Diversity-Oriented A3-Macrocyclization for Studying Influences of Ring-Size and Shape of Cyclic Peptides: CD36 Receptor Modulators

Ring-Size and Shape of Cyclic Peptides: CD36 Receptor Modulators

Article

Diversity-Oriented A ‑Macrocyclization for Studying Inﬂuences of

Ragnhild G. Ohm, Mukandila Mulumba, Ramesh M. Chingle, Ahsanullah, Jinqiang Zhang,

Sylvain Chemtob, Huy Ong,* and William D. Lubell*

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sı Supporting Information

ABSTRACT: Cyclic peptide diversity has been broadened by elaborating the A³-

macrocyclization to include various di-amino carboxylate components with diﬀerent

N -amine substituents. Triple-bond reduction provided new cyclic peptide macro-

cycles with Z-oleﬁn and completely saturated structures. Moreover, cyclic

azasulfurylpeptides were prepared by exchanging the propargylglycine (Pra)

component for an amino sulfamide surrogate. Examination of such diversity-oriented

methods on potent cyclic azapeptide modulators of the cluster of diﬀerentiation 36

receptor (CD36) identiﬁed the importance of the triple bond as well as the N -allyl

lysine and azaPra residues for high CD36 binding aﬃnity. Cyclic azapeptides which

engaged CD36 eﬀectively reduced pro-inﬂammatory nitric oxide and downstream cytokine and chemokine production in

macrophages stimulated with a Toll-like receptor-2 agonist. Studying the triple bond and amine components in the multiple-

component A -macrocyclization has given a diverse array of macrocycles and pertinent information to guide the development of

ideal CD36 modulators with biomedical potential for curbing macrophage-driven inﬂammation.

INTRODUCTION

amine substituent, di-amino carboxylate chain length, and

carbonyl linchpin.

The cluster of diﬀerentiation 36 receptor (CD36) is a

membrane-bound glycoprotein expressed in many cell types:

skeletal, cardiac muscle, microvascular endothelial, and retinal

■

Cyclic peptides are a proven class of clinically used drugs due

in part to their success in stabilizing active secondary

structures, improving metabolic stability, and enhancing

cellular penetration.

Eﬀorts to make side-chain-to-side-

cells, as well as mononuclear phagocytes such as macrophages

chain cross-linked (so-called bridged or stapled) peptides

have intensiﬁed as their promise to act as inhibitors of

intracellular protein−protein interactions becomes realized

with stapled-α-helix peptides advancing in clinical trials.

Cyclic peptides can achieve bioavailability in spite of violating

rules typically associated with small-molecules, but few have

14−16

and microglia cells.

The single 472-amino acid peptide

chain of CD36 is cross-linked by three disulﬁde bonds and

post-translationally modiﬁed by signiﬁcant glycosylation and

phosphorylation. Implicated in the regulation of physio-

logical processes vital for cardiovascular biology and innate

immunity, CD36 functions as a class B scavenger receptor and

binds a broad variety of endogenous ligands: long-chain fatty

acids, thrombospondin 1, oxidative low-density lipoprotein

(oxLDL), apoptotic cells, and photoreceptor outer seg-

exhibited signiﬁcant oral absorption. The uniformity of

contemporary cross-linking chemistry, however, limits the

product variety and may undermine the eﬀorts to develop

drug-like cyclic peptides.

14−16,18−20

ments.

In macrophage-driven inﬂammation, CD36

Diversity-oriented methods for making cyclic peptides have

garnered signiﬁcant interest because of their potential to

provide a variety of macrocycles from related chemical

acts as a co-receptor and sustains the signaling of the Toll-like

receptor (TLR)-2/6 heterodimer assembly on the surface of

21−23

membranes of mononuclear phagocytes.

Ligands that

−10

bind and modulate the activity of CD36 oﬀer potential for

reactions and common intermediates.

In the so-called

A -macrocyclization”, a multiple-component reaction takes

place between alkyne, aldehyde, and amine components

treating cardiovascular, metabolic, and immunological disor-

“

ders.

(

Figure 1). The amine and aldehyde condense to form an

imine, which is attacked by an acetylide nucleophile from

Received: April 7, 2021

Published: June 23, 2021

activation of the alkyne by the metal catalyst. Preorganization

from metal chelation of the acetylene and amine components

may reduce entropic costs of folding to favor A -macro-

cyclization. Diversity can in principle be introduced in the

A -macrocyclization by modiﬁcation of the peptide sequence,

2021 American Chemical Society

Journal of Medicinal Chemistry

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J. Med. Chem. 2021, 64, 9365−9380

which linear counterparts were active.

Article

Figure 1. Diversity-oriented A -macrocyclization may be favored by metal chelation of the acetylene and amine components: R , R , and R =

diverse amine and carbonyl substituents, R CO−, −NHR , and Xaa = peptide chains, n = diﬀerent di-amino acid side-chain lengths.

In eﬀorts to develop CD36 ligands based on the linear lead

analysis using NMR spectroscopy and computational analysis

revealed that the semicarbazide sp -conﬁguration provided the

growth hormone-releasing peptide-6 (GHRP-6, H-His-D-Trp-

Ala-Trp-D-Phe-Lys-NH ), A -macrocyclization was conceived

best representation of the aza-residue geometry in the active

and employed to synthesize both cyclic peptide and azapeptide

macrocycles. Moreover, the active conformer of azacyclopep-

tide 7a was deduced to have a compact topology featuring a

type II′ β-turn geometry centered about the D-Trp-Ala

dipeptide possessing an additional bridging hydrogen bond

between the C-terminal amide NH and D-Trp carbonyl

analogues (Figure 2). Potent CD36 ligands, cyclic azapep-

tides (e.g., 3a and 7a, Figure 2), exhibited signiﬁcant binding

aﬃnity and eﬃcacy in reducing TLR-2 agonist-induced nitric

oxide (NO) overproduction and in diminishing pro-inﬂam-

matory cytokine and chemokine production in macrophages at

oxygen.

−

Previously, cyclic (aza)peptides having 16-, 19-, 22-, and 25-

concentrations that were 10-fold lower (10 ) than those at

11,24,25

atom ring sizes were, respectively, synthesized using A -

Conformational

macrocyclization and formaldehyde to cross-link diﬀerent N -

substituted lysine side chains with (R)- and (S)-propargylgly-

11,24−26

cine (Pra) and their azaPra counterpart (Figure 2).

The

semicarbazide served as an amino amide surrogate in the

azapeptides. Examining the scope of the A -macrocyclization

more deeply, the diversity of the di-amino carboxylate

component has now been expanded. The signiﬁcance of the

allyl substituent on the N -amine of lysine (Lys) has been

investigated using cyclopropylmethyl and n-propyl groups.

Shorter side-chain lengths have been studied using ornithine

(

Orn), diaminobutyrate (Dab), and diaminopropionate (Dap).

Moreover, macrocycles have been produced by reduction of

the triple bond to Z-oleﬁn and completely saturated counter-

parts. In addition, the azaPra residue was substituted by an

amino-sulfamide (azasulfurylpropargylglycine, AsPra) to syn-

thesize a cyclic azasulfurylpeptide isostere.

The analogues were initially examined for their modulatory

eﬃciency in reducing NO production induced by the TLR-2

agonist ﬁbroblast-stimulating lipopeptide (R-FSL-1) in macro-

phage cells. Promising macrocycles, which reduced NO

production, were subsequently evaluated in a competition

binding assay using the photo-activated agonist [¹I]-Tyr-Bpa-

Ala-hexarelin as a radiotracer to characterize the binding

aﬃnity. Moreover, the capacity to modulate R-FSL-1-induced

pro-inﬂammatory cytokine and chemokine release was

examined by measuring the reduction of secreted levels of

tumor necrosis factor-α (TNFα), interleukin-1β (IL-1β), and

monocyte chemoattractant protein-1 (MCP1, CCL-2) in cell

culture media of one of two macrophage cell types: RAW

64.7 cells for TNFα and CCL-2; bone marrow-derived

Figure 2. Representative examples of previously synthesized cyclic

macrophages (BMA 3.1 A7) primed to M1 phenotype for IL-

(

aza)peptide CD36 ligands from A -macrocyclization.

1β.

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The utility of A -macrocyclization for the diversity-oriented

density of the N -allyl group for the potency of cyclic

synthesis of cyclic peptide analogues has been further

elaborated by broadening the scope of di-amino acid and Pra

components. Diﬀerent azacyclopeptide CD36 modulators have

been identiﬁed exhibiting interesting promise for mitigating

TLR-2 agonist-induced pro-inﬂammatory responses. Inves-

tigating their structure−activity relationships (SARs) has

provided information for designing more potent CD36

modulators with therapeutic potential and illustrated the

eﬀectiveness of this diversity-oriented method for peptide-

based drug discovery.

azapeptides (e.g., 7a) was explored by the synthesis of N -

cyclopropylmethyl and n-propyl Lys derivatives 18g and 18h

using Mitsunobu reactions with the respective alcohols on

resin 15a.

Elongation of Dap, Dab, Orn, and Lys resins 18a,d−h using

standard Fmoc-based solid-phase peptide synthesis gave tetra-

and pentapeptide resins 21 and 22 (Scheme 2). The azaPra

Scheme 2. Synthesis of Azacyclopeptides 3a,e,f and 7a,e−h

RESULTS AND DISCUSSION

■

A series of macrocyclic GHRP-6 analogues was previously

synthesized by A -macrocyclization of linear GHRP-6 ana-

logues possessing N -alkyl-Lys and an azaPra residue, which

was systematically moved from the 1- to the 4-position in the

peptide (e.g., 1−3, 5−7, and 9). The Mitsunobu reaction on

the N -o-nitrobenzenesulfonamido (o-NBS)-Lys residue

proved eﬀective for installing a set of N -substituents (Me,

allyl, and i-Pr). Moreover, macrocycles 10−13 were prepared

by inserting a second Lys residue into the sequence.

The length of the di-amino carboxylate in A macro-

cyclization has now been examined by replacement of Lys

with lower homologues Orn, Dab, and Dap (Scheme 1). As

Scheme 1. Anchoring of Diﬀerent Di-amino Carboxylates

on Rink Amide Resin

residue was introduced by employing N-Fmoc-azaPra-Cl (23),

previously described for the synthesis of Fmoc-Lys(o-NBS,

allyl) resin 18a, Fmoc-Dap(o-NBS)-OH (14d) was coupled to

Rink amide resin using N,N′-diisopropylcarbodiimide (DIC)

which was prepared from the corresponding carbazate with

bis(trichloromethyl)carbonate (BTC). After Fmoc group

removal, semicarbazides 26a and 27a,f−h were coupled to the

symmetric anhydride from Boc-Ala-OH to furnish azapeptide

and 1-hydroxybenzotriazole (HOBt) and converted to N -allyl

derivative 18d under Mitsunobu reaction conditions.

28a and 29a,f−h. Semicarbazides 26e,f and 27e were reacted

Attempts to couple Fmoc-Dab(o-NBS)-OH (14e) and

Fmoc-Orn(o-NBS)-OH (14f) to the Rink amide resin gave

under microwave irradiation at 60 °C with N-Fmoc-Ala-Cl,

which was generated using BTC and collidine in THF, to give

Fmoc-protected linear peptides, which were converted to Boc

counterparts 28e,f and 29e.

however signiﬁcantly lower loadings of 18e and 18f (0.10 and

.19 mmol/g) likely due to intramolecular cyclization on N -

sulfonamide to aﬀord the corresponding pyrrolidinone and

piperidinone side products. Improved resin loadings (18e =

After removal of the o-NBS protection with 2-mercaptoe-

thanol and DBU, cyclic azapeptides 3a,e,f and 7a,e−h were

.34 and 18f = 0.33 mmol/g) were obtained from couplings of

Fmoc-Dab(o-NBS, allyl)-OH and Fmoc-Orn(o-NBS, allyl)-

OH (17e and 17f), which were prepared from acids 14e and

prepared from linear counterparts 30a,e,f and 31a,e−h by A -

macrocyclization using aqueous formaldehyde and CuI in

DMSO, cleaved from the resin using a TFA/TES/H O

4f by treatment with allyl alcohol, DIAD, and triphenylphos-

cocktail, and puriﬁed by HPLC (Table 1). On the other hand,

attempts to perform A -macrocyclization on Dap azapeptides

phine in THF, followed by ester hydrolysis without Fmoc

removal using NaOH and CaCl in a solution of i-PrOH/

H O/MeCN. In addition, the importance of the electron

30d and 31d failed to give the corresponding azacyclopeptides.

A cursory attempt to increase the azaPra length using N′-

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Table 1. Purity, Retention Times, and Mass of Cyclic Azapeptides

R_t(min)

MeOH MeCN

MS [M + 1]

purity at

214 nm

m/z

cyclic azapeptide

(calc.)

m/z (obs.)

c-{[azaPra(δC)−CH -Dab(γN)]H-Ala-azaPra-Ala-Trp-D-Phe-Dab(allyl)-NH }

5.51

5.72

6.91

6.85

8.12

4.16

4.26

4.87

4.79

8.09

>99

>97

741.3831

755.3988

927.4624

963.4600

969.5094

741.3842

755.3990

927.4643

963.4573

969.5057

c-{[azaPra(δC)−CH -Orn(δN)]H-Ala-azaPra-Ala-Trp-D-Phe-Orn(allyl)-NH }

c-{[azaPra(δC)−CH -Dab(γN)]H-Ala-azaPra-D-Trp-Ala-Trp-D-Phe-Dab(allyl)-NH }

c-{[azaPra(δC)−CH -Orn(δN)]H-Ala-azaPra-D-Trp-Ala-Trp-D-Phe-Orn(allyl)-NH }

c-{[azaPra(δC)−CH -Lys(εN)]H-Ala-azaPra-D-Trp-Ala-Trp-D-Phe-Lys[CH CH(CH ) ]-

NH }

c-{[azaPra(δC)−CH -Lys(εN)]H-Ala-azaPra-D-Trp-Ala-Trp-D-Phe-Lys(n-Pr)-NH }

8.01

6.04

6.64

5.86

5.71

4.93

>99

955.5094

961.5407

971.5250

955.5100

961.5391

971.5254

c-{[azaGly(αN)−(CH ) −Lys(εN)]H-Ala-azaGly-D-Trp-Ala-Trp-D-Phe-Lys(n-Pr)-NH }

c-{[aza-hexenylglycine(εC)-Lys(εN)]H-Ala-Z-aza-hexenylglycinyl-D-Trp-Ala-Trp-D-Phe-

Lys(cyclopropylmethyl)-NH }

c-{[AsPra(δC)−CH -Lys(εN)]H-Ala-AsPra-D-Trp-Ala-Trp-D-Phe-Lys(allyl)-NH }

8.12

5.99

>99

991.4607

991.4657

Isolated purity ascertained by LC−MS using gradients of X−Y% [MeOH (0.1% FA)/H O (0.1% FA)] or [MeCN (0.1% FA)/H O (0.1% FA)]

over Z min. 10−90%/10. 5−50%/9.

homopropargyl-ﬂourenylmethyl-carbazate proved more chal-

lenging than anticipated and was abandoned.

