orthogonal chemistry for the synthesis of thiocoraline triostin …. med. chem. 2013, 56,...

Orthogonal Chemistry for the Synthesis of Thiocoraline−TriostinHybrids. Exploring their Structure−Activity RelationshipJudit Tulla-Puche,*,†,‡ Sara Auriemma,† Chiara Falciani,† and Fernando Albericio*,†,‡,§,⊥

†Institute for Research in Biomedicine Barcelona, Baldiri Reixac 10, 08028 Barcelona, Spain‡CIBER-BBN, Networking Centre on Bioengineering, Biomaterials, and Nanomedicine, Baldiri Reixac 10, 08028 Barcelona§Department of Organic Chemistry, University of Barcelona, Martí i Franques 1, 08028 Barcelona, Spain⊥University of KwaZulu Natal, 4001 Durban, South Africa

*S Supporting Information

ABSTRACT: The natural compounds triostin and thiocora-line are potent antitumor agents that act as DNAbisintercalators. From a pharmaceutical point of view, thesecompounds are highly attractive although they present a lowpharmacokinetic profile, in part due to their low solubility.Synthetically, they represent a tour de force because no robuststrategies have been developed to access a broad range of thesebicyclic (depsi)peptides in a straightforward manner. Here wedescribe solid-phase strategies to synthesize new bisintercala-tors, such as thiocoraline−triostin hybrids, as well as analoguesbearing soluble tags. Orthogonal protection schemes (up tofive from: Fmoc, Boc Alloc, pNZ, o-NBS, and Troc), togetherwith the right concourse of the coupling reagents (HOSu,HOBt, HOAt, Oxyma, EDC, DIPCDI, PyAOP, PyBOP, HATU, COMU), were crucial to establish the synthetic plan. In vitrostudies and structure−activity relationships have been shown trends in the structure−activity relationship that will facilitate thedesign of new bisintercalators.

■ INTRODUCTION

The family of bisintercalator natural products assemblesnonribosomally biosynthesized peptides with either a cyclic,2-fold symmetrical bicyclic or a pseudosymmetric bicyclic core.1

Luzopeptins,2 quinoxapeptins,3 quinaldopeptin,4 and sandra-mycin5 have a cyclic scaffold, while triostin6 and thiocoraline7

hold a 2-fold symmetrical bicyclic structure. Finally, a thioacetalbridge prevents echinomycin8 from adopting a symmetricalarrangement. Our research group focuses on thiocoraline andtriostin, as they have demonstrated a high antitumor activityand a fascinating architecture from a synthetic point of view.These two depsipeptides share the same scaffold (Figure 1),which comprises a bicyclic structure formed by a disulfidebridge flanked by two thioester or ester moieties, respectively,and two heterocyclic chromophores, which are responsible forDNA bisintercalation. They also embrace the same degree ofN-methylation but differ in their amino acid sequence. Whilethiocoraline holds a NMe-Cys(Me) and a Gly residue, triostinhas a NMe-Val and an Ala. Furthermore, both triostin andthiocoraline exhibit a clear preference for binding to GC-richsequences.9,10 Interestingly, this specificity is altered in thedemethylated analogue of triostin A, TANDEM, which bindspreferentially to AT-rich sequences.11 These compounds bindto DNA by inserting the chromophores planar in between thebases and positioning the peptidic core in the minor groove of

DNA.12 Many studies have addressed the biosynthesis oftriostin A,13 while that of thiocoraline is under way.14

Bisintercalators exhibit high potency, as they display higherDNA binding affinity than their monomeric counterparts,15 andthey are therefore of particular interest because of theirpotential use as therapeutics.16 The search for newbisintercalators, including those that are not based on naturalproducts, is thus an active field of research. Compounds such asbis-acridines,17 bis-naphtalimides,18 bis-porphyrines,19 and bis-anthracyclines20 have been developed. Regarding naturalbisintercalators, which present a more complex structure,advances in synthetic methods have led to the identificationof new candidates.21−24 More recently, biosynthetic platformshave also allowed the discovery of new compounds.25 Ourgroup has devoted the research efforts of many years toaccessing analogues with better pharmacokinetic properties,such as increased stability and solubility.26 In this regard,substitution of the thioester bridges in thiocoraline by bridgedNMe-amides resulted in a more stable analogue that retainedthe same level of activity. The synthesis of these kinds ofdepsipeptides requires considerable effort, as they bearconsecutive NMe amino acids as well as numerous Cys

Received: April 24, 2013Published: June 7, 2013

Article

pubs.acs.org/jmc

© 2013 American Chemical Society 5587 dx.doi.org/10.1021/jm4006093 | J. Med. Chem. 2013, 56, 5587−5600

pubs.acs.org/jmc

Figure 1. Structures of thiocoraline (1) and triostin (2).

Figure 2. Library of (A) thiocoraline−triostin hybrid analogues; (B) soluble analogues based on NMe-azathiocoraline.

Journal of Medicinal Chemistry Article

dx.doi.org/10.1021/jm4006093 | J. Med. Chem. 2013, 56, 5587−56005588

residues and also heterocyclic units. Strategies relying on solid-phase synthesis have been developed, although each particularanalogue requires fine-tuning of the synthetic scheme. As afurther step in our bisintecalator program, here we describe thesynthesis and biological activity of hybrid bisintercalators basedon triostin A and thiocoraline and of thiocoraline analogueswith decreased hydrophobicity. In designing these analogues,three main approaches were pursued: the formation ofthiocoraline−triostin hybrids, by using either sequences ofone compound and the heterocycle of the other, replacing thenatural thioester and also ester moieties by either esters, in thefirst case, or bridged NMe amides, and finally, the introductionof “solubilizing residues” by replacing the NMe-Cys(Me)residue.

■ RESULTS AND DISCUSSIONDesign of the Analogues. As mentioned previously, three

key aspects of the parent compounds were combined in orderto design the hybrid analogues. These were: (i) the ester orbridged NMe amide moiety, (ii) the amino acid sequence, and(iii) the intercalating chromophore. These parameters were alsoused to name the analogues. Therefore, a compound bearingester moieties, the amino acid sequence belonging tothiocoraline, and the 3-hydroxyquinaldic acid is named oxa-thio-3-hqa. A small library was synthesized (Figure 2A). Inaddition, and in order to improve solubility of thiocoraline, theNMe-Cys(Me) moiety, which is thought not to participate inthe binding,10 was replaced by an amino side-chain-containingresidue [such as diaminopropionic (Dap) or diaminobutyric(Dab) acids], which could be left as a free amino or be coupledto a PEG group (Figure 2B). This replacement was carried outtaking NMe-thio-3hqa (or NMe-azathiocoraline) as a parent

compound, which retains the same degree of activity as thenatural product (IC50: 10

−9 M).Synthetic Strategies. The high versatility of solid-phase

synthesis makes this strategy the most appropriate for thesecompounds. On the basis of preceding work done by our groupand to achieve the target compounds, three strategies could beapplied: (a) a stepwise elongation, (b) a 4 + 4 fragmentcoupling, and (c) the formation of an intermolecular dimer.The symmetrical nature of these compounds allowed theapplication of approaches (b) and (c), which would beotherwise inadequate. In seeking the correct syntheticapproach, the main feature considered was the presence ofeither ester moieties or NMe-amides. Therefore, in synthesizingthe bridged NMe derivatives, the first choice was to use a 4 + 4coupling approach, developed earlier for NMe-azathiocoraline.Similarly, dimer formation was attempted first in order toobtain the oxa analogues. Nevertheless, differences in thesequence proved to be crucial, especially when performing a 4 +4 fragment coupling approach, and sometimes a change in thesynthetic strategy was required. Scheme 1 summarizes the threepossible approaches, exemplified for NMe-triostin-qxa (orNMe-triostin) (in red the key intermediates that are definedby the strategy chosen). For this analogue, after loading Fmoc-Ala-OH onto a 2-chlorotrityl (2-CTC) resin27 and removingthe Fmoc group with piperidine−DMF (1:4), either Boc-D-Dap(Fmoc)-OH (4 + 4 and dimer strategies) or Fmoc-D-Dap(Alloc)-OH (stepwise strategy) was coupled. The secondderivative would allow the incorporation of the intercalatingchromophore on the solid phase after removal of the Fmocgroup. The side-chain protecting group was cleaved and thefree amino group was N-methylated, and the two last aminoacids were introduced sequentially to obtain the protected

