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European Journal of Medicinal Chemistry 71 (2014) 306e315

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European Journal of Medicinal Chemistry

journal homepage: http: / /www.elsevier .com/locate/ejmech

Original article

Synthesis of novel pyrano[3,2-f]quinoline, phenanthroline derivativesand studies of their interactions with proteins: An application inmammalian cell imaging

K.C. Majumdar a,*, Sudipta Ponra a, Tapas Ghosh a, Ratan Sadhukhan b, Utpal Ghosh b

aDepartment of Chemistry, University of Kalyani, Kalyani 741235, W.B., IndiabDepartment of Biochemistry & Biophysics, University of Kalyani, Kalyani 741235, W.B., India

a r t i c l e i n f o

Article history:Received 10 June 2013Received in revised form3 September 2013Accepted 26 October 2013Available online 2 November 2013

Keywords:Pyrano[3,2-f]quinolinePhenanthrolineFluorophoreProtein bindingHeLa cell

* Corresponding author. Tel.: þ91 33 25828750; faxE-mail address: kcm@klyuniv.ac.in (K.C. Majumda

0223-5234/$ e see front matter � 2013 Published byhttp://dx.doi.org/10.1016/j.ejmech.2013.10.067

a b s t r a c t

A series of tri-cyclic pyrano[3,2-f]quinoline and phenanthroline derivatives have been synthesized by aHCl-mediated 6-‘endo-trig’ Michael type ring closure reaction of 6-amino-5-(3-hydroxy-3-methylbut-1-ynyl)-2H-chromen-2-one in excellent yields. The process is very simple, facile and inexpensive and canprovide a diverse range of substituted quinoline derivatives from simple and easily available startingmaterials. Moreover, the synthesized derivatives exhibit staining property to the cultured HeLa cells afterfixing and can be used as fluorophores which can bind with protein molecule.

� 2013 Published by Elsevier Masson SAS.

A large number of biologically active natural products andpharmaceuticals contain quinoline as structural frameworks andquinoline represent an important class of alkaloids among variousheterocyclic systems [1]. 2-Aryl-2,3-dihydroquinoline-4-(1H)-onesare valuable precursors for the synthesis of medicinally importantcompounds [2]. Pyranoquinoline derivatives containing bothquinoline ring and pyran moieties, afford exceptional biologicalactivities, such as psychotropic activity [3], antiallergic activity [4],anti-inflammatory activity [5], estrogenic activity [6] and are alsoused as potential pharmaceuticals [7]. Natural products such ashelietidine, dutadrupine, and geibalansine [8] contain pyr-anoquinoline core structure. 4,7-Phenanthroline derivatives andtheir analogs exhibit antibacterial activity and can be used in thetreatment of gastrointestinal disease [9e15].

Fluorophores provide flexible tools for probing environmentsand living systems. There has been continuous search for newmolecules with improved fluorescence properties [16] due to theirpotential applications as probes for biological uses [17], as molec-ular sensors [18] and in optoelectronic devices [19]. BODIPY,rhodamine, fluorescein, coumarin and cyanine derivatives are small

: þ91 33 25828282.r).

Elsevier Masson SAS.

molecular organic dyes and widely used in medical diagnosis,bioimaging, environmental detection and biosensing [20]. There-fore, design and synthesis of fluorescent species with a heterocyclicbackbone is still an area of current research interest.

In view of the importance of pyranoquinoline and pyridopyr-imidine and their derivatives, several methods were developed forthe syntheses of quinoline or pyranoquinoline and phenanthrolinederivatives [21]. However, many of these synthetic protocols re-ported so far suffer from some disadvantages, such as harsh reac-tion conditions [21,22], multistep reaction [23], expensive reagents[24] and longer reaction time [21], require large amount of catalystand giving lower yields [24]. Therefore, a newprotocol with reagenteconomy, one-pot reaction, cheaper catalyst and improved yields inshorter reaction time is desirable. As a part of our continuing efforttowards the development of new protocols for the expeditioussynthesis of biologically relevant heterocyclic compounds [25], webecame interested to explore the possibility of developing newermethodologies for the synthesis of highly bioactive pyranoquino-line and phenanthroline derivatives. Here in we report our results.

The starting materials 3 for this study were prepared from easilyavailable starting materials according to the reactions outlined inScheme 1.

The process involves Sonogashira coupling of 2 with 2-methylbut-3-yn-2-ol followed by a Brönsted acid-catalyzed cyclization

XNHR

OX

NHR

O Br

X

NHR

OOH

X = O, NMe, NEtR = H, Me, Et, Ac

(i)

(ii)

OH

1 2 3

Scheme 1. Preparation of starting materials.

Table 1Acid mediated cyclization of 3ae4a.

Entry Acid H4 equiv. Solvent Heating Time (min) Yield (%)

K.C. Majumdar et al. / European Journal of Medicinal Chemistry 71 (2014) 306e315 307

of the resulting 3a to give 4a in 98% yield (Scheme 2). 2-methyl but-3-yn-2-ol has been used widely as a readily available, cheap, non-volatile, protected form of acetylene which is unmasked via ther-molysis in the presence of a base with evolution of acetone.

Our studies started with a model reaction of 6-amino-5-(3-hydroxy-3-methylbut-1-ynyl)-2H-chromen-2-one with HCl underdifferent reaction conditions. As shown in Table 1, the cyclizationdoes not occur at room temperature, even using an excess of HCl(entry 1). The optimized condition for the reactionwas determinedto be the use of 1 equiv. acid at 100 �C for 30 min to achievecompletion of the reaction. Microwave heating has emerged as aversatile method for enhancing the rate of many chemical pro-cesses, affording cleaner reactions, and delivering high yields in fewminutes. We decided to try our reaction under microwave irradi-ation (entry 5). Under 250 W microwave irradiation completecyclization of 3a took place in just 1 min at 100 �C to affordexcellent yield of 4a (optimized condition).

To determine the scope of this methodology a range of hetero-cyclic amines were investigated under this acid-mediated cycliza-tion condition. Various pyrano[3,2-f]quinoline and phenanthrolinederivatives have been synthesized in excellent yields (Table 2).From various examples it is evident that substituent on the het-erocyclic ring or on the amine group have no significant effect onthe cyclization. But in case of starting materials 3j, 3k (whereR ¼ Ac) gave exclusively cyclized products 4a, 4g (Scheme 3) sameas obtained from the starting materials 3a, 3g (where R ¼ H). Inthese cases acid-catalyzed de-acetylation occurred to give 3a, gfollowed by acid-mediated cyclization to give 4a, 4g.