The scope of the propargylglycine component was further

explored in A -macrocyclization by employing azasulfurylpro-

The chemistry of propargyl amines oﬀers rich potential for

pargylglycine (AsPra; As = azasulfuryl). Previously, replace-

ment of the semicarbazide in the linear analogue [aza-(4-

supplementary modiﬁcations after A -macrocyclization.

Partial saturation and full saturation of the triple bond were

both explored to alter the macrocycle ring-shape. Azapeptide

F)F ]-GHRP-6 by an amino-sulfamide gave [As-(4-F)F ]-

GHRP-6, which had comparable CD36 binding aﬃnity and

exhibited similar activity in suppressing TLR-agonist-induced

pro-inﬂammatory NO, cytokine, and chemokine production in

macrophages and reducing retinal inﬂammation by activated

mononuclear phagocytes upon photo-oxidative stress in a

a was completely saturated by hydrogenation using Pd/C as a

catalyst in MeOH to give macrocycle 32 in 74% yield (Scheme

). Partial reduction of the triple bond of the cyclopropyl

Scheme 3. Full and Partial Acetylene Saturation by

Macrocycle Hydrogenation

mouse model. In contrast to [aza-(4-F)F ]-GHRP-6, which

inhibited microvascular sprouting mediated through CD36 in

the mouse choroidal explant model, the azasulfurylpeptide

counterpart preserved microvascular survival. Subtle chem-

ical modiﬁcation from a carbonyl to a sulfuryl residue eﬀected

signiﬁcantly speciﬁc aspects of CD36-mediated chemical

biology. In comparison to the planar urea in the semi-

carbazide moiety, the sulfamide adopts tetrahedral geometry,

increases NH Brønsted acidity, and adds a second Lewis basic

oxygen. In crystal structures of model analogues, such

structural changes altered the β-turn conformation adopted by

azapeptides to a γ-turn geometry in azasulfurypeptides. The

impact of such a change has now been investigated by the

synthesis of cyclic azasulfurylpeptide 36 (Scheme 4).

Azasulfuryl tripeptide 42 was ﬁrst prepared in solution by

coupling Boc-Ala-OH to H-azaPra-OFm (37) using isobutyl

chloroformate and N-methylmorpholine in THF to prepare

Boc-Ala-azaPra-OFm (38) in 72% yield. After Fmoc group

removal, hydrazide 39 was reacted with sulfamidate 40 and

triethylamine in dichloroethane under microwave irradiation to

provide Boc-Ala-AsPra-D-Trp(Boc)-ODmb (41). Selective

Dmb ester hydrolysis was achieved without removal of acid

labile Boc groups using 1% TFA in DCM to provide acid 42.

Coupling of acid 42 to tetrapeptide resin 43 using DIC and

HOBt furnished linear azasulfurylpeptide resin 44. After o-NBS

removal, cyclic azasulfurylpeptide 36 was obtained unevent-

fully by A -macrocyclization of azasulfurylheptapeptide 45,

followed by resin and protecting group cleavage and HPLC

puriﬁcation (Table 1).

methyl analogue 7g using the Lindlar catalyst and quinoline in

MeOH gave Z-oleﬁn 33 in 33% yield after HPLC puriﬁcation.

Partial hydrogenation of cyclic alkynes 7a, 7e, and 7f without

terminal oleﬁn saturation was attempted using the Lindlar

catalytic conditions but produced inseparable mixtures of allyl

and n-propyl analogues 34 and 35.

Biology. In earlier studies of linear and cyclic azapeptides,

CD36 modulator activity was initially evaluated by the ability

to reduce overproduction of NO in macrophage cells induced

with the TLR-2 agonist R-FSL-1.

phages to battle against pathogenic microbes but potentially

destructive to host tissue, NO is a useful marker of the

24,34

Produced by macro-

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Scheme 4. Synthesis of Cyclic Azasulfurylpeptide 36

cytokines and chemokines induced by R-FSL-1 in macrophage

cells.

Among the ﬁve diﬀerent macrocycle ring sizes previously

studied (e.g., 1−13, Figure 2), the larger 22- and 25-membered

cyclic azapeptides (e.g. 3, 4, 6−9) gave consistently higher

percentages of NO reduction compared to the 16- and 19-

membered ring systems (e.g., 1, 2, and 10−13). A basic

protonated amine at the N-terminal appeared key for reducing

NO overproduction by the 25-membered cyclic azapeptides

−

(

10 M, Table 2): N-terminal alanine analogue 7a (35%) >

N-acetyl [for example, 9a (23%) > 6a (18%)] ≈ N-terminal

semicarbazide 5a (5%), which is likely not protonated under

physiologic conditions. The N -substituent of the Lys

component of the 25-membered cyclic azapeptides has now

been shown to inﬂuence the ability to reduce NO over-

production. A comparison of N -allyl (7a, 35%), cyclo-

propylmethyl (7g, 14%), and n-propyl (7h, 4%) derivatives

illustrates that the saturated analogues gave signiﬁcantly lower

inhibitory activity and binding aﬃnity compared to the

unsaturated derivative. The intermediate abilities to reduce

NO overproduction shown by cyclic azapeptide 7g may be due

to the greater electron density of the cyclopropyl “bent” bonds,

which are situated between the σ- and π-bonds in 7h and 7a

(

Figure 3).

The binding aﬃnity (IC ) of analogues exhibiting high

activity in reducing NO overproduction was evaluated in a

competition binding assay using the photo-activated agonist

[

I]-Tyr-Bpa-Ala-hexarelin as the radiotracer and rat heart

membrane preparation as the source of CD36 binding sites

(

Table 2 and Figure 4). Some correlation of the binding

aﬃnity of the 25-membered macrocycles with their NO

inhibitory activity was observed (r = −0.55; P = 0.0159,

Supporting Information): 7a (35%, 0.1 μM) > 9a (23%, 0.67

μM) > 6a (18%, 0.89 μM) > 7g (14%, 3.06 μM) > 7h (4%,

1.86 μM) (Figure 4A,B). Moreover, the related 22-membered

macrocycle 3a (25%, 0.24 μM) with an N-terminal alanine

featured relatively high NO inhibitory potency and binding

modulation of the inﬂammatory pathway and can be eﬀectively

measured by formation of a ﬂuorescence naphthotriazole

adduct using 2,3-diaminonaphthalene. Reduction of NO

overproduction by CD36 ligands was correlated with binding

aﬃnity and with decreased production of pro-inﬂammatory

Table 2. Cyclic Peptide Binding Aﬃnity and Modulatory Eﬀect on R-FSL-1-Induced Release of NO, TNFα, and CCL-2 in

RAW 264.7 Macrophage Cells and of IL-1β in Bone Marrow Macrophages BMA 3.1 A7

TNF-α

IC₅₀(μM) mean ± SD (pmol)

851 ± 22.08

CCL-2

IL-1β

mean ± SD (μM)

mean ± SD (pmol)

basal

R-FSL-1 stimulation as control

0.774 ± 0.235

2.821 ± 0.1

2.302 ± 0.137

2.506 ± 0.356

2.812 ± 0.241

2.723 ± 0.241

2.451 ± 0.177

2.113 ± 0.284

2.26 ± 0.216

2.52 ± 0.428

2.533 ± 0.181

2.748 ± 0.21

238 ± 127

71 ± 9

4966 ± 35

3837 ± 221

4848 ± 79

1097 ± 26

758 ± 114

954 ± 177

1028 ± 179

1042 ± 92

410 ± 40

291 ± 17

0.24

10.2

22.8

>50

0.89

0.1

4.75

13.1

3.06

1.86

0.67

2.26

9.05

10.2

389 ± 31

381 ± 33

388 ± 34

351 ± 7

253 ± 28

271 ± 24

4756 ± 165

4901 ± 163

4558 ± 146

3943 ± 109

4178 ± 108

4654 ± 135

4623 ± 150

4462 ± 405

4042 ± 167

4113 ± 141

4823 ± 296

5120 ± 133

785 ± 178

672 ± 34

857 ± 54

971 ± 167

984 ± 69

918 ± 78

411 ± 33

366 ± 38

313 ± 33

304 ± 18

312 ± 17

2.346 ± 0.197

2.078 ± 0.236

2.566 ± 0.085

2.739 ± 0.273

707 ± 98

802 ± 49

903 ± 73

969 ± 89

312 ± 17

378 ± 37

−4

SD: standard deviation. Statistical evaluation for cyclic azapeptide comparison was performed using one-way ANOVA with post hoc Dunnett’s

test. P < 0.05. P < 0.01. P < 0.001. P < 0.0001 vs R-FSL-1 stimulatory condition as control. N = 4 replicates per experiment. % of inhibition

relative to R-FSL-1 as control.

369

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J. Med. Chem. 2021, 64, 9365−9380

focused on modifying cyclic azapeptides 3a and 7a by

Article

shortening the di-amino carboxylate component, reducing

the triple bond, and swapping of the urea for a sulfamide

moiety. These structural modiﬁcations had signiﬁcant con-

sequences on pharmacological activity and binding aﬃnity. For

example, replacement of the Lys residue by the shorter di-

amino acids, Dab (3e and 7e) and Orn (3f and 7f), caused

profound drops in NO inhibitory potency. Substitution of Lys

in macrocycles 7a and 3a by Orn resulted, respectively, in 131-

fold (7f) and 95-fold (3f) losses of binding aﬃnity, illustrating

the signiﬁcant importance of the di-amino acid chain length

(

Figure 4B,D).

Complete hydrogenation of the acetylene and N -allyl

groups of cyclic azapeptide 7a gave saturated N -propyl

analogue 32, which exhibited a similar NO inhibitory potency

but a 22.6-fold loss in binding aﬃnity. Compared to N -propyl

cyclic alkyne 7h, saturated N -propyl analogue 32 had a

ninefold better potency on NO inhibition but 1.2-fold less

binding aﬃnity (Table 2 and Figure 4B,C). Partial hydro-

genation of the acetylene of the N -cyclopropylmethyl

Figure 3. Cyclic azapeptides evaluated for CD36 binding aﬃnity and

biological activity.

macrocycle 7g to Z-oleﬁn 33 caused only a subtle drop in

NO inhibitory potency but a threefold loss in binding aﬃnity.

In the cyclic alkyne series, the N -allyl analogue 7a exhibited,

aﬃnity. On the other hand, cyclic azapeptide 5a with a N-

terminal semicarbazide did not show any competitive binding

aﬃnity or eﬀect on cytokine and chemokine expression (vide

infra) and has been suggested to likely reduce NO production

respectively, 2.5- and 8.75-fold higher NO inhibitory potencies

as well as 30.6- and 18.6-fold better binding aﬃnities relative to

the N -n-propyl and N -cyclopropylmethyl analogues 7h and

7g (Figure 4B). The losses in binding aﬃnity caused by

hydrogenation to saturated and Z-oleﬁn analogues 32 and 33

by an oﬀ-target mechanism.

Considering the high NO inhibitory potency and binding

aﬃnity of the 22- and 25-membered macrocycles, eﬀorts were

relative to their corresponding N -n-propyl and N -cyclo-

propylmethyl alkyne analogues 7h and 7g highlight the

signiﬁcance of the triple bond for binding aﬃnity. On the

Figure 4. A−D) Assessment of cyclic azapeptides’ binding aﬃnity for CD36 in the presence of [¹²⁵I]Tyr-Bpa-Ala-hexarelin.

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other hand, improved and retained NO inhibitory potency of

CONCLUSIONS

■

2 and 33 relative to 7h and 7g indicates that increased

Diversity-oriented synthesis of cyclic peptides by way of A -

macrocyclization oﬀers power to provide an array of cyclic

peptides with subtle diﬀerences in macrocycle substitution,

ﬂexibility may give rise to alternative active conformers.

In linear aza- and azasulfurylpeptide analogues, such as [aza-

(

4-F)F ]- and [As-(4-F)F ]-GHRP-6, the exchange of the urea

size, and shape for studying SAR. A -macrocyclization could be

moiety by a sulfamide had a limited eﬀect on binding aﬃnity.

applicable for developing high-aﬃnity binders for various

biological targets. Moreover, the methods described for

selective alkyne reduction may be of value for further

exploration of cyclic peptide analogues possessing unsaturated

In contrast, replacement of semicarbazide 7a by N-amino

sulfamide 36 caused a 102-fold drop in binding aﬃnity.

Conformational analyses of cyclic azapeptide 7a and cyclic

peptide counterparts (R)- and (S)-8a using NMR spectroscopy

and computation have previously indicated that the semi-

40,41

cross-linkers.

