Scholarly article on FMOC-ALPHA-ME-LEU-OH 312624-65-0 from Journal of Medicinal Chemistry p. 6408

Article

pubs.acs.org/jmc

Synthetic Modiﬁcation within the “RPRL” Region of Apelin Peptides:

Impact on Cardiovascular Activity and Stability to Neprilysin and

Plasma Degradation

†

‡,§

†

Shaun M. K. McKinnie, Wang Wang, Conrad Fischer, Tyler McDonald, Kevin R. Kalin,

Xavier Iturrioz,

∥

,⊥,#

∥,⊥,#

‡,§

,†

Catherine Llorens-Cortes,

Gavin Y. Oudit, and John C. Vederas*

†

Department of Chemistry, University of Alberta, 11227 Saskatchewan Drive NW, Edmonton, Alberta T6G 2G2, Canada

Department of Medicine, University of Alberta, 8440-112 Street NW, Edmonton, Alberta T6G 2B7, Canada

Mazankowski Alberta Heart Institute, University of Alberta, 8440-112 Street NW, Edmonton, Alberta T6G 2B7, Canada

‡

§

∥

Laboratory of Central Neuropeptides in the Regulation of Body Fluid Homeostasis and Cardiovascular Functions, INSERM, U1050,

Paris, F-75005, France

⊥

Center for Interdisciplinary Research in Biology (CIRB), College de France, Paris, F-75005, France

#

CNRS, UMR 7241, Paris, F-75005, France

S Supporting Information

*

ABSTRACT: Apelin is an important mammalian peptide hormone with a range of physiological roles, especially in the car-

diovascular system. The apelinergic system is a promising target for treatment of disease, but this remains to be realized due to

rapid proteolysis of apelin-derived peptides by proteases, including neprilysin (NEP). The synthetic analogues modiﬁed within

the NEP degradation site (“RPRL” motif) showed improved in vitro proteolytic stability while maintaining receptor-binding

aﬃnities, with three candidate peptides retaining full cardiovascular activities for potential therapeutic application. Many such

analogues proved physiologically inactive even with relatively conservative modiﬁcations, highlighting the importance of this

region for full agonist activity of this peptide hormone.

INTRODUCTION

system, such as initiating vasodilation through a NO-mediated

■

17−21

2,22

23

mechanism,

positive inotropy,

angiogenesis, ₄and the

Apelin is an endogenous mammalian peptide hormone

2

1

prevention of myocardial ischemic reperfusion injury.

discovered in 1998. Genetically encoded as a 77 amino acid

Despite the potent and beneﬁcial physiological eﬀects of the

apelinergic system, the life span of apelin peptides is heavily

regulated via proteolysis. Angiotensin converting enzyme 2

prepropeptide, apelin is degraded into three major isoforms:

pyr-1-apelin-13 (1), apelin-17 (2), and apelin-36 (3), which all

possess conserved C-terminal 12 amino acids (Figure 1). These

(

ACE2) is a well-known monocarboxypeptidase that eﬃciently

ⁱ^s^o^f^o^r^m^s^h^a^v^e^dⁱ^ﬀ^e^r^eⁿ^tⁱ^a^l^dⁱ^s^t^rⁱ^b^u^tⁱ^oⁿ^sⁱⁿ^t^h^e^o^r^g^aⁿ^s^y^s^t^e^m^s2^,

catalyzes the removal of the conserved C-terminal phenyl-

with 1 being the most dominant in the cardiovascular system.

25

26

alanine from apelin isoforms in vitro and in vivo. Des-

phenylalanine apelin isoforms behave as biased agonists by

retaining native binding and forskolin-induced cAMP inhibition

but abolishing apelin receptor internalization and β-arrestin

The apelinergic system has generated scientiﬁc and thera-

peutic interest due to its diverse tissue distribution and numer-

ous roles in human physiology and pathology. When bound

to its cognate apelin receptor (APJ), a GPCR with close

homology to the angiotensin receptor, this peptide hormone

3

−6

2

7,28

7

recruitment.

Truncated analogues show a diminished

8

,9

has a role in body ﬂuid regulation, energy metabolism and

10−14

15,16

the adipoinsular axis,

and inhibiting HIV infection.

Received: May 19, 2017

Apelin has also a strong beneﬁcial role in the cardiovascular

©

XXXX American Chemical Society

A

DOI: 10.1021/acs.jmedchem.7b00723

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Journal of Medicinal Chemistry

Article

Figure 1. Bioactive apelin isoforms 1−3, with identiﬁed sites of NEP and ACE2 proteolytic degradation highlighted.

capacity to lower blood pressure and have no ability to pro-

RESULTS AND DISCUSSION

■

2

6,28

tect against myocardial ischemic reperfusion injury,

making

Chemistry. To negate the impact of ACE2 cleavage, the

the C-terminal Phe residue essential for full agonist activity.

To negate the impact of ACE2 degradation on apelin iso-

forms, we and others developed resistant analogues (apelin

ACE2 resistant “A2” C-terminus was incorporated into all

Arg/Leu modiﬁed analogues. To eﬃciently synthesize a variety of

novel analogues, Fmoc-p-BrPhe-OH was loaded onto 2-chlorotrityl

chloride resin in 1.0 mmol batches (6) and extended by solid

phase peptide synthesis until heptapeptide 7 was obtained.

For each novel analogue series, 0.2 mmol of 7 was extended

to the dodecapeptide stage, incorporating the targeted Arg/Leu

modiﬁcation at the appropriate position as protected amino

acids, dipeptides, or tripeptides. The resin was subsequently

halved for the divergent syntheses of pyr-1-apelin-13 A2 or

apelin-17 A2 analogues (Scheme 1).

“

A2” analogues) with C-terminal Nle11, Aib12, and para-

24,29

BrPhe13 amino acid substitutions.

Both pyr-1-apelin-13 A2

and apelin-17 A2 analogues (4, 5) showed complete in vitro

ACE2 stability, retained full beneﬁcial cardiovascular activity,

and improved pharmacokinetics through in vitro plasma sta-

2

6

bility. However, analogues such as 4 and 5 were still degraded

through additional proteolytic events at unique positions

that mirrored previously reported in vitro apelin degradation

3

0,31

trends.

To enhance A2 peptides to NEP stability, we proposed to

use a variety of substitutions known to enhance proteolytic

stability at amino acid residues surrounding the cleavage site.

We selected to incorporate the following synthetic modiﬁca-

tions into both the pyr-1-apelin-13 and apelin-17 A2 scaﬀolds:

D-amino acids, N-methylations, α-methylations, and aza-peptide

isosteres to generate a total of 12 novel analogues (Figure 2).

D-Substituted apelin A2 analogues were easily accessible

due to the commercial availability of Fmoc-protected D-amino

acids. Previous D-scan experiments highlighted Leu5 as the

only residue within the NEP proteolytic region of pyr-1-apelin-

Through the use of a FRET-based assay and activity-guided

fractionation of human blood plasma, we recently discovered

that metalloprotease neutral endopeptidase 24.11 (neprilysin,

32

NEP) has a role in the in vitro proteolysis of apelin. NEP is a

signiﬁcant protease implicated in the degradation and

3

34

inactivation of vasoactive peptides bradykinin, angiotensins,

35

36

atrial naturatic factor (ANP), and endothelins in addition to

37

many other peptide hormones in other organ systems. Pre-

vious structure−activity relationship studies on apelin have

highlighted the essential nature of the amino acid residues

surrounding the site of NEP proteolysis (the “RPRL” motif).

18,27,29

16,18

29

1

3 that retained modest APJ receptor binding compared to

Alanine substitution,

epimerization, or truncation

29

native 1. Following the Fmoc-SPPS strategy previously out-

lined in Scheme 1, D-Leu analogues of pyr-1-apelin-13 A2 (8)

and apelin-17 A2 (9) were synthesized.

of amino acids within this region is deleterious to receptor

binding and subsequent GPCR activation and internalization.

Although apelin isoforms show no dominant secondary struc-

ture in aqueous solutions by circular dichroism, NMR structural

characterization of 2 has shown that this “RPRL” motif has an

enhanced rigidity and is suggested to induce a β-turn con-

N-methylated Leu dipeptide 20 was synthesized through the

HATU coupling of commercially available N-methylleucine

benzyl ester p-TsOH salt with Fmoc-Orn(Boc)-OH (Scheme 2).

The ornithine δ-amine was deprotected under acidic conditions

and then guanidinylated with 1,3-di-Boc-2-(triﬂuoromethylsulf-

onyl)guanidine to produce di-Boc protected arginine dipeptide

21. Benzyl ester removal under Pd-catalyzed hydrogenolysis

generated free acid 22, which was incorporated into N-MeLeu

apelin 13 and 17 A2 analogues 10 and 11, respectively.

Steric occlusion of the susceptible amide bond through

α-methylation had previously been successful in conferring

resistance to ACE2 by the incorporation of Aib N-terminal to

38

formation upon binding. The two Arg residues have been

proposed to form key electrostatic interactions with acidic

amino acids on the exterior of the apelin receptor to facilitate

ligand binding. This has been supported through site-directed

39

mutagenesis experiments and NMR structural elucidation

of the N-terminal tail and ﬁrst transmembrane helix of the

4

0

receptor. This indicates that the site of NEP proteolysis is also

structurally and functionally an essential feature for apelin

binding and subsequent physiological activity. The preparation

of analogues with enhanced stability to NEP could not only

generate potent therapeutic peptides but also facilitate inves-

tigation into the physiological role of this enzyme in apelin

degradation and give further insight into the importance of this

24

the monocarboxypeptidase cleavage site. Ala-Ni(II)-(S)-BPB

Schiﬀ base complex 23 was used to enantioselectively prepare

target α-methylated amino acids due to ease of synthesis, recy-

clability of the chiral ligand, and ability to separate individual

41,42

“RPRL” region of the peptide for its activity.

diastereomers by ﬂash chromatography.

Complex 23 was

B

DOI: 10.1021/acs.jmedchem.7b00723

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Scheme 1. General SPPS Approach for the Divergent Syntheses of Arg/Leu Modiﬁed Apelin A2 Analogues, with Areas of

Synthetic Modiﬁcation Shown in Red

4

1

synthesized according to literature protocol and was used to

divergently synthesize both α-methylarginine and α-methyl-

leucine amino acids.

the SPPS-compatible dipeptide 29 for the eventual preparation

of αMeArg analogues 12 and 13 (Scheme 3).

To prepare α-methylleucine, 23 was alkylated with 2-methyl-

1-iodopropane to generate the desired (S,S)-diastereomer 30,

with the absolute stereochemistry con ﬁ rmed by X-ray crystal-

lography (CCDC 1528625, Supporting Information Figure S2).

Acid hydrolysis followed by Fmoc-protection produced amino

For the synthesis of α-methylarginine, 23 was deprotonated

t

43

with KO Bu and alkylated with 1-azido-3-iodopropane (24)

to generate α,α-disubstituted Ni-complex 25. The desired

S,S)-diastereomer was puriﬁed by silica ﬂash chromatography

in 69% yield, and the absolute stereochemistry was con ﬁ rmed by

X-ray crystallography (CCDC 1528624, Supporting Information

Figure S1 ). Complex 25 was hydrolyzed under acidic condi-

tions, and the resulting amino acid was Fmoc-protected (26).

A HATU-mediated peptide coupling with H-Leu-OMe HCl

salt aﬀorded dipeptide 27, which was subsequently converted

to αMe-Arg containing dipeptide 28 through azide hydro-

(

t

acid 31, which was subsequently coupled to H-Ser( Bu)-OMe

44

to give dipeptide 32. Methyl ester deprotection gave the

SPPS-compatible dipeptide 33, which was used for αMeLeu

apelin analogues 14 and 15 (Scheme 4).

Azadipeptide 34 was prepared by the sequential addition

t

of disuccinimidyl carbonate, then a solution of H-Leu-O Bu in

DIPEA to benzophenone hydrazone analogous to previously

44

45,46

genolysis and guanidinylation. Methyl ester deprotection gave

established literature protocol.

Regioselective semicarbazone

C

DOI: 10.1021/acs.jmedchem.7b00723

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Article

Figure 2. Targeted synthetic modiﬁcations to improve apelin proteolytic stability against NEP.

Scheme 2. Synthesis of Dipeptide 22 for the Preparation of N-Methylleucine Apelin A2 Analogues 10 and 11

alkylation was achieved with tetraethylammonium hydroxide

and 3-chloro-1-bromopropane to yield chloroalkylated prod-

uct 35. Displacement of the primary alkyl chloride with sodium

azide aﬀorded 36, which was deprotected to semicarbazide 37

with hydroxylamine hydrochloride. Fmoc-protection of 37 was

unsuccessful in our hands, but fortunately the semicarbazide

could be aminoacylated with the next N-terminal amino acid

(Fmoc-Pro-OH) to generate azatripeptide 38. The SPPS com-

patible tripeptide 39 was synthesized following tert-butyl ester

deprotection, hydrogenolysis of the azide, and guanidinyla-

tion of the primary amine. Tripeptide 39 was incorporated into

azaArg apelin analogues 16 and 17 (Scheme 5).

D

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Scheme 3. Synthesis of Dipeptide 29 for the Preparation of α-Methylarginine Apelin A2 Analogues 12 and 13

Scheme 4. Synthesis of Dipeptide 33 for the Preparation of α-Methylleucine Apelin A2 Analogues 14 and 15

To prepare a synthetic azaLeu tripeptide, 40 was pre-

In Vitro NEP Stability. We initially compared the in vitro

NEP stability of these Arg/Leu analogues to native apelin

isoforms and their A2 substituted counterparts. Analogues were

incubated with recombinant human NEP (rhNEP, Sino

Biological) at 37 °C for up to 72 h, and the extent of degrada-

tion was analyzed via LC−MS because of the overlapping

elution patterns of peptide fragments (Figure 3). Native apelin

isoforms (1, 2) and their respective A2 substituted analogues

(4, 5) were degraded by NEP at rates comparable to those pre-

pared by the sequential addition of disuccinimidyl carbonate,

t

then a solution of H-Ser( Bu)-O Bu in DIPEA to benzophe-

none hydrazone. Semicarbazone alkylation was accomplished

using tetraethylammonium hydroxide and 3-bromo-2-methyl-

propene to yield 41. Concomitant Pd-catalyzed hydrogena-

tion and hydrogenolysis aﬀorded semicarbazide 42, which

was coupled to Fmoc-Orn(2-Cl-Cbz)-OH to generate azatri-

peptide 43. Chemoselective tert-butyl ester deprotection to

free acid 44 was accomplished by addition of silica in reﬂux-

ing toluene to protected azatripeptide 43. Subsequent 2-chloro-

Cbz deprotection and guanidinylation of the resultant pri-

mary amine yielded azatripeptide 45, which was further

employed to generate azaLeu-containing analogues 18 and 19

32

viously reported. Analogue 5 interestingly showed increased

susceptibility to NEP despite all synthetic modiﬁcations being

at minimum 6 amino acids (P6′) away from the site of endo-

protease cleavage. Distal amino acid substitutions have been

shown to modulate NEP proteolysis in the breakdown of

47

48

(

Scheme 6).

Aβ and synthetic substrates, and previous in vitro plasma

E

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Scheme 5. Synthesis of Azatripeptide 39 for the Preparation of Aza-arginine Apelin A2 Analogues 16 and 17

Scheme 6. Synthesis of Azatripeptide 45 for the Preparation of Aza-leucine Apelin A2 Analogues 18 and 19

degradation experiments observed that C-terminal modiﬁca-

tions to apelin analogues markedly aﬀected the presence of key

peptide fragments.

When examining the impact of Arg/Leu-substitution, all

analogues 8−19 showed dramatically improved proteolytic

stability to NEP compared to either native peptide (1, 2) or the

corresponding A2 substituted analogues (4, 5). Overall, Arg/

Leu-substituted apelin-17 A2 analogues showed an enhanced

stability compared to the pyr-1-apelin-13 A2 analogues. How-

ever, the proteolysis of these analogues was not due to degrada-

tion at either location associated with NEP endoprotease activ-

ity. Instead, analogues were slowly degraded between Nle-Aib

31

F

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Figure 3. In vitro NEP degradation trends for (a) pyr-1-apelin-13 analogues and (b) apelin-17 analogues, comparing native apelin isoforms (1, 2),

ACE2-resistant analogues (4, 5), and novel Arg/Leu substituted analogues 8−19.

Table 1. Remaining Percentage of Apelin Analogues after in Vitro Human Plasma Incubation

pyr-1-apelin-13 analogue

% apelin, 30 min

apelin-17 analogue

% apelin, 30 min

Arg-Leu modiﬁcation

analogue

% apelin, 60 min

analogue

% apelin, 60 min

2

6

none (native)

1

45 ± 8

77 ± 19

32 ± 2

77 ± 3

88 ± 4

45 ± 6

83 ± 3

19 ± 5

12 ± 2

65 ± 14

22 ± 1

59 ± 6

62 ± 6

38 ± 3

57 ± 1

2 ± 1

2

51 ± 17

43 ± 1

80 ± 3

49 ± 10

61 ± 10

77 ± 4

96 ± 6

82 ± 1

39 ± 7

35 ± 5

80 ± 4

47 ± 7

55 ± 4

48 ± 7

94 ± 3

68 ± 4

2

6

none (A2)

D-Leu

4

5

8

9

N-MeLeu

α-MeArg

α-MeLeu

azaArg

10

12

14

16

18

11

13

15

17

19

azaLeu

Table 2. Aﬃnities of Pyr-1-apelin-13 and Apelin-17 Analogues toward Rat APJ Determined by Radioligand Binding of

a

Pyr-1-apelin-13 and Apelin-17 Analogues

pyr-1-apelin-13 analogues

apelin-17 analogues

Arg-Leu modiﬁcation

analogue

aﬃnity, K (nM)

analogue

aﬃnity, K (nM)

i

2

6

none (native)

1

0.3 ± 0.07 (4)

2 ± 0.3 (3)

2

0.053 ± 0.009 (4)

0.19 ± 0.025 (3)

0.14 ± 0.056 (3)

0.45 ± 0.110 (3)

0.13 ± 0.032 (3)

0.18 ± 0.079 (3)

0.14 ± 0.009 (3)

0.15 ± 0.015 (3)

26

none (A2)

D-Leu

4

5

8

0.12 ± 0.035 (3)

0.10 ± 0.048 (3)

0.56 ± 0.033 (3)

0.17 ± 0.050 (3)

1 ± 0.10 (3)

9

N-MeLeu

α-MeArg

α-MeLeu

azaArg

10

12

14

16

18

11

13

15

17

19

azaLeu

0.13 ± 0.063 (3)

a

Aﬃnity values are the mean ± SE (n), with n representing the number of independent experiments performed in duplicate.

within the A2-modiﬁed C-terminus (Supporting Information

Figure S3 ). NEP does have signiﬁcant carboxydipeptidase activ-

ity as exempliﬁed in the degradation of neurological enkephalin

nearly identical in either the presence or absence of analogues

(Supporting Information Figure S4), suggesting that in vitro

proteolytic activity is not signiﬁcantly impacted.

3

7

pentapeptides, and key amino acids have been implicated in

binding and orienting the C-terminus of the peptide substrate

In Vitro Analogue Stability: Plasma. We next explored

the in vitro plasma stability of synthetic apelin analogues 8−19.

Triplicate assessments were performed in human blood plasma

49,50

to facilitate proteolysis.

