Identification of First-in-Class Inhibitors of Kallikrein-Related Peptidase 6 That Promote Oligodendrocyte Differentiation

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Identiﬁcation of First-in-Class Inhibitors of Kallikrein-Related

Peptidase 6 That Promote Oligodendrocyte Diﬀerentiation

Sabrina A ï t Amiri, Cyrille Deboux, Feryel Soualmia, Nancy Chaaya, Maxime Louet, Eric Duplus,

Sandrine Betuing, Brahim Nait Oumesmar,* Nicolas Masurier,* and Chahrazade El Amri *

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ABSTRACT: Multiple sclerosis (MS) is an autoimmune demyeli-

nating disease of the central nervous system (CNS) that causes

severe motor, sensory, and cognitive impairments. Kallikrein-

related peptidase (KLK)6 is the most abundant serine protease

secreted in the CNS, mainly by oligodendrocytes, the myelin-

producing cells of the CNS, and KLK6 is assumed to be a robust

biomarker of MS, since it is highly increased in the cerebrospinal

ﬂuid (CSF) of MS patients. Here, we report the design and

biological evaluation of KLK6’s low-molecular-weight inhibitors,

para-aminobenzyl derivatives. Interestingly, selected hit com-

pounds were selective of the KLK6 proteolytic network

encompassing KLK1 and plasmin that also participate in the

development of MS physiopathology. Moreover, hits were found noncytotoxic on primary cultures of murine neurons and

oligodendrocyte precursor cells (OPCs). Among them, two compounds (32 and 42) were shown to promote the diﬀerentiation of

OPCs into mature oligodendrocytes in vitro constituting thus emerging leads for the development of regenerative therapies.

inﬂammation and promoting myelin regeneration for more

complete health care of MS patients. There is thus an urgent

need to identify and validate novel targets to reach this issue.

In adults, under normal physiological conditions, kallikrein-

related peptidase 6 (KLK6) is strongly expressed by

oligodendrocytes and can be detected in neurons, whereas it

is very weakly expressed in astrocytes and resting microglia.⁹

However, in response to a CNS lesion such as glutamate-

induced excitotoxicity, inﬂammation, or traumatic injury, the

level of KLK6 expression rises in oligodendrocytes and

neurons, and its expression is induced in reactive astrocytes

and microglia.¹⁰Among the 15 tissue kallikreins, KLK6 is the

most abundant in the CNS, and is arguably the most abundant

serine proteases produced in the CNS.¹¹

INTRODUCTION

■

Multiple sclerosis (MS) is a chronic inﬂammatory demyelinat-

ing disease of the central nervous system (CNS). MS aﬀects

more than two million people worldwide.¹In young adults, MS

is the most common neurological disorder and the leading

cause of severe nontraumatic disability. The main target of MS

is the myelin sheath surrounding CNS axons in the brain,

spinal cord, and optic nerve.¹The myelin sheath is synthesized

by oligodendrocytes (OLs) that play a crucial role in the

maintenance and functioning of neuronal networks. MS is

associated with an inﬂammatory reaction of the CNS that

results in degradation of the myelin sheath, therefore leading to

diﬀuse demyelinating lesions. The consequences of this

demyelination are multiple: nerve conduction is impaired

and neurodegeneration occurs with disease progression.²

Moreover, myelin debris exacerbates the activation of immune

pathways triggering demyelination.³Clinically, these distur-

bances result in severe motor, sensory, and cognitive deﬁcits.

The lack of a regenerative therapy that prevents disease

progression represents the greatest unmet medical in MS.

Disease-modifying drugs in the market are mainly targeting the

inﬂammatory components of the pathology, but have little

impact on oligodendrocyte regeneration and remyelination.⁴⁻⁶

The identiﬁcation of new therapeutic compounds for MS-

enhancing myelin repair is thus a critical public health issue.^7,8

A special eﬀort may be put on the search for pharmacological

compounds, capable of simultaneously modulating neuro-

KLK6 has a versatile action within the CNS.⁹Interestingly,

KLK6 cleaves myelin proteins, such as the myelin basic protein

(MBP). KLK6 is a major protease of the process of

demyelination/remyelination of axons.^9,10,12In addition to

cleaving myelin proteins, KLK6 can also degrade blood−brain

barrier components such as laminin, ﬁbronectin, and collagen,

Received: December 16, 2020

Published: May 5, 2021

https://doi.org/10.1021/acs.jmedchem.0c02175

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Scheme 1. Putative KLK6’s Pathological Proteolytic Network

Chart 1. Main KLK6 Inhibitors Reported in the Literature

and therefore induce inﬂammation via protease-activated

receptors (PAR) activation and underlying intracellular Ca²⁺

mobilization. It should also be noted that the use of

recombinant KLK6 in vitro contributes to neuronal lesions

and oligodendrogliopathies.¹³Some proteases have also been

suggested for the activation of KLK6 in vivo, including plasmin,

a serine protease involved in ﬁbrinolysis. Finally, KLK6 is

involved in oligodendrocyte diﬀerentiation and myelination

processes. Diﬀerent studies support that KLK6 is involved in

the pathophysiology of MS.¹²⁻¹⁷First, patients with MS have

abnormally high KLK6 levels in serum, cerebrospinal ﬂuid

(CSF), and demyelinating lesions, which demonstrate the

implication of this protease at diﬀerent levels of the

pathology.¹⁸Second, KLK6 induces demyelination, through

both excessive cleavage of myelin proteins and deleterious

eﬀects on oligodendrocyte-process outgrowth and diﬀer-

entiation, thus leading to a drastic reduction in the number

of myelinating oligodendrocytes.¹⁹Third, KLK6 participates in

the neuroinﬂammatory process through the overactivation of

PAR receptors and also by increasing the expression of

proinﬂammatory cytokines. Therefore, KLK6 participates in

the establishment of a toxic environment that leads to

demyelination and neurodegeneration, the main causes of

disability progression in MS patients.¹⁹KLK6 and KLK1 may

also serve as serological markers of MS and may contribute

directly to neurodegeneration. Furthermore, the immunization

of mice with recombinant KLK6 antibodies signiﬁcantly

delayed onset and severity of clinical deﬁcits in an experimental

autoimmune encephalomyelitis (EAE) mouse model of

MS.^12,20It is also well established that the potential

signiﬁcance of proteolytic activity in MS not only relates to

their use as potential biomarkers but also as prospective

therapeutic targets. The range of potential involvement of

proteolytic activity in MS pathogenesis extends from

parenchymal degenerative events, including myelin destruction

and axon injury, to release of antigenic self-epitopes, immune

cell activation, and permeabilization of the blood−brain

barrier. Scarisbrick and co-workers have proposed the term

of MS degradome to encompass the set of proteases,

substrates, and endogenous protease inhibitors involved in

the development and progression of MS.¹⁹

Scheme 1 summarizes the central role of KLK6 and its

crosstalk with thrombolytic proteolytic pathways, thus

illustrating the early concept of MS degradome.²¹Particularly,

it has been recently shown that plasminogen and plasmin-

mediated ﬁbrinolysis are key modiﬁers of the onset of

neuroinﬂammatory demyelination.^22,23KLK1 was early

shown to be a potential serological marker of progressive MS

and contribute directly to the development of neurological

disability by promoting axonal injury and neuron apoptosis.¹⁹

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Figure 1. Rational design of para-amidobenzyl derivatives. (A) Key interactions of compound V in the active site of KLK6 (H bonds are shown as

dashed lines); (B) positioning of compound V in the S1 and S1′ pockets of KLK6; and (C) structures of studied compounds.

a

Scheme 2. Synthesis of Compounds 1−53

a

Reagents and conditions: (i) Boc₂O, triethylamine (TEA), dichloromethane (DCM), room temperature (RT), 2 h, 80%; (ii) H₂, 10% Pd/C,

AcOEt, RT, 4 h, qt; (iii) ArCO₂H, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDCI), tetrahydrofuran (THF), reﬂux, 12 h,

12−82%; (iv) (1) Compound 26, EtOH, reﬂux, 6 h; (2) NaBH₄, RT, 1.5 h, 64% and (v) 6 N HCl gas in 1,4-dioxane, RT, 1.5 h, 44−99%.

In this context, the development of innovative inhibitors of

KLK6 activity and its associated proteolytic network appears as

a new therapeutic avenue.

organic inhibitors for KLK6 and its proteolytic network. The

designed low-molecular-weight inhibitors are potent and

reversible toward KLK6, and their inhibitory potency was

also evaluated on a large panel of proteases. We also provided a

detailed structure−activity relationship (SAR) and dissected

out the chemical basis for optimal inhibition. Hit compounds

were found devoid of cytotoxic eﬀects toward both primary

cultures of mouse neurons and oligodendrocyte precursor cells

(OPCs) and displayed favorable druglike characteristics.

Interestingly, some hit compounds promoted OPC diﬀer-

entiation in vitro. These selected compounds constitute

promising leads for the development of innovative myelination

therapy.

The aim of this study is thus to identify potent KLK6

inhibitors encompassing the following properties: (i) reversi-

bility, indeed noncovalent inhibitors, which are devoid of a

reactive group, do not have the drawbacks generally associated

with the presence of a warhead, such as lack of speciﬁcity,

instability, and excessive reactivity. Considering the physio-

logical implication of KLK6 in the CNS homeostasis reversible

inhibition would be safer, and (ii) able to inhibit other

proteases involved in the KLK6 proteolysis network, i.e., KLK1

and plasmin. Herein, we identiﬁed and optimized original

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a

Table 1. Eﬃcacy of para-Aminobenzyl Compounds toward KLK6

a

For each compound, the inhibitory eﬀect was quantiﬁed either by the percentage inhibition of KLK6 at 10 μM or by the IC₅₀for compound hits.

To determine the IC₅₀, compounds at diﬀerent concentrations (concentration ranges adjusted depending on the inhibitory potency) are

preincubated for 15 min at 37 °C with KLK6 (2 nM). The enzymatic reaction is triggered by the addition of the Boc-QAR-AMC substrate (100

μM) in 50 mM Tris buﬀer, 1 M citrate 0.05% Brij-35; pH 7.4 at 37 °C. NA: not applicable. NI: noninhibitor. The data result from at least three

independent experiments in duplicate. The IC₅₀values were calculated by ﬁtting the experimental data to eq 2a or eq 2b and expressed as geometric

standard deviation.

A common approach for the development of inhibitors has

been to use natural peptide inhibitors as starting points,

exploiting combinatorial or designed mutations to achieve

selectivity toward the targeted KLK (e.g., compound I, Chart

1).^24,28However, the development of subtype-selective KLK

inhibitors remains challenging. Papo and collaborators

developed a yeast-displayed mutant library of the human

RESULTS AND DISCUSSION

■

Rational Design of para-Benzylamine Derivatives.

While the ﬁeld of KLK’s inhibitor discovery is relatively

underdeveloped despite very huge interest in several

therapeutic areas, recent reviews highlight the interest and

need for further works.²⁴⁻²⁸

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amyloid precursor protein Kunitz protease inhibitor (APPI)

domain to derive serine protease inhibitors especially of

KLK6.²⁹Recently, Miller and co-workers have reported

depsipeptides as potent KLK6’s inhibitors to derive activity-

based probes (compound II, Chart 1).³⁰Our team has also

recently identiﬁed 6-substituted coumarin-3-carboxylate de-

rivatives as mechanism-based inhibitors (suicide substrates,

compound III, Chart 1).³¹Several small molecules were also

reported as reversible inhibitors of KLK6. Among them,

benzamidine and benzylamine derivatives are probably the

most potent inhibitors (e.g., compounds IV and V, Chart

1).^32,33Especially, N-(4-aminomethyl-phenyl)-2-hydroxy-ben-

zamide derivatives reported by Liang and co-workers are easily

accessible and can be subjected to a large chemical

diversiﬁcation for structure−activity relationship (SAR)

studies. Moreover, these compounds can potentially provide

access to submicromolar inhibitors. In this study, nine

analogues were described and the 5-tert-butyl derivative

(compound V, Chart 1) revealed to be the most potent

inhibitor of this series (half-maximal inhibitory concentration

(IC₅₀) = 0.3 μM) constituting an initial hit.³³Substitution of

the methylene group of the benzylamine as well as alkylation at

the amino nitrogen led to inactive compounds, which clearly

demonstrates that the amino group of the benzylamine is

mandatory for the KLK6 inhibition. X-ray structure obtained

with one of the derivatives of this series revealed that the

benzylamine group bound into the S1 pocket of KLK6 by

sharing a primary amine H-bonding with the side chain of

N189. The side chain of I218 is also an important factor to the

binding aﬃnity through hydrophobic interaction with the

phenyl ring of the benzylamine moiety. The hydroxyl group of

the phenol occupied the center of the oxyanion hole, forming

an H-bonding network with the backbone NH groups of G193

and S195 (Figure 1A,B). Keeping constant these key elements

involved in KLK6 interaction (Figure 1C), we synthesized and

evaluated novel series of N-(4-aminomethyl-phenyl)-2-hy-

droxy-benzamide and hydroxynaphthamide derivatives and

identiﬁed druglike reversible organic inhibitors of KLK6 and its

associated proteolytic network.

KLK6 at a concentration of 10 μM and then determined their

inhibitory potency through the quantiﬁcation of IC₅₀.

Starting from reference compound 28 (Table 1) previously

identiﬁed by Liang et al.,³³several modiﬁcations were studied

in the phenol series, to determine key structural elements for

KLK6 inhibition. Suppression of the carbonyl group

(compound 29) as well as the tert-butyl group (compound

30) led to the loss of the activity. Then, sensitivity to the

position of the substituent carried out by the phenol group was

observed in the methyl series (compounds 31−34). Sub-

stitution of position 4 and position 5, to a less extent, was most

favorable to observe an optimal inhibition of KLK6. Several

other groups were then introduced into C4 and C5. These

positions must be substituted by an apolar or bulky group such

as an isopropyl (compound 37) or a methyl group (compound

32) for C4-substituted derivatives and a tert-butyl group

(compound 28) for C5-substituted derivatives. Interestingly,

the substitution of the C5 position by a halogen (compound

35) or by a methoxy group (compound 36) is strongly

unfavorable for the inhibition of KLK6.

When the inhibitor contains a naphthol group, a slight

sensitivity with respect to the position of the hydroxyl group is

observed. Indeed, it must be substituted at positions 1 or 3 for

optimal inhibition of KLK6 (compounds 40 and 42,

respectively). More generally, the hydroxyl group is essential

because the inhibition of KLK6 decreases drastically when the

inhibitor contains a naphthyl (compound 43) instead of a

naphthol. This result is not surprising because, in the phenol

series, the hydroxyl group was reported to be involved in key

interactions notably with the S195 residue of KLK6. There is

also a lack of inhibitory eﬀect when the compound contains an

indolyl (compound 44) or an oxoquinoline ring (compound

45) in place of the naphthol ring, indicating that the hydroxyl

group could not be replaced by another hydrogen donor or

acceptor (NH or ketone). Moreover, we noted the importance

of the benzylamine group. Indeed, we observed a signiﬁcant

decrease in the inhibition when this same position is occupied

by a piperidinyl group (compound 53). Finally, several

substitutions into the naphthol ring were studied (compounds

47−51, see the Supporting Information (SI)). However, all

studied modiﬁcations led to autoﬂuorescent compounds that

could not be evaluated, except when a methoxy group was

introduced on position 6 (compound 47). In this case, a slight

loss of activity compared to its unsubstituted analogue

(compound 40) was observed.

Thus, among the newly synthesized compounds, ﬁve new

hits were identiﬁed in the series of para-aminobenzyl

compounds (molecules 32, 37, 40, 42, and 47) (Table 1),

displaying IC₅₀equal or below 5 μM. The IC₅₀accounts for

the eﬀectiveness of the inhibitor with respect to the enzyme

and allows a prioritization according to the inhibitory power.

The IC₅₀values place compounds 32 and 42 at the top, while

reference compound 28 (IC₅₀= 9.06 μM) has one of the

lowest eﬃcacies with respect to KLK6 among the selected

inhibitors.

Then, we assessed the inhibitory potency of our hit

compounds (32, 37, 40, 42, and 47) and of reference

compound 28 toward KLK1 and plasmin, as these two

proteases are thought to be part of the proteolytic network of

KLK6 in MS.¹⁹All of these para-aminobenzyl derivatives lead

to at least 50% inhibition of the plasmin activity except

inhibitor 28 (45%) while all of the tested inhibitors lead to an

inhibition of at least 50% of KLK1 activity at a concentration

Targeted compounds, as well as the reference compound V

(now named compound 28) were synthesized as reported in

Scheme 2.

