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European Journal of Medicinal Chemistry 45 (2010) 3607e3616

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European Journal of Medicinal Chemistry

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

Original article

Substituted dihydronaphthalenes as efflux pump inhibitors ofStaphylococcus aureus

Niranjan Thota a, Mallepally V. Reddy a, Ashwani Kumar b, Inshad A. Khan b, Payare L. Sangwan a,Nitin P. Kalia b, Jawahir L. Koul a, Surrinder Koul a,*aBioorganic Chemistry Division, Indian Institute of Integrative Medicine (CSIR), Canal Road, Jammu Tawi-180001, IndiabClinical Microbiology Unit, Indian Institute of Integrative Medicine (CSIR), Canal Road, Jammu Tawi-180001, India

a r t i c l e i n f o

Article history:Received 7 January 2010Received in revised form3 May 2010Accepted 5 May 2010Available online 12 May 2010

Keywords:Bacterial NorA efflux pump inhibitorsEugenolTetraloneCiprofloxacinStaphylococcus aureusEthidium bromide

* Corresponding author. Tel.: þ91 191 2569021; faxE-mail address: skoul@iiim.res.in (S. Koul).

0223-5234/$ e see front matter � 2010 Elsevier Masdoi:10.1016/j.ejmech.2010.05.006

a b s t r a c t

A new series of 3-(substituted-3,4-dihydronaphthyl)-2-propenoic acid amides has been preparedthrough convergent synthetic strategies and tested in combination with ciprofloxacin against NorAoverexpressing Staphylococcus aureus 1199B as test strain for potentiating of the drug activity. Out of 24compounds evaluated, 12 compounds potentiated the activity of ciprofloxacin and resulted in 2e16 foldreduction in the MIC (4e0.5 mg/mL) of the drug. The failure of these efflux pump inhibitors (EPIs) topotentiate the activity of ciprofloxacin when tested against NorA knock out S. aureus SA-K1758 estab-lished their identity as NorA inhibitors. The structure of all these newly synthesised compounds wasconfirmed by spectral data. The present communication describes the synthesis, bioevaluation, structureactivity relationship and mechanism of action of these EPIs.

� 2010 Elsevier Masson SAS. All rights reserved.

1. Introduction

In recent years bacterial resistance to antimicrobial agents hasrisen dramatically, resulting in a serious public health problem.Mechanisms by which this occurs include enzymatic inactivation,target modification, and reduction of drug accumulationwithin thecell by the reduced permeability of the bacterial envelope and/orextrusion of the drug from the cell by way of membrane-basedefflux proteins [1e5]. Efflux pumps are found in both Gram-positiveand Gram-negative pathogens [6,7]. Some of these efflux pumpsconfer multiple-drug resistance (MDR), and the NorA protein ofStaphylococcus aureus is one such pump [8,9]. It belongs to themajor facilitator superfamily (MFS) of transport proteins, one of themost studied MDR pump, and its substrates include antimicrobialagents and dyes such as Ciprofloxacin, Norfloxacin, Ethidiumbromide and Acriflavine [10]. Efforts are underway to generate newantibacterial agents capable of avoiding drug efflux by synthesizingcompounds that are poor pump substrates [11], or alternatively,identify compounds that reduce or block the efflux pump activity of

: þ91 191 2569333.

son SAS. All rights reserved.

the antiinfective/s and possibly reduce the emergence of target-based resistance mechanisms. The latter approach would representa significant advance in antibacterial chemotherapy [12]. Thesecompounds, called efflux pump inhibitors (EPIs), could becombined with substrate antimicrobial agents similar to the b-lactam-b-lactamase combinations already in clinical use [13]. Thereare ongoing efforts to develop formulations of existing drugs withEPIs, and one such example is that of an aerosolized ciprofloxacin/EPI combination for the treatment of cystic fibrosis [14]. Thedevelopment of novel bacterial EPIs has been reported by severalgroups and many of these compounds are reported to substantiallylower MICs of clinically relevant drugs [15e18]. The presentcommunication describes the preparation and identification ofpotent EPIs, demonstration of their abilities to reduce MICs whencombined with the antibacterial drug ciprofloxacin and the struc-ture activity relationship (SAR) studies.

2. Results and discussion

2.1. Chemistry

Twenty four compounds (5e10 and 19e36) selected forbiological evaluationwere prepared as described in Schemes1 and2.

MeO

HO

MeO

ROR = CH3 (2)R = C3H5 (2a)

MeO

ROR = CH3 (3)R = C3H5 (3a)

a bO

MeO

RO

R = CH3 (4)R = C3H5 (4a)

OH

O

c

dMeO

RO R1

5-10

N

O

R = CH3, R1 =

5

O

N

O

6

HN

O6

7

HN

O

8

R = C3H5 , R1 =N

O

9

HN

O

10

1

Scheme 1. Synthetic methods for the preparation of 5e10: Reagents: (a) RX/K2CO3

(2, 85%; 2a, 81%); (b) DMF/POCl3 (3, 26%; 3a, 23%); (c) (i) P(Ph)3/BrCH2COOEt/NaH/Benzene; (ii) NaOH/MeOH/HCl (4, 86%; 4a, 60%); (d) SOCl2/DCM; NHR1R2(amines)/C6H6.

R = H, R1 = NO

HN

O20

N

O

R = H, R1 = NO

HN

O34 35

R = Ph, R

19 21

NO

R = Ph, R1 =

24

HN

O25

N

O26

NO

R = CH2Ph, R1 =

29

HN

O

30

N

O3

a

b

d

b

b

c

11

12 13

14 15

16 17

Route-A

Route-B

Route-C

O

OH

O

OCl O

HO

Scheme 2. Synthetic methods for the preparation of 19e36: Reagents: (a) NaBH4/MeOH, 9183%; (d) PhCH2Br/Mg/THF, 71.5%; (e) Malonic acid/base/HCl or P(Ph)3/BrCH2COOEt/NaH/(amines)/C6H6; (g) H2/PdeC.

N. Thota et al. / European Journal of Medicinal Chemistry 45 (2010) 3607e36163608

The synthesis of compounds 5e10 was accomplished by alkylationof naturally occurring plant isolate eugenol 1 and subjecting thealkylated products 2 and 2a [19] to Vilsmeier reaction to giveformylated products 3 and 3a [20]. Wittig reaction of formylatedproducts 3 and 3a and subsequent saponification of the productsafforded the acids 3-(3,4-dihydronphthalene)-2-propenoic acids 4and4a respectively [21]. The acid chloride of 4 and4awas allowed toreact with appropriate amines to afford compounds 5e10 in 20%over all yields (Scheme 1).

Compounds 19e23 were prepared via route-A in Scheme 2 bytaking a-tetralone 11 as a starting material and carrying out itsreduction with sodium borohydride followed by Vilsmeier reactionto give formylated product 13 [22]. Wittig reaction of 13 andsubsequent saponification afforded 3-(3,4-dihydronaphthyl)-2-propenoic acid 18. Acid chloride of 18 was allowed to condensewith different amines to give compounds 19e23 in 87e92% yields.

Compounds 24e28 were prepared via route-B in Scheme 2 bySuzuki coupling [23] of compound 14 (obtained by Vilsmeierreaction of 11 [24]) followed by subjecting the product 15 toKnoevenagel reaction with malonic acid to afford the acid 18a.Condensation of acid chloride of 18a with appropriate amines toafford compounds 24e28 in 70e92% yield.

Compounds 29e33 were prepared via route-C in Scheme 2which involved Grignard reaction of 11 with benzyl halide toafford compound 16. Vilsmeier reaction of 16 provided formylderivative 17, which on Wittig reaction and subsequent hydrolysis

HN OMe

O

22

HN

O

NO

1 =

23

N

O

27

O

HN

28

32

N

O

O

O

HN

O

331

36

f

e

e

e

19-23 R = H24-28 R = Ph29-33 R = CH2Ph

g

34,35 R = H36 R = Ph

18 R = H18a R = Ph 18b R = CH2Ph

O

R O

OH

RR1

RR1

%; (b) DMF/POCl3 (13, 96%; 14, 93%; 17, 87%); (c) Phenyl boronic acid, K2CO3/Pd(PPh3)4,Benzene/NaOH/MeOH/HCl, (18, 94.1%; 18a, 70%; 18b, 78%); (f) SOCl2/DCM, NHR1R2

N. Thota et al. / European Journal of Medicinal Chemistry 45 (2010) 3607e3616 3609

afforded the acid 18b. Condensation of acid chloride of 18bwith appropriate amines furnished the amides 29e33 in 66e82%yield.

2.2. Bioevaluation studies

In our earlier studies, we found that in comparison to EPIs suchas 5-(3,4-methylenedioxy-5-yl)-2E,4E-pentadienoic acid amides(Piperine and its analogues), incorporation of an alkyl group at theC-4 position of the Piperine improved EPI activity and showedmany fold decrease in the MIC of the drug [25e28]. These obser-vations prompted us to question the effect of substitution of anethyl group at position C-4 on EPI activity if it is fused to the C-6position of the phenyl ring of the molecule. We found that thefusion leads to the generation of a dihydronaphthyl ring system(Fig. 1). Therefore, preparation of 3-(dihydronaphthyl)-propenoicacid amides and their subsequent evaluation for EPI activityappeared to be a viable option.

Evaluation of compounds 5e10 in combination with cipro-floxacin against bacterial pathogens S. aureus 1199 and 1199Bresulted in the identification of compounds 8 and 10 as EPIs specificto S. aureus 1199B but non-potentiating against the parentalS. aureus 1199 strain, which is not surprising keeping in mind thatS. aureus 1199B is a NorA-overexpressing mutant. Since thebioevaluation studies revealed low EPI activity of 3-(3,4-dialky-lated-dihydronaphthalene)-2-propenoic acid amides, (Table 1),further modifications were attempted in a bid to improve upon theEPI activity and fifteen more derivatives were synthesizedfollowing route A, B and C with a-tetralone 11 as the startingmaterial (Scheme 2).

