Constraining the Side Chain of C-Terminal Amino Acids in Apelin-13 Greatly Increases Affinity, Modulates Signaling, and Improves the Pharmacokinetic Profile

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Constraining the Side Chain of C‑Terminal Amino Acids in Apelin-13

Greatly Increases Aﬃnity, Modulates Signaling, and Improves the

Pharmacokinetic Proﬁle

̂

Kien Tran, Robin Van Den Hauwe, Xavier Sainsily, Pierre Couvineau, J é rome Cot é , Louise Simard,

Marco Echevarria, Alexandre Murza, Alexandra Serre, L é a Th é roux, Sabrina Saibi, Lounes Haroune,

̀

Jean-Michel Longpr é , Olivier Lesur, Mannix Auger-Messier, Claude Spino, Michel Bouvier,

Philippe Sarret,* Steven Ballet, and Eric Marsault

́

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ABSTRACT: Side-chain-constrained amino acids are useful tools

to modulate the biological properties of peptides. In this study, we

applied side-chain constraints to apelin-13 (Ape13) by substituting

the Pro12 and Phe13 positions, aﬀecting the binding aﬃnity and

signaling proﬁle on the apelin receptor (APJ). The residues 1Nal,

Trp, and Aia were found to be beneﬁcial substitutions for Pro12,

and the resulting analogues displayed high aﬃnity for APJ (K_i

0.08−0.18 nM vs Ape13 K_i0.7 nM). Besides, constrained (D-Tic)

or α,α-disubstituted residues (Db_zg; D-α-Me-Tyr(OBn)) were

favorable for the Phe13 position. Compounds 47 (Pro12-Phe13 replaced by Aia-Phe, K_i0.08 nM) and 53 (Pro12-Phe13 replaced by

1Nal−Db_zg, K_i0.08 nM) are the most potent Ape13 analogues activating the Gα₁₂pathways (53, EC₅₀Gα₁₂2.8 nM vs Ape13, EC₅₀

43 nM) known to date, displaying high aﬃnity, resistance to ACE2 cleavage as well as improved pharmacokinetics in vitro (t_1/25.8−

7.3 h in rat plasma) and in vivo.

Human apelin peptides include Pyr-apelin-13, apelin-17, and

apelin-36, all derived from the proteolytic cleavage of a 77-mer

prepropeptide, with Pyr-apelin-13 (Ape13) being predominant

in both heart and plasma.^20,21The downstream signaling

cascades triggered upon APJ activation are highly complex, and

their physiological impacts are not fully understood.¹¹The

apelin receptor mediates its physiological responses through

the Gα_i/o(in particular Gα_i1and Gα_i2) and Gα_12/13

proteins.^11,22The Gα_i-dependent pathway primarily inhibits

cyclic adenosine monophosphate (cAMP) production and

activates the extracellular-signal-regulated kinase (ERK) signal-

ing cascade.^4,23Several studies suggest that the Gα_ipathway

and ERK phosphorylation could correlate with some

bioactivities of the apelinergic ligand, such as adipogenesis

suppression,²⁴body ﬂuid homeostasis,²⁵or protective eﬀects in

brain ischemia/reperfusion injury;²⁶however, a direct link

between those eﬀects and Gα_iactivation still needs to be

demonstrated. The Gα_12/13pathway was also reported to be

INTRODUCTION

■

Both apelin and the apelin receptor (APJ), a G-protein-

coupled receptor (GPCR), have gained signiﬁcant interest due

to their involvement in a wide array of physiological processes

like body ﬂuid homeostasis, hypothalamic−pituitary−adrenal

(HPA) axis regulation, and carbohydrate and lipid metabo-

lism.¹⁻⁵To date, potential applications of the apelinergic

system have been largely centered on cardiovascular actions.

As a strong inodilator, apelin exerts potent hypotensive action

and positive inotropic eﬀects, which concertedly reduce

peripheral vascular resistance and increase cardiac output.⁶⁻¹¹

Apelin and its receptor are widely distributed in the vasculature

and peripheral organs, including kidneys, lung, and heart.¹¹⁻¹⁵

Immunocytochemical localization revealed the presence of a

high expression of APJ on human cardiomyocytes, vascular

smooth muscle cells, and endothelial cells.¹⁶In the central

nervous system, it is mainly expressed in the hypothalamic

supraoptic and paraventricular nuclei, critical for body ﬂuid

homeostasis.^17,18Even though much research has been

dedicated to the apelinergic system, to date no apelin

(receptor)-based pharmaceuticals are on the market, despite

having been covered in multiple patents to date.¹⁹The

majority of patents report the discovery of small-molecule

agonists, yet clinical validation remains sparse and continued

research is needed.¹⁹

Received: November 9, 2020

Published: February 1, 2021

https://dx.doi.org/10.1021/acs.jmedchem.0c01941

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Figure 1. Conformationally constrained amino acids used in this study. Most unnatural amino acids were synthesized according to known

procedures. Tic and ^D-Tic were purchased from commercial sources.

activated by apelin, leading to the activation of the MEF2

transcription factor and playing an important role in

cardiovascular development in mice.²²Besides, recent

discovery showed that its signaling is inﬂuenced by the

formation of APJ homodimer-oligomers or even heterodimer

complexes with other GPCRs, such as kappa opioid (KOR),

neurotensin type 1 (NTS1), angiotensin II type 1 (AT1), or

bradykinin B2 (B2R) receptors.²⁷⁻³¹

Upon activation, APJ is rapidly phosphorylated by GPCR

kinases (GRK2 and GRK5), thereby increasing its aﬃnity for

β-arrestins.³²Chronic activation may lead to receptor internal-

ization due to the β-arrestin-mediated process, eventually

resulting in desensitization and loss of therapeutic eﬃcacy.³²

However, β-arrestin1/2 activation also plays an important role

in GPCR signaling, acting as a “multifunctional scaﬀold” to

recruit other intracellular signaling cascades.³³For APJ, β-

arrestin1/2 recruitment likely leads to the activation of the

eNOS/NO pathway^7,34,35and correlates with the hypotensive

action of apelin^36,37since ligands biased in disfavor of β-

arrestin recruitment abolish both eNOS activation and

vasodilation eﬀect.^34,37,38Other downstream signaling partners

have been reported, such as PI3K/Akt, FoxO1/3, AMPK,^11,39

and the BK_Capotassium channel,⁴⁰although their coupling

mechanisms and functions relative to APJ remain to be

elucidated.

Interestingly, rapid internalization is promoted via inter-

actions of the receptor with the C-terminal phenylalanine

residue of apelin-13 analogues.⁴¹Indeed, deletion of Phe13

biases the receptor in favor of the Gα_iover the β-arrestin

pathway.³⁷The corresponding Pyr-apelin-13[1−12] sequence

retains most of the native peptide biological activity, yet

structure−activity relationship (SAR) studies reveal that the

introduction of unnatural amino acids at the C-terminus of the

apelin peptide can signiﬁcantly improve aﬃnity and signaling

potency.⁴²

identiﬁed to cleave apelin. These include the angiotensin-

converting enzyme 2 (ACE2),^44,46prolyl carboxypeptidase

(PRCP),⁴⁷neprilysin,⁴⁸and human plasma kallikrein

(KLKB1).⁴⁹Excellent reviews discussing the biological

implications of apelin analogues and their cognate receptor

are available.^{11,19,50−56}Of relevance to the current work, C-

terminal modiﬁcations (Pro12 and Phe13) in apelin have been

shown to drastically impact binding aﬃnity and protease

stability in both plasma and cerebrospinal ﬂuid.^43,57The Phe13

C-terminus is the master switch for binding aﬃnity and

receptor activation.^37,38,42Its mutation into Ala reduces the

binding aﬃnity, and the resulting compound (F13A) could

antagonize the Ape13 eﬀect on blood pressure.³⁸Moreover,

previous work by our group has shown that replacing Phe13

with p-benzoyl-^L-phenylalanine (Bpa) or α-MePhe provides

potent analogues with aﬃnities in the sub-nM range and 30-

fold improvement in cAMP production inhibition. Addition-

ally, replacement of Phe13 with Tyr(OBn) resulted in the

highest aﬃnity APJ ligands reported to date (K_i0.02 nM).⁴²

For the Pro12 position, D-scan and Ala-scan showed that it

does not play an important role in ligand binding.^58,59

However, substitution in this position by proline mimetics

(Aib, Acpc) may aﬀect peptide stability since several enzymes

(ACE2, PRCP) cleave the amide bond between Pro12 and

Phe13.^44,58,59Altogether, the C-terminal extremity of Ape13

emerges as a critical determinant of aﬃnity, signaling, and

stability. To ﬁne-tune these properties and provide compounds

with further improved properties, we describe herein the

synthesis of modiﬁed Ape13 peptides in which the Pro12 and

Phe13 residues were substituted with a large set of unnatural,

conformationally constrained amino acids and measured the

impact of the modiﬁcations on the resulting pharmacological

properties.

The aﬃnity of modiﬁed analogues for APJ was determined

by a competitive radioligand binding assay against [¹²⁵I]

[Nle⁷⁵, Tyr⁷⁷]Pyr-apelin-13. Their ability to trigger the

activation of the Gα_i1, Gα₁₂, and β-arrestin2 pathways was

assessed using bioluminescence resonance energy transfer

Apelin and Elabela, the other endogenous ligands of the

apelin receptor, are prone to fast proteolytic degradation with

half-lives under 5 min in vivo.⁴³⁻⁴⁵Several proteases have been

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(BRET)-based biosensors using HEK293 cells.⁶⁰The

proteolytic stability of selected compounds was evaluated

separately against rat plasma and recombinant rat ACE2

enzyme. Finally, potent analogues were chosen for further

pharmacokinetic studies and an assessment on blood pressure

in vivo.

Scheme 2). Linkage of the aromatic side chain to the amine of

the next amino acid in the sequence not only allows χ-space

Scheme 2. Synthesis of Fmoc-Aia-Gly-OH 11a and Fmoc-

a

Aia-Phe 11b

RESULTS AND DISCUSSION

■

Synthesis. Unnatural amino acids were selected as close

analogues of Phe or Pro possessing side chains constrained in

diﬀerent ways (Figure 1). This includes the introduction of

substituents at the α- or β-position (e.g., α,α-dibenzylglycine

(Db_zg), α-MeTyr(OBn), β-MePhe), formation of a methylene

bridge with the α-amine of the peptide backbone (e.g., Tic,

Ana, or Aia derivatives), or side-chain derivatization (e.g., Bip,

cypTyr(OBn)), all synthesized according to known proce-

dures.⁶¹⁻⁶⁴The description of their syntheses can be found in

the Electronic Supporting Information section of this manu-

script (ESI, Sections 2 −4). Additionally, several unnatural

amino acids are reported for the ﬁrst time in this manuscript,

such as β-diMeTic (6), cypTyr(OBn) (15a), and dcypTyr-

(OBn) (15b). Their syntheses are described in the

Experimental Section. Moreover, a new asymmetric synthesis

for ^D-α-MeTyr(OBn) (22) is also presented.

a

(a) cat. H₂SO₄formalin, rt, 4 h, 49%; (b) Fmoc-OSu, Na₂CO₃,

acetone/H₂O, rt, 16 h, 71%; (c) SeO₂, dioxane, reﬂux, 2 h, 45%; (d)

NaCNBH₃, CH₂Cl₂, HCl.H₂N-Gly-OMe or HCl.H₂N-Phe-OMe,

NMM, AcOH, rt; (e) EDC HCl, ACN/H₂O, pyridine, rt, 48 h; (f)

1 N HCl/acetone, 90 °C, 16 h. 11a: 40% (three steps for 11a), 11b:

21% (three steps for 11b).

Constrained amino acids 5 and 6 were prepared starting

from a modiﬁed literature procedure (Scheme 1).⁶²First,

Scheme 1. Synthesis of Fmoc-β-dimethyl-Phe/Tic-OH (5/

a

6)

screening, it is also a great tool to increase metabolic stability

of the corresponding analogues.⁶³Hence, we synthesized three

diﬀerent azepinones-containing dipeptides Aia-Gly, Aia-Phe,

and 1-Ana-Gly (Figure 1) following known procedures.⁶⁴

Synthesis of the indoloazepinone-constrained Aia started by

ring-closing ^L-Trp using the Pictet−Spengler reaction, followed

by Fmoc-protection of the amine to deliver 9. Formation of the

corresponding aldehyde 10 enabled incorporation of a second

amino acid via reductive amination. After formation of the

secondary amine, ring closure using EDC·HCl by intra-

molecular coupling yielded the indoloazepinone scaﬀold.

Dipeptides 11a and 11b were obtained after ﬁnal ester

hydrolysis. The synthesis of Fmoc-1-Ana-Gly-OH (not shown)

was completely performed as described previously.⁶⁵

In past works, Tyr(OBn) substitution of Phe13 on Ape13

was shown to increase aﬃnity down to the pM level.⁴²For this

reason, the Tyr(OBn) residue was used as a scaﬀold to

implement conformational constraints. Constrained analogues

Tic(7-OBn) and m-Tyr(OBn) were easily prepared from

commercially available starting materials (Figure 1; see the

Supporting Information for syntheses). On the other hand,

analogues cypTyr(OBn) and dcypTyr(OBn) were synthesized

from unprotected ^L-tyrosine (Scheme 3A). First, 3′-C-

alkylation of ^L-Tyr 12 was performed with cyclopentanol in

H₃PO₄85% at 100 °C.⁶⁶This reaction yielded a mixture of

mono- and dialkylated products, cypTyr 13a and dcypTyr 13b,

which were separated after subsequent N-Alloc protection. O-

alkylation and hydrolysis were then carried out to deliver N-

Alloc-cypTyr(OBn) 15a and N-Alloc-dcypTyr(OBn) 15b,

used as building blocks in solid-phase synthesis. Peptidic

analogues containing dcypTyr(OH) were generated from

those having dcypTyr(OBn) by increasing the peptide cleavage

time up to 4 h with a mixture of triﬂuoroacetic acid (TFA)/

triisopropylsilane (TIPS)/H₂O (95:2.5:2.5).

a

(a) MeI, NaOH, THF rt, 89%; (b) SOCl₂, MeOH reﬂux, 90%; (c)

NH₂OH.HCl, MeOH, 65 °C, 94%; (d) Zn, 30% H₂SO₄, AcOH,

reﬂux, 66%; (e) 1 N HCl/acetone, reﬂux, 99%; (f) 12 N HCl,

formalin, reﬂux, 72%; (g) Fmoc-OSu, Na₂CO₃, acetone/water, rt,

76% for 5 and 71% for 6.

methylation at the β-position of commercial phenylpyruvic

acid 1 was performed using MeI and NaOH in tetrahydrofuran

(THF). Subsequent esteriﬁcation was achieved with thionyl-

chloride in methanol, followed by oxime synthesis using

hydroxylamine as a nucleophile in dry MeOH. Reduction of

oxime 3 with zinc powder provided the corresponding amino

ester, which was saponiﬁed in standard conditions to deliver

the pivotal amino acid intermediate 4 as a racemate. The latter

was Fmoc-protected to give Fmoc-βdiMe-Phe-OH 5 or

underwent a Pictet−Spengler reaction to cleanly deliver the

corresponding six-membered isoquinoline. Crystallization of

this intermediate was achieved upon cooling, and ﬁnal Fmoc-

protection provided Fmoc-βdiMe-Tic-OH 6.

