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Synthesis of Cyclic Peptides as Potential Anti-Malarials Catherine Fagundez, Diver Sellanes, and Gloria Lourdes Serra ACS Comb. Sci., Just Accepted Manuscript • DOI: 10.1021/acscombsci.7b00154 • Publication Date (Web): 15 Feb 2018 Downloaded from http://pubs.acs.org on February 20, 2018

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ACS Combinatorial Science

Synthesis of Cyclic Peptides as Potential Anti-Malarials Catherine Fagundez, a Diver Sellanes, a Gloria Serra. a* a Cátedra de Química Farmacéutica, (DQO), Facultad de Química, Universidad de la República, Gral. Flores 2124, CP 11800, Montevideo, Uruguay. KEYWORDS: Cyclopeptides, Solid Phase Peptide Synthesis, macrocyclization, malaria

ABSTRACT. The results from the synthesis of peptides by Fmoc/SPPS on a 2-CTC resin and then lactamization in solution or solid phase for the preparation of cyclopeptides are presented. Both procedures allow the synthesis of the desired compounds in good to very good yield and with high cyclization efficiency for on-resin macrocyclization. In addition, the activities of the corresponding cyclopeptides against the chloroquine-resistant K1 strain of Plasmodium falciparum were evaluated. Cyclo-Cys(Trt)-Gly-Thr(tBu)-Gly-Cys(Trt)-Gly showed potent in vitro and selective activity against this parasite, EC50 = 28 nM.

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INTRODUCTION Cyclic peptides have demonstrated advantageous properties as candidates for drug discovery. They usually have structural characteristics promoting bioactive conformations, often show high selectivity in binding to target receptors, and exhibit good metabolic stability. In addition, increased hydrophobicity associated with the removal of polar N- and C-termini allows better membrane permeation compared with linear analogs.1 Natural cyclic peptides have proved to have a wide spectrum of biological activities, including antimicrobial, antiviral, cytotoxic, anti-inflammatory, immunosuppressive, insecticidal, and antifungal effects. Among examples of antiparasitic compounds are the cyclic heptapeptides mahafacyclin B and chevalierin A, isolated from the latex of Jatropha, which showed IC50 = 2.2 and 8.9 µM, respectively, against P. falciparum.2 Selective inhibitory activity of certain cyclohexapeptides has also been reported, such as cyclo [Trp-D-Phe-Pro-Phe-Phe-Lys(Z)-] with an IC50 = 3µM against triose phosphate isomerase of Trypanosoma brucei. 3 As part of a search for compounds with antimalarial activity, we have previously reported the synthesis and biological evaluation of analogs of the antiparasitics aerucyclamide B (1), C (2) and D


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(3),4 Figure 1. We obtained non azolic-cyclohexapeptides containing cysteine or threonine/serine amino acids, which are biomimetic precursors of thiazoline/ thiazole or oxazoline/oxazole, respectively, such as compounds 4 and 5 (Figure 1).5 These present interesting anti-plasmodial activity (EC50 =0.19 ± 0.02 µM and 0.8 ± 0.2 µM, respectively) and were not toxic to murine macrophages showing excellent selectivity indices. These activities are in the same order of magnitude than that of aerucyclamides,4 showing that cyclic hexapeptides composed by alternating amino acids with alcohol or thiol groups in the lateral chain and hydrophobic amino acids are potential antiparasitic compounds. We describe here the improvement of synthetic methods for this type of compounds, and an exploration of structureactivity relationships that led to candidates with increased potency in vitro.

Figure 1. Aerucyclamides and analogs with anti-plamodial activity Cyclic peptides are most commonly made by Fmoc-based solid phase peptide synthesis (SPPS),6 followed by macrocyclization either on-resin or in solution. However, extensive comparisons between the cyclization efficiencies of solution vs. on-resin macrolactamization are not frequently reported. One


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example was described for cyclopentapeptides and cyclohexapeptides by Sewald et al.,7 using a dual syringe pump method for solution cyclization. They obtained better yields for the solution-phase method, 9-36%, than for on-resin cyclization, 1-22%. In contrast, Jeong and co-workers, reported solution macrocyclization of a pentapetide in 7% yield and on-resin lactamization of an hexapeptide in 35% yield.8 However, both procedures were not applied for the preparation of cyclopeptides with the same size and sequence. It is well known that high dilution in solution (1-10 mM) and moderate density of loading on resins (the latter to enhance the “pseudo-dilution” provided by attachment to a support9) promote macrolactamization relative to unwanted intermolecular reactions such as polymerization. Due to long reaction times and difficulties in purification inherent in the solution-phase method,5, 10 we embarked on a search for methods to reduce these problems. We chose to take advantage of an on-resin head-to-tail lactamization design in which the peptide is anchored to the resin through a side chain of a trifunctional amino acid. At least three dimensions of orthogonality are necessary to construct the linear peptide,11 allowing for subsequent cyclization, washing, and release of the pure product by cleavage of the resin connection. Various supports have been employed for this purpose, including Wang,12 Rink amide,13 BrWang,14 and 2-CTC (2-chlorotritylchloride) resins.15 In the present work, we have compared solution- and solid-phase ring-closing reactions following linear SPPS on a 2-CTC resin, chosen in the hope that the steric nature of the 2-CTC linkage would help to minimize racemization during the on-resin macrocyclization process.16 Sequences were chosen based on structures 4 and 5, which are composed by alternating amino acids with alcohol or thiol groups in the lateral chain and hydrophobic amino acids. In addition, Ser, Thr or Cys and hydrophobic amino acids were changed by Glu or Arg and more hydrophilic ones, respectively. Moreover, two


