Recent Advances in Macrocyclic Drugs and Microwave-Assisted and/or Solid-Supported Synthesis of Macrocycles

Macrocycles represent attractive candidates in organic synthesis and drug discovery. Since 2014, nineteen macrocyclic drugs, including three radiopharmaceuticals, have been approved by FDA for the treatment of bacterial and viral infections, cancer, obesity, immunosuppression, etc. As such, new synthetic methodologies and high throughput chemistry (e.g., microwave-assisted and/or solid-phase synthesis) to access various macrocycle entities have attracted great interest in this chemical space. This article serves as an update on our previous review related to macrocyclic drugs and new synthetic strategies toward macrocycles (Molecules, 2013, 18, 6230). In this work, I first reviewed recent FDA-approved macrocyclic drugs since 2014, followed by new advances in macrocycle synthesis using high throughput chemistry, including microwave-assisted and/or solid-supported macrocyclization strategies. Examples and highlights of macrocyclization include macrolactonization and macrolactamization, transition-metal catalyzed olefin ring-closure metathesis, intramolecular C–C and C–heteroatom cross-coupling, copper- or ruthenium-catalyzed azide–alkyne cycloaddition, intramolecular SNAr or SN2 nucleophilic substitution, condensation reaction, and multi-component reaction-mediated macrocyclization, and covering the literature since 2010.


Introduction and Recently Approved Macrocyclic Drugs
Macrocycles continue to serve as an important class of compounds and have had a profound impact on chemistry, biology, and medicine [1][2][3]. Because of their cyclic nature, conformational and configurational characteristics, along with template-induced preorganization associated with macrocycles [4], compared to small molecule drugs and large biologics, macrocycles can offer unique drug-like profiles such as favorable pharmacokinetic and pharmacodynamic (PK/PD) parameters and improved oral bioavailability, enhanced metabolic stability and cell permeability, increased binding affinity, and desirable conformational rigidity [1,5]. As such, new synthetic strategies and structure-activity relationship (SAR) studies of macrocyclic chemical entities and related natural products remain an attractive research area in this field [6][7][8].

Macrolactonization and Macrolactamization
Caporale et al. reported the synthesis and evaluation of a novel, cyclic parathyroid hormone fragment analog via side-chain cyclization of serine residues in 2010 [35]. On-resin diester bridge formation of 1 was performed using adipic acid via acylation of the hydroxyl groups of Serines 6 and 10 (Scheme 1). Specifically, following selective deprotection of the trityl groups from the Ser residues in 1 with 1% TFA/CH2Cl2, solid-supported peptide 2 with a cross-linked side chain was obtained via esterification with adipic acid upon in situ activation in the presence of HATU/HOAt/collidine [35]. Scheme 1. On-resin synthesis of a cyclic peptide 2 via side-chain diester cyclization strategy [35].
In order to extend their previous work in the synthesis of aza crowns [36], in 2012, Rostami et al. reported the MW-assisted synthesis of new aza thia crowns [37]. Specifically, aza thia crowns 5 were synthesized from α, α-bis(bromomethyl) benzene 3, and methylthioglycolate, followed by the reaction of this diester 4 and diamines in ethyleneglycol under MW irradiation at 100 • C for 9 min in 74-91% yields (Scheme 2). In contrast, conventional reflux in methanol for 24 h gave much lower yields (9-19%) [37]. Cini et al. reported the MW-assisted synthesis of conformationally constrained peptidomimetics in 2012 [38]. The cyclopentapeptide analog 7 bearing an arginylglycylaspartic acid (RGD) motif, along with an enantiopure 