To further explore the impact of C-terminal modiﬁcations,

two azepinone-based constraints were implemented (11a, 11b,

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a

Scheme 3. Synthetic Scheme of Tyr(OBn) Analogues

a

(A) (a) cyclopentanol, H₃PO₄85%, 100 °C, 16 h; (b) allyl chloroformate (1.2 equiv), NaHCO₃, THF-H₂O (1:1), rt, 15 min, 55%; (c) (i) BnBr,

K₂CO₃, acetonitrile reﬂux, 1 h, 73−77%; (ii) KOH 2 M, 1.2 equiv, EtOH-H₂O (5:1), 70 °C, 15 min, 83−85%; (B) (d) tetramethylsilane (TMS)−

acetylene, CuI, K₂CO₃, tetrabutylammonium iodide (TBAI), acetonitrile, 40 °C, 24 h, 94%; (e) MeOH, K₂CO₃, rt, 1.5 h, 88%; (f) (i), AlMe₃,

ZrCp₂Cl₂, dichloromethane (DCM), rt, 16 h; (ii), p-menthyl-3-carboxaldehyde, THF, −78 °C, 2 h, 47%; (g) (i), trichloroacetyl isocyanate, DCM,

0 °C, 1 h; (ii), MeOH, K₂CO₃, rt, 16 h, 72%; (h) TFAA, TEA, DCM, 0 °C, 15 min, 95%; (i) Ti(OtBu)₄, 9-ﬂuorenemethanol, benzene, 45 °C, 3 h,

75%; (j) (i), ozone, PPh₃, DCM, rt, 15 h; (ii), NaOCl, tert-butanol-H₂O (1:1), rt, 16 h, 67%.

Figure 2. Unnatural amino acids used to modify Phe13 of Pyr-apelin-13.

The L-α-MeTyr(OBn) residue was prepared from commer-

cially available L-α-MeTyr, whereas the D-α-MeTyr(OBn)

analogue was synthesized from 4-(benzyloxy)benzyl chloride

16 using p-menthyl-3-carboxaldehyde as a chiral auxiliary

(Scheme 3B).⁶⁷Critical steps in this synthesis are the Zr-

catalyzed carboalumination of the triple bond of intermediate

17b, followed by trapping of the vinylalane intermediate with

p-menthyl-3-carboxaldehyde to produce chiral allylic alcohol

18 as a single detected diastereomer. The α-nitrogen was

introduced using trichloroacetyl isocyanate, ﬁrst forming

carbamate 19, followed by [3,3]-sigmatropic rearrangement

to construct the asymmetric center of the α-amino acid

precursor in the form of isocyanate 20. The latter was

converted to Fmoc-protected amine 21 by reaction with 9-

ﬂuorenemethanol. Finally, the chiral auxiliary was cleaved to

give the desired Fmoc-^D-α-MeTyr(OBn) 22 ready for

incorporation in Ape13.

Impact of Modiﬁcations on Binding Aﬃnity and

Functional Activities. Impact of Modiﬁcations of Phe13.

Phe13 is an important residue for activation of the downstream

signaling pathway of the apelin receptor.³⁷As receptor binding

is a dynamic process, the side chain of Phe13 may adopt

several conformations as suggested by molecular dynamics

simulations.^41,68For this reason, we decided to constrain its

side chain in diﬀerent ways (Figure 2 and Table 1) to explore

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Table 1. Aﬃnity and Functional Activities of Phe13-Modiﬁed Pyr-apelin-13 Analogues

a

b

c

d

peptide sequence

binding K_i(nM)

Gα_i1EC₅₀(nM)

Gα₁₂EC₅₀(nM)

β-arr2 EC₅₀(nM)

Ape13

23

24

25

26

27

28

29

30

Pyr-R-P-R-L-S-H-K-G-P-M-P-F

0.7 0.1

0.37 0.04

0.36 0.01

1.1 0.1

0.07 0.03

0.04 0.01

0.35 0.01

0.31 0.01

2.8 0.1

1.1 0.1

1.0 0.2

2.4 0.3

2.5 0.7

1.0 0.1

1.5 0.4

2.1 0.4

1.4 0.4

8.6 2.8

6.4 1.1

6.8 1.6

43

108

67

88

23

10

21

24

142

74

1

6

3

4

3

2

1

4

40

69

4

7

Pyr-R-P-R-L-S-H-K-G-P-Nle-P-(β-MePhe)-OH

Pyr-R-P-R-L-S-H-K-G-P-Nle-P-(β-diMePhe)-OH

Pyr-R-P-R-L-S-H-K-G-P-Nle-P-Tic-OH

Pyr-R-P-R-L-S-H-K-G-P-Nle-P-(d-Tic)-OH

Pyr-R-P-R-L-S-H-K-G-P-Nle-P-Db_zg-OH

Pyr-R-P-R-L-S-H-K-G-P-Nle-P-(4-Bip)-OH

Pyr-R-P-R-L-S-H-K-G-P-Nle-P-(3-Bip)-OH

Pyr-R-P-R-L-S-H-K-G-P-Nle-P-(2-Bip)-OH

Pyr-R-P-R-L-S-H-K-G-P-Nle-P-(1-Ana-Gly)-OH

Pyr-R-P-R-L-S-H-K-G-P-Nle-P-Aia-Gly-OH

67 10

10

19

64

47

391 11

221 34

311 82

2

4

31

32

1.7 0.5

4.3 0.3

481 12

a

K_iwas calculated from experimental IC₅₀values (the concentration of ligand that displaces 50% of radiolabeled [¹²⁵I][Nle⁷⁵, Tyr⁷⁷]Pyr-apelin-13)

using the Cheng−Prusoﬀ equation.⁶⁹Values represent the mean standard error of the mean (SEM) of two to three experiments, each performed

in duplicate. EC₅₀corresponds to the concentration of ligand that produces 50% dissociation of Gα_i1from the Gβγ subunits. EC₅₀corresponds to

the concentration of ligand that produces 50% recruitment of the Gα₁₂eﬀector (PDZ-RhoGEF) to the cell membrane. EC₅₀is the concentration

b

c

d

of ligand that produces 50% recruitment of β-arrestin2 to the apelin receptor. Values represent the mean SEM of three experiments, each

performed in triplicate.

Figure 3. BRET dose−response curves of Gα_i1, Gα₁₂, and β-arrestin2 pathways of Ape13 analogues with modiﬁcation of Phe13.

how the modulation of this side chain impacts the binding

aﬃnity and signaling proﬁle.

endogenous ligand (28, K_i0.35 nM; 29, K_i0.31 nM), while

2-Bip substitution slightly reduces the binding aﬃnity (30, K_i

2.8 nM vs Ape13, K_i0.7 nM). Altogether, these results suggest

that there is signiﬁcant room to accommodate aromatic

residues around the Phe aryl group. This is congruent with

previous results consisting of replacing Phe13 by 1-Nal, 2-Nal,

or Tyr(OBn).⁵⁸

First, methyl substitutions were introduced at the β-position

of Phe13 to restrict rotations of its side chain in the χ space.⁶⁵

Mono- or dimethyl substitutions on racemic Phe showed little

eﬀect on ligand binding proﬁles (23, K_i0.37 nM; 24, K_i0.36

nM vs Ape13, K_i0.7 nM), and no signiﬁcant impact on

signaling was observed. Despite a slight increase in aﬃnity of

this epimeric mixture (<2-fold), the possibility that one epimer

may provide better aﬃnity than the other cannot be ruled out.

In contrast, cyclic constraint of the aromatic side chain into Tic

analogues produced interesting eﬀects. ^L-Tic substitution

resulted in a slight decrease in binding aﬃnity (25, K_i1.1

nM), while ^D-Tic led to a 10-fold increase in binding (26, K_i

0.07 nM vs Ape13, K_i0.7 nM). Our previous study showed

that chiral inversion of Phe13 to ^D-Phe on Ape13 results in a

loss of binding aﬃnity to the apelin receptor,⁵⁸which is quite

diﬀerent compared to this result. It is plausible that the

structural constraint of ^D-Tic may keep the side chain in a

favorable conformation for receptor binding, which is less

accessible by free rotation in the ^D-Phe side chain or,

alternatively, impacts the peptide backbone’s conformation.

Substitution at the α-position also brings beneﬁts, as observed

with the α,α-dibenzylglycine analogue, which displays a 17-fold

increase in binding aﬃnity (27, K_i0.04 nM).

Side-chain-constrained dipeptide analogues, such as 1-Ana-

Gly (31) or Aia-Gly (32), were also introduced at the Phe13

position. They both allow the carboxylate group to extend

deeper into the binding pocket since an additional glycine is

inserted. Although the aﬃnity of these analogues does not

increase, it stays in the low nM level (31, K_i1.7 nM; 32, K_i4.3

nM vs Ape13, K_i0.7 nM), underlining that the binding pocket

has a considerable degree of ﬂexibility and is able to

accommodate additional residues at the C-terminus of Ape13.

Regarding signaling, compounds showing good binding

aﬃnity, such as 26 (K_i0.07 nM) and 27 (K_i0.04 nM), also

display excellent potency in activating signaling pathways

(Figure 3). Indeed, compound 27 is around fourfold superior

at Gα₁₂activation (EC₅₀Gα₁₂10 nM) compared to the

endogenous ligand (Ape13, EC₅₀Gα₁₂43 nM). Moreover,

analogues 26 (Phe13 → ^D-Tic) and 27 (Phe13 → Db_zg)

exhibit EC₅₀on β-arrestin2 recruitment at 10 and 19 nM,

respectively, which is two- to fourfold better than that of

Ape13 (EC₅₀β-arr2 40 nM) and makes them some of the most

potent activators of the β-arrestin2 pathway. Despite such

improvements in binding and Gα₁₂and β-arrestin pathways,

the potency of Gα_i1activation of 26 and 27 stays at the same

To probe the space around the aromatic ring of Phe13, we

also introduced a phenyl substituent at the ortho-, meta-, and

para-positions of this residue. Analogues containing 3-Bip and

4-Bip demonstrate similar activities compared to the

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Figure 4. Unnatural analogues of Tyr(OBn) used to replace Phe13.

Table 2. Aﬃnity and Functional Activities of Pyr-apelin-13 Analogues Containing Tyr(OBn) Surrogates

a

b

c

d

peptide sequence

Pyr-R-P-R-L-S-H-K-G-P-M-P-F

binding K_i(nM)

Gα_i1EC₅₀(nM)

Gα₁₂EC₅₀(nM)

β-arr2 EC₅₀(nM)

Ape13

33

34

35

36

37

38

39

40

0.7 0.1

0.02 0.003

0.19 0.06

0.41 0.13

13 0.4

1.6 0.2

0.46 0.05

6.1 0.3

1.1 0.1

0.5 0.2

3.4 1.0

5.7 3.1

43

11

22

53

1

6

9

40

14

45

4

6

Pyr-R-P-R-L-S-H-K-G-P-Nle-P-Tyr(OBn)-OH

Pyr-R-P-R-L-S-H-K-G-P-Nle-P-Tic(7-OBn)-OH

Pyr-R-P-R-L-S-H-K-G-P-Nle-P-cypTyr(OBn)-OH

Pyr-R-P-R-L-S-H-K-G-P-Nle-P-dcypTyr(OBn)-OH

Pyr-R-P-R-L-S-H-K-G-P-Nle-P-dcypTyr(OH)-OH

Pyr-R-P-R-L-S-H-K-G-P-Nle-P-(m-Tyr(OBn))-OH

Pyr-R-P-R-L-S-H-K-G-P-Nle-P-Hyp(4-OBn)-OH

Pyr-R-P-R-L-S-H-K-G-P-Nle-P-(l-α-MeTyr(OBn))-OH

Pyr-R-P-R-L-S-H-K-G-P-Nle-P-(d-α-MeTyr(OBn))-OH

69 14

367 128

e

22

16

8

5

141 30

111 30

361

7

3.8 0.4

6.4 1.8

45

63 27

11

4.4 0.5

9

106 12

192 61

571 57

1.0 0.2

0.12 0.08

19

9

1

f

41

2.7 0.9

30

1

a

K_iwas calculated from experimental IC₅₀values (the concentration of ligand that displaces 50% of radiolabeled [¹²⁵I][Nle⁷⁵, Tyr⁷⁷]Pyr-apelin-13)

using the Cheng−Prusoﬀ equation.⁶⁹Values represent the mean

corresponds to the concentration of ligand that produces 50% dissociation of Gα_i1from the Gβγ subunits. EC₅₀corresponds to the concentration

of ligand that produces 50% recruitment of the Gα₁₂eﬀector (PDZ-RhoGEF) to the cell membrane. EC₅₀is the concentration of ligand that

SEM of two to three experiments, each performed in duplicate. EC₅₀

b

c

d

produces 50% recruitment of β-arrestin2 to the apelin receptor. Values represent the mean SEM of three experiments, each performed in

e

f

triplicate. Most compounds showed E_max> 75%, except for 36 in the β-arrestin2 pathway and 41 in the Gα₁₂pathway. E_max53%. E_max46%.

Figure 5. BRET dose−response curves of Gα_i1, Gα₁₂, and β-arrestin2 pathways of Ape13 analogues with Tyr(OBn) derivatives on the Phe13

position.

level as those of Ape13 (EC₅₀Gα_i11−1.5 nM). In contrast,

Aia-Gly (32, K_i4.3 nM) or 2-Bip (30, K_i2.8 nM) substitution

displays 6- to 10-fold lower potency in all examined signaling

pathways, which goes in the same direction as their aﬃnity.

In conclusion, although the binding pocket of Phe13

displays a high tolerance for side-chain modiﬁcations and

even Phe13 replacement by dipeptide moieties, it still shows

preference for a certain type of constrained residues, such as

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Figure 6. Unnatural amino acids used to modify Pro12 of Pyr-apelin-13.

Table 3. Aﬃnity and Functional Activities of Pro12-Modiﬁed Pyr-apelin-13 Analogues

a

b

c

d

peptide sequence

binding K_i(nM)

Gα_i1EC₅₀(nM)

Gα₁₂EC₅₀(nM)

β-arr2 EC₅₀(nM)

Ape13

42

43

44

45

Pyr-R-P-R-L-S-H-K-G-P-M-P-F

0.7 0.1

2.1 0.3

4.2 0.7

1.1 0.1

5.6 0.6

43

54

48

67

9.2 0.2

17

1

2

40

100

4

9

Pyr-R-P-R-L-S-H-K-G-P-Nle-Hyp(4-OBn)-F-OH

Pyr-R-P-R-L-S-H-K-G-P-Nle-Pc3Phe-F-OH

Pyr-R-P-R-L-S-H-K-G-P-Nle-Tic-F-OH

Pyr-R-P-R-L-S-H-K-G-P-Nle-1Nal-F-OH

Pyr-R-P-R-L-S-H-K-G-P-Nle-Trp-F-OH

Pyr-R-P-R-L-S-H-K-G-P-Nle-Aia-F-OH

17

3

454 75

142 18

41

38

25

9.7 0.8

9.6 5.1

4.4 0.6

3.8 0.6

2.5 0.3

0.12 0.01

0.18 0.02

0.08 0.01

4

2

5

46

47

3

1

11

a

K_iwas calculated from experimental IC₅₀values (the concentration of ligand that displaces 50% of radiolabeled [¹²⁵I][Nle⁷⁵, Tyr⁷⁷]Pyr-apelin-13)

using the Cheng−Prusoﬀ equation.⁶⁹Values represent the mean

corresponds to the concentration of ligand that produces 50% dissociation of Gα_i1from the Gβγ subunits. EC₅₀corresponds to the concentration

of ligand that produces 50% recruitment of the Gα₁₂eﬀector (PDZ-RhoGEF) to the cell membrane. EC₅₀is the concentration of ligand that

SEM of two to three experiments, each performed in duplicate. EC₅₀

b

c

d

produces 50% recruitment of β-arrestin2 to the apelin receptor. Values represent the mean SEM of three experiments, each performed in

triplicate.

Figure 7. BRET dose−response curves of Gα_i1, Gα₁₂, and β-arrestin2 pathways of Ape13 analogues with modiﬁcation on Pro12.

constrained D-amino acid (D-Tic) or the α,α-disubstituted

analogue Db_zg.

well as the orientation of the carboxylate pharmacophore on

the C-terminal tail.

