Synthesis, SAR and biological studies of sugar amino acid-based almiramide analogues: N-methylation leads the way

Article abstract of DOI:10.1039/c6ob02610a

Leishmaniasis, caused by the protozoan parasites of the genus Leishmania, is one of the most neglected diseases endemic in many continents posing enormous global health threats and therefore the discovery of new antileishmanial compounds is of utmost urgency. The antileishmanial activities of a library of sugar amino acid-based linear lipopeptide analogues were examined with the aim to identify potential drug candidates to treat visceral leishmaniasis. It was found that among the synthesized analogues, most of the permethylated compounds exhibited more activity in in vitro studies against intra-macrophagic amastigotes than the non-methylated analogues. SAR and NMR studies revealed that introduction of the N-methyl groups inhibited the formation of any turn structure in these molecules, which led to their improved activities.

Full text of DOI:10.1039/c6ob02610a

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Synthesis, SAR and biological studies of sugar amino acid based
almiramide analogues: N-methylation leads the way
Received 00th January 20xx,
Accepted 00th January 20xx
Dipendu Das,a Hina P. A. Khan,b Rahul Shivahare,c Suman Gupta,c Jayanta Sarkar,d Mohd. Imran
Siddiqui,e Ravi Sankar Ampapathi,*f Tushar Kanti Chakraborty*b
DOI: 10.1039/x0xx00000x
Leishmaniasis, caused by the protozoan
www.rsc.org/
parasites of the genus Leishmania, is one of
the most neglected diseases endemic in
many continents posing enormous global
health threats and therefore, the discovery
of new antileishmanial compounds is of
utmost urgency. Antileishmanial activities of a library of sugar amino acid based linear lipopeptide analogues have been
examined with the aim to identify potential drug candidates to treat visceral leishmaniasis and we were pleased to find
that among the synthesized analogues most of the permethylated compounds exhibited more activity in in vitro studies
against intra-macrophagic amastigotes than its non-methylated analogues. SAR and NMR studies revealed that
introduction of the N-methyl groups inhibited the formation of any turn structure in these molecules that led to improved
activities.
paromomycine.5 However, the treatment with these existing
drugs suffers from several limitations such as cost, high
toxicity, difficulty in administration and spread of drug
resistance.6 Therefore, there is still a crying need of new
efficacious and safe drugs based on new molecular scaffold in
the absence of any upcoming vaccine.7 Among the different
heterocyclic structures that have been disclosed, the nitrogen
containing compounds found in many natural products are the
source of some important lead molecules.8
Introduction
Leishmaniasis, one of the “most neglected diseases”1 caused
by protozoan parasites belonging to the genus Leishmania, is
transmitted to human through the bite of female
phlebotomine sand flies infected with the parasites.2 It is a
geographically widespread disease prevalent in many parts of
the tropical and subtropical world causing significant morbidity
or mortality.3 This disease is a severe public health problem in
many developing countries of East Africa, the Indian
subcontinent and Latin America.
Antileishmanial therapy relies on handful of drugs such as,
the first-line treatment option involving the administration of
meglumine antimonate (MA), sodium stibogluconate (SSG) and
amphotericin B.4 Second-line drugs include miltefosine, and
The N-methylated lipopeptides, almiramides A-C (1-3
)
(Figure 1) are marine natural products, originally isolated from
a Panamian collection of Lyngbya majuscula in conjunction
with the Panama International Cooperative Biodiversity Group
(ICBG).8c,9 This class of natural products was found to be active
against Leishmania donovani, the causative agent of
leishmaniasis and also showed activity against the related
kinetoplastid parasite Trypanosoma brucei, the causative
agent of human African trypanosomiasis. Target identification
studies indicated that the almiramides likely perturb
glycosomal function through disruption of membrane
assembly machinery.9
The encouraging pharmacological profiles of almiramides
as peptide based lead molecules having antileishmanial activity
prompted us to target almiramides for further drug
development and also to explore the SARs by preparing their
analogues.
a.Medicinal & Process Chemistry Division, CSIR-Central Drug Research Institute,
Sector 10, Jankipuram Extension, Sitapur Road, Lucknow 226031, India.
b.Department of Organic Chemistry, Indian Institute of Science, CV Raman Road,
Bengaluru 560012, India.
c. Parasitology Division, CSIR-Central Drug Research Institute, Sector 10, Jankipuram
Extension, Sitapur Road, Lucknow 226031, India.
d.Biochemistry Division, CSIR-Central Drug Research Institute, Sector 10, Jankipuram
Extension, Sitapur Road, Lucknow 226031, India.
e. Molecular and Structural Biology Division, CSIR-Central Drug Research Institute,
Sector 10, Jankipuram Extension, Sitapur Road, Lucknow 226031, India.
f. Centre for Nuclear Magnetic Resonance, SAIF, CSIR-Central Drug Research
Institute, Sector 10, Jankipuram Extension, Sitapur Road, Lucknow 226031, India
Electronic Supplementary Information (ESI) available: NMR spectra and molecular
dynamics. See DOI: 10.1039/x0xx00000x
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In recent years, the interest in rational design of
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DOI: 10.1039/C6OB02610A
peptidomimetics has steadily grown due to the
pharmacological limitations of bioactive natural peptides. A
large variety of peptide modifications have been used for
conformationally directed drug design to investigate the active
peptide-receptor binding interactions. For this purpose, wide
ranging structurally rigid non-peptidic molecular scaffolds have
been designed. Insertion of these moieties in appropriate sites
to restrict the conformational degrees of freedom in peptides
produces the specific 3D structures required for binding their
receptors. Sugar amino acids (SAA) belong to one such class of
conformationally constrained templates that have been used
extensively in many peptidomimetic studies.10 Linington and
co-workers have synthesized a library of synthetic analogues of
Compound
R1
R2
H
R3
m,n
1,1
1,1
1,1
4A
4B
4C
OMe
NH2
H
Me
NMe2
almiramides
1-3 and checked their biological activity in parallel
4D
4E
H
H
OMe
NH2
1,0
1,0
with the original lead compounds, and identified several new
4F
4G
4H
4I
H
H
OMe
NH2
0,1
0,1
0,1
0,1
0,1
0,1
0,1
Me
H
NMe2
OMe
NH2
4J
H
4K
4L
Me
H
NMe2
OMe
4M
4N
4O
4P
H
Me
H
NH2
NMe2
OMe
NH2
0,1
0,1
0,1
0,1
0,1
0,1
H
Figure 1. Molecular structures of almiramides A-C (1-3
)
4Q
4R
Me
H
NMe2
OMe
and dolastatin 10 analogues.
structures with comparable activities to the original natural
products.8c,9
4S
4T
H
NH2
0,1
0,1
Our interests in the use of SAAs as dipeptide isosteres in
peptidomimetic studies11 prompted us to synthesize a small
library of almiramide analogues by incorporating furanoid
sugar amino acid in the backbone replacing some of its
constituent amino acids to prove the viability of this template
in discovering antileishmanial leads for future medicinal
chemistry and drug development.
