Melatonin- And Ferulic Acid-Based HDAC6 Selective Inhibitors Exhibit Pronounced Immunomodulatory Effects in Vitro and Neuroprotective Effects in a Pharmacological Alzheimer's Disease Mouse Model

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Melatonin- and Ferulic Acid-Based HDAC6 Selective Inhibitors

Exhibit Pronounced Immunomodulatory Eﬀects In Vitro and

Neuroprotective Eﬀects in a Pharmacological Alzheimer’s Disease

Mouse Model

Feng He, C. James Chou, Matthias Scheiner, Eleonora Poeta, Natalia Yuan Chen, Sandra Gunesch,

Matthias Ho ﬀ mann, Christoph Sotri ﬀ er, Barbara Monti, Tangui Maurice,* and Michael Decker *

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ABSTRACT: The structures of melatonin and ferulic acid were merged into tertiary amide-based histone deacetylase 6 (HDAC6)

inhibitors to develop multi-target-directed inhibitors for neurodegenerative diseases to incorporate antioxidant eﬀects without losing

aﬃnity and selectivity at HDAC6. Structure-activity relationships led to compound 10b as a hybrid molecule showing pronounced

and selective inhibition of HDAC6 (IC50 = 30.7 nM, > 25-fold selectivity over other subtypes). This compound shows comparable

DPPH radical scavenging ability to ferulic acid, comparable ORAC value to melatonin and comparable Cu²⁺chelating ability to

EDTA. It also lacks neurotoxicity on HT-22 cells, exhibits a pronounced immunomodulatory eﬀect, and is active in vivo showing

signiﬁcantly higher eﬃcacy in an AD mouse model to prevent both Aβ25−35-induced spatial working and long-term memory

dysfunction at lower dose (0.3 mg/kg) compared to positive control HDAC6 inhibitor ACY1215 and an equimolar mixture of the

three entities ACY1215, melatonin and ferulic acid, suggesting potentially disease-modifying properties.

modulates tau protein phosphorylation, which aggregates to

intracellular neuroﬁbrillary tangles (NFTs) in AD patients.⁷

Phenylhydroxamic acid-based selective HDAC6 inhibitors can

activate Nrf2 and hypoxic adaptive response, which contribute

to neuroprotection in vitro and in vivo.⁸5-Aroylindole-based

HDAC6 inhibitors showed neuroprotective activity by

triggering ubiquitination and ameliorated the cognition

impairment on a scopolamine-induced AD model in rats, a

model reﬂecting the hypocholinergy seen in the pathology.⁹

Lastly, given the importance in AD progression of a sustained

INTRODUCTION

■

Histone deacetylases (HDACs) are responsible for controlling

gene expression by modulating the acetylation status of histone

and some nonhistone proteins.¹HDACs play essential roles in

neurodevelopment, memory formation, and cognitive pro-

cesses, and HDAC inhibitors have been validated as innovative

agents for the treatment of neurodegenerative disorders, such

as Alzheimer’s disease (AD).²Among all 18 subtypes, HDAC6

is of particular interest as it contains two catalytic domains and

is mainly cytoplasmic. Moreover, due to its cytoplasmic

localization, HDAC6 rather targets nonhistone proteins, such

as HSP-90, α-tubulin, β-catenin, and tau protein, which

renders HDAC6 to be an interesting target for treatment of

neurodegenerative diseases and several rare diseases like Rett

syndrome and idiopathic pulmonary ﬁbrosis.³⁻⁵It is overex-

pressed in the brain of AD patients, especially in the cortex and

hippocampus.⁶HDAC6 enables tau protein deacetylation and

Received: November 9, 2020

Published: March 26, 2021

https://doi.org/10.1021/acs.jmedchem.0c01940

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Figure 1. Design of melatonin- and ferulic acid-based HDAC inhibitors.

immune response (inﬂammation) and microglia-related

mechanisms in Aβ clearance and production,¹⁰the potential

immunomodulatory activity of HDACs through control of

release of cytokines, as well as alteration of activity or function

of CNS’ macrophages (microglia) and dendritic cells, has been

widely investigated.¹¹Recent studies have demonstrated that

manipulation of HDAC6 by tubastatin A (TBSA) alleviates

LPS-induced neuroinﬂammation, representing a new strategy

to counteract inﬂammation and consequently neurodegener-

ative progression in mice.¹²

Given the multifactorial nature of AD, multitarget

compounds being able to simultaneously modulate multiple

targets involved in the onset of the disease are promising to

restore the physiological balance and generate suﬃcient

therapeutic eﬃcacy. There are several recent reviews covering

the design of multitarget compounds.¹³⁻¹⁶As a new approach

for AD treatment, the pharmacophore model of HDAC

inhibitors has been designed into multitarget compounds

addressing several other related targets, which has been

extensively discussed in several comprehensive reviews.¹⁷⁻²⁰

Generally, HDAC inhibitors comprise a zinc binding group

(ZBG, e.g., hydroxamic acid), a linker mimicking the lysine

chain, and a cap group, usually an aromatic group that interacts

with the external surface of the enzyme.²¹Due to the high

compatibility of the cap group, it can be replaced by diﬀerent

pharmacophores addressing a second target. The Oyarzabal

group has reported HDAC and phosphodiesterase 5 (PDE5)

dual-target inhibitor CM-414, which can rescue the impaired

long-term potentiation evident in hippocampal slices from

APP/PS1 mice and reverse cognitive deﬁcits in Tg2576

mice.²²⁻²⁴Simone et al. designed the ﬁrst-in-class GSK-3β-

HDAC dual inhibitor showing disease-modifying eﬀects in

vitro for AD.²⁵The Wu group recently published memantine-

based HDAC-NMDAR and tacrine-based HDAC-ChEs multi-

target inhibitors, which exhibit potent neuroprotection,

antioxidant, and anti-Aβ-aggregation activities.^26,27Tseng et

al. designed HDAC-AChE multitarget inhibitors that enhance

neurite outgrowth and show signiﬁcant anti-Aβ-aggregation

activity.²⁸

Natural products melatonin and ferulic acid have shown

promising pharmacological eﬀects potentially beneﬁcial for AD

treatment, such as antioxidant eﬀects, neuroprotection, and

neurogenesis promotion.²⁹⁻³²In the hippocampus of AD

patients, increased melatonin receptor 1 (MT1) and decreased

melatonin receptor 2 (MT2) expression has been ob-

served.^33,34However, ferulic acid has low bio-availability and

poor blood-brain-barrier penetration, melatonin possesses very

short biological half time and lack of selectivity, which are

present limitations for further clinical application.^29,30Never-

theless, pan-HDAC inhibitors based on ferulic acid, trypt-

amine, or tryptophan all show potent inhibitory activities.³⁵⁻³⁸

For instance, the tryptamine-based HDAC inhibitor panobino-

stat (LBH589) was approved by the FDA for the treatment of

multiple myeloma in 2015.³⁹In addition, we have recently

reported that hybrids of ferulic and cinnamic acids with

ﬂavonoids shown pronounced neuroprotection in vitro, which

also translates to in vivo activity. These activities are due to

speciﬁc interactions with distinct mitochondrial targets.^40,41

In this study, we chemically merged the structures of

melatonin and ferulic acid into HDAC6 inhibitor A to develop

novel multitarget-directed drug candidates for AD with the aim

to combine antioxidant eﬀects in a synergistic manner without

losing aﬃnity at and selectivity toward HDAC6. Accordingly,

15 target compounds have been designed (Figure 1),

synthesized, and bioevaluated. Compounds 3a and 3b were

designed with two aromatic linker groups to replace the acetyl

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a

Scheme 1. Synthesis of Target Compounds 3a−3b, 5a−5d and 8a−8c from 5-Methoxytryptamine (1)

a

Reagents and conditions: (a) methyl (E)-3-(4-(bromomethyl)phenyl)acrylate for 2a, methyl 4-(bromomethyl)benzoate for 2b, K₂CO₃, THF,

H₂O, reﬂux; (b) NH₂OH·HCl, KOH, MeOH, r.t.; (c) (E)-4-(3-methoxy-3-oxoprop-1-en-1-yl)benzoic acid for 4a, 4-(methoxycarbonyl)benzoic

acid for 4b, (E)-3-(4-(methoxycarbonyl)-phenyl)acrylic acid for 4c, 6-methoxy-6-oxohexanoic acid for 4d, TBTU, TEA, THF, r.t.; (d) acetic

anhydride, DCM, 0 °C to r.t.; (e) (i) methyl (E)-3-(4-(bromomethyl)phenyl)acrylate for 7a, methyl 4-(bromomethyl)benzoate for 7b, methyl 6-

bromohexanoate for 7c, t-BuOK, THF, reﬂux; (ii) SOCl₂, MeOH, r.t.; (f) ferulic acid, ethyl chloroformate, TEA, dried THF, 0 °C; (g) Boc-Gly-

OH, EDCI, DMAP, TEA, THF, r.t.; (h) 10% TFA in DCM, r.t.

a

Scheme 2. Synthesis of Target Compounds 10a−10c and 15a−15c from 5-Methoxytryptamine (1)

a

Reagents and conditions: (a) methyl (E)-3-(4-(bromomethyl)phenyl)acrylate for 13a, methyl 4-(bromomethyl)benzoate for 13b, methyl 6-

bromohexanoate for 13c, K₂CO₃, THF, H₂O, reﬂux; (b) ferulic acid, ethyl chloroformate, TEA, dried THF, 0 °C; (c) NH₂OH·HCl, KOH, MeOH,

r.t.; (d) Boc-Gly-OH, EDCI, DMAP, TEA, THF, r.t.; (e) 10% TFA in DCM, r.t.

group; similarly, compounds 5a−5d incorporate four linker

groups in the same position and maintain the amide linkage to

study SARs and evaluate the importance of the amide linkage.

The linker groups of compounds 8a−8c were introduced at

the indole nitrogen atom, this design makes the linker groups

not as long as the linker group in ethylamine position, and the

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Table 1. Structures, In Vitro HDAC (HDAC3 and HDAC6) Inhibitory, DPPH Radical Scavenging Activities, and ORAC

Values of Target Compounds

a

b

c

Data from ref 48. Data are the mean (n = 3) SD. Data are expressed as Trolox equivalents and are the mean (n = 3) SD.

structures are less ﬂexible, which is supposed to produce higher

HDAC6 selective inhibition. Melatonin and ferulic acid as the

cap group were merged into one entity for compounds 10a−

10c and 15a−15c aiming to produce stronger HDAC6

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Table 2. Inhibitory Activities of Compounds 8b and 10b at HDAC1−4 and HDAC6−9

8b

10b

HDAC classes subtype IC₅₀(nM) (pIC₅₀SEM) (n = 2) selectivity ratio to HDAC6 IC₅₀(nM) (pIC₅₀SEM) (n = 2) selectivity ratio to HDAC6

class I

HDAC1

HDAC2

HDAC3

HDAC8

HDAC4

HDAC7

HDAC9

HDAC6

262 (6.58 0.07)

336 (6.47 0.09)

641 (6.19 0.09)

838 (6.08 0.23)

3431 (5.47 0.06)

1653 (5.78 0.10)

362 (6.44 0.11)

11.2 (7.59 0.06)

23

30

57

75

306

148

32

771 (6.11 0.09)

2851 (5.55 0.41)

1613 (5.79 0.16)

3590 (5.45 0.54)

>10 μM

30.7 (7.51 0.06)

25

93

53

117

>326

class IIa

class IIb

Figure 2. Proposed binding modes of compounds 8b (A) and 10b (B, C) at the active site of HDAC6. The two alternative binding modes shown

for 10b correspond to the best-ranked result (B) and the most populated cluster of the docking results (C). Distances are given in Å; selected

interactions are highlighted with dashed lines. The Zn²⁺ion is represented as a grey sphere. Loops 1 and 2 are denoted as L1 and L2. The deep

active-site pocket is visualized with a transparent light-grey surface.

selective inhibition and more pronounced antioxidant

capacity.²¹

15a−15c were obtained through procedures similar to the

synthesis of compounds 10a−10c. All ester intermediates 2a,

2b, 4a−4d, 7a−7c, 9a−9c, and 14a−14c were converted to

hydroxamic acid compounds 3a, 3b, 5a−5d, 8a−8c, 10a−10c,

and 15a−15c, respectively, by NH₂OH-KOH in methanol.

Puriﬁcation of target compounds proved challenging because

of a carboxylic acid byproduct formed and absorbed on silica

gel.

Due to the tertiary amide groups of compounds 10a−10c

and 15a−15c, the occurrence of cis/trans amide bond rotamers

was observed, which possess two sets of NMR signals, arising

from the restricted rotation around the tertiary amide bond for

each rotamer. Since this phenomenon had already been

discussed in detail in several publications,⁴²⁻⁴⁴variable

temperature NMR studies (VT-NMR) for these compounds

did not deem necessary.

Inhibition of HDACs. To evaluate selectivity toward

HDAC6 and extent of inhibition, a ﬂuorescence assay against

HDAC3 and HDAC6 was applied to test the target molecules

(Table 1).⁴⁵At 1 μM, compounds 3b, 5a, 5d, and 15b show

less potent inhibition of both HDAC3 and HDAC6 than all

other target compounds. Compounds 3b, 5a, and 5d share

similar lengths of linker groups, which are shorter than the

cinnamate group in compounds 3a, 5b, and 5c, indicating that

the longer linker (like a cinnamate group) is preferable. For

comparison, compounds 8b and 10b show the highest

selectivity values regarding HDAC6 inhibition, namely, 57-

fold and 53-fold, respectively, over HDAC3. Interestingly,

compounds 8b and 10b share the same phenylhydroxamic acid

with diﬀerent bulky capping groups, being in line with the

other reported selective HDAC6 inhibitors.²¹Also, due to the

longer linker and its high ﬂexibility, compounds 3a, 3b, and

RESULTS AND DISCUSSION

■

Chemistry. In Scheme 1, the syntheses of target

compounds 3a, 3b, 5a−5d, and 8a−8c are described starting

from 5-methoxytryptamine (1). Corresponding linker groups

were introduced to the ethylamine position of 5-methoxytrypt-

amine (1) by stirring with potassium carbonate in a mixture of

THF and water to aﬀord intermediates 2a and 2b. The

formation of dual-substituted byproducts lowered the yields of

this step, especially the synthesis of intermediate 2c presented

in Scheme 2. Amide condensation to form intermediates 4a−

4d were carried out after corresponding carboxylic acids had

been activated by TBTU and stirred with compound 1, which

gave reasonable yields of 60−79%. Melatonin (6) formed by

acetylation of compound 1 was reﬂuxed with corresponding

bromides and t-BuOK in THF; meanwhile, the ester groups

were hydrolyzed. The obtained crude carboxyl intermediates

were therefore esteriﬁed in methanol after activation with

thionyl chloride to aﬀord intermediates 7a−7c.

The key step to synthesize compounds 10a−10c was the

introduction of a ferulic acid moiety, which was introduced

into intermediates 2a−2c to produce the tertiary amides

(Scheme 2). First, the carboxyl group of ferulic acid was

activated by excess ethyl chloroformate; then the phenolic

hydroxyl group was esteriﬁed and could be hydrolyzed in

NH₂OH-KOH methanol solution during the formation of the

hydroxamate group. BOC-Gly-OH reacted with 5-methoxy-

tryptamine (1) after it had been activated with EDCI, and the

Boc protecting group was removed in 10% TFA in dichloro-

methane to aﬀord intermediate 12. Subsequently, compounds

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5a−5d exhibit less selective inhibition of HDAC6 compared to

the other compounds. On the contrary, compounds 3a and 5b

show the most potent inhibition to HDAC3 with IC₅₀values of

5.1 and 3.6 nM, respectively, which is more pronounced than

their inhibition of HDAC6. Compounds 5b and 3a with or

without the amide linkage showed no diﬀerences in the extent

of HDAC inhibition.

To further conﬁrm selective inhibition of compounds 8b

and 10b over other HDACs, their IC₅₀s at other Zn²⁺-

dependent HDACs (HDAC1− 9) were measured (Table 2).

Both compounds show highly selective inhibition (more than

23-fold over other subtypes) toward HDAC6, and their

selectivity values are higher than those of reference compound

ACY1215. The results further validate the importance of

phenylhydroxamic acid with bulky capping groups to yield

pronounced HDAC6 selective inhibition.

Figure 3. Western blot analysis of acetylated tubulin (AcTubulin),

acetylated histone H3 (AcHH3), acetylated histone H4 (AcHH4),

and Hsp90 in HT22 cell lines after 24 h treatment with compounds

10b (500, 100, and 20 nM) and 8b (500 nM) using ACY1215 (500

nM) as a positive control. Tubulin was used as a loading control.