Impact of Replacements of Phe13 by Tyr(OBn) Ana-

logues. We reported previously that the Tyr(OBn) sub-

stitution is favorable at the Phe13 position of Ape13, providing

a 60-fold increase in binding aﬃnity (33, K_i0.02 nM).⁴²To

explore more thoroughly the binding pocket around this key

residue and ﬁnd out whether close derivatives of Tyr(OBn)

bring similar beneﬁts, we developed several analogues where

the side chain is either constrained, such as Tic(7-OBn) (34,

K_i0.19 nM), or bears sterically hindered groups in the ortho-

position of the benzyloxy moiety, such as cypTyr(OBn) (35, K_i

0.41 nM), dcypTyr(OBn) (36, K_i13 nM), or dcypTyr(OH)

(37, K_i1.6 nM) (Figure 4 and Table 2). These modiﬁcations

were in general well-tolerated with the exception of dcypTyr-

(OBn), which seems too bulky for the binding pocket.

Similarly, substitution of Phe13 with m-Tyr(OBn) (38, K_i0.46

nM) does not provide the same improvement as Tyr(OBn)

(33, K_i0.02 nM), while replacement by 4-benzyloxyproline

Hyp(4-OBn) (39, K_i6.1 nM) seems less tolerated. The aﬃnity

reduction of 39 compared to its analogue 33 could be

explained by the fact that Hyp(4-OBn) substitution changes

the benzyloxy group position, the backbone conformation, as

The eﬀect of substitution on the α-carbon of the C-terminal

Tyr(OBn) analogue was also investigated. Introduction of a

methyl group at this position, providing the ^L-α-MeTyr(OBn)

analogue, did not produce sub-nM aﬃnity ligands as expected

(40, K_i1.0 nM vs Tyr(OBn), 33, K_i0.02 nM). However,

stereoinversion to ^D-α-MeTyr(OBn) leads to analogue 41

displaying an aﬃnity constant at the sub-nM range (41, K_i0.12

nM). Remarkably, the two epimers display a 10-fold diﬀerence

in binding aﬃnity.

Substitution of Phe13 with Tyr(OBn) analogues signiﬁ-

cantly alters the signaling proﬁle of the resulting ligands. The

α-substituted analogues 40 (K_i1.0 nM) and 41 (K_i0.12 nM)

display excellent potencies at Gα₁₂activation (EC₅₀Gα₁₂11

and 4.4 nM, respectively), equal to or slightly better than the

reference analogue with Tyr(OBn) (33, EC₅₀Gα₁₂11 nM)

and clearly superior to Ape13 (EC₅₀43 nM). While analogue

40 provides good potency on Gα₁₂pathways, it is much

weaker on both the Gα_i1and β-arrestin2 pathways, suggesting

a biased proﬁle in favor of Gα₁₂. Besides, the maximum eﬃcacy

of 41 on the Gα₁₂pathway is signiﬁcantly reduced, reaching

only 46% of the maximum response (E_max) of Ape13, as

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Figure 8. Unnatural amino acids used to modify Phe13 in combination with the substitution of 1-Nal on Pro12.

Table 4. Aﬃnity and Functional Activities of Pyr-apelin-13 Analogues with Combined Modiﬁcation of Pro12 and Phe13

a

b

c

d

peptide sequence

Pyr-R-P-R-L-S-H-K-G-P-M-P-F

Pyr-R-P-R-L-S-H-K-G-P-Nle-P-Tyr(OBn)-OH

Pyr-R-P-R-L-S-H-K-G-P-Nle-1Nal-F-OH

Pyr-R-P-R-L-S-H-K-G-P-Nle-1Nal-Tic-OH

Pyr-R-P-R-L-S-H-K-G-P-Nle-2Nal-Tic-OH

Pyr-R-P-R-L-S-H-K-G-P-Nle-1Nal-(d-Tic)-OH

Pyr-R-P-R-L-S-H-K-G-P-Nle-1Nal-(β-MeTic)-OH

Pyr-R-P-R-L-S-H-K-G-P-Nle-1Nal-(β-diMeTic)-OH

Pyr-R-P-R-L-S-H-K-G-P-Nle-1Nal-Tcc-OH

Pyr-R-P-R-L-S-H-K-G-P-Nle-1Nal-Db_zg-OH

Pyr-R-P-R-L-S-H-K-G-P-Nle-1Nal-dcypTyr(OH)-OH

binding K_i(nM)

Gα_i1EC₅₀(nM)

Gα₁₂EC₅₀(nM)

β-arr2 EC₅₀(nM)

Ape13

33

45

48a

48b

49

50

51

52

53

0.7 0.1

1.1 0.1

0.5 0.2

4.4 0.6

1.6 0.3

4.3 0.3

4.2 0.9

4.3 0.5

4.1 0.9

6.0 0.8

3.8 0.9

43

11

1

6

40

14

41

32

94

33

44

34

96

36

4

6

4

2

9

4

2

6

1

0.02 0.003

0.12 0.01

0.09 0.01

0.37 0.01

0.08 0.01

0.40 0.17

0.16 0.04

1.01 0.43

0.08 0.01

1.1 0.1

9.2 0.2

15

n/a

28

2

1

e

111

10

25

6

1

2.8 0.1

178 13

54

10

2

95 14

a

K_iwas calculated from experimental IC₅₀values (the concentration of ligand that displaces 50% of radiolabeled [¹²⁵I][Nle⁷⁵, Tyr⁷⁷]Pyr-apelin-13)

using the Cheng−Prusoﬀ equation.⁶⁹Values represent the mean

corresponds to the concentration of ligand that produces 50% dissociation of Gα_i1from the Gβγ subunits. EC₅₀corresponds to the concentration

of ligand that produces 50% recruitment of the Gα₁₂eﬀector (PDZ-RhoGEF) to the cell membrane. EC₅₀is the concentration of ligand that

SEM of two to three experiments, each performed in duplicate. EC₅₀

b

c

d

produces 50% recruitment of β-arrestin2 to the apelin receptor. Values represent the mean SEM of three experiments, each performed in

e

triplicate. Not tested yet.

Figure 9. BRET dose−response curves of Gα_i1, Gα₁₂, and β-arrestin2 pathways of Ape13 analogues with combined modiﬁcation on Pro12 and

Phe13.

opposed to the other analogues (Figure 5), suggesting that the

high-aﬃnity complex of 41 with the apelin receptor may not

eﬃciently trigger Gα₁₂activation. Interestingly though, 41

remains a full agonist, having similar potency as Ape13 on the

Gα_i1and β-arrestin2 pathways (Figure 5). Regarding β-

arrestin2 recruitment, the bulky dcypTyr(OBn) and dcypTyr-

(OH) substitutions elicit lower potencies (36, β-arr2 EC₅₀367

nM; 37 EC₅₀361 nM vs Ape13, EC₅₀40 nM) and analogue 36

behaves as a partial agonist on the β-arrestin2 pathway,

producing only half of E_maxat the highest concentration

(Figure 5).

To summarize, most substitutions of Tyr(OBn) did not

further improve the binding and signaling proﬁles of analogue

33, except for ^L/D-α-MeTyr(OBn). Those results follow the

same trend observed in Phe13 modiﬁcations, highlighting the

advantage of α,α-disubstituted analogues at the C-terminal

position of Ape13. Of particular interest, 41 emerges as a

partial agonist on the Gα₁₂pathway and 36 for the β-arrestin2

pathway.

Impact of Modiﬁcations on Pro12. The Pro12 position has

been studied previously using proline mimetics,⁵⁸which

improved both stability and aﬃnity. The X-ray structure of

APJ complexes with apelin-17 analogues reveals that the Pro12

residue is surrounded by a hydrophobic environment with

available space for structural derivatization (Figure 1S,

Supporting Information).⁷⁰For this reason, we substituted

Pro12 with bulky proline derivatives (e.g., Hyp(4-OBn),

P3cPhe, Tic) and aromatic residues (e.g., 1-Nal, Trp) (Figure

6 and Table 3). Binding results conﬁrm that the Pro binding

pocket is large and can accommodate bulky residues without

major loss in receptor binding aﬃnity (e.g., Hyp(4-OBn), 42,

K_i2.1 nM vs Ape13, K_i0.7 nM). However, side-chain-

constrained residues, such as P3cPhe and Tic, may impact the

peptide backbone conformation since these substitutions result

in 6- to 14-fold loss in aﬃnity, respectively (43, K_i4.2 nM; 44,

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a

K_i9.7 nM). Surprisingly, replacement of Pro12 with an

unconstrained aromatic amino acid (1-Nal and Trp) leads to a

signiﬁcant improvement in binding aﬃnity (45, K_i0.12 nM;

46, K_i0.18 nM). Aia substitution also follows the same trend,

further improving aﬃnity (47, K_i0.08 nM).

Scheme 4. Synthesis of Fmoc-α,α-dibenzylglycine

Regarding signaling, compounds 43 and 44 having the

proline analogue Pc3Phe or constraint residue Tic substitution

display 9- to 17-fold loss in the Gα_i1pathways and 3- to 11-fold

loss in β-arrestin recruitment, while the Gα₁₂pathway remains

comparable to the endogenous ligand (Figure 7). In contrast,

compound 45 (Pro12 → 1Nal, EC₅₀Gα₁₂9.2 nM) or 47

(Pro12-Phe13 → Aia-Phe, EC₅₀Gα₁₂11 nM) is comparable

on Gα_i1activation and β-arrestin recruitment compared to

Ape13 but fourfold more potent on the Gα₁₂pathway (Ape13,

EC₅₀Gα₁₂43 nM). These results suggest that proline is not

mandatory for this position and aromatic residues, such as

1Nal or Aia, could be a favorable replacement. This is

consistent with the presence of a hydrophobic and aromatic

environment around position Pro12 in the APJ binding pocket.

Impact of Combined Modiﬁcations of Pro12 and Phe13.

The 1Nal substitution of Pro12 shows a great beneﬁt for

binding aﬃnity as well as signaling potency. Indeed, 45 (Pro12

→ 1Nal) possesses an excellent binding aﬃnity (45, K_i0.12

nM) and high potency across the diﬀerent signaling pathways

tested. In contrast with Aia, this residue was easily

incorporated with other constrained amino acids at the

Phe13 position. For this reason, 45 was chosen as a starting

point for further modiﬁcations to test the potential for additive

or synergistic eﬀects (Figure 8 and Table 4). The results show

that the combined modiﬁcations between 1-Nal at Pro12 and

Tic (48a, K_i0.09 nM) and D-Tic (49, K_i0.08 nM) at Phe13

are beneﬁcial, leading to compounds with sub-nM aﬃnities.

The aﬃnity contribution of 1-Nal at the Pro12 position seems

pivotal when Tic (48a) and ^D-Tic (49) residues are placed in

the Phe13 position, as the latter no longer inﬂuence the

binding constant (cf. compounds 25 and 26, Table 1).

Remarkably, replacement of 1-Nal by its regioisomer 2-Nal

nulliﬁes improvements in binding aﬃnity (48b, K_i0.37 nM vs

48a, K_i0.09 nM), again conﬁrming the beneﬁcial role of the 1-

Nal residue. Introduction of a methyl substituent at the β-

position of Tic reduces the aﬃnity by two- to ﬁvefold (50, K_i

0.40 nM; 51, K_i0.16 nM). This result may be explained by the

proximity of β-methyl substitution on Tic residues to the C-

terminal carboxylate group, hindering its interaction with

Lys268 or Arg168 of APJ.⁷⁰While the C-terminal carboxylate

is important for binding, such steric hindrance could be

detrimental.¹³The combination of 1-Nal and Db_zg generated

compound 53 (K_i0.08 nM), which is as potent as the previous

analogue containing Db_zg (i.e., 5, K_i0.04 nM), conﬁrming the

consistent beneﬁt of α,α-disubstitution at the level of Phe13.

As a key residue, Fmoc-Db_zg-OH could be easily obtained

from ethyl 2-nitroacetate through a four-step synthesis

(Scheme 4).

a

(a) BnBr, N,N-diisopropylethylamine (DIPEA), TBAI, dry N,N-

dimethylformamide (DMF), 0−25 °C, ovn, 35%; (b) Zn, trace HCl 1

M, AcOH, rt, 2 h, 70%; (c) KOH 2 M, EtOH, reﬂux, 2 h, 90%; (d)

(i), TMSCl, DIPEA, DCM, rt, 10 min; (ii), Fmoc-Cl, DCM, rt, ovn,

73% over two steps.

nM). Among these, 53 (K_i0.08 nM, Pro12-Phe13 → 1Nal-

Db_zg) possesses the highest potency on the Gα₁₂pathway

(EC₅₀Gα₁₂2.8 nM), becoming the most potent Gα₁₂activator

known to date of the Ape13-modiﬁed analogues.

In Vitro Stability in Rat Plasma and ACE2 Degrada-

tion. Analogues with high aﬃnity for the apelin receptor were

assessed for their in vitro stability in rat plasma (Table 5).

Table 5. Stability of Ape13 Analogues in Rat Plasma

rat plasma t_1/2

a

peptide sequence

(h)

Ape13 Pyr-R-P-R-L-S-H-K-G-P-M-P-F

0.5 0.1

1.3 0.1

0.8 0.2

0.5 0.1

0.9 0.3

25

26

27

41

Pyr-R-P-R-L-S-H-K-G-P-Nle-P-Tic-OH

Pyr-R-P-R-L-S-H-K-G-P-Nle-P-(d-Tic)-OH

Pyr-R-P-R-L-S-H-K-G-P-Nle-P-Db_zg-OH

Pyr-R-P-R-L-S-H-K-G-P-Nle-P-(d-α-

MeTyr(OBn))-OH

45

46

47

48a

49

50

Pyr-R-P-R-L-S-H-K-G-P-Nle-1Nal-F-OH

Pyr-R-P-R-L-S-H-K-G-P-Nle-W-F-OH

Pyr-R-P-R-L-S-H-K-G-P-Nle-(Aia)-F-OH

Pyr-R-P-R-L-S-H-K-G-P-Nle-1Nal-Tic-OH

Pyr-R-P-R-L-S-H-K-G-P-Nle-1Nal-(d-Tic)-OH

0.4 0.1

0.2 0.1

5.8 1.6

3.4 0.3

6.1 1.2

5.3 0.5

Pyr-R-P-R-L-S-H-K-G-P-Nle-1Nal-(β-MeTic)-

OH

51

Pyr-R-P-R-L-S-H-K-G-P-Nle-1Nal-(β-

diMeTic)-OH

6.3 2.1

52

53

54

Pyr-R-P-R-L-S-H-K-G-P-Nle-1Nal-Tcc-OH

Pyr-R-P-R-L-S-H-K-G-P-Nle-1Nal-Db_zg-OH

7.8 0.9

7.3 1.1

>24 h

Pyr-R-P-R-L-S-H-K-G-P-Nle-1Nal-

dcypTyr(OH)-OH

a

Values represent the mean

SEM of three determinations. The

Improvements in binding aﬃnity do not necessarily translate

into improvements in signaling potency (Table 4 and Figure

9). For analogues 48a−54, they manifest similar or slightly

lower potency on the Gα_i1pathway (EC₅₀1.6−10 nM) and

the β-arrestin recruitment (EC₅₀32−95 nM) compared to

Ape13 (EC₅₀Gα_i11.1 nM, EC₅₀β-arr2 40 nM). The most

signiﬁcant change in Gα₁₂activation was observed with several

potent analogues, such as 48a (bearing 1Nal-Tic), 51 (1Nal-

(β-diMeTic)), and 53 (1Nal-Db_zg), which display 3−15-fold

potency increases (EC₅₀2.8−10 nM vs Ape13 EC₅₀Gα_i143

most abundant fragments detected at the time point near t_1/2are

underlined.

Among them, the two most promising compounds (47, 53)

were additionally tested for their stability against recombinant

rat ACE2 compared with 46 and Ape13. Indeed, the fact that

Ape13 is cleaved between Pro12 and Phe13 by ACE2 and

PRCP proteases^46,47suggests that modifying either residue

could improve resistance to proteolysis. Single modiﬁcation on

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Phe13 slightly increases plasma stability, in agreement with

previous ﬁndings (25, t_1/21.3 h; 26, t_1/20.8 h vs Ape13, t_1/20.5

h).⁴³Regarding Pro12, substitution with 1-Nal or Trp does not

change the half-life (45, t_1/20.4 h; 46, t_1/20.2 h). The analogue

with Pro12 → Trp substitution (46) showed some level of

resistance to ACE2, whereas Ape13 was rapidly cleaved

(Figure 10). However, the cleavage of 46 by ACE2 was still

well as complete resistance to ACE2 cleavage (Figure 10) and

high stability against plasma proteases, with an in vitro half-life

of 7.3 h.