Me
NMe2
4U
4V
H
H
OMe
NH2
0,1
0,1
0,1
4W
Me
NMe2
Figure 2. Structures of the designed analogues (4A-W).
Results and Discussion
Chemistry. A series of 23 compounds with a general structure 4
(4A-W, Figure 2) having hybrid sequences of natural amino acids
(AAs), unnatural (4R,5S)-4-amino-5-methylheptanoic acid (AMH)
and a mannose-derived sugar amino acid (MAA) were synthesized
to screen against L. donovani. These compounds were derived from
our earlier reported potent dolastatin 10 analogues (Figure 1) that
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were shown to have excellent tubulin binding properties strongly only one of these building blocks – 4A-C having bVoitehw AMrticAleAOnalninde
suppressing the dynamics of microtubule assembly in vitro.12 The AMH, 4D-E having only AMH and 4F-W having only MAA.
DOI: 10.1039/C6OB02610A
fact that mammalian tubulin binding cytotoxic molecules have been
widely screened against kinetoplastid tubulin for discovering anti-
Scheme 3. Attachment of the lipophilic chains
leishmanial compounds13 prompted us to screen our dolastatin 10
analogues for their antileishmanial activities as well. In addition,
these dolastatin 10 analogues also showed synergistic effect with
colchicine lending further support to screen them for
antileishmanial activities as colchicine is known for its strong
binding with β-tubulin of Leishmania spp.14 Subsequent discovery of
almiramides A-C was of immense help for remodelling the
dolastatin 10 analogues into almiramide look-alikes for the present
study. The basic peptide skeleton of the parent dolastatin 10
analogues was retained in three compounds 4A-C with insertions of
only the N-acyl groups. The dipeptide isosteric MAA essentially
Scheme 1. Synthesis of the modified isoleucine unit 8
Synthesis of the monomeric building blocks. For the synthesis
of the almiramide analogues, represented by a general
formula
4
(Figure 2), the differentially protected
(Schemes 1 and 2) and
peptidomimetic building blocks
8, 9
other natural amino acids were employed.
The synthesis of the protected monomeric unit Boc-AMH-
OMe (
Scheme 1).13 First, reduction of the ester group by LAH
provided the alcohol which was oxidized using Swern
condition15 followed by 2-carbon olefination to furnish the α,β-
unsaturated ester 7 16
Hydrogenation of delivered the
required monomeric unit . Synthesis of the methyl N-Boc-6-
8) started from the Boc-L-isoleucine methyl ester (5,
replaced the Val-Ala segment of almiramides. As Val is known to
maximally stabilize secondary structures in peptides, replacement
of only one Val was assumed not to cause any major
conformational change. The presence of Phe in the C-terminus of
dolastain 10 analogues suitably conformed to the C-terminus of
almiramides.
6
.
7
8
To assess the effects of the unnatural amino acids, the
conformationally restrained δ-amino acid MAA and flexible γ-amino
acid, (4R,5S)-4-amino-5-methylheptanoic acid (AMH) on the
activities of these molecules, analogues were made with both or
Scheme 4. Synthesis of the amide terminus and
permethylated peptide backbone
Scheme 2. Synthesis of the linear tetrapeptide 14
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Scheme 5. Synthesis of the pentapeptide backbone 4A
amino-2,5-anhydro-3,4-di-O-methyl-6-deoxy-D-mannonate,
corresponding carboxylic acids, which were directly used after
Boc-MAA(Me)2-OMe (9), was accomplished in 11 steps using aqueous work-up for the next peptide coupling reaction
D-mannitol as the starting material following the reported without further purification or characterization.
procedure.11c,d MAA(Me)2 was used to replace the Val-Ala
spacer segment of almiramides.
Incorporation of the lipophilic side chains. Having successfully
prepared the tetrapeptide 14 with C-terminal methyl ester, we
Synthesis of the linear tetrapeptide 14. Having prepared the turned our attention to incorporate the lipid side chains (chain
unnatural monomeric units required for the synthesis of the length is variable and ranges from 6 to 10 carbons, Figure 2) to
analogues, the stage was set for the synthesis of the target create the structural diversity at the N-terminus.
peptides. The linear tetrapeptide 14 was synthesized by
The tetrapeptide 14 upon treatment with TFA in DCM gave the
coupling MAA (
9
) with L-Phe and then attaching Val-Val17 to corresponding TFA salt 15 (Scheme 3). Coupling of 15 with various
the N-terminus of MAA-Phe following linear peptide coupling acids having the lipophilic side chains under standard peptide
method as depicted in the Scheme 2. Standard solution phase coupling conditions by sequential addition of HOBt and EDCI
peptide
synthesis
method
using
1-ethyl-3-(3- followed by DIPEA in DCM gave the lipid-attached peptides with a
dimethylamino)propyl)carbodiimide hydrochloride (EDCI) and general structure 16 as shown in Scheme
1-hydroxybenzotriazole (HOBt) as coupling agents, DIPEA as compounds 4F, 4I, 4L, 4O, 4R and 4U.
base and DCM as solvent was followed to build these peptides.
3 – comprising
While the Boc group was used for N-protection, the C-terminal
Scheme 7. Synthesis of the congeners without sugar amino
acid scaffold
was protected as methyl esters. Deprotection of the former
was carried out with trifluoroacetic acid (TFA) in DCM to
furnish the TFA salt and saponification of the methyl ester was
achieved with LiOH.H2O in THF/MeOH/H2O (3:1:1) to give the
Scheme 6. Synthesis of the amide 4B and permethylated
compound 4C from 4A.
