Influences of Histidine-1 and Azaphenylalanine-4 on the Affinity, Anti-inflammatory, and Antiangiogenic Activities of Azapeptide Cluster of Differentiation 36 Receptor Modulators

Article abstract of DOI:10.1021/acs.jmedchem.7b01209

Azapeptide analogues of growth hormone releasing peptide-6 (GHRP-6) exhibit promising affinity, selectivity, and modulator activity on the cluster of differentiation 36 receptor (CD36). For example, [A¹, azaF⁴]- and [azaY⁴

Full text of DOI:10.1021/acs.jmedchem.7b01209

Subscriber access provided by Gothenburg University Library
Article
Influences of Histidine-1 and Azaphenylalanine-4 on the
Affinity, Anti-inflammatory and Antiangiogenic Activities of
Azapeptide Cluster of Differentiation 36 Receptor Modulators
Kelvine Chignen Possi, Mukandila Mulumba, Samy Omri, Yesica Garcia-
Ramos, Houda Tahiri, Sylvain Chemtob, Huy Ong, and William D. Lubell
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01209 • Publication Date (Web): 13 Oct 2017
Downloaded from http://pubs.acs.org on October 15, 2017
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 41
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Influences of Histidineꢀ1 and Azaphenylalanineꢀ4 on
the Affinity, Antiꢀinflammatory and Antiangiogenic
Activities of Azapeptide Cluster of Differentiation 36
Receptor Modulators
Kelvine Chignen Possi †, Mukandila Mulumba §, Samy Omri ‡, Yesica GarciaꢀRamos †
Sylvain Chemtob ‡, Huy Ong §, William D. Lubell †*
,
Houda Tahiri ‡,
‡
§
†Département de Chimie, Département de Pédiatrie and Faculté de Pharmacie, Université de
Montréal, C.P. 6128, Succursale, CentreꢀVille, Montréal, Québec H3C 3J7, Canada
ABSTRACT Azapeptide analogs of growth hormone releasing peptideꢀ6 (GHRPꢀ6) exhibit promising
affinity, selectivity and modulator activity on the cluster of differentiation 36 receptor (CD36). For
example, [A1, azaF4]ꢀ and [azaY4]ꢀGHRPꢀ6 (1a and 2b) were previously shown to bind selectively to
CD36, and exhibited respectively significant antiangiogenic and slight angiogenic activities in a
microvascular sprouting assay using choroid explants. The influences of the 1ꢀ and 4ꢀposition residues
on the affinity, antiꢀinflammatory and antiangiogenic activity of these azapeptides have now been
studied in detail by the synthesis and analysis of a set of 25 analogs featuring Ala1 or His1 and a variety
aromatic side chains at the azaꢀamino acid residue in the 4ꢀposition. Although their binding affinities
differed only by a factor of 17, the analogs exhibited significant differences in ability to modulate
production of nitric oxide (NO) in macrophages and choroidal neovascularization.
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 41
1
2
3
4
5
6
7
INTRODUCTION
8
9
The cluster of differentiation 36 receptor (CD36) is a glycoprotein expressed in the membranes of
various cells, including red blood cells and macrophages.1,2 Classified as a class B scavenger receptor,
CD36 acts in normal cellular function by binding to a variety of endogenous ligands, including
oxidized lowꢀdensity lipoproteins and thrombospondin.2 Moreover, CD36 is coꢀexpressed with the
Tollꢀlike receptorꢀ2 (TLRꢀ2)–TLRꢀ6 complex that plays a significant role in innate immunity.3,4 In the
light of such interactions, CD36 is a promising therapeutic target for the treatment of various
indications including atherosclerosis,5 angiogenesis,6 and ageꢀrelated macular degeneration.7,8,9,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Certain synthetic peptide ligands, such as growth hormone releasing peptideꢀ6 (GHRPꢀ6, HisꢀDꢀTrpꢀ
AlaꢀTrpꢀDꢀPheꢀLysꢀNH2), exhibit binding affinity for CD36, and also serve as agonists of the growth
hormone secretagogue receptorꢀ1a (GHSꢀR1a) and elicit growth hormone release upon binding to this
Gꢀprotein coupled receptor.10,11 To achieve selectivity towards CD36, we have found that introduction
of semicarbazide residues within the GHRPꢀ6 sequence can provide azapeptide analogs with similar
binding affinity towards CD36 as the GHRPꢀ6 prototype, but without binding affinity to the GHSꢀR1a
receptor.10,12 Their ligandꢀreceptor interaction has also been supported by photoaffinity13 and surface
plasmon resonance studies.14 In particular, respective replacement of azaPhe and azaTyr for the Trp4
residue of GHRPꢀ6 in [A1, azaF4]ꢀ and [azaY4]ꢀGHRPꢀ6 (1a and 2b) preserved binding affinity to
CD36 and decreased by a thousandꢀfold binding affinity to GHSꢀR1a.8 Conformational analysis of
azaꢀGHRP analogs, such as 1a and 2b, using circular dichroism (CD) and nuclear magnetic resonance
(NMR) spectroscopy in solution indicated that in contrast to the random coil conformation presented
by GHRPꢀ6, the azapeptides exhibited curve shapes, solventꢀshielded amide NHs, and throughꢀspace
2
ACS Paragon Plus Environment

Page 3 of 41
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
rotating frame nuclear Overhauser effects (ROE) indicative of turn conformations, which may be
responsible for their selectivity and affinity.10,12 The turn geometry of the azapeptides is expected to
arise from the combination of urea planarity and lone pairꢀlone pair repulsion between the adjacent
nitrogen of the diacylhydrazine in the semicarbazide residue.15,16
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Examination of the influence of azapeptide GHRPꢀ6 analogs on neovascularization relative to control
in a microvascular sprouting assay on mouse choroidal explants demonstrated that 1a and 2b exhibited
divergent behavior.10,17 Relative to control, [A1, azaF4]ꢀ and [azaY4]ꢀGHRPꢀ6 (1a and 2b) caused
respectively a slight increase and significant reduction in neovascularization. Considering the similar
CD curve shapes for 1a and 2b, their common conformational preferences were inconsistent with their
divergent behavior on angiogenesis. The combination of the CD36 selectivity and divergent activity
on neovascularization in the microvascular sprouting assay made azapeptides 1a and 2b interesting
leads for exploring the importance of the Nꢀterminal and 4ꢀposition residues on azapeptide biological
activity.
Here, we report the synthesis and the biological activity of twentyꢀfive azapeptide GHRPꢀ6 analogs in
which alanine and histidine were respectively introduced at the first position and various aromatic side
chains were installed on the azaꢀamino acid at the fourth position in the sequence. In addition to their
CD36 binding affinity and their activities on vascular sprouting, the 25 azaꢀGHRPꢀ6 analogs were first
examined for potential to curb macrophageꢀdriven inflammation. Capacity to modulate CD36
mediated overproduction of nitric oxide (NO) in macrophage cells was measured after treatment with
the TLRꢀ2ꢀagonist fibroblastꢀstimulating lipopeptide (RꢀFSLꢀ1). A mediator of inflammatory
responses in macrophages, NO is used against pathogenic microbes, but may damage host tissues.18
Nitric oxide can be effectively measured as nitrite as a marker of the modulation of the inflammatory
pathway. Moreover, in studies of cyclic azapeptides,19 reduction of nitrite production was correlated
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 41
1
2
3
4
5
6
7
8
9
with binding affinity, as well as decreased production of proꢀinflammatory cytokines and chemokines
in macrophages. The important role of CD36 in the phagocytosis of debris (e.g., modified lipoproteins
and drusen),20 and in the activation of macrophages,21,22 indicate that targeting macrophage signaling
pathways may offer promise for countering the chronic inflammation underlying diseases such as
cancer, atherosclerosis and ageꢀrelated macular degeneration.23 Assessment of their ability to reduce
NO overproduction was thus employed for selecting modulators that were subsequently examined for
their binding affinity and their activity on neovascularization.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
RESULTS AND DISCUSSION
Chemistry. Replacement of the Trp4 residue in the GHRPꢀ6 sequence for azaPhe derivatives was
accomplished using two different approaches involving respectively the alkylation of a resinꢀbound
azaꢀglycine semicarbazone with different aromatic alkyl halides, and the use of a solutionꢀphase
method in which azaꢀdipeptide building blocks were assembled prior to incorporation into the peptide
sequence by solidꢀphase synthesis (Scheme 1).11,24 In addition, azaꢀ1,2,3ꢀtriazoleꢀ3ꢀalanine4ꢀGHRPꢀ6
(3) was prepared by a solidꢀphase route featuring copperꢀcatalyzed 1,3ꢀdipolar cycloaddition of
trimethylsilylazide onto an azaꢀprogargylglycine residue (Scheme 2).25 Key for the introduction of the
azaꢀresidue in all of these sequences was the use of the soꢀcalled submonomer azapeptide synthesis in
which an activated methylidene carbazate was used to acylate an amino acid residue to provide a
semicarbazone intermediate that could be selectively deprotonated and alkylated.26 Subsequent
removal of the hydrazone and acylation of the resulting semicarbazide was then used to elongate the
azapeptide sequence.
Benzhydrylidene carbazate 4 was synthesized by activating benzophenone hydrazone with N,N′ꢀ
disuccinimidyl carbonate (DSC) and coupled respectively to DꢀphenylalaninylꢀNεꢀ(Boc)lysinamide
4
ACS Paragon Plus Environment

