125I-Radiolabeled Morpholine-Containing ArginineminusGlycineminusAspartate (RGD) Ligand of αvβ3 Integrin As a Molecular ImagingProbe for AngiogenesisFrancesca BianchinidaggerDagger Nicoletta Cinisect Andrea Trabocchi∥ Anna Bottoncettisect Silvia Raspanti

Eleonora Vanzi Gloria Menchi∥ Antonio Guarna∥ Alberto PupiDagger and Lido CalorinidaggerDagger

daggerDepartment of Experimental Pathology and Oncology University of Florence Viale Morgagni 50 50134 Florence ItalyDaggerIstituto Toscano Tumori (ITT) Via T Alderotti 26N 50139 Florence ItalysectAzienda Ospedaliera Universitaria Careggi (AOUC) Largo Brambilla 3 50134 Florence Italy∥Department of Chemistry ldquoUgo Schiffrdquo University of Florence Via della Lastruccia 13 50019 Sesto Fiorentino Florence ItalyInterdepartmental Center for Preclinical Development of Molecular Imaging (CISPIM) University of Florence Viale Morgagni 8550134 Florence ItalyDepartment of Clinical Physiopathology Nuclear Medicine Unit University of Florence Viale Morgagni 85 50134 Florence

S Supporting Information

ABSTRACT In this paper using a hybrid small-animal Micro SPECTCT imaging system we report that a new 125I-Cilengitide-like RGD-cyclopentapeptide containing D-morpholine-3-carboxylic acid interacts in vivo with αvβ3 integrinexpressed by melanoma cells Images clearly show that the 125I-compound has the capacity to monitor the growth of a melanomaxenograft Indeed retention of the labeled ligand in the tumor mass has a good tumorbackground ratio and a significantreduction of its uptake was observed after injection of unlabeled ligand These results suggest that the use of 125I-labeledmorpholine-based RGD-cyclopentapeptides targeting αvβ3 positive tumors may play a role in future therapeutic strategies

INTRODUCTIONAs pointed out by the Food and Drug Agency1 a majorproblem with the health system of western societies is theslowness of drug development This is because of the increasingcost of new drug pipelines which at present exceeds 15 billiondollars2 Use of biomarkers as molecular imaging tools in earlynonclinical and clinical development provides a more informedscientific basis for the design of pivotal trials and has beenalready proven useful in simplifying the development of targettherapies3 Indeed the method implemented in preclinicalphases of drug development by molecular imaging can betranslated into clinical trials In addition using biomarkers inmolecular imaging of small-animal models has become a newimportant scientific field because the animals can besequentially used but not sacrificed At the same time newmolecular imaging technology is extending the sensitivity fordefining treatment strategies Among these strategies newtumor therapies include Cilengitide a RGD (arginineminusglycineminusaspartic acid) mimetic integrin inhibitor with anti-

angiogenic property which is under investigation in phase II4

and III5 for the treatment of glioblastomaIntegrins are heterodimeric glycoprotein transmembrane

receptors consisting of various combinations of α and βsubunits These receptors regulate biological processes such asapoptosis cell adhesion and migration cell differentiation andangiogenesis and are key points to determine invasiveness andmetastatic dissemination of tumor cells6

Integrins on the surface of tumor cells are quite specific anddictate tumor progression High expression of integrinscharacterizes various malignant tumors such as breast andprostate tumors ovarian carcinoma glioblastoma and malig-nant melanoma During the transition from a benignmelanocytic lesion to a metastatic malignant melanomamelanocytes undergo a series of changes in the expression ofcell-surface integrins Among these integrins the vitronectin

Received November 30 2011Published May 23 2012

Article

pubsacsorgjmc

copy 2012 American Chemical Society 5024 dxdoiorg101021jm2016232 | J Med Chem 2012 55 5024minus5033

receptor αvβ3 plays a fundamental role in melanoma growthand dissemination αvβ3 integrin overexpression is associatedwith transition of melanoma cells from the radial to verticalgrowth phase and characterizes cells at the invasive front of atumor7 In particular αvβ3 integrin favors the localization ofMMP-2 on the surface of invasive tumor cells8 and it has beendemonstrated that the expression of membrane type-1 MMPon the cell surface requires αvβ3 integrin

9 A recent analysis ofthe gene-expression profile of 34 vertical growth phase ofhuman melanoma indicates that proteins of epithelial-mesenchymal transition (EMT) program namely αvβ3 integrinand MMP-2 are significantly correlated with the metastaticdisease10

It is noteworthy that the expression of αvβ3 integrin is alsocharacteristic of endothelial cells of tumor vasculature andparticipates in tumor angiogenesis Recently together with αvβ3integrin an additional RGD integrin the fibronectin receptorα5β1 which plays a role in tumor angiogenesis has beenfound11

The relevance of RGD-dependent integrins in tumorprogression has stimulated the search of RGD mimetics totarget growth and dissemination of cancer cells and to inhibittumor angiogenesis12

The interaction between natural ligands and the RGDrecognition site of integrins can be inhibited by antagonists thatcontain the RGD sequence Inhibition of different RGD-integrins can be modulated by the sequence and the structureof antagonists containing or mimicking the RGD sequenceRecently many efforts have been dedicated in developingPET13 and SPECT14 radiopharmaceuticals addressing the RGDmoietyRegarding radionuclides for molecular imaging 125I is gaining

interest as a suitable SPECT agent because it is a readilyavailable radionuclide with a half-life of 602 days and emissionenergy in the range of 27minus35 KeV 125I-labeled tracers arecommonly used in molecular biology and many 125I-labeledradiopharmaceuticals are commercially available In additionseveral 125I-labeled imaging probes can be prepared with the aid

of commercially available iodination reagents Moleculeslabeled with iodine represent a convenient tool for animalmodels up to humans because substitution of 125I with 123I(159-KeV γ rays with half-life 132 h) provides a probe with apath-length suitable for imaging in humans Furthermoremolecular imaging of iodine-labeled compounds by PET can beaccomplished with the positron emitter 124I and the translationto radionuclide therapy using 131I (emitting β- and γ-rays) canbe easily assessed High-resolution SPECT systems for in vivoimaging of 125I-labeled biomolecules are important fordeveloping new imaging probes for diagnostic and therapeuticapproaches

RESULTS

We recently synthesized and tested a RGD-based cyclo-pentapeptide containing D-morpholine-3-carboxylic acid(Mor) as a replacement for N-methyl-valine as found inCilengitide (Figure 1 compound 1) This compound expressedhigh binding capacity for αvβ3αvβ5 integrins

15 In this work wehave modified compound 1 by replacing a D-Phe with a D-Tyr(compound 2) and introducing an iodine atom on the aromaticring of D-Tyr (compound 3) with the aim of assessing whetherthe modified Cilengitide-like inhibitor 3 could be suitable forSPECT imaging using 125I as the radionuclide We have alsodemonstrated that the morpholine-based RGD-cyclopentapep-tides 1minus3 interact with αvβ3 integrin in vitro and in vivo andthat the 125I-labeled compound 125I-3 may be used to monitormelanoma xenografts with a hybrid small-animal MicroSPECTCT imaging system

Synthesis Compound 2 was prepared as previouslyreported15 with direct labeling with NaI (11 molar ratio)using chloramine-T as the oxidizing reagent in phosphatebuffered saline solution (PBS) The reaction was quenchedwith sodium metabisulfite and the crude product was purifiedby semipreparative HPLC to give compound 3 (47 mg 32)Compound 125I-3 was prepared starting from compound 2 andNa125I (181 molar ratio) (as for compound 3) The crudereaction mixture was purified by preparative HPLC and the

Figure 1 Morpholine-based RGD-cyclopeptides D-Mor-containing compound 1 D-Mor-D-Tyr-containing compound 2 127I-D-Mor-D-Tyr-containing compound 3 125I-D-Mor-D-Tyr-containing compound 125I-3

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radioactivity was monitored with a γ-counter (see Figure S1Supporting Information) The radiolabeling procedure toobtain 125I-3 resulted in a clean iodination reaction with aradiochemical yield of 78 for the monoiodinated peptideIntegrin Binding Assays The ability of cyclopentapeptide

3 to compete with [125I]echistatin for binding to pure αvβ3integrin isolated from human placenta was evaluated in solidphase assays15 IC50 values showed a binding affinity ofcompound 3 toward αvβ3 integrin of 480 plusmn 12 nM slightlylower than that of peptide 1 (IC50 65 plusmn 20 nM) (Figure 2)15

Data analyses indicates that compound 3 binds to αvβ3 integrinaccording to a one-site binding model (Hill slope minus092 forone-site model) whereas cyclopeptide 1 showed binding toαvβ3 integrin according to a two-site binding model (Hill slopeminus056 for one-site model) thus suggesting that the iodo-tyrosine-derived ligand 3 displays a different affinity profiletoward αvβ3 integrin

15

Molecular Modeling To understand the potential differ-ences in binding mode of cyclopeptide 3 to αvβ3 integrin ascompared to ligand 1 a docking simulation was carried out15

The crystal structure of the complex formed by c[RGDf(Me)V]and the extracellular fragment of αvβ3 integrin (PDB code1L5G) provides a general mode of interaction between theintegrin and its ligands16 Asp carboxylate and Arg guanidiniummoieties of RGD-based ligands are key structural elements forreceptor recognition The carboxylate group interacts with themetal ion-dependent adhesion site (MIDAS) of the Mn2+ ion

and Ser121Ser123 whereas the Arg guanidinium group isresponsible for salt bridge interactions with Asp218 and Asp150side chains Additional ligandminusreceptor contacts occur betweenTyr122 in hydrophobic π-stacking and Asn215 The dockingprogram Autodock 40117 was used to evaluate the bindingenergies of selected conformations of ligand 318 Dockedconformations were analyzed by taking into account thebinding interactions as observed in the crystal structure of thebound ligandminusprotein complex Docking calculations resultedin a cluster of conformations characterized by key interactionswith Asp218 and the MIDAS site of the integrin thusconfirming these interactions as necessary for the molecularrecognition of ArgminusGlyminusAsp-containing ligands (Figure 3)

Ligand 3 showed that the Asp side chain is located in theMIDAS site and interacts with Ser121 and guanidinium groupof Arg undergoes a salt-bridge interaction with Asp218 and amonodentate interaction with Asp150 The main conformationof the cluster showed a different orientation of the D-Tyraromatic ring not facing Tyr-122 in a π-stacking interaction asobserved for compound 1 but displaying a hydrogen-bondbetween the Tyr OH group of the aromatic side chain and theOH group of Tyr-178 as hydrogen-bond acceptor The NHgroup of D-Tyr showed a hydrogen bond with the Tyr-122 COgroup Thus a different binding mode geometry was assessedfor compound 3 as a result of D-Tyr side chain possibly relatingto a different binding profile with respect to compound 1(Figure 2)

Cell Biology Assays Adhesion and Migration of A375 MMelanoma Cells in the Presence of Morpholine-Based RGD-Cyclopeptides 1minus3 A375 M human melanoma cells express awide variety of integrin subunits (αV β3 β5 α5 β1) and a highlevel of αvβ3 heterodimer12g Figure 4 shows the ability ofligands 1 2 and 3 used at final concentrations of 10 10 or 01μM to inhibit the binding of A375 M melanoma cells tovarious RGD-containing substrates such as vitronectin andfibronectin Tests were performed in the presence of 2 mmolLMnCl2 in order to switch integrins of tumor cells into theactivated form6c Figure 4 also shows that ligand 1 inhibits thebinding of melanoma cells to vitronectin to a great extent

Figure 2 Morpholine-based RGD-cyclopeptides D-Mor-containingcompound 1 127I-D-Mor-D-Tyr-containing compound 3 Data arepresented as means plusmn SD from three independent experiments anderror bars represent 95 confidence interval

Figure 3 RGD ligand 3 (cyan) docked into the binding region of αvβ3integrin highlighting protein residues (green) that form keyinteractions (dotted lines) Asp218 vs Arg Ser121 and Mn2+ vs AspTyr122 and Tyr178 vs D-I-Tyr Nonpolar hydrogen atoms are omittedfor clarity

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whereas the inhibition of cell adhesion to fibronectin is lesseffective Ligands 2 and 3 expressed an inhibitory ability ontumor cell adhesion comparable to that of ligand 1 These

results suggest that ligands 1 2 and 3 display a quitecomparable affinity for αvβ3 integrin and the addition of iodinedoes not modify the affinity in living cells A ldquoscratch woundrdquo

Figure 4 Adhesion of A375 M melanoma cells HUVECs and dermal fibroblasts to vitronectin and fibronectin in the presence of compounds 1 2or 3 The values are expressed as inhibition plusmn SEM of cell adhesion relative to control (untreated cells) Significantly different at p lt 005 ascompared to compound 1

Figure 5 Radioactivity biodistribution evaluated in a healthy mouse at 60 120 and 300 min and 24 h after 125I-3 injection

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model was used to examine the effect of D-morpholine-containing RGD-ligands on in vitro migration of melanomacells Tumor cells exposed to ligands 1 2 and 3 did not modifytheir migration rate with respect to nonexposed tumor cells(control tumor cells) The migration rate (as represented bythe reduction of scratch width) and the morphology of tumorcells exposed to D-morpholine-containing RGD-ligands werefound comparable at 10 μM 1 μM and 10 nM concentrationof the ligand with respect to control tumor cells (Figure S2Supporting Information)Adhesion and in Vitro Tubulogenesis of HUVEC in the

Presence of Morpholine-Based RGD-Cyclopeptides 1minus3 IL-1β-stimulated HUVECs express integrin subunits (αv β3 β5 α5β1) and αvβ3 heterodimer Ligands 1 2 and 3 suppressed theadhesion of IL-1β-stimulated HUVECs to vitronectin whereasthey did not completely suppress the adhesion of activatedendothelial cells to fibronectin (Figure 4) We can speculatethat D-morpholine-based RGD-cyclopeptides act preferentiallyon the endothelial αvβ3 integrin instead of the α5β1 integrin Toassess whether ligands 1 2 and 3 were able to exert anantiangiogenic activity we examined the ability of thesepeptides to repress in vitro tube formation by HUVEC seededon a Matrigel layer Figure S3 of Supporting Information showsthat tube formation by HUVEC was not inhibited by D-morpholine-containing RGD-ligandsAdhesion of HDFs in the Presence of Morpholine-Based

RGD-Cyclopeptides As shown in Figure 4 ligand 1 suppressedthe adhesion of HDFs to vitronectin and fibronectin with ahigh efficiency whereas ligand 2 and 3 expressed a significantlyreduced efficiencyBiodistribution and Melanoma Imaging The radio-

activity distribution was evaluated in a nontumor-bearingmouse at 60 120 and 300 min and 24 h post injection (pi)(Figure 5) The accumulation in gallbladder and kidneysremained consistent for at least two hours pi and the tracer wasexcreted via renal and hepatobiliary routes allowing for a fastclearance of nonspecific bound tracer Accumulation in thespleen might be due to the structural similarity of integrin αvβ3to αIIββ3 integrin expressed on spleen-enriched platelets Therelease of the 125I atom from the molecule and its accumulationin the thyroid gland increased with time a phenomenon thatcan be avoided by the preadministration of a solution of KI (4mgL) At 24 h pi little radioactivity remained in the organismmainly in the thyroid and bowelFigure 6 shows the localization of the 125I-3 compound in the

site of the implanted tumor as determined by micro SPECT-CT imaging The images clearly show that the 125I-3 ligand hasthe capacity to monitor in vivo the growth of αvβ3 integrin-positive melanoma cells Binding studies showed a gooddisplacement of the radiotracer by the parent nonradioactivecompound with a mean ratio of saturated to displacedreceptors in tumor tissue of 5753 plusmn 06 and having thecontrolateral muscle as the reference tissue

DISCUSSION AND CONCLUSIONSAs reported αvβ3 integrin-targeted radiotracers are able toreveal a tumor mass and to monitor growth and diffusion oftumor cells19 We tested D-morpholine-based RGD-cyclo-pentapeptides 1 2 and 3 in in vitro and in vivo assays andthey were found to inhibit the adhesion of human melanomacells to RGD substrates such as vitronectin and fibronectionThese ligands proved to be more specific for αvβ3 than α5β1integrin ldquoScratch woundrdquo experiments revealed that melanoma

cells exposed to ligands 1 2 and 3 did not modify theirmigration rate as compared to nonexposed tumor cells Severalinteractions operate between tumor cells and the extracellularmatrix thus the inhibition of αvβ3 integrin-mediated celladhesion per se might not be sufficient to influence tumor cellmigration6c20 Ligands 1 2 and 3 also repressed the adhesionof IL-1β-stimulated HUVEC to vitronectin whereas inhibitionof endothelial cell adhesion on fibronectin was not significantAt the same time ligands 1 2 and 3 did not block in vitrocapillary-like tube formation by HUVEC We assume that thereduced affinity of D-morpholine-based RGD-cyclopentapep-tides for α5β1 integrin fails to influence in vitro angiogenesisIndeed it has been found that only simultaneous inhibition ofboth integrins reduces capillary tube formation1112h21

Tumor cells recruit host inflammatory and stromal cellswhich generate a microenvironment able to promote growthand dissemination of cancer cells22 Because fibroblastsrepresent the most abundant host cells within a tumormicroenvironment23 we injected a suspension of tumor cellsand αvβ3 integrin expressing HDF into immunodeficient miceto test in vivo behavior of 125I-3 ligand by micro-SPECTCTanalysis We found a high tracer retention within the tumormass with a satisfying tumor to background ratio Co-injectionof 125I-3 and the corresponding unlabeled ligand 3 showed highselectivity of this ligand for αvβ3 expressing tumor cellsThus the 125I-3 ligand may represent a new radio-

pharmaceutical with the potential of early detection of atumor mass and of monitoring the therapeutic responseMoreover high affinity of this radiotracer for αvβ3 integrinwhich is characteristic of tumor endothelial cells can be avaluable diagnostic tool in a large population of cancersincluding tumors with low level of αvβ3-expressing cells Theseconsiderations extend the applications of 125I-3 from melanomato several tumor histotypesFinally the D-Mor-containing cyclopentapeptide ligand 3

might be used in novel therapeutic strategies upon labeling with131I radioisotope

EXPERIMENTAL SECTIONGeneral H-Gly-2-Cl-Trt resin (11 mmolg) was purchased from

Fluka Chromatographic separations were performed on silica gel

Figure 6 Transaxial slices through mouse chest at implantedmelanoma tumor level (arrows) In upper panels CT images inlower panels fused CT and SPECT images Images were acquired at 40min after tail vein injection of 125I-3 (250 μCi in 02 mL of saline) Thedisplacement study was achieved with an excess of cold ligand (18 mgkg) injected 10 min before the tracer

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(Kieselgel 60 Merck) using flash-column techniques Rf values refer toTLC carried out on 25 mm silica gel plates (Merck F254) with sameeluant as for column chromatography Solid-phase reactions werecarried out on a shaker using solvents of HPLC quality BromophenolBlue (BB) test was performed as follows a small amount of resinbeads were suspended in a vial with 05 mL of DMF two drops of a1 solution of bromophenol blue in dimethylacetamide were addedand the sample was observed The BB test was considered positive(presence of free amino groups) when the resin beads turned blue andnegative (absence of free amino groups) when the beads remainedcolorless ESI mass spectra were carried out on a ion-trap doublequadrupole mass spectrometer using electrospray (ES+) ionizationtechniques and a normalized collision energy within the range of 25minus32 eV for MSMS experiments Ligands 2 and 3 were purified byBeckman-Gold HPLC system equipped with a reverse-phase column(Alltima C18 10 μm 250 mm times 10 mm Alltech) using H2OCH3CNgradient eluant buffered with 01 TFA (flow rate 25 mLmin λ 254nm gradient eluant CH3CN 105 min CH3CN 10minus9025 min)Analytical HPLC analyses were performed on Dionex Ulltimate 3000system equipped with a reverse-phase column (Alltima C18 5 μm 250mm times 46 mm Alltech) and the same gradient eluant Ligand 125I-3was purified by Beckman-Gold HPLC system equipped with a reverse-phase column (C-8 Waters Spherisorb S5 250 mm times 46 mm) and thesame gradient eluant Na125I in 02N NaOH solution was purchasedfrom PerkinElmer (10 μL 100 mCimL 17 Ci (629GBq)mg 125I97 126I lt 3 radiochemical purity gt99 carrier free) HPLCanalysis was used to determine purity and all tested compoundspossessed gt95 purityc[RGDy-(3R)-carboxymorpholine] (2) H-Gly-2-Cl-Trt resin

(466 mg 05 mmol) was used as starting material Fmoc-deprotectionsand completion of each coupling reaction were assessed by performinga BB test Coupling was performed with a solution consisting of N-Fmoc-Arg(Pbf)-OH (973 mg 15 mmol 3 equiv) HOBt (203 mg 15mmol 3 equiv) in DMF (3 mL) and DIC (203 μL 15 mmol 3equiv) was added dropwise at 0 degC The resulting mixture was stirredfor 10 min at 0 degC and for a further 10 min at room temperature andthen was added to the resin This mixture was shaken at roomtemperature for 24 h The solution was drained and the resin wassequentially washed with 5 DIPEA in DMF (3 times 5 mL) and DMF (5times 5 mL) Fmoc deprotection was performed with 30 piperidine inDMF (10 mL) for 30 min followed by resin washings with DMF (3 times10 mL) After deprotection H-Arg(Pbf)-Gly-2-Cl-Trt resin wassuspended in a solution of HOBt (3568 mg 264 mmol 6 equiv)DIC (4156 μL 264 mmol 6 equiv) and N-Fmoc-(R)-morpholine-3-carboxylic acid (465 mg 132 mmol 264 equiv) in DMF (3 mL) andshaken for two days The solution was drained and resin was washedwith 5 DIPEA in DMF (3 times 5 mL) and DMF (5 times 5 mL) After

deprotection H-(3R)-carboxymorpholine-Arg(Pbf)-Gly-2-Cl-Trt resinwas suspended in a solution of HOBt (405 mg 3 mmol 6 equiv) DIC(472 μL 3 mmol 6 equiv) and N-Fmoc-D-Tyr(t-Bu)-OH (689 mg15 mmol 3 equiv) in DMF (3 mL) and shaken for three days Thesolution was drained and the resin washed with 5 DIPEA in DMF (3times 5 mL) and DMF (5 times 5 mL) After deprotection H-D-Tyr(t-Bu)-(3R)-carboxymorpholine-Arg(Pbf)-Gly-2-Cl-Trt resin was suspendedin a solution of TBTU (8027 mg 25 mmol 5 equiv) N-Fmoc-Asp(t-Bu)-OH (1028 g 25 mmol 5 equiv) and DIPEA (428 μL 25 mmol5 equiv) in DMF (3 mL) and shaken for three days The solution wasdrained and resin washed with 5 DIPEA in DMF (3 times 5 mL) andDMF (5 times 5 mL) N-Fmoc-Asp(t-Bu)-D-Tyr(t-Bu)-(3R)-carboxymor-pholine-Arg(Pbf)-Gly-2-Cl-Trt resin was deprotected and washed withDMF (5 times 5 mL) and DCM (5 times 5 mL) In a solid-phase reactionvessel H-Asp(t-Bu)-D-Phe-(3R)-carboxymorpholine-Arg(Pbf)-Gly-2-Cl-Trt resin was treated with 5 mL of 1 TFADCM solution (10times 2 min) The filtrates were immediately neutralized with a 10pyridineMeOH solution (1 mL) and then the resin was washed withDCM (3 times 5 mL) Fractions (TLC eluant 41 DCMMeOH) werecombined and concentrated under reduced pressure to yield a residuewhich was suspended in H2O and purified from the pyridinium salts bysize-exclusion chromatography (AMBERLITE XAD2 resin H2O thenMeOH) Evaporation of combined MeOH fractions containing theproduct afforded the side-chain protected compound (493 mg 33)as a white solid material The linear peptide (493 mg 05 mmol) wasdissolved under a nitrogen atmosphere in DMF (120 mL) HATU(1521 mg 04 mmol 08 equiv) and TBTU (354 mg 11 mmol 22equiv) and DIPEA (257 μL 15 mmol 3 equiv) were added Theresulting mixture was stirred for two days at room temperature Thesolvent was distilled off under reduced pressure and the residue wasdissolved in H2O (60 mL) and extracted with EtOAc (3 times 80 mL)The organic phase was washed twice with 5 NaHCO3 dried withNa2SO4 and evaporated under reduced pressure The crude residuewas purified by flash column chromatography (91 DCMminusMeOH Rf037) to give the protected cyclic peptide (140 mg 30) as a yellowsolid material HPLC tR = 2430 purity 97 Side chain protectedcyclic peptide (140 mg 015 mmol) was treated with 952525 TFAH2OTIS mixture (20 mL) for 2 h The reaction mixture wasevaporated under reduced pressure and the residue was dissolved inH2O (60 mL) The aqueous phase was washed with iPr2O (3 times 60mL) and freeze-dried The crude residue was purified by semi-preparative HPLC to give the side-chain deprotected 2 (30 mg 34)as a white solid Trifluoroacetate ion was replaced with chloride ion byion-exchange chromatography (AMBERLITE IRA-96 resin chlorideform) See Table 1 for 1H and 13C NMR data ESI-MS mz 60514 (M+ H+ 100) ESI-MSMS mz 6055 (M + H+ 1) 5880 (100) 5770(30) 5700 (17) 5599 (42) 5460 (11) 533 (12) 5200 (72) Anal

