Beyond Basicity: Discovery of Nonbasic DENV-2 Protease Inhibitors with Potent Activity in Cell Culture

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Beyond Basicity: Discovery of Nonbasic DENV‑2 Protease Inhibitors

with Potent Activity in Cell Culture

̈

Nikos Kuhl, Mila M. Leuthold, Mira A. M. Behnam, and Christian D. Klein*

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ABSTRACT: The viral serine protease NS2B-NS3 is one of the promising

targets for drug discovery against dengue virus and other ﬂaviviruses. The

molecular recognition preferences of the protease favor basic, positively charged

moieties as substrates and inhibitors, which leads to pharmacokinetic liabilities

and oﬀ-target interactions with host proteases such as thrombin. We here

present the results of eﬀorts that were aimed speciﬁcally at the discovery and

development of noncharged, small-molecular inhibitors of the ﬂaviviral

proteases. A key factor in the discovery of these compounds was a cellular

reporter gene assay for the dengue protease, the DENV2proHeLa system.

Extensive structure−activity relationship explorations resulted in novel

benzamide derivatives with submicromolar activities in viral replication assays

(EC₅₀0.24 μM), selectivity against oﬀ-target proteases, and negligible cytotoxicity. This structural class has increased drug-likeness

compared to most of the previously published active-site-directed ﬂaviviral protease inhibitors and includes promising candidates for

further preclinical development.

arginine or arginine mimetics.^10,15−18This accumulation of

positive charge is problematic with respect to pharmacokinetics

and oﬀ-target interactions with host proteases such as trypsin,

thrombin, and generally with proteases from the blood clotting

INTRODUCTION

■

Dengue is a mosquito-borne viral disease characterized by high

fever, headache, muscle pain, and rash.^1,2There are four

distinct serotypes of dengue viruses (DENV) that cause

dengue fever. Recovery from infection provides lifelong

immunity against the involved serotype. Subsequent infections

by other serotypes increase the risk of developing a severe form

of dengue fever, which can threaten patients’ lives.^3,4The

global incidence of dengue has grown dramatically in recent

years.^3,4This can be explained by population growth and

density, inadequate public health, increased travel, and growing

vector distribution.^1,5,6Speciﬁc antiviral drugs against

ﬂaviviruses are not available, and the treatment remains

supportive, focused on pain and fever control as well as ﬂuid

management.⁷A recently approved vaccine (Dengvaxia)

performs diﬀerently in seropositive and seronegative patients,

and its use is therefore highly restricted.^8,9

Like other ﬂaviviruses, dengue virus contains a positive-sense

single-stranded RNA that is translated by host cell proteins

into a single polyprotein. The viral serine protease NS2B-NS3

processes the viral polyprotein into functional structural and

nonstructural proteins^10,11and is therefore considered as a

promising drug target.^10,12

cascade.

Recently, we reported a novel class of compounds with

lower molecular mass than previously published DENV

protease inhibitors that incorporate a single cationic moiety.¹⁹

The most active compounds contain a guanidine moiety (see

Figure 1).²⁰Oﬀ-target activities of this compound class were

signiﬁcantly reduced in comparison to peptidic inhibitors, but

the pharmacokinetic properties (passive membrane perme-

ation) remained problematic.

Eﬀorts to increase activity in NS2B-NS3 drug discovery, and

in medicinal chemistry in general, are frequently associated

with increased molecular weight. In addition, inhibitors of

ﬂaviviral proteases frequently incorporate highly charged

guanidino or similar functional groups because of the

recognition preference of the enzyme for basic residues.

Therefore, a challenge arises to reconcile the two opposing

development aims of pharmacodynamic and pharmacokinetic

optimization, where basicity can be considered a “friend and

The hydrophilic domain of the membrane-associated

nonstructural protein 2B is required as a cofactor for the

protease activity in NS3. The protease has a strong preference

for substrates with dibasic sequences in P₁and P₂.^10,13,14Since

most of the known peptidic or peptide-hybrid inhibitors are

based on those sequences, the existing chemotypes of

inhibitors feature two or more basic functionalities, frequently

Received: November 24, 2020

Published: April 14, 2021

https://doi.org/10.1021/acs.jmedchem.0c02042

J. Med. Chem. 2021, 64, 4567−4587

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Figure 1. Recently published basic DENV NS2B-NS3 inhibitors¹⁹and the novel uncharged inhibitor series presented here.

a

Scheme 1. Synthesis of Various Cap Moieties

a

(a) Benzyl bromide, K₂CO₃, DMF, rt to 80 °C, 72 h; (b) 2 M NaOH/MeOH (1:1), 50 °C; (c) HCl; (d) SOCl₂, MeOH, 0 °C to reﬂux,

overnight; (e) respective phenylboronic acid, Cu(OAc)₂, pyridine, 4 Å sieves, DCM; (f) LiOH, THF/H₂O (2:1); (g) 1-bromo-3-methylbutane,

K₂CO₃, DMF, 100 °C, overnight; (h) 2 M NaOH, 60 °C, overnight.

foe”, respectively. This was also noticed in our own attempts,

and the present work was therefore particularly aimed at a

reduction of molecular size and polarity in order to approach

the expected criteria for oral bioavailability, as described in the

Lipinski rule of ﬁve and similar concepts.^21,22Extensive

structure−activity relationship (SAR) studies were conducted

to identify structural elements that would allow the reduction

of basicity while maintaining or improving the inhibitory

activity. We herein present the synthesis, cellular structure−

activity relationships, and in vitro pharmacokinetic and oﬀ-

target evaluation of this inhibitor series.

in detail before,¹⁹to be a crucial factor in the discovery and

development of this compound class. The stable cotransfection

of the DENV-2 NS2B-NS3 protease and an ODD-luciferase

reporter construct allows high-throughput determination of

intracellular DENV protease inhibition. Treatment of

DENV2proHeLa cells with DENV-2 protease inhibitors

decreases the Renilla reniformis luciferase signal signiﬁcantly.

In our previous work, we noticed a signiﬁcant noncorrelation

of biochemical assay DENV protease inhibitory values vs both

antiviral eﬃcacy and activity in the DENV2proHeLa reporter

gene system. In contrast, the DENV2proHeLa system showed

a relatively good correlation to antiviral eﬃcacy in viral

replication assays, the latter being considered the “gold

We consider the application of a cellular DENV-2 protease

reporter gene assay (DENV2proHeLa), which was described

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a

Scheme 2. Synthesis of Phenoxybenzoic Acid Cap Moieties

a

(a) SOCl₂, MeOH, 0−50 °C, 5 h; (b) respective phenol derivative, Cu(OAc)₂, pyridine, 4 Å sieves, DCM; (c) LiOH, THF/H₂O (2:1); (d) HCl.

a

Scheme 3. Exemplary Solid-Phase Peptide Synthesis on Rink Amide Resin

a

(a) Piperidine, DMF; (b) NaHCO₃, Fmoc-OSu, H₂O/ACN, 0 °C to rt; (c) COMU, TMP, DMF; (d) Fmoc-4-benzyloxy-L-phenylglycine,

COMU, TMP, DMF; (e) 4-phenoxybenzoic acid, COMU, TMP, DMF; (f) TFA, H₂O, TIPS (95:4:1, v/v).

standard” for antiviral drug discovery. In other words,

DENV2proHeLa appears to be a suitable surrogate for the

tedious, time-consuming, and low-throughput viral replication

assay. These observations, and the promising discovery of the

lead compound with the DENV2proHeLa system, led us to

favor this cellular assay as the primary SAR guidance tool in the

present work.

benzyl bromide at 80 °C for 72 h and treatment with 2 M

NaOH/MeOH (1:1) at 50 °C. In a similar reaction, 1-bromo-

3-methylbutane was used for O-alkylation of 90 at 100 °C

overnight.

The 4-benzyloxyphenylglycine derivatives were connected

via amide bonds to various carboxylic acids and amines. The

synthesis was executed by solid-phase peptide synthesis

according to the Fmoc protocol or in solution. Synthetic

routes are outlined in Schemes 3 and 4. Phenylglycine

racemization was prevented by COMU/TMP conditions for

all peptide coupling reactions on resin.²⁷Benzamide

compounds were synthesized using Rink amide resin as

presented in Scheme 3. Amine building blocks were N-Fmoc

protected in solution using Fmoc-OSu (see the Supporting

Information, compounds 143−151) before coupling to the

resin under COMU/TMP conditions overnight.

Chlorotrityl chloride (CTC) resin was used for direct

syntheses of compound 25 and the intermediates 101 and 102.

Compound 102 was coupled to the respective amine building

block by peptide coupling in solution with HATU/TMP. The

reaction was quenched with 5% TFA in DCM after two hours

to avoid racemization of 4-benzyloxyphenylglycine (see

Scheme 4). CTC resin was also used for syntheses of the

hydroxyl compounds 9 and 10. Therefore, the respective

hydroxyl building blocks were N-Fmoc protected in solution

RESULTS AND DISCUSSION

■

Chemistry. 4-Hydroxyphenylglycine was O-alkylated using

alkyl halogenides as previously described.^16,19Synthetic

procedures and analytical data for the phenylglycine derivatives

are provided in the Supporting Information (compounds 115−

135). 4-Phenoxyphenylglycine of compound 66 was synthe-

sized by copper-mediated O-arylation with phenylboronic acid

as described by Evans et al.²³4-Phenoxybenzoic acid

derivatives were synthesized via the same procedure (Schemes

1 and 2): 4-Hydroxybenzoic acid and 4-carboxyphenylboronic

acid were protected as methyl esters with thionyl chloride at

increased temperature (compounds 91 and 92). The O-

arylation of the intermediates was performed with copper-

catalyzed Chan−Evans−Lam coupling and can be conducted

in air at room temperature.²³⁻²⁶LiOH in THF/H₂O (2:1) was

used to cleave the methyl ester to yield the free carboxylic acid

compounds. Cap 89 was synthesized by O-alkylation with

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a

Scheme 4. Exemplary Solid-Phase Peptide Synthesis on Chlorotrityl Chloride Resin and Peptide Coupling in Solution

a

(a) Fmoc-4-benzyloxy-L-phenylglycine, TMP, DMF; (b) DCM/MeOH/TMP (80:15:5, v/v); (c) piperidine, DMF; (d) hexanoic acid, COMU,

TMP, DMF; (e) TFA, DCM; (f) HATU, respective amine derivative, TMP, DCM/DMF, 0 °C to rt; (g) TFA in DCM (5%, v/v), (7−92% yield).

with Fmoc-OSu (see the Supporting Information) before

coupling to the solid support in pyridine overnight.

for the guanidine moiety while maintaining or increasing

cellular antiviral activity and selectivity against oﬀ-targets. As

outlined in Table 1, a variety of nonbasic replacements for the

guanidinophenyl moiety were studied, based on the lead

compound with a 4-benzyloxyphenylglycine moiety and a

hexanoic acid cap. Whereas the 4-chlorobenzyl (compound 4),

4-methoxyphenyl (5), and unsubstituted benzyl (2) derivatives

had no relevant activity, the 4-methoxybenzyl (6), 4-

hydroxyphenethyl (10), 4-(phenyl)methanol (9), and 4-

acetamidobenzyl (21) derivatives had activities (EC₅₀)

between 10 and 20 μM. Moving into the structural vicinity

of the guanidino group, para- and meta-substituted benzamides

yielded one-digit micromolar inhibition values and were

therefore considered potential noncharged guanidine replace-

ments at this position (compounds 14−19). This represents an

analogy to earlier reports on meta-benzamide derivatives

described as guanidine isosters to achieve binding in the S1

pockets of factor Xa³⁴and the tissue factor/factor VIIa

complex.^35,36The urea, hydrazide, and hydroxamic acid

analogs 22, 23, and 24 are also similar to the guanidine

moiety, but only minor inhibition was detected. No relevant

activity was detected for the benzenesulfonamide (26) and

benzoic acid compound (25). Hence, no negative charge and

no large groups at this position like 4-benzyloxyphenyl (8) and

N-phenylpiperidine-4-carboxamide (12) were tolerated. The

cycloalkyl derivatives 11 and 13 demonstrated low activity,

except piperidine 1 with an EC₅₀of around 13 μM. Cytotoxic

eﬀects were low or absent, with the exception of compound 3,

which could therefore not be studied in cellular assays.

Analysis by chiral HPLC demonstrated that racemization

during synthesis of the phenylglycine derivatives was negligible

for this inhibitor set (see the Supporting Information for

further details).

Synthesis of compounds 22 and 23 was described before.¹⁹

Synthesis of precursors used for compounds 8, 12, and 26, cap

moieties, and other building blocks is described in detail in the

Supporting Information.

Structure−Activity Relationships. Three series of

compounds were evaluated in the DENV protease serotype 2

cellular reporter gene assay (DENV2proHeLa). This approach

oﬀers the opportunity to determine cellular activity, perme-

ability, metabolic stability, and interaction with host factors in

a single cellular assay. Another advantage of this assay is the

use of a full-length construct of the DENV2 NS3 with its NS2B

cofactor instead of a truncated protease with an artiﬁcial

glycine-linker, which is used in most of the biochemical assays

of DENV protease and in HTS campaigns.^10,28,29Inhibitory

activity assays were limited to one serotype. DENV serotype 2

was chosen since this serotype has the highest rate of severe

manifestations like dengue shock syndrome.³⁰⁻³²Compounds

MB-53,¹⁶NK-189,¹⁹and tolcapone³³were included in the

assays as reference inhibitors.

Furthermore, all compounds were tested against thrombin

and trypsin in biochemical assays to evaluate their selectivity.

The SAR explorations were focused on replacing the guanidine

function with a less polar, and preferably noncharged, moiety.

In addition, 4-benzyloxyphenylglycine derivatives and lip-

ophilic cap moieties were evaluated.

Merging of Fragments. The benzamides, as the most

active fragments, were merged with diﬀerent 4-benzyloxyphe-

nylglycine derivatives and diﬀerent cap moieties to further

increase their activity. 4-Benzamide and 3-benzamide deriva-

Nonbasic Guanidine Replacements. The primary goal

of this work was to identify a less basic or neutral alternative

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Table 1. Inhibitory Activity of Compounds with Diﬀerent Nonbasic Moieties in the Cellular DENV Protease Reporter Gene

Assay (DENV2proHeLa) and Cytotoxicity in HeLa Cells

a

b

Values against the DENV serotype 2 protease in the reporter gene assay in HeLa cells. Measured in HeLa cells. If inhibition ≤ 10%, then there is

no inhibition (n.i.). n.d. = not determined. All measurements were carried out in triplicate.

tives were evaluated (Tables 2 and 3, respectively). Whereas

polar caps like 2-(2-methoxyethoxy)acetic acid (compound

47), 6-aminohexanoic acid (compound 49), or 4-

(aminomethyl)benzoic acid (50) decreased the activity, long

alkyl chains and ring systems maintained or even increased it.

Thiophene ring systems, 4-phenoxy- and 4-benzyloxybenzoic

acid cap moieties, led to potent inhibitors. Secondary amines

like the alkyl compound (46) and the benzyl derivative (53)

decreased the cellular activity, possibly due to the additional

positive charge. Further extension of the 4-benzyloxy residue

was not well-tolerated. Only the 3-methoxy-, 4-(triﬂuorometh-

yl), and 1,3-dichloro derivatives had comparable activities to

compound 18. ^L-Conﬁguration at the Phg chiral center is

preferred, as demonstrated before for the basic derivatives.¹⁹

Removal of the benzyloxy fragment (R₂in compounds 27 and

29) and its exchange to a 4-triﬂuoromethyl moiety or an amino

acid exchange to phenylalanine (compound 30) resulted in

loss of activity.

and demonstrated slightly higher activity. Similarly, all other

meta-substituted derivatives caused no relevant cytotoxicity

and had a signiﬁcantly higher selectivity index than the

previously published series of compounds.¹⁹Most of the

evaluated phenoxyphenyl or biphenyl caps resulted in high

inhibitory activities with EC₅₀values at about 0.6−2 μM

(except compound 86), whereas the 2-naphthoic acid cap (62)

and the 2,3-dihydrobenzo[b][1,4]dioxine-6-carboxylic acid cap

(63) resulted in lower activities around 5 μM. The 4-

benzoylbenzoic acid capped compound (77) as well as

compounds 67 and 72 revealed high nanomolar inhibition

and an excellent selectivity index (SI > 140). Substitution of

the phenoxy group of compound 67 was mostly tolerated: 2-

Methoxy, 4-cyano, 4-chloro, 3-chloro, and 4-triﬂuoromethyl

caps resulted in comparable to 3-fold higher EC₅₀values

relative to compound 67. 4-(Benzo[d][1,3]dioxol-5-yloxy)-

benzoic acid (74) showed about 2-fold higher activity

compared to next homolog 4-(2,3-dihydrobenzo[b][1,4]-

dioxin-6-yloxy)benzoic acid (75). The (morpholine-4-

carbonyl)benzoic acid derivatives 79 and 80 were designed

in analogy to compound 77 with a polar morpholine moiety,

but the activity decreased signiﬁcantly. Compound 66 contains

a 4-phenoxyphenylglycine core, which slightly decreased the

Compound 57 caused 50% inhibition of DENV protease

activity at 1 μM but also showed relevant cytotoxicity (CC₅₀,

18 μM; SI, 18). To overcome this problem, meta-substituted

derivatives were evaluated (Table 3). Compound 67 in

contrast to compound 57 was not cytotoxic up to 100 μM

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Table 2. Inhibitory Activity of 4-Benzamide Compounds with Diﬀerent 4-Benzyloxyphenylglycine Derivatives and Diﬀerent

Cap Moieties in the Cellular DENV Protease Reporter Gene Assay (DENV2proHeLa) and Cytotoxicity in HeLa Cells

a

b

Values against the DENV serotype 2 protease in the reporter gene assay in HeLa cells. Measured in HeLa cells. If inhibition ≤ 10%, then there is

no inhibition (n.i.). n.d. = not determined. All measurements were carried out in triplicate.

activity compared to the 4-benzyloxyphenylglycine derivatives.

5-(2,5-Dimethylphenoxy)-2,2-dimethylpentanoic acid, the cap

in compound 82, is a known drug (INN: Gemﬁbrozil) for

treatment of hyperlipidemia and hypertriglyceridemia.³⁷

Phenylpiperidine compound 87 revealed low inhibition,

which could be explained with the basic nature of the cap

moiety and the expected lower membrane penetration of the

compound. Other ring exchanges in the cap moiety to

heterocycles like furan (compound 85) and morpholine

(compound 88) and substitution at the para position

(compound 84) were well-tolerated.

Inhibitory Activity against Isolated DENV-2 and WNV

Proteases. Surprisingly, only low to moderate inhibition was

observed against the isolated DENV-2 and WNV proteases in

biochemical assays (see the Supporting Information for

details). This inconsistency was observed for other DENV

protease inhibitors before.^19,38−41We interpret this phenom-

enon as a consequence of two factors: ﬁrst, the artiﬁcial

composition of the biochemical assay. The assay is usually

performed at basic pH values of 9.0 or 8.5, and high

concentrations of polyols are needed for adequate activity of

the protease.^10,28,42Addition of physiological salt concen-

trations to the assay buﬀer leads to a signiﬁcant decrease in

proteolytic activity^43,44and lowers the interaction with the

cofactor NS2B.⁴⁵Furthermore, other assay components could

lead to false negative results against isolated DENV and WNV

proteases as observed previously for nonionic detergents.⁴⁶⁻⁴⁸

Upon exchange of the nonionic detergent Brij 58 to the ionic

detergent CHAPS, we observed increased inhibitory activity of

the compounds at the isolated targets (Table S2). The most

active derivatives showed potency in the low micromolar IC₅₀

range against DENV-2 and WNV proteases. Both viral

proteases have been described before as sensitive toward

buﬀer composition and detergents in particular.^46,47Li et al.

described a ZIKV split luciferase complementary assay, in

which nonionic detergents interfered strongly with the signal

and revealed inhibitory eﬀects.⁴⁹It may be hypothesized that

certain structural features, like the aliphatic hexadecyl chain in

Brij 58, occupy lipophilic binding sites on the target proteins

and thereby interfere with target recognition of the present

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Table 3. Inhibitory Activity of 3-Benzamide Compounds with Diﬀerent 4-Benzyloxyphenylglycine Derivatives and Diﬀerent

Cap Moieties in the Cellular DENV Protease Reporter Gene Assay (DENV2proHeLa) and Cytotoxicity in HeLa Cells

a

b

Values against the DENV serotype 2 protease in the reporter gene assay in HeLa cells. Measured in HeLa cells. If inhibition ≤ 10%, then there is

no inhibition (n.i.). n.d. = not determined. All measurements were carried out in triplicate.

series of compounds. The inhibitors BP2109 and BP13944

consist of long alkyl chain residues, and their inhibition against

the NS3 domain was proven by selection for resistance

experiments.^39,40This hypothesis, however, is diﬃcult to align

with the substrate-competitive binding mode shown for

selected compounds (see below). Second, the cellular

DENV2proHeLa assay incorporates a full-length and not a

truncated viral protease with covalent a Gly-Ser NS2B linker as

in most of the biochemical studies. The transmembrane helices

of NS2B that anchor the active protease complex to the

endoplasmic reticulum are missing in the isolated protease

construct, and the cofactor is covalently linked to the protease

domain by an artiﬁcial linker sequence.⁴⁴Furthermore, an

intracellular or subcellular accumulation of the compounds

may lead to increased activity in cellular assay systems, as

pointed out by others before.³⁸Taken together, the

biochemical assay, with its presence of non-natural detergents

and buﬀers, may not resemble the plasticity and functional

behavior of the full-length protease complex within its in vivo

environment, and the predictive value of biochemical assays for

antiviral eﬃcacy in cells appears to be limited. In contrast, we

recently found a relatively good correlation between eﬃcacies

in the cellular DENV-2 reporter gene (DENV2proHeLa) and

the “gold standard” DENV-2 titer reduction assays.¹⁹

Selectivity against Oﬀ-Target Proteases and Binding

Mode. Several oﬀ-target serine proteases, in particular

thrombin and trypsin, have a recognition preference for basic

moieties in S1. They are therefore potential oﬀ-targets, and

their interaction with ﬂaviviral protease inhibitors, who have a

similar property proﬁle, should be studied. To determine the

activity of the compound against potential oﬀ-targets,

biochemical thrombin and trypsin assays were performed.