Page 5 of 41
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
linked to Rink amide resin and tertꢀbutyl Dꢀphenylalaninate to obtained azaꢀglycine resinꢀbound
tripeptide 5 and dipeptide 7 (Scheme 1).27 As previously reported, application of DSC instead of
phosgene or paraꢀnitrophenyl chloroformate minimized formation of side products such as symmetric
urea to provide better yields of azaꢀglycinyl dipeptide.28,29
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 1. Synthesis of [A1, azaF4]ꢀGHRPꢀ6 and [azaF4]ꢀGHRPꢀ6 analogs 1 and 2.
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 41
1
2
3
4
5
6
7
8
9
Ph
H
N
O
NHBoc
H-D-Phe-Lys(Boc)-NH
O
H
N
H
H
N
N
NH2
Ph
DSC,
DCM
O
Ph
DMF, 0°C to rt
N
N
N
N
N
H
O
O
O
Ph
Ph
Ph
Ph
4
5
O
40% aq. Et4NOH,
THF, rt; BrCH2Ar
Ph
CO2t-Bu
NH
1. CF3CO2H, CH2Cl2
2. HBTU, DIEA, DMF,
H-Lys(Boc)-NH
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
H-D-Phe-Ot-Bu
DMF, 0°C to rt
Ar
O
O
O
NHBoc
Ph
H
N
H
N R
Ph
N
N
Ph
N
N
N
H
O
Ph
Ph
6
40% aq. Et4NOH,THF, rt;
ArCH2Br
7: R = H
8: R = CH2Ar
NH2OH.HCl,
pyridine, 60°C
Fmoc-Ala-OH,
DIC,
DCM, DMF
Ar
Ar
O
O
O
NHBoc
O
NHBoc
H
N
H
H
H
FmocHN
N
N
N
N
N
N
N
H
H2N
N
H
H
CH3
O
O
O
Ph
Ph
10
9
Ro2
Elongation
Rp
Deprotection
Cleavage
Ar =
Ro1
Ro1 Ro2
Rm
Rm Rp
a
b
c
d
e
f
g
h
i
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OH
H
H
OH
F
Cl
Br
Ph
O
R
O
CH3
O
O
H
H
N
H
N
N
H2N
N
N
N
H
NH2
NH2
I
H
O
O
OMe
OiPr
OnPr
NO2
CF3
H
F
F
H
OMe
Cl
NH
Ar
j
k
l
N
H
H
1: R = H
2: R =
Me Me
Me Me
F
H
Cl
OMe
N
H
m
n
o
p
q
F
H
Cl
Cl
H
Resinꢀbound azaꢀglycine 5 was treated with tetraethylammonium hydroxide (40% aq. solution) in THF
using the respective alkyl halide: benzyl bromide, and 4ꢀtertꢀbutyldimethylsilyloxyꢀ, 4ꢀbromoꢀ, 4ꢀ
6
ACS Paragon Plus Environment

Page 7 of 41
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
fluoroꢀ, 4ꢀchloroꢀ, 4ꢀiodo, 4ꢀnitroꢀ, 4ꢀtrifluoromethylꢀ, 4ꢀmethoxyꢀ, 2,6ꢀdimethylꢀ, 2,6ꢀdimethylꢀ4ꢀ
fluoroꢀ, 2,4,6ꢀtifluoroꢀ and 3ꢀ(tertꢀbutyldimethylsilyloxy)ꢀbenzyl bromides or 4ꢀmethoxybenzyl
chloride. The respective azatripeptides 6 were ascertained to be produced in 20ꢀ90% conversion by
cleavage of a resin aliquot with a freshly made solution of TFA/H2O/triethylsilane (TES; 95/2.5/2.5),
removal of the resin by filtration, and analysis of the residue by LCꢀMS.17
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Alternatively, in solution, azadipeptide 7 was treated with tetraethylammonium hydroxide (40% aq.
solution) in THF and alkylated with the respective benzyl bromide: 4ꢀpropoxyꢀ, 4ꢀisopropoxyꢀ, 2,6ꢀ
dichloꢀ4ꢀmethoxyꢀ and 2,4ꢀdichloroꢀ6ꢀmethoxybenzyl bromides. 2,6ꢀDichloroꢀ4ꢀmethoxyꢀ and 2,4ꢀ
dichloroꢀ6ꢀmethoxybenzyl bromides were synthesized by chloromethylation of 5,5ꢀdichloroanisole,
followed by hydrolysis to provide separable benzyl alcohols,30 and conversion to the respective
bromides using phosphorus tribromide in dichloromethane [see supporting information (SI)].31 Azaꢀ
phenylalanine dipeptides 8h, 8i, 8p and 8q were isolated in 90ꢀ96% yields respectively after
chromatography. The solutionꢀphase approach facilitated monitoring of the alkylation and provided
building blocks for incorporation onto the resinꢀbond peptide. In the cases of 8h and 8i, the alkylation
was subsequently performed on resin as described above.
Esters 8p and 8q were quantitatively converted to the corresponding acids with 50% trifluoroacetic
acid in dichloromethane. Although benzhydrylideneꢀazaꢀ4ꢀethoxyꢀ and 2,6ꢀdimethylꢀ4ꢀmethoxyꢀ
phenylalaninylꢀDꢀphenylalanine tertꢀbutyl esters were also synthesized in 99% and 96% yields
respectively, ester solvolysis with TFA caused concomitant side chain removal to give the
corresponding azaꢀglycinylꢀphenylalanines.15 Azaꢀdipeptides were coupled onto Nεꢀ(Boc)ꢀlysinamide
Rink amide resin using 2ꢀ(1Hꢀbenzotriazolꢀ1ꢀyl)ꢀ1,1,3,3ꢀtetramethyluronium hexafluorophosphate
(HBTU) and Hünig's base in DMF to furnish azaꢀtripeptides 6p and 6q.
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 41
1
2
3
4
5
6
7
8
9
Semicarbazone resins 6 were converted to their respective semicarbazides 9 by sonication with
hydroxylamine hydrochloride in pyridine at 60°C for 16h.17 Acylation of semicarbazide resin 9
swollen in DMF was performed using the symmetric anhydride that was formed on treatment of Nꢀ
(Fmoc)ꢀalanine with diisopropylcarbodiimide (DIC) in dichloromethane. Azaꢀtetrapeptides 10 were
subsequently elongated by standard Fmocꢀbased solidꢀphase peptide synthesis.32
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Final peptides were cleaved from the resin on treatment with TFA:H2O:TES (95: 2.5: 2.5 v/v/v) for 2h.
The resin was filtered and washed several times with TFA, and the combined filtration and washings
were concentrated, and diluted with Et2O to precipitate azapeptides, that were dissolved in 1:9
MeOH/H2O, freezeꢀdried, and analyzed and purified by HPLC on a C18 column (Table 1). Low yields
of [Oꢀnꢀ and iꢀpropylꢀazaY4]ꢀGHRPꢀ6 analogs 2h and 2i were obtained likely because of loss of the
respective 4ꢀalkyloxybenzyl carbocations during resin cleavage.33
Table 1: Purity, retention times and mass of [azaF4]ꢀGHRPꢀ6 analogues
Compounds
RT (min ) in
CH3OH CH3CN
Purity at
214 nm
MS [M+1]
m/z (calc.)
m/z (obs.)
778.4 10
1a
1b
1d
1e
1f
HꢀAlaꢀDꢀTrpꢀAlaꢀazaPheꢀDꢀPheꢀLysꢀNH2
10.6
13.4a
7.3h
15.9
4.0d
7.0i
˃ 99
778.4
HꢀAlaꢀDꢀTrpꢀAlaꢀazaTyrꢀDꢀPheꢀLysꢀNH2
˃ 99
˃ 99
˃ 99
˃ 99
˃ 99
˃ 99
˃ 99
˃ 99
˃ 99
˃ 99
˃ 99
˃ 99
785.4093
825.3574
869.3069
895.3110
821.4069
836.3814
837.4018
835.4362
851.4311
853.4268
869.3972
457.1770
785.4103
825.3584
869.3052
895.3105
821.4052
836.3809
837.4032
835.4374
851.4322
853.4269
869.3967
457.1766
HꢀAlaꢀDꢀTrpꢀAlaꢀaza (4ꢀchloro)PheꢀDꢀPheꢀLysꢀNH2
HꢀAlaꢀDꢀTrpꢀAlaꢀaza(4ꢀbromo)PheꢀDꢀPheꢀLysꢀNH2
HꢀAlaꢀDꢀTrpꢀAlaꢀaza(4ꢀiodo)PheꢀDꢀPheꢀLysꢀNH2
HꢀAlaꢀDꢀTrpꢀAlaꢀaza(4ꢀmethoxy)PheꢀDꢀPheꢀLysꢀNH2
HꢀAlaꢀDꢀTrpꢀAlaꢀaza(4ꢀnitro)PheꢀDꢀPheꢀLysꢀNH2
HꢀAlaꢀDꢀTrpꢀAlaꢀaza(4ꢀtrifluoromethyl)PheꢀDꢀPheꢀLysꢀNH2
HꢀHisꢀDꢀTrpꢀAlaꢀazaPheꢀDꢀPheꢀLysꢀNH2
7.8h
5.4j
7.8m
13.4p
7.2s
8.2i
1g
1j
9.0j
13.9 t
11.8j
15.1b
8.2c
5.1e
4.8g
6.1k
1k
2a
2b
2c
2d
2e
11.3
8.9 a
8.2 a
11.8a
6.7f
HꢀHisꢀDꢀTrpꢀAlaꢀazaTyrꢀDꢀPheꢀLysꢀNH2
HꢀHisꢀDꢀTrpꢀAlaꢀaza(4ꢀfluoro)PheꢀDꢀPheꢀLysꢀNH2
HꢀHisꢀDꢀTrpꢀAlaꢀaza(4ꢀchloro)PheꢀDꢀPheꢀLysꢀNH2
HꢀHisꢀDꢀTrpꢀAlaꢀaza(4ꢀbromo)PheꢀDꢀPheꢀLysꢀNH2
3.5h
8
ACS Paragon Plus Environment