C-terminal dipeptide excision was

26

not observed with native isoforms 1−3, likely due to the

unfavorable geometry of proline in the P1′ position. Although

Pro to Aib substitution in the A2 analogue series enabled an

additional NEP in vitro proteolytic site, we are proposing that

this dipeptide excision is negligible in comparison to in vivo

peptide hormone degradation and metabolism.

and analyzed by RP-HPLC (Table 1). Arg/Leu substitution

had a varying impact on the diﬀerent apelin isoforms. With the

exception of 11, apelin-17 A2 analogues showed enhanced sta-

bility compared to native 2 or A2-substitued analogue 5. Most

notably, azaArg8 analogue 17 showed very little degradation

during the time course examined. In contrast, there was a much

greater range of in vitro stabilities when examining the Arg/Leu

substituted pyr-1-apelin-13 A2 analogues. Analogues substi-

tuted at the Arg4 position (12, 16) or directly on the suscep-

tible amide bond (10) showed comparable stability to that of 4,

while Leu5-modiﬁed analogues (8, 14, 18) showed enhanced

proteolysis compared to native 1 or A2-modiﬁed analogue 4.

We additionally investigated the ability of synthetic ana-

logues 8−19 to inhibit the inherent proteolytic activity of NEP.

51

Although NEP inhibition via sacubitril (Entresto) is currently

a clinically approved method for the treatment of heart disease,

our focus was to minimize analogue interference with this

important regulatory protease. Following preincubation of NEP

with equimolar concentrations of 8−19, degradation of 1 was

32

On the basis of our previous kinetic studies and combined

G

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Figure 4. In vivo systolic (SBP, A/E), diastolic (DBP, B/F), mean arterial blood pressure (MABP, C/G), and heart rate analyses (HR, D/H)

following injection of pyr-1-apelin-13 A2 (left) and apelin-17 A2 (right) analogues in anesthetized mice.

in vitro NEP and plasma pharmacokinetics, NEP proteolysis

appears to be more signiﬁcant in the degradation of apelin-17

isoforms. However, the enhanced in vitro stabilities of Arg/Leu-

modiﬁed analogues to both NEP and nonspeciﬁc plasma pro-

teolysis were encouraging prior to interrogating their individual

physiological activities.

with inhibitory constant (K ) values of 0.3 and 0.05 nM, respec-

i

tively (Table 2). Pyr-1-apelin-13 analogues 8, 10, 14, and 18

showed a modest improvement in receptor binding, while

parent analogue 4 and azaArg4- and αMeArg4-substituted

analogues (16 and 12, respectively) exhibited a slightly weaker

aﬃnity compared to 1 (by a factor of 6, 3, and 2, respectively).

In contrast, Arg/Leu substitution slightly reduced the binding

aﬃnity of all apelin-17 analogues compared to native 2, exhib-

Binding Aﬃnity of Apelin Analogues. To examine the

impact of synthetic modiﬁcation within the critical RPRL motif

of the apelin peptide analogues, we evaluated the ability of

novel analogues to bind to the wild-type rat apelin receptor.

Membrane preparations from CHO cells stably expressing the

wild-type apelin receptor tagged at its C-terminus with EGFP

iting K values in the subnanomolar range, about 3 times less

i

potent. However, these data showed no drastic eﬀect on in vitro

apelin receptor binding, thus supporting our hypothesis that

our selected synthetic modiﬁcations within the RPRL motif of

apelin were not deleterious for receptor binding.

Physiological Experiments. Synthetic analogues 8−19

were tested for blood pressure lowering abilities in anesthetized

mice. Apelin peptide analogues were delivered systemically via the

right internal jugular vein while blood pressure (BP) and heart

39

were generated as previously described, and the aﬃnities of

native isoforms 1 and 2, parent analogues 4 and 5, and Arg/

Leu-analogues 8−19 were determined by their abilities to

displace [¹I]-pyr-1-apelin-13 (0.2 nM). Native isoforms 1 and

25

2

inhibited speciﬁc binding to the wild-type rat apelin receptor

H

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Figure 5. Langendorﬀ experiments following reperfusion of saline (negative control, sham), native 2 (positive control) and analogues 11, 17−19

after a 30 min period of ischemia, assessing heart rate (HR, A), maximum derivative of change in systolic pressure over time (max dP/dt, B), left

ventricle developed pressure (LVDP, C), minimum derivative of change in diastolic pressure over time (min dP/dt, D), and rate−pressure product

(

RPP, E).

rate (HR) were continuously monitored in the aorta cannulated

via the right carotid artery. Surprisingly, the potent physiological

eﬀects of 4 were not observed with any of the Arg/Leu-sub-

stituted pyr-1-apelin-13 A2 analogues (Figure 4A). Conserva-

tive substitutions such as epimerization (8) or N-methylation

(max dP/dt), minimum derivative of change in diastolic pressure

over time (min dP/dt), and rate-pressure product (RPP)) indi-

cative of general heart function and performance (Figure 5).

Following Langendorﬀ experiments, the potent hypotensive

apelin-17 A2 analogues 11, 17, and 19 showed an equivalent

ability to rescue the heart from ischemia−reperfusion injury

compared to native isoform 2. In all assessed parameters, these

analogues showed full potency, with 17 behaving very ana-

logously to 2, while 11 and 19 showed a slight improvement

over the native isoform in max/min dP/dt, LVDP and RPP.

Unsurprisingly, the inactive pyr-1-apelin-13 A2 analogue 18

behaved comparably to that of the negative saline control, having

no beneﬁcial cardiovascular eﬀects following periods of ischemia.

Synthetic substitution of the site of NEP proteolysis gave rise

to three potent analogues (11, 17, and 19) with full beneﬁcial

cardiovascular activities, subnanomolar receptor aﬃnities, and

improved in vitro NEP and plasma stabilities. As well, these

analogues showed no ability to inhibit the inherent activity of

NEP. Further optimization of the two apelin-17 A2 azapeptide

derivatives (17, 19) due to their improved pharmacokinetics is

encouraging in the pursuit of an apelin analogue with practical

therapeutic applicability. This study additionally provides

insight into the limited tolerance to modiﬁcation within the

“RPRL” motif and further supports its signiﬁcant role in the

apelinergic system. Most notably, any Arg/Leu modiﬁcation

abolished full agonistic activity within the pyr-1-apelin-13 A2

scaﬀold. Even conservative substitutions employed such as

epimerization, N- or α-methylations, or α-carbon replacement

with nitrogen failed to retain full physiological activity despite

having comparable in vitro apelin receptor binding. We propose

the inactivity of the Arg/Leu modiﬁcations within the 13A2

scaﬀold is due to a shorter N-terminal region that has a lesser

ability to be induced into the proposed active β-turn-like

(10) at the Leu5 position showed no ability to decrease blood

pressure in this assay. However, select analogues (10, 14, 16)

did have a signiﬁcant and pronounced eﬀect of increasing the

murine HR following injection. Tachycardia is directly cor-

52

related with native apelin isoforms binding to the receptor

and suggests that these analogues, while not fully functional,

still have some inherent physiological activities.

This diﬀerential physiological activity was further observed

when investigating the impact of the same substitutions on the

apelin-17 A2 isoform. All Arg/Leu modiﬁcations, even those

inactive in the 13 A2 isoform, showed an ability to induce

tachycardia. Most signiﬁcantly, three analogues (11, 17, 19)

showed potent hypotensive activity that rivaled or bested that

of native 2 or A2 variant 5 (Figure 4B). Analogues that retained

activity involved N-methyl Leu9 analogue 11 and both confor-

mationally ﬂexible aza-analogues 17 and 19, suggesting that

these synthetic modiﬁcations in the apelin-17 framework were

ﬂexible or conservative enough to induce full biological activity

upon binding to the apelin receptor.

Analogues that showed hypotensive activity were further

examined for their abilities to prevent myocardial ischemic

reperfusion injury using the Langendorﬀ protocol. Stable iso-

lated rat hearts were exposed to a 30 min period of ischemia,

then reperfused with saline (vehicle); native 2; vasoactive

analogues 11, 17, 19; or inactive 18; in a postconditioning pro-

tocol. The ex vivo heart was assessed for a variety of parameters

(

heart rate (HR), left ventricle developed pressure (LVDP),

maximum derivative of change in systolic pressure over time

I

DOI: 10.1021/acs.jmedchem.7b00723

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

4

0

conformation in the presence of the apelin receptor. How-

ever, the fact that some of these same modiﬁcations show

potency in the apelin-17 A2 series suggests that the longer

N-terminal extension is capable of facilitating the appropriate

receptor interactions and internalization necessary to induce

full physiological activity.

hormone. These active and robust apelin analogues will assist in

harnessing the potent cardiovascular beneﬁts of the apelinergic

system for future therapeutic applications.

EXPERIMENTAL SECTION

■

General Information. Reagents, Solvents, and Puriﬁcation.

Commercially available chemical and biological reagents were

purchased from Sigma-Aldrich Canada, Chem-Impex International

Inc., Fisher Scientiﬁc Ltd., Alfa Aesar Ltd., R&D Systems, Tocris

Bioscience, Harvard Apparatus, Caledon, or VWR International and

used without further puriﬁcation unless otherwise stated. All solvents

were of American Chemical Society (ACS) grade and were used

without further puriﬁcation. All anhydrous reactions were conducted

under positive pressure of argon using ﬂame-dried glassware. Solvents

for anhydrous reactions were distilled prior to use: dichloromethane

and dichloroethane were distilled over calcium hydride, tetrahydrofur-

an and diethyl ether were distilled over sodium with benzophenone as an

indicator, and methanol was distilled over magnesium. HPLC grade

acetonitrile, dimethylformamide, 2-propanol, hexanes, and methanol

were used without further puriﬁcation. Commercially available ACS

grade solvents (>99.0% purity) were used for column chromatography

without further puriﬁcation. Deionized water was obtained from a

Milli-Q reagent water ﬁltration system (Millipore Co., Milford, MA).

All reactions and fractions from column chromatography were

monitored by thin layer chromatography (TLC) using glass plates

The inactivity of both D-Leu substituted analogues 8 and 9

was particularly surprising because of literature precedent that

D-Leu pyr-1-apelin-13 retained acceptable abilities to bind

(

∼10-fold decrease in aﬃnity) and inhibit forskolin-induced

29

cAMP accumulation. Epimerization within or adjacent to an

inducible structural element such as a β-turn would likely be

detrimental to activity. However, a recent apelin-17 analogue

(

P92) with full agonistic activity has been reported with mul-

tiple epimerizations, including two ﬂanking both the N- (D-Gln)

and C- (D-Leu) termini of the “RPRL” region. This analogue

has additional substitutions that make direct comparison with

our analogues diﬃcult, but it is encouraging to know that

physiological activity and stability can be enhanced through

epimerizations.

5

3

Select pyr-1-apelin-13 A2 (10, 14, 16) and apelin-17 A2

9, 13, 15) analogues had an impact on the heart rate of mice

(

following perfusion without inducing any vasoactive eﬀects.

This may be rationalized through the biased agonism of these

analogues for G-protein pathways instead of being internalized.

It is known that β-arrestin recruitment and internaliza-

tion initiates vasodilation and additional cardioprotective eﬀects

of apelin, and the C-terminal phenylalanine of apelin plays

with a UV ﬂuorescent indicator (normal SiO , Merck 60 F₂₅₄).

2

Visualization was performed following UV absorption by ﬂuores-

cence quenching, staining with phosphomolybdic acid in ethanol

(

/

10 g/100 mL), ninhydrin (ninhydrin/acetic acid/n-butanol, 0.6 g/6 mL

200 mL) or potassium permanganate (KMnO /K CO /NaOH/H O,

4

2

3

2

27,28,54

a critical role in this process.

However, much less is

1.5 g/10 g/0.12 g/200 mL). Flash chromatography was performed

using Merck type 60, 230−400 mesh silica gel. Semipreparative

and analytical scale high performance liquid chromatography (HPLC)

was performed on a Gilson chromatograph equipped with model

322 pump heads, a model 171 diode array detector, a FC 203B fraction

collector, and a Rheodyne 7725i injector ﬁtted with a 1000 μL sample

loop. HPLC solvents were ﬁltered through a Millipore ﬁltration system

under vacuum prior to use. Peptides were puriﬁed to >95% purity as

assessed by analytical reinjection.

understood about how tachycardia is initiated. The vasoac-

tive and inotropic activities of apelin have previously been

decoupled through the pharmacological inhibition of neuronal

ganglion in rats, and additional studies have suggested that

the Gα pathway is responsible for the cardiac contractility

associated with apelin. A comparable eﬀect has been seen

with a synthetic apelin analogue tested in humans, where the

critical “RPRL” motif is constrained within an intramolecular

disulﬁde bridge. This compound shows a potent increase in

cardiac output and slight increase in heart rate without inducing

vasoactive eﬀects. Although we have not observed an equiv-

alent physiological activity to what has been reported, these

results suggest that these analogues are not wholly inactive.

These physiological results clearly highlight the signiﬁcant role

of the “RPRL” motif within apelin peptide and show that it has

limited ability to be manipulated synthetically to produce fully

active analogues.

5

2

q

55

Characterization. Optical rotations were measured on a PerkinElmer

241 polarimeter with a microcell (10 cm, 1 mL) at ambient tem-

−1

2

−1

perature and are reported in units of 10 deg cm g . All reported

optical rotations were referenced against air and measured at the

sodium D line (λ = 589.3 nm).

5

6

Infrared spectra (IR) were recorded on a Nicolet Magna 750 or a

0SX FT-IR spectrometer. Cast refers to the evaporation of a solution

2

on a NaCl plate.

Nuclear magnetic resonance (NMR) spectra were recorded on an

Agilent/Varian Inova 600, Inova 400, Inova 300, DD2 MR 400,

1

VNMRS 700, or Unity 500 spectrometer at 27 °C. For H (300, 400,

5

00, 600, or 700 MHz) spectra, δ values were referenced to CDCl

3

1

3

CONCLUSION

(7.26 ppm), D O (4.79 ppm), or CD OD (3.30 ppm), and for

C

2

3

■

(

100, 125, 150, or 175 MHz) spectra, δ values were referenced to

The continued investigation of the diverse roles of apelin pep-

tides in mammalian physiology has strengthened their thera-

peutic potential for the treatment of cardiovascular disease and

other pathologies. However, the susceptibility of native apelin

CDCl (77.0 ppm) or CD OD (49.0 ppm) as the solvents. Reported

3

splitting patterns are abbreviated as s = singlet, d = doublet, t = triplet,

q = quartet, sept = septet, m = multiplet.

Mass spectra (MS) were recorded on a Kratos AEIMS-50, Bruker

9.4 T Apex-Qe FTICR (high resolution, HRMS) or on a Perspective

Biosystems Voyager Elite MALDI-TOF MS using 4-hydroxy-α-

cyanocinnamic acid (HCCA) as a matrix. MS/MS was performed

on a Bruker Ultraﬂextreme MALDI/TOF/TOF. LC−MS was per-

formed on an Agilent Technologies 6130 LCMS instrument.

25,26

32

isoforms to proteases such as ACE2,

NEP, and prolyl

5

7

carboxypeptidase dramatically limits their direct use. The

preparation of analogues with improved proteolytic stabilities

that conserve beneﬁcial interactions with the cognate APJ

receptor enables potential therapeutic application. In this

manuscript, the preparation of 12 novel analogues, modiﬁed

surrounding the NEP proteolytic site, aﬀorded three potent

compounds with full cardiovascular activities and improved

in vitro pharmacokinetic stabilities. The diﬀerentiation of activ-

ities of these Arg/Leu-substituted analogues adds insight into

the signiﬁcant physiological role of this region of the peptide

1. General Apelin Analogue SPPS Elongation Method.

A suspension of resin in DMF (10 mL) was bubbled under Ar gas

for 15 min to swell. The N-terminal Fmoc group was removed by

bubbling a solution of 20% piperidine in DMF (3 × 10 mL) for 3 min,

washing considerably with DMF (3 × 10 mL) after each deprotection.

Fmoc-deprotection was monitored by TLC, with the dibenzofulvene−

piperidine adduct appearing as a bright purple spot under UV.

J

DOI: 10.1021/acs.jmedchem.7b00723

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

DIPEA (6.0 equiv) was added to a solution of Fmoc-protected amino

acid (3.0 equiv compared to resin loading), HATU (3.0 equiv), and

HOAt (3.0 equiv) in DMF (10 mL) and stirred for 5 min. The activ-

ated amino acid solution was added to the resin and bubbled under

Ar gas for 1−3 h. The resin was washed with DMF (3 × 10 mL), and

the extent of coupling was assessed by either (i) cleaving a small sample

Fmoc-Pro-OH, Fmoc-Arg(Pmc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-

Arg(Pmc)-OH, Fmoc-Arg(Pmc)-OH, Fmoc-Phe-OH, and Fmoc-

Lys(Boc)-OH. A portion (0.05 mmol) of resin-bound peptide

was cleaved and puriﬁed using a C₁₈RP-HPLC analytical column

(method A), eluting at 11.2 min. The desired peptide was isolated as a

white solid after lyophilization (9.9 mg, 9.1%). Monoisotopic MW

calculated for C H BrN O 547.2947, found high resolution

of resin with a solution of 95:2.5:2.5 TFA/TIPS/H O for 1 h and

2

96 161

34 20

4+

MALDI-TOF analysis or (ii) performing a Kaiser test to detect the

(FTICR-ESI-MS) 547.2942 (M + 4H) .

presence of free amines. A solution of 20% acetic anhydride in DMF

N-Methylleucine A2 Analogues. Benzyl N-((S)-2-((((9H-Fluo-

ren-9-yl)methoxy)carbonyl)amino)-5-((tert-butoxycarbonyl)-

amino)pentanoyl)-N-methyl-L-leucinate (20). Fmoc-Orn(Boc)-OH

(

10 mL) was added to the resin and bubbled under Ar gas for 10 min

to end-cap any unreacted amines. The resin was rinsed thoroughly

with DMF (3 × 10 mL) and subjected to Fmoc-deprotection to

further elongate the peptide or rinsed with CH Cl (3 × 10 mL) and

(1.23 g, 2.70 mmol), HATU (1.03 g, 2.70 mmol), HOAt (4.5 mL [0.6 M

solution in DMF], 2.70 mmol), and DIPEA (1.28 mL, 7.36 mmol)

were dissolved in dry DMF (10 mL) and stirred for 5 min to

preactivate the amino acid. A solution of N-Me-Leu-OBn p-TsOH salt

2

MeOH (3 × 10 mL), dried thoroughly, and stored under Ar gas at

20 °C.

. General Method for Apelin Analogue Resin Cleavage.

Resin-bound apelin analogue (0.05 mmol) was suspended in

5:2.5:2.5 TFA/TIPS/H O with shaking under an Ar atmosphere

−

2

(1.00 g, 2.45 mmol) and DIPEA (0.43 mL, 2.45 mmol) in dry CH

(10 mL) was added, and the reaction was stirred for 17 h at room

temperature. The reaction was washed with 10% aqueous NaHCO

(20 mL), 10% aqueous citric acid (20 mL), and brine (20 mL). The

organic layer was dried over Na SO , ﬁltered, and concentrated in

2 2

Cl

9

3

2

for 2−3 h. The resin was removed via ﬁltration through glass wool,

rinsed with TFA, and the solution was concentrated in vacuo. Cold

diethyl ether (2 × 5 mL) was added to triturate the crude peptide. The

diethyl ether was decanted into a 15 mL Falcon tube and brieﬂy

centrifuged to pellet any residual peptide. The diethyl ether pellet and

triturated crude residue were pooled together and dissolved in 10%

aqueous acetonitrile, 0.1% TFA.