The scavenger receptor CD36 plays roles in the regulation of

the TLR2/6 heterodimer assembly, the activation of tran-

scription of proinﬂammatory cytokines, and the production of

NO and reactive oxygen species.

notable potency for reducing TLR-2 agonist-induced pro-

carbazide sp -conﬁguration provided a better representation of

the aza-residue geometry in the active macrocycle than the

pseudo-sp (R)- and (S)-hybridizations. The preferred sp

37,42

Cyclic azapeptides oﬀer

geometry of the N-amino sulfamide may account in part for

the diminished binding aﬃnity of cyclic azasulfurylpeptide 36

inﬂammatory cytokines and chemokines by binding CD36 and

(

Figure 4C).

inducing dissociation from the TLR complex. The employ-

Cyclic azapeptide with macrocycles of 22- (3a) and 25-

ment of A -macrocyclization has provided a set of cyclic

members (6a, 7a, 7g, 7h, 9a, and 33) were further tested for

capacity to modulate R-FSL-1-induced production of pro-

inﬂammatory cytokines (TNFα, IL-1β) and chemokine (CCL-

azapeptide CD36 modulators. Employing diﬀerent N -

substituents on Lys as well as shorter Dab and Orn

homologues, substituent electron density and macrocycle size

were, respectively, shown to be signiﬁcant for CD36 binding

aﬃnity and NO inhibitory potency. By reducing the cyclic

alkyne to Z-oleﬁn and saturated counterparts, the macrocycle

shape and conformational ﬂexibility were similarly demon-

strated to inﬂuence CD36 binding aﬃnity and anti-

inﬂammatory activity. Moreover, the relevance of the semi-

carbazide geometry for binding and activity was illustrated by

replacement with an amino-sulfamide counterpart through the

) in macrophage-type cell models. Cultured cells were treated

−

with cyclic azapeptide at 10 M, and levels of TNF-α and

CCL-2 were measured by ELISA kit assays on the collected

cell culture supernatants. Secreted levels of IL-1β were

ascertained in BMA macrophages by measuring the activity of

the NLRP3-caspase-1 inﬂammasome cascade of the innate

7−39

immune system.

Modiﬁcations on macrocycles that led to loss of binding

aﬃnity resulted typically in the loss of inhibitory eﬀect on NO

as well as reduced ability to inhibit the release of pro-

inﬂammatory cytokines (TNF-α and IL-1β) and the chemo-

kine (CCL2) induced by the TLR-2 agonist in macrophages. A

negative correlation was, respectively, observed between the

binding aﬃnity and the inhibitory eﬀects of the cyclic

azapeptides on TLR2-mediated secretion of pro-inﬂammatory

cytokines IL-1β and TNF-α and chemokine CCL-2 (r = −0.61,

r = −0.57 and r = −0.69 Supporting Information). Inhibitory

potency on NO production strongly correlated with the

inhibition of pro-inﬂammatory cytokines (IL-1β, r = 0.71 and

TNF-α , r = 0.82) and chemokine (CCL-2, r = 0.83, Supporting

Information). For example, the drop of inhibitory potency on

NO release upon replacement of Lys (3a) by Dab (3e) and

Orn (3f) caused between 2.3-fold and 9-fold diminishment of

inhibitory eﬀects on TNF-α, IL-1β, and CCL-2. Substitution of

Lys (7a) by Orn (7f) precipitated, respectively, the inhibitory

eﬀects on the release of CCL2, IL-1β, and TNF-α by 3.3-, 9.3-,

and 3.1-fold. In contrast, the corresponding Dab modiﬁcation

synthesis of azasulfurylpeptide 36 using A -macrocyclization.

Notably, cyclic azapeptides exhibited a correlation between

CD36 binding aﬃnity and modulation of anti-inﬂammatory

responses. Further study is planned to examine the inﬂuences

of such subtle structural changes on other CD36-mediated

actions, such as cholesterol eﬄux from macrophages and lipid

metabolism. The diversity-oriented utility of A -macrocycliza-

tion for generating cyclic peptide ligands has been further

validated by the production of a valuable set of CD36 ligands

exhibiting biomedical potential for modulating both immune

and metabolic responses.

EXPERIMENTAL SECTION

■

General Protocols. Chromatography was performed on 230−400

mesh silica gel. Analytical thin-layer chromatography (TLC) was

performed on glass-backed silica gel plates (Merck 60 F254).

Visualization of TLC plates was performed by UV absorbance or

staining with potassium permanganate stain. H and C NMR spectra

were, respectively, measured in CDCl or DMSO-d at 500 (125)

MHz and referenced to CDCl [7.26 (77.16) ppm] and DMSO-d

2.50 (39.52) ppm]. Coupling constant J values were measured in

(

7e) attenuated moderately inhibitory potency on the release

[

of proinﬂammatory cytokines and chemokine. Similarly, the

critical role of the allyl N -substituent of 7a was evidenced by

replacement with N -cyclopropylmethyl (7g) and n-propyl

Hertz (Hz) and chemical shift values in parts per million (ppm).

^S^p^e^cⁱ^ﬁ^c^r^o^t^a^tⁱ^oⁿ^s^,^[^α^]D values, were measured at 25 °C at the

speciﬁed concentrations (c in g/100 mL) using a 1 dm cell length on a

PerkinElmer Polarimeter 589 and expressed using the general

(

7h) groups, which decreased by 1.6-fold to 6.25-fold the

inhibitory eﬀects on TNF-α, CCL-2, and IL-1β (Table 2).

Finally, saturated N -propyl analogue 32 exhibited activity in

formula: [α]₅= (100 × α)/(d × c). High-resolution mass

spectrometric analyses were performed at the Centre Regional de

Spectrometrie de Masse de l’Universite de Montreal. Protonated

molecular ions [M + H] and sodium adducts [M + Na] were used

for empirical formula conﬁrmation.

Unless otherwise speciﬁed, reagents were used as received.

Polystyrene Rink amide resin (0.5 mmol/g) and HOBt were

purchased from Advanced Chemtech, Boc-Ala-OH, Fmoc-Ala-OH,

Fmoc-Dab(Boc)-OH, Fmoc-Dap(Boc)-OH, Fmoc-Lys(Boc)-OH,

Fmoc-Lys-OH, Fmoc-Orn(Boc)-OH, Fmoc-D-Phe-OH, Fmoc-D-Trp-

(Boc)-OH, Fmoc-Trp(Boc)-OH, Fmoc-chloride 1,8-

reducing TNF-α, CCL-2, and IL-1β by 21, 34, and 29%,

respectively. These inhibitory levels were similar to that of N -

allyl cyclic alkyne 7a and higher than that of N -propyl cyclic

alkyne 7h. The subtle eﬀects of the cyclic peptides in reducing

pro-inﬂammatory cytokine and chemokine production high-

lighted the importance of N-terminal and N -substituents, ring

size, and ﬂexibility on downstream signaling once eﬀective

CD36 binding aﬃnity is achieved.

371

J. Med. Chem. 2021, 64, 9365−9380

Journal of Medicinal Chemistry

Article

washed sequentially with AcOH/H O/DMF (5:15:80, v/v/v, ×3),

DMF (×3), THF (×3), MeOH (×3), and DCM (×3). Examination

by LCMS of a cleaved resin sample (5 mg) showed complete

conversion and a peak with a molecular ion consistent with cyclic

azapeptide 3f: LCMS R = 4.50 min [10−90% MeOH (0.1% FA) in

water (0.1% FA) over 10 min], MS m/z: calcd for C H N O [M-

2Boc + H] , 755.4; found, 755.5. Resin-bound Boc-protected cyclic

azahexapeptide was deprotected and cleaved from the support using a

51 10

obtained from VWR International. Anhydrous solvents (CH Cl and

freshly made solution of TFA/H O/TES (95:2.5:2.5, v/v/v, 5 mL) at

THF) were obtained by passage through solvent ﬁltration systems

GlassContour, Irvine, CA). Fmoc-Lys(o-NBS)-OH and Fmoc-

Dap(o-NBS)-OH were synthesized according to literature proce-

room temperature for 2 h. The resin was ﬁltered and rinsed with TFA

(5 mL). The ﬁltrate and rinses were concentrated to an oil, from

which a precipitate was obtained by addition of cold ether (10 mL).

After centrifugation (1200 rpm for 10 min), the supernatant was

removed, and the precipitate was taken up in aqueous MeOH (10%

v/v) and freeze-dried prior to puriﬁcation. The resulting light brown

foam was puriﬁed by preparative HPLC to give cyclic azahexapeptide

(

dures. Cyclic azapeptides 3a, 5a, 6a, 7a, and 9a were synthesized as

11,26

previously described.

Cyclic Peptide Nomenclature. Side-chain-to-side-chain cross-

linked cyclic peptides are described as follows: c-{[N-terminal residue

(

atom linked)linkerC-terminal residue (atom linked)]linear

3f (1.6 mg, 1%) as a white foam: HPLC, R = 5.72 min [10−90% of

sequence without linker}. No linker is given in cases in which the

N- and C-terminal residues are directly linked.

MeOH (0.1% FA) in H O (0.1% FA) over 10 min and then 10%

MeOH (0.1% FA) for 5 min]; R = 4.26 min [10−90% MeCN (0.1%

Peptide Puriﬁcation and Analysis. Azapeptides were puriﬁed

on a semipreparative column (C18 Gemini column) using the

appropriate gradient from 10% to a higher % of MeOH (0.1% FA) in

water (0.1% FA) at a ﬂow rate of 10 mL/min. Purity (>95%) was

assessed using analytical HPLC on a 5 μM, 50 mm × 4.6 mm C18

Phenomenex Gemini column with a ﬂow rate of 0.5 mL/min using

the appropriate gradient in two diﬀerent solvent systems: MeCN

FA) in H O (0.1% FA) over 10 min and then 10% MeCN (0.1% FA)

for 5 min]; HRMS m/z: calcd for C H N O [M + H] , 755.3988;

51 10

found, 755.3990.

c-{[azaPra(δC)−CH −Dab(γN)]H-Ala-azaPra-D-Trp-Ala-Trp-D-

Phe-Dab(allyl)-NH } (7e). Azapeptide 7e was prepared according to

the protocol described for cyclic azapeptide 7f from linear

azaheptapeptide resin 31e (∼500 mg, 0.164 mmol) using CuI (6.0

mg, 0.032 mmol) and aq. formaldehyde (70 μL, 0.94 mmol, 37% in

(0.1% FA) in water (0.1% FA) and MeOH (0.1% FA) in water (0.1%

FA).

H O). Examination by LCMS of a cleaved resin sample (5 mg)

Solid-Phase Chemistry, Removal of Fmoc Protection, and

showed complete conversion and a peak with a molecular ion

Peptide Analysis. Fmoc-based peptide synthesis was performed on

an automated shaker using the polystyrene Rink amide resin (0.5

mmol/g, 75−100 mesh) and standard conditions. The loading was

calculated from the UV absorbance for Fmoc-deprotection after the

coupling of the ﬁrst amino acid. Couplings of amino acids (3 equiv)

were performed in DMF using DIC (3 equiv) and HOBt (3 equiv) for

consistent with cyclic azapeptide 7f: LCMS R = 5.36 min [10−90%

MeOH (0.1% FA) in water (0.1% FA) over 10 min]; MS m/z: calcd

for C H N O [M-3Boc + H] , 927.5; found, 927.5. Resin

49 59 12 7

cleavage using a freshly made solution of TFA/H O/TES (95:2.5:2.5,

v/v/v, 5 mL) and puriﬁcation by preparative HPLC gave cyclic

azaheptapeptide 7e (3.4 mg, 2%) as a white foam: HPLC R = 6.91

−6 h. Fmoc-deprotections were performed by treating the resin with

min [10−90% of MeOH (0.1% FA) in H O (0.1% FA) over 10 min

0% piperidine in DMF for 30 min. The resin was washed after each

and then 90% MeOH (0.1% FA) for 5 min]; R = 4.87 min [10−90%

coupling and the deprotection step performed sequentially with DMF

×3), MeOH (×3), THF (×3), and CH Cl (×3). The reaction

MeCN (0.1% FA) in H O (0.1% FA) over 10 min and then 90%

(

MeCN (0.1% FA) for 5 min]; HRMS m/z: calcd for C H N O

49 59 12

quality was assessed with a Kaiser test for the coupling of the amino

acids. Coupling reaction conversion was assessed by LC−MS analysis

on a cleaved 2−5 mg dried resin sample after treatment with TFA/

[M + H] , 927.4624; found, 927.4643.

c-{[azaPra(δC)−CH −Orn(δN)]H-Ala-azaPra-D-Trp-Ala-Trp-D-

Phe-Orn(allyl)-NH } (7f). In a plastic syringe tube equipped with a

TES/H O (95:2.5:2.5, v/v/v, 0.5 mL) for 15 min and ﬁltration. The

Teﬂon ﬁlter, stopcock, and stopper, resin 31f (∼1 g, 0.20 mmol) was

swollen in DMSO (8 mL) for 30 min, treated with CuI (7.0 mg, 0.04

ﬁltrate was evaporated under reduced pressure, dissolved in MeOH,

evaporated to dryness, and then examined by LC−MS.

mmol) and aq. formaldehyde (90 μL, 1.2 mmol, 37% in H O), shaken

c-{[azaPra(δC)−CH −Dab(γN)]H-Ala-azaPra-Ala-Trp-D-Phe-

on an automated shaker for 31 h, ﬁltered, and washed sequentially

Dab(allyl)-NH } (3e). Cyclic azapeptide 3e was prepared from linear

azahexapeptide resin 30e (∼500 mg, 0.18 mmol) according to the

protocol described for the synthesis of cyclic azapeptide 3f using CuI

with AcOH/H O/DMF (5:15:80, v/v/v, ×3), DMF (×3), THF

(×3), MeOH (×3), and DCM (×3). Examination by MS of a cleaved

resin sample (5 mg) showed a peak with a molecular ion consistent

(6.0 mg, 0.032 mmol) and aqueous formaldehyde (80 μL, 1.07 mmol,

with cyclic azaheptapeptide 7f: MS m/z: calcd for C H N O [M

61 12

7% in H O). Examination by LCMS of a cleaved resin sample (5

+ H] , 941.5, found 941.4. The resin was treated with a freshly made

mg) showed complete conversion and a peak with molecular ion

solution of TFA/H O/TES (95:2.5:2.5, v/v/v, 5 mL) at room

consistent with cyclic azapeptide 3e: LCMS R = 4.76 min [10−90%

temperature for 2 h, ﬁltered, and washed with TFA (5 mL). The

ﬁltrate and washes were combined and concentrated to an oil, from

which a precipitate was obtained by addition of cold ether (10 mL).