The efflux pump inhibitory activity of compounds 19e23 wastested in combinationwith ciprofloxacin against S. aureus 1199 and1199B. Bioevaluation results revealed low potentiating effectagainst S. aureus 1199 showing MIC reductions of only two tofourfold (Table 1), but showed significant EPI activity against S.aureus SA-1199B. Sixteen fold reduction of the minimum inhibitoryconcentration of ciprofloxacin was observed for the compounds 20and 21 (MIC 0.5 mg/mL), an eightfold reduction for compounds 19and 22 (MIC 1.0 mg/mL), and fourfold reduction for compound 23(MIC 2.0 mg/mL) against the pathogen S. aureus 1199B. Compounds24e33 when tested against the test pathogens showed eight foldMIC reduction for compound 24 (MIC 1.0 mg/mL), fourfold forcompound 29 (MIC 2.0 mg/mL) and the rest proved to be poor ornon-potentiator of the antibiotic ciprofloxacin (Table 1). From thesestudies, it was observed that replacement of hydrogen atom/s bymethoxy/allyloxy substituents at the 6,7-position of 3,4-dihydronaphthalene system leads to low activity and/or inactivation of theEPIs, while molecules without a substitution in the partially satu-rated ring of the dihydronaphthyl moiety proved to be betterpotentiators of ciprofloxacin.

To investigate the role of double bonds on the EPI activity, threeEPIs (compound 19, 20 and 24) were selected and hydrogenated(H2/Pd/C) (Scheme 2) to give compounds 34e36. Saturation of thedouble bonds substantially lowered the potentiating activity of theEPIs as shown by the very low MIC reduction observed for thesecompounds (2 fold) against their unsaturated analogs (19, 20 and24) where MIC reduction ranged from 8 to 16 fold, emphasizing,

R1

R2

R

R1

R2

R

Fig. 1. Proposed model for the synthesis of dihydronaphthalene derivatives.

thereby, that the presence of double bonds is essential for theexhibition of high EPI activity (Table 1).

In order to establish that these compounds are NorA inhibitors,we also included S. aureus SA-K1758, in which NorA gene wasknocked out resulting in non- functional NorA protein. Due to theabsence of functional NorA protein, this strain exhibited MIC of0.12 mg/mL, which is 2-fold lower than the MIC of S. aureus 1199(wild type). As expected, no change was observed in the MICof Ciprofloxacin in the absence or presence of these EPI (Includingthe known inhibitors such as Piperine, Reserpine, Verapamil andCarsonic acid) which conclusively established that thesecompounds are NorA inhibitors (Table 1).

The inhibitory mechanism of EPIs was further confirmed by anefflux inhibition assay using ethidium bromide as the substrate ofNorA efflux pump along with the most potent inhibitor 20, theinactive EPI 5 (negative control), and the known S. aureus NorAEPI Reserpine (positive control). These EPIs were tested at theirminimum effective concentration (MEC). All the experimentswere performed in triplicate. Since ethidium bromide fluores-cences only when it is bound to nucleic acids, NorA mediatedefflux resulted in a rapid decrease in fluorescence. As can beobserved from Fig. 2, only those cells not exposed to EPIs or inpresence of the inactive EPI 5 extruded ethidium bromide,resulting in a significant decrease in fluorescence over the courseof the assay. The potent inhibitor 20 effectively preventedethidium bromide efflux.

3. Conclusion

In conclusion, a new series of 24 compounds has beensynthesised and several of them identified as potent EPIs capableof reducing the MIC of Ciprofloxacin by sixteen fold against a NorA-overexpressing strain of S. aureus. SAR studies revealed thatsubstituents such as alkyloxy groups in the phenyl ring of thedihydronaphthyl moiety lower the potentiating activity of the drugdrastically, while substituents such as phenyl and benzyl substit-uents at C-1position in the partially saturated ring of dihy-dronaphthyl moiety show marginally better potentiating activity.Unsubstituted 3-(3,4-dihydronaphthyl)-2-propenoic acid amidesproved to be the most potent EPIs. Unsaturated amides displayedbetter activity than their saturated analogs. The fact that the activeEPIs are targeting the NorA protein of S. aureus was confirmedwhen these were tested against NorA knock out strain (S. aureusSA-K1758), where the loss of target was reflected in no change inthe MIC of ciprofloxacin. The inhibitory effect of these EPIs wasfurther demonstrated by their ability to block the efflux of ethidiumbromide in NorA overexpressing S. aureus strain (1199B).Compounds capable of potentiating the effect of antimicrobialagents may allow use of drugs previously discarded as a result ofresistance. They also may prevent the emergence of de novoresistance by reducing MICs of target organisms. The present studyprovided the basis for further development of robust EPIs ofpotential clinical importance, an effort which has been initiated inour laboratory.

4. Experimental procedure

4.1. Chemistry methods

All reagents for chemical synthesis were obtained from SigmaeAldrich. The starting material eugenol was isolated from lemongrass oil. All the solvents used in reactions were distilled and driedbefore use. All reactions were monitored by TLC on 0.25 mm silicagel 60 F254 plates (E. Merck) using UV light, or ceric sulfate solutionfor visualization of the spots. Silica gel 60e120 mesh was used for

Table 1Activity of Ciprofloxacin against S. aureus 1199B, S. aureus 1199 and NorA knock out S. aureus K1758 respectively in combination with synthesized molecules.

S. No. MECa of EPI S. aureus 1199B MICb of Cipro. (mg/mL) S. aureus 1199 MICb of Cipro. (mg/mL) S. aureus K1758 MICb of Cipro. (mg/mL)

Without EPI With EPI Fold reduction Without EPI With EPI Fold reduction Without EPI With EPI Fold reduction

5 >100 8 8 0 0.25 0.25 0 0.125 0.125 06 >100 8 8 0 0.25 0.25 0 0.125 0.125 07 >100 8 8 0 0.25 0.25 0 0.125 0.125 08 12.5 8 4 2 0.25 0.25 0 0.125 0.125 09 >100 8 8 0 0.25 0.25 0 0.125 0.125 010 12.5 8 4 2 0.25 0.25 0 0.125 0.125 019 25 8 1 8 0.25 0.12 2 0.125 0.125 020 12.5 8 0.5 16 0.25 0.06 4 0.125 0.125 021 25 8 0.5 16 0.25 0.06 4 0.125 0.125 022 25 8 1 8 0.25 0.25 0 0.125 0.125 023 25 8 2 4 0.25 0.25 0 0.125 0.125 024 12.5 8 1 8 0.25 0.12 2 0.125 0.125 025 >100 8 8 0 0.25 0.25 0 0.125 0.125 026 >100 8 8 0 0.25 0.25 0 0.125 0.125 027 >100 8 8 0 0.25 0.25 0 0.125 0.125 028 >100 8 8 0 0.25 0.25 0 0.125 0.125 029 25 8 2 4 0.25 0.25 0 0.125 0.125 030 >100 8 8 0 0.25 0.25 0 0.125 0.125 031 >100 8 8 0 0.25 0.25 0 0.125 0.125 032 >100 8 8 0 0.25 0.25 0 0.125 0.125 033 >100 8 8 0 0.25 0.25 0 0.125 0.125 034 25 8 4 2 0.25 0.25 0 0.125 0.125 035 25 8 4 2 0.25 0.25 0 0.125 0.125 036 25 8 4 2 0.25 0.25 0 0.125 0.125 0Piperine 50 8 4 2 0.25 0.12 2 0.125 0.125 0Reserpine 25 8 2 4 0.25 0.12 2 0.125 0.125 0Carsonic acid 25 8 4 2 0.25 0.12 2 0.125 0.125 0Verapamil 50 8 4 2 0.25 0.12 2 0.125 0.125 0

The bold values are shown for those compounds which have proved to be active EPIs and those in normal font represent inactive/ least significant.1. No antibacterial activity of EPIs was observed at 100 mg/mL that was the highest concentration tested.2. For determining potentiation of Ciprofloxacin, EPI were tested at concentration range of 50e0.8 mg/mL.

a MEC ¼ Minimum effective concentration.b MIC ¼ Minimum inhibitory concentration.

N. Thota et al. / European Journal of Medicinal Chemistry 45 (2010) 3607e36163610

column chromatography. 1H NMR and 13C NMR spectra wererecorded on Bruker DPX 200 and 500 instrument using CDCl3 as thesolvent with TMS as internal standard. Mass spectra were recordedon ESI-esquire 3000 Bruker Daltonics instrument and IR spectrarecorded on Bruker Vector 22 instrument. Melting points wererecorded on Buchi-510 instrument.

Fig. 2. Ethidium bromide efflux inhibition assay, S. aureus 1199B. Cells were loadedwith ethidium bromide efflux was allowed to occur in the absence of EPI (control) or inthe presence of EPIs. Each time point represents the mean log10 �SD of threeexperiments.

4.2. Synthesis

4.2.1. 4-Allyl-1,2-dimethoxy benzene (2)A solution of eugenol (1) (20 g,121.20mmol) and CH3I (18mL) in

dry acetone (100 mL) was refluxed at 60 �C for 24 h. The reactionmixture reduced to one fourth of its volume on a rotavapor, thecontents cooled and diluted with water (150 mL) and extractedwith ethyl acetate (3 � 100 mL). The combined organic layerwashed with water (3 � 50 mL), dried over anhydrous sodiumsulfate and concentrated to give crude product, which on purifi-cation by column chromatography on silica gel with pet. ether60e80 �C as an eluent afforded 4-allyl-1,2-dimethoxy benzene (2)(18.6 g, yield 85.2%) as an oil, Anal. Calcd for C11H14O2: C, 74.13; H,7.92%. Found: C, 75.03; H, 7.99%. MS (%) Mþþ1 at m/z 179. 1H NMR:d 3.22 (2H, d, J ¼ 6.6 Hz, AreCH2), 3.83 (6H, br s, 2 � OCH3),5.05e5.12 (2H, m, CH]CH2), 5.88e6.04 (1H, m, CH2CH]CH2),6.69e6.72 (2H, m, 2� AreH), and 6.85 (1H, d, J ¼ 8.6 Hz, AreH).

