Bioorganic & Medicinal Chemistry 21 (2013) 5470–5479

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Bioorganic & Medicinal Chemistry

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Antiproliferative activity on human prostate carcinoma cell lines ofnew peptidomimetics containing the spiroazepinoindolinonescaffold

0968-0896/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.bmc.2013.06.006

⇑ Corresponding author. Tel.: +39 0250314468/0250314481; fax: +390250314476.

E-mail address: sara.pellegrino@unimi.it (S. Pellegrino).� These authors contributed equally to this work.

Sara Pellegrino a,⇑,�, Massimiliano Ruscica b,�, Paolo Magni b,�, Giulio Vistoli c,�, Maria Luisa Gelmi a,�

a DISFARM, sezione di Chimica Generale e Organica ‘‘A. Marchesini’’, Università degli Studi di Milano, via Venezian 21, Milano 20133, Italyb Department of Pharmacological and Biomolecular Sciences, Università degli Studi di Milano, via Balzaretti 9, Milano 20133, Italyc DISFARM, sezione di Chimica Farmaceutica ‘‘P. Pratesi’’, Università degli Studi di Milano, via Mangiagalli 25, Milano 20133, Italy

a r t i c l e i n f o

Article history:Received 21 November 2012Revised 30 May 2013Accepted 4 June 2013Available online 13 June 2013

Keywords:GhrelinProstate cancerPeptidomimeticsDocking

a b s t r a c t

Peptidomimetics containing the spiroazepinoindolinone scaffold were designed and synthesized in orderto ascertain their antiproliferative activity on the DU-145 human prostatic carcinoma cell line. Ethyl20-oxa-1,2,3,5,6,7-hexahydrospiro[4H-azepine-4,30-3H-indole]-10-carboxylate scaffold was functional-ized at nitrogen azepino ring with Aib-(L/D)Trp-OH dipeptides. Combining the different stereochemistriesof the scaffold and the tryptophan, diastereoisomeric peptidomimetics were prepared and tested. Theirbiological activity was evaluated by proliferation studies proving that the isomer containing S spiroaze-pino-indolinone scaffold and L tryptophan is the most active compound. Docking studies confirmed thatthe active peptidomimetic could bind the GHSR-1a receptor with docking scores comparable with thoseof well-known agonists even though with a somewhat different binding mode.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Prostate cancer is the third most frequent cancer in men world-wide, representing about 11% of all male cancers in Europe andaccounting for 9% of all cancer deaths among men. Recent findingssuggest an involvement of the ghrelin system in prostate cancer,being ghrelin itself and some synthetic derivatives able to modu-late cell proliferation in different androgen dependent or indepen-dent prostatic carcinoma cell lines.1

Ghrelin is a multifunctional 28 residue peptide hormone char-acterized by an octanoyl moiety on Ser-3. Its biological activity ismediated by the interaction with a specific G-protein coupledreceptor, GHSR-1a, which is characterized by a marked constitutiveactivity and has been demonstrated to be expressed both in centraland peripheral tissues. Beside the regulation of growth hormonerelease, ghrelin can influence widespread important physiologicfunctions, ranging from body weight, metabolism, appetite, glu-cose homeostasis and insulin secretion thus indicating that ghrelinanalogues can find manifold therapeutic applications.2 In the lastyears, there is also growing evidence of its role in the control of cellproliferation and cancer progression, although the available dataon ghrelin actions on the different stages of cancer progression still

remain conflicting, and ghrelin may, according to specific condi-tions, both promote and/or antagonize tumor progression.3 Recentstudies reported however that expression of ghrelin is correlatedwith favorable outcome in human breast cancer,3b thus giving arationale to the further clarification of these effects. Consideringthe potent orexigenic effect of ghrelin peptide, GHSR-1a agonistsshould also have a beneficial role in cancer cachexia, which is asso-ciated with anorexia, weight loss, weakness and fatigue and ac-counts for up to 30% of cancer-related deaths.3a Therefore, itcomes as no surprise that several peptidomimetics have been re-ported as ligands of GHS-R1a acting as agonists of ghrelin.2,4 Therational design of these ligands is based on the observation thatshort ghrelin analogues, including the first 4-6 residues (e.g., Gly-Ser-Ser(n-octanoyl)-Phe-NH2, EC50 = 72 nM),3d are able to elicitthe GHSR-1a agonism.

Among the proposed GHSR-1a ligands, some very active com-pounds, like ibutamoren (EC50: 1.3 ± 0.09 nM, in rat pituitarycells,4h Fig. 1), which reached the clinical trials, include the spiro-piperidine scaffold.

The reported derivatives indicate that the Ser(n-octanoyl) resi-due can be conveniently replaced by benzyloxy moiety or, in thecase of compound 1 (EC50 = 0.6 nM),4f by indole, conserving an effi-cient GHSR-1a activity. In these compounds, the spiro system,which replaces the terminal Phe-NH2 residue, is decorated by H-bonding functions which should mimic the terminal carbamidemoiety. Homology GHSR-1a models, generated by our researchgroup using a fully fragmental approach in its open and close

N

N

SO2Me

HN

NH2

O

H

OCH2Ph

O

MK-0677Ibutamoren

HN

NH2

NH

O

O

HO

N

L-163,0211

Figure 1. Ligands of hGHS-R1a acting as agonists of ghrelin.

S. Pellegrino et al. / Bioorg. Med. Chem. 21 (2013) 5470–5479 5471

states,5 revealed the specific roles of each ligand moieties evidenc-ing a certain degree of interchangeability between the two aro-matic systems of the spiro derivatives. Depending on theconfiguration and steric hindrances, the spiropiperidine systemcan be also harbored in the subcavity that normally interacts withthe octanoyl residue. Notably, this interchangeability is paralleledby the architecture of the binding site since apolar residues evenlyflank both subcavities with a marked abundance of aromatic sidechains (see Fig. 6 below).

With a view to investigating in depth the specific role of spirosystem, the present study describes the design and synthesis ofnew putative ghrelin agonists of general formula 2 (Fig. 2).

In detail, the Aib-Trp dipeptide chain substitution was chosen,as it is present into highly potent ligands.4b

Considering the richness of aromatic residues lining the GHSR-1a binding site, we selected the indolinone moiety both for itscapacity to elicit H-bonds and its enhanced ability to stabilize ex-tended p–p stacking if compared to the hydroxy indane of com-pound 1.

The replacement of piperidine ring with azepino one increasesthe aliphatic character of this moiety, adding a stereocenter tothe molecule. As reported for known derivatives4g the configura-tion of the spiro moieties could have a dramatic influence on bio-logical results. Furthermore, even though (D)-amino acids led tothe better activity in most GHSR-1a ligands,4b the stereochemistryof the Trp amino acid may also affect the activity of the consideredligands. The four possible diastereoisomers (see below) can be seenas structurally constrained geometries allowing the relations be-tween ligand conformation and bioactivity to be analysed ascer-taining the mutual influence of the stereocenters on thebiological activity.

N

H

O

NTrp AibNH2

R2

Figure 2. General formula of synthesized putative ghrelin agonists.

The possibility to introduce a H-bond acceptor functional groupon the aromatic ring, such as the nitro group, have been also con-sidered. We selected a strong electron-drawing substituent with aview to analysing its effects on p–p interactions that should bestrengthened by reducing electrostatic repulsion between p cloudsaccording to Hunter–Sanders rules,6 even though recent studies re-vealed that the substituent effects on p–p stacking could markedlyvary depending on the chemical features and reciprocal arrange-ment of the two interacting aromatic systems.7

Finally, by considering the key role exerted by the ion-pair be-tween ligand protonated amine terminus and Glu124, the N-acetylanalogue (�)-16 (see below) was also synthesized, as a negativecontrol, in order to further ascertain the biological profile of thehere proposed derivatives.

Since ghrelin and some of its synthetic agonists showed anti-proliferative activity on prostatic carcinoma cell lines, the pro-posed derivatives were tested on DU-145 human prostatecarcinoma cells,8 showing an androgen independent phenotypeand expressing the GHS-R1a receptor.

Lastly and considering the heterogeneity of the recently re-solved GPCR structures, we decided to generate a new model forGHSR-1a with a view to taking advantages from these recent X-ray structures to derive more reliable docking results.

2. Chemistry

Peptidomimetics of general formula 2 were synthesized takingadvantage from the recently prepared spiroazepinoindoline deriv-atives 3.9 The chemoselective reduction of the lactam function of3a to amino one was achieved via transformation of the carbonylgroup to thiolactam using Lawesson’s reagent10 (1.2 equiv) indichloromethane at 25 �C affording compound 4 (85% yield).(Scheme 1) The thioamide was then alkylated using MeI in THFobtaining compound 5, that was reduced to amino compound 6awith NaBH3CN in EtOH in the presence of AcOH (pH 4) at 25 �C fol-lowed by urethane deprotection with NaOH (80% overall yield).Similarly, starting from enantiopure (+)-3a,9 azepino derivative(�)-6a was obtained in 66% overall yield via intermediates (+)-4and 5 (Scheme 1).

