Influence of N-methylation and conformation on almiramide anti-leishmanial activity

Article abstract of DOI:10.3390/molecules26123606

The almiramide N-methylated lipopeptides exhibit promising activity against trypanosomatid parasites. A structure–activity relationship study has been performed to examine the influences of N-methylation and conformation on activity against various strains of leishmaniasis protozoan and on cytotoxicity. The synthesis and biological analysis of twenty-five analogs demonstrated that derivatives with a single methyl group on either the first or fifth residue amide nitrogen exhibited greater activity than the permethylated peptides and relatively high potency against resistant strains. Replacement of amino amide residues in the peptide, by turn inducing α-amino γ-lactam (Agl) and N-aminoimidazalone (Nai) counterparts, reduced typically anti-parasitic activity; however, peptide amides possessing Agl residues at the second residue retained significant potency in the unmethylated and permethylated series. Systematic study of the effects of methylation and turn geometry on anti-parasitic activity indicated the relevance of an extended conformer about the central residues, and conformational mobility by tertiary amide isomerization and turn geometry at the extremities of the active peptides.

Full text of DOI:10.3390/molecules26123606

molecules
Article
Influence of N-Methylation and Conformation on Almiramide
Anti-Leishmanial Activity
Anh Minh Thao Nguyen 1, Skye Brettell 1, Noélie Douanne 2, Claudia Duquette 2, Audrey Corbeil 2,
Emanuella F. Fajardo 3, Martin Olivier 3, Christopher Fernandez-Prada 2 and William D. Lubell 1,
*
1
Départements de chimie, Université de Montréal, C.P. 6128, Succursale Centre-Ville,
Montréal, QC H3C 3J7, Canada; anh.minh.thao.nguyen@umontreal.ca (A.M.T.N.);
2196732B@student.gla.ac.uk (S.B.)
2
Département de Pathologie et Microbiologie, Université de Montréal, C.P. 6128, Succursale Centre-Ville,
Montréal, QC H3C 3J7, Canada; noelie.douanne@umontreal.ca (N.D.); claudia.duquette@umontreal.ca (C.D.);
audrey.corbeil@umontreal.ca (A.C.); christopher.fernandez.prada@umontreal.ca (C.F.-P.)
Department of Microbiology and Immunology, McGill University, Montréal, QC H3A 2B4, Canada;
emanuella_fajardo@yahoo.com.br (E.F.F.); martin.olivier@mcgill.ca (M.O.)
3
*
Correspondence: william.lubell@umontreal.ca; Tel.: +1-514-343-7339; Fax: +1-202-513-8788
Abstract: The almiramide N-methylated lipopeptides exhibit promising activity against trypanoso-
matid parasites. A structure–activity relationship study has been performed to examine the influences
of N-methylation and conformation on activity against various strains of leishmaniasis protozoan
and on cytotoxicity. The synthesis and biological analysis of twenty-five analogs demonstrated that
derivatives with a single methyl group on either the first or fifth residue amide nitrogen exhibited
greater activity than the permethylated peptides and relatively high potency against resistant strains.
Replacement of amino amide residues in the peptide, by turn inducing α amino γ lactam (Agl) and
N-aminoimidazalone (Nai) counterparts, reduced typically anti-parasitic activity; however, peptide
amides possessing Agl residues at the second residue retained significant potency in the unmethy-
lated and permethylated series. Systematic study of the effects of methylation and turn geometry on
anti-parasitic activity indicated the relevance of an extended conformer about the central residues,
and conformational mobility by tertiary amide isomerization and turn geometry at the extremities of
the active peptides.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Nguyen, A.M.T.; Brettell, S.;
Douanne, N.; Duquette, C.;
Corbeil, A.; Fajardo, E.F.; Olivier, M.;
Fernandez-Prada, C.; Lubell, W.D.
Influence of N-Methylation and
Conformation on Almiramide
Anti-Leishmanial Activity. Molecules
2021, 26, 3606. https://doi.org/
10.3390/molecules26123606
Keywords: almiramide; leishmaniasis; N-methylated peptide
Academic Editor: Carla Boga
Received: 22 May 2021
Accepted: 9 June 2021
Published: 12 June 2021
1. Introduction
Neglected tropical diseases caused by trypanosomatid protozoan infections, such as
human African trypanosomiasis, Chagas disease and leishmaniasis, impact significantly
on public health, especially in tropical countries, where their influences are compounded
due to poverty, environment, and drug resistance [
intracellular protozoan belonging to the genus Leishmania (L.) and transmitted by the
phlebotomine sandflies [ ]. Upon mammal host infection by way of a sand fly bite, the
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with regard to jurisdictional claims in
published maps and institutional affil-
iations.
1–3]. Leishmaniasis is caused by an
4
parasites enter the blood cells in the so-called promastigote stage, then begin to multiply in
the amastigote stage spreading to other cells and tissues. Depending on the species, the
parasites may disperse via blood and lymph fluid to other body sites, such as the skin and
major organs.
Copyright:
© 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
With over 1 million new cases occurring annually, around 12 to 15 million people
worldwide are infected with leishmaniasis [
neous, mucocutaneous, visceral and post Kala-azar dermal leishmaniasis (CL, MCL, VL
and PKDL) [ ]. Leishmaniasis causes enlargement of the spleen and liver, anemia [ ], skin
lesions [ ], and destruction of mucous membranes [10]. In addition, controlling co-infection
with HIV and visceral leishmaniasis has become a serious challenge [11].
5,6], which presents as four main forms: cuta-
7
8
9
Molecules 2021, 26, 3606. https://doi.org/10.3390/molecules26123606
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 3606
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In the absence of a vaccine, chemotherapy is the main tool to treat leishmaniasis, but
only a few drugs are used clinically [12]. Pentavalent antimonial drugs, such as sodium
stibogluconate and meglumine antimoniate, were first-line defenses against leishmania
since the 1940s [13], but are now failing as treatments due to resistance [14]. Ampho-
tericin B, which was discovered in the late 1950s, became an alternative drug for treating
leishmaniasis patients [15]. Miltefosine was the first oral anti-leishmanial agent and has
been used to treat VL patients since 2002 [16]. High cost and side effects have; however,
limited the use of amphotericin B and miltefosine [17]. The aminoglycoside antibiotic paro-
momycin has been used to treat leishmaniasis [18], with variations in efficacy contingent
upon geographic region [19].
The need for new anti-leishmanial agents is critical due to limitations in availability of
effective drugs and the rise of drug resistant strains of leishmaniasis [20]. In the search for
antiparasitic agents, selectivity and potency (µM) against certain Leishmania strains have
been observed using chromone natural products [21] and artemisinin-inspired analogs [22].
Moreover, notable differences in ribosomal structure were exploited to conceive paro-
momycin derivatives with potent anti-leishmanial activity and low ototoxicity [23]. Other
anti-leishmanial agents under investigation include mitochondrial cytochrome bc1 and
pteridine reductase 1 inhibitors, as well as aryl nicotinic acid derivatives [20]. In addition,
the kinetoplastid-selective proteasome inhibitor LXE408 which exhibited efficacy in murine
CL and VL models has recently entered Phase 1 human clinical trials [24].
Almiramides A-C (
the marine cyanobacterium Lyngbya majuscula [25]. Certain almiramide analogs have ex-
hibited low M anti-parasitic activity against the causative pathogen of fatal leishmaniasis,
1–3, Figure 1) are N-methylated linear lipopeptides isolated from
µ
as well as a high therapeutic index [25]. Almiramide derivatives are attractive leads for
developing agents against leishmaniasis, in part due to their likely mechanism of action
involving disruption of the vital energy machinery proteins of the glycosome [26]. A
peroxisome-related organelle without mammalian counterpart, the glycosome performs vi-
tal metabolic processes in trypanosomatids [27
the interface of two key transport proteins [peroxin (PEX)14
pyrazolo[4,3-c]pyridines which killed effectively the causative trypanosomatids of Human
,
28]. In search of glycosome targeting agents,
PEX5] has been blocked by
−
African trypanosomiasis and Chagas disease, Trypanosoma brucei and Trypanosoma cruzi,
and had low mammalian cell cytotoxicity [29,30]. Moreover, an examination of almiramide
analogs in affinity capture, and fluorescent microscopy experiments on T. brucei, identified
the glycosome proteins PEX11 and glycosomal integral membrane protein-5a (GIM5A) as
likely targets [26].
Preliminary structure–activity relationship studies on the almiramides have indicated
the importance of the unsaturated lipophilic terminus. Almiramides B and C (
Figure 1) with 2-methyl-7-octynoyl and -7-octenoyl tails were reported to exhibit low
M activity against Leishmania donovani, but their 2-methyl-7-oxooctanoyl counterpart
almiramide A ( ) was inactive [25]. Employment of a 6-heptynyl lipophilic terminus and
N-methy1 valine3 in acid 12 and dimethyl amide 13 gave peptides with
M IC50 values
against L. donovani, and improved therapeutic indices with relatively lower cytotoxicity
(CC50) against mammalian Vero kidney cells: selectivity index (CC50/EC50) 12 13 (50.2)
(21.8) > (17.4) [25]. In this series, the significance of the peptide C-terminal was
2 and 3,
µ
1
µ
=
>
2
3
illustrated by the methyl ester counterpart (e.g., 14) of peptide acid 12, which exhibited a
significant loss of anti-parasitic activity and a relative gain in Vero cell cytotoxicity [25]. In
addition, peptide
2 and novel almiramides D-H (4–8) possessing the common 2-methyl-7-
octynoyl N-terminus and a distinct N-(Me)Ile4 residue were isolated together from marine
benthic cyanobacteria in a study that found
gingival fibroblast cells [31].
2 and 9 exhibited toxicity against human
The active almiramide conformation has been examined by replacement of the Val3-
Ala4 dipeptide moiety with a mannose-derived furanose amino acid in analogs
Sugar-peptide hybrids 11 exhibited a similar activity and selectivity index as miltefosine
against intra-macrophage amastigotes of L. donovani and Vero cells [32]. Conformational
9–11.
9–