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Synthesis of the primary amide C-terminus and the fully analogues 18 (Scheme 4) with fully methylateVdiewbAarcticklbe oOnnlienes
20
DOI: 10.1039/C6OB02610A
methylated peptide backbone. After the N-terminal lipid chain represented by 4H, 4K, 4N, 4Q, 4T and 4W.
insertion, we next sought to prepare the C-terminal primary
Synthesis of 4A-C containing the γ-amino acid (AMH, 8) and
MAA (9). After that, we decided to undertake the synthesis of a
small library of pentapeptides 4A-C, containing both the γ-
Table 1. Bioactivities of the synthetic analogues
amino acid AMH (8) and δ-amino acid MAA (9), to access
Compound Antiamastigote
activity
Cytotoxicity on
Vero cells
(CC50, µM)
Selectivity
Index*
(CC50 / IC50)
another type of structural diversity mainly focusing on the
number of amino acids (i.e. five, same as in almiramides)
instead of the total number of atoms in the linear peptidic
chain. Compound 4A was synthesized from the appropriate
amino acid precursor as described in Scheme 5. Then it was
converted to its amide 4B and the permethylated compound
4C as shown in Scheme 6.
(IC50, µM)
(MQ/amast.
model)
4A
4B
4C
4D
4E
4F
>100
>100
26.41
>100
>25
295.47
>400
79.43
210.25
125.21
>400
>400
79.51
>400
>400
>400
>400
>400
24.07
>400
>400
14.53
>400
>400
58.35
>400
>400
95.31
51.27
<2.95
4.0
3.0
To further optimize our library, the 2nd generation
modification was aimed to devise analogues 4D-E which were
different from the others due to the absence of any SAA, to
prove the viability of the SAA based scaffold in the design and
activity (Scheme 7).
Biological evaluations: antileishmanial activities. After
successful preparation of the compounds, we were next
interested to identify new and potent antileishmanial leads
from the library. For that, all the compounds were screened
against L. donovani. Each candidate was evaluated for its in
vitro activity against intra-macrophagic amastigotes and also
evaluated for their cytotoxic properties over mammalian Vero
cells (kidney fibroblast cells).
<2.10
<5.0
>4.33
4.0
92.18
>100
56.48
>100
>100
>100
60.67
>100
26.67
68.48
>100
10.10
>50
4G
4H
4I
1.40
4.0
4J
4.0
4K
4L
4.0
>6.59
4.0
4M
4N
4O
4P
4Q
4R
4S
0.90
5.84
4.0
Gratifyingly, the compounds 4C
exhibited moderate micromolar activity, having poor to
moderate selectivity profiles with IC50 values ranging from
10.1 to 26.67 M (Table 1). A progressive enhancement of the
, 4E, 4N, 4Q, 4T, 4U and 4W
,
1.44
8.0
μ
activity was observed in the case of the compounds having the
MAA scaffold. It was also observed that, overall variations in
the functional groups at the C-terminus as well as at the N-
terminus have significant effect on activity. Close investigation
of the activity of each series of peptides revealed that the
compounds which contained C-terminal primary amide
derivatives were completely inactive as compared to the
compounds containing either methyl ester or tertiary amide.
This observation was contrasting with the biological data
reported by Linington et al.8c,9 where the C-terminal methyl
ester derivatives were largely inactive in the screen. The
improvement in the present study might be due to the fact
that the esters were better tolerated in our compounds and
also, due to the less polarity, they had better membrane
permeability as compared to the more polar amides.
Importantly, our permethylated compounds displayed better
activity presumably due to their increased membrane
permeabilities. Although their selectivity profiles still remained
moderate, they were comparable to that of the standard
antileishmanial drug, miltefosine (last entry, Table 1). The
permethylated compounds were also explored earlier by
Linington et al.8c,9 and some of their analogues showed
superior activity and cytotoxicity profiles.
>100
10.92
22.86
>100
13.63
8.48
4.0
4T
4U
4V
4W
5.34
>17.49
4.0
6.99
6.05
†
MF
* Selectivity index (SI) is defined as the ratio of CC50 on
mammalian kidney fibroblast cells (Vero cell line) to IC50 on L.
donovani intracellular amastigotes. † MF (miltefosine) was used as
a reference antileishmanial drug.
amide and its corresponding permethylated analogues as N-
methylation of peptides is known to lead to improved
membrane permeability, pharmacokinetic properties and
better biological activities.18 Preparation of these compounds
is shown in Scheme 4. Saponification of 16 with LiOH.H2O in
THF/MeOH/H2O (3:1:1) was carried out and the resulting
carboxylic acids were converted to their amides by treatment
with aq. NH3 in presence of ethyl chloroformate and Et3N to
furnish C-terminus amides with general structure 17 (Scheme
Having confirmed the importance of the permethylated
4), representing 4G
permethylation using Ag2O and MeI in DMF generated the derivatives with variable lipid chains and found that the
, 4J, 4M, 4P, . Next, analogues, we next screened various permethylated
4S and 4V 19
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Molecules were drawn and minimized in MOE2V1iefwoAllroticwleeOdnlibnye
compounds 4D, 4E, 4I, 4J and 4K were inactive, indicating the
22
DOI: 10.1039/C6OB02610A
intolerance of the alkyne moiety at the peptide terminus. partial charge calculation using AMBER99. Structures were
Further, it is to be noted here that the compounds with subsequently subjected to molecular dynamics simulation at 300 K
saturated alkyl chain 4Q and the olefin terminus 4T showed and AMBER99 force field in vacuum with 100 ps of equilibrium run
comparable activity with better selectivity for the later, in followed by production run for 500 ps. Resulting structures were
conflict with the earlier result9 which endorsed the analysed in Chimera.23
Peptidomimetics adopt specific conformation in solution to
interact with the corresponding target proteins. In this work, as the
phi1 vs psi1 graph
target was not known, SAR studies were performed using molecular
dynamics simulations on the given set of peptidomimetic
compounds to examine if there was any conformational pattern
that could be correlated to their antileishmanial activities. From
three dimensional arrangements of the atoms in these molecules, a
clear conformational preference of the torsional angles (phi and psi
80
60
40
20
Actives
Inactives
0
-150
-100
-50
0
50
100
150
-20
-40
-60
angles) around first peptide bond for the active molecules over the
inactive ones was observed (Figure 3). Peptidomimetic molecules
with both positive phi and psi values for the first residue were
found to be relatively more active while inactive molecules could
have negative values for either or both phi and psi values for the
first residue. These differences demonstrate the H-bond forming
ability of the first residue in each molecule. Therefore, preferred
conformation of the molecules can be prioritized using phi-psi
angles to establish the blueprint for biological activity. Moreover,
profound effect of N-methylation of peptide bonds can be
distinguished clearly from the visual analysis of the molecular
structures. However, from the listings of phi and psi values for the
compounds (see Table S1, Supporting Information), it is seen that
compound 4U, which is not N-methylated and, therefore, having
negative phi and psi values, is still considerably active. Activity of
this compound could be attributed to the larger exposed non-polar
surface area due to the presence of the phenyl ring containing long
aliphatic chain at N-terminus as discussed below.