Brieﬂy, compound 2 was obtained in two steps from 4-

nitrobenzylamine hydrochloride after tert-butyloxycarbonyl

(Boc) protection of the amine group, followed by the

reduction of the nitro function. Compound 2 was then

coupled with the appropriate salicylic or naphthoic acid in the

presence of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide

hydrochloride (EDCI) to oﬀer compounds 3−25 in moderate

yields. Compound 27 was synthesized by reductive amination

with aldehyde 26 in the presence of sodium borohydride.

Reaction between 1-naphthoic acid, 1-Boc-4-aminopiperidine

and EDCI oﬀered compound 52 in 64% yields. Finally, Boc

group was removed using a mixture of hydrochloric acid in 1,4-

dioxane to oﬀer compounds 28−51 and 53 as hydrochloride

salts.

Inhibitory Potency Hit Compounds on KLK6 and on

Its Proteolytic Network. Newly synthesized compounds

28−51 and 53 were ﬁrst screened on KLK6 through the

evaluation of the percentage inhibition at 10 μM. This allows

highlighting the promising compounds for the inhibition of

KLK6. Among the 25 designed compounds, we preselected

those raising an inhibition of at least 50% of the activity of

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a

Table 2. Eﬃcacy of KLK6 para-Aminobenzyl Hit Inhibitors on Plasmin and KLK1

a

For each compound, the inhibitory eﬀect was quantiﬁed by the IC₅₀. To determine IC₅₀, compounds at diﬀerent concentrations (from 100 to 1.56

μM) are preincubated for 15 min at 37 °C with plasmin (3 nM) or KLK1 (0,75 nM) in 50 mM Tris buﬀer, 1 M citrate 0.05% Brij-35; pH 7.4 at 37

°C. The enzymatic reaction is then triggered by the addition of the Boc-QAR-AMC substrate (100 μM) for plasmin and H-PFR-AMC substrate

(100 μM) for KLK1. The data result from at least three independent experiments in duplicate. The IC₅₀values were calculated by ﬁtting the

experimental data to eq 2a or eq 2b and expressed as geometric standard deviation.

of 10 μM (data not shown). The inhibitory potency (IC₅₀) of

hit compounds was then determined. The IC₅₀values with

respect to plasmin place compound 47 (IC₅₀= 3.3 μM) at the

top while reference compound 28 (IC₅₀= 21.3 μM) has one of

the lowest eﬃciencies (Table 2). At the same time, compound

42 is characterized by an IC₅₀value of 5.1 μM toward KLK1,

while other compounds showed lower inhibitory potency and

were thus not selected for further mechanistic studies (Table

2).

Hit compounds (32, 37, 40, 42, 47, 49) and reference

compound 28 were assessed by the evaluation of their

selectivity spectrum against a set of serine proteases and

proteases involved in CNS homeostasis. Table 3 provides an

overview of putative cross-inhibition within a large set of

proteases, both serine proteases challenging in the CNS

(KLK8, tissue-type plasminogen activator (tPA), thrombin,

trypsin, trypsin-3, KLK11) and other kallikreins (KLK3, KLK4,

KLK5, KLK14), matriptase, and diverse proteases involved in

CNS inﬂammation (caspase-2, caspase-3, caspase-6, cathepsin

L). Overall, this screening shows that hit compounds 32 and

42 have little eﬀect on these selected challenging proteases in

contrast to the other identiﬁed inhibitors (28, 37, 40, 47, 49)

that display signiﬁcant inhibition on several proteases. This

allows us to conclude that compounds 32 and 42 are quite

speciﬁc to KLK6 and its proximal proteolytic network.

Mechanism of Inhibition: Hit Compounds. Mechanistic

studies were only performed for hit compound that display an

ideal balance between potency and selectivity proﬁles toward

KLK6 and its proximal proteolytic network (KLK1 and

plasmin) as well as the reference compound 28 for

comparison. The reversibility of the inhibitions exerted by

the compounds on KLK6 and its proteolytic network (plasmin

and KLK1) were demonstrated by the dilution method as

described in the Experimental Section. This experiment

distinguishes an irreversible covalent inhibitor from a reversible

one. Whatever the compound tested, a 1/100 dilution of the

enzyme−inhibitor complex allowed restoring the initial activity

of KLK6 to more than 80%. Hence, hit compounds are

reversible inhibitors (Figure 2). The mechanisms were

determined using representations of Dixon for hits and

reference compounds. The results are shown in Figure 2.

The Dixon graphs obtained for compounds 28, 32, and 40

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Table 3. Selectivity of the Inhibition of Hit Compounds toward Selected CNS Concurrent Proteases (Inhibition Percentage at

a

10 μM, %)

a

Each inhibitor (10 μM) is preincubated with the enzyme at the optimal concentration for 15 min at 37 °C. The reaction is triggered by the

addition of the speciﬁc 7-amino-4-methylcoumarin (AMC) substrate in the appropriate buﬀer (see the Selectivity Proﬁling section). The data result

from at least three independent experiments with a standard deviation <10%. Proteases implicated in the CNS physiological processes. Gray light:

b

signiﬁcative percentage of inhibition.

Figure 2. Mechanisms of inhibition toward KLK6. Dixon plots for inhibitors 28 (A), 32 (B), 40 (C), and 42 (D). Inhibitors at diﬀerent

concentrations (1/4; 1/2; 1; 2; 4 of the IC₅₀value) were tested using 2 nM KLK6 with substrate Boc-QAR-AMC at diﬀerent concentrations (125;

62.5; 31.25; 15.625 μM) in 50 mM Tris, 1 M citrate, 0.05% Brij-35, pH 7 buﬀer at 37 °C.

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Figure 3. Mechanisms of inhibition toward KLK1 and plasmin. Dixon plots for inhibitors 28 (A), 40 (B), and 42 (C) toward plasmin. (D) Dixon

plot for inhibitor 42 toward KLK1. Inhibitors at concentrations (1/4; 1/2; 1; 2; 4 of the IC₅₀value) were tested on plasmin (3 nM) or KLK1 (0,75

nM) with, respectively, Boc-QAR-AMC or H-PFR-AMC substrates at concentrations (125; 62.5; 31.25; 15.625 μM) in 50 mM Tris buﬀer, 1 M

citrate, 0.05% Brij-35, pH 7 at 37 °C.

Table 4. Mechanisms of Inhibition and K_iValues of Hit Compounds on KLK6 and Its Associated Proteolytic Network (KLK1

a

and Plasmin)

KLK6

type of inhibition

plasmin

type of inhibition

KLK1

type of inhibition

compound

K_i(μM)

28

32

37

40

42

47

competitive

noncompetitive

competitive

4.3 1.6

competitive

NA

competitive

NA

14.5 1.2

NA

1.5 0.5

1.3 0.53

NA

competitive

NA

2.4 0.5

NA

2

0.5

1.5 0.2

2.4 0.1

0.8 0.3

1

0.3

a

NA, not applicable.

(Figure 2A−C) are typical of competitive inhibitors.

Compounds 28, 32, and 40 have K_ivalues of 4.3, 2, and 2.4

μM, respectively. Surprisingly the mechanistic proﬁle of

compound 42 was found compatible with a noncompetitive

inhibition with a K_ivalue of 0.8 μM which may be indicative of

a more complex binding (Figure 2D). The Dixon plots for

compounds 37 and 47 are provided in the Supporting

Information (Figure S1).

Concerning plasmin and KLK1, all compounds are

reversible inhibitors as shown using the dilution method.

Dixon plots obtained for compounds 28, 40, and 42 (Figure

3A−C) are typical of competitive inhibitors toward plasmin.

Compounds 28, 40, and 42 have K_ivalues of 14.5, 1.5, and 1.3

μM, respectively. The Dixon plot for KLK1 obtained with

inhibitor 42 is also typical of a competitive inhibitor (K_i= 2.4

μM) (Figure 3D and Table 4). Hence, it is shown that some of

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Figure 4. Cytotoxicity of hit inhibitors (32, 40, and 42) and reference compound 28 against primary cultures of cortical and striatal neurons. (A)

Cortical neurons. (B) Striatal neurons. Primary cultures were treated with inhibitors in a concentration range from 10 to 100 μM, with the vehicle

(dimethyl sulfoxide (DMSO) 1%, “V”), or with rotenone 50 μM (CTRL) for 24 h (N = 3). Cell survival was then measured using XTT assay. All

data sets were compared to the vehicle condition (V) for the generation of the p-values. *p-value <0.05; **p-value <0.01; ***p-value <0.001

(Kruskal−Wallis test). NT: no treatment.

our hit KLK6 inhibitors also target key serine proteases of

KLK6 proteolytic network with a relatively good aﬃnity, which

may constitute an interesting perspective for a polypharmaco-

logical strategy.

Evaluation of the Cytotoxicity of Hit Compounds

toward Neural Cells. To ensure that hit compounds (32, 40,

and 42) may constitute good starting points for further

applications, their potential cytotoxicity toward neural cells was

ﬁrst evaluated in comparison to reference compound 28 on

primary cultures of mouse cortical and striatal neurons at 10,

25, 50, and 100 μM (Figure 4A). Compounds 32 and 42

showed similar eﬀects on both cortical and striatal neurons. A

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Figure 5. KLK6 inhibitors promote oligodendrocyte diﬀerentiation on the CG4 mCherry/GFP cell line. Schematic representation of the diﬀerent

developmental stages of the CG4 cell line (A). Diﬀerentiation of the mCherry/GFP double-ﬂuorescent CG4 cell line, after 4 days of diﬀerentiation

in basal medium (B, C). GFP ﬂuorescence is shown in (B) and mCherry ﬂuorescence in (C). Note that the diﬀerentiation of CG4 cells is not

synchronous in these cultures and the expression of mCherry occurs speciﬁcally at the mature oligodendrocyte stage. Graphs of the fold change of

the number of mCherry+ cells in N1 alone, N1 + compound 28 (D), N1 + compound 32 (E), N1 + compound 40 (F), or N1 + compound 42

(G), after 4 days of diﬀerentiation. The treatment with KLK6 inhibitors 32 and 42 induces a signiﬁcant increase of CG4 cell diﬀerentiation at 8 and

5 μM, respectively, relative to the N1 basal medium. Note that CG4 cells treated with compound 28 at 2 μM lead to a signiﬁcant increase in the

number of mCherry+ oligodendrocytes. However, at higher concentrations with this compound (ranging from 10 to 30 μM), cells were lost

presumably due to a cytotoxicity. Wilcoxon−Mann−Whitney test: *p ≤ 0.05, **p ≤ 0.01. 9-cis-retinoic acid (9-cis-RA, 1 μM) was used as a positive

control. Scale bar (B, C): 50 μm.

slight eﬀect is observed from a concentration of 50 μM of

inhibitors 32 and 42, with percentages of cell survival around

65−70%. In contrast, compound 40 is the only compound

showing a cytotoxic eﬀect on striatal neurons from a

concentration of 10 μM, this cytotoxicity is stable over the

concentration range, meaning that a plateau may be reached

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Figure 6. KLK6 inhibitors promote oligodendrocyte diﬀerentiation of primary OPC cultures. Primary OPC cultures, stained for MBP (green) and

Olig2 (red), after 4 days of diﬀerentiation in basal medium (A), in the presence of compound 42 ((B) 5 μM) or compound 32 ((C) 8 μM). The

treatment with KLK6 inhibitors induces a signiﬁcant increase of MBP+ oligodendrocytes with respect to control. N = 3 independent experiments;

Wilcoxon−Mann−Whitney test: *p ≤ 0.05. Scale bar (A−C): 100 μm.

(Figure 4B). Thus, these newly identiﬁed inhibitors are all very

poorly cytotoxic in comparison to the reference inhibitor 28,

which displays a noticeable cytotoxic eﬀect, even at 25 μM,

very close to the treatment with rotenone vehicle at 50 μM.

Hence, since compounds 32, 40, and 42 did not show toxic

eﬀects toward neurons, they were selected for further studies in

oligodendrocyte precursor cell (OPC) cultures as a relevant

biological model for MS.

CG4 cells start to diﬀerentiate and expressed mCherry (Figure

5B,C). Fluorescence imaging (90 image ﬁelds were acquired

for each condition) and quantiﬁcation were performed with

the ArrayScan XTI Imaging System. For each concentration

tested, we quantiﬁed the number of mCherry+ cells and data

were normalized relative to control (N1 basal medium). 9-cis-

Retinoic acid (9-cis-RA), a well-known compound promoting

OPC diﬀerentiation was used a positive control.⁵³

Pro-diﬀerentiating Eﬀect of Hit Compounds. Oligo-

dendrocytes are derived from OPCs, a class of progenitors

highly abundant during development but also persistent in the

adult CNS, where they contribute to myelin remodeling and

remyelination following acute demyelination. Although

eﬃcient remyelination occurs in the early stages of MS, it

becomes ineﬃcient and ultimately fails with disease pro-

gression. Remyelination requires the generation of new mature

oligodendrocytes (OLs) from OPCs that must be recruited to

the demyelinated lesions. Over the last decade, there has been

huge interest in developing medicines to improve remyelina-

tion in MS.³⁴The approaches range widely, from developing

novel medicines and repurposing existing drugs. We thus

assayed hit compounds on CG4 cell line and rat OPC primary

cultures to check for their ability to induce diﬀerentiation. To

monitor OPC diﬀerentiation, we developed a stable CG4 cell

line expressing the green ﬂuorescent protein (GFP) reporter at

all stages of the oligodendroglial lineage cells and the mCherry

reporter only in mature oligodendrocytes (Figure 5A). This

innovative cellular assay allows the screening of pro-

myelinating compounds based on the detection of ﬂuorescence

parameters. Indeed, both morphological changes and gen-

eration of mature oligodendrocytes can be monitored in a

single assay, based on enhanced GFP (EGFP) and mCherry

ﬂuorescence, respectively. After few days in basal medium,

Our data revealed no statistically signiﬁcant change in the

number of mCherry+ cells after treatments with compounds

40 (Figure 5F). For compound 28, a signiﬁcant increase in the

number of mCherry+ oligodendrocytes was observed at 2 μM,

while treatments at higher concentrations ranging from 10 to

30 μM lead to a complete loss of cells (Figure 5D), presumably

due to cytotoxicity. In contrast, for compound 32 (Figure 5E)

a signiﬁcant increase of diﬀerentiation was observed with

respect to basal control at concentrations of 6 μM (p ≤ 0.05)

and 8 μM (p ≤ 0.01, Mann−Whitney test), and for compound

42 (Figure 5G) at 5 μM (p ≤ 0.05, Mann−Whitney test).

Thus, compounds 32 and 42 promote the diﬀerentiation of

CG4 cells into mature oligodendrocytes. In view of their

pharmacological and biological proﬁles, compounds 32 and 42

appear particularly suitable. Compounds 32 and 42 were also

evaluated on primary cultures of rat OPCs (Figure 6). In this

model, the diﬀerentiation rate was measured by the

quantiﬁcation of the number of MBP-expressing oligoden-

drocytes. MBP is a major constituent of myelin and is

commonly used as a marker of mature oligodendrocytes.

Compounds 32 and 42 promote the diﬀerentiation at

concentrations of 8 and 5 μM, respectively. Overall, these

ﬁndings support the pro-myelinating potential of para-

aminobenzyl-hit derivatives.

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Figure 7. Anti-inﬂammatory potential of KLK6 inhibitors on a primary culture of microglia. Changes in TNF-α (A, B) and IL-1 β (C, D) secretions

after 24 h of LPS exposure (10 ng/mL) and treatment by either inhibitor 32 (left) or 42 (right) in the concentration range [0.5−10 μM]. Cytokine

secretion was measured by enzyme-linked immunosorbent assay (ELISA). The treatment with inhibitor 42 induces a signiﬁcant decrease of TNFα

and IL1-β secretions. Control: no treatment. Dexamethasone (100 nM): positive control (n = 3) (graph, *p ≤ 0.05).

Anti-inﬂammatory Properties of Hit Compounds. In

MS, chronic activation of microglia may contribute to

neurodegeneration and neuroinﬂammation.³⁵Thus, we also

examined the impact of hit compounds 32 and 42 on

microglial activation and proinﬂammatory cytokines. These

experiments were carried out in primary microglial cells

isolated from newborn rat brain.³⁶Eﬀects of selected

compounds on microglia were analyzed after treatment with

lipopolysaccharide (LPS, 10 ng/mL) alone or in the presence

of diﬀerent concentrations framing IC₅₀values of compounds

32 and 42, using dexamethasone as a positive control (Figure

7). Using quantitative reverse transcription polymerase chain

reaction (RT-qPCR) of pro- and anti-inﬂammatory cytokine

gene expression, we showed that compound 42 signiﬁcantly

lowered the expression of tumor necrosis factor (TNF)α and

interleukin (IL)-1 β proinﬂammatory cytokines while com-

pound 32 had no eﬀect. This result illustrates potential distinct

dual therapeutical eﬀects and/or mechanism of action for these

hit compounds.

