molecules

Review Recent Reports of Solid-Phase Cyclohexapeptide Synthesis and Applications

Allan M. Prior, Taylor Hori, Ashriel Fishman and Dianqing Sun * ID

Department of Pharmaceutical Sciences, The Daniel K. Inouye College of Pharmacy, University of Hawaii at Hilo, 34 Rainbow Drive, Hilo, Hawaii, HI 96720, USA; [email protected] (A.M.P.); [email protected] (T.H.); [email protected] (A.F.) * Correspondence: [email protected]; Tel.: +1-808-933-2960

Academic Editor: Viktor Krchnak


Received: 16 May 2018; Accepted: 16 June 2018; Published: 18 June 2018


Abstract: Macrocyclic peptides are privileged scaffolds for drug development and constitute a significant portion of macrocyclic drugs on the market today in fields spanning from infectious disease to oncology. Developing orally bioavailable peptide-based drugs remains a challenging task; however, macrocyclization of linear peptides can be an effective strategy to improve membrane permeability, proteolytic stability, oral bioavailability, and overall drug-like characteristics for this class. Significant advances in solid-phase peptide synthesis (SPPS) have enabled the efficient construction of macrocyclic peptide and peptidomimetic libraries with macrolactamization being performed on-resin or in solution phase. The primary goal of this review is to summarize solid-phase cyclohexapeptide synthesis using the on-resin and solution-phase macrocyclization methodologies published since 2013. We also highlight their broad applications ranging from natural product total synthesis, synthetic methodology development, and medicinal chemistry, to drug development and analyses of conformational and physiochemical properties.

Keywords: cyclohexapeptide; solid-phase synthesis; total synthesis; macrocyclization; macrolactamization; structure–activity relationship; natural products; on-resin cyclization; solution-phase cyclization

1. Introduction Macrocyclic peptides constitute a significant portion of macrocyclic drugs on the market today and are used in many fields ranging from infectious disease to oncology [1,2]. Recently, reports of antibacterial macrocyclic peptide natural products have demonstrated that macrocyclic peptides are privileged scaffolds for drug development [3]. Historically, the pharmaceutical industry has been cautious with developing macrocyclic drugs because of concerns of higher cost and synthetic challenges associated with lead optimization and scale-up campaigns [1]. Head-to-tail cyclization of linear peptides of three to eight amino acids can be challenging, particularly for peptides containing exclusively the L-configuration [4]. The formation of linear as well as cyclic dimers and oligomers during the cyclization step can compete with head-to-tail cyclization [5]. Additionally, epimerization of the C-terminal amino acid is commonly encountered during the C-terminal activation step before the cyclization reaction takes place. To date, significant progress has been made in the synthesis of macrocycles, making their efficient construction more feasible [1]. A common strategy to improve head-to-tail cyclization of small peptides involves the incorporation of the “turn-inducing” functionality into a linear peptide sequence such as glycine, proline, pseudoproline, N-alkyl, or D-amino acid residue [4,6–8]. In the case of the pseudoproline method, a pseudoproline residue, synthesized by the condensation of serine, threonine, or cysteine with an aldehyde or ketone, is

Molecules 2018, 23, 1475; doi:10.3390/molecules23061475 www.mdpi.com/journal/molecules Molecules 2018, 23, 1475 2 of 25 incorporated into the peptide sequence pre-cyclization. The pseudoproline residue can then be deprotected post cyclization [6,7]. Coupling reagents in the azabenzotriazole class are the most commonly employed for cyclization of linear peptide precursors, as they give faster rates of cyclization with lower amounts of epimerization, typically less than 10% [4]. Moreover, linear peptides with the D-configuration at their C-terminal residue have favorable cyclization kinetics [4]. This may be a result of less steric hindrance during the formation of the peptide bond occurring between the D- and L-amino acids, particularly when bulky side chains are present. Cyclodepsipeptides, with one ester linkage in the macrocyclic backbone [9], have been utilized in the epimerization-free synthesis of cyclopeptides by employing a key O-N-acyl migration reaction at a serine residue [10,11]. Macrocyclic peptides are known to possess some improved membrane permeability [12,13], proteolytic stability [13–16], oral bioavailability [17,18], and overall drug-like characteristics [13,19,20] over their linear analogues. Moreover, peptide macrocyclization is a way of locking the peptide sequence in a β-strand conformation [15,16], a conformation often recognized by their enzyme targets, such as proteases [15,16,21]. Macrocycles are not completely rigid but still have a degree of flexibility, which facilitates interactions with their receptors [1]. Additionally, the entropic cost of receptor binding may be reduced for macrocyclic drugs compared to their linear equivalents as a result of conformational pre-organization [1]. N-Methylation of the cyclic peptide backbone has been shown in some cases to improve peptide metabolic stability [14,22] and oral bioavailability [23] as a result of changes in peptide conformation or steric hindrance [22]. The immunosuppressant drug ciclosporin is a macrocyclic undecapeptide bearing seven N-methylated motifs and can be administered as an oral formulation [1,22]. Modification of the cyclohexapeptide backbone to form cyclohexapeptoids can, in some cases, lead to an increase to cell permeability [24]. Most cyclic peptide drugs on the market today are administered parenterally, and only few are orally bioavailable [1], highlighting an ongoing challenge. For this reason, the synthesis and evaluation of novel cyclopeptide scaffolds to expand our understanding of their pharmacokinetic (PK) properties is still at the forefront of many research programs [2,12,23]. Solid-phase peptide synthesis (SPPS) was first described in 1963 by Merrifield [25], whereby a growing linear peptide was synthesized in a step-wise fashion while covalently attached to a solid support (resin). In general, excess reagents are used in solid-phase synthesis to help to drive reactions to completion. The solid-supported methodology allows excess reagents to be removed after each step by employing a simple filtration, and the final desired product is obtained after cleavage from the resin [25]. To date, many new advances in SPPS have allowed for the rapid and efficient construction of peptide or peptidomimetic [26] libraries for subsequent biological evaluation and high-throughput screening [27,28]. The current literature base surrounding natural and synthetic macrocyclic peptides is extensive. The structures of these macrocycles have a wide-ranging incorporation of natural and unnatural amino acids as well as different ring sizes. Interestingly, cyclohexapeptides—macrocyclic peptides comprising six amino acids in the ring—are one of the most ubiquitous classes of macrocyclic peptides synthesized by SPPS. Macrocyclic hexapeptides can be obtained directly from on-resin or solution-phase cyclization following the cleavage of a linear hexapeptide precursor from resin (Figure1). This brief review summarizes the preceding five years’ worth of solid-phase cyclohexapeptide synthesis and its applications, which span from natural product total synthesis, synthetic methodology development, and medicinal chemistry, to drug development and analyses of conformational and physiochemical properties. Molecules 2018, 23, 1475 3 of 25 Molecules 2018, 23, x FOR PEER REVIEW 3 of 25 Molecules 2018, 23, x FOR PEER REVIEW 3 of 25

Figure 1 1.. (A(A).). Illustration Illustration of of solid solid-phase-phase cyclohexapeptide cyclohexapeptide synthesis. synthesis. (B). (RBesins). Resins highlighted highlighted in this in Figurereview.this review. 1 . (A). Illustration of solid-phase cyclohexapeptide synthesis. (B). Resins highlighted in this review. 2. Solid-Phase Synthesis of Cyclohexapeptides Using Solution-Phase Cyclization 2. Solid-Phase Synthesis of Cyclohexapeptides Using Solution-Phase Cyclization 2. Solid-Phase Synthesis of Cyclohexapeptides Using Solution-Phase Cyclization In 2013, Wu et al. reported the structures of two new cyclohexapeptides, nocardiamides A (1) In 2013, Wu et al. reported the structures of two new cyclohexapeptides, nocardiamides A (1) and BIn (2013,2), that Wu were et al. isolated reported from the a structures culture broth of two of anew CNX037 cyclohexapeptides, strain of actinomycete nocardiamides, which A is ( 1a) and B (2), that were isolated from a culture broth of a CNX037 strain of actinomycete, which is a andNocardiopsis B (2), that species were (Figure isolated 2 ) from[29]. a culture broth of a CNX037 strain of actinomycete, which is a Nocardiopsis species (Figure2)[29]. Nocardiopsis species (Figure 2) [29].

Figure 2. Structures of naturally occurring nocardiamides A (1) and B (2). FigureFigure 2 2.. StructuresStructures of of naturally occurring nocardiamides nocardiamides A ( 1) and B (2). Their structures were confirmed through an independent total synthesis, which helped to confirmThe their structures location of were the two confirmed D- and L through-Val resi dues. an independent The synthesis total of 1 synthesisand 2 is shown, which in helpedScheme to1. confirmTheir synthesis the location used of 2 -thechlorotrityl two D- and chloride L-Val re(2s-CTCidues.) resin The synthesisas a solid of support 1 and 2 tois shownconstruct in Schemethe linear 1. Theirhexapeptides synthesis 3 andused 4 using2-chlorotrityl classical chloride SPPS, followed (2-CTC )by resin cleavage as a solidfrom supportthe resin to using construct a trifluoroacetic the linear hexapeptides 3 and 4 using classical SPPS, followed by cleavage from the resin using a trifluoroacetic

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Their structures were confirmed through an independent total synthesis, which helped to confirm the location of the two D- and L-Val residues. The synthesis of 1 and 2 is shown in Scheme1. Their synthesis used 2-chlorotrityl chloride (2-CTC) resin as a solid supportMolecules 2018 to,construct 23, x FOR PEER the REVIEW linear hexapeptides 3 and 4 using classical SPPS, followed by cleavage4 of 25 from the resin using a trifluoroacetic acid TFA-based cleavage cocktail solution. Subsequent acid TFA-based cleavage cocktail solution. Subsequent N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1- N,N,N0,N0-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU)-mediated yl)uronium hexafluorophosphate (HBTU)-mediated solution-phase cyclization of 3 or 4 provided solution-phase cyclization of 3 or 4 provided cyclohexapeptides 1 and 2 in 7.2% and 10.7% yields, cyclohexapeptides 1 and 2 in 7.2% and 10.7% yields, respectively. Antimicrobial evaluation of 1–4 respectively. Antimicrobial evaluation of 1–4 was performed against Escherichia coli, Staphylococcus was performed against Escherichia coli, Staphylococcus aureus, Enterococcus faecalis, Bacillus aureus, Enterococcus faecalis, Bacillus thuringiensis, Bacillus subtilis, Micrococcus luteus, and Candida albicans; thuringiensis, Bacillus subtilis, Micrococcus luteus, and Candida albicans; however, only negligible however, only negligible activity was found. Compounds 1 and 2 were also screened against a human activity was found. Compounds 1 and 2 were also screened against a human colon carcinoma cell colon carcinoma cell line, HCT-116, and no cell cytotoxicity was observed [29]. line, HCT-116, and no cell cytotoxicity was observed [29].

Scheme 1. Solid-phaseSolid-phase synthesis of nocardiamides A ( 1) and B ( 2) on 2-chlorotrityl2-chlorotrityl chloride ( (2-CTC)2-CTC) resin. Reagents Reagents and and conditions: conditions: (a (a)) Fmoc Fmoc-Tyr(-Tyr(tBu)tBu)-OH;-OH; (b) (b) HBTU, HBTU, DIPEA, DIPEA, and and DMF DMF (r.t., (r.t., 1.5 1.5h); ( h);c) (c)MeOH MeOH (r.t., (r.t., 0.5 0.5h); ( h);d) (d)20% 20% piper piperidine/DMFidine/DMF (r.t., (r.t.,20 min 20) min);; (e) Fmoc (e) Fmoc-AA-OH,-AA-OH, HBTU, HBTU, DIPEA, DIPEA, and DMF and

DMF(r.t., 0.5 (r.t., h); 0.5 (f) h);TFA/thioanisole/PhOH/ (f) TFA/thioanisole/PhOH/1,2-ethanedithiol/H1,2-ethanedithiol/H2O (82.52/O5/ (82.5/5/5/2.5/5)5/2.5/5) (r.t.; 31.1% (r.t.; yield 31.1% for 3 yield and for47.8%3 and for 47.8% 4 after for 4 RPafter-HPLC RP-HPLC purification purification);); (g) HBTU, (g) HBTU, DIPEA, DIPEA, and and DMF DMF (r.t. (r.t.;; 7.2% 7.2% yiel yieldd for nocardiamide A (1)) and 10.7%10.7% for nocardiamide BB ((22)) afterafter RP-HPLCRP-HPLC purification).purification).

In 2013, 2013, Cochrane Cochrane et et al. al. reported reported the thesynthesis synthesis of the of first the members first members of a new of aclass new of class cyclic of- cyclic-peptide-containingpeptide-containing hemicryptophanes hemicryptophanes (e.g., 8 in (e.g., Scheme8 in 2) Scheme[30]. From2)[ this30 ].work, From the solid this- work,supported the solid-supportedlinear hexapeptide linear 5hexapeptide was prepared5 was from prepared 2-CTC from resin 2-CTC by resin standard by standard microwave microwave-assisted-assisted 9- 9-fluorenylmethoxycarbonylfluorenylmethoxycarbonyl (Fmoc (Fmoc)) SPPS SPPS (Scheme (Scheme 2). Cleavage2). Cleavage of the of hexapeptide the hexapeptide from from the resin the resin was wasdone doneusing using5% TFA 5% in TFA CH3 inCN CH/H32OCN/H (4:1),2 Oaffording (4:1), affording the unprotected the unprotected linear peptide linear 6 in peptide 85% yield.6 in 85%Subsequent yield. Subsequent cyclization cyclization of 6 of 6usingusing (benzotriazol-1-yloxy)tripyrrolidinophosphonium(benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphatehexafluorophosphate ( (PyBOP)PyBOP) in dimethylformamidedimethylformamide ( (DMF)DMF) gave the cyclohexapeptide 7 in quantitative yield after 1 h.h. Interesting Interestingly,ly, cyclization cyclization of of 6 proceeded mmuchuch faster than that of its corresponding O--tert-butyltert-butyl protected linear peptide counterpart. Cyclic Cyclic hexapeptide hexapeptide 7 was used to produce a new class of cyclohexapeptide cyclohexapeptide-containing-containing hemicryptophanes hemicryptophanes,, which were investigatedinvestigated for their enantioselective binding propertiesproperties by complexation withwith carnitinecarnitine [[30]30]..