Table 1 1H and 13C Chemical Shifts of 2 and 3 in DMSO-d6 Solutions at 298 K1H 2 1H 3 13C 2 13C 3

H-2 428 337 428 334 C-2 688 690H-3 459 466 C-3 535 537H-5 308 316 308 C-5 668 664H-6 393 370 390 372 C-6 682 684Gly NH 873 861Arg NH 740 856Asp NH 865 866D-Tyr NH 719 735Gly H-α 407 399 310 Gly C-α 445 441Arg H-α 399 401 Arg C-α 533 537Arg H-βγ 165 150 163 143 Arg C-βγ 249 220 248 212Arg H-δ 304 306 Arg C-δ 405 401Asp H-α 478 471 Asp C-α 570 567Asp H-β 282 270 288 256 Asp C-β 396 398D-Tyr H-α 447 451 D-Tyr C-α 494 504D-Tyr H-β 214 232 D-Tyr C-β 287 358D-Tyr H-Ar 695 660 766 734 724 D-Tyr C-Ar 1153 1301 1158 1298 1391

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Calcd for C26H37ClN8O9 C 4871 H 582 N 1748 Found C4888 H 591 N 1732 HPLC tR = 121 95 purityc[RGD-(3primeI-y)-(3R)-carboxymorpholine] (3) A solution of

ligand 2 (120 mg 0020 mmol) in PBS (2 mL) was added to sodiumiodide (307 mg 0020 mmol) followed by chloramine-T hydrate(466 mg 0020 mmol) The reaction mixture was stirred at roomtemperature for 20 min and then quenched with a solution of 5sodium metabisulphite (08 mL) The mixture was extracted withDCM (3 times 5 mL) and loaded on a preconditioned C18 Sep-pakcolumn The C18 column was washed with water (6 mL) andsubsequently eluted with MeOH The crude product was purified bysemipreparative HPLC to obtain ligand 3 (467 mg 32) See Table 1for 1H and 13C NMR data ESI-MS mz 73125 (M + H+ 100) ESI-MSMS mz 7312 (M + H+ 15) 7141 (100) 6859 (39) 6721 (22)6461 (76) Anal Calcd for C26H36ClIN8O9 C 4072 H 473 N1461 Found C 4081 H 483 N 1412 HPLC tR = 152 96purityc[RGD-(3prime125I-y)-(3R)-carboxymorpholine] (125I-3) A solution

of PBS (100 μL 005 M) containing Na125I (20 μL 2 mCi 0918 nM)peptide 2 (10 μL of a 005 M PBS solution 165 nmol) andchloramine-T (188 μL of a freshly prepared 005 M PBS solution 824nmol 9 equiv) was prepared The reaction mixture was stirred at roomtemperature for 20 min and then quenched by adding a solution ofsodium metabisulphite (218 μL of a freshly prepared 005 M PBSsolution 824 nmol 9 equiv) The mixture was extracted with DCM (3times 05 mL) and purified by HPLC and the radioactivity of fractionscollected every 30 s was determined with a γ-counter (SupportingInformation Figure S1) Fractions containing the chemically pure 125I-3 were pooled together (155 mCi 78 radiochemical yield) and usedfor in vivo experiments after solvent removal HPLC tR = 152 98radiochemical purity gt99 chemical puritySolid-Phase Integrin Binding Assay [125I]-Echistatin labeled by

the lactoperoxidase method24 to a specific activity of 2000 Cimmolwas purchased from GE Healthcare and integrin αvβ3 from humanplacenta was purchased from Chemicon International Inc TemeculaCAPurified αvβ3 integrin was diluted in coating buffer (20 mM Tris

(pH 74) 150 mM NaCl 2 mM CaCl2 1 mM MgCl2 1 mM MnCl2)at concentrations of 500 or 1000 ngmL An aliquot of diluted integrin(100 μLwell) was added to a 96-well microtiter plate (Optiplate-96HB PerkinElmer Life Sciences Boston MA) and plates wereincubated overnight at 4 degC Plates were washed once withblockingbinding buffer (20 mM Tris (pH 74) 150 mM NaCl 2mM CaCl2 1 mM MgCl2 1 mM MnCl2 1 BSA) and incubated atroom temperature for additional 2 h Plates were rinsed twice withsame buffer and then competition binding assays were performed witha constant concentration of [125I]-Echistatin (005 nM) Concen-trations of tested compound ranged from 001 to 100 nM All assayswere performed in triplicate in a final volume of 02 mL eachcontaining the following species 005 mL of [125I]-Echistatin 004 mLof tested compound and 011 mL of blockingbinding bufferNonspecific binding was defined as [125I]-Echistatin bound in presenceof an excess (1 μM) of unlabeled echistatin After incubation at roomtemperature for 3 h plates were washed three times with blockingbinding buffer and counted in a Top-Count NXT microplatescintillation counter (PerkinElmer Life Sciences Boston MA) using200 μLwell of MicroScint-40 liquid scintillation (PerkinElmer LifeSciences Boston MA)Data are shown as means plusmn SD from three independent

experiments IC50 values were determined by fitting binding inhibitiondata by nonlinear regression using GraphPad Prism 40 SoftwarePackage (GraphPad Prism San Diego CA)Cell Lines and Culture Conditions The A375 M human

melanoma cell line was obtained from the American Type CultureCollection (ATCC Rockville MD)12g Human dermal skin fibroblasts(HDF) were kindly donated by Dr Daniela Monti Department ofExperimental Pathology and Oncology University of FlorenceMelanoma cells and HDF were grown in Dulbeccorsquos Modified EagleMedium containing 4500 mgL glucose (DMEM 4500 GIBCO)supplemented with 10 fetal calf serum (FCS) at 37 degC in a

humidified incubator containing 10 CO2 Then 50 times 105 melanomacells or 1 times 106 HDF were seeded in 100 mm dishes and propagatedevery 3 days by incubation with a trypsinminusEDTA solution Cultureswere periodically monitored for mycoplasma contamination Primarycultures of human umbilical vein endothelial cells (HUVEC) wereobtained from Lonza (USA) HUVEC grown in endothelial cellgrowth medium-2 (EGM-2) containing endothelial cell growthsupplements were subcultivated using a trypsin-EDTA solution 13split ratio Cells between passages 2 and 4 grown to confluence inplates coated with 1 bovine gelatin (Sigma St Louis) were used inthe experiments

RNA Isolation and Polymerase Chain Reaction (PCR) TotalRNA extracted using RNAgents (Total RNA Isolation SystemPromega Madison WI) was determined spectrophotometricallyComplementary DNA (cDNA) was synthesized from 2 μg of totalRNA using 1 μL of ImProm-II reverse transcriptase (Promega)Aliquots of 2 μL of the cDNA were used for PCR amplification Thespecific primers used for the identification of human αv α5 β1 β3 β5and GAPDH are reported in Table S1 of Supporting Informations AllPCR experiments were conducted using 005 UμL of Go-TaqPolymerase (Promega) Amplification was carried out on a Perkin-Elmer Thermal cycler Then 8 μL of each PCR products werevisualized after electrophoresis in a 2 agarose gel containing 05 μgmL of ethidium bromide cDNA products were evaluated on the basisof a standard PCR marker

Flow Cytometry Assay Tumor cells were detached by gentletreatment with Accutase a 05 mM EDTA solution washed andincubated for 1 h at 4 degC in the presence of anti αvβ3 monoclonalantibody (1 μg50 μL Anti-Integrin αvβ3 clone LM609 Millipore)Cells were then washed and incubated for 1 h at 4 degC with a secondaryantibody 5 μgmL goat antimouse IgG conjugated with FITC (SantaCruz Biotechnology Inc Santa Cruz CA) αvβ3 -positive cells wereanalyzed at 488 nm on the flow cytometer FACScan system (BD-FACS Canto)

Cell Adhesion Assay Plates (96 wells) were coated withfibronectin (1 μgmL) or vitronectin (10 μgmL) by overnightincubation at 4 degC and then incubated at 37 degC for 1 h with 1 BSA inPBS Melanoma cells IL-1β (400 UmL)-stimulated HUVECs orhuman dermal fibroblasts (HDF) were resuspended in serum-freemedium and exposed to ligands 1 2 or 3 (final concentration was 011 or 10 μM) at 37 degC for 30 min to allow for ligandminusintegrinequilibrium to be reached Melanoma cell adhesion assays wereperformed in the presence or in the absence of 2 mmolL MnCl2Cells were plated (4minus5 times 104 cellswell) and incubated at 37 degC for 2h Nonadherent cells were removed with PBS and adherent cells werestained with 05 crystal violet solution in 20 methanol After 2 h ofincubation at 4 degC plates were examined at 540 nm in a counterELX800 (Biotek Instruments) Experiments were conducted intriplicate and repeated at least three times Data are presented asmeans plusmn SD from three independent experiments

Animal Model and Study Design In vivo experiments wereconducted in accordance with national guidelines and approved by theEthical Committee of the Animal Welfare Office of the Italian WorkMinistry and conforming to legal mandates and Italian guidelines forcare and maintenance of laboratory animals Eighteen-week-old SCIDbgbg mice (Charles River Laboratories International) were injectedsubcutaneously in the shoulder area with a suspension of melanomacells and 20 ngmL TGFβ-activated fibroblasts Then 1 times106 A375 Mmelanoma cells and 05 times 106 HDF were previously resuspended in100 μL of PBS diluted 11 with a Matrigel solution (BD Biosciences)Animals were kept under pathogen-free conditions and allowed to feedfreely Tumor growth was monitored daily

Small-Animal SPECT and CT Studies In vivo studies wereconducted using a ldquohybridrdquo imaging system for small laboratoryanimals allowing for the study of biological processes using minimallyinvasive procedures Engrafted tumor tracer uptake was studied atdifferent time-points after tail vein injection Binding specificity tointegrin receptors was evaluated by in vivo displacement studies inwhich a high dose of cold tracer was injected 10 min beforeradiolabeled one Imaging studies were performed using the Flex

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dxdoiorg101021jm2016232 | J Med Chem 2012 55 5024minus50335030

Triumph preclinical imaging system which combines positron (PET)and single-photon emission (SPECT) tomography with the emissiontomography (CT) for anatomic coregistration (GE HealthcareGamma Medica-Ideas Waukesha WI) 125I-iodinated tracers 125I-3was studied in vivo by SPECT allowing for use directly the in vitrotracer data to the in vivo experimentation Imaging studies requiredmonitoring of anesthetized animals throughout the imaging procedureand anesthesia was conducted in accordance with National Guidelinesand approved by the Ethical Committee Given that anesthesia ingeneral causes hypothermia in small animals appropriate warming wasprovided during imaging procedure by using a heating bed set at 37degC Physiological monitoring was achieved by measuring respirationrate with a pressure pad placed under the mouse abdomen Saturationexperiments (SE) to visualize tumors and displacement experiments(DE) were performed In SE 125I-3 (7minus10 MBq in 200 μL ofphysiological saline) was injected via tail vein into tumor bearinganimals kept under anesthesia After 30 min a CT aquisition wasperformed followed by SPECT scan starting at 40 min pi CT scanwas acquired with 50 kV and 320 μA tube settings The magnificationfactor was set to 2 resulting in an axial field of view of 592 mm 512projections 500 μs each were acquired on a circular orbit in 256 swith an acquisition matrix of 1184 times 1120 100 μm pixel SPECTprojections were acquired with a 5-pinhole high-resolution collimator(1 mm hole diameter 75 mm focal distance) with radius of rotation of40 mm resulting in an axial field of view of 529 mm then 64projections 20 s each were acquired on a circular orbit for a totalacquisition time of about 22 min Acquisition matrix was 80 times 80 15mm pixel size Energy window was set on the 125I photopeak rangingfrom 20 to 36 KeV The DE were performed by injecting 18 mgkg ofcompound 3 10 min before injection of tracer Once scanning wascomplete the animals were recovered in a housing unit and monitoreduntil awakeData Analysis of Small-Animal SPECT and CT Studies CT

image data set was reconstructed using a cone-beam FBP algorithm(FDK) and an optimum noise filter Reconstruction matrix was 512 times512 times 512 180 μm pixel size SPECT images were reconstructed withordered subset expectation maximization algorithm (8 subsets and 5iterations) and a reconstruction matrix of 60 times 60 times 60 15 mm pixelsize No correction for radiation attenuation and scatter was appliedFused images were visually analyzed to detect the engrafted tumormass and regional SPECT activities were measured using the analysissoftware Vivid (Gamma Medica-Ideas Northridge CA) by delineatingthe region of interest (ROI) of tumor in fused CT images A similarROI was generated for muscle tissue on contralateral side of tumorTumor ROI activity was normalized (nTA) with the followingformula

= minusnTA

TA BABA

where TA is tumor activity and BA is background activity measured inmuscle tissue contralateral to tumorDocking Calculations Automated docking studies were carried

out by Autodock 401 program17 using the Lamarckian GeneticAlgorithm (LGA) as a search engine The AutoDockTools 145(ADT) graphical interface25 was used to prepare integrin and ligandsPDBQT files Coordinates of ligand 3 were retrieved from lowestenergy conformers resulting from molecular mechanics calculationsstarting from geometry of ligand 1 whereas coordinates of αvβ3receptor were retrieved from the Protein Data Bank (PDB code1L5G) and ligandminusprotein complex was unmerged for achieving freereceptor structure Water molecules were removed For proteinreceptor and ligand 3 all hydrogens were added Gasteiger chargeswere computed and nonpolar hydrogens were merged A charge valueof +20 to each Mn atom of protein receptor was successively addedThree-dimensional energy scoring grids of 0375 Aring resolution and 40Aring times 40 Aring times 40 Aring dimensions were computed Center of grid was set tobe coincident with mass center of ligands preliminary fitted on the X-ray structure of c[RGDf(Me)V] in the αvβ3 complex (1L5G) A totalof 50 runs with a maximum of 2500000 energy evaluations werecarried out for each ligand using the default parameters for LGA

Cluster analysis was performed on docked results using a root-mean-square (rms) tolerance of 15 Aring Analysis of binding mode calculationof binding energy and prediction of binding activity of dockedconformations were carried out using PyMol Autodock Tools pluginwithin PyMol software26

ASSOCIATED CONTENTS Supporting InformationAdditional experimental methods radiochromatogram of 125I-3and additional cell biology assays data This material is availablefree of charge via the Internet at httppubsacsorg

AUTHOR INFORMATIONCorresponding AuthorFor AG phone +39 055 4573481 fax +39 055 4573569 E-mail antonioguarnaunifiit For AP phone +39 0557947527 fax +39 055 4224392 E-mail apupidfcunifiitFor LC phone +39 055 4598207 fax +39 055 4598900 E-mail lidocaloriniunifiit

NotesThe authors declare no competing financial interest

ACKNOWLEDGMENTSThis work has received financial support from the IstitutoTo s c ano Tumor i a nd MIUR (PR IN08 G r an t2008J4YNJY_003) Also financial contributions from CINM-PIS University of Florence and Fondazione Roma areacknowledged Dr Alessandro Sisti is acknowledged forcarrying out preliminary labeling experiments Ente Cassa diRisparmio is acknowledged for the granting the MicroPETSPECTCT instrument

ABBREVIATIONS USEDSPECT single positron emission computed tomography PETpositon emission tomography RGD Arg-Gly-Asp VGPvertical growth phase CT computed tomography VNvitronectin FN fibronectin HUVEC human umbilical veinendothelial cells IL-1β interleukin-1β ESI-MSMS electroprayionization tandem mass spectrometry EGM-2 endothelial cellgrowth medium-2 EDTA ethylenediaminetetraacetic acidGAPDH glyceraldehyde 3-phosphate dehydrogenase FACSfluorescence-activated cell sorting FITC fluorescein isothio-cyanate PBS phosphate buffered saline HUVEC humanumbilical vein endothelial cells HDF human dermal skinfibroblasts EMT epithelial-mesenchymal transition TGFtransform grown factor pi post injection

REFERENCES(1) Challenges and Opportunities Report US Department of Healthand Human Services Food and Drug Administration Silver SpringMD March 2004 httpwwwfdagovScienceResearchSpecialTopicsCriticalPathInitiativeCriticalPathOpportunities-Reports-ucm077262htm(2) (a) Backgrounder How New Drugs Move Through the Developmentand Approval Process Tufts Center for the Study of DrugDevelopment Boston November 1 2001 (b) Gilbert J HenskeP Singh A Rebuilding Big Pharmarsquos Business Model The Business ampMedicine Report Windhover Information November 2003 Vol 21 no10 httpwwwbaincomImagesrebuilding_big_pharmapdf(3) Innovative drug development approaches-project Final report fromTHEEMEACHMP-THINK-TANK group on innovative drug develop-ment EMEA 2007 pp 1minus26 httpwwwemaeuropaeudocsen_GBdocument_libraryOther200910WC500004913pdf

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dxdoiorg101021jm2016232 | J Med Chem 2012 55 5024minus50335031

radioactivity was monitored with a γ-counter (see Figure S1Supporting Information) The radiolabeling procedure toobtain 125I-3 resulted in a clean iodination reaction with aradiochemical yield of 78 for the monoiodinated peptideIntegrin Binding Assays The ability of cyclopentapeptide

3 to compete with [125I]echistatin for binding to pure αvβ3integrin isolated from human placenta was evaluated in solidphase assays15 IC50 values showed a binding affinity ofcompound 3 toward αvβ3 integrin of 480 plusmn 12 nM slightlylower than that of peptide 1 (IC50 65 plusmn 20 nM) (Figure 2)15

Data analyses indicates that compound 3 binds to αvβ3 integrinaccording to a one-site binding model (Hill slope minus092 forone-site model) whereas cyclopeptide 1 showed binding toαvβ3 integrin according to a two-site binding model (Hill slopeminus056 for one-site model) thus suggesting that the iodo-tyrosine-derived ligand 3 displays a different affinity profiletoward αvβ3 integrin

15

Molecular Modeling To understand the potential differ-ences in binding mode of cyclopeptide 3 to αvβ3 integrin ascompared to ligand 1 a docking simulation was carried out15

The crystal structure of the complex formed by c[RGDf(Me)V]and the extracellular fragment of αvβ3 integrin (PDB code1L5G) provides a general mode of interaction between theintegrin and its ligands16 Asp carboxylate and Arg guanidiniummoieties of RGD-based ligands are key structural elements forreceptor recognition The carboxylate group interacts with themetal ion-dependent adhesion site (MIDAS) of the Mn2+ ion

and Ser121Ser123 whereas the Arg guanidinium group isresponsible for salt bridge interactions with Asp218 and Asp150side chains Additional ligandminusreceptor contacts occur betweenTyr122 in hydrophobic π-stacking and Asn215 The dockingprogram Autodock 40117 was used to evaluate the bindingenergies of selected conformations of ligand 318 Dockedconformations were analyzed by taking into account thebinding interactions as observed in the crystal structure of thebound ligandminusprotein complex Docking calculations resultedin a cluster of conformations characterized by key interactionswith Asp218 and the MIDAS site of the integrin thusconfirming these interactions as necessary for the molecularrecognition of ArgminusGlyminusAsp-containing ligands (Figure 3)

Ligand 3 showed that the Asp side chain is located in theMIDAS site and interacts with Ser121 and guanidinium groupof Arg undergoes a salt-bridge interaction with Asp218 and amonodentate interaction with Asp150 The main conformationof the cluster showed a different orientation of the D-Tyraromatic ring not facing Tyr-122 in a π-stacking interaction asobserved for compound 1 but displaying a hydrogen-bondbetween the Tyr OH group of the aromatic side chain and theOH group of Tyr-178 as hydrogen-bond acceptor The NHgroup of D-Tyr showed a hydrogen bond with the Tyr-122 COgroup Thus a different binding mode geometry was assessedfor compound 3 as a result of D-Tyr side chain possibly relatingto a different binding profile with respect to compound 1(Figure 2)

Cell Biology Assays Adhesion and Migration of A375 MMelanoma Cells in the Presence of Morpholine-Based RGD-Cyclopeptides 1minus3 A375 M human melanoma cells express awide variety of integrin subunits (αV β3 β5 α5 β1) and a highlevel of αvβ3 heterodimer12g Figure 4 shows the ability ofligands 1 2 and 3 used at final concentrations of 10 10 or 01μM to inhibit the binding of A375 M melanoma cells tovarious RGD-containing substrates such as vitronectin andfibronectin Tests were performed in the presence of 2 mmolLMnCl2 in order to switch integrins of tumor cells into theactivated form6c Figure 4 also shows that ligand 1 inhibits thebinding of melanoma cells to vitronectin to a great extent

Figure 2 Morpholine-based RGD-cyclopeptides D-Mor-containingcompound 1 127I-D-Mor-D-Tyr-containing compound 3 Data arepresented as means plusmn SD from three independent experiments anderror bars represent 95 confidence interval

Figure 3 RGD ligand 3 (cyan) docked into the binding region of αvβ3integrin highlighting protein residues (green) that form keyinteractions (dotted lines) Asp218 vs Arg Ser121 and Mn2+ vs AspTyr122 and Tyr178 vs D-I-Tyr Nonpolar hydrogen atoms are omittedfor clarity

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whereas the inhibition of cell adhesion to fibronectin is lesseffective Ligands 2 and 3 expressed an inhibitory ability ontumor cell adhesion comparable to that of ligand 1 These

results suggest that ligands 1 2 and 3 display a quitecomparable affinity for αvβ3 integrin and the addition of iodinedoes not modify the affinity in living cells A ldquoscratch woundrdquo

Figure 4 Adhesion of A375 M melanoma cells HUVECs and dermal fibroblasts to vitronectin and fibronectin in the presence of compounds 1 2or 3 The values are expressed as inhibition plusmn SEM of cell adhesion relative to control (untreated cells) Significantly different at p lt 005 ascompared to compound 1

Figure 5 Radioactivity biodistribution evaluated in a healthy mouse at 60 120 and 300 min and 24 h after 125I-3 injection

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model was used to examine the effect of D-morpholine-containing RGD-ligands on in vitro migration of melanomacells Tumor cells exposed to ligands 1 2 and 3 did not modifytheir migration rate with respect to nonexposed tumor cells(control tumor cells) The migration rate (as represented bythe reduction of scratch width) and the morphology of tumorcells exposed to D-morpholine-containing RGD-ligands werefound comparable at 10 μM 1 μM and 10 nM concentrationof the ligand with respect to control tumor cells (Figure S2Supporting Information)Adhesion and in Vitro Tubulogenesis of HUVEC in the

Presence of Morpholine-Based RGD-Cyclopeptides 1minus3 IL-1β-stimulated HUVECs express integrin subunits (αv β3 β5 α5β1) and αvβ3 heterodimer Ligands 1 2 and 3 suppressed theadhesion of IL-1β-stimulated HUVECs to vitronectin whereasthey did not completely suppress the adhesion of activatedendothelial cells to fibronectin (Figure 4) We can speculatethat D-morpholine-based RGD-cyclopeptides act preferentiallyon the endothelial αvβ3 integrin instead of the α5β1 integrin Toassess whether ligands 1 2 and 3 were able to exert anantiangiogenic activity we examined the ability of thesepeptides to repress in vitro tube formation by HUVEC seededon a Matrigel layer Figure S3 of Supporting Information showsthat tube formation by HUVEC was not inhibited by D-morpholine-containing RGD-ligandsAdhesion of HDFs in the Presence of Morpholine-Based

