Next Generation Diprovocims with Potent Human and Murine TLR1/TLR2 Agonist Activity That Activate the Innate and Adaptive Immune Response

Abstract

The diprovocims, a new class of Toll-like receptor (TLR) agonists, bear no similarity to prior TLR agonists, act by a well-defined mechanism (TLR1/TLR2 agonist), exhibit exquisite structure–activity relationships, and display in vivo adjuvant activity. They possess potent and efficacious agonist activity towards human TLR1/TLR2 but modest agonism towards the murine receptor. A manner by which diprovocims can be functionalized without impacting hTLR1/TLR2 activity is detailed, permitting future linkage to antigenic, targeting, or delivery moieties. Improvements in both potency and its low efficacy in the murine system were also achieved, permitting more effective use in animal models while maintaining the hTLR1/TLR2 activity. The prototypical member diprovocim-X exhibits the excellent potency/efficacy of diprovocim-1 in human cells, displays substantially improved potency/efficacy in mouse macrophages, and serves as an adjuvant in mice when co-administrated with a non-immunogenic antigen, indicating stimulation of the adaptive as well as innate immune response.

Graphical Abstract

Introduction

Controlled manipulation of both the inflammatory innate immune response and ensuing adaptive immune response is necessary for precision therapeutics targeting infectious diseases and cancer while avoiding toxic side effects. Immunostimulatory small molecules with defined specificities that activate both innate and adaptive immune responses are therefore highly desirable but are still quite rare, and even more so those with comprehensively characterized molecular and cellular mechanisms. Most are mimics or modifications of antigenic microbial or viral components that act as ligands for innate immune receptors (e.g.; LPS, lipopeptides, nucleic acids); as such their actions are separated, either or both spatially and temporally, from adaptive immune activation by co-administered antigens. Furthermore, they represent structurally unattractive starting points for drug discovery, being difficult to structurally modify and chemically prepare. However, the endogenous protein targets on which they act and the intricate atomic-level mechanisms they activate provide powerful opportunities for the development of new therapeutics.^1,2

The Toll-like receptors (TLRs) comprise one family of innate immune receptors, collectively mediate the recognition of most microbes, and elicit intracellular signaling leading to the release of inflammatory cytokines and chemokines that provide critical activating signals to adaptive immune cells. TLR agonists are attractive adjuvants for use in prophylactic vaccination against either bacterial or viral pathogens,^3–8 and as immunostimulators in the field of cancer immunotherapy where the adaptive immune system is exploited to not only identify and eradicate cancer cells based on their expression of neo-antigens but also to form a long lasting systemic anti-tumor memory response (antigen-specific anti-tumor immunity).^9–11

Studies^12–15 conducted over 30 years ago discovered the immune activating N-terminal segments of bacterial lipoproteins and lipopeptides that were later shown to act by heterodimerization of TLR1/TLR2.^16–20 Such agonists based on the lipoproteins are effective vaccine adjuvants and continue to be widely studied today.^20–24 TLR2 requires heterodimerization with either TLR1 or TLR6 for activation. Bacterial triacylated proteins or peptides activate TLR1/TLR2 (e.g.; Pam3CSK4,¹³ Figure 1) whereas diacylated lipopolypeptides stimulate TLR2/TLR6 (e.g.; MALP-2,¹⁴ Figure 1).^16–20 Complementary to recent studies that disclosed the only other known and rare small molecule TLR2/TLR1 agonists^25–29 and through screening a compound library designed to promote cell surface receptor dimerization,^30–32 we discovered and characterized the diprovocims as a new and especially potent class of synthetic small molecule TLR2/TLR1 agonists that bear no structural similarity to any other natural or synthetic TLR agonist.^33–35 The most potent diprovocims elicit agonist activity at extraordinarily low concentrations (EC₅₀ = 110 pM) in human cells, being more potent than any known small molecule TLR agonist. Moreover, the efficacy of the class matches that of the lipopeptide-based synthetic TLR agonists such as Pam3CSK4 but are more potent. A representative of the series, compound 1 (diprovocim-1, Figure 1), was shown to act as an effective adjuvant in mice when co-administered by conventional intramuscular (i.m.) injection (vaccination) with the antigen ovalbumin (OVA), which is non-immunogenic in the unadjuvanted state.³³ It was further shown to act synergistically with a checkpoint inhibitor (anti-PD-L1), where the combination treatment eradicated tumors in mice implanted with an immunogen bearing murine melanoma (B16-OVA) and protected mice from tumor rechallenge.³³ This impressive in vivo activity was observed with diprovocim/OVA co-administration i.m., rather than with intratumor adjuvant administration that has been a convention in recent studies.

Figure 1.

Naturally-derived agonists Pam3CSK4 (TLR1/TLR2), MALP-2 (TLR6/TLR2) and the synthetic diprovocim-1 (1, TLR1/TLR2).

Herein, we detail studies that led to the identification of a site and manner by which the diprovocims could be functionalized, permitting potential covalent linkage to candidate protein and peptide antigens or coupling with a targeting or delivery moiety,³⁶ without impacting TLR1/TLR2 activity. Because the potency and efficacy of the diprovocims on human receptors are superb, whereas their activity is significantly weaker in murine systems typically employed in initial in vivo studies, a second and equally important advance disclosed is the identification of substantial improvements in both potency and especially efficacy on mTLR1/TLR2 while maintaining full activity on hTLR1/TLR2.

Results and Discussion

TLR1/TLR2 preferentially binds triacyl lipopeptides but also binds diacyl lipopeptides weakly, whereas TLR2/TLR6 only binds diacyl lipopeptides. X-ray crystallographic structures of Pam3CSK4 (three lipid chains) bound to hTLR1/TLR2¹⁹ and Pam2CSK4 (two lipid chains) bound to mTRL2/TLR6¹⁸ have been disclosed.²⁰ These studies revealed that the amide lipid chain of Pam3CSK4 inserts into a TLR1 hydrophobic pocket and the remaining two ester lipid chains bind in a TLR2 hydrophobic channel, filling a continuous hydrophobic pocket spanning both proteins at the TLR1/TLR2 heterodimer interface. Three of the diprovocim amide side chains serve the same role as the three lipid chains of Pam3CSK4, extending into the hydrophobic pocket spanning TLR1/TLR2 and that found in TLR1. However, the symmetrical diprovocims (e.g., diprovocim-1) contain four and not three such groups. Models of diprovocim-1 bound to TLR1/TLR2 based on a related X-ray crystal structure and a complementary mutagenesis analysis of the binding site indicate that three of the side chains mimic the three lipid chains of the natural agonists and bind the hydrophobic pockets of TLR1/TLR2 whereas the fourth binds the exterior of the heterodimer interface and extends toward the surrounding solvent.³⁵ This fourth side chain was chosen to achieve our objectives, providing both a potential functionalized conjugation site and site that might improve murine activity without impacting the already superb human activity.

The initial basis for the studies rested with our prior observation that one of the four side chains in diprovocim-1 could be replaced with a hydrophobic linear aliphatic side chain. That side chain displayed a well-behaved chain length dependence (Figure 2), culminating in optimal potency and efficacy with chain lengths of 12–14 carbons that matched the activity of diprovocim-1 (1).³⁴

Figure 2.

Single side chain replacements in diprovocim-1 previously disclosed (ref 34).

The candidate TLR1/TLR2 agonists were assessed for potency (EC₅₀) and efficacy (amount relative to diprovocim-1 as 100%) by measurement of the stimulated release of TNF-α from both differentiated THP-1 cells (human) and thioglycolate-elicited mouse peritoneal macrophages (murine) as detailed in prior studies leading the discovery and characterization of the diprovocims.³⁴ In initial studies, two series of side chain replacements of varied length that terminate in either a methyl ester or primary alcohol were prepared by coupling the carboxylic acid 2³⁴ with the corresponding side chain free amine (Figure 3). Each series recapitulated the observation that potency improved significantly with increasing hydrophobic chain length (12 > 10 > 8 > 6) with each 3 and 7 (12 carbon length) matching the activity of diprovocim-1 (Figure 3). Although neither the potency or efficacy improved, it was significant that useful polar functionality could be incorporated at the side chain terminus. Examination of the corresponding carboxylic acid 11 led to a substantial reduction in activity, whereas simple amides (12 and 13) and the corresponding amine 16 led only to modest reductions in the activity. Notably, and analogous to subsequent observations, the Boc protected amine 17 also exhibited good activity approaching that of diprovocim-1 (1) and the methyl ester and alcohol (3 and 7). Collectively, these studies indicate that a hydrophobic side chain bearing a terminal ester, alcohol, amine, carbamate, and perhaps amide maintain the activity of the potent diprovocims and offer opportunities for further functionalization or extension.

Figure 3.

Linker chain and impact of terminal functionalization.

In addition, three functional groups commonly used in conjugation studies (19–21; alkyne, azide, and thiol) were found to be well tolerated at the terminus of the 12-carbon hydrophobic chain where the alkyne 19 not only matched or perhaps exceeded the potency of diprovocim-1, but also exhibited a small improvement in the response efficacy (Figure 3). Although not extensively examined, incorporation of a hydrophobic aryl versus straight chain linker was also well tolerated although this was only briefly explored (22–27, Figure 3). Within this small series, the activity improved with increasing side chain length with 26 and 27 approaching the activity of diprovocim-1 and further functionalized with a terminal free phenol or aniline capable of conjugation. Notably, the efficacy in the murine system employing mouse macrophages exhibited a welcomed substantial increase in efficacy with both 26 and 27. Finally, introduction of polar atoms in the linker chain with 28–31, incorporating three repeating ethylene glycol units into the 12-atom linker that were also prepared from 2 by amide coupling with the corresponding amine, led to substantial losses in activity (>1000-fold), highlighting the preferential hydrophobic character required of the side chain region (Figure 4).

Figure 4.

Impact of incorporating polar atoms in the linker chain.

The alcohol 7 was chosen as the functionalized side chain to examine in detail for further derivatization (Figure 5) by direct acylation. Simple acetylation with 32 had little further impact on potency although a small improvement was observed in the human THP-1 cell line. Notably, a small improvement was observed in the efficacy (extent of response) in both the human and murine cell lines, suggesting this may provide an avenue to improve the murine efficacy. Among the earliest such compounds examined, acylation with glycine protected as its Boc derivative to provide 35 afforded an exciting new lead. Its activity and efficacy essentially match that of diprovocim-1 in human THP-1 cells, exhibiting an EC₅₀ of 140 pM with an efficacy of 100% that of diprovocim-1. Even more significantly, it displayed a superb EC₅₀ of 750 pM in mouse macrophages, being 2-fold more active than diprovocim-1, and it exhibited a remarkably improved efficacy of 550% that of diprovocim-1, achieving a key objective of the studies. As a result, and because it became a key compound in the studies, we have come to refer to 35 as diprovocim-X (diprovocim-10), reflecting its chronological discovery.³⁴ A series of additional glycine derivatives 36–42 all exhibited superb potency and good or improved murine efficacy with the exception of the large Fmoc derivative 38. None exceeded the combined properties of diprovocim-X (35) although the structurally related Troc derivative 37 most closely approached the combined properties, suggesting the size and hydrophobic character of the group is responsible for the improved murine efficacy. Even the acylated glycine as its free amine (43), its dimethyl derivative (44), the corresponding alcohol 45, and its benzyl ether (46) displayed superb or even improved potency [EC₅₀ 50–200 pM (THP-1) and 0.7–5 nM (mouse macrophages)] while maintaining (THP-1) or slightly improving (mouse macrophages) on the efficacy of diprovocim-1. Non hydrolytically labile variations on diprovocim-X derived by removing the ester carbonyl affording the ether 48, examination of the similar ether 49, and replacement of the ester with the carbamate 50 provided interesting analogues that deserve comparative examination in instances where questions on the in vivo stability of 35 or its conjugates might arise. Interestingly, the amide variant 17 of diprovocim-X did not display this increased or improved murine activity, highlighting the importance of the ester O versus an amide NH. Finally, our studies have shown that even large substituents at this terminal region are well tolerated (not shown) and the 4-substitued benzyl ethers 51 and 52 are representative of this feature, displaying superb activity and maintained or improved efficacy.

Figure 5.

Diprovocim-X (35) and related analogues.

Consistent with this wider tolerability for substituents extending the side chain beyond the functionalized C12 side chain, introduction of a defined ethylene glycol chain often enlisted with conjugation efforts either through acylation of the free alcohol 7 or amide bond formation on the terminal carboxylate of 3/11 provided potent TLR1/TLR2 agonists (Figure 6). Notably, 55 suffered only a two-fold reduction in activity relative to diprovocim-1, approaching the activity of the free alcohol 7 itself, and exhibited a modest increase in the efficacy of the murine response.

Figure 6.

Impact of incorporating polar atoms in further linker chain extensions.

Given the impact of the BocNH-glycine extension of the free alcohol 7 that provided diprovocim-X (35), the acylation of the free alcohol with a full set of BocNH-amino acids was conducted (Figure 7). The results of the study provided a clear indication that the BocNH-glycine group within 35 plays an integral role in the activity enhancement. Only its replacement with BocNH-l-alanine with 57 approached the activity of 35. All others generally resulted in >10-fold reductions in potency and only sporadic improvements in murine efficacy although all maintained the superb human efficacy. Even the conservative modifications of N-methylation of 35 to provide 70 and replacement of the glycine methylene with a NH to provide the BocNH acyl hydrazide 71 also resulted in this reduction in potency and efficacy.

Figure 7.

Integral role of the BocNH-glycine group.

As the forgoing studies were being conducted, additional functionalization studies of the alcohol 7, the glycine derivative 35/43, and its ether variant 48 were pursued in parallel in exploration of candidate linker conjugate constructs and/or the consequences of their cleavage. Notably, the SK4 region of Pam3CSK4 has been widely used for such conjugation. It binds the same manner/region as our functionalized fourth side chain, extending out of the TLR2/TLR1 complex toward and into solvent, and provides the basis for confident success in this as a conjugation site. The results of these studies are summarized in Figure 8. Acylation of the free alcohol 7 with functionalized p-substituted benzoic acids provided a series of diprovocims, one of which exhibited superb human and murine potency and efficacy (75) and several of which displayed superb improvements in the murine efficacy (73–75). Although the examples are limited, those terminating in a polar substituent proved substantially more potent than those bearing the paired masked polar substituent, suggesting this region may extend into the surrounding solvent rather than remain in intimate contact with the dimerized receptor. Acylation of the glycine residue of 35 as well as its ether variant 48 with bridging groups that would permit sequential amide bond formations for conjugation with its protein/peptide partners provided analogous observations (79–86).

Figure 8.

Further functionalized spacers.

Although it does not appear to be a substituent that engages the TLR1 or TLR2 lipid chain hydrophobic pockets, the hydrophobic nature, the length, and terminal functionalization of the fourth side chain can substantially impact the potency and greatly improve the modest murine efficacy (species activity). Most all further side chain extensions beyond that found in diprovocim-X (35) are well tolerated consistent with expectations that this region extends beyond the intimate TLR1/TLR2 dimerization interface extending into the surrounding solvent.

Extensive in vitro characterization of diprovocim-1 (1) as a TLR1/TLR2 agonist and its downstream signaling was disclosed along with its discovery,³³ indicating that it effectively elicits the innate immune response. Since TLR1/TLR2 signaling plays an essential role in the generation of both innate and adaptive immune responses, we investigated whether diprovocim-X (35), like diprovocim-1 (1), can also serve as an adjuvant in vivo in mice, indicating stimulation of the adaptive immune response, and whether its improved murine in vitro potency and efficacy translates into a further improved murine in vivo response (Figure 9). Intramuscular immunization of C57BL/6J mice with ovalbumin (OVA) plus diprovocim-1 or diprovocim-X induced strong in vivo cytotoxicity directed against C57BL/6J splenocytes loaded with the target peptide SIINFEKL. In vivo cytotoxicity was measured twice: 9 days after the initial immunization and 9 days after a boost, and was notably stronger if two immunizing injections were performed than if only one was performed (Figure 9B). While no response to OVA alone was measurable after a single injection, a relatively weak response was evident after the boost. Resistance to B16-OVA melanoma, known to be mediated by cytotoxic T lymphocytes, was notably enhanced when inoculation was performed 10 days after the second challenge (Figure 9C). Fourteen days after a single immunization, OVA-specific circulating antibodies of different subtypes were measured by ELISA (Figure 9D–F). These showed strong responses to OVA administered in the presence of diprovocim-X and diprovocim-1, but not by itself. The data demonstrate that diprovocim-1 and diprovocim-X exhibit comparable adjuvant activity in both CD4- and CD-8 dependent responses. Taken together, these data indicate that diprovocim-X (35) is an adjuvant in mice, matching or surpassing the in vivo activity of diprovocim-1, and serves to stimulate the adaptive immune response. Significantly, the study indicates that compounds in this class are of a caliber that exhibit in vivo activity, being active not only on its target proteins and in functional cellular assays, but in vivo in mice as well.

Figure 9.

In vivo adjuvant activity of diprovocim-X. (A) Timeline for in vivo testing of diprovocim-X adjuvant activity. Mice were immunized i.m. with saline, ovalbumin (OVA), diprovocim-1 + OVA, or diprovocim-X + OVA; diprovocims and OVA were administered at 200 μg (10 mg/kg) and 100 μg (5 mg/kg) per mouse, respectively. Immunizations (#1, initial; #2, boost) are indicated in yellow. Assays are indicated in blue. (B) Cytotoxic T lymphocyte (CTL) assay. CD8 T cell cytotoxic activity towards target cells (SIINFEKL-loaded C57BL/6J splenocytes) measured 9 days after initial immunization (open circles) and 9 days after a boost (filled circles). (C) B16-OVA tumor volume measured on the indicated days after B16-OVA administration (N = 8, 8, 8, 5 mice respectively for saline, OVA, diprovocim-1 + OVA, diprovocim-X + OVA). P values represent significance of difference on day 22 compared to saline. (D–F) Serum OVA-specific IgG (D), IgG1 (E), and IgG2b (F) concentrations measured by ELISA 14 days after initial immunization. Data points represent individual mice (B, D–F). Mean ± SD are plotted (B–F). P values were determined by one-way ANOVA and post-hoc Tukey multiple comparisons test; * P=0.0502, **** P<0.0001.

Finally, and like diprovocim-1,³³ the diprovocim-X stimulated release of TNF-α from (human) differentiated THP-1 cells proved dependent on both TLR2 and TLR1, but notably independent of TLR6 (Figure 10). Diprovocim-1 and Diprovocim-X both induced TLR4-deficient C3H/HeJ cells and TLR4-sufficient C3H/HeOuJ macrophages to release TNF-α in equal amounts (Figure 10).

Figure 10.

TLR dependence. (A) Diprovocim-X (5 nM) stimulated release of TNF-α from differentiated human THP-1 cells is dependent on TLR1 and TLR2 and independent of TLR6, established by neutralizing antibody cotreatment. NT = no antibody cotreatment (control); anti-TLR1, anti-TLR2, and anti-TLR6 refer to the TLR neutralizing antibody cotreatment (20 μg/mL, 1 h pretreatment). (B) Stimulated release of TNF-α from peritoneal macrophages of Tlr4-WT C3H/HeOuJ (black) and Tlr4-mutant C3H/HeJ (red) mice (which express the inactive P712H variant of TLR4) by diprovocim-X and diprovocim-1 (at 500 nM each). LPS used as positive control (not shown). The data indicate no TLR4 dependence and highlight the greater efficacy of diprovocim-X versus diprovocim-1 as a TNF inducer in mouse macrophage cultures.

Conclusions

The diprovocims, which originally emerged from screening a unique chemical library designed to permit cell surface receptor dimerization,^30–32 stimulate the innate and adaptive immune responses to co-administered non-immunogenic antigens, act by a well-defined mechanism (TLR1/TLR2 agonist), are easy to produce and structurally manipulate, exhibit exquisite structure–activity relationships, are remarkably potent and efficacious in human and now murine systems, and bear no structural similarity to any known natural or synthetic TLR agonist.^33–35 Herein, we described studies demonstrating that the fourth side chain of the diprovocims may be replaced with a hydrophobic side chain of a well-defined length, and terminally functionalized in a manner that results in improvements in potency as well as enhancements in efficacy towards the murine receptor (mTLR1/TLR2), while maintaining remarkable potency and efficacy towards the human receptor (hTLR1/TLR2) as measured by TNF production by macrophages from each species. We showed that the improved potency and efficacy in vitro in the murine system with the prototype diprovocim-X relative to diprovcim-1 translated into a maintained or further improved murine in vivo response. We also introduce a site and convenient manner by which covalent linkage to candidate antigens or coupling with targeting or delivery moieties may be conducted without impacting TLR1/TLR2 activation by the diprovocims. These modifications bring powerful capabilities to the diprovocims; in particular, the ability to simultaneously deliver defined adjuvant activity and antigenic peptides in close proximity, thereby minimizing toxicity (e.g., by traditional adjuvant mixtures) and off-target effects (due to stimulation of adaptive immune cells distant from immunostimulated innate immune cells). The precise immune activation afforded by improvements to the diprovocims described in this work may ultimately permit this adjuvant to be delivered at extremely low but highly effective, defined doses, and to be optimized for targeting to antigen presenting cells in lymphoid tissues. The diprovocims, including diprovocim-1 (1) and now diprovocim-X (35) detailed herein, represent both user-friendly and highly useful additions to the limited number of TLR agonists. They are attractive starting points for drug discovery, and simple to structurally modify and chemically prepare.³⁶ As a result, we expect many others will find the diprovocims an attractive class of TLR agonists to work with.

Experimental

General Information.

Unless otherwise noted, reactions were performed under an atmosphere of Ar using oven-dried glassware. Stainless steel syringes were used to transfer air- and moisture-sensitive liquid reagents. Reactions were monitored by thin-layer chromatography (TLC) on UNIPLATE Silica Gel HLF plates, visualized by UV or fluorescence, or by staining with KMnO₄, anisaldehyde, phosphomolybdic acid or iodine. Column chromatography and preparative thin layer chromatography (PTLC) were used to purify products. Unless otherwise noted, reagents and anhydrous solvents including DMF, CH₂Cl₂, THF and MeOH were purchased from commercial sources, and used as received. The abbreviation rt = room temperature. Proton nuclear magnetic resonance (¹H NMR) spectra and carbon nuclear magnetic resonance (¹³C NMR) spectra were recorded on Bruker 400 AVANCE spectrometer (400 and 100 MHz, respectively), Bruker 500 AVANCE spectrometer (500 and 125 MHz, respectively) or Bruker 600 AVANCE spectrometer (600 and 150 MHz, respectively). Chemical shifts (δ) for protons are reported in parts per million (ppm) downfield from tetramethylsilane and are referenced to the proton resonance of a residual proton in the NMR solvent (CDCl₃: δ = 7.26, CD₃OD: δ = 3.31 or DMSO-d₆: δ = 2.50). Chemical shifts (δ) for carbon are reported in ppm downfield from tetramethylsilane and are referenced to the carbon resonances of the solvent peak (CDCl₃: δ = 77.23, CD₃OD: δ = 49.15 or DMSO-d₆: δ = 39.51). Fluorine nuclear magnetic resonance (¹⁹F NMR) spectra were recorded on a Bruker 400 AVANCE spectrometer (376 MHz). ¹⁹F NMR chemical shifts (δ) are reported in ppm upfield from trichlorofluoromethane (0 ppm). NMR data are represented as follows: chemical shift (ppm), multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, p = pent, m = multiplet), coupling constant (J) in Hertz (Hz), integration. High-resolution mass determinations were obtained either by electrospray ionization (ESI) on a Waters LCT Premier^™ mass spectrometer or by matrix-assisted laser desorption/ionization (MALDI). Infrared spectra were measured at a Shimadzu FTIR-8400S Fourier Transform Infrared Spectrometer. Uncorrected melting points were measured on Thomas Hoover Capillary Melting Point apparatus. All tested compounds were >95% pure by HPLC analysis. Compounds were prepared from commercially available (1S,2R)-2-phenylcyclopropylamine (99% purity, 99% ee, eNovation Chemicals) and precursors containing the (S,S)-pyrrolidine-3,4-dicarboxylic acid core (>99% ee) prepared as detailed.³⁴

Diprovocim-X (12-((3S,4S)-1-(4-((3S,4S)-3,4-Bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)-pyrrolidine-1-carbonyl)benzoyl)-4-(((1S,2R)-2-phenylcyclopropyl)carbamoyl)-pyrrolidine-3-carboxamido)dodecyl-2-((tert-butoxycarbonyl)amino)acetate) (35).

