Development of Macrocyclic Peptide-Based Proteasome Inhibitors with Enhanced Blood-Brain Barrier Penetration for Treating Brain Neoplasms

Jehad Almaliti; Momen Al-Hindy; Pavla Fajtová; Evgenia Glukhov; Theodore Berger; Yan Graf; Cesar Emiliano Hoffmann da Silva; Chandler Huang; Narek Azizyan; Yuxian He; Anthony J O’Donoghue; William H Gerwick

doi:10.1021/acs.jmedchem.5c00544

. Author manuscript; available in PMC: 2026 Oct 1.

Published in final edited form as: J Med Chem. 2025 Sep 8;68(18):18908–18929. doi: 10.1021/acs.jmedchem.5c00544

Development of Macrocyclic Peptide-Based Proteasome Inhibitors with Enhanced Blood-Brain Barrier Penetration for Treating Brain Neoplasms

Jehad Almaliti ^a,^b, Momen Al-Hindy ^a, Pavla Fajtová ^c, Evgenia Glukhov ^a, Theodore Berger ^a, Yan Graf ^a, Cesar Emiliano Hoffmann da Silva ^d, Chandler Huang ^a, Narek Azizyan ^a, Yuxian He ^a, Anthony J O’Donoghue ^c, William H Gerwick ^a,^c

PMCID: PMC13626384 NIHMSID: NIHMS2206153 PMID: 40922058

Abstract

Proteasome inhibitors are effective in treating hematologic cancers but have limited utility in brain tumors due to poor blood–brain barrier (BBB) penetration and metabolic instability. In this study, we developed novel macrocyclic peptide epoxyketone inhibitors with improved drug-like properties. Compounds were screened for cytotoxicity against brain cancer cell lines, permeability (PAMPA-BBB and Caco-2), and metabolic stability. Lead compound 10 demonstrated potent in vitro activity (IC₅₀ < 100 nM), low P-gp efflux, and favorable microsomal and plasma stability. In vivo pharmacokinetic studies in mice showed that compound 10 maintained therapeutic plasma levels and achieved measurable brain concentrations without toxicity. Co-administration of a P-gp inhibitor significantly enhanced brain exposure of compound 35, confirming efflux as a key parameter. The incorporation of fluorinated phenyl and α,α-dimethylglycine moieties contributed to improved BBB permeability and metabolic stability. These findings support further development of macrocyclic epoxyketone inhibitors as promising candidates for brain cancer therapy.

INTRODUCTION

Proteasomes represent attractive drug targets for treatment of cancer, autoimmune diseases and infectious diseases.^1,2 These enzymes are multi-component protease complexes crucial for cellular degradation of intracellular protein substrates and regulation of essential processes like the cell cycle and antigen presentation. Proteasomes consist of two stacked rings of seven β subunits sandwiched between two rings of seven α subunits. This 28-subunit core complex is commonly referred to as the 20S proteasome while the combination of the 20S core and regulatory proteins is referred to as the 26S proteasome. The subunits responsible for peptide hydrolysis are located in the β-ring, namely β1, β2, and β5, each of which has a distinct substrate specificity profile. While the constitutive 20S proteasome (c20S) is expressed in most cells, antigen-presenting cells produce an immunoproteasome 20S (i20S) with alternative catalytic subunits β1i, β2i, and β5i.³

Proteasome inhibitors are currently approved for treatment of multiple myeloma and include bortezomib, carfilzomib, and ixazomib.⁴ These inhibitors preferentially target the β5 and β5i subunits and only inactivate the β1/β1i and β2/β2i subunits at higher concentration. Other inhibitors in clinical trials include zetomipzomib (KZR-616), a first-in-class selective i20S inhibitor that is being evaluated for treatment of lupus nephritis.^5,6

Most proteasome inhibitors share common structural features, including a linear peptide backbone and a C-terminal electrophile that forms a covalent bond with the N-terminal threonine, the catalytic residue in each of the proteasome subunits.⁶ While inhibition of all subunits can contribute to efficacy, targeting the β5 or β5i subunit is believed to be particularly important for clinical benefits against cancer.⁷ However, these linear peptides have poor blood-brain barrier permeability and therefore do not have value in the treatment of glioblastoma or other brain cancers.⁸ Previously, we reported the discovery of carmaphycin B, a potent proteasome inhibitor isolated from a marine cyanobacterium.^5,9 Carmaphycin B consists of a synthetically tractable tripeptide-epoxy ketone. Consequently, we have made several hundred analogs of carmaphycin B and have found numerous of these that are potent and selective for several parasite proteasomes.

Other proteasome inhibitors in clinical trials include the β-lactone marizomib (salinosporamide A, NPI-0052), that is being evaluated for treatment of glioblastoma.^10–14 Unlike other proteasome inhibitors, marizomib inhibits all three of the enzymatic sites of the proteasome.^15,16 Moreover, preclinical studies demonstrated its ability to cross the blood–brain barrier and show anti-glioma effects in animal models. Consistent with these findings, marizomib has also shown clinical efficacy in multiple myeloma patients with CNS tumor involvement.¹⁷ Based on these promising results, a phase 1/2 clinical trial was conducted for patients with recurrent glioblastoma, testing marizomib as both a monotherapy and in combination with bevacizumab, with preliminary outcomes indicating particularly strong efficacy in patients with O6-methylguanine-DNA-methyltransferase (MGMT) promoter-unmethylated tumors.¹⁸ However, a recent phase 3 clinical study on marizomib found that adding it to standard radiochemotherapy failed to improve overall survival or progression-free survival in patients with newly diagnosed glioblastoma.¹⁴ In general, glioblastoma patients have few treatment options, all of which are of limited efficacy.

Nevertheless, building on the initially promising results of marizomib as a first-in-class blood-brain barrier (BBB) penetrant proteasome inhibitor, we sought to design and synthesize additional BBB-penetrant inhibitors to potentially treat brain neoplasms. Here we present new macrocyclic proteasome inhibitors that are brain-penetrable and exhibit potent activity against cell lines of medulloblastoma (D283-med), glioblastoma (SF188) and non-small cell lung cancer (H460). In contrast to marizomib, which is non-selective for proteasome subunits, the new inhibitors are highly potent and selective for the β5 subunit with more moderate activity against β2 and no activity against β1. Therefore, the new macrocyclic brain penetrant proteasome inhibitors have considerable potential in the treatment of brain cancers.

Peptides, especially linear peptides, face challenges in terms of metabolic stability and cellular permeability,¹⁹ and these limitations often hinder their development as drug candidates. A number of clinically used peptide drugs suffer from short half-lives in vivo and are susceptible to metabolism, thus restricting their applicability and the types of cancers they can effectively treat.²⁰ However, macrocyclic or ‘stapled’ peptides offer several advantages over linear peptides. Macrocyclization can enhance cellular penetrance by providing additional conformational stability.^19,21 Macrocyclic peptides also possess greater resistance to proteases in vitro and in vivo, leading to their improved oral bioavailability.²¹ Based on these findings, we hypothesized that converting a linear peptide proteasome inhibitor, such as carmaphycin B, into a macrocyclic structure, would improve its pharmacological properties and expand its potential for treating various cancers by enhancing cell permeability and metabolic stability.

RESULTS

Cytotoxicity of designed analogues

Carfilzomib, a clinically approved proteasome inhibitor, contains an epoxyketone moiety and can be divided into five distinct sub-structures P1-P5 (Figure 1). However, its efficacy is hampered by poor cellular permeability and a short half-life, restricting its application to injectable formulations for treating multiple myeloma.²² Our research aims to develop more efficacious proteasome inhibitors with improved drug-like properties for a broader range of cancers, including malignant brain neoplasms. We designed an initial library of epoxyketone-based analogues incorporating non-natural amino acids predicted to enhance cellular permeability, metabolic stability, and BBB penetration. Given the established role of fluorine in optimizing pharmacokinetic profiles, and the documented benefits of perfluoroaryl groups in peptide conjugation for improved cellular and BBB permeability^23,24, we explored the use of pentafluorophenyl sulfides. Wong et al., demonstrated that a pentafluorophenyl sulfide containing macrocycle, substantially increased albumin binding and extended in vivo circulation half-life (Figure 2A).²⁵ Inspired by these findings, we designed a library of proteasome inhibitors incorporating this motif to potentially improve drug-like properties. Our library consists of linear peptide analogues (1, 2, 3, and 4) incorporating a leucine epoxyketone warhead similar to that of carfilzomib and the natural product carmaphycin B, and either pentafluorophenyl l-cysteine in the P2 position (Figure 2B) or a pentafluorophenyl acetic acid moiety in the P4 position (Figure 2B and 2C). The N-cap group (P4 moiety) groups included linear hexanoyl chains (1), phenyl propanoyl groups (2) or halogenated phenyls (3 and 4). To mitigate the peptide character of these compounds, we also synthesized dipeptide analogs (5, 6, 7, and 8) by eliminating the P3 l-valine residue (Figure 2D).

Figure 1. — A. Structures of FDA-approved proteasome inhibitors carfilzomib, bortezomib, ixazomib used for treatment of multiple myeloma. B. The marine natural product carmaphycin B isolated from a cyanobacterium is a template for numerous anti-parasitic proteasome inhibitors. C. Zetomipzomib, a selective inhibitor of i20S in clinical trials for treatment of lupus nephritis. D. Marizomib, a marine natural product and clinical candidate for treatment of glioblastoma.

Figure 2. — A. Structural features used by Wong et al. to enhance drug-like proprieties of peptides. B. Pentafluorophenyl-cysteine, a non-natural amino acid incorporated in the linear peptide analogues. C. Linear peptide analogues incorporating a pentafluorophenyl-cysteine residue with various N-cap (P4) groups. D. Dipeptide analogues that lack the P3 valine residue.

To evaluate cytotoxicity of these compounds, we tested them against three cancer cell lines; D283-med, a medulloblastoma cell line derived from a pediatric cerebellar tumor; SF188, a human glioblastoma cell line from an aggressive adult brain tumor; and H460, derived from a non-small cell lung cancer cell line (Table 1).^26,27 Carmaphycin B served as control linear peptide proteasome inhibitor.⁹ Marizomib, currently in phase 3 clinical trials for glioblastoma, was used as a positive control to benchmark our analogs against a leading proteasome inhibitor. In addition, quisinostat, an experimental histone deacetylase inhibitor that is used for treating various cancer types, was included as an additional chemotherapeutic control.

Table 1.

IC₅₀s of the linear peptide analogues against D283-med, H460, and SF188 cell lines.

Compound	D283-med IC₅₀ nM	SF188 IC₅₀ nM	H460 IC₅₀ nM
Quisinostat	37 ± 2	43 ± 2	30 ± 3
Carfilzomib	205 ± 24	706 ± 82	56 ± 5
Marizomib	70 ± 3	22 ± 4	73 ± 6
Carmaphycin B	13 ± 2	19 ± 2	111 ± 30
1	236 ± 27	143 ± 18	1134 ± 216
2	67 ± 8	132 ± 22	632 ± 167
3	102 ± 7	213 ± 10	723 ± 958
4	57 ± 13	192 ± 70	1752 ± 160
5	>10,000	>10,000	>10,000
6	>10,000	4080 ± 770	6670 ± 910
7	>10,000	>10,000	>10,000
8	3700 ± 270	532 ± 23	9104 ± 400

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Quisinostat demonstrated consistent activity across all three cell lines with IC₅₀ values in the 30–43 nM range confirming the general sensitivity of these cell lines to chemotherapy. The cells were also sensitive to the broad-spectrum proteasome inhibitor marizomib, with SF188 cells showing ~3.5-fold greater sensitive compared to D283-med and H460 (Table 1). Interestingly, the tripeptide-epoxyketone inhibitor carmaphycin B exhibited 6- to 8-fold greater potency against the brain cancer cell lines (D283-med and SF188) relative to the lung cancer cell line (H460). This suggests a potential for selective targeting of brain cancer with peptide epoxyketone inhibitors, prompting us to focus on developing analogues with improved drug-like properties while preserving this selectivity. Notably, analogues 2 and 4 demonstrated the most promising activity with IC₅₀ values below 200 nM against D283-med and SF188 but above 600 nM against H460 cells. To assess the importance of the P3 residue, we synthesized truncated dipeptide analogues 5, 6, 7, and 8, lacking the P3 L-valine residue. These dipeptides exhibited significantly reduced activity with IC₅₀ values in the micromolar range and a loss of selectivity for the brain cancer cell lines. This underscores the critical role of the tripeptide structure to enable essential hydrogen bonding interactions within the proteasome’s active site and facilitating optimal positioning of amino acid side chains within the binding pockets for robust irreversible inhibition.

Early assessment of BBB permeability can significantly streamline the development of new brain cancer chemotherapeutics. Following cytotoxic screening, we evaluated the BBB permeability potential of the most promising compounds (1, 2, 3 and 4) using the parallel artificial membrane permeability assay (PAMPA-BBB). PAMPA-BBB is a well-established, high-throughput non-cell-based permeation model developed by Kansy and colleagues.²⁸ It is widely utilized in early drug discovery to predict the passive diffusion of drug molecules across phospholipid membranes. Unfortunately, the PAMPA-BBB assay revealed that the four compounds exhibited very low permeability, with mean Pe values < 1 (10⁻⁶ cm/sec) and recovery rates less than 50%. These results suggest that linear perfluoroaryl-containing peptides are unlikely to cross the BBB and therefore were not pursued as lead candidates.

To enhance the drug-like properties and potential for BBB permeability of the lead molecules, we employed a macrocyclization strategy, incorporating non-natural amino acids and various conjugation strategies. Macrocyclic peptides are known to exhibit superior cell permeability and metabolic stability compared to their linear counterparts.^19,29 Therefore, we synthesized a library of analogues, focusing on cyclizing different segments of the linear peptide to yield macrocyclic proteasome inhibitors. We hypothesized that cyclizing between the P2 and P4 amino acids would be the most favorable and least disruptive for binding to the proteasome. Computational docking studies supported this hypothesis, predicting that P2-P4 cyclization would yield active compounds.³⁰ In contrast, other cyclization points, such as P1-P3, P2-P3, and P3-P4, were predicted to result in analogs with less optimal binding to the proteasome. To further enhance their metabolic stability, cell permeability, and BBB permeability, we again incorporated a perfluoroaryl moiety into these macrocycles. Fadzen and colleagues demonstrated that synthetic peptide macrocycles containing perfluoroaryl-cysteine components have improved ability to traverse the BBB.²⁹ Additionally, a Pt(IV)-conjugate of these brain-penetrant perfluoroaryl macrocyclic peptides exhibited preclinical efficacy in a glioblastoma mouse model.³¹ Wong and colleagues also reported enhanced albumin binding and extended in vivo circulation half-life with macrocyclic peptides possessing pentafluorophenyl sulfide residues.²⁵ These findings inspired us to incorporate perfluoroaryl moieties into our P2-P4 macrocyclic peptides.

Our initial investigations with P2-P4 macrocycles focused on evaluating the impact of macrocyclization via perfluoroaryl-sulfide SNAr chemistry on membrane translocation and other biological properties^25,29 The first analogue, 9, contained the l-leucine epoxyketone warhead at P1, l-valine at P3, and a 17-member perfluoroaryl-sulfide macrocycle formed via SNAr chemistry of pentafluorophenyl alanine to the thiol in 3-mercaptophenylacetic acid. When compared to 2, the closest linear analog, cyclization resulted in increased potency against brain cancer cell lines D282-med (IC₅₀ = 91 nM) and SF188 (87 nM), but lower potency against the lung cancer cell line (754 nM). This data suggested that macrocyclic peptides may be more potent at targeting brain cell lines over other cell lines. We then synthesized 10, substituting 4-mercaptophenylacetic acid with 3-mercaptophenylacetic acid. This modification led to a 5-fold increase in potency against D283-med cells (17 nM) but a 3-fold decrease against H460 cells (2,587 nM). The conversion of sulfide in 10 to sulfone in 11 was achieved by emulating the natural product proteasome inhibitor, carmaphycin B, which contains a methionine sulfone moiety in P2 and is also selective for the brain cancer cell lines. The sulfone analogue displayed even greater cytotoxicity against the two brain cancer cell lines, while exhibiting approximately 10-fold lower cytotoxicity against the lung cancer cell lines, H460.

Motivated by the potent cytotoxic activity observed for compounds 10 and 11, we synthesized a library of related analogues (Table 2) exploring substitutions in the P3 position. We incorporated various natural and non-natural amino acids, including l-tert-leucine, l-threonine, O-acetyl-l-threonine, l-hydroxyvaline, N-Methyl-l-valine, d-homo-β-valine, l-homo-β-valine, glycine, and α,α-dimethylglycine. Incorporation of l-tert-leucine (12) led to a 3- to 20-fold reduction in cytotoxicity against D283-med and SF188 compared to l-valine (10), suggesting that bulky lipophilic side chains at the P3 moiety are poorly tolerated for binding to the proteasome S3 pocket. Similarly, substituting l-valine with a bulky cyclopropyl-proline moiety (13) that is found in the COVID protease inhibitor Nirmatrelvir, led to a complete loss of efficacy across all three cell lines.

Table 2.

IC₅₀ values of the perfluoroaryl-sulfide macrocyclic analogues to the D283-med, H460, and SF188 cell lines.


Compound	R	n	D283-med IC₅₀ nM	SF188 IC₅₀ nM	H460 IC₅₀ nM
10		1	17 ± 7	123 ± 8	2,587 ± 262
12		1	432 ± 74	507 ± 134	2,474 ± 241
13		1	>15,000	>15,000	>15,000
14		1	20 ± 1	40 ± 9	186 ± 52
15		1	52.7 ± 1	179 ± 35	484 ± 48
16		1	6,094 ± 1,848	>15,000	>15,000
17		1	1,799 ± 40	2,461 ± 142	5,381 ± 1,239
18		2	232 ± 12	239 ± 10	1,077 ± 55
19		1	10,780 ± 1,637	12,300 ± 526	>15,000
20		1	662 ± 44	611 ± 85	828 ± 57
21		1	4,996 ± 578	>15,000	>15,000

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Conversely, analogues with l-threonine (14) and l-hydroxyvaline (15) at P3 exhibited potency comparable to 10 against D283-med and SF188. Moreover, these analogues were 4- to 5-fold more potent than 10 against H460 cancer cells, indicating good tolerance for a hydroxy moiety in the P3 residue. We also explored modifications to the amide linkage between P3 and P4. Replacing the l-valine moiety with N-Methyl-l-valine (19), d-homo-β-valine (16) and l-homo-β-valine (17) generally yielded less active compounds with low or sub-micromolar IC₅₀ values across all three cell lines. However, substituting a bulky α,α-dimethyl glycine derivative (20) that is known to reduce peptide hydrolysis and enhance metabolic stability, displayed high nanomolar inhibitory activity against all three cell lines. This underscores the essential role of the amide linkage between the P3-P4 residues, possibly involving hydrogen bonding with residues that make up the proteasome substrate binding pocket. Additionally, substituting the P3 l-valine with glycine (21) also reduced potency against all three cell lines. In summary, l-valine (10), l-hydroxyvaline (15) or l-threonine (14) appear to be the most suitable P3 residues in this series of fluorinated macrocycles for potent inhibition of the brain cancer cell lines.

Similar to the conversion of 10 to 11, we synthesized the oxidized sulfone version of all of the P3 analogues described above. Consistent with the 10 to 11 trend, these sulfone analogues exhibiting 3- to 20-fold enhanced potency across all three cell lines (Table 3). However, the selectivity of these analogues for brain derived cell lines was reduced compared to 10 and the other sulfide analogues. Notably, 27 emerged as one of the most active of these sulfone analogues with low nanomolar IC₅₀ value against D283-med (IC₅₀ = 28 nM) and SF188 (IC₅₀ = 72 nM), while exhibiting lower cytotoxicity against the H460 cells (IC₅₀ = 360 nM). This data confirms that the sulfone functionality within this macrocycle series is beneficial to the selectivity of these compounds to brain cancer cells. We also evaluated several sulfone analogs (11, 22, and 31) for cytotoxicity against the human foreskin fibroblasts (HFF-1) cell line, a model for evaluating selective toxicity between cancerous and non-cancerous cell lines. Importantly, these compounds exhibited similarly reduced toxicity toward HFF-1 cells, mirroring the results observed with the H460 cancer cell line.

Table 3.

IC₅₀ values of the perfluoroaryl-sulfone macrocyclic analogues against D283-med, SF188, H460, and HFF-1 cell lines.