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Scheme 2.2. SolidSolid-phase-phase s synthesisynthesis of of cyclohexapeptide cyclohexapeptide 77 on 2 2-chlorotrityl-chlorotrityl chloride ((2-CTC)2-CTC) resin. Reagents and conditions: conditions: ( (a)a) 1. 1. Fmoc Fmoc-Gly-OH,-Gly-OH, DIPEA, DIPEA, and and DCM/DMF DCM/DMF (4/1) (4/1) (r.t., (r.t., 16 h 16); 2. h); MeOH 2. MeOH (r.t., t (r.t.,20 min 20 min);); (b) (b)two two treatments treatments with with 20% 20% piper piperidine/DMFidine/DMF (r.t., (r.t., 5 5 min min);); (c (c)) Fmoc Fmoc-Tyr(-Tyr(tBu)Bu)-OH,-OH, HATU (0.5M),(0.5M), DIPEA,DIPEA, and DMF (70(70 °C,◦C, 5 5 min, min, microwave microwave heating heating);); ( (d)d) Fmoc Fmoc-Gly-OH,-Gly-OH, HATU HATU (0.5 (0.5M),M), DIPEA, DIPEA, and DMF (70 °C,◦ 5 min, microwave heating); (e) three treatments with 5% TFA in CH3CN/H2O (4:1) and DMF (70 C, 5 min, microwave heating); (e) three treatments with 5% TFA in CH3CN/H2O (4:1)(r.t., (r.t., 20 min, 20 min, 85% 85%);); (f) PyBOP (f) PyBOP and and DMF DMF (r.t., (r.t., 1 1 h; h; quant quantitativeitative yield yield after after purification purification by flashflash chromatography (silica(silica gel)).gel)).

Peptide-based therapeutics are notorious for their poor oral bioavailability profiles and low Peptide-based therapeutics are notorious for their poor oral bioavailability profiles and low plasma stability, which have limited their use as orally delivered drugs. Although N-methylation of plasma stability, which have limited their use as orally delivered drugs. Although N-methylation cyclohexapeptides was shown to improve oral bioavailability [23], Hill et al. demonstrated that of cyclohexapeptides was shown to improve oral bioavailability [23], Hill et al. demonstrated that cyclohexaleucine peptides 9 and 10 without N-methylation functionality showed some degree of oral cyclohexaleucine peptides 9 and 10 without N-methylation functionality showed some degree of oral bioavailability: 17% and 9%, respectively [12]. Interestingly, the epimer 10 showed ~2-fold lower oral bioavailability: 17% and 9%, respectively [12]. Interestingly, the epimer 10 showed ~2-fold lower oral bioavailability than 9 as a result of its notably higher plasma clearance rate (24.1 versus 4.7 bioavailability than 9 as a result of its notably higher plasma clearance rate (24.1 versus 4.7 mL/min/kg). mL/min/kg). The reason was suggested to be caused by differences in solvent exposure to the peptide The reason was suggested to be caused by differences in solvent exposure to the peptide backbone backbone resulting from the conformational change at a Leu residue. Membrane permeability was resulting from the conformational change at a Leu residue. Membrane permeability was measured measured using RRCK and CACO-2 cell monolayers, the former having a lack of active transporters. using RRCK and CACO-2 cell monolayers, the former having a lack of active transporters. In the In the RRCK assay, compounds 9 and 10 showed 2–3-fold greater membrane permeability than the RRCK assay, compounds 9 and 10 showed 2–3-fold greater membrane permeability than the control control standard cyclosporin A (CsA). In the CACO-2 assay, 9 and 10 showed similar permeability to standard cyclosporin A (CsA). In the CACO-2 assay, 9 and 10 showed similar permeability to CsA CsA (Papp ≈ 5 × 10−6 cm·s−1) [12]. As shown in Scheme 3, the cyclohexaleucine peptide 9 was synthesized (P ≈ 5 × 10−6 cm·s−1)[12]. As shown in Scheme3, the cyclohexaleucine peptide 9 was synthesized usingapp SPPS on 2-CTC resin as a linear hexapeptide precursor, followed by cleavage from resin and using SPPS on 2-CTC resin as a linear hexapeptide precursor, followed by cleavage from resin solution-phase cyclization under dilute conditions. Epimer 10 was formed during the final cyclization and solution-phase cyclization under dilute conditions. Epimer 10 was formed during the final step but could be successfully separated out during purification using reverse-phase high- cyclization step but could be successfully separated out during purification using reverse-phase performance liquid chromatography (RP-HPLC). The observed epimerization was due to a well- high-performance liquid chromatography (RP-HPLC). The observed epimerization was due to a known epimerization process that takes place during the carboxylic acid activation step prior to well-known epimerization process that takes place during the carboxylic acid activation step prior to cyclization [31,32]. In this example, the epimer ratio of 9 and 10 was 85:15 [12]. cyclization [31,32]. In this example, the epimer ratio of 9 and 10 was 85:15 [12].

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Scheme 3 3.. SolidSolid-phase-phase synthesis synthesis of of 9 9andand 1010 onon 2-chlorotrityl 2-chlorotrityl chloride chloride (2-CTC (2-CTC)) resin resin.. Reagents Reagents and andconditions: conditions: (a) Fmoc (a)- Fmoc-Leu-OH,Leu-OH, DIPEA, DIPEA, and DCM and DCM(r.t., overnight(r.t., overnight);); (b) treated (b) treated twice twice with with50% 50%piperi piperidine/DMFdine/DMF (r.t., 10 (r.t., min 10); min); (c) Fmoc (c)- Fmoc-Leu-OH,Leu-OH, HATU, HATU, DIPEA DIPEA,, and andDMF DMF(r.t., (r.t., 2 h); 2 ( h);d)

(d)TFA/TIPS/H TFA/TIPS/H2O (952O/2.5 (95/2.5/2.5)/2.5) (r.t., 2(r.t., h); 2 (e h);) dropwise (e) dropwise addition addition of linear of linear peptide peptide solution solution in DMFin DMF to tosolution solution of ofPyBOP, PyBOP, DIPEA, DIPEA, and and DMF DMF (over (over 3 3h, h, r.t. r.t.,, overnight overnight;; 17% 17% yield yield for for 99 andand 3% 3% for 10 after preparative RP-HPLCRP-HPLC purification).purification).

In 2014, Masuda et al. reported the total synthesis and insecticidal activity of the In 2014, Masuda et al. reported the total synthesis and insecticidal activity of the cyclohexapeptide cyclohexapeptide natural products PF1171A (11), C (12), F (13), and G (14) (Figure 3) [33]. The natural products PF1171A (11), C (12), F (13), and G (14) (Figure3)[ 33]. The producing organisms producing organisms are commonly fungi, such as Hamigera avellanea [34], Acremonium [35] or are commonly fungi, such as Hamigera avellanea [34], Acremonium [35] or Penicillium [36] species. Penicillium [36] species. Of particular interest is the high degree of D-amino acid incorporation (D- Of particular interest is the high degree of D-amino acid incorporation (D-Ala, D-Aba, D-allo-Ile, Ala, D-Aba, D-allo-Ile, and D-Val) within the macrocyclic scaffold. These natural products also and D-Val) within the macrocyclic scaffold. These natural products also contain non-proteinogenic contain non-proteinogenic anthranilic acid (Ant) and L-pipecolinic acid (Pip) residues, suggesting anthranilic acid (Ant) and L-pipecolinic acid (Pip) residues, suggesting that a non-ribosomal that a non-ribosomal biosynthetic pathway may have been used by the producing organisms. The biosynthetic pathway may have been used by the producing organisms. The enhanced bioavailability enhanced bioavailability of these natural products can be attributed to improved cell permeability of these natural products can be attributed to improved cell permeability (via passive diffusion) and (via passive diffusion) and higher metabolic stability due to their cyclic nature and incorporation of higher metabolic stability due to their cyclic nature and incorporation of D-amino acids [33]. The D-amino acids [33]. The synthesis of 11–14 was achieved via the initial construction of linear synthesis of 11–14 was achieved via the initial construction of linear hexapeptides by SPPS using hexapeptides by SPPS using a trityl alcohol SynPhase Lantern solid support. A representative a trityl alcohol SynPhase Lantern solid support. A representative synthesis for the construction synthesis for the construction of cyclohexapeptide 11 is shown in Scheme 4. The SPPS started by of cyclohexapeptide 11 is shown in Scheme4. The SPPS started by attaching Fmoc-D-Ala-OH attaching Fmoc-D-Ala-OH for 11–13 or Fmoc-D-Aba-OH for 14, as this allowed the final solution- for 11–13 or Fmoc-D-Aba-OH for 14, as this allowed the final solution-phase HBTU-mediated phase HBTU-mediated macrocyclization step to proceed with the least amount of steric hindrance macrocyclization step to proceed with the least amount of steric hindrance [33]. The N-methyl-Leu, [33]. The N-methyl-Leu, L-pipecolinic acid, and anthranilic acid functionality seen in 11-14 was L-pipecolinic acid, and anthranilic acid functionality seen in 11-14 was incorporated into the incorporated into the peptide sequence employing Fmoc-L-MeLeu-OH, Fmoc-L-Pip-OH or Fmoc- peptide sequence employing Fmoc-L-MeLeu-OH, Fmoc-L-Pip-OH or Fmoc-Ant-OH during SPPS, Ant-OH during SPPS, respectively. When coupling to the weakly nucleophilic amino function of the respectively. When coupling to the weakly nucleophilic amino function of the N-terminal anthranilic N-terminal anthranilic acid residue, the Fmoc-amino acid chloride of D-allo-Ile or D-Val was acid residue, the Fmoc-amino acid chloride of D-allo-Ile or D-Val was generated in situ using generated in situ using triphosgene prior to coupling [33]. The linear hexapeptides were cleaved from triphosgene prior to coupling [33]. The linear hexapeptides were cleaved from the resin using the resin using 30% hexafluoroisopropanol (HFIP) in dichloromethane (DCM) and cyclized in 30% hexafluoroisopropanol (HFIP) in dichloromethane (DCM) and cyclized in solution using solution using 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate hexafluorophosphate (HATU) and N,N-diisopropylethylamine (DIPEA) in DCM at room (HATU) and N,N-diisopropylethylamine (DIPEA) in DCM at room temperature for 3 h to afford 11–14. temperature for 3 h to afford 11–14. The natural products 11–14 were tested in a fourth-instar larvae The natural products 11–14 were tested in a fourth-instar larvae assay and were all found to have assay and were all found to have paralytic activity against silkworm larvae. The stereochemistry of paralytic activity against silkworm larvae. The stereochemistry of the D-Ala side chain was found the D-Ala side chain was found to be crucial for paralytic activity, as side-chain epimers of 11 and 12 to be crucial for paralytic activity, as side-chain epimers of 11 and 12 having L-Ala in place of D-Ala having L-Ala in place of D-Ala showed little activity. showed little activity.

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Figure 3. Structures of naturally occurring cyclohexapeptides 11–14. FigureFigure 3 3.. StructuresStructures of of naturally occurring cyclohexapeptides cyclohexapeptides 11–14.