phi angles
Figure 3. Conformational preferences of the torsional
angles. Phi-1 and psi-1 are the dihedral angles of the
first residue in the peptidomimetic compounds.
requirement of an unsaturated terminus on the side chain
over saturated one for better activity. We next explored the
importance of the length of the lipid chain which was varied
from 6 to 10 carbons and the results suggested that the
bioactivity increases with the increase in the chain length.
Further, increment of the total peptide length by incorporating
one extra amino acid residue that led to compounds 4A-C
showed no significant improvement in activity.
Computational approach to Structure-Activity Relationship
(SAR) studies. The encouraging results of the analogues prompted
us to explore SAR analysis to find out the role of each of the
components of the peptide backbone.
Observation of the three dimensional structures of the
compounds (Figure 4) highlighted that the amide proton containing
Figure 4. Effect of the H-bonding on the structural changes in the peptides. Unlike active molecules (4W), inactive
molecules (4V) are characterized by the presence of β-turn due to the formation of H-bond.
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molecules form the hydrogen bond between the carbonyl oxygen of residue and MAACδH, suggesting that the bent turn VsiterwuAcrttuicrleeOmnlianye
DOI: 10.1039/C6OB02610A
the first acyl residue and amide proton of the third residue which also be there as seen in their counterparts.
adopt a particular tight 10-membered β-turn. This results into
Molecular dynamics simulation based on NMR data. Energy
minimization and simulated molecular dynamics (MD) calculations
were performed on Discovery studio 3.0 version,24a using CHARMm
force field24b with default parameters throughout the simulation
with the aid of distance dependent dielectric constant with ε = 46.7
(dielectric constant for DMSO). Distance restraints used in the MD
were calculated from the volume integrals of the cross peaks in the
ROESY spectra by categorizing them as strong, medium and weak
peaks based on their volume integrals. Force constants of 10 K
cal/Å, 5 K cal/Å were employed for distance and torsional restraints
respectively. Minimization was done with steepest descent
algorithm followed by conjugate gradient methods for maximum
1000 iterations each iterations or RMS deviation of 0.001 Kcal /mol,
whichever was earlier. The molecules were initially equilibrated for
5 pS and then subjected to 1 nS production run. Starting from 50 K,
they were heated to 300 K in five steps, increasing the temperature
50 K at each step. 20 structures were stored from the production
run and were again energy minimized with the above-mentioned
protocol. Superposition was done on to the average structure of
these minimized structures. The superimposed 10 energy
minimized structures of 4P and 4V are shown in Figure 5. The
reduced flexibility of the N-terminal hydrophobic chains which turn
around the backbone leading to the disruption of the coiled
secondary structure and also in the reduction of the hydrophobic
surface area. Moreover, beside H-bond, hydrophobicity may also be
playing the role of one of the determining factors for the activity.
Long hydrophobic chain at N-terminus is probably more favorable
for the antileishmanial activity. As obvious from the activity data, 4K
and 4H have relatively poor activity although having N-methylated
amide but low hydrophobic surface area. Whereas, among the N-
protonated peptides, 4U and 4O (to a lesser extent) were found to
be somewhat active due to the presence of long chain hydrophobic
groups. It seems that the presence of long aliphatic/aromatic
groups at the N-terminal end causes steric clashes with the C-
terminal hydrophobic groups and, therefore, forces them to turn
away from the backbone, leading to an increase in the total
hydrophobic surface area. Additionally, presence of polar groups
like –NH2 group at the other N-terminal end also reduces the
activity to some extent as evident from compounds 4P and 4V in
comparison to 4O and 4U, respectively, having the same structure
except the C-terminal groups.
Conformational analysis by NMR. Solution conformational structures of 4P and 4V based on NMR data, although appear
analyses on selected peptides 4P, 4Q, 4V and 4W were undertaken different than those derived computationally (Figure 4), bear a
in an attempt to decipher the underlying structural implications commonality in brining the C-terminus close to the lipophilic end of
that might have led to much improved biological activities found in the fatty acid chain leading to a more condensed hydrophobic
the permethylated analogues 4Q and 4W over their inactive surface.
unmethylated counterparts 4P and 4V, respectively. NMR studies
were carried out in DMSO-d6 with 5-10 mM concentration of the
samples. For peptides 4P and 4V, single set of resonance observed
Conclusions
in the proton spectrum suggested that these peptides exist in single
conformation in the NMR time scale. The variable temperature (VT)
study carried out in DMSO-d6, suggested minimal Δδ/ΔT value for
PheNH supporting its participation in hydrogen bonding. Wide
dispersion of the amide protons suggested stable secondary
structure for these peptides, and further the characteristic nOes
In conclusion, inspired by the interesting biological activities of
almiramides, we have synthesized a small library of 23 linear
lipopeptides by incorporating a mannose-derived furanoid
PheNH
↔ MaaCδH, PheNH ↔ MaaCεH (see Figure S7 in
supporting information) and minimal Δδ/ΔT value for PheNH
2
suggest a 10-membered β-turn structure involving PheNH and Val
carbonyl, in contrast to the 10-membered β-turn structure involving
MAANH and C-terminus acyl carbonyl theoretically observed in SAR
analysis.