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cyclopentapeptides were prepared to study the influence of the cyclopeptide size and those substitutions in the activity against the chloroquine-resistant K1 strain of Plasmodium falciparum. RESULTS AND DISCUSSIONS In the following discussion, method A refers to the synthesis of the peptide sequence on the resin, followed by cleavage and macrocyclization in solution, whereas method B refers to synthesis and macrocyclization both performed on-resin. The general procedure A is shown in Scheme 1. The 2-CTC resin was employed to minimize the formation of diketopiperazines due to the large size of the chlorotrityl group,17 and the very mild acidic cleavage conditions allowed several other acid-labile protecting groups to be used in the peptide synthesis. Unreacted reactive sites after the first amino acid attachment were capped with MeOH,18 followed by standard SPPS using HCTU or HBTU and DIPEA in most cases. A combination of DIC and Cl-HOBt was used when the next amino acid was Fmoc-L-Cys(Trt)-OH, because this coupling agent does not require the addition of a base, so as to decrease the possibility of racemization, a common problem when amide bonds are formed from a Cys carboxyl.19


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Scheme 1. General procedure (A): Fmoc-strategy SPPS and solution phase macrocyclization Following resin cleavage, solution macrocyclization was performed at 1-5 mM concentration by activation with HBTU or HATU, using DIPEA in DCM, whereby the desired compounds were obtained in moderate to good yields (26-80%), (Table 1). The ring size and the amino acids involved are the most important factors governing the success of the head-to-tail peptide macrocyclization. It has been reported that cyclization of peptides with less than seven amino acids is troublesome, the E-geometry of the peptide bond prevents the peptides to adopt the ring-like conformation conducive to cyclization. 20 Even though, small cyclic peptides containing a β− turn such as a proline, a D- aminoacid, a thiazole or oxazole ring, etc. can be prepared in good yields. If the peptide does not contain a turn-inducing, the cyclization is very dependent on its sequence. To minimize racemization, and thereby the formation of diastereomers, during ring closure,21 we started most peptide sequences with Glycine at the C-terminus, which minimizes steric hindrance during the macrocyclization process.


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Eleven cyclic peptides, were synthesized using method A in 23 to 63% overall yield (Figure 2). In some compounds, Ser or Thr present in 4 or 5 were substituted by Glu or Arg (10-14), and hydrophobic amino acids were substituted by others as Gly, Phe, Met or by Arg (6-9), in order to obtain more soluble compounds in several cases, and to study the influence of those substitutions in the bioactivities. In addition, as we were interested in evaluating if the ring size affects the antimalarial activity, two cyclic pentapeptides were prepared. The processes were monitored by HPLC analyses to determine the reaction progress. No racemization was observed, including for compound 8,22 formed from a peptide with Cys at the carboxyl end. The best macrocyclization yield (80%) was obtained for the single cyclohexapeptide which presents only one hydrophilic amino acid (Glu) and five hydrophobic residues (14). Lower macrocyclization yields and longer reaction times were associated with the presence of Arg(Pbf) in the peptide sequence (compounds 9 and 10), perhaps because of steric hindrance of the macrocyclic process.23


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Figure 2. Cyclopeptides synthesized in this work by methodology A.


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Peptide precursor

Product

Time (days)

H-L-Cys(Trt)-Gly-L-Thr(tBu)-Gly-L-Cys(Trt)-Gly-OH

6

H-L-Ser(tBu)-L-Phe-L-Cys(Trt)-L-Met-L-Cys(Trt)-Gly-OH H-L-Val-L-Ser(tBu)-L-Phe-L-Cys(Trt)-L-Ile-L-Cys(Trt)-OH t

H-L-Cys(Trt)-L-Arg(Pbf)-L-Cys(Trt)-L-Phe-L-Ser( Bu)-Gly-OH

3

Macrocycliz. Yieldc/Purityd (%) 34a/98

Overall yielde (%) 32

7

3

38a/85

31

8

3

43a/82

42

3

a

27 /80

22

a

9

H-L-Cys(Trt)-L-Ile-L-Cys(Trt)-L-Phe-L-Arg(Pbf)-Gly-OH

10

3

26 /93

23

H-L-Cys(Trt)-L-Ile-L-Cys(Trt)-L-Phe-L-Glu(γ-tBu)-Gly-OH

11

1

46a/89 -59b/85

43a/55b

H-L-Cys(Trt)-L-Cys(Trt)-L-Ile-L-Phe-L-Glu(tBu)-Gly-OH

12

2

59a/83

51

3

a

34 /93

32

a

t

t

t

H-L-Ser( Bu)-L-Ile-L-Ser( Bu)-L-Phe-L-Glu( Bu)- Gly-OH t

13

H- L-Met-L-Phe-L-Phe-L-Glu( Bu)- L-Met-Gly-OH

14

2

80 /93

63

H-L-Cys(Trt)-L-Cys(Trt)-L-Phe-L-Ser(tBu)-Gly-OH

15

1

54a/94

51

H-L-Cys(Trt)-L-Cys(Trt)-L-Ile-L-Ser(tBu)-Gly-OH

16

1

69a/96

58

a

HBTU was used as coupling reagent for macrocyclization; b HATU was used as coupling reagent for macrocyclization; c for the purified final compounds; ddetermined by HPLC analysis; e yields for the linear peptides were based on the determination of the resin loading.