Page 9 of 41
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
2f
2g
2h
2i
HꢀHisꢀDꢀTrpꢀAlaꢀaza(4ꢀiodo)PheꢀDꢀPheꢀLysꢀNH2
HꢀHisꢀDꢀTrpꢀAlaꢀaza(4ꢀmethoxy)PheꢀDꢀPheꢀLysꢀNH2
HꢀHisꢀDꢀTrpꢀAlaꢀaza(4ꢀisopropoxy)PheꢀDꢀPheꢀLysꢀNH2
HꢀHisꢀDꢀTrpꢀAlaꢀaza(4ꢀnꢀpropoxy)PheꢀDꢀPheꢀLysꢀNH2
HꢀHisꢀDꢀTrpꢀAlaꢀaza(4ꢀnitro)PheꢀDꢀPheꢀLysꢀNH2
HꢀHisꢀDꢀTrpꢀAlaꢀaza(4ꢀtrifluoromethyl)PheꢀDꢀPheꢀLysꢀNH2
HꢀHisꢀDꢀTrpꢀAlaꢀaza(2,6ꢀmethyl)PheꢀDꢀPheꢀLysꢀNH2
8.4l
7.6n
5.3f
4.7f
7.6r
14.0p
5.3u
5.5f
6.2f
5.5f
5.0f
6.4f
7.7w
5.7d
6.9o
3.8g
4.2q
3.9c
4.9d
6.2v
5.6g
6.0g
2.6g
6.0g
6.3g
8.8x
˃ 95
˃ 99
˃ 95
˃ 99
˃ 99
˃ 99
˃ 99
˃ 99
˃ 99
˃ 99
˃ 99
˃ 99
˃ 99
983.3148
865.4468
893.4781
447.2427
880.4213
903.4236
885.4494
441.2327
445.2076
426.2192
467.18881
467.18881
826.4220
983.314y
865.4467
893.4764
447.2442z
880.4196
903.4238
885.4489 y
441.2348
445.2092z
426.2208z
467.1902z
467.1900z
826.4211
2j
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2k
2l
2m HꢀHisꢀDꢀTrpꢀAlaꢀaza(4ꢀfluoroꢀ2,6ꢀmethyl)PheꢀDꢀPheꢀLysꢀNH2
2n
2o
2p
2q
3
HꢀHisꢀDꢀTrpꢀAlaꢀaza(2,4,6ꢀtrifluoro)PheꢀDꢀPheꢀLysꢀNH2
HꢀHisꢀDꢀTrpꢀAlaꢀaza(3ꢀhydroxy)PheꢀDꢀPheꢀLysꢀNH2
HꢀHisꢀDꢀTrpꢀAlaꢀaza(2,6ꢀdichloroꢀ4ꢀmethoxy)PheꢀDꢀPheꢀLysꢀNH2
HꢀHisꢀDꢀTrpꢀAlaꢀaza(2,4ꢀdichloroꢀ6ꢀmethoxy)PheꢀDꢀPheꢀLysꢀNH2
HꢀHisꢀDꢀTrpꢀAlaꢀazaꢀ1,2,3ꢀtriazoleꢀ3ꢀAlaꢀDꢀPheꢀLysꢀNH2
Isolated purity ascertained by LCꢀMS using gradients of XꢀY% A [H2O (0.1% FA)/MeOH (0.1% FA)] or B [H2O (0.1%
a
b
c
d
e
f
FA)/MeCN (0.1% FA)] over Z min: 20ꢀ50% B/20; 15ꢀ30% B/20; 10ꢀ40% B/14; 10ꢀ50% B/14; 5ꢀ50% B/14; 10ꢀ80%
A/14; g10ꢀ80% B/14; h25ꢀ50% A/14; i25ꢀ45% B/14, j20ꢀ40% B/14; k25ꢀ35% B/14; l30ꢀ50% A/20; m40ꢀ60% A/20; n25ꢀ45%
o
p
q
r
s
t
u
A/14; 10ꢀ35% B/14 min; 10ꢀ50% A/20; 5ꢀ80% B/14; 10ꢀ30% A/14; 35ꢀ55% A/20; 20ꢀ30% A/14min 30ꢀ60% A/14
min, v15ꢀ40% B/12, w0ꢀ50% A/14; x0ꢀ30% B/14. y[M+Na]+; z[M+2H]+/2.
[Azaꢀ1,2,3ꢀtriazoleꢀ3ꢀalanine4]ꢀGHRPꢀ6 (3) was synthesized by a route featuring propargylation of
azapeptide resin 5 and copper catalyzed azide alkyne cycloaddition (Scheme 2).25,34 Benzhydrylidene
azaꢀpropargylglycine 11 was prepared by alkylation of resin 5 swollen in THF using
tetraethylammonium hydroxide (40% aq. solution) and propargyl bromide.28 Azaꢀ1,2,3ꢀtriazoleꢀ3ꢀ
alanine 12 was obtained by reacting azaꢀpropargylglycine resin 11 with azidotrimethylsilane in the
presence of copper iodide, Lꢀproline and trimethylamine in DMF, followed by triazole protection with
(Boc)2O in THF.25,35ꢀ37 Azaꢀtriazoleꢀalanine4ꢀGHRPꢀ6 3 was isolated after removal of the
benzhydrylidene protection, acylation of the resulting semicarbazide 13, azapeptide elongation,
cleavage and purification, all which were performed as discussed above for the synthesis of analogs 2
(Table 1).
9
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 41
1
2
3
4
Scheme 2: Synthesis of [azaꢀ1,2,3ꢀtriazoleꢀ3ꢀalanine]ꢀGHRPꢀ6 (3)
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Biological Results. The structureꢀactivity relationships dictating the divergent behavior of [A1,
azaF4]ꢀ and [azaY4]ꢀGHRPꢀ6 (1a and 2b) on angiogenesis were probed by the synthesis and
evaluation of twentyꢀfive azaPhe4ꢀGHRPꢀ6 analogs possessing respectively His1 or Ala1. In
addition to the relevance of the imidazole at the one position, modifications of the azaPhe4 side
chain were performed to explore the influence of location, size, electronegativity, polarizability
and hydrogen bonding potential of the aromatic ring substituents. Moreover, analogs possessing
2,6ꢀdimethyl substituents were prepared to study the influence of the χꢀdihedral angle on
10
ACS Paragon Plus Environment