Moreover, the synthesis of 4aei can also be accomplished undermicrowave irradiation. The reaction time was reduced from severalmin. to 1 min for the cyclization step under microwave irradiation.As shown in Table 2 various tetrahydroquinoline derivatives weresynthesized in high yields. In general, slightly better yields wereobtained under microwave irradiation although the major advan-tage is in the significant reduction of reaction time.

Although the starting materials 3 were easily prepared bySonogashira cross coupling of 2 with 2-methyl but-3-yn-2-ol, wewondered if 4a could be prepared by a one-pot, two step procedurestarting from 1. To this end 2awas treated with 2-methyl but-3-yn-2-ol under standard Sonogashira reaction condition at 90 �C for 3 hfollowed by addition of 2 equiv. of HCl to the reaction mixture andheating the reaction mixture at the same temperature for further30min (Scheme 4). Gratifyingly the product 4awas obtained in 94%overall yield without isolation of the intermediate 3a.

The scope of this one-pot procedure was established by syn-thesizing various tetrahydroquinoline derivatives in satisfactory tohigh yields (Table 3). Moreover, this one-pot procedure is

O

NH

O O

O

NH2

OOH

aorb

Scheme 2. Synthesis of substituted quinoline derivative.

applicable to gram-scale synthesis of various pyrano[3,2-f]quino-line and phenanthroline derivatives.

A probable pathway is outlined in Scheme 5. Initially the aldols Iare formed via addition of a water molecule to the alkyne moiety.Next the a,b unsaturated ketones II are formed from I by regiose-lective dehydrative rearrangement that on 6-‘endo-trig’ Michael-type ring closure give the products 4.

The synthesized compounds showed high fluorescence in nakedeye (Fig. 1). The interesting feature of these compounds is theappearance of two emission peaks in the range of 360 nm and 474e500 nm. The emission peak appeared in the range of 474e500 nm isgenerally due to coumarin and quinoline motifs. Fig 2 and Fig. 3show the emission spectra of the compounds 4aei upon excita-tion at 450 nm. Fig. 3 shows that, there is a red shift (w20 nm) forthe compounds 4b, c, e, f, h, i. The emission peak centered at500 nm for the compound 4c shows the highest intensity. This isdue to the inhibition of electron transfer (ET) from the nitrogenatom to a photo excited fluorophore.

The fluorescence spectra of all of the compounds were investi-gated in different solvents (Fig. 4) such as CH3CN, CH2Cl2, THF,dioxane, methanol, methanol:H2O (1:1) upon excitation at 450 nm.All the synthesized compounds showed intense green fluorescenceeven in strong polar solvents and showed highest intensity indichloromethane. All the compounds displayed positive solventeffect and slight red shift was observed in the fluorescence spectraon increasing the solvent polarity.

1. Protein binding study by intrinsic protein fluorescencequenching and evaluation of binding constant

Serum albumin is one of the important constituent proteins ofblood plasma and it plays crucial role in drug transport and drugmetabolism. We chose bovine serum albumin (BSA) as standardserum protein and studied its interaction with the newly

1. HCl 2 H2O r.t. 180 02. HCl 2 H2O Reflux 30 963. HCl 1 H2O Reflux 30 984. HC1 0.5 H2O Reflux 30 915. HCl 1 H2O M.W. 1 996. H2SO4 1 H2O Reflux 30 947. HNO3 1 H2O Reflux 30 938. AcOH 1 H2O Reflux 30 909. TsOH 1 H2O Reflux 30 95

Bold values highlight the cyclization

Table 2Acid mediated synthesis of various tetrahydroquinoline derivatives.

Entry Starting X R Heating Time (min.) Product Yield (%)

1. 3a O H D 30 4a 982. 3a O H M.W. 1 4a 993. 3b O Me D 35 4b 964. 3b O Me M.W. 1 4b 975. 3c O Et D 30 4c 956. 3c O Et M.W. 1.5 4c 957. 3d NMe H D 40 4d 968. 3d NMe H M.W. 1.5 4d 969. 3e NMe Me D 30 4e 9510. 3e NMe Me M.W. 1 4e 9611. 3f NMe Et D 30 4f 9412. 3f NMe Et M.W. 1 4f 9413. 3g NEt H D 25 4g 9714. 3g NEt H M.W. 1 4g 9715. 3h NEt Me D 30 4h 9416. 3h NEt Me M.W. 1.5 4h 9517. 3i NEt Et D 35 4i 8918. 3i NEt Et M.W. 1 4i 90

X

NH

OO

X

NH

O OH

3 4

HCl / H2OO 100 oC, 30 mins.

Entry

1.

2.

Starting

3j

3k

X

O

NEt

Product

4a

4g

Yield (%)

89

81

Scheme 3. Acid mediated deacetylation & cyclization reactions.

K.C. Majumdar et al. / European Journal of Medicinal Chemistry 71 (2014) 306e315308

synthesized compounds using intrinsic protein fluorescencequenching. As stated in methods, 1 mM BSA was titrated with thecompounds resulting successive quenching of the protein fluores-cence spectra and from the titration profile we calculated bindingconstants for each compound as shown in Fig. 5A�I. In each figureProtein spectra have been shown at different concentrations of thesynthesized compounds and the double reciprocal plot [1/DF vs 1/(CL � CP)] are shown in the inset. From the double reciprocal plot Kd

O

NH2

O

Br2-methyl but-3-yn-2-ol, PDMF (7 ml.), NEt3 (3 ml.

HCl (2 equiv.), another

2a

Scheme 4. One-pot synthesis of pyrano[3,2-

values were calculated and summarized in Table 4. From the table itis clear that compound 4a, 4e, 4f and 4g has comparable Kd valuesbut compound 4e has minimum value. Therefore, the affinityconstant for those four compounds are comparable but complex 4fhas highest affinity to the BSA protein. The compound 4d havinghighest Kd value indicating its lowest affinity to the BSA protein.

