Mining Natural Products for Macrocycles to Drug Difficult Targets

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Mining Natural Products for Macrocycles to Drug Diﬃcult Targets

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Fabio Begnini, Vasanthanathan Poongavanam, Bjorn Over, Marie Castaldo, Stefan Geschwindner,

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Patrik Johansson, Mohit Tyagi, Christian Tyrchan, Lisa Wissler, Peter Sjo, Stefan Schiesser,*

and Jan Kihlberg *

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ABSTRACT: Lead generation for diﬃcult-to-drug targets that

have large, featureless, and highly lipophilic or highly polar and/or

ﬂexible binding sites is highly challenging. Here, we describe how

cores of macrocyclic natural products can serve as a high-quality in

silico screening library that provides leads for diﬃcult-to-drug

targets. Two iterative rounds of docking of a carefully selected set

of natural-product-derived cores led to the discovery of an

uncharged macrocyclic inhibitor of the Keap1-Nrf2 protein−

protein interaction, a particularly challenging target due to its

highly polar binding site. The inhibitor displays cellular eﬃcacy

and is well-positioned for further optimization based on the

structure of its complex with Keap1 and synthetic access. We believe that our work will spur interest in using macrocyclic cores for in

silico-based lead generation and also inspire the design of future macrocycle screening collections.

cores would then be useful for in silico screening against targets

such as PPIs, with subsequent optimization providing novel

leads. In addition, such a collection could inspire the design of

natural-product-like macrocycle screening libraries.

INTRODUCTION

■

More than half of all targets predicted to be involved in human

disease are considered to be diﬃcult to modulate with traditional

small-molecule drugs that obey Lipinski’s rule of 5 (Ro5).¹

Protein−protein interactions (PPIs), proteases, some kinases as

well as transferases and isomerases are important examples.^2,3

These diﬃcult-to-drug targets often have binding sites that are

large, featureless, highly lipophilic or highly polar, and/or

ﬂexible.²⁻⁴Finding orally bioavailable drugs that reach intra-

cellular diﬃcult-to-drug targets is a daunting task that often

requires discovery of ligands in the uncharted chemical space

beyond the Ro5 (bRo5).^2,5,6Macrocycles⁷are enriched among

oral drugs in the bRo5 space⁵because they oﬀer superior

binding to targets that have large and featureless binding sites as

compared to traditional small molecules.^2,8In addition,

macrocyclization may improve plasma stability, cell perme-

ability, and oral absorption.⁹However, macrocycles are often

under-represented in screening collections, limiting their use for

lead generation. For instance, the AstraZeneca collection

contains less than 17 000 macrocycles out of a total of more

than 3.8 million compounds.

Despite a growing number of bRo5 compounds entering

clinical trials, lead generation for diﬃcult-to-drug targets has not

been systematically investigated and is challenging as the

chemical space expands dramatically with increasing molecular

size.¹⁰Natural products are an important source of drugs,¹¹and

we hypothesized that the cores of macrocycles may be seen as

nature’s privileged substructures and could play a similar role in

lead generation for diﬃcult-to-drug targets as fragments do for

traditional targets.^12,13A collection of lead-like macrocyclic

Here, we report how two sets of macrocyclic cores that may be

used to discover leads for diﬃcult-to-drug targets were

generated by a comprehensive investigation of macrocyclic

natural product chemical space. Docking of the smaller and

more lead-like set into the highly charged binding site for Nrf2

on Keap1 identiﬁed the core of cyclothialidine (1) as a potential

inhibitor of Keap1. A second round of docking studies using

structurally simpliﬁed analogues of the core, followed by

synthesis, identiﬁed a weak inhibitor of the Keap1-Nrf2 PPI.

Optimization of the hit via synthesis of an additional nine

compounds led to an inhibitor (14), which has a potency in the

low μM range and displays cellular activity. The crystal structure

of the complex of Keap1 and 14 reveals how the uncharged

macrocycle 14 binds to the charged binding site of Keap1 and

provides a platform for its further optimization.

Received: September 8, 2020

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Figure 1. Mining the Dictionary of Natural Products for macrocycle cores. (a) Principal component analysis comparing the chemical space of all

compounds in the Dictionary of Natural Products¹⁴(white circles) to that of orally and parenterally administered drugs from DrugBank¹⁶(red and

blue circles, respectively). The ﬁrst two principal components explain 90% of the variance in the data set (R²= 0.90, Q²= 0.70). The positions of the

core of cyclothialidine (1, yellow circle) and macrocycle cores 2−5 (circles in cyan) are indicated. The contribution of descriptors used to characterize

the drugs to each of the principal components is indicated by their position on each of the axes. Descriptors: cLogP, calculated lipophilicity; HBA,

hydrogen-bond acceptor; HBD, hydrogen-bond donor; MW, molecular weight; NRotB, number of rotatable bonds; TPSA, topological polar surface

area. (b) Funnel describing how the Dictionary of Natural Products was ﬁltered and clustered to give one large set of 217 macrocycle cores and one

smaller set of 41 lead-like cores. The identiﬁcation of the core of cyclothialidine (1) by docking of the lead-like cores into the binding site of Keap1 is

also indicated. (c) Structures of the ﬁve top-ranked macrocycles obtained by docking into Keap1. The bonds of the docked cores are in black and their

heteroatoms are colored, while side chains that were pruned from the original natural product have bonds and heteroatoms in gray. (d) Pose obtained

from docking of the core of cyclothialidine (1) into the binding site of Keap1 (PDB ID: 4IQK), in which the substituted phenylene group protrudes

into the Kelch channel. Key residues in the Keap1 binding site are highlighted.

heavy atoms or functional groups connected to the macrocycle

using the ﬁrst two steps of the fragment generation algorithm

reported previously.¹²In this way, fragmentation of the

macrocycle ring was avoided. The resulting extended Murcko

scaﬀolds,¹⁵herein denoted macrocycle cores, were ﬁltered to

contain at least a total of three oxygen and nitrogen atoms and

<15 rotatable bonds, thereby removing highly lipophilic and/or

ﬂexible cores unlikely to be developable into drugs. Close to

2200 structurally diﬀerent cores were identiﬁed in this

macrocycle collection, which were clustered into a set of 217

representative cores. Cores that contained a few remaining

reactive groups (i.e., disulﬁdes, peroxides, and alkyl halides), had

an overwhelming synthetic complexity, or that were oligomeric

(oligopeptides and oligosaccharides) were removed by visual

inspection. This provided a smaller set of 41 more lead-like cores

that to a large extent adhered to Lipinski’s rule of 5 (Table S1

and Figure S3). As molecular weight and complexity may be

expected to increase during optimization, they were judged to be

suitable starting points for development into cell-permeable

inhibitors of diﬃcult-to-drug targets. This set of cores originates

from 35 natural products for which the absolute stereochemistry

RESULTS

■

Mining the Dictionary of Natural Products. The

Dictionary of Natural Products (DNP)¹⁴contains >150 000

compounds that cover a vast chemical space. This space includes

that of approved oral and parenteral drugs but also extends far

beyond (Figures 1a and S1). Therefore, we considered the DNP

as an attractive source for the identiﬁcation of macrocyclic cores

as promising starting points for diﬃcult-to-drug targets. First,

duplicates and natural products containing known toxicophores

and/or very reactive groups were removed from the DNP using

the HTS ﬁlter implemented in Pipeline Pilot,¹²which removes

several functionalities including acyl halides, anhydrides, diazo

groups, and hydrazides. Then, the remaining compounds were

ﬁltered to contain at least one macrocyclic ring, not more than

ten smaller rings, and to have a molecular weight (MW) <2500

Da (Figures 1b and S2). This removed all nonmacrocycles and

large macrocycles, e.g., of polysaccharidic or polypeptidic

nature, which are unlikely to reach intracellular targets and

provided a data set of 3657 macrocycles. The side chains of this

set were pruned to attachment points consisting of at least two

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has been reported and from three natural products for which

only the relative stereochemistry is known, which were included

as enantiomeric pairs. The two sets of cores constitute unique in

silico collections that can be used in eﬀorts to ﬁnd novel starting

points in drug discovery (Supporting Excel Sheet). The

structural complexity varies between the cores in the two sets;

some can be readily synthesized, while simpliﬁed analogues may

be more attractive starting points for other macrocycles.

Keap1-Nrf2 PPI (Table S4). No synthetic routes have been

reported for 3 and 4, the routes to 2 and 5 are long,^27,28while a

route suitable for synthesis of analogues has been reported for

the core of 1.²⁹Overall, these considerations revealed the

cyclothialidine core as the most promising starting point for

discovery of a novel Keap1 inhibitor.

Induced-ﬁt docking suggested that simpliﬁed analogues of

cyclothialidine core, such as stereoisomers 6−9 where the

hydroxyl and methyl groups of the phenylene group and the

hydroxyl of the proline have been removed (Figure 2a), would

also bind to Keap1 (Figure S6). In addition, the docking also

indicated that 8 and 9 were more likely to bind stronger than 6

and 7. Satisfactorily, 9 was found to inhibit the binding of Keap1

to an immobilized peptide derived from Nrf2 (K_D= 237 μM) in

a surface plasmon resonance (SPR)-based inhibition in solution

assay (ISA)³⁰(Figure 2a). Inversion of the stereochemistry of

the ^L-proline moiety to give 10 led to a reduced binding aﬃnity

that revealed its importance for inhibition of Keap1, while

hydroxylation of ^L-proline (11) maintained the K_D(Figures 2a

and S7). Importantly, replacement of the methyl ester with

diﬀerent amides (12−15) provided dimethylamide 14, which is

close to 2 orders of magnitude more potent than 9. Ring-opened

16 was drastically less active than 14; similarly, macrocycles ring

expanded by one and three atoms, respectively (17 and 18), also

lost binding aﬃnity, conﬁrming the critical role of the

macrocycle for inhibition of Keap1. The activity of 14 in the

ISA was conﬁrmed in a direct SPR binding assay³¹and by

isothermal titration calorimetry (ITC), which provided almost

identical aﬃnities for Keap1 (Figure 2b,c). Macrocycle 14

displayed cellular activity as it induced Nrf2 translocation into

the nucleus by inhibiting binding to Keap1 (Figure 2d).

Moreover, 14 has high aqueous solubility, low-to-moderate

permeability across Caco-2 cell monolayers, and a medium in

vitro microsomal clearance. It therefore constitutes a promising

lead compound for further optimization into an uncharged,

nonacidic Keap1 advanced lead.

Syntheses of Compounds 6−18. Compounds 6−18 were

synthesized in ﬁve to seven steps from commercially available

starting materials, using a modiﬁed version of the route reported

for cyclothialidine (Scheme 1).²⁹The synthesis of macrocycles

6−9 (Scheme 1A) was initiated with the protection of 2-

(bromomethyl)benzoic acid as a 2,2,2-trichloroethyl (Tce) ester

to give 19. Benzyl bromide 19 was used to alkylate the thiol of ^L-

and ^D-cysteine methyl ester, respectively, providing 20a and

20b, which were then coupled with ^L- and D-Boc-serine aﬀording

dipeptides 21a−d. The Tce protecting group was cleaved using

zinc powder to obtain acids 22a−d, which were subjected to an

intramolecular Mitsunobu reaction to obtain macrocycles 23a−

d. To our delight, the macrocyclization conditions proved

robust, providing satisfactory yields for all four diastereoisomers

23a−d. Removal of the Boc group using acidic conditions,

followed by coupling of the resulting free amine with Ac-^L-Pro-

OH, provided compounds 6−9.

Novel Class of Inhibitors of the Keap1-Nrf2 PPI. We

chose the PPI between Kelch-like ECH-associated protein 1

(Keap1) and nuclear factor erythroid 2-related factor 2 (Nrf2),

to evaluate our sets of macrocyclic cores. The Keap1-Nrf2

system is an important cellular response mechanism for

oxidative stress, which is involved in many chronic, age-related,

or inﬂammatory diseases.¹⁷Inhibitors of this PPI are of major

interest for drug discovery as Nrf2 controls the expression of

several cytoprotective genes but is negatively regulated through

complexation with Keap1, leading to its ubiquitination and

degradation. The Keap1-Nrf2 PPI consists of a highly polar

pocket on Keap1, which binds the peptide motif DEETGE on

Nrf2, and other negatively charged ligands, with high

aﬃnity.^18,19This renders it challenging to ﬁnd cell-permeable

and orally absorbed ligands that reach and inhibit the Keap1-

Nrf2 PPI,¹⁹⁻²¹just as for other targets that have polar binding

sites.^22,23Targeting the Keap1-Nrf2 PPI in CNS disorders is

particularly challenging.^19,20We hypothesize that macrocycles,

due to their conformational restriction, may provide an optimal

ﬁt in the polar binding site of Keap1. Thus, they may be ideal for

the discovery of novel uncharged inhibitors with improved cell

permeability and oral absorption as compared to current Keap1

inhibitors, the majority of which are acidic.^20,21

The binding site on Keap1 for Nrf2 shows a certain degree of

conformational mobility.²¹In particular, the side chain of

Arg415 adopts its conformation so that the binding site is more

open in the apo form and more closed when bound to small-

molecule inhibitors. To avoid bias toward a particular binding

site conformation when docking the macrocyclic cores, we

selected four high-resolution crystal structures of Keap1 that

showed variation in the binding site: two with a bound small-

molecule ligand (PDB ID: 4IQK²⁴and 3VNG) and two apo

structures (PDB ID: 4IFJ and 1ZGK²⁵) (Figure S4). Docking of

the 41 cores of the lead-like set into the binding site was then

performed using ﬂexible docking in Glide.²⁶The ten cores that

docked best into each Keap1 structure were identiﬁed by their

GlideScores (Table S2). Then, the cores that had docked into

three or four of the Keap1 structures were identiﬁed from the

combined top-ten lists and selected as potential hits as we

anticipated them to be more likely to bind to Keap1 than cores

that docked well into only one or two crystal structures (Table

S3). Interestingly, the resulting top-ﬁve macrocycles displayed a

large structural diversity (Figure 1c). Inspection of the docked

poses revealed that the core of cyclothialidine (1), which docked

well into all four Keap1 structures, was able to bind deep in the

binding site and reached into the Kelch channel (Figure 1d).

The cores of piperazinomycin (2), 8-ethoxy-3-oxo-1,2-dehy-

droretrorsine (3), numismine (4), and iriomoteolide 3a (5),

which docked well into only three of the Keap1 structures,

bound closer to the periphery, providing fewer opportunities for

interactions with Keap1 (Figure S5). To the best of our

knowledge, the natural product precursors of cores 1−5 have

not been reported to bind to Keap1. However, cytotoxicity has

been reported for the natural product precursor of core 5, which

may make 5 less suitable for development of an inhibitor of the

Macrocycle 23d, the key intermediate in the synthetic

sequence, was used to access compounds 10−15. After cleavage

of its Boc group, coupling with Ac-^D-Pro-OH or Ac-L-Hyp-OH

provided compounds 10 and 11, respectively (Scheme 1B). The

methyl ester of compound 23d was cleaved using Me₃SnOH, as

the use of traditional alkaline metal hydroxides led to partial

opening of the lactone ring. The resulting crude acid was directly

coupled with four diﬀerent amines to give amides 24−27

(Scheme 1C). In a similar fashion as for compounds 6−9,

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Figure 2. Characterization of cyclothialidine analogues. (a) Synthesized analogues of the cyclothialidine core that were evaluated as inhibitors of

binding of Keap1 to an immobilized peptide derived from Nrf2 by surface plasmon resonance using an inhibition in solution assay (ISA) format.

Dissociation constants, reported as mean values standard deviation, from three measurements on three distinct samples are given for each analogue.

(b) Interaction kinetic analysis of a dilution series of macrocycle 14 in a direct binding assay using immobilized Keap1 (left). Determination of the

dissociation constant (K_D) for 14 by ﬁtting of the data to a two-parametric sigmoidal equation (right). The dissociation constant was obtained from

three measurements on three distinct samples and is reported as the mean value standard deviation. (c) Determination of K_Dfor the binding of 14 to

Keap1 by isothermal titration calorimetry. The raw heat signals from the exothermic binding reaction (left) have been integrated to yield a binding

isotherm (right) from which the thermodynamic parameters were extracted (insert). The dissociation constant was obtained from three measurements

on three distinct samples and is reported as the mean value standard deviation. (d) Characterization of macrocycle 14 by calculated descriptors (MW

and TPSA), solubility in phosphate-buﬀered saline at 25 °C and pH 7.4, eﬄux-inhibited permeability across a Caco-2 cell monolayer (P_appAB + inh),

human microsomal metabolism (Cl_int), and induction of Nrf2 translocation into the nucleus (Nrf2 transl) at 256 μM. The values for solubility, cell

permeability, and human microsomal metabolism are mean values standard deviation from three measurements on three distinct samples. The Nrf2

translocation into the nucleus is the mean from two measurements on two distinct samples.

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a

Scheme 1. Syntheses of Compounds 6−18

a

Reagents and conditions: (a) 2,2,2-trichloroethanol, EDC·HCl, 4-dimethylaminopyridine (DMAP), dichloromethane (DCM), rt, 16 h; (b) H-(D/

L)-Cys-OMe·HCl, Et₃N, DMSO, rt, 2 h; (c) Boc-(D/L)-Ser-OH, EDC·HCl, MeCN, rt, 1 h; (d) Zn dust, aq. NH₄OAc, tetrahydrofuran (THF), rt, 2

h; (e) PPh₃, di-tert-butylazodicarboxylate, toluene:DMSO 95:5, rt, 4 h; (f) HCl in 1,4-dioxane, rt, 1 h, then Ac-L-Pro-OH, EDC·HCl, N,N-

diisopropylethylamine (DIPEA), dimethyl sulfoxide (DMSO), rt, 2 h; (g) HCl in 1,4-dioxane, rt, 1 h, then Ac-^D-Pro-OH, EDC·HCl, DIPEA,

DMSO, rt, 2 h; (h) HCl in 1,4-dioxane, rt, 1 h, then Ac-^L-Hyp-OH, EDC·HCl, DIPEA, DMSO, rt, 2 h; (i) Me₃SnOH, 1,2-DCE, 83 °C, 45 min,

then R₂NH, EDC·HCl, HOBt·xH₂O, N,N-dimethylformamide (DMF), rt, 2 h; (j) MeI, DIPEA, DMSO, rt, 2 h, then Boc-D-Ser-OH, EDC·HCl,

MeCN, rt, 1 h; (k) BzCl, DMAP, Et₃N, DCM, 0 °C to rt, 1 h; (l) Me₃SnOH, 1,2-DCE, 83 °C, 45 min, then Me₂N·HCl, EDC·HCl, HOBt·xH₂O,

DIPEA, DMF, rt, 2 h; (m) Boc-^D-Cys-OH, Et₃N, THF:DMF 4:1, rt, 16 h, then Me₂N·HCl, EDC·HCl, HOBt·xH₂O, DIPEA, DMF, rt, 2 h; (n)

HCl in 1,4-dioxane, rt, 1 h, then Boc-^D-Homoser-OH, EDC·HCl, DIPEA, MeCN, rt, 1 h; (o) aq. LiOH, MeOH, 35 °C, 16 h; (p) HCl in 1,4-

dioxane, rt, 1 h, then Boc-Gly-OH, EDC·HCl, MeCN, rt, 1 h. DMAP, 4-dimethylaminopyridine; EDC, N-(3-dimethylaminopropyl)-N′-

ethylcarbodiimide; HOBt, 1-hydroxybenzotriazole; Tce, 2,2,2-trichloroethyl.

