Passive Membrane Permeability of Macrocycles Can Be Controlled by

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Passive membrane permeability of macrocycles can be controlled by exocyclic amide bonds Jennifer L. Hickey, Serge Zaretsky, Megan A. St. Denis, Sai Kumar Chakka, M. Monzur Morshed, Conor C. G. Scully, Andrew L. Roughton, and Andrei K Yudin J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00222 • Publication Date (Web): 27 Apr 2016 Downloaded from http://pubs.acs.org on April 29, 2016

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Journal of Medicinal Chemistry

Passive membrane permeability of macrocycles can be controlled by exocyclic amide bonds Jennifer L. Hickey,†,‡ Serge Zaretsky,† Megan A. St. Denis,†,‡ Sai Kumar Chakka,†,‡ M. Monzur Morshed,†,‡ Conor C. G. Scully,† Andrew L. Roughton,‡ and Andrei K. Yudin†* † Davenport Research Laboratories, Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, Ontario M5S 3H6, Canada ‡ Encycle Therapeutics Inc., 101 College Street, Suite 314, Toronto, Ontario M5G 1L7, Canada

ABSTRACT

We have developed a strategy to synthesize passively permeable peptidomimetic macrocycles. The cyclization chemistry centers on using aziridine aldehydes in a multicomponent reaction with peptides and isocyanides. The linker region in the resulting product contains an exocyclic amide positioned α to the peptide backbone, an arrangement that is not found among natural amino acids. This amide provides structural rigidity within the cyclic peptidomimetic and promotes the creation of a stabilizing intramolecular hydrogen bonding network. This exocyclic control element also contributes to the increased membrane permeability exhibited by multicomponent-derived macrocycles with respect to their homodetic counterparts. The exocyclic control element is employed along with a strategic placement of N-methyl and D-amino acids to produce passively

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permeable peptides, which contain multiple polar residues. This strategy should be applicable in the pursuit of synthesizing therapeutically relevant macrocycles.

INTRODUCTION The discovery of new biologically active molecules is complicated by the need to not only consider binding and specificity towards the intended biomolecular target, but also the properties required for pharmacological activity in vivo. Peptide ligands often exhibit high binding affinity toward their target receptors; however, they have insufficient stability resulting in limited therapeutic application.1,2 Linear peptides typically undergo enzymatic degradation prior to executing their pharmaceutical objective, which has made cyclization an attractive modification.3 It has been shown that peptide cyclization can have a beneficial impact on essential drug-like properties such as membrane permeability,4–6 metabolic stability,7 and overall pharmacokinetics.8,9 Evidence suggests that limited flexibility within the backbone enables the formation of intramolecular hydrogen bonds, contributing to increased passive permeability and improving overall oral bioavailability.8,10–12 Moreover, the structural preorganization may also reduce the entropy cost of receptor binding by eliminating unproductive conformations, subsequently increasing binding affinity compared to linear analogues.8,13–16 Consequently, macrocyclic peptides have been the subject of sustained interest as therapeutically relevant molecules. Macrocycles typically have high molecular weights (> 500 Da), moving them outside of what has traditionally been considered orally bioavailable drug space. The conventional metrics for “drug-likeness” are of limited value in addressing macrocycles as the guidelines have been constructed exclusively through the evaluation of small molecule drug candidates.17–20 Therefore,


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understanding the drug-like properties required for oral bioavailability when it comes to beyond the “Rule of Five” molecules continues to be a work in progress. Modified sets of desired property ranges for the design of oral macrocyclic drugs have recently been proposed.8,21–23 Although the data sets are small, it is apparent that macrocycles display consistent and clearly distinct properties from those observed for conventional drugs. Aside from a few rare instances, cell-permeable macrocycles do not contain more than one polar side chain.21,24,25 In fact, a large proportion of passively permeable peptide macrocycles consist solely of lipophilic amino acids, such as leucine or alanine, limiting their therapeutic relevance.10,11,26–29 These compounds are often poorly water-soluble and require complex formulation strategies prior to administration. As approximately 50% of drug candidates fail due to poor drug-like properties/pharmacokinetics, mostly attributable to poor water solubility,30–34 the significant challenge for peptide macrocycle drug development is to maintain membrane permeability while increasing water solubility. We recently reported the discovery of a structural control element, an unnatural exocyclic amide motif,35 that is integrated into peptide macrocycles via aziridine aldehyde-mediated macrocyclization (Scheme 1).36–38 This exocyclic control element (ECE) has been shown to set the intramolecular H-bonding network within the peptide backbone, and contribute to the overall rigidification of the macrocyclic structure. In this manuscript, we uncover a conserved backbone scaffold, formed during the multicomponent cyclization of 18-membered ring macrocycles. The ECE set by the cyclization linker is also shown to improve the passive membrane permeability of macrocyclic peptides containing polar side chains. By masking the polar surface area (PSA), this structural motif allows for a broader range of amino acid residues to be utilized while maintaining favourable pharmacokinetic properties.


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Scheme 1. Aziridine aldehyde-mediated macrocyclization. Resulting macrocycle with cyclization linker region and ECE highlighted in red

RESULTS AND DISCUSSION 18-membered ring macrocycles are the most commonly found motif in nature.39–43 We were interested in comparing the intramolecular hydrogen bonding pattern of 18-membered rings with an ECE,35,44 to the traditional homodetic cyclic hexapeptide (Figure 1). The structural difference between 1 and 2 resided in the replacement of an sp2 amide carbonyl with an sp3 alkyl carbon centre. More specifically, the alanine amide carbonyl group in 2 was substituted with a peptidomimetic linker region containing an exocyclic tert-butyl amide. Variable temperature NMR comparison of backbone intramolecular H-bonds VT NMR was the first method we used for structural comparison of the macrocyclic peptides. Analysis of the temperature dependence of amide proton chemical shifts through VT NMR studies is a common method utilized to detect the presence of intramolecular H-bonding,7,45,46 and more recently has been used as a strategy to improve the oral bioavailability of peptides.2,47 The 1H NMR spectra were acquired from 25-65 °C at 10 °C increments in DMSO-d6. A cutoff of


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≥ -3 ppb/K was selected for a strong intramolecular hydrogen bonding interaction, and temperature coefficients of < -4.5 ppb/K were indicative of solvent exposed protons. Intermediate values suggested some reduction in solvent-accessibility presumably due to a weak/strained H-bonding interaction.48,49 We analyzed four different macrocycles: 1 and its homodetic counterpart 2 as well as two additional peptides 3 and 4 (Figure 1). In 3, the aziridine ring was opened using thiophenol as the nucleophile, while 4 still contained the intact acyl aziridine ring. When comparing the experimental H-bonding pattern, we noted that the macrocycles with an ECE had well-defined backbone structures with four amide protons involved in intramolecular H-bonds (Table 1, see Supporting Information for VT 1H NMR traces). Whether a large thiophenol nucleophile was present (3) or not (1), the basic scaffold of these 18-membered rings was unaffected. The 2nd and 3rd amides, the linker amide, which was revealed upon aziridine ring-opening, and the exocyclic tert-butyl amide were all critical for this H-bonding network. However, the 4th and 5th amides in the macrocycle were solvent exposed in both cases.


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Figure 1. Comparison of 18-membered ring macrocycles: macrocycle 1, c*[PS-DLeu-FF]; comparable homodetic hexapeptide 2, cyclo[PS-DLeu-FFA]; thiophenol ring-opened macrocycle 3, c*[PS-DLeu-FF]a; and acyl aziridine-containing macrocycle 4, c[P-S(OtBu)-DLeu-FF]. Comparable linker regions highlighted in red and amino acid residue assignments in blue.

