4H), 4.86 (s, 1H, pyridine CH), 4.99 (s, 2H, NCH2), 5.16 (s, 2H, OCH2), 6.83–6.89 (m, 1H, ArH), 7.16–7.23 (m, 1H, Ar-H), 7.58 (s, 1H, Ar-H), 7.77 (s, 1H, pyridine NH), 7.92 (s, 1H, CONH), 8.31 (s, 1H, triazole H). MS (ESI) m/z 644 [M+H]+; HR-MS (ESI) calcd for C30H38O6N5Br [M+H]+: 644.2078. Found: 644.2082. 4.3.2. Diethyl 4-(5-bromo-2-((1-(2-oxo-2-(5-(trifluoromethyl)1,3,4-thiadiazol-2-ylamino)ethyl)-1H-1,2,3-triazol-4-yl)meth oxy)phenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5dicarboxylate 6b White solid, mp = 160 °C, IR (KBr) mmax: 3349, 2935, 1690, 1216, 1039 cm 1; 1H NMR (DMSO, 300 MHz): d = 1.11 (t, 6H, J = 6.9 Hz, CO2CH2Me), 2.28 (s, 6H, Me), 3.88–4.15 (m, 4H), 4.89 (s, 1H, pyridine CH), 5.09 (s, 2H, OCH2), 5.28 (s, 2H, NCH2), 6.86–6.93 (m, 1H, Ar-H), 7.18–7.26 (m, 1H, Ar-H), 7.58–7.63 (m, 1H, Ar-H), 7.81 (s, 1H, pyridine NH), 7.98 (s, 1H, CONH), 8.39 (s, 1H, triazole H). MS (ESI) m/z 715 [M+H]+; HR-MS (ESI) calcd for C26H27O6N8BrF3S [M+H]+: 714.0826. Found: 714.0784.
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4.3.3. 3-(2-(4-((2-(3,5-Bis(ethoxycarbonyl)-2,6-dimethyl-1,4dihydropyridin-4-yl)-4,6-dibromophenoxy)methyl)-1H-1,2,3triazol-1-yl)acetamido)benzoic acid 6c White solid, mp = 158 °C, IR (KBr) mmax: 3316, 2979, 1684, 1208, 1122 cm 1; 1H NMR (DMSO, 300 MHz): d = 1.08 (t, 6H, J = 6.9 Hz, CO2CH2Me), 2.25 (s, 6H, Me), 3.81–4.10 (m, 4H), 4.85 (s, 1H, pyridine CH), 5.11 (s, 2H, OCH2), 5.32 (s, 2H, NCH2), 6.91–6.98 (m, 4H, Ar-H), 7.22–7.29 (m, 2H, Ar-H), 7.79 (s, 1H, pyridine NH), 7.97 (s, 1H, CONH), 8.43 (s, 1H, Triazole H), 10.48 (s, 1H, COOH). MS (ESI) m/z 762 [M+H]+; HR-MS (ESI) calcd for C31H31Br2N5O8 [M+H]+: 761.9935. Found: 761.9915. 4.3.4. Diethyl4-(3,5-dibromo-2-((1-(2-oxo-2-(4-(trifluoromethoxy)benzylamino)ethyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate 6d Pale yellow solid, mp = 100 °C, IR (KBr) mmax: 3320, 2975, 1685, 1213, 1021 cm 1; 1H NMR (DMSO, 500 MHz): d = 1.08 (t, 6H, J = 6.9 Hz, CO2CH2Me), 2.19 (s, 6H, Me), 3.88–4.19 (m, 4H), 4.42 (d, 2H, J = 5.9 Hz, benzyl H), 4.81 (s, 1H, pyridine CH), 5.13 (s, 2H, OCH2), 5.23 (s, 2H, NCH2), 7.16–7.24 (m, 2H, Ar-H), 7.32–7.42 (m, 2H, Ar-H), 7.52 (s, 1H, pyridine NH), 7.72–7.80 (m, 2H, Ar-H), 8.05 (s, 1H, triazole H), 8.86 (t, 1H, J = 5.9 Hz, CONH). MS (ESI) m/ z 815 [M+H]+; HR-MS (ESI) calcd for C32H33O7N2Br2F4 [M+H]+: 814.0483. Found: 814.0525. 