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cleavage of the Boc group from 24−27 and coupling with Ac-L-

Pro-OH provided compounds 12−15.

The synthesis of ring-opened compound 16 (Scheme 1D)

started with the methylation of the sulfur of ^D-cysteine methyl

ester, followed by coupling with Boc-^D-Ser to provide dipeptide

28, the hydroxyl group of which was acylated with benzoyl

chloride in the presence of DMAP leading to compound 29.

After Boc removal and coupling with Ac-^L-Pro-OH tripeptide 30

was obtained. Cleavage of the methyl ester followed by coupling

of the resulting free acid with dimethylamine aﬀorded the linear

control 16.

The preparation of the ring-expanded macrocycles 17 and 18

(Scheme 1E,F) is based on the common intermediate 31, which

was prepared by alkylation of the thiol moiety of Boc-^D-Cys with

commercially available 2-bromomethyl methyl benzoate,

followed by coupling of the crude carboxylic acid with

dimethylamine. The Boc protecting group was then cleaved,

the liberated amine was coupled with Boc-^D-homoserine to give

compound 32, followed by saponiﬁcation of the methyl ester,

which gives compounds 33. The same conditions employed for

the Mitsunobu reaction with acids 23a−d were successfully

applied to form the 13-membered lactone of 34. Finally, Boc

removal and coupling with Ac-^L-Pro-OH provided macrocycle

17. For the preparation of compound 18 (Scheme 1F), the Boc

group of 31 was cleaved followed by coupling with Boc-glycine

to obtain dipeptide 35. The newly introduced Boc group was

cleaved and the liberated amine was coupled with Boc-^D-serine

to aﬀord tripeptide 36. After cleavage of the methyl ester, the

obtained acid 37 was subjected to a Mitsunobu reaction to form

the 15-membered core of compound 38, followed by Boc

cleavage and coupling with Ac-^L-Pro-OH to give macrocycle 18.

Structure of Inhibitor 14 in Complex with Keap1. We

determined the structure of the complex of macrocycle 14 and

Keap1 at 2.4 Å resolution to understand how uncharged 14

binds in the charged and polar binding site of Keap1. The crystal

structure conﬁrms that 14 binds to the same polar binding site in

Keap1 as Nrf2 (Figure 3a, Table S5, and Figure S8). Inspection

of the structure shows that the phenylene group and parts of the

macrocycle are wedged between R415 and A556 in a fairly

hydrophobic pocket that reaches toward the Kelch channel,

conﬁrming that 14 is bound similarly to the docked poses of

analogue 9 (Figure S9). The complex is stabilized only by a few

polar interactions in addition to the cation−π interaction

between R415 and 14. The carbonyl group of Ser in 14 forms a

hydrogen bond to the side chain of S602 in Keap1, similar to that

of the main-chain carbonyl group of T80 in Nrf2.³²In addition, a

chloride ion bridges the NH of Ser in 14 with three residues in

Keap1, in a similar manner as in the complex with a highly potent

inhibitor identiﬁed by fragment-based drug discovery.³³As

physiological salt conditions were used in the SPR and ITC

assays, as well as in the cellular Nrf2 translocation assay, we

believe that formation of this complex also occurs in biologically

relevant environments. The ability of macrocycle 14 to adopt a

compact and well-deﬁned conformation in the binding site

explains why the ring-opened and ring-expanded analogues 16−

18 have a signiﬁcantly lower aﬃnity for Keap1. The solvent

exposure of part of Pro and its N-acetyl group is in line with the

fact that replacement with hydroxyproline (compound 11) does

not aﬀect the inhibitory potency.

Figure 3. Characterization of the Keap1−14 complex. (a) Crystal

structure of the complex between macrocyclic dimethylamide 14 and

Keap1 determined at 2.4 Å resolution (PDB ID: 6Z6A). Residues in

Keap1 that make key contacts with 14 are shown in black text. The

chloride ion that links R415, N414, and N382 to the NH of the serine

moiety of macrocycle 14 is shown as a green sphere. An active site water

that mediates formation of the protein−ligand complex is shown as a

red sphere. The Kelch channel (dark gray) projects down from the

phenylene group of 14. The nonclassical intramolecular hydrogen

bonds that stabilize the bound structure of 14 are indicated with dashed

orange lines. (b) Comparison of dissociation constants (K_D) with

ligand binding aﬃnities (ΔG) calculated with Prime MM-GBSA³⁴for

the binding of 9, 12, 13, 14, and 15 to Keap1 (top). Energy components

that contribute to the binding aﬃnities of 9, 12, 13, 14, and 15 in their

complexes with Keap1 calculated with Prime MM-GBSA (bottom).³⁴

Compounds 9, 13, and 15, which are marked with asterisks, may bind to

Keap1 with their OMe, NHMe, and NEt₂groups in diﬀerent

conformations, e.g., the cis- and trans-orientations about the C-

terminal amide bond of 13. Mean values for the energy components and

standard deviations are shown here; values for each conformation are

found in Tables S6 −S7. Energy components: Solv GB, generalized

Born electrostatic solvation energy; SelfCont, self-contact correction;

Covalent, covalent binding energy; Coulomb, Coulomb energy; vdW,

van der Waals energy; Lipo, lipophilic energy; Hbond, hydrogen-

bonding correction; Packing, π−π packing correction.

Ligand binding aﬃnity calculations using Prime MM-GBSA,³⁴

which is frequently used to estimate the free energy of protein−

ligand complexes,³⁵provided insights both into the underlying

reasons for the potency diﬀerences and into what forces stabilize

The C-terminal dimethylamide of 14 is well-deﬁned by the

electron density of the X-ray structure and stacks against Y572,

but inspection of the complex does not explain the large increase

in potency as compared to ester 9 and amides 12, 13, and 15.

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the complex between 14 and Keap1 (Figure 3b, Tables S6 −S7,

Figure S10). In agreement with the experimental dissociation

constants, macrocycle 14 was predicted to have the highest

aﬃnity for Keap1, with 9 and 13 being predicted as intermediate

and 12 and 15 as weak. Nonpolar van der Waals and lipophilic

interactions were found to be the main contributors to the

binding aﬃnity of 14 and to that it had a higher aﬃnity than

inhibitors 9 and 13. According to the calculations, the complex

of the inactive 12 is stabilized by stronger polar (Coulomb)

interactions, but this is oﬀset by a larger desolvation penalty,

while inactive 15 forms the weakest polar (Coulomb) and van

der Waals interactions among the ﬁve compounds investigated.

Interestingly, the distances between the two N-methyl groups of

14 and the carbonyl groups of Pro and the N-acetyl group show

that two intramolecular nonclassical hydrogen bonds³⁶are

formed between these residues (Figure 3a), an observation that

was supported by quantum mechanical calculations (see

Supporting Information, Computational Procedure 2). Non-

classical hydrogen bonds are weaker than hydrogen bonds,³⁷but

the two intramolecular bonds are likely to provide additional

conformational restriction and stabilization of the Keap1-bound

conformation of 14.

Comparison of 14 to Reported Keap1 Inhibitors. We

assembled two sets of inhibitors of the Keap1-Nrf2 PPI to

generate an overview of the diversity of the Keap1 inhibitor

space and to investigate whether macrocycle 14 occupies a

unique position in this space. The ﬁrst set, termed “PubChem”,

was obtained from the 528 unique compounds reported as active

in the PubChem Bioassay database after testing of >337 000

compounds.³⁸Removal of compounds that did not pass a pan

assay interference compounds (PAINS) ﬁlter³⁹from the actives

gave the PubChem set consisting of 375 inhibitors (Table S8). A

diﬀerent set of “validated” inhibitors was assembled from two

sources. First, nine compounds reported²⁰to show reproducible

activity in a triad of orthogonal assays after resynthesis were

included. Then, compounds bound in the binding site of Keap1

for Nrf2 were retrieved from the Protein Data Bank (PDB).

Peptides were excluded from this set, providing a “validated” set

of 35 inhibitors, including six fragments that had a MW < 205 Da

(Table S9).

The Tanimoto coeﬃcient,⁴⁰used to describe structural

diversity, was calculated from seven ﬁngerprints and revealed

that 14 had a low similarity to the compounds in both the

PubChem and the validated set (Figure 4a and S11). The

diversity of the Keap1 inhibitor space was investigated by

networklike similarity graphs derived from substructure frag-

ment ﬁngerprints⁴¹calculated for 14, the combined validated

and PubChem sets, and the validated set alone. The graph for

the combined set, just as the one for the validated set, conﬁrmed

that macrocycle 14 occupies a unique position in chemical space

with no structurally similar neighbors (cf. color and size of

spheres and the background, in Figure 4b,c). The overview of

the combined set also revealed the Keap1 inhibitor space to be

fairly diverse and that most inhibitors have few structural

neighbors, with the exception of a large cluster located in the left

center of the graph. This cluster contains a series of phthalimides

represented by the validated inhibitor²⁰V1 (cf. structure in

Figure 4c and Table S9). 1,4-Diaminonaphtalene V3, cyclic

sulfonamide V7, and diazole V8 (Figure 4c) are the most potent

reported Keap1 inhibitors and were obtained after medicinal

chemistry optimization. In contrast to 14, they all contain

carboxylic acids. In summary, both Tanimoto coeﬃcients and

the networklike similarity graphs highlight the potential of using

Figure 4. Structural similarity of inhibitor 14 to reported inhibitors of

the Keap1-Nrf2 PPI. Comparisons have been made to a set of inhibitors

retrieved from PubChem (n = 375) and to a validated set (n = 35). (a)

Histograms of Tanimoto coeﬃcients⁴⁰(Tc), calculated from seven

structural ﬁngerprints, comparing 14 to the PubChem and validated

inhibitor sets. Inhibitors that have large structural diﬀerences as

compared to 14 have Tc values close to 0. (b, c) Network-like similarity

graphs illustrating the structural diversity of the Keap1 inhibitor

landscape and structural similarities, using 14 as the reference. The

graphs were obtained after calculation of FragFp substructure fragment

ﬁngerprints⁴¹for the compounds in the combined PubChem +

validated set (panel b) and for the validated set only (panel c).

Compounds that are similar to 14 are indicated by spheres that are

similar in size and color to 14 (blue sphere). Connecting lines indicate

clusters of inhibitors that have similar structures, and the number of

similar neighbors is also highlighted with the background color (red,

low number; blue, high number). Structures of four inhibitors from the

validated set are shown together with compound 14 in panel c.

natural-product-derived cores as a source of structurally unique

Keap1 inhibitors, belonging to a chemical space very diﬀerent

from that of previously reported inhibitors.

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been designed by combination and fusion of natural-product-

derived fragments.⁴⁷However, natural products and their

derivatives often have complex structures. We demonstrated

how two rounds of docking of our smaller set of lead-like cores

ﬁrst identiﬁed the cyclothialidine core and then tailored it to its

target Keap1 via docking of 6−9 while simultaneously reducing

the structural complexity. In this manner, one of the major

shortcomings of natural products, i.e., that they are often

prepared via long synthetic routes, was mitigated. Similarly, the

larger set of 217 structurally more complex macrocyclic cores

may provide inspiration for design of collections having

simpliﬁed structures that can be prepared through shorter

routes than the original natural products. In conclusion, the

cores reported herein provide another example of how nature’s

privileged structures and their diversity can be capitalized on as a

rich source of quality leads for drug discovery.¹¹

DISCUSSION AND CONCLUSIONS

■

Lead generation is very challenging for targets that have diﬃcult-

to-drug binding sites,²⁻⁴such as protein−protein interactions

(PPIs).⁴²High-throughput screening (HTS) has often failed to

identify useful hits for such targets as screening collections do

not provide suﬃcient coverage of relevant chemical space due to

their limited size and history of assembly.⁴²Fragment-based lead

discovery allows a more comprehensive search of chemical space

and has shown success in some cases where HTS has failed.¹³

Structure-based docking constitutes an alternative that may be

particularly appealing for diﬃcult-to-drug targets, as platforms

for docking of ultralarge libraries are now being developed.^43,44

Diﬃcult-to-drug targets that have ﬂat and featureless binding

sites are often modulated by macrocyclic drugs, many of which

originate from natural products.^2,8We therefore mined the

macrocycles in the Dictionary of Natural Products to facilitate

the identiﬁcation of novel chemical matter for modulation of

diﬃcult-to-drug targets. This provided a smaller set of 41 lead-

like macrocyclic cores and a larger set of 217 cores with more

complex structures (Supporting Excel Sheet). Docking of the

smaller set of cores into the positively charged and polar binding

site of Keap1 led to the discovery of the 4 μM inhibitor 14, which

originates from the core of cyclothialidine (1), after synthesis of

only 13 compounds. Macrocycle 14 constitutes an uncharged

inhibitor of Keap1, indicating that macrocycles may be used to

discover promising starting points for diﬃcult-to-drug targets

that have polar binding sites, in addition to targets with ﬂat and

featureless sites. In contrast to 14, the majority of the reported

Keap1 inhibitors contain acidic groups, which may be

detrimental to cell permeability and oral absorption,²¹and are

expected to prevent CNS permeability.¹⁹Compound 14 has

druglike properties and shows cellular potency in an Nrf2

translocation assay, making it suitable for further lead

optimization. The structure of its complex with Keap1 illustrates

how cation−π interactions may allow an uncharged ligand to

bind in a charged binding site and provides a ﬁrst indication of

how an uncharged inhibitor can be tailored to ﬁt the charged

binding site.

It is interesting to contrast our results to those obtained in a

virtual screen of 1.3 billion compounds from Enamine’s REAL

space library and the ZINC library,⁴⁴as well as with fragment-

based lead generation.^33,45The ultralarge screen identiﬁed

Keap1 inhibitors with 100 nM potencies as determined by

surface plasmon resonance,⁴⁴but which contain potentially

reactive or toxic groups. In addition, the structures were similar

to previously reported inhibitors and appeared to provide

limited opportunities for optimization into drug candidates.

Obviously, screening of large libraries provides immense

opportunities for drug discovery, but our results reiterate the

importance of the structural diversity and quality of the libraries,

just as for regular HTS compound collections. One of the most

advanced Keap1 inhibitors was obtained by merging informa-

tion from three fragments that bound in diﬀerent subsites of the

Keap1 binding site.^33,45While highly potent in in vitro (K_D1.3

nM) and active in cellular and in vivo models, the oral

bioavailability in rats was low (7%), potentially because of the

presence of a carboxylic acid moiety originating from one of the

fragments.

EXPERIMENTAL SECTION

■

Chemistry. General Methods. All reagents were purchased from

Sigma-Aldrich, Fluorochem, and VWR International. DCM, DMSO,

hexane, DMF, and acetonitrile were purchased from VWR Interna-

tional, while 1,2-DCE, toluene, and THF were purchased from Sigma-

Aldrich. All nonaqueous reactions were performed in oven-dried

glassware under an argon atmosphere. The Buchi rotary evaporator R-

̈

114 was used to remove solvents in vacuo. Reactions were generally

monitored by liquid chromatography−mass spectrometry (LC-MS)

with an Agilent 1100 series high-performance liquid chromatography

(HPLC) with a C18 Atlantis T3 column (3.0 mm × 50 mm, 5 μm)

using acetonitrile−water (ﬂow rate 0.75 mL/min over 6 min) as the

mobile phase and a Waters micromass ZQ (model code: MM1) mass

spectrometer with the electrospray ionization mode as the detector.

Alternatively, TLC silica gel 60 F254 plates from VWR International

were used and visualization was done using UV light (254 nm) or by

staining with a KMnO₄solution (2% m/v in water). Silica gel (43−63

μm, VWR international) was used for puriﬁcation of compounds with

ﬂash column chromatography. Preparative reversed-phase HPLC was

performed on a Kromasil C8 column (250 mm × 21.2 mm, 5 μm) on a

Gilson HPLC equipped with a Gilson 322 pump, a UV−visible-156

detector, and a 202 collector using acetonitrile−water gradients as

eluents with a ﬂow rate of 15 mL/min and detection at 214 or 254 nm.

¹H, ¹³C, COSY, HSQC, and HMBC NMR spectra for synthesized

compounds were recorded at 298 K on an Agilent Technologies 400

MR spectrometer at 400 or 100 MHz or on an OXFORD AS500

spectrometer at 500 or 126 MHz or on a Bruker Avance III

spectrometer at 600 MHz or at 151 MHz. The residual peak of the

respective solvent was used as the internal standard [CDCl₃(CHCl₃

δH 7.26 ppm, CDCl₃δC 77.0 ppm), DMSO-d₆(CD₂HSOCD₃δH 2.50

ppm, CD₃SOCD₃δC 39.5 ppm), CD₃OD (CD₂HOD δH 3.31 ppm,

CD₃OD δC 49.0 ppm), CD₃CN (CD₂HCN δH 1.94 ppm, CD₃CN δC

1.3 and 118.3 ppm), acetone-d₆(CD₂HCOCD₃δH 2.05 ppm,

CD₃COCD₃δC 29.8 and 206.3 ppm)]. HRMS for all new compounds

were recorded in the electrospray ionization (ESI) mode on an S3 LCT

Premier connected to a Waters acquity UPLC I-class with acetonitrile−

water used as the mobile phase (1:1, with a ﬂow rate of 0.25 mL/min).

The purity of compounds 6−18 is ≥95% as determined using a Waters

LCT Premiere mass spectrometer coupled to a Waters Acquity UPLC.

The Waters Acquity UPLC was equipped with either a BEH C18

column (1.7 μm, 2.1 mm × 50 mm, at 45 °C using a gradient from 5 to

90% acetonitrile modiﬁed with 40 mM ammonia and 5 mM H₂CO₃, pH

10 within 2.5 or 3 min, detection at 210 nm) or a CSH C18 column (1.7

μm, 2.1 mm × 50 mm at 45 °C using a gradient from 5 to 90%

acetonitrile modiﬁed with 10 mM formic acid and 1 mM ammonium

formate, pH 3, within 2.5 or 3 min, detection at 230 nm).

2,2,2-Trichloroethyl 2-(bromomethyl)benzoate (19). 2-Methyl-

benzoic acid (8.00 g, 37.2 mmol, 1.0 equiv) was dissolved in DCM (30

mL). EDC·HCl (10.7 g, 55.8 mmol, 1.5 equiv), DMAP (454 mg, 3.72

mmol, 0.1 equiv), and 2,2,2-trichloroethanol (4.65 mL, 48.4 mmol, 1.3

equiv) were then added, and the mixture was stirred at rt for 16 h. The

Recently, natural products have also been utilized as a source

of three-dimensional (3D) fragments¹²and as inspiration for

design of compound collections prepared by diversity-oriented

synthesis (DOS).⁴⁶In addition, pseudo-natural products have

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mixture was diluted with DCM (150 mL) and washed with 1 M

aqueous HCl solution (2 × 100 mL) and saturated aqueous NaHCO₃

solution (2 × 100 mL). The organic phase was dried over MgSO₄,

ﬁltered, and then concentrated under reduced pressure. The crude

mixture was puriﬁed by ﬂash chromatography on a silica gel column

using 2−10% EtOAc in hexane as the eluent to give 19 (5.41 g, 15.6

mmol, 42%) as a colorless oil. The ¹H NMR spectrum is in accordance

with the literature data.⁴⁸

2,2,2-Trichloroethyl-(R)-2-(((2-amino-3-methoxy-3-oxopropyl)-

thio)methyl)benzoate (20a). 2,2,2-Trichloroethyl 2-(bromomethyl)-

benzoate 19 (3.46 g, 10.0 mmol, 1.0 equiv) was dissolved in DMSO (50

mL), ^L-cysteine methyl ester hydrochloride (2.06 g, 12.0 mmol, 1.2

equiv) and triethylamine (3.10 mL, 22.0 mmol, 2.2 equiv) were added,

and the mixture was stirred for 2 h at rt. The mixture was diluted with

EtOAc (250 mL) and washed with saturated aqueous NaHCO₃

solution (2 × 100 mL) and brine (100 mL). The organic phase was

dried over MgSO₄, ﬁltered, and then removed under reduced pressure.