Molecules 2021, 26, 3606
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analysis of sugar-peptide hybrids 9 and 11, by a combination of computational and NMR
spectroscopic methods, concluded a potential bent structure from a characteristic nuclear
Overhauser effect correlation between the phenylalanine N-methyl group and the d-proton
of the furanose amino acid residue [32].
R4 R5
N
CH3 CH3
N
O
O
O
H
N
R1
N
N
NH2
R6
R2
O
O
CH3
O
R3
R1
-CH2-CO-CH3 Me
-CH2
CH Me
-CH2 CH=CH2 Me
R2
R3
R4
Me
Me
Me
R5
Me
Me
Me
H
R6
Bn
Bn
Bn
Me
Me
Almiramide
A (1)
H
H
H
B (2)
C
C (3)
D (4)
Me Me sec-butyl
-CH2
C
CH
H
Me
Me Me
Me
H
E (5)
sec-butyl
sec-butyl
sec-butyl
-CH2
-CH2
-CH2
C
C
C
CH
CH
CH
F (6)
H
CH2OH
Me
G (7)
H
H
H (8)
Me Me sec-butyl
H3CO OCH3
H
H
-CH2
C
CH
O
O
N
R
N
O
N
NMe2
N
O
O
O
Ph
R = 9, -(CH2)3CH3; 10, -(CH2)2CH=CH2; 11, Ph
CH3
N
O
CH3
N
O
CH3 O
N
N
N
X
O
O
CH3 O
CH3
Ph
X = 12, OH; 13, N(CH3)2; 14, OCH3
Figure 1. Natural almiramides A-H (1–8) and carbohydrate and C-terminal derivatives 9–14.
The intriguing potential of the almiramides for the development of anti-parasitic
agents has prompted a more detailed study to ascertain the features responsible for their
potency and selectivity. In spite earlier efforts, limited knowledge exists of the relevance of
N-methylation and conformation for almiramide activity against parasite and host cells.
Among the reported almiramide analogs, peptides having only two N-methyl residues
[e.g., 5
, Val(Me)1 and Ile(Me)4] to completely permethylated amides (e.g., acid 12) have
been respectively isolated and synthesized. To the best of our knowledge, the activity of
unmethylated and singly methylated almiramide peptides has yet to be reported nor has the
effectiveness of almiramide analogs against resistant strains of Leishmania been examined.

Molecules 2021, 26, 3606
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A systematic study of almiramide peptides has now been performed to shine light on
the importance of tertiary amides and turn conformers for activity against Leishmania and
resistant strains.
2. Results and Discussion
2.1. Chemistry
Commencing with the potent and selective lead peptide 12, a systematic study of the
influence of N-methyl groups on each of the five amides was begun by the preparation
of the corresponding unmethylated peptide acid 15 (Figure 2). Considering that long
chain secondary amide derivatives of 12 retained significant anti-parasitic activity and
offered potential for the synthesis of conjugates to study mechanism of action [26], the
corresponding 1,6-hexanediamine amides 16 and 17 were examined for comparison with
permethylated and unmethylated acids 12 and 15. Subsequently, an N-methyl scan was
performed on amide 16 to provide singly methylated analogs 18–22. For systematic solid-
phase syntheses of N-methyl peptides 18–22, the required N-methyl amino acids (Fmoc-
Ala(Me)-OH, Fmoc-Phe(Me)-OH and Fmoc-Val(Me)-OH) were respectively prepared in
solution, according to literature protocols featuring acid-catalyzed condensation of an
Fmoc-protected amino acid with paraformaldehyde, to form an oxazolidinone followed by
reduction with triethyl silane and trifluoroacetic acid [33].
R1
N
R3
N
R5
N
O
O
O
N
N
X
R4
R2
O
O
O
Ph
R1
H
R2
R3
R4
R5
X
Peptide
15
16
17
18
19
20
21
22
H
H
H
H
H
OH
NH(CH2)6NH2
H
H
H
H
Me Me Me Me Me NH(CH2)6NH2
Me
H
H
Me
H
H
H
H
H
H
H
NH(CH2)6NH2
NH(CH2)6NH2
NH(CH2)6NH2
NH(CH2)6NH2
NH(CH2)6NH2
H
Me
H
H
H
H
H
Me
H
H
H
H
H
Me
Figure 2. N-Methyl scan of almiramide analogs.
In the study of biologically active peptides,
Figure 2) so-called Freidinger-Veber lactams have been commonly used to restrict backbone
and dihedral angles to favor -turn conformers, in which the Agl residue situates at
the i + 1 position [34 35]. The related N-amino-imidazol-2-one (Nai) residues offer similar
backbone constraint with potential to add substituents at the heterocycle 4- and 5-positions
to mimic side chain function with constrained dihedral angle geometry [35 36]. A
biologically active almiramide bent conformer has been proposed based on the activity
of sugar-peptide hybrids 11 32]. To probe this hypothesis further, Agl and (5-Me)Nai
α-amino γ-lactam residues (Agl residues,
ω
ψ
β
,
χ
,
9–
[