Aiming to functionalize the aromatic ring with the nitro group,several attempts were performed. First 3a was transformed in thecorresponding nitro derivative 3b (45%) using the mixture H2SO4/HNO3 in CHCl3 at 50 �C. Unfortunately, the transformation of 3bin the corresponding thiolactam (Lawesson’s reagent or P4S10)failed. The direct nitration of thiolactam 4, operating in the abovecondition, gave only trace amount of the expected compound,being the nitro lactam 3b the main product. The direct nitrationof amine 6a was then studied performing the reaction in differentconditions but in any cases failed.11

Finally, compound 7b was successfully obtained (64%) from N-acylamine 7a (obtained from 6a and Ac2O operating in CH2Cl2 inthe presence of DIPEA at 25 �C, 54%) by nitration with NaNO3 in tri-fluoroacetic acid at 0 �C. The nitrogen atom was then deprotectedby hydrolysis with 6 N HCl at 120 �C and compound 6b was iso-lated in quantitative yield. (Scheme 1)

All compounds 4–7 were characterized by analytical and spec-troscopic analyses. Interestingly, 1H NMR spectrum of the azepinoderivatives 6 and 7 showed resonances corresponding to two con-formers. Since this phenomenon was present in all the synthesizedpeptides 8–16, we performed dynamic NMR experiments on theacetamido derivative 7a (DMSO-d6; temperature range: 300–383 K). Two conformers were present in the 1H NMR spectrum of7a at 300 K in 40:60 ratio, being NH present as two singlets thatresonate at d 10.39 and 10.36, respectively. The coalescencetemperature is at 363 K where a single conformer is present. Asdepicted in Figure 3 and Figure FSI1 (Supplementary data), 10 ns

Figure 3. Conformational equilibrium shown by the seven-membered ring of 7awhich can assume two stable boat conformations which can be clearly defined bythe two synclinal geometries assumed by the displayed s torsion

NCO2Et

O

NH

O

NCO2Et

O

NH

S

NCO2Et

O

NH+ I-

SMe

NHO

NH

(±)-3a(+)-S-3a

(±)-4(+)-S-4

Lawesson

CH2Cl2

MeI, THF

(±)-7a

(±)-6a(-)-S-6a

NCO2Et

O

NH

O

(±)-3b

O2N

H2SO4/HNO3,CHCl3, 50 °C

NHO

NCOMe

O2N

NHO

NCOMe

5

NaBH3CN, AcOHEtOH

then NaOH (2N)

Ac2O/DIPEACH2Cl2, 25 °C

(±)-7b

CF3CO2H, NaNO3

CHCl3, 25 °CNHO

NH2HCl

O2N

(±)-6b

6N HCl

Scheme 1. Synthesis of hexahydrospiro-4H-azepine-4,30-3H-indoles 6.

5472 S. Pellegrino et al. / Bioorg. Med. Chem. 21 (2013) 5470–5479

MD simulations at increasing temperature revealed that the seven-membered ring shows an equilibrium between two boat confor-mations differing for the torsion angle defined by C2 and C3 atomswhich can assume both synclinal geometries, while the amidofunction constantly conserves the same geometry during all simu-lations. Interestingly, the MD run at 300 K shows only five transi-tions in 10 ns and this can explain the presence of two well-defined conformations, while the number of observed transitionssignificantly increases by heating the system at 370 K in line withthe observed coalescence temperature.

Semiempirical PM6 calculations showed that the conformationcharacterized by a positive synclinal geometry is slightly favoredwith a DE = 1.19 Kcal/mol in agreement with the 40:60 ratio (ascomputed by Boltzmann distribution). Interestingly, the samebehavior was observed for the seven membered derivative 6,unsubstituted at nitrogen atom, thus further confirming that thecis–trans isomerization of amido group is not involved in the ob-served conformational equilibrium (Supplementary data, Fig. FSI2).

In order to prepare the enantiopure intermediates 8/9, (L)-Boc-tryptophane was used as both reagent and resolving agent. Thecondensation reaction was performed using EDC�HCl, HOBt and DI-PEA in CH2Cl2 at 0 �C. The reaction of (±)-6a afforded a mixture ofdiastereoisomers (�)-8a and (+)-9a (65%) that were separated byflash chromatography. Instead, the diastereoisomeric derivatives8b and 9b, (92% yield from (±)-6b) were found not separable.(Scheme 2) The deprotection of N-Boc on compounds (�)-8a and(+)-9a was achieved operating in TFA/CH2Cl2 (from 0 to 35 �C),obtaining (�)-10a (67%) and (+)-11a (73%), respectively. Peptidom-

imetics (�)-12a (64%), (+)-13a (35%) were obtained from pure 10aor 11a and N-Boc Aib, according to the above condensative proto-col. Their deprotection at nitrogen atom gave (�)-14a (89%) and(+)-15a (37%), respectively. Compound (�)-14a was finally acyl-ated to the N-terminus (Ac2O, DIPEA, THF, 25 �C) affording (�)-16 (75%). (Scheme 2)

Aiming to assign to each diastereoisomer of the a series the cor-rect stereochemistry at the spiro center, enantiopure compound(�)-14a was prepared. In this case, we coupled directly the freeamine (�)-6a with previously synthesized Boc-Aib-(L)-Trp-OHdipeptide using the above condensation conditions followed byN-Boc deprotection (76% overall yield, Scheme 2). ½a�25

D values ofcompound 14a, obtained in the two different synthetic procedures,allowed to assign the S-stereochemistry to the spiro center forcompounds 8a, 10a, 12a, 14a and 16. As a consequence, R-spirocarbon stereochemistry characterizes compounds 9a, 11a, 13aand 15a.

Starting from racemic 6a, the (D)-Trp series was prepared usingthe above condensation conditions with previously synthesizedBoc-Aib-(D)Trp-OH dipeptide followed by N-Boc deprotectionobtaining (+)-14a and (�)-15a isomers (65% overall yield,Scheme 2), which were then separated using preparative HPLCand their purity was checked by comparing their ½a�25

D values withthe previously obtained (L)-Trp series.

As reported above, the separation of diastereoisomeric com-pounds 8b and 9b failed and for this reason, to prevent further syn-thetic steps, we planned the direct synthesis of the final nitro-substituted peptidomimetics 14b/15b, by direct condensation of(±)-6b with Boc-Aib-(L)TrpOH according to condensation/deprotec-tion standard protocols. (Scheme 2) The above compounds wereobtained in 45% overall yield but, in this case too, any attempt toseparate the diastereoisomers failed and they were used in mixturefor the biological tests.

3. Cell proliferation studies and effect on ERK1/2phosphorylation

The DU145 cell line was selected as the in vitro model to testour ghrelin analogues, since these cells have been previouslyshown to express GHS-R1a.8b,12 Furthermore, ghrelin and different

Figure 4a. Effects of ghrelin and ghrelin analogue (�)-14a on BrdU-incorporationon DU145 cells.

NH

O

NH

NH

O

N NHR

NH

HO

NH

O

N NHR

NH

HO

NH

O

NHN

NH

HO

NHR

O

NH

O

N

NH

HO

HN NHR

O

(±)-6a, (-)-S-6a, (±)-6b

Y

Y Y

a Y = Hb Y = NO2

i

iii

S R

Y Y

(-)-8a, 8b: R = Boc(-)-10a: R = H . CF3CO2Hii (+)-9a, 9b: R = Boc

(+)-11a: R = H . CF3CO2Hii

(+)-13a, 13b: R = Boc(+)-15a, 15b: R = H . CF3CO2Hii(-)-12a, 12b: R = Boc

(-)-14a, 14b: R = H . CF3CO2H(-)-16: R = Ac .

ii

v

iv

iii

NH

O

NHN

NH

HO

NHR

O

NH

O

N

NH

HO

HN NHR

OY Y

vi,ii

(+)-14a (-)-15a

S

R

R

S

L L

L L

D D

Scheme 2. Synthesis of spiroazepinoindolinone peptidomimetics. Reagents and conditions: (i) NBoc-(L)-Trp, HOBt (2.5 equiv), EDC (2.5 equiv), DIPEA (2.5 equiv), CH2Cl2,25 �C; (ii) CF3CO2H/CH2Cl2 (1:1), 0 �C then 25 �C; (iii) NBoc-Aib, HOBt (2.5 equiv), EDC (2.5 equiv), DIPEA (2.5 equiv), CH2Cl2, 25 �C; (iv) NBoc-Aib-(L)-Trp, HOBt (1.2 equiv),EDC (1.2 equiv), DIPEA (4 equiv), CH2Cl2, 25 �C; (v) Ac2O, DIPEA, THF, 25 �C (vi) NBoc-Aib-(D)-Trp, HOBt (1.2 equiv), EDC (1.2 equiv), DIPEA (4 equiv), CH2Cl2, 25 �C.