None of the compounds had activity against thrombin and

trypsin at compound concentrations of 25 and 50 μM,

respectively (see Table S3).

IC₅₀values of compounds 67, 71, 77, and 84 were

determined against the isolated DENV-2 protease at diﬀerent

substrate concentrations, resulting in the Cheng−Prusoﬀ plots

provided in Figure S1. All plots indicate a competitive

inhibition of the viral protease. Compounds 67, 71, 77, and

84 have K_ivalues of 6.1, 6.3, 3.5, and 6.9 μM, respectively.

Additionally, Michaelis−Menten studies with diﬀerent con-

centrations of compounds 67 and 84 were performed (Figure

S3). Both compounds showed no signiﬁcant change for the

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V_maxvalue but an increase of the K_mvalue, presenting the

behavior of competitive inhibitors. The K_ivalues derived from

Michaelis−Menten plots for compounds 67 and 84 were 5.2

and 4.1 μM, respectively.

and this compound is one of the most active viral protease

inhibitors in a ﬂaviviral titer reduction assay reported to date.

The 4-benzoylbenzoic acid (compound 77), 4′-chlorobiphen-

yl-4-carboxylic acid (compound 84), and 4-morpholinobenzoic

acid caps (compound 88) led to EC₅₀values of 0.85 μM. The

EC₉₀values in the low micromolar range provide further

evidence for the high antiviral activity of the compounds.

Membrane Permeability and Metabolic Stability. By

using the precoated trilayer parallel artiﬁcial membrane

permeability assay (PAMPA),^50,51passive membrane perme-

ability of selected compounds and references was evaluated

(Table 5). The passive permeability for the references was in

accordance with literature values.⁵⁰Whereas compound 18

showed no permeability, compounds 57, 67, 70, 71, 74, 77, 84,

85, and 88 revealed signiﬁcant passive permeability. The tested

set of benzamide compounds showed signiﬁcantly higher

passive membrane penetration than the previously described

guanidine compound set, which explains the higher intra-

cellular antiviral activity of the benzamides.¹⁹Compounds 67,

77, and 88 showed even higher passive membrane penetration

than guanidine compound NK-189, which previously had

shown the most promising value. The measured passive

permeability values are near the range of approved drugs.⁵⁰

An important factor to consider during the early stages of

drug discovery is compound stability against liver and

pancreatic enzymes. Many compounds with promising in

vitro activities never become drugs due to poor metabolic

proﬁles. The metabolic stability of compounds 67, 71, 77, 84,

and 88 as well as NK-189 and reference compound

testosterone was assessed using liver microsomes from rats.

Liver microsomes contain phase 1 metabolic enzymes that

catalyze typical phase 1 metabolic reactions, such as reduction,

oxidation, and hydrolysis of compounds.⁵²As described

before,^16,18,53samples were incubated for 60 min at 37 °C,

and the depletion of the parent compound was monitored

(Figure 4). Testosterone (t_1/2= 16 min) and compounds NK-

189 (t_1/2= 55 min) and 88 (t_1/2= 27 min) showed signiﬁcant

depletion of the parent compound, whereas compounds 71

and 84 were stable over the whole test period. Except

compound 88, the newly described benzamide inhibitors had

signiﬁcantly higher stability against liver microsomes than

compound NK-189. All assessed inhibitors demonstrated

remarkable stability compared to previously published DENV

protease inhibitors.¹⁸

Tryptophan quenching experiments⁴⁸were performed with

compounds 56 and 85, which both incorporate caps with a

signiﬁcant quenching eﬀect. Both compounds were active in

the cellular assay (DENV2proHeLa) in the low micromolar

range. The addition of the compounds to the isolated DENV-2

protease resulted in a concentration-dependent quenching of

the ﬂuorescence of tryptophan residues of the protease (Figure

S4). The active site inhibitor aprotinin restored ﬂuorescence,

which is interpreted to result from displacement of 56 and 58,

indicating a binding of the compounds in the same region

(active site) as aprotinin. The 2,2′-bithiophene-5-carboxylic

acid cap (compound 56) was used as a quenching moiety for

this application before,^16,19whereas the 4-(furan-2-yl)benzoic

acid cap (compound 85) is newly described here.

Antiviral Activity in Virus Titer Reduction Assay. To

conﬁrm the high cellular activity of the compounds in the

DENV2proHela assay, selected compounds and reference

inhibitor MB-53 were further tested in a DENV2 titer

reduction assay. Huh-7 cells were infected with DENV2

(MOI of 1) and treated with various concentrations of the

compounds as indicated. The supernatants were collected at 48

h post-infection and analyzed by a plaque assay. The tested

compounds revealed high reduction of the viral titer even at

the low screening concentration of 12.5 μM (see Figure 2).

Figure 2. Inhibitory activity and cytotoxicity of selected compounds

and reference compound MB-53 at a concentration of 12.5 μM in the

DENV-2 virus titer reduction assay.

The newly described structures include carboxamide bonds,

which are prone to cleavage by pancreatic enzymes such as α-

chymotrypsin and trypsin upon oral administration. Therefore,

the stability of selected compounds (NK-189, 67, 71, 77, 84,

and 88) was assessed against those enzymes as previously

reported.^53,54Compound MB-53 was examined before⁵³and

was included as a reference compound. Compounds were

incubated with the respective enzyme in buﬀer for 120 min at

37 °C, and the depletion of the parent compound was

monitored. Except for reference compound MB-53 and 88, all

compounds demonstrated stability against bovine trypsin

(maximum 25% depletion of parent compounds over 120

min) (Figure S6). Reference compound MB-53 showed

comparable half-life time against α-chymotrypsin to previous

published data (t_1/2= 25 min).⁵³70−80% of compounds 67,

71, 77, and 84 remained after 120 min incubation with α-

chymotrypsin (Figure S7). Compounds NK-189 and 88 were

less stable (approximately 40−50% compound cleavage). The

incorporation of a 4-morpholinobenzoic acid cap and 4-

The inhibitors did not show cytotoxicity in Huh-7 cells at this

concentration except compound 57. It should be noted that

the most active compounds inhibit up to 100% of the viral

titers at the tested concentration. Compounds 18 and 85

demonstrated lower activity than in the DENV2proHeLa

assay, whereas the high activity of the other inhibitors is in

accordance with their DENV2proHeLa screening results.

Antiviral dose−response curves were obtained for the most

active derivatives (Figure 3). Residual plots, showing the

diﬀerences between observed values and curve ﬁtting results

for all viral titer reduction curves, are provided in the

Supporting Information (Figure S5). All tested compounds

resulted in dose-dependent reduction of viral titers, with EC₅₀

values ranging from 0.24 to 2.2 μM (Table 4). Unsubstituted

compound 67 as well as the 4-cyano compound (70) had EC₅₀

values of 0.66 μM, whereas the 4-(benzo[d][1,3]dioxol-5-

yloxy)benzoic acid compound (74) showed 3-fold lower

activity. A 4-triﬂuoromethyl substitution (71) increased the

activity more than 2-fold to a very promising EC₅₀of 0.24 μM,

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Figure 3. Viral titer reduction assay: concentration-dependent DENV-2 titer reduction for selected compounds, including reference compound

MB-53 (chart H).

Table 4. EC₅₀and EC₉₀Values for Selected Compounds in

DENV-2 Titer Reduction Assay

CONCLUSIONS

■

a

The aim of the present work was to increase the drug-likeness

and cellular activity of DENV NS2B-NS3 inhibitors, with the

activity in the DENV2proHeLa reporter gene assay as the main

SAR guidance tool. The charged guanidine moiety, which is

present in peptidic and nonpeptidic compounds reported

before,^{15−17,55−58}was successfully replaced by nonbasic

groups. Optimization of the substituents on the P₁phenyl

position of lead compound NK-189¹⁹led to benzamides as

noncharged alternatives. By further optimization of the cap

moiety, several highly potent and selective DENV inhibitors

were discovered. Those noncharged compounds are valuable

and novel leads in the exploration of selective ﬂaviviral

inhibitors. The presented inhibitors show low micromolar to

submicromolar activities in the DENV2proHeLa assay as well

as in the DENV-2 titer reduction assay and lead to complete

compound

EC₅₀[μM]

EC₉₀[μM]

67

70

71

74

77

84

88

MB-53¹⁶

0.66 0.08

0.66 0.17

0.24 0.07

2.2 0.2

1.1 0.2

1.6 0.2

7.0 0.1

8.0 0.1

4.8 0.1

6.8 0.1

4.6 0.1

4.7 0.1

15 0.2

n.d.

0.85 0.51

8.6 3.5

a

n.d. = not determined. All measurements were carried out in

duplicate.

phenylguanidine appears to decrease the stability of the

presented compound series against chymotrypsin.

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Table 5. Passive Membrane Permeability of Selected Compounds Determined by PAMPA

a

b

c

d

cpd.

caﬀeine

carbamazepine

M (g/mol)

C_Acc(μM)

C_Don(μM)

P_e(10⁻⁶cm/s)

R (%)

195

236

252

645

502

488

586

572

597

640

616

584

590

546

565

33

42

25

4

6

2

173 25

7.3

8.9

5.7

2.4

−7.6

8.7

187

166

4

7

phenytoin

MB-53¹⁶

not permeable at a detectable range

171 11 1.6

not permeable at a detectable range

NK-189¹⁹

7.5 0.7

12

18

57

67

70

71

74

77

84

85

88

3.0 1.4

9.5 0.9

4.5 1.0

6.9 1.0

3.7 1.3

8.6 1.0

3.7 0.7

2.1 0.2

8.1 1.7

180 15

164 12

0.61

2.2

1.0

1.4

0.8

1.8

0.7

0.45

1.7

8.9

15

177

188

180

178

187

169

181

7

8

7

9

3

9.9

3.7

8.7

8.1

5.5

15

7.1

a

b

Concentration of the compound in the acceptor plate after 5 h of incubation. Concentration of the compound in the donor plate after 5 h of

incubation. Permeability of the compound calculated according to the literature.⁵⁰Mass retention of the compound calculated according to the

literature.⁵⁰

c

d

resin (200−400 mesh; loading capacity, 1.6 mmol Cl⁻/g resin; cross-

linked with 1% DVB) were purchased from Carbolution Chemicals

(Germany). Fmoc-Rink amide resin (75−150 mesh; loading capacity,

0.68 mmol/g resin) was purchased from Iris Biotech (Germany).

Solvents were used as obtained from the commercial suppliers.

Equipment and Analytical Methods. The progress of the

reactions was determined by thin-layer chromatography (TLC) on

Merck silica gel plates 60 F₂₅₄(UV detection). Flash chromatography

was performed on a Biotage Isolera One puriﬁcation system using

silica gel (0.060−0.200 mm) cartridges (KP-Sil) and UV monitoring

at 254 and 280 nm. NMR spectra were recorded on a Varian NMR

instrument at 300 or 500 MHz, 300 K; chemical shifts (δ) are given in

parts per million (ppm). Residual peaks of nondeuterated solvents

were used as an internal standard: CDCl₃(δ ppm, 7.26), CD₃OD (δ

ppm, 3.31), DMSO-d₆(δ ppm, 2.50), CD₃CN (δ ppm, 1.94), and

acetone-d₆(δ ppm, 2.05). Coupling constants (J) are given in hertz

Figure 4. Degradation of compounds and reference substance

testosterone in the presence of rat liver microsomes.

(Hz). Multiplicity is reported as s (singlet), d (doublet), t (triplet), q

inhibition of viral replication at a concentration of 12.5 μM.

Cytotoxic activity and interference with oﬀ-target serine

proteases are absent from most of the compounds, resulting

in excellent selectivity indices. Most of the compounds are

capable of permeating through phospholipid membranes and

have remarkable stability against rat liver microsomes as well as

against the pancreatic enzymes α-chymotrypsin and trypsin.

The present study also indicates a need to further investigate

the weak or absent correlation of biochemical assay results that

use isolated ﬂaviviral proteases versus the cellular antiviral

activities, which has likely contributed to the failure of

antiﬂaviviral drug discovery in the past. A reconsideration of

screening procedures (e.g., addition of nonionic detergents)

and optimization aims may be required in the ﬁeld of ﬂaviviral

proteases. Since other working groups reported similar

observations,³⁸⁻⁴¹future studies will be directed on the

investigation of this issue.

(quartet), p (pentet), dd (doublet of doublet), m (multiplet), and br

(broad). Mass spectra (HR-ESI) of all compounds were measured on

a Bruker micrOTOF-Q II instrument. Analysis of the compounds and

intermediates was carried out using solutions in water, methanol,

acetonitrile, or water/methanol. Purity of inhibitors was determined

by HPLC on a Jasco HPLC system with a Jasco UV-2070 Plus

Intelligent UV/VIS detector on an RP-18 column (ReproSil-Pur-

ODS-3, Dr. Maisch GmbH, Germany; 5 μm, 50 mm × 2 mm). Purity

for the ﬁnal compounds is ≥95% unless indicated otherwise (see

Table S4).

4-(Benzyloxy)benzoic Acid (89). To a suspension of 4-hydrox-

ybenzoic acid (499 mg, 3.6 mmol) and K₂CO₃(2501 mg, 18.1 mmol)

in DMF was added respective benzyl bromide (0.903 mL, 7.6 mmol).

The solution was warmed to 80 °C and was stirred for 2−3 days. The

reaction mixture was diluted with ethyl acetate and washed with 1 N

HCl, saturated NaHCO₃solution, and water. The organic layer was

dried over anhydrous MgSO₄and concentrated to give an oil. This

was dissolved in a mixture of MeOH and 2 N aqueous NaOH (1:1)

and was stirred at 50 °C for 2 h. After the pH was adjusted to 2 with

concentrated HCl solution, the resulting precipitate was collected by

1

EXPERIMENTAL SECTION

ﬁltration and dried under reduced pressure (771 mg, 93% yield). H

■

NMR (300 MHz, DMSO-d₆): δ 12.64 (s, 1H), 7.91 (d, J = 8.7 Hz,

2H), 7.53−7.29 (m, 5H), 7.09 (d, J = 8.7 Hz, 2H), 5.17 (s, 2H). ¹³C

NMR (APT, 75 MHz, DMSO-d₆): δ 167.0, 161.9, 136.5, 131.4, 128.5,

127.8, 123.2, 114.6, 69.5. HRMS (ESI): m/z [M−H]⁻calcd for

C₁₄H₁₁O₃, 227.0714; found, 227.0725.

4-(Isopentyloxy)benzoic Acid (90). 1-Bromo-3-methylbutane

(2.290 mL, 19.1 mmol), 4-hydroxybenzoic acid (1202 mg, 8.7

mmol), and K₂CO₃(2644 mg, 19.1 mmol) were mixed in DMF (25

Reagents and Solvents. All chemicals for the synthesis of

precursors were obtained from Sigma-Aldrich (Germany), Alfa Aesar

(Germany), Thermo Fisher Scientiﬁc (Germany/United States),

Acros Organics (Belgium), TCI Europe (Belgium), and Carbolution

Chemicals (Germany) and were of analytical grade. The amino acids

were purchased from Carbolution Chemicals (Germany), Alfa Aesar

(Germany), and TCI Europe (Belgium) or synthesized according to

procedures described. COMU, HATU, and 2-chlorotrityl chloride

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mL) and stirred at 100 °C overnight. The reaction mixture was

poured into ice water (25 mL) and was extracted 3 times with ethyl

acetate. The combined organic layers were washed with saturated

sodium bicarbonate solution, water, and brine and dried over

anhydrous MgSO₄. After concentration of the product under reduced

pressure, the resulting residue was suspended in 2 N NaOH (25 mL)

and was stirred at 60 °C overnight. The mixture was acidiﬁed to pH

1−2, and the resulting precipitate was collected by ﬁltration to yield a

white solid (1006 mg, 56% yield). ¹H NMR (300 MHz, DMSO-d₆): δ

12.25 (s, 1H), 7.87 (d, J = 8.5 Hz, 2H), 6.98 (d, J = 8.5 Hz, 2H), 4.00

(t, J = 6.4 Hz, 2H), 1.78−1.65 (m, 1H), 1.57−1.47 (m, 2H), 0.90 (s,

6H). ¹³C NMR (APT, 75 MHz, DMSO-d₆): δ 165.3, 162.5, 131.1,

122.0, 114.3, 66.2, 37.2, 24.7, 22.3. HRMS (ESI): m/z [M−H]⁻calcd

for C₁₂H₁₅O₃, 207.1027; found, 207.1005.

Methyl 4-Hydroxybenzoate (91). To a solution of 4-hydrox-

ybenzoic acid (2505 mg, 18.1 mmol) in dry MeOH (20 mL) was

added SOCl₂(3.944 mL, 54.3 mmol) dropwise at 0 °C. The mixture

was heated to reﬂux and was stirred for 24 h. The solution was

concentrated in vacuo to aﬀord a white solid (2740 mg, 99% yield).

¹H NMR (300 MHz, DMSO-d₆): δ 10.30 (s, 1H), 7.81 (d, J = 8.6 Hz,

mg, 3.4 mmol), Cu(OAc)₂(609 mg, 3.4 mmol), and pyridine (0.541

mL, 6.7 mmol) were reacted together to give the respective methyl

ester (104) as a yellow oil (285 mg, 67% yield). The deprotection of

carboxylic acid was performed as described in the general procedure.

In short, 104 (196 mg, 0.8 mmol) and a solution of LiOH (46 mg, 1.9

mmol) were reacted together to aﬀord 94 as a white solid (169 mg,

1

91% yield). H NMR (300 MHz, DMSO-d₆): δ 12.80 (s, 1H), 8.01

(s, J = 8.5 Hz, 2H), 7.89 (d, J = 8.4 Hz, 2H), 7.23 (d, J = 8.9 Hz, 2H),

7.19 (d, J = 8.7 Hz, 2H). ¹³C NMR (APT, 75 MHz, DMSO-d₆): δ

166.6, 159.7, 158.7, 134.8, 131.8, 127.0, 119.2, 118.6, 116.4, 106.3.

HRMS (ESI): m/z [M−H]⁻calcd for C₁₄H₈NO₃, 238.0510; found,

238.0510.

4-(4-(Triﬂuoromethyl)phenoxy)benzoic Acid (95). Following the

general procedure, 91 (101 mg, 0.7 mmol), 4-(triﬂuoromethyl)-

phenylboronic acid (248 mg, 1.3 mmol), Cu(OAc)₂(239 mg, 1.3

mmol), and pyridine (0.212 mL, 4.0 mmol) were reacted together to

give the respective methyl ester (105) as a yellow oil (126 mg, 65%

yield). The deprotection of carboxylic acid was performed as

described in the general procedure. In short, 105 (90 mg, 0.3

mmol) and a solution of LiOH (18 mg, 0.8 mmol) were reacted

1

2H), 6.84 (d, J = 8.6 Hz, 2H), 3.77 (s, 3H). ¹³C NMR (APT, 75

MHz, DMSO-d₆): δ 166.1, 162.0, 131.4, 120.3, 115.3, 51.6. HRMS

(ESI): m/z [M−H]⁻calcd for C₈H₇O₃, 151.0401; found, 151.0379.

4-(Methoxycarbonyl)phenylboronic Acid (92). To a solution of 4-

boronobenzoic acid (554 mg, 3,3 mmol) in dry MeOH (15 mL) was

added SOCl₂(0.727 mL, 10.0 mmol) dropwise at 0 °C. The mixture

was heated to 50 °C and was stirred for 5 h. The solution was

concentrated in vacuo to aﬀord a white solid (590 mg, 98% yield). ¹H

NMR (300 MHz, DMSO-d₆): δ 9.18 (s, 2H), 8.00 (d, J = 5.8 Hz,

2H), 7.90 (d, J = 5.4 Hz, 2H), 3.85 (s, 3H). ¹³C NMR (APT, 75

MHz, DMSO-d₆): δ 166.5, 134.1, 130.9, 128.0, 52.1. HRMS (ESI):

m/z [M−H]⁻calcd for C₈H₈BO₄, 179.0518; found, 179.0523.