Molecules 2021, 26, 3606
5 of 23
residues were used to replace systematically the first four residues in the sequences of
peptides 12 and 15–17 (Figure 3). For example, six Agl almiramide derivatives (e.g., 23–28)
were respectively prepared by replacing valine (e.g., Val1, Val2, Val3) with the heterocycle
in acids 12 and 15. The first four residues of permethylated and unmethylated peptide
amides 16 and 17 were also systematically replaced by Agl residues to provide peptides
29–
35. Moreover, (5-Me)Nai residues were respectively used to replace Val1 and Val2 of
peptide acid 15 in peptides 36 and 37.
H3C
N
N
N
= Agl residue
= (5-Me)Nai residue
N
N
H
H
O
O
23: HCC(CH2)4CO-Agl-Val-Val-Ala-Phe-OH
24: HCC(CH2)4CO-Val-Agl-Val-Ala-Phe-OH
25: HCC(CH2)4CO-Val-Val-Agl-Ala-Phe-OH
26: HCC(CH2)4CO-Agl(Me)-Val-Val(Me)-Ala(Me)-Phe(Me)-OH
27: HCC(CH2)4CO-Val(Me)-Agl(Me)-Val-Ala(Me)-Phe(Me)-OH
28: HCC(CH2)4CO-Val(Me)-Val(Me)-Agl(Me)-Ala-Phe(Me)-OH
29: HCC(CH2)4CO-Agl-Val-Val-Ala-Phe-NH(CH2)6NH2
30: HCC(CH2)4CO-Val-Agl-Val-Ala-Phe-NH(CH2)6NH2
31: HCC(CH2)4CO-Val-Val-Agl-Ala-Phe-NH(CH2)6NH2
32: HCC(CH2)4CO-Val-Val-Val-Agl-Phe-NH(CH2)6NH2
33: HCC(CH2)4CO-Agl(Me)-Val-Val(Me)-Ala(Me)-Phe(Me)-NH(CH2)6NH2
34: HCC(CH2)4CO-Val(Me)-Agl(Me)-Val-Ala(Me)-Phe(Me)-NH(CH2)6NH2
35: HCC(CH2)4CO-Val(Me)-Val(Me)-Agl(Me)-Ala-Phe(Me)-NH(CH2)6NH2
36: HCC(CH2)4CO-(5-Me)Nai-Val-Val-Ala-Phe-OH
37: HCC(CH2)4CO-Val-(5-Me)Nai-Val-Ala-Phe-OH
Figure 3. Examination of turn conformation using Agl and Nai residues.
For the Agl scan, Fmoc-Agl dipeptides [Fmoc-Agl-Val-OH, Fmoc-Agl-Ala-OH and
Fmoc-Agl-Phe-OH (38a
modification of the original Freidinger-Veber protocol, as recently reported for the prepara-
tion of the valine dipeptide [34 37]. In brief, N-(Boc)methionyl dipeptide tert-butyl esters
–c), supporting information] were respectively synthesized by a
,
were treated with iodomethane to prepare the corresponding sulfonium ion intermedi-
ate, which on treatment with NaH underwent intramolecular N-alkylation to furnish
N-(Boc)Agl dipeptide tert-butyl esters (e.g., 39a–c, supporting information). The carbamate
and ester groups were removed using trifluoroacetic acid (TFA) in dichloromethane and the
Fmoc group was installed using N-(9-fluorenylmethoxycarbonyloxy)succinimide (Fmoc-
OSu) and sodium carbonate in aqueous acetone to provide Agl dipeptides 38. During
the cyclization to form Boc-Agl-Phe-Ot-Bu (39c, supporting information), ester epimeriza-
tion occurred providing an inseparable 2:1 mixture of diastereomers, which was used to
prepare the separable mixture of Agl-Phe peptide 32 and Agl-D-Phe isomer R-32. The cor-
responding Nai-dipeptide ester [Fmoc-(5-Me)Nai-Val-Ot-Bu (40, supporting information)]
was synthesized from Fmoc-azaGly-Val-Ot-Bu (41, supporting information), by a route
featuring oxidation to the corresponding azopeptide using N-bromosuccinimide (NBS)
and lutidine in dichloromethane, proline-catalyzed alkylation with propionaldehyde, and
dehydration using p-toluenesulfonic acid in chloroform [38]. Ester solvolysis using TFA
in dichloromethane gave the Fmoc-(5-Me)Nai-Val-OH which without purification was
coupled onto resin.
As illustrated by the synthesis of Agl analog 24, peptides 15
using conventional Fmoc/t-Bu solid phase peptide synthesis (SPPS) methods starting from
chlorotrityl resin (CTC resin, Scheme 1) [25 39]. N-Methyl peptide amides 18 22 were
–37, all were synthesized
,
–

Molecules 2021, 26, 3606
6 of 23
synthesized on CTC resin modified with a 1,6-diaminohexane linker. 1,6-Diaminohexane
was loaded onto the CTC resin using N,N0-diisopropylethylamine.
= a, O-CTC resin;
b, HN(CH2)6NH-CTC resin
H-Ala-Phe-
DIC, HOBt,
NMP
Fmoc-Agl-Val-OH
Fmoc-Agl-Val-Ala-Phe-
38a
42a,b
1) 20% piperidine, DMF
2) Fmoc-Val-OH, DIC, HOBt, NMP
3) 20% piperidine, DMF
H-Val-Agl-Val-Ala-Phe-
43a,b
HCC(CH2)4CO2H, DIC, HOBt, NMP
F3CO2H:Et3SiH:H2O
HCC(CH2)4CO-Val-Agl-Val-Ala-Phe-
44a,b
HCC(CH2)4CO-Val-Agl-Val-Ala-Phe-OH
(95:2.5:2.5)
24
HCC(CH2)4CO-Val-Agl-Val-Val-Phe-NH(CH2)6NH2
30
Scheme 1. Representative SPPS (solid phase peptide synthesis) synthesis of almiramides
derivatives 24 and 30.
N-(9-Fluorenylmethoxycarbonyl)phenylalanine was coupled to CTC resin using
N,N’-diisopropylethylamine. Alternatively, Fmoc-Phe-OH and Fmoc-Phe(Me)-OH were
coupled to 1,6-diaminohexane CTC resin using N,N’-diisopropylcarbodiimide (DIC) and
1-hydroxybenzotriazole (HOBt) in NMP. After Fmoc group removals with a 20% solu-
tion of piperidine in DMF, Fmoc-Ala-OH, Fmoc-Val-OH, Fmoc-Agl dipeptide acids 38a-c,
Fmoc-(5-Me)Nai-Val-OH and 6-heptynoic acid, all were respectively coupled using a
similar DIC/HOBt protocol. The N-methyl residues (e.g., Fmoc-Ala(Me)-OH and Fmoc-
Val(Me)-OH) were coupled using 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-
b]pyridinium 3-oxide hexafluorophosphate (HATU) and N-methylmorpholine in DMF [40].
Peptide cleavage was performed using a TFA/TES/H2O (95:2.5:2.5) cocktail. Final peptides
were precipitated with diethyl ether (Et2O) and purified by HPLC on a C18 column (Table 1).
Peptide acids (e.g. 24) were permethylated using sodium hydride and iodomethane in
THF (Scheme 2) [25]. As illustrated for the synthesis of amide 34, unmethylated and
permethylated peptide amides 16 and 17, as well as Agl peptide amides 29-35, all were
prepared by coupling the corresponding peptide acids (e.g., 27) to 1,6-diaminohexane using
N,N0-dicyclohexylcarbodiimide and 4-dimethylaminopyridine in dichloromethane.
Table 1. Purity, retention times, and mass of almiramide derivatives.
RT
MS [M + 1]
m/z (calcd)
Sequences
(R- = HCC(CH2)4CO-)
Purity at 214 nm
#
CH3CN b
7.19
CH3OH
6.83 a
m/z (obsd)
734.4468
642.3865
740.5069
R-Val(Me)-Val(Me)-Val(Me)-Ala(Me)-
Phe(Me)-OH
12
15
16
>99
>99
>99
734.4463
642.3861
740.5067
R-Val-Val-Val-Ala-Phe-OH
8.83 a
7.44
R-Val-Val-Val-Ala-Phe-
NH(CH2)6NH2
7.33 a
6.52
R-Val(Me)-Val(Me)-Val(Me)-Ala(Me)-
Phe(Me)-NH(CH2)6NH2
17
8.95 a
7.81
>99
810.5852
810.5851

Molecules 2021, 26, 3606
7 of 23
Table 1. Cont.
RT
MS [M + 1]
m/z (calcd) m/z (obsd)
Sequences
(R- = HCC(CH2)4CO-)
Purity at 214 nm
#
CH3CN b
CH3OH
R-Val(Me)-Val-Val-Ala-Phe-
NH(CH2)6NH2
18
19
20
21
22
8.42 a
7.86 a
7.91 a
7.61 a
7.62 a
6.90
6.15
6.57
6.44
6.62
>99
>99
>99
>99
>99
754.5225
754.5225
754.5225
754.5225
754.5225
754.5232
754.5235
754.5229
754.5225
754.5225
R-Val-Val(Me)-Val-Ala-Phe-
NH(CH2)6NH2
R-Val-Val-Val(Me)-Ala-Phe-
NH(CH2)6NH2
R-Val-Val-Val-Ala(Me)-Phe-
NH(CH2)6NH2
R-Val-Val-Val-Ala-Phe(Me)-
NH(CH2)6NH2
23
24
25
R-Agl-Val-Val-Ala-Phe-OH
R-Val-Agl-Val-Ala-Phe-OH
R-Val-Val-Agl-Ala-Phe-OH
7.11 c
7.55 c
7.94 c
6.71
6.87
7.17
>99
>99
>99
626.3548
626.3548
626.3548
626.3523
626.3529
626.3521
R-Agl(Me)-Val-Val(Me)-Ala(Me)-
Phe(Me)-OH
26
27
8.69 c
9.12 c
9.01 c
6.72 a
6.83 a
7.50 c
5.31 b
5.07 b
6.72 c
6.86 c
6.80 c
7.95
8.26
8.43
6.00
5.98
6.44
6.42
6.42
7.03
7.09
7.18
>99
>99
>99
>94
>96
>99
>92
>94
>99
>99
>99
682.4174
704.3993 d
704.3993 d
724.4756
724.4756
724.4756
752.5069
752.5069
780.5382
780.5382
780.5382
682.4149
704.3999 d
704.3964 d
724.4763
724.4774
724.4768
752.5082
752.5072
780.5389
780.5383
780.5394
R-Val(Me)-Agl(Me)-Val-Ala(Me)-
Phe(Me)-OH
R-Val(Me)-Val(Me)-Agl(Me)-Ala-
Phe(Me)-OH
28
R-Agl-Val-Val-Ala-Phe-
NH(CH2)6NH2
29
R-Val-Agl-Val-Ala-Phe-
NH(CH2)6NH2
30
R-Val-Val-Agl-Ala-Phe-
NH(CH2)6NH2
31
R-Val-Val-Val-Agl-Phe-
NH(CH2)6NH2
32
R-Val-Val-Val-Agl-D-Phe-
NH(CH2)6NH2
R-32
33
R-Agl(Me)-Val-Val(Me)-Ala(Me)-
Phe(Me)-NH(CH2)6NH2
R-Val(Me)-Agl(Me)-Val-Ala(Me)-
Phe(Me)-NH(CH2)6NH2
34
R-Val(Me)-Val(Me)-Agl(Me)-Ala-
Phe(Me)-NH(CH2)6NH2
35
36
37
R-(5-Me)Nai-Val-Val-Ala-Phe-OH
R-Val-(5-Me)Nai-Val-Ala-Phe-OH
5.34 c
4.41 c
6.48
8.68
>99
>96
639.3501
639.3501
639.3474
-
Isolated purity ascertained by LC-MS analysis using gradients of X
−
Y% A [H2O (0.1% FA)/MeOH (0.1% FA)] or B [H2O (0.1% FA)/MeCN
a
b
d
(0.1% FA)] over Z min: 30−95% A/14. 10−90% B/14; c 50−90% A/14. [M + Na].
2.2. Bioactivity
The biological activity of peptides 15–37 was assessed against the L. infantum wild-
type stain (WT), as well as mutants resistant to the common anti-leishmanial agents, such
as antimony (SbIII), amphotericin B (AmB) and miltefosine (MF): Sb2000.1, AmB1000.1
and MF200.5. Activity against Leishmania promastigotes was determined by monitoring
the replication of parasites after 72 h of incubation at 25 ◦C in the presence of increasing
concentrations of the different peptides 12–37 and reported as EC50 values (Table 2). In