2. Cell staining with the complex

We have also investigated for whether these compounds couldbe used to stain the cultured mammalian cells since these com-pounds can bind protein molecule. We observed that the com-pounds have intrinsic fluorescence in the sky-blue region around505 nmwith excitation near 365 nm. Hoechst dye is the one of thestandard dyes used for staining the nucleus of the cultured cells andhas the fluorescence in the sky-blue region. Hoechst is well knownDNA binding compound and hence the cells can be observed underfluorescence microscope with UV filter with excitation near365 nm. The cell stained with Hoechst was shown in Fig 6A. Wehave tested the compound’s staining ability of cultured cells asdescribed in methods and observed that all the compounds exceptcompound 4b & 4d were able to stain the cultured HeLa cells afterfixing. But, none of them was able to stain the live cells. Fixingmakes the cell porous and the compounds were able to penetrateinto cells after fixing and gave fluorescence with excitation at UVrange near 365 nm as observed under fluorescence microscope.

The compound 4g itself has intrinsic fluorescence with lemmaxima 505 nm which is the sky-blue range. The same kind ofspectra obtained when the complex is boundwith standard proteinBSA. The HeLa cells stainedwith compounds 4a, 4c, 4eei are shownin Fig. 6BeH respectively. All the images of cells were sky-blue. Wedo not know the interacting partner from HeLa cell component(s)that could bind the compounds and produced such fluorescence.Possibly, the compounds can bind the cellular protein componentsand thus making the cells visible under fluorescence microscopedue to their intrinsic fluorescence. The cell image intensity for allthe compounds as evident from the figures that compound 4g (Fig6F) gave highest intensity which is almost comparable to ourstandard control Hoechst fluorescence (Fig 6A). However, thefluorescence intensity for other complexes was observed to be lessthan that of our standard control. So, these complexes could beused as dye for staining cultured cells.

In conclusion, we have developed an efficient, rapid, highyielding procedure for the construction of potentially bioactivepyranoquinoline and phenanthroline derivatives from easilyavailable starting materials. This method is simple, easy to handleand does not require transition metal catalyst and the synthesizedcompounds exhibit high fluorescence and bind with proteinmolecule. Almost all the synthesized heterocyclic compound isable to strain cultured Hela cell after fixing and give fluorescenceat the sky blue region. This methodology will be readily applicableto the synthesis of various substituted pyranoquinoline and phe-nanthroline derivatives for potential use as dye for stainingcultured cell.

d(PPh3)2Cl2, CuI), 90 oC, 180 min.30 minutes

ONH

OO

4a

f ]quinoline-3,10(7H)-dione derivatives.

Table 3One-pot synthesis of various tetrahydroquinoline derivatives.

Entry Starting X R Time (min.) Product Yield (%)

1. 2a O H 210 4a 942. 2b O Me 215 4b 923. 2c O Et 210 4c 924. 2d NMe H 220 4d 915. 2e NMe Me 210 4e 896. 2f NMe Et 210 4f 877. 2g NEt H 205 4g 938. 2h NEt Me 210 4h 909. 2i NEt Et 215 4i 85

X

NHR

O

OH

3

H2O, H+

X

NHR

O

H2O

X

NHR

O

HO

I II

H

X

NR

O

O

4

HOOH2

Scheme 5. Possible pathway.

Fig. 1. Compound 4a, 4d, 4g in open eye.

300 350 400

200

400

600

4a 4b 4c 4d 4e 4f 4g 4h 4 i

Flu

ores

cenc

e In

tens

ity

(a.u

.)

Wavelength (nm)

Fig. 2. Compounds 4ei 1st peak.

K.C. Majumdar et al. / European Journal of Medicinal Chemistry 71 (2014) 306e315 309

3. Experimental

Melting points were determined in open capillaries and areuncorrected. IR spectra were run for KBr discs (and neat for liquidsamples) on a PerkineElmer 120-000A apparatus (ymax in cm�1)and 1H NMR and 13C NMR spectra were determined for solutions inCDCl3 with TMS as internal standard on a Bruker DPX-400 instru-ment. CHN was recorded on a Perkin Elmer 2400 series II CHNanalyzer. Mass spectra were recorded on a Qtof Micro instrument.Silica gel (60e120 mesh) and (230e400 mesh) were used forchromatographic separation. Silica gel-G [E-Mark (India)] was usedfor TLC. Petroleum-ether refers to the fraction between 60 �C and80 �C. Hoechst dye and BSAwere purchased from Sigma Chemicals

500 600

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4a

4b

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4d

4e

4f

4g

4h

4i

Flu

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(a.u

.)

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Fig. 3. Compounds 4aei 2nd peak 1% catenated.

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--- Acetonitrile--- DCM--- THF--- Dioxane--- Methanol--- Methanol:Water

Fig. 4. Fluorescence spectra (right) of compounds 4a, 4c, 4d and 4g in different solvents (c ¼ 1 � 10�5 M).

K.C. Majumdar et al. / European Journal of Medicinal Chemistry 71 (2014) 306e315310

(USA). Cell culture medium (MEM) was procured from HiMedia,Mumbai, India. Other molecular biology grade fine chemicals wereprocured locally.

3.1. General procedure for synthesis of 3aek

To a stirred solution of 6-amino-5-bromo-2H-chromen-2-one or6-amino-5-bromo-1-methylquinolin-2(1H)-one or 6-amino-5-bromo-1-ethylquinolin-2(1H)-one or N-(5-bromo-2-oxo-2H-chro-men-6-yl)acetamide (1 equiv.) in DMF (7 mL) and NEt3 (3 mL) 2-methyl but-3-yn-2-ol (1.2 equiv.) was added at room tempera-ture. Then Pd(PPh3)2Cl2 (0.05 equiv.) and CuI (0.05 equiv.) wasadded under reaction mixture in nitrogen atmosphere and stirredat 90 �C for 3 h. After completion of the reaction (monitored by TLC)the reaction mixture was cooled and extracted with dichloro-methane (25 mL � 3). The combined organic extract was washedwith brine (25 mL � 4) and dried over Na2SO4. The solvent wasdistilled off. The resulting crude product was purified by filtrationthrough a pad of silica gel (60e120 mesh) using petroleum ethereethyl acetate mixture as eluent to give the pure compound (3).

