A Novel N-Substituted Valine Derivative with Unique Peroxisome Proliferator-Activated Receptor γbinding Properties and Biological Activities

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A Novel N‑Substituted Valine Derivative with Unique Peroxisome

Proliferator-Activated Receptor γ Binding Properties and Biological

Activities

,∇

∇

́

Franck Peiretti,* Roberta Montanari, Davide Capelli, Bernadette Bonardo, Cecilia Colson,

Ez-Zoubir Amri, Marina Grimaldi, Patrick Balaguer, Keiichi Ito, Robert G. Roeder, Giorgio Pochetti,^∇

and Jean Michel Brunel^∇

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ABSTRACT: A proprietary library of novel N-aryl-substituted amino acid derivatives bearing a hydroxamate head group allowed

the identiﬁcation of compound 3a that possesses weak proadipogenic and peroxisome proliferator-activated receptor γ (PPARγ)

activating properties. The systematic optimization of 3a, in order to improve its PPARγ agonist activity, led to the synthesis of

compound 7j (N-aryl-substituted valine derivative) that possesses dual PPARγ/PPARα agonistic activity. Structural and kinetic

analyses reveal that 7j occupies the typical ligand binding domain of the PPARγ agonists with, however, a unique high-aﬃnity

binding mode. Furthermore, 7j is highly eﬀective in preventing cyclin-dependent kinase 5-mediated phosphorylation of PPARγ

serine 273. Although less proadipogenic than rosiglitazone, 7j signiﬁcantly increases adipocyte insulin-stimulated glucose uptake and

eﬃciently promotes white-to-brown adipocyte conversion. In addition, 7j prevents oleic acid-induced lipid accumulation in

hepatoma cells. The unique biochemical properties and biological activities of compound 7j suggest that it would be a promising

candidate for the development of compounds to reduce insulin resistance, obesity, and nonalcoholic fatty liver disease.

(NAFLD) characterized by the accumulation of triglycerides

in hepatocytes.⁷However, ﬁbrates increase markers of

cardiovascular and renal disease and that of liver dysfunction,

which underlines their ability to trigger adverse eﬀects.⁸

Therefore, eﬀorts are being made to develop PPARα agonists

with improved clinical eﬃcacy, penaﬁbrate being one of these

new generation agonists.⁹

PPARδ is involved in the regulation of fatty acid oxidation

and mitochondrial respiration predominantly in skeletal

muscle, liver, and adipose tissue.¹⁰Therefore, agonists

targeting PPARδ may be considered as potential therapeutic

agents for insulin resistance-related conditions. PPARδ

INTRODUCTION

■

Peroxisome proliferator-activated receptors (PPARs) are

transcription factors belonging to the nuclear receptor

superfamily and activated by ligands such as dietary fatty

acids, particularly polyunsaturated fatty acids. The three PPAR

subtypes: PPARα, PPARγ, and PPARδ (β), have diﬀerent, yet

overlapping, tissue expression patterns¹and exert major roles

in the regulation of speciﬁc physiological functions including

glucose and lipid metabolism and energy homeostasis.²⁻⁴

These features make PPARs important molecular targets for

the development of drugs for metabolic diseases.

PPARα is expressed in all metabolic tissues but predom-

inantly in the liver where it is involved in the regulation of the

uptake and oxidation of fatty acids and lipoprotein

metabolism.⁵The ﬁbrate family of drugs (cloﬁbrate and

fenoﬁbrate) is pharmacologically weak agonists of PPARα that

are used to treat dyslipidemia as they lower plasma

triglycerides and increase high-density lipoprotein cholesterol

levels.⁶The PPARα agonist fenoﬁbrate has also a beneﬁcial

eﬀect in patient with nonalcoholic fatty liver disease

Received: September 6, 2020

https://dx.doi.org/10.1021/acs.jmedchem.0c01555

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Figure 1. Chemical structures and PPARγ agonist activity of 3a and its derivatives. (A) Amino acid core, (B) hydrophobic capping group, and

(C) binding head group of 3a were modiﬁed (left panels), and PPARγ transactivation activity of each molecule (5 μM) was measured (right

panels). Color ﬁlls accentuate the amino acid core (gray), hydrophobic capping group (green), and binding head group (yellow) of 3a. The

percentage indicates the yield of the synthesis. PPARγ transactivation values are means SD expressed relative to the mean of control values,

which was set to 1.

agonists have been developed and used in research,¹¹but

none of them are currently approved for clinical use.

modulators (SPPARγMs) with higher therapeutic proﬁles than

full agonists.^15,16The peculiar properties of SPPARγM are

being explained by the ability of PPARγ to adopt ligand-

speciﬁc conformations with diﬀerent transcriptional signatures.

In addition, it has been shown that the clinical beneﬁt of

PPARγ partial agonists and SPPARγMs also involves their

ability to inhibit the cyclin-dependent kinase 5 (CDK5)-

mediated PPARγ phosphorylation at serine 273.^17,18The

other concept for developing safe antidiabetic drugs targeting

PPAR considers that beneﬁcial eﬀects of their activation could

counteract their harmful eﬀects. PPARα/γ dual agonists, so-

called glitazars, that combine the insulin sensitizing eﬀect of

PPARγ agonists with the beneﬁcial eﬀect of PPARα agonists

on the lipid proﬁle are representative of this class of drugs.^19,20

Saroglitazar is approved in India for the treatment of peculiar

type of diabetic dyslipidemia and hypertriglyceridemia.^21,22

Moreover, dual α/δ and γ/δ PPAR agonists as well as “pan”

PPARγ is considered the master regulator of adipogenesis

via its promotion of lipid production and storage.

Thiazolidinediones, including rosiglitazone (Rosi) and piogli-

tazone, are the most eﬀective PPARγ activating drugs that

were widely prescribed for the treatment of type 2 diabetes.¹²

However, their strong agonist activities are partly responsible

for unwanted harmful side eﬀects such as weight gain, ﬂuid

retention, osteoporosis, heart failure, and cancer,^13,14which

precipitated their withdrawal from the market.

The quest for antidiabetic compounds targeting PPAR with

good therapeutic potential and reduced adverse eﬀects has

followed two main directions. The one based on the

observation that a moderate, rather than full, activation of

PPARγ dissociates the deleterious from the therapeutic eﬀects

of the agonist has led to the generation of selective PPARγ

B

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Figure 2. Chemical structures and PPARγ agonist activity of 7a and its derivatives. (A−C) Absolute conformations of the valine core (A), the

amino acid core (B), or the hydrophobic capping group (C) of 7a were modiﬁed (left panels), and PPARγ transactivation activity of each

molecule (5 μM) was measured (right panels). The percentage indicates the yield of the synthesis. PPARγ transactivations are expressed as shown

in Figure 1.

agonists acting on all three isoforms are the subject of intense

investigations²³⁻²⁵that could lead to the generation of

molecules with potential additional therapeutic indications.

This work reports a mild two-step synthesis of a library of

new N-aryl-substituted amino acid derivatives from commer-

cially easily available and inexpensive reagents. The eﬀect of

these compounds, in particular the inﬂuence of substitutions

on their phenyl group, was evaluated on PPAR activity and led

to the development of a new balanced and potent dual

PPARα/γ agonist with unique ligand binding properties and

singular biological activities that would be expected for a

potential therapeutic candidate to reduce insulin resistance,

obesity, and NAFLD.

tains an ^L-valine (core) and a six-carbon atom chain (capping

group) (Figure 1A and Figure S1), was identiﬁed as a weak

activator of adipocyte diﬀerentiation as compared with Rosi

(Figure S2). We hypothesized that 3a increased adipocyte

diﬀerentiation by activating the master regulator of adipo-

genesis: PPARγ. The PPARγ agonistic activity of 3a was

conﬁrmed by the use of a luminescence-based cell-based

PPARγ transactivation assay in which a wild-type PPARγ

ligand binding domain (PPARγ-LBD) is fused to the GAL4

DNA binding domain (PPARγ-LBD-GAL4) and the Fireﬂy

luciferase reporter gene is under the control of GAL4 binding

elements (Figure 1A). Importantly, 3a increased PPARγ

activity by a factor of 2.8, while 1 μM Rosi (a full PPARγ

agonist) increased it by a factor of 25

7. A thorough

RESULTS AND DISCUSSION

comparison of the PPARγ activation properties of the newly

synthesized compounds with that of Rosi will be carried out at

the later stage. We initiated a process to identify the domains

of 3a involved in PPARγ activation that would ideally lead to

an optimization of its PPARγ agonist activity.

Eﬀect of the Amino Acid Core. Analogues of the 3a hit

were designed and synthesized through a reaction involving a

two-step synthesis procedure in dichloromethane from amino

■

New N-aryl-substituted amino acid derivatives bearing a

hydroxamate head group (head), initially designed to identify

MMP inhibitors, were screened on their ability to induce

spontaneous (in the absence of any other inductor) adipocyte

diﬀerentiation of 3T3-L1 cells by measuring intracellular lipid

accumulation. The molecule named 3a (4-hexyloxy-N-((S)-1-

hydroxycarbamoyl-2-methyl-propyl)-benzamide), which con-

C

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Figure 3. PPAR agonist activity of 3a, 7a, and 7j. (A) Concentration-dependent PPARγ transactivation activities of 3a, 7a, and 7j were compared

to that of Rosi using the PPARγ-LBD-GAL4 chimera assay. Values are means SD expressed as % of the maximal response measured with Rosi

(5 μM). (B) HEK293 cells were transfected with PPRE-driven Fireﬂy luciferase and SV40-driven Renilla luciferase coding vectors together with

an empty plasmid (−PPARγ) or with the PPARγ expression vector (+PPARγ). After 36 h of post-transfection, cells were incubated for 17 h with

3a, 7a, 7j (1 μM), or Rosi (0.1 μM). PPRE promoter activity was calculated as the ratio of Fireﬂy/Renilla luciferase. Values are means SD

expressed relative to the control situation (*p < 0.05 and **p < 0.01 (one-way ANOVA followed by Bonferroni’s post-hoc test). (C)

Concentration-dependent PPARα transactivation activities of 7j, GW 7647, and bezaﬁbrate were measured using the PPARα-LBD-GAL4 chimera

assay. Values are means SD expressed as % of the maximal response measured with GW 7647 (1 μM).

acid methyl ester hydrochloride 1a−1h, easily prepared from

amino acids. By using a BOP reagent as an eﬃcient and

versatile reagent for the coupling of alkyloxybenzoic acid with

1a−1h, the synthesis of various substituted amino acid ester

derivatives 2a−2h in high chemical yields of up to 90% was

achieved. It is noteworthy that the transient ester species are

successfully transformed into their corresponding hydroxamic

acid parent derivatives 3a−3h by using hydroxylamine (40%

H₂O) in MeOH at reﬂux for 24 h in yields varying from 36 to

72% (Figure 1A). Changing the ^L-valine core by any other

amino acid decreased the PPARγ activation potency of the

molecule (Figure 1A) perhaps due to the larger steric

hindrance generated by the isopropyl moiety with respect to

a benzyl or hydroxy methyl group.