A reaction solution of 7 (0.39 g, 0.40 mmol), BocNH-Gly-OH (70 mg, 0.40 mmol), DMAP (49 mg, 0.40 mmol), and EDCI•HCl (0.11 g, 0.60 mmol) in anhydrous DMF (2.0 mL) and CH₂Cl₂ (2.0 mL) was stirred at 40 °C (or rt) for 8 h (or overnight). The reaction was quenched by the addition of aqueous 1 N HCl (1 mL) and the mixture was diluted with H₂O (8 mL). The mixture was extracted with EtOAc (10 mL × 3), and the combined organic phase was dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. Flash chromatography (SiO₂, 8–10% MeOH–CH₂Cl₂ gradient) afforded 35 (0.39 g, 86%) as a white solid. [a]_D²⁵ +44 (c 0.3, HFIP). ¹H NMR (CDCl₃, 400 MHz) δ 7.55−7.51 (m, 4H), 7.38 (br,1H), 7.29−7.07 (m, 15H), 6.78 (br, 1H), 6.69 (br, 1H), 5.07 (br, 1H), 4.14−4.10 (m, 2H), 3.89−3.88 (m, 2H), 3.81−3.61 (m, 8H), 3.30−3.21 (m, 4H), 3.03−2.82 (m, 5H), 2.06−1.95 (m, 3H), 1.62−1.60 (m, 2H), 1.44 (s, 11H), 1.28−1.07 (m, 22H). ¹³C NMR (CDCl₃, 150 MHz) δ 171.24, 171.21, 171.17, 170.7, 170.6, 170.1, 170.0, 169.9, 169.3, 167.95, 167.92, 167.84, 167.80, 155.3, 139.8, 137.43, 137.38, 137.24, 137.19, 127.98, 127.96, 127.0, 126.9, 125.9, 125.8, 125.7, 79.5, 65.1, 51.5, 51.4, 48.4, 48.3, 48.2, 47.2, 47.1, 47.0, 45.2, 45.1, 45.0, 44.8, 42.0, 39.41, 39.39, 31.8, 31.74, 31.69, 29.08, 29.03, 28.97, 28.90, 28.84, 28.78, 28.7, 28.0, 27.9, 26.4, 25.3, 24.3, 24.21, 24.18, 24.1, 15.49, 15.46, 15.2. IR (film) 3279, 2927, 2855, 1716, 1642, 1546, 1435, 1391, 1260, 1168, 1057, 1031, 863, 736, 696 cm⁻¹. HRMS (ESI-TOF) m/z calcd for C₆₆H₈₄N₇O₁₀ [M+H]⁺ 1134.6274, found 1134.6272. HPLC purity analysis (98.65% pure) provided in Supporting Information (Figure S1).

Methyl 12-((3S,4S)-1-(4-((3S,4S)-3,4-bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-1-carbonyl)benzoyl)-4-(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-3-carboxamido)-dodecanoate (3).

A solution of 2³⁴ (119 mg, 0.150 mmol), methyl 12-aminododecanoate hydrochloride³⁷ (58.5 mg, 0.220 mmol), HOAt (24.5 mg, 0.180 mmol), and 2,6-lutidine (87 μL, 0.75 mmol) in anhydrous DMF (2.0 mL) was cooled to 0 °C and treated with EDCI•HCl (42.2 mg, 0.220 mmol) in one portion. The reaction solution was stirred at 0 °C for 30 min, warmed to rt and stirred for another 3 h. The reaction was quenched by the addition of aqueous 1 N HCl (1 mL) and diluted with H₂O (2 mL). The mixture was extracted with EtOAc (5 mL × 3), and the combined organic phase was dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. Flash chromatography (SiO₂, 1–10% MeOH–CH₂Cl₂ gradient) afforded 3 (112 mg, 74%) as a white solid. ¹H NMR (CDCl₃, 400 MHz) δ 7.52 (s, 4H), 7.37 (br, 1H), 7.28−7.07 (m, 15H), 6.78 (br, 1H), 6.68 (br, 1H), 3.79−3.61 (m, 11H), 3.28−2.98 (m, 6H), 2.89−2.84 (m, 3H), 2.31−2.26 (m, 2H), 2.04−1.98 (m, 3H), 1.62−1.58 (m, 2H), 1.46−1.42 (m, 2H), 1.26−1.11 (m, 20H). HRMS (ESI-TOF) m/z calcd for C₆₀H₇₃N₆O₈ [M+H]⁺ 1005.5484, found 1005.5484.

Methyl 10-((3S,4S)-1-(4-((3S,4S)-3,4-bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-1-carbonyl)benzoyl)-4-(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-3-carboxamido)-decanoate (4).

A solution of 2³⁴ (20 mg, 0.025 mmol), methyl 10-aminodecanoate hydrochloride (8.9 mg, 0.0375 mmol), HOAt (4.1 mg, 0.030 mmol), and 2,6-lutidine (15 μL, 0.125 mmol) in anhydrous DMF (0.5 mL) was cooled to 0 °C and treated with EDCI•HCl (7.2 mg, 0.0375 mmol) in one portion. The reaction solution was stirred at 0 °C for 30 min, warmed to rt and stirred for another 3 h. The reaction was quenched by the addition of aqueous 1 N HCl (0.5 mL) and diluted with H₂O (2 mL). The mixture was extracted with EtOAc (4 mL × 3), and the combined organic phase was dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. Flash chromatography (SiO₂, 1–10% MeOH–CH₂Cl₂ gradient) afforded 4 (11 mg, 45%) as a white solid. ¹H NMR (CDCl₃, 400 MHz) δ 7.53 (s, 4H), 7.40 (br, 1H), 7.32 (br, 1H), 7.27−7.07 (m, 15H), 6.80 (br, 1H), 6.73 (br, 1H), 3.80−3.63 (m, 11H), 3.31−2.97 (m, 6H), 2.91−2.82 (m, 3H), 2.30−2.25 (m, 2H), 2.05−1.95 (m, 3H), 1.62−1.56 (m, 2H), 1.49−1.41 (m, 2H), 1.27−1.07 (m, 16H). HRMS (ESI-TOF) m/z calcd for C₅₈H₆₉N₆O₈ [M+H]⁺ 977.5171, found 977.5181.

Methyl 8-((3S,4S)-1-(4-((3S,4S)-3,4-bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-1-carbonyl)benzoyl)-4-(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-3-carboxamido)-octanoate (5).

A solution of 2³⁴ (20 mg, 0.025 mmol), methyl 8-aminooctanoate hydrochloride³⁸ (7.9 mg, 0.0375 mmol), HOAt (4.1 mg, 0.030 mmol), and 2,6-lutidine (15 μL, 0.125 mmol) in anhydrous DMF (0.5 mL) was cooled to 0 °C and treated with EDCI•HCl (7.2 mg, 0.0375 mmol) in one portion. The reaction solution was stirred at 0 °C for 30 min, warmed to rt and stirred for another 3 h. The reaction was quenched by the addition of aqueous 1 N HCl (0.5 mL) and the solution was diluted with H₂O (2 mL). The mixture was extracted with EtOAc (4 mL × 3), and the combined organic phase was dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. Flash chromatography (SiO₂, 1–10% MeOH–CH₂Cl₂ gradient) afforded 5 (17 mg, 72%) as a white solid. ¹H NMR (CDCl₃, 400 MHz) δ 7.53 (s, 4H), 7.45 (br, 1H), 7.40−7.33 (m, 2H), 7.27−7.06 (m, 15H), 6.86−6.81 (m, 1H), 3.79−3.61 (m, 11H), 3.31−2.96 (m, 6H), 2.90−2.80 (m, 3H), 2.28 (q, J = 7.6 Hz, 2H), 2.05−1.94 (m, 3H), 1.62−1.56 (m, 2H), 1.48−1.40 (m, 2H), 1.30−1.06 (m, 12H). HRMS (ESI-TOF) m/z calcd for C₅₆H₆₅N₆O₈ [M+H]⁺ 949.4858, found 949.4856.

Methyl 6-((3S,4S)-1-(4-((3S,4S)-3,4-bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-1-carbonyl)benzoyl)-4-(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-3-carboxamido)-hexanoate (6).

A solution of 2³⁴ (20 mg, 0.025 mmol), methyl 6-aminohexanoate hydrochloride³⁹ (6.8 mg, 0.0375 mmol), HOAt (4.1 mg, 0.030 mmol), and 2,6-lutidine (15 μL, 0.125 mmol) in anhydrous DMF (0.5 mL) was cooled to 0 °C and treated with EDCI•HCl (7.2 mg, 0.0375 mmol) in one portion. The reaction solution was stirred at 0 °C for 30 min, warmed to rt and stirred for another 3 h. The reaction was quenched by the addition of aqueous 1 N HCl (0.5 mL) and diluted with H₂O (2 mL). The mixture was extracted with EtOAc (4 mL × 3), and the combined organic phase was dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. Flash chromatography (SiO₂, 1–10% MeOH–CH₂Cl₂ gradient) afforded 6 (17 mg, 74%) as a white solid. ¹H NMR (CDCl₃, 400 MHz) δ 7.53 (s, 4H), 7.44 (br, 1H), 7.34 (br, 2H), 7.27−7.06 (m, 15H), 6.94−6.90 (m, 1H), 3.80−3.59 (m, 11H), 3.30−2.96 (m, 6H), 2.91−2.81 (m, 3H), 2.32−2.26 (m, 2H), 2.05−1.94 (m, 3H), 1.64−1.57 (m, 2H), 1.51−1.43 (m, 2H), 1.37−1.06 (m, 8H). HRMS (ESI-TOF) m/z calcd for C₅₄H₆₁N₆O₈ [M+H]⁺ 921.4545, found 921.4563.

(3S,4S)-1-(4-((3S,4S)-3,4-Bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-1-carbonyl)-benzoyl)-N³-(12-hydroxydodecyl)-N⁴-((1S,2R)-2-phenylcyclopropyl)pyrrolidine-3,4-dicarboxamide (7).

A solution of 2³⁴ (127 mg, 0.160 mmol), 12-aminododecan-1-ol hydrochloride (57.1 mg, 0.240 mmol), HOAt (25.9 mg, 0.190 mmol), and 2,6-lutidine (93 μL, 0.80 mmol) in anhydrous DMF (2.0 mL) was cooled to 0 °C and treated with EDCI•HCl (46.0 mg, 0.240 mmol) in one portion. The reaction solution was stirred at 0 °C for 30 min, warmed to rt and stirred for another 3 h. The reaction was quenched by the addition of aqueous1 N HCl (1 mL) and diluted with H₂O (2 mL). The mixture was extracted with EtOAc (5 mL × 3), and the combined organic phase was dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. Flash chromatography (SiO₂, 1–10% MeOH–CH₂Cl₂ gradient) afforded 7 (110 mg, 70%) as a white solid. ¹H NMR (CD₃OD, 400 MHz) δ 7.62 (s, 4H), 7.27−7.06 (m, 15H), 4.03−3.96 (m, 2H), 3.79−3.61 (m, 6H), 3.52 (t, J = 6.7 Hz, 2H), 3.34−3.01 (m, 6H), 2.91−2.78 (m, 3H), 2.07−1.92 (m, 3H), 1.53−1.08 (m, 26H). HRMS (ESI-TOF) m/z calcd for C₅₉H₇₃N₆O₇ [M+H]⁺ 977.5535, found 977.5544.

(3S,4S)-1-(4-((3S,4S)-3,4-Bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-1-carbonyl)benzoyl)-N³-(10-hydroxydecyl)-N⁴-((1S,2R)-2-phenylcyclopropyl)pyrrolidine-3,4-dicarboxamide (8).

A solution of 2³⁴ (20 mg, 0.025 mmol), 10-aminodecan-1-ol hydrochloride⁴⁰ (6.5 mg, 0.0375 mmol), HOAt (4.1 mg, 0.030 mmol), and 2,6-lutidine (15 μL, 0.125 mmol) in anhydrous DMF (0.5 mL) was cooled to 0 °C and treated with EDCI•HCl (7.2 mg, 0.0375 mmol) in one portion. The reaction solution was stirred at 0 °C for 30 min, warmed to rt and stirred for another 3 h. The reaction was quenched by the addition of aqueous 1 N HCl (0.5 mL) and diluted with H₂O (2 mL). The mixture was extracted with EtOAc (4 mL × 3), and the combined organic phase was dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. Flash chromatography (SiO₂, 1–10% MeOH–CH₂Cl₂ gradient) afforded 8 (15 mg, 63%) as a white solid. ¹H NMR (CD₃OD, 400 MHz) δ 7.62 (s, 4H), 7.27−7.06 (m, 15H), 4.03−3.97 (m, 2H), 3.79−3.60 (m, 6H), 3.54−3.50 (m, 2H), 3.34−3.02 (m, 6H), 2.91−2.78 (m, 3H), 2.08−1.91 (m, 3H), 1.53−1.08 (m, 22H). HRMS (ESI-TOF) m/z calcd for C₅₇H₆₉N₆O₇ [M+H]⁺ 949.5222, found 949.5231.

(3S,4S)-1-(4-((3S,4S)-3,4-Bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-1-carbonyl)-benzoyl)-N³-(8-hydroxyoctyl)-N⁴-((1S,2R)-2-phenylcyclopropyl)pyrrolidine-3,4-dicarboxamide (9).

A solution of 2³⁴ (20 mg, 0.025 mmol), 8-aminooctan-1-ol hydrochloride (5.4 mg, 0.0375 mmol), HOAt (4.1 mg, 0.030 mmol), and 2,6-lutidine (15 μL, 0.125 mmol) in anhydrous DMF (0.5 mL) was cooled to 0 °C and treated with EDCI•HCl (7.2 mg, 0.0375 mmol) in one portion. The reaction solution was stirred at 0 °C for 30 min, warmed to rt and stirred for another 3 h. The reaction was quenched by the addition of aqueous 1 N HCl (0.5 mL) and diluted with H₂O (2 mL). The mixture was extracted with EtOAc (4 mL × 3), and the combined organic phase was dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. Flash chromatography (SiO₂, 1–10% MeOH–CH₂Cl₂ gradient) afforded 9 (17 mg, 74%) as a white solid. ¹H NMR (CD₃OD, 400 MHz) δ 7.62 (s, 4H), 7.27−7.06 (m, 15H), 4.03−3.97 (m, 2H), 3.79−3.61 (m, 7H), 3.52 (q, J = 7.1 Hz, 2H), 3.34−3.02 (m, 6H), 2.91−2.77 (m, 3H), 2.08−1.93 (m, 3H), 1.55−1.08 (m, 18H). HRMS (ESI-TOF) m/z calcd for C₅₅H₆₅N₆O₇ [M+H]⁺ 921.4909, found 921.4909.

(3S,4S)-1-(4-((3S,4S)-3,4-Bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-1-carbonyl)-benzoyl)-N³-(6-hydroxyhexyl)-N⁴-((1S,2R)-2-phenylcyclopropyl)pyrrolidine-3,4-dicarboxamide (10).

A solution of 2³⁴ (20 mg, 0.025 mmol), 6-aminohexan-1-ol hydrochloride (4.4 mg, 0.0375 mmol), HOAt (4.1 mg, 0.030 mmol), and 2,6-lutidine (15 μL, 0.125 mmol) in anhydrous DMF (0.5 mL) was cooled to 0 °C and treated with EDCI•HCl (7.2 mg, 0.0375 mmol) in one portion. The reaction solution was stirred at 0 °C for 30 min, warmed to rt and stirred for another 3 h. The reaction was quenched by the addition of aqueous 1 N HCl (0.5 mL) and diluted with H₂O (2 mL). The mixture was extracted with EtOAc (4 mL × 3), and the combined organic phase was dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. Flash chromatography (SiO₂, 1–10% MeOH–CH₂Cl₂ gradient) afforded 10 (18 mg, 81%) as a white solid. ¹H NMR (CD₃OD, 400 MHz) δ 7.62 (s, 4H), 7.27−7.06 (m, 15H), 4.03−3.98 (m, 2H), 3.79−3.61 (m, 7H), 3.56−3.49 (m, 2H), 3.34−3.06 (m, 6H), 2.91−2.79 (m, 3H), 2.09−1.93 (m, 3H), 1.55−1.08 (m, 14H). HRMS (ESI-TOF) m/z calcd for C₅₃H₆₁N₆O₇ [M+H]⁺ 893.4596, found 893.4619.

12-((3S,4S)-1-(4-((3S,4S)-3,4-Bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-1-carbonyl)benzoyl)-4-(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-3-carboxamido)-dodecanoic acid (11).

Compound 3 (2.2 mg, 0.00219 mmol) was dissolved in THF/MeOH/H₂O (0.4 mL/0.1 mL/0.1 mL). LiOH (1 mg) was added at 0 °C. After the reaction mixture was stirred at room temperature for 3 h, it was poured into aqueous 1 N HCl (2 mL) and EtOAc (3 mL). The aqueous phase was extracted with EtOAc (3 mL), and the combined organic phases were washed with saturated aqueous NaCl (2 mL). The organic phase was dried over Na₂SO₄, filtered and concentrated. PTLC (SiO₂, 8% MeOH/0.05% formic acid/CH₂Cl₂) provided 11 (1.9 mg, 86%). ¹H NMR (600 MHz, DMSO-d₆, mixture of rotamers) δ 8.44−8.27 (m, 3H), 8.06−7.89 (m, 1H), 7.55 (s, 4H), 7.28−7.20 (m, 6H), 7.18−7.09 (m, 6H), 7.08−7.04 (m, 3H), 3.82−3.76 (m, 2H), 3.67−3.61 (m, 2H), 3.54−3.45 (m, 4H), 3.23−2.88 (m, 4H), 2.86−2.72 (m, 3H), 2.20−2.14 (m, 2H), 1.99−1.82 (m, 3H), 1.50−1.43 (m, 2H), 1.42−1.30 (m, 2H), 1.27−1.06 (m, 22H). HRMS (ESI-TOF) m/z calcd for C₅₉H₇₁N₆O₈ [M+H]⁺ 991.5328, found 991.5329.

(3S,4S)-N³-(12-Amino-12-oxododecyl)-1-(4-((3S,4S)-3,4-bis(((1S,2R)-2-phenylcyclopropyl)-carbamoyl)pyrrolidine-1-carbonyl)benzoyl)-N⁴-((1S,2R)-2-phenylcyclopropyl)pyrrolidine-3,4-dicarboxamide (12).

Compound 11 (2.7 mg, 0.00272 mmol), ammonium chloride (0.3 mg, 0.00544 mmol), HOAt (0.6 mg, 0.00408 mmol), and 2,6-lutidine (2.9 mg, 0.0272 mmol) were dissolved in anhydrous DMF (0.1 mL). Upon dissolution of the reagents (ca. 5 min) at 0 °C, EDCI•HCl (1.6 mg, 0.00816 mmol) was added in one portion at 0 °C, and the reaction mixture was stirred at the same temperature for 30 min. The reaction mixture was warmed to rt and stirred for 23 h, after which it was poured into aqueous 1 N HCl (2 mL) and EtOAc (3 mL). The aqueous phase was extracted twice with EtOAc (3 mL), and the combined organic phases were washed sequentially with aqueous 1 N HCl (2 mL), saturated aqueous NaHCO₃ (2 mL), and saturated aqueous NaCl (2 mL). The organic phase was dried over Na₂SO₄, filtered and concentrated. PTLC (SiO₂, 10% MeOH–CH₂Cl₂ gradient) provided 12 (1.8 mg, 67%). ¹H NMR (500 MHz, DMSO-d₆, mixture of rotamers) δ 8.43−8.26 (m, 3H), 8.06−7.88 (m, 1H), 7.56 (s, 4H), 7.28−7.04 (m, 16H), 6.66 (br, 1H) 3.83−3.76 (m, 2H), 3.68−3.61 (m, 2H), 3.55−3.45 (m, 4H), 3.23−2.88 (m, 4H), 2.87−2.72 (m, 3H), 2.03−1.82 (m, 5H), 1.49−1.29 (m, 4H), 1.27−1.06 (m, 22H). HRMS (ESI-TOF) m/z calcd for C₅₉H₇₂N₇O₇ [M+H]⁺ 990.5487, found 990.5484.

(3S,4S)-1-(4-((3S,4S)-3,4-Bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-1-carbonyl)-benzoyl)-N³-(12-(methylamino)-12-oxododecyl)-N⁴-((1S,2R)-2-phenylcyclopropyl)pyrrolidine-3,4-dicarboxamide (13).

Compound 11 (2.8 mg, 0.00282 mmol), methylamine hydrochloride (0.4 mg, 0.00564 mmol), HOAt (0.6 mg, 0.00423 mmol), and 2,6-lutidine (3.0 mg, 0.0282 mmol) were dissolved in anhydrous DMF (0.1 mL). Upon dissolution of the reagents (ca. 5 min) at 0 °C, EDCI•HCl (1.6 mg, 0.00846 mmol) was added in one portion at 0 °C, and the reaction mixture was stirred at the same temperature for 30 min. The reaction mixture was warmed to rt and stirred for 23 h, after which it was poured into aqueous 1 N HCl (2 mL) and EtOAc (3 mL). The aqueous phase was extracted twice with EtOAc (3 mL), and the combined organic phases were washed sequentially with aqueous 1 N HCl (2 mL), saturated aqueous NaHCO₃ (2 mL), and saturated aqueous NaCl (2 mL). The organic phase was dried over Na₂SO₄, filtered and concentrated. PTLC (SiO₂, 10% MeOH–CH₂Cl₂) provided 13 (1.9 mg, 68%). ¹H NMR (500 MHz, CDCl₃, mixture of rotamers) δ 7.54 (s, 4H), 7.31−7.08 (m, 18H), 6.84−6.62 (m, 1H), 5.65−5.56 (m, 1H), 3.86−3.63 (m, 7H), 3.33−3.13 (m, 3H), 3.07−2.96 (m, 2H), 2.94−2.83 (m, 3H), 2.76−2.73 (m, 3H), 2.16−1.96 (m, 5H), 1.63−1.41 (m, 4H), 1.32−1.06 (m, 22H). HRMS (ESI-TOF) m/z calcd for C₆₀H₇₄N₇O₇ [M+H]⁺ 1004.5644, found 1004.5643.

tert-Butyl (12-((3S,4S)-1-(4-((3S,4S)-3,4-bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)-pyrrolidine-1-carbonyl)-benzoyl)-4-(((1S,2R)-2-phenylcyclopropyl)carbamoyl)-pyrrolidine-3-carboxamido)dodecanoyl)glycinate (14).