After centrifugation (1200 rpm for 10 min), the supernatant was

decanted and the precipitate was taken up in aqueous MeOH (10% v/

v) and freeze-dried to a light brown foam, which was puriﬁed by

preparative HPLC to give cyclic azaheptapeptide 7f (1.3 mg, 1%) as a

MeOH (0.1% FA) in water (0.1% FA) over 10 min]; MS m/z: calcd

for C H N O [M-2Boc + H] , 741.4; found, 741.5. Resin

49 10

cleavage using a freshly made solution of TFA/H O/TES (95:2.5:2.5,

v/v/v, 5 mL) and puriﬁcation by preparative HPLC gave cyclic

azahexapeptide 3e (2.5 mg, 2%) as a white foam: HPLC R = 5.51 min

[

10−90% of MeOH (0.1% FA) in H O (0.1% FA) over 10 min and

then 10% MeOH (0.1% FA) for 5 min,]; R = 4.16 min [10−90%

MeCN (0.1% FA) in H O (0.1% FA) over 10 min and then 10%

MeCN (0.1% FA) for 5 min]; HRMS m/z: calcd for C H N O

[

white foam: HPLC R = 6.85 min [10−90% of MeOH (0.1% FA) in

H O (0.1% FA) over 10 min and then 10% MeOH (0.1% FA) for 5

min]; R = 4.79 min [10−90% MeCN (0.1% FA) in H O (0.1% FA)

49 10

M + H] , 741.3831; found, 741.3842.

over 10 min and then 10% MeCN (0.1% FA) for 5 min]; HRMS m/z:

c-{[azaPra(δC)−CH −Orn(δN)]H-Ala-azaPra-Ala-Trp-D-Phe-

calcd for C H N O Na [M + Na] , 963.4600; found, 963.4573.

50 60 12 7

Orn(allyl)-NH } (3f). In a plastic syringe tube equipped with a Teﬂon

c-{[azaPra(δC)−CH −Lys(εN)]H-Ala-azaPra-D-Trp-Ala-Trp-D-

ﬁlter, stopcock, and stopper, Boc-Ala-azaPra-Ala-Trp(Boc)-D-Phe-

Orn(allyl) Rink amide resin 30f (∼500 mg, 0.15 mmol) was swollen

in DMSO (6 mL) for 30 min, treated with CuI (6.0 mg, 0.032 mmol)

Phe-Lys[CH CH(CH ) ]-NH } (7g). Cyclic azapeptide 7g was

2 2

prepared from resin 18g according to protocols described for the

synthesis of 7f. Puriﬁcation provided a 2.1% yield of material of 99%

and aq. formaldehyde (70 μL, 0.94 mmol, 37% in H O), shaken on an

automated shaker for 42 h, and ﬁltered. After ﬁltration, the resin was

purity: LCMS analysis R = 8.12 min [5−50% of MeOH (0.1% FA) in

H O (0.1% FA) over 9 min and then 5% MeOH for 5 min]; R = 8.09

372

J. Med. Chem. 2021, 64, 9365−9380

Journal of Medicinal Chemistry

Article

min [5−50% of MeCN (0.1% FA) in H O (0.1% FA) over 9 min and

colorless oil (1.81 g, 66%): [α] 5.9 (c 0.011, MeOH); H NMR (300

then 5% MeCN for 5 min]; HRMS m/z calcd for C H N O [M +

MHz, CDCl ): δ 8.02−7.96 (m, 1H), 7.78 (d, J = 7.4 Hz, 2H), 7.71−

62 12

H] , 969.5094; found, 969.5057

7.55 (m, 5H), 7.41 (t, J = 7.4 Hz, 2H), 7.32 (dd, J = 8.1, 6.9 Hz, 2H),

5.92 (qd, J = 11.0, 5.8 Hz, 1H), 5.68 (ddt, J = 16.5, 9.9, 6.5 Hz, 1H),

5.52 (d, J = 8.1 Hz, 1H), 5.41−5.15 (m, 4H), 4.67 (d, J = 5.6 Hz,

c-{[azaPra(δC)−CH −Lys(εN)]H-Ala-azaPra-D-Trp-Ala-Trp-D-

Phe-Lys(n-Pr)-NH } (7h). Cyclic azapeptide 7h was prepared from

resin 18h according to protocols described for the synthesis of 7f.

Puriﬁcation provided a 1.9% yield of material of 99% purity: LCMS

analysis R = 8.01 min [5−50% of MeOH containing 0.1% FA in H O

H), 4.39 (d, J = 7.6 Hz, 3H), 4.24 (t, J = 7.1 Hz, 1H), 4.03−3.86 (m,

H), 3.53−3.31 (m, 2H), 2.29−2.13 (m, 1H), 2.10−1.95 (m, 1H).

C NMR (75 MHz, CDCl ): δ 171.3, 156.0, 148.2, 144.0, 143.9,

(

0.1% FA) over 9 min and then at 5% MeOH for 5 min]; R = 5.86

141.5, 133.8, 133.4, 132.5, 131.8, 131.5, 131.0, 127.9, 127.3, 125.3,

124.4, 120.1, 119.4, 67.4, 66.6, 52.0, 50.5, 47.2, 43.5, 30.8. LCMS R =

11.01 min [10−90% MeOH (0.1% FA) in water (0.1% FA) over 10

min]; ESI-HRMS m/z: calcd for C H N O S [M + H] , 606.1905;

min [5−50% of MeCN (0.1% FA) in H O (0.1% FA) over 9 min and

then 5% MeCN for 5 min]; HRMS m/z calcd for C H N O [M +

H] , 957.5094; found, 955.5100.

65 12

Fmoc-Dab(o-NBS)-OH (14e). A solution of Fmoc-Dab(Boc)-OH

found, 606.1923.

(

1.01 g, 2.29 mmol) in CH Cl (30 mL) was treated with TFA (20

Fmoc-Orn(allyl, o-NBS)-OCH CHCH (16f). Ester 16f was

mL), stirred at room temperature for 3 h, and evaporated on a rotary

evaporator. The resulting yellow oil was dissolved in MeOH and

water and lyophilized. The resulting white foam was dissolved in THF

synthesized using the protocol described above for 16e from Fmoc-

Orn(o-NBS)-OH (14f, 2.4 g, 4.44 mmol), PPh (3.46 g, 13.2 mmol),

allyl alcohol (1.8 mL, 26.5 mmol), and DIAD (2.6 mL, 13.2 mmol).

After column chromatography using 20−50% EtOAc in petroleum

ether as the eluent, evaporation of the collected fractions provided

(25 mL) and water (25 mL), treated with DIEA (4.00 mL, 23.0

mmol) and o-NBS-Cl (577 g, 2.60 mmol), stirred at room

temperature for 4 h, and diluted with EtOAc (50 mL). The organic

phase was washed with aqueous HCl (1 M, 50 mL × 3), water (50

mL), and brine (50 mL). The organic layer was dried over MgSO₄,

amino ester 16f (1.70 g, 62%) as a pale-yellow oil: [α] −2.6 (c

.0115, MeOH). H NMR (300 MHz, CDCl ): δ 8.05−7.99 (m,

H), 7.78 (d, J = 7.4 Hz, 2H), 7.69−7.55 (m, 5H), 7.41 (t, J = 7.4 Hz,

H), 7.32 (td, J = 7.4, 1.1 Hz, 2H), 5.99−5.84 (m, 1H), 5.76−5.59

ﬁltered, and evaporated to give 14e as a light-yellow gum (1.15 g,

6%): [α] −6.9 (c 0.026, MeOH); H NMR (300 MHz, DMSO): δ

(

m, 1H), 5.39−5.31 (m, 3H), 5.28−5.20 (m, 1H), 5.19−5.13 (m,

.18 (t, J = 5.4 Hz, 1H), 7.97 (dq, J = 5.7, 3.1 Hz, 2H), 7.92−7.81 (m,

H), 7.68 (dd, J = 13.4, 7.8 Hz, 3H), 7.41 (t, J = 7.1 Hz, 2H), 7.31 (t,

H), 4.65 (d, J = 5.7 Hz, 2H), 4.46−4.32 (m, 3H), 4.23 (t, J = 7.0 Hz,

H), 3.91 (d, J = 6.1 Hz, 2H), 3.32 (t, J = 6.3 Hz, 2H), 1.85 (d, J =

J = 7.4 Hz, 2H), 4.32−4.17 (m, 3H), 4.02 (tt, J = 8.9, 4.4 Hz, 1H),

0.1 Hz, 1H), 1.70−1.55 (m, 3H). C NMR (75 MHz, CDCl ): δ

171.9, 156.0, 148.1, 144.0, 143.9, 141.5, 133.7, 133.6, 132.7, 131.8,

31.5, 131.1, 127.9, 127.2, 125.2, 124.3, 120.1, 119.5, 119.3, 67.2,

6.3, 53.6, 50.1, 47.3, 46.7, 29.8, 23.9. LCMS [10−90% MeOH (0.1%

FA) in water (0.1% FA) over 10 min]; R = 10.01 min. ESI-HRMS m/

z: calcd for C32H N O SNa [M + Na] , 642.1881; found, 642.1898.

.97 (dd, J = 14.2, 7.6 Hz, 2H), 2.02−1.91 (m, 1H), 1.86−1.70 (m,

_H₎_.13C NMR (75 MHz, DMSO): δ 173.4, 172.0, 156.1, 147.8,

43.8, 140.7, 134.0, 132.6, 129.4, 127.7, 127.1, 125.3, 124.5, 120.1,

5.7, 51.3, 46.6, 30.8. LCMS, R = 10.01 min [10−90% MeOH (0.1%

FA) in water (0.1% FA) over 10 min]; ESI-HRMS m/z: calcd for

C H N O SNa [M + Na] , 548.1098; found, 548.1105.

33 3 8

Fmoc-Dab(allyl, o-NBS)-OH (17e). A solution of Fmoc-Dab-

allyl, o-NBS)-OCH CHCH (16e, 1.44 g, 2.38 mmol) in MeCN

Fmoc-Orn(o-NBS)-OH (14f). Sulfonamide 14f was prepared

according to the protocol for the synthesis of 14e above from

Fmoc-Orn(Boc)-OH (2.02 g, 4.44 mmol) using o-NBSCl (1.13 g,

(

10 mL) was treated with a 0.8 M CaCl solution in 7:3 i-PrOH:H O

60 mL), followed by aqueous NaOH (1 M, 2.4 mL, 2.4 mmol),

.08 mmol) and obtained as a light-yellow solid (2.4 g, quantitative):

stirred at room temperature for 6 h, and diluted with EtOAc (100

mL). The organic phase was washed with aqueous HCl (1 M, 100

mL). The aqueous layer was extracted with EtOAc (100 mL). The

combined organic layers were washed with brine (100 mL), dried

over MgSO , ﬁltered, and evaporated to a residue that was puriﬁed by

chromatography using a CombiFlash instrument and 0−100% EtOAc

in hexane. Evaporation of the collected fractions gave acid 17e (850

mp 108−110 °C; [α] −1.6 (c 0.036, MeOH); H NMR (300 MHz,

DMSO): δ 8.10 (t, J = 5.6 Hz, 1H), 8.03−7.92 (m, 2H), 7.92−7.81

(m, 4H), 7.72 (d, J = 7.4 Hz, 2H), 7.61 (d, J = 8.0 Hz, 1H), 7.41 (t, J

7.2 Hz, 2H), 7.32 (t, J = 7.1 Hz, 2H), 4.34−4.16 (m, 3H), 3.89 (td,

J = 8.7, 4.6 Hz, 1H), 2.90 (q, J = 6.3 Hz, 2H), 1.73 (s, 1H), 1.65−1.43

_m_,₃_H₎_.₁3C NMR (75 MHz, DMSO): δ 173.7, 156.1, 147.8, 143.8,

(

40.7, 134.0, 132.7, 132.6, 129.4, 127.7, 127.1, 125.3, 124.4, 120.1,

mg, 63%) as a light-yellow oil: [α] −15.2 (c 0.01, MeOH); H NMR

5.6, 53.5, 46.7, 42.3, 27.9, 26.0. LCMS, R = 11.04 min {10−90%

(

300 MHz, DMSO): δ 8.01 (ddd, J = 16.9, 7.7, 1.4 Hz, 2H), 7.92−

7.77 (m, 4H), 7.74−7.60 (m, 3H), 7.41 (t, J = 7.4 Hz, 2H), 7.31 (t, J

7.4 Hz, 2H), 5.66 (dq, J = 10.4, 6.2 Hz, 1H), 5.19 (dd, J = 25.1, 13.7

Hz, 2H), 4.33−4.17 (m, 3H), 4.00−3.87 (m, 3H), 3.46−3.19 (m,

MeOH [0.1% formic acid (FA)] in water (0.1% FA) over 10 min}.

ESI-HRMS m/z: calcd for C H N O S [M + H] , 540.1435;

found, 540.1449.

Fmoc-Lys(o-NBS)-Rink Amide Resin 15a. In a syringe ﬁtted

with a Teﬂon ﬁlter, stopcock, and stopper, the Fmoc group was

removed from the Rink amide resin (3.00 g) using 20% piperidine in

DMF solution for 30 min. The resin was ﬁltered and washed

sequentially with DMF (×3), MeOH (×3) and CH Cl (×3). A

H), 2.05−1.91 (m, 1H), 1.91−1.77 (m, 1H). C NMR (75 MHz,

DMSO): δ 173.7, 156.5, 147.9, 144.2, 141.2, 135.1, 133.2, 133.0,

32.2, 130.4, 128.1, 127.5, 125.7, 124.8, 120.6, 119.7, 66.1, 51.9, 50.3,

7.1, 44.8, 30.0. LCMS R = 10.54 min [10−90% MeOH (0.1% FA)

in water (0.1% FA) over 10 min]. ESI-HRMS m/z: calcd for

C H N O S [M + H] , 566.1592; found, 566.1608.