Eﬀect of the Carbon Atom Chain Capping Group.

The diﬀerent derivatives 4a−4h were synthesized according to

a similar procedure than for compounds 3a−3h in good-to-

excellent yields, varying from 32 to 75% (Figure 1B). Altering

the number of carbons in the polycarbon chain capping group

of the aryl moiety showed that a linear six-carbon chain was

optimal for PPARγ activation (Figure 1B). This result suggests

that the steric hindrance and the hydrophobic nature of the

polycarbon chain are important factors to consider when

designing most potent analogues.

Eﬀect of the Head Group. Substitution of the

hydroxamate head moiety in 3a (Figure 1C) revealed that

the preferred functions for PPARγ activation are OH >

NHOH ≥ NH₂> COOMe (Figure 1C). The analogue of 3a,

with a carboxylic acid moiety in place of the hydroxamate

head group that optimally activated PPARγ, was named 7a

(Figure S3).

Optimization of PPARγ Agonist Activity of 7a.

Changing the absolute conformation of the core valine from

L to D abolished the PPARγ activation capacity of 7a (Figure

2A). Furthermore, substitution of the amino acid core (^L-

valine) by other amino acid (L-form) and/or reducing the

length of the polycarbon chain decreased the PPARγ

activation eﬃciency of 7a (Figure 2B). The PPARγ trans-

activation activity of 7a was gradually enhanced by the

extension of the polycarbon chain up to eight carbon atoms

(Figure 2C). The analogue of 7a with the eight-carbon atom

chain in place of the six-carbon atom chain, which optimally

activates PPARγ, was named 7j (Figure S4). It should be

noted that the presence of a chain with 10 carbon atoms in

the molecule 7k did not further enhance its ability to activate

PPARγ.

Typical PPAR agonists are known to consist of three parts:

a polar head group (usually bearing a carboxylic acid

functionality), a hydrophobic tail moiety, and a linker,

which consists of ﬂexible methylene units and an aromatic

ring.²⁶Interestingly, 7a and 7j closely meet these elementary

criteria.

Potency and Eﬃcacy of PPARγ Activation. The

concentration-dependent activation of PPARγ by the “hit”

compound 3a and its two “lead” derivatives 7a and 7j was

compared to that of the PPARγ full agonist Rosi using the

PPARγ-LBD-GAL4 chimera assay (Figure 3A).

The potencies of the compounds were ranked as follows: 3a

≈ 7a < 7j < Rosi (Table 1). Regarding their eﬃcacy (maximal

PPARγ transactivation), compounds were ranked as follows:

3a < 7a < 7j ≤ Rosi (Table 1).

Table 1. EC₅₀and Maximal Activation of PPARγ and

a

PPARα

compound

PPAR

EC₅₀(nM)

activity (%)

Rosi

7j

7a

3a

GW 7647

7j

bezaﬁbrate

γ

76 30

400 150

1940 940

1840 1020

100

γ

66.36 3.41

25.06 2.03

6.33 1.03

100

75.6 3.83

35.4 3.81

γ

α

5.4

1

1882 665

31,700 5410

a

Activities are expressed as % of the maximal response measured with

5 μM Rosi (for PPARγ) or with 1 μM GW 7647 (for PPARα). Values

are means SD.

PPARγ activation elicited by 7j was around 66% of that

obtained with Rosi. However, this value is certainly over-

estimated. Indeed, in this speciﬁc cell-based assay, we

observed that the Renilla luciferase expression (Figure S5A),

which is used to normalize reporter gene values for variations

inherent to transfection eﬃciency and sample handling, was

more reduced by 7j concentrations that induce maximal

D

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PPARγ activity (>10⁻⁶M) than by Rosi. As a consequence,

inclusion of Renilla luciferase expression values in the

calculation of PPARγ activation by 7j leads to an “artiﬁcial

rise” of the maximum response (Figure S5B). Interestingly,

the measurement of ATP cell content revealed that the

number of viable HEK293 cells was no more reduced by 7j

than by Rosi, ruling out that the 7j-dependent decrease in

Renilla luciferase expression is due to a reduction in the

number of viable cells (Figure S5C).

found that 7j concentration dependently bound to PPARα

with a K_dvalue of 3.1 μM (Table 2).

PPAR forms permissive heterodimers with RXR, thus

allowing both the ligands of PPAR and RXR to regulate the

transcription of PPAR target genes. Using a mammalian two-

hybrid system, we showed that 7j was less potent and less

eﬃcient than Rosi in inducing PPARγ/RXRα heterodimeriza-

tion (Figure S8A). Moreover, using the RXRα-LBD-GAL4

chimera assay (Figure S8B) and RXR responsive element

(RXRE)-based luciferase assay (Figure S8C), we demon-

strated that 7j is not a direct RXR agonist.

Taken together, these data allow us to deﬁne 3a and 7a as

partial agonists of PPARγ and 7j as a strong partial agonist of

PPARγ.

Therefore, 7j is a dual PPARγ/α agonist (glitazar), and as

such, it could theoretically have beneﬁcial synergistic activities

on glucose and lipid homeostasis.^19,20

A PPAR responsive element (PPRE)-based luciferase assay

was carried out to assess the ability of 3a, 7a, and 7j to

transactivate genes controlled by the binding of PPARγ to

PPRE. The three molecules increased the expression of PPRE-

driven luciferase only when full-length PPARγ was expressed

(Figure 3B), showing that these molecules stimulate the actual

transcriptional activity of PPARγ. Interestingly, the trans-

activation eﬃciencies were in agreement with those measured

by the PPARγ-LBD-GAL4 chimera assay, i.e., 3a ≈ 7a < 7j. In

addition, and as expected, 1 μM 7j and 0.1 μM Rosi gave

similar activation levels in both the PPARγ-LBD-GAL4

chimera assay and PPRE-based assay.

To attest that 3a, 7a, and 7j are actual PPARγ ligands, their

binding aﬃnity (K_d) and rate constants (k_onand k_off) for

PPARγ were compared to those of the reference ligand Rosi

by surface plasmon resonance technology-based experiments

(Table 2 and Figure S6).

Binding of 7j, 7a, and 3a to PPARγ. The crystal

structure of the complex of PPARγ LBD with the ligand 7j

was solved, collecting diﬀraction data from apo-crystals soaked

for 3 days in the presence of the ligand at 0.5 mM. Initial

diﬀerence Fourier maps revealed clear electron density for the

ligand (Figure S9), showing that it occupies the typical LBD

region of the PPARγ agonists (Figure 4A and Figure S10),

similar to that of the full agonist Rosi (Figure 4B and Figure

S11), with its carboxylate group directly interacting through H

bonds with Y473 of helix 12 (H12), at a short distance of 2.3

Å, the two histidines (H323 and H449) of the canonical triad,

and S289 (Figure 4A). The NH of the 7j amide bond is also

involved in the H bond with S289, and the CO makes a H

bond with Y327. The terminal aliphatic chain of 7j is in

equilibrium between two diﬀerent conformations (occupation

factors of 0.6 and 0.4) and makes vdW contacts with several

residues of the internal strand of the β-sheet (Figure 4A and

Figure S12) with a consequent eﬀective stabilization of this

domain. The isopropyl terminal group of 7j, makes vdW

interactions in the hydrophobic pocket formed by the residues

F282, C285, Q286, L453, and L469 (belonging to H3, H11,

and H12), contributing, in this way, to a tighter binding of the

ligand (Figure 4C and Figure S13).

The thorough analysis of the eﬀective binding network of 7j

into the PPARγ LBD indicates that the ligand is very tightly

locked into the LBD, with its carboxylate group strongly

interacting with Y473 of H12 (2.3 Å), but in a slightly

distorted mode, with respect to Rosi. As a consequence, there

is an adjustment of the conformation of helix 12 (H12), the

loop 11/12, and the beginning of helix 11 (H11) (Figure 4D

and Figure S14). Importantly, H12 is a critical regulatory

structural element in the activation function-2 (AF-2)

coregulator binding surface, which determines the transcrip-

tional output of PPARγ through diﬀerential recruitment of

coregulators.

Comparison of the crystal structures of PPARγ in complex

with 7j, 7a, or 3a²⁸shows no diﬀerence in the binding mode

of these ligands, regardless of the presence of a carboxylic or

hydroxamic head (Figure S15). However, the shorter aliphatic

chains of 7a and 3a accommodate in a diﬀerent part of the

PPARγ LBD with respect to 7j, farther away from the β-sheet

(Figure S15). Moreover, it is known that partial agonists of

PPARγ preferentially stabilize the β-sheet region of the LBD.

In the PPARγ/7j structure, the long aliphatic chain of the

ligand strongly contributes to the stabilization of this region,

through vdW interactions, with both its observed conforma-

tions facing residues of the innermost β-strand. The solvent

entropic gain arising from a more eﬃcacious displacement of

water molecules, known to occupy the β-sheet pocket, could

Table 2. Aﬃnity (K_d) and Rate Constants (k_onand k_off) for

a

PPARγ/Ligand and PPARα/Ligand Interactions

interaction

k_on(M⁻¹s⁻¹

)

k_off(s⁻¹

)

K_d(nM)

PPARγ/Rosi

PPARγ/7j

PPARγ/7a

PPARγ/3a

PPARα/7j

3.45 (0.02) × 10⁵

5.65 (0.06) × 10⁵

1.52 (0.02) × 10⁵

1.55 (0.02) × 10⁵

1.01 (0.03) × 10⁴

0.0164 (0.0001)

0.0170 (0.0001)

0.0143 (0.0001)

0.0190 (0.0001)

0.0312 (0.0005)

47.6 (0.3)

30.1 (0.3)

94 (1)

123 (2)

3100 (50)

a

Experimental error is reported in parentheses.

The aﬃnities of 3a and 7a for PPARγ were lower than

those of 7j and Rosi, which is consistent with their low PPARγ

transactivation capacity measured in cell-based assays.

Remarkably, the partial agonist 7j shows an aﬃnity for

PPARγ similar or higher to that of the full agonist Rosi. It was

shown that some compounds with moderate PPARγ agonist

activity but with high binding aﬃnity to PPARγ retain

signiﬁcant antidiabetic activity while having fewer and/or less

severe adverse events than full PPARγ agonists.²⁷Therefore,

7j can reasonably be considered as the scaﬀold molecule to

develop new antidiabetic drugs.