The permethylated peptides 4Q and 4W that showed the best
antileishmanial activities, exhibited either broad peaks or multiple
peaks in DMSO-d6 which may be as
a result of multiple
conformations that are either in the intermediate or slow exchange
regime in the NMR time scale. The chemical shift assignments for
the major conformer were made and are tabulated in the
supporting information. Though there may be lack of H-bonding as
because of the absence of amide protons, yet the turn structures
Figure 5. Stereo view of the 15 superimposed energy
minimized structures of 4P (top) and 4V (bottom).
were evident among these peptides as we have seen
a
characteristic nOe correlation observed from the NMe of the Phe
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3.5 mmol) in EtOH (10 mL), 10% Pd/C (164 mg) was added and the
DOI: 10.1039/C6OB02610A
mixture was hydrogenated at room temperature using a H2-filled
sugar amino acid as a novel scaffold along with other building
blocks and assessed them against intra-macrophagic
amastigotes of L. donovani. A number of such compounds
exhibited in vitro activity comparable to the other current
antileishmanials. To the best of our knowledge, peptides with
sugar amino acid based scaffolds and their antileishmanial
activities have never been reported to date. The SAR and NMR
studies revealed that among all the synthesized compounds,
the MAA-containing permethylated analogues having longer
hydrophobic chains at the N-terminus were more active than
their unmethylated counterparts possibly due to the improved
cell permeability of the former resulting from their condensed
hydrophobic surface. Hence, the SAA based scaffolds can serve
as a useful building block for further antileishmanial lead
development. Especially explorations with the other
stereoisomeric variants of this multifunctional hybrid
molecule, their incorporation into the lead sequences found in
the present study and evaluation of their biological profiles
could lead to the prospective antileishmanials of tomorrow.
View Article Online
balloon for 2 h. The solution was then filtered through a short pad
of Celite and the filter cake was washed with EtOAc. The filtrate and
washings were combined and concentrated in vacuo. Flash column
chromatography (Silica gel, 8% EtOAc in Hexane) furnished 8 (984
mg, 98%) as colorless oil.
Rf = 0.45 (Silica gel, 15% EtOAc in Hexane); 1H NMR (400 MHz,
CDCl3): δ 4.35 (d, J = 9.6 Hz, 1H), 4.11 (q, J = 7.0 Hz, 2H), 3.48 (m,
1H), 2.33 (t, J = 7.6 Hz, 2H), 1.85 – 1.75 (m, 1H), 1.60 – 1.35 (m,
12H), 1.24 (t, J = 7.1 Hz, 3H), 1.15 – 1.03 (m, 1H), 0.95 – 0.80 (m,
6H); 13C NMR (100 MHz, CDCl3): δ 173.84, 155.79 78.95, 60.38,
54.43, 39.25, 31.44, 28.37, 26.47, 25.25, 14.89, 14.19, 11.74; IR νmax
(neat, cm–1): 3366, 2968, 2930, 2876, 1711, 1597, 1521, 1453, 1368,
1249, 1174, 1019; MS (ESIMS/[M+Na]+): m/z(%) 310; HRMS
(ESI/[M+Na]+): Calcd for C15H29NO4Na+ : 310.1989, found 310.1987.
General Procedure for the Saponification of Ester: To a
solution of compound in THF/MeOH/H2O (3:1:1, 3 mL/mmol) at 0 °C
was added LiOH.H2O (3 eq) and the mixture was stirred at room
temperature for 1 h. The mixture was then acidified to pH 2 with 1N
HCl at 0 °C. The reaction mixture was extracted with EtOAc, washed
with water and brine, dried (Na2SO4), filtered and concentrated in
vacuo to obtain the corresponding acid. The crude acid was used
directly in the next step without further purification.
Experimental Section
General Experimental Details: All the reactions were carried out
under an inert atmosphere in oven-dried glassware using dry
solvents, unless otherwise stated. All chemicals purchased from
commercial suppliers were used as received unless otherwise
stated. Reactions and chromatography fractions were monitored by
Merck silica gel 60 F-254 glass TLC plates and visualized using UV
General Procedure for Deprotection of N-Boc Group: To the
light, 7% ethanolic phosphomolybdic acid-heat, 2.5% ethanolic compound in DCM (3 mL/mmol) at 0 °C, was added trifluoroacetic
anisaldehyde (with 1% AcOH and 3.3% conc. H2SO4), ninhydrin or acid (TFA; 1 mL/mmol) and the mixture was stirred for 30 min at
chlorine/o-tolidine-heat as developing agents. Flash column room temperature. The reaction mixture was then concentrated in
chromatography was performed with 100-200 mesh silica gel and vacuo, followed by azeotroping with DCM for 3 times to obtain the
yields refer to chromatographically and spectroscopically pure trifluoroacetate salt which was directly used in the next step.
compounds.
All NMR spectra were recorded in CDCl3 or in DMSO-d6 on a 300,
400 and 500 MHz instruments at 300 K and are calibrated to
residual solvent peaks (CHCl3 7.26 ppm and 77.0 ppm, DMSO 2.50
ppm and 40.0 ppm). Multiplicities are abbreviated as: s = singlet; d
= doublet; t = triplet; q = quartet; m = multiplate. All IR data were
recorded as neat liquid or KBr pellets using a Perkin Elmer’s RX I
FTIR spectrophotometer. Mass spectra were obtained under
electron spray ionisation (ESI) and HRMS spectra were taken with a
3000 mass spectrometer using Waters Agilent 6520-Q-TofMS/MS
system and JEOL-AccuTOF JMS-T100 LC. RP-HPLC was performed on
a Waters HPLC system and Shimadzu's ISO 9001 HPLC system
(model no. LC-20AD) equipped with a 5 µ SunFire C18 column (4.6 ×
250 mm) and 5 µ Shimadzu's C18 column (4.6 × 250 mm)
respectively in combination with eluants A (H2O) and B (MeCN) with
flow rate: 0.8 mL/min and photodiode array detector setting of λ =
210-254 nm.
General Procedure for Peptide Coupling: To a stirring solution
of the acid in DCM (3 mL/mmol) or DMF (3 mL/mmol) at 0 °C were
sequentially added 1-hydroxybenzotriazole (HOBt, 1.5 eq) and 1-
ethyl-3-(3-(dimethylamino)-propyl)carbodimide
hydrochloride
(EDCI, 1.5 eq). After 10 min, the previously prepared
trifluoroacetate salt was cannulated into the reaction mixture
followed by the addition of DIPEA (5 eq). The reaction mixture was
brought to room temperature and continuously stirred for 12 h. The
reaction mixture was then extracted with EtOAc and washed with
1N HCl, saturated aqueous NaHCO3 solution, water and brine, dried
(Na2SO4), filtered and concentrated in vacuo. Purification by column
chromatography (100-200 mesh silica gel, 0.8-1.5% MeOH in
chloroform as eluant for compounds 4A, 4D, 4F, 4I, 4L, 4O, 4R and
4U) afforded the coupling product.