In contrast, VT NMR data for homodetic peptide 2 revealed a drastic increase in structural flexibility. 2 existed as a mixture of two major conformers at room temperature. Upon heating, the amides broadened until accurate measurements were not possible. These observations did not necessarily indicate a complete absence of hydrogen bonding, but were consistent with a conformationally flexible backbone. This sort of conformational flux has previously been observed in the context of 15-22 membered cyclic peptides, which commonly exist in solution as an equilibrium mixture of multiple conformers.50


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Table 1. Variable temperature NMR values in ppb/K for the amide NHs found in compounds 1 – 4, with blue highlighted cells indicating the presence of an intramolecular hydrogen bond ppb/K Amide

c*[PS-DLeu-FF], cyclo[PS-DLeu-FFA], c*[PS-DLeu-FF]a, c[P-S(OtBu)-DLeu-FF]b, 1 2 3 4

2

- 1.75

---

- 1.0

- 0.75

3

-1

---

- 3.25

- 5.0

4

-5

---

- 5.5

- 6.5

5

- 5.75

---

- 5.75

- 5.75

Linker/6

- 3.75

---

0.5

Exo

-1

a

thiophenol nucleophile;

- 3.0 b

- 5.75

c[P-S(OtBu)-DLeu-FF] with an intact acyl aziridine ring was not

deprotected with TFA

On the other hand, we observed only a single H-bond for compound 4 upon VT NMR analysis. We hypothesized that the presence of the more rotationally flexible aziridine amide perturbed the backbone geometry such that the hydrogen bonding pattern was no longer accessible. Even though the exocyclic amide was present, the absence of the linker NH destabilized the overall H-bonding network. This suggested to us that the ECE was composed of both the exocyclic amide and the linker backbone amide. We concluded that both features were important for the conformational homogeneity and stable H-bonding network found in our 18-membered macrocycles.


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Passive membrane permeability The ability to form intramolecular hydrogen bonds is believed to be critical for passive membrane permeability and can be the distinguishing factor among structurally related compounds as it decreases solvent-exposed PSA by shielding polar backbone amides.10,11,16,24,25,51–54 In addition to the VT NMR studies, we also wanted to determine the influence of the ECE on the extent of passive membrane permeability for macrocycles 1, 2, and 3. The three compounds were analyzed using a parallel artificial membrane permeability assay (PAMPA) with an output –log Pe value of 6 or less being indicative of a passively permeable compound.55–57 For compounds 1 and 3, we observed –log Pe values of 5.6, indicating that these molecules were permeable under the assay conditions (Table 2). In contrast, a –log Pe value of 7.2 was detected for homodetic peptide 2, indicating that it was not passively permeable. We noted an increase in both cLogP and topological PSA (tPSA) for compounds 1 and 3 over that of 2, which suggested that these calculated parameters were too simplistic to individually account for the complex set of properties dictating the passive permeability of our macrocycles (see Supporting Information for cLogP and PSA calculations). We then looked to calculate PSA of the modelled solutionphase structures of 1 – 3 to see if we could obtain a better rationale for the permeability results. Indeed, we observed from the solution structure PSA that compounds 1 and 3 exhibited a distinct decrease in PSA, compared to that of 2. Accordingly, we hypothesized that the permeability difference was primarily due to the presence of the ECEs in 1 and 3 as observed through Hbonding in VT NMR. The notion that backbone structural motifs are potentially more integral in promoting membrane permeability than physical properties such as solvent exposed NHs, lipophilicity, and number and position of N-methyl groups has been reported.54,58 In our case, the


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exocyclic amide was the sole structural difference between our molecules and the homodetic peptide, leading us to believe that it played a critical structural role in masking PSA and enabling passive permeability. We also analyzed a linear control (Ac-PS-DLeu-FF-NH2, 5), which contained the same peptide sequence but in a much more flexible arrangement. Linear control 5 did not display a hydrogen bonding network and was not passively permeable. We included two additional literature reported molecules in our permeability study. We synthesized and analyzed 6, a compound previously reported by Lokey and co-workers as it was a well-known 18-membered ring compound and an applicable comparison to our macrocycles. 6 was reported to be PAMPA permeable and 28% orally bioavailable in rat.25 We also analyzed cyclosporin A (CSA, 7), which, like our ECE-bearing macrocycles, contains four intramolecular hydrogen bonds.29,51 CSA was reported to be passively permeable and depending on formulation, achieved oral bioavailability between 15-50%.18 While slightly different PAMPA conditions prevent a true comparison to the previously reported values, it can be said that 1 and 3 are more passively permeable than both 6 and CSA (7).


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Table 2. PAMPA data, with –Log Pe values ≤ 6.0 being indicative of passively permeable compounds PAMPA (-Log Pe)

Abbreviated Sequence

a

c*[PS-DLeu-FF], 1

5.6

cyclo[PS-DLeu-FFA], 2

7.2

c*[PS-DLeu-FF]a, 3

5.6

Ac-PS-DLeu-FF-NH2, 5

7.4

cyclo[DPro-MeTyr-Leu-DMeLeu-MeLeu-Leu], 6

6.4

CSA, 7

6.2

thiophenol nucleophile

The stark difference between 1 and 2 raised two important questions: was the passive permeability we observed due to the structured H-bonding network enforced by the ECEs of the macrocycles? And would any 18-membered ring derived from our multicomponent chemistry yield the same H-bonding pattern? To answer these questions, we synthesized a library of 18membered ring macrocycles and endeavored to analyze the ECE-induced H-bonding networks via VT NMR, as well as the passive permeability of the macrocycles through PAMPA analysis. VT NMR on a library subset Ten compounds from the library were analyzed by VT NMR as shown in Table 3. We attempted to synthesize and study a diverse set of compounds, with a wide range of amino acid residues as well as D-amino acid and N-methylation permutations. Upon VT NMR analysis we noted that the ECE-induced H-bonding network present within these considerably diverse macrocycles was


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very similar (Figure 2). Only one out of the ten model substrates strayed from the consistent Hbonding pattern, with the exocyclic amide not being involved in H-bonding (15, Table 3). In this case, the system was greatly perturbed by two adjacent N-methylated amino acids at the Cterminus. The consistent pattern demonstrated that these compounds are, for the most part, remarkably tolerant to substitution while maintaining a robust H-bonding network. The common H-bonding pattern also suggested to us that we have uncovered a scaffold, which existed within the backbone of these 18-membered peptide macrocycles.

Figure 2. a) H-bonding network of a typical 18-membered macrocycle with an ECE, highlighting four intramolecular hydrogen bonds and two solvent exposed amides; b) Map of NHs showing the consistent H-bonding pattern (blue circles highlight the amide protons involved in intramolecular H-bonding interactions; red circle highlight the amides which were solvent exposed).


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Table 3. VT NMR temperature coefficient table with positive and negative indications for the presence of an intramolecular H-bond Amide involved in H-bondc Sequence

a

2

3

4

5

Linker

Exo

c*[PLLGF], 8

Yes

Yes

No

No

Yes

Yes

c*[PSLYF], 9

Yes

Yes

No

No

Yes

Yes

c*[PTLYF], 10

Yes

Yes

No

No

Yes

Yes

c*[PT-DLeu-YF], 11

Yes

Yes

No

No

Yes

Yes

c*[PLLGF]a, 12

Yes

Yes

No

No

Yes

Yes

c*[PLL-Sar-F]a, 13

Yes

Yes

No

Yes

Yes

c*[PLLG-MePhe]a,b, 14

Yes

Yes

Yes

Yes

c*[PLL-Sar-MePhe]a,b, 15

Yes

Yes

Yes

No

c*[PT-MeLeu-YF], 16

Yes

No

No

Yes

Yes

c*[PT-DMeLeu-YF], 17

Yes

No

No

Yes

Yes

No

thiophenol nucleophile; b conformers were present at room temp. VT NMR analysis

performed on the major conformer; c grey box denotes the absence of an amide NH in the amino acid residue

The incorporation of sarcosine at position four (13 and 15, Table 2) did not have any detrimental effects on cyclization nor did it disrupt the H-bonding pattern of the final product. However, upon addition of the N-methyl phenylalanine residue at position five (14 and 15), there were two conformers present in the NMR spectra of the final macrocycle. This was most likely due to steric interactions caused by the presence of the adjacent phenyl sulfide, introduced through thiophenol ring-opening of the acyl aziridine. We also observed that when both the 4th and the 5th


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amide NHs were blocked as N-methyls (15), the exocyclic amide was no longer involved in a strong H-bonding interaction. This could have also been a result of the adjacent bulky phenyl sulfide group. Another interesting finding was the incorporation of an N-methyl amino acid in position three (16 and 17), an amide position which was shown to be consistently involved in intramolecular H-bonding. When position three was N-methylated and blocked from H-bond donation, the same H-bonding pattern was retained, while the 4th and 5th amides continued to be solvent exposed. PAMPA trends Through PAMPA analysis, we studied a broad set of 75 compounds with the hopes of uncovering trends with respect to structural features that could potentially be enabling for passive permeability. The major trend that we observed was the correlation between the number of polar side chains present in the macrocycle and permeability (Figure 3).