Speciﬁcity. The concentration-dependent activations of

PPARδ and PPARα by 3a, 7a, and 7j were analyzed using the

appropriate PPAR-LBD-GAL4 chimera assays. None of the

molecules activated PPARδ (Figure S7). Only 7j trans-

activated PPARα (Figure 3C and Table 1), with potency

between bezaﬁbrate (a weak pan agonist for all three PPAR

isoforms) and GW 7647 (a full PPARα agonist). 7j appeared

to be a good PPARα agonist. In order to conﬁrm that 7j is a

PPARα ligand, their direct physical binding was measured by

surface plasmon resonance technology-based experiments. We

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Figure 4. Binding of 7j to PPARγ. (A) Hydrogen-bond network of 7j in the PPARγ LBD. (B) Superimposition of 7j (yellow) and Rosi (green)

structures. (C) vdW network of 7j with residues belonging to H3, H11, and H12 of the PPARγ LBD. (D) Superimposition of 7j (yellow) and

Rosi (green) with the coactivator SRC-1 (magenta); the PPARγ/Rosi structure is colored in green, and the PPARg/7j structure is colored in gray

(PDB codes: 6QJ5 for PPARγ/7j and 2PRG for PPARγ/Rosi).

Figure 5. Ligand-speciﬁc coregulator binding proﬁles. (A−D) HEK293 cells were transfected with GAL4-NCoR (A), GAL4-SMRT (B), GAL4-

TIF2 (C), GAL4-MED1 (D), and VP16-PPARγ-LBD expression vectors in a mammalian two-hybrid setting and treated with vehicle or various

concentrations of 7j or Rosi. Values are means SD expressed as % of the maximal response measured.

play an important role in lowering the free energy of the

binding. The higher aﬃnity of 7j for PPARγ with respect to

those of 7a and 3a (Table 2) demonstrates that the aﬃnity is

largely aﬀected by the length of the aliphatic chain, rather than

the character of the acidic head.

Interestingly, a similar unique binding network was

observed in the PPARγ crystal structure with the partial

agonist SR2067 (Figure S16).²⁹Both ligands share a common

amide group that forms two hydrogen bonds with Y327 and

S289, interactions that are not possible with Rosi. However,

unlike 7j, SR2067 does not interact with Y473.

Interaction of Coregulators with PPARγ. Since Rosi

and 7j stabilize diﬀerent conformations of the AF2 coregulator

binding surface of PPARγ, we analyzed how these molecules

modulate the interaction of PPARγ with some of its

coregulators. In the mammalian two-hybrid system, 7j was

less potent than Rosi in inducing displacement of the NCoR

and SMRT corepressors from PPARγ and in inducing

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Figure 6. Eﬀect of 7j on PPARγ phosphorylation. (A) Percentage of in vitro PPARγ Ser273 phosphorylation by CDK5 in the presence of 0.1 μM

of 7j or Rosi. (B) Phosphorylation of PPARγ Ser273 in 3T3-L1 adipocyte incubated for 60 min with 5 μM Rosi, 7j, or roscovitine (Rsv) before

TNFα stimulation (50 ng mL⁻¹; 90 min). Two independent experiments are shown. Phosphospeciﬁc antibodies were from Rockland or New

England Peptide (NEP). Arrow heads indicate phosphorylated PPARγ and PPARγ2. (C) RT-PCR analysis of the expression levels of a selection

of genes known to be regulated by CDK5-dependent phosphorylation of PPARγ. Values are means SD (n = 5) expressed relative to the mean

of control. **p < 0.01 and ***p < 0.001 vs control (one-way ANOVA followed by Dunnett’s post-hoc test).

recruitment of TIF2 and MED1 coactivators to PPARγ

(Figure 5).

Adipocyte Diﬀerentiation and Glucose Uptake.

Adipogenesis-mediated weight gain is a major side eﬀect of

PPARγ full agonists.³⁰Partial PPARγ agonists are expected to

have a reduced eﬀect on lipid storage while maintaining a

signiﬁcant insulin sensitization eﬀect. Therefore, the proadipo-

genic properties of 3a, 7a, and 7j were compared to that of

Rosi. 3T3-L1 ﬁbroblast cells were incubated in the presence of

PPARγ agonists as the sole inducer of adipocyte diﬀer-

entiation. Only 7j and Rosi (the strongest PPARγ agonists)

signiﬁcantly increased the intracellular lipid content (Figure

7A and Figure S17A), showing that these two molecules

stimulate adipocyte diﬀerentiation. However, for the same

concentration, the proadipogenic property of 7j was

signiﬁcantly lower than that of Rosi. To ascertain that the

proadipogenic eﬀect of 7j involved PPARγ activation, 3T3-L1

cells were coincubated with the speciﬁc PPARγ antagonist

GW 9662 and with 7j; then, intracellular lipid accumulation

was measured. This approach has been successfully used to

demonstrate the involvement of PPARγ activation in the

proadipogenic eﬀect of Rosi.³¹Because of its short half-life,

GW 9662 was added every day, and to simplify the

experiment, the duration of the treatment was reduced to 4

days instead of 6 days, thus limiting the intracellular lipid

accumulation. Antagonizing PPARγ activation signiﬁcantly

prevented 7j- and Rosi-induced adipogenesis (Figure 7B and

Figure S17B), showing that PPARγ activation is necessary for

the proadipogenic eﬀect of these molecules. When 3T3-L1

ﬁbroblast cells were primed for adipocyte diﬀerentiation using

the conventional adipogenic cocktail (insulin, dexamethasone,

and IBMX) and then treated for 7 days with 3a, 7a, 7j or

Rosi, mRNA levels of the markers of adipocyte diﬀerentiation

were all increased in proportion to the ability of the molecules

to activate PPARγ (Figure 7C).

The diﬀerences between the proﬁles of the concentration−

response curves of 7j and Rosi to modulate the interaction of

PPARγ with its partners (NCoR, SMRT, TIF2, MED1,

mediator, and also RXRα) suggest that these two molecules

induce distinct patterns of coregulatory protein recruitment.

CDK5-Mediated Phosphorylation of PPARγ. Part of

the antidiabetic eﬀects of PPARγ partial agonists has been

associated with their ability to prevent the CDK5-mediated

phosphorylation of the PPARγ serine 273 residue.^17,18

Therefore, we assessed the ability of 7j to inhibit such

phosphorylation. An ELISA protocol was optimized to

quantify the phosphorylation of PPARγ triggered in vitro by

recombinant CDK5. 7j prevented the phosphorylation of

PPARγ serine 273 at least as eﬃciently as Rosi (Figure 6A).

Furthermore, pretreatment of 3T3-L1 adipocytes with 7j or

Rosi reduced the TNFα-stimulated phosphorylation of PPARγ

serine 273 (Figure 6B). The involvement of CDK5 in this

phosphorylation was attested by the fact that it was impeded

by treatment of cells with roscovitine, a CDK5 inhibitor.

Moreover, out of 10 genes known to be signiﬁcantly

controlled by CDK5-dependent PPARγ phosphorylation in

fully diﬀerentiated adipocytes,^17,18seven were regulated in the

same way by 7j and Rosi (Figure 6C). Two genes were only

regulated by Rosi, and the expression of one gene was not

signiﬁcantly modiﬁed by 7j and Rosi, although both molecules

tended to alter this expression in the same direction.

These results underline the ability of 7j to reduce CDK5-

mediated phosphorylation of PPARγ serine 273 and to alter

the expression of genes controlled by PPARγ phosphorylation.

This feature supports a potential antidiabetic eﬀect of 7j.

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suggesting that partial PPARγ agonists, which retain signiﬁcant

white fat browning ability, may also have therapeutic beneﬁts

in the treatment of obesity and diabetes. We therefore studied

the ability of 3a, 7a, and 7j to induce brite/brown-like

adipocytes in 3T3-L1 cells.

Only 7j (and Rosi) upregulated mRNA levels of genes

considered as brite/brown adipocyte-selective transcript

(Figure 9A,B).³⁶Immunoblotting analysis conﬁrmed that 7j

and Rosi increased the expression of uncoupling protein 1

(UCP1), which is a functional marker of brown adipocytes

(Figure 9B inset). Coherent with their adipocyte browning

eﬀect, Rosi and 7j increased mRNA levels of additional genes

involved in (or associated with) mitochondrial biogenesis

(Figure 9C)³⁷and in accordance with these two molecules

signiﬁcantly increased the selective labeling of active

mitochondria by the MitoTracker dye (Figure 9D). We

have previously shown that Rosi induced the conversion of

hMADS white adipocytes into brite adipocytes as evidenced

by the strong expression of UCP1.³⁸In order to test whether

compounds 3a, 7a, and 7j were able to substitute for Rosi,

hMADS cells ﬁrst diﬀerentiated into white adipocytes were

treated with these compounds between days 14 and 18. UCP1

expression was analyzed as an indicator of the degree of white-

to-brown adipocyte conversion. UCP1 mRNA levels were

increased in 3a, 7a and 7j-treated cells compared to untreated

cells, and 7j was found to be the most potent compound

(Figure 9E), although less potent than Rosi (Figure 9F). The

PPARγ agonists also increased the levels of UCP1 antigen,

which is consistent with observations of its mRNA levels

(Figure 9G).

Figure 7. Adipogenic eﬀect of the compounds. (A, B) 3T3-L1

ﬁbroblasts were incubated for 6 days (A) or for 4 days (B) with

insulin (350 nM), the indicated PPARγ agonists (1 μM), and the

PPARγ antagonist GW 9662 (5 μM). Intracellular lipids were stained

with Oil Red O, and then colored lipids were quantiﬁed. (C) 3T3-L1

adipocyte diﬀerentiation was triggered by the standard mixture of

inductors, and then cells were incubated for 7 days with 3a, 7a, 7j (1

μM), or Rosi (0.1 μM). The expression levels of adipogenesis-related

genes were measured by RT-PCR. Values are means SD expressed

as fold relative to untreated situation. *p < 0.05, **p < 0.01, and

***p < 0.001 vs control (one-way ANOVA followed by Dunnett’s

‡

post-hoc test); p < 0.05 and ^‡‡p < 0.01 (t-test).

The expression of the adipogenic marker perilipin was not

modiﬁed by Rosi or 7j (Figure S18A), whereas the expression

of adiponectin, a PPARγ-responsive gene, was more eﬃciently

increased by Rosi than by 7j (Figure S18B), which conﬁrms

that the PPARγ agonist activity of 7j is lower than that of

Rosi.

As expected for partial PPARγ agonists, 3a, 7a, and 7j have

reduced proadipogenic properties compared with the full

agonist Rosi.

It has been shown that the ability of PPARγ ligands to

increase cellular glucose uptake is not necessarily related to

their transactivation activity or proadipogenic potential.³²

Therefore, glucose uptake was measured in fully diﬀerentiated

3T3-L1 adipocytes after an acute treatment with PPARγ

agonists. The insulin-dependent glucose uptake was increased

by a short-term treatment with 7j or Rosi (Figure 8), denoting

that part of their insulin sensitizing eﬀect is independent of

their ability to increase adipocyte diﬀerentiation.