RGD-Cyclopeptides As shown in Figure 4 ligand 1 suppressedthe adhesion of HDFs to vitronectin and fibronectin with ahigh efficiency whereas ligand 2 and 3 expressed a significantlyreduced efficiencyBiodistribution and Melanoma Imaging The radio-

activity distribution was evaluated in a nontumor-bearingmouse at 60 120 and 300 min and 24 h post injection (pi)(Figure 5) The accumulation in gallbladder and kidneysremained consistent for at least two hours pi and the tracer wasexcreted via renal and hepatobiliary routes allowing for a fastclearance of nonspecific bound tracer Accumulation in thespleen might be due to the structural similarity of integrin αvβ3to αIIββ3 integrin expressed on spleen-enriched platelets Therelease of the 125I atom from the molecule and its accumulationin the thyroid gland increased with time a phenomenon thatcan be avoided by the preadministration of a solution of KI (4mgL) At 24 h pi little radioactivity remained in the organismmainly in the thyroid and bowelFigure 6 shows the localization of the 125I-3 compound in the

site of the implanted tumor as determined by micro SPECT-CT imaging The images clearly show that the 125I-3 ligand hasthe capacity to monitor in vivo the growth of αvβ3 integrin-positive melanoma cells Binding studies showed a gooddisplacement of the radiotracer by the parent nonradioactivecompound with a mean ratio of saturated to displacedreceptors in tumor tissue of 5753 plusmn 06 and having thecontrolateral muscle as the reference tissue

DISCUSSION AND CONCLUSIONSAs reported αvβ3 integrin-targeted radiotracers are able toreveal a tumor mass and to monitor growth and diffusion oftumor cells19 We tested D-morpholine-based RGD-cyclo-pentapeptides 1 2 and 3 in in vitro and in vivo assays andthey were found to inhibit the adhesion of human melanomacells to RGD substrates such as vitronectin and fibronectionThese ligands proved to be more specific for αvβ3 than α5β1integrin ldquoScratch woundrdquo experiments revealed that melanoma

cells exposed to ligands 1 2 and 3 did not modify theirmigration rate as compared to nonexposed tumor cells Severalinteractions operate between tumor cells and the extracellularmatrix thus the inhibition of αvβ3 integrin-mediated celladhesion per se might not be sufficient to influence tumor cellmigration6c20 Ligands 1 2 and 3 also repressed the adhesionof IL-1β-stimulated HUVEC to vitronectin whereas inhibitionof endothelial cell adhesion on fibronectin was not significantAt the same time ligands 1 2 and 3 did not block in vitrocapillary-like tube formation by HUVEC We assume that thereduced affinity of D-morpholine-based RGD-cyclopentapep-tides for α5β1 integrin fails to influence in vitro angiogenesisIndeed it has been found that only simultaneous inhibition ofboth integrins reduces capillary tube formation1112h21

Tumor cells recruit host inflammatory and stromal cellswhich generate a microenvironment able to promote growthand dissemination of cancer cells22 Because fibroblastsrepresent the most abundant host cells within a tumormicroenvironment23 we injected a suspension of tumor cellsand αvβ3 integrin expressing HDF into immunodeficient miceto test in vivo behavior of 125I-3 ligand by micro-SPECTCTanalysis We found a high tracer retention within the tumormass with a satisfying tumor to background ratio Co-injectionof 125I-3 and the corresponding unlabeled ligand 3 showed highselectivity of this ligand for αvβ3 expressing tumor cellsThus the 125I-3 ligand may represent a new radio-

pharmaceutical with the potential of early detection of atumor mass and of monitoring the therapeutic responseMoreover high affinity of this radiotracer for αvβ3 integrinwhich is characteristic of tumor endothelial cells can be avaluable diagnostic tool in a large population of cancersincluding tumors with low level of αvβ3-expressing cells Theseconsiderations extend the applications of 125I-3 from melanomato several tumor histotypesFinally the D-Mor-containing cyclopentapeptide ligand 3

might be used in novel therapeutic strategies upon labeling with131I radioisotope

EXPERIMENTAL SECTIONGeneral H-Gly-2-Cl-Trt resin (11 mmolg) was purchased from

Fluka Chromatographic separations were performed on silica gel

Figure 6 Transaxial slices through mouse chest at implantedmelanoma tumor level (arrows) In upper panels CT images inlower panels fused CT and SPECT images Images were acquired at 40min after tail vein injection of 125I-3 (250 μCi in 02 mL of saline) Thedisplacement study was achieved with an excess of cold ligand (18 mgkg) injected 10 min before the tracer

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dxdoiorg101021jm2016232 | J Med Chem 2012 55 5024minus50335028

(Kieselgel 60 Merck) using flash-column techniques Rf values refer toTLC carried out on 25 mm silica gel plates (Merck F254) with sameeluant as for column chromatography Solid-phase reactions werecarried out on a shaker using solvents of HPLC quality BromophenolBlue (BB) test was performed as follows a small amount of resinbeads were suspended in a vial with 05 mL of DMF two drops of a1 solution of bromophenol blue in dimethylacetamide were addedand the sample was observed The BB test was considered positive(presence of free amino groups) when the resin beads turned blue andnegative (absence of free amino groups) when the beads remainedcolorless ESI mass spectra were carried out on a ion-trap doublequadrupole mass spectrometer using electrospray (ES+) ionizationtechniques and a normalized collision energy within the range of 25minus32 eV for MSMS experiments Ligands 2 and 3 were purified byBeckman-Gold HPLC system equipped with a reverse-phase column(Alltima C18 10 μm 250 mm times 10 mm Alltech) using H2OCH3CNgradient eluant buffered with 01 TFA (flow rate 25 mLmin λ 254nm gradient eluant CH3CN 105 min CH3CN 10minus9025 min)Analytical HPLC analyses were performed on Dionex Ulltimate 3000system equipped with a reverse-phase column (Alltima C18 5 μm 250mm times 46 mm Alltech) and the same gradient eluant Ligand 125I-3was purified by Beckman-Gold HPLC system equipped with a reverse-phase column (C-8 Waters Spherisorb S5 250 mm times 46 mm) and thesame gradient eluant Na125I in 02N NaOH solution was purchasedfrom PerkinElmer (10 μL 100 mCimL 17 Ci (629GBq)mg 125I97 126I lt 3 radiochemical purity gt99 carrier free) HPLCanalysis was used to determine purity and all tested compoundspossessed gt95 purityc[RGDy-(3R)-carboxymorpholine] (2) H-Gly-2-Cl-Trt resin

(466 mg 05 mmol) was used as starting material Fmoc-deprotectionsand completion of each coupling reaction were assessed by performinga BB test Coupling was performed with a solution consisting of N-Fmoc-Arg(Pbf)-OH (973 mg 15 mmol 3 equiv) HOBt (203 mg 15mmol 3 equiv) in DMF (3 mL) and DIC (203 μL 15 mmol 3equiv) was added dropwise at 0 degC The resulting mixture was stirredfor 10 min at 0 degC and for a further 10 min at room temperature andthen was added to the resin This mixture was shaken at roomtemperature for 24 h The solution was drained and the resin wassequentially washed with 5 DIPEA in DMF (3 times 5 mL) and DMF (5times 5 mL) Fmoc deprotection was performed with 30 piperidine inDMF (10 mL) for 30 min followed by resin washings with DMF (3 times10 mL) After deprotection H-Arg(Pbf)-Gly-2-Cl-Trt resin wassuspended in a solution of HOBt (3568 mg 264 mmol 6 equiv)DIC (4156 μL 264 mmol 6 equiv) and N-Fmoc-(R)-morpholine-3-carboxylic acid (465 mg 132 mmol 264 equiv) in DMF (3 mL) andshaken for two days The solution was drained and resin was washedwith 5 DIPEA in DMF (3 times 5 mL) and DMF (5 times 5 mL) After

deprotection H-(3R)-carboxymorpholine-Arg(Pbf)-Gly-2-Cl-Trt resinwas suspended in a solution of HOBt (405 mg 3 mmol 6 equiv) DIC(472 μL 3 mmol 6 equiv) and N-Fmoc-D-Tyr(t-Bu)-OH (689 mg15 mmol 3 equiv) in DMF (3 mL) and shaken for three days Thesolution was drained and the resin washed with 5 DIPEA in DMF (3times 5 mL) and DMF (5 times 5 mL) After deprotection H-D-Tyr(t-Bu)-(3R)-carboxymorpholine-Arg(Pbf)-Gly-2-Cl-Trt resin was suspendedin a solution of TBTU (8027 mg 25 mmol 5 equiv) N-Fmoc-Asp(t-Bu)-OH (1028 g 25 mmol 5 equiv) and DIPEA (428 μL 25 mmol5 equiv) in DMF (3 mL) and shaken for three days The solution wasdrained and resin washed with 5 DIPEA in DMF (3 times 5 mL) andDMF (5 times 5 mL) N-Fmoc-Asp(t-Bu)-D-Tyr(t-Bu)-(3R)-carboxymor-pholine-Arg(Pbf)-Gly-2-Cl-Trt resin was deprotected and washed withDMF (5 times 5 mL) and DCM (5 times 5 mL) In a solid-phase reactionvessel H-Asp(t-Bu)-D-Phe-(3R)-carboxymorpholine-Arg(Pbf)-Gly-2-Cl-Trt resin was treated with 5 mL of 1 TFADCM solution (10times 2 min) The filtrates were immediately neutralized with a 10pyridineMeOH solution (1 mL) and then the resin was washed withDCM (3 times 5 mL) Fractions (TLC eluant 41 DCMMeOH) werecombined and concentrated under reduced pressure to yield a residuewhich was suspended in H2O and purified from the pyridinium salts bysize-exclusion chromatography (AMBERLITE XAD2 resin H2O thenMeOH) Evaporation of combined MeOH fractions containing theproduct afforded the side-chain protected compound (493 mg 33)as a white solid material The linear peptide (493 mg 05 mmol) wasdissolved under a nitrogen atmosphere in DMF (120 mL) HATU(1521 mg 04 mmol 08 equiv) and TBTU (354 mg 11 mmol 22equiv) and DIPEA (257 μL 15 mmol 3 equiv) were added Theresulting mixture was stirred for two days at room temperature Thesolvent was distilled off under reduced pressure and the residue wasdissolved in H2O (60 mL) and extracted with EtOAc (3 times 80 mL)The organic phase was washed twice with 5 NaHCO3 dried withNa2SO4 and evaporated under reduced pressure The crude residuewas purified by flash column chromatography (91 DCMminusMeOH Rf037) to give the protected cyclic peptide (140 mg 30) as a yellowsolid material HPLC tR = 2430 purity 97 Side chain protectedcyclic peptide (140 mg 015 mmol) was treated with 952525 TFAH2OTIS mixture (20 mL) for 2 h The reaction mixture wasevaporated under reduced pressure and the residue was dissolved inH2O (60 mL) The aqueous phase was washed with iPr2O (3 times 60mL) and freeze-dried The crude residue was purified by semi-preparative HPLC to give the side-chain deprotected 2 (30 mg 34)as a white solid Trifluoroacetate ion was replaced with chloride ion byion-exchange chromatography (AMBERLITE IRA-96 resin chlorideform) See Table 1 for 1H and 13C NMR data ESI-MS mz 60514 (M+ H+ 100) ESI-MSMS mz 6055 (M + H+ 1) 5880 (100) 5770(30) 5700 (17) 5599 (42) 5460 (11) 533 (12) 5200 (72) Anal

Table 1 1H and 13C Chemical Shifts of 2 and 3 in DMSO-d6 Solutions at 298 K1H 2 1H 3 13C 2 13C 3

H-2 428 337 428 334 C-2 688 690H-3 459 466 C-3 535 537H-5 308 316 308 C-5 668 664H-6 393 370 390 372 C-6 682 684Gly NH 873 861Arg NH 740 856Asp NH 865 866D-Tyr NH 719 735Gly H-α 407 399 310 Gly C-α 445 441Arg H-α 399 401 Arg C-α 533 537Arg H-βγ 165 150 163 143 Arg C-βγ 249 220 248 212Arg H-δ 304 306 Arg C-δ 405 401Asp H-α 478 471 Asp C-α 570 567Asp H-β 282 270 288 256 Asp C-β 396 398D-Tyr H-α 447 451 D-Tyr C-α 494 504D-Tyr H-β 214 232 D-Tyr C-β 287 358D-Tyr H-Ar 695 660 766 734 724 D-Tyr C-Ar 1153 1301 1158 1298 1391

Journal of Medicinal Chemistry Article

dxdoiorg101021jm2016232 | J Med Chem 2012 55 5024minus50335029

Calcd for C26H37ClN8O9 C 4871 H 582 N 1748 Found C4888 H 591 N 1732 HPLC tR = 121 95 purityc[RGD-(3primeI-y)-(3R)-carboxymorpholine] (3) A solution of

ligand 2 (120 mg 0020 mmol) in PBS (2 mL) was added to sodiumiodide (307 mg 0020 mmol) followed by chloramine-T hydrate(466 mg 0020 mmol) The reaction mixture was stirred at roomtemperature for 20 min and then quenched with a solution of 5sodium metabisulphite (08 mL) The mixture was extracted withDCM (3 times 5 mL) and loaded on a preconditioned C18 Sep-pakcolumn The C18 column was washed with water (6 mL) andsubsequently eluted with MeOH The crude product was purified bysemipreparative HPLC to obtain ligand 3 (467 mg 32) See Table 1for 1H and 13C NMR data ESI-MS mz 73125 (M + H+ 100) ESI-MSMS mz 7312 (M + H+ 15) 7141 (100) 6859 (39) 6721 (22)6461 (76) Anal Calcd for C26H36ClIN8O9 C 4072 H 473 N1461 Found C 4081 H 483 N 1412 HPLC tR = 152 96purityc[RGD-(3prime125I-y)-(3R)-carboxymorpholine] (125I-3) A solution

of PBS (100 μL 005 M) containing Na125I (20 μL 2 mCi 0918 nM)peptide 2 (10 μL of a 005 M PBS solution 165 nmol) andchloramine-T (188 μL of a freshly prepared 005 M PBS solution 824nmol 9 equiv) was prepared The reaction mixture was stirred at roomtemperature for 20 min and then quenched by adding a solution ofsodium metabisulphite (218 μL of a freshly prepared 005 M PBSsolution 824 nmol 9 equiv) The mixture was extracted with DCM (3times 05 mL) and purified by HPLC and the radioactivity of fractionscollected every 30 s was determined with a γ-counter (SupportingInformation Figure S1) Fractions containing the chemically pure 125I-3 were pooled together (155 mCi 78 radiochemical yield) and usedfor in vivo experiments after solvent removal HPLC tR = 152 98radiochemical purity gt99 chemical puritySolid-Phase Integrin Binding Assay [125I]-Echistatin labeled by

the lactoperoxidase method24 to a specific activity of 2000 Cimmolwas purchased from GE Healthcare and integrin αvβ3 from humanplacenta was purchased from Chemicon International Inc TemeculaCAPurified αvβ3 integrin was diluted in coating buffer (20 mM Tris

(pH 74) 150 mM NaCl 2 mM CaCl2 1 mM MgCl2 1 mM MnCl2)at concentrations of 500 or 1000 ngmL An aliquot of diluted integrin(100 μLwell) was added to a 96-well microtiter plate (Optiplate-96HB PerkinElmer Life Sciences Boston MA) and plates wereincubated overnight at 4 degC Plates were washed once withblockingbinding buffer (20 mM Tris (pH 74) 150 mM NaCl 2mM CaCl2 1 mM MgCl2 1 mM MnCl2 1 BSA) and incubated atroom temperature for additional 2 h Plates were rinsed twice withsame buffer and then competition binding assays were performed witha constant concentration of [125I]-Echistatin (005 nM) Concen-trations of tested compound ranged from 001 to 100 nM All assayswere performed in triplicate in a final volume of 02 mL eachcontaining the following species 005 mL of [125I]-Echistatin 004 mLof tested compound and 011 mL of blockingbinding bufferNonspecific binding was defined as [125I]-Echistatin bound in presenceof an excess (1 μM) of unlabeled echistatin After incubation at roomtemperature for 3 h plates were washed three times with blockingbinding buffer and counted in a Top-Count NXT microplatescintillation counter (PerkinElmer Life Sciences Boston MA) using200 μLwell of MicroScint-40 liquid scintillation (PerkinElmer LifeSciences Boston MA)Data are shown as means plusmn SD from three independent

experiments IC50 values were determined by fitting binding inhibitiondata by nonlinear regression using GraphPad Prism 40 SoftwarePackage (GraphPad Prism San Diego CA)Cell Lines and Culture Conditions The A375 M human

melanoma cell line was obtained from the American Type CultureCollection (ATCC Rockville MD)12g Human dermal skin fibroblasts(HDF) were kindly donated by Dr Daniela Monti Department ofExperimental Pathology and Oncology University of FlorenceMelanoma cells and HDF were grown in Dulbeccorsquos Modified EagleMedium containing 4500 mgL glucose (DMEM 4500 GIBCO)supplemented with 10 fetal calf serum (FCS) at 37 degC in a

humidified incubator containing 10 CO2 Then 50 times 105 melanomacells or 1 times 106 HDF were seeded in 100 mm dishes and propagatedevery 3 days by incubation with a trypsinminusEDTA solution Cultureswere periodically monitored for mycoplasma contamination Primarycultures of human umbilical vein endothelial cells (HUVEC) wereobtained from Lonza (USA) HUVEC grown in endothelial cellgrowth medium-2 (EGM-2) containing endothelial cell growthsupplements were subcultivated using a trypsin-EDTA solution 13split ratio Cells between passages 2 and 4 grown to confluence inplates coated with 1 bovine gelatin (Sigma St Louis) were used inthe experiments

RNA Isolation and Polymerase Chain Reaction (PCR) TotalRNA extracted using RNAgents (Total RNA Isolation SystemPromega Madison WI) was determined spectrophotometricallyComplementary DNA (cDNA) was synthesized from 2 μg of totalRNA using 1 μL of ImProm-II reverse transcriptase (Promega)Aliquots of 2 μL of the cDNA were used for PCR amplification Thespecific primers used for the identification of human αv α5 β1 β3 β5and GAPDH are reported in Table S1 of Supporting Informations AllPCR experiments were conducted using 005 UμL of Go-TaqPolymerase (Promega) Amplification was carried out on a Perkin-Elmer Thermal cycler Then 8 μL of each PCR products werevisualized after electrophoresis in a 2 agarose gel containing 05 μgmL of ethidium bromide cDNA products were evaluated on the basisof a standard PCR marker

Flow Cytometry Assay Tumor cells were detached by gentletreatment with Accutase a 05 mM EDTA solution washed andincubated for 1 h at 4 degC in the presence of anti αvβ3 monoclonalantibody (1 μg50 μL Anti-Integrin αvβ3 clone LM609 Millipore)Cells were then washed and incubated for 1 h at 4 degC with a secondaryantibody 5 μgmL goat antimouse IgG conjugated with FITC (SantaCruz Biotechnology Inc Santa Cruz CA) αvβ3 -positive cells wereanalyzed at 488 nm on the flow cytometer FACScan system (BD-FACS Canto)

Cell Adhesion Assay Plates (96 wells) were coated withfibronectin (1 μgmL) or vitronectin (10 μgmL) by overnightincubation at 4 degC and then incubated at 37 degC for 1 h with 1 BSA inPBS Melanoma cells IL-1β (400 UmL)-stimulated HUVECs orhuman dermal fibroblasts (HDF) were resuspended in serum-freemedium and exposed to ligands 1 2 or 3 (final concentration was 011 or 10 μM) at 37 degC for 30 min to allow for ligandminusintegrinequilibrium to be reached Melanoma cell adhesion assays wereperformed in the presence or in the absence of 2 mmolL MnCl2Cells were plated (4minus5 times 104 cellswell) and incubated at 37 degC for 2h Nonadherent cells were removed with PBS and adherent cells werestained with 05 crystal violet solution in 20 methanol After 2 h ofincubation at 4 degC plates were examined at 540 nm in a counterELX800 (Biotek Instruments) Experiments were conducted intriplicate and repeated at least three times Data are presented asmeans plusmn SD from three independent experiments

Animal Model and Study Design In vivo experiments wereconducted in accordance with national guidelines and approved by theEthical Committee of the Animal Welfare Office of the Italian WorkMinistry and conforming to legal mandates and Italian guidelines forcare and maintenance of laboratory animals Eighteen-week-old SCIDbgbg mice (Charles River Laboratories International) were injectedsubcutaneously in the shoulder area with a suspension of melanomacells and 20 ngmL TGFβ-activated fibroblasts Then 1 times106 A375 Mmelanoma cells and 05 times 106 HDF were previously resuspended in100 μL of PBS diluted 11 with a Matrigel solution (BD Biosciences)Animals were kept under pathogen-free conditions and allowed to feedfreely Tumor growth was monitored daily

Small-Animal SPECT and CT Studies In vivo studies wereconducted using a ldquohybridrdquo imaging system for small laboratoryanimals allowing for the study of biological processes using minimallyinvasive procedures Engrafted tumor tracer uptake was studied atdifferent time-points after tail vein injection Binding specificity tointegrin receptors was evaluated by in vivo displacement studies inwhich a high dose of cold tracer was injected 10 min beforeradiolabeled one Imaging studies were performed using the Flex

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Triumph preclinical imaging system which combines positron (PET)and single-photon emission (SPECT) tomography with the emissiontomography (CT) for anatomic coregistration (GE HealthcareGamma Medica-Ideas Waukesha WI) 125I-iodinated tracers 125I-3was studied in vivo by SPECT allowing for use directly the in vitrotracer data to the in vivo experimentation Imaging studies requiredmonitoring of anesthetized animals throughout the imaging procedureand anesthesia was conducted in accordance with National Guidelinesand approved by the Ethical Committee Given that anesthesia ingeneral causes hypothermia in small animals appropriate warming wasprovided during imaging procedure by using a heating bed set at 37degC Physiological monitoring was achieved by measuring respirationrate with a pressure pad placed under the mouse abdomen Saturationexperiments (SE) to visualize tumors and displacement experiments(DE) were performed In SE 125I-3 (7minus10 MBq in 200 μL ofphysiological saline) was injected via tail vein into tumor bearinganimals kept under anesthesia After 30 min a CT aquisition wasperformed followed by SPECT scan starting at 40 min pi CT scanwas acquired with 50 kV and 320 μA tube settings The magnificationfactor was set to 2 resulting in an axial field of view of 592 mm 512projections 500 μs each were acquired on a circular orbit in 256 swith an acquisition matrix of 1184 times 1120 100 μm pixel SPECTprojections were acquired with a 5-pinhole high-resolution collimator(1 mm hole diameter 75 mm focal distance) with radius of rotation of40 mm resulting in an axial field of view of 529 mm then 64projections 20 s each were acquired on a circular orbit for a totalacquisition time of about 22 min Acquisition matrix was 80 times 80 15mm pixel size Energy window was set on the 125I photopeak rangingfrom 20 to 36 KeV The DE were performed by injecting 18 mgkg ofcompound 3 10 min before injection of tracer Once scanning wascomplete the animals were recovered in a housing unit and monitoreduntil awakeData Analysis of Small-Animal SPECT and CT Studies CT

image data set was reconstructed using a cone-beam FBP algorithm(FDK) and an optimum noise filter Reconstruction matrix was 512 times512 times 512 180 μm pixel size SPECT images were reconstructed withordered subset expectation maximization algorithm (8 subsets and 5iterations) and a reconstruction matrix of 60 times 60 times 60 15 mm pixelsize No correction for radiation attenuation and scatter was appliedFused images were visually analyzed to detect the engrafted tumormass and regional SPECT activities were measured using the analysissoftware Vivid (Gamma Medica-Ideas Northridge CA) by delineatingthe region of interest (ROI) of tumor in fused CT images A similarROI was generated for muscle tissue on contralateral side of tumorTumor ROI activity was normalized (nTA) with the followingformula