7-substituted azepane-2-carboxylic acid (ACA) linker, was synthesized from a linear counterpart 6 in the presence of HATU/DIEA in dichloromethane (0.7 mM) at 75 • C under MW irradiation (25 W) for 25 min in 79% yield, followed by global side-chain deprotection in TFA/thioanisole/H 2 O (90/5/5) at room temperature for 3 h (Scheme 3) [38]. Macrocycle 7 showed low micromolar affinity towards α ν β 3 and α ν β 5 receptors with IC 50 values of 1.8 and 2.9 µM, respectively [38]. Ferrie et al. reported the solid-phase synthesis and a comparative protease stability study of macrocyclic hexapeptides to mimic two endocrine hormones in 2013 [39]. Cyclohexapeptides 11 were synthesized using Rink resin and standard Fmoc amino acid coupling protocol, deprotection of the allyl group in 8, on-resin macrolactamization of 9, resin cleavage to provide 11 (Scheme 4) [39]. These endocrine peptide mimics showed improved stability profiles relative to naturally occurring vasopressin, oxytocin, and a linear control peptide. Tao et al. reported the synthesis of azole cyclopeptide analogs via an on-resin cyclization-cleavage strategy in 2013 [40]. A selected example 13 from solid-supported linear precursor 12 is shown in Scheme 5, and using this developed solid-phase-based cyclitive-cleavage strategy, a chemical library was synthesized efficiently to produce >100 diverse azole cyclopeptide derivatives with various ring sizes, as modulators of multidrug resistance efflux pumps Choi et al. reported a highly efficient pre-activation cyclization of the long peptide via succinimidyl ester-amine reaction strategy in 2015 [41]. After the formation of a preactivated succinimidyl ester precursor, on-resin macrocyclization of the 25 AA peptide 15 was achieved effectively to yield cyclopeptide 16 (Scheme 6) [41]. . The Zr(IV) macrocycle complex, 23, showed good antibacterial activities against extended-spectrum β-lactamase (ESBL)-producing E. coli strains [42].
Moreira et al. reported the synthesis of macrocyclic daptomycin analogs via replacing the ester with an amide in 2019 [44]. A representative example is shown in Scheme 9. Specifically, macrocyclization of peptide 27 was performed using PyAOP/HOAt/2,4,6collidine in DMF/DCM (3/1) containing 1% Triton™ X-100, followed by simultaneous resin cleavage and global deprotection to provide daptomycin analog 28 [44]. Previously, in 2016, Lohani et al. reported an alternative solid-supported synthesis of a daptomycin analog DapE12W13 only using single α-azido amino acid and on-resin macrocyclization was performed prior to the construction of the side chain containing the decanoyl tail [45]. In 2019, Itoh and Inoue reviewed solid-supported total synthesis of macrocyclic natural peptides with branched chains (e.g., polymyxin E2 and daptomycin) via four-dimensionally orthogonal protective group strategies [46]. Arbour et al. reported the on-resin and self-cleaving head-to-tail macrolactamization of unprotected peptides via mild N-acyl urea activation in 2019 [47]. The macrocyclization of unprotected N-acyl urea-linked peptides 29 was mediated by N-terminal cysteine (Scheme 10). The S-to-N acyl transfer reaction, a robust and high-yielding chemoselective ligation methodology to form an amide bond, is widely used in organic synthesis, medicinal chemistry, and chemical biology [48]. Specifically, this efficient S-to-N acyl transfer cascade process involves the initial formation of a reactive thioester intermediate, followed by the acyl transfer to the nucleophilic NH 2 group. In this work, diverse macrocycles such as tetraand pentapeptides 30 and 31 and the intramolecular disulfide-linked 14-AA-peptides 32 (sunflower trypsin inhibitor 1) were synthesized and demonstrated, preventing head-to-tail dimer formation and hydrolysis of most substrates [47].

Scheme 9.