4.2.2. 6,7-Dimethoxy-2-formyl-3,4-dihydro-naphthalene (3)To a chilled solution of 4-allyl-1,2-dimethoxy benzene (2)

(18.0 g, 101.12 mmol) in dry DMF (50 mL), POCl3 (26 mL) was addeddropwise at 0e5 �C for 1 h and the resulting mixture was stirred atroom temperature for 90 h. The reaction mixture was poured intoice-cold water (150 mL) and pH 7.0 attained by adding 1 N NaOHsolution. The reaction mixture was extracted with ethyl acetate(5 � 100 mL) and the combined organic layer washed with water(3� 50mL), dried over anhydrous sodium sulfate and concentratedon rotavapour under reduced pressure to give crude product, whichwas purified on silica gel column using pet. ether 60e80 �C:ethylacetate (4:1) as an eluent to afford 3 (6.4 g, yield 26.1%) as a solidmp 101e102 �C. Anal. Calcd for C13H14O3: C, 71.54; H, 6.67%. Found:C, 72.44; H, 6.71%. MS (%) Mþ at m/z 218. 1H NMR: d 2.55 (2H, t,

N. Thota et al. / European Journal of Medicinal Chemistry 45 (2010) 3607e3616 3611

J ¼ 8.3 Hz, AreCH2CH2), 2.82 (2H, t, J ¼ 8.3 Hz, AreCH2CH2), 3.90and 3.93 (3H each, s, 2� OCH3), 6.74 (1H, s, AreH), 6.81 (1H, s,AreH), 7.20 (1H, s, AreCH]), 9.61 (1H, s, CHO). 13C NMR: d 19.26,26.91, 55.86, 55.97, 111.06, 111.58, 125.06, 132.00, 136.92, 145.98,147.65, 150.83, and 191.78.

4.2.3. 3-(6,7-Dimethoxy-3,4-dihydronaphth-2-yl)-propenoic acid(4)

To a stirring benzene solution of ylide (11.5 g) (prepared fromtriphenylphosphine and ethyl bromoacetate) containing sodiumhydride (1.5 g) added benzene solution of 3 (5.5 g, 25.2 mmol) at0e5 �C for 30 min and stirred the contents for 24 h and an addi-tional amount of sodium hydride (0.5 g) was added. The reactionmixture was stirred for 72 h at 40 �C. On cooling, the contents werediluted with ethyl acetate (100 mL) to quench unused sodiumhydride, and then diluted with water (200 mL), organic layerseparated and the aqueous layer extracted with ethyl acetate(2 � 100 mL). The combined organic layer washed with water(2 � 50 mL) and concentrated on a rotavapor under reducedpressure. The crude 3-(6,7-dimethoxy-3,4-dihydronaphth-2-yl)-propenoiate intermediate obtained was without purificationhydrolysed in 10% methanolic potassium hydroxide solution andafter usual work up, the acid obtained was purified on silica gelcolumn using pet. ether 60e80 �C:ethyl acetate (3:1) as an eluent togive compound 4 (5.59 g, yield 85.3%) as a greyish white solid mp147 �C. Anal. Calcd for C15H16O4: C. 69.22; H, 6.2%. Found: C, 70.12;H, 6.27%. MS (%) Mþþ1 atm/z 260. IR (KBr Pellet). 3421, 2997, 2935,1682,1634,1538, 1420,1315,1237,1123,1008, 891, 751, 679 cm�1 1HNMR: d 2.49 (2H, t, J ¼ 8.2 Hz, AreCH2CH2), 2.84 (2H, t, J ¼ 8.2 Hz,AreCH2CH2), 3.88 and 3.91 (3H each, s, 2� OCH3), 5.94 (1H, d,J¼ 15.5 Hz, CH]CHCO), 6.71e6.80 (3H, m, 2� AreH and AreCH]),7.56 (1H, d, J ¼ 15.5 Hz, CH]CHCO).13C NMR: d 22.73, 27.25, 56.03,56.11, 111.12, 114.68, 126.24, 126.92, 127.72, 133.21, 136.26, 147.62,148.38, 149.52, and 172.10.

4.2.4. 3-(6,7-Dimethoxy-3,4-dihydronaphth-2-yl)-propenoic acidpiperidide (5)

To the compound 3-(6,7-dimethoxy-3,4-dihydronaphth-2-yl)-acrylic acid (4) (500 mg, 1.92 mmol) in dry benzene (50 mL) addedfreshly distilled thionyl chloride (2.0 mL excess) and refluxed for1 h. Excess of thionyl chloride removed under reduced pressureand the acid chloride reconstituted in benzene and condensedwith benzene solution of piperidine (1.0 mL), the contents stirredfor 30 min. The organic layer washed with water (2 � 25 mL),dried over anhydrous sodium sulfate and concentrated on rota-vapor to give crude product, which was purified on silica gelcolumn using pet. ether:ethyl acetate (9:1) as an eluent to give thecompound (5) (590 mg, yield 93%) as a brown solid mp 118-119 �C.Anal. Calcd for C20H25NO3: C, 73.37; H, 7.70; N, 4.28%. Found: C,74.21; H, 7.78; N, 4.31%. MS (%) Mþþ1 at m/z 328. IR (KBr Pellet).2998, 2935, 1630, 1587, 1515, 1405, 1236, 1218, 1118, 985, 764,729 cm�1 1H NMR: d 1.60 (6H, br s, eNeCH2(CH2)3), 2.47 (2H, t,J ¼ 8.15 Hz, AreCH2CH2), 2.86 (2H, t, J ¼ 8.15 Hz, AreCH2CH2), 3.59(4H, br s, eN(CH2)2), 3.82 and 3.88 (3H each, s, 2� OCH3), 6.39 (1H,d, J ¼ 15.1 Hz, CH]CHCO), 6.66e6.72 (3H, m, 2� AreH and CH]CeCH), 7.41 (1H, d, J ¼ 15.1 Hz, CH]CHCO). 13C NMR: d 23.16,24.69, 26.18, 27.08, 27.37, 42.29, 45.28, 55.99, 56.01, 110.85, 111.01,114.57, 121.87, 124.49, 133.08, 133.43, 143.80, 147.02, 148.44, and164.44.

4.2.5. 3-(6,7-Dimethoxy-3,4-dihydronaphth-2-yl)-propenoic acidmorpholide (6)

This was prepared in 93% yield from 4 and morpholine by theprocedure as described for compound 5 as a brick red gummymass.Anal. Calcd for C19H23NO4: C, 69.28; H, 7.04; N, 4.25%. Found: C,

70.11; H, 7.10; N, 4.31%. MS (%) Mþþ1 at m/z 330. IR (KBr Pellet).2960, 2926, 1630, 1589, 1455, 1331, 1288, 1259, 1223, 1116, 977, 859,729 cm�1 1H NMR: d 2.47 (2H, t, J¼ 8.2 Hz, AreCH2CH2), 2.82 (2H, t,J ¼ 8.2 Hz, AreCH2CH2), 3.31 (4H, br s, eNe(CH2)2), 3.73 (4H, brs, eOe(CH2)2), 3.82 and 3.89 (3H each, s, 2�OCH3), 6.32 (1H, d,J ¼ 15.1 Hz, CH]CHCO), 6.66e6.69 (3H, m, 2� AreH and CH]CeCH]), 7.66 (1H, d, J ¼ 15.1 Hz, CH]CHCO). 13C NMR: d 23.19,27.39, 56.06, 56.12, 66.93 (4�CH2), 111.01, 111.12, 113.76, 126.84,127.98, 133.22, 134.34, 145.06, 147.30, 148.59, and 164.3.

4.2.6. 3-(6,7-Dimethoxy-3,4-dihydronaphth-2-yl)-propenoic acidn-octylamide (7)

This was prepared in 84% yield from 4 and n-octylamine by theprocedure as described for compound 5 as a brown resinous mass.Anal. Calcd for C23H33NO3: C, 74.36; H, 8.95; N, 3.77%. Found: C,74.97; H, 9.02; N, 3.81%. MS (%) Mþþ1 at m/z 372. IR (KBr Pellet).3240, 3039, 2927, 2855, 1644, 1608, 1521, 1438, 1335, 1292, 1239,1219, 1187, 1033, 899, 861, 824, 760 cm�1 1H NMR: d 0.88 (3H, t,J¼ 6.3 Hz, CH2CH3), 1.22e1.26 (10H, m,eNCH2CH2(CH2)5), 1.54 (2H,br s, eNCH2CH2), 2.42 (2H, t, J ¼ 8.2 Hz, AreCH2CH2), 2.82 (2H, t,J ¼ 8.2 Hz, AreCH2CH2), 3.35 (2H, t, J ¼ 6.30 Hz, eNCH2), 3.82 and3.86 (3H each, s, 2� OCH3), 5.90 (1H, d, J ¼ 15.2 Hz, CH]CHCO),6.64e6.68 (3H, m, 2� AreH and CH]C-CH), 7.41 (1H, d, J¼ 15.2 Hz,CH]CHCO). 13C NMR: d 13.09, 21.63, 22.01, 25.99, 26.10, 26.35,28.22, 28.69, 30.79, 43.66, 54.98, 55.01, 109.83, 110.01, 116.73,125.82, 128.91, 132.07, 132.59, 145.07, 147.35, 148.28, and 169.80.