= minusnTA

TA BABA

where TA is tumor activity and BA is background activity measured inmuscle tissue contralateral to tumorDocking Calculations Automated docking studies were carried

out by Autodock 401 program17 using the Lamarckian GeneticAlgorithm (LGA) as a search engine The AutoDockTools 145(ADT) graphical interface25 was used to prepare integrin and ligandsPDBQT files Coordinates of ligand 3 were retrieved from lowestenergy conformers resulting from molecular mechanics calculationsstarting from geometry of ligand 1 whereas coordinates of αvβ3receptor were retrieved from the Protein Data Bank (PDB code1L5G) and ligandminusprotein complex was unmerged for achieving freereceptor structure Water molecules were removed For proteinreceptor and ligand 3 all hydrogens were added Gasteiger chargeswere computed and nonpolar hydrogens were merged A charge valueof +20 to each Mn atom of protein receptor was successively addedThree-dimensional energy scoring grids of 0375 Aring resolution and 40Aring times 40 Aring times 40 Aring dimensions were computed Center of grid was set tobe coincident with mass center of ligands preliminary fitted on the X-ray structure of c[RGDf(Me)V] in the αvβ3 complex (1L5G) A totalof 50 runs with a maximum of 2500000 energy evaluations werecarried out for each ligand using the default parameters for LGA

Cluster analysis was performed on docked results using a root-mean-square (rms) tolerance of 15 Aring Analysis of binding mode calculationof binding energy and prediction of binding activity of dockedconformations were carried out using PyMol Autodock Tools pluginwithin PyMol software26

ASSOCIATED CONTENTS Supporting InformationAdditional experimental methods radiochromatogram of 125I-3and additional cell biology assays data This material is availablefree of charge via the Internet at httppubsacsorg

AUTHOR INFORMATIONCorresponding AuthorFor AG phone +39 055 4573481 fax +39 055 4573569 E-mail antonioguarnaunifiit For AP phone +39 0557947527 fax +39 055 4224392 E-mail apupidfcunifiitFor LC phone +39 055 4598207 fax +39 055 4598900 E-mail lidocaloriniunifiit

NotesThe authors declare no competing financial interest

ACKNOWLEDGMENTSThis work has received financial support from the IstitutoTo s c ano Tumor i a nd MIUR (PR IN08 G r an t2008J4YNJY_003) Also financial contributions from CINM-PIS University of Florence and Fondazione Roma areacknowledged Dr Alessandro Sisti is acknowledged forcarrying out preliminary labeling experiments Ente Cassa diRisparmio is acknowledged for the granting the MicroPETSPECTCT instrument

ABBREVIATIONS USEDSPECT single positron emission computed tomography PETpositon emission tomography RGD Arg-Gly-Asp VGPvertical growth phase CT computed tomography VNvitronectin FN fibronectin HUVEC human umbilical veinendothelial cells IL-1β interleukin-1β ESI-MSMS electroprayionization tandem mass spectrometry EGM-2 endothelial cellgrowth medium-2 EDTA ethylenediaminetetraacetic acidGAPDH glyceraldehyde 3-phosphate dehydrogenase FACSfluorescence-activated cell sorting FITC fluorescein isothio-cyanate PBS phosphate buffered saline HUVEC humanumbilical vein endothelial cells HDF human dermal skinfibroblasts EMT epithelial-mesenchymal transition TGFtransform grown factor pi post injection

REFERENCES(1) Challenges and Opportunities Report US Department of Healthand Human Services Food and Drug Administration Silver SpringMD March 2004 httpwwwfdagovScienceResearchSpecialTopicsCriticalPathInitiativeCriticalPathOpportunities-Reports-ucm077262htm(2) (a) Backgrounder How New Drugs Move Through the Developmentand Approval Process Tufts Center for the Study of DrugDevelopment Boston November 1 2001 (b) Gilbert J HenskeP Singh A Rebuilding Big Pharmarsquos Business Model The Business ampMedicine Report Windhover Information November 2003 Vol 21 no10 httpwwwbaincomImagesrebuilding_big_pharmapdf(3) Innovative drug development approaches-project Final report fromTHEEMEACHMP-THINK-TANK group on innovative drug develop-ment EMEA 2007 pp 1minus26 httpwwwemaeuropaeudocsen_GBdocument_libraryOther200910WC500004913pdf

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dxdoiorg101021jm2016232 | J Med Chem 2012 55 5024minus50335031

whereas the inhibition of cell adhesion to fibronectin is lesseffective Ligands 2 and 3 expressed an inhibitory ability ontumor cell adhesion comparable to that of ligand 1 These

results suggest that ligands 1 2 and 3 display a quitecomparable affinity for αvβ3 integrin and the addition of iodinedoes not modify the affinity in living cells A ldquoscratch woundrdquo

Figure 4 Adhesion of A375 M melanoma cells HUVECs and dermal fibroblasts to vitronectin and fibronectin in the presence of compounds 1 2or 3 The values are expressed as inhibition plusmn SEM of cell adhesion relative to control (untreated cells) Significantly different at p lt 005 ascompared to compound 1

Figure 5 Radioactivity biodistribution evaluated in a healthy mouse at 60 120 and 300 min and 24 h after 125I-3 injection

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model was used to examine the effect of D-morpholine-containing RGD-ligands on in vitro migration of melanomacells Tumor cells exposed to ligands 1 2 and 3 did not modifytheir migration rate with respect to nonexposed tumor cells(control tumor cells) The migration rate (as represented bythe reduction of scratch width) and the morphology of tumorcells exposed to D-morpholine-containing RGD-ligands werefound comparable at 10 μM 1 μM and 10 nM concentrationof the ligand with respect to control tumor cells (Figure S2Supporting Information)Adhesion and in Vitro Tubulogenesis of HUVEC in the

Presence of Morpholine-Based RGD-Cyclopeptides 1minus3 IL-1β-stimulated HUVECs express integrin subunits (αv β3 β5 α5β1) and αvβ3 heterodimer Ligands 1 2 and 3 suppressed theadhesion of IL-1β-stimulated HUVECs to vitronectin whereasthey did not completely suppress the adhesion of activatedendothelial cells to fibronectin (Figure 4) We can speculatethat D-morpholine-based RGD-cyclopeptides act preferentiallyon the endothelial αvβ3 integrin instead of the α5β1 integrin Toassess whether ligands 1 2 and 3 were able to exert anantiangiogenic activity we examined the ability of thesepeptides to repress in vitro tube formation by HUVEC seededon a Matrigel layer Figure S3 of Supporting Information showsthat tube formation by HUVEC was not inhibited by D-morpholine-containing RGD-ligandsAdhesion of HDFs in the Presence of Morpholine-Based

RGD-Cyclopeptides As shown in Figure 4 ligand 1 suppressedthe adhesion of HDFs to vitronectin and fibronectin with ahigh efficiency whereas ligand 2 and 3 expressed a significantlyreduced efficiencyBiodistribution and Melanoma Imaging The radio-

activity distribution was evaluated in a nontumor-bearingmouse at 60 120 and 300 min and 24 h post injection (pi)(Figure 5) The accumulation in gallbladder and kidneysremained consistent for at least two hours pi and the tracer wasexcreted via renal and hepatobiliary routes allowing for a fastclearance of nonspecific bound tracer Accumulation in thespleen might be due to the structural similarity of integrin αvβ3to αIIββ3 integrin expressed on spleen-enriched platelets Therelease of the 125I atom from the molecule and its accumulationin the thyroid gland increased with time a phenomenon thatcan be avoided by the preadministration of a solution of KI (4mgL) At 24 h pi little radioactivity remained in the organismmainly in the thyroid and bowelFigure 6 shows the localization of the 125I-3 compound in the

site of the implanted tumor as determined by micro SPECT-CT imaging The images clearly show that the 125I-3 ligand hasthe capacity to monitor in vivo the growth of αvβ3 integrin-positive melanoma cells Binding studies showed a gooddisplacement of the radiotracer by the parent nonradioactivecompound with a mean ratio of saturated to displacedreceptors in tumor tissue of 5753 plusmn 06 and having thecontrolateral muscle as the reference tissue

DISCUSSION AND CONCLUSIONSAs reported αvβ3 integrin-targeted radiotracers are able toreveal a tumor mass and to monitor growth and diffusion oftumor cells19 We tested D-morpholine-based RGD-cyclo-pentapeptides 1 2 and 3 in in vitro and in vivo assays andthey were found to inhibit the adhesion of human melanomacells to RGD substrates such as vitronectin and fibronectionThese ligands proved to be more specific for αvβ3 than α5β1integrin ldquoScratch woundrdquo experiments revealed that melanoma

cells exposed to ligands 1 2 and 3 did not modify theirmigration rate as compared to nonexposed tumor cells Severalinteractions operate between tumor cells and the extracellularmatrix thus the inhibition of αvβ3 integrin-mediated celladhesion per se might not be sufficient to influence tumor cellmigration6c20 Ligands 1 2 and 3 also repressed the adhesionof IL-1β-stimulated HUVEC to vitronectin whereas inhibitionof endothelial cell adhesion on fibronectin was not significantAt the same time ligands 1 2 and 3 did not block in vitrocapillary-like tube formation by HUVEC We assume that thereduced affinity of D-morpholine-based RGD-cyclopentapep-tides for α5β1 integrin fails to influence in vitro angiogenesisIndeed it has been found that only simultaneous inhibition ofboth integrins reduces capillary tube formation1112h21

Tumor cells recruit host inflammatory and stromal cellswhich generate a microenvironment able to promote growthand dissemination of cancer cells22 Because fibroblastsrepresent the most abundant host cells within a tumormicroenvironment23 we injected a suspension of tumor cellsand αvβ3 integrin expressing HDF into immunodeficient miceto test in vivo behavior of 125I-3 ligand by micro-SPECTCTanalysis We found a high tracer retention within the tumormass with a satisfying tumor to background ratio Co-injectionof 125I-3 and the corresponding unlabeled ligand 3 showed highselectivity of this ligand for αvβ3 expressing tumor cellsThus the 125I-3 ligand may represent a new radio-

pharmaceutical with the potential of early detection of atumor mass and of monitoring the therapeutic responseMoreover high affinity of this radiotracer for αvβ3 integrinwhich is characteristic of tumor endothelial cells can be avaluable diagnostic tool in a large population of cancersincluding tumors with low level of αvβ3-expressing cells Theseconsiderations extend the applications of 125I-3 from melanomato several tumor histotypesFinally the D-Mor-containing cyclopentapeptide ligand 3

might be used in novel therapeutic strategies upon labeling with131I radioisotope

EXPERIMENTAL SECTIONGeneral H-Gly-2-Cl-Trt resin (11 mmolg) was purchased from

Fluka Chromatographic separations were performed on silica gel

Figure 6 Transaxial slices through mouse chest at implantedmelanoma tumor level (arrows) In upper panels CT images inlower panels fused CT and SPECT images Images were acquired at 40min after tail vein injection of 125I-3 (250 μCi in 02 mL of saline) Thedisplacement study was achieved with an excess of cold ligand (18 mgkg) injected 10 min before the tracer

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(Kieselgel 60 Merck) using flash-column techniques Rf values refer toTLC carried out on 25 mm silica gel plates (Merck F254) with sameeluant as for column chromatography Solid-phase reactions werecarried out on a shaker using solvents of HPLC quality BromophenolBlue (BB) test was performed as follows a small amount of resinbeads were suspended in a vial with 05 mL of DMF two drops of a1 solution of bromophenol blue in dimethylacetamide were addedand the sample was observed The BB test was considered positive(presence of free amino groups) when the resin beads turned blue andnegative (absence of free amino groups) when the beads remainedcolorless ESI mass spectra were carried out on a ion-trap doublequadrupole mass spectrometer using electrospray (ES+) ionizationtechniques and a normalized collision energy within the range of 25minus32 eV for MSMS experiments Ligands 2 and 3 were purified byBeckman-Gold HPLC system equipped with a reverse-phase column(Alltima C18 10 μm 250 mm times 10 mm Alltech) using H2OCH3CNgradient eluant buffered with 01 TFA (flow rate 25 mLmin λ 254nm gradient eluant CH3CN 105 min CH3CN 10minus9025 min)Analytical HPLC analyses were performed on Dionex Ulltimate 3000system equipped with a reverse-phase column (Alltima C18 5 μm 250mm times 46 mm Alltech) and the same gradient eluant Ligand 125I-3was purified by Beckman-Gold HPLC system equipped with a reverse-phase column (C-8 Waters Spherisorb S5 250 mm times 46 mm) and thesame gradient eluant Na125I in 02N NaOH solution was purchasedfrom PerkinElmer (10 μL 100 mCimL 17 Ci (629GBq)mg 125I97 126I lt 3 radiochemical purity gt99 carrier free) HPLCanalysis was used to determine purity and all tested compoundspossessed gt95 purityc[RGDy-(3R)-carboxymorpholine] (2) H-Gly-2-Cl-Trt resin

(466 mg 05 mmol) was used as starting material Fmoc-deprotectionsand completion of each coupling reaction were assessed by performinga BB test Coupling was performed with a solution consisting of N-Fmoc-Arg(Pbf)-OH (973 mg 15 mmol 3 equiv) HOBt (203 mg 15mmol 3 equiv) in DMF (3 mL) and DIC (203 μL 15 mmol 3equiv) was added dropwise at 0 degC The resulting mixture was stirredfor 10 min at 0 degC and for a further 10 min at room temperature andthen was added to the resin This mixture was shaken at roomtemperature for 24 h The solution was drained and the resin wassequentially washed with 5 DIPEA in DMF (3 times 5 mL) and DMF (5times 5 mL) Fmoc deprotection was performed with 30 piperidine inDMF (10 mL) for 30 min followed by resin washings with DMF (3 times10 mL) After deprotection H-Arg(Pbf)-Gly-2-Cl-Trt resin wassuspended in a solution of HOBt (3568 mg 264 mmol 6 equiv)DIC (4156 μL 264 mmol 6 equiv) and N-Fmoc-(R)-morpholine-3-carboxylic acid (465 mg 132 mmol 264 equiv) in DMF (3 mL) andshaken for two days The solution was drained and resin was washedwith 5 DIPEA in DMF (3 times 5 mL) and DMF (5 times 5 mL) After

deprotection H-(3R)-carboxymorpholine-Arg(Pbf)-Gly-2-Cl-Trt resinwas suspended in a solution of HOBt (405 mg 3 mmol 6 equiv) DIC(472 μL 3 mmol 6 equiv) and N-Fmoc-D-Tyr(t-Bu)-OH (689 mg15 mmol 3 equiv) in DMF (3 mL) and shaken for three days Thesolution was drained and the resin washed with 5 DIPEA in DMF (3times 5 mL) and DMF (5 times 5 mL) After deprotection H-D-Tyr(t-Bu)-(3R)-carboxymorpholine-Arg(Pbf)-Gly-2-Cl-Trt resin was suspendedin a solution of TBTU (8027 mg 25 mmol 5 equiv) N-Fmoc-Asp(t-Bu)-OH (1028 g 25 mmol 5 equiv) and DIPEA (428 μL 25 mmol5 equiv) in DMF (3 mL) and shaken for three days The solution wasdrained and resin washed with 5 DIPEA in DMF (3 times 5 mL) andDMF (5 times 5 mL) N-Fmoc-Asp(t-Bu)-D-Tyr(t-Bu)-(3R)-carboxymor-pholine-Arg(Pbf)-Gly-2-Cl-Trt resin was deprotected and washed withDMF (5 times 5 mL) and DCM (5 times 5 mL) In a solid-phase reactionvessel H-Asp(t-Bu)-D-Phe-(3R)-carboxymorpholine-Arg(Pbf)-Gly-2-Cl-Trt resin was treated with 5 mL of 1 TFADCM solution (10times 2 min) The filtrates were immediately neutralized with a 10pyridineMeOH solution (1 mL) and then the resin was washed withDCM (3 times 5 mL) Fractions (TLC eluant 41 DCMMeOH) werecombined and concentrated under reduced pressure to yield a residuewhich was suspended in H2O and purified from the pyridinium salts bysize-exclusion chromatography (AMBERLITE XAD2 resin H2O thenMeOH) Evaporation of combined MeOH fractions containing theproduct afforded the side-chain protected compound (493 mg 33)as a white solid material The linear peptide (493 mg 05 mmol) wasdissolved under a nitrogen atmosphere in DMF (120 mL) HATU(1521 mg 04 mmol 08 equiv) and TBTU (354 mg 11 mmol 22equiv) and DIPEA (257 μL 15 mmol 3 equiv) were added Theresulting mixture was stirred for two days at room temperature Thesolvent was distilled off under reduced pressure and the residue wasdissolved in H2O (60 mL) and extracted with EtOAc (3 times 80 mL)The organic phase was washed twice with 5 NaHCO3 dried withNa2SO4 and evaporated under reduced pressure The crude residuewas purified by flash column chromatography (91 DCMminusMeOH Rf037) to give the protected cyclic peptide (140 mg 30) as a yellowsolid material HPLC tR = 2430 purity 97 Side chain protectedcyclic peptide (140 mg 015 mmol) was treated with 952525 TFAH2OTIS mixture (20 mL) for 2 h The reaction mixture wasevaporated under reduced pressure and the residue was dissolved inH2O (60 mL) The aqueous phase was washed with iPr2O (3 times 60mL) and freeze-dried The crude residue was purified by semi-preparative HPLC to give the side-chain deprotected 2 (30 mg 34)as a white solid Trifluoroacetate ion was replaced with chloride ion byion-exchange chromatography (AMBERLITE IRA-96 resin chlorideform) See Table 1 for 1H and 13C NMR data ESI-MS mz 60514 (M+ H+ 100) ESI-MSMS mz 6055 (M + H+ 1) 5880 (100) 5770(30) 5700 (17) 5599 (42) 5460 (11) 533 (12) 5200 (72) Anal

Table 1 1H and 13C Chemical Shifts of 2 and 3 in DMSO-d6 Solutions at 298 K1H 2 1H 3 13C 2 13C 3

H-2 428 337 428 334 C-2 688 690H-3 459 466 C-3 535 537H-5 308 316 308 C-5 668 664H-6 393 370 390 372 C-6 682 684Gly NH 873 861Arg NH 740 856Asp NH 865 866D-Tyr NH 719 735Gly H-α 407 399 310 Gly C-α 445 441Arg H-α 399 401 Arg C-α 533 537Arg H-βγ 165 150 163 143 Arg C-βγ 249 220 248 212Arg H-δ 304 306 Arg C-δ 405 401Asp H-α 478 471 Asp C-α 570 567Asp H-β 282 270 288 256 Asp C-β 396 398D-Tyr H-α 447 451 D-Tyr C-α 494 504D-Tyr H-β 214 232 D-Tyr C-β 287 358D-Tyr H-Ar 695 660 766 734 724 D-Tyr C-Ar 1153 1301 1158 1298 1391

Journal of Medicinal Chemistry Article

dxdoiorg101021jm2016232 | J Med Chem 2012 55 5024minus50335029

Calcd for C26H37ClN8O9 C 4871 H 582 N 1748 Found C4888 H 591 N 1732 HPLC tR = 121 95 purityc[RGD-(3primeI-y)-(3R)-carboxymorpholine] (3) A solution of

ligand 2 (120 mg 0020 mmol) in PBS (2 mL) was added to sodiumiodide (307 mg 0020 mmol) followed by chloramine-T hydrate(466 mg 0020 mmol) The reaction mixture was stirred at roomtemperature for 20 min and then quenched with a solution of 5sodium metabisulphite (08 mL) The mixture was extracted withDCM (3 times 5 mL) and loaded on a preconditioned C18 Sep-pakcolumn The C18 column was washed with water (6 mL) andsubsequently eluted with MeOH The crude product was purified bysemipreparative HPLC to obtain ligand 3 (467 mg 32) See Table 1for 1H and 13C NMR data ESI-MS mz 73125 (M + H+ 100) ESI-MSMS mz 7312 (M + H+ 15) 7141 (100) 6859 (39) 6721 (22)6461 (76) Anal Calcd for C26H36ClIN8O9 C 4072 H 473 N1461 Found C 4081 H 483 N 1412 HPLC tR = 152 96purityc[RGD-(3prime125I-y)-(3R)-carboxymorpholine] (125I-3) A solution

of PBS (100 μL 005 M) containing Na125I (20 μL 2 mCi 0918 nM)peptide 2 (10 μL of a 005 M PBS solution 165 nmol) andchloramine-T (188 μL of a freshly prepared 005 M PBS solution 824nmol 9 equiv) was prepared The reaction mixture was stirred at roomtemperature for 20 min and then quenched by adding a solution ofsodium metabisulphite (218 μL of a freshly prepared 005 M PBSsolution 824 nmol 9 equiv) The mixture was extracted with DCM (3times 05 mL) and purified by HPLC and the radioactivity of fractionscollected every 30 s was determined with a γ-counter (SupportingInformation Figure S1) Fractions containing the chemically pure 125I-3 were pooled together (155 mCi 78 radiochemical yield) and usedfor in vivo experiments after solvent removal HPLC tR = 152 98radiochemical purity gt99 chemical puritySolid-Phase Integrin Binding Assay [125I]-Echistatin labeled by

the lactoperoxidase method24 to a specific activity of 2000 Cimmolwas purchased from GE Healthcare and integrin αvβ3 from humanplacenta was purchased from Chemicon International Inc TemeculaCAPurified αvβ3 integrin was diluted in coating buffer (20 mM Tris

(pH 74) 150 mM NaCl 2 mM CaCl2 1 mM MgCl2 1 mM MnCl2)at concentrations of 500 or 1000 ngmL An aliquot of diluted integrin(100 μLwell) was added to a 96-well microtiter plate (Optiplate-96HB PerkinElmer Life Sciences Boston MA) and plates wereincubated overnight at 4 degC Plates were washed once withblockingbinding buffer (20 mM Tris (pH 74) 150 mM NaCl 2mM CaCl2 1 mM MgCl2 1 mM MnCl2 1 BSA) and incubated atroom temperature for additional 2 h Plates were rinsed twice withsame buffer and then competition binding assays were performed witha constant concentration of [125I]-Echistatin (005 nM) Concen-trations of tested compound ranged from 001 to 100 nM All assayswere performed in triplicate in a final volume of 02 mL eachcontaining the following species 005 mL of [125I]-Echistatin 004 mLof tested compound and 011 mL of blockingbinding bufferNonspecific binding was defined as [125I]-Echistatin bound in presenceof an excess (1 μM) of unlabeled echistatin After incubation at roomtemperature for 3 h plates were washed three times with blockingbinding buffer and counted in a Top-Count NXT microplatescintillation counter (PerkinElmer Life Sciences Boston MA) using200 μLwell of MicroScint-40 liquid scintillation (PerkinElmer LifeSciences Boston MA)Data are shown as means plusmn SD from three independent

experiments IC50 values were determined by fitting binding inhibitiondata by nonlinear regression using GraphPad Prism 40 SoftwarePackage (GraphPad Prism San Diego CA)Cell Lines and Culture Conditions The A375 M human

melanoma cell line was obtained from the American Type CultureCollection (ATCC Rockville MD)12g Human dermal skin fibroblasts(HDF) were kindly donated by Dr Daniela Monti Department ofExperimental Pathology and Oncology University of FlorenceMelanoma cells and HDF were grown in Dulbeccorsquos Modified EagleMedium containing 4500 mgL glucose (DMEM 4500 GIBCO)supplemented with 10 fetal calf serum (FCS) at 37 degC in a

humidified incubator containing 10 CO2 Then 50 times 105 melanomacells or 1 times 106 HDF were seeded in 100 mm dishes and propagatedevery 3 days by incubation with a trypsinminusEDTA solution Cultureswere periodically monitored for mycoplasma contamination Primarycultures of human umbilical vein endothelial cells (HUVEC) wereobtained from Lonza (USA) HUVEC grown in endothelial cellgrowth medium-2 (EGM-2) containing endothelial cell growthsupplements were subcultivated using a trypsin-EDTA solution 13split ratio Cells between passages 2 and 4 grown to confluence inplates coated with 1 bovine gelatin (Sigma St Louis) were used inthe experiments