Compound	R	n	D283-med IC₅₀ nM	SF188 IC₅₀ nM	H460 IC₅₀ nM	HFF-1 IC₅₀ nM
11		1	6 ± 2	9.8 ± 1.2	79 ± 20	85 ± 7
22		1	155 ± 8	318 ± 51	679 ± 42	448 ± 47
23 (Sulfoxide)		1	176.2 ± 2	205 ± 21	1636 ± 87	nd
24 (Sulfone)		1	54 ± 2	100 ± 7	410 ± 59	nd
25		1	>15,000	>15,000	>15,000	nd
26		1	472 ± 36	673 ± 50	2,357 ± 50	nd
27		1	28 ± 10	72 ± 10	360 ± 56	nd
28		1	44 ± 6	148 ± 35	549 ± 21	nd
29		2	146 ± 29	150 ± 12	470 ± 154	nd
30		1	1,467 ± 432	1,677 ± 509	3,196 ± 326	nd
31		1	57 ± 14	24 ± 9	175 ± 16	122 ± 14
32		1	563 ± 46	7,233 ± 113	4,541 ± 2,065	nd

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In a separate series of epoxyketone-containing macrocycles, we explored analogues with tyrosine fused to hexanoate as the macrocycle core, eliminating the fluorinated phenyl sulfide moiety (Figure 4A). Analogue 33 with l-valine as the P3 residue, showed potent cytotoxicity against the brain cancer cells with an IC₅₀ around 10 nM (Table 4), and 5-fold lower cytotoxicity against H460 cells. Two additional variants were synthesized with glycine (34) or α,α-dimethylglycine (35) at P3. 34 was weakly cytotoxic to all three cell lines with IC₅₀ values in the micromolar range, whereas 35 showed moderate cytotoxicity against the brain cancer cells D283-med (IC₅₀ = 332 nM) and SF118 (IC₅₀ = 806 nM). These results, consistent with our findings in the fluorinated macrocyclic series (20 and 21), demonstrate that replacing l-valine with glycine or α,α-dimethylglycine significantly reduces potency. We also investigated the effect of replacing the phenyl ether linkage between P2 and P4 moieties with a sulfide (36) or sulfone bridge (37 and 38). Of these compounds, the presence of l-valine in P3 of 38 demonstrated the most potent activity. This result matches with the activity of other compounds that have l-valine in the P3 position.

Figure 4. — A. Aromatic (non-fluorinated) macrocycles. B. Linear (non-aromatic) macrocycles.

Table 4.

IC₅₀ values of the aromatic (non-fluorinated) macrocycles and linear (non-aromatic) macrocycles against D283-med, H460, and SF188 cell lines.

Compound	D283-med cell line IC₅₀ nM	SF188 cell line IC₅₀ nM	H460 cell line IC₅₀ nM
33	10.1 ± 2.1	12.2 ± 0.4	53 ± 15
34	2,080 ± 177	2,906 ± 1,547	4,520 ± 867
35	332 ± 12	806 ± 388	853 ± 90
36	1,443 ± 103	2,260 ± 1,484	2,332 ± 275
37	590 ± 20	4,424 ± 385	1,717 ± 164
38	16 ± 2	157 ± 45	287 ± 42
39	14.8 ± 2.3	20 ± 3	79 ± 21
40	2.26 ± 0.26	6.7 ± 0.3	12 ± 1
41	90 ± 8	113 ± 9	430 ± 30

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We next explored non-aromatic macrocycles for their cytotoxic properties (Figure 4B). Analogue 39 fuses the P2-P4 positions with cysteine and decanoic acid, while maintaining l-valine at the P3 moiety and the leucine-epoxyketone warhead. This analogue exhibited exceptional potency against all three cell lines (Table 4). Consequently, we synthesized the oxidized analogue 40, which further enhanced potency, achieving single-digit IC₅₀ values against D283-med (2.3 nM) and SF118 (6.7 nM). Notably, this compound represents the most cytotoxic proteasome inhibitor reported in the literature, while also possessing a macrocyclic peptide structure. However, both 39 and 40 exhibit high lipophilicity with clogP values of 4.6 and 6.5, respectively, potentially leading to solubility challenges. To address this, we designed a di-ether version of this analogue (41) with reduced lipophilicity (clogP = 2.7) that demonstrated potent cytotoxicity to the brain cancer cell lines (Table 4).

Chemistry

The synthesis of linear analogues 1–8 was carried out following a previously reported procedure. Compounds 9-32 were synthesized following the route outlined in Scheme 1. Methyl protection of 2,3,4,5,6-pentafluoro-L-phenylalanine was achieved using methanol and thionyl chloride, after which it was coupled with an N-Boc-protected amino acid bearing variable R₁, R₂, and R₃ substituents (serving as the P3 moiety) to afford intermediate II. This intermediate then underwent a nucleophilic aromatic substitution (SNAr) reaction with 3-mercaptophenylacetic acid, using tris(hydroxymethyl)aminomethane as the base, to yield carboxylic acid intermediate III. The Boc protecting group was removed from the P3 moiety using TFA/DCM, and the resulting amine underwent a cyclization reaction under dilute conditions (≤2 mM). When required, the cyclic core (intermediate V) was oxidized using m-CPBA to generate sulfoxide or sulfone analogues. The resulting sulfide, sulfoxide, or sulfone intermediates were then hydrogenated to remove the benzyl group and subsequently coupled with leucine epoxyketone, furnishing the final compounds 9-32.

Scheme 1. — General synthetic route of analogues **9–32**

(a) SOCl₂, MeOH, rt, quant; (b) HBTU, DIPEA, DCM, rt, 93–99%; (c) tris(hydroxymethyl)aminomethane, DMF, rt; (d) TFA, DCM, rt; (e) m-CPBA, DCM, rt (Only for sulfoxide/sulfone analogues); (f) LiOH, Dioxane, rt.

Compounds **36–38** were synthesized as illustrated in Scheme 3. The formation of a C–S bond via a Pd-catalyzed thiol–aryl cross-coupling reaction (Buchwald-type C–S coupling) between N-Boc-4-iodo-l-phenylalanine methyl ester and tert-butyl-6-sulfanylhexanoic acid, affording intermediate **XIV**. Subsequent Boc deprotection of **XIV** was followed by amide coupling with the appropriate Boc-protected l-amino acid. A one-pot deprotection of both the Boc and tert-butyl ester groups was then performed, followed by cyclization under dilute conditions (≤2 mM) to yield the cyclic intermediate **XVIII**. For analogues 37 and 38, the cyclic sulfide intermediate **XVIII** was further oxidized using m-CPBA to obtain the corresponding sulfoxide (intermediate **XIX**). Both the sulfide (**XVIII**) and sulfone (**XIX**) intermediates were then hydrogenated to remove the benzyl protecting group, followed by coupling with leucine epoxyketone, affording the final compounds **36–38**.

The non-fluorinated cyclic aromatic analogues 33–35 were synthesized as outlined in Scheme 2. The synthesis commenced with H-Tyr-OBzl as the starting material, which was coupled with the appropriate Boc-protected l-amino acid corresponding to each analogue to afford intermediate VIII. Alkylation of the phenolic hydroxy group of tyrosine was performed using tert-butyl 6-bromohexanoate in the presence of potassium carbonate and DMF, yielding intermediate X in high yield. Simultaneous removal of the Boc and tert-butyl protecting groups was achieved using 80% TFA in DCM, followed by dilution-driven cyclization under high-dilution conditions (≤2 mM). The resulting cyclized product was converted to the corresponding carboxylic acid via hydrogenolysis, and subsequent amide coupling with the leucine-derived epoxyketone furnished the final compounds 33–35.

Compounds 36–38 were synthesized as illustrated in Scheme 3. The formation of a C–S bond via a Pd-catalyzed thiol–aryl cross-coupling reaction (Buchwald-type C–S coupling) between N-Boc-4-iodo-l-phenylalanine methyl ester and tert-butyl-6-sulfanylhexanoic acid, affording intermediate XIV. Subsequent Boc deprotection of XIV was followed by amide coupling with the appropriate Boc-protected l-amino acid. A one-pot deprotection of both the Boc and tert-butyl ester groups was then performed, followed by cyclization under dilute conditions (≤2 mM) to yield the cyclic intermediate XVIII. For analogues 37 and 38, the cyclic sulfide intermediate XVIII was further oxidized using m-CPBA to obtain the corresponding sulfoxide (intermediate XIX). Both the sulfide (XVIII) and sulfone (XIX) intermediates were then hydrogenated to remove the benzyl protecting group, followed by coupling with leucine epoxyketone, affording the final compounds 36–38.

The synthesis of the non-aromatic cyclic analogues 39–41 is depicted in Scheme 4. The sequence began with the coupling of l-cysteine benzyl ester with Boc-l-valine, followed by alkylation using alkyl bromides of varying chain lengths and substituents to afford intermediate XXIII. Subsequent simultaneous deprotection of the N- and C-terminal protecting groups in one-pot, followed by cyclization under high-dilution conditions (≤2 mM), furnished the cyclic intermediate XXIV. For analogue 40, the sulfide in XXIV was oxidized to the corresponding sulfone XXV. The resulting sulfide (XXIV) or sulfone (XXV) cyclic core was then subjected to hydrogenolysis to remove the benzyl ester, followed by amide coupling with leucine epoxyketone, yielding the final compounds 39–41.

Screening with the PAMPA-BBB permeability assay

The PAMPA-BBB assay is a widely used method for assessing passive diffusion of molecules across the BBB. Compounds with effective permeability (Pe) exceeding 3.0 × 10⁻⁶ cm s⁻¹ are considered to have favorable BBB penetration and are categorized as a CNS (+). We initially screened compounds with IC₅₀ values below 400 nM against either of the two brain cancer cell lines in the PAMPA-BBB assay. Those compounds active in the PAMPA assay were then identified for further in vitro testing. All linear peptide proteasome inhibitors (1, 2, 3, and 4) exhibited low permeability in the PAMPA-BBB assay, with mean Pe values below 1 × 10⁻⁶ cm s⁻¹ (Table 5). Additionally, these compounds have low recovery rates, suggesting potential degradation or physical interaction with the PAMPA-BBB layer, rendering them as unsuitable candidates for BBB-permeable proteasome inhibitors. Conversely, most of our macrocyclic analogues displayed improved Pe values, accompanied by higher recovery rates exceeding 50% in most cases. This suggests that conformational constraints imposed by the macrocycle enhance both the permeability and stability of these molecules. Notably, 10, 14, and 15 emerged as the most permeable compounds among the fluorinated macrocycle analogues, with Pe values around 4 × 10⁻⁶ cm s⁻¹, classifying them as CNS (+) molecules. It is worth noting that all three compounds are sulfide analogs, which is expected, as higher lipophilicity generally leads to an increased likelihood of permeability in the PAMPA-BBB assay.

Table 5.

PAMPA-BBB permeability and recovery of the most potently cytotoxic analogues.

Compound	Mean Permeability (10⁻⁶ cm/sec)	% Recovery	Compound	Mean Permeability (10⁻⁶ cm/sec)	% Recovery
Midazolam (+ve control)	6.80	84	15	4.03	53
Marizomib	0.13	2	24	2.06	79
Carfilzomib	1.43	41	27	1.40	88
Theophylline (-ve control)	0.1	96	31	3.70	64
1	0.63	16	33	1.49	87
2	0.38	10	35	6.81	100
3	0.05	52	38	0.27	99
4	0.18	8	39	3.43	44
10	3.48	49	40	3.34	77
11	2.15	64	41	0.22	98%
14	3.75	53

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Furthermore, non-aromatic macrocycle compounds 39 and 40 also exhibited Pe values around 4 × 10⁻⁶ cm s⁻¹ in the PAMPA-BBB assay. Compound 35, containing α,α-dimethylglycine as the P3 moiety, displayed the highest permeability among all tested compounds, with a Pe value of 6.8 × 10⁻⁶ cm s⁻¹ and 100% recovery. Although this compound had lower cytotoxicity compared to the most potent analogues, its improved permeability makes it a promising candidate for brain cancer treatment. Interestingly, marizomib, a compound recently evaluated in clinical trials for glioblastoma, exhibited a very low Pe of 0.13 × 10⁻⁶ cm s⁻¹ and a minimal recovery rate of 2% in the PAMPA-BBB assay. These findings for marizomib may help explain the recent reports that marizomib offers no significant benefit over the standard of therapeutic care.³²

Evaluation of in vitro plasma and microsomal stability

To assess the potential for in vivo efficacy, we determined the stability of several of our potent and CNS (+) compounds in mouse plasma and microsomes. Compounds 2, 4, J-14, 10, 11, 14, 15, 39, 40, and 35 all exhibited exceptional stability after incubation in mouse plasma, with more than 75% remaining after 5 hours (Table 6). Additionally, the majority of these molecules retained more than 95% of their integrity after the initial two hours of incubation. This level of stability indicates the potential for these compounds to effectively reach target cells in the brain.

Table 6.

Microsomal and plasma stability of selected compounds.

Compound	Microsomal Stability			Plasma Stability
Compound	t_1/2 (min)	mCLint (μL/min/mg)	Clearance Category	% remaining (hr)
2	<5	NA	High	> 99 after 2 hr
4	<5	NA	High	> 99 after 2 hr
10	10	202	High	98 after 2 hr
11	32	43	Medium	88 after 2 hr and 78 after 5 hr
15	8	160	High	92 after 2 hr
39	42	33	Medium	87 after 2 hr
40	<5	323	High	94 after 2 hr
35	87	10	Low	64 after 2 hr
31	8	182	High	81 after 2 hr
Carfilzomib	<5	NA	High	99 after 2 hr
Marizomib	<5	NA	High	20 after 2 hr

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Liver microsomal stability is a critical parameter in drug discovery and is frequently assessed during the early stages of drug development. Hence, we evaluated the same 10 compounds that were evaluated in mouse plasma for their microsomal stability (Table 6). The linear analogues 2 and 4 had a short half-life of less than 5 minutes, indicating high susceptibility to oxidative metabolism. However, the macrocyclic analogue 10 exhibited a longer half-life of approximately 10 minutes. 11, differing from 10 only by the oxidation of the sulfide to sulfone, displayed a half-life of 32 minutes. 39 demonstrated a promising half-life of 42 minutes, whereas 35 emerged as the most metabolically stable compound in this study with a half-life of approximately 90 minutes. For comparison, both carfilzomib and marizomib exhibit short half-lives of less than 5 minutes in this assay. In summary, our macrocyclic analogs generally exhibit greater metabolic stability than our control agents in liver microsomes, likely due to their constrained conformations, and this may translate to enhanced in vivo stability.

Caco-2 Cell permeability evaluation

Caco-2 cells are commonly used as a model for studying human transepithelial drug transportation.³³ When cultured as a monolayer, these cells differentiate to form tight junctions, simulating paracellular movement across the intestinal epithelium. Additionally, Caco-2 cells express transporter proteins, efflux proteins, and Phase II conjugation enzymes, enabling them to model various transcellular pathways and the metabolic transformation of test compounds. We evaluated nine of our most promising compounds in Caco-2 cells to assess their susceptibility to P-glycoprotein (P-gp) efflux and their cellular permeability. The linear peptide 4 displayed minimal permeability across Caco-2 cells, coupled with low recovery (Table 7). In contrast, macrocyclic analogues 10, 11, 15, 35, 39, 40 and 31 all exhibited significant permeability, efficiently traversing from the apical to basolateral layers (A→B) of the Caco-2 cells. Furthermore, analogues 10 and 39 were less affected by P-gp efflux whereas 11, 15, 31, 35 and 40 demonstrated significantly more efflux, perhaps due to their greater polarity. These findings indicate high passive permeability for the macrocyclic analogues, albeit with cellular permeability potentially influenced by efflux for some of the analogues. The macrocyclic analogues also showed high recovery rates, indicating their relative stability during incubation with Caco-2 cells. Given the covalent, irreversible mechanism of action of these potent proteasome inhibitors, they are less likely to be negatively impacted by efflux once they have permeated the cells and reached their target.³⁴

Table 7.

Caco-2 cell permeability data for selected compounds.

Compound	Pe (A-B) (10⁻⁶ cm/sec)	Pe (B-A) (10⁻⁶ cm/sec)	Efflux Ratio	% Recovery (A-B)/(B-A)
Digoxin (+ve control)	1.3	13.0	9.9	91/94
4	ND	1.3	ND	27/42
10	25.1	34.4	1.4	83/75
11	7.2	45.4	6.3	91/97
15	5.7	47.9	8.4	91/96
41	0.75	20.8	27.6	102/101
35	18.6	46.5	2.5	98/93
39	9.8	14.6	1.5	67/74
40	10.1	48.8	4.8	91/89
31	3.6	63.0	17.6	94/73
Carfilzomib	NA	NA	28.6*	NA

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Literature data

ND: Not determined due to poor permeability < 0.1 x 10⁻⁶ cm/sec

NA: Not available

Proteasome Inhibition Evaluation

We evaluated the most promising compounds for their inhibitory activity against the three catalytic subunits of the human constitutive and immunoproteasome (Table 8). 39 and 40 were the most potent compounds that targeted the β5 and β2 subunits of the constitutive proteasome, surpassing even the potency of the control compound, marizomib (Table 8). Notably, they also showed the potent inhibition of the immunoproteasome β5 and β2 subunits. We hypothesize that potent inhibition of β5 and β2 with weaker inhibition of β1, may play a role in the observed activity of these molecules toward brain cancer cell lines. 41, the diether analogue of 39, exhibited notably weaker β5 inhibition but retained some potency against the β2 subunit of the constitutive proteasome while also inhibiting β1. Its potent cytotoxicity against the two brain cancer cell lines highlights that dual inhibition of proteasome subunits can be highly effective. Furthermore, 33 emerged as a potent inhibitor of both β5 and β2 of the constitutive and immunoproteasome, correlating with its potent cytotoxicity across all three cancer cell lines.

Table 8.

Determination of minimal half inhibitory concentration (IC₅₀ ± SD) of selected compounds on human constitutive and immunoproteasome (Values are in nM).

Compound	c20S β5	c20S β2	c20S β1	i20S β5	i20S β2	i20S β1
Marizomib	16.5 ± 1.5	236.7 ± 37.7	336.3 ± 27.2	16.8 ± 0.9	207.4 ± 22.3	40.5 ± 1.7
10	139.3 ± 17.7	1047.0 ± 105.1	690.9 ± 66.0	532.4 ± 56.9	463.5 ± 50.1	163.3 ± 14.9
11	50.8 ± 6.3	1471.0 ± 401.1	4204.0 ± 1315.0	829.5 ± 260.2	828.8 ± 187.4	379.4 ± 47.7
14	172.9 ± 17.7	2370.0 ± 452.0	2971.0 ± 723.2	858.1 ± 341.8	1063.0 ± 170.9	309.9 ± 39.5
15	349.3 ± 46.3	3335.0 ± 623.7	8850.0 ± 987.3	3927.0 ± 2362.0	3998.0 ± 288.5	1158.0 ± 113.5
31	150.9 ± 16.5	2752.0 ± 267.6	6008.0 ± 411.9	763.3 ± 198.9	1063.0 ± 121.0	206.7 ± 14.3
33	23.0 ± 2.0	64.1 ± 13.0	924.1 ± 185.0	32.9 ± 5.9	67.4 ± 8.4	81.7 ± 8.6
35	1396.0 ± 213.6	1673.0 ± 386.6	803.9 ± 115.7	1905.0 ± 587.0	1752.0 ± 239.9	78.2 ± 5.3
39	9.4 ± 1.3	28.6 ± 3.4	360.4 ± 34.3	63.9 ± 7.2	45.3 ± 3.1	99.0 ± 8.4
40	7.5 ± 0.6	39.4 ± 6.3	766.9 ± 176.9	16.0 ± 2.0	23.0 ± 1.6	144.5 ± 9.2
41	389.9 ± 50.7	109.6 ± 20.3	484.0 ± 33.0	4900.0 ± 2159.0	276.9 ± 49.1	49.0 ± 2.2

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Compound 11 is a potent inhibitor of the β5 subunit (IC₅₀ = 50.8 nM) and has 29-fold and 82-fold lower potency for β2 and β1, respectively, making it a relatively selective β5 inhibitor. Other fluorinated macrocycles like 31, 14, and 15 also displayed selective inhibition of the β5 subunit of c20S but were much less potent. While these compounds exhibited potent cytotoxicity against the brain cancer cell lines, presumably due to their inhibition of the β5 subunit, studies have shown that inhibitors are more cytotoxic when they target two subunits, usually β5 and β2.³⁵ This appears to be particularly important for achieving potency against H460 cell lines, as our data show that the most cytotoxic compounds to H460 cells, such as compounds 33, 39, and 40, exhibit significantly stronger inhibition of the β2 subunit of the c20S proteasome. In fact, many studies have shown that H460 cell lines are less sensitive to proteasome inhibitors, and this may provide an additional reason for the weaker inhibition of some of our epoxyketone compounds to these cell lines.³⁶ Moreover, inhibiting two subunits of the proteasome reduces the potential for development of resistance.³⁷ While the potency of most compounds is in the order of β5>β2>β1, compound 10 was different in that its potency for β1 was higher than β2 (essentially β5>β1>β2) and therefore while it as not as potent as other compounds, it was evaluated in subsequent in vivo PK assays.