Computational docking studies were carried out to identify

putative binding modes of compounds 8b and 10b for

HDAC6. The protein structure from the crystallographically

determined complex of HDAC6 with reference compound A

(PDB: 6DVM)²¹was used for this purpose. For compound 8b,

a well-clustered top-ranked docking result was obtained in

which the phenylhydroxamate binds to the deep active-site

tunnel in a previously reported conformation.^21,46,47The

hydroxamate shows a monodentate coordination to the zinc-

ion at 2.5 Å and a hydrogen-bonding interaction with Gly582

at 2.8 Å distance (Figure 2A). The phenyl linker is sandwiched

in the tunnel between Phe583 and Phe643 at 4.1 and 4.5 Å,

respectively. The melatonin-based capping group is positioned

in a pocket at the outer rim of the active site, ﬂanked by the L1

loop. The indole moiety is oriented toward His463 and

Pro464, while the extended amide might accept a hydrogen

bond from Asn457. For compound 10b, two binding modes

appear feasible for the capping groups, which are placed above

the pocket ﬂanked by loop L1 but can be switched in their

orientation (Figure 2B,C). In the best binding pose according

to the DSX scoring function⁴⁹(cf. the Supporting Information

for further details), aromatic interactions of the indole moiety

with His463 appear to be feasible, whereas the hydroxy-

methoxyphenyl ring shows van der Waals interactions with

Leu712. In the switched orientation, which is somewhat less

favorably scored by DSX but found more frequently in the

docking runs, the indole is placed above Leu712, whereas the

hydroxy group of the ferulic acid-based moiety can reach

Asp460 for a hydrogen-bond interaction. Based on the docking

results for both compounds, binding of the capping groups to

the L1 loop pocket can be assumed to be the likely reason for

HDAC6 selectivity, in line with the elegant crystallographic

analyses presented by Porter et al.²¹

To examine the cellular potency and selectivity of

compounds 10b and 8b, acetylated tubulin and acetylated

histone H3 and H4 were examined in HT-22 cells (Figure 3).

As expected, 10b strongly induced the increase of acetylated

tubulin (AcTub), while a little-to-no increase was observed for

acetylated histone H3 and H4 (AcHH3 and AcHH4).

Compound 8b also selectively increases the level of AcTub

at 500 μM. The Western blot data indicate that both 10b and

8b are selectively inhibiting HDAC6 activity but not the class I

HDACs targeting histone acetylation.

Antioxidant Activities. By combination of antioxidants

melatonin and ferulic acid, compounds were designed to

maintain their antioxidant capacity. Their antioxidant

capacities were evaluated using both 2,2-diphenyl-1-picrylhy-

drazyl radical (DPPH) and oxygen radical absorbance capacity

(ORAC) assays. These assays are well-established techniques

for determining the radical scavenging activity (RSA) of a test

compound. Reactive oxygen species (ROS), such as the

hydroxyl or superoxide radicals, cause lipid peroxidation,

protein oxidation, and DNA damage, which can lead to

organelles malfunction and further cellular disorders.⁵⁰

Melatonin is inactive regarding inactivation of the DPPH

radical, and HDAC6 inhibitor ACY1215 shows only weak

scavenging activities. Among all target compounds, tertiary

amide-based compounds 10a, 10c, and 15a show the highest

radical scavenging ability, and compound 10b is comparable to

ferulic acid. Various linker groups also aﬀect radical scavenging

activities: the compounds bearing the cinnamate group (e.g.

compounds 3a, 5b and 8a) exhibit stronger scavenging

activities than compounds bearing a phenylene (e.g. com-

pounds 3b, 5a and 8b) or analkylene chain (e.g. compounds

5d and 8c), respectively. ACY1215 also shows higher radical

scavenging activity than melatonin indicating that the

hydroxamic acid group is supportive to scavenge the DPPH

radical. The ORAC values shown in Table 1 are expressed as

trolox (water-soluble analogue of vitamin E) equivalents (TE),

in relation to radical scavenging properties of trolox. Ferulic

acid and melatonin were used as positive controls showing

ORAC values of 3.1 and 2.0, respectively. All evaluated

compounds exhibit strong radical scavenging properties with

TE values ranging from 1.0 (10a) to 3.2 (15c). HDAC6

selective compound 10b possesses a TE value of 2.1, which is

comparable to melatonin. Compounds 8a−8c show lower TE

values than melatonin and most other compounds tested,

indicating the importance of unsubstituted indole-N to

maintain the antioxidant capacity of melatonin. There is no

obvious eﬀect for amide linkage regarding DPPH scavenging

or TE values in compounds 5a and 3a, 5b and 3b. Compounds

15a−15c show no signiﬁcant diﬀerence to compounds 10a−

10c, which means that the presence of an acetyl group is not

necessary. Introduction of one alkylene chain is much more

eﬀective than aromatic linkers, as seen from the TE values of

compounds 5d, 10c, and 15c, respectively. Also, the presence

of ferulic acid moiety is poorly correlated with the observed TE

values unlike for the DPPH radical scavenging capacity.

Cerebral biometals (such as copper, zinc, and iron) play

crucial roles in Aβ aggregation and neurotoxicity, being a

driving force for neurodegeneration in AD.⁵¹As shown in

Figure S1, compounds 8b, 10b, and ACY1215 exhibit

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comparable chelating abilities to each other at all concen-

trations tested and also comparable abilities to EDTA when

chelating copper ions. In contrast to nonselective chelation of

EDTA, compounds 8b, 10b, and ACY1215, respectively, show

higher selectivity to the copper ion over ferrous and zinc ions,

which might render them superior regarding copper-ion

dysregulation-related neurodegeneration diseases like AD.⁵²

In a summary, compounds 8b and 10b show the best

selective HDAC6 inhibition and additional potent total

antioxidant capacities, especially, compound 10b exhibits

comparable radical scavenging capacities to both ferulic acid

and melatonin in DPPH assay and ORAC assay, respectively.

Neurotoxicity. HDAC inhibitors usually possess high

cytotoxicity, which might counteract their application as

CNS-targeting therapeutics. Hence, it was important to

investigate their neurotoxicity proﬁle. A survival assay was

performed, treating murine hippocampal neuronal HT-22 cells

with 10 μM of each of the target compounds (Figure 4).

neurotoxic M1 to the neuroprotective M2, of HDACs

inhibitors 8b, 10b, and control ACY1215, N9 microglial cells

were treated with increasing concentration of compounds (0.1,

0.5, 1, 2.5, 5 μM) in the presence or absence of 100 ng/mL

LPS (lipopolysaccharide), which induces an M1 activation

state. After 24 h of treatment, to investigate nitric oxide and

IL1β release, markers of M1 microglia, conditioned media

were collected and evaluated by a colorimetric assay based on

the Griess reaction and Western blot analysis, respectively. In

parallel, cell lysates were analyzed by Western blot to measure

the expression of the inducible nitric oxide synthase (iNOS,

M1 marker), TREM2 and TGFβ2, both markers of M2

microglia. As shown in Figure 5, compound 10b strongly

reduces in dose-dependent manner nitrite accumulation (F)

and IL1β release (I) compared to compound 8b and control

ACY1215, as well as iNOS expression induced by LPS-

mediated microglia activation (L). Additionally, compound

10b leads to an increase in the phagocytic protein TREM2 (O)

and TGFβ2 (R) expression, suggesting an immunomodulatory

eﬀect, i.e., a switch from neurotoxic M1 to neuroprotective M2

microglia when cells are treated with high concentration of the

compound (2.5, 5 μM). Diﬀerently, no change in TREM2 and

TGFB2 expression was observed in the presence of increasing

concentration of compound 8b (E, H, K, N, Q).

In Vivo Studies. Due to its promising in vitro properties,

compound 10b was chosen to evaluate its neuroprotective

properties in an in vivo model of AD in mice. The HDAC6

inhibitor ACY1215 was also tested as a comparator. Mice

received an intracerebroventricular (icv) injection of theoligo-

merized Aβ₂₅₋₃₅peptide into the brain on day 1. Compounds

were then injected intraperitoneally (ip) once daily (o.d.) from

days 1 to 7. Spatial working memory was evaluated using the

spontaneous alternation behavior when mice explored a Y

maze, on day 8. Nonspatial long-term memory was analyzed

using the step-through passive avoidance response, with

training on day 9 and retention on day 10. In this

administration scheme, not symptomatic eﬀects, but neuro-

protectivity can be assessed in vivo.⁵⁴⁻⁵⁷

As shown in Figure S3 (cf. Supporting Information), none of

the treatment (DMSO containing a vehicle or each

compound) signiﬁcantly aﬀected the mouse body weight

gain during the week of administration showing good

tolerability. A transient weight loss was observed on day 2,

related to the icv injection of the peptide under light gazeous

anesthesia, but animals recovered during the following days.

As shown in Figure 6A,C, compound 10b signiﬁcantly

attenuated the Aβ₂₅₋₃₅-induced spontaneous alternation

deﬁcits at a lower dose, 0.3 mg/kg, compared to ACY1215,

active at 1 mg/kg. The compound 10b was active in the 0.3−1

mg/kg dose-range, reﬂecting a more potent neuroprotective

eﬀect on working memory deﬁcits. The hybrid molecule 10b

was compared to an equimolar (0.7 μM) mixture of the three

entities ACY-1215, melatonin, and ferulic acid (Figure 6E).

Melatonin did not prevent Aß₂₅₋₃₅-induced alternation deﬁcits

at this low, 0.16 mg/kg dose, and neither did the mixture.

None of the treatment aﬀected the exploratory response,

measured in terms of number of arms entered during the

session (Figure 6B,D,F).

Figure 4. Neurotoxicity of target compounds in HT-22 cells at 10

μM. Statistical analysis was performed applying One-way ANOVA

followed by Dunnett’s multiple comparison post-test. Levels of

signiﬁcance: ***p < 0.001, *p < 0.05 referring to control cells treated

with DMSO.

Generally, the compounds proved to be safe and showed no

neurotoxicity. Compound 10b showed no signiﬁcant neuro-

toxicity, and slight neurotoxic eﬀects were observed for

compound 8b and reference compound ACY1215.

Interestingly, compounds 3a and 5b also showed some

neurotoxic eﬀects with 50 and 60% cell survival, respectively.

This is not too surprising due to their potent inhibition of

HDAC3 and might even be beneﬁcial for further anticancer

research. With 10 μM, a relatively high concentration was

tested and the viability rates should not counteract beneﬁcial

eﬀects.

As shown in Figure S2 (cf. Supporting Information), the

neuroprotection of target compounds against glutamate-

induced oxytosis and iodoacetic acid-induced ATP depletion

in HT-22 cells has been tested; only compound 10a revealed

signiﬁcant neuroprotection at 50 μM.

Immunomodulatory Eﬀect. Activation of microglia cells,

i.e., CNS’ macrophages, is a hallmark of neuroinﬂammation, a

characteristic feature of neurodegenerative conditions. De-

pending on brain microenvironment, microglia acquire diﬀer-

ent phenotypes: the neurotoxic M1 phenotype, generally

characterized by production of proinﬂammatory cytokines

(TNF-α, IL-1β) or reactive oxygen species (ROS), or the

neuroprotective M2 phenotype distinguished by the expression

of phagocytic protein TREM2 and of growth factors, such as

TGFβ2.⁵³To analyze the possible immunomodulatory activity,

i.e., the ability to shift the microglial phenotype from the

In the passive avoidance test, compound ACY1215

attenuated the Aβ₂₅₋₃₅-induced deﬁcit at all doses tested, but

not signiﬁcantly vs the Aβ₂₅₋₃₅-injected group (Figure 7A).

Compound 10b attenuated the deﬁcit at both 0.1 and 0.3 mg/

kg, signiﬁcantly at the lowest dose tested, but was not active at

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Figure 5. Eﬀect of compounds 8b, 10b and control ACY1215 on murine N9 cells induced in a M1 activation state by LPS (100ng/mL) treatment.

IL1β release in N9-conditioned medium and expression of iNOS, TREM2 and TGFβ2 were analyzed by western blot after 24h treatment with LPS

in presence of increasing concentrations (0.1, 0.5, 1, 2.5 and 5μM) of compounds ACY1215 (A), 8a (B) and 10b (C). Nitric Oxide release was

evaluated through Griess reaction in media conditioned for 24h by microglial cells treated with LPS in presence of compounds ACY1215 (D), 8a

(E) and 10b (F); as shown in ﬁgure F, only compound 10b induced a signiﬁcative reduction of NO release, with the strongest eﬀect at 5μM.

Additionally, IL1β release and iNOS expression, both markers of M1 neurotoxic microglia, strongly decreased in the presence of increasing

concentrations of compound 10b compared to LPS-treated control (I, L); conversely, no signiﬁcant eﬀects where observed on N9 cells following

ACY1215 (G, J), and 8a (H, K) treatment. In parallel, expression of M2 microglial marker TREM2 increased in cells co-treated by LPS-10b (O)

and LPS-ACY1215 (M) compounds, whereas no signiﬁcant eﬀects in TREM2 expression were obtained following LPS-8a treatment (N). In

addition, compared to ACY1215 (P) and 8a (Q), in LPS-10b treated cells TGFβ2 expression turned out to increase (R) suggesting, together with

TREM expression (O), a shift from neurotoxic M1 to neuroprotective M2 microglia. All quantitative data are presented as means S.E. from at

last 3 independent experiments. Statistical signiﬁcance between diﬀerent treatments was calculated with GRAPHPAD PRISM 6 (La Jolla,

California, USA) by using one-way analysis of variance (ANOVA) followed by post-hoc comparison through Bonferroni's test. *p<0.05; **p<0.01

compared to un-treated control; #p<0.05; ##p<0.01compared to LPS-treated control.

1 mg/kg (Figure 7B). These bell-shaped dose−response eﬀects

are commonly observed in cognitive and neuroprotection

responses in vivo, as shown recently with the NMDAR

antagonist memantine in the same Aß₂₅₋₃₅mouse model of

AD.⁵⁷They are here observed in our study for both ACY1215

and 10b at the 1 mg/kg dose, suggesting that this biphasic

eﬀect relies on HDAC6 inhibition. Melatonin or the equimolar

mixture of the three compounds failed to attenuate Aβ₂₅₋₃₅

-

induced passive avoidance deﬁcit, again showing the

pronounced eﬀect of the hybrid molecule (Figure 7C).

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CONCLUSIONS

■

In this work, we designed hybrid HDAC inhibitors by

chemically merging the structures of melatonin and ferulic

acid into the HDAC6 inhibitor to develop novel multitarget-

directed drug candidates for AD with the aim to combine

eﬀects in a synergistic manner without losing aﬃnity and

selectivity at HDAC6. Altogether, 15 target compounds have

been designed, synthesized, and evaluated comprehensively in

several in vitro and in vivo assays.

The newly synthesized compounds were ﬁrstly evaluated for

inhibition of HDACs. Melatonin-based compound 8b and

melatonin-ferulic acid-based compound 10b show the highest

HDAC6 inhibition with more than 20-fold selectivity over

other subtypes and maintaining two-digit nanomolar IC₅₀s.

Compound 8b and 10b also maintain the antioxidant

capacities from melatonin and ferulic acid, especially

compound 10b, which scavenges the DPPH radical com-

parable to ferulic acid and possesses the comparable ORAC

value with regard to melatonin. Compound 10b does not

exhibit neurotoxicity at 10 μM and comparable Cu²⁺chelating

ability to the positive control and potent chelator EDTA,

which indicates beneﬁcial eﬀects for reduction of metal-

mediated oxidative stress, tau, and Aβ aggregation.⁵²

Compound 10b shows an immunomodulatory eﬀect,

leading to a reduction in LPS-induced microglia inﬂammation

and to a switch from neurotoxic M1 to the neuroprotective M2

microglial phenotype. Furthermore, it shows pronounced

attenuation of spatial working memory and long-term memory

alteration in an in vivo AD mouse model induced by icv

Aβ₂₅₋₃₅peptide injection, at very low doses, thereby proving

neuroprotective properties. The superior in vivo eﬃcacy of

compound 10b may indicate a synergistic eﬀect between

HDAC6 inhibition and its antioxidant activities. In addition or

alternatively, interaction with MT receptors is possible since

expression levels of both MT receptor subtypes are altered in

AD and melatonin was reported to rescue neurogenesis

impairment by multiple modes of action,³¹⁻³⁴as well as

beneﬁcial pharmacokinetic properties. In any case, the tertiary

amide-based HDAC6 selective inhibitor described herein

shows that HDAC6 inhibitors cannot only be merged with

melatonin and ferulic acid moieties and also maintain potent

HDAC6 selective inhibition and the antioxidant properties but

also represent a suitable scaﬀold to develop neuroprotective

compounds in vivo, with potentially neurodegenerative disease-

modifying properties.

Figure 6. Eﬀects of compound on the Aβ₂₅₋₃₅-induced spontaneous

alternation deﬁcit in mice. Mice received Aβ₂₅₋₃₅(9 nmol icv) or

vehicle solution (3 μL icv) on day 1 and compounds (A, B) ACY1215

or (C, D) 10b, in the 0.1−3 mg/kg ip dose range, o.d., from days 1 to

7. In (E, F), the eﬀect of a drug combination with equimolar doses of

ACY-1215 (0.3 mg/kg), melatonin (0.16 mg/kg), and ferulic acid

(0.13 mg/kg) was compared to the eﬃcacy of the hybrid molecule.

Mice were then tested for spontaneous alternation performance in the

YMT in day 8. Top panel (A, C, and E): spontaneous alternation

performance; lower panel (B, D, and F): number of arm entries. Data

show mean SEM with n = 12−22 per group. ANOVA: F_(4,76)

=

2.81, p < 0.05 in (A); F_(4,86)= 2.68, p < 0.05 in (B); F_(4,76)= 1.16, p >

0.05 in (C); F_(4,86)= 0.581, p > 0.05 in (D); F_(5,76)= 6.20, p < 0.0001

in (E); F_(5,76)= 0.554, p > 0.05 in (F). *p < 0.05, **p < 0.01, ***p <

#

0.001 vs (V + V)-treated group. p < 0.05, ^##p < 0.01 vs (Aβ₂₅₋₃₅

+

V)-treated group; Dunnett′s test.