Molecules 2021, 26, 3606
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general, almiramide peptides 12
strains in the range of 5–300
against the amphotericin B resistant strain (e.g., AmB1000.1).
–
37 exhibited activity against wild type and resistant
µM. The greatest potencies were observed using analogs
Scheme 2. Representative permethylation of peptide acid 24 and coupling to 1,6-diaminohexane to prepare peptide
amide 34.
Examining peptide potency (EC50) against wild type L. infantum, the 1,6-diaminohexane
amides (e.g., 16 and 17) were respectively more potent than the acid counterparts (e.g., 15
and 12). In the Agl series, amides 29
acid counterparts 23 25 and 26 28. Moreover, the unmethylated analogs (e.g., 15
and 31) were typically more potent than the respective permethylated counterparts (e.g.,
12 17
33 and 35), except in the case of Agl2 analogs 30 and 34. Comparison of the activity
–
31 and 33
–
35 were similarly more active than their
–
–
,
16, 29
,
,
of unmethylated 1,6-diaminohexane amide 16 with singly methylated counterparts 18–22
illustrated decreasing potency in the order of 16 > MePhe5 22 > MeVal1 18 > MeVal3 20
> MeAla4 21 > MeVal2 19. Considering the Agl analogs as constrained versions of the
corresponding N-methyl dipeptide amides, except in the case of MePhe 22 which was
significantly more potent than Agl-Phe 32, the more restricted Agl analogs 29
slightly more active than their N-methyl counterparts 19 21. Activity against Leishmania
28) and their (5-Me)Nai
–31 were
–
dropped significantly in the case of the Agl peptide acids (23
counterparts (36 and 37).
–
Many of the structure–activity relationships, that were observed in the wild-type
strain, were also found in the resistant strains. For example, the amides were more potent
than their acid counterparts. The nonmethylated analogs were also typically more potent
than their permethylated analogs; however, in the case of the amphotericin B resistant
strain (e.g., AmB1000.1), permethylated analogs 17 and 35 were respectively 4- and 1.4-fold
more active than nonmethylated counterparts 16 and 31, and in the case of the miltefosine
resistant strain (e.g., MF200.5), 35 was 1.5-fold more active than 31. Although the order
of potency for nonmethylated 16, MeVal1 18 and MePhe5 22 was contingent on the tested
resistant strain, analogs with no tertiary amide and methylation at the C- and N-terminal
residues were consistently more active than peptides 19–21 with N-methylation in the
central residues. The potency of MePhe5 22 was greater than Agl-Phe5 32 for all strains
tested. The Agl constraint at other positions tended to slightly favor activity compared
to the tertiary amide counterpart in the antimony resistant strain (e.g., Sb2000.1) as was
observed in wild type; however, the lactam analogs were significantly less active than the
respective N-methyl counterparts against the miltefosine and amphotericin B strains. In
addition, preliminary investigations of peptides (e.g., 12–22), which had notable potency
against Leishmania (EC50 = 5–90
(data not shown).
µM), detected no activity against T. brucei and T. cruzi
The host cytotoxicity was evaluated by monitoring the survival rates of murine
LM-1 macrophage at different concentrations of peptides 15 37 (0.0001 to 100 mM) over
–

Molecules 2021, 26, 3606
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24 h. Most analogs exhibited CC50 values between 170–450
µM. Exceptionally, MeAla
peptide amide 21 was 3.2- to 8-fold less toxic than the other peptide analogs. Comparing
macrophage toxicity with potency against L. infantum, the selectivity index (SI) for MeAla
peptide amide 21 was relatively high (SI = 19.1) and comparable with the most potent
peptide (e.g., 16, SI = 19.2). In comparisons of macrophage toxicity with potency against
the AmB resistant Leishmania strain, permethylated and MeVal1 peptide amides 17 and 18
exhibited, respectively, therapeutic indices of 64 and 36.
Table 2. Structure, bioactivity of almiramide derivatives on different strains of Leishmania (EC50) and cytotoxicity of
peptides 12–39.
Sequence
(R- = HCC(CH2)4CO-)
Ldi WT 1
(µM)
Ldi Sb-Res 2
Ldi MF-Res 3
Ldi AmB-Res 4
CC50
(µM)
Selectivity
Index
#
(µM)
(µM)
(µM)
R-Val(Me)-Val(Me)-Val(Me)-
Ala(Me)-Phe(Me)-OH
68.33
70.69
75.60
79.59
12
15
16
17
18
19
20
21
22
23
281
316
446
338
177
316
281
1440
446
363
4.1
7.0
[61.53, 75.00]
[65.62, 75.89]
[71.05, 79.15]
[73.92, 85.46]
44.69
[34.60, 58.13]
60.26
[54.09, 66.33]
50.77
[44.46, 57.93]
46.38
[41.63, 51.59]
R-Val-Val-Val-Ala-Phe-OH
R-Val-Val-Val-Ala-Phe-
NH(CH2)6NH2
23.38
[17.49, 32.79]
30.34
[24.31, 38.73]
18.09
[16.25, 20.15]
20.76
[18.22, 23.64]
19.0
6.9
R-Val(Me)-Val(Me)-Val(Me)-
Ala(Me)-Phe(Me)-NH(CH2)6NH2
48.42
[44.44, 52.60]
60.18
[55.50, 65.25]
25.46
[21.71, 29.97]
5.60
[5.06, 6.16]
R-Val(Me)-Val-Val-Ala-Phe-
36.70
[33.61, 40.00]
39.65
[36.65, 42.83]
12.12
[10.87, 13.58]
5.03
[4.71, 5.34]
4.8
NH(CH2)6NH2
R-Val-Val(Me)-Val-Ala-Phe-
81.17
[75.54, 87.30]
78.91
[74.53, 83.48]
89.23
[83.11, 95.96]
70.26
[65.91, 75.02]
3.9
NH(CH2)6NH2
R-Val-Val-Val(Me)-Ala-Phe-
53.86
[46.63, 62.51]
40.25
[34.98, 46.35]
60.64
[55.44, 66.22]
57.18
[52.23, 62.35]
5.2
NH(CH2)6NH2
R-Val-Val-Val-Ala(Me)-Phe-
75.83
[70.34, 81.57]
79.78
[74.41, 85.68]
63.53
[58.45, 69.01]
65.66
[59.68, 72.35]
18.9
15.3
1.61
NH(CH2)6NH2
R-Val-Val-Val-Ala-Phe(Me)-
29.04
[24.49, 34.38]
25.02
[20.45, 30.70]
27.76
[23.00, 33.13]
25.91
[21.00, 31.54]
NH(CH2)6NH2
225.1
[201.5, 261.6]
240.6
[209.0, 299.3]
231.5
[203.1, 280.7]
211.7
[193.1, 238.8]
R-Agl-Val-Val-Ala-Phe-OH
248.1
251.1
237.5
261.1
24
25
26
R-Val-Agl-Val-Ala-Phe-OH
R-Val-Val-Agl-Ala-Phe-OH
398
416
316
1.6
-
[231.3, 270.5]
[233.4, 274.7]
[218.6, 263.7]
[233.1, 304.7]
N.D.
N.D.
N.D.
N.D.
R-Agl(Me)-Val-Val(Me)-Ala(Me)-
217.00
[204.5, 232.8]
220.4
[209.9, 233.0]
223.7
[211.1, 239.1]
252.3
[229.1, 284.9]
1.46
Phe(Me)-OH
R-Val(Me)-Agl(Me)-Val-Ala(Me)-
Phe(Me)-OH
283.5
295.8
27
28
N.D.
N.D.
N.D.
295
245
416
407
389
389
338
407
398
371
1.0
0.9
5.5
8.6
6.0
4.3
3.6
3.4
12.0
4.7
[261.8, 314.1]
[275.1, 324.4]
R-Val(Me)-Val(Me)-Agl(Me)-Ala-
254.7
[238.4, 274.5]
250.2
[232.1, 272.7]
281.5
[269.8, 295.7]
Phe(Me)-OH
R-Agl-Val-Val-Ala-Phe-
74.98
[69.52, 80.41]
90.39
[86.16, 94.65]
67.89
[64.58, 71.27]
71.98
[69.28, 74.69]
29
NH(CH2)6NH2
R-Val-Agl-Val-Ala-Phe-
47.43
[40.21, 55.05]
37.65
[31.00, 45.19]
129.3
[125.3, 133.6]
114.4
[111.4, 117.5]
30
NH(CH2)6NH2
R-Val-Val-Agl-Ala-Phe-
64.64
[59.94, 69.44]
48.78
[41.26, 56.36]
126.4
[122.8, 130.1]
130.20
[128.1, 132.6]
31
NH(CH2)6NH2
R-Val-Val-Val-Agl-Phe-
91.17
[88.95, 93.47]
87.78
[85.60, 90.02]
84.21
[82.07, 86.39]
90.08
[88.22, 91.99]
32
NH(CH2)6NH2
R-Val-Val-Val-Agl-D-Phe-
92.56
[90.51, 94.66]
110.8
[106.2, 115.5]
123.1
[120.5, 125.8]
117.6
[114.8, 120.4]
R-32
33
NH(CH2)6NH2
R-Agl(Me)-Val-Val(Me)-Ala(Me)-
119.4
[116.2, 122.8]
140.4
[138.4, 142.4]
191.3
[185.1, 198.4]
183.7
[177.1, 191.1]
Phe(Me)-NH(CH2)6NH2
R-Val(Me)-Agl(Me)-Val-Ala(Me)-
33.22
[31.06, 35.49]
35.75
33.73, 37.88]
23.13
[20.72, 25.80]
24.75
[22.12, 27.67]
34
Phe(Me)-NH(CH2)6NH2
R-Val(Me)-Val(Me)-Agl(Me)-Ala-
78.56
[74.91, 82.31]
81.52
[78.04, 85.14]
85.30
[80.97, 89.88]
92.04
[88.01, 96.27]
35
Phe(Me)-NH(CH2)6NH2
36
37
R-(5-Me)Nai-Val-Val-Ala-Phe-OH
R-Val-(5-Me)Nai-Val-Ala-Phe-OH
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
436
407
-
-
1 = Leishmania infantum (MHOM/MA/67/ITMAP-263) wild-type strain. 2 = Leishmania infantum (MHOM/MA/67/ITMAP-263) resistant
to 2 mM trivalent (and pentavalent) antimonials. 3 = Leishmania infantum (MHOM/MA/67/ITMAP-263) resistant to 200 M miltefosine.
4 = Leishmania infantum (MHOM/MA/67/ITMAP-263) resistant to 1 M amphotericin B. 95% confidence intervals for EC50 determinations
are reported within brackets (n = 3). SI = selectivity index = CC50/EC50 Ldi WT. N.D. = Non disponible = >300 µM.
µ
µ