Table 1Cell proliferation assay for ghrelin and compounds 14–16

Compounds Cell proliferation (% variation vs untreated cells)

10�6 M 10�9 M

Ghrelin �25 (p <0.05) �38 (p <0.01)(�)-(S,L)-14a �27 (p <0.01) �20 (p <0.05)(+)-(R,D)-14a +9.7 (ns) �2.4 (ns)(�)-(S,D)-15a �5.5 (ns) �4.8 (ns)(+)-(R,L)-15a �15 (ns) �10 (ns)14b/15b +5 (ns) +4 (ns)(�)-(S,L)-16 +2.1 (ns) +1.2 (ns)

S. Pellegrino et al. / Bioorg. Med. Chem. 21 (2013) 5470–5479 5473

growth hormone secretagogues, such as ibutamoren, were foundto exert antiproliferative effects in this cell model.8b

DU145 cells were subjected to a 48-h dose-response study(10�6 and 10�9 M) with ghrelin or compounds (�)-14a, (+)-14a,(�)-15a, (+)-15a and (�)-16 or with the diastereomeric mixture14b/15b. (Table 1)

Among all the substances tested, only ghrelin and compound(�)-14a were found to inhibit DU145 cell proliferation. In particu-lar, the native ligand ghrelin was significantly effective in inhibit-ing cell growth (�25% at 10�6 M (p <0.05) and �38% at 10�9 M(p <0.01)) (Fig. 4a).

Compound (�)-14a was able to reduce DU145 cell growth by27% at 10�6 M (p <0.01) and 20% at 10-9M (p <0.05) (Fig. 4a). The

addition of the GHS-R1a antagonist D-Lys3-GHRP-6 counteractedthe antiproliferative effect of 10�6 and 10�9 M ghrelin and (�)-14a (Fig. 4b). Of particular interest the inactivity of acetylderiva-tive (�)-16a, which underlines the relevance of the protonatedamine terminus of Aib for biological activity.

Figure 4b. Effects of the GHS-R1a antagonist D-Lys3-GHRP-6 on (�)-14a-inducedBrdU-incorporation on DU145 cells.

5474 S. Pellegrino et al. / Bioorg. Med. Chem. 21 (2013) 5470–5479

The effect of ghrelin and ghrelin analogue (�)-14a on the acti-vation of the ERK1/2 pathway was then tested, since ghrelin isknown to modulate this pathway in cell-based prostate cancermodels.13 After a 16-h serum starvation, DU145 cells were exposedto ghrelin and (�)-14a (both at 10�6 or 10�9 M) for 10 min and theextent of phosphorylated ERK1/2 (pERK1/2) was analyzed by Wes-tern blot. pERK1/2 levels are very low in untreated cells, whereasghrelin and (�)-14a were found to increase pERK1/2 at each dosetested (Fig. 5a). To further confirm that ghrelin and (�)-14a acti-vated ERK1/2 phosphorylation via GHS-R1a involvement, the

Figure 5a. Effects of ghrelin and ghrelin analogue (�)-14a on ERK1/2 phosphor-ylation in DU145 cells.

Figure 5b. Effects of the GHS-R1a antagonist D-Lys3-GHRP-6 on (�)-14a-inducedERK1/2 phosphorylation in DU145 cells.

above described experiments were repeated in the presence ofGHS-R1a antagonist D-Lys3-GHRP-6. As shown in Fig. 5b, pretreat-ment (1 h) with 10�6 M D-Lys3-GHRP-6 fully counteracted (�)-14a-driven ERK phosphorylation.

The reported data on the antiproliferative effect of (�)-14a arein agreement with previous observations obtained, using the sameDU145 cell line, with the growth hormone secretagogue ibutamo-ren, which also resulted maximally effective at the 10�6 M concen-tration.8b The specific involvement of GHSR-1a in this activation ofthe ERK1/2 pathway by (�)-14a, as well as in the antiproliferativeeffect of this compound in DU145 cells, is confirmed by the clear-cut counteracting effect exerted by D-Lys3-GHRP-6, a well-knownGHSR-1a antagonist (Fìgs. 5a and b).

4. Docking results

In order to gain a deeper insight in receptor binding, dockingstudies have been then performed and can be subdivided intotwo parts. The first part involved docking simulations for the twoenantiomers of ibutamorem, a well-known GHSR-1a agonist thatcan be seen as the reference compound for the novel derivativesproposed in the present study. These preliminary calculations havethe objective of validating the architecture of the GHSR-1a bindingsite revealing the main residues involved in ligand recognition.Based on the obtained results, the second part concerned dockinganalyses on the here proposed compounds with a view to rational-izing their activity and the observed inversion of stereoselectivity.

Thus, Figure 6a shows the main contacts stabilizing the bestcomplex with (D)-ibutamoren and emphasizes the pivotal role ofthe polar interactions which the ammonium head stabilizes withGlu124, Thr127, Tyr128 and which can vastly counteract the elec-trostatic repulsion exerted by Arg283. Yet again, the sulphonylgroup elicits H-bonds with Ser123, Tyr313, and to minor extentwith Gln120. Finally, the complex is stabilized by an extendedset of p–p interactions, which involve the dihydro indole systemwith Phe279, Phe312 and Tyr313 as well as the phenyl ring withPhe220, Phe221, Phe222, His280 and Tyr284. The analysis of allgenerated poses for (D)-ibutamoren reveals a second significantpose that is characterized by an inverted arrangement of the aro-matic moieties. While conserving the key contacts elicited by theammonium head, such a pose is clearly disfavored, as shown inTable 2, since it stabilizes the already described set of p–p interac-tions but loses the crucial H-bonds involving the sulfonyl group.Notably, these two possible poses can explain the observed stere-oselectivity of ibutamoren; indeed, the inverted pose appears to bethe favored one for the (L)-isomer, allegedly because the steric hin-drance between the ammonium head and the sulfonyl group pre-vents the entire interaction pattern to be stabilized whenassuming the normal pose. Collectively, these preliminary resultsemphasize that (D)-ibutamoren can assume both poses, the normalpose being the favored one (Table 2); whereas, the (L)-ibutamorenassume preferentially the less favored inverted pose thus explain-ing its lower activity.

Docking analyses on the here proposed derivatives confirmedthe relevance of the two observed poses despite having a differentenergy profile as reported in Table 2. In detail, the inverted posesappear to be the preferred ones regardless of their configurationfor the a series. This result can be explained by considering thatthe indolinone ring has a reduced capacity to accept H-bonds(compared to the sulfonyl group) and the enlarged spiroazepinering bumps against Glu124 and Arg283 thus distorting the finearchitecture of the entire binding site when the ligands assumethe normal pose. Another key element influencing the ligand poseis the intramolecular H-bonds between the Aib peptide bond andthe indoline carbonyl group which further stabilizes the ligandconformation when assuming the inverted pose.

Table 2AutoDock binding score of the minimized normal and inverted poses for ibutamorenand derivatives 14 and 15 (all values are expressed in Kcal/mol)

Compounda AutoDock Score normalpose

AutoDock Score invertedpose

(D) Ibutamoren �19.92

�12.40(L) Ibutamoren �11.27 �13.40(�)-(S,L)-14a �15.9 �17.08(+)-(R,D)-14a �9.88 �10.56(S,L)-14b �13.94 �12.16(R,D)-14b �10.85 �10.32(+)-(R,L)-15a �12.40 �13.09(�)-(S,D)-15a �11.52 �13.98(S,D)-15b �11.71 �10.78(R,L)-15b �12.96 �10.47

a R/S is referred to the stereochemistry of the spiro center, L/D to the stereo-chemistry of Trp.

Figure 6. Main interactions stabilized the so called normal pose for ibutamoren (D)(a) as well as the inverted pose for (�)-14a (S,L) (b).

