A New Series of 1,3-Dihidro-Imidazo[1,5-c]thiazole-5,7-Dione Derivatives: Synthesis andInteraction with Ab(25-35) Amyloid Peptide

Pietro Campiglia1, Mario Scrima1, ManuelaGrimaldi1, Giuseppina Cioffi1,Alessia Bertamino2, Marina Sala1,Claudio Aquino2, Isabel Gomez-Monterrey2, Paolo Grieco2, EttoreNovellino2 and Anna Maria D’Ursi1,*

1Dipartimento di Scienze Farmaceutiche, Universit� di Salerno, viaPonte Don Melillo 11c, 84084 Fisciano, Italy2Dipartimento di Chimica Farmaceutica e Tossicologica, Universit� diNapoli ``Federico II'', Via D. Montesano, 49, 80131 Napoli, Italy*Corresponding author: Anna Maria D'Ursi, dursi@unisa.it

Deposition of senile plaques composed of fibrillaraggregates of Ab-amyloid peptide is a characteris-tic hallmark of Alzheimer’s disease. A widelyemployed approach in the study of anti-Alzheimeragents involves the identification of substancesable to prevent amyloid aggregation, or to disag-gregate the amyloid fibrils through a directstructural interaction with the soluble or aggre-gated forms of the peptide. Here, we report thesynthesis of a set of 1,3-dihydro-3,6-disubstituted-imidazo[1,5-c]thiazole-5,7-dione derivatives sup-porting different alkyl, aryl and alkylamine sidechains. The ability of these compounds to interactwith the Ab(25-35) peptide was evaluated usingcircular dichroism, nuclear magnetic resonanceand thioflavin fluorescence spectroscopy. Amolecular model for Ab(25-35)–ligand interactionswas calculated by molecular docking procedures.Our data show that the ability of the synthesizedcompounds to modify the structural behaviour ofAb(25-35) varies as a function of the overall struc-tural features of the ligands rather contributionsfrom specific individual substituents.

Key words: Ab(25-35), b-amyloid peptide, fibril modulators, interac-tion

Received 22 December 2008, revised 18 June 2009 and accepted forpublication 28 June 2009

Alzheimer's disease (AD) is a progressive neurodegenerative disor-der, and is the most common cause of dementia in adults. Themechanisms underlying the disease are still poorly understood, butthe deposition of senile plaques composed of fibrillar aggregates of

amyloid b peptide (Ab) is a characteristic hallmark of the pathologyand is believed to play a fundamental role in disease pathogenesis(1–4). Amyloid plaques are primarily composed of b-amyloid pep-tides (Ab)39-43 amino acids in length. These b-amyloid peptidesare produced by the proteolytic cleavage of amyloid precursor pro-tein (APP) by b- and c-secretases (5). Ab(1-40) and Ab(1-42) areparticularly significant in that they are the main components of theplaques found in the brain tissue of AD patients (6). Depending onconditions, amyloid peptides undergo a conformational transitionfrom random coil or a-helical monomers to the highly toxic b-sheetoligomers, which form the mature fibrils (7–9). The plaques exist inequilibrium with soluble monomers, and increasing evidence showsthat both plaques and soluble monomers are neurotoxic (10).

A widely employed approach in the research of anti-Alzheimeragents involves the identification of substances able to preventamyloid aggregation, or to disaggregate the amyloid fibrils througha direct interaction with either soluble or aggregated peptide (11–14). Stains et al. (15) recently classified a series of structurallyunrelated compounds, such as congo red and analogues, rifampicinand derivatives, tetracyclines, quinones, acridones, benzofurans andquaternary ammonium derivatives, according to their ability to inter-act with Ab conformers involved in oligomerization and ⁄ or fibrilla-tion processes. A selective mode of interaction of these compoundswith soluble oligomers or amyloid aggregates has still not beenclearly established. The development of small molecules able tointeract with amyloid peptides may be a useful tool to investigatethe structural parameters that could lead to Ab stabilization and ⁄ oraggregation.

In this context, we recently reported both a conformational analysisof the synthetic Ab(25–35) segment under several solution condi-tions and the modulation of the conformational behaviour of thisfragment by interaction with nicotine (16,17). Ab(25-35) representsthe biologically active region of Ab, as it includes the shortest frag-ment showing the ability to form large b-sheet aggregates. Thisfragment also preserves the toxicity of the full-length peptide (18–20). More recently, we reported a study of the conformational sta-bility of the Ab(25-35) fragment in the presence of thiazolidinederivatives using circular dichroism (CD) and nuclear magnetic reso-nance (NMR) techniques (21). Thiazolidine-4-carboxylic acid and2-(4-pyridin)-thiazolidin-4-carboxylic acid showed a significant abilityto modify the conformational behaviour of the b-amyloid peptide. Inthis paper, we describe a novel series of 1,3-dihydro-3,6-disubsti-tuted-imidazo[1,5-c]thiazole-5,7-dione derivatives characterized by a