Molecules 2021, 26, 3606
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3. Discussion
The structure–activity relationship studies obtained in wild type and resistant strains
of L. infantum reflect likely a combination of ability to engage the target and pharmacoki-
netic properties that may influence peptide availability. The molecular targets of almi-
ramide C have been studied using a combination of photo-affinity and fluorescent probes
in T. brucei, and suggested to include integral membrane proteins found in glycosomes
(e.g., GIM5 and PEX11), which are specific to kinetoplastid parasites [26]. Responsible
for the first seven steps of glycolysis, the glycosome is a peroxisome-related organelle
essential for parasite survival in the bloodstream stage [41]. Notably, translocation across
the glycosomal membrane implicates transporter and pore-forming proteins [42], which
may differ contingent on species and be modified in resistant strains.
Resistant strains of Leishmania emerge by different mechanisms which contingent on
the drug act commonly to reduce the active concentration inside the parasite by either
decreasing uptake, increasing efflux or inhibiting activity [43]. Leishmania antimonial
resistance is associated with thiol metabolism to prevent reduction of the SbV to more
active SbIII, and to sequester antimony in thiolate complexes amenable for efflux [44].
Miltefosine resistance is commonly associated with mutations in the Leishmania miltefosine
transporter (LMT), a P-type ATPase responsible for the translocation of phospholipids,
as well as overexpression of ABC transporters [44]. Amphotericin B resistant L. donovani
promastigotes have been shown to feature substitution of ergosterol for another sterol,
which alters the fluidity and AmB binding affinity in the cell membrane [45].
Although the conformational preferences of the almiramides alone and target bound
have yet to be described, information gleaned from related peptides and their N-methyl
and lactam counterparts offers a lens through which to interpret the structure–activity
relationships. For example, the circular dichroism spectrum of the (Val-Val-Val-Ala)n
oligomer in a mixture of hexafluoro-2-propanol and trifluoro ethanol indicated a curve
shape typical of an extended
region in nonmethylated almiramide analogs 15 and 16, as well as MePhe analog 22 may
likely adopt an extended -strand conformer. Introduction of N-methyl groups causes
β-sheet structure [46]. The corresponding Val-Val-Val-Ala
β
significant consequences on peptide conformation, due in part to creation of a tertiary
amide which loses a potential NH hydrogen-bond donor and may adopt energetically
similar cis and trans isomers [47]. Computational analysis of the N’-methyl amides of
N-acetyl-N-methyl- and N-acetyl-alanine indicated that repulsive interactions between the
N-methyl and carbonyl oxygen moieties of the former abolished the low-energy minimum
β
-conformer adopted by the latter [48]. In cyclic peptides, N-methyl residues have also
induced backbone f and dihedral angles consistent with - and -turn conformers [49],
as well as altered side chain geometry [50]. N-Alkylation of the central amide of the
ψ
β
γ
χ
hairpin inducing D-Pro-Aib dipeptide has also been shown by variable temperature CD
spectroscopy to reinforce the central turn conformer and enhance the stability of the
folded
causes likely a shift from an extended sequence to a dynamic series of cis- and trans-
amide conformers exhibiting a preference for turn geometry, which in peptides 19 21
β-sheet peptide [51]. Introduction of N-methyl residues within the peptide chain
–
reduces activity. In MeVal1 analog 18 and permethylated analogs 12 and 17, methylation
at the N-terminal enables a cis-amide conformer in which the lipid tail may fold in the
direction of the peptide. Such a geometry may improve membrane transport by hiding
hydrophilic NH moieties and may account for the significantly improved activity of 17
and 18 against the amphotericin B resistant strains. To further examine the influence of
N-methylation on conformation, the 1H NMR spectra of nonmethylated peptide 16 and
N-methyl counterpart 18 were examined in DMSO-d6 (supporting information). In contrast
to peptide 16, which exhibited a narrow distribution of amide protons signals between 7.55
and 7.95 ppm characteristic of a linear conformer, the corresponding peaks were downfield
shifted, dispersed between 7.65 and 8.35 ppm, and existed in isomeric pairs for MeVal1
peptide 18, likely due the tertiary amides favoring cis- and trans-amide isomers of similar
energy and local turn conformers with intramolecular hydrogen bonds.