2

4

vacuo. The dipeptide was puriﬁed using ﬂash chromatography (silica

gel, 25% EtOAc in hexanes), obtaining 20 as a crunchy white solid

2

6

(1.25 g, 76%) (R

(c 1.0, CH Cl ); IR (CH

1714, 1645, 1513, 1451, 1251, 1172 cm . H (CDCl

= 0.9 on SiO

, 1:3 hexanes/EtOAc); [α]

−17.4

f

2

D

Cl cast) 3315, 3066, 3037, 2957, 2870,

2

−1 1

, 500 MHz):

3

. Apelin Peptide Puriﬁcation Methods. Peptides were puriﬁed

δ 7.76 (d, J = 7.5 Hz, 2H, Ar-H), 7.59 (d, J = 7.5 Hz, 2H, Ar-H),

7.43−7.27 (m, 9H, Ar-H), 5.65 (d, J = 8.5 Hz, 1H, Fmoc-NH), 5.35

using C₁₈RP-HPLC using aqueous 0.1% TFA (solvent A) and 0.1%

TFA in acetonitrile (solvent B) as eluents. The following puriﬁcation

methods were used:

Method A (semiprep puriﬁcation, 3 mL/min, Phenomenex Luna

u, C18(2) 100 Å, 5 μm, 10.00 mm × 250 mm): 10% B (3 min),

(dd, J = 10.8, 5.0 Hz, 1H, Leu-CHα), 5.19−5.06 (m, 2H, Bn-OCH ),

2

4.68 (ddd, J = 7.7, 7.7, 4.8 Hz, 1H, Orn-CHα), 4.51 (s, 1H, Boc-NH),

4.37 (ddd, J = 10.6, 7.1, 7.1 Hz, 2H, Fmoc-CH ), 4.21 (t, J = 7.1 Hz,

1H, Fmoc-CH), 3.09−3.04 (m, 2H, Orn-CH δ), 2.93 (s, 3H, N-CH ),

1.81−1.65 (m, 3H, 2 × Leu-CH β, Orn-CH β), 1.56−1.46 (m, 4H,

Orn-CH β, 2 × Orn-CH γ, Leu-CH(CH ), 1.44 (s, 9H, -C(CH ),

1.01−0.84 (m, 6H, -CH(CH ). C (CDCl , 125 MHz): δ 172.6,

2

5

1

2

3

0−25% B (1.5 min), 25−42% B (17 min), 42−95% B (2.5 min), 95%

2

B (3 min), 95−10% B (0.5 min), 10% B (2.5 min).

2

3

)

2

)

3 3

13

Method B (analytical reinjection, 1 mL/min, Alltech Prosphere

3

)

2

3

(

00 Å, 5 μm, 4.6 mm × 250 mm): 27% B (3 min), 27−35% B

10 min), 35% B (2 min), 35−27% B (1 min), 27% B (2 min).

. Apelin Analogue Syntheses. Reference Compounds. The

syntheses and characterization of ACE2-resistant analogues 4 and 5

171.3, 156.0, 155.9, 143.9, 143.8, 141.3, 141.3, 135.4, 128.7, 128.5,

128.4, 127.7, 127.1, 127.1, 125.2, 120.0, 79.1, 67.1, 67.0, 54.9, 50.7,

4

47.2, 40.1, 36.8, 31.1, 30.0, 28.5, 25.4, 24.9, 23.2, 21.4. HRMS (ES)

+

calculated for C39

H

N

50

O

672.3643, found 672.3642 (M + H) .

3

7

24,26

41

have been previously described.

Synthetic compounds 23 and

were prepared according to literature protocols.

Fmoc-pBrPhe-2-Cl-Trt (6). 2-Chlorotrityl chloride resin (1.05 mmol)

was transferred to a SPPS vessel, washed with CH Cl (2 × 10 mL),

Benzyl (11S,14S,E)-11-((((9H-Fluoren-9-yl)methoxy)carbonyl)-

amino)-6-((tert-butoxycarbonyl)amino)-14-isobutyl-2,2,13-trimeth-

yl-4,12-dioxo-3-oxa-5,7,13-triazapentadec-5-en-15-oate (21). The

43

2

4

58

guanidinylation reaction was adapted from a literature procedure.

TFA (5 mL) was added to a solution of dipeptide 20 (0.701 g,

.04 mmol) in dry CH Cl (10 mL) and stirred for 1 h. The reaction

2

DMF (2 × 10 mL), and ﬁnally suspended in DMF and bubbled under

Ar gas for 10 min. Fmoc-p-bromo-L-phenylalanine (1.0 mmol) was

suspended in dry CH Cl (10 mL) with DIPEA (5.0 equiv) and added

to the swollen resin. This solution was bubbled under Ar gas for 2.5 h.

The functionalized resin was washed considerably with CH Cl

1

2

was concentrated in vacuo, using diethyl ether coevaporations to

remove residual TFA. The yellow oil was resuspended in dry CH Cl

2

(20 mL), and 1,3-di-Boc-2-(triﬂuoromethylsulfonyl)guanidine (0.449 g,

2

1

.15 mmol) and triethylamine (0.32 mL, 2.30 mmol) were added and

(

3 × 10 mL) and DMF (3 × 10 mL), and then unreacted sites

were capped by bubbling with dry MeOH (5 mL) under Ar gas for

stirred for 80 min. The reaction was washed with 2 N aqueous sodium

bisulfate (10 mL) and 10% NaHCO (10 mL), dried over Na SO ,

4

0

5 min. The resin was dried thoroughly under Ar, weighed, divided into

.2 mmol portions, and stored under an Ar atmosphere at −20 °C.

Fmoc-His(Trt)-Lys(Boc)-Gly-Pro-Nle-Aib-pBrPhe-2-Cl-Trt (7). Func-

tionalized resin 6 was elongated by manual SPPS, introducing amino

acids in the following order: Fmoc-Aib-OH, Fmoc-Nle-OH, Fmoc-

Pro-OH, Fmoc-Gly-OH, Fmoc-Lys(Boc)-OH, and Fmoc-His(Trt)-

OH. A small sample of the resin was cleaved and analyzed by MALDI-

TOF: calculated for C H BrN O 1083.4, found 1083.0 (M + H) .

D-Leu Substituted A2 Analogues. D-Leu5-pyr-1-apelin-13 A2

8). Advanced intermediate 7 (0.1 mmol) was subjected to manual

SPPS, introducing amino acids in the following order: Fmoc-Ser( Bu)-

OH, Fmoc-D-Leu-OH, Fmoc-Arg(Pmc)-OH, Fmoc-Pro-OH, Fmoc-

Arg(Pmc)-OH, and pyr-Glu-OH. A portion (0.05 mmol) of resin-

bound peptide was cleaved and puriﬁed using a C₁₈RP-HPLC

analytical column (method A), eluting at 20.6 min. The desired pep-

tide was isolated as a white solid after lyophilization (11.3 mg, 14%).

Monoisotopic MW calculated for C H BrN O 791.3860, found

high resolution (FTICR-ESI-MS) 791.3846 (M + 2H) .

D-Leu9-apelin-17 A2 (9). Advanced intermediate 7 (0.1 mmol) was

subjected to manual SPPS, introducing amino acids in the following

order: Fmoc-Ser( Bu)-OH, Fmoc-D-Leu-OH, Fmoc-Arg(Pmc)-OH,

3

2

4

ﬁltered, and concentrated in vacuo. The dipeptide was puriﬁed using

ﬂash chromatography (silica gel, 20% EtOAc in hexanes), obtaining 21

as a crunchy white solid (0.820 g, 97%) (R = 0.5 on SiO , 3:1

f

2

26

^h^e^x^aⁿ^e^s^/^E^t^O^A^c⁾^;^[^α^]D −12.5 (c 1.21 CHCl ); IR (CHCl cast)

331, 2959, 2871, 1721, 1640, 1451, 1415, 1330, 1156, 1134 cm .

H (CDCl , 500 MHz): δ 8.29 (t, J = 5.4 Hz, 1H, Arg-NHε), 7.76 (d,

3

−1

3

1

3

+

J = 7.6 Hz, 2H, Ar-H), 7.60 (d, J = 7.6 Hz, 2H, Ar-H), 7.43−7.27 (m,

53

68

10 10

9H, Ar-H), 5.68 (d, J = 8.5 Hz, 1H, Fmoc-NH), 5.37−5.28 (m, 1H,

(

Leu-CHα), 5.23−5.05 (m, 2H, -OCH

Arg-CHα), 4.44−4.31 (m, 2H, Fmoc-CH

Fmoc-CH), 3.36 (ddd, J = 7.4, 5.0, 3.2 Hz, 2H, Arg-CH

(s, 3H, N-CH ), 1.82−1.75 (m, 1H, Leu-CH β), 1.75−1.66 (m, 2H,

Leu-CH β, Arg-CH β), 1.63−1.55 (m, 3H, Arg-CH β, 2 × Arg-CH γ)

1.49 (m, 10H, Leu-CH(CH ) , -C(CH ) ), 1.48 (s, 9H, -C(CH ) ),

2

Ph), 4.67 (td, J = 8.1, 4.3 Hz, 1H,

), 4.21 (t, J = 6.9 Hz, 1H,

δ), 2.93

t

2

3

2

3

2

3

3 3

0.93 (d, J = 6.6 Hz, 3H, Leu-CH(CH ) ), 0.90 (d, J = 6.5 Hz, 3H,

3

2

13

Leu-CH(CH ) ). C (CDCl , 125 MHz): δ 172.5, 171.2, 163.6, 156.2,

69

111

22 16

3

2

3

2+

156.0, 153.3, 143.9, 143.8, 141.3, 141.3, 135.4, 128.7, 128.6, 128.4, 127.7,

127.1, 127.1, 125.2, 125.2, 120.0, 83.1, 79.2, 67.1, 67.0, 54.9, 50.8, 47.2,

40.3, 36.8, 31.2, 29.9, 28.3, 28.1, 27.9, 27.9, 24.9, 23.2, 21.4. HRMS (ES)

t

+

calculated for C H N O 814.4386, found 814.4378 (M + H) .

45

60

5

9

K

DOI: 10.1021/acs.jmedchem.7b00723

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

(

11S,14S,E)-11-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-6-

(2S)-2-(3′-Azidopropyl)-2-methylglycine-Ni(II)-(S)-BPB (25).

(

(tert-butoxycarbonyl)amino)-14-isobutyl-2,2,13-trimethyl-4,12-

dioxo-3-oxa-5,7,13-triazapentadec-5-en-15-oic Acid (22). A solu-

tion of 21 (0.501 g, 0.62 mmol) was dissolved in methanol (50 mL)

and had 10% Pd/C (20 mg) added. The suspension was stirred under

hydrogen gas for 22 h, ﬁltered through a pad of Celite, and concen-

trated in vacuo. The crude residue was puriﬁed using ﬂash chro-

matography (silica gel, 5% MeOH in CH Cl , 0.1% AcOH), yielding

2

2 as a white solid (0.378 g, 85%) (R = 0.2 on SiO , 1% AcOH in

f

2

4

3

26

Complex 25 was prepared according to literature procedure. Nickel

complex 23 (6.49 g, 12.7 mmol) and potassium tert-butoxide (2.13 g,

19.0 mmol) were added to an RBF and dissolved in dry DMF (50 mL)

at 0 °C under an Ar atmosphere. This solution was stirred for 3 min,

followed by the addition of a solution of 24 (5.01 g, 19.0 mmol) in dry

DMF (10 mL). The reaction was stirred at 0 °C for 45 min, then

warmed to room temperature for 75 min. 5% aqueous acetic acid

EtOAc); [α]_D−2.8 (c 1.0, CH Cl ); IR (CH Cl cast) 3326, 3140,

2

3

1

066, 2959, 2872, 1721, 1641, 1618, 1451, 1415, 1331, 1253,

136 cm . H (CDCl , 500 MHz): δ 8.48 (s, 1H, Arg-NHε), 7.76

−1 1

3

(

7

(

d, J = 7.5 Hz, 2H, Ar-H), 7.61 (d, J = 7.5 Hz, 2H, Ar-H), 7.40 (t, J =

.5 Hz, 2H, Ar-H), 7.32 (tdd, J = 7.5, 3.4, 1.2 Hz, 2H, Ar-H), 6.05

d, J = 7.6 Hz, 1H, Fmoc-N-H), 5.37 (dd, J = 10.7, 5.1 Hz, 1H,

Leu-CHα), 4.80 (dd, J = 5.4, 5.3 Hz, 1H, Arg-CHα), 4.38 (dd, J =

(

200 mL) was added to quench the reaction, followed by extraction

with CH Cl (3 × 75 mL). Pooled organic layers were washed with

brine (2 × 75 mL), dried over Na SO , ﬁltered, and concentrated in

vacuo. The desired diastereomer was puriﬁed by ﬂash chromatography

silica gel; 2.5% MeOH in EtOAc), yielding 25 as a red solid (5.18 g,

9%). Crystals of 25 were obtained after dissolution in minimal

CH Cl , dilution with hexanes, and slow evaporation (Supporting

Information Figure S1 ) (R = 0.6 on SiO , 9:1 EtOAc/MeOH); [α]

853.4 (c 1.0, CH Cl ); IR (CH Cl cast) 3060, 2937, 2869, 2097,

2 2 2 2

10.6, 7.2 Hz, 2H, Fmoc-CH ), 4.21 (t, J = 7.1 Hz, 1H, Fmoc-CH),

2

3

.55−3.45 (m, 1H, Arg-CH δ), 3.14−3.05 (m, 1H, Arg-CH δ), 2.98

2

(

s, 3H, N-CH ), 1.92−1.56 (m, 6H, 2 × Leu-CH β, 2 × Arg-CH β,

2

4

3

2

× Arg-CH γ), 1.52 (s, 9H, -C(CH ) ), 1.49 (m, 10H, Leu-

2 3 3

(

6

CH(CH ) , -C(CH ) ), 0.96 (d, J = 6.6 Hz, 3H, Leu-CH(CH ) ),

3

2

3

3 2

1

3

0

1

5

.93 (d, J = 6.5 Hz, 3H, Leu-CH(CH ) ). C (CDCl , 125 MHz): δ

3 2 3

72.3, 172.1, 162.8, 156.3, 155.7, 153.2, 144.9, 143.8, 141.4, 141.3,

27.7, 127.7, 127.1, 127.1, 125.2, 125.2, 120.0, 119.9, 83.1, 80.6, 66.9,

4.7, 51.1, 47.3, 40.6, 36.1, 30.7, 28.4, 28.1, 28.1, 24.8, 23.2, 21.4.

2

6

f

2

D

1

−

1

673, 1639, 1574, 1439, 1359, 1253, 1165 cm .^H⁽^C^D^C^l3^,

), 8.05 (dd, J = 8.7, 0.9 Hz,

, Ar-H ), 7.47−7.41 (m, 3H,

, Ar-H11), 7.35−7.28 (m, 2H, Ar-H , Ar-H10), 7.16 (ddd, J = 8.5,

5.8, 2.7 Hz, 1H, Ar-H ), 7.08 (dt, J = 7.6, 1.3 Hz, 1H Ar-H12),

6.69−6.62 (m, 2H, Ar-H , Ar-H ), 4.50 (d, J = 12.7 Hz, 1H,

Ph-CH -N), 3.70 (dd, J = 18.7, 11.8 Hz, 2H, Ph-CH -N, Pro-CH δ),

3.51−3.42 (m, 2H, Pro-CHα, -CH ), 3.32−3.20 (m, 2H, -CH

Pro-CH γ), 2.71 (dddd, J = 12.8, 7.1, 5.8, 2.6 Hz, 1H, Pro-CH

2.58−2.43 (m, 2H, Pro-CH β, -CH CH ), 2.26−2.04 (m, 3H,

-CH CH , Pro-CH γ, Pro-CH δ), 1.86 (ddd, J = 13.8, 12.4, 4.2 Hz,

1H, -CH CH CH ), 1.76 (ddd, J = 13.7, 12.4, 4.6 Hz, 1H,

-CH CH CH ), 1.28 (s, 3H, -CH

HRMS (ES) calculated for C H N O 724.3916, found 724.3924

38

54

5

9

+

(

M + H) .

500 MHz): δ 8.12−8.07 (m, 2H, Ar-H

1H, Ar-H ), 7.55−7.48 (m, 2H, Ar-H

Ar-H

3

1

N-MeLeu5-pyr-1-apelin-13 A2 (10). Advanced intermediate 7

4

8

9

(

0.1 mmol) was subjected to manual SPPS, introducing amino acids

2

t

in the following order: Fmoc-Ser( Bu)-OH,22, Fmoc-Pro-OH, Fmoc-

Arg(Pmc)-OH. The resin was split into half, and Fmoc-SPPS was

continued on 0.1 mmol scale, coupling pyr-Glu-OH. No end-capping

was performed following addition of 22. A portion (0.05 mmol) of

resin-bound peptide was cleaved as previously described and puriﬁed

using a C₁₈RP-HPLC analytical column (method A), eluting at

5

6

7

2

N

N ,

2 3

β),

2

3

2

N

2 3

2

1

1.2 min. The desired peptide was isolated as a white solid after

2

N

3

2

lyophilization (6.0 mg, 7%). Monoisotopic MW calculated for

C H BrN O 532.5983, found high resolution (FTICR-ESI-MS)

2

N

2 3

1

3

N

); C (CDCl

, 125 MHz): δ

70

114

22 16

2

3

3+

532.5972 (M + 3H) .

182.0, 180.6, 172.8, 141.6, 136.4, 133.4, 133.4, 131.8, 131.7, 130.2,

129.6, 129.1, 129.0, 128.5, 128.1, 127.3, 127.1, 124.0, 120.8, 77.4, 70.0,

N-MeLeu9-apelin-17 A2 (11). The remaining 0.1 mmol carried

over from the synthesis of 10 was coupled with Fmoc-Gln(Trt)-OH,

Fmoc-Arg(Pmc)-OH, Fmoc-Arg(Pmc)-OH, Fmoc-Phe-OH, and

Fmoc-Lys(Boc)-OH. No end-capping was performed following addi-

tion of 22. A portion (0.05 mmol) of resin-bound peptide was cleaved

as previously described and puriﬁed using a C₁₈RP-HPLC analytical

column (method A), eluting at 11.3 min. The desired peptide was

isolated as a white solid after lyophilization (23.8 mg, 22%).

Monoisotopic MW calculated for C H BrN O 550.7986, found

63.5, 57.2, 51.3, 37.5, 30.7, 29.4, 25.5, 23.4. HRMS (ES) calculated for

+

C

H

32

N

NaNiO

617.1782, found 617.1785 (M + Na) .

31

6

3

(S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-5-azido-2-

methylpentanoic Acid (26). A solution of 3 N HCl/MeOH (1:1,

40 mL) was heated to 70 °C and had 25 (2.60 g, 4.37 mmol) dissolved

in MeOH (10 mL) added dropwise over 5 min. The reaction mixture

was stirred for 50 min at 70 °C, turning from dark red to green as the

reaction proceeded. The reaction was cooled to room temperature,

concentrated in vacuo, and resuspended in 10% aqueous sodium

carbonate (20 mL). EDTA disodium salt (3.25 g, 8.73 mmol) was

added and stirred for 0.5 h at room temperature. The reaction was

cooled to 0 °C and had a solution of Fmoc-OSu (1.62 g, 4.80 mmol)

dissolved in acetone (20 mL) added to it. This reaction was slowly

warmed to room temperature and stirred for 16 h. The following day,

the reaction was diluted with EtOAc (50 mL) and washed with 1 N

97

163

34 20

4+

high resolution (FTICR-ESI-MS) 550.7983 (M + 4H) .

α-Methylarginine A2 Analogues. 1-Azido-3-iodopropane (24).

4

3

Compound 24 was prepared according to literature procedures.