General Procedure for the Synthesis of 4-Phenyloxybenzoic Acid

Derivatives (Procedure A). Methyl 4-phenoxybenzoate derivatives

were synthesized according to a procedure modiﬁed from the

literature.²³Molecular sieves (4 Å) were given to a mixture of 91 (1

equiv), respective arylboronic acid (2 equiv), and Cu(OAc)₂(2

equiv). DCM (10−15 mL) was added, and the resulting colored

suspension was treated with 4 equiv of pyridine. After stirring the

reaction mixture at an ambient atmosphere overnight, the suspension

was dried under reduced pressure. The resulting residue was

suspended in H₂O and ethyl acetate. The suspension was ﬁltered,

and the ﬁltrate was washed 2 times with 1 N HCl, 0.2 N NaOH, and

brine. The organic layer was dried over anhydrous MgSO₄, ﬁltered,

and concentrated in vacuo.

The methyl ester of carboxylic acid was cleaved by dissolving the

compound in a solution of LiOH (2−3 equiv) in THF/H₂O (2:1, 10

mL). The solution was stirred for 3 h. The mixture was adjusted to

pH 2 with concentrated HCl, and a precipitate was formed. The

precipitate was ﬁltered, washed with several portions of H₂O, and

dried under reduced pressure. The crude mixture was puriﬁed by ﬂash

chromatography if necessary or used for subsequent synthetic steps

without further puriﬁcation.

4-(2-Methoxyphenoxy)benzoic Acid (93). Following the general

procedure, 91 (228 mg, 1.5 mmol), 2-methoxyphenylboronic acid

(455 mg, 3.0 mmol), Cu(OAc)₂(544 mg, 3.0 mmol), and pyridine

(0.484 mL, 6.0 mmol) were reacted together to give the respective

methyl ester (103) as a yellow oil (165 mg, 43% yield). The

deprotection of carboxylic acid was performed as described in the

general procedure. In short, 103 (161 mg, 0.6 mmol) and a solution

of LiOH (39 mg, 1.6 mmol) were reacted together to aﬀord 93 as a

pale yellow solid (141 mg, 93% yield). ¹H NMR (300 MHz, DMSO-

d₆): δ 12.69 (s, 1H), 7.89 (d, J = 8.9 Hz, 2H), 7.21 (t, J = 8.6 Hz, 1H),

7.12 (t, J = 8.5 Hz, 1H), 7.02 (d, J = 8.9 Hz, 1H), 6.87 (d, J = 8.9 Hz,

2H), 6.72 (d, J = 8.8 Hz, 1H), 3.79 (s, 3H). ¹³C NMR (APT, 75

MHz, DMSO-d₆): δ 167.3, 162.1, 151.5, 142.2, 131.5, 123.2, 122.6,

121.5, 119.3, 115.5, 113.9, 55.8. HRMS (ESI): m/z [M−H]⁻calcd

for C₁₄H₁₁O₄, 243.0663; found, 243.0676.

together to aﬀord 95 as a white solid (86 mg, 92% yield). H NMR

(300 MHz, acetone-d₆): δ 12.76 (s, 1H), 8.11 (d, J = 8.9 Hz, 2H),

7.79 (d, J = 8.6 Hz, 2H), 7.28 (d, J = 8.4 Hz, 2H), 7.18 (d, J = 8.8 Hz,

2H). ¹³C NMR (APT, 75 MHz, acetone-d₆): δ 167.8, 163.1, 160.8,

132.9, 128.4, 128.4, 128.0, 120.3, 120.2, 119.6. HRMS (ESI): m/z

[M−H]⁻calcd for C₁₄H₈F₃O₃, 281.0431; found, 281.0441.

4-(Benzo[d][1,3]dioxol-5-yloxy)benzoic Acid (96). Following the

general procedure, 91 (181 mg, 1.2 mmol), benzo[d][1,3]dioxol-5-

ylboronic acid (393 mg, 2.4 mmol), Cu(OAc)₂(428 mg, 2.4 mmol),

and pyridine (0.382 mL, 4.7 mmol) were reacted together to give the

respective methyl ester (106) as a brown oil (108 mg, 34% yield).

The deprotection of carboxylic acid was performed as described in the

general procedure. In short, 106 (107 mg, 0.4 mmol) and a solution

of LiOH (24 mg, 1.0 mmol) were reacted together to aﬀord 96 as a

brown solid (89 mg, 87% yield). ¹H NMR (300 MHz, acetone-d₆): δ

12.80 (s, 1H), 8.02 (d, J = 8.8 Hz, 2H), 7.00 (d, J = 8.6 Hz, 2H), 6.90

(d, J = 8.4 Hz, 1H), 6.80 (d, J = 8.4 Hz, 1H), 6.70 (s, 1H), 6.06 (s,

2H). ¹³C NMR (APT, 75 MHz, acetone-d₆): δ 170.5, 163.5, 150.7,

150.6, 149.7, 132.3, 125.4, 117.3, 115.9, 113.8, 103.5, 101.8. HRMS

(ESI): m/z [M−H]⁻calcd for C₁₄H₉O₅, 257.0455; found, 257.0443.

4-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yloxy)benzoic Acid (97).

Following the general procedure, 91 (214 mg, 1.4 mmol), 2,3-

dihydrobenzo[b][1,4]dioxin-6-ylboronic acid (506 mg, 2.8 mmol),

Cu(OAc)₂(511 mg, 2.8 mmol), and pyridine (0.454 mL, 5.6 mmol)

were reacted together to give the respective methyl ester (107) as a

brown oil (198 mg, 49% yield). The deprotection of carboxylic acid

was performed as described in the general procedure. In short, 107

(157 mg, 0.5 mmol) and a solution of LiOH (33 mg, 1.4 mmol) were

reacted together to aﬀord 97 as a brown solid (137 mg, 92% yield).

¹H NMR (300 MHz, DMSO-d₆): δ 12.69 (s, 1H), 7.93 (d, J = 8.6 Hz,

2H), 6.99 (d, J = 8.6 Hz, 2H), 6.92 (d, J = 8.7 Hz, 1H), 6.82 (d, J =

8.5 Hz, 1H), 6.67 (s, 1H), 4.25 (s, 4H). ¹³C NMR (APT, 75 MHz,

DMSO-d₆): δ 165.7, 162.1, 151.0, 148.1, 144.1, 131.5, 123.5, 116.4,

113.2, 111.2, 107.3, 64.2, 64.2. HRMS (ESI): m/z [M−H]⁻calcd for

C₁₅H₁₁O₅, 271.0612; found, 271.0610.

General Procedure for the Synthesis of 4-Phenyloxybenzoic Acid

Derivatives (Procedure B). Methyl 4-phenoxybenzoate derivatives

were synthesized according to a procedure modiﬁed from the

literature.²³Molecular sieves (4 Å) were given to a mixture of 92 (2

equiv), the respective phenol derivative (1 equiv), and Cu(OAc)₂(2

equiv). DCM (15 mL) was added, and the resulting colored

suspension was treated with 4 equiv of pyridine. After stirring the

reaction mixture at an ambient atmosphere for two days, the

suspension was dried under reduced pressure. The resulting residue

was suspended in H₂O and ethyl acetate. The suspension was ﬁltered,

and the ﬁltrate was washed 2 times with 1 N HCl, 0.2 N NaOH, and

brine. The organic layer was dried over anhydrous MgSO₄, ﬁltered,

and concentrated in vacuo.

4-(4-Cyanophenoxy)benzoic Acid (94). Following the general

procedure, 91 (255 mg, 1.7 mmol), 4-cyanophenylboronic acid (493

The methyl ester of carboxylic acid was cleaved by dissolving the

compound in a solution of LiOH (3.5 equiv) in THF/H₂O (2:1, 10

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mL) at 0 °C. The solution was stirred at room temperature overnight.

The mixture was adjusted to pH 2 with concentrated HCl, and a

precipitate was formed. The precipitate was ﬁltered, washed with

several portions of H₂O, and dried under reduced pressure. The crude

mixture was puriﬁed by ﬂash chromatography if necessary or used for

subsequent synthetic steps without further puriﬁcation.

5.86−5.78 (m, 1H), 5.11 (s, 2H), 3.47−3.27 (m, 4 H), 2.26−2.10 (m,

2H), 1.64−1.13 (m, 12H), 0.85 (t, J = 6.8 Hz, 3H). ¹³C NMR (APT,

126 MHz, DMSO-d₆): δ 171.4, 167.9, 157.7, 137.0, 130.4, 129.1,

128.4, 127.8, 127.6, 114.7, 69.2, 52.2, 45.8, 42.6, 34.8, 30.8, 25.5, 25.2,

24.9, 23.8, 21.8, 13.8. HRMS (ESI): m/z [M + H]⁺calcd for

C₂₆H₃₅N₂O₃, 423.2642; found, 423.2658.

4-(3-Oxo-2,3-dihydro-1H-inden-4-yloxy)benzoic Acid (98). Fol-

lowing the general procedure B, 92 (270 mg, 1.5 mmol), 7-hydroxy-

2,3-dihydro-1H-inden-1-one (111 mg, 0.8 mmol), Cu(OAc)₂(273

mg, 1.5 mmol), and pyridine (0.242 mL, 3.0 mmol) were reacted

together to give the respective methyl ester (108) as a brown oil (226

mg, 53% yield). The deprotection of carboxylic acid was performed as

described in the general procedure. In short, 108 (198 mg, 0.7 mmol)

and a solution of LiOH (59 mg, 2.5 mmol) were reacted together to

(S)-N-(2-(Benzylamino)-1-(4-(benzyloxy)phenyl)-2-oxoethyl)-

hexanamide (2). Compound 2 was synthesized according to the

general procedure for peptide coupling in solution from 102 (21 mg,

0.06 mmol), HATU (27 mg, 0.07 mmol), benzylamine (0.008 mL,

0.07 mmol), and TMP (0.010 mL, 0.08 mmol) and was obtained as a

white powder (14 mg, 54% yield). ¹H NMR (300 MHz, acetone-d₆):

δ 7.83 (t, J = 5.9 Hz, 1H), 7.53 (d, J = 7.6 Hz, 1H), 7.47 (d, J = 6.7

Hz, 2H), 7.43−7.29 (m, 5H), 7.29−7.15 (m, 5H), 6.96 (d, J = 8.7 Hz,

2H), 5.51 (d, J = 7.7 Hz, 1H), 5.12 (s, 2H), 4.39 (d, J = 6.2 Hz, 2H),

2.25 (t, J = 7.5 Hz, 2H), 1.66−1.52 (m, 2H), 1.36−1.21 (m, 4H),

0.86 (t, J = 6.8 Hz, 3H). ¹³C NMR (APT, 126 MHz, DMSO-d₆): δ

171.9, 170.3, 157.7, 139.1, 137.1, 131.2, 128.4, 128.2, 127.8, 127.6,

127.0, 126.7, 114.5, 69.1, 55.6, 42.0, 34.9, 30.9, 24.9, 21.9, 13.9.

HRMS (ESI): m/z [M + H]⁺calcd for C₂₈H₃₃N₂O₃, 445.2486; found,

445.2487.

1

aﬀord 98 as a pale red solid (180 mg, 96% yield). H NMR (300

MHz, DMSO-d₆): δ 12.46 (s, 1H), 7.79 (d, J = 8.7 Hz, 2H), 7.43 (t, J

= 7.8 Hz, 1H), 7.01 (d, J = 8.5 Hz, 1H), 6.82 (d, J = 8.7 Hz, 2H), 6.72

(d, J = 8.1 Hz, 1H), 3.15−2.96 (m, 2H), 2.64−2.52 (m, 2H). ¹³C

NMR (APT, 75 MHz, DMSO-d₆): δ 205.7, 167.2, 161.6, 156.6, 156.2,

134.1, 131.5, 128.1, 123.3, 121.4, 115.1, 113.7, 36.1, 25.2. HRMS

(ESI): m/z [M−H]⁻calcd for C₁₆H₁₃O₄, 267.0663; found, 267.0699.

4-(4-Chlorophenoxy)benzoic Acid (99). Following the general

procedure B, 92 (159 mg, 0.9 mmol), 4-chlorophenol (57 mg, 0.4

mmol), Cu(OAc)₂(160 mg, 0.9 mmol), and pyridine (0.143 mL, 1.8

mmol) were reacted together to give the respective methyl ester

(109) as a yellow oil (162 mg, 70% yield). The deprotection of

carboxylic acid was performed as described in the general procedure.

In short, 109 (152 mg, 0.6 mmol) and a solution of LiOH (49 mg, 2.0

mmol) were reacted together to aﬀord 99 as a white solid (127 mg,

(S)-N-(1-(4-(Benzyloxy)phenyl)-2-oxo-2-(phenethylamino)ethyl)-

hexanamide (3). Compound 3 was synthesized according to the

general procedure for peptide coupling in solution from 102 (27 mg,

0.08 mmol), HATU (34 mg, 0.09 mmol), phenylethylamine·HCl (14

mg, 0.09 mmol), and TMP (0.023 mL, 0.17 mmol) and was obtained

as a white powder (19 mg, 56% yield). ¹H NMR (300 MHz, DMSO-

d₆): δ 8.30 (d, J = 8.1 Hz, 1H), 8.23 (t, J = 5.3 Hz, 1H), 7.47−7.31

(m, 5H), 7.27−7.15 (m, 5H), 7.09 (d, J = 6.6 Hz, 2H), 6.94 (d, J =

8.6 Hz, 2H), 5.35 (d, J = 8.1 Hz, 1H), 5.10 (s, 2H), 3.28−3.18 (m,

2H), 2.66 (t, J = 7.1 Hz, 2H), 2.16 (t, J = 7.4 Hz, 2H), 1.46 (p, J = 7.2

Hz, 2H), 1.33−1.14 (m, 4H), 0.84 (t, J = 6.9 Hz, 3H). HRMS (ESI):

m/z [M + H]⁺calcd for C₂₉H₃₅N₂O₃, 459.2642; found, 459.2641.

(S)-N-(1-(4-(Benzyloxy)phenyl)-2-(4-chlorobenzylamino)-2-

oxoethyl)hexanamide (4). Compound 4 was synthesized according

to the general procedure for peptide coupling in solution from 102

(21 mg, 0.06 mmol), HATU (27 mg, 0.07 mmol), 4-chlorobenzyl-

amine (0.009 mL, 0.07 mmol), and TMP (0.010 mL, 0.08 mmol) and

1

88% yield). H NMR (300 MHz, DMSO-d₆): δ 12.79 (s, 1H), 7.95

(d, J = 8.8 Hz, 2H), 7.49 (d, J = 8.9 Hz, 2H), 7.15 (d, J = 8.9 Hz, 2H),

7.06 (d, J = 8.8 Hz, 2H). ¹³C NMR (APT, 75 MHz, DMSO-d₆): δ

166.7, 160.5, 154.1, 131.7, 130.2, 128.5, 125.7, 121.6, 117.5. HRMS

(ESI): m/z [M−H]⁻calcd for C₁₃H₁₀ClO₃, 247.0167; found,

247.0169.

4-(3-Chlorophenoxy)benzoic Acid (100). Following the general

procedure B, 92 (156 mg, 0.9 mmol), 3-chlorophenol (56 mg, 0.4

mmol), Cu(OAc)₂(157 mg, 0.9 mmol), and pyridine (0.140 mL, 1.7

mmol) were reacted together to give the respective methyl ester

(110) as a yellow oil (203 mg, 89% yield). The deprotection of

carboxylic acid was performed as described in the general procedure.

In short, 110 (193 mg, 0.7 mmol) and a solution of LiOH (62 mg, 2.6

mmol) were reacted together to aﬀord 100 as a white solid (127 mg,

1

was obtained as a white powder (15 mg, 54% yield). H NMR (300

MHz, acetone-d₆): δ 7.97 (t, J = 5.4 Hz, 1H), 7.59 (d, J = 7.6 Hz,

1H), 7.47 (d, J = 7.1 Hz, 2H), 7.43−7.31 (m, 5H), 7.30−7.17 (m,

4H), 6.97 (d, J = 8.7 Hz, 2H), 5.49 (d, J = 7.4 Hz, 1H), 5.12 (s, 2H),

4.37 (d, J = 6.1 Hz, 2H), 2.25 (t, J = 7.5 Hz, 2H), 1.64−1.51 (m, 2H),

1.35−1.20 (m, 4H), 0.85 (t, J = 6.9 Hz, 3H). ¹³C NMR (APT, 126

MHz, DMSO-d₆): δ 171.9, 170.4, 157.7, 138.3, 137.1, 131.2, 131.0,

128.8, 128.4, 128.1, 127.8, 127.6, 114.5, 69.1, 55.7, 41.3, 34.8, 30.9,

24.9, 21.9, 13.9. HRMS (ESI): m/z [M + H]⁺calcd for

C₂₈H₃₂ClN₂O₃, 479.2096; found, 479.2081.

(S)-N-(1-(4-(Benzyloxy)phenyl)-2-(4-methoxyphenylamino)-2-

oxoethyl)hexanamide (5). Compound 5 was synthesized according

to the general procedure for peptide coupling in solution from 102

(28 mg, 0.08 mmol), HATU (35 mg, 0.09 mmol), 4-methoxyaniline

(0.011 mL, 0.09 mmol), and TMP (0.018 mL, 0.14 mmol) and was

obtained as a white powder (33 mg, 92% yield). ¹H NMR (300 MHz,

DMSO-d₆): δ 10.12 (s, 1H), 8.47 (d, J = 7.9 Hz, 1H), 7.48 (d, J = 9.0

Hz, 2H), 7.44−7.30 (m, 7H), 6.99 (d, J = 8.6 Hz, 2H), 6.86 (d, J =

8.8 Hz, 2H), 5.54 (d, J = 7.9 Hz, 1H), 5.09 (s, 2H), 3.70 (s, 3H), 2.20

(t, J = 7.4 Hz, 2H), 1.49 (p, J = 7.2 Hz, 2H), 1.34−1.15 (m, 4H), 0.84

(t, J = 6.7 Hz, 3H). ¹³C NMR (APT, 75 MHz, DMSO-d₆): δ 172.0,

168.6, 157.8, 155.3, 137.0, 132.0, 130.7, 128.44, 128.41, 127.8, 127.6,

120.6, 114.7, 113.9, 69.2, 56.1, 55.1, 34.8, 30.9, 24.9, 21.9, 13.9.

HRMS (ESI): m/z [M + H]⁺calcd for C₂₈H₃₃N₂O₄, 461.2435; found,

461.2431.

1

69% yield). H NMR (300 MHz, DMSO-d₆): δ 12.84 (s, 1H), 7.97

(d, J = 8.8 Hz, 2H), 7.46 (t, J = 8.1 Hz, 1H), 7.29 (d, J = 8.1 Hz, 1H),

7.22 (s, 1H), 7.13−7.05 (m, 3H). ¹³C NMR (APT, 75 MHz, DMSO-

d₆): δ 166.7, 160.1, 156.3, 134.2, 131.8, 131.7, 126.0, 124.5, 119.8,

118.4, 117.8. HRMS (ESI): m/z [M−H]⁻calcd for C₁₃H₁₀ClO₃,

247.0167; found, 247.0158.

General Procedure for Peptide Coupling in Solution. Respective

carboxylic acid (1.0 equiv) and HATU (1.2 equiv) were suspended in

DCM (2−10 mL). The mixture was cooled to 0 °C, and the

respective amine (1.2 equiv) was added prior to the dropwise addition

of TMP. DMF was added until all solids were dissolved, and the

mixture was stirred at room temperature for about 2 h. The reaction

was quenched with the addition of a solution of TFA in DCM (5%, 5

mL). All solvents were removed in vacuo, and the resulting residue

was dissolved in ethyl acetate. The ethyl acetate phase was washed 2

times with 1 N HCl, 0.05 N NaOH, and water. The combined organic

phase was dried over anhydrous MgSO₄, concentrated under reduced

pressure, and puriﬁed by preparative HPLC. After puriﬁcation, all

organic solvents were evaporated, and the compounds were freeze-

dried in H₂O/ACN.