The crude reaction mixture was puriﬁed by ﬂash chromatography on a

silica gel column using 0−10% MeOH in DCM as the eluent to give 20a

(3.09 g, 7.70 mmol, 77%) as a yellow oil. HRMS (ESI) m/z calcd for

C₁₄H₁₇Cl₃NO₄S [M + H]⁺399.9944, found 399.9953. ¹H NMR (400

MHz, CDCl₃) δ 8.08 (dd, J = 7.8, 1.4 Hz, 1H), 7.52 (td, J = 7.8, 1.4 Hz,

1H), 7.42−7.32 (m, 2H), 4.97 (s, 2H), 4.23 (d, J = 13.2 Hz, 1H), 4.16

(d, J = 13.2 Hz, 1H), 3.72 (s, 3H), 3.63 (dd, J = 7.6, 4.6 Hz, 1H), 2.87

(dd, J = 13.6, 4.6 Hz, 1H), 2.68 (dd, J = 13.6, 7.6 Hz, 1H), 1.79 (br s,

2H). ¹³C NMR (101 MHz, CDCl₃) δ 174.3, 165.2, 141.0, 132.8, 131.8,

131.3, 127.7, 127.5, 95.0, 74.6, 54.2, 52.2, 36.9, 34.8.

2,2,2-Trichloroethyl 2-((4R,7S)-7-(Hydroxymethyl)-4-(methoxy-

carbonyl)-11,11-dimethyl-6,9-dioxo-10-oxa-2-thia-5,8-

diazadodecyl)benzoate (21a). Compound 20a (1.25 g, 3.13 mmol,

1.0 equiv) was dissolved in acetonitrile (18 mL), Boc-^L-Ser-OH (0.96 g,

4.71 mmol, 1.5 equiv) and EDC·HCl (0.89 g, 4.71 mmol, 1.5 equiv)

were added, and the reaction was stirred for 1 h at rt. EtOAc (150 mL)

was then added, and the mixture was washed with 1 M aqueous HCl

solution (100 mL), saturated aqueous NaHCO₃solution (100 mL),

and brine (100 mL). The organic phase was dried over MgSO₄, ﬁltered,

and then concentrated under reduced pressure. The crude reaction

mixture was puriﬁed by ﬂash chromatography on a silica gel column

using 0−5% MeOH in DCM as the eluent to give 21a (1.35 g, 2.31

mmol, 74%) as a colorless foam. HRMS (ESI) m/z calcd for

C₂₂H₃₀Cl₃N₂O₈S [M + H]⁺587.0788, found 578.0794. ¹H NMR

(400 MHz, CDCl₃) δ 8.08 (d, J = 8.0 Hz, 1H), 7.50 (t, J = 8.0 Hz, 1H),

7.40−7.34 (m, 3H), 5.62 (d, J = 7.4 Hz, 1H), 4.96 (s, 2H), 4.85−4.75

(m, 1H), 4.29−4.25 (m, 1H), 4.23 (d, J = 12.8 Hz, 1H), 4.10 (d, J = 12.8

Hz, 1H), 4.03 (dd, J = 11.5, 3.6 Hz, 1H), 3.71 (s, 3H), 3.69−3.64 (m,

1H), 3.20 (br s, 1H), 2.94 (dd, J = 14.0, 4.8 Hz, 1H), 2.86 (dd, J = 14.0,

6.3 Hz, 1H), 1.43 (s, 9H). ¹³C NMR (101 MHz, CDCl₃) δ 171.2, 170.9,

165.2, 155.8, 140.4, 133.0, 131.8, 131.4, 127.7, 127.6, 94.9, 80.9, 74.6,

63.1, 55.3, 52.8, 52.1, 34.8, 33.5, 28.3.

2-((4R,7S)-7-(Hydroxymethyl)-4-(methoxycarbonyl)-11,11-di-

methyl-6,9-dioxo-10-oxa-2-thia-5,8-diazadodecyl)benzoic Acid

(22a). Compound 21a (1.35 g, 2.31 mmol, 1.0 equiv) was dissolved

in THF (19 mL) and aqueous 1 M NH₄OAc (4 mL). Zn dust (1.51 g,

23.1 mmol, 10 equiv) was added, and the reaction was stirred for 2 h at

rt. The mixture was diluted with MeOH (250 mL), ﬁltered through a

pad of Celite, and concentrated under reduced pressure. The crude

reaction mixture was puriﬁed by ﬂash chromatography on a silica gel

column using 0−20% MeOH/EtOAc as the eluent to give 22a (642 mg,

1.41 mmol, 61%) as a colorless powder. The ¹H NMR spectrum is in

accordance with the literature data.²⁹

23a (313 mg, 0.71 mmol, 51%) as a colorless powder. The ¹H NMR

spectrum was in accordance with the literature data.²⁹

Methyl-(4R,7S)-7-((S)-1-acetylpyrrolidine-2-carboxamido)-6,10-

dioxo-1,3,4,5,6,7,8,10-octahydrobenzo[j][1]oxa[8]thia[5]-

azacyclododecine-4-carboxylate (6). Compound 6 (100 mg, 0.21

mmol, 1.0 equiv) was dissolved in 4 M HCl in dioxane (5 mL) and

stirred for 1 h at rt. After evaporation of the solvent under reduced

pressure, the resulting salt was dissolved in DMSO (4 mL); Ac-^L-Pro-

OH (70 mg, 0.42 mmol, 2.0 equiv), EDC·HCl (86 mg, 0.42 mmol, 2.0

equiv), and DIPEA (39 μL, 0.21 mmol, 1.0 equiv) were added, and the

reaction was stirred for 2 h at rt. The crude product was puriﬁed by

reverse-phase HPLC using a gradient from 30 to 75% acetonitrile in

water to give 6 (43 mg, 0.18 mmol, 40%) as a colorless powder. HRMS

(ESI) m/z calcd for C₂₂H₂₈N₃O₇S [M + H]⁺478.1648, found

478.1652. ¹H NMR (400 MHz, CDCl₃) δ 7.73 (dd, J = 7.8, 1.4 Hz, 1H),

7.68 (d, J = 8.8 Hz, 1H), 7.52−7.42 (m, 2H), 7.37 (dd, J = 7.6, 1.4 Hz,

1H), 7.32 (td, J = 7.8, 1.4 Hz, 1H), 5.00−4.86 (m, 3H), 4.68−4.55 (m,

1H), 4.51 (dd, J = 7.4, 5.3 Hz, 1H), 4.02 (d, J = 10.3 Hz, 1H), 3.81 (d, J

= 10.3 Hz, 1H), 3.75 (s, 3H), 3.73−3.66 (m, 1H), 3.55−3.44 (m, 1H),

3.26 (dd, J = 14.5, 4.8 Hz, 1H), 2.96 (dd, J = 14.5, 9.7 Hz, 1H), 2.27−

2.16 (m, 2H), 2.11−1.94 (m, 2H), 2.03 (s, 3H). ¹³C NMR (101 MHz,

CDCl₃) δ 172.0, 171.1, 170.8, 169.6, 168.9, 136.4, 132.3, 131.8, 130.8,

130.7, 127.5, 67.1, 61.1, 53.8, 52.7, 51.6, 48.5, 34.7, 34.4, 29.2, 25.0,

22.8.

2,2,2-Trichloroethyl (S)-2-(((2-Amino-3-methoxy-3-oxopropyl)-

thio)methyl)benzoate (20b). Compound 20b was prepared following

the procedure described for the synthesis of compound 20a using 2,2,2-

trichloroethyl 2-(bromomethyl)benzoate 19 (1.70 g, 4.91 mmol, 1.0

equiv), ^D-cysteine methyl ester hydrochloride (1.01 g, 5.89 mmol, 1.2

equiv), and triethylamine (1.53 mL, 10.8 mmol, 2.2 equiv). The crude

reaction mixture was puriﬁed by ﬂash chromatography on a silica gel

column using 0−10% MeOH in DCM as the eluent to give 20b (1.37 g,

3.43 mmol, 70%) as a yellow oil. HRMS (ESI) m/z calcd for

1

C₁₄H₁₆Cl₃NNaO₄S [M + Na]⁺421.9763, found 421.9770. H NMR

(400 MHz, CDCl₃) δ 8.07 (dd, J = 7.7, 1.5 Hz, 1H), 7.52−7.47 (m,

1H), 7.43−7.28 (m, 2H), 4.95 (s, 2H), 4.21 (d, J = 13.2 Hz, 1H), 4.14

(d, J = 13.2 Hz, 1H), 3.70 (s, 3H), 3.60 (dd, J = 7.6, 4.6 Hz, 1H), 2.85

(dd, J = 13.6, 4.6 Hz, 1H), 2.66 (dd, J = 13.6, 7.6 Hz, 1H), 1.74 (br s,

2H). ¹³C NMR (101 MHz, CDCl₃) δ 174.3, 165.2, 141.0, 132.8, 131.8,

131.3, 127.7, 127.5, 95.0, 74.6, 54.1, 52.2, 36.9, 34.8.

2,2,2-Trichloroethyl 2-((4S,7S)-7-(Hydroxymethyl)-4-(methoxy-

carbonyl)-11,11-dimethyl-6,9-dioxo-10-oxa-2-thia-5,8-

diazadodecyl)benzoate (21b). Compound 21b was prepared

following the procedure described for the synthesis of compound 21a

using 20b (1.30 g, 3.25 mmol, 1.0 equiv), Boc-^L-Ser-OH (990 mg, 4.87

mmol, 1.5 equiv), and EDC·HCl (932 mg, 4.88 mmol, 1.5 equiv). The

crude reaction mixture was puriﬁed by ﬂash chromatography on a silica

gel column using 0−5% MeOH in DCM as the eluent to give 21b (1.45

g, 2.47 mmol, 76%) as a colorless foam. HRMS (ESI) m/z calcd for

C₂₂H₃₀Cl₃N₂O₈S [M + H]⁺587.0788, found 587.0773. ¹H NMR (400

MHz, CDCl₃) δ 8.08 (dd, J = 8.0, 1.4 Hz, 1H), 7.50 (td, J = 8.0, 1.4 Hz,

1H), 7.40−7.34 (m, 2H), 7.36−7.31 (m, 1H), 5.62 (d, J = 7.4 Hz, 1H),

4.99 (d, J = 12.0 Hz, 1H), 4.96 (d, J = 12.0 Hz, 1H), 4.85−4.75 (m, 1H),

4.27−4.18 (m, 2H), 4.10 (d, J = 12.8 Hz, 1H), 4.09−4.04 (m, 1H), 3.71

(s, 3H), 3.68 (dd, J = 11.6, 4.9 Hz, 1H), 2.97−2.84 (m, 2H), 2.38 (br s,

1H), 1.43 (s, 9H). ¹³C NMR (101 MHz, CDCl₃) δ 171.5, 171.0, 165.2,

156.0, 140.4, 133.0, 131.8, 131.4, 127.7, 127.6, 94.9, 80.5, 74.6, 63.1,

55.3, 52.8, 52.0, 34.8, 33.4, 28.3.

2-((4S,7S)-7-(Hydroxymethyl)-4-(methoxycarbonyl)-11,11-di-

methyl-6,9-dioxo-10-oxa-2-thia-5,8-diazadodecyl)benzoic Acid

(22b). Compound 22b was prepared following the procedure described

for the synthesis of compound 22a using 21b (1.20 g, 2.04 mmol, 1.0

equiv) and Zn dust (1.33 g, 20.4 mmol, 10 equiv). The crude reaction

mixture was puriﬁed by ﬂash chromatography on a silica gel column

using 0−20% MeOH in EtOAc as the eluent to give 22b (567 mg, 1.24

mmol, 61%) as a colorless powder. HRMS (ESI) m/z calcd for

Methyl (4R,7S)-7-((tert-Butoxycarbonyl)amino)-6,10-dioxo-

1,3,4,5,6,7,8,10-octahydrobenzo[j][1]oxa[8]thia[5]-

azacyclododecine-4-carboxylate (23a). Compound 22a (640 mg,

1.40 mmol, 1.0 equiv) was dissolved in toluene (33 mL) and DMSO (2

mL). Triphenylphosphine (550 mg, 2.10 mmol, 1.5 equiv) and di-tert-

butyl azodicarboxylate (483 mg, 2.10 mmol, 1.5 equiv) were then

added, and the mixture was stirred for 4 h at rt. After evaporation of the

solvent, the crude product was puriﬁed by ﬂash chromatography on a

silica gel column using 40−60% EtOAc in hexane as the eluent to give

1

C₂₀H₂₉N₂O₈S [M + H]⁺457.1645, found 457.1637. H NMR (400

MHz, DMSO-d₆) δ 12.93 (br s, 1H), 8.27 (d, J = 7.8 Hz, 1H), 7.82 (dd,

J = 8.0, 1.7 Hz, 1H), 7.46 (td, J = 7.6, 1.7 Hz, 1H), 7.41−7.29 (m, 2H),

6.63 (d, J = 8.1 Hz, 1H), 4.79 (br s, 1H), 4.55−4.42 (m, 1H), 4.07 (s,

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2H), 4.06−4.03 (m, 1H), 3.60 (s, 3H), 3.59−3.41 (m, 2H), 2.74 (dd, J

= 13.8, 5.8 Hz, 1H), 2.66 (dd, J = 13.8, 7.6 Hz, 1H), 1.35 (s, 9H). ¹³C

NMR (101 MHz, DMSO-d₆) δ 171.4, 170.9, 168.8, 155.6, 140.3, 132.0,

131.4, 131.3, 130.5, 127.6, 78.6, 62.3, 57.1, 52.5, 52.5, 34.1, 32.8, 28.6.

Methyl-(4S,7S)-7-((tert-butoxycarbonyl)amino)-6,10-dioxo-

1,3,4,5,6,7,8,10-octahydrobenzo[j][1]oxa[8]thia[5]-

azacyclododecine-4-carboxylate (23b). Compound 23b was pre-

pared following the procedure described for the synthesis of compound

23a using 22b (350 mg, 0.76 mmol, 1.0 equiv), triphenylphosphine

(300 mg, 1.14 mmol, 1.5 equiv), and di-tert-butyl azodicarboxylate (263

mg, 1.14 mmol, 1.5 equiv). The crude product was puriﬁed by ﬂash

chromatography on a silica gel column using 40−60% EtOAc in hexane

as the eluent to give 23b (146 mg, 0.33 mmol, 44%) as a colorless

powder. HRMS (ESI) m/z calcd for C₂₀H₂₆N₂NaO₇S [M + Na]⁺

461.1358, found 461.1356. ¹H NMR (400 MHz, DMSO-d₆) δ 9.01 (d, J

= 9.3 Hz, 1H), 7.74 (d, J = 7.8 Hz, 1H), 7.51 (td, J = 7.8, 1.5 Hz, 1H),

7.45−7.33 (m, 2H), 7.12 (d, J = 8.3 Hz, 1H), 4.77 (td, J = 10.4, 4.0 Hz,

1H), 4.60−4.52 (m, 1H), 4.50−4.35 (m, 2H), 4.29 (dd, J = 10.7, 4.6

Hz, 1H), 3.84 (d, J = 10.3 Hz, 1H), 3.66 (s, 3H), 3.08 (dd, J = 14.5, 4.0

Hz, 1H), 2.70 (dd, J = 14.5, 10.9 Hz, 1H), 1.37 (s, 9H). ¹³C NMR (101

MHz, DMSO-d₆) δ 171.4, 170.1, 167.6, 155.3, 137.3, 132.9, 132.4,

131.6, 130.4, 128.1, 78.9, 65.8, 52.7, 52.6, 52.3, 35.9, 33.9, 28.6.

Methyl-(4S,7S)-7-((S)-1-acetylpyrrolidine-2-carboxamido)-6,10-

dioxo-1,3,4,5,6,7,8,10-octahydrobenzo[j][1]oxa[8]thia[5]-

azacyclododecine-4-carboxylate (7). Compound 7 was prepared

following the procedure described for the synthesis of compound 6

using 23b (100 mg, 0.22 mmol, 1.0 equiv), Ac-^L-Pro-OH (69 mg, 0.44

mmol, 2.0 equiv), EDC·HCl (84 mg, 0.44 mmol, 2.0 equiv), and

DIPEA (39 μL, 0.22 mmol, 1.0 equiv). The crude product was puriﬁed

by reverse-phase HPLC using a gradient from 30 to 75% acetonitrile in

water to give 7 (62 mg, 0.13 mmol, 59%) as a colorless powder. HRMS

(ESI) m/z calcd for C₂₂H₂₈N₃O₇S [M + H]⁺478.1648, found

478.1651. ¹H NMR (400 MHz, CDCl₃) δ 7.79 (dd, J = 7.7, 1.4 Hz, 1H),

7.61 (d, J = 7.7 Hz, 1H), 7.43 (td, J = 7.5, 1.4 Hz, 1H), 7.34−7.26 (m,

2H), 7.20 (d, J = 7.5 Hz, 1H), 4.83 (td, J = 7.3, 4.6 Hz, 1H), 4.69−4.58

(m, 2H), 4.56−4.48 (m, 2H), 4.15 (d, J = 11.1 Hz, 1H), 4.06 (d, J = 11.1

Hz, 1H), 3.74 (s, 3H), 3.71−3.61 (m, 1H), 3.52−3.40 (m, 1H), 3.21

(dd, J = 14.3, 4.8 Hz, 1H), 3.14 (dd, J = 14.3, 7.1 Hz, 1H), 2.33−2.21

(m, 1H), 2.13−1.96 (m, 3H), 2.06 (s, 3H). ¹³C NMR (101 MHz,

CDCl₃) δ 171.2, 171.0, 170.3, 168.6, 168.6, 137.5, 132.3, 131.2, 131.1,

130.1, 127.6, 65.0, 60.2, 52.8, 52.5, 52.5, 48.3, 36.3, 34.5, 28.4, 25.0,

22.4.

