Self-Masked Aldehyde Inhibitors: A Novel Strategy for Inhibiting Cysteine Proteases

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Self-Masked Aldehyde Inhibitors: A Novel Strategy for Inhibiting

Cysteine Proteases

Linfeng Li, Bala C. Chenna, Kai S. Yang, Taylor R. Cole, Zachary T. Goodall, Miriam Giardini,

Zahra Moghadamchargari, Elizabeth A. Hernandez, Jana Gomez, Claudia M. Calvet, Jean A. Bernatchez,

Drake M. Mellott, Jiyun Zhu, Andrew Rademacher, Diane Thomas, Lauren R. Blankenship,

Aleksandra Drelich, Arthur Laganowsky, Chien-Te K. Tseng, Wenshe R. Liu, A. Joshua Wand,

Jorge Cruz-Reyes, Jair L. Siqueira-Neto, and Thomas D. Meek *

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ABSTRACT: Cysteine proteases comprise an important class of drug

targets, especially for infectious diseases such as Chagas disease

(cruzain) and COVID-19 (3CL protease, cathepsin L). Peptide

aldehydes have proven to be potent inhibitors for all of these proteases.

However, the intrinsic, high electrophilicity of the aldehyde group is

associated with safety concerns and metabolic instability, limiting the

use of aldehyde inhibitors as drugs. We have developed a novel class of

self-masked aldehyde inhibitors (SMAIs) for cruzain, the major cysteine

protease of the causative agent of Chagas diseaseTrypanosoma cruzi.

These SMAIs exerted potent, reversible inhibition of cruzain (K_i* = 18−

350 nM) while apparently protecting the free aldehyde in cell-based

assays. We synthesized prodrugs of the SMAIs that could potentially

improve their pharmacokinetic properties. We also elucidated the kinetic

and chemical mechanism of SMAIs and applied this strategy to the

design of anti-SARS-CoV-2 inhibitors.

require lengthy regimens. Accordingly, an emerging trend is

the development of reversible covalent inhibitors which provide

potent inhibition of the desired target, but will not permanently

inactivate oﬀ-target proteins.¹⁰Among the reversible covalent

warheads explored for cruzain to date, an aldehyde group often,

if not always, aﬀords outstanding inhibitory potency compared

to α-keto amides, nitriles, oximes, heterocycles, and so on.¹¹⁻¹⁴

On the other hand, the inherent electrophilicity of aldehydes

contributes to their rapid metabolism, immunotoxicities, and

untoward drug−drug interactions,¹⁵which have all but

disqualiﬁed their use in drug design. Theoretically, these issues

can be mitigated by conﬁning the exposure of the aldehyde

solely to the germane protein target while eschewing oﬀ-target

proteins; however, such approaches have been challenging to

implement.⁹

INTRODUCTION

■

Cysteine proteases are prospective drug targets for a variety of

diseases caused by viruses (e.g., picornaviruses and coronavi-

ruses),^1,2bacteria (e.g., Salmonella and Listeria),³and

particularly, many species of parasitic protozoa.⁴One of these,

Trypanosoma cruzi, is the causative agent of Chagas disease,

which aﬀects an estimated 6−7 million people worldwide.⁵

During the life cycle of T. cruzi, its major cysteine protease,

known as cruzain, plays a multifaceted role involving nutrient

processing, morphological transformation, invasion into host

cells, and evasion of the immune response.⁶Consequently,

cruzain has been widely studied and also serves as a paradigm for

inhibitor design for other cysteine proteases.

Substrate−analogue peptidomimetic compounds containing

a suitable electrophilic warhead comprise the most common

classes of inhibitors and inactivators of cruzain, as well as other

cysteine proteases.⁷A notable example is K777 (or K11777), a

dipeptide analogue containing a vinyl sulfone warhead, which

undergoes irreversible thia-Michael addition to the active-site

Cys₂₅of cruzain,⁶and other cysteine proteases including

cathepsins K, L, and B (Figure 1A).⁸However, irreversible

binding of small molecules to protein targets is associated with

idiosyncratic immunotoxicity,⁹especially for drugs which

Received: April 6, 2021

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A

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Figure 1. Inactivator and inhibitors of cysteine proteases. (A) Structure of K777 and its mechanism of covalent inactivation of cysteine proteases. (B)

1,2,4-trioxolane inhibitor PG4b undergoes Fe(II)-catalyzed fragmentation to elaborate aldehyde PG3b, a potent inhibitor of falcipain, and a radical

byproduct. (C) γ-lactol inhibitor MN3 is a masked aldehyde of SJA0617. It exhibits higher transcorneal permeability and releases the free aldehyde to

inhibit μ-calpain.

An alternative approach to incorporate aldehydes into drug

design is to mask the aldehyde using another functional group.

The antimalarial aldehyde inhibitor of falcipain PG3b was

“masked” by a 1,2,4-trioxolane structure (Figure 1B), resulting

in the prodrug PG4b from which the active aldehyde is liberated

upon reaction with Fe(II) inside the vacuoles of the plasmodial

parasite.¹⁶Unfortunately, this clever masked aldehyde resulted

in toxicity to the host cells, apparently from the action of free

radical byproduct(s). Its utility is also limited by the variable

availability of Fe(II) in target cells. The calpain inhibitor MN3 is

another type of masked aldehyde in which a homoserine at the

P₁position¹⁷spontaneously forms a cyclic hemiacetal (Figure

1C). Compared to the potent free aldehyde SJA6017 (with Leu

at P_1,IC₅₀= 22 nM), protection of the aldehyde in MN3 greatly

improved transcorneal permeability despite lower potency

against calpain (IC₅₀= 880 nM).^18,19Herein, we describe an

unprecedented class of self-masked aldehyde inhibitors

(SMAIs) for cruzain, their kinetic and chemical mechanisms

of reversible covalent inhibition, and their eﬀects on

trypanosomal infection in cell cultures. In addition, we

developed prodrugs of these SMAIs to improve their metabolic

stability to optimize their pharmacokinetic properties. We

adapted this concept to develop a novel inhibitor for the main

cysteine protease (3CL protease) of SARS-CoV-2.

converting the P₁group to an ortho-tyrosine (2, Cbz-Phe-o-Tyr-

H).The oxygen of the phenol group is in close proximity to the

aldehydic carbon permitting a nucleophilic addition reaction to

occur, producing a cyclic hemiacetal (δ-lactol) similar to the γ-

lactol found in calpain inhibitor MN3.¹⁹We anticipated that a

SMAI would remain “locked” in its δ-lactol form before binding

to cruzain, after which enzyme catalysis would elaborate the free

aldehyde followed by formation of a hemithioacetal adduct with

cruzain (Figure 2A). If true, then the potential advantages of

SMAIs include: (a) the intramolecular nature of the δ-lactol

likely provides sustained protection of the aldehyde outside of an

enzyme active site; (b) compared to the 1,2,3-trioxolane masked

aldehyde,¹⁶the cleavage of the hemiacetal does not produce

reactive byproducts; and (c) the introduced hydroxyl group on

the o-tyrosine sidechain is small enough so as to impose minimal

perturbation of the original binding mode of the parent aldehyde

1.

The ﬁrst question arising from our hypothesis is: could the

lactol form of 2 bind to cruzain and remain closed? To

preliminarily address this question, we employed molecular

modeling using docking methods conforming to the binding of

compound 2 in both its noncovalent, lactol form (Figure 2B)

and its free aldehyde form which was docked as a hemithioacetal

adduct with the catalytic Cys₂₅(Figure 2C). The covalent

adduct of 2 is predicted to bind in a manner similar to that of the

covalent inactivator K777 as seen in the cruzain-K777 cocrystal

structure.²⁰As expected, the phenoxy substituent of the

covalently bound inhibitor 2 is well tolerated in the cruzain

active site and may form hydrogen bonds with Gln₁₉, His₁₆₂, or

Trp₁₈₄, similar to the sulfone oxygens of K777. Although the

Cbz-Phe group in Figure 2B adopts a similar orientation to the

open form (Figure 2C), the lactol displays a puckered

RESULTS AND DISCUSSION

■

Design of SMAIs. The dipeptide aldehyde 1 (Cbz-Phe-Phe-

H, Figure 2A) was ﬁrst prepared for use as an intermediate in the

synthesis of other protease inhibitors and proved to be an

extraordinarily potent inhibitor of cruzain, as discussed below.

To modify its P₁structure to aﬀord a self-masked aldehyde, we

added a hydroxyl group to the 2′ position of the phenyl ring,

B

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Figure 2. Rationale for the design of SMAIs. (A) In compound 2, the aldehyde group is expected to be masked by the 2′-phenol group via spontaneous

formation of a lactol. It is anticipated that the SMAI will undergo enzyme-catalyzed opening of the lactol ring and subsequently form the hemithioacetal

adduct with Cys₂₅. The scheme describes a two-step inhibition mechanism in which rapid formation of an EI complex precedes isomerization to EI*,

which slowly converts back to EI. (B) Lactol form of 2 (green) noncovalently docked to cruzain. (C) Opened form of 2 covalently docked to form a

hemithioacetal with Cys₂₅. Both structures are superimposed with a covalently bound K777 (white) from the crystal structure (PDB ID: 2OZ2).²⁰

Colored dashed lines represent corresponding cruzain−inhibitor interactions.

conformation in the active site. The bound lactol has a poorer

binding free energy (ΔG_predicted= −5.08 kcal/mol) than that of

the hemithioacetal (ΔG_predicted= −7.24 kcal/mol), apparently

owing to the bicyclic lactol moiety. One may infer that the

recognition of the Cbz-Phe scaﬀold of 2 by cruzain assists in the

binding of the lactol group and orients it for enzyme-catalyzed

ring-opening to yield the high-aﬃnity aldehyde.

Kinetic Analysis. A series of SMAIs and related compounds

1−12 were prepared and evaluated as inhibitors of cruzain

(Table 1). Time-course data for inhibition of cruzain by many of

these compounds conformed to the kinetic scheme shown in

Figure 2A, that is, initiation of reaction by adding enzyme to

substrate and inhibitor led to curvilinear time courses (Figure

3A,C) in which reaction rates demonstrably decreased as the EI

complex (characterized by K_i) progressed to the tighter EI*

complex (characterized by K_i*). The observation of time

dependence may or may not indicate that covalent bond

formation has occurred, but it does reﬂect that K_i* < K_i, arising

from either the formation of a hemithioacetal between the

enzyme and inhibitor or a slow isomerization step of EI to EI*

not involving covalent bond formation.

concentrations of inhibitors for which reactions were initiated

by the addition of enzyme. Inhibition of cruzain by the free

aldehyde 1 was characterized by downward-concave curvilinear

time courses, often referred to as “burst kinetics” (Figure 3A),

indicative of time-dependent inhibition for which equilibrium

between cruzain and 1 was slowly established over 30 min. Each

curve was ﬁtted to eq 1, and the resulting values of k_obswere

replotted versus inhibitor concentration [1] (Figure 3A, inset).

The replot was best ﬁtted to eq 3, implying a lack of saturation of

the EI complex by 1 for which K_i> > K_i*. The unimolecular rate

constant for conversion of EI* back to EI was extremely slow [k₄

= (7.6 0.6) × 10⁻⁴s⁻¹]. The low value of k₄is likely the reason

for the slow onset of inhibition and potency of aldehyde 1.

Unlike aldehyde 1, inhibition of cruzain by 2 exhibited almost

linear time courses with only slight curvature observed at early

stages (Figure 3B), as was also observed for SMAIs 6−10 (all

containing the Cbz-Phe scaﬀold, Figure S1A). The nominal

burst phase was only observed within the ﬁrst 1−2 min, after

which an apparent steady-state reaction was established,

indicating a signiﬁcantly faster rate (greater k₄) for conversion

of EI* back to EI than for compound 1. This diﬀerence suggests

that, compared to a relatively stable hemithioacetal intermediate

cruzain-1 species, the reverse reaction of this intermediate for a

We ﬁrst evaluated cruzain inhibition by time-course data

containing ﬁxed concentrations of substrates and variable

C

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a b c

, ,

Table 1. Inhibition Data of SMAIs and Related Compounds with Cruzain

a

To the assay buﬀer (50 mM MES, 50 mM TAPSO, 1 mM CHAPS, 1 mM Na₂EDTA, 5 mM DTT, 10% DMSO (v/v), and pH 7.5) containing

variable concentrations of inhibitors (0.02−20 μM) and 10 μM of substrate Cbz-Phe-Arg-AMC (K_m= 0.9 μM) was added cruzain (ﬁnal

b

concentration of 0.2 nM), and changes in ﬂuorescence were monitored for 20−60 min. Values of K_i* (n ≥ 2) were obtained as described.

3

c

contains two diastereomeric anti products, and only one is shown here. Reported as an apparent IC₅₀in ref 48.

SMAI is likely facilitated by the attack of its phenoxy group on

the hemithioacetal (Figure 3D). To characterize these

inhibitors, we obtained steady-state rates of v_sand v₀at assay

times ≥20 min. For instance, upon plotting v_s/v₀versus [2]

(Figure 3B, inset) and ﬁtting using eq 4, we obtained a value of

the overall inhibition constant (K_i*) of 49 2 nM. For free

aldehyde 11 and SMAI 12, both of which incorporated the N-

methylpiperazinyl-Phe (NMePip-Phe) scaﬀold as found in

K777, the ratio of their activities as cruzain inhibitors

[K_i*(12)/K_i*(11) = 94] was almost identical to that of their

D

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Figure 3. Kinetic analysis of inhibition of cruzain by 1, 2, and 12. (A−C) Time courses of cruzain inhibition by 1, 2, and 12. Insets for (A) and (C): the

k

_obsvalues were obtained by ﬁtting progress curves to eq 1. Replot of k_obsvs [I] with the line drawn through data points from ﬁtting to eq 3. 1: k₄= (7.6

0.6) × 10⁻⁴s⁻¹; 12: k₄= (1.8 0.7) × 10⁻⁴s⁻¹. Inset for (B): the steady-state rates with (v_s) and without inhibitor (v₀) were obtained at t ≥ 20 min.

Plot of v_s/v₀vs [2] with the line drawn through data points from ﬁtting to eq 4. (D) Proposed mechanism of phenoxy-assisted conversion of EI* back to

EI.

Figure 4. Rapid dilution assay for SMAIs. (A) Dilution scheme for testing the reversibility of a SMAI. Upon 100-fold dilution, the inhibitor

concentration decreases from 10-fold > K_i*^app(91% inhibition) to 10-fold < K_i*^app(9% inhibition). (B, C) Cruzain activity rapidly recovered from

inhibition by 2 and 6−10. (D−F) Cruzain activity recovery showed a signiﬁcant lag for 1, 11, and 12. All curves were ﬁtted to eq 1 to provide k_obsvalues

as listed in Table S1.

Cbz-Phe-containing counterparts 1 and 2 [K_i*(2)/K_i*(1) =

110]. Similar to aldehyde 1, slow onset of inhibition was

observed for 11 (Figure S1B). Surprisingly, unlike SMAIs

containing the Cbz-Phe scaﬀold, 12 also exhibited time-

dependent inhibition (Figure 3C).