Scheme 1. Synthetic Strategies for NMe-triostin-qxa



tetrapeptides (see Supporting Information). Although a 4 + 4approach was undertaken first, neither PyAOP nor HATU/HOAt as coupling reagent afforded the desired octapeptide.Instead, the target peptide was achieved by the other two

strategies: a stepwise elongation and the formation of anintermolecular dimer (Scheme 1). Formation of the dimer wasobtained by treatment with I2 in DMF, and after cleavage of thedimer from the resin, a double cyclization was performed.Finally, the two Boc groups were removed, and the finalheterocycles were introduced. As for the stepwise approach,elongation proceeded following the same steps as thetetrapeptide until reaching the octapeptide. Afterward, disulfideformation was achieved on solid-phase, and after cleavage of thepeptide from the resin, cyclization, which was more difficultthan for the other analogues, took place, rendering the desiredNMe-triostin-qxa.The successful strategies for the thiocoraline−triostin hybrids

are summarized in Table 1.

In the case of the soluble analogues (Figure 2B), thestrategies became more complex because of the need for anadditional protecting group for the side-chain of the Dap orDab residues. The choice of this protecting group depends onseveral aspects. In the case of the free amino derivatives, theprotecting group will be removed at the last stage of thesynthesis, therefore a Boc or a pNZ28 group may beappropriate; however, in this case, the choice of the protectinggroup that masks the heterocycle needs to be reconsidered. Asfor the PEG-containing analogues, the alternatives depend onwhether the introduction of PEG is performed on solid-phaseor in solution, and this also depends on the stage of thesynthetic elongation at which the heterocycle is introduced.Another feature that needs to be re-evaluated is the resin used,as more steps are required for these syntheses, especially N-methylation steps, which may give partial cleavage on 2-CTCresin. Therefore, 4-methylbenzhydryl bromide (MBHBr) resin,a more resistant solid support cleavable by 6−8% TFA, waschosen.The strategy to synthesize 9 started by anchoring Fmoc-Gly-

OH on the MBHBr resin with the aid of CsI and DIEA(Scheme 2). Next, the Fmoc group was removed in the usualconditions, and Fmoc-D-Dap(Alloc)-OH was introduced usingCOMU/Oxyma and DIEA as the coupling system. Aftercleavage of the Fmoc group, the free amino was reprotectedwith Z-OSu and DIEA in DMF for 1 h. The ninhydrin testshowed complete coupling. At this point, the Alloc side-chainprotecting group was removed by the action of [Pd(PPh3)4]and PhSiH3, and the free amino was reprotected with o-nitrobenzenylsulfonyl (o-NBS)-Cl and collidine29 in order tocarry an N-methylation reaction. After this step, Fmoc-L-Dap(Boc)-OH was incorporated, again by treatment withCOMU/OxymaPure and DIEA, in DMF. This Boc group

masks the free amino group present in the final molecule. TheFmoc group was then cleaved, and the three-step N-methylation reaction was repeated on this residue. Alloc-NMe-Cys(Acm)-OH was next coupled with difficulty (threecouplings were necessary) to give the protected tetrapeptide.After cleavage of the Alloc group, stepwise elongation, using thesame steps as for the tetrapeptide, was followed until assemblyof the unprotected octapeptide took place. The HPLCchromatogram showed a complex crude product, with a mainpeak corresponding to the desired compound but accompaniedby many impurities. The major problems arose when removingthe o-NBS groups at the tripeptide and heptapeptide stage(harsh conditions such as β-mercaptoethanol and DBU couldgive β-elimination of the Dap residue) and the incorporation ofthe Alloc-NMe-Cys(Acm)-OH residue at the tetrapeptide andoctapeptide level. Nevertheless, synthesis was continued byforming the disulfide bridge on solid-phase with I2 in DMF, andthe cyclic peptide was cleaved from the resin by means of asolution of TFA−CH2Cl2 (8:92).After evaporation and lyophilization, the crude peptide was

subjected to a first prepurification before cyclization. Thecyclization proceeded with PyBOP and HOAt and wascompleted in 4 h. The bycyclic product was treated with H2and Pd/C to remove the Z groups, but neither the applicationof pressure nor the treatment with neat TFA/anisole wassufficient to remove the Z groups. However, although in lowyields, the desired product was obtained after the incorporationof the 3-hqa acid and final Boc cleavage.Given the results obtained for 9, the strategy to synthesize 11

discarded the Z group. In addition to the Fmoc and Allocgroups for chain elongation, a first approach included the Bocand the p-nitrobenzyl (pNZ) group masking the heterocycleand the PEG30 moiety, respectively. Therefore, Fmoc-Gly-OHwas loaded as before on the MBHBr resin (Scheme 3). TheFmoc group was removed in the usual conditions, and Fmocquantification showed a loading of 0.55 mmol/g. Next, Boc-D-Dap(Fmoc)-OH was coupled and the side-chain Fmoc groupwas cleaved and then N-methylated following Mitsunobuconditions. Next, Fmoc-Dap(Alloc)-OH was coupled to theunprotected N-methylated side-chain, the Fmoc group wascleaved, and the free amino was safely reprotected with the o-NBS group. Instead of carrying out the N-methylation step atthis stage of the elongation, the side-chain Alloc protectinggroup was removed and the free amino was reprotected as well,but this time with the pNZ group. Synthesis continued by N-methylating the Dap residue and coupling Alloc-NMe-Cys-(Acm)-OH to build the tetrapeptide. This coupling over theDap(pNZ) residue proved difficult and could not becompleted, presumably because of the steric hindrance of theside-chain of this preceding amino acid.At this point, a portion of the resin was cleaved (TFA−

CH2Cl2 6:94) and lyophilized, while another portion wastreated with [Pd(PPh3)4] and PhSiH3 to cleave the Alloc group.A 4 + 4 fragment coupling approach also resulted unsuccessful.Removal of the pNZ group at the tetrapeptide stage, andincorporation of the PEG moiety, did not improve the 4 + 4fragment coupling. Therefore, a stepwise assembly was thenconsidered.In addition to the Fmoc and Alloc groups for chain

elongation, the second strategy (Scheme 4) included thepNZ group to mask the amino function that holds theheterocycle. Specifically, Fmoc-Gly-OH was loaded on theMBHBr resin, as described before, for 5 h. MeOH was then

Table 1. Synthetic Strategies and Choice of HeterocycleCoupling in the Hybrid Compounds

analogue strategy heterocycle coupling

NMe-thio-3hqa 4 + 4 solutionNMe-thio-qxa dimer solid-phaseNMe-triostin-qxa stepwise/dimer solution/solid-phaseoxa-triostin-qxa dimer solid-phaseoxa-triostin-3hqa dimer solutionoxa-thio-3hqa dimer solution



added and shaken for 1 h to cap any unreacted sites. Aftercleavage and quantification of the Fmoc group (a loading of0.85 mmol/g was obtained), Fmoc-D-Dap(Alloc)-OH wascoupled instead of Boc-D-Dap(Fmoc)-OH. After removal ofthe Fmoc group, the α-amino was reprotected with the pNZgroup.The Alloc group was then removed, and the same steps as in

the previous strategy were followed to achieve the N-methylated o-NBS-protected tripeptide. After removal of theAlloc group from the side-chain, the PEG chain was introducedon solid-phase. The o-NBS group proved difficult to remove(five treatments), possibly because of the steric hindrance ofthe PEG chain, and Alloc-NMe-Cys(Acm)-OH was thenintroduced (two couplings HATU and one with PyBOP).After removal of the Alloc group, stepwise elongation with thesame set of steps was repeated until achieving the unprotectedoctapeptide. The pNZ group was then cleaved by treatmentwith SnCl2 and cat. HCl in DMF, and the 3-hydroxyquinaldicunits were introduced on solid phase with DIPCDI and HOBt.With the linear assembly completed, the disulfide bridge wasformed on solid-phase by exposing the peptidyl resin to asolution of I2 in DMF. After cleavage of the peptide from theresin, the peptide was prepurified before the cyclization insolution, which was accomplished with PyBOP/HOAt,