2,2,2-Trichloroethyl 2-((4R,7R)-7-(Hydroxymethyl)-4-(methoxy-

carbonyl)-11,11-dimethyl-6,9-dioxo-10-oxa-2-thia-5,8-

diazadodecyl)benzoate (21c). Compound 21c was prepared follow-

ing the procedure described for the synthesis of compound 21a using

20a (2.01 g, 5.81 mmol, 1.0 equiv), Boc-^D-Ser-OH (1.78 g, 8.72 mmol,

1.5 equiv), and EDC·HCl (1.64 g, 8.72 mmol, 1.5 equiv). The crude

reaction mixture was puriﬁed by ﬂash chromatography on a silica gel

column using 0−5% MeOH in DCM as the eluent to give 21c (2.55 g,

4.34 mmol, 75%) as a colorless oil. HRMS (ESI) m/z calcd for

C₂₂H₂₉Cl₃N₂NaO₈S [M + Na]⁺609.0608, found 609.0612. ¹H NMR

(400 MHz, CDCl₃) δ 8.07 (dd, J = 8.3, 1.4 Hz, 1H), 7.53−7.48 (m,

1H), 7.45−7.38 (m, 1H), 7.39−7.33 (m, 2H), 5.67 (d, J = 7.3 Hz, 1H),

5.00−4.92 (m, 2H), 4.78 (ddd, J = 8.3, 6.4, 5.0 Hz, 1H), 4.23 (d, J =

13.1 Hz, 1H), 4.11 (d, J = 13.0 Hz, 1H), 4.08−4.01 (m, 1H), 3.71 (s,

3H), 3.68−3.65 (m, 1H), 3.29−3.22 (m, 1H), 2.91 (dd, J = 14.0, 5.0

Hz, 1H), 2.86 (dd, J = 14.0, 6.4 Hz, 1H), 2.10 (br s, 1H), 1.42 (s, 9H).

¹³C NMR (101 MHz, CDCl₃) δ 171.4, 171.0, 165.2, 156.0, 140.5,

J = 8.2, 1.5 Hz, 1H), 7.45 (td, J = 7.5, 1.5 Hz, 1H), 7.37−7.26 (m, 2H),

6.62 (d, J = 8.3 Hz, 1H), 4.50−4.43 (m, 1H), 4.07 (s, 2H), 4.04−3.99

(m, 1H), 3.59 (s, 3H), 3.58−3.43 (m, 2H), 2.74 (dd, J = 13.8, 5.8 Hz,

1H), 2.65 (dd, J = 13.8, 7.5 Hz, 1H), 1.34 (s, 9H). The CH₂OHproton

was not detectable in this spectrum. ¹³C NMR (101 MHz, DMSO-d₆) δ

171.4, 170.9, 168.8, 155.6, 140.3, 132.0, 131.4, 131.3, 130.6, 127.6, 79.6,

78.6, 62.3, 57.1, 52.5, 34.1, 32.8, 28.6.

Methyl-(4R,7R)-7-((tert-butoxycarbonyl)amino)-6,10-dioxo-

1,3,4,5,6,7,8,10-octahydrobenzo[j][1]oxa[8]thia[5]-

azacyclododecine-4-carboxylate (23c). Compound 23c was prepared

following the procedure described for the synthesis of compound 23a

using 22c (500 mg, 1.09 mmol, 1.0 equiv), triphenylphosphine (429

mg, 1.64 mmol, 1.5 equiv), and di-tert-butyl azodicarboxylate (377 mg,

1.64 mmol, 1.5 equiv). The crude product was puriﬁed by ﬂash

chromatography on a silica gel column using 40−60% EtOAc in hexane

as the eluent to give 23c (253 mg, 0.57 mmol, 53%) as a colorless

powder. HRMS (ESI) m/z calcd for C₂₀H₂₆N₂NaO₇S [M + Na]⁺

461.1358, found 461.1349. ¹H NMR (400 MHz, CDCl₃) δ 7.79 (d, J =

7.5 Hz, 1H), 7.42 (td, J = 7.5, 1.3 Hz, 1H), 7.36− 7.22 (m, 2H), 6.96 (d,

J = 7.0 Hz, 1H), 5.51 (d, J = 6.5 Hz, 1H), 4.92−4.85 (m, 1H), 4.72−

4.60 (m, 2H), 4.47−4.38 (m, 1H), 4.14−4.02 (m, 2H), 3.75 (s, 3H),

3.15 (dd, J = 14.4, 4.7 Hz, 1H), 3.05 (dd, J = 14.4, 5.9 Hz, 1H), 1.45 (s,

9H). ¹³C NMR (101 MHz, CDCl₃) δ 170.4, 169.1, 167.8, 154.9, 137.2,

132.2, 131.3, 131.0, 130.2, 127.7, 80.4, 65.3, 52.9, 52.6, 51.9, 35.9, 34.8,

28.3.

Methyl-(4R,7R)-7-((S)-1-acetylpyrrolidine-2-carboxamido)-6,10-

dioxo-1,3,4,5,6,7,8,10-octahydrobenzo[j][1]oxa[8]thia[5]-

azacyclododecine-4-carboxylate (8). Compound 8 was prepared

following the procedure described for the synthesis of compound 6

using 23c (95 mg, 0.21 mmol, 1.0 equiv), Ac-^L-Pro-OH (66 mg, 0.42

mmol, 2.0 equiv), EDC·HCl (80 mg, 0.42 mmol, 2.0 equiv), and

DIPEA (47 μL, 0.27 mmol, 1.0 equiv). The crude product was puriﬁed

by reverse-phase HPLC using a gradient from 30 to 75% acetonitrile in

water to give 8 (45 mg, 94 μmol, 45%) as a colorless powder. HRMS

(ESI) m/z calcd for C₂₂H₂₈N₃O₇S [M + H]⁺478.1648, found

478.1648. ¹H NMR (400 MHz, CDCl₃) δ 7.78 (dd, J = 7.7, 1.5 Hz, 1H),

7.60 (d, J = 7.0 Hz, 1H), 7.42 (td, J = 7.5, 1.4 Hz, 1H), 7.34−7.27 (m,

3H), 4.87 (ddd, J = 7.4, 6.6, 4.5 Hz, 1H), 4.75−4.64 (m, 2H), 4.60−

4.42 (m, 2H), 4.18 (s, 2H), 3.75 (s, 3H), 3.64−3.58 (m, 1H), 3.55−

3.37 (m, 1H), 3.19 (dd, J = 14.3, 4.5 Hz, 1H), 3.12 (dd, J = 14.3, 6.6 Hz,

1H), 2.34−2.28 (m, 1H), 2.20−2.06 (m, 1H), 2.10 (s, 3H), 2.04−1.91

(m, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 171.4, 170.8, 170.5, 168.7,

168.0, 137.4, 132.2, 131.2, 131.1, 130.3, 127.6, 65.0, 59.9, 52.8, 52.4,

52.0, 48.3, 36.3, 34.6, 28.3, 25.0, 22.5.

2,2,2-Trichloroethyl 2-((4S,7R)-7-(Hydroxymethyl)-4-(methoxy-

carbonyl)-11,11-dimethyl-6,9-dioxo-10-oxa-2-thia-5,8-

diazadodecyl)benzoate (21d). Compound 21d was prepared

following the procedure described for the synthesis of compound 21a

using 20b (1.30 g, 3.25 mmol, 1.0 equiv), Boc-^D-Ser-OH (990 mg, 4.87

mmol, 1.5 equiv), and EDC·HCl (932 mg, 4.88 mmol, 1.5 equiv). The

crude reaction mixture was puriﬁed by ﬂash chromatography on a silica

gel column using 0−5% MeOH in DCM as the eluent to give 21d (1.37

g, 2.34 mmol, 72%) as a colorless oil. HRMS (ESI) m/z calcd for

C₂₂H₂₉Cl₃N₂O₈SNa [M + Na]⁺609.0608, found 609.0594. ¹H NMR

(400 MHz, CDCl₃) δ 8.09 (dd, J = 7.7, 1.5 Hz, 1H), 7.52 (td, J = 7.5, 1.4

Hz, 1H), 7.41−7.36 (m, 2H), 7.30 (d, J = 7.0 Hz, 1H), 5.57 (d, J = 7.5

Hz, 1H), 4.98 (s, 2H), 4.83−4.77 (m, 1H), 4.31−4.20 (m, 2H), 4.10 (d,

J = 12.8 Hz, 1H), 4.08−4.01 (m, 1H), 3.73 (s, 3H), 3.68 (dd, J = 11.4,

5.2 Hz, 1H), 2.96 (dd, J = 14.1, 4.7 Hz, 1H), 2.88 (dd, J = 14.1, 6.2 Hz,

1H), 2.79 (br s, 1H), 1.44 (s, 9H). ¹³C NMR (101 MHz, CDCl₃) δ

171.2, 170.9, 165.3, 155.8, 140.4, 133.0, 131.8, 131.4, 127.7, 127.6, 94.9,

80.5, 74.7, 63.3, 55.2, 52.8, 52.0, 34.9, 33.6, 28.3.

133.0, 131.8, 131.4, 127.7, 127.6, 94.9, 80.5, 74.6, 63.0, 55.5, 52.8, 52.0,

34.7, 33.4, 28.3.

2-((4R,7R)-7-(Hydroxymethyl)-4-(methoxycarbonyl)-11,11-di-

methyl-6,9-dioxo-10-oxa-2-thia-5,8-diazadodecyl)benzoic Acid

(22c). Compound 22c was prepared following the procedure described

for the synthesis of compound 22a using 21c (1.58 g, 2.69 mmol, 1.0

equiv) and Zn dust (1.75 g, 26.9 mmol, 10 equiv). The crude reaction

mixture was puriﬁed by ﬂash chromatography on a silica gel column

using 0−15% MeOH/EtOAc as the eluent to give 22c (834 mg, 1.83

mmol, 68%) as a colorless powder. HRMS (ESI) m/z calcd for

2-((4S,7R)-7-(Hydroxymethyl)-4-(methoxycarbonyl)-11,11-di-

methyl-6,9-dioxo-10-oxa-2-thia-5,8-diazadodecyl)benzoic Acid

(22d). Compound 22d was prepared following the procedure described

for the synthesis of compound 22a using 21d (1.05 g, 1.78 mmol, 1.0

equiv) and Zn dust (1.16 g, 17.8 mmol, 10 equiv). The crude reaction

mixture was puriﬁed by ﬂash chromatography on a silica gel column

using 0−20% MeOH/EtOAc as the eluent to give 22d (478 mg, 1.05

mmol, 59%) as a colorless powder. HRMS (ESI) m/z calcd for

1

C₂₀H₂₉N₂O₈S [M + H]⁺457.1645, found 457.1633. H NMR (400

1

MHz, DMSO-d₆) δ 12.86 (br s, 1H), 8.26 (d, J = 7.9 Hz, 1H), 7.81 (dd,

C₂₀H₂₉N₂O₈S [M + H]⁺457.1645, found 457.1635. H NMR (400

J

https://dx.doi.org/10.1021/acs.jmedchem.0c01569

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

MHz, DMSO-d₆) δ 8.25 (d, J = 7.7 Hz, 1H), 7.82 (d, J = 7.7 Hz, 1H),

7.37 (t, J = 7.7 Hz, 1H), 7.33−7.19 (m, 2H), 6.69 (d, J = 7.9 Hz, 1H),

4.53−4.38 (m, 1H), 4.18 (d, J = 13.0 Hz, 1H), 4.13 (d, J = 13.0 Hz, 1H),

4.09−3.96 (m, 1H), 3.58 (s, 3H), 3.56−3.41 (m, 2H), 2.77 (dd, J =

13.8, 5.8 Hz, 1H), 2.68 (dd, J = 13.8, 7.2 Hz, 1H), 1.36 (s, 9H). The

OHprotons were not detectable in this spectrum. ¹³C NMR (126 MHz,

DMSO-d₆) δ 171.4, 171.0, 168.8, 155.7, 140.4, 132.0, 131.5, 131.4,

130.6, 127.7, 78.7, 62.2, 57.2, 52.6, 52.6, 34.1, 32.7, 28.6.

Methyl-(4S,7R)-7-((tert-butoxycarbonyl)amino)-6,10-dioxo-

1,3,4,5,6,7,8,10-octahydrobenzo[j][1]oxa[8]thia[5]-

azacyclododecine-4-carboxylate (23d). Compound 23d was pre-

pared following the procedure described for the synthesis of compound

23a using 22d (401 mg, 0.87 mmol, 1.0 equiv), triphenylphosphine

(344 mg, 1.32 mmol, 1.5 equiv), and di-tert-butyl azodicarboxylate (303

mg, 1.32 mmol, 1.5 equiv). The crude product was puriﬁed by ﬂash

chromatography on a silica gel column using 40−65% EtOAc in hexane

as the eluent to give 23d (186 mg, 0.43 mmol, 49%) as a colorless

powder. HRMS (ESI) m/z calcd for C₂₀H₂₆N₂NaO₇S [M + Na]⁺

461.1358, found 461.1360. ¹H NMR (400 MHz, CDCl₃) δ 7.82 (d, J =

7.4 Hz, 1H), 7.45 (t, J = 7.5 Hz, 1H), 7.35− 7.29 (m, 3H), 5.75−5.62

(m, 1H), 4.96 (dd, J = 11.4, 3.0 Hz, 1H), 4.89−4.79 (m, 1H), 4.60−

4.56 (m, 2H), 4.10−4.00 (m, 2H), 3.74 (s, 3H), 3.30−3.25 (m, 1H),

3.20−3.12 (m, 1H), 1.47 (s, 9H). ¹³C NMR (101 MHz, CDCl₃) δ

170.4, 169.2, 168.5, 155.0, 137.3, 132.5, 131.4, 131.4, 129.8, 127.6, 81.1,

66.7, 55.3, 52.8, 52.1, 36.3, 34.9, 28.2.

Methyl-(4S,7R)-7-((S)-1-acetylpyrrolidine-2-carboxamido)-6,10-

dioxo-1,3,4,5,6,7,8,10-octahydrobenzo[j][1]oxa[8]thia[5]-

azacyclododecine-4-carboxylate (9). Compound 9 was prepared

following the procedure described for the synthesis of compound 6

using 23d (120 mg, 0.27 mmol, 1.0 equiv), Ac-^L-Pro-OH (84 mg, 0.54

mmol, 2.0 equiv), EDC·HCl (103 mg, 0.54 mmol, 2.0 equiv), and

DIPEA (47 μL, 0.27 mmol, 1.0 equiv). The crude product was puriﬁed

by reverse-phase HPLC using a gradient from 30 to 75% acetonitrile in

water to give 9 (62 mg, 0.13 mmol, 48%) as a colorless powder. HRMS

(ESI) m/z calcd for C₂₂H₂₆N₃O₇S [M − H]⁻476.1491, found

476.1503. ¹H NMR (400 MHz, CDCl₃) δ 7.89 (dd, J = 7.7, 1.2 Hz, 1H),

7.78 (d, J = 8.8 Hz, 1H), 7.71 (d, J = 8.8 Hz, 1H), 7.50− 7.42 (m, 1H),

7.39 (dd, J = 7.7, 1.2 Hz, 1H), 7.32 (td, J = 7.7, 1.5 Hz, 1H), 5.09 (dd, J =

11.1, 2.9 Hz, 1H), 4.91 (td, J = 8.9, 4.5 Hz, 1H), 4.88−4.83 (m, 1H),

4.50−4.37 (m, 2H), 4.30 (d, J = 10.5 Hz, 1H), 3.74−3.66 (m, 1H), 3.92

(d, J = 10.5 Hz, 1H), 3.71 (s, 3H), 3.53−3.41 (m, 1H), 3.21 (dd, J =

14.6, 4.5 Hz, 1H), 2.94 (dd, J = 14.6, 9.0 Hz, 1H), 2.41−2.20 (m, 2H),

2.08 (s, 3H), 2.05−1.87 (m, 2H). ¹³C NMR (101 MHz, CDCl₃) δ

171.6, 171.5, 170.8, 168.9, 167.9, 137.2, 132.5, 132.2, 131.8, 129.8,

127.5, 66.9, 60.2, 53.4, 52.5, 52.4, 48.4, 36.2, 34.8, 28.0, 25.3, 22.6.

Methyl-(4S,7R)-7-((R)-1-acetylpyrrolidine-2-carboxamido)-6,10-

dioxo-1,3,4,5,6,7,8,10-octahydrobenzo[j][1]oxa[8]thia[5]-

azacyclododecine-4-carboxylate (10). Compound 10 was prepared

following the procedure described for the synthesis of compound 6

using 23d (27 mg, 61 μmol, 1.0 equiv), Ac-^D-Pro-OH (19 mg, 0.12

mmol, 2.0 equiv), EDC·HCl (23 mg, 0.12 mmol, 2.0 equiv), and

DIPEA (21 μL, 0.12 mmol, 2.0 equiv). The crude product was puriﬁed

by reverse-phase HPLC using a gradient from 30 to 75% acetonitrile in

water to give 10 (16 mg, 33 μmol, 54%) as a colorless powder. HRMS

(ESI) m/z calcd for C₂₂H₂₈N₃O₇S [M + H]⁺478.1648, found

478.1663. ¹H NMR (400 MHz, CD₃CN) δ 8.07 (d, J = 9.0 Hz, 1H),

7.96 (dd, J = 7.8, 1.2 Hz, 1H), 7.55 (td, J = 7.8, 1.2 Hz, 1H), 7.46 (dd, J =

7.8, 1.4 Hz, 1H), 7.42 (td, J = 7.8, 1.4 Hz, 1H), 7.30 (d, J = 7.4 Hz, 1H),

4.92 (dd, J = 11.3, 3.0 Hz, 1H), 4.84 (ddd, J = 11.7, 9.0, 4.0 Hz, 1H),

4.68−4.62 (m, 1H), 4.40 (dd, J = 11.4, 2.0 Hz, 1H), 4.35 (d, J = 9.3 Hz,

1H), 4.28−4.23 (m, 1H), 3.86 (d, J = 9.3 Hz, 1H), 3.71 (s, 3H), 3.55−

3.43 (m, 1H), 3.25 (dd, J = 14.6, 4.0 Hz, 1H), 2.91 (dd, J = 14.6, 11.7

Hz, 1H), 2.30−2.22 (m, 1H), 2.02 (s, 3H), 2.01−1.94 (m, 4H). ¹³C

NMR (101 MHz, CDCl₃) δ 172.0, 171.1, 170.7, 169.6, 168.9, 136.4,

132.3, 131.8, 130.8, 130.7, 127.5, 67.1, 61.1, 53.8, 52.7, 51.6, 48.6, 34.7,

34.4, 29.3, 25.0, 22.8.