To explore the reversibility of cruzain inhibition by these

compounds, we ﬁrst preincubated each inhibitor with cruzain

followed by a 100-fold rapid dilution accompanying the addition

of substrate (Figure 4A). For inhibitors 2 and 6−10 (Figure

4B,C), 91% recovery of cruzain activity was observed over the

E

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Figure 5. Studies on the chemical mechanism of SMAI binding to cruzain. (A) Expansion of the superposed ¹H-¹³C HSQC NMR spectra of ¹³C-

labeled compound 12 with (blue) and without (red) an approximately equimolar concentration of cruzain, which were obtained at 800 MHz (¹H) at

25 °C. (B) Time course of phenylhydrazone formation by treating 0.2 mM 11 (aldehyde) or 12 (lactol) with 1 mM phenylhydrazine, with a control

sample containing DMSO. Data for the curve with compound 11 were ﬁtted to Abs_{282 nm}= a[1−exp.(−k_obst)] + C from which a = 0.279 0.002, k_obs

=

(2.61 0.05) × 10⁻⁴s⁻¹, and C = 2.63, while ﬁtting the other data to this expression led to negligible values of k_obs

.

course of minutes, wherein the residual concentration of

inhibitor was 0.1× K_i*^app, capable of aﬀording 9% inhibition,

thereby demonstrating that binding of these inhibitors was fully

reversible. In comparison, the activity of cruzain preincubated

with aldehydes 1, 11, or SMAI 12 was restored at much slower

rates (Figure 4D−F). After ﬁtting these “lag” time courses to eq

1, the calculated residence times (τ = 1/k_obs) for compounds 2

and 6−10 ranged from 2.5 to 5.5 min, whereas 1, 11, and 12

exhibited signiﬁcantly longer residence times of 52, 45, and 26

min, respectively (Table S1). While longer residence times were

expected for aldehydes 1 and 11, compound 12 was the only

SMAI to eﬀect signiﬁcant time-dependent inhibition as

evidenced by the observation of both burst and lag kinetic

time courses, and the rate of conversion of the EI* complex to EI

was comparable to that of aldehyde 11. Considering that

compound 12 was equipotent to 2, one could infer that the

peptidomimetic scaﬀold of 12 might also aﬀect the adduct

formation so that k₃/k₄remained nearly unchanged for both the

SMAI and the free aldehyde. Therefore, a covalent docking

study was conducted for 12, which predicted a binding free

energy (ΔG_predicted= −7.15 kcal/mol) that was close to that of

compound 2 (ΔG_predicted= −7.24 kcal/mol) as formerly

mentioned, which was in accordance with their similar K_i*

values. Interestingly, an intramolecular hydrogen bond between

the P₁phenol group and P₃carbonyl group has dragged the two

moieties away from their binding subsites (Figure S2). The

consequence of this conformation is that the phenol group in 12

is less likely to take part in the conversion of EI* back to EI

(Figure 3D), leading to slower dissociation from cruzain and its

unique lag kinetics compared to 2.

Substitution of the aldehyde of compound 2 with a primary

alcohol also resulted in the poor inhibitor 4, which corroborates

the importance of the formation of carbon−sulfur bonds in the

cruzain-SMAI complexes. In the free aldehyde 5, the phenoxy

group was methylated, aﬀording an inhibitor that is 50-fold less

active than aldehyde 1, implying that a bulkier 2′ substituent

sterically hinders the formation of the hemithioacetal.

Another potential feature of SMAIs is the ability to tune the

reactivity of the phenol group by installing substituents on the

benzene ring. We introduced several substituents at the 5′

position of the ortho-tyrosine, including electron-donating

groups (i.e., -OMe and −Me), weak electron-withdrawing

groups (i.e., -F and -Cl), and moderate-to-strong electron

withdrawing group (i.e., -CO₂Me). The electron-donating

substituents on 6 (K_i* = 350 nM) and 7 (K_i* = 100 nM)

appear to undermine their potencies, as these inhibitors bind,

respectively, 7- and 20-fold more weakly than un-substituted

SMAI 2. The stronger electron-withdrawing capability of the

ester substituent (10; K_i* = 18 nM) provided a 3-fold increase in

potency versus 2, suggesting that the 5′-methyl ester facilitates

the opening of the lactol ring. In addition to the electronic eﬀect,

a steric eﬀect is likely operative. The methoxy group of

compound 6 is bulkier than the methyl group of 7, which may

contribute to its over 3-fold lower activity. Chlorine (0.79 Å, 8)

has a slightly larger radius than ﬂuorine (0.42 Å, 9), and ﬂuorine

is similar in size to a hydrogen (0.53 Å, 2) (note: the C−F is

longer than the C−H bond so that, at that increased length, the

ﬂuorine is eﬀectively the same radius as hydrogen),²¹consistent

with the diﬀerences between their K_i* values. It is interesting

that a steric eﬀect apparently dictates the diﬀerences in potency

between compounds 8 and 9, probably because ﬂuorine and

chlorine are only weakly electron-withdrawing. The methyl ester

of 10, while comprising the bulkiest substituent among this

series of compounds, provided the SMAI of the highest potency

in this study, apparently because the electron-withdrawing eﬀect

aﬀorded by this large substituent overcomes any steric eﬀect.

These results suggested that appropriate substitution of the P₁

phenyl ring in SMAIs could optimize their potencies, and

further, may aﬀect the stability of enzyme-bound lactol.

Structure−Activity Relationships of SMAIs. Inhibition

constants for all compounds prepared for this study are shown in

Table 1. The equipotency of SMAIs 2 and 12 at ∼50 nM

suggests that their diﬀerent P₃sidechains have no impact on

their inhibition. We prepared compound 3, an analogue of SMAI

12 in which its ether oxygen was replaced with a methylene

group, and the resulting compound containing the stable

tetrahydro-naphthol did not inhibit cruzain at ≤100 μM, despite

its similarity to the chroman-2-ol group of the SMAIs.

F

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Chemical Mechanism of SMAI Binding to Cruzain.

Next, we prepared compound 12 in which the aldehydic carbon

was ¹³C-labeled, to be used in a study of ¹H-¹³C heteronuclear

single quantum correlation (HSQC) nuclear magnetic reso-

nance (NMR) to elucidate the chemical specie(s) of 12 that are

bound to cruzain. First, 0.4 mM of ¹³C-labeled 12 in phosphate

buﬀer (pH 7.5) was analyzed by HSQC NMR. No discernable

peak was present at δ(¹³C) >180 ppm, ruling out the existence of

minute concentrations of free aldehyde in this aqueous solution

(see the Supporting Information for a full-view spectrum).²²

The two salient peaks, A and B, occurring near δ(¹³C) 90.8 ppm,

were consistent with the signal of a carbon occurring in a

hemiacetal (Figure 5A, red). These signals could not be assigned

to an aldehyde hydrate in view of the fact that analysis by liquid

chromatography−mass spectrometry (LC−MS) displayed a

molecular ion peak corresponding to the hemiacetal but not the

hydrate (Figure S3). We propose that peaks A and B are

associated with two δ-lactol anomers spontaneously generated

during lactol formation. The ratio of anomer A to anomer B is

1:1.32 based on peak volumes (Table S2), yet the exact

stereochemistry cannot be assigned at this point.

(BSFs) of Trypanosoma brucei brucei (T. b. brucei), which are

found in infected mammals (Table 2, Figure S5). The most

Table 2. Antitrypanosomal Activities of SMAIs and their

Prodrugs

cruzain

T. cruzi-infected

compound

number

inhibition K_i* T. b. brucei BSFs

cardiomyoblasts EC₅₀

a

(nM)

EC₅₀(μM)

(μM)

1

2

6

7

8

9

10

12

13

14

15

16

17

0.44 0.02

3.3 2.1

6.8 1.1

2.6 1.1

0.5 0.2

5.8 3.4

2.7 0.2

4.0 0.2

0.6 0.1

3.7 0.2

3.1 0.2

4.0 0.2

12 0.1

14 0.9

0.09 0.06

0.5 0.4

2.9 0.2

ND

5.7 0.6

ND

3.5 0.6

3.5 0.3

>20

49

350 30

103

74 10

48

18 0.5

47

2

d

5

2

b

∼1000

>5000

13

2

b

ND

10

b

3

b

ND

0.04 0.01

b

c

To the inhibitor sample was added an approximately

stoichiometric amount of cruzain, and a new spectrum was

acquired after 1 h (Figure 5A, blue). The apparent hemiacetal

peak B was eliminated while a trace of peak A remained, which

indicates a slight preference of cruzain for anomer B over A, in

consideration of the ratio of integrated volumes (A:B = 1:1.32).

The fact that the cross peaks shifted considerably upon addition

of cruzain is strongly indicative of hemithioacetal formation.

This is because the new signals observed at δ(¹³C) 76.2 and 79.7

ppm are very similar to those observed for the formation of a

hemithioacetal adduct between N-acetyl-L-phenylalanine-

[2-¹³C]glycinal and papain (δ(¹³C) 75.1 ppm).²³The broad-

ening of peaks A’ and B′ also suggested a protein-bound ligand,

because of reduced tumbling and the increased relaxation time

of the cruzain-bound ¹³C-labeled 12. In addition, mass

spectrometric experiments of cruzain preincubated with 12 as

well as three SMAIs with the Cbz-Phe- scaﬀold (2, 7, and 9)

clearly revealed the formation of corresponding enzyme−

inhibitor adducts as indicated by the mass changes relative to

apo-cruzain (Figure S4), serving as orthogonal evidence of

covalent bond formation.

As phenylhydrazine readily forms phenylhydrazone adducts

with aldehydes, we treated 0.2 mM of aldehyde 11 and SMAI 12

with 1 mM phenylhydrazine in the same buﬀer (lacking DTT)

used in the NMR study (Figure 5B) to determine the fraction of

free aldehyde found in 12. While aldehyde 11 was rapidly, and

apparently, completely converted to phenylhydrazone, 12

remained intact after 2 h. This ﬁnding showed that even an

excess amount of phenylhydrazine cannot drive the equilibrium

of 12 toward the formation of open-form aldehyde, which not

only corroborated the chemical stability of lactol, but also

indicated that the ring-opening is likely an enzyme-catalyzed

process. Therefore, we conclude that SMAI 2 predominantly

maintains its lactol form in aqueous solution in the absence of

cruzain, while its binding to cruzain apparently promotes

opening of its ring followed by the formation of a covalent bond

with Cys₂₅.

K777

0.2

a

Trypanocidal activities of compounds in axenic cultures of T. b.

brucei BSFs and in cardiomyoblasts with T. cruzi infection were all

measured as EC₅₀values (half maximal eﬀective concentration), along

with SEMs from at least two replicates. Typical test concentrations of

inhibitors were 0.5−20 μM for T. b. brucei BSF assays and 0.02−40

b

μM for T. cruzi-infected cardiomyoblast assays. Structures of 13−17

are shown in Figure 6B. Reported as an apparent IC₅₀in ref 48. ND,

not determined.

c

d

active inhibitors for BSFs, 7 and 12, exhibited respective EC₅₀

values of 0.5 and 0.6 μM, which are more potent than the

antitrypanosomal drug, diminazene (Berenil, EC₅₀= 0.99 μM in

T. b. brucei BSFs).²⁵Notably, the free aldehyde 1, despite its 100-

fold higher potency versus cruzain, was equally or less potent in

inhibiting T. b. brucei BSFs compared to the SMAIs. Considering

that aldehyde 1 is nearly four orders of magnitude less eﬀective

versus T. b. brucei than versus puriﬁed cruzain (EC₅₀/K_i* =

7500), while the average value of EC₅₀/K_i* for all SMAIs is 74,

one may speculate that the SMAIs are more accessible to its

cellular target(s) than free aldehyde 1 in cell culture media. In

this sense, masking of the active aldehyde within the SMAIs

aﬀords signiﬁcant protection of its electrophilic group.

Additionally, aldehyde 1, SMAIs 2, 7, 9, 10, 12, 13, and 15

(the latter two compounds are discussed in the next section)

were analyzed in a murine cardiomyoblast model of T. cruzi

infection in which the disease-relevant amastigote forms of the

parasite were evaluated (Table 2, Figure S6). The parent

aldehyde 1 was the most potent inhibitor in this series (EC₅₀

=

0.4 μM), yet it still lost most of its activity considering its

subnanomolar activity against cruzain. Compounds 2, 7, 9, and

10 killed T. cruzi at EC₅₀values ranging from 2.9−5.7 μM, which

were similar in potency to benznidazole, a ﬁrst-line antichagasic

drug with an EC₅₀value of 4.0 2.0 μM in this assay (Figure

S6). Surprisingly, 12 exhibited much weaker trypanocidal

activity in this assay in contrast to its excellent activity in T. b.

brucei, probably because its peptidomimetic scaﬀold, with higher

polarity, had diﬃculty in penetrating the host cell membrane.

Apart from 12, these SMAIs were essentially equipotent in

axenic cultures of T. b. brucei BSFs and T. cruzi-infected

cardiomyoblasts, suggesting that they target a cysteine protease

homologue of cruzain in T. b. brucei. Additionally, they exerted

SMAIs Display Antitrypanosomal Activity. Subspecies of

pathogenic Trypanosoma brucei have essential cysteine proteases

(TbCatL, brucipain, or rhodesain) which are close homologs of

cruzain and perform similar biological functions.²⁴We evaluated

our compounds in axenic cultures of the bloodstream forms

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Figure 6. Development and assessment of SMAI prodrugs. (A) Pralnacasan is a SMAI prodrug for VRT-18858, an inhibitor for caspase-1. (B)

Proposed metabolic routes of O-derivatized SMAIs to 12. (C) Time course of remaining 13−15 in reactions with or without addition of porcine

esterase. (D) High-performance liquid chromatography (HPLC) traces of compound 15 in buﬀer (control). (E) HPLC traces of 15 treated with

esterase.

no toxicity against the host cardiomyoblasts at up to 40 μM

where both aldehyde 1 and K777 exhibited signiﬁcant

cytotoxicity (Figure S6). Accordingly, the self-masking of the

aldehyde group in SMAIs provides the apparent delivery of these

otherwise reactive compounds to trypanosomes harbored within

mammalian cardiomyoblasts, with no apparent untoward eﬀects

on the host cells. These results encouraged progressing the

SMAIs to preclinical analysis, as well as the development of

prodrug forms of SMAIs.

Prodrugs of SMAIs. The pharmacokinetic evaluation of

compound 12 in mice by intravenous administration indicated a

short half-life arising from apparent ﬁrst-pass metabolism (data

not shown). Its analogue, K777, has superior pharmacokinetic

properties to 12, and it was shown that the sites of oxidative

metabolism on K777 were largely conﬁned to the N-

methylpiperazine ring, common to both inhibitors, and the

homophenylalanyl sidechain.²⁶Therefore, metabolism of the

cyclic hemiacetal of 12 may be responsible for its rapid clearance

in mice.

esterases via formation of a hemiacetal intermediate (Figure

6A).²⁷We designed two types of derivatization of the hemiacetal

hydroxyl group, that is, via O-acylation and O-alkylation (Figure

6B). The O-acylated compounds should be hydrolyzed to

compound 12 by the action of cellular esterases, which are

ubiquitous in the ER lumen of mammalian tissues.²⁸For the O-

alkylated compounds, the enzymatic oxidation is likely to occur

by the action of liver cytochrome P450 enzymes, thereby

elaborating a new hemiacetal that subsequently collapses to

form compound 12.