4.2.7. 3-(6,7-Dimethoxy-3,4-dihydronaphth-2-yl)-propenoic acidisobutyl amide (8)

This was prepared in 88% yield from 4 and isobutyl amine by theprocedure as described for compound 5 as a red gummymass. Anal.Calcd for C19H25NO3: C, 72.35; H, 7.99; N, 4.44%. Found: C 72.99 H8.08 N 4.49%.MS (%)Mþ þNa atm/z 338. IR (KBr Pellet). 3067, 2935,1660, 1599, 1385, 1370, 1217, 1159, 1007, 847, 572 cm�1 1H NMR:d 0.96 (6H, d, J ¼ 6.3 Hz, CH(CH3)2), 1.53e1.89 (1H, m, CH(CH3)2),2.44 (2H, t, J ¼ 8.3 Hz, AreCH2CH2), 2.77 (2H, t, J ¼ 8.3 Hz,AreCH2CH2), 3.17e3.24 (2H, m, eNCH2), 3.82 and 3.89 (3H each, s,2� OCH3), 5.90 (1H, d, J ¼ 15.3 Hz, CH]CHCO), 6.66e6.69 (3H, m,2� AreH and CH]CeCH), 7.88 (1H, d, J ¼ 15.3 Hz, CH]CHCO). 13CNMR: d 20.20, 20.50, 23.21, 27.61, 28.89, 47.21, 56.23, 56.31, 111.24,111.48, 117.24, 128.59, 129.45, 131.64, 132.15, 143.23, 147.42, 148.29,and 165.54.

4.2.8. 4-Allyl-(1-allyloxy)-2-methoxybenzene (2a).This was prepared in 81% yield from 1 and allyl bromide by the

procedure as described for compound 2 as a brown gummy mass.Anal. Calcd for C13H16O2: C, 76.44; H, 7.90%. Found: C, 76.99; H,8.08%. MS (%) Mþ þNa at m/z 205. 1H NMR: d 3.32 (2H, d,J ¼ 6.64 Hz, AreCH2), 3.85 (3H, s, OCH3), 4.59 (2H, d, J ¼ 6.65 Hz,OCH2), 5.02e5.12 (2H, m, AreCH2eCH]CH2), 5.23e5.43 (2H, m,OCH2eCH]CH2), 5.94e6.06 (2H, m, 2� CH2CH), 6.66e6.79 (2H, m,2� AreH), and 6.83 (1H, d, J ¼ 7.8 Hz, AreH).

4.2.9. 6-Methoxy-7-allyloxy-2-formyl-3,4-dihydro-naphthalene(3a)

This was prepared in 23% yield from 2a by the procedure asdescribed for compound 2 as a brown gummy mass. Anal. Calcd forC15H16O3: C, 73.75; H, 6.60%. Found: C, 74.29; H, 6.88%. MS (%)Mþ þNa atm/z 246. 1H NMR: d 2.54 (2H, t, J¼ 8.21 Hz, AreCH2CH2),2.82 (2H, t, J ¼ 8.21 Hz, AreCH2CH2), 3.92 (3H, s, eOCH3), 4.62 (2H,d, J¼ 6.64 Hz, eOCH2), 5.28e5.37 (2H, m, CH]CH2), 5.99e6.16 (1H,m, CH2CH), 6.74 and 6.82 (1H each, s, 2� AreH), 7.18 (1H, s, AreCH),9.61 (1H, s, eCHO). 13C NMR: d 28.67, 39.90, 54.84, 68.74, 105.51,108.14, 117.66, 126.21, 131.30, 131.53, 132.26, 137.73, 148.18, 151.24,and 191.16.

N. Thota et al. / European Journal of Medicinal Chemistry 45 (2010) 3607e36163612

4.2.10. 3-(6-Methoxy-7-allyloxy-3,4-dihydronaphth-2-yl)-propenoic acid (4a)

This was prepared in 60% yield from 3a by the procedure asdescribed for compound 3 as a brick red gummy mass. Anal. Calcdfor C17H18O4: C, 71.31; H, 6.34%. Found: C, 71.79; H, 6.68%. MS (%)Mþ þNa atm/z 287. 1H NMR: d 2.53 (2H, t, J¼ 8.21 Hz, AreCH2CH2),2.82 (2H, t, J ¼ 8.21 Hz, AreCH2CH2), 3.92 (3H, s, eOCH3), 4.67 (2H,d, J ¼ 6.54 Hz, OCH2), 5.28e5.39 (2H, m, CH]CH2), 6.07 (1H, d,J ¼ 15.14 Hz, CH]CHCO), 6.09e6.16 (1H, m, CH2CH), 6.74 (1H, s,AreH), 6.85 (1H, s, AreCH), 7.10 (1H, s, AreH), 7.56 (1H, d,J ¼ 15.14 Hz, CH]CHCO).13C NMR: d 22.63, 27.48, 56.08, 68.59,111.24, 114.86, 116.63, 117.62, 126.45, 129.85, 131.36, 133.31, 136.48,140.74, 148.46, 149.64, and 167.26.

4.2.11. 3-(6-Methoxy-7-allyloxy-3,4-dihydronaphth-2-yl)-propenoic acid piperidide (9)

This was prepared in 67% yield from 4a and piperidine by theprocedure as described for compound 5 as a yellow semisolid. Anal.Calcd for C22H27NO3: C, 74.76; H, 7.70%. Found; C, 75.56; H, 7.98; MS(%) Mþ þNa at m/z 355. 1H NMR: d 1.60 (6H, br s, eNeCH2(CH2)3),2.53 (2H, t, J ¼ 8.31 Hz, AreCH2CH2), 2.82 (2H, t, J ¼ 8.31 Hz,AreCH2CH2), 3.59 (4H, br s, eN(CH2)2), 3.92 (3H, s, eOCH3), 4.67(2H, d, J ¼ 6.54 Hz, eOCH2), 5.28e5.39 (2H, m, CH]CH2), 6.07 (1H,d, J ¼ 15.14 Hz, CH]CHCO), 6.09e6.16 (1H, m, CH2 CH), 6.74 (1H, s,AreH), 6.85 (1H, s, AreCH), 7.10 (1H, s, AreH), 7.56 (1H, d,J ¼ 15.14 Hz, CH]CHCO).13C NMR: d 23.21, 24.43, 25.57, 26.56,27.45, 42.45, 45.38, 55.92, 68.57, 111.19, 114.17, 116.51, 122.37, 126.57,129.34, 132.53, 133.17, 136.47, 140.06, 147.08, 148.28, and 164.53.

4.2.12. 3-(6-Methoxy-7-allyloxy-3,4-dihydronaphth-2-yl)-propenoic acid isobutyl amide (10)

This was prepared in 88% yield from 4a and isobutyl amine bythe procedure as described for compound 5 as a gummymass. Anal.Calcd for C21H27NO3: C, 73.87; H, 7.97; N, 4.10%. Found: C, 74.79; H,8.08; N, 4.49%. MS (%) Mþ þ Na at m/z 342. IR (KBr Pallet). 3066,2929, 1669, 1599, 1385, 1370, 1217, 1159, 1007, 847, 572 cm�1 1HNMR: d 0.96 (6H, d, J ¼ 6.5 Hz, CH(CH3)2), 1.58e1.88 (1H, m, CH(CH3)2), 2.44 (2H, t, J¼ 8.31 Hz, AreCH2CH2), 2.87 (2H, t, J¼ 8.31 Hz,AreCH2CH2), 3.18e3.24 (2H, m, eNCH2), 3.87 (3H, s, OCH3), 4.61(2H, d, J ¼ 6.44 Hz, eOCH2), 5.27e5.38 (2H, m, CH]CH2), 5.90 (1H,d, J ¼ 15.3 Hz, CH]CHCO), 5.92e6.09 (1H, m, CH]CH2), 6.68e6.71(3H, m, 2� AreH and CH]CeCH), 7.58 (1H, d, J ¼ 15.3 Hz, CH]CHCO). 13C NMR: d 20.13 (2�CH3), 24.58, 26.96, 28.07, 47.51, 56.32,68.91, 111.23, 114.16, 116.93, 121.93, 126.71, 129.69, 132.81, 133.14,136.23, 139.96, 147.21, 148.23, and 164.45.

4.2.13. 1,2,3,4-Tetrahydronaphth-1-ol (12)To a chilled solution of a-tetralone (11) (8.0 g, 54.79 mmol) in

methanol (100 mL), sodium borohydride (500 mg) was added insmall instalments in 2 h maintaining temperature 0e5 �C and afterthe completion of the reaction (monitored by TLC), the volume ofthe contents was reduced to one fifth, diluted with ice-cold waterand extracted with solvent ether, the organic layer washed withwater and dried over anhydrous sodium sulfate, concentrated ona rotavapor under reduced pressure to give 12 (7.1 g, 91.3%). asa colourless viscous mass. Anal. Calcd for C10H12O: C, 81.01; H,8.16%. Found: C, 82.11; H, 8.21%. MS (%) Mþ þNa atm/z 171. 1H NMR:d 1.55e1.88 (4H, m, AreCH2(CH2)2), 2.86 (2H, br s, AreCH2), 4.66(1H, br s, AreCHOH), 6.97e7.15 (3H, m, 3� AreH), 7.25 (1H, d,J ¼ 8.54 Hz, AreH).