3.1.1. 6-Amino-5-(3-hydroxy-3-methylbut-1-ynyl)-2H-chromen-2-one: (3a)

Yield ¼ 82%, yellow colored solid, mp 152e154 �C. IR (KBr):nmax ¼ 1675, 1715, 2224, 2974, 3464 cm�1. 1H NMR (400 MHz,CDCl3): dH ¼ 1.70 (s, 6H), 4.30 (bs, 2H), 6.41 (d, 1H, J ¼ 9.6 Hz), 6.87(d, 1H, J ¼ 9.2 Hz), 7.10 (d, 1H, J¼ 8.8 Hz), 7.96 (d, 1H, J ¼ 9.6 Hz). 13CNMR (100MHz, CDCl3): dC¼ 31.7, 65.7, 74.8,105.9,116.9,117.8,118.5,119.4, 128.6, 132.0, 141.8, 145.2, 146.8, 161.2. MS: m/z ¼ 265.13[M þ Na]þ. Anal. Calcd. for C14H13NO3: C, 69.12; H, 5.39; N, 5.76;Found: C, 69.34; H, 5.45; N, 5.68.

3.1.2. 5-(3-Hydroxy-3-methylbut-1-ynyl)-6-(methylamino)-2H-chromen-2-one: (3b)

Yield ¼ 88%, yellow colored solid, mp 126e128 �C. IR (KBr):nmax ¼ 1595, 1646, 2106, 2974, 3528 cm�1. 1H NMR (300 MHz,CDCl3): dH ¼ 1.71 (s, 6H), 2.80 (bs, 1H), 2.93 (s, 3H), 4.62 (bs, 1H),6.40 (d, 1H, J ¼ 9.6 Hz), 6.75 (d, 1H, J ¼ 9.0 Hz), 7.16 (d, 1H,J ¼ 9.0 Hz), 7.92 (d, 1H, J ¼ 9.6 Hz). 13C NMR (100 MHz, CDCl3):dC ¼ 30.6, 31.8, 65.8, 74.8, 102.4, 106.4, 113.1, 117.0, 117.8, 119.6, 141.9,145.7, 147.3, 161.4. MS: m/z ¼ 280.09 [M þ Na]þ. Anal. Calcd. forC15H15NO3: C, 70.02; H, 5.88; N, 5.44; Found: C, 69.84; H, 5.92; N,5.55.

3.1.3. 6-(Ethylamino)-5-(3-hydroxy-3-methylbut-1-ynyl)-2H-chromen-2-one: (3c)

Yield ¼ 91%, yellow colored solid, mp 72e74 �C. IR (KBr):nmax ¼ 1701, 2978, 3481 cm�1. 1H NMR (400 MHz, CDCl3): dH ¼ 1.31(t, 3H, J ¼ 7.2 Hz), 1.71 (s, 6H), 2.81 (bs, 1H), 3.23 (q, 2H, J ¼ 7.2 Hz),4.47 (bs, 1H), 6.40 (d, 1H, J ¼ 9.6 Hz), 6.75 (d, 1H, J ¼ 9.2 Hz), 7.14 (d,1H, J ¼ 9.2 Hz), 7.93 (d, 1H, J ¼ 9.6 Hz). 13C NMR (100 MHz, CDCl3):dC ¼ 14.7, 31.0, 31.7, 38.3, 65.8, 74.9, 102.3, 106.4, 113.6, 117.0, 117.8,119.6, 141.9, 145.6, 146.4, 161.3. MS: m/z ¼ 294.06 [M þ Na]þ. Anal.Calcd. for C16H17NO3: C, 70.83; H, 6.32; N, 5.16; Found: C, 70.68; H,6.35; N, 5.27.

3.1.4. 6-Amino-5-(3-hydroxy-3-methylbut-1-ynyl)-1-methylquinolin-2(1H)-one: (3d)

Yield ¼ 77%, light gray colored solid, mp 148e150 �C. IR (KBr):nmax ¼ 1648, 2209, 2978, 3392 cm�1. 1H NMR (400 MHz, CDCl3):dH ¼ 1.71 (s, 6H), 3.66 (s, 3H), 4.18 (bs, 1H), 6.71 (d, 1H, J ¼ 9.6 Hz),6.86 (d, 1H, J ¼ 9.2 Hz), 7.06 (d, 1H, J ¼ 8.8 Hz), 7.87 (d, 1H,J ¼ 9.6 Hz). 13C NMR (100 MHz, CDCl3): dC ¼ 29.7, 31.7, 65.4, 75.0,

K.C. Majumdar et al. / European Journal of Medicinal Chemistry 71 (2014) 306e315 311

103.7, 106.3, 115.2, 118.1, 121.3, 122.2, 132.7, 136.7, 143.3, 161.7. MS:m/z ¼ 279.0 [M þ Na]þ. Anal. Calcd. for C15H16N2O2: C, 70.29; H,6.29; N, 10.93; Found: C, 70.43; H, 6.21; N, 10.85.

3.1.5. 5-(3-Hydroxy-3-methylbut-1-ynyl)-1-methyl-6-(methylamino)quinolin-2(1H)-one: (3e)

Yield¼ 84%, light yellow colored solid, mp 210e212 �C. IR (KBr):nmax¼ 1592,1644, 2206, 2970, 3296, 3390 cm�1. 1H NMR (400MHz,CDCl3): dH ¼ 1.71 (s, 6H), 2.95 (s, 3H), 3.66 (s, 3H), 6.72 (d, 1H,J ¼ 10.0 Hz), 6.82 (d, 1H, J ¼ 9.2 Hz), 7.16 (d, 1H, J ¼ 9.2 Hz), 7.90 (d,1H, J¼ 9.6 Hz). 13C NMR (100MHz, CDCl3): dC¼ 29.5, 30.7, 31.7, 65.6,75.2, 102.8, 106.5, 112.9, 115.4, 121.6, 122.4, 131.6, 136.6, 145.7, 161.6.MS: m/z ¼ 271 [M þ H]þ. Anal. Calcd. for C16H18N2O2: C, 71.09; H,6.71; N, 10.36; Found: C, 70.89; H, 6.63; N, 10.45.