Page 11 of 41
Journal of Medicinal Chemistry
1
2
activity.38 The scope of azaꢀresidues with electron rich aromatic rings was however limited due
loss of the side chain as a stabilized carbocation under the acidic conditions employed for
peptide cleavage.33
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Initially, the 25 analogues were first examined for their ability to modulate NO overproduction in
macrophage cells elicited by the TLRꢀ2 agonist RꢀFSLꢀ1.19 Based on their efficacy in reducing
NO production, the top 15 analogs were then selected and their binding affinity for CD36 was
examined using a competition binding assay against photoactivatable [125I]ꢀTyrꢀBpaꢀAlaꢀ
hexarelin (HꢀHisꢀDꢀ2ꢀmethylꢀTrpꢀAlaꢀTrpꢀDꢀPheꢀLysꢀNH2) as radiotracer.10,19 The influence on
angiogenesis of the top 15 analogues was finally examined to document specific structureꢀ
activity relationships (SAR) using a modified bioassay from that previously reported.10 The
sclera/choroid/RPE complex was employed in the bioassay to document their modulatory effect
on angiogenic activity. This angiogenic bioassay that uses the total choroid/RPE complex is
more physiologically relevant than that which employs the choroid alone, because the
RPE/choroid interaction is essential for the regulation, maintenance and integrity of the
choroid.39,40
The ability of the [azaF4]ꢀGHRPꢀ6 analogs to inhibit NO production induced by the TLRꢀ2
agonist RꢀFSLꢀ1 was performed in both RAW and J774 macrophage cell lines (Figure 1). The
[A1, azaF4]ꢀGHRPꢀ6 analogs 1 exhibited an inhibitory effect over RꢀFSLꢀ1ꢀinduced nitrite
production in macrophages except for the 4ꢀiodo and 4ꢀtrifluoromethyl analogs 1f and 1k
(Figures 1A and 1B). Similarly, many of the [azaF4]ꢀGHRPꢀ6 analogs 2 exhibited noteworthy
inhibition of RꢀFSLꢀ1ꢀinduced NO production relative to the controls, except for the parent
analog 2a, and its 4ꢀiodo (2f), 4ꢀisoꢀpropoxy (2h), 4ꢀnitro (2j), 4ꢀtrifluoromethyl (2k), 2,4,6ꢀ
trifluoro (2n), and dichloroꢀmethoxy (2p and 2q) counterparts, which did not show significant
11
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 41
1
2
inhibitory effect. In addition, azaꢀtriazoleꢀalanine4ꢀGHRPꢀ6 3 had no influence on NO
3
4
5
6
production.
7
8
9
A
B
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
C
D
Figure 1: Effects of [azaF4]ꢀGHRPꢀ6 analogues on the overproduction of NO induced by the
TLRꢀ2 agonist RꢀFSLꢀ1 in J774 (A) and RAW (BꢀD) macrophage cell line. *, P < 0.05; **, P <
0.01 and ***, P < 0.001 vs RꢀFSLꢀ1 control
Analogs possessing Ala1 and His1 reduced NO production contingent on the azaPhe4 side chain,
with a preference for small electronegative substituents. The location of the hydroxyl substituent
in the paraꢀ or metaꢀposition (e.g., 2b and 2o) did not influence ability to reduce NO production.
Moreover, 2,6ꢀdimethyl substituents, which may restrain χꢀspace favoured reduction of NO
12
ACS Paragon Plus Environment

Page 13 of 41
Journal of Medicinal Chemistry
1
2
production (e.g., 2l and 2m). Strikingly, the parent azaPhe4 analogs 1a and 2a exhibited
divergent behavior: the former exhibited significant capacity to reduce NO production and the
latter was without activity. In addition to significantly active analogs, such as 1a, the binding
affinity and activity on vascularization of 2a were thus further examined.
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The ability of [azaF4]ꢀGHRPꢀ6 analogs to compete with the radiolabeled [125I]ꢀTyrꢀBpaꢀAlaꢀ
hexarelin for CD36 binding was tested on purified membranes from rat heart as source of CD36
(Figures 2 and 3). The binding affinity differed only by a factor of 17 among these analogs
having relatively similar structures. Among the best CD36 binders, [azaY4]ꢀGHRPꢀ6 (2b) and
its Oꢀnꢀpropyl counterpart 2i displayed similar affinity as hexarelin, with IC50 values of 1.93 and
1.65 µM, respectively (Table 2 and Figures 3A and 3C).
A
B
Figure 2: Competition curves of [A1, azaF4]ꢀGHRPꢀ6 analogs 1 for binding of photoactivable
[125I]ꢀTyrꢀBpaꢀAlaꢀhexarelin in rat cardiac membrane
13
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 41
1
2
3
4
A
B
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
C
Figure 3: Competition binding curves of [azaF4]ꢀGHRPꢀ6 analogs 2 for binding of photoactivable
[125I]ꢀTyrꢀBpaꢀAlaꢀhexarelin in rat cardiac membrane
The imidazole at the 1ꢀposition enhanced typically CD36 affinity by a factor of 2ꢀ5 as shown in
comparisons of the Ala1 and His1 analogs possessing azaPhe4, azaTyr4 and aza(pꢀBr)Phe4
residues 1a,b,e and 2a,b,e (Table 2). On the other hand, in the case of aza(pꢀCl)Phe4, the Ala1
analog 1d exhibited 2ꢀfold better affinity than its His1 counterpart 2d. The nature of the paraꢀ
substituent influenced affinity differently in the Ala1 (Cl > OH > Br > H) and His1 (OnꢀPr > OH
> Br > F > OMe > Cl > H) series, but typically improved binding affinity. Moreover, the ring
position influenced dramatically affinity as observed in the nearly 15ꢀfold difference in the
affinities of the para and meta hydroxyl analogs 2b and 2o (IC50 1.9 vs 28.2 µM). Finally, the
favourable impact on binding of the 2,6ꢀdimethyl substituents, which may restrict motion about
14
ACS Paragon Plus Environment

Page 15 of 41
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
the side chain χꢀdihedral angles, was tempered by the presence of a paraꢀsubstituent. Adding
2,6ꢀdimethyl substituents gave a 2ꢀfold enhancement in binding affinity in azaPhe4 analogs (2l vs
2a; IC50 5.51 vs 10.6 µM), yet had little impact on the aza(pꢀF)Phe4 derivatives (2m vs 2c; IC50
5.58 vs 6.06 µM).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 2: IC50 binding values for CD36 receptors and angiogenic activity of selected analogues
Number
Azapeptides
IC50 (µM)
factor
Angiogenic
activity
Reference
Hexarelin
18.1
17.1
14.8
1.56 ± 0.25
1.65 ± 0.33
1,90 ± 0.42
2i
HꢀHisꢀDꢀTrpꢀAlaꢀaza(4ꢀnꢀpropoxy)PheꢀDꢀPheꢀLysꢀNH2
(+)
(–)
2b
HꢀHisꢀDꢀTrpꢀAlaꢀazaTyrꢀDꢀPheꢀLysꢀNH2
2e
1d
2l
HꢀHisꢀDꢀTrpꢀAlaꢀaza(4ꢀbromo)PheꢀDꢀPheꢀLysꢀNH2
HꢀAlaꢀDꢀTrpꢀAlaꢀaza(4ꢀchloro)PheꢀDꢀPheꢀLysꢀNH2
HꢀHisꢀDꢀTrpꢀAlaꢀaza(2,6ꢀmethyl)PheꢀDꢀPheꢀLysꢀNH2
HꢀHisꢀDꢀTrpꢀAlaꢀaza(4ꢀfluoroꢀ2,6ꢀmethyl)PheꢀDꢀPheꢀLysꢀNH2
HꢀHisꢀDꢀTrpꢀAlaꢀaza(4ꢀfluoro)PheꢀDꢀPheꢀLysꢀNH2
HꢀHisꢀDꢀTrpꢀAlaꢀaza(4ꢀmethoxy)PheꢀDꢀPheꢀLysꢀNH2
HꢀHisꢀDꢀTrpꢀAlaꢀaza(4ꢀchloro)PheꢀDꢀPheꢀLysꢀNH2
HꢀAlaꢀDꢀTrpꢀAlaꢀazaTyrꢀDꢀPheꢀLysꢀNH2
7.8
5.7
5.1
5.0
4.6
3.7
3.4
3.1
2.8
(t–)
(t–)
(na)
(na)
(–)
3.60 ± 1.34
4.94 ± 1.64
5.51 ± 1.27
5.58 ± 2.99
6.06 ± 2.81
7.51 ± 1.42
8.36 ± 3.74
9.19 ± 2.56
10.06 ± 3.32
2m
2c
2g
2d
1b
2a
(t–)
(t+)
(na)
(na)
HꢀHisꢀDꢀTrpꢀAlaꢀazaPheꢀDꢀPheꢀLysꢀNH2
1e
1a
HꢀAlaꢀDꢀTrpꢀAlaꢀaza(4ꢀbromo)PheꢀDꢀPheꢀLysꢀNH2
HꢀAlaꢀDꢀTrpꢀAlaꢀazaPheꢀDꢀPheꢀLysꢀNH2
2.2
1.5
(na)
(na)
12.5 ± 4.53
19.0 ± 4.46
2o
HꢀHisꢀDꢀTrpꢀAlaꢀaza(3ꢀhydroxy)PheꢀDꢀPheꢀLysꢀNH2
1.0
(na)
28.2 ± 11.3
(+) = proꢀangiogenic; (–) = antiꢀangiogenic; (na) = did not affect chroidal vascular growing; (t+) = proꢀangiogenic
trend; (t–) = antiꢀangiogenic trend
Examination of the effect of azapeptides on angiogenic activity on the sclera/choroid/RPE
complex illustrated the importance of the combination of a small electronegative paraꢀ
substituent on the azaPhe4 residue and a His1 residue for reducing angiogenic sprouting (Figure
15
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 41
1
2
3
4
5
6
7
8
9
4, Table 2). For example, analogs without a small electronegative paraꢀsubstituent (e.g., 1a, 2a,
2l and 2o) did not affect choroidal vascular growth. Moreover, [A1, azaY4]ꢀGHRPꢀ6 (1b)
exhibited no influence on vascular sprouting, in sharp contrast to [azaY4]ꢀGHRPꢀ6 (2b) which
exhibited significant antiangiogenic effect. Although [aza(pꢀF)F4]ꢀGHRPꢀ6 (2c) reduced
angiogenic sprouting, the related [A1, aza(pꢀCl)F4]ꢀGHRPꢀ6 (1d), [aza(pꢀBr)F4]ꢀGHRPꢀ6 (2e),
and [aza(pꢀMeO)F4]ꢀGHRPꢀ6 (2g) exhibited only trends to reduce vascular sprouting. The
azapeptides [aza(pꢀCl)F4]ꢀGHRPꢀ6 (2d) and [aza(pꢀnPrO)F4]ꢀGHRPꢀ6 (2i) demonstrated
respectively a proangiogenic trend and a proangiogenic effect on vascular sprouting. The
relatively larger chloride, bromide, methoxy and nꢀpropoxy substituents were thus all found to
perturb the antiangiogenic effect. Moreover, [A1, aza(pꢀBr)F4]ꢀGHRPꢀ6 (1e) and [aza(4ꢀfluoroꢀ
2,6ꢀdimethyl)F4]ꢀGHRPꢀ6 (2m), both did not affect choroidal vascular growth suggesting that
the larger bromide and the extra steric bulk of the dimethyl substituents also interfered
respectively with antiangiogenic activity. Notably, [A1, azaF4]ꢀGHRPꢀ6 (1a) had previously
enhanced slightly angiogenesis in the sclera/choroid model,3 but showed no influence on
vascular sprouting in the sclera/choroid/RPE complex likely due to the influence of angiogenic
factors produced by the RPE.41,42,43,44
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
16
ACS Paragon Plus Environment