In Vivo Pharmacokinetics in Rats. The two most

promising apelin analogues in this series, i.e., 47 (K_i0.08

nM, t_1/25.8 h) and 53 (K_i0.08 nM, t_1/27.3 h), were selected

for in vivo pharmacokinetics in rats (Figure 11). In these

Figure 11. Trace of the plasma concentration for compounds 47, 53,

and Ape13 in male Sprague−Dawley rats (n = 3).

Figure 10. Stability of analogues 46, 47, and 53 against rat ACE2

cleavage, compared to Pyr-apelin-13. The Ape13 analogues (ﬁnal

concentration 50 μM) were incubated with 20 nM enzyme (n = 3).

experiments, animals received the test compounds at a dose of

3 mg/kg i.v. The plasma concentration of these compounds

was monitored at 5, 10, 15, 20, 30, 60, 120, and 240 min after

injection by liquid chromatography and tandem mass

spectrometry (LC-MS/MS) analysis. Results show that

analogues 47 and 53 are deﬁnitely more stable than Ape13,

displaying half-lives of 20−26 min in vivo (compared to <1 min

for Ape13) and signiﬁcant improvement in target exposure

(AUC 0.12−0.13 mg*(min/mL) vs AUC 0.24.10⁻³mg*(min/

mL) of Ape13). The clearance of these analogues is medium,

with values around 23−27 mL/(min kg), yet shows drastic

improvements compared to Ape13. In conclusion, these results

conﬁrm that Pyr-apelin-13 analogues 47 and 53 are not only

more stable in the rat plasma in vitro assay but also possess

improved pharmacokinetic proﬁles in vivo (Table 6).

clearly observed at 24 h. The consensus recognition sequence

of this enzyme is Pro-X-Pro-hydrophobic, yet several

exceptions have been reported.⁴⁶Moreover, this analogue

could be targeted by other proteases, indicating that single

mutation in the sequence may not be suﬃcient to protect the

peptide against enzymatic degradation. When the peptide bond

between Pro12 and Phe13 of apelin is prone to proteolysis,

constraining this particular bond represents an elegant way to

improve stability. Consequently, the side-chain-constrained

version of analogue 46 was prepared (with Aia-Phe, 47, K_i0.08

nM), showing an excellent binding aﬃnity and a great

improvement in plasma stability (47, t_1/25.8 h vs Ape13, t_1/2

0.5 h). As expected, 47 also displayed complete resistance

against ACE2 cleavage after 24 h (Figure 10).

The combined modiﬁcation of Pro12 and Phe13 provides

improved peptide resistance to proteolytic degradation. All

compounds with double modiﬁcations (48a to 54; t_1/23.4 to

>24 h) consistently display higher half-lives compared to those

with single modiﬁcations (25−27, 45, 46; t_1/20.2−1.3 h),

except for analogue 47.

Table 6. In Vivo Pharmacokinetic Parameters for

Compounds 47, 53, and Ape13 in Male Sprague−Dawley

a

Rats (n = 3)

compounds AUC_totmin*(mg/mL) t_1/2(min) clearance mL/(kg min)

47

53

0.13 0.01

0.12 0.03

20

26

1

23

27

2

7

The cleavage fragments were detected by comparing MS

spectra between 0 h and the time point near the half-life of the

compound. By conﬁrming the presence of those fragments, we

identiﬁed several cleavage sites, such as the Leu5-Ser6 peptide

bond (attributed to neprilysin)⁴⁸and the Ser6-His7 one for

analogues possessing Phe13 substitution (25, 26, 27, Table 5).

Regarding Pro12-substituted analogues, the dominant cleavage

site is between Nle11-1Nal and Nle11-Trp12 (45, 46, Table

5). The C-terminal fragments were usually detected as the

remaining part for compounds with signiﬁcantly improved half-

lives (47, 49−54), whereas N-terminal fragments remained

dominantly present for analogues with lower half-lives (Ape13,

25, 45, 46). These results suggest that stabilizing the C-

terminal end of apelin-13 improves peptide stability, in

agreement with previous studies.⁵⁷

Ape13

0.0002 0.0001

<1 min

>100

a

Analogues were administered by i.v. bolus injection at 3 mg/kg.

In Vivo Blood Pressure Measurement. Finally, to

compare in vivo eﬀects of modiﬁed analogues with Ape13,

compounds 36, 41, 47, and 53 were administered i.v. to rats at

a dose of 19.6 nmol/kg while the mean arterial blood pressure

(MABP) was monitored (Figure 12). Ape13 is known to

induce a blood pressure drop following an i.v. bolus that

reaches the maximum eﬀect at the tested dose.⁷¹Analogues 41,

47, and 53 produced a similar blood pressure drop as Ape13

(around −30 mmHg after 1 min), conﬁrming that the

compounds are active in vivo. In contrast, analogue 36 has

no eﬀect on blood pressure, even with a higher dose (65 nmol/

kg). Since the hypotensive eﬀect of activated APJ is correlated

with the recruitment of β-arrestins,³⁶those results might be

explained by the fact that analogues 41, 47, and 53 have

In this series, 53 is the overall lead in terms of receptor

binding (53, K_i0.08 nM vs Ape13, K_i0.7 nM) and signaling

potency (53, Gα₁₂EC₅₀2.8 nM vs Ape13, EC₅₀43 nM), as

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pharmacokinetic proﬁles of the two most promising analogues

47 (Aia-Phe) and 53 (Db_zg) were characterized, showing half-

lives in vivo of around 20−26 min (>15-fold improvement

compared to Ape13) and complete resistance to ACE2

cleavage. Those stable and reﬁned analogues, along with

those possessing diverse signaling pathways, represent unique

tools to further unravel the pharmacology of the apelinergic

system.

EXPERIMENTAL SECTION

■

Synthetic Procedures and Compound Characterization

(Solution-Phase Synthesis). Thin-layer chromatography (TLC)

was performed on glass plates precoated with silica gel 60F254

(Merck, Darmstadt, Germany) using the mentioned solvent systems.

Visualization of the products on TLC plates was realized using UV

light (254 nm) and KMnO₄spray. Puriﬁcation of organic molecules

was performed via silica gel column chromatography (Davisil LC60A

or SiliCycle SILIAFLASH P60, 40−63 μm) on a Biotage Isolera One

system (Charlotte, North Carolina). Mass spectrometry (MS) was

performed on a Micromass Q-Tof Micro spectrometer with

electrospray ionization (ESI). High-resolution electrospray mass

spectroscopy (HRMS) data were recorded with a Micromass

QTOFmicro system or a maXis ESI-Q-Tof apparatus (Billerica).

Analytical reversed-phase high-performance liquid chromatography

(RP-HPLC) was performed using a Chromaster system (VWR

Hitachi) or ultrahigh-performance liquid chromatography (UPLC)-

MS system from Waters. Data collection and spectrum analysis were

done with Masslynx software. Preparative RP-HPLC puriﬁcation was

done on a Gilson (Middleton, WI) HPLC system or a preparative

HPLC from Waters (Milford) (detail in Supporting Information). All

fractions were lyophilized using a Flexy-Dry lyophilizer (FTS Systems,

Warminster, PA). ¹H and ¹³C NMR spectra (298 K) were recorded at

250 and 63 MHz on a Bruker Avance DRX 250 spectrometer, at 400

and 100 MHz, respectively, on Bruker Ascend 400 or at 500 MHz and

126 MHz, respectively, on a Bruker Avance II 500 spectrometer.

Chemical shifts are in parts per million (ppm). Tetramethylsilane

(TMS) or residual solvent signals were used as internal standards.

The solvent used is mentioned in all cases, and the abbreviations used

are as follows: s (singlet), br s (broad singlet), d (doublet), dd

(double doublet), t (triplet), t* (pseudo triplet), q (quadruplet), and

m (multiplet). The assignments were made using one-dimensional

Figure 12. Tracing of blood pressure variation of male Sprague−

Dawley rats after administration of compounds 47, 53, and Ape13 at

19.6 nmol/kg (n = 5).

comparable potencies and eﬃcacies for the recruitment of β-

arrestin2 as Ape13 (similar EC₅₀values and E_max), while 36 is

only a partial agonist (E_max53%) and possesses ∼10-fold lower

potency on this pathway compared to other analogues. On the

other hand, a partial agonist on the Gα₁₂pathway such as 41

produces an eﬀect on blood pressure, suggesting that this

pathway may not be responsible for the vasodilator eﬀect of

apelin. Although 47 and 53 possess much higher half-lives and

exposure in vivo than Ape13, their eﬀects on blood pressure are

only marginally longer (130−150 vs 100 s to normalization of

blood pressure), testifying the eﬃciency of the baroreﬂex⁷²to

counteract the hypotensive eﬀect of systemically administered

apelin agonists.

CONCLUSIONS

■

The C-terminus of Ape13 was previously suggested to

represent a crucial site to modulate the signaling of APJ.^37,42

In this work, the Pro12 and Phe13 of Ape13 were modiﬁed

using a series of side-chain-constrained residues, which extends

SAR knowledge of this bioactive peptide. Careful modiﬁcations

with the help of >30 derivatives led to signiﬁcant improve-

ments not only in binding aﬃnity but also in terms of signaling

proﬁles and stability. As such, single modiﬁcations of Pro12

with 1-Nal, Trp, or Aia increased aﬃnity and potency on the

Gα₁₂pathway, while substitution of Phe13 with Db_zg, D-α-

MeTyr(OBn), or ^D-Tic improved potency for both the Gα₁₂

and β-arrestin2 pathways, providing one of the most potent

Ape13 analogues on β-arrestin2 recruitment (27, EC₅₀10

nM). Several analogues possess sub-nM aﬃnity (40−90 pM);

among them, compound 27 bearing Db_zg (K_i40 pM) displays

the highest aﬃnity in this series. The peptide bond between

Pro12 and Phe13 is known to be cleaved by ACE2 and by

PRCP.^46,47Single modiﬁcation of the C-terminal residues

provides limited protection of the apelin peptide against

proteolytic degradation. In contrast, the combined modiﬁca-

tions of Pro12 and Phe13 or constraining the cleaved bond in a

dipeptide moiety (e.g., 47, Aia-Phe, K_i0.08 nM) dramatically

increases stability in rat plasma. The resulting analogues show

a half-life ranging from 3 to 7 h. The combined modiﬁcations

also result in the most potent Gα₁₂agonist in this series, with a

15-fold improvement (53, K_i0.08 nM, EC₅₀Gα₁₂2.8 nM)

compared to the endogenous ligand (Ape13, K_i0.7 nM, EC₅₀

Gα₁₂43 nM), as well as partial agonists of the Gα₁₂(41, E_max

46%) and the β-arrestin (36, E_max53%) pathway. The

1

(1D) H and ¹³C spectra or two-dimensional (2D) HSQC, HMBC,

and COSY spectra. All commercial reagents and solvents were used

without further puriﬁcation, unless otherwise stated. Solvents were

dried over microwave-activated 4 Å molecular sieves, degassed with a

gaseous N₂ﬂow, and stored under an argon atmosphere.

β-DiMe-Pyruvic Acid (1). 3-Phenylpyruvic acid (4 g, 24.4 mmol, 1

equiv) was dissolved in a mixture of THF/water (40 mL, 4:1 v/vol)

solution, and the mixture was subsequently cooled by means of an ice-

bath. After putting the reaction under an Ar atmosphere, methyliodide

(4.5 mL, 73 mmol, 3 equiv) and an aqueous solution of NaOH (5 M,

15 mL) were added. The ice-bath was removed, and the resulting

mixture was reﬂuxed for 4 h. Additional MeI and NaOH were added

after allowing the mixture to cool down to room temperature. Stirring

was continued overnight at room temperature. Upon reaction

completion, the mixture was concentrated in vacuo and redissolved

in water. The aqueous phase was washed with EtOAc, acidiﬁed with

an aqueous solution of HCl (1 N), and subsequently extracted with

EtOAc. The combined organic layers were washed with brine, dried

over MgSO₄, ﬁltered, and concentrated in vacuo. The desired brown

oil was used in the next step without puriﬁcation. 89% (4.17 g); MS

(ES⁻): 191 [M − H]⁻; ¹H NMR (CDCl₃, 250 MHz): δ (ppm) = 10.6

(br. s, 1H), 7.33−7.23 (m, 5H), 1.59 (s, 6H); ¹³C NMR (CDCl₃, 63

MHz): δ (ppm) = 197.5, 162.0, 141.4, 128.9, 127.4, 126.0, 50.1, 25.2.

β-DiMe-pyruvic Methyl Ester (2). Thionylchloride (5 mL, 69

mmol, 6.6 equiv) was added dropwise to a solution of 3-methyl-2-oxo-

3-phenylbutanoic acid (2 g, 10.4 mmol, 1 equiv) in dry methanol (52

mL), which was cooled by means of an ice-bath. After completing the

addition, the mixture was heated to reﬂux and stirring was continued

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overnight. The volatiles were removed under reduced pressure. No

additional washings or puriﬁcations were performed, and the brown

oil was used in the next step without puriﬁcation. 90% (1.93 g);

Fmoc-βdiMe-L/D-Tic-OH (6). This product was synthesized

following the procedure for the synthesis of Fmoc-βdiMe-Phe-OH

from the unprotected amine. White solid, 71% (0.520 g); MS(ES⁺):

428 [M + H]⁺, 450 [M + Na]⁺; HRMS (ESP⁺): found 450.1661 m/z

1

MS(ES⁺): no ionization observed; H NMR (CDCl₃, 250 MHz): δ

1

[M + H]⁺, [C₂₇H₂₅NO₄]⁺; calculated 450.1676; H NMR (CDCl₃,

(ppm) = 7.38−7.24 (m, 5H); 3.59 (s, 3H), 1.62 (s, 6H); ¹³C NMR

(CDCl₃, 63 MHz): δ (ppm) = 197.88, 162.55, 141.78, 128.77, 127.30,

126.18, 52.22, 50.48, 25.19.

250 MHz, rotamers visible, ratio: 57/43): δ (ppm) = 7.78−7.05 (m,

12H), 4.48 (m, 6H), 1.56 (s, 1.7H), 1.48 (s, 1.3H), 1.24 (s, 1.7H),

1.14 (s, 1.3H); ¹³C NMR (CDCl₃, 63 MHz): δ (ppm) = 175.67,

156.51, 155.52, 143.71, 141.39, 139.55, 138.94, 131.32, 131.11,

127.80, 127.14, 126.69, 126.05, 124.97, 124.18, 123.99, 120.01, 68.00,

67.84, 62.92, 62.67, 47.17, 45.12, 37.41, 31.05, 24.37.

Methyl (E)-2-(Hydroxyimino)-3-methyl-3-phenylbutanoate (3).

Methyl 3-methyl-2-oxo-3-phenylbutanoate (1 g, 4.84 mmol, 1

equiv) was dissolved in dry methanol (18 mL), followed by the

addition of hydroxylamine hydrochloride (1 g, 14.5 mmol, 3 equiv).

