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European Journal of Medicinal Chemistry 46 (2011) 3581e3589

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European Journal of Medicinal Chemistry

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

Original article

In-vitro metabolic inhibition and antifertility effect facilitated by membranealteration: Search for novel antifertility agent using nifedipine analogues

Abhijeet Waghmare a, Meena Kanyalkar a, Mamata Joshi b, Sudha Srivastava b,*

a Prin K. M. Kundnani College of Pharmacy, Cuffe Parade, Mumbai 400005, IndiabNational Facility for High Field NMR, Tata Institute of Fundamental Research, Homi Bhabha Road, Mumbai 400005, India

a r t i c l e i n f o

Article history:Received 20 December 2010Received in revised form10 May 2011Accepted 10 May 2011Available online 19 May 2011

Keywords:AntifertilityNifedipineSpermatozoaMotilityMetabolismModel membrane

* Corresponding author.E-mail address: sudha@tifr.res.in (S. Srivastava).

0223-5234/$ e see front matter � 2011 Elsevier Masdoi:10.1016/j.ejmech.2011.05.022

a b s t r a c t

In search of non-hormonal male contraceptives, analogues of nifedipine, which causes reversible infer-tility, have been synthesized and their interaction at molecular level with model membrane has beenprobed. Analogues act differently with respect to their antifertility action. This is achieved by altering thecell metabolism thereby directly affecting the motility which is responsible for fertility. Secondly, thesedrugs bind differently to the interior of the cell-membrane affecting the membrane fluidity, architectureand dynamics. Sulfasalazine and D4 interact to a larger extent and alter the lipid bilayer phase toa hexagonal. D1, D2 and D3 do not have considerable effect. D4 is the most promising candidate as a leadcompound for the development of novel non-hormonal male antifertility agents.

� 2011 Elsevier Masson SAS. All rights reserved.

1. Introduction

Considering the fact that several steroid and peptide hormonesplay an essential role in the process of spermatogenesis, use ofhormonal methods as contraceptives has been the choice for thedevelopment of oral contraceptives. However, looking at its adverseside effects and undesired pathological circumstances [1] such asincrease in the incidence of cancer and hormonal imbalance, thereis a growing interest in the development of non-hormonal oralcontraceptives [2]. This focuses our research towards exploration ofnon-hormonal male contraceptives which might elicit a more rapidonset of infertility as compared to hormonal approaches [3]. Anti-fertility drugs such as gossypol and other compounds isolated fromthe plant Tripterygiumwilffordi have irreversible effects and toxicityissues [4]. Thus, there is a need to develop reversible, non-hormonal antifertility agents with minimal side effects towardssafe and effective contraceptive approach.

Sperm motility plays an important role in normal fertilizationprocess. Ionic fluxes through ion channels are crucial in spermmetabolism, maturation, capacitation and in initiating the processof gamete interaction. Acrosomal reaction of spermatozoa is highly

son SAS. All rights reserved.

associated with L-type calcium channels [5]. The importance ofCaþ2 ions in regulating diverse processes in sperm, includingmotility and acrosome reaction has been reported [6]. Attention toion channels as drug targets for contraception has grown with therealization that there are sperm specific ion channels located on thesperm tail [7].

Nifedipine (N) (Fig. 1(A)), L-type Caþ2 channel blocker antihy-pertensive drug, apart from its well known cardiotherapeuticactivity, is known to have effect on reproductive functions in malerats and is known to cause reversible infertility by altering spermlipid metabolism [8,9]. However, if used as antifertility agent, itsantihypertensive effect will be undesirable [2]. In view of this, wehave selected nifedipine as a prototype lead and synthesized its fouranalogues (D1eD4) (Fig. 1(C)) to carry out comparative analysis atmolecular level. The results have also been comparedwith the anti-inflammatory drug sulfasalazine (S) (Fig. 1(B)), which is also knownto affect acrosome reaction [10]. Thus the side effects of nifedipineand sulfasalazine, primarily known for different therapeutic actionhave been optimized. Also this provides a model of comparison inorder to develop a newer series of antifertility agents with greaterpromise. Analogues of nifedipine have been synthesized by modi-fying the carboxyl ester chain on dihydropyridyl nucleus alongwithsubstitution on aryl nucleus. Structure activity relationship (SAR)suggests that para substitution of 1, 4-dihydropyridine hinderscardio therapeutic activity [11]. To support this hypothesis we have