Scheme 4. Solid-phase peptide synthesis of 11 on trityl alcohol lantern. Reagents and conditions: (a) Scheme 4 4.. SolidSolid-phase-phase peptide peptide synthesis synthesis of of 1111 onon trityl trityl alcohol alcohol lantern lantern.. Reagents Reagents and and conditions: conditions: (a) AcCl and DCM (r.t., 4 h); (b) Fmoc-D-Ala-OH, DIPEA, and DCM (r.t., 12 h); (c) 20% piperidine/DMF AcCl(a) AcCl and and DCM DCM (r.t., (r.t., 4 h 4); h); (b) (b) Fmoc Fmoc-D-Ala-OH,-D-Ala-OH, DIPEA, DIPEA, and and DCM DCM (r.t., (r.t., 12 12 h h);); (c) (c) 20% piperidine/DMFpiperidine/DMF (r.t., 1 h); (d) Fmoc-AA-OH, DIC, HOBt, and DMF (r.t., 12 h); (e) 30% hexafluoroisopropanol ((r.t.,r.t., 1 h);h); (d) Fmoc Fmoc-AA-OH,-AA-OH, DIC, HOBtHOBt,, and DMF ( (r.t.,r.t., 12 h);h); (e) 30% hexafluoroisopropanolhexafluoroisopropanol (HFIP)/DCM (r.t., 1 h); (f) HATU, DIPEA, and DCM (r.t., 3 h); yields (over 13 steps): 29% (11), 41% ((HFIP)/DCMHFIP)/DCM (r.t., (r.t., 1 1h) h);; (f) (f) HATU, HATU, DIPEA, DIPEA, and and DCM DCM (r.t., (r.t., 3 h 3); h);yields yields (over (over 13 steps) 13 steps):: 29% ( 29%11), 41% (11), (12), 39% (13), and 29% (14)). (41%12), (39%12), ( 39%13), (and13), 29% and ( 29%14)). ( 14)). In 2014, Peña et al. reported the synthesis and antimalarial and antitrypanosomal activity of In 2014, Peña et al. reported the synthesis and antimalarial and antitrypanosomal activity of sevenIn novel 2014, cyclohexapeptides Peña et al. reported 15 the–21 synthesis with 20 andand antimalarial21 incorporating and antitrypanosomal interesting thiazol activitye functionality of seven seven novel cyclohexapeptides 15–21 with 20 and 21 incorporating interesting thiazole functionality [37]novel. The cyclohexapeptides study was prompted15–21 bywith earlier20 and reports21 incorporating of cyanobacteriu interestingm Microcystis thiazole aeruginosa functionality PCC 7806 [37]. [37]. The study was prompted by earlier reports of cyanobacterium Microcystis aeruginosa PCC 7806 Thenatural study products, was prompted aerucyclamide by earliers A reports–D (Figure of cyanobacterium 4), which displayedMicrocystis promising aeruginosa microPCCmolar 7806 IC natural50 (50% natural products, aerucyclamides A–D (Figure 4), which displayed promising micromolar IC50 (50% products,of maximal aerucyclamides inhibitory concentration A–D (Figure) values4), which against displayed a K1 chloroquine promising-resistant micromolar strain of IC Plasmodium50 (50% of of maximal inhibitory concentration) values against a K1 chloroquine-resistant strain of Plasmodium falciparummaximalinhibitory [38,39]. The concentration) SPPS of linear values hexapeptide against precursors a K1 chloroquine-resistant was conducted using strain 2-CTC of Plasmodium resin and falciparum [38,39]. The SPPS of linear hexapeptide precursors was conducted using 2-CTC resin and standardfalciparum Fmoc[38,39 chemistry]. The SPPS (Scheme of linear 5). hexapeptide The first Fmoc precursors-amino was acid conducted was loaded using onto 2-CTC 2-CTC resin resin and in standard Fmoc chemistry (Scheme 5). The first Fmoc-amino acid was loaded onto 2-CTC resin in DCMstandard in the Fmoc presence chemistry of DIPEA, (Scheme followed5). The by first the Fmoc-aminocapping of unreacted acid was resin loaded sites onto with 2-CTC methanol. resin For in DCM in the presence of DIPEA, followed by the capping of unreacted resin sites with methanol. For theDCM synthesis in the presenceof 20 and of21 DIPEA,, the 2-CTC followed resin was by thefirst capping loaded with of unreacted an Fmoc- resinprotected sites thiazole with methanol. residue the synthesis of 20 and 21, the 2-CTC resin was first loaded with an Fmoc-protected thiazole residue (FmocFor the-Thz synthesis-OH) using of 20 analogouand 21, thes conditions. 2-CTC resin Subsequent was first loaded amino acids with an were Fmoc-protected installed via iterative thiazole (Fmoc-Thz-OH) using analogous conditions. Subsequent amino acids were installed via iterative deprotectionresidue (Fmoc-Thz-OH) (20% piperidine using in analogous DMF) and conditions. coupling (N,N' Subsequent-diisopropylcarbodiimide amino acids were ( installedDIC) and via 3- deprotection (20% piperidine in DMF) and coupling (N,N'-diisopropylcarbodiimide (DIC) and 3- hiterativeydroxytriazolo[4,5 deprotection-b]pyridine (20% piperidine (HOAt) inin DMF)DMF) andsteps. coupling After cleavage (N,N'-diisopropylcarbodiimide of linear hexapeptides from (DIC) the hydroxytriazolo[4,5-b]pyridine (HOAt) in DMF) steps. After cleavage of linear hexapeptides from the resinand 3-hydroxytriazolo[4,5- using 1% TFA in DCMb]pyridine, macrocyclizations (HOAt) in were DMF) performed steps. After using cleavage HBTU, of DIPEA linear,hexapeptides and catalytic resin using 1% TFA in DCM, macrocyclizations were performed using HBTU, DIPEA, and catalytic 4from-dimethylaminopyridine the resin using 1% TFA (DMAP in DCM,) in DCM macrocyclizations under dilute wereconditions performed (1–5 mM). using The HBTU, coupling DIPEA, site and for 4-dimethylaminopyridine (DMAP) in DCM under dilute conditions (1–5 mM). The coupling site for macroccatalyticyclization 4-dimethylaminopyridine was chosen to occur (DMAP) at N-terminal in DCM underglycine dilute for 15 conditions, C-terminal (1–5 glycine mM). Thefor 17 coupling–19, or macrocyclization was chosen to occur at N-terminal glycine for 15, C-terminal glycine for 17–19, or Csite-terminal for macrocyclization thiazole for 20 was–21, chosenas this to provided occur at theN-terminal least degree glycine of stericfor 15 , hindranceC-terminal, resulting glycine for in C-terminal thiazole for 20–21, as this provided the least degree of steric hindrance, resulting in 17favorable–19, or C yields-terminal [37] thiazole. Moreover, for 20 cyclization–21, as this of provided linear precursors the least degree to afford of steric 17–21 hindrance, took place resulting at a C- favorable yields [37]. Moreover, cyclization of linear precursors to afford 17–21 took place at a C- terminalin favorable glycine yields or thiazole [37]. Moreover, to prevent cyclization epimerization. of linear precursors to afford 17–21 took place at a terminal glycine or thiazole to prevent epimerization. C-terminal glycine or thiazole to prevent epimerization.

Molecules 2018, 23, 1475 8 of 25

Molecules 2018, 23, x FOR PEER REVIEW 8 of 25 Molecules 2018, 23, x FOR PEER REVIEW 8 of 25

Figure 4. Structures of aerucyclamides A–D. Figure 4. Structures of aerucyclamides A–D. Figure 4. Structures of aerucyclamides A–D.

Scheme 5. Solid-phase peptide synthesis of 15–21 on 2-chlorotrityl chloride (2-CTC) resin. Reagents andScheme conditions: 5. Solid (a)-phase Fmoc peptide-AA-OH synthesis (or Fmoc of-Thz 15–-21OH on in 2 the-chlorotrityl synthesis chlorideof 20 and (2 21-CTC), DIPEA,) resin .and Reagents DCM Scheme 5. Solid-phase peptide synthesis of 15–21 on 2-chlorotrityl chloride (2-CTC) resin. Reagents (andr.t., conditions: 1 h, capped (a) with Fmoc MeOH,-AA-OH r.t., ( or 0.5 Fmoc h); (b)-Thz 1.- OHdeprotection: in the synthesis 20% piperidineof 20 and 21/DMF), DIPEA, (r.t., 2and × 5 DCM min and conditions:followed(r.t., 1 h (a), bycap Fmoc-AA-OH 1ped × 10 with min) MeOH,; 2. coupling: (or r.t., Fmoc-Thz-OH 0.5 Fmoc h); (b)-AA 1. -dOH,eprotection: in HOAt, the synthesisDIC, 20% and piperidine DMF of 20 (r.t.,/DMFand 2 h21()r.t.,; ),3. DIPEA,2steps × 5 min1–2 and DCM (r.t., 1 h, cappedrepeatedfollowed; with by (c) 1c ×leavage: MeOH, 10 min) four; r.t.,2. coupling: treatments 0.5 h); Fmoc (b) with 1.-AA deprotection:1%-OH, TFA/DCM HOAt, DIC, (r.t., 20% and 3 piperidine/DMFminDMF); ( (d)r.t., m2 acrocyclization:h); 3. steps (r.t., 1–2 2 × 5 min followed by(concentrationrepeated 1 ×; 10 (c) min); cofleavage: 1–5 2.mM) four coupling: HBTU, treatments DIPEA, Fmoc-AA-OH, withDMAP, 1% and TFA/DCM DCM HOAt, ( r.t.,(r.t., 3– DIC,5 3 days min and;) ;cyclization (d) DMFmacrocyclization: yields (r.t.,: 259% h); 3. steps ((concentration15), 55% (16), 48% of 1 –(175 mM)), 66% HBTU, (18), 54% DIPEA, (19), 83%DMAP, (20), and and DCM 40% ((21r.t.,)). 3–5 days; cyclization yields: 59% 1–2 repeated; (c) cleavage: four treatments with 1% TFA/DCM (r.t., 3 min); (d) macrocyclization: (15), 55% (16), 48% (17), 66% (18), 54% (19), 83% (20), and 40% (21)). (concentrationCyclohexapeptides of 1–5 mM) HBTU, 15–21 were DIPEA, screened DMAP, with and P. DCMfalciparum (r.t., K1 3–5 and days; infective cyclization T. b. brucei yields: assays. 59% (15), 55% (16Of), particular 48%Cyclohexapeptides (17), interest 66% (18 was), 15 54% – the21 werepromising (19), 83%screened ( antimalarial20), with and P. 40% falciparum activity (21)). shown K1 and by infective 17, 20, T.and b. 21brucei against assays. P. falciparumOf particular K1, interestwith EC was50 (5 0% the of promising maximal effective antimalarial concentration activity shown) values by of 17 0.19,, 20 , 0.19and, and21 against 0.41 µM P., respectivelyfalciparum K1, and, with no EC observed50 (50% ofcell maximal cytotoxicity effective against concentration murine macrophages) values of 0.19, [37] .0.19 Also, and noteworthy 0.41 µM, Cyclohexapeptideswasrespectively the anti,trypanosomal and 15no– observed21 were activity cell screened ofcytotoxicity 15 and with 19 –against21P. against falciparum murine T. b. macrophagesbruceiK1 and, with infective EC [37]50 values. AlsoT. noteworthyof b. 1.06, brucei 2.1, assays. Of particular3.0was interest, and the 2.8anti was µMtrypanosomal, the respectively promising activity [37]. antimalarial of 15 and 19– activity21 against shown T. b. brucei by, 17with, 20 EC, and50 values21 againstof 1.06, 2.1,P. falciparum In 2015, Wong et al. reported the total synthesis of dichotomin A (22) (Figure 5) from linear K1, with EC3.050, and(50% 2.8 µM of maximal, respectively effective [37]. concentration) values of 0.19, 0.19, and 0.41 µM, respectively, peptide precursors 23 and 24 that contain penicillamine-derived pseudoproline residue (Scheme 6) and no observedIn 2015, cell Wong cytotoxicity et al. reported against the total murine synthesis macrophages of dichotomin A [ 37 (22].) (Figure Also 5) noteworthy from linear was the [8]peptide. The utilizationprecursors of23 a and pseudoproline 24 that contain residue penicillamine in place of-derived the proteinogenic pseudoproline Val residue (Schemeduring the 6) antitrypanosomalsynthesis[8]. The utilization of activity dichotomin of ofa pseudoproline 15 A wasand a 19 protec– 21residuetingagainst groupin place T. stratergy of b. the brucei proteinogenic and , a with way to ECVal improve50 residuevalues head during of-to- 1.06,tailthe 2.1, 3.0, and 2.8 µM,cyclizationsynthesis respectively of of dichotominlinear [37 peptide]. A wasprecursors a protec becauseting group of the stratergy ability of and pseudoproline a way to improve to induce head a turn-to-tail or In 2015,“kink”cyclization Wong in the of et peptidelinear al. peptide reported backbone precursors theas well total asbecause to synthesis aid of peptide the ability of solubility dichotomin of pseudoproline [8]. The A pseudoproline (22 to )induce (Figure a residueturn5) fromor linear was deprotected and converted into the desired Val residue post cyclization to afford dichotomin A peptide precursors“kink” in the23 peptideand 24 backbonethat contain as well penicillamine-derived as to aid peptide solubility pseudoproline [8]. The pseudoproline residue residue (Scheme 6)[ 8]. was deprotected and converted into the desired Val residue post cyclization to afford dichotomin A The utilization of a pseudoproline residue in place of the proteinogenic Val residue during the synthesis of dichotomin A was a protecting group stratergy and a way to improve head-to-tail cyclization of linear peptide precursors because of the ability of pseudoproline to induce a turn or “kink” in the peptide backbone as well as to aid peptide solubility [8]. The pseudoproline residue was deprotected and converted into the desired Val residue post cyclization to afford dichotomin A (22). Dichotomin A was prepared from two different disconnection sites within the macrocyclic ring (Scheme6). The first linear peptide precursor 23 contained a non-epimerizable C-terminal glycine and an N-terminal O-tert-butyl protected threonine residue. The second peptide precursor 24 contained an epimerizable MoleculesMolecules2018, 23 2018, 1475, 23, x FOR PEER REVIEW 9 of 25 9 of 25 Molecules 2018, 23, x FOR PEER REVIEW 9 of 25 (22). Dichotomin A was prepared from two different disconnection sites within the macrocyclic ring N-terminal(Scheme(22). Dichotomin leucine 6). The and firstA awas Clinear-terminal prepared peptide from phenylalanine, precursor two different 23 contained disconnection creating a non a more- epimerizablesites within sterically the C- macrocyclicterminal hindered glycine ring cyclization site andand(Scheme providing an N6-).terminal The a wayfirst O linear to-tert test-butyl peptide the protected robustness precursor threonine 23 of contained this residue. methodology a non Th-eepimerizable second [8]. peptideLinear C-terminal peptides precursor glycine 2324 and 24 containedand an N - aterminaln epimerizable O-tert- butylN-terminal protected leucine threonine and a residue.C-terminal Th phenylalaninee second peptide, creating precursor a more 24 containing pseudoproline residues were synthesized on 2-CTC resin using standard Fmoc-strategy stericallycontained hindered an epimerizable cyclization N -siteterminal and providing leucine and a way a C to-terminal test the phenylalaninerobustness of ,this creating methodology a more SPPS with[8]sterically. HBTULinear hindered peptides as the coupling cyclization23 and 24 reagent containingsite and andproviding pseudoproline 10% piperidine a way residuesto test in the DMF were robustness forsynthesized Fmoc of deprotection.this on methodology 2-CTC resin Cleavage from resinusing[8]. Linear was standard donepeptides Fmoc using 23-strategy and HFIP 24 SPPScontaining in DCMwith HBTU pseudoproline while as keepingthe coupling residues the reagent side-chainwere andsynthesized 10% protecting piperidine on 2-CTC in groups DMFresin intact. The conformationsforusing Fmoc standard deprotection. ofFmoc23-strategyand Cleavage24 SPPSwere from wit establishedresinh HBTU was as done the using couplingusing 1HFIPH reagent nuclear in DCM and magnetic while10% piperidine keeping resonance the in DMFside- (NMR) spectroscopychainfor Fmoc protecting and deprotection. rotating-frame groups Cleavage intact. Overhauser The from conformations resin was effect done of spectroscopy 23 using and HFIP24 were in (ROESY) DCMestablished while experiments, keepingusing 1H the nuclear side which- also 1 confirmedmagneticchain a protecting single resonance set groups of resonance(NMR intact.) spectroscopy The structures conformations and for rotating eachof 23 peptide.and-frame 24 were Overhauser Macrocycle established effect 25using was spectroscopy H obtainednuclear from (magneticROESY) experimentsresonance ,( NMRwhich) also spectroscopy confirmed anda single rotating set of-frame resonance Overhauser structures effect for each spectroscopy peptide. linear peptides 23 or 24 containing a pseudoproline residue in less than 3 h and in good to excellent Macrocycle(ROESY) experiments 25 was obtained, which from also linear confirmed peptides a single 23 or set24 containingof resonance a pseudoproline structures for residueeach peptide. in less yields (78–88%)thanMacrocycle 3 h usingand 25 was in 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholiniumgood obtained to excellentfrom linear yields peptides (78– 88%)23 or 24 using containing 4-(4,6- daimethoxy pseudoproline-1,3,5- triazinresidue tetrafluoroborate-2 in-yl) less-4- (DMTMMmethylmorpholiniumthan·BF 3 4 h). and In contrast, in good tetrafluoroborate to cyclization excellent yields of (DMTMM·BF analogous (78–88%)4). linear usingIn contrast, 4 peptide-(4,6 -cyclizationdimethoxy precursors -1,3,5of analogous-triazin containing-2 -linearyl)-4- a native Val residuepeptidemethylmorpholinium had precursors much slower con tetrafluoroboratetaining reaction a native kinetics, Val(DMTMM·BF residue requiring had4). Inmuch contrast, 3 daysslower cyclization for reaction reaction kinetics of analogous completion, requir linearing and3 with lower yieldsdayspeptide for (33–36%). precursorsreaction completion The containing pseudoproline and a native with lowerVal and residue yieldsO-tert-butyl-Thr had(33– 36%).much Theslower residuespseudoproline reaction in kinetics25 andwere O, requir-tert simultaneously-butyling 3- Thrdays residuesfor reaction in completion25 were and simultaneously with lower yields deprotected (33–36%). using The pseudoprolinetrifluoromethanesulfonic and O-tert- butyl acid- deprotected using trifluoromethanesulfonic acid (TFMSA)/water (2:1, v/v) to give 26 followed by (ThrTFMSA residues)/water in(2:1 25, v /vwere) to give simultaneously 26 followed by deprotecteddesulfurization using using trifluoromethanesulfonic NiCl2 and NaBH4 in MeOH ac toid desulfurization using NiCl and NaBH in MeOH to afford dichotomin A (22) in 24% yield. afford(TFMSA dichotomin)/water (2:1 A, (v22/2v) toin give24% yield.26 followed4 by desulfurization using NiCl2 and NaBH4 in MeOH to afford dichotomin A (22) in 24% yield.