Table 1. Synthesis of macrocycles by methodology A. Using methodology B, Scheme 2, first Fmoc-L-Glu-OAll was loaded onto 2-CTC resin with an excess of DIPEA, and then a capping step with MeOH was realized. The peptide elongation for the synthesis of the linear peptides was according the Fmoc SPPS protocol used in methodology A. Lim and co-workers, reported the use of allyl-based terminal protecting groups prior to macrocyclization in their solid-phase total synthesis of coibamide A.16 In contrast, we decided to use the readily available, Fmoc protected amino acids at the N-termini and perform the removal of the allyl ester using [Pd(PPh3)4] in a solution of 10% piperidine in THF,24 which allows the preparation of

the desired peptide with the free

carboxylic group at C-terminal and the unprotected amino group at N-terminal.


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Scheme 2. General procedure (B): Fmoc-strategy SPPS and on resin macrocyclization. Then on-resin macrocyclization was realized using DIC/Cl-HOBt to obtain four cyclopeptides (1720), Figure 3. The macrocyclizations were completed overnight for the four cyclopeptides. In addition, no racemization was detected by analysis of the HPLC chromatograms and the NMR spectra of the crude,23 consequently the desired macrocyles were obtained in yields from 63 to 99%, and purities from 72 to 83%, Table 2. Further purification rendered the isolated macrocycles in yields from 46 to 80% and purities from 93 to 95%, Table 3. Contrary to our expectations the best macrocyclization yield was not for the synthesis of 17, 18 or 19 which their precursors present an N-terminal Gly with the advantage of non-steric hindrance. In the other hand, coupling between the amino group of Met and the carboxyl of Glu, to obtain 20, was a very efficient process as its crude was obtained in 99% yield and 83% purity. It is worthy to note, that compounds obtained by methodology B, present a free carboxylic acid which allows the synthesis of new derivatives, with different physicochemical properties for their possible use as drugs candidates.


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In order to determine if on-resin macrocyclization yield can be improved by the use of different coupling reagents, HATU/DIPEA and HBTU/DIPEA in DMF:CH2Cl2 (8:2), overnight, were tested to obtain compound 17, Figure 3. By the use of HATU, 17 was obtained in 100% yield but in lower purity (59%). In the other hand, using HBTU, it was obtained in 70 % yield and 80% purity showing that, there is no

significant difference with the use of DIC/Cl-HOBt.

Figure 3. Cyclohexapeptides synthesized in this work by Methodology B.

Peptide precursor

H-Gly-L-Cys(Trt)-L-Ile-L-Cys(Trt)-L-Phe-L-GluOH H-Gly-L-Cys(Trt)-L-Cys(Trt)-L-Ile-L-Phe-L-GluOH H-Gly- L-Ser(tBu)-L-Ile-L-Ser(tBu)-L-Phe-L-GluOH H-L-Met-Gly-L-Met-L-Phe-L-Phe-L-Glu-OH

Product

Crude

Time (h)

17

Yielda/Purityb (%) 68/81

Isolated Macrocycle Yielda/Purityb (%) 51/95

18

98/72

63/95

16

19

63/78

46/93

16

20

99/83

80/95

16

16


11


a

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Yield based on the determination of resin loading; bdetermined by HPLC analysis

Table 2. Synthesis of macrocycles by methodology B using DIC/Cl-HOBt.

Compounds 11, 12, 13 and 14 which were obtained by solution macrocyclization differ from 17, 18, 19 and 20, respectively, by the presence of tBu protecting group in the γ carboxylic of Glu. In order to compare both procedures, A and B, yield and purity for the purified macrocycles are taken into account, Table 3. For the last three sequences (compounds 12 and 18, 13 and 19, 14 and 20), methodology B resulted in a more efficient process than methodology A. In the case of 11, it was obtained in a higher yield but in poor purity than 17. Moreover, it is worth to note, that 17, 18, 19 and 20 were obtained by draining and washing the resin in acceptable purity, (see Table 2). The highest yield of each methodology A and B is for compounds 14 and 20, respectively, which present the same sequence. In the same manner, the lowest yield for methodology A and B is for 13 and 19, respectively. The above results, are in agreement with previous reports indicating that yield of the cyclization step is strongly sequence dependent.24