35, the least cytotoxic compound, also showed the weakest inhibition of all three proteasome subunits in both the constitutive and immunoproteasome. 40, 39, and 33, exhibiting potent inhibition of both the constitutive and immunoproteasome, high microsomal and plasma stability, and PAMPA-BBB permeability, represent promising lead compounds for the potential treatment of brain cancer.

In vivo Pharmacokinetics of Selected Compounds in Mice

The in vivo pharmacokinetics (PK) of four selected compounds 10, 11, 35, and 40 were investigated in mice to assess their distribution in plasma and brain tissues (Figure 5 and Table 10). These compounds were selected based on their potent cytotoxicity against brain cancer cell lines D283-Med and SF188, as well as favorable in vitro stability and cell permeability profiles. Compound 10 demonstrated superior selectivity toward the two brain tumor cell lines, a low P-gp efflux ratio, and moderate permeability in the PAMPA-BBB assay (Table 9). Compounds 11 and 40 showed potent cytotoxic activity and favorable microsomal stability; however, both appeared to be susceptible to P-gp-mediated efflux. Compound 35 was particularly notable for its exceptional microsomal stability (t_1/2 = 87 min, and low clearance) and high permeability in the PAMPA-BBB assay. While it also showed moderate (sub-micromolar) cytotoxicity, it was the most permeable compound in this series. However, it demonstrated susceptibility to P-gp-mediated efflux despite its permeability in the Caco-2 assay. Nevertheless, based on these profiles these four compounds were selected for in vivo PK studies with analysis of both plasma and brain tissues. Given the likelihood that some of these compounds are P-gp substrates, two treatment arms were designed for each: intravenous administration of the test compound alone, and co-administration with the known P-gp inhibitor tariquidar (13 mg/kg, i.p.). This approach was specifically applied to compounds 11 and 35, which exhibited significant efflux ratios (>2) in the Caco-2 assay along with favorable plasma exposure profiles (Figure 5).

Figure 5. — Plasma Concentration versus Time Profiles of Compounds 10, 11, and 35 in Male C57BL/6 Mice.

Table 10.

Brain Concentrations of Compounds 10 and 35 Following Intravenous Administration.

Compound	Time point (h)	Mean Brain Con (ng/g)*	Mean Plasma Conc. (ng/mL)	Mean B/P ratio
10	1.0	12.0	16.8	0.78
10	4.0	BLQ	5.4	NA
35	1.0	26.3	330	0.08
35	2.0	BLQ	5.3	NA
35 (pretreated with tariquidar)	0.5	137.0	165.7	0.80
35 (pretreated with tariquidar)	2.0	BLQ	5.0	NA

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LLOQ: 2.00 ng/mL; BLQ-Below limit of quantitation, NA: Not applicable

Table 9.

Summary of In Vitro Properties of Compounds 10, 11, 35, and 40

	Cytotoxicity IC50s			PAMPA-BBB	Microsomal Stability		Plasma Stability	Caco-2 permeability
Compound	D283-m IC₅₀ nM	SF188 IC₅₀ nM	H460 IC₅₀ nM	Mean Permeability (10^-6 cm/sec)	t_1/2 (min)	Clearance Category	% remaining after 2 hr	Pe (A-B) (10^-6 cm/sec)	Efflux Ratio
10	17	123	2,587	3.48	10	High	98	25.1	1.4
11	6	9.8	79	2.15	32	Medium	88	7.2	6.3
35	332	806	853	6.81	87	Low	64	18.6	2.5
40	2.26	6.7	12	3.34	<5	High	94	10.1	4.8

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Compound 10 was administered intravenously (i.v.) at 1 mg/kg. Plasma concentrations were measured at 0, 2, 4, 6, 8, and 24 hours post-injection, while brain concentrations were assessed at 1 and 4 hours. Compound 10 exhibited favorable plasma stability, maintaining concentrations above its IC₅₀ for up to 8 hours (Fig 5). The Cmax was 423 ng/mL at the earliest time point. For irreversible inhibitors like 10, a high Cmax and short half-life are preferred to achieve optimal therapeutic efficacy.³⁸ Significant brain uptake was observed at 1 hour, but concentrations fell below the limit of quantification by 4 hours (Table 10). The brain-to-plasma ratio at 1 hour was 0.78, indicating comparable CNS and plasma exposure. Importantly, no signs of toxicity were observed in any of the 12 mice at the tested dose, suggesting that compound 10 is well tolerated and capable of achieving therapeutically relevant brain concentrations.

Compound 40, administered i.v. at 1 mg/kg, showed negligible plasma concentrations beyond 5 minutes post-dosing and failed to achieve detectable levels in brain tissues. Hence, the dosing level was increased to 10 mg/kg; however, the compound still did not exhibit significant plasma concentrations beyond 5 minutes post-administration. This prompted further evaluation of its stability in whole blood. While previous plasma stability assays indicated >90% compound retention after 2 hours, whole blood incubation revealed only ~15% retention over the same period, highlighting its instability in whole blood. Due to this poor PK profile, compound 40 was excluded from further development and was not evaluated in the tariquidar treatment group.

Given the lack of toxicity of compounds 10 and 40 at 1 mg/kg, a higher dose of 10 mg/kg was used for compounds 11 and 35 to enhance brain exposure. Compound 11 was administered i.v. with and without tariquidar, and plasma levels were measured at 0, 2, 4, 6, 8, and 24 hours. Brain concentrations were assessed at 30 minutes and 2 hours. The compound displayed high plasma Cmax and a moderate half-life (1.2–1.4 hours) in both groups. However, no quantifiable concentrations were detected in brain tissue at either time point, suggesting that compound 11 does not effectively cross the BBB under the tested conditions.

Compound 35 was also tested at 10 mg/kg i.v. with and without tariquidar. Plasma concentrations were measured at the standard time points, and brain levels were analyzed at 30 minutes and 2 hours. This compound exhibited a high plasma Cmax (average 2453 ng/mL) and a short half-life (~0.25 hours), consistent with in vitro plasma stability data. Encouragingly, compound 35 demonstrated the highest brain concentration among all tested compounds at the 30-minute mark (137 ng/mL in the tariquidar group). The brain-to-plasma ratio in the tariquidar group was 0.85, compared to only 0.08 in the compound 35 by itself treated group (26 ng/mL), indicating significant enhancement in CNS retention with P-gp inhibition. By 2 hours, compound 35 levels in the brain had fallen below the quantification limit, which corresponds to the observed rapid plasma clearance. Although the 30-minute brain concentration was below the compound’s in vitro IC₅₀, it is plausible that in vivo efficacy may still be achieved due to the irreversible nature of its inhibitory mechanism. Future studies will incorporate earlier time points to better characterize brain exposure kinetics.

DISCUSSION

Linear peptide proteasome inhibitors, despite their potent in vitro cytotoxicity against cancer cell lines, have shown limited utility for drug development due to inadequate cellular and organ permeability, as well as chemical instability. This is especially problematic for targeting solid cancers including those of the CNS and cerebrum.³⁹ The FDA-approved proteasome inhibitors are not efficacious against glioblastoma as well as many other solid tumors, presumably as a result of a combination of stability, BBB-penetrance, cell permeability, and p-gp-mediated efflux characteristics. Our findings reported herein further confirm that linear peptide proteasome inhibitors may not be suitable for treating malignant solid tumors including those in brain tissues.

Therefore, we have focused on discovering novel, potent, cell permeable, metabolically stable, and BBB-permeable proteasome inhibitors. To achieve this, we employed a multilayer approach (Figure 6). Initially, we designed novel analogs and tested them against brain cancer cell lines as well as non-brain cancer cells with a goal of discovering potent and brain cancer-targeting agents. The most promising compounds with IC₅₀ values < 400 nM were further screened using the PAMPA-BBB assay to evaluate their potential for crossing the BBB in vivo. Compounds identified as PAMPA-BBB positive were subsequently assessed for microsomal and plasma stability, with some also evaluated for their Caco-2 permeability. The most promising candidates from these tests were further examined for their proteasome inhibitory activity. Based on these profiles, we selected four compounds for in vivo studies to assess their BBB permeability. This progressive screening process facilitated the discovery of a novel proteasome inhibitors 10, 11, 35 and 40 with drug-like properties.

Figure 6. — Flowchart for the discovery of BBB penetrant proteasome inhibitors.

Cyclic proteasome inhibitors developed in this study, particularly those incorporating fluorinated phenyl moieties (e.g., 10, 11, 15), exhibited favorable cell permeability, high PAMPA-BBB permeability, and potent cytotoxicity toward brain cancer cells. The presence of fluorine appears to enhance not only permeability but also metabolic stability, as supported by Caco-2 and PAMPA-BBB data. Additionally, the macrocyclic scaffolds themselves may contribute to improved permeability, likely due to their conformational rigidity and ability to traverse cellular membranes more efficiently. While these compounds primarily target the β5 subunit of the constitutive proteasome, with some showing additional activity at β2 and β1, their strong cytotoxicity against brain tumor cells may be more closely tied to their superior permeability profiles than to subunit selectivity alone. In this regard, several fluorinated macrocycles demonstrated only moderate proteasome inhibition but still achieved potent cytotoxic effects, suggesting cell permeability in these brain tumor cell lines may be critical contributors to their biological activity.

Macrocyclic analogues with non-fluorinated aromatic rings, such as compound 33, also demonstrated potent in vitro activity. Compound 33 inhibited both β5 and β2 subunits of the constitutive and immunoproteasome and showed broad cytotoxicity across all cancer cell lines tested. However, its lower PAMPA-BBB permeability compared to fluorinated analogues raises the possibility that limited permeability may restrict its effectiveness in brain tumors in vivo. In contrast, compound 35, which incorporated an α,α-dimethylglycine residue, showed improved PAMPA-BBB permeability and microsomal stability, likely due to its conformational features. Though compound 35 displayed relatively weak proteasome inhibition and moderate in vitro cytotoxicity, its high cell permeability and metabolic stability made it a compelling candidate for in vivo PK testing where it achieved substantial brain concentrations. This highlights that tissue and cellular permeability, rather than potency against isolated proteasomal subunits, may be a more important determinant of tissue-level drug exposure and therapeutic potential in CNS cancers. The covalent, irreversible mechanism of proteasome inhibition may lessen the consequences of suboptimal drug concentrations in the brain as covalent target engagement can still occur with even limited exposure.

Non-aromatic macrocyclic inhibitors such as 39, 40, and 41 also exhibited low-nanomolar cytotoxicity and favorable PAMPA-BBB permeability. Compounds 39 and 40 potently inhibited β5 and β2 subunits of both the constitutive and immunoproteasome and demonstrated stronger subunit inhibition than marizomib. Although these compounds had only moderate PAMPA-BBB values, their lipophilic character and good Caco-2 permeability suggested they would likely penetrate the BBB in vivo. However, increased permeability maybe offset by susceptibility to P-gp–mediated efflux, as seen in several of our macrocycles. For example, compound 40 showed improved Caco-2 permeability but was significantly affected by efflux. It did not achieve any detectable brain concentration levels, possibly due to its susceptibility to P-gp-mediated efflux as well as its poor stability in whole blood stability assays. This again shows that beyond subunit selectivity and proteasome potency, factors like permeability and stability can be just as crucial.

Importantly, data from Caco-2 permeability studies further support the link between cellular permeability and selective brain tumor cell cytotoxicity. Most macrocyclic analogues tested (e.g., 10, 11, 15, 31, 35, 39, 40) demonstrated significant apical-to-basolateral transport, suggesting high passive permeability; compounds 10 and 35 stood out with the highest permeability in the A→B direction. Compound 10 also showed the strongest selectivity toward brain tumor cell lines, providing an additional correlation between permeability and in vitro cytotoxicity. Compounds 10 and 35 also reached substantial brain tissue concentrations in vivo and exhibited a favorable brain-to-plasma ratio, validating their permeability and retention as key drivers of CNS exposure.

While several analogues showed efflux susceptibility, their irreversible mechanism of action may reduce the need for sustained intracellular concentrations. Nonetheless, minimizing efflux liability remains desirable for CNS applications. Indeed, the interplay between passive permeability, P-gp efflux susceptibility, and metabolic stability appears to play a critical role in determining compound distribution, particularly across the blood–brain barrier. In light of these findings, proteasome subunit selectivity, while relevant, may not be the dominant factor dictating in vitro cytotoxicity or in vivo efficacy in brain cancer models. Future development efforts will focus on refining these lead structures to balance proteasome targeting with optimal permeability, efflux avoidance, and metabolic stability, in order to maximize therapeutic efficacy in CNS malignancies.

Recent studies have explored various other proteasome inhibitors for brain cancer treatment. CEP-18770, a boronic acid derivative, has demonstrated efficacy against medulloblastoma. However, like bortezomib, these compounds are highly hydrophilic, making effective BBB penetration challenging. In fact, multiple studies have shown that bortezomib is unable to cross the BBB effectively.^16,40 Achieving sufficient CNS exposure would require significantly higher doses, increasing the risk of systemic toxicity and further limiting their therapeutic potential for brain neoplasms. Consequently, boronic acid-based proteasome inhibitors are unlikely to be promising candidates for future brain cancer treatments.

In contrast, epoxyketone-based proteasome inhibitors demonstrate superior BBB permeability, making them promising candidates for CNS applications. A study by Park et al. introduced brain-permeable macrocyclic peptide epoxyketones designed to target the immunoproteasome for potential treatment of Alzheimer’s disease, and demonstrated that this scaffold enhances metabolic stability and cell permeability compared to linear peptides.⁴¹ However, these inhibitors were specifically optimized for neurodegenerative disorders rather than brain neoplasms, and their selective targeting of the immunoproteasome limits their broader applicability in oncology. Additionally, their study did not evaluate activity against the constitutive proteasome, which is essential for minimizing toxicity in healthy brain cells. Notably, the doses used in their study (40–150 mg/kg) are significantly higher than those typically used for chemotherapeutics, making it less likely that these molecules can be repurposed for anticancer indications. By contrast, our studies have focused on macrocyclic peptide epoxyketones as potential treatments for brain neoplasms. Our inhibitors not only demonstrate significant BBB permeability but also incorporate novel moieties, such as fluorinated phenyl groups and α,α-dimethylglycine residues that enhance metabolic stability, providing a distinct advantage over prior studies. Furthermore, their notable selectivity allows for the potential in vivo dosing at higher levels (beyond the 10 mg/kg used in our PK evaluation) to achieve optimal treatment of brain cancers. These features position our inhibitors as strong candidates for brain cancer treatment, addressing key limitations of previously developed proteasome inhibitors.

CONCLUSIONS

Linear peptide proteasome inhibitors, while potent, have limited therapeutic utility against malignant solid tumors due to poor metabolic stability, low permeability, and inadequate tissue retention. In contrast, macrocyclic peptides, particularly those modified with fluorinated or non-fluorinated phenyl moieties, show improved drug-like properties across these parameters. Our study identified several macrocyclic inhibitors with exceptional potency, permeability, and the potential to overcome challenges faced by current proteasome inhibitors. Notably, compounds 10 and 40 demonstrated the ability to cross the BBB and be retained in brain tissue in a pharmacokinetic study conducted on mice, corroborating our in vitro findings. Continuing studies are focused on evaluation of these compounds as therapeutic treatments of brain neoplasms and other solid cancers.

EXPERIMENTAL SECTION

Inhibitor Subunit Specificity

Human constitutive proteasome (c20S) and immunoproteasome (i20S) were purchased from R&D Systems. Fluorogenic substrates Suc-LLVY-AMC, Z-VLR-AMC, and Z-LLE-AMC were obtained from Cayman Chemical. Substrates were dissolved in DMSO at a concentration of 10 mM and stored at −20°C. MZB and h20S were aliquoted and stored at −80°C.

Half-Maximal Inhibitory Concentration (IC₅₀) Assays

The inhibition of individual 20S proteasome subunits was assessed using subunit-specific fluorogenic substrates: Z-LLE-AMC (β1, β1i), Ac-PAL-AMC (β2/β2i), Z-VLR-AMC (β2/β2i), and Suc-LLVY-AMC (β5/β5i). Kinetic assays were performed in 384-well plates, using 1 nM c20S or i20S, preincubated with 100 nM PA28α human activator. The PA28α activator was expressed in E. coli using the pSumo vector, as previously described. Reactions were conducted in 50 mM HEPES (pH 7.5) and 1 mM DTT, with the following substrate concentrations: 80 μM Z-LLE-AMC, 30 μM Z-VLR-AMC, and 65 μM Ac-LLVY-AMC for c20S, and 230 μM Ac-PAL-AMC, 35 μM Z-VLR-AMC, and 140 μM Ac-LLVY-AMC for i20S. The final reaction volume per well was 8 μL. Inhibitors were dispensed using an Echo650 liquid handler (Beckman Coulter). For the irreversible inhibitor, pre-steady-state kinetic measurements were performed, and IC50 values were determined 60 to 90 minutes after reaction initiation. All assays were conducted in triplicate in 384-well black plates (Nunc) at 37°C. Fluorescence measurements were recorded using a Synergy HTX Multi-Mode Microplate Reader (BioTek, Winooski, VT) with excitation and emission wavelengths of 360 nm and 460 nm, respectively. Data analysis was performed using GraphPad Prism software.

Ethical Statement

Male C57BL/6 mice (6–8 weeks old) were obtained from the Laboratory Animal Centre of Huazhong Agricultural University (Wuhan, China). All animal studies were conducted in accordance with all applicable regulations and guidelines of the Institutional Animal Care and Use Committee (IACUC) (approval: IACUC No. 00001476).

Chemistry.

General.

Unless stated otherwise, chemicals and solvents used were purchased from commercial suppliers and used without further purification. HPLC was conducted with an Agilent 1260 infinity using C18 columns (5 μm, 250 × 10 mm). NMR spectra were recorded on a Varian 500 MHz spectrometer (500 and 125 MHz for the ¹H and ¹³C nuclei, respectively) using CDCl₃, (CD₃)₂SO or CD₃OD as solvents from Cambridge Isotope Laboratories, Inc. Spectra were referenced to residual CDCl₃, CD₃OD, or DMSO solvent as the internal standard (for CDCl₃ δH 7.26, and δC 77.1; and for CD₃OD δH 3.31 and δC 49.2; for (CD₃)₂SO δH 2.54, and δC 40.45). LC-HRMS data for analysis of compounds were obtained on an Agilent 6239 HR-ESI-TOFMS equipped with a Phenomenex Luna 5 μm C18 100 Å column (4.6 × 250 mm). LCMS data for purity analysis of the synthesized compounds were obtained with a Thermo Finnigan Surveyor Autosampler-Plus/LC-PumpPlus/PDA-Plus system and a Thermo Finnigan LCQ Advantage Max mass spectrometer (monitoring 200–600 nm and m/z 150–2000 in positive ion mode) using a linear gradient of 30 – 100% H₂O/acetonitrile; flow rate of 0.750 mL/min. Structural integrity and purity of the test compounds were determined from the composite of ¹H and ¹³C NMR, HRMS, and HPLC, and rigorous LCMS analysis confirmed >95% purity for all target compounds. Chemical shifts (δ) are given in parts per million (ppm) and coupling constants (J) are reported in Hertz (Hz). The compounds are named according to their IUPAC name as determined using ChemBioDraw Ultra (version 16.0).

General Synthetic Procedures - General Procedure for Coupling Amino Acids

A solution of 1 equivalent of the acid and the amine in DCM was prepared, followed by the addition of 1.1 equivalents of HBTU and 3 equivalents of DIPEA. The reaction mixture was stirred overnight. After evaporation of DCM, the crude product was diluted with ethyl acetate and transferred to a separatory funnel. The organic layer was sequentially washed twice with 1 M HCl, saturated sodium bicarbonate solution, and brine. It was then dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The resulting material was purified using automated flash chromatography.

General Synthetic Procedures - Nucleophilic Aromatic Substitution (SNAr)

A solution of 1 equivalent of the pentafluorophenyl-alanine peptide derivative in DMF (0.1 M) was prepared, followed by the addition of 2 equivalents of an alkyl or aryl halide and 5 equivalents of Tris(hydroxymethyl)aminomethane. The reaction mixture was stirred overnight, and product formation was confirmed by LCMS analysis. The reaction was then diluted with 1 M HCl to adjust the pH to 2, resulting in the appearance of a white suspension. The aqueous phase was extracted twice with ethyl acetate, and the combined organic layers were washed with brine, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The crude product was subsequently purified by automated flash chromatography using a gradient of 0 to 5% MeOH in DCM.