S. Pellegrino et al. / Bioorg. Med. Chem. 21 (2013) 5470–5479 5475

The completely different energetic profile (Table 2) of the twopossible poses reflects on the different stereoselectivity since, asdocumented by docking scores and in line with what was observedfor ibutamoren, the (L)-isomer can assume more stably the

inverted pose. Similarly, the stereoselectivity observed for the chi-ral center belonging to the spirocycle emphasizes the role of p–pinteractions stabilized by the indolinone ring which finely dependson the arrangement of the entire spirocycle. As example, Figure 6breports the main interactions stabilizing the complex of the mostactive derivative (�)-14a showing that it conserves the majorinteractions stabilized by the ammonium head with Glu124,Thr127, Tyr128, as well as the set of p–p interactions involvingthe indole ring with Phe279, Phe309, Phe312 and Tyr313 as wellas the indolinone moiety with Phe221, Phe222, Phe223, His280,Tyr284. Clearly, (�)-14a is unable to elicit H-bonds with Phe312and Ser123 as observed with (D) ibutamoren.

The role of H-bonding groups in influencing the assumed posesis further emphasized by nitro derivatives 14b and 15b for whichthe normal pose was found to be the preferred one regardless oftheir configuration. Docking results for all diastereoisomers (Ta-ble 2) showed indeed the completely negative role of nitro groupwhen the ligand assumes the inverted pose since it is inserted inan apolar subpocket where it weakens the key p–p interactionsand detrimentally approaches Leu227. When the ligands assumethe normal pose, the nitro group is inserted in a more polar subcav-ity where it can stabilize H-bonds with the NH backbone groups ofPhe312 and Tyr313 even though this affects the precise arrange-ment of the aromatic residues around the indolinone ring. Themarked decrease of docking scores especially for the invertedposes suggests that here the steric hindrance exerted by nitrogroup negatively prevails on any beneficial electronic effect (asmentioned in the Introduction) thus justifying the reported biolog-ical results of the nitro derivatives.

Taken together, the obtained results emphasize the key role ofp–p interactions that the ligand’s aromatic moieties stabilize inthe two described subpockets. These cavities show a comparablecapacity to elicit p–p stacking but greatly differ in the ability togive H-bonds as evidenced by the binding mode and stereoselec-tivity of ibutamoren. When the two ligand aromatic moieties havea modest and similar H-bonding capacity, the preferred bindingmode accommodates the bulkier aromatic system in the subcavitylined by Phe221, Phe222, Phe223, His280, Tyr284 to minimize thesteric interferences with the mandatory contacts stabilized by theammonium thus explaining the inversion of stereoselectivity forthe here proposed derivatives.

5. Conclusions

In conclusion, a series of peptidomimetics containing the newspiroazepinoindolinone scaffold 6, characterized by S or R stereo-chemistry to the spiro center, and Aib-(L/D)Trp-OH sequenceslinked to nitrogen atom of the ring were prepared. Their synthesiswas achieved in good yields and their biological activity was eval-uated by DU145 proliferation studies.

Beside their clear structural similarity with known ghrelinagonists, several lines of evidence concur to define GHSR-1a astheir main biological target. First, the reported data on theantiproliferative effect of (�)-14a are in agreement with previousobservation for analogues.8b,14 Second, the activation of theERK1/2 pathway, as well as the counteracting effect of GHS-R1aantagonist D-Lys3-GHRP-6, clearly suggest the direct involvementof the GHSR-1a. Third, the inactivity of acetylderivative (�)-16aunderlines the relevance of the protonated amine terminus ofAib which plays a key role in GHSR-1a recognition.

Lastly, docking simulations reveal that the here proposed li-gands assume reasonable poses within the GHSR-1a binding sitestabilizing a set of interactions which are in line with thoseobserved for ibutamoren and able to rationalize the observedinversion of enantioselectivity concerning tryptophan residue.

5476 S. Pellegrino et al. / Bioorg. Med. Chem. 21 (2013) 5470–5479

6. Experimental

6.1. Materials

Starting materials 3 were prepared as described in literature.9

N-Boc-Aib-(L)-Trp-OH and NBoc-Aib-(D)-Trp-OH were preparedon 2-chloro-trytil chloride resin using conventional solid phasepeptide synthesis technique. All reagents and solvents were pro-vided by Sigma–Aldrich. Ghrelin and the GHS-R1a antagonist D-Lys3-GHRP-6 were purchased from AnaSpec (Fremont, CA).

Reactions were monitored by thin-layer chromatography on0.25 mm silica gel plates (60 F254) using UV light as a visualizingagent. Infrared Spectra were recorded on a Fourier transform IRspectrometer, and values are reported in cm�1 units. Molecularweights were identified on a MAT LCQ ion trap mass spectrometerequipped with a Microsoft Window NT data system (ESI) from Ther-mo Finningan. C, H, N analyses were performed on Perkin-ElmerCHN Analyzer Series II 2400. NMR spectra were obtained on a VarianMercury 200 MHz, on a Bruker Avance 300 and 500 MHz. Couplingconstants (J) are given in Hertz. HPLC analyses were performed on aJasco bs-997-01 equipment. Preparative RP-HPLC analyses wereperformed using a DENALI C-18 column from GRACE VYDAC(10 lm, 250 � 22 mm). Two mobile phases were used: A = 95%Water, 4.9% ACN, 0.1% TFA; B = 95% ACN, 4.9% Water, 0.1% TFA.

6.2. Chemical synthesis of spiroazepinoindolinonepeptidomimetics

6.2.1. Ethyl 20-oxa-7-thiooxa-1,2,3,5,6,7-hexahydrospiro[4H-azepine-4,30-3H-indole]-10-carboxylate (4)

Compound (±)-3a (8.5 g, 28.2 mmol) was dissolved in CH2Cl2

(700 mL). Lawesson’s reagent (6.5 g, 16.0 mmol) was added at25 �C and the solution was stirred for 17 h (TLC: CH2Cl2/MeOH,10:1). The solvent was evaporated and the crude mixture waschromatographed on silica gel (CH2Cl2/MeOH, from 1:0 to 100:3).After crystallization pure compound (±)-4 (7.6 g, 85%) was isolated.Operating in the same reaction conditions, starting from (+)-3a(200 mg, 0.65 mmol), pure compound (+)-4 (160 mg, 77%) was ob-tained. Mp 160 �C (CH2Cl2/iPr2O); ½a�25

D +45 (c 0.4, CHCl3); IR (KBr)mmax 3436, 1788, 1755, 1734 cm�1; 1H NMR (500 MHz, CDCl3) d8.79 (br s, 1H, exch.), 7.91 (d, J 8.1, 1H), 7.38–7.35 (m, 1H), 7.26–7.20 (m, 2H), 4.56–4.52 (m, 1H), 4.52 (q, J 7.1, 2 H), 3.98–3.93(m, 1H), 3.33–3.27 (m, 1H), 3.12–3.07 (m, 1H), 2.11–1.95 (m,4H), 1.49 (t, J 7.1, 3H); 13C NMR (500 MHz, CDCl3) d 209.7, 177.6,150.8, 137.6, 132.9, 128.8, 125.3, 122.5, 115.3, 63.7, 47.1, 40.8,38.3, 35.9, 32.7, 14.3; MS, m/z (ESI) 341.4 (M++Na, 100%), 319.5(M++1). Found: C, 60.18; H, 5.82; N, 8.67. Calcd for C16H18N2O3S:C, 60.36; H, 5.70; N, 8.80.

6.2.2. 20-Oxa-1,2,3,5,6,7-hexahydrospiro-4H-azepine-4,30-3H-indole (6a)

Operating under nitrogen atmosphere, thioamide (±)-4 (7.6 g,23.9 mmol) was dissolved in anhydrous THF (380 mL). MeI(17.0 g, 119.4 mmol) was added to the solution and the stirringwas maintained for 17 h at 25 �C (TLC: CH2Cl2/MeOH, 20:1). A yel-low solid was formed. After solvent evaporation crude compound 5was obtained. The crude reaction mixture was dissolved in EtOH(120 mL) and AcOH (3 mL) and NaBH3CN (750 mg, 11.9 mmol)were added at 25 �C. The mixture was stirred overnight (TLC:CH2Cl2/MeOH, 10:1). The solvent was removed and the crude mix-ture was taken up with AcOEt (100 mL). 2 N NaOH was added untilpH 9 and the mixture was stirred overnight at 25 �C. 2 N HCl wasadded until pH 7 and the solvent was evaporated. The mixturewas chromatographed on silica gel (CH2Cl2/MeOH, from 1:0 to5:1) and pure amine (±)-6a (4 g, 80%) was isolated after crystalliza-

tion. Operating in the same reaction conditions, starting from or(+)-3 (100 mg, 0.50 mmol), pure compound (�)-6a (90 mg, 86%)was obtained.