Fig. 1. Molecular structure of (A) nifedipine, (B) sulfasalazine and (C) nifedipine analogues.

A. Waghmare et al. / European Journal of Medicinal Chemistry 46 (2011) 3581e35893582

synthesized para and ortho analogues to optimize their antifertilityaction. This allows probing the optimum requirement of the groupsfor maximum antifertility action.

Insertion of lipophilic calcium ion antagonists into the spermplasma membrane has been shown to have an inhibitory effect onsperm fertilization [12]. In view of this, we have probed the inser-tion/interaction of thesemolecules intomodel membrane preparedfrom dipalmitoyl phosphatidyl choline (DPPC). The polymorphism,dynamics and thermotropic behavior of the system has beenanalyzed at molecular level using multinuclear NMR and differen-tial scanning calorimetric (DSC) techniques. The antifertility effectof nifedipine, its analogues and sulfasalazine, has been evaluated bymonitoring anaerobic glycolysis (lactate signal, the end product) ofintact sperm cells with time using 13C NMR spectroscopy [13].These combined results are expected to provide basis for selecting

Fig. 2. DSC plots of DPPC (50 mM) incorporated with (N) nifedipine, D1, D2, D3, D4 and (S) s

the best analogue which might act as lead molecule in the devel-opment of anti-fertility agent which is non-hormonal, reversibleand may act like a lipophilic calcium ion antagonist.

2. Results

2.1. AlogP calculation

Hydrophobicity/hydrophilicity of the drug influences thebehavior of a molecule in a living organism, affecting its bioavail-ability, transport, reactivity, toxicity, metabolic stability and manyother properties. We have therefore calculated the AlogP values forthe molecules used in our study using VG method in Marrin SketchVersion 5.2 of ChemAxon software. The AlogP values are nifedipine1.766, D1 2.569, D2 2.327, D3 3.234,D4 2.327 and sulfasalazine 3.463.

ulfasalazine. The additives: lipid molar ratios are (a) 0:100, (b) 1:5, (c) 1:2 and (d) 1:1.

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Thus the hydrophilicity of these molecules decreases in thefollowing order. Nifedipine > D4 ¼ D2 > D1 > D3 > sulfasalazine.From the logP values, it is anticipated that greater the hydropho-bicity higher would be the affinity of the molecules for themembrane.

2.2. Binding studies with MLVs

The apparent binding constants measured for different mole-cules are nifedipine- 0.08434M�1, D1- 0.0203M�1, D2- 0.00338M�1,D3- 0.0109 M�1, D4- 0.5017 M�1 and sulfasalazine- 0.0638 M�1. Theextent of binding is in the order D4 > nifedipine¼ sulfasalazine > D1 ¼ D3 > D2. These results indicate that thesemolecules bind to theMLVswith variable degree of affinity. Thismaybedue to the structural differences anddifferences inpolarity as alsoindicated by LogP values. The analogue D4 exhibits potential withregard to its binding with model membrane structure.

It may be noted that the blood-testis barrier is an importantfactor to consider in studies related to male antifertility agents.Hydrophobic (lipid-soluble) compounds are known to penetratereadily into the somatic cells which form a bloodetestis barrier ascompared to hydrophilic molecules which penetrate slowly [14,15].Further, It is likely that any contraceptive agent that affects spermmotility would influence spermatozoa indirectly through disrup-tion of epididymal epithelial cell function or act directly on thespermatozoa by affecting their enzymes [16].