Page 17 of 41
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 4: Angiogenic effects of CD36 ligands in mouse choroidal explant model
Representative microvascular sprouting from Matrigelꢀembedded choroidal explants treated 24h with azapeptides
(10ꢀ6 M). Histogram represents the quantification of the variation of choroidal sprouting areas according to the ratio
(T24ꢀT0)/T0. Values are mean ± SEM *P < 0.05 compared to vehicle (n = 3ꢀ4).
CONCLUSION
In conclusion, submonomer synthesis has provided access to a series of azaPhe4ꢀGHRPꢀ6
analogs to examine the importance of the Nꢀterminal residue and aromatic side chain of the azaꢀ
residue for binding affinity, and antiꢀinflammatory and antiꢀangiogenic activity. Although
removal of the imidazole moiety by replacement of His1 with Ala1 gave analogs exhibiting
capacity to reduce NO production in macrophages treated with a TLRꢀ2 agonist as well as
binding affinity towards CD36 (e.g., 1d, IC50 4.94 µM), their ability to inhibit neovascular
sprouting was significantly impaired. Small electronegative substituents at the paraꢀposition of
the aromatic side chain of the azaꢀresidue tended to favour affinity and activity in both the [A1,
azaF4]ꢀ and [azaF4]ꢀGHRPꢀ6 series. Combining these positive benefits, the His1 analogs
[azaY4]ꢀGHRPꢀ6 (2b) and [aza(4ꢀF)F4]ꢀGHRPꢀ6 (2c) exhibited high binding affinity to CD36
17
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 41
1
2
3
4
5
6
7
8
9
(IC50 1.90 and 6.06 µM), as well as significant ability to reduce NO production in macrophages
and to inhibit choroidal vascular sprouting. The potential for the hydroxyl group of the azaꢀ
tyrosine residue to form a hydrogen bond with the receptor may account for its greater binding
affinity than the less polarizable fluoride of the paraꢀfluorophenylalanine residue.45 This study
has thus provided fundamental insight into the structural requirements for CD36 modulation as
well as two important lead compounds [azaY4]ꢀ and [aza(4ꢀF)F4]ꢀGHRPꢀ6 (2b and 2c) towards
the search for therapeutics to target macrophageꢀdriven inflammation and angiogenesis. The
chemicalꢀbiology underlying the mechanism of action of these CD36 modulators as well as their
therapeutic potential in animal models are currently under investigation and will be reported in
due time.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
EXPERIMENTAL SECTION
Materials and Methods. Unless specified, all reactions in solution were run under argon at
ambient atmosphere. Chromatography was on 230ꢀ400 mesh silica gel. Analytical thinꢀlayer
chromatography (TLC) was performed on glassꢀbacked silica gel plates (Merck 60 F254).
Visualization of the developed chromatogram was performed by UV absorbance or staining with
ceric ammonium molybdate. 1H NMR spectra were measured in CDCl3 at 400 MHz and
13
referenced to CDCl3 (7.26 ppm); C NMR spectra were measured in CDCl3 at 75 MHz and
referenced to CDCl3 (77.0 ppm). Coupling constants, J values were measured in Hertz (Hz) and
chemical shift values in parts per million (ppm). Specific rotations, [α]D were measured at 25°C
at the specified concentrations (c in g/100 mL) using a 1 dm cell on a Perkin Elmer Polarimeter
589 and expressed using the general formula: [αD25 = (100 x α)/(d x c). High resolution mass
spectral analyses were obtained by the Centre régional de spectroscopie de masse de l'Université
18
ACS Paragon Plus Environment

Page 19 of 41
Journal of Medicinal Chemistry
1
2
de Montréal. Either protonated molecular ions [M+H]+, [M+2H]/2+ or sodium adducts [M+Na]+
were used for empirical formula confirmation.
3
4
5
6
7
8
9
Polystyrene Rink Amide resin (0.5 mmol/g) was purchased from Advanced Chemtech™, and the
manufacturer’s reported loading of the resin was used in the calculation of the yields of the final
products. Reagents such as benzophenone hydrazone, benzyl bromide, 4ꢀbromobenzyl bromide,
4ꢀfluorobenzyl bromide, 4ꢀchlorobenzyl bromide, 4ꢀiodobenzyl bromide, 4ꢀnitrobenzyl bromide,
4ꢀtrifluoromethylbenzyl bromide, 4ꢀmethoxybenzyl bromide or 4ꢀmethoxybenzyl chloride,
iodopropane, 2ꢀiodopropane, 80% propargyl bromide in toluene, 2,6ꢀbenzylaldehyde, 3,5ꢀ
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
dichlorophenol,
4ꢀhydroxybenzaldehyde,
3ꢀhydroxybenzaldehyde,
1ꢀfluoroꢀ3,5ꢀ
dimethylbenzene, 2,4,6ꢀtrifluorobenzaldehyde, hydroxylamine hydrochloride, pyridine,
piperidine, formic acid (FA), N,Nꢀdiisopropylethylamine and N,N′ꢀdisuccinimidyl carbonate
(DSC), all were purchased from Aldrich and used without further purification. 4ꢀ(tertꢀ
Butyldimethylsilyloxy)benzyl bromide,31 2,4,6ꢀtrifluorobenzylbromide,46 2,6ꢀdimethylbenzyl
bromide,47 4ꢀpropoxybenzyl bromide,48 ethoxybenzyl bromide,48 4ꢀisopropoxybenzyl bromide 49
and 2,6ꢀdimethylꢀ4ꢀfluorobenyl bromide,50 all were synthesized according to the literature (see
SI for representative examples).
Thinꢀlayer chromatography was performed on silica gel 60 F254 plates from Merck™. All the
amino acids and coupling reagents such as HBTU and diisopropylcarbodiimide (DIC) were
purchased from GL Biochem™ and used as received. All solvents were obtained from VWR
international. Anhydrous solvents (THF, DMF, CH2Cl2, and CH3OH) were obtained by passage
through solvent filtration systems (GlassContour, Irvine, CA).
19
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 41
1
2
3
4
5
6
7
8
9
Azapeptide purification and analysis. Azapeptides were purified on a semiꢀpreparative column
(C18 Gemini column™) using the appropriate gradient from pure water (0.1% FA) to mixtures
with MeOH (0.1% FA) at a flow rate of 10mL/min. Purity of azapeptides (>95%) was assessed
using analytical LCꢀMS on a 5 ꢁM, 50 mm x 4.6 mm C18 Phenomenex Gemini column™ with a
flow rate of 0.5 mL/min using the appropriate linear gradient in two different solvent systems:
water (0.1% FA) with CH3CN (0.1% FA) and water (0.1% FA) with MeOH (0.1% FA).
Fmoc deprotection and peptides analysis. Peptide synthesis was performed on an automated
shaker using polystyrene Rink amide resin (0.5 mmol/g 75ꢀ100 mesh). Removal of Fmoc group
was performed by treating the resin with 20% piperidine in DMF for 30 min. The resin was
washed sequentially with DMF (3 x 10 mL), MeOH (3 x 10 mL), THF (3 x 10 mL), and DCM (3
x 10 mL). The quality of the reaction was assessed with a Kaiser Test for the coupling of the first
and second amino acids to provide DꢀPheꢀLys(Boc) Rink amide resin.9 Conversion in all
remaining coupling reactions were ascertained by LCꢀMS analysis after dissolution of the dry
peptide in methanol (0.5mL). To obtain sample of deprotected peptides for analysis, unless
described otherwise, a 5mg aliquot of resin was treated with TFA/TES/H2O (95:2.5:2.5, v/v/v,
0.5 mL) on the shaker for 30 min, filtered, and the filtrate was evaporated under reduced pressure
dissolved in methanol and evaporated to dryness.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Representative deprotection, cleavage and purification of azapeptide: HꢀHisꢀDꢀTrpꢀ
Alaꢀaza(2,6ꢀdichloroꢀ4ꢀmethoxy)PheꢀDꢀPheꢀLysꢀNH2 (2p). In a plastic syringe tube
equipped with Teflon™ filter, stopper and stopcock, NꢀBocꢀHis(Trt)ꢀDꢀTrp(Boc)ꢀAlaꢀaza(2,6ꢀ
dichloroꢀ4ꢀmethoxy)PheꢀDꢀPheꢀLys(Boc)ꢀNHꢀRink amide resin (1618 mg, 0.53 mmol) was
treated with a freshly made solution of TFA/H2O/TES (95:2.5:2.5, v/v/v, 10 mL). The syringe
was closed properly and shaken for 2 h at room temperature, and filtered. The filtrate was
20
ACS Paragon Plus Environment