Molecules 2021, 26, 3606
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The propensity for Agl bearing peptides to adopt type II and II’
β-turns contingent on
α
-carbon stereochemistry has been demonstrated using spectroscopic and computational
methods, as well as X-ray diffraction, which has also characterized crystals of lactam
analog in extended conformers [52]. Relatively diminished activities of Agl analogs 29 35
compared to nonmethylated peptide 16 may again be due to the favored turn geometry.
The slightly better activity exhibited by Agl analogs 29 31 in comparison to N-methyl
counterparts 19 21 in wild type L. infantum may be attributed to the capacity of the
–
–
–
amino lactam to stabilize trans-amide isomers. On the other hand, the notably better
activity of MePhe analog 22 relative to Agl counterpart 32 indicates the attributes of greater
conformational flexibility at the C-terminal. Similarly, greater conformational dynamics
of N-methyl analogs compared to the Agl counterparts appear to be important for the
relatively better activity of the former in resistant Leishmania strains.
4. Materials and Methods
4.1. Experimental Section
4.1.1. Leishmania Cultures and Antileishmanial Activity Determination
The L. infantum (MHOM/MA/67/ITMAP-263) wild-type stain (WT), as well as the
resistant mutants Sb2000.1, AmB1000.1 and MF200.5 [53
amphotericin B (AmB) and miltefosine (MF), respectively, were grown in M199 medium
at 25 ◦C supplemented with 10% fetal bovine serum, 5
g/mL of haemin at pH 7.0 and
2000 µM Sb (Potassium antimonyl tartrate, Sigma-Aldrich), 200 M of MF (Miltefosine,
Cayman Chem., Ann Arbor, MI, USA) or 1 M AmB (Amphotericin B solution, Sigma,
–
58], resistant to antimony (SbIII),
µ
µ
µ
Oakville, ON, Canada). Antileishmanial values were determined in Leishmania p◦romastig-
otes by monitoring the replication of parasites after 72 h of incubation at 25 C in the
presence of increasing concentrations of the different peptides by measuring A600 using
a Cytation 5 machine (BioTek, Winooski, VT, USA). EC50 values were calculated based
on dose–response curves analyzed by non-linear regression with GraphPad Prism 8.4.3
software (GraphPad Software, La Jolla, CA, USA). An average of at least three independent
biological replicates from independent cultures was performed for each determination.
4.1.2. LM-1 Macrophages and Cytotoxicity Determination
The LM1- macrophages were grown in DMEM supplemented with 10% heat inac-
tivated FBS. Cells at the concentration of 100,000 cells/mL were cultivated for 24 h in
a 96-well plate (37 ◦C, 5% CO2). The culture medium was removed and fresh medium
containing the appropriate drug and concentration was added to the cells, which were
incubated for 24 h (37 ◦C, 5% CO2). Seven different concentrations were tested (100, 10,
1, 0.1, 0.01, 0.001 and 0.0001 mM) as well as controls (without drugs). After 24 h, the
culture medium was removed and replaced for fresh medium containing 10% Alamar Blue
◦
(Thermo Fisher Scientific, Waltham, MA, USA) and incubated for 2 h (37 C, 5% CO2).
Readings at 570 and 600 nm (Asys UVM340 Plate reader, Biochrom, Cambridge, UK) were
taken and analyzed according to the manufacturer protocol. Survival rates at different
drug concentrations and CC50 values were calculated using the Excel software.
4.2. Materials
Anhydrous solvents (THF, DMF, CH2Cl2, and NMP) were obtained by passage
through solvent filtration systems (GlassContour, Irvine, CA, USA). Unless specified other-
wise, all reagents from commercial sources were used as received. 2-Chlorotrityl chloride
(CTC) resin (1.46 mmol/g, 100–200 mesh), N-(9-fluorenylmethoxycarbonyloxy)succinimide
(Fmoc-Osu), N,N0-diisopropylcarbodiimide (DIC), all were purchased from ChemImpex;
4-dimethylaminopyridine (DMAP), N,N0-dicyclohexylcarbodiimide (DCC), iodomethane,
N,N-diisopropylethylamine (DIEA), formic acid (FA), all were purchased from Aldrich;
amino acids, such as Fmoc-Phe-OH, Fmoc-Ala-OH, Fmoc-Val-OH, and 6-heptynoic acid
were purchased from GL Biochem, ChemImpex and Combi-blocks; solvents were obtained
from Fisher. Sodium hydride (60% dispersion in mineral oil) was washed with hexane

Molecules 2021, 26, 3606
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three times to remove oil prior to use. The N-methyl amino acids, Fmoc-Phe(Me)-OH,
Fmoc-Ala(Me)-OH and FmocVal(Me)-OH, were prepared by according to the literature
procedure and exhibited 1H NMR spectra data identical to that previously reported [33].
The Agl dipeptide, Fmoc-Agl-Val-OH was prepared according to the literature procedure
and exhibited a 1H NMR spectrum identical to that previously reported [37].
Chromatography was on 230 400 mesh silica gel. Analytical thin-layer chromatogra-
−
phy (TLC) was performed on glass-backed silica gel plates (Merck 60 F254). Visualization
of the developed chromatogram was performed by UV absorbance or staining with nin-
1
hydrin. H and 13C NMR spectra were measured respectively in CDCl3 at 500 MHz and
126 MHz, and referenced to CDCl3 (7.26 and 77.0 ppm). Coupling constants, J values were
measured in Hertz (Hz) and chemical shift values in parts per million (ppm). Specific
◦
rotations, [
α
]
were measured at 25 C at the specified concentrations (c in g/100 mL)
D
using a 1 dm cell on a PerkinElmer Polarimeter 589 and expressed using the general for-
mula: [αD25] = (100 × α)/(d
×
c). High resolution mass spectral analyses were obtained
by the Centre Régional de Spectrométrie de Masse de l’Université de Montréal. Proto-
nated molecular ions [M + H]+ and sodium adducts [M + Na]+ were used for empirical
formula confirmation.
Almiramide peptide analog synthesis was performed using Fmoc-based solid-phase
synthesis in an automated shaker commencing with 2-chlorotrityl chloride resin. All final
peptides were purified on a preparative column (C18 Gemini column) using a gradient from
pure water (0.1% FA) to mixtures with MeOH (0.1% FA) at a flow rate of 10 mL/min. Purity
of peptides (>95%) was evaluated using analytical LC
−MS on a 5 µM 50 mm × 4.6 mm
C18 Phenomenex Gemini column in two different solvent systems: water (0.1% FA) with
CH3CN (0.1% FA) and water (0.1% FA) with MeOH (0.1% FA) at a flow rate of 0.5 mL/min
using the appropriate linear gradient.
12
HCC(CH2)4CO-Val(Me)-Val(Me)-Val(Me)-Ala(Me)-Phe(Me)-OH (12) Under argon,
◦
peptide 15 (200 mg) was dissolved in THF (40 mL), cooled to 0 C, treated with NaH
(600 mg, 80 eq.), stirred for 5 min, and treated dropwise with iodomethane (0.6 mL, 9 mmol,
30 eq.). The cooling bath was removed. The reaction mixture warmed to room temperature.
After 2 h, more iodomethane (0.6 mL, 30 eq.) was added to the reaction mixture, which
was stirred for 20 h. The suspension was quenched with water, concentrated to a reduced
volume, and acidified to pH = 1 using 10% aqueous HCl. The acidified mixture was
extracted 3 times with EtOAc. The organic layers were combined, dried over Na2SO4,
filtered and evaporated to 174 mg of residue, from which part (87 mg) was used to make
peptide 17 as described below, and the remainder was purified by HPLC on a C18 column
using a gradient of 30% to 90% MeOH in H2O to obtain peptide 12 (16 mg, 13%), which
was shown to be >99% pure by LC-MS analysis [30
FA), 14 min, RT 6.83 min].
−
95% MeOH (0.1% FA) in H2O (0.1%