2.3. DSC studies

The thermotropic aspect of drugelipid interaction has beenstudied using DSC technique by examining the changes in themelting point and the shape of the DSC trace [17e19]. Fig. 2 showsDSC curves of lipid bilayer prepared from DPPC incorporated withvarying concentrations of nifedipine, its analogues and sulfasala-zine. At zero drug concentration (trace a) multilamellar bilayers ofDPPC showa pre-transition at 33.74 �C and amain transition (Tm) at41.34 �C due to themobility of the polar choline head group and thealkyl chain, respectively. On addition of nifedipine, its analoguesand sulfasalazine, at 1:5 M ratios, there is a change in both pre-transition and main-transition temperatures. Noticeably, largerchange in the pre-transition temperature is observed in all the

Fig. 3. Transition temperature (Tm �C) values (average of three different samples), inheating mode, of hydrated DPPC vesicles containing (N) nifedipine, D1, D2, D3, D4 and(S) sulfasalazine, as a function of their mol fractions.

cases as compared to the main transition temperature (pre-tran-sition peak broadens in case of D3 and D4). This indicates that drugsinduce the fluidizing effect due to the binding to polar head groupof the lipid bilayer. Pre-transition peaks broaden at higher molarratios. In addition, decrease in themain transition temperature (Tm)is observed in all the cases with maximum of 4.25 �C in case of D4.These observations may be largely explained in terms of fluidizingeffect due to the introduction of lipophilic molecules into theordered structure of the lipid bilayer [20]. The fluidizing effect is inthe order D4>D2>D1>N>D3> S indicatingmaximum fluidizingeffect by D4 analogue. This indicates the effect of eOH substitutionat ortho or para is larger as compared to -Cl substitution. . It may benoted that the Tm shift elicited by these molecules followed thistrend only for low molar ratio (1:5 drug:lipid molar ratio), buta different behavior is observed at higher drug concentrations. Onincreasing concentrations of these molecules into the DPPC bilayer(trace b-d) (1:2 M ratio or higher), both the transition temperaturestend to shift to a higher value (Fig. 3 (indicated for Tm)). Thisbehavior indicates the type of distribution thesemolecules attain inlipid bilayer, at higher molar ratios. It is well known that attemperatures below the Tm, the drug molecules are less soluble inthe rigid gel phase of the lipid bilayer leading to the aggregation[21]. Therefore, at high concentrations the drug molecules maytend to aggregate with each other leading to their segregation into

Fig. 4. 125.7 MHz 13C NMR spectra of (Nd) nifedipine in DMSOd6, (D1eD4) D1, D2, D3

and D4 incorporated into DPPC unilamellar vesicles (1:5 additive: lipid molar ratio) (C)DPPC (50 mM) unilamellar vesicles. All experiments are at 323 K.

Fig. 5. Representative 2D NOESY spectrum of DPPC unilamellar vesicles incorporated with Nifedipine (1:5 N:lipid molar ratio). The experiment was carried out with a mixing timeof 400 ms.

A. Waghmare et al. / European Journal of Medicinal Chemistry 46 (2011) 3581e35893584

isle-like clusters [22]. As a result these drug molecules start oozingout of from the homogeneous lipid-drug dispersion, causing anincrease of Tm value towards the values shown for unperturbed/ordered bilayer structure of pure DPPC liposomes [23]. However itis observed that the extent of this effect varies with the type ofsubstituents present in the drug molecular structure. This clearlydemonstrates that the type of substituents play an important rolein the stabilization of membrane.