6.2.2.1. Compound 5 (crude compound). IR (KBr) mmax 3436,1761, 1732 cm�1; 1H NMR (200 MHz, CDCl3) d 11.8 (br s, exch.),7.87 (d, J 8.1, 1H), 7.47–7.20 (m), 4.56–4.44 (m, 3H), 4.23–4.15(dd, J 14.3, 12.1, 1H), 3.20 (s, 4H), 2.74 (dd, J 15.0, 5.8, 1H), 2.50–2.40 (m. 1H), 2.23 (dd, J 15.0, 11.4, 1H), 2.20–1.60 (m, 2H), 1.47(t, J 7.0, 3H); 13C NMR (200 MHz, CDCl3) d 192.5, 177.5, 150.6,137.6, 132.0, 129.5, 125.9, 123.5, 115.5, 64.1, 46.7, 42.8, 33.8,31.0, 30.7, 19.6, 14.4; MS, m/z (ESI) 333.2 (M++1, 100%).

6.2.2.2. Compound (�)-6a. ½a�25D �18 (c 0.3, MeOH); Mp

219 �C (CH2Cl2/iPr2O); IR (KBr) mmax 3436, 1697 cm�1; 1H NMR(300 MHz, CD3OD) d 7.40 (d, J 7.4, 1H), 7.25 (dd, J 7.7, 7.0, 1H),7.08 (dd, J 7.5, 7.4, 1H), 6.92 (d, J 7.7, 1H), 3.70 (ddd, J 13.7, 10.0,2.8, 1H), 3.51–3.40 (m, 3H), 2.47–2.30 (m, 2H), 2.18–1.95 (m,4H); 13C NMR (300 MHz, CDCl3) d 181.3, 138.9, 135.2, 129.1,123.9, 122.2, 111.6, 50.6, 45.5, 42.7, 37.4, 31.2, 22.8; MS, m/z(ESI) 217.4 (M+ +1, 100%). Found: C, 71.93; H, 7.59; N, 12.81. Calcdfor C13H16N2O: C, 72.19; H, 7.46; N, 12.95.

6.2.3. 1-Acyl-20-oxa-1,2,3,5,6,7-hexahydrospiro-4H-azepine-4,30-3H-indole ((±)-7a)

Compound (±)-6a (1.0 g, 4.6 mmol) was suspended in CH2Cl2

(80 mL). Ac2O (874 lL, 9.24 mmol) and DIPEA (690 lL, 4.62 mmol)were added and the stirring was continued for 2.30 h at 25 �C (TLC:CH2Cl2/MeOH, 10:1). The organic mixture was washed with 2 NHCl (4 � 40 mL), with 2 N NaOH (40 mL) and then with brine(40 mL). Pure compound (±)-7a (640 mg, 54%) was isolated afterpurification on silica gel column chromatography (CH2Cl2/MeOH,from 1:0 to 100:6) and crystallization. Mp 254 �C (CH2Cl2/iPr2O);IR (KBr) mmax 3500–3100, 1720, 1625 cm�1; 1H NMR (500 MHz,DMSO-d6, 363 K) d 10.0 (s, 1H), 7.27 (br s, 1H), 7.18 (dd, J 7.6,7.5, 1H), 6.96 (dd, J 7.5, 7.4, 1H), 6.88 (d, J 7.6, 1H), 3.85–3.47 (m,4H), 2.21 (br s, 1H), 2.08 (s, 3H), 2.01–1.67 (m, 5H); 13C NMR(500 MHz, DMSO-d6, 363 K) d 181.2, 168.9, 140.6, 135.1, 127.2,123.0, 121.1, 109.2, 49.4, 48.2, 43.9 (43.4), 35.7 (35.5), 35.0(35.1), 23.4 (22.8), 22.0 (21.8); MS, m/z (ESI) 259.1 (M++1, 100%).Found: C, 69.60; H, 7.11; N, 10.70. Calcd for C15H18N2O2: C,69.74; H, 7.02; N, 10.84.

6.2.4. 1-Acyl-50-nitro-20-oxa-1,2,3,5,6,7-hexahydrospiro-4H-azepine-4,30-3H-indole ((±)-7b)

Compound (±)-7a (189 mg, 0.7 mmol) was dissolved inCF3CO2H (3 mL) and the solution was cooled at 0 �C. NaNO3

(62.5 mg, 0.7 mmol) was added under stirring. The solution turnedred. After 2 h (TLC: AcOEt) the mixture was treated with 2 N NaOH(3 mL) and extracted with AcOEt (3 � 15 mL). The organic layerswere dried over Na2SO4 and the solvent was evaporated. Pure com-pound (±)-7b (140 mg, 64%) was obtained after column chroma-tography (CH2Cl2/MeOH 15:1).

6.2.4.1. Compound (±)-7b. Mp 197 �C dec. (CH2Cl2/iPr2O); IR(KBr) mmax 3500–3100, 1726, 1623 cm�1; 1H NMR (DMSO-d6,298 K) d 11.1 (br s, 1H), 8.26–8.15 (m, 2H), 7.07–6.97 (m, 1H),3.90–3.41 (m, 4H), 2.20–1.70 (m, 6H), 2.06 (2.04) (s, 3H); 13CNMR (200 MHz, DMSO-d6, 298 K) d 182.5 (182.3), 170.1 (170.2),148.1, 142.8, 136.9 (136.6), 125.8, 119.8 (120.1), 110.2, 49.2(49.4), 48.3, 44.4 (43.3), 35.6 (35.8), 34.5 (35.0), 22.8 (22.6), 22.7(22.4); MS, m/z (ESI) 326.2 (M++Na, 100%), 304.1 (M++1). Found:C, 59.15; H, 5.81; N, 13.66. Calcd for C15H17N3O4: C, 59.40; H,5.65; N, 13.85.

S. Pellegrino et al. / Bioorg. Med. Chem. 21 (2013) 5470–5479 5477

6.2.5. 50-Nitro-20-oxa-1,2,3,5,6,7-hexahydrospiro-4H-azepine-4,30-3H-indole.HCl ((±)-6b)

Operating in a sealed tube, compound (±)-7b (100 mg,0.33 mmol) was suspended in 6 N HCl (5 mL) and the mixturewas heated at 120 �C for 72 h (TLC: CH2Cl2/MeOH, 10:1). The sol-vent was removed affording pure compound (±)-6b (95 mg, 99%).Mp 221 �C dec. (iPr2O); IR (KBr) mmax 3434, 3202, 1728,1623 cm�1; 1H NMR (300 MHz, CD3OD) d 8.31 (d, J 2.2, 1H), 8.25(dd, J 8.6, 2.2, 1H,), 7.11 (d, J 8.6, 1H), 3.76–3.77 (ddd, J 14.2,10.2, 2.2, 1H), 3.48 (ddd, J 14.2, 6.6, 2.7, 1H), 3.45–3.40 (m, 2H),2.47 (ddd, J 16.3, 10.2, 2.7, 1H), 2.49–2.35 (m, 1H), 2.27–2.05 (m,4H); 13C NMR (300 MHz, CD3OD) d 182.4, 146.9, 143.8, 136.7,125.5, 118.7, 110.0, 48.8, 47.1, 41.7, 35.7, 32.0, 21.1. MS, m/z(ESI) 262.3 (M+, 100%). Found: C, 52.27; H, 5.69; N, 13.89. Calcdfor C13H16ClN3O3: C, 52.44; H, 5.42; N, 14.11.

6.2.6. General procedure for the coupling reactionN-Boc protected amino acid or N-Boc-Aib-TrpOH (0.57 mmol)

was suspended in CH2Cl2 (2.5 mL) at 0 �C. EDC�HCl (248 mg,1.3 mmol) and HOBt (175 mg, 1.3 mmol) were added to the mix-ture that was stirred for 30 min. Compound 6a or 6b or 10a or11a (0.52 mmol), suspended in CH2Cl2 (2.5 mL) and DIPEA(222 lL 0.57 mmol), were added in sequence to the reaction mix-ture containing activated amino acid and the mixture was warmedat 25 �C under stirring for 15 h (TLC: CH2Cl2/MeOH, 10:1). The or-ganic layer was washed with H2O (1.5 mL) and dried over Na2SO4.The solvent was evaporated. The crude mixture was purified onsilica gel (CH2Cl2/MeOH, from 1:0 to 100:1). Yields are reportedin Table 3. It is possible to separate the mixture of (�)-8a/(+)-9ausing flash chromatography (cyclohexane/AcOEt, 1:1), while anyattempt to separate 8b/9b and 12b/13b mixture failed.