Adipocyte Browning. Brown fat is a target for antiobesity

and antidiabetes experimental therapies that aim to increase

energy expenditure.³³Interestingly, strong PPARγ agonists

activate the “browning” of white adipose tissues,^34,35

These data suggest that 7j can eﬃciently promote the

conversion from white to brown adipocytes.

Lipid Accumulation in Hepatocytes. As PPARα

agonists decrease hepatic steatosis,^5,7we compared the eﬀect

of 7j (dual PPARα/γ agonist) to those of GW 7647 (PPARα

agonist) and Rosi (PPARγ agonist) on the lipid accumulation

into hepatocytes. In primary rat hepatocytes, only GW 7647

prevented the basal intracellular lipid accumulation and oleic

acid-induced lipid accumulation was prevented by Rosi, 7j,

and GW 7647 (Figure 10 A). In human HuH7 hepatoma

cells, 7j and GW 7647 similarly prevented the oleic acid-

stimulated lipid accumulation (Figure 10B). These results

suggest that 7j is a promising compound to prevent hepatic

steatosis.

Cytotoxicity of 7j. We conﬁrmed, as previously

reported,³⁹⁻⁴¹that viability, membrane integrity, and

apoptosis of primary hepatocytes were not signiﬁcantly altered

by concentrations of Rosi up to 10 μM. These parameters

were also not altered by the 7j compound (Figure S19).

CONCLUSIONS

■

We have described the synthesis and optimization of

compound 7j: a new N-aryl-substituted valine derivative

with a balanced agonist activity on PPARα and PPARγ.

Compound 7j occupies the typical LBD region of the PPARγ

agonists with a unique high-aﬃnity binding mode and

eﬃciently prevents CDK5-mediated phosphorylation of

Figure 8. Adipocyte glucose uptake. 3T3-L1 adipocytes were treated

with 7j (1 μM) or Rosi (0.1 μM) for 16 h prior to insulin stimulation

(50 nM, 10 min), and cellular glucose uptake was determined. Values

are mean SD expressed as fold relative to the control situation. **p

< 0.01 and ***p < 0.001 vs control (one-way ANOVA followed by

Dunnett’s post-hoc test).

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Figure 9. Adipocyte browning eﬀect of the compounds. 3T3-L1 cells were treated as in Figure 7C. (A−C) Expression levels of beige (A) and

brown (B) adipocyte markers as well as those of genes involved in mitochondrial biogenesis (C) were measured by RT-PCR. Inset in panel B:

immunodetection of UCP1 and β-actin (loading control) in lysates of 3T3-L1 cells treated with 7j and Rosi. (D) Adipocyte mitochondria content

was evaluated by ﬂow cytometry after their selective labeling with MitoTracker dye. (E, F) hMADS white adipocytes were treated with the

indicated compounds, and UCP1 mRNA levels were measured by RT-PCR. (G) Immunodetection of UCP1 and β-actin (loading control) in

lysates of hMADS treated with the indicated concentrations of 7j, Rosi (0.1 μM), 3a, and 7a (1 μM). Values are mean SD expressed as fold

relative to the control (cont) situation. *p < 0.05, **p < 0.01, and ***p < 0.001 vs control (one-way ANOVA followed by Dunnett’s post-hoc

test).

Figure 10. Lipid accumulation in hepatocytes. (A) Primary rat hepatocytes and (B) human HuH7 hepatoma cells were treated with 7j, Rosi, or

GW 7647 (1 μM) for 8 h before addition of oleic acid (0.25 mM). Twenty-four hours later, cellular lipid content was measured by Oil Red O

staining. Values are mean SD expressed as fold relative to the control. *p < 0.05, **p < 0.01, and ***p < 0.001 vs control (Kruskal−Wallis

followed by Dunn’s post-hoc test).

PPARγ. Meanwhile, poorly proadipogenic, compound 7j

increases adipocyte insulin-stimulated glucose uptake and

eﬃciently promotes white-to-brown adipocyte conversion. In

addition, compound 7j prevents the oleic acid-induced lipid

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4-Hexyloxy-N-[(S)-1-hydroxycarbamoyl-2-(3H-imidazol-4-yl)-

ethyl]-benzamide (3b). The procedure is similar to that applied for

the preparation of 3a.

accumulation in hepatocytes. The unique biochemical proper-

ties of compound 7j, its speciﬁc biological activities, and its

low toxicity make it a promising candidate for the develop-

ment of compounds to reduce insulin resistance, obesity, and

NAFLD.

1

Pale yellow solid. H NMR (DMSO-d₆): δ = 7.52−7.89 (m, 3H),

6.89−7.42 (m, 5H), 5.55 (s, 2H), 4.75−4.73 (m, 2H), 4.10−4.19

(m, 2H), 1.22−1.92 (m, 9H), 0.89−0.96 (m, 3H). ¹³C NMR

(DMSO-d₆): δ = 174.19, 164.07, 162.03, 133.17, 129.03, 123.47,

118.22, 115.35, 67.56, 52.96, 30.99, 30.91, 29.05, 25.49, 22.54, 14.25.

MS (ESI) m/z: calcd for C₁₉H₂₆N₄O₄, 375.2014 (100%, (M + H⁺)).

4-Hexyloxy-N-[(S)-1-hydroxycarbamoyl-2-(1H-indol-2-yl)-ethyl]-

benzamide (3c). The procedure is similar to that applied for the

preparation of 3a.

EXPERIMENTAL SECTION

■

Chemistry. All solvents were puriﬁed according to reported

procedures, and reagents were used as commercially available.

Methanol, ethyl acetate, dichloromethane, ammonia, and petroleum

ether (35−60 °C) were purchased from VWR and used without

further puriﬁcation. Column chromatography was performed on

White solid. ¹H NMR (DMSO-d₆): δ = 7.32−7.62 (m, 5H),

6.29−7.25 (m, 7H), 3.92−4.03 (m, 2H), 2.89−3.34 (m, 4H), 1.20−

1.79 (m, 6H), 0.88−0.92 (m, 3H). ¹³C NMR (DMSO-d₆): δ =

172.22, 168.98, 161.25, 139.48, 131.05, 129.14, 126.29, 120.15,

118.78, 113.69, 110.36, 104.25, 71.24, 53.32, 30.21, 28.28, 25.14,

22.04, 14.13. MS (ESI) m/z: calcd for C₂₄H₂₉N₃O₄, 424.2236 (100%,

(M + H⁺)).

1

VWR silica gel (70−230 mesh). H NMR and ¹³C NMR spectra

were recorded in CDCl₃or DMSO-d₆on a Bruker AC 300

spectrometer working at 300 and 75 MHz, respectively (the usual

abbreviations are used: s, singlet; d, doublet; t, triplet; q, quadruplet;

and m, multiplet). Tetramethylsilane was used as the internal

standard. All chemical shifts are given in ppm. Purity of all the new

compounds evaluated by HPLC (Agilent 1100, C18) analysis was

>95%.

4-Hexyloxy-N-((S)-2-hydroxy-1-hydroxycarbamoyl-ethyl)-benza-

mide (3d). The procedure is similar to that applied for the

preparation of 3a.

White solid. ¹H NMR (DMSO-d₆): δ = 7.02−7.51 (m, 4H),

3.62−4.20 (m, 5H), 1.31−1.82 (m, 9H), 0.89−0.92 (m, 3H). ¹³C

NMR (DMSO-d₆): δ = 170.36, 168.24, 160.89, 130.14, 125.34,

113.47, 68.78, 61.24, 53.89, 31.01, 28.14, 25.98, 21.33, 13.88. MS

(ESI) m/z: calcd for C₁₆H₂₄N₂O₅, 325.1768 (100%, (M + H⁺)).

4-Hexyloxy-N-((S)-1-hydroxycarbamoyl-2-phenyl-ethyl)-benza-

mide (3e). The procedure is similar to that applied for the

preparation of 3a.

Typical Procedure for the Synthesis of Amino Acid Methyl

Ester Hydrochloride Derivatives. Synthesis of ^L-Valine Methyl

Ester Hydrochloride. In a two-neck round ﬂask equipped with a

condenser was placed at room temperature 3 g of ^L-valine (2.56 ×

10⁻²mol) in 20 mL of methanol. The mixture was placed under

stirring at 0 °C, and 3.4 mL of thionyl chloride (4.7 × 10⁻³mol) was

slowly added. After removal of the solvents, diethyl ether was added

and the product precipitates as a white solid. After ﬁltration, the

product was dried under vacuum to aﬀord the expected ^L-valine

methyl ester hydrochloride in 86% yield.

White solid. ¹H NMR (DMSO-d₆): δ = 7.66−7.68 (m, 2H),

6.47−7.16 (m, 2H), 3.55−3.97 (m, 2H), 2.89−3.23 (m, 2H), 1.35−

1.74 (m, 8H), 0.90−0.91 (m, 3H). ¹³C NMR (DMSO-d₆): δ =

178.45, 168.32, 161.25, 137.47, 129.02, 125.68, 114.98, 60.01, 53.87,

40.36, 30.23, 28.14, 24.12, 22.98, 13.48. MS (ESI) m/z: calcd for

C₂₂H₂₈N₂O₄, 385.2057 (100%, (M + H⁺)).

1

White solid. H NMR (D₂O): δ = 4.14−4.17 (m, 1H), 3.80−3.85

(m, 3H), 2.50−2.56 (m, 1H), 1.12−1.15 (m, 6H). ¹³C NMR (D₂O):

δ = 168.91, 58.57, 53.02, 29.95, 18.15.

(S)-2-(4-Hexyloxy-benzoylamino)-3-methyl-butyric Acid Methyl

Ester (6). In a two-neck round ﬂask equipped with a condenser were

placed at room temperature 2.34 g of benzotriazol-1-yloxytris-

(dimethylamino)phosphonium hexaﬂuorophosphate (BOP; 5.26 ×

10⁻³mol), 2.34 mL of diisopropylethylamine (1.8 × 10⁻²mol), 1.2 g

of 4-(hexyloxy)benzoic acid (5.40 × 10⁻³mol), and 0.88 g of L-valine

methyl ester hydrochloride (5.25 × 10⁻³mol) in 15 mL of CH₂Cl₂.

The mixture was placed under stirring at room temperature for 24 h.

Water was added to allow phase separation. The bottom phase layer

was washed with NaHCO₃(10%) solution, dried over Na₂SO₄,

ﬁltered, and concentrated in vacuo. After removal of the solvents, the

crude residue was puriﬁed by chromatography on a silica gel column

using CH₂Cl₂/ethyl acetate (1/1) as the eluent, aﬀording the

expected product 6 in 85% yield.

N-[(S)-2-(3,4-Dihydroxy-phenyl)-1-hydroxycarbamoyl-ethyl]-4-

hexyloxy-benzamide (3f). The procedure is similar to that applied

for the preparation of 3a.