To explore this prodrug approach, we prepared O-acylated

compounds 13−15 and O-alkylated compounds 16 and 17

(structures shown in Figure 6B). These compounds exhibited

negligible inhibition against cruzain, with the exception of 13,

which weakly inhibited cruzain in a time-dependent fashion (K_i*

= 1 μM, Figure S1C). Compounds 13−15 were treated with

porcine esterase in buﬀer (pH 7.5), and the hydrolysis of these

prodrugs was monitored by LC−MS (Figure 6C). In control

samples without esterase, these compounds largely remained

intact. Compound 13 was an exception and degraded about 10%

in buﬀer over 3 h, which likely explains the observed inhibition

of cruzain by 13 because of the formation of 12. Upon addition

The cyclic α-keto-acetal prodrug for caspase-1, pralnacasan

(VX-740), can be regarded as an SMAI, as it is rapidly converted

to the active aldehyde inhibitor (VRT-18858) by plasma

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Figure 7. Compound 18 is a potential SMAI for SARS-CoV-2 3CL^pro. (A) 2-Pyridone is a surrogate for the P₁γ-lactam. (B) Structure and activity of

compound 18. (C) Surface representation of the 3CL^pro-18 complex (PDB ID: 7M2P). The 2F_o−F_celectron density map contoured at 1.0 σ indicated

C−S bond formation. (D) Interaction between 3CL^proand 18. Dashed lines depict hydrogen bonds; the oxyanion hole is circled.

of the esterase, compounds 13−15 were all converted to

compound 12 at variable rates of reaction with respective half-

lives of 48, 24, and 16 min (by ﬁtting curves in Figure 6C to eq 6)

and displayed a trend of increased hydrolysis with the increasing

steric bulk of the acyl groups. A reasonable interpretation is that

the main mammalian esterase, carboxylesterase-1 (CES1), has a

preference for larger acyl groups.²⁸Accordingly, modifying the

acyl group of O-acylated compounds to obtain a suitable half-life

can potentially overcome the apparent ﬁrst-pass metabolism of

12. As observed for compound 15 (Figure 6D,E), there are two

peaks in the chromatograph that presumably correspond to the

two anomers, but the conversion of one peak (A, t_R= 4.38 min)

to 12 is faster than the other (B, t_R= 4.52 min). Peak A is largely

eliminated after 30 min, while peak B, though it constitutes a

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a

Scheme 1. Synthesis of 2−12

a

(i) DBU, DCM, (ii) Pd/C, H₂, MeOH, (iii) TFA, DCM, (iv) Cbz-Phe-OH or NMePip-Phe-OH (S1j), DIPEA, T3P, DCM, (v) TBSCl,

imidazole, DCM, (vi) K₂CO₃, CH₃I, DMF, (vii) NaBH₄, MeOH, (viii) Dess-Martin periodinane, NaHCO₃, DCM, 0 °C, (ix) TBAF, THF, 0 °C,

(x) Et₃N, THF, 0 °C, (xi) Pd/C, H₂, MeOH, (xii) mCPBA, chloroform, (xiii) NaN₃, MeOH/H₂O, 60 °C, (xiv) Pd/C, H₂, MeOH, (xv) NMePip-

Phe-OH (S1j), DIPEA, T3P, DCM, (xvi) NMePip-Phe-OH (S1j), DIPEA, T3P, DCM, (xvii) NaBH₄, MeOH, and (xviii) Dess-Martin

periodinane, NaHCO₃, DCM, 0 °C. *S1l contains two diastereomeric anti products, and only one is drawn here, so do S1m and 3.

smaller proportion of untreated compound, does not decom-

pose completely even after 3 h, suggesting that the anomer in

peak A is the more speciﬁc substrate for esterase. These results

provide a proof of concept that the use of O-acylated prodrugs of

SMAIs will provide a means to deliver these inhibitors in vivo.

Compound 12 was also modiﬁed to yield two mixed acetals

16 and 17 (Figure 6B) to yield prodrugs that did not inhibit

cruzain. Because cytochrome P450 forms 3A4, 2D6, and 2C9

together account for over 60% of drug-metabolizing P450

isoforms,²⁹we used these enzymes to conduct in vitro assays to

determine if 16 and 17 could be transformed to compound 12 as

expected. Both compounds were incubated with diﬀerent CYPs

in the presence of a NADPH-regenerating system for up to 4 h.

Unfortunately, no transformation to 12 was observed for either

compound by any of the CYPs. However, the apparent stability

versus these puriﬁed P450s does not mean that (an)other

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a

Scheme 2. Synthesis of 13−17

a

(i) acetic/propionic/isobutyric anhydride, Et₃N, DMAP, DCM and (ii) BF₃OEt₂, EtOH/iPrOH.

microsomal oxidase(s) cannot release the active SMAIs from

these prodrug forms.

catalyzes essential cleavage of the coronaviral Spike protein.^8,32

As a result, compound 18 has the potential to be a dual-acting

inhibitor for two enzymes which are critical to the infection of

human cells by SARS-CoV-2.

These compounds were tested in cell assays (Table 2).

Although less active than their parent compound 12,

compounds 13−15 showed promising trypanocidal activity

We also obtained a high-resolution (1.70 Å) crystal structure

of SARS-CoV-2 3CL^proin complex with 18. The well-deﬁned

electron density (Figure 7C) conﬁrmed the formation of a

hemithioacetal of 18 with active-site Cys₁₄₅, and the resulting

hydroxyl group of the hemithioacetal is stabilized by an oxyanion

hole provided by Gly₁₄₃and Cys₁₄₅(Figure 7D). The P₁2-

pyridone establishes essential binding interactions with 3CL^pro

analogous to that of the γ-lactam³⁰in which the carbonyl oxygen

of the 2-pyridone accepts two hydrogen bonds from His₁₆₃and

Ser₁₄₄, while the amide nitrogen acts as a hydrogen bond donor

to Glu₁₆₆and Phe₁₄₀. These interactions clearly demonstrate that

the γ-lactam can be bioisosterically replaced by the 2-pyridone

moiety, which is a potential SMAI substructure. As with cruzain,

3CL^prois apparently capable of catalyzing ring-opening of the

putative masked aldehyde of 18.

against axenic T. b. brucei comparable to other SMAIs (EC₅₀

=

3−4 μM). Moreover, 13 and 15 were also tested in the T. cruzi-

infected model. It is notable that they showed moderate anti-T.

cruzi eﬀects (Table 2, EC₅₀= 13 and 10 μM, respectively), which

were more potent than 12, indicating that their acyl groups

helped them enter host cells probably by increasing the

hydrophobicity of these molecules. These results also

demonstrated that the O-acylated prodrug forms could well be

converted to 12 inside host cells or parasites by esterases.

Interestingly, compounds 16 and 17 were able to kill the T. b.

brucei in spite of lower activity (K_i* > 5 μM). Because treatment

with P450s did not lead to transformation of these prodrugs, the

data implied the existence of either a drug-metabolizing enzyme

or another potential activating enzyme in T. b. brucei.

Application of SMAIs to SARS-CoV-2 3CL^pro. The SMAI

strategy is not limited to cruzain and may also ﬁnd application in

other cysteine proteases. To this end, we attempted to develop

SMAIs for SARS-CoV-2 main protease (3CL^pro), a drug target

for COVID-19.³⁰Numerous inhibitors of the homologous

cysteine proteases of SARS-CoV, MERS-CoV, and some

picornaviruses have shown diﬀerent levels of inhibition against

SARS-CoV-2. Peptidomimetic aldehyde inhibitors of 3CL^pro

contain a nearly invariant 2-oxo-pyrrolidin-2-yl group (γ-

lactam) as the P₁side chain. We propose that 2-pyridone can

act as a surrogate for the γ-lactam (Figure 7A). Apart from their

similarity in size and heteroatom substitution, the tautomeriza-

tion of 2-pyridone toward 2-hydroxypyridine has been well

characterized.³¹Although the 2-pyridone tautomer is preferred

in aqueous solution, the presence of the C-terminal aldehyde

could well form a SMAI with the 2-hydroxypyridine.

A 2-pyridone compound 18 was synthesized (Figure 7B,

Scheme 4), and the proton NMR of 18 in organic solvents

conﬁrmed the apparent absence of aldehyde, suggesting that it

forms a δ-lactol, though it is unclear what the exact species is in

aqueous buﬀers. Compound 18 was such a potent inhibitor of

3CL^prothat its K_i* (9 nM) was a result of apparent titration of

the enzyme (40 nM used in the assay). Anti-CoV-2 activity

(EC₅₀= 5 μM) in SARS-CoV-2-infected A549/ACE2 cells was

also demonstrated (Table S3). Interestingly, 18 also inhibited

human cathepsin L, which was recently shown to be essential to

the penetrance of SARS-CoV-2 into mammalian cells in which it

Chemistry. The synthesis of SMAIs and their analogues

involved the preparation of various P₁building blocks and

subsequent coupling with P₃-P₂scaﬀolds (Scheme 1). A variety

of 5-substituted salicylaldehydes were coupled with N-Boc-2-

phosphonoglycine trimethyl ester in Horner−Wadsworth−

Emmons reactions. The formed double bond in S1a was

reduced to aﬀord the Boc-protected methyl ester of substituted

ortho-tyrosine (S1b) as a mixture of enantiomers, which was

then deprotected and reacted with the carboxylic acids of

corresponding P₃-P₂fragments, Cbz-Phe-OH or NMePip-Phe-

OH. Cbz-Phe-OH was commercially available, while NMePip-

Phe-OH (S1j) was simply prepared in two steps including amide

coupling and hydrogenolytic cleavage of the benzyl group. The

resulting S1d and subsequent intermediates were diastereomers

that could be mostly separated on a silica gel column. Because

the phenol group is relatively reactive that may interfere with

future reactions, a methyl group was originally used for its

protection and eventually resulted in compound 5. In our eﬀort

to remove the methyl group, we experienced unwanted side

reactions because of harsh conditions such as the use of BBr₃.

Instead, a TBS group was employed as the protecting group.

Next, the methyl ester of S1e was successively reduced to alcohol

using NaBH₄(S1f and 4) and oxidized to aldehyde using Dess-

Martin periodinane (S1g). Finally, eﬃcient removal of TBS by

TBAF exposed the phenol group toward the reaction with

aldehyde, leading to in situ formation of the lactol ring (S1h). It

should be noted that each SMAI was a mixture of anomers that

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a

Scheme 3. Synthesis of ¹³C-labeled 12

a

(i) 1-methylimidazole, MsCl, DCM, 45 °C, (ii) [1-¹³C] glycine, Ni(NO₃)₂·6H₂O, KOH, MeOH, 60 °C, (iii) (2-(benzyloxy)phenyl)methanol,

cyanomethylenetributylphosphorane (CMBP), toluene, 120 °C, (iv) 8-quinolinol, MeCN/H₂O, 40 °C, (v) Et₃N, Boc anhydride, dioxane/H₂O,

(vi) N,O-dimethylhydroxylamine, DIPEA, T3P, DCM, (vii) TFA, DCM; NMePip-Phe-OH, DIPEA, T3P, DCM, (viii) LAH, THF, 0 °C, and (ix)

Pd/C, H₂, MeOH.

a

Scheme 4. Synthesis of 18

a

(i) DBU, DCM, (ii) Pd/C, H₂, MeOH, (iii) LiOH, MeOH/H₂O, (iv) N,O-dimethylhydroxylamine, DIPEA, T3P, DCM, (v) TFA, DCM; Cbz-

Val-Cha-OH, DIPEA, T3P, DCM, and (vi) LAH, THF, 0 °C.

could be observed by HPLC or NMR (see the Supporting

Information). Because the spontaneous interconversion existed

in between the anomers, it was neither necessary nor possible to

chromatographically isolate them.

The P₁building block of 3 was synthesized using a diﬀerent

strategy. 1,4-Dihydronaphthalene was subjected to epoxidation

(S1k) followed by nucleophilic attack by NaN₃to produce S1l.

The azide was then converted to the amino group which was

coupled with NMePip-Phe-OH to yield compound 3. Notably,

3 contains two diastereomeric anti products as the P₁NH and

OH are not on the same side of the ring. The preparation of free

aldehyde 11 was similar to other SMAIs, except for that the latter

required one more step for TBS removal.

triﬂuoride etherate in the presence of corresponding alcohols

(S2b).

Because ¹³C-lableled N-Boc-2-phosphonoglycine trimethyl

ester was not purchasable, we adopted a Ni(II) complex-based

synthetic route for ¹³C-labeled 12 (Scheme 3).³³A chiral

auxiliary S3a (2-(N-benzylprolyl)aminobenzophenone, BPB)

was synthesized and then treated with [1-¹³C] glycine and

nickel(II) nitrate hexahydrate under strongly alkaline conditions

to form the Ni(II) complex (S3b), which could increase the

acidity of α-protons in the glycine. Next, a Mitsunobu−Tsunoda

reaction using CMBP was performed to introduce the side chain

onto glycine with high stereoselectivity (S3c).³³We employed

8-quinolinol for the decomplexation to release the ¹³C-labeled

benzylated o-Tyr (S3d). After protecting the amine with Boc

(S3e), the carboxylic acid was converted to a Weinreb amide

(S3f). The latter was deprotected and coupled to NMePip-Phe-

OH (S3g) followed by reduction to aldehyde with an equimolar

The derivatizations of the hydroxy group of 12 are

summarized in Scheme 2. Acylation was achieved by reacting

12 with corresponding acid anhydrides under basic conditions

(S2a); alkylation was performed by treating 12 with boron

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puriﬁed by semipreparative HPLC (Prep-HPLC) on the same UltiMate

3000 HPLC system, which was connected to a fraction collector. The

typical settings for Prep-HPLC were as follows: Column used:

Phenomenex Luna 5 μm C18(2) 100 Å, 21.2 mm × 250 mm; mobile

phases A and B were the same as those for analytical HPLC; method:

gradient elution at 10−100% B over 25 min, then isocratic elution at

100% B for 5 min; ﬂow rate: 21.2 mL/min. ¹H and ¹³C NMR spectra of

compounds, except for ¹³C-labeled compound 12, were recorded on a

Bruker AVANCE III 400 MHz using tetramethylsilane (TMS, 0.00

ppm) or residual solvent (CDCl₃, 7.26 ppm; CD₃OD, 3.31 ppm;

(CD₃)₂SO, 2.50 ppm) as the internal standards. All ﬁnal compounds

used for testing in assays had purities that were determined to be >95%

as evaluated by their NMR spectra and/or their LC−MS.

amount of lithium aluminum hydride (S3h) and removal of the

benzyl-protecting group, resulting in ¹³C-labeled 12.

Similar to Scheme 1, the 2-pyridone inhibitor 18 was also

synthesized from 2-oxo-1,2-dihydro-3-pyridinecarbaldehyde

(Scheme 4). After hydrogenation of S4a, the methyl ester of

S4b was hydrolyzed (S4c) and converted to Weinreb amide S4d.

The incorporation of the Cbz-Val-Cha-scaﬀold resulted in S4e

as diastereomers that could be separated on a silica gel column.

It turned out that no protection was needed for the 2-pyridone/

2-hydroxypyridine, so that the LAH reduction of the Weinreb

amide aﬀorded the ﬁnal product 18. Although there is no

apparent aldehydic peak in NMR spectra when using CDCl₃as

the solvent, the two inherent equilibria (lactol/aldehyde and

pyridone/hydroxypyridine) have led to greater complexity. The

exact species of 18 are dependent on multiple factors including

solvents, temperature, pH, and so on, as exempliﬁed by its

HPLC−MS data (see the Supporting Information): there are

several crowded peaks in HPLC, yet all of which correspond to

the mass of 18 (m/z 552.29).

Synthetic Procedures. Detailed procedures and characterization

of compounds for all reactions in Schemes 1234 were as follows. Some

of them were general procedures, which were described by

representative compounds.