Data for 11: Scale of reaction 1.0 g, 3.13 mmol; yield 1.21 g, 83%
(Purification by column chromatography in 100-200 mesh silica gel,
35% EtOAc in hexane as eluant).
Rf = 0.50 (Silica gel, 70% EtOAc in Hexane); 1H NMR (400 MHz,
Experimental Procedures and Characterization Data:
CDCl3): δ 7.3 – 7.2 (m, 3H), 7.16 – 7.11 (m, 2H), 7.06 (d, J = 8.2 Hz,
Preparation of 8: Compounds 6 and 7 were synthesized according 1H), 4.94 (m, 1H), 4.89 – 4.83 (m, 1H), 4.41 (d, J = 1.5 Hz, 1H), 4.06
to the literature procedures 16. To a solution of the ester 7 (1.0 g, (t, J = 1.4 Hz, 1H), 4.04 – 3.98 (m, 1H), 3.69 (s, 3H), 3.57 (dd, J = 2.2,
8 | J. Name., 2012, 00, 1-3
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1.5 Hz, 1H), 3.41 (s, 3H), 3.38 – 3.31 (m, 1H), 3.29 (s, 3H), 3.27 – 52.61, 52.17, 41.10, 38.57, 37.92, 33.18, 29.58, 28.17, 27.25, 25.56,
View Article Online
1
3.19 (m, 1H), 3.13 – 3.01 (m, 2H), 1.45 (s, 9H); 13C NMR (100 MHz, 19.67, 19.22, 17.65, 17.24, 14.96, 11.39; H NMR (400 MHz, DMSO-
CDCl3): δ 171.48, 169.86, 155.92, 135.67, 129.32, 128.55, 127.15, d6): δ 8.01 (d, J = 8.4 Hz, 1H), 7.91 (t, J = 5.7 Hz, 1H), 7.69 – 7.60 (m,
86.88, 85.62, 83.89, 82.76, 79.39, 77.20, 57.33, 52.56, 52.27, 42.24, 2H), 7.27 – 7.17 (m, 5H), 6.82 (d, J = 8.9 Hz, 1H), 4.58 – 4.52 (m, 1H),
38.03, 28.35; IR νmax (neat, cm–1): 3405, 3350, 2977, 2933, 2828, 4.29 (d, J = 2.2 Hz, 1H), 4.17 – 4.12 (m, 1H), 3.90 – 3.85 (m, 1H), 3.81
1744, 1710, 1519, 1447, 1365, 1274, 1251, 1172, 1112, 1088, 1020; – 3.74 (m, 2H), 3.63 (s, 3H), 3.61 – 3.56 (m, 2H), 3.27 (s, 3H), 3.24 –
MS (ESIMS/[M+Na]+): m/z(%) 489; HRMS (ESI/[M+Na]+): Calcd for 3.17 (m, 2H), 3.14 (s, 3H), 3.05 – 3.00 (m, 2H), 2.17 – 1.87 (m, 4H),
DOI: 10.1039/C6OB02610A
C23H34N2O8Na+ : 489.2207, found 489.2315.
1.72 – 1.62 (m, 1H), 1.55 – 1.34 (m, 11H), 1.13 – 0.94 (m, 2H), 0.87 –
Data for 14: Scale of reaction 1.2 g, 2.57 mmol; yield 1.33 g, 78% 0.76 (m, 18H); 13C NMR (100 MHz, DMSO-d6): δ 172.72, 172.09,
(Purification by column chromatography in 100-200 mesh silica gel, 171.63, 170.97, 170.41, 155.91, 137.59, 129.61, 128.71, 126.99,
2% MeOH in chloroform as eluant).
88.18, 85.66, 82.49, 82.40, 78.49, 60.57, 58.34, 57.30, 57.09, 53.33,
Rf = 0.52 (Silica gel, 5% MeOH in CHCl3); 1H NMR (400 MHz, CDCl3): 52.66, 52.52, 40.87, 38.49, 36.79, 32.91, 30.55, 28.64, 26.75, 25.40,
δ 7.29 – 7.20 (m, 3H), 7.15 – 7.08 (m, 3H), 6.83 (t, J = 5.7 Hz, 1H), 19.89, 19.69, 18.75, 18.63, 15.43, 11.93.
6.64 (d, J = 8.5 Hz, 1H), 5.16 (d, J = 7.2 Hz, 1H), 4.89 – 4.82 (m, 1H), IR νmax (neat, cm–1): 3403, 3326, 3272, 2926, 1639, 1522, 1449,
4.42 (bs, 1H), 4.26 (dd, J = 8.2, 6.3 Hz, 1H), 4.06 (bs, 2H), 3.89 (t, J = 1368, 1219, 1019; MS (ESIMS/[M+Na]+): m/z(%) 828; HRMS
6.6 Hz, 1H), 3.68 (s, 3H), 3.61 (m, 1H), 3.43 (m, 5H), 3.26 (s, 3H), (ESI/[M+Na]+): Calcd for C41H67N5O11Na+
: 828.4729, found
3.13 – 3.01 (m, 2H), 2.26 – 2.16 (m, 1H), 2.14 – 2.07 (m, 1H), 1.42 (s, 828.4737. HPLC data: Linear gradient 50→90% B in 10 mins, total
9H), 0.96 – 0.85 (m, 12H); 13C NMR (100 MHz, CDCl3): δ 171.78, run 20 mins, tR= 12.425 min, 80% purity.
171.43, 171.08, 169.77, 156.10, 135.68, 129.29, 128.50, 127.10, Data for 4A: Scale of reaction 390 mg, 0.48 mmol; yield 328 mg,
86.87, 85.71, 83.07, 82.59, 80.21, 60.51, 58.47, 57.36, 57.26, 52.61, 83%.
52.26, 41.04, 37.98, 28.20, 19.32, 19.29, 17.79, 17.59; IR νmax (neat, Rf = 0.51 (Silica gel, 5% MeOH in CHCl3); 1H NMR (300 MHz, CDCl3):
cm–1): 3313, 2961, 2928, 1648, 1566, 1518, 1443, 1365, 1290, 1219, δ 7.37 – 7.07 (m, 7H), 7.06 – 6.76 (m, 3H), 4.94 – 4.81 (m, 1H), 4.63
1154, 1086, 1018; MS (ESIMS/[M+Na]+): m/z(%) 687; HRMS – 4.48 (m, 1H), 4.41 (bs, 1H), 4.37 – 4.11 (m, 2H), 4.10 – 3.96 (m,
(ESI/[M+Na]+): Calcd for C33H52N4O10Na+
687.3581.