White solid. ¹H NMR (DMSO-d₆): δ = 7.71−7.73 (m, 2H),

6.59−6.93 (m, 4H), 6.32 (s, 1H), 5.87 (s, 1H), 3.99−4.02 (m, 2H),

2.95−2.99 (m, 2H), 1.17−2.02 (m, 9H), 0.87−0.89 (m, 3H). ¹³C

NMR (DMSO-d₆): δ = 171.25, 168.33, 160.58, 146.01, 143.69,

128.69, 126.47, 123.74, 116.36, 115.47, 69.71, 52.34, 38.38, 28.47,

22.14, 25.69, 22.47, 13.12. MS (ESI) m/z: calcd for C₂₂H₂₈N₂O₆,

417.1934 (100%, (M + H⁺)).

4-Hexyloxy-N-((S)-1-hydroxycarbamoyl-4-methylsulfanyl-butyl)-

benzamide (3g). The procedure is similar to that applied for the

preparation of 3a.

1

White solid. ¹H NMR (DMSO-d₆): δ = 6.91−7.69 (m, 5H),

1.17−2.74 (m, 20H), 0.87−0.88 (m, 3H). ¹³C NMR (DMSO-d₆): δ

= 170.78, 168.65, 162.21, 128.12, 125.14, 117.77, 69.66, 51.45, 34.12,

31.78, 28.02, 25.63, 22.33, 15.15, 13.19. MS (ESI) m/z: calcd for

C₁₉H₃₀N₂O₄S, 383.1935 (100%, (M + H⁺)).

White solid. H NMR (CDCl₃): δ = 8.06 (m, 2H), 7.15 (m, 2H),

4.15−4.22 (m, 4H), 3.71−3.75 (m, 3H), 1.81−2.05 (m, 3H), 1.21−

1.37 (m, 6H), 0.69−0.92 (m, 9H). ¹³C NMR (CDCl₃): δ = 172.90,

166.54, 160.52, 133.08, 130.21, 130.01, 114.55, 68.22, 57.56, 52.34,

33.49, 31.32, 29.34, 25.67, 22.61, 18.48, 14.03. MS (ESI) m/z: calcd

for C₁₉H₂₉NO₄, 336.2146 (100%, (M + H⁺)).

4-(Hexyloxy)-N-((2S,3S)-3-hydroxy-1-(hydroxyamino)-1-oxobu-

tan-2-yl)benzamide (3h). The procedure is similar to that applied

for the preparation of 3a.

4-Hexyloxy-N-((S)-1-hydroxycarbamoyl-2-methyl-propyl)-benza-

mide (3a). In a 25 mL round ﬂask was placed at room temperature

0.6 g of 6 (1.78 × 10⁻³mol) in 15 mL of ethanol. A hydroxylamine

solution (40%, 2 mL) was subsequently added, and the mixture was

allowed to stir at reﬂux for 24 h. After removal of the solvents, the

crude residue was puriﬁed by chromatography on a silica gel column

using petroleum ether/ethyl acetate (1/1) and methanol/ethyl

acetate (1/1) as eluents, aﬀording the expected product 3a in 72%

yield.

White solid. ¹H NMR (DMSO-d₆): δ = 7.71−7.73 (m, 2H),

6.98−7.11 (m, 3H), 5.32 (s, 1H), 4.60−4.65 (m, 1H), 4.06−4.44

(m, 3H), 1.80−1.92 (m, 1H), 1.07−1.37 (m, 12H). ¹³C NMR

(DMSO-d₆): δ = 169.75, 167.63, 162.54, 132.53, 125.82, 114.51,

68.78, 67.63, 59.34, 31.88, 28.02, 24.89, 22.33, 15.12, 13.89. MS

(ESI) m/z: calcd for C₁₇H₂₆N₂O₅, 339.1879 (100%, (M + H⁺)).

(S)-N-(1-(Hydroxyamino)-3-methyl-1-oxobutan-2-yl)-4-methox-

ybenzamide (4a). White solid. ¹H NMR (DMSO-d₆): δ = 0.91−0.96

(m, 6H), 2.16−2.23 (m, 1H), 3.84 (s, 3H), 4.33−4.45 (m, 1H),

6.98−7.53 (m, 2H), 7.83−7.89 (m, 2H), 8.79 (s, 2H). ¹³C NMR

(DMSO-d₆): δ = 174.44, 170.42, 164.51, 130.95, 127.71, 115.17,

56.40, 52.96, 32.19, 20.12, 19.65. MS (ESI) m/z: calcd for

C₁₃H₁₈N₂O₄, 267.1386 (100%, (M + H⁺)).

1

White solid. H NMR (DMSO-d₆): δ = 8.01 (m, 2H), 7.09 (m,

2H), 3.95−3.98 (m, 3H), 1.78−1.82 (m, 2H), 1.01−1.31 (m, 6H),

0.71−0.93 (m, 9H). ¹³C NMR (DMSO-d₆): δ = 167.20, 166.23,

161.1, 133.41, 129.80, 113.98, 69.41, 57.31, 31.03, 29.42, 25.76,

22.43, 18.62, 13.89. MS (ESI) m/z: calcd for C₁₈H₂₈N₂O₄, 337.2045

(100%, (M + H⁺)).

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(S)-4-Ethoxy-N-(1-(hydroxyamino)-3-methyl-1-oxobutan-2-yl)-

64.36, 31.32, 31.04, 29.14, 25.31, 22.14, 18.47, 13.96. MS (ESI) m/z:

calcd for C₁₈H₂₇NO₄, 322.1932 (100%, (M + H⁺)).

1

benzamide (4b). White solid. H NMR (DMSO-d₆): δ = 0.91−0.95

(m, 6H), 1.49 (t, J = 7.0 Hz, 3H), 1.68−1.75 (m, 1H), 4.01−4.11

(m, 2H), 4.31−4.41 (m, 1H), 6.94−6.99 (m, 2H), 7.80−7.85 (m,

2H), 8.59 (s, 2H). ¹³C NMR (DMSO-d₆): δ = 171.10, 166.47,

166.91, 132.01, 129.30, 114.59, 63.42, 56.95, 30.10, 18.64, 14.69. MS

(ESI) m/z: calcd for C₁₄H₂₀N₂O₄, 281.1412 (100%, (M + H⁺)).

(S)-N-(1-(Hydroxyamino)-3-methyl-1-oxobutan-2-yl)-4-propoxy-

(S)-2-(4-Hexyloxy-benzoylamino)-3-phenyl-propionic Acid (7b).

The procedure is similar to that applied for the preparation of 7a.

White solid. ¹H NMR (CDCl₃): δ = 10.98 (s, 1H), 7.89−8.01 (m,

2H), 6.95−7.21 (m, 7H), 4.83−4.85 (m, 11H), 3.89−3.92 (m, 2H),

3.01−3.03 (m, 2H), 0.95−1.61 (m, 12H). ¹³C NMR (CDCl₃): δ =

176.15, 168.13, 161.14, 140.03, 129.18, 128.82, 123.52, 123.42,

115.22, 73.34, 61.56, 38.78, 33.14, 31.42, 26.14. MS (ESI) m/z: calcd

for C₂₃H₃₀NO₄, 385.2221 (100%, (M + H⁺)).

1

benzamide (4c). White solid. H NMR (DMSO-d₆): δ = 0.92−1.12

(m, 9H), 1.69−1.89 (m, 2H), 3.27−3.38 (m, 2H), 3.90−4.02 (m,

2H), 6.90−7.07 (m, 2H), 7.76−7.89 (m, 2H). ¹³C NMR (DMSO-

d₆): δ = 171.14, 165.95, 162.22, 130.95, 128.03, 115.09, 70.35, 57.03,

30.75, 22.63, 18.64, 10.63. MS (ESI) m/z: calcd for C₁₅H₂₂N₂O₄,

294.1634 (100%, (M + H⁺)).

(S)-2-(4-Hexyloxy-benzoylamino)-3-(3H-imidazol-4-yl)-propionic

Acid (7c). The procedure is similar to that applied for the preparation

of 7a.

1

Yellow solid. H NMR (CDCl₃): δ = 7.93 (s, 1H), 7.63−7.65 (m,

2H), 6.75−6.92 (m, 4H), 4.69−4.72 (m, 2H), 4.12−3.92 (m, 2H),

2.67−2.78 (m, 2H), 0.76−1.52 (m, 11H). ¹³C NMR (CDCl₃): δ =

174.75, 169.70, 161.08, 132.04, 131.03, 129.92, 125.75, 121.56,

111.04, 68.13, 52.67, 31.14, 29.57, 29.15, 25.56, 22.59, 13.82. MS

(ESI) m/z: calcd for C₁₉H₂₅N₃O₄, 360.1834 (100%, (M + H⁺)).

(S)-2-(4-Hexyloxy-benzoylamino)-3-hydroxy-propionic Acid

(7d). The procedure is similar to that applied for the preparation

of 7a.

(S)-4-Butoxy-N-(1-(hydroxyamino)-3-methyl-1-oxobutan-2-yl)-

1

benzamide (4d). White solid. H NMR (DMSO-d₆): δ = 0.90−0.98

(m, 9H), 1.52−1.62 (m, 2H), 1.77−1.83 (m, 2H), 2.16−2.22 (m,

2H), 3.97−4.02 (m, 2H), 4.37−4.42 (m, 1H), 7.75−7.77 (m, 2H),

7.98−8.02 (m, 2H), 8.53 (m, 1H). ¹³C NMR (DMSO-d₆): δ =

171.49, 166.14, 166.16, 133.37, 130.18, 122.86, 69.16, 57.04, 30.74,

30.69, 18.48, 13.75. MS (ESI) m/z: calcd for C₁₆H₂₄N₂O₄, 309.1701

(100%, (M + H⁺)).

1

White solid. H NMR (DMSO-d₆): δ = 7.68 (s, 2H), 6.92−6.96

(S)-N-(1-(Hydroxyamino)-3-methyl-1-oxobutan-2-yl)-4-

1

(m, 2H), 3.92−4.54 (m, 6H), 1.20−1.95 (m, 9H), 0.89−0.92 (m,

3H). ¹³C NMR (DMSO-d₆): δ = 173.32, 166.74, 161.44, 128.45,

125.18, 111.14, 68.84, 62.30, 56.14, 31.12, 29.11, 24.13, 22.59, 14.13.

MS (ESI) m/z: calcd for C₁₆H₂₃NO₅, 310.1667 (100%, (M + H⁺)).

(4-Hexyloxy-benzoylamino)-acetic Acid (7e). The procedure is

similar to that applied for the preparation of 7a.