Molecular Docking Modeling to the Study Structural

Basis of the Inhibition. In silico analyses using molecular

docking with the AutoDock Vina software highlighted some

characteristic structural bases on the inhibition of hit

compounds 42 and 32 (Figure 8A). Figure 8B,C illustrates

their positioning for the best docking poses. Overall, the

orientation of both compounds within the active site is

identical with the benzylamine group pointing to the S1 pocket

and the phenol/naphthol groups to the catalytic triad.

Inhibitors 32 (Figure 8B) and 42 (Figure 8A) share several

polar contacts with some active site residues, namely, H57 and

S195. In particular, the NH of the amide bond of compound

32 establishes a direct H-bond with the catalytic S195. We do

not exclude a certain ﬂexibility in this region, given the

obtained results, which could allow the NH of the amide bond

of compound 42 to behave similarly, this behavior may in part

explain the noncompetitive mechanism underlined by Dixon

plots. The OH hydroxyl group of phenols or naphthols could

also contribute to a polar bond with Q192 side chain as well as

the NH backbone of G193. Finally, the primary amine shows

strong polar contact in both cases with D189 and S190 side

chains and with W215 backbone. The visual inspection of

other docking poses with lower scores informed us about the

certain degree of freedom (dof) of the benzylamine moiety

inside the S1 pocket, the benzyl group having enough space to

slightly rotate around its axis, even suggested by the two best

poses of compounds 32 and 42. These poses are quite

compatible with their mechanism of inhibition.

Pharmacological Proﬁle and Evaluation of Druglike

Properties. To gain more insights on the druglike potential of

the two selected hit compounds, diﬀerent evaluations were

performed to assess their: (i) their putative eﬀect on

coagulation; (ii) their cross-reactivity toward key pharmaco-

logical targets using the SafetyScreen44 Panel from Euroﬁns;³⁷

(iii) some absorption, distribution, metabolism, excretion, and

toxicity (ADME-Tox) properties: solution properties (aqueous

solubility in simulated ﬂuids and plasma protein binding), in

vitro absorption (Caco2 cell line); in vitro metabolism

(intrinsic clearance using human liver microsomes). More

relevant parameters are summarized in Table S2, Supporting

Information. Altogether, these evaluations demonstrated that

hit compounds 32 and 42 bear a very favorable pharmaco-

logical proﬁle. They were shown to have no toxicity on

coagulation after incubation with human blood (neither the

activated partial thromboplastin time nor the prothrombin

ratio was modiﬁed) and to possess acceptable aqueous

solubility (>150 μM, except for 42 at pH ≥ 7.4), permeability

(>90%), and clearance properties (half-life >120 min)

compared to reference substances. Interestingly, among the

44 screened targets, both receptors and enzymes, very few were

inhibited by our lead compounds. No activity at ion channels

̀

(including human ether-a-go-go-related gene (hERG)) was

detected meaning weak toxicity. The only signiﬁcant detected

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CNS tissue; they are thus primarily eﬀective at the acute stage

of disease, but much less so at the chronic stage. An MS

therapy that has both immunomodulatory and neuro-

regenerative eﬀects would be highly beneﬁcial. Novel targets

aiming to promote such therapeutic strategies are thus of great

interest.⁸KLK6 and its proximal proteolytic network may

constitute a new opportunity to develop new therapeutics or

molecular tools for MS physiopathology. A series of para-

aminobenzyl derivatives incorporating a phenol or a naphthol

group was thus designed, synthesized, and studied for their

properties to inhibit KLK6 activity and proteases that may be

involved in the KLK6’s pathological network in MS, namely,

inhibitions of both KLK1 and plasmin may be interesting in a

polypharmacological perspective.

Several designed low-molecular-weight inhibitors are low-

micromolar potency and reversible toward KLK6. In the

phenol series, introduction of an alkyl group at the C4 position

led to more potent compounds (32 and 37), whereas in the

naphthol series, the best activity was obtained with the 1- and

3-naphthol derivatives (40 and 42). Interestingly, these hit

compounds were selective of KLK6 and its associated network

over a large set of CNS concurrent proteases, and devoid of

cytotoxic eﬀects on primary cultures of mouse cortical and

striatal neurons and oligodendrocytes. Most importantly, we

show for the ﬁrst time that synthetic inhibitors of KLK6 and

proximal proteolytic network, a key actor of MS physiopathol-

ogy, are able to promote the diﬀerentiation of OPCs into

mature oligodendrocytes in vitro, which is as far as we know

the ﬁrst report of such properties. Indeed, the presence of

oligodendrocyte progenitors (OPCs) in the adult CNS, which

are able to migrate and regenerate remyelinating oligoden-

drocytes in demyelinating lesions, represents the main cellular

target for the development of pharmacological strategy aiming

to promote remyelination. In line with this idea, compounds

32 and 42 were also tested for their potential eﬀect on

neuroinﬂammation using primary cultures of microglia.

Compound 42 decreased signiﬁcantly the expression of

proinﬂammatory cytokines. Hence, compound 42 in contrast

to compound 32 appears to be a good candidate as a ﬁrst-in-

class KLK6 inhibitor bearing both remyelinating and anti-

inﬂammatory potentials. There is a growing interest to

decipher mechanisms regulating OPC diﬀerentiation for the

identiﬁcation of novel speciﬁc pharmacological targets for

remyelination-enhancing therapy.³⁴On the other hand, there

also a growing interest for pro-diﬀerentiating compounds

selected ﬁrst through screening campaigns and more recently

using rational design as illustrated in up-to-date reports.

Namely, spiroindolines were shown as novel inducers of OPC

diﬀerentiation.³⁹Recently, benzothiazoles LRRK2 inhibitors

were shown to promote OPC proliferation and diﬀerentiation

through Wnt/β-catenin signaling pathway.⁴⁰Moreover,

modiﬁed ﬂavonoids were reported as selective inhibitors of

hyaluronidase activity and could promote OPC maturation,

making them excellent candidates to accelerate myelination or

promote remyelination following perinatal and adult CNS

insults.⁴¹Thus, in view of their pharmacological and biological

properties, para-aminobenzylamine derivatives 32 and 42

appear particularly valuable starting points for the development

of original pharmacological strategies aiming to promote

remyelination in demyelinating diseases, such as MS.

Figure 8. Structure of lead compounds 42 and 32 (A) and pose

predictions by molecular docking of 42 (B) and 32 (C) in KLK6.

Most relevant interactions were depicted by dashed lines together

with distances in angstrom. Compounds as well as residues of the

proteins mainly involved in ligand interactions are shown in licorice,

while the full proteins are shown in transparent-white cartoon. The

color code for the atoms is as follows: carbon in gray, oxygen in red,

nitrogen in blue, and hydrogen in white. The side chains that make no

contact with 42 and 32, as well as hydrogens of the protein and

nonpolar hydrogens of 42 and 32 were not represented for clarity.

oﬀ-targets were of interest for both CNS diseases (5-HT_2A/_2B

serotonin receptors) and inﬂammation (COX-2).³⁸Hence, the

selected compounds display favorable druglike properties and

thus constitute excellent starting point to derive drugs for

regenerative therapy of MS and related diseases associated with

a deregulation of KLK6 and proximal proteolytic network.

CONCLUSIONS

Current MS therapeutics are mainly immunomodulatory,

having very less eﬀect on the neuro-regeneration of damaged

■

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ﬁltrate was evaporated under reduced pressure to yield compound 2

as a pale orange solid, m = 880 mg, quantitative yield; mp 72−75 °C

(lit. 72−75 °C);⁴⁴¹H NMR (CDCl₃, 300 MHz): δ ppm 1.43 (s, 9H),

3.60 (bs, 2H), 4.14 (d, 2H, J = 5.6 Hz), 4.74 (bs, 1H), 6.60 (d, 2H, J

= 8.4 Hz), 7.02 (d, 2H, J = 8.4 Hz); ¹³C NMR (CDCl₃, 75 MHz): δ

ppm 28.5, 44.5, 79.4, 115.3, 128.9, 135.1, 145.8, 155.9; HPLC, RT =

0.93 min; MS (ESI+): m/z 223.3 [M + H]⁺.

EXPERIMENTAL SECTION

■

All reagents were of analytical grade. Distilled water was ﬁltered and

deionized through a Millipore water puriﬁcation system.

Chemistry Materials and General Procedures. Commercially

available reagents and solvents were used without further puriﬁcation.

Reactions were monitored by an analytical Waters Alliance 2690 high-

performance liquid chromatography (HPLC) instrument, equipped

with a photodiode array and an analytical Chromolith SpeedROD RP-

C18 185 Pm column (50 × 4.6 mm², 5 mm), at a ﬂow rate of 3.0 mL/

min, and gradients of 100/0 to 0/100 eluents A/B for 5 min (eluent A

= H₂O/0.1% triﬂuoroacetic acid (TFA) and B = CH₃CN/0.1% TFA).

General Procedure for the Synthesis of Compounds 3−28. To a

solution of 100 mg of compound 2 (0.45 mmol) in 2.5 mL of THF

were added 1 equiv of the appropriate carboxylic acid derivative (0.45

mmol) and 103 mg of N-(3-dimethylaminopropyl)-N′-ethylcarbodii-

mide hydrochloride (1 equiv, 0.45 mmol). The solution was stirred at

reﬂux for 12 h. After cooling to room temperature, the solution was

evaporated to dryness. The crude product was dissolved in 20 mL of

ethyl acetate, and the solution was washed with 1 N hydrochloric acid

solution (3 x 20 mL) and then by 10% aqueous sodium carbonate

solution. The organic layer was dried over Na₂SO₄, ﬁltered, and the

solvent was evaporated in vacuo. The resulting crude mixture was

washed with Et₂O for compounds 4, 10, and 23. Compounds 13, 16−

21, and 24 were used without further puriﬁcation in the subsequent

step. Other compounds were puriﬁed by chromatography on silica gel.

t e r t - B u t y l ( 4 - ( 5 - t e r t - B u t y l - 2 - h y d r o x y b e n z o y l ) ) -

aminobenzylcarbamate (3). Elution: DCM/EtOH 98.5/1.5 v/v;

Detection was performed at 214 nm. H and ¹³C NMR spectra were

1

recorded on Bruker spectrometers (300 or 400 MHz) at room

temperature in deuterated solvents. Chemical shifts (δ) are expressed

in parts per million (ppm), relative to the resonance of CDCl₃= 7.26

ppm for ¹H (77.16 ppm for ¹³C), CD₃OD = 3.31 ppm for ¹H (49.00

ppm for ¹³C) or DMSO-d₆= 2.50 ppm for H (39.52 ppm for ¹³C).

1

The following abbreviations were used: singlet (s), doublet (d), triplet

(t), quartet (q), multiplet (m), broad singlet (bs). Analytical thin-

layer chromatography (TLC) was carried out on aluminum plates

covered with 0.2 mm of silica, and column chromatography was

performed on silica gel 60 (70−230 mesh).

1

The liquid chromatography/mass spectrometry (LC/MS) system

consisted of a Waters Alliance 2695 HPLC, coupled to a Micromass

(Manchester, U.K.) ZQ spectrometer (positive electrospray ionization

mode, ESI+). All of the analyses were carried out using a Merck

Chromolith SpeedROD C18, 25 × 4.6 mm²reversed-phase column. A

ﬂow rate of 3 mL/min and a gradient of 0−100% B over 3 min (or

over 15 min) were used. Eluent A: water/0.1% HCO₂H; eluent B:

acetonitrile/0.1% HCO₂H. Retention times (RT) are given in

minutes. Nitrogen was used for both the nebulizing and drying gas.

The data were obtained in a scan mode ranging from 100 to 1000 m/z

in 0.1 s intervals; 10 scans were summed up to get the ﬁnal spectrum.

High-resolution mass spectrometry (HRMS) analyses were performed

with a Waters Synapt G2-S time-of-ﬂight mass spectrometer ﬁtted

with an electrospray ionization source. All measurements were

performed in the positive ion mode. Melting points (mp) are

uncorrected and were recorded on a Stuart capillary melting point

apparatus SMP3. All compounds tested for biological activity showed

>95% purity, as assessed by reversed-phase HPLC (RP-HPLC)

(Chromolith SpeedROD RP-C18 185 Pm column 50 × 4.6 mm², 5

μm; ﬂow rate: 5.0 mL/min; gradients from 100/0 to 0/100 eluents

A/B over 5 min, in which eluent A = H₂O/0.1% TFA and B =

CH₃CN/0.1% TFA; detection was done at 214 nm).

white solid (m = 58 mg, 32% yield); mp: 177−178 °C; H NMR

(CDCl₃, 300 MHz): δ ppm 1.31 (s, 9H), 1.44 (s, 9H), 4.26 (d, 2H, J

= 5.9 Hz), 4.89 (bs, 1H), 6.93 (d, 1H, J = 8.7 Hz), 7.24 (m, 2H), 7.48

(m, 4H), 8.24 (bs, 1H), 11.0 (s, 1H); ¹³C NMR (CDCl₃, 75 MHz): δ

ppm 28.6, 31.4, 31.6, 44.4, 79.9, 114.4, 118.4, 121.9, 122.1, 128.2,

132.3, 136.0, 136.1, 142.0, 156.1, 159.3, 168.7; HPLC, RT = 2.10

min; MS (ESI+): m/z 399.3 [M + H]⁺; HRMS calcd for C₂₃H₃₁N₂O₄

399.2278, found 399.2272.

tert-Butyl (4-(2-Hydroxybenzoyl))aminobenzylcarbamate (4).

1

White solid (m = 113 mg, 73% yield); mp: 107−171 °C; H NMR

(DMSO-d₆, 300 MHz): δ ppm 1.40 (s, 9H), 3.30 (bs, 1H), 4.10 (d,

2H, J = 6.0 Hz), 6.96 (m, 2H), 7.23 (d, 2H, J = 8.4 Hz), 7.33 (bt, 1H,

J = 6.0 Hz), 7.44 (td, 1H, J = 8.5, 1.6 Hz), 7.62 (d, 2H, J = 8.4 Hz),

7.97 (dd, 1H, J = 8.5, 1.6 Hz), 10.35 (s, 1H); ¹³C NMR (DMSO-d₆,

75 MHz): δ ppm 28.2, 43.0, 77.7, 117.2, 118.9, 120.9, 127.3, 128.9,

133.6, 136.0, 136.6, 155.7, 158.6, 166.5; HPLC, RT = 1.78 min; MS

(ESI+): m/z 365.0 [M + Na]⁺, 287.1 [M + H − t-Bu]⁺; HRMS calcd

for C₁₉H₂₃N₂O₄343.1652, found 343.1655.

t e r t - B u t y l ( 4 - ( 3 - M e t h y l - 2 - h y d r o x y b e n z o y l ) ) -

aminobenzylcarbamate (5). Elution: DCM/EtOH 92/2 v/v; light

1

yellow solid (m = 125 mg, 78% yield); mp: 166.2−166.9 °C; H

NMR (CDCl₃, 400 MHz): δ ppm 1.45 (s, 9H), 2.27 (s, 3H), 4.28 (d,

2H, J = 5.7 Hz), 4.85 (bs, 1H), 6.79 (t, 1H, J = 7.7 Hz), 7.28 (m, 3H),

7.37 (d, 1H, J = 7.7 Hz), 7.50 (d, 2H, J = 8.4 Hz), 8.00 (bs, 1H), 12.3

(s, 1H); ¹³C NMR (CDCl₃, 100 MHz): δ ppm 16.0, 28.6, 44.4, 79.9,

113.8, 118.4, 121.7, 123.1, 128.3, 128.4, 135.6, 136.1, 136.2, 156.1,

160.5, 169.1; HPLC, RT = 2.01 min; MS (ESI+): m/z 379.2 [M +

Na]⁺, 301.2 [M + H − t-Bu]⁺; HRMS calcd for C₂₀H₂₅N₂O₄

357.1809, found 357.1811.

Compound 26 was synthesized according to the procedure

described by Darensbourg et al.⁴²and its physical characteristics

were in agreement with the published data. Carboxylic acids were

commercially available or synthesized according to the procedures

described in SI.