The mixture was reﬂuxed for 16 h. Upon reaction completion, the

volatiles were removed in vacuo. The residue was redissolved in

EtOAc and washed with brine, dried over MgSO₄, and ﬁltered to yield

the desired oxime. The brown oil was used in the following step

Fmoc-^L-Aia-L-Phe-OH (11b). This product was synthesized

following the procedure for the synthesis of Fmoc-βdiMe-Phe-OH

starting from the unprotected amine. White solid, 21% (0.320 g) over

three steps: reductive amination/cyclization, hydrolysis, and Fmoc-

protection. MS(ES⁺): 586 [M + H]⁺, 608 [M + Na]⁺, HRMS (ESP

+): found 608.2133 m/z [M + H]⁺, [C₂₇H₂₅NO₄Na]⁺calculated

608.2156; ¹H NMR (DMSO-d₆, 500 MHz, 348.15 K, rotamers

visible): δ (ppm) = 10.60 (s, 1H), 7.86−7.72 (m, 4H), 7.42−7.30 (m,

6H), 7.13−7.07 (m, 6H), 7.04−6.98 (m, 2H), 5.22 (dd, J = 8.2 Hz, J

= 6.2 Hz, 1H), 4.9 (br. s, 2H), 4.41−4.31 (m, 3H), 4.25 (m, 1H),

3.29 (dd, J = 14.4 Hz, J = 6 Hz, 1H), 3.05 (m, 2H), 2.78 (ps t, J =

13.2 Hz, 1H); ¹³C NMR (DMSO-d₆, 125 MHz, 298 K, rotamers

visible): δ (ppm) = 172.56, 171.81, 155.76, 144.44, 144.24, 141.19,

137.80, 134.88, 131.68, 129.19, 128.55, 128.12, 128.00, 127.57,

126.63, 125.89, 125.81, 121.45, 120.57, 118.96, 117.89, 111.46,

107.05, 66.26, 60.01, 51.85, 47.14, 42.49, 34.92, 27.97.

Alloc-cypTyr-OH and Alloc-dcypTyr-OH (14a and 14b). ^L-Tyr-

OH with weight 4 g was weighed in a round-bottom ﬂask, and around

20 mL of H₃PO₄85% (20 mL) and 4 mL of cyclopentanol were

added. The mixture was stirred at 100 °C overnight. After 16 h, a

small sample of the mixture was collected in a test tube, neutralized

with NaHCO₃, derivated with a few drops of allyl chloroformate

(mixed and stirred for 15 min), extracted, and analyzed using LC/MS

analysis to conﬁrm the presence of the desired product. The reaction

produced a mixture of cypTyr-OH (13a, monoalkylation) and

dcypTyr-OH (13b, dialkylation). For the workup, the reaction

mixture was poured slowly into 100 mL of ice water and neutralized

with 30 g of NaHCO₃(CAUTION: vigorous reaction). A small

amount of NaHCO₃was added to adjust the pH of this mixture to 7,

and the product was precipitated. The suspension was ﬁltered and

washed with cold water and dried under a fume hood for 1 day,

obtaining 2.89 g of crude product (UPLC provided below, Figure S2).

The product was used as such in the next reaction without further

characterization.

1

without any puriﬁcation. 94% (1 g); MS(ES+): 243 [M + Na]⁺, H

NMR (CDCl₃, 250 MHz): δ (ppm) = 8.83 (s, 1H), 7.37−7.25 (m,

5H), 3.62 (s, 3H), 1.59 (s, 6H); ¹³C NMR (CDCl₃, 63 MHz): δ

(ppm) = 163.94, 158.81, 143.89, 128.31, 126.86, 126.32, 125.21,

51.88, 42.83, 27.05.

HCl·H-βdiMe-^L/D-Phe-OH (4). The oxime (0.75 g, 4.85 mmol, 1

equiv) was dissolved in acetic acid (7.5 mL), and the mixture was

allowed to cool by means of an ice-saltwater bath. An aqueous

solution of H₂SO₄(30%, 7.5 mL) was added followed by a

portionwise addition of powdered zinc (0.66 g, 10.17 mmol, 3

equiv). Upon addition completion, the ice-saltwater bath was

removed and the mixture stirred for 16 h at room temperature. The

volatiles were removed in vacuo followed by extracting the aqueous

solution with EtOAc. Basifying the aqueous layer with a saturated

NaHCO₃solution allowed extraction with EtOAc. The combined

organic layers were dried over MgSO₄, ﬁltered, and concentrated in

vacuo to aﬀord a white foam. Then, the ester (1 equiv) was dissolved

in a 1 N HCl/acetone (1:1 v/v, 0.05 M) solution. The mixture was

stirred for 6 h under reﬂux conditions in an oil bath at 90 °C. The

acetone/water mixture was evaporated in vacuo when the reaction was

complete. Compound HCl·H-βdiMe-Phe-OH was obtained as a

white solid without any further puriﬁcation. 65% (over two steps,

0.506 g); MS(ES⁺): 194 [M + H]⁺; ¹H NMR (DMSO-d₆, 250 MHz):

δ (ppm) = 7.47 (m, 5H), 4.34 (s, 1H), 1.50 (s, 3H),1.46 (s, 3H); ¹³C

NMR (DMSO-d₆, 63 MHz): δ (ppm) = 169.82, 143.02, 129.20,

127.89, 126.38, 53.33, 40.05, 25.64, 22.65.

Fmoc-βdiMe-Phe-OH (5). The N-unprotected compound was

dissolved in a water/acetone mixture (1:1, v/v, 0.1 M). Fmoc-OSu (1

equiv) and Na₂CO₃(1.1 equiv) were added. After stirring for 3−4 h,

the acetone was evaporated and the mixture was acidiﬁed with HCl (1

N) and extracted with EtOAc (3 times). The organic phases were

combined, washed with brine, dried over MgSO₄, and concentrated in

vacuo. The crude product was puriﬁed using automated ﬂash

chromatography (CH₂Cl₂(1% AcOH): MeOH (1% AcOH) 0−

20% gradient 30 min) and was obtained as a white solid. 76% (0.54

g); MS(ES⁺): 438 [M + Na]⁺; ¹H NMR (CDCl₃, 250 MHz): δ

(ppm) = 7.76 (d, J = 7.4 Hz, 1H), 7.56−7.51 (m, 1H), 7. 42−7. 20

(m, 11H), 5.22 (d, J = 9.5 Hz, 1H), 4.58 (d, J = 9.5 Hz, 1H), 4.43 (m,

1H), 4.28 (m, 1H), 4.17 (m, 1H), 1.45 (s, 3H), 1.40 (s, 3H); ¹³C

NMR (CDCl₃, 63 MHz): δ (ppm) = 171.47, 155.94, 144.40, 143.78,

141.32, 128.22, 127.71, 127.06, 126.28, 125.01, 119.97, 66.98, 62.54,

51.77, 41.70, 25.43, 25.02.

The mixture of cypTyr-OH (13a) and dcypTyr-OH (13b) (2.89 g)

was suspended in 40 mL of THF-H₂O (1:1, v/v), and 1.29 g of

NaHCO₃was added. Allyl chloroformate (1.63 mL, 1,2 equiv) was

dropped slowly to the mixture for 5 min at 0 °C, and the mixture was

stirred for 15 min at room temperature. After the TLC conﬁrmed

complete consumption of the starting materials, the reaction mixture

was acidiﬁed with HCl (1 M) until pH 4−5 (around 20 mL). Around

30 mL of NaCl saturated solution was added, and the product was

extracted with EtOAc (3 × 20 mL). The organic phases were

combined, dried on MgSO₄, and evaporated in vacuo to yield the

crude product. The alloc-cypTyr-OH (14a) and alloc-dcypTyr-OH

(14b) were separated using ﬂash chromatography (gradient 15−40%

EtOAc in hexane + 0.25% AcOH) with a total yield of 55%. Alloc-

cypTyr-OH (14a): HRMS: found 332.1509 [M − H]⁻, calculated

332.1503. ¹H NMR (CDCl_3,400 MHz, 298 K) δ (ppm) 6.96 (d, J =

1.6 Hz, 1H), 6.82 (dd, J = 8.0, 1.3 Hz, 1H), 6.64 (d, J = 8.1 Hz, 1H),

5.87 (m, 1H), 5.28 (d, J = 17.1 Hz, 1H), 5.20 (m, 2H, proton of

ethylene + amide proton), 4.63 (dd, J = 13.8, 5.8 Hz, 1H), 4.56 (d, J =

5.6 Hz, 2H), 3.28−3.14 (m, 1H), 3.07 (qd, J = 14.1, 5.7 Hz, 2H), 1.99

(m, 2H), 1.84−1.42 (m, 6H); ¹³C NMR (CDCl_3,101 MHz, 298 K) δ

(ppm) 176.38, 155.99, 152.86, 132.53, 132.50, 128.23, 127.44,

127.35, 118.18, 115.66, 66.22, 54.78, 38.95, 37.31, 33.06, 32.99, 25.49,

25.47. Alloc-dcypTyr-OH (14b): HRMS: found 400.2137 [M − H]⁻,

HCl·H-βdiMe-^L/D-Tic-OH. The phenylalanine derivative (0.5 g, 2.17

mmol, 1 equiv) was ﬁrst fully dissolved in an aqueous solution of HCl

(3 mL, 12 N) followed by the addition of formalin (1 mL). The

mixture was reﬂuxed for 2 h and was allowed to cool to room

temperature upon completion. The formed white crystals were ﬁltered

oﬀ and used in the next reaction without further puriﬁcation. 72%

(0.40 g); MS(ES⁺): 206 [M + H]⁺; HRMS (ESP+): found 206.1166

1

m/z [M + H]⁺, [C₁₂H₁₅NO₂]⁺calculated 206.1176; H NMR (D₂O,

250 MHz): δ (ppm) = 7.56 (m, 1H), 7.39 (m, 1H), 7.29 (m, 1H),

7.18 (m, 1H), 4.40 (m, 2H), 4.15 (s, 1H), 1.61 (s, 3H), 1.33 (s, 3H);

¹³C NMR (D₂O, 63 MHz): δ (ppm) = 170.92, 140.65, 128.88,

127.21, 126.92, 126.50, 125.70, 64.46, 44.60, 35.86, 27.40, 25.71.

1

calculated 400.2129. H NMR (CDCl₃, 400 MHz, 298 K) δ (ppm):

6.82 (s, 2H), 5.88 (s, 1H), 5.28 (d, J = 17.2 Hz, 1H), 5.20 (d, J = 10.4

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Hz, 1H), 5.10 (d, J = 8.1 Hz, 1H), 4.66−4.58 (m, 1H), 4.56 (d, J =

4.6 Hz, 2H), 3.22−3.00 (m, 4H), 2.08−1.93 (m, 4H), 1.85−1.48 (m,

12H); ¹³C NMR (CDCl₃, 101 MHz, 298 K) δ (ppm): 177.00, 155.82,

150.64, 132.59, 131.72, 126.89, 125.15, 118.00, 66.07, 54.79, 39.11,

37.49, 33.07, 25.46, 25.44.

was puriﬁed by silica gel ﬂash column chromatography using EtOAc-

hexane (1: 9, v/v) as the eluant to aﬀord the desired product as a

colorless oil. Yield: 94% (3.61 g); chemical formula: C₁₉H₂₂OSi; MW:

294.4690 g/mol; HRMS m/z: [M + Na]⁺calculated for C₁₉H₂₂OSi

1

294.1440, found 294.1427; H NMR (300 MHz, CDCl₃) δ (ppm)

Alloc-dcypTyr(OBn)-OH and Alloc-cypTyr(OBn)-OH (15a and

15b). Alloc-cypTyr-OH (1.67 g, 4.76 mmol, 1 equiv) was dissolved in

ACN (25 mL), and K₂CO₃(1.98 g, 14.3 mmol, 3 equiv) was added.

Benzyl bromide (1.24 mL, 10.4 mmol, 2.2 equiv) was added dropwise

while stirring. The reaction mixture was heated at 90 °C for 1 h and

overnight at room temperature to completely alkylate both COOH

and OH functions. Once the reaction was done, solid particles (KBr,

K₂CO₃) were remove by ﬁltration and ACN was evaporated in vacuo.

Water (around 50 mL) was added, and the product was extracted

with EtOAc (50 mL). The organic phase was combined and washed

with water and brine to remove residual base. The product was

puriﬁed by ﬂash chromatography (20−35% EtOAc in hexane for 10

CV) to give the intermediate Alloc-cypTyr(OBn)-OBn. HRMS:

7.46−7.30 (m, 5H), 7.25 (d, J = 8.8 Hz, 2H), 6.94 (dt, J = 8.7, 3 Hz,

2H), 5.06 (s, 2H), 3.60 (s, 2H), 0.19 (t, J = 3.6 Hz, 9H). ¹³C NMR

(300 MHz, CDCl₃) δ (ppm) 157.65, 137.19, 128.95, 128.73, 128.66,

128.00, 127.52, 114.97, 104.88, 86.67, 70.10, 25.43, 0.26.

1-(Benzyloxy)-4-(prop-2-ynyl)benzene (17b). To a stirred solution

of (3-(4-(benzyloxy)phenyl)prop-1-ynyl)trimethylsilane (1.6 g, 1.0

equiv, 5.43 mol) in dry THF (0.09 M, 32 mL) and methanol (0.09 M,

32 mL) was added anhydrous K₂CO₃(3.83 g, 5.1 equiv, 27.71 mmol).

Stirring was continued at room temperature for 1.5 h. The solvent was

removed under reduced pressure, and the residue was extracted with

diethyl ether (3 × 50 mL). The organic extract was washed with brine

(75 mL), dried over MgSO₄, ﬁltered, and concentrated under reduced

pressure. The crude product was puriﬁed by silica gel ﬂash column

chromatography using EtOAc-hexanes (2:98, v/v) as eluents to aﬀord

17b as a white solid in 88% (1.06 g) yield. The product was used as

such in the next reaction without further characterization.

1

found 514.2596 [M + H]⁺, calculated 514.2488. H NMR (CDCl₃,

400 MHz, 298 K) δ (ppm): 7.48−7.27 (m, 10H), 6.99 (d, J = 1.8 Hz,

1H), 6.82 (dd, J = 8.2, 1.6 Hz, 1H), 6.77 (d, J = 8.3 Hz, 1H), 5.92

(ddd, J = 22.6, 10.8, 5.6 Hz, 1H), 5.37−5.26 (m, 1H), 5.26−5.19 (m,

2H), 5.16 (s, 2H), 5.06 (s, 2H), 4.69 (dd, J = 14.0, 5.8 Hz, 1H), 4.58

(d, J = 5.5 Hz, 2H), 3.43−3.32 (m, 1H), 3.09 (d, J = 5.8 Hz, 2H),

2.05−1.96 (m, 2H), 1.82−1.72 (m, 2H), 1.72−1.62 (m, 2H), 1.61−

1.50 (m, 2H). ¹³C NMR (CDCl_3,101 MHz, 298 K) δ (ppm): 171.68,

155.82, 155.60, 137.58, 135.25, 132.73, 128.68, 128.62, 128.52,

128.38, 127.96, 127.84, 127.57, 127.31, 127.22, 117.86, 111.86, 70.18,

67.20, 65.87, 55.02, 39.13, 37.67, 33.09, 33.06, 25.57. Alloc-

cypTyr(OBn)-OBn (1.93 g, 3.67 mmol) was dissolved in EtOH

(20 mL), and an aqueous solution of KOH (2.2 mL, 2 M) was added.