Compound 11 (5.1 mg, 0.00515 mmol), tert-butyl glycinate hydrochloride (1.7 mg, 0.0103 mmol), HOAt (1.1 mg, 0.00773 mmol), and 2,6-lutidine (5.5 mg, 0.0515 mmol) were dissolved in anhydrous DMF (0.1 mL). Upon dissolution of the reagents (ca. 5 min) at 0 °C, EDCI•HCl (3.0 mg, 0.0155 mmol) was added in one portion at 0 °C, and the reaction mixture was stirred at the same temperature for 30 min. The reaction mixture was warmed to rt and stirred for 23 h, after which it was poured into aqueous 1 N HCl (2 mL) and EtOAc (3 mL). The aqueous phase was extracted twice with EtOAc (3 mL), and the combined organic phases were washed sequentially with aqueous 1 N HCl (2 mL), saturated aqueous NaHCO₃ (2 mL), and saturated aqueous NaCl (2 mL). The organic phase was dried over Na₂SO₄, filtered and concentrated. PTLC (SiO₂, 10% MeOH–CH₂Cl₂) provided 14 (3.6 mg, 63%). ¹H NMR (500 MHz, CDCl₃, mixture of rotamers) δ 7.57 (s, 4H), 7.31−7.05 (m, 18H), 6.81−6.63 (m, 1H), 6.10−6.03 (m, 1H), 3.96−3.89 (m, 2H), 3.85−3.65 (m, 7H), 3.35−3.12 (m, 3H), 3.08−2.97 (m, 2H), 2.96−2.84 (m, 3H), 2.26−2.18 (m, 2H), 2.11−1.98 (m, 3H), 1.68−1.57 (m, 2H), 1.54−1.42 (m, 11H), 1.37−1.08 (m, 22H). HRMS (ESI-TOF) m/z calcd for C₆₅H₈₂N₇O₉ [M+H]⁺ 1104.6168, found 1104.6157.

(12-((3S,4S)-1-(4-((3S,4S)-3,4-Bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-1-carbonyl)benzoyl)-4-(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-3-carboxamido)-dodecanoyl)glycine (15).

Compound 14 (2.1 mg, 0.00190 mmol) was dissolved in CH₂Cl₂ (0.2 mL) at rt. TFA (0.2 mL) was added at 0 °C, and the mixture was stirred at rt for 2 h. The solvent was removed by a N₂ stream. The residual solids were suspended in MeCN and the solvents removed (repeat twice) to ensure complete removal of the TFA. This process was repeated with CH₂Cl₂. PTLC (SiO₂, 8% MeOH/0.05% formic acid/CH₂Cl₂) provided 15 (2.1 mg, quant). ¹H NMR (500 MHz, DMSO-d₆, mixture of rotamers) δ 8.49−8.31 (m, 3H), 8.10−7.92 (m, 1H), 7.86 (br, 1H), 7.55 (s, 4H), 7.28−7.20 (m, 6H), 7.18−7.03 (m, 9H), 3.82−3.75 (m, 2H), 3.68−3.60 (m, 4H), 3.55−3.45 (m, 4H), 3.24−2.88 (m, 4H), 2.87−2.72 (m, 3H), 2.11−2.06 (m, 2H), 1.99−1.82 (m, 3H), 1.50−1.37 (m, 3H), 1.35−1.29 (m, 1H), 1.27−1.02 (m, 22H). HRMS (ESI-TOF) m/z calcd for C₆₁H₇₄N₇O₉ [M+H]⁺ 1048.5542, found 1048.5524.

(3S,4S)-N³-(12-Aminododecyl)-1-(4-((3S,4S)-3,4-bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)-pyrrolidine-1-carbonyl)benzoyl)-N⁴-((1S,2R)-2-phenylcyclopropyl)pyrrolidine-3,4-dicarboxamide (16).

A solution of 17 (21 mg, 0.020 mmol) in 4 N HCl/1,4-dioxane (1.0 mL) was stirred at rt for 1 h. The solvent was removed, and the residue was purified by HPLC using the gradient 0.07% TFA-water/CH₃CN (40%→70%) eluting system to afford the TFA salt of 16 as a white solid. ¹H NMR (CD₃OD, 500 MHz) δ 8.54 (d, J = 3.9 Hz, 1H), 8.37 (br, 1H), 7.62 (s, 4H), 7.27−7.06 (m, 15H), 4.02−3.97 (m, 2H), 3.79−3.60 (m, 6H), 3.35−3.03 (m, 6H), 2.91−2.78 (m, 5H), 2.08−1.92 (m, 3H), 1.66−1.59 (m, 2H), 1.53−1.43 (m, 2H), 1.38−1.08 (m, 22H). HRMS (ESI-TOF) m/z calcd for C₅₉H₇₄N₇O₆ [M+H]⁺ 976.5701, found 976.5708.

tert-Butyl (12-((3S,4S)-1-(4-((3S,4S)-3,4-bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)-pyrrolidine-1-carbonyl)benzoyl)-4-(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-3-carboxamido)dodecyl)carbamate (17).

A solution of 2³⁴ (20 mg, 0.025 mmol), tert-butyl (12-aminododecyl)carbamate (11 mg, 0.0375 mmol), HOAt (4.1 mg, 0.030 mmol), and 2,6-lutidine (15 μL, 0.125 mmol) in anhydrous DMF (0.5 mL) was cooled to 0 °C and treated with EDCI•HCl (7.2 mg, 0.0375 mmol) in one portion. The reaction solution was stirred at 0 °C for 30 min, warmed to rt and stirred for another 3 h. The reaction was quenched by the addition of aqueous 1 N HCl (0.5 mL) and diluted with H₂O (2 mL). The mixture was extracted with EtOAc (4 mL × 3), and the combined organic phase was dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. Flash chromatography (SiO₂, 1–10% MeOH–CH₂Cl₂ gradient) afforded 17 (22 mg, 82%) as a white solid. ¹H NMR (CDCl₃, 500 MHz) δ 7.54 (d, J = 2.8 Hz, 4H), 7.28−7.22 (m, 5H), 7.19−7.08 (m, 10H), 3.80−3.59 (m, 8H), 3.32−2.94 (m, 8H), 2.91−2.82 (m, 3H), 2.06−1.96 (m, 3H), 1.46−1.40 (m, 11H), 1.28−1.07 (m, 24H). HRMS (ESI-TOF) m/z calcd for C₆₄H₈₂N₇O₈ [M+H]⁺ 1076.6225, found 1076.6230.

tert-Butyl (2-((12-((3S,4S)-1-(4-((3S,4S)-3,4-bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)-pyrrolidine-1-carbonyl)-benzoyl)-4-(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-3-carboxamido)dodecyl)amino)-2-oxoethyl)carbamate (18).

Step 1: A solution of 17 (21 mg, 0.020 mmol) in MeOH (0.1 mL), CH₂Cl₂ (0.1 mL), and 4 N HCl/1,4-dioxane (1.0 mL) was stirred at rt for 30 min. The solvents were removed under reduced pressure, and the residue was further dried under high vacuum to afford the amine hydrochloride. Step 2: A solution of the amine hydrochloride, N-(tert-butoxycarbonyl)glycine (4.2 mg, 0.024 mmol), HOAt (3.3 mg, 0.024 mmol), and 2,6-lutidine (12 μL, 0.10 mmol) in anhydrous DMF (0.5 mL) was cooled to 0 °C and treated with EDCI•HCl (5.7 mg, 0.030 mmol) in one portion. The reaction solution was stirred at 0 °C for 20 min, warmed to rt and stirred for another 4 h. The reaction was quenched by the addition of aqueous 1 N HCl (0.5 mL) and diluted with H₂O (2 mL). The mixture was extracted with EtOAc (4 mL × 3), and the combined organic phase was dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. Flash chromatography (SiO₂, 1–10% MeOH–CH₂Cl₂ gradient) afforded 18 (17 mg, 75%) as a white solid. ¹H NMR (CD₃OD, 400 MHz) δ 8.18 (t, J = 5.7 Hz, 1H), 8.01 (t, J = 5.7 Hz, 1H), 7.82 (br, 1H), 7.62 (s, 4H), 7.27−7.06 (m, 15H), 6.79 (br, 1H), 4.03−3.97 (m, 2H), 3.79−3.60 (m, 8H), 3.34−3.00 (m, 8H), 2.91−2.78 (m, 3H), 2.09−1.91 (m, 3H), 1.53−1.41 (m, 13H), 1.34−1.07 (m, 22H). HRMS (ESI-TOF) m/z calcd for C₆₆H₈₅N₈O₉ [M+H]⁺ 1133.6440, found 1133.6439.

(3S,4S)-1-(4-((3S,4S)-3,4-Bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-1-carbonyl)-benzoyl)-N³-((1S,2R)-2-phenylcyclopropyl)-N⁴-(tetradec-13-yn-1-yl)pyrrolidine-3,4-dicarboxamide (19).

A solution of 2³⁴ (20 mg, 0.025 mmol), tetradec-13-yn-1-amine⁴¹ (7.8 mg, 0.0375 mmol), HOAt (4.1 mg, 0.030 mmol), and 2,6-lutidine (15 μL, 0.125 mmol) in anhydrous DMF (0.5 mL) was cooled to 0 °C and treated with EDCI•HCl (7.2 mg, 0.0375 mmol) in one portion. The reaction solution was stirred at 0 °C for 30 min, warmed to rt and stirred for another 3 h. The reaction was quenched by the addition of aqueous 1 N HCl (0.5 mL) and diluted with H₂O (2 mL). The mixture was extracted with EtOAc (4 mL × 3), and the combined organic phase was dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. Flash chromatography (SiO₂, 5–10% MeOH–CH₂Cl₂ gradient) afforded 19 (18 mg, 73%) as a white solid. ¹H NMR (CDCl₃, 500 MHz) δ 7.56−7.52 (m, 4H), 7.28−7.08 (m, 15H), 3.84−3.62 (m, 8H), 3.32−3.10 (m, 4H), 3.06−2.95 (m, 2H), 2.91−2.82 (m, 3H), 2.19−2.15 (m, 2H), 2.06−1.96 (m, 3H), 1.93 (t, J = 2.7 Hz, 1H), 1.53−1.06 (m, 26H). HRMS (ESI-TOF) m/z calcd for C₆₁H₇₃N₆O₆ [M+H]⁺ 985.5592, found 985.5602.

(3S,4S)-N³-(12-Azidododecyl)-1-(4-((3S,4S)-3,4-bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)-pyrrolidine-1-carbonyl)benzoyl)-N⁴-((1S,2R)-2-phenylcyclopropyl)pyrrolidine-3,4-dicarboxamide (20).

A solution of 2³⁴ (20 mg, 0.025 mmol), 12-azidododecan-1-amine⁴¹ (8.5 mg, 0.0375 mmol), HOAt (4.1 mg, 0.030 mmol), and 2,6-lutidine (15 μL, 0.125 mmol) in anhydrous DMF (0.5 mL) was cooled to 0 °C and treated with EDCI•HCl (7.2 mg, 0.0375 mmol) in one portion. The reaction solution was stirred at 0 °C for 30 min, warmed to rt and stirred for another 3 h. The reaction was quenched by the addition of aqueous 1 N HCl (0.5 mL) and diluted with H₂O (2 mL). The mixture was extracted with EtOAc (4 mL × 3), and the combined organic phase was dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. PTLC (SiO₂, 10% MeOH–CH₂Cl₂) afforded 20 (16 mg, 64%) as a white solid. ¹H NMR (CD₃OD, 400 MHz) δ 7.61 (s, 4H), 7.27−7.06 (m, 15H), 4.03−3.96 (m, 2H), 3.79−3.60 (m, 6H), 3.35−3.00 (m, 6H), 2.91−2.77 (m, 3H), 2.08−1.91 (m, 3H), 1.58−1.43 (m, 4H), 1.37−1.07 (m, 22H). HRMS (ESI-TOF) m/z calcd for C₅₉H₇₂N₉O₆ [M+H]⁺ 1002.5600, found 1002.5601.

(3S,4S)-1-(4-((3S,4S)-3,4-Bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-1-carbonyl)-benzoyl)-N³-(12-mercaptododecyl)-N⁴-((1S,2R)-2-phenylcyclopropyl)pyrrolidine-3,4-dicarboxamide (21).

A solution of 2³⁴ (20 mg, 0.025 mmol), 12-aminododecane-1-thiol hydrochloride⁴¹ (9.5 mg, 0.0375 mmol), HOAt (4.1 mg, 0.030 mmol), and 2,6-lutidine (15 μL, 0.125 mmol) in anhydrous DMF (0.5 mL) was cooled to 0 °C and treated with EDCI•HCl (7.2 mg, 0.0375 mmol) in one portion. The reaction solution was stirred at 0 °C for 30 min, warmed to rt and stirred for another 3 h. The reaction was quenched by the addition of aqueous 1 N HCl (0.5 mL) and diluted with H₂O (2 mL). The mixture was extracted with EtOAc (4 mL × 3), and the combined organic phase was dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. PTLC (SiO₂, 10% MeOH–CH₂Cl₂) afforded 21 (9.0 mg, 36%) as a goose yellow solid. ¹H NMR (CDCl₃, 400 MHz) δ 7.56−7.51 (m, 4H), 7.32 (br, 1H), 7.28−7.08 (m, 15H), 6.81 (d, J = 8.1 Hz, 1H), 6.71 (br, 1H), 6.60 (br, 1H), 3.85−3.61 (m, 8H), 3.32−3.09 (m, 4H), 3.06−2.81 (m, 5H), 2.54−2.47 (m, 2H), 2.06−1.96 (m, 3H), 1.62−1.05 (m, 26H). HRMS (ESI-TOF) m/z calcd for C₅₉H₇₃N₆O₆S [M+H]⁺ 993.5312, found 993.5317.

(3S,4S)-N³-Benzyl-1-(4-((3S,4S)-3,4-bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-1-carbonyl)benzoyl)-N⁴-((1S,2R)-2-phenylcyclopropyl)pyrrolidine-3,4-dicarboxamide (22).

A solution of 2³⁴ (20 mg, 0.025 mmol), phenylmethanamine (8.2 μL, 0.075 mmol), HOAt (4.1 mg, 0.030 mmol), and 2,6-lutidine (15 μL, 0.125 mmol) in anhydrous DMF (0.5 mL) was cooled to 0 °C and treated with EDCI•HCl (7.2 mg, 0.0375 mmol) in one portion. The reaction solution was stirred at 0 °C for 30 min, warmed to rt and stirred for another 3 h. The reaction was quenched by the addition of aqueous 1 N HCl (0.5 mL) and diluted with H₂O (2 mL). The mixture was extracted with EtOAc (4 mL × 3), and the combined organic phase was dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. Flash chromatography (SiO₂, 1–10% MeOH–CH₂Cl₂ gradient) afforded 22 (18 mg, 81%) as a white solid. ¹H NMR (CD₃OD, 400 MHz) δ 8.67 (br, 1H), 8.54 (br, 1H), 8.48 (br, 1H), 8.37 (br, 1H), 7.61 (s, 4H), 7.35−7.05 (m, 20H), 4.52−4.20 (m, 2H), 4.05−3.97 (m, 2H), 3.80−3.61 (m, 6H), 3.42−3.18 (m, 4H), 2.92−2.74 (m, 3H), 2.09−1.87 (m, 3H), 1.26−1.01 (m, 6H). HRMS (ESI-TOF) m/z calcd for C₅₄H₅₅N₆O₆ [M+H]⁺ 883.4183, found 883.4184.

(3S,4S)-1-(4-((3S,4S)-3,4-Bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-1-carbonyl)-benzoyl)-N³-phenethyl-N⁴-((1S,2R)-2-phenylcyclopropyl)pyrrolidine-3,4-dicarboxamide (23).

A solution of 2³⁴ (20 mg, 0.025 mmol), 2-phenylethanamine (9.4 μL, 0.075 mmol), HOAt (4.1 mg, 0.030 mmol), and 2,6-lutidine (15 μL, 0.125 mmol) in anhydrous DMF (0.5 mL) was cooled to 0 °C and treated with EDCI•HCl (7.2 mg, 0.0375 mmol) in one portion. The reaction solution was stirred at 0 °C for 30 min, warmed to rt and stirred for another 3 h. The reaction was quenched by the addition of aqueous 1 N HCl (0.5 mL) and diluted with H₂O (2 mL). The mixture was extracted with EtOAc (4 mL × 3), and the combined organic phase was dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. Flash chromatography (SiO₂, 1–10% MeOH–CH₂Cl₂ gradient) afforded 23 (18 mg, 80%) as a white solid. ¹H NMR (CD₃OD, 400 MHz) δ 8.54 (d, J = 3.8 Hz, 1H), 8.37 (t, J = 4.1 Hz, 1H), 8.24 (t, J = 5.7 Hz, 1H), 8.04 (t, J = 5.8 Hz, 1H), 7.64−7.58 (m, 4H), 7.30−7.05 (m, 20H), 4.04−3.92 (m, 2H), 3.80−3.57 (m, 6H), 3.51−3.38 (m, 2H), 3.36−3.15 (m, 4H), 2.92−2.65 (m, 5H), 2.08−1.89 (m, 3H), 1.25−1.05 (m, 6H). HRMS (ESI-TOF) m/z calcd for C₅₅H₅₇N₆O₆ [M+H]⁺ 897.4340, found 897.4338.

(3S,4S)-1-(4-((3S,4S)-3,4-Bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-1-carbonyl)-benzoyl)-N³-(4-fluorobenzyl)-N⁴-((1S,2R)-2-phenylcyclopropyl)pyrrolidine-3,4-dicarboxamide (24).

A solution of 2³⁴ (20 mg, 0.025 mmol), (4-fluorophenyl)methanamine (8.6 μL, 0.075 mmol), HOAt (4.1 mg, 0.030 mmol), and 2,6-lutidine (15 μL, 0.125 mmol) in anhydrous DMF (0.5 mL) was cooled to 0 °C and treated with EDCI•HCl (7.2 mg, 0.0375 mmol) in one portion. The reaction solution was stirred at 0 °C for 30 min, warmed to rt and stirred for another 3 h. The reaction was quenched by the addition of aqueous 1 N HCl (0.5 mL) and diluted with H₂O (2 mL). The mixture was extracted with EtOAc (4 mL × 3), and the combined organic phase was dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. Flash chromatography (SiO₂, 1–10% MeOH–CH₂Cl₂ gradient) afforded 24 (18 mg, 80%) as a white solid. ¹H NMR (CD₃OD, 400 MHz) δ 7.60 (s, 4H), 7.32−6.98 (m, 19H), 4.47−4.17 (m, 2H), 4.03−3.98 (m, 2H), 3.79−3.61 (m, 6H), 3.41−3.19 (m, 4H), 2.90−2.72 (m, 3H), 2.08−1.86 (m, 3H), 1.25−0.99 (m, 6H). HRMS (ESI-TOF) m/z calcd for C₅₄H₅₄FN₆O₆ [M+H]⁺ 901.4089, found 901.4105.

(3S,4S)-1-(4-((3S,4S)-3,4-Bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-1-carbonyl)-benzoyl)-N³-(4-fluorophenethyl)-N⁴-((1S,2R)-2-phenylcyclopropyl)pyrrolidine-3,4-dicarboxamide (25).

A solution of 2³⁴ (20 mg, 0.025 mmol), 2-(4-fluorophenyl)ethanamine (9.8 μL, 0.075 mmol), HOAt (4.1 mg, 0.030 mmol), and 2,6-lutidine (15 μL, 0.125 mmol) in anhydrous DMF (0.5 mL) was cooled to 0 °C and treated with EDCI•HCl (7.2 mg, 0.0375 mmol) in one portion. The reaction solution was stirred at 0 °C for 30 min, warmed to rt and stirred for another 3 h. The reaction was quenched by the addition of aqueous 1 N HCl (0.5 mL) and diluted with H₂O (2 mL). The mixture was extracted with EtOAc (4 mL × 3), and the combined organic phase was dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. Flash chromatography (SiO₂, 1–10% MeOH–CH₂Cl₂ gradient) afforded 25 (18 mg, 79%) as a white solid. ¹H NMR (CD₃OD, 400 MHz) δ 7.64−7.58 (m, 4H), 7.27−6.89 (m, 19H), 4.04−3.93 (m, 2H), 3.80−3.48 (m, 6H), 3.43 (t, J = 7.2 Hz, 1H), 3.38−3.15 (m, 5H), 2.90−2.64 (m, 5H), 2.08−1.89 (m, 3H), 1.25−1.05 (m, 6H). HRMS (ESI-TOF) m/z calcd for C₅₅H₅₆FN₆O₆ [M+H]⁺ 915.4245, found 915.4246.

(3S,4S)-1-(4-((3S,4S)-3,4-Bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-1-carbonyl)-benzoyl)-N³-(8-(4-hydroxyphenyl)octyl)-N⁴-((1S,2R)-2-phenylcyclopropyl)pyrrolidine-3,4-dicarboxamide (26).

A solution of 2³⁴ (20 mg, 0.025 mmol), 4-(8-aminooctyl)phenol⁴¹ (8.3 mg, 0.0375 mmol), HOAt (4.1 mg, 0.030 mmol), and 2,6-lutidine (15 μL, 0.125 mmol) in anhydrous DMF (0.5 mL) was cooled to 0 °C and treated with EDCI•HCl (7.2 mg, 0.0375 mmol) in one portion. The reaction solution was stirred at 0 °C for 30 min, warmed to rt and stirred for another 3 h. The reaction was quenched by the addition of aqueous 1 N HCl (0.5 mL) and diluted with H₂O (2 mL). The mixture was extracted with EtOAc (4 mL × 3), and the combined organic phase was dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. Flash chromatography (SiO₂, 1–10% MeOH–CH₂Cl₂ gradient) afforded 26 (20 mg, 80%) as a white solid. ¹H NMR (CD₃OD, 400 MHz) δ 7.60 (s, 4H), 7.26−7.03 (m, 15H), 6.94 (dd, J = 8.3, 4.5 Hz, 2H), 6.67 (d, J = 8.5 Hz, 2H), 4.03−3.96 (m, 2H), 3.77−3.59 (m, 6H), 3.34−3.00 (m, 6H), 2.90−2.76 (m, 3H), 2.46 (q, J = 8.1 Hz, 2H), 2.08−1.90 (m, 3H), 1.55−1.39 (m, 4H), 1.31−1.06 (m, 14H). HRMS (ESI-TOF) m/z calcd for C₆₁H₆₉N₆O₇ [M+H]⁺ 997.5228, found 997.5220.

(3S,4S)-N³-(8-(4-Aminophenyl)octyl)-1-(4-((3S,4S)-3,4-bis(((1S,2R)-2-phenylcyclopropyl)-carbamoyl)pyrrolidine-1-carbonyl)benzoyl)-N⁴-((1S,2R)-2-phenylcyclopropyl)pyrrolidine-3,4-dicarboxamide (27).

A solution of 2³⁴ (20 mg, 0.025 mmol), 4-(8-aminooctyl)aniline⁴¹ (8.3 mg, 0.0375 mmol), HOAt (4.1 mg, 0.030 mmol), and 2,6-lutidine (15 μL, 0.125 mmol) in anhydrous DMF (0.5 mL) was cooled to 0 °C and treated with EDCI•HCl (7.2 mg, 0.0375 mmol) in one portion. The reaction solution was stirred at 0 °C for 30 min, warmed to rt and stirred for another 3 h. The reaction was quenched by the addition of aqueous 1 N HCl (0.5 mL) and diluted with H₂O (2 mL). The mixture was extracted with EtOAc (4 mL × 3), and the combined organic phase was dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. Flash chromatography (SiO₂, 1–10% MeOH–CH₂Cl₂ gradient) afforded 27 (9.0 mg, 36%) as a white solid. ¹H NMR (CD₃OD, 400 MHz) δ 7.61 (d, J = 1.9 Hz, 4H), 7.27−7.05 (m, 15H), 6.93 (dd, J = 8.0, 5.6 Hz, 2H), 6.71 (dd, J = 8.3, 2.6 Hz, 2H), 4.03−3.96 (m, 2H), 3.78−3.59 (m, 6H), 3.34−3.02 (m, 6H), 2.91−2.77 (m, 3H), 2.46 (q, J = 8.4 Hz, 2H), 2.09−1.90 (m, 3H), 1.59−1.40 (m, 4H), 1.32−1.06 (m, 14H). HRMS (ESI-TOF) m/z calcd for C₆₁H₇₀N₇O₆ [M+H]⁺ 996.5388, found 996.5397.

tert-Butyl 1-((3S,4S)-1-(4-((3S,4S)-3,4-bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-1-carbonyl)-benzoyl)-4-(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidin-3-yl)-1-oxo-5,8,11-trioxa-2-azatetradecan-14-oate (28).