Synthesis of tert-Butyl (4-Nitrobenzyl)carbamate (1). To a

solution of 4-nitrobenzylamine hydrochloride (1 g, 5.3 mmol) in 10

mL of dichloromethane were added 480 μL of triethylamine (348 mg,

3.44 mmol, 1.3 equiv) and 790 μL of tert-butyl dicarbonate (751 mg,

3.44 mmol, 1.3 equiv). The solution was stirred at room temperature

for 2 h. Then, 0.3 equiv of aminomethyl polystyrene resin was added

and the suspension was stirred for 1 h. After ﬁltration, the solution

was washed with 1 M aqueous potassium hydrogenosulfate solution

(2 × 15 mL) and brine (15 mL). The organic layer was dried over

Na₂SO₄, ﬁltered, and the solvent was evaporated in vacuo to oﬀer

compound 1 as a white solid (m = 1.06 g, 80% yield); mp 109−111

°C (lit. 109−110 °C);⁴³¹H NMR (CDCl₃, 300 MHz): δ ppm 1.43 (s,

9H), 4.37 (d, 2H, J = 6.2 Hz), 5.03 (bs, 1H), 7.41 (d, 2H, J = 8.8 Hz),

8.14 (d, 2H, J = 8.8 Hz); ¹³C NMR (CDCl₃, 75 MHz): δ ppm 27.5,

44.2, 85.3, 123.9, 127.9, 146.9, 147.4, 156.0; HPLC, RT = 1.68 min.

Synthesis of tert-Butyl (4-Aminobenzyl)carbamate (2). To a

solution of compound 1 (1 g, 4.0 mmol) in 20 mL of ethyl acetate

was added 10% palladium activated on charcoal (∼20 mg). The

suspension was hydrogenated at atmospheric pressure for 4 h at room

temperature. The suspension was then ﬁltered on celite, and the

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

aminobenzylcarbamate (6). Elution: n-Hex/AcOEt 2/1 v/v; light

1

yellow solid (m = 131 mg, 82% yield); mp: 191.8−192.8 °C; H

NMR (CDCl₃, 400 MHz): δ ppm 1.45 (s, 9H), 2.33 (s, 3H), 4.28 (d,

2H, J = 5.5 Hz), 4.84 (bs, 1H), 6.70 (dd, 1H, J = 8.1, 1.1 Hz), 6.82 (d,

1H, J = 1.1 Hz), 7.27 (d, 2H, J = 8.4 Hz), 7.39 (d, 1H, J = 8.1 Hz),

7.50 (d, 2H, J = 8.4 Hz), 7.93 (bs, 1H), 11.94 (s, 1H); ¹³C NMR

(CDCl₃, 100 MHz): δ ppm 21.9, 28.6, 44.4, 79.9, 112.1, 119.3, 120.3,

121.6, 125.5, 128.4, 136.1, 136.2, 146.1, 156.1, 162.1, 168.6; HPLC,

RT = 1.90 min; MS (ESI+): m/z 357.2 [M + H]⁺, 379.1 [M + Na]⁺,

301.2 [M + H − t-Bu]⁺; HRMS calcd for C₂₀H₂₅N₂O₄357.1809,

found 357.1809.

t e r t - B u t y l ( 4 - ( 5 - M e t h y l - 2 - h y d r o x y b e n z o y l ) ) -

aminobenzylcarbamate (7). Elution: n-Hex/AcOEt 2/1 v/v. Light

yellow solid (m = 68 mg, 42% yield); mp: 174.5−175.5 °C; ¹H NMR

(CDCl₃, 400 MHz): δ ppm 1.45 (s, 9H), 2.32 (s, 3H), 4.28 (d, 2H, J

= 5.4 Hz), 4.84 (bs, 1H), 6.91 (d, 1H, J = 8.4 Hz), 7.22−7.29 (m,

4H), 7.52 (d, 2H, J = 8.4 Hz), 7.96 (bs, 1H), 11.71 (s, 1H); ¹³C NMR

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(CDCl₃, 100 MHz): δ ppm 20.8, 28.6, 44.4, 79.9, 114.4, 115.5, 118.8,

121.6, 123.3, 125.7, 128.4, 135.8, 136.1, 156.1, 159.8, 168.6; HPLC,

RT = 1.90 min; MS (ESI+): m/z 379.2 [M + Na]⁺, 301.2 [M + H −

t-Bu]⁺; HRMS calcd for C₂₀H₂₅N₂O₄357.1809, found 357.1810.

t e r t - B u t y l ( 4 - ( 6 - M e t h y l - 2 - h y d r o x y b e n z o y l ) ) -

aminobenzylcarbamate (8). Elution: n-Hex/AcOEt 2/1 v/v. Light

yellow solid (m = 33 mg, 21% yield); mp: 149.0−150.0 °C; ¹H NMR

(CDCl₃, 300 MHz): δ ppm 1.46 (s, 9H), 2.60 (s, 3H), 4.28 (d, 2H, J

= 5.4 Hz), 4.91 (bs, 1H), 6.76 (d, 1H, J = 7.5 Hz), 6.84 (d, 1H, J = 8.2

Hz), 7.23 (dd, 1H, J = 8.2, 7.5 Hz), 7.27 (d, 2H, J = 8.0 Hz), 7.52 (d,

2H, J = 8.0 Hz), 7.72 (s, 1H), 9.85 (bs, 1H); ¹³C NMR (CDCl₃, 75

MHz): δ ppm 22.1, 28.5, 44.3, 78.3, 115.7, 119.0, 121.0, 123.1, 128.4,

132.5, 135.5, 136.1, 136.2, 156.1, 159.2, 168.4; HPLC, RT = 1.60

min; MS (ESI+): m/z 379.1 [M + Na]⁺, 301.2 [M + H − t-Bu]⁺;

HRMS calcd for C₂₀H₂₅N₂O₄357.1809, found 357.1808.

tert-Butyl (4-(1-Hydroxy-2-naphthoyl))aminobenzylcarbamate

(14). Elution: n-Hex/AcOEt 3/1 v/v; white solid (m = 40 mg, 23%

1

yield); mp: 145−146 °C; H NMR (DMSO-d₆, 300 MHz): δ ppm

1.40 (s, 9H), 4.13 (d, 2H, J = 5.9 Hz), 7.27 (d, 2H, J = 8.1 Hz), 7.40

(bt, 1H, J = 5.9 Hz), 7.46 (d, 1H, J = 8.9 Hz), 7.58 (dd, 1H, J = 8.1,

7.1 Hz), 7.68 (m, 3H), 7.92 (d, 1H, J = 8.1 Hz), 8.10 (d, 1H, J = 9.0

Hz), 8.31 (d, 1H, J = 8.1 Hz), 10.92 (bs, 1H); ¹³C NMR (DMSO-d₆,

75 MHz): δ ppm 28.2, 43.0, 77.8, 107.5, 117.7, 121.1, 123.0, 124.7,

125.9, 127.2, 127.5, 129.1, 136.0, 136.1, 136.7, 155.8, 160.0, 169.4;

HPLC, RT = 2.13 min; MS (ESI+): m/z 415.1 [M + Na]⁺, 337.0 [M

+ H − t-Bu]⁺; HRMS calcd for C₂₃H₂₅N₂O₄393.1809, found

393.1807.

tert-Butyl (4-(2-Hydroxy-1-naphthoyl))aminobenzylcarbamate

(15). Elution: DCM/EtOH 98.5/1.5 v/v; white solid (m = 90 mg,

1

51% yield); mp: 188.5−189.5 °C; H NMR (CDCl₃, 300 MHz): δ

ppm 1.45 (s, 9H), 4.28 (d, 2H, J = 5.6 Hz), 4.88 (bs, 1H), 7.16 (d,

1H, J = 9.0 Hz), 7.29 (d, 2H, J = 8.8 Hz), 7.36 (dd, 1H, J = 7.4, 6.8

Hz), 7.54 (m, 3H), 7.81 (m, 2H), 8.01 (bs, 1H), 8.14 (d, 1H, J = 8.6

Hz), 10.92 (bs, 1H); ¹³C NMR (CDCl₃, 75 MHz): δ ppm 28.6, 44.4,

79.9, 110.3, 119.5, 121.0, 122.6, 123.8, 128.4, 128.6, 129.0, 129.7,

130.7, 134.4, 136.2, 136.3, 156.0, 159.7, 168.5; HPLC, RT = 1.74

min; MS (ESI+): m/z 415.1 [M + Na]⁺, 337.0 [M + H − t-Bu]⁺;

HRMS calcd for C₂₃H₂₅N₂O₄393.1809, found 393.1801.

t e r t - B u t y l ( 4 - ( 5 - B r o m o - 2 - h y d r o x y b e n z o y l ) ) -

aminobenzylcarbamate (9). Elution: n-Hex/AcOEt 4/1 v/v; white

1

solid (m = 24 mg, 12% yield); mp: >300 °C; H NMR (DMSO-d₆,

400 MHz): δ ppm 1.93 (s, 9H), 4.10 (d, 2H, J = 6.0 Hz), 6.95 (d, 1H,

J = 8.8 Hz), 7.23 (d, 2H, J = 8.3 Hz), 7.37 (t, 1H, J = 6.8 Hz), 7.57

(dd, 1H, J = 8.8, 2.6 Hz), 7.62 (d, 2H, J = 8.3 Hz), 8.08 (d, 1H, J =

2.6 Hz), 10.44 (s, 1H), 11.93 (bs, 1H); ¹³C NMR (CDCl₃, 100

MHz): δ ppm 28.2, 43.0, 77.8, 110.0, 119.6, 119.9, 120.8, 127.3,

131.1, 135.8, 136.2, 136.5, 155.8, 157.6, 164.9; HPLC, RT = 2.00

min; MS (ESI+): m/z 443.0 [M + Na]⁺, 445.0 [M + 2 + Na]⁺, 367.0

[M + H − t-Bu]⁺, 365.0 [M + H + 2− t-Bu]⁺; HRMS calcd for

C₁₉H₂₁BrN₂NaO₄443.0577, found 443.0575.

tert-Butyl (4-(3-Hydroxy-2-naphthoyl))aminobenzylcarbamate

1

(16). White solid (m = 56 mg, 32% yield); mp: 199−200 °C; H

NMR (DMSO-d₆, 400 MHz): δ ppm 1.40 (s, 9H), 4.11 (d, 2H, J =

6.1 Hz), 7.25 (d, 2H, J = 8.4 Hz), 7.32 (s, 1H), 7.35 (m, 2H), 7.50

(ddd, 1H, J = 8.4, 6.8, 1.0 Hz), 7.69 (d, 2H, J = 8.4 Hz), 7.75 (d, 1H, J

= 8.2 Hz), 7.92 (d, 1H, J = 8.2 Hz), 8.51 (s, 1H), 10.65 (bs, 1H); ¹³C

NMR (DMSO-d₆, 100 MHz): δ ppm 28.2, 43.0, 77.7, 110.6, 120.5,

121.6, 123.7, 125.7, 126.8, 127.4, 128.1, 128.7 130.4, 135.8, 135.9,

137.0, 154.0, 155.8, 165.6; HPLC, RT = 1.99 min; MS (ESI+): m/z

415.2 [M + Na]⁺, 337.1 [M + H − t-Bu]⁺; HRMS calcd for

C₂₃H₂₅N₂O₄393.1809, found 393.1810.

t e r t - B u t y l ( 4 - ( 5 - M e t h o x y - 2 - h y d r o x y b e n z o y l ) ) -

aminobenzylcarbamate (10). White solid (m = 75 mg, 45%

1

yield); H NMR (DMSO-d₆, 300 MHz): δ ppm 1.39 (s, 9H), 3.76

(s, 3H), 4.10 (d, 2H, J = 5.9 Hz), 6.92 (d, 1H, J = 8.9 Hz), 7.06 (ddd,

1H, J = 9.0, 9.0, 3.0 Hz), 7.23 (d, 2H, J = 8.3 Hz), 7.37 (bt, 1H, J =

5.9 Hz), 7.50 (d, 1H, J = 3.0 Hz), 7.62 (d, 2H, J = 8.3 Hz), 10.90 (s,

1H); ¹³C NMR (DMSO-d₆, 75 MHz): δ ppm 28.2, 43.0, 55.7, 77.8,

112.5, 117.3, 118.1, 120.6, 121.0, 127.4, 136.1, 136.6, 151.8, 152.3,

155.8, 166.0;; HPLC, RT = 1.78 min; MS (ESI+): m/z 395.1 [M +

Na]⁺, 373.1 [M + H]⁺; 317.1 [M + H − t-Bu]⁺.

tert-Butyl (4-(2-Naphthoyl))aminobenzylcarbamate (17). White

solid (m = 61 mg, 36% yield); mp: 182.5−183.5 °C; ¹H NMR

(CDCl₃, 400 MHz): δ ppm 1.45 (s, 9H), 4.28 (d, 2H, J = 5.3 Hz),

4.86 (bs, 1H), 7.28 (d, 2H, J = 8.4 Hz), 7.56 (m, 2H), 7.63 (d, 2H, J =

8.4 Hz), 7.91 (m, 4H), 8.03 (bs, 1H), 8.36 (s, 1H); ¹³C NMR

(CDCl₃, 100 MHz): δ ppm 28.6, 44.5, 79.8, 120.7, 123.8, 127.2,

127.8, 128.0, 128.1, 128.5, 129.0, 129.2, 132.3, 132.8, 135.1, 135.4,

137.4, 156.1, 166.0; HPLC, RT = 1.91 min; MS (ESI+): m/z 377.1

[M + H]⁺, 399.1 [M + Na]⁺, 321.3 [M + H − t-Bu]⁺; HRMS calcd for

C₂₃H₂₅N₂O₃377.1860, found 377.1855.

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

aminobenzylcarbamate (11). Elution: n-Hex/AcOEt 3/1 v/v;

1

white solid (m = 53 mg, 31% yield); H NMR (CDCl₃, 400 MHz):

δ ppm 1.60 (d, 6H, J = 6.7 Hz), 1.83 (s, 9H), 3.24 (hept, 1H, J = 6.7

Hz), 4.64 (d, 2H, J = 4.0 Hz), 5.25 (bs, 1H), 7.12 (d, 1H, J = 7.1 Hz),

7.60 (d, 2H, J = 7.6 Hz), 7.86 (m, 3H), 8.47 (bs, 1H), 12.30 (bs, 1H);

¹³C NMR (CDCl₃, 100 MHz): δ ppm 23.7, 28.6, 34.4, 44.4, 79.9,

tert-Butyl (1H-2-Indolylcarbonyl)aminobenzylcarbamate (18).

White solid (m = 67 mg, 41% yield); mp: 207−208 °C; H NMR

112.5, 116.5, 117.9, 121.7, 125.8, 128.3, 136.0, 136.2, 156.2, 156.9,

162.1, 168.6; HPLC, RT = 2.09 min; MS (ESI+): m/z 407.1 [M +

Na]⁺, 385.2 [M + H]⁺.

1

(DMSO-d₆, 400 MHz): δ ppm 1.40 (s, 9H), 4.10 (s, 2H), 7.06 (t, 1H,

J = 7.5 Hz), 7.22 (m, 3H), 7.36 (bs, 1H), 7.40 (s, 1H), 7.46 (d, 1H, J

= 8.3 Hz), 7.66 (d, 1H, J = 7.5 Hz), 7.72 (d, 2H, J = 8.3 Hz), 10.2 (bs,

1H), 11.76 (bs, 1H); ¹³C NMR (DMSO-d₆, 100 MHz): δ ppm 28.3,

40.0, 77.7, 103.8, 112.4, 119.8, 120.1, 121.7, 123.7, 127.0, 127.3,

131.6, 135.3, 136.9, 137.5, 155.7, 159.7; HPLC, RT = 1.82 min; MS

(ESI+): m/z 388.2 [M + Na]⁺, 310.2 [M + H − t-Bu]⁺; HRMS calcd

for C₂₁H₂₄N₃O₃366.1812, found 366.1812.

tert-Butyl (4-(4-Triﬂuoromethyl-2-hydroxybenzoyl))-

aminobenzylcarbamate (12). Elution DCM/EtOH 99/1 v/v;

white solid (m = 114 mg, 62% yield); mp: 179.5−180.5 °C; ¹H

NMR (CD₃OD, 400 MHz): δ ppm 1.46 (s, 9H), 4.22 (s, 2H), 4.76

(bs, 1H), 7.22 (m, 2H), 7.29 (d, 2H, J = 8.4 Hz), 7.63 (d, 2H, J = 8.4

Hz), 8.11 (d, 1H, J = 8.6 Hz); ¹³C NMR (CD₃OD, 100 MHz): δ ppm

28.8, 44.6, 80.2, 115.2 (d, J = 3.7 Hz), 116.5 (d, J = 3.7 Hz), 122.0,

122.5, 126.3, 128.7, 131.2, 135.8, 137.8, 158.6, 160.4, 165.2, 167.4;

HPLC, RT = 1.86 min; MS (ESI+): m/z 433.0 [M + Na]⁺, 411.1 [M

+ H]⁺, 355.0 [M + H − t-Bu]⁺; HRMS calcd for C₂₀H₂₂F₃N₂O₄

411.1526, found 411.1537.