:
687.3575, found 2H), 3.87 – 3.74 (m, 1H), 3.69 (s, 3H), 3.64 – 3.55 (m, 1H), 3.47 –
3.35 (m, 5H), 3.32 – 3.24 (m, 3H), 3.07 (d, J = 5.6 Hz, 2H), 2.48 (t, J =
Data for 19: Scale of reaction 400 mg, 0.86 mmol; yield 422 mg, 6.7 Hz, 2H), 2.34 – 2.08 (m, 9H), 1.96 – 1.82 (m, 4H), 1.57 – 1.37 (m,
81% (Purification by column chromatography in 100-200 mesh silica 2H), 1.03 – 0.82 (m, 18H); 13C NMR (75 MHz, CDCl3): δ 208.49,
gel, 50% EtOAc in hexane as eluant).
173.79, 173.37, 172.85, 171.72, 171.63, 171.44, 169.99, 135.83,
Rf = 0.52 (Silica gel, 80% EtOAc in Hexane); 1H NMR (400 MHz, 129.25, 128.45, 127.01, 87.19, 85.93, 83.29, 82.73, 57.34, 57.15,
CDCl3): δ 7.29 – 7.19 (m, 3H), 7.15 – 7.11 (m, 2H), 7.08 (d, J = 8.2 Hz, 52.65, 52.22, 42.48, 41.07, 39.04, 37.94, 34.89, 31.84, 29.80, 29.61,
1H), 6.45 (t, J = 5.5 Hz, 1H), 4.89 – 4.83 (m, 1H), 4.48 (d, J = 9.9 Hz, 25.47, 22.60, 19.88, 19.39, 18.97, 18.69, 18.28, 14.92, 14.03, 11.59;
1H), 4.43 (bs, 1H), 4.09 – 4.04 (m, 2H), 3.69 (s, 3H), 3.63 (bs, 1H), 1H NMR (400 MHz, DMSO-d6): δ 8.01 (d, J = 8.3 Hz, 1H), 7.94 – 7.83
3.52 – 3.35 (m, 6H), 3.28 (s, 3H), 3.13 – 3.00 (m, 2H), 2.44 – 2.10 (m, (m, 2H), 7.72 (d, J = 9.0 Hz, 1H), 7.59 (d, J = 8.9 Hz, 1H), 7.28 – 7.17
3H), 1.89 – 1.78 (m, 1H), 1.60 – 1.36 (m, 11H), 1.14 – 1.03 (m, 1H), (m, 5H), 4.59 – 4.52 (m, 1H), 4.29 (d, J = 2.2 Hz, 1H), 4.21 – 4.07 (m,
0.90 – 0.83 (m, 6H); 13C NMR (100 MHz, CDCl3): δ 173.29, 171.45, 2H), 3.92 – 3.84 (m, 1H), 3.76 (t, J = 1.9 Hz, 1H), 3.63 (s, 3H), 3.61 –
169.79, 156.34, 135.65, 129.31, 128.50, 127.11, 86.71, 85.51, 83.71, 3.55 (m, 2H), 3.27 (s, 3H), 3.24 – 3.16 (m, 2H), 3.14 (s, 3H), 3.07 –
82.82, 79.15, 57.31, 57.25, 54.30, 52.58, 52.25, 41.05, 39.29, 38.01, 3.00 (m, 2H), 2.39 (t, J = 7.3 Hz, 2H), 2.17 – 2.09 (m, 3H), 2.05 (s,
33.65, 28.35, 27.87, 25.22, 15.00, 11.72; IR νmax (neat, cm–1): 3335, 3H), 1.97 – 1.88 (m, 2H), 1.70 – 1.61 (m, 2H), 1.55 – 1.31 (m, 4H),
2962, 2928, 1744, 1666, 1524, 1449, 1366, 1219, 1171, 1111, 1018; 1.10 – 0.99 (m, 2H), 0.88 – 0.76 (m, 18H); 13C NMR (100 MHz,
MS (ESIMS/[M+Na]+): m/z(%) 630; HRMS (ESI/[M+Na]+): Calcd for DMSO-d6): δ 208.59, 172.72, 172.35, 172.06, 171.40, 170.97,
C31H49N3O9Na+ : 630.3361, found 630.3367.
170.39, 137.58, 129.59, 128.69, 126.97, 88.17, 85.65, 82.49, 82.39,
Data for 20: Scale of reaction 410 mg, 0.65 mmol; yield 398 mg, 58.62, 58.28, 57.29, 57.07, 53.31, 52.65, 52.49, 42.44, 40.85, 38.47,
76% (Purification by column chromatography in 100-200 mesh silica 36.79, 34.65, 30.88, 30.58, 30.19, 25.31, 20.09, 19.85, 19.63, 18.70,
gel, 2% MeOH in chloroform as eluant).
18.67, 15.46, 11.90; IR νmax (neat, cm–1): 3277, 2959, 2925, 1633,
Rf = 0.49 (Silica gel, 8% MeOH in CHCl3); 1H NMR (400 MHz, CDCl3): 1545, 1451, 1219, 1114, 1018; MS (ESIMS/[M+H]+): m/z(%) 818;
δ 7.28 – 7.17 (m, 4H), 7.16 – 7.10 (m, 2H), 6.91 (t, J = 6.1 Hz, 1H), HRMS (ESI/[M+H]+): Calcd for C42H68N5O11 : 818.4910, found
+
6.73 – 6.61 (m, 2H), 5.23 (d, J = 5.2 Hz, 1H), 4.88 – 4.80 (m, 1H), 818.4900. HPLC data: Linear gradient 50→90% B in 10 mins, total
4.40 (bs, 1H), 4.20 – 4.13 (m, 1H), 4.07 – 3.98 (m, 2H), 3.84 (t, J =5.2 run 20 mins, tR= 7.863 min, 89% purity.