Figure 7. Eﬀects of compounds on Aβ₂₅₋₃₅-induced passive avoidance

deﬁcits in mice. Mice received Aβ₂₅₋₃₅(9 nmol icv) or vehicle

solution (3 μL icv) on day 1 and compounds (A) ACY1215 and (B)

10b, in the 0.1−3 mg/kg ip dose range, o.d., from days 1 to 7.

Animals were trained in the passive avoidance test on day 9, and

retention (step-through latency) was analyzed on day 10. Data show

the median and interquartile range with n = 10−21 per group.

Kruskal−Wallis ANOVA: H = 10.76, p < 0.05 in (A); H = 16.59, p <

0.01 in (B); H = 14.25, p < 0.05 in (C). *p < 0.05, **p < 0.01, ***p <

EXPERIMENTAL SECTION

■

General Chemistry. Common reagents and solvents were

purchased from commercial suppliers and used without further

puriﬁcation unless otherwise stated. Tetrahydrofuran (THF) was

distilled from sodium-benzophenone under an argon atmosphere.

Reaction progress was monitored using analytical thin-layer

chromatography (TLC) on precoated silica gel GF254 plates

#

0.001 vs (V + V)-treated group. p < 0.05 vs (Aβ₂₅₋₃₅+ V)-treated

group; Dunn’s test.

(Macherey Nagel GmbH & Co. KG, Duren, Germany), and spots

̈

were detected under UV light (254 and 366 nm). Compounds were

puriﬁed with ﬂash column chromatography with a silica gel and

particle size of 40−63 μM (VWR chemicals, Leuven, Belgium) as the

stationary phase and petroleum ether/ethyl acetate or dichloro-

methane/methanol mixtures as eluent systems. Nuclear magnetic

resonance spectra were measured on a Bruker AV-400 NMR

instrument (Bruker, Karlsruhe, Germany) in deuterated solvents

(DMSO-d₆, CDCl₃, MeOD-d₄). Chemical shifts are expressed in ppm

Compound 10b therefore exhibited a remarkably eﬀective

neuroprotective properties in vivo at sub-mg/kg active dose-

range against Aβ₂₅₋₃₅-induced memory dysfunction, suggesting

that HDAC6 inhibition could be an eﬀective neuroprotective

strategy for the treatment of AD.

1

relative to DMSO-d₆, CDCl₃, or MeOD-d₄(2.50/7.26/3.31 for H;

39.52/77.16/49.00 for ¹³C). ¹H NMR signals marked with an asterisk

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(∗) correspond to peaks assigned to the minor rotamer conformation.

Infrared spectra were measured using a Jasco FT/IR-4100, absorption

values are expressed as wavenumbers (cm⁻¹). Uncorrected melting

points were measured using a Stuart melting point apparatus SMP30.

Measurements for veriﬁcation and purity of the compounds were

performed by LC/MS (from Shimadzu), comprising a DGU-20A3R

controller, pump LC-20AB, degasser DGU-20A, and SPD-20A UV/

Vis detector. ESI ionization was accomplished by an LCMS-2020

single quadrupol mass spectrometer. As a stationary phase, for

analytical purpose, a Synergi 4U fusion-RP 80 Å (150 × 4.6 mm)

column and, for preparative purpose, a Synergi 4 U fusion-RP 80 Å

(250 × 10.0 mm) were used. As a mobile phase, a gradient of MeOH/

water (both containing 0.1% formic acid) (phase 1/phase 2) was

used. The compounds were dissolved in MeOH and ﬁltered through

syringe ﬁlters. Method A: V(1) / (V(1) + V(2)) = from 0 to 60% over

10 min, V(1) / (V(1) + V(2)) = 60% for 5 min, V(1) / (V(1) +

V(2)) = from 60 to 0% over 3 min. Method B: V(1) / (V(1) + V(2))

= from 5 to 90% over 10 min, V(1) / (V(1) + V(2)) = 90% for 5 min,

V(1) / (V(1) + V(2)) = from 90 to 5% over 3 min. Methods were

performed with a ﬂow rate of 1.0 mL/min. Compounds were detected

at λ = 254 nm, and target compounds were ≥95% pure.

(E)-N-Hydroxy-3-(4-(((2-(5-methoxy-1H-indol-3-yl)-ethyl)amino)-

methyl)phenyl)acrylamide (3a). To a stirred solution of NH₂OH·

HCl (23.35 g, 343 mmol) in 120 mL of MeOH was added KOH (28

g, 509 mmol) in 70 mL of MeOH at r.t. After 30 min, a white

precipitate was ﬁltered out to aﬀord NH₂OK methanol solution. 2a

(80 mg, 0.22 mmol) was added to 5 mL of NH₂OK methanol

solution, the mixture was stirred at room temperature for 5 min. The

solvent was evaporated under vacuum; the residue was redissolved

with 5 mL of water and then adjusted to pH 6−7 by hydrochloric

acid. The precipitate was ﬁltered and puriﬁed with column

chromatography to aﬀord light red solid 44 mg, yield 29%. ¹H

NMR (400 MHz, DMSO-d₆): δ 10.88 (brs, 1H) 10.74 (s, 1H), 8.98

(brs, 1H), 7.58 (d, J = 8.1 Hz, 2H), 7.53 (d, J = 8.1 Hz, 2H), 7.45 (d,

J = 15.8 Hz, 1H), 7.24 (d, J = 8.7 Hz, 1H), 7.14 (d, J = 2.2 Hz, 1H),

7.03 (d, J = 2.3 Hz, 1H), 6.73 (dd, J = 8.7, 2.4 Hz, 1H), 6.51 (d, J =

15.8 Hz, 1H), 4.07 (s, 2H), 3.75 (s, 3H), 3.01 (s, 4H) ppm (one

exchangeable proton not observed). ¹³C NMR (101 MHz, DMSO-

d₆): δ 164.3, 153.5, 146.3, 137.9, 135.2, 131.9, 130.4 (2C), 127.9

(2C), 127.7, 124.1, 120.0, 112.6, 111.7, 110.6, 100.7, 55.9, 50.9, 48.1,

23.4 ppm. IR (ﬁlm): ν 3174, 2918, 2823, 1651, 1613, 1579, 1481,

1450, 1435, 1418, 1338, 1288, 1208, 1162, 1064, 1038, 1017, 964,

818, 796, 745, 698 cm⁻¹. LRMS (ESI+) m/z calcd for

[C₂₁H₂₄N₃O₃]⁺: 366.17, found: 339.05 [M + H]⁺. HPLC (method

A): t_R= 8.77 min, purity = 96.1%. mp = 166−168 °C.

All designed target compounds did not show any promiscuous

moieties in the pan-assay interference compound assay (PAINS)

using two diﬀerent in silico ﬁlters (http://www.cbligand.org/PAINS/

and http://fafdrugs4.mti.univ-paris-diderot.fr/).

N-Hydroxy-4-(((2-(5-methoxy-1H-indol-3-yl)ethyl)-amino)-

methyl)benzamide (3b). Compound 3b was prepared from 2b in a

similar manner as described for compound 3a. Light red solid, yield

Methyl (E)-3-(4-(((2-(5-Methoxy-1H-indol-3-yl)ethyl)-amino)-

methyl)phenyl)acrylate (2a). K₂CO₃(0.25 g, 1.78 mmol) and

methyl (E)-3-(4-(bromomethyl)phenyl)-acrylate (0.23 g, 0.89 mmol)

were added in portions to a solution of 5-methoxytryptamine 1 (0.17

g, 0.89 mmol) in 5 mL of THF and 0.5 mL of H₂O at 70 °C and

stirred for 2 h. Excess MgSO₄was added to the mixture and stirred for

30 min; the precipitation was ﬁltered out and washed with THF twice.

THF solution was combined and concentrated under vacuum and

then puriﬁed with column chromatography to aﬀord 0.16 g of red oil,

1

48%. H NMR (400 MHz, DMSO-d₆): δ 11.30 (brs, 1H), 10.79 (s,

1H), 9.13 (brs, 1H), 7.80 (d, J = 8.3 Hz, 2H), 7.66 (d, J = 8.3 Hz,

2H), 7.25 (d, J = 8.8 Hz, 1H), 7.17 (d, J = 2.4 Hz, 1H), 7.09 (d, J =

2.4 Hz, 1H), 6.74 (dd, J = 8.7, 2.4 Hz, 1H), 4.21 (s, 2H), 3.76 (s,

3H), 3.10 (s, 4H) ppm (one exchangeable proton not observed). ¹³C

NMR (101 MHz, DMSO-d₆) δ 164.0, 153.6, 136.2, 133.4, 131.9,

130.3 (2C), 127.6, 127.5 (2C), 124.3, 112.7, 111.7, 109.8, 100.7, 56.0,

50.0, 47.5, 22.4 ppm. IR (ﬁlm): ν 3197, 2928, 2861, 1623, 1579, 1553,

1487, 1460, 1442, 1384, 1317, 1232, 1210, 1182, 1165, 1111, 1036,

1017, 897, 836, 791, 699 cm⁻¹. LRMS (ESI+) m/z calcd for

[C₁₉H₂₂N₃O₃]⁺: 340.16, found: 340.10 [M + H]⁺. HPLC (method

A): t_R= 7.50 min, purity = 99.4%. mp = 190−192 °C.

1

yield 49%. H NMR (400 MHz, DMSO-d₆): δ 10.60 (s, 1H), 7.71−

7.62 (m, 3H), 7.40 (d, J = 8.1 Hz, 2H), 7.21 (d, J = 8.7 Hz, 1H), 7.08

(d, J = 2.3 Hz, 1H), 6.94 (d, J = 2.4 Hz, 1H), 6.70 (dd, J = 8.7, 2.4 Hz,

1H), 6.61 (d, J = 16.1 Hz, 1H), 3.80 (s, 2H), 3.73 (s, 6H), 2.86−2.75

(m, 4H) ppm (one exchangeable proton not observed). ¹³C NMR

(101 MHz, DMSO-d₆): δ 167.2, 153.4, 145.0, 132.9, 131.9, 128.9

(2C), 128.8, 128.7 (2C), 128.0, 123.7, 117.6, 112.7, 112.4, 111.5,

100.6, 55.8, 52.8, 51.9, 49.7, 25.8 ppm. LRMS (ESI+) m/z calcd for

[C₂₂H₂₅N₂O₃]⁺: 365.18, found: 365.20 [M + H]⁺. HPLC (method

B): t_R= 8.71 min.

Methyl 4-((2-(5-Methoxy-1H-indol-3-yl)ethyl)-carbamoyl)-

benzoate (4a). TEA (0.48 g, 4.74 mmol) and TBTU (0.51 g, 1.58

mmol) were added to a solution of 4-(methoxycarbonyl)benzoic acid

(0.28 g, 1.58 mmol) in 10 mL of THF and then stirred at r.t. for 1 h.

5-Methoxytryptamine 1 (0.3 g, 1.58 mmol) was added to the mixture

and stirred for 2 h. By subsequently washing with water and brine,

drying over MgSO₄, and ﬁltering, then, the solvent was removed

under vacuum. The raw product was puriﬁed with column

chromatography to aﬀord red oil (0.37 g, yield 67%). ESI-MS m/z:

Methyl 4-(((2-(5-Methoxy-1H-indol-3-yl)ethyl)-amino)methyl)-

benzoate (2b). Intermediate 2b was prepared from 5-methoxytrypt-

amine 1 and methyl 4-(bromomethyl)benzoate in a similar manner as

described for intermediate 2a. Brown solid, yield 62%. ¹H NMR (400

MHz, DMSO-d₆): δ 10.59 (s, 1H), 7.90 (d, J = 8.2 Hz, 2H), 7.49 (d, J

= 8.2 Hz, 2H), 7.21 (d, J = 8.7 Hz, 1H), 7.08 (d, J = 2.3 Hz, 1H), 6.94

(d, J = 2.4 Hz, 1H), 6.70 (dd, J = 8.7, 2.4 Hz, 1H), 3.85 (s, 3H), 3.84

(s, 2H), 3.73 (s, 3H), 2.87−2.74 (m, 4H) ppm (one exchangeable

proton not observed). ¹³C NMR (101 MHz, DMSO-d₆): δ 166.7,

153.3, 147.3, 131.9, 129.5, 128.6, 128.4, 128.0, 123.7, 112.7, 112.4,

111.4, 100.6, 55.8, 52.8, 52.5, 49.9, 25.9 ppm. LRMS (ESI+) m/z

calcd for [C₂₀H₂₃N₂O₃]⁺: 339.16, found: 339.15 [M + H]⁺. HPLC

(method B): t_R= 8.05 min.

Methyl 6-((2-(5-Methoxy-1H-indol-3-yl)ethyl)-amino)hexanoate

(2c). Intermediate 2c was prepared from 5-methoxytryptamine 1 and

methyl 6-bromo-hexanoate in a similar manner as described for

intermediate 2a. Brown solid, yield 35%. ¹H NMR (400 MHz,

CDCl₃): δ 8.52 (s, 1H), 7.24 (d, J = 8.8 Hz, 1H), 7.08 (d, J = 2.3 Hz,

1H), 7.00 (d, J = 1.3 Hz, 1H), 6.87 (dd, J = 8.8, 2.3 Hz, 1H), 3.87 (s,

3H), 3.67 (s, 3H), 2.97 (s, 4H), 2.65 (t, J = 7.3 Hz, 2H), 2.30 (t, J =

7.5 Hz, 2H), 1.68−1.58 (m, 2H), 1.56−1.45 (m, 3H), 1.38−1.27 (m,

2H) ppm. ¹³C NMR (101 MHz, CDCl₃): δ 174.2, 153.8, 131.5,

127.8, 122.8, 113.3, 112.1, 111.9, 100.7, 56.0, 51.5, 49.8, 49.5, 34.0,

29.6, 26.9, 25.6, 24.8 ppm. LRMS (ESI+) m/z calcd for

[C₁₈H₂₇N₂O₃]⁺: 319.19, found: 319.20 [M + H]⁺. HPLC (method

B): t_R= 7.52 min.

1

353.14 [M + H]⁺. H NMR (400 MHz, CDCl₃): δ 7.96 (d, J = 8.2

Hz, 2H), 7.64 (d, J = 8.2 Hz, 2H), 7.19 (s, 1H), 7.02−6.94 (m, 2H),

6.80 (dd, J = 8.8, 2.3 Hz, 1H), 6.23 (brs, 1H), 3.85 (s, 3H), 3.76−3.68

(m, 5H), 3.00 (t, J = 6.5 Hz, 2H) ppm (one exchangeable proton not

observed). ¹³C NMR (101 MHz, CDCl₃): δ 166.6, 166.3, 154.2,

138.6, 132.6, 131.6, 129.8 (2C), 127.7, 126.9 (2C), 122.9, 112.7

(2C), 112.1, 100.4, 55.9, 52.4, 40.5, 25.2 ppm. LRMS (ESI+) m/z

calcd for [C₂₀H₂₁N₂O₄]⁺: 353.14, found: 353.15 [M + H]⁺. HPLC

(method B): t_R= 11.28 min.

Methyl (E)-3-(4-((2-(5-Methoxy-1H-indol-3-yl)ethyl)-carbamoyl)-

phenyl)acrylate (4b). Intermediate 4b was prepared from 5-

methoxytryptamine 1 and (E)-4-(3-methoxy-3-oxoprop-1-en-1-yl)-

benzoic acid in a similar manner as described for intermediate 4a.

1

Faint yellow solid, yield 71%. H NMR (400 MHz, DMSO-d₆): δ

10.69 (s, 1H), 8.72 (t, J = 5.6 Hz, 1H), 7.94 (d, J = 8.3 Hz, 2H), 7.87

(d, J = 8.3 Hz, 2H), 7.75 (d, J = 16.1 Hz, 1H), 7.28 (d, J = 8.7 Hz,

1H), 7.19 (d, J = 2.0 Hz, 1H), 7.11 (d, J = 2.2 Hz, 1H), 6.83−6.74

(m, 2H), 3.80 (s, 3H), 3.77 (s, 3H), 3.60 (q, J = 6.9 Hz, 2H), 2.98 (t,

J = 7.4 Hz, 2H) ppm. ¹³C NMR (101 MHz, DMSO-d₆): δ 167.0,

165.9, 153.5, 144.0, 136.9, 136.5, 131.9, 128.7 (2C), 128.13 (2C),

128.09, 123.8, 119.8, 112.5, 112.2, 111.5, 100.6, 55.8, 52.1, 40.8, 25.6

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ppm. LRMS (ESI+) m/z calcd for [C₂₂H₂₃N₂O₄]⁺: 379.16, found:

379.20 [M + H]⁺. HPLC (method B): t_R= 11.69 min.