a

Macrocycle Methodology A

Yield /Purity

Macrocycle Methodology B 17

Yield/Purity

11

59a/85

12

59/83

18

63/95

13

34/93

19

46/93

14

80/93

20

80/95

51/95

HATU was used as coupling reagent for macrocyclization

Table 3. Comparison between methodology A and B


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The novel synthesized and purified macrocycles were evaluated in vitro as antimalarial agents. The anti-plasmodium activities were determined against the chloroquine-resistant K1 strain of P. falciparum by using the [3H]-hypoxanthine incorporation method reported by Desjardins et al.,25 the results are shown in Table 4. As compounds 6, 7, 11, 16, 17 and 18 showed sub-micromolar EC50 as the previously reported compounds 4 and 5,5 cytotoxicity evaluation against HepG2 cells was performed. The compounds were not toxic, showing EC50> 250 µM. The substitution of the Ser(tBu) in 4, by a Glu(tBu) or Glu in 11 and 17 respectively has little effect in the bioactivity. By comparison of the EC50 for compound 11 and 17, it could be concluded that the tBu ester and the free carboxylic acid in Glu, have the same effect in the antiplasmodium activity for this amino acids sequence. In contrast, 18, which differs from 12 by the presence of a Glu instead of a Glu(tBu), respectively, resulted in a more active compound. The other macrocycles containing Glu, resulted inactive against P. falciparum. The substitution of the two Cys(Trt) in 17 by Ser(tBu) in 19 impacts negatively in the biological activity by decreasing 100-folds the potency towards P. falciparum. The substitution of Ile or Ser(tBu) in 4 by Arg (Pbf) in 9 or 10, respectively, decreases the activity. The removal of Ile in cyclohexapeptide 4 to obtain cyclopentapeptide 16 decreases the activity in almost 4-fold (EC50 = 0.19 and 0.73 µM respectively). Macrocycle 6, which presents two Gly instead of Ile or Phe presents in 4, is the most active compound reported by our group against P. falciparum K1, EC50 = 28 nM. This evaluation was repeated two times in order to confirm it. Moreover, it was not toxic against HepG2 cells (EC50> 250 µM) showing a selectivity index (SI) against the parasite > 8900. Even though, it is less active than artesunate, EC50 = 3 nM, it is worthy to note that its activity is in the same order of magnitude than artemisinin, EC50 = 20 nM.


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Compound

EC 50

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Compound

EC 50

P. falciparum K1(µM)a

P. falciparum K1(µM)a

6

0.028 ± 0.006

14

4.6 ± 0.8

7

0.68 ± 0.04

15

8.4 ± 0.7

8

6.50 ± 0.05

16

0.73± 0.06

9

16.8 ± 0.9

17

0.47 ± 0.04

10

43.1 ± 0.8

18

0.42 ± 0.05

11

0.42 ± 0.03

19

>50

12

>10

20

>50

13

3.8 ± 0.6

a

Control: chloroquine: EC50 = 0.47 µM, Artemisinin: EC50 = 0.02 µM, Artesunate: EC50 = 0.003 µM; nt: not tested

Table 4. Anti-plasmodium activities of macrocycles CONCLUSIONS Taking into account the results for both procedures, we can conclude that methodology B, on resin macrocyclization, is the most convenient as large quantities of the desired compounds can be obtained in higher yield and acceptable purity. Comparing our results for on-resin peptide cyclization, with those obtained by other authors, our procedure has high cyclization efficiency.8,

9, 24, 26

The cyclopeptides

obtained by this methodology present a free carboxyl group in the side chain of one of the amino acids which is an excellent point to mark or to provide them the required properties for their possible use as drugs candidates.


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The two methodologies allow working with the same protecting groups, as the same strategy was used. However, methodology A allows to obtain more diverse cyclic peptides as the anchoring to the resin is performed by the C-terminal carboxyl group, and contrary to method B, it is not necessary to have amino acids containing a lateral chain with carboxyl group in their sequence. As a consequence, the selection of the procedure A or B, depends of the desired cyclopeptides. If methodology B can be used, cyclopeptides possessing very diverse physicochemical properties could be obtained quickly and in good yields, facilitating the approaching to the best candidate with desirable biological activity. Six of the obtained compounds showed sub-micromolar activity against P. falciparum K1. Two of them containing a free carboxylic group from Glu, 17 and 18, allow the preparation of derivatives or more soluble salts. The obtained cyclic pentapeptides are less active than cyclic hexapeptides. In addition, we have prepared a very active and selective compound, 6, EC50 = 28 nM and concluded that substitutions of the larger hydrophobic amino acids Phe, Met, Ile, presents in 4 and 5 by Gly maintaining one Thr and two Cys, have a great impact in the biological activities. New compounds presenting this structural requirement will be prepared for additional evaluations and will be reported in the due course. As compound 6 is active in the nanomolar scale, it would be selected as candidate for further investigations on mechanism of action.

EXPERIMENTAL PROCEDURES Optical rotation was measured using a Kruss Optronic GmbH P8000 polarimeter with a 0.5 mL cell. IR spectra were recorded on a Shimadzu FTIR 8101A spectrophotometer. 1H NMR and

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C NMR spectra

were recorded on Bruker Advance DPX-400. Chemical shifts are related to TMS as an internal standard. High resolution mass spectra (HRMS) were obtained on a micro Q-TOF (ESI) (Bruker Daltonics) and


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LTQ-FT Ultra (NanoESI) (Thermo Scientific). Flash column chromatography was carried out with Silica gel 60 (J.T. Baker, 40 µm average particle diameter). All solution phase reactions and chromatographic separations were monitored by TLC, conducted on 0.25 mm Silica gel plastic sheets (Macherey/Nagel, Polygram_ SIL G/UV 254). TLC plates were analyzed under 254 nm UV light, iodine vapor, p-hydroxybenzaldehyde spray or ninhydrine spray. Yields are reported for chromatographically and spectroscopically (1H and 13C NMR) pure compounds. Analytical HPLC was performed on a Shimadzu (LC-10AT Pump) equipped with a Waters µBondapakTM C18 column (150 x 3.9 mm, 10 µm) and a SPD20A Prominence UV/Vis detector. All solvents were purified according to literature procedures.27 All reactions were carried out in dry, freshly distilled solvents under anhydrous conditions unless otherwise stated.