Figure 5. Structure of dichotomin A (22). Figure 5. Structure of dichotomin A (22). Figure 5. Structure of dichotomin A (22).

Scheme 6. Solid-phase peptide synthesis of dichotomin A (22) on 2-chlorotrityl chloride (2-CTC) resin. Reagents and conditions: (a) 1. Fmoc-Gly-OH (for 23) or Fmoc-Phe-OH (for 24), DIPEA, and DCM; 2. DCM/MeOH/DIPEA (17:2:1); (b) 10% piperidine/DMF; (c) Fmoc-AA-OH, HBTU, DIPEA, and

NMP; (d) 20% HFIP/DCM; (e) (concentration of 1 mM) DMTMM·BF4, DIPEA, and DMF (r.t., 3 h; yield of 25: 84% from 23 and 87% from 24)); (f) TFMSA/water (2:1) (r.t., 4 h, 33%); (g) NiCl2, NaBH4, and MeOH (0 ◦C, 0.5 h, 24%). Molecules 2018, 23, x FOR PEER REVIEW 10 of 25 Molecules 2018, 23, x FOR PEER REVIEW 10 of 25 Scheme 6. Solid-phase peptide synthesis of dichotomin A (22) on 2-chlorotrityl chloride (2-CTC) resin. Scheme 6. Solid-phase peptide synthesis of dichotomin A (22) on 2-chlorotrityl chloride (2-CTC) resin. Reagents and conditions: (a) 1. Fmoc-Gly-OH (for 23) or Fmoc-Phe-OH (for 24), DIPEA, and DCM; 2. Reagents and conditions: (a) 1. Fmoc-Gly-OH (for 23) or Fmoc-Phe-OH (for 24), DIPEA, and DCM; 2. DCM/MeOH/DIPEA (17:2:1); (b) 10% piperidine/DMF; (c) Fmoc-AA-OH, HBTU, DIPEA, and NMP; DCM/MeOH/DIPEA (17:2:1); (b) 10% piperidine/DMF; (c) Fmoc-AA-OH, HBTU, DIPEA, and NMP; (d) 20% HFIP/DCM; (e) (concentration of 1 mM) DMTMM·BF4, DIPEA, and DMF (r.t., 3 h; yield of 25: (d) 20% HFIP/DCM; (e) (concentration of 1 mM) DMTMM·BF4, DIPEA, and DMF (r.t., 3 h; yield of 25: Molecules84%2018 from, 23 ,2 14753 and 87% from 24)); (f) TFMSA/water (2:1) (r.t., 4 h, 33%); (g) NiCl2, NaBH4, and MeOH10 of 25 84% from 23 and 87% from 24)); (f) TFMSA/water (2:1) (r.t., 4 h, 33%); (g) NiCl2, NaBH4, and MeOH (0 °C, 0.5 h, 24%). (0 °C, 0.5 h, 24%). In 2015, 2015, Prompanya Prompanya et et al. al. isolated isolated a cyclohexapeptide a cyclohexapeptide from a from culture a culture of the marine of the sponge marine- In 2015, Prompanya et al. isolated a cyclohexapeptide from a culture of the marine sponge- sponge-associatedassociated fungus Aspergillus fungus Aspergillus similanensis similanensis KUFA 0013KUFA and named 0013 it and similanamide named it ( similanamide27) (Figure 6) [40] (27). associated fungus Aspergillus similanensis KUFA 0013 and named it similanamide (27) (Figure 6) [40]. (FigureMasuda6 )[et al40. ].undertook Masuda the et al.total undertook synthesis of the similanamide total synthesis (27 of) in similanamide 2015 [41] but noticed (27) in discrepancies 2015 [41] but Masuda et al. undertook the total synthesis of1 similanamide13 (27) in 2015 [41] but noticed discrepancies noticedwhen comparing discrepancies the 1H when and comparing13C-NMR data the ofH their and synthesizedC-NMR data similanamide of their synthesized (27) with similanamidereported data when comparing the 1H and 13C-NMR data of their synthesized similanamide (27) with reported data ([40]27).with The reported reported data data [ 40 for]. The similanamide reported data (27) for did similanamide, however, agree (27) did, favorably however, with agree a previously favorably [40]. The reported data for similanamide (27) did, however, agree favorably with a previously withsynthesized a previously diastereomer synthesized 28 diastereomer[41]. This led28 to[41 the]. This structural led to the revision structural of similanamide revision of similanamide (27) to the synthesized diastereomer 28 [41]. This led to the structural revision of similanamide (27) to the (structurally27) to the structurally similar diastereomer similar diastereomer 28 in 2015 28[41]in. 2015 [41]. structurally similar diastereomer 28 in 2015 [41].

Figure 6.6. Structures of cyclohexapeptides 27 and 28.. Figure 6. Structures of cyclohexapeptides 27 and 28. In 2016, Amso et al. reported the synthesis of cyclohexapeptide dianthin G (cyclo-Pro-Leu-Thr- In 2016, 2016, Amso Amso et al. etreported al. the reported synthesis the of cyclohexapeptide synthesis of cyclohexapeptide dianthin G (cyclo-Pro dianthin-Leu-Thr G- Leu-Phe-Gly, 29) as well as nine of its analogues, including Nα-methylated derivativesα (31–35) and (cyclo-Pro-Leu-Thr-Leu-Phe-Gly,Leu-Phe-Gly, 29) as well as nine of29 its) asanalog wellue ass, nineincluding of its N analogues,α-methylated including derivativesN -methylated(31–35) and conformationally constrained cyclic “dicarba” bridged analogues (e.g., 30) (Figure 7) [42,43]. The in conformationallyderivatives (31–35 constrained) and conformationally cyclic “dicarba” constrained bridged cyclicanalog “dicarba”ues (e.g., 3 bridged0) (Figure analogues 7) [42,43] (e.g.,. The30 in) vitro osteoblast proliferation activity of the synthesized compounds was determined. Dianthin G (29) (Figurevitro osteoblast7)[ 42,43 proliferation]. The in vitro activityosteoblast of the proliferationsynthesized compounds activity of the was synthesized determined. compounds Dianthin G was(29) and its Nα-methylated derivatives (31α–35) were synthesized by SPPS followed by cleavage and anddetermined. its Nα-methylated Dianthin Gderivatives (29) and its (31N–35-methylated) were synthesized derivatives by ( 31SPPS–35) followed were synthesized by cleavage by SPPS and solution-phase head-to-tail macrolactamization. solutionfollowed-phase by cleavage head-to and-tail solution-phase macrolactamization. head-to-tail macrolactamization.

Figure 7. Structures of dianthin G (29), “dicarba” analogue 30, and N-methyl analogues 31–35. Figure 7. StructuresStructures of dianthin G ( 29), “dicarba” analogue 30, and N--methylmethyl analogues 31–35. The SPPS of native cyclohexapeptide dianthin G (29) was reported previously [43] but was later The SPPS of native cyclohexapeptide dianthin G (29) was reported previously [43] but was later adaptedThe for SPPS the synthesis of native of m cyclohexapeptideethylated analogue dianthins 31–34 (Scheme G (29) 7). was The reportedNα-methylated previously derivatives [43] adapted for the synthesis of methylated analogues 31–34 (Scheme 7). The Nα-methylated derivatives but was later adapted for the synthesis of methylated analogues 31–34 (Scheme7). α The N -methylated derivatives 31–34 were synthesized from aminomethyl polystyrene resin and the 3-(4-hydroxymethylphenoxy)propionic acid) (HMPP) linker; however, the synthesis of 35 required the use of a more hindered 2-CTC resin to prevent the formation of undesired diketopiperazine by-products [42]. A representative synthesis of cyclohexapeptide 31 is shown in Molecules 2018, 23, x FOR PEER REVIEW 11 of 25 Molecules 2018, 23, 1475 11 of 25 31–34 were synthesized from aminomethyl polystyrene resin and the 3-(4- hydroxymethylphenoxy)propionic acid) (HMPP) linker; however, the synthesis of 35 required the Schemeuse7 . of The a more aminomethyl hindered 2 polystyrene-CTC resin to resin prevent was thefirst formation loaded with of undesired Fmoc-Gly-HMPP-OH diketopiperazine to form by- 36. The solid-supportedproducts [42]. A linear representa pentapeptidetive synthesis37 was of constructed cyclohexapeptide on-resin 31 usingis shown classical in Scheme SPPS. The 7. Theterminal aminoaminomethyl function was polystyrene activated resin by reacting was first with loaded 2-nitrobenzenesulfonyl with Fmoc-Gly-HMPP- chlorideOH to form to form36. The38 ,solid followed- by methylationsupported linear and deprotectionpentapeptide 37 to was afford constructed39. After on attaching-resin using the classical final prolineSPPS. The residue, terminal hexapeptide amino 40 wasfunction cleaved was from activated the resin by reacting to form with41, which 2-nitrobenzenesulfonyl was cyclized using chloride HBTU-mediated to form 38, followed solution-phase by cyclizationmethylation to afford and 31deprotection. to afford 39. After attaching the final proline residue, hexapeptide 40 was cleaved from the resin to form 41, which was cyclized using HBTU-mediated solution-phase Cyclic dicarba analogues contained a non-native dicarba bridge, for example, 30, and were cyclization to afford 31. synthesized by on-resin Grubbs’ ring-closing metathesis (RCM) and then cleaved from resin to form Cyclic dicarba analogues contained a non-native dicarba bridgeα , for example, 30, and were 30 assynthesized an inseparable by on mixture-resin Grubbs’ of cis ring and-closing trans metathesis isomers [ 42(RCM)]. An andN then-methyl cleaved amide from bondresin to scan form of the synthesized30 as an dianthininseparable G, mixture which wasof cis done and trans to investigate isomers [42] the. An effect Nα- thatmethyl altering amide amidebond scan bonds of the had on osteoblastsynthesized proliferation, dianthin G, found which that was all done native to investigate peptide bondsthe effect contained that altering in the amide primary bonds sequencehad on of dianthinosteoblast G (29 )proliferation, were of importance found that for all osteoblast native peptide proliferation bonds contained activity. in From the primaryin vitro sequencestudies, of native dianthindianthin G (29 G) ( and29) were a dicarba of importance bridged for analogue osteoblast (at proliferation 10−8 M) were activity. found From to in increase vitro studies, the numbers native of humandianthin osteoblasts G (29) withoutand a dicarba having bridged a significant analogue effect(at 10− on8 M) osteoclast were found differentiation to increase the or numbers development. of An inseparablehuman osteoblasts Z/E mixture without of having olefins a insignificant a 2:1 ratio effect with on aosteoclastβ-sheet-like differentiation secondary or structure development. similar to An inseparable Z/E mixture of olefins in a 2:1 ratio with a -sheet-like secondary structure similar to that of native dianthin G (29) was determined through spectroscopic analysis of the dicarba analogue. that of native dianthin G (29) was determined through spectroscopic analysis of the dicarba analogue. It was suggested that this secondary structure is important for the bone activity associated with It was suggested that this secondary structure is important for the bone activity associated with dianthindianthin peptides. peptides.