224

Chem Biol Drug Des 2009; 74: 224–233

Research Article

ª 2009 John Wiley & Sons A/S

doi: 10.1111/j.1747-0285.2009.00853.x

higher molecular complexity than thiazolidine-4-carboxylic acid or2-(4-pyridin)-thiazolidin-4-carboxylic acid. The aim of our research isto develop a better understanding of the critical structural require-ments for these molecules' interaction with the Ab(25-35) peptideand its subsequent stabilization and ⁄ or aggregation. In order todetermine how the steric and electronic properties of the substitu-ent at the C-3 and N-6 positions may influence the conformationalstability of the Ab(25-35) peptide, we synthesized several deriva-tives supporting alkyl, aryl, donor and acceptor electron groups atC-3, and (N,N-dimethyl)amino ethyl, (N,N-diethyl)aminopropyl, 2-mor-pholinoethyl and (3,4,5-trimethoxy)benzyl as side chains at N-6. Theability of these compounds to interact with Ab(25-35) peptide wasevaluated using a combined approach based on CD, NMR and spec-troscopy. The results were analysed in terms of molecular interac-tions using molecular dynamic and molecular docking calculations.

Experimental Section

General procedure for the synthesis of 1,3-dihydro-3,6-disubstituted-imidazo[1,5-c]thiazole-5,7-dione derivatives (trans 1-6a–d, cis1a, 2d)Triphosgene (118 mg, 0.4 mmol) was added to a solution of thiazoli-dine derivatives (1–6, 1 mmol) in dry tetrahydrofurane (THF, 25 mL)at room temperature. Then triethylamine (TEA, 0.3 mL, 2 mmol) wasadded dropwise over 5 min, and stirring was continued for 10 addi-tional minutes. Afterwards, a solution of (N,N-dimethyl)ethylenedia-mine (a), or (N,N-dimethyl)propylenediamine (b), or 2-morpholinoethylamine (c), or (3,4,5-trimethoxy)benzylamine (d) (1.1 Eq), in dryTHF (5 mL) and TEA (0.3 mL, 2 mmol) was added, and the mixturewas stirred for 1 h. Then, the reaction mixture was diluted withchloroform, washed with water, dried over sodium sulphate andevaporated to dryness. The residue was dissolved into ethanol(20 mL) and TEA (0.15 mL, 1 mmol) was added to this solution. Thereaction mixture was stirred for 1–3 h at reflux temperature, thenneutralized with 1 N HCl and diluted with chloroform. The organicextract was washed with 10% sodium hydrocarbonate and water,dried over sodium sulphate and evaporated to dryness. Flash chro-matography of the residues, using ethyl acetate or dichloromethaneas eluents yielded the corresponding bicyclic derivatives in eachcase (for analytical and spectroscopic data of all synthesized com-pounds see 'Supporting Information').

Peptide synthesisThe Ab(25-35) amyloid peptide, GSNKGAIIGLM, was manually syn-thesized by conventional solid-phase chemistry using the Fmoc ⁄ t-Bustrategy (22). The Na-Fmoc-protected amino acids were coupledusing 1-hydroxybenzo triazole and O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexa-fuorophosphate (fourfold excess) as couplingreagents in the presence of a sixfold excess of N,N-diisopropylethyl-amine. Peptide-resin cleavage and side-chain deprotection reactionswere carried out in 90% trifluoroacetic acid (TFA), 5% water and5% triisopropylsilane. The resin was filtered and the solution addeddropwise to cold diethyl ether to precipitate the peptide. High-performance liquid chromatographic analysis and purification werecarried out on a Waters 600E (Milford, MA, USA) system controller

using a Jupiter (Phenomenex, Anzola Emilia, Bologna, Italy) C18 col-umn (25 cm · 4.6 cm, 5 l, 300 � pore size) for analytical runs anda Vydac C18 column (25 cm · 10 cm, 5 l, 300 � pore size; Grace,Deerfield, IL, USA) for purification. Linear gradients of CH3CN inwater containing 0.1% TFA were used. The peptide was character-ized on a Finnigan LCQ Deca ion trap instrument equipped with anelectrospray source (LCQ Deca Finnigan, San Jose, CA, USA); thesample was directly infused into the electrospray ionization sourceby using a syringe pump at the flow rate of 5 lL ⁄ min. Data wereanalysed with XCALIBUR software (Thermo Scientific, Waltham, MA,USA).

Sample preparationIt has been shown that a TFA pretreatment renders Ab peptideseasily soluble in aqueous solutions and in organic solvents. TheTFA-treated Ab exhibits the properties of monomeric, random coilstructures and lacks preaggregated material (23). Thus, in order toensure sample reproducibility and removal of aggregated stateswhich can be present, dry peptide was pretreated with neat TFA(from Fluka; St. Louis, MO, USA) for 3 h, followed by 10-fold dilu-tion with MilliQ water and lyophilization (Millipore, Billerica, MA,USA). This procedure was adopted for all CD and NMR samples,immediately before dissolution in the appropriate solvent. Thisprocedure was also adopted before using the seed-dependentextension reaction (24) to prepare Ab(25-35) amyloid fibrils.