6.2.6.1. Compound (�)-8a. Mp 174 �C (n-hexane/AcOEt);½a�25

D �8 (c 0.3 CH2Cl2); IR (KBr) mmax 3010, 1709, 1620 cm�1; 1HNMR (300 MHz, DMSO-d6; 373 K) d 10.52 (s, 1H, exch.), 9.90 (s,1H, exch.), 7.59 (d, J 7.8, 1H), 7.35 (d, J 7.9, 1H), 7.17–7.18 (m,3H), 7.07 (dd, J 7.8, 7.1, 1H), 6.99 (dd, J 7.5, 7.0, 1H), 6.91 (dd, J7.4, 6.9, 1H), 6.84 (d, J 7.7, 1H), 6.30 (d, J 8.1, 1H, exch.), 4.78 (brs, 1H), 3.78–3.35 (m, 4H), 3.19 (dd, J 14.3, 7.4, 1H), 3.02 (dd, J14.3, 6.9, 1H), 2.25–2.00 (m, 1H), 1.95–1.50 (m, 5H), 1.36 (s, 9H);13C NMR (300 MHz, DMSO-d6; 373 K) d 182.2, 175.0, 162.7,141.6, 137.3, 136.3, 128.6, 128.2, 124.6, 123.9, 122.2, 121.7,119.2, 119.0, 112.2, 111.1, 110.2, 79.2, 52.4, 49.5, 44 (C �2, overl.),36.3, 35.1, 29.4, 29.1, 24.4, Found: C, 69.05; H, 7.09; N, 10.88. Calcdfor C29H34N4O4: C, 69.30; H, 6.82; N, 11.15.

6.2.6.2. Compound (+)-9a. Mp 181 �C (n-hexane/AcOEt); ½a�25D

+28 (c 0.2 CH2Cl2); IR (KBr) mmax 3007, 1707, 1619 cm�1; 1H NMR(300 MHz, DMSO-d6; 373 K) d 10.5 (s, 1H, exch.), 9.89 (s 1H, exch.),7.56 (d, J 7.7, 1H), 7.35 (d, J 8.1, 1H), 7.18–7.10 (m, 3H), 7.09 (dd, J7.0, 6.3, 1H), 7.00 (t, J 7.0, 1H), 6.89 (t, J 7.5, 1H), 6.84 (d, J 7.7, 1H),6.33 (d, J 8.3, 1H, exch.), 4.77 (dd, J 15.1, 6.9, 1H), 3.88–3.68 (m,2H), 3.68–3.45 (m, 1H), 3.45–3.25 (m, 1H), 3.18 (dd, J 14.2, 6,7,1H), 3.02 (dd, J 14.2, 6,7, 1H), 2.19–2.02 (m, 1H), 1.92–1.55 (m,

Table 3Yield of synthesized peptidomimetics 8–13

Amine N-Boc-amino acid Product Yield (%)

(±)-6a N-Boc-(L)Trp-OH (�)-8a/(+)-9a 65(�)-6a N-Boc-Aib-(L)TrpOH (�)-12a 86(�)-10a N-Boc-Aib-OH (�)-12a 64(+)-11a N-Boc-Aib-OH (+)-13a 35(±)-6b N-Boc-(L)Trp-OH 8b/9b 92(±)-6b N-Boc-Aib-(L)TrpOH 12b/13b 50(±)-6a N-Boc-Aib-(D)TrpOH (+)-12a/(�)-13a —a

a Not isolated; directly deprotected at nitrogen atom.

5H), 1.38 (s, 9H); 13C NMR (300 MHz, DMSO-d6; 373 K) d 182.3,172.4, 155.8, 141.6, 137.3, 136.3, 128.6, 128.2, 124.6, 123.9,122.2, 121.7, 119.2, 119.0, 112.2, 111.1, 110.2, 79.2, 52.3, 49.5,46.94, 42.4, 36.1, 35.3, 29.3, 29.1, 23.6. MS, m/z (ESI) 525.2(M++Na, 100%), 503.3 (M++1). Found: C, 69.02; H, 7.02; N, 10.89.Calcd for C29H34N4O4: C, 69.30; H, 6.82; N, 11.15.

6.2.6.3. Compound 8b/9b. IR (nujol) mmax 3600–3060, 1718,1625 cm�1; 1H NMR (500 MHz, DMSO-d6, 363 K) d 10.69 (s, 1H,exch.), 10.51 (s, 1H, exch.), 8.13 (d, J 5.6, 1H), 8.01 (br s, 1H),7.60–7.50 (m, 1H), 7.40–7.30 (m, 1H), 7.18–7.12 (m, 1H), 7.10–6.93 (m, 3H), 6.37 (br s, 1H, exch.), 4.76 (br s, 1H), 4.00–3.30 (m,4H), 3.25–3.00 (m, 2H), 2.30–2.10 (m, 1H), 2.00–1.60 (m, 5H),1.36 (s, 9H); 13C NMR (500 MHz, DMSO-d6, 300 K) d 181.9 (182.0,182.3), 172.2 (172.8, 172.3), 156.1 (156.9, 155.6, 155.5), 147.7(147.8, 147.5), 142.8 (142.7, 142.6), 136.5 (137.6, 136.4), 127.8(127.9, 127.7), 1257 (125.6, 125.5), 124.6 (124.4), 121.3 (121.4,121.2), 119.3–118.6 (7s �4C), 111.9 (111.8), 110.5 (110.7, 110.6,110.4), 110.0 (109.9), 78.6 (78.7, 78.5), 51.8 (52.3, 51.4), 48.5(48.8, 48.6), 46.6 (45.9) 45.0, 35.2 (35.3, 35.0, 34.7), 33.6 (33.8),28.6 (�3), 27.9 (27.7), 22.5 (21.8, 21.6, 20.7). MS, m/z (ESI) 570.2(M+23, 100%). Found: C, 63.37; H, 6.28; N, 12.60. Calcd forC29H33N5O6: C, 63.61; H, 6.07; N, 12.79.

6.2.6.4. Compound (�)-12a. Mixture of conformers. Mp207 �C (CH2Cl2); ½a�25

D �12 (c 0.4 CH2Cl2); IR (KBr) mmax 3334,1710, 1625 cm�1; 1H NMR (200 MHz, CDCl3, 298 K) d 9.95–8.30(m, 1H, exch.), 8.80 (8.60) (s, 1H, exch.), 7.90–6.50 (m, 10H),5.58–5.00 (m, 1H + 1H exch.), 4.10–3.00 (m, 6H), 2.30–1.00 (m,21H); 13C NMR (200 MHz, CDCl3, 298 K) d 182.9 (183.1), 175.5(176.0, 175.9), 172.3 (172.9), 155.8 (155.4, 155.3), 140.4 (140.2),136.9 (136.8), 135.4 (135.3), 127.7, 127.6 (127.2), 126.1, 123.7(123.0), 122.2 (122.4), 121.5 (121.4), 118.9 (118.7), 118.3 (118.0,117.4), 111.4 (111.2), 109.7, 109.4 (109.5), 79.2, 56.2 (55.6), 49.7(50.4), 48.7 (46.2), 45.8 (45.2), 42.3 (40.8), 35.4 (36.0), 33.8(34.5), 29.0, 27.5 (�3), 24.6 (�2), 21.9 (21.1). Found: C, 67.19; H,7.21; N, 11.77. Calcd for C33H41N5O5: C, 67.44; H, 7.03; N, 11.92.

6.2.6.5. Compound (+)-13a. Mixture of conformers. Mp210 �C (CH2Cl2); ½a�25

D +26 (c 0.5 CH2Cl2); IR (KBr) mmax 3600–3200, 1705, 1619 cm�1; 1H NMR (200 MHz, CDCl3, 298 K) d 9.05–8.60 (m, 2H, exch.), 7.85–6.70 (m, 10H), 5.40–5.10 (m, 1H + 1Hexch.), 3.90–2.80 (m, 6H), 2.25–1.20 (m, 21H); 13C NMR(200 MHz, CDCl3, 298 K) d 182.9 (182.4), 175.1 (175.0), 172.6(172.5), 154.8, 139.7 (139.4), 136.5 (136.4), 135.3, 128.0, 127.7,126.9 (126.6), 123.8, 122.9 (122.7), 122.5 (122.2), 119.9 (119.7),119.2 (118.7), 117.4, 111.6 (111.5), 110.1 (109.9), 80.3, 56.9, 50.1(50.5), 49.3 (48.2), 47.4 (46.3), 41.3 (43.4), 35.6 (36.3), 34.1(34.9), 29.9 (29.6), 28.5 (�3), 25.83 (25.84 �2), 22.3 (21.1); MS,m/z (ESI) 610.3 (M++Na, 100%), 588.2, (M++1), Found: C, 67.21; H,7.20; N, 11.80. Calcd for C33H41N5O5: C, 67.44; H, 7.03; N, 11.92.