Fmoc-Orn(allyl, o-NBS)-OH (17f). Acid 17f was synthesized

from ester 16f (1.70 g, 2.75 mmol) using the protocol described for

17e with a 0.8 M CaCl solution in 7:3 i-PrOH:H O (70 mL) and

2 2

NaOH (1 M, 2.7 mL, 2.7 mmol). The residue was puriﬁed twice by

column chromatography using EtOAc/n-hexane (4:1) and then

MeOH/CH Cl (1:9) to give amino acid 17f (623 mg, 39%) as a

light-yellow oil: [α] 12.0 (c 0.0075, MeOH); H NMR (300 MHz,

separate solution of Fmoc-Lys(o-NBS)-OH (1.62 g, 2.93 mmol) in

DMF (20 mL) was treated with DIC (0.7 mL, 4.52 mmol) and HOBt

28 28

(611 mg, 4.52 mmol), stirred for 3 min, and added to the syringe

containing the resin. The resin mixture was shaken for 14 h, ﬁltered,

and sequentially washed with DMF (×3), MeOH (×3), and CH Cl

(

×3) and dried. The loading of resin 15a was measured at 0.345

mmol/g resin.

Fmoc-Dab(allyl, o-NBS)-OCH CHCH (16e). Under an argon

2 2

atmosphere, Fmoc-Dab(o-NBS)-OH (14e, 2.39 g, 4.54 mmol) was

dissolved in THF (dry, 50 mL), treated sequentially with PPh (3.58

g, 13.7 mmol), allyl alcohol (1.9 mL, 27.9 mmol), and DIAD (2.7 mL,

DMSO): δ 8.04 (dd, J = 7.5, 1.6 Hz, 1H), 7.96 (dd, J = 7.6, 1.5 Hz,

1H), 7.92−7.78 (m, 4H), 7.72 (d, J = 7.1 Hz, 2H), 7.60 (d, J = 7.9

Hz, 1H), 7.42 (t, J = 7.3 Hz, 2H), 7.32 (t, J = 7.3 Hz, 2H), 5.71−5.56

(m, 1H), 5.27−5.10 (m, 2H), 4.33−4.17 (m, 3H), 3.90 (d, J = 6.0 Hz,

2H), 3.50−3.15 (m, 3H), 1.74−1.45 (m, 4H). C NMR (75 MHz,

DMSO): δ 173.6, 156.1, 147.5, 143.9, 143.8, 140.7, 134.5, 132.8,

132.5, 131.9, 129.9, 127.6, 127.1, 125.3, 124.2, 120.1, 119.1, 65.6,

53.6, 49.3, 46.7, 27.9, 24.1. LC−MS [30−95% MeOH (0.1% FA) in

3.7 mmol), stirred at room temperature for 2 h, and diluted with

EtOAc (100 mL). The organic phase was washed with water (100 mL

3) and brine (100 mL), dried over MgSO , ﬁltered, and evaporated

to a residue, which was puriﬁed by chromatography using a

CombiFlash instrument and 0−100% EtOAc in hexane as the eluent.

Evaporation of the collected fractions gave amino ester 16e as a

373

J. Med. Chem. 2021, 64, 9365−9380

Journal of Medicinal Chemistry

Article

water (0.1% FA) over 10 min]; R = 8.85 min. ESI-HRMS m/z: calcd

warmed to rt, stirred for 30 min, and evaporated. The residue was

dissolved in CH Cl (10 mL) and evaporated. The resulting white

for C H N O S [M + H] , 580.1748; found, 580.1763.

Fmoc-Lys(o-NBS, allyl)-Rink Amide Resin 18a. In a syringe

ﬁtted with a Teﬂon ﬁlter, vacuum dried Fmoc-Lys(o-NBS)-resin 15a

0.441 mmol) was swollen in dry THF (5 mL), treated sequentially

with solutions of allyl alcohol (206 μL, 3.03 mmol) in THF (dry, 1

solid was dissolved in dry CH Cl (6 mL), added to H-Ala-Trp(Boc)-

2 2

D-Phe-Orn(o-NBS, allyl) Rink amide resin 21f (∼600 mg, 0.167

mmol) swollen in DIEA (0.13 mL, 0.746 mmol) in a plastic syringe

equipped with a Teﬂon ﬁlter, stopcock, and stopper, and shaken for

18 h. The resin was ﬁltered and washed sequentially with DMF (×3),

MeOH (×3), THF (×3), and CH Cl (×3). Examination by LCMS

(

mL), PPh (397 mg, 1.51 mmol) in THF (dry, 1 mL), and DIAD

(

298 μL, 1.51 mmol) in THF (dry, 1 mL), shaken for 90 min, ﬁltered,

and sequentially washed with DMF (×3), MeOH (×3), THF (×3),

of a cleaved resin sample (5 mg) showed complete coupling and

and CH Cl (×3). Examination by LCMS of a cleaved resin sample (5

formation of azapeptide: LCMS R = 9.25 min [30−95% MeOH

mg) showed complete allylation: LCMS R = 8.65 min [30−95%

(0.1% FA) in water (0.1% FA) over 10 min]; ESI-MS m/z: calcd for

MeOH (0.1% FA) in water (0.1% FA) over 10 min]; ESI-MS m/z:

11S [M-Boc + H] , 1079.4; found, 1079.3.

calcd for C H N O S [M + H] , 593.2; found, 593.2.

Fmoc-azaPra-D-Trp(Boc)-Ala-Trp(Boc)-D-Phe-Dab(o-NBS,

Fmoc-Dab(allyl, o-NBS) Rink Amide Resin 18e. In a plastic

syringe equipped with a stopcock and Teﬂon ﬁlter, the Fmoc group

was removed from the Rink amide resin (850 mg) using 20%

piperidine in DMF solution for 30 min. The resin was ﬁltered and

washed sequentially with DMF (×3), MeOH (×3), and CH Cl

allyl) Rink Amide Resin 25e. Semicarbazide 25e was prepared from

pentapeptide 22e (∼400 mg, 0.164 mmol) using the protocol

described for the synthesis of azapeptide 25f and Fmoc-azaPra-Cl

from N′-propargyl-ﬂuorenylmethylcarbazate (100 mg, 0.342 mmol)

and phosgene in toluene (0.35 mL, 0.49 mmol). Examination by

LCMS of a cleaved resin showed complete coupling to provide the

(

×3). A solution of Fmoc-Dab(allyl,o-NBS)-OH (17e, 450 mg, 0.796

mmol) in DMF (10 mL) was treated in a separate ﬂask with DIC

0.19 mL, 1.23 mmol) and HOBt (166 mg, 1.30 mmol), stirred for 3

azapeptide: LCMS R = 7.89 min [50−90% MeOH (0.1% FA) in

water (0.1% FA) over 10 min] ESI-MS m/z: calcd for

(

C H N O S [M-2Boc +2H] 626.2; found, 626.3.

min, and transferred to the syringe containing the swollen resin. The

resin mixture was shaken for 23 h, ﬁltered, washed sequentially with

DMF (×3), MeOH (×3) and CH Cl (×3), and was dried. The

66 67 12 12

Fmoc-azaPra-D-Trp(Boc)-Ala-Trp(Boc)-D-Phe-Orn(o-NBS,

allyl) Rink Amide Resin 25f. N′-Propargyl-ﬂuorenylmethyl

carbazate (248 mg, 0.849 mmol, prepared according to ref 26) was

dissolved in CH Cl (dry, 40 mL) under an argon atmosphere, cooled

loading was measured to be 0.327 mmol/g resin.

Fmoc-Orn(allyl, o-NBS) Rink Amide Resin 18f. As described

for resin 18e, the Fmoc group was removed from the Rink amide resin

to 0 °C, and treated with a 20% solution of phosgene in toluene (1

mL, 1.87 mmol). The ice bath was removed. The reaction mixture

was warmed to rt with stirring for 50 min and evaporated to a residue,

which was dissolved in CH Cl (10 mL) and evaporated. The

(

902 mg), which was then reacted with a premixed mixture of Fmoc-

Orn(allyl, o-NBS)-OH (17f, 510 mg, 0.879 mmol), DIC (0.2 mL,

.29 mmol), and HOBt (178 mg, 1.32 mmol) in DMF (8 mL),

resulting white solid was dissolved in dry CH Cl (7 mL), added to a

shaken for 18 h, ﬁltered, washed, and dried to give a resin with a

loading of 0.338 mmol/g.

solution of H-D-Trp(Boc)-Ala-Trp(Boc)-D-Phe-Orn(o-NBS, allyl)

Rink amide resin 22f (∼1 g, 0.20 mmol) swollen in DIEA (0.07

mL, 0.402 mmol) in a plastic syringe equipped with a Teﬂon ﬁlter,

stopcock, and stopper, and shaken for 28 h. The resin was ﬁltered and

washed sequentially with DMF (×3), MeOH (×3), THF (×3), and

CH Cl (×3). Examination by LCMS of a cleaved resin showed

Fmoc-Lys(o-NBS, cyclopropylmethyl) Rink Amide Resin

8g. Resin 18g was prepared using cyclopropylmethanol in the

protocol described for the synthesis of 18a: LCMS R = 10.46 min

[

10−95% MeOH (0.1% FA) in water (0.1% FA) over 10 min]; ESI-

MS m/z calcd for C H N O S [M + H] , 607.2; found, 607.2.

complete coupling to provide the azapeptide: LCMS R = 8.26 min

Fmoc-Lys(o-NBS, propyl)-Rink Amide Resin 18h. Resin 18g

was prepared using n-propanol in the protocol described to make

[

30−95% MeOH (0.1% FA) in water (0.1% FA) over 10 min]; ESI-

MS m/z: calcd for C H N O S [M-Fmoc-2Boc + H] , 1043.4;

resin 18a: LCMS R = 8.74 min [10−95% MeOH (0.1% FA) in water

52 59 12 10

found, 1043.3.

(

0.1% FA) over 10 min]; ESI-MS m/z calcd for C H N O S [M +

30 34 4 7

Boc-Ala-azaPra-Ala-Trp-D-Phe-Dab(o-NBS, allyl) Rink Amide

Resin 28e. Azapeptide 28e was prepared from Fmoc-deprotected

vacuum-dried azapeptide 26e (∼400 mg, 0.18 mmol) according to

the protocol for the synthesis of azapeptide 28f using Fmoc-Ala-OH

H] , 595.2; found, 595.2.

Peptide Elongation. Peptides were elongated by standard Fmoc

peptide synthesis methods.

Fmoc-Ala-Trp-D-Phe-Dab(o-NBS, allyl) Rink Amide Resin

(

279 mg, 0.897 mmol), BTC (95 mg, 0.320 mmol), and 2,4,6-

(

9e. LCMS R = 6.63 min [30−95% MeOH (0.1% FA) in water

collidine (0.24 mL, 1.82 mmol). Examination by LCMS of a cleaved

0.1% FA) over 10 min]; ESI-MS m/z: calcd for C H N O S [M-

resin showed complete coupling: LCMS R = 9.12 min [30−95%

Fmoc-2Boc + H] , 747.3; found, 747.2.

MeOH (0.1% FA) in water (0.1% FA) over 10 min]; ESI-MS m/z:

Fmoc-D-Trp(Boc)-Ala-Trp(Boc)-D-Phe-Dab(o-NBS,allyl) Rink

Amide Resin 20e. LCMS R = 9.58 min [30−95% MeOH (0.1%

calcd for C58

Fmoc group was removed and the resin (∼400 mg) was treated with

Boc O (218 mg, 1.0 mmol).

H N O12S [M-Boc + H] 1136.4; found, 1136.3. The

62 11

FA) in water (0.1% FA) over 10 min]; ESI-MS m/z: calcd for

C H N O S [M-2Boc + H] , 1155.4; found, 1155.4.

63 10 11

Fmoc-D-Trp(Boc)-Ala-Trp(Boc)-D-Phe-Orn(o-NBS, allyl) Rink

Boc-Ala-azaPra-Ala-Trp(Boc)-D-Phe-Orn(o-NBS, allyl) Rink

Amide Resin 28f. Dry THF (10 mL) was added to a mixture of

Fmoc-Ala-OH (265 mg, 0.852 mmol) and BTC (85 mg, 0.286 mmol)

in a microwave vial containing a magnetic stirring bar. The vial was

sealed, cooled to 0 °C, treated with 2,4,6-collidine (0.23 mL, 1.74

mmol), and stirred for 1 min. The vial was opened to remove the

magnetic stirring bar, treated with Fmoc-deprotected vacuum-dried

azapeptide resin 26f (∼600 mg, 0.167 mmol), sealed, heated with

microwave irradiation at 60 °C for 1 h, cooled, and opened. The resin

was transferred to a plastic syringe equipped with a Teﬂon ﬁlter,

stopcock, and stopper, then washed sequentially with DMF (×3),

Amide Resin 20f. LCMS R = 6.13 min [30−95% MeOH (0.1% FA)

in water (0.1% FA) over 10 min]; ESI-MS m/z: calcd for

C H N O S [M-Fmoc-2Boc + H] , 947.4; found, 947.3.

55 10

Fmoc-azaPra-Ala-Trp-D-Phe-Dab(o-NBS, allyl) Rink Amide

Resin 24e. Semicarbazide 24e was prepared from pentapeptide 21e

(

∼400 mg, 0.18 mmol) using the protocol described for the synthesis

of azapeptide 24f and Fmoc-azaPra-Cl from N′-propargyl-ﬂuorenyl-

methylcarbazate (100 mg, 0.342 mmol) and phosgene in toluene

(

0.35 mL, 0.49 mmol). Examination by LCMS of a cleaved resin

sample (5 mg) showed complete coupling: LCMS R = 7.46 min [50−

0% MeOH (0.1% FA) in water (0.1% FA) over 10 min]; ESI-MS m/

MeOH (×3), THF (×3), and CH

of a cleaved resin showed complete coupling and a new azapeptide:

LCMS R = 9.22 min [30−95% MeOH (0.1% FA) in water (0.1%

FA) over 10 min]; ESI-MS m/z: calcd for C H N O S [M-Boc +

2 2

Cl (×3). Examination by LCMS

z: calcd for C H N O S [M-Boc + H] 1065.4; found, 1065.3.

57 10 11

Fmoc-azaPra-Ala-Trp(Boc)-D-Phe-Orn(o-NBS, allyl) Rink

Amide Resin 24f. N′-Propargyl-ﬂuorenylmethylcarbazate (110 mg,

64 11 12

.377 mmol, prepared according to ref 26) was dissolved in dry

H] , 1150.5; found, 1150.3.