Methyl 2-((tert-butoxycarbonyl)amino)-3-(2-hydroxy-5-

methylphenyl)acrylate (S1a, R = Me). To a solution of ( )-Boc-α-

phosphonoglycine trimethyl ester (4.22 g, 14.2 mmol, 1.2 eq) in DCM

(20 mL) was added DBU (2.12 mL, 14.2 mmol, 1.2 eq) dropwise at

−10 °C, and the resulting mixture was stirred for 20 min. Then 4-

methylsalicylaldehyde (1.61 g, 11.8 mmol, 1.0 eq) in DCM (10 mL)

was slowly added to the mixture over 10 min. The reaction mixture was

stirred at room temperature overnight. The resulting mixture was

concentrated under reduced pressure, diluted with EtOAc (100 mL),

and washed successively with saturated aqueous NH₄Cl (20 mL),

saturated aqueous NaHCO₃, (20 mL), and brine (20 mL). The organic

layer was dried over Na₂SO₄, ﬁltered, and concentrated under reduced

pressure. The obtained crude product was puriﬁed by FCC (30−50%

CONCLUSIONS

■

While the aldehyde group has frequently been employed in

chemical probes in drug discovery, rarely has this reactive

functionality been advanced in earnest to drug discovery

campaigns. However, the masking of aldehydes in drugs and

clinical agents has provided a few examples of the successful

“masking” of aldehydes for drug discovery. Here, we sequestered

the potent free aldehyde inhibitor 1 into an intramolecular

masked aldehyde by the introduction of a 2-hydroxyl group onto

the P₁benzyl group, which resulted in a surprisingly stable δ-

lactol derivative that eﬀected potent yet reversible inhibition of

cruzain. The apparent stability of δ-lactol in examples of these

SMAIs suggests that the observed potent inhibition of cruzain by

these SMAIs most likely arises from cruzain-catalyzed ring-

opening of the δ-lactol followed by formation of a hemi-

thioacetal. Where evaluated, these self-masked aldehyde

inhibitors inhibited axenic and infected cell cultures of

trypanosomes with potency equaling or exceeding that of the

parent aldehydes. Furthermore, the O-acylation of SMAIs

yielded prodrugs that might possess improved metabolic

stability and pharmacokinetic properties. Finally, we developed

1

EtOAc in hexane, v/v) to give S1a (R = Me, 2.91 g, 80%). H NMR

(400 MHz, CD₃OD) δ 1.45 (s, 9H, Boc), 2.25 (s, 3H, PhCH₃), 3.83 (s,

3H, COOCH₃), 6.77 (d, J = 8.3 Hz, 1H, oleﬁn), 7.02 (dd, J = 2.2, 8.4

Hz, 1H, Ph), 7.41 (d, J = 2.2 Hz, 1H, Ph), 7.53 (s, 1H, Ph). ¹³C NMR

(100 MHz, CD₃OD) δ 19.22, 27.18, 51.36, 79.97, 115.23, 120.46,

124.58, 128.22, 129.63, 131.02, 153.42, 154.76, 166.73. LC−MS: t_R=

+

4.93 min; C₁₁H₁₄NO₃[M + H - Boc]⁺, m/z calcd 208.10, found

208.08.

Methyl 2-((tert-butoxycarbonyl)amino)-3-(2-hydroxy-5-

methylphenyl)propanoate (S1b, R = Me). S1a (R = Me, 1.15 g,

3.74 mmol, 1.0 eq) was placed in a two-necked round bottom and

charged with N₂gas. 10% palladium on carbon powder (Pd/C, 110 mg,

cat.) was quickly added to the ﬂask followed by addition of MeOH (20

mL). The ﬂask was degassed and backﬁlled with H₂for three cycles.

The reaction mixture was stirred at room temperature overnight with a

balloon of H₂for replenishment. The balloon was removed, and the

mixture was ﬁltered under reduced pressure. Note that the operation

should be rapid, and the Pd/C powder must be kept wet to avoid

catching ﬁre and was appropriately disposed in a water-ﬁlled, cap-closed

container. The ﬁltrate was concentrated under reduced pressure and

puriﬁed by FCC (30% EtOAc in hexane, then 6% MeOH in DCM) to

give S1b (R = Me, 1.1 g, 95%). ¹H NMR (400 MHz, CDCl₃) δ 1.39 (s,

9H, Boc), 2.36 (s, 3H, PhCH₃), 2.79−3.02 (m, 1H, βCH₂), 3.07 (m,

1H, βCH₂), 3.75 (s, 3H, COOCH₃), 4.43 (d, J = 6.2 Hz, 1H, αCH),

5.54 (d, J = 7.4 Hz, 1H, NH), 6.65−6.73 (m, 2H, Ph), 6.73−6.79 (m,

1H, Ph). ¹³C NMR (100 MHz, CDCl₃) δ 20.57, 28.24, 32.84, 51.98,

54.97, 79.50, 113.30, 116.22, 119.28, 127.84, 147.51, 153.95, 155.33,

and characterized a novel SMAI of SARS-CoV-2 3CL^pro

,

suggesting a broad applicability of the SMAI strategy to other

cysteine proteases.

EXPERIMENTAL SECTION

■

General Information of Chemistry. Unless otherwise noted, all

starting materials, reagents, and solvents were obtained commercially

and used without further puriﬁcation or distillation. Reactions were

conducted under an inert atmosphere (Ar or N₂), and reaction progress

was monitored by thin layer chromatography (TLC) or HPLC−MS.

TLC experiments were performed with silica gel plates on aluminum

foil (Sigma-Aldrich, cat. no. 60778). HPLC analysis was conducted on

an UltiMate 3000 HPLC system coupled to MS analysis by an ISQ EM

single quadrupole mass spectrometer. The typical settings for HPLC−

MS were as follows. Column used: Phenomenex Luna 5 μm C18(2)

100 Å, 4.6 mm × 50 mm; mobile phase A: water containing 0.1% formic

acid (v/v); mobile phase B: MeCN containing 0.1% formic acid (v/v);

method: gradient elution at 10−100% B over 6 min, then isocratic

elution at 100% B for 2 min; ﬂow rate: 1 mL/min; MS parameters:

HESI, vaporizer temperature 350 °C, source CID voltage 20 V, and gas

ﬂow: sheath = 40−50 psi, aux = sweep = 2.0 psi. Most compounds were

puriﬁed by ﬂash column chromatography (FCC) on silica gel (200−

300 mesh) with diﬀerent solvent systems. Some compounds were

+

172.70. LC−MS: t_R= 4.98 min; C₁₆H₂₄NO₅[M + H]⁺, m/z calcd

310.16, found 310.14.

Methyl 2-amino-3-(2-hydroxy-5-methylphenyl)propanoate TFA

Salt (S1c, R = Me). To a suspension of S1b (R = Me, 1.1 g, 3.56 mmol,

1.0 eq) in DCM (6 mL) was added TFA (3 mL) at 0 °C, and the

resulting mixture was stirred for 30 min. The mixture was then

concentrated with toluene three times to remove most of the residual

TFA. The obtained crude product S1c (R = Me) was used without

further puriﬁcation.

Methyl (S)-2-((S)-2-(((benzyloxy)carbonyl)amino)-3-phenylpro-

panamido)-3-(2-hydroxy-5-methylphenyl)propanoate (S1d, R =

Me, R₁= BnO). A mixed suspension of S1c (R = Me, ∼1.15 g, 3.56

mmol, 1.0 eq), Cbz-Phe-OH (1.07 g, 3.56 mmol, 1.0 eq), and DIPEA

M

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(2.17 mL, 12.46 mmol, 3.5 eq) in DCM (20 mL) was cooled to 0 °C

followed by dropwise addition of T3P (>50% in MeCN, 3.53 mL, 5.34

mmol, 1.5 eq). The resulting mixture was stirred at room temperature

for 2 h. The reaction mixture was concentrated under reduced pressure,

diluted with EtOAc (75 mL), and washed successively with 5% citric

acid (15 mL), saturated aqueous NaHCO₃(15 mL), and brine (15

mL). The organic layer was dried over Na₂SO₄, ﬁltered, and

concentrated under reduced pressure. The obtained crude product

was puriﬁed by FCC (40% EtOAc in hexane) to give S1d (R = Me, R₁=

BnO, 1.11 g, 63% for two steps). ¹H NMR (400 MHz, CDCl₃) δ 2.30 (s,

3H, PhCH₃), 2.83−3.16 (m, 4H, 2 × βCH₂), 3.69 (s, 3H, COOCH₃),

4.50 (s, 1H, αCH), 4.75 (s, 1H, αCH), 4.98−5.14 (m, 2H, Cbz CH₂),

5.70 (s, 1H, OH), 6.68 (t, J = 8.8 Hz, 1H, NH), 6.91−7.10 (m, 3H, Ph),

7.22 (dd, J = 20.7, 26.3 Hz, 7H, Ph), 7.33 (s, 3H, Ph), 8.08 (s, 1H, NH).

¹³C NMR (100 MHz, CDCl₃) δ 20.66, 33.10, 38.37, 52.47, 53.19,

56.10, 67.24, 117.11, 124.49, 127.01, 127.88, 128.22, 128.32, 128.42,

128.53, 128.61, 129.25, 130.87, 135.98, 136.14, 153.57, 156.27, 171.72,

(10 mL) was added NaBH₄(1.2 g, 31.7 mmol, >20 eq) in multiple

portions every 30 min. The reaction mixture was stirred at room

temperature for another 2 h and then quenched by addition of saturated

aqueous NH₄Cl (10 mL). The mixture was concentrated under

reduced pressure and diluted with EtOAc (50 mL). The organic layer

was washed with saturated aqueous NaHCO₃(10 mL) and brine (10

mL) and was dried over Na₂SO₄, ﬁltered, and concentrated under

reduced pressure. The obtained crude product was puriﬁed by FCC

(35% EtOAc in hexane) to give S1f (R = Me, R₁= BnO, R₂= TBS, 450

mg, 53%). ¹H NMR (400 MHz, CDCl₃) δ 0.26 (s, 6H, TBS Si(CH₃)₂),

1.03 (s, 9H, TBS C(CH₃)₃), 2.40 (s, 3H, PhCH₃), 2.74 (dt, J = 7.0, 13.1

Hz, 2H, βCH₂), 2.97 (dd, J = 6.8, 40.5 Hz, 2H, βCH₂), 3.25−3.60 (m,

2H, CH₂OH), 4.02−4.15 (m, 1H, αCH), 4.39 (s, 1H, αCH), 4.97−

5.15 (m, 2H, Cbz CH₂), 5.69 (dd, J = 7.8, 66.8 Hz, 1H, NH), 6.54 (dd, J

= 6.8, 121.3 Hz, 1H, Ph), 6.72−6.81 (m, 1H, Ph), 7.04−7.14 (m, 2H,

Ph), 7.15−7.30 (m, 6H, Ph & NH), 7.34 (q, J = 6.2, 7.1 Hz, 4H, Ph).

¹³C NMR (100 MHz, CDCl₃) δ −4.05, 18.27, 21.02, 25.87, 31.17,

+

172.08. LC−MS: t_R= 5.39 min; C₂₈H₃₁N₂O₆[M + H]⁺, m/z calcd

39.00, 52.34, 56.69, 63.66, 67.03, 119.96, 126.28, 127.00, 127.56,

127.93, 128.03, 128.16, 128.47, 128.65, 129.29, 130.27, 131.00, 136.24,

136.53, 152.38, 155.94, 170.98, 171.35. LC−MS: t_R= 6.90 min;

C₃₃H₄₅N₂O₅Si⁺[M + H]⁺, m/z calcd 577.31, found 577.37.

491.22, found 491.19.

Methyl (S)-2-((S)-2-(((benzyloxy)carbonyl)amino)-3-phenylpro-

panamido)-3-(2-((tert-butyldimethylsilyl)oxy)-5-methylphenyl)-

propanoate (S1e, R = Me, R₁= BnO, R₂= TBS). A mixed solution of

S1d (R = Me, R₁= BnO, 990 mg, 2.02 mmol, 1.0 eq), TBSCl (609 mg,

4.04 mmol, 2.0 eq), and imidazole (412 mg, 6.06 mmol, 3.0 eq) was

stirred at room temperature overnight. The reaction mixture was

quenched by addition of 0.5 M HCl (10 mL) and was stirred for

another 15 min. The mixture was concentrated under reduced pressure

and was partitioned between EtOAc (50 mL) and water (10 mL). The

organic layer was washed with saturated aqueous NaHCO₃(10 mL)

and brine (10 mL) and was dried over Na₂SO₄, ﬁltered, and

concentrated under reduced pressure. The obtained crude product

was puriﬁed by FCC (20% EtOAc in hexane) to give S1e (R = Me, R₁=

BnO, R₂= TBS, 897 mg, 73%). ¹H NMR (400 MHz, CDCl₃) δ 0.16 (s,

6H, TBS Si(CH₃)₂), 0.93 (s, 9H, TBS C(CH₃)₃), 2.37 (s, 3H, PhCH₃),

2.59−2.87 (m, 2H, βCH₂), 2.89−3.05 (m, 2H, βCH₂), 3.54 (d, J = 4.3

Hz, 3H, COOCH₃), 4.36 (d, J = 22.6 Hz, 1H, αCH), 4.64 (q, J = 8.3 Hz,

1H, αCH), 4.95 (p, J = 12.1 Hz, 2H, Cbz CH₂), 5.32−5.55 (m, 1H,

NH), 6.56−6.65 (m, 1H, Ph), 6.83−7.02 (m, 3H, Ph), 7.05−7.25 (m,

9H, Ph). ¹³C NMR (100 MHz, CDCl₃) δ −3.50, 18.27, 20.53, 25.81,

32.94, 38.57, 52.20, 52.57, 56.08, 66.96, 119.79, 125.91, 126.90, 127.89,

127.95, 128.02, 128.04, 128.07, 128.14, 128.44, 128.47, 128.57, 128.71,

129.28, 129.35, 130.82, 136.25, 136.47, 152.62, 155.90, 170.76, 171.62,

172.00. LC−MS: t_R= 7.23 min; C₃₄H₄₅N₂O₆Si⁺[M + H]⁺, m/z calcd

605.30, found 605.24.

Benzyl ((S)-1-(((S)-1-(2-((tert-butyldimethylsilyl)oxy)-5-methyl-

phenyl)-3-oxopropan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-

carbamate (S1g, R = Me, R₁= BnO, R₂= TBS). To a solution of S1f (R

= Me, R₁= BnO, R₂= TBS, 151 mg, .26 mmol, 1.0 eq) in DCM (8 mL)

at 0 °C were added Dess-Martin periodinane (133 mg, 0.31 mmol, 1.2

eq) and NaHCO₃powder (55 mg, 0.65 mmol, 2.5 eq). The resulting

mixture was stirred at 0 °C for 1 h and then quenched by addition of

saturated aqueous Na₂S₂O₃(2 mL). The reaction mixture was

concentrated under reduced pressure, diluted with EtOAc (50 mL),

and washed with brine (3 × 10 mL). The organic layer was dried over

Na₂SO₄, ﬁltered, and concentrated under reduced pressure. The

obtained crude product was puriﬁed by FCC (25% EtOAc in hexane) to

give S1g (R = Me, R₁= BnO, R₂= TBS, 68 mg, 45%). ¹H NMR (400

MHz, CDCl₃) δ 0.24 (d, J = 4.6 Hz, 6H, TBS Si(CH₃)₂), 1.01 (s, 9H,

TBS C(CH₃)₃), 2.24 (s, 3H, PhCH₃), 2.77−3.20 (m, 4H, 2 × βCH₂),

4.51 (q, J = 6.7 Hz, 1H, αCH), 5.02−5.15 (m, 2H, Cbz CH₂), 5.35 (s,

1H, αCH), 6.52 (d, J = 5.5 Hz, 1H, NH), 6.71 (d, J = 8.2 Hz, 1H, Ph),

6.85 (d, J = 2.2 Hz, 1H, Ph), 6.94 (dd, J = 2.3, 8.2 Hz, 1H, Ph), 7.16 (d, J

= 7.1 Hz, 2H, Ph), 7.21−7.28 (m, 3H, Ph), 7.35 (dt, J = 4.7, 6.9 Hz, 5H,

Ph), 9.41 (s, 1H, CHO). ¹³C NMR (100 MHz, CDCl₃) δ −4.14, 18.28,

20.43, 25.90, 29.92, 38.80, 56.11, 59.57, 67.01, 118.77, 125.68, 127.09,

128.01, 128.16, 128.50, 128.67, 128.91, 129.26, 130.94, 131.91, 136.13,

151.28, 170.96, 198.75. LC−MS: t_R= 6.81 min; C₃₃H₄₃N₂O₅Si⁺[M +

H]⁺, m/z calcd 575.29, found 575.36.