RNA Isolation and Polymerase Chain Reaction (PCR) TotalRNA extracted using RNAgents (Total RNA Isolation SystemPromega Madison WI) was determined spectrophotometricallyComplementary DNA (cDNA) was synthesized from 2 μg of totalRNA using 1 μL of ImProm-II reverse transcriptase (Promega)Aliquots of 2 μL of the cDNA were used for PCR amplification Thespecific primers used for the identification of human αv α5 β1 β3 β5and GAPDH are reported in Table S1 of Supporting Informations AllPCR experiments were conducted using 005 UμL of Go-TaqPolymerase (Promega) Amplification was carried out on a Perkin-Elmer Thermal cycler Then 8 μL of each PCR products werevisualized after electrophoresis in a 2 agarose gel containing 05 μgmL of ethidium bromide cDNA products were evaluated on the basisof a standard PCR marker

Flow Cytometry Assay Tumor cells were detached by gentletreatment with Accutase a 05 mM EDTA solution washed andincubated for 1 h at 4 degC in the presence of anti αvβ3 monoclonalantibody (1 μg50 μL Anti-Integrin αvβ3 clone LM609 Millipore)Cells were then washed and incubated for 1 h at 4 degC with a secondaryantibody 5 μgmL goat antimouse IgG conjugated with FITC (SantaCruz Biotechnology Inc Santa Cruz CA) αvβ3 -positive cells wereanalyzed at 488 nm on the flow cytometer FACScan system (BD-FACS Canto)

Cell Adhesion Assay Plates (96 wells) were coated withfibronectin (1 μgmL) or vitronectin (10 μgmL) by overnightincubation at 4 degC and then incubated at 37 degC for 1 h with 1 BSA inPBS Melanoma cells IL-1β (400 UmL)-stimulated HUVECs orhuman dermal fibroblasts (HDF) were resuspended in serum-freemedium and exposed to ligands 1 2 or 3 (final concentration was 011 or 10 μM) at 37 degC for 30 min to allow for ligandminusintegrinequilibrium to be reached Melanoma cell adhesion assays wereperformed in the presence or in the absence of 2 mmolL MnCl2Cells were plated (4minus5 times 104 cellswell) and incubated at 37 degC for 2h Nonadherent cells were removed with PBS and adherent cells werestained with 05 crystal violet solution in 20 methanol After 2 h ofincubation at 4 degC plates were examined at 540 nm in a counterELX800 (Biotek Instruments) Experiments were conducted intriplicate and repeated at least three times Data are presented asmeans plusmn SD from three independent experiments

Animal Model and Study Design In vivo experiments wereconducted in accordance with national guidelines and approved by theEthical Committee of the Animal Welfare Office of the Italian WorkMinistry and conforming to legal mandates and Italian guidelines forcare and maintenance of laboratory animals Eighteen-week-old SCIDbgbg mice (Charles River Laboratories International) were injectedsubcutaneously in the shoulder area with a suspension of melanomacells and 20 ngmL TGFβ-activated fibroblasts Then 1 times106 A375 Mmelanoma cells and 05 times 106 HDF were previously resuspended in100 μL of PBS diluted 11 with a Matrigel solution (BD Biosciences)Animals were kept under pathogen-free conditions and allowed to feedfreely Tumor growth was monitored daily

Small-Animal SPECT and CT Studies In vivo studies wereconducted using a ldquohybridrdquo imaging system for small laboratoryanimals allowing for the study of biological processes using minimallyinvasive procedures Engrafted tumor tracer uptake was studied atdifferent time-points after tail vein injection Binding specificity tointegrin receptors was evaluated by in vivo displacement studies inwhich a high dose of cold tracer was injected 10 min beforeradiolabeled one Imaging studies were performed using the Flex

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dxdoiorg101021jm2016232 | J Med Chem 2012 55 5024minus50335030

Triumph preclinical imaging system which combines positron (PET)and single-photon emission (SPECT) tomography with the emissiontomography (CT) for anatomic coregistration (GE HealthcareGamma Medica-Ideas Waukesha WI) 125I-iodinated tracers 125I-3was studied in vivo by SPECT allowing for use directly the in vitrotracer data to the in vivo experimentation Imaging studies requiredmonitoring of anesthetized animals throughout the imaging procedureand anesthesia was conducted in accordance with National Guidelinesand approved by the Ethical Committee Given that anesthesia ingeneral causes hypothermia in small animals appropriate warming wasprovided during imaging procedure by using a heating bed set at 37degC Physiological monitoring was achieved by measuring respirationrate with a pressure pad placed under the mouse abdomen Saturationexperiments (SE) to visualize tumors and displacement experiments(DE) were performed In SE 125I-3 (7minus10 MBq in 200 μL ofphysiological saline) was injected via tail vein into tumor bearinganimals kept under anesthesia After 30 min a CT aquisition wasperformed followed by SPECT scan starting at 40 min pi CT scanwas acquired with 50 kV and 320 μA tube settings The magnificationfactor was set to 2 resulting in an axial field of view of 592 mm 512projections 500 μs each were acquired on a circular orbit in 256 swith an acquisition matrix of 1184 times 1120 100 μm pixel SPECTprojections were acquired with a 5-pinhole high-resolution collimator(1 mm hole diameter 75 mm focal distance) with radius of rotation of40 mm resulting in an axial field of view of 529 mm then 64projections 20 s each were acquired on a circular orbit for a totalacquisition time of about 22 min Acquisition matrix was 80 times 80 15mm pixel size Energy window was set on the 125I photopeak rangingfrom 20 to 36 KeV The DE were performed by injecting 18 mgkg ofcompound 3 10 min before injection of tracer Once scanning wascomplete the animals were recovered in a housing unit and monitoreduntil awakeData Analysis of Small-Animal SPECT and CT Studies CT

image data set was reconstructed using a cone-beam FBP algorithm(FDK) and an optimum noise filter Reconstruction matrix was 512 times512 times 512 180 μm pixel size SPECT images were reconstructed withordered subset expectation maximization algorithm (8 subsets and 5iterations) and a reconstruction matrix of 60 times 60 times 60 15 mm pixelsize No correction for radiation attenuation and scatter was appliedFused images were visually analyzed to detect the engrafted tumormass and regional SPECT activities were measured using the analysissoftware Vivid (Gamma Medica-Ideas Northridge CA) by delineatingthe region of interest (ROI) of tumor in fused CT images A similarROI was generated for muscle tissue on contralateral side of tumorTumor ROI activity was normalized (nTA) with the followingformula

= minusnTA

TA BABA

where TA is tumor activity and BA is background activity measured inmuscle tissue contralateral to tumorDocking Calculations Automated docking studies were carried

out by Autodock 401 program17 using the Lamarckian GeneticAlgorithm (LGA) as a search engine The AutoDockTools 145(ADT) graphical interface25 was used to prepare integrin and ligandsPDBQT files Coordinates of ligand 3 were retrieved from lowestenergy conformers resulting from molecular mechanics calculationsstarting from geometry of ligand 1 whereas coordinates of αvβ3receptor were retrieved from the Protein Data Bank (PDB code1L5G) and ligandminusprotein complex was unmerged for achieving freereceptor structure Water molecules were removed For proteinreceptor and ligand 3 all hydrogens were added Gasteiger chargeswere computed and nonpolar hydrogens were merged A charge valueof +20 to each Mn atom of protein receptor was successively addedThree-dimensional energy scoring grids of 0375 Aring resolution and 40Aring times 40 Aring times 40 Aring dimensions were computed Center of grid was set tobe coincident with mass center of ligands preliminary fitted on the X-ray structure of c[RGDf(Me)V] in the αvβ3 complex (1L5G) A totalof 50 runs with a maximum of 2500000 energy evaluations werecarried out for each ligand using the default parameters for LGA

Cluster analysis was performed on docked results using a root-mean-square (rms) tolerance of 15 Aring Analysis of binding mode calculationof binding energy and prediction of binding activity of dockedconformations were carried out using PyMol Autodock Tools pluginwithin PyMol software26

ASSOCIATED CONTENTS Supporting InformationAdditional experimental methods radiochromatogram of 125I-3and additional cell biology assays data This material is availablefree of charge via the Internet at httppubsacsorg

AUTHOR INFORMATIONCorresponding AuthorFor AG phone +39 055 4573481 fax +39 055 4573569 E-mail antonioguarnaunifiit For AP phone +39 0557947527 fax +39 055 4224392 E-mail apupidfcunifiitFor LC phone +39 055 4598207 fax +39 055 4598900 E-mail lidocaloriniunifiit

NotesThe authors declare no competing financial interest

ACKNOWLEDGMENTSThis work has received financial support from the IstitutoTo s c ano Tumor i a nd MIUR (PR IN08 G r an t2008J4YNJY_003) Also financial contributions from CINM-PIS University of Florence and Fondazione Roma areacknowledged Dr Alessandro Sisti is acknowledged forcarrying out preliminary labeling experiments Ente Cassa diRisparmio is acknowledged for the granting the MicroPETSPECTCT instrument

ABBREVIATIONS USEDSPECT single positron emission computed tomography PETpositon emission tomography RGD Arg-Gly-Asp VGPvertical growth phase CT computed tomography VNvitronectin FN fibronectin HUVEC human umbilical veinendothelial cells IL-1β interleukin-1β ESI-MSMS electroprayionization tandem mass spectrometry EGM-2 endothelial cellgrowth medium-2 EDTA ethylenediaminetetraacetic acidGAPDH glyceraldehyde 3-phosphate dehydrogenase FACSfluorescence-activated cell sorting FITC fluorescein isothio-cyanate PBS phosphate buffered saline HUVEC humanumbilical vein endothelial cells HDF human dermal skinfibroblasts EMT epithelial-mesenchymal transition TGFtransform grown factor pi post injection

REFERENCES(1) Challenges and Opportunities Report US Department of Healthand Human Services Food and Drug Administration Silver SpringMD March 2004 httpwwwfdagovScienceResearchSpecialTopicsCriticalPathInitiativeCriticalPathOpportunities-Reports-ucm077262htm(2) (a) Backgrounder How New Drugs Move Through the Developmentand Approval Process Tufts Center for the Study of DrugDevelopment Boston November 1 2001 (b) Gilbert J HenskeP Singh A Rebuilding Big Pharmarsquos Business Model The Business ampMedicine Report Windhover Information November 2003 Vol 21 no10 httpwwwbaincomImagesrebuilding_big_pharmapdf(3) Innovative drug development approaches-project Final report fromTHEEMEACHMP-THINK-TANK group on innovative drug develop-ment EMEA 2007 pp 1minus26 httpwwwemaeuropaeudocsen_GBdocument_libraryOther200910WC500004913pdf

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dxdoiorg101021jm2016232 | J Med Chem 2012 55 5024minus50335031

model was used to examine the effect of D-morpholine-containing RGD-ligands on in vitro migration of melanomacells Tumor cells exposed to ligands 1 2 and 3 did not modifytheir migration rate with respect to nonexposed tumor cells(control tumor cells) The migration rate (as represented bythe reduction of scratch width) and the morphology of tumorcells exposed to D-morpholine-containing RGD-ligands werefound comparable at 10 μM 1 μM and 10 nM concentrationof the ligand with respect to control tumor cells (Figure S2Supporting Information)Adhesion and in Vitro Tubulogenesis of HUVEC in the

Presence of Morpholine-Based RGD-Cyclopeptides 1minus3 IL-1β-stimulated HUVECs express integrin subunits (αv β3 β5 α5β1) and αvβ3 heterodimer Ligands 1 2 and 3 suppressed theadhesion of IL-1β-stimulated HUVECs to vitronectin whereasthey did not completely suppress the adhesion of activatedendothelial cells to fibronectin (Figure 4) We can speculatethat D-morpholine-based RGD-cyclopeptides act preferentiallyon the endothelial αvβ3 integrin instead of the α5β1 integrin Toassess whether ligands 1 2 and 3 were able to exert anantiangiogenic activity we examined the ability of thesepeptides to repress in vitro tube formation by HUVEC seededon a Matrigel layer Figure S3 of Supporting Information showsthat tube formation by HUVEC was not inhibited by D-morpholine-containing RGD-ligandsAdhesion of HDFs in the Presence of Morpholine-Based

RGD-Cyclopeptides As shown in Figure 4 ligand 1 suppressedthe adhesion of HDFs to vitronectin and fibronectin with ahigh efficiency whereas ligand 2 and 3 expressed a significantlyreduced efficiencyBiodistribution and Melanoma Imaging The radio-

activity distribution was evaluated in a nontumor-bearingmouse at 60 120 and 300 min and 24 h post injection (pi)(Figure 5) The accumulation in gallbladder and kidneysremained consistent for at least two hours pi and the tracer wasexcreted via renal and hepatobiliary routes allowing for a fastclearance of nonspecific bound tracer Accumulation in thespleen might be due to the structural similarity of integrin αvβ3to αIIββ3 integrin expressed on spleen-enriched platelets Therelease of the 125I atom from the molecule and its accumulationin the thyroid gland increased with time a phenomenon thatcan be avoided by the preadministration of a solution of KI (4mgL) At 24 h pi little radioactivity remained in the organismmainly in the thyroid and bowelFigure 6 shows the localization of the 125I-3 compound in the

site of the implanted tumor as determined by micro SPECT-CT imaging The images clearly show that the 125I-3 ligand hasthe capacity to monitor in vivo the growth of αvβ3 integrin-positive melanoma cells Binding studies showed a gooddisplacement of the radiotracer by the parent nonradioactivecompound with a mean ratio of saturated to displacedreceptors in tumor tissue of 5753 plusmn 06 and having thecontrolateral muscle as the reference tissue

DISCUSSION AND CONCLUSIONSAs reported αvβ3 integrin-targeted radiotracers are able toreveal a tumor mass and to monitor growth and diffusion oftumor cells19 We tested D-morpholine-based RGD-cyclo-pentapeptides 1 2 and 3 in in vitro and in vivo assays andthey were found to inhibit the adhesion of human melanomacells to RGD substrates such as vitronectin and fibronectionThese ligands proved to be more specific for αvβ3 than α5β1integrin ldquoScratch woundrdquo experiments revealed that melanoma

cells exposed to ligands 1 2 and 3 did not modify theirmigration rate as compared to nonexposed tumor cells Severalinteractions operate between tumor cells and the extracellularmatrix thus the inhibition of αvβ3 integrin-mediated celladhesion per se might not be sufficient to influence tumor cellmigration6c20 Ligands 1 2 and 3 also repressed the adhesionof IL-1β-stimulated HUVEC to vitronectin whereas inhibitionof endothelial cell adhesion on fibronectin was not significantAt the same time ligands 1 2 and 3 did not block in vitrocapillary-like tube formation by HUVEC We assume that thereduced affinity of D-morpholine-based RGD-cyclopentapep-tides for α5β1 integrin fails to influence in vitro angiogenesisIndeed it has been found that only simultaneous inhibition ofboth integrins reduces capillary tube formation1112h21

Tumor cells recruit host inflammatory and stromal cellswhich generate a microenvironment able to promote growthand dissemination of cancer cells22 Because fibroblastsrepresent the most abundant host cells within a tumormicroenvironment23 we injected a suspension of tumor cellsand αvβ3 integrin expressing HDF into immunodeficient miceto test in vivo behavior of 125I-3 ligand by micro-SPECTCTanalysis We found a high tracer retention within the tumormass with a satisfying tumor to background ratio Co-injectionof 125I-3 and the corresponding unlabeled ligand 3 showed highselectivity of this ligand for αvβ3 expressing tumor cellsThus the 125I-3 ligand may represent a new radio-

pharmaceutical with the potential of early detection of atumor mass and of monitoring the therapeutic responseMoreover high affinity of this radiotracer for αvβ3 integrinwhich is characteristic of tumor endothelial cells can be avaluable diagnostic tool in a large population of cancersincluding tumors with low level of αvβ3-expressing cells Theseconsiderations extend the applications of 125I-3 from melanomato several tumor histotypesFinally the D-Mor-containing cyclopentapeptide ligand 3

might be used in novel therapeutic strategies upon labeling with131I radioisotope

EXPERIMENTAL SECTIONGeneral H-Gly-2-Cl-Trt resin (11 mmolg) was purchased from

Fluka Chromatographic separations were performed on silica gel

Figure 6 Transaxial slices through mouse chest at implantedmelanoma tumor level (arrows) In upper panels CT images inlower panels fused CT and SPECT images Images were acquired at 40min after tail vein injection of 125I-3 (250 μCi in 02 mL of saline) Thedisplacement study was achieved with an excess of cold ligand (18 mgkg) injected 10 min before the tracer

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dxdoiorg101021jm2016232 | J Med Chem 2012 55 5024minus50335028

(Kieselgel 60 Merck) using flash-column techniques Rf values refer toTLC carried out on 25 mm silica gel plates (Merck F254) with sameeluant as for column chromatography Solid-phase reactions werecarried out on a shaker using solvents of HPLC quality BromophenolBlue (BB) test was performed as follows a small amount of resinbeads were suspended in a vial with 05 mL of DMF two drops of a1 solution of bromophenol blue in dimethylacetamide were addedand the sample was observed The BB test was considered positive(presence of free amino groups) when the resin beads turned blue andnegative (absence of free amino groups) when the beads remainedcolorless ESI mass spectra were carried out on a ion-trap doublequadrupole mass spectrometer using electrospray (ES+) ionizationtechniques and a normalized collision energy within the range of 25minus32 eV for MSMS experiments Ligands 2 and 3 were purified byBeckman-Gold HPLC system equipped with a reverse-phase column(Alltima C18 10 μm 250 mm times 10 mm Alltech) using H2OCH3CNgradient eluant buffered with 01 TFA (flow rate 25 mLmin λ 254nm gradient eluant CH3CN 105 min CH3CN 10minus9025 min)Analytical HPLC analyses were performed on Dionex Ulltimate 3000system equipped with a reverse-phase column (Alltima C18 5 μm 250mm times 46 mm Alltech) and the same gradient eluant Ligand 125I-3was purified by Beckman-Gold HPLC system equipped with a reverse-phase column (C-8 Waters Spherisorb S5 250 mm times 46 mm) and thesame gradient eluant Na125I in 02N NaOH solution was purchasedfrom PerkinElmer (10 μL 100 mCimL 17 Ci (629GBq)mg 125I97 126I lt 3 radiochemical purity gt99 carrier free) HPLCanalysis was used to determine purity and all tested compoundspossessed gt95 purityc[RGDy-(3R)-carboxymorpholine] (2) H-Gly-2-Cl-Trt resin

(466 mg 05 mmol) was used as starting material Fmoc-deprotectionsand completion of each coupling reaction were assessed by performinga BB test Coupling was performed with a solution consisting of N-Fmoc-Arg(Pbf)-OH (973 mg 15 mmol 3 equiv) HOBt (203 mg 15mmol 3 equiv) in DMF (3 mL) and DIC (203 μL 15 mmol 3equiv) was added dropwise at 0 degC The resulting mixture was stirredfor 10 min at 0 degC and for a further 10 min at room temperature andthen was added to the resin This mixture was shaken at roomtemperature for 24 h The solution was drained and the resin wassequentially washed with 5 DIPEA in DMF (3 times 5 mL) and DMF (5times 5 mL) Fmoc deprotection was performed with 30 piperidine inDMF (10 mL) for 30 min followed by resin washings with DMF (3 times10 mL) After deprotection H-Arg(Pbf)-Gly-2-Cl-Trt resin wassuspended in a solution of HOBt (3568 mg 264 mmol 6 equiv)DIC (4156 μL 264 mmol 6 equiv) and N-Fmoc-(R)-morpholine-3-carboxylic acid (465 mg 132 mmol 264 equiv) in DMF (3 mL) andshaken for two days The solution was drained and resin was washedwith 5 DIPEA in DMF (3 times 5 mL) and DMF (5 times 5 mL) After

deprotection H-(3R)-carboxymorpholine-Arg(Pbf)-Gly-2-Cl-Trt resinwas suspended in a solution of HOBt (405 mg 3 mmol 6 equiv) DIC(472 μL 3 mmol 6 equiv) and N-Fmoc-D-Tyr(t-Bu)-OH (689 mg15 mmol 3 equiv) in DMF (3 mL) and shaken for three days Thesolution was drained and the resin washed with 5 DIPEA in DMF (3times 5 mL) and DMF (5 times 5 mL) After deprotection H-D-Tyr(t-Bu)-(3R)-carboxymorpholine-Arg(Pbf)-Gly-2-Cl-Trt resin was suspendedin a solution of TBTU (8027 mg 25 mmol 5 equiv) N-Fmoc-Asp(t-Bu)-OH (1028 g 25 mmol 5 equiv) and DIPEA (428 μL 25 mmol5 equiv) in DMF (3 mL) and shaken for three days The solution wasdrained and resin washed with 5 DIPEA in DMF (3 times 5 mL) andDMF (5 times 5 mL) N-Fmoc-Asp(t-Bu)-D-Tyr(t-Bu)-(3R)-carboxymor-pholine-Arg(Pbf)-Gly-2-Cl-Trt resin was deprotected and washed withDMF (5 times 5 mL) and DCM (5 times 5 mL) In a solid-phase reactionvessel H-Asp(t-Bu)-D-Phe-(3R)-carboxymorpholine-Arg(Pbf)-Gly-2-Cl-Trt resin was treated with 5 mL of 1 TFADCM solution (10times 2 min) The filtrates were immediately neutralized with a 10pyridineMeOH solution (1 mL) and then the resin was washed withDCM (3 times 5 mL) Fractions (TLC eluant 41 DCMMeOH) werecombined and concentrated under reduced pressure to yield a residuewhich was suspended in H2O and purified from the pyridinium salts bysize-exclusion chromatography (AMBERLITE XAD2 resin H2O thenMeOH) Evaporation of combined MeOH fractions containing theproduct afforded the side-chain protected compound (493 mg 33)as a white solid material The linear peptide (493 mg 05 mmol) wasdissolved under a nitrogen atmosphere in DMF (120 mL) HATU(1521 mg 04 mmol 08 equiv) and TBTU (354 mg 11 mmol 22equiv) and DIPEA (257 μL 15 mmol 3 equiv) were added Theresulting mixture was stirred for two days at room temperature Thesolvent was distilled off under reduced pressure and the residue wasdissolved in H2O (60 mL) and extracted with EtOAc (3 times 80 mL)The organic phase was washed twice with 5 NaHCO3 dried withNa2SO4 and evaporated under reduced pressure The crude residuewas purified by flash column chromatography (91 DCMminusMeOH Rf037) to give the protected cyclic peptide (140 mg 30) as a yellowsolid material HPLC tR = 2430 purity 97 Side chain protectedcyclic peptide (140 mg 015 mmol) was treated with 952525 TFAH2OTIS mixture (20 mL) for 2 h The reaction mixture wasevaporated under reduced pressure and the residue was dissolved inH2O (60 mL) The aqueous phase was washed with iPr2O (3 times 60mL) and freeze-dried The crude residue was purified by semi-preparative HPLC to give the side-chain deprotected 2 (30 mg 34)as a white solid Trifluoroacetate ion was replaced with chloride ion byion-exchange chromatography (AMBERLITE IRA-96 resin chlorideform) See Table 1 for 1H and 13C NMR data ESI-MS mz 60514 (M+ H+ 100) ESI-MSMS mz 6055 (M + H+ 1) 5880 (100) 5770(30) 5700 (17) 5599 (42) 5460 (11) 533 (12) 5200 (72) Anal

Table 1 1H and 13C Chemical Shifts of 2 and 3 in DMSO-d6 Solutions at 298 K1H 2 1H 3 13C 2 13C 3

H-2 428 337 428 334 C-2 688 690H-3 459 466 C-3 535 537H-5 308 316 308 C-5 668 664H-6 393 370 390 372 C-6 682 684Gly NH 873 861Arg NH 740 856Asp NH 865 866D-Tyr NH 719 735Gly H-α 407 399 310 Gly C-α 445 441Arg H-α 399 401 Arg C-α 533 537Arg H-βγ 165 150 163 143 Arg C-βγ 249 220 248 212Arg H-δ 304 306 Arg C-δ 405 401Asp H-α 478 471 Asp C-α 570 567Asp H-β 282 270 288 256 Asp C-β 396 398D-Tyr H-α 447 451 D-Tyr C-α 494 504D-Tyr H-β 214 232 D-Tyr C-β 287 358D-Tyr H-Ar 695 660 766 734 724 D-Tyr C-Ar 1153 1301 1158 1298 1391