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

aminobenzylcarbamate (13). Elution DCM/EtOH 99/1 v/v;

white solid (m = 37 mg, 23% yield); mp: 155−5−156.5 °C; ¹H

NMR (CD₃OD, 400 MHz): δ ppm 1.45 (s, 9H), 4.21 (s, 2H), 6.64−

6.70 (m, 2H), 7.26 (d, 2H, J = 8.4 Hz), 7.59 (d, 2H, J = 8.4 Hz), 7.98

(dd, 1H, J = 9.4, 8.4 Hz); ¹⁹F NMR (CD₃OD, 400 MHz): δ ppm

−107.1; ¹³C NMR (CD₃OD, 100 MHz): δ ppm 28.9, 44.8, 80.3,

105.2 (d, J = 24.2 Hz), 107.6 (d, J = 24.2 Hz), 114.8, 122.7, 128.8,

132.1 (d, J = 11.1 Hz), 132.5, 137.5, 138.0, 163.3 (d, J = 13.3 Hz),

167.4 (d, J = 251.3 Hz), 168.4; HPLC, RT = 1.88 min; MS (ESI+):

m/z 383.1 [M + Na]⁺, 361.1 [M + H]⁺, 305.1 [M + H − t-Bu]⁺;

HRMS calcd for C₁₉H₂₂FN₂O₄361.1558, found 361.1554.

tert-Butyl 4-[(4-Oxo-1,4-dihydroquinolin-3-yl)carbonyl]-

aminobenzylcarbamate (19). White solid (m = 48 mg, 27%

1

yield); mp: 238−239 °C; H NMR (DMSO-d₆, 400 MHz): δ ppm

1.40 (s, 9H), 4.08 (d, 2H, J = 6.0 Hz), 7.17 (d, 2H, J = 8.3 Hz), 7.24

(td, 1H, J = 8.1, 1.1 Hz), 7.33 (t, 1H, J = 6.1 Hz), 7.50 (td, 1H, J =

8.1, 1.5 Hz), 7.59 (d, 1H, J = 8.1 Hz), 7.66 (d, 2H, J = 8.3 Hz), 8.24

(dd, 1H, J = 8.1, 1.1 Hz), 8.87 (s, 1H), 13.64 (s, 1H); ¹³C NMR

(DMSO-d₆, 100 MHz): δ ppm 28.3, 43.1, 77.7, 108.6, 118.9, 122.2,

124.7, 126.7, 127.5, 127.6, 129.3, 133.5, 139.0, 149.3, 151.6, 155.8,

166.1, 174.0; HPLC, RT = 1.67 min; MS (ESI+): m/z 394.2 [M +

H]⁺, 338.2 [M + H − t-Bu]⁺; HRMS calcd for C₂₂H₂₄N₃O₄394.1761,

found 394.1756.

tert-Butyl (4-(3-Hydroxypyridin-2-yl))aminobenzylcarbamate

(20). Light yellow solid (m = 30 mg, 19% yield); mp: 90−92 °C;

¹H NMR (CDCl₃, 400 MHz): δ ppm 1.45 (s, 9H), 4.29 (d, 2H, J =

5.7 Hz), 7.30 (d, 2H, J = 8.4 Hz), 7.31−7.37 (m, 2H), 7.65 (d, 2H, J

5681

https://doi.org/10.1021/acs.jmedchem.0c02175

J. Med. Chem. 2021, 64, 5667−5688

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

= 8.4 Hz), 8.06 (bs, 1H), 8.10 (dd, 1H, J = 4.1, 1.6 Hz), 9.90 (bs,

1H), 11.89 (s, 1H); HPLC, RT = 1.85 min; MS (ESI+): m/z 366.1

[M + Na]⁺.

1.29 (s, 9H), 1.44 (s, 9H), 4.19 (d, 2H, J = 5.6 Hz), 4.35 (s, 2H), 4.81

(bs, 2H), 6.76 (d, 2H, J = 8.4 Hz), 6.81 (d, 1H), 7.12 (m, 3H), 7.21

(dd, 1H, J = 8.4, 2.4 Hz), 11.2 (s, 1H); ¹³C NMR (CDCl₃, 75 MHz):

δ ppm 28.6, 31.7, 44.4, 48.8, 64.9, 79.6, 115.8, 116.1, 122.4, 124.9,

125.8, 126.1, 128.8, 130.9, 143.0, 146.9, 154.2; HPLC, RT = 3.57

min; MS (ESI+): m/z 329.1 [M + H − t-Bu]⁺, 385.1 [M + H]⁺, 407.1

[M + Na]⁺; HRMS calcd for C₂₃H₃₃N₂O₃385.2486, found 385.2499.

Synthesis of tert-Butyl 4-[(1-Hydroxy-2-naphthoyl)amino]-

piperidine-1-carboxylate (52). To a solution of 91 mg of compound

1-Boc-4-aminopiperidine (0.45 mmol) in 2.5 mL of THF were added

86 mg of 1-hydroxy-2-naphthoic acid (1 equiv, 0.45 mmol) and 103

mg of EDCI (1 equiv, 0.45 mmol). The solution was stirred at reﬂux

for 12 h. After cooling to room temperature, the solution was

evaporated to dryness. The crude product was dissolved in 20 mL of

ethyl acetate, and the solution was washed with 1 N hydrochloric acid

solution (3 × 20 mL) and then by 10% aqueous sodium carbonate

solution. The organic layer was dried over Na₂SO₄, ﬁltered, and the

solvent was evaporated in vacuo. The resulting crude mixture was

puriﬁed by chromatography on silica gel and eluted by n-Hex/AcOEt

3/1 v/v to oﬀer compound 52 as a white solid (m = 38 mg, 23%

yield); ¹H NMR (CDCl₃, 400 MHz): δ ppm 1.23 (m, 1H), 1.42 (m,

1H), 1.46 (s, 9H), 2.04 (m, 2H), 2.90 (t, 2H, J = 12.1 Hz), 4.07−4.19

(m, 3H), 6.25 (d, 1H, J = 7.7 Hz), 7.23 (d, 1H, J = 8.8 Hz), 7.28 (d,

1H, J = 8.9 Hz), 7.50 (td, 1H, J = 8.2, 1.3 Hz), 7.56 (td, 1H, J = 8.2,

1.3 Hz), 7.74 (d, 1H, J = 7.8 Hz), 8.40 (d, 1H, J = 8.2 Hz), 13.75 (s,

1H); ¹³C NMR (CDCl₃, 100 MHz): δ ppm 28.6, 29.8, 32.2, 47.3,

80.0, 106.6, 118.3, 120.9, 124.0, 125.8, 126.0, 127.4, 129.1, 136.4,

154.8, 160.9, 170.2; HPLC, RT = 2.16 min; MS (ESI+): m/z 371.2

[M + H]⁺, 315.2 [M + H − t-Bu]⁺.

tert-Butyl (4-(6-Methoxy-1-hydroxy-2-naphthoyl))-

aminobenzylcarbamate (21). White solid (m = 34 mg, 18%

1

yield); mp: 205.8−206.3 °C; H NMR (CDCl₃, 300 MHz): δ ppm

1.48 (s, 9H), 3.94 (s, 3H), 4.30 (d, 2H, J = 5.4 Hz), 4.87 (bs, 1H),

7.07 (d, 1H, J = 2.3 Hz), 7.16 (dd, 1H, J = 9.1, 2.4 Hz), 7.20 (d, 1H, J

= 8.1 Hz), 7.30 (d, 2H, J = 8.3 Hz), 7.44 (d, 1H, J = 9.1 Hz), 7.55 (d,

2H, J = 8.3 Hz), 7.98 (bs, 1H), 8.35 (d, 1H, J = 9.1 Hz); ¹³C NMR

(CDCl₃, 75 MHz): δ ppm 28.6, 44.4, 55.5, 79.3, 105.4, 106.2, 117.6,

118.2, 121.0, 121.6, 121.7, 125.9, 128.4, 136.0, 136.2, 138.6, 142.7,

143.1, 160.6, 161.6; HPLC, RT = 2.14 min; MS (ESI+): m/z 445.2

[M + Na]⁺, 367.1 [M + H − t-Bu]⁺; HRMS calcd for C₂₄H₂₇N₂O₅

423.1914, found 423.1911.

tert-Butyl (4-(7-Methoxy-1-hydroxy-2-naphthoyl))-

aminobenzylcarbamate (22). White solid (m = 30 mg, 16%

1

yield); H NMR (CDCl₃, 400 MHz): δ ppm 1.40 (s, 9H), 3.89 (s,

3H), 4,24 (d, 2H, J = 5.4 Hz), 4.81 (bs, 1H), 7.17−7.29 (m, 6H),

7.49 (d, 1H, J = 8.3 Hz), 7.60 (d, 1H, J = 8.9 Hz), 7.65 (d, 1H, J = 2.4

Hz), 7.98 (bs, 1H), 13.38 (s, 1H); ¹³C NMR (CDCl₃, 400 MHz): δ

ppm 28.6, 29.9, 44.4, 55.7, 79.9, 102.2, 107.4, 118.5, 118.6, 121.7,

122.0, 126.9, 128.5, 129.1, 131.9, 136.2, 156.1, 158.3, 160.3, 169.5;

HPLC, RT = 2,12 min; MS (ESI+): m/z 423.1 [M + H]⁺, m/z 845.3

[2M + H]⁺, m/z 440.2 [M + NH₄]⁺.

tert-Butyl (4-(7-Methyl-1-hydroxy-2-naphthoyl))-

aminobenzylcarbamate (23). Elution: n-Hex/AcOEt 4/1 v/v;

white solid (m = 140 mg, 77% yield); mp: 188.3−188.9 °C; ¹H

NMR (CDCl₃, 500 MHz): δ ppm 1.41 (s, 9H), 2.47 (s, 3H), 4.22 (d,

2H, J = 5.8 Hz), 4.84 (bt, 1H), 7.20 (m, 3H), 7.34 (d, 1H, J = 9.0

Hz), 7.36 (dd, 1H, J = 8.4, 1.4 Hz), 7.46 (d, 1H, J = 8.1 Hz), 7.60 (d,

1H, J = 8.4 Hz), 8.05 (bs, 1H), 8.15 (s, 1H), 13.45 (s, 1H); ¹³C NMR

(CDCl₃, 125 MHz): δ ppm 22.0, 28.6, 44.3, 79.9, 107.0, 118.5, 120.0,

121.8, 123.0, 125.9, 127.4, 128.4, 131.6, 134.8, 136.0, 136.1, 136.2,

156.1, 161.0, 169.5; HPLC, RT = 2.23 min; MS (ESI+): m/z 429.1

[M + Na]⁺, 407.2 [M + H]⁺, 351.1 [M + H − t-Bu]⁺; HRMS calcd for

C₂₄H₂₇N₂O₄407.1965, found 407.1962.

General Procedure for the Synthesis of Compounds 28−51 and

53. Compound 3−25, 27, or 52 (0.2 mmol) in 5 mL of 6 N HCl in

dioxane was stirred a room temperature for 1 h 30 min. The dioxane

was evaporated, and the crude product was washed with diethyl ether

to oﬀer a deprotected compound as a hydrochloride salt.

N-(4-(Aminomethyl)phenyl)-5-tert-butyl-2-hydroxybenzamide

Hydrochloride (28). White solid (m = 54 mg, 80% yield); mp: 268−

1

269 °C; H NMR (DMSO-d₆, 300 MHz): δ ppm 1.30 (s, 9H), 3.56

tert-Butyl (4-(7-Chloro-1-hydroxy-2-naphthoyl))-

(s, 1H), 3.99 (s, 2H), 6.97 (d, 1H, J = 8.2 Hz), 7.50 (m, 3H), 7.72 (d,

2H, J = 7.0 Hz), 7.94 (s, 1H), 8.42 (bs, 3H), 10.52 (s, 1H); ¹³C NMR

(DMSO-d₆, 75 MHz): δ ppm 31.2, 41.8, 66.3, 116.7, 116.9, 120.9,

122.8, 125.5, 129.4, 130.6, 138.3, 141.3, 155.7, 166.3; HPLC, RT =

1.45 min; HRMS calcd for C₁₈H₂₃N₂O₂299.1754, found 299.1752.

(2-(4-Aminomethylphenyl)aminomethyl)-4-tert-butylphenol Hy-

drochloride (29). White solid (m = 52 mg, 73% yield); mp: 258−260

°C (dec.); ¹H NMR (DMSO-d₆, 300 MHz): δ ppm 1.20 (s, 9H), 3.93

(d, 2H, J = 5.2 Hz), 4.31 (bs, 2H), 6.81 (d, 1H, J = 8.4 Hz), 7.15 (m,

3H), 7.42 (m, 3H), 8.45 (bs, 3H), 10.43 (s, 1H); ¹³C NMR (DMSO-

d₆, 75 MHz): δ ppm 31.4, 33.7, 41.7, 46.3, 114.7, 119.0, 120.0, 123.2,

125.8, 127.5, 130.0, 130.3, 140.9, 153.3; HPLC, RT = 1.39 min;

HRMS calcd for C₁₈H₂₅N₂O 285.1961, found 285.1954.

N-(4-(Aminomethyl)phenyl)-2-hydroxybenzamide Hydrochlor-

ide (30). White solid (m = 45 mg, 81% yield); mp: 275−276 °C

(dec.); ¹H NMR (DMSO-d₆, 300 MHz): δ ppm 3.99 (d, 2H, J = 4.9

Hz), 6.96 (t, 1H, J = 7.5 Hz), 7.04 (d, 1H, J = 8.4 Hz), 7.43 (dd, 1H, J

= 8.4, 7.2 Hz), 7.49 (d, 2H, J = 8.4 Hz), 7.75 (d, 2H, J = 8.4 Hz), 8.01

(d, 1H, J = 7.2 Hz), 8.45 (bs, 3H), 10.51 (s, 1H); ¹³C NMR (DMSO-

d₆, 75 MHz): δ ppm 41.7, 117.2, 117.5, 119.0, 120.8, 129.2, 129.4,

129.5, 133.6, 138.3, 158.2, 166.4; HPLC, RT = 1.05 min; MS (ESI+):

m/z 244.1 [M + H + 1]⁺; HRMS calcd for C₁₄H₁₅N₂O₂243.1128,

found 243.1129.

aminobenzylcarbamate (24). White solid (m = 101 mg, 53%

1

yield); H NMR (CDCl₃, 400 MHz): δ ppm 1.58 (s, 9H), 4.32 (d,

2H, J = 5.7 Hz), 4.88 (bs, 1H), 7.33 (m, 3H), 7.48 (d, 1H, J = 8.8

Hz), 7.55 (dd, 1H, J = 8.8, 2.2 Hz), 7.57 (d, 2H, J = 8.3 Hz), 7.72 (d,

1H, J = 8.7 Hz), 8.00 (s, 1H), 8.43 (d, 1H, J = 2.2 Hz), 13.51 (s, 1H);

¹³C NMR (CDCl₃, 400 MHz): δ ppm 28.6, 44.4, 79.9, 107.7, 118.4,

121.1, 121.7, 123.4, 126.6, 128.5, 129.2, 130.2, 132.4, 134.7, 135.9,

136.4, 156.1, 160.6, 169.0; HPLC, RT = 2,12 min; MS (ESI+): m/z

371.1 [M + H − t-Bu]⁺, 373.1 [M + H − t-Bu + 2]⁺, 444.1 [M +

NH₄]⁺, 446.1 [M + NH₄+ 2]⁺, 427.1 [M + H]⁺, 429.1 [M + H + 2]⁺.

tert-Butyl (4-(6,7-Dimethoxy-1-hydroxy-2-naphthoyl))-

aminobenzylcarbamate (25). Elution: n-Hex/AcOEt 2/1 v/v;

white solid (m = 140 mg, 69% yield); ¹H NMR (CDCl₃, 400

MHz): δ ppm 1.47 (s, 9H), 4.02 (s, 3H), 4.04 (s, 3H), 4.31 (d, 2H, J

= 5.4 Hz), 4.87 (bs, 1H), 7.07 (s, 1H), 7.18 (d, 1H, J = 8.7 Hz), 7.30

(d, 2H, J = 8.3 Hz), 7.36 (d, 1H, J = 8.8 Hz), 7.56 (d, 2H, J = 8.3 Hz),

7.70 (s, 1H), 8.01 (s, 1H), 13.44 (s, 1H); ¹³C NMR (CDCl₃, 100

MHz): δ ppm 28.6, 44.4, 56.2, 56.3, 79.8, 102.9, 106.1, 106.5, 117.3,

119.5, 120.7, 121.7, 128.5, 131.1, 133.0, 136.0, 136.3, 149.6, 152.1,

160.2, 169.5; HPLC, RT = 3.81 min; MS (ESI+): m/z 397.2 [M + H

− t-Bu]⁺, 453.3 [M + H]⁺, 475.3 [M + Na]⁺.