PAMPA permeability (-log Pe)

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


10

8

6

4 0

1

2

3

# of polar side chains

Figure 3. PAMPA permeability versus the number of polar side chains in the peptide sequence (– log Pe ≤ 6 indicates passive permeability).


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We observed comparable passive permeability for sequences containing zero or one polar side chain. Encouragingly, the PAMPA permeability was not necessarily achieved at the expense of solubility as we were able to observe greater than 300 µM aqueous solubility for lipophilic compounds such as 8. Conversely, the solubility of compounds 6 and 7 were almost an order of magnitude lower (see Supporting Information for solubility data). The highly soluble, yet liphophilic, nature of our compounds could be attributed to the weakly basic nitrogen contained in the linker portion of the ECE-bearing macrocycles. Solubility remained high once there were two polar side chains present; however, the availability of four intramolecular H-bonds was not sufficient to mask the increased PSA. There were also a few unsurprising outliers present in the PAMPA data. If there was a single aspartic acid or lysine residue present, the permeability was greatly diminished, irrespective of the total number of polar side chains. Table 4. PAMPA data, cLogP and PSA calculations for c*[PT-DLeu-YF], 11, series of polar compounds with a –Log Pe value ≤ 6.0 being indicative of a passively permeable compound PAMPA (-Log Pe)

cLogPa

tPSAa

PSAb

c*[PT-DLeu-YF], 11

8.8

4.51

218.30

216.44

c*[PT-DMeLeu-YF], 17

8.4

5.23

209.51

216.13

c*[PT-DMeLeu-DTyr-F], 18

6.1

5.23

209.51

204.61

c*[PT-DMeLeu-DTyr-MePhe], 19

5.4

5.88

200.72

198.36

Abbreviated Sequence

a

topological PSA calculated using JChem; b calculated based on modelled solution

structures using QikProp


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Based on the PAMPA data, we expected that a sequence with two polar side chains would likely not be permeable; however, knowing that N-methylation could improve the membrane permeability of peptides,5,27,59 we sought to convert a non-permeable polar macrocycle to an analogue with favourable PAMPA behaviour. Starting with 11, a macrocycle containing two polar side chains and a –log Pe value of 8.8 (Table 4), we attempted to N-methylate the D-Leu in the 3rd position (17) with the hopes of reducing overall PSA. We knew from the VT data that the H-bonding pattern remained consistent even upon blocking the 3rd position NH from H-bond donation. This was confirmed to also show no detrimental effect on passive permeability. We then decided to alter the solvent exposed 4th amino acid by switching it from an L-amino acid to a D-amino

acid (18). By inverting the stereochemistry, we were attempting to alter the backbone

conformation in an effort to strengthen the intramolecular H-bonds, and in turn further mask PSA and improve permeability. To our delight, this permutation drastically improved permeability, resulting in a compound that was right on the cusp of the –log Pe value of 6 cutoff. As per our earlier hypothesis, we observed that PSA based on the solution structure was more predictive and indicative of passive permeability trends than the general tPSA and cLogP calculations. Neither cLogP nor tPSA took into account the stereochemical or conformational differences between compounds 17 and 18, yet a significant improvement in the PAMPA results were observed and supported by the solution structure PSA data. Finally, we swapped the Phe residue at the C-terminus of the peptide for an N-methyl-Phe residue (19) and achieved passive permeability. CONCLUSIONS The ability to manipulate an impermeable sequence and convert it into a passively permeable compound is a significant challenge. The difficulty of this goal is further underscored by the


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need to balance membrane permeability with solubility. Whereas prominent literature methods have centered on the use of hydrophobic residues and N-methylation strategies, we have attempted a rational approach to increasing permeability of cyclic peptides while maintaining the more functionally important and solvable polar amino acids. By studying the structures and Hbonding networks of 18-membered macrocycles derived through aziridine aldehyde-based macrocyclization, we have found that a single scaffold was predominant amongst all of the study peptides. We believe a crucial part of the stable scaffold design is the presence of an ECE, which is derived from the macrocyclization chemistry. In a broad PAMPA screen of these compounds, we found that one polar side chain could be tolerated and we used this knowledge to rationally improve c*[PT-DLeu-YF], 11, a non-permeable peptide, to an effectively permeable compound c*[PT-DMeLeu-DTyr-MePhe], 19. The results herein clearly demonstrate a viable route to optimizing cyclic peptide permeability, enabling the study and optimization of H-bonding networks in macrocyclic scaffolds. More broadly, these findings lend further support to the importance of incorporating an ECE, such as the unnatural α-amide group present in our macrocyclic peptides, in order to create molecules with increased rigidity and lower apparent polar character. The generation of permeable, drug-like macrocycles may open up new avenues for therapeutic intervention, including the potential to generate orally bioavailable modulators of protein-protein interactions. EXPERIMENTAL SECTION General information. All chemicals were purchased from commercial sources and used without further purification unless otherwise noted. (S)-aziridine-2-carboxaldehyde dimer was prepared as per literature protocol.60,61 Analytical HPLC was performed on an Agilent Technologies 1200 series HPLC paired to a 6130 Mass Spectrometer. Compounds were analyzed on an Agilent


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Poroshell 120 EC-C18, 2.7 µm, 4.6 x 50 mm2 column at room temperature with a flow rate of 1 mL/min. The gradient consisted of eluents A (0.1% formic acid in double distilled water) and B (0.1% formic acid in HPLC-grade acetonitrile). The gradient method started at 5% of B followed by a linear gradient from 5% to 95% B in 4.0 minutes. The column was then washed with 95 % B for 1.0 minute and equilibrated at 5% B for 1.5 minutes. Purifications were performed on a Teledyne Isco CombiFlash system under reverse-phase conditions using a 30 g RediSep C18 Gold Column. The gradient consisted of eluents A (0.1% formic acid in double distilled water) and B (0.1% formic acid in HPLC-grade acetonitrile) at a flow rate of 35 mL/min. The purity of all final compounds was determined to be ≥ 95% via analytical HPLC/MS. NMR data was obtained on an Agilent 700 MHz, Agilent 500 MHz, or a Varian 600 MHz instrument in d6DMSO with the chemical shifts referenced to solvent signals (d6-DMSO, 1H 2.50 ppm and 13C 39.5 ppm) relative to TMS. Peak multiplicities are designated by the following abbreviations: s, singlet; bs, broad singlet; d, doublet; t, triplet; q, quartet; m, multiplet; ds, doublet of singlets; dd, doublet of doublets; ddd, doublet of doublet of doublets; dt, doublet of triplets; bt, broad triplet; td, triplet of doublets; tdd, triplet of doublets of doublets. General peptide synthesis. Fully protected resin-bound peptides were synthesized via standard Fmoc solid-phase peptide chemistry using manual peptide synthesis methods. 2-Chlorotrityl resin (loading 1.2 mmol/g) was utilized as the solid support. All N-Fmoc amino acids were employed. Fmoc removal was achieved by treatment with 20% piperidine in NMP for five and 20 minutes with consecutive DMF and NMP washes after each addition. For all Fmoc amino acid couplings, the resin was treated with 3 eq. of Fmoc amino acids, 3 eq. of HATU and 6 eq. of DIPEA in NMP for one hour. Once the linear sequence was assembled a final Fmoc deprotection was performed followed by cleavage from the resin by treatment with a 25:75 mixture of HFIP