3.1.6. 6-(Ethylamino)-5-(3-hydroxy-3-methylbut-1-ynyl)-1-methylquinolin-2(1H)-one: (3f)

Yield ¼ 83%, light yellow colored solid, mp 180e182 �C. IR(KBr): nmax ¼ 1603, 1646, 2924, 3393 cm�1. 1H NMR (300 MHz,CDCl3): dH ¼ 1.30 (t, 3H, J ¼ 7.2 Hz), 1.70 (s, 6H), 3.23 (q, 2H,J ¼ 7.2 Hz), 3.62 (s, 3H), 4.14 (bs, 1H), 4.41 (bs, 1H), 6.68 (d, 1H,J ¼ 9.6 Hz), 6.72 (d, 1H, J ¼ 9.3 Hz), 7.00 (d, 1H, J ¼ 9.3 Hz), 7.79 (d,1H, J ¼ 9.6 Hz). 13C NMR (100 MHz, CDCl3): dC ¼ 15.0, 29.6, 31.7,38.4, 65.3, 75.0, 102.9, 107.1, 113.4, 115.2, 121.5, 122.2, 131.5, 136.9,

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1/(C -C )

Complound-4a (Fig 5A)

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1/ΔF

1/ΔF

Fig. 5. Each fig. shows the intrinsic protein spectra of BSA (lex ¼ 280 nm) titrated with the inof concentration of the complex. Inset picture shows the double-reciprocal plot for determ

144.8, 161.8. HRMS: m/z calcd for C17H20N2O2 [M þ Na]þ: 307.1423;found; 307.1419.

3.1.7. 6-Amino-1-ethyl-5-(3-hydroxy-3-methylbut-1-ynyl)quinolin-2(1H)-one: (3g)

Yield ¼ 79%, gray colored solid, mp 142e144 �C. IR (KBr):nmax ¼ 1623, 1644, 2210, 2977, 3199, 3340, 3459 cm�1. 1H NMR(400 MHz, CDCl3): dH ¼ 1.30 (t, J ¼ 7.2 Hz, 3H), 4.24 (bs, 1H), 4.28 (q,J¼ 7.2Hz, 2H), 6.71 (d,1H, J¼ 9.6Hz), 6.90 (d,1H, J¼ 9.2Hz), 7.11 (d,1H,J¼ 8.8Hz), 7.92 (d,1H, J¼9.6Hz).13CNMR(100MHz, CDCl3): dC¼ 13.0,31.7, 37.5, 65.3, 75.1, 103.9, 106.4, 115.1, 118.3, 121.6, 122.0, 131.5, 136.7,143.4, 161.2. MS: m/z ¼ 271 [M þ H]þ. Anal. Calcd. for C16H18N2O2: C,71.09; H, 6.71; N, 10.36; Found: C, 70.85; H, 6.67; N, 10.48.

3.1.8. 1-Ethyl-5-(3-hydroxy-3-methylbut-1-ynyl)-6-(methylamino)quinolin-2(1H)-one: (3h)

Yield¼ 85%, light yellow colored solid, mp 138e140 �C. IR (KBr):nmax ¼ 1598, 1646, 2210, 2979, 3290 cm�1. 1H NMR (400 MHz,CDCl3): dH¼ 1.30 (t, 3H, J¼ 7.2 Hz),1.71 (s, 6H), 2.93 (s, 3H), 3.71 (bs,1H), 4.27 (q, 2H, J¼ 7.2 Hz), 4.58 (bs,1H), 6.69 (d,1H, J¼ 9.6 Hz), 6.81(d, 1H, J ¼ 9.2 Hz), 7.15 (d, 1H, J ¼ 9.2 Hz), 7.89 (d, 1H, J ¼ 9.6 Hz). 13CNMR (100 MHz, CDCl3): dC ¼ 13.0, 30.6, 31.7, 37.3, 65.5, 75.3, 103.1,106.7,113.0,115.3,121.9,122.3,130.4,132.1,136.7,145.6,161.1. HRMS:m/z calcd for C17H20N2O2 [M þ Na]þ: 307.1423; Found: 307.1419.

300 320 340 360 380 400 420 440 4600

20

40

60

80

100

120

Kd = 7.09

Pro

tein

fluo

resc

ence

(a.

u)

[Wavelength], nm

1/(C -C )

compound – 4b (Fig5B)

320 360 400 440

0

40

80

120

0.0 0.1 0.2

0.00

0.04

0.08

0.12

Kd = 34.83

Pro

tein

flu

orescen

ce (a.u

)

[Wavelength], nm

1/ΔF

1/(C -C )

Compound – 4d (Fig 5D)

1/ΔF

creasing concentration of complex. The peak fluorescence decreased with the increaseining Kd value.

320 360 400 440

0

40

80

120

0.00 0.04 0.08 0.12 0.16

0.00

0.01

0.02

0.03

0.04

0.05

0.06

Kd = 5.20

Pro

tein

flu

orescen

ce (a.u

)

[Wavelength], nm

1/ΔF

1/(C -C )

320 360 400 440

0

40

80

120

0.00 0.04 0.08 0.12 0.16 0.20

0.000

0.025

0.050

0.075

Kd = 5.24

Pro

tein

flu

orescen

ce (

a.u

)

[Wavelength], nm

1/(C -C )

320 360 400 440 480

0

20

40

60

80

100

120

0.00 0.04 0.08 0.12 0.16

0.000

0.025

0.050

0.075

Kd = 5.57

Pro

tein

flu

orescen

ce (

a.u

)

[Wavelength], nm

1/(C -C )

Compound 4g (Fig 5G)

320 360 400 440

0

100

200

300

400

0.00 0.05 0.10 0.15

0.000

0.005

0.010

0.015

0.020

Kd = 12.20

Pro

tein

flu

orescen

ce (a.u

)

[Wavelength], nm

1/(C -C )

300 320 340 360 380 400 420 440

0

20

40

60

80

100

120

Kd = 7.75

Pro

tein

fluo

resc

ence

(a.

u)

[Wavelength], nm

1/(C -C )

1/ΔF

1/ΔF

1/ΔF

1/ΔF

Compound − 4h (Fig 5H) Compound − 4i (Fig 5I)

Compound − 4e (Fig 5E) Compound − 4f (Fig 5F)

Fig. 5. (continued).