Synthesis of tert-Butyl ((4-(5-tert-Butyl-2-hydroxybenzyl)amino)-

benzyl)carbamate (29). To a solution of 100 mg of compound 2

(0.45 mmol) in 2.5 mL of ethanol was added 80 mg of aldehyde 26

(0.45 mmol, 1 equiv). The solution was stirred at reﬂux for 6 h. After

cooling to room temperature, 9 mg of sodium borohydride (0.235

mmol, 0.5 equiv) was added and the solution was stirred at room

temperature for 1 h 30 min. Then, 20 mL of water was added and the

solution was extracted with ethyl acetate (3 × 20 mL). The organic

layer was dried over Na₂SO₄, ﬁltered, and the solvent was evaporated

in vacuo to oﬀer compound 30 as a white solid (m = 110 mg, 64%

N-(4-(Aminomethyl)phenyl)-3-methyl-2-hydroxybenzamide Hy-

drochloride (31). Beige solid (m = 54 mg, 92% yield); mp: 275−276

1

°C; H NMR (DMSO-d₆, 400 MHz): δ ppm 2.20 (s, 3H), 4.00 (q,

2H, J = 5.6 Hz), 6.88 (t, 1H, J = 7.7 Hz), 7.38 (d, 1H, J = 7.7 Hz),

7.51 (d, 2H, J = 8.4 Hz), 7.75 (d, 2H, J = 8.4 Hz), 8.00 (d, 1H, J = 7.7

Hz), 8.45 (bs, 3H), 10.59 (s, 1H), 12.53 (s, 1H); ¹³C NMR (DMSO-

d₆, 100 MHz): δ ppm 15.5, 41.7, 114.1, 118.0, 121.9, 125.5, 126.1,

129.4, 130.1, 135.1, 137.8, 159.2, 169.2; HPLC, RT = 1.22 min;

HRMS calcd for C₁₅H₁₇N₂O₂257.1285, found 257.1292.

1

yield); mp 102.0−103.0 °C; H NMR (CDCl₃, 300 MHz): δ ppm

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J. Med. Chem. 2021, 64, 5667−5688

Journal of Medicinal Chemistry

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Article

N-(4-(Aminomethyl)phenyl)-4-methyl-2-hydroxybenzamide Hy-

drochloride (32). Beige solid (m = 53 mg, 90% yield); mp: 282−283

°C (dec.); ¹H NMR (DMSO-d₆, 400 MHz): δ ppm 2.30 (s, 3H), 3.98

(q, 2H, J = 5.6 Hz), 6.78 (d, 1H, J = 8.2 Hz), 6.84 (s, 1H), 7.48 (d,

2H, J = 8.4 Hz), 7.74 (d, 2H, J = 8.4 Hz), 7.94 (d, 1H, J = 8.2 Hz),

8.43 (bs, 3H), 10.46 (s, 1H); ¹³C NMR (DMSO-d₆, 100 MHz): δ

ppm 21.1, 41.8, 114.2, 117.5, 120.1, 120.9, 129.0, 129.5, 130.3, 138.3,

144.4, 158.8, 166.7; HPLC, RT = 1.11 min; HRMS calcd for

C₁₅H₁₇N₂O₂257.1285, found 257.1293.

2 N-(4-(Aminomethyl)phenyl)-5-methyl-2-hydroxybenzamide

Hydrochloride (33). White solid (m = 58 mg, 99% yield); mp:

292−293 °C (dec.); ¹H NMR (DMSO-d₆, 400 MHz): δ ppm 2.28 (s,

3H), 3.98 (q, 2H, J = 5.4 Hz), 6.92 (d, 1H, J = 8.3 Hz), 7.24 (d, 1H, J

= 8.3 Hz), 7.48 (d, 2H, J = 8.4 Hz), 7.74 (d, 2H, J = 8.4 Hz), 7.82 (s,

1H), 8.42 (bs, 3H), 10.49 (s, 1H), 11.61 (s, 1H); ¹³C NMR (DMSO-

d₆, 100 MHz): δ ppm 20.0, 41.8, 117.1, 120.7, 127.7, 129.1, 129.5,

130.2, 134.3, 138.4, 156.1, 166.5; HPLC, RT = 1.13 min; HRMS

calcd for C₁₅H₁₇N₂O₂257.1285, found 257.1289.

d₆, 100 MHz): δ ppm 41.8, 103.8 (d, J = 24.2 Hz), 106.5 (d, J = 24.2

Hz), 114.5, 120.9, 129.4, 129.6, 131.5 (d, J = 11 Hz), 138.2, 160.4 (d,

J = 13.2 Hz), 164.8 (d, J = 249 Hz), 165.7; HPLC, RT = 1.09 min;

HRMS calcd for C₁₄H₁₄FN₂O₂261.1034, found 261.1034.

N-(4-(Aminomethyl)phenyl)-1-hydroxy-2-naphthamide Hydro-

chloride (40). White solid (m = 63 mg, 99% yield); mp: 277−278

°C (dec.); ¹H NMR (DMSO-d₆, 300 MHz): δ ppm 4.02 (s, 2H), 7.46

(d, 1H, J = 8.9 Hz), 7.53 (d, 2H, J = 8.3 Hz), 7.59 (d, 1H, J = 7.5 Hz),

7.68 (t, 1H, J = 7.5 Hz), 7.80 (d, 2H, J = 8.3 Hz), 7.92 (d, 1H, J = 8.0

Hz), 8.22 (d, 1H, J = 8.9 Hz), 8.31 (d, 1H, J = 8.3 Hz), 8.48 (bs, 3H),

10.43 (s, 1H); ¹³C NMR (DMSO-d₆, 75 MHz): δ ppm 41.8, 107.5,

117.8, 122.1, 123.1, 123.2, 124.6, 125.9, 127.5, 129.2, 129.4, 130.2,

136.0, 137.8, 160.0, 169.6; HPLC, RT = 1.45 min; MS (ESI+): m/z

294.1 [M + H + 1]⁺; HRMS calcd for C₁₈H₁₇N₂O₂293.1290, found

293.1289.

N-(4-(Aminomethyl)phenyl)-2-hydroxy-1-naphthamide Hydro-

chloride (41). White powder (m = 57 mg, 97% yield); mp: 248−

249 °C; ¹H NMR (DMSO-d₆, 300 MHz): δ ppm 3.98 (d, 2H, J = 5.3

Hz), 7.32 (m, 2H), 7.45 (m, 3H), 7.67 (d, 1H, J = 8.3 Hz), 7.84 (m,

4H), 8.42 (bs, 3H), 10.22 (bs, 1H), 10.43 (s, 1H); ¹³C NMR

(DMSO-d₆, 75 MHz): δ ppm 41.9, 118.4, 119.1, 122.9, 123.2, 126.9,

127.3, 127.9, 128.6, 129.5, 130.1, 131.3, 139.7, 151.7, 165.8; HPLC,

RT = 1.11 min; MS (ESI+): m/z 294.1 [M + H + 1]⁺; HRMS calcd

for C₁₈H₁₇N₂O₂293.1285, found 293.1293.

N-(4-(Aminomethyl)phenyl)-6-methyl-2-hydroxybenzamide Hy-

drochloride (34). White solid (m = 58 mg, 99% yield); mp: 251−252

°C (dec.); ¹H NMR (CD₃OD, 300 MHz): δ ppm 2.32 (s, 3H), 4.10

(s, 2H), 6.72 (d, 1H, J = 8.0 Hz), 6.74 (d, 1H, J = 8.0 Hz), 7.13 (t,

1H, J = 8.0 Hz), 7.44 (d, 2H, J = 8.5 Hz), 7.78 (d, 2H, J = 8.5 Hz);

¹³C NMR (DMSO-d₆, 100 MHz): δ ppm 19.2, 44.0, 114.0, 121.7,

N-(4-(Aminomethyl)phenyl)-3-hydroxy-2-naphthamide Hydro-

chloride (42). White powder (m = 40 mg, 61% yield); mp: 299−

300 °C (dec.); ¹H NMR (DMSO-d₆, 400 MHz): δ ppm 4.00 (s, 2H),

7.37 (m, 2H), 7.51 (m, 3H), 7.76 (d, 1H, J = 8.3 Hz), 7.80 (d, 2H, J =

8.4 Hz), 7.94 (d, 1H, J = 8.3 Hz), 8.35 (bs, 3H), 8.53 (s, 1H), 10.70

(s, 1H), 11.39 (bs, 1H); ¹³C NMR (DMSO-d₆, 100 MHz): δ ppm

41.8, 110.6, 120.4, 121.8, 123.7, 125.8, 126.9, 128.1, 128.7, 129.4,

129.6, 130.6, 135.8, 138.7, 153.6, 165.6; HPLC, RT = 1.26 min;

HRMS calcd for C₁₈H₁₇N₂O₂293.1285, found 293.1286.

122.1, 126.6, 129,9, 130.6, 131.1, 137.5, 141.0, 155.5, 170.1; HPLC,

RT = 0.88 min; HRMS calcd for C₁₅H₁₇N₂O₂257.1285, found

257.1287.

N-(4-(Aminomethyl)phenyl)-5-bromo-2-hydroxybenzamide Hy-

drochloride (35). Yellow solid (m = 71 mg, 99% yield); mp: 309−

1

310 °C; H NMR (DMSO-d₆, 400 MHz): δ ppm 4.00 (s, 2H), 7.01

(d, 1H, J = 8.8 Hz), 7.47 (d, 2H, J = 8.6 Hz), 7.58 (dd, 1H, J = 8.8,

2.4 Hz), 7.73 (d, 2H, J = 8.6 Hz), 8.07 (d, 1H, J = 2.4 Hz), 8.32 (bs,

3H), 10.50 (s, 1H), 11.90 (bs, 1H); ¹³C NMR (CDCl₃, 100 MHz): δ

ppm 41.8, 110.2, 119.5, 120.7, 129.5, 129.7, 131.4, 135.9, 138.2,

157.1, 164.8; HPLC, RT = 1.21 min; HRMS calcd for C₁₄H₁₄N₂O₂Br

321.0233, found 321.0236.

N-(4-(Aminomethyl)phenyl)-2-naphthamide Hydrochloride (43).

White powder (m = 26 mg, 61% yield); mp: 285−286 °C (dec.): ¹H

NMR (DMSO-d₆, 500 MHz): δ ppm 4.00 (q, 2H, J = 6.0 Hz), 7.50

(d, 2H, J = 8.5 Hz), 7.64 (m, 2H), 7.88 (d, 2H, J = 8.5 Hz), 8.01 (dd,

1H, J = 7.6, 1.4 Hz), 8.05 (m, 2H), 8.10 (dd, 1H, J = 7.3, 1.3 Hz),

8.47 (bs, 3H), 8.64 (s, 1H), 10.63 (s, 1H); ¹³C NMR (DMSO-d₆, 125

MHz): δ ppm 41.9, 120.3, 124.5, 126.9, 127.7, 128.0, 128.1, 128.2,

129.0, 129.2, 129.5, 132.0, 132.1, 134.3, 139.4, 165.7; HPLC, RT =

1.21 min; HRMS calcd for C₁₈H₁₇N₂O₂277.1335, found 277.1326.

N-[4-(Aminomethyl)phenyl]-1H-indole-2-carboxamide (44).

N-(4-(Aminomethyl)phenyl)-5-methoxy-2-hydroxybenzamide

Hydrochloride (36). White solid (m = 55 mg, 90% yield); mp: 277.5−

278.5 °C; ¹H NMR (DMSO-d₆, 300 MHz): δ ppm 3.76 (s, 3H), 4.00

(s, 2H), 6.95 (d, 1H, J = 8.8 Hz), 7.06 (dd, 1H, J = 8.8, 2.2 Hz), 7.47

(d, 2H, J = 8.1 Hz), 7.50 (d, 1H, J = 2.2 Hz), 7.74 (d, 2H, J = 8.1 Hz),

8.22 (bs, 3H), 10.54 (s, 1H); ¹³C NMR (DMSO-d₆, 75 MHz): δ ppm

41.8, 55.7, 112.7, 117.5, 118.2, 120.6, 120.8, 129.5, 129.6, 138.3,

151.8, 152.0, 165.9; HPLC, RT = 1.10 min; HRMS calcd for

C₁₅H₁₇N₂O₃273.1234, found 273.1240.

1

White solid (m = 40 mg, 76% yield); mp: 291−292 °C (dec.); H

NMR (DMSO-d₆, 300 MHz): δ ppm 3.98 (q, 2H, J = 5.7 Hz), 7.07

(t, 1H, J = 7.8 Hz), 7.22 (t, 1H, J = 7.8 Hz), 7.47 (m, 4H), 7.67 (d,

1H, J = 8.0 Hz), 7.87 (d, 2H, J = 8.6 Hz), 8.40 (bs, 3H), 10.47 (s,

1H), 11.88 (s, 1H); ¹³C NMR (DMSO-d₆, 100 MHz): δ ppm 41.9,

104.4, 112.4, 119.9, 120.0, 121.8, 123.9, 127.0, 128.9, 129.5, 131.4,

136.8, 139.2, 159.7; HPLC, RT = 1.82 min; HRMS calcd for

C₁₆H₁₆N₃O 266.1288, found 266.1284.

N-(4-(Aminomethyl)phenyl)-4-isopropyl-2-hydroxybenzamide

Hydrochloride (37). White solid (m = 63 mg, 56% yield); mp: 282−

283 °C; ¹H NMR (DMSO-d₆, 400 MHz): δ ppm 1.20 (d, 6H, J = 6.8

Hz), 2.87 (hept, 1H, J = 6.8 Hz), 3.99 (d, 2H, J = 3.9 Hz), 6.87 (m,

2H), 7.48 (d, 2H, J = 8.3 Hz), 7.74 (d, 2H, J = 8.3 Hz), 7.95 (d, 1H, J

= 7.9 Hz), 8.39 (bs, 3H), 10.5 (s, 1H), 11.97 (bs, 1H); ¹³C NMR

(DMSO-d₆, 100 MHz): δ ppm 23.3, 33.3, 41.8, 114.6, 114.7, 117.4,

120.8, 129.0, 129.4, 138.3, 155.0, 158.8, 166.7; HPLC, RT = 1.33

min; HRMS calcd for C₁₇H₂₁N₂O₂285.1598, found 285.1603.

N-(4-(Aminomethyl)phenyl)-4-triﬂuoromethyl-2-hydroxybenza-

mide Hydrochloride (38). White solid (m = 67 mg, 96% yield); mp:

N-[4-(Aminomethyl)phenyl]-4-oxo-1,4-dihydroquinoline-3-car-

boxamide (45). White solid (m = 65 mg, 99% yield); mp: 287−289

1

°C (dec.); H NMR (DMSO-d₆, 400 MHz): δ ppm 3.96 (q, 2H, J =

5.5 Hz), 7.50 (d, 2H, J = 8.4 Hz), 7.53 (td, 1H, J = 7.9, 1.3 Hz), 7.76

(d, 2H, J = 8.4 Hz), 7.79−7.86 (m, 2H), 8.31 (d, 1H, J = 7.9 Hz),

8.53 (bs, 3H), 8.82 (d, 1H, J = 6.7 Hz), 12.56 (s, 1H), 13.62 (d, 1H, J

= 6.4 Hz); ¹³C NMR (DMSO-d₆, 100 MHz): δ ppm 41.8, 110.3,

119.2, 119.5, 125.3, 125.4, 125.9, 128.9, 129.9, 133.0, 138.9, 139.1,

143.9, 163.0, 176.3; HPLC, RT = 1.04 min; HRMS calcd for

C₁₇H₁₆N₃O₂294.1237, found 294.1247.

1

270−272 °C (dec.); H NMR (DMSO-d₆, 400 MHz): δ ppm 3.99

(bs, 2H), 7.28 (d, 1H, J = 7.9 Hz), 7.39 (s, 1H), 7.48 (d, 2H, J = 8.1

Hz), 7.76 (d, 2H, J = 8.1 Hz), 8.03 (d, 1H, J = 7.9 Hz), 8.38 (bs, 3H),

10.54 (s, 1H), 11.97 (bs, 1H); ¹⁹F (DMSO-d₆, 400 MHz): −61.77;

¹³C NMR (DMSO-d₆, 100 MHz): δ ppm 41.8, 113.4, 115.3, 120.3,

122.1, 123.5, 129.5, 130.7, 132.3, 132.6, 138.4, 157.1, 164.5; HPLC,

RT = 1.29 min; MS (ESI+): m/z 312.1 [M + H]⁺, 294.1 [M + H −

NH₃]⁺; HRMS calcd for C₁₅H₁₄F₃N₂O₂311.1002, found 311.1005.