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and CH2Cl2 for 30 minutes (x2). The cleavage cocktail was concentrated under reduced pressure and the resultant peptide was then precipitated using TBME, and collected after sonication, centrifugation and decantation. Peptide Macrocyclization General cyclization procedure. The linear peptide (0.15 mmol) was placed in a two-dram vial followed by TFE (1.05 mL) and a 0.2 M solution of (S)-aziridine-2-carboxaldehyde dimer in TFE (450 µL, 1.2 eq. as monomer). tert-Butyl isocyanide (20.5 µL, 1.2 eq.) was added and the reaction was left to stir at room temperature for four hours. Nucleophilic ring-opening procedures Thiobenzoic acid ring-opening. Following the four hour cyclization, thiobenzoic acid (53 µL, 3 eq.) was added directly to the cyclization reaction mixture. Following two more hours of stirring at room temperature, Raney®-Nickel was added as a slurry (approx. 2 mL), capped tightly, and left to stir overnight. Then, the mixture was filtered through Celite with EtOAc and MeOH, and evaporated under reduced pressure. Thiophenol ring-opening. Alternatively, following the four hour cyclization, the reaction mixture was concentrated under a stream of N2. The residue was then dissolved in 3 mL of CH2Cl2, to which was added thiophenol (46 µL, 3 eq.) and DIPEA (78.4 µL, 3 eq.). The reaction was stirred for an additional two hours followed by concentration under a stream of N2. The resultant crude macrocycle was then precipitated using TBME or diethyl ether, and collected after sonication, centrifugation and decantation.


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Global deprotection and purification. Macrocycles containing polar residues with side chain protecting groups are then treated with TFA containing water (2.5% v/v) and TIS (2.5% v/v) as scavengers for two hours. The TFA solution was concentrated under a stream of N2. The resultant peptide was then precipitated using TBME, and collected after sonication, centrifugation and decantation. The final product can be isolated via reverse-phase HPLC purification or C18 flash purification. (3S,6R,9S,12S,15S,16S,20aS)-9,12-dibenzyl-N-(tert-butyl)-3-(hydroxymethyl)-6-isobutyl-15methyl-1,4,7,10,13-pentaoxoicosahydropyrrolo[1,2a][1,4,7,10,13,16]hexaazacyclooctadecine-16-carboxamide – (1) ESI MS [M+H]+ expected: 748.4, experimental: 748.4. LC-MS retention time: 4.2 min. Yield: 16.8 mg (14%). 1H NMR (500 MHz, d6-DMSO) δ 8.82 (d, J = 5.6 Hz, 1H), 8.72 (d, J = 7.6 Hz, 1H), 7.70 (d, J = 9.3 Hz, 1H), 7.32 – 7.23 (m, 4H), 7.22 – 7.17 (m, 5H), 7.17 – 7.12 (m, 2H), 7.01 (s, 1H), 6.70 (d, J = 8.9 Hz, 1H), 5.00 (t, J = 5.0 Hz, 1H), 4.47 (q, J = 7.0 Hz, 1H), 4.28 (dt, J = 9.3, 4.0 Hz, 1H), 4. 09 (m, 1H), 4.02 (ddd, J = 11.2, 5.6, 3.8 Hz, 1H), 3.92 – 3.80 (m, 2H), 3.43 (ddd, J = 15.1, 9.7, 6.5 Hz, 2H), 3.32 – 3.27 (m, 1H), 3.08 (dd, J = 13.8, 11.7 Hz, 1H), 2.98 (d, J = 10.8 Hz, 1H), 2.93 – 2.85 (m, 1H), 2.71 (dd, J = 14.2, 11.2 Hz, 1H), 2.61 (dd, J = 14.2, 3.8 Hz, 1H), 2.09 – 1.97 (m, 1H), 1.67 (m, 3H), 1.25 (t, J = 6.8 Hz, 2H), 1.22 (s, 9H), 1.22 – 1.10 (m, 1H), 1.07 (d, J = 6.4 Hz, 3H), 0.67 (d, J = 6.5 Hz, 3H), 0.63 (d, J = 6.5 Hz, 3H).13C NMR (126 MHz, d6-DMSO) δ 173.5, 173.1, 171.6, 169.3, 169.3, 168.6, 139.2, 137.5, 129.2, 128.7, 128.2, 128.2, 126.4, 126.2, 65.3, 64.2, 60.0, 56.8, 55.9, 53.2, 50.6, 45.6, 45.6, 42.2, 35.8, 34.7, 30.0, 28.2, 23.9, 23.9, 22.6, 22.4, 18.3.


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(3S,6R,9S,12S,15S,20aS)-9,12-dibenzyl-3-(hydroxymethyl)-6-isobutyl-15methyltetradecahydropyrrolo[1,2-a][1,4,7,10,13,16]hexaazacyclooctadecine-1,4,7,10,13,16hexaone – (2) ESI MS [M+H]+ expected: 663.4, experimental: 663.4. LC-MS retention time: 4.0 min. Yield: 6.5 mg (5%). Mixture of conformers, 1:0.7 ratio: 1H NMR (500 MHz, d6-DMSO) δ 8.82 (d, J = 7.9 Hz, 1H), 8.64 – 8.46 (m, 3.1H), 8.20 (d, J = 8.0 Hz, 1H), 7.81 (d, J = 20.4 Hz, 0.7H), 7.58 (d, J = 6.3 Hz, 1H), 7.38 – 7.09 (m, 18H), 6.66 (d, J = 6.7 Hz, 0.7H), 5.00 (s, 0.7H), 4.79 (s, 1H), 4.52 (td, J = 8.3, 5.0 Hz, 1H), 4.43 – 4.31 (m, 4.4H), 4.31 – 4.23 (m, 0.7H), 4.20 – 4.11 (m, 2.4H), 4.03 (ddd, J = 11.6, 8.0, 3.6 Hz, 1H), 3.89 (dt, J = 9.2, 4.6 Hz, 1H), 3.77 – 3.66 (m, 1.4H), 3.57 (q, J = 8.5 Hz, 0.7H), 3.54 – 3.39 (m, 3.7H), 3.40 – 3.24 (m, 1.7H), 3.12 (dd, J = 13.8, 5.0 Hz, 1H), 3.01 (t, J = 12.6 Hz, 0.7H), 2.86 (dd, J = 13.8, 3.7 Hz, 1H), 2.82 – 2.67 (m, 1.7H), 2.67 – 2.56 (m, 1.7H), 2.22 – 2.15 (m, 1.7H), 2.13 – 2.01 (m, 1H), 1.98 – 1.88 (m, 0.7H), 1.87 – 1.77 (m, 1.7H), 1.75 – 1.59 (m, 1.7H), 1.31 (d, J = 7.0 Hz, 2.1H), 1.29 – 1.23 (m, 1.7H), 1.21 (d, J = 7.0 Hz, 3H), 1.14 (dt, J = 13.5, 7.2 Hz, 1.4H), 1.02 – 0.87 (m, 1.7H), 0.76 – 0.69 (m, 5.1H), 0.64 (dd, J = 6.6, 1.6 Hz, 5.1H). 13C NMR (126 MHz, d6-DMSO) δ 171.8, 171.4, 171.2, 171.1, 171.0, 171.0, 170.8, 170.2, 169.9, 169.6, 168.2, 138.9, 138.3, 138.0, 137.2, 129.7, 129.2, 129.1, 129.0, 128.8, 128.0, 127.9, 126.3, 126.1, 126.0, 61.7, 61.5, 61.0, 60.6, 56.1, 55.1, 54.8, 54.4, 52.1, 51.9, 50.8, 48.2, 46.9, 46.2, 40.6, 38.5, 36.4, 31.8, 25.1, 23.7, 22.8, 22.1, 21.7, 16.2, 15.4. (3S,6R,9S,12S,15R,16S,20aS)-9,12-dibenzyl-N-(tert-butyl)-3-(hydroxymethyl)-6-isobutyl1,4,7,10,13-pentaoxo-15-((phenylthio)methyl)icosahydropyrrolo[1,2a][1,4,7,10,13,16]hexaazacyclooctadecine-16-carboxamide – (3)