Benzyl ((2S)-1-(((3S)-2-hydroxy-6-methylchroman-3-yl)amino)-

1-oxo-3-phenylpropan-2-yl)carbamate (S1h, 7, R = Me, R₁= BnO,

R₂= TBS). To a solution of S1g (R = Me, R₁= BnO, R₂= TBS, 68 mg,

0.12 mmol, 1.0 eq) in THF (3 mL) was slowly added 1.0 M TBAF in

THF (131 μL, 0.13 mmol, 1.1 eq) at 0 °C. The resulting mixture was

stirred at 0 °C for 1 h and concentrated under reduced pressure. The

residue was diluted with EtOAc (50 mL) and washed with saturated

aqueous NH₄Cl (10 mL) and brine (10 mL). The organic layer was

dried over Na₂SO₄, ﬁltered, and concentrated under reduced pressure.

The obtained crude product was puriﬁed by FCC (35% EtOAc in

hexane) to give S1h (R = Me, R₁= BnO, R₂= TBS, i.e., compound 7, 20

mg, 36%). ¹H NMR (400 MHz, CDCl₃) δ 2.10−2.18 (m, 3H, PhCH₃),

2.37−3.10 (m, 4H, 2 × βCH₂), 4.10−4.45 (m, 2H, αCH & NH), 4.71−

5.10 (m, 3H, Cbz CH₂& NH), 5.53 (dd, J = 7.2, 51.9 Hz, 1H, αCH),

5.97−6.41 (m, 1H, lactol C(O)OH), 6.59 (td, J = 10.3, 31.2, 32.4 Hz,

2H, Ph), 6.78 (dd, J = 8.2, 18.7 Hz, 1H, Ph), 6.98−7.28 (m, 10H, Ph).

¹³C NMR (100 MHz, CDCl₃) δ 20.46, 25.96, 39.10, 46.02, 56.24,

Methyl (S)-2-((S)-2-(((benzyloxy)carbonyl)amino)-3-phenylpro-

panamido)-3-(2-methoxyphenyl)propanoate (S1e, R = H, R₁

=

BnO, R₂= Me). To a solution of S1d (R = H, R₁= BnO, 172 mg, 0.36

mmol, 1.0 eq) in DMF (2 mL) were successively added K₂CO₃(100

mg, 0.72 mmol, 2.0 eq) and iodomethane (67 μL, 1.08 mmol, 3.0 eq) at

0 °C. The resulting mixture was stirred at room temperature for 20 h

during which the system should be kept securely sealed to avoid

evaporation of iodomethane. The mixture was diluted with EtOAc (50

mL) and washed with water extensively (5 × 10 mL). The organic layer

was dried over Na₂SO₄, ﬁltered, and concentrated under reduced

pressure. The obtained crude product was puriﬁed by FCC (30%

EtOAc in hexane) to give S1e (R = H, R₁= BnO, R₂= Me, 167 mg,

95%). ¹H NMR (400 MHz, CDCl₃) δ 3.02 (qd, J = 5.7, 13.6, 16.9 Hz,

4H, 2 × βCH₂), 3.61 (s, 3H, COOCH₃), 3.93 (s, 3H, PhOCH₃), 4.48

(s, 1H, αCH), 4.72 (s, 1H, αCH), 4.88−5.12 (m, 2H, Cbz CH₂), 5.69

(d, J = 8.2 Hz, 1H, NH), 6.64−6.88 (m, 2H, Ph), 6.92−7.06 (m, 3H,

Ph), 7.08−7.18 (m, 4H, Ph), 7.24 (d, J = 18.6 Hz, 5H, Ph), 7.77 (d, J =

43.8 Hz, 1H, NH). ¹³C NMR (100 MHz, CDCl₃) δ 32.65, 32.96, 38.40,

52.33, 52.40, 53.70, 56.04, 67.11, 115.84, 120.35, 122.73, 126.88,

126.90, 127.88, 128.16, 128.52, 128.72, 129.30, 131.30, 136.14, 154.71,

67.20, 91.04, 91.93, 116.68, 118.34, 119.01, 119.23, 128.04, 128.12,

128.20, 128.52, 128.67, 128.77, 129.19, 129.32, 129.64, 130.50, 130.63,

136.02, 136.26, 148.52, 156.15, 171.23. LC−MS: t_R= 5.32 min;

C₂₇H₂₉N₂O₅⁺[M + H]⁺, m/z calcd 461.21, found 461.27.

+

156.21, 156.23, 171.98, 172.34. LC−MS: t_R= 5.71 min; C₂₈H₃₁N₂O₆

[M + H]⁺, m/z calcd 491.22, found 491.33.

Benzyl (4-methylpiperazine-1-carbonyl)-L-phenylalaninate (S1i).

To a solution of 4-methylpiperazine-1-carbonyl chloride hydrochloride

(1.365 g, 6.86 mmol, 1.1 eq) in THF (20 mL) at −10 °C was added

Et₃N (2.08 mL, 14.96 mmol, 2.4 eq) dropwise. The resulting mixture

Benzyl ((S)-1-(((S)-1-(2-((tert-butyldimethylsilyl)oxy)-5-methyl-

phenyl)-3-hydroxypropan-2-yl)amino)-1-oxo-3-phenylpropan-2-

yl)carbamate (S1f, R = Me, R₁= BnO, R₂= TBS). To a solution of S1e

(R = Me, R₁= BnO, R₂= TBS, 897 mg, 1.48 mmol, 1.0 eq) in MeOH

N

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was stirred for 15 min, and then a solution of benzyl L-phenylalaninate

hydrochloride (1.82 g, 6.23 mmol, 1.0 eq) was added into THF (20

mL) dropwise. The reaction mixture was stirred at room temperature

overnight, quenched by addition of water (10 mL), and concentrated

under reduced pressure. The residue was diluted with EtOAc (100 mL)

and washed successively with saturated aqueous NH₄Cl (20 mL),

saturated aqueous NaHCO₃(20 mL), and brine (20 mL). The organic

layer was dried over Na₂SO₄, ﬁltered, and concentrated under reduced

pressure. The obtained crude product was puriﬁed by FCC (5−10%

1H). ¹³C NMR (100 MHz, DMSO-d₆) δ 32.84, 35.71, 38.13, 43.63,

45.94, 50.11, 54.62, 56.33, 67.15, 126.12, 126.24, 126.48, 128.35,

128.99, 129.38, 129.73, 134.39, 134.78, 139.14, 157.37, 163.56, 172.89.

4-Methyl-N-((S)-1-oxo-1-(((S)-1-oxo-4-phenylbutan-2-yl)amino)-

3-phenylpropan-2-yl)piperazine-1-carboxamide (11). The prepara-

tion of S1n, S1o, and 11 was similar to the procedures for S1d, S1f, and

S1g, respectively. ¹H NMR (400 MHz, CDCl₃) δ 1.82 (dt, J = 7.6, 14.6

Hz, 1H, piperazinyl CH₂), 2.09−2.17 (m, 1H, piperazinyl CH₂), 2.28

(s, 3H, NCH₃), 2.34 (dt, J = 3.8, 7.1 Hz, 4H, piperazinyl CH₂&

homoPhe βCH₂), 2.55 (dt, J = 7.9, 27.5 Hz, 2H, homoPhe γCH₂), 3.11

(p, J = 5.9, 6.5 Hz, 2H, Phe βCH₂), 3.29−3.39 (m, 4H, 2 × piperazinyl

CH₂), 4.33 (td, J = 5.3, 7.5 Hz, 1H, homoPhe αCH), 4.64 (q, J = 7.3 Hz,

1H, Phe αCH), 5.02 (d, J = 7.3 Hz, 1H, NH), 6.75 (d, J = 7.1 Hz, 1H,

NH), 7.08−7.13 (m, 2H, Ph), 7.18−7.25 (m, 5H, Ph), 7.27−7.31 (m,

1

MeOH in DCM) to give S1i (1.55 g, 60%). H NMR (400 MHz,

CDCl₃) δ 2.31 (s, 3H, NCH₃), 2.37 (t, J = 5.1 Hz, 4H, 2 × piperazinyl

CH₂), 3.14 (d, J = 4.2 Hz, 2H, βCH₂), 3.37 (q, J = 4.9 Hz, 4H, 2 ×

piperazinyl CH₂), 4.87 (d, J = 5.1 Hz, 2H, Cbz CH₂), 5.05−5.28 (m,

2H, αCH & NH), 7.02 (dd, J = 2.9, 6.5 Hz, 2H, Ph), 7.20−7.27 (m, 3H,

Ph), 7.30−7.42 (m, 5H, Ph). ¹³C NMR (100 MHz, CDCl₃) δ 38.31,

43.69, 46.09, 54.32, 54.59, 67.11, 126.93, 128.44, 128.53, 128.57,

129.38, 135.26, 136.11, 156.46, 172.45. LC−MS: t_R= 3.42 min;

C₂₂H₂₈N₃O₃⁺[M + H]⁺, m/z calcd 382.21, found 382.2.

+

3H, Ph), 9.43 (s, 1H, CHO). C₂₅H₃₃N₄O₃[M + H]⁺, m/z calcd

437.25, found 481.21.

(3S)-3-((S)-2-(4-methylpiperazine-1-carboxamido)-3-

phenylpropanamido)chroman-2-yl acetate (S2a, 13, R₁= Me). To a

solution of compound 12 (50 mg, 0.114 mmol, 1.0 eq) in DCM (3 mL)

were added acetic anhydride (32 μL, 0.342 mmol, 3.0 eq), Et₃N (48 μL,

0.342 mmol, 3.0 eq), and DMAP (2.8 mg, 0.023 mmol, 0.2 eq). The

resulting mixture was stirred at room temperature overnight and

concentrated under reduced pressure. The residue was diluted with

EtOAc (50 mL) and washed successively with saturated aqueous

NH₄Cl (10 mL), saturated aqueous NaHCO₃(10 mL), and brine (10

mL). The organic layer was dried over Na₂SO₄, ﬁltered, and

concentrated under reduced pressure. The obtained crude product

was puriﬁed by FCC (10% MeOH in DCM) to give S2a (R₁= Me, i.e.,

compound 13, 31 mg, 57%). ¹H NMR (400 MHz, CDCl₃) δ 2.01 (d, J =

23.4 Hz, 3H, CH₃CO), 2.29 (d, J = 3.5 Hz, 3H, NCH₃), 2.30−2.38 (m,

4H, 2 × piperazinyl CH₂), 2.69−2.97 (m, 2H, lactol βCH₂), 2.98−3.15

(m, 2H, Phe βCH₂), 3.25−3.41 (m, 4H, 2 × piperazinyl CH₂), 4.49 (dt,

J = 6.9, 13.8 Hz, 1H, αCH), 5.18 (dd, J = 7.4, 40.0 Hz, 1H, Phe αCH),

6.12 (dd, J = 2.5, 17.4 Hz, 1H, lactol CH), 6.31−6.54 (m, 1H, NH),

6.82−6.91 (m, 1H, Ph), 6.91−7.07 (m, 2H, Ph), 7.07−7.19 (m, 2H,

Ph), 7.19 (s, 3H, Ph & NH), 7.26−7.34 (m, 2H, Ph). ¹³C NMR (100

MHz, CDCl₃) δ 20.99, 26.61, 29.67, 38.69, 43.71, 45.99, 54.48, 56.23,

88.88, 89.33, 117.07, 119.40, 121.89, 127.09, 128.06, 128.70, 128.74,

129.15, 129.20, 136.94, 150.22, 157.02, 169.38, 171.96. LC−MS: t_R=

3.29 min; C₂₆H₃₃N₄O₅⁺[M + H]⁺, m/z calcd 481.24, found 481.3.

N-((2S)-1-(((3S)-2-ethoxychroman-3-yl)amino)-1-oxo-3-phenyl-

propan-2-yl)-4-methylpiperazine-1-carboxamide (S2b, 16, R₂= Et).

To a solution of compound 12 (40 mg, 0.091 mmol, 1.0 eq) in EtOH (2

mL) was added BF₃OEt₂(300 μL, 2.43 mmol, >20 eq) dropwise at 0

°C. The resulting mixture was stirred at room temperature overnight

and quenched by addition of saturated aqueous NH₄Cl (2 mL). The

reaction mixture was concentrated under reduced pressure and then

partitioned between DCM (20 mL) and water (20 mL). The water

layer was further washed with DCM (2 × 20 mL). The combined

organic layers were dried over Na₂SO₄, ﬁltered, and concentrated under

reduced pressure. The obtained crude product was puriﬁed by FCC

(8% MeOH in DCM) to give S2b (R₂= Et, i.e., compound 16, 18.3 mg,

43%). ¹H NMR (400 MHz, CDCl₃) δ 1.00 (t, 3H, OCH₂CH₃), 2.16−

2.36 (m, 7H, NCH₃& 2 × piperazinyl CH₂), 2.69 (dd, J = 9.2, 14.2 Hz,

1H, βCH₂), 2.83−2.95 (m, 1H, βCH₂), 3.10 (dt, J = 7.2, 13.4 Hz, 1H,

Phe βCH₂), 3.21 (dd, J = 4.0, 6.1 Hz, 1H, Phe βCH₂), 3.31 (dt, J = 6.0,

11.1 Hz, 3H, OCH₂CH₃& piperazinyl CH₂), 3.58−3.76 (m, 1H,

piperazinyl CH₂), 4.13−4.27 (m, 1H, piperazinyl CH₂), 4.41 (q, J = 7.3

Hz, 1H, piperazinyl CH₂), 4.68 (dd, J = 2.3, 49.7 Hz, 1H, αCH), 4.90−

5.15 (m, 1H, Phe αCH), 5.78 (dd, J = 8.9, 24.8 Hz, 1H, lactol CH), 6.73

(d, J = 8.1 Hz, 1H, Ph), 6.80 (q, J = 6.8, 7.4 Hz, 1H, Ph), 6.90 (d, J = 7.2

Hz, 1H, Ph), 7.03 (t, J = 7.2 Hz, 1H, Ph), 7.07−7.30 (m, 5H, Ph). ¹³C

NMR (100 MHz, CDCl₃) δ 14.94, 14.98, 27.08, 39.59, 43.72, 45.10,

46.06, 54.58, 56.30, 64.01, 96.02, 96.34, 116.79, 120.48, 121.16, 126.99,

127.67, 128.65, 128.75, 129.20, 129.33, 136.84, 137.17, 150.58, 150.70,

(4-methylpiperazine-1-carbonyl)-L-phenylalanine (S1j). The

compound was prepared from S1i in a similar procedure as described

for S1b. The obtained crude product S1j was used without further

puriﬁcation.