(S)-N-(1-(4-(Benzyloxy)phenyl)-2-(4-methoxybenzylamino)-2-

oxoethyl)hexanamide (6). Compound 6 was synthesized according

to the general procedure for peptide coupling in solution from 102

(21 mg, 0.06 mmol), HATU (27 mg, 0.07 mmol), 4-methoxybenzyl-

amine (0.009 mL, 0.07 mmol), and TMP (0.010 mL, 0.08 mmol) and

(S)-N-(1-(4-(Benzyloxy)phenyl)-2-oxo-2-(piperidin-1-yl)ethyl)-

hexanamide (1). Compound 1 was synthesized according to the

general procedure for peptide coupling in solution from 102 (27 mg,

0.08 mmol), HATU (33 mg, 0.09 mmol), piperidine·HCl (11 mg,

0.09 mmol), and TMP (0.023 mL, 0.17 mmol) and was obtained as a

1

yellow oil (18 mg, 56% yield). H NMR (300 MHz, acetone-d₆): δ

was obtained as a white powder (18 mg, 64% yield). H NMR (300

7.54−7.43 (m, 3H), 7.43−7.27 (m, 5H), 6.98 (d, J = 8.7 Hz, 2H),

MHz, acetone-d₆): δ 7.74 (t, J = 6.0 Hz, 1H), 7.57−7.44 (m, 3H),

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7.44−7.28 (m, 5H), 7.13 (d, J = 8.6 Hz, 2H), 6.96 (d, J = 8.7 Hz,

2H), 6.81 (d, J = 8.6 Hz, 2H), 5.49 (d, J = 7.6 Hz, 1H), 5.11 (s, 2H),

4.31 (d, J = 5.8 Hz, 2H), 3.75 (s, 3H), 2.25 (t, J = 7.5 Hz, 2H), 1.63−

1.51 (m, 2H), 1.34−1.21 (m, 4H), 0.86 (t, J = 6.8 Hz, 3H). ¹³C NMR

(APT, 75 MHz, DMSO-d₆): δ 171.8, 170.1, 158.2, 157.7, 137.1,

131.3, 131.0, 128.8, 128.4, 127.8, 127.6, 126.7, 114.5, 113.6, 69.2,

55.6, 55.0, 41.5, 34.9, 30.9, 25.0, 21.9, 13.9. HRMS (ESI): m/z [M +

H]⁺calcd for C₂₉H₃₅N₂O₄, 497.2411; found, 497.2409.

puriﬁed with reversed-phase liquid chromatography and freeze-dried

in H₂O/ACN to aﬀord a white powder (16 mg, 59% yield). ¹H NMR

(300 MHz, DMSO-d₆): δ 9.87 (s, 1H), 8.61 (t, J = 5.7 Hz, 1H), 8.35

(d, J = 8.0 Hz, 1H), 7.48−7.28 (m, 9H), 7.06 (d, J = 8.4 Hz, 2H),

6.96 (d, J = 8.6 Hz, 2H), 5.41 (d, J = 8.0 Hz, 1H), 5.09 (s, 2H), 4.27−

4.10 (m, 2H), 2.17 (t, J = 7.3 Hz, 2H), 2.01 (s, 3H), 1.47 (p, J = 7.3

Hz, 2H), 1.30−1.14 (m, 4H), 0.83 (t, J = 6.9 Hz, 3H). ¹³C NMR

(APT, 75 MHz, DMSO-d₆): δ 171.9, 170.2, 168.1, 157.7, 138.0,

137.1, 133.6, 131.2, 128.42, 128.39, 127.8, 127.6, 127.4, 118.8, 114.5,

69.2, 55.6, 41.6, 34.9, 30.9, 25.0, 24.0, 21.9, 13.9. HRMS (ESI): m/z

[M + H]⁺calcd for C₃₀H₃₆N₃O₄, 502.2700; found, 502.2691.

(S)-4-((2-(4-(Benzyloxy)phenyl)-2-hexanamidoacetamido)-

methyl)-N-hydroxybenzamide (24). Compound 24 was synthesized

according to a procedure modiﬁed from the literature.⁵⁹25 (28 mg,

0.06 mmol) was dissolved in dry THF (1 mL) under a nitrogen

atmosphere. To this solution, carbonyldiimidazole (14 mg, 0.09

mmol) in dry THF (0.5 mL) was added. The mixture was stirred for 1

h at room temperature, and hydroxylamine hydrochloride (8 mg, 0.11

mmol) in dry THF (1 mL) was added. The mixture was further

stirred overnight, and the mixture was diluted with 5% aqueous

KHSO₄. The resulting suspension was extracted with ethyl acetate

and was washed 2 times with brine and water. The organic phase was

concentrated in vacuo, ﬁltered over anhydrous MgSO₄, and was

further puriﬁed with reversed-phase liquid chromatography. The

puriﬁed product was redissolved in water and freeze-dried to yield a

white powder (6 mg, 21% yield). ¹H NMR (300 MHz, acetone-d₆): δ

10.68 (br s, 1H), 7.97−7.87 (m, 2H), 7.72 (d, J = 8.5 Hz, 2H), 7.57−

7.51 (m, 1H), 7.48 (d, J = 7.5 Hz, 2H), 7.43−7.25 (m, 7H), 6.98 (d, J

= 6.9 Hz, 2H), 5.53−5.47 (m, 1H), 5.13 (s, 2H), 4.50−4.41 (m, 2H),

2.26 (t, J = 7.7 Hz, 2H), 1.64−1.51 (m, 2H), 1.35−1.24 (m, 4H),

0.86 (t, J = 6.9 Hz, 3H). HRMS (ESI): m/z [M + H]⁺calcd for

C₂₉H₃₃N₃NaO₅, 526.2312; found, 526.2316.

(S)-4-((2-(4-(Benzyloxy)phenyl)-2-(hexylamino)acetamido)-

methyl)benzamide (46). The peptide synthesis for 46 was done

according to the general procedure for the synthesis of peptides on

Rink amide resin. After drying the resin in vacuo (with compound 40

attached), a reductive amination was performed on resin. Therefore,

the resin (130 mg) was reswollen in DMF for 30 min, hexanal was

added (0.024 mL), and the resin was shaken for 3 h. The resin was

washed with DMF, DCM, and dry MeOH. NaBH₃CN (10 mg) in 1

mL of dry MeOH was added to the resin and was shaken overnight.

The resin was washed with MeOH, DMF, DCM, and diethyl ether. It

was dried under reduced pressure overnight, and the peptide was

cleaved oﬀ the resin and puriﬁed by the standard procedure. ¹H NMR

(300 MHz, acetone-d₆): δ 8.70 (t, J = 5.8 Hz, 1H), 7.81 (d, J = 8.1

Hz, 2H), 7.59 (d, J = 8.7 Hz, 2H), 7.48 (d, J = 7.0 Hz, 2H), 7.44−

7.30 (m, 3H), 7.26 (d, J = 8.1 Hz, 2H), 7.04 (d, J = 8.7 Hz, 2H), 6.83

(br s, 2H), 5.39 (s, 1H), 5.13 (s, 2H), 4.58−4.35 (m, 2H), 3.11−2.83

(m, 2H), 1.84−1.67 (m, 2H), 1.33−1.11 (m, 6H), 0.82 (t, J = 6.7 Hz,

3H). ¹³C NMR (APT, 75 MHz, DMSO-d₆): δ 167.5, 167.0, 159.3,

141.7, 136.8, 133.0, 130.1, 128.5, 127.9, 127.7, 127.5, 126.7, 124.1,

115.2, 69.3, 61.9, 45.4, 42.0, 30.6, 25.6, 25.1, 21.8, 13.8. HRMS (ESI):

m/z [M + H]⁺calcd for C₂₉H₃₆N₃O₃, 474.2751; found, 474.2761.

(S)-4-((2-(Benzylamino)-2-(4-(benzyloxy)phenyl)acetamido)-

methyl)benzamide (53). The peptide synthesis for 53 was done

according to the general procedure for the synthesis of peptides on

Rink amide resin. After drying the resin in vacuo (with compound 40

attached), a reductive amination was performed on resin. Therefore,

the resin (130 mg) was reswollen in DMF for 30 min, benzaldehyde

was added (0.026 mL), and the resin was shaken for 3 h. The resin

was washed with DMF, DCM, and dry MeOH. NaBH₃CN (10 mg) in

1 mL of dry MeOH was added to the resin and was shaken overnight.

The resin was washed with MeOH, DMF, DCM, and diethyl ether. It

was dried under reduced pressure overnight, and the peptide was

cleaved oﬀ the resin and puriﬁed by the standard procedure. ¹H NMR

(300 MHz, acetone-d₆): δ 8.58 (t, J = 5.7 Hz, 1H), 7.80 (d, J = 7.8

Hz, 2H), 7.59 (d, J = 8.3 Hz, 2H), 7.52−7.30 (m, 10H), 7.25 (d, J =

7.8 Hz, 2H), 7.06 (d, J = 7.7 Hz, 2H), 6.79 (br s, 2H), 5.33 (s, 1H),

5.14 (s, 2H), 4.56−4.36 (m, 2H), 4.35−4.15 (m, 2H). ¹³C NMR

(APT, 75 MHz, DMSO-d₆): δ 167.5, 166.9, 159.2, 141.6, 136.8,

(S)-N-(1-(4-(Benzyloxy)phenyl)-2-(3-methoxybenzylamino)-2-

oxoethyl)hexanamide (7). Compound 7 was synthesized according

to the general procedure for peptide coupling in solution from 102

(28 mg, 0.08 mmol), HATU (35 mg, 0.09 mmol), (3-

methoxyphenyl)methanamine (0.012 mL, 0.09 mmol), and TMP

(0.018 mL, 0.14 mmol) and was obtained as a white powder (24 mg,

1

65% yield). H NMR (300 MHz, DMSO-d₆): δ 8.66 (t, J = 6.0 Hz,

1H), 8.37 (d, J = 7.9 Hz, 1H), 7.48−7.28 (m, 7H), 7.15 (t, J = 7.8 Hz,

1H), 6.97 (d, J = 8.7 Hz, 2H), 6.80−6.68 (m, 3H), 5.43 (d, J = 7.9

Hz, 1H), 5.09 (s, 2H), 4.24 (d, J = 5.8 Hz, 2H), 3.65 (s, 3H), 2.18 (t,

J = 7.4 Hz, 2H), 1.47 (p, J = 7.2 Hz, 2H), 1.32−1.13 (m, 4H), 0.83 (t,

J = 6.9 Hz, 3H). ¹³C NMR (APT, 75 MHz, DMSO-d₆): δ 171.9,

170.4, 159.3, 157.7, 140.8, 137.1, 131.2, 129.2, 128.4, 127.8, 127.6,

119.1, 114.5, 112.6, 112.0, 69.2, 55.7, 54.8, 41.9, 34.9, 30.9, 24.9, 21.9,

13.9. HRMS (ESI): m/z [M + H]⁺calcd for C₂₉H₃₅N₂O₄, 475.2591;

found, 475.2606.

(S)-N-(1-(4-(Benzyloxy)phenyl)-2-(4-(benzyloxy)phenylamino)-2-

oxoethyl)hexanamide (8). Compound 8 was synthesized according

to the general procedure for peptide coupling in solution from 102

(28 mg, 0.08 mmol), HATU (35 mg, 0.09 mmol), 111 (22 mg, 0.09

mmol), and TMP (0.013 mL, 0.10 mmol) and was obtained as a

1

white powder (30 mg, 71% yield). H NMR (300 MHz, CDCl₃): δ

7.47−7.28 (m, 14H), 6.98−6.85 (m, 4H), 5.64 (s, 1H), 5.03 (s, 2H),

5.02 (s, 2H), 2.25 (t, J = 7.6 Hz, 2H), 1.68−1.56 (m, 2H), 1.33−1.21

(m, 4H), 0.86 (t, J = 6.6 Hz, 3H). ¹³C NMR (APT, 75 MHz, DMSO-

d₆): δ 172.0, 168.7, 157.8, 154.3, 137.1, 137.0, 132.2, 130.7, 128.44,

128.41, 128.37, 127.78, 127.75, 127.64, 127.57, 120.5, 114.9, 114.7,

69.3, 69.2, 56.1, 34.8, 30.9, 24.9, 21.9, 13.9. HRMS (ESI): m/z [M +

H]⁺calcd for C₃₄H₃₇N₂O₄, 537.2748; found, 537.2752.

(S)-1-(2-(4-(Benzyloxy)phenyl)-2-hexanamidoacetyl)-N-phenyl-

piperidine-4-carboxamide (12). Compound 12 was synthesized

according to the general procedure for peptide coupling in solution

from 102 (28 mg, 0.08 mmol), HATU (35 mg, 0.09 mmol), 112 (19

mg, 0.09 mmol), and TMP (0.013 mL, 0.10 mmol) and was obtained

as a white powder (27 mg, 64% yield). ¹H NMR (300 MHz, CDCl₃):

δ 7.55−7.28 (m, 9H), 7.16−6.89 (m, 5H), 5.83 (s, 1H), 5.03 (s, 2H),

3.56−3.28 (m, 4H), 2.73−2.60 (m, 1H), 2.30−1.93 (m, 6H), 1.71−

1.53 (m, 2H), 1.37−1.19 (m, 4H), 0.85 (t, J = 6.9 Hz, 3H). HRMS

(ESI): m/z [M + H]⁺calcd for C₃₃H₄₀N₃O₄, 542.3013; found,

542.3000.

( S ) - N - ( 1 - ( 4 - ( B e n z y l o x y ) p h e n y l ) - 2 - o x o - 2 - ( 4 -

sulfamoylphenylamino)ethyl)hexanamide (26). Compound 26 was

synthesized according to the general procedure for peptide coupling

in solution from 102 (20 mg, 0.06 mmol), HATU (26 mg, 0.07

mmol), 113 (12 mg, 0.07 mmol), and TMP (0.010 mL, 0.07 mmol)

1

and was obtained as a pale yellow solid (2 mg, 7% yield). H NMR

(300 MHz, DMSO-d₆): δ 10.19 (s, 1H), 8.49 (d, J = 7.8 Hz, 1H),

7.89−7.47 (m, 4H), 7.47−7.25 (m, 7H), 7.00 (d, J = 8.7 Hz, 2H),

5.53 (d, J = 7.8 Hz, 1H), 5.10 (s, 2H), 2.21 (t, J = 7.3 Hz, 2H), 1.57−

1.26 (m, 2H), 1.36−1.13 (m, 4H), 0.84 (t, J = 6.8 Hz, 3H). HRMS

(ESI): m/z [M + H]⁺calcd for C₂₇H₃₂N₃O₅S, 510.2057; found,

510.2069.

(S)-N-(2-(4-Acetamidobenzylamino)-1-(4-(benzyloxy)phenyl)-2-

oxoethyl)hexanamide (21). 101 (31 mg, 0.05 mmol) was dissolved

in DCM (5 mL) and cooled to 0 °C with an ice-water bath.

Triethylamine (0.009 mL, 0.07 mmol) was added to the solution prior

to the addition of acetyl chloride (0.004 mL, 0.06 mmol). The

mixture was warmed to room temperature and stirred overnight

before the solvent was removed under reduced pressure. The residue

was dissolved in ethyl acetate and washed 2 times with saturated

sodium bicarbonate solution and brine. The organic layer was dried

over anhydrous MgSO₄and concentrated in vacuo. The residue was

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133.0, 130.2, 128.6, 128.5, 127.9, 127.7, 127.5, 126.8, 115.2, 69.3,

61.4, 48.9, 42.0. HRMS (ESI): m/z [M + H]⁺calcd for C₃₀H₃₀N₃O₃,

480.2282; found, 480.2280.

DMF and was handled like the Rink amide resin in the next coupling

steps. The resin loaded with the ﬁnished sequence was washed 5×

with diethyl ether and dried under reduced pressure. The ﬁnal

product was cleaved oﬀ the resin 3 times with TFA/DCM solution

(5:95, 1−2 mL per 100 mg of resin), and the mixture was shaken each

time for at least 30 min. The cleavage solution was dispensed into a

round-bottom ﬂask, washed two times with a small amount of water,

dried under reduced pressure, and coevaporated with toluene several

times.

(S)-4-((2-(4-(Benzyloxy)phenyl)-2-(heptylsulfonamido)-

acetamido)methyl)benzamide (45). Sodium heptanesulfonate (50

mg, 0.28 mmol) was reﬂuxed in thionyl chloride (0.5 mL) under a

nitrogen atmosphere. After 6 h, the mixture was cooled to room

temperature, the volatiles were removed under reduced pressure, and

the resulting yellow oil was dissolved in dry DCM under a nitrogen

atmosphere. The solution was cooled with an ice-water bath, and

NEt₃(0.058 mL, 0.42 mmol) and 40 (110 mg, 0.28 mmol) were

added slowly as a suspension in dry DCM. The mixture was allowed

to warm to room temperature and was stirred for 16 h. DCM (10

mL) was added, and the mixture was washed with 1 N HCl, saturated

NaHCO₃solution, and water. The organic phase was dried over

anhydrous MgSO₄and concentrated in vacuo to yield an orange oil.

The product was further puriﬁed with RP-HPLC, redissolved in H₂O/

All ﬁnal compounds were puriﬁed by preparative RP-HPLC on an

AKTA Puriﬁer, GE Healthcare (Germany), with an RP-18 pre and

̈

main column (Rephospher, Dr. Maisch GmbH, Germany; C18-DE, 5

μm, 30 mm × 16 mm, and 120 mm × 16 mm). The following

conditions were used: eluent A, water (0.1% TFA); eluent B,

methanol (0.1% TFA) or eluent A, water (0.1% TFA); eluent B,

acetonitrile (0.1% TFA); ﬂow rate, 8 mL/min; and gradient, 10% B

(2.5 min), 100% B (23.5 min), 100% B (26 min), 10% B (26.1 min),

and 10% B (30 min). Detection was performed at 214, 254, and 280

nm. After puriﬁcation, the organic solvent was evaporated, and the

compounds and intermediates were freeze-dried in H₂O/ACN and

stored at −20 °C.

(S)-N-(1-(4-(Benzyloxy)phenyl)-2-(4-(hydroxymethyl)-

phenylamino)-2-oxoethyl)hexanamide (9). ¹H NMR (300 MHz,

CD₃OD/D₂O): δ 7.50 (d, J = 8.4 Hz, 2H), 7.44−7.26 (m, 9H), 7.00

(d, J = 8.6 Hz, 2H), 5.51 (s, 1H), 5.09 (s, 2H), 4.54 (s, 2H), 2.29 (t, J

= 7.5 Hz, 2H), 1.68−1.53 (m, 2H), 1.36−1.25 (m, 4H), 0.88 (t, J =

6.9 Hz, 3H). ¹³C NMR (APT, 75 MHz, DMSO-d₆): δ 172.1, 169.0,

157.9, 137.6, 137.4, 137.0, 130.5, 128.5, 128.4, 127.8, 127.6, 126.9,

118.8, 114.7, 69.2, 62.5, 56.3, 34.8, 30.9, 24.9, 21.9, 13.9. HRMS

(ESI): m/z [M + H]⁺calcd for C₂₈H₃₃N₂O₄, 461.2435; found,

461.2421.

(S)-N-(1-(4-(Benzyloxy)phenyl)-2-(4-hydroxyphenethylamino)-2-

oxoethyl)hexanamide (10). ¹H NMR (300 MHz, acetone-d₆): δ 8.13

(br s, 1H), 7.51−7.26 (m, 9H), 6.99−6.88 (m, 4H), 6.70 (d, J = 8.5

Hz, 2H), 5.41 (d, J = 7.7 Hz, 1H), 5.12 (s, 2H), 3.50−3.21 (m, 2H),

2.63 (t, J = 7.1 Hz, 2H), 2.29−2.19 (m, 2H), 1.65−1.51 (m, 2H),

1.35−1.22 (m, 4H), 0.92−0.82 (m, 3H). ¹³C NMR (APT, 126 MHz,

DMSO-d₆): δ 171.7, 170.0, 157.6, 155.6, 137.1, 131.4, 129.5, 129.3,

128.4, 128.2, 127.8, 127.6, 115.0, 114.4, 69.2, 55.4, 40.6, 34.9, 34.0,

30.9, 24.9, 21.8, 13.9. HRMS (ESI): m/z [M + H]⁺calcd for

C₂₉H₃₅N₂O₄, 475.2591; found, 475.2581.

1

ACN, and freeze-dried to yield a yellow solid (3 mg, 21% yield). H

NMR (300 MHz, MeOH-d₄/D₂O): δ 7.77 (d, J = 8.5 Hz, 2H), 7.45−

7.32 (m, 7H), 7.28 (d, J = 8.2 Hz, 2H), 7.02 (d, J = 8.0 Hz, 2H), 5.35

(s, 1H), 5.12 (s, 2H), 4.43 (s, 2H), 3.24−3.15 (m, 2H), 1.72−1.50

(m, 2H), 1.40−1.13 (m, 8H), 0.86 (t, J = 6.9 Hz, 3H). HRMS (ESI):

m/z [M + H]⁺calcd for C₃₀H₃₈N₃O₅S, 552.2527; found, 552.2513.

General Procedure for the Synthesis of Inhibitors and

Intermediates on a Solid Support. All sequences and intermediates

were assembled by stepwise solid-phase synthesis on 2-chlorotrityl

chloride resin or Rink amid resin using the standard Fmoc strategy, as

previously described.^17,42,60Solid-phase synthesis was done manually

in plastic syringes equipped with a frit; all steps were performed at

room temperature under continuous shaking. The Rink amide resin

was preswollen in DCM for at least 20 min and then washed 3× with

DMF. For Fmoc deprotection, a piperidine solution (10−20% in

DMF) was added 2× for 10 and 5 min. Following each deprotection

or coupling step, the resin was washed 3× with DMF, 3× with DCM,

and again 3× with DMF. COMU and TMP were used for coupling

steps. In detail, the coupling solution contained the Nα-Fmoc-

protected amino acid or the respective building block (2.0−3.0

equiv), COMU (2.0−3.0 equiv), and TMP (2.3−3.9 equiv) in DMF

(1.0 mL per 100 mg of resin). The solution was added to the resin,

and the mixture was shaken for 60−120 min. Afterward, the resin was

washed as described before. Fmoc deprotection and coupling steps

were iteratively repeated until the desired sequence was obtained. For

the sequence containing 4-(OH)-phenylglycine, a ﬁnal treatment with

piperidine solution (20% in DMF) for 30 min was carried out after

coupling of the cap to cleave any formed esters at the unprotected

hydroxyl group. The resin loaded with the ﬁnished peptide or peptide

hybrid was washed 5× with diethyl ether and dried under reduced

pressure. The ﬁnal product was cleaved 2× oﬀ the resin with TFA/

TIPS/H₂O solution (95:1:4, 1−2 mL per 100 mg of resin) or TFA/

H₂O/DCM solution (95:2.5:2.5, 1−2 mL per 100 mg of resin), and

the mixture was shaken for 2 h. The cleavage solution was dispensed

into cold diethyl ether or hexane (35 mL per 100 mg of resin), and

the resulting precipitate was centrifuged (4000g, 10 min), washed

with diethyl ether/hexane, and dried under reduced pressure. If the

peptides were soluble in the organic solvent, then the organic phase

was washed 2 times with a small amount of water, and all solvents

were removed in vacuo. The resulting residue was coevaporated with

toluene several times.