4.3.5. Diethyl4-(3,5-dibromo-2-((1-(2-oxo-2-(4-(trifluoromethyl thio)phenylamino)ethyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate 6e White solid, mp = 134 °C, IR (KBr) mmax: 3334, 2968, 1681, 1209, 1026 cm 1; 1H NMR (DMSO, 300 MHz): d = 1.24 (t, 6H, J = 6.9 Hz, CO2CH2Me), 2.18 (s, 6H, Me), 3.91–4.16 (m, 4H), 4.85 (s, 1H, pyridine CH) 5.17 (s, 2H, OCH2), 5.38 (s, 2H, NCH2), 7.49–7.79 (m, 6H, Ar-H), 7.85 (s, 1H, pyridine NH), 8.13 (s, 1H, triazole H), 10.66 (s, 1H, CONH). MS (ESI) m/z 818 [M+H] +; HR-MS (ESI) calcd for C31H30O7N4Br2F3S [M+H]+: 818.0284. Found: 818.0226. 4.3.6. Diethyl 4-(3,5-dibromo-2-((1-(2-(cyclopropylamino)-2oxoethyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-2,6-dimethyl1,4-dihydropyridine-3,5-dicarboxylate 6f Yellow solid, mp = 76 °C, IR (KBr) mmax: 3321, 2944, 1678, 1211, 1029 cm 1; 1H NMR (CDCl3, 500 MHz): d = 0.43–0.60 (m, 2H, cyclopropyl CH2), 0.70–0.91 (m, 2H, cyclopropyl CH2), 1.04 (t, 6H, J = 6.9 Hz, CO2CH2Me), 2.63 (s, 6H, Me), 2.72 (m, 1H, cyclopropyl CH), 3.88–4.21 (m, 4H), 4.88 (s, 1H, pyridine CH), 5.18 (s, 2H, OCH2), 5.40 (s, 2H, NCH2), 7.35–7.42 (m, 2H, Ar-H), 7.83 (s, 1H, pyridine NH), 8.11 (s, 1H, triazole H), 10.63 (s, 1H, CONH). MS (ESI) m/z 682 [M+H]+; HR-MS (ESI) calcd for C27H31Br2N5O6 [M+H]+: 681.0122. Found: 681.0132. 4.3.7. 3-(2-(4-((2-(3,5-Bis(ethoxycarbonyl)-2,6-dimethyl-1,4dihydropyridin-4-yl)-4,6-dibromo phenoxy)methyl)-1H-1,2,3triazol-1-yl)acetamido)propanoic acid 6g Yellow solid, mp = 78 °C, IR (KBr) mmax: 3332, 2932, 1727, 1234, 1105 cm 1; 1H NMR (DMSO, 500 MHz): d = 1.21 (t, 6H, J = 6.9 Hz, CO2CH2Me), 2.24 (s, 6H, Me), 2.42 (t, 2H, J = 7.2 Hz), 3.48–3.58 (m, 2H), 3.89–4.18 (m, 4H), 4.89 (s, 1H, pyridine CH), 5.20 (s, 2H, OCH2), 5.42 (s, 2H, NCH2CO), 7.52–7.68 (m, 2H, Ar-H), 7.89 (s, 1H, pyridine NH), 8.15 (s, 1H, triazole H), 10.58 (s, 1H, CONH), 13.20 (s, 1H, COOH). MS (ESI) m/z 714 [M+H]+; HR-MS (ESI) calcd for C27H31Br2N5O8 [M+H]+: 713.0154. Found: 713.0143.