(1aR,7aS)-1a,2,7,7a-tetrahydronaphtho[2,3-b]oxirene (S1k). To a

solution of 1,4-dihydronaphthalene (2.09 mg, 16.1 mmol, 1.0 eq) in

chloroform (40 mL) was slowly added 70% mCPBA (4.74 g, 19.3

mmol, 1.2 eq) at 0 °C. The resulting mixture was stirred at room

temperature overnight and quenched by addition of 2 M KOH (60

mL). The reaction mixture was concentrated under reduced pressure

and diluted with EtOAc (100 mL). The organic layer was washed with

brine (3 × 20 mL) and was dried over Na₂SO₄, ﬁltered, and

concentrated under reduced pressure. The obtained crude product

was puriﬁed by FCC (10% EtOAc in hexane) to give S1k (2.03 g, 86%)

¹H NMR (400 MHz, CDCl₃) δ 3.05 (d, J = 17.7 Hz, 2H, CH₂), 3.18 (d,

J = 16.8 Hz, 2H, CH₂), 3.29−3.36 (m, 2H, 2 × epoxide CH), 6.95 (dd, J

= 3.5, 5.6 Hz, 2H, Ph), 7.06 (dd, J = 3.4, 5.7 Hz, 2H, Ph). ¹³C NMR

(100 MHz, CDCl₃) δ 29.84, 51.71, 126.57, 129.33, 131.74.

(2R,3R)-3-azido-1,2,3,4-tetrahydronaphthalen-2-ol and (2S,3S)-

3-azido-1,2,3,4-tetrahydronaphthalen-2-ol (S1l). To a solution of

S1k (2.02 g, 13.8 mmol, 1.0 eq) in a mixture of MeOH (30 mL) and

H₂O (10 mL) were added NaN₃(1.8 g, 27.7 mmol, 2.0 eq) and NH₄Cl

(1.11 g, 20.8 mmol, 1.5 eq). The resulting mixture was heated to 60 °C

and stirred overnight. The reaction mixture was cooled to room

temperature, concentrated under reduced pressure, and diluted with

EtOAc (100 mL). The organic layer was washed with brine (3 × 20

mL) and was dried over Na₂SO₄, ﬁltered, and concentrated under

reduced pressure. The obtained crude product S1l (2.22 g, 85%) was

used without further puriﬁcation. ¹H NMR (400 MHz, CDCl₃) δ 2.77−

2.86 (m, 2H, CH₂), 3.17 (ddd, J = 2.9, 5.8, 16.5 Hz, 2H, CH₂), 3.66 (td,

1H, CH-N₃), 3.87 (td, J = 5.8, 9.4 Hz, 1H, CH−OH), 7.04−7.09 (m,

2H, Ph), 7.09−7.16 (m, 2H, Ph). ¹³C NMR (100 MHz, CDCl₃) δ

33.64, 36.57, 63.62, 70.47, 126.50, 126.68, 128.59, 128.99, 132.62,

133.41.

(2R,3R)-3-amino-1,2,3,4-tetrahydronaphthalen-2-ol and (2S,3S)-

3-amino-1,2,3,4-tetrahydronaphthalen-2-ol (S1m). The title com-

pound was prepared from S1l in a similar procedure as described for

S1b. The obtained crude product S1m was used without further

puriﬁcation.

N-((S)-1-(((2S,3S)-3-hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)-

amino)-1-oxo-3-phenylpropan-2-yl)-4-methylpiperazine-1-carbox-

amide and N-((S)-1-(((2R,3R)-3-hydroxy-1,2,3,4-tetrahydronaph-

thalen-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-4-methylpipera-

zine-1-carboxamide (3). The title compound was prepared from S1j

and S1m in a similar procedure as described for S1d. The obtained

crude product was puriﬁed by FCC (10% MeOH in DCM) to give

compound 3. ¹H NMR (400 MHz, DMSO-d₆) δ 2.17 (s, 3H), 2.20 (q, J

= 3.3 Hz, 3H) 2.61 (ddd, J = 6.7, 16.9, 27.1 Hz, 2H), 2.83 (dd, J = 10.2,

13.6 Hz, 1H), 2.95 (ddd, J = 4.7, 8.9, 12.8 Hz, 2H), 3.04−3.33 (m, 5H),

3.79 (td, J = 4.8, 6.8 Hz, 1H), 3.93 (p, J = 7.0 Hz, 1H), 4.33 (ddd, J = 4.6,

8.3, 10.1 Hz, 1H), 6.48 (d, J = 8.3 Hz, 1H), 7.05−7.10 (m, 3H), 7.13−

7.21 (m, 1H), 7.22−7.27 (m, 3H), 7.77 (d, J = 7.8 Hz, 1H), 8.15 (s,

+

156.56, 156.68, 171.26, 171.66. LC−MS: t_R= 3.53 min; C₂₆H₃₅N₄O₄

[M + H]⁺, m/z calcd 467.27, found 467.3.

(S)-N-(2-benzoylphenyl)-1-benzylpyrrolidine-2-carboxamide

(S3a). To a mixed solution of benzyl-L-proline (1.03 g, 5.0 mmol, 1.0

eq) and 1-methylimidazole (877 μL, 11.0 mmol, 2.2 eq) in DCM (10

O

https://doi.org/10.1021/acs.jmedchem.1c00628

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

mL) was added MsCl (387 μL, 5.0 mmol, 1.0 eq) dropwise at 0 °C. The

resulting mixture was stirred at room temperature for 10 min, and then

was added a solution of 2-aminobenzophenone (888 mg, 4.5 mmol, 0.9

eq) in DCM (10 mL). The reaction mixture was heated to 45 °C and

stirred overnight. The reaction mixture was quenched by addition of

NH₄Cl (15 mL) and concentrated under reduced pressure. The residue

was diluted with EtOAc (75 mL) and washed with saturated aqueous

NaHCO₃(15 mL) and brine (15 mL). The organic layer was dried over

Na₂SO₄, ﬁltered, and concentrated under reduced pressure. The

obtained crude product was puriﬁed by FCC (15−25% EtOAc in

(m, 2H, Ph), 6.96−7.22 (m, 9H, Ph), 7.28 (d, J = 7.6 Hz, 2H, Ph),

7.33−7.46 (m, 4H, Ph), 7.95−8.08 (m, 2H, Ph), 8.35 (dd, J = 1.1, 8.7

Hz, 1H, Ph). ¹³C NMR (100 MHz, CDCl₃) δ 22.35, 25.60, 28.10,

59.19, 63.38, 68.12, 69.68, 71.86, 112.85, 122.38, 123.33, 123.36,

124.10, 127.39, 127.74, 127.80, 128.06, 128.35, 128.51, 128.73, 128.88,

129.20, 129.27, 129.61, 130.06, 130.30, 130.32, 134.10, 134.82, 137.07,

145.11, 155.82, 158.83, 170.83, 175.71. LC−MS: t_R= 4.70 min;

C₄₀¹³CH₃₈N₃NiO₄⁺[M + H]⁺, m/z calcd 695.22, found 694.96.

(S)-2-amino-3-(2-benzyloxy)phenyl)propanoic-1-¹³C Acid (S3d).

A mixed solution of S3c (522 mg, 0.75 mmol, 1.0 eq) and 8-quinolinol

(272 mg, 1.88 mmol, 2.5 eq) in MeCN (10 mL) and H₂O (1 mL) was

heated to 40 °C and stirred overnight. The reaction mixture was ﬁltered,

and the ﬁltrate was concentrated. The residue was diluted with DCM

(30 mL) and washed with water (3 × 30 mL). The organic layer

containing the retrieved BPB was concentrated and saved for future use.

The combined water layers were concentrated to a small volume that

was subsequently puriﬁed by Prep-HPLC to give S3d (87 mg, 43%). ¹H

NMR (400 MHz, CDCl₃) δ 2.96 (ddd, J = 2.5, 9.4, 14.3 Hz, 1H, βCH₂),

3.49 (ddd, J = 2.9, 4.5, 14.3 Hz, 1H, βCH₂), 3.93 (dt, J = 4.7, 9.4 Hz, 1H,

αCH), 5.20 (s, 2H, OBn CH₂), 6.92 (td, J = 1.1, 7.4 Hz, 1H, Ph), 7.05

(d, J = 8.1 Hz, 1H, Ph), 7.24 (dd, J = 6.7, 8.2 Hz, 2H, Ph), 7.28−7.34

(m, 1H, Ph), 7.38 (dd, J = 6.6, 8.3 Hz, 2H, Ph), 7.45−7.53 (m, 2H, Ph).

¹³C NMR (100 MHz, CDCl₃) δ 33.52, 55.91, 71.50, 114.97, 120.74,

1

hexane) to give S3a (1.344 g, 70%). H NMR (400 MHz, CDCl₃) δ

1.72−1.85 (m, 2H, Pro γCH₂), 1.91−2.01 (m, 1H, Pro βCH₂), 2.17−

2.31 (m, 1H, Pro βCH₂), 2.40 (td, J = 6.8, 9.5 Hz, 1H, Pro δCH₂), 3.20

(ddd, J = 2.4, 6.4, 9.1 Hz, 1H, Pro δCH₂), 3.31 (dd, J = 4.8, 10.1 Hz, 1H,

Pro αCH), 3.58 (d, J = 12.9 Hz, 1H, Bn CH₂), 3.91 (d, J = 12.9 Hz, 1H,

Bn CH₂), 7.07 (td, J = 1.1, 7.6 Hz, 1H, Ph), 7.10−7.17 (m, 3H, Ph),

7.33−7.40 (m, 2H, Ph), 7.49 (qd, J = 6.5, 7.9 Hz, 4H, Ph), 7.56−7.61

(m, 1H, Ph), 7.73−7.83 (m, 2H, Ph), 8.57 (dd, J = 1.0, 8.4 Hz, 1H, Ph),

11.50 (s, 1H, NH). ¹³C NMR (100 MHz, CDCl₃) δ 24.18, 31.03, 53.89,

59.87, 68.32, 121.53, 122.19, 125.36, 127.06, 128.16, 128.31, 129.13,

130.10, 132.45, 132.54, 133.35, 138.15, 138.59, 139.22, 174.60, 197.99.

+

LC−MS: t_R= 3.50 min; C₂₅H₂₅N₂O₂[M + H]⁺, m/z calcd 385.19,

found 384.85.

(S,E)-2-(((2-(1-benzylpyrrolidine-2-carboxamido)phenyl)-

(phenyl)methylene)amino)acetate-1-¹³C in Complex with Ni(II)

(S3b). To a mixed suspension of S3a (860 mg, 2.24 mmol, 1.0 eq),

[1-¹³C] glycine (425 mg, 5.59 mmol, 2.5 eq), and Ni(NO₃)₂·6H₂O

(1.30 g, 4.47 mmol, 2.0 eq) in MeOH (7 mL) at 45 °C was added a

solution of ground KOH (752 mg, 13.4 mmol, 6.0 eq) in MeOH (3

mL). The resulting mixture was stirred at 60 °C for 1 h (note that

prolonged heating might lead to racemization).The reaction mixture

was neutralized by addition of acetic acid (800 μL) and diluted with

water to a volume of 50 mL. The reaction mixture was concentrated

under reduced pressure and extracted with DCM (75 mL). The organic

layer was dried over Na₂SO₄, ﬁltered, and concentrated under reduced

pressure. The obtained crude product was puriﬁed by FCC (20−30%

124.67, 127.65, 127.97, 128.07, 128.32, 130.64, 139.00, 157.44, 173.03.

LC−MS: t_R= 2.70 min; C₁₅¹³CH₁₈NO₃⁺[M + H]⁺, m/z calcd 273.13,

found 272.75.

(S)-3-(2-(benzyloxy)phenyl)-2-((tert-butoxycarbonyl)amino)-

propanoic-1-¹³C Acid (S3e). To a solution of S3d (87 mg, 0.32 mmol,

1.0 eq) in dioxane (5 mL) and H₂O (5 mL) were added Et₃N (134 μL,

0.96 mmol, 3.0 eq) and Boc anhydride (84 mg, 0.38 mmol, 1.2 eq) at 0

°C. The resulting mixture was stirred at room temperature for 2.5 h and

then concentrated under reduced pressure. The residue was acidiﬁed by

addition of 0.1 M HCl to pH 2−3 and then diluted with EtOAc (50

mL). The organic layer was washed with brine (3 × 10 mL), dried over

Na₂SO₄, ﬁltered, and concentrated under reduced pressure. The

obtained crude product S3e (113 mg, 94%) was used without further

puriﬁcation. ¹H NMR (400 MHz, CDCl₃) δ 1.28 (s, 9H, Boc), 2.74−

3.06 (m, 1H, βCH₂), 3.15 (d, J = 14.5 Hz, 1H, βCH₂), 4.33−4.55 (m,

1H, αCH), 5.00 (d, J = 6.7 Hz, 2H, OBn CH₂), 6.82 (d, J = 8.0 Hz, 2H,

Ph), 7.04−7.15 (m, 2H, Ph), 7.21 (t, J = 7.3 Hz, 1H, Ph), 7.28 (d, J =

14.8 Hz, 2H, Ph), 7.36 (d, J = 7.4 Hz, 2H, Ph), 9.86 (s, 1H, COOH).

¹³C NMR (100 MHz, CDCl₃) δ 28.30, 29.71, 32.44, 54.81, 70.26,

1

acetone in DCM) to give S3b (715 mg, 64%). H NMR (400 MHz,

CDCl₃) δ 2.07 (dddd, J = 2.4, 5.6, 8.5, 15.1 Hz, 1H, Pro γCH₂), 2.12−

2.21 (m, 1H, Pro γCH₂), 2.42 (dddd, J = 8.3, 9.5, 10.7, 13.4 Hz, 1H, Pro

βCH₂), 2.57 (dddd, J = 2.6, 6.3, 9.2, 14.4 Hz, 1H, Pro βCH₂), 3.25−

3.40 (m, 1H, Pro δCH₂), 3.46 (dd, J = 5.4, 10.7 Hz, 1H, Pro δCH₂),

3.61−3.74 (m, 3H, N-Bn CH₂& Pro αCH), 3.77 (dd, J = 5.5, 20.1 Hz,

1H, Gly CH₂), 4.48 (d, J = 12.7 Hz, 1H, Gly CH₂), 6.69 (ddd, J = 1.2,

6.9, 8.1 Hz, 1H, Ph), 6.80 (dd, J = 1.7, 8.3 Hz, 1H, Ph), 6.92−7.04 (m,

1H, Ph), 7.05−7.13 (m, 1H, Ph), 7.20 (ddd, J = 1.7, 6.9, 8.7 Hz, 1H,

Ph), 7.28−7.33 (m, 1H, Ph), 7.42 (t, J = 7.6 Hz, 2H, Ph), 7.52 (ddd, J =

3.4, 7.7, 18.9 Hz, 3H, Ph), 8.01−8.12 (m, 2H, Ph), 8.31 (dd, J = 1.1, 8.6

Hz, 1H, Ph). ¹³C NMR (100 MHz, CDCl₃) δ 23.69, 30.76, 57.56,

61.57, 63.16, 69.94, 124.26, 125.16, 126.27, 128.91, 129.11, 129.34,

129.59, 131.73, 132.22, 132.77, 133.17, 133.37, 134.67, 142.62, 143.81,

177.21, 179.27. LC−MS: t_R= 3.71 min; C₂₆¹³CH₂₆N₃NiO₃⁺[M + H]⁺,

m/z calcd 499.14, found 498.83.

111.95, 121.12, 127.22, 127.93, 128.46, 128.64, 131.40, 176.93. LC−

MS: t_R= 4.28 min; C₂₀¹³CH₂₅NNaO₅⁺[M + Na]⁺, m/z calcd 395.17,

+

found 394.83; C₁₅¹³CH₁₈NO₃[M + H - Boc]⁺, m/z calcd 273.13,

found 272.82.