SPPS general procedure for elongation of the peptide chain The synthesis was done in a plastic syringe equipped with Teflon filters. The 2-chlorotritylchloride resin (100-300 mesh, 1.20 mmol/g) was swelled in CH2Cl2 (3X30 sec). A solution of the first protected amino acid (Fmoc-AA-OH 2.0 eq) in CH2Cl2 and DIPEA (3.0 eq) was gently shaken for 10 min, then an extra 7.0 eq. of DIPEA was added and shaking was continued for 45 min. MeOH (0.8 mL/g of resin) was then added in order to cap unreacted functional groups on the resin; the mixture was then shaken for 10 min. The resin was filtered, and then washed with CH2Cl2 (X3), DMF (X3) and CH2Cl2 (X3). The Nterminus was deprotected using 20% piperidine in DMF (2X 5 min and 1X 10 min). The resin was then washed with DMF (X3), CH2Cl2 (X3) and DMF (X3). A solution of Fmoc-AA-OH (3.0 eq.) and DIPEA (6 eq.) in DMF was added to the resin, followed by a solution of HBTU or HCTU (2.9 eq) in DMF; or,


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if the amino acid is Fmoc-Cys(Trt)-OH, a solution of Fmoc-AA-OH (3.0 eq.), DIC (2.9 eq.) and ClHOBt (2.9 eq) in DMF was added to the resin. The mixture was stirred for 90 min. After the coupling was completed, the resin was washed with DMF (X3) and CH2Cl2 (X3). Deprotection and coupling cycles were repeated with the appropriate amino acids to provide the desired compound. The peptide was cleaved from the resin by treatment with 1% TFA in CH2Cl2 for 2-3 minutes at room temperature followed by filtration and collection of the filtrate in MeOH. The treatment was repeated three times and then the resin washed with CH2Cl2 (X5). Solvents were removed in vacuo to obtain the crude peptide. General procedure for macrocyclization in solution phase. Macrocyclization reaction was performed in diluted conditions (1-5 mM) using HBTU or HATU (1.5 eq.), DIPEA (3 eq.), 4-DMAP (catalytic) in dried CH2Cl2 at room temperature during 1-3 days. The reaction mixture was washed with HCl 5%, saturated aqueous NaHCO3, dried over MgSO4, filtered and concentrated in vacuo. The crude was purified by flash chromatography to obtain the macrocycle. Cyclo-L-Cys(Trt)-L-Ile-L-Cys(Trt)-L-Phe-L-Glu(γ-tBu)-Gly (11) The trifluoroacetate salt of H2N-L-Cys(Trt)-L-Ile-L-Cys(Trt)-L-Phe-L-Glu(γ-tBu)-Gly-OH was obtained following the general SPPS procedure, yielded 537 mg (93% yield) as a white solid. The purity (88 %) was determined by HPLC (linear gradient: 8 to 100% acetonitrile in H2O/ 0.003m TFA over 20 min; flow rate = 1.5 mL/min; tR = 9.64 min). Macrocyclization reaction was performed following the general procedure (dilution 5 mM, 1 day), starting from the trifluoroacetate salt of the amino acid H2N-L-Cys(Trt)-L-Ile-L-Cys(Trt)-L-Phe-L-Glu(γt

Bu)-Gly-OH (150 mg, 0.124 mmol). Further purification by flash chromatography, rendered the desired

macrocycle (88 mg, 0.074 mmol) in 59% yield and 85% purity. Cyclo-L-Cys(Trt)-L-Ile-L-Cys(Trt)-L-Phe-L-Glu(γ-tBu)-Gly (11): White solid (55% overall yield). Rf= 0.3 (EtOAc:PE, 3:1). [α]D25= -52.5 (c 0.25, DCM). 1H NMR (400 MHz, CDCl3) δ (ppm): 0.81-


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0.92 (m, 6H), 1.00-1.14 (m, 1H), 1.47 (s, 9H), 1.41-1.52 (m, 1H), 1.79-2.03 (m, 2H), 2.11-2.25 (m, 3H), 2.45 (dd, J=5.4, 12.8 Hz, 1H), 2.56 (dd, J=5.0, 13.2 Hz, 1H), 2.69-2.82 (m, 2H), 2.97-3.12 (m, 2H), 3.14-3.29 (m, 2H), 3.95 (t, J=7.4 Hz, 1H), 4.00-4.12 (m, 2H), 4.22 (m, 1H), 4.27-4.36 (m, 1H), 6.436.54 (m, 1H), 6.68-6.79 (m, 1H), 6.89-7.00 (m, 1H), 7.08-7.48 (m, 38H). 13C NMR (100 MHz, CDCl3) δ(ppm): 11.2, 15.5, 25.0, 25.4, 28.2 (3C), 31.7, 31.9, 32.5, 36.1, 36.5, 43.9, 52.9, 53.5, 54.3, 56.3, 58.3, 67.4, 67.5, 80.6, 127.0 (6C), 128.0, 128.1 (6C), 128.2 (6C), 128.8 (2C), 129.2 (2C), 129.5 (12C), 136.9, 144.2 (6C), 170.0, 170.1, 170.4, 171.3, 171.4, 172.3, 172.4. HRMS m/z calc. for C70H76N6O8S2 ([M+Na]+) 1215.5058, found 1215.5062.