(pentyloxy)benzamide (4e). White solid. H NMR (DMSO-d₆): δ =

0.90−0.98 (m, 9H), 1.52−1.62 (m, 2H), 1.77−1.83 (m, 2H), 2.16−

2.22 (m, 2H), 3.97−4.02 (m, 2H), 4.37−4.42 (m, 1H), 7.75−7.77

(m, 2H), 7.98−8.02 (m, 2H), 8.53 (m, 1H). ¹³C NMR (DMSO-d₆):

δ = 169.71, 167.51, 162.83, 132.86, 126.86, 119.98, 68.72, 58.13,

31.14, 30.98, 29.34, 28.14, 18.54, 14.16. MS (ESI) m/z: calcd for

C₁₇H₂₆N₂O₄, 323.1934 (100%, (M + H⁺)).

White solid. ¹H NMR (DMSO-d₆): δ = 7.68 (s, 2H), 6.98 (s, 2H),

4.10−4.12 (m, 2H), 3.78 (m, 2H), 1.49−1.76 (m, 8H), 0.89−0.92

(m, 3H). ¹³C NMR (DMSO-d₆): δ = 172.31, 168.28, 161.02, 130.06,

114.55, 114.53, 66.42, 42.85, 34.56, 32.12, 29.14, 23.12, 21.45, 14.14.

MS (ESI) m/z: calcd for C₁₅H₂₁NO₄, 280.1511 (100%, (M + H⁺)).

(S)-2-(4-Methoxy-benzoylamino)-3-methyl-butyric Acid (7f).

The procedure is similar to that applied for the preparation of 7a.

White solid. ¹H NMR (DMSO-d₆): δ = 7.53−7.55 (m, 2H),

6.95−6.97 (m, 2H), 6.05 (s, 1H), 4.75−4.77 (m, 1H), 3.72 (s, 3H),

1.75−1.87 (m, 1H), 1.04−1.06 (m, 6H). ¹³C NMR (DMSO-d₆): δ =

172.33, 167.28, 160.14, 131.12, 127.30, 110.15, 58.66, 55.65, 30.93,

19.03. MS (ESI) m/z: calcd for C₁₃H₁₇NO₄, 251.1232 (100%, (M +

H⁺)).

(S)-4-(Heptyloxy)-N-(1-(hydroxyamino)-3-methyl-1-oxobutan-2-

1

yl)benzamide (4f). White solid. H NMR (DMSO-d₆): δ = 0.90−

0.96 (m, 9H), 1.28−1.74 (m, 10H), 2.02−2.16 (m, 1H), 3.97−4.07

(m, 2H), 4.37−4.42 (m, 1H), 7.03−7.08 (m, 2H), 8.02−8.09 (m,

2H), 8.53 (m, 1H). ¹³C NMR (DMSO-d₆): δ = 171.09, 166.42,

162.02, 130.07, 129.03, 115.18, 68.15, 57.63, 31.80, 29.18, 26.04,

22.14, 18.74, 13.98. MS (ESI) m/z: calcd for C₁₉H₃₀N₂O₄, 351.2245

(100%, (M + H⁺)).

(S)-N-(1-(Hydroxyamino)-3-methyl-1-oxobutan-2-yl)-4-

1

(octyloxy)benzamide (4g). White solid. H NMR (DMSO-d₆): δ =

0.90−0.97 (m, 9H), 1.29−1.82 (m, 12H), 2.06−2.15 (m, 1H), 3.95−

4.07 (m, 2H), 4.32−4.41 (m, 1H), 7.00−7.08 (m, 2H), 8.01−8.10

(m, 2H), 8.51 (m, 1H). ¹³C NMR (DMSO-d₆): δ = 171.10, 166.35,

162.35, 131.37, 129.90, 115.11, 68.24, 57.52, 31.79, 30.95, 29.31,

26.82, 22.47, 18.63, 14.21. MS (ESI) m/z: calcd for C₂₀H₃₂N₂O₄,

365.2423 (100%, (M + H⁺)).

(S)-2-(4-Hexyloxy-benzoylamino)-3-methyl-butyric Acid (7a). In

a 25 mL round ﬂask was placed at room temperature 0.6 g of 6 (1.78

× 10⁻³mol) in 15 mL of ethanol. A sodium hydroxide solution

(10%, 2 mL) was subsequently added, and the mixture was allowed

to stir at room temperature for 24 h. The bottom phase layer was

discarded, and the aqueous phase was acidiﬁed with HCl (1 N).

After extraction with ethyl acetate, the organic phase was dried over

Na₂SO₄, ﬁltered, and concentrated in vacuo. The crude residue was

puriﬁed by chromatography on a silica gel column using petroleum

ether/ethyl acetate (1/1) as the eluent, aﬀording the expected

product 7a in 69% yield.

(S)-2-(4-Ethoxy-benzoylamino)-3-methyl-butyric Acid (7g). The

procedure is similar to that applied for the preparation of 7a.

1

White solid. H NMR (DMSO-d₆): δ = 7.59 (s, 2H), 7.02−7.05

(m, 2H), 4.16−4.40 (m, 3H), 1.87−1.92 (m, 1H), 1.32−1.36 (m,

3H), 0.98−1.03 (m, 6H). ¹³C NMR (DMSO-d₆): δ = 173.15,

167.10, 160.83, 129.98, 127.04, 113.21, 61.89, 57.93, 29.98, 19.19,

14.31. MS (ESI) m/z: calcd for C₁₄H₁₉NO₄, 266.1342 (100%, (M +

H⁺)).

(S)-3-Methyl-2-(4-propoxy-benzoylamino)-butyric Acid (7h).

The procedure is similar to that applied for the preparation of 7a.

1

White solid. H NMR (DMSO-d₆): δ = 6.95−7.32 (m, 4H), 6.75

(s, 1H), 3.98−4.05 (m, 3H), 1.54−1.89 (m, 3H), 1.12−1.17 (m,

3H), 0.97−1.02 (m, 6H). ¹³C NMR (DMSO-d₆): δ = 172.34,

167.42, 160.15, 128.64, 127.42, 112.45, 69.37, 58.62, 29.89, 21.12,

18.82, 10.29. MS (ESI) m/z: calcd for C₁₅H₂₁NO₄, 280.1534 (100%,

(M + H⁺)).

(4-(Heptyloxy)benzoyl)-^L-valine (7i). White solid. ¹H NMR

(DMSO-d₆): δ = 0.93−1.41 (m, 19H), 1.41−1.46 (m, 2H), 2.23−

2.35 (m, 1H), 3.87−3.92 (t, J = 5 Hz, 2H), 4.46−4.69 (m, 1H),

6.82−6.89 (m, 2H), 7.66−7.69 (m, 2H). ¹³C NMR (DMSO-d₆): δ =

175.24, 167.12, 162.78, 129.02, 114.39, 68.28, 57.84, 31.80, 31.27,

29.14, 29.07, 25.98, 22.64, 19.15, 17.92, 14.12. MS (ESI) m/z: calcd

for C₁₉H₂₉NO₄, 336.2165 (100%, (M + H⁺)).

White solid. 1H NMR (CDCl₃): δ = 7.80−7.84 (m, 2H), 6.95−

7.04 (m, 2H), 4.44−4.46 (m, 1H), 4.02−4.05 (m, 2H), 2.19−2.35

(m, 1H), 1.73−1.84 (m, 2H), 1.43−1.57 (m, 2H), 1.26−1.35 (m,

5H), 1.04 (s, 3H), 1.03 (s, 3H), 0.91−0.94 (m, 3H). ¹³C NMR

(CDCl₃): δ = 174.59, 168.63, 162.17, 128.97, 125.94, 113.84, 67.86,

58.88, 31.35, 30.54, 28.87, 25.42, 22.27, 18.43, 17.52, 12.98. MS

(ESI) m/z: calcd for C₁₈H₂₇NO₄, 322.1932 (100%, (M + H⁺)).

(R)-2-(4-Hexyloxy-benzoylamino)-3-methyl-butyric Acid (7a′).

The procedure is similar to that applied for the preparation of 7a.

(4-(Octyloxy)benzoyl)-^L-valine (7j). White solid. ¹H NMR

(DMSO-d₆): δ = 0.91−1.34 (m, 10H), 1.38−1.54 (m, 11H),

1.76−1.87 (m, 2H), 2.23−2.37 (m, 1H), 4.0.3−4.08 (t, J = 5 Hz,

2H), 4.48−4.52 (dd, J = 5 Hz, 1H), 6.98−7.01 (m, 2H), 7.83−8.86

(m, 2H). ¹³C NMR (DMSO-d₆): δ = 175.31, 170.16, 163.59, 130.46,

1

White solid. H NMR (CDCl₃): δ = 6.55−7.19 (m, 4H), 3.89−

3.99 (m, 3H), 1.50−2.11 (m, 7H), 0.89−1.32 (m, 11H). ¹³C NMR

(CDCl₃): δ = 177.56, 166.34, 159.95, 132.36, 130.12, 114.08, 72.30,

K

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127.28, 115.21, 69.24, 59.88, 33.04, 31.82, 30.53, 30.46, 30.34, 27.19,

23.77, 19.79, 18.97, 14.49. MS (ESI) m/z: calcd for C₂₀H₃₁NO₄,

350.2315 (100%, (M + H⁺)).

solution for 20 min, then washed again, and stained with 0.35% Oil

Red O solution (Sigma-Aldrich) in 60% isopropanol for 20 min.

Then, cells were washed with water, and photographs were taken.

The stain from the cells was eluted using 100% isopropanol, and the

absorbance of the eluted stain was read at 490 nm. Experiments were

performed ﬁve times in duplicate for cell lines and twice in triplicate

for primary cells.

AdipoRed assay. 3T3-L1 preadipocytes were cultured in a black

96-well plate. Two days post-conﬂuency, cells were incubated with

the test molecules (10 μM) for 7 additional days. Cells were washed

with PBS, and then a solution of AdipoRed reagent (1/40 in PBS)

was added into each well. After 10 min at room temperature and in

the dark, ﬂuorescence was measured at 485 and 572 nm for

excitation and emission, respectively, on an EnSight microplate

reader (PerkinElmer) in the scanning mode. Since this technique was

used for screening purposes, the experiment was performed once in

duplicate.

2-Deoxy-^D-glucose Uptake Assay. Glucose uptake activity of

fully diﬀerentiated 3T3-L1 adipocytes was measured by the

chemiluminescent assay⁴³using a Glucofax kit as described by the

manufacturer (Yelen, Ensues la Redonne, France). Brieﬂy, 3T3-L1

adipocytes (80% of the cells displaying the characteristic lipid-ﬁlled

phenotype) were gently detached from the plate by Accutase

(Thermo Fisher Scientiﬁc) treatment, seeded (3.5 × 10⁴cells well⁻¹)

at conﬂuence in 96-well culture dishes, and cultured for 2 more days

in DMEM supplemented with 10% FBS. Adipocytes were then

incubated in glucose-free DMEM for 4 h, then washed twice with

Krebs-Ringer-phosphate-Hepes (KRPH) buﬀer (20 mM Hepes, 5

mM KH₂PO₄, 1 mM MgSO₄, 1 mM CaCl₂, 136 mM NaCl, and 4.7

mM KCl at pH 7.4) containing 0.2% BSA, and incubated 30 min in

100 μL of KRPH/BSA. KRPH buﬀer was removed, and cells were

incubated for 20 min with 170 μL of 100 nM insulin diluted in

KRBH buﬀer; then, 19 μL of 10 mM 2-deoxy-^D-glucose (Sigma-

Aldrich) was added and the cells were incubated for 20 min. Cells

were then washed four times with cold PBS and lysed with 60 μL of

reagent I of the Glucofax kit. After 60 min of incubation at 37 °C, 20

μL of cell lysates was collected and transferred into a white 96-well

plate. Then, 100 μL of reagent II was added, and after 10 min of

incubation, the chemiluminescence was recorded on the EnSight

multimode reader (PerkinElmer). Experiments were performed three

times in sextuplet.