General Synthetic Procedures - Deprotection of Boc and Tert-butyl Protecting Groups

A solution of the protected amino acid or peptide (0.1 M) in DCM was combined with an equal volume of trifluoroacetic acid (TFA) and stirred at room temperature for 0.5 to 6 hours, with reaction progress monitored by LCMS. The solvent was then removed under reduced pressure, followed by the addition of 5 to 10 mL of toluene and re-evaporation to ensure complete TFA removal. The resulting product was used directly in the subsequent reaction without further purification.

General Synthetic Procedures - Hydrogenation of Benzyl-Esters

A solution of the benzyl-protected product (1 eq) in methanol was treated with 0.1 equivalent of 10% w/w palladium on carbon. The reaction vessel was sealed and purged with hydrogen at 1 atm, then stirred overnight with progress monitored by TLC. Upon complete deprotection, the mixture was filtered through Celite and concentrated using a rotary evaporator. The resulting product was used directly in next steps without further purification.

General Synthetic Procedures - Hydrolysis of Methyl Esters

A solution of the methyl-protected peptide (0.2 M) in THF was treated with 1.5 equivalents of LiOH (0.5 M) dissolved in water. The reaction was stirred at room temperature for 1–3 hours, with monitoring for completion by LC/MS. The mixture was then acidified to pH 2 by adding 1M HCl, followed by three extractions with ethyl acetate. The combined organic layers were dried over anhydrous magnesium sulfate, filtered, and evaporated to yield the product for next steps.

General Synthetic Procedures - Methyl Protection of the Carboxylic Acid.

A round-bottom flask was charged with 1 equivalent of amino acid, sealed, and purged with argon. Anhydrous methanol (0.1 M) was then added, followed by the dropwise addition of 10 equivalent of thionyl chloride. The reaction mixture was left overnight, after which the solvent was evaporated under reduced pressure to yield a solid material; this material was used directly in subsequent reactions without further purification.

General Synthetic Procedures - Oxidation of Sulfide to Sulfone

To a solution of the cyclized peptide (0.4 M, 1 equiv) in CH₂Cl₂, mCPBA (2 equiv) was added, and the mixture was stirred overnight at room temperature. Reaction progress was monitored by LCMS to confirm complete oxidation. The reaction mixture was then diluted with ethyl acetate, washed sequentially with saturated sodium bicarbonate solution and brine, and dried over anhydrous sodium sulfate. The organic phase was filtered and concentrated under reduced pressure to afford a crude solid, which was subsequently purified by flash chromatography.

General Synthetic Procedures - Macrocyclization Through Amide Formation

A solution of the deprotected acyclic precursor (1 eq, 2 mM) in CH₂Cl₂ was prepared, followed by the addition of 1.5 equivalents of HBTU and 3 equivalents of DIPEA. The reaction mixture was stirred overnight. After evaporating the DCM, the crude product was diluted with ethyl acetate and transferred to a separatory funnel. The organic layer was washed twice sequentially with 1 M HCl, saturated sodium bicarbonate solution, and brine. It was then dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The resulting material was purified using automated flash chromatography.

N-((S)-3-Methyl-1-(((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-((perfluorophenyl)thio)propan-2-yl)amino)-1-oxobutan-2-yl)hexanamide (1).

¹H NMR (500 MHz, CDCl₃) δ 7.17 (d, J = 7.8 Hz, 1H), 6.99 (d, J = 8.0 Hz, 1H), 6.21 (d, J = 8.4 Hz, 1H), 4.59 – 4.47 (m, 2H), 4.33 (dd, J = 8.4, 6.8 Hz, 1H), 3.27–3.21 (m, 2H), 3.16 (dd, J = 13.9, 6.9 Hz, 1H), 2.91 (d, J = 4.9 Hz, 1H), 2.26 (dd, J = 8.7, 6.7 Hz, 2H), 2.08 (h, J = 6.8 Hz, 1H), 1.63 (dtt, J = 16.0, 6.4, 3.5 Hz, 2H), 1.58 – 1.54 (m, 1H), 1.52 (s, 3H), 1.40 – 1.24 (m, 6H), 0.96 – 0.87 (m, 14H). ¹³C NMR (126 MHz, CDCl₃) δ 208.03, 173.66, 171.51, 169.13, 153.18, 148.66, 142.83, 133.50, 59.08, 58.35, 52.32, 52.12, 50.63, 40.00, 36.61, 36.23, 31.45, 30.88, 25.41, 25.16, 23.34, 22.39, 21.20, 19.19, 18.16, 16.68, 13.95. ESI-HRMS [M + H]⁺ m/z 638.2683 (calculated for C₂₉H₄₀F₅N₃O₅S 638.2687).

(S)-3-Methyl-N-((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-((perfluorophenyl)thio)propan-2-yl)-2-(3-phenylpropanamido)butanamide (2)

¹H NMR (500 MHz, CDCl₃) δ 7.25 (q, J = 6.0 Hz, 6H), 7.18 (d, J = 7.4 Hz, 7H), 7.07 (d, J = 7.9 Hz, 1H), 6.93 (d, J = 8.0 Hz, 1H), 6.55 (s, 1H), 6.15 (d, J = 8.2 Hz, 1H), 4.54 – 4.49 (m, 1H), 4.46 (q, J = 6.9 Hz, 1H), 4.36 (t, J = 7.5 Hz, 1H), 4.24 (t, J = 7.5 Hz, 1H), 3.83 (t, J = 1.4 Hz, 2H), 3.16 (ddd, J = 24.0, 12.8, 5.7 Hz, 3H), 2.95 (tt, J = 7.8, 4.8 Hz, 4H), 2.55 (tt, J = 14.5, 7.0 Hz, 4H), 2.09 – 1.94 (m, 2H), 1.61 – 1.52 (m, 1H), 1.49 (d, J = 2.9 Hz, 3H), 0.84 (ddd, J = 37.3, 16.1, 6.3 Hz, 19H). ¹³C NMR (126 MHz, CD₃OD) δ 207.85, 173.82, 172.15, 169.99, 163.99, 148.71, 140.76, 140.63, 128.07, 128.02, 128.01, 125.80, 125.78, 109.64, 59.44, 58.61, 52.32, 51.62, 50.33, 38.72, 37.19, 37.07, 31.44, 30.60, 29.98, 24.77, 22.35, 19.95, 18.19, 17.20, 15.62. ESI-HRMS [M + H]⁺ m/z 671.2452 (calculated for C₃₂H₃₈F₅N₃O₅S 671.2452).

(S)-3-Methyl-N-((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-((perfluorophenyl)thio)propan-2-yl)-2-(2-(perfluorophenyl)acetamido)butanamide (3).

¹H NMR (500 MHz, CDCl₃) δ 4.47 (td, J = 10.5, 3.2 Hz, 1H), 4.38 (dd, J = 8.5, 5.4 Hz, 1H), 4.22 (d, J = 7.1 Hz, 1H), 3.80 (d, J = 2.7 Hz, 2H), 3.37–3.32 (m, 2H), 3.18 (dd, J = 8.4, 5.1 Hz, 1H), 3.09 – 2.98 (m, 1H), 2.91 (dd, J = 7.8, 5.0 Hz, 1H), 2.06 (dp, J = 13.7, 6.8 Hz, 1H), 1.63 (dq, J = 9.4, 4.2 Hz, 1H), 1.45 (s, 3H), 1.28 (td, J = 7.4, 3.7 Hz, 1H), 0.95 (t, J = 6.5 Hz, 6H), 0.90 (d, J = 6.6 Hz, 3H), 0.86 (d, J = 6.5 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 207.88, 171.84, 170.00, 168.70, 58.84, 58.61, 52.29, 51.53, 50.34, 38.72, 35.24, 30.87, 24.77, 22.30, 19.92, 18.30, 17.13, 15.48. ESI-HRMS [M + H]⁺ m/z 748.1903 (calculated for C₃₁H₃₂F₁₀N₃O₅S 748.1903).

2,5-Dichloro-N-((S)-3-methyl-1-(((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-((perfluorophenyl)thio)propan-2-yl)amino)-1-oxobutan-2-yl)benzamide (4)

1H NMR (500 MHz, CDCl₃) δ 7.64 (t, J = 1.4 Hz, 1H), 7.35 (d, J = 1.5 Hz, 2H), 6.99 (d, J = 7.7 Hz, 1H), 6.82 (d, J = 8.0 Hz, 1H), 6.77 (d, J = 7.9 Hz, 1H), 4.49 (tdd, J = 13.2, 10.8, 5.2 Hz, 3H), 3.27 (dd, J = 14.0, 5.6 Hz, 1H), 3.20 (d, J = 5.0 Hz, 1H), 3.13 (dd, J = 14.0, 6.9 Hz, 1H), 2.88 (d, J = 5.0 Hz, 1H), 2.24 (h, J = 6.8 Hz, 1H), 1.69 – 1.57 (m, 2H), 1.49 (s, 3H), 1.32 – 1.21 (m, 1H), 1.01 (dd, J = 10.2, 6.8 Hz, 6H), 0.89 (dd, J = 13.8, 6.3 Hz, 6H). ¹³C NMR (126 MHz, CD₃OD) δ 207.92, 171.69, 169.99, 167.05, 137.47, 132.50, 131.18, 131.03, 130.83, 130.66, 129.11, 128.74, 128.58, 59.43, 58.64, 52.21, 51.56, 50.40, 38.67, 35.44, 30.70, 24.79, 22.31, 19.93, 18.45, 17.51, 15.49. ESI-HRMS [M + H]⁺ m/z 711.1360 (calculated for C₃₀H₃₂Cl₂F₅N₃O₅S 711.1360).

(S)-N-((S)-4-Methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)-2-(2-(perfluorophenyl)acetamido)-3-((perfluorophenyl)thio)propenamide (5)

¹H NMR (500 MHz, CDCl₃) δ 6.63 (dd, J = 12.6, 7.7 Hz, 2H), 4.51 (ddd, J = 10.3, 7.9, 2.6 Hz, 1H), 4.44 – 4.37 (m, 1H), 3.62 (s, 2H), 3.23 (dd, J = 14.0, 5.0 Hz, 1H), 3.18 (d, J = 4.9 Hz, 1H), 3.06 (dd, J = 14.1, 7.5 Hz, 1H), 2.90 (d, J = 4.9 Hz, 1H), 1.72 – 1.58 (m, 2H), 1.50 (s, 3H), 1.31 – 1.19 (m, 2H), 0.93 (p, J = 3.2 Hz, 7H). ¹³C NMR (126 MHz, CDCl₃) δ 207.77, 168.99, 167.07, 148.77, 148.76, 146.32, 141.69, 139.74, 136.69, 107.99, 59.14, 52.37, 52.11, 51.17, 39.78, 36.52, 25.28, 23.26, 21.08, 16.70. ESI-HRMS [M + H]⁺ m/z 648.1143 (calculated for C₂₆H₂₂F₁₀N₂O₄S 648.1141).

2,5-Dichloro-N-((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-((perfluorophenyl)thio)propan-2-yl)benzamide (6)

¹H NMR (500 MHz, CDCl₃) δ 7.61 (dd, J = 2.2, 0.7 Hz, 1H), 7.39 – 7.31 (m, 2H), 6.71 (d, J = 8.0 Hz, 1H), 3.38 (dd, J = 14.1, 4.7 Hz, 1H), 3.22 (d, J = 5.0 Hz, 1H), 3.18 (dd, J = 14.1, 7.4 Hz, 1H), 2.90 (d, J = 5.0 Hz, 1H), 1.60 – 1.53 (m, 4H), 1.52 (s, 3H), 1.31 (ddd, J = 14.0, 10.5, 4.0 Hz, 1H), 0.94 (dd, J = 6.5, 2.1 Hz, 6H). ¹³C NMR (126 MHz, CD₃OD) δ 207.98, 169.96, 166.78, 136.74, 132.48, 131.25, 130.96, 129.37, 128.61, 58.68, 52.90, 51.60, 50.60, 38.67, 35.33, 24.86, 22.34, 19.95, 15.53. ESI-HRMS [M + H]⁺ m/z 612.0668 (calculated for C₂₅H₂₃Cl₂F₅N₂O₄S 612.0676).

N-((S)-4-Methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)-2-(2-(perfluorophenyl)acetamido)acetamide (7)

¹H NMR (500 MHz, CDCl₃) δ 7.05 – 6.89 (m, 2H), 4.60 (ddt, J = 10.8, 8.2, 2.6 Hz, 1H), 4.06 – 3.92 (m, 2H), 3.71 – 3.61 (m, 2H), 3.25 (dd, J = 5.0, 2.1 Hz, 1H), 2.91 (dd, J = 5.2, 2.3 Hz, 1H), 1.62 (tt, J = 10.4, 4.6 Hz, 1H), 1.56 – 1.46 (m, 5H), 1.18 (dddd, J = 14.6, 10.7, 4.1, 1.8 Hz, 1H), 0.93 (dd, J = 6.6, 2.1 Hz, 2H), 0.90 (dd, J = 6.5, 2.1 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 208.51, 168.60, 167.74, 146.31, 144.37, 144.34, 144.27, 139.55, 138.49, 136.50, 108.54, 108.50, 59.09, 52.38, 50.49, 43.16, 39.84, 29.10, 25.16, 23.23, 21.10, 16.67. ESI-HRMS [M + H]⁺ m/z 437.1500 (calculated for C₁₉H₂₂F₅N₂O₄ 437.1500).

2,5-Dichloro-N-(2-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-2-oxoethyl)benzamide (8)

¹H NMR (500 MHz, CDCl₃) δ 7.68 (t, J = 1.5 Hz, 1H), 7.36 (d, J = 1.5 Hz, 2H), 7.04 (q, J = 6.5 Hz, 1H), 6.50 (d, J = 8.2 Hz, 1H), 4.67 (ddd, J = 10.8, 8.1, 3.0 Hz, 1H), 4.26 – 4.10 (m, 2H), 3.30 (d, J = 4.9 Hz, 1H), 2.93 (d, J = 4.9 Hz, 1H), 1.76 – 1.64 (m, 1H), 1.61 – 1.56 (m, 1H), 1.53 (s, 3H), 1.34 – 1.28 (m, 1H), 0.98 (d, J = 6.5 Hz, 3H), 0.94 (d, J = 6.6 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 208.50, 168.15, 165.33, 135.31, 133.33, 131.71, 131.54, 130.23, 129.09, 59.12, 52.47, 50.54, 43.47, 40.19, 25.19, 23.39, 21.21, 16.75. ESI-HRMS [M + H]⁺ m/z 401.1035 (calculated for C₁₈H₂₃Cl₂N₂O₄ 401.1035).

(5S,8S)-3²,3³,3⁵,3⁶-Tetrafluoro-8-isopropyl-N-((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)-7,10-dioxo-2-thia-6,9-diaza-1,3(1,4)-dibenzenacycloundecaphane-5-carboxamide (9)

¹H NMR (500 MHz, CDCl₃) δ 7.68 (ddd, J = 8.1, 3.5, 2.0 Hz, 1H), 7.57 (dt, J = 8.0, 1.7 Hz, 1H), 7.24 (dd, J = 8.1, 2.1 Hz, 1H), 7.17 (dd, J = 8.0, 2.2 Hz, 1H), 6.30 (d, J = 8.3 Hz, 1H), 6.09 (d, J = 8.7 Hz, 1H), 5.82 (d, J = 6.8 Hz, 1H), 4.79 (ddd, J = 12.0, 8.6, 3.6 Hz, 1H), 4.64 (tdd, J = 10.6, 8.4, 2.8 Hz, 1H), 3.88 – 3.79 (m, 1H), 3.59 – 3.41 (m, 2H), 3.25 (d, J = 4.9 Hz, 1H), 3.07 – 2.99 (m, 1H), 2.95 (d, J = 4.9 Hz, 1H), 2.93 – 2.86 (m, 2H), 2.06 – 2.00 (m, 1H), 1.65 – 1.59 (m, 1H), 1.55 (s, 3H), 1.52 (s, 1H), 1.31 (d, J = 10.1 Hz, 1H), 0.94 (t, J = 6.1 Hz, 6H), 0.79 (dd, J = 6.9, 4.6 Hz, 6H). ¹³C NMR (126 MHz, CDCl₃) δ 208.31, 170.36, 170.08, 169.65, 138.03, 136.05, 135.89, 133.99, 129.78, 129.34, 117.31, 116.42, 116.28, 116.14, 59.13, 58.15, 52.46, 51.83, 50.99, 44.78, 39.92, 32.18, 27.24, 25.30, 23.28, 21.12, 18.24, 17.98, 16.75. ESI-HRMS [M + H]⁺ m/z 638.2318 (calculated for C₃₁H₃₆F₄N₃O₅S 638.2312).

(5S,8S)-3²,3³,3⁵,3⁶-Tetrafluoro-8-isopropyl-N-((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)-7,10-dioxo-2-thia-6,9-diaza-1(1,3),3(1,4)-dibenzenacycloundecaphane-5-carboxamide (10)

¹H NMR (500 MHz, CD₃OD) δ 7.35 (d, J = 7.9 Hz, 1H), 7.28 (t, J = 7.8 Hz, 1H), 7.08 (d, J = 7.5 Hz, 1H), 6.57 (s, 1H), 5.15 – 5.06 (m, 2H), 4.57 (dd, J = 11.1, 2.8 Hz, 1H), 4.28 (dd, J = 6.7, 1.4 Hz, 1H), 3.73 (d, J = 14.6 Hz, 1H), 3.34 (s, 1H), 3.18–3.12 (m, 3H), 2.98 (dd, J = 5.1, 1.3 Hz, 1H), 2.93 (dd, J = 5.2, 1.3 Hz, 1H), 1.89 (dt, J = 13.4, 6.7 Hz, 1H), 1.79 – 1.65 (m, 2H), 1.51 (s, 3H), 0.97 (d, J = 6.7 Hz, 3H), 0.94 (d, J = 6.5 Hz, 3H), 0.89 – 0.83 (m, 6H). ¹³C NMR (126 MHz, CD₃OD ) δ 212.01, 175.54, 175.40, 174.31, 150.59, 148.59, 141.82, 138.77, 132.59, 131.56, 129.57, 128.72, 121.80, 114.96, 62.71, 61.24, 55.62, 54.78, 54.34, 45.34, 42.23, 35.90, 29.89, 28.91, 26.19, 23.75, 22.17, 20.87, 19.56. ESI-HRMS [M + H]⁺ m/z 638.2311 (calculated for C₃₁H₃₆F₄N₃O₅S 638.2312).

(5S,8S)-3²,3³,3⁵,3⁶-Tetrafluoro-8-isopropyl-N-((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)-7,10-dioxo-2-thia-6,9-diaza-1(1,3),3(1,4)-dibenzenacycloundecaphane-5-carboxamide 2,2-dioxide (11)

¹H NMR (500 MHz, CDCl₃) δ 8.05 (dt, J = 7.4, 1.8 Hz, 1H), 7.66 (t, J = 1.8 Hz, 1H), 7.60 – 7.50 (m, 2H), 6.71 (d, J = 7.4 Hz, 1H), 6.67 (d, J = 8.8 Hz, 1H), 6.38 (d, J = 7.9 Hz, 1H), 4.98 (ddd, J = 12.6, 8.8, 4.1 Hz, 1H), 4.66 (ddd, J = 10.3, 8.1, 2.4 Hz, 1H), 4.18 – 4.09 (m, 2H), 3.61 (d, J = 14.7 Hz, 1H), 3.55 (d, J = 14.8 Hz, 1H), 3.28 (d, J = 4.9 Hz, 1H), 3.16 (ddd, J = 14.0, 4.2, 1.7 Hz, 1H), 3.07 (t, J = 13.0 Hz, 1H), 2.96 (d, J = 4.9 Hz, 1H), 1.97 – 1.88 (m, 1H), 1.66 – 1.57 (m, 1H), 1.55 (s, 3H), 0.94 (d, J = 5.9 Hz, 3H), 0.93 – 0.91 (m, 3H), 0.82 (d, J = 6.9 Hz, 3H), 0.79 (d, J = 6.7 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 208.29, 170.55, 169.77, 169.39, 147.12, 144.35, 142.17, 137.54, 135.35, 129.71, 127.06, 125.36, 121.59, 119.86, 59.21, 57.85, 52.45, 51.12, 50.58, 43.12, 39.80, 32.09, 29.73, 27.53, 25.30, 23.27, 18.32, 18.25, 16.78. ESI-HRMS [M + H]⁺ m/z 670.2215 (calculated for C₃₁H₃₆F₄N₃O₇S 670.2210).