Page 21 of 41
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
concentrated, dissolved in Et2O, cooled to –30 °C and centrifuged (1200 RPM) for 10 min. The
solution was decanted and the pellet was dissolved in water and freezeꢀdryed to provide
azapeptide, that was dissolved in a 10% H2O/methanol solution and analyzed by LCꢀMS on a
GeminiTM C18 reverseꢀphase column (150 × 4.60 mm, 5 ꢁm) using a flow rate of 0.5 mL/min
and gradient between 5ꢀ80% MeOH (0.1% FA) in water (0.1% FA). The azapeptide was
purified on a WatersTM PrepLC instrument equipped with a reverseꢀphase GeminiTM C18
column (250 × 21.2 mm, 5 ꢁm) using a binary solvent system consisting of [5ꢀ80% methanol
(0.1% FA) in water (0.1% FA) over 60 min] at a flow rate of 10 mL/min with UV detection at
214 nm. The collected fractions were combined, and freezeꢀdried to a white powder and
analyzed for purity (Table 1) by LCꢀMS on a GeminiTM C18 reverseꢀphase column (150 × 4.60
mm, 5 ꢁm), using a flow rate of 0.5 mL/min and a gradient of 0ꢀ95% CH3CN (0.1% FA) or 10ꢀ
80% MeOH (0.1% FA) in water (0.1% FA).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
BenzhydrylideneꢀazaꢀglycinylꢀDꢀphenylalaninylꢀNεꢀ(Boc)lysine Rink amide resin (5).
Benzophenone hydrazone (3.3 eq., 0.589 g, 3 mmol) in dichloromethane (4 mL) was cooled to
0°C, and treated dropwise over 15 min with a solution of N,N'ꢀdisuccinimidyl carbonate (3 eq.,
0.699 g, 2.73 mmol) in DMF (3.9 mL) and DCM (8 mL). After stirring for 30 min, the ice bath
was removed and the reaction mixture was let warm to room temperature with stirring for 1h.
The resulting solution of 1ꢀ(2,5ꢀdioxopyrrolidinꢀ1ꢀyl)ꢀN'ꢀ(diphenylmethylidene) carbazate 4 was
cooled to 0 °C, and treated with DIEA (6 eq., 0.901 mL, 5.45 mmol). The resulting suspension
was quickly transferred to a plastic syringe tube equipped with Teflon™ filter, stopper and
stopcock containing DꢀPheꢀLys(Boc) amide resin (1 eq., 2.4 g, 1 mmol, synthesized as
previously described7), and agitated on an automated shaker for 16 h at room temperature. The
resin was filtered, washed with DMF (3 × 10 mL), MeOH (3 × 10 mL), THF (3 × 10 mL), and
21
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 41
1
2
3
4
5
6
7
8
9
DCM (3 ×10 mL), dried under vacuum, and stored in the fridge. Complete reaction conversion
was ascertained by LCꢀMS [20ꢀ80% MeOH (0.1 % FA)/H2O (0.1 % FA), 14 min, RT 7.3 min]
on a sample from evaporation of the filtrate after filtration of the cleavage product from
treatment an aliquot (5 mg) using 1 mL of TFA/TES/H2O (95:2.5:2.5, v/v/v).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
BenzhydrylideneꢀazaꢀphenylalaninylꢀDꢀphenylalaninylꢀNεꢀ(Boc)lysine Rink amide resin
(6a). Azaꢀglycine resin 5 (1 eq, 0.5 mmol) in a 40 mL syringe equipped with Teflon™ filter,
stopcock and cap, was swollen in THF (10 mL), treated with a 40% aqueous solution of
tetraethylammonium hydroxide (9 eq., 2.7 M, 0.6 mL, 4.5 mmol), and agitated for 30 min at 0°C.
The resin mixture was treated with benzylbromide (9 eq, 4.5 mmol, 765 mg) and shaken at room
temperature overnight. The resin was filtered, washed and analyzed as decribed for resin 5
above, which indicated quantitative conversion to benzhydrylideneꢀazaꢀphenylalaninylꢀDꢀ
phenylalaninylꢀlysine amide 6a [20ꢀ80% MeOH (0.1 % FA)/H2O (0.1 % FA), 14 min, RT
8.3].
Benzhydrylideneꢀazaꢀ4ꢀ(tertꢀbutyldimethylsilyloxy)ꢀphenylalaninylꢀDꢀphenylalaninylꢀNεꢀ
(Boc)lysine Rink amide resin (6b). Employing the protocol described above for the synthesis
of resin 6a using 4ꢀ(tertꢀbutyldimethylsilyloxy)ꢀbenzyl bromide (9 eq, 4.5 mmol, 1.35 g),
azapeptide resin 6b was analyzed by LCꢀMS: [20ꢀ80% MeOH (0.1 % FA)/H2O (0.1 % FA),
14min, RT 7.22 min].
Benzhydrylideneꢀazaꢀ4ꢀfluorophenylalaninylꢀDꢀphenylalaninylꢀNεꢀ(Boc)lysine Rink amide
resin (6c). Employing the protocol described above for the synthesis of resin 6a using 4ꢀ
fluorobenzyl bromide (6 eq, 3 mmol, 564 mg), azapeptide resin 6c was shown by LCꢀMS
analysis [10ꢀ80% MeOH (0.1 % FA)/H2O (0.1 % FA), 14 min, RT 8.99 min] to be quantitatively
obtained.
22
ACS Paragon Plus Environment