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ESI MS [M+H]+ expected: 856.4, experimental: 856.4. LC-MS retention time: 4.5 min. Yield: 6.8 mg (5%). 1H NMR (500 MHz, d6-DMSO) δ 8.58 (d, J = 6.1 Hz, 1H), 8.40 (d, J = 8.4 Hz, 1H), 7.57 (d, J = 9.3 Hz, 1H), 7.50 – 7.39 (m, 3H), 7.39 – 7.14 (m, 11H), 7.13 – 7.07 (m, 2H), 6.95 (s, 1H), 6.75 (d, J = 9.4 Hz, 1H), 4.98 (s, 1H), 4.48 – 4.35 (m, 2H), 4.27 (qd, J = 9.5, 3.5 Hz, 1H), 4.18 – 4.08 (m, 1H), 4.00 (dt, J = 9.5, 6.0 Hz, 1H), 3.68 (dd, J = 10.8, 5.2 Hz, 1H), 3.58 (dd, J = 10.8, 5.3 Hz, 1H), 3.41 – 3.34 (m, 1H), 3.28 – 3.06 (m, 3H), 3.05 – 2.95 (m, 2H), 2.80 – 2.73 (m, 1H), 2.68 – 2.61 (m, 2H), 2.06 – 1.97 (m, 1H), 1.71 – 1.58 (m, 3H), 1.34 – 1.21 (m, 3H), 1.19 (s, 9H), 0.74 (d, J = 6.3 Hz, 3H), 0.66 (d, J = 6.3 Hz, 3H). (4S,7S,10R,13S,15aS,20S,20aS)-4,7-dibenzyl-13-(tert-butoxymethyl)-N-(tert-butyl)-10isobutyl-3,6,9,12,15-pentaoxooctadecahydro-1H,3H-azirino[1,2-a]pyrrolo[1,2d][1,4,7,10,13,16]hexaazacyclooctadecine-20-carboxamide – (4) ESI MS [M+H]+ expected: 802.5, experimental: 802.5. LC-MS retention time: 4.8 min. Yield: 20.4 mg (16%). 1H NMR (500 MHz, d6-DMSO) δ 8.36 (d, J = 7.9 Hz, 1H), 8.32 (d, J = 9.1 Hz, 1H), 8.04 (d, J = 8.4 Hz, 1H), 7.63 (d, J = 8.7 Hz, 1H), 7.38 (s, 1H), 7.34 – 7.11 (m, 8H), 7.11 – 7.05 (m, 2H), 4.52 (ddd, J = 9.7, 8.4, 5.6 Hz, 1H), 4.34 (dt, J = 8.7, 6.6 Hz, 1H), 4.27 (dt, J = 8.9, 7.8 Hz, 1H), 4.16 (ddd, J = 9.9, 7.8, 5.3 Hz, 1H), 3.39 (dd, J = 10.2, 3.8 Hz, 1H), 3.30 – 3.27 (m, 1H), 3.20 (dd, J = 13.8, 5.6 Hz, 1H), 3.05 (t, J = 7.0 Hz, 1H), 2.98 – 2.89 (m, 2H), 2.73 (td, J = 9.2, 5.9 Hz, 1H), 2.57 (d, J = 7.8 Hz, 2H), 2.45 – 2.36 (m, 2H), 2.06 – 1.94 (m, 2H), 1.77 – 1.50 (m, 3H), 1.28 (m, 1H), 1.25 (s, 9H), 1.18 (m, 1H), 1.07 (s, 9H), 0.88 – 0.79 (m, 1H), 0.74 (dd, J = 8.9, 6.6 Hz, 6H).13C NMR (126 MHz, d6-DMSO) δ 183.4, 173.7, 172.3, 171.7, 170.3, 168.6, 138.3, 138.0, 129.7, 129.4, 128.6, 128.5, 126.8, 126.8, 73.0, 67.5, 65.7, 62.2, 56.2, 55.5, 52.3, 51.8, 50.9, 49.3, 39.9, 37.9, 36.8, 36.1, 32.5, 31.0, 28.9, 27.5, 24.7, 24.1, 23.3, 22.2.


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(S)-1-acetyl-N-((S)-1-(((R)-1-(((S)-1-(((S)-1-amino-1-oxo-3-phenylpropan-2-yl)amino)-1oxo-3-phenylpropan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)amino)-3-hydroxy-1oxopropan-2-yl)pyrrolidine-2-carboxamide – (5) ESI MS [M+H]+ expected: 651.4, experimental: 651.4. LC-MS retention time: 3.6 min. Yield: 10.5 mg (8%). Mixture of conformers, 1:0.6 ratio. 1H NMR (500 MHz, d6-DMSO) δ 8.30 (d, J = 8.5 Hz, 0.6H), 8.26 (d, J = 8.2 Hz, 1H), 8.12 (d, J = 8.0 Hz, 0.6H), 8.06 (d, J = 8.3 Hz, 0.6H), 8.02 (d, J = 8.3 Hz, 1H), 7.97 (d, J = 7.8 Hz, 0.6H), 7.94 (d, J = 7.7 Hz, 1H), 7.75 (d, J = 7.2 Hz, 1H), 7.32 – 7.11 (m, 17.2H), 7.08 (m, 2H), 4.93 (t, J = 5.4, 5.4 Hz, 0.6H), 4.85 (t, J = 5.6, 5.6 Hz, 1H), 4.51 – 4.33 (m, 4.4H), 4.32 – 4.19 (m, 2.6H), 4.13 (dt, J = 8.8, 6.5, 6.5 Hz, 1H), 3.56 (m, 2.6H), 3.45 (dt, J = 9.8, 6.9, 6.9 Hz, 1H), 3.40 – 3.27 (m, 1.2H), 3.11 – 2.94 (m, 3.2H), 2.87 (m, 1.6H), 2.65 (dd, J = 13.8, 10.8 Hz, 1.6H), 2.16 – 2.07 (m, 0.6H), 2.07 – 1.98 (m, 1H), 1.94 (s, 3H), 1.87 – 1.76 (m, 5.4H), 1.74 – 1.65 (m, 1.2H), 1.31 – 1.05 (m, 4.8H), 0.77 – 0.62 (m, 9.6H). (3S,6S,12S,15S,16S,20aS)-12-benzyl-N-(tert-butyl)-3,6-diisobutyl-15-methyl-1,4,7,10,13pentaoxoicosahydropyrrolo[1,2-a][1,4,7,10,13,16]hexaazacyclooctadecine-16-carboxamide – (8) ESI MS [M+H]+ expected: 684.4, experimental: 684.4. LC-MS retention time: 4.4 min. Yield: 33.0 mg (30%). 1H NMR (500 MHz, d6-DMSO) δ 8.94 (t, J = 5.0 Hz, 1H), 8.50 (d, J = 8.3 Hz, 1H), 7.75 (d, J = 8.8 Hz, 1H), 7.38 (d, J = 9.1 Hz, 1H), 7.33 – 7.23 (m, 3H), 7.23 – 7.15 (m, 3H), 6.32 (s, 1H), 4.39 – 4.31 (m, 1H), 4.30 – 4.13 (m, 3H), 3.63 (dd, J = 15.1, 4.1 Hz, 1H), 3.47 – 3.37 (m, 2H), 3.36 – 3.33 (m, 1H), 3.13 (t, J = 7.9 Hz, 1H), 2.97 (ddd, J = 8.6, 7.0, 4.4 Hz, 1H), 2.84 – 2.73 (m, 2H), 1.99 (dtd, J = 11.9, 7.9, 5.8 Hz, 1H), 1.76 – 1.63 (m, 3H), 1.62 – 1.43 (m, 6H), 1.36 – 1.31 (m, 3H), 1.21 (s, 9H), 0.89 (dd, J = 6.3, 3.7 Hz, 9H), 0.82 (d, J = 6.4 Hz, 3H).