153.5, 138.1, 137.9, 133.7, 131.9, 128.0, 127.92 (2C), 127.87 (2C),

124.5, 123.8, 112.5, 112.1, 111.6, 100.6, 55.8, 49.1, 25.7 ppm. IR

(ﬁlm): ν 3198, 2894, 1659, 1565, 1536, 1491, 1459, 1356, 1317, 1224,

1204, 1174, 1029, 1013, 986, 895, 855, 801, 779, 713, 675 cm⁻¹.

LRMS (ESI+) m/z calcd for [C₂₁H₂₂N₃O₄]⁺: 380.15, found: 380.10

[M + H]⁺. HPLC (method A): t_R= 12.24 min, purity = 97.1%. mp =

140−142 °C.

Methyl (E)-4-(3-((2-(5-Methoxy-1H-indol-3-yl)ethyl)-amino)-3-

oxoprop-1-en-1-yl)benzoate (4c). Intermediate 4c was prepared

from 5-methoxytryptamine 1 and (E)-3-(4-(methoxycarbonyl)-

phenyl)acrylic acid in a similar manner as described for intermediate

4a. Yellow solid, yield 79%. ESI-MS m/z: 379.15 [M + H]⁺. ¹H NMR

(400 MHz, DMSO-d₆): δ 10.65 (s, 1H), 8.30 (t, J = 5.7 Hz, 1H), 7.99

(d, J = 8.4 Hz, 2H), 7.70 (d, J = 8.4 Hz, 2H), 7.50 (d, J = 15.8 Hz,

1H), 7.23 (d, J = 8.7 Hz, 1H), 7.14 (d, J = 2.3 Hz, 1H), 7.05 (d, J =

2.4 Hz, 1H), 6.78 (d, J = 15.8 Hz, 1H), 6.72 (dd, J = 8.7, 2.4 Hz, 1H),

3.87 (s, 3H), 3.76 (s, 3H), 3.49 (q, J = 7.1 Hz, 2H), 2.88 (t, J = 7.2

Hz, 2H), 2.70 (s, 4H) ppm. ¹³C NMR (101 MHz, DMSO-d₆): δ

166.3, 165.0, 153.5, 140.1, 137.6, 131.9, 130.4, 130.2, 128.2, 128.0,

125.6, 123.9, 112.5, 112.1, 111.6, 100.6, 55.8, 52.7, 38.7, 25.6 ppm.

LRMS (ESI+) m/z calcd for [C₂₂H₂₃N₂O₄]⁺: 379.16, found: 379.20

[M + H]⁺. HPLC (method B): t_R= 11.81 min.

Methyl 6-((2-(5-Methoxy-1H-indol-3-yl)ethyl)-amino)-6-oxohex-

anoate (4d). Intermediate 4d was prepared from 5-methoxytrypt-

amine 1 and 6-methoxy-6-oxohexanoic acid in a similar manner as

described for intermediate 4a. Colorless oil, yield 60%. ESI-MS m/z:

319.15 [M + H]⁺. ¹H NMR (400 MHz, DMSO-d₆): δ 10.62 (s, 1H),

7.88 (t, J = 5.6 Hz, 1H), 7.22 (d, J = 8.7 Hz, 1H), 7.09 (d, J = 2.2 Hz,

1H), 7.02 (d, J = 2.4 Hz, 1H), 6.72 (dd, J = 8.7, 2.4 Hz, 1H), 3.76 (s,

3H), 3.59 (s, 3H), 3.35−3.27 (m, 2H), 2.77 (t, J = 7.4 Hz, 2H), 2.21

(t, J = 6.9 Hz, 2H), 2.07 (t, J = 7.0 Hz, 2H), 1.54−1.45 (m, 4H) ppm.

¹³C NMR (101 MHz, DMSO-d₆): δ 173.7, 172.2, 153.4, 131.9, 128.0,

N¹-Hydroxy-N⁶-(2-(5-methoxy-1H-indol-3-yl)ethyl)-adipamide

(5d). Compound 5d was prepared from 4d in a similar manner as

1

described for compound 3a. White solid, yield 17%. H NMR (400

MHz, DMSO-d₆): δ 10.62 (s, 1H), 10.33 (s, 1H), 8.65 (s, 1H), 7.87

(t, J = 5.4 Hz, 1H), 7.22 (d, J = 8.7 Hz, 1H), 7.09 (d, J = 2.1 Hz, 1H),

7.02 (d, J = 2.3 Hz, 1H), 6.71 (dd, J = 8.7, 2.4 Hz, 1H), 3.76 (s, 3H),

3.31 (q, J = 7.4 Hz, 2H), 2.77 (t, J = 7.4 Hz, 2H), 2.06 (t, J = 8.0 Hz,

2H), 1.94 (t, J = 8.0 Hz, 2H), 1.51−1.43 (m, 4H) ppm. ¹³C NMR

(101 MHz, DMSO-d₆): δ 172.3, 169.5, 153.4, 131.9, 128.0, 123.7,

112.4, 112.1, 111.5, 100.6, 98.7, 55.8, 35.8, 32.6, 25.8, 25.5, 25.4 ppm.

IR (ﬁlm): ν 3386, 3318, 3189, 2972, 2871, 1637, 1616, 1586, 1549,

1485, 1466, 1452, 1434, 1416, 1288, 1213, 1170, 1036, 1020, 951,

923, 840, 812, 773, 702, 667 cm⁻¹. LRMS (ESI+) m/z calcd for

[C₁₇H₂₄N₃O₄]⁺: 334.17, found: 334.05 [M + H]⁺. HPLC (method

A): t_R= 10.64 min, purity = 96.5%. mp = 138−139 °C.

N-(2-(5-Methoxy-1H-indol-3-yl)ethyl)acetamide (Melatonin, 6).

5-Methoxytryptamine 1 (2.0 g, 10.5 mmol) was suspended in 25 mL

of DCM, and acetic anhydride (4.4 g, 42.11 mmol) in 5 mL of DCM

was added in drops to the solution at 0 °C and then stirred at r.t. for 2

h. The mixture was washed with sat. NaHCO₃aq., water, and brine;

dried with MgSO₄; ﬁltered; and removed the solvent to aﬀord brown

solid 2.0 g, yield 82%. ¹H NMR (400 MHz, CDCl₃): δ 8.00 (brs, 1H),

7.20 (d, J = 8.7 Hz, 1H), 6.97 (d, J = 2.3 Hz, 1H), 6.95 (s, 1H), 6.80

(dd, J = 8.8, 2.2 Hz, 1H), 5.53 (brs, 1H), 3.79 (s, 3H), 3.52 (q, J = 6.4

Hz, 2H), 2.86 (t, J = 6.7 Hz, 2H), 1.86 (s, 3H) ppm. LRMS (ESI+)

m/z calcd for [C₁₃H₁₇N₂O₂]⁺: 233.12, found: 233.15 [M + H]⁺.

HPLC (method B): t_R= 8.98 min, purity 98.7%.

123.7, 112.4, 112.1, 111.5, 100.6, 55.8, 51.7, 35.6, 33.5, 25.8, 25.2,

24.6 ppm. LRMS (ESI+) m/z calcd for [C₁₈H₂₅N₂O₄]⁺: 333.17,

found: 333.20 [M + H]⁺. HPLC (method B): t_R= 10.31 min.

N¹-Hydroxy-N⁴-(2-(5-methoxy-1H-indol-3-yl)ethyl)-terephthala-

mide (5a). Compound 5a was prepared from 4a in a similar manner

1

as described for compound 3a. Gray solid, yield 19%. H NMR (400

MHz, DMSO-d₆): δ 11.32 (s, 1H), 10.65 (s, 1H), 9.11 (s, 1H), 8.70

(t, J = 5.6 Hz, 1H), 7.91 (d, J = 8.4 Hz, 2H), 7.82 (d, J = 8.4 Hz, 2H),

7.23 (d, J = 8.7 Hz, 1H), 7.15 (d, J = 2.1 Hz, 1H), 7.06 (d, J = 2.3 Hz,

1H), 6.72 (dd, J = 8.7, 2.4 Hz, 1H), 3.73 (s, 3H), 3.55 (q, J = 7.3 Hz,

2H), 2.93 (t, J = 7.4 Hz, 2H) ppm. ¹³C NMR (101 MHz, DMSO-d₆):

δ 165.9, 164.0, 153.5, 137.4, 135.4, 131.9, 128.1, 127.7 (2C), 127.3

(2C), 123.8, 112.5, 112.2, 111.5, 100.6, 55.8, 40.8, 25.6 ppm. IR

(ﬁlm): ν 3258, 2921, 2833, 1667, 1608, 1541, 1482, 1443, 1420, 1332,

1303, 1291, 1214, 1178, 1153, 1030, 1013, 838, 795, 723, 692, 677

cm⁻¹. LRMS (ESI+) m/z calcd for [C₁₉H₂₀N₃O₄]⁺: 354.14, found:

354.00 [M + H]⁺. HPLC (method A): t_R= 11.36 min, purity = 99.9%.

mp = 179−181 °C.

(E)-4-(3-(Hydroxyamino)-3-oxoprop-1-en-1-yl)-N-(2-(5-methoxy-

1H-indol-3-yl)ethyl)benzamide (5b). Compound 5b was prepared

from 4b in a similar manner as described for compound 3a. Red solid,

yield 30%. ¹H NMR (400 MHz, DMSO-d₆): δ 10.82 (brs, 1H), 10.65

(s, 1H), 8.65 (t, J = 5.5 Hz, 1H), 7.88 (d, J = 8.3 Hz, 2H), 7.64 (d, J =

8.2 Hz, 2H), 7.50 (d, J = 15.8 Hz, 1H), 7.23 (d, J = 8.7 Hz, 1H), 7.14

(d, J = 2.0 Hz, 1H), 7.06 (d, J = 2.3 Hz, 1H), 6.72 (dd, J = 8.7, 2.4 Hz,

1H), 6.56 (d, J = 15.8 Hz, 1H), 3.73 (s, 3H), 3.54 (q, J = 6.8 Hz, 2H),

2.93 (t, J = 7.4 Hz, 2H) ppm (one exchangeable proton not

observed). ¹³C NMR (101 MHz, DMSO-d₆): δ 166.0, 162.9, 153.5,

137.8, 137.8, 135.7, 131.9, 128.2 (2C), 128.1, 127.8 (2C), 123.8,

121.2, 112.5, 112.2, 111.5, 100.7, 55.8, 40.7, 25.6 ppm. IR (ﬁlm): ν

3186, 2883, 2814, 1609, 1525, 1487, 1478, 1440, 1427, 1284, 1207,

1163, 1043, 1021, 974, 831, 792, 755, 704, 660 cm⁻¹. LRMS (ESI+)

m/z calcd for [C₂₁H₂₂N₃O₄]⁺: 380.15, found: 380.05 [M + H]⁺.

HPLC (method A): t_R= 12.13 min, purity = 99.1%. mp = 90−92 °C.

(E)-N-Hydroxy-4-(3-((2-(5-methoxy-1H-indol-3-yl)-ethyl)amino)-

3-oxoprop-1-en-1-yl)benzamide (5c). Compound 5c was prepared

from 4c in a similar manner as described for compound 3a. Yellow

solid, yield 18%. ¹H NMR (400 MHz, DMSO-d₆): δ 11.26 (brs, 1H),

10.65 (s, 1H), 9.07 (brs, 1H), 8.25 (t, J = 5.7 Hz, 1H), 7.78 (d, J = 8.3

Hz, 2H), 7.63 (d, J = 8.3 Hz, 2H), 7.47 (d, J = 15.8 Hz, 1H), 7.23 (d,

J = 8.7 Hz, 1H), 7.14 (d, J = 2.2 Hz, 1H), 7.05 (d, J = 2.3 Hz, 1H),

6.77−6.65 (m, 2H), 3.76 (s, 3H), 3.48 (q, J = 7.0 Hz, 2H), 2.87 (t, J =

7.2 Hz, 2H) ppm. ¹³C NMR (101 MHz, DMSO-d₆): δ 165.5, 165.1,

Methyl (E)-3-(4-((3-(2-Acetamidoethyl)-5-methoxy-1H-indol-1-

yl)methyl)phenyl)acrylate (7a). Melatonin (0.5 g, 2.2 mmol) and t-

BuOK (0.49 g, 4.4 mmol) were stirred in 15 mL of THF at 70 °C for

40 min; then methyl (E)-3-(4-(bromomethyl)phenyl)acrylate (0.66 g,

2.6 mmol) was added and stirred for 12 h. The mixture was cooled to

r.t. and concentrated under vacuum; the residue was redissolved in

DCM and then washed with 0.1 M HCl, water, and brine. The

organic phase was dried over MgSO₄and ﬁltered, the solvent was

removed and then redissolved in MeOH, and SOCl₂(0.79 g, 6.6

mmol) was added in drop with an ice bath and after that stirred at r.t.

overnight. The solvent was removed under vacuum and redissolved

with DCM, then washed with 0.1 M NaOH, water, and brine and

ﬁnally puriﬁed with column chromatography to aﬀord yellow solid

0.83 g, yield 66%. ¹H NMR (400 MHz, DMSO-d₆): δ 7.94 (t, J = 5.5

Hz, 1H), 7.64 (d, J = 8.3 Hz, 2H), 7.61 (d, J = 16.4 Hz, 1H), 7.27 (d,

J = 8.8 Hz, 1H), 7.27 (s, 1H), 7.18 (d, J = 8.1 Hz, 2H), 7.06 (d, J =

2.3 Hz, 1H), 6.73 (dd, J = 8.8, 2.3 Hz, 1H), 6.58 (d, J = 16.0 Hz, 1H),

5.34 (s, 2H), 3.76 (s, 3H), 3.71 (s, 3H), 3.32 (q, J = 7.4 Hz, 2H), 2.79

(t, J = 7.4 Hz, 2H), 1.81 (s, 3H) ppm. ¹³C NMR (101 MHz, DMSO-

d₆): δ 169.5, 167.1, 153.8, 144.6, 141.6, 133.5, 131.8, 129.0 (2C),

128.7, 127.9 (2C), 127.6, 118.1, 112.2, 111.7, 111.2, 101.2, 55.9, 51.9,

49.3, 25.6, 23.2 ppm. LRMS (ESI+) m/z calcd for [C₂₄H₂₇N₂O₄]⁺:

407.19, found: 407.20 [M + H]⁺. HPLC (method B): t_R= 12.48 min.

Methyl 4-((3-(2-Acetamidoethyl)-5-methoxy-1H-indol-1-yl)-

methyl)benzoate (7b). Intermediate 7b was prepared from melatonin

and methyl 4-(bromomethyl)-benzoate in a similar manner as

1

described for intermediate 7a. Oﬀ-white solid, yield 63%. H NMR

(400 MHz, DMSO-d₆): δ 7.93 (t, J = 5.0 Hz, 1H), 7.88 (d, J = 8.3 Hz,

2H), 7.27 (s, 1H), 7.25 (d, J = 8.3 Hz, 2H), 7.24 (d, J = 8.8 Hz, 1H),

7.06 (d, J = 2.4 Hz, 1H), 6.73 (dd, J = 8.8, 2.4 Hz, 1H), 5.42 (s, 2H),

3.82 (s, 3H), 3.76 (s, 3H), 3.32 (q, J = 7.4 Hz, 2H), 2.80 (t, J = 7.4

Hz, 2H), 1.80 (s, 3H) ppm. ¹³C NMR (101 MHz, DMSO-d₆): δ

169.5, 166.4, 153.8, 144.6, 131.8, 129.9, 129.0, 128.7, 127.7, 127.5,

112.3, 111.8, 111.2, 101.2, 55.9, 52.6, 49.2, 25.6, 23.2 ppm. LRMS

(ESI+) m/z calcd for [C₂₂H₂₅N₂O₄]⁺: 381.17, found: 381.20 [M +

H]⁺. HPLC (method B): t_R= 12.08 min.

3804

https://doi.org/10.1021/acs.jmedchem.0c01940

J. Med. Chem. 2021, 64, 3794−3812

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Methyl 6-(3-(2-Acetamidoethyl)-5-methoxy-1H-indol-1-yl)-

hexanoate (7c). Intermediate 7c was prepared from melatonin and

methyl 6-bromohexanoate in a similar manner as described for

intermediate 7a. Yellow oil, yield 57%. ¹H NMR (400 MHz, CDCl₃):

δ 7.13 (d, J = 8.9 Hz, 1H), 6.94 (d, J = 2.1 Hz, 1H), 6.84 (s, 1H), 6.81

(dd, J = 8.9, 2.1 Hz, 1H), 6.05 (brs, 1H), 3.97 (t, J = 6.9 Hz, 2H),

3.79 (s, 3H), 3.60 (s, 3H), 3.56 (s, 3H), 3.54−3.47 (m, 2H), 2.87 (t, J

= 6.0 Hz, 2H), 2.21 (t, J = 7.3 Hz, 2H), 1.78−1.70 (m, 2H), 1.57−

1.51 (m, 2H), 1.30−1.20 (m, 2H) ppm. ¹³C NMR (101 MHz,

CDCl₃): δ 169.2, 165.4, 149.1, 127.0, 123.4, 121.6, 107.2, 106.2,

105.5, 95.9, 51.2, 46.8, 41.4, 35.3, 29.0, 25.2, 21.7, 20.5, 19.7, 18.6

ppm. LRMS (ESI+) m/z calcd for [C₂₀H₂₉N₂O₄]⁺: 361.20, found:

361.20 [M + H]⁺. HPLC (method B): t_R= 11.83 min.