Journal of Medicinal Chemistry Article

dxdoiorg101021jm2016232 | J Med Chem 2012 55 5024minus50335029

Calcd for C26H37ClN8O9 C 4871 H 582 N 1748 Found C4888 H 591 N 1732 HPLC tR = 121 95 purityc[RGD-(3primeI-y)-(3R)-carboxymorpholine] (3) A solution of

ligand 2 (120 mg 0020 mmol) in PBS (2 mL) was added to sodiumiodide (307 mg 0020 mmol) followed by chloramine-T hydrate(466 mg 0020 mmol) The reaction mixture was stirred at roomtemperature for 20 min and then quenched with a solution of 5sodium metabisulphite (08 mL) The mixture was extracted withDCM (3 times 5 mL) and loaded on a preconditioned C18 Sep-pakcolumn The C18 column was washed with water (6 mL) andsubsequently eluted with MeOH The crude product was purified bysemipreparative HPLC to obtain ligand 3 (467 mg 32) See Table 1for 1H and 13C NMR data ESI-MS mz 73125 (M + H+ 100) ESI-MSMS mz 7312 (M + H+ 15) 7141 (100) 6859 (39) 6721 (22)6461 (76) Anal Calcd for C26H36ClIN8O9 C 4072 H 473 N1461 Found C 4081 H 483 N 1412 HPLC tR = 152 96purityc[RGD-(3prime125I-y)-(3R)-carboxymorpholine] (125I-3) A solution

of PBS (100 μL 005 M) containing Na125I (20 μL 2 mCi 0918 nM)peptide 2 (10 μL of a 005 M PBS solution 165 nmol) andchloramine-T (188 μL of a freshly prepared 005 M PBS solution 824nmol 9 equiv) was prepared The reaction mixture was stirred at roomtemperature for 20 min and then quenched by adding a solution ofsodium metabisulphite (218 μL of a freshly prepared 005 M PBSsolution 824 nmol 9 equiv) The mixture was extracted with DCM (3times 05 mL) and purified by HPLC and the radioactivity of fractionscollected every 30 s was determined with a γ-counter (SupportingInformation Figure S1) Fractions containing the chemically pure 125I-3 were pooled together (155 mCi 78 radiochemical yield) and usedfor in vivo experiments after solvent removal HPLC tR = 152 98radiochemical purity gt99 chemical puritySolid-Phase Integrin Binding Assay [125I]-Echistatin labeled by

the lactoperoxidase method24 to a specific activity of 2000 Cimmolwas purchased from GE Healthcare and integrin αvβ3 from humanplacenta was purchased from Chemicon International Inc TemeculaCAPurified αvβ3 integrin was diluted in coating buffer (20 mM Tris

(pH 74) 150 mM NaCl 2 mM CaCl2 1 mM MgCl2 1 mM MnCl2)at concentrations of 500 or 1000 ngmL An aliquot of diluted integrin(100 μLwell) was added to a 96-well microtiter plate (Optiplate-96HB PerkinElmer Life Sciences Boston MA) and plates wereincubated overnight at 4 degC Plates were washed once withblockingbinding buffer (20 mM Tris (pH 74) 150 mM NaCl 2mM CaCl2 1 mM MgCl2 1 mM MnCl2 1 BSA) and incubated atroom temperature for additional 2 h Plates were rinsed twice withsame buffer and then competition binding assays were performed witha constant concentration of [125I]-Echistatin (005 nM) Concen-trations of tested compound ranged from 001 to 100 nM All assayswere performed in triplicate in a final volume of 02 mL eachcontaining the following species 005 mL of [125I]-Echistatin 004 mLof tested compound and 011 mL of blockingbinding bufferNonspecific binding was defined as [125I]-Echistatin bound in presenceof an excess (1 μM) of unlabeled echistatin After incubation at roomtemperature for 3 h plates were washed three times with blockingbinding buffer and counted in a Top-Count NXT microplatescintillation counter (PerkinElmer Life Sciences Boston MA) using200 μLwell of MicroScint-40 liquid scintillation (PerkinElmer LifeSciences Boston MA)Data are shown as means plusmn SD from three independent

experiments IC50 values were determined by fitting binding inhibitiondata by nonlinear regression using GraphPad Prism 40 SoftwarePackage (GraphPad Prism San Diego CA)Cell Lines and Culture Conditions The A375 M human

melanoma cell line was obtained from the American Type CultureCollection (ATCC Rockville MD)12g Human dermal skin fibroblasts(HDF) were kindly donated by Dr Daniela Monti Department ofExperimental Pathology and Oncology University of FlorenceMelanoma cells and HDF were grown in Dulbeccorsquos Modified EagleMedium containing 4500 mgL glucose (DMEM 4500 GIBCO)supplemented with 10 fetal calf serum (FCS) at 37 degC in a

humidified incubator containing 10 CO2 Then 50 times 105 melanomacells or 1 times 106 HDF were seeded in 100 mm dishes and propagatedevery 3 days by incubation with a trypsinminusEDTA solution Cultureswere periodically monitored for mycoplasma contamination Primarycultures of human umbilical vein endothelial cells (HUVEC) wereobtained from Lonza (USA) HUVEC grown in endothelial cellgrowth medium-2 (EGM-2) containing endothelial cell growthsupplements were subcultivated using a trypsin-EDTA solution 13split ratio Cells between passages 2 and 4 grown to confluence inplates coated with 1 bovine gelatin (Sigma St Louis) were used inthe experiments

RNA Isolation and Polymerase Chain Reaction (PCR) TotalRNA extracted using RNAgents (Total RNA Isolation SystemPromega Madison WI) was determined spectrophotometricallyComplementary DNA (cDNA) was synthesized from 2 μg of totalRNA using 1 μL of ImProm-II reverse transcriptase (Promega)Aliquots of 2 μL of the cDNA were used for PCR amplification Thespecific primers used for the identification of human αv α5 β1 β3 β5and GAPDH are reported in Table S1 of Supporting Informations AllPCR experiments were conducted using 005 UμL of Go-TaqPolymerase (Promega) Amplification was carried out on a Perkin-Elmer Thermal cycler Then 8 μL of each PCR products werevisualized after electrophoresis in a 2 agarose gel containing 05 μgmL of ethidium bromide cDNA products were evaluated on the basisof a standard PCR marker

Flow Cytometry Assay Tumor cells were detached by gentletreatment with Accutase a 05 mM EDTA solution washed andincubated for 1 h at 4 degC in the presence of anti αvβ3 monoclonalantibody (1 μg50 μL Anti-Integrin αvβ3 clone LM609 Millipore)Cells were then washed and incubated for 1 h at 4 degC with a secondaryantibody 5 μgmL goat antimouse IgG conjugated with FITC (SantaCruz Biotechnology Inc Santa Cruz CA) αvβ3 -positive cells wereanalyzed at 488 nm on the flow cytometer FACScan system (BD-FACS Canto)

Cell Adhesion Assay Plates (96 wells) were coated withfibronectin (1 μgmL) or vitronectin (10 μgmL) by overnightincubation at 4 degC and then incubated at 37 degC for 1 h with 1 BSA inPBS Melanoma cells IL-1β (400 UmL)-stimulated HUVECs orhuman dermal fibroblasts (HDF) were resuspended in serum-freemedium and exposed to ligands 1 2 or 3 (final concentration was 011 or 10 μM) at 37 degC for 30 min to allow for ligandminusintegrinequilibrium to be reached Melanoma cell adhesion assays wereperformed in the presence or in the absence of 2 mmolL MnCl2Cells were plated (4minus5 times 104 cellswell) and incubated at 37 degC for 2h Nonadherent cells were removed with PBS and adherent cells werestained with 05 crystal violet solution in 20 methanol After 2 h ofincubation at 4 degC plates were examined at 540 nm in a counterELX800 (Biotek Instruments) Experiments were conducted intriplicate and repeated at least three times Data are presented asmeans plusmn SD from three independent experiments

Animal Model and Study Design In vivo experiments wereconducted in accordance with national guidelines and approved by theEthical Committee of the Animal Welfare Office of the Italian WorkMinistry and conforming to legal mandates and Italian guidelines forcare and maintenance of laboratory animals Eighteen-week-old SCIDbgbg mice (Charles River Laboratories International) were injectedsubcutaneously in the shoulder area with a suspension of melanomacells and 20 ngmL TGFβ-activated fibroblasts Then 1 times106 A375 Mmelanoma cells and 05 times 106 HDF were previously resuspended in100 μL of PBS diluted 11 with a Matrigel solution (BD Biosciences)Animals were kept under pathogen-free conditions and allowed to feedfreely Tumor growth was monitored daily

Small-Animal SPECT and CT Studies In vivo studies wereconducted using a ldquohybridrdquo imaging system for small laboratoryanimals allowing for the study of biological processes using minimallyinvasive procedures Engrafted tumor tracer uptake was studied atdifferent time-points after tail vein injection Binding specificity tointegrin receptors was evaluated by in vivo displacement studies inwhich a high dose of cold tracer was injected 10 min beforeradiolabeled one Imaging studies were performed using the Flex

Journal of Medicinal Chemistry Article

dxdoiorg101021jm2016232 | J Med Chem 2012 55 5024minus50335030

Triumph preclinical imaging system which combines positron (PET)and single-photon emission (SPECT) tomography with the emissiontomography (CT) for anatomic coregistration (GE HealthcareGamma Medica-Ideas Waukesha WI) 125I-iodinated tracers 125I-3was studied in vivo by SPECT allowing for use directly the in vitrotracer data to the in vivo experimentation Imaging studies requiredmonitoring of anesthetized animals throughout the imaging procedureand anesthesia was conducted in accordance with National Guidelinesand approved by the Ethical Committee Given that anesthesia ingeneral causes hypothermia in small animals appropriate warming wasprovided during imaging procedure by using a heating bed set at 37degC Physiological monitoring was achieved by measuring respirationrate with a pressure pad placed under the mouse abdomen Saturationexperiments (SE) to visualize tumors and displacement experiments(DE) were performed In SE 125I-3 (7minus10 MBq in 200 μL ofphysiological saline) was injected via tail vein into tumor bearinganimals kept under anesthesia After 30 min a CT aquisition wasperformed followed by SPECT scan starting at 40 min pi CT scanwas acquired with 50 kV and 320 μA tube settings The magnificationfactor was set to 2 resulting in an axial field of view of 592 mm 512projections 500 μs each were acquired on a circular orbit in 256 swith an acquisition matrix of 1184 times 1120 100 μm pixel SPECTprojections were acquired with a 5-pinhole high-resolution collimator(1 mm hole diameter 75 mm focal distance) with radius of rotation of40 mm resulting in an axial field of view of 529 mm then 64projections 20 s each were acquired on a circular orbit for a totalacquisition time of about 22 min Acquisition matrix was 80 times 80 15mm pixel size Energy window was set on the 125I photopeak rangingfrom 20 to 36 KeV The DE were performed by injecting 18 mgkg ofcompound 3 10 min before injection of tracer Once scanning wascomplete the animals were recovered in a housing unit and monitoreduntil awakeData Analysis of Small-Animal SPECT and CT Studies CT

image data set was reconstructed using a cone-beam FBP algorithm(FDK) and an optimum noise filter Reconstruction matrix was 512 times512 times 512 180 μm pixel size SPECT images were reconstructed withordered subset expectation maximization algorithm (8 subsets and 5iterations) and a reconstruction matrix of 60 times 60 times 60 15 mm pixelsize No correction for radiation attenuation and scatter was appliedFused images were visually analyzed to detect the engrafted tumormass and regional SPECT activities were measured using the analysissoftware Vivid (Gamma Medica-Ideas Northridge CA) by delineatingthe region of interest (ROI) of tumor in fused CT images A similarROI was generated for muscle tissue on contralateral side of tumorTumor ROI activity was normalized (nTA) with the followingformula

= minusnTA

TA BABA

where TA is tumor activity and BA is background activity measured inmuscle tissue contralateral to tumorDocking Calculations Automated docking studies were carried

out by Autodock 401 program17 using the Lamarckian GeneticAlgorithm (LGA) as a search engine The AutoDockTools 145(ADT) graphical interface25 was used to prepare integrin and ligandsPDBQT files Coordinates of ligand 3 were retrieved from lowestenergy conformers resulting from molecular mechanics calculationsstarting from geometry of ligand 1 whereas coordinates of αvβ3receptor were retrieved from the Protein Data Bank (PDB code1L5G) and ligandminusprotein complex was unmerged for achieving freereceptor structure Water molecules were removed For proteinreceptor and ligand 3 all hydrogens were added Gasteiger chargeswere computed and nonpolar hydrogens were merged A charge valueof +20 to each Mn atom of protein receptor was successively addedThree-dimensional energy scoring grids of 0375 Aring resolution and 40Aring times 40 Aring times 40 Aring dimensions were computed Center of grid was set tobe coincident with mass center of ligands preliminary fitted on the X-ray structure of c[RGDf(Me)V] in the αvβ3 complex (1L5G) A totalof 50 runs with a maximum of 2500000 energy evaluations werecarried out for each ligand using the default parameters for LGA

Cluster analysis was performed on docked results using a root-mean-square (rms) tolerance of 15 Aring Analysis of binding mode calculationof binding energy and prediction of binding activity of dockedconformations were carried out using PyMol Autodock Tools pluginwithin PyMol software26

ASSOCIATED CONTENTS Supporting InformationAdditional experimental methods radiochromatogram of 125I-3and additional cell biology assays data This material is availablefree of charge via the Internet at httppubsacsorg

AUTHOR INFORMATIONCorresponding AuthorFor AG phone +39 055 4573481 fax +39 055 4573569 E-mail antonioguarnaunifiit For AP phone +39 0557947527 fax +39 055 4224392 E-mail apupidfcunifiitFor LC phone +39 055 4598207 fax +39 055 4598900 E-mail lidocaloriniunifiit

NotesThe authors declare no competing financial interest

ACKNOWLEDGMENTSThis work has received financial support from the IstitutoTo s c ano Tumor i a nd MIUR (PR IN08 G r an t2008J4YNJY_003) Also financial contributions from CINM-PIS University of Florence and Fondazione Roma areacknowledged Dr Alessandro Sisti is acknowledged forcarrying out preliminary labeling experiments Ente Cassa diRisparmio is acknowledged for the granting the MicroPETSPECTCT instrument

ABBREVIATIONS USEDSPECT single positron emission computed tomography PETpositon emission tomography RGD Arg-Gly-Asp VGPvertical growth phase CT computed tomography VNvitronectin FN fibronectin HUVEC human umbilical veinendothelial cells IL-1β interleukin-1β ESI-MSMS electroprayionization tandem mass spectrometry EGM-2 endothelial cellgrowth medium-2 EDTA ethylenediaminetetraacetic acidGAPDH glyceraldehyde 3-phosphate dehydrogenase FACSfluorescence-activated cell sorting FITC fluorescein isothio-cyanate PBS phosphate buffered saline HUVEC humanumbilical vein endothelial cells HDF human dermal skinfibroblasts EMT epithelial-mesenchymal transition TGFtransform grown factor pi post injection

REFERENCES(1) Challenges and Opportunities Report US Department of Healthand Human Services Food and Drug Administration Silver SpringMD March 2004 httpwwwfdagovScienceResearchSpecialTopicsCriticalPathInitiativeCriticalPathOpportunities-Reports-ucm077262htm(2) (a) Backgrounder How New Drugs Move Through the Developmentand Approval Process Tufts Center for the Study of DrugDevelopment Boston November 1 2001 (b) Gilbert J HenskeP Singh A Rebuilding Big Pharmarsquos Business Model The Business ampMedicine Report Windhover Information November 2003 Vol 21 no10 httpwwwbaincomImagesrebuilding_big_pharmapdf(3) Innovative drug development approaches-project Final report fromTHEEMEACHMP-THINK-TANK group on innovative drug develop-ment EMEA 2007 pp 1minus26 httpwwwemaeuropaeudocsen_GBdocument_libraryOther200910WC500004913pdf

Journal of Medicinal Chemistry Article

dxdoiorg101021jm2016232 | J Med Chem 2012 55 5024minus50335031

(Kieselgel 60 Merck) using flash-column techniques Rf values refer toTLC carried out on 25 mm silica gel plates (Merck F254) with sameeluant as for column chromatography Solid-phase reactions werecarried out on a shaker using solvents of HPLC quality BromophenolBlue (BB) test was performed as follows a small amount of resinbeads were suspended in a vial with 05 mL of DMF two drops of a1 solution of bromophenol blue in dimethylacetamide were addedand the sample was observed The BB test was considered positive(presence of free amino groups) when the resin beads turned blue andnegative (absence of free amino groups) when the beads remainedcolorless ESI mass spectra were carried out on a ion-trap doublequadrupole mass spectrometer using electrospray (ES+) ionizationtechniques and a normalized collision energy within the range of 25minus32 eV for MSMS experiments Ligands 2 and 3 were purified byBeckman-Gold HPLC system equipped with a reverse-phase column(Alltima C18 10 μm 250 mm times 10 mm Alltech) using H2OCH3CNgradient eluant buffered with 01 TFA (flow rate 25 mLmin λ 254nm gradient eluant CH3CN 105 min CH3CN 10minus9025 min)Analytical HPLC analyses were performed on Dionex Ulltimate 3000system equipped with a reverse-phase column (Alltima C18 5 μm 250mm times 46 mm Alltech) and the same gradient eluant Ligand 125I-3was purified by Beckman-Gold HPLC system equipped with a reverse-phase column (C-8 Waters Spherisorb S5 250 mm times 46 mm) and thesame gradient eluant Na125I in 02N NaOH solution was purchasedfrom PerkinElmer (10 μL 100 mCimL 17 Ci (629GBq)mg 125I97 126I lt 3 radiochemical purity gt99 carrier free) HPLCanalysis was used to determine purity and all tested compoundspossessed gt95 purityc[RGDy-(3R)-carboxymorpholine] (2) H-Gly-2-Cl-Trt resin

(466 mg 05 mmol) was used as starting material Fmoc-deprotectionsand completion of each coupling reaction were assessed by performinga BB test Coupling was performed with a solution consisting of N-Fmoc-Arg(Pbf)-OH (973 mg 15 mmol 3 equiv) HOBt (203 mg 15mmol 3 equiv) in DMF (3 mL) and DIC (203 μL 15 mmol 3equiv) was added dropwise at 0 degC The resulting mixture was stirredfor 10 min at 0 degC and for a further 10 min at room temperature andthen was added to the resin This mixture was shaken at roomtemperature for 24 h The solution was drained and the resin wassequentially washed with 5 DIPEA in DMF (3 times 5 mL) and DMF (5times 5 mL) Fmoc deprotection was performed with 30 piperidine inDMF (10 mL) for 30 min followed by resin washings with DMF (3 times10 mL) After deprotection H-Arg(Pbf)-Gly-2-Cl-Trt resin wassuspended in a solution of HOBt (3568 mg 264 mmol 6 equiv)DIC (4156 μL 264 mmol 6 equiv) and N-Fmoc-(R)-morpholine-3-carboxylic acid (465 mg 132 mmol 264 equiv) in DMF (3 mL) andshaken for two days The solution was drained and resin was washedwith 5 DIPEA in DMF (3 times 5 mL) and DMF (5 times 5 mL) After

deprotection H-(3R)-carboxymorpholine-Arg(Pbf)-Gly-2-Cl-Trt resinwas suspended in a solution of HOBt (405 mg 3 mmol 6 equiv) DIC(472 μL 3 mmol 6 equiv) and N-Fmoc-D-Tyr(t-Bu)-OH (689 mg15 mmol 3 equiv) in DMF (3 mL) and shaken for three days Thesolution was drained and the resin washed with 5 DIPEA in DMF (3times 5 mL) and DMF (5 times 5 mL) After deprotection H-D-Tyr(t-Bu)-(3R)-carboxymorpholine-Arg(Pbf)-Gly-2-Cl-Trt resin was suspendedin a solution of TBTU (8027 mg 25 mmol 5 equiv) N-Fmoc-Asp(t-Bu)-OH (1028 g 25 mmol 5 equiv) and DIPEA (428 μL 25 mmol5 equiv) in DMF (3 mL) and shaken for three days The solution wasdrained and resin washed with 5 DIPEA in DMF (3 times 5 mL) andDMF (5 times 5 mL) N-Fmoc-Asp(t-Bu)-D-Tyr(t-Bu)-(3R)-carboxymor-pholine-Arg(Pbf)-Gly-2-Cl-Trt resin was deprotected and washed withDMF (5 times 5 mL) and DCM (5 times 5 mL) In a solid-phase reactionvessel H-Asp(t-Bu)-D-Phe-(3R)-carboxymorpholine-Arg(Pbf)-Gly-2-Cl-Trt resin was treated with 5 mL of 1 TFADCM solution (10times 2 min) The filtrates were immediately neutralized with a 10pyridineMeOH solution (1 mL) and then the resin was washed withDCM (3 times 5 mL) Fractions (TLC eluant 41 DCMMeOH) werecombined and concentrated under reduced pressure to yield a residuewhich was suspended in H2O and purified from the pyridinium salts bysize-exclusion chromatography (AMBERLITE XAD2 resin H2O thenMeOH) Evaporation of combined MeOH fractions containing theproduct afforded the side-chain protected compound (493 mg 33)as a white solid material The linear peptide (493 mg 05 mmol) wasdissolved under a nitrogen atmosphere in DMF (120 mL) HATU(1521 mg 04 mmol 08 equiv) and TBTU (354 mg 11 mmol 22equiv) and DIPEA (257 μL 15 mmol 3 equiv) were added Theresulting mixture was stirred for two days at room temperature Thesolvent was distilled off under reduced pressure and the residue wasdissolved in H2O (60 mL) and extracted with EtOAc (3 times 80 mL)The organic phase was washed twice with 5 NaHCO3 dried withNa2SO4 and evaporated under reduced pressure The crude residuewas purified by flash column chromatography (91 DCMminusMeOH Rf037) to give the protected cyclic peptide (140 mg 30) as a yellowsolid material HPLC tR = 2430 purity 97 Side chain protectedcyclic peptide (140 mg 015 mmol) was treated with 952525 TFAH2OTIS mixture (20 mL) for 2 h The reaction mixture wasevaporated under reduced pressure and the residue was dissolved inH2O (60 mL) The aqueous phase was washed with iPr2O (3 times 60mL) and freeze-dried The crude residue was purified by semi-preparative HPLC to give the side-chain deprotected 2 (30 mg 34)as a white solid Trifluoroacetate ion was replaced with chloride ion byion-exchange chromatography (AMBERLITE IRA-96 resin chlorideform) See Table 1 for 1H and 13C NMR data ESI-MS mz 60514 (M+ H+ 100) ESI-MSMS mz 6055 (M + H+ 1) 5880 (100) 5770(30) 5700 (17) 5599 (42) 5460 (11) 533 (12) 5200 (72) Anal

Table 1 1H and 13C Chemical Shifts of 2 and 3 in DMSO-d6 Solutions at 298 K1H 2 1H 3 13C 2 13C 3

H-2 428 337 428 334 C-2 688 690H-3 459 466 C-3 535 537H-5 308 316 308 C-5 668 664H-6 393 370 390 372 C-6 682 684Gly NH 873 861Arg NH 740 856Asp NH 865 866D-Tyr NH 719 735Gly H-α 407 399 310 Gly C-α 445 441Arg H-α 399 401 Arg C-α 533 537Arg H-βγ 165 150 163 143 Arg C-βγ 249 220 248 212Arg H-δ 304 306 Arg C-δ 405 401Asp H-α 478 471 Asp C-α 570 567Asp H-β 282 270 288 256 Asp C-β 396 398D-Tyr H-α 447 451 D-Tyr C-α 494 504D-Tyr H-β 214 232 D-Tyr C-β 287 358D-Tyr H-Ar 695 660 766 734 724 D-Tyr C-Ar 1153 1301 1158 1298 1391