CD measurementsCircular dichroism measurements were performed on a JASCOJ-810 spectropolarimeter (Jasco, Cremella, Italy) equipped with athermostated cell holder, using a quartz cell with a 1.0-mm pathlength. Spectra were collected over the wavelength range from260–190 nm with a bandwidth of 2.0 nm and a time constant of8 s. The samples for CD analysis were prepared by dissolvingTFA-pretreated Ab(25-35) in water solution (pH 5) to give a finalpeptide concentration of approximately 100 lM.

The CD spectrum of Ab(25-35) in the absence of ligands wasrecorded for comparison. Circular dichroism spectra of Ab(25-35) inwater solution containing all ligands (1.25 equivalents inwater ⁄ methanol, 50:50 v:v) were recorded over 48 h. Each spectrumwas corrected for the contribution of the buffer containing the pureligand (125 lM in water ⁄ methanol, 50:50 v:v)

For estimation of secondary structure content, CD spectra wereanalysed using the CONTINN algorithm of the DICHROWEB on-lineserver (25).

NMR measurements1H-NMR experiments were performed on a 600 MHz Bruker Avancespectrometer (Milan, Italy) equipped with cryoprobe, at a probe tem-perature of 300 K. The Ab(25-35) peptide was dissolved in 0.5 mL ofH2O ⁄ D2O (90:10 v:v) solution (pH 5) at a concentration of 2 mM. Titra-tion was performed by adding 5 lL of the CD3OD solution containing3a, 3c, 4a, 4c, 5a and 5c compounds (120 mM) to the peptide sam-ple and recording 1H-NMR one-dimensional (1D) spectrum under the

Synthesis and Interaction with Ab(25-35) Amyloid Peptide

Chem Biol Drug Des 2009; 74: 224–233 225

same experimental conditions. After each addition, there was a 20-min wait to ensure equilibrium had been reached. One-dimensionalNMR spectra were recorded in the Fourier mode with quadraturedetection, and the water signal was suppressed by low-power selec-tive irradiation in the homogated mode. Data block sizes were 16 Kpoints in t2. Before Fourier transformation, the time domain data FIDswere multiplied by shifted squared sine bell functions. Double-quan-tum filtered-correlation spectroscopy (DQF-COSY), total correlatedspectroscopy (TOCSY) and nuclear Overhauser effect spectroscopy(NOESY) (26–28) experiments were run in the phase-sensitive modeusing quadrature detection in x1 by time-proportional phase incre-mentation of the initial pulse NOESY. Data block sizes comprised2048 addresses in t2 and 512 equidistant t1 values. Before Fouriertransformation, the time domain data matrices were multiplied byshifted sine (2) functions in both dimensions. A mixing time of 70 mswas used for the TOCSY experiments. The qualitative and quanti-tative analysis of DQF-COSY, TOCSY and NOESY spectra wasobtained using the SPARKY interactive program package (29) (UCSF,San Francisco, CA, USA).

Thioflavin assayFluorescence spectra were recorded using a 2 · 10-mm path lengthquartz cuvette and a Perkin-Elmer LS 55 spectrofluorimeter (Welles-ley, MA, USA) equipped with a thermostated cell holder attachedto a Haake F8 water bath (Karlsruhe, Germany). The excitation andemission wavelengths were 450 and 485 nm, respectively.

Ab(25-35) amyloid fibrils for thioflavin (ThT) assay were preparedusing the seed-dependent extension reaction (24). For the prepara-tion of seeds, monomeric Ab(25-35) was incubated in polymeriza-tion buffer (50-mM sodium phosphate buffer at pH 7.5 and 100-mM

NaCl) for 1 week at 37 �C. The seed fibrils were added at a finalconcentration of 5 mg ⁄ mL to 100-mM monomeric Ab(25-35) in thepolymerization buffer at 37 �C. There was no agitation of solutionduring the fibril formation.

To perform the ThT assay, amyloid fibrils were mixed, sonicatedand left overnight. Then, 12-lL fibril solution containing 1-mM

Ab(25-35) was added to 3 mL of 10-mM phosphate buffer (pH7.4) containing 20 lL of 5.0-mM ThT. Six different aliquots(500 lL) of this solution, each of them containing compounds3a, 3c, 4a, 4c, 5a and 5c (4 lL, 5 mM in methanol ⁄ water5:5), were incubated at 25 �C for 3 h. Single ThT readings weretaken every 10 min; each experiment was run at least threetimes, evidencing small but reproducible increase of fluorescenceintensity. Fluorescence spectra of ThT in buffer phosphate and inamyloid fibril solution were recorded as references. Fluorescencespectra of the amyloid fibril solution containing 3a, 3c, 4a, 4c,5a and 5c without ThT were recorded to exclude possiblefluorescence interference because of the ligands.

Molecular mechanics studiesMolecular mechanics (MM) calculations were performed on a Pen-tium D 3.2 GHz using the macromodel 8.5 software packagea, theMMFFs (Marr Molecular Force Field) force field (30) and water-likeimplicit solvent. Subsequently, the conformational search for a full

exploration of the conformational space was obtained by the molec-ular dynamics (MD) method. A temperature of 750 K was used dur-ing the dynamic simulations and a standard constant temperaturevelocity-Verlet algorithm was used to integrate the equations ofmotions. A constant dielectric term (e = 1), mimicking the presenceof water, was used in the calculations to reduce the artefacts asso-ciated with the absence of solvent. Molecular dynamics runs werecarried out for 20 ns. The last step of the MM studies was charac-terized by obtained geometries optimization by a minimizationmethod using a Polak–Ribiere conjugate gradient (50 000 steps,convergence threshold 0.005 kJ ⁄ mol �).