rendering the target peptide, which was finally purified byreversed-phase HPLC.Limitations of this strategy arise mainly from the difficulty in

removing the o-NBS group, which gives rise to many impurities,including products from a β-elimination of the Dap residue, asit had also been observed in the case of 9. Therefore, theredesign of the analogues included the replacement of the Dapresidue by Dab, which should be less prone to suffer this side-reaction.The strategy to synthesize 10 used 2-CTC resin because we

intended to apply a 4 + 4 fragment coupling approach thatwould need only two N-methylation steps. In terms ofprotecting groups, Fmoc and Alloc would be used for chainelongation, the Boc group would mask the final heterocycle,and the Troc group would be tested at the side-chain of theDab residue to render the final free amino moiety. In thisregard, Fmoc-Gly-OH was loaded on the 2-CTC resin usingthe usual conditions (Scheme 5). After cleavage of the Fmocgroup (Fmoc quantification: 0.98 mmol/g), Boc-D-Dap-(Fmoc)-OH was incorporated with HATU/HOAt and DIEAin DMF for 1 h. Cleavage of the Fmoc group, subsequent N-methylation, and incorporation of the previously synthesizedFmoc-Dab(Troc)-OH proceeded smoothly. After cleavage ofthe Fmoc group, the N-methylation sequence was repeated. No

Scheme 2. Synthesis of Dap-NMe-thio-3-hqa



difficulties were observed when removing the o-NBS group, andthe standard treatment (3 × 15 min) was sufficient to fullycleave it. Analysis by HPLC also showed an improvement withrespect to the previous strategies where the Dap residue hadbeen used. Incorporation of Alloc-NMe-Cys(Acm)-OH,however, continued to show complexity, and several couplingsand the application of temperature were required. At this point,3/4 of the peptidyl resin was cleaved with TFA−CH2Cl2(1.5:98.5), and the Alloc group in the remaining portion wasremoved using standard conditions. The protected tetrapeptidewas lyophilized and subjected to prepurification with a Porapakcolumn using H2O−ACN as eluents. With this tetrapeptide inhand, a 4 + 4 fragment coupling was performed using PyAOP/DIEA in DMF. After 3 h, HPLC analysis showed that thereaction had proceeded up to 66%. More exposure to freshlyadded coupling reagents did not improve the extent of thereaction. The following steps consisted of the removal of the N-terminal Alloc group, the formation of the disulfide bridge onsolid-phase, and the cleavage from the resin. Next, the peptidewas cyclized with EDC/HOAt in DMF−DCM (1:9) at pH 7,the Boc groups were cleaved, and the 3-hydroxyquinaldic acidwas introduced by means of EDC and HOSu in DCM. The useof a more reactive coupling system leads to auto overacylation.Finally, the Troc groups were removed and the final peptidewas purified by reversed-phase HPLC.

The synthesis of 12 took advantage of the previous strategyand was assembled in a very similar way (Scheme 5). The onlydifference was that a previously synthesized Fmoc-Dab(PEG)-OH was introduced at the tripeptide level. Purities of 69% forthe unprotected tetrapeptide and of 61% for the protectedoctapeptide after the fragment coupling were obtained.Removal of the N-terminal Alloc group, disulfide formation,cleavage from the resin, cyclization, and final introduction ofthe 3-hydroxyquinaldic acid rendered the desired compound.Therefore, a synthetic scheme has been developed andoptimized to reach these complex bicycles bearing free aminoor PEG groups pending at the side-chain of Dab.

Biological Activity. The activity of the analogues wastested in three human cancer cell lines (Table 2).Replacement of 3-hydroquinaldic acid by quinoxaline-2-

carboxylic acid as chromophore in NMe-thio (5) resulted in aloss of an order of magnitude, although the activity of 3 was stillconsiderable. In contrast, the activity of oxa-triostin bearing thenatural quinoxaline-2-carboxylic acid (7) was higher than whenit was replaced by the 3-hqa acid (6). These results suggest thatthe sequence plays a crucial role in activity and that activity isnot attributable exclusively to the chromophore. This is alsoillustrated by the activities obtained for NMe-thio-qxa (3) andNMe-triostin-qxa (4), where the first analogue was 2 orders ofmagnitude more active than the second. The same trend was

Scheme 3. Synthesis of Dap(PEG)-NMe-thio-3hqa: Strategy 1



observed in the oxa analogues, where oxa-thio-3hqa (8) wasmore active than oxa-triostin-3hqa (6).In comparing the bridged-NMe-amide-analogues with the

oxa ones, opposite results were obtained for the triostin andthiocoraline families. In the latter, NMe-thio-3hqa (6) showedmuch higher activity than oxa-thio-3hqa (8). Conversely, NMe-triostin-qxa was less active than the parent compound oxa-triostin-qxa (4). These observations could be explained by therigidity imposed by the NMe group in the final molecule. The

triostin sequence is less flexible than that of thiocoralinebecause of the presence of NMe-Val and Ala. The presence of aGly residue in thiocoraline is thought to confer a certain degreeof flexibility.10 Thus, this compound can easily accommodate anextra NMe moiety in the near environment. This explanationmatches the results obtained experimentally, where cyclizationof NMe-triostin-qxa was extremely difficult. Structural changesmay have taken place that result in a less favored arrangementto bind to DNA, thereby accounting for the loss of activity.

Scheme 4. Synthesis of Dap(PEG)-NMe-thio-3hqa: Strategy 2



Regarding the “soluble analogues”, compounds 9−12, the invitro activity decreased dramatically. In principle, thereplacement of the natural NMe-Cys(Me) residue should not

affect binding with DNA, as it is located on the outer part of thestaple-like shape that these compounds adopt when theyintercalate.10 However, the presence of free amino groups could

Scheme 5. Synthesis of Dab-NMe-thio-3-hqa and Dab(PEG)-NMe-thio-3-hqa



change the hydrogen bond pattern of the molecules as well asthe hydrophobic interactions, preventing them from position-ing themselves in the preferred conformation for binding.Similarly, the steric hindrance of the PEG groups may alsojeopardize the correct arrangement for DNA bisintercalation.Thus, examining the molecular structure of the DNA−triostinA complex (1VS2 of the PDB),9b the Val side-chains, whichwould be the analogue positions for Dap or Dab(PEG), are inclose proximity to the heterocycles. A long chain at thisposition could preclude an efficient bisintercalation. In addition,several papers point out the importance of the stacking andhydrophobic interactions for binding, as well as the “hydro-

phobic potential of the drug”.31 Therefore, the addition of polargroups such as PEG or amino moieties may result in loss ofcrucial hydrophobic interactions. BE-22179, an analogue ofthiocoraline bearing a didehydroalanine in the place of NMe-Cys(Me), exhibits still potent activity, but the hydrophobicityin this case may be maintained.32

■ CONCLUSIONS

In summary, here we studied solid-phase approaches to accesscomplex bicyclic thiocoraline−triostin hybrids. Robust strat-egies to achieve amino- and PEG-derivatives were developedbased on the concourse of six amino protecting groups: Fmoc,