4.3.8. Diethyl 4-(2-((1-(2-(6-fluorobenzo[d]thiazol-2-ylamino)2-oxoethyl)-1H-1,2,3-triazol-4-yl)methoxy)-3-methoxyphenyl)2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate 6h Pale green solid, mp = 100 °C, IR (KBr) mmax: 3334, 2936, 1701, 1231, 1078 cm 1; 1H NMR (DMSO, 500 MHz): d = 1.26 (t, 6H, J = 6.9 Hz, CO2CH2Me), 2.31 (s, 6H,Me), 3.28 (s, 3H, OCH3), 3.62–4.18 (m, 4H), 4.91 (s, 1H, pyridine CH), 5.22 (s, 2H, OCH2), 5.45 (s, 2H, NCH2), 6.57–7.47 (m, 3H, Ar-H), 7.51–7.83 (m, 3H, Ar-H), 8.0 (s, 1H, pyridine NH), 8.13 (s, 1H, triazole H), 10.24 (s, 1H, CONH). MS (ESI) m/z 665 [M+H]+; HR-MS (ESI) calcd for C32H34O7N6FS [M+H]+: 665.2188. Found: 665.2193. 4.3.9. Diethyl4-(3-methoxy-2-((1-(2-oxo-2-(5-(trifluoromethyl)1,3,4-thiadiazol-2-ylamino)ethyl)-1H-1,2,3-triazol-4-yl)meth oxy) phenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate 6i White solid, mp = 132 °C, IR (KBr) mmax: 3351, 2933, 1689, 1215, 1039 cm 1; 1H NMR (DMSO, 500 MHz): d = 1.14 (t, 6H, J = 6.9 Hz, CO2CH2Me), 2.15 (s, 6H, Me), 3.82 (s, 3H, OCH3), 3.89–4.13 (m, 4H), 5.0 (s, 1H, pyridine CH), 5.17 (s, 2H, OCH2), 5.53 (s, 2H, NCH2), 6.65–7.17 (m, 3H, Ar-H), 7.43 (s, 1H, pyridine NH), 8.03 (s, 1H, triazole H), 10.92 (s, 1H, CONH). MS (ESI) m/z 666 [M+H]+; HR-MS (ESI) calcd for C28H29O7N7F3S [M+H]+: 664.1795. Found: 664.1802. 4.3.10. Diethyl 4-(2-((1-(3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10heptadecafluorodecyl)-1H-1,2,3-triazol-4-yl)methoxy)-3methoxyphenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5dicarboxylate 6j Yellow viscous liquid, IR (neat) mmax: 3353, 2939, 1679, 1228, 1054 cm 1; 1H NMR (DMSO, 300 MHz): d = 1.15 (t, 6H, J = 6.9 Hz, CO2CH2Me), 2.26 (s, 6H, Me), 2.85 (t, 2H, J = 7.6 Hz, CF2CH2), 3.84 (s, 3H, OCH3), 3.94–4.26 (m, 4H), 4.72 (t, 2H, J = 7.6 Hz, NCH2), 5.08 (s, 1H, pyridine CH), 5.20 (s, 2H, OCH2), 6.66–7.12 (m, 3H, Ar-H), 7.27 (s, 1H, pyridine NH), 7.91 (s, 1H, triazole H). MS (ESI) m/z 903 [M+H]+; HR-MS (ESI) calcd for C33H30O6N4F17 [M+H]+: 901.1888. Found: 901.1871. 4.4. General procedure for the preparation of compound (7) To a solution of terminal acetylene (2 mmol) in THF was added secondary amine (2.1 mmol), 37% formaldehyde (2 mmol), CuI (0.2 mmol) and 3 g neutral alumina. This reaction mixture was stirred at room temperature for 12 h. The progress of reaction was monitored by TLC. Solvent was removed under reduced pressure and extracted with ethyl acetate, washed with water, brine and dried over Na2SO4. Product was purified by column chromatography using (ethyl acetate/n-hexane, 50:50) as eluent to afford the pure product 7. 4.4.1. Diethyl4-(5-bromo-2-(4-morpholinobut-2-ynyloxy) phenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate 7a Brown solid, mp = 73 °C, IR (KBr) mmax: 3324, 2935, 1692, 1213 cm 1; 1H NMR (CDCl3, 500 MHz): d = 1.31 (t, 6H, J = 6.9 Hz, CO2CH2Me), 2.39 (s, 6H, Me), 2.61 (m, 4H), 3.38 (s, 2H, NCH2), 3.82 (m, 4H, morpholine), 4.14 (m, 4H, morpholine), 4.73 (s, 2H, OCH2), 5.21 (s, 1H, pyridine CH), 5.94 (s, 1H, pyridine NH), 7.20–7.57 (m, 3H, Ar-H). MS (ESI) m/z 562 [M+H]+; HR-MS (ESI) calcd for C27H33O6N2Br [M+H]+: 561.1595. Found: 561.1578.