On-resin synthesis of macrocyclic daptomycin analog via the amide coupling strategy [44]. Qu et al. reported the synthesis of disulfide surrogate peptides with large-span surrogate bridges via a native-chemical-ligation (NCL)-assisted diaminodiacid (DADA) approach in 2020 [49]. As an example, on-resin macrocyclization of linear peptide 33 was performed using PyAOP/HOAt/NMM at 37 • C for 3 h (2 times) to provide resin-bound 16-AA-peptide 34 with a thioether linkage as a complex mixture (Scheme 11) [49]. The failure of this on-resin DADA cyclization strategy was presumably due to its large 15AA ring size. Subsequently, a more effective NCL-assisted DADA strategy was developed to achieve large ring macrocyclization in the solution phase and with an alternative cyclization site (Trp8-Cys9) [49]. Bérubé et al. reported the solid-phase total synthesis and antimalarial evaluation of macrocyclic octapeptide dominicin, isolated from a marine sponge in 2020 [50]. The on-resin cyclization−cleavage reaction of 35 was performed using LiBr (5 eq) and DIPEA (2.5 eq) in CH 2 Cl 2 /THF (20:1), providing 36a in a 58% yield as a cyclic monomer (>99%), based on the initial resin loading (Scheme 12). Subsequent catalytic Pd/C hydrogenation in acetic acid to remove the benzyl group on the side chain provided dominicin (36b) in a 53% yield after preparative HPLC purification. An alternative methodology was also developed using entirely on-resin synthesis and biorthogonal protection in N-Boc-L-Thr(OtBu)-OH [50]. In 2018, Bérubé et al. reported the on-resin synthesis of pseudacyclins A-E via a similar head-to-side chain concomitant cyclization-cleavage strategy [51]. On-resin macrocyclizations of hexapeptides were previously reviewed by Prior et al. [52]; additional cyclopeptides include those synthesized via standard macrocyclization amide coupling strategy [53][54][55][56][57][58][59], synthetic peptide macrocycles with two-strand forming segments [60], on-resin macrolactamization [61], diaminodiacid-based macrocyclopeptides with a 1,2,3-triazole linker to mimic the disulfide bond [62], a cyclic peptide with a side chain-to-side chain cyclization and a central warhead residue [63], and solid-phase synthesis of radiolabeled somatostatin octapeptide analogs with macrocyclic disulfide linkage and conjugated to a tetraazacyclododecane chelator motif [64]. In 2013, Kumarn et al. reported the synthesis of cycloheptapeptide integerrimide A via an on-resin tandem Fmocdeprotection-macrocyclization strategy [65]. Moreover, in 2013, Thakkar et al. analyzed >2 million peptides to investigate on-resin cyclization efficiency with regard to various ring sizes, peptide sequence, and solvent [66].

Transition-Metal Catalyzed Olefin Ring-Closure Metathesis (RCM) Macrocyclization
Khan et al. reported the MW-assisted solid-phase synthesis of cyclic peptoids via the RCM strategy in 2011 [80]. Synthesis of 38 was performed from the model peptoid 37, a heptamer with 3-buten-1-amine at the first and last positions, using Hoveyda-Grubbs second-generation catalyst (2 mol%) in 1,2-dichlorobenzene under MW irradiation for 2 min (4 × 30 s) (Scheme 13). The HPLC yield of the cyclic peptoid 38 as a mixture of E/Z isomers was~80% following the resin cleavage in 92% TFA [80]. Accordingly, cyclopeptoids 40 with different ring sizes were synthesized from 39 in 70-85% HPLC yields using the same MW RCM protocol. In contrast, the HPLC yields were slightly higher when the reactions were performed under conventional heating in dichloromethane at 40 • C for 2 h, but with longer reaction times [80].  [81]. Macrocycles 43-(E) and 45-(E), the most potent IRAP inhibitors with high stability against proteolysis by metallopeptidases, were synthesized from 41 and 44 on solid and solution phase, respectively (Scheme 14). The Wang resin was used in the MW-assisted solid-phase synthesis of 41, and the on-resin RCM was performed at 140 • C using the Hoveyda-Grubbs second-generation catalyst and 1,2-dichloroethane (DCE) as solvent under microwave heating [81]. Interestingly, 43 was isolated from a complex reaction mixture and obtained in 2% yield, following isomerization of 41, ring contraction, and subsequent double bond migration [81]. Accordingly, macrocycles 45-(E) and 45-(Z) were obtained in 28% and 6% yields, respectively.

Scheme 14.