(5S,8S)-8-(Tert-butyl)-3²,3³,3⁵,3⁶-tetrafluoro-N-((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)-7,10-dioxo-2-thia-6,9-diaza-1(1,3),3(1,4)-dibenzenacycloundecaphane-5-carboxamide (12)

¹H NMR (500 MHz, CDCl₃) δ 7.35 (ddd, J = 8.0, 2.0, 1.0 Hz, 1H), 7.23 (t, J = 7.8 Hz, 1H), 7.05 – 7.00 (m, 1H), 6.80 (d, J = 9.0 Hz, 2H), 6.56 (d, J = 1.9 Hz, 1H), 6.46 (d, J = 9.3 Hz, 1H), 5.07 (q, J = 8.6 Hz, 1H), 4.72 (td, J = 8.0, 4.3 Hz, 1H), 4.29 (d, J = 9.3 Hz, 1H), 3.45 (s, 2H), 3.29 (d, J = 4.9 Hz, 1H), 3.14 (d, J = 8.3 Hz, 2H), 2.91 (d, J = 4.8 Hz, 1H), 1.72 – 1.58 (m, 2H), 1.52 (s, 5H), 1.29 – 1.20 (m, 1H), 0.97 – 0.92 (m, 6H), 0.90 (s, 9H). ¹³C NMR (126 MHz, CDCl₃) δ 208.57, 170.38, 169.73, 169.70, 146.48, 144.37, 137.05, 135.04, 129.13, 128.24, 127.18, 126.40, 116.45, 112.49, 60.06, 59.11, 52.46, 50.71, 50.06, 43.19, 40.33, 35.47, 26.58, 25.24, 23.29, 21.24, 16.69. ESI-HRMS [M + H]⁺ m/z 652.2461 (calculated for C₃₂H₃₈F₄N₃O₅S 652.2468).

(61S,64S,65R,9S)-1²,1³,1⁵,1⁶-Tetrafluoro-6⁶,6⁶-dimethyl-N-((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)-5,7-dioxo-2-thia-6³,8-diaza-6(3,4)-bicyclo[3.1.0]hexana-1(1,4),3(1,3)-dibenzenacyclodecaphane-9-carboxamide (13)

¹H NMR (500 MHz, CDCl₃) δ 7.35 (ddt, J = 8.0, 1.7, 0.8 Hz, 1H), 7.19 (t, J = 7.7 Hz, 1H), 6.91 – 6.86 (m, 1H), 6.52 (dd, J = 8.0, 4.7 Hz, 2H), 6.45 (d, J = 1.9 Hz, 1H), 4.82 (q, J = 7.8 Hz, 1H), 4.69 – 4.60 (m, 1H), 4.18 (s, 1H), 3.79 (dd, J = 10.1, 5.4 Hz, 1H), 3.60 (d, J = 17.6 Hz, 1H), 3.44 (d, J = 10.1 Hz, 1H), 3.34 (ddd, J = 12.4, 5.0, 0.7 Hz, 2H), 3.18 (d, J = 7.9 Hz, 2H), 2.96 (d, J = 5.0 Hz, 1H), 1.73 – 1.61 (m, 2H), 1.56 (s, 3H), 1.35 (ddd, J = 13.8, 10.6, 3.7 Hz, 1H), 1.30 (d, J = 7.6 Hz, 1H), 1.03 (s, 3H), 0.99 (d, J = 6.4 Hz, 3H), 0.98 – 0.93 (m, 7H), 0.90 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 208.19, 171.74, 170.21, 168.81, 147.85, 145.90, 135.16, 134.60, 130.17, 128.37, 127.31, 126.47, 116.32, 113.58, 60.78, 59.20, 59.08, 52.51, 50.71, 50.11, 47.16, 40.36, 39.96, 39.92, 30.59, 28.02, 26.25, 25.11, 23.41, 21.33, 19.18. ESI-HRMS [M + H]⁺ m/z 676.2465 (calculated for C₃₄H₃₈F₄N₃O₅S 676.2468).

(5S,8S)-3²,3³,3⁵,3⁶-Tetrafluoro-8-((R)-1-hydroxyethyl)-N-((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)-7,10-dioxo-2-thia-6,9-diaza-1(1,3),3(1,4)-dibenzenacycloundecaphane-5-carboxamide (14)

¹H NMR (500 MHz, CDCl₃) δ 7.41 (ddd, J = 7.9, 2.0, 1.0 Hz, 1H), 7.25 (t, J = 7.7 Hz, 1H), 7.10 – 7.01 (m, 1H), 6.90 – 6.85 (m, 1H), 6.65 (d, J = 4.9 Hz, 2H), 5.09 (ddd, J = 12.3, 9.6, 4.1 Hz, 1H), 4.62 (ddd, J = 10.6, 7.7, 3.0 Hz, 1H), 4.49 (dd, J = 7.6, 4.0 Hz, 1H), 4.08 (dt, J = 10.6, 5.2 Hz, 1H), 3.45 (d, J = 9.1 Hz, 2H), 3.37 (d, J = 5.0 Hz, 1H), 2.95 (d, J = 5.0 Hz, 1H), 2.83 (s, 2H), 1.71–1.66 (m, 1H), 1.54 (s, 3H), 1.31–1.28 (m, 2H), 1.08 (d, J = 6.4 Hz, 3H), 0.95 (d, J = 6.5 Hz, 3H), 0.92 (d, J = 6.6 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 208.39, 171.17, 169.55, 169.16, 147.76, 144.76, 136.56, 135.10, 129.37, 128.43, 128.06, 126.68, 117.24, 112.42, 67.88, 59.26, 57.26, 52.47, 50.92, 50.87, 43.30, 39.79, 25.22, 23.34, 21.30, 17.64, 16.78. ESI-HRMS [M + H]⁺ m/z 640.2104 (calculated for C₃₀H₃₄F₄N₃O₆S 640.2104).

(5S,8S)-3²,3³,3⁵,36-Tetrafluoro-8-(2-hydroxypropan-2-yl)-N-((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)-7,10-dioxo-2-thia-6,9-diaza-1(1,3),3(1,4)-dibenzenacycloundecaphane-5-carboxamide (15)

¹H NMR (500 MHz, CDCl₃) δ 7.39 – 7.31 (m, 1H), 7.21 (t, J = 7.7 Hz, 1H), 7.11 (d, J = 9.6 Hz, 1H), 7.05 (d, J = 8.2 Hz, 1H), 6.99 (dt, J = 7.6, 1.3 Hz, 1H), 6.70 (d, J = 8.7 Hz, 1H), 6.51 (d, J = 2.2 Hz, 1H), 5.07 (ddd, J = 13.3, 9.8, 4.0 Hz, 1H), 4.73 – 4.62 (m, 1H), 4.47 (d, J = 8.8 Hz, 1H), 3.43 (s, 2H), 3.33 (q, J = 5.3 Hz, 2H), 2.98 (d, J = 13.3 Hz, 1H), 2.93 (d, J = 5.0 Hz, 1H), 1.67 – 1.61 (m, 1H), 1.58 (dd, J = 13.3, 3.3 Hz, 1H), 1.54 (s, 3H), 1.32 – 1.27 (m, 1H), 1.26 (s, 3H), 1.14 (s, 3H), 0.95 (d, J = 6.3 Hz, 3H), 0.92 (d, J = 6.4 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 208.49, 170.84, 170.30, 169.57, 147.45, 144.34, 136.70, 135.05, 129.21, 128.16, 127.35, 126.22, 117.09, 112.34, 72.99, 59.58, 59.55, 52.46, 51.33, 50.78, 42.96, 40.11, 27.39, 25.83, 25.23, 25.08, 23.34, 21.29. ESI-HRMS [M + H]⁺ m/z 654.2261 (calculated for C₃₁H₃₆F₄N₃O₆S 654.2261).

(5S,9S)-3²,3³,3⁵,3⁶-Tetrafluoro-9-isopropyl-N-((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)-7,11-dioxo-2-thia-6,10-diaza-1(1,3),3(1,4)-dibenzenacyclododecaphane-5-carboxamide (16):

(69.3% yield). ¹H NMR (500 MHz, CDCl₃) δ 7.46 (dt, J = 8.0, 1.5 Hz, 1H), 7.14 (dt, J = 7.7, 1.3 Hz, 1H), 6.86 (d, J = 1.9 Hz, 1H), 6.50 (d, J = 7.8 Hz, 1H), 6.48 – 6.41 (m, 2H), 4.85 (ddd, J = 11.1, 7.7, 2.9 Hz, 1H), 4.61 (ddd, J = 10.7, 7.9, 2.8 Hz, 1H), 3.69 (dt, J = 8.8, 4.4 Hz, 1H), 3.44 (d, J = 14.2 Hz, 1H), 3.37 (d, J = 14.2 Hz, 1H), 3.26 (d, J = 5.0 Hz, 1H), 3.15 (d, J = 13.9 Hz, 1H), 3.02 (t, J = 12.9 Hz, 1H), 2.94 (d, J = 5.0 Hz, 1H), 2.20 (qd, J = 15.0, 4.7 Hz, 2H), 1.72 – 1.63 (m, 1H), 1.63 – 1.58 (m, 1H), 1.34 (ddd, J = 13.9, 10.6, 3.7 Hz, 1H), 1.24 (s, 4H), 0.96 (t, J = 6.9 Hz, 6H), 0.79 (d, J = 6.6 Hz, 3H), 0.43 (d, J = 6.7 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 208.43, 171.71, 170.16, 169.87, 137.66, 135.41, 129.66, 129.06, 128.70, 128.54, 118.33, 59.44, 52.67, 52.09, 51.71, 51.49, 44.81, 39.81, 36.67, 32.81, 29.91, 25.46, 23.53, 21.28, 19.79, 18.74, 16.97. ESI-HRMS [M + H]⁺ m/z - (calculated for C₃₂H₃₇F₄N₃O₅S 651.2390).

(5S,9R)-3²,3³,3⁵,3⁶-Tetrafluoro-9-isopropyl-N-((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)-7,11-dioxo-2-thia-6,10-diaza-1(1,3),3(1,4)-dibenzenacyclododecaphane-5-carboxamide: (17): (75.5% yield).

¹H NMR (500 MHz, CDCl₃) δ 7.43 (dt, J = 7.7, 1.6 Hz, 1H), 7.25 – 7.18 (m, 2H), 7.17 (d, J = 9.6 Hz, 1H), 4.88 (ddd, J = 12.5, 9.3, 3.5 Hz, 1H), 4.65 – 4.58 (m, 2H), 3.66 (tt, J = 9.6, 3.7 Hz, 1H), 3.38 (d, J = 13.1 Hz, 1H), 3.32 (d, J = 5.0 Hz, 1H), 3.21 – 3.14 (m, 1H), 2.92 (d, J = 25.9 Hz, 1H), 2.87 (d, J = 5.0 Hz, 1H), 2.36 (dd, J = 16.6, 3.2 Hz, 1H), 1.73 – 1.54 (m, 4H), 1.52 (s, 3H), 1.31 (ddd, J = 13.8, 10.5, 3.7 Hz, 1H), 0.96 – 0.93 (m, 6H), 0.84 (d, J = 6.6 Hz, 3H), 0.79 (d, J = 6.7 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 209.57, 171.73, 170.40, 169.93, 137.70, 134.74, 129.63, 129.20, 128.70, 128.50, 117.53, 112.90, 59.25, 52.64, 52.01, 51.38, 50.35, 44.77, 40.50, 39.84, 38.82, 36.32, 31.20, 25.39, 23.57, 21.51, 20.04, 16.88. ESI-HRMS [M + H]⁺ m/z 651.2389 - (calculated for C₃₂H₃₇F₄N₃O₅S 651.2390).

(5S,8S)-32,33,35,36-Tetrafluoro-8-isopropyl-N-((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)-7,10-dioxo-2-thia-6,9-diaza-1,3(1,4)-dibenzenacyclododecaphane-5-carboxamide (18): (69.7% yield).

¹H NMR (800 MHz, DMSO-d₆) δ 8.57 (d, J = 7.1 Hz, 1H), 8.03 (d, J = 9.6 Hz, 1H), 7.40 (d, J = 7.8 Hz, 2H), 7.17 (d, J = 8.8 Hz, 1H), 7.13 (d, J = 7.8 Hz, 2H), 4.92 – 4.87 (m, 1H), 4.35 (q, J = 7.1 Hz, 1H), 4.03 (dd, J = 9.0, 4.0 Hz, 1H), 3.19 (d, J = 5.4 Hz, 1H), 3.04 (d, J = 5.2 Hz, 1H), 2.95 – 2.91 (m, 2H), 2.75 – 2.71 (m, 3H), 2.24 (dd, J = 10.8, 3.6 Hz, 1H), 1.74 (h, J = 6.8 Hz, 1H), 1.63 (dq, J = 13.8, 7.0 Hz, 1H), 1.42 (s, 3H), 0.88 (d, J = 6.7 Hz, 3H), 0.81 (d, J = 6.5 Hz, 3H), 0.64 (d, J = 6.9 Hz, 3H), 0.56 (d, J = 6.9 Hz, 3H). ¹³C NMR (201 MHz, DMSO-d₆) δ 208.53, 170.78, 170.59, 170.21, 142.80, 134.15, 131.07, 130.75, 118.34, 115.53, 59.50, 55.32, 52.19, 50.67, 49.07, 38.23, 35.70, 33.31, 30.61, 29.48, 25.04, 23.61, 21.07, 19.52, 17.24, 17.00. ESI-HRMS [M + H]⁺ 652.2472 m/z - (calculated for C₃₂H₃₈F₄N₃O₅S 652.2468).

(5S,8S)-3²,3³,3⁵,3⁶-Tetrafluoro-8-isopropyl-9-methyl-N-((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)-7,10-dioxo-2-thia-6,9-diaza-1(1,3),3(1,4)-dibenzenacycloundecaphane-5-carboxamide (19): (77.2% yield).

¹H NMR (500 MHz, CDCl₃) δ 7.36 (d, J = 7.9 Hz, 1H), 7.22 (t, J = 7.7 Hz, 1H), 6.95 (d, J = 7.5 Hz, 1H), 6.79 (d, J = 7.8 Hz, 1H), 6.43 (s, 1H), 4.75 – 4.65 (m, 1H), 4.62 (ddd, J = 10.6, 7.8, 3.1 Hz, 1H), 4.36 (d, J = 11.1 Hz, 1H), 3.73 (d, J = 16.2 Hz, 1H), 3.48 (d, J = 16.3 Hz, 1H), 3.33 – 3.31 (m, 1H), 3.26 – 3.08 (m, 2H), 3.00 – 2.95 (m, 3H), 2.94 (d, J = 4.9 Hz, 1H), 2.18 (dp, J = 11.0, 6.6 Hz, 1H), 1.64 (dtd, J = 13.1, 7.1, 6.5, 3.9 Hz, 1H), 1.54 (s, 3H), 1.32 – 1.21 (m, 2H), 0.97 – 0.89 (m, 9H), 0.84 (d, J = 6.6 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 171.91, 171.76, 170.05, 136.17, 135.39, 129.05, 128.08, 127.47, 126.85, 62.75, 59.35, 52.68, 51.99, 50.77, 40.41, 40.17, 31.18, 27.47, 25.24, 24.31, 23.59, 21.31, 19.41, 19.36, 16.96. ESI-HRMS [M + H]⁺ 652.2474 m/z - (calculated for C₃₂H₃₈F₄N₃O₅S 652.2468).

(S)-3²,3³,3⁵,3⁶-Tetrafluoro-8,8-dimethyl-N-((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)-7,10-dioxo-2-thia-6,9-diaza-1(1,3),3(1,4)-dibenzenacycloundecaphane-5-carboxamide (20). (62.8% yield).

¹H NMR (500 MHz, CDCl₃) δ 7.55 (d, J = 8.0 Hz, 1H), 7.29 (d, J = 7.8 Hz, 1H), 7.13 (d, J = 7.7 Hz, 1H), 6.99 (s, 1H), 6.46 (s, 1H), 6.40 (d, J = 8.4 Hz, 1H), 6.31 (d, J = 7.9 Hz, 1H), 4.87 (ddd, J = 12.1, 8.5, 3.9 Hz, 1H), 4.61 (ddd, J = 10.6, 7.9, 2.8 Hz, 1H), 3.50 – 3.37 (m, 2H), 3.34 – 3.28 (m, 1H), 3.24 – 3.17 (m, 1H), 3.02 (t, J = 12.7 Hz, 1H), 2.97 (d, J = 4.9 Hz, 1H), 1.69 (t, J = 3.2 Hz, 1H), 1.68 – 1.59 (m, 1H), 1.56 (s, 3H), 1.50 (s, 3H), 1.43 (s, 3H), 1.41 – 1.30 (m, 1H), 0.98 (dd, J = 7.2, 6.4 Hz, 6H).¹³C NMR (126 MHz, CDCl₃) δ 208.39, 174.64, 169.86, 169.77, 137.01, 134.95, 131.38, 130.27, 129.66, 116.72, 114.42, 76.98, 60.66, 59.46, 57.17, 52.69, 51.48, 45.15, 39.70, 25.86, 25.46, 23.54, 22.70, 21.25, 16.96, 14.40. ESI-HRMS [M + H]⁺ 624.2158 m/z - (calculated for C₃₀H₃₃F₄N₃O₅S 624.2155).

(S)-32,33,35,36-Tetrafluoro-N-((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)-7,10-dioxo-2-thia-6,9-diaza-1(1,3),3(1,4)-dibenzenacycloundecaphane-5-carboxamide (21): (72.4% yield).

¹H NMR (500 MHz, DMSO-d₆) δ 7.37 (d, J = 7.8 Hz, 1H), 7.28 (t, J = 7.7 Hz, 1H), 7.06 (d, J = 7.5 Hz, 1H), 6.51 (s, 1H), 4.96 (ddd, J = 12.7, 9.1, 3.9 Hz, 1H), 4.35 (dt, J = 10.9, 5.5 Hz, 1H), 4.19 (dd, J = 16.9, 8.6 Hz, 1H), 3.72 (d, J = 14.3 Hz, 1H), 3.29 (d, J = 14.3 Hz, 1H), 3.25 – 3.15 (m, 2H), 3.15 – 3.10 (m, 1H), 3.07 (d, J = 5.3 Hz, 1H), 2.86 (t, J = 12.9 Hz, 1H), 1.73 (s, 1H), 1.43 (s, 3H), 1.37 (dd, J = 9.7, 4.8 Hz, 2H), 0.89 (dd, J = 20.4, 6.5 Hz, 6H). ¹³C NMR (126 MHz, DMSO-d₆) δ 209.13, 170.52, 170.51, 170.07, 138.14, 136.21, 129.78, 128.88, 128.36, 125.83, 59.46, 59.44, 52.17, 50.30, 49.96, 41.84, 38.85, 25.17, 23.69, 22.61, 21.41, 16.99. ESI-HRMS [M + H]⁺ 596.1843 m/z - (calculated for C₂₈H₃₀F₄N₃O₅S 596.1842).

(5S,8S)-8-(Tert-butyl)-3²,3³,3⁵,3⁶-tetrafluoro-N-((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)-7,10-dioxo-2-thia-6,9-diaza-1(1,3),3(1,4)-dibenzenacycloundecaphane-5-carboxamide 2,2-dioxide (22)

¹H NMR (500 MHz, CDCl₃) δ 8.07 (dd, J = 7.8, 1.7 Hz, 1H), 7.65 – 7.52 (m, 3H), 6.31 (dd, J = 27.1, 8.4 Hz, 1H), 6.10 (d, J = 9.0 Hz, 1H), 5.78 (d, J = 8.6 Hz, 2H), 4.98 – 4.87 (m, 1H), 4.70 – 4.61 (m, 2H), 4.05 (d, J = 8.8 Hz, 1H), 3.41 – 3.33 (m, 2H), 3.26 (d, J = 4.9 Hz, 1H), 2.96 (d, J = 5.0 Hz, 1H), 2.91 (d, J = 5.1 Hz, 1H), 2.23 (d, J = 7.8 Hz, 1H), 2.05 – 2.01 (m, 1H), 1.71 (d, J = 4.2 Hz, 1H), 1.55 (s, 3H), 1.00 – 0.94 (m, 15H). ¹³C NMR (126 MHz, CDCl₃) δ 209.32, 170.36, 169.15, 169.13, 160.88, 152.19, 142.34, 137.32, 135.26, 129.83, 126.52, 125.47, 121.37, 120.45, 76.47, 60.46, 59.15, 52.48, 50.07, 43.43, 40.40, 35.82, 29.72, 26.62, 25.19, 23.42, 21.33, 16.72. ESI-HRMS [M + H]⁺ m/z 684.2367 (calculated for C₃₂H₃₈F₄N₃O₇S 684.2367).