N-(4-(Aminomethyl)phenyl)-4-ﬂuoro-2-hydroxybenzamide Hy-

drochloride (39). White solid (m = 45 mg, 76% yield); mp: 280−

281 °C; ¹H NMR (DMSO-d₆, 400 MHz): δ ppm 3.99 (s, 2H), 6.88−

6.80 (m, 2H), 7.48 (d, 2H, J = 8.4 Hz), 7.74 (d, 2H, J = 8.4 Hz), 8.08

(dd, 1H, J = 8.7, 8.7 Hz), 8.36 (bs, 3H), 10.47 (s, 1H), 12.36 (bs,

1H); ¹⁹F NMR (DMSO-d₆, 400 MHz): −105.70; ¹³C NMR (DMSO-

N-(4-(Aminomethyl)phenyl)-3-hydroxypyridine-2-carboxamide

Hydrochloride (46). Beige solid (m = 28 mg, 44% yield); mp: 251−

252 °C; ¹H NMR (DMSO-d₆, 300 MHz): δ ppm 4.00 (q, 2H, J = 5.4

Hz), 4.78 (bs, 3H), 7.50 (m, 3H), 7.61 (dd, 1H, J = 8.5, 4.3 Hz), 7.87

(d, 2H, J = 8.6 Hz), 8.27 (dd, 1H, J = 4.3, 1.1 Hz), 8.35 (bs, 2H),

10.99 (s, 1H); ¹³C NMR (DMSO-d₆, 75 MHz): δ ppm 41.8, 121.3,

126.5, 129.4, 129.6, 130.2, 131.2, 13 7.3, 139.8, 157.6, 167.3; HPLC,

RT = 0.95 min; HRMS calcd for C₁₃H₁₄N₃O₂244.1081, found

244.1102.

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N-(4-(Aminomethyl)phenyl)-6-methoxy-1-hydroxy-2-naphtha-

mide Hydrochloride (47). Beige solid (m = 65 mg, 91% yield); mp:

256−257 °C (dec.); ¹H NMR (DMSO-d₆, 300 MHz): δ ppm 3.91 (s,

3H), 4.02 (s, 2H), 7.19 (dd, 1H, J = 9.2, 2.5 Hz), 7.35 (m, 2H), 7.51

(d, 2H, J = 8.6 Hz), 7.77 (d, 2H, J = 8.6 Hz), 8.14 (d, 1H, J = 9.2 Hz),

8.20 (d, 1H, J = 9.2 Hz), 8.36 (bs, 3H), 10.55 (bs, 1H), 14.00 (s,

1H); ¹³C NMR (DMSO-d₆, 75 MHz): δ ppm 41.8, 55.4, 105.7, 106.4,

117.0, 117.9, 119.4, 122.0, 123.9, 125.0, 129.4, 130.1, 137.9, 138.1,

159.9, 160.3, 169.7; HPLC, RT = 1.36 min; HRMS calcd for

C₁₉H₁₉N₂O₃323.1390, found 323.1397.

N-(4-(Aminomethyl)phenyl)-7-methoxy-1-hydroxy-2-naphtha-

mide Hydrochloride (48). White solid (m = 55 mg, 77% yield); mp:

250−251 °C; ¹H NMR (DMSO-d₆, 400 MHz): δ ppm 3.91 (s, 3H),

4.02 (q, 2H, J = 5.0 Hz), 7.32 (dd, 1H, J = 8.9, 2.6 Hz), 7.42 (d, 1H, J

= 8.8 Hz), 7.51 (d, 2H, J = 8.6 Hz), 7.60 (d, 1H, J = 2.6 Hz), 7.79 (d,

2H, J = 8.6 Hz), 7.85 (d, 1H, J = 8.9 Hz), 8.02 (d, 1H, J = 8.9 Hz),

8.36 (bs, 3H), 10.59 (s, 1H), 13.86 (s, 1H); ¹³C NMR (DMSO-d₆,

100 MHz): δ ppm 41.8, 55.2, 101.5, 107.9, 117.7, 120.6, 121.2, 122.1,

125.6, 129.2, 129.4, 130.1, 131.3, 137.8, 157.4, 158.8, 169.7; HPLC,

RT = 1.37 min; HRMS calcd for C₁₉H₁₉N₂O₃323.1390, found

323.1403.

enediaminetetraacetic acid (EDTA) (Sigma-Aldrich) to obtain an

active KLK1 stock at the same concentration of 100 μg/mL. Plasmin

(Sigma-Aldrich) and caspase-2, caspase-3, and caspase-6 (Enzo Life

Sciences) were purchased in mature and active forms. All enzyme

stocks were stored at −20 °C. KLK4, KLK5, KLK7, KLK8, KLK11,

KLK13, thrombin, cathepsin L, matriptase, trypsin, trypsin-3, and tPA

were purchased from R&D Systems as pro-enzymes (KLK4, KLK7,

KLK8, KLK11, KLK13) or as mature and active forms (KLK5,

thrombin, cathepsin L, matriptase, trypsin-3, tPA). Activation of

KLK4, KLK7, KLK8, KLK11, and KLK13 is performed in optimized

buﬀers. The activation reactions for KLK4, KLK7, and KLK11 are

initiated bacterial thermolysin at 0.4 μg/mL (Sigma-Aldrich) and

stopped by the addition of 100 mM EDTA (Sigma-Aldrich) to obtain

active stocks. All enzyme stocks were stored at −20 °C. H-PFR-AMC

(KLK1), Boc-VPR-AMC (KLK4, KLK5, KLK8, KLK13, KLK14,

Thrombin), Boc-QAR-AMC (KLK6, KLK11, matriptase, trypsin,

trypsin-3, plasmin), Ac-VDVAD-AMC (caspase-2), Ac-DEVD-AMC

(caspase-3), Ac-VEID-AMC (caspase-6), Z-LR-AMC (cathepsin L),

and Z-GGR-AMC (tPA) substrates were purchased from Bachem.

MeO-Suc-RPY-AMC (KLK7) substrate was purchased from AAT

Bioquest.

N-(4-(Aminomethyl)phenyl)-7-methyl-1-hydroxy-2-naphtha-

mide Hydrochloride (49). White solid (m = 53 mg, 77% yield); mp:

276−278 °C (dec.); ¹H NMR (DMSO-d₆, 500 MHz): δ ppm 3.34 (s,

3H), 4.01 (s, 2H), 7.40 (d, 1H, J = 8.7 Hz), 7.51 (m, 3H), 7.80 (m,

3H), 8.08 (s, 1H), 8.12 (d, 1H, J = 8.7 Hz), 8.44 (bs, 3H), 10.62 (s,

1H), 13.94 (s, 1H); ¹³C NMR (DMSO-d₆, 125 MHz): δ ppm 21.4,

41.8, 107.5, 117.6, 122.0, 122.1, 122.2, 124.7, 127.4, 129.4, 130.2,

131.2, 134.2, 135.4, 137.8, 159.6, 169.7; HPLC, RT = 1.43 min;

HRMS calcd for C₁₉H₁₉N₂O₂307.1441, found 307.1452.

N-(4-(Aminomethyl)phenyl)-7-chloro-1-hydroxy-2-naphthamide

Hydrochloride (50). White solid (m = 55 mg, 76%); mp: 277−278

°C; ¹H NMR (DMSO-d₆, 500 MHz): δ ppm 4.02 (q, 2H, J = 5.6 Hz),

7.53 (m, 3H), 7.70 (dd, 1H, J = 8.8, 2.2 Hz), 7.79 (d, 2H, J = 8.6 Hz),

7.99 (d, 1H, J = 8.8 Hz), 8.24 (m, 2H), 8.39 (bs, 3H), 10.73 (s, 1H);

¹³C NMR (DMSO-d₆, 100 MHz): δ ppm 108.7, 117.8, 121.8, 122.2,

Kinetics Assays. Compounds were screened on KLK6, KLK1,

and plasmin using a BMG Fluostar microplate reader (black 96-well

microplates). The proteases are preincubated for 15 min with each

compound (10 and 50 μM) or with DMSO (<2%, negative control)

in a total volume of 100 μL of 50 mM Tris buﬀer, 1 M citrate, 0.05%

Brij-35, pH 7, 37 °C. The reaction is triggered by adding the Boc-

QAR-AMC (KLK6, plasmin) or H-PFR-AMC (KLK1) ﬂuorogenic

substrate (100 μM) and followed for 30 min at 37 °C. The release of

the AMC ﬂuorescent group is detected using the following

wavelengths: λ_ex= 360 nm for the excitation and λ_em= 460 nm for

the measurement of the emission. The percent inhibition is calculated

from eq 1, where V₀is the initial rate of the DMSO control and V_iis

the initial rate in the presence of the inhibitor.

% inhibition = (1 − (V/V )) × 100)

(1)

i

0

123.8, 125.4, 129.5, 129.6, 129.9, 130.4, 130.8, 134.4, 137.7, 158.8,

169.3; HPLC, RT = 1.49 min; HRMS calcd for C₁₈H₁₆N₂O₂Cl

327.0895, found 327.0890.

The selected compounds are those for which the inhibition is greater

than 50% at a concentration of 10 μM; then, IC₅₀is determined. The

inhibitory eﬀect of the compound (%) as a function of its

concentration generally follows eq 2a,b, which results in a hyperbole

or a sigmoid. The equation is entered in the Kaleidagraph 4.5 software

for curve ﬁtting f([I]) = % inhibition, where [I] is the inhibitor

concentration. The concentration ranges of inhibitor were adjusted to

inhibitory potency as detected in preliminary screening tests; [I] was

from 0.1 to 100 μM.

N-(4-(Aminomethyl)phenyl)-6,7-dimethoxy-1-hydroxy-2-naph-

thamide Hydrochloride (51). White solid (m = 62 mg, 80%); mp:

289−291 °C (dec.); ¹H NMR (DMSO-d₆, 400 MHz): δ ppm 3.90 (s,

3H), 3.92 (s, 3H), 4.01 (q, 2H, J = 6.0 Hz), 7.31 (d, 1H, J = 8.8 Hz),

7.33 (s, 1H), 7.51 (d, 2H, J = 8.6 Hz), 7.55 (s, 1H), 7.79 (d, 2H, J =

8.6 Hz), 8.03 (d, 1H, J = 8.8 Hz), 8.45 (bs, 3H), 10.53 (s, 1H), 13.85

(s, 1H); ¹³C NMR (DMSO-d₆, 100 MHz): δ ppm 55.4, 55.6, 66.3,

101.8, 106.1, 106.7, 116.6, 119.2, 121.4, 121.9, 129.4, 130.0, 132.5,

137.9, 149.0, 151.6, 158.8, 169.8; HPLC, RT = 2.28 min; HRMS

calcd for C₂₀H₂₁N₂O₄353.1496, found 353.1496.

The inhibitory activity of compounds was expressed as IC₅₀

(inhibitor concentrations giving 50% inhibition). The values of IC₅₀

were calculated by ﬁtting the experimental data to eq 2a,b

% inhibition = 100 × (1 − V/V ) = 100[I]₀/(IC₅₀+ [I]₀)

i

0

1-Hydroxy-N-piperidin-4-yl-2-naphthamide (53). White solid (m

1

(2a)

(2b)

= 43 mg, 70% yield); mp: 279−280 °C; H NMR (DMSO-d₆, 400

MHz): δ ppm 1.89−2.02 (m, 4H), 3.02 (m, 2H), 3.33 (d, 2H, J =

12.8 Hz), 4.19 (m, 1H), 7.37 (d, 1H, J = 8.8 Hz), 7.55 (td, 1H, J =

8.1, 1.1 Hz), 7.63 (td, 1H, J = 8.1, 1.2 Hz), 7.87 (d, 1H, J = 8.1 Hz),

8.03 (d, 1H, J = 8.8 Hz), 8.25 (d, 1H, J = 8.1 Hz), 8.98 (d, 1H, J = 8.1

Hz), 9.08 (bs, 2H), 14.50 (s, 1H); ¹³C NMR (DMSO-d₆, 100 MHz):

δ ppm 27.9, 44.5, 66.3, 106.9, 117.4, 122.9, 123.0, 124.6, 125.7, 127.4,

128.9, 135.8, 159.7, 170.1; HPLC, RT = 1.28 min; HRMS calcd for

C₁₆H₁₉N₂O₂271.1441, found 271.1439.

n_H

% inhibition = 100[I]₀/(IC₅₀+ [I]₀

where n_His the Hill number.

)

Reversibility was analyzed by diluting the reaction mixtures

(dilution factor of 100) after 15 and 60 min preincubation of the

enzyme with inhibitor. Aliquots of reaction mixtures (2.5 μL) were

added to 97.5 μL of buﬀer containing the ﬂuorogenic substrate

(experimental conditions identical to the routine protocol used for a

given enzyme). The mechanism of inhibition was determined by

varying substrate and inhibitor concentrations, type of inhibition, and

inhibition parameter (K_i), which are determined by Dixon’s plots.

Selectivity Proﬁling. Hit compounds were screened on the

selected group of CNS proteases using optimized concentrations and

buﬀers (KLK4 9.6 nM; KLK5 0.85 nM; KLK7 19 nM; KLK8 0.88

nM; KLK11 67 nM; KLK13 0.2 nM; KLK14 0.6 nM; caspase-2 0.2

nM; caspase-3 0.1 nM; caspase-6 68.5 mU/μL; cathepsin L 0.12 nM;

matriptase 0.69 nM; thrombin 25 pM; tPA 6.7 nM; trypsin 0.63 pM;

trypsin-3 0.8 pM; plasmin 3 nM). The reaction is triggered by the

appropriate substrate at the optimized concentration (H-PFR-AMC

Enzymes and Chemicals. KLK6 was purchased from R&D

Systems as pro-KLK6. Activation of KLK6 is performed in 50 mM

Tris, 0.05% (w/v) Brij-35, pH 8.0. The activation reaction is initiated

by lysyl-endopeptidase at 2.5 mU/mL (Wako Bioproducts). The

activation reaction is stopped by diluting KLK6 to 0.5 μg/mL in assay

buﬀer (50 mM Tris, 1 M citrate, 0.05% (w/v) Brij-35, pH 7.4) to

obtain an active KLK6 stock. KLK1 was purchased from R&D

Systems as pro-KLK1. Activation of KLK1 is performed in 50 mM

Tris−HCl, 10 mM CaCl₂, 150 mM NaCl, 0.05% Brij-35, pH 7.5. The

activation reaction is initiated by bacterial thermolysin at 0.4 μg/mL

(Sigma-Aldrich) and stopped by the addition of 100 mM ethyl-

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100 μM; Boc-VPR-AMC 100 μM; Boc-QAR-AMC 100 μM; Meo-

Suc-RPY-AMC 100 μM; Ac-VDVAD-AMC 25 μM; Ac-DEVD-AMC

10 μM; Ac-VEID-AMC 100 μM; Z-LR-AMC 50 μM; Z-GGR-AMC

100 μM) and followed for 30 min at 37 °C. Percentages of inhibition

at 10 μM of each compound are determined as described in the

previous paragraph. Results are given from three independent

experiments with a standard error below 10%.

Evaluation of the Eﬀect of Compounds on Oligodendrocyte

Diﬀerentiation. B104 Cell Culture. B104 cells were cultured in ﬂasks

(TPP) containing Dulbecco’s modiﬁed Eagle’s medium (DMEM)

(Gibco) supplemented with 1% penicillin−streptomycin (Gibco), 1%

nonessential amino acids (NEAA) solution (Gibco), and 10% fetal

bovine serum (FBS; Gibco) and passaged every week using trypsin

0.25% (Gibco; 25200056). This medium was changed after 4 days for

N1 medium w/o biotin. This B104-conditioned medium containing

growth factors was collected after 3 days and used to prepare N1B104

medium.