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4.4.2. Diethyl 4-(5-bromo-2-(4-(4-(pyridin-2-yl)piperazin-1yl)but-2-ynyloxy)phenyl)-2,6-dimethyl-1,4-dihydropyridine3,5-dicarboxylate 7b Brown solid, mp = 76 °C, IR(KBr) mmax: 3329, 2929, 1691, 1437, 1212 cm 1; 1H NMR (DMSO, 300 MHz): d = 1.21 (t, 6H, J = 6.9 Hz, CO2CH2Me), 2.29 (s, 6H, Me), 2.64 (m, 4H), 3.02 (s, 2H, NCH2), 3.55 (m, 4H, piperazine H), 4.04 (m, 4H, piperazine H), 4.62 (s, 2H, OCH2), 5.11 (s, 1H, pyridine CH), 5.94 (s, 1H, pyridine NH), 6.43–6.86 (m, 3H, Ar-H), 7.07–7.69 (m, 3H, Ar-H), 8.19 (m, 1H, Ar-H). MS (ESI) m/z 638 [M+H]+; HR-MS (ESI) calcd for C37H26O3N7 [M+H]+: 637.1989. Found: 637.2004. 4.4.3. Diethyl4-(3,5-dibromo-2-(4-(4-(2-hydroxyethyl)piperazin -1-yl)but-2-ynyloxy)phenyl)-2,6-dimethyl-1,4-dihydropyridine3,5-dicarboxylate 7c Brown solid, mp = 74 °C, IR (KBr) mmax: 3412, 3327, 2932, 1694, 1441, 1215 cm 1; 1H NMR (CDCl3, 500 MHz): d = 1.05 (t, 6H, J = 6.9 Hz, CO2CH2Me), 2.28 (s, 6H, Me), 2.62 (s, 8H, piperazine H), 2.69 (t, 2H, J = 5.9 Hz), 3.41 (s, 2H, NCH2), 3.48 (m, 2H), 3.66 (s, 1H), 4.10 (m, 4H), 4.36 (s, 2H, OCH2), 4.76 (s, 1H, pyridine CH), 5.13 (s, 1H, pyridine NH), 7.36 (d, 1H, J = 2.3 Hz, Ar-H), 7.50 (d, 1H, J = 2.5 Hz, Ar-H). MS (ESI) m/z 682 [M+H] +; HR-MS (ESI) calcd for C32H28O7N4Br [M+H]+: 682.1034. Found: 682.0975. 4.4.4. Diethyl4-(3,5-dibromo-2-(4-(4-methylpiperazin-1-yl)but2-ynyloxy)phenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5dicarboxylate 7d Brown solid, mp = 81 °C, IR (KBr) mmax: 3328, 2931, 1696, 1439, 1214 cm 1; 1H NMR (CDCl3, 300 MHz): d = 1.21 (t, 6H, J = 6.9 Hz, CO2CH2Me), 2.31 (s, 9H), 2.36 (s, 8H), 3.39 (s, 2H, NCH2), 4.05 (m, 4H), 4.59 (s, 2H, OCH2), 5.08 (s, 1H, pyridine CH), 6.98 (s, 1H, pyridine NH), 7.36 (d, 1H, J = 2.3 Hz, Ar-H), 7.50 (d, 1H, J = 2.5 Hz, Ar-H). MS (ESI) m/z 654 [M+H]+; HR-MS (ESI) calcd for C28H38O5N3 Br2 [M+H]+: 654.1173. Found: 654.1006. 4.4.5. Diethyl 4-(3,5-dibromo-2-(4-(4-(4-fluorophenyl)piperazin -1-yl)but-2-ynyloxy)phenyl)-2,6-dimethyl-1,4-dihydropyridine3,5-dicarboxylate 7e Brown solid, mp = 70 °C, IR (KBr) mmax: 3331, 2930, 1693, 1440, 1215 cm 1; 1H NMR (CDCl3, 300 MHz): d = 1.22 (t, 6H, J = 6.9 Hz, CO2CH2Me), 2.31 (s, 6H, Me), 2.60–2.82 (m, 4H), 2.99–3.26 (m, 4H), 3.41 (s, 2H, NCH2), 3.92–4.20 (m, 4H), 4.69 (s, 2H, OCH2), 5.11 (s, 1H, pyridine CH), 6.37 (s, 1H, pyridine NH), 6.75–7.08 (m, 4H, Ar-H), 7.34 (d, 1H, J = 2.3 Hz, Ar-H), 7.49 (d, 1H, J = 2.5 Hz, ArH). MS (ESI) m/z 734 [M+H]+; HR-MS (ESI) calcd for C33H37O6N2Br2F [M+H]+: 734.0997. Found: 734.1058. 