Quantum mechanical studiesThe best empirical geometries carried out by the MM studies wereoptimized by DFT (Density Functional Theory) calculations at theB3LYP level, using the 6-31G(d) basis set and the GAUSSIAN 03software package (31) (Gaussian Inc., Wallingford, CT, USA). Sub-sequently, the charges of ligands were calculated with the ChelpGmethod (charges from electrostatic potentials using a grid basedsystem) (32) at the B3LYP ⁄ 6-31G(d) level of population analysis. Thecalculated charges were then used for docking calculations.

Docking studiesAUTODOCK 3.0.5 (33) was used for all docking calculations. For all thedocking calculations, a grid box size of 40 · 40 · 52 with spacing of0.303 � between the grid points, centred between 30Ala and 35Metcovering the region. For all the docked structures, all bonds weretreated as active torsional bonds. For an exhaustive exploration ofconformational space, six docking calculations consisting of 256 runswere performed, obtaining 1536 structures (256 · 6). A Lamarkiangenetic algorithm was used for docking calculations. An initial popu-lation of 150 randomly placed individuals, a maximum number of2.5 · 105 energy evaluations, and a maximum number of 2.7 · 104

generations were taken into account. Mutation and crossover ratesused were 0.02 and 0.8, respectively. The obtained data, differing byless than 1.5 � in positional root mean square deviation (RMSD),were clustered together and represented by the result with the mostfavourable free energy of binding. All the 3D models were depictedusing PYTHON software (34) and UCSF CHIMERA software (35). Molecularsurfaces are rendered using maximal speed molecular surface (36).

Results

ChemistryOur synthetic approach involves the condensation of the corre-sponding 3-chlorocarbonyl derived from thiazolidines, with differentamines and subsequent intramolecular lactamization. We selectedrepresentative 2-substituted-thiazolidines containing alkyl, aryl,donor and acceptor electron groups, and the amines (N,N-dimethyl)ethylenediamine (a), (N,N-diethyl)propylenediamine (b),2-morpholinoethylamine (c) and (3,4,5-trimethoxy)benzylamine (d).

Scheme 1 shows the key synthetic strategy that permits the con-struction of substituted bicyclic hydantoins in a general manner.The initial thiazolidines (1–6) were obtained by a condensation

Campiglia et al.

226 Chem Biol Drug Des 2009; 74: 224–233

reaction between L-Cys-OEt and the corresponding aldehyde deriva-tives (37), with 80–90% yields as (2R:2S) epimeric mixtures rangingbetween 60:40 and 40:60 ratios.

The reaction of these thiazolidines with triphosgene (38) in thepresence of TEA was followed by the in situ addition of amines a–

d, yielding the corresponding N-carbamoyl derivatives 1¢a–d to6¢a–d. Preliminary 1H-NMR analysis of the crude products showedthat the reaction of 2-alkyl-thiazolidines (1 and 2) with amines a–d

produced (2R,4R:2S,4R) diastereoisomeric mixtures of the N-carbam-oyl derivatives (1¢a–d, 2¢a–d) in a 2:1 ratio, which were notresolved. The reaction of 2-aryl-thiazolidines 3–6 with the sameamines gave the N-carbamoyl derivatives (3¢a–d to 6¢a–d) with apreferential formation of the (2R,4R) diastereoisomers versus the(2S,4R) isomers (8:1 ratio) (39). The intramolecular cyclization ofthese derivatives was performed in ethanol in the presence of TEA,and the corresponding 1,3-dihydro-3,6-disubstitued-imidazo[1,5-c]thi-azole-5,7-dione derivatives (1a–d to 6a–d) were obtained with50–60% overall yields. The 1H-NMR analysis of the crude productsrevealed different results depending on the nature of the R substit-uents at C-3. Lactamization of the 2-alkyl-N-carbamoyl derivatives(1¢a–d, 2¢a–d) produced initially (trans:cis) diastereoisomeric mix-tures of the bicyclic derivatives 1a–d, 2a–d in variable ratios(7:1–4:1). Interestingly, during the chromatographic resolution pro-cess, the epimerization of cis 1b, 1c, 1d, 2a, 2b and 2c deriva-tives to corresponding trans-isomers was complete. The compoundcis 1a slowly epimerises to trans 1a at room temperature in CDCl3

solution. Under the same conditions and after 30 days, both iso-lated propyl derivative trans and cis 2d were recovered unchanged.Epimerization must proceed through an initial ring opening at C-3and subsequent epimerization ⁄ ring closure, as previously describedby Miskolczi et al. (40). However, the cyclization of the 2-aryl-N-car-bamoyl derivatives (3¢a–d to 6¢a–d) was stereospecific towardsthe respective H-3 ⁄ H-7a trans-isomers (3a–d to 6a–d), and nocorresponding epimers at C-3 (H-3 ⁄ H-7a cis-isomers) were detectedin the reaction mixture. The final compounds 1a–c, 6a–c contain-ing (N,N-dimethyl)aminoethyl, (N,N-dimethyl)aminopropyl and 2-mor-pholinoethyl side chains were obtained as hydrochloride salts aftertreatment of the corresponding free bases with a solution of hydro-chloric acid in diethyl ether.