Molecules 2021, 26, 3606
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15
HCC(CH2)4CO-Val-Val-Val-Ala-Phe-OH (15) Fmoc-Phe-OH (848 mg, 1.5 eq.) and
DIEA (0.8 mL, 3 eq.) were added to a suspension of 2-chlorotrityl chloride resin (1 g,
200–400 mesh, 1% DBV) swollen in CH2Cl2 (25 mL). The mixture was shaken for 18 h,
filtered and washed sequentially with CH2Cl2 (3 times for 1 min/wash) and DMF (3 times
for 1 min/wash). The Fmoc group was removed upon treatment twice for 20 min with a 20%
solution of piperidine in DMF (20 mL/g resin). Subsequently, Fmoc-Ala-OH (3 eq.) was
coupled to the resin swollen in NMP (25 mL/g resin) using DIC (3 eq.) and HOBt (3 eq.).
After shaking for 16 h, the coupling mixture was filtered, and the resin was washed as
described above. Subsequent Fmoc group removal and coupling of Fmoc protected amino
acid residues were performed using the above protocols. Complete coupling reactions
were confirmed by LC–MS monitoring. After coupling of the last residue, the 6-heptynoic
acid (3 eq.), DIC (3 eq.) and HOBt (3 eq.) were added to the resin. The mixture was shaken
for 18 h, filtered and washed as previously described. Then, the linear peptide was cleaved
from the solid support using a solution of TFA/TES/H2O (95/2.5/2.5). The volatiles were
evaporated. The reduced volume was treated with diethyl ether. The precipitate was
collected by centrifugation (1200 rpm), washed with ether and recollected by centrifugation
(3
×
10 min). Removal of diethyl ether afforded a colorless solid [2], which gave 387 mg of
residue, from which 200 mg was used to make peptide 12 as described below, and 60 mg
purified by HPLC on C18 column using a gradient of 30% to 90% MeOH in H2O to obtain
peptide 15 (24 mg, 12%), which was shown to be >99% pure by LC-MS analysis [30 95%
−
MeOH (0.1% FA) in H2O (0.1% FA), 14 min, RT 8.33 min].
16
HCC(CH2)4CO-Val-Val-Val-Ala-Phe-NH(CH2)6NH2 (16) A suspension of 2-chlorotrityl
chloride resin (250 mg, 200–400 mesh, 1% DBV) swollen in CH2Cl2 (25 mL) was treated
with 1,6-diaminohexane (51 mg, 1.2 eq.) and DIEA (0.2 mL, 3 eq.), shaken for 24 h, filtered
and washed sequentially with CH2Cl2 (3 times for 1 min/wash) and DMF (3 times for
1 min/wash). Subsequently, Fmoc-Phe-OH (3 eq.) was coupled to the resin swollen in
NMP (25 mL/g resin) using DIC (3 eq.) and HOBt (3 eq.). After shaking for 16 h, the
coupling mixture was filtered, and the resin was washed as described above. Subsequent
Fmoc group removals, couplings of Fmoc protected amino acid residues, and acylation
with 6-heptynoic acid, all were performed using the protocols described for the synthesis
of peptide 15. The linear peptide was cleaved from the solid support using a solution
of TFA/TES/H2O (95/2.5/2.5). Removal of the volatiles gave a colorless oil, which was
purified by HPLC on a C18 column using a gradient of 30% to 90% MeOH in H2O to obtain
peptide 16 as a white solid (8.8 mg, 3%), which was shown to be >99% pure by LC-MS
analysis [30−95% MeOH (0.1% FA) in H2O (0.1% FA), 14 min, RT 7.33 min].

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17
HCC(CH2)4CO-Val(Me)-Val(Me)-Val(Me)-Ala(Me)-Phe(Me)-NH(CH2)6NH2 (17)
A
solution of permethylated peptide 12 (87 mg) in dry CH2Cl2 (15 mL) was treated with
1,6-hexamethylenediamine (17 mg, 1.2 eq.), DMAP (4 mg, 0.3 eq.), and DCC (30 mg, 1.2 eq.),
and stirred for 48 h at room temperature. The reaction mixture was filtered through Celite
™
and washed with CH2Cl2. The filtrate and washings were evaporated under reduced
pressure. The residue was purified by HPLC on a C18 column using a gradient of 30% to
90% MeOH in H2O. Evaporation of the collected fractions afforded amino hexanamide
17 as white solid (17 mg, 14%), which was shown to be >99% pure by LC-MS analysis
[30−95% MeOH (0.1% FA) in H2O (0.1% FA), 14 min, RT 9.01 min].
18
HCC(CH2)4CO-Val(Me)-Val-Val-Ala-Phe-NH(CH2)6NH2 (18) As described for the
synthesis of peptide 16, 1,6-diaminohexane (51 mg, 1.2 eq.) and DIEA (0.2 mL, 3 eq.) were
reacted with 2-chlorotrityl chloride resin (250 mg, 200–400 mesh, 1% DBV) in CH2Cl2
(25 mL). The resulting amine resin was swollen in NMP (25 mL/g resin), treated with
Fmoc-Phe-OH (3 eq.), DIC (3 eq.) and HOBt (3 eq.), shaken for 16 h, filtered, and washed
as described above. The Fmoc group was removed upon treatment twice for 20 min with
a 20% solution of piperidine in DMF (20 mL/g resin). After Fmoc group removal, the
peptide was elongated, coupled to 6-heptynoic acid using HATU (3 eq.) in 0.4 NMM in
DMF (25 mL/g resin), cleaved and purified as described for the synthesis of peptide 16
.
Evaporation of the collected fractions gave peptide 18 (8.2 mg, 3%), which was prepared
and shown to be >99% pure by LC-MS analysis [30
FA), 14 min, RT 8.42 min].
−
95% MeOH (0.1% FA) in H2O (0.1%
19
HCC(CH2)4CO-Val-Val(Me)-Val-Ala-Phe-NH(CH2)6NH2 (19) Employing the proto-
col described above for the synthesis of peptide 18 using Fmoc-Val(Me)-OH (386 mg, 3 eq.)
in the fourth coupling, peptide 21 (6.4 mg, 2%) was prepared and shown to be >99% pure
by LC-MS analysis [30−95% MeOH (0.1% FA) in H2O (0.1% FA), 14 min, RT 7.86 min].

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20
HCC(CH2)4CO-Val-Val-Val(Me)-Ala-Phe-NH(CH2)6NH2 (20) Employing the proto-
col described above for the synthesis of peptide 18 using Fmoc-Val(Me)-OH (386 mg, 3 eq.)
in the third coupling, peptide 20 (5.8 mg, 2%) was prepared and shown to be >99% pure by
LC-MS analysis [30−95% MeOH (0.1% FA) in H2O (0.1% FA), 14 min, RT 7.91 min].
21
HCC(CH2)4CO-Val-Val-Val-Ala(Me)-Phe-NH(CH2)6NH2 (21) Employing the proto-
col described above for the synthesis of peptide 18 using Fmoc-Ala(Me)-OH (355 mg, 3 eq.),
peptide 21 (7.8 mg, 3%) was prepared and shown to be >99% pure by LC-MS analysis
[30−95% MeOH (0.1% FA) in H2O (0.1% FA), 14 min, RT 7.61 min]
22
HCC(CH2)4CO-Val-Val-Val-Ala-Phe(Me)-NH(CH2)6NH2 (22) Employing the proto-
col described above for the synthesis of peptide 22 using Fmoc-Phe(Me)-OH (528 mg, 3 eq.),
peptide 22 (4.5 mg, 2%) was prepared and shown to be >99% pure by LC-MS analysis
[30−95% MeOH (0.1% FA) in H2O (0.1% FA), 14 min, RT 7.62 min].
23
HCC(CH2)4CO-Agl-Val-Val-Ala-Phe-OH (23) Employing the protocol described above
for the synthesis of peptide 15 using Fmoc-Agl-Val-OH (462 mg, 3 eq.) in the third coupling,
peptide 23 (7.5 mg, 3%) was prepared and shown to be >99% pure by LC-MS analysis
[50−95% MeOH (0.1% FA) in H2O (0.1% FA), 14 min, RT 7.11 min].

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24
HCC(CH2)4CO-Val-Agl-Val-Ala-Phe-OH (24) Employing the protocol described above
for the synthesis of peptide 15 using Fmoc-Agl-Val-OH (462 mg, 3 eq.) in the second
coupling, peptide 24 (8.4 mg, 3%) was prepared and shown to be >99% pure LC-MS
analysis [50−95% MeOH (0.1% FA) in H2O (0.1% FA), 14 min, RT 7.55 min].
25
HCC(CH2)4CO-Val-Val-Agl-Ala-Phe-OH (25) Employing the protocol described above
for the synthesis of peptide 15 using Fmoc-Agl-Ala-OH (359 mg, 3 eq.) in the first coupling,
peptide 25 (9.1 mg, 3%) was prepared and shown to be >99% pure by LC-MS analysis
[30−95% MeOH (0.1% FA) in H2O (0.1% FA), 14 min, RT 7.94 min].
26
HCC(CH2)4CO-Agl(Me)-Val-Val(Me)-Ala-Phe(Me)-OH (26) Employing the proto-
col described above for the synthesis of peptide 12 using Fmoc-Agl-Val-OH (462 mg, 3 eq.)
in the third coupling, followed by permethylation, peptide 26 (9.5 mg, 3%) was prepared
and shown to be >95% pure by LC-MS analysis [50
FA), 14 min, RT 8.69 min].
−
90% MeOH (0.1% FA) in H2O (0.1%
27
HCC(CH2)4CO-Val(Me)-Agl(Me)-Val-Ala(Me)-Phe(Me)-OH (27) Employing the pro-
tocol described above for the synthesis of peptide 12 using Fmoc-Agl-Val-OH (462 mg,
3 eq.) in the second coupling, followed by permethylation, peptide 27 (8.3 mg, 3%) was