4.4.6. Diethyl 4-(3,5-dibromo-2-(4-(4-(pyridin-2-yl)piperazin-1yl)but-2-ynyloxy)phenyl)-2,6-dimethyl-1,4-dihydropyridine3,5-dicarboxylate 7f Yellow solid, mp = 75 °C, IR (KBr) mmax: 3329, 2928, 1692, 1437, 1211 cm 1; 1H NMR (CDCl3, 300 MHz): d = 1.22 (t, 6H, J = 6.9 Hz, CO2CH2Me), 2.28 (s, 6H, Me), 2.55–2.76 (m, 4H, piperazine H), 3.40 (s, 2H, NCH2), 3.48–3.62 (m, 4H, piperazine H), 3.93–4.17 (m, 4H), 4.65 (s, 2H, OCH2), 5.09 (s, 1H, pyridine CH), 6.23 (s, 1H, pyridine NH), 6.54–6.75 (m, 3H, Ar-H), 7.34 (d, 1H, J = 2.3 Hz, ArH), 7.49 (d, 1H, J = 2.5 Hz, Ar-H), 8.15–8.22 (m, 1H, Ar-H). MS (ESI) m/z 717 [M+H]+; HR-MS (ESI) calcd for C33H39O6N2Br2 [M+H]+: 717.1169. Found: 717.1118. 4.4.7. Diethyl 4-(3-methoxy-2-(4-(pyrrolidin-1-yl)but-2-ynyloxy) phenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate 7g Brown solid, mp = 122 °C, IR (KBr) mmax: 3334, 2938, 1693, 1210 cm 1; 1H NMR (CDCl3, 300 MHz): d = 1.19 (t, 6H, J = 6.9 Hz, CO2CH2Me), 1.62–1.93 (m, 4H), 2.30 (s, 6H, Me), 2.46–2.74 (m, 4H), 3.41 (s, 2H, NCH2), 3.79 (s, 3H, OCH3), 3.93–4.13 (m, 4H),
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4.60 (s, 2H, OCH2), 5.09 (s, 1H, pyridine CH), 6.49 (s, 1H, pyridine NH), 6.70 (dd, 1H, J = 2.5 Hz, Ar-H), 6.83–6.95 (m, 2H, Ar-H). MS (ESI) m/z 497 [M+H]+; HR-MS (ESI) calcd for C28H37O6N2 [M+H]+: 497.2646. Found: 497.2628. 4.4.8. Diethyl 4-(3-methoxy-2-(4-morpholinobut-2-ynyloxy) phenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate 7h Yellow solid, mp = 80 °C, IR (KBr) mmax: 3324, 2934, 1692, 1215 cm 1; 1H NMR (CDCl3, 300 MHz): d = 1.20 (t, 6H, J = 6.9 Hz, CO2CH2Me), 2.29 (s, 6H, Me), 2.50 (m, 4H), 3.30 (s, 2H, NCH2), 3.72 (t, 4H, J = 4.5 Hz), 3.81 (s, 3H, OCH3), 3.98–4.10 (m, 4H), 4.65 (s, 2H, OCH2), 5.12 (s, 1H, pyridine CH), 6.17 (s, 1H, pyridine NH), 6.70 (dd, 1H, J = 2.5 Hz, Ar-H), 6.86–6.93 (m, 2H, Ar-H). MS (ESI) m/z 513 [M+H]+; HR-MS (ESI) calcd for C28H37O7N2 [M+H]+: 513.2595. Found: 513.2586. 4.4.9. Diethyl 4-(3-methoxy-2-(4-(4-methylpiperazin-1-yl)but2-ynyloxy)phenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate 7i Yellow solid, mp = 118 °C, IR (KBr) mmax: 3328, 2930, 1695, 1439, 1214 cm 1; 1H NMR (CDCl3, 300 MHz): d = 1.29 (t, 6H, J = 6.9 Hz, CO2CH2Me), 2.29 (s, 9H), 2.37 (s, 8H), 3.37 (s, 2H, NCH2), 3.80 (s, 3H, OCH3), 3.96–4.08 (m, 4H), 4.57 (s, 2H, OCH2), 5.08 (s, 1H, pyridine CH), 6.49 (s, 1H, pyridine NH), 6.70 (dd, 1H, J = 2.5 Hz, Ar-H), 6.83–6.95 (m, 2H, Ar-H). MS (ESI) m/z 526 [M+H]+; HR-MS (ESI) calcd for C29H40O6N3 [M+H]+: 526.2912. Found: 526.2904. 4.4.10. Diethyl 4-(3-methoxy-2-(4-(4-phenylpiperazin-1-yl)but2-ynyloxy)phenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate 7j Brown solid, mp = 82 °C, IR (KBr) mmax: 3335, 2937, 1692, 1438, 1216 cm 1; 1H NMR (CDCl3, 300 MHz): d = 1.19 (t, 6H, J = 6.9 Hz, CO2CH2Me), 2.28 (s, 6H, Me), 2.45–2.87 (m, 4H), 2.95–3.30 (m, 4H), 3.38 (s, 2H, NCH2), 3.79 (s, 3H, OCH3), 3.94–4.12 (m, 4H), 4.65 (s, 2H, OCH2), 5.12 (s, 1H, pyridine CH), 6.32 (s, 1H, pyridine NH), 6.65–7.05 (m, 5H, Ar-H), 7.18–7.40 (m, 3H, Ar-H). MS (ESI) m/z 588 [M+H]+; HR-MS (ESI) calcd for C34H42O6N3 [M+H]+: 588.3068. Found: 588.3069. 