Compound 2³⁴ (6.0 mg, 0.0076 mmol), tert-butyl 3-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)propanoate (3.2 mg, 0.0114 mmol), HOAt (1.2 mg, 0.00912 mmol), and 2,6-lutidine (4.1 mg, 0.0380 mmol) were dissolved in anhydrous DMF (0.1 mL). Upon dissolution of the reagents (ca. 5 min) at 0 °C, EDCI•HCl (2.2 mg, 0.0114 mmol) was added in one portion at 0 °C, and the reaction mixture was stirred at the same temperature for 30 min. The reaction mixture was warmed to rt and stirred for 3 h, after which it was poured into aqueous 1 N HCl (2 mL) and EtOAc (3 mL). The aqueous phase was extracted with EtOAc (3 mL), and the combined organic phases were washed sequentially with aqueous 1 N HCl (2 mL), saturated aqueous NaHCO₃ (2 mL), and saturated aqueous NaCl (2 mL). The organic phase was dried over Na₂SO₄, filtered and concentrated. PTLC (SiO₂, 10% MeOH–CH₂Cl₂) provided 28 (6.3 mg, 79%). ¹H NMR (500 MHz, CDCl₃, mixture of rotamers) δ 7.57−7.51 (m, 4H), 7.35−6.99 (m, 19H), 3.97−3.41 (m 22H), 3.34−2.96 (m, 4H), 2.93−2.79 (m, 3H), 2.52−2.43 (m, 2H), 2.09−1.95 (m, 3H), 1.46−1.41 (m, 9H), 1.28−1.06 (m, 6H). HRMS (ESI-TOF) m/z calcd for C₆₀H₇₃N₆O₁₁ [M+H]⁺ 1053.5322, found 1053.5334.

Methyl 1-((3S,4S)-1-(4-((3S,4S)-3,4-bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-1-carbonyl)benzoyl)-4-(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidin-3-yl)-1-oxo-5,8,11-trioxa-2-azatetradecan-14-oate (29).

A solution of 2³⁴ (20 mg, 0.025 mmol), methyl 3-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)propanoate hydrochloride⁴² (10 mg, 0.0375 mmol), HOAt (4.1 mg, 0.030 mmol), and 2,6-lutidine (15 μL, 0.125 mmol) in anhydrous DMF (0.5 mL) was cooled to 0 °C and treated with EDCI•HCl (7.2 mg, 0.0375 mmol) in one portion. The reaction solution was stirred at 0 °C for 20 min, warmed to rt and stirred for another 4 h. The reaction was quenched by the addition of aqueous 1 N HCl (0.5 mL) and diluted with H₂O (2 mL). The mixture was extracted with EtOAc (4 mL × 3), and the combined organic phase was dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. Flash chromatography (SiO₂, 1–10% MeOH–CH₂Cl₂ gradient) afforded 29 (16 mg, 63%) as a white solid. ¹H NMR (CD₃OD, 400 MHz) δ 7.62 (s, 4H), 7.27−7.06 (m, 15H), 4.03−3.97 (m, 2H), 3.80−3.45 (m, 23H), 3.43−3.17 (m, 4H), 2.91−2.78 (m, 3H), 2.58−2.52 (m, 2H), 2.09−1.92 (m, 3H), 1.28−1.08 (m, 6H). HRMS (ESI-TOF) m/z calcd for C₅₇H₆₇N₆O₁₁ [M+H]⁺ 1011.4868, found 1011.4876.

(3S,4S)-1-(4-((3S,4S)-3,4-Bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-1-carbonyl)-benzoyl)-N³-(2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl)-N⁴-((1S,2R)-2-phenylcyclopropyl)-pyrrolidine-3,4-dicarboxamide (30).

A solution of 2³⁴ (20 mg, 0.025 mmol), 2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethanol (7.2 mg, 0.0375 mmol), HOAt (4.1 mg, 0.030 mmol), and 2,6-lutidine (15 μL, 0.125 mmol) in anhydrous DMF (0.5 mL) was cooled to 0 °C and treated with EDCI•HCl (7.2 mg, 0.0375 mmol) in one portion. The reaction solution was stirred at 0 °C for 20 min, warmed to rt and stirred for another 4 h. The reaction was quenched by the addition of aqueous 1 N HCl (0.5 mL) and diluted with H₂O (2 mL). The mixture was extracted with EtOAc (4 mL × 3), and the combined organic phase was dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. Flash chromatography (SiO₂, 1–10% MeOH–CH₂Cl₂ gradient) afforded 30 (19 mg, 78%) as a white solid.¹H NMR (CD₃OD, 400 MHz) δ 7.62 (s, 4H), 7.27−7.06 (m, 15H), 4.03−3.98 (m, 2H), 3.79−3.47 (m, 22H), 3.44−3.17 (m, 4H), 2.91−2.78 (m, 3H), 2.08−1.92 (m, 3H), 1.33−1.10 (m, 6H). HRMS (ESI-TOF) m/z calcd for C₅₅H₆₅N₆O₁₀ [M+H]⁺ 969.4762, found 969.4762.

1-((3S,4S)-1-(4-((3S,4S)-3,4-Bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-1-carbonyl)benzoyl)-4-(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidin-3-yl)-1-oxo-5,8,11-trioxa-2-azatetradecan-14-oic acid (31).

Compound 28 (2.2 mg, 0.00209 mmol) was dissolved in CH₂Cl₂ (0.4 mL) at rt. TFA (0.38 mL), triisopropylsilane (0.02 mL) and H₂O (0.02 mL) was added at 0 °C, and the mixture was stirred at rt for 2 h. The solvent was removed by a N₂ stream. The residual solid was suspended in MeCN and the solvent evaporated (repeated twice) to ensure complete removal of the TFA. This process was repeated with CH₂Cl₂. PTLC (SiO₂, 8% MeOH/0.05% formic acid/CH₂Cl₂) provided 31 (2.1 mg, quant). ¹H NMR (500 MHz, DMSO-d₆, mixture of rotamers) δ 8.44−8.27 (m, 3H), 8.18−8.00 (m, 1H), 7.55 (s, 4H), 7.28−7.20 (m, 6H), 7.18−7.10 (m, 6H), 7.08−7.04 (m, 3H), 3.82−3.75 (m, 2H), 3.68−3.62 (m, 2H), 3.61−3.55 (m, 3H), 3.54−3.33 (m, 13H), 3.27−3.07 (m, 6H), 2.87−2.73 (m, 3H), 2.45−2.39 (m, 2H), 1.99−1.82 (m, 3H), 1.20−1.07 (m, 6H). HRMS (ESI-TOF) m/z calcd for C₅₆H₆₅N₆O₁₁ [M+H]⁺ 997.4706, found 997.4708.

12-((3S,4S)-1-(4-((3S,4S)-3,4-Bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-1-carbonyl)benzoyl)-4-(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-3-carboxamido)-dodecyl acetate (32).

A solution of 7 (9.8 mg, 0.010 mmol) and DMAP (3.7 mg, 0.030 mmol) in anhydrous pyridine (50 μL) was treated with acetic anhydride (50 μL) dropwise at rt. The reaction mixture was stirred at 50 °C overnight, and cooled to rt. The reaction mixture was quenched by the addition of aqueous 10% citric acid (0.5 mL) and diluted with H₂O (0.5 mL). The mixture was extracted with EtOAc (1 mL × 3), and the combined organic phase was dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. PTLC (SiO₂, 10% MeOH–CH₂Cl₂) afforded 32 (8.0 mg, 78%) as a white solid. ¹H NMR (CDCl₃, 400 MHz) δ 7.54 (s, 4H), 7.34 (br, 1H), 7.28−7.08 (m, 15H), 6.80 (br, 1H), 6.73 (br, 1H), 6.63 (br, 1H), 4.06−4.02 (m, 2H), 3.85−3.61 (m, 8H), 3.33−3.08 (m, 4H), 3.05−2.81 (m, 5H), 2.06−1.95 (m, 6H), 1.63−1.40 (m, 4H), 1.34−1.05 (m, 22H). HRMS (ESI-TOF) m/z calcd for C₆₁H₇₅N₆O₈ [M+H]⁺ 1019.5646, found 1019.5661.

12-((3S,4S)-1-(4-((3S,4S)-3,4-Bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-1-carbonyl)benzoyl)-4-(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-3-carboxamido)-dodecyl 2-cyanoacetate (33).

A solution of 2-cyanoacetic acid (2.6 mg, 0.030 mmol) in anhydrous THF (0.14 mL) was treated with Et3N (8.4 μL, 0.060 mmol) and 2,4,6-trichlorobenzoyl chloride (7.8 μL, 0.050 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 2 h, before being transferred to a suspension of 7 (9.8 mg, 0.010 mmol) and DMAP (1.2 mg, 0.010 mmol) in anhydrous THF (0.20 mL) at 0 °C. The resulting reaction mixture was warmed to rt and stirred for 8 h (or overnight). The reaction mixture was quenched by the addition of aqueous 1 N HCl (0.2 mL) and diluted with H₂O (1 mL). The mixture was extracted with EtOAc (1.5 mL × 4), and the combined organic phase was dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. PTLC (SiO₂, 10% MeOH–CH₂Cl₂) afforded 33 (7.1 mg, 68%) as a white solid. ¹H NMR (CD₃OD, 400 MHz) δ 7.62 (s, 4H), 7.27−7.07 (m, 15H), 4.18−4.14 (m, 2H), 4.03−3.96 (m, 2H), 3.79−3.60 (m, 6H), 3.35−3.01 (m, 8H), 2.91−2.78 (m, 3H), 2.09−1.91 (m, 3H), 1.69−1.61 (m, 2H), 1.55−1.42 (m, 2H), 1.38−1.07 (m, 22H). HRMS (ESI-TOF) m/z calcd for C₆₂H₇₄N₇O₈ [M+H]⁺ 1044.5599, found 1044.5603.

12-((3S,4S)-1-(4-((3S,4S)-3,4-Bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-1-carbonyl)benzoyl)-4-(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-3-carboxamido)-dodecyl 2-azidoacetate (34).

A solution of 2-azidoacetic acid⁴³ (1.4 mg, 0.014 mmol) in anhydrous THF (0.14 mL) was treated with Et3N (3.8 μL, 0.027 mmol) and 2,4,6-trichlorobenzoyl chloride (3.4 μL, 0.022 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 2 h, before being transferred to a suspension of 7 (9.8 mg, 0.010 mmol) and DMAP (1.2 mg, 0.010 mmol) in anhydrous THF (0.20 mL) at 0 °C. The resulting reaction mixture was warmed to rt and stirred for 8 h (or overnight). The reaction mixture was quenched by the addition of aqueous 1 N HCl (0.2 mL) and diluted with H₂O (1 mL). The mixture was extracted with EtOAc (1.5 mL × 4), and the combined organic phase was dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. PTLC (SiO₂, 10% MeOH–CH₂Cl₂) afforded 34 (8.8 mg, 83%) as a white solid. ¹H NMR (CD₃OD, 400 MHz) δ 7.62 (s, 4H), 7.27−7.06 (m, 15H), 4.17 (t, J = 6.6 Hz, 2H), 4.03−3.96 (m, 2H), 3.93 (s, 2H), 3.79−3.60 (m, 6H), 3.35−3.01 (m, 6H), 2.91−2.77 (m, 3H), 2.08−1.91 (m, 3H), 1.68−1.61 (m, 2H), 1.55−1.42 (m, 2H), 1.37−1.07 (m, 22H). HRMS (ESI-TOF) m/z calcd for C₆₁H₇₄N₉O₈ [M+H]⁺ 1060.5660, found 1060.5658.

12-((3S,4S)-1-(4-((3S,4S)-3,4-Bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-1-carbonyl)benzoyl)-4-(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-3-carboxamido)-dodecyl 2-(((benzyloxy)carbonyl)amino)acetate (36).

A reaction solution of 7 (9.8 mg, 0.010 mmol), CbzNH-Gly-OH (2.1 mg, 0.010 mmol), DMAP (1.2 mg, 0.010 mmol), and EDCI•HCl (2.9 mg, 0.015 mmol) in anhydrous DMF (0.1 mL) and CH₂Cl₂ (0.1 mL) was stirred at 40 °C (or rt) for 8 h (or overnight). The reaction was quenched by the addition of aqueous 1 N HCl (0.1 mL) and the solution was diluted with H₂O (1 mL). The mixture was extracted with EtOAc (1.5 mL × 3), and the combined organic phase was dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. PTLC (SiO₂, 10% MeOH–CH₂Cl₂) afforded 36 (7.9 mg, 68%) as a white solid. ¹H NMR (CDCl₃, 400 MHz) δ 7.56−7.51 (m, 4H), 7.35−7.30 (m, 5H), 7.28−7.08 (m, 15H), 6.75 (br, 1H), 6.64 (br, 1H), 5.43−5.35 (m, 1H), 5.12 (s, 2H), 4.14−4.10 (m, 2H), 3.97−3.95 (m, 2H), 3.81−3.61 (m, 8H), 3.29−3.12 (m, 4H), 3.05−2.82 (m, 5H), 2.06−1.95 (m, 3H), 1.66−1.57 (m, 2H), 1.50−1.40 (m, 2H), 1.28−1.06 (m, 22H). HRMS (ESI-TOF) m/z calcd for C₆₉H₈₂N₇O₁₀ [M+H]⁺ 1168.6123, found 1168.6108.

12-((3S,4S)-1-(4-((3S,4S)-3,4-Bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-1-carbonyl)benzoyl)-4-(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-3-carboxamido)-dodecyl 2-(((2,2,2-trichloroethoxy)carbonyl)amino)acetate (37).

A reaction solution of 7 (9.8 mg, 0.010 mmol), TrocNH-Gly-OH⁴⁴ (2.5 mg, 0.010 mmol), DMAP (1.2 mg, 0.010 mmol), and EDCI•HCl (2.9 mg, 0.015 mmol) in anhydrous DMF (0.1 mL) and CH₂Cl₂ (0.1 mL) was stirred at 40 °C (or rt) for 8 h (or overnight). The reaction was quenched by the addition of 1 N HCl (0.1 mL) and the solution was diluted with H₂O (1 mL). The mixture was extracted with EtOAc (1.5 mL × 3), and the combined organic phase was dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. PTLC (SiO₂, 10% MeOH–CH₂Cl₂) afforded 37 (7.8 mg, 64%) as a white solid. ¹H NMR (CDCl₃, 400 MHz) δ 7.56−7.52 (m, 4H), 7.32 (br, 1H), 7.28−7.08 (m, 15H), 6.74 (br, 1H), 6.64 (br, 1H), 5.69−5.61 (m, 1H), 4.74 (s, 2H), 4.17−4.12 (m, 2H), 4.00−3.98 (m, 2H), 3.82−3.61 (m, 8H), 3.32−3.12 (m, 4H), 3.02−2.82 (m, 5H), 2.07−1.95 (m, 3H), 1.66−1.60 (m, 2H), 1.51−1.40 (m, 2H), 1.28−1.07 (m, 22H). HRMS (ESI-TOF) m/z calcd for C₆₄H₇₇Cl₃N₇O₁₀ [M+H]⁺ 1208.4797, found 1208.4785.

12-((3S,4S)-1-(4-((3S,4S)-3,4-Bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-1-carbonyl)benzoyl)-4-(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-3-carboxamido)-dodecyl 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetate (38).

A solution of FmocNH-Gly-OH (4.2 mg, 0.014 mmol) in anhydrous THF (0.14 mL) was treated with Et₃N (1.4 μL, 0.010 mmol) and 2,4,6-trichlorobenzoyl chloride (3.4 μL, 0.022 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 2 h, before being transferred to a suspension of 7 (9.8 mg, 0.010 mmol) and DMAP (1.2 mg, 0.010 mmol) in anhydrous THF (0.20 mL) at 0 °C. The resulting reaction mixture was warmed to rt and stirred at 40 °C (or rt) for 8 h (or overnight). The reaction was quenched by the addition of aqueous 1 N HCl (0.2 mL) and the solution was diluted with H₂O (1 mL). The mixture was extracted with EtOAc (1.5 mL × 4), and the combined organic phase was dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. PTLC (SiO₂, 10% MeOH–CH₂Cl₂) afforded 38 (9.8 mg, 78%) as a white solid. ¹H NMR (CDCl₃, 400 MHz) δ 7.76 (d, J = 7.5 Hz, 2H), 7.59 (d, J = 7.6 Hz, 2H), 7.55−7.51 (m, 4H), 7.39 (t, J = 7.5 Hz, 2H), 7.34−7.07 (m, 17H), 5.46−5.39 (m, 1H), 4.39 (d, J = 7.1 Hz, 2H), 4.23 (t, J = 7.2 Hz, 1H), 4.16−4.12 (m, 2H), 3.98−3.96 (m, 2H), 3.87−3.61 (m, 8H), 3.29−3.13 (m, 4H), 3.05−2.82 (m, 5H), 2.06−1.96 (m, 3H), 1.66−1.58 (m, 2H), 1.51−1.39 (m, 2H), 1.35−1.07 (m, 22H). HRMS (ESI-TOF) m/z calcd for C₇₆H₈₆N₇O₁₀ [M+H]⁺ 1256.6436, found 1256.6462.

12-((3S,4S)-1-(4-((3S,4S)-3,4-Bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-1-carbonyl)benzoyl)-4-(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-3-carboxamido)-dodecyl 2-((methoxycarbonyl)amino)acetate (39).

A reaction solution of 7 (9.8 mg, 0.010 mmol), N-(methoxycarbonyl)glycine (1.3 mg, 0.010 mmol), DMAP (1.2 mg, 0.010 mmol), and EDCI•HCl (2.9 mg, 0.015 mmol) in anhydrous DMF (0.1 mL) and CH₂Cl₂ (0.1 mL) was stirred at 40 °C (or rt) for 8 h (or overnight). The reaction was quenched by the addition of aqueous 1 N HCl (0.1 mL) and the solution was diluted with H₂O (1 mL). The mixture was extracted with EtOAc (1.5 mL × 3), and the combined organic phase was dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. PTLC (SiO₂, 10% MeOH–CH₂Cl₂) afforded 39 (7.8 mg, 71%) as a white solid. ¹H NMR (CDCl₃, 400 MHz) δ 7.56−7.52 (m, 4H), 7.33 (br, 1H), 7.28−7.08 (m, 15H), 6.76 (br, 1H), 6.66 (br, 1H), 5.36 (br, 1H), 4.15−4.11 (m, 2H), 3.95−3.93 (m, 2H), 3.82−3.62 (m, 11H), 3.32−3.12 (m, 4H), 3.06−2.82 (m, 5H), 2.05−1.96 (m, 3H), 1.66−1.61 (m, 2H), 1.48−1.43 (m, 2H), 1.28−1.06 (m, 22H). HRMS (ESI-TOF) m/z calcd for C₆₃H₇₈N₇O₁₀ [M+H]⁺ 1092.5810, found 1092.5787.

12-((3S,4S)-1-(4-((3S,4S)-3,4-Bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-1-carbonyl)benzoyl)-4-(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-3-carboxamido)-dodecyl 2-acetamidoacetate (40).

A solution of 2³⁴ (48 mg, 0.060 mmol), 12-bromododecan-1-amine hydrobromide⁴⁵ (31 mg, 0.090 mmol), HOAt (9.8 mg, 0.072 mmol), and 2,6-lutidine (35 μL, 0.30 mmol) in anhydrous DMF (0.6 mL) was cooled to 0 °C and treated with EDCI•HCl (17 mg, 0.090 mmol) in one portion. The reaction solution was stirred at 0 °C for 30 min, warmed to rt and stirred for another 3 h. The reaction was quenched by the addition of aqueous 1 N HCl (0.6 mL) and the solution was diluted with H₂O (3 mL). The mixture was extracted with EtOAc (6 mL × 3), and the combined organic phase was dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. PTLC (SiO₂, 1–10% MeOH–CH₂Cl₂ gradient) afforded the intermediate coupled bromide (39 mg, 62%, compound A) as a white solid. ¹H NMR (CDCl₃, 400 MHz) δ 7.53 (s, 4H), 7.43 (br, 1H), 7.35 (br, 1H), 7.27−7.06 (m, 15H), 6.82 (br, 1H), 6.74 (br, 1H), 3.84−3.59 (m, 8H), 3.51 (td, J = 6.7, 2.5 Hz, 1H), 3.39 (td, J = 6.9, 2.4 Hz, 1H), 3.33−2.93 (m, 6H), 2.92−2.80 (m, 3H), 2.06−1.94 (m, 3H), 1.86−1.70 (m, 2H), 1.49−1.04 (m, 24H). HRMS (ESI-TOF) m/z calcd for C₅₉H₇₂BrN₆O₆ [M+H]⁺ 1039.4697, found 1039.4679.

A reaction solution of this material (16 mg, 0.015 mmol), N-acetyl glycine (3.5 mg, 0.030 mmol), NaI (9.0 mg, 0.060 mmol), and K₂CO₃ (4.1 mg, 0.030 mmol) in anhydrous DMF (0.2 mL) was stirred at 60 °C for overnight. The reaction solution was cooled to rt and quenched with the addition of H₂O (1 mL). The mixture was extracted with EtOAc (1.5 mL × 3), and the combined organic phase was dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. PTLC (SiO₂, 1–10% MeOH–CH₂Cl₂ gradient) afforded 40 (10 mg, 62%) as a white solid. ¹H NMR (CDCl₃, 400 MHz) δ 7.55−7.51 (m, 4H), 7.43−7.38 (m, 1H), 7.35−7.33 (m, 1H), 7.28−7.07 (m, 15H), 6.88−6.77 (m, 1H), 6.25−6.17 (m, 1H), 4.15−4.10 (m, 2H), 4.01−3.98 (m, 2H), 3.84−3.58 (m, 8H), 3.35−3.08 (m, 4H), 3.06−2.80 (m, 5H), 2.06−1.94 (m, 6H), 1.65−1.58 (m, 2H), 1.51−1.40 (m, 2H), 1.34−1.04 (m, 22H). HRMS (ESI-TOF) m/z calcd for C₆₃H₇₈N₇O₉ [M+H]⁺ 1076.5861, found 1076.5845.

12-((3S,4S)-1-(4-((3S,4S)-3,4-Bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-1-carbonyl)benzoyl)-4-(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-3-carboxamido)-dodecyl 2-(2,2,2-trifluoroacetamido)acetate (41).