A solution of 1-chloro-3-iodopropane (2.63 mL, 24.5 mmol) and

sodium azide (1.91 g, 29.4 mmol) in dry DMF (60 mL) was stirred at

room temperature for 24 h. The reaction was diluted by the addition

of water (100 mL), and diethyl ether washes (2 × 100 mL) were used

to extract organic components. The diethyl ether fractions were dried

over Na SO , ﬁltered, and concentrated in vacuo until a small volume

HCl (3 × 20 mL). The organic layer was dried over MgSO

and concentrated in vacuo. The product was puriﬁed by ﬂash

chromatography (silica gel, 1−5% MeOH in CH Cl gradient),

yielding 26 as a white solid (1.64 g, 95%) (R = 0.4 on SiO , 9:1

, 500 MHz): δ 7.77 (d, J = 7.5 Hz, 2H,

, ﬁltered,

4

2

4

(

∼10 mL) of ether remained. The reaction was diluted in acetone

2

60 mL), and following the addition of sodium iodide (5.50 g,

f

2

1

36.7 mmol), the reaction was heated at 60 °C for 24 h. The solution

EtOAc/MeOH). H (CDCl

3

was concentrated in vacuo until a small volume (∼10 mL) remained,

Ar-H), 7.58 (d, J = 7.5 Hz, 2H, Ar-H), 7.40 (td, J = 7.4, 3.1 Hz, 2H,

Ar-H), 7.32 (tdd, J = 7.4, 2.6, 1.1 Hz, 2H, Ar-H), 5.57 (br s, 1H,

then diluted with diethyl ether (100 mL) and washed with H O

2

(

2 × 100 mL). The organic layer was dried over Na SO , vacuum

Fmoc-NH), 4.48−4.36 (m, 2H, Fmoc-CH

2

), 4.21 (t, J = 6.2 Hz, 1H,

2

4

ﬁltered through a small plug of alumina, and concentrated in vacuo,

obtaining the desired product as a light yellow oil (3.91 g, 76%).

IR (CH Cl cast) 2927, 2869, 2098, 1449, 1428, 1347, 1290, 1224,

Fmoc-CH), 3.32−3.19 (m, 2H, -CH N ), 2.28−2.17 (m, 1H,

2

3

Orn-CH γ), 2.02−1.90 (m, 1H, Orn-CH γ), 1.60 (br s, 3H, -CH ),

2

3

13

1.50−1.39 (m, 2H, Orn-CH β). C (CDCl , 125 MHz): δ 170.1,

153.9, 143.8, 141.4, 127.7, 127.1, 125.0, 120.0, 66.6, 51.1, 47.2, 34.0,

33.4, 23.7, 23.5. HRMS (ES) calculated for C H N O 393.1568,

2

3

−1

1

173 cm . H (CDCl , 500 MHz): δ 3.44 (t, J = 6.4 Hz, 2H,

3

-CH N ), 3.25 (t, J = 6.6 Hz, 2H, -CH I), 2.04 (app pentet, J = 6.5 Hz,

2

3

2

21 21

4

13

−

2

H, -CH -). C (CDCl , 125 MHz): δ 51.5, 32.4, 2.3.

found 393.1566 (M − H) .

2

3

L

DOI: 10.1021/acs.jmedchem.7b00723

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

1

Methyl ((S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-5-

H (CD OD, 500 MHz): δ 7.89 (s, 1H, Arg-NHε), 7.78 (dt, J = 7.6,

3

azido-2-methylpentanoyl)-L-leucinate (27). Compound 26 (1.61 g,

4

0.9 Hz, 2H, Ar-H), 7.70−7.64 (m, 2H, Ar-H), 7.41−7.34 (m, 2H,

Ar-H), 7.30 (td, J = 7.4, 1.1 Hz, 3H, Ar-H), 4.38 (d, J = 6.6 Hz, 2H,

.09 mmol), HATU (1.55 g, 4.09 mmol), HOAt (0.68 mL [0.6 M

solution in DMF], 0.41 mmol), and DIPEA (2.85 mL, 16.4 mmol)

were dissolved in dry DMF (25 mL) and stirred for 5 min to

preactivate the amino acid. A solution of H-Leu-OMe·HCl (0.780 g,

Fmoc-CH ), 4.25−4.19 (m, 2H, Fmoc-CH, Leu-CHα), 3.33−3.27 (m,

2

2H, Arg-CH δ), 2.03−1.94 (m, 1H, Arg-CH β), 1.84−1.75 (m, 1H,

2

Arg-CH β), 1.68−1.52 (m, 4H, -CH(CH ) , 2 × Leu-CH β,

2

3

2

4

.29 mmol) in dry CH Cl (25 mL) was added, and the reaction was

Arg-CH γ), 1.50 (d, J = 3.7 Hz, 10H, -C(CH ) , Arg-CH γ), 1.44

2

3

2

stirred for 16 h at room temperature. The reaction was concentrated in

vacuo, resuspended in EtOAc (50 mL), and washed with 10% citric

acid, water, and brine (50 mL). The organic layer was dried over

Na SO , ﬁltered, and concentrated in vacuo. Crude 27 was puriﬁed

(s, 9H, -C(CH ) ), 1.40 (s, 3H, Arg-CH ), 0.88 (d, J = 6.4 Hz, 3H,

3 3 3

1

3

-CH(CH ) ), 0.87 (d, J = 6.4 Hz, 3H, -CH(CH ) ). C (CDCl₃,

3

2

3 2

125 MHz): δ 176.3, 172.4, 164.6, 157.5, 154.1, 145.3, 142.7, 128.9,

128.2, 126.2, 121.0, 84.5, 80.4, 79.5, 67.8, 60.6, 49.6, 49.3, 41.8, 28.6,

28.3, 26.1, 24.7, 23.7, 22.1. HRMS (ES) calculated for C H N O

9

2

4

using ﬂash chromatography (silica gel, 33% EtOAc in hexanes),

3

8

54

5

+

obtaining the desired dipeptide as a yellow oil (1.22 g, 57%) (R = 0.5

724.3916, found 724.3913 (M + H) .

f

26

on SiO , 2:1 hexane/EtOAc); [α]_D−1.2 (c 1.0, CHCl ); IR (CHCl

cast) 3342, 2956, 2872, 2097, 1731, 1664, 1495, 1450, 1249,

089 cm . H (CDCl , 500 MHz): δ 7.76 (dd, J = 7.4, 1.0 Hz, 2H,

α-MeArg4-pyr-1-apelin-13 A2 (12). Advanced intermediate 7

2

3

(0.2 mmol) was subjected to manual SPPS, introducing amino acids

−1

1

t

1

in the following order: Fmoc-Ser( Bu)-OH,29, Fmoc-Pro-OH, Fmoc-

3

Ar-H), 7.59 (ddd, J = 7.5, 4.0, 1.0 Hz, 2H, Ar-H), 7.44−7.35 (m, 2H,

Ar-H), 7.32 (dddd, J = 7.5, 7.5, 2.2, 1.2 Hz, 2H, Ar-H), 6.38 (br s, 1H,

Leu-NH), 5.83 (br s, 1H, Fmoc-NH), 4.60 (ddd, J = 8.6, 8.0, 4.7 Hz,

Arg(Pmc)-OH. The resin was split into half, and Fmoc-SPPS was

continued on 0.1 mmol scale, coupling pyr-Glu-OH. No end-capping

was performed following addition of 29. A portion (0.05 mmol) of

resin-bound peptide was cleaved as previously described and puriﬁed

using a C₁₈RP-HPLC analytical column (method A), eluting at

13.4 min. The desired peptide was isolated as a white solid after

lyophilization (8.5 mg, 11%). Monoisotopic MW calculated for

C H BrN O 532.5983, found high resolution (FTICR-ESI-MS)

1

2

1

H, Leu-CHα), 4.49−4.35 (m, 2H, Fmoc-CH ), 4.20 (t, J = 6.6 Hz,

2

H, Fmoc-CH), 3.72 (s, 3H, -OCH ), 3.34−3.18 (m, 2H, Arg-CH δ),

3

2

.47−2.23 (m, 1H, Arg-CH β), 1.84−1.76 (m, 1H, Arg-CH β),

2

.70−1.40 (m, 8H, Arg-CH , 2 × Arg-CH γ, 2 × Leu-CH β,

3

2

13

-CH(CH ) ), 0.93 (m, 6H, -CH(CH ) ). C (CDCl , 125 MHz): δ

3 2 3 2 3

70 114

22 16

3+

1

2

5

73.4, 173.1, 154.6, 143.9, 143.9, 141.4, 127.7, 127.1, 127.1, 125.0,

20.0, 66.5, 59.4, 52.4, 51.2, 51.1, 47.3, 41.3, 33.9, 25.0, 24.0, 23.5,

2.8, 21.9. HRMS (ES) calculated for C H N NaO 544.2530, found

532.5973 (M + 3H) .

α-MeArg8-apelin-17 A2 (13). The remaining 0.1 mmol carried

over from the synthesis of 12 was coupled with Fmoc-Gln(Trt)-OH,

Fmoc-Arg(Pmc)-OH, Fmoc-Arg(Pmc)-OH, Fmoc-Phe-OH, and

Fmoc-Lys(Boc)-OH. No end-capping was performed following

addition of 29. A portion (0.05 mmol) of resin-bound peptide was

^c^l^e^a^v^e^d^a^s^p^r^e^vⁱ^o^u^s^l^y^d^e^s^c^rⁱ^b^e^d^aⁿ^d^p^u^rⁱ^ﬁ^e^d^u^sⁱⁿ^g^a^C18 RP-HPLC

analytical column (method A), eluting at 13.0 min. The desired pep-

tide was isolated as a white solid after lyophilization (12.5 mg, 11%).

Monoisotopic MW calculated for C H BrN O 550.7986, found

28

35

5

+

44.2527 (M + Na) .

Methyl ((S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-5-

(Z)-2,3-bis(tert-butoxycarbonyl)guanidino)-2-methylpentanoyl)-L-

leucinate (28). The guanidinylation reaction was adapted from a

(

58

literature procedure. Dipeptide 27 (1.16 g, 2.23 mmol) was dissolved

in MeOH (50 mL) and had 10% Pd/C (20 mg) added. The sus-

pension was stirred under hydrogen gas for 2 h, ﬁltered through a pad

of Celite, and concentrated in vacuo. The crude residue was suspended

in CH Cl (30 mL), and 1,3-di-Boc-2-(triﬂuoromethylsulfonyl)gua-

97

163

34 20

4+

high resolution (FTICR-ESI-MS) 550.7974 (M + 4H) .

α-Methylleucine A2 Analogues. (2S)-2-(2′-Methylpropyl)-2-

methylglycine-Ni(II)-(S)-BPB (30).

2

nidine (0.961 g, 2.45 mmol) and triethylamine (0.69 mL, 4.91 mmol)

were added. This reaction was stirred at room temperature under Ar

for 18 h, concentrated in vacuo, and puriﬁed using ﬂash chromatography

(

6

silica gel, 35% EtOAc in hexanes), yielding 28 as a white solid (1.08 g,

26

6%) (R = 0.4 on SiO , 3:1 hexane/EtOAc); [α] 3.2 (c 1.0, CHCl );

f 2 D 3

IR (CHCl cast) 3332, 2959, 1723, 1644, 1619, 1495, 1451, 1369,

3

−1 1

1

332, 1234, 1155, 1135, 1057 cm . H (CDCl , 500 MHz): δ 8.28

3

(

s, 1H, Arg-NHε), 7.76 (dt, J = 7.5, 0.9 Hz, 2H, Ar-H), 7.60 (ddq,

J = 7.7, 5.0, 1.0 Hz, 2H, Ar-H), 7.46−7.36 (m, 2H, Ar-H), 7.32 (dddd,

J = 7.4, 7.4, 3.8, 1.2 Hz, 2H, Ar-H), 6.39 (br s, 1H, Leu-NH), 5.82

(

2

br s, 1H, Fmoc-NH), 4.62−4.53 (m, 1H, Leu-CHα), 4.46−4.36 (m,

H, Fmoc-CH ), 4.21 (t, J = 6.7 Hz, 1H, Fmoc-CH ), 3.71 (s, 3H,

2

4

2

Alkylation conditions were adapted from literature protocol. Nickel

-

OCH ), 3.44−3.32 (m, 2H, Arg-CH δ), 2.35−2.19 (m, 1H, Arg-

3

2

complex 23 (3.20 g, 6.25 mmol) and potassium tert-butoxide (1.05 g,

.37 mmol) were added to an RBF and dissolved in dry DMF (25 mL)

at 0 °C under an Ar atmosphere. This solution was stirred for 3 min,

followed by the addition of 1-iodo-2-methylpropane (2.16 mL,

8.7 mmol). The reaction was stirred at 0 °C for 30 min, then

warmed to room temperature for an additional 75 min. 5% aqueous

acetic acid (100 mL) was added to quench the reaction, followed by

extraction with CH Cl (3 × 75 mL). The pooled organic layers were

washed with brine (2 × 75 mL), dried over Na SO , ﬁltered, and

concentrated in vacuo. The desired diastereomer was puriﬁed by ﬂash

chromatography (silica gel; 2.5% MeOH in EtOAc), yielding 30 as a

red solid (2.43 g, 69%). Crystals of 30 were obtained after dissolution

in minimal CH Cl , dilution with hexanes, and slow evaporation

CH β), 1.82−1.71 (m, 1H, Arg-CH β), 1.70−1.51 (m, 8H, Arg-CH ,

2

1

(

1

2

3

9

× Arg-CH γ, 2 × Leu-CH β, -CH(CH ) ), 1.49 (s, 9H, -C(CH ) ),

2 2 3 2 3 3

13

.48 (s, 9H, -C(CH ) ), 0.93 (d, J = 6.2 Hz, 6H, -CH(CH ) ).

C

3

3 2

CDCl , 125 MHz): δ 173.5, 173.2, 163.5, 156.2, 153.2, 143.9, 143.9,

3

1

41.3, 127.7, 127.1, 127.1, 125.0, 120.0, 83.1, 79.3, 66.5, 59.5, 52.4,

5

(

1.1, 47.3, 41.2, 40.6, 28.3, 28.1, 25.0, 24.0, 23.7, 22.8, 21.8. HRMS

+

ES) calculated for C H N O 738.4073, found 738.4069 (M + H) .

39

56

5

9

(

(S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-5-((Z)-2,3-

bis(tert-butoxycarbonyl)guanidino)-2-methylpentanoyl)-L-leucine

29). Methyl ester deprotection conditions were adapted from a lit-

2

2 4

(

44

erature protocol. Dipeptide 28 (1.06 g, 1.44 mmol) was dissolved in

aqueous 2-propanol (70% 2-propanol in water, 50 mL) and had

CaCl ·2H O (5.88 g) added to generate a 0.8 M Ca solution.

2+

2

Sodium hydroxide (0.115 g, 2.87 mmol) was added, and the reaction

was stirred for 3.5 h. Acetic acid (0.164 mL, 2.87 mmol) was added to

neutralize residual base, and the reaction was concentrated in vacuo.

The crude residue was dissolved in minimal MeOH and the dipeptide

(Supporting Information Figure S2 ) (R = 0.6 on SiO , 9:1 EtOAc/

f 2

MeOH); [α]_D²⁶1683.7 (c 1.0, CHCl ); IR (CHCl cast) 2959, 2870,

3

−1

1

1668, 1639, 1535, 1439, 1360, 1253, 1170 cm . H (CDCl₃,

500 MHz): δ 8.12−8.06 (m, 3H Ar-H , Ar-H ), 7.51−7.43 (m, 2H,

1

4

was precipitated by the addition of H O and collected by vacuum

Ar-H , Ar-H ), 7.42−7.36 (m, 3H, Ar-H , Ar-H ), 7.34−7.22 (m, 2H,

2

8

9

2

11

ﬁltration, yielding 29 as a white solid (0.982 g, 94%) (R = 0.05 on

Ar-H , Ar-H ), 7.12 (ddd, J = 8.6, 6.7, 1.9 Hz, 1H, Ar-H ), 7.06−7.00

f

3

10

5

26

SiO , 1:1 hexane/EtOAc); [α]_D−6.0 (c 1.0 MeOH); IR (MeOH

cast) 3347, 2957, 1722, 1641, 1450, 1416, 1368, 1331, 1135 cm .

(m, 1H, Ar-H ), 6.64−6.53 (m, 2H, Ar-H , Ar-H ), 4.48 (d, J =

2

12

6

7

−1

12.7 Hz, 1H, Ph-CH -N), 3.74−3.64 (m, 2H, Ph-CH -N, Pro-CH δ),

2

M

DOI: 10.1021/acs.jmedchem.7b00723

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

3

.45 (dd, J = 10.6, 6.1 Hz, 1H, Pro-CHα), 3.26−3.10 (m, 1H, Pro-

reaction was concentrated in vacuo. The crude residue was puriﬁed by

ﬂash chromatography (silica gel, 50% EtOAc in hexanes 0.1% AcOH),

CH γ), 2.74−2.65 (m, 1H, Pro-CH β), 2.56−2.37 (m, 2H, Pro-CH β,

2

Leu-CHγ), 2.13−2.00 (m, 2H, Pro-CH γ, Pro-CH δ), 1.73−1.60

yielding dipeptide 33 as a white sticky solid (0.368 g, 44%) (R = 0.3

2

f

26

(

m, 2H, Leu-CH β), 1.27 (d, J = 6.6 Hz, 3H, Leu-CH δ), 1.22 (s, 3H,

on SiO , 0.1% AcOH in EtOAc); [α]_D27.0 (c 1.0, CHCl ); IR

2

3

2

3

13

Leu-CH β), 1.14 (d, J = 6.7 Hz, 3H, Leu-CH δ). C (CDCl₃,

(CHCl cast) 3386, 3320, 3068, 3018, 2974, 2876, 1727, 1665, 1497,

3

−1 1

1

7

25 MHz): δ 182.9, 180.6, 172.3, 141.7, 136.8, 133.6, 133.5, 131.7,

31.6, 130.5, 129.4, 129.0, 128.9, 128.4, 127.8, 126.9, 123.8, 120.6,

7.5, 70.2, 63.6, 57.1, 48.5, 30.7, 30.7, 25.6, 24.5, 23.3, 23.3. HRMS (ES)

1450, 1240, 1194, 1105 cm . H (CDCl , 500 MHz): δ 7.76 (dd, J =

3

7.7, 0.9 Hz, 2H, Ar-H), 7.59 (ddd, J = 7.5, 2.0, 1.0 Hz, 2H, Ar-H), 7.40

(ddd, J = 7.5, 7.5, 0.9 Hz, 2H, Ar-H), 7.31 (ddd, J = 7.7, 7.5, 1.2, Hz,

2H, Ar-H), 6.78 (br s, 1H, Ser-NH), 5.73 (s, 1H, Fmoc-NH),

+

calculated for C H N NiO 568.2105, found 568.2100 (M + H) .

32

36

3

(

S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-2,4-dimethyl-

4.67−4.56 (m, 1H, Ser-CHα), 4.48−4.34 (m, 2H, Fmoc-CH ), 4.20

2

pentanoic Acid (31). A solution of 3 N HCl/MeOH (1:1, 10 mL) was

heated to 70 °C and had 30 (2.36 g, 4.16 mmol) dissolved in MeOH

(t, J = 6.6 Hz, 1H, Fmoc-CH), 3.95−3.88 (m, 1H, Ser-CH β),

2

3.54−3.48 (m, 1H, Ser-CH β), 2.19−2.07 (m, 1H, Leu-CH β),

2

(

5 mL) added dropwise over 5 min. The reaction mixture was stirred

1.72−1.59 (m, 2H, Leu-CH β, Leu-CHγ), 1.54 (s, 3H, Leu-CH β),

2

3

1

3

for 40 min, turning from dark red to green as the reaction proceeded.