(E)-3-(4-((3-(2-Acetamidoethyl)-5-methoxy-1H-indol-1-yl)-

methyl)phenyl)-N-hydroxyacrylamide (8a). Compound 8a was

prepared from 7a in a similar manner as described for compound

3a. Red solid, yield 34%. ¹H NMR (400 MHz, DMSO-d₆): δ 8.21 (s,

2H), 7.94 (t, J = 5.3 Hz, 1H), 7.48 (d, J = 8.0 Hz, 2H), 7.40 (d, J =

15.8 Hz, 1H), 7.27 (d, J = 7.6 Hz, 1H), 7.26 (s, 1H), 7.18 (d, J = 8.1

Hz, 2H), 7.05 (d, J = 2.3 Hz, 1H), 6.73 (dd, J = 8.8, 2.4 Hz, 1H), 6.40

(d, J = 15.8 Hz, 1H), 5.33 (s, 2H), 3.76 (s, 3H), 3.31 (q, J = 7.4 Hz,

2H), 2.79 (t, J = 7.4 Hz, 2H), 1.80 (s, 3H) ppm. ¹³C NMR (101

MHz, DMSO-d₆): δ 169.5, 165.5, 153.8, 140.4, 138.3, 134.3, 131.8,

128.7, 128.1 (2C), 127.9 (2C), 127.6, 119.4, 112.1, 111.7, 111.2,

101.2, 55.9, 49.3, 39.9, 25.6, 23.2 ppm. IR (ﬁlm): ν 3276, 2919, 2839,

1662, 1631, 1562, 1492, 1455, 1440, 1358, 1303, 1222, 1178, 1040,

975, 831, 795, 738, 669 cm⁻¹. LRMS (ESI+) m/z calcd for

[C₂₃H₂₆N₃O₄]⁺: 408.18, found: 408.10 [M + H]⁺. HPLC (method

A): t_R= 13.31 min, purity = 95.2%. mp = 187−189 °C.

4-((3-(2-Acetamidoethyl)-5-methoxy-1H-indol-1-yl)-methyl)-N-

hydroxybenzamide (8b). Compound 8b was prepared from 7b in a

similar manner as described for compound 3a. Oﬀ-white solid, yield

14%. ¹H NMR (400 MHz, DMSO-d₆) δ 11.13 (s, 1H), 8.99 (s, 1H),

7.94 (t, J = 5.6 Hz, 1H), 7.66 (d, J = 8.2 Hz, 2H), 7.27 (s, 1H), 7.26

(d, J = 8.3 Hz, 1H), 7.20 (d, J = 8.2 Hz, 2H), 7.05 (d, J = 2.4 Hz, 1H),

6.73 (dd, J = 8.8, 2.4 Hz, 1H), 5.36 (s, 2H), 3.76 (s, 3H), 3.31 (q, J =

7.3 Hz, 2H), 2.79 (t, J = 7.4 Hz, 2H), 1.80 (s, 3H). ¹³C NMR (101

MHz, DMSO-d₆) δ 169.5, 164.4, 153.8, 142.2, 132.3, 131.7, 128.7,

127.6, 127.5 (2C), 127.3 (2C), 112.1, 111.7, 111.2, 101.2, 55.9, 49.2,

39.9, 25.6, 23.2. IR (ﬁlm): ν 3204, 3012, 2812, 2633, 2531, 1646,

1627, 1585, 1574, 1495, 1453, 1411, 1356, 1224, 1040, 1030, 844,

837, 807, 773, 715 cm⁻¹. LRMS (ESI+) m/z calcd for

[C₂₁H₂₄N₃O₄]⁺: 382.17, found: 382.05 [M + H]⁺. HPLC (method

A): t_R= 12.14 min, purity = 98.0%. mp = 90−92 °C.

7.72*/7.71 (d, J = 8.1 Hz, 2H), 7.66/7.64* (d, J = 15.9 Hz, 1H),

7.62*/7.40 (d, J = 15.4 Hz, 1H), 7.46*/7.28 (d, J = 1.9 Hz, 1H),

7.35/7.31* (d, J = 8.1 Hz, 2H), 7.22*/7.18 (d, J = 8.8 Hz, 1H),

7.20*/7.11 (d, J = 8.8 Hz, 1H), 7.09 (d, J = 2.2 Hz, 1H), 7.04*/6.99

(d, J = 2.2 Hz, 1H), 6.85 (dd, J = 8.8, 1.6 Hz, 1H), 6.82 (d, J = 15.7

Hz, 1H), 6.71 (dd, J = 8.7, 2.3 Hz, 1H), 6.63/6.62* (d, J = 16.0 Hz,

1H), 4.87*/4.72 (s, 2H), 4.30−4.19 (m, 2H), 3.83*/3.80 (s, 3H),

3.75*/3.73 (s, 3H), 3.71 (s, 3H), 3.71−3.58 (m, 2H), 3.02−2.84 (m,

2H), 1.29/1.27* (t, J = 7.1 Hz, 3H) ppm (the minor rotamer: 35%).

¹³C NMR (101 MHz, DMSO-d₆): δ 167.2, 167.1, 166.3, 153.6, 153.5,

152.8, 151.4, 144.7, 141.6, 140.8, 140.6, 134.8, 133.3, 131.9, 129.2,

129.0, 128.7, 128.0, 127.8, 124.7, 120.4, 119.5, 118.0, 112.9, 112.6,

111.6, 110.9, 100.4, 100.0, 65.2, 56.6, 56.4, 55.79, 55.75, 51.9, 48.5,

48.0, 25.1, 14.5 ppm. LRMS (ESI+) m/z calcd for [C₃₅H₃₇N₂O₈]⁺:

613.25, found: 613.30 [M + H]⁺. HPLC (method B): t_R= 13.33 min.

Methyl (E)-4-((3-(4-((Ethoxycarbonyl)oxy)-3-meth-oxyphenyl)-N-

(2-(5-methoxy-1H-indol-3-yl)ethyl)acry-lamido)methyl)benzoate

(9b). Intermediate 9b was prepared from 2b and ferulic acid in a

similar manner as described for intermediate 9a. Yellow solid, yield

90%. ¹H NMR (400 MHz, DMSO-d₆): δ 10.66/10.64* (d, J = 1.5 Hz,

1H), 7.95 (d, J = 8.2 Hz, 2H), 7.62*/7.40 (d, J = 15.2 Hz, 1H), 7.44

(d, J = 8.2 Hz, 2H), 7.41 (d, J = 8.4 Hz, 1H), 7.28/7.26* (d, J = 1.7

Hz, 1H), 7.24*/6.82 (d, J = 15.6 Hz, 1H) 7.22*/7.17 (d, J = 8.8 Hz,

1H), 7.20*/7.11 (d, J = 8.8 Hz, 1H), 7.08 (d, J = 2.4 Hz, 1H), 7.03*/

6.99 (d, J = 2.2 Hz, 1H), 6.85 (dd, J = 8.8, 1.6 Hz, 1H), 6.71 (dd, J =

8.7, 2.3 Hz, 1H), 4.93*/4.78 (s, 2H), 4.29−4.20 (m, 2H), 3.85/3.83*

(s, 3H), 3.83*/3.80 (s, 3H), 3.74*/3.71 (s, 3H), 3.70−3.58 (m, 2H),

3.00−2.84 (m, 2H), 1.29/1.28* (t, J = 7.0 Hz, 3H) ppm (the minor

rotamer: 32%). ¹³C NMR (101 MHz, DMSO-d₆): δ 166.8, 166.7,

166.6, 166.5, 153.7, 153.5, 149.0, 148.8, 148.3, 148.2, 144.9, 144.8,

143.1, 142.5, 131.9, 130.0, 129.8, 129.1, 128.8, 128.3, 128.0, 127.9,

127.6, 127.12, 127.05, 124.6, 123.9, 122.8, 121.9, 116.1, 115.3, 112.6,

112.5, 112.3, 111.9, 111.8, 111.6, 111.0, 100.6, 100.4, 56.2, 56.1, 55.8,

55.4, 52.5, 51.1, 48.7, 48.1, 47.7, 25.2, 23.9, 14.3 ppm. LRMS (ESI+)

m/z calcd for [C₃₃H₃₅N₂O₈]⁺: 587.23, found: 587.25 [M + H]⁺.

HPLC (method B): t_R= 13.13 min.

Methyl (E)-6-(3-(4-((Ethoxycarbonyl)oxy)-3-meth-oxyphenyl)-N-

(2-(5-methoxy-1H-indol-3-yl)ethyl)acry-lamido)hexanoate (9c). In-

termediate 9c was prepared from 2c and ferulic acid in a similar

1

manner as described for intermediate 9a. Yellow oil, yield 49%. H

NMR (400 MHz, DMSO-d₆): δ 10.66/10.64* (d, J = 1.5 Hz, 1H),

7.55*/7.30 (d, J = 15.6 Hz, 1H), 7.48*/7.03 (d, J = 2.2 Hz, 1H),

7.33*/6.82 (dd, J = 8.3, 1.5 Hz, 1H), 7.25−7.22 (m, 1H), 7.18 (d, J =

8.7 Hz, 1H), 7.16−7.11 (m, 2H), 7.09 (d, J = 8.2 Hz, 1H), 6.76−6.69

(m, 1H), 4.25*/4.24 (q, J = 7.1 Hz, 2H), 3.87*/3.79 (s, 3H), 3.77*/

3.72 (s, 3H), 3.72*/3.62 (t, J = 7.3 Hz, 2H), 3.59/3.54* (s, 3H),

3.48*/3.39 (t, J = 7.3 Hz, 2H), 2.94/2.92* (t, J = 7.0 Hz, 2H), 2.32/

2.27* (t, J = 7.4 Hz, 2H), 1.65−1.48 (m, 4H), 1.36−1.23 (m, 5H)

ppm (the minor rotamer: 42%). ¹³C NMR (101 MHz, DMSO-d₆): δ

173.8, 173.7, 165.7, 165.6, 153.6, 152.9, 151.5, 151.4, 140.8, 140.5,

139.9, 135.0, 131.8, 127.9, 124.7, 123.8, 122.9, 121.0120.3, 120.1,

119.9, 112.8, 112.6, 112.5, 112.0, 111.54, 111.50, 111.0, 100.8, 100.4,

65.2, 56.5, 56.4, 55.8, 55.7, 51.6, 51.6, 48.2, 45.8, 33.7, 33.6, 29.6,

27.6, 26.5, 26.0, 25.4, 24.7, 24.6, 24.1, 14.5 ppm. LRMS (ESI+) m/z

calcd for [C₃₁H₃₉N₂O₈]⁺: 567.26, found: 567.30 [M + H]⁺. HPLC

(method B): t_R= 12.90 min.

(E)-3-(4-Hydroxy-3-methoxyphenyl)-N-(4-((E)-3-(hydroxyamino)-

3-oxoprop-1-en-1-yl)benzyl)-N-(2-(5-methoxy-1H-indol-3-yl)ethyl)-

acrylamide (10a). Compound 10a was prepared from 9a in a similar

manner as described for compound 3a. Gray solid 0.16 g, yield 60%.

¹H NMR (400 MHz, DMSO-d₆): δ 10.75 (s, 1H), 10.67/10.64* (s,

1H), 9.40 (s, 1H), 9.03 (s, 1H), 7.54 (d, J = 7.5 Hz, 2H), 7.45/7.44*

(d, J = 15.8 Hz, 1H), 7.38 (d, J = 15.3 Hz, 1H), 7.33/7.29* (d, J = 8.1

Hz, 2H), 7.24 (d, J = 3.5 Hz, 1H), 7.21 (d, J = 8.6 Hz, 1H), 7.12−

7.06 (m, 2H), 7.03*/7.00 (d, J = 1.9 Hz, 1H), 6.81−6.64 (m, 3H),

6.45/6.44* (d, J = 15.8 Hz, 1H), 4.83*/4.70 (s, 2H), 3.80*/3.77 (s,

3H), 3.74*/3.73 (s, 3H), 3.68/3.61* (t, J = 6.8 Hz, 2H), 3.01−2.85

(m, 2H) ppm (the minor rotamer: 38%). ¹³C NMR (101 MHz,

DMSO-d₆): δ 166.7, 166.6, 163.3, 153.6, 153.5, 149.0, 148.8, 148.3,

148.1, 142.9, 142.3, 140.6, 138.5, 134.1, 131.9, 128.7 (2C), 128.3,

6-(3-(2-Acetamidoethyl)-5-methoxy-1H-indol-1-yl)-N-hydroxy-

hexanamide (8c). Compound 8c was prepared from 7c in a similar

manner as described for compound 3a. Pale yellow solid, yield 26%.

¹H NMR (400 MHz, DMSO-d₆): δ 10.31 (s, 1H), 8.64 (s, 1H), 7.93

(t, J = 5.4 Hz, 1H), 7.30 (d, J = 8.8 Hz, 1H), 7.12 (s, 1H), 7.02 (d, J =

2.3 Hz, 1H), 6.76 (dd, J = 8.8, 2.4 Hz, 1H), 4.04 (t, J = 7.0 Hz, 2H),

3.77 (s, 3H), 3.31 (q, J = 7.4 Hz, 2H), 2.76 (t, J = 7.4 Hz, 2H), 1.92

(t, J = 7.4 Hz, 2H), 1.81 (s, 3H), 1.74−1.65 (m, 2H), 1.56−1.44 (m,

2H), 1.26−1.15 (m, 2H) ppm. ¹³C NMR (101 MHz, DMSO-d₆): δ

169.5, 169.4, 153.5, 131.7, 128.4, 127.0, 111.5, 111.3, 110.8, 101.0,

55.9, 45.7, 39.9, 32.6, 30.1, 26.4, 25.6, 25.2, 23.2 ppm. IR (ﬁlm): ν

3298, 3265, 2933, 2879, 1667, 1659, 1596, 1504, 1461, 1398, 1332,

1231, 1185, 1094, 1040, 966, 849, 791, 769, 676 cm⁻¹. LRMS (ESI+)

m/z calcd for [C₁₉H₂₈N₃O₄]⁺: 362.20, found: 362.10 [M + H]⁺.

HPLC (method A): t_R= 12.28 min, purity = 99.5%. mp = 130−131

°C.

Methyl (E)-3-(4-(((E)-3-(4-((Ethoxycarbonyl)oxy)-3-methoxy-

phenyl)-N-(2-(5-methoxy-1H-indol-3-yl)-ethyl)acrylamido)methyl)-

phenyl)acrylate (9a). TEA (0.33 g, 3.3 mmol) and ethyl

chloroformate (0.28 g, 2.6 mmol) were added to a solution of ferulic

acid (0.26 g, 1.3 mmol) in 15 mL of THF and then stirred at room

temperature for 5 min. 2a (0.40 g, 1.1 mmol) was added to the

mixture in portions and then stirred at r.t. for 30 min. The solvent was

removed under vacuum, and the residue was puriﬁed with column

chromatography to aﬀord pale-yellow solid 0.40 g, yield 60%. ¹H

NMR (400 MHz, DMSO-d₆): δ 10.66/10.64* (d, J = 1.6 Hz, 1H),

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128.1 (2C), 128.0, 127.8, 127.13, 127.07, 124.6, 123.8, 122.8, 121.9,

119.4, 119.2, 116.04, 115.98, 115.5, 112.6, 112.3, 111.9, 111.8, 111.6,

111.0, 100.6, 100.4, 56.24, 56.15, 55.8, 48.4, 47.9, 47.5, 25.1, 23.9

ppm. IR (ﬁlm): ν 3229, 2952, 2885, 1626, 1572, 1505, 1472, 1450,

1434, 1412, 1352, 1259, 1230, 1204, 1171, 1148, 1120, 1022, 976,

835, 816, 792, 748, 701, 656 cm⁻¹. LRMS (ESI+) m/z calcd for

[C₃₁H₃₂N₃O₆]⁺: 542.22, found: 542.30 [M + H]⁺. HPLC (method

B): t_R= 11.20 min, purity = 99.8%. mp = 135−137 °C.

MHz, DMSO-d₆): δ 10.65 (s, 1H), 8.01 (s, 1H), 7.23 (d, J = 8.7 Hz,

1H), 7.12 (d, J = 2.3 Hz, 1H), 7.05 (d, J = 2.4 Hz, 1H), 6.72 (dd, J =

8.7, 2.4 Hz, 1H), 3.77 (s, 3H), 3.38 (q, J = 7.1 Hz, 2H), 3.11 (s, 2H),

2.81 (t, J = 7.4 Hz, 2H), 2.29 (brs, 2H) ppm. ¹³C NMR (101 MHz,

DMSO-d₆): δ 173.0, 153.5, 131.9, 128.0, 123.8, 112.5, 111.9, 111.5,

100.7, 55.8, 55.4, 44.7, 25.7 ppm. LRMS (ESI+) m/z calcd for

[C₁₃H₁₈N₃O₂]⁺: 248.13, found: 248.20 [M + H]⁺. HPLC (method

B): t_R= 6.15 min.