(5S,8S)-3²,3³,3⁵,3⁶-Tetrafluoro-8-(2-hydroxypropan-2-yl)-N-((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)-7,10-dioxo-2-thia-6,9-diaza-1(1,3),3(1,4)-dibenzenacycloundecaphane-5-carboxamide 2-oxide (23)

¹H NMR (500 MHz, CDCl₃₃) δ 7.92 (ddd, J = 7.9, 1.9, 1.0 Hz, 1H), 7.59 (t, J = 7.8 Hz, 1H), 7.40 (dt, J = 7.7, 1.3 Hz, 1H), 7.03 (d, J = 8.5 Hz, 1H), 6.95 (d, J = 9.5 Hz, 1H), 6.83 (d, J = 1.8 Hz, 1H), 6.60 (d, J = 8.3 Hz, 1H), 5.84 (d, J = 8.5 Hz, 1H), 5.05 (ddd, J = 13.0, 9.4, 3.9 Hz, 1H), 4.71 – 4.61 (m, 2H), 4.25 (d, J = 8.3 Hz, 1H), 3.49 – 3.41 (m, 2H), 3.32 (ddd, J = 18.8, 5.0, 0.7 Hz, 2H), 2.94 (d, J = 4.8 Hz, 1H), 2.90 (d, J = 5.0 Hz, 1H), 1.71 – 1.62 (m, 1H), 1.62 – 1.55 (m, 1H), 1.54 (s, 3H), 1.32 – 1.28 (m, 1H), 0.98 – 0.91 (m, 12H). ¹³C NMR (126 MHz, CDCl₃) δ 208.49, 170.50, 170.22, 169.15, 146.81, 145.89, 143.99, 136.62, 132.52, 129.95, 124.09, 123.62, 122.87, 120.78, 73.26, 59.97, 59.07, 52.49, 50.78, 50.12, 43.41, 40.18, 27.68, 25.24, 24.97, 23.41, 23.13, 21.26, 16.72. ESI-HRMS [M + H]⁺ m/z 670.2211 (calculated for C₃₁H₃₆F₄N₃O₇S 670.2210 ).

(5S,8S)-3²,3³,3⁵,3⁶-Tetrafluoro-8-(2-hydroxypropan-2-yl)-N-((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)-7,10-dioxo-2-thia-6,9-diaza-1(1,3),3(1,4)-dibenzenacycloundecaphane-5-carboxamide 2,2-dioxide (24)

¹H NMR (500 MHz, CDCl₃) δ 8.10 – 8.03 (m, 1H), 7.61 – 7.47 (m, 3H), 7.03 – 6.96 (m, 1H), 6.92 (d, J = 9.9 Hz, 1H), 6.53 (d, J = 8.1 Hz, 1H), 4.96 (ddd, J = 12.9, 9.3, 3.9 Hz, 1H), 4.65 (ddd, J = 10.9, 8.3, 3.0 Hz, 1H), 4.32 (dd, J = 13.7, 8.2 Hz, 1H), 3.63 – 3.47 (m, 2H), 3.41 – 3.34 (m, 1H), 3.28 – 3.24 (m, 1H), 2.98 (t, J = 13.2 Hz, 1H), 2.93 (dd, J = 4.9, 2.9 Hz, 1H), 1.72 – 1.54 (m, 2H), 1.52 (s, 3H), 1.36 – 1.27 (m, 1H), 1.02 – 0.89 (m, 12H). ¹³C NMR (126 MHz, CDCl₃) δ 208.48, 170.47, 170.06, 168.92, 142.35, 137.13, 135.27, 129.82, 129.81, 126.33, 126.30, 125.53, 122.31, 119.66, 73.50, 73.41, 59.88, 59.10, 52.42, 50.75, 43.01, 40.17, 27.93, 25.22, 24.73, 23.38, 21.18, 16.70. ESI-HRMS [M + H]⁺ m/z 686.2155 (calculated for C₃₁H₃₆F₄N₃O₈S 686.2159 ).

(5S,9S)-3²,3³,3⁵,3⁶-Tetrafluoro-9-isopropyl-N-((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)-7,11-dioxo-2-thia-6,10-diaza-1(1,3),3(1,4)-dibenzenacyclododecaphane-5-carboxamide 2,2-dioxide (25): (65.1% yield).

¹H NMR (500 MHz, DMSO-d₆) δ 7.92 (dt, J = 6.1, 2.4 Hz, 1H), 7.82 (d, J = 8.4 Hz, 1H), 7.74 (d, J = 2.1 Hz, 1H), 7.62 (dd, J = 4.7, 2.2 Hz, 2H), 4.71 (dd, J = 12.2, 3.9 Hz, 1H), 4.28 (dd, J = 9.6, 4.3 Hz, 1H), 3.71 (tdd, J = 8.6, 4.5, 2.1 Hz, 1H), 3.48 (d, J = 12.2 Hz, 1H), 3.25 (d, J = 19.6 Hz, 1H), 3.20 (d, J = 5.7 Hz, 1H), 3.07 – 3.01 (m, 1H), 3.00 (d, J = 5.3 Hz, 1H), 2.81 (t, J = 12.9 Hz, 1H), 2.17 (dd, J = 17.6, 2.1 Hz, 1H), 1.87 (dd, J = 17.5, 8.5 Hz, 1H), 1.61 (dddd, J = 13.8, 11.5, 6.9, 4.7 Hz, 1H), 1.38 (s, 3H), 1.35 – 1.28 (m, 2H), 0.85 (d, J = 6.7 Hz, 3H), 0.81 (d, J = 6.6 Hz, 3H), 0.69 (dd, J = 9.0, 6.9 Hz, 6H). ¹³C NMR (126 MHz, DMSO-d₆) δ 208.30, 170.05, 169.91, 167.85, 140.70, 139.28, 135.92, 129.29, 126.92, 124.45, 59.15, 51.76, 50.46, 49.42, 48.99, 43.03, 37.58, 34.98, 32.20, 27.05, 24.60, 23.14, 20.96, 18.25, 17.87, 16.54^. ESI-HRMS [M + H]⁺ 684.2369 m/z - (calculated for C₃₂H₃₈F₄N₃O₇S 684.2367).

(5S,9R)-3²,3³,3⁵,3⁶-Tetrafluoro-9-isopropyl-N-((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)-7,11-dioxo-2-thia-6,10-diaza-1(1,3),3(1,4)-dibenzenacyclododecaphane-5-carboxamide 2,2-dioxide (26): (61.9% yield).

¹H NMR (500 MHz, CD₃OD) δ 8.06 (dt, J = 7.9, 1.6 Hz, 1H), 7.78 – 7.71 (m, 2H), 7.66 (t, J = 7.8 Hz, 1H), 4.91 – 4.84 (m, 2H), 4.71 (d, J = 6.0 Hz, 1H), 4.54 (dd, J = 11.1, 2.8 Hz, 1H), 3.51 (d, J = 13.3 Hz, 2H), 3.30 (d, J = 5.3 Hz, 2H), 3.16 – 3.12 (m, 1H), 3.08 – 2.99 (m, 1H), 2.97 (d, J = 5.1 Hz, 1H), 2.51 (dd, J = 16.6, 3.9 Hz, 1H), 1.72 (ddt, J = 14.3, 10.2, 3.4 Hz, 3H), 1.57 – 1.51 (m, 1H), 1.49 (s, 3H), 1.39 (ddd, J = 14.1, 11.0, 3.9 Hz, 1H), 0.94 (dd, J = 11.8, 6.6 Hz, 6H), 0.87 (d, J = 6.6 Hz, 3H), 0.81 (d, J = 6.8 Hz, 3H). ¹³C NMR (126 MHz, CD₃OD) δ 209.74, 172.96, 172.11, 171.45, 143.61, 139.77, 137.04, 130.77, 128.10, 126.75, 125.23, 60.42, 54.15, 53.33, 52.54, 52.14, 43.96, 39.85, 37.02, 31.38, 28.47, 26.59, 23.86, 21.44, 20.86, 20.27, 17.22. ESI-HRMS [M + H]⁺ 684.2368 m/z - (calculated for C₃₂H₃₈F₄N₃O₇S 684.2369).

(5S,8S)-3²,3³,3⁵,3⁶-Tetrafluoro-8-((S)-1-hydroxyethyl)-N-((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)-7,10-dioxo-2-thia-6,9-diaza-1(1,3),3(1,4)-dibenzenacycloundecaphane-5-carboxamide 2,2-dioxide (27): (69.4% yield).

¹H NMR (500 MHz, CDCl₃) δ 8.09 (d, J = 7.9 Hz, 1H), 7.61 (dd, J = 14.4, 6.7 Hz, 2H), 7.54 (d, J = 7.5 Hz, 1H), 7.14 (d, J = 9.2 Hz, 1H), 6.80 (d, J = 7.9 Hz, 1H), 6.40 (d, J = 6.6 Hz, 1H), 4.97 (ddd, J = 12.8, 9.1, 4.0 Hz, 1H), 4.67 – 4.56 (m, 2H), 4.28 (dd, J = 6.6, 4.1 Hz, 1H), 3.62 (s, 2H), 3.36 – 3.34 (m, 1H), 3.32 – 3.29 (m, 1H), 2.99 (t, J = 13.1 Hz, 1H), 2.94 (d, J = 4.9 Hz, 1H), 1.70 – 1.61 (m, 2H), 1.60 – 1.53 (m, 1H), 1.52 (s, 3H), 1.01 (d, J = 6.3 Hz, 3H), 0.97 (d, J = 6.5 Hz, 3H), 0.91 (d, J = 6.6 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 208.55, 171.12, 169.14, 168.98, 142.57, 137.31, 135.55, 130.21, 126.91, 126.06, 67.69, 59.41, 57.75, 52.68, 51.23, 50.29, 40.54, 39.91, 25.37, 23.55, 21.49, 21.32, 17.45, 16.91. ESI-HRMS [M + H]⁺ 672.2000 m/z - (calculated for for C₃₀H₃₄F₄N₃O₈S 672.2003).

(S)-1-((5S,8S)-3²,3³,3⁵,3⁶-Tetrafluoro-5-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)carbamoyl)-2,2-dioxido-7,10-dioxo-2-thia-6,9-diaza-1(1,3),3(1,4)-dibenzenacycloundecaphane-8-yl)ethyl acetate (28): (71.4% yield).

¹H NMR (500 MHz, CDCl₃) δ 8.10 – 8.05 (m, 1H), 7.70 (s, 1H), 7.63 – 7.58 (m, 2H), 6.79 (t, J = 9.3 Hz, 2H), 6.41 (d, J = 7.5 Hz, 1H), 5.14 (td, J = 6.5, 3.7 Hz, 1H), 4.92 (ddd, J = 12.6, 8.8, 4.1 Hz, 1H), 4.58 (ddd, J = 10.6, 7.7, 2.6 Hz, 1H), 4.35 (dd, J = 7.7, 3.7 Hz, 1H), 3.70 – 3.58 (m, 2H), 3.32 (d, J = 5.1 Hz, 1H), 3.21 (dd, J = 14.9, 3.7 Hz, 1H), 3.02 (t, J = 13.2 Hz, 1H), 2.95 (d, J = 5.0 Hz, 1H), 2.04 (d, J = 1.0 Hz, 3H), 1.62 – 1.56 (m, 2H), 1.55 (s, 3H), 1.40 (ddd, J = 14.0, 10.6, 3.8 Hz, 1H), 1.13 (d, J = 6.5 Hz, 3H), 0.97 (d, J = 6.2 Hz, 3H), 0.93 (d, J = 6.7 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 207.97, 170.28, 170.21, 169.09, 168.41, 152.53, 142.26, 137.34, 135.49, 129.87, 126.81, 125.76, 69.85, 59.33, 55.46, 52.53, 51.40, 50.63, 43.20, 39.21, 27.03, 25.43, 23.27, 21.10, 16.84, 16.31, 14.23. ESI-HRMS [M + H]⁺ 714.2108 m/z - (calculated for C₃₂H₃₆F₄N₃O₉S 714.2108).

(5S,8S)-3²,3³,3⁵,3⁶-Tetrafluoro-8-isopropyl-N-((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)-7,10-dioxo-2-thia-6,9-diaza-1,3(1,4)-dibenzenacyclododecaphane-5-carboxamide 2,2-dioxide (29): (74.1% yield).

¹H NMR (500 MHz, DMSO-d₆) δ 8.70 (d, J = 7.0 Hz, 1H), 7.80 (d, J = 8.1 Hz, 2H), 7.44 (s, 2H), 4.86 (dd, J = 11.6, 5.5 Hz, 1H), 4.33 – 4.27 (m, 1H), 4.01 (dd, J = 4.0, 1.0 Hz, 1H), 3.17 (d, J = 5.2 Hz, 1H), 3.03 (d, J = 5.2 Hz, 1H), 2.96 – 2.91 (m, 2H), 2.87 – 2.82 (m, 2H), 2.81 – 2.74 (m, 1H), 2.31 – 2.25 (m, 1H), 1.69 (dh, J = 10.8, 3.9, 3.3 Hz, 1H), 1.63 – 1.56 (m, 1H), 1.40 (s, 3H), 1.32 (dd, J = 8.2, 5.4 Hz, 2H), 0.85 (d, J = 6.6 Hz, 3H), 0.79 (d, J = 6.5 Hz, 3H), 0.60 (d, J = 6.8 Hz, 3H), 0.50 (d, J = 6.9 Hz, 3H).¹³C NMR (126 MHz, DMSO-d₆) δ 208.22, 170.58, 169.83, 169.50, 149.01, 137.89, 125.90, 125.83, 119.62, 59.15, 54.64, 51.79, 50.33, 49.04, 38.86, 37.68, 35.17, 33.00, 30.74, 26.25, 24.64, 23.24, 20.59, 19.17, 16.58. ESI-HRMS [M + H]⁺ 684.2369 m/z - (calculated for C₃₂H₃₈F₄N₃O₇S 684.2367).

(5S,8S)-3²,3³,3⁵,3⁶-Tetrafluoro-8-isopropyl-9-methyl-N-((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)-7,10-dioxo-2-thia-6,9-diaza-1(1,3),3(1,4)-dibenzenacycloundecaphane-5-carboxamide 2,2-dioxide (30): (75.7% yield).

¹H NMR (500 MHz, CD₃OD) δ 7.98 (dt, J = 7.3, 1.8 Hz, 1H), 7.72 – 7.59 (m, 2H), 7.57 (d, J = 1.9 Hz, 1H), 4.58 – 4.50 (m, 2H), 4.03 (d, J = 14.5 Hz, 1H), 3.59 (d, J = 14.4 Hz, 1H), 3.29 (d, J = 4.9 Hz, 1H), 3.24 – 3.11 (m, 2H), 2.98 (s, 3H), 2.96 (d, J = 5.1 Hz, 1H), 2.07 (dq, J = 10.8, 6.6 Hz, 1H), 1.70 (dtt, J = 13.4, 6.6, 3.8 Hz, 1H), 1.49 (s, 3H), 1.41 – 1.30 (m, 2H), 1.00 – 0.95 (m, 3H), 0.94 (d, J = 6.7 Hz, 3H), 0.90 (dd, J = 6.6, 5.1 Hz, 6H), 0.87 (d, J = 6.6 Hz, 3H). ¹³C NMR (126 MHz, CD₃OD) δ 209.64, 173.88, 173.46, 172.08, 144.00, 138.79, 136.57, 130.79, 125.40, 124.42, 63.09, 60.38, 53.26, 52.47, 51.31, 41.22, 39.83, 32.48, 29.50, 26.87, 26.46, 23.90, 21.35, 20.22, 19.80, 17.23. ESI-HRMS [M + H]⁺ 628.1740 m/z - (calculated for C₂₈H₃₀F₄N₃O₇S 628.1741).

(S)-3²,3³,3⁵,3⁶-Tetrafluoro-8,8-dimethyl-N-((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)-7,10-dioxo-2-thia-6,9-diaza-1(1,3),3(1,4)-dibenzenacycloundecaphane-5-carboxamide (31). (62.8% yield).

¹H NMR (500 MHz, CDCl₃) δ 8.07 – 8.00 (m, 1H), 7.63 (s, 1H), 7.53 (t, J = 7.8 Hz, 1H), 7.45 (d, J = 7.7 Hz, 1H), 6.32 (d, J = 8.0 Hz, 1H), 6.09 (s, 1H), 6.05 (d, J = 7.7 Hz, 1H), 4.57 (ddd, J = 11.9, 7.9, 3.6 Hz, 1H), 4.49 (ddd, J = 10.1, 7.9, 2.3 Hz, 1H), 3.59 (d, J = 16.5 Hz, 1H), 3.49 (d, J = 16.5 Hz, 1H), 3.17 (d, J = 5.0 Hz, 1H), 3.11 (d, J = 15.4 Hz, 1H), 2.91 (d, J = 12.9 Hz, 1H), 2.88 (d, J = 4.9 Hz, 1H), 1.56 – 1.47 (m, 2H), 1.46 (s, 3H), 1.44 (s, 3H), 1.32 (s, 3H), 1.28 – 1.21 (m, 1H), 0.88 (dd, J = 6.1, 2.7 Hz, 6H).¹³C NMR (126 MHz, DMSO-d₆) δ 208.64, 170.04, 170.03, 169.58, 137.65, 135.73, 129.30, 128.39, 127.87, 125.35, 58.97, 58.96, 51.68, 49.81, 49.47, 41.35, 38.36, 24.68, 23.20, 22.12, 20.92, 16.51. ESI-HRMS [M + H]⁺ 656.2052 m/z - (calculated for C₃₀H₃₄F₄N₃O₇S 656.2054).

(S)-32,33,35,36-Tetrafluoro-N-((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)-7,10-dioxo-2-thia-6,9-diaza-1(1,3),3(1,4)-dibenzenacycloundecaphane-5-carboxamide 2,2-dioxide (32): (61.8% yield).

¹H NMR (500 MHz, DMSO-d₆) δ 8.73 (d, J = 6.6 Hz, 1H), 8.46 (d, J = 9.0 Hz, 1H), 8.17 (d, J = 7.3 Hz, 2H), 7.93 (dt, J = 7.3, 1.9 Hz, 1H), 7.63 – 7.61 (m, 1H), 4.88 (ddd, J = 12.7, 9.0, 3.8 Hz, 1H), 4.35 – 4.33 (m, 1H), 3.89 (dd, J = 16.7, 7.5 Hz, 1H), 3.73 (d, J = 13.0 Hz, 1H), 3.33 (d, J = 12.9 Hz, 1H), 3.30 – 3.23 (m, 1H), 3.22 (d, J = 5.4 Hz, 1H), 3.16 – 3.04 (m, 1H), 2.99 (d, J = 5.3 Hz, 1H), 2.84 (t, J = 12.9 Hz, 1H), 1.64 (qd, J = 6.6, 3.4 Hz, 1H), 1.42 (s, 3H), 1.29 (td, J = 10.4, 9.8, 4.6 Hz, 2H), 0.91 (s, 3H), 0.84 (s, 3H). ¹³C NMR (126 MHz, DMSO-d₆) δ 208.89, 169.51, 169.43, 168.80, 139.24, 135.51, 129.45, 125.97, 123.59, 59.16, 58.94, 51.65, 50.49, 49.43, 41.40, 38.37, 24.57, 23.18, 22.11, 20.96, 16.49. ESI-HRMS [M + H]⁺ 628.1744 m/z - (calculated C₂₈H₃₀F₄N₃O₇S 628.1741).

(10S,13S)-10-Isopropyl-N-((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)-8,11-dioxo-2-oxa-9,12-diaza-1(1,4)-benzenacyclotetradecaphane-13-carboxamide (33): (78.7% yield).

¹H NMR (500 MHz, CDCl₃) δ 6.98 (s, 2H), 6.76 (d, J = 8.2 Hz, 2H), 4.85 (ddd, J = 11.1, 9.3, 4.4 Hz, 1H), 4.66 (ddd, J = 10.7, 8.4, 2.9 Hz, 1H), 4.21 – 4.13 (m, 3H), 3.34 (d, J = 5.0 Hz, 1H), 3.08 (dd, J = 14.1, 4.3 Hz, 1H), 2.92 (d, J = 4.9 Hz, 1H), 2.81 – 2.74 (m, 1H), 2.29 – 2.23 (m, 1H), 1.89 (dq, J = 13.5, 6.8 Hz, 1H), 1.67 (dt, J = 9.8, 5.5 Hz, 2H), 1.61 – 1.56 (m, 1H), 1.53 (s, 3H), 1.48 (d, J = 1.7 Hz, 1H), 1.36 – 1.32 (m, 1H), 1.24 (t, J = 7.2 Hz, 5H), 0.93 (d, J = 6.0 Hz, 3H), 0.90 (d, J = 6.3 Hz, 3H), 0.83 (d, J = 6.7 Hz, 6H).¹³C NMR (126 MHz, CDCl₃) δ 208.55, 172.30, 171.64, 171.15, 157.84, 130.56, 128.50, 116.83, 68.00, 60.61, 59.35, 58.23, 53.69, 52.64, 50.67, 40.09, 37.67, 35.62, 31.79, 29.59, 25.41, 24.59, 23.56, 21.52, 19.11, 18.64, 17.01, 14.39. ESI-HRMS [M + H]⁺ 530.3231 m/z - (calculated for C₂₉H₄₄N3O₆ 530.3230).