Table 2. In Vitro Activities of Compounds 3−12

analogue study breast MDA-MB-231 NSCLC (lung) A549 colon HT-29

thiocoraline, 1 GI50 2.07 × 10−9 6.22 × 10−9 4.15 × 10−8

TGI 9.50 × 10−9 2.42 × 10−8 2.68 × 10−8

LC50 3.46 × 10−8 9.50 × 10−8 1.38 × 10−7

triostin, 2 GI50 2.02 × 10−7 3.40 × 10−7 2.30 × 10−7

TGI 5.15 × 10−7 1.66 × 10−6 2.85 × 10−6

LC50 1.56 × 10−6 >9.20 × 10−6 >9.20 × 10−6

NME-thio-qxa, 3 GI50 4.73 × 10−8 7.13 × 10−8 3.57 × 10−7

TGI 1.43 × 10−6 >8.92 × 10−6 >8.92 × 10−6

LC50 >8.92 × 10−6 >8.92 × 10−6 >8.92 × 10−6

NMe-triostin-qxa, 4 GI50 5.93 × 10−6 >8.98 × 10−6 8.44 × 10−6

TGI >8.98 × 10−6 >8.98 × 10−6 >8.98 × 10−6

LC50 >8.98 × 10−6 >8.98 × 10−6 >8.98 × 10−6

NMe-thio-3hqa, 5 GI50 4.08 × 10−9 3.39 × 10−9 2.08 × 10−8

TGI 4.26 × 10−8 2.00 × 10−8 1.13 × 10−8

LC50 3.73 × 10−7 1.65 × 10−7 7.47 × 10−7

oxa-triostin-3hqa, 6 GI50 >8.92 × 10−6 >8.92 × 10−6 >8.92 × 10−6

TGI >8.92 × 10−6 >8.92 × 10−6 >8.92 × 10−6

LC50 >8.92 × 10−6 >8.92 × 10−6 >8.92 × 10−6

oxa-triostin-qxa, 7 GI50 2.02 × 10−7 3.40 × 10−7 2.30 × 10−7

TGI 5.15 × 10−7 1.66 × 10−6 2.85 × 10−6

LC50 1.56 × 10−6 >9.20 × 10−6 >9.20 × 10−6

oxa-thio-3hqa, 8 GI50 4.62 × 10−7 3.11 × 10−7 4.00 × 10−7

TGI 2.75 × 10−6 2.75 × 10−6 3.55 × 10−6

LC50 6.40 × 10−6 7.91 × 10−6 7.55 × 10−6

Dap-NMe-thio-3hqa, 9 GI50 8.95 × 10−6 8.95 × 10−6 8.95 × 10−6

TGI 8.95 × 10−6 8.95 × 10−6 8.95 × 10−6

LC50 8.95 × 10−6 8.95 × 10−6 8.95 × 10−6

Dab-NMe-thio-3hqa, 10 GI50 8.95 × 10−6 8.95 × 10−6 8.95 × 10−6

TGI 8.95 × 10−6 8.95 × 10−6 8.95 × 10−6

LC50 8.95 × 10−6 8.95 × 10−6 8.95 × 10−6

Dap(PEG)-NME-thio-3hqa, 11 GI50 >7.09 × 10−6 >7.09 × 10−6 >7.09 × 10−6

TGI >7.09 × 10−6 >7.09 × 10−6 >7.09 × 10−6

LC50 >7.09 × 10−6 >7.09 × 10−6 >7.09 × 10−6

Dab(PEG)-NMe-thio-3hqa, 12 GI50 >6.96 × 10−6 >6.96 × 10−6 >6.96 × 10−6

TGI >6.96 × 10−6 >6.96 × 10−6 >6.96 × 10−6

LC50 >6.96 × 10−6 >6.96 × 10−6 >6.96 × 10−6



Boc Alloc, pNZ, o-NBS, and Troc. The last group proveddecisive in masking the side-chain amino-function of the Dabresidue. Furthermore, the concourse of a broad range of activespecies from the less reactive OSu, to prevent overacylation, tothe most reactive OAt, have been used. In this sense, aminiumsalt coupling reagents (COMU and HATU) for the stepwiseelongation, phosphonium salts (PyBOP and PyAOP) for thefragment couplings and cyclization, and carbodiimides (EDC,DIPCDI) for the incorporation of the heterocycle, were appliedto finely tune all the coupling steps. The slightly greater stabilityof the MBHBr resin vs CTC was decisive for the preparation ofseveral analogues. The use of Dab instead of Dap minimized β-elimination side reactions. Furthermore, the in vitro activities ofthese compounds revealed relevant trends in the structure−activity relationship that will facilitate the design of newbisintercalators. Thus, it has been demonstrated the importanceof maintaining the hydrogen bond pattern as well as thehydrophobic interactions to favor the correct conformation forthe binding.

■ EXPERIMENTAL SECTIONGeneral. Solid-phase syntheses were performed in polypropylene

syringes (2, 5 mL) fitted with a polyethylene porous disc. Solvents andsoluble reagents were removed by suction. Washings betweendeprotection, coupling, and final deprotection steps were carried outwith DMF (5 × 1 min) and CH2Cl2 (5 × 1 min) using 5 mLsolvent·g−1 resin for each wash. Peptide synthesis transformations andwashes were performed at 25 °C. Purity for each compound wasdetermined by reversed-phase analytical HPLC at a wavelength of 220nm, at the conditions specified in each case. All final compoundsshowed purities ≥95%.Representative Syntheses of Each Strategy. NMe-thio-qxa

(3): Dimer Strategy. 2-Qxa-D-Dap(Alloc)-Gly-O-CTC-PS. CTC resin(200 mg, 1.6 mmol/g) was placed in a 10 mL polypropylene syringeand washed with DMF (5 × 1 min) and DCM (3 × 1 min). A solutionof Fmoc-Gly-OH (60 mg, 0.2 mmol) and DIEA (234 μL, 1.32 mmol,6.6 equiv) in DCM was added. After 10 min, more DIEA (117 μL,0.66 mmol, 3.3 equiv) was added, and the mixture was stirred for 50min at room temperature. The reaction was quenched by addition ofMeOH (200 μL) and the mixture stirred for further 10 min. Afterfiltration, the peptide resin was washed with DCM (3 × 1 min), DMF(3 × 1 min), and piperidine−DMF (1:4; 2 × 1 min, 2 × 5 min). Aloading of 0.98 mmol/g was obtained. Next, Fmoc-D-Dap(Alloc)-OH(201 mg, 0.49 mmol, 2.5 equiv) was introduced with HATU (186 mg,0.49 mmol, 2.5 equiv), HOAt (66.6 mg, 0.49 mmol, 2.5 equiv), andDIEA (166 μL, 0.98 mmol, 6 equiv) as coupling reagents in DMF. Thepeptide resin was washed with DMF (3 × 0.5 min), DCM (3 × 0.5min), DMF (3 × 0.5 min), and DCM (3 × 0.5 min), and afteracetylation with Ac2O (184 μL, 1.96 mmol, 10 equiv) and DIEA (340μL, 1.96 mmol, 10 equiv) for 30 min, the peptidyl resin was washedwith DCM (3 × 1 min), DMF (3 × 1 min), and piperidine−DMF(1:4; 2 × 1 min, 2 × 5 min), and again with DMF (5 × 0.5 min) andDCM (3 × 0.5 min). Next, 2-quinoxaline-carboxylic acid (70 mg,0.392 mmol, 2 equiv) was introduced by reaction with PyBOP (408mg, 0.784 mmol, 4 equiv), HOAt (106.6 mg, 0.784 mmol, 4 equiv),and DIEA (276 μL, 1.55 mmol, 8 equiv) in DMF for 90 min. Kaiser’stest showed that the coupling reaction was incomplete after 2 h. SoPyBOP (204 mg, 0.392 mmol, 2 equiv), HOAt (53 mg, 0.392 mmol, 2equiv), and DIEA (138 μL, 1.55 mmol, 4 equiv) were added, and thereaction was shaken for 2 h. An aliquot of the resin was cleaved andanalyzed by HPLC [tR = 8.64 min; gradient, 0:100 to 100:0 (ACN/H2O) in 15 min] and by HPLC-ESMS [tR = 8.59 min; gradient, 0:100to 100:0 (ACN/H2O) in 15 min; m/z calcd for C18H19N5O6 401.13,found 401.89 [M + H]+].{[Boc-NMe-Cys(Acm)-NMe-Cys(Me)&][Qxa-D-Dap(Me&)-Gly-O-