To confirm the relative configuration of the heterocyclic core struc-tures, extensive NMR studies including COrrelation SpectroscopY(1H COSY), rotating frame overhauser effect spectroscopY (ROESY)(41), heteronuclear single quantum coherence (1H–13C HSQC) (42,43)and heteronuclear multiple bond correlation (1H–13C HMBC) experi-ments were carried out on trans and cis 2d compounds. Here, theassignment of the stereochemistry at C-3 and C-7a positions forboth epimers was attempted on the basis of the significant NOEcorrelations observed in the ROESY 2D spectra (Figure 1). In particu-lar, a significant NOE interaction between H-3 and H-7a protonswas observed for the minor isomer cis 2d. In contrast, magnetiza-tion exchanges among H-3 and H-7a were not observed in com-pound trans 2d.

NHS

COOEt

R

NS

COOEt

R

HN

OR1 NS

N

R

R1

O

O

H

H

1) TriphosgenTEA, THF, rt2) R1-NH2

(a-d)

EtOH, TEA, Δ

3' a-d4' a-d5' a-d6' a-d

HNS

N

R

R1

O

O

H

H

+

trans 1a-dtrans 2a-d

1-6

cis 1acis 2d

1

3

67a

3

1, 2

3-6

NS

COOEt

R

HN

OR1

NS

N

R

R1

O

O

H

H

EtOH, TEA, Δ

H

1

3

67a

trans 3a-dtrans 4a-dtrans 5a-dtrans 6a-d

1' a-d2' a-d

H

1 R = CH3

2 R =

CH3

Cl

NO2

OCH3

OCH3

3 R =

4 R =

5 R =

6 R =

a R1 =

b R1 =

c R1 =

d R1 =

N O

OCH3

OCH3

OCH3

N

N

Scheme 1: Synthesis of1,3-dihydro-3,6-disubstituted-imidazo[1,5-c]thiazole-5,7(3H,6H)-dione derivatives.

Synthesis and Interaction with Ab(25-35) Amyloid Peptide

Chem Biol Drug Des 2009; 74: 224–233 227

As the synthesis starts from L-Cys-OEt, the absolute configurationat C-7a is R. Consequently we assigned the 3S configuration tocompound trans 2d and 3R to compound cis 2d. In the 1H-NMRspectra, the main difference between 3S- and 3R-epimers wasfound for the chemical shifts of H-3. Thus, H-3 resonance in com-pound cis 2d having a 3R configuration appeared shielded whencompared with the same proton in the 3S-epimer trans 2d

(0.5 ppm). In addition, C-3 resonance in the 3S derivative appearsat higher fields than the corresponding carbon of the 3R-epimer (d:63.7 and 66.8 ppm, respectively). The stereochemical assignment ofcompounds trans 1a–d, 6a–d and cis 1a was made by correlatingtheir 1H- and 13C-NMR spectra with those of trans 2d and cis 2d.

Circular dichroism analysisSmall peptides in solution are present in different conformerpopulations, whose abundance varies according to the conformationalpreferences of the peptide. Circular dichroism spectroscopy is a suit-able technique to measure the composition of conformer populations.

To explore the ability of the imidazo[1,5-c]thiazole-5,7(3H,6H)-dionederivative to modify the conformational preferences of Ab((25-35),CD spectra of the peptide were recorded in water solution in thepresence and absence of all ligands. The CD spectrum of mono-meric Ab((25-35) (100 lM aqueous solution), prepared according to

the Zagorski method (23) (see 'Experimental Section'), was recordedfor comparison (Figure 2). Blank CD spectra of the pure imidazo[1,5-c]thiazole-5,7(3H,6H)-dione derivatives were recorded in metha-nol ⁄ water solution (50:50, v:v). Final CD spectra of Ab((25-35)ligands were obtained after subtraction of the blank spectra for thepure ligands.

As is evident from the CD spectrum of Ab(25-35) in water (Figure 2),the most common conformation of Ab(25-35) under the examinedconditions is random coil (57% of the total conformer population asshown in Table S1). Circular dichroism spectra recorded after theaddition of compounds 3a, 3b, 3c, 4a, 4b, 4c, 5a, 5b and 5c

indicate that these compounds modify Ab(25-35) conformationalpreferences. (Table S1). Figure 2 shows the CD spectra of Ab((25-35) recorded in the presence of compounds 3a, 4a and 5a. Thesecompounds were chosen as representative of series 3, 4 and 5,

respectively because, according to the qualitative and quantitativeevaluation of the CD curves, molecules belonging to the same ser-ies a–d, induce similar conformational changes. (Table S1). Analysisof CD spectra shows that the addition of compounds 4a–c

decreases the fraction of Ab((25-35) in a random coil conformationand increases the b-strand population (Table 1 b-strand populationvaries from 14% in the absence of compound 4a to �40% in itspresence). Addition of compounds 3a–c featuring a 4-CH3-phenylreduces the population of random coil conformers and populatesb-turn and b-strand conformations. The presence of compounds car-rying electron-rich moieties, such as 4-NO2-phenyl in derivatives5a–c, reduces the fraction of b-strand conformers while stabilizingturn-helical and random coil structures.