Methyl (E)-3-(4-(((2-((2-(5-Methoxy-1H-indol-3-yl)-ethyl)amino)-

2-oxoethyl)amino)methyl)phenyl)acry-late (13a). Intermediate 13a

was prepared from intermediate 12 and methyl (E)-3-(4-(bromo-

methyl)-phenyl)acrylate in a similar manner as described for

intermediate 2a. Yellow oil, yield 30%. ¹H NMR (400 MHz,

DMSO-d₆): δ 10.66 (s, 1H), 7.90 (t, J = 5.7 Hz, 1H), 7.65 (d, J =

15.7 Hz, 1H), 7.64 (d, J = 8.4 Hz, 2H), 7.32 (d, J = 8.1 Hz, 2H), 7.24

(d, J = 8.7 Hz, 1H), 7.12 (d, J = 2.3 Hz, 1H), 7.05 (d, J = 2.4 Hz, 1H),

6.73 (dd, J = 8.7, 2.4 Hz, 1H), 6.62 (d, J = 16.1 Hz, 1H), 3.76 (s, 3H),

3.73 (s, 3H), 3.66 (s, 2H), 3.40 (q, J = 7.0 Hz, 2H), 3.07 (s, 2H), 2.81

(t, J = 7.2 Hz, 2H) ppm (one exchangeable proton not observed). ¹³C

NMR (101 MHz, DMSO-d₆): δ 171.0, 167.2, 153.5, 144.9, 143.3,

133.0, 131.9, 129.0 (2C), 128.7 (2C), 128.0, 123.8, 117.7, 112.5,

111.9, 111.5, 100.7, 55.8, 55.4, 52.7, 51.9, 51.8, 25.7 ppm. LRMS (ESI

+) m/z calcd for [C₂₄H₂₈N₃O₄]⁺: 422.20, found: 422.20 [M + H]⁺.

HPLC (method B): t_R= 8.69 min.

(E)-N-Hydroxy-4-((3-(4-hydroxy-3-methoxyphenyl)-N-(2-(5-me-

thoxy-1H-indol-3-yl)ethyl)acrylamido)-methyl)benzamide (10b).

Compound 10b was prepared from 9b in a similar manner as

1

described for compound 3a. Orange oil, yield 46%. H NMR (400

MHz, DMSO-d₆): δ 11.18 (s, 1H), 10.68/10.64* (s, 1H), 9.42*/9.40

(s, 1H), 9.01 (brs, 1H), 7.77−7.69 (m, 2H), 7.58−7.16 (m, 5H),

7.12−6.98 (m, 3H), 6.82−6.64 (m, 3H), 4.85*/4.71 (s, 2H), 3.79*/

3.77 (s, 3H), 3.74*/3.73 (s, 3H), 3.69/3.61* (t, J = 6.6 Hz, 2H),

3.00−2.85 (m, 2H) ppm (the minor rotamer: 38%). ¹³C NMR (101

MHz, DMSO-d₆): δ 169.2, 166.8, 153.6, 153.49, 153.46, 148.8, 148.1,

147.8, 147.6, 142.4, 142.3, 132.8, 131.9, 128.4, 128.0, 127.6, 127.5,

127.3, 127.3, 127.0, 124.6, 124.1, 123.8, 122.4, 121.9, 116.0, 115.8,

115.4, 113.0, 112.6, 112.3, 111.6, 111.0, 110.9, 100.4, 100.2, 56.2,

56.1, 56.0, 55.9, 55.8, 55.8, 55.4, 49.1, 25.1, 21.0 ppm. IR (ﬁlm): ν

3201, 2930, 2856, 1639, 1574, 1513, 1482, 1452, 1366, 1268, 1211,

1155, 1124, 1070, 1025, 1013, 973, 894, 815, 796, 747 cm⁻¹. LRMS

(ESI+) m/z calcd for [C₂₉H₃₀N₃O₆]⁺: 516.21, found: 516.25 [M +

H]⁺. HPLC (method B): t_R= 10.77 min, purity = 95.7%. mp = 86−88

°C.

(E)-N-Hydroxy-6-(3-(4-hydroxy-3-methoxyphenyl)-N-(2-(5-me-

thoxy-1H-indol-3-yl)ethyl)acrylamido)-hexanamide (10c). Com-

pound 10c was prepared from 9c in a similar manner as described

for compound 3a. White solid, yield 33%. ¹H NMR (400 MHz,

DMSO-d₆): δ 10.67/10.63* (s, 1H), 10.34/10.32* (s, 1H), 9.35 (brs,

1H), 8.66 (s, 1H), 7.48*/7.27 (d, J = 15.2 Hz, 1H), 7.26−7.09 (m,

1H), 7.16−7.10 (s, 1H), 7.03 (s, 1H), 6.91*/6.58 (d, J = 15.3 Hz,

1H), 6.81*/6.73 (d, J = 8.1 Hz, 1H), 6.76−6.67 (m, 2H), 6.18−6.14

(m, 1H), 3.83*/3.77 (s, 3H), 3.75*/3.74 (s, 3H), 3.70/3.60* (t, J =

7.2 Hz 2H), 3.45*/3.36 (t, J = 7.2 Hz 2H), 2.94/2.90* (t, J = 6.80

Hz, 2H), 2.02−1.90 (m, 2H), 1.62−1.46 (m, 4H), 1.34−1.18 (m,

2H) ppm (the minor rotamer: 40%). ¹³C NMR (101 MHz, DMSO-

d₆) δ 169.6, 169.5, 166.1, 166.0, 163.0, 153.6, 153.5, 148.8, 148.6,

148.3, 148.1, 142.1, 141.4, 131.9, 127.9, 127.3, 124.6, 123.8, 122.4,

121.7, 116.1, 115.8, 112.6, 112.2, 112.0, 111.5, 111.5, 111.1, 100.8,

100.4, 56.3, 56.1, 55.79, 55.75, 48.2, 47.5, 45.9, 32.7, 29.7, 27.8, 26.7,

26.2, 25.5, 25.3 ppm. IR (ﬁlm): ν 3304, 3199, 2922, 2851, 1637, 1582,

1513, 1481, 1454, 1427, 1372, 1268, 1214, 1161, 1124, 1101, 1064,

1028, 972, 917, 816, 795 cm⁻¹. LRMS (ESI+) m/z calcd for

[C₂₇H₃₄N₃O₆]⁺: 496.24, found: 496.25 [M + H]⁺. HPLC (method

B): t_R= 10.77 min, purity = 97.6%. mp = 85−87 °C.

Methyl 4-(((2-((2-(5-Methoxy-1H-indol-3-yl)ethyl)-amino)-2-

oxoethyl)amino)methyl)benzoate (13b). Intermediate 13b was

prepared from intermediate 12 and methyl 4-(bromomethyl)benzoate

in a similar manner as described for intermediate 2a. Pale yellow oil,

1

yield 16%. H NMR (400 MHz, DMSO-d₆): δ 10.66 (s, 1H), 7.93−

7.84 (m, 3H), 7.41 (d, J = 8.2 Hz, 2H), 7.23 (d, J = 8.7 Hz, 1H), 7.12

(d, J = 2.2 Hz, 1H), 7.05 (d, J = 2.3 Hz, 1H), 6.73 (dd, J = 8.7, 2.4 Hz,

1H), 3.85 (s, 3H), 3.76 (s, 3H), 3.69 (s, 2H), 3.40 (q, 7.0 Hz, 2H),

3.07 (s, 2H), 2.81 (t, J = 7.2 Hz, 2H) ppm (one exchangeable proton

not observed). ¹³C NMR (101 MHz, DMSO-d₆): δ 171.1, 166.7,

153.5, 146.6, 131.9, 129.5 (2C), 128.6 (2C), 128.5, 128.0, 123.8,

112.5, 112.0, 111.5, 100.7, 55.8, 52.7, 52.5, 52.0, 25.7 ppm. LRMS

(ESI+) m/z calcd for [C₂₂H₂₆N₃O₄]⁺: 396.18, found: 396.20 [M +

H]⁺. HPLC (method B): t_R= 8.30 min.

Methyl 6-((2-((2-(5-Methoxy-1H-indol-3-yl)ethyl)-amino)-2-

oxoethyl)amino)hexanoate (13c). Intermediate 13c was prepared

from intermediate 12 and methyl 6-bromohexanoate in a similar

1

manner as described for intermediate 2a. Yellow oil, yield 42%. H

NMR (400 MHz, MeOD-d₄): δ 7.24 (d, J = 8.8 Hz, 1H), 7.07 (s,

1H), 7.07 (d, J = 2.4 Hz, 1H), 6.77 (dd, J = 8.8, 2.4 Hz, 1H), 3.84 (s,

3H), 3.74 (s, 2H), 3.67 (s, 3H), 3.61−3.54 (m, 2H), 2.95 (t, J = 7.0

Hz, 2H), 2.77 (t, J = 7.6 Hz, 2H), 2.34 (t, J = 7.3 Hz, 2H), 1.93−1.83

(m, 2H), 1.74−1.52 (m, 2H), 1.43−1.23 (m, 2H) ppm (exchangeable

protons not observed). ¹³C NMR (101 MHz, MeOD-d₄): δ 174.2,

167.6153.6, 131.0, 127.3, 122.9, 112.2, 111.7, 111.4, 99.8, 54.6, 52.5,

50.7, 49.8, 48.6, 33.2, 28.2, 26.3, 24.9, 24.6 ppm. LRMS (ESI+) m/z

calcd for [C₂₀H₃₀N₃O₄]⁺: 376.22, found: 376.20 [M + H]⁺. HPLC

(method B): t_R= 7.96 min.

tert-Butyl (2-((2-(5-Methoxy-1H-indol-3-yl)ethyl)-amino)-2-

oxoethyl)carbamate (11). TEA (1.07 g, 10.50 mmol), EDCI (2.40

g, 12.60 mmol), and DMAP (1.28 g, 10.5 mmol) were added to a

solution of Boc-Glu-OH (2.20 g, 12.60 mmol) in 40 mL of THF; then

5-methoxytryptamine 1 (2.00 g, 10.50 mmol) was added to the

mixture and, after that, stirred overnight at r.t. THF was removed, and

the residue was redissolved in DCM, washed with 0.1 M HCl, water,

and brine, dried over MgSO₄, and puriﬁed with column chromatog-

Methyl (E)-3-(4-(((E)-3-(4-((Ethoxycarbonyl)oxy)-3-methoxy-

phenyl)-N-(2-((2-(5-methoxy-1H-indol-3-yl)-ethyl)amino)-2-

oxoethyl)acrylamido)methyl)phenyl)-acrylate (14a). Intermediate

14a was prepared from intermediate 13a and ferulic acid in a similar

1

manner as described for intermediate 9a. White solid, yield 44%. H

raphy to aﬀord a 3.20 g white solid, yield 88%. H NMR (400 MHz,

NMR (400 MHz, CDCl₃): δ 8.22/8.20* (s, 1H), 7.73*/7.67 (d, J =

15.2 Hz, 1H), 7.65/7.60* (d, J = 16.0 Hz, 1H), 7.45/7.31* (d, J = 8.0

Hz, 2H), 7.22/7.15* (d, J = 8.8 Hz, 2H), 7.19−7.01 (m, 5H), 6.99/

6.95* (s, 1H), 6.88−6.76 (m, 1H), 6.65/6.62* (d, J = 15.2 Hz, 1H),

6.55/6.01* (t, J = 5.4 Hz, 1H), 6.42/6.37* (d, J = 16.1 Hz, 1H), 4.60/

4.58* (s, 2H), 4.40−4.28 (m, 2H), 4.03/4.02* (s, 2H), 3.85 (s, 3H),

3.84 (s, 3H), 3.82 (s, 3H), 3.58/3.52* (q, J = 6.4 Hz, 2H), 2.95/2.86*

(t, J = 6.6 Hz, 2H), 1.46−1.35 (m, 3H) ppm (the minor rotamer:

31%). ¹³C NMR (101 MHz, CDCl₃): δ 168.9, 167.7, 167.3, 167.3,

154.1, 153.0, 151.6, 151.5, 143.9, 143.8, 141.5, 138.3, 134.2, 133.8,

128.9, 128.7, 128.5, 127.9, 127.1, 123.2, 122.8, 120.9, 120.6, 118.3,

116.5, 112.3, 112.3, 112.0, 112.0, 100.5, 100.4, 65.2, 65.2, 56.1, 56.0,

55.9, 52.2, 51.8, 51.0, 46.9, 39.7, 39.2, 24.9, 14.2, 8.6 ppm. LRMS

CDCl₃): δ 7.99 (brs, 1H), 7.18 (d, J = 7.9 Hz 1H), 6.95 (d, J = 2.3

Hz, 1H), 6.93 (s, 1H), 6.79 (dd, J = 8.8, 2.4 Hz, 1H), 6.13 (brs, 1H),

5.03 (brs, 1H), 3.79 (s, 3H), 3.66 (s, 2H), 3.53 (q, J = 6.1 Hz, 2H),

2.87 (t, J = 6.7 Hz, 2H), 1.35 (s, 9H) ppm. ¹³C NMR (101 MHz,

CDCl₃): δ 172.2, 169.3, 154.1, 131.6, 127.7, 122.9, 112.4 (2C), 112.0,

100.5, 56.0, 44.5, 39.5, 28.3 (3C), 25.3 ppm. LRMS (ESI+) m/z calcd

for [C₁₈H₂₆N₃O₄]⁺: 348.18, found: 348.20 [M + H]⁺. HPLC (method

B): t_R= 10.74 min.

2-Amino-N-(2-(5-methoxy-1H-indol-3-yl)ethyl)acet-amide (12).

11 (2.5 g, 7.20 mmol) was dissolved in 30 mL of DCM; then 8

mL of TFA was added and stirred at r.t. for 30 min. After that, the

solvent was removed under vacuum and puriﬁed with column

chromatography to aﬀord light red oil 1.6 g, yield 90%. ¹H NMR (400

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(ESI+) m/z calcd for [C₃₇H₄₀N₃O₉]⁺: 670.27, found: 670.30 [M +

H]⁺. HPLC (method B): t_R= 12.72 min.

111.60, 111.4, 111.1, 110.5, 100.0, 55.2, 55.1, 55.03, 54.99, 52.6, 51.9,

50.5, 50.1, 40.1, 39.9, 24.8, 24.6 ppm. IR (ﬁlm): ν 3229, 2933, 2830,

1641, 1595, 1581, 1521, 1491, 1462, 1447, 1283, 1253, 1211, 1173,

1127, 1024, 1013, 894, 793, 708 cm⁻¹. LRMS (ESI+) m/z calcd for

[C₃₁H₃₃N₄O₇]⁺: 573.23, found: 573.30 [M + H]⁺. HPLC (method

B): t_R= 9.91 min, purity = 97.0%. mp = 98−100 °C.

(E)-N-Hydroxy-6-(3-(4-hydroxy-3-methoxyphenyl)-N-(2-((2-(5-

methoxy-1H-indol-3-yl)ethyl)amino)-2-oxoethyl)acrylamido)-

hexanamide (15c). Compound 15c was prepared from 14c in a

similar manner as described for compound 3a. Red solid, yield 60%.

¹H NMR (400 MHz, MeOD-d₄): δ 7.57−7.18 (m, 2H), 7.17−7.03

Methyl (E)-4-((3-(4-((Ethoxycarbonyl)oxy)-3-meth-oxyphenyl)-N-

(2-((2-(5-methoxy-1H-indol-3-yl)ethyl)-amino)-2-oxoethyl)-

acrylamido)methyl)benzoate (14b). Intermediate 14b was prepared

from intermediate 13b and ferulic acid in a similar manner as

described for intermediate 9a. White solid, yield 71%. ¹H NMR (400

MHz, CDCl₃): δ 8.05/8.02* (s, 1H), 7.91/7.81* (d, J = 8.1 Hz, 2H),

7.67−7.49 (m, 1H), 7.17−7.08 (m, 3H), 7.08−6.98 (m, 2H), 6.98−

6.91 (m, 2H), 6.88 (s, 1H), 6.81−6.69 (m, 1H), 6.52*/6.50 (d, J =

15.3 Hz, 1H), 6.44/5.94* (t, J = 5.1 Hz, 1H), 4.54/4.52* (s, 2H),

4.32−4.15 (m, 2H), 3.95/3.91* (s, 2H), 3.84 (s, 3H), 3.80*/3.76 (s,

3H), 3.77*/3.75 (s, 3H), 3.59−3.39 (m, 2H), 2.87/2.79* (t, J = 6.7

Hz, 2H), 1.36−1.26 (m, 3H) ppm (the minor rotamer: 33%). ¹³C

NMR (101 MHz, CDCl₃): δ 169.3, 168.8, 167.7, 166.5, 154.1, 153.0,

151.5, 143.9, 141.5, 141.3, 133.8, 131.5, 130.3, 130.1, 129.9, 128.2,

126.5, 123.1, 122.9, 122.8, 121.4, 120.6, 118.0, 116.4, 116.2, 112.4,

112.3, 112.0, 111.9, 100.5, 65.2, 65.2, 56.0, 55.9, 52.2, 52.2, 51.2, 46.7,

39.7, 39.3, 24.9, 24.9, 14.2, 8.6 ppm. LRMS (ESI+) m/z calcd for

[C₃₅H₃₈N₃O₉]⁺: 644.25, found: 644.30[M + H]⁺. HPLC (method B):

t_R= 12.40 min.