The reaction was cooled to room temperature, concentrated in vacuo,

and resuspended in 10% aqueous sodium carbonate (25 mL). EDTA

disodium salt (3.09 g, 8.31 mmol) was added and stirred for 0.5 h at

room temperature. The reaction was cooled to 0 °C and had a solution

of Fmoc-OSu (1.54 g, 4.57 mmol) dissolved in acetone (25 mL)

added to it. This reaction was slowly warmed to room temperature and

stirred for 22 h. The following day, the reaction was diluted with

EtOAc (50 mL) and washed with 1 N HCl (3 × 50 mL). The organic

1.19 (s, 9H, -C(CH ) ), 0.94−0.82 (m, 6H, 2 × Leu-CH δ).

(CDCl , 125 MHz): δ 174.3, 172.0, 154.9, 143.8, 141.4, 129.1, 128.2,

C

3

127.7, 127.1, 125.0, 120.0, 74.7, 66.6, 61.0, 59.9, 52.7, 47.2, 45.8, 27.7,

24.4, 24.0, 23.6. HRMS (ES) calculated for C H N O 509.2657,

29

37

2

6

−

found 509.2657 (M − H) .

α-MeLeu5-pyr-1-apelin-13 A2 (14). Advanced intermediate 7

(0.2 mmol) was subjected to manual SPPS, introducing amino acids

in the following order: 33, Fmoc-Pro-OH, and Fmoc-Arg(Pmc)-OH.

The resin was split into half, and Fmoc-SPPS was continued on

0.1 mmol scale, coupling pyr-Glu-OH. No end-capping was performed

following addition of 33. A portion (0.05 mmol) of resin-bound pep-

tide was cleaved as previously described and puriﬁed using a C₁₈

RP-HPLC analytical column (method A), eluting at 13.7 min. The

desired peptide was isolated as a white solid after lyophilization (8.5 mg,

11%). Monoisotopic MW calculated for C H BrN O 532.5983,

layer was dried over MgSO , ﬁltered, and concentrated in vacuo. The

product was puriﬁed by ﬂash chromatography (silica gel, 1−5%

MeOH in CH Cl gradient), yielding 31 as a white solid (1.45 g, 95%)

4

2

1

(

R = 0.35 on SiO , 7.5% MeOH in CH Cl ). H (CDCl , 500 MHz):

f 2 2 2 3

δ 7.80−7.74 (m, 2H, Ar-H), 7.64−7.57 (m, 2H, Ar-H), 7.43−7.38

(

m, 2H, Ar-H), 7.37−7.29 (m, 2H, Ar-H), 5.92 (br s, 1H, N-H),

70

114

22 16

3

+

4

2

1

.46−4.33 (m, 2H, Fmoc-CH ), 4.32 (t, 1H, J = 6.0 Hz Fmoc-CH),

found high resolution (FTICR-ESI-MS) 532.5972 (M + 3H) .

α-MeLeu9-apelin-17 A2 (15). The remaining 0.1 mmol carried

over from the synthesis of 14 was coupled with Fmoc-Gln(Trt)-OH,

Fmoc-Arg(Pmc)-OH, Fmoc-Arg(Pmc)-OH, Fmoc-Phe-OH, and

Fmoc-Lys(Boc)-OH. A portion (0.05 mmol) of resin-bound peptide

was cleaved as previously described and puriﬁed using a C₁₈RP-HPLC

analytical column (method A), eluting at 11.5 min. The desired pep-

tide was isolated as a white solid after lyophilization (14.3 mg, 13%).

Monoisotopic MW calculated for C H BrN O 550.7986, found

2

.26−2.19 (m, 1H, Leu-CH β), 1.83−1.73 (m, 1H, Leu-CH β),

2

.70−1.55 (m, 4H, Leu-CHγ, Leu-CH β), 0.96−0.82 (m, 6H, 2 ×

3

13

Leu-CH δ). C (CDCl , 125 MHz): δ 179.4, 155.2, 143.9, 141.6,

3

1

(

27.8, 127.1, 124.8, 120.0, 65.1, 47.3, 24.9, 24.7, 23.8, 23.0. HRMS

−

ES) calculated for C H NO 366.1711, found 366.1706 (M − H) .

Methyl N-((S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-

,4-dimethylpentanoyl)-O-(tert-butyl)-L-serinate (32). Amino acid

1 (1.53 g, 4.16 mmol), HATU (1.58 g, 4.16 mmol), HOAt (0.69 mL

0.6 M solution in DMF], 0.42 mmol), and DIPEA (2.90 mL,

6.6 mmol) were dissolved in dry DMF (25 mL) and stirred for 5 min

to preactivate the amino acid. A solution of H-Ser( Bu)-OMe·HCl

0.925 g, 4.37 mmol) in dry CH Cl (25 mL) was added, and the

reaction was stirred for 20 h at room temperature. The reaction was

concentrated in vacuo, resuspended in EtOAc (50 mL), and washed

with 10% citric acid, water, and brine (50 mL). The organic layer was

dried over Na SO , ﬁltered, and concentrated in vacuo. Crude 32 was

22

24

4

2

3

[

1

9

7

163

34 20

4

+

high resolution (FTICR-ESI-MS) 550.7974 (M + 4H) .

Aza-arginine A2 Analogues. tert-Butyl (2-(Diphenylmeth-

t

ylene)hydrazine-1-carbonyl)-L-leucinate (34). Semicarbazone 34

4

6

(

was prepared by modifying a literature procedure. A solution of

benzophenone hydrazone (4.39 g, 22.4 mmol) in dry CH Cl (50 mL)

2

was cooled to 0 °C and cannulated into a 0 °C solution of dis-

uccinimidyl carbonate (7.41 g [85% purity], 24.6 mmol) in dry

CH Cl (50 mL) and dry DMF (10 mL). The reaction was warmed to

2

4

2

puriﬁed using ﬂash chromatography (silica gel, 25% EtOAc in

room temperature for 45 min, then cooled down to 0 °C. A 0 °C solu-

t

hexanes), obtaining the desired product as a white solid (0.872 g,

tion of H-Leu-O Bu·HCl (5.00 g, 22.4 mmol) and DIPEA (7.79 mL,

26

D

4

0%) (R = 0.2 on SiO , 3:1 hexanes/EtOAc); [α]

20.4 (c 1.0,

44.7 mmol) in dry CH Cl (50 mL) was then cannulated into the

f

2

CHCl ); IR (CHCl cast) 3440, 3384, 2973, 2956, 2872, 1730, 1669,

reaction vessel and allowed to slowly come up to room temperature

over 16 h. The reaction was concentrated in vacuo and puriﬁed by

ﬂash chromatography (silica gel, 20% EtOAc in hexanes), yielding a

light yellow sticky solid (8.29 g, 91%) (R = 0.8 on SiO , 1:1 hexanes/

3

−1 1

1

7

6

493, 1364, 1241, 1099 cm . H (CDCl , 500 MHz): δ 7.76 (dd, J =

.6, 0.9 Hz, 2H, Ar-H), 7.64−7.58 (m, 2H, Ar-H), 7.39 (ddd, J = 6.8,

.8, 0.8 Hz, 2H, Ar-H), 7.31 (ddd, J = 7.4, 6.8, 1.1 Hz, 2H, Ar-H), 6.64

3

f

2

EtOAc); [α]_D²⁶36.7 (c 1.27 CHCl ); IR (CHCl cast) 3414, 3355,

(

br s, 1H, Ser-NH), 6.09 (s, 1H, Fmoc-NH), 4.74−4.68 (m, 1H,

3 3

Ser-CHα), 4.43−4.30 (m, 2H, Fmoc-CH ), 4.22 (t, J = 6.8 Hz, 1H,

Fmoc-CH), 3.81 (d, J = 8.8 Hz, 1H, Ser-CH β), 3.75 (s, 3H, -OCH ),

3188, 3062, 2957, 2871, 1732, 1682, 1519, 1446, 1368, 1153,

2

−1

1

1113 cm . H (CDCl , 400 MHz): δ 7.59 (s, 1H, = N-NH),

2

3

.57 (d, J = 8.8 Hz, 1H, Ser-CH β), 2.44−2.31 (m, 1H, Leu-CH β),

7.57−7.46 (m, 5H, Ar-H), 7.40−7.29 (m, 3H, Ar-H), 7.29−7.22

(m, 2H, Ar-H), 6.68 (d, J = 9.0 Hz, 1H, Leu-NH), 4.52 (ddd, J = 8.8,

8.8, 5.6 Hz, 1H, Leu-CHα), 1.85−1.74 (m, 1H, Leu-CHγ), 1.74−1.60

(m, 2H, Leu-CH β), 1.50 (s, 9H, -C(CH ) ), 1.00 (d, J = 6.5 Hz, 6H,

2

1

.73−1.52 (m, 5H, Leu-CH β, Leu-CHγ, Leu-CH β), 1.13 (s, 9H,

2

3

1

3

-C(CH ) ), 0.94−0.82 (m, 6H, 2 × Leu-CH δ). C (CDCl , 125

3

MHz): δ 173.9, 170.7, 154.4, 144.1, 144.0, 141.3, 127.6, 127.1, 125.1,

2

3 3

1

3

1

2

5

20.0, 73.4, 66.4, 61.9, 59.6, 53.1, 52.4, 47.3, 45.1, 27.3, 25.2, 24.7,

3.7, 23.6. HRMS (ES) calculated for C H N O 525.2959, found

25.2956 (M + H) .

N-((S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-2,4-

dimethylpentanoyl)-O-(tert-butyl)-L-serine (33). Methyl ester depro-

2 × Leu-CH δ); C (CDCl , 125 MHz): δ 172.6, 154.9, 148.2, 137.0,

3 3

131.9, 129.8, 129.7, 129.4, 129.3, 128.8, 128.5, 128.3, 128.1, 127.2,

126.5, 81.7, 51.9, 42.4, 28.1, 25.0, 23.0, 22.1. HRMS (ES) calculated

30

41

2

6

+

for C H N O 410.2438, found 410.2438 (M + H) .

24

32

3

tert-Butyl (1-(3-Chloropropyl)-2-(diphenylmethylene)hydrazine-

4

tection conditions were adapted from literature procedure. Dipeptide

3

1-carbonyl)-L-leucinate (35). Alkylation conditions were initially

4

5

2 (0.853 g, 1.63 mmol) was dissolved in aqueous 2-propanol (70%

adapted from a literature procedure. Semicarbazone 34 (2.47 g,

6.03 mmol) was dissolved in THF (50 mL) and cooled to 0 °C.

Aqueous tetraethylammonium hydroxide (22.2 mL [20% solution],

30.1 mmol) was added and stirred for 30 min, followed by the addi-

tion of 1-bromo-3-chloropropane (4.47 mL, 45.2 mmol) at 0 °C.

2-propanol in water, 50 mL) and had CaCl ·2H O (5.88 g) added

2

2+

to generate a 0.8 M Ca solution. Sodium hydroxide (0.130 g,

.25 mmol) was added, and the reaction was stirred for 4 h. Acetic acid

0.186 mL, 3.25 mmol) was added to neutralize residual base, and the

3

(

N

DOI: 10.1021/acs.jmedchem.7b00723

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

The reaction was slowly warmed to room temperature and was

quenched after 72 h by the addition of 10% citric acid (20 mL)

followed by brine (20 mL). Organic components were extracted with

EtOAc (3 × 100 mL), dried over Na SO , ﬁltered, and concentrated in

addition of a solution of semicarbazide 37 (1.34 g, 4.07 mmol) in dry

CH Cl (20 mL). The reaction mixture was stirred under Ar gas for

2

18 h and then concentrated in vacuo. The crude reaction mixture was

resuspended in EtOAc (75 mL) and washed with 10% citric acid

(50 mL), water (50 mL), and brine (50 mL). Pooled aqueous layers

were washed with EtOAc (2 × 75 mL), and pooled organic fractions

were dried over Na SO , ﬁltered, and concentrated in vacuo. The

2

4

vacuo. Alkylated semicarbazone 204 was puriﬁed by ﬂash chromatog-

raphy (silica gel, 15% EtOAc in hexanes) and was isolated as a yellow

26

D

oil (2.16 g, 74%) (R = 0.85 on SiO , 2:1 hexanes/EtOAc); [α] 67.5

f

2

4

(

1

c 1.42 CHCl ); IR (CHCl cast) 3414, 3061, 2959, 2870, 1734, 1682,

product was puriﬁed by ﬂash chromatography (silica gel, 50% EtOAc

3

−1 1

499, 1446, 1368, 1154 cm . H (CDCl , 500 MHz): δ 7.55−7.41

in hexanes) yielding azatripeptide 38 as a white solid (1.74 g, 66%)

3

2

6

(

m, 6H, Ar-H), 7.41−7.30 (m, 4H, Ar-H), 6.69 (d, J = 8.7 Hz, 1H,

Leu-NH), 4.47 (ddd, J = 8.8, 8.7, 5.6 Hz, 1H, Leu-CHα), 3.46 (dt, J =

4.7, 6.8 Hz, 1H, N-CH ), 3.38−3.27 (m, 3H, N-CH , 2 ×

(R = 0.4 on SiO , 1:1 hexanes/EtOAc); [α] −7.2 (c 0.83 CHCl );

f

2

D

3

IR (CHCl cast) 3357, 3229, 2957, 2872, 2097, 1690, 1529, 1452,

1424, 1367, 1257, 1155 cm . H (CDCl , 500 MHz): δ 8.20 (s, 1H,

3

−1 1

1

2

N-CH CH ), 1.80−1.72 (m, 3H, 2 × -CH Cl, Leu-CHγ), 1.66

(

Leu-CH β), 1.48 (s, 9H, -C(CH ) ), 0.98 (d, J = 6.6 Hz, 6H, 2 ×

C(O)NH-N), 7.80−7.74 (m, 2H, Ar-H), 7.61−7.54 (m, 2H, Ar-H),

7.47−7.37 (m, 2H, Ar-H), 7.31 (t, J = 7.5 Hz, 2H, Ar-H), 6.04 (d, J =

2

ddd, J = 13.6, 8.0, 5.6 Hz, 1H, Leu-CH β), 1.61−1.51 (m, 1H,

2

8.2 Hz, 1H, Leu-NH), 4.47 (dd, J = 10.5, 7.1 Hz, 1H, Fmoc-CH

),

, Leu-CHα), 4.25 (t, J = 6.8 Hz, 1H,

Fmoc-CH), 4.19 (dd, J = 7.4, 4.5 Hz, 1H, Pro-CHα), 3.67−3.54

(m, 3H, 2 × N-CH , Pro-CH δ), 3.55−3.46 (m, 1H, Pro-CH δ),

3.44−3.33 (m, 2H, -CH ), 2.27 (ddd, J = 11.5, 5.3, 4.9 Hz, 1H,

Pro-CH β), 2.17−2.06 (m, 2H, Pro-CH β, Pro-CH γ), 2.01−1.93

2

3 3

2

13

Leu-CH δ). C (CDCl , 125 MHz): δ 172.8, 159.4, 158.3, 138.6,

1

4

4.42−4.29 (m, 2H, Fmoc-CH

2

3

35.7, 130.2, 129.8, 128.9, 128.8, 128.6, 128.2, 81.3, 52.7, 44.1, 42.4,

2.1, 30.0, 28.1, 25.1, 22.9, 22.2. HRMS (ES) calculated for

2

+

C H ClN O 486.2518, found 486.2518 (M + H) .

N

2 3

27

37

3

tert-Butyl (1-(3-Azidopropyl)-2-(diphenylmethylene)hydrazine-1-

2

carbonyl)-L-leucinate (36). This reaction was adapted from a literature

procedure. Chloroalkylated semicarbazone 35 (2.08 g, 4.27 mmol)

and sodium azide (0.833 g, 12.8 mmol) were dissolved in DMF

(

(m, 1H, Pro-CH γ), 1.82−1.72 (m, 2H, -CH CH N ), 1.63 (ddd,

2

3

45

J = 13.4, 6.7, 6.5 Hz, 1H, Leu-CHγ), 1.54 (ddd, J = 7.1, 6.7, 6.7 Hz,

1H, Leu-CH β), 1.48−1.38 (m, 10H, Leu-CH β, -C(CH ) ), 0.84

2

3 3

1

3

50 mL) and heated at 60 °C for 20 h. The reaction was cooled and

(d, J = 6.5, 3H, Leu-CH δ), 0.83 (d, J = 6.5, 3H, Leu-CH δ).

C

3

had H O (150 mL) added to it, followed by EtOAc (3 × 150 mL)

(CDCl , 125 MHz): δ 173.5, 170.9, 156.8, 156.0, 143.7, 141.4, 127.9,

127.1, 125.0, 120.1, 81.3, 68.0, 59.2, 52.6, 49.2, 47.2, 47.1, 46.2, 41.9,

2

3

washes to extract organic components. Pooled EtOAc layers were

dried over Na SO , ﬁltered, and concentrated in vacuo. The product

was puriﬁed using ﬂash chromatography (silica gel, 10% EtOAc in

28.8, 28.0, 27.2, 25.0, 24.7, 22.8, 22.1. HRMS (ES) calculated for

2

4

+

C H N O 648.3504, found 648.3503 (M + H) .

34

46

7

6

hexanes), generating azide 36 as a yellow oil (1.90 g, 90%) (R = 0.2 on

(2-((S)-1-(((9H-Fluoren-9-yl)methoxy)carbonyl)pyrrolidine-2-car-

boxamido)-7-((tert-butoxycarbonyl)amino)-11,11-dimethyl-9-oxo-

0-oxa-2,6,8-triazadodec-7-enoyl)-L-leucine (39). A solution of

azatripeptide 38 (0.803 g, 1.24 mmol) in dry CH Cl (20 mL) was

cooled to 0 °C and had TFA (10 mL) added, and the mixture was

stirred for 15 min. The reaction was warmed to room temperature

for 90 min and concentrated in vacuo, using toluene coevaporations

to remove residual TFA. The product was resuspended in MeOH

f

26

SiO , 9:1 hexanes/EtOAc); [α]_D53.8 (c 1.00 CHCl ); IR (CHCl₃

2

3

1

cast) 3411, 3061, 2958, 2871, 2097, 1734, 1683, 1499, 1446, 1368,

−

1 1

1

7

(

257, 1154 cm . H (CDCl , 500 MHz): δ 7.54−7.40 (m, 6H, Ar-H),

2

3

.40−7.26 (m, 4H, Ar-H), 6.74 (d, J = 8.7 Hz, 1H, Leu-NH), 4.47

ddd, J = 8.8, 8.7, 5.6 Hz, 1H, Leu-CHα), 3.42 (dt, J = 14.7, 7.0 Hz,

1

H, N-CH ), 3.27 (dt, J = 14.7, 6.8 Hz, 1H, N-CH ), 3.05 (t, J =

2

7

.1 Hz, 2H, -CH N ), 1.76 (ddsept, J = 12.9, 7.8, 6.5 Hz, 1H,

2

3

(20 mL), 10% Pd/C (25 mg) was added, and the reaction was stirred

Leu-CHγ), 1.70−1.62 (m, 1H, Leu-CH β), 1.61−1.55 (m, 1H,

2

under an atmosphere of hydrogen gas for 75 min. The reaction

mixture was ﬁltered through a pad of Celite, concentrated in vacuo to

dryness, and resuspended in dry CH Cl (20 mL) with triethylamine

Leu-CH β), 1.56−1.49 (m, 2H, -CH CH N ), 1.48 (s, 9H,

2

3

-

C(CH ) ), 0.99 (d, J = 6.6 Hz, 3H, Leu-CH δ), 0.98 (d, J =

3

13

6

1

4

.6 Hz, 3H, Leu-CH δ). C (CDCl , 125 MHz): δ 172.8, 158.5,

2

3

(0.76 mL, 5.45 mmol) and 1,3-di-Boc-2-(triﬂuoromethylsulfonyl)-

58.4, 138.6, 135.8, 130.2, 129.8, 128.9, 128.8, 128.5, 128.3, 81.3, 52.7,

guanidine (0.801 g, 2.05 mmol) for 40 h. Solvents were removed in

vacuo, and 39 was puriﬁed using ﬂash chromatography (silica gel, 90%

8.9, 43.4, 42.1, 28.1, 26.2, 25.1, 22.9, 22.2. HRMS (ES) calculated for

+

C H N O 493.2922, found 493.2922 (M + H) .