SchemeScheme 7. Representative 7. Representative solid-phase solid-phase peptide peptide synthesis of of 3131 onon aminomethyl aminomethyl polystyrene polystyrene resin.resin. Reagents and conditions: (a) Fmoc-Gly-O-HMMP-OH, DIC, DCM, and DMF (r.t., 4 h); (b) Reagents and conditions: (a) Fmoc-Gly-O-HMMP-OH, DIC, DCM, and DMF (r.t., 4 h); (b) deprotection: deprotection: 20% piperidine/DMF (r.t., 2 × 5 min); (c) coupling: Fmoc-AA-OH, HATU, DIPEA, and 20% piperidine/DMF (r.t., 2 × 5 min); (c) coupling: Fmoc-AA-OH, HATU, DIPEA, and DMF (r.t., DMF (r.t., 45 min); (d) 2-nitrobenzenesulfonyl chloride, sym-collidine, and NMP (r.t., 2 × 15 min); (e) × 45 min);dimethyl (d) 2-nitrobenzenesulfonyl sulfate, DBU, and NMP chloride,(r.t., 2 × 5 sym-collidine,min); (f) 2-mercaptoethanol, and NMP (r.t., DBU, 2 and15 NMP min); (r.t., (e) 2 dimethyl × 5 × × sulfate,min DBU,); (g) TFA and/TIPS NMP/H2O (r.t., (95/ 22.5/2.5)5 min); (r.t., 3 (f)h); 2-mercaptoethanol,(h) HBTU, 6-Cl-HOBt, DBU,DIPEA and, DCM, NMP and (r.t.,DMF 2(r.t., 536 min); (g) TFA/TIPS/Hh; overall yields2O: 52% (95/2.5/2.5) (29), 38% ( (r.t.,31), 22% 3 h); (32 (h)), HBTU,51% (33), 6-Cl-HOBt, 43% (34), 39% DIPEA, (35)). DCM, and DMF (r.t., 36 h; overall yields: 52% (29), 38% (31), 22% (32), 51% (33), 43% (34), 39% (35)). In 2017, Asfaw et al. reported the synthesis of cyclohexapeptide wollamide B (42) and 24 of its analogues [44]. The Fmoc-based SPPS of a linear hexapeptide precursor was followed by solution- In 2017, Asfaw et al. reported the synthesis of cyclohexapeptide wollamide B (42) and 24 of its phase macrocyclization and cleavage of protecting groups, as described in Scheme 8. The first amino analogues [44]. The Fmoc-based SPPS of a linear hexapeptide precursor was followed by solution-phase acid (Fmoc-Leu-OH) was loaded onto 2-CTC resin by using DIPEA/DCM followed by capping with macrocyclizationmethanol. HATU and and cleavage DIPEA ofin protectingNMP were used groups, during as describedthe coupling in steps Scheme to elongate8. The the first peptides. amino acid (Fmoc-Leu-OH)The N-terminal was Fmoc loaded group onto was 2-CTCdeprotected resin after by each using round DIPEA/DCM of coupling followedusing 20% bypiperidi cappingne in with methanol.DMF. The HATU resin and was DIPEA treated inwith NMP 20% were HFIP used in DCM during to complete the coupling cleavage steps of the to linear elongate hexapeptide the peptides. The N -terminal Fmoc group was deprotected after each round of coupling using 20% piperidine in DMF. The resin was treated with 20% HFIP in DCM to complete cleavage of the linear hexapeptide precursors. Macrocyclization was done using HATU, hydroxybenzotriazole (HOBt), and DIPEA in DMF to produce a crude cyclic hexapeptide that was then purified via column chromatography. Lastly, the Trt and Boc side-chain protecting groups were removed using a TFA/triisopropylsilane Molecules 2018, 23, x FOR PEER REVIEW 12 of 25

Moleculesprecursor2018, 23s,. 1475 Macrocyclization was done using HATU, hydroxybenzotriazole (HOBt), and DIPEA in12 of 25 DMFMolecules to 2018 produce, 23, x FOR a crudePEER REVIEW cyclic hexapeptide that was then purified via column chromatography.12 of 25 Lastly, the Trt and Boc side-chain protecting groups were removed using a TFA/triisopropylsilane (TIPS)/H O solution (95/2.5/2.5) to afford wollamide B (47) in 91% yield after purification by silica (precursorTIPS2)/H2Os. solutionMacrocyclization (95/2.5/2.5) was to afforddone using wollamide HATU, B (hydroxybenzotriazole47) in 91% yield after purification (HOBt), and by DIPEA silica gel in gel chromatography.chromatographyDMF to produce. a crude cyclic hexapeptide that was then purified via column chromatography. Lastly, the Trt and Boc side-chain protecting groups were removed using a TFA/triisopropylsilane (TIPS)/H2O solution (95/2.5/2.5) to afford wollamide B (47) in 91% yield after purification by silica gel chromatography.

Scheme 8. Solid-phase peptide synthesis of 42 on 2-chlorotrityl chloride (2-CTC) resin. Reagents and Scheme 8. Solid-phase peptide synthesis of 42 on 2-chlorotrityl chloride (2-CTC) resin. Reagents conditions: (a) Fmoc-Leu-OH, DIPEA, and DCM (r.t., 2 h); (b) 20% piperidine/DMF (r.t., 2 × 10 min); and conditions: (a) Fmoc-Leu-OH, DIPEA, and DCM (r.t., 2 h); (b) 20% piperidine/DMF (r.t., (c) Fmoc-AA-OH, HBTU, DIPEA, and NMP (r.t., 1 h); (d) 20% HFIP/DCM (r.t., 1 h); (e) (concentration × 2 10ofScheme min1 mM)); 8 (c). SolidHATU, Fmoc-AA-OH,-phase HOBt, peptide DIPEA, HBTU, synthesis and DIPEA, DMF of 42 (on0 and °C2-chlorotrityl to NMP r.t.; (r.t.,r.t., chloride 3 1 days h); (d); (cyclization2-CTC 20%) resin. HFIP/DCM yield Reagents: 68%) (r.t., ;and (f) 1 h); ◦ (e) (concentrationTFAconditions:/TIPS/H (2aO) of Fmoc(95/2.5/2.5) 1 mM)-Leu HATU,-OH, (r.t. ,DIPEA, 3 h HOBt,; yield and DIPEA,of DCM42: 91%). (r.t., and 2 DMFh); (b) (0 20%C piperidine/DMF to r.t.; r.t., 3 days; (r.t., cyclization 2 × 10 min) yield:; 68%);(c (f)) Fmoc TFA/TIPS/H-AA-OH, HBTU,2O (95/2.5/2.5) DIPEA, and (r.t., NMP 3 h;(r.t., yield 1 h); of (d42) 20%: 91%). HFIP/DCM (r.t., 1 h); (e) (concentration Theof 1 antimycobacterialmM) HATU, HOBt, activities DIPEA, and as wellDMF as(0 the°C to in r.t.vitro; r.t., dru 3 g days metabolism; cyclization and yield PK: 68%)(absorption; (f) , TFA/TIPS/H2O (95/2.5/2.5) (r.t., 3 h; yield of 42: 91%). Thedistribution antimycobacterial, metabolism, activitiesand excretion as well—ADME) as the profilesin vitro ofdrug 42 and metabolism structural and analog PKue (absorption,s were investigated. Wollamide B (42) was found to have notable aqueous solubility, moderate affinity for distribution, metabolism, and excretion—ADME) profiles of 42 and structural analogues were plasmaThe protein antimycobacterial albumin, activities modest lipophilicity, as well as the poor in vitro passive drug metabolism permeability and through PK (absorption artificial, investigated. Wollamide B (42) was found to have notable aqueous solubility, moderate affinity for membranes,distribution, significantmetabolism in, vitroand plasmaexcretion stability,—ADME) high profiles microsomal of 42 metabolic and structural stability, analog and noue toxicitys were plasmaininvestigated. protein HepG2 cells albumin, W ollamide for concentrations modest B (42 lipophilicity,) was of found up to to poor 50have µM. passive notable Notabl permeabilityaqueousy, five of solubility, the throughsynthesized moderate artificial wollamide affinit membranes,y for B significantanalogplasmainue proteins vitro (43–plasma47 ; albumin, Figure stability, 8 modest) displayed high lipophilicity,microsomal potent antimycobacterial poor metabolic passive stability, permeability activity and (m inimum no through toxicity inhibitory artificial in HepG2 cellsconcentrationmembranes, for concentrations significant (MIC) of of ≤3.1in up vitro µ toM) plasma 50 andµM. no stability, toxicity Notably, highin fiveHepG2 microsomal of cell thes synthesizedfor metabolic concentrations stability, wollamide of and up tono B100 toxicity analogues µM. (43–47Cin;ompounds Figure HepG28 )cells displayed 43 and for concentrations46 potentalso showed antimycobacterial of an up optimal to 50 µM.balance activity Notabl between (minimumy, five anti of mycobacter the inhibitory synthesizedial concentration activity wollamide and PK (MIC)B of ≤3.1properties.analogµM)ue ands ( Overall,43 no–47 toxicity; Figure the synthesizedin 8HepG2) displayed cells wollamides for potent concentrations antimycobacterial displayed notable of up toactivity plasma 100 µ M. stability (m Compoundsinimum and inhibitory aqueous43 and 46 concentration (MIC) of ≤3.1 µM) and no toxicity in HepG2 cells for concentrations of up to 100 µM. also showedsolubility an with optimal moderate balance to low between metabolic antimycobacterial stability. activity and PK properties. Overall, the Compounds 43 and 46 also showed an optimal balance between antimycobacterial activity and PK synthesized wollamides displayed notable plasma stability and aqueous solubility with moderate to properties. Overall, the synthesized wollamides displayed notable plasma stability and aqueous low metabolicsolubility with stability. moderate to low metabolic stability.

Figure 8. Structures of cyclohexapeptides 43–47.

In 2017, our group reported the total synthesis of the natural products w ollamides A (48) and B (42) and desotamide B (49Figure) Figure(Figure 8. 8.Structures Structures 9) using of of SPPS cyclohexapeptidescyclohexapeptides of linear hexapeptide 4343–47–47. . precursors followed by cleavage and solution-phase cyclization [45]. A representative synthesis that was used to access In 2017, our group reported the total synthesis of the natural products wollamides A (48) and B In(42 2017,) and ourdesotamide group reported B (49) (Figure the total 9) using synthesis SPPS of of the linear natural hexapeptide products precursors wollamides followed A (48 ) by and B (42) andcleavage desotamide and solu Btion (49)-phase (Figure cyclization9) using SPPS [45]. ofA linear representative hexapeptide synthesis precursors that was followed used to by access cleavage and solution-phase cyclization [45]. A representative synthesis that was used to access wollamide B(42) is highlighted in Scheme9. The first Fmoc amino acid (Fmoc-Asn(Trt)-OH) was loaded onto 2-CTC resin with the aid of DIPEA in DCM. The hexapeptide sequence corresponding to wollamide B MoleculesMolecules2018, 23 2018, 1475, 23, x FOR PEER REVIEW 13 of 25 13 of 25 Molecules 2018, 23, x FOR PEER REVIEW 13 of 25 wollamide B (42) is highlighted in Scheme 9. The first Fmoc amino acid (Fmoc-Asn(Trt)-OH) was (D-Orn-Trp-Leu-D-Leu-Val-Asn)loadedwollamide onto B 2 (-42CTC) is resin highlighted with the was in aid Scheme synthesized of DIPEA 9. The in on-resinDCM.first Fmoc Thevia hexapepamino repeated acidtide ( Fmocsequence coupling-Asn( correspondingTrt) and-OH deprotection) was loaded onto 2-CTC resin with the aid of DIPEA in DCM. The hexapeptide sequence corresponding steps, asto w shownollamide in B Scheme(D-Orn-Trp9.-Leu Cleavage-D-Leu-Val of- theAsn) linear was synthesized hexapeptide on-resin from via repeated the resin coupling using and the mild deprotectionto wollamide stepsB (D-,Or asn shown-Trp-Leu in- DScheme-Leu-Val 9. -CleavageAsn) was synthesizedof the linear onhexapeptide-resin via repeated from the coupling resin using and cleavage reagent HFIP resulted in 50 having side-chain residue protecting groups still attached. The thedeprotection mild cleavage steps , reagentas shown HFIP in Scheme resulted 9 . inCleavage 50 having of the side line-chainar hexapeptide residue protecting from the groupsresin using still solution-phaseattached.the mild macrocyclizationThe cleavage solution reagent-phase HFIP macrocyclization was resulted done using in 50was HBTUhaving done using and side- DIPEAchainHBTU residueand in DIPEA DMF, protecting providingin DMF groups, provid the stilling protected cyclohexapeptidetattached.he protected The51 solutioncyclohexapeptide. Wollamide-phase macrocyclization B ( 4251.) Wollamide was obtained was B (42 done after) was using removalobtained HBTU ofafter and the DIPEAremoval side-chain in of DMF the protecting , sideprovid-chaining groups of 51 andprotectingthe final protected purification groups cyclohexapeptide of 51 using and final flash 51 purification. Wollamide column chromatographyusing B (42 flash) was column obtained chromatography on after silica removal gel. An ofon the optimizationsilica side gel.-chain An study investigatedoptimizationprotecting the efficiencygroups study of investigated 51 of and the final macrocyclization the purification efficiency usingof the step flash macrocyclization occurring column chromatography at each step occurring of the sixon at silica peptide each gel. of bondtheAn sites. optimization study investigated the efficiency of the macrocyclization step occurring at each of the This determinedsix peptide that bond macrocyclization sites. This determined of the that linear macrocyclization hexapeptide of precursor the linear betweenhexapeptide terminal precursor L-Leu and betweensix peptide terminal bond sites.L-Leu This and determinedD-Leu residues that providedmacrocyclization the most of efficient the linear macrocyclization hexapeptide precursor without D-Leu residuesanybetween detect providedterminalable epimerization L- theLeu mostand [45]D efficient-Leu. residues macrocyclization provided the most without efficient any macrocyclization detectable epimerization without [45]. any detectable epimerization [45].

Figure 9. Wollamides A (48), and B (42) and desotamide B (49). Figure 9. Wollamides A (48), and B (42) and desotamide B (49). Figure 9. Wollamides A (48), and B (42) and desotamide B (49).