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(3S,6S,9S,12S,15S,16S,20aS)-12-benzyl-N-(tert-butyl)-9-(4-hydroxybenzyl)-3(hydroxymethyl)-6-isobutyl-15-methyl-1,4,7,10,13-pentaoxoicosahydropyrrolo[1,2a][1,4,7,10,13,16]hexaazacyclooctadecine-16-carboxamide – (9) ESI MS [M+H]+ expected: 764.4, experimental: 764.4. LC-MS retention time: 3.7 min. Yield: 16.8 mg (14%). 1H NMR (500 MHz, d6-DMSO) δ 9.22 (s, 1H), 8.49 (s, 1H), 7.78 (d, J = 8.9 Hz, 1H), 7.71 (s, 1H), 7.29 – 7.22 (m, 2H), 7.26 – 7.15 (m, 1H), 7.12 – 7.06 (m, 3H), 7.03 (d, J = 7.9 Hz, 1H), 6.90 – 6.85 (m, 2H), 6.67 – 6.61 (m, 2H), 6.50 (s, 1H), 4.31 (ddd, J = 11.6, 7.7, 3.6 Hz, 1H), 4.24 (dt, J = 9.1, 4.7 Hz, 1H), 4.10 (ddd, J = 10.2, 8.7, 6.5 Hz, 1H), 4.05 (m, 1H), 3.98 (dt, J = 9.1, 4.8 Hz, 1H), 3.74 (dd, J = 10.9, 4.2 Hz, 1H), 3.58 (dd, J = 10.9, 4.2 Hz, 1H), 3.33 – 3.05 (m, 5H), 2.95 – 2.90 (m, 1H), 2.84 (dd, J = 14.5, 8.9 Hz, 1H), 2.64 (dd, J = 14.6, 5.1 Hz, 1H), 1.99 (dt, J = 11.0, 4.5 Hz, 1H), 1.74 – 1.55 (m, 5H), 1.40 – 1.28 (m, 1H), 1.22 (s, 9H), 1.11 (d, J = 6.5 Hz, 3H), 0.89 (d, J = 6.1 Hz, 3H), 0.84 (d, J = 6.3 Hz, 3H).13C NMR (126 MHz, d6-DMSO) δ 173.8, 173.4, 170.9, 169.6, 169.4, 168.7, 156.0, 138.7, 129.8, 129.1, 128.2, 127.1, 126.3, 115.0, 65.5, 65.0, 61.1, 57.5, 55.6, 55.5, 50.5, 50.3, 45.7, 45.3, 41.6, 34. 8, 34.7, 29. 6, 28.1, 24.1, 23.7, 23.2, 21.2, 18.6. (3S,6S,9S,12S,15S,16S,20aS)-12-benzyl-N-(tert-butyl)-9-(4-hydroxybenzyl)-3-((R)-1hydroxyethyl)-6-isobutyl-15-methyl-1,4,7,10,13-pentaoxoicosahydropyrrolo[1,2a][1,4,7,10,13,16]hexaazacyclooctadecine-16-carboxamide – (10) ESI MS [M+H]+ expected: 778.4, experimental: 778.4. LC-MS retention time: 3.7 min. Yield: 3.0 mg (2%). 1H NMR (500 MHz, d6-DMSO) δ 9.31 (s, 1H), 8.60 (s, 1H), 7.75 (s, 1H), 7.62 (d, J = 9.5 Hz, 1H), 7.26 (dd, J = 8.1, 6.8 Hz, 2H), 7.22 – 7.16 (m, 1H), 7.14 – 7.07 (m, 3H), 7.04 (d, J = 7.7 Hz, 1H), 6.88 (d, J = 8.3 Hz, 2H), 6.69 – 6.60 (m, 2H), 6.57 (s, 1H), 5.17 (d, J = 5.2 Hz,


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1H), 4.31 (td, J = 9.0, 7.4, 3.6 Hz, 1H), 4.20 – 4.11 (m, 2H), 4.08 (dd, J = 9.5, 2.9 Hz, 1H), 4.04 – 3.93 (m, 1H), 3.35 (d, J = 8.8 Hz, 1H), 3.24 (dd, J = 9.2, 5.6 Hz, 2H), 3.19 – 3.05 (m, 2H), 2.98 – 2.89 (m, 1H), 2.85 (dd, J = 14.5, 8.9 Hz, 1H), 2.69 – 2.60 (m, 1H), 2.09 – 1.99 (m, 1H), 1.79 – 1.55 (m, 5H), 1.35 – 1.25 (m, 1H), 1.22 (s, 9H), 1.11 (d, J = 6.5 Hz, 3H), 1.02 (d, J = 6.3 Hz, 3H), 0.90 (d, J = 6.1 Hz, 3H), 0.82 (d, J = 6.3 Hz, 3H).

13

C NMR (126 MHz, d6-DMSO) δ 173.8,

173.5, 170.9, 169.7, 168.6, 165.8, 156.0, 138.7, 129.7, 128.9, 128.2, 127.0, 126.3, 115.0, 65.7, 65.2, 65.1, 58.6, 57.5, 50.6, 50.2, 47.2, 45.9, 45.2, 42.0, 34.7, 34.7, 30.1, 28.1, 23.9, 23.7, 23.5, 21.4, 20.7, 18.5. (3S,6R,9S,12S,15S,16S,20aS)-12-benzyl-N-(tert-butyl)-9-(4-hydroxybenzyl)-3-((R)-1hydroxyethyl)-6-isobutyl-15-methyl-1,4,7,10,13-pentaoxoicosahydropyrrolo[1,2a][1,4,7,10,13,16]hexaazacyclooctadecine-16-carboxamide – (11) ESI MS [M+H]+ expected: 778.4, experimental: 778.4. LC-MS retention time: 3.7 min. Yield: 28.1 mg (23%). 1H NMR (700 MHz, d6-DMSO) δ 9.19 (s, 1H), 8.67 (d, J = 5.0 Hz, 2H), 7.72 (d, J = 9.7 Hz, 1H), 7.28 (t, J = 7.6 Hz, 2H), 7.22 – 7.18 (m, 1H), 7.18 – 7.14 (m, 2H), 7.10 (d, J = 7.6 Hz, 1H), 7.02 (s, 1H), 6.93 (d, J = 8.1 Hz, 2H), 6.74 (d, J = 8.4 Hz, 1H), 6.68 – 6.59 (m, 2H), 4.48 (q, J = 6.9 Hz, 1H), 4.35 (qd, J = 6.3, 2.3 Hz, 1H), 4.19 – 4.05 (m, 2H), 3.96 – 3.90 (m, 1H), 3.86 (s, 1H), 3.42 – 3.35 (m, 2H), 3.31 (dd, J = 14.0, 4.2 Hz, 1H), 3.06 (t, J = 11.5 Hz, 2H), 2.91 (ddd, J = 9.4, 5.9, 3.3 Hz, 1H), 2.60 (dd, J = 14.3, 11.0 Hz, 1H), 2.53 – 2.50 (m, 1H), 2.05 (ddd, J = 12.4, 9.4, 6.0 Hz, 1H), 1.80 – 1.66 (m, 2H), 1.66 – 1.57 (m, 1H), 1.28 (p, J = 6.4 Hz, 2H), 1.22 (s, 9H), 1.20 – 1.11 (m, 1H), 1.06 (d, J = 6.4 Hz, 3H), 0.97 (d, J = 6.4 Hz, 3H), 0.69 (dd, J = 17.2, 6.5 Hz, 6H). 13C NMR (126 MHz, d6-DMSO) δ 173.6, 173.3, 169.4, 169.1, 168.6, 165.8, 156.0, 139.3, 129.7, 129.2, 128.1, 127.3, 126.1, 114.9, 65.2, 64.1, 63.9, 58.6, 57.1, 56.8, 50.6, 50.5, 45.7, 45.5, 42.5, 35.1, 30.4, 28.3, 24.0, 23.9, 22.6, 22.4, 20.5, 18.4.