2.4. NMR experiments

2.4.1. 13C NMRTo get insight into the nature of the intermolecular interactions

between nifedipine, its analogues and sulfasalazine with the lipidmembrane, 13C NMR experiments on these molecules incorporatedinto the lipid bilayers have been carried out (Fig. 4). The spectrumof the DPPC bilayers (Fig. 4(C)) has been assigned as reported in theliterature [24]. The spectral features of nifedipine (Fig. 4(Nd)) andits analogs (not shown) are nearly identical except for the signalsarising from the respective substituents. The assignments havebeen made from the multiplicity pattern of the resonances. It isobserved that on incorporating these molecules into lipid bilayersat 1:5 M ratio, the signals fromDPPC remain sharp to a large extent.Also, in case of D2 incorporated into bilayers, some resonances fromD2, in particular from aromatic ring (between 110 and 140 ppm)remain sharp (Fig. 4(D2)) indicating a fast exchange between thebound and free forms [25]. This could be due to the presence of OH

Table 1Intermolecular NOE’s observed from the lipid protons of DPPC unilamellar vesicles with th

DPPC Nifedipine D1 D2

CH3 CH3(13,14) CH3(13,14) e

(CH2)n CH3(13,14), CH3(16) CH3(13,14), CH2(16) CH3(13,

N(CH3)3 CH3(13,14) CH3(13,14) e

CH2b CH3(13,14) CH3(13,14) CH3(13,

CH2a e CH3(13,14) CH3(13,CH b e CH3(13,14), CH2(16) e

group at ortho position in the ring R1 which provides a hydrophiliccharacter and enables an easy access of this molecule to the exteriorof the bilayer. On the other hand, in case of nifedipine (Fig. 4(N))and its analogs D1, D3 and D4 (Fig. 4) on incorporation into thebilayer, all the signals are broadened to baseline. The broadening ofthe signals arises due to an exchange at intermediate time scalebetween the bound and the free form of these molecules. Similarobservation is made in case of sulfasalazine also (figure not shown).Due to the broad nature of the signals it is not possible to measureboth the spin lattice relaxation time (T1) and the spin espinrelaxation time (T2) which is a measure of the overall tumblingbehavior and segmental motion of the molecule [26]. In the fasttumbling range, both the T1 and T2 are large, of the order of a fewseconds. The estimate of T2 from the line widths which is about 100ms indicates that the molecules lose their mobility and becomestrongly bound to lipid bilayer resulting in loss of motionalfreedom. This also indicates an increase in the motional ordering ofthe lipid acyl chain and the head group. The results thus indicatethat binding of thesemolecules to DPPC is greatly dependent on thenature of the derivative. Except D2 which shows different motionalcharacteristic, nifedipine D1, D3, D4 and sulfasalazine show identicalbinding characteristics and strongly bound to the lipid bilayerloosing their motional freedom.

2.4.2. 2D NOESYThe 2D NOESY spectrum contains a wealth of information on

intermolecular interactions as well as the conformation (from

e protons of different molecules incorporated in it. Numbering scheme follows Fig. 1.

D3 D4 Sulfasalazine

CH3(13,14) CH3(13,14) e

14) CH3(13,14), CH(8,12),CH(9,11)

CH3(13,14),CH2(16)

CH(4), CH(20),CH(21), CH(22)

CH3(13,14) e e

14) CH3(13,14), CH(8,12),CH(9,11)

e e

14) CH3(13,14) e e

e e e

Fig. 6. 202.4 MHz 31P NMR spectra of DPPC (100 mM) multilamellar vesicles incor-porated with (C) no drug, (D1eD4) nifedipine analogues (S) sulfasalazine. The addi-tives: lipid molar ratio is 1:2. All experiments are at 323K.

Fig. 8. 125.7 MHz 13C NMR spectrum of spermatozoa obtained from goat epididymis.Spermatozoa were incubated with 1-13C-labeled glucose, and the spectrum wasrecorded after 2 h of glucose addition. The assignments of glucose (a þ b anomers) andlactate signals have been indicated.