CH Cl (10 mL) under an argon atmosphere, cooled to 0 °C, treated

After the Fmoc group removal, the resin (∼500 mg, 0.15 mmol)

with a 15% solution of phosgene in toluene (0.5 mL, 0.70 mmol),

was treated with a solution of Boc O (170 mg, 0.779 mmol) in dry

374

J. Med. Chem. 2021, 64, 9365−9380

Journal of Medicinal Chemistry

Article

THF (5 mL) in a plastic syringe equipped with a Teﬂon ﬁlter,

stopcock, and stopper. The resin was shaken for 1 h, ﬁltered, and

washed sequentially with DMF (×3), MeOH (×3), THF (×3), and

CH Cl (×3). The Kaiser test showed complete Boc-protection.

Boc-Ala-azaPra-D-Trp(Boc)-Ala-Trp(Boc)-D-Phe-Dab(o-NBS,

allyl) Rink Amide Resin 29e. Azapeptide 29e was prepared from

Fmoc-deprotected azapeptide resin 27e (∼400 mg, 0.164 mmol)

according to the protocol to make resin 28f using Fmoc-Ala-OH (270

mg, 0.868 mmol), BTC (85 mg, 0.286 mmol), and 2,4,6-collidine

74%) as a white foam: HPLC R = 6.04 min [10−90% of MeOH

(0.1% FA) in H O (0.1% FA) over 10 min and then 90% MeOH

(0.1% FA) for 5 min]; R = 5.71 min [10−90% MeCN (0.1% FA) in

H O (0.1% FA) over 10 min and then 90% MeCN (0.1% FA) for 5

min]; HRMS m/z: calcd for C H N O [M + H] , 961.5407;

51 69 12 7

found, 961.5391.

c-{[Aza-hexenylglycine(εC)-Lys(εN)]H-Ala-Z-aza-hexenyl

glycinyl-D-Trp-Ala-Trp-D-Phe-Lys(cyclopropylmethyl)-NH

(33). A solution of c-{[azaPra(δC)−CH −Lys(εN)]H-Ala-azaPra-D-

Trp-Ala-Trp-D-Phe-Lys(cyclopropylmethyl)-NH } (7g, 6 mg, 6.2

}

(0.23 mL, 1.74 mmol). Examination by LCMS of a cleaved resin

showed complete coupling: LCMS R = 9.59 min [30−95% MeOH

μmol) in MeOH (2 mL) was mixed with the Lindlar catalyst (5

mg,, prepared according to ref 45). The vessel containing the

suspension was ﬂushed with argon. Quinoline (50 μL, 0.42 mmol)

was added to the suspension, which was placed under an atmosphere

of hydrogen and stirred at room temperature for 1 h. The atmosphere

was exchanged for argon. The suspension was ﬁltered through a plug

of Celite. The ﬁlter cake was washed with MeOH. The ﬁltrate and

washings were combined, treated with water, and freeze-dried. The

resulting light-yellow foam was puriﬁed by preparative HPLC to give

(

0.1% FA) in water (0.1% FA) over 10 min]; ESI-MS m/z: calcd for

C H N O S [M-2Boc +2H] 661.8; found, 661.8. After Fmoc

72 13 13

removal, the resin was treated with Boc O (182 mg, 0.834 mmol).

Boc-Ala-azaPra-D-Trp(Boc)-Ala-Trp(Boc)-D-Phe-Orn(o-NBS,

allyl) Rink Amide Resin 29f. The Fmoc group was removed from

semicarbazide 25f (∼1 g, 0.20 mmol) and resin 27f was treated twice

with symmetric anhydride that was generated from Boc-Ala-OH

according to the protocol described in ref 26. Examination by LCMS

of a cleaved resin sample (5 mg) showed complete coupling: LCMS

R = 6.39 min [30−95% MeOH (0.1% FA) in H O (0.1% FA) over

cyclic azapeptide 33 (2 mg, 33%) as a white foam: HPLC R

min [10−90% of MeOH (0.1% FA) in H O (0.1% FA) over 10 min

and then 90% MeOH (0.1% FA) for 5 min]; R = 4.93 min [10−90%

MeCN (0.1% FA) in H O (0.1% FA) over 10 min and then 90%

= 6.64

0 min]; ESI-MS m/z: calcd for C H N O S [M-3Boc + H] ,

64 13 11

114.5; found, 1114.4.

Boc-Ala-azaPra-Ala-Trp-D-Phe-Dab(allyl) Rink Amide Resin

MeCN (0.1% FA) for 5 min]; HRMS m/z: calcd for C52

[M + H] , 971.5250; found, 971.5254.

N O

0e. Azapeptide 30e was prepared from azapeptide 28e (∼400 mg,

.18 mmol) according to the protocol to make resin 30f using DBU

c-{[AsPra(δC)−CH −Lys(εN)]H-Ala-AsPra-D-Trp-Ala-Trp-D-

(270 μL, 1.81 mmol) and 2-mercaptoethanol (60 μL, 0.86 mmol).

Phe-Lys(allyl)-NH } (36). Cyclic azasulfurylpeptide 36 was prepared

Examination by LCMS of a cleaved resin sample (5 mg) showed

according to the protocol described for cyclic azapeptide 7f using

linear azasulfurylpeptide resin 45 (1 equiv, 282 mg, 0.0925 mmol),

CuI (0.2 equiv, 3.52 mg, 0.0185 mmol), and aq. formaldehyde (6

complete o-NBS-removal: LCMS R = 4.52 min [10−90% MeOH

(

0.1% FA) in water (0.1% FA) over 10 min]; ESI-MS m/z: calcd for

C H N O [M-2Boc + H] , 729.4; found, 729.6.

equiv, 41.6 μL, 0.555 mmol): LCMS R = 7.75 [10−90% MeOH

49 10

Boc-Ala-azaPra-Ala-Trp(Boc)-D-Phe-Orn(allyl) Rink Amide

Resin 30f. In the plastic syringe, o-NBS-protected azapeptide resin

(0.1% FA) in water (0.1% FA) over 10 min and then in 10% MeOH

(0.1% FA) in water (0.1% FA) for 5 min]; ESI-MS m/z calcd for

8f (∼500 mg, 0.15 mmol) was swollen in DMF (6 mL), treated with

S [M + H] , 991.45; found, 991.4. Resin cleavage using

DBU (220 μL, 1.47 mmol) and 2-mercaptoethanol (50 μL, 0.71

mmol), shaken for 3 h, ﬁltered, and sequentially washed with DMF

a freshly made solution of TFA/H O/TES (95:2.5:2.5, v/v/v, 5 mL)

at 4 °C for 3 h and puriﬁcation of the light brown foam by preparative

RP-HPLC on a Gemini 5 μm C18 110A column (Phenomenex Inc.,

250 × 21.2 mm, 5 μm) using a gradient of 5−60% MeOH (0.1% FA)

in water (0.1% FA) with a ﬂow rate of 10.0 mL/min and UV

detection at 214 nm gave cyclic azasulfurylpeptide 36 (1.0 mg, 1%) as

(

×3), MeOH (×3), THF (×3), and CH Cl (×3). Examination by

2 2

LCMS of a cleaved resin sample (5 mg) showed complete o-NBS-

removal and a new azapeptide: LCMS R = 3.54 min [10−90%

MeOH (0.1% FA) in water (0.1% FA) over 10 min]; ESI-MS m/z:

calcd for C H N O [M-2Boc + H] , 743.4; found, 743.5.

a white foam, which was analyzed on a Sunﬁre column: LCMS R 8.12

51 10

Boc-Ala-azaPra-D-Trp(Boc)-Ala-Trp(Boc)-D-Phe-Dab(allyl)

Rink Amide Resin 31e. Azapeptide 31e was prepared according to

the protocol to make azapeptide 31f using resin 29e (∼400 mg, 0.164

mmol), DBU (250 μL, 1.67 mmol), and 2-mercaptoethanol (60 μL,

min [5−50% MeOH (0.1% FA) in water (0.1% FA) over 9 min and

then 5% MeOH (0.1% FA) in water (0.1% FA) for 5.0 min] >99%

purity; R 5.99 min [5−50% MeCN (0.1% FA) over 9 min and then

5% MeCN (0.1% FA) for 5.0 min] >99% purity; HRMS m/z calcd for

.86 mmol): LCMS R = 5.10 min [10−90% MeOH (0.1% FA) in

[M + H] C H N O S, 991.4607; found, 991.4657.

50 63 12

water (0.1% FA) over 10 min]; ESI-MS m/z: calcd for C H N O

N-Propargyl Fluorenylmethyl Carbazate (H-azaPra-Ofm,

37). A solution of tert-butyl carbazate (1 equiv, 2 g, 15.1 mmol) in

9:1 THF/DMF (30 mL) was treated with powdered K CO (1.5

2 3

equiv, 3.14 g, 22.7 mmol), cooled to 0 °C, and treated dropwise with

a solution of propargyl bromide (0.8 equiv, 1.3 mL, 12.1 mmol, 80%

in toluene) in THF (10 mL). The ice bath was removed. After

warming to room temperature with stirring overnight, the reaction

59 12

[

M-3Boc + H] , 915.5; found, 915.5.

Boc-Ala-azaPra-D-Trp(Boc)-Ala-Trp(Boc)-D-Phe-Orn(allyl)

Rink Amide Resin 31f. In a plastic syringe equipped with a Teﬂon

ﬁlter, stopcock, and stopper, o-NBS-protected azapeptide 29f (∼1 g,

.20 mmol) was swollen in DMF (6 mL), treated with DBU (300 μL,

.01 mmol) and 2-mercaptoethanol (70 μL, 1.00 mmol), shaken for 1

h, ﬁltered, and washed sequentially with DMF (×3), MeOH (×3),

mixture was washed with H O (100 mL) and brine (100 mL), dried

THF (×3), and CH Cl (×3). Examination by LCMS of a cleaved

over Na SO , ﬁltered, and evaporated. The reside was puriﬁed by

resin sample (5 mg) showed complete o-NBS-removal and a new

column chromatography using 30−40% EtOAc/hexane as the eluent

azapeptide: LCMS R = 4.49 min [30−95% MeOH (0.1% FA) in

to give N′-propargyl tert-butyl carbazate (1.91 g, 6.54 mmol, 54%) as

water (0.1% FA) over 10 min]; ESI-MS m/z: calcd for C H N O

a white solid: mp 54−55 °C; R

= 0.24 (EtOAc/hexane 1:4); H

61 12

[

M-3Boc+2Na]²⁺487.2; found, 487.3.

NMR (500 MHz, CDCl ): δ 6.20 (s, 1H), 4.07 (s, 1H), 3.62 (d, J =

2.5 Hz, 2H), 2.24 (t, J = 2.5 Hz, 1H), 1.46 (s, 9H); C NMR (125

c-{[azaGly(αN)−(CH ) −Lys(εN)]H-Ala-azaGly-D-Trp-Ala-Trp-

D-Phe-Lys(n-Pr)-NH } (32). A solution of c-{[azaPra(δC)−CH −

MHz, CD OD): δ 156.5, 81.0, 80.1, 72.5, 41.4, 28.5; HRMS m/z

Lys(εN)]H-Ala-azaPra-D-Trp-Ala-Trp-D-Phe-Lys(allyl)-NH } (7a, 5

calcd for C H N O [M + H] , 171.1128; found, 171.1122.

mg, 5.0 μmol) in MeOH (2 mL) was treated with Pd/C (10 wt %, 4

mg), placed under an argon atmosphere, treated with hydrogen gas

bubbles, and stirred at room temperature for 1 h under an atmosphere

of hydrogen. The atmosphere was exchanged for argon. The

suspension was ﬁltered through a plug of Celite. The ﬁlter cake was

washed with MeOH. The ﬁltrate and washings were combined,

treated with water, and freeze-dried to a light-yellow foam, which was

puriﬁed by preparative HPLC to give cyclic azapeptide 32 (3.7 mg,

A solution of N′-propargyl tert-butyl carbazate (1 equiv, 0.57 g,

3.35 mmol) in CH Cl (40 mL) was cooled to −40 °C and treated

with DIEA (1.5 equiv, 0.83 mL, 5.02 mmol), followed dropwise by a

solution of Fmoc-Cl (1.1 equiv, 0.95 g, 3.68 mmol) in CH Cl (10

mL). The cooling bath was removed. After warming to room

temperature with stirring overnight, the reaction mixture was washed

with H O (100 mL) and brine (100 mL), dried over Na SO , ﬁltered,

and evaporated. The reside was puriﬁed by column chromatography

375

J. Med. Chem. 2021, 64, 9365−9380

Journal of Medicinal Chemistry

Article

using 10−15% EtOAc/hexane as the eluent to give N′-propargyl N′-

(m, 2H), 7.33−7.26 (m, 3H), 7.21 (t, J = 7.3 Hz, 1H), 7.11 (d, J = 8.0

Hz, 1H), 6.46−6.39 (m, 2H), 5.44 (d, J = 8.0 Hz, 1H), 5.12 (s, 2H),

4.78 (dd, J = 13.6, 5.4 Hz, 1H), 4.40−4.28 (m, 2H), 4.19 (t, J = 7.3

Hz, 1H), 3.80 (s, 3H), 3.79 (s, 3H), 3.33−3.20 (m, 2H), 1.65 (s,

Fmoc tert-butyl carbazate (1.24 g, 3.15 mmol, 94%) as a white solid:

mp 93−94 °C; R = 0.4 (EtOAc/hexane 1:4). H NMR (500 MHz,

CDCl ): δ 7.77 (d, J = 7.6 Hz, 2H), 7.62 (d, J = 7.5 Hz, 2H), 7.41 (t, J

7.5 Hz, 2H), 7.34−7.29 (m, 2H), 6.63 (s, 1H), 4.41 (br s, 4H), 4.26

9H); C NMR (125 MHz, CDCl ): δ 171.7, 161.7, 159.2, 155.8,

(br s, 1H), 2.30 (s, 1H), 1.50 (s, 9H); C NMR (125 MHz, CDCl ):

149.7, 144.0, 143.9, 141.4 (2C), 131.7 (2C), 127.8 (2C), 127.2 (2C),

125.3 (2C), 124.6 (2C), 124.5, 124.4, 122.8, 120.1, 119.0, 115.8,

115.4, 115.1, 104.2, 98.7, 83.8, 67.3, 63.4, 55.5 (2C), 54.4, 47.3, 28.3

δ 156.7, 154.4, 143.7 (2C), 141.4 (2C), 128.0 (2C), 127.3 (2C),

25.4 (2C), 120.2 (2C), 82.2, 78.1, 72.9, 69.1, 47.1, 39.9, 28.3 (3C);

HRMS m/z calcd for C H N O [M + Na] , 415.1628; found,

(3C), 28.0; HRMS m/z calcd for C H N O [M + H] , 677.2857;

40 41

415.1621.

found, 677.2859.