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(3S,6S,12S,15R,16S,20aS)-12-benzyl-N-(tert-butyl)-3,6-diisobutyl-1,4,7,10,13-pentaoxo-15((phenylthio)methyl)icosahydropyrrolo[1,2-a][1,4,7,10,13,16]hexaazacyclooctadecine-16carboxamide – (12) ESI MS [M+H]+ expected: 792.4, experimental: 792.4. LC-MS retention time: 5.2 min. Yield: 32.1 mg (26%). 1H NMR (500 MHz, d6-DMSO) δ 9.11 (dd, J = 6.4, 3.3 Hz, 1H), 8.78 (d, J = 8.0 Hz, 1H), 7.83 (d, J = 8.7 Hz, 1H), 7.61 (d, J = 9.4 Hz, 1H), 7.52 – 7.45 (m, 2H), 7.41 – 7.33 (m, 2H), 7.31 – 7.25 (m, 3H), 7.25 – 7.21 (m, 2H), 7.21 – 7.17 (m, 1H), 7.12 (d, J = 8.2 Hz, 1H), 6.29 (s, 1H), 4.51 – 4.39 (m, 1H), 4.32 – 4.16 (m, 3H), 3.68 (td, J = 15.2, 2.9 Hz, 2H), 3.51 – 3.27 (m, 4H), 3.10 (t, J = 8.1 Hz, 1H), 2.87 – 2.63 (m, 3H), 2.06 – 1.93 (m, 1H), 1.77 – 1.62 (m, 3H), 1.61 – 1.41 (m, 6H), 1.21 (s, 9H), 0.89 (dd, J = 6.5, 4.3 Hz, 9H), 0.81 (d, J = 6.4 Hz, 3H). (3S,6S,12S,15R,16S,20aS)-12-benzyl-N-(tert-butyl)-3,6-diisobutyl-8-methyl-1,4,7,10,13pentaoxo-15-((phenylthio)methyl)icosahydropyrrolo[1,2a][1,4,7,10,13,16]hexaazacyclooctadecine-16-carboxamide – (13) ESI MS [M+H]+ expected: 806.5, experimental: 806.4. LC-MS retention time: 4.8 min. Yield: 22.5 mg (18%). 1H NMR (500 MHz, d6-DMSO) δ 8.74 (d, J = 8.1 Hz, 1H), 7.83 (d, J = 8.8 Hz, 1H), 7.56 – 7.44 (m, 3H), 7.40 – 7.34 (m, 2H), 7.33 – 7.25 (m, 3H), 7.23 – 7.17 (m, 3H), 7.14 (d, J = 7.8 Hz, 1H), 6.22 (s, 1H), 4.87 (td, J = 8.2, 3.3 Hz, 1H), 4.35 – 4.26 (m, 1H), 4.26 – 4.15 (m, 1H), 3.96 (d, J = 15.0 Hz, 1H), 3.69 (dd, J = 13.9, 2.5 Hz, 1H), 3.61 (d, J = 14.9 Hz, 1H), 3.44 (dd, J = 14.4, 2.8 Hz, 1H), 3.40 – 3.23 (m, 4H), 3.15 (s, 3H), 3.10 (t, J = 8.1 Hz, 1H), 2.83 – 2.69 (m, 3H), 2.05 – 1.93 (m, 1H), 1.75 – 1.61 (m, 2H), 1.59 – 1.41 (m, 6H), 1.20 (s, 9H), 0.93 – 0.84 (m, 9H), 0.81 (d, J = 6.3 Hz, 3H).


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(3S,6S,12S,15R,16S,20aS)-12-benzyl-N-(tert-butyl)-3,6-diisobutyl-11-methyl-1,4,7,10,13pentaoxo-15-((phenylthio)methyl)icosahydropyrrolo[1,2a][1,4,7,10,13,16]hexaazacyclooctadecine-16-carboxamide – (14) ESI MS [M+H]+ expected: 806.5, experimental: 806.4. LC-MS retention time: 4.8 min. Yield: 6.1 mg (5%). Mixture of conformers, 3:1 ratio. 1H NMR (500 MHz, d6-DMSO) δ 9.10 (t, J = 5.4 Hz, 0.35H), 9.05 (s, 1H), 8.45 (d, J = 9.7 Hz, 0.35H), 7.80 (d, J = 9.0 Hz, 1H), 7.71 (d, J = 8.6 Hz, 0.7H), 7.50 – 7.45 (m, 2H), 7.43 – 7.06 (m, 14.5H), 6.84 (s, 0.35H), 4.79 (dd, J = 9.0, 6.1 Hz, 0.35H), 4.50 – 4.40 (m, 1H), 4.35 – 4.24 (m, 2.35H), 4.24 – 4.07 (m, 1.05H), 4.07 – 3.97 (m, 1H), 3.56 (d, J = 13.8 Hz, 1H), 3.49 (dd, J = 14.3, 2.7 Hz, 0.35H), 3.42 – 3.34 (m, 1H), 3.32 (s, 3H), 3.30 – 3.18 (m, 3.7H), 3.17 (d, J = 5.0 Hz, 0.35H), 3.14 – 3.04 (m, 1.7H), 2.99 – 2.89 (m, 3.35H), 2.83 – 2.74 (m, 1.35H), 2.73 (s, 1H), 2.47 – 2.40 (m, 0.35H), 2.07 – 1.91 (m, 1.35H), 1.74 – 1.61 (m, 5.4H), 1.60 – 1.41 (m, 6.75H), 1.22 – 1.16 (m, 12.15H), 0.92 – 0.84 (m, 12H), 0.82 (dd, J = 7.9, 6.1 Hz, 4.2H). (3S,6S,12S,15R,16S,20aS)-12-benzyl-N-(tert-butyl)-3,6-diisobutyl-8,11-dimethyl1,4,7,10,13-pentaoxo-15-((phenylthio)methyl)icosahydropyrrolo[1,2a][1,4,7,10,13,16]hexaazacyclooctadecine-16-carboxamide – (15) ESI MS [M+H]+ expected: 820.5, experimental: 820.4. LC-MS retention time: 5.0 min. Yield: 8.5 mg (7%). 1H NMR (500 MHz, d6-DMSO) δ 8.64 (d, J = 9.7 Hz, 1H), 7.86 – 7.79 (m, 2H), 7.61 (d, J = 7.9 Hz, 1H), 7.54 – 7.07 (m, 21H), 7.00 (s, 1H), 6.92 (d, J = 9.6 Hz, 1H), 6.35 (s, 1H), 5.36 (d, J = 8.3 Hz, 1H), 4.91 – 4.83 (m, 1H), 4.82 – 4.58 (m, 3H), 4.42 – 4.23 (m, 5H), 4.13 (q, J = 10.1 Hz, 1H), 3.60 (d, J = 13.7 Hz, 1H), 3.53 (d, J = 14.4 Hz, 1H), 3.44 (d, J = 15.2 Hz, 1H), 3.38 – 3.26 (m, 6H), 3.25 – 3.18 (d, J = 10.4 Hz, 1H), 3.17 – 3.02 (m, 9H), 2.96 (d, J =