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intramolecular NOEs) of the molecules in the lipid bilayer. TheNOESY spectrum of nifedipine incorporated into DPPC unilamellarvesicles as a representative spectrum is shown in Fig. 5. Theassignments for nifedipine and lipid signals are indicated in thefigure along the F2 and F1 dimensions respectively. The intermo-lecular NOE’s observed between the lipid and nifedipine, itsanalogues and sulfasalazine has been presented in Table 1. The dataindicates that molecules show intermolecular interactions with

Fig. 7. Percent sperm motility in cells obtained from the cauda region of goatepididymis in presence of (N) nifedipine, (D1eD4) nifedipine analogues and (S) sul-fasalazine. Open bar- motility count after 1 h, dashed bar- motility count after 2 h ofincubation.

lipid bilayers which includes both hydrophobic as well as hydro-philic. For example, nifedipine, D1 and D3 show NOEs with bothhead as well as tail ((CH2) n-CH3) region of the lipid bilayer. This ismore likely of hydrophobic interaction with the lipid bilayer chainand hydrophilic interaction with the head group. On the otherhand, in case of D2, interactions with both head group (N(CH3)3) aswell as with terminal tail methyl group of lipid are lacking. Incontrast, D4 and sulfasalazine show NOEs only with hydrophobictail region of lipid bilayer. In these two molecules any hydrophilicinteraction is missing. This indicates that D4 and sulfasalazine getburied in the hydrophobic core of the membrane altering thebilayer phase to a larger extent. These results support the obser-vations made in binding studies as well as those in DSCexperiments.

Fig. 9. Rate of lactate production by spermatozoa obtained from goat epididymisincubated with 1.25 mM of (N) nifedipine, (D1eD4) nifedipine analogues and (S) sul-fasalazine. A rising exponential equation was used for the calculation of the rate oflactate production. Inset shows time course of lactate production for (C) control, (N)nifedipine, D4 and (S) sulfasalazine.

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2.4.3. 31P NMR31P NMR spectroscopy is sensitive to local motions and the

orientation of the phosphate group in the membrane [27], makingit a well suited tool for monitoring structural changes and detectingpolymorphism in model membrane [28]. The 31P NMR resonanceline shape is determined by the chemical shift anisotropy (CSA) ofthe phosphate group coupled with the molecular motions near thehead groups [29]. The effect of nifedipine, its analogues and sulfa-salazine on the 31P NMR line shape was measured as a function ofconcentration (Fig. 6) as well as temperature (figure not shown). At1:2 drug:lipid molar ratio, the bilayer characteristics of 31P NMRline shape remain intact for nifedipine, D1, D2 and D3 with a smallchange in CSA parameter as seen by the sharpening of the parallelcomponent at �20 ppm. This is due to the increased motionalfreedom/decreased order of the phospholipid head group. In case ofD4 and sulfasalazine, on the other hand, the peak at 0 ppm sharpenswith increase in intensity. The main peak at �20 ppm broadens.This is characteristic of the transformation of the bilayer phase tothe hexagonal phase [19,30e32]. These results thus indicate thatboth sulfasalazine and D4 tend to transform the original multi-lamellar bilayer phase into hexagonal which is accompanied withdecreased motional freedom/enhanced acyl chain ordering.Hexagonal phase transition leads to dynamic heterogeneityproducing weakening of the mechanical properties i.e. compress-ibility and bending rigidity of the bilayer. The overall changes in the

Scheme 1. Hantzsch synthesis of dihydropyridine analogues of nifedipine using an aryl alde

membrane properties affect the association and binding of theproteins and enzymes and thereby their function [33].

2.5. Sperm motility

Spermmotility of cells was measured at 1 h and 2 h interval. It isobserved that during this time interval cells remain motile so thateffect of drugs can be monitored. The motility of control samplesdecreases to 50% and to 20% after 1 h and 2 h, respectively. Effect ofnifedipine, its analogues and sulfasalazine has been also monitoredfor the same time duration. Results indicate that different mole-cules/drugs render spermatozoa immotile to a different extent(Fig. 7). Nifedipine leads to a complete inhibition after 2 h whereasin case of D4 similar extent of inhibition is achieved in 1 h only.Effect of D1 and D3 is identical about 25%e30% loss in motility. D2has slow effect on motility. These observations point out to the factthat D4 analogue, although is comparable to nifedipine with regardto its effect on cell motility, unlike nifedipine, it may have less or noside effects due to its para substitution.