A solution of N′-propargyl N′-Fmoc tert-butyl carbazate (1 equiv,

A solution of Fmoc-D-Trp(Boc)-Odmb (1 equiv, 4.2 g, 6.21 mmol)

in MeCN (62 mL) was treated with diethylamine (18.3 mL, 334

mmol), stirred at room temperature for 2 h, and evaporated to a

.1 g, 2.8 mmol) in CH Cl (50 mL) was treated with bubbles of dry

HCl gas for 2 h when complete consumption of the starting material

was observed by TLC. The volatiles were removed under reduced

residue that was puriﬁed by ﬂash chromatography on silica gel

pressure to give N-propargyl ﬂuorenylmethyl carbazate (37, 1.09 g,

eluting with 40% EtOAc in hexane containing 2% triethylamine to

aﬀord H-D-Trp(Boc)-Odmb (2.48 mg, 5.46 mmol, 88%) as an oil: H

.77 mmol, 99%) as a white solid: mp 176 °C; H NMR (500 MHz,

CD OD): δ 7.82 (d, J = 7.6 Hz, 2H), 7.66 (dd, J = 7.5, 0.8 Hz, 2H),

NMR (500 MHz, CDCl ): δ 8.12 (s, 1H), 7.55 (d, J = 7.6 Hz, 1H),

.45−7.39 (m, 2H), 7.36−7.31 (m, 2H), 4.64 (d, J = 6.3 Hz, 2H),

7.47 (s, 1H), 7.33−7.28 (m, 1H), 7.25−7.20 (m, 1H), 7.14 (d, J = 8.1

Hz, 1H), 6.47−6.43 (m, 2.3 Hz, 2H), 5.12 (s, 2H), 3.84 (dd, J = 7.7,

5.0 Hz, 1H), 3.81 (s, 3H), 3.81 (s, 3H), 3.23−3.15 (m, 1H), 3.01−

.37−4.31 (m, 3H), 3.03 (t, J = 2.5 Hz, 1H); C NMR (125 MHz,

CD OD): δ 155.7, 144.5 (2C), 142.7 (2C), 129.1 (2C), 128.3 (2C),

26.0 (2C), 121.1 (2C), 77.1, 76.2, 70.9, 48.0, 39.7; HRMS m/z calcd

2.94 (m, 1H), 1.66 (s, 9H); C NMR (125 MHz, CDCl ): δ 175.3,

for C H N O [M + H] , 293.1285; found, 293.1274.

161.5, 159.2, 149.7, 135.6, 131.6, 130.6, 124.5, 124.3, 122.6, 119.1,

Boc-Ala-azaPra-Ofm (38). A −15 °C solution of Boc-Ala-OH

1.1 equiv, 0.513 g, 2.71 mmol) in THF (10 mL) was treated

116.4, 116.2, 115.4, 104.1, 98.7, 83.6, 62.6, 55.53, 55.50, 54.7, 30.5,

(

28.3 (3C); HRMS m/z calcd for C25

[M + H] , 455.2177;

sequentially with isobutyl chloroformate (1.1 equiv, 0.353 mL, 2.71

mmol) and N-methylmorpholine (2 equiv, 0.542 mL, 4.93 mmol),

stirred for 15 min, treated with a solution of H-azaPra-Ofm (37, 1

equiv, 0.72 g, 2.46 mmol) in THF (15 mL), followed by DIEA (1

equiv, 0.407 mL, 2.46 mmol), and stirred for 1 h. The volatiles were

removed by rotary evaporation. The residue was puriﬁed by column

chromatography eluting with 30−35% EtOAc/hexane. Evaporation of

the collected fractions gave Boc-Ala-azaPra-Ofm (38, 0.82 g, 1.77

mmol, 72%) as a white solid: mp 78−79 °C; R = 0.33 (EtOAc/

hexane 2:3). H NMR (500 MHz, CDCl ): δ 8.39 (br s, 1H), 7.76 (d,

J = 7.5 Hz, 2H), 7.57 (s, 2H), 7.40 (t, J = 7.4 Hz, 2H), 7.34−7.27 (m,

found, 455.2172.

A −78 °C solution of 4-nitrophenyl chlorosulfate (2 equiv, 2.3 g,

9.68 mmol, prepared according to ref 33) in DCM (25 mL) was

treated dropwise with a solution of H-D-Trp(Boc)-Odmb (1 equiv,

2.2 g, 4.84 mmol), 4-nitrophenol (3 equiv, 2.02 g, 14.5 mmol), and

triethylamine (TEA, 6 equiv, 4.04 mL, 29 mmol) in DCM (100 mL)

and stirred for 1.5 h. The cooling bath was removed. After warming to

room temperature with stirring for 1 h, the reaction mixture was

evaporated to a residue that was puriﬁed by ﬂash chromatography

eluting with 40% Et O in petroleum ether to aﬀord fractions

contaminated with 4-nitrophenol. The collected fractions were

evaporated, dissolved in DCM (25 mL), washed with sat. NaHCO

(aq, 3 × 25 mL), dried over MgSO , ﬁltered, and evaporated to aﬀord

sulfamidate 40 (1.08 g, 1.65 mmol, 34%) as a white solid: mp 48−50

H), 4.81 (br s, 1H), 4.56−4.42 (m, 2H), 4.37 (s, 2H), 4.23 (br s,

H), 2.25 (br s, 1H), 1.44 (s, 9H), 1.31 (br s, 3H); ¹³C NMR (125

MHz, CDCl ): δ 180.1, 156.0, 143.7, 143.6 (2C), 141.4 (2C), 128.0

(

2C), 127.3 (2C), 125.2 (2C), 120.2 (2C), 80.9, 77.7, 73.2, 68.5,

8.5, 47.1, 39.4, 28.5 (3C), 17.4; HRMS m/z calcd for C H N O

°C; R

0.30 (40% Et O in petroleum ether); H NMR (500 MHz,

f 2

CDCl ): δ 8.11−8.10 (m, 1H), 8.10−8.09 (m, 1H), 7.50 (d, J = 7.7

[

M + H] , 464.2180; found, 464.2178.

Hz, 1H), 7.45 (s, 1H), 7.35−7.30 (m, 1H), 7.26−7.18 (m, 3H), 7.10

(d, J = 8.0 Hz, 1H), 6.92−6.88 (m, 1H), 6.46−6.41 (m, 2H), 5.63 (d,

J = 8.5 Hz, 1H), 5.17−5.08 (m, 2H), 4.59−4.53 (m, 1H), 3.82 (s,

N-(Boc)Alanine N′-Propargyl Hydrazide (39). A solution of

Boc-Ala-azaPra-Ofm (38, 1 equiv, 0.82 g, 1.77 mmol) in MeCN (15

mL) was treated with diethylamine (27.4 equiv, 3.55 g, 5 mL, 48.5

mmol), stirred at room temperature for 1.5 h, and evaporated to a

residue that was puriﬁed by ﬂash chromatography on silica gel

eluting with a gradient of 60−80% EtOAc in hexanes containing 2%

triethylamine to aﬀord hydrazide 39 (0.265 g, 1.1 mmol, 62%) as a

white solid: mp 75−76 °C; R 0.21 (40% EtOAc in hexanes

containing 2% triethylamine); H NMR (500 MHz, CDCl ): δ 7.96

(

3H), 3.79 (s, 3H), 3.32−3.22 (m, 2H), 1.67 (s, 9H); C NMR (125

MHz, CDCl ): δ 170.6, 162.0, 161.5, 159.4, 154.4, 149.6, 146.0,

132.2, 130.2, 126.4, 125.5, 124.91, 124.86, 122.9, 122.3, 118.9, 115.8,

115.5, 115.2, 113.7, 104.3, 98.7, 84.3, 64.2, 57.2, 55.59, 55.55, 28.8,

28.3 (3C). HRMS m/z calcd for C31

678.1728; found, 678.1720.

Boc-Ala-AsPra-D-Trp(Boc)-Odmb (41). To a microwave vessel

containing a solution of sulfamidate 40 (1 equiv, 0.453 g, 0.691

mmol) in dichloroethane (4.5 mL), hydrazide 39 (1.2 equiv, 0.2 g,

0.829 mmol) was added, followed by TEA (2 equiv, 0.14 g, 0.192 mL,

1.38 mmol), at which point the solution turned yellow. The vessel was

sealed, heated to 60 °C using microwave irradiation for 3 h, and

cooled to room temperature. The volatiles were evaporated. The

11S [M + Na] ,

s, 1H), 4.99 (br s, 1H), 4.71 (s, 1H), 4.16 (s, 1H), 3.61 (s, 2H), 2.23

(

t, J = 2.5 Hz, 1H), 1.44 (s, 9H), 1.37 (d, J = 7.1 Hz, 3H); ¹³C NMR

(125 MHz, CDCl ): δ 172.4, 155.6, 80.6, 79.7, 72.7, 48.9, 41.2, 28.5

(3C), 18.2; HRMS m/z calcd for C H N O [M + H] , 242.1499;

found, 242.1494.

,4-Dimethoxybenzyl N -(Boc)-D-tryptophan 4-Nitrophenyl

Sulfamidate (40). A solution of Fmoc-D-Trp(Boc)-OH (1 equiv, 4.5

g, 8.55 mmol), TBTU (1 equiv, 2.74 g, 8.55 mmol), and DIEA (2

equiv, 2.82 mL, 17.1 mmol) in DMF (20 mL) under an argon

atmosphere was stirred at room temperature for 30 min, treated

dropwise with a solution of 2,4-dimethoxybenzyl alcohol (1.1 equiv,

residue was puriﬁed by ﬂash chromatography on silica gel eluting

with a solution of 40−50% EtOAc in hexane. The collected fractions

were evaporated to a residue, which was dissolved in DCM (25 mL),

washed with sat. NaHCO (3 × 25 mL), dried over MgSO , ﬁltered,

and evaporated to aﬀord azasulfuryl tripeptide 41 (0.29 g, 0.383

.58 g, 9.4 mmol) in DMF (5 mL), stirred at room temperature for 4

h, and diluted with EtOAc. The organic phase was washed with water

2 × 100 mL) and brine (2 × 100 mL), dried over Na SO , ﬁltered,

mmol, 55%) as a white solid: R 0.25 (40% EtOAc in hexane); mp 79

°C; H NMR (500 MHz, CDCl ): δ 8.30 (s, 1H), 8.08 (br s, 1H),

(

7.58 (d, J = 7.7 Hz, 1H), 7.45 (s, 1H), 7.29 (t, J = 7.3 Hz, 1H), 7.22

(t, J = 7.2 Hz, 1H), 7.12 (d, J = 7.8 Hz, 1H), 6.46−6.41 (m, 2H), 5.47

(d, J = 7.0 Hz, 1H), 5.08 (d, J = 2.7 Hz, 2H), 4.95 (br s, 1H), 4.65−

4.58 (m, 1H), 4.32−4.24 (m, 1H), 4.23−4.16 (m, 1H), 4.16−4.08

(m, 1H), 3.82 (s, 3H), 3.80 (s, 3H), 3.27 (t, J = 4.5 Hz, 2H), 2.32 (t, J

= 2.4 Hz, 1H), 1.66 (s, 9H), 1.42 (s, 9H), 1.33 (d, J = 7.1 Hz, 3H);

and evaporated to a residue that was puriﬁed by ﬂash chromatog-

raphy on silica gel eluting with 20% EtOAc in hexane. Evaporation

of the collected fractions aﬀorded Fmoc-D-Trp(Boc)-Odmb (4.97 g,

EtOAc in hexane); H NMR (500 MHz, CDCl ): δ 8.11 (br s, 1H),

.35 mmol, 86%) as a white solid: mp 67−68 °C; R 0.29 (20%

.76 (d, J = 7.6 Hz, 2H), 7.58−7.48 (m, 3H), 7.44 (s, 1H), 7.42−7.36

C NMR (125 MHz, CDCl ): δ 171.8, 171.6, 161.7, 159.2, 149.7,

376

J. Med. Chem. 2021, 64, 9365−9380

Journal of Medicinal Chemistry

Article

31.9, 130.7 126.3, 124.8, 124.6, 122.8, 119.4, 115.8, 115.5, 115.3,

14.4, 104.3, 98.7, 83.8, 81.0, 76.4, 75.0, 63.9, 56.4, 55.5 (2C), 49.2,

and the plate was read using a ﬂuorescence plate reader (TECAN

Saﬁre, λ : 365 nm and λ : 430 nm).

exc

2.0, 28.49, 28.42 (3C), 28.35 (3C), 17.3; HRMS m/z calcd for

Radiolabeling of the Photoactivatable Ligand as the

Radiotracer for the Receptor-Binding Assay. The radioiodina-

tion of Tyr-Bpa-Ala-hexarelin was performed as previously described

in ref 47. Brieﬂy, 10 nmol of Tyr-Bpa-Ala-hexarelin was mixed with

C H N O S [M + H] , 758.3066; found, 758.3061.