4.4.11. Diethyl 4-(2-(4-(4-(ethoxycarbonyl)piperidin-1-yl)but-2ynyloxy)-3-methoxyphenyl)-2,6-dimethyl-1,4-dihydropyridine3,5-dicarboxylate 7k Brown solid, mp = 84 °C, IR (KBr) mmax: 3339, 2936, 1693, 1436, 1218 cm 1; 1H NMR (CDCl3, 300 MHz): d = 1.19 (t, 6H, J = 6.9 Hz, CO2CH2Me), 1.26 (t, 3H, J = 7.6 Hz), 1.63–2.01 (m, 4H), 2.30 (s, 6H, Me), 2.34–2.51 (m, 1H), 2.59–2.97 (m, 4H), 3.30 (s, 2H, NCH2), 3.80 (s, 3H, OCH3), 4.03 (q, 2H, J = 6.8 Hz), 4.08–4.22 (m, 4H), 4.62 (s, 2H, OCH2), 5.10 (s, 1H, pyridine CH), 6.36 (s, 1H, pyridine NH), 6.70 (dd, 1H, J = 3.0 Hz, Ar-H), 6.85–6.93 (m, 2H, Ar-H). MS (ESI) m/z 583 [M+H]+; HR-MS (ESI) calcd for C32H43O8N2 [M+H]+: 583.3014. Found: 583.3017. 5. ACE inhibition assay ACE inhibition assay was performed using the method described by Jimsheena and Gowda.25 Rabbit lung acetone powder (Sigma Aldrich, USA) was used as a source of ACE enzyme. 1 gm of rabbit lung acetone powder was incubated with 10 mL of 0.05 M sodium borate buffer pH 8.2 containing 0.3 M NaCl and 0.5% Triton X-100 at 4 °C for 24 h followed by centrifugation at 4 °C, 12,000 rpm for 30 min. The supernatant was then collected and stored in aliquots and was used as a source of ACE enzyme. ACE activity was assayed by monitoring the release of Hippuric acid (HA) from the hydrolysis of hippuryl-histidyl-leucine (HHL) (Sigma–Aldrich, USA). ACE solution was preincubated with test
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and standard drug solution for 10 min at 37 °C. The enzyme reaction was started by adding 0.05 M sodium borate buffer (pH 8.2) containing 0.3 M NaCl and 5 mM substrate (HHL) followed by incubation at 37 °C for 30 min. The reaction was arrested by the addition of 100 lL of 0.1 M HCl. After stopping the reaction, 200 lL of pyridine (SD Fine chemical, India) was added followed by 100 lL of benzyl sulfonyl chloride (BSC) (SD Fine chemical, India). The solution was mixed by inversion for 1 min and cooled on ice. The yellow color developed was measured at 410 nm. The decreased concentration of HA in the test reaction compared with the control reaction was expressed as percentage inhibition and calculated from the equation: Inhibition% = 100 [T/C] 100, where T = absorbance of test reaction and C = absorbance of control reaction. The therapeutic drug Lisinopril was used as reference ACE inhibitor. 