(S)-10,10-Dimethyl-N-((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)-8,11-dioxo-2-oxa-9,12-diaza-1(1,4)-benzenacyclotetradecaphane-13-carboxamide (34): (83.1% yield).

¹H NMR (500 MHz, CDCl₃) δ 7.44 (d, J = 8.0 Hz, 1H), 7.04 – 6.97 (m, 2H), 6.94 (dd, J = 8.5, 2.6 Hz, 1H), 6.88 – 6.80 (m, 1H), 6.07 (s, 1H), 6.00 (d, J = 8.5 Hz, 1H), 4.63 (ddd, J = 11.1, 7.9, 2.8 Hz, 1H), 4.56 (ddd, J = 8.8, 6.0, 2.9 Hz, 1H), 4.37 (dt, J = 12.1, 4.7 Hz, 1H), 4.14 (ddd, J = 12.4, 9.3, 3.6 Hz, 1H), 3.54 (dd, J = 13.7, 3.0 Hz, 1H), 3.41 (d, J = 5.1 Hz, 1H), 2.88 (d, J = 5.1 Hz, 1H), 2.71 (dd, J = 13.8, 6.1 Hz, 1H), 2.16 (ddd, J = 15.1, 8.5, 6.2 Hz, 1H), 1.92 (dddd, J = 18.0, 15.0, 8.7, 5.5 Hz, 2H), 1.55 (s, 3H), 1.54 – 1.49 (m, 2H), 1.48 (s, 3H), 1.43 (s, 3H), 1.42 – 1.33 (m, 1H), 1.27 – 1.12 (m, 3H), 1.09 – 1.04 (m, 1H), 1.02 (d, J = 6.4 Hz, 3H), 0.90 (d, J = 6.4 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 208.73, 173.44, 171.72, 168.77, 157.99, 128.60, 116.89, 68.31, 59.39, 53.85, 52.59, 50.62, 42.77, 39.86, 35.64, 29.22, 25.33, 25.12, 24.66, 23.58, 21.41, 16.97. ESI-HRMS [M + H]⁺ 488.2761 m/z - (calculated for C₂₆H₃₈N₃O₆ 488.2761).

S)-N-((S)-4-Methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)-8,11-dioxo-2-oxa-9,12-diaza-1(1,4)-benzenacyclotetradecaphane-13-carboxamide (35): (84.0% yield).

¹H NMR (500 MHz, CDCl₃) δ 7.20 – 7.08 (m, 1H), 7.02 (s, 1H), 7.00 – 6.91 (m, 1H), 6.82 (d, J = 8.1 Hz, 2H), 6.59 (s, 1H), 4.94 (td, J = 9.6, 4.7 Hz, 1H), 4.65 (ddd, J = 10.8, 8.0, 3.2 Hz, 1H), 4.20 (dddd, J = 20.0, 16.1, 11.2, 4.9 Hz, 3H), 3.47 (dd, J = 16.7, 3.3 Hz, 1H), 3.35 (d, J = 5.0 Hz, 1H), 3.08 (dd, J = 13.7, 4.7 Hz, 1H), 2.94 (d, J = 5.0 Hz, 1H), 2.80 (dd, J = 13.7, 9.7 Hz, 1H), 2.32 – 2.11 (m, 2H), 1.75 – 1.61 (m, 3H), 1.61 – 1.57 (m, 1H), 1.54 (s, 3H), 1.53 – 1.48 (m, 1H), 1.46 – 1.33 (m, 2H), 1.28 (ddd, J = 14.8, 7.3, 3.9 Hz, 2H), 0.96 (dd, J = 13.0, 6.4 Hz, 6H). ¹³C NMR (126 MHz, CDCl₃) δ 208.23, 174.07, 173.16, 171.55, 159.55, 130.89, 130.09, 128.45, 119.18, 117.88, 70.02, 59.41, 57.85, 53.37, 52.50, 50.53, 38.77, 36.40, 35.70, 28.97, 28.18, 25.75, 24.71, 23.85, 23.42, 23.15, 20.89, 16.99. ESI-HRMS [M + H]⁺ 516.3074 m/z - (calculated for C28H₄₂N₃O₆ 516.3074).

(S)-10,10-Dimethyl-N-((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)-8,11-dioxo-2-thia-9,12-diaza-1(1,4)-benzenacyclotetradecaphane-13-carboxamide (36): (78.1% yield).

¹H NMR (500 MHz, DMSO-d₆) δ 7.98 (d, J = 1.9 Hz, 1H), 7.65 (d, J = 7.5 Hz, 1H), 7.41 (d, J = 7.8 Hz, 2H), 7.08 (d, J = 7.7 Hz, 2H), 6.29 (d, J = 7.7 Hz, 1H), 4.41 (ddd, J = 10.7, 7.5, 3.3 Hz, 1H), 4.35 (dt, J = 7.7, 5.4 Hz, 1H), 3.24 (d, J = 5.3 Hz, 1H), 3.12 (dd, J = 13.8, 5.8 Hz, 1H), 3.02 (q, J = 5.3, 4.7 Hz, 2H), 2.86 (dd, J = 13.8, 5.1 Hz, 1H), 2.75 (ddd, J = 14.5, 8.6, 5.9 Hz, 1H), 1.91 (ddd, J = 15.6, 9.8, 5.8 Hz, 1H), 1.77 (ddd, J = 15.6, 9.8, 5.6 Hz, 1H), 1.67 (ddd, J = 14.1, 8.0, 5.8 Hz, 1H), 1.54 – 1.45 (m, 2H), 1.40 (s, 3H), 1.34 (dt, J = 11.0, 3.8 Hz, 1H), 1.30 (d, J = 4.0 Hz, 6H), 1.28 – 1.21 (m, 1H), 1.16 (tq, J = 12.0, 6.5 Hz, 2H), 0.97 – 0.92 (m, 1H), 0.90 (d, J = 6.5 Hz, 3H), 0.85 (d, J = 6.6 Hz, 3H), 0.79 (qd, J = 7.7, 6.1, 2.9 Hz, 1H). ¹³C NMR (126 MHz, DMSO-d₆) δ 207.80, 173.32, 172.90, 171.12, 135.27, 135.25, 133.22, 129.95, 58.97, 56.07, 52.55, 51.62, 49.67, 38.15, 35.52, 35.20, 34.99, 30.01, 26.04, 25.96, 24.22, 24.10, 23.44, 22.39, 20.79, 16.55^.. ESI-HRMS [M + H]⁺ 532.2841 m/z - (calculated for C₂₈H₄₂N₃O₅S 532.2845).

(S)-10,10-Dimethyl-N-((R)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)-8,11-dioxo-2-thia-9,12-diaza-1(1,4)-benzenacyclotetradecaphane-13-carboxamide 2,2-dioxide (37): (47.8% yield).

¹H NMR (500 MHz, DMSO-d₆) δ 7.87 (d, J = 7.5 Hz, 2H), 7.75 (d, J = 8.0 Hz, 2H), 6.61 (d, J = 7.6 Hz, 1H), 4.49 (td, J = 7.2, 4.8 Hz, 1H), 4.38 (ddd, J = 10.8, 7.4, 3.3 Hz, 1H), 3.36 – 3.29 (m, 1H), 3.28 (d, J = 5.1 Hz, 1H), 3.26 – 3.11 (m, 2H), 3.06 (d, J = 5.1 Hz, 1H), 3.03 (dd, J = 14.1, 4.8 Hz, 1H), 1.90 (p, J = 7.2 Hz, 1H), 1.63 (dt, J = 13.6, 6.9 Hz, 2H), 1.54 – 1.46 (m, 1H), 1.43 (s, 3H), 1.38 (t, J = 3.8 Hz, 1H), 1.36 – 1.30 (m, 1H), 1.28 (s, 3H), 1.25 (s, 3H), 1.16 (t, J = 7.1 Hz, 1H), 0.98 (d, J = 7.1 Hz, 2H), 0.88 (dd, J = 6.7, 2.5 Hz, 2H), 0.85 (d, J = 6.6 Hz, 6H). ¹³C NMR (126 MHz, DMSO-d₆) δ 208.01, 173.86, 172.27, 170.92, 142.51, 137.01, 128.02, 58.96, 55.88, 52.58, 51.72, 49.46, 38.41, 36.03, 34.24, 26.25, 25.19, 24.65, 24.51, 23.14, 22.68, 22.10, 20.97, 16.46, 13.77. ESI-HRMS [M + H]⁺ 564.2748 m/z - (calculated for C₂₈H₄₂N₃O₇S 564.2743).

(13S)-10-Isopropyl-N-((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)-8,11-dioxo-2-thia-9,12-diaza-1(1,4)-benzenacyclotetradecaphane-13-carboxamide 2,2-dioxide (38): (76.3% yield).

¹H NMR (500 MHz, DMSO-d₆) δ 8.14 (d, J = 7.3 Hz, 1H), 8.05 (d, J = 9.5 Hz, 1H), 7.26 (d, J = 9.1 Hz, 1H), 7.19 (d, J = 8.3 Hz, 2H), 7.15 (d, J = 8.1 Hz, 2H), 4.67 – 4.59 (m, 1H), 4.39 (ddd, J = 10.7, 7.3, 3.3 Hz, 1H), 4.02 (dd, J = 9.1, 7.4 Hz, 1H), 3.21 (d, J = 5.2 Hz, 1H), 3.02 (s, 1H), 3.02 – 2.97 (m, 1H), 2.87 – 2.83 (m, 2H), 2.11 – 2.03 (m, 1H), 1.95 (dt, J = 15.1, 5.0 Hz, 1H), 1.71 (dt, J = 13.7, 6.8 Hz, 1H), 1.67 (s, 1H), 1.53 (dt, J = 10.5, 6.0 Hz, 1H), 1.41 (s, 3H), 1.40 – 1.36 (m, 1H), 1.34 – 1.27 (m, 1H), 1.18 – 1.08 (m, 2H), 1.05 (d, J = 7.7 Hz, 1H), 0.91 (d, J = 6.6 Hz, 3H), 0.86 (d, J = 6.5 Hz, 3H), 0.76 (d, J = 6.6 Hz, 3H), 0.72 (d, J = 6.8 Hz, 3H). ESI-HRMS [M + H]+ 578.2900 m/z - (calculated for C₂₉H₄₄N₃O₇S 578.2900).

3R,6S)-6-Isopropyl-N-((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)-5,8-dioxo-1-thia-4,7-diazacycloheptadecane-3-carboxamide (39): (84.2% yield).

¹H NMR (500 MHz, CD₃OD) δ 4.59 (dt, J = 7.8, 3.9 Hz, 1H), 4.57 – 4.50 (m, 1H), 4.24 (ddd, J = 15.0, 7.5, 5.4 Hz, 1H), 3.26 (d, J = 5.0 Hz, 1H), 2.94 (s, 1H), 2.89 (dd, J = 14.4, 5.3 Hz, 1H), 2.77 – 2.68 (m, 1H), 2.64 (dd, J = 14.4, 9.0 Hz, 1H), 2.57 (dd, J = 12.5, 6.4 Hz, 1H), 2.35 – 2.30 (m, 1H), 2.28 – 2.15 (m, 1H), 2.05 – 1.93 (m, 1H), 1.73 (dddd, J = 16.6, 13.2, 8.3, 4.8 Hz, 2H), 1.62 (tdd, J = 12.2, 7.1, 5.1 Hz, 1H), 1.56 – 1.48 (m, 1H), 1.49 – 1.46 (m, 4H), 1.35 (dtd, J = 23.6, 14.2, 12.5, 4.8 Hz, 12H), 0.96 – 0.91 (m, 12H). ¹³C NMR (126 MHz, CD₃OD) δ 208.05, 174.88, 172.21, 171.49 (d, J = 11.1 Hz), 77.91, 58.70, 58.56, 51.92, 50.26, 38.74, 31.03, 30.91, 28.03, 27.42, 27.29, 26.44, 26.30, 24.92, 22.39, 20.03, 19.98, 18.40, 18.35, 17.63, 17.58, 15.70, 15.61. ESI-HRMS [M + H]⁺ 526.3315 m/z - (calculated for C₂₇H₄₈N₃O₅S 526.3315).

(3R,6S)-6-Isopropyl-N-((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)-5,8-dioxo-1-thia-4,7-diazacycloheptadecane-3-carboxamide 1,1-dioxide (40): (79.1% yield).

¹H NMR (500 MHz, CD₃OD) δ 5.07 – 4.99 (m, 1H), 4.52 (dd, J = 10.9, 2.9 Hz, 1H), 4.22 (d, J = 7.1 Hz, 1H), 3.43 (dd, J = 6.1, 1.7 Hz, 2H), 3.26 (d, J = 5.1 Hz, 1H), 3.21 (ddd, J = 13.8, 9.0, 6.3 Hz, 1H), 3.07 (ddd, J = 13.8, 9.2, 6.5 Hz, 1H), 2.94 (d, J = 5.0 Hz, 1H), 2.37 (ddd, J = 14.3, 10.9, 3.5 Hz, 1H), 2.23 (ddd, J = 14.1, 6.5, 3.7 Hz, 1H), 2.01 (h, J = 6.9 Hz, 1H), 1.89 – 1.82 (m, 2H), 1.78 – 1.73 (m, 1H), 1.54 – 1.50 (m, 2H), 1.48 (s, 3H), 1.41 – 1.26 (m, 12H), 0.97 – 0.92 (m, 12H). ¹³C NMR (126 MHz, CD₃OD) δ 209.43, 176.42, 173.72, 171.05, 60.27, 60.02, 56.19, 53.21, 52.67, 52.18, 49.43, 40.27, 36.13, 32.65, 28.13, 28.11, 27.95, 27.63, 27.25, 26.41, 25.85, 23.92, 21.92, 21.53, 19.88, 18.98, 17.14. ESI-HRMS [M + H]⁺ 558.3217 m/z - (calculated for C₂₇H₄₈N₃O₇S 558.3213).

(9R,12S)-12-Isopropyl-N-((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)-11,14-dioxo-1,4-dioxa-7-thia-10,13-diazacyclohexadecane-9-carboxamide (41)

¹H NMR (500 MHz, CDCl₃) δ 7.46 (dd, J = 18.9, 7.3 Hz, 0H), 7.15 (d, J = 8.9 Hz, 1H), 7.01 (d, J = 8.7 Hz, 2H), 6.96 (d, J = 8.8 Hz, 1H), 4.62 (td, J = 7.4, 4.9 Hz, 1H), 4.54 (ddd, J = 11.1, 7.9, 3.3 Hz, 1H), 4.32 – 4.25 (m, 2H), 3.92 (tt, J = 9.8, 4.8 Hz, 1H), 3.76 – 3.66 (m, 4H), 3.67 – 3.60 (m, 3H), 3.35 – 3.22 (m, 2H), 2.91 – 2.81 (m, 3H), 2.76 (tdd, J = 16.6, 7.0, 3.4 Hz, 2H), 2.65 – 2.58 (m, 1H), 2.54 – 2.45 (m, 1H), 2.31 (p, J = 7.1 Hz, 1H), 1.65 (ddt, J = 13.8, 10.9, 5.2 Hz, 1H), 1.50 – 1.36 (m, 5H), 1.07 – 0.87 (m, 12H). ¹³C NMR (126 MHz, CDCl₃) δ 207.83, 172.80, 171.52, 170.39, 71.10, 70.09, 69.90, 67.08, 59.69, 59.21, 52.76, 52.44, 50.59, 39.42, 36.79, 33.77, 31.79, 29.49, 25.20, 23.40, 21.20, 19.63, 18.05, 16.83. ESI-HRMS [M + H]⁺ m/z 516.2745 (calculated for C₂₄H₄₂N₃O₇S 516.2743).

NMR Data for Selected Intermediates

Methyl-(5S,8S)-3²,3³,3⁵,3⁶-tetrafluoro-8-isopropyl-7,10-dioxo-2-thia-6,9-diaza-1,3(1,4)-dibenzenacyclododecaphane-5-carboxylate: (81.6% yield).

¹H NMR (500 MHz, CDCl₃) δ 7.46 (d, J = 7.8 Hz, 2H), 7.00 (d, J = 8.1 Hz, 2H), 5.89 (d, J = 7.1 Hz, 1H), 5.64 (d, J = 9.7 Hz, 1H), 5.01 (ddd, J = 12.7, 9.7, 4.4 Hz, 1H), 3.86 – 3.82 (m, 1H), 3.74 (s, 3H), 3.25 (ddd, J = 14.1, 4.8, 2.1 Hz, 1H), 2.93 – 2.83 (m, 2H), 2.76 – 2.71 (m, 1H), 2.53 (d, J = 14.1 Hz, 1H), 2.15 (ddd, J = 14.2, 13.1, 4.2 Hz, 1H), 1.93 (ddq, J = 10.1, 6.8, 3.4 Hz, 1H), 0.75 (d, J = 6.9 Hz, 3H), 0.71 (d, J = 6.9 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 170.88, 170.34, 170.08, 141.67, 131.64, 129.94, 115.75, 115.61, 56.51, 53.04, 32.55, 30.68, 25.67, 18.06, 17.42.

Methyl-(S)-2-(2-((tert-butoxycarbonyl)amino)acetamido)-3-(perfluorophenyl)propanoate: (62.8% yield).

¹H NMR (500 MHz, CDCl₃) δ 4.83 (td, J = 7.5, 5.8 Hz, 1H), 3.75 (dd, J = 15.7, 5.1 Hz, 1H), 3.72 (s, 3H), 3.67 (dd, J = 16.8, 5.8 Hz, 1H), 3.34 – 3.25 (m, 1H), 3.11 – 3.03 (m, 1H), 1.37 (s, 9H).

Methyl-(S)-2-((2S,3S)-3-acetoxy-2-aminobutanamido)-3-(perfluorophenyl)propanoate: (92.7% yield).

¹H NMR (500 MHz, CDCl₃) δ 5.33 (dd, J = 6.5, 3.5 Hz, 1H), 4.81 (d, J = 6.5 Hz, 1H), 4.20 (dd, J = 8.7, 3.4 Hz, 1H), 3.77 (s, 3H), 3.40 – 3.23 (m, 1H), 3.11 (dd, J = 14.2, 6.7 Hz, 1H), 2.03 (s, 3H), 1.22 (d, J = 6.4 Hz, 3H).

2-(3-((4-((S)-2-((2S,3S)-3-Acetoxy-2-((tert-butoxycarbonyl)amino)butanamido)-3-methoxy-3-oxopropyl)-2,3,5,6-tetrafluorophenyl)thio)phenyl)acetic acid: (54.9% yield).

¹H NMR (500 MHz, CD₃OD) δ 7.26 (t, J = 7.7 Hz, 1H), 7.20 (d, J = 8.6 Hz, 1H), 7.17 – 7.13 (m, 1H), 5.14 (dd, J = 6.4, 4.4 Hz, 1H), 4.71 – 4.69 (m, 1H), 4.17 (d, J = 4.4 Hz, 1H), 3.71 (s, 3H), 3.55 (s, 2H), 3.35 (dd, J = 14.0, 5.5 Hz, 1H), 3.20 (dd, J = 14.0, 9.3 Hz, 1H), 1.96 (s, 3H), 1.38 (s, 9H), 1.18 (d, J = 6.4 Hz, 3H).

Methyl-(5S,8S)-8-((S)-1-acetoxyethyl)-3²,3³,3⁵,3⁶-tetrafluoro-7,10-dioxo-2-thia-6,9-diaza-1(1,3),3(1,4)-dibenzenacycloundecaphane-5-carboxylate: (70.0% yield).

¹H NMR (500 MHz, CDCl₃) δ 7.45 (ddd, J = 7.9, 1.9, 1.1 Hz, 1H), 7.28 (d, J = 7.4 Hz, 2H), 7.12 – 7.06 (m, 1H), 5.18 – 5.07 (m, 2H), 4.47 (dd, J = 7.5, 4.0 Hz, 1H), 3.90 (s, 3H), 3.50 (s, 2H), 3.44 – 3.37 (m, 1H), 3.01 – 2.93 (m, 1H), 2.09 (s, 3H), 1.12 (d, J = 6.4 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 170.82, 170.37, 169.74, 168.42, 136.80, 135.28, 129.59, 128.92, 128.58, 127.60, 116.56 (d, J = 17.4 Hz), 113.19, 69.84, 55.42, 53.50, 50.47, 43.56, 21.23, 15.56.

Benzyl-(10S,13S)-10-isopropyl-8,11-dioxo-2-oxa-9,12-diaza-1(1,4)-benzenacyclotetradecaphane-13-carboxylate: (51.1% yield).