Molecules 2021, 26, 3606
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prepared and shown to be >95% pure by LC-MS analysis [50
H2O (0.1% FA), 14 min, RT 9.12 min].
−
90% MeOH (0.1% FA) in
28
HCC(CH2)4CO-Val(Me)-Val(Me)-Agl(Me)-Ala-Phe(Me)-OH (28) Employing the proto-
col described above for the synthesis of peptide 12 using Fmoc-Agl-Ala-OH (359 mg, 3 eq.)
in the second coupling, followed by permethylation, peptide 28 (6.7 mg, 2%) was prepared
and shown to be >95% pure by LC-MS analysis [50
FA), 14 min, RT 9.01 min].
−
90% MeOH (0.1% FA) in H2O (0.1%
29
HCC(CH2)4CO-Agl-Val-Val-Ala-Phe-NH(CH2)6NH2 (29) Employing the protocol
described above for the synthesis of peptide 18 using Fmoc-Agl-Val-OH (462 mg, 3 eq.) in
the fourth coupling, peptide 29 (7.5 mg, 3%) was prepared and shown to be >95% pure by
LC-MS analysis [30−95% MeOH (0.1% FA) in H2O (0.1% FA), 14 min, RT 6.72 min].
30
HCC(CH2)4CO-Val-Agl-Val-Ala-Phe-NH(CH2)6NH2 (30) Employing the protocol
described above for the synthesis of peptide 18 using Fmoc-Agl-Val-OH (462 mg, 3 eq.)
in the third coupling, peptide 30 (8.4 mg, 3%) was prepared and shown to be >95% pure
LC-MS analysis [30−95% MeOH (0.1% FA) in H2O (0.1% FA), 14 min, RT 6.83 min].
31

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HCC(CH2)4CO-Val-Val-Agl-Ala-Phe-NH(CH2)6NH2 (31) Employing the protocol
described above for the synthesis of peptide 18 using Fmoc-Agl-Ala-OH (359 mg, 3 eq.) in
the second coupling, peptide 31 (9.1 mg, 3%) was prepared and shown to be >99% pure by
LC-MS analysis [30−95% MeOH (0.1% FA) in H2O (0.1% FA), 14 min, RT 7.42 min].
32
R-32
HCC(CH2)4CO-Val-Val-Val-Agl-Phe-NH(CH2)6NH2 (32) and HCC(CH2)4CO-Val-
Val-Val-Agl-D-Phe-NH(CH2)6NH2 (R-32) Employing the protocol described above for
the synthesis of peptide 18 using Fmoc-Agl-(D,L)-Phe-OH (1.03 g, 3 eq.), the mixture of two
diastereoisomers was prepared and separated by HPLC on C18 column using a gradient
of 50% to 90% MeOH in H2O to obtain peptide 32 (1 mg, 1%), which was shown to be
>95% pure by LC-MS analysis [50 90% MeOH (0.1% FA) in H2O (0.1% FA), 14 min, RT
−
5.31 min] and peptide 39 (0.9 mg, 1%), which was shown to be >95% pure by LC-MS
analysis [50−90% MeOH (0.1% FA) in H2O (0.1% FA), 14 min, RT 5.07 min].
33
HCC(CH2)4CO-Agl(Me)-Val-Val(Me)-Ala(Me)-Phe(Me)-NH(CH2)6NH2 (33) Employ-
ing the protocol described above for the synthesis of peptide 17 using Fmoc-Agl-Val-OH
(462 mg, 3 eq.) in the fourth coupling, followed by permethylation and coupling to 1,6-
diaminohexane, peptide 33 (9.5 mg, 3%) was prepared and shown to be >99% pure by
LC-MS analysis [50−90% MeOH (0.1% FA) in H2O (0.1% FA), 14 min, RT 6.72 min].

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34
HCC(CH2)4CO-Val(Me)-Agl(Me)-Val-Ala(Me)-Phe(Me)-NH(CH2)6NH2 (34) Employ-
ing the protocol described above for the synthesis of peptide 17 using Fmoc-Agl-Val-OH
(462 mg, 3 eq.) in the third coupling, followed by permethylation and coupling to 1,6-
diaminohexane, peptide 34 (8.3 mg, 3%) was prepared and shown to be >95% pure by
LC-MS analysis [50−90% MeOH (0.1% FA) in H2O (0.1% FA), 14 min, RT 6.86 min].
35
HCC(CH2)4CO-Val(Me)-Val(Me)-Agl(Me)-Ala-Phe(Me)-NH(CH2)6NH2 (35) Employ-
ing the protocol described above for the synthesis of peptide 17 using Fmoc-Agl-Ala-OH
(359 mg, 3 eq.) in the second coupling, followed by permethylation and coupling to 1,6-
diaminohexane, peptide 35 (6.7 mg, 2%) was prepared and shown to be >95% pure by
LC-MS analysis [50−90% MeOH (0.1% FA) in H2O (0.1% FA), 14 min, RT 6.80 min].
HCC(CH2)4CO-(5-Me)Nai-Val-Val-Ala-Phe-OH (36) Employing the protocol described
above for the synthesis of peptide 15 using Fmoc-(5-Me)Nai-Val-OH (318 mg, 2 eq.) in
the third coupling, peptide 36 (8.8 mg, 9%) was prepared and shown to be >99% pure by
LC-MS analysis [50−90% MeOH (0.1% FA) in H2O (0.1% FA), 14 min, RT 5.34 min].
HCC(CH2)4CO-Val-(5-Me)Nai-Val-Ala-Phe-OH (37) Employing the protocol described
above for the synthesis of peptide 15 using Fmoc-(5-Me)Nai-Val-OH (318 mg, 2 eq.) in the
second coupling, peptide 37 (4.7 mg, 4%) was prepared and shown to be >99% pure by
LC-MS analysis [50−90% MeOH (0.1% FA) in H2O (0.1% FA), 14 min, RT 4.41 min].

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5. Conclusions
The unsaturated lipid tail and the C-terminal acid and carboxamide functions of
almiramide peptides have previously been shown to have relevance for activity against
trypanosomatid parasites [24,25]. Moreover, active almiramide analogs possessing sugar
amino acids were suggested to adopt bent structures [31]. The influences on anti-parasite ac-
tivity of amide N-methylation and turn-inducing Agl and Nai residues within almiramide
peptides have now been investigated in wild type and resistant strains of L. infatum and
compared with macrophage cytotoxicity. Peptide amides exhibited consistently better activ-
ity than their C-terminal acid counterparts. Within a set of almiramide 1,6-diaminohexane
amides, more potent peptides with relatively high selectivity were typically obtained with-
out methylation (e.g., 16) and with a single methyl group in MePhe5 almiramide 22 than
with analogs possessing a methyl amide at other positions and with permethylated analog
17. On the other hand, permethylated and MeVal1 peptide amides 17 and 18 exhibited
µM
inhibitory activity against the amphotericin B resistant strain with high therapeutic indices.
Replacement of amino acid residues by turn-inducing counterparts caused typically losses
of activity against the L. infatum strains; however, permethylated Agl2 amide had similar ac-
tivity and better selectivity against wild type L. infatum compared to permethylated analog
17. Although studies of the consequences of such structural modifications on metabolism
and mechanism of action are in progress, conformers extended about the central residues
and mobile at the extremities of the peptide may favor almiramide activity.
Supplementary Materials: The following are available online, synthesis procedure of Agl and Nai
dipeptides; NMR Spectra; Ascertainment of purity by HPLC; Dose-response curves of Leishmania
strains and LM-1 mac-rophage survival rate in the presence of peptides 12–37.
Author Contributions: Conceptualization, methodology, validation, resources, writing—review and
editing, visualization, supervision, project administration, funding acquisition: M.O., C.F.-P. and
W.D.L.; formal analysis, data curation: A.M.T.N.; investigation: A.M.T.N., S.B., N.D., C.D., A.C.
and E.F.F.; writing—original draft preparation: A.M.T.N.; All authors have read and agreed to the
published version of the manuscript.
Funding: This work was supported by a New Frontiers in Research Fund Exploration Grant NFRFE-
2018-00766 awarded to C.F.-P., M.O. and W.D.L.; the Natural Sciences and Engineering Research
Council (NSERC) of Canada Discovery Grant Program Projects #06647 (WL) and RGPIN-2017-04480
(CFP); the FRQNT Centre in Green Chemistry and Catalysis, Project #171310 (WL). The Canada
Foundation for Innovation, grant number 37324 (CFP). AC and ND were supported by the Fonds de
recherche du Québec—Nature et technologies (FRQNT).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: Authors want to thank M. Ouellette for the kind gift of the Sb2000.1, MF200.5
and AmB1000.1 drug-resistant strains to the CFP lab. We acknowledge the assistance of members of
the Université de Montreal facilities: A. Fürtös, M.-C. Tang, and L. Mahrouche (mass spectrometry,
HPLC analyses), C. Malveau, P. Aguiar, and S. Bilodeau (NMR spectroscopy).
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of the compounds are not available from the authors.
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