General procedure for on-resin macrocyclization. Macrocyclization reaction was performed on-resin using DIC (4.0 eq.), Cl-HOBt (4.0 eq.), 4-DMAP (catalytic) in dried DMF:CH2Cl2 (8:2) at room temperature overnight. After the macrocyclization was completed, the resin was filtered and then washed with DMF (X3), CH2Cl2 (X3). The macrocycle was cleaved from the resin by treatment with 1% TFA in CH2Cl2 for 2-3 minutes at room temperature followed by filtration and collection of the filtrate in MeOH. The treatment was repeated three times and then the resin washed with CH2Cl2 (X5). Solvents were removed in vacuo to obtain the crude macrocycle. Cyclo-Gly-L-Cys(Trt)-L-Ile-L-Cys(Trt)-L-Phe-L-Glu (17) Cyclopeptide was obtained following the general SPPS and on-resin macrocyclization procedures, yielded 139 mg (68% yield) of the crude macrocycle as a yellow solid. The purity (81%) was determined by HPLC (linear gradient: 8 to 100% acetonitrile in H2O/ 0.003M TFA over 20 min); flow


18

Page 19 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


rate = 1.5 mL/min; tR = 10.13 min). Further purification by flash chromatography, rendered the desired macrocycle (104 mg) in 51% yield and 95% purity. Cyclo-Gly-L-Cys(Trt)-L-Ile-L-Cys(Trt)-L-Phe-L-Glu (17): Yellow solid, tR = 10.13 min (linear gradient: 8 to 100% acetonitrile in H2O/ 0.003M TFA over 20 min). [α]D25= -34.6 (c 0.56, MeOH). 1H NMR (400 MHz, (CD3)2CO) δ (ppm): 0.79-0.91 (m, 6H), 1.01-1.13 (m, 1H), 1.42-1.56 (m, 2H), 1.882.00 (m, 3H), 2.17-2.28 (m, 1H), 2.30-2.38 (m, 2H), 2.49-2.59 (m, 1H), 2.62-2.72 (m, 2H), 2.94-3.04 (m, 2H), 3.05-3.15 (m, 1H), 3.16-3.27 (m, 1H), 3.37 (d, J=15.1 Hz, 1H), 4.08 (d, J=6.8 Hz, 1H), 4.114.16 (m, 1H), 4.19 (d, J=15.1 Hz, 1H), 4.25-4.39 (m, 1H), 4.36-4.43 (m, 1H), 7.12-7.45 (m, 41H). 13C NMR (100 MHz, (CD3)2CO) δ(ppm): 10.7, 15.1, 24.6, 26.1, 30.0, 31.9, 33.2, 35.7, 37.0, 38.7, 43.4, 52.5, 53.5, 54.4, 56.6, 57.9, 66.6, 66.9, 126.4, 126.8 (6C), 128.0 (12C), 128.4 (2C), 129.1 (2C), 129.5 (12C), 138.4, 144.6 (6C), 169.5, 169.6, 170.9, 171.4, 174.5, 171.8, 173.6. HRMS m/z calc. for C66H68N6O8S2 ([M+K]+) 1175.4177, found 1175.3783. Supporting

Information.

General

characterization data (1H and

methods,

synthetic

procedures,

anti-plasmodium

assay,

13

C NMR and HRMS) and HPLC chromatograms for the obtained

macrocycles are included in the SI file. Corresponding Author * Cátedra de Química Farmacéutica, (DQO), Facultad de Química, Universidad de la República, Gral. Flores 2124, CP 11800, Montevideo, Uruguay. [email protected]

Funding Sources British Embassy in Uruguay, ANII, CSIC-Grupos, PEDECIBA


19


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ACKNOWLEDGMENT This work was supported by Grants from British Embassy in Uruguay, CSIC Grupos 983 (Universidad de la República) and PEDECIBA (Uruguay). The authors acknowledge the collaboration of Dr. Vanessa Yardley and Lindsay Stewart from London School of Hygiene and Tropical Medicines for performing the biological evaluations, a PhD fellowship from ANII (Agencia Nacional de Investigación e Innovación) (Catherine Fagundez), and an internship supported by PEDECIBA References

1

a) Giordanetto, F.; Kihlberg J. Macrocyclic drugs and clinical candidates: what can medicinal chemists

learn from their properties? J. Med. Chem. 2014, 57, 278. b) Mallinson, J.; Collins, I. Macrocycles in new drug discovery. Future Med. Chem. 2012, 4, 1409. c) Peña, S.; Scarone, L.; Serra, G. Macrocycles as potential therapeutic agents in neglected diseases. Future Med. Chem. 2015, 7, 355. d) Driggers, E.; Hale, S.; lee, J; Terret, N. Nat. Rev. Drug. Discov. The exploration of macrocycles for drug discovery-an underexploited structural class. 2008, 7, 608. e) Davies, J. S. The cyclization of peptides and depsipeptides. J. Pept. Sci. 2003, 9, 471. f) Skropeta, D.; Jolliffe, A. K.; Turner, P. Pseudoprolines as removable turn inducers: tools for the cyclization of small peptides. J. Org. Chem. 2004, 69, 8804. g) Wipf, P. Synthetic studies of biologically active marine cyclopeptides. Chem. Rev. 1995, 95, 2115. 2

a) Baraguey, C.; Blond, A.; Cavelier, F.; Pousset, J.-L.; Bodo, B.; Auvin-Guette, C. Isolation, structure

and synthesis of mahafacyclin B, a cyclic heptapeptide from the latex of Jatropha mahafalensis. J. Chem. Soc., Perkin Trans. 2001, 1, 2098. b) Baraguey, C.; Auvin-Guette, C.; Blond, A.; Cavelier, F; Lezenven, F.; Pousset, J. L.; Bodo, B. Isolation, structure and synthesis of chevalierins A, B and C, cyclic peptides from the latex of Jatropha chevalieri. J. Chem. Soc., Perkin Trans. 1998, 1, 3033. 3

a) Kuntz, D. A.; Osowski, R.; Schudok, M.; Wierenga, R., Muller, K., Kessler, H.; Opperdoes, F. R.