6. Cytotoxicity assay in cell lines A549-cells (Human Lung Adenocarcinoma Epithelial Cell Line) and HEK293 cells (Human Embryonic Kidney Cell Line) were purchased from National Centre for Cell Science (NCCS), Pune. Both cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Sigma, USA) with 10% heat-inactivated fetal bovine serum (FBS, Lonza), 100 U/mL penicillin, 100 U/mL streptomycin, and 2 mM L-glutamine at 37 °C in a humidified atmosphere of 5% CO2. Cells were passaged every 2–3 days to maintain exponential growth. 7. In vitro cytotoxicity assay The cytotoxicity of the different compounds (Table 1) was studied by means of a colorimetric micro culture assay using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide.10 1 104 cells/well were seeded in 100 lL DMEM, supplemented with 10% FBS in each well of 96-well micro culture plates and incubated for 24 h at 37 °C in a CO2 incubator. The desired concentrations (10 lM–1000 lM) of the compounds were made and added to the wells with respective vehicle control. After 24 h of incubation, 10 lL MTT (5 mg/mL) was added to each well and the plates were further incubated for 4 h. Then the supernatant from each well was carefully removed, formazan crystals were dissolved in 100 lL of DMSO, and absorbance was recorded at 540-nm wavelength. Cell survival rate was determined by comparing the absorbance value of treated cells with that in the control cells. 50% effective concentration (EC50) values were decided by probit analysis. Acknowledgments We are thankful to the Director of IICT for providing facilities. U.B.K., P.K.B., A.K. and K. A. thank for Council of Scientific and Industrial research (CSIR) and T.L.D. thank for UGC, New Delhi,
for the award of a fellowship and R.M.K thanks S.E.R.C., Department of Science & Technology, Government of India for financial assistance under the Fast Track Scheme for young scientists (SR/FTP/CS-031/2010). SKB is thankful to Department of Biotechnology (DBT) for providing Ramalingaswami Fellowship. References and notes 1. 2. 3. 4. 5. 6. 7. 8.
9.
10. 11.
12. 13. 14. 15.
16.
17. 18. 19. 20.
21.
22. 23.
24. 25.
Bhuyan, B. J.; Mugesh, G. Org. Biomol. Chem. 2011, 9, 5185. Hall, J. E. Clin. Cardiol. 1991, 114, 6. Hall, J. E.; Coleman, T. G.; Guyton, A. C. J. Am. Geriatr. Soc. 1989, 37, 801. Parish, R. C.; Miller, L. J. Drug Safety 1992, 7, 14. Sweitzer, Nancy. K. Circulation 2003, 108, 16. Migdalof, B. H.; Antonaccio, M. J.; McKinstry, D. N.; Singhvi, S. M.; Lan, S. J.; Egli, P.; Kripalani, K. J. Drug Metab. Rev. 1984, 15, 841. Izzo, J. L.; Weir, M. R. J. Clin. Hypertens. (Greenwich) 2011, 13, 667. (a) Hassan, H. A.; Abdel Aziz, M.; Abuo Rahma Gel, D; Farag, H. H. Bioorg. Med. Chem. 2009, 15, 1681; (b) Czyrak, A.; Mogilnicka, E.; Maj, J. Neuropharmacology 1989, 28, 229. (a) Gaudio, A. C.; Korolkovas, A.; Takahata, Y. J. Pharm. Sci. 1994, 83, 1110; (b) Schleifer, K. J. J. Med. Chem. 1999, 42, 2204; (c) Visentin, S.; Amiel, P.; Frittero, R.; Boschi, D.; Roussel, C.; Giusta, L. J. Med. Chem. 1999, 42, 1422. Carlos Sunkel, E.; Fau de Casa Juana, Miguel; Cillero, Francisco Javier; Priego, Jaime G.; Pilar Ortega, M. J. Med. Chem. 1988, 31, 1886. (a) Surendra Kumar, R.; Idhayadhulla, A.; Jamal Abdul Nasser, A.; Kavimani, S.; Indumathy, S. Indian J. Pharm. Sci. 2010, 72, 719; (b) Subudhi, B. B.; Panda, P. K.; Swain, S. P.; Sarangi, P. Acta Pol. Pharm. 