27

37

6

3

EtOAc in hexanes, 0.1% AcOH), yielding a white solid (0.413 g, 41%)

tert-Butyl (1-(3-Azidopropyl)hydrazine-1-carbonyl)-L-leucinate

4

6,59

26

(

37). This procedure was adapted from literature protocols.

(R

CH

1419, 1335, 1156, 1136 cm . H (CDCl

f

= 0.05 on SiO

2

, 0.1% AcOH in EtOAc); [α]

D

−42.4 (c 1.00

Azide 36 (1.89 g, 3.84 mmol) was dissolved in a solution of hydro-

xylamine hydrochloride (1.07 g, 15.4 mmol) in pyridine (75 mL) and

heated to 60 °C for 22 h. The reaction was cooled to room tem-

OH); IR (CH

3

OH cast) 3328, 2959, 1721, 1688, 1642, 1530,

−1 1

, 500 MHz): δ 9.47 (br s,

3

1H, Arg-NHε), 8.53 (s, 1H, C(O)NH-N), 7.75 (d, J = 7.5 Hz, 2H,

Ar-H), 7.56 (d, J = 7.5 Hz, 1H, Ar-H), 7.54 (d, J = 7.5 Hz, 1H, Ar-H),

7.39 (app t, J = 7.4 Hz, 2H, Ar-H), 7.30 (app t, J = 7.5 Hz, 2H, Ar-H),

perature, then concentrated in vacuo, using CH Cl₂and EtOAc

2

coevaporations to remove residual pyridine. The product was puriﬁed

using ﬂash chromatography (silica gel, 60% EtOAc in hexanes,

6.47 (br s, 1H, Leu-NH), 4.39 (dd, J = 10.4, 7.1 Hz, 1H, Fmoc-CH

4.30−4.23 (m, 2H, Fmoc-CH2, Leu-CHα), 4.23−4.17 (m, 2H, Fmoc-

CH, Pro-CHα), 3.84−3.69 (m, 1H, Arg-N-CH β), 3.64−3.57 (m, 1H,

Pro-CH δ), 3.56−3.48 (m, 2H, Pro-CH δ, Arg-CH δN), 3.46−3.38

),

2

0

.1% DIPEA) yielding semicarbazide 37 as a yellow oil (1.19 g, 94%)

26

(R = 0.5 on SiO , 1:1 hexanes/EtOAc); [α] 5.0 (c 0.92 CHCl ); IR

2

f

2

D

3

(

CHCl cast) 3406, 3335, 3216, 2958, 2934, 2871, 2098, 1732, 1654,

2

3

−1 1

(

1

m, 1H, Pro-CH δ), 3.35−3.26 (m, 1H, Arg-CH δN), 2.21−2.09

1

8

3

2

510, 1368, 1257, 1155 cm . H (CDCl , 500 MHz): δ 6.67 (d, J =

2

3

m, 2H, Pro-CH β, Arg-CH γ), 2.09−2.01 (m, 1H, Arg-CH γ),

.8 Hz, 1H, Leu-NH), 4.33 (ddd, J = 8.9, 8.8, 5.5 Hz, 1H, Leu-CHα),

.64 (s, 2H, H N-N), 3.62−3.51 (m, 2H, N-CH ), 3.36 (t, J = 6.7 Hz,

2

.93−1.86 (m, 1H, Pro-CH β), 1.84−1.72 (m, 2H, Pro-CH γ),

2

.70−1.54 (m, 3H, Leu-CHγ, 2 × Leu-CH β), 1.49 (m, 9H,

H, -CH N ), 1.84 (app p, J = 6.8 Hz, 2H, -CH CH N ), 1.71

2

3

2

3

-C(CH ) ), 1.46 (s, 9H, -C(CH ) ), 0.95−0.77 (m, 6H, 2 × Leu-

(

5

ddsept, J = 8.2, 8.0, 6.5 Hz, 1H, Leu-CHγ), 1.59 (ddd, J = 13.6, 8.1,

.5 Hz, 1H, Leu-CH β), 1.53−1.47 (m, 1H, Leu-CH β), 1.45 (s, 9H,

3

3 3

13

CH δ). C (CDCl , 125 MHz): δ 171.8, 163.1, 158.7, 156.5, 155.3,

3

2

1

3

1

2

53.1, 143.7, 141.3, 129.0, 128.2, 127.8, 127.1, 127.1, 125.3, 125.0,

20.0, 83.4, 79.8, 67.8, 58.7, 53.1, 47.1, 44.9, 39.9, 37.9, 29.7, 28.3,

-

C(CH ) ), 0.95 (d, J = 6.6 Hz, 6H, 2 × Leu-CH δ). C (CDCl , 125

3

MHz): δ 173.5, 158.7, 81.3, 52.3, 49.2, 48.0, 42.4, 28.1, 26.3, 25.0, 22.9,

8.1, 27.2, 24.9, 24.5, 22.8, 21.8. HRMS (ES) calculated for

2

3

2.1. HRMS (ES) calculated for C H N O 329.2296, found

29.2290 (M + H) .

14 29 6 3

−

+

C H N O 806.4094, found 806.4088 (M-H) .

41 56

7

10

azaArg4-pyr-1-apelin-13 A2 (16). Advanced intermediate 7

(

9H-Fluoren-9-yl)methyl (S)-2-(2-(3-Azidopropyl)-2-(((S)-1-(tert-

butoxy)-4-methyl-1-oxopentan-2-yl)carbamoyl)hydrazine-1-

(0.2 mmol) was subjected to manual SPPS, introducing amino acids

t

carbonyl)pyrrolidine-1-carboxylate (38). A solution of Fmoc-Pro-OH

in the following order: Fmoc-Ser( Bu)-OH, 39, and Fmoc-Arg(Pmc)-

(

[

1.65 g, 4.88 mmol), HATU (1.86 g, 4.88 mmol), HOAt (0.81 mL

0.6 M solution], 0.49 mmol), and DIPEA (2.13 mL, 12.2 mmol) were

dissolved in dry DMF (20 mL) and preactivated for 5 min before the

OH. The resin was split into half, and Fmoc-SPPS was continued on

0.1 mmol scale, coupling pyr-Glu-OH. No end-capping was performed

following addition of 39. A portion (0.05 mmol) of resin-bound

O

DOI: 10.1021/acs.jmedchem.7b00723

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

peptide was cleaved as previously described and puriﬁed using a C₁₈

RP-HPLC analytical column (method A), eluting at 13.5 min. The

desired peptide was isolated as a white solid after lyophilization (5.5 mg,

using ﬂash chromatography (silica gel, 50% EtOAc in hexanes),

yielding semicarbazide 42 as a yellow oil (1.37 g, 93%) (R = 0.6 on

f

26

SiO , 3:1 hexanes/EtOAc); [α]_D20.8 (c 0.45 CH Cl ); IR (CH Cl

2

7

%). Monoisotopic MW calculated for C H BrN O 528.2582,

cast) 3420, 3331, 3217, 2974, 2934, 2873, 1741, 1656, 1509, 1366,

68

111

23 16

3+

−1 1

found high resolution (FTICR-ESI-MS) 528.2571 (M + 3H) .

azaArg8-apelin-17 A2 (17). The remaining 0.1 mmol carried

over from the synthesis of 16 was coupled with Fmoc-Gln(Trt)-OH,

Fmoc-Arg(Pmc)-OH, Fmoc-Arg(Pmc)-OH, Fmoc-Phe-OH, and

Fmoc-Lys(Boc)-OH. A portion (0.05 mmol) of resin-bound peptide

was cleaved as previously described and puriﬁed using a C₁₈RP-HPLC

analytical column (method A), eluting at 11.1 min. The desired pep-

tide was isolated as a white solid after lyophilization (15.6 mg, 14%).

Monoisotopic MW calculated for C H BrN O 547.5435, found

1232, 1157, 1100 cm . H (CDCl , 500 MHz): δ 7.04 (d, J = 9.0 Hz,

3

1H, Ser-NH), 4.45 (ddd, J = 9.0, 3.1, 3.0 Hz, 1H, Ser-CHα), 3.78

(dd, J = 8.6, 3.1 Hz, 1H, Ser-CH β), 3.56 (s, 2H, H N-N), 3.52

2

(dd, J = 8.6, 3.2 Hz, 1H, Ser-CH β), 3.36 (dd, J = 13.9, 7.7 Hz, 1H,

2

Leu-NCH β), 3.26 (dd, J = 13.9, 7.4 Hz, 1H, Leu-NCH β), 2.01−1.89

2

(m, 1H, Leu-CHγ), 1.46 (s, 9H, C(O)OC(CH ) ), 1.14 (s, 9H,

3

-CH OC(CH ) ), 0.92 (d, J = 6.7 Hz, 3H, Leu-CH δ), 0.91 (d, J =

2

3

13

6.7 Hz, 3H, Leu-CH δ). C (CDCl , 125 MHz): δ 170.8, 159.1, 81.1,

3

72.8, 63.1, 57.4, 54.4, 28.1, 27.4, 26.1, 19.9. HRMS (ES) calculated for

95

160

35 20

4+

+

high resolution (FTICR-ESI-MS) 547.5430 (M + 4H) .

C H N O 332.2544, found 332.2539 (M + H) .

16

34

3

4

Aza-leucine A2 Analogues. tert-Butyl O-(tert-Butyl)-N-(2-

tert-Butyl N-(2-((S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)-

(

diphenylmethylene)hydrazine-1-carbonyl)-L-serinate (40). This

amino)-5-((((2-chlorobenzyl)oxy)carbonyl)amino)pentanoyl)-1-

isobutylhydrazine-1-carbonyl)-O-(tert-butyl)-L-serinate (43). A solu-

tion of Fmoc-Orn(2-Cl-Cbz)-OH (2.59 g, 4.91 mmol), HATU

(1.87 g, 4.91 mmol), HOAt (0.82 mL [0.6 M solution], 0.49 mmol),

and DIPEA (2.14 mL, 12.3 mmol) was dissolved in dry DMF (20 mL)

and preactivated for 5 min before the addition of a solution of

4

6

molecule was prepared by adapting a literature procedure. A solu-

tion of benzophenone hydrazone (1.55 g, 7.88 mmol) in dry CH Cl₂

2

(

30 mL) was cooled to 0 °C and cannulated into a 0 °C solution of

disuccinimidyl carbonate (2.61 g [85% purity], 8.67 mmol) in dry

CH Cl (30 mL) and dry DMF (5 mL). The reaction was warmed to

2

room temperature for 45 min, then cooled down to 0 °C. A 0 °C

semicarbazide 42 (1.36 g, 4.09 mmol) in dry CH Cl (20 mL). This

2 2

t

solution of H-Ser( Bu)-O Bu·HCl (2.00 g, 7.88 mmol) and DIPEA

2.75 mL, 15.8 mmol) in dry CH Cl (20 mL) was then cannulated

reaction was stirred under Ar for 30 h and then concentrated in vacuo.

The crude reaction was resuspended in EtOAc (100 mL) and washed

with 10% citric acid (2 × 100 mL) and brine (100 mL). Pooled

aqueous layers were washed with EtOAc (2 × 100 mL), and pooled

(

2

into the reaction vessel and allowed to slowly come up to room

temperature over 24 h. The reaction was concentrated in vacuo and

puriﬁed by ﬂash chromatography (silica gel, 25% EtOAc in hexanes),

yielding a light yellow sticky solid (2.94 g, 83%) (R = 0.4 on SiO , 1:1

organic fractions were dried over Na SO , ﬁltered, and concentrated in

2 4

vacuo. The reaction was puriﬁed by ﬂash chromatography (silica

gel, 40% EtOAc in hexanes) yielding azatripeptide 43 as a white solid

f

2

hexanes/EtOAc); [α]_D²⁶28.0 (c 1.0, CH Cl ); IR (CH Cl cast) 3425,

2

−1

3

173, 3059, 2975, 2932, 1751, 1743, 1693, 1518, 1367, 1161 cm .

(2.64 g, 77%), with a 17% recovery of 42 (R

f

= 0.2 on SiO

); IR (CHCl cast) 3321,

3007, 2974, 2934, 2873, 1700, 1657, 1523, 1450, 1367, 1247,

2

, 1:1

1

26

H (CDCl , 500 MHz): δ 7.61 (s, 1H, = N-NH), 7.58−7.45 (m, 5H,

hexanes/EtOAc); [α]

D

11.8 (c 1.00 CHCl

3

Ar-H), 7.38−7.29 (m, 3H, Ar-H), 7.29−7.23 (m, 2H, Ar-H), 7.17

−1

1

(

d, J = 8.9 Hz, 1H, Ser-NH), 4.55 (ddd, J = 8.9, 3.3, 2.9 Hz, 1H,

1157 cm . H (CDCl , 500 MHz): δ 8.31 (s, 1H, C(O)NH-N),

3

Ser-CHα), 3.88 (dd, J = 8.6, 2.9 Hz, 1H, Ser-CH β), 3.64 (dd, J = 8.6,

7.79−7.74 (m, 2H, Fmoc-Ar-H), 7.59 (d, J = 7.5 Hz, 2H, Fmoc-Ar-H),

7.39 (m, 4H, 2 × Fmoc-Ar-H, 2 × 2-Cl-Ar-H), 7.34−7.28 (m, 2H,

Fmoc-Ar-H), 7.27−7.23 (m, 2H, 2 × 2-Cl-Ar-H), 5.82 (d, J = 8.2 Hz,

1H, Ser-NH), 5.53 (d, J = 7.8 Hz, 1H, Orn-NHα), 5.27−5.20

2

3

.3 Hz, 1H, Ser-CH β), 1.50 (s, 9H, C(O)OC(CH ) ), 1.22 (s, 9H,

2

3

1

3

-CH OC(CH ) ); C (CDCl , 125 MHz): δ 169.9, 155.2, 147.9,

2 3 3 3

1

2

4

37.1, 131.9, 129.7, 129.2, 128.5, 128.2, 127.1, 81.6, 73.1, 62.7, 53.8,

8.1, 27.5. HRMS (ES) calculated for C H N O 440.2544, found

(m, 2H, -OCH

-OCH (2-Cl)Ph), 4.48−4.38 (m, 3H, Ser-CHα, Orn-CHα,

Fmoc-CH ), 4.21 (t, J = 7.0 Hz,

(2-Cl)Ph, Orn-NHε), 5.16 (d, J = 13.0 Hz, 1H,

25

34

3

4

2

+

40.2549 (M + H) .

tert-Butyl O-(tert-Butyl)-N-(2-(diphenylmethylene)-1-(2-meth-

ylallyl)hydrazine-1-carbonyl)-L-serinate (41). Alkylation conditions

2

), 4.38−4.32 (m, 1H, Fmoc-CH

2

1H, Fmoc-CH), 3.71 (dd, J = 8.8, 2.8 Hz, 1H, Ser-CH β), 3.54−3.45

2

45

were adapted from a literature procedure. Semicarbazone 40 (0.945 g,

.15 mmol) was dissolved in THF (10 mL) and cooled to 0 °C.

Aqueous tetraethylammonium hydroxide (7.91 mL [20% solution],

0.8 mmol) was added and stirred for 30 min, followed by the addition

(m, 2H, Ser-CH β, Orn-CH δ), 3.44−3.36 (m, 1H, Leu-NCH β),

2

3.33−3.24 (m, 1H, Leu-NCH β), 3.24−3.15 (m, 1H, Orn-CH δ),

2

2.00−1.90 (m, 1H, Orn-CH β), 1.86−1.77 (m, 1H, Leu-CHγ), 1.75−

2

1

1.56 (m, 3H, Orn-CH β, 2 × Orn-CH γ), 1.41 (s, 9H, C(O)OC-

2

of 3-bromo-2-methylpropene (1.63 mL, 16.1 mmol) at 0 °C. The reac-

tion was slowly warmed to room temperature and was quenched

after 60 h by the addition of 10% citric acid (15 mL) followed by

brine (15 mL). Organic components were extracted with EtOAc

(CH ) ), 1.08 (s, 9H, -CH OC(CH ) ), 0.89 (d, J = 6.6 Hz, 6H, 2 ×

3 3 2 3 3

13

Leu-CH δ). C (CDCl , 125 MHz): δ 173.7, 170.2, 157.1, 156.7,

3

156.4, 143.6, 141.3, 134.1, 133.5, 129.7, 129.5, 129.4, 127.8, 127.1,

126.8, 125.1, 120.0, 81.6, 72.9, 67.3, 64.1, 62.6, 55.4, 54.5, 52.1, 47.1,

39.7, 29.8, 28.0, 27.3, 26.9, 26.2, 20.0, 20.0. HRMS (ES) calculated for

(

3 × 75 mL), dried over Na SO , ﬁltered, and concentrated in vacuo.

2 4

+

Alkylated semicarbazone 41 was puriﬁed by ﬂash chromatography

C H ClN O 836.3996, found 836.4008 (M + H) .

44

59

5

9

(

1

silica gel, 20% EtOAc in hexanes) and was isolated as a yellow oil

N-(2-((S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-5-((((2-

26

1.06 g, 99%) (R = 0.6 on SiO , 3:1 hexanes/EtOAc); [α]

6.1

chlorobenzyl)oxy)carbonyl)amino)pentanoyl)-1-isobutylhydrazine-

1-carbonyl)-O-(tert-butyl)-L-serine (44). Azatripeptide 43 (1.21 g,

1.44 mmol) was dissolved in toluene (100 mL) and had ﬂash-grade

silica (25.0 g) added. This suspension was reﬂuxed at 115 °C for

95 min, checking TLC and LC−MS every 30 min. The reaction was

f

2

D

c 0.75 CH Cl ); IR (CH Cl cast) 3421, 3062, 2974, 2935, 2877,

2

−1 1

745, 1687, 1496, 1366, 1154, 1098 cm . H (CDCl , 500 MHz):

3

δ 7.50−7.39 (m, 6H, Ar-H, Ser-NH), 7.39−7.34 (m, 1H, Ar-H),

7

.32−7.27 (m, 4H, Ar-H), 4.71 (dq, J = 1.4, 1.1 Hz, 1H, CHH),

4

.58 (ddd, J = 8.9, 3.2, 3.2 Hz, 1H, Ser-CHα), 4.48−4.43 (m, 1H, 

cooled to room temperature and a 10% MeOH in CH Cl solution

2 2

CHH), 3.98 (d, J = 17.0 Hz, 1H, Leu-NCH β), 3.88−3.79 (m, 2H,

(100 mL) was added, then ﬁltered through a pad of Celite. The reac-

tion was concentrated in vacuo and puriﬁed by ﬂash chromatography

(silica gel, 0−2% MeOH in EtOAc, 0.1% AcOH). The desired product

2

Ser-CH β, Leu-NCH β), 3.62 (dd, J = 8.6, 3.3 Hz, 1H, Ser-CH β),

2

1

.48 (s, 9H, C(O)OC(CH ) ), 1.41−1.32 (m, 3H, -CH ), 1.17 (s, 9H,

3

13

-CH OC(CH ) ). C (CDCl , 125 MHz): δ 170.2, 158.5, 154.1,

was obtained as a sticky white solid (0.770 g, 69%) (R

f

= 0.3 on SiO

14.5 (c 1.00 CHCl ); IR

cast) 3313, 3018, 2971, 2874, 1697, 1528, 1450, 1249, 1193,

3

2

,

2

3

26

1

6

39.8, 139.2, 135.9, 129.5, 129.4, 128.3, 128.2, 128.0, 111.8, 81.3, 72.9,

1:4 hexanes/EtOAc, 0.1% AcOH); [α]

(CHCl

D

3

2.8, 54.7, 50.6, 28.1, 27.4 19.5. HRMS (ES) calculated for

+

−1 1

C H N O 494.3013, found 494.3010 (M + H) .