2.6. Cell metabolism

In NMR spectrum of intact cells and tissues, signals arising frommacromolecules such as proteins, nucleic acids, lipid assembliesand carbohydrates are broad and do not provide useful

hyde, acetoacetic acid ester (ethyl acetoacetate) and ammonia in the molar ratio 1:2:1.

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informations. However, 1H, 13C and 31P signals from the lowmolecular weight compounds are sharp and can be used to look atcell metabolism [13,34]. It is particularly profitable to use 13Clabeled substrates. When cells are fed with labeled substrate, thelabel appears at the corresponding carbon atom in variousmetabolites. In current study, sperm cells have been incubated withglucose labeled with 13C at C-1 position, as substrate. The a,b signals of glucose are observed between 90 and 100 ppm (Fig. 8).13C NMR spectra monitored as a function of time, show decrease inglucose (a and b isomers) and increase in lactate signals (arisingfrom C-3 methyl of lactate) intensities. Experiments performed onspermatozoa incubated with different molecules under investiga-tion, indicate that the rates of glucose consumption and lactategeneration, changes monotonically with an increase in the drugconcentration. A positive correlation between the rate of substrateconsumption and the degree of motility has been established andreported [35]. These results led to important information on theeffect of different substituents on the dihydropyridine aryl nucleusof nifedipine on sperm metabolism and thereby fertility. It isobserved that cells incubated with 1.25 mM drug show a decreasedlactate production (metabolic inhibition) monitored after 2 h(Fig. 9). Metabolic inhibition is observed maximum for D4 amongstall the analogues. A trend of lactate production with time is shownin inset Fig. 9 for threemost active drugs. In all the three cases thereis initial increase in lactate intensity which subsequently decreaseswith time indicating metabolic inhibition.

3. Discussion

The present study indicates that the analogues act differentlywith respect to their antifertility action. This is achieved by firstlyaltering the metabolism (lactate production) which directly affectsthe cell motility which is responsible factor for the cells to be fertile.Secondly, these drugs affect the membrane fluidity as well asarchitecture thereby change in membrane dynamics. This in turnaffects the binding of these molecules to the interior of cell-membrane resulting in cell damage with regard to the fertility.

It is observed from NOESY experiments that both sulfasalazineand D4 bind to the interior of the lipid bilayer membrane, dis-turbing the strong hydrophobic interactions between the lipidmolecules. On the other hand, the broadening of peak and loweringof transition temperature in the DSC trace demonstrates that boththe size and packing of bilayers are altered, and the systembecomes disordered. Both these molecules affect the cell motilitydrastically. Our studies also indicate that D4 in parallel with sulfa-salazine interacts with the lipid bilayer to a larger extent ascompared to nifedipine and changes the lipid bilayer phase tohexagonal phase. On the other hand, the DSC and 31P NMR resultshighlight that D1, D2 and D3 analogs of nifedipine do not disturb thebilayer phase of the MLVs.

These results are further supported by 13C NMR results on in-vivo metabolism where sulfasalazine and D4 are found to inhibitlactate production in much more effective manner/to a largerextent than nifedipine. Thus, it can be seen that sulfasalazine andthe D4 analogue of nifedipine are analogous with respect to theiraction on antifertilty activity enhancement. These results are wellsupported by the predictions for sulfasalazine which is known tohave antifertility action by decreasing the epididymal weight [36].The D3 analog is less effective but comparable to nifedipine. D1 andD2 are the least effective.

Further it may be noted that the para substituted analogues ofnifedipine show better promise than the ortho substitutedanalogues. Interestingly it is reported that para substitution ondihydropyridine compounds decreases the cardiotherapeuticactivity [11]. Our studies using DSC, NMR and in-vivo metabolic

activity testing highlight that para-hydroxy substituted dihy-dropyridine analogue shows better promise as antifertility agentwhich might have less or no cardiotherapeutic effect.