CTC-PS]}. The peptidyl resin was then treated with [Pd(PPh3)4] (23mg, 0.02 mmol, 0.1 equiv) and PhSiH3 (241 μL, 1.96 mmol, 10 equiv)in DCM (3 × 15 min) and then washed with DCM (3 × 0.5 min),

DMF (3 × 0.5 min), and DCM (3 × 0.5 min). Next, a solution of o-NBS-Cl (174 mg, 0.78 mmol, 4 equiv) and DIEA (348 μL, 1.96 mmol,10 equiv) in DCM was added and the mixture stirred for 90 min. Afterfiltration and washing with DCM (3 × 0.5 min), an aliquot of resinwas cleaved and analyzed by HPLC [tR = 9.55 min; gradient, 0:100 to100:0 (ACN/H2O) in 15 min] and by HPLC-ESMS [tR = 9.59 min;gradient, 0:100 to 100:0 (ACN/H2O) in 15 min; m/z calcd forC20H18N6O8S 502.09, found, 502.74 [M + H]+]. The peptidyl resinwas then washed with DMF (3 × 0.5 min), DCM (3 × 0.5 min), anddry THF (3 × 0.5 min), and a solution of PPh3 (257 mg, 0.98 mmol, 5equiv) and MeOH (80 μL, 1.96 mmol, 10 equiv) in dry THF and asolution of DIAD (190 μL, 0.98 mmol, 5 equiv) in dry THF weremixed and added to the peptidyl resin. After the solution had beenstirred for 1 h and filtered, the outcome of reaction was checked. Thereaction of methylation was repeated once. An aliquot of resin wascleaved and analyzed by HPLC [tR = 9.91 min; gradient, 0:100 to100:0 (ACN/H2O) in 15 min] and by HPLC-ESMS [tR = 9.94 min;gradient, 0:100 to 100:0 (ACN/H2O) in 15 min); m/z calcd forC21H20N6O8S 516.11, found, 516.92 [M + H]+]. After treatments (2 ×15 min) with DBU (147 μL, 0.98 mmol, 5 equiv) and β-mercaptoethanol (144 μL, 1.96 mmol, 10 equiv) in DMF, the resinwas washed with DMF (3 × 0.5 min), DCM (3 × 0.5 min), and DMF(3 × 0.5 min). An aliquot of resin was cleaved and analyzed by HPLC[tR = 5.72 min; gradient, 0:100 to 100:0 (ACN/H2O) in 15 min] andby HPLC-ESMS [tR = 4.98 min; gradient, 0:100 to 100:0 (ACN/H2O)in 15 min]; m/z calcd for C15H17N5O4 331.13, found, 331.89, [M +H]+]. The elongation of the peptide chain was performed by additionof Alloc-NMe-Cys(Me)-OH in the presence of HATU (298 mg, 0.784mmol, 4 equiv), HOAt (106.6 mg, 0.784 mmol, 4 equiv), and DIEA(280 μL, 1.57 mmol, 8 equiv) in DMF for 35 min, and after filtration,washings with DMF (3 × 0.5 min) and DCM (3 × 0.5 min) wereperformed. An aliquot of resin was cleaved and analyzed by HPLC [tR= 9.79 min; gradient, 0:100 to 100:0 (ACN/H2O) in 15 min; 92%purity] and by HPLC-ESMS [tR = 9.94 min; gradient, 0:100 to 100:0(ACN/H2O) in 15 min; m/z calcd for C24H30N6O7S 546.19, found547.02 [M + H]+]. Then the peptide resin was treated (3 × 15 min)with [Pd(PPh3)4] (23 mg, 0.02 mmol, 0.1 equiv) and PhSiH3 (241 μL,1.96 mmol, 10 equiv) in DCM and washed with DCM (3 × 0.5 min),DMF (3 × 0.5 min), DCM (3 × 0.5 min), and DMF (3 × 0.5 min).Coupling of the peptidyl-resin with Boc-NMe-Cys(Acm)-OH (180mg, 0.58 mmol, 3 equiv) was performed first with HATU (223 mg,0.588 mmol, 3 equiv), HOAt (80 mg, 0.588 mmol, 3 equiv), and DIEA(210 μL, 1.18 mmol, 6 equiv) in DMF for 35 min. The couplingreaction was repeated once (240 mg aa, 0.784 mmol, 4 equiv), PyAOP(408 mg, 0.784 mmol, 4 equiv), HOAt (107 mg, 0.784 mmol, 4equiv), and DIEA (280 μL, 1.57 mmol, 8 equiv) in DMF for 90 min.After an additional 2 h, PyAOP, HOAt, and DIEA were added in thesame conditions described above, and the reaction was shaken for 2 h.Filtration and washings of the resin with DCM (3 × 0.5 min), DMF (3× 0.5 min), DCM (3 × 0.5 min), and DMF (3 × 0.5 min) followed.An aliquot of resin was cleaved and analyzed by HPLC [mixture of twoconformers tR = 9.57 (minor) min and 10.2 min (major); gradient,0:100 to 100:0 (ACN/H2O) in 15 min); 65% purity] and by HPLC-ESMS [mixture of two conformers tR = 9.59 min (minor) and 10.3min (major); gradient, 0:100 to 100:0 (ACN/H2O) in 15 min; m/zcalcd for C32H46N8O9S2 750.28, found 751.75 [M + H]+, 651.57 [M −Boc + H]+].

{[NMe-Cys(&1)-NMe-Cys(Me)&2] [Qxa-D-Dap(Me&2)-Gly-OH]}2.Dimer formation was achieved by treatments (2 × 10 min) with asolution of I2 (247 mg, 0.98 mmol, 5 equiv) in DMF (10 mL),followed by washing with DMF (3 × 0.5 min), DCM (3 × 0.5 min),DMF (3 × 0.5 min), and DCM (3 × 0.5 min). HPLC analysis of acleaved aliquot showed the completion of reaction. Next, the peptidewas cleaved from the resin using a TFA−DCM solution (2:98, 5 × 1min), and the filtrates were collected in presence of H2O, dried, andlyophilized. HPLC [mixture of two conformers, tR= 12.3 min (minor)and 12.8 min (major); gradient, 0:100 to 100:0 (ACN/H2O) in 15min; 40% purity] and HPLC-MS [mixture of two conformers, tR =12.5 (minor) and 13.1 min (major); gradient, 0:100 to 100:0 (ACN/



H2O) in 15 min; m/z calcd for C58H80N14O16S4 1356.48, found1357.53 [M + H]+].The Boc-protected dimer was then treated with a solution of TFA−

DCM−H2O (35:5:60) for 45 min and the crude lyophilized product.HPLC: mixture of conformers tR = 7.19−7.27 min (gradient. 0:100 to100:0 (ACN/H2O) in 15 min). HPLC-ESMS: mixture of threeconformers, tR = 6.12, 6.29, 6.43 min; gradient, 0:100 to 100:0 (ACN/H2O) in 15 min; m/z calcd for C48H64N14O12S4 1156.37, found1157.20, 1157.33, 1157.07 [M + H]+ .NMe-thio-qxa. An aliquot of the unprotected dimer (28 mg, 0.024

mmol, 1 equiv, 0.2 mM) was dissolved in DCM/DMF and added to asolution of HOAt (26 mg, 0.2 mmol, 8 equiv) in DMF. At neutral pH,a solution of PyBOP (99 mg, 0.2 mmol, 8 equiv) in DMF was added,followed by DIEA until reaching pH 8−9. The reaction was stirred atroom temperature for 4 h. Then PyBOP (50 mg, 0.1 mmol, 4 equiv)in DMF was added, and the reaction was stirred for a further 20 h atroom temperature. The solvent was then evaporated, and the crudebicycle was redissolved in DCM. The organic layers were washed withsaturated NH4Cl (2 × 50 mL) and brine (2 × 50 mL), dried withMgSO4, evaporated under vacuum, and lyophilized. The peptide waspurified by analytical HPLC: tR = 22.25 min; gradient. 25:75 to 55:45(ACN/H2O). HPLC-MS: m/z calcd for C48H60N14O10S4 1120.35,found 1121.40 [M + H]+, 1143.27 [M + Na]+. HR-MS: m/z calcd forC48H61N14O10S4 1121.3578 [M + H]+, found 1121.3591.NMe-triostin-qxa (4): Stepwise Strategy. Qxa-D-Dap(Alloc)-Ala-