Page 23 of 41
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Benzhydrylideneꢀazaꢀ4ꢀchlorophenylalaninylꢀDꢀphenylalaninylꢀNεꢀ(Boc)lysine Rink amide
resin (6d). Employing the protocol described above for the synthesis of resin 6a using 4ꢀ
chlorobenzylbromide (6 eq, 3 mmol, 609 mg ), azapeptide resin 6d was shown by LCꢀMS
analysis [20ꢀ80% MeOH (0.1 % FA)/H2O (0.1 % FA), 14 min, RT 8.27 min] to be quantitatively
obtained.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Benzhydrylideneꢀazaꢀ4ꢀbromophenylalaninylꢀDꢀphenylalaninylꢀNεꢀ(Boc)lysine Rink amide
resin (6e). Employing the protocol described above for the synthesis of resin 6a using 4ꢀ
bromobenzyl bromide (6 eq, 3 mmol, 741 mg), azapeptide resin 6e was shown by LCꢀMS
analysis [10ꢀ80% MeOH (0.1 % FA)/H2O (0.1 % FA), 14 min, RT 8.79 min] to be quantitatively
obtained.
Benzhydrylideneꢀazaꢀ4ꢀiodophenylalaninylꢀDꢀphenylalaninylꢀNεꢀ(Boc)lysine Rink amide
resin (6f). Employing the protocol described above for the synthesis of resin 6a using 4ꢀ
iodobenzyl bromide (6 eq, 0.92 mmol, 272 mg), azapeptide resin 6f was shown by LCꢀMS
analysis [10ꢀ90% MeOH (0.1 % FA)/H2O (0.1 % FA), 20 min, RT 12.65 min] to be
quantitatively obtained.
Benzhydrylideneꢀazaꢀ4ꢀmethoxyphenylalaninylꢀDꢀphenylalaninylꢀNεꢀ(Boc)lysine
Rink
amide resin (6g). Employing the protocol described above for the synthesis of resin 6a using 4ꢀ
methoxybenzyl bromide (9 eq, 4.5 mmol, 899 mg) azapeptide resin 6g was analyzed by LCꢀ
MS [10ꢀ80% MeOH (0.1 % FA)/H2O (0.1 % FA), 14 min, RT 8.80 min], which showed
complete disappearance of azaꢀglycine resin 5 (RT 8.1 min).
Benzhydrylideneꢀazaꢀ4ꢀisoꢀpropoxyphenylalaninylꢀDꢀphenylalaninylꢀNεꢀ(Boc)lysine Rink
amide resin (6h). Employing the protocol described above for the synthesis of resin 6a using 4ꢀ
isoꢀpropoxybenzyl bromide (9 eq, 4.5 mmol, 1.03 g), azapeptide resin 6h was analyzed by
23
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 41
1
2
3
4
LCꢀMS [30ꢀ80% MeOH (0.1 % FA)/H2O (0.1 % FA), 14 min, RT 7.04 min], which showed
5
6
complete disappearance of azaꢀglycine resin 5 (RT 6.90 min).
7
8
9
Benzhydrylideneꢀazaꢀ4ꢀpropoxyphenylalaninylꢀDꢀphenylalaninylꢀNεꢀ(Boc)lysine
Rink
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
amide resin (6i). Employing the protocol described above for the synthesis of resin 6a using
40% aqueous solution of tetraethylammonium hydroxide (9 eq., 2.7 M, 0.6 mL, 4.5 mmol) and
4ꢀnꢀpropoxybenzyl bromide (9 eq, 4.5 mmol, 1031 mg), azapeptide resin 6i was analyzed by
LCꢀMS [30ꢀ80% MeOH (0.1 % FA)/H2O (0.1 % FA), 14 min, RT 9.77 min], which showed
complete disappearance of azaꢀglycine resin 5 [10ꢀ80% MeOH (0.1 % FA)/H2O (0.1 % FA), 14
min, RT 8.01 min].
Benzhydrylideneꢀazaꢀ4ꢀ(trifluoromethyl)lphenylalaninylꢀDꢀphenylalaninylꢀNεꢀ(Boc)lysine
Rink amide resin (6k). Employing the protocol described above for the synthesis of resin 6a
using 4ꢀtrifluoromethylbenzyl bromide (6 eq, 3 mmol, 710 mg), azapeptide resin 6k was
analyzed by LCꢀMS [20ꢀ90% MeOH (0.1 % FA)/H2O (0.1 % FA), 20 min, RT 12.57 min],
which showed quantitative conversion from azaꢀglycine resin 5 (RT 10.61 min).
Benzhydrylideneꢀazaꢀ2,6ꢀdimethylphenylalaninylꢀDꢀphenylalaninylꢀNεꢀ(Boc)lysine
Rink
amide resin (6l). Employing the protocol described above for the synthesis of resin 6a using
2,6ꢀdimethylbenzyl bromide (6 eq, 3 mmol, 593 mg), azapeptide resin 6l was analyzed by LCꢀ
MS [50ꢀ90% MeOH (0.1 % FA)/H2O (0.1 % FA), 20 min, RT 8.66 min], which showed
quantitative conversion from azaꢀglycine resin 5 (RT 2.82 min).
Benzhydrylideneꢀazaꢀ4ꢀfluoroꢀ2,6ꢀdimethylphenylalaninylꢀDꢀphenylalaninylꢀNεꢀ(Boc)lysine
Rink amide resin (6m). Employing the protocol described above for the synthesis of resin 6a
using 4ꢀfluoro2,6ꢀdimethylbenzyl bromide (6eq, 3 mmol, 645 mg), azapeptide resin 6m was
24
ACS Paragon Plus Environment

Page 25 of 41
Journal of Medicinal Chemistry
1
2
3
4
5
6
analyzed by LCꢀMS [20ꢀ80% MeOH (0.1 % FA)/H2O (0.1 % FA), 14 min, RT 9.24 min], which
showed quantitative conversion from azaꢀglycine resin 5 (RT 8.04 min).
7
Benzhydrylideneꢀazaꢀ2,4,6ꢀtrifluorophenylalaninylꢀDꢀphenylalaninylꢀNεꢀ(Boc)lysine Rink
amide resin (6n). Employing the protocol described above for the synthesis of resin 6a using
2,4,6ꢀtrifluorobenzyl bromide (6 eq, 3 mmol, 671 mg), azapeptide resin 6n was analyzed by
LCꢀMS [30ꢀ80% MeOH (0.1 % FA)/H2O (0.1 % FA), 14 min, RT 7.97 min], which showed
quantitative conversion from azaꢀglycine resin 5 (RT 6.90 min).
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Benzhydrylideneꢀazaꢀ3ꢀ(tertꢀbutyldimethylsilyloxy)phenylalaninylꢀDꢀphenylalaninylꢀNεꢀ
(Boc)lysine Rink amide resin (6o). Employing the protocol described above for the synthesis
of resin 6a using 2,4,6ꢀtrifluorobenzyl bromide (9 eq, 4.5 mmol, 1.35 g), azapeptide resin 6o
was analyzed by LCꢀMS [10ꢀ80% MeOH (0.1 % FA)/H2O (0.1 % FA), 14 min, RT 8.14 min],
which showed quantitative conversion from azaꢀglycine resin 5 (RT 8.01 min).
Azadipeptide coupling onto NεꢀBocꢀlysine Rink amide resin: benzhydrylideneꢀazaꢀ
(2,6ꢀdichloroꢀ4ꢀmethoxy)phenylalaninylꢀDꢀphenylalaninylꢀNεꢀBocꢀlysine Rink amide
resin (6p). Benzhydrylideneꢀazaꢀ(2,6ꢀdichloroꢀ4ꢀmethoxy)phenylalaninylꢀDꢀphenylalanine
tertꢀbutyl ester (8p, 1 g, 1,58 mmol) was added to a solution of 20 mL of 1:1 TFA/DCM, stirred
at room temperature for 2 h, and the volatiles were evaporated under reduced pressure. The
residue was dissolved and evaporated twice from DCM (20 mL). The recovered oil (911 mg;
1.58 mmol) was used without further purification, dissolved in DMF (3 mL), treated with DIEA
(6 eq., 0.48 mL, 3.2 mmol), treated with a solution of HBTU (3 eq., 594.5 mg, 1.58 mmol) in
DMF (3 mL) for 30 sec, and transferred as a yellow solution to a syringe containing Nεꢀ
(Boc)lysine Rink amide resin (1 eq., 1010 mg, 0.53 mmol) swollen in DMF. The syringe was
closed properly and shaken for 16 h, and as described for resin 5, washed and analyzed by LCꢀ
25
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 41
1
2
3
4
MS [30ꢀ80% MeOH (0.1% FA)/water (0.1% FA) over 14 min] which indicated complete
5
6
7
conversion
to
benzhydrylideneꢀazaꢀ2,6ꢀdichloroꢀ4ꢀmethoxyphenylalaninylꢀDꢀ
8
9
phenylalaninylꢀlysinamide 6p (RT 9.3 min)
.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Benzhydrylideneꢀazaꢀ(2,4ꢀdichloroꢀ6ꢀmethoxy)phenylalaninylꢀDꢀphenylalaninylꢀNεꢀ
(Boc)lysine Rink amide resin (6q). Employing the protocol described for the synthesis of resin
6p, ester 8q (1 g, 1,58 mmol) was converted to the corresponding acid and coupled to NεꢀBocꢀ
lysine Rink amide resin to provide complete conversion to resin 6q, as ascertained by LCꢀMS
analysis [30ꢀ80% MeOH (0.1% FA)/water (0.1% FA) over 14 min, RT 8.75 min].
tertꢀButyl
benzhydrylideneꢀazaꢀ2,6ꢀdichloroꢀ4ꢀmethoxyphenylalaninylꢀDꢀ
phenylalanine (8p) Benzhydrylidene azaꢀglycinylꢀDꢀphenylalanine tertꢀbutyl ester (7, 1
eq., 925 mg, 2.09 mmol, synthesized as previously described28) was dissolved in THF
(51.3 mL), cooled to 0°C, treated with 40% aqueous tetraethylammonium hydroxide (3
eq., 2.72 M, 2.3 mL, 6.26 mmol), stirred for 30 min, treated with 2,6ꢀdichloroꢀ4ꢀ
methoxybenzyl bromide (3 eq., 6.26 mmol, 1.7 g) and stirred at the room temperature
overnight. The volatiles were evaporated. The residue was partitioned between H2O (50 mL)
and EtOAc (50 mL). The phases were separated, and the aqueous phase was extracted twice
with EtOAc. The organic phases were combined, dried with MgSO4, filtered, and evaporated to
a residue that was purified by flash chromatography using a gradient of 10ꢀ20% EtOAc in
hexane. Evaporation of the collected fractions gave azaꢀdipeptide 8p (1300 mg, 2.06 mmol, 96%
25
1
yield): [α]D 6.1o; Rf 0.43 (8:2 hexane/EtOAc); H NMR (300 MHz, CDCl3) δ 7.55 – 7.41 (m,
3H), 7.40 – 7.30 (m, 3H), 7.29 – 7.10 (m, 6H), 6.95 (d, J = 14.5, 13.0 Hz, 2H), 6.63 (s, 2H), 6.19
(d, J = 8.4 Hz, 1H), 4.91 – 4.66 (m, 3H), 3.81 (s, 1H), 3.72 (s, 3H), 3.09 (d, J = 6.2 Hz, 2H), 1.38
26
ACS Paragon Plus Environment