The derivatives containing an alkylic function at C-3 (trans 1a–c,cis 1a and trans 2a–c), as well as compounds 6a–c carrying adisubstituted phenyl group at the same position, were not effectivein modifying the Ab((25-35) CD spectra.

–30

–25

–20

–15

–10

–5

0

5

10

15

190 210 230 250

Wavelength (nm)

Mea

n r

esid

ue

ellip

icit

y d

eg c

m2 /

dm

ol

Ab(2535)_water

Ab(2535)_3a

Ab(2535)_4a

Ab(2535)_5a Figure 2: CD spectra ofAb(25-35) in water pH5, recordedin absence (yellow) and presenceof compounds 3a (grey), 4a

(black), 5a (red).

(S)N(R)

S

N

O

O

H

H

trans 2d cis 2d

OMe

OMe

OMe (R)N(R)

S

N

O

O

H

H

OMe

OMe

OMe

Figure 1: Significant NOEs observed for compounds 2d.

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228 Chem Biol Drug Des 2009; 74: 224–233

Finally, CD spectra of Ab(25-35) in aqueous solution are notmodified by the addition of compounds trans 1d–6d or the diaste-reoisomer cis 2d, which is characterized by the presence of a3,4,5-trimethoxyphenyl ring at position N-6. For these compoundsan underestimation of the conformational effects should be hypo-thesized because of the low solubility of these compounds underthe experimental conditions.

Based on the similar conformational behaviour and the high struc-tural similarity of compounds belonging to series a and b, respec-tively featuring a N,N-(dimethyl)ethyl and a N,N-(diethyl)propylgroup at N-6, we decided to continue the NMR, thioflavin fluores-cence analysis and molecular modelling studies on compounds 3a,3c, 4a, 4c, 5a and 5c.

NMR analysisIn order to explore the possibility that 1,3-dihydro-3,6-disubstituted-imidazo[1,5-c]thiazole-5,7-dione derivatives could bind to the Ab(25-35) peptide, we used 1D and 2D 1H-NMR spectroscopy. The solu-tion NMR approach has the advantage of specifically identifyingthe interaction sites between peptides and small molecules by mon-itoring chemical shift modifications. In our case, we monitoredthese modifications following addition of increasing amounts of 3a,3c, 4a, 4c, 5a and 5c derivatives to Ab(25-35) (see 'ExperimentalSection').

1D and 2D proton spectra were recorded in aqueous solution of1 mM Ab(25-35). Complete proton resonance assignments wereachieved by the Wuthrich procedure (44). The systematic analysisof DQF-COSY, TOCSY and NOESY (26–28) experiments was car-ried out using the SPARKY software package (29). The protonchemical shifts of Ab(25-35) in water solutions are included inthe supplementary material. A preliminary inspection of the NO-ESY spectra did not show an adequate number of NOE connec-tivities for Ab(25-35) structure calculation; therefore, CD evidencesuggesting the prevalence of Ab(25-35) random coil structure inwater was confirmed. Comparison of the downfield spectralregions of Ab(25-35) in water, recorded in the presence andabsence of the compounds under study, revealed a change inthe chemical shifts of the Ab(25-35) NH peaks after addition ofcompounds 4a and 4c. In particular, the NH protons of 32Ileand 35Met undergo an appreciable high-field shift, suggestingpossible participation of 32Ile and 35Met residues in the interac-tion between Ab(25-35) and the 4a and 4c ligands. No chemicalshift changes were observable upon the addition of the remain-ing compounds.

Thioflavin assaysThioflavin binds rapidly to amyloid fibrils with a dramatic increasein fluorescence at around 485 nm when excited at 455 nm (45–47).

Circular dichroism data showed that compounds 3a, 3c, 4a, 4c,5a and 5c are able to modify the conformational preferences ofAb(25-35). This effect is potentially subsequent to the interaction ofthe ligands with Ab(25-35) soluble monomers. To verify the abilityof the ligands to interact not only with Ab(25-35) monomers but

also with its fibrillar aggregates, fluorescence spectra of thiazoli-dine were recorded in the presence of pre-formed Ab(25-35) fibrils.

Ab(25-35) fibril solutions, containing ThT (5 mM) as a fluorescentprobe, were incubated in the presence and absence of compounds3a, 3c, 4a, 4c, 5a or 5c for 3 h. Fluorescence spectra wererecorded every 10 min. Under these conditions, the addition ofligands 4a and 4c induced 55% and 72% increases in ThT fluores-cence, respectively. The addition of 3a and 3c induced 5% and3% fluorescence increases, whereas 5a and 5c induced 13% and17% fluorescence decreases (Figure 3). At least three similar exper-iments were run, the results shown, are from a single representa-tive experiment.