Ac-^L-Pro-OH (31 mg, 0.18 mmol, 2.0 equiv), EDC·HCl (35 mg, 0.18

mmol, 2.0 equiv), and DIPEA (31 μL, 0.18 mmol, 2.0 equiv). The crude

product was puriﬁed by reverse-phase HPLC using a gradient from 20

to 70% acetonitrile in water to give 11 (14 mg, 28 μmol, 32%) as a

colorless powder. HRMS (ESI) m/z calcd for C₂₂H₂₇N₃NaO₈S [M +

Na]⁺516.1417, found 516.1407. ¹H NMR (400 MHz, CD₃CN) δ 8.47

(d, J = 9.7 Hz, 1H), 7.98 (dd, J = 7.7, 1.4 Hz, 1H), 7.69 (d, J = 7.3 Hz,

1H), 7.56 (td, J = 7.5, 1.4 Hz, 1H), 7.48 (dd, J = 7.5, 1.4 Hz, 1H), 7.41

(td, J = 7.7, 1.4 Hz, 1H), 5.02 (dd, J = 11.1, 2.5 Hz, 1H), 4.84 (ddd, J =

11.7, 9.7, 3.9 Hz, 1H), 4.77 (d, J = 9.4 Hz, 1H), 4.57−4.52 (m, 1H),

4.52−4.48 (m, 1H), 4.46−4.40 (m, 1H), 4.38 (dd, J = 11.2, 2.2 Hz,

1H), 3.85 (d, J = 9.4 Hz, 1H), 3.75−3.68 (m, 1H), 3.66 (s, 3H), 3.48

(dt, J = 11.1, 1.7 Hz, 1H), 3.24 (dd, J = 14.8, 3.9 Hz, 1H), 2.95 (dd, J =

14.8, 11.7 Hz, 1H), 2.16 (ddd, J = 13.1, 8.6, 4.5 Hz, 2H), 2.10−2.02 (m,

1H), 2.00 (s, 3H). ¹³C NMR (101 MHz, CD₃CN) δ 172.6, 170.7,

170.7, 169.3, 167.1, 137.5, 132.8, 132.6, 132.0, 129.8, 127.7, 69.8, 66.0,

58.9, 56.4, 53.7, 53.4, 51.9, 37.3, 37.2, 34.5, 21.9.

tert-Butyl-((4S,7R)-4-carbamoyl-6,10-dioxo-1,3,4,5,6,7,8,10-

octahydrobenzo[j][1]oxa[8]thia[5]azacyclododecin-7-yl)-

carbamate (24). Compound 23d (84 mg, 0.19 mmol, 1.0 equiv) was

dissolved in 1,2-dichloroethane (2 mL); Me₃SnOH (143 mg, 0.76

mmol, 4.0 equiv) was added, and the mixture was stirred for 45 min at

83 °C. The reaction was allowed to cool to rt, acidiﬁed with 1 M

aqueous HCl solution (5 mL), and extracted with DCM (3 × 25 mL).

The combined organic phase was dried over MgSO₄, ﬁltered, and then

concentrated under reduced pressure, and the obtained crude mixture

was dissolved in DMF (5 mL). HOBt·xH₂O (39 mg, 0.29 mmol, 1.5

equiv), EDC·HCl (72 mg, 0.38 mmol, 2.0 equiv), and ammonia (7 M in

MeOH, 54 μL, 0.38 mmol, 2.0 equiv) were added, and the mixture was

stirred for 2 h at rt. EtOAc (50 mL) was then added, and the mixture

was washed with 1 M aqueous HCl solution (25 mL), saturated

aqueous NaHCO₃solution (25 mL), and brine (25 mL). The organic

phase was dried over MgSO₄, ﬁltered, and then concentrated under

reduced pressure. The crude product was puriﬁed by reverse-phase

HPLC using a gradient from 35 to 70% acetonitrile in water to give 24

(39 mg, 91 μmol, 48%) as a colorless powder. HRMS (ESI) m/z calcd

for C₁₉H₂₅N₃NaO₆S [M + Na]⁺446.1362, found 446.1377. ¹H NMR

(500 MHz, CD₃OD) δ 7.93 (d, J = 7.8 Hz, 1H), 7.50 (t, J = 7.5 Hz, 1H),

7.42 (d, J = 7.5 Hz, 1H), 7.36 (t, J = 7.8 Hz, 1H), 5.02−4.96 (m, 1H),

4.73 (dd, J = 10.5, 4.5 Hz, 1H), 4.60 (d, J = 10.0 Hz, 1H), 4.42 (d, J =

10.0 Hz, 1H), 4.40−4.36 (m, 1H), 3.83 (d, J = 9.7 Hz, 1H), 3.29−3.23

(m, 1H), 2.94−2.85 (dd, J = 14.6, 10.8 Hz, 1H), 1.48 (s, 9H). The NH

protons were not detectable in this spectrum. ¹³C NMR (126 MHz,

CD₃OD) δ 173.6, 171.4, 167.6, 157.2, 137.5, 132.3, 132.1, 131.7, 129.5,

127.2, 80.3, 65.7, 55.7, 53.7, 36.8, 34.0, 27.2.

(4S,7R)-7-((S)-1-Acetylpyrrolidine-2-carboxamido)-6,10-dioxo-

1,3,4,5,6,7,8,10-octahydrobenzo[j][1]oxa[8]thia[5]-

azacyclododecine-4-carboxamide (12). Compound 12 was prepared

following the procedure described for the synthesis of compound 6

using 24 (26 mg, 62 μmol, 1.0 equiv), Ac-^L-Pro-OH (19 mg, 0.12 mmol,

2.0 equiv), EDC·HCl (23 mg, 0.12 mmol, 2.0 equiv), and DIPEA (20

μL, 0.12 mmol, 2.0 equiv). The crude product was puriﬁed by reverse-

phase HPLC using a gradient from 20 to 70% acetonitrile in water to

give 12 (14 mg, 32 μmol, 52%) as a colorless powder. HRMS (ESI) m/z

calcd for C₂₁H₂₆N₄O₆S [M + H]⁺463.1651, found 463.1647. ¹H NMR

(400 MHz, DMSO-d₆) δ 9.39 (d, J = 5.1 Hz, 1H), 8.13 (d, J = 9.7 Hz,

1H), 7.92 (d, J = 7.5 Hz, 1H), 7.54 (t, J = 7.3 Hz, 1H), 7.46 (d, J = 7.3

Hz, 1H), 7.40 (t, J = 7.5 Hz, 1H), 7.26 (br s, 1H), 6.48 (br s, 1H), 4.80−

4.74 (m, 2H), 4.45−4.36 (m, 2H), 4.36−4.30 (m, 2H), 3.79 (d, J = 9.2

Hz, 1H), 3.63−3.53 (m, 1H), 3.52−3.45 (m, 1H), 3.17 (dd, J = 14.7,

3.8 Hz, 1H), 2.77 (dd, J = 14.7, 12.1 Hz, 1H), 2.11−2.01 (m, 2H), 1.95

(s, 3H), 1.90−1.81 (m, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 174.9,

172.4, 170.4, 169.0, 167.1, 137.8, 133.3, 133.1, 132.3, 129.6, 128.1, 65.8,

59.7, 55.5, 54.5, 48.5, 37.0, 34.5, 29.2, 25.3, 22.7.

tert-Butyl-((4S,7R)-4-(methylcarbamoyl)-6,10-dioxo-

1,3,4,5,6,7,8,10-octahydrobenzo[j][1]oxa[8]thia[5]-

azacyclododecin-7-yl)carbamate (25). Compound 25 was prepared

following the procedure described for the synthesis of compound 24

using 23d (136 mg, 0.31 mmol, 1.0 equiv), Me₃SnOH (233 mg, 1.24

mmol, 4.0 equiv), HOBt·xH₂O (63 mg, 0.47 mmol, 1.5 equiv), EDC·

Methyl-(4S,7R)-7-((2S,4R)-1-acetyl-4-hydroxypyrrolidine-2-car-

boxamido)-6,10-dioxo-1,3,4,5,6,7,8,10-octahydrobenzo[j][1]oxa-

[8]thia[5]azacyclododecine-4-carboxylate (11). Compound 11 was

prepared following the procedure described for the synthesis of

compound 6 using 23d (39 mg, 89 μmol, 1.0 equiv), 4-trans-hydroxy-

K

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HCl (118 mg, 0.62 mmol, 2.0 equiv), and methylamine (2 M in THF,

171 μL, 0.34 mmol, 1.1 equiv). The crude reaction mixture was puriﬁed

by ﬂash chromatography on a silica gel column using 0−5% MeOH in

DCM as the eluent to give 25 (78 mg, 0.18 mmol, 58%) as a colorless

powder. HRMS (ESI) m/z calcd for C₂₀H₂₈N₃O₆S [M + H]⁺438.1699,

found 438.1687. ¹H NMR (400 MHz, CDCl₃) δ 7.83 (d, J = 7.8 Hz,

1H), 7.48 (t, J = 7.5 Hz, 1H), 7.42−7.31 (m, 2H), 7.19 (br s, 1H), 6.58

(br s, 1H), 5.58 (br s, 1H), 4.90−4.84 (m, 1H), 4.71−4.57, (m, 2H),

4.56−4.49 (m, 1H), 4.01 (d, J = 9.8 Hz, 1H), 3.80 (d, J = 9.8 Hz, 1H),

3.37 (dd, J = 14.8, 5.3 Hz, 1H), 2.97 (dd, J = 14.8, 9.8 Hz, 1H), 2.80 (d, J

= 4.1 Hz, 3H), 1.46 (s, 9H). ¹³C NMR (126 MHz, CDCl₃) δ 170.6,

170.4, 168.4, 156.4, 136.2, 132.7, 132.1, 131.6, 129.9, 127.9, 81.7, 66.2,

55.9, 50.9, 34.4, 33.6, 28.2, 26.4.

tert-Butyl-((4S,7R)-4-(diethylcarbamoyl)-6,10-dioxo-

1,3,4,5,6,7,8,10-octahydrobenzo[j][1]oxa[8]thia[5]-

azacyclododecin-7-yl)carbamate (27). Compound 27 was prepared

following the procedure described for the synthesis of compound 24,

using 23d (153 mg, 0.35 mmol, 1.0 equiv), Me₃SnOH (263 mg, 1.40

mmol, 4.0 equiv), HOBt·xH₂O (71 mg, 0.53 mmol, 1.5 equiv), EDC·

HCl (133 mg, 0.70 mmol, 2.0 equiv), and diethylamine (39 μL, 0.38

mmol, 1.1 equiv). The crude reaction mixture was puriﬁed by ﬂash

chromatography on a silica gel column using 0−10% MeOH in DCM as

the eluent to give 27 (55 mg, 0.12 mmol, 33%) as a colorless oil. HRMS

(ESI) m/z calcd for C₂₃H₃₄N₃O₆S [M + H]⁺480.2168, found

480.2184. ¹H NMR (400 MHz, CDCl₃) δ 7.80 (d, J = 7.4 Hz, 1H), 7.41

(t, J = 7.4 Hz, 1H), 7.32 (t, J = 7.5 Hz, 1H), 7.29−7.21 (m, 2H), 5.53−

5.46 (m, 1H), 5.18−5.08 (m, 1H), 4.70−4.53 (m, 2H), 4.56−4.45 (m,

1H), 4.14 (d, J = 10.8 Hz, 1H), 4.07 (d, J = 10.8 Hz, 1H), 3.55−3.33 (m,

3H), 3.33−3.17 (m, 1H), 3.01 (dd, J = 14.2, 3.4 Hz, 1H), 2.79 (dd, J =

14.2, 8.7 Hz, 1H), 1.44 (s, 9H), 1.24 (t, J = 7.0 Hz, 3H), 1.11 (t, J = 7.0

Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 169.0, 169.0, 167.8, 154.8,

137.0, 132.1, 131.3, 131.3, 130.5, 127.7, 80.2, 65.5, 52.8, 48.8, 42.1,

40.8, 37.0, 36.1, 28.3, 14.6, 12.8.

(4S,7R)-7-((S)-1-Acetylpyrrolidine-2-carboxamido)-N,N-diethyl-

6,10-dioxo-1,3,4,5,6,7,8,10-octahydrobenzo[j][1]oxa[8]thia[5]-

azacyclododecine-4-carboxamide (15). Compound 15 was prepared

following the procedure described for the synthesis of compound 6

using 27 (35 mg, 72 μmol, 1.0 equiv), Ac-^L-Pro-OH (22 mg, 0.14 mmol,

2.0 equiv), EDC·HCl (27 mg, 0.14 mmol, 2.0 equiv), and DIPEA (24

μL, 0.14 mmol, 2.0 equiv). The crude product was puriﬁed by reverse-

phase HPLC using a gradient from 5 to 95% acetonitrile in water to give

15 (9 mg, 37 μmol, 24%) as a colorless powder. HRMS (ESI) m/z calcd

for C₂₅H₃₅N₄O₆S [M + H]⁺519.2277, found 519.2286. ¹H NMR (600

MHz, DMSO-d₆, mixture of two rotamers in the ratio 6:4) δ 9.14 (d, J =

3.8 Hz, 0.6H), 9.13 (d, J = 3.8 Hz, 0.4H), 8.61 (d, J = 8.2 Hz, 0.4H),

8.18 (d, J = 8.2 Hz, 0.6H), 7.78 (t, J = 7.6 Hz, 1H), 7.53 (t, J = 7.8 Hz

1H), 7.46−7.43 (m, 1H), 7.41 (t, J = 7.6 Hz, 1H), 5.04−4.97 (m, 1H),

4.97−4.92 (m, 1H), 4.63 (d, J = 9.7 Hz, 0.6H), 4.54−4.50 (m, 0.6H),

4.48−4.43 (m, 1.2H), 4.38 (dd, J = 8.5, 3.4 Hz, 0.6H), 4.33 (dd, J =

10.4, 4.5 Hz, 0.4H), 4.17 (dd, J = 10.2, 4.5 Hz, 0.6H), 3.89 (d, J = 9.8

Hz, 0.4H), 3.85 (d, J = 9.8 Hz, 0.6H), 3.62−3.51 (m, 1H), 3.49−3.43

(m, 1H), 3.41−3.34 (m, 2H), 3.33−3.27 (m, 1H), 3.22−3.18 (m, 1H),

3.02−2.93 (m, 1H), 2.80 (dd, J = 14.6, 11.4 Hz, 0.4H), 2.76 (dd, J =

14.6, 11.2 Hz, 0.6H), 2.23−2.15 (m, 0.4H), 2.05−1.98 (m, 0.6H), 1.95

(s, 1.8H), 1.93−1.88 (m, 1H), 1.88−1.71 (m, 2H), 1.76 (s, 1.2H), 1.14

(t, J = 7.0 Hz, 1.8H), 1.11 (t, J = 7.0 Hz, 1.2H), 1.04 (t, J = 7.1 Hz,

1.8H), 1.02 (t, J = 7.1 Hz, 1.2H). ¹³C NMR (151 MHz, DMSO-d₆,

mixture of two rotamers) δ 172.6 and 172.3 (1C), 169.5 (1C), 169.4

(1C), 168.9 and 168.8 (1C), 167.7 and 167.6 (1C), 137.5 and 137.4

(1C), 133.1 and 133.0 (1C), 132.6 and 132.4 (1C), 131.9 and 131.8

(1C), 130.4 and 130.3 (1C), 128.2 (1C), 65.6 and 65.5 (1C), 60.2 and

59.3 (1C), 50.9 and 50.7 (1C), 50.1 and 49.6 (1C), 48.1 and 46.7 (1C),

41.7 and 41.6 (1C), 37.3 and 37.0 (1C), 35.4 and 35.3 (1C), 32.4 and

30.3 (1C), 24.7 (1C), 23.0 and 22.9 (1C), 22.7 and 22.4 (1C), 14.9 and

14.8 (1C), 13.4 and 13.3 (1C).

(4S,7R)-7-((S)-1-Acetylpyrrolidine-2-carboxamido)-N-methyl-

6,10-dioxo-1,3,4,5,6,7,8,10-octahydrobenzo[j][1]oxa[8]thia[5]-

azacyclododecine-4-carboxamide (13). Compound 13 was prepared

following the procedure described for the synthesis of compound 6

using 25 (50 mg, 0.11 mmol, 1.0 equiv), Ac-^L-Pro-OH (34 mg, 0.22

mmol, 2.0 equiv), EDC·HCl (42 mg, 0.22 mmol, 2.0 equiv), and

DIPEA (19 μL, 0.11 mmol, 1.0 equiv). The crude product was puriﬁed

by reverse-phase HPLC using a gradient from 20 to 70% acetonitrile in

water to give 13 (17 mg, 37 μmol, 34%) as a colorless powder. HRMS

(ESI) m/z calcd for C₂₂H₂₉N₄O₆S [M + H]⁺477.1808, found

477.1802. ¹H NMR (400 MHz, CDCl₃) δ 8.23 (d, J = 9.1 Hz, 1H), 7.82

(d, J = 7.5 Hz, 1H), 7.45 (t, J = 7.8 Hz, 1H), 7.39 (d, J = 7.8 Hz, 1H),

7.32 (t, J = 7.5 Hz, 1H), 7.23 (d, J = 7.1 Hz, 1H), 6.58 (br s, 1H), 5.08−

5.01 (m, 1H), 4.77−4.67 (m, 2H), 4.59−4.53 (m, 1H), 4.21−4.13 (m,

1H), 3.99 (d, J = 9.5 Hz, 1H), 3.94 (d, J = 9.5 Hz, 1H), 3.68−3.59 (m,

1H), 3.59−3.51 (m, 2H), 2.91−2.81 (m, 1H), 2.76 (d, J = 4.0 Hz, 3H),

2.32−2.09 (m, 4H), 2.00 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ

173.4, 171.1, 170.4, 169.4, 168.5, 136.4, 132.5, 132.4, 131.4, 130.1,

127.6, 66.0, 61.0, 54.5, 53.3, 48.6, 34.8, 34.6, 29.2, 26.3, 25.4, 22.4.

tert-Butyl-((4S,7R)-4-(dimethylcarbamoyl)-6,10-dioxo-

1,3,4,5,6,7,8,10-octahydrobenzo[j][1]oxa[8]thia[5]-

azacyclododecin-7-yl)carbamate (26). Compound 26 was prepared

following the procedure described for the synthesis of compound 24,

using 23d (82 mg, 0.19 mmol, 1.0 equiv), Me₃SnOH (143 mg, 0.76

mmol, 4.0 equiv), HOBt·xH₂O (38 mg, 0.28 mmol, 1.5 equiv), EDC·

HCl (72 mg, 0.38 mmol, 2.0 equiv), and dimethylamine (2 M in THF,

105 μL, 0.21 mmol, 1.1 equiv). The crude product was puriﬁed by

reverse-phase HPLC using a gradient from 20 to 75% acetonitrile in

water to give 26 (40 mg, 89 μmol, 47%) as a colorless powder. HRMS

(ESI) m/z calcd for C₂₁H₂₉N₃NaO₆S [M + Na]⁺474.1675, found

474.1667. ¹H NMR (500 MHz, CDCl₃) δ 7.86 (dd, J = 7.8, 1.4 Hz, 1H),

7.47 (td, J = 7.5, 1.5 Hz, 1H), 7.39−7.31 (m, 3H), 5.58−5.50 (m, 1H),

5.21 (ddd, J = 8.6, 6.7, 5.0 Hz, 1H), 5.06 (dd, J = 11.3, 2.8 Hz, 1H),

4.62−4.55 (m, 1H), 4.50 (dd, J = 11.3, 2.2 Hz, 1H), 4.10−4.05 (m,

1H), 3.99 (d, J = 10.2 Hz, 1H), 3.14−3.09 (m, 1H), 3.11 (s, 3H), 3.03

(dd, J = 14.7, 6.7 Hz, 1H), 2.95 (s, 3H), 1.49 (s, 9H). ¹³C NMR (126

MHz, CDCl₃) δ 169.5, 169.2, 168.1, 155.1, 137.1, 132.4, 131.8, 131.6,

130.0, 127.6, 81.3, 66.1, 55.3, 48.9, 37.6, 37.1, 35.9, 35.5, 28.2.