2009, 66, 147. Beyer, T.; Gansohr, N.; Gjörstrup, P.; Ravens, U. Naunyn Schmiedebergs Arch Pharmacol. 1986, 334, 488. Refat, Hala M.; Fadda, A. A. Eur. J. Med. Chem. 2013, 70, 419. Chu, C. K.; Bhadti, V. S.; Doshi, K. J.; Etse, J. T.; Gallo, J. M.; Boudinot, F. D.; Schinazi, R. F. J. Med. Chem. 1990, 33, 2188. (a) Miri, R.; Javidnia, K.; Mirkhani, H.; Kazemi, F.; Hemmateenejad, B.; Edraki, N.; Mehdipour, A. R. DARU 2008, 16, 263; (b) Kumar, R. S.; Idhayadhulla, A.; Abdul Nasser, A. J.; Selvin, J. Eur. J. Med. Chem. 2011, 46, 804. (a) Choi, S. J.; Cho, J. H.; Im, I.; Lee, S. D.; Jang, J. Y.; Oh, Y. M.; Jung, Y. K.; Jeon, E. S.; Kim, Y. C. Eur. J. Med. Chem. 2010, 45, 2578; (b) Al-Nadaf, A.; Abu Sheikha, G.; Taha, M. O. Bioorg. Med. Chem. 2010, 1, 9. (a) Bodor, N.; Buchwald, P. Adv. Drug Deliver. Rev. 1999, 36, 229; (b) Bodor, N.; Buchwald, P. Drug Discovery Today 2002, 7, 766. Wang, P.; Song, L.; Yi, H.; Zhang, M.; Zhu, S.; Deng, H.; Shao, M. Tetrahedron Lett. 2010, 51, 3975. Matucci, R.; Ottaviani, M. F.; Barbieri, M.; Cerbai, E.; Mugelli, A. Br. J. Pharmacol. 1997, 122, 1353. (a) van Amsterdam, F. T.; Roveri, A.; Maiorino, M.; Ratti, E.; Ursini, F. Free Radic. Biol. Med. 1992, 12, 183; (b) Wang, Fan; Chou, Ann; Segatori, Laura Chem. Biol. 2011, 18, 766. Cominacini, Luciano; Fratta Pasini, Anna; Garbin, Ulisse; Pastorino, Antonio M.; Davoli, Anna; Nava, Cristina; Campagnola, Mario; Rossato, Paolo; Lo Cascio, V. Biochem. Biophys. Res. Commun. 2003, 302, 679; (b) Tirzitis, Gunars; Tirzite, Dace; Hyvonen Czech, Zhanna J. Food Sci. 2000, 19, 81. Wirtz, Susanne; Herzig Br, Stefan J. Pharmacol. 2004, 142, 275. (a) Ravindra Kumbhare, M.; Janaki Ramaiah, M.; Dadmal, Tulshiram; Pushpavalli, S. N. C. V. L.; Mukhopadhyay, Debasmita; Divya, B.; Anjana Devi, T.; Kosurkar, Umesh; Pal Bhadra, Manika Eur. J. Med. Chem. 2011, 46, 4258; (b) Ravindra Kumbhare, M.; Kosurkar, Umesh B.; Janaki Ramaiah, M.; Dadmal, Tulshiram L.; Pushpavalli, S. N. C. V. L.; Pal Bhadra, Manika Bioorg. Med. Chem. Lett. 2012, 22, 5424; (c) Kantevari, Srinivas; Addla, Dinesh; Bagul, Pankaj K.; Sridhar, Balasubramanian; Banerjee, Sanjay K. Bioorg. Med. Chem. 2011, 19, 4772. Sharifi, Ali; Farhangian, Hossein; Mohsenzadeh, Farshid; Naimi-Jamal, M. R. Monatsh. Chem. 2002, 133, 199. (a) Jimsheena, V. K.; Gowda, L. R. Anal. Chem 2009, 81, 9388; (b) Jimsheena, V. K.; Gowda, L. R. Peptides 2010, 31, 1165.