H-Ala-Trp(Boc)-D-Phe-Lys(o-NBS, allyl) Rink Amide Resin

(43). In a plastic syringe equipped with a Teﬂon ﬁlter, stopcock, and

stopper, a vacuum-dried Fmoc-Ala-Trp(Boc)-D-Phe-Lys(o-NBS) Rink

amide resin (1 equiv, 4.26 g, 1.5 mmol, synthesized according to the

protocol in ref 11) was swollen in THF (42.6 mL), placed under

argon, treated sequentially with solutions of allyl alcohol (10 equiv,

100 ng of lactoperoxidase and 1 mCi of Na I in a volume of 30 μL

of 0.1 M sodium acetate buﬀer, pH 5.6. The reaction was started by

adding 3 nmol of H O over 5 min at room temperature. This step

was repeated twice with a period of 5 min of incubation for each

addition. The reaction was stopped by diluting the mixture with 1 mL

of 0.1% TFA. The radio-iodinated peptide was puriﬁed by HPLC on a

reverse-phase Vydac C₁₈column with a 60 min linear gradient from

1.02 mL, 15 mmol) in THF (14.2 mL), PPh (5 equiv, 1.97 g, 7.5

mmol) in THF (14.2 mL), and DIAD (5 equiv, 1.49 mL, 7.5 mmol)

in THF (14.2 mL), shaken for 30 min, ﬁltered, and washed

sequentially using 15 s agitations with DMF (×3), MeOH (×3), and

DCM (×3). The resin was vacuumdried. A 0.5 g resin sample was

treated with 20% piperidine in DMF (10 mL) to aﬀord resin 43.

Examination by LCMS of a cleaved resin sample (2−3 mg) showed

complete allylation: LCMS R = 7.94 min [10−90% MeOH (0.1%

FA) in water (0.1% FA) over 9 min and then 10% MeOH (0.1% FA)

in water (0.1% FA) for 5 min]; ESI-MS m/z: calcd for C H N O S

0 to 50% of acetonitrile (0.1% TFA) in H O (0.1% TFA). The

eluted radio-labeled tracer was collected, aliquoted, and stored at −80

Competition Binding Curves. The receptor binding assay of the

photoactivatable ligand was performed as follows: Isolated mem-

branes from rat hearts (50 μg) were incubated in the dark in the

125

presence of 250,000 cpm of [ I]-Tyr-Bpa-Ala-hexarelin in buﬀer (50

[

M-Boc + H] , 775.32; found, 775.3.

mM Tris-HCl pH 7.4 containing 2 mM EGTA and 0.05% Bacitracin)

and increasing concentrations from 0.1 to 10 μM of competition

ligands: hexarelin (as the reference standard), cyclic azapeptides, and

cyclic azasulfurylpeptides. Nonspeciﬁc binding was deﬁned as binding

not displaced by 50 μM of the corresponding peptide. After an

incubation period of 60 min at 22 °C (vortexed every 15 min),

membranes were subjected to irradiation with UV lamps (365 nm)

for 15 min at 4 °C. After centrifugation at 12,000g for 15 min, the

pellets were resuspended in 40 μL of sample buﬀer (62 mM Tris-HCl,

pH 6.8, 2% SDS, 10% glycerol, 15% 2-mercaptoethanol, and 0.05%

bromophenol blue) and proteins were separated on a 7.5% SDS-

PAGE Bio-Rad Mini-Protean electrophoresis system (150 V for 1 h).

The gels resulting from SDS/PAGE were ﬁxed, colored in Coomassie

Brilliant Blue R-250, dried, exposed to a storage phosphor intensifying

screen (Amersham Biosciences), and analyzed using a Typhoon

Phosphorimager (GE Healthcare Life Sciences). Protein bands were

quantiﬁed by densitometry and the covalent binding signal of 87 kDa

was analyzed by densitometry and ImageLab 6.1 software (Bio-Rad

Laboratories Inc.) to set competition curves. The curves were

analyzed using the Graphpad Prism version 7.05 software package.

Modulation of R-FSL-1-Induced Pro-inﬂammatory Cytokine

Boc-Ala-AsPra-D-Trp(Boc)-Ala-Trp(Boc)-D-Phe-Lys(o-NBS,

allyl) Rink Amide Resin (44). 2,4-Dimethoxybenzyl ester 41 (180

mg, 0.238 mmol) was stirred in a solution of 1% TFA in DCM (5

mL) at room temperature for 1 h. The volatiles were evaporated. The

residue was treated with Et O (5 mL) to provide a precipitate that

was ﬁltered. The ﬁltrate was evaporated to aﬀord Boc-Ala-AsPra-D-

Trp(Boc)-OH (42). A solution of tripeptide 42 (1.2 equiv, 126 mg,

.208 mmol) and HOBt (1.2 equiv, 28.1 mg, 0.208 mmol) in DMF (5

mL) was stirred for 5 min, treated with DIC (1.2 equiv, 32.4 μL,

.208 mmol), stirred for 5 min, and transferred to a plastic syringe

equipped with a Teﬂon ﬁlter, stopcock, and stopper containing

swollen H-Ala-Trp(Boc)-D-Phe-Lys(o-NBS, allyl) Rink amide resin

3 (1 equiv, 460 mg, 0.173 mmol) in DMF (5 mL). The resin was

shaken for 18 h at room temperature, ﬁltered, and washed sequentially

using 15 s agitations with DMF (×3), MeOH (×3), and DCM (×3).

Examination by LCMS of a cleaved resin sample (2−3 mg) showed

complete coupling to give azasulfurylpeptide 44: LCMS R = 6.25 min

[

50−90% MeOH (0.1% FA) in water (0.1% FA) over 8.5 min and

then 50% MeOH (0.1% FA) in water (0.1% FA) for 5.5 min]; ESI-

MS m/z: calcd for C H N O S [M-3Boc + H] , 1164.43; found,

66 13 12

164.3.

Boc-Ala-AsPra-D-Trp(Boc)-Ala-Trp(Boc)-D-Phe-Lys(allyl) Rink

Amide Resin (45). Azasulfurylpeptide resin 44 (1 equiv, 300 mg,

.0925 mmol) was swollen in DMF (6 mL) for 30 min, treated with

(

TNF-α, IL-1β) and Chemokine (CCL-2) Release by Cyclic

Azapeptides in a Macrophage Model. The murine RAW264.7

macrophage cell line (American Type Cell Collection, ATCC #TIB-

1) was seeded at 1.5 × 10 cells/well in a 48-well plate (Costar

DBU (10 equiv, 138 μL, 0.925 mmol) and 2-mercaptoethanol (5

equiv, 32.5 μL, 0.462 mmol), shaken for 30 min, ﬁltered, and washed

sequentially using 15 s agitations with DMF (×3), MeOH (×3), and

DCM (×3). Examination by LCMS of a cleaved resin sample (2−3

3548) and weaned overnight in medium (DMEM supplemented

with 1% penicillin/streptomycin) at 37 °C with 5% CO . The next

day, the medium was changed for DMEM-1% penicillin/streptomycin

supplemented with 0.2% BSA. The cells were ﬁrst pretreated with

mg) showed complete removal of the o-NBS group: LCMS R = 7.46

either linear azapeptide [azaTyr ]-GHRP-6 as the reference standard

min [10−90% MeOH (0.1% FA) in water (0.1% FA) over 10 min

−

or cyclic azapeptide at the concentration of 10 M for 30 min and

and then 10% MeOH (0.1% FA) in water (0.1% FA) for 5 min]; ESI-

then treated with R-FSL-1 (300 ng/mL ﬁnal concentration, R-FSL-1,

MS m/z: calcd for C H N O S [M-3Boc + H] , 979.45; found,

63 13

Invivogen #L7022) for 4 h at 37 °C with 5% CO for assessment of

79.4.

Eﬀects of Cyclic Azapeptide and Cyclic Azasulfurylpeptide

on the Overproduction of NO Induced by R-FSL-1 in the RAW

64.7 Macrophage Cell Line. The murine RAW 264.7 macrophage

TNF-α and CCL-2 or 19 h for nitrite assay. At the end of the

incubation period, the media was collected and assayed for the nitrites

4,35

as previously described,

or stored at −80 °C for determination of

cell line (American Type Cell Collection, ATCC #TIB-71) was

TNF-α and CCL-2 levels using ELISA kits (EbioScience #88-7391

seeded at 1.5 × 10 cells/well in DMEM supplemented with penicillin

and #88-7324).

and streptomycin on a 48-well plate and incubated at 37 °C with 5%

For the IL-1β assay, murine bone marrow-derived macrophage cells

(BMA 3.1 A7, kindly provided by Dr. K. Rock, University of

CO . After 2 h, the medium of adhered cells was changed to DMEM-

Pen/Strep containing 0.2% of bovine serum albumin (BSA) and

supplemented with either the cyclic azapeptide or cyclic azasulfur-

ylpeptide at a concentration of 10⁻M. After 1 h of pre-incubation,

the cells were stimulated overnight with R-FSL-1 (Invivogen

Massachusetts Medical School) were seeded at 5 × 10 cells/well in a

48-well plate (Costar #3548) in DMEM medium (supplemented with

1% penicillin/streptomycin and 10% of fetal bovine serum). The next

day, cells were ﬁrst primed with LPS (200 ng/mL) for 2 h and washed

with phosphate-buﬀered saline. Then, cells were preincubated with

cyclic azapeptide for 15 min and stimulated with R-FSL-1 (300 ng/

mL) for 4 h. Thirty minutes before the end of incubation time,

adenosine triphosphate (0.5 mM ATP, Sigma-Aldrich # A2383) was

added to culture media to induce IL-1β release. At the end of the

L7022). Supernatants were collected for ﬂuorescence determination

of nitrite using 2,3-diaminonaphthalene (DAN). Brieﬂy, 25 μL of the

sample was incubated with 0.5 μg of DAN in a 100 μL ﬁnal volume of

phosphate buﬀer (50 mM, pH 7.5) at room temperature in the dark.

After 15 min, the reaction was stopped with 20 μL of NaOH (2.8 N),

377

J. Med. Chem. 2021, 64, 9365−9380

Journal of Medicinal Chemistry

The Supporting Information is available free of charge at

Article

incubation period, the media was collected and stored at −80 °C for

IL-1β analysis using an ELISA kit (EbioScience # 88-7013-88).

The percentages of inhibition (%) of cytokines and chemokine

released by the cycloazapeptide treatment were determined by the

ratio: mean levels induced by R-FSL-1 (control) minus that detected

in the presence of corresponding cycloazapeptides/mean level

induced by R-FSL-1 minus mean level detected in basal condition

X100.

Statistical data were analyzed by analysis of variance (ANOVA)

with post hoc comparisons using Dunnett’s test with statistical

software package and statistical evaluation of correlation between

variables was assessed by a Pearson normality test (Graph Pad Prism

version 7.05). P < 0.05 was considered statistically signiﬁcant.

performed by W.D.L., H.O., S.C., R.G.O., R.M.C., A., M.M.,

and J.Z.; methodology was studied by R.G.O., R.M.C., A.,

M.M., and J.Z.; funding acquisition, project administration,

resources, supervision, and validation were completed by

W.D.L., H.O., and S.C.; writing the original draft was done by

R.G.O., R.C., and W.D.L. The manuscript was written through

contributions of all authors. All authors have given approval to

the ﬁnal version of the manuscript.

Funding

This project was supported by funding from the Natural

Sciences and Engineering Research Council (NSERC) of

Canada (Discovery Research Project #04079), the Canadian

Institutes of Health Research (CIHR; MOP89716 and PPP-

ASSOCIATED CONTENT

sı Supporting Information

https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00642.

■

0157), the NSERC-CIHR for funding the Collaborative

Health Research Project “Azacyclopeptide Modulators of

Immuno-metabolism to Treat Age-Related Macular Degener-

ation” No. 163973, the Minister

e du Dev

Economique, de l’Innovation et de l’Exportation du Gouverne-

ment provincial du Quebec (PSVT3-21396) in partnership

eloppement

Details of synthesis, spectroscopic data, and HPLC

chromatograms for compound identiﬁcation, and

correlations between cyclic azapeptide inhibitory eﬀects

with Amorchem Holdings Inc., and Mperia Therapeutics Inc.

(PDF )

Notes

Molecular formula strings (CSV )

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

AUTHOR INFORMATION

Corresponding Authors

Huy Ong − Faculté de Pharmacie, Université de Montréal,

Montréal, Québec H3C 3J7, Canada ; Phone: 514-343-

■

We thank Dr. Alexandra Furtos-Matei, Marie-Christine Tang,

Louiza Mahrouche, and Simon Comtois-Marotte for assistance

in high-resolution mass spectrometric analyses.

460; Email: huy.ong@umontreal.ca

William D. Lubell − Département de Chimie, Université de

Montréal, Montréal, Québec H3C 3J7, Canada;

orcid.org/0000-0002-3080-2712 ; Phone: 514-343-7339;

Email: william.lubell@umontreal.ca; Fax: 514-343-7586

ABBREVIATIONS

■

azaPra, aza-propargylglycine; AsPra, azasulfurylpropargylgly-

cine; Boc, tert-butyloxycarbonyl; BTC, bis(trichloromethyl)-

carbonate; CCL-2, monocyte chemoattractant protein-1;

CD36, cluster of diﬀerentiation 36; Dab, diaminobutyrate;

Dap, diaminopropionate; DIC, N,N′-diisopropylcarbodiimide;

FA, formic acid; GHRP-6, growth hormone releasing peptide

Authors

Ragnhild G. Ohm − Département de Chimie, Université de

Montréal, Montréal, Québec H3C 3J7, Canada;

; HOBt, 1-hydroxybenzotriazole; IL-1β, interleukin-1β; o-

orcid.org/0000-0002-1928-3819

NBS, o-nitrobenzenesulfonamido; NMR, nuclear magnetic

resonance; NO, nitric oxide; Orn, ornithine; oxLDL, oxidized

low-density lipoproteins; Pra, propargylglycine; R-FSL-1,

ﬁbroblast-stimulating lipopeptide; SAR, structure−activity

relationships; SI, Supporting Information; TBTU, 2-(1H-

benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetraﬂuorobo-

rate; TFA, triﬂuoroacetic acid; TLR, Toll-like receptor;

TNFα, tumor necrosis factor-α

Mukandila Mulumba − Faculté de Pharmacie, Université de

Montréal, Montréal, Québec H3C 3J7, Canada

Ramesh M. Chingle − Département de Chimie, Université de

Montréal, Montréal, Québec H3C 3J7, Canada; Present

Address: Chemical Biology Laboratory, Center for Cancer

Research, National Cancer Institute, National Institutes of

Health, Frederick, Maryland, 21702 United States.;

orcid.org/0000-0003-1175-2454

Ahsanullah − Département de Chimie, Université de Montréal,

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