O-(Br-PS). A solution of Fmoc-Ala-OH (176 mg, 0.57 mmol), CsI(147 mg, 0.57 mmol), and DIEA (96 μL, 0.57 mmol) in DMF wasadded to a previously washed MBHBr resin (200 mg, 1.41 mmol/g),and the suspension was shaken for 14 h. Then MeOH (160 μL) wasadded, and the resin was shaken for 2 h. After washing the resin withDMF (5 × 1 min) and DCM (3 × 1 min), the Fmoc group wascleaved with a solution of piperidine−DMF (1:4), and Fmoc-D-Dap(Alloc)−OH was incorporated as described previously forcompound 3. The Fmoc group was cleaved again with piperidine−DMF (1:4), and 2-quinoxaline carboxylic acid (50 mg, 0.29 mmol, 2equiv) was incorporated using PyBOP (150 mg, 0.29 mmol), HOAt(39.4 mg, 0.29 mmol), and DIEA (98 μL, 0.58 mmol) in DMF for 4 h.{[Alloc-NMe-Cys(Acm)-NMe-Val&][Qxa-D-Dap(Me&)-Ala-O-(Br-

PS)}. To remove the Alloc group, the peptidyl-resin was then treatedwith [Pd(PPh3)4] (20 mg, 0.017 mmol, 0.1 equiv) and PhSiH3 (197μL, 1.7 mmol), (3 × 15 min), and the free amino group wasreprotected with o-NBS-Cl (113 mg, 0.51 mmol) and DIEA (289 μL,1.7 mmol) in DCM for 90 min. The peptidyl-resin was then washedwith DMF (3 × 0.5 min) and DCM (3 × 0.5 min). An aliquot wascleaved and analyzed by HPLC [tR = 6.00 min; gradient, 5:95 to 100:0(ACN/H2O) in 8 min] and HPLC-MS [tR = 7.61 min; gradient, 5:95to 100:0 (ACN/H2O) in 8 min; m/z calcd for C21H20N6O8S 516.11,found 517.20 [M + H]+]. The resin was washed with dry THF (3 ×0.5 min) and a solution of PPh3 (223 mg, 0.85 mmol), and MeOH (69μL, 1.7 mmol) in dry THF were added to the resin. DIAD (165 μL,0.85 mmol) in dry THF was then added to the peptidyl-resin, whichwas shaken for 1 h. An aliquot was cleaved and analyzed by HPLC [tR= 6.97 min; gradient, 5:95 to 100:0 (ACN/H2O) in 8 min] andHPLC-MS [tR = 7.87 min; gradient, 5:95 to 100:0 (ACN/H2O) in 8min; m/z calcd for C22H22N6O8S 530.12, found 531.21 [M + H]+]. Toremove the o-NBS group, a solution of β-mercaptoethanol (119 μL,1.7 mmol) and DBU (127 μL, 0.85 mmol) in DMF was added to theresin and shaken for 15 min. The treatment was repeated twice more.An aliquot was cleaved and analyzed by HPLC [tR = 5.64 min;gradient, 5:95 to 100:0 (ACN/H2O) in 8 min] and HPLC-MS [tR =4.69 min; gradient, 5:95 to 100:0 (ACN/H2O) in 8 min; m/z calcd forC16H19N5O4 345.14, found, 346.12 [M + H]+]. Next, Fmoc-NMe-Val-OH (300 mg, 0.85 mmol) was introduced with HATU (323 mg, 0.85mmol), HOAt (116 mg, 0.85 mmol), and DIEA (289 μL, 1.7 mmol)in DMF for 70 min. A second coupling with Fmoc-NMe-Val-OH (180mg, 0.51 mmol), HATU (194 mg, 0.51 mmol), HOAt (70 mg, 0.51mmol), and DIEA (173 μL, 1.0 mmol) for 1 h was performed. Analiquot was cleaved and analyzed by HPLC [tR = 7.95 min; gradient,5:95 to 100:0 (ACN/H2O) in 8 min] and HPLC-MS [tR = 9.61 min;gradient, 5:95 to 100:0 (ACN/H2O) in 8 min; m/z calcd for

C37H40N6O7 680.30, found 681.24 [M + H]+]. After cleavage of theFmoc group in the usual conditions, piperidine−DMF (1:4) (2 × 1min, 2 × 5 min) and Alloc-NMe-Cys(Acm)-OH (197 mg, 0.68 mmol)was incorporated with HATU (259 mg, 0.68 mmol), HOAt (93 mg,0.68 mmol), and DIEA (231 μL, 1.36 mmol) in DMF for 70 min. Thecoupling was repeated again in the same conditions and a third timewith PyAOP/DIEA for 2 h. An aliquot was cleaved and analyzed byHPLC [tR = 5.57 min; gradient, 5:95 to 100:0 (ACN/H2O) in 8 min]and HPLC-MS [tR = 7.78 min; gradient, 5:95 to 100:0 (ACN/H2O) in8 min; m/z calcd for C33H46N8O9S 730.31, found 731.28 [M + H]+].

[Qxa-D-Dap(Me&1)-Ala-NMe-Cys(Acm)-NMe-Val&2][Alloc-NMe-Cys(Acm)-NMe-Val&1][qxa-D-Dap(Me&2)-Ala-O-Br-PS. The Allocgroup was removed with [Pd(PPh3)4] (20 mg, 0.017 mmol) andPhSiH3 (197 μL, 1.7 mmol) in DCM (3 × 15 min). An aliquot wascleaved and analyzed by HPLC [mixture of three conformers, tR =3.70, 3.84, 4.0 min; gradient, 5:95 to 100:0 (ACN/H2O) in 8 min] andHPLC-MS [mixture of three conformers, tR = 5.37, 5.66, 5.92 min;gradient, 5:95 to 100:0 (ACN/H2O) in 8 min; m/z calcd forC29H42N8O7S 646.29, found 647.27, 648.29 [M + H]+]. Next, Fmoc-Ala-OH (211 mg, 0.68 mmol) was introduced with HATU (259 mg,0.68 mmol), HOAt (92 mg, 0.68 mmol), and DIEA (231 μL, 1.36mmol) in DMF for 1 h. The coupling was repeated once more. Analiquot was cleaved and analyzed by HPLC [mixture of twoconformers, tR = 6.45, 6.68 min; gradient, 5:95 to 100:0 (ACN/H2O) in 8 min] and HPLC-MS [mixture of two conformers, tR = 8.61,9.10 min; gradient, 5:95 to 100:0 (ACN/H2O) in 8 min; m/z calcd forC47H57N9O10S 939.39, found, 940.41 [M + H]+]. Steps to reach theoctapeptide (coupling of Fmoc-D-Dap(Alloc)-OH and 2-quinoxalinecarboxylic acid, N-methylation, coupling of Fmoc-NMe-Val-OH andAlloc-NMe-Cys(Acm)-OH, and cleavages of Fmoc and Alloc groups)were performed in the same way as described above for thetetrapeptide. An aliquot was cleaved and analyzed by HPLC [tR =7.97 min; gradient, 5:95 to 100:0 (ACN/H2O) in 8 min] and HPLC-MS [tR = 7.97 min; gradient, 5:95 to 100:0 (ACN/H2O) in 8 min; m/z calcd for C62H86N16O15S2 1358.59, found, 1359.56 [M + H]+].