Inhibition of triosephosphate isomerase from. Trypanosoma brucei with cyclic hexapeptides. Eur. J. Biochem. 1992, 207,441. b) Callens, M.; Van Roy, J.; Zeelen, J. Ph.; Borchert, T. V.; Nalis, D.; Wierenga, R.; Opperdoes, F. R. Selective interaction of glycosomal enzymes from Trypanosoma brucei with hydrophobic cyclic hexapeptides. Biochem. Biophis. Res. Comm. 1993, 195, 667.


20

Page 21 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

4


a) Portmann, C.; Blom, J. F.; Gademann, K.; Jüttner, F. Aerucyclamides A and B: isolation and

synthesis of toxic ribosomal heterocyclic peptides from the cyanobacterium Microcystis aeruginosa PCC 7806. J. Nat. Prod. 2008, 71, 1193. b) Portmann, C.; Blom, J. F.; Kaiser, M.; Brun, R.; Jüttner, F.; Gademann, K. Isolation of aerucyclamides C and D and structure revision of microcyclamide 7806A: heterocyclic ribosomal peptides from Microcystis aeruginosa PCC 7806 and their antiparasite evaluation. J. Nat. Prod. 2008, 71,1891. 5

Peña, S.; Fagundez, C.; Medeiros, A.; Comini, M.; Scarone, L.; Sellanes, D.; Manta, E.; Tulla-Puche,

J.; Albericio, F.; Stewart, L.; Yarley, V.; Serra, G. Synthesis of cyclohexapeptides as antimalarial and anti-trypanosomal agents. Med. Chem. Commun. 2014, 5, 1309. 6

a) Merrifield, R. Solid Phase Peptide Synthesis. I. The Synthesis of a Tetrapeptide. J. Am. Chem. Soc.

1963, 85, 2149. b) Merrifield, R., Solid Phase Synthesis. Science, 1986, 232, 341. c) Albericio, F., Zompra, A., Galanis, A., Werbitzky, O. Manufacturing peptides as active pharmaceutical ingredients. Future Med. Chem. 2009, 1, 361. 7

Malesevic,

M.; Strijowski, U.; Bachle, D.; Sewald, N.

An improved method for the solution

cyclization of peptides under pseudo-high dilution conditions. J. Biotech. 2004, 112, 73. 8

Kim, J.; Hong, I-K.; Kim, H-J.; Jeong, H-J.; Choi, M-J; Yoon, C-N.; Jeong, J-H. Efficient

macrocyclization for cyclicpeptide using solid-phase reaction. Arch. Pharm. Res. 2002, 25, 801. 9

a) Scott, L. T.; Rebek, J.; Ovsyanko, L. Organic chemistry on the solid phase. Site-site interactions on

functionalized polystyrene. J. Am. Chem. Soc. 1977, 99, 625. b) Mazur, S.; Jayalekshmy, P. Chemistry of polymer-bound o-benzyne. Frequency of encounter between substituents on crosslinked polystyrenes. J. Am. Chem. Soc. 1979, 101, 677. 10

a) Peña, S.; Scarone, L.; Manta, E.; Stewart, L.; Yardley, V.; Croft, S.; Serra, G. Synthesis of a

Microcystis aeruginosa predicted metabolite with antimalarial activity. Bioorg. Med. Chem. Lett., 2012, 22, 4994. b) Peña, S.; Scarone, L.; Manta, E.; Serra, G. First Total Synthesis of Aerucyclamide B. Tetrahedron Lett., 2013, 54, 2806. c) Peña, S.; Scarone, L.; Medeiros, A.; Manta, E.; Comini, M.; Serra, G. Synthesis of precursors and macrocycle analogs of aerucyclamides as anti-trypanosomal agents. Med. Chem. Commun., 2012, 3, 1443. 11

a) Isied, S.; Kuehn, C. G.; Lyon, J. M.; Merrifield, R. B. Specific peptide sequences for metal ion

coordination. 1. Solid-phase synthesis of cyclo-(Gly-His)3. J. Am. Chem. Soc. 1982, 104, 2632. b)