Real-Time PCR Analysis. Total RNA was extracted using a

NucleoSpin RNA kit (Macherey-Nagel, Hoerdt, France), and cDNA

was synthesized from 0.5 μg of RNA using Moloney murine leukemia

virus reverse transcriptase (Thermo Fisher Scientiﬁc) and used for

PCR ampliﬁcation. Real-time PCR (RT-PCR) was performed on the

LightCycler 480 instrument (Roche Applied Science, Basel, Switzer-

land) using the EvaGreen master mix (Euromedex, Souﬀelweyer-

sheim, France). The comparative Ct method (2^−(ΔΔCt)) was used to

calculate the relative diﬀerences in mRNA expression. The acidic

ribosomal phosphoprotein P0 (Rplp0) was used as the housekeeping

gene. Changes were normalized to the mean of control values, which

was set to 1. Primers were synthesized by Eurogentec (Seraing,

Belgium), and their sequences were previously published.^17,36,42

Experiments were performed ﬁve times in duplicate.

(4-(Decyloxy)benzoyl)-^L-valine (7k). White solid. ¹H NMR

(DMSO-d₆): δ = 0.92−1.32 (m, 10H), 1.44−1.54 (m, 15H),

1.82−1.88 (m, 2H), 2.33−2.46 (m, 1H), 3.94−4.06 (m, 2H), 4.82−

4.87 (m, 1H), 6.86−6.94 (m, 2H), 7.73−7.83 (m, 2H) ¹³C NMR

(DMSO-d₆): δ = 175.36, 167.71, 162.20, 129.31, 129.10, 125.71,

114.33, 114.15, 68.25, 57.62, 31.94, 31.43, 29.61, 29.43, 29.37, 29.16,

26.03, 22.73, 19.09, 17.91, 14.18. MS (ESI) m/z: calcd for

C₂₂H₃₅NO₄, 378.2645 (100%, (M + H⁺)).

Chemicals. Rosiglitazone, bezaﬁbrate, and roscovitine were from

Sigma-Aldrich (Saint-Quentin-Fallavier, France). GW 7647 was from

Axon Medchem (Groningen, The Netherlands). GW 9662 and SR

11235 were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Antibodies. PPARγ antibody (E-8) was from Santa Cruz

Biotechnology. PPARγ phospho Ser273 antibodies were from

Rockland (Limerick, PA, USA) or custom produced by New

England Peptide (Gardner, MA, USA). UCP1 antibodies

(ab10983) were from Abcam (Cambridge, MA, USA). β-actin

(13E5) was from Cell Signaling Technology (Danvers, MA, USA).

Cell Culture. Media and cell culture reagents were from Thermo

Fisher Scientiﬁc (Illkirch-Graﬀenstaden, France). HEK293 cells

(GripTite 293 MSR) were from Thermo Fisher Scientiﬁc. 3T3-L1

cells (from ATCC) were routinely cultured in DMEM with 4 mM ^L-

glutamine, 4.5 g L⁻¹glucose, and 0.11 g L⁻¹sodium pyruvate and

supplemented with 10% fetal bovine serum and antibiotics. Two days

after conﬂuence, adipocyte diﬀerentiation was triggered by changing

the conventional induction mixture (0.1 μM dexamethasone, 500 μM

3-isobutyl-1-methylxanthine, and 174.5 nM insulin from Sigma-

Aldrich, L’Isle d’Abeau Chesnes, France). After 48 h, the medium

was removed and replaced by a fresh medium containing only 174.5

nM insulin. HuH7 hepatoma cells from the Japanese Cancer

Research Resources Bank were cultured in DMEM containing 10%

fetal bovine serum (FBS). At the conﬂuence, cells were treated for 24

h with 0.25 mM oleic acid (Sigma-Aldrich) complexed with BSA.

The establishment, characterization, and culture protocols of human

multipotent adipose-derived stem (hMADS) cells have been

described previously.⁴²Brieﬂy, conﬂuent cells were submitted to

diﬀerentiation medium (DMEM/Ham’s F12 media containing 10 μg

mL⁻¹transferrin, 10 nM insulin, and 0.2 nM triiodothyronine from

Sigma-Aldrich) supplemented with 1 μM dexamethasone and 500

μM isobutyl-methylxanthine. Two days later, the medium was

changed, dexamethasone and isobutyl-methylxanthine were omitted,

and 100 nM Rosi (Sigma-Aldrich) was added for the indicated

periods. Cells were treated between days 2 and 9 with Rosi to enable

white adipocyte diﬀerentiation to take place. After 5 days in the

absence of Rosi, brite adipocyte conversion was induced by adding

compounds to be tested (day 14). The medium was changed every

other day, and cells were used at day 18. Pooled plateable

cryopreserved primary rat hepatocytes (Male, Sprague−Dawley)

from XenoTech (Kansas city, KS, USA) were plated and cultured as

described by the manufacturer.

Cell Viability, Toxicity, and Apoptosis Assay. Cell viability

was monitored using the CellTiter-Blue cell viability assay (Promega,

Madison, WI) based on the ability of living cells to convert resazurin

into the ﬂuorescent end product resoruﬁn and by measuring cellular

ATP levels, which decline rapidly when cells undergo necrosis or

apoptosis, using an ATPLite 1step luminescence assay system

(PerkinElmer, Waltham, MA, USA) according to the manufacturer’s

instructions. Cytotoxicity was determined by measuring the lactate

dehydrogenase (LDH) released into the culture medium upon

plasma membrane damage using the LDH-Glo cytotoxicity assay

(Promega). Apoptosis was determined by measuring caspase 3/7-

dependent cleavage of a luminogenic substrate using the Caspase-Glo

3/7 Assay (Promega). Experiments were performed two to three

times in triplicate.

MitoTracker Staining. 3T3-L1 adipocytes were trypsinized and

centrifuged at 300g at 4 °C for 5 min. Cells were suspended in

KRPH containing 0.5% BSA and incubated with 0.1 μM MitoTracker

Green FM (Thermo Fisher Scientiﬁc) for 30 min at 37 °C. Cells

were spun at 300g at 4 °C for 5 min and suspended in 400 μL of

fresh KRPH; then, 50,000 cells were analyzed using a BD Accuri C6

ﬂow cytometer (BD Biosciences). Experiments were performed three

times in triplicate.

Cell-Based PPAR and RXRα Transactivation Assay. The

PPARγ-LBD-Gal4 or PPARα-LBD-Gal4 expression vector (given by

Dr. Teruo Kawada, Kyoto University, Japan) was transfected along

with the SV40-driven Renilla luciferase expression vector in HEK293

cells stably expressing the Gal4 response element-driven Fireﬂy

luciferase reporter (pGL4.35[luc2P/9XGAL4UAS/Hygro] vector

from Promega, Madison, WI, USA). Thirty-six hours after trans-

Cellular Lipid Content Staining and Measurement. Oil Red

O staining. 3T3-L1 cells, HuH7 hepatoma cells, or primary rat

hepatocytes were washed with PBS and ﬁxed with 4% formaldehyde

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fection, cells were exposed to the tested compounds for additional 16

h; then, Fireﬂy and Renilla luciferase activities were measured in the

cell lysates using the reagents Genofax A and C (Yelen) in an

EnSight multimode reader (PerkinElmer). PPAR transactivation

activity of the compounds is calculated as the ratio of Fireﬂy to

Renilla luciferase activity. Experiments were performed at least three

times in duplicate (allowing the calculation of EC₅₀and maximal

activation, Table 1), and a representative experiment is shown in

Figure 3 and Figure S5. For the measure of PPARδ transactivation,

HG5LN-GAL-PPARδ reporter cell line was previously described.⁴⁴

Reporter cells were seeded at a density of 20,000 cells well⁻¹in 96-

well white opaque tissue culture plates and maintained in phenol red-

free Dulbecco’s modiﬁed Eagle’s medium (DMEM) supplemented

with 5% dextran-coated, charcoal-treated fetal calf serum. Twenty-

four later, the culture medium was replaced with DMEM containing

tested compounds. After 16 h, exposure media were replaced with

media containing 0.3 mM luciferin. Luminescence was measured in

intact living cells for 2 s in a MicroBeta Wallac luminometer

(PerkinElmer). Experiments were performed twice in duplicate. The

measurement of RXRα transactivation was performed as described

for PPARγ except that the RXRα-LBD-Gal4 expression vector was

transfected.

Mammalian Two-Hybrid Assay. HEK293 cells stably express-

ing the GAL4 response element-driven Fireﬂy luciferase reporter

were transfected with the SV40-driven Renilla luciferase expression

vector together with the expression vector of a fusion protein of the

VP16 activation domain to PPARγ-LBD and with the expression

vector of a fusion protein of the GAL4-DNA binding domain to

NCoR1, SMRT, TIF2, MED1, or RXRα. Then, cells were treated as

described for the cell-based PPAR transactivation assay. Experiments

were performed two to three times in duplicate.

Immunoblot. 3T3-L1 adipocytes were incubated 24 h with 1%

SVF and then treated with PPARγ agonists for 60 min followed by

90 min of stimulation with murine TNF (50 ng mL⁻¹). Cells were

lysed in the presence of a cocktail of protease and phosphatase

inhibitor. Identical amounts of total protein were heat-denatured and

reduced (70 °C; 10 min), then submitted to SDS-PAGE separation

on 4−12% gradient (Thermo Fisher Scientiﬁc), and transferred to

polyvinylidene ﬂuoride membranes. Membranes were blocked for 1 h

in 5% BSA solution and incubated with the appropriate primary and

HRP-conjugated secondary antibodies (1/1000 and 1/10,000

dilutions, respectively). Immunodetections were performed using

the ECL reagent, and image acquisition was performed by using a

chemiluminescent CCD imager ImageQuant LAS 4000 (GE

Healthcare, Velizy-Villacoublay, France).