Molecular Docking. Molecular docking experiments were con-

ducted to propose interaction models of the hit para-aminobenzyl

derivatives 32 and 42 with KLK6. The protonation states of the

ligands were calculated using MarvinSketch at pH = 7. The major

microspecies described the primary amine function charged, while the

rest of the molecules were neutral (protonated alcohol function). The

three-dimensional (3D) conformations of both ligands were

generated by MarvinSketch. The structure of the target was retrieved

from the Protein Data Bank (PDB) and chose due to the presence of

an orthosteric inhibitor similar in structure to the para-aminobenzyl

derivatives to shape the binding pocket to these two compounds

(PDB id: 4D8N^33,45). The protonation state of the target was

predicted with Propka.⁴⁶The catalytic histidine was found charged

and protonated on both nitrogens, while other residues and termini

were found in their canonical state at pH 7. The ligands and target

were prepared using Autodock Tools 4⁴⁷before ﬂexible molecular

docking with Autodock Vina.⁴⁸Q192 and S195 side chains were

found in diﬀerent orientations in other KLK6 structures. These two

residues were set ﬂexible in the molecular docking routine to increase

the reliability of the results. Including this protein ﬂexibility, the

number of degrees of freedom (dof) was still in an acceptable range

for Vina (10 dof, while Vina is designed to converge properly until 12

dof). The box was centered near the geometrical center of the S1

binding pocket (residue: H57, D189 to S195, S214 to N217, C220),

with a ﬁnal size of 18 × 19 × 18 Å³, which largely included S1, S1′, S2,

and S2′. Twenty poses were asked with an exhaustiveness of 128. The

poses were then rescored using Convex-PL,⁴⁹and the best poses

according to this new scoring function of both compounds into KLK6

were further analyzed. The coordinate ﬁles of both poses are available

in the online version.

Neuronal Cytotoxicity of Hit Compounds. All animals were

ethically maintained and used in compliance with the European Policy

on Ethics. Cortices and striatum were microdissected from E14

embryos of Swiss mice (Janvier, Labs Le Genest Saint Isle, France) in

Dulbecco’s phosphate-buﬀered saline (DPBS) supplemented with

0.1% (w/w) glucose (Thermo Fisher Scientiﬁc; 15023-021).

Dissected structures were digested with trypsin/EDTA (Thermo

Fisher Scientiﬁc; R001100) for 15 min at room temperature. After

trypsin inactivation with 10% (v/v) fetal bovine serum (FBS)

(10500056; Thermo Fisher Scientiﬁc), structures were mechanically

dissociated with a pipette in Neurobasal media (Thermo Fisher

Scientiﬁc; 2110349) supplemented with DNAse I (Thermo Fisher

Scientiﬁc; EN0525), B27 (Thermo Fisher Scientiﬁc; 17504−044), ^L-

glutamine (Thermo Fisher Scientiﬁc; 25030-024), and penicillin/

streptomycin (Thermo Fisher Scientiﬁc, 1540-122). Cortical and

striatal cells were then seeded at the density of 100 000 cells/well in

sterile transparent 96-well plates previously coated. The cells are

cultured for 7 days at 37 °C in a 5% CO₂atmosphere. The cells were

treated for 24 h with inhibitors at diﬀerent concentrations (10, 25, 50,

or 100 μM), with the vehicle DMSO 1% (negative control, vehicle),

or with rotenone 50 μM (positive control). The treatment of primary

cultures of neurons by rotenone is a standard compound used to

induce neuronal death. The medium is then replaced with 50 μL of

XTT (0.3 mg/mL) for 3 h to carry out the cell viability test (Merck;

11465015001). XTT is a derivative of tetrazolium salt whose

reduction by mitochondrial dehydrogenase viable cells shows a

yellow-orange coloring. The activity of the mitochondria is

determined by measuring the absorbance at 485 nm. The percentage

of cell survival is calculated by the ratio of the absorbance in the

presence of the inhibitor to the negative control condition (DMSO

1%). Statistical analyses of diﬀerences between treatments were

assessed by a Kruskal−Wallis test using GraphPad Prism 7.03. For all

analysis, *p-value <0.05; **p-value <0.01; ***p-value <0.001.

CG4 Cell Line Culture. CG4 cells were cultured in culture ﬂasks

(TPP) coated with 0.01% poly-^L-ornithine (Sigma) with ﬁltered

N1B104 medium to ensure proliferation. The cells were passaged with

trypsin 0.05% (Gibco) when reaching 60−70% conﬂuence.⁵²The

CG4 line used in this study expressed the ﬂuorescent reporters GFP

at all developmental stages of the oligodendroglial cell lineage and

mCherry only in diﬀerentiated and mature oligodendrocytes.

For diﬀerentiation assay, CG4 cells were seeded in N1B104

medium in 96-well plates (Nunc) at the density of 6500 cell/cm²for a

few hours. The cells were then switched in a diﬀerentiation medium

(DMEM/F12 1:1 (Gibco) supplemented with 2% B27 (Gibco); 1%

laminin (Sigma) and 1% N1 biotin) with or without the compounds.

The 9-cis-retinoic acid at 1 μM (Sigma) was used as a positive control.

The cells were diﬀerentiated for 5 days before image acquisition and

quantiﬁcation on an Arrayscan XTI System (Thermo Scientiﬁc).

Oligodendrocyte Precursor Cell (OPCs) Primary Cell Cultures.

Primary OPCs cultured were derived from Wistar rats’ cerebral

cortices at P1, as previously described.⁵⁰Brieﬂy, the cerebral cortices

were dissected and the meninges were removed in DME, 1%

penicillin−streptomycin, and 1% MEM NEAA. Tissues were

dissociated enzymatically using trypsin−EDTA solution for 10 min

at 37 °C. The cell suspension was ﬁltered with 70 μm nylon cell

strainer, centrifuged at 1000 rpm for 10 min, and resuspended in an

appropriate volume of DMEM/10% FBS for seeding (10 mL per

ﬂask/two brains in each) in culture ﬂasks coated with 0.01% poly-^L-

ornithine and incubated at 5% CO₂and 37 °C for approximately 2

weeks. The medium was changed on day 7. After 14 days, the cells

were shaken for 2 h at 250 rpm to remove microglia.⁵¹An additional

shaking is performed overnight at 250 rpm to detach and collect

OPCs. Collected OPCs were plated on polyornithine coated 24-well

plates, at a density of 25 000 cells/well. OPCs were kept in

proliferation medium containing DMEM/F12 (1:1) and supple-

mented with 2% B27 (all purchased from Gibco), FGF, and PDGF

(10 ng/mL each) to allow adhesion. The medium is then changed for

a diﬀerentiation medium with or without the compounds. As for the

CG4 cultures, 9-cis-retinoic acid at 1 μM was used as a positive

control. The cells were kept in diﬀerentiation medium for 5 days,

before image acquisition and quantiﬁcation on an Arrayscan XTI

System (Thermo Scientiﬁc).

Immunostaining. Cells were ﬁxed with 2% paraformaldehyde

(electron microscopy) for 5 min at room temperature, washed with

phosphate-buﬀered saline (PBS) 1×, and stored at 4 °C prior to

staining. They were incubated for 1 h at RT in 4% bovine serum

albumin (BSA) (Sigma), 1× PBS, 0.1% Triton X-100, and primary

antibodies. They were next rinsed three times with PBS 1× and

incubated for 45 min at RT in 4% BSA in 1× PBS, 1 μg/mL Hoechst

dye (Sigma, B2261), and secondary antibodies. After incubation,

plates were washed three times with PBS 1×. Image acquisitions and

quantiﬁcation were performed as described above.

The line used is a rat oligodendrocyte precursor (CG4) CG4 cell

line, stably transduced with the GFP reporter gene under the control

of the CMV promoter and mCherry under the control of a

diﬀerentiated oligodendrocyte-speciﬁc promoter. The mCherry

marker is only expressed at the diﬀerentiated oligodendrocyte stage

and thus makes it possible to test compounds capable of inducing

diﬀerentiation of OPCs. The cells are cultured in the proliferation

medium described by Louis et al.⁵²at the time of their enumeration,

then in a medium of diﬀerentiation (DMEM/F12, B27, N1, biotin,

laminin). The cells are treated with diﬀerent inhibitor concentrations

for 72 h in 96-well plates and then screened using an automated

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inverted microscope. The experimental controls used are a basal

control containing only the diﬀerentiation medium and a positive

control, 9-cis-retinoic acid, known for its eﬀect in inducing

diﬀerentiation of OPCs.⁵³To evaluate the eﬀects of the compounds

on OPC diﬀerentiation, we quantiﬁed the number of mCherry+ and

GFP+ cells under each experimental condition relative to the basal

control. An eﬀect in favor of diﬀerentiation is reﬂected in a signiﬁcant

increase in the number of mCherry+ cells, in contrast to an eﬀect to

the detriment of diﬀerentiation. The results are analyzed using the

Mann−Whitney statistical test.

Paris, France; Email: brahim.nait_oumesmar@ sorbonne-

universite.fr

Nicolas Masurier − Institut des Biomolécules Max Mousseron,

Université de Montpellier, CNRS, ENSCM, F-34093

Montpellier, France; Email: nicolas.masurier@

umontpellier.fr

Chahrazade El Amri − Faculty of Sciences and Engineering,

IBPS, UMR 8256 CNRS-UPMC, ERL INSERM U1164,

Biological Adaptation and Ageing, Sorbonne Université, F-

75252 Paris, France; orcid.org/0000-0002-1028-5113;

Email: chahrazade.el_amri@sorbonne-universite.fr

Anti-inﬂammatory Potential. The impact of compounds on

microglial activation and proinﬂammatory cytokines was assessed in

puriﬁed primary microglial cultures isolated from newborn rat brain,

as previously described.³⁶All animals used were handled in

accordance with European standards for ethics and animal welfare.

The newborn WISTAR rats (Janvier Labs, Le Genest Saint Isle,

France) at stage P1 are sacriﬁced by decapitation. After removal of the

meninges, the cortical structures are removed in DMEM containing

1% penicillin/streptomycin mix (Thermo Fisher, 15070063) and 1%

nonessential amino acids (MEM NEAA, Thermo Fisher, 11140050).

Tissues are enzymatically dissociated by the addition of trypsin−

EDTA (Gibco, 25200056) for 10 min at 37 °C. The cell suspension is

then passed through a nylon ﬁlter 70 μm in diameter, centrifuged at

1000 rpm for 10 min, and then resuspended in 10 mL of DMEM

supplemented with 10% fetal calf serum (FCS) (Thermo Fisher

Scientiﬁc, 10500056) in a ﬂask previously coated with 0.01% poly-^L-

ornithine (Sigma-Aldrich, P4957). The cells are cultured for 2 weeks

at 37 °C in a humid atmosphere (5% CO₂) with a change of medium

on the 7th day. At the end of this period, the cells are stirred for 2 h at

250 rpm to detach and recover the microglia. The cells are inoculated

into sterile transparent 96-well FALCON plates at a density of 50 000

cells/well in DMEM/F12 medium supplemented with 1% N₂mix and

then cultured for 24 h at 37 °C in a humid atmosphere (5% CO₂).

The microglia is treated with lipopolysaccharide (LPS, 10 ng/mL,

Sigma-Aldrich) to create an inﬂammatory environment to be

activated. Concomitantly, the cells are treated with inhibitors at

diﬀerent concentrations deﬁned according to the IC₅₀values (0.5−10

μM), with 1% DMSO (negative control), or with 100 nM

dexamethasone (positive anti-inﬂammatory control, Sigma-Aldrich,

D4902) for 3 h at 37 °C in a humid atmosphere (5% CO₂). At the

end of this incubation, supernatants are recovered for assays of

proinﬂammatory cytokines TNF-α and IL1-β by ELISA (Rat TNF-α

ELISA MAXTM Deluxe Set # 438204, rat IL1-β ELISA kit

ab100768).

Authors

Sabrina Aït Amiri − Faculty of Sciences and Engineering,

IBPS, UMR 8256 CNRS-UPMC, ERL INSERM U1164,

Biological Adaptation and Ageing, Sorbonne Université, F-

75252 Paris, France

Cyrille Deboux − Institut du Cerveau, Inserm U 1127, CNRS

UMR 7725, Sorbonne Université, F-75013 Paris, France

Feryel Soualmia − Faculty of Sciences and Engineering, IBPS,

UMR 8256 CNRS-UPMC, ERL INSERM U1164,

Biological Adaptation and Ageing, Sorbonne Université, F-

75252 Paris, France

Nancy Chaaya − Faculty of Sciences and Engineering, IBPS,

UMR 8256 CNRS-UPMC, ERL INSERM U1164,

Biological Adaptation and Ageing, Sorbonne Université, F-

75252 Paris, France

Maxime Louet − Institut des Biomolécules Max Mousseron,

Université de Montpellier, CNRS, ENSCM, F-34093

Montpellier, France

Eric Duplus − Faculty of Sciences and Engineering, IBPS,

UMR 8256 CNRS-UPMC, ERL INSERM U1164,

Biological Adaptation and Ageing, Sorbonne Université, F-

75252 Paris, France

Sandrine Betuing − Faculty of Sciences and Engineering, IBPS,

UMR 8246-CNRS/INSERM U1130, Neurosciences Paris

Seine, Sorbonne Université, F-75252 Paris, France

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.0c02175

Author Contributions

ASSOCIATED CONTENT

■

The research was designed by C.E., N.M., and B.N.O.

Chemical synthesis was performed by N.M. Enzymology

assays were performed by S.A.A., N.C., and F.S.; biological

assays were performed with neural cells, OPCs, and microglial

cells by S.A.A. and C.D. Docking models were determined by

M.L. E.D. and S.B. contributed to experiments on primary

cultures of neural cells. Data analysis and manuscript

preparation were done by C.E.A., N.M., and B.N.O. All of

the authors have given approval the ﬁnal version of the

manuscript.

sı

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c02175.

Compounds’ synthesis and characterization data; Dixon

plot of compounds 37 and 47 toward KLK6 (Figure

S1); structures of synthesized compounds (Table S1);

and comparison of ADME and pharmacological

parameters for hit compounds 32 and 42 (Table S2)

(PDF)

Notes

Molecular formula strings of compounds described in

this study (CSV)

The authors declare no competing ﬁnancial interest.

Models of compound 32 on 4D8N KLK6 (PDB)

ACKNOWLEDGMENTS

■

Models of compound 42 docked on 4D8N KLK6

(PDB)

The authors are grateful to Sorbonne Université, Université of

Montpellier, Institut National pour la Recherche Médicale

(INSERM), Centre National de la Recherche Scientiﬁque

(CNRS), Fondation pour la recherche sur la sclérose en

plaques (ARSEP Fondation), as well as the SATT Lutech for

research funding. The authors thank Dr. David Akbar (ICM

Cell Culture Facility, CELIS) for his advice and technical

AUTHOR INFORMATION

■

Corresponding Authors

Brahim Nait Oumesmar − Institut du Cerveau, Inserm U

1127, CNRS UMR 7725, Sorbonne Université, F-75013

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activated receptor 2 controls myelin development, resiliency and

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(17) Panos, M.; Christophi, G. P.; Rodriguez, M.; Scarisbrick, I. A.

Differential expression of multiple kallikreins in a viral model of

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assistance on ﬂuorescence imaging and quantitative analysis.

This study was supported by the Investissements d’Avenir

ANR-10-IAIHU-06 (IHU-A-ICM) and ANR-11-INBS-0011

(NeurATRIS) (to B.N.O.). The authors thank the French

Ministry of Research and Education for S.A.A. and F.S. Ph.D.

fellowships.

ABBREVIATIONS

■

ADMET, absorption diﬀusion metabolism, elimination, and

toxicity; CNS, central nervous system; DMEM, Dulbecco’s

modiﬁed Eagle’s medium; DMSO, dimethyl sulfoxide; EDCI,

N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochlor-

ide; IL1β, interleukin 1 β; KLK, tissue kallikreins; KLK1,

kallikrein-related peptidase 1; KLK6, kallikrein-related pepti-

dase 6; LPS, lipopolysaccharide; MBP, myelin basic protein;

MS, multiple sclerosis; OPC, oligodendrocyte precurcor cell;

PAR, protease-activated receptors; TNF β, tumor necrosis

factor

(19) Scarisbrick, I. A. The multiple sclerosis degradome: enzymatic

cascades in development and progression of central nervous system

inflammatory disease. Curr. Top. Microbiol. Immunol. 2008, 318, 133−

175.

(20) Blaber, S. I.; Ciric, B.; Christophi, G. P.; Bernett, M. J.; Blaber,

M.; Rodriguez, M.; Scarisbrick, I. A. Targeting kallikrein 6 proteolysis

attenuates CNS inflammatory disease. FASEB J. 2004, 18, 920−932.

(21) Yoon, H.; Blaber, S. I.; Evans, D. M.; Trim, J.; Juliano, M. A.;

Scarisbrick, I. A.; Blaber, M. Activation profiles of human kallikrein-

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J. Med. Chem. 2021, 64, 5667−5688

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Identification of First-in-Class Inhibitors of Kallikrein-Related Peptidase 6 That Promote Oligodendrocyte Differentiation

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