In case the mixture was still transparent, more water was added until it

turned cloudy. The suspension was heated to 70 °C for 15 min. The

mixture was acidiﬁed with 5 mL of HCl 1 M and extracted with

EtOAc. The organic phase was dried and the crude product was

puriﬁed using ﬂash chromatography (20−30% AcOEt in hexane +

0.25% AcOH in 10 CV) to obtain 15a. Alloc-dcypTyr(OBn)-OH

(S,E)-4-(4-(Benzyloxy)phenyl)-1-((1R,2S,5R)-2-isopropyl-5-meth-

ylcyclohexyl)-3-methylbut-2-en-1-ol (18). To a stirred suspension of

bis-(cyclopentadienyl) zirconium(IV) dichloride (0.61 g, 0.22 equiv,

2.08 mmol) in dry DCM (0.22 M, 33 mL) at 0 °C under argon was

added dropwise a solution of trimethyl aluminum (2 M in toluene,

14.7 mL, 3.1 equiv, 29.3 mmol), and the solution was stirred for 15

min. A solution of alkyne (2.1 g, 1.0 equiv, 9.45 mmol) in DCM (10

mL) was added dropwise; the reaction mixture was stirred at room

temperature for 16 h and then cooled to −78 °C before a solution of

freshly distilled p-menthane-3-carboxaldehyde, synthesis described in

Supporting Information, (2.07 g, 1.3 equiv, 12.28 mmol) in THF (0.4

M, 24 mL) was added dropwise. The reaction mixture was stirred at

the same temperature for 2 h and then allowed to return to room

temperature. The reaction mixture was cooled to 0 °C and quenched

with a cold K₂CO₃0.5 M solution (80 mL). The phases were

separated, and the aqueous phase was extracted with Et₂O (3 × 70

mL). All organic phases were combined and washed with brine, dried

over MgSO₄, ﬁltered, and concentrated under reduced pressure. The

crude compound was puriﬁed by ﬂash chromatography using

EtOAc:hexane (4:96 to 20:80% in 10 CV) as the eluant to aﬀord

the desired compound 18 as a colorless oil (1.79 g, 47% yield), (<5%

of the other diastereomer by ¹H NMR). Yield: 47% (1.79 g);

chemical formula: C₂₈H₃₈O₂; MW: 406.61 g/mol; HRMS m/z: [M +

1

(15a): HRMS: found 422.1981 [M − H]⁻, calculated 422.1973. H

NMR (CDCl₃, 400 MHz, 298 K) δ (ppm): 7.46−7.29 (m, 5H), 7.04

(d, J = 1.1 Hz, 1H), 6.94 (dd, J = 8.3, 2.0 Hz, 1H), 6.83 (d, J = 8.3 Hz,

1H), 5.90 (ddd, J = 22.6, 10.8, 5.6 Hz, 1H), 5.35−5.11 (m, 3H), 5.05

(s, 2H), 4.65 (dd, J = 13.6, 5.9 Hz, 1H), 4.57 (d, J = 5.4 Hz, 2H),

3.46−3.33 (m, 1H), 3.15 (dd, J = 14.0, 5.4 Hz, 1H), 3.07 (dd, J =

14.1, 6.2 Hz, 1H), 2.06−1.96 (m, 2H), 1.81−1.50 (m, 6H). ¹³C NMR

(CDCl₃, 101 MHz, 298 K) δ (ppm): 176.94, 155.90, 155.86, 137.55,

135.47, 132.58, 128.64, 128.02, 127.86, 127.45, 127.27 (peak

overlap), 118.06, 111.95, 70.22, 66.10, 54.78, 39.08, 37.23, 33.10,

25.60 (25.59). The same protocol was used for synthesis of Alloc-

dcypTyr(OBn)-OH (15b): HRMS: found 492.2758 [M + H]⁺,

1

Na]⁺calculated for C₂₈H₃₈O₂: 406.2872, found 406.2868; H NMR

(CDCl₃, 300 MHz, 298 K) δ (ppm) 7.45−7.26 (m, 5H), 7.09 (d, J =

8,64 Hz, 2H), 6.90 (d, J = 9 Hz, 2H), 5.43 (dd, J = 8.07, 1.14 Hz,

1H), 5.05 (s, 2H), 4.68 (d, J = 8.07 Hz, 1H), 3.26 (s, 2H), 2.16 (m,

1H), 1.92 (m, 1H), 1.75−1.63 (m, 2H), 1.60 (d, J = 1.15 Hz, 3H),

1.35−1.27 (m, 3H), 1.26 (t, J = 7.2 Hz, 1H), 0.98−0.86 (m, 9H),

0.78 (d, J = 6.91 Hz, 3H); ¹³C NMR (CDCl₃, 300 MHz, 298 K) δ

(ppm) 157.4, 137.29, 136.27, 132.18, 129.90, 128.92, 128.64, 127.98,

127.56, 114.81, 70.15, 67.97, 45.53, 45.08, 43.26, 35.26, 34.29, 32.95,

26.45, 24.38, 23.06, 21.75, 16.41, 15.69.

(S,E)-4-(4-(Benzyloxy)phenyl)-1-((1R,2S,5R)-2-isopropyl-5-meth-

ylcyclohexyl)-3-methylbut-2-enyl Carbamate (19). To a stirred

solution of alcohol (1.9 g, 1.0 equiv, 4.67 mmol) in DCM (0.12 M, 40

mL) at 0 °C under argon was added dropwise trichloroacetyl

isocyanate (2.64 g, 3.0 equiv, 14 mmol), and the solution was stirred

for 1 h at 0 °C (consumption of the alcohol was monitored by TLC).

The solvent was evaporated under reduced pressure, and the crude

intermediate was dissolved in methanol (0.15 M, 31 mL) and water

(0.8 M, 16 mL) followed by addition of potassium carbonate (1.94 g,

3.0 eq, 14 mmol) at 0 °C. The reaction mixture was then stirred at

room temperature overnight (∼16 h). Methanol from the reaction

mixture was evaporated, and the aqueous phase was extracted with

DCM (3 × 50 mL). The combined organic phases were washed with

brine (70 mL), dried over anhydrous MgSO₄, ﬁltered, and

concentrated under reduced pressure. The crude compound was

puriﬁed by silica gel ﬂash chromatography with EtOAc-hexanes (5:95

1

calculated 492.2745. H NMR (CDCl₃, 400 MHz, 298 K) δ (ppm):

7.36−7.13 (m, 5H), 6.75 (s, 2H), 5.74 (ddd, J = 22.4, 10.8, 5.6 Hz,

1H), 5.25−4.89 (m, 3H), 4.65 (s, 2H), 4.58−4.45 (m, 1H), 4.42 (d, J

= 1.4 Hz, 2H), 3.33−3.14 (m, 2H), 3.02 (dd, J = 14.0, 5.3 Hz, 1H),

2.93 (dd, J = 14.0, 6.2 Hz, 1H), 1.94−1.80 (m, J = 13.0, 11.5, 5.7 Hz,

4H), 1.72−1.58 (m, 4H), 1.56−1.44 (m, 4H), 1.44−1.30 (m, 4H).

¹³C NMR (CDCl₃,101 MHz, 298 K) δ (ppm): 177.26, 156.07,

154.43, 140.36, 138.14, 132.87, 131.74, 128.99, 128.37, 127.87,

126.07, 118.35, 76.92, 66.41, 54.96, 38.71, 38.04, 35.73, 35.71, 26.35.

Synthesis of (3-(4-(Benzyloxy)phenyl)prop-1-ynyl)trimethylsilane

(17a). To a stirred solution of 4-benzyloxybenzyl chloride (3.05 g, 1.0

equiv, 13.11 mmol) in dry acetonitrile (0.15 M, 87 mL) at room

temperature under argon, puriﬁed CuI (2.5 g, 1.0 equiv, 13.11 mmol)

was added, followed by K₂CO₃(3.62 g, 2.0 equiv, 26.21 mmol) and

tetrabutylammonium iodide (4.84 g, 1.0 equiv, 13.11 mmol). After 5

min of stirring, ethynyltrimethylsilane (2.22 mL, 1.2 equiv, 15.75

mmol) was added dropwise as a neat liquid and the mixture was

stirred at 40 °C for 24 h. The reaction mixture was diluted with a

saturated ammonium chloride solution (50 mL) and extracted with

diethyl ether (3 × 50 mL). The organic phase was dried over MgSO₄,

ﬁltered, and concentrated under reduced pressure. The crude product

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to 20:80) as eluants to aﬀord the desired compound 19 as a colorless

oil.

(0.75 g, 4.0 equiv, 8.28 mmol) and sodium hydrogen phosphate (1.18

g, 4.0 equiv, 8.28 mmol) in distilled water (20 mL) was added, and

stirring was continued at room temperature for 15 h (the

consumption of aldehyde was monitored by TLC). The mixture

was concentrated under reduced pressure, and the residue was diluted

with brine (50 mL) and extracted with DCM (3 × 50 mL). The

combined organic phases were dried over MgSO₄, ﬁltered, and

concentrated under reduced pressure. The crude compound (yellow

oil) was puriﬁed by silica gel ﬂash column chromatography using

methanol-DCM (2/98) to aﬀord the title compound 22 as a white

solid. HRMS m/z: [M + Na]⁺calculated for C₃₂H₂₉NO₅507.2046,

(S,E)-4-(4-(Benzyloxy)phenyl)-1-((R,E)-2-isocyanato-4-

((1S,2S,5R)-2-isopropyl-5-methylcyclo hexyl)-2-methylbut-3-enyl)-

benzene (20). To a stirred solution of carbamate 19 (1.5 g, 1.0

equiv, 3.34 mmol) in CH₂Cl₂(0.13 M, 26 mL) at 0 °C under argon

was added triethylamine (1.39 mL, 3.0 equiv, 10 mmol) followed by

dropwise addition of triﬂuoroacetic anhydride (0.47 mL, 1.0 equiv,

3.34 mmol) (freshly distilled over P₂O₅). The solution was stirred for

15 min at 0 °C (consumption of carbamate 19 was monitored by

TLC), and then, the reaction mixture was quenched with a saturated

NH₄Cl solution (50 mL). The phases were separated, and the

aqueous phase was extracted with Et₂O (3 × 20 mL). The combined

organic phases were washed with brine (50 mL), dried over MgSO₄,

ﬁltered, and concentrated under reduced pressure to aﬀord the

isocyanate as a pale-yellow oil. HRMS m/z: [M + Na]⁺calculated for

C₂₉H₃₇NO₂: 431.2824, found 431.2814; ¹H NMR (CDCl₃, 300 MHz,

298 K) δ (ppm) 7.46−7.30 (m, 5H), 7.10 (d, J = 8.7 Hz, 2H), 6.91

(d, J = 8.7 Hz, 2H), 5.38 (d, J = 9.7 Hz, 1H), 5.36 (s, 1H), 5.05 (s,

2H), 2.77 (s, 2H), 1.97−1.85 (m, 1H), 1.75−1.69 (m, 2H), 1.63−

1.51 (m, 2H), 1.38 (s, 3H), 1.35−1.26 (m, 1H), 1.08−0.90 (m, 4H),

0.88 (d, J = 1.90 Hz, 3H), 0.86 (d, J = 2.43 Hz, 3H), 0.70 (d, 3H, J =

6.89 Hz). ¹³C NMR (CDCl₃, 300 MHz, 298 K) δ (ppm) 157.98,

137.18, 134.21, 133.00, 131.82, 128.64, 128.00, 127.58, 123.41,

114.40, 70.08, 61.82, 48.85, 47.24, 44.24, 43.14, 35.21, 32.52, 28.77,

28.22, 24.26, 22.69, 21.49, 15.52.

1

found 507.2038; H NMR (CDCl₃, 300 MHz, 298 K) δ (ppm) 7.77

(d, J = 7.50 Hz, 2H), 7.58 (t, J = 7.16 Hz, 2H) 7.46−7.34 (m, 9H),

6.95 (s, 2H), 6.83 (d, J = 8.46 Hz, 2H), 5.32 (s, 1H), 4.99 (s, 2H),

4.47 (s, 2H), 4.25 (t, J = 6.38 Hz, 1H), 3.23 (s, 2H), 1.61 (s, 3H); ¹³C

NMR (CDCl₃, 300 MHz, 298 K) δ (ppm) 178.13, 157.91, 143.90,

143.79, 141.39, 137.03, 131.05, 128.59, 127.97, 127.73, 127.50,

127.12, 125.00, 120.01, 114.70, 69.99, 66.53, 60.51, 47.31, 40.59,

23.52.

Ethyl 2-Benzyl-2-nitro-3-phenylpropanoate (56, NO₂-Db_zg-OEt).

To a solution of ethyl 2-nitro-acetate (5 mL, 45.04 mmol, 1.0 equiv)

in 30 mL of anhydrous DMF under argon were added DIPEA (16.1

mL, 92.33 mmol, 2.0 equiv), tetrabutylammonium iodide (TBAI, 1.7

g, 4.5 mmol, 0.1 equiv), and benzyl bromide (11 mL, 92.33 mmol, 2.0

equiv) at 0 °C. The ice-bath was removed, and the reaction mixture

was stirred overnight at room temperature. Around 50 mL of HCl 0.1

M was added to quench the reaction. The product was extracted 3

times with 50 mL of Et₂O-hexane (1:1). The organic phase was

washed with HCl 0.1 M, dried, and concentrated under a vacuum.

The product was puriﬁed by column chromatography on silica gel.

Yield: 35% (5 g). HRMS: found 336.1211 [M + Na]⁺, calculated

(9H-Fluoren-9-yl)methyl(R,E)-1-(4-(benzyloxy)phenyl)-4-

((1S,2S,5R)-2-isopropyl-5-methylcyclohexyl)-2-methylbut-3-en-2-

ylcarbamate (21). To a solution of the isocyanate 20 (1.36 g, 1.0

equiv, 3.16 mmol) in dry benzene (0.07 M, 48.5 mL) at room

temperature under argon was added 9-ﬂuorenemethanol (0.93 g, 1.5

equiv, 4.75 mmol) as a solid followed by dropwise addition of

titanium(IV) tert-butoxide (0.24 mL, 0.2 equiv, 0.63 mmol). The

reaction mixture was then heated at 45 °C for 3 h and quenched with

a saturated NH₄Cl solution. The phases were separated, and the

aqueous phase was extracted with diethyl ether (3 × 50 mL). The

combined organic phases were washed with brine (75 mL), dried over

MgSO₄, ﬁltered, and concentrated under reduced pressure. The crude

compound was puriﬁed by silica gel ﬂash column chromatography

using diethyl ether-hexanes (8:92 to 2:8 in 10 CV) to aﬀord the

desired product as a white solid. HRMS m/z: [M + Na]⁺calculated

1

336.1206. H NMR (CDCl₃, 400 MHz, 298 K) δ (ppm): 7.35−7.28

(m, 6H), 7.22−7.15 (m, 4H), 4.13 (q, J = 7.2 Hz, 2H), 3.49 (s, 4H),

1.13 (t, J = 7.2 Hz, 3H); ¹³C NMR (CDCl₃, 101 MHz, 298 K) δ

(ppm): 166.48, 133.37, 130.27, 128.79, 128.01, 97.35, 62.89, 40.20,

13.70.

Ethyl 2-Amino-2-benzyl-3-phenylpropanoate (57, H₂N-Db_zg-

OEt). To a solution of NO₂-Db_zg-OEt (56, 200 mg, 0.64 mmol, 1

equiv) in 3 mL of AcOH, Zn dust (376 mg, 5.75 mmol, 9 equiv, size

<10 mm) was added for a period of 15 min. Five drops of HCl 1 M

were added to accelerate the reaction. After the reaction was ﬁnished

(followed up by TLC), the mixture was ﬁltered over Celite (wetted

with EtOH), and the solid was washed several times with EtOH. The

combined solution was concentrated under a vacuum, resulting in

precipitation of acetate salt. Saturated NaHCO₃was added to liberate

the free base compound, and this intermediate was extracted with

EtOAc. The organic phase was washed with brine, dried with MgSO₄,

ﬁltered, and evaporated to dryness to aﬀord the pure product without

further puriﬁcation. Yield: 70% (126 mg). HRMS: found 284.1649

[M + H]⁺, calculated 284.1645. ¹H NMR (MeOD-d₄, 400 MHz, 298

K) δ (ppm): 7.46−7.06 (m, 10H), 4.08 (q, J = 7.2 Hz, 2H), 3.35 (d, J

= 13.3 Hz, 2H), 2.83 (d, J = 13.3 Hz, 2H), 1.22 (t, J = 7.2 Hz, 3H);

¹³C NMR (MeOD-d₄, 101 MHz, 298 K) δ (ppm): 176.39, 137.24,

1

for C₄₃H₄₉NO₃: 627.3712, found: 627.3708; H NMR (CDCl₃, 300

MHz, 298 K) δ (ppm) 7.77 (d, 2H, J = 7.46 Hz), 7.60 (d, 2H, J =

7.37 Hz) 7.46−7.29 (m, 9H), 6.99 (s, 2H), 6.84 (d, 2H, J = 8.51 Hz),

5.56 (d, 1H, J = 15.00 Hz), 5.21 (dd, 1H, J = 15.57, 9.57 Hz), 5.02 (s,

2H), 4.60 (s, 1H), 4.43 (s, 2H), 4.25 (t, 1H, J = 6.57 Hz), 2.98 (s,

2H), 1.89 (d, 1H, J = 9.37 Hz), 1.72 (d, 2H, J =12.49 Hz), 1.59 (m,

3H), 1.37 (s, 3H), 1.32−1.25 (m, 1H), 1.05−0.94 (m, 3H), 0.87 (d,

3H, J = 2.09Hz), 0.85 (d, 3H, J = 2.64 Hz), 0.72 (d, 3H, J = 6.85 Hz).