Tert-Butyl (S)-(3-(2-(benzyloxy)phenyl)-1-(methoxy(methyl)-

amino)-1-oxopropan-2-yl-1-¹³C)carbamate (S3f). The title com-

pound was prepared from S3e (113 mg, 0.30 mmol, 1.0 eq) and N,O-

dimethylhydroxylamine hydrochloride (44.4 mg, 0.45 mmol, 1.5 eq) in

a similar procedure as described for S1d. The obtained crude product

was puriﬁed by FCC (25% EtOAc in hexane) to give S3f (136 mg,

100%). ¹H NMR (400 MHz, CDCl₃) δ 1.25 (s, 9H, Boc), 2.79−2.96

(m, 2H, o-Tyr βCH₂), 2.99 (d, J = 5.6 Hz, 3H, OCH₃), 3.44 (s, 3H,

NCH₃), 4.90 (s, 1H, αCH), 4.99 (d, J = 4.1 Hz, 2H, OBn CH₂), 5.21 (d,

J = 8.9 Hz, 1H, NH), 6.79 (t, J = 7.7 Hz, 2H, Ph), 7.00−7.14 (m, 2H,

Ph), 7.18−7.32 (m, 3H, Ph), 7.38 (d, J = 7.5 Hz, 2H, Ph). ¹³C NMR

(100 MHz, CDCl₃) δ 28.30, 29.66, 32.03, 45.85, 61.26, 70.23, 79.09,

111.68, 120.70, 125.50, 127.52, 127.87, 128.15, 128.54, 131.42, 137.08,

(S)-3-(2-(benzyloxy)phenyl)-2-(((E)-(2-((S)-1-benzylpyrrolidine-2-

carboxamido)phenyl)(phenyl)methylene)amino)propanoate-1-¹³C

in Complex with Ni(II) (S3c). A mixed slurry of S3b (715 mg, 1.43

mmol, 1.0 eq), (2-(benzyloxy)phenyl)methanol (613 mg, 2.86 mmol,

2.0 eq), and CMBP (750 μL, 2.86 mmol, 2.0 eq) in toluene (5 mL) was

heated to 120 °C and stirred overnight. The reaction mixture was

cooled to room temperature and concentrated under reduced pressure.

The obtained crude product was puriﬁed by FCC (10% acetone in

+

155.20, 157.10, 172.86. LC−MS: t_R= 4.60 min; C₂₂¹³CH₃₀N₂NaO₅

1

[M + Na]⁺, m/z calcd 438.21, found 437.81; C₁₇¹³CH₂₂N₂O₅⁺[M + H -

Boc]⁺, m/z calcd 316.17, found 315.73.

DCM) to give S3c (417 mg, 40%). H NMR (400 MHz, CDCl₃) δ

1.48−1.59 (m, 1H, Pro γCH₂), 1.99 (ddd, J = 6.5, 8.4, 11.1 Hz, 1H, Pro

δCH₂), 2.04−2.14 (m, 1H, Pro γCH₂), 2.31 (tdd, J = 2.9, 6.7, 9.7 Hz,

2H, Pro βCH₂), 2.76 (ddd, J = 1.6, 4.4, 13.6 Hz, 1H, o-Tyr βCH₂),

2.90−3.05 (m, 1H, Pro δCH₂), 3.22 (ddd, J = 5.0, 7.2, 13.6 Hz, 1H, o-

Tyr βCH₂), 3.33 (dd, J = 8.0, 9.3 Hz, 1H, Pro αH), 3.44 (d, J = 12.6 Hz,

1H, N-Bn CH₂), 4.18 (q, J = 4.4 Hz, 1H, o-Tyr αCH), 4.23 (d, J = 12.6

Hz, 1H, N-Bn CH₂), 4.55 (d, J = 10.8 Hz, 1H, OBn CH₂), 4.90 (d, J =

10.8 Hz, 1H, OBn CH₂), 6.14 (dt, J = 1.5, 7.7 Hz, 1H, Ph), 6.41 (dd, J =

1.7, 8.2 Hz, 1H, Ph), 6.63 (ddd, J = 1.2, 6.9, 8.2 Hz, 1H, Ph), 6.83−6.94

N-((S)-1-(((S)-3-(2-(benzyloxy)phenyl)-1-(methoxy(methyl)-

amino)-1-oxopropan-2-yl-1-¹³C)amino)-1-oxo-3-phenylpropan-2-

yl)-4-methylpiperazine-1-carboxamide (S3g). The Boc group of S3f

(136 mg, 0.33 mmol, 1.0 eq) was ﬁrst deprotected in a similar

procedure as described for S1c. Subsequently, the intermediate product

was coupled to NMePip-Phe-OH (114 mg, 0.39 mmol, 1.2 eq) in a

similar procedure as described for S1d. The obtained crude product was

puriﬁed by FCC (8% MeOH in DCM) to give S3g (107 mg, 55%).¹H

P

https://doi.org/10.1021/acs.jmedchem.1c00628

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

NMR (400 MHz, CDCl₃) δ 2.27 (s, 3H, piperazinyl NCH₃), 2.30 (dt, J

= 3.7, 6.5 Hz, 4H, 2 × piperazinyl CH₂), 2.96 (dd, J = 2.9, 6.3 Hz, 3H,

OCH₃), 3.07 (s, 4H, 2 x βCH₂), 3.28 (ddd, J = 7.7, 13.5, 17.9 Hz, 4H, 2

× piperazinyl CH₂), 3.48 (s, 3H, NCH₃), 4.49 (q, J = 6.5 Hz, 1H, αCH),

4.99 (d, J = 7.0 Hz, 1H, αCH), 5.05 (s, 2H, OBn CH₂), 5.19 (s, 1H,

NH), 6.62 (s, 1H, NH), 6.79 (t, J = 7.3 Hz, 1H, Ph), 6.90 (dd, J = 7.3,

19.1 Hz, 2H, Ph), 7.12−7.25 (m, 6H, Ph), 7.30 (t, J = 7.3 Hz, 1H, Ph),

7.37 (t, J = 7.4 Hz, 2H, Ph), 7.46 (d, J = 7.2 Hz, 2H, Ph). ¹³C NMR (100

MHz, CDCl₃) δ 32.03, 32.92, 38.74, 43.62, 46.03, 49.23, 54.53, 55.10,

61.22, 70.22, 111.82, 120.68, 125.09, 126.70, 127.57, 127.97, 128.30,

128.36, 128.59, 129.68, 131.43, 136.95, 137.02, 156.53, 156.99, 171.19,

171.76. LC−MS: t_R= 3.72 min; C₃₂¹³CH₄₂N₅O₅⁺[M + H]⁺, m/z calcd

589.32, found 589.02.

modiﬁed from our lab protocol and described brieﬂy as follows.³⁵All

enzyme assays were conducted in 96-well, clear bottom, black plates at

25 °C, and ﬂuorescence emanating from product formation was

measured using either a SpectraMax M2 (Molecular Devices) or a

Synergy HTX (Biotek) microplate reader, with excitation at λ_ex= 360

nm and emission at λ_em= 460 nm. For evaluation of inhibitors that

exhibited time-dependent time courses (“burst kinetics),” variable

concentrations of inhibitor and a ﬁxed concentration of substrate

[typically, for cruzain: 10 μM Cbz-Phe-Arg-AMC (K_m= 0.9 μM), for

3CL^pro: 40 μM ACC-SAVLQSGFRK(DNP)-NH₂(K_m= 26 μM)] were

ﬁrst added into assay buﬀer [for cruzain: 50 mM MES (pH 7.5), 50 mM

TAPSO, 1 mM CHAPS, 1 mM Na₂EDTA, 5 mM DTT, and 10%

DMSO (v/v); for 3CL^pro: 20 mM Tris·HCl (pH 7.5), 150 mM NaCl,

0.1 mM EDTA-Na₂, 2 mM DTT, and 10% DMSO (v/v)]. Reactions

were started with addition of enzyme (typically, to a ﬁnal concentration

of 0.2 nM for cruzain or 40 nM for 3CL^pro), and the time courses were

recorded for 0.5−1 h. In a rapid dilution assay, 100X concentrations of

the enzyme and inhibitor were preincubated for 1−2 h and then diluted

by 100-fold into assay buﬀer containing standard concentrations of the

substrates followed by data collection.

In Vitro Cell Assays. BSF T. b. brucei strain SM427 was grown in

HM1−9 medium that included fetal bovine serum (10%) and

antibiotics (100 U/mL penicillin−streptomycin, Gibco). Inhibitors

dissolved in 100% (v/v) DMSO stock solutions were added at ﬁnal

concentrations of 0.5−20 μM (ﬁnal concentrations of DMSO were

<1% (v/v)). T. b. brucei were seeded at ∼1 × 10⁶cells at 37 °C and

diluted daily to maintain a log phase of growth for up to 96 h. Treated

cells were typically grown for 4 days. After each dilution, the cell

cultures were supplemented with inhibitor or DMSO (control) to keep

concentrations constant. Cell density was determined and graphed as

previously reported.³⁶Trypanosomes were quantiﬁed using a Z2

Coulter Counter, and values of EC₅₀were determined after 3−4 days of

treatment by the interpolation method of Huber and Koella.³⁷

T. cruzi infection was evaluated in C2C12 murine cardiomyoblast

cells. Three hundred and eighty four-microwell plates containing

C2C12 murine cardiomyoblast cells were infected with 10³and 10⁴

cells of T. cruzi (Ca-I/72), to which was added 3-fold serial dilutions of

inhibitor with maximal concentrations of 40 μM followed by incubation

at 37 °C for 2 days. The wells were ﬁxed with 2% PFA in PBS and

stained by 5 μg/mL of DAPI. After >30 min of incubation in a dark

environment, the plates were read on an ImageXpress MicroXL

microscope (Molecular Devices). The images were analyzed to quantify

parasites as well host cells.

N-((S)-1-(((S)-1-(2-(benzyloxy)phenyl)-3-oxopropan-2-yl-3-¹³C)-

amino)-1-oxo-3-phenylpropan-2-yl)-4-methylpiperazine-1-carbox-

amide (S3h). To a solution of S3g (107 mg, 0.182 mmol, 1.0 eq) in

THF (8 mL) at −10 °C was added 2.0 M LAH in THF (218 μL, 0.218

mmol, 1.2 eq) dropwise. The resulting mixture was stirred at 0 °C for 1

h and then quenched using tiny pieces of ice. The reaction mixture was

concentrated under reduced pressure and diluted with EtOAc (50 mL).

The organic layer was washed successively with a saturated solution of

Rochelle salt (15 mL), NaHCO₃(15 mL), and brine (15 mL), dried

over Na₂SO₄, ﬁltered, and concentrated under reduced pressure. The

obtained crude product was puriﬁed by FCC (10% MeOH in DCM) to

give S3h (81 mg, 84%). ¹H NMR (400 MHz, CDCl₃) δ 2.28 (s, 3H,

piperazinyl NCH₃), 2.31 (t, J = 4.2 Hz, 4H, 2 × piperazinyl CH₂), 2.95−

3.13 (m, 4H, 2 × βCH₂), 3.29 (dt, J = 5.2, 9.7 Hz, 4H, 2 × piperazinyl

CH₂), 4.52 (m, 2H, αCH & NH), 4.95 (d, J = 7.3 Hz, 1H, αCH), 5.04

(dd, J = 3.6, 10.5 Hz, 2H, OBn CH₂), 6.82 (s, 3H, Ph & NH), 7.10−

7.24 (m, 6H, Ph), 7.29−7.50 (m, 6H, Ph), 9.36 (s, 1H, CHO). ¹³C

NMR (100 MHz, CDCl₃) δ 29.66, 38.59, 43.67, 46.04, 54.52, 55.31,

59.49, 70.25, 97.62, 111.96, 121.04, 126.93, 127.55, 128.19, 128.55,

128.60, 128.73, 129.33, 129.43, 131.61, 136.52, 136.76, 156.39, 156.60,

+

171.89, 198.37. LC−MS: t_R= 3.76 min; C₃₀¹³CH₃₆N₄O₄[M + H]⁺,

m/z calcd 530.28, found 530.03.

N-((2S)-1-(((3S)-2-hydroxychroman-3-yl-2-¹³C)amino)-1-oxo-3-

phenylpropan-2-yl)-4-methylpiperazine-1-carboxamide (¹³C-la-

beled 12). The title compound was prepared from S3h (81 mg,

0.152 mmol, 1.0 eq) in a similar procedure as described for S1b. The

obtained crude product was puriﬁed by Prep-HPLC to give ¹³C-labeled

compound 12 (45 mg, 67%). ¹H NMR (400 MHz, CDCl₃) δ 1.28 (s,

1H, lactol OH), 2.45 (s, 3H, NCH₃), 2.65 (p, J = 6.5 Hz, 4H, 2 ×

piperazinyl CH₂), 3.03 (dt, J = 7.6, 21.2 Hz, 2H, o-Tyr βCH₂), 3.40−

3.60 (m, 4H, 2 x piperazinyl CH₂), 4.21−4.40 (m, 1H, Phe βCH₂), 4.64

(dq, J = 7.7, 46.1 Hz, 1H, Phe βCH₂), 5.38 (dd, J = 30.9, 170.9 Hz, 1H,

o-Tyr αCH), 6.16 (d, J = 8.0 Hz, 1H, Phe αCH), 6.50 (dd, J = 8.0, 141.5

Hz, 1H, lactol CH), 6.86−7.08 (m, 3H, Ph), 7.13 (dd, J = 3.0, 6.9 Hz,

1H, Ph), 7.20 (dt, J = 2.3, 6.5 Hz, 2H, Ph), 7.25 (d, J = 6.7 Hz, 2H, Ph),

7.71 (s, 2H, Ph & NH), 8.27 (s, 1H, NH). ¹³C NMR (100 MHz, CDCl₃,

scanned for only a few times) δ 91.69, 92.25. LC−MS: t_R= 2.77 min;

C₂₃¹³CH₃₁N₄O₄⁺[M + H]⁺, m/z calcd 440.24, found 439.94.

Evaluation of the anti-CoV-2 activity of compound 18 using human

A549 cells transduced with the ACE2 viral receptor (A549/ACE2) was

as described in our recent publication.⁸

Evaluation of SMAI Prodrugs. Esterase from porcine liver was

obtained as an ammonium sulfate suspension (Sigma-Aldrich, E2884,

10 U/μL). Compounds 13−15 in 100% (v/v) DMSO solutions were

diluted by 10-fold into cruzain assay buﬀer to make 1 mL samples, each

containing 2 mM compound. For the experimental sample, 4 μL of

esterase suspension was added to the reaction mixtures (40 U/mL),

which was immediately placed on a low-speed orbital shaker in a 37 °C

incubator. In the meantime, a negative control (lacking esterase) and a

blank control (buﬀer only) were included. Aliquots (140 μL) of the

reaction mixtures were removed at 0, 15, 30, 60, 120, 180, and 300 min,

and reaction mixtures were quenched by the addition of MeCN (260

μL). The resulting solution was vortexed followed by centrifugation to

precipitate the protein, and the resulting compounds in the supernatant

were analyzed by HPLC−MS with detection at 270 nm.

Commercially available CYPs 3A4, 2D6, and 2C9 (CypExpress;

Sigma-Aldrich, MTOXCE3A4, MTOXCE3D6, and MTOXCE2C9)

containing glucose-6-phosphate dehydrogenase (G6PDH) and Mg²⁺

were used to evaluate the stability of acetal prodrugs to these oxidases.

The following components were combined in 800 μL reaction

mixtures: 0.5 mM 16 or 17, 5 mM sodium glucose 6-phosphate, 2

mM NADP⁺, cruzain assay buﬀer (pH 7.5), and 2.5% DMSO (v/v). For

the experimental samples, 64 mg of CypExpress P450 as a lyophilized

powder was added to the reaction vial (80 mg/mL), which was gently

shaken to dissolve the solid, and the mixtures were stirred at 30 °C in an

In Scheme 4, the preparation of S4a and S4b was similar to the

procedures for S1a and S1b, respectively; the preparation of S4d, S4e,

and S4f was similar to the procedures for S3f, S3g, and S3h, respectively.