(4S,7R)-7-((S)-1-Acetylpyrrolidine-2-carboxamido)-N,N-dimeth-

yl-6,10-dioxo-1,3,4,5,6,7,8,10-octahydrobenzo[j][1]oxa[8]thia[5]-

azacyclododecine-4-carboxamide (14). Compound 14 was prepared

following the procedure described for the synthesis of compound 6

using 26 (38 mg, 84 μmol, 1.0 equiv), Ac-^L-Pro-OH (27 mg, 0.17 mmol,

2.0 equiv), EDC·HCl (32 mg, 0.17 mmol, 2.0 equiv), and DIPEA (27

μL, 0.17 mmol, 2.0 equiv). The crude product was puriﬁed by reverse-

phase HPLC using a gradient from 25 to 75% acetonitrile in water to

give 14 (13 mg, 26 μmol, 32%) as a colorless powder. HRMS (ESI) m/z

calcd for C₂₃H₃₁N₄O₆S [M + H]⁺491.1964, found 491.1941. ¹H NMR

(500 MHz, CDCl₃) δ 7.90 (d, J = 7.3 Hz, 2H), 7.54 (d, J = 9.0 Hz, 1H),

7.39 (t, J = 7.4 Hz, 1H), 7.32 (d, J = 7.4 Hz, 1H), 7.27 (t, J = 7.6 Hz,

1H), 5.13−5.01 (m, 2H), 4.87−4.83 (m, 1H), 4.52−4.47 (m, 2H), 4.44

(d, J = 11.0 Hz, 1H), 3.90 (d, J = 11.0 Hz, 1H), 3.77−3.65 (m, 1H),

3.50−3.42 (m, 1H), 3.06 (s, 3H), 3.04−2.94 (m, 2H), 2.92 (s, 3H),

2.35−2.26 (m, 1H), 2.22−2.12 (m, 1H), 2.15 (s, 3H), 1.96−1.84 (m,

2H). ¹³C NMR (126 MHz, CDCl₃) δ 172.0, 171.4, 169.5, 168.7, 167.7,

137.2, 132.6, 132.3, 132.2, 129.6, 127.6, 66.8, 60.0, 53.3, 49.9, 48.4,

38.4, 37.1, 35.9, 35.8, 27.6, 25.2, 22.6.

Methyl N-((tert-Butoxycarbonyl)-^D-seryl)-S-methyl-D-cysteinate

(28). ^D-Cysteine methyl ester hydrochloride (1.02 g, 5.90 mmol, 1.0

equiv) was dissolved in DMF (12 mL); methyl iodide (404 μL, 6.51

mmol, 1.1 equiv) and DIPEA (2.01 mL, 11.8 mmol, 2.0 equiv) were

then added, and the mixture stirred at rt for 2 h. The mixture was diluted

with EtOAc (100 mL) and washed with saturated aqueous NaHCO₃

solution (2 × 50 mL) and brine (100 mL). The organic phase was dried

over MgSO₄, ﬁltered, and then concentrated under reduced pressure.

The obtained crude mixture was dissolved in acetonitrile (10 mL); Boc-

D-Ser-OH (1.21 g, 5.90 mmol, 1.0 equiv) and EDC·HCl (1.12 g, 5.90

mmol, 1.0 equiv) were added, and the reaction was stirred for 1 h at rt.

EtOAc (100 mL) was then added, and the mixture was washed with 1 M

aqueous HCl solution (50 mL), saturated aqueous NaHCO₃solution

(50 mL), and brine (100 mL). The organic phase was dried over

MgSO₄, ﬁltered, and then concentrated under reduced pressure. The

obtained colorless oil (867 mg, 2.60 mmol, 44% over two steps) was

employed in the next step without further puriﬁcation. HRMS (ESI)

m/z calcd for C₁₃H₂₅N₂O₆S [M + H]⁺337.1433, found 337.1428. ¹H

L

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NMR (400 MHz, CDCl₃) δ 7.31 (br s, 1H), 5.56 (d, J = 6.7 Hz, 1H),

4.84−4.74 (m, 1H), 4.23 (br s, 1H), 4.07 (m, 1H), 3.76 (s, 3H), 3.67

(dd, J = 11.6, 5.3 Hz, 1H), 3.00 (dd, J = 14.0, 4.8 Hz, 1H), 2.90 (dd, J =

14.1, 6.5 Hz, 1H), 2.10 (s, 3H), 1.45 (s, 9H). The CH₂OH proton was

not detectable in this spectrum. ¹³C NMR (101 MHz, CDCl₃) δ 171.3,

171.1, 155.9, 80.5, 63.0, 55.2, 52.8, 51.6, 36.1, 28.3, 16.0.

3.09 (s, 3H), 2.93 (s, 3H), 2.85 (dd, J = 13.5, 6.7 Hz, 1H), 2.72 (dd, J =

13.5, 6.3 Hz, 1H), 2.41−2.33 (m, 1H), 2.18 (s, 3H), 2.02 (s, 3H), 1.97−

1.79 (m, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 171.8, 171.6, 169.9,

168.2, 166.4, 133.2, 130.0, 130.0, 129.5, 128.4, 128.4, 64.5, 59.9, 52.9,

48.3, 48.3, 37.8, 36.9, 35.9, 27.4, 25.2, 22.6, 16.3.

Methyl-(S)-2-(((2-((tert-butoxycarbonyl)amino)-3-(dimethylami-

no)-3-oxopropyl)thio)methyl)benzoate (31). Boc-^D-Cys-OH (6.81 g,

30.8 mmol, 1.5 equiv) was dissolved in DMF (20 mL) and THF (80

mL). Methyl 2-bromomethylbenzoate (4.72 g, 20.5 mmol, 1.0 equiv)

and triethylamine (6.38 mL, 45.1 mmol, 2.2 equiv) were added, and the

mixture was stirred for 16 h at rt. After evaporation of THF under

reduced pressure, the mixture was diluted with EtOAc (250 mL) and

washed with 1 M aqueous HCl solution (100 mL) and brine (2 × 100

mL). The organic phase was dried over MgSO₄, ﬁltered, and then

concentrated under reduced pressure, and the obtained oil was

dissolved in DMF (50 mL). Dimethylamine hydrochloride (3.34 g, 41.0

mmol, 2.0 equiv), EDC·HCl (7.86 g, 41.0 mmol, 2.0 equiv), HOBt·

xH₂O (4.15 g, 30.8 mmol, 1.5 equiv), and DIPEA (7.16 mL, 41.0 mmol,

2.0 equiv) were then added, and the mixture was stirred for 2 additional

hours at rt. The mixture was diluted with EtOAc (250 mL) and washed

with 1 M aqueous HCl solution (100 mL), saturated aqueous NaHCO₃

solution (100 mL), and brine (100 mL). The organic phase was dried

over MgSO₄, ﬁltered, and then concentrated under reduced pressure.

The crude reaction mixture was puriﬁed by ﬂash chromatography on a

silica gel column using 10−100% EtOAc in hexane as the eluent to give

31 (3.90 g, 9.84 mmol, 48% over two steps) as a yellow oil. HRMS

(ESI) m/z calcd for C₁₉H₂₉N₂O₅S [M + H]⁺397.1797, found

397.1804. ¹H NMR (500 MHz, CDCl₃) δ 7.90 (d, J = 7.7 Hz, 1H), 7.42

(t, J = 7.3 Hz, 1H), 7.38 (d, J = 7.3 Hz, 1H), 7.30 (t, J = 7.6 Hz, 1H),

5.37 (d, J = 8.7 Hz, 1H), 4.81−4.75 (m, 1H), 4.18−4.08 (m, 2H), 3.89

(s, 3H), 3.06 (s, 3H), 2.95 (s, 3H), 2.76 (dd, J = 13.7, 7.4 Hz, 1H), 2.63

(dd, J = 13.7, 6.0 Hz, 1H), 1.43 (s, 9H). ¹³C NMR (126 MHz, CDCl₃) δ

171.0, 167.7, 155.1, 140.2, 131.9, 131.2, 131.2, 129.4, 127.2, 79.8, 52.2,

49.6, 37.4, 35.9, 34.9, 34.6, 28.3.

Methyl-2-((4S,7R)-4-(dimethylcarbamoyl)-7-(2-hydroxyethyl)-

11,11-dimethyl-6,9-dioxo-10-oxa-2-thia-5,8-diazadodecyl)-

benzoate (32). Compound 31 (1.18 g, 3.01 mmol, 1.0 equiv) was

dissolved in 4 M HCl in dioxane (10 mL) and stirred for 1 h at rt. After

evaporation of the solvent under reduced pressure, the resulting salt was

dissolved in acetonitrile (15 mL). Boc-^D-Homoser-OH (1.99 g, 9.03

mmol, 3.0 equiv), EDC·HCl (1.72 g, 9.03 mmol, 3.0 equiv), and DIPEA

(524 μL, 3.01 mmol, 1.0 equiv) were added, and the reaction was stirred

for 2 h at rt. EtOAc (100 mL) was then added, and the mixture was

washed with 1 M aqueous HCl solution (50 mL), saturated aqueous

NaHCO₃solution (50 mL), and brine (50 mL). The organic phase was

dried over MgSO₄, ﬁltered, and then concentrated under reduced

pressure. The reaction crude was puriﬁed by ﬂash chromatography on a

silica gel column using 0−10% MeOH in DCM as the eluent to give 32

(500 mg, 1.01 mmol, 33% over two steps) as a white foam. HRMS

(ESI) m/z calcd for C₂₃H₃₅N₃O₇NaS [M+Na]⁺520.2093, found

520.2079. ¹H NMR (500 MHz, CDCl₃) δ 7.89 (dd, J = 7.8, 1.2 Hz, 1H),

7.44 (td, J = 7.7, 1.2 Hz, 1H), 7.37 (dd, J = 7.7, 1.2 Hz, 1H), 7.31 (td, J =

7.8, 1.2 Hz, 1H), 7.21−7.13 (m, 1H), 5.61 (d, J = 7.4 Hz, 1H), 5.06−

4.99 (m, 1H), 4.40−4.33 (m, 1H), 4.18 (d, J = 13.2 Hz, 1H), 4.08 (d, J =

13.2 Hz, 1H), 3.90 (s, 3H), 3.75−3.66 (m, 2H), 3.02 (s, 3H), 2.94 (s,

3H), 2.80 (dd, J = 13.7, 6.6 Hz, 1H), 2.69 (dd, J = 13.7, 6.4 Hz, 1H),

2.04−1.95 (m, 1H), 1.83−1.73 (m, 1H), 1.44 (s, 9H). The

CH₂OHproton was not detectable in this spectrum. ¹³C NMR (126

MHz, CDCl₃) δ 171.5, 170.2, 167.9, 156.3, 140.0, 132.0, 131.2, 131.2,

129.5, 127.4, 80.3, 58.8, 52.3, 52.0, 48.6, 37.4, 36.0, 36.0, 35.1, 34.1,

28.3.

(R)-2-((tert-Butoxycarbonyl)amino)-3-(((S)-1-methoxy-3-(meth-

ylthio)-1-oxopropan-2-yl)amino)-3-oxopropyl Benzoate (29). Com-

pound 28 (215 mg, 0.64 mmol, 1.0 equiv) was dissolved in DCM (10

mL) and cooled to 0 °C. Benzoyl chloride (148 μL, 1.28 mmol, 2.0

equiv), triethylamine (181 μL, 1.28 mmol, 2.0 equiv), and DMAP (15

mg, 0.13 mmol, 0.2 equiv) were then added, and the reaction was

allowed to warm to rt and stirred for 1 h. The mixture was diluted with

EtOAc (100 mL) and washed with saturated aqueous NaHCO₃

solution (2 × 25 mL) and brine (25 mL). The organic phase was

dried over MgSO₄, ﬁltered, and then concentrated under reduced

pressure. The crude reaction mixture was puriﬁed by ﬂash

chromatography on a silica gel column using 50−70% EtOAc in

hexane as the eluent to give 29 (237 mg, 0.54 mmol, 84%) as a yellow

oil. HRMS (ESI) m/z calcd for C₂₀H₂₈N₂O₇SNa [M + Na]⁺463.1515,

found 463.1523. ¹H NMR (400 MHz, CDCl₃) δ 8.05−7.99 (m, 2H),

7.55 (t, J = 7.7 Hz, 1H), 7.42 (t, J = 7.7 Hz, 2H), 7.14 (d, J = 7.7 Hz,

1H), 5.46 (br s, 1H), 4.85−4.79 (m, 1H), 4.73−4.59 (m, 2H), 4.55 (dd,

J = 10.8, 4.1 Hz, 1H), 3.70 (s, 3H), 2.99 (dd, J = 14.1, 4.9 Hz, 1H), 2.92

(dd, J = 14.0, 5.6 Hz, 1H), 2.04 (s, 3H), 1.44 (s, 9H). ¹³C NMR (101

MHz, CDCl₃) δ 170.8, 169.1, 166.3, 155.4, 133.3, 129.8, 129.8, 128.4,

128.4, 128.3, 80.8, 64.6, 54.0, 52.7, 51.8, 36.3, 28.2, 16.1.

(R)-2-((S)-1-Acetylpyrrolidine-2-carboxamido)-3-(((S)-1-me-

thoxy-3-(methylthio)-1-oxopropan-2-yl)amino)-3-oxopropyl Ben-

zoate (30). Compound 30 was prepared following the procedure

described for the synthesis of compound 6 using 29 (200 mg, 0.46

mmol, 1.0 equiv), Ac-^L-Pro-OH (144 mg, 0.92 mmol, 2.0 equiv), EDC·

HCl (174 mg, 0.92 mmol, 2.0 equiv), and DIPEA (0.16 mL, 0.92 mmol,

2.0 equiv). The crude reaction mixture was puriﬁed by ﬂash

chromatography on a silica gel column using 0−10% MeOH in

DCM as the eluent to give 30 (92 mg, 0.19 mmol, 41%) as a colorless

powder. HRMS (ESI) m/z calcd for C₂₂H₂₉N₃NaO₇S [M + Na]⁺

502.1624, found 502.1616. ¹H NMR (500 MHz, CDCl₃) δ 8.03 (d, J =

7.8 Hz, 2H), 7.67 (d, J = 8.2 Hz, 1H), 7.55−7.44 (m, 2H), 7.36 (t, J =

7.7 Hz, 2H), 4.86−4.80 (m, 2H), 4.70−4.65 (m, 1H), 4.48−4.44 (m,

1H), 4.43−4.34 (m, 1H), 3.65 (s, 3H), 3.63−3.58 (m, 1H), 3.46−3.38

(m, 1H), 2.96−2.83 (m, 2H), 2.32−2.22 (m, 1H), 2.20−2.10 (m, 1H),

2.06 (s, 3H), 1.95 (s, 3H), 1.93−1.82 (m, 2H). ¹³C NMR (126 MHz,

CDCl₃) δ 171.7, 171.4, 171.0, 168.7, 166.4, 133.3, 130.0, 130.0, 129.5,

128.4, 128.4, 64.3, 60.1, 52.7, 52.5, 51.9, 48.4, 35.9, 27.8, 25.2, 22.6,

15.9.

(R)-2-((S)-1-Acetylpyrrolidine-2-carboxamido)-3-(((S)-1-(dime-

thylamino)-3-(methylthio)-1-oxopropan-2-yl)amino)-3-oxopropyl

Benzoate (16). Compound 30 (54 mg, 0.11 mmol, 1.0 equiv) was

dissolved in 1,2-dichloroethane (3 mL). Me₃SnOH (83 mg, 0.44 mmol,

4.0 equiv) was added, and the mixture was stirred for 45 min at 83 °C.

The reaction was allowed to cool to rt, acidiﬁed with 1 M aqueous HCl

solution (5 mL), and extracted with DCM (3 × 40 mL). The organic

phase was dried over MgSO₄, ﬁltered, and then concentrated under

reduced pressure. The obtained crude was dissolved in DMF (5 mL);

HOBt·xH₂O (23 mg, 0.17 mmol, 1.5 equiv), EDC·HCl (42 mg, 0.22

mmol, 2.0 equiv), and dimethylamine (2 M in THF, 110 μL, 0.22

mmol, 2.0 equiv) were added, and the mixture was stirred for 2 h at rt.

EtOAc (30 mL) was then added, and the mixture was washed with 1 M

aqueous HCl solution (25 mL), saturated aqueous NaHCO₃solution

(25 mL), and brine (25 mL). The organic phase was dried over MgSO₄,

ﬁltered, and then concentrated under reduced pressure. The crude

product was puriﬁed by reverse-phase HPLC using a gradient from 25

to 75% acetonitrile in water to give 45 (9 mg, 20 μmol, 18% over two

steps) as a colorless powder. HRMS (ESI) m/z calcd for C₂₃H₃₃N₄O₆S

[M + H]⁺493.2121, found 493.2119. ¹H NMR (400 MHz, CDCl₃) δ

8.15−8.07 (m, 2H), 8.07−8.02 (m, 1H), 7.59−7.52 (m, 1H), 7.48−

7.39 (m, 3H), 5.07−5.00 (m, 1H), 4.89 (dd, J = 11.2, 4.3 Hz, 1H),

4.83−4.77 (m, 1H), 4.54 (dd, J = 7.9, 2.8 Hz, 1H), 4.49 (dd, J = 11.2,

3.5 Hz, 1H), 3.74 (ddd, J = 9.8, 8.1, 3.5 Hz, 1H), 3.48−3.41(m, 1H),

2-((4S,7R)-4-(Dimethylcarbamoyl)-7-(2-hydroxyethyl)-11,11-di-

methyl-6,9-dioxo-10-oxa-2-thia-5,8-diazadodecyl)benzoic Acid

(33). Compound 33 (480 mg, 0.97 mmol, 1.0 equiv) was dissolved in

MeOH (5 mL) and 0.1 M aqueous LiOH (20 mL), and the reaction

mixture was stirred at 35 °C for 16 h. The reaction was then allowed to

cool to rt, acidiﬁed with 1 M aqueous HCl solution (5 mL), and

extracted with DCM (3 × 50 mL). The organic phase was dried over

MgSO₄, ﬁltered, and then concentrated under reduced pressure

providing compound 33 (302 mg, 0.63 mmol, 64%) as a colorless

M

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solid. HRMS (ESI) m/z calcd for C₂₂H₃₄N₃O₇S [M + H]⁺484.2117,

found 484.2120. ¹H NMR (600 MHz, DMSO-d₆) δ 12.94 (br s, 1H),

8.01 (d, J = 8.5 Hz, 1H), 7.84 (dd, J = 7.8, 1.4 Hz, 1H), 7.47 (td, J = 7.5,

1.4 Hz, 1H), 7.40 (dd, J = 7.5, 1.4 Hz, 1H), 7.36 (td, J = 7.8, 1.4 Hz,

1H), 6.87 (d, J = 8.0 Hz, 1H), 4.88−4.82 (m, 1H), 4.53−4.45 (m, 1H),

4.14 (d, J = 13.0 Hz, 1H), 4.05 (d, J = 13.0 Hz, 1H), 4.95−4.03 (m, 1H),

3.48−3.36 (m, 2H), 2.94 (s, 3H), 2.82 (s, 3H), 2.75 (dd, J = 13.6, 7.7

Hz, 1H), 2.46 (dd, J = 13.6, 5.9 Hz, 1H), 1.80−1.71 (m, 1H), 1.69−

1.58 (m, 1H), 1.38 (s, 9H). ¹³C NMR (151 MHz, DMSO-d₆) δ 172.3,

170.1, 168.8, 155.7, 140.6, 131.9, 131.4, 131.3, 130.6, 127.6, 78.6, 58.0,

52.4, 48.6, 37.0, 35.7, 35.4, 34.3, 33.4, 28.6.

tert-Butyl-((4S,7R)-4-(dimethylcarbamoyl)-6,11-dioxo-

1,4,5,6,7,8,9,11-octahydro-3H-benzo[c][1]oxa[6]thia[9]-

azacyclotridecin-7-yl)carbamate (34). Compound 34 was prepared

following the procedure described for the synthesis of compound 23a

using 33 (163 mg, 0.33 mmol, 1.0 equiv), triphenylphosphine (173 mg,

0.66 mmol, 2.0 equiv), and di-tert-butyl azodicarboxylate (152 mg, 0.66

mmol, 2.0 equiv). The crude product was puriﬁed by reverse-phase

HPLC using a gradient from 20 to 70% acetonitrile in water to give 34

(32 mg, 69 μmol, 21%) as a colorless powder. HRMS (ESI) m/z calcd

for C₂₂H₃₂N₃O₆S [M + H]⁺466.2012. found 466.2003. ¹H NMR (500

MHz, CDCl₃) δ 7.67 (d, J = 7.2 Hz, 1H), 7.46−7.38 (m, 2H), 7.33−

7.27 (m, 1H), 7.22 (d, J = 7.9 Hz, 1H), 5.74 (d, J = 9.2 Hz, 1H), 4.92

(td, J = 8.5, 2.7 Hz, 1H), 4.70−4.63 (m, 1H), 4.59−4.52 (m, 1H),

4.42−4.43 (m, 1H), 4.21 (d, J = 13.6 Hz, 1H), 3.98 (d, J = 13.6 Hz, 1H),

2.98 (s, 3H), 2.93 (s, 3H), 2.82−2.70 (m, 2H), 2.39 (dd, J = 14.2, 8.5

Hz, 1H), 2.08−1.99 (m, 1H), 1.48 (s, 9H). ¹³C NMR (126 MHz,

CDCl₃) δ 170.6, 169.5, 168.9, 156.4, 138.9, 131.9, 131.7, 131.6, 129.4,

127.4, 80.8, 63.7, 51.0, 49.5, 37.1, 36.0, 34.2, 32.8, 30.5, 28.4.