(S)-1-(2-(4-(Benzyloxy)phenyl)-2-hexanamidoacetyl)piperidine-

1

4-carboxamide (11). H NMR (300 MHz, DMSO-d₆): δ 8.32−8.19

(m, 1H), 7.56−7.16 (m, 9H), 6.97 (d, J = 7.6 Hz, 2H), 5.78 (d, J =

8.0 Hz, 1H), 5.09 (s, 2H), 4.33 (t, J = 14.5 Hz, 1H), 3.89 (t, J = 14.5

Hz, 1H), 3.11−2.54 (m, 2H), 2.35−2.19 (m, 1H), 2.18−2.06 (m,

2H), 1.73−1.08 (m, 10H), 0.82 (t, J = 6.9 Hz, 3H). HRMS (ESI): m/

z [M + H]⁺calcd for C₂₇H₃₆N₃O₄, 466.2700; found, 466.2699.

( 1 S , 4 r ) - 4 - ( ( ( S ) - 2 - ( 4 - ( B e n z y l o x y ) p h e n y l ) - 2 -

1

hexanamidoacetamido)methyl)cyclohexanecarboxamide (13). H

NMR (300 MHz, CDCl₃): δ 7.44−7.27 (m, 7H), 6.94 (d, J = 8.7 Hz,

2H), 5.68 (s, 1H), 5.05 (s, 2H), 3.11 (s, 2H), 2.21 (t, J = 7.6 Hz, 2H),

2.06−1.84 (m, 3H), 1.75−1.54 (m, 6H), 1.48−1.34 (m, 3H), 1.32−

1.19 (m, 4H), 0.86 (t, J = 6.5 Hz, 3H). ¹³C NMR (APT, 75 MHz,

DMSO-d₆): δ 177.2, 171.8, 170.1, 159.8, 137.1, 131.6, 128.4, 128.2,

127.8, 127.6, 114.4, 69.1, 55.4, 43.7, 42.8, 37.0, 34.9, 30.9, 29.5, 28.7,

25.0, 21.9, 13.9. HRMS (ESI): m/z [M + H]⁺calcd for C₂₉H₄₀N₃O₄,

494.3013; found, 494.3010.

The peptide synthesis with 2-chlorotrityl chloride resin was

performed as described with Rink amid resin with the exception of

the loading and cleavage step. The respective Fmoc-protected ﬁrst

building block (1.5−3.0 equiv) was added to the resin in the syringe,

and 1 mL of DMF and TMP (3 equiv) was added. If a hydroxyl

functional group was coupled in the loading step, then 1 mL of

pyridine (per 100 mg resin) was added instead of the DMF/TMP

mixture. The resin was shaken overnight and capped before the Fmoc

deprotection step. Therefore, the resin was washed with 3× DMF, 3×

DCM, and again 3× DMF, and the capping solution (DCM/MeOH/

TMP, 80:15:5 (v/v), 1 mL per 100 mg of resin) was added 2× for 20

min. The resin was washed with 3× DMF, 3× DCM, and again 3×

(S)-4-(2-(4-(Benzyloxy)phenyl)-2-hexanamidoacetamido)-

1

benzamide (14). H NMR (300 MHz, DMSO-d₆): δ 10.45 (s, 1H),

8.51 (d, J = 7.0 Hz, 1H), 7.92−7.72 (m, 4H), 7.62 (d, J = 8.5 Hz,

2H), 7.49−7.20 (m, 7H), 7.00 (d, J = 8.1 Hz, 2H), 5.55 (d, J = 7.1

Hz, 1H), 5.09 (s, 2H), 2.20 (t, J = 7.1 Hz, 2H), 1.58−1.41 (m, 2H),

1.30−1.18 (m, 4H), 0.84 (t, J = 6.3 Hz, 3H). HRMS (ESI): m/z [M +

H]⁺calcd for C₂₈H₃₂N₃O₄, 474.2387; found, 474.2386.

(S)-2-(2-(4-(Benzyloxy)phenyl)-2-hexanamidoacetamido)-

1

benzamide (16). H NMR (300 MHz, DMSO-d₆): δ 12.27 (s, 1H),

8.64 (d, J = 6.4 Hz, 1H), 8.48 (d, J = 8.5 Hz, 1H), 8.25 (s, 2H), 7.80

(d, J = 7.2 Hz, 1H), 7.71 (s, 1H), 7.51−7.28 (m, 7H), 7.10 (t, J = 7.4

Hz, 1H), 7.00 (d, J = 8.7 Hz, 2H), 5.23 (d, J = 6.4 Hz, 1H), 5.09 (s,

4580

https://doi.org/10.1021/acs.jmedchem.0c02042

J. Med. Chem. 2021, 64, 4567−4587

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

2H), 2.30−2.20 (m, 2H), 1.50 (p, J = 7.2 Hz, 2H), 1.29−1.18 (m,

4H), 0.83 (t, J = 6.7 Hz, 3H). HRMS (ESI): m/z [M + H]⁺calcd for

C₂₈H₃₂N₃O₄, 474.2387; found, 474.2379.

HRMS (ESI): m/z [M + H]⁺calcd for C₂₃H₃₀N₃O₃, 396.2282; found,

396.2287.

(R)-4-((2-(4-(Benzyloxy)phenyl)-2-hexanamidoacetamido)-

methyl)benzamide (31). ¹H NMR (300 MHz, DMSO-d₆): δ 8.71 (t,

J = 5.8 Hz, 1H), 8.38 (d, J = 7.8 Hz, 1H), 7.91 (s, 2H), 7.77 (d, J =

8.3 Hz, 2H), 7.46−7.28 (m, 7H), 7.20 (d, J = 8.2 Hz, 2H), 6.98 (d, J

= 8.8 Hz, 2H), 5.43 (d, J = 7.8 Hz, 1H), 5.10 (s, 2H), 4.30 (d, J = 5.9

Hz, 2H), 2.18 (t, J = 7.4 Hz, 2H), 1.48 (p, J = 7.3 Hz, 2H), 1.30−1.16

(m, 4H), 0.83 (t, J = 6.9 Hz, 3H). ¹³C NMR (APT, 75 MHz, DMSO-

d₆): δ 171.9, 170.5, 167.6, 157.7, 142.5, 137.1, 132.7, 131.1, 128.4,

128.3, 127.8, 127.6, 127.4, 126.6, 114.5, 69.2, 55.7, 41.7, 34.9, 30.9,

24.9, 21.9, 13.9. HRMS (ESI): m/z [M + H]⁺calcd for C₂₉H₃₄N₃O₄,

488.2544; found, 488.2538.

(S)-4-((2-Hexanamido-2-(4-(4-(triﬂuoromethyl)benzyloxy)-

phenyl)acetamido)methyl)benzamide (32). ¹H NMR (300 MHz,

acetone-d₆): δ 7.96 (t, J = 5.7 Hz, 1H), 7.83 (d, J = 8.0 Hz, 2H), 7.73

(q, J = 8.6 Hz, 4H), 7.59 (d, J = 7.8 Hz, 1H), 7.39 (d, J = 8.6 Hz, 2H),

7.27 (d, J = 7.8 Hz, 2H), 6.99 (d, J = 8.4 Hz, 2H), 6.57 (s, 2H), 5.53

(d, J = 7.5 Hz, 1H), 5.25 (s, 2H), 4.45 (d, J = 6.0 Hz, 2H), 2.26 (t, J =

7.5 Hz, 2H), 1.66−1.48 (m, 2H), 1.35−1.20 (m, 4H), 0.85 (t, J = 5.9

Hz, 3H). HRMS (ESI): m/z [M + H]⁺calcd for C₃₀H₃₃F₃N₃O₄,

556.2418; found, 556.2423.

(R)-4-((2-Hexanamido-2-(4-(4-(triﬂuoromethyl)benzyloxy)-

phenyl)acetamido)methyl)benzamide (33). ¹H NMR (300 MHz,

DMSO-d₆): δ 8.72 (t, J = 5.7 Hz, 1H), 8.38 (d, J = 7.8 Hz, 1H), 7.90

(s, 2H), 7.77 (d, J = 8.1 Hz, 4H), 7.66 (d, J = 8.0 Hz, 2H), 7.35 (d, J =

8.5 Hz, 2H), 7.20 (d, J = 8.1 Hz, 2H), 6.99 (d, J = 8.5 Hz, 2H), 5.43

(d, J = 7.8 Hz, 1H), 5.23 (s, 2H), 4.30 (d, J = 5.7 Hz, 2H), 2.18 (t, J =

7.3 Hz, 2H), 1.48 (p, J = 7.3 Hz, 2H), 1.30−1.14 (m, 4H), 0.83 (t, J =

6.8 Hz, 3H). HRMS (ESI): m/z [M + H]⁺calcd for C₃₀H₃₃F₃N₃O₄,

556.2418; found, 556.2406.

( S ) - 4 - ( ( 2 - ( 4 - ( 4 - t e r t - B u t y l b e n z y l o x y ) p h e n y l ) - 2 -

hexanamidoacetamido)methyl)benzamide (34). ¹H NMR (300

MHz, DMSO-d₆): δ 8.66 (t, J = 5.9 Hz, 1H), 8.30 (d, J = 7.5 Hz, 1H),

7.90 (s, 2H), 7.76 (d, J = 7.7 Hz, 2H), 7.45−7.01 (m, 8H), 6.86 (d, J

= 7.9 Hz, 2H), 5.36 (d, J = 7.4 Hz, 1H), 5.13 (s, 2H), 4.30 (d, J = 4.8

Hz, 2H), 2.17 (t, J = 6.9 Hz, 2H), 1.55−1.41 (m, 2H), 1.32−1.11 (m,

13H), 0.83 (t, J = 6.9 Hz, 3H). HRMS (ESI): m/z [M + H]⁺calcd for

C₃₃H₄₂N₃O₄, 544.3170; found, 544.3161.

(S)-N-(2-(4-(2-Amino-2-oxoethyl)phenylamino)-1-(4-

(benzyloxy)phenyl)-2-oxoethyl)hexanamide (17). ¹H NMR (300

MHz, DMSO-d₆): δ 10.12 (s, 1H), 8.36 (d, J = 7.7 Hz, 1H), 7.45 (d, J

= 7.8 Hz, 2H), 7.28−7.11 (m, 9H), 7.07 (d, J = 8.5 Hz, 2H), 6.82 (d,

J = 8.0 Hz, 2H), 5.45 (d, J = 7.7 Hz, 1H), 5.11 (s, 1H), 3.83 (s, 2H),

2.17 (t, J = 7.1 Hz, 2H), 1.47 (p, J = 7.2 Hz, 2H), 1.30−1.15 (m, 4H),

0.84 (t, J = 6.6 Hz, 3H). HRMS (ESI): m/z [M + H]⁺calcd for

C₂₉H₃₄N₃O₄, 488.2544; found, 488.2549.

(S)-4-((2-(4-(Benzyloxy)phenyl)-2-hexanamidoacetamido)-

methyl)benzamide (18). ¹H NMR (500 MHz, DMSO-d₆): δ 8.71 (t,

J = 5.9 Hz, 1H), 8.37 (d, J = 7.9 Hz, 1H), 7.90 (s, 2H), 7.76 (d, J =

8.1 Hz, 2H), 7.44 (d, J = 7.3 Hz, 2H), 7.39 (t, J = 7.5 Hz, 2H), 7.33

(d, J = 8.6 Hz, 2H), 7.31−7.27 (m, 1H), 7.20 (d, J = 8.1 Hz, 2H),

6.98 (d, J = 8.6 Hz, 2H), 5.42 (d, J = 7.8 Hz, 1H), 5.10 (s, 2H), 4.30

(d, J = 5.7 Hz, 2H), 2.18 (t, J = 7.4 Hz, 2H), 1.48 (p, J = 7.4 Hz, 2H),

1.24−1.16 (m, 4H), 0.83 (t, J = 7.1 Hz, 3H). ¹³C NMR (APT, 126

MHz, DMSO-d₆): δ 171.9, 170.4, 167.6, 157.7, 142.5, 137.1, 132.7,

131.1, 128.4, 128.4, 127.8, 127.6, 127.4, 126.6, 114.5, 69.2, 55.7, 41.7,

34.9, 30.9, 24.9, 21.8, 13.9. HRMS (ESI): m/z [M + H]⁺calcd for

C₂₉H₃₄N₃O₄, 488.2544; found, 488.2537.

(S)-4-(2-(2-(4-(Benzyloxy)phenyl)-2-hexanamidoacetyl)-

hydrazinyl)benzamide (19). ¹H NMR (300 MHz, acetone-d₆): δ

9.16 (br s, 1H), 7.71 (d, J = 8.8 Hz, 2H), 7.61−7.54 (m, 1H), 7.49 (d,

J = 7.2 Hz, 2H), 7.46−7.22 (m, 6H), 7.02 (d, J = 8.8 Hz, 2H), 6.73

(d, J = 8.7 Hz, 2H), 6.28 (s, 2H), 5.59−5.54 (m, 1H), 5.15 (s, 2H),

2.27 (t, J = 7.4 Hz, 2H), 1.66−1.53 (m, 2H), 1.36−1.21 (m, 4H),

0.86 (t, J = 6.9 Hz, 3H). HRMS (ESI): m/z [M−H]⁻calcd for

C₂₈H₃₁N₄O₄, 487.2351; found, 487.2368.

(S)-4-(2-(2-(4-(Benzyloxy)phenyl)-2-hexanamidoacetamido)-

1

ethyl)benzamide (20). H NMR (300 MHz, CD₃OD/D₂O): δ 7.71

(d, J = 8.6 Hz, 2H), 7.44 (d, J = 6.8 Hz, 2H), 7.40−7.28 (m, 3H),

7.25−7.13 (m, 4H), 6.94 (d, J = 8.8 Hz, 2H), 5.27 (s, 1H), 5.10 (s,

2H), 3.63−3.49 (m, 2H), 2.81 (t, J = 6.8 Hz, 2H), 2.25 (t, J = 7.6 Hz,

2H), 1.66−1.53 (m, 2H), 1.39−1.21 (m, 4H), 0.88 (t, J = 6.7 Hz,

3H). HRMS (ESI): m/z [M + H]⁺calcd for C₃₀H₃₆N₃O₄, 502.2700;

found, 502.2697.

( S ) - 4 - ( ( 2 - ( 4 - ( 4 - C y a n o b e n z y l o x y ) p h e n y l ) - 2 -

hexanamidoacetamido)methyl)benzamide (35). ¹H NMR (300

MHz, acetone-d₆): δ 7.92 (t, J = 5.9 Hz, 1H), 7.82 (d, J = 6.7 Hz,

2H), 7.70 (d, J = 7.3 Hz, 2H), 7.55 (d, J = 6.7 Hz, 1H), 7.39 (d, J =

7.9 Hz, 2H), 7.27 (d, J = 7.1 Hz, 2H), 7.14 (d, J = 7.7 Hz, 2H), 6.99

(d, J = 7.8 Hz, 2H), 6.53 (br s, 2H), 5.52 (d, J = 5.8 Hz, 1H), 5.26 (s,

2H), 4.45 (d, J = 4.2 Hz, 2H), 2.26 (t, J = 7.6 Hz, 2H), 1.69−1.49 (m,

2H), 1.37−1.20 (m, 4H), 0.86 (t, J = 5.8 Hz, 3H). ¹³C NMR (APT,

126 MHz, DMSO-d₆): δ 171.9, 170.4, 167.6, 157.3, 143.0, 142.5,

132.7, 132.4, 131.4, 128.0, 127.4, 126.6, 118.7, 114.5, 110.4, 68.2,

55.7, 41.7, 34.8, 30.9, 24.9, 21.8, 13.9. HRMS (ESI): m/z [M + H]⁺

calcd for C₃₀H₃₃N₄O₄, 513.2496; found, 513.2499.

(S)-4-((2-(4-(Benzyloxy)phenyl)-2-hexanamidoacetamido)-

methyl)benzoic Acid (25). ¹H NMR (300 MHz, acetone-d₆): δ 7.99−

7.88 (m, 3H), 7.55 (d, J = 7.4 Hz, 1H), 7.48 (d, J = 7.1 Hz, 2H),

7.43−7.28 (m, 7H), 6.98 (d, J = 8.8 Hz, 2H), 5.51 (d, J = 7.5 Hz,

1H), 5.13 (s, 2H), 4.48 (d, J = 6.3 Hz, 2H), 2.26 (t, J = 7.5 Hz, 2H),

1.66−1.52 (m, 2H), 1.34−1.22 (m, 4H), 0.86 (t, J = 6.9 Hz, 3H).

HRMS (ESI): m/z [M−H]⁻calcd for C₂₉H₃₁N₂O₅, 487.2238; found,

487.2266.

(S)-4-((2-Hexanamido-2-phenylacetamido)methyl)benzamide

1

(27). H NMR (300 MHz, DMSO-d₆): δ 8.80 (t, J = 5.7 Hz, 1H),

8.46 (d, J = 7.9 Hz, 1H), 7.90 (s, 2H), 7.76 (d, J = 8.0 Hz, 2H), 7.43

(d, J = 7.0 Hz, 2H), 7.37−7.28 (m, 3H), 7.19 (d, J = 8.0 Hz, 2H),

5.51 (d, J = 7.9 Hz, 1H), 4.31 (d, J = 5.6 Hz, 2H), 2.20 (t, J = 7.3 Hz,

2H), 1.49 (p, J = 7.1 Hz, 2H), 1.32−1.14 (m, 4H), 0.83 (t, J = 6.8 Hz,

3H). HRMS (ESI): m/z [M + H]⁺calcd for C₂₂H₂₈N₃O₃, 382.2136;

found, 382.2143.

(S)-4-((2-Hexanamido-2-(4-(triﬂuoromethyl)phenyl)acetamido)-

methyl)benzamide (28). ¹H NMR (300 MHz, DMSO-d₆): δ 8.92 (t,

J = 5.6 Hz, 1H), 8.63 (d, J = 7.9 Hz, 1H), 7.90 (s, 2H), 7.82−7.70 (m,

4H), 7.64 (d, J = 7.8 Hz, 2H), 7.21 (d, J = 8.0 Hz, 2H), 5.63 (d, J =

7.9 Hz, 1H), 4.44−4.21 (m, 2H), 2.21 (t, J = 7.3 Hz, 2H), 1.54−1.41

(m, 2H), 1.32−1.15 (m, 4H), 0.83 (t, J = 6.8 Hz, 3H). HRMS (ESI):

m/z [M + H]⁺calcd for C₂₃H₂₇F₃N₃O₃, 450.1999; found, 450.1994.

(S)-4-((2-Hexanamido-3-phenylpropanamido)methyl)-

benzamide (30). ¹H NMR (300 MHz, DMSO-d₆): δ 8.51 (t, J = 5.6

Hz, 1H), 8.07 (d, J = 8.3 Hz, 1H), 7.90 (s, 2H), 7.78 (d, J = 8.1 Hz,

2H), 7.40−7.13 (m, 7H), 4.61−4.50 (m, 1H), 4.30 (d, J = 5.6 Hz,

2H), 3.05−2.68 (m, 2H), 2.04 (t, J = 7.5 Hz, 2H), 1.45−1.29 (m,

2H), 1.27−0.99 (m, 4H), 0.80 (t, J = 7.1 Hz, 3H). ¹³C NMR (APT,

75 MHz, DMSO-d₆): δ 172.1, 171.4, 167.6, 142.6, 138.0, 132.7, 129.1,

128.0, 127.4, 126.7, 126.2, 54.0, 41.7, 37.7, 35.1, 30.7, 24.9, 21.9, 13.8.

(S)-4-((2-Hexanamido-2-(4-(3-methoxybenzyloxy)phenyl)-

1

acetamido)methyl)benzamide (36). H NMR (300 MHz, DMSO-

d₆): δ 8.71 (t, J = 5.6 Hz, 1H), 8.37 (d, J = 7.8 Hz, 1H), 7.90 (s, 2H),

7.77 (d, J = 8.0 Hz, 2H), 7.39−7.24 (m, 4H), 7.20 (d, J = 8.0 Hz,

2H), 7.06−6.94 (m, 3H), 6.88 (d, J = 8.4 Hz, 1H), 5.42 (d, J = 7.8

Hz, 1H), 5.08 (s, 2H), 4.30 (d, J = 5.4 Hz, 2H), 3.76 (s, 3H), 2.18 (t,

J = 7.2 Hz, 2H), 1.48 (p, J = 7.0 Hz, 2H), 1.30−1.15 (m, 4H), 0.83 (t,

J = 6.8 Hz, 3H). HRMS (ESI): m/z [M + H]⁺calcd for C₃₀H₃₆N₃O₅,

518.2649; found, 518.2648.