K.C. Majumdar et al. / European Journal of Medicinal Chemistry 71 (2014) 306e315312

3.1.9. 1-Ethyl-6-(ethylamino)-5-(3-hydroxy-3-methylbut-1-ynyl)quinolin-2(1H)-one: (3i)

Yield¼ 83%, light yellow colored solid, mp 128e130 �C. IR (KBr):nmax ¼ 1597, 1642, 2208, 2972, 3292, 3338 cm�1. 1H NMR (300MHz,CDCl3): dH ¼ 1.32 (t, 6H, J ¼ 6.9 Hz), 1.72 (s, 6H), 3.25 (q, 2H,J ¼ 7.2 Hz), 4.29 (q, 2H, J ¼ 6.9 Hz), 6.71 (d, 1H, J ¼ 9.6 Hz), 6.86 (d,1H, J ¼ 9.3 Hz), 7.18 (d, 1H, J ¼ 9.3 Hz), 7.94 (d, 1H, J ¼ 9.6 Hz). 13CNMR (100 MHz, CDCl3): dC ¼ 13.0, 14.9, 31.7, 37.4, 38.3, 65.2, 75.1,

103.1, 107.0, 113.5, 115.1, 121.8, 122.1, 130.3, 132.0, 136.8, 144.7, 161.2.MS: m/z ¼ 299 [M þ H]þ. Anal. Calcd. for C18H22N2O2: C, 72.46; H,7.43; N, 9.39; Found: C, 72.63; H, 7.40; N, 9.48.

3.1.10. N-(5-(3-Hydroxy-3-methylbut-1-ynyl)-2-oxo-2H-chromen-6-yl)acetamide: (3j)

Yield ¼ 78%, light gray colored solid, mp 134e136 �C. IR (KBr):nmax ¼ 1676, 1668, 1737, 2219, 3295 cm�1. 1H NMR (400 MHz,

Table 4The Kd values of the compounds for binding withBSA (1 mM).

Compound Kd

4a 5.384b 7.094c 7.444d 34.834e 5.204f 5.244g 5.574h 12.204i 7.75

K.C. Majumdar et al. / European Journal of Medicinal Chemistry 71 (2014) 306e315 313

CDCl3): dH ¼ 1.71 (s, 6H), 2.23 (s, 3H), 3.48 (bs, 1H), 6.41 (d, 1H,J ¼ 9.6 Hz), 7.21 (d, 1H, J ¼ 8.8 Hz), 7.90 (d, 1H, J ¼ 9.6 Hz), 7.97 (s,1H), 8.43 (d, 1H, J ¼ 9.2 Hz). 13C NMR (100 MHz, CDCl3): dC ¼ 24.8,31.4, 65.7, 73.5, 108.2, 109.5, 117.5, 119.0, 123.6, 136.2, 141.3, 149.9,160.4, 168.7. MS:m/z¼ 286 [Mþ H]þ. Anal. Calcd. for C16H15NO4: C,67.36; H, 5.30; N, 4.91; Found: C, 67.19; H, 5.35; N, 4.99.

3.1.11. N-(1-Ethyl-5-(3-hydroxy-3-methylbut-1-ynyl)-2-oxo-1,2-dihydroquinolin-6-yl)acetamide: (3k)

Yield ¼ 79%, light gray colored solid, mp 162e164 �C. IR (KBr):nmax ¼ 1672, 1660, 1712, 2214, 3292 cm�1. 1H NMR (400 MHz,CDCl3): dH ¼ 1.27 (t, 3H, J ¼ 7.2 Hz), 1.74 (s, 6H), 2.26 (s, 3H), 4.22 (q,2H, J ¼ 6.8 Hz), 6.71 (d, 1H, J ¼ 9.6 Hz), 7.14 (d, 1H, J ¼ 9.6 Hz), 7.89(d, 1H, J ¼ 9.6 Hz), 8.04 (s, 1H), 8.41 (d, 1H, J ¼ 9.2 Hz). 13C NMR(100 MHz, CDCl3): dC ¼ 12.9, 24.7, 31.5, 37.6, 65.3, 74.0, 108.5, 109.9,114.7, 120.7, 122.5, 122.6, 134.4, 134.8, 136.7, 161.5, 168.6. MS: m/z ¼ 313 [M þ H]þ. Anal. Calcd. for C18H20N2O3: C, 69.21; H, 6.45; N,8.97; Found: C, 69.40; H, 6.48; N, 8.92.

3.2. General procedure for the preparation of compound 4aei

Amixture of 3 (1 equiv.) and conc. HCl (1 equiv.) was refluxed inwater for 30e40 min or irradiated under microwave for just 1e1.5 min in 250 W, 100 �C and without any pressure. After comple-tion of the reaction as monitored by TLC, the reaction mixture wascooled extracted with ethylacetate (3 � 25 mL). The combinedorganic extract was washed with saturated solution of sodium bi-carbonate followed by brine solution and dried over anhydrous

Fig. 6. HeLa cells stained with standard Hoechst dye and our synthesized compounds after fiA, Hoechst dye, BeH, stained with compound 4a, 4c, 4eei respectively.

Na2SO4. The solvent was distilled off. The crude product was pu-rified by column chromatography over silica gel (60e120 mesh)using petroleum ethereethyl acetate (75:25) mixture as eluent togive compounds (4aei).

3.2.1. 8,8-Dimethyl-8,9-dihydro-3H-pyrano[3,2-f]quinoline-3,10(7H)-dione: (4a)

Yield ¼ 99%, light yellow colored solid, mp 172e174 �C. IR (KBr):nmax ¼ 1554, 1719, 2955, 3108 cm�1. 1H NMR (400 MHz, CDCl3):dH ¼ 1.34 (s, 6H), 2.64 (s, 2H), 4.60 (s, 1H), 6.48 (d, 1H, J ¼ 10.0 Hz),6.85 (d, 1H, J ¼ 9.2 Hz), 7.27 (d, 1H, J ¼ 9.2 Hz), 9.35 (d, 1H,J ¼ 10.0 Hz). 13C NMR (100 MHz, CDCl3): dC ¼ 27.1, 51.4, 53.4, 109.1,117.5, 117.8, 121.3, 124.2, 142.4, 147.3, 148.6, 160.7, 195.2. MS: m/z ¼ 265.13 [M þ Na]þ. Anal. Calcd. For C14H13NO3: C, 69.12; H, 5.39;N, 5.76; Found: C, 69.29; H, 5.44; N, 5.66.