4.2.14. 2-Formyl-3,4-dihydronaphthalene (13)To a chilled solution of 12 (7 g, 47.3 mmol) in dry DMF (25.0 mL),

POCl3 (12.0 mL) was added drop wise at 0e5 �C for 1 h and theresulting mixture was stirred at room temperature for 24 h. The

reaction mixture was poured into ice-cold water and pH-7 attainedby adding 0.5N NaOH solution and the reaction mixture extractedwith ethyl acetate. The combined organic layer washed with water,dried over anhydrous sodium sulfate and evaporated on rotavaporto give crude product, whichwas purified on silica gel column usingpet. ether 60e80 �C as an eluent to give 13 (7.2 g, 96.3%) as a paleyellow solid, mp 107e108 �C. Anal. Calcd for C11H10O: C, 83.53; H,6.37%. Found: C, 84.44; H, 6.41%. 1H NMR: d 2.56 (2H, t, J ¼ 8.2 Hz,AreCH2CH2), 2.87 (2H, t, J ¼ 8.2 Hz, AreCH2CH2), 7.18e7.35 (5H, m,4� AreH and AreCH), and 9.66 (1H, s, CHO).

4.2.15. 3-(3,4-Dihydronaphth-2-yl)-propenoic acid (18)To a mixture of 13 (7.0 g, 44.3 mmol) in pyridine (20 mL) and

piperidine (3 mL), malonic acid (9.2 g, 2 eq) was added and thereaction mixture stirred at room temperature for 52 h and thenheated on water bath for 6 h. The contents cooled and diluted withwater and acidified with 5% HCl solution. The resulting precipitatefiltered, washed with water and crystallized in pet. ether:ethylacetate (19:1) to yield 18 (8.1g. 91.4%) as a light brown solid, mp186e189 �C, Anal. Calcd for C13H12O2: C, 77.98; H, 6.04%. Found; C,78.76; H, 6.11%. MS (%) Mþþ1 at m/z 201. 1H NMR: d 2.52 (2H, t,J ¼ 8.24 Hz, AreCH2CH2), 2.92 (2H, t, J ¼ 8.23 Hz, AreCH2CH2), 6.0(1H, d, J ¼ 15.6 Hz, CH]CHCO), 6.81 (1H, s, AreCH]), 7.14e7.23(4H, m, 4� AreH), 7.58 (1H, d, J ¼ 15.6 Hz, CH]CHCO). 13C NMR:d 20.60, 27.32, 116.56, 126.66, 127.40, 127.55, 128.50, 133.50, 135.30,135.46, 136.30, 146.94, and 169.80.

4.2.16. 3-(3,4-Dihydronaphth-2-yl)-propenoic acid piperidide (19)This was prepared in 87% yield from 18 and piperidine by the

procedure as described for compound 5 as a light brown crystallinesolid, mp 183e184. �C. Anal. Calcd for C18H21NO: C, 80.86; H, 7.92;N, 5.24%. Found: C, 81.77; H, 7.99; N, 5.28%. MS (%) (Mþþ1) at m/z268. IR (KBr pellet). 3025, 2928, 1637, 1587, 1438, 1366, 1227, 1192,804, 584 cm�1 1H NMR: d 1.61 (6H, br s,eNeCH2(CH2)3), 2.51 (2H, t,J ¼ 8.23 Hz, AreCH2CH2), 2.89 (2H, t, J ¼ 8.22 Hz, AreCH2CH2),3.49e3.63 (4H, m, 2� eN(CH2), 6.44 (1H, d, J ¼ 15.17 Hz, CH]CHCO), 6.79 (1H, s, AreCH]), 7.13e7.19 (4H, m, 4� AreH), 7.51 (1H,d, J ¼ 15.17 Hz, CH]CHCO). 13C NMR: d 23.02, 24.59, 25.55, 26.67,27.48, 42.38, 45.71, 116.05, 126.62, 127.25, 127.26, 127.90, 133.45,133.93, 135.77, 135.93, 143.70, and 165.70.

4.2.17. 3-(3,4-Dihydronapth-2-yl)-propenoic acid isobutyl amide(20)

This was prepared in 92% yield from 18 and isobutyl amine bythe procedure as described for compound 5 as a colourless solid,mp 141e143 �C. Anal. Calcd for C17H21NO: C, 79.96; H, 8.29; N,5.49%. Found: C, 80.84; H, 8.33; N, 5.55%. MS (%) (Mþþ1) atm/z 256.IR (KBr Pellet). 3138, 3011, 2943, 1675, 1610, 1410, 1336, 1310, 1280,1030, 929, 865, 733, 710, 547 cm�1 1H NMR: d 0.94 (6H, d,J ¼ 6.70 Hz, CH(CH3)2), 1.59e1.91 (1H, m, CH(CH3)2), 2.47 (2H, t,J ¼ 8.21 Hz, AreCH2CH2), 2.88 (2H, t, J ¼ 8.21 Hz, AreCH2CH2),3.17e3.24 (2H, m, eNHCH2), 5.96 (1H, d, J ¼ 15.29 Hz, CH]CHCO),6.73 (1H, s, AreCH]), 7.12e7.21 (4H, m, 4� AreH), 7.43 (1H, d,J ¼ 15.29 Hz, CH]CHCO). 13C NMR: d 20.06 (2� CH3), 22.91, 27.45,28.57, 46.95, 119.33, 126.64, 127.29 (2� CH), 128.05, 133.81, 133.89,135.24, 135.96, 142.29, and 166.27.

4.2.18. 3-(3,4-Dihydronaphth-2-yl)-propenoic acid N,N-diisopropylamide (21)

This was prepared in 90% yield from 18 and N,N-diisopropylamine by the procedure as described for compound 5 as a gummymass. Anal. Calcd for C19H25NO: C, 80.52; H, 8.89; N, 4.90%. Found:C, 81.43; H, 8.93; N, 4.97%. MS (%) (Mþþ1) atm/z 284. IR (KBr Pellet).3017, 2968, 1632, 1591, 1438, 1404, 1337, 1208, 1152, 979, 755,613 cm�1 1H NMR: d 1.33 (12H, br s, 2� CH(CH3)2), 2.50 (2H, t,

N. Thota et al. / European Journal of Medicinal Chemistry 45 (2010) 3607e3616 3613

J ¼ 8.2 Hz, AreCH2CH2), 2.89 (2H, t, J ¼ 8.21 Hz, AreCH2CH2), 3.97(2H, br s, 2� N-(CH), 6.39 (1H, d, J¼ 15.14 Hz, CH]CHCO), 6.68 (1H,s, AreCH]), 7.09e7.19 (4H, m, 4� AreH), 7.40 (1H, d, J ¼ 15.14 Hz,CH]CHCO). 13C NMR: d 20.09 (2� CH3), 20.11 (2� CH3), 22.94,27.51, 46.31, 48.72, 119.43, 126.59, 127.53 (2� CH), 128.11, 133.89,134.03, 135.31, 135.93, 143.10, and 165.27.

4.2.19. 3-(3,4-Dihydronaphth-2-yl)-propenoic acid p-methoxyanilide (22)

This was prepared in 92% yield from 18 and p-methoxy anilineby the procedure as described for compound 5 as a colourlessgummymass, Anal. Calcd for C20H19NO2: C, 78.66; H, 6.27, N, 4.59%.Found: C, 78.70; H, 6.29; N, 4.63%. MS (%) Mþþ1 atm/z 306. IR (KBrPellet). 3065, 2932, 1650, 1607, 1454, 1411, 1273, 1106, 892, 780,554 cm�1 1H NMR: d 2.54 (2H, t, J ¼ 8.21 Hz, AreCH2CH2), 2.88 (2H,t, J ¼ 8.21 Hz, AreCH2CH2), 3.79 (3H, s, eOCH3), 6.12 (1H, d,J ¼ 15.16 Hz, CH]CHCO), 6.73 (1H, s, AreCH]), 6.85 (2H, d,J ¼ 8.9 Hz, 2� Ar0eH), 7.21e7.29 (4H, m, 4� AreH), 7.41 (1H, d,J ¼ 15.16 Hz, CH]CHCO), 7.64 (2H, d, J ¼ 8.9 Hz, 2� Ar0eH). 13CNMR: d 23.44, 27.92, 55.90, 114.64 (2� CH), 119.88, 122.06 (2� CH),127.18, 127.82, 127.93, 128.71, 132.16, 134.93, 135.08, 135.68, 136.54,143.88, 157.73, and 166.23.

4.2.20. 3-(3,4-Dihydronaphth-2-yl)-propenoic acid o-methylanilide (23)

This was prepared in 90% yield from 18 and o-methyl aniline bythe procedure as described for compound 5 as a colourless solid,mp 162e163 �C. Anal. Calcd for C20H19NO: C, 83.01; H, 6.62; N,4.84%. Found: C, 83.08; H, 6.69; N, 4.88%. MS (%) Mþþ2 at m/z 291.IR (KBr Pellet). 3237, 2931, 1651, 1609, 1530, 1488, 1403, 1339, 1160,1045, 750, 591 cm�1 1H NMR: d 2.30 (3H, s, eCH3), 2.51 (2H, t,J ¼ 8.15 Hz, AreCH2CH2), 2.91 (2H, t, J ¼ 8.15 Hz, AreCH2CH2), 6.12(1H, d, J ¼ 15.25 Hz, CH]CHCO), 6.78 (1H, s, AreCH]), 7.07e7.25(8H, m, 4� AreH and 4� Ar0eH), 7.56 (1H, d, J ¼ 15.25 Hz, CH]CHCO). 13C NMR: d 17.81, 22.94, 27.43, 126.70, 126.78 (2� CH),127.35 (2� CH), 127.48 (2� CH), 128.27 (2� CH), 130.43, 133.70,134.82, 135.16, 135.80, 136.08, 144.06, and 165.49.

4.2.21. 1-Chloro-2-formyl-3,4-dihydronaphthalene (14)This was prepared in 93% yield from 11 by the procedure as

described for compound 13 as pale yellowgummymass. Anal. Calcdfor C11H9ClO: C, 68.58; H, 4.71; Cl, 18.40%. Found: C, 69.04; H, 4.72;Cl, 18.41%. MS (%) Mþ at m/z 193. 1H NMR: 2.61 (2H, t, J ¼ 8.21 Hz,AreCH2CH2), 2.85 (2H, t, J ¼ 8.21 Hz, AreCH2CH2), 7.20e7.42 (3H,m, 3� AreH), 7.87 (1H, d, J ¼ 6.6 Hz, AreH), 10.39 (1H, s, CHO). 13CNMR: d 22.71, 27.64, 125.52, 126.34, 127.77, 129.81, 137.58, 138.33,143.37, 148.91, and 192.64.