6.2.6.6. Compound 12b/13b. IR (nujol) 3500–3150, 1709,1620 cm�1; 1H NMR (500 MHz, CDCl3; 300 K) d 8.69 (8.62; 2s,1H, exch.), 8.50–8.25 (br s, 1H, exch.), 8.21–8.10 (m, 1H), 7.92–7.74 (m, 2H), 7.70–6.92 (m, 6H), 5.68–5.60 (5.34–5.28, 5.28, 5.22;3 m, 1H), 5.20–5.00 (m, 1H), 4.00–3.00 (m, 6H), 2.40–1.00 (s,21H); 13C NMR (500 MHz, CDCl3; 300 K) d 180.9 (180.8), 173.9,171.8 (172.4), 144.5 (144.6), 142.6 (142.5), 139.4, 135.9 (135.7,135.6, 135.3), 127.7 (126.8), 125.9 (125.4), 124.5 (124.3), 121.8(122.6, 122.3, 121.4), 119.4–117.8 (7s, �4C), 116.2, 110.7 (110.8,110.5, 110.4), 108.7, 76 (overlapped), 56.0 (55.6), 49.1 (47.7),47.1 (47.0, 46.9), 45.6 (46.2, 44.5), 40.6 (40.7, 39.8, 38.7), 35.8(36.1, 34.7), 32.4 (33.4, 33.0, 32.7), 29.0 (29.2), 27.6 (27.5, �3C),24.6 (�2), 20.7 (19.9, 19.5). MS, m/z (ESI) 633.2, (M++1, 100%).

5478 S. Pellegrino et al. / Bioorg. Med. Chem. 21 (2013) 5470–5479

6.2.7. General procedure for nitrogen deprotectionCompound (�)-8a or (+)-9a or (�)-12a or (+)-13a or 12b/13b or

(+)-12a/(�)-13a mixture (0.124 mmol) was dissolved in CH2Cl2

(6 mL) at 0 �C. TFA (600 lL) was added and the reaction mixturewas stirred at 25 �C. After 3 h (TLC: CH2Cl2/MeOH, 10:1) the sol-vent was removed and the crude mixture was crystallized. It ispossible to separate (+)-14a from (�)-15a using RP-HPLC (80% Ato 30% A gradient over 30 min, 20 mL min�1 flow).

6.2.7.1. Compound (�)-10a. CF3CO2H: mixture of conform-ers, 67%. Mp 156 �C (CH2Cl2/Et2O); ½a�25

D �11 (c 0.6, MeOH); IR(KBr) mmax cm�1 3600–3050, 1686; 1H NMR (200 MHz, CD3OD,298 K) d 7.65–6.80 (m, 9H), 4.79–4.50 (m, 1H), 4.00–3.60 (m,2H), 3.60–2.80 (m, 4H), 2.26–1.80 (m, 6H); 13C NMR (200 MHz,CD3OD, 298 K) d 183.1 (182.8), 169.0, 140.1 (140.0), 136.9, 135.6(135.5), 127.7 (127.9), 127.3 (127.2), 124.5, 123.0 (122.8), 122.3,121.9 (121.8), 119.4 (119.1), 117.8 (117.7), 111.7 (111.6), 109.6(109.7), 107.0 (106.7), 50.6 (51.4), 49.1 (48.8), 46.2 (45.8), 40.9(41.5), 35.1, 34.1 (36.3), 27.6, 21.6 (21.0). Found: C, 60.20; H,5.44; N, 10.59. Calcd for C26H27F3N4O4: C, 60.46; H, 5.27; N, 10.85.

6.2.7.2. Compound (+)-11a. CF3CO2H: 73%. Mp 164 �C(CH2Cl2). ½a�25

D +7 (c 0.5, MeOH); IR (nujol) 3600–3200, 1782,1687, cm�1; 1H NMR (200 MHz, CD3OD, 298 K) d 7.60–6.80 (m,9H), 4.80–4.60 (m, 1H), 3.95–3.40 (m, 4H), 3.14–2.87 (m, 2H),2.20–1.47 (m, 6H); 13C NMR (200 MHz, CD3OD, 298 K) d 183.2(182.7), 169.2 (169.0), 140.3 (139.9), 136.9 (136.8), 135.7 (135.5),127.82 (127.81), 127.3 (127.2), 124.53 (127.52), 123.0 (122.8),122.3 (122.2), 121.82 (121.80), 119.2, 117.9, 111.7 (111.6),109.72 (109.71), 107.0 (106.7), 51.1, 48.9 (48.8), 45.6, 42.2 (41.2),35.2 (35.1), 35.0 (34.1), 27.8 (27.5), 21.9 (21.3). MS, m/z (ESI)403.5 (M+1, 100%). Found: C, 60.19; H, 5.49; N, 10.56. Calcd forC26H27F3N4O4: C, 60.46; H, 5.27; N, 10.85.

6.2.7.3. Compound (�)-14ª. CF3CO2H: 89%. Mp 185 �C dec.(MeOH/Et2O). ½a�25

D �13 (c 0.6 MeOH); IR (nujol) 3500–3100,1725, 1670 cm�1; 1H NMR (500 MHz, DMSO-d6; 363 K) d 10.64(s, 1H, exch.), 10.00 (s, 1H, exch.), 8.24 (br s, 1H, exch.), 7.60–7.55 (m, 1H), 7.45–7.35 (m, 1H), 7.36 (d, J 7.7, 1H), 7.25–7.15 (m,2H), 7.08 (dd, 7.7, 6.8, 1H), 7.02–6.90 (m, 2H), 6.85 (d, J 7.7, 1H),5.50–5.00 (m, 1H), 4.00–3.50 (m, 4H), 3.40–3.30, 3.20–3.10 (twom, 2H), 2.20–2.00 (m, 1H), 1.90–1.50 (m, 5H), 1.48 (s, 6H); Signalsof a second conformer are at d 7.94 (d, J 8.4, 1H), 7.89 (brs, 1H,exch.), 7.61 (d, J 6.8, 1H), 7.50 (dd, 8.4, 7.8, 1H); 13C NMR(200 MHz, CD3OD; 300 K) d 182.6, 173.2 (172.4, 171.8), 172.2(171.2, 171.0), 161.3, 139.9 (139.8), 136.5, 135.5, 135.1, 128.2-109.4 (10 C), 57.0 (56.9), 53.7, 51.9 (50.5), 48.9 (48.5), 45.4, 42.6(42.5, 40.8), 35.4 (35.3), 34.5 (33.9), 29.6 (28.1, 27.0), 23.1 (22.9,22.8; �2), 22.0 (21.2). MS, m/z (ESI) 488.4 (M++1, 100%). Found:C, 59.71; H, 5.85; N, 11.39. Calcd for C30H34F3N5O5: C, 59.89; H,5.70; N, 11.64.

6.2.7.4. Compound (+)-14a. CF3CO2H: 30% (overall yield from(±)-6a). Mp 191 �C dec. (MeOH/Et2O). ½a�25

D +15 (c 0.3 MeOH).

6.2.7.5. Compound (+)-15a. CF3CO2H: 37%. Mp 178-185 �Cdec. (Et2O). ½a�25

D +23 (c 0.2, MeOH); IR (nujol) 3600–3100, 1720,1668 cm�1; 1H NMR (300 MHz, CD3OD, 298 K) d 8.01–7.83 (m,2H), 7.42–6.84 (m, 6H), 5.66–5.42 (m, 3H), 4.33–3.28 (m, 10H),1.75–1.63 (m, 5H), 1.51–1.34 (m, 7H); 13C NMR (300 MHz, CD3OD,300 K) d 183.5 (183.1), 172.8 (172.6), 171.8 (171.6), 162, 142.0,140.6 (140.2), 137.1, 135.5 (135.3), 128.5–109.5 (10 C), 57.2(57.1), 51.5, 51.2, 49.5, 46.0, 42.4 (41.7), 36.4 (35.0), 34.3 (34.7),29.7 (29.1, 28.5), 23.2 (23.0, 22.8, 22.6; x2), 21.3. MS, m/z (ESI)488.2 (M+1, 100%). Found: C, 59.74; H, 5.86; N, 11.44. Calcd forC30H34F3N5O5: C, 59.89; H, 5.70; N, 11.64.

6.2.7.6. Compound (�)-15a. CF3CO2H: 35% (overall yieldfrom (±)-6a). Mp 187 �C dec. (Et2O). ½a�25

D �22 (c 0.4, MeOH).