3.2.2. 7,8,8-Trimethyl-8,9-dihydro-3H-pyrano[3,2-f]quinoline-3,10(7H)-dione: (4b)

Yield¼ 97%, light yellow colored solid, mp 160e162 �C. IR (KBr):nmax ¼ 1517, 1723, 2958, 3109 cm�1. 1H NMR (400 MHz, CDCl3):dH ¼ 1.33 (s, 6H), 2.70 (s, 2H), 2.97 (s, 3H), 6.47 (d, 1H, J ¼ 10.0 Hz),7.03 (d, 1H, J ¼ 9.6 Hz), 7.35 (d, 1H, J ¼ 9.2 Hz), 9.36 (d, 1H,J ¼ 10.0 Hz). 13C NMR (100 MHz, CDCl3): dC ¼ 24.1, 32.1, 52.7, 58.2,111.5, 117.4, 118.1, 118.6, 124.0, 142.4, 146.5, 149.6, 160.4, 195.0. MS:m/z ¼ 280 [M þ Na]þ. Anal. Calcd. For C15H15NO3: C, 70.02; H, 5.88;N, 5.44; Found: C, 69.81; H, 5.82; N, 5.51.

3.2.3. 7-Ethyl-8,8-dimethyl-8,9-dihydro-3H-pyrano[3,2-f]quinoline-3,10(7H)-dione: (4c)

Yield ¼ 95%, light yellow colored solid, mp 156e158 �C. IR (KBr):nmax ¼ 1514, 1720, 2954, 3108 cm�1. 1H NMR (400 MHz, CDCl3):dH ¼ 1.32 (t, 3H, J ¼ 6.8 Hz), 1.35 (s, 6H), 2.66 (s, 2H), 3.43 (q, 2H,J¼ 7.36Hz), 6.47 (d,1H, J¼ 10.0Hz), 7.00 (d,1H, J¼ 9.6Hz), 7.36 (d,1H,J ¼ 9.6 Hz), 9.38 (d, 1H, J ¼ 10.0 Hz). 13C NMR (100 MHz, CDCl3):dC ¼ 15.3, 24.7, 40.5, 53.0, 58.2, 111.2, 117.7, 118.1, 118.2, 124.2, 142.5,146.5, 148.8, 160.5, 195.0. MS: m/z ¼ 294 [M þ Na]þ. Anal. Calcd. ForC16H17NO3: C, 70.83; H, 6.32; N, 5.16; Found: C, 70.66;H, 6.38; N, 5.25.

3.2.4. 3,3,7-Trimethyl-3,4-dihydro-4,7-phenanthroline-1,8(2H,7H)-dione: (4d)

Yield¼ 96%, light yellow colored solid, mp 248e250 �C. IR (KBr):nmax ¼ 1606, 1650, 2966, 3256 cm�1. 1H NMR (400 MHz, CDCl3):

xing and visualized under fluorescence microscope (Carl Zeiss) with UV excitation filter.

K.C. Majumdar et al. / European Journal of Medicinal Chemistry 71 (2014) 306e315314

dH ¼ 1.36 (s, 6H), 2.66 (s, 2H), 3.72 (s, 3H), 4.95 (s, 1H), 6.78 (d, 1H,J ¼ 10.0 Hz), 6.97 (d, 1H, J ¼ 9.2 Hz), 7.42 (d, 1H, J ¼ 9.6 Hz), 9.39 (d,1H, J ¼ 10.0 Hz). 13C NMR (100 MHz, CDCl3): dC ¼ 27.2, 29.8, 51.9,53.4, 109.8, 119.6, 120.8, 122.0, 123.4, 133.1, 136.6, 147.4, 161.1, 195.4.MS:m/z ¼ 279 [M þ Na]þ. Anal. Calcd. For C15H16N2O2: C, 70.29; H,6.29; N, 10.93; Found: C, 70.11; H, 6.24; N, 11.01.

3.2.5. 3,3,4,7-Tetramethyl-3,4-dihydro-4,7-phenanthroline-1,8(2H,7H)-dione: (4e)

Yield ¼ 96%, light yellow colored solid, mp 158e160 �C. IR(KBr): nmax ¼ 1607, 1653, 2966, 3073 cm�1. 1H NMR (400 MHz,CDCl3): dH ¼ 1.33 (s, 6H), 2.73 (s, 2H), 2.99 (s, 3H), 3.72 (s, 3H),6.79 (d, 1H, J ¼ 10.0 Hz), 7.11 (d, 1H, J ¼ 10.0 Hz), 7.50 (d, 1H,J ¼ 9.6 Hz), 9.38 (d, 1H, J ¼ 10.0 Hz). 13C NMR (100 MHz, CDCl3):dC ¼ 24.2, 29.7, 32.1, 53.3, 58.2, 112.6, 118.0, 119.8, 121.8, 124.0,132.4, 136.7, 148.4, 161.1, 195.4. MS: m/z ¼ 271 [M þ H]þ. Anal.Calcd. For C16H18N2O2: C, 71.09; H, 6.71; N, 10.36; Found: C,70.86; H, 6.67; N, 10.46.

3.2.6. 4-Ethyl-3,3,7-trimethyl-3,4-dihydro-4,7-phenanthroline-1,8(2H,7H)-dione: (4f)

Yield¼ 94%, light yellow colored solid, mp 168e170 �C. IR (KBr):nmax ¼ 1609, 1649, 2967, 3077 cm�1. 1H NMR (400 MHz, CDCl3):dH ¼ 1.34 (m, 9H), 2.66 (s, 2H), 3.44 (q, 2H, J ¼ 6.8 Hz), 3.70 (s, 3H),6.75 (d, 1H, J ¼ 10.4 Hz), 7.08 (d, 1H, J ¼ 9.6 Hz), 7.49 (d, 1H,J ¼ 10.0 Hz), 9.37 (d, 1H, J ¼ 10.0 Hz). 13C NMR (100 MHz, CDCl3):dC ¼ 15.3, 24.6, 29.6, 40.3, 53.3, 58.0, 111.6, 117.7, 119.7, 121.9, 123.4,131.8, 136.7, 147.5, 160.9, 195.2. HRMS: m/z calcd for C17H20N2O2[M þ Na]þ: 307.1423; Found: 307.1664.