4.2.22. 1-Phenyl-2-formyl-3,4-dihydronaphthalene (15)To Pd(PPh3)4 (0.6 g, 0.52 mmol) was added a deoxygenated

solution of 14 (1.0 g, 5.2 mmol) in DMF (20 mL). This was followedby addition of a deoxygenated solution of boronic acid (0.952 g,7.8 mmol) in ethanol (10 mL), and then a deoxygenated solutionof aqueous sodium carbonate (4.41g, 41.6 mmol in 16.2 mLwater). The resultant mixture was heated under reflux undernitrogen for 4 h. After allowing the mixture to cool to roomtemperature, it was quenched with water (75 mL) and the organicmaterial was then extracted with CH2Cl2 (3 � 100 mL). Theresultant organic extracts were combined, dried (MgSO4), andfiltered through a celite plug and the excess solvent removedusing a rota vapor. The crude product (an oil) was purified bycolumn chromatography (30% ethyl acetate/hexane) to afford thedesired product 15 (4.80 g, 83%) as a brown solid, mp 110e111 �C.Anal. Calcd for C17H14O: C, 87.15; H, 6.02%. Found: C, 87.18; H,6.05%. 1H NMR: d 2.63 (2H, t, J ¼ 8.23 Hz, AreCH2CH2), 2.91 (2H, t,

J ¼ 8.23 Hz, AreCH2CH2), 6.86 (1H, d, J ¼ 7.6, AreH), 7.10e7.16(1H, m, Ar0eH), 7.24e7.33 (3H, m, 3� AreH), 7.44e7.47 (4H, m,Ar0eH), and 9.68 (1H, s, CHO).

4.2.23. 3-(1-Phenyl-3,4-dihydronaphth-2-yl)-propenoic acid (18a)This was prepared in 70% yield from 18 by the procedure as

described for compound 3 as a light brown gummy mass. Anal.Calcd for C19H16O2: C, 82.58; H, 5.84%. Found: C, 82.62; H, 5.88%. 1HNMR: d 2.64 (2H, t, J¼ 8.23 Hz, AreCH2CH2), 2.90 (2H, t, J¼ 8.23 Hz,AreCH2CH2), 5.99 (1H, d, J ¼ 15.7 Hz, CH]CHCO), 6.70 (1H, d,J ¼ 7.7, AreH), 7.03e7.08 (1H, m, Ar0eH), 7.14e7.21 (3H, m, AreH),7.38e7.41 (4H, m, Ar0eH), 7.49 (1H, d, J ¼ 15.7 Hz, CH]CHCO). 13CNMR: d 21.94, 25.81, 116.64, 123.96, 124.64, 125.13, 125.65, 126.12(2� CH), 126.52, 126.29 (2� CH), 129.91, 133.55, 135.05, 137.11(2� C), 141.15, and 167.76.

4.2.24. 3-(1-Phenyl-3,4-dihydronaphth-2-yl)-propenoic acidpiperidide (24)

This was prepared in 87% yield from 18a and piperidine by theprocedure as described for compound 5 as a brown gummy mass.Anal. Calcd for C24H25NO: C, 83.93; H, 7.34; N, 4.08%. Found: C,83.98; H, 7.38; N, 4.09%. MS (%) Mþ atm/z 343. IR (KBr Pellet). 3020,2959, 2870, 1637, 1586, 1440, 1366, 1143, 1072, 983, 845, 770, 702,665, 615 cm�1 1H NMR: d 1.59 (6H, br s,eNeCH2(CH2)3), 2.64 (2H, t,J ¼ 8.23 Hz, AreCH2CH2), 2.96 (2H, t, J ¼ 8.23 Hz, AreCH2CH2),3.49e3.55 (4H, m, eN(CH2)2), 6.43 (1H, d, J ¼ 15.3 Hz, CH]CHCO),6.70 (1H, d, J ¼ 7.6 Hz, AreH), 7.11e3.16 (1H, m, Ar0eH), 7.18e7.34(8H, m, 3� AreH, 4� Ar0eH and CH]CHCO). 13C NMR: d 24.92,25.07, 25.99, 28.45, 30.11, 43.56, 47.41, 117.58, 126.81, 127.55, 127.92(2� CH), 128.11, 128.77 (2� CH), 130.85 (2� CH), 132.28, 136.86,136.99, 138.40, 141.74, 143.14, and 166.48.

4.2.25. 3-(1-Phenyl-3,4-dihydronaphth-2-yl)-propenoic acidisobutyl amide (25)

This was prepared in 92% yield from 18a and isobutyl amine bythe procedure as described for compound 5 as a yellowish gummymass. Anal. Calcd for C23H25NO: C, 83.34; H, 7.60; N, 4.23%. Found:C, 83.39; H, 7.66; N, 4.28%. MS (%) Mþþ1 at m/z 332. IR (KBr Pellet).3024, 2927, 1642, 1604, 1483, 1440, 1368, 1273, 1195, 928, 786, 729,665, 595 cm�1 1H NMR: d 1.22 (6H, d, J ¼ 6.6 Hz, CH(CH3)2),1.59e1.71 (1H, m, CH(CH3)2), 2.55 (2H, t, J ¼ 8.21 Hz, AreCH2CH2),2.87 (2H, t, J ¼ 8.23 Hz, AreCH2CH2), 3.04 (2H, t, J ¼ 6.37 Hz,eNCH2), 5.92 (1H, d, J¼ 15.14 Hz, CH]CHCO), 6.62 (1H, d, J¼ 7.4 Hz,AreH), 6.91e6.95 (1H, m, Ar0eH), 6.99e7.35 (8H, m, 3� AreH, 4�Ar0eH, CH]CHCO). 13C NMR: d 20.19 (2� CH3), 24.46, 28.07, 28.65,47.03, 120.34, 126.50, 127.23, 127.64, 127.68, 127.90, 128.4 (2� CH),130.50 (2� CH), 131.44, 136.40, 136.69, 137.89, 140.12, 143.33, and166.58.

4.2.26. 3-(1-Phenyl-3,4-dihydronaphth-2-yl)-propenoic acid N,N-diisopropyl amine (26)

This was prepared in 92% yield from 18a and N,N-diisopropylamine by the procedure as described for compound 5 as a lightbrown gummy mass. Anal. Calcd for C25H29NO: C, 83.52; H, 8.13; N,3.90%. Found: C, 83.56; H, 8.19; N, 3.99%. MS (%) Mþþ1 at m/z 360.IR (KBr Pallet). 3059, 2965, 2930, 1632, 1584, 1370, 1260, 1212, 1132,1072, 1044, 928, 844, 782, 731, 667, 615, 570 cm�1 1H NMR: d 1.23(12H, d, J ¼ 6.7 Hz, 2� CH(CH3)2), 2.57 (2H, t, J ¼ 8.24 Hz,AreCH2CH2), 2.88 (2H, t, J ¼ 8.24 Hz, AreCH2CH2), 3.67e3.78 (2H,m, 2� CH(CH3)2), 6.31 (1H, d, J ¼ 15.3 Hz, CH]CHCO), 6.59 (1H, d,J ¼ 7.4 Hz, AreH), 6.97e7.07 (1H, m, Ar0eH), 7.09e7.34 (8H, m, 3�AreH, 4� Ar0eH and CH]CHCO). 13C NMR: d 21.57 (4� CH3), 24.95,28.49, 46.18, 48.41, 120.71, 126.79, 127.51, 127.85, 127.93, 128.00,128.82 (2� CH), 130.83 (2� CH), 131.91, 137.36, 137.38, 138.51,140.28, 143.51, and 166.54.

N. Thota et al. / European Journal of Medicinal Chemistry 45 (2010) 3607e36163614

4.2.27. 3-(1-Phenyl-3,4-dihydronaphth-2-yl)-propenoic acid N,N-diisobutyl amide (27)

This was prepared in 90% yield from 18a and N,N-diisobutylamine by the procedure as described for compound 5 as a gummymass. Anal. Calcd for C27H33NO: C, 83.68; H, 8.58; N, 3.61%. Found:C, 83.74; H, 8.62; N, 3.66%. MS (%) Mþþ1 atm/z 388. IR (neat). 3018,2934, 1633, 1586, 1430, 1352, 1289, 1221, 1160, 1123, 1020, 929, 851,770, 666, 592 cm�1 1H NMR: d 0.93 (12H, d, J ¼ 6.3 Hz, 2� CH(CH3)2), 1.99e2.06 (2H, m, 2� eNCH2(CH), 2.69 (2H, t, J ¼ 8.3 Hz,AreCH2CH2), 3.01 (2H, t, J¼ 8.3 Hz, AreCH2CH2), 3.22e3.26 (4H, m,2� NCH2), 6.48 (1H, d, J ¼ 15.1 Hz, CH]CHCO), 6.74 (1H, d,J ¼ 7.6 Hz, AreH), 7.03e7.06 (1H, m, Ar0eH), 7.08e7.42 (8H, m,3� AreH, 4� Ar0eH, CH]CHCO). 13C NMR: d 20.27 (2� CH3), 20.43(2� CH3), 24.58, 26.96, 28.06, 29.03, 54.91, 56.37, 117.33, 126.42,127.13, 127.55 (2� CH), 127.72, 128.38 (2� CH), 130.43 (2� CH),131.81, 136.50, 136.56, 137.90, 141.40, 143.01, and 167.21.