¹H NMR (500 MHz, CDCl₃) δ 7.36 – 7.24 (m, 5H), 6.96 (d, J = 13.7 Hz, 2H), 6.76 – 6.66 (m, 2H), 5.18 (d, J = 12.1 Hz, 1H), 5.10 (d, J = 12.1 Hz, 1H), 4.96 (ddd, J = 12.2, 9.6, 4.7 Hz, 1H), 4.17 (ddd, J = 12.1, 6.3, 4.0 Hz, 1H), 4.08 (ddd, J = 12.0, 7.8, 3.9 Hz, 1H), 3.91 (dd, J = 8.6, 6.6 Hz, 1H), 3.32 (dd, J = 14.0, 4.7 Hz, 1H), 2.47 (dd, J = 14.0, 12.2 Hz, 1H), 2.18 (ddd, J = 14.7, 6.0, 4.3 Hz, 1H), 1.87 – 1.77 (m, 2H), 1.69 – 1.61 (m, 2H), 1.47 – 1.37 (m, 1H), 1.30 – 1.19 (m, 1H), 1.15 – 1.03 (m, 2H), 0.76 (dd, J = 6.8, 5.0 Hz, 6H).¹³C NMR (126 MHz, Benzene-d₆) δ 171.98, 171.60, 170.41, 157.61, 153.11, 135.22, 128.92, 128.71, 127.93, 116.59, 67.93, 67.70, 57.84, 52.61, 38.14, 35.78, 32.16, 29.39, 25.29, 24.80, 18.93, 18.46.

Methyl-(S)-2-((R)-2-((tert-butoxycarbonyl)(methyl)amino)-3-methylbutanamido)-3-(perfluorophenyl)propanoate: (95.7% yield).

¹H NMR (500 MHz, CDCl₃) δ 4.80 (td, J = 8.6, 4.8 Hz, 1H), 4.02 (d, J = 11.1 Hz, 1H), 3.76 (s, 3H), 3.34 – 3.21 (m, 1H), 3.03 (dd, J = 14.1, 9.1 Hz, 1H), 2.61 (s, 3H), 2.19 – 2.11 (m, 1H), 1.44 (s, 9H), 0.82 (dd, J = 24.7, 6.6 Hz, 6H).

2-(3-((4-((S)-2-((S)-2-((tert-butoxycarbonyl)(methyl)amino)-3-methylbutanamido)-3-methoxy-3-oxopropyl)-2,3,5,6-tetrafluorophenyl)thio)phenyl)acetic acid: (48.7% yield).

¹H NMR (500 MHz, CD₃OD) δ 7.26 (d, J = 2.7 Hz, 1H), 7.24 (d, J = 7.6 Hz, 1H), 7.19 (d, J = 7.5 Hz, 2H), 4.08 (d, J = 11.2 Hz, 1H), 3.75 (s, 3H), 3.55 (s, 2H), 3.36 (dd, J = 13.9, 4.3 Hz, 1H), 3.19 – 3.08 (m, 1H), 2.41 (s, 2H), 2.05 (dq, J = 12.0, 6.5 Hz, 1H), 1.43 (s, 9H), 0.83 (d, J = 6.5 Hz, 3H), 0.75 (d, J = 6.7 Hz, 3H).

Methyl-(5S,8S)-3²,3³,3⁵,3⁶-tetrafluoro-8-isopropyl-9-methyl-7,10-dioxo-2-thia-6,9-diaza-1(1,3),3(1,4)-dibenzenacycloundecaphane-5-carboxylate: (68.3% yield).

¹H NMR (500 MHz, CDCl₃) δ 7.25 (ddd, J = 7.9, 2.0, 1.0 Hz, 1H), 7.13 (t, J = 7.7 Hz, 1H), 6.87 (ddd, J = 6.7, 1.8, 1.0 Hz, 1H), 6.35 (s, 1H), 4.82 (ddd, J = 12.7, 8.3, 4.4 Hz, 1H), 4.33 (d, J = 11.2 Hz, 1H), 3.73 (s, 3H), 3.64 (d, J = 16.0 Hz, 1H), 3.43 (d, J = 16.1 Hz, 1H), 3.36 – 3.28 (m, 1H), 3.03 (t, J = 13.3 Hz, 1H), 2.86 (s, 3H), 2.05 – 1.96 (m, 1H), 0.90 (d, J = 6.5 Hz, 3H), 0.75 (d, J = 6.6 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 171.63, 171.53, 171.01, 136.32, 129.08, 127.51, 127.25, 126.70, 116.93, 116.79, 113.17, 53.65, 53.04, 50.80, 40.36, 31.15, 27.53, 24.95, 19.55, 19.33.

Methyl-(S)-2-(2-((tert-butoxycarbonyl)amino)-2-methylpropanamido)-3-(perfluorophenyl)propanoate: (92.5% yield).

¹H NMR (500 MHz, CDCl₃) δ 4.79 (td, J = 7.3, 5.8 Hz, 1H), 3.71 (s, 3H), 3.28 – 3.22 (m, 1H), 3.06 (ddt, J = 14.0, 7.0, 1.5 Hz, 1H), 1.38 (s, 3H), 1.34 (s, 12H).

Supplementary Material

NIHMS2206153-supplement-Supplementary_Material.docx^{(41MB, docx)}

Compound molecular formula strings and properties

NIHMS2206153-supplement-Compound_molecular_formula_strings_and_properties.csv^{(8.2KB, csv)}

ASSOCIATED CONTENT

Supporting Information

The Supporting Information is available free of charge at https://pubs.acs.org/

Proteasome inhibition assay protocol and IC₅₀ determination using fluorogenic substrates; IC₅₀ figures for selected compounds; pharmacokinetic evaluations for compounds 10, 11, 35, and 40; LCMS chromatograms of final products; and ¹H and ¹³C NMR spectra of synthetic products (PDF)

Compound molecular formula strings and properties (CSV)

Figure 3. — A. Conceptual macrocyclic ring formation through coupling between P2 and P4 moieties. B. Analogues 9, 10, **11,** the first set of fluorinated macrocycles.

ACKNOWLEDGMENTS

J.A. acknowledges the St. Baldrick’s Foundation for the International Scholar award 2022–2025 (Award # 940892) and the deanship of scientific research at the University of Jordan for his scientific sabbatical leave. P.F. received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No [846688]. C.E.H.S. would like to thank the Brazilian agency, CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico), for the grant received under process nº 200018/2023–0. The research was supported by NIH awards AI158612, AI171824 and AI146387 to A.J.O. and GM107550 to W.H.G.

ABBREVIATIONS

BBB: blood–brain barrier
BLQ: below limit of quantification
Boc: tert-butoxycarbonyl
Caco-2: human colon adenocarcinoma cell line
c20S: constitutive 20S proteasome
CNS: central nervous system
DCM: dichloromethane
DIPEA: N,N-diisopropylethylamine
DMF: dimethylformamide
dppf: 1,1′-bis(diphenylphosphino)ferrocene
HBTU: hexafluorophosphate benzotriazole tetramethyl uronium
HFF-1: human foreskin fibroblast cell line
i20S: immunoproteasome 20S
LLOQ: lower limit of quantification
m-CPBA: meta-chloroperoxybenzoic acid
MGMT: O6-methylguanine-DNA-methyltransferase
NA: not applicable
ND: not determined
PAMPA-BBB: parallel artificial membrane permeability assay for BBB
Pd₂(dba)₃: tris(dibenzylideneacetone)dipalladium(0)
P-gp: P-glycoprotein
PK: pharmacokinetics
SNAr: nucleophilic aromatic substitution
TFA: trifluoroacetic acid

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material

NIHMS2206153-supplement-Supplementary_Material.docx^{(41MB, docx)}

Compound molecular formula strings and properties

NIHMS2206153-supplement-Compound_molecular_formula_strings_and_properties.csv^{(8.2KB, csv)}

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PERMALINK

Development of Macrocyclic Peptide-Based Proteasome Inhibitors with Enhanced Blood-Brain Barrier Penetration for Treating Brain Neoplasms

Jehad Almaliti

Momen Al-Hindy

Pavla Fajtová

Evgenia Glukhov

Theodore Berger

Yan Graf

Cesar Emiliano Hoffmann da Silva

Chandler Huang

Narek Azizyan

Yuxian He

Anthony J O’Donoghue

William H Gerwick

Abstract

INTRODUCTION

RESULTS

Cytotoxicity of designed analogues

Figure 1.

Figure 2.

Table 1.

Table 2.

Table 3.

Figure 4.

Table 4.

Chemistry

Scheme 1.

Scheme 2.

Scheme 3.

Scheme 4.

Screening with the PAMPA-BBB permeability assay

Table 5.

Evaluation of in vitro plasma and microsomal stability

Table 6.

Caco-2 Cell permeability evaluation

Table 7.

Proteasome Inhibition Evaluation

Table 8.

In vivo Pharmacokinetics of Selected Compounds in Mice

Figure 5.

Table 10.

Table 9.

DISCUSSION

Figure 6.

CONCLUSIONS

EXPERIMENTAL SECTION

Inhibitor Subunit Specificity

Half-Maximal Inhibitory Concentration (IC50) Assays

Ethical Statement

Chemistry.

General.

General Synthetic Procedures - General Procedure for Coupling Amino Acids

General Synthetic Procedures - Nucleophilic Aromatic Substitution (SNAr)

General Synthetic Procedures - Deprotection of Boc and Tert-butyl Protecting Groups

General Synthetic Procedures - Hydrogenation of Benzyl-Esters

General Synthetic Procedures - Hydrolysis of Methyl Esters

General Synthetic Procedures - Methyl Protection of the Carboxylic Acid.

General Synthetic Procedures - Oxidation of Sulfide to Sulfone

General Synthetic Procedures - Macrocyclization Through Amide Formation

N-((S)-3-Methyl-1-(((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-((perfluorophenyl)thio)propan-2-yl)amino)-1-oxobutan-2-yl)hexanamide (1).

(S)-3-Methyl-N-((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-((perfluorophenyl)thio)propan-2-yl)-2-(3-phenylpropanamido)butanamide (2)

(S)-3-Methyl-N-((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-((perfluorophenyl)thio)propan-2-yl)-2-(2-(perfluorophenyl)acetamido)butanamide (3).

2,5-Dichloro-N-((S)-3-methyl-1-(((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-((perfluorophenyl)thio)propan-2-yl)amino)-1-oxobutan-2-yl)benzamide (4)

(S)-N-((S)-4-Methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)-2-(2-(perfluorophenyl)acetamido)-3-((perfluorophenyl)thio)propenamide (5)

2,5-Dichloro-N-((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-((perfluorophenyl)thio)propan-2-yl)benzamide (6)

N-((S)-4-Methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)-2-(2-(perfluorophenyl)acetamido)acetamide (7)

2,5-Dichloro-N-(2-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-2-oxoethyl)benzamide (8)

(5S,8S)-32,33,35,36-Tetrafluoro-8-isopropyl-N-((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)-7,10-dioxo-2-thia-6,9-diaza-1,3(1,4)-dibenzenacycloundecaphane-5-carboxamide (9)

(5S,8S)-32,33,35,36-Tetrafluoro-8-isopropyl-N-((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)-7,10-dioxo-2-thia-6,9-diaza-1(1,3),3(1,4)-dibenzenacycloundecaphane-5-carboxamide (10)

(5S,8S)-32,33,35,36-Tetrafluoro-8-isopropyl-N-((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)-7,10-dioxo-2-thia-6,9-diaza-1(1,3),3(1,4)-dibenzenacycloundecaphane-5-carboxamide 2,2-dioxide (11)

(5S,8S)-8-(Tert-butyl)-32,33,35,36-tetrafluoro-N-((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)-7,10-dioxo-2-thia-6,9-diaza-1(1,3),3(1,4)-dibenzenacycloundecaphane-5-carboxamide (12)

(61S,64S,65R,9S)-12,13,15,16-Tetrafluoro-66,66-dimethyl-N-((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)-5,7-dioxo-2-thia-63,8-diaza-6(3,4)-bicyclo[3.1.0]hexana-1(1,4),3(1,3)-dibenzenacyclodecaphane-9-carboxamide (13)

(5S,8S)-32,33,35,36-Tetrafluoro-8-((R)-1-hydroxyethyl)-N-((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)-7,10-dioxo-2-thia-6,9-diaza-1(1,3),3(1,4)-dibenzenacycloundecaphane-5-carboxamide (14)

(5S,8S)-32,33,35,36-Tetrafluoro-8-(2-hydroxypropan-2-yl)-N-((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)-7,10-dioxo-2-thia-6,9-diaza-1(1,3),3(1,4)-dibenzenacycloundecaphane-5-carboxamide (15)

(5S,9S)-32,33,35,36-Tetrafluoro-9-isopropyl-N-((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)-7,11-dioxo-2-thia-6,10-diaza-1(1,3),3(1,4)-dibenzenacyclododecaphane-5-carboxamide (16):

(5S,9R)-32,33,35,36-Tetrafluoro-9-isopropyl-N-((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)-7,11-dioxo-2-thia-6,10-diaza-1(1,3),3(1,4)-dibenzenacyclododecaphane-5-carboxamide: (17): (75.5% yield).

(5S,8S)-32,33,35,36-Tetrafluoro-8-isopropyl-N-((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)-7,10-dioxo-2-thia-6,9-diaza-1,3(1,4)-dibenzenacyclododecaphane-5-carboxamide (18): (69.7% yield).

(5S,8S)-32,33,35,36-Tetrafluoro-8-isopropyl-9-methyl-N-((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)-7,10-dioxo-2-thia-6,9-diaza-1(1,3),3(1,4)-dibenzenacycloundecaphane-5-carboxamide (19): (77.2% yield).

(S)-32,33,35,36-Tetrafluoro-8,8-dimethyl-N-((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)-7,10-dioxo-2-thia-6,9-diaza-1(1,3),3(1,4)-dibenzenacycloundecaphane-5-carboxamide (20). (62.8% yield).

(S)-32,33,35,36-Tetrafluoro-N-((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)-7,10-dioxo-2-thia-6,9-diaza-1(1,3),3(1,4)-dibenzenacycloundecaphane-5-carboxamide (21): (72.4% yield).

Half-Maximal Inhibitory Concentration (IC₅₀) Assays

(5S,8S)-3²,3³,3⁵,3⁶-Tetrafluoro-8-isopropyl-N-((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)-7,10-dioxo-2-thia-6,9-diaza-1,3(1,4)-dibenzenacycloundecaphane-5-carboxamide (9)

(5S,8S)-3²,3³,3⁵,3⁶-Tetrafluoro-8-isopropyl-N-((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)-7,10-dioxo-2-thia-6,9-diaza-1(1,3),3(1,4)-dibenzenacycloundecaphane-5-carboxamide (10)

(5S,8S)-3²,3³,3⁵,3⁶-Tetrafluoro-8-isopropyl-N-((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)-7,10-dioxo-2-thia-6,9-diaza-1(1,3),3(1,4)-dibenzenacycloundecaphane-5-carboxamide 2,2-dioxide (11)

(5S,8S)-8-(Tert-butyl)-3²,3³,3⁵,3⁶-tetrafluoro-N-((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)-7,10-dioxo-2-thia-6,9-diaza-1(1,3),3(1,4)-dibenzenacycloundecaphane-5-carboxamide (12)

(61S,64S,65R,9S)-1²,1³,1⁵,1⁶-Tetrafluoro-6⁶,6⁶-dimethyl-N-((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)-5,7-dioxo-2-thia-6³,8-diaza-6(3,4)-bicyclo[3.1.0]hexana-1(1,4),3(1,3)-dibenzenacyclodecaphane-9-carboxamide (13)

(5S,8S)-3²,3³,3⁵,3⁶-Tetrafluoro-8-((R)-1-hydroxyethyl)-N-((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)-7,10-dioxo-2-thia-6,9-diaza-1(1,3),3(1,4)-dibenzenacycloundecaphane-5-carboxamide (14)

(5S,8S)-3²,3³,3⁵,36-Tetrafluoro-8-(2-hydroxypropan-2-yl)-N-((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)-7,10-dioxo-2-thia-6,9-diaza-1(1,3),3(1,4)-dibenzenacycloundecaphane-5-carboxamide (15)

(5S,9S)-3²,3³,3⁵,3⁶-Tetrafluoro-9-isopropyl-N-((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)-7,11-dioxo-2-thia-6,10-diaza-1(1,3),3(1,4)-dibenzenacyclododecaphane-5-carboxamide (16):

(5S,9R)-3²,3³,3⁵,3⁶-Tetrafluoro-9-isopropyl-N-((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)-7,11-dioxo-2-thia-6,10-diaza-1(1,3),3(1,4)-dibenzenacyclododecaphane-5-carboxamide: (17): (75.5% yield).

(5S,8S)-3²,3³,3⁵,3⁶-Tetrafluoro-8-isopropyl-9-methyl-N-((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)-7,10-dioxo-2-thia-6,9-diaza-1(1,3),3(1,4)-dibenzenacycloundecaphane-5-carboxamide (19): (77.2% yield).

(S)-3²,3³,3⁵,3⁶-Tetrafluoro-8,8-dimethyl-N-((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)-7,10-dioxo-2-thia-6,9-diaza-1(1,3),3(1,4)-dibenzenacycloundecaphane-5-carboxamide (20). (62.8% yield).

(5S,8S)-8-(Tert-butyl)-3²,3³,3⁵,3⁶-tetrafluoro-N-((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)-7,10-dioxo-2-thia-6,9-diaza-1(1,3),3(1,4)-dibenzenacycloundecaphane-5-carboxamide 2,2-dioxide (22)

(5S,8S)-3²,3³,3⁵,3⁶-Tetrafluoro-8-(2-hydroxypropan-2-yl)-N-((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)-7,10-dioxo-2-thia-6,9-diaza-1(1,3),3(1,4)-dibenzenacycloundecaphane-5-carboxamide 2-oxide (23)

(5S,8S)-3²,3³,3⁵,3⁶-Tetrafluoro-8-(2-hydroxypropan-2-yl)-N-((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)-7,10-dioxo-2-thia-6,9-diaza-1(1,3),3(1,4)-dibenzenacycloundecaphane-5-carboxamide 2,2-dioxide (24)

(5S,9S)-3²,3³,3⁵,3⁶-Tetrafluoro-9-isopropyl-N-((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)-7,11-dioxo-2-thia-6,10-diaza-1(1,3),3(1,4)-dibenzenacyclododecaphane-5-carboxamide 2,2-dioxide (25): (65.1% yield).

(5S,9R)-3²,3³,3⁵,3⁶-Tetrafluoro-9-isopropyl-N-((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)-7,11-dioxo-2-thia-6,10-diaza-1(1,3),3(1,4)-dibenzenacyclododecaphane-5-carboxamide 2,2-dioxide (26): (61.9% yield).

(5S,8S)-3²,3³,3⁵,3⁶-Tetrafluoro-8-((S)-1-hydroxyethyl)-N-((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)-7,10-dioxo-2-thia-6,9-diaza-1(1,3),3(1,4)-dibenzenacycloundecaphane-5-carboxamide 2,2-dioxide (27): (69.4% yield).

(S)-1-((5S,8S)-3²,3³,3⁵,3⁶-Tetrafluoro-5-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)carbamoyl)-2,2-dioxido-7,10-dioxo-2-thia-6,9-diaza-1(1,3),3(1,4)-dibenzenacycloundecaphane-8-yl)ethyl acetate (28): (71.4% yield).

(5S,8S)-3²,3³,3⁵,3⁶-Tetrafluoro-8-isopropyl-N-((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)-7,10-dioxo-2-thia-6,9-diaza-1,3(1,4)-dibenzenacyclododecaphane-5-carboxamide 2,2-dioxide (29): (74.1% yield).

(5S,8S)-3²,3³,3⁵,3⁶-Tetrafluoro-8-isopropyl-9-methyl-N-((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)-7,10-dioxo-2-thia-6,9-diaza-1(1,3),3(1,4)-dibenzenacycloundecaphane-5-carboxamide 2,2-dioxide (30): (75.7% yield).

(S)-3²,3³,3⁵,3⁶-Tetrafluoro-8,8-dimethyl-N-((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)-7,10-dioxo-2-thia-6,9-diaza-1(1,3),3(1,4)-dibenzenacycloundecaphane-5-carboxamide (31). (62.8% yield).

Methyl-(5S,8S)-3²,3³,3⁵,3⁶-tetrafluoro-8-isopropyl-7,10-dioxo-2-thia-6,9-diaza-1,3(1,4)-dibenzenacyclododecaphane-5-carboxylate: (81.6% yield).

Methyl-(5S,8S)-8-((S)-1-acetoxyethyl)-3²,3³,3⁵,3⁶-tetrafluoro-7,10-dioxo-2-thia-6,9-diaza-1(1,3),3(1,4)-dibenzenacycloundecaphane-5-carboxylate: (70.0% yield).

Methyl-(5S,8S)-3²,3³,3⁵,3⁶-tetrafluoro-8-isopropyl-9-methyl-7,10-dioxo-2-thia-6,9-diaza-1(1,3),3(1,4)-dibenzenacycloundecaphane-5-carboxylate: (68.3% yield).