Ab(25-35) ⁄ ligands binding modeOur previous NMR investigation of Ab(25-35) under different envi-ronmental conditions (16) showed that Ab(25-35) assumes a b-turnconformation which involves 30Ala-33Gly and 32Ile-35Met residueswhile retaining an extended, random coil structure at the N-termi-nus (16). In order to rationalize the secondary structure modifica-tions evidenced by CD and NMR data in the context of molecularinteractions, we carried out docking calculations (AUTODOCK 3.0.5software) (33) of compounds 3a, 3c, 4a, 4c, 5a and 5c on theNMR structure of the Ab(25-35) peptide. The starting conformationsfor docking studies were obtained by MM and quantum mechanicscalculations (see 'Experimental Section').

The Ab(25-35) 3D model was downloaded from the Brookhaven Pro-tein Data Bank (PDB code 1QWP) (16). The partial charges of theligands were calculated at the DFT B3LYP level and using the

600 3600 7200 10 800

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110

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Flu

ore

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ce in

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sity 5a

5c4a4c3a3c

Figure 3: Kinetics of the increase in the fluorescence emissionof ThT (excitation at 450 nm) incubated with self-aggregatedAb(25-35), in the presence and absence of compounds 3a, 3c, 4a,4c, 5a and 5c.

Synthesis and Interaction with Ab(25-35) Amyloid Peptide

Chem Biol Drug Des 2009; 74: 224–233 229

6-31G(d) basis set. The ChelpG method (32) was used for populationanalysis in the subsequent docking calculations.

The dynamic stability of each docked conformation was monitoredby computing the RMSD of the ligands relative to their initialdocked orientation. AUTODOCK 3.0.5 provided well-clustered dockingresults for all compounds. The independent docking runs carried outfor the ligands converged to a small number of different positions('clusters' of results differing by < 1.5 � RMSD). Generally, the top-ranking clusters (i.e. those with the most favourable DGbind) werealso associated with the highest frequency of occurrence, whichsuggested a good convergence of the search algorithm. AUTODOCK

3.05 software found different binding poses for analysed com-pounds, with highly populated clusters (30 ⁄ 1536 on average)and estimated binding free energy ranging between -5.01 and-3.67 kcal ⁄ mol. Significant distances in the 3D docking models,binding energies, docking energies and inhibition constants arelisted in the 'Supporting Information'.

Molecular docking studies indicate that 3a, 3c, 4a, 4c, 5a and5c exhibit common preferential orientation with respect to Ab(25-35) model (Figure 4). They show extended structures interactingwith C-terminal turns of Ab(25-35), via several sites along the30Ala-35Met segment.

An overall analysis of the most representative binding modes indi-cates that substituents at the N-6 position occupy the turn formed by30Ala-33Gly. In particular, N,N-dimethyl or morpholine moieties areclose to (less than 4.0 �) the 30Ala b-methyl, and the ethyl linker isclose to the 33Gly and 34Leu backbone atoms. A 4-substituted phenylring at the 3-position is located within the 32Ile-35Met turn. Thiazoli-dine and imidazolidindione rings, although with different orientations,are in contact with both the 30Ala-33Gly and 32Ile-35Met turns.

The list of short distances, reported in the supplementary material,shows that in Ab(25-35) ⁄ ligand complexes, 32Ile and 35Met (of the

target) and the phenyl ring (of the ligand) participate most in thebinding. This is particularly evident for 4a, 4c, 5a and 5c com-pounds. 3a and 3c bind Ab(25-35) through a predominant partici-pation of the imidazothiazole bicyclic rings.

Molecular dynamics calculations (data not shown) were carried outto explore the conformational preferences of the docked ligands.The dynamic outputs show that compounds 3a, 3c, 4a, 4c, 5a

and 5c have a tendency to assume a more or less extended struc-ture according to the most prevalent relative positioning of the phe-nyl and thiazolidine rings. In particular, compounds 3a and 5a

(Figure 5B) have a less extended structure because of the staggeredarrangement of the thiazolidine and imidazolidindione rings. Com-pound 4a (Figure 5A) preferentially exhibits a flatter structurebecause of the preferential coplanar orientation of the phenyl andthiazolidine rings.

Figure 4: Binding mode of Ab(25-35) peptide and ligands 3a

(violet), 3c (cyan), 4a (green), 4c (yellow), 5a (red), 5c (blue). Thepeptide is represented as a wire model. Van der Waals volume, cor-responding to 30Ala-35Met residues, is displayed as a transparentgrey surface.

Figure 5: (A) Top – Preferential conformations of compound 4a

as derived from molecular dynamic calculations. The phenyl (P) andthiazolidine ring (T) are coplanar with respect to the xy plane. (B)Bottom – Preferential conformations of compound 5a as derivedfrom molecular dynamic calculations. The phenyl (P), thiazolidine (T)and imidazolidindione (I) rings have different orientations withrespect to the xy plane.

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230 Chem Biol Drug Des 2009; 74: 224–233

Discussion

b-Amyloid plaques in the brain are considered to be the hallmarkof AD, and substantial evidence shows that they are the origin ofinflammatory processes which cause the loss of neuronal plasticityand functionality. One of the strategies in fighting AD is the identi-fication of new entities able to reduce amyloid plaque formationthrough efficacious interaction with a b-amyloid peptide (11–14).