On-resin or solution phase MW-assisted RCM cyclization of 14-membered 43 and 45 [81]. Baron et al. reported the MW-assisted synthesis of macrocyclic pseudopeptides via RCM cyclization-cleavage strategy using a cis-5-aminopent-3-enoic acid (cis-Apa) linker in 2011 [82]. Macrocycle 47 with an RGD motif was synthesized from 46 using Grubbs' second-generation catalyst in dichloromethane at 100 • C under MW irradiation in 64% purity (Scheme 15). In contrast, when this reaction was performed at 60 • C for 5 h under MW irradiation without the catalyst, 47 was obtained in a higher 73% purity. Following reverse-phase chromatography purification, 47 was isolated as two configurational E and Z-isomers (3/1 ratio) in an overall 25% yield from the resin and a 58% cyclization yield from 46. In addition, compared to conventional heating, MW heating is more efficient with higher yields and shorter reaction times [82]. Macrocyclic RGD peptides may have the potential to function as potent and/or selective α 5 β 1 and α ν β 3 integrin antagonists [83]. Lampa et al. reported the RCM macrocyclization of the P2 phenylglycine and the alkenylic P1 and HCV NS3 protease inhibition in 2011 [84]. As an example, the RCM of diastereomeric starting material 48 using Hoveyda-Grubbs' second-generation catalyst in dichloroethane at 130 • C under MW irradiation is shown in Scheme 16. p-Benzoquinone was also added in the reaction to minimize the formations of ring-contracted and double bond migration side products. Four RCM products, 49-S, 49-R, 50-S, and 50-R, were obtained as stereoisomers R/S and configurational E and Z-isomers. In order to evaluate the reaction outcome, the impact of different methods (e.g., increasing temperature and MW irradiation) was measured, and it was found that the substrate appeared to play a more important role in affecting reaction outcomes relative to the cyclization method [84]. Raymond et al. reported the MW-assisted macrocyclic olefin metathesis at high concentrations using a phase-separation strategy in 2014 [85]. In this work, a protocol to promote macrocyclic olefin metathesis was developed at relatively high concentrations (up to 60 mM) under MW irradiation. As examples, diverse macrocyclic scaffolds such as 52, 54, and 56 with different alkyl, aryl, or amino acid spacers were synthesized from their respective diene substrates in the presence of the Grubbs-Hoveyda second-generation catalyst and a mixture of PEG 500 (OMe 2 )/methyl t-butyl ether (MTBE) at 100 • C under MW heating for 2 h in 60-78% yields (Scheme 17).
Qian et al. reported the synthesis of imidazolium-containing phosphopeptide macrocycles via neighbor-directed histidine N(τ)-alkylation in 2015 [86]. MW-assisted RCM reaction of 57 was performed on a solid support and by leveraging the Pro residue with a pentenyloxy side chain and the adjacent N(τ)-alkenyl group to produce various 20-, 22-, or 24-membered macrocycles 58, using second-generation Hoveyda-Grubbs catalyst in CH 2 Cl 2 /DMF (10/1) under microwave irradiation at 120 • C for 30 min (Scheme 18). For this reaction, a solvent combination of DMF and CH 2 Cl 2 is important for desirable peptide solubility, MW absorption, and resin swelling [86]. Syntheses of charge-masked macrocyclic phosphopeptides 58, 60, and 62 and subsequent derivatives bearing the bisalkyl-His imidazolium ring have the application potential toward biologically important protein-protein interactions. Sousbie et al. reported the discovery, synthesis, and chemistry optimization of macrocyclic neurotensin analogs with potent analgesic effects in 2018 [88,89]. RCM macrocyclizations of 66 and 68 were performed under microwave heating at 50 • C using Hoveyda−Grubbs second-generation catalyst and p-benzoquinone in DCE for 1 h, followed by the acetyl deprotection for 66 using 20% piperidine in DMF and simultaneous deprotection of the nosyl and acetyl groups for 68 using 2-mercaptoethanol and DBU, and final resin cleavage and side-chain deprotection in TFA/DCM/TIPS (Scheme 20). Two promising macrocycle candidates, 67 and 69, with low nanomolar potency and/or good plasma stability, showed in vivo efficacy in two rodent pain models [88].
Morin et al. reported the synthesis and evaluation of a renewable macrocyclic musk using batch, MW, and continuous flow methodologies in 2019 [90]. At 10 mM concentration of the diene 70 in toluene, macrocycle 71 was synthesized in 32% yield under MW irradiation in the presence of Stewart-Grubbs catalyst at 70 • C for 60 min (Scheme 21). In contrast, batch synthesis at 21 • C for 5 days and continuous flow strategy at 150 • C for 5 min gave 47% and 32% yields, respectively, offering the advantage of facile scale-up (>1 g) in both cases [90].
Guo et al. reported the synthesis and evaluation of rapamycin-inspired macrocycles with new target specificity in 2019 [91]. A general synthetic route to rapafucin is shown in Scheme 22. Specifically, on-resin MW-assisted RCM cyclization-cleavage was performed from 72 using Hoveyda-Grubbs catalyst II (30 mol%) in 1,2-dichloroethane at 140 • C for 0.5 h to provide macrocycles 73 [91]. Following the Fmoc and side-chain deprotection and resin cleavage, target peptide 81 was obtained. Accordingly, after the removal of allyl and allyloxycarbonyl protecting groups of 82 using Pd(PPh 3 ) 4 , on-resin lactamization of 83 was then performed using the HCTU coupling reagent, followed by the global deprotection and simultaneous resin cleavage with TFA to provide the lactamized peptide 82 [93].