( S ) - 4 - ( ( 2 - ( 4 - ( 2 , 6 - D i c h l o r o b e n z y l o x y ) p h e n y l ) - 2 -

hexanamidoacetamido)methyl)benzamide (37). ¹H NMR (300

MHz, acetone-d₆): δ 7.95−7.87 (m, 1H), 7.83 (d, J = 7.3 Hz, 2H),

7.58−7.38 (m, 6H), 7.30 (d, J = 7.4 Hz, 2H), 7.04 (d, J = 7.7 Hz,

2H), 6.52 (s, 2H), 5.53 (d, J = 6.8 Hz, 1H), 5.32 (s, 2H), 4.47 (d, J =

4.7 Hz, 2H), 2.47−2.31 (m, 2H), 1.68−1.49 (m, 2H), 1.37−1.22 (m,

4H), 0.86 (t, J = 5.8 Hz, 3H). HRMS (ESI): m/z [M + H]⁺calcd for

C₂₉H₃₂Cl₂N₃O₄, 556.1764; found, 556.1754.

( R ) - 4 - ( ( 2 - ( 4 - ( 3 , 4 - D i c h l o r o b e n z y l o x y ) p h e n y l ) - 2 -

hexanamidoacetamido)methyl)benzamide (38). ¹H NMR (300

MHz, DMSO-d₆): δ 8.73 (t, J = 5.6 Hz, 1H), 8.39 (d, J = 7.8 Hz, 1H),

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7.91 (s, 2H), 7.77 (d, J = 8.0 Hz, 2H), 7.72 (s, 1H), 7.66 (d, J = 8.2

Hz, 1H), 7.46−7.30 (m, 3H), 7.20 (d, J = 7.9 Hz, 2H), 6.99 (d, J =

8.3 Hz, 2H), 5.43 (d, J = 7.8 Hz, 1H), 5.12 (s, 2H), 4.30 (d, J = 5.3

Hz, 2H), 2.18 (t, J = 7.2 Hz, 2H), 1.48 (p, J = 7.2 Hz, 2H), 1.31−1.13

(m, 4H), 0.83 (t, J = 6.7 Hz, 3H). HRMS (ESI): m/z [M + H]⁺calcd

for C₂₉H₃₂Cl₂N₃O₄, 556.1764; found, 556.1755.

4-(((S)-2-((S)-2-Acetamidohexanamido)-2-(4-(benzyloxy)-

phenyl)acetamido)methyl)benzamide (39). ¹H NMR (300 MHz,

DMSO-d₆): δ 8.70 (t, J = 5.9 Hz, 1H), 8.36 (d, J = 7.5 Hz, 1H), 7.99

(d, J = 8.0 Hz, 1H), 7.90 (s, 2H), 7.76 (d, J = 7.9 Hz, 2H), 7.49−7.25

(m, 7H), 7.18 (d, J = 7.9 Hz, 2H), 6.99 (d, J = 8.3 Hz, 2H), 5.39 (d, J

= 7.5 Hz, 1H), 5.11 (s, 2H), 4.40−4.23 (m, 3H), 1.83 (s, 3H), 1.69−

1.21 (m, 6H), 0.84 (t, J = 6.5 Hz, 3H). HRMS (ESI): m/z [M + H]⁺

calcd for C₃₁H₃₇N₄O₅, 545.2758; found, 545.2740.

δ 8.72 (t, J = 5.8 Hz, 1H), 8.36 (d, J = 6.5 Hz, 1H), 7.76 (d, J = 8.2

Hz, 2H), 7.47−7.29 (m, 7H), 7.26 (d, J = 8.2 Hz, 2H), 7.00 (d, J =

8.8 Hz, 2H), 5.40−5.34 (m, 1H), 5.10 (s, 2H), 4.54−4.30 (m, 2H),

2.90 (t, J = 7.6 Hz, 2H), 2.41−2.22 (m, 2H), 1.74−1.56 (m, 4H),

1.46−1.32 (m, 2H). ¹³C NMR (APT, 75 MHz, DMSO-d₆): δ 171.7,

170.4, 167.6, 157.8, 142.5, 137.1, 132.7, 131.0, 128.5, 128.4, 127.8,

127.6, 127.4, 126.6, 114.6, 69.2, 55.8, 41.7, 34.6, 26.8, 25.5, 24.7.

HRMS (ESI): m/z [M + H]⁺calcd for C₂₉H₃₅N₄O₄, 503.2653; found,

503.2663.

(S)-4-(Aminomethyl)-N-(1-(4-(benzyloxy)phenyl)-2-(4-carba-

moylbenzylamino)-2-oxoethyl)benzamide (50). ¹H NMR (300

MHz, DMSO-d₆): δ 8.83 (d, J = 7.7 Hz, 1H), 8.77 (t, J = 5.8 Hz,

1H), 8.29 (br s, 3H), 7.97 (d, J = 8.2 Hz, 2H), 7.92 (s, 2H), 7.78 (d, J

= 8.2 Hz, 2H), 7.53 (d, J = 8.3 Hz, 2H), 7.48−7.29 (m, 7H), 7.23 (d,

J = 8.2 Hz, 2H), 7.01 (d, J = 8.7 Hz, 2H), 5.65 (d, J = 7.5 Hz, 1H),

5.12 (s, 2H), 4.35 (d, J = 5.6 Hz, 2H), 4.11 (s, 2H). HRMS (ESI): m/

z [M + H]⁺calcd for C₃₁H₃₁N₄O₄, 523.2340; found, 523.2366.

(S)-4-((2-(4-(Benzyloxy)phenyl)-2-(3-phenylpropanamido)-

(S)-4-((2-(4-(Benzyloxy)phenyl)-2-(cyclobutanecarboxamido)-

1

acetamido)methyl)benzamide (41). H NMR (300 MHz, DMSO-

d₆): δ 8.71 (t, J = 5.8 Hz, 1H), 8.21 (d, J = 7.8 Hz, 1H), 7.91 (s, 2H),

7.77 (d, J = 8.2 Hz, 2H), 7.50−7.26 (m, 7H), 7.19 (d, J = 8.2 Hz,

2H), 6.98 (d, J = 8.8 Hz, 2H), 5.42 (d, J = 7.8 Hz, 1H), 5.10 (s, 2H),

4.30 (d, J = 5.8 Hz, 2H), 3.21 (p, J = 8.0 Hz, 1H), 2.29−1.66 (m,

6H). HRMS (ESI): m/z [M + H]⁺calcd for C₂₈H₃₀N₃O₄, 472.2231;

found, 472.2228.

(S)-4-((2-(4-(Benzyloxy)phenyl)-2-pentanamidoacetamido)-

methyl)benzamide (42). ¹H NMR (300 MHz, acetone-d₆): δ 7.90 (t,

J = 6.7 Hz, 1H), 7.82 (d, J = 7.5 Hz, 2H), 7.53 (d, J = 7.6 Hz, 1H),

7.47 (d, J = 7.4 Hz, 2H), 7.44−7.30 (m, 5H), 7.27 (d, J = 7.9 Hz,

2H), 6.97 (d, J = 7.1 Hz, 2H), 6.56 (s, 2H), 5.51 (d, J = 6.3 Hz, 1H),

5.12 (s, 2H), 4.45 (d, J = 5.7 Hz, 2H), 2.26 (t, J = 6.7 Hz, 2H), 1.56

(p, J = 7.4 Hz, 2H), 1.38−1.23 (m, 2H), 0.87 (t, J = 7.2 Hz, 3H). ¹³C

NMR (APT, 75 MHz, DMSO-d₆): δ 171.9, 170.5, 167.6, 157.7, 142.5,

137.1, 132.7, 131.0, 128.44, 128.42, 127.8, 127.6, 127.4, 126.6, 114.5,

69.2, 55.7, 41.7, 34.6, 27.4, 21.8, 13.8. HRMS (ESI): m/z [M + H]⁺

calcd for C₂₈H₃₂N₃O₄, 474.2387; found, 474.2393.

1

acetamido)methyl)benzamide (51). H NMR (300 MHz, DMSO-

d₆): δ 8.73 (t, J = 6.0 Hz, 1H), 8.46 (d, J = 7.8 Hz, 1H), 7.91 (s, 2H),

7.78 (d, J = 8.3 Hz, 2H), 7.46−7.17 (m, 14H), 6.97 (d, J = 8.8 Hz,

2H), 5.43 (d, J = 7.8 Hz, 1H), 5.10 (s, 2H), 4.31 (d, J = 5.7 Hz, 2H),

2.80 (t, J = 7.8 Hz, 2H), 2.59−2.51 (m, 2H). ¹³C NMR (APT, 75

MHz, DMSO-d₆): δ 171.1, 170.3, 167.6, 157.7, 142.5, 141.3, 137.1,

132.7, 131.0, 128.4, 128.4, 128.2, 127.8, 127.6, 127.4, 126.7, 125.8,

114.5, 69.2, 55.8, 41.7, 36.5, 31.1. HRMS (ESI): m/z [M + H]⁺calcd

for C₃₂H₃₂N₃O₄, 522.2387; found, 522.2395.

(S,E)-4-((2-(4-(Benzyloxy)phenyl)-2-cinnamamidoacetamido)-

methyl)benzamide (52). ¹H NMR (300 MHz, DMSO-d₆): δ 8.83 (t,

J = 5.6 Hz, 1H), 8.70 (d, J = 7.7 Hz, 1H), 7.91 (s, 2H), 7.78 (d, J =

7.9 Hz, 2H), 7.55 (d, J = 6.7 Hz, 2H), 7.45−7.29 (m, 11H), 7.22 (d, J

= 7.9 Hz, 2H), 7.01 (d, J = 8.3 Hz, 2H), 6.94 (d, J = 15.8 Hz, 1H),

5.57 (d, J = 7.7 Hz, 1H), 5.11 (s, 2H), 4.33 (d, J = 4.3 Hz, 2H). ¹³C

NMR (75 MHz, DMSO-d₆): δ 170.3, 167.6, 164.5, 157.9, 142.5,

139.1, 137.1, 134.9, 132.8, 130.9, 129.5, 128.9, 128.5, 128.4, 127.8,

127.6, 127.5, 126.7, 122.1, 114.6, 69.2, 56.0, 41.8. HRMS (ESI): m/z

[M + H]⁺calcd for C₃₂H₃₀N₃O₄, 520.2231; found, 520.2228.

(S)-N-(1-(4-(Benzyloxy)phenyl)-2-(4-carbamoylbenzylamino)-2-

oxoethyl)thiophene-2-carboxamide (54). ¹H NMR (300 MHz,

DMSO-d₆): δ 8.84 (d, J = 7.9 Hz, 1H), 8.76 (t, J = 5.8 Hz, 1H), 8.02

(d, J = 3.4 Hz, 1H), 7.91 (s, 2H), 7.82−7.73 (m, 3H), 7.46−7.29 (m,

7H), 7.23 (d, J = 8.1 Hz, 2H), 7.16−7.12 (m, 1H), 7.01 (d, J = 8.5

Hz, 2H), 5.61 (d, J = 7.6 Hz, 1H), 5.12 (s, 2H), 4.34 (d, J = 5.5 Hz,

2H). HRMS (ESI): m/z [M + H]⁺calcd for C₂₈H₂₆N₃O₄S, 500.1639;

found, 500.1641.

(S)-N-(1-(4-(Benzyloxy)phenyl)-2-(4-carbamoylbenzylamino)-2-

oxoethyl)benzo[b]thiophene-2-carboxamide (55). ¹H NMR (300

MHz, DMSO-d₆): δ 9.12 (d, J = 7.5 Hz, 1H), 8.80 (t, J = 5.8 Hz, 1H),

8.38 (s, 1H), 8.01 (d, J = 8.5 Hz, 1H), 7.91 (s, 2H), 7.79 (d, J = 8.2

Hz, 2H), 7.49−7.29 (m, 10H), 7.24 (d, J = 8.2 Hz, 2H), 7.03 (d, J =

8.7 Hz, 2H), 5.64 (d, J = 7.6 Hz, 1H), 5.13 (s, 2H), 4.36 (d, J = 5.7

Hz, 2H). HRMS (ESI): m/z [M + H]⁺calcd for C₃₂H₂₈N₃O₄S,

550.1795; found, 550.1792.

(S)-N-(1-(4-(Benzyloxy)phenyl)-2-(4-carbamoylbenzylamino)-2-

oxoethyl)-2,2′-bithiophene-5-carboxamide (56). ¹H NMR (300

MHz, acetone-d₆): δ 8.11−7.99 (m, 2H), 7.86−7.79 (m, 3H),

7.52−7.28 (m, 11H), 7.26 (d, J = 3.9 Hz, 1H), 7.11 (dd, J = 5.1, 3.7

Hz, 1H), 7.01 (d, J = 8.8 Hz, 2H), 6.55 (br s, 2H), 5.69 (d, J = 7.3 Hz,

1H), 5.14 (s, 2H), 4.50 (d, J = 6.0 Hz, 2H). HRMS (ESI): m/z [M +

H]⁺calcd for C₃₂H₂₈N₃O₄S₂, 582.1516; found, 582.1513.

(S)-4-((2-(4-(Benzyloxy)phenyl)-2-heptanamidoacetamido)-

1

methyl)benzamide (43). H NMR (300 MHz, acetone-d₆): δ 7.95−

7.88 (m, 1H), 7.82 (d, J = 7.0 Hz, 2H), 7.56−7.51 (m, 1H), 7.49 (d, J

= 7.0 Hz, 2H), 7.43−7.30 (m, 5H), 7.28 (d, J = 7.7 Hz, 2H), 6.97 (d,

J = 9.0 Hz, 2H), 6.52 (s, 2H), 5.53−5.47 (m, 1H), 5.12 (s, 2H), 4.45

(d, J = 6.2 Hz, 2H), 2.30−2.22 (m, 2H), 1.65−1.50 (m, 2H), 1.34−

1.22 (m, 6H), 0.92−0.82 (m, 3H). ¹³C NMR (APT, 126 MHz,

DMSO-d₆): δ 171.9, 170.4, 167.6, 157.7, 142.5, 137.1, 132.7, 131.1,

128.43, 128.41, 127.8, 127.6, 127.4, 126.6, 114.5, 69.2, 55.7, 41.7,

34.9, 31.0, 28.3, 25.2, 22.0, 13.9. HRMS (ESI): m/z [M + H]⁺calcd

for C₃₀H₃₆N₃O₄, 502.2700; found, 502.2704.

(S)-4-((2-(4-(Benzyloxy)phenyl)-2-octanamidoacetamido)-

methyl)benzamide (44). ¹H NMR (300 MHz, acetone-d₆): δ 7.90 (t,

J = 5.7 Hz, 1H), 7.82 (d, J = 8.1 Hz, 2H), 7.53 (d, J = 7.2 Hz, 1H),

7.47 (d, J = 7.4 Hz, 2H), 7.43−7.33 (m, 5H), 7.28 (d, J = 8.1 Hz,

2H), 6.97 (d, J = 8.6 Hz, 2H), 6.52 (s, 2H), 5.50 (d, J = 7.4 Hz, 1H),

5.12 (s, 2H), 4.45 (d, J = 6.0 Hz, 2H), 2.26 (t, J = 7.4 Hz, 2H), 1.66−

1.51 (m, 2H), 1.36−1.22 (m, 8H), 0.86 (t, J = 5.7 Hz, 3H). HRMS

(ESI): m/z [M + H]⁺calcd for C₃₁H₃₈N₃O₄, 516.2857; found,

516.2833.

(S)-4-(4-(4-(Benzyloxy)phenyl)-3,6-dioxo-8,11-dioxa-2,5-

diazadodecyl)benzamide (47). ¹H NMR (300 MHz, CD₃OD/

D₂O): δ 7.73 (d, J = 8.1 Hz, 2H), 7.45−7.29 (m, 7H), 7.24 (d, J = 8.1

Hz, 2H), 7.01 (d, J = 8.5 Hz, 2H), 5.40 (s, 1H), 5.12 (s, 2H), 4.55−

4.30 (m, 2H), 4.15−3.96 (m, 2H), 3.78−3.48 (m, 4H), 3.21 (s, 3H).

¹³C NMR (APT, 75 MHz, DMSO-d₆): δ 169.9, 168.7, 167.6, 157.9,

142.3, 137.0, 132.8, 130.8, 128.4, 128.3, 127.8, 127.6, 127.5, 126.7,

114.7, 71.0, 70.2, 69.6, 69.2, 58.1, 55.1, 41.8. HRMS (ESI): m/z [M +

H]⁺calcd for C₂₈H₃₂N₃O₆, 506.2286; found, 506.2280.

(S)-N-(1-(4-(Benzyloxy)phenyl)-2-(4-carbamoylbenzylamino)-2-

oxoethyl)-4-phenoxybenzamide (57). ¹H NMR (300 MHz, acetone-

d₆): δ 8.08−7.93 (m, 4H), 7.83 (d, J = 8.1 Hz, 2H), 7.52−7.26 (m,

11H), 7.20 (t, J = 7.3 Hz, 1H), 7.08 (d, J = 7.8 Hz, 2H), 7.05−6.96

(m, 4H), 6.53 (br s, 2H), 5.72 (d, J = 7.0 Hz, 1H), 5.13 (s, 2H), 4.49

(d, J = 5.9 Hz, 2H). ¹³C NMR (APT, 75 MHz, DMSO-d₆): δ 170.3,

168.3, 167.6, 159.5, 157.9, 155.7, 142.6, 137.1, 132.7, 130.6, 130.2,

130.0, 129.0, 128.6, 128.4, 127.8, 127.7, 127.4, 126.7, 124.2, 119.4,

117.3, 114.5, 69.2, 56.7, 41.9. HRMS (ESI): m/z [M + H]⁺calcd for

C₃₆H₃₂N₃O₅, 586.2336; found, 586.2329.

4-(((2S)-2-(4-(Benzyloxy)phenyl)-2-(2-hydroxypentanamido)-

1

acetamido)methyl)benzamide (48). H NMR (300 MHz, CD₃OD/

D₂O): δ 7.75 (d, J = 7.9 Hz, 2H), 7.46−7.30 (m, 7H), 7.24 (d, J = 7.7

Hz, 2H), 7.00 (d, J = 8.1 Hz, 2H), 5.41 (s, 1H), 5.11 (s, 2H), 4.38 (s,

2H), 4.10−4.02 (m, 1H), 1.76−1.26 (m, 4H), 0.91 (t, J = 7.6 Hz,

3H). HRMS (ESI): m/z [M + H]⁺calcd for C₂₈H₃₂N₃O₅, 490.2336;

found, 490.2343.

(S)-4-((2-(6-Aminohexanamido)-2-(4-(benzyloxy)phenyl)-

acetamido)methyl)benzamide (49). ¹H NMR (300 MHz, CD₃OD):

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(S)-4-(Benzyloxy)-N-(1-(4-(benzyloxy)phenyl)-2-(4-carbamoyl-

benzylamino)-2-oxoethyl)benzamide (58). ¹H NMR (300 MHz,

acetone-d₆): δ 7.99 (t, J = 5.9 Hz, 1H), 7.96−7.88 (m, 3H), 7.83 (d, J

= 8.1 Hz, 2H), 7.51−7.34 (m, 12H), 7.30 (d, J = 8.3 Hz, 2H), 7.07 (d,

J = 8.7 Hz, 2H), 7.00 (d, J = 8.6 Hz, 2H), 6.53 (s, 2H), 5.70 (d, J =

7.1 Hz, 1H), 5.19 (s, 2H), 5.13 (s, 2H), 4.49 (d, J = 6.0 Hz, 2H).

HRMS (ESI): m/z [M + H]⁺calcd for C₃₇H₃₄N₃O₅, 600.2493; found,

600.2474.

(d, J = 8.0 Hz, 1H), 7.56−7.45 (m, 3H), 7.42 (d, J = 7.7 Hz, 2H),

7.37 (d, J = 7.7 Hz, 1H), 7.35−7.25 (m, 2H), 7.21 (t, J = 7.4 Hz, 1H),

7.08 (d, J = 7.8 Hz, 2H), 7.05−6.96 (m, 5H), 6.88 (d, J = 8.1 Hz,

1H), 5.73 (d, J = 7.2 Hz, 1H), 5.08 (s, 2H), 3.74 (s, 3H). ¹³C NMR

(APT, 75 MHz, DMSO-d₆): δ 169.2, 167.8, 165.6, 159.6, 159.3,

158.0, 157.9, 138.9, 138.6, 135.1, 133.9, 130.2, 130.0, 129.9, 129.5,

129.4, 129.2, 128.6, 124.3, 121.8, 119.6, 119.4, 118.8, 117.2, 114.7,

113.2, 113.1, 69.0, 57.4, 55.0. HRMS (ESI): m/z [M + H]⁺calcd for

C₃₆H₃₂N₃O₆, 602.2286; found, 602.2268.