A reaction solution of 7 (19 mg, 0.020 mmol), 2-(2,2,2-trifluoroacetamido)acetic acid (3.4 mg, 0.020 mmol), DMAP (2.4 mg, 0.020 mmol), and EDCI•HCl (5.8 mg, 0.030 mmol) in anhydrous DMF (0.2 mL) and CH₂Cl₂ (0.2 mL) was stirred at 40 °C (or rt) for 8 h (or overnight). The reaction was quenched by the addition of aqueous 1 N HCl (0.1 mL) and the solution was diluted with H₂O (1 mL). The mixture was extracted with EtOAc (1.5 mL × 3), and the combined organic phase was dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. PTLC (SiO₂, 10% MeOH–CH₂Cl₂) afforded 41 (10 mg, 44%) as a white solid. ¹H NMR (CDCl₃, 400 MHz) δ 7.56−7.51 (m, 4H), 7.34 (br, 1H), 7.30 (br, 1H), 7.28−7.07 (m, 15H), 6.78 (br, 1H), 6.69 (br, 1H), 4.19−4.15 (m, 2H), 4.09−4.07 (m, 2H), 3.84−3.60 (m, 8H), 3.35−3.09 (m, 4H), 3.04−2.80 (m, 5H), 2.06−1.94 (m, 3H), 1.66−1.59 (m, 2H), 1.50−1.40 (m, 2H), 1.33−1.04 (m, 22H). ¹⁹F NMR (CDCl₃, 376 MHz) δ –75.9 (d, J = 13.9 Hz, 3F). HRMS (ESI-TOF) m/z calcd for C₆₃H₇₅F₃N₇O₉ [M+H]⁺ 1130.5578, found 1130.5551.

12-((3S,4S)-1-(4-((3S,4S)-3,4-Bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-1-carbonyl)benzoyl)-4-(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-3-carboxamido)-dodecyl 2-pivalamidoacetate (42).

A reaction solution of intermediate compound A detailed above (38 mg, 0.037 mmol), 2-pivalamidoacetic acid (12 mg, 0.073 mmol), NaI (22 mg, 0.146 mmol), and K₂CO₃ (10 mg, 0.073 mmol) in anhydrous DMF (0.3 mL) was stirred at 60 °C for overnight. The reaction solution was cooled to rt and quenched with the addition of H₂O (1 mL). The mixture was extracted with EtOAc (1.5 mL × 3), and the combined organic phase was dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. PTLC (SiO₂, 1–10% MeOH–CH₂Cl₂ gradient) afforded 42 (19 mg, 46%) as a white solid. ¹H NMR (CDCl₃, 400 MHz) δ 7.53 (s, 4H), 7.43 (br, 1H), 7.36−7.06 (m, 17H), 6.87−6.75 (m, 1H), 6.25−6.20 (m, 1H), 4.15−4.11 (m, 2H), 4.00−3.98 (m, 2H), 3.84−3.59 (m, 8H), 3.35−3.08 (m, 4H), 3.06−2.80 (m, 5H), 2.05−1.94 (m, 3H), 1.65−1.58 (m, 2H), 1.50−1.40 (m, 2H), 1.33−1.06 (m, 31H). HRMS (ESI-TOF) m/z calcd for C₆₆H₈₄N₇O₉ [M+H]⁺ 1118.6331, found 1118.6316.

12-((3S,4S)-1-(4-((3S,4S)-3,4-Bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-1-carbonyl)benzoyl)-4-(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-3-carboxamido)-dodecyl glycinate (43).

A solution of 35 (diprovocim-X, 11 mg, 0.010 mmol) in 4 N HCl/1,4-dioxane (0.50 mL) was stirred at rt for 1 h. The solvent was removed and the residue was purified by HPLC using the gradient 0.07% TFA-water/CH₃CN (40%→70%) elution system to afford 43 (11 mg, 96%) as a white solid. ¹H NMR (CD₃OD, 500 MHz) δ 7.62 (s, 4H), 7.27−7.20 (m, 5H), 7.17−7.06 (m, 10H), 4.23 (td, J = 6.7, 2.7 Hz, 2H), 4.02−3.97 (m, 2H), 3.82 (s, 2H), 3.79−3.60 (m, 6H), 3.35−3.02 (m, 6H), 2.91−2.78 (m, 3H), 2.08−1.92 (m, 3H), 1.70−1.64 (m, 2H), 1.53−1.42 (m, 2H), 1.37−1.08 (m, 22H). HRMS (ESI-TOF) m/z calcd for C₆₁H₇₆N₇O₈ [M+H]⁺ 1034.5755, found 1034.5746.

12-((3S,4S)-1-(4-((3S,4S)-3,4-Bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-1-carbonyl)benzoyl)-4-(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-3-carboxamido)-dodecyl 2-(dimethylamino)acetate (44).

A solution of N,N-dimethylglycine hydrochloride (2.0 mg, 0.014 mmol) in anhydrous THF (0.14 mL) was treated with Et₃N (3.8 μL, 0.027 mmol) and 2,4,6-trichlorobenzoyl chloride (3.4 μL, 0.022 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 2 h, before being transferred to a suspension of 7 (9.8 mg, 0.010 mmol) and DMAP (1.2 mg, 0.010 mmol) in anhydrous THF (0.20 mL) at 0 °C. The resulting reaction mixture was warmed to rt and stirred for 8 h (or overnight). The reaction mixture was quenched by the addition of aqueous 1 N HCl (0.2 mL) and diluted with H₂O (1 mL). The mixture was extracted with EtOAc (1.5 mL × 4), and the combined organic phase was dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. PTLC (SiO₂, 10% MeOH–CH₂Cl₂) afforded 44 (10 mg, 94%) as a white solid. ¹H NMR (CD₃OD, 400 MHz) δ 7.62 (s, 4H), 7.27−7.07 (m, 15H), 4.11 (td, J = 6.7, 1.7 Hz, 2H), 4.03−3.97 (m, 2H), 3.79−3.60 (m, 6H), 3.35−3.01 (m, 8H), 2.91−2.78 (m, 3H), 2.34 (s, 6H), 2.09−1.91 (m, 3H), 1.67−1.59 (m, 2H), 1.55−1.42 (m, 2H), 1.36−1.07 (m, 22H). HRMS (ESI-TOF) m/z calcd for C₆₃H₈₀N₇O₈ [M+H]⁺ 1062.6068, found 1062.6075.

12-((3S,4S)-1-(4-((3S,4S)-3,4-Bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-1-carbonyl)benzoyl)-4-(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-3-carboxamido)-dodecyl 2-hydroxyacetate (45).

A solution of 47 (10 mg, 0.0092 mmol) in TFA (0.15 mL) and anhydrous CH₂Cl₂ (0.15 mL) was stirred at rt for 8 h. The liquids were concentrated under reduced pressure. PTLC (SiO₂, 15% MeOH–CH₂Cl₂) afforded 45 (6.5 mg, 68%) as a white solid. ¹H NMR (CDCl₃, 500 MHz) δ 7.56−7.52 (m, 4H), 7.27−7.07 (m, 15H), 4.19−4.15 (m, 2H), 4.14 (s, 2H), 3.81−3.61 (m, 8H), 3.32−3.11 (m, 4H), 3.05−2.96 (m, 2H), 2.90−2.81 (m, 3H), 2.05−1.96 (m, 3H), 1.65−1.60 (m, 2H), 1.49−1.40 (m, 2H), 1.33−1.05 (m, 22H). HRMS (ESI-TOF) m/z calcd for C₆₁H₇₅N₆O₉ [M+H]⁺ 1035.5596, found 1035.5593.

12-((3S,4S)-1-(4-((3S,4S)-3,4-Bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-1-carbonyl)benzoyl)-4-(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-3-carboxamido)-dodecyl 2-(benzyloxy)acetate (46).

A solution of 2-benzyloxyacetic acid (2.3 mg, 0.014 mmol) in anhydrous THF (0.14 mL) was treated with Et₃N (3.8 μL, 0.027 mmol) and 2,4,6-trichlorobenzoyl chloride (3.4 μL, 0.022 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 2 h, before being transferred to a suspension of 7 (9.8 mg, 0.010 mmol) and DMAP (1.2 mg, 0.010 mmol) in anhydrous THF (0.20 mL) at 0 °C. The resulting reaction mixture was warmed to rt and stirred for 8 h (or overnight). The reaction mixture was quenched by the addition of aqueous 1 N HCl (0.2 mL) and diluted with H₂O (1 mL). The mixture was extracted with EtOAc (1.5 mL × 4), and the combined organic phase was dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. PTLC (SiO₂, 10% MeOH–CH₂Cl₂) afforded 46 (10 mg, 89%) as a white solid. ¹H NMR (CD₃OD, 400 MHz) δ 7.52 (s, 4H), 7.36−7.07 (m, 20H), 6.81−6.69 (m, 2H), 4.63 (s, 2H), 4.16−4.12 (m, 2H), 4.09 (s, 2H), 3.81−3.60 (m, 8H), 3.35−2.95 (m, 6H), 2.90−2.81 (m, 3H), 2.05−1.94 (m, 3H), 1.65−1.59 (m, 2H), 1.50−1.40 (m, 2H), 1.35−1.05 (m, 22H). HRMS (ESI-TOF) m/z calcd for C₆₈H₈₁N₆O₉ [M+H]⁺ 1125.6065, found 1125.6074.

12-((3S,4S)-1-(4-((3S,4S)-3,4-Bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-1-carbonyl)benzoyl)-4-(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-3-carboxamido)-dodecyl 2-(tert-butoxy)acetate (47).

A reaction solution of 7 (14.7 mg, 0.015 mmol), 2-t-butyloxyacetic acid (2.0 mg, 0.015 mmol), DMAP (1.8 mg, 0.015 mmol), and EDCI•HCl (4.4 mg, 0.023 mmol) in anhydrous DMF (0.15 mL) and CH₂Cl₂ (0.15 mL) was stirred at 40 °C (or rt) for 8 h (or overnight). The reaction was quenched by the addition of aqueous 1 N HCl (0.1 mL) and the solution was diluted with H₂O (1 mL). The mixture was extracted with EtOAc (1.5 mL × 3), and the combined organic phase was dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. PTLC (SiO₂, 10% MeOH–CH₂Cl₂) afforded 47 (10 mg, 61%) as a white solid. ¹H NMR (CDCl₃, 500 MHz) δ 7.56−7.52 (m, 4H), 7.28−7.22 (m, 4H), 7.19−7.09 (m, 11H), 4.16−4.10 (m, 2H), 4.01 (s, 2H), 3.82−3.63 (m, 8H), 3.32−3.10 (m, 4H), 3.04−2.96 (m, 2H), 2.91−2.82 (m, 3H), 2.06−1.96 (m, 3H), 1.64−1.60 (m, 2H), 1.48−1.44 (m, 2H), 1.28−1.07 (m, 31H). HRMS (ESI-TOF) m/z calcd for C₆₅H₈₃N₆O₉ [M+H]⁺ 1091.6222, found 1091.6227.

tert-Butyl (2-((12-((3S,4S)-1-(4-((3S,4S)-3,4-bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)-pyrrolidine-1-carbonyl)-benzoyl)-4-(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-3-carboxamido)dodecyl)oxy)ethyl)carbamate (48).

A solution of 2³⁴ (63 mg, 0.080 mmol), tert-butyl (2-((12-aminododecyl)oxy)ethyl)carbamate⁴¹ (41 mg, 0.12 mmol), HOAt (13 mg, 0.096 mmol), and 2,6-lutidine (47 μL, 0.40 mmol) in anhydrous DMF (1.0 mL) was cooled to 0 °C and treated with EDCI•HCl (23 mg, 0.12 mmol) in one portion. The reaction solution was stirred at 0 °C for 30 min, warmed to rt and stirred for overnight. The reaction was quenched by the addition of aqueous 1 N HCl (1 mL) and the solution was diluted with H₂O (5 mL). The mixture was extracted with EtOAc (10 mL × 3), and the combined organic phase was dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. Flash chromatography (SiO₂, 1–10% MeOH–CH₂Cl₂ gradient) afforded 48 (63 mg, 70%) as a white solid. ¹H NMR (CDCl₃, 500 MHz) δ 7.54−7.50 (m, 4H), 7.49 (br, 1H), 7.41−7.34 (m, 2H), 7.25−7.05 (m, 15H), 6.88−6.80 (m, 1H), 4.90 (br, 1H), 3.82−3.59 (m, 8H), 3.46−3.38 (m, 4H), 3.33−3.07 (m, 6H), 3.04−2.94 (m, 2H), 2.90−2.81 (m, 3H), 2.04−1.94 (m, 3H), 1.55−1.52 (m, 2H), 1.48−1.40 (m, 11H), 1.30−1.05 (m, 22H). ¹³C NMR (CDCl₃, 150 MHz) δ 171.9, 171.8, 171.3, 170.5, 170.0, 168.6, 168.5, 168.4, 156.2, 140.4, 138.1, 138.0, 137.9, 137.8, 128.6, 127.6, 127.5, 126.5, 126.4, 126.3, 79.4, 71.4, 69.8, 53.6, 52.2, 48.9, 47.9, 47.8, 47.7, 47.6, 45.8, 45.6, 45.5, 45.3, 40.6, 40.0, 32.4, 32.3, 29.9, 29.8, 29.6, 29.5, 28.6, 27.1, 26.3, 24.9, 24.8, 24.7, 16.1, 15.8. HRMS (ESI-TOF) m/z calcd for C₆₆H₈₆N₇O₉ [M+H]⁺ 1120.6487, found 1120.6506.

tert-Butyl 2-((12-((3S,4S)-1-(4-((3S,4S)-3,4-bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)-pyrrolidine-1-carbonyl)benzoyl)-4-(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-3-carboxamido)dodecyl)oxy)acetate (49).

A solution of 2³⁴ (16 mg, 0.020 mmol), tert-butyl 2-((12-aminododecyl)oxy)acetate⁴¹ (22 mg, 0.070 mmol), HOAt (3.3 mg, 0.024 mmol), and 2,6-lutidine (12 μL, 0.10 mmol) in anhydrous DMF (0.2 mL) was cooled to 0 °C and treated with EDCI•HCl (5.7 mg, 0.030 mmol) in one portion. The reaction solution was stirred at 0 °C for 30 min, warmed to rt and stirred for overnight. The reaction was quenched by the addition of aqueous 1 N HCl (0.1 mL) and the solution was diluted with H₂O (0.5 mL). The mixture was extracted with EtOAc (1 mL × 3), and the combined organic phase was dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. Flash chromatography (SiO₂, 1–10% MeOH-CH₂Cl₂ gradient) afforded 49 (7.0 mg, 32%) as a white solid. ¹H NMR (CDCl₃, 500 MHz) δ 7.56−7.52 (m, 4H), 7.28−7.08 (m, 15H), 3.95 (s, 2H), 3.84−3.61 (m, 8H), 3.49 (q, J = 6.4 Hz, 2H), 3.34−3.08 (m, 4H), 3.06−2.95 (m, 2H), 2.92−2.83 (m, 3H), 2.07−1.96 (m, 3H), 1.64−1.56 (m, 2H), 1.51−1.43 (m, 11H), 1.36−1.07 (m, 22H). HRMS (ESI-TOF) m/z calcd for C₆₅H₈₃N₆O₉ [M+H]⁺ 1091.6222, found 1091.6202.

Methyl 2-((((12-((3S,4S)-1-(4-((3S,4S)-3,4-bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)-pyrrolidine-1-carbonyl)benzoyl)-4-(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-3-carboxamido)dodecyl)oxy)carbonyl)amino)acetate (50).

A solution of 7 (9.8 mg, 0.010 mmol) in anhydrous CH2Cl2 (0.2 mL) was treated with anhydrous pyridine (3.2 μL, 0.040 mmol) and 4-nitrophenyl chloroformate (4.0 mg, 0.020 mmol) at rt. The reaction mixture was stirred at 30 °C for overnight and then diluted with EtOAc (1 mL). The organic layer was washed with aqueous 10% citric acid (1 mL) and saturated aqueous NaCl (1 mL), and dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. PTLC (SiO₂, 10% MeOH–CH₂Cl₂) afforded the activated carbonate (9.0 mg, 79%, compound B) as a white solid. ¹H NMR (CDCl₃, 500 MHz) δ 8.27 (d, J = 9.1 Hz, 2H), 7.56−7.52 (m, 4H), 7.37 (dd, J = 9.1, 1.8 Hz, 2H), 7.27−7.08 (m, 15H), 4.27 (td, J = 6.7, 2.6 Hz, 2H), 3.84−3.61 (m, 8H), 3.32−3.10 (m, 4H), 3.04−2.95 (m, 2H), 2.91−2.82 (m, 3H), 2.06−1.96 (m, 3H), 1.77−1.71 (m, 2H), 1.48−1.06 (m, 24H). HRMS (ESI-TOF) m/z calcd for C₆₆H₇₆N₇O₁₁ [M+H]⁺ 1142.5597, found 1142.5586.

A solution of this intermediate (0.01 mmol) and glycine methyl ester hydrochloride (2.5 mg, 0.020 mmol) in anhydrous CH₂Cl₂ (0.2 mL) and DMF (0.2 mL) was treated with Et₃N (2.8 μL, 0.020 mmol) at rt. The reaction mixture was stirred at 45 °C for overnight and then cooled to rt. The reaction was quenched by the addition of aqueous 1 N HCl (0.2 mL) and the solution was diluted with H₂O (1 mL). The mixture was extracted with EtOAc (1 mL × 3), and the combined organic phase was dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. Flash chromatography (SiO₂, 1–10% MeOH–CH₂Cl₂ gradient) afforded 50 (5.6 mg, 51%) as a white solid. ¹H NMR (CD₃OD, 400 MHz) δ 7.62 (s, 4H), 7.27−7.07 (m, 15H), 4.05−3.97 (m, 4H), 3.83−3.60 (m, 11H), 3.35−3.02 (m, 6H), 2.91−2.78 (m, 3H), 2.09−1.91 (m, 3H), 1.60−1.42 (m, 4H), 1.36−1.07 (m, 22H). HRMS (ESI-TOF) m/z calcd for C₆₃H₇₈N₇O₁₀ [M+H]⁺ 1092.5810, found 1092.5802

(3S,4S)-1-(4-((3S,4S)-3,4-Bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-1-carbonyl)-benzoyl)-N³-(12-((4-nitrobenzyl)oxy)dodecyl)-N⁴-((1S,2R)-2-phenylcyclopropyl)pyrrolidine-3,4-dicarboxamide (51).

A solution of 2³⁴ (32 mg, 0.040 mmol), 12-((4-nitrobenzyl)oxy)dodecan-1-amine hydrogen chloride⁴¹ (22 mg, 0.060 mmol), HOAt (6.5 mg, 0.048 mmol), and 2,6-lutidine (23 μL, 0.20 mmol) in anhydrous DMF (0.8 mL) was cooled to 0 °C and treated with EDCI•HCl (12 mg, 0.060 mmol) in one portion. The reaction solution was stirred at 0 °C for 30 min, warmed to rt and stirred for another 4 h. The reaction was quenched by the addition of aqueous 1 N HCl (1.0 mL) and the solution was diluted with H₂O (4 mL). The mixture was extracted with EtOAc (8 mL × 3), and the combined organic phase was dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. PTLC (SiO₂, 10% MeOH–CH₂Cl₂) afforded 51 (36 mg, 81%) as a white solid. ¹H NMR (CDCl₃, 500 MHz) δ 8.19 (d, J = 7.2 Hz, 2H), 7.56−7.52 (m, 4H), 7.49 (d, J = 7.8 Hz, 2H), 7.27−7.22 (m, 5H), 7.18−7.08 (m, 10H), 4.58 (s, 2H), 3.85−3.62 (m, 8H), 3.50 (td, J = 6.7, 3.1 Hz, 2H), 3.32−3.08 (m, 4H), 3.04−2.94 (m, 2H), 2.91−2.81 (m, 3H), 2.06−1.95 (m, 3H), 1.66−1.59 (m, 2H), 1.48−1.42 (m, 2H), 1.38−1.34 (m, 2H), 1.28−1.05 (m, 20H). HRMS (ESI-TOF) m/z calcd for C₆₆H₇₈N₇O₉ [M+H]⁺ 1082.6114, found 1082.6105.

(3S,4S)-N³-(12-((4-Aminobenzyl)oxy)dodecyl)-1-(4-((3S,4S)-3,4-bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-1-carbonyl)benzoyl)-N⁴-((1S,2R)-2-phenylcyclopropyl)-pyrrolidine-3,4-dicarboxamide (52).

Step 1: A solution of 2³⁴ (53 mg, 0.067 mmol), tert-butyl (4-(((12-aminododecyl)oxy)methyl)phenyl)carbamate⁴¹ (41 mg, 0.10 mmol), HOAt (11 mg, 0.080 mmol), and 2,6-lutidine (38 μL, 0.33 mmol) in anhydrous DMF (1.2 mL) was cooled to 0 °C and treated with EDCI•HCl (19 mg, 0.10 mmol) in one portion. The reaction solution was stirred at 0 °C for 30 min, warmed to rt and stirred for another 8 h. The reaction was quenched by the addition of aqueous 1 N HCl (2.0 mL) and the solution diluted with H₂O (8 mL). The mixture was extracted with EtOAc (16 mL × 3), and the combined organic phase was dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. PTLC (SiO₂, 10% MeOH–CH₂Cl₂) afforded the NHBoc derivative of 52 (51 mg, 64%) as a white solid. ¹H NMR (CDCl₃, 500 MHz) δ 7.55−7.51 (m, 4H), 7.32 (d, J = 8.2 Hz, 2H), 7.27−7.06 (m, 17H), 4.42 (s, 2H), 3.83−3.60 (m, 8H), 3.41 (td, J = 6.6, 3.2 Hz, 2H), 3.32−3.08 (m, 4H), 3.04−2.93 (m, 2H), 2.91−2.82 (m, 3H), 2.05−1.95 (m, 3H), 1.60−1.53 (m, 2H), 1.51−1.41 (m, 11H), 1.32−1.07 (m, 22H). HRMS (ESI-TOF) m/z calcd for C₇₁H₈₈N₇O₉ [M+H]⁺ 1182.6638, found 1182.6638. Step 2: A solution of this material (24 mg, 0.020 mmol) in 4 N HCl/1,4-dioxane (0.30 mL) was stirred at rt for 1 h. The solvent was removed and the residue was purified by PTLC (SiO₂, 10% MeOH–CH₂Cl₂) afforded 52 (10 mg, 46%) as a white solid. ¹H NMR (CDCl₃, 500 MHz) δ 7.53 (s, 4H), 7.27−7.05 (m, 17H), 6.72 (s, 2H), 4.37 (s, 2H), 3.84−3.61 (m, 8H), 3.43−3.38 (m, 2H), 3.32−3.00 (m, 6H), 2.91−2.80 (m, 3H), 2.06−1.95 (m, 3H), 1.60−1.53 (m, 2H), 1.50−1.40 (m, 2H), 1.33−1.07 (m, 22H). HRMS (ESI-TOF) m/z calcd for C₆₆H₈₀N₇O₇ [M+H]⁺ 1082.6114, found 1082.6116.

12-((3S,4S)-1-(4-((3S,4S)-3,4-Bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-1-carbonyl)benzoyl)-4-(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-3-carboxamido)-dodecyl 2-(methyl(prop-2-yn-1-yl)amino)acetate (53).

A solution of 2-(methyl(prop-2-yn-1-yl)amino)trifluoroacetic acid^46,47 (3.4 mg, 0.014 mmol) in anhydrous THF (0.14 mL) was treated with Et3N (3.8 μL, 0.027 mmol) and 2,4,6-trichlorobenzoyl chloride (3.4 μL, 0.022 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 2 h, before being transferred to a suspension of 7 (9.8 mg, 0.010 mmol) and DMAP (1.2 mg, 0.010 mmol) in anhydrous THF (0.20 mL) at 0 °C. The resulting reaction mixture was warmed to rt and stirred for 8 h (or overnight). The reaction was quenched by the addition of aqueous 1 N HCl (0.2 mL) and the solution was diluted with H₂O (1 mL). The mixture was extracted with EtOAc (1.5 mL × 4), and the combined organic phase was dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. PTLC (SiO₂, 10% MeOH–CH₂Cl₂) afforded 53 (5.4 mg, 50%) as a white solid. ¹H NMR (CDCl₃, 400 MHz) δ 7.50 (m, 4H), 7.26−7.04 (m, 15H), 4.14−4.10 (m, 2H), 3.91−3.74 (m, 4H), 3.71−3.66 (m, 3H), 3.63 (s, 2H), 3.49 (s, 1H), 3.45 (s, 2H), 3.32−3.06 (m, 6H), 2.96−2.80 (m, 3H), 2.53 (s, 3H), 2.32 (s, 1H), 2.05−1.93 (m, 3H), 1.65−1.59 (m, 2H), 1.47−1.38 (m, 2H), 1.33−1.04 (m, 22H). HRMS (ESI-TOF) m/z calcd for C₆₅H₈₀N₇O₈ [M+H]⁺ 1086.6063, found 1086.6069.