4. Conclusion

Sperm membrane integrity is vital for the process of fertiliza-tion. Disruption of the membrane may lead to decreased spermmotility thereby impairment in fertility. Nifedipine is reported toalter the sperm lipid composition [8,9] thereby causing membranedisordering. Sulfasalazine interacts with the lipid bilayer to a largerextent as compared to nifedipine and changes the lipid bilayerphase to a hexagonal phase. Analogue D4 imparts similar effects.However, D1, D2 and D3 analogues neither have considerable effecton cell metabolism nor on membrane architecture. On the basis ofthese results, it may be concluded that of the four analogues, thepara substituted analogue, D4 is the most promising candidatewhich can act as a lead compound for the development of novelnon-hormonal male antifertility agents.

5. Materials and methods

5.1. Materials

Nifedipine and sulfasalazine were gift samples from Unichemand IPCA Laboratories, India, respectively. 4-Hydroxy benzaldehydewas obtained from LOBA Chemie. All other solvents used in thesynthesis were of LR grade.

5.2. Synthesis of nifedipine analogues

For the synthesis of dihyrdropyridine compounds, Hantzschreaction method was used [37e39]. This involves a multi-component condensation of an aldehyde (1 mol) with a 1, 3-dicarbonyl compound (2 mol) and ammonia by refluxing withmethanol. Four different analogues of nifedipine have beensynthesized by modifying the aryl nucleus and increasing the alkylester side chain without altering the dihydropyridine nucleus. Theroute of synthesis of these molecules is shown in Scheme 1.

Nifedipine analogues D1, D2, D3 and D4 (Fig. 1(C)) were preparedby using benzaldehyde (10.415 g, 0.0982 mol), salicylaldehyde(22.92 g, 0.187 mol), 4-chloro benzaldehyde (11.9 g, 0.0843 mol)and 4-hydroxybenzaldeyde (5 g, 0.0410 mol), respectively, as thestarting materials. The quantities of ethyl acetoacetate andammoniawere varied as per themolar ratios. The resultingmixturewas then refluxed for 7e8 h using methanol as the solvent. Theprogress of reaction was monitored by TLC. The reaction mixturewas filtered and the residue was subjected to washings to removeany water soluble impurities. The solid obtained was recrystallizedusing methanol for D1 and D4 and chloroform for D2 and D3. Thefinal yield of D1 and D2 was 80% and that of D3 and D4 was 77% and70% respectively. The purity of the final compounds was confirmedby physical constants, TLC and structure verification was doneusing IR and NMR spectroscopy.

5.3. DSC experiments

DSC measurements were carried out on differential scanningcalorimeter VP-DSC (Microcal, Northhampton MA, USA). Thesamples were degassed under vacuum before being loaded into thereference and sample cells. A scan rate of 10 �C/h was employed.Data was analyzed with the software ORIGIN provided byMicroCal.All the experiments were carried out in the temperature range20 �Ce60 �C. Repeated scans for the same sample were generallysuperimposable.

A. Waghmare et al. / European Journal of Medicinal Chemistry 46 (2011) 3581e35893588

5.4. NMR experiments

NMR experiments were recorded on BRUKER AVANCE 500 and700 MHz NMR spectrometers. NOESY (Nuclear Overhauser effectspectroscopy) spectra were recorded at 400 ms mixing time usingstandard pulse program [40]. 31P and 13C NMR experiments werecarried out with a relaxation delay of 2 s and broadband protondecoupling. The NMR data was processed with Bruker Topspin 2.1.External standard used was TSP and inorganic phosphate forreferencing 13C and 31P NMR spectra, respectively.