2.94 (s, 3H), 2.80 (dd, J = 14.0, 6.8 Hz, 1H), 2.69 (dd, J = 14.0, 5.6 Hz,

1H), 1.44 (s, 9H). ¹³C NMR (126 MHz, CDCl₃) δ 170.3, 168.8, 167.8,

155.8, 140.1, 132.0, 131.2, 131.2, 129.4, 127.3, 80.2, 52.3, 48.3, 44.1,

37.4, 35.9, 35.2, 34.2, 28.3.

Methyl 2-((4S,10R)-4-(Dimethylcarbamoyl)-10-(hydroxymethyl)-

14,14-dimethyl-6,9,12-trioxo-13-oxa-2-thia-5,8,11-

triazapentadecyl)benzoate (36). Compound 35 (3.80 g, 8.40 mmol,

1.0 equiv) was dissolved in 4 M HCl in dioxane (15 mL) and stirred for

1 h at rt. After evaporation of the solvent under reduced pressure, the

resulting salt was dissolved in acetonitrile (25 mL). Boc-^D-Ser-OH

(3.44 g, 16.8 mmol, 2.0 equiv), EDC·HCl (3.21 g, 16.8 mmol, 2.0

equiv), and DIPEA (1.46 mL, 8.40 mmol, 1.0 equiv) were added, and

the reaction was stirred for 2 h at rt. EtOAc (150 mL) was then added,

and the mixture was washed with 1 M aqueous HCl solution (50 mL),

saturated aqueous NaHCO₃solution (50 mL), and brine (50 mL). The

organic phase was dried over MgSO₄, ﬁltered, and then concentrated

under reduced pressure. The crude reaction mixture was puriﬁed by

ﬂash chromatography on a silica gel column using 0−15% MeOH in

DCM as the eluent to give 36 (1.50 g, 2.77 mmol, 33% over two steps)

as a colorless oil. HRMS (ESI) m/z calcd for C₂₄H₃₆N₄O₈NaS [M +

Na]⁺563.2152, found 563.2145. ¹H NMR (500 MHz, CDCl₃) δ 7.89

(d, J = 9.4 Hz, 1H), 7.52 (d, J = 8.2 Hz, 1H), 7.46−7.42 (m, 1H), 7.36−

7.29 (m, 2H), 7.22−7.15 (m, 1H), 5.62 (d, J = 8.6 Hz, 1H), 5.06−4.95

(m, 1H), 4.32−4.22 (m, 2H), 4.12−4.03 (m, 3H), 3.92−3.90 (m, 1H),

3.90 (s, 3H), 3.71−3.60 (m, 1H), 3.03 (s, 3H), 2.91 (s, 3H), 2.75 (dd, J

= 13.9, 7.2 Hz, 1H), 2.66 (dd, J = 13.9, 6.1 Hz, 1H), 1.43 (s, 9H). ¹³C

NMR (126 MHz, CDCl₃) δ 172.0, 170.7, 168.5, 168.0, 155.6, 140.0,

132.1, 131.2, 131.2, 129.5, 127.4, 80.3, 63.2, 55.8, 52.3, 48.4, 43.1, 37.5,

36.1, 35.3, 33.9, 28.3.

(4S,7R)-7-((S)-1-Acetylpyrrolidine-2-carboxamido)-N,N-dimeth-

yl-6,11-dioxo-1,4,5,6,7,8,9,11-octahydro-3H-benzo[c][1]oxa[6]thia-

[9]azacyclotridecine-4-carboxamide (17). Compound 17 was pre-

pared following the procedure described for the synthesis of compound

6 using 34 (25 mg, 54 μmol, 1.0 equiv), Ac-^L-Pro-OH (25 mg, 0.16

mmol, 3.0 equiv), EDC·HCl (31 mg, 0.16 mmol, 3.0 equiv), and

DIPEA (28 μL, 0.16 mmol, 3.0 equiv). The crude product was puriﬁed

by reverse-phase HPLC using a gradient from 20 to 70% acetonitrile in

water to give 17 (13 mg, 25 μmol, 46%) as a colorless powder. HRMS

(ESI) m/z calcd for C₂₄H₃₃N₄O₆S [M + H]⁺505.2121, found

505.2121. ¹H NMR (600 MHz, CDCl₃) δ 8.12 (d, J = 9.9 Hz, 1H), 7.73

(d, J = 7.6 Hz, 1H), 7.57 (d, J = 6.9 Hz, 1H), 7.55 (d, J = 7.7 Hz, 1H),

7.44 (t, J = 7.7 Hz, 1H), 7.29−7.25 (m, 1H), 4.89−4.83 (m, 2H), 4.75

(td, J = 11.5, 2.5 Hz, 1H), 4.58−4.55 (m, 1H), 4.54 (d, J = 13.9 Hz, 1H),

4.32 (dt, J = 12.2, 4.3 Hz, 1H), 3.90−3.85 (m, 1H), 3.82 (d, J = 13.9 Hz,

1H), 3.53 (td, J = 9.4, 6.6 Hz, 1H), 2.93 (s, 6H), 2.90−2.86 (m, 1H),

2.65− 2.56 (m, 2H), 2.44−2.38 (m, 1H), 2.36−2.28 (m, 1H), 2.13 (s,

3H), 2.11−2.03 (m, 2H), 2.00−1.93 (m, 1H). ¹³C NMR (151 MHz,

CDCl₃) δ 172.0, 171.6, 170.2, 169.3, 169.1, 139.2, 132.2, 131.5, 131.2,

130.1, 127.0, 61.5, 60.7, 50.8, 49.4, 47.7, 36.8, 35.8, 32.7, 31.7, 29.3,

28.7, 24.9, 22.6.

2-((4S,10R)-4-(Dimethylcarbamoyl)-10-(hydroxymethyl)-14,14-

dimethyl-6,9,12-trioxo-13-oxa-2-thia-5,8,11-triazapentadecyl)-

benzoic Acid (37). Compound 36 (502 mg, 0.93 mmol, 1.0 equiv) was

dissolved in MeOH (5 mL) and 0.1 M aqueous LiOH (20 mL). After

being stirred at 35 °C for 16 h, the reaction was acidiﬁed with 1 M

aqueous HCl solution (5 mL) and extracted with DCM (3 × 50 mL).

The organic phase was dried over MgSO₄, ﬁltered, and then

concentrated under reduced pressure. The crude reaction mixture

was puriﬁed by ﬂash chromatography on a silica gel column using 0−

15% MeOH in DCM as the eluent to give 37 (240 mg, 0.46 mmol,

49%) as a colorless powder. HRMS (ESI) m/z calcd for C₂₃H₃₃N₄O₈S

[M − H]⁻525.2019, found 525.2023. ¹H NMR (500 MHz, DMSO-d₆)

δ 12.77 (br s, 1H), 8.20 (d, J = 8.3 Hz, 1H), 8.16−8.04 (m, 1H), 7.83

(dd, J = 7.6, 1.4 Hz, 1H), 7.48 (td, J = 7.6, 1.3 Hz, 1H), 7.40 (dd, J = 7.6,

1.3 Hz, 1H), 7.35 (td, J = 7.6, 1.4 Hz, 1H), 6.72 (d, J = 7.8 Hz, 1H),

4.88−4.70 (m, 1H), 4.13 (d, J = 13.0 Hz, 1H), 4.09 (d, J = 13.0 Hz, 1H),

4.01−3.96 (m, 1H), 3.79−3.65 (m, 2H), 3.56 (d, J = 5.5 Hz, 2H), 2.96

(s, 3H), 2.83 (s, 3H), 2.75 (dd, J = 13.5, 7.9 Hz, 1H), 2.48−2.42 (m,

1H), 1.39 (s, 9H). ¹³C NMR (126 MHz, DMSO-d₆) δ 171.2, 170.0,

169.0, 168.8, 155.7, 140.5, 131.9, 131.4, 131.4, 131.2, 127.6, 78.7, 62.4,

57.1, 48.7, 42.3, 37.1, 35.8, 34.4, 33.6, 28.6.

Methyl-(S)-2-(4-(dimethylcarbamoyl)-11,11-dimethyl-6,9-dioxo-

10-oxa-2-thia-5,8-diazadodecyl)benzoate (35). Compound 31 (4.16

g, 10.5 mmol, 1.0 equiv) was dissolved in 4 M HCl in dioxane (15 mL)

and stirred for 1 h at rt. After evaporation of the solvent under reduced

pressure, the resulting salt was dissolved in acetonitrile (35 mL). Boc-

Gly-OH (2.76 g, 15.8 mmol, 1.5 equiv), EDC·HCl (3.03 g, 15.8 mmol,

1.5 equiv), and DIPEA (1.83 mL, 10.5 mmol, 1.0 equiv) were added,

and the reaction was stirred for 2 h at rt. EtOAc (200 mL) was then

added, and the mixture was washed with 1 M aqueous HCl solution

(100 mL), saturated aqueous NaHCO₃solution (100 mL), and brine

(50 mL). The organic phase was dried over MgSO₄, ﬁltered, and then

concentrated under reduced pressure. The crude reaction mixture was

puriﬁed by ﬂash chromatography on a silica gel column using 0−10%

MeOH in DCM as the eluent to give 35 (3.81 g, 8.41 mmol, 80% over

two steps) as a colorless oil. HRMS (ESI) m/z calcd for C₂₁H₃₂N₃O₆S

[M + H]⁺454.2012, found 454.2002. ¹H NMR (400 MHz, CDCl₃) δ

7.90 (dd, J = 7.8, 1.3 Hz, 1H), 7.44 (td, J = 7.6, 1.3 Hz, 1H), 7.37 (dd, J =

7.6, 1.3 Hz, 1H), 7.31 (td, J = 7.8, 7.3 Hz, 1H), 6.90 (d, J = 7.1 Hz, 1H),

5.24−5.14 (m, 1H), 5.12−5.02 (m, 1H), 4.17 (d, J = 13.1 Hz, 1H), 4.07

(d, J = 13.1 Hz, 1H), 3.90 (s, 3H), 3.85−3.73 (m, 2H), 3.03 (s, 3H),

tert-Butyl-((4S,10R)-4-(dimethylcarbamoyl)-6,9,13-trioxo-

1,4,5,6,7,8,9,10,11,13-decahydro-3H-benzo[m][1]oxa[11]thia[5,8]-

diazacyclopentadecin-10-yl)carbamate (38). Compound 38 was

prepared following the procedure described for the synthesis of

compound 23a using 37 (130 mg, 0.25 mmol, 1.0 equiv),

triphenylphosphine (131 mg, 0.50 mmol, 2.0 equiv), and di-tert-butyl

azodicarboxylate (115 mg, 0.50 mmol, 2.0 equiv). The crude product

was puriﬁed by reverse-phase HPLC using a gradient from 20 to 70%

acetonitrile in water to give 38 (25 mg, 50 μmol, 20%) as a colorless

powder. HRMS (ESI) m/z calcd for C₂₃H₃₃N₄O₇S [M + H]⁺509.2070,

found 509.2068. ¹H NMR (500 MHz, CDCl₃) δ 7.72 (d, J = 7.5 Hz,

1H), 7.56−7.47 (m, 1H), 7.41−7.36 (m, 1H), 7.36−7.26 (m, 3H), 5.87

(d, J = 6.7 Hz, 1H), 4.93−4.86 (m, 1H), 4.84 (d, J = 11.0 Hz, 1H),

4.69−4.61 (m, 1H), 4.39 (dd, J = 11.1, 5.7 Hz, 1H), 4.23−4.13 (m,

1H), 4.09 (d, J = 13.3 Hz, 1H), 3.93 (d, J = 13.6 Hz, 1H), 3.63−3.55 (m,

1H), 3.08 (s, 3H), 2.93 (s, 3H), 2.84−2.75 (m, 1H), 2.66−2.55 (m,

1H), 1.45 (s, 9H). ¹³C NMR (126 MHz, CDCl₃) δ 170.1, 169.9, 168.8,

167.4, 155.3, 139.1, 131.7, 130.7, 130.5, 130.2, 127.4, 80.5, 65.2, 53.1,

48.8, 43.7, 37.3, 36.1, 35.4, 33.9, 28.3.

N

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(4S,10R)-10-((S)-1-Acetylpyrrolidine-2-carboxamido)-N,N-di-

methyl-6,9,13-trioxo-1,4,5,6,7,8,9,10,11,13-decahydro-3H-benzo-

[m][1]oxa[11]thia[5,8]diazacyclopentadecine-4-carboxamide (18).

Compound 18 was prepared following the procedure described for the

synthesis of compound 6 using 38 (20 mg, 39 μmol, 1.0 equiv), Ac-^L-

Pro-OH (19 mg, 0.12 mmol, 3.0 equiv), EDC·HCl (23 mg, 0.12 mmol,

3.0 equiv), and DIPEA (21 μL, 0.12 mmol, 3.0 equiv). The crude

product was puriﬁed by reverse-phase HPLC using a gradient from 25

to 75% acetonitrile in water to give 18 (9 mg, 16 μmol, 41%) as a

colorless powder. HRMS (ESI) m/z calcd for C₂₅H₃₄N₅O₇S [M + H]⁺

548.2179, found 548.2128. ¹H NMR (500 MHz, CDCl₃) δ 8.20 (dd, J =

7.6, 4.7 Hz, 1H), 7.88 (dd, J = 7.8, 1.5 Hz, 1H), 7.59 (d, J = 9.3 Hz, 1H),

7.50 (d, J = 7.7 Hz, 1H), 7.47 (td, J = 7.5, 1.5 Hz, 1H), 7.40 (dd, J = 7.7,

1.3 Hz, 1H), 7.36 (td, J = 7.5, 1.4 Hz, 1H), 5.18 (dd, J = 11.3, 2.3 Hz,

1H), 4.94 (dt, J = 9.3, 2.8 Hz, 1H), 4.86 (td, J = 8.1, 4.6 Hz, 1H), 4.47

(dd, J = 11.2, 3.3 Hz, 1H), 4.37−4.25 (m, 3H), 3.82 (d, J = 13.5 Hz,

1H), 3.72−3.66 (m, 1H), 3.62−3.49 (m, 2H), 3.13 (s, 3H), 2.94 (s,

3H), 2.89−2.80 (m, 2H), 2.29−2.14 (m, 3H), 2.12 (s, 3H), 2.03−1.94

(m, 1H). ¹³C NMR (126 MHz, CDCl₃) δ 172.7, 170.6, 170.4, 169.1,

168.9, 168.2, 139.1, 132.2, 131.4, 130.8, 129.7, 127.6, 64.3, 61.2 52.4,

48.6, 48.0, 44.0, 37.4, 36.2, 36.1, 34.9, 30.0, 25.4, 22.5.

Calculation of Descriptors and PCA Analysis. Two-dimensional

structures from the DNP and DrugBank¹⁶databases were imported

into MOE (version 2015.10, Chemical Computing Group, www.

chemcomp.com).⁴⁹Both data sets were cleaned by addition of explicit

hydrogen atoms, removal of organometallic compounds, and adjust-

ment of protonation states (i.e., strong acids were deprotonated and

strong bases were protonated). Duplicate structures were eliminated

using the 2D SMILES string of each compound. Molecular descriptors

were calculated for all compounds in the DNP and in DrugBank, and for

the 41 lead-like macrocycle cores identiﬁed from the DNP, using the

MOE descriptor module.⁵⁰Principal component analysis (PCA) was

performed using SIMCA (version 14.1)⁵¹to identify clusters and

investigate the coverage of the chemical space of the DNP and

DrugBank.

Extraction of Macrocyclic Cores from the DNP. The structures

of the natural products contained in the sd-ﬁle version of the Dictionary

of Natural Products (DNP) were cleaned as described previously,

whereby natural products containing toxic or reactive groups are

removed using the knowledge-based HTS ﬁlter in Pipeline Pilot.¹²

Further ﬁltering in Pipeline Pilot was performed to retain macrocycles

(≥12 and ≤30 atoms in the ring) having <10 rings in total and a MW <

2500 Da to provide 3657 macrocyclic natural products.

The side chains of the macrocyclic natural products were then

pruned to provide macrocycle cores using the ﬁrst two steps of the

fragment generation algorithm reported previously,¹²i.e., deglycosyla-

tion and extraction of extended Murcko frameworks¹⁵decorated with

attachment points (Figure S2). In these two steps, macrocycles were

not fragmented and all ring systems and linker atoms were kept while

linear side chains were pruned, but not removed. This provided

macrocycle cores, i.e., macrocycles decorated with attachment points,

which are functional groups linked directly to the ring system or pruned

side chains. Thus, the algorithm was designed to retain functional

groups and only cut side chains at carbon−carbon bonds at a distance of

>2 atoms from the ring.

connected to a heteroatom by a double bond, the immediate next

neighbor atom in the other branch will be investigated using the same

rules as for a linear side chain, irrespective of its distance from the ring.