Journal of Medicinal Chemistry Article

dxdoiorg101021jm2016232 | J Med Chem 2012 55 5024minus50335029

Calcd for C26H37ClN8O9 C 4871 H 582 N 1748 Found C4888 H 591 N 1732 HPLC tR = 121 95 purityc[RGD-(3primeI-y)-(3R)-carboxymorpholine] (3) A solution of

ligand 2 (120 mg 0020 mmol) in PBS (2 mL) was added to sodiumiodide (307 mg 0020 mmol) followed by chloramine-T hydrate(466 mg 0020 mmol) The reaction mixture was stirred at roomtemperature for 20 min and then quenched with a solution of 5sodium metabisulphite (08 mL) The mixture was extracted withDCM (3 times 5 mL) and loaded on a preconditioned C18 Sep-pakcolumn The C18 column was washed with water (6 mL) andsubsequently eluted with MeOH The crude product was purified bysemipreparative HPLC to obtain ligand 3 (467 mg 32) See Table 1for 1H and 13C NMR data ESI-MS mz 73125 (M + H+ 100) ESI-MSMS mz 7312 (M + H+ 15) 7141 (100) 6859 (39) 6721 (22)6461 (76) Anal Calcd for C26H36ClIN8O9 C 4072 H 473 N1461 Found C 4081 H 483 N 1412 HPLC tR = 152 96purityc[RGD-(3prime125I-y)-(3R)-carboxymorpholine] (125I-3) A solution

of PBS (100 μL 005 M) containing Na125I (20 μL 2 mCi 0918 nM)peptide 2 (10 μL of a 005 M PBS solution 165 nmol) andchloramine-T (188 μL of a freshly prepared 005 M PBS solution 824nmol 9 equiv) was prepared The reaction mixture was stirred at roomtemperature for 20 min and then quenched by adding a solution ofsodium metabisulphite (218 μL of a freshly prepared 005 M PBSsolution 824 nmol 9 equiv) The mixture was extracted with DCM (3times 05 mL) and purified by HPLC and the radioactivity of fractionscollected every 30 s was determined with a γ-counter (SupportingInformation Figure S1) Fractions containing the chemically pure 125I-3 were pooled together (155 mCi 78 radiochemical yield) and usedfor in vivo experiments after solvent removal HPLC tR = 152 98radiochemical purity gt99 chemical puritySolid-Phase Integrin Binding Assay [125I]-Echistatin labeled by

the lactoperoxidase method24 to a specific activity of 2000 Cimmolwas purchased from GE Healthcare and integrin αvβ3 from humanplacenta was purchased from Chemicon International Inc TemeculaCAPurified αvβ3 integrin was diluted in coating buffer (20 mM Tris

(pH 74) 150 mM NaCl 2 mM CaCl2 1 mM MgCl2 1 mM MnCl2)at concentrations of 500 or 1000 ngmL An aliquot of diluted integrin(100 μLwell) was added to a 96-well microtiter plate (Optiplate-96HB PerkinElmer Life Sciences Boston MA) and plates wereincubated overnight at 4 degC Plates were washed once withblockingbinding buffer (20 mM Tris (pH 74) 150 mM NaCl 2mM CaCl2 1 mM MgCl2 1 mM MnCl2 1 BSA) and incubated atroom temperature for additional 2 h Plates were rinsed twice withsame buffer and then competition binding assays were performed witha constant concentration of [125I]-Echistatin (005 nM) Concen-trations of tested compound ranged from 001 to 100 nM All assayswere performed in triplicate in a final volume of 02 mL eachcontaining the following species 005 mL of [125I]-Echistatin 004 mLof tested compound and 011 mL of blockingbinding bufferNonspecific binding was defined as [125I]-Echistatin bound in presenceof an excess (1 μM) of unlabeled echistatin After incubation at roomtemperature for 3 h plates were washed three times with blockingbinding buffer and counted in a Top-Count NXT microplatescintillation counter (PerkinElmer Life Sciences Boston MA) using200 μLwell of MicroScint-40 liquid scintillation (PerkinElmer LifeSciences Boston MA)Data are shown as means plusmn SD from three independent

experiments IC50 values were determined by fitting binding inhibitiondata by nonlinear regression using GraphPad Prism 40 SoftwarePackage (GraphPad Prism San Diego CA)Cell Lines and Culture Conditions The A375 M human

melanoma cell line was obtained from the American Type CultureCollection (ATCC Rockville MD)12g Human dermal skin fibroblasts(HDF) were kindly donated by Dr Daniela Monti Department ofExperimental Pathology and Oncology University of FlorenceMelanoma cells and HDF were grown in Dulbeccorsquos Modified EagleMedium containing 4500 mgL glucose (DMEM 4500 GIBCO)supplemented with 10 fetal calf serum (FCS) at 37 degC in a

humidified incubator containing 10 CO2 Then 50 times 105 melanomacells or 1 times 106 HDF were seeded in 100 mm dishes and propagatedevery 3 days by incubation with a trypsinminusEDTA solution Cultureswere periodically monitored for mycoplasma contamination Primarycultures of human umbilical vein endothelial cells (HUVEC) wereobtained from Lonza (USA) HUVEC grown in endothelial cellgrowth medium-2 (EGM-2) containing endothelial cell growthsupplements were subcultivated using a trypsin-EDTA solution 13split ratio Cells between passages 2 and 4 grown to confluence inplates coated with 1 bovine gelatin (Sigma St Louis) were used inthe experiments

RNA Isolation and Polymerase Chain Reaction (PCR) TotalRNA extracted using RNAgents (Total RNA Isolation SystemPromega Madison WI) was determined spectrophotometricallyComplementary DNA (cDNA) was synthesized from 2 μg of totalRNA using 1 μL of ImProm-II reverse transcriptase (Promega)Aliquots of 2 μL of the cDNA were used for PCR amplification Thespecific primers used for the identification of human αv α5 β1 β3 β5and GAPDH are reported in Table S1 of Supporting Informations AllPCR experiments were conducted using 005 UμL of Go-TaqPolymerase (Promega) Amplification was carried out on a Perkin-Elmer Thermal cycler Then 8 μL of each PCR products werevisualized after electrophoresis in a 2 agarose gel containing 05 μgmL of ethidium bromide cDNA products were evaluated on the basisof a standard PCR marker

Flow Cytometry Assay Tumor cells were detached by gentletreatment with Accutase a 05 mM EDTA solution washed andincubated for 1 h at 4 degC in the presence of anti αvβ3 monoclonalantibody (1 μg50 μL Anti-Integrin αvβ3 clone LM609 Millipore)Cells were then washed and incubated for 1 h at 4 degC with a secondaryantibody 5 μgmL goat antimouse IgG conjugated with FITC (SantaCruz Biotechnology Inc Santa Cruz CA) αvβ3 -positive cells wereanalyzed at 488 nm on the flow cytometer FACScan system (BD-FACS Canto)

Cell Adhesion Assay Plates (96 wells) were coated withfibronectin (1 μgmL) or vitronectin (10 μgmL) by overnightincubation at 4 degC and then incubated at 37 degC for 1 h with 1 BSA inPBS Melanoma cells IL-1β (400 UmL)-stimulated HUVECs orhuman dermal fibroblasts (HDF) were resuspended in serum-freemedium and exposed to ligands 1 2 or 3 (final concentration was 011 or 10 μM) at 37 degC for 30 min to allow for ligandminusintegrinequilibrium to be reached Melanoma cell adhesion assays wereperformed in the presence or in the absence of 2 mmolL MnCl2Cells were plated (4minus5 times 104 cellswell) and incubated at 37 degC for 2h Nonadherent cells were removed with PBS and adherent cells werestained with 05 crystal violet solution in 20 methanol After 2 h ofincubation at 4 degC plates were examined at 540 nm in a counterELX800 (Biotek Instruments) Experiments were conducted intriplicate and repeated at least three times Data are presented asmeans plusmn SD from three independent experiments

Animal Model and Study Design In vivo experiments wereconducted in accordance with national guidelines and approved by theEthical Committee of the Animal Welfare Office of the Italian WorkMinistry and conforming to legal mandates and Italian guidelines forcare and maintenance of laboratory animals Eighteen-week-old SCIDbgbg mice (Charles River Laboratories International) were injectedsubcutaneously in the shoulder area with a suspension of melanomacells and 20 ngmL TGFβ-activated fibroblasts Then 1 times106 A375 Mmelanoma cells and 05 times 106 HDF were previously resuspended in100 μL of PBS diluted 11 with a Matrigel solution (BD Biosciences)Animals were kept under pathogen-free conditions and allowed to feedfreely Tumor growth was monitored daily

Small-Animal SPECT and CT Studies In vivo studies wereconducted using a ldquohybridrdquo imaging system for small laboratoryanimals allowing for the study of biological processes using minimallyinvasive procedures Engrafted tumor tracer uptake was studied atdifferent time-points after tail vein injection Binding specificity tointegrin receptors was evaluated by in vivo displacement studies inwhich a high dose of cold tracer was injected 10 min beforeradiolabeled one Imaging studies were performed using the Flex

Journal of Medicinal Chemistry Article

dxdoiorg101021jm2016232 | J Med Chem 2012 55 5024minus50335030

Triumph preclinical imaging system which combines positron (PET)and single-photon emission (SPECT) tomography with the emissiontomography (CT) for anatomic coregistration (GE HealthcareGamma Medica-Ideas Waukesha WI) 125I-iodinated tracers 125I-3was studied in vivo by SPECT allowing for use directly the in vitrotracer data to the in vivo experimentation Imaging studies requiredmonitoring of anesthetized animals throughout the imaging procedureand anesthesia was conducted in accordance with National Guidelinesand approved by the Ethical Committee Given that anesthesia ingeneral causes hypothermia in small animals appropriate warming wasprovided during imaging procedure by using a heating bed set at 37degC Physiological monitoring was achieved by measuring respirationrate with a pressure pad placed under the mouse abdomen Saturationexperiments (SE) to visualize tumors and displacement experiments(DE) were performed In SE 125I-3 (7minus10 MBq in 200 μL ofphysiological saline) was injected via tail vein into tumor bearinganimals kept under anesthesia After 30 min a CT aquisition wasperformed followed by SPECT scan starting at 40 min pi CT scanwas acquired with 50 kV and 320 μA tube settings The magnificationfactor was set to 2 resulting in an axial field of view of 592 mm 512projections 500 μs each were acquired on a circular orbit in 256 swith an acquisition matrix of 1184 times 1120 100 μm pixel SPECTprojections were acquired with a 5-pinhole high-resolution collimator(1 mm hole diameter 75 mm focal distance) with radius of rotation of40 mm resulting in an axial field of view of 529 mm then 64projections 20 s each were acquired on a circular orbit for a totalacquisition time of about 22 min Acquisition matrix was 80 times 80 15mm pixel size Energy window was set on the 125I photopeak rangingfrom 20 to 36 KeV The DE were performed by injecting 18 mgkg ofcompound 3 10 min before injection of tracer Once scanning wascomplete the animals were recovered in a housing unit and monitoreduntil awakeData Analysis of Small-Animal SPECT and CT Studies CT

image data set was reconstructed using a cone-beam FBP algorithm(FDK) and an optimum noise filter Reconstruction matrix was 512 times512 times 512 180 μm pixel size SPECT images were reconstructed withordered subset expectation maximization algorithm (8 subsets and 5iterations) and a reconstruction matrix of 60 times 60 times 60 15 mm pixelsize No correction for radiation attenuation and scatter was appliedFused images were visually analyzed to detect the engrafted tumormass and regional SPECT activities were measured using the analysissoftware Vivid (Gamma Medica-Ideas Northridge CA) by delineatingthe region of interest (ROI) of tumor in fused CT images A similarROI was generated for muscle tissue on contralateral side of tumorTumor ROI activity was normalized (nTA) with the followingformula

= minusnTA

TA BABA

where TA is tumor activity and BA is background activity measured inmuscle tissue contralateral to tumorDocking Calculations Automated docking studies were carried

out by Autodock 401 program17 using the Lamarckian GeneticAlgorithm (LGA) as a search engine The AutoDockTools 145(ADT) graphical interface25 was used to prepare integrin and ligandsPDBQT files Coordinates of ligand 3 were retrieved from lowestenergy conformers resulting from molecular mechanics calculationsstarting from geometry of ligand 1 whereas coordinates of αvβ3receptor were retrieved from the Protein Data Bank (PDB code1L5G) and ligandminusprotein complex was unmerged for achieving freereceptor structure Water molecules were removed For proteinreceptor and ligand 3 all hydrogens were added Gasteiger chargeswere computed and nonpolar hydrogens were merged A charge valueof +20 to each Mn atom of protein receptor was successively addedThree-dimensional energy scoring grids of 0375 Aring resolution and 40Aring times 40 Aring times 40 Aring dimensions were computed Center of grid was set tobe coincident with mass center of ligands preliminary fitted on the X-ray structure of c[RGDf(Me)V] in the αvβ3 complex (1L5G) A totalof 50 runs with a maximum of 2500000 energy evaluations werecarried out for each ligand using the default parameters for LGA

Cluster analysis was performed on docked results using a root-mean-square (rms) tolerance of 15 Aring Analysis of binding mode calculationof binding energy and prediction of binding activity of dockedconformations were carried out using PyMol Autodock Tools pluginwithin PyMol software26

ASSOCIATED CONTENTS Supporting InformationAdditional experimental methods radiochromatogram of 125I-3and additional cell biology assays data This material is availablefree of charge via the Internet at httppubsacsorg

AUTHOR INFORMATIONCorresponding AuthorFor AG phone +39 055 4573481 fax +39 055 4573569 E-mail antonioguarnaunifiit For AP phone +39 0557947527 fax +39 055 4224392 E-mail apupidfcunifiitFor LC phone +39 055 4598207 fax +39 055 4598900 E-mail lidocaloriniunifiit

NotesThe authors declare no competing financial interest

ACKNOWLEDGMENTSThis work has received financial support from the IstitutoTo s c ano Tumor i a nd MIUR (PR IN08 G r an t2008J4YNJY_003) Also financial contributions from CINM-PIS University of Florence and Fondazione Roma areacknowledged Dr Alessandro Sisti is acknowledged forcarrying out preliminary labeling experiments Ente Cassa diRisparmio is acknowledged for the granting the MicroPETSPECTCT instrument

ABBREVIATIONS USEDSPECT single positron emission computed tomography PETpositon emission tomography RGD Arg-Gly-Asp VGPvertical growth phase CT computed tomography VNvitronectin FN fibronectin HUVEC human umbilical veinendothelial cells IL-1β interleukin-1β ESI-MSMS electroprayionization tandem mass spectrometry EGM-2 endothelial cellgrowth medium-2 EDTA ethylenediaminetetraacetic acidGAPDH glyceraldehyde 3-phosphate dehydrogenase FACSfluorescence-activated cell sorting FITC fluorescein isothio-cyanate PBS phosphate buffered saline HUVEC humanumbilical vein endothelial cells HDF human dermal skinfibroblasts EMT epithelial-mesenchymal transition TGFtransform grown factor pi post injection

REFERENCES(1) Challenges and Opportunities Report US Department of Healthand Human Services Food and Drug Administration Silver SpringMD March 2004 httpwwwfdagovScienceResearchSpecialTopicsCriticalPathInitiativeCriticalPathOpportunities-Reports-ucm077262htm(2) (a) Backgrounder How New Drugs Move Through the Developmentand Approval Process Tufts Center for the Study of DrugDevelopment Boston November 1 2001 (b) Gilbert J HenskeP Singh A Rebuilding Big Pharmarsquos Business Model The Business ampMedicine Report Windhover Information November 2003 Vol 21 no10 httpwwwbaincomImagesrebuilding_big_pharmapdf(3) Innovative drug development approaches-project Final report fromTHEEMEACHMP-THINK-TANK group on innovative drug develop-ment EMEA 2007 pp 1minus26 httpwwwemaeuropaeudocsen_GBdocument_libraryOther200910WC500004913pdf

Journal of Medicinal Chemistry Article

dxdoiorg101021jm2016232 | J Med Chem 2012 55 5024minus50335031

Calcd for C26H37ClN8O9 C 4871 H 582 N 1748 Found C4888 H 591 N 1732 HPLC tR = 121 95 purityc[RGD-(3primeI-y)-(3R)-carboxymorpholine] (3) A solution of

ligand 2 (120 mg 0020 mmol) in PBS (2 mL) was added to sodiumiodide (307 mg 0020 mmol) followed by chloramine-T hydrate(466 mg 0020 mmol) The reaction mixture was stirred at roomtemperature for 20 min and then quenched with a solution of 5sodium metabisulphite (08 mL) The mixture was extracted withDCM (3 times 5 mL) and loaded on a preconditioned C18 Sep-pakcolumn The C18 column was washed with water (6 mL) andsubsequently eluted with MeOH The crude product was purified bysemipreparative HPLC to obtain ligand 3 (467 mg 32) See Table 1for 1H and 13C NMR data ESI-MS mz 73125 (M + H+ 100) ESI-MSMS mz 7312 (M + H+ 15) 7141 (100) 6859 (39) 6721 (22)6461 (76) Anal Calcd for C26H36ClIN8O9 C 4072 H 473 N1461 Found C 4081 H 483 N 1412 HPLC tR = 152 96purityc[RGD-(3prime125I-y)-(3R)-carboxymorpholine] (125I-3) A solution

of PBS (100 μL 005 M) containing Na125I (20 μL 2 mCi 0918 nM)peptide 2 (10 μL of a 005 M PBS solution 165 nmol) andchloramine-T (188 μL of a freshly prepared 005 M PBS solution 824nmol 9 equiv) was prepared The reaction mixture was stirred at roomtemperature for 20 min and then quenched by adding a solution ofsodium metabisulphite (218 μL of a freshly prepared 005 M PBSsolution 824 nmol 9 equiv) The mixture was extracted with DCM (3times 05 mL) and purified by HPLC and the radioactivity of fractionscollected every 30 s was determined with a γ-counter (SupportingInformation Figure S1) Fractions containing the chemically pure 125I-3 were pooled together (155 mCi 78 radiochemical yield) and usedfor in vivo experiments after solvent removal HPLC tR = 152 98radiochemical purity gt99 chemical puritySolid-Phase Integrin Binding Assay [125I]-Echistatin labeled by

the lactoperoxidase method24 to a specific activity of 2000 Cimmolwas purchased from GE Healthcare and integrin αvβ3 from humanplacenta was purchased from Chemicon International Inc TemeculaCAPurified αvβ3 integrin was diluted in coating buffer (20 mM Tris

(pH 74) 150 mM NaCl 2 mM CaCl2 1 mM MgCl2 1 mM MnCl2)at concentrations of 500 or 1000 ngmL An aliquot of diluted integrin(100 μLwell) was added to a 96-well microtiter plate (Optiplate-96HB PerkinElmer Life Sciences Boston MA) and plates wereincubated overnight at 4 degC Plates were washed once withblockingbinding buffer (20 mM Tris (pH 74) 150 mM NaCl 2mM CaCl2 1 mM MgCl2 1 mM MnCl2 1 BSA) and incubated atroom temperature for additional 2 h Plates were rinsed twice withsame buffer and then competition binding assays were performed witha constant concentration of [125I]-Echistatin (005 nM) Concen-trations of tested compound ranged from 001 to 100 nM All assayswere performed in triplicate in a final volume of 02 mL eachcontaining the following species 005 mL of [125I]-Echistatin 004 mLof tested compound and 011 mL of blockingbinding bufferNonspecific binding was defined as [125I]-Echistatin bound in presenceof an excess (1 μM) of unlabeled echistatin After incubation at roomtemperature for 3 h plates were washed three times with blockingbinding buffer and counted in a Top-Count NXT microplatescintillation counter (PerkinElmer Life Sciences Boston MA) using200 μLwell of MicroScint-40 liquid scintillation (PerkinElmer LifeSciences Boston MA)Data are shown as means plusmn SD from three independent

experiments IC50 values were determined by fitting binding inhibitiondata by nonlinear regression using GraphPad Prism 40 SoftwarePackage (GraphPad Prism San Diego CA)Cell Lines and Culture Conditions The A375 M human

melanoma cell line was obtained from the American Type CultureCollection (ATCC Rockville MD)12g Human dermal skin fibroblasts(HDF) were kindly donated by Dr Daniela Monti Department ofExperimental Pathology and Oncology University of FlorenceMelanoma cells and HDF were grown in Dulbeccorsquos Modified EagleMedium containing 4500 mgL glucose (DMEM 4500 GIBCO)supplemented with 10 fetal calf serum (FCS) at 37 degC in a

humidified incubator containing 10 CO2 Then 50 times 105 melanomacells or 1 times 106 HDF were seeded in 100 mm dishes and propagatedevery 3 days by incubation with a trypsinminusEDTA solution Cultureswere periodically monitored for mycoplasma contamination Primarycultures of human umbilical vein endothelial cells (HUVEC) wereobtained from Lonza (USA) HUVEC grown in endothelial cellgrowth medium-2 (EGM-2) containing endothelial cell growthsupplements were subcultivated using a trypsin-EDTA solution 13split ratio Cells between passages 2 and 4 grown to confluence inplates coated with 1 bovine gelatin (Sigma St Louis) were used inthe experiments

RNA Isolation and Polymerase Chain Reaction (PCR) TotalRNA extracted using RNAgents (Total RNA Isolation SystemPromega Madison WI) was determined spectrophotometricallyComplementary DNA (cDNA) was synthesized from 2 μg of totalRNA using 1 μL of ImProm-II reverse transcriptase (Promega)Aliquots of 2 μL of the cDNA were used for PCR amplification Thespecific primers used for the identification of human αv α5 β1 β3 β5and GAPDH are reported in Table S1 of Supporting Informations AllPCR experiments were conducted using 005 UμL of Go-TaqPolymerase (Promega) Amplification was carried out on a Perkin-Elmer Thermal cycler Then 8 μL of each PCR products werevisualized after electrophoresis in a 2 agarose gel containing 05 μgmL of ethidium bromide cDNA products were evaluated on the basisof a standard PCR marker

Flow Cytometry Assay Tumor cells were detached by gentletreatment with Accutase a 05 mM EDTA solution washed andincubated for 1 h at 4 degC in the presence of anti αvβ3 monoclonalantibody (1 μg50 μL Anti-Integrin αvβ3 clone LM609 Millipore)Cells were then washed and incubated for 1 h at 4 degC with a secondaryantibody 5 μgmL goat antimouse IgG conjugated with FITC (SantaCruz Biotechnology Inc Santa Cruz CA) αvβ3 -positive cells wereanalyzed at 488 nm on the flow cytometer FACScan system (BD-FACS Canto)

Cell Adhesion Assay Plates (96 wells) were coated withfibronectin (1 μgmL) or vitronectin (10 μgmL) by overnightincubation at 4 degC and then incubated at 37 degC for 1 h with 1 BSA inPBS Melanoma cells IL-1β (400 UmL)-stimulated HUVECs orhuman dermal fibroblasts (HDF) were resuspended in serum-freemedium and exposed to ligands 1 2 or 3 (final concentration was 011 or 10 μM) at 37 degC for 30 min to allow for ligandminusintegrinequilibrium to be reached Melanoma cell adhesion assays wereperformed in the presence or in the absence of 2 mmolL MnCl2Cells were plated (4minus5 times 104 cellswell) and incubated at 37 degC for 2h Nonadherent cells were removed with PBS and adherent cells werestained with 05 crystal violet solution in 20 methanol After 2 h ofincubation at 4 degC plates were examined at 540 nm in a counterELX800 (Biotek Instruments) Experiments were conducted intriplicate and repeated at least three times Data are presented asmeans plusmn SD from three independent experiments

Animal Model and Study Design In vivo experiments wereconducted in accordance with national guidelines and approved by theEthical Committee of the Animal Welfare Office of the Italian WorkMinistry and conforming to legal mandates and Italian guidelines forcare and maintenance of laboratory animals Eighteen-week-old SCIDbgbg mice (Charles River Laboratories International) were injectedsubcutaneously in the shoulder area with a suspension of melanomacells and 20 ngmL TGFβ-activated fibroblasts Then 1 times106 A375 Mmelanoma cells and 05 times 106 HDF were previously resuspended in100 μL of PBS diluted 11 with a Matrigel solution (BD Biosciences)Animals were kept under pathogen-free conditions and allowed to feedfreely Tumor growth was monitored daily

Small-Animal SPECT and CT Studies In vivo studies wereconducted using a ldquohybridrdquo imaging system for small laboratoryanimals allowing for the study of biological processes using minimallyinvasive procedures Engrafted tumor tracer uptake was studied atdifferent time-points after tail vein injection Binding specificity tointegrin receptors was evaluated by in vivo displacement studies inwhich a high dose of cold tracer was injected 10 min beforeradiolabeled one Imaging studies were performed using the Flex