12-((3S,4S)-1-(4-((3S,4S)-3,4-Bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-1-carbonyl)benzoyl)-4-(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-3-carboxamido)-dodecyl 2-(prop-2-yn-1-yloxy)acetate (54).

A solution of 2-(prop-2-yn-1-yloxy)acetic acid⁴⁸ (1.6 mg, 0.014 mmol) in anhydrous THF (0.14 mL) was treated with Et₃N (3.8 μL, 0.027 mmol) and 2,4,6-trichlorobenzoyl chloride (3.4 μL, 0.022 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 2 h, before being transferred to a suspension of 7 (9.8 mg, 0.010 mmol) and DMAP (1.2 mg, 0.010 mmol) in anhydrous THF (0.20 mL) at 0 °C. The resulting reaction mixture was warmed to rt and stirred for 8 h (or overnight). The reaction was quenched by the addition of aqueous 1 N HCl (0.2 mL) and the solution was diluted with H₂O (1 mL). The mixture was extracted with EtOAc (1.5 mL × 4), and the combined organic phase was dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. PTLC (SiO₂, 10% MeOH–CH₂Cl₂) afforded 54 (10 mg, 93%) as a white solid. ¹H NMR (CD₃OD, 400 MHz) δ 7.62 (s, 4H), 7.27−7.06 (m, 15H), 4.27 (d, J = 2.3 Hz, 2H), 4.18 (s, 2H), 4.14 (t, J = 6.7 Hz, 2H), 4.03−3.96 (m, 2H), 3.79−3.60 (m, 6H), 3.35−3.01 (m, 6H), 2.92−2.78 (m, 4H), 2.09−1.91 (m, 3H), 1.67−1.59 (m, 2H), 1.54−1.41 (m, 2H), 1.37−1.07 (m, 22H). HRMS (ESI-TOF) m/z calcd for C₆₄H₇₇N₆O₉ [M+H]⁺ 1073.5746, found 1073.5739.

12-((3S,4S)-1-(4-((3S,4S)-3,4-Bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-1-carbonyl)benzoyl)-4-(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-3-carboxamido)-dodecyl 2,2-dimethyl-4-oxo-3,8,11,14,17-pentaoxa-5-azaicosan-20-oate (55).

A solution of 2,2-dimethyl-4-oxo-3,8,11,14,17-pentaoxa-5-azaicosan-20-oic acid (5.1 mg, 0.014 mmol) in anhydrous THF (0.14 mL) was treated with Et3N (3.8 μL, 0.027 mmol) and 2,4,6-trichlorobenzoyl chloride (3.4 μL, 0.022 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 2 h, before being transferred to a suspension of 7 (9.8 mg, 0.010 mmol) and DMAP (1.2 mg, 0.010 mmol) in anhydrous THF (0.20 mL) at 0 °C. The resulting reaction mixture was warmed to rt and stirred for 8 h (or overnight). The reaction was quenched by the addition of aqueous 1 N HCl (0.2 mL) and the solution was diluted with H₂O (1 mL). The mixture was extracted with EtOAc (1.5 mL × 4), and the combined organic phase was dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. PTLC (SiO₂, 10% MeOH–CH₂Cl₂) afforded 55 (9.8 mg, 74%) as a white solid. ¹H NMR (CD₃OD, 400 MHz) δ 7.62 (s, 4H), 7.27−7.07 (m, 15H), 4.07 (t, J = 6.6 Hz, 2H), 4.03−3.96 (m, 2H), 3.79−3.60 (m, 20H), 3.50 (t, J = 5.6 Hz, 2H), 3.35−3.01 (m, 8H), 2.91−2.78 (m, 3H), 2.56 (t, J = 6.2 Hz, 2H), 2.09−1.91 (m, 3H), 1.65−1.49 (m, 4H), 1.43 (s, 9H), 1.36−1.07 (m, 22H). HRMS (ESI-TOF) m/z calcd for C₇₅H₁₀₂N₇O₁₄ [M+H]⁺ 1324.7485, found 1324.7457.

tert-Butyl 1-((3S,4S)-1-(4-((3S,4S)-3,4-bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-1carbonyl)benzoyl)-4-(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidin-3-yl)-1,14-dioxo-18,21,24-trioxa-2,15-diazaheptacosan-27-oate (56).

Step 1: A solution of 3 (36 mg, 0.036 mmol) in THF (0.40 mL), MeOH (0.10 mL), and H₂O (0.10 mL) was treated with LiOH•H₂O (6.0 mg, 0.144 mmol) at 0 °C, and stirred at rt for 2 h. The reaction solution was diluted with H₂O (1 mL) and acidified with aqueous 1 N HCl to pH 3. The mixture was extracted with EtOAc (2 mL × 3), and the combined organic phase was dried over anhydrous Na₂SO₄, filtered, and concentrated in vacuo to afford the carboxylic acid 11 (35 mg, 98%) as a white solid, which was used without further purification. Step 2: A solution of 11 (9.9 mg, 0.010 mmol), H₂N(CH₂CH₂O)₃CH₂CH₂CO₂tBu (4.2 mg, 0.015 mmol), HOAt (1.6 mg, 0.012 mmol), and 2,6-lutidine (5.8 μL, 0.050 mmol) in anhydrous DMF (0.2 mL) was cooled to 0 °C and treated with EDCI•HCl (2.9 mg, 0.015 mmol) in one portion. The reaction solution was stirred at 0 °C for 30 min, warmed to rt and stirred for another 3 h. The reaction was quenched by the addition of aqueous 1 N HCl (0.3 mL) and the solution was diluted with H₂O (1 mL). The mixture was extracted with EtOAc (2 mL × 3), and the combined organic phase was dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. PTLC (SiO₂, 10% MeOH–CH₂Cl₂) afforded 56 (9.0 mg, 72%) as a white solid. ¹H NMR (CD₃OD, 400 MHz) δ 7.62 (s, 4H), 7.27−7.07 (m, 15H), 4.03−3.97 (m, 2H), 3.79−3.58 (m, 16H), 3.52 (t, J = 5.5 Hz, 2H), 3.35−3.01 (m, 8H), 2.91−2.78 (m, 3H), 2.47 (t, J = 6.3 Hz, 2H), 2.18 (t, J = 7.6 Hz, 2H), 2.09–1.91 (m, 3H), 1.62–1.41 (m, 13H), 1.34–1.08 (m, 22H). HRMS (ESI-TOF) m/z calcd for C₇₂H₉₆N₇O₁₂ [M+H]⁺ 1250.7117, found 1250.7095.

(S)-12-((3S,4S)-1-(4-((3S,4S)-3,4-Bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-1-carbonyl)benzoyl)-4-(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-3-carboxamido)-dodecyl 2-((tert-butoxycarbonyl)amino)propanoate (57).

A solution of BocNH-L-Ala-OH (2.6 mg, 0.014 mmol) in anhydrous THF (0.14 mL) was treated with Et₃N (3.8 μL, 0.027 mmol) and 2,4,6-trichlorobenzoyl chloride (3.4 μL, 0.022 mmol) at 0°C. The reaction mixture was stirred at 0 °C for 2 h, before being transferred to a suspension of 7 (9.8 mg, 0.010 mmol) and DMAP (1.2 mg, 0.010 mmol) in anhydrous THF (0.20 mL) at 0 °C. The resulting reaction mixture was warmed to rt and stirred at 40 °C (or rt) for 8 h (or overnight). The reaction was quenched by the addition of aqueous 1 N HCl (0.2 mL) and the solution was diluted with H₂O (1 mL). The mixture was extracted with EtOAc (1.5 mL × 4), and the combined organic phase was dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. PTLC (SiO₂, 10% MeOH–CH₂Cl₂) afforded 57 (8.6 mg, 75%) as a white solid. ¹H NMR (CDCl₃, 400 MHz) δ 7.56−7.52 (m, 4H), 7.35 (br, 1H), 7.28−7.08 (m, 15H), 6.75 (br, 1H), 6.65 (br, 1H), 5.09 (br, 1H), 4.33−4.25 (m, 1H), 4.14−4.08 (m, 2H), 3.85−3.61 (m, 8H), 3.35−3.08 (m, 4H), 3.05− 2.82 (m, 5H), 2.04−1.96 (m, 3H), 1.65−1.58 (m, 2H), 1.51−1.41 (m, 11H), 1.37 (d, J = 7.2 Hz, 3H), 1.32−1.05 (m, 22H). HRMS (ESI-TOF) m/z calcd for C₆₇H₈₆N₇O₁₀ [M+H]⁺ 1148.6436, found 1148.6454.

12-((3S,4S)-1-(4-((3S,4S)-3,4-Bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-1-carbonyl)benzoyl)-4-(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-3-carboxamido)-dodecyl (tert-butoxycarbonyl)-D-alaninate (58).

A reaction solution of 7 (9.8 mg, 0.010 mmol), BocNH-D-Ala-OH (1.9 mg, 0.010 mmol), DMAP (1.2 mg, 0.010 mmol), and EDCI•HCl (2.9 mg, 0.015 mmol) in anhydrous DMF (0.1 mL) and CH₂Cl₂ (0.1 mL) was stirred at 40 °C (or rt) for 8 h (or overnight). The reaction was quenched by the addition of aqueous 1 N HCl (0.1 mL) and the solution was diluted with H₂O (1 mL). The mixture was extracted with EtOAc (1.5 mL × 3), and the combined organic phase was dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. PTLC (SiO₂, 10% MeOH–CH₂Cl₂) afforded 58 (7.7 mg, 67%) as a white solid. ¹H NMR (CDCl₃, 500 MHz) δ 7.56−7.52 (m, 4H), 7.27−7.22 (m, 5H), 7.18−7.07 (m, 10H), 5.10 (br, 1H), 4.32−4.25 (m, 1H), 4.16−4.06 (m, 2H), 3.81−3.62 (m, 8H), 3.32−3.08 (m, 4H), 3.06−2.94 (m, 2H), 2.91−2.82 (m, 3H), 2.05−1.95 (m, 3H), 1.65−1.58 (m, 2H), 1.50−1.44 (m, 11H), 1.37 (d, J = 7.1 Hz, 3H), 1.31−1.06 (m, 22H). HRMS (ESI-TOF) m/z calcd for C₆₇H₈₆N₇O₁₀ [M+H]⁺ 1148.6436, found 1148.6407.

(S)-12-((3S,4S)-1-(4-((3S,4S)-3,4-Bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-1-carbonyl)benzoyl)-4-(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-3-carboxamido)-dodecyl 2-((tert-butoxycarbonyl)amino)-3-methylbutanoate (59).

A solution of 7 (9.8 mg, 0.010 mmol), BocNH-L-Val-OH (2.2 mg, 0.010 mmol), DMAP (1.2 mg, 0.010 mmol), and EDCI•HCl (2.9 mg, 0.015 mmol) in anhydrous DMF (0.1 mL) and CH₂Cl₂ (0.1 mL) was stirred at 40 °C (or rt) for 8 h (or overnight). The reaction was quenched by the addition of aqueous 1 N HCl (0.1 mL) and the solution was diluted with H₂O (1 mL). The mixture was extracted with EtOAc (1.5 mL × 3), and the combined organic phase was dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. PTLC (SiO₂, 10% MeOH–CH₂Cl₂) afforded 59 (5.8 mg, 49%) as a white solid. ¹H NMR (CDCl₃, 400 MHz) δ 7.56−7.52 (m, 4H), 7.31 (br, 1H), 7.28−7.08 (m, 15H), 6.70 (br, 1H), 6.60 (br, 1H), 5.07−5.01 (m, 1H), 4.23−4.17 (m, 1H), 4.15−4.04 (m, 2H), 3.85−3.62 (m, 8H), 3.32−3.08 (m, 4H), 3.06−2.80 (m, 5H), 2.17−2.08 (m, 1H), 2.07−1.95 (m, 3H), 1.63−1.56 (m, 2H), 1.51−1.38 (m, 11H), 1.35−1.04 (m, 22H), 0.95 (d, J = 6.8 Hz, 3H), 0.88 (d, J = 6.8 Hz, 3H). HRMS (ESI-TOF) m/z calcd for C₆₉H₉₀N₇O₁₀ [M+H]⁺ 1176.6749, found 1176.6725.

(S)-2-(12-((3S,4S)-1-(4-((3S,4S)-3,4-Bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-1-carbonyl)benzoyl)-4-(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-3-carboxamido)-dodecyl) 1-tert-butyl pyrrolidine-1,2-dicarboxylate (60).

A reaction solution of 7 (9.8 mg, 0.010 mmol), Boc-L-Pro-OH (2.1 mg, 0.010 mmol), DMAP (1.2 mg, 0.010 mmol), and EDCI•HCl (2.9 mg, 0.015 mmol) in anhydrous DMF (0.1 mL) and CH₂Cl₂ (0.1 mL) was stirred at 40 °C (or rt) for 8 h (or overnight). The reaction was quenched by the addition of aqueous 1 N HCl (0.1 mL) and the solution was diluted with H₂O (1 mL). The mixture was extracted with EtOAc (1.5 mL × 3), and the combined organic phase was dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. PTLC (SiO₂, 10% MeOH–CH₂Cl₂) afforded 60 (7.4 mg, 63%) as a white solid. ¹H NMR (CDCl₃, 400 MHz) δ 7.56−7.51 (m, 4H), 7.33 (br, 1H), 7.28−7.08 (m, 15H), 6.74 (br, 1H), 6.65 (br, 1H), 4.32−4.27 (m, 1H), 4.21 (dd, J = 8.6, 3.8 Hz, 1H), 4.15−4.01 (m, 2H), 3.85−3.59 (m, 8H), 3.57−3.34 (m, 2H), 3.33−3.08 (m, 4H), 3.05−2.81 (m, 5H), 2.26−2.11 (m, 1H), 2.07−1.80 (m, 6H), 1.64−1.56 (m, 2H), 1.51−1.38 (m, 11H), 1.34−1.04 (m, 22H). HRMS (ESI-TOF) m/z calcd for C₆₉H₈₈N₇O₁₀ [M+H]⁺ 1174.6593, found 1174.6575.

(2S,3S)-12-((3S,4S)-1-(4-((3S,4S)-3,4-Bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-1-carbonyl)benzoyl)-4-(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-3-carboxamido)-dodecyl 2-((tert-butoxycarbonyl)amino)-3-methylpentanoate (61).

A solution of BocNH-L-IIe-OH (2.3 mg, 0.010 mmol) in anhydrous THF (0.14 mL) was treated with Et₃N (1.4 μL, 0.010 mmol) and 2,4,6-trichlorobenzoyl chloride (1.7 μL, 0.011 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 2 h, before being transferred to a suspension of 7 (9.8 mg, 0.010 mmol) and DMAP (0.61 mg, 0.0050 mmol) in anhydrous THF (0.20 mL) at 0 °C. The resulting reaction mixture was warmed to rt and stirred at 40 °C (or rt) for 8 h (or overnight). The reaction was quenched by the addition of aqueous 1 N HCl (0.2 mL) and the solution was diluted with H₂O (1 mL). The mixture was extracted with EtOAc (1.5 mL × 4), and the combined organic phase was dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. PTLC (SiO₂, 10% MeOH–CH₂Cl₂) afforded 61 (6.6 mg, 55%) as a white solid. ¹H NMR (CDCl₃, 400 MHz) δ 7.56–7.52 (m, 4H), 7.32 (br, 1H), 7.28−7.08 (m, 15H), 6.71 (br, 1H), 6.61 (br, 1H), 5.06 (d, J = 9.0 Hz, 1H), 4.28−4.21 (m, 1H), 4.16−4.03 (m, 2H), 3.85−3.62 (m, 8H), 3.33−3.08 (m, 4H), 3.05−2.81 (m, 5H), 2.04−1.95 (m, 3H), 1.90−1.79 (m, 1H), 1.65−1.57 (m, 2H), 1.52−1.37 (m, 11H), 1.34−1.05 (m, 24H), 0.92−0.88 (m, 6H). HRMS (ESI-TOF) m/z calcd for C₇₀H₉₂N₇O₁₀ [M+H]⁺ 1190.6906, found 1190.6896.

12-((3S,4S)-1-(4-((3S,4S)-3,4-Bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-1-carbonyl)benzoyl)-4-(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-3-carboxamido)-dodecyl 1-((tert-butoxycarbonyl)amino)cyclopropanecarboxylate (62).

A solution of 1-(N-Boc-amino)cyclopropanecarboxylic acid (2.8 mg, 0.014 mmol) in anhydrous THF (0.14 mL) was treated with Et3N (3.8 μL, 0.027 mmol) and 2,4,6-trichlorobenzoyl chloride (3.4 μL, 0.022 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 2 h, before being transferred to a suspension of 7 (9.8 mg, 0.010 mmol) and DMAP (1.2 mg, 0.010 mmol) in anhydrous THF (0.20 mL) at 0 °C. The resulting reaction mixture was warmed to rt and stirred at 40 °C (or rt) for 8 h (or overnight). The reaction was quenched by the addition of aqueous 1 N HCl (0.2 mL) and the solution was diluted with H₂O (1 mL). The mixture was extracted with EtOAc (1.5 mL × 4), and the combined organic phase was dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. PTLC (SiO₂, 10% MeOH–CH₂Cl₂) afforded 62 (9.0 mg, 78%) as a white solid. ¹H NMR (CDCl₃, 400 MHz) δ 7.54 (s, 4H), 7.31 (br, 1H), 7.28−7.08 (m, 15H), 4.08−4.04 (m, 2H), 3.82−3.62 (m, 8H), 3.30−3.12 (m, 4H), 3.07−2.81 (m, 5H), 2.04−1.96 (m, 3H), 1.63−1.54 (m, 2H), 1.52−1.40 (m, 11H), 1.37−1.05 (m, 26H). HRMS (ESI-TOF) m/z calcd for C₆₈H₈₆N₇O₁₀ [M+H]⁺ 1060.6436, found 1060.6449.

(S)-12-((3S,4S)-1-(4-((3S,4S)-3,4-Bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-1-carbonyl)benzoyl)-4-(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-3-carboxamido)-dodecyl 2-((tert-butoxycarbonyl)amino)-3-phenylpropanoate (63).

A solution of BocNH-L-Phe-OH (3.7 mg, 0.014 mmol) in anhydrous THF (0.14 mL) was treated with Et₃N (3.8 μL, 0.027 mmol) and 2,4,6-trichlorobenzoyl chloride (3.4 μL, 0.022 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 2 h, before being transferred to a suspension of 7 (9.8 mg, 0.010 mmol) and DMAP (1.2 mg, 0.010 mmol) in anhydrous THF (0.20 mL) at 0 °C. The resulting reaction mixture was warmed to rt and stirred at 40 °C (or rt) for 8 h (or overnight). The reaction was quenched by the addition of aqueous 1 N HCl (0.2 mL) and the solution was diluted with H₂O (1 mL). The mixture was extracted with EtOAc (1.5 mL × 4), and the combined organic phase was dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. PTLC (SiO₂, 10% MeOH–CH₂Cl₂) afforded 63 (9.8 mg, 80%) as a white solid. ¹H NMR (CDCl₃, 400 MHz) δ 7.56−7.52 (m, 4H), 7.33 (br, 1H), 7.29−7.08 (m, 20H), 6.72 (br, 1H), 6.61 (br, 1H), 5.03−4.97 (m, 1H), 4.59−4.52 (m, 1H), 4.09−4.05 (m, 2H), 3.85−3.62 (m, 8H), 3.33−3.16 (m, 4H), 3.15−2.81 (m, 7H), 2.06−1.95 (m, 3H), 1.61−1.53 (m, 2H), 1.51−1.35 (m, 11H), 1.31−1.05 (m, 22H). HRMS (ESI-TOF) m/z calcd for C₇₃H₉₀N₇O₁₀ [M+H]⁺ 1224.6749, found 1224.6765.

(S)-12-((3S,4S)-1-(4-((3S,4S)-3,4-Bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-1-carbonyl)benzoyl)-4-(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-3-carboxamido)-dodecyl 3-(benzyloxy)-2-((tert-butoxycarbonyl)amino)propanoate (64).

A reaction solution of 7 (9.8 mg, 0.010 mmol), BocNH-L-Ser(OBn)-OH (2.9 mg, 0.010 mmol), DMAP (1.2 mg, 0.010 mmol), and EDCI•HCl (2.9 mg, 0.015 mmol) in anhydrous DMF (0.1 mL) and CH2Cl2 (0.1 mL) was stirred at 40 °C (or rt) for 8 h (or overnight). The reaction was quenched by the addition of aqueous 1 N HCl (0.1 mL) and the solution was diluted with H₂O (1 mL). The mixture was extracted with EtOAc (1.5 mL × 3), and the combined organic phase was dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. PTLC (SiO₂, 10% MeOH–CH₂Cl₂) afforded 64 (8.5 mg, 68%) as a white solid. ¹H NMR (CDCl₃, 400 MHz) δ 7.56−7.51 (m, 4H), 7.34−7.08 (m, 20H), 6.71 (br, 1H), 6.62 (br, 1H), 5.41 (d, J = 8.8 Hz, 1H), 4.55−4.46 (m, 2H), 4.44−4.39 (m, 1H), 4.15−4.09 (m, 2H), 3.88−3.61 (m, 10H), 3.33−3.08 (m, 4H), 3.06−2.81 (m, 5H), 2.06−1.95 (m, 3H), 1.62−1.54 (m, 2H), 1.51−1.37 (m, 11H), 1.33−1.04 (m, 22H). HRMS (ESI-TOF) m/z calcd for C₇₄H₉₂N₇O₁₁ [M+H]⁺ 1254.6855, found 1254.6851.

(R)-12-((3S,4S)-1-(4-((3S,4S)-3,4-Bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-1-carbonyl)benzoyl)-4-(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-3-carboxamido)-dodecyl 3-(benzylthio)-2-((tert-butoxycarbonyl)amino)propanoate (65).

A reaction solution of 7 (9.8 mg, 0.010 mmol), BocNH-L-Cys(SBn)-OH (3.1 mg, 0.010 mmol), DMAP (1.2 mg, 0.010 mmol), and EDCI•HCl (2.9 mg, 0.015 mmol) in anhydrous DMF (0.1 mL) and CH2Cl2 (0.1 mL) was stirred at 40 °C (or rt) for 8 h (or overnight). The reaction was quenched by the addition of aqueous 1 N HCl (0.1 mL) and the solution was diluted with H₂O (1 mL). The mixture was extracted with EtOAc (1.5 mL × 3), and the combined organic phase was dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. PTLC (SiO₂, 10% MeOH–CH₂Cl₂) afforded 65 (8.7 mg, 68%) as a white solid. ¹H NMR (CDCl₃, 400 MHz) δ 7.56−7.52 (m, 4H), 7.32 (br, 1H), 7.30−7.08 (m, 20H), 6.71 (br, 1H), 6.61 (br, 1H), 5.33−5.28 (m, 1H), 4.54−4.47 (m, 1H), 4.13−4.09 (m, 2H), 3.85−3.59 (m, 10H), 3.33−3.08 (m, 4H), 3.05−2.76 (m, 7H), 2.06−1.95 (m, 3H), 1.63−1.56 (m, 2H), 1.51−1.36 (m, 11H), 1.33−1.04 (m, 22H). HRMS (ESI-TOF) m/z calcd for C₇₄H₉₂N₇O₁₀S [M+H]⁺ 1270.6626, found 1270.6611.