4.2.28. 3-(1-Phenyl-3,4-dihydronaphth-2-yl)-propenoic acidnaphthyl amide (28)

This was prepared in 92% yield from 18a and naphthyl amine bythe procedure as described for compound 5 as a colourless gummymass. Anal. Calcd for C29H23NO: C, 86.75; H, 5.77; N, 3.49%. Found:C, 86.79; H, 5.77; N, 3.56%. MS (%) Mþþ1 atm/z 402. IR (KBr Pellet).3381, 3057, 2854, 2362, 1648, 1601, 1537, 1499, 1401, 1349, 1295,1249, 1021, 765, 699 cm�1 1H NMR: d 2.67 (2H, t, J ¼ 8.23 Hz,AreCH2CH2), 2.97 (2H, t, J ¼ 8.23 Hz, AreCH2CH2), 6.26 (1H, d,J ¼ 15.68 Hz, CH]CHCO), 6.77 (1H, d, J ¼ 7.5 Hz, AreH), 7.03e7.06(1H, m, Ar0eH), 7.08e7.92 (15H, m, 3� AreH, 4� Ar0eH, 7� Ar00eHand CH]CHCO). 13C NMR: d 23.47, 27.08, 112.53, 119.83, 120.72,124.60, 125.07, 125.22, 125.54, 126.31, 126.75, 126.76, 127.17, 127.43(2� CH), 127.50, 127.63, 129.55 (2� CH), 130.65, 131.79, 133.44,133.25, 135.34, 135.92, 136.97, 141.67, 143.28, and 166.62.

4.2.29. 1-(Benzyl)-tetrahydro-naphth-1-ol (16)To an ethereal solution of Grignard reagent (prepared from

magnesium metal and benzyl bromide), was added a-tetralone(15.0g,102.7mmol) in 1 h and then the reactionmixtureworked upby pouring the contents into 1% aqueous ammonium chloridesolution, the organic layer separated and the aqueous layerextracted with solvent ether (4 � 100 mL). The combined organiclayer washed with water (3� 20mL), dried over anhydrous sodiumsulfate, concentrated on rotavapor to give 16 as a gummy mass,(17.5g, 71.5%), Anal. Calcd for C17H18O: C, 85.67; H, 7.61%. Found: C,85.95; H, 7.69%. 1H NMR: d 1.61e1.67 (2H, m, C-CH2), 1.79e1.97 (2H,m, AreCH2CH2), 2.76e2.82 (2H, m, AreCH2), 3.02 and 3.16 (1H each,d, J¼ 13.7 Hz, Ar0eCH2), 7.22e7.34 (8H, m, 3� AreH and 5� Ar0eH),and 7.60 (1H, d, J ¼ 6.80 Hz, AreH).

4.2.30. 1-Benzyl-2-formyl-3,4-dihydronaphthalene (17)To a chilled solution of 16 (10 g, 42 mmol) in dry DMF (35 mL),

POCl3 (16.2 mL) was added dropwise at 0e5 �C for 1 h and theresulting mixture was stirred at room temperature for 45 h. Thereactionmixturewas poured into ice-coldwater (250mL) containingsodiumacetate (15 g). The contentswere extractedwith ethyl acetate(3�100mL). The organic layerwashedwithwater (3� 50mL), driedover anhydrous sodiumsulfate and concentratedon rotavapor to givecrude product, which was purified on silica gel column using pet.ether 60e80 �C:ethyl acetate (9:1) as aneluent to give yellowgummymass (9.1 g, 87.3%), Anal. Calcd for C18H16O: C, 87.06; H, 6.49%. Found:C, 87.08; H, 6.53%. MS (%) Mþ at m/z 248. 1H NMR: d 2.63 (2H, t,J¼ 8.3 Hz, AreCH2CH2), 2.80 (2H, t, J¼ 8.3 Hz, AreCH2CH2), 4.42 (2H,s, AreCH2), 7.11e7.42 (8H, m, 3� AreH, 5�Ar0eH), 7.44 (1H, d,J ¼ 7.61 Hz, AreH), and 10.30 (1H, s, CHO). 13C NMR: d 20.25, 27.73,32.26, 125.92,126.64, 126.84,127.83 (2� CH),128.00,128.88 (2�CH),129.98, 135.23, 135.39, 138.89, 139.28, 149.92, and 191.22.

4.2.31. 3-(1-Benzyl-3,4-dihydronaphth-2-yl)-propenoic acid (18b)To a mixture of 17 (8.5 g, 29.31 mmol) in pyridine (25 mL) and

piperidine (5.0 mL), malonic acid (7.12 g, 2 eq.) was added, and thereaction mixture stirred at room temperature for 72 h, followed byheating on water bath for 6 h, and the contents cooled, dilutedwith cold water followed by addition of 5% HCl solution. Theprecipitate filtered, dried and crystallized from pet. ether:ethylacetate (97:3) to yield yellow crystalline compound 18b (7.5g,78%), Anal. Calcd for C20H18O2: C, 82.73; H, 6.25%. Found: C, 82.78;H, 6.30%. 1H NMR: d 2.59 (2H, t, J ¼ 8.2 Hz, AreCH2CH2), 2.87 (2H,t, J ¼ 8.2 Hz, AreCH2CH2), 4.18 (2H, s, AreCH2), 6.11 (1H, d,J ¼ 15.4 Hz, CH]CHCO), 7.16e7.32 (9H, m, 4� AreH, 5� Ar0eH),and 8.06 (1H, d, J ¼ 15.4 Hz, CH]CHCO). 13C NMR: d 21.52, 25.39,31.18, 115.54, 122.86, 123.54, 124.03, 124.77, 125.27 (2� CH),125.42, 125.99 (2� CH), 129.81, 132.65, 134.95, 137.01 (2� C),140.25, and 167.26.

4.2.32. 3-(1-Benzyl-3,4-dihydronaphth-2-yl)-propenoic acidpiperidide (29)

This was prepared in 70% yield from 18b and piperidine by theprocedure as described for compound 5 as a light brown gummymass. Anal. Calcd for C25H27NO: C, 83.99; H, 7.61; N, 3.92%. Found:C, 84.08; H, 7.66; N, 3.99%. MS (%) Mþþ1 at m/z 358. IR (KBr pellet)3060, 2936, 2855, 1715, 1585, 1493, 1366, 1270, 1219, 1138, 1121,1076, 969, 852, 752, 622 cm�1 1H NMR: d 1.59 (6H, br s,eNCH2(CH2)3), 2.59 (2H, t, J ¼ 8.24 Hz, AreCH2CH2), 2.88 (2H, t,J¼ 8.24 Hz, AreCH2CH2), 3.47e3.54 (4H, m, 2�eN(CH2), 4.18 (2H, s,Ar0eCH2), 6.68 (1H, d, J ¼ 15.07 Hz, CH]CHCO), 7.09e7.30 (9H, m,4� AreH and 5�Ar0eH), 7.92 (1H, d, J ¼ 15.14 Hz, CH]CHCO). 13CNMR: d 23.54, 23.69, 25.65, 27.11, 28.65, 32.71, 42.23, 46.01, 116.78,124.18, 124.94, 125.57, 126.18, 126.45, 126.90 (2� CH), 127.53(2� CH), 131.11, 131.80, 134.62, 136.13, 136.47, 138.79, and 165.04.

4.2.33. 3-(1-Benzyl-3,4-dihydronaphth-2-yl)-propenoic acidisobutyl amide (30)

This was prepared in 78% yield from 18b and isobutyl amine bythe procedure as described for compound 5 as a brown solid, mp174e176 �C. Anal. Calcd for C24H27NO: C, 83.44; H, 7.88; N, 4.05%.Found: C, 83.91; H, 7.92; N, 4.11%. MS (%) Mþþ1 at m/z 346. IR (KBrPellet). 3282, 3026, 2958, 1643, 1603, 1548, 1452, 1368, 1306, 1274,1220, 1119, 1076, 973, 802, 698, 581 cm�1 1H NMR: d 0.92 (6H, d,J ¼ 6.6 Hz, 2� CH(CH3), 1.65e1.83 (1H, m, CH(CH3)2), 2.55 (2H, t,J ¼ 8.24 Hz, AreCH2CH2), 2.89 (2H, t, J ¼ 8.24 Hz, AreCH2CH2),3.14e3.20 (2H, m, eNCH2), 4.17 (2H, s, Ar0eCH2), 6.08 (1H, d,J ¼ 15.13 Hz, CH]CHCO), 7.07e7.30 (9H, m, 4� AreH and 5�Ar0eH), 7.90 (1H, d, J ¼ 15.13 Hz CH]CHCO). 13C NMR: d 19.24 (2�CH3), 23.53, 27.21, 27.70, 32.81, 46.13, 120.10, 124.35, 125.11, 125.71,126.32, 126.67, 127.01(2� CH), 127.62 (2�CH), 131.93, 134.62,136.30, 137.17, 137.50, 138.85, and 165.57.

4.2.34. 3-(1-Benzyl-3,4-dihydronaphth-2-yl)-propenoic acid N,N-diisopropylamide (31)

This was prepared in 82% yield from 18b and N,N-diisopropylamine by the procedure as described for compound 5 as a light brownsolid, mp 127-130 �C, Anal. Calcd for C26H31NO: C, 83.60; H, 8.37; N,3.75%. Found: C, 83.66; H, 8.39; N, 4.79%. MS (%) Mþþ1 atm/z 374. IR(KBr Pellet). 3291, 3061, 2967,1631,1582,1493,1370,1281,1210,1153,1076,1030, 898, 804, 701, 664, 701, 582 cm�1 1H NMR: d 1.28 (12H, d,J¼6.5Hz,2�CH(CH3)2), 2.58 (2H, t, J¼8.24Hz,AreCH2CH2), 2.90 (2H,t, J¼8.24Hz,AreCH2CH2), 3.76e4.02(2H,m,2�CH(CH3)2), 4.17 (2H, s,Ar0eCH2), 6.55 (1H, d, J¼ 15.07 Hz, CH]CHCO), 7.08e7.24 (9H, m, 4�AreH, and 5� Ar0eH), 7.89 (1H, d, J¼ 15.07 Hz CH]CHCO). 13C NMR:d 19.23 (2� CH3), 19.28 (2� CH3), 23.65, 27.16, 32.71, 46.83, 48.21,117.26, 124.23, 124.93, 125.59, 126.20, 126.45, 126.94 (2� CH), 127.45(2� CH), 131.81, 134.61, 136.22, 136.63, 138.68, 138.79, and 166.21.