3.2.7. 7-Ethyl-3,3-dimethyl-3,4-dihydro-4,7-phenanthroline-1,8(2H,7H)-dione: (4g)

Yield¼ 97%, light yellow colored solid, mp 136e138 �C. IR (KBr):nmax ¼ 1591, 1648, 2979 cm�1. 1H NMR (400 MHz, CDCl3): dH ¼ 1.34(t, J ¼ 7.2 Hz, 3H), 1.35 (s, 6H), 2.66 (s, 2H), 4.35 (d, 2H, J ¼ 7.2 Hz),4.45 (s, 1H), 6.79 (d, 1H, J ¼ 10.0 Hz), 6.91 (d, 1H, J ¼ 9.6 Hz), 7.42 (d,1H, J ¼ 9.2 Hz), 9.39 (d, 1H, J ¼ 10.4 Hz). 13C NMR (100 MHz, CDCl3):dC ¼ 13.4, 27.3, 37.5, 52.0, 53.5, 110.4, 120.1, 120.6, 121.9, 123.7, 132.3,136.5, 140.1, 160.6, 195.3. MS: m/z ¼ 271 [M þ H]þ. Anal. Calcd. ForC16H18N2O2: C, 71.09; H, 6.71; N, 10.36; Found: C, 70.96; H, 6.77; N,10.45.

3.2.8. 7-Ethyl-3,3,4-trimethyl-3,4-dihydro-4,7-phenanthroline-1,8(2H,7H)-dione: (4h)

Yield ¼ 95%, light yellow colored solid, mp 126e128 �C. IR(KBr): nmax ¼ 1595, 1646, 2974, 3528 cm�1. 1H NMR (400 MHz,CDCl3): dH ¼ 1.32 (s, 6H), 1.36 (t, J ¼ 7.2 Hz, 3H), 2.72 (s, 2H), 2.98(s, 3H), 4.36 (t, 2H, J ¼ 7.2 Hz), 6.78 (d, 1H, J ¼ 10.0 Hz), 7.11 (d,1H, J ¼ 10.0 Hz), 7.51 (d, 1H, J ¼ 10.0 Hz), 9.38 (d, 1H, J ¼ 10.0 Hz).13C NMR (100 MHz, CDCl3): dC ¼ 13.3, 24.1, 32.1, 37.3, 53.3, 58.2,112.6, 118.0, 120.1, 121.7, 123.9, 131.2, 136.6, 148.3, 160.5, 195.3.HRMS: m/z calcd for C17H20N2O2 [M þ Na]þ: 307.1423; found;307.1664.

3.2.9. 4,7-Diethyl-3,3-dimethyl-3,4-dihydro-4,7-phenanthroline-1,8(2H,7H)-dione: (4i)

Yield¼ 90%, light yellow colored solid, mp 164e166 �C. IR (KBr):nmax ¼ 1608, 1644, 2969, 3074 cm�1. 1H NMR (400 MHz, CDCl3):dH ¼ 1.33 (m,12H), 2.65 (s, 2H), 3.42 (q, 2H, J¼ 7.32 Hz), 4.33 (q, 2H,J¼ 7.32 Hz), 6.75 (d, 1H, J¼ 10.0 Hz), 7.07 (d, 1H, J¼ 9.6 Hz), 7.48 (d,1H, J¼ 10.0 Hz), 9.37 (d,1H, J¼ 10.0 Hz). 13C NMR (100MHz, CDCl3):dC ¼ 13.4, 15.5, 24.7, 37.3, 40.4, 53.5, 58.1, 112.1, 117.7, 120.2, 121.9,123.8,131.0,136.7,147.4,160.6,195.2. MS:m/z¼ 299 [MþH]þ. Anal.Calcd. For C18H22N2O2: C, 72.46; H, 7.43; N, 9.39; Found: C, 72.23; H,7.46; N, 9.47.

3.3. Cell culture

Human cervical cancer cell line (HeLa) was obtained fromNational Centre for Cell Sciences, Pune, India. HeLa cells weregrown in MEM supplemented with 10% bovine serum (completemedium) at 37 �C in humidified atmosphere containing 5% CO2[26].

3.4. Protein binding study and evaluation of binding constants

To check the binding of the compound 4 with cellular compo-nents, if any, we have chosen bovine serum albumin (BSA) a stan-dard protein. We took 1 mM BSA in quartz cuvette with 1 cm pathlength and measured protein fluorescence with excitation at285 nm in 970-CRT spectrofluorimeter (Agilant, Shanghai). Theband pass was 5 nm for both excitation and emission channels.Small aliquots of methanolic stock of the complex were added andeach step protein fluorescence spectra was observed. The protein-free PBS containing different concentration of compounds wasused as blank and subtracted from the protein bound complexspectra. All the measurements were done at 25 �C and repeated theexperiment thrice.

Progressive quenching of protein fluorescence upon addingsmall aliquots of complex was noted at emission 346 nm withexcitation at 285 and used to calculate the dissociation constant(Kd) of the protein-compound interaction using the standard for-mula as given below Equation (1) [26].

1=DF ¼ 1=DFmax þ Kd=½DFmaxðCL � CPÞ� (1)

where DF is the change of protein fluorescence at each concentra-tion of complex, DFmax is the maximum change of fluorescence i.e.when total protein was saturated with the complex, CL is theconcentration of compound and CP is the concentration of protein.This relation is valid when CL >> CP.

3.5. Cell staining with the compounds

The cells were processed for staining following our protocolwith slight modification [27]. In brief, the HeLa cells were grownover cover slip for 24 h and washed thrice with PBS to remove themedium. Then cells over the cover slip were fixed with methanol-acetone (1:1) at 4 �C for 1 h. Then the cover slip was washed twicewith PBS and incubated with 1 mM of the compound in PBS for10 min in dark. After washing with PBS the cover slip was placedupside down over the glass slide so that the cells touched the glassslide. Then the slides were observed under fluorescence micro-scope (Carl Zeiss) with UV excitation as well as in normal lightmode [28e30].

Acknowledgments

One of us (KCM) is thankful to UGC, (New Delhi) for a UGCEmeritus Fellowship, and two of us (SP) and (TG) are grateful toCSIR, (New Delhi) for their senior research fellowships. We alsothank the DST (New Delhi) for providing Bruker NMR spectrometer(400 MHz) and PerkineElmer CHN analyzer, UVeVIS spectrometerand PerkineElmer FT-IR under FIST program.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.ejmech.2013.10.067.

K.C. Majumdar et al. / European Journal of Medicinal Chemistry 71 (2014) 306e315 315

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