Methyl (E)-6-(3-(4-((Ethoxycarbonyl)oxy)-3-meth-oxyphenyl)-N-

(2-((2-(5-methoxy-1H-indol-3-yl)ethyl)-amino)-2-oxoethyl)-

acrylamido)hexanoate (14c). Intermediate 14c was prepared from

intermediate 13c and ferulic acid in a similar manner as described for

intermediate 9a. Colorless oil, yield 35%. ¹H NMR (400 MHz,

CDCl₃): δ 8.18/8.16* (brs, 1H), 7.91−7.78 (m, 2H), 7.53−7.40 (m,

1H), 7.20−7.10 (m, 2H), 7.02−6.90 (m, 2H), 6.88−6.78 (m, 1H),

6.75*/6.71 (d, J = 15.2 Hz, 1H), 6.45/6.00* (t, J = 5.2 Hz, 1H),

4.30−4.15 (m, 2H), 3.89/3.86* (s, 2H), 3.82−3.58 (m, 9H), 3.55−

3.47 (m, 2H), 2.83−2.67 (m, 4H), 2.52−2.23 (m, 2H), 1.66−1.45

(m, 4H), 1.35−1.20 (m, 5H) ppm (the minor rotamer: 34%). ¹³C

NMR (101 MHz, CDCl₃): δ 173.1, 173.0, 171.2, 168.7, 167.1, 165.2,

153.7, 148.5, 148.1, 143.5, 134.3, 128.2, 127.9, 127.3, 123.12, 123.08,

122.6, 120.4, 116.7, 115.1, 113.4, 112.0, 110.9, 99.3, 63.02, 62.96,

56.2, 55.9, 55.4, 55.2, 41.2, 31.9, 28.2, 27.4, 26.9, 25.9, 25.7, 25.6,

24.9, 24.8, 24.7, 24.6, 14.3, 8.6 ppm. LRMS (ESI+) m/z calcd for

[C₃₃H₄₂N₃O₉]⁺: 624.28, found: 624.30[M + H]⁺. HPLC (method B):

t_R= 12.19 min.

(E)-3-(4-Hydroxy-3-methoxyphenyl)-N-(4-((E)-3-(hydroxyamino)-

3-oxoprop-1-en-1-yl)benzyl)-N-(2-((2-(5-methoxy-1H-indol-3-yl)-

ethyl)amino)-2-oxoethyl)-acrylamide (15a). Compound 15a was

prepared from 14a in a similar manner as described for compound 3a.

Oﬀ-white solid, yield 44%. ¹H NMR (400 MHz, MeOD-d₄): δ 7.67−

7.30 (m, 4H), 7.27−7.12 (m, 3H), 7.12−6.93 (m, 4H), 6.90−6.60

(m, 3H), 6.54−6.36 (m, 1H), 4.75−4.50 (m, 2H), 4.17−3.91 (m,

2H), 3.89−3.73 (m, 6H), 3.51/3.47* (t, J = 7.3 Hz, 1H), 3.00−2.82

(m, 2H) ppm (exchangeable protons not observed) (the minor

rotamer: 22%). ¹³C NMR (101 MHz, MeOD-d₄): δ 171.2, 169.5,

169.3, 168.9, 164.9, 153.6, 148.9, 147.9, 144.3, 144.2, 139.7, 138.8,

134.3, 132.0, 128.7, 128.3, 127.9, 127.8, 127.7, 127.3, 126.9, 123.1,

122.9, 122.4, 117.3, 115.1, 113.5, 113.3, 111.8, 111.6, 111.5, 111.2,

110.5, 110.5, 100.0, 55.1, 54.98, 54.95, 51.9, 50.0, 49.6, 39.9, 39.8,

24.7, 24.6 ppm. IR (ﬁlm): ν 3222, 2934, 2831, 1644, 1591, 1516,

1489, 1459, 1444, 1423, 1279, 1208, 1174, 1126, 1029, 972, 792, 743,

706, 662 cm⁻¹. LRMS (ESI+) m/z calcd for [C₃₃H₃₅N₄O₇]⁺: 599.24,

found: 599.30 [M + H]⁺. HPLC (method B): t_R= 10.38 min, purity =

98.9%. mp = 140−142 °C.

(m, 3H), 7.01−6.41 (m, 4H), 4.24−3.75 (m, 8H), 3.60−3.50 (m,

2H), 2.95/2.90* (t, J = 6.6 Hz, 2H), 2.12/2.00* (t, J = 7.0 Hz, 2H)

1.74−1.43 (m, 4H), 1.41−1.04 (m, 4H) ppm (exchangeable protons

not observed) (the minor rotamer: 17%). ¹³C NMR (101 MHz,

MeOD-d₄): δ 171.0, 170.1, 170.0, 167.7, 167.0, 164.7, 153.6, 148.8,

147.9, 143.8, 132.0, 127.81, 127.77, 127.0, 123.1, 122.2, 119.1, 115.1,

114.0, 111.6, 111.2, 110.7, 99.9, 63.0, 62.9, 55.2, 55.1, 55.01, 54.97,

40.0, 32.1, 28.3, 27.4, 26.6, 25.9, 25.72, 25.66, 24.91, 24.85, 24.7, 24.6

ppm. IR (ﬁlm): ν 3174, 3074, 3020, 2936, 2881, 1632, 1575, 1511,

1480, 1450, 1433, 1370, 1273, 1209, 1195, 1162, 1123, 1070, 1024,

996, 974, 815, 792, 663 cm⁻¹. LRMS (ESI+) m/z calcd for

[C₂₉H₃₇N₄O₇]⁺: 553.26, found: 553.25 [M + H]⁺. HPLC (method

B): t_R= 9.97 min, purity = 97.7%. mp = 55−58 °C.

In Vitro HDAC Inhibition Fluorescence Assay. All the HDAC

enzymes were bought from BPS Bioscience. In vitro HDAC inhibition

assays were conducted as previously described with minor

modiﬁcations.⁵⁸In brief, 20 μL of recombinant HDAC enzyme

solution (HDAC1−4, 6−9) was mixed with various concentrations of

tested compound (20 μL). The mixture was incubated at 30 °C for 1

h; then 10 μL of ﬂuorogenic substrate (Boc-Lys (acetyl)-AMC for

HDAC1, 2, 3, and 6, Boc-Lys (triﬂuoroacetyl)-AMC for HDAC 4, 7,

8 and 9) was added. After incubation at 30 °C for 2 h, the catalysis

was stopped by the addition of 10 μL of developer containing trypsin.

Thirty minutes later, ﬂuorescence intensity was measured using a

microplate reader at excitation and emission wavelengths of 360 and

460 nm, respectively. All samples were prepared in duplicate, and the

minimum of two independent assays was performed for each

compound. The inhibition ratios were calculated from the

ﬂuorescence intensity readings of tested wells relative to those of

control wells, and the IC50 curves and values were determined by

GraphPad Prism 7.0, using the “log(inhibitor) versus normalized

response - variable slope” function.

Western Blot Analysis of Cellular HDAC Inhibition. HT22

cells (1,500,000 per well) were plated in ﬂat-bottom six-well plates

and allowed to grow for 12 h and then treated with diﬀerent

concentrations of compounds. Twenty-four hours later, cells were

harvested, washed by PBS, and lysed with RIPA buﬀer, which

comprised 50 mM Tris base, 150 mM NaCl, 5 mM EDTA, 0.1% (v/

v) SDS, 0.5% (v/v) sodium deoxycholate, and 1% (v/v) Triton-x-100.

After lysing, the suspension was ultrasonicated and centrifuged at

14,000 rpm for 15 min at 4 °C. The mixture of 75 μL of the

supernatant and 25 μL of β-mercaptoethanol/LDS solution (15:85)

was heated at 90 °C for 15 min and normalization according to BCA

test carried out before loading. Lysates were run on Invitrogen

NuPAGE 4−12% Bis-Tris 15 well gels at 170 V for approximately 60

min in MES buﬀer. Gels were transferred to methylcellulose and run

at 30 V for 3 h. Membranes were incubated at 4 °C overnight with 1/

1000 primary antibodies, which were diluted in 2.5% (w/v) milk or

5% (w/v) bovine serum albumin. The membrane was washed twice

with TBST buﬀer before being incubated with secondary antibodies.

Images were acquired using a GE ImageQuant LAS 4000.

(E)-N-Hydroxy-4-((3-(4-hydroxy-3-methoxyphenyl)-N-(2-((2-(5-

methoxy-1H-indol-3-yl)ethyl)amino)-2-oxoethyl)acrylamido)-

methyl)benzamide (15b). Compound 15b was prepared from 14b in

a similar manner as described for compound 3a. Golden yellow solid,

1

yield 22%. H NMR (400 MHz, MeOD-d₄): δ 8.15−7.54 (m, 3H),

7.50−7.19 (m, 3H), 7.19−6.92 (m, 4H), 6.88−6.48 (m, 3H), 4.80−

4.54 (m, 2H), 4.23−3.92 (m, 2H), 3.91−3.68 (m, 6H), 3.52/3.47* (t,

J = 6.8 Hz, 2H), 2.99−2.82 (m, 2H) ppm (exchangeable protons not

observed) (the minor rotamer: 19%). ¹³C NMR (101 MHz, MeOD-

d₄): δ 171.5, 171.3, 169.6, 169.1, 168.5, 166.7, 153.6, 149.0, 148.9,

147.9, 147.4, 144.7, 144.4, 141.3, 134.8, 132.0, 130.9, 129.3, 128.3,

128.0, 127.82, 127.75, 127.5, 127.4, 127.23, 127.15, 126.8, 123.1,

123.0, 122.9, 122.5, 122.0, 119.3, 115.1, 114.8, 113.0, 111.9, 111.62,

Computational Docking. Docking studies for compounds 8b

and 10b were conducted based on the crystal structure of HDAC6

complexed with compound A (PDB: 6DVM).²¹The preparation of

the structures and the setup of the docking calculations with

GOLD^59,60are described in detail in the Supporting Information.

Figures were prepared with the PyMOL molecular graphics system

̈

(Schrodinger-LLC, version 2.4.1).

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DPPH Assay. For determination of antioxidative properties, a 2,2-

diphenyl-1-picrylhydrazyl (DPPH) radical assay was used as

previously described.⁶¹A stock solution (200 μM) of DPPH was

freshly prepared in MeOH. Compounds were dissolved in MeOH (5

mM) as stock solution. To start the reaction, 50 μL of the DPPH

stock solution was added to a 100 μL diluted compound or pure

methanol as a negative control. The 96-well plate was incubated at

room temperature in the dark for 30 min, and absorbance was then

determined with a microplate reader at 517 nm. The experiments

were performed in triplicates. A dilution series of compound served as

a blank to the particular compound and was subtracted from each

measurement point. The scavenging potency is expressed as the

concentration that scavenged 50% of the DPPH free radicals (EC₅₀)

determined via a nonlinear regression curve. Ferulic acid was used as

references under the same assay conditions.

compound and Fe²⁺aq. served as a blank to the particular compound

and was subtracted from each measurement point. EDTA·2Na in

water was diluted with MeOH, which was served as a reference under

the same assay conditions.

The ability of compounds to chelate Cu²⁺was assessed using the

method as Santos et al.⁶⁴described and partially modiﬁed that

employs pyrocatechol violet (PV) as the chromogen agent. In

aqueous medium buﬀered at slightly acidic medium (pH 6.0). There

is usually a leftover of Cu²⁺in the reactional medium because not all

the copper is bound by chemical compounds present in a test sample.

Thereafter, the remaining Cu²⁺reacts with pyrocatechol violet at a

proportion of 2:1, forming a blue-colored complex that can be

monitored at 632 nm. However, the dark color turns into yellow in

the presence of chelating agents that dissociate the complex, and the

chelating activity can thus be estimated by the measurement of the

rate of color reduction. Brieﬂy, in each well, 50 μL of Cu²⁺aq. (400

μM in buﬀer) was added to 100 μL diluted compounds-MeOH

solution or pure methanol as negative control. The 96-well-plate was

incubated at room temperature for 2 min, 50 μL of PV aq. (400 μM in

buﬀer) was added to each well (avoid the light) and then incubated at

room temperature for 10 min, and absorbance was then determined

with a microplate reader at 632 nm. The experiments were performed

in duplicates. EDTA·2Na in water was diluted with MeOH, which was

served as a reference under the same assay conditions.

ORAC Assay. To study the antioxidant capacities of target

compounds and the SAR of melatonin, we used the ORAC-

Fluorescein method of Ou et al.⁶²partially modiﬁed by Davalos et

́

al.⁶³that uses ﬂuorescein (FL) as a ﬂuorescent probe, ( )-6-hydroxy-

2,5,7,8-tetramethyl-chromane-2-carboxylic acid (Trolox) as a refer-

ence, and compound 2,2′-azobis(amidinopropane) dihydrochloride

(AAPH) as a free-radical generator. When the free-radical generator is

added, production of free radicals starts, and those oxidize ﬂuorescein

decreasing its ﬂuorescence. When an antioxidant is present, the

scavenger traps free radicals and the ﬂuorescence remains unchanged.

Reactions were performed in 75 mM PBS (pH 7.4), and the ﬁnal

reaction mixture volume was 200 μL at 37 °C. Derivatives at the

desired concentrations (20 μL) and FL (120 μL, 70 nM, ﬁnal

concentration) solutions were mixed in a black 96-well microplate

(SARSTEDT) and preincubated for 15 min at 37 °C. Subsequently,

AAPH solution (60 μL, 12 mM, ﬁnal concentration) was added

rapidly and the plate was placed in a TECAN microplate reader to

measure the ﬂuorescence (485 nm excitation and 510 nm emission

ﬁlters) intensity every 2 min for 90 min. Samples were measured at

two diﬀerent concentrations (2 and 4 μM). A blank (FL + AAPH in

PBS) and a calibration curve of Trolox (1, 2, 4, 6, 8, 10, 12, and 15

μM) were included in each experiment. All samples were prepared in

duplicate, and three independent assays were performed for each

compound. The area under the ﬂuorescence decay curve (ﬂuo-

rescence vs time) (AUC) was calculated as

The chelating ability to zinc ions was carried out as Catapano et

al.⁶⁵described and partially modiﬁed. Stock solutions of target

compounds (10 mM) were prepared in DMSO, whereas that of zinc

chloride (10 mM) and EDTA (10 mM) were prepared in ultrapure

water. Dithizone (10 mM) as the chromogen agent was prepared in

DMSO. NaAc buﬀer was prepared (50 mM, pH 6.0). Firstly, zinc-ion

solutions (50 μL, 60 μM) or water was added to the buﬀers (150 μL).

Thereafter, 50 μL of diluted compounds-DMSO solution or pure

DMSO was added. After 2 min of mixing, 50 μL of dithizone-DMSO

solution (250 μM) was added, and the absorbance was measured

immediately at 540 nm.

The chelation ratios were calculated from the absorbance intensity

readings of tested wells relative to those of control wells, data are

expressed as means SEM of two diﬀerent independent experiments.

Neurotoxicity Assay. HT-22 cells were grown in Dulbecco’s

modiﬁed Eagle medium (DMEM, Sigma-Aldrich, Munich Germany)

supplemented with 10% (v/v) heat-inactivated fetal calf serum (FCS)

and 1% (v/v) penicillin−streptomycin. Cells were subcultured every 2

days and incubated at 37 °C with 5% CO₂in a humidiﬁed incubator.

Compounds were dissolved in DMSO (Sigma-Aldrich, Munich,

Germany) and diluted with a medium. Generally, 80% conﬂuent cells

were seeded with 5000 cells per well into sterile 96-well plates and

were incubated for 24 h. For the neurotoxicity assay, the previous

medium was removed and 10 μM of the compound was added to the

wells, DMSO (0.05%) in DMEM served as a control. Cells were

incubated for 24 h. MTT solution (4 mg/mL in PBS) was diluted

1:10 with medium and added to the wells after the removal of the

previous medium. Cells were incubated for 3 h when the supernatant

was removed, and lysis buﬀer (10% SDS) was applied. The next day,

absorbance at 560 nm was determined with a multiwell plate

photometer (Tecan−SpectraMax 250). Experiments were performed

in triplicates. Results are presented as percentage to untreated control

i=45

AUC = 1 +

f /f

i 0

∑

i=1

where f₀is the initial ﬂuorescence reading at 0 min and f_iis the

ﬂuorescence reading at time 2i. The net AUC corresponding to a

sample was calculated by subtracting the AUC corresponding to the

blank (Net AUC = AUC_antioxidant− AUC_blank). Linear regression

equations were calculated by plotting the net AUC against the trolox

antioxidant standard concentration (R²> 0.99). ORAC-FL values

were expressed as trolox equivalents by using the standard curve

calculated for each assay, where the ORAC-FL value of Trolox was

taken as 1, plot the Trolox standard curve, and interpolate it to

determine sample ORAC values.

Metal Chelating Assays. The chelating ability to the ferrous ion

was carried out as Santos et al.⁶⁴described and partially modiﬁed. The

ferrous ion in ferrous sulfate readily reacts with ferrozoine (3-(2-

Pyridyl)-5,6-diphenyl-1,2,4-triazine-p,p′-disulfonic acid monosodium

salt hydrate) to form a Fe²⁺-ferrozoine complex, a purple-colored

chromogen whose intensity is measured at 562 nm. The presence of

antioxidant chelators reduces the formation of Fe²⁺-ferrozoine

complex. Lower absorbance indicates higher metal-chelating activity.