Therefore, only the preparation of S4c was described in detail as below.

2-((tert-butoxycarbonyl)amino)-3-(2-oxo-1,2-dihydropyridin-3-

yl)propanoic Acid (S4c). A mixed solution of S4b (1.18 g, 4.0 mmol,

1.0 eq) and LiOH (192 mg, 8.0 mmol, 2.0 eq) in MeOH (10 mL) and

H₂O (5 mL) was stirred at room temperature for 2 h. The reaction

mixture was acidiﬁed by addition of 0.1 M HCl to pH 2−3 and then

concentrated under reduced pressure. The residue was extracted with

DCM (3 × 50 mL). The organic layers were combined, dried over

Na₂SO₄, ﬁltered, and concentrated under reduced pressure. The

obtained crude product S4c was used without further puriﬁcation. ¹H

NMR (400 MHz, CDCl₃) δ 1.39 (s, 9H, Boc), 2.86 (dd, J = 8.9, 14.0

Hz, 1H, βCH₂), 3.11 (dd, J = 4.3, 13.9 Hz, 1H, βCH₂), 4.42 (dd, J = 4.3,

8.8 Hz, 1H, αCH), 6.32 (t, J = 6.7 Hz, 1H, pyridine), 7.30 (dd, J = 2.0,

6.6 Hz, 1H, pyridine), 7.39−7.54 (m, 1H, pyridine).

Enzyme Assays. The preparation of cruzain and 3CL^prowas

described in previous publications.^8,34The evaluation of inhibitors was

Q

https://doi.org/10.1021/acs.jmedchem.1c00628

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

incubator. Sample vials were capped loosely with aluminum foil to allow

aeration as well as to reduce evaporation. Additionally, a negative

control lacking CypExpress P450 and a blank control containing only

buﬀer were included. Aliquots (65 μL) were removed from mixtures at

0, 30, 60, 120, 180, and 240 min, and reaction mixtures were quenched

with the addition of MeCN (65 μL). The resulting solution was

vortexed followed by centrifugation to precipitate the protein, and the

resulting compounds in the supernatant were analyzed by HPLC−MS

with detection at 270 nm.

2D NMR Study. Wilmad precision NMR tubes (541-PP-8, thin wall,

800 MHz, 5 mm × 8 inch) were used for this experiment. The total

volume of sample in each tube was 660 μL, and the ﬁnal concentrations

of the components in the NMR buﬀer were as follows: 18 mM

phosphate (K₂HPO₄/KH₂PO₄), 45 mM NaCl, 4.5 mM DTT, pH 7.5,

and 10% DMSO-d₆(v/v). A 10 mM stock of ¹³C-labeled 12 in DMSO-

d₆was diluted into the NMR buﬀer to speciﬁed concentrations. A

control sample contained 0.6 mM of ¹³C-labeled 12, and an

experimental sample contained 0.4 mM of ¹³C-labeled 12 and 0.44

mM of cruzain freshly puriﬁed by size exclusion chromatography,

directly after thawing from a −80 °C stock to minimize autoproteolysis.

Both samples were analyzed on a Bruker AVANCE III HD 800 MHz

(18.8 Tesla) equipped with a 5 mm triple resonance cryoprobe. ¹H-¹³C

HSQC NMR spectra were acquired at 25 °C with 72 scans for the

enzyme-free control sample and 128 scans for the experimental sample

containing cruzain. Spectral widths were obtained at 9615 and 28,179

Hz in the ¹³C and ¹H dimensions, respectively. Spectra were processed

using NMRpipe,³⁸and cross-peak intensities were analyzed using

Sparky³⁹and MestreNova.⁴⁰

reactive residue. The binding aﬃnity was scored, and the best three

poses were visually inspected.

Kinetic Data Analysis. All time-course data were ﬁtted to eq 1,

wherein P is the ﬂuorescence generated by formation of AMC or

liberated ACC, C is the background constant, v_iand v_sare, respectively,

initial-state and steady-state reaction rates, t is time, and k_obsis the

observed rate of conversion of the initial inhibited rate to the ﬁnal

inhibited rate.⁴⁶

i

j

y

z

v − v

k_obs

i

s

−k_obs

j

z

P = vt + j

z(1 − e

^t) + C

s

j

z

(1)

k

{

Values of k_obsvs [I] were then replotted and ﬁtted to eq 2, wherein k₃

and k₄represent the respective rates of formation and dissolution of the

EI* complex as depicted in Figure 2A.

k₃[I]

k_obs= k₄+

[S]

K_m

K_i1 +

+ [I]

(

)

(2)

As described in the main text, the linearity of k_obsversus [I] of

compounds 1 and 12 arises when K_i> > K_i*. Under these conditions,

the concentration of inhibitor required to observe time-dependent

inhibition would be much less than the value of K_ifor the EI complex. In

this case, eq 2 reduces to eq 3, which is a linear function with a slope =

k₄/K_i*^appand an y-intercept = k₄. For the rapid dilution assay, [I] was

ﬁxed at 0.1 × K_i*^app, and after dilution, k₄could be estimated as k_obs/1.1

according to eq 3.

Ä

É

Detection of Aldehyde Content Using Phenylhydrazine. In a

96-well clear microplate, compounds 11 and 12 in DMSO solutions, or

pure DMSO as a control, were diluted into the above NMR buﬀer

without addition of DTT to make 250 μL samples, each containing 0.2

mM of compounds and 10% DMSO (v/v). Upon addition of 1 M

phenylhydrazine (0.25 μL), the formation of phenylhydrazone in these

wells was continuously monitored for 2 h by measuring UV absorbance

at a maximal wavelength of 282 nm.

X-ray Crystallography. The procedure of crystallization for the

3CL^pro-18 complex is based on the previous publication.⁴¹The crystals

were obtained at 25 °C from 0.2 M ammonium phosphate dibasic, 17%

w/v PEG3350, pH 8.0, with a protein concentration of 14 mg/mL.

Soaking was performed to produce 3CL^pro-18 complex crystals. The

crystals were washed three times with reservoir solution plus 0.5 mM

inhibitor and 2% DMSO (Inhibitor was dissolved to 25 mM in 100%

DMSO). The mixture was incubated at 25 °C for 48 h. The cryo-

protectant solution contained mother liquor plus 30% glycerol, 0.5 mM

inhibitor, and 2% DMSO. Cryo-protected crystals were ﬁshed for data

collection.

Å

Ñ

Å

Ñ

Å

Ñ

Å

Ñ

[I]

Å

Ñ

k_obs= k 1 +

Å

4

Å

Ñ

[S]

*

K_i1 +

(

)

K_m

(3)

Å

Ç

Ñ

Ö

Overall inhibition constants (K_i* values) were obtained by ﬁtting v_s/

v₀to eq 4, wherein v_sand v₀are steady-state reaction rates in the

presence and absence of the inhibitor, respectively, [S] and [I]

represent the ﬁxed concentration of the substrate and variable inhibitor,

and K_mis the Michaelis constant of the substrate.

v

v₀

1

s

=

Ä

Å

É

Ñ

[S]

K_m

*

1 + [I]

K

1 +

i

(

)

Å

Ñ

(4)

Å

Ç

Ñ

Ö

As a tight-binding inhibitor, the K_i* values of 1 for cruzain and 18 for

3CL^prowere obtained by ﬁtting v_i/v₀data to eq 5 in which v_iand v₀are

the initial rates obtained with or without the inhibitor, respectively, [E]_t,

[I]_t, [S], K_i*, and K_m, are, respectively, concentrations of enzyme,

inhibitor, and substrate, and K_i* and K_mare the inhibition and

Michaelis constants, respectively.⁴⁷

The 3CL^pro-18 data were collected at 1.70 Å on a Rigaku R-AXIS IV+

+ image plate detector. Reﬂections were indexed, integrated, scaled, and

merged to space group I 1 2 1 using iMosﬂm.⁴²The structure was

solved by molecular replacement using the Phaser module of the Phenix

package using a high-resolution structure of the apo SARS-CoV-2

3CL^pro(PDB accession code: 6Y2E) as the search model.^43,44The

atomic coordinates and geometric restraints for the inhibitor were

generated in the CCP4 suite.⁴²The inhibitor was built into the F_o−F_c

electron density omit maps using Coot.⁴⁵Structural reﬁnement was

performed with the Real-space Reﬁnement module of the Phenix

package. Crystallographic data were summarized in Table S4. All

structural ﬁgures were generated with PyMOL (https://www.pymol.

org).

l

o

i

j

y

z

v

v₀

1

2[E]_t

[S]

i

*

=

m[E]_t− [I]_t− K_ij1 +

z

j

o

K_m

k

{

o

n

|

o

2

i

y

i

j

y

j

[S] z

z

*

i

+

[E] + [I] + K j1 +

− 4[E]_t[I]_t}

t

j

o

K_m

k

{

k

{

(5)

~

Rates of deacylation reactions of SMAI prodrugs 13−15 were

obtained upon evaluation of the ﬁrst-order loss of reagent as ﬁtted to eq

6 in which A₀and C are the initial and ﬁnal fraction of the unhydrolyzed

compound, respectively, and k is the pseudo-ﬁrst-order rate constant

from which the half-life was t_1/2= ln(2)/k.

Molecular Modeling. Docking studies of compound 2 binding to

̈

cruzain were carried out using the Schrodinger Suite program package.

The receptor ﬁle (PDB accession code: 2OZ2) was processed using the

Protein Preparation Wizard function, and the compound was

energetically minimized by the LigPrep function. Noncovalent docking

was executed and scored by the Glide module. For covalent docking, a

cubic box of 18 Å³was set to encompass any simulated poses with K777

as a reference ligand at its centroid. The SMARTS pattern for 2 was

manually deﬁned as [C;H1] = [O], and Cys₂₅was checked as the

A = (A₀− C)e^−kt+ C

PDB ID Code. The cocrystal structure of 3CL^pro-18 is deposited in

Protein Data Bank under the ID code 7M2P. Authors will release the

atomic coordinates and experimental data upon article publication.

(6)

R

https://doi.org/10.1021/acs.jmedchem.1c00628

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Andrew Rademacher − Department of Biochemistry and

Biophysics, Texas A&M University, College Station, Texas

77843, United States

Diane Thomas − Skaggs School of Pharmacy and

Pharmaceutical Sciences, University of California San Diego,

La Jolla, California 92093, United States

Lauren R. Blankenship − Department of Chemistry, Texas

A&M University, College Station, Texas 77843, United States

Aleksandra Drelich − Department of Microbiology and

Immunology, University of Texas Medical Branch, Galveston,

Texas 77555, United States

Arthur Laganowsky − Department of Chemistry, Texas A&M

University, College Station, Texas 77843, United States;

orcid.org/0000-0001-5012-5547

ASSOCIATED CONTENT

■

sı

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00628.

Kinetic parameters obtained from rapid dilution assays,

details of peaks in ¹H-¹³C HSQC NMR, statistic summary

of the cocrystal structure of 3CL^pro-18, LC-MS of ¹³C-

labeled 12, kinetic analysis of cruzain inhibition by 11 and

13, growth curves for T. b. brucei BSFs, cell viability of T.

cruzi-infected murine cardiomyoblasts, and NMR spectra

of the synthesized compounds (PDF)

Molecular formula strings (CSV)

Model coordinates for 2 bound to cruzain in its free

aldehyde form (PDB) and its lactol form (PDB)

Chien-Te K. Tseng − Department of Microbiology and

Immunology, University of Texas Medical Branch, Galveston,

Texas 77555, United States

Wenshe R. Liu − Department of Biochemistry and Biophysics,

Texas A&M University, College Station, Texas 77843, United

States; Department of Chemistry, Texas A&M University,

College Station, Texas 77843, United States

A. Joshua Wand − Department of Biochemistry and Biophysics,

Texas A&M University, College Station, Texas 77843, United

States; orcid.org/0000-0001-8341-0782

AUTHOR INFORMATION

■

Corresponding Author

Thomas D. Meek − Department of Biochemistry and

Biophysics, Texas A&M University, College Station, Texas

77843, United States; orcid.org/0000-0002-1931-8073;

Phone: +1-979-458-9789; Email: tdmeek@tamu.edu

Jorge Cruz-Reyes − Department of Biochemistry and

Biophysics, Texas A&M University, College Station, Texas

77843, United States

Jair L. Siqueira-Neto − Skaggs School of Pharmacy and

Pharmaceutical Sciences, University of California San Diego,

La Jolla, California 92093, United States; orcid.org/0000-

0001-9574-8174

Authors

Linfeng Li − Department of Biochemistry and Biophysics, Texas

A&M University, College Station, Texas 77843, United

States; orcid.org/0000-0001-6185-4999

Bala C. Chenna − Department of Biochemistry and Biophysics,

Texas A&M University, College Station, Texas 77843, United

States; orcid.org/0000-0002-2660-5302

Kai S. Yang − Department of Chemistry, Texas A&M

University, College Station, Texas 77843, United States;

orcid.org/0000-0002-1890-4169

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.1c00628

Taylor R. Cole − Department of Biochemistry and Biophysics,

Texas A&M University, College Station, Texas 77843, United

States

Notes

The authors declare no competing ﬁnancial interest.

Zachary T. Goodall − Department of Biochemistry and

Biophysics, Texas A&M University, College Station, Texas

77843, United States

Miriam Giardini − Skaggs School of Pharmacy and

Pharmaceutical Sciences, University of California San Diego,

La Jolla, California 92093, United States

Zahra Moghadamchargari − Department of Chemistry, Texas

A&M University, College Station, Texas 77843, United States

Elizabeth A. Hernandez − Department of Biochemistry and

Biophysics, Texas A&M University, College Station, Texas

77843, United States

Jana Gomez − Department of Biochemistry and Biophysics,

Texas A&M University, College Station, Texas 77843, United

States

Claudia M. Calvet − Skaggs School of Pharmacy and

Pharmaceutical Sciences, University of California San Diego,

La Jolla, California 92093, United States

Jean A. Bernatchez − Skaggs School of Pharmacy and

Pharmaceutical Sciences, University of California San Diego,

La Jolla, California 92093, United States; orcid.org/0000-

0002-8309-1627

Drake M. Mellott − Department of Biochemistry and

Biophysics, Texas A&M University, College Station, Texas

77843, United States; orcid.org/0000-0002-8043-8170

Jiyun Zhu − Department of Biochemistry and Biophysics, Texas

A&M University, College Station, Texas 77843, United States

ACKNOWLEDGMENTS

■

Financial support for the research was provided by NIH grants

R21 AI127634, R01 GM129076, and Texas A&M AgriLife

Research.

ABBREVIATIONS

■

ACC, 7-amino-4-carbamoylmethylcoumarin; AMC, 7-amino-4-

methylcoumarin; BSF, bloodstream form; CES, carboxylester-

ase; CHAPS, 3-[3-(cholamidopropyl)dimethylammonio]-1-

propanesulfonate; CMBP, cyanomethylenetributylphosphor-

ane; DIPEA, N,N-diisopropylethylamine; ER, endoplasmic

reticulum; FCC, ﬂash column chromatography; hPhe, homo-

phenylalanyl; MES, 2-(N-morpholino)ethanesulfonic acid;

NMePip, N-methlypiperazinyl; SMAI, self-masked aldehyde

inhibitor; T3P, propylphosphonic anhydride; TAPSO, 3-[N-

tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic

acid

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