5.5. Sample preparation for NMR and DSC experiments

Multilamellar vesicles (MLV) were prepared using standardprocedure [41] wherein the desired quantity of DPPC and synthe-sized molecules were dissolved in chloroform. The solvent wasthen evaporated with a stream of nitrogen gas so as to deposita thin film on the walls of the container. The last traces of thesolvent were removed using vacuum for 2 h. The lipid film washydratedwith the required amount of D2O; this was then incubatedfor half an hour in a water bath at 50 �C with repeated vortexing.The lipid concentrations were maintained at 100 mM for the NMRand 50 mM for the DSC experiments. Unilamellar vesicles (ULV)were prepared by sonicating the above dispersions with a Bransonsonicator (Model 450) at 50% duty cycles till the solution wasoptically clear.

5.6. Determination of MLV-drug (nifedipine, its analogues andsulfasalazine) binding

Binding constants were determined by the centrifugationmethod [42]. Optical density of 100 mM solution of the drugmolecules was measured using spectrophotometer at a wavelengthrange of 220e400l. MLVs were prepared by varying lipid concen-tration systematically from 0.25 mg/ml to 2.0 mg/ml and fixed drugconcentration of 100 mM. The resulting solutions were incubatedfor 2 h at 50 �C and subsequently transferred into ultracentrifugetubes. Separation of liposomes from the aqueous phase was ach-ieved by centrifugation at 40,000 rpm for 2 h. The drug concen-tration in the supernatant was determined by measuring theoptical density. The fraction of drug bound to liposomes wasdetermined from the difference. The drug-liposome apparentbinding constant (k) was analyzed using the double reciprocal plot.A plot of 1/(fraction bound) vs. 1/(lipid concentration) yieldsa straight line with slope 1/k.

5.7. Measurement of anti-fertility activity by monitoring glycolysis

5.7.1. Cell sample preparationNMR measurements were conducted in real time on viable

sperm under anaerobic conditions. Cells were collected from caudaregion of goat epididymis by gentle mincing and tweezing in Dul-becco buffer. Tissue pieces were removed by allowing the cellsuspension to settle for 5 min. The cells were washed, made intoa pellet by centrifugation, and then suspended in an appropriatequantity of buffer to attain desired concentration.

5.7.2. Motility analysisThe effect of drugs and analogues on sperm motility was

measured using the SandereCramer test [43]. Concentration of thecell was adjusted to 3 � 102 cells/ml by using Dulbecco buffer(control) or with 1.25 mM drug solution. This sample (50 ml) wasplaced on a slide and covered with a cover glass (18 mm � 18 mm)and at least six microscopic fields were examined. Each slide wasevaluated twice. Motility data has been collected at two time

intervals i.e. after 1 h and 2 h of incubation. Sperm motility inhi-bition was expressed as a percentage scale.

5.7.3. Metabolic profile measurementThe 13C NMR experiments were carried out on Bruker 500 MHz

spectrometer using 25,000 Hz spectral width, 60� flip angle and 2srelaxation delay with power gated broadband proton decoupling.Dulbecco buffer with 10% D2O was used for NMR field-frequencylocking. Glucose labeled with 13C at C-1 position was used as thesubstrate for glycolytic reaction. 13C NMR spectra monitored asa function of time show a decrease in glucose signal (substrateconsumption) intensity and an increase in lactate signal (buildup)intensities. The progress of glycolysis has been monitored bydirectly measuring glucose consumption and lactate productionwith time [13,44e46]. The effect of nifedipine, its analogues andsulfasalazine on the metabolism of sperm cells was compared bymonitoring the lactate signal build up with time and thus esti-mating their glycolytic inhibitory potency.

All data were analysed by Anova and Bonferroni tests andexpressed as mean � SEM (n ¼ 6). A value of P < 0.05 wasconsidered statistically significant.

Acknowledgements

The authors thank Unichem and IPCA laboratories for the giftsample of nifedipine and sulfasalazine respectively. The Nationalfacility for High Field NMR located at TIFR is gratefully acknowl-edged for their invaluable support.

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