6.2.7.7. Compound 14b/15b. CF3CO2H: 90%. IR (nujol) 3600–3100, 1676 cm�1; 1H NMR (300 MHz, CD3OD, 300 K) d 8.19 (br s,1H), 7.90–6.55 (m, 7H), 5.37–5.32 (5.21–4.95; 2 m, 1H), 4.20–2.60 (m, 6H), 2.40–0.90 (m, 12H); 13C NMR (300 MHz, CD3OD,300 K) d 183.2 (182.22, 182.9), 173.3 (173.0, 172.7, 172.1, 171.7),162.2, 146.9, 143.5, 137.4 (137.0), 127.8 (128.4, 127.7), 125.2(125.1), 123.8, (124.1, 124.0), 121.6 (121.9), 119.7–118.0 (8s,�5C), 111.7 (111.5), 109.5 (110.4), 57.2, 52.6 (51.9), 51.4 (50.7),46.5 (45.3), 41.2 (42.2, 41.7, 40.1), 34.8 (36.2, 35.3, 34.6), 34.0(33.8, 33.7), 29.7 (28.5, 28.3, 27.6) 23.9 (23.2, 23.0, 22.9 �2C),21.6 (20.9, 20.2). MS, m/z (ESI) 556.3 (M++Na, 100%). Found: C,55.44; H, 5.32; N, 13.77. Calcd for C30H33F3N6O7: C, 55.72; H,5.14; N, 13.00.

6.2.8. Acetylation of compound ((�)-14a)Compound (�)-14a (15 mg, 0.025 mmol) was dissolved in THF

(1 mL). Acetic anhydride (18 lL, 0.1 mmol) and DIPEA (12 lL,0.125 mmol) were added and the solution was left at 25 �C understirring for 3 h. The solvent was removed and the crude mixturewas purified using preparative HPLC (DENALI C-18 column,10 lm, 250 � 22 mm, gradient: H2O/MeCN/TFA 95:5:0.1 to H2O/MeCN/TFA 50:50:0.1 in 20 min) obtaining pure compound (�)-16(10 mg, 75%). Mp 150 �C (hexane/AcOEt). ½a�25

D �16 (c 0.2, MeOH);mixture of two conformers: 1H NMR (300 MHz, CD3OD; 300�K) d7.77–7.48 (4d, 2H), 7.38–3.31 (m, 1H), 7.22 (s, 1H), 7.20–6.70 (m,6H), 5.44–5.37 (5.21–5.15) (m, 1H), 4.08–3.10 (m, 6H), 2.30–1.37(m, 6H), 1.99 (1.95) (s, 3H), 1.50, 1.48 (1.47, 1.44) (two s, 6 H);13C NMR (300 MHz, CD3OD; 300�K) d 179.6, 172.7 (172.6), 172.0(172.9), 158.2, 140.5, 138.9, 136.9, 135.5, 127.7 (127.6), 123.5-109.6 (9C), 57.3, 50.9, 50.2, 45.6, 42.2 (40.9), 35.9 (35.4), 34.7(34.0), 29.3 (28.8), 24.7 (24.4, 24.3 �2), 22.3 (21.9, 21.4), 17.1.MS, m/z (ESI) 552.4 (M++23, 100%). Found: C, 67.88; H, 6.75; N,13.05. Calcd for C30H35N5O4: C, 68.03; H, 6.66; N, 13.22.

6.2.9. Cell proliferation assays of peptidomimetics on DU1456.2.9.1. Cell cultures. DU145, a human androgen-indepen-dent prostate cancer cells, from American Type Culture Collection(Rockville, MD), were grown at 37 �C in a humidified CO2 incubatorin monolayer. The culture medium was RPMI 1640, with 10 mg/Lphenol red (Biochrom, Berlin, Germany), and 5% FBS (Gibco, GrandIsland, NY). Subconfluent cells were harvested with 0.05% trypsin/0.02% EDTA (Biochrom, Berlin, Germany) and were seeded in Petridishes (Becton-Dickinson, Plymouth, UK) or in 96 well-plates(Viewplate-96; Perkin–Elmer, Milan, Italy) depending on theexperiments.

6.2.9.2. Cell proliferation studies. To estimate the effect ofhuman ghrelin and ghrelin analogs ((�)-14a, (+)-14a, (�)-15a,(+)-15a, 14b/15b, and (�)-16) on DU145 cell proliferation,5 � 104 DU145 cells were seeded in a 96-well plates in a final vol-ume of 200 lL per well and incubated at 37 �C in a humidified 5%CO2 atmosphere for 24 h. The culture medium was replaced byadding 180 lL of experimental medium (RPMI 1640/5% charcoalstripped FBS) containing human ghrelin and tested compounds(all at doses of 10�6 and 10�9 M). The GHS-R1a, at the doses of10�6 M, was used alone or in combination with ghrelin and com-pound (�)-14a. Twenty microliter of 100 lM 50-bromo-20deoxyur-idine (BrdU labeling solution, Perkin–Elmer, Milan, Italy) wereadded to the cultures 4 h before the end of the incubation, accord-ing to previously published procedures.15 Cells were subsequentlyfixed and incubated with a primary monoclonal antibody againstBrdU (0.5 lg/mL) conjugated with Europium (Eu). Finally, theEu-fluorescence was measured in a time-resolved fluorometer. To

S. Pellegrino et al. / Bioorg. Med. Chem. 21 (2013) 5470–5479 5479

exclude any unspecific binding of both BrdU and anti-BrdU-Eu, forevery experiment a row without cells (only medium) has been ana-lyzed. Results (Eu-counts) were obtained by determining the meanvalue of at least 3 experiments in 8 replicates.

6.2.9.3. Western blot analysis. Cells were serum starved for16 h, treated with compounds and then collected in 100 lL T-PERbuffer (Thermo Scientific, Rockford, IL) containing 1% proteaseinhibitor cocktail. The procedure followed a previously publishedprotocol.16 Briefly, the cell suspension was centrifuged at13,000 rpm for 15 min at 4 �C and protein concentration was quan-tified with the BCA Protein Assay kit (Thermo Scientific). Proteinsamples (suspended in Laemmli sample buffer) and molecularmass markers (Life Technologies Europe, Milan, Italy) were sepa-rated on a sodium dodecylsulfate–polyacrylamide gel. Proteinswere transferred from the gel to a nitrocellulose membrane over-night at 4 �C. The membrane was blocked with 5% dry milk in TBSTfor 1 h at room temperature. The blot was then incubated over-night at 4 �C with a diluted solution of the primary antibody(anti-pERK1/2, 1:150; anti-ERK1/2, 1:1000; Santa Cruz Biotechnol-ogy, Inc., Santa Cruz, CA).17 The subsequent incubation with a sec-ondary antibody conjugated with peroxidase was performed atroom temperature for 2 h. Immunoreactivity was detected by theSuperSignal West Pico Substrate working solution (Thermo Scien-tific) and exposure of the membrane to photographic film at roomtemperature for the required time.

6.2.9.4. Analysis of the data. Statistical analysis was per-formed using the Prism statistical analysis package (GraphPadSoftware, San Diego, CA). Data are given as Mean ± SD of threeindependent experiments. Differences between treatment groupswere evaluated by one way ANOVA and were considered signifi-cant at p <0.05.

6.2.10. Computational details6.2.10.1. Generation of the hGHSR-1a model. The hGHSR-1amodel was generated by Modeller9.1018 using the as the template(PDB Id: 4EA3). Such a choice is justified by the satisfactory homol-ogy between the two sequences (equal to 63.3%) as well as by theobservation that both receptors recognize peptide ligands and thissuggests a similar architecture of their binding sites. Among thegenerated models, the best model was selected by consideringthe Modeller scores as well as the percentage of residues fallingwithin the allowed regions of the Ramachandran plot.

After adding hydrogen atoms, the model was minimized keep-ing fixed the backbone atoms to preserve the predicted foldingand the so obtained structure was utilized in the following dockingsimulations. The quality of the homology model was assessed byProcheck19 and is substantiated by (a) percentage of residues fall-ing within the allowed regions of the Ramachandran plot (cor-e + allowed) equal to 98.6, (b) percentage of residues falling withthe allowed chi-space equal to 98.5%, and (c) bond lengths, bondangles and planar rings within geometric limits in more than 95%.

The considered ligands were constructed in their ionized formsand their conformational profile was explored by a clusterd Monte-Carlo calculation (as implemented in VEGA suite of programs)20

which produces 1000 minimized conformers and the lowest en-ergy structure underwent docking simulations. Docking analyseswere carried out by AutoDock4.0 focusing the search in a sphereof 12.0 Å radius around Glu124, so encompassing the entire bind-ing cavity. The resolution of the grid was 64 � 64 � 64 points witha grid spacing of 0.380 Å. The ligands were then docked into thisgrid with the Lamarckian algorithm as implemented in AutoDockand the flexible bonds of the ligand were left free to rotate. The ge-netic-based algorithm ran 100 simulations per substrate with

2,000,000 energy evaluations and a maximum number of genera-tions of 27,000. The crossover rate was increased to 0.8, and thenumber of individuals in each population to 150. All other param-eters were left at the AutoDock default settings.21 The best com-plexes were finally minimized keeping fixed the atoms outside a15 Å radius sphere around the bound ligand and then used to recal-culate docking scores.

Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.bmc.2013.06.006.

References and notes

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