The next upcoming carbon atom is kept, as the terminating carbon, but

the rest of the side chain is pruned. If the branching atom is a

heteroatom, the immediate next neighbor atoms will be investigated in

the same manner, irrespective of their distance from the ring. As a result

of this pruning process, the original functional group motif of the

macrocyclic part of the natural product remains intact in the core. In

addition, functional groups close to the macrocycle ring in side chains

remain intact. All generated macrocycle cores are stored in a database

for further processing.

If identical macrocyclic cores were obtained after side-chain pruning

of diﬀerent natural products, only one was kept. Cores with ≤3 O or N

atoms or >15 rotatable bonds, without a heteroatom in the macrocycle,

and/or having more than one macrocycle were also removed in Pipeline

Pilot. This resulted in 2175 natural-product-derived macrocycle cores,

which are based on 764 diﬀerent Murcko scaﬀolds. The 2175 cores

were clustered in Pipeline Pilot using an FCFP6 pharmacophore

ﬁngerprint⁵²into 217 clusters (Supporting Excel Sheet). The cluster

centers were visually inspected by three or more experienced medicinal

chemists, and 41 cluster centers or near neighbors were selected by

exclusion of cores containing reactive or potentially redox-sensitive

groups, excessively complex structures including >6 rings, or

oligomeric/pseudo-oligomeric structures (Supporting Excel Sheet).

Docking of Macrocycle Cores into Keap1. The atomic

coordinates of four crystal structures of human Keap1, in complex

with a small molecule [PDB ID: 4IQK²⁴(1.97 Å) and 3VNG (2.1 Å)]

or in the apo form [PDB ID: 4IFJ (1.8 Å) and 1ZGK²⁵(1.35 Å)], were

obtained from the Protein Data Bank. Each crystal structure was

imported and reﬁned with Protein Preparation Wizard⁵³using the

54

̈

Schrodinger Suite. Structure reﬁnement included adding hydrogen

atoms, assigning bond orders, building disulﬁde bonds, and removal of

water molecules beyond 5 Å from the ligand atoms. The PROPKA tool

from Protein Preparation Wizard was used to predict the protonation

states of the ionizable residues at pH 7.0.⁵⁵Subsequently, the positions

of the hydrogen atoms in the Keap1 structure were energy-minimized

using the OPLS3 force ﬁeld.⁵⁶

The receptor grid generation module of Glide²⁶was used to deﬁne

the active site for the docking experiments. The active site of Keap1 was

deﬁned either by the bound ligand or by key residues in the active site

(R415, R483, Y525, Y572, A556, S603), which were used as the

centroid of the grid box (the radius of the active site was 15 Å from the

centroid). The docking protocol was ﬁrst validated by comparing the

conformation (the pose) of the bound ligands as obtained from docking

with the one determined by X-ray crystallography for the two ligand-

bound structures (PDB ID: 4IQK²⁴and 3VNG; see the Supporting

Information, Computational Procedure 1). Subsequently, the set of 41

cores was preprocessed using LigPrep, which included generating

possible ionization states (at pH 7) using the Epik tool⁵⁷and structure

minimization using the OPLS3 force ﬁeld.⁵⁶During preprocessing,

input chirality was retained.

The 41 lead-like macrocyclic cores (Table S1) were then docked into

each of the four reﬁned Keap1 structures using Glide, which treats the

cores as fully ﬂexible while Keap1 remains rigid during the docking.

Docking poses for each macrocycle core were ranked according to the

GlideScore from the standard precision mode, and for each core, the

docked pose that had the lowest GlideScore for binding to each of the

Keap1 structures was identiﬁed (Table S2). The resulting combined list

of the top-10 cores for each Keap1 structure was then used to rank the

cores by the number of Keap1 structures each core bound to (Table

S3), after which the docked poses of the top-ﬁve cores in each of the

four crystal structure were inspected visually (Figure S5).

The algorithm deﬁnes an attachment point by traversing through the

side chain of the macrocycle through two heavy atoms, starting from the

ring-based atom. For each heavy atom thereafter it decides, based on a

set of rules,¹²whether the atom is kept, whether that atoms neighboring

atoms are examined, or if the side chain is cut after the second heavy

atom. The algorithm keeps aliphatic carbon atoms as long as they are

within two atoms from the ring, and longer aliphatic side chains are

pruned to ethyl groups. If the algorithm encounters a carbon atom

linked to a non-carbon and non-hydrogen atom, it will keep the

heteroatom and examine the immediate neighbor atom, irrespective of

its distance from the ring. The algorithm prunes after the next

upcoming carbon atom as long as this is not double-bonded to a

heteroatom.

Induced-ﬁt docking (IFD)⁵⁸of stereoisomeric macrocycles 6−9 was

dinger suite.^58,59Three-

̈

performed as implemented in the Schro

dimensional structures of compounds 6−9 were built with Maestro

dinger suite⁶⁰and energy-minimized using the OPLS3

̈

from the Schro

force ﬁeld. A grid centered at the bound ligand (PDB ID: 4IQK²⁴) with

the box size of 15 Å was deﬁned. Subsequently, 6−9 were docked and

scored using the standard precision mode with an extended sampling

In branched side chains, the algorithm traverses the branches

separately, within the distance limit. If the branching carbon is

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protocol, which will generate up to 80 poses per ligand from several

docking runs. The cyclothialidine core was included for comparison. A

total of 286 binding poses were analyzed with regard to if the phenyl

ring of 1 and 6−9 reached deep into the Kelch channel of the Keap1

binding pocket (Figure S6).

Binding Aﬃnity Calculations. The atomic coordinates of Keap1

in complex with compound 14 were preprocessed as described above in

the docking section. The chloride ion in the ligand binding pocket was

included in the binding aﬃnity calculations as it is coordinated with

residues in Keap1 (N382, N414, and R415) and with 14. Methyl ester

(9), amide (12), monomethyl amide (13), and diethylamide (15) were

modeled into the Keap1 binding site using the bound conformation of

14 as a template. For compounds 9 and 13, two alternative

conformations of the methyl ester and methyl amide (cis- and trans-

forms) were explored, while ﬁve alternative conformations were

explored for the diethylamide of 15 (Tables S6 and S7).

of 50 μM Nrf2-peptide in 10 mM Na-acetate, pH 4.0 for 2 min. Any

reactive groups still present on the surface were deactivated by a 7 min

injection of 0.1 M ethanolamine hydrochloride-NaOH, pH 8.5. Peptide

immobilization levels were typically between 400 and 600 RU to ensure

mass transport limitation and thus a protein-concentration-dependent

response.

For the detection of active compounds, a solution of 25 nM Keap1

(Kelch domain = aa321−aa609) was prepared in running buﬀer and

preincubated with compounds at either constant or varying

concentrations. These mixtures were subsequently injected over the

peptide-modiﬁed biosensor, and control samples devoid of compounds

were used to determine the remaining free protein concentration of

Keap1 in response to compound binding. For this, the initial association

slopes of the binding sensorgrams have been measured (interval 5−15 s

after sample injection) for each sample, followed by a regeneration of

the sensor for the next cycle through a 45 s injection of 50 mM Tris/Cl,

0.25% SDS, 5 mM TCEP, pH 7.5. Results have been reported as K_D-

values for concentration-response experiments. This was achieved by a

nonlinear regression analysis of the concentration-response data, which

is normalized by the control samples representing 0% inhibition.

SPR Direct Binding Assay (DBA). The SPR DBA was performed

as described previously,³¹with the diﬀerence that HBS-P [10 mM

HEPES, 150 mM NaCl, 0.05% (v/v) Tween 20, 1.0 mM TCEP, pH

7.40] was used as a continuous ﬂow buﬀer and that the contact and

dissociation times were 45 and 60 s, respectively.

Prior to aﬃnity calculations, the ligand binding site was deﬁned using

the receptor grid generation module available in Glide as described in

the docking section above. Subsequently, the initial input ligand

coordinates for 9 and 12−15 were optimized in the receptor, the ligand

binding pose was scored, and poses were used for ligand binding aﬃnity

calculations in Prime MM-GBSA³⁴as implemented in the Schrodinger

̈

suite. The ligand and a 5 Å region of the protein around the ligand were

treated as ﬂexible in the binding aﬃnity calculation. Other settings were

chosen as defaults.

Similarity to Reported Inhibitors of the Keap1-Nrf2 PPI. A set

of inhibitors of the Keap1-Nrf2 PPI, termed “PubChem”, was retrieved

from the PubChem Bioassay database (PubChem Bioassay AIDs:

504523; 588683; 651798; 651801; 651806; 651807; 651823; 651829;

651833; 651834).³⁸Duplicate entries, compounds that lacked

complete stereochemical information, and solvents were removed

from the 1127 compounds reported as active in the 10 listed bioassays.

Compounds that did not pass the PAINS ﬁlter³⁹were also removed,

providing a PubChem set consisting of 375 inhibitors (Table S8).

Another set of “validated” inhibitors was assembled from two sources.

First, nine compounds, reported to show reproducible activity in a triad

of orthogonal assays by Tran et al.,²⁰were selected. Second, crystal

structures that had a compound bound in the binding site for Nrf2 of

Keap1 were retrieved from the Protein Data Bank (PDB). Peptides

were excluded, while fragments were retained, providing a validated set

of 35 compounds (Table S9).

Isothermal Titration Calorimetry (ITC). Isothermal titration

calorimetry experiments were carried out on a MicroCal ITC-200

system (GE Healthcare) using Keap1 protein (Kelch domain = aa321−

aa609) that had been passed through a PD-10 column (GE Healthcare)

equilibrated with 10 mM HEPES, 150 mM NaCl, 0.05% (v/v) Tween

20, 1 mM TCEP, pH 7.40, and 1% DMSO. Complete titration of 60 μM

Keap1 was typically achieved by injecting 19 × 2 μL of aliquots of a 0.6

mM compound at a temperature of 25 °C and a spacing interval of 90 s

between injections. The thermodynamic binding parameters were

extracted by nonlinear regression analysis of the binding isotherms

(Microcal Origin version 7.0 software package). A single site binding

model was applied, yielding binding enthalpy (ΔH), stoichiometry (n),

entropy (ΔS), and association constant (K_a).

Aqueous Solubility. The aqueous solubility of compound 14 was

determined at AstraZeneca as reported previously.⁶²

Cell Permeability. The eﬄux-inhibited permeability of compound

14 across Caco-2 cell monolayers was determined in the presence of a

cocktail of inhibitors of eﬄux transporters (50 μM quinidine, 30 μM

benzbromarone, and 20 μM sulfasalazine) by the DMPK department at

Pharmaron as reported previously.⁶³

Clearance by Human Liver Microsomes. The clearance of

compound 14 by human liver microsomes (CL_int) was determined at

AstraZeneca as reported previously.⁶²

The Tanimoto coeﬃcient⁴⁰was calculated to determine the

similarity between compound 14 and the Keap1 inhibitors in the

validated and PubChem sets, respectively, using seven commonly used

structural ﬁngerprints (Figure S11). The similarity was also calculated

based on all ﬁngerprints. All substructure similarity analyses were

performed with the Canvas tool (version 3.1.011).⁶¹The molecular

similarity landscape (a so-called networklike similarity graph) was

investigated for the merged validated and PubChem sets and for the

validated set alone, using compound 14 as a reference. The structures of

all compounds were imported, and a substructure fragment ﬁngerprint

(FragFp)-based similarity landscape was computed using DataWarrior

(version 5.2.1).⁴¹FragFp contains more than 500 predeﬁned binary

structural ﬁngerprints. DataWarrior calculates a similarity matrix for

each compound in the data set and allocates each compound to the

most similar neighbor space using the Rubberbanding force-ﬁeld

approach.⁴¹Compounds with a structural similarity >0.95 were

clustered for the analysis.

SPR Inhibition in Solution Assay (ISA). The SPR ISA was

performed in analogy to the protocol developed by Chen et al.³⁰with

the following diﬀerences. Instead of a biotinylated peptide, a lysine-

tagged version of the Nrf2-peptide (KKKKAFFAQLQLDEETGEFL)

was utilized for tethering to the sensor surface. For the covalent

tethering, a CM5 biosensor (GE Healthcare, research grade) was

employed using HBS-P [10 mM HEPES, 150 mM NaCl, 0.05% (v/v)

Tween 20, 1.0 mM TCEP, pH 7.40] as the continuous ﬂow buﬀer at a

rate of 10 μL/min at 20 °C on a BIAcore 3000 optical biosensor unit

(GE Healthcare). The surface was activated by injecting a mixture of 1-

ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)

and N-hydroxysuccinimide (NHS) for 7 min, followed by an injection

Cell Assay for 14. The cellular activity of macrocycle 14 was

determined by DiscoveRx using their PathHunter U2OS Keap1-Nrf2

functional assay,⁶⁴which quantiﬁes the translocation of Nrf2 into the

nucleus due to inhibition of formation of the Keap1-Nrf2 complex.

Keap1 Expression, Puriﬁcation, and Crystallization with 14.

The DNA coding domain for human Keap1 (6 × His- TCS-Keap1

(A321-T609) [E540A, E542S]) was cloned into pET24 and expressed

in Escherichia coli BL21(DE3)-Star in LB media at 291 K. After harvest,

the cells were resuspended in 20 mM Tris/HCl, pH 7.5, 200 mM NaCl,

5% glycerol, 1 mM DTT, protease inhibitors, disrupted with a high-

pressure homogenizer, and clariﬁed by centrifugation. Puriﬁcation was

made by aﬃnity chromatography using Ni Sepharose FF (GE

Healthcare), eluted in one step with 300 mM imidazole followed by

SEC (Superdex200, GE Healthcare). The puriﬁed protein was

detagged at room temperature with a thrombin cleave kit (Sigma)

and ﬁnalized by a second SEC step (Superdex200, GE Healthcare) in

buﬀer 20 mM Tris/HCl, pH 7.5, 5 mM DTT before concentration

(Amicon cells, 10 kDa cutoﬀ). Crystals were grown at 20 °C in sitting

drops using 200 nL of protein (11−13 mg/mL) and 200 nL of well

solution (3.7−4.1 M ammonium acetate, 0.09 M sodium acetate pH

4.6, and 10 mM cadmium chloride). Crystals appeared after one day but

continued to grow for approximately one week. Compound 14 was

P

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soaked by incubating crystals for 1 h with 1 mM compound in 5 M

ammonium acetate and 0.1 M sodium acetate at pH 4.6. Crystals were

subsequently frozen in liquid nitrogen using a soaking solution

supplemented with 20% glycerol as the cryoprotectant prior to data

collection.

AstraZeneca, 43183 Mölndal, Sweden; orcid.org/0000-

0002-2154-8345

Patrik Johansson − Structure, Biophysics & Fragment-Based

Lead Generation, Discovery Sciences, R&D, AstraZeneca,

43183 Mölndal, Sweden

Data Collection, Structure Solution, and Reﬁnement. X-ray

diﬀraction data was collected at the European Synchrotron Facility,

beamline ID29, using a Pilatus 6M-F pixel detector to a resolution of

2.37 Å. The data were indexed and integrated with MOSFLM⁶⁵and

scaled with SCALA⁶⁶in the space group P2₁2₁2₁with cell dimensions of

75.7 75.8 203.1 Å. Two Keap1 molecules were identiﬁed by PHASER⁶⁷

using a published Keap1 structure (PDB ID: 1ZGK²⁵) as a search

model. The Keap1 macrocycle complex was further reﬁned by

alternative cycles of model rebuilding in Coot⁶⁸and reﬁnement in

AutoBuster 2.11.6 (Global Phasing Ltd, Cambridge U.K.). A ﬁnal

model was reﬁned to an R/Rfree of 19/21%. Full data collection and

reﬁnement statistics can be found in Table S5, and the 2Fo-Fc electron

density of compound 14 is in Figure S8. The coordinates and

corresponding structure factors have been deposited to the Protein

Data Bank with accession code 6Z6A.

Mohit Tyagi − Department of Chemistry - BMC, Uppsala

University, 75123 Uppsala, Sweden

Christian Tyrchan − Department of Medicinal Chemistry,

Research and Early Development, Respiratory and

Immunology, BioPharmaceuticals R&D, AstraZeneca, 43183

Mölndal, Sweden

Lisa Wissler − Structure, Biophysics & Fragment-Based Lead

Generation, Discovery Sciences, R&D, AstraZeneca, 43183

Mölndal, Sweden

̈

Peter Sjo − Drugs for Neglected Diseases initiative (DNDi),

1202 Geneva, Switzerland

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.0c01569

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01569.

Author Contributions

■

^#F.B., V.P., and B.O. made equal contributions to the

manuscript. The manuscript was written through contributions

of all authors. All authors have given approval to the ﬁnal version

of the manuscript.

sı

Supporting tables, supporting ﬁgures, computational

procedures, NMR spectra of all new compounds, purity

reports for compounds 6−18 (PDF)

Structures (SMILES codes) for the sets of 41 and 217

macrocyclic cores (XLSX)

Funding

This work was funded by grants from the Swedish Research

Council (grant no. 2016-05160) and AstraZeneca.

Notes

The authors declare the following competing ﬁnancial

interest(s): M.C., S.G., P.J., C.T., L.W., and S.S. are employees

and shareholders of AstraZeneca. P.S. is an employee of Drugs

for Neglected Diseases initiative (DNDi).

Structures (SMILES codes) and biological properties for

compounds 6−18, (CSV)

Accession Codes

Coordinates and structure factors for the complex of Keap1 with

compound 14 have been deposited with the PDB with accession

code 6Z6A. The authors will release the atomic coordinates and

experimental data upon article publication.

ACKNOWLEDGMENTS

■

We thank Dr. Matthew Perry at AstraZeneca for contributions

to the selection of the 41 lead-like macrocycle cores and for

comments on the manuscript and Dr. Werngard Czechtizky at

AstraZeneca for providing valuable comments on the manu-

script.

AUTHOR INFORMATION

■

Corresponding Authors

Stefan Schiesser − Department of Medicinal Chemistry,

Research and Early Development, Respiratory and

Immunology, BioPharmaceuticals R&D, AstraZeneca, 43183

Mölndal, Sweden; orcid.org/0000-0002-8668-2844;

Email: stefan.schiesser@astrazeneca.com

Jan Kihlberg − Department of Chemistry - BMC, Uppsala

University, 75123 Uppsala, Sweden; orcid.org/0000-

0002-4205-6040; Email: jan.kihlberg@kemi.uu.se

ABBREVIATIONS

■

bRo5, beyond the rule of 5; Cl_int, human microsomal

metabolism; cLogS, calculated solubility; DNP, Dictionary of

Natural Products; ISA, inhibition in solution assay; ITC,

isothermal titration calorimetry; Keap1, Kelch-like ECH

Associated Protein-1; Nrf2, nuclear factor, erythroid 2 Like 2;

NRotB, number of rotatable bonds; P_appAB + inh, eﬄux-

inhibited permeability across a Caco-2 cell monolayer; PPI,

protein−protein interaction; SPR, surface plasmon resonance;

TCE, 2,2,2-trichloroethyl; TPSA, topological polar surface area

Authors

Fabio Begnini − Department of Chemistry - BMC, Uppsala

University, 75123 Uppsala, Sweden

Vasanthanathan Poongavanam − Department of Chemistry -

BMC, Uppsala University, 75123 Uppsala, Sweden;

orcid.org/0000-0002-8880-9247

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