(S)-5-Benzyl 1-(12-((3S,4S)-1-(4-((3S,4S)-3,4-bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)-pyrrolidine-1-carbonyl)-benzoyl)-4-(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-3-carboxamido)dodecyl) 2-((tert-butoxycarbonyl)amino)pentanedioate (66).

A reaction solution of 7 (9.8 mg, 0.010 mmol), BocNH-L-Glu(OBn)-OH (3.4 mg, 0.010 mmol), DMAP (1.2 mg, 0.010 mmol), and EDCI•HCl (2.9 mg, 0.015 mmol) in anhydrous DMF (0.1 mL) and CH2Cl2 (0.1 mL) was stirred at 40 °C (or rt) for 8 h (or overnight). The reaction was quenched by the addition of aqueous 1 N HCl (0.1 mL) and diluted with H₂O (1 mL). The mixture was extracted with EtOAc (1.5 mL × 3), and the combined organic phase was dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. PTLC (SiO₂, 10% MeOH–CH₂Cl₂) afforded 66 (8.8 mg, 68%) as a white solid. ¹H NMR (CDCl₃, 400 MHz) δ 7.56−7.52 (m, 4H), 7.38−7.30 (m, 5H), 7.28−7.08 (m, 15H), 6.70 (br, 1H), 6.60 (br, 1H), 5.19−5.11 (br, 1H), 5.11 (s, 2H), 4.36−4.27 (m, 1H), 4.12−4.08 (m, 2H), 3.85−3.62 (m, 8H), 3.32−3.08 (m, 4H), 3.05−2.81 (m, 5H), 2.52−2.31 (m, 2H), 2.25−2.12 (m, 1H), 2.07−1.90 (m, 4H), 1.63−1.56 (m, 2H), 1.51−1.37 (m, 11H), 1.33−1.05 (m, 22H). HRMS (ESI-TOF) m/z calcd for C₇₆H₉₄N₇O₁₂ [M+H]⁺ 1296.6960, found 1296.6932.

(S)-12-((3S,4S)-1-(4-((3S,4S)-3,4-Bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-1-carbonyl)benzoyl)-4-(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-3-carboxamido)-dodecyl 2-((tert-butoxycarbonyl)amino)-4-(methylthio)butanoate (67).

A reaction solution of 7 (9.8 mg, 0.010 mmol), BocNH-L-Met-OH (2.5 mg, 0.010 mmol), DMAP (1.2 mg, 0.010 mmol), and EDCI•HCl (2.9 mg, 0.015 mmol) in anhydrous DMF (0.1 mL) and CH₂Cl₂ (0.1 mL) was stirred at 40 °C (or rt) for 8 h (or overnight). The reaction was quenched by the addition of aqueous 1 N HCl (0.1 mL) and the solution was diluted with H₂O (1 mL). The mixture was extracted with EtOAc (1.5 mL × 3), and the combined organic phase was dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. PTLC (SiO₂, 10% MeOH–CH₂Cl₂) afforded 67 (7.0 mg, 58%) as a white solid. ¹H NMR (CDCl₃, 400 MHz) δ 7.56−7.52 (m, 4H), 7.34 (br, 1H), 7.28−7.08 (m, 15H), 6.74 (br, 1H), 6.63 (br, 1H), 5.22−5.13 (m, 1H), 4.43−4.35 (m, 1H), 4.14−4.10 (m, 2H), 3.85−3.62 (m, 8H), 3.33−3.09 (m, 4H), 3.05−2.81 (m, 5H), 2.53 (t, J = 7.4 Hz, 2H), 2.18−1.87 (m, 8H), 1.65−1.56 (m, 2H), 1.53−1.38 (m, 11H), 1.35−1.04 (m, 22H). HRMS (ESI-TOF) m/z calcd for C₆₉H₉₀N₇O₁₀S [M+H]⁺ 1208.6470, found 1208.6461.

(S)-12-((3S,4S)-1-(4-((3S,4S)-3,4-Bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-1-carbonyl)benzoyl)-4-(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-3-carboxamido)-dodecyl 2-((tert-butoxycarbonyl)amino)-3-(1-tosyl-1H-imidazol-4-yl)propanoate (68).

A reaction solution of 7 (15 mg, 0.015 mmol), BocNH-L-His(Tos)-OH (6.1 mg, 0.015 mmol), DMAP (1.8 mg, 0.015 mmol), and EDCI•HCl (4.4 mg, 0.023 mmol) in anhydrous DMF (0.15 mL) and CH2Cl2 (0.15 mL) was stirred at 40 °C (or rt) for 8 h (or overnight). The reaction was quenched by the addition of aqueous 1 N HCl (0.1 mL) and the solution was diluted with H₂O (1 mL). The mixture was extracted with EtOAc (1.5 mL × 3), and the combined organic phase was dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. PTLC (SiO₂, 10% MeOH–CH₂Cl₂) afforded 68 (10 mg, 49%) as a white solid. ¹H NMR (CDCl₃, 400 MHz) δ 7.90 (s, 1H), 7.78 (d, J = 8.1 Hz, 2H), 7.56−7.51 (m, 4H), 7.33 (d, J = 7.7 Hz, 3H), 7.28−7.05 (m, 15H), 6.73 (br, 1H), 6.63 (br, 1H), 5.56 (d, J = 8.4 Hz, 1H), 4.55−4.48 (m, 1H), 4.02−3.99 (m, 2H), 3.85−3.62 (m, 8H), 3.35−3.08 (m, 4H), 3.06−2.81 (m, 7H), 2.42 (s, 3H), 2.05−1.95 (m, 3H), 1.57−1.36 (m, 13H), 1.30−1.05 (m, 22H). HRMS (ESI-TOF) m/z calcd for C₇₇H₉₄N₉O₁₂S [M+H]⁺ 1368.6743, found 1368.6766.

(S)-12-((3S,4S)-1-(4-((3S,4S)-3,4-Bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-1-carbonyl)benzoyl)-4-(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-3-carboxamido)-dodecyl 6-((((9H-fluoren-9-yl)methoxy)-carbonyl)amino)-2-((tert-butoxycarbonyl)amino)-hexanoate (69).

A solution of BocNH-L-Lys(Fmoc)-OH (6.6 mg, 0.014 mmol) in anhydrous THF (0.14 mL) was treated with Et3N (1.4 μL, 0.010 mmol) and 2,4,6-trichlorobenzoyl chloride (3.4 μL, 0.022 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 2 h, before being transferred to a suspension of 7 (9.8 mg, 0.010 mmol) and DMAP (1.2 mg, 0.010 mmol) in anhydrous THF (0.20 mL) at 0 °C. The resulting reaction mixture was warmed to rt and stirred at 40 °C (or rt) for 8 h (or overnight). The reaction was quenched by the addition of aqueous 1 N HCl (0.2 mL) and the solution was diluted with H₂O (1 mL). The mixture was extracted with EtOAc (1.5 mL × 4), and the combined organic phase was dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. PTLC (SiO₂, 10% MeOH–CH₂Cl₂) afforded 69 (9.9 mg, 69%) as a white solid. ¹H NMR (CDCl₃, 400 MHz) δ 7.76 (d, J = 7.5 Hz, 2H), 7.58 (d, J = 7.5 Hz, 2H), 7.53 (s, 4H), 7.41−7.07 (m, 19H), 5.12 (br, 1H), 4.94 (br, 1H), 4.48−4.33 (m, 2H), 4.31−4.17 (m, 2H), 4.13−4.06 (m, 2H), 3.81−3.61 (m, 8H), 3.33−2.94 (m, 8H), 2.92−2.80 (m, 3H), 2.06−1.94 (m, 3H), 1.91−1.69 (m, 2H), 1.67−1.57 (m, 2H), 1.52−1.35 (m, 13H), 1.31−1.04 (m, 24H). HRMS (ESI-TOF) m/z calcd for C₈₅H₁₀₃N₈O₁₂ [M+H]⁺ 1427.7695, found 1427.7743.

12-((3S,4S)-1-(4-((3S,4S)-3,4-Bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-1-carbonyl)benzoyl)-4-(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-3-carboxamido)-dodecyl 2-((tert-butoxycarbonyl)(methyl)amino)acetate (70).

A reaction solution of 7 (9.8 mg, 0.010 mmol), Boc-Sar-OH (1.9 mg, 0.010 mmol), DMAP (1.2 mg, 0.010 mmol), and EDCI•HCl (2.9 mg, 0.015 mmol) in anhydrous DMF (0.1 mL) and CH₂Cl₂ (0.1 mL) was stirred at 40 °C (or rt) for 8 h (or overnight). The reaction was quenched by the addition of aqueous 1 N HCl (0.1 mL) and the solution was diluted with H₂O (1 mL). The mixture was extracted with EtOAc (1.5 mL × 3), and the combined organic phase was dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. PTLC (SiO₂, 10% MeOH–CH₂Cl₂) afforded 70 (8.3 mg, 72%) as a white solid. ¹H NMR (CDCl₃, 400 MHz) δ 7.56−7.51 (m, 4H), 7.34 (br, 1H), 7.28−7.08 (m, 15H), 6.73 (br, 1H), 6.64 (br, 1H), 4.14−4.08 (m, 2H), 3.96 (d, J = 2.3 Hz, 1H), 3.88 (s, 1H), 3.82−3.61 (m, 8H), 3.30−3.09 (m, 4H), 3.04−2.82 (m, 8H), 2.06−1.95 (m, 3H), 1.64−1.56 (m, 2H), 1.49−1.42 (m, 11H), 1.27−1.04 (m, 22H). HRMS (ESI-TOF) m/z calcd for C₆₇H₈₆N₇O₁₀ [M+H]⁺ 1148.6436, found 1148.6406.

1-(12-((3S,4S)-1-(4-((3S,4S)-3,4-Bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-1-carbonyl)benzoyl)-4-(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-3-carboxamido)-dodecyl) 2-tert-butyl hydrazine-1,2-dicarboxylate (71).

A reaction solution of the intermediate activated carbonate prepared by treatment of 7 with 4-nitrophenyl chloroformate as detailed earlier (compound B, 19 mg, 0.017 mmol), tert-butyl carbazate (4.5 mg, 0.034 mmol), and Et₃N (4.7 μL, 0.034 mmol) in anhydrous DMF (0.2 mL) and CH₂Cl₂ (0.2 mL) was stirred at 45 °C for overnight. The reaction was quenched by the addition of aqueous 1 N HCl (0.1 mL) and the solution was diluted with H₂O (1 mL). The mixture was extracted with EtOAc (1.5 mL × 3), and the combined organic phase was dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. PTLC (SiO₂, 10% MeOH–CH₂Cl₂) afforded 71 (12 mg, 62%) as a white solid. ¹H NMR (CDCl₃, 400 MHz) δ 7.53 (s, 4H), 7.47 (br, 1H), 7.45 (br, 1H), 7.39 (br, 1H), 7.33 (br, 1H), 7.27−7.06 (m, 15H), 6.98 (br, 1H), 6.90 (br, 1H), 4.12−4.08 (m, 2H), 3.85−3.59 (m, 8H), 3.34−2.97 (m, 6H), 2.93−2.80 (m, 3H), 2.06−1.95 (m, 3H), 1.63−1.55 (m, 2H), 1.52−1.37 (m, 11H), 1.33−1.06 (m, 22H). HRMS (ESI-TOF) m/z calcd for C₆₅H₈₃N₈O₁₀ [M+H]⁺ 1135.6232, found 1135.6251.

12-((3S,4S)-1-(4-((3S,4S)-3,4-Bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-1-carbonyl)benzoyl)-4-(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-3-carboxamido)-dodecyl 2-(2-((tert-butoxycarbonyl)amino)acetamido)acetate (72).

A reaction solution of 7 (9.8 mg, 0.010 mmol), BocNH-Gly-Gly-OH (2.3 mg, 0.010 mmol), DMAP (1.2 mg, 0.010 mmol), and EDCI•HCl (2.9 mg, 0.015 mmol) in anhydrous DMF (0.1 mL) and CH₂Cl₂ (0.1 mL) was stirred at 40 °C (or rt) for 8 h (or overnight). The reaction was quenched by the addition of aqueous 1 N HCl (0.1 mL) and the solution was diluted with H₂O (1 mL). The mixture was extracted with EtOAc (1.5 mL × 3), and the combined organic phase was dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. PTLC (SiO₂, 10% MeOH–CH₂Cl₂) afforded 72 (5.4 mg, 45%) as a white solid. ¹H NMR (CDCl₃, 400 MHz) δ 7.54 (s, 4H), 7.31 (br, 1H), 7.28−7.08 (m, 15H), 6.91 (br, 1H), 6.81 (br, 1H), 6.72 (br, 1H), 5.36 (br, 1H), 4.15−4.10 (m, 2H), 4.03−3.97 (m, 2H), 3.87−3.62 (m, 10H), 3.33−3.12 (m, 4H), 3.10−2.81 (m, 5H), 2.08−1.95 (m, 3H), 1.68−1.57 (m, 4H), 1.45 (s, 9H), 1.33−1.06 (m, 22H). HRMS (ESI-TOF) m/z calcd for C₆₈H₈₇N₈O₁₁ [M+H]⁺ 1191.6494, found 1191.6470.

12-((3S,4S)-1-(4-((3S,4S)-3,4-Bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-1-carbonyl)benzoyl)-4-(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-3-carboxamido)-dodecyl 4-((tert-butoxycarbonyl)amino)benzoate (73).

A reaction solution of 7 (9.8 mg, 0.010 mmol), 4-((tert-butoxycarbonyl)amino)benzoic acid (3.6 mg, 0.015 mmol), DMAP (1.2 mg, 0.010 mmol), and EDCI•HCl (2.9 mg, 0.015 mmol) in anhydrous DMF (0.1 mL) and CH2Cl2 (0.1 mL) was stirred at 40 °C (or rt) for 8 h (or overnight). The reaction was quenched by the addition of aqueous 1 N HCl (0.1 mL) and the solution was diluted with H₂O (1 mL). The mixture was extracted with EtOAc (1.5 mL × 3), and the combined organic phase was dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. PTLC (SiO₂, 10% MeOH–CH₂Cl₂) afforded 73 (6.7 mg, 56%) as a white solid. ¹H NMR (CDCl₃, 400 MHz) δ 7.95 (dd, J = 8.9, 2.5 Hz, 2H), 7.55−7.50 (m, 4H), 7.42 (d, J = 8.5 Hz, 2H), 7.34 (br, 1H), 7.28−7.08 (m, 15H), 6.97 (br, 1H), 6.76 (br, 1H), 6.65 (br, 1H), 4.29−4.25 (m, 2H), 3.85−3.61 (m, 8H), 3.33−3.08 (m, 4H), 3.06−2.81 (m, 5H), 2.06−1.96 (m, 3H), 1.77−1.70 (m, 2H), 1.52 (s, 9H), 1.47−1.38 (m, 2H), 1.32−1.06 (m, 22H). HRMS (ESI-TOF) m/z calcd for C₇₁H₈₆N₇O₁₀ [M+H]⁺ 1196.6436, found 1196.6453.

12-((3S,4S)-1-(4-((3S,4S)-3,4-Bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-1-carbonyl)benzoyl)-4-(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-3-carboxamido)-dodecyl 4-aminobenzoate (74).

A reaction solution of 73 (9.3 mg, 0.0078 mmol) in 4 N HCl/1,4-dioxane (0.2 mL) was stirred at rt for 2 h. The reaction was quenched by the addition of H₂O (1 mL) and the solution was adjusted to pH 7 with the addition of saturated aqueous NaHCO₃. The mixture was extracted with EtOAc (1.5 mL × 3), and the combined organic phase was dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. PTLC (SiO₂, 10% MeOH–CH₂Cl₂) afforded 74 (6.3 mg, 74%) as a white solid. ¹H NMR (CD₃OD, 400 MHz) δ 7.72 (d, J = 8.4 Hz, 2H), 7.61 (s, 4H), 7.27−7.05 (m, 15H), 6.63 (d, J = 8.4 Hz, 2H), 4.21 (t, J = 6.6 Hz, 2H), 4.03−3.96 (m, 2H), 3.79−3.60 (m, 6H), 3.34−3.01 (m, 6H), 2.91−2.77 (m, 3H), 2.09−1.91 (m, 3H), 1.75−1.67 (m, 2H), 1.53−1.07 (m, 24H). HRMS (ESI-TOF) m/z calcd for C₆₆H₇₈N₇O₈ [M+H]⁺ 1096.5912, found 1096.5931.

12-((3S,4S)-1-(4-((3S,4S)-3,4-Bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-1-carbonyl)benzoyl)-4-(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-3-carboxamido)-dodecyl 4-(tert-butoxy)benzoate (76).

A reaction solution of 7 (19 mg, 0.020 mmol), 4-tert-butoxybenzoic acid (3.9 mg, 0.020 mmol), DMAP (2.4 mg, 0.020 mmol), and EDCI•HCl (5.7 mg, 0.030 mmol) in anhydrous DMF (0.2 mL) and CH₂Cl₂ (0.2 mL) was stirred at 40 °C (or rt) for 8 h (or overnight). The reaction was quenched by the addition of aqueous 1 N HCl (0.2 mL) and the solution was diluted with H₂O (2 mL). The mixture was extracted with EtOAc (3.0 mL × 3), and the combined organic phase was dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. PTLC (SiO₂, 10% MeOH–CH₂Cl₂) afforded 76 (13 mg, 56%) as a white solid. ¹H NMR (CDCl₃, 500 MHz) δ 7.95 (dd, J = 8.8, 2.1 Hz, 2H), 7.55−7.51 (m, 4H), 7.27−7.07 (m, 15H), 7.00 (dd, J = 8.7, 1.4 Hz, 2H), 4.28−4.25 (m, 2H), 3.80−3.60 (m, 8H), 3.33−3.09 (m, 4H), 3.04−2.93 (m, 2H), 2.90−2.81 (m, 3H), 2.05−1.95 (m, 3H), 1.75−1.69 (m, 2H), 1.48−1.38 (m, 11H), 1.33−1.06 (m, 22H). HRMS (ESI-TOF) m/z calcd for C₇₀H₈₅N₆O₉ [M+H]⁺ 1153.6378, found 1153.6407.

12-((3S,4S)-1-(4-((3S,4S)-3,4-Bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-1-carbonyl)benzoyl)-4-(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-3-carboxamido)-dodecyl 4-hydroxybenzoate (75).

A solution of 76 (23 mg, 0.020 mmol) in TFA (0.20 mL) and anhydrous CH₂Cl₂ (0.20 mL) was stirred at rt for 8 h. The liquids were concentrated under reduced pressure. PTLC (SiO₂, 10% MeOH–CH₂Cl₂) afforded 75 (15 mg, 68%) as a white solid. ¹H NMR (CDCl₃, 500 MHz) δ 7.85 (dd, J = 8.7, 2.5 Hz, 2H), 7.54−7.49 (m, 4H), 7.27−7.03 (m, 15H), 6.83 (d, J = 8.7 Hz, 2H), 4.29−4.25 (m, 2H), 3.82−3.56 (m, 8H), 3.35−2.95 (m, 6H), 2.90−2.81 (m, 3H), 2.05−1.93 (m, 3H), 1.73−1.68 (m, 2H), 1.45−1.05 (m, 24H). HRMS (ESI-TOF) m/z calcd for C₆₆H₇₇N₆O₉ [M+H]⁺ 1097.5752, found 1097.5784.

12-((3S,4S)-1-(4-((3S,4S)-3,4-Bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-1-carbonyl)benzoyl)-4-(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-3-carboxamido)-dodecyl tert-butyl terephthalate (77).

A reaction solution of 7 (9.8 mg, 0.010 mmol), 4-(tert-butoxycarbonyl)benzoic acid (3.3 mg, 0.015 mmol), DMAP (1.2 mg, 0.010 mmol), and EDCI•HCl (2.9 mg, 0.015 mmol) in anhydrous DMF (0.1 mL) and CH₂Cl₂ (0.1 mL) was stirred at 40 °C (or rt) for 8 h (or overnight). The reaction was quenched by the addition of aqueous 1 N HCl (0.1 mL) and the solution was diluted with H₂O (1 mL). The mixture was extracted with EtOAc (1.5 mL × 3), and the combined organic phase was dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. PTLC (SiO₂, 10% MeOH–CH₂Cl₂) afforded 77 (8.1 mg, 69%) as a white solid. ¹H NMR (CDCl₃, 400 MHz) δ 8.09−8.02 (m, 4H), 7.56−7.51 (m, 4H), 7.34 (br, 1H), 7.28−7.08 (m, 15H), 6.73 (br, 1H), 6.64 (br, 1H), 4.33−4.30 (m, 2H), 3.85−3.61 (m, 8H), 3.33−3.07 (m, 4H), 3.05−2.80 (m, 5H), 2.05−1.95 (m, 3H), 1.80−1.72 (m, 2H), 1.60 (s, 9H), 1.49−1.04 (m, 24H). HRMS (ESI-TOF) m/z calcd for C₇₁H₈₅N₆O₁₀ [M+H]⁺ 1181.6327, found 1181.6324.

4-(((12-((3S,4S)-1-(4-((3S,4S)-3,4-Bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-1-carbonyl)benzoyl)-4-(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-3-carboxamido)-dodecyl)oxy)carbonyl)benzoic acid (78).

A reaction solution of 77 (8.0 mg, 0.0068 mmol) in TFA (0.2 mL) and CH₂Cl₂ (0.2 mL) was stirred at rt for 8 h. The solvents were removed under reduced pressure. PTLC (SiO₂, 15% MeOH–CH₂Cl₂) afforded 78 (3.4 mg, 44%) as a white solid. ¹H NMR (CD₃OD, 400 MHz) δ 8.11−8.07 (m, 4H), 7.62 (s, 4H), 7.27−7.05 (m, 15H), 4.33 (td, J = 6.5, 1.2 Hz, 2H), 4.03−3.97 (m, 2H), 3.79−3.60 (m, 6H), 3.36−3.01 (m, 6H), 2.91−2.77 (m, 3H), 2.09−1.91 (m, 3H), 1.81−1.73 (m, 2H), 1.54−1.07 (m, 24H). HRMS (ESI-TOF) m/z calcd for C₆₇H₇₇N₆O₁₀ [M+H]⁺ 1125.5701, found 1125.5719.

tert-Butyl 4-((2-((12-((3S,4S)-1-(4-((3S,4S)-3,4-bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)-pyrrolidine-1-carbonyl)benzoyl)-4-(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-3-carboxamido)dodecyl)oxy)-2-oxoethyl)carbamoyl)benzoate (79).