1104 cm . H (CDCl , 500 MHz): δ 8.10 (s, 1H, C(O)NH-N), 7.71

3

29

40

3

4

tert-Butyl O-(tert-Butyl)-N-(1-isobutylhydrazine-1-carbonyl)-L-

(d, J = 7.6 Hz, 2H, Fmoc-Ar-H), 7.54 (d, J = 7.5 Hz, 2H, Fmoc-Ar-H),

7.40−7.29 (m, 4H, 2 × Fmoc-Ar-H, 2 × 2-Cl-Ar-H), 7.29−7.16

(m, 4H, 2 × Fmoc-Ar-H, 2 × 2-Cl-Ar-H), 6.05 (br s, 1H, Ser-NH),

serinate (42). Alkylated semicarbazone 41 (2.19 g, 4.43 mmol) was

dissolved in MeOH (50 mL) and had 10% Pd/C (20 mg) added. The

suspension was stirred under hydrogen gas for 20 h, ﬁltered through a

pad of Celite, and concentrated in vacuo. The crude residue puriﬁed

5.53 (br s, 1H, Orn-NHα), 5.22−5.11 (m, 2H, -OCH (2-Cl)Ph),

2

4.42−4.37 (m, 2H, Ser-CHα, Fmoc-CH ), 4.35−4.27 (m, 1H, Orn-CHα),

2

P

DOI: 10.1021/acs.jmedchem.7b00723

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

4

.21 (dd, J = 10.1, 8.7 Hz, 1H, Fmoc-CH ), 4.14 (t, J = 8.3 Hz, 1H,

(10 μL), diluted to 100 μL with Milli-Q water, and analyzed by

LC−MS. Assays were performed in triplicate (n = 3) for each time

point. Integrated peak areas of the parent apelin peptide and internal

standard derived from LC−MS analyses were extracted using Mass

Hunter software (version B.04.00) and compared in positive extracted

ion count modus (+EIC). The extent of proteolysis was determined by

comparing the initial (0 h) ratio of the integrated extracted ions of the

parent apelin peptide to the internal dansyl-YVG standard (apelin/

dansyl-YVG) to the same ratio at the diﬀerent assay time points. The

decrease in apelin/dansyl-YVG ratio was converted to a percentage of

cleaved peptide over time.

2

Fmoc-CH), 3.76−3.68 (m, 1H, Ser-CH β), 3.52−3.46 (m, 1H,

2

Ser-CH β), 3.33−3.13 (m, 4H, 2 × Orn-CH δ, 2 × Leu-NCH β),

2

1

.89−1.80 (m, 1H, Orn-CH β), 1.79−1.65 (m, 2H, Orn-CH β,

2

Leu-CHγ), 1.62−1.54 (m, 2H, Orn-CH γ), 1.02 (s, 9H, -CH OC-

2

(

CH ) ), 0.83 (d, J = 6.5 Hz, 3H, Leu-CH δ), 0.81 (d, J = 6.5 Hz, 3H,

3

13

Leu-CH δ). C (CDCl , 125 MHz): δ 171.8, 170.4, 159.7, 158.3,

3

156.6, 143.6, 141.2, 134.2, 133.4, 129.6, 129.5, 129.3, 127.8, 127.1,

126.9, 125.1, 120.0, 73.6, 67.3, 63.9, 62.1, 55.7, 55.2, 52.5, 47.0, 39.9,

29.7, 27.3, 27.0, 25.9, 20.0. HRMS (ES) calculated for C H ClN O

7

40

49

5

9

−

78.3224, found 778.3208 (M − H) .

N-((S)-5-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-10-((tert-

Neprilysin Inhibition Assays. A solution of rhNEP (3.1 nM in

butoxycarbonyl)amino)-2-isobutyl-14,14-dimethyl-4,12-dioxo-13-

oxa-2,3,9,11-tetraazapentadec-10-enoyl)-O-(tert-butyl)-L-serine

100 mM Tris, 100 mM NaCl, 10 μM ZnCl , pH 7.5) was preincubated

2

with 5 μL of apelin analogue (1 mM) and 1.5 μL of internal standard

dansyl-Tyr-Val-Gly-OH (1 mM, dansyl-YVG-OH, Sigma-Aldrich) at

(45). A solution of 44 (0.730 g, 0.94 mmol) in MeOH (50 mL) had

1

0% Pd/C (25 mg) added, and the suspension was stirred under an

3

7 °C for 10 min prior to the addition of 5 μL of pyr-1-apelin-13

atmosphere of hydrogen gas for 135 min. The reaction mixture was

ﬁltered through a pad of Celite, concentrated in vacuo to dryness, and

resuspended in dry CH Cl (30 mL) with triethylamine (0.59 mL,

(1 mM). Aliquots (10 μL) were removed at 0, 1, 4, 24, and 72 h and

quenched and analyzed as previously described. Assays were

performed in duplicate (n = 2) for each time point.

2

4

.21 mmol) and 1,3-di-Boc-2-(triﬂuoromethylsulfonyl)guanidine

0.549 g, 1.40 mmol) for 30 h. Solvents were removed in vacuo and

the azatripeptide product was puriﬁed using ﬂash chromatography

silica gel, 90% EtOAc in hexanes, 0.1% AcOH), yielding white solid

5 (0.161 g, 20%) (R = 0.1 on SiO , 1:4 hexanes/EtOAc, 0.1%

6. Isolation and Quantiﬁcation of Apelin Peptides from

(

Plasma. 20 μL of plasma was portioned into microfuge tubes and

prewarmed to 37 °C. 5 μL of apelin peptide (400 μM) was added and

incubated at 37 °C for varying lengths of time. Experiments were

quenched by the addition of 20 μL of 10% aqueous TFA. 5 μL of

internal standard (1 mM dansyl-Tyr-Val-Gly-OH) was added, and

experiments were diluted up to 100 μL with 0.1% aqueous TFA. These

assays were loaded onto a pre-equilibrated C₁₈spin column (Harvard

Apparatus), which had previously been wet with 2 × 300 μL of 50%

acetonitrile in 0.1% aqueous TFA and 2 × 300 μL of 0.1% aqueous

TFA respectively, centrifuging at 300g for 2 min between each 300 μL

aliquot. Quenched plasma assays were centrifuged at 300g until the

sample was loaded. The resultant ﬁltrate was reloaded onto the

column along with 100 μL of 0.1% aqueous TFA and centrifuged at

300g two additional times. The desired plasma peptides were washed

by the addition of 2 × 300 μL of 0.1% aqueous TFA and centrifuged at

300g for 2 min, discarding the ﬁltrate after each wash. Desired peptides

were eluted by the addition of 300 μL of 40% acetonitrile (human

plasma) in 0.1% aqueous TFA and centrifugation at 300g for 2 min.

Eluted samples were diluted with 0.1% aqueous triﬂuoroacetic acid

prior to analysis by C₁₈RP-HPLC. To analyze the remaining per-

centage of apelin peptides in plasma, incubations of pyr-1-apelin-13 or

apelin-17 in plasma were immediately quenched, worked up, and

analyzed by C₁₈RP-HPLC as previously described, and the ratio of

apelin peptide to internal standard was calculated based on the area

under the peaks. This 0 min incubation ratio was used to compare the

apelin peptide/internal standard ratios for the time experiments.

7. APJ Radioligand Binding Experiments. Membrane prepara-

tions from CHO cells stably expressing the wild-type rat apelin

(

4

f

2

26

AcOH); [α]_D2.3 (c 0.85 CHCl ); IR (CHCl cast) 3326, 2978,

3

−1 1

2

5

7

934, 1722, 1644, 1531, 1368, 1331, 1136, 1054 cm . H (CDCl ,

3

00 MHz): δ 8.94 (br s, 1H, C(O)NH-N), 8.46 (br s, 1H, Arg-NHε),

.75 (d, J = 7.6 Hz, 2H, Ar-H), 7.58 (d, J = 7.6 Hz, 2H, Ar-H), 7.39

(

t, J = 7.5 Hz, 2H, Ar-H), 7.30 (t, J = 7.5 Hz, 2H, Ar-H), 6.16 (br s,

1

H, Arg-NHα), 6.08 (s, 1H, Ser-NH), 4.49−4.40 (m, 3H, Arg-CHα,

Ser-CHα, Fmoc-CH ), 4.34−4.28 (m, 1H, Fmoc-CH ), 4.18 (t, J =

2

7

1

2

.0 Hz, 1H, Fmoc-CH), 3.81−3.75 (m, 1H, Ser-CH β), 3.61−3.49 (m,

2

H, Ser-CH β), 3.49−3.24 (m, 4H, 2 × Arg-CH δ, 2 × Leu-NCH β),

.92−1.83 (m, 1H, Arg-CH β), 1.81−1.60 (m, 4H, Arg-CH β,

2

× Arg-CH γ, Leu-CHγ), 1.50 (s, 9H, Arg-C(CH ) ), 1.47 (s, 9H,

2

3 3

Arg-C(CH ) ), 1.12 (s, 9H, -CH OC(CH ) ), 0.91−0.85 (m, 6H,

3

2

3 3

13

2

1

7

2

× Leu-CH δ). C (CDCl , 125 MHz): δ 171.1, 170.2, 163.3, 162.4,

3 3

57.7, 156.5, 153.2, 143.7, 141.3, 127.8, 127.1, 125.0, 120.0, 83.6, 79.9,

4.7, 67.3, 61.5, 55.8, 54.0, 53.1, 47.1, 40.4, 29.4, 28.3, 28.0, 27.3, 27.0,

5.5, 20.2, 20.1. HRMS (ES) calculated for C H N O 854.4658,

43

64

7

11

+

found 854.4654 (M + H) .

azaLeu5-pyr-1-apelin-13 A2 (18). Advanced intermediate 7

(

0.2 mmol) was subjected to manual SPPS, introducing amino acids

in the following order: 45, Fmoc-Pro-OH, and Fmoc-Arg(Pmc)-OH.

The resin was split into half, and Fmoc-SPPS was continued on

0

following addition of 45. A portion (0.05 mmol) of resin-bound pep-

tide was cleaved as previously described and puriﬁed using a C₁₈

RP-HPLC analytical column (method A), eluting at 13.3 min. The

desired peptide was isolated as a white solid after lyophilization (4.8 mg,

.1 mmol scale, coupling pyr-Glu-OH. No end-capping was performed

39,54

receptor-EGFP were prepared as previously described.

Crude

membrane preparations (1 μg total mass of membranes/assay) were

125

6%). Monoisotopic MW calculated for C H BrN O 528.2582,

incubated for 3 h at 20 °C with 0.2 nM [ I]-pyr-1-apelin-13

68

111

23 16

3+

8

found high resolution (FTICR-ESI-MS) 528.2576 (M + 3H) .

azaLeu9-pyr-1-apelin-13 A2 (19). The remaining 0.1 mmol carried

over from the synthesis of 18 was coupled with Fmoc-Gln(Trt)-OH,

Fmoc-Arg(Pmc)-OH, Fmoc-Arg(Pmc)-OH, Fmoc-Phe-OH, and

Fmoc-Lys(Boc)-OH. A portion (0.05 mmol) of resin-bound peptide

was cleaved as previously described and puriﬁed using a C₁₈RP-HPLC

analytical column (method A), eluting at 11.3 min. The desired pep-

tide was isolated as a white solid after lyophilization (4.4 mg, 4%).

Monoisotopic MW calculated for C H BrN O 547.5435, found

high resolution (FTICR-ESI-MS) 547.5420 (M + 4H) .

. In Vitro Protease Experiments. Neprilysin Degradation

Assays. Apelin peptides were dissolved in Milli-Q water (1 mM).

Recombinant human neprilysin (rhNEP, Sino Biological) was recon-

stituted in Tris buﬀer (100 mM Tris, 100 mM NaCl, 10 μM ZnCl₂,

pH 7.5). To initiate the assay, rhNEP was diluted in Tris buﬀer

(monoiodinated on Lys with Bolton−Hunter reagent, PerkinElmer,

Wellesley, MA, USA) in binding buﬀer (50 mM HEPES, 5 mM

MgCl , pH 7.5, BSA 1%) alone or in the presence of the diﬀerent

2

compounds at various concentrations. The reaction was stopped by

adding 4 mL of cold binding buﬀer, ﬁltered on Whatman GF/C ﬁlters,

and washed with 5 mL of cold binding buﬀer. Radioactivity was then

counted using a Wizard 1470 Wallac γ counter (PerkinElmer, Turku,

Finland). Binding experiment data were analyzed with GraphPad

Prism.

8. Blood Pressure Assays. Mice were anesthetized with 1.5%

isoﬂurane/oxygen, and body temperature was monitored and

maintained at 36 °C by a heating pad. The aorta was cannulated via

the right carotid artery using a PV loop catheter (model 1.2F from

Scisense, Transonic) in order to continuously record arterial blood

pressure and heart rate (LabScribe 2.0, Scisense). Peptides 1, 2, 4, 5,

8−19 (1.4 μM/kg body weight) or the same volume of saline was

injected via the right jugular vein. Results are reported as systolic blood

pressure (SBP), diastolic blood pressure (DBP), mean arterial blood

pressure (MABP), and heart rate (HR).

95

160

35 20

4+

5

(

3.1 nM ﬁnal concentration) and preincubated at 37 °C for 10 min

prior to the addition of 5 μL of apelin peptide (1 mM) and 1.5 μL of

internal standard dansyl-Tyr-Val-Gly-OH (1 mM). Aliquots (10 μL)

were removed at 0, 1, 4, 24, and 72 h, quenched with 0.1 M EDTA

Q

DOI: 10.1021/acs.jmedchem.7b00723

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

9

. Langendorﬀ Isolated Heart Technique Experiments.

mean arterial blood pressure; max dP/dt, maximum derivative

of change in systolic pressure over time; min dP/dt, minimum

derivative of change in diastolic pressure over time; NEP,

neprilysin; Nle, norleucine; Orn, ornithine; p-TsOH, para-

toluenesulfonic acid; Pmc, 2,2,5,7,8-pentamethylchroman-6-

sulfonyl; rh, recombinant human; RP, reversed phase; RPP,

rate-pressure product; SPPS, solid phase peptide synthesis; Trt,

trityl

Langendorﬀ heart perfusion and ischemia−reperfusion injury were

prepared, and cardiac function was measured. Mice were heparinized

and anesthetized with 1.5−2% isoﬂurane inhalation. The heart was

excised, mounted on a Langendorﬀ system, and perfused at a con-

sistent pressure of 80 mmHg with modiﬁed Krebs−Henseleit solution

116 mM NaCl, 3.2 mM KCl, 2.0 mM CaCl , 1.2 mM MgSO , 25 mM

NaHCO , 1.2 mM KH PO , 11 mM glucose, 0.5 mM EDTA, and

mM pyruvate), which was kept at 37 °C and continuously oxy-

genated with 95% O and 5% CO which maintained the perfusion

(

2

4

3

2

4

2

buﬀer at pH 7.4. After stabilization and 10 min of baseline recording,

global ischemia was induced for 30 min followed by 40 min of

reperfusion. Apelin 17 (2) or apelin analogues (11, 17, 18, or 19) were

given at the start of reperfusion for 10 min at a concentration of 1 μM.

Left ventricular functions were obtained continuously by PowerLab

system (ADInstruments, Australia). Data were reported as mean value

of every 5 min. Left ventricular functions were reported as left ven-

tricular developed pressure (LVDP), heart rate (HR), maximum deriv-

ative of change in systolic pressure over time (max dP/dt), minimum

derivative of change in diastolic pressure over time (min dP/dt), and

rate pressure product (RPP).

REFERENCES

■

(

1) Tatemoto, K.; Hosoya, M.; Habata, Y.; Fujii, R.; Kakegawa, T.;

Zou, M.-X.; Kawamata, Y.; Fukusumi, S.; Hinuma, S.; Kitada, C.;

Kurokawa, T.; Onda, H.; Fujino, M. Isolation and characterization of a

novel endogenous peptide ligand for the human APJ receptor.

Biochem. Biophys. Res. Commun. 1998, 251, 471−476.

2) Maguire, J. J.; Kleinz, M. J.; Pitkin, S. L.; Davenport, A. P.

Pyr1]apelin-13 identified as the predominant apelin isoform in the

human heart: vasoactive mechanisms and inotropic action in disease.

Hypertension 2009, 54, 598−604.

3) Pitkin, S. L.; Maguire, J. J.; Bonner, T. I.; Davenport, A. P.

(

[

(

International Union of Basic and Clinical Pharmacology. LXXIV.

Apelin receptor nomenclature, distribution, pharmacology, and

function. Pharmacol. Rev. 2010, 62, 331−342.

ASSOCIATED CONTENT

Supporting Information

The Supporting Information is available free of charge on the

ACS Publications website at DOI: 10.1021/acs.jmed-

chem.7b00723.

■

*

S

(

4) Narayanan, S.; Harris, D. L.; Maitra, R.; Runyon, S. P. Regulation

of the apelinergic system and its potential in cardiovascular disease:

Peptides and small molecules as tools for discovery. J. Med. Chem.

2

(

015, 58, 7913−7927.

Crystal structures of 25 and 30, LCMS chromatograms,

MS proﬁles, and results of degradation studies (PDF )

Crystallographic information for C H N NiO (CIF )

5) Zhong, J.-C.; Zhang, Z.-Z.; Wang, W.; McKinnie, S. M. K.;

Vederas, J. C.; Oudit, G. Y. Targeting the apelin pathway as a novel

therapeutic approach for cardiovascular diseases. Biochim. Biophys.

Acta, Mol. Basis Dis. 2017, 1863, 1942−1950.

3

1

32

6

3

Crystallographic information for C H N NiO (CIF )

3

2

35

3

Molecular formula strings (CSV )

(6) Yang, P.; Maguire, J. J.; Davenport, A. P. Apelin, Elabela/Toddler,

and biased agonists as novel therapeutic agents in the cardiovascular

system. Trends Pharmacol. Sci. 2015, 36, 560−567.

(7) O’Dowd, B. F.; Heiber, M.; Chan, A.; Heng, H. H. Q.; Tsui, L.-

C.; Kennedy, J. L.; Shi, X.; Petronis, A.; George, S. R.; Nguyen, T. A

human gene that shows identity with the gene encoding the

angiotensin receptor is located on chromosome 11. Gene 1993, 136,

AUTHOR INFORMATION

Corresponding Author

E-mail: john.vederas@ualberta.ca.

■

*

ORCID

3

(

55−360.

John C. Vederas: 0000-0002-2996-0326

8) Reaux-Le Goazigo, A.; De Mota, N.; Skultetyova, I.; Lenkei, Z.; El

Notes

Messari, S.; Gallatz, K.; Corvol, P.; Palkovits, M.; Llorens-Cortes, C.