Scheme 9. Solid-phase peptide synthesis of 42 on 2-chlorotrityl chloride (2-CTC) resin. Reagents and conditions:Scheme 9. Solid(a) Fmoc-phase-Asn( peptideTrt)-OH, synthesis DIPEA, of and42 on DCM 2-chlorotrityl (r.t., 3 h); (chlorideb) 25% 4 (-2methylpiperidine-CTC) resin. Reagents/DMF; and (c) Scheme 9. Solid-phase peptide synthesis of 42 on 2-chlorotrityl chloride (2-CTC) resin. Reagents and Fmocconditions:-Val-OH, (a) Fmoc DIC, - HOBt,Asn(Trt) and-OH, DMF/DCM DIPEA, and (1:1 DCM) (r.t., (r.t., 4 h 3) ; h ()d; )( b Fmoc) 25%- D4--methylpiperidineLeu-OH, DIC, HOBt,/DMF; and (c) conditions:DMF/DCMFmoc (a)-Val Fmoc-Asn(Trt)-OH,-OH, (1:1 ) DIC, (r.t., HOBt,4 h); ( eand) Fmoc DMF/DCM DIPEA,-Leu-OH, and ( 1:1DIC, DCM) ( r.t.,HOBt, 4 (r.t., h and); ( 3d DMF/DCM) h); Fmoc (b)-D 25%-Leu (1:1- 4-methylpiperidine/DMF;OH,) (r.t., DIC, 4 h ) HOBt,; (f) Fmoc and- (c) Fmoc-Val-OH,Trp(DMF/DCMBoc)-OH, (1:1 DIC,DIC,) (r.t., HOBt, HOBt, 4 h) ;and (e and) FmocDMF/DCM DMF/DCM-Leu-OH, (1:1 )DIC, (r.t., (1:1) HOBt,4 h) (r.t.,; (g and) Fmoc 4 DMF/DCM h);-D (d)-Orn( Fmoc-D-Leu-OH,Boc (1:1)-OH,) (r.t., DIC, 4 h )HOBt,; (f) Fmoc DIC, and- HOBt, and DMF/DCMTrp(Boc)-OH, (1:1) DIC, (r.t., HOBt, 4 and h); DMF/DCM (e) Fmoc-Leu-OH, (1:1) (r.t., 4 h DIC,); (g) Fmoc HOBt,-D-Orn( andBoc DMF/DCM)-OH, DIC, HOBt, (1:1) and (r.t., 4 h); (f) Fmoc-Trp(Boc)-OH, DIC, HOBt, and DMF/DCM (1:1) (r.t., 4 h); (g) Fmoc-D-Orn(Boc)-OH, DIC, HOBt, and DMF/DCM (1:1) (r.t., 4 h); (h) HFIP/DCM (1:4) (r.t., 0.5 h); (i) (concentration of 1 mM) HBTU, DIPEA, and DMF (r.t., 0.5 h; cyclization yield of 51: 72%); (j) TFA/TIPS/DCM (50:5:45) (r.t., 0.5 h, 42%).

In 2018, our group reported the antitubercular and antibacterial activities of wollamides A (48) and B (42) and desotamide B (49) as well as structural analogues thereof [46]. The optimized synthetic Molecules 2018, 23, 1475 14 of 25 route to access these compounds was described previously [45]. The 27 peptides’ library was screened against a panel of Gram-positive and -negative bacterial pathogens, which discovered that wollamides A(48) and B (42) and the position-II L-Ile analogue 52 exhibited promising antibacterial activity against Mycobacterium tuberculosis, with MIC values of 1.56 µg/mL and favorable selectivity indexes (SIs) of >100 (Figure 10)[46]. The cyclic nature of the wollamide cyclohexapeptides was crucial for their antibacterial activity, as the corresponding linear hexapeptide precursor did not show activity even at the highest concentration tested (200 µg/mL) [46]. The residues at positions II and VI were found to have a major impact on the activity and selectivity, and hence further structure–activity relationship (SAR) studies that focus on optimizing these residues are highly warranted. Molecules 2018, 23, x FOR PEER REVIEW 15 of 25

Figure 10. Synthesis and structure–activity relationship (SAR) studies of cyclohexapeptides 48, 52, Figure 10.andSynthesis 42. and structure–activity relationship (SAR) studies of cyclohexapeptides 48, 52, and 42. 3. Solid-Phase Cyclohexapeptide Synthesis Using On-Resin Cyclization

Molecules 2018, 23, 1475 15 of 25

Molecules 2018, 23, x FOR PEER REVIEW 16 of 25 3. Solid-Phase Cyclohexapeptide Synthesis Using On-Resin Cyclization In 2015, Lewis et al. synthesized two cyclic hexapeptides, 53 and 54 (Figure 11), in order to probe theirIn cell 2015, permeability Lewis et al. and synthesized PK properties, two cyclicincluding hexapeptides, oral bioavailability53 and 54 [23](Figure. They 11 ),demonstrated in order to probe that theirthe tri cell-N permeability-methylated peptideand PK properties,54 experienced including increased oral bioavailability cell permeability, [23]. They higher demonstrated plasma protein that thebinding tri-N,-methylated and decreased peptide clearance54 experienced rates compared increased to the cell non permeability,-methylated higher variant plasma 53 [23] protein. This resulted binding, andin a decreasedfavorable clearance bioavailability rates compared of 30% for to the the non-methylatedtri-N-methylated variant cyclohexapeptide53 [23]. This 54 resulted. The cyclic in a favorablehexapeptides bioavailability cyclo-Leu- ofD- 30%Leu– forLeu the-Leu tri--DN--methylatedPro-Tyr (53) cyclohexapeptide and cyclo-Leu-NMe54. The-D-Leu cyclic-NMe hexapeptides-Leu-Leu- cyclo-Leu-D-Leu–Leu-Leu-D-Pro-TyrD-Pro-NMe-Tyr (54) were synthesized (53 using) and traditional cyclo-Leu-NMe-D-Leu-NMe-Leu-Leu-D-Pro-NMe-Tyr SPPS starting from a trityl resin preloaded (with54) were allyl ester synthesized Fmoc-Tyr using, which traditional was resin SPPS-linked starting via the from Tyr aside trityl-chain resin hydroxyl preloaded group with (Scheme allyl ester 10). Fmoc-Tyr,The resin which-linked was linear resin-linked hexapeptide via the 55 Tyr was side-chain constructed hydroxyl using group a (Scheme sequence 10 ). of The coupling resin-linked and lineardeprotection hexapeptide steps.55 Thewas allyl constructed and N-terminal using a Fmoc sequence group ofs coupling were removed and deprotection using Pd(PPh steps.3)4 Theand allyl10% andpiperidine/THF,N-terminal Fmoc respectively, groups were to give removed 56, which using was Pd(PPh cyclized3)4 and on- 10%resin piperidine/THF, using HATU/HOBt respectively, to give the to giveresin56-attached, which wascyclohexapeptide cyclized on-resin 57 using. Cleavage HATU/HOBt of 57 from to give the the resin resin-attached using TFA cyclohexapeptide provided 53. 57Cyclohexapeptide. Cleavage of 57 5from4 was thesynthesized resin using from TFA 57 providedvia the global53. Cyclohexapeptideand selective introduction54 was synthesizedof N-methyl t fromgroups57 usingvia the a globalLiOtBu and base selective, followed introduction by MeI to ofprovideN-methyl resin groups-attached using trimethylated a LiO Bu base, derivative followed 58 by(Scheme MeI to 10 provide). Cleavage resin-attached of 58 from trimethylated the resin afforded derivative 54. An58 (Scheme alternative 10). synthesis Cleavage of of 5458 fromwas also the resininvestigated afforded and54 . involved An alternative a stepwise synthesis construction of 54 was of the also trimethylated investigated andlinear involved hexapeptide a stepwise using constructiontraditional SPPS ofthe followed trimethylated by cleavage linear and hexapeptide solution-phase using macrocyclization. traditional SPPS Although followed two by routes cleavage for andthe synthesis solution-phase of 54 macrocyclization.were investigated, Although the first tworoute routes involving for the global synthesis N-methylation of 54 were investigated, of the resin- thebound first cyclohexapeptide route involving global57 followedN-methylation by cleavage of was the resin-boundmore efficient cyclohexapeptide and provided crude57 followed54 in higher by cleavagepurity before was morefinal purification. efficient and provided crude 54 in higher purity before final purification.

Figure 11.11. Structures of cyclohexapeptides 53 andand 54..

In 2015, Wodtke et al. reported the design and synthesis of cyclohexapeptide analogues 59–61 containing an amino acid sequence inspired by the Asp-Glu-Lys-Ser (DEKS) motif of N-terminal telopeptide of type I collagen [47]. It was suggested that the DEKS motif adopts a β-turn conformation upon docking with its receptor; in addition, the β-turn conformation of the DEKS motif can be stabilized by incorporating it into a cyclized peptide backbone along with strategic introduction of D-Pro and Lys(4-fluorobenzoyl) residues [47]. The presence of the 4-fluorobenzoyl group in the second Lys of 59–61 could have an application as a radiolabeling site, if required, by incorporating fluorine-18 [47]. The cyclohexapeptide 59 was resistant to bovine trypsin-mediated degradation over 30 min, during which time its corresponding linear analogue was completely digested. This demonstrated how cyclized peptides offer enhanced metabolic stability over their linear counterparts [47].

Molecules 2018, 23, 1475 16 of 25 Molecules 2018, 23, x FOR PEER REVIEW 17 of 25

Scheme 10.10. SolidSolid-phase-phase synthesis synthesis of of 5353 andand 5454 onon trityl trityl resin resin.. Reagents Reagents and and conditions conditions:: (a) (a)20% 20% 4- 4-methylpiperidine/DMFmethylpiperidine/DMF (r.t., (r.t., 2 × 5 2 min× 5); min);(b) Fmoc (b)-AA Fmoc-AA-OH,-OH, PyOxim, PyOxim, DIPEA, DIPEA,and DMF/NMP and DMF/NMP (1:1) (r.t., 45 min); (c) Pd(PPh3)4 and 10% piperidine/THF (r.t., 2 h); (d) HATU, HOBt, DIPEA, and DMF/NMP (1:1) (r.t., 45 min); (c) Pd(PPh3)4 and 10% piperidine/THF (r.t., 2 h); (d) HATU, HOBt, DIPEA, and t DMF/NMP(1:1) (r.t., 18 (1:1)h); (e (r.t.,) 10% 18 TFA h); (e)/DCM 10% ( TFA/DCMr.t., 4 × 5 min (r.t.,); ( 4f) ×LiO5 min);Bu and (f) THF LiOt Bu(r.t., and 0.5THF h); (g (r.t.,) MeI 0.5 and h); (g)DMSO MeI and(r.t., DMSO0.5 h). (r.t., 0.5 h).

In 2015, Wodtke et al. reported the design and synthesis of cyclohexapeptide analogues 59–61 The synthesis of cyclohexapeptide 59–61 was carried out according to Scheme 11. Firstly, containing an amino acid sequence inspired by the Asp-Glu-Lys-Ser (DEKS) motif of N-terminal Fmoc-Lys-OAll or Fmoc-Hnl-OAll was loaded onto 2-CTC resin (attached via side chain) in THF telopeptide of type I collagen [47]. It was suggested that the DEKS motif adopts a β-turn conformation and DIPEA followed by capping unreacted resin sites with methanol. The subsequent Fmoc-amino upon docking with its receptor; in addition, the β-turn conformation of the DEKS motif can be acids were added to the sequence using standard microwave-assisted SPPS, 20% piperidine, stabilized by incorporating it into a cyclized peptide backbone along with strategic introduction of and 0.1M HOBt in DMF for the Fmoc deprotection steps and Fmoc-amino acid, HBTU, and D-Pro and Lys(4-fluorobenzoyl) residues [47]. The presence of the 4-fluorobenzoyl group in the DIPEA in NMP for the coupling steps. The resin-bound linear hexapeptide was cyclized on-resin second Lys of 59–61 could have an application as a radiolabeling site, if required, by incorporating using HATU and DIPEA in DMF after deprotection of the C-terminal allyl and N-terminal Fmoc fluorine-18 [47]. The cyclohexapeptide 59 was resistant to bovine trypsin-mediated degradation over groups with Pd(PPh ) and 20% piperidine in DMF, respectively. The 4-fluorobenzoyl moiety 30 min, during which3 4 time its corresponding linear analogue was completely digested. This was installed onto the lysine side chain using 4-fluorobenzoyl chloride after firstly removing demonstrated how cyclized peptides offer enhanced metabolic stability over their linear counterparts the 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-ethyl (Dde) protecting group with hydrazine. [47]. The cyclohexapeptide was cleaved from the resin with simultaneous removal of the side-chain The synthesis of cyclohexapeptide 59–61 was carried out according to Scheme 11. Firstly, Fmoc- protecting groups using a cleavage cocktail solution comprising TFA/triethylsilane (TES) (95/2.5/2.5) Lys-OAll or Fmoc-Hnl-OAll was loaded onto 2-CTC resin (attached via side chain) in THF and to give 59–60 in good yields. Compound 61 was synthesized by cleaving the cyclohexapeptide 62 DIPEA followed by capping unreacted resin sites with methanol. The subsequent Fmoc-amino acids from the resin using mild acid conditions for TFA/TES/DCM (1:5:94) to give 62 with side-chain were added to the sequence using standard microwave-assisted SPPS, 20% piperidine, and 0.1M protecting groups attached. Finally, Dess–Martin periodinane oxidation of 62 followed by removal of HOBt in DMF for the Fmoc deprotection steps and Fmoc-amino acid, HBTU, and DIPEA in NMP for the side-chain protecting groups provided 61 in good yield. the coupling steps. The resin-bound linear hexapeptide was cyclized on-resin using HATU and DIPEA in DMF after deprotection of the C-terminal allyl and N-terminal Fmoc groups with Pd(PPh3)4 and 20% piperidine in DMF, respectively. The 4-fluorobenzoyl moiety was installed onto the lysine side chain using 4-fluorobenzoyl chloride after firstly removing the 1-(4,4-dimethyl-2,6- dioxocyclohex-1-ylidene)-3-ethyl (Dde) protecting group with hydrazine. The cyclohexapeptide was cleaved from the resin with simultaneous removal of the side-chain protecting groups using a cleavage cocktail solution comprising TFA/triethylsilane (TES) (95/2.5/2.5) to give 59–60 in good yields. Compound 61 was synthesized by cleaving the cyclohexapeptide 62 from the resin using mild acid conditions for TFA/TES/DCM (1:5:94) to give 62 with side-chain protecting groups attached.