{[Qxa-D-Dap(Me&1)-Ala-NMe-Cys(&2)-NMe-Val&3][NMe-Cys(&2)-NMe-Val&1][Qxa-D-Dap(Me&3)-Ala-O-(Br-PS)]}. The Alloc group wasremoved by treatment with [Pd(PPh3)4] (20 mg, 0.017 mmol) andPhSiH3 (197 μL, 1.7 mmol) in DCM (3 × 15 min). An aliquot wascleaved and analyzed by HPLC [tR = 5.10 min; gradient, 5:95 to 100:0(ACN/H2O) in 8 min] and HPLC-MS [tR = 5.94 min; gradient, 5:95to 100:0 (ACN/H2O) in 8 min; m/z calcd for C58H82N16O13S21274.57, found, 1275.51 [M + H]+]. Next, the disulfide bridge wasaccomplished by treating the peptidyl resin with a solution of I2 (184mg, 0.72 mmol) in DMF (2 × 15 min). The peptide was cleaved fromthe resin by treatment with TFA−DCM (8:92), and the filtrate wasevaporated and lyophilized. HPLC [tR = 5.09 min; gradient, 5:95 to100:0 (ACN/H2O) in 8 min] and HPLC-MS [tR = 6.35 min; gradient,10:90 to 70:30 (ACN/H2O) in 8 min; m/z calcd for C52H70N14O11S21130.48, found 1131.41 [M + H]+].

NMe-triostin-qxa. The peptide (0.19 mmol) and HOAt (210 mg,1.54 mmol, 8 equiv) were dissolved in the minimum amount of DMF,and then DCM (945 mL) was added in order to obtain a 0.2 mMsolution. Addition of DIEA until pH 8 and PyBOP (803 mg, 1.54mmol, 8 equiv) started the reaction. The reaction was stirred at roomtemperature overnight, and HPLC-ESMS analysis was used to revealthe completion of the reaction. The organic layer was thenconcentrated under reduced pressure and washed with saturatedNH4Cl (3 × 50 mL) and brine (2 × 50 mL), dried with Na2SO4, andevaporated under vacuum. The peptide was prepurified with a Porapakcolumn previously washed with MeOH and equilibrated with H2O,and the peptide was eluted with increasing percentages of ACN. Thefraction eluted with 40% ACN showed the desired peptide, which wasfurther purified by reversed-phase HPLC. HPLC [tR = 6.0 min;gradient, 5:95 to 100:0 (ACN/H2O) in 8 min] and HPLC-MS[mixture of two isomers, tR = 8.11, 8.36 min; gradient, 20:80 to 100:0(ACN/H2O) in 8 min; m/z calcd for C52H68N14O10S 1112.47, found1113.21 [M + H]+]. MALDI-TOF: 1114.49. HR-MS: m/z calcd forC52H69N14O10S 1113.4763 [M + H]+, found 1113.4751.



Dab-NMe-thio-3hqa (10): 4 +4 Strategy. Boc-D-Dap(Fmoc)-Gly-O-CTC-PS. 2-CTC resin (400 mg, 1.6 mmol/g) was placed in a 10 mLpolypropylene syringe and washed with DMF (5 × 1 min) and DCM(3 × 1 min). A solution of Fmoc-Gly-OH (119 mg, 0.4 mmol) andDIEA (455 μL) in DCM was added to the resin. After 5 min, moreDIEA (227 μL) was added, and the resin was shaken for 45 min.MeOH (320 μL) was added to cap any unreacted sites, and the resinwas shaken for 10 min. Cleavage of the Fmoc group in the usualconditions with piperidine−DMF (1:4) showed a loading of 0.98mmol/g. Introduction of Boc-D-Dap(Fmoc)-OH (501 mg, 1.18mmol) was performed with HATU (447 mg, 1.18 mg), HOAt (160mg, 1.18 mmol), and DIEA (400 μL, 2.35 mmol) in DMF for 1 h.Kaiser’s test indicated completion of the coupling,{[Alloc-NMe-Cys(Acm)-NMe-Dab(Troc)&][Boc-D-Dap(Me&)-Gly-O-

CTC-PS]}. After cleaving the Fmoc group by treatment withpiperidine−DMF (1:4), a solution of o-NBS-Cl (261 mg, 1.18mmol) and collidine (260 μL, 1.96 mmol) in DCM was added to theresin, and the mixture was stirred for 45 min. The resin was filteredand washed with DCM (3 × 0.5 min), DMF (3 × 0.5 min), DCM (3× 0.5 min), and THF (3 × 0.5 min), a solution of PPh3 (514 mg, 1.96mmol) and MeOH (158 μL, 3.92 mmol) in THF, and a solution ofDIAD (400 μL, 1.96 mmol) in THF were mixed and added to thepeptide resin. After stirring the resin for 1 h, it was washed with THF(3 × 0.5 min), DCM (3 × 0.5 min), and DMF (3 × 0.5 min). Thereaction was repeated once for 45 min. An aliquot was cleaved andanalyzed by analytical HPLC [tR = 6.09 min; gradient, 5:95 to 100:0(ACN/H2O) in 8 min] and HPLC-MS [tR = 7.90 min; gradient, 5:95to 100:0 (ACN/H2O) in 8 min; m/z calcd for C17H24N4O9S 460.13l,found 461.14 [M + H]+]. After treatments (2 × 15 min) with DBU(275 μL, 1.96 mmol) and β-mercaptoethanol (275 μL, 3.92 mmol) inDMF, the resin was washed with DMF (3 × 0.5 min), DCM (3 × 0.5min), and DMF (3 × 0.5 min). An aliquot was cleaved and analyzed byanalytical HPLC [tR = 1.96 min; gradient, 5:95 to 100:0 (ACN/H2O)in 8 min] and HPLC-MS [tR = 2.22 min; gradient, 5:95 to 100:0(ACN/H2O) in 8 min; m/z calcd for C11H21N3O5 275.15, found276.19 [M + H]+]. Next, previously prepared Fmoc-Dab(Troc)-OH(403 mg, 0.78 mmol) was incorporated with HATU (298 mg, 0.78mmol), HOAt (107 mg, 0.78 mmol), and DIEA (333 μL, 1.96 mmol)in DMF for 75 min. An aliquot was cleaved and analyzed by analyticalHPLC [tR = 7.98 min; gradient, 5:95 to 100:0 (ACN/H2O) in 8 min].After cleaving the Fmoc group in the usual conditions, the peptidylresin was treated with o-NBS-Cl (261 mg, 1.18 mmol) and collidine(260 μL, 1.96 mmol) for 45 min. A second treatment was performed.After washes of the resin, an aliquot was cleaved and analyzed byHPLC [tR = 6.89 min; gradient, 5:95 to 100:0 (ACN/H2O) in 8 min]and HPLC-MS [tR = 8.92 min; gradient, 5:95 to 100:0 (ACN/H2O) in8 min; m/z calcd for C24H33Cl3N6O12S 734.09, found 735.04 [M +H]+]. Then, a solution of PPh3 (514 mg, 1.96 mmol) and MeOH (158μL, 3.92 mmol) in THF was added to the resin, followed by DIAD(380 μL, 1.96 mmol) in THF. After 1 h, the N-methylation reactionwas complete. An aliquot of the resin was cleaved and analyzed byHPLC [tR = 7.01 min; gradient, 5:95 to 100:0 (ACN/H2O) in 8 min]and HPLC-MS [tR = 9.06 min; gradient, 5:95 to 100:0 (ACN/H2O) in8 min; m/z calcd for C25H35Cl3N6O12S 748.11, found 748.98 [M +H]+]. Next, to cleave the o-NBS group, a solution of β-mercaptoethanol (275 μL, 3.92 mmol) and DBU (294 μL, 1.96mmol) in DMF was added to the resin and it was shaken for 15 min.The treatment was repeated twice more. After washing the resin withDMF (3 × 15 min) and DCM (3 × 15 min), an aliquot of resin wascleaved and analyzed by HPLC [tR = 4.57 min; gradient, 5:95 to 100:0(ACN/H2O) in 8 min] and HPLC-MS [tR = 5.92 min; gradient, 5:95to 100:0 (ACN/H2O) in 8 min; m/z calcd for C19H32Cl3N5O8 563.13,found 564.11 [M + H]+]. Next, Alloc-NMe-Cys(Acm)-OH (455 mg,1.57 mmol) was introduced with HATU (596 mg, 1.57 mmol), HOAt(214 mg, 1.57 mmol), and DIEA (533 μL, 3.14 mmol) in DMF, andthe pH was further adjusted with DIEA (400 μL) to reach pH 8. Thepeptidyl-resin was shaken for 1 h at 25 °C. A second coupling withAlloc-NMe-Cys(Acm)-OH (307 mg, 1.06 mmol), PyAOP (450 mg,0.86 mmol), HOAt (144 mg, 1.06 mmol), and DIEA (360 μL, 2.12mmol) in DMF at 35 °C for 90 min was performed. An aliquot of

resin was cleaved and analyzed by HPLC [tR = 6.59 min; gradient, 5:95to 100:0 (ACN/H2O) in 8 min] and HPLC-MS [tR = 8.44 min;gradient, 5:95 to 100:0 (ACN/H2O) in 8 min; m/z calcd forC30H48Cl3N7O12S 835.21, found 836.10 [M + H]+].