¹³C NMR (CDCl₃, 300 MHz, 298 K) δ (ppm) 157.60, 144.19,

141.45, 137.26, 134.00, 133.70, 131.70, 129.71, 128.66, 128.01,

127.73, 127.55, 127.12, 125.13, 120.06, 114.34, 70.08, 66.04, 56.41,

47.53, 47.29, 44.68, 44.46, 43.29, 35.25, 32.54, 28.16, 25.26, 24.22,

22.72, 21.57, 15.58.

131.09, 129.52, 128.22, 64.08, 62.34, 46.92, 14.46.

Fmoc-^D-α-MeTyr(OBn)-OH (22). To a stirred solution of the

carbamate 21 (1.3 g, 1.0 equiv, 2.07 mmol) and Sudan(III) (1 mg)

(used as indicator) in CH₂Cl₂(41.5 mL, 0.05 M) at −78 °C was

bubbled ozone until the red-colored solution became orange (∼20

min). Triphenylphosphine (2.72 g, 5.0 equiv, 10.35 mmol) was added

to the intermediate ozonide solution at 0 °C and stirred at room

temperature for 15 h. The solvent was evaporated under reduced

pressure, and the orange solid obtained was ﬁltered through a small

silica gel bed eluting with hexane and diethyl ether (80:20). The

ﬁltrate was concentrated under reduced pressure to aﬀord the

intermediate aldehyde (49 in Supporting Information) (1.04 g) as a

colorless oil.

Fmoc-Db_zg-OH (59). To a solution of H₂N-Db_zg-OEt (57, 1.81 g,

6.4 mmol) dissolved in 21 mL of EtOH, 42 mL of KOH 2 M was

added. The mixture was heated to reﬂux for 2 h. The mixture was

concentrated in vacuo until half the volume, and 37% HCl was added

dropwise at 0 °C until the product precipitates (pI ∼ 6.5). The

product was ﬁltered and rinsed with iced water. Then, 1.46 g of 58

(NH₂-Db_zg-OH) was obtained in a yield of 90%. The product was

dried under a fume hood and was used as such in the next reaction.

Free amino acid NH₂-Db_zg-OH (58, 1.46 g, 5.73 mmol, 1 equiv) was

suspended in CH₂Cl₂(10 mL), and DIPEA (3 mL, 17.18 mmol, 3

equiv) was added. Chlorotrimethylsilan (1.5 mL, 11.45 mmol, 2

equiv) was added slowly to the mixture (smoke appear) followed by

10 mL of DCM. This mixture was stirred for 10 min (until a clear

solution was obtained). Fmoc-Cl (1.48 g, 5.73 mmol, 1 equiv) was

added, and the mixture was stirred for 24 h and monitored by LC-MS

(sample prepared in MeOH, which allows one to cleave the TMS-

[Pinnick oxidation]: To a stirred solution of the intermediate

aldehyde (1.04 g, 2.07 mmol) in t-butanol (0.1 M, 20 mL) was added

2-methyl-2-butene (4.4 mL, 20.0 equiv, 41.4 mmol) as a neat liquid,

and the mixture was cooled to 0 °C. A solution of sodium chlorite

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protecting group). When the reaction remained unchanged, the

organic phase was washed with 1 M HCl. The solvent was evaporated

in vacuo, and the mixture was puriﬁed with MeOH-DCM (5:95) +

0.25% CH₃COOH to provide 2 g of product, yield 73%. HRMS:

incubated at 37 °C under a humid atmosphere maintaining 5% CO₂.

G418 (400 μg/mL) was used to create a selection pressure for cells

expressing APJ, and penicillin/streptomycin (0.1%) was added to

prevent bacteria contamination.

1

found 500.1842 [M + Na]⁺, calculated 500.1832. H NMR (CDCl₃,

Binding Experiments. Binding experiments were carried out on

HEK293 cell membranes stably expressing the YFP-tagged human

APJ receptor. Cells were frozen at −80 °C for storage and only

thawed right before the experiments. For membrane extraction, cells

were suspended in 4 mL of EDTA solution (1 mM EDTA and 50

mM Tris-HCl, pH 7.4), transferred to a 10 mL falcon tube, and

centrifuged at 3500 rpm for 15 min at 4 °C. The precipitate was

suspended in binding buﬀer (50 mM Tris-HCl, 0.2% BSA, pH 7.4).

Binding assay was carried out in 96-well plates: 15 μg of membrane

proteins was incubated with 0.2 nM radiolabeled [¹²⁵I][Nle75,

Tyr77]Pyr-apelin-13 (820 Ci/mmol)⁷⁴and the test ligand with a

range of concentrations from 10⁻⁵to 10⁻¹¹M or 10⁻⁷to 10⁻¹³M in a

total volume of 200 μL for 1 h at room temperature. The incubation

mixtures were ﬁltered through a glass ﬁber ﬁlter (Millipore,

preabsorbed of PEI 0.5% for 2 h at 4 °C) to remove unbound

ligands, and the ﬁltered membranes were washed three times with 170

μL of cold binding buﬀer (4 °C). The γ emission was measured using

a γ-counter 1470 Wizard from PerkinElmer (Waltham) (80%

eﬃciency). Nonspeciﬁc binding did not exceed over 5% of the total

signal (determined by incubation with 10⁻⁵M unlabeled Pyr-apelin-

13). IC₅₀values, determined from those results using GraphPad Prism

8, represent the concentration of the tested ligand displacing 50% of

the radiolabeled ligand from the receptor. The K_Dof Pyr-apelin-13 is

1.8 nM, determined by the saturation binding assay. The dissociation

constant K_ivalue was calculated from IC₅₀using the Cheng−Prusoﬀ

equation, and results were displayed as mean SEM of two to three

independent experiments, each done in duplicate.⁶⁹

BRET Assays for Gα_i1Activation and β-Arrestin2 Recruitment.

HEK293 cells seeded in T175 ﬂasks were allowed to grow in high-

glucose DMEM supplemented with 10% FBS, 100 U/mL penicillin/

streptomycin, 2 mM glutamine, and 20 mM 4-(2-hydroxyethyl)-1-

piperazineethanesulfonic acid (HEPES) at 37 °C in a humidiﬁed

chamber at 5% CO₂. All transfections were carried out with PEI. After

24 h, cells were transfected with the plasmids coding for hAPJ, Gα_i1-

RlucII(91), GFP10-Gγ₂, and G_β1(from cDNA.org) (for the BRET-

based Gα_i1activation assay) or coding for hAPJ-GFP10 and RlucII-β-

arrestin2.^42,75,76To perform BRET assays, cells were transferred into

white 96-well plates BD Bioscience (Mississauga, Canada) at a

concentration of 50 000 cells/well 24 h after transfection and

incubated at 37 °C overnight. Cells were then washed with

phosphate-buﬀered saline (PBS), and 90 μL of Hanks’ balanced salt

solution (HBSS) was added to each well. Then, cells were stimulated

with analogues at concentrations ranging from 10⁻⁵M to 10⁻¹¹M for

5 min at 37 °C (Gα_i1) or for 30 min at room temperature (β-

arrestin2). After stimulation, 5 μM coelanterazine 400A was added to

each well and the plate was read using the BRET²ﬁlter set of a

GeniosPro plate reader (Tecan, Austria). The BRET²ratio was

determined as GFP10_em/RlucII_em. Data were plotted, and EC₅₀values

were determined using GraphPad Prism 8. Each data point represents

the mean SEM of at least three diﬀerent experiments each done in

triplicate.

400 MHz, 298 K) δ (ppm): 7.81 (d, J = 7.5 Hz, 2H), 7.57 (d, J = 7.5

Hz, 2H), 7.44 (t, J = 7.4 Hz, 2H), 7.33 (td, J = 7.5, 0.8 Hz, 2H),

7.29−7.20 (m, 6H), 7.18−7.09 (m, 4H), 5.52 (s, 1H, amide proton),

4.51 (d, J = 7.0 Hz, 2H), 4.30 (t, J = 6.9 Hz, 1H), 3.91 (d, J = 13.7 Hz,

2H), 3.27 (d, J = 13.6 Hz, 2H); ¹³C NMR (CDCl₃, 101 MHz, 298 K)

δ (ppm): 177.27, 154.76, 143.93, 141.47, 135.88, 129.85, 128.58,

127.89 (two peaks superposed), 127.25, 125.31, 120.13, 66.82, 66.67,

47.40, 41.24.

Synthetic Procedures and Compound Characterization

(Solid-Phase Synthesis). Materials. 2-Chlorotrityl chloride resin

(maximum loading 0.85 mmol/g, particle size 100−200 mesh), Wang

resin (loading 0.3−1 mmol/g, particle size 100−200 mesh), and O-

(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexaﬂuorophos-

phate (HATU) were obtained from Matrix Innovation (Quebec,

Canada). N,N-diisopropylethylamine (DIPEA), triﬂuoroacetic acid

(TFA), and Fmoc-protected amino acids were purchased from Chem-

Impex International (Wood Dale) or Combi-Blocks (San Diego).

Polypropylene cartridges 12 mL with 20 μm PE frit were purchased

from Applied Separation (Allentown). Other reagents and solvents

were purchased from Sigma-Aldrich (Missouri) and Fisher Scientiﬁc

(Hampton). Reagents and solvents were used as received.

Solid-Phase Synthesis. The present study employs unnatural and

conformationally constrained Pro12 and Phe13 mimetics to screen

the binding space in the Ape13 sequence. All noncommercial amino

acids were synthesized in solution and incorporated into the peptide

using conventional Fmoc-based solid-phase peptide synthesis (SPPS).

All peptides were synthesized manually via Fmoc-based solid-phase

peptide synthesis (SPPS) on 2-chlorotrityl chloride resin. First, the

residue was loaded into the resin using 1.2 equiv of Fmoc-AA-OH and

2.5 equiv of DIPEA in DCM overnight (loading 0.45−0.70 mmol/g).

Attempts to load Fmoc-Db_zg-OH on 2-chlorotrityl chloride resin were

unsuccessful; thus, it was loaded to the Wang resin as described in the

Supporting Information. Peptide couplings were performed with 3

equiv of Fmoc-AA-OH, 3 equiv of HATU, and 5 equiv of DIPEA for

30 min at room temperature. The reaction was monitored by the

standard Kaiser test.⁷³Fmoc deprotection was carried out by

treatment of the resin with 4-methylpiperidine or piperidine in

DMF (20:80 v/v) for 2 × 15 min. After every coupling and

deprotection step, the resin was washed with DMF (3×) and CH₂Cl₂

(3×). Final cleavage (and side-chain deprotection) of the peptides

from the resin was performed for 3−4 h in TFA/triethylsilane (TES)/

H₂O (95:2.5:2.5 v/v) at room temperature. The resin was ﬁltered,

and the ﬁltrate was concentrated in vacuo. Final peptides were puriﬁed

via preparative RP-HPLC and immediately lyophilized. The peptides

were obtained as white powders with a purity of >95% as determined

by analytical RP-HPLC or UPLC-MS, except for 44 with purity 92%.

Purity assessment was carried out on a UPLC-MS system from

Waters (Milford) (column Acquity UPLC CSH C₁₈(2.1 mm × 50

mm) packed with 1.7 μm particles) with the following gradient:

acetonitrile and water with 0.1% formic acid (0 → 0.2 min: 5%

acetonitrile; 0.2 → 1.5 min: 5% → 95%; 1.5 → 1.8 min: 95%; 1.8 →

2.0 min: 95% → 5%; 2.0 → 2.5 min: 5%). The structures were

conﬁrmed by high-resolution electrospray mass spectrometry (maXis

ESI-Q-Tof apparatus from Bruker, Billerica) and after preparing a

stock solution were used as such in in vitro and in vivo assays.

In Vitro and In Cellulo Assays. Materials. High-glucose

Dulbecco’s modiﬁed Eagle’s medium (DMEM), G418, and

penicillin/streptomycin were obtained from Invitrogen Life Tech-

nologies (Carlsbad). Fetal bovine serum (FBS) was purchased from

Wisent (Saint-Jean-Baptiste, Canada), and bovine serum albumin

(BSA) was from BioShop (Burlington, Canada). Polyethylenimine

(PEI) was ordered from Polysciences (Warminster).

BRET Assay for Gα₁₂Activation. The pcDNA3.1 plasmid encoding

human APJ, human Gα₁₂, human G_β1, and human G_γ1was purchased

from Origene (Rockville, MD). PDZ-Rho-Gef-RLucII and rGFP-

CAAX have been previously described.⁷⁷HEK293T cells were seeded

in 150 cm²ﬂasks (Corning, Corning, NY), and 48 h before the

experiments, they were transiently transfected with the hAPJ in

combination with BRET-based biosensor cDNA in 96-well culture

plates (Corning, Corning, NY) (35 000 cells/well). Transient

transfections were performed using PEI at a ratio of 3:1 PEI/DNA.

The total amount of transfected DNA was kept constant (100 ng/

well) by the addition of salmon sperm DNA. Measurement. Gα₁₂

activation was assessed by monitoring ebBRET between PDZ-

RhoGEF-RLucII and rGFP-CAAX coexpressed with Gα₁₂, Gβ₁, and

Gγ₁. Forty-eight hours following transfection with the appropriate

biosensors and hAPJ, cells were stimulated with the ligands for 5 min.

Cell Culture. Human embryonic kidney cells (HEK293) stably

expressing yellow ﬂuorescent protein (YFP)-tagged human APJ were

cultured using the DMEM medium with 10% FBS. Cells were

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The luciferase substrate, coelenterazine 400A (2.5 μM), was added 5

min before the reads. BRET was monitored with a Mithras LB 940

(Berthold Technologies, Bad Wildbad, Germany) equipped with a

410/70 nm donor ﬁlter and a 515/20 nm acceptor ﬁlter. The data

were analyzed in GraphPad Prism 8 using “dose−response-

stimulation log(agonist) vs normalized response-variable slope” with

the constraint of sharing the Hill slope across all data sets. Data were

presented as mean SEM of eight diﬀerent experiments each done in

simplicate.

Rat Plasma Stability. In a 96-well plate, 6 μL of compound (1 mM

aqueous solution) was incubated with 27 μL of plasma (from male

Sprague−Dawley rat) at 37 °C for 0, 1, 2, 4, 7, and 24 h (or 0, 5, 10,

20, 60, 120 min for less stable analogues). Degradation was quenched

by adding 140 μL of acetonitrile-ethanol (1:1) solution containing

N,N-dimethylbenzamide 0.25 mM (internal standard).⁷⁸This mixture

was transferred to a ﬁlter plate Impact Protein Precipitation

(Phenomenex, California), and another 96-well UPLC plate was

placed at the bottom to collect ﬁltrates. Both plates were centrifuged

at 500 g for 10 min at 4 °C. The ﬁltrates were diluted with 80 μL of

distilled water and analyzed using an Acquity UPLC-MS system class

H (column Acquity UPLC protein BEH C4 (2.1 mm × 50 mm), 1.7

μm particles with pore 300 Å). Peptide half-life was calculated from

the degradation curve using the exponential one-phase decay function

in GraphPad Prism 8. Mass spectra at the time point near half-life

were compared with those at 0 min (plasma-inactivated before adding

the compound) to identify cleavage fragments. Data were presented

as mean SEM of at least three diﬀerent experiments each done in

simplicate.