Molecules 2018, 23, x FOR PEER REVIEW 18 of 25

Finally,Molecules Dess2018–Martin, 23, 1475 periodinane oxidation of 62 followed by removal of the side-chain protecting17 of 25 groups provided 61 in good yield.

Scheme 11. Solid-phase peptide synthesis of 59–62 on 2-chlorotrityl chloride (2-CTC) resin. Reagents Scheme 11. Solid-phase peptide synthesis of 59–62 on 2-chlorotrityl chloride (2-CTC) resin. Reagents and conditions: (a) 1. (for X=NH) Fmoc-Lys-OAll, DIPEA, and THF (r.t., 2 h); 2. DCM/MeOH/DIPEA and conditions: (a) 1. (for X=NH) Fmoc-Lys-OAll, DIPEA, and THF (r.t., 2 h); 2. DCM/MeOH/DIPEA (17:1:2) (r.t., 3 × 2 min); 1. (for X=O), Fmoc-Hln-OAll, pyridine, and DCM/DMF (1:1) (r.t., 64 h); 2. (17:1:2) (r.t., 3 × 2 min); 1. (for X=O), Fmoc-Hln-OAll, pyridine, and DCM/DMF (1:1) (r.t., 64 h); DCM/MeOH/DIPEA (17:1:2) (r.t., 3 × 2 min); (b) 1. deprotection: 20% piperidine, 0.1M HOBt, and 2. DCM/MeOH/DIPEA (17:1:2) (r.t., 3 × 2 min); (b) 1. deprotection: 20% piperidine, 0.1M HOBt, DMF (microwave irradiation: 35 W, 75 °C, 30 s, followed by 44 W, 75 °C, 3 min); 2. coupling: Fmoc- and DMF (microwave irradiation: 35 W, 75 ◦C, 30 s, followed by 44 W, 75 ◦C, 3 min); 2. coupling: AA-OH, HBTU, DIPEA, and DMF (microwave irradiation: 21 W, 75 °C, 5 min); 3. steps 1–2 repeated; Fmoc-AA-OH, HBTU, DIPEA, and DMF (microwave irradiation: 21 W, 75 ◦C, 5 min); 3. steps 1–2 (c) Pd(PPh3)4, and DCM/NMM/acetic acid (8:2:1) (r.t., 4 h); (d) 20% piperidine/DMF (r.t., 2 × 8 min); repeated; (c) Pd(PPh3)4, and DCM/NMM/acetic acid (8:2:1) (r.t., 4 h); (d) 20% piperidine/DMF (e) HATU, DIPEA, and DMF (r.t., 4 h); (f) 2% N2H4 and DMF (r.t., 5–12 × 5 min); (g) 4-fluorobenzoyl (r.t., 2 × 8 min); (e) HATU, DIPEA, and DMF (r.t., 4 h); (f) 2% N2H4 and DMF (r.t., 5–12 × 5 min); chloride, NEt3, and DCM (r.t., 2 h); (h) TFA/TES/H2O (95/2.5/2.5) (r.t., 3 h); (i) TFA/TES/DCM (1:5:94) (g) 4-fluorobenzoyl chloride, NEt , and DCM (r.t., 2 h); (h) TFA/TES/H O (95/2.5/2.5) (r.t., 3 h); (r.t., 0.5 h); (j) Dess–Martin periodinane3 and DCM (r.t., 3 h); (k) TFA/DCM (9:1) (2r.t., 1 h; overall yields: (i) TFA/TES/DCM (1:5:94) (r.t., 0.5 h); (j) Dess–Martin periodinane and DCM (r.t., 3 h); (k) TFA/DCM 80% (59), 30% (60), and 25% (61)). (9:1) (r.t., 1 h; overall yields: 80% (59), 30% (60), and 25% (61)). In 2016, Jikyo reported the synthesis of cyclic hexapeptides 65a–d using an on-resin head-to-tail cyclizationIn 2016, strategy Jikyo on reported trichloroacetimidate the synthesis Wang of cyclic resin hexapeptides (Scheme 165a2) [48]–d using. The anD-Ser on-resin side chain head-to-tail of Fmoccyclization-D-Ser-OAll strategy was anchored on trichloroacetimidate to the trichloroacetimidate Wang resin Wang (Scheme resin 12 using)[48 ].BF3 The·O Et D-Ser2 in dry side TFA chain. Iterativeof Fmoc-D-Ser-OAll coupling and wasdeprotection anchored steps to the using trichloroacetimidate BOP/HOBt/DIPEA Wang and resin 20% using piperidine/DMF BF3·OEt2 in, dry respectivelyTFA. Iterative, for coupling five cycles and constructed deprotection resin steps-bound using BOP/HOBt/DIPEAlinear hexapeptide andintermediates 20% piperidine/DMF, having Fmoc/Boc/OAllrespectively, protecting for five cycles groups constructed intact. Pd(PPh resin-bound3)4 in CHCl linear3/AcOH/ hexapeptideN-methylmorpholine intermediates (NMM having) wasFmoc/Boc/OAll used under anhydrous protecting conditions groups intact. to complete Pd(PPh3) 4Cin-terminal CHCl3/AcOH/ O-allyl N deprotection-methylmorpholine before (NMM) the additionwas used of 20% under piperidine anhydrous in conditions DMF to remove to complete N-terminalC-terminal FmocO,-allyl which deprotection afforded anchored before the linear addition peptidesof 20% 63 piperidinea–d having in deprotected DMF to remove C- andN -terminalN-terminals Fmoc,. On- whichresin c affordedyclization anchored to afford linear 64a–d peptides was achieved63a–d withhaving PyBOP/HOBt/DIPEA deprotected C- and N-asterminals. the coupling On-resin agent cyclization, and cleavage to afford from64a the– dresinwas using achieved 95% with TFAPyBOP/HOBt/DIPEA resulted in cyclohexapeptides as the coupling 65a–d in agent, 13–63% and yields cleavage. Interestingly, from the resin on-resin using cyclization 95% TFA resultedof 64c, in havingcyclohexapeptides two D-Pro residues65a– dinin the 13–63% chain, yields. gave the Interestingly, highest yield on-resin of cyclized cyclization product of 64c65c, with having a yield two D-Proof 63%.residues in the chain, gave the highest yield of cyclized product 65c with a yield of 63%.

Molecules 2018, 23, 1475 18 of 25 Molecules 2018, 23, x FOR PEER REVIEW 19 of 25

SchemeScheme 12.12Solid-phase. Solid-phase peptide peptide synthesis synthesis (SPPS) (SPPS) of cyclohexapeptides of cyclohexapeptides65a–d. Reagents 65a–d. andReagents conditions: and conditions: (a) Fmoc-D-Ser-OAll, BF3·OEt2, and dry TFA (r.t., 1 h); (b) SPPS for five cycles: (a) Fmoc-D-Ser-OAll, BF3·OEt2, and dry TFA (r.t., 1 h); (b) SPPS for five cycles: deprotection: 20% deprotection: 20% piperidine/DMF; coupling: Fmoc-AA-OH, BOP, HOBt, DIPEA, and DMF; (c) piperidine/DMF; coupling: Fmoc-AA-OH, BOP, HOBt, DIPEA, and DMF; (c) Pd(PPh3)4 and PhSiH3; (d)Pd(PPh 20%3 piperidine/DMF;)4 and PhSiH3; (d) (e) 20% PyBOP, piperidine/DMF HOBt, DIPEA,; (e) andPyBOP DMF, HOBt (r.t., 17, DIPEA h); (f), 95% and TFADMF (r.t., (r.t 2., h;17 yields: h); (f) . 15%95% (TFA65a), ( 38%r.t., 2 ( 65bh; yield), 63%s: 15% (65c ),(65a and), 13%38% ((65d65b).), 63% (65c), and 13% (65d)

In 2018, Chen et al. reported the synthesis and antibacterial evaluation of cyclohexapeptides In 2018, Chen et al. reported the synthesis and antibacterial evaluation of cyclohexapeptides desotamide B (49) and wollamide B (42) in addition to a series of their structural analogues by desotamide B (49) and wollamide B (42) in addition to a series of their structural analogues utilizing an on-resin head-to-tail cyclization strategy (Scheme 13) [49]. Fmoc-Asp-OAll was first by utilizing an on-resin head-to-tail cyclization strategy (Scheme 13)[49]. Fmoc-Asp-OAll anchored to the Rink Amide AM resin through the use of the coupling reagent O-(1H-6- was first anchored to the Rink Amide AM resin through the use of the coupling reagent chlorobenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU)/DIPEA to form O-(1H-6-chlorobenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU)/DIPEA the resin-bond amino acid 66. SPPS was used to form the linear peptide 67, which contained different to form the resin-bond amino acid 66. SPPS was used to form the linear peptide 67, which contained Fmoc protected amino acids. The allyl group was uncapped with Pd(PPh3)4 and phenylsilane. The N- different Fmoc protected amino acids. The allyl group was uncapped with Pd(PPh ) and phenylsilane. terminal amino group was then released by using a treatment of 20% piperidine/DMF3 4 to form the The N-terminal amino group was then released by using a treatment of 20% piperidine/DMF linear peptide precursor 68. The on-resin cyclization step included treatment with (7-azabenzotriazol- to form the linear peptide precursor 68. The on-resin cyclization step included treatment with 1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP)/HOAt/NMM in NMP for 12 h to (7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP)/HOAt/NMM produce the protected cyclohexapeptide 69 on-resin. The target cyclohexapeptides were produced in NMP for 12 h to produce the protected cyclohexapeptide 69 on-resin. The target cyclohexapeptides after final cleavage and global deprotection with TFA/phenol/water/TIPS (88:5:5:2, v/v/v/v). were produced after final cleavage and global deprotection with TFA/phenol/water/TIPS (88:5:5:2, v/v/v/v).

Molecules 2018, 23, 1475 19 of 25 Molecules 2018, 23, x FOR PEER REVIEW 20 of 25 Molecules 2018, 23, x FOR PEER REVIEW 20 of 25

Scheme 13. Representative solid-phase peptide synthesis (SPPS) of 49 on Rink amide resin. Reagents Scheme 13. Representative solid-phase peptide synthesis (SPPS) of 49 on Rink amide resin. Reagents andScheme conditions: 13. Representative (a) 20% piperidine solid-phase/DMF peptide (r.t., 5 synthesis× 10 min) (SPPS); (b) Fmoc of 49-Aspon Rink-OAll, amide HCTU, resin. DIPEA Reagents, and and conditions: (a) 20% piperidine/DMF (r.t., 5 × 10 min); (b) Fmoc-Asp-OAll, HCTU, DIPEA, and DMFand conditions: (r.t., 1 h); ( (a)c) SPPS 20%: piperidine/DMFcoupling: Fmoc-AA (r.t.,-OH, 5 × HCTU,10 min); DIPEA (b) Fmoc-Asp-OAll,, and DMF (r.t., HCTU, 1 h); d DIPEA,eprotection: and DMF (r.t., 1 h); (c) SPPS: coupling: Fmoc-AA-OH, HCTU, DIPEA, and DMF (r.t., 1 h); deprotection: 20%DMF (r.t.,piperidine 1 h); (c)/DMF SPPS: ( coupling:r.t.; 5 × Fmoc-AA-OH,10 min); coupling/deprotection HCTU, DIPEA, and DMFsteps (r.t.,repeated. 1 h); deprotection: (d) Pd(PPh 20%3)4, 20%piperidine/DMF piperidine/DMF (r.t.; 5 (×r.t.;10 min);5 × 10 coupling/deprotection min); coupling/deprotection steps repeated. steps (d) repeated. Pd(PPh )(d, phenylsilane,) Pd(PPh3)4, phenylsilane, and DCM; (e) PyAOP, HOAt, NMM, and NMP (r.t., 12 h); (f) TFA/H3 24O/phenol/TIPS phenylsilane, and DCM; (e) PyAOP, HOAt, NMM, and NMP (r.t., 12 h); (f) TFA/H2O/phenol/TIPS (88:5:5:2,and DCM; v/v/v/v (e) PyAOP,) (r.t., 2 HOAt, h). NMM, and NMP (r.t., 12 h); (f) TFA/H2O/phenol/TIPS (88:5:5:2, v/v/v/v) (88:5:5:2,(r.t., 2 h). v/v/v/v) (r.t., 2 h). Cyclohexapeptide 71 (Figure 12) showed inhibitory antibacterial activity for methicillin-resistant Cyclohexapeptide 71 (Figure 12) showed inhibitory antibacterial activity for methicillin-resistant S. aureusCyclohexapeptide (MRSA)2 (MIC71 = (Figure128 µg/mL) 12) showed, MRSA4 inhibitory (MIC = 32 antibacterial µg/mL), MRSA5 activity (MIC for methicillin-resistant= 64 µg/mL), and S. S. aureus (MRSA)2 (MIC = 128 µg/mL), MRSA4 (MIC = 32 µg/mL), MRSA5 (MIC = 64 µg/mL), and S. aureusS. aureus (MIC(MRSA)2 = 64 µg/mL), (MIC = which 128 µ g/mL),was about MRSA4 2-fold (MIC increase = 32 inµ g/mL),activity MRSA5compared (MIC to desotamide = 64 µg/mL), B ( and49). aureus (MIC = 64 µg/mL), which was about 2-fold increase in activity compared to desotamide B (49). ItS. was aureus therefore(MIC = suggested 64 µg/mL), that which replacing was about Val with 2-fold Ile increase improves in activitybioactivity. compared Compounds to desotamide 70 and 7 B2 It was therefore suggested that replacing Val with Ile improves bioactivity. Compounds 70 and 72 showed(49). It was a loss therefore of activity suggested (MIC that> 128 replacing µg/mL), Val suggest withing Ile improvesthat D-Leu bioactivity. is necessary Compounds for antibacterial70 and showed a loss of activity (MIC > 128 µg/mL), suggesting that D-Leu is necessary for antibacterial activity.72 showed The a loss of activity of (MIC 73 suggest > 128 µedg/mL), that D suggesting-Orn may be that required D-Leu isfor necessary the antibacterial for antibacterial activity activity. The loss of activity of 73 suggested that D-Orn may be required for the antibacterial activity ofactivity. wollamide The loss B of (42 activity). However, of 73 suggested it was also that suggested D-Orn may that be requiredD-Orn may for the increase antibacterial cytotoxicity activity, as of of wollamide B (42). However, it was also suggested that D-Orn may increase cytotoxicity, as wollamide B (42).) showed However, higher it was cytotoxicity also suggested than that 73 in D-Orn MCF- may7 and increase HepG- cytotoxicity,2 assays. Nearly as wollamide all of the wollamide B (42) showed higher cytotoxicity than 73 in MCF-7 and HepG-2 assays. Nearly all of the syB(nthesized42) showed cyclopeptides higher cytotoxicity lacked thancytotoxicity73 in MCF-7 (IC50 > and 100 HepG-2 µM) against assays both. Nearly human all oftumor the synthesized cells MCF- synthesized cyclopeptides lacked cytotoxicity (IC50 > 100 µM) against both human tumor cells MCF- 7cyclopeptides and HepG-2, lacked except cytotoxicity for wollamide (IC50 B >(42 100), whichµM) against exhibited both cytotoxicity human tumor against cells MCF-7HepG-2 and (IC HepG-2,50 = 79.2 7 and HepG-2, except for wollamide B (42), which exhibited cytotoxicity against HepG-2 (IC50 = 79.2 µM).except for wollamide B (42), which exhibited cytotoxicity against HepG-2 (IC50 = 79.2 µM). µM).