Page 27 of 41
Journal of Medicinal Chemistry
1
2
13
3
(s, 9H); C NMR (75 MHz, CDCl3) δ 171.2, 168.2, 158.8, 157.5, 138.2, 136.9, 136.6, 134.8,
130.5, 129.5, 129.4, 129.3, 128.9, 128.3, 128.0, 126.7, 114.2, 81.6, 55.6, 54.6, 47.8, 38.8, 27.9.
IR (neat) vmax/cmꢀ1 1671, 1494, 1148, 1061, 1038, 912, 731, 697. HRMS (ESI) calcd for
C35H36N3O4 [M+H]+, 632.2077 found 632.2068.
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Benzhydrylideneꢀazaꢀ2,4ꢀdichloroꢀ6ꢀmethoxyphenylalaninylꢀDꢀphenylalanine
tertꢀbutyl
ester (8q). Employing the protocol described above for the synthesis of azaꢀdipeptide ester 8p
using azaꢀglycine 7 (1 eq., 925 mg, 2.09 mmol), azaꢀdipeptide ester 8q (1292 mg, 2.04 mmol, 98
1
% yield) was isolated: [α]D25 –56°; Rf 0.81 (7:3 hexane/EtOAc); H NMR (300 MHz, CDCl3) δ
7.48 – 7.24 (m, 8H), 7.18 (s, 4H), 7.08 – 7.01 (m, 2H), 6.81 (dd, J = 3.4, 2.0 Hz, 1H), 6.53 (d, J
= 1.9 Hz, 1H), 6.38 (d, J = 8.3 Hz, 1H), 4.80 – 4.63 (m, 3H), 3.88 (s, 1H), 3.58 (s, 3H), 3.22 –
13
2.94 (m, 2H), 1.39 (s, 9H); C NMR (75 MHz, CDCl3) δ 171.3, 159.4, 157.8, 138.5, 136.7,
136.1, 134.8, 133.7, 130.3, 129.5, 129.3, 129.2, 129.1 128.3, 128.0, 126.7, 121.6, 121.3, 109.5,
81.6, 55.8, 54.7, 44.1, 38.8, 28.0; IR (neat) vmax/cmꢀ1 1569, 1497, 1399, 1219, 1148, 1101, 1045,
1011, 889, 731, 697. HRMS (ESI) calcd for C35H36N3O4 [M+H]+, 632.2077 found 632.2054.
Benzhydrylideneꢀazaꢀ(1ꢀBocꢀ1,2,3ꢀtriazolyl)ꢀ3ꢀalanineꢀDꢀphenylalaninylꢀNεꢀ(Boc)lysine
amide (12) In a syringe tube equipped with stopper, Teflon™ filter and stopcock, azaꢀ
propargylglycinyl dipeptide resin 11 (1 eq., 195 mg, 0.3 mmol, prepared as previously
reported51) was swollen in a mixture of DMF (6961 ꢁL) and MeOH (455 ꢁL), treated
sequentially with CuI (5 eq., 285 mg, 1.5 mmol), Lꢀproline (5 eq., 172 mg, 1.5 mmol),
trimethylsilyl azide (5 eq., 197 ꢁL, 1.5 mmol), triethylamine (5 eq., 208 ꢁL, 1.5 mmol) and
molecular sieves (4A°, 500 mg). The syringe tube was flushed with argon, capped, sealed with
parafilm, and heated in a water bath with sonication at 80°C for 24 h. The resin mixture was
cooled, filtered and as described above for resin 5, washed, and analyzed by LCꢀMS [10ꢀ80%
27
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 41
1
2
3
4
5
6
7
8
9
MeOH (0.1% FA)/water (0.1% FA) over 14 min] which indicated quantitative conversion of azaꢀ
propargylglycinyl resin 11 (RT 8.44 min) into triazole resin (RT 7.04 min). Triazole resin (1 eq.,
208 mg, 0.3 mmol) was swollen in THF (6 mL) treated with Et3N (3 eq., 0.125 mL, 0.9 mmol)
and Boc2O (3 eq., 196 mg, 0.9 mmol), and stirred for 2 h at room temperature to give
benzhydrylideneꢀazaꢀ(1ꢀBocꢀ1,2,3ꢀtriazolyl)ꢀ3ꢀalanineꢀDꢀphenylalaninylꢀNεꢀ(Boc)lysine amide
12. The resin was filtered and washed with DMF (3x10 mL), DMF/0.1 N HCl (3:1, 3x10 mL),
H2O (3x10 mL), MeOH (3x10 mL), THF (3x10 mL), and DCM (3x10 mL).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Semicarbazone deprotection, semicarbazide acylation, and azapeptide elongation, all were
performed as previously described.12
Modulatory effect of azapeptide on the overproduction of NO induced by the TLRꢀ2
agonist R FSLꢀ1 in macrophage cell line
The murine RAW1 (Cat# TIBꢀ71) and J774A.1 (Cat# TIBꢀ67) macrophage cell lines were
obtained from American Type Cell Collection macrophage cell line, seeded at 1.5 x 105
cells/well in DMEM medium supplemented with penicillin and streptomycin on a 48ꢀwell plate,
and incubated at 37 °C with 5% CO2. After 2 h, the medium of adhered cells was changed to
DMEMꢀPen/Strep with 0.2% of bovine serum albumin (BSA), containing either azapeptides (1a,
1b, 1d–1g, 1j, 1k, 2aꢀ2q and 3) or [azaLys6]ꢀGHRPꢀ6 as negative control at final concentration
of 10ꢀ6 M. After a 1 h preꢀincubation period, the cells were stimulated overnight with TLRꢀ2
ligand fibroblastꢀstimulating lipopeptide (RꢀFSLꢀ1, 300 ng/mL, Invivogen® #L7022).
Supernatants were collected for nitrite determination by fluorescence using 2,3ꢀ
diaminonaphthalene (DAN). Briefly, 25 µL of 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
28
ACS Paragon Plus Environment

Page 29 of 41
Journal of Medicinal Chemistry
1
2
3
4
5
6
15 min, the reaction was stopped with 20 µL of NaOH (2.8N) and fluorescence was read using a
fluorescence plate reader (TECAN® Safire, λexc 365 nm and λem 430 nm).
7
8
9
CD36 affinity studies of azapeptide analogs
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Rat heart membrane preparation as source of CD36 for binding study
The hearts from Sprague Dawley rats (300gꢀ325g, Charles River) were sliced into small pieces
and homogenized with a Polytron at low speed in buffer A (10 mM NaHCO3, 5 mM NaN3, 10
ꢁM Pefabloc, 0.1 ꢁM Aprotinin, 1 ꢁM Pepstatin A, 1 ꢁM Leupeptin, pH 7.0). The homogenate
was centrifuged at 8,700 x g for 10 min and the supernatant was collected on ice. The
supernatant was centrifuged at 35,000 x g for 20 min to provide a pellet fraction, that was
suspended in Buffer B (20 mM Trisꢀmaleate, 0.6M KCl, pH 6.8) using a glass homogenizer with
a Teflon pestle and centrifuged again at 35,000 x g for 60 min. The resulting pellet fraction was
suspended in Buffer C (10 mM Tris/HCl buffer pH 7.4) and submitted to another centrifugation
at 35,000 x g for 60 min. The collected pellet was suspended in 50 mM Tris/HCl buffer pH 7.4)
containing 2 mM EGTA. The membrane preparation obtained was frozen at –80°C. Protein
concentration was determined using the bicinchoninic acid (BCA) method, using BSA as
standard.
Radioiodination of photoactivatable ligand tracer for receptor binding assay
The radiodination of TyrꢀBpaꢀAlaꢀhexarelin was performed as previously described.52 Briefly,
10 nmol of TyrꢀBpaꢀAlaꢀhexarelin was mixed with 100 ng of lactoperoxidase and 1mCi of
Na125I in a volume of 30 µL of 0.1M sodium acetate buffer pH 5.6. The reaction was started by
adding 3 nmol of H2O2 for 5 min at room temperature. This step was repeated twice with a 5ꢀmin
incubation period between each addition. The reaction was stopped by diluting the mixture with
1 mL of 0.1% TFA. The radiodinated peptide was purified by HPLC on reverseꢀphase Vydac
29
ACS Paragon Plus Environment