In Vivo Pharmacokinetics. Male Sprague−Dawley rats weighing

250−300 g, of 8−10 weeks of age, were used in this study. A jugular

vein catheter (Silastic Laboratory tubing; 0.02 in I.D. × 0.037 in

O.D.) was surgically inserted 24 h prior to dosing to minimize

possible anesthetic eﬀects. This catheter was used for intravenous

injections (i.v., n = 3 for analogue 47 or 53 in saline solution 0.9%,

≈350 μL) and to withdraw blood. Injection volumes were determined

according to the weight of the animals (3 mg/kg). Animals were

placed in a containment chamber prior to i.v. injection to facilitate

blood sampling. Blood samples (0.2 mL to obtain about 0.1 mL of

plasma after centrifugation) were taken at 5, 10, 30, 60, 120, and 240

min (1, 2, 5, 10, 15 min for Pyr-apelin-13) following i.v.

administration. Blood sample collection was made in K2-EDTA

microtainer tubes (Sarstedt, Numbrecht, Germany) and immediately

̈

placed on crushed ice before being centrifuged at 13 000 rpm for 5

min at 4 °C to isolate plasma from blood cells. The resultant plasma

was then separated and transferred to polypropylene tubes and

immediately frozen at −80 °C.

Sample Preparation. Plasma extraction of analogues 47 and 53

was performed by a combination of a protein precipitation and a

solid-phase extraction step by LC/MS/MS analysis. The plasma

sample was defrosted on ice. After vortex agitation (60 s), 100 μL was

withdrawn and 300 μL of cold acetonitrile was added for protein

precipitation. The sample was then vortexed (60 s) and centrifuged at

4500 rpm at 4 °C for 10 min. The supernatant was then withdrawn

and directly passed through an HLB prime for additional cleanup.

The ﬁltrate was diluted 10 times in eluent A or B according to its

solubility and then ﬁltered through a 0.22 μm syringe ﬁlter before

LC/MS/MS analysis.

Stability against ACE2. Recombinant rat ACE2 (Sino Biological)

was reconstituted in Milli-Q water and diluted to a ﬁnal concentration

of 20 nM in an assay buﬀer (100 mM Tris-HCl, 100 mM NaCl, 10

μM ZnCl2, pH 7.4). First, 1.7 μL of analogue (1 mM) was mixed with

1.7 μL of N,N-dimethylbenzamide 5 mM (internal standard) in a 96-

well plate. To initiate the enzymatic reaction, 30 μL of enzyme

solution was added to the mixture and incubated for 1, 2, and 24 h at

37 °C on an orbital shaker (300 rpm). At the corresponding time

point, the enzyme was inactivated by adding 30 μL of EDTA 0.1 M

(prepared in Milli-Q water and ﬁltered). The blank (t = 0 min) for the

ACE2 assay was prepared with similar components, but the enzyme

was inactivated before adding to the mixture. The remaining peptide

after incubation was quantiﬁed using a UPLC-MS system from Waters

(Milford) (Acquity UPLC Protein BEH C4 column (2.1 mm × 50

mm) packed with 1.7 μm particles, pore 300 Å) with the following

gradient: acetonitrile and water with 0.1% HCOOH (0 → 0.2 min:

5% acetonitrile; 0.2 → 1.5 min: 5% → 95%; 1.5 → 1.8 min: 95%; 1.8

→ 2.0 min: 95% → 5%; 2.0 → 2.5 min: 5%), and the MS detector was

set to the single-ion mode (SIM). Trp and Aia residues were oxidized

(+16, +32) in the presence of EDTA-Zn, and a sum of oxidized and

nonoxidized form was used for calculation. Experiments were

repeated three times.

Mass Spectrometry Analysis. Analysis of analogue 47 or 53 was

performed on a Sciex Qtrap 6500+ equipped with microﬂow liquid

chromatography (Eksigient M3 microﬂow). A UPLC HSS-T3 column

(1 mm × 100 mm, 1.8 μm, equipped with a 0.2 μm fritted preﬁlter)

was used for the chromatographic separation. The solvent ﬂow rate

was set to 50 μL/min, and the column temperature was kept at 40 °C.

The injection volume was 3 μL. The mobile phase was 0.1% formic

acid/water (A) and 0.1% formic acid/acetonitrile (B), with an elution

gradient starting with 2% of eluent B, increasing to 95% in 8 min,

maintaining at 95% for 2 min, and then back to initial conditions in 2

min for a total run time of 13 min. Optimized parameters were

obtained by direct infusion of Ape13, 47, and 53 analytical standard

solutions at 100 ng/mL. Two daughter traces (transitions) were used

(Table 1S). The most abundant transition was used for quantiﬁcation

and the second most abundant for conﬁrmation.

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01941.

■

sı

Synthetic schemes for unnatural amino acids, HRMS

spectra and analytical UPLC-MS spectra, and structures

of the investigated compounds (PDF)

Molecular formula strings of the investigated com-

pounds (CSV)

In Vivo Blood Pressure Measurement. Animals. Adult male

Sprague−Dawley rats, 8−10 weeks of age (Charles River

Laboratories, St-Constant, Quebec, Canada), were kept on a 12 h

light/12 h dark cycle with access to food and water ad libitum. The

animal experimental protocols in this study were approved by the

́

Animal Care Committee of Universite de Sherbrooke and complied

with policies and directives of the Canadian Council on Animal Care.

Blood Pressure Test. Male Sprague−Dawley rats (250−300 g, 8−

10 weeks of age) anesthetized with ketamine/xylazine injection (87/

13 mg/kg i.m.) were placed in supine position on a thermostatic pad.

A blood pressure monitoring catheter (PE 50 ﬁlled with heparinized

saline) was inserted into the right carotid artery and connected to a

Micro-Med transducer (model TDX-300, Calabasas) and a Micro-

Med blood pressure analyzer (model BPA-100c). Bolus injection of

vehicle (isotonic saline) followed 5 min later by the injection of

Ape13, compound 47, or compound 53 (given at 19.5 nmol/kg;

volume of 0.25 mL over 10 s) was carried out through another

catheter (PE10) inserted into the left jugular vein. To remove residual

injected substances, this i.v. catheter was ﬂushed with saline (0.2 mL)

immediately after each injection.

AUTHOR INFORMATION

Corresponding Author

■

Philippe Sarret − Département de Pharmacologie-Physiologie,

Faculté de Médecine et des Sciences de la Santé, Université de

Sherbrooke, Sherbrooke J1H 5N4, Québec, Canada; Institut

de Pharmacologie de Sherbrooke, Sherbrooke J1H 5N4,

Québec, Canada; orcid.org/0000-0002-7627-701X;

Email: Philippe.Sarret@USherbrooke.ca

Authors

̂

Kien Tran − Département de Pharmacologie-Physiologie,

Faculté de Médecine et des Sciences de la Santé, Université de

5360

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Article

Sherbrooke, Sherbrooke J1H 5N4, Québec, Canada; Institut

de Pharmacologie de Sherbrooke, Sherbrooke J1H 5N4,

Québec, Canada

Robin Van Den Hauwe − Research Group of Organic

Chemistry, Departments of Chemistry and Bioengineering

Sciences, Vrije Universiteit Brussel, Pleinlaan 2 1050,

Brussels, Belgium

Xavier Sainsily − Département de Pharmacologie-Physiologie,

Faculté de Médecine et des Sciences de la Santé, Université de

Sherbrooke, Sherbrooke J1H 5N4, Québec, Canada; Institut

de Pharmacologie de Sherbrooke, Sherbrooke J1H 5N4,

Québec, Canada

Pierre Couvineau − Institut de Recherche en Immunologie et

en Cancérologie (IRIC), Université de Montréal, Montréal

H3T 1J4, Québec, Canada

Claude Spino − Institut de Pharmacologie de Sherbrooke,

Sherbrooke J1H 5N4, Québec, Canada; Département de

Chimie, Faculté de Science, Université de Sherbrooke,

Sherbrooke J1K 2R1, Québec, Canada; orcid.org/0000-

0001-6249-5908

Michel Bouvier − Institut de Recherche en Immunologie et en

Cancérologie (IRIC), Université de Montréal, Montréal H3T

1J4, Québec, Canada; orcid.org/0000-0003-1128-0100

Steven Ballet − Research Group of Organic Chemistry,

Departments of Chemistry and Bioengineering Sciences, Vrije

Universiteit Brussel, Pleinlaan 2 1050, Brussels, Belgium;

orcid.org/0000-0003-4123-1641

Eric Marsault − Département de Pharmacologie-Physiologie,

‡

́

Faculté de Médecine et des Sciences de la Santé, Université de

Sherbrooke, Sherbrooke J1H 5N4, Québec, Canada; Institut

de Pharmacologie de Sherbrooke, Sherbrooke J1H 5N4,

Québec, Canada; orcid.org/0000-0002-5305-8762

̂

Jérome Coté − Département de Pharmacologie-Physiologie,

Faculté de Médecine et des Sciences de la Santé, Université de

Sherbrooke, Sherbrooke J1H 5N4, Québec, Canada; Institut

de Pharmacologie de Sherbrooke, Sherbrooke J1H 5N4,

Québec, Canada

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.0c01941

Louise Simard − Institut de Pharmacologie de Sherbrooke,

Sherbrooke J1H 5N4, Québec, Canada; Département de

Chimie, Faculté de Science, Université de Sherbrooke,

Sherbrooke J1K 2R1, Québec, Canada

Marco Echevarria − Institut de Pharmacologie de Sherbrooke,

Sherbrooke J1H 5N4, Québec, Canada; Département de

Chimie, Faculté de Science, Université de Sherbrooke,

Sherbrooke J1K 2R1, Québec, Canada

Alexandre Murza − Département de Pharmacologie-

Physiologie, Faculté de Médecine et des Sciences de la Santé,

Université de Sherbrooke, Sherbrooke J1H 5N4, Québec,

Canada; Institut de Pharmacologie de Sherbrooke,

Sherbrooke J1H 5N4, Québec, Canada

Alexandra Serre − Département de Pharmacologie-Physiologie,

Faculté de Médecine et des Sciences de la Santé, Université de

Sherbrooke, Sherbrooke J1H 5N4, Québec, Canada; Institut

de Pharmacologie de Sherbrooke, Sherbrooke J1H 5N4,

Québec, Canada

Author Contributions

K.T. and R.V.D. H. contributed equally to this study. E.M.,

P.S., and S.B. contributed equally to directing this study.

Notes

The authors declare no competing ﬁnancial interest.

^‡In memory of our close colleague, best friend and research

́

director, Professor Eric Marsault who passed away far too

early.

ACKNOWLEDGMENTS

■

Financial support from Université de Sherbrooke, the

Canadian Institutes of Health Research, the Canada

Foundation for Innovation, and the Fonds de la Recherche

du Québec en Santé (FRQS) is gratefully acknowledged. The

Canadian Francophonie Scholarship Program (PCBF),

MITACS, and FRQS are also acknowledged for scholarship

grants to K.T., A.M., and X.S., respectively. M.A.-M. is the

recipient of a Heart and Stroke Foundation of Canada (HFSC)

New Investigator award and a research scholar from the Fonds

de la Recherche du Québec en Santé (FRQS). P.S. is the

recipient of the Canada Research Chair in Neurophysiophar-

macology of Chronic Pain. E.M. is a member of the FRQNT-

funded Proteo Network and the FRQS-funded Réseau

Québécois de Recherche sur le Médicament. R.V.D.H. and

S.B. thank the Research Council of the Vrije Universiteit

Brussel for the ﬁnancial support. S.B. also acknowledges the

Spearhead Research Program of VUB for continuous support.

Léa Théroux − Département de Pharmacologie-Physiologie,

Faculté de Médecine et des Sciences de la Santé, Université de

Sherbrooke, Sherbrooke J1H 5N4, Québec, Canada; Institut

de Pharmacologie de Sherbrooke, Sherbrooke J1H 5N4,

Québec, Canada

Sabrina Saibi − Institut de Pharmacologie de Sherbrooke,

Sherbrooke J1H 5N4, Québec, Canada

̀

Lounes Haroune − Institut de Pharmacologie de Sherbrooke,

Sherbrooke J1H 5N4, Québec, Canada; orcid.org/0000-

0001-5420-6619

Jean-Michel Longpré − Département de Pharmacologie-

Physiologie, Faculté de Médecine et des Sciences de la Santé,

Université de Sherbrooke, Sherbrooke J1H 5N4, Québec,

Canada; Institut de Pharmacologie de Sherbrooke,

Sherbrooke J1H 5N4, Québec, Canada

Olivier Lesur − Institut de Pharmacologie de Sherbrooke,

Sherbrooke J1H 5N4, Québec, Canada; Département de

Médecine spécialisé, Faculté de Médecine et des Sciences de la

Santé, Université de Sherbrooke, Sherbrooke J1H 5N4,

Québec, Canada

Mannix Auger-Messier − Institut de Pharmacologie de

Sherbrooke, Sherbrooke J1H 5N4, Québec, Canada;

Département de Médecine spécialisé, Faculté de Médecine et

des Sciences de la Santé, Université de Sherbrooke, Sherbrooke

J1H 5N4, Québec, Canada

ABBREVIATIONS

■

Ape-13, pyr-apelin-13; Ana, amino-naphthoazepinone; Aia,

amino-indoloazepinone; BRET, bioluminescence resonance

energy transfer; Bn, benzyl; DCM, dichloromethane; DIPEA,

N,N-diisopropylethylamine; cAMP, cyclic adenosine mono-

phosphate; cyp, cyclopentyl; Db_zg, α,α-dibenzylglycine; dcyp,

dicyclopentyl; DMF, N,N-dimethylformamide; EDC, 1-ethyl-

3-(3-dimethylaminopropyl)carbodiimide; ebBRET, enhanced

bystander Bioluminescence Resonance Energy Transfer; E_max

,

maximum eﬃcacy; ERK, extracellular-signal-regulated kinase;

GPCR, G-protein-coupled receptor; HATU, O-(7-azabenzo-

triazol-1-yl)-1,1,3,3-tetramethyluronium hexaﬂuorophosphate;

HBSS, Hanks’ balanced salt solution; HEK, human embryonic

kidney; HRMS, high-resolution mass spectrometry; iPrOH,

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Rajadas, J.; Kwei, S. L.; Li, M. O.; Morrison, A. R.; Quertermous, T.;

Mani, A.; Red-Horse, K.; Chun, H. J. Endothelial APLNR Regulates

Tissue Fatty Acid Uptake and Is Essential for Apelin ’s Glucose-

Lowering Effects. Sci. Transl. Med. 2017, 9, No. eaad4000.

(15) Zhao, H.; Tian, X.; He, L.; Li, Y.; Pu, W.; Liu, Q.; Tang, J.; Wu,

J.; Cheng, X.; Liu, Y.; Zhou, Q.; Tan, Z.; Bai, F.; Xu, F.; Smart, N.;

Zhou, B. Apj+ Vessels Drive Tumor Growth and Represent a

Tractable Therapeutic Target. Cell Rep. 2018, 25, 1241−1254.e5.

(16) Kleinz, M. J.; Skepper, J. N.; Davenport, A. P. Immunocy-

tochemical Localisation of the Apelin Receptor, APJ, to Human

Cardiomyocytes, Vascular Smooth Muscle and Endothelial Cells.

Regul. Pept. 2005, 126, 233−240.

(17) O’Carroll, A.-M.; Selby, T. L.; Palkovits, M.; Lolait, S. J.

Distribution of MRNA Encoding B78/Apj, the Rat Homologue of the

Human APJ Receptor, and Its Endogenous Ligand Apelin in Brain

and Peripheral Tissues. Biochim. Biophys. Acta, Gene Struct. Expression

2000, 1492, 72−80.

isopropanol; PBS, phosphate-buﬀered saline; PEI, polyethyle-

nimine; RlucII, Renilla luciferase; Pyr, pyroglutamic acid; SAR,

structure−activity relationship; SPPS, solid-phase peptide

synthesis; TBME, tert-butyl methyl ether; Tcc, 1,2,3,4-

tetrahydro-β-carboline-3-carboxylic acid; TFA, triﬂuoroacetic

acid; Tic, tetrahydroisoquinoline carboxylic acid; TIPS,

triisopropylsilane; TES, triethylsilane; UPLC, ultrahigh-per-

formance liquid chromatography; YFP, yellow ﬂuorescent

protein

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Constraining the Side Chain of C-Terminal Amino Acids in Apelin-13 Greatly Increases Affinity, Modulates Signaling, and Improves the Pharmacokinetic Profile

DOI: 10.1021/acs.jmedchem.0c01941

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