N. Thota et al. / European Journal of Medicinal Chemistry 45 (2010) 3607e3616 3615

4.2.35. 3-(1-Benzyl-3,4-dihydronaphth-2-yl)-propenoic acid N,N-diisobutylamine (32)

This was prepared in 70% yield from 18b and N,N-diisobutylamine by the procedure as described for compound 5 as a yellowsolid, mp 146e149 �C, Anal. Calcd for C28H35NO: C, 83.74; H, 8.78; N,3.49%. Found: C, 83.79; H, 8.79; N, 3.56%. MS (%) Mþþ1 at m/z 402.IR (KBr Pellet). 3061, 3025, 2928, 1637, 1587, 1465, 1438, 1386, 1297,1227, 1144, 1120, 1029, 972, 845, 804, 747, 696, 584 cm�1 1H NMR:d 0.91 (12H, d, J ¼ 6.30 Hz, 2� CH(CH3)2), 1.83e2.04 (2H, m,2� NCH2(CH), 2.60 (2H, t, J ¼ 8.3 Hz, AreCH2CH2), 2.88 (2H, t,J ¼ 8.3 Hz, AreCH2CH2), 3.18e3.28 (4H, m, 2� NCH2), 4.17 (2H, s,Ar0eCH2), 6.56 (1H, d, J ¼ 15.13 Hz, CH]CHCO), 7.05e7.22 (9H, m,4� AreH, 5� Ar0eH), 7.91 (1H, d, J¼ 15.13 Hz, CH]CHCO). 13C NMR:d 19.27 (2� CH3), 19.43 (2� CH3), 23.58, 25.96, 27.07, 28.03, 32.76,53.91, 55.37, 117.31, 124.28, 124.98, 125.62, 126.25, 126.51, 126.99(2� CH), 127.51 (2� CH), 131.86, 134.66, 136.27, 136.69, 138.74,138.85, and 166.27.

4.2.36. 3-(1-Benzyl-3,4-dihydronaphth-2-yl)-propenoic acid 30,40-methelenedioxy anilide (33)

This was prepared in 70% yield from 18b and 3,4-meth-elenedioxy aniline by the procedure as described for compound 5as a colourless gummymass. Anal. Calcd for C27H23NO3: C, 79.20; H,5.66; N, 3.42%. Found: C, 79.29; H, 5.69; N, 3.46%. MS (%) Mþ1 atm/z410. IR (KBr Pellet). 3165, 2926, 1648, 1636, 1558, 1501, 1403, 1205,1038, 802, 600 cm�1 1H NMR: d 2.59 (2H, t, J ¼ 8.3 Hz, AreCH2CH2),2.86 (2H, t, J ¼ 8.3 Hz, AreCH2CH2), 4.19 (2H, s, Ar0eCH2), 5.90 (2H,s, eOCH2O-), 6.16 (1H, d, J ¼ 15.12 Hz, CH]CHCO), 6.74 (1H, d,J ¼ 8.2 Hz, Ar00eH), 7.10e7.30 (11H, m, 4� AreH, 5� Ar0eH and 2�Ar00eH), 8.01 (1H, d, J ¼ 15.12 Hz, CH]CHCO). 13C NMR: d 23.24,26.98, 32.57, 101.38, 106.81, 113.03, 117.43, 120.11, 124.19, 124.85,125.49, 126.36, 126.57, 127.13 (2� CH), 127.56 (2� CH), 127.78,131.76, 134.63, 136.32, 136.65, 138.79, 138.83, 144.02, 147.61, and166.57.

4.2.37. 3-(1,2,3,4-Tetrahydronaphth-2-yl)-propanoic acidpiperidide (34)

A mixture of methanolic solution of amide 19 (50 mg) and 5%PdeC (20 mg) was hydrogenated at 30 psi for 5 h and the contentswere filtered, washedwith excess of methanol and concentrated ona rotavapor at reduced pressure to give crude product which on CCover silica gel and elution with pet. ether:ethyl acetate (80:20)afforded compound 34 (77% yield) as a gummymass, Anal. Calcd forC18H25NO: C, 79.66; H, 9.28; N, 5.16%. Found: C, 79.95; H, 9.21; N,5.48%. MS (%) Mþþ1 at m/z 271 1H NMR: d 1.29e1.74 (10H, m,eNCH2(CH2)3 and CH(CH2)2), 1.92e1.96 (1H, m, CH(CH2)2), 2.27(2H, t, J ¼ 7.80 Hz, COCH2), 2.39e2.47 and 2.72e2.85 (4H, m,2� AreCH2), 3.20e3.55 (4H, m, 2� eN(CH2), and 7.05e7.09 (4H, m,4� AreH). 13C NMR: d 24.57, 25.57, 26.59, 27.51, 29.06, 30.92, 31.95,34.11, 36.00, 42.65, 46.68, 124.43, 125.36, 126.28, 127.86, 133.27,135.60, and 170.45.

4.2.38. 3-(1,2,3,4-Tetrahydronapth-2-yl)-propanoic acid isobutylamide (35)

This was prepared in 69% yield from 20 by the procedure asdescribed for compound 34 as a ccolourless gummy mass. Anal.Calcd for C17H25NO: C, 78.72; H, 9.71; N 5.40%. Found: C, 78.84; H,9.83; N, 5.55%. MS (%) Mþþ1 at m/z 261. 1H NMR: d 0.91 (6H, d,J ¼ 6.70 Hz, CH(CH3)2), 1.42e1.78 (5H, m, CH(CH2)2), 1.88e1.93 (1H,m, CH(CH3)2), 2.29 (2H, t, J ¼ 7.40 Hz, COCH2), 2.47e2.56 and2.78e2.89 (4H, m, 2� AreCH2), 3.06e3.12 (2H, m, eNHCH2), and7.12-7-21 (4H, m, 4� AreH). 13C NMR: d 20.13 (2�CH3), 27.64, 28.63,29.14, 30.98, 31.97, 34.17, 36.07, 46.98,124.45,125.39,126.29,127.89,133.29, 135.57, and 170.34.

4.2.39. 3-(1-Phenyl-1,2,3,4-tetrahydronaphth-2-yl)-propanoic acidpiperidide (36)

This was prepared in 66% yield from 24 by the procedure asdescribed for compound 34 as a brown gummy mass. Anal. Calcdfor C24H29NO: C, 82.95; H, 8.41; N, 4.03%. Found: C, 83.18; H, 8.58;N, 4.09% MS (%) Mþþ2 at m/z 349. 1H NMR: d 1.41e1.71 (10H,m, eNCH2(CH2)3 and CH(CH2)2), 1.96e2.08 (1H, m, CH(CH2)2), 2.36(2H, t, J ¼ 9.60 Hz, COCH2), 2.84e2.96 (2H, m, AreCH2), 3.29e3.36(4H, m, eN(CH2)2), 4.17 (1H, d, J ¼ 5.0 Hz, AreH), and 6.99e7.32,(9H, m, 4� AreH, 5� Ar0eH). 13C NMR: d 22.14, 23.51, 24.53, 25.52,28.01, 28.16, 30.27, 37.52, 41.63, 45.71, 48.25, 124.69, 124.95, 125.01,126.67 (2� CH), 127.76, 129.29 (2� CH), 129.59, 135.51, 138.67,142.14, and 170.38.

4.3. Biology

4.3.1. Determination of minimum effective concentration (MEC) ofthe EPIs

The inhibitory potential of the EPIs was tested through thedetermination of MEC of these EPIs against S. aureus 1199 (wildtype), S. aureus 1199B (NorA overexpressing) and S. aureus SAK-1758 (NorA knock out). The MIC of ciprofloxacin was determinedagainst these cultures in MullereHinton broth in the presence ofincreasing amounts of efflux pump inhibitors by broth checker-board synergy method in microtiter plates using two fold serialdilutions [29]. Seven serial dilutions of EPIs (50e0.8 mg/mL) weretested in combination with ten serial dilutions of ciprofloxacin(64e0.12 mg/mL). The plates were incubated at 37 �C for 24 h, andwere read visually after incubation at 37 �C for 24 h. The lowestconcentration well in each row showing no turbidity was recordedas MIC. The minimal concentration of EPI that produced themaximal reduction in ciprofloxacin was recorded as MEC [30].

4.3.2. Efflux mechanism studiesThe mechanism of inhibition of bacterial efflux pumps by these

EPIs was determined by the measure of the levels of ethidiumbromide accumulation and efflux in S. aureus 1199B using themethod described by Brenwald and co-workers [31]. The bacterialsuspension was exposed to 2 mg/mL ethidium bromide in presenceof the most active EPI (20 at 12.5 mg/mL concentration), inactive EPI5 (50 mg/mL) and standard EPI Reserpine (25 mg/mL) for 30 min at37 �C. The cells were pelleted down by centrifugation and re-suspended in fresh buffer. The loss of fluorescence was recorded for30 min at 5 min interval at excitation and emission wavelengths of530 and 600 nm in a spectrophotometer (PerkineElmer modelLS50).

Acknowledgements

The authors thank Prof. G. W. Kaatz of Wayne State UniversitySchool of Medicine, Detroit, Michigan, USA for providing S. aureus1199, 1199B and SA-K1758.

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