Chartier et al. reported the design, synthesis, and evaluation of the first selective macrocyclic neurotensin (NT) receptor type 2 non-opioid analgesic in 2021 [94]. Macrocycle 88, the most potent IRAP inhibitor with high stability against proteolysis by metallopeptidases, was synthesized from 86 on solid-phase (Scheme 25). The 2-chlorotrityl chloride resin was used in the MW-assisted solid-phase synthesis of 86, and the on-resin RCM was performed between two allylglycine residues at 70 °C using the second generation Grubbs-Hoveyda catalyst and DCE as solvent under microwave heating in ~70% yield, judged by UPLC-MS [94]. Based on this developed methodology, a series of new NT macrocyclic analogs with various side chain to side chain cyclizations and therapeutic potential were synthesized to probe the chemical space associated with NT receptor binding, as well as the in vivo efficacy study in rodent pain models [94]. Chartier et al. reported the design, synthesis, and evaluation of the first selective macrocyclic neurotensin (NT) receptor type 2 non-opioid analgesic in 2021 [94]. Macrocycle 88, the most potent IRAP inhibitor with high stability against proteolysis by metallopeptidases, was synthesized from 86 on solid-phase (Scheme 25). The 2-chlorotrityl chloride resin was used in the MW-assisted solid-phase synthesis of 86, and the on-resin RCM was performed between two allylglycine residues at 70 • C using the second generation Grubbs-Hoveyda catalyst and DCE as solvent under microwave heating in~70% yield, judged by UPLC-MS [94]. Based on this developed methodology, a series of new NT macrocyclic analogs with various side chain to side chain cyclizations and therapeutic potential were synthesized to probe the chemical space associated with NT receptor binding, as well as the in vivo efficacy study in rodent pain models [94].
Trân et al. reported the synthesis and evaluation of a series of apelin-13 based macrocyclic peptide analogs as modulators and pharmacological tools in the cardiovascular system in 2018 [95]. As an example, MW-assisted RCM reaction of 89 was performed on a solid support to produce a 17-membered macrocycle 90 using second-generation Hoveyda-Grubbs catalyst in DCE under microwave irradiation at 120 • C for 10 min (Scheme 26) [95]. Notably, the cleaved and side-chain deprotected compound shows a comparable binding affinity and a longer half-life in vivo relative to its linear peptide analog [95]. Other on-resin RCM reactions include the synthesis of a linked amino acid mimetic macrocycle under MW heating by Maxwell et al. in 2013 [96], diverse macrocyclization strategies using on-resin RCM, lactamization and thiol alkylation [97], synthesis of novel echinocandin analogs via on-resin RCM or disulfide formation strategy [98], the synthesis of peptides with olefin crosslinks [99], synthesis of peptide thioureas and thiazole-containing macrocycles via Ru-catalyzed RCM [100], and Grb2 SH3 domain-binding peptides [101].
In order to build on their previous work employing MW-assisted Suzuki-Miyaura macrocyclization reaction [111], in 2020, Ng-Choi et al. reported the MW-assisted synthesis of biaryl cyclopeptides via on-resin intramolecular Suzuki-Miyaura coupling [112]. Suzuki-Miyaura macrocyclization of 113 was performed on solid-support using Pd 2 (dba) 3 (0.2 eq), SPhos (0.4 eq), and KF (4 eq) in DME/EtOH/H 2 O (9/9/2) at 120 or 140 • C under MW irradiation for 30 min, followed by simultaneous resin cleavage and side-chain deprotections in TFA/TIS/H 2 O to provide 114 (Scheme 33). By using this developed methodology, several other analogs, including cyclolipopeptides 115 and 116, were synthesized [112]. ; the MW-assisted Ullmann cyclizations performed more efficiently with higher yields and shorter reaction times compared to sealed pressure tube as well as conventional heating [113,114]. This class of macrocyclic diarylheptanoid analogs and an extended macrocycle library, produced via MW-assisted intramolecular Ullmann coupling, were tested for antibacterial activity, and several macrocycles with phenethyl-and n-hexylamino moieties showed antibacterial activity with minimum inhibitory concentrations (MICs) of 12.5-25 µg/mL against M. tuberculosis, and Gram-positive organisms [115].