Journal of Medicinal Chemistry Article

dxdoiorg101021jm2016232 | J Med Chem 2012 55 5024minus50335030

Triumph preclinical imaging system which combines positron (PET)and single-photon emission (SPECT) tomography with the emissiontomography (CT) for anatomic coregistration (GE HealthcareGamma Medica-Ideas Waukesha WI) 125I-iodinated tracers 125I-3was studied in vivo by SPECT allowing for use directly the in vitrotracer data to the in vivo experimentation Imaging studies requiredmonitoring of anesthetized animals throughout the imaging procedureand anesthesia was conducted in accordance with National Guidelinesand approved by the Ethical Committee Given that anesthesia ingeneral causes hypothermia in small animals appropriate warming wasprovided during imaging procedure by using a heating bed set at 37degC Physiological monitoring was achieved by measuring respirationrate with a pressure pad placed under the mouse abdomen Saturationexperiments (SE) to visualize tumors and displacement experiments(DE) were performed In SE 125I-3 (7minus10 MBq in 200 μL ofphysiological saline) was injected via tail vein into tumor bearinganimals kept under anesthesia After 30 min a CT aquisition wasperformed followed by SPECT scan starting at 40 min pi CT scanwas acquired with 50 kV and 320 μA tube settings The magnificationfactor was set to 2 resulting in an axial field of view of 592 mm 512projections 500 μs each were acquired on a circular orbit in 256 swith an acquisition matrix of 1184 times 1120 100 μm pixel SPECTprojections were acquired with a 5-pinhole high-resolution collimator(1 mm hole diameter 75 mm focal distance) with radius of rotation of40 mm resulting in an axial field of view of 529 mm then 64projections 20 s each were acquired on a circular orbit for a totalacquisition time of about 22 min Acquisition matrix was 80 times 80 15mm pixel size Energy window was set on the 125I photopeak rangingfrom 20 to 36 KeV The DE were performed by injecting 18 mgkg ofcompound 3 10 min before injection of tracer Once scanning wascomplete the animals were recovered in a housing unit and monitoreduntil awakeData Analysis of Small-Animal SPECT and CT Studies CT

image data set was reconstructed using a cone-beam FBP algorithm(FDK) and an optimum noise filter Reconstruction matrix was 512 times512 times 512 180 μm pixel size SPECT images were reconstructed withordered subset expectation maximization algorithm (8 subsets and 5iterations) and a reconstruction matrix of 60 times 60 times 60 15 mm pixelsize No correction for radiation attenuation and scatter was appliedFused images were visually analyzed to detect the engrafted tumormass and regional SPECT activities were measured using the analysissoftware Vivid (Gamma Medica-Ideas Northridge CA) by delineatingthe region of interest (ROI) of tumor in fused CT images A similarROI was generated for muscle tissue on contralateral side of tumorTumor ROI activity was normalized (nTA) with the followingformula

= minusnTA

TA BABA

where TA is tumor activity and BA is background activity measured inmuscle tissue contralateral to tumorDocking Calculations Automated docking studies were carried

out by Autodock 401 program17 using the Lamarckian GeneticAlgorithm (LGA) as a search engine The AutoDockTools 145(ADT) graphical interface25 was used to prepare integrin and ligandsPDBQT files Coordinates of ligand 3 were retrieved from lowestenergy conformers resulting from molecular mechanics calculationsstarting from geometry of ligand 1 whereas coordinates of αvβ3receptor were retrieved from the Protein Data Bank (PDB code1L5G) and ligandminusprotein complex was unmerged for achieving freereceptor structure Water molecules were removed For proteinreceptor and ligand 3 all hydrogens were added Gasteiger chargeswere computed and nonpolar hydrogens were merged A charge valueof +20 to each Mn atom of protein receptor was successively addedThree-dimensional energy scoring grids of 0375 Aring resolution and 40Aring times 40 Aring times 40 Aring dimensions were computed Center of grid was set tobe coincident with mass center of ligands preliminary fitted on the X-ray structure of c[RGDf(Me)V] in the αvβ3 complex (1L5G) A totalof 50 runs with a maximum of 2500000 energy evaluations werecarried out for each ligand using the default parameters for LGA

Cluster analysis was performed on docked results using a root-mean-square (rms) tolerance of 15 Aring Analysis of binding mode calculationof binding energy and prediction of binding activity of dockedconformations were carried out using PyMol Autodock Tools pluginwithin PyMol software26

ASSOCIATED CONTENTS Supporting InformationAdditional experimental methods radiochromatogram of 125I-3and additional cell biology assays data This material is availablefree of charge via the Internet at httppubsacsorg

AUTHOR INFORMATIONCorresponding AuthorFor AG phone +39 055 4573481 fax +39 055 4573569 E-mail antonioguarnaunifiit For AP phone +39 0557947527 fax +39 055 4224392 E-mail apupidfcunifiitFor LC phone +39 055 4598207 fax +39 055 4598900 E-mail lidocaloriniunifiit

NotesThe authors declare no competing financial interest

ACKNOWLEDGMENTSThis work has received financial support from the IstitutoTo s c ano Tumor i a nd MIUR (PR IN08 G r an t2008J4YNJY_003) Also financial contributions from CINM-PIS University of Florence and Fondazione Roma areacknowledged Dr Alessandro Sisti is acknowledged forcarrying out preliminary labeling experiments Ente Cassa diRisparmio is acknowledged for the granting the MicroPETSPECTCT instrument

ABBREVIATIONS USEDSPECT single positron emission computed tomography PETpositon emission tomography RGD Arg-Gly-Asp VGPvertical growth phase CT computed tomography VNvitronectin FN fibronectin HUVEC human umbilical veinendothelial cells IL-1β interleukin-1β ESI-MSMS electroprayionization tandem mass spectrometry EGM-2 endothelial cellgrowth medium-2 EDTA ethylenediaminetetraacetic acidGAPDH glyceraldehyde 3-phosphate dehydrogenase FACSfluorescence-activated cell sorting FITC fluorescein isothio-cyanate PBS phosphate buffered saline HUVEC humanumbilical vein endothelial cells HDF human dermal skinfibroblasts EMT epithelial-mesenchymal transition TGFtransform grown factor pi post injection

REFERENCES(1) Challenges and Opportunities Report US Department of Healthand Human Services Food and Drug Administration Silver SpringMD March 2004 httpwwwfdagovScienceResearchSpecialTopicsCriticalPathInitiativeCriticalPathOpportunities-Reports-ucm077262htm(2) (a) Backgrounder How New Drugs Move Through the Developmentand Approval Process Tufts Center for the Study of DrugDevelopment Boston November 1 2001 (b) Gilbert J HenskeP Singh A Rebuilding Big Pharmarsquos Business Model The Business ampMedicine Report Windhover Information November 2003 Vol 21 no10 httpwwwbaincomImagesrebuilding_big_pharmapdf(3) Innovative drug development approaches-project Final report fromTHEEMEACHMP-THINK-TANK group on innovative drug develop-ment EMEA 2007 pp 1minus26 httpwwwemaeuropaeudocsen_GBdocument_libraryOther200910WC500004913pdf

Journal of Medicinal Chemistry Article

dxdoiorg101021jm2016232 | J Med Chem 2012 55 5024minus50335031

Triumph preclinical imaging system which combines positron (PET)and single-photon emission (SPECT) tomography with the emissiontomography (CT) for anatomic coregistration (GE HealthcareGamma Medica-Ideas Waukesha WI) 125I-iodinated tracers 125I-3was studied in vivo by SPECT allowing for use directly the in vitrotracer data to the in vivo experimentation Imaging studies requiredmonitoring of anesthetized animals throughout the imaging procedureand anesthesia was conducted in accordance with National Guidelinesand approved by the Ethical Committee Given that anesthesia ingeneral causes hypothermia in small animals appropriate warming wasprovided during imaging procedure by using a heating bed set at 37degC Physiological monitoring was achieved by measuring respirationrate with a pressure pad placed under the mouse abdomen Saturationexperiments (SE) to visualize tumors and displacement experiments(DE) were performed In SE 125I-3 (7minus10 MBq in 200 μL ofphysiological saline) was injected via tail vein into tumor bearinganimals kept under anesthesia After 30 min a CT aquisition wasperformed followed by SPECT scan starting at 40 min pi CT scanwas acquired with 50 kV and 320 μA tube settings The magnificationfactor was set to 2 resulting in an axial field of view of 592 mm 512projections 500 μs each were acquired on a circular orbit in 256 swith an acquisition matrix of 1184 times 1120 100 μm pixel SPECTprojections were acquired with a 5-pinhole high-resolution collimator(1 mm hole diameter 75 mm focal distance) with radius of rotation of40 mm resulting in an axial field of view of 529 mm then 64projections 20 s each were acquired on a circular orbit for a totalacquisition time of about 22 min Acquisition matrix was 80 times 80 15mm pixel size Energy window was set on the 125I photopeak rangingfrom 20 to 36 KeV The DE were performed by injecting 18 mgkg ofcompound 3 10 min before injection of tracer Once scanning wascomplete the animals were recovered in a housing unit and monitoreduntil awakeData Analysis of Small-Animal SPECT and CT Studies CT

image data set was reconstructed using a cone-beam FBP algorithm(FDK) and an optimum noise filter Reconstruction matrix was 512 times512 times 512 180 μm pixel size SPECT images were reconstructed withordered subset expectation maximization algorithm (8 subsets and 5iterations) and a reconstruction matrix of 60 times 60 times 60 15 mm pixelsize No correction for radiation attenuation and scatter was appliedFused images were visually analyzed to detect the engrafted tumormass and regional SPECT activities were measured using the analysissoftware Vivid (Gamma Medica-Ideas Northridge CA) by delineatingthe region of interest (ROI) of tumor in fused CT images A similarROI was generated for muscle tissue on contralateral side of tumorTumor ROI activity was normalized (nTA) with the followingformula

= minusnTA

TA BABA

where TA is tumor activity and BA is background activity measured inmuscle tissue contralateral to tumorDocking Calculations Automated docking studies were carried

out by Autodock 401 program17 using the Lamarckian GeneticAlgorithm (LGA) as a search engine The AutoDockTools 145(ADT) graphical interface25 was used to prepare integrin and ligandsPDBQT files Coordinates of ligand 3 were retrieved from lowestenergy conformers resulting from molecular mechanics calculationsstarting from geometry of ligand 1 whereas coordinates of αvβ3receptor were retrieved from the Protein Data Bank (PDB code1L5G) and ligandminusprotein complex was unmerged for achieving freereceptor structure Water molecules were removed For proteinreceptor and ligand 3 all hydrogens were added Gasteiger chargeswere computed and nonpolar hydrogens were merged A charge valueof +20 to each Mn atom of protein receptor was successively addedThree-dimensional energy scoring grids of 0375 Aring resolution and 40Aring times 40 Aring times 40 Aring dimensions were computed Center of grid was set tobe coincident with mass center of ligands preliminary fitted on the X-ray structure of c[RGDf(Me)V] in the αvβ3 complex (1L5G) A totalof 50 runs with a maximum of 2500000 energy evaluations werecarried out for each ligand using the default parameters for LGA

Cluster analysis was performed on docked results using a root-mean-square (rms) tolerance of 15 Aring Analysis of binding mode calculationof binding energy and prediction of binding activity of dockedconformations were carried out using PyMol Autodock Tools pluginwithin PyMol software26

ASSOCIATED CONTENTS Supporting InformationAdditional experimental methods radiochromatogram of 125I-3and additional cell biology assays data This material is availablefree of charge via the Internet at httppubsacsorg

AUTHOR INFORMATIONCorresponding AuthorFor AG phone +39 055 4573481 fax +39 055 4573569 E-mail antonioguarnaunifiit For AP phone +39 0557947527 fax +39 055 4224392 E-mail apupidfcunifiitFor LC phone +39 055 4598207 fax +39 055 4598900 E-mail lidocaloriniunifiit

NotesThe authors declare no competing financial interest

ACKNOWLEDGMENTSThis work has received financial support from the IstitutoTo s c ano Tumor i a nd MIUR (PR IN08 G r an t2008J4YNJY_003) Also financial contributions from CINM-PIS University of Florence and Fondazione Roma areacknowledged Dr Alessandro Sisti is acknowledged forcarrying out preliminary labeling experiments Ente Cassa diRisparmio is acknowledged for the granting the MicroPETSPECTCT instrument

ABBREVIATIONS USEDSPECT single positron emission computed tomography PETpositon emission tomography RGD Arg-Gly-Asp VGPvertical growth phase CT computed tomography VNvitronectin FN fibronectin HUVEC human umbilical veinendothelial cells IL-1β interleukin-1β ESI-MSMS electroprayionization tandem mass spectrometry EGM-2 endothelial cellgrowth medium-2 EDTA ethylenediaminetetraacetic acidGAPDH glyceraldehyde 3-phosphate dehydrogenase FACSfluorescence-activated cell sorting FITC fluorescein isothio-cyanate PBS phosphate buffered saline HUVEC humanumbilical vein endothelial cells HDF human dermal skinfibroblasts EMT epithelial-mesenchymal transition TGFtransform grown factor pi post injection

REFERENCES(1) Challenges and Opportunities Report US Department of Healthand Human Services Food and Drug Administration Silver SpringMD March 2004 httpwwwfdagovScienceResearchSpecialTopicsCriticalPathInitiativeCriticalPathOpportunities-Reports-ucm077262htm(2) (a) Backgrounder How New Drugs Move Through the Developmentand Approval Process Tufts Center for the Study of DrugDevelopment Boston November 1 2001 (b) Gilbert J HenskeP Singh A Rebuilding Big Pharmarsquos Business Model The Business ampMedicine Report Windhover Information November 2003 Vol 21 no10 httpwwwbaincomImagesrebuilding_big_pharmapdf(3) Innovative drug development approaches-project Final report fromTHEEMEACHMP-THINK-TANK group on innovative drug develop-ment EMEA 2007 pp 1minus26 httpwwwemaeuropaeudocsen_GBdocument_libraryOther200910WC500004913pdf

Journal of Medicinal Chemistry Article

dxdoiorg101021jm2016232 | J Med Chem 2012 55 5024minus50335031

(4) Gilbert M R Kuhn J Lamborn K R Lieberman F Wen PY Mehta M Cloughesy T Lassman A B Deangelis L MChang S Prados M Cilengitide in patients with recurrentglioblastoma the results of NABTC 03minus02 a phase II trial withmeasures of treatment delivery J Neurooncol 2012 106 147minus153(5) Cilengitide Temozolomide and Radiation Therapy in TreatingPatients With Newly Diagnosed Glioblastoma and Methylated GenePromoter Status (CENTRIC) National Institutes of Health BethesdaMD httpclinicaltrialsgovct2showNCT00689221 httpclinicaltrialsgovct2showNCT00085254(6) For reviews see (a) Arnaout M A Mahalingam B Xiong J PIntegrin structure allostery andbidirectional signaling Annu Rev CellDev Biol 2005 21 381minus410 (b) Cabodi S del Pilar Camacho-LealM Di Stefano P Defilippi P Integrin signalling adaptors not onlyfigurants in the cancer story Nature Rev Cancer 2010 10 858minus870(c) Shattil S J Kim C Ginsberg M H The final steps of integrinactivation the end game Nature Rev Mol Cell Biol 2010 11 288minus300 (d) Desgrosellier J S Cheresh D A Integrins in cancerbiological implications and therapeutic opportunities Nature RevCancer 2010 10 9minus22 (e) Hanahan D Weinberg R A Hallmarksof cancer the next generation Cell 2011 144 646minus674(7) (a) Albelda S M Mette S A Elder D E Stewart RDamjanovich L Herlyn M Buck C A Integrin distribution inmalignant melanoma association of the beta 3 subunit with tumorprogression Cancer Res 1990 50 6757minus6764 (b) Seftor R E Roleof the beta3 integrin subunit in human primary melanomaprogression multifunctional activities associated with alpha(v)beta3integrin expression Am J Pathol 1998 153 1347minus1351 (c) VanBelle P A Elenitsas R Satyamoorthy K Wolfe J T Guerry DIV Schuchter L Van Belle T J Albelda S Tahin P Herlyn MElder D E Progression-related expression of beta3 integrin inmelanomas and nevi Hum Pathol 1999 30 562minus567 (d) McGary EC Lev D C Bar-Eli M Cellular adhesion pathways and metastaticpotential of human melanoma Cancer Biol Ther 2002 1 459minus465(8) (a) Brooks P C Stromblad S Sanders L C von Schalscha TL Aimes R T Stetler-Stevenson W G Quigley J P Cheresh DA Localization of matrix metalloproteinase MMP-2 to the surface ofinvasive cells by interaction with integrin alpha v beta 3 Cell 1996 85683minus693 (b) Hofmann U B Westphal J R Waas E T Becker JC Ruiter D J van Muijen G N Coexpression of integrinalpha(v)beta3 and matrix metalloproteinase-2 (MMP-2) coincideswith MMP-2 activation correlation with melanoma progression JInvest Dermatol 2000 115 625minus632(9) Galvez B G Matiacuteas-Roman S Yanez-Mo M Sanchez-MadridF Arroyo A G ECM regulates MT1-MMP localization with beta1 oralphavbeta3 integrins at distinct cell compartments modulating itsinternalization and activity on human endothelial cells J Cell Biol2002 159 509minus521(10) Alonso S R Tracey L Ortiz P Perez-Gomez B Palacios JPollan M Linares J Serrano S Saez-Castillo A I Sanchez LPajares R Sanchez-Aguilera A Artiga M J Piris M A Rodriacuteguez-Peralto J L A high-throughput study in melanoma identifiesepithelialminusmesenchymal transition as a major determinant of meta-stasis Cancer Res 2007 67 3450minus3460(11) Hynes R O A reevaluation of integrins as regulators ofangiogenesis Nature Med 2002 8 918minus921(12) (a) Haubner R Finsinger D Kessler H Stereoisomericpeptide libraries and peptidomimetics for designing selective inhibitorsof the alpha(V)beta(3) integrin for a new cancer therapy AngewChem Int Ed Engl 1997 36 1374minus1389 (b) MacDonald T JTaga T Shimada H Tabrizi P Zlokovic B V Cheresh D ALaug W E Preferential susceptibility of brain tumors to theantiangiogenic effects of an alpha(v) integrin antagonist Neurosurgery2001 48 151minus157 (c) Kumar C C Malkowski M Yin ZTanghetti E Yaremko B Nechuta T Varner J Liu M Smith EM Neustadt B Presta M Armstrong L Inhibition of angiogenesisand tumor growth by SCH221153 a dual alpha(v)beta3 andalpha(v)beta5 integrinreceptor antagonist Cancer Res 2001 612232minus2238 (d) Burke P A De Nardo S J Miers L A

Lamborn K L Matzku S De Nardo G L Cilengitide Targeting ofavb3 Integrin Receptor Synergizes with Radioimmunotherapy toIncrease Efficacy and Apoptosis in Breast Cancer Xenografts CancerRes 2002 62 4263minus4272 (e) Belvisi L Riccioni T Marcellini MVesci L Chiarucci I Efrati D Potenza D Scolastico C ManzoniL Lombardo K Stasi M A Orlandi A Ciucci A Nico BRibatti D Giannini G Presta M Carminati P Pisano CBiological and molecular properties of a new alpha(v)beta3alpha(v)-beta5 integrin antagonist Mol Cancer Ther 2005 4 1670minus1680(f) Clezardin P Teti A Bone metastasis pathogenesis andtherapeutic implications Clin Exp Metastasis 2007 24 599minus608(g) Trabocchi A Menchi G Cini N Bianchini F Raspanti SBottoncetti A Pupi A Calorini L Guarna A Click-chemistry-derived triazole ligands of arginine-glycine-aspartate (RGD) integrinswith a broad capacity to inhibit adhesion of melanoma cells and bothin vitro and in vivo angiogenesis J Med Chem 2010 53 7119minus7128(h) Gentilucci L Cardillo G Spampinato S Tolomelli ASquassabia F De Marco R Bedini A Baiula M Belvisi L CiveraM Antiangiogenic effect of dualselective alpha(5)beta(1)alpha(v)-beta(3) integrin antagonists designed on partiallymodified retro-inverso cyclotetrapeptide mimetics J Med Chem 2010 53 106minus118(13) For reviews see (a) Liu S Radiolabeled Multimeric CyclicRGD Peptides as Integrin αvβ3 Targeted Radiotracers for TumorImaging Mol Pharmaceutics 2006 3 472minus486 (b) Beer A JSchwaiger M Imaging of integrin αvβ3 expression Cancer MetastasisRev 2008 27 631minus644 (c) Haubner R Decristoforo CRadiolabelled RGD peptides and peptidomimetics for tumor targetingFront Biosci 2009 14 872minus886 (d) Liu L Radiolabeled Cyclic RGDPeptides as Integrin αvβ3-Targeted Radiotracers Maximizing BindingAffinity via Bivalency Bioconjugate Chem 2009 20 2199minus2213(f) Beer A J Kessler H Wester H J Schwaiger M PET Imagingof Integrin αvβ3 Expression Theranostic 2011 1 48minus57(14) (a) Dijkgraaf I Beer A J Wester H J Application of RGD-containing peptides as imaging probes for alphavbeta3 expressionFront Biosci 2009 14 887minus899 (b) Schottelius M Laufer BKessler H Wester H J Ligands for Mapping αvβ3-IntegrinExpression in Vivo Acc Chem Res 2009 42 969minus980 (c) Li Z BChen X MicroPET MicroSPECT and NIR Fluorescence Imaging ofBiomolecules in Vivo Methods Mol Biol 2009 544 461minus481(d) Zhou Y Chakraborty S Liu S Radiolabeled Cyclic RGDPeptides as Radiotracers for Imaging Tumors and Thrombosis bySPECT Theranostic 2011 1 58minus62(15) Cini N Trabocchi A Menchi G Bottoncetti A RaspantiS Pupi A Guarna A Morpholine-based RGD-cyclopentapeptides asalphavbeta3alphavbeta5 integrin ligands role of configurationtowards receptor binding affinity Bioorg Med Chem 2009 171542minus1549(16) Xiong J P Stehle T Zhang R Joachimiak A Frech MGoodman S L Arnaout M A Crystal Structure of the extracellulardomain of integrin alphaV beta3 in complex with an Arg-Gly-Aspligand Science 2002 296 151minus155(17) Morris G M Goodsell D S Halliday R S Huey R HartW E Belew R K Olson A J Automated docking using Lamarckiangenetic algorithm and an empirical binding free energy function JComput Chem 1998 19 1639minus1662(18) We assumed 3prime-iodo-D-Tyr side chain not to influence theconformational preferences of the cyclopeptide backbone asconfirmed by conformational analysis by molecular mechanicsresulting in a similar geometry as for 1 possessing the cis-amidebond between D-Tyr and D-morpholine-3-carboxylic acid(19) (a) Benaron D A The future of cancer imaging CancerMetastasis Rev 2002 21 45minus78 (b) Beer A J Schwaiger M Imagingof integrin alphavbeta3 expression Cancer Metastasis Rev 2008 27631minus644(20) Christofori G New signals from the invasive front Nature2006 441 444minus450(21) Laurens N Engelse M A Jungerius C Lowik C W vanHinsbergh V W Koolwijk P Single and combined effects of avb3 and

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a5b1 integrins on capillary tube formationin a human fibrinous matrixAngiogenesis 2009 12 275minus285(22) Witz I P Tumor-microenvironment interactions dangerousliaisons Adv Cancer Res 2008 100 203minus229(23) Kalluri R Zeisberg M Fibroblasts in cancer Nature RevCancer 2006 6 392minus401(24) Kumar C C Nie H Armstrong L Zhang R Vijay-KumarS Tsarbopoulos A Chloramine T-induced structural and biochemicalchanges in echistatin FEBS Lett 1998 429 239minus248(25) Gillet A Sanner M Stoffler D Olson A Tangible Interfacesfor Structural Molecular Biology Structure 2005 13 483minus491(26) DeLano W L The PyMOL Molecular Graphics System DeLanoScientific LLC San Carlos CA [httpwwwpymolorg]

Journal of Medicinal Chemistry Article

dxdoiorg101021jm2016232 | J Med Chem 2012 55 5024minus50335033