Figure 12. Structures of desotamide B (49), wollamide B (42), and cyclohexapeptide analogues 70– FigureFigure 12. 12.Structures Structures of of desotamide desotamide B B ( 49(49),), wollamide wollamide B B ( 42(42),), and and cyclohexapeptide cyclohexapeptide analogues analogues70 70–73– . 73. 73. In 2018, Fagundez et al. synthesized cyclohexapeptides via Fmoc/SPPS by using 2-CTC resin In 2018, Fagundez et al. synthesized cyclohexapeptides via Fmoc/SPPS by using 2-CTC resin followedIn 2018, by macrolactamization Fagundez et al. synthesized either on-resin cyclohexapeptides (75–78) (Figure via 13 ) Fmoc/SPPS or in solution by phase using after 2-CTC cleavage resin followed by macrolactamization either on-resin (75–78) (Figure 13) or in solution phase after cleavage followedof linear by peptide macrolactamization precursors from either resin on-resin [50]. (7 A5– general78) (Figure method 13) or in for solution the on-resin phase after cyclization cleavage of of linear peptide precursors from resin [50]. A general method for the on-resin cyclization of of linear peptide precursors from resin [50]. A general method for the on-resin cyclization of cyclohexapeptides 75–78 is shown in Scheme 14. The synthesis of 74 consists of the peptide sequence cyclohexapeptides 75–78 is shown in Scheme 14. The synthesis of 74 consists of the peptide sequence

Molecules 2018, 23, x FOR PEER REVIEW 21 of 25 Molecules 2018, 23, x FOR PEER REVIEW 21 of 25 being synthesized on the resin in a similar fashion as is discussed above and being cleaved before being synthesized on the resin in a similar fashion as is discussed above and being cleaved before undergoing macrocyclization in the solution phase. The compounds produced through on-resin undergoing macrocyclization in the solution phase. The compounds produced through on-resin cyclization allowed new derivatives to be synthesized as a result of the presence of a free carboxylic cyclization allowed new derivatives to be synthesized as a result of the presence of a free carboxylic acid. Solution-phase cyclization resulted in the ability to produce more diverse cyclic peptides; acid. Solution-phase cyclization resulted in the ability to produce more diverse cyclic peptides; however, the on-resin route was more convenient, as larger amounts of product with higher yield however, the on-resin route was more convenient, as larger amounts of product with higher yield and acceptable purity could be produced. and acceptable purity could be produced. The activity against the chloroquine-resistant K1 strain of P. falciparum for each of the The activity against the chloroquine-resistant K1 strain of P. falciparum for each of the synthesized cyclopeptides was determined. Two cyclopentapeptides, which were also synthesized synthesized cyclopeptides was determined. Two cyclopentapeptides, which were also synthesized and compared to the cyclic hexapeptides, were found to have less activity. Six of the synthesized andMolecules compared2018, 23, 1475to the cyclic hexapeptides, were found to have less activity. Six of the synthesized20 of 25 cyclic hexapeptides exhibited sub-micromolar activity against P. falciparum K1. Hexapeptides 75 and cyclic hexapeptides exhibited sub-micromolar activity against P. falciparum K1. Hexapeptides 75 and 76 exhibited a free carboxylic group from Glu, which permitted the production of derivatives or 76 exhibited a free carboxylic group from Glu, which permitted the production of derivatives or productscyclohexapeptides with more75 soluble–78 is shown salts. inCyclic Scheme hexapeptide 14. The synthesis 74 cyclo of-Cys(Trt)74 consists-Gly of-Thr( thetBu) peptide-Gly-Cys(Trt) sequence- productsbeing synthesized with more on soluble the resin salts. in aCyclic similar hexapeptide fashion as is74discussed cyclo-Cys(Trt) above-Gly and-Thr( beingtBu) cleaved-Gly-Cys(Trt) before- Gly was determined to be very active as well as selective against P. falciparum (EC50 = 28 nM). It was Gly was determined to be very active as well as selective against P. falciparum (EC50 = 28 nM). It was suggestedundergoing that macrocyclization the biological activity in the solutionof 74 was phase. affected The by compounds substitution produced of the larger through hydrophobic on-resin suggested that the biological activity of 74 was affected by substitution of the larger hydrophobic aminocyclization acids allowed Phe, Met, new and derivatives Ile contained to be in synthesized 17 and 19 by as Gly a result, as well of the as presence by the retention of a free of carboxylic one Thr amino acids Phe, Met, and Ile contained in 17 and 19 by Gly, as well as by the retention of one Thr andacid. two Solution-phase Cys. cyclization resulted in the ability to produce more diverse cyclic peptides; andhowever, two Cys. the on-resin route was more convenient, as larger amounts of product with higher yield and acceptable purity could be produced.

Figure 13. Structures of cyclohexapeptides 74–78. Figure 13 13.. Structures of cyclohexapeptides 7474–7878.

Scheme 14. Solid-phase peptide synthesis of 75–78 on 2-chlorotrityl chloride (2-CTC) resin. Reagents Scheme 14.14. SolidSolid-phase-phase peptide synthesis of 7755–7788 on 2 2-chlorotrityl-chlorotrityl chloride (2-CTC)(2-CTC) resin. Reagents and conditions: (a) Fmoc-Glu-OAll, DIPEA, and DMF; (b) capped with MeOH; (c) deprotection: 20% and conditions: ( (a)a) Fmoc Fmoc-Glu-OAll,-Glu-OAll, DIPEA, and DMF; ( (b)b) c cappedapped with MeOH; ( (c)c) d deprotection:eprotection: 20% piperidine/DMF; (d) coupling: Fmoc-AA-OH, HBTU, DIPEA, and DMF (r.t., 1–2 h); (e) Pd(PPh3)4 and piperidine/DMF;piperidine/DMF; (d (d)) coupling: coupling: Fmoc Fmoc-AA-OH,-AA-OH, HBTU, HBTU, DIPEA DIPEA,, and and DMF DMF (r.t., (r.t., 1–2 1–2 h); ( h);e) Pd(PPh (e) Pd(PPh3)4 and) 10% piperidine/THF (r.t., 3 h); (f) DIC, Cl-HOBt, and DMF/DCM (8:2) (r.t., overnight); (g) 1%3 4 10%and 10% piperidine piperidine/THF/THF (r.t., (r.t., 3 h) 3; h);(f) (f) DIC, DIC, Cl Cl-HOBt,-HOBt, and and DMF DMF/DCM/DCM (8:2) (8:2) (r.t., (r.t., overnight overnight);); ( (g)g) 1% TFA/DCM (r.t., 2–3 min; overall yield: 51% (76), 63% (77), 46% (78), and 80% (79)). TFA/DCMTFA/DCM ( (r.t.,r.t., 2 2–3–3 min min;; overall yield yield:: 51 51%% ( 76),), 63 63%% ( (77), 46 46%% ( (78), and 80 80%% ( 79))).).

The activity against the chloroquine-resistant K1 strain of P. falciparum for each of the synthesized cyclopeptides was determined. Two cyclopentapeptides, which were also synthesized and compared to the cyclic hexapeptides, were found to have less activity. Six of the synthesized cyclic hexapeptides exhibited sub-micromolar activity against P. falciparum K1. Hexapeptides 75 and 76 exhibited a free carboxylic group from Glu, which permitted the production of derivatives or products with more soluble salts. Cyclic hexapeptide 74 cyclo-Cys(Trt)-Gly-Thr(tBu)-Gly-Cys(Trt)-Gly was determined to be very active as well as selective against P. falciparum (EC50 = 28 nM). It was suggested that the Molecules 2018, 23, 1475 21 of 25 biological activity of 74 was affected by substitution of the larger hydrophobic amino acids Phe, Met, and Ile contained in 17 and 19 by Gly, as well as by the retention of one Thr and two Cys. Together, in the on-resin head-to-tail cyclization strategy, the peptide stays anchored to the resin throughout the final cyclization step, whereas in classical methods reported in previous studies, cleavage of the on-resin linear precursors occurred before cyclization in the solution phase. It was suggested that the on-resin head-to-tail cyclization strategy could improve the process of synthesizing cyclic peptides as well as reduce the quantity of product lost during synthesis.

4. Conclusions In conclusion, cyclohexapeptides represent an important class of natural products and medicinal molecules. Compared to their linear hexapeptide counterparts, macrocyclic hexapeptides often possess improved cell permeability, higher metabolic stability, and enhanced bioavailability as a result of their cyclic nature and incorporation of unnatural amino acids. SPPS has enabled the rapid, efficient synthesis of hexapeptide and peptidomimetic libraries for subsequent biological evaluation and high-throughput screening. In this review, we summarize recent methods used in the successful construction of cyclohexapeptides using SPPS followed by on-resin or solution-phase cyclization. We also highlight recent advances in solid-phase hexapeptide synthesis and their applications, ranging from natural product total synthesis, synthetic methodology development, medicinal activities, and drug development, to analyses of conformational and physiochemical properties. Head-to-tail cyclization of linear peptides can be accomplished by either on-resin or solution-phase macrolactamization once they are released from the resin. The advantage of on-resin cyclization is that the formation of linear and/or cyclic oligomeric side products can be minimized. In addition, cyclization on-resin can shorten the synthetic route and minimize purification steps, although the reaction progress is more challenging to monitor. In the case of solution-phase cyclization, performing the reaction under dilute conditions (1–5 mM) can favor cyclization over dimer/oligomer formation. In some examples, adding the linear peptide precursor in a dropwise fashion to maintain high dilution was used successfully. Utilizing a turn-inducing pseudoproline residue can greatly enhance cyclization rates, although it is required to be deprotected post cyclization, with additional reaction step(s). On the other hand, epimerization of the C-terminal amino acid during cyclization can be attenuated by employing efficient coupling reagents, such as the azabenzotriazole class. Furthermore, epimerization can be completely avoided by choosing to cyclize at a non-epimerizable residue such as C-terminal Gly (when applicable) or by employing the method of O-N-acyl migration on cyclodepsipeptides precursors. Among various solid supports, 2-CTC resin remains one of the most popular resins in SPPS because of its mild acidolytic cleavage conditions as well as steric effect, preventing diketopiperazine formation.

Funding: This work was partly supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award No. P20GM103466. A.F. was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award No. R25GM113747. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Conflicts of Interest: The authors declare no conflict of interest. Molecules 2018, 23, 1475 22 of 25

Abbreviations The following abbreviations are used in this manuscript:

ADME Absorption, distribution, metabolism and excretion BOP (Benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate CsA Cyclosporin A 2-CTC 2-Chlorotrityl chloride DBU 1,8-Diazabicyclo[5.4.0]undec-7-ene DCM Dichloromethane Dde 1-(4,4-Dimethyl-2,6-dioxocyclohex-1-ylidene)-3-ethyl DIC N,N0-Diisopropylcarbodiimide DIPEA N,N-Diisopropylethylamine DMAP 4-Dimethylaminopyridine DMF Dimethylformamide DMTMM.BF4 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium tetrafluoroborate EC50 50% of Maximal effective concentration Fmoc 9-Fluorenylmethoxycarbonyl Fmoc-AA-OH Fmoc protected amino acid 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide HATU hexafluorophosphate HBTU N,N,N0,N0-Tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate O-(1H-6-Chlorobenzotriazole-1-yl)-1,1,3,3-tetramethyluronium HCTU hexafluorophosphate HFIP 1,1,1,3,3,3-Hexafluoro-2-propanol HMPP 3-(4-Hydroxymethylphenoxy)propionic acid HOAt 3-Hydroxytriazolo[4,5-b]pyridine HOBt Hydroxybenzotriazole IC50 50% of Maximal inhibitory concentration MIC Minimum inhibitory concentration MRSA Methicillin-resistant S. aureus NMM N-Methylmorpholine NMP N-Methyl-2-pyrrolidone NMR Nuclear magnetic resonance PK Pharmacokinetic PyAOP (7-Azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate PyBOP (Benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate [Ethyl cyano(hydroxyimino)acetato-O2]tri-1-pyrrolidinylphosphonium PyOxim hexafluorophosphate RP-HPLC Reverse phase-high-performance liquid chromatography r.t. Room temperature SAR Structure-activity relationship SI Selectivity index SPPS Solid-phase peptide synthesis TES Triethylsilane TFA Trifluoroacetic acid TFMSA Trifluoromethanesulfonic acid THF Tetrahydrofuran TIPS Triisopropylsilane WA Wollamide A WB Wollamide B Molecules 2018, 23, 1475 23 of 25

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