(S)-N-(2-(3-Carbamoylphenylamino)-2-oxo-1-(4-

phenoxyphenyl)ethyl)-4-phenoxybenzamide (66). ¹H NMR (300

MHz, DMSO-d₆): δ 10.48 (s, 1H), 8.92 (d, J = 7.6 Hz, 1H), 8.06 (s,

1H), 7.99 (d, J = 8.2 Hz, 2H), 7.95 (s, 2H), 7.79 (d, J = 7.7 Hz, 1H),

7.59 (d, J = 8.4 Hz, 2H), 7.55−7.28 (m, 7H), 7.25−6.95 (m, 9H),

5.81 (d, J = 7.4 Hz, 1H). ¹³C NMR (APT, 75 MHz, DMSO-d₆): δ

169.0, 167.7, 165.7, 159.6, 156.4, 155.6, 138.8, 135.1, 132.5, 132.0,

130.2, 130.0, 129.9, 129.9, 129.7, 128.6, 128.4, 124.3, 123.6, 122.2,

121.8, 119.4, 118.7, 118.4, 117.2, 57.4. HRMS (ESI): m/z [M + H]⁺

calcd for C₃₄H₂₈N₃O₅, 558.2023; found, 558.2021.

(S)-N-(1-(4-(Benzyloxy)phenyl)-2-(3-carbamoylphenylamino)-2-

oxoethyl)-4-phenoxybenzamide (67). ¹H NMR (500 MHz, DMSO-

d₆): δ 10.40 (s, 1H), 8.84 (d, J = 7.4 Hz, 1H), 8.04 (s, 1H), 7.97 (d, J

= 8.8 Hz, 2H), 7.95 (d, J = 8.9 Hz, 1H), 7.93 (s, 2H), 7.77 (d, J = 8.0

Hz, 1H), 7.53 (d, J = 7.8 Hz, 1H), 7.49 (d, J = 8.7 Hz, 2H), 7.46−

7.29 (m, 7H), 7.21 (t, J = 6.9 Hz, 1H), 7.08 (d, J = 8.5 Hz, 2H),

7.05−6.99 (m, 4H), 5.74 (d, J = 7.3 Hz, 1H), 5.10 (s, 2H). ¹³C NMR

(APT, 126 MHz, DMSO-d₆): δ 172.1, 169.2, 167.8, 159.6, 158.0,

155.6, 138.9, 137.0, 135.1, 130.2, 130.0, 129.7, 129.4, 129.2, 128.6,

128.5, 128.4, 127.8, 127.6, 124.2, 122.1, 121.8, 119.4, 117.2, 114.7,

69.2, 57.4. HRMS (ESI): m/z [M + H]⁺calcd for C₃₅H₃₀N₃O₅,

572.2180; found, 572.2162.

(S)-N-(1-(4-(Benzyloxy)phenyl)-2-(3-carbamoylphenylamino)-2-

oxoethyl)-3-phenoxybenzamide (68). ¹H NMR (300 MHz, DMSO-

d₆): δ 10.39 (s, 1H), 8.99 (d, J = 7.1 Hz, 1H), 8.03 (s, 1H), 7.93 (s,

2H), 7.75 (t, J = 7.0 Hz, 2H), 7.59−7.30 (m, 13H), 7.23−7.12 (m,

2H), 7.08−6.97 (m, 4H), 5.71 (d, J = 7.1 Hz, 1H), 5.10 (s, 2H). ¹³C

NMR (APT, 75 MHz, DMSO-d₆): δ 169.1, 167.8, 165.6, 158.1, 156.5,

138.9, 137.0, 135.7, 135.1, 130.1, 129.9, 129.4, 129.3, 128.6, 128.4,

127.8, 127.6, 123.6, 122.9, 122.1, 121.8, 118.8, 118.6, 117.9, 114.7,

69.2, 57.5. HRMS (ESI): m/z [M + H]⁺calcd for C₃₅H₃₀N₃O₅,

572.2180; found, 572.2162.

(S)-N-(1-(4-(Benzyloxy)phenyl)-2-(3-carbamoylphenylamino)-2-

oxoethyl)-4-(2-methoxyphenoxy)benzamide (69). ¹H NMR (300

MHz, acetone-d₆): δ 9.66 (s, 1H), 8.16 (s, 2H), 8.03 (d, J = 6.7 Hz,

1H), 7.94 (d, J = 8.0 Hz, 2H), 7.85 (d, J = 7.9 Hz, 1H), 7.60 (d, J =

7.6 Hz, 1H), 7.56−7.28 (m, 9H), 7.24 (d, J = 7.6 Hz, 1H), 7.17 (d, J

= 8.0 Hz, 1H), 7.11 (d, J = 7.6 Hz, 1H), 7.06−6.96 (m, 3H), 6.87 (d,

J = 8.3 Hz, 2H), 5.82 (d, J = 7.0 Hz, 1H), 5.13 (s, 2H), 3.76 (s, 3H).

HRMS (ESI): m/z [M + H]⁺calcd for C₃₆H₃₂N₃O₆, 602.2286; found,

602.2302.

(S)-N-(1-(4-(Benzyloxy)phenyl)-2-(3-carbamoylphenylamino)-2-

oxoethyl)-4-(4-cyanophenoxy)benzamide (70). ¹H NMR (300

MHz, DMSO-d₆): δ 10.41 (s, 1H), 8.94 (d, J = 7.2 Hz, 1H), 8.09−

7.99 (m, 2H), 7.92 (s, 2H), 7.88 (d, J = 8.5 Hz, 2H), 7.77 (d, J = 7.8

Hz, 1H), 7.57−7.27 (m, 10H), 7.23−7.14 (m, 4H), 7.03 (d, J = 8.4

Hz, 2H), 5.74 (d, J = 7.0 Hz, 1H), 5.11 (s, 2H). HRMS (ESI): m/z

[M + H]⁺calcd for C₃₆H₂₉N₄O₅, 597.2132; found, 597.2121.

(S)-N-(1-(4-(Benzyloxy)phenyl)-2-(3-carbamoylphenylamino)-2-

oxoethyl)-4-(4-(triﬂuoromethyl)phenoxy)benzamide (71). ¹H NMR

(300 MHz, DMSO-d₆): δ 10.41 (s, 1H), 8.93 (d, J = 7.3 Hz, 1H),

8.03 (d, J = 8.5 Hz, 2H), 8.01−7.97 (m, 1H), 7.93 (s, 2H), 7.82−7.73

(m, 3H), 7.56−7.29 (m, 10H), 7.26−7.14 (m, 4H), 7.03 (d, J = 8.7

Hz, 2H), 5.75 (d, J = 7.1 Hz, 1H), 5.11 (s, 2H). HRMS (ESI): m/z

[M + H]⁺calcd for C₃₆H₂₉F₃N₃O₅, 640.2054; found, 640.2041.

(S)-N-(1-(4-(Benzyloxy)phenyl)-2-(3-carbamoylphenylamino)-2-

oxoethyl)-4-(4-chlorophenoxy)benzamide (72). ¹H NMR (300

MHz, DMSO-d₆): δ 10.40 (s, 1H), 8.87 (d, J = 7.2 Hz, 1H), 8.04

(s, 1H), 7.99 (d, J = 8.5 Hz, 2H), 7.93 (s, 2H), 7.77 (d, J = 8.0 Hz,

1H), 7.57−7.27 (m, 11H), 7.11 (d, J = 8.7 Hz, 2H), 7.08−6.98 (m,

4H), 5.74 (d, J = 7.2 Hz, 1H), 5.10 (s, 2H). ¹³C NMR (APT, 75

(S)-4-((2-(4-(Benzyloxy)phenyl)-2-(2-(biphenyl-4-yl)acetamido)-

1

acetamido)methyl)benzamide (59). H NMR (300 MHz, acetone-

d₆): δ 7.94 (t, J = 6.0 Hz, 1H), 7.82 (d, J = 8.1 Hz, 2H), 7.72−7.67

(m, 1H), 7.64 (d, J = 7.4 Hz, 2H), 7.58 (d, J = 8.0 Hz, 2H), 7.50−

7.32 (m, 12H), 7.26 (d, J = 8.2 Hz, 2H), 6.96 (d, J = 8.6 Hz, 2H),

6.47 (s, 2H), 5.48 (d, J = 7.4 Hz, 1H), 5.11 (s, 2H), 4.44 (d, J = 5.8

Hz, 2H), 3.67 (s, 2H). HRMS (ESI): m/z [M + Na]⁺calcd for

C₃₇H₃₃N₃NaO₄, 606.2363; found, 606.2376.

( S ) - 3 - ( 2 - ( 4 - ( 2 , 6 - D i c h l o r o b e n z y l o x y ) p h e n y l ) - 2 -

hexanamidoacetamido)benzamide (60). ¹H NMR (300 MHz,

CD₃OD): δ 8.04 (s, 1H), 7.71 (d, J = 8.0 Hz, 1H), 7.57 (d, J =

8.0 Hz, 1H), 7.50−7.41 (m, 4H), 7.41−7.31 (m, 2H), 7.06 (d, J = 8.7

Hz, 2H), 5.54 (s, 1H), 5.30 (s, 2H), 2.31 (t, J = 7.5 Hz, 2H), 1.69 (p,

J = 7.4 Hz, 2H), 1.40−1.25 (m, 4H), 0.90 (t, J = 6.9 Hz, 3H). ¹³C

NMR (APT, 75 MHz, CD₃OD): δ 176.0, 172.1, 171.4, 160.5, 139.9,

138.0, 136.0, 133.5, 132.0, 131.1, 130.2, 130.0, 129.7, 124.6, 124.3,

120.6, 116.2, 66.3, 58.9, 49.0, 36.6, 32.5, 26.6, 23.5, 14.3. HRMS

(ESI): m/z [M + H]⁺calcd for C₂₈H₃₀Cl₂N₃O₄, 542.1608; found,

542.1590.

(S)-3-(2-(4-(Benzyloxy)phenyl)-2-octanamidoacetamido)-

1

benzamide (61). H NMR (300 MHz, DMSO-d₆): δ 10.35 (s, 1H),

8.48 (d, J = 7.7 Hz, 1H), 8.01 (s, 1H), 7.91 (s, 2H), 7.74 (d, J = 8.1

Hz, 1H), 7.52 (d, J = 7.9 Hz, 1H), 7.45−7.26 (m, 8H), 7.00 (d, J =

8.8 Hz, 2H), 5.54 (d, J = 7.8 Hz, 1H), 5.09 (s, 2H), 2.20 (t, J = 7.4

Hz, 2H), 1.48 (p, J = 7.1 Hz, 2H), 1.33−1.16 (m, 8H), 0.84 (t, J = 6.7

Hz, 3H). ¹³C NMR (APT, 75 MHz, DMSO-d₆): δ 172.2, 169.4,

167.7, 157.9, 138.8, 137.0, 135.1, 130.5, 130.2, 128.6, 128.4, 127.8,

127.6, 125.1, 121.8, 118.7, 114.7, 69.2, 56.4, 34.8, 31.2, 28.6, 28.4,

25.2, 22.0, 13.9. HRMS (ESI): m/z [M + H]⁺calcd for C₃₀H₃₆N₃O₄,

502.2706; found, 502.2684.

(S)-N-(1-(4-(Benzyloxy)phenyl)-2-(3-carbamoylphenylamino)-2-

1

oxoethyl)-2-naphthamide (62). H NMR (300 MHz, DMSO-d₆): δ

10.45 (s, 1H), 9.06 (d, J = 7.2 Hz, 1H), 8.59 (s, 1H), 8.09−7.89 (m,

7H), 7.79 (d, J = 8.0 Hz, 1H), 7.67−7.57 (m, 2H), 7.55 (d, J = 8.6

Hz, 2H), 7.49−7.27 (m, 7H), 7.05 (d, J = 8.5 Hz, 2H), 5.81 (d, J =

7.2 Hz, 1H), 5.12 (s, 2H). ¹³C NMR (APT, 75 MHz, DMSO-d₆): δ

169.3, 167.8, 166.5, 158.1, 138.9, 137.0, 135.1, 134.2, 132.0, 131.0,

129.6, 129.5, 129.3, 128.9, 128.6, 128.4, 128.1, 127.8, 127.7, 127.6,

126.7, 124.6, 124.5, 122.1, 121.8, 118.8, 114.7, 69.2, 57.5. HRMS

(ESI): m/z [M + H]⁺calcd for C₃₃H₂₈N₃O₄, 530.2074; found,

530.2071.

(S)-N-(1-(4-(Benzyloxy)phenyl)-2-(3-carbamoylphenylamino)-2-

oxoethyl)-2,3-dihydrobenzo[b][1,4]dioxine-6-carboxamide (63). ¹H

NMR (300 MHz, DMSO-d₆): δ 10.36 (s, 1H), 8.71 (d, J = 7.1 Hz,

1H), 8.03 (s, 1H), 7.92 (s, 2H), 7.76 (d, J = 7.8 Hz, 1H), 7.57−7.27

(m, 12H), 7.02 (d, J = 8.2 Hz, 2H), 5.70 (d, J = 7.1 Hz, 1H), 5.10 (s,

2H), 4.27 (s, 4H). HRMS (ESI): m/z [M + H]⁺calcd for

C₃₁H₂₈N₃O₆, 538.1973; found, 538.1969.

( S ) - N - ( 2 - ( 3 - C a r b a m o y l p h e n y l a m i n o ) - 1 - ( 4 - ( 2 , 6 -

dichlorobenzyloxy)phenyl)-2-oxoethyl)-4-phenoxybenzamide (64).

¹H NMR (300 MHz, DMSO-d₆): δ 10.42 (s, 1H), 8.86 (d, J = 7.4 Hz,

1H), 8.06 (s, 1H), 7.99 (d, J = 8.7 Hz, 2H), 7.93 (s, 2H), 7.79 (d, J =

8.3 Hz, 1H), 7.59−7.30 (m, 9H), 7.21 (t, J = 7.5 Hz, 1H), 7.12−7.06

(m, 4H), 7.03 (d, J = 8.8 Hz, 2H), 5.76 (d, J = 7.2 Hz, 1H), 5.22 (s,

2H). ¹³C NMR (APT, 75 MHz, DMSO-d₆): δ 169.2, 167.8, 165.7,

159.6, 158.2, 155.6, 138.9, 136.0, 135.1, 131.7, 131.6, 130.2, 130.0,

129.9, 129.5, 129.3, 129.2, 128.8, 128.6, 128.5, 124.8, 119.5, 118.8,

117.3, 114.4, 64.9, 57.5. HRMS (ESI): m/z [M + H]⁺calcd for

C₃₅H₂₈Cl₂N₃O₅, 640.1401; found, 640.1389.

( S ) - N - ( 2 - ( 3 - C a r b a m o y l p h e n y l a m i n o ) - 1 - ( 4 - ( 3 -

methoxybenzyloxy)phenyl)-2-oxoethyl)-4-phenoxybenzamide

1

(65). H NMR (300 MHz, DMSO-d₆): δ 10.40 (s, 1H), 8.83 (d, J =

7.3 Hz, 1H), 8.04 (s, 1H), 7.97 (d, J = 8.6 Hz, 2H), 7.93 (s, 2H), 7.77

4583

https://doi.org/10.1021/acs.jmedchem.0c02042

J. Med. Chem. 2021, 64, 4567−4587

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

MHz, DMSO-d₆): δ 169.2, 167.8, 165.6, 159.1, 158.0, 154.7, 138.9,

137.0, 135.1, 130.1, 129.6, 129.4, 129.2, 128.9, 128.6, 128.4, 128.0,

127.8, 127.6, 122.1, 121.8, 121.1, 118.8, 117.5, 114.7, 69.2, 57.4.

HRMS (ESI): m/z [M + H]⁺calcd for C₃₅H₂₉ClN₃O₅, 606.1790;

found, 606.1790.

(S)-N-(1-(4-(Benzyloxy)phenyl)-2-(3-carbamoylphenylamino)-2-

oxoethyl)-4-(3-chlorophenoxy)benzamide (73). ¹H NMR (300

MHz, DMSO-d₆): δ 10.41 (s, 1H), 8.89 (d, J = 6.7 Hz, 1H), 8.13−

7.89 (m, 3H), 7.78 (d, J = 7.3 Hz, 1H), 7.60−7.21 (m, 11H), 7.17−

6.93 (m, 6H), 5.74 (d, J = 6.6 Hz, 1H), 5.11 (s, 2H). ¹³C NMR (APT,

75 MHz, DMSO-d₆): δ 169.2, 167.8, 165.6, 158.6, 158.1, 157.0, 138.9,

137.0, 135.1, 134.1, 131.6, 130.2, 129.6, 129.3, 129.2, 128.6, 128.4,

127.8, 127.6, 124.0, 122.1, 121.8, 119.1, 118.8, 118.0, 117.8, 114.7,

69.2, 57.5. HRMS (ESI): m/z [M + H]⁺calcd for C₃₅H₂₉ClN₃O₅,

606.1790; found, 606.1761.

(S)-4-(Benzo[d][1,3]dioxol-5-yloxy)-N-(1-(4-(benzyloxy)phenyl)-

2-(3-carbamoylphenylamino)-2-oxoethyl)benzamide (74). ¹H

NMR (300 MHz, DMSO-d₆): δ 10.39 (s, 1H), 8.80 (d, J = 7.2 Hz,

1H), 8.03 (s, 1H), 8.00−7.84 (m, 3H), 7.77 (d, J = 7.7 Hz, 1H),

7.57−7.27 (m, 10H), 7.08−6.90 (m, 5H), 6.79 (s, 1H), 6.56 (d, J =

8.3 Hz, 1H), 6.06 (s, 2H), 5.73 (d, J = 7.2 Hz, 1H), 5.10 (s, 2H). ¹³C

NMR (APT, 75 MHz, DMSO-d₆): δ 168.7, 167.8, 165.5, 160.6, 158.2,

150.6, 149.6, 146.9, 140.1, 137.1, 133.2, 129.9, 129.2, 128.4, 127.9,

127.8, 127.6, 122.4, 118.6, 116.2, 114.7, 114.1, 112.6, 108.6, 102.6,

69.2, 56.5. HRMS (ESI): m/z [M + H]⁺calcd for C₃₆H₃₀N₃O₇,

638.1898; found, 638.1880.

= 8.0 Hz, 1H), 7.57−7.27 (m, 9H), 7.03 (d, J = 8.6 Hz, 2H), 5.75 (d,

J = 7.3 Hz, 1H), 5.11 (s, 2H), 3.80−3.40 (m, 8H). ¹³C NMR (APT,

75 MHz, DMSO-d₆): δ 169.1, 168.4, 167.8, 165.9, 158.1, 138.9, 138.3,

137.0, 135.1, 134.6, 129.5, 129.2, 128.6, 128.4, 128.0, 127.8, 127.6,

126.8, 122.1, 121.8, 118.8, 114.7, 69.2, 66.0, 57.5. Signals of C-3 and

C-5 of morpholine were not observed in ¹³C NMR. HRMS (ESI): m/

z [M + H]⁺calcd for C₃₄H₃₃N₄O₆, 593.2395; found, 593.2982.

(S)-N-(1-(4-(Benzyloxy)phenyl)-2-(3-carbamoylphenylamino)-2-

oxoethyl)-3-(morpholine-4-carbonyl)benzamide (80). ¹H NMR

(300 MHz, DMSO-d₆): δ 10.42 (s, 1H), 9.06 (d, J = 7.1 Hz, 1H),

8.48 (s, 1H), 8.09−7.96 (m, 3H), 7.93 (s, 2H), 7.77 (d, J = 7.7 Hz,

1H), 7.58−7.27 (m, 10H), 7.03 (d, J = 8.6 Hz, 2H), 5.75 (d, J = 7.0

Hz, 1H), 5.11 (s, 2H), 3.80−3.41 (m, 8H). ¹³C NMR (APT, 75 MHz,

DMSO-d₆): δ 169.1, 168.5, 167.8, 165.7, 158.1, 138.9, 137.0, 135.6,

135.1, 134.0, 129.7, 129.5, 129.3, 128.9, 128.6, 128.4, 128.4, 127.8,

127.6, 126.4, 122.2, 121.8, 118.8, 114.7, 69.2, 66.1, 57.5. Signals of C-

3 and C-5 of morpholine were not observed in ¹³C NMR. HRMS

(ESI): m/z [M + H]⁺calcd for C₃₄H₃₃N₄O₆, 593.2395; found,

593.2988.

(S)-N-(1-(4-(Benzyloxy)phenyl)-2-(3-carbamoylphenylamino)-2-

oxoethyl)-4-(isopentyloxy)benzamide (81). ¹H NMR (300 MHz,

CD₃CN): δ 8.85 (s, 1H), 8.04 (s, 1H), 7.81 (d, J = 8.7 Hz, 2H),

7.78−7.69 (m, 2H), 7.56 (d, J = 6.4 Hz, 1H), 7.54−7.29 (m, 10H),

7.01 (d, J = 8.6 Hz, 2H), 6.96 (d, J = 8.7 Hz, 2H), 5.62 (d, J = 6.6 Hz,

2H), 5.10 (s, 2H), 4.07 (t, J = 6.7 Hz, 2H), 1.84−1.74 (m, 1H),

1.69−1.50 (m, 2H), 0.97 (s, 3H), 0.95 (s, 3H). HRMS (ESI): m/z

[M + H]⁺calcd for C₃₄H₃₆N₃O₅, 566.2649; found, 566.2630.