Among numerous click reactions and applications, the radical-mediated thiol-ene reaction remains an attractive and robust click strategy in organic synthesis and polymer science because of its simplicity, high efficiency, and rapid conversion rate [121]. In 2010, Aimetti et al. reported on-resin peptide photocyclization via thiol-ene click strategy [122].

Intramolecular S N Ar or S N 2 Nucleophilic Substitution
Li et al. reported the MW-assisted concise synthesis and Ca 2+ -mobilizing activity in T-lymphocytes of new nucleobase-simplified cyclic ADP-ribose (cADPR) analogs in 2010 [123]. Synthesis of 135 was performed from 134 in pyridine and I 2 under MW irradiation at 90 • C for 15 min in 95% yield (Scheme 40) [123]. Notably, under the optimized conditions and MW heating, the yield of the intermolecular pyrophosphoration byproduct 136 decreased to < 3%. Following the acetal deprotection of 135 in 50% HCOOH, the resultant free, cyclic nucleotide bearing the 2' and 3'-OH groups functions as a membrane-permeable cADPR mimic with calcium release activity in intact T-lymphocytes [123]. By using similar MWassisted intramolecular pyrophosphorylation methodology, some novel cADPR structural analogs were subsequently synthesized and evaluated for Ca 2+ -mobilizing activity [124,125]. Hickey et al. reported the design and synthesis of 99m Tc/Re-containing macrocyclic peptides as integrated metal-centric peptidomimetic imaging probes in 2015 [129]. For example, the macrocyclic peptide 148 was synthesized on-resin from 147 and 2,6-bis(aminomethyl) pyridinein DMF with TEA via a pyridyl tridentate chelation core strategy, followed by side chain deprotection, resin cleavage, and metal chelation with [ 99m Tc(CO) 3 [130]. The Weinreb amide resin-bound tetrapeptides 150 were cyclized via an intramolecular S N Ar mechanism, followed by the resin cleavage using lithium aluminum hydride (LiAlH 4 ) to provide peptide aldehydes 151 (Scheme 45) [130]. The biaryl ether macrocycles 151 represent the first macrocyclic peptide aldehydes with high potency (Ki = 54.5 and 241 nM), cellular stability, and specificity for the proteasome. Kheirabadi et al. reported the synthesis of thioether-or amine-bridged macrocyclic peptides via a "catch-release" strategy in 2018 [131]. This innovative enabling technology was highly effective, producing three macrocyclic peptide libraries containing 5-20 amino acids and thioether-or amine-based macrocyclic linkers (Scheme 46) [131]. Following resin release in 0.1 M NH 3 /MeOH, 5-20 AA membered cyclopeptides 154 were provided with very good purity. Scheme 46. On-resin synthesis of cyclopeptides via a "catch-release" strategy [131].
Other diverse on-resin intramolecular S N Ar or S N 2 nucleophilic halide substitution reactions include synthesis of macrocyclic peptides with both α-helix and polyproline helix motifs [132], synthesis of cyclic peptides via an S N 2 intramolecular thioalkylation [133], synthesis of 19-membered thioether cyclic peptidomimetics [134], synthesis of thiazolecontaining cyclopeptides via on-resin intramolecular thioalkylation [135], on-resin synthesis of lipidated macrocyclic, and bicyclic peptides via convenient and intramolecular halide substitution by a diamino acid [136]. In 2018, Zhang et al. reported the on-resin MWassisted macrocyclization and discovery of bisthioether-stapled peptides with excellent proteolytic stability and potent inhibition of PRC2 catalytic activity [137]. In 2021, Roy et al. reported a powerful, high-throughput quality control assay for on-resin synthesis and analysis of macrocyclic DNA-encoded libraries via thioalkylation strategy [138].