NaAc buﬀer was prepared (50 mM, pH 6.0). To start the reaction, 50

μL of Fe²⁺aq. (80 μM in buﬀer) was added to 100 μL diluted

compounds-MeOH solution or pure methanol as a negative control.

The 96-well plate was incubated at room temperature for 2 min, 50

μL of ferrozine aq. (250 μM in buﬀer) was added to each well and

then incubated at room temperature for 10 min, and absorbance was

then determined with a microplate reader at 562 nm. The experiments

were performed in duplicates. The mixture of a dilution series of

cells. Data are expressed as means

SD of three diﬀerent

independent experiments. Analysis was accomplished using GraphPad

Prism 7 Software applying oneway ANOVA followed by Dunnett’s

multiple comparison posttest. Levels of signiﬁcance: *p < 0.05; **p <

0.01; ***p < 0.001.

For the oxytosis assay, 5 mM glutamate (monosodium-^L-glutamate,

Sigma-Aldrich, Munich, Germany) together with respective com-

pounds was added to the cells and incubated for 24 h. Quercetin (25

μM) (Sigma-Aldrich, Munich, Germany) together with 5 mM

glutamate served as a positive control. After 24 h incubation, cell

viability was determined using a colorimetric MTT assay as described

above. Results are presented as percentage of untreated control cells.

Data is expressed as means SEM of two independent experiments.

Analysis was accomplished using GraphPad Prism 7 Software applying

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Oneway ANOVA followed by Dunnett’s multiple comparison

posttest. Levels of signiﬁcance: ***p < 0.001.

peptide in the mouse. Compounds were injected intraperitoneally

(ip) o.d. between days 1 and 7, the oligomerized Aβ₂₅₋₃₅peptide was

injected icv on the ﬁrst day of the study, and behavioral evaluation

was performed between days 8 and 10 followed by sacriﬁce on day 11.

Brain samples were collected and stored at −80 °C awaiting further

biochemical analyses.

Animals. Male Swiss mice, 6 weeks old, weighing 31−36 g, from

JANVIER (Saint Berthevin, France), were kept for housing, and

experiments took place within the animal facility building of the

University of Montpellier (CECEMA, Oﬃce of Veterinary Services

agreement # B-34-172-23). Upon arrival, animals were divided into

groups and housed with access to food and water ad libitum, except

during behavioral experiments. Mice were kept in a temperature- and

humidity-controlled animal facility on a 12 h/12 h light/dark cycle

(lights oﬀ at 07:00 PM). All animal procedures were conducted in

strict adherence to the European Union directive of September 22,

2010 (2010/63/UE) and the ARRIVE guidelines. The project was

authorized by the French National Ethic Committee (APAFIS #1485-

15034).

Preparation of Compound Injection. Compounds were weighed,

dissolved in pure DMSO at a concentration of 1 mg/mL, and diluted

into ﬁnal test concentrations with saline. The ﬁnal percentage of

DMSO in saline was 60% for all compounds. Vehicle solution used for

control groups was 60% DMSO in saline. General behavior of the

mice in the home cage was checked visually after injections. In

particular, weight gain was checked every day. As shown in Figure S2,

the icv injection aﬀected animals on day 1, but then weight gain was

regular and not signiﬁcantly diﬀerent from control. All treatments

showed good compound and vehicle solution tolerability. Moreover,

animals were tested in behavioral tests 24 h after the last compound/

vehicle administration.

Amyloid Peptide Preparation and icv Injection. Mice were

anesthetized with isoﬂurane (2.5%) and icv injected with the Aβ₂₅₋₃₅

peptide (9 nmol/mouse), and homogeneous oligomeric preparation

of the Aβ₂₅₋₃₅peptide was performed as described by Maurice et al.⁵⁴

The control group received bidistilled water icv as we previously

reported that injection of a control peptide does not result in memory

deﬁcits or toxicity.

Spontaneous Alternation Performances in the Y Maze. On day 8,

all animals were tested for spontaneous alternation performance in the

Y maze to evaluate their spatial working memory. The Y maze is made

of gray poly(vinyl chloride). Each arm is 40 cm long, 13 cm high, 3

cm wide at the bottom, 10 cm wide at the top, and converging at an

equal angle. Each mouse is placed at the end of one arm and allowed

to explore the maze freely for 8 min. The sequence of arm entries

(including possible returns into the same arm) was checked visually

and noted down. If the mouse enters all three arms on consecutive

occasion, this is deﬁned as an alternation. Therefore, the total number

of arm entries minus two is also the maximum number of alternations.

The percentage of alternation was calculated as (actual alternations/

maximum alternations) × 100. Parameters for the evaluation of

behavior are given as the percentage of alternation (memory index)

and the total number of arm entries (exploration index). When an

extreme behavior (alternation percentage < 20% or > 90% or a

number of arm entries of <10) was observed, animals were excluded

from the calculations, which corresponded to a 4.7% attrition in this

study.

Passive Avoidance Test. On days 9 and 10 of the study, a passive

avoidance test was performed to measure nonspatial long-term

memory, as described previously.⁵⁴⁻⁵⁷The setup for the experiment

consists of a two-compartment (15 × 20 × 15 cm high) poly(vinyl

chloride) box, whereas one compartment is white and illuminated

with a bulb (60 W, 40 cm above the apparatus), and the other is black

with a cover and grid ﬂoor. A guillotine door separates the two

compartments. On day 9, during the training session, each animal was

placed in the white compartment with the door closed. After 5 s, the

door was opened, and the mouse was allowed to enter the dark

compartment. When it had placed all its paws on the grid ﬂoor, the

door was closed, and a foot shock was delivered (0.3 mA) for 3 s

using a scrambled shock generator (Lafayette Instruments, Lafayette,

For the ATP depletion assay, 3 × 10³cells per well were seeded

into sterile 96-well plates and incubated overnight. The next day

medium was exchanged with fresh medium. Iodoacetic acid (20 μM)

(IAA) was added with a vehicle (DMSO) as a negative control, or

together with respective compounds for protection. After 2 h

incubation at 37 °C in the incubator, the medium was aspirated,

the fresh medium was applied, and only the compounds at the same

respective concentrations were added without IAA. After 24 h, cell

viability was determined using a colorimetric MTT assay. Results are

presented as percentage of untreated control cells.

Immunomodulation Analysis. Mouse N9-microglial cells were

cultured in Dulbecco modiﬁed eagle medium (DMEM) supple-

mented with 10% heat inactivated Fetal Bovine Serum (FBS), 1%

penicillin/streptomycin, and 2 mM glutamine (all from Aurogene Srl,

Rome, Italy). To evaluate the immunomodulatory eﬀect, 2.5 × 10⁵

microglial cells were plated in a 35 mm dish in the presence or

absence of 100 ng/mL LPS (lipopolysaccharide) with increasing

concentrations of compounds. After 24 h of treatment, the microglial

conditioned medium was collected and used for nitrite measurement,

or concentrated using Microcon YM-3 (Millipore, Billerica, MA), and

resuspended in 4× loading buﬀer (0.2 M Tris−HCL pH 6.8; sodium

dodecyl sulfate; 40% glycerol; 0.4% bromophenol blue and 0.4 M

dithiothreitol; Sigma-Aldrich) for Western blot analysis. In parallel,

microglial cells were collected in lysis buﬀer (1% SDS, 50 Mm Tris

pH 7.4, 1 mM EDTA, 10 μL/mL protease inhibitors and 10 μL/mL

phosphatase inhibitors), and protein content was determined by using

the Lowry protein assay.

For Western blot analysis, media and cell samples were brieﬂy

sonicated and loaded into 12% sodium-dodecyl sulfate-polyacryla-

mide gels (SDS-PAGE; Bio-Rad). After electrophoresis and transfer

onto nitrocellulose membranes (GE Healthcare, Milano, Italy),

membranes were blocked 1 h in blocking solution PBS-0.1%

Tween-20 (Sigma-Aldrich), 4% non-fat dry milk (Bio-Rad), and

incubated overnight at 4 °C with primary antibodies in PBS-0.1%

Tween-20: rabbit anti-iNOS (1:1000, Cell Signaling Technology,

Danvers, Massachusetts, USA), rabbit anti-TREM2 (1:1000, Cell

Signaling Technology), mouse anti-TGFβ2 (1:1000 Abcam, Cam-

bridge, UK), and mouse anti-GAPDH (1:20,000, Dako North

America, Agilent Technologies, USA). The next day, membranes

were then incubated with speciﬁc antibodies conjugated to horse-

radish peroxidase (goat anti-rabbit and goat-anti mouse, 1:5000,

Jackson ImmunoResearch, Cambridge, UK) for 90 min at RT in PBS-

0.1% Tween-20. Proteins were visualized by using the Clarity Western

ECL Substrate (Bio-Rad) and detected using Bio-Rad Image Lab

Software with a ChemiDoc imaging system (Bio-Rad).

For nitrite measurement, accumulation of NO in microglial

conditioned media was quantiﬁed by a colorimetric assay based on

Greiss reaction in a 96-well-plate. A nitrate standard curve was

performed with NaNO₂at known concentrations. To start the

reaction, 5 mM sulfanilamide (Sigma-Aldrich) was added to the

culture medium and standard curve; it reacts with nitrite under acidic

conditions to form a diazonium cation, which subsequently couples to

N-1-naphthylethylenediamine dihydrochloride 40 mM (NEDA;

Sigma-Aldrich) to produce a colored azo dye. After 15 min of

incubation at RT in the dark, absorbance was read at 540 nm with a

multiplate spectrophotometric reader (Bio-rad Laboratories Srl,

Milano, Italy).

All quantitative data are presented as means

S.E. from at last

three independent experiments. Statistical signiﬁcance between

diﬀerent treatments was calculated with GRAPHPAD PRISM6 (L

Jolla, California, USA) using one-way analysis of variance (ANOVA)

followed by post-hoc comparison Bonferroni’s test. A value of p <

0.05 was considered statistically signiﬁcant.

In Vivo Studies. Protocols for the behavioral experiments were

established previously.⁵⁴⁻⁵⁷Compound 10b and the control

ACY1215 were tested for their neuroprotective properties in the in

vivo pharmacological model of Alzheimer’s disease induced by

intracerebroventricular (icv) injection of the oligomerized Aβ₂₅₋₃₅

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USA). The time spent to enter the dark compartment (step-through

latency) and the level of sensitivity to the shock were evaluated (no

sign = 0, ﬂinching reactions = 1, vocalizations = 2). None of the

treatments aﬀected the step-through latency or shock sensitivity in the

present study (data not shown). The retention test was carried out on

day 10. Five seconds after the mouse was placed in the white

compartment, the door was opened, and the mouse was allowed to

explore the box. The time spent to enter the dark compartment (step-

through latency) was measured up to 300 s. Animals showing

latencies during the training and retention session lower than 10 s and

shock sensitivity = 0 were considered as failing to learn the task and

discarded from calculations. In this study, it corresponded to 0.6%

attrition.

Sandra Gunesch − Pharmaceutical and Medicinal Chemistry,

Institute of Pharmacy and Food Chemistry, Julius

Maximilian University of Wurzburg, Wurzburg 97074,

̈ ̈

Germany

Matthias Hoﬀmann − Pharmaceutical and Medicinal

Chemistry, Institute of Pharmacy and Food Chemistry, Julius

Maximilian University of Wurzburg, Wurzburg 97074,

̈ ̈

Germany

Christoph Sotriﬀer − Pharmaceutical and Medicinal

Chemistry, Institute of Pharmacy and Food Chemistry, Julius

Maximilian University of Wu

Germany

rzburg, Wurzburg 97074,

̈ ̈

Statistical Analyses. All values, except passive avoidance latencies,

are expressed as mean SEM. Statistical analyses were performed on

the diﬀerent conditions using one-way ANOVA (F value), followed

by Dunnett’s post hoc multiple comparison test. Passive avoidance

latencies do not follow a Gaussian distribution since upper cutoﬀ

times (300 s) are deﬁned. Therefore, they were analyzed using a

Kruskal−Wallis nonparametric ANOVA (H value) followed by

Dunn’s multiple comparison test. p < 0.05 was considered as

statistically signiﬁcant.

Barbara Monti − Department of Pharmacy and

Biotechnologies, University of Bologna, Bologna 40126, Italy;

orcid.org/0000-0003-0330-482X

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.0c01940

Author Contributions

The manuscript was written through the contributions of all

authors. Synthesis of the target compounds, DPPH assay,

ORAC assay, and metal chelation assay were performed by

F.H. HDAC inhibition assay was performed by C.J.C. The

docking study was conducted by N.Y.C. and C.S. The

neurotoxicity experiment was performed by S.G. The

immunomodulation study was performed by E.P. and B.M.

In vivo experiments were performed by M.S., M.H., and T.M.

M.D. was responsible for the oversight and development of the

whole project.

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01940.

■

sı

HPLC-chromatograms of target compounds, details of

computational docking, metal chelation, neuroprotec-

tion against oxytosis and ATP depletion, and weight of

animals during in vivo studies (PDF)

Funding

Molecular formula strings (CVS)

M.D. acknowledges the German Research Council (Deutsche

Forschungsgemeinschaft) under DFG DE 1546/6-3. M.D. and

T.M. acknowledge support from Campus France (PHC

Procope) and the German Academic Exchange Service

(DAAD) with funds of the Federal Ministry of Education

and Research (BMBF) under Grant # 57387204, as well as

support from BayFrance (Franco-Bavarian University cooper-

ation center) under Grant # FK03-2020. F.H. acknowledges

the Ph.D. scholarship from the China Scholarship Council

(CSC). N.Y.C. acknowledges support from the German

Academic Exchange Service (DAAD). M.H. acknowledges

the German Academic Scholarship Foundation (“Studien-

stiftung des deutschen Volkes”) for a Ph.D. fellowship and the

Elite Network of Bavaria (International Doctoral Program

“Receptor Dynamics”) for support.

AUTHOR INFORMATION

Corresponding Authors

Tangui Maurice − MMDN, Univ Montpellier, EPHE,

INSERM, Montpellier 34095, France;

Email: tangui.maurice@umontpellier.fr

Michael Decker − Pharmaceutical and Medicinal Chemistry,

Institute of Pharmacy and Food Chemistry, Julius

■

Maximilian University of Wurzburg, Wurzburg 97074,

̈ ̈

Germany; orcid.org/0000-0002-6773-6245;

Phone: +49-93131-89676; Email: michael.decker@uni-

wuerzburg.de; Fax: +49-931 31-85494

Authors

Feng He − Pharmaceutical and Medicinal Chemistry, Institute

of Pharmacy and Food Chemistry, Julius Maximilian

̈ ̈

University of Wurzburg, Wurzburg 97074, Germany

Notes

The authors declare no competing ﬁnancial interest.

C. James Chou − Department of Drug Discovery and

Biomedical Sciences, South Carolina College of Pharmacy,

Medical University of South Carolina, Charleston, South

Carolina 29425, United States; orcid.org/0000-0001-

6808-9089

Matthias Scheiner − Pharmaceutical and Medicinal

Chemistry, Institute of Pharmacy and Food Chemistry, Julius

ABBREVIATIONS

■

AAPH, 2,2′-azobis(amidinopropane) dihydrochloride; Aβ,

amyloid-beta; AChE, acetylcholinesterase; AD, Alzheimer’s

disease; ANOVA, analysis of variance; APP, amyloid precursor

protein; ChEs, cholinesterases; DCM, dichloromethane;

DMSO, dimethylsulfoxide; EDTA, ethylenediaminetetraacetic

acid; FL, ﬂuorescein; HDAC, histone deacetylase; HSP-90, hot

shock protein 90; Il-1β, interleukin 1 beta; iNOS, inducible

nitric oxide synthase; LPS, lipopolysaccharides; MIF, macro-

phage migration inhibitory factor; MTT, 3-(4,5-dimethylth-

iazol-2-yl)-2,5-diphenyltetrazolium bromide; M.P., melting

point; NFTs, neuroﬁbrillary tangles; NMDAR, N-methyl-D-

aspartate receptor; PBS, phosphate buﬀered saline; PDE5,

phosphodiesterase 5 (PDE5); ROS, reactive oxygen species;

Maximilian University of Wu

Germany

rzburg, Wurzburg 97074,

̈ ̈

Eleonora Poeta − Department of Pharmacy and

Biotechnologies, University of Bologna, Bologna 40126, Italy

Natalia Yuan Chen − Pharmaceutical and Medicinal

Chemistry, Institute of Pharmacy and Food Chemistry, Julius

Maximilian University of Wurzburg, Wurzburg 97074,

̈ ̈

Germany

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Journal of Medicinal Chemistry

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tetrahydrofuran; TREM2, triggering receptor expressed on

myeloid cells 2; Trolox, ( )-6-hydroxy-2,5,7,8-tetramethyl-

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Article Doi

Melatonin- And Ferulic Acid-Based HDAC6 Selective Inhibitors Exhibit Pronounced Immunomodulatory Effects in Vitro and Neuroprotective Effects in a Pharmacological Alzheimer's Disease Mouse Model

DOI: 10.1021/acs.jmedchem.0c01940

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