We recently demonstrated that the thiazolidine moiety, able to sta-bilize the Ab(25-35) conformation, could be a valuable starting pointto fine-tune interactions with amyloid peptides (21). In the presentwork, we have reported the synthesis of a set of imidazothiazolederivatives and the analysis of their interaction with Ab(25-35)using CD, NMR and ThT fluorescence spectroscopy.

Circular dichroism experiments (Figure 2) show that 3a–c, 4a–c

and 5a–c are able to affect the conformational behaviour ofAb(25-35), depending on the nature of R at the 3-position (seeScheme 1). NMR experiments showed that the ability of compoundsincluding 4-Cl phenyl ring (4a, 4b and 4c) at C-3 to stabilizeAb(25-35) b-structures involves the participation of 32Ile and35Met amide protons.

A great deal of evidence shows that b-amyloid is present as a mix-ture of monomers, oligomers and prefibrillar aggregates. The transi-tion of b-amyloid peptide to the fibril form is a complicatedprocess involving soluble amyloid oligomers in random coil ⁄ turnconformation, or prefibril intermediates in b-strand structure. Bothconformers can be primary toxic species (14,48,49). On these bases,the search for molecules able to interact with amyloid peptideshould take into account the potential for ligands to interact withthe Ab-peptide in the monomeric and ⁄ or the aggregated form(14,48,49). Molecules targeting b-amyloid in the monomeric formcan be studied as anti-aggregating agents, and molecules targetingb-amyloid fibrils can be studied as dis-aggregating agents.

Circular dichroism data proved compounds 3a–c, 4a–c and 5a–c

are able to interact with Ab(25-35) in solution. To verify the possi-bility that these compounds could also bind peptide aggregates, werecorded ThT fluorescence spectra in the presence of Ab(25-35)preformed fibrils. The results of the ThT assays were consistentwith the CD data, demonstrating that compounds 4a and 4c

increased both Ab(25-35) b-strand structures (CD data) and fibrilaggregates (ThT fluorescence). Circular dichroism data indicatedthat compounds 5a and 5c stabilized Ab(25-35) in random coil–turn conformations, and fluorescence assays indicated that thesecompounds decreased fibril aggregates (Figure 3). Compounds 3a

and 3c, which CD spectra indicated favour a similar partition ofconformers, were shown to weakly interact with Ab(25-35) byinducing a slight increase in the b-strand population.

Based on the preliminary CD, fluorescence and NMR data, molecu-lar docking procedures were carried out to build a molecular modelof Ab(25-35)–ligand interaction. The results of docking calculationsshowed that the docked compounds 3a, 3c, 4a, 4c, 5a and 5c,irrespective of the 4-phenyl substituent, are potentially able to bind

Ab(25-35) peptide according to a common preferential bindingmode. Therefore, based on the hypothesis that the interactionbetween the single ligands and Ab(25-35) could be modulated by acomplex mechanism involving more than the contribution of thespecific molecular moieties, we performed molecular dynamicscalculations to explore the conformational properties of the fullmolecules. The results indicate that 4a and 4c, – on the one hand– and 3a, 3c, 5a and 5c – on the contrary – are essentiallydifferent in the shape of their surface (Figure 5A,B). While com-pounds 4a and 4c preferentially exhibit flat surfaces because ofthe prevalent coplanar orientation of phenyl and thiazolidine rings,this planarity is not observable in compounds belonging to series3a, 3c, 5a and 5c where the staggered positioning of thiazolidineand imidazolidindione rings is more typical. Accordingly, these datasuggest a model where the effects of the two series of compounds– 4a, 4c, and 3a, 3c, 5a, 5c – in stabilizing Ab(25-35) b-strandconformers rather than random coil conformations, depends oninteractions involving the full molecular shape of the ligand ratherthan single specific substituents present on the compounds.

Based on the data that extended molecular structures could interactwith aggregates of amyloid in b-structures, catalysing in severalcases the formation of fibrillar aggregates (15), our data suggest amodel wherein a mixture of soluble amyloid oligomers (in randomcoil ⁄ turn conformations) or prefibril intermediates (in b-strand struc-tures) (14,48,49) and imidazothiazole derivatives belonging to the 4

series (exhibiting extended molecular surfaces) preferentially inter-act with prefibrillar aggregate intermediates. This interaction maypromote the formation of new beta fibrils. Imidazothiazole deriva-tives belonging to the 3 and 5 series, which prevalently exhibitnon-planar molecular shape, preferentially interact with soluble pep-tide monomers, possibly by stabilizing random coil–turn conforma-tions and preventing oligomerization and aggregation.

These initial results suggest that the imidazothiazole scaffold couldbe used to explore the structural parameters involved in the modu-lation of the fine conformational equilibrium of Ab peptides.

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Supporting Information

Additional Supporting Information may be found in the onlineversion of this article:

Table S1.

Please note: Wiley-Blackwell is not responsible for the content orfunctionality of any supporting materials supplied by the authors.Any queries (other than missing material) should be directed to thecorresponding author for the article.

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