Measure of In Vitro PPARγ Phosphorylation. In vitro

phosphorylation was performed on wild-type PPARγ in the apo

form and in complex with 7j and Rosi. Stock solutions of ligands

were prepared by diluting with 100% DMSO to a concentration of

50 mM. The stock solutions were further diluted with 50 mM Tris−

HCl (pH 7.5) up to the ﬁnal concentrations of 0.1, 1, and 10 μM

and pre-equilibrated overnight at 4 °C with the protein.

Phosphorylation was carried out at 30 °C for 3.5 h in 300 μL of

buﬀer containing 50 mM Tris−HCl (pH 7.5), 7.2 μg mL⁻¹PPARγ,

0.1 μM, 1 μM, 10 μM ligand, 25 mM MgCl₂, 50 μM DTT, 2 mM

ATP, and 0.66 ng mL⁻¹CDK5/p35 (Sigma-Aldrich code no.

SRP5011). Then, polystyrene microwell plates were coated overnight

at 4 °C with the reaction mixture, then washed three times with PBS

+ Tween 0.005%, and left to block in PBS containing 1% bovine

serum for 90 min at 37 °C. The wells were washed three times and

incubated for 60 min at 37 °C with 100 μL of antiphospho-Ser/The-

Pro antibody (SigmaAldrich code no. A05368) diluted 1/500 in PBS.

After three washes, 100 μL of antimouse IgG-peroxidase antibody

produced in goat (Sigma Aldrich code no. A4416; 1/1000 in PBS)

were added to the wells and incubated 60 min at 37 °C. The wells

were washed, and 200 μL of o-phenylenediamine dihydrochloride

(SIGMAFAST OPD code no. P9187) dissolved in water was added

to the wells. Optical density was measured at 450 nm using an

ApplyScan Thermo Fisher reader, and the data were processed using

Excel.

Experiments were performed three times in triplicates.

Protein Expression and Puriﬁcation. PPARγ LBD was

expressed as N-terminal His-tagged proteins using a pET28 vector

and puriﬁed as previously described.⁴⁵Brieﬂy, freshly transformed

Escherichia coli BL21 DE3 were grown in the LB medium with 30 μg

of kanamycin mL⁻¹at 310 K to an OD of 0.6. The culture was then

induced with 0.2 mM isopropyl-β-^D-thio-galactopyranoside and

further incubated at 291 K for 20 h. Cells were harvested and

resuspended in a 20 mL L⁻¹culture of buﬀer A (20 mM Tris−HCl,

150 mM NaCl, 10% glycerol, 1 mM Tris 2-carboxyethylphosphine

HCL (TCEP), pH 8) in the presence of protease inhibitors

(cOmplete Mini EDTA-free; Roche Applied Science). Cells were

sonicated, and the soluble fraction was isolated by centrifugation

(35,000g for 45 min). The supernatant was loaded onto a Ni²⁺-

nitrilotriacetic acid column (GE Healthcare) and eluted with a

gradient of imidazole 0−500 mM in buﬀer A (20 mM Tris−HCl, 20

mM NaCl, 10% glycerol, 1 mM TCEP, pH 8) (batch method). The

pure protein was identiﬁed by SDS PAGE. The protein was then

dialyzed over buﬀer A to remove imidazole, and it was cleaved with

thrombin protease (Sigma-Aldrich Life Science) (10 units mg⁻¹) at

room temperature for 2 h. The digested mixture was reloaded onto a

Ni²⁺-nitrilotriacetic acid column to remove the His tag and the

undigested protein. The ﬂow-through was dialyzed with buﬀer B (20

mM Tris−HCl, 10% glycerol, 1 mM TCEP, pH 8) to remove NaCl,

loaded onto a Q Sepharose HP column (GE Healthcare), and eluted

with a gradient of NaCl 0−500 mM in buﬀer B with a BioLogic

DuoFlow FPLC system (Bio-Rad Laboratories, Italy). Finally, the

protein was puriﬁed by gel ﬁltration chromatography on a HiLoad

Superdex 75 column (GE Healthcare) and eluted with buﬀer C (20

mM Tris−HCl, 1 mM TCEP, 0.5 mM EDTA, pH 8). The protein

was then concentrated at 8 mg mL⁻¹using Amicon centrifugal

concentrators with a 10 kDa cutoﬀ membrane (Millipore, USA).

Crystallization and Data Collection. Crystals of apo-PPARγ

were obtained by vapor diﬀusion at 18 °C using a sitting drop made

by mixing 2 μL of protein solution with 2 μL of reservoir solution

(0.8 M Na citrate, 0.15 M Tris, pH 8.0). The crystals were soaked for

3 days in a storage solution (1.2 M Na citrate, 0.15 M Tris, pH 8.0)

containing the ligand 7j or 7a (0.5 mM). The ligand dissolved in

DMSO (50 mM) was diluted in the storage solution so that the ﬁnal

concentration of DMSO was 1%. The storage solution with glycerol

20% (v/v) was used as the cryoprotectant. Crystals (0.15 × 0.15

mm) of PPARγ/7j and PPARγ/7a belong to the space group C2

with cell parameters shown in Table S1.

Structure Determination and Reﬁnement. X-ray data sets

were collected at 100 K under a nitrogen stream using synchrotron

radiation (beamline ID30B at ESRF, Grenoble, France). The

collected data were processed using the programs Mosﬂm and

Scala.⁴⁶Structure solution was performed with AMoRe,⁴⁷using the

coordinates of PPARγ/LT175R (27) (PDB code: 3D6D) as the

starting model. The coordinates were then reﬁned with CNS⁴⁸and

with PHENIX⁴⁹including data between 58.2 and 2.0 Å for PPARγ/

7j (57.4−1.8 Å for PPARγ/7a). The statistics of crystallographic data

and reﬁnement is summarized in Table S1. The coordinates and

structure factors described here have been deposited in the PDB

under accession numbers 6QJ5 and 6ZLY for PPARγ/7j and

PPARγ/7a, respectively.

Surface Plasmon Resonance. Surface plasmon resonance

analyses were performed by using a Pioneer AE optical biosensor

equipped with COOH5 chips (SensiQ). PPARγ surfaces were

prepared by using standard amine-coupling procedures⁵⁰and HBS

(Hepes-buﬀered saline: 10 mM Hepes, 150 mM NaCl, 0.005% P20,

DMSO 1%, pH 7.4) as the running buﬀer. Flow cells were activated

for 7 min by injecting 140 μL of 50 mM N-hydroxysuccinimide

(NHS)/200 mM ethyl-3(3-dimethylamino) propylcarbodiimide

(EDC). A 0.25 mg mL⁻¹PPARγ solution (150 μL, in 10 mM

NaOAc, pH 5.0) was injected for 15 min at 10 μL min⁻¹on channels

1 and 3 (channel 2 was used as the reference, for a duplicate

experiment) followed by a 70 μL injection of ethanolamine to block

any remaining activated groups on the surface. RU (11,320 and

11,220) of protein was immobilized on channels 1 and 3,

M

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J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

respectively. The screening of the analytes was performed using HBS,

with 1% DMSO. To collect details, a dilution protocol was used,

injecting diﬀerent concentrations of the analytes at a ﬂow rate of 50

μL min⁻¹over the two channels at 20 °C (association phase of 60 s).

A similar protocol was followed for the experiments with PPARα,

using a PCH chip (Pall ForteBio).

Four buﬀer blanks were injected for double referencing. The

regeneration of the surfaces between binding cycles was not

necessary because all the analytes dissociate quickly in the 120 s

dissociation phase. A DMSO calibration plot was constructed (buﬀer

sample containing 0−2% (vol/vol) DMSO) to correct for bulk

refractive index shifts. All sensorgrams were processed by using

double referencing. To obtain kinetic rate constants and aﬃnity

constants, the corrected response data were ﬁt in the program

QDAT. A kinetic analysis of each ligand/analyte interaction was

obtained by ﬁtting the response data to a 1/1 bimolecular interaction

model. The equilibrium dissociation constant (K_d) was determined

by the ratio of k_off/k_on.

Robert G. Roeder − Laboratory of Biochemistry and Molecular

Biology, The Rockefeller University, New York, New York

10065, United States

Giorgio Pochetti − Istituto di Cristallograﬁa, Consiglio

Nazionale delle Ricerche, 00015 Rome, Italy; orcid.org/

0000-0002-3980-3180

Jean Michel Brunel − Aix Marseille University, INSERM, SSA,

MCT, 13385 Marseille, France; orcid.org/0000-0002-

9355-8980

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.0c01555

Author Contributions

^∇F.P. and R.M. contributed equally to this work; G.P. and

J.M.B. contributed equally to this work.

Notes

Statistical Analyses. Statistical signiﬁcance was estimated with

one-way ANOVA followed by the Bonferroni or Dunnett post-hoc

test, with the two-tailed t-test or with the F-test (statistical test is

speciﬁed in ﬁgure legends) using GraphPad Prism version 5.0

(GraphPad Software, San Diego, CA). Diﬀerences with p values of

less than 0.05 were considered statistically signiﬁcant.

The authors declare no competing ﬁnancial interest.

Coordinates and structure factors of the PPARγ complexes

with the compounds 7j and 7a have been deposited in the

Protein Data Bank under the accession codes 6QJ5 and 6ZLY,

respectively. Authors will release the atomic coordinates and

experimental data upon article publication.

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01555.

Genes are identiﬁed by the symbols approved by the Human

Genome Organization.

■

sı

ACKNOWLEDGMENTS

■

This work was supported by funds from Aix Marseille

University, Inserm, CNRS, ANR and National Institutes of

Health grant DK071900 to R.G.R.

PDB data of the compounds (PDB)

NMR and HPLC spectra of 3a, 7a, and 7j; X-ray crystal

structures of 3a, 7a, 7j, rosiglitazone and SR2067 in

PPARγ LBD; SPR sensorgrams of 3a, 7a, and 7j;

adipogenic eﬀect of the compounds; PPARγ, PPARδ,

and RXRα transactivation; PPARγ/RXRα heterodime-

rization; toxicity of 7j; statistics of crystallographic data

and reﬁnement (PDF)

ABBREVIATIONS

■

BOP, benzotriazol-1-yloxytris(dimethylamino)phosphonium

hexaﬂuorophosphate; CDK5, cyclin-dependent kinase 5;

hMADS, human multipotent adipose-derived Stem; LBD,

ligand binding domain; NAFLD, nonalcoholic fatty liver

disease; PPRE, PPAR responsive element; Rosi, rosiglitazone;

SPPARγMs, selective PPARγ modulators

Molecular formula string for 3a, 7a, and 7j (CSV)

AUTHOR INFORMATION

Corresponding Author

Franck Peiretti − Aix Marseille University, INSERM, INRAE,

C2VN, 13385 Marseille, France; orcid.org/0000-0001-

7198-0534; Email: franck.peiretti@univ-amu.fr

■

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A Novel N-Substituted Valine Derivative with Unique Peroxisome Proliferator-Activated Receptor γbinding Properties and Biological Activities

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