(S)-3-(2-(4-(Benzyloxy)phenyl)-2-(5-(2,5-dimethylphenoxy)-2,2-

(S)-N-(1-(4-(Benzyloxy)phenyl)-2-(3-carbamoylphenylamino)-2-

oxoethyl)-4-(2,3-dihydrobenzo[b][1,4]dioxin-6-yloxy)benzamide

1

(75). H NMR (300 MHz, DMSO-d₆): δ 10.39 (s, 1H), 8.79 (d, J =

dimethylpentanamido)acetamido)benzamide (82). H NMR (300

7.3 Hz, 1H), 8.03 (s, 1H), 7.99−7.88 (m, 3H), 7.76 (d, J = 7.9 Hz,

1H), 7.57−7.27 (m, 10H), 7.06−6.99 (m, 2H), 6.96 (d, J = 8.5 Hz,

2H), 6.90 (d, J = 8.7 Hz, 1H), 6.63 (s, 1H), 6.57 (d, J = 8.5 Hz, 1H),

5.72 (d, J = 7.0 Hz, 1H), 5.10 (s, 2H), 4.25 (s, 4H). HRMS (ESI): m/

z [M + H]⁺calcd for C₃₇H₃₂N₃O₇, 630.2235; found, 630.2220.

(S)-N-(1-(4-(Benzyloxy)phenyl)-2-(3-carbamoylphenylamino)-2-

oxoethyl)-4-(3-oxo-2,3-dihydro-1H-inden-4-yloxy)benzamide (76).

¹H NMR (300 MHz, DMSO-d₆): δ 10.40 (s, 1H), 8.87 (d, J = 7.1 Hz,

1H), 8.03 (s, 1H), 7.95 (d, J = 8.5 Hz, 2H), 7.92 (s, 2H), 7.77 (d, J =

7.6 Hz, 1H), 7.57−7.27 (m, 12H), 7.11 (d, J = 8.0 Hz, 1H), 7.07−

6.95 (m, 3H), 5.73 (d, J = 7.2 Hz, 1H), 5.10 (s, 2H), 3.19−3.07 (m,

2H), 2.67−1.57 (m, 2H). HRMS (ESI): m/z [M + H]⁺calcd for

C₃₈H₃₂N₃O₆, 626.2286; found, 626.2268.

MHz, DMSO-d₆): δ 10.33 (s, 1H), 8.02 (s, 1H), 7.92 (s, 2H), 7.77−

7.69 (m, 2H), 7.53 (d, J = 7.9 Hz, 1H), 7.45−7.27 (m, 8H), 7.02−

6.93 (m, 3H), 6.68 (s, 1H), 6.61 (d, J = 7.5 Hz, 1H), 5.55 (d, J = 7.3

Hz, 1H), 5.07 (s, 2H), 3.85 (t, J = 5.6 Hz, 2H), 2.23 (s, 3H), 2.06 (s,

3H), 1.77−1.52 (m, 4H), 1.16 (s, 6H). ¹³C NMR (APT, 75 MHz,

DMSO-d₆): δ 176.4, 169.3, 167.7, 157.9, 156.5, 138.8, 137.0, 136.0,

135.1, 133.6, 130.2, 130.0, 129.6, 128.6, 128.4, 127.8, 127.6, 127.5,

122.5, 120.5, 118.8, 114.7, 112.1, 69.2, 67.8, 56.7, 41.2, 36.8, 25.2,

25.1, 24.5, 21.0, 15.6. HRMS (ESI): m/z [M + H]⁺calcd for

C₃₇H₄₂N₃O₅, 608.3119; found, 608.3115.

(S)-N-(1-(4-(Benzyloxy)phenyl)-2-(3-carbamoylphenylamino)-2-

oxoethyl)biphenyl-4-carboxamide (83). ¹H NMR (300 MHz,

DMSO-d₆): δ 10.42 (s, 1H), 8.96 (d, J = 7.3 Hz, 1H), 8.04 (d, J =

8.3 Hz, 2H), 7.93 (s, 2H), 7.82−7.69 (m, 5H), 7.59−7.27 (m, 13H),

7.04 (d, J = 8.4 Hz, 2H), 5.77 (d, J = 7.2 Hz, 1H), 5.11 (s, 2H). ¹³C

NMR (APT, 75 MHz, DMSO-d₆): δ 169.2, 167.8, 166.1, 158.1, 142.9,

139.2, 138.9, 137.1, 135.1, 132.6, 129.7, 129.2, 129.0, 128.6, 128.5,

128.4, 128.1, 127.8, 127.6, 126.9, 126.4, 122.2, 121.8, 118.8, 114.7,

69.2, 57.5. HRMS (ESI): m/z [M + H]⁺calcd for C₃₅H₃₀N₃O₄,

556.2231; found, 556.2219.

(S)-4-Benzoyl-N-(1-(4-(benzyloxy)phenyl)-2-(3-carbamoylpheny-

1

lamino)-2-oxoethyl)benzamide (77). H NMR (300 MHz, DMSO-

d₆): δ 10.44 (s, 1H), 9.15 (d, J = 7.2 Hz, 1H), 8.08 (d, J = 8.1 Hz,

2H), 8.05 (s, 1H), 7.93 (s, 2H), 7.83−7.72 (m, 5H), 7.69 (d, J = 7.2

Hz, 1H), 7.59 (d, J = 7.5 Hz, 2H), 7.56−7.48 (m, 3H), 7.46−7.29 (m,

6H), 7.04 (d, J = 8.4 Hz, 2H), 5.77 (d, J = 7.1 Hz, 1H), 5.11 (s, 2H).

¹³C NMR (APT, 75 MHz, DMSO-d₆): δ 195.4, 169.0, 167.8, 165.8,

158.1, 139.3, 138.9, 137.1, 137.0, 136.7, 135.1, 133.0, 129.7, 129.4,

129.3, 129.2, 128.7, 128.6, 128.4, 128.0, 127.8, 127.6, 122.2, 121.8,

118.8, 114.7, 69.2, 57.6. HRMS (ESI): m/z [M + H]⁺calcd for

C₃₆H₃₀N₃O₅, 584.2180; found, 584.2170.

(S)-N-(1-(4-(Benzyloxy)phenyl)-2-(3-carbamoylphenylamino)-2-

oxoethyl)-4′-chlorobiphenyl-4-carboxamide (84). ¹H NMR (300

MHz, DMSO-d₆): δ 10.42 (s, 1H), 8.98 (d, J = 7.2 Hz, 1H), 8.04 (d, J

= 7.7 Hz, 2H), 7.93 (s, 2H), 7.85−7.70 (m, 5H), 7.59−7.47 (m, 5H),

7.46−7.27 (m, 7H), 7.03 (d, J = 8.4 Hz, 2H), 5.77 (d, J = 7.1 Hz,

1H), 5.11 (s, 2H). ¹³C NMR (APT, 75 MHz, DMSO-d₆): δ 169.2,

167.7, 165.9, 158.0, 141.5, 138.8, 137.9, 137.0, 135.1, 132.91, 132.86,

129.6, 129.2, 128.9, 128.60, 128.56, 128.5, 128.4, 127.8, 127.5, 126.3,

122.1, 121.7, 118.7, 114.7, 69.1, 57.4. HRMS (ESI): m/z [M + H]⁺

calcd for C₃₅H₂₉ClN₃O₄, 590.1841; found, 590.1823.

(S)-4-Benzoyl-N-(2-(3-carbamoylphenylamino)-1-(4-(3-

methoxybenzyloxy)phenyl)-2-oxoethyl)benzamide (78). ¹H NMR

(300 MHz, DMSO-d₆): δ 10.44 (s, 1H), 9.15 (d, J = 7.1 Hz, 1H),

8.08 (d, J = 8.2 Hz, 2H), 8.05 (s, 1H), 7.93 (s, 2H), 7.83−7.66 (m,

5H), 7.77 (d, J = 7.1 Hz, 1H), 7.59 (d, J = 7.4 Hz, 2H), 7.57−7.48

(m, 2H), 7.41−7.24 (m, 4H), 7.03 (d, J = 8.8 Hz, 2H), 7.01−6.97 (m,

1H), 6.88 (d, J = 8.1 Hz, 1H), 5.77 (d, J = 7.1 Hz, 1H), 5.09 (s, 2H),

3.74 (s, 3H). ¹³C NMR (APT, 75 MHz, DMSO-d₆): δ 195.4, 169.1,

167.8, 165.8, 159.3, 158.1, 139.3, 138.9, 138.9, 138.6, 137.1, 136.7,

135.2, 135.1, 133.0, 129.7, 129.6, 129.3, 129.3, 128.7, 128.0, 122.1,

121.8, 119.6, 118.8, 114.7, 113.1, 113.1, 69.0, 57.6, 55.0. HRMS

(ESI): m/z [M + H]⁺calcd for C₃₇H₃₂N₃O₆, 614.2286; found,

614.2278.

(S)-N-(1-(4-(Benzyloxy)phenyl)-2-(3-carbamoylphenylamino)-2-

oxoethyl)-4-(furan-2-yl)benzamide (85). ¹H NMR (300 MHz,

DMSO-d₆): δ 10.41 (s, 1H), 8.99−8.85 (m, 1H), 8.12−7.88 (m,

3H), 7.84−7.74 (m, 3H), 7.69 (d, J = 8.2 Hz, 1H), 7.57−7.28 (m,

11H), 7.23 (d, J = 8.2 Hz, 1H), 7.03 (d, J = 8.3 Hz, 2H), 6.69−6.60

(m, 1H), 5.75 (d, J = 5.1 Hz, 1H), 5.11 (s, 2H). HRMS (ESI): m/z

[M + H]⁺calcd for C₃₃H₂₈N₃O₅, 546.2023; found, 546.2010.

(S)-N-(1-(4-(Benzyloxy)phenyl)-2-(3-carbamoylphenylamino)-2-

oxoethyl)-4-(morpholine-4-carbonyl)benzamide (79). ¹H NMR

(300 MHz, DMSO-d₆): δ 10.41 (s, 1H), 9.02 (d, J = 7.2 Hz, 1H),

8.09−8.02 (m, 3H), 7.99 (d, J = 8.1 Hz, 2H), 7.93 (s, 2H), 7.77 (d, J

1

oxoethyl)-4-(pyrimidin-5-yl)benzamide (86). H NMR (300 MHz,

DMSO-d₆): δ 10.43 (s, 1H), 10.40 (s, 1H), 9.28 (s, 2H), 9.06 (d, J =

7.3 Hz, 1H), 8.10 (d, J = 8.4 Hz, 2H), 8.06 (s, 1H), 7.94 (d, J = 8.2

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Hz, 2H), 7.87−7.72 (m, 2H), 7.52 (d, J = 8.6 Hz, 2H), 7.48−7.30 (m,

8H), 7.04 (d, J = 8.7 Hz, 2H), 5.78 (d, J = 7.2 Hz, 1H), 5.11 (s, 2H).

¹³C NMR (APT, 75 MHz, DMSO-d₆): δ 169.2, 167.8, 165.8, 158.1,

154.9, 150.2, 138.9, 137.0, 136.6, 135.1, 133.9, 129.5, 129.3, 129.2,

128.7, 128.6, 128.4, 127.8, 127.6, 126.7, 124.5, 121.8, 118.8, 114.7,

69.2, 57.5. HRMS (ESI): m/z [M + H]⁺calcd for C₃₃H₂₈N₅O₄,

558.2136; found, 558.2128.

reduction assay with analogous treatment. Each concentration was

assayed in triplicates.

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c02042.

■

sı

(S)-N-(1-(4-(Benzyloxy)phenyl)-2-(3-carbamoylphenylamino)-2-

1

oxoethyl)-1-phenylpiperidine-4-carboxamide (87). H NMR (300

Molecular formula strings and activity data (CSV)

Expression and puriﬁcation of the viral proteases,

inhibitory activity of compounds against isolated viral

proteases, characterization of compounds 56 and 85,

HPLC-based DENV and WNV protease assays with

compounds 56 and 85, enzyme kinetic studies: Cheng−

Prusoﬀ plots, Abz calibration curve, Michaelis−Menten

plots, tryptophan ﬂuorescence quenching assay, inhib-

itory activity of compounds against thrombin and

trypsin, residual plots for DENV-2 titer reduction

curves, PAMPA, metabolic stability against liver micro-

somes, stability against pancreatic enzymes, FRET

substrate synthesis, synthesis and analytical data of

precursors, synthesis and analytical data of inhibitors,

HPLC purity of inhibitors, enantiomeric purity of

MHz, acetone-d₆/D₂O): δ 10.82 (s, 1H), 8.41 (s, 1H), 8.00−7.81 (m,

2H), 7.77−7.52 (m, 4H), 7.46 (d, J = 7.0 Hz, 2H), 7.42−7.25 (m,

5H), 7.13−6.94 (m, 4H), 6.82−6.59 (m, 3H), 5.71 (s, 1H), 5.12 (s,

2H), 3.93−2.99 (m, 9H). HRMS (ESI): m/z [M + H]⁺calcd for

C₃₄H₃₅N₄O₄, 563.2653; found, 563.2659.

(S)-N-(1-(4-(Benzyloxy)phenyl)-2-(3-carbamoylphenylamino)-2-

oxoethyl)-4-morpholinobenzamide (88). ¹H NMR (300 MHz,

DMSO-d₆): δ 10.37 (s, 1H), 8.56 (d, J = 7.1 Hz, 1H), 8.04 (s,

1H), 7.92 (s, 2H), 7.85 (d, J = 8.4 Hz, 2H), 7.77 (d, J = 7.9 Hz, 1H),

7.55−7.28 (m, 9H), 7.02 (d, J = 8.3 Hz, 2H), 6.96 (d, J = 8.5 Hz,

2H), 5.73 (d, J = 7.2 Hz, 1H), 5.10 (s, 2H), 3.78−3.69 (m, 4H),

3.28−3.14 (m, 4H). ¹³C NMR (APT, 75 MHz, DMSO-d₆): δ 169.5,

167.8, 165.9, 158.0, 153.0, 138.9, 137.0, 135.1, 130.0, 129.1, 129.1,

128.6, 128.4, 127.8, 127.6, 123.3, 122.1, 121.8, 118.7, 114.7, 113.2,

69.2, 65.9, 57.2, 47.3. HRMS (ESI): m/z [M + H]⁺calcd for

C₃₃H₃₃N₄O₅, 565.2445; found, 565.2451.

1

compounds, and H und ¹³C NMR spectra (PDF)

Cell Culture. If not stated otherwise, HeLa, Huh-7, and Vero E6

cells were maintained in DMEM supplemented with 100 U/mL

penicillin, 100 μg/mL streptomycin, and 10% heat-inactivated FCS.

During infection of Huh-7 cells, DMEM was supplemented with 10

mM HEPES.

AUTHOR INFORMATION

Corresponding Author

■

Christian D. Klein − Medicinal Chemistry, Institute of

Pharmacy and Molecular Biotechnology IPMB, Heidelberg

University, 69120 Heidelberg, Germany; orcid.org/0000-

0003-3522-9182; Phone: +49 6221 544875;

DENV2 Reporter Gene Assay (DENV2proHeLa). Stable cells

were seeded into 96-well plates with a density of 2 × 10⁴cells per well

and treated immediately with inhibitors of the dengue virus protease

in a ﬁnal volume of 100 μL. After incubation for 24 h at 37 °C, the

medium was removed, and cells were lysed by adding 25 μL of lysis

buﬀer (Promega) for 15 min at room temperature. Luciferase activity

was recorded using a FLUOstar omega plate reader (BMG Labtech)

with injections of 100 μL per well of coelenterazine (2.75 μM in

PBS). Luminescence was recorded for 5 s. Each concentration was

assayed in triplicates. Percent inhibition was calculated in relation to

an untreated control. For EC₅₀and EC₉₀calculations, data were ﬁtted

and calculated with Prism 6.01 (GraphPad Software, Inc.) using a 4-

parameter nonlinear dose−response curve with background sub-

traction (wells without cells).

Virus Titer Reduction Assay. Huh-7 cells were seeded into 96-

well plates at a density of 1 × 10⁴cells in 50 μL per well and

incubated overnight. The next day, cells were infected with DENV

serotype 2 for 2 h at an MOI of 1, before medium change and

compound addition. Infected cells were then incubated for 48 h in the

presence of compounds in triplicate wells. In order to determine the

EC₅₀, a range of serial diluted compound concentrations starting at 50

μM was used. After 48 h incubation, supernatants were harvested, and

triplicates were pooled. These virus-containing supernatants were

used to determine the virus titer (reduction) by a plaque assay, as

described elsewhere.⁶¹Brieﬂy, VeroE6 cells were seeded into 24-well

plates at a density of 2.5 × 10⁵cells/well and the next day infected for

1 h with 10-fold serial dilutions of the virus supernatant ranging from

10⁻¹to 10⁻⁶. After medium exchange and addition of the plaque

medium, the VeroE6 cells were incubated for 7 days. Cells were

subsequently ﬁxed with formaldehyde, and plaques were visualized by

crystal violet staining. At a suitable dilution, plaques were counted,

and the virus titer was calculated and plotted against the respective

compound concentration. EC₅₀values were derived from ﬁtting data

to a dose−response curve (variable-slope, nonlinear regression

model) using Prism 6.01 (GraphPad Software, Inc.).

Email: c.klein@uni-heidelberg.de; Fax: +49 6221 546430

Authors

Nikos Kuhl − Medicinal Chemistry, Institute of Pharmacy and

̈

Molecular Biotechnology IPMB, Heidelberg University,

69120 Heidelberg, Germany; orcid.org/0000-0001-

9025-7245

Mila M. Leuthold − Medicinal Chemistry, Institute of

Pharmacy and Molecular Biotechnology IPMB, Heidelberg

University, 69120 Heidelberg, Germany; orcid.org/0000-

0001-7698-2360

Mira A. M. Behnam − Medicinal Chemistry, Institute of

Pharmacy and Molecular Biotechnology IPMB, Heidelberg

University, 69120 Heidelberg, Germany; orcid.org/0000-

0002-5839-7675

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.0c02042

Notes

The authors declare the following competing ﬁnancial

interest(s): A patent application which covers the chemical

structures and medical application of the compounds described

in this work was ﬁled by Heidelberg University (PCT/

EP2020/056001).

ACKNOWLEDGMENTS

■

This work was supported by the Deutsche Forschungsgemein-

schaft under grant no. 1356/3-2. We thank Heiko Rudy for

measuring ESI high-resolution spectra and Natascha Stefan

and Tobias Timmermann for technical support. Prof. Ralf

Bartenschlager and Marie Bartenschlager kindly provided

stocks of the DENV-2 virus and other experimental inputs.

Cytotoxicity. Cell viability in Huh-7 or Hela cells in the presence

of compound dilutions was determined using CellTiter-Blue

(Promega) according to the manufacturer’s instructions. Plates were

prepared in parallel to the cell-based DENV reporter or virus titer

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Tully, D. C.; Williams, J. A.; Jiricek, J.; Priestle, J. P.; Harris, J. L.;

Vasudevan, S. G. Functional Profiling of Recombinant NS3 Proteases

from All Four Serotypes of Dengue Virus Using Tetrapeptide and

Octapeptide Substrate Libraries. J. Biol. Chem. 2005, 280, 28766−

28774.

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Susanne Oberbillig and Christoph Pfaﬀendorf assisted in

peptide synthesis.

ABBREVIATIONS USED

■

Boc, tert-butoxycarbonyl; CHAPS, 3-[(3-cholamidopropyl)-

dimethylammonio]-1-propanesulfonate; COMU, (1-cyano-2-

ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-

carbenium hexaﬂuorophosphate; CTC, 2-chlorotrityl chloride;

DCM, dichloromethane; DENV, dengue virus; DMEM,

Dulbecco’s modiﬁed Eagle medium; DMF, dimethylforma-

mide; FCS, fetal bovine serum; Fmoc, ﬂuorenylmethylox-

ycarbonyl; Fmoc-OSu, 9-ﬂuorenylmethyl N-succinimidyl car-

bonate; HATU, 1-[bis(dimethylamino)methylene]-1H-1,2,3-

triazolo[4,5-b]pyridinium 3-oxide hexaﬂuorophosphate;

HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid;

MOI, multiplicity of infection; PBS, phosphate-buﬀered saline;

PHG, phenylglycine; TFA, triﬂuoroacetic acid; THF, tetrahy-

drofuran; TIPS, triisopropylsilane; TMP, 2,4,6-trimethylpyr-

idine; WNV, West Nile virus

(18) Weigel, L. F.; Nitsche, C.; Graf, D.; Bartenschlager, R.; Klein,

C. D. Phenylalanine and Phenylglycine Analogues as Arginine

Mimetics in Dengue Protease Inhibitors. J. Med. Chem. 2015, 58,

7719−7733.

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https://doi.org/10.1021/acs.jmedchem.0c02042

J. Med. Chem. 2021, 64, 4567−4587

Article Doi

Beyond Basicity: Discovery of Nonbasic DENV-2 Protease Inhibitors with Potent Activity in Cell Culture

DOI: 10.1021/acs.jmedchem.0c02042

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Full text of DOI:10.1021/acs.jmedchem.0c02042

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