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7.4 Hz, 2H), 2.93 – 2.82 (m, 5H), 2.81 – 2.62 (m, 6H), 2.06 – 1.93 (m, 2H), 1.80 – 1.59 (m, 6H), 1.58 – 1.42 (m, 8H), 1.41 – 1.33 (m, 1H), 1.17 (s, 18H), 1.00 – 0.76 (m, 26H). (3S,6S,9S,12S,15S,16S,20aS)-12-benzyl-N-(tert-butyl)-9-(4-hydroxybenzyl)-3-((R)-1hydroxyethyl)-6-isobutyl-5,15-dimethyl-1,4,7,10,13-pentaoxoicosahydropyrrolo[1,2a][1,4,7,10,13,16]hexaazacyclooctadecine-16-carboxamide – (16) ESI MS [M+H]+ expected: 792.5, experimental: 792.5. LC-MS retention time: 3.7 min. Yield: 21.5 mg (17%). 1H NMR (700 MHz, d6-DMSO) δ 9.20 (s, 1H), 7.67 (s, 1H), 7.53 – 7.50 (m, 1H), 7.31 – 7.23 (m, 3H), 7.22 – 7.10 (m, 3H), 7.00 (d, J = 8.0 Hz, 1H), 6.85 (d, J = 8.0 Hz, 1H), 6.71 – 6.53 (m, 3H), 6.34 (s, 1H), 4.65 (bs, 1H), 4.21 (bs, 1H), 4.06 – 3.99 (m, 1H), 3.93 (bs, 1H), 3.83 – 3.71 (m, 1H), 3.60 (bs, 1H), 3.30 – 3.22 (m, 1H), 3.16 (s, 3H), 3.12 – 3.03 (m, 3H), 2.87 (bs, 1H), 2.84 – 2.74 (m, 2H), 2.69 – 2.52 (m, 1H), 2.24 (bs, 1H), 2.00 (bs, 1H), 1.76 – 1.56 (m, 4H), 1.52 (m, 1H), 1.19 (s, 9H), 1.16 (d, J = 6.4 Hz, 3H), 1.03 (d, J = 6.2 Hz, 3H), 0.95 – 0.88 (m, 6H). (3S,6R,9S,12S,15S,16S,20aS)-12-benzyl-N-(tert-butyl)-9-(4-hydroxybenzyl)-3-((R)-1hydroxyethyl)-6-isobutyl-5,15-dimethyl-1,4,7,10,13-pentaoxoicosahydropyrrolo[1,2a][1,4,7,10,13,16]hexaazacyclooctadecine-16-carboxamide – (17) ESI MS [M+H]+ expected: 792.5, experimental: 792.4. LC-MS retention time: 3.8 min. Yield: 15.8 mg (13%). 1H NMR (700 MHz, d6-DMSO) δ 9.19 (bs, 1H), 8.51 (bs, 1H), 8.27 (bs, 1H), 7.29 (t, J = 7.6 Hz, 2H), 7.26 – 7.17 (m, 4H), 6.85 (d, J = 8.0 Hz, 2H), 6.68 (s, 1H), 6.65 – 6.56 (m, 2H), 6.42 (d, J = 8.9 Hz, 1H), 5.38 – 5.29 (m, 1H), 4.67 (dd, J = 10.8, 6.9 Hz, 1H), 4.08 – 3.94 (m, 2H), 3.92 – 3.85 (m, 1H), 3.80 (dt, J = 10.4, 5.2 Hz, 1H), 3.45 – 3.40 (m, 1H), 3.33 – 3.28 (m, 1H), 3.11 (t, J = 7.7 Hz, 1H), 3.02 (t, J = 13.0 Hz, 1H), 2.95 – 2.88 (m, 1H), 2.83 (s,


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3H), 2.71 – 2.61 (m, 2H), 2.42 (dd, J = 14.1, 4.5 Hz, 1H), 2.09 – 1.96 (m, 1H), 1.80 – 1.64 (m, 3H), 1.64 – 1.51 (m, 2H), 1.36 – 1.26 (m, 1H), 1.17 (s, 9H), 1.03 (d, J = 6.3 Hz, 3H), 0.98 (d, J = 6.2 Hz, 3H), 0.88 – 0.83 (m, 6H). 13C NMR (126 MHz, d6-DMSO) δ 173.3, 172.1, 171.6, 169.7, 169.1, 166.2, 155.9, 139.1, 129.8, 129.2, 128.1, 127.8, 126.2, 114.9, 66.2, 64.9, 64.2, 53.6, 52.8, 50.5, 47.2, 45.2, 45.1, 37.2, 35.3, 34.9, 32.4, 31.1, 29.6, 28.1, 24.8, 23.4, 23.1, 20.4, 19.3, 18.3. (3S,6R,9R,12S,15S,16S,20aS)-12-benzyl-N-(tert-butyl)-9-(4-hydroxybenzyl)-3-((R)-1hydroxyethyl)-6-isobutyl-5,15-dimethyl-1,4,7,10,13-pentaoxoicosahydropyrrolo[1,2a][1,4,7,10,13,16]hexaazacyclooctadecine-16-carboxamide – (18) ESI MS [M+H]+ expected: 792.5, experimental: 792.4. LC-MS retention time: 3.8 min. Yield: 8.2 mg (7%). 1H NMR (500 MHz, d6-DMSO) δ 9.15 (s, 1H), 8.28 (d, J = 9.2 Hz, 1H), 8.21 (d, J = 6.8 Hz, 1H), 7.29 – 7.14 (m, 6H), 6.88 – 6.80 (m, 2H), 6.69 – 6.62 (m, 2H), 6.55 (d, J = 8.9 Hz, 1H), 6.40 (s, 1H), 5.33 (dd, J = 12.5, 4.4 Hz, 1H), 4.81 (d, J = 4.6 Hz, 1H), 4.73 (dd, J = 10.6, 7.2 Hz, 1H), 4.48 (ddd, J = 12.4, 9.2, 3.4 Hz, 1H), 4.06 (ddd, J = 10.9, 9.0, 6.4 Hz, 1H), 3.85 (td, J = 6.8, 4.7 Hz, 1H), 3.57 (ddd, J = 11.4, 6.6, 4.9 Hz, 1H), 3.38 (q, J = 7.6 Hz, 1H), 3.11 (t, J = 7.9 Hz, 1H), 3.01 (dd, J = 14.2, 4.9 Hz, 1H), 2.93 – 2.82 (m, 5H), 2.78 – 2.67 (m, 2H), 2.10 – 1.97 (m, 1H), 1.80 – 1.64 (m, 3H), 1.43 – 1.30 (m, 2H), 1.25 (s, 9H), 1.20 – 1.11 (m, 1H), 1.09 (d, J = 6.5 Hz, 3H), 0.98 (d, J = 6.2 Hz, 3H), 0.78 (dd, J = 9.8, 6.4 Hz, 6H). (3S,6R,9R,12S,15S,16S,20aS)-12-benzyl-N-(tert-butyl)-9-(4-hydroxybenzyl)-3-((R)-1hydroxyethyl)-6-isobutyl-5,11,15-trimethyl-1,4,7,10,13-pentaoxoicosahydropyrrolo[1,2a][1,4,7,10,13,16]hexaazacyclooctadecine-16-carboxamide – (19) ESI MS [M+H]+ expected: 806.5, experimental: 806.4. LC-MS retention time: 4.0 min. Yield: 1.0 mg (1%).


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ASSOCIATED CONTENT Supporting Information Additional information detailing the PAMPA methodology and results, aqueous solubility, cLogP and PSA calculations, NMR spectra, and X-ray crystallography data. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *Phone: 416-946-5042. E-mail: [email protected] ACKNOWLEDGMENT We would like to thank CQDM - Québec Consortium for Drug Discovery, the Natural Sciences and Engineering Research Council of Canada, Ontario Genomics Institute, and Canadian Institutes of Health Research for financial support. We would like to thank Dr. D. Burns and D. Pichugin for their assistance with NMR spectroscopic experiments and Dr. A. J. Lough for acquiring and solving X-ray crystal structures. Dr. J. Frost is acknowledged for his assistance in editing the manuscript. The authors also wish to acknowledge the Canadian Foundation for Innovation, project number 19119, and the Ontario Research Fund for funding of the Centre for Spectroscopic Investigation of Complex Organic Molecules and Polymers. ABBREVIATIONS DIPEA, N,N-diisopropylethylamine; DMF, N,N-dimethylformamide; DMSO, dimethyl sulfoxide; ECE, exocyclic control element; Fmoc, 9-fluorenylmethoxycarbonyl; HATU, O-(7Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate; HFIP, 1,1,1,3,3,3-


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hexafluoro-2-propanol; NMP, N-methylpyrrolidinone; PAMPA, parallel artificial membrane permeability assay; PSA, polar surface area; TBME, tert-butyl methyl ether; TFA, trifluoroacetic acid; TFE, 2,2,2-trifluoroethanol; TIS, triisopropylsilane; TMS, tetramethylsilane; tPSA, topological polar surface area; VT, variable temperature. REFERENCES (1)

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Passive Membrane Permeability of Macrocycles Can Be Controlled by

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