21


Page 22 of 23

Rovero, P.; Quartara, L.; Fabbri, G. Synthesis of cyclic-peptides on solid support. Tetrahedron Lett. 1991, 32, 2639. c) Trzeciak, A.; Bannwarth, W. Synthesis of ‘head-to-tail’ cyclized peptides on solid support by Fmoc chemistry. Tetrahedron Lett. 1992, 33, 4557. d) Kates, S. A.; Solé, N.A.; Johnson, C. R.; Hudson, D.; Barany, G.; Albericio, F. A novel, convenient, three-dimensional orthogonal strategy for solid-phase synthesis of cyclic peptides. Tetrahedron Lett. 1993, 34, 1549. e) Alsina, J.; Chiva, C.; Ortiz, M.; Rabanal, F.; Giralt, E.; Albericio, F. Active carbonate resins for solid phase synthesis through the anchoring of a hydroxyl function. Synthesis of cyclic and alcohol peptides. Tetrahedron Lett. 1997, 38, 883. f) Alsina, J.; Rabanal, F.; Giralt, E.; Albericio, F. Solid-phase synthesis of “Head-to-Tail” cyclic peptides vía lysine side-chain anchoring. Tetrahedron Lett. 1994, 35, 9633. g) García, O.; Nicolás, E.; Albericio, F. Solid-phase synthesis: A linker for side-chain anchoring of arginine. Tetrahedron Lett. 2003, 44, 5319. h) Cabrele, C.; Langer, M.; Beck- Sickinger, A. Amino acid side chain attachment approach and its application to the synthesis of tyrosine-containing cyclic peptides. J. Org. Chem. 1999, 64, 4353. 12

a) Myer, J. P.; Yan, L. Z.; Edwards, P.; Flora, D. Synthesis of cyclic peptides through hydroxyl side-

chain anchoring. Tetrahedron Lett. 2004, 45, 923. 13

Wang, P.; Miranda, L.P. Fmoc-Protein synthesis: preparation of peptide thioesters using a side-chain

anchoring strategy. Int. J. Pept. Res. Therap. 2005, 11, 117. 14

Wong, C.; Ficht, S.; Payne, R.; Guy, R. Solid-phase synthesis of peptide and glycopeptide thioesters

through side-chain-anchoring strategies. Chem. Eur. J. 2008, 14, 3620. 15

a) Schutkowski, M.; Bernhardt, A.; Drewello, M. The solid-phase synthesis of side-chain-

phosphorylated peptide-4-nitroanilides. J. Peptide Res. 1997, 50,143. b) Rizzi, L.; Cendic, K.; Vaiana, N.; Romeo, S. Alcohols immobilization onto 2‐chlorotritylchloride resin under microwave irradiation. Tetrahedron Lett. 2011, 52, 2808. 16

Sable, G. A.; Park, J.: Kim, H.: Lim, S.-J.: Jang, S.; Lim, D. Solid-phase total synthesis of the

proposed structure of coibamide A and its derivative: highly methylated cyclic depsipeptides Eur. J. Org. Chem. 2015, 7043. 17

Barlos, K.; Gatos, D.; Schäfer, W. Synthesis of prothymosin a (ProTo)—a protein consisting of 109

amino acid residues. Angew. Chem. Int. Ed. Engl. 1991, 30, 590.


22

Page 23 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

18


López-Maciá, A.; Jiménez, J. C.; Royo, M.; Giralt, E.; Albericio, F. Synthesis and structure

determination of kahalalide F (1,2). J. Am. Chem. Soc. 2001, 123, 11398. 19

a) Hood, A.C; Fuentes, G.; Patel, H.; Page, K.; Menakuru, M.; Park, J. H. Fast conventional Fmoc

solid-phase peptide synthesis with HCTU. J. Pept. Sci. 2008, 14, 97. b) Marder, O.; Albericio, F. Industrial application of coupling reagents in peptides. Chim. Oggi 2003, 21, 6. c) “Fmoc Solid Phase Peptide Synthesis, A Practical Approach”, Chan, W. C.; White P. D. Eds., Oxford University Press, 2004, 9.. 20

a) Schmidt, U.; Langner, J. Cyclotetrapeptides and cyclopentapeptides: occurrence and synthesis. J.

Pep. Res. 1997, 49, 67. b) White, C.; Yudin, A. Contemporary strategies for peptide macrocyclization. Nat. Chem. 2011, 3, 509. 21

Humphrey, J.; Chamberlin, A. Chemical synthesis of natural product peptides: coupling methods for

the incorporation of noncoded amino acids into peptides. Chem. Rev. 1997, 97, 2243. 22

See Supporting Information.

23

Thakkar, A.; Trinh, T. B.; Pei, D. Global analysis of peptide cyclization efficiency. ACS Comb.

Sci. 2013, 15, 120. 24

Rand, A. C.; Leung, S. F.; Eng, H.; Rotter, C. J.; Sharma, R.; Kalgutkar, A. S.; Zhang, Y.; Varma,

M.; Farley, K. A.; Khunte, B.; Limberakis, C.; Price, D. A.; Liras, S.; Mathiowetz, A. M.; Jacobson, M. P.; Scott, R. Optimizing PK properties of cyclic peptides: the effect of side chain substitutions on permeability and clearance. Med. Chem. Commun. 2012, 3, 1282. 25

a) Desjardins, R. E.; Canfield, C. J.; Haynes D. E.; Chulay, J. D. Quantitative assessment of

antimalarial activity in vitro by a semiautomated microdilution technique. Antimicrob. Agents Chemother. 1979, 16, 710. b) See Supporting Information. 26

Sable, G. A.; Park, J.; Lim, S. J.; Lim, D. Solid-phase total synthesis of amide analogues of coibamide

a: azacoibamide a and O-desmethyl azacoibamide A. Bull. Korean Chem. Soc. 2016, 37, 330. 27

Perrin, D. D.; Armarego, W. L. F. “Purification of Laboratory Chemicals”, 3th Ed. Pergamon Press, Oxford, 1988.


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