Condensation Reactions
Ahmed et al. reported the MW-assisted synthesis and antibacterial evaluation of metal complexes with Schiff base 2, 6-pyridinedicarboxaldehydethiosemicarbazone (PDCTC) in 2014 [139]. Specifically, macrocyclic ligand 157 was synthesized from 2, 6-pyridinedicarboxaldehyde 155 and thiosemicarbazone 156 in dry ethanol under MW irradiation for 4-5 min, followed by the formation of Cu, Co, and Ni (II), and Cr (III) metal complexes 158 of these macrocycle ligands (Scheme 47). The metal complexes are generally more active than their parent Schiff base ligand in antibacterial testing [139].  8 (µ 3 -Cl) 2 ]Cl 2 ]n were also synthesized from 159 by replacing the nitrate ions with bromide, iodide or by reacting 160 with HgCl 2 , respectively, and investigated in this work [140]. Wilson et al. reported the solid-phase total synthesis of scytonemide A employing Weinreb AM resin in 2018 [141]. Following Fmoc deprotection, macrocyclization of the protected heptapeptide 161 was achieved via spontaneous imine formation upon resin cleavage using a reducing agent lithium aluminum hydride (LAH) and subsequent aqueous workup (Scheme 49). The final deprotection of 162 was performed under TBAF in methanol, providing scytonemide A (163) in a 51% yield [141]. Guéret et al. reported the solid-phase synthesis of macrocyclic modalities containing peptide epitopes and natural product motifs via macrocyclization by imine formation and subsequent stereoselective 1,3-dipolar cycloaddition in 2020 [143]. Specifically, a one-pot solid-phase synthesis protocol was developed to deprotect the Fmoc group in 169 and generate Schiff base 170 using trimethyl orthoformate as the dehydrating agent. Subsequent intramolecular cyclization of 170 was performed to provide 171 via azomethine ylide formation and 1,3-dipolar cycloaddition in the presence of lithium bromide (Scheme 51). Following the removal of side-chain protecting groups and simultaneous release from the resin, the major diastereomer 172 of the desired cycloadducts was produced in overall 8−14% purified yields [143].  [144]. On-resin macrocyclization of 173 was achieved using aziridine aldehyde dimer and an appropriate isocyanide, providing 174, which was subsequently functionalized by the nucleophilic opening of the aziridine ring (Scheme 52). This developed methodology enables the rapid parallel synthesis of macrocyclic peptides 175 [144]. Scheme 52. On-resin synthesis of macrocyclic peptidomimetics via three-component coupling strategy [144].
Morejón et al. reported the solution-and solid-phase synthesis of N-aryl-bridged cyclolipopeptides via the Ugi-Smiles multicomponent methodology in 2016 [145]. Solidsupported Ugi-Smiles macrocyclization of 3-nitrotyrosine-containing peptide 176 took place effectively in the presence of an isocyanide and an aldehyde, followed by simultaneous resin cleavage and side-chain deprotections in TFA/TIS/H 2 O to provide 177 in good yields (Scheme 53) [145]. Solid-supported Ugi-4-component macrocyclization methodology was subsequently reported to synthesize polycationic cyclolipopeptides with stabilized helical structures and antimicrobial properties [146], short and medium-size canonical cyclopeptides with turn-inducing moieties [147], stabilized cyclic β-hairpins, and N-alkylated peptides with various exo-cyclic functionalization such as cationic or hydrophobic tails and bioconjugation handles to install fluorescent and affinity tags, etc. [148]. In 2019, Reguera and Rivera reviewed the applications of the MCR toolbox for peptide macrocyclization and stapling [149]. In 2021, Rivera et al. reviewed solid-phase multicomponent protocols for biopolymer assembly and derivatization (e.g., the use of MCRs in the traceless on-resin synthesis of cyclic peptides) [150]. New synthetic methodology via on-resin Ugi reactionbased macrocyclization of peptide side chains to exocyclic functionalized helical peptides was subsequently developed by the same research team [151].  [152]. A representative example is shown in Scheme 54. Specifically, the Petasis borono-Mannich macrocyclization was carried out from 178 and glyoxylic acid in DCM/HFIP at room temperature for 24 h, followed by resin cleavage in TFA/TIPS/H 2 O at room temperature for 1 h to provide the desired product 180 [152]. Ohm et al. reported the diversity-oriented A 3 -macrocyclization of cyclic peptides with different substitution, ring size, and shape as CD36 receptor modulators in 2021 [153]. Specifically, cyclic azapeptides 182 were synthesized from linear precursors 181 using A 3macrocyclization strategy in aqueous formaldehyde and CuI in DMSO, followed by the resin cleavage and side-chain deprotection using a TFA/TES/H 2 O cocktail (Scheme 55) [153]. Previously, the same research group studied the dynamic chirality of this cyclic azapeptide class of allosteric CD36 modulators of macrophage-driven inflammation using the same macrocyclization approach [154], aza-propargylglycine installation via aza-amino acylation strategy [155], and diverse cyclic azapeptides as CD36-modulating peptidomimetics via A3-macrocyclization approach [156]. Scheme 55. Diversity-oriented on-resin synthesis of cyclic azapeptides via A 3 -macrocyclization strategy [153].