Physiological role of a novel neuropeptide, apelin, and its receptor in

the rat brain. J. Neurochem. 2001, 77, 1085−1096.

The authors declare no competing ﬁnancial interest.

(9) De Mota, N.; Reaux-Le Goazigo, A.; El Messari, S.; Chartrel, N.;

ACKNOWLEDGMENTS

■

Roesch, D.; Dujardin, C.; Kordon, C.; Vaudry, H.; Moos, F.; Llorens-

Cortes, C. Apelin, a potent diuretic neuropeptide counteracting

vasopressin actions through inhibition of vasopressin neuron activity

and vasopressin release. Proc. Natl. Acad. Sci. U. S. A. 2004, 101,

10464−10469.

(10) Boucher, J.; Masri, B.; Daviaud, D.; Gesta, S.; Guigne, C.;

Mazzucotelli, A.; Castan-Laurell, I.; Tack, I.; Knibiehler, B.; Carpene,

C.; Audigier, Y.; Saulnier-Blache, J. S.; Valet, P. Apelin, a newly

identified adipokine up-regulated by insulin and obesity. Endocrinology

We thank Jing Zheng, Bela Reiz, Dr. Angie Morales-Izquierdo,

and Dr. Randy Whittal (University of Alberta Mass Spec-

trometry Facility) for assistance with mass spectrometry

characterization and analyses of peptides and peptide frag-

ments, Wayne Moﬀat and Jennifer Jones (University of Alberta

Analytical Laboratory) for assistance with IR and optical

rotation experiments, Dr. Bob McDonald (University of Alberta

X-ray Crystallography Laboratory) for assistance with small

molecule crystallography, and Isaac Jones for preliminary

synthetic work. This work was supported by the Canadian

Institutes of Health Research (CIHR Grant 136921), the

Natural Sciences and Engineering Council of Canada

2

(

005, 146, 1764−1771.

11) Heinonen, M. V.; Purhonen, A. K.; Miettinen, P.; Paakkonen,

M.; Pirinen, E.; Alhava, E.; Akerman, K.; Herzig, K. H. Apelin, orexin-

A and leptin plasma levels in morbid obesity and effect of gastric

banding. Regul. Pept. 2005, 130, 7−13.

(

NSERC), Alberta Innovates Health Solutions (AIHS), the

Institut National de la Sante et de la Recherche Medicale

INSERM), and the College de France.

(12) Yue, P.; Jin, H.; Aillaud, M.; Deng, A. C.; Azuma, J.; Asagami,

T.; Kundu, R. K.; Reaven, G. M.; Quertermous, T.; Tsao, P. S. Apelin

is necessary for the maintenance of insulin sensitivity. Am. J. Physiol.:

Endocrinol. Metab. 2010, 298, E59−E67.

́

(

(13) Chen, H.; Zheng, C.; Zhang, X.; Li, J.; Li, J.; Zheng, L.; Huang,

ABBREVIATIONS USED

■

K. Apelin alleviates diabetes-associated endoplasmic reticulum stress in

ACE2, angiotensin-converting enzyme 2; Aib, aminoisobutyric

acid; APJ, apelin receptor; DIPEA, N,N-diisopropylethylamine;

EIC, extracted-ion chromatogram; HOAt, 1-hydroxy-7-azaben-

zotriazole; LVDP, left ventricle developed pressure; MABP,

the pancreas of Akita mice. Peptides 2011, 32, 1634−1639.

(

́

14) Attane, C.; Foussal, C.; Le Gonidec, S.; Benani, A.; Daviaud, D.;

́

Wanecq, E.; Guzman-Ruiz, R.; Dray, C.; Bezaire, V.; Rancoule, C.;

Kuba, K.; Ruiz-Gayo, M.; Levade, T.; Penninger, J. M.; Burcelin, R.;

R

DOI: 10.1021/acs.jmedchem.7b00723

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Pen

́

icaud, L.; Valet, P.; Castan-Laurell, I. Apelin treatment increases

apelin fragment lacking the C-terminal phenylalanine. J. Biol. Chem.

2014, 289, 24599−24610.

(29) Murza, A.; Parent, A.; Besserer-Offroy, E.; Tremblay, H.;

Karadereye, F.; Beaudet, N.; Leduc, R.; Sarret, P.; Marsault, E.

Elucidation of the structure-activity relationships of apelin: influence of

unnatural amino acids on binding, signaling, and plasma stability.

ChemMedChem 2012, 7, 318−325.

complete fatty acid oxidation, mitochondrial oxidative capacity, and

biogenesis in muscle of insulin-resistant mice. Diabetes 2012, 61, 310−

320.

(

15) Choe, H.; Farzan, M.; Konkel, M.; Martin, K.; Sun, Y.; Marcon,

L.; Cayabyab, M.; Berman, M.; Dorf, M. E.; Gerard, N.; Gerard, C.;

Sodroski, J. The orphan seven-transmembrane receptor Apj supports

the entry of primary T-cell-line-tropic and dualtropic human

immunodeficiency virus type 1. J. Virol. 1998, 72, 6113−6118.

(30) Zhang, Y.; Maitra, R.; Harris, D. L.; Dhungana, S.; Snyder, R.;

Runyon, S. P. Identifying structural determinants of potency for

analogs of apelin-13: integration of C-terminal truncation with

structure-activity. Bioorg. Med. Chem. 2014, 22, 2992−2997.

(

16) Fan, X.; Zhou, N.; Zhang, X.; Mukhtar, M.; Lu, Z.; Fang, J.;

DuBois, G. C.; Pomerantz, R. J. Structural and functional study of the

apelin-13 peptide, an endogenous ligand of the HIV-1 coreceptor, APJ.

Biochemistry 2003, 42, 10163−10168.

(31) Murza, A.; Belleville, K.; Longpre, J. M.; Sarret, P.; Marsault, E.

Stability and degradation patterns of chemically modified analogs of

apelin-13 in plasma and cerebrospinal fluid. Biopolymers 2014, 102,

(

17) Lee, D. K.; Cheng, R.; Nguyen, T.; Fan, T.; Kariyawasam, A. P.;

Liu, Y.; Osmond, D. H.; George, S. R.; O’Dowd, B. F. Characterization

of apelin, the ligand for the APJ receptor. J. Neurochem. 2000, 74, 34−

2

(

97−303.

32) McKinnie, S. M. K.; Fischer, C.; Tran, K. M. H.; Wang, W.;

41.

Mosquera, F.; Oudit, G. Y.; Vederas, J. C. The metalloprotease

neprilysin degrades and inactivates apelin peptides. ChemBioChem

(

18) Medhurst, A. D.; Jennings, C. A.; Robbins, M. J.; Davis, R. P.;

Ellis, C.; Winborn, K. Y.; Lawrie, K. W. M.; Hervieu, G.; Riley, G.;

Bolaky, J. E.; Herrity, N. C.; Murdock, P.; Darker, J. G.

Pharmacological and immunohistochemical characterization of the

APJ receptor and its endogenous ligand apelin. J. Neurochem. 2003, 84,

2

(

016, 17, 1495−1498.

33) Gafford, J. T.; Skidgel, R. A.; Erdos, E. G.; Hersh, L. B. Human

̈

kidney "enkephalinase", a neutral metalloendopeptidase that cleaves

active peptides. Biochemistry 1983, 22, 3265−3271.

1

(

162−1172.

(34) Rice, G. I.; Thomas, D. A.; Grant, P. J.; Turner, A. J.; Hooper, N.

19) Japp, A. G.; Cruden, N. L.; Amer, D. A.; Li, V. K.; Goudie, E. B.;

M. Evaluation of angiotensin-converting enzyme (ACE), its

homologue ACE2 and neprilysin in angiotensin peptide metabolism.

Biochem. J. 2004, 383, 45−51.

Johnston, N. R.; Sharma, S.; Neilson, I.; Webb, D. J.; Megson, I. L.;

Flapan, A. D.; Newby, D. E. Vascular effects of apelin in vivo in man. J.

Am. Coll. Cardiol. 2008, 52, 908−913.

(35) Watanabe, Y.; Nakajima, K.; Shimamori, Y.; Fujimoto, Y.

(

20) Barnes, G. D.; Alam, S.; Carter, G.; Pedersen, C. M.; Lee, K. M.;

Comparison of the hydrolysis of the three types of natriuretic peptides

by human kidney neutral endopeptidase 24.11. Biochem. Mol. Med.

Hubbard, T. J.; Veitch, S.; Jeong, H.; White, A.; Cruden, N. L.; Huson,

L.; Japp, A. G.; Newby, D. E. Sustained cardiovascular actions of APJ

agonism during renin-angiotensin system activation and in patients

with heart failure. Circ.: Heart Failure 2013, 6, 482−491.

1

(

997, 61, 47−51.

36) Vijayaraghavan, J.; Scicli, A. G.; Carretero, O. A.; Slaughter, C.;

Moomaw, C.; Hersh, L. B. Hydrolysis of endothelins by neutral

endopeptidase 24.11 (enkephalinase). J. Biol. Chem. 1990, 265,

(

21) Japp, A. G.; Cruden, N. L.; Barnes, G.; van Gemeren, N.;

Mathews, J.; Adamson, J.; Johnston, N. R.; Denvir, M. A.; Megson, I.

L.; Flapan, A. D.; Newby, D. E. Acute cardiovascular effects of apelin in

humans: potential role in patients with chronic heart failure.

Circulation 2010, 121, 1818−1827.

1

(

4150−14155.

37) Nalivaeva, N. N.; Turner, A. J., Neprilysin. In Handbook of

Proteolytic Enzymes, 3rd ed.; Rawlings, N. D., Salvesen, G. S., Eds.;

Academic Press,: London, UK, 2013; Vol. 1, pp 612−619.

(

22) Szokodi, I.; Tavi, P.; Fo

̈

ldes, G.; Voutilainen-Myllyla,

̈

S.; Ilves,

h, M.;

(38) Langelaan, D. N.; Bebbington, E. M.; Reddy, T.; Rainey, J. K.

M.; Tokola, H.; Pikkarainen, S.; Piuhola, J.; Rysa,

Ruskoaho, H. Apelin, the novel endogenous ligand of the orphan

̈

H.; Tot

́

Structural insight into G-protein coupled receptor binding by apelin.

Biochemistry 2009, 48, 537−548.

receptor APJ, regulates cardiac contractility. Circ. Res. 2002, 91, 434−

(39) Gerbier, R.; Leroux, V.; Couvineau, P.; Alvear-Perez, R.;

440.

Maigret, B.; Llorens-Cortes, C.; Iturrioz, X. New structural insights

into the apelin receptor: identification of key residues for apelin

binding. FASEB J. 2015, 29, 314−322.

(

23) Kidoya, H.; Ueno, M.; Yamada, Y.; Mochizuki, N.; Nakata, M.;

Yano, T.; Fujii, R.; Takakura, N. Spatial and temporal role of the

apelin/APJ system in the caliber size regulation of blood vessels during

angiogenesis. EMBO J. 2008, 27, 522−534.

(40) Langelaan, D. N.; Reddy, T.; Banks, A. W.; Dellaire, G.; Dupre,

D. J.; Rainey, J. K. Structural features of the apelin receptor N-terminal

tail and first transmembrane segment implicated in ligand binding and

receptor trafficking. Biochim. Biophys. Acta, Biomembr. 2013, 1828,

(

24) Wang, W.; McKinnie, S. M.; Patel, V. B.; Haddad, G.; Wang, Z.;

Zhabyeyev, P.; Das, S. K.; Basu, R.; McLean, B.; Kandalam, V.;

Penninger, J. M.; Kassiri, Z.; Vederas, J. C.; Murray, A. G.; Oudit, G. Y.

Loss of apelin exacerbates myocardial infarction adverse remodeling

and ischemia-reperfusion injury: therapeutic potential of synthetic

apelin analogues. J. Am. Heart Assoc. 2013, 2, e000249.

1

(

471−1483.

41) Belokon’, Y. N.; Tararov, V. I.; Maleev, V. I.; Savel’eva, T. F.;

Ryzhov, M. G. Improved procedures for the synthesis of (S)-2-[N-(N′-

benzyl-prolyl)amino]benzophenone (BPB) and Ni(II) complexes of

Schiff’s bases derived from BPB and amino acids. Tetrahedron:

Asymmetry 1998, 9, 4249−4252.

(

25) Vickers, C.; Hales, P.; Kaushik, V.; Dick, L.; Gavin, J.; Tang, J.;

Godbout, K.; Parsons, T.; Baronas, E.; Hsieh, F.; Acton, S.; Patane, M.;

Nichols, A.; Tummino, P. Hydrolysis of biological peptides by human

angiotensin-converting enzyme-related carboxypeptidase. J. Biol. Chem.

(42) Gu, X.; Ndungu, J. M.; Qiu, W.; Ying, J.; Carducci, M. D.;

Wooden, H.; Hruby, V. J. Large scale enantiomeric synthesis,

purification, and characterization of ω-unsaturated amino acids via a

Gly-Ni(II)-BPB-complex. Tetrahedron 2004, 60, 8233−8243.

(43) Nash, H. M.; Annis, D. A.; Guerlavais, V.; Licklider, L. Improved

Peptidomimetic Macrocycles. WO 2011047215, April 21, 2011.

(44) Pascal, R.; Sola, R. Preservation of the Fmoc protective group

under alkaline conditions by using CaCl2. Applications in peptide

synthesis. Tetrahedron Lett. 1998, 39, 5031−5034.

(45) Traore, M.; Doan, N. D.; Lubell, W. D. Diversity-oriented

synthesis of azapeptides with basic amino acid residues: aza-lysine, aza-

ornithine, and aza-arginine. Org. Lett. 2014, 16, 3588−3591.

(46) Garcia-Ramos, Y.; Lubell, W. D. Synthesis and alkylation of aza-

glycinyl dipeptide building blocks. J. Pept. Sci. 2013, 19, 725−729.

2

(

002, 277, 14838−14843.

26) Wang, W.; McKinnie, S. M.; Farhan, M.; Paul, M.; McDonald,

T.; McLean, B.; Llorens-Cortes, C.; Hazra, S.; Murray, A. G.; Vederas,

J. C.; Oudit, G. Y. Angiotensin converting enzyme 2 metabolizes and

partially inactivates pyr-apelin-13 and apelin-17: Physiological effects

in the cardiovascular system. Hypertension 2016, 68, 365−377.

(

27) El Messari, S.; Iturrioz, X.; Fassot, C.; De Mota, N.; Roesch, D.;

Llorens-Cortes, C. Functional dissociation of apelin receptor signaling

and endocytosis: implications for the effects of apelin on arterial blood

pressure. J. Neurochem. 2004, 90, 1290−1301.

(

28) Ceraudo, E.; Galanth, C.; Carpentier, E.; Banegas-Font, I.;

Schonegge, A. M.; Alvear-Perez, R.; Iturrioz, X.; Bouvier, M.; Llorens-

Cortes, C. Biased signaling favoring Gi over β-arrestin promoted by an

S

DOI: 10.1021/acs.jmedchem.7b00723

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

(

47) Betts, V.; Leissring, M. A.; Dolios, G.; Wang, R.; Selkoe, D. J.;

Walsh, D. M. Aggregation and catabolism of disease-associated intra-

Aβ mutations: reduced proteolysis of AβA21G by neprilysin.

Neurobiol. Dis. 2008, 31, 442−450.

(

48) Almenoff, J.; Orlowski, M. Membrane-bound kidney neutral

metalloendopeptidase: Interaction with synthetic substrates, natural

peptides, and inhibitors. Biochemistry 1983, 22, 590−599.

(

49) Dion, N.; Le Moual, H.; Fournie-Zaluski, M. C.; Roques, B. P.;

Crine, P.; Boileau, G. Evidence that Asn542 of neprilysin (EC

.4.24.11) is involved in binding of the P2′ residue of substrates and

inhibitors. Biochem. J. 1995, 311, 623−627.

50) Bateman, R. C., Jr.; Jackson, D.; Slaughter, C. A.; Unnithan, S.;

3

(

Chai, Y. G.; Moomaw, C.; Hersh, L. B. Identification of the active-site

arginine in rat neutral endopeptidase 24.11 (enkephalinase) as arginine

1

6

(

02 and analysis of a glutamine 102 mutant. J. Biol. Chem. 1989, 264,

151−6157.

51) McMurray, J. J.; Packer, M.; Desai, A. S.; Gong, J.; Lefkowitz, M.

P.; Rizkala, A. R.; Rouleau, J. L.; Shi, V. C.; Solomon, S. D.; Swedberg,

K.; Zile, M. R. PARADIGM-HF Investigators and Committees.

Angiotensin-neprilysin inhibition versus enalapril in heart failure. N.

Engl. J. Med. 2014, 371, 993−1004.

(

52) Cheng, X.; Cheng, X. S.; Pang, C. C. Y. Venous dilator effect of

apelin, an endogenous peptide ligand for the orphan APJ receptor, in

conscious rats. Eur. J. Pharmacol. 2003, 470, 171−175.

(

53) Gerbier, R.; Alvear-Perez, R.; Margathe, J. F.; Flahault, A.;

Couvineau, P.; Gao, J.; De Mota, N.; Dabire, H.; Li, B.; Ceraudo, E.;

Hus-Citharel, A.; Esteoulle, L.; Bisoo, C.; Hibert, M.; Berdeaux, A.;

Iturrioz, X.; Bonnet, D.; Llorens-Cortes, C. Development of original

metabolically-stable apelin-17 analogs with diuretic and cardiovascular

effects. FASEB J. 2017, 31, 687−700.

(

54) Iturrioz, X.; Gerbier, R.; Leroux, V.; Alvear-Perez, R.; Maigret,

B.; Llorens-Cortes, C. By interacting with the C-terminal Phe of

apelin, Phe255 and Trp259 in helix VI of the apelin receptor are

critical for internalization. J. Biol. Chem. 2010, 285, 32627−32637.

(

55) Perjes, A.; Skoumal, R.; Tenhunen, O.; Konyi, A.; Simon, M.;

Horvath, I. G.; Kerkela, R.; Ruskoaho, H.; Szokodi, I. Apelin increases

cardiac contractility via protein kinase C epsilon- and extracellular

signal-regulated kinase-dependent mechanisms. PLoS One 2014, 9,

e93473.

(

56) Brame, A. L.; Maguire, J. J.; Yang, P.; Dyson, A.; Torella, R.;

Cheriyan, J.; Singer, M.; Glen, R. C.; Wilkinson, I. B.; Davenport, A. P.

Design, characterization, and first-in-human study of the vascular

actions of a novel biased apelin receptor agonist. Hypertension 2015,

6

(

5, 834−840.

57) Kehoe, K.; Van Elzen, R.; Verkerk, R.; Sim, Y.; Van der Veken,

P.; Lambeir, A. M.; De Meester, I. Prolyl carboxypeptidase purified

from human placenta: its characterization and identification as an

apelin-cleaving enzyme. Biochim. Biophys. Acta, Proteins Proteomics

2

(

016, 1864, 1481−1488.

58) Baker, T. J.; Tomioka, M.; Goodman, M. Preparation and use of

N,N′-di-Boc-N″-triflylguanidine. Org. Synth. 2002, 78, 91.

59) Bourguet, C. B.; Proulx, C.; Klocek, S.; Sabatino, D.; Lubell, W.

(

D. Solution-phase submonomer diversification of aza-dipeptide

building blocks and their application in aza-peptide and aza-DKP

synthesis. J. Pept. Sci. 2010, 16, 284−296.

T

DOI: 10.1021/acs.jmedchem.7b00723

J. Med. Chem. XXXX, XXX, XXX−XXX

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