[Boc-D-Dap(Me&1)-Gly-NMe-Cys(&2)-NMe-Dab(Troc)&3][Alloc-NMe-Cys(&2)-NMe-Dab(Troc)&1][Boc-D-Dap(Me&3)-Gly-O-CTC-PS].The protected tetrapeptide was split in two portions: 1/4 of the resinwas treated with a solution of PhSiH3 (121 μL, 0.98 mmol) in DCM,and then [Pd(PPh3)4] (11.3 mg, 0.01 mmol) was further added, andthe resin was shaken for 15 min. After washings with DCM (3 × 15min), the treatment was repeated twice. An aliquot of resin wascleaved and analyzed by HPLC [tR = 3.52 min; gradient, 20:80 to100:0 (ACN/H2O) in 8 min] and HPLC-MS [tR = 4.65 min; gradient,20:80 to 100:0 (ACN/H2O) in 8 min; m/z calcd for C26H44Cl3N7O10S751.19, found 752.16 [M + H]+]. The remaining resin (3/4) wascleaved with a solution of TFA−DCM (1.5:98.5), (10 × 1 min), andthe filtrates were collected H2O at pH 8. The organic solvent wasevaporated and the aqueous filtrates were lyophilized. A prepur-ification of the protected tetrapeptide was carried out with a Porapakcolumn with increasing percentages of ACN. The protectedtetrapeptide eluted at 50% ACN, and this filtrate was lyophilized.To the unprotected peptidyl-resin was added the protectedtetrapeptide (30 mg) with PyAOP (51 mg, 0.098 mmol) and DIEA(20 μL, 0.12 mmol) in DMF, and the peptidyl-resin was shaken for 3h. After washing the resin with DMF (3 × 15 min) and DCM (3 × 15min), an aliquot of resin was cleaved and analyzed by HPLC [mixtureof isomers: tR = 6.43, 6.60 min; gradient, 20:80 to 100:0 (ACN/H2O)in 8 min] and HPLC-MS [tR = 8.26, 8.47 min; gradient, 20:80 to 100:0(ACN/H2O) in 8 min; m/z calcd for C56H90Cl6N14O21S2 1568.40,found 1569.70 [M + H]+]. The Alloc group was then cleaved withPhSiH3 (121 μL, 0.98 mmol) and [Pd(PPh3)4] (11.3 mg, 0.01 mmol)in DCM (3 × 15 min). An aliquot was cleaved and analyzed by HPLC[tR = 3.63 min; gradient, 20:80 to 100:0 (ACN/H2O) in 8 min] andHPLC-MS [tR = 5.82 min; gradient, 20:80 to 100:0 (ACN/H2O) in 8min; m/z calcd for C52H86Cl6N14O19S2 1484.38, found 1485.68 [M +H]+]. Disulfide formation was accomplished with I2 (124 mg, 0.49mmol) in DMF (2 × 10 min). After washing the peptidyl resin withDMF (3 × 1 min) and DCM (3 × 1 min), the peptide was cleavedfrom the resin with TFA−DCM (1.5:98.5), collecting the filtrate overH2O. The solvent was evaporated, and the aqueous solution waslyophilized. HPLC [tR = 4.99 min; gradient and HPLC-MS [tR = 6.36min; gradient, 20:80 to 100:0 (ACN/H2O) in 8 min; m/z calcd forC46H74Cl6N12O17S2 1340.29, found 1341.36 [M + H]+].

Dab-NMe-thio-3hqa. HOAt (16 mg, 0.12 mmol) was dissolved inthe minimum amount of DMF, and the crude peptide (32 mg, 0.024mmol) dissolved in DMF−DCM (1:9, 40 mL) was added. The pHwas adjusted to 7 with DIEA (20 μL), and cyclization started byadding EDC·HCl (23 mg, 0.12 mmol). After 4 h, the reaction waswashed with H2O, and the solvent was evaporated. HPLC [tR = 8.32min; gradient, 5:95 to 100:0 (ACN/H2O) in 8 min] and HPLC-MS[tR = 9.55 min; gradient, 20:80 to 100:0 (ACN/H2O) in 8 min; m/zcalcd for C46H73N12O16S2 1323.28, found 1325.45]. The crude peptidewas subjected to TFA−DCM (1:3) for 1 h to cleave the Boc groups,and the solvent was evaporated and lyophilized in HCl 0.01 N. Tointroduce the heterocycles, 3-hydroxyquinaldic acid (10 mg, 0.053mmol) was dissolved in DCM (5 mL) with HOSu (6.1 mg, 0.053mmol) and, after 15 min, the crude peptide was added. EDC·Cl (10mg, 0.053 mmol) was then added, and the reaction was stirred for 16h. H2O was added, and the organic phase was extracted andevaporated. HPLC: tR = 6.96 min; gradient, 5:95 to 100:0 (ACN/H2O) in 8 min; HPLC-MS, tR = 10.18 min; gradient, 20:80 to 100:0(ACN/H2O) in 8 min; m/z calcd for C56H67Cl6N14O16S2 1465.24,found 1467.37. Finally, the Troc group was cleaved with Zn inaqueous 90% AcOH. The crude peptide was prepurified on a Porapakcolumn and then purified by reversed-phase HPLC (linear gradient,10:90 to 30:70 (ACN/H2O) in 20 min). HR-ESMS: m/z calcd forC50H65N14O12S2 1117.4342 [M + H]+, found 1117.4315.



■ ASSOCIATED CONTENT*S Supporting InformationExperimental procedures and characterization data. Thismaterial is available free of charge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author*Phone: +34 93 4037088. Fax: +34 93 4037126. E-mail:[email protected] (F.A.); [email protected](J.T.-P.).NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThis work was partially supported by CICYT (CTQ2012-30930), the Generalitat de Catalunya (2009SGR1024), and theInstitute for Research in Biomedicine Barcelona (IRBBarcelona). We gratefully acknowledge PharmaMar SA forperforming the biological tests and Dr. Andres M. Franceschand Dr. Carmen Cuevas for encouraging this work. J.T.-P.thanks the MEC for a Juan de la Cierva contract.

■ ABBREVIATIONS USEDa

ACN, acetonitrile; Alloc, allyloxycarbonyl; Boc, t-butyloxycar-bonyl; CTC, chlorotrityl chloride (Barlos) resin; DIEA, N,N-diisopropylethylamine; DIPCDI, N,N′-diisopropylcarbodii-mide; DKP, diketopiperazine; DMF, N,N-dimethylformamide;ESMS, electrospray mass spectrometry; EtOAc, ethyl acetate;HATU, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo-[4,5-b]pyridinium hexafluorophosphate 3-oxide; HOAt, 1-hydroxy-7-azabenzotriazole; HOBt, 1-hydroxybenzotriazole;HPLC, high performance liquid chromatography; MS, massspectrometry; TFA, trifluoroacetic acid; TBME, t-butyl methylether; TIS, triisopropylsilane; Trt, trityl

■ ADDITIONAL NOTEaAbbreviations used for amino acids and the designations ofpeptides follow the rules of the IUPAC-IUB Commission ofBiochemical Nomenclature in J. Biol. Chem. 1982, 247, 977−983. Amino acid symbols denote L-configuration unlessindicated otherwise. All reported solvent ratios are expressedas v/v, unless otherwise stated.

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mailto:[email protected]

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