Conclusions
Macrocycles offer some key attributes and advantages over small molecules and natural products such as favorable PK/PD parameters and improved oral bioavailability, enhanced metabolic stability, and cell permeability, and thus play an important role in chemical biology and medicinal chemistry. Since 2014, nineteen macrocyclic drugs and radiopharmaceuticals have been approved by FDA in the therapeutic areas of bacterial and viral infections, oncology, immunosuppression, etc. Remarkably, among them, five macrocyclic NS3/4A protease inhibitors have been approved for the treatment of HCV infections either alone or in combination with other viral medications. However, several of these agents were discontinued from the US due to clinical practice changes or other reasons in the arena of chronic HCV treatment, including newer, safer, and/or more effective treatment options.
Undoubtedly, macrocycles continue to represent an exciting chemical class in clinical drug development pipelines, exemplified by a tri-macrocyclic phase II clinical candidate, a proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitor, for lipid-lowering therapy [157]. This tricyclic peptide PCSK9 inhibitor has the potential to be developed as an oral once-daily lipid-lowering agent as the only other two FDA-approved PCSK9 drugs, Praluent (alirocumab) and Repatha (evolocumab), are monoclonal antibodies (biologics), which are administered subcutaneously. Notably, it remains a challenge to develop macrocyclic peptides with oral bioavailability for systemic use. Other macrocyclic orally bioavailable peptide drugs include immunosuppressants Gengraf, Neoral, Sandimmune (cyclosporine), and Lupkynis (voclosporin). In the case of macrocyclic peptide drug Trulance (plecanatide) with two intramolecular disulfide linkages, it is taken orally but with minimal GI absorption and thus used for the treatment of GI-related disorders (e.g., CIC and IBS-C). In this context, such other recently approved macrocyclic oral drugs with minimal absorption include antibacterial Aemcolo (rifamycin SV) for managing and treating travelers' diarrhea and Dificid (fidaxomicin) for the treatment of adult patients with C. difficile infections [13]. In addition, several advanced macrocycle clinical candidates were developed using the macrocycle-antibiotic hybrid strategy [5].
Moreover, high throughput chemistry, modular synthesis, and new macrocyclization methodology enable facile, rapid synthesis, SAR, and new advances in macrocycle drug discovery. Specifically, microwave-assisted and/or on-resin macrocyclizations have a profoundly accelerated generation of macrocycle libraries. These were highlighted via various recent MW-assisted and/or solid-supported macrocyclization strategies, including macrolactonization and macrolactamization, transition-metal catalyzed olefin ring-closure metathesis, intramolecular C-C and C-heteroatom cross-coupling, copper-or rutheniumcatalyzed azide-alkyne cycloaddition, intramolecular S N Ar or S N 2 nucleophilic substitution, condensation reactions, and multi-component reaction-mediated macrocyclization. Depending on the nature of reaction substrates and types, both MW irradiation and solidphase synthesis can shorten reaction times and synthetic routes, enhance reaction efficiency with higher yields and fewer side products, improve catalyst performance, consume less solvent, and/or streamline the synthesis and overall purification process. Though in some cases, significant optimization studies are required to find the most optimal reaction conditions and reagents. In multiple cases, on-resin MW-assisted macrocyclizations were effectively applied to the macrolactamization of peptide and peptidomimetics, RCM, triazole click chemistry, and intramolecular C-C coupling. Other innovative macrocycle entities and new advances in synthetic methodology development include the MCR-mediated macrocyclizations. Collectively, this work reviews and showcases some exciting advances in the macrocycle space, and its significance and impact will continue to thrive in the areas of chemistry, biology, and medicine.