Step 1: A solution of 35 (57 mg, 0.050 mmol) in 4 N HCl/1,4-dioxane (2.0 mL) was stirred at rt for 2 h. The solvent was removed and the residue was further dried under high vacuum to afford the amine 43 hydrochloride salt. HRMS (ESI-TOF) m/z calcd for C₆₁H₇₆N₇O₈ [M-Cl]⁺ 1034.5755, found 1034.5759. Step 2: A solution of the amine hydrochloride salt (0.010 mmol), 4-(tert-butoxycarbonyl)benzoic acid (11 mg, 0.050 mmol), DMAP (3.0 mg, 0.025 mmol), Et3N (10 μL, 0.075 mmol), and EDCI•HCl (14 mg, 0.075 mmol) in anhydrous DMF (0.25 mL) and CH₂Cl₂ (0.25 mL) was stirred at 40 °C for overnight. The reaction was quenched by the addition of H₂O (1 mL), and the mixture was extracted with EtOAc (1.5 mL × 3). The combined organic phase was dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. PTLC (SiO₂, 10% MeOH–CH₂Cl₂) afforded 79 (40 mg, 65%) as a white solid. ¹H NMR (CDCl₃, 400 MHz) δ 8.03 (dd, J = 8.4, 1.9 Hz, 2H), 7.84 (d, J = 8.2 Hz, 2H), 7.55−7.50 (m, 4H), 7.34 (br, 1H), 7.27−7.07 (m, 15H), 6.95 (br, 1H), 6.90 (br, 1H), 6.78 (br, 1H), 6.69 (br, 1H), 4.22−4.16 (m, 4H), 3.84−3.60 (m, 8H), 3.33−3.08 (m, 4H), 3.05−2.80 (m, 5H), 2.04−1.95 (m, 3H), 1.69−1.57 (m, 11H), 1.49−1.38 (m, 2H), 1.35−1.06 (m, 22H). HRMS (ESI-TOF) m/z calcd for C₇₃H₈₈N₇O₁₁ [M+H]⁺ 1238.6542, found 1238.6539.

4-((2-((12-((3S,4S)-1-(4-((3S,4S)-3,4-Bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-1-carbonyl)benzoyl)-4-(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-3-carboxamido)-dodecyl)oxy)-2-oxoethyl)carbamoyl)benzoic acid (80).

A solution of 79 (15 mg, 0.012 mmol) in TFA (0.15 mL) and anhydrous CH₂Cl₂ (0.15 mL) was stirred at rt for overnight. The liquids were concentrated under reduced pressure. PTLC (SiO₂, 15% MeOH–CH₂Cl₂) afforded 80 (9.6 mg, 68%) as a white solid. ¹H NMR (CD₃OD, 400 MHz) δ 8.10 (dd, J = 8.4, 1.6 Hz, 2H), 7.92 (d, J = 8.4 Hz, 2H), 7.62 (s, 4H), 7.27−7.05 (m, 15H), 4.16−4.13 (m, 2H), 4.11 (s, 2H), 4.03−3.97 (m, 2H), 3.80−3.60 (m, 6H), 3.35−3.02 (m, 6H), 2.91−2.77 (m, 3H), 2.09−1.91 (m, 3H), 1.67−1.59 (m, 2H), 1.54−1.40 (m, 2H), 1.36−1.07 (m, 22H). ¹³C NMR (CD₃OD, 150 MHz) δ 174.51, 174.48, 173.9, 173.85, 173.82, 173.1, 172.5, 171.5, 170.9, 169.8, 169.5, 142.24, 142.21, 142.15, 142.14, 142.11, 139.3, 138.8, 135.9, 131.0, 129.5, 128.6, 127.4, 127.3, 127.22, 127.20, 66.6, 53.4, 53.3, 50.6, 50.5, 50.4, 47.8, 47.7, 47.5, 42.9, 40.8, 40.7, 35.1, 33.6, 33.5, 33.2, 30.91, 30.90, 30.85, 30.83, 30.82, 30.77, 30.7, 30.61, 30.56, 30.50, 30.47, 30.46, 30.4, 29.8, 28.10, 28.06, 27.1, 26.3, 25.5, 25.43, 25.39, 25.3, 23.9, 16.31, 16.29, 16.26, 14.6. HRMS (ESI-TOF) m/z calcd for C₆₉H₈₀N₇O₁₁ [M+H]⁺ 1182.5916, found 1182.5912.

(E)-tert-Butyl 4-((2-((12-((3S,4S)-1-(4-((3S,4S)-3,4-bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)-pyrrolidine-1-carbonyl)benzoyl)-4-(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-3-carboxamido)dodecyl)oxy)-2-oxoethyl)amino)-4-oxobut-2-enoate (81).

Step 1: A solution of 35 (57 mg, 0.050 mmol) in 4 N HCl/1,4-dioxane (2.0 mL) was stirred at rt for 2 h. The solvent was removed and the residue was further dried under high vacuum to afford the amine 43 hydrochloride salt. Step 2: A solution of the amine hydrochloride salt (0.050 mmol), (E)-4-(tert-butoxy)-4-oxobut-2-enoic acid (8.6 mg, 0.050 mmol), DMAP (3.0 mg, 0.025 mmol), Et₃N (10 μL, 0.075 mmol), and EDCI•HCl (14 mg, 0.075 mmol) in anhydrous DMF (0.25 mL) and CH₂Cl₂ (0.25 mL) was stirred at 40 °C for overnight. The reaction was quenched by the addition of H₂O (1 mL), and the mixture was extracted with EtOAc (1.5 mL × 3). The combined organic phase was dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. PTLC (SiO₂, 10% MeOH–CH₂Cl₂) afforded 81 (33 mg, 55%) as a white solid. ¹H NMR (CDCl₃, 400 MHz) δ 7.56−7.51 (m, 4H), 7.34 (br, 1H), 7.31 (br, 1H), 7.28−7.07 (m, 15H), 6.93−6.87 (m, 2H), 6.81 (br, 1H), 6.76−6.72 (m, 2H), 4.14 (q, J = 6.4 Hz, 2H), 4.08−4.04 (m, 2H), 3.82−3.60 (m, 8H), 3.35−3.11 (m, 4H), 3.07−2.81 (m, 5H), 2.06−1.95 (m, 3H), 1.64−1.58 (m, 2H), 1.49−1.40 (m, 11H), 1.32−1.05 (m, 22H). HRMS (ESI-TOF) m/z calcd for C₆₉H₈₆N₇O₁₁ [M+H]⁺ 1188.6385, found 1188.6371.

(E)-4-((2-((12-((3S,4S)-1-(4-((3S,4S)-3,4-Bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)-pyrrolidine-1-carbonyl)benzoyl)-4-(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-3-carboxamido)dodecyl)oxy)-2-oxoethyl)amino)-4-oxobut-2-enoic acid (82).

A solution of 81 (22 mg, 0.018 mmol) in TFA (0.18 mL) and anhydrous CH₂Cl₂ (0.18 mL) was stirred at rt for overnight. The liquids were concentrated under reduced pressure. PTLC (SiO₂, 15% MeOH–CH₂Cl₂) afforded 82 (8.0 mg, 39%) as a white solid. ¹H NMR (CD₃OD, 400 MHz) δ 7.62 (s, 4H), 7.27−7.06 (m, 15H), 6.91 (d, J = 15.5 Hz, 1H), 6.76 (d, J = 15.4 Hz, 1H), 4.14−4.10 (m, 2H), 4.03−3.96 (m, 4H), 3.79−3.60 (m, 6H), 3.36−3.02 (m, 6H), 2.91−2.78 (m, 3H), 2.09−1.91 (m, 3H), 1.66−1.59 (m, 2H), 1.54−1.41 (m, 2H), 1.33−1.08 (m, 22H). ¹³C NMR (CD₃OD, 150 MHz) δ 174.53, 174.51, 173.9, 173.8, 173.1, 172.5, 171.2, 170.9, 167.53, 142.3, 142.22, 142.17, 142.15, 142.13, 139.33, 139.32, 135.1, 134.9, 129.5, 128.6, 127.4, 127.3, 127.23, 127.20, 66.6, 53.4, 53.3, 50.54, 50.51, 50.4, 49.7, 47.9, 47.7, 47.5, 42.4, 40.8, 40.7, 35.2, 33.6, 33.5, 33.2, 30.91, 30.90, 30.85, 30.82, 30.79, 30.77, 30.7, 30.61, 30.56, 30.50, 30.46, 30.45, 30.40, 29.8, 28.11, 28.07, 27.1, 26.3, 25.5, 25.43, 25.40, 25.36, 23.9, 16.3, 16.25, 16.23, 14.6. HRMS (ESI-TOF) m/z calcd for C₆₅H₇₈N₇O₁₁ [M+H]⁺ 1132.5759, found 1132.5737.

tert-Butyl 2-((2-((12-((3S,4S)-1-(4-((3S,4S)-3,4-bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)-pyrrolidine-1-carbonyl)benzoyl)-4-(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-3-carboxamido)dodecyl)oxy)-2-oxoethyl)amino)-2-oxoacetate (83).

Step 1: A solution of 35 (48 mg, 0.0423 mmol) in 4 N HCl/1,4-dioxane (1.2 mL) was stirred at rt for 1 h. The solvent was removed and the residue was further dried under high vacuum to afford the amine 43 hydrochloride salt. Step 2: A solution of the amine hydrochloride salt (0.0423 mmol) in anhydrous pyridine (0.4 mL) was treated with Et₃N (5.9 μL) and tert-butyl 2-chloro-2-oxoacetate (10 mg, 0.0634 mmol) dissolved in anhydrous CH₂Cl₂ (0.4 mL). The reaction solution was stirred at 40 °C for several hours and then quenched by the addition of H₂O (1 mL), and the mixture was extracted with EtOAc (3.0 mL × 3). The combined organic phase was dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. PTLC (SiO₂, 10% MeOH–CH₂Cl₂) afforded 83 (25 mg, 51%) as a white solid. ¹H NMR (CDCl₃, 600 MHz) δ 7.58 (br, 1H), 7.55−7.51 (m, 4H), 7.44−7.30 (m, 3H), 7.25−7.06 (m, 15H), 6.86−6.79 (m, 1H), 4.16−4.13 (m, 2H), 4.07−4.06 (m, 2H), 3.82−3.60 (m, 8H), 3.32−3.09 (m, 4H), 3.04−2.94 (m, 2H), 2.89−2.81 (m, 3H), 2.04−1.95 (m, 3H), 1.64−1.61 (m, 2H), 1.54 (s, 9H), 1.48−1.41 (m, 2H), 1.32−1.06 (m, 22H). HRMS (ESI-TOF) m/z calcd for C₆₇H₈₄N₇O₁₁ [M+H]⁺ 1162.6229, found 1162.6265.

2-((2-((12-((3S,4S)-1-(4-((3S,4S)-3,4-Bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-1-carbonyl)benzoyl)-4-(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-3-carboxamido)-dodecyl)oxy)-2-oxoethyl)amino)-2-oxoacetic acid (84).

A solution of 83 (20 mg, 0.017 mmol) in TFA (0.17 mL) and anhydrous CH₂Cl₂ (0.17 mL) was stirred at rt for overnight. The liquids were concentrated under reduced pressure. PTLC (SiO₂, 15% MeOH–CH₂Cl₂) afforded 84 (12 mg, 64%) as a white solid. ¹H NMR (CD₃OD, 600 MHz) δ 7.62 (s, 4H), 7.26−7.20 (m, 6H), 7.16−7.11 (m, 6H), 7.09−7.06 (m, 3H), 4.12−4.10 (m, 2H), 4.01−3.97 (m, 4H), 3.79−3.61 (m, 6H), 3.35−3.03 (m, 6H), 2.90−2.78 (m, 3H), 2.08−1.93 (m, 3H), 1.64−1.59 (m, 2H), 1.53−1.43 (m, 2H), 1.33−1.10 (m, 22H). ¹³C NMR (CD₃OD, 150 MHz) δ 174.5, 173.9, 173.3, 172.7, 171.3, 170.9, 142.3, 142.2, 142.1, 139.3, 129.5, 128.6, 127.4, 127.3, 127.2, 66.5, 53.3, 50.5, 50.4, 47.8, 47.7, 47.5, 42.4, 40.8, 40.7, 33.6, 33.5, 30.9, 30.8, 30.7, 30.6, 30.5, 30.4, 29.8, 28.1, 27.0, 25.5, 25.4, 25.3, 16.3, 16.2. HRMS (ESI-TOF) m/z calcd for C₆₃H₇₆N₇O₁₁ [M+H]⁺ 1106.5603, found 1106.5630.

tert-Butyl 4-((2-((12-((3S,4S)-1-(4-((3S,4S)-3,4-bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)-pyrrolidine-1-carbonyl)benzoyl)-4-(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-3-carboxamido)dodecyl)oxy)ethyl)carbamoyl)benzoate (85).

Step 1: A solution of 48 (42 mg, 0.037 mmol) in 4 N HCl/1,4-dioxane (0.8 mL) was stirred at rt for 1 h. The solvent was removed and the residue was further dried under high vacuum to afford the amine hydrochloride salt. Step 2: A solution of the amine hydrochloride salt (0.037 mmol), 4-(tert-butoxycarbonyl)benzoic acid (8.2 mg, 0.037 mmol), DMAP (2.2 mg, 0.018 mmol), Et3N (8.0 μL, 0.055 mmol), and EDCI•HCl (11 mg, 0.055 mmol) in anhydrous DMF (0.20 mL) and CH₂Cl₂ (0.20 mL) was stirred at 40 °C for overnight. The reaction was quenched by the addition of H₂O (1 mL), and the mixture was extracted with EtOAc (3.0 mL × 3). The combined organic phase was dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. PTLC (SiO₂, 10% MeOH–CH₂Cl₂) afforded 85 (32 mg, 71%) as a white solid. ¹H NMR (CDCl₃, 600 MHz) δ 8.02 (dd, J = 8.2, 3.8 Hz, 2H), 7.79 (d, J = 8.0 Hz, 2H), 7.53−7.50 (m, 4H), 7.44−7.39 (m, 2H), 7.25−7.05 (m, 15H), 6.92−6.85 (m, 1H), 6.73−6.69 (m, 1H), 3.80−3.57 (m, 12H), 3.46−3.43 (m, 2H), 3.33−3.24 (m, 2H), 3.21−3.08 (m, 2H), 3.04−2.96 (m, 2H), 2.89−2.81 (m, 3H), 2.04−1.93 (m, 3H), 1.59−1.53 (m, 11H), 1.48−1.38 (m, 2H), 1.33−1.05 (m, 22H). ¹³C NMR (CDCl₃, 150 MHz) δ 171.9, 171.8, 171.3, 170.5, 170.0, 168.6, 168.5, 168.4, 166.9, 165.1, 140.4, 138.1, 138.0, 137.8, 134.7, 129.8, 128.6, 127.6, 127.5, 127.0, 126.5, 126.4, 126.3, 81.8, 71.5, 69.1, 52.1, 49.0, 48.9, 47.8, 47.7, 47.6, 45.7, 45.6, 45.5, 45.3, 40.1, 40.0, 32.4, 32.3, 29.9, 29.7, 29.6, 29.5, 29.4, 28.3, 27.1, 26.3, 24.9, 24.8, 24.7, 16.1, 15.8. HRMS (ESI-TOF) m/z calcd for C₇₃H₉₀N₇O₁₀ [M+H]⁺ 1224.6749, found 1224.6774.

4-((2-((12-((3S,4S)-1-(4-((3S,4S)-3,4-Bis(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-1-carbonyl)benzoyl)-4-(((1S,2R)-2-phenylcyclopropyl)carbamoyl)pyrrolidine-3-carboxamido)-dodecyl)oxy)ethyl)carbamoyl)benzoic acid (86).

A solution of 85 (67 mg, 0.055 mmol) in TFA (0.55 mL) and anhydrous CH₂Cl₂ (0.55 mL) was stirred at rt for overnight. The liquids were concentrated under reduced pressure. PTLC (SiO₂, 15% MeOH–CH₂Cl₂) afforded 86 (40 mg, 62%) as a white solid. ¹H NMR (CD₃OD, 500 MHz) δ 8.07 (dd, J = 8.4, 2.6 Hz, 2H), 7.88 (d, J = 8.0 Hz, 2H), 7.61 (s, 4H), 7.26−7.04 (m, 15H), 4.02−3.97 (m, 2H), 3.78−3.68 (m, 4H), 3.65−3.55 (m, 6H), 3.47 (t, J = 6.6 Hz, 2H), 3.35−3.02 (m, 6H), 2.90−2.77 (m, 3H), 2.08−1.91 (m, 3H), 1.56−1.41 (m, 4H), 1.35−1.07 (m, 22H). ¹³C NMR (CD₃OD, 150 MHz) δ 174.5, 173.8, 173.1, 172.5, 170.9, 169.9, 169.6, 142.25, 142.22, 142.15, 142.12, 139.4, 139.3, 136.1, 130.9, 129.5, 128.6, 128.5, 127.4, 127.3, 127.2, 72.2, 70.1, 53.4, 53.3, 50.5, 50.4, 47.8, 47.7, 47.5, 41.2, 40.8, 40.7, 33.6, 33.5, 30.91, 30.87, 30.8, 30.7, 30.63, 30.57, 30.5, 28.11, 28.07, 27.4, 25.5, 25.43, 25.39, 25.3, 16.30, 16.26. HRMS (ESI-TOF) m/z calcd for C₆₉H₈₂N₇O₁₀ [M+H]⁺ 1168.6123, found 1168.6138.

Compound Assay

EC₅₀ determination:

THP-1 (American Type Culture Collection) cells, a human monocytic leukemia cell line, were differentiated by treatment with 100 nM PMA (Sigma) in Roswell Park Memorial Institute (RPMI) cell culture medium [RPMI containing 10% vol/vol FBS (Gemini Bio Products), 1% penicillin and streptomycin (Life Technologies)] for 24 h. After that, the cells were washed with PBS and cultured in fresh RPMI cell culture medium for 24 h before use in experiments. PMA-differentiated THP-1 cells were seeded onto 96-well plates at 0.5 × 10⁵ cells per well and treated with systematically varied concentrations of the candidate compounds (dissolved in DMSO, and the final assay DMSO concentration (0.1%) was kept constant in all experiments) for 4 h. Ultra-pure LPS (dissolved in H₂O, Enzo Life Sciences) was assessed alongside the compounds as a positive control and DMSO treated control was used as a negative control. Human TNF-α in the supernatants was measured by ELISA (eBioscience). This assay was used for establishing dose-response curves and measured EC₅₀’s. EC₅₀’s were calculated by Graphpad Prism. All data is the mean of duplicates and SD is within ±10% of mean.

EC₅₀ Measurement of TNF-α release from mouse macrophages:

Thioglycolate-elicited macrophages were recovered 4 d after i.p. injection of 2 mL BBL thioglycolate medium, brewer modified (4%; BD Biosciences) by peritoneal lavage with 5 mL phosphate buffered saline (PBS). The peritoneal macrophages were cultured in DMEM cell culture medium [DMEM containing 10% FBS (Gemini Bio Products), 1% penicillin and streptomycin (Life Technologies)] at 37 °C and 95% air/5% CO₂. Cells were seeded onto 96-well plates at 1 × 10⁵ cells per well and treated with systematically varied concentrations of the candidate compounds (dissolved in DMSO, and final assay DMSO concentrations (≤0.5%) were kept constant in all experiments) for 4 h. Ultra-pure LPS (dissolved in H₂O, Enzo Life Sciences) was assessed alongside the compounds as a positive control and DMSO treated controls. 1% DMSO or less does not induce TNF-α release from mouse macrophages. It has been reported that DMSO (1%) enhances LPS induced IL-1β release, but has no effect on LPS-induced TNF-α and IL-6 release or NF-κB activation. See: Xing, L.; Remick, D. G. Mechanisms of dimethyl sulfoxide augmentation of IL-1β production. J. Immunol. 2005, 174, 6195–6202. Mouse TNF-α in the supernatants was measured by ELISA kits according to the manufacturer’s instructions (eBioscience). EC50’s were calculated by Graphpad Prism. All data is mean of duplicates and SD is within ±10% of mean. Mouse cells were from wild type C57BL/6J mice.

Immunization and In Vivo Assays of CTL Activity, Antibody Responses, and Tumor Growth

C57BL/6J mice were purchased from The Jackson Laboratory. All experimental procedures using mice were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Texas Southwestern Medical Center and were conducted in accordance with institutionally approved protocols and guidelines for animal care and use. All the mice were maintained at the University of Texas Southwestern Medical Center in accordance with institutionally approved protocols. Female 8 week old C57BL/6J mice (ca. 20 g) were injected i.m. on day 0 and day 28 (Figure 9A) with vehicle alone (DMSO:Tween 80:saline = 1:1:8) (8 mice), vehicle mixed with 100 μg OVA (5 mg/kg) (8 mice), or with 100 μg OVA (5 mg/kg) further mixed with 200 μg (10 mg/kg) diprovocim-1 (1, 8 mice) or diprovocim-X (35, 5 mice) per mouse. EndoFit ovalbumin (OVA) with ≥98% purity minimum (SDS-PAGE) and endotoxin levels <1 EU/mg was purchased from Invivogen.

CTL assay:

On day 9 and day 37 (Figure 9A), naive C57BL/6J mice were killed, and splenocytes were collected. Half of the splenocytes were left unpulsed, and half were pulsed with OVA peptide (SIINFEKL) for 2 h in complete medium [RPMI containing 10% vol/vol FBS, 1% penicillin and streptomycin] at 37 °C. The unpulsed and peptide-pulsed cells were labeled, respectively, with 0.5 μM (“low”) or 5 μM (“high”) CellTrace Violet (Invitrogen) in serum-free medium for 20 min. Equal numbers (2×10⁶) of CellTrace Violet^high (OVA pulsed) and CellTrace Violet^low (unpulsed) cells were mixed together and injected intravenously into the immunized mice. After 48 h, blood from treated mice was collected and subjected to flow cytometry analysis. The numbers of remaining live CellTrace Violet^high and CellTrace Violet^low cells were determined and used to calculate the percentage of OVA peptide-pulsed target cells killed. Specific killing was defined as the ratio = CellTrace Violet^low cells/CellTrace Violet^high cells. The percentage of target cell lysis = [1 − unimmunized ratio/immunized ratio] × 100.

Antibody response assay:

On day 14 (Figure 9A), serum titers of OVA-specific IgG, IgG1, or IgG2b (SouthernBiotech) were measured by ELISA. Nunc MaxiSorp flat-bottom 96-well microplates (Thermo Fisher Scientific) were coated with 5 μg/mL soluble OVA (Invivogen) and incubated at 4°C overnight. Plates were washed four times with washing buffer (0.05% [vol/vol] Tween-20 in PBS), then blocked with 1% (vol/vol) BSA in PBS for 1 h at room temperature. Serum samples diluted in 1% (vol/vol) BSA were added to the prepared plates and incubated for 2 h. The plates were washed eight times with washing buffer and then incubated with HRP-conjugated goat anti-mouse IgG, IgG1, or IgG2b for 1 h at room temperature. Plates were washed eight times with washing buffer, then developed with SureBlue TMB Microwell Peroxidase Substrate and TMB Stop Solution (KPL). Absorbance was measured at 450 nm on a Synergy Neo2 Plate Reader (BioTek).

Tumor growth assay:

B16-OVA cells (B16F10 melanoma cells stably expressing chicken ovalbumin) were grown in DMEM containing 10% vol/vol FBS. On day 38 (Figure 9A), a total of 2×10⁵ B16-OVA cells in 100 μL PBS were injected s.c. into the right flank of the C57BL/6J mice to establish tumors. On days 41, 44, and 47, the mice were injected i.p. with 200 μg checkpoint inhibitor (anti-mPD-L1, BioXcell) in 100 μL saline. Tumors were measured with a digital caliper (Fisher) and the tumor sizes were calculated using the following formula: volume = 0.5 × length × width². Mice were sacrificed when the tumor length or width reached 2 cm.

Abbreviations Used

Abu

aminobutyric acid

DME

dimethoxyethane

DMF

dimethylformamide

DMSO

dimethylsulfoxide

DMAP

4-(dimethylamino)pyridine

EDCI

1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide

HOAt

1-hydroxy-7-azabenzotriazole

HOBt

1-hydroxybenzotriazole

IFN

interferon

IL

interleukin

LPS

lipopolysaccharide

Met

methionine NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells

OVA

ovalbumin

rt

room temperature

TLR

Toll-like receptor

TNF-α

tumor necrosis factor alpha