Hz, 1H), 3.80 – 3.72 (m, 1H), 3.66 (s, 3H), 3.64 – 3.60 (bs, 1H), 3.42 – Data for 21: Scale of reaction 250 mg, 0.87 mmol; yield 314 mg,
3.36 (m, 5H), 3.25 (s, 3H), 3.05 (d, J =5.7 Hz, 2H), 2.40 – 2.06 (m, 86% (Purification by column chromatography in 100-200 mesh silica
4H), 2.01 – 1.87 (m, 1H), 1.63 – 1.34 (m, 11H), 1.14 – 1.01 (m, 2H), gel, 25% EtOAc in hexane as eluant).
0.99 – 0.76 (m, 18H); 13C NMR (100 MHz, CDCl3): δ 173.51, 171.96, Rf = 0.6 (Silica gel, 60% EtOAc in Hexane); 1H NMR (400 MHz,
171.57, 170.86, 170.12, 156.54, 135.77, 129.22, 128.47, 127.00, CDCl3): δ 7.31 – 7.20 (m, 3H), 7.19 – 7.14 (m, 2H), 6.86 (d, J = 7.1 Hz,
87.11, 85.52, 83.37, 82.69, 80.64, 61.56, 59.45, 57.28, 57.14, 52.96, 1H), 4.83 – 4.74 (m, 1H), 4.46 (d, J = 9.8 Hz, 1H), 3.72 (s, 3H), 3.67 –
This journal is © The Royal Society of Chemistry 20xx
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3.57 (m, 1H), 3.16 (dd, J = 13.9, 5.3 Hz, 1H), 3.05 (dd, J = 13.9, 7.5 IR νmax (neat, cm–1): 3274, 2958, 2927, 1745, 1631, 1545, 1450,
View Article Online
Hz, 1H), 2.25 – 2.12 (m, 2H), 1.86 – 1.70 (m, 2H), 1.55 – 1.39 (m, 1113, 1018; MS (ESIMS/[M+Na]+): m/z(%) 685; HRMS
DOI: 10.1039/C6OB02610A
11H), 1.16 – 1.02 (m, 1H), 0.94 – 0.82 (m, 6H); 13C NMR (100 MHz, (ESI/[M+Na]+): Calcd for C34H54N4O9Na+ : 685.3783, found 685.3773.
CDCl3): δ 173.04, 172.21, 156.59, 136.29, 129.21, 128.47, 126.94, HPLC data: Linear gradient 50→90% B in 10 mins, total run 20 mins,
79.29, 54.07, 53.63, 52.21, 39.26, 37.67, 33.28, 28.37, 28.32, 25.22, tR= 7.758 min, 88% purity.
15.08, 11.71; IR νmax (neat, cm–1): 3310, 2963, 2927, 2874, 1747, Data for 4I: Scale of reaction 135 mg, 0.18 mmol; yield 93 mg, 78%.
1681, 1656, 1529, 1449, 1365, 1249, 1217, 1171, 1018; MS Rf = 0.6 (Silica gel, 6% MeOH in CHCl3); 1H NMR (400 MHz, CDCl3): δ
(ESIMS/[M+Na]+): m/z(%) 443; HRMS (ESI/[M+H]+): Calcd for 7.28 – 7.19 (m, 4H), 7.17 – 7.10 (m, 3H), 6.95 – 6.82 (m, 1H), 6.80
C23H37N2O5+ : 421.2697, found 421.2692.
– 6.61 (m, 1H), 4.90 – 4.82 (m, 1H), 4.50 – 4.36 (m, 2H), 4.26 (t, J =
Data for 22: Scale of reaction 300 mg, 0.71 mmol; yield 349 mg, 8.0 Hz, 1H), 4.10 – 3.98 (m, 2H), 3.69 (s, 3H), 3.62 – 3.57 (m, 1H),
79% (Purification by column chromatography in 100-200 mesh silica 3.49 – 3.36 (m, 5H), 3.26 (s, 3H), 3.13 – 3.03 (m, 2H), 2.43 – 2.31 (m,
gel, 1.5% MeOH in chloroform as eluant).
2H), 2.26 – 2.19 (m, 2H), 2.16 – 1.94 (m, 3H), 1.91 – 1.78 (m, 2H),
Rf = 0.4 (Silica gel, 5% MeOH in CHCl3); 1H NMR (400 MHz, CDCl3): δ 0.98 – 0.84 (m, 12H); 13C NMR (100 MHz, CDCl3): δ 172.52, 171.72,
7.29 – 7.09 (m, 6H), 6.64 – 6.49 (m, 2H), 5.04 (d, J = 5.6 Hz, 1H), 4.82 171.57, 171.24, 169.77, 135.77, 129.30, 128.49, 127.09, 86.80,
– 4.75 (m, 1H), 4.21 – 4.12 (m, 1H), 3.81 – 3.66 (m, 5H), 3.16 (dd, J = 85.96, 83.41, 83.19, 82.64, 69.21, 58.83, 58.38, 57.42, 57.28, 52.66,
13.8, 5.7 Hz, 1H), 3.03 (dd, J = 13.8, 7.4 Hz, 1H), 2.38 – 2.06 (m, 4H), 52.30, 41.03, 37.97, 34.89, 31.28, 30.66, 24.22, 19.22, 19.13, 18.32,
1.98 – 1.86 (m, 1H), 1.62 – 1.39 (m, 12H), 1.16 – 1.03 (m, 1H), 1.00 – 18.17, 17.81; IR νmax (neat, cm–1): 3275, 2959, 2929, 1745, 1674,
0.79 (m, 18H); 13C NMR (100 MHz, CDCl3): δ 173.17, 172.29, 171.85, 1631, 1545, 1220, 1114, 1018; MS (ESIMS/[M+Na]+): m/z(%) 681;
170.89, 156.39, 136.51, 129.26, 128.36, 126.75, 80.72, 60.98, 59.40, HRMS (ESI/[M+Na]+): Calcd for C34H50N4O9Na+ : 681.3470, found
53.55, 53.36, 52.15, 38.39, 37.74, 33.04, 29.85, 29.25, 28.21, 27.73, 681.3472. HPLC data: Isocratic (50%), total run 25 mins, tR= 7.910
25.62, 19.69, 19.31, 17.62, 17.37, 15.14, 11.29; IR νmax (neat, cm–1): min, 92% purity.
3280, 2962, 2929, 2873, 1750, 1689, 1641, 1547, 1523, 1371, 1220, Data for 4L: Scale of reaction 283 mg, 0.43 mmol; yield 209 mg, 71
1171, 1018; MS (ESIMS/[M+Na]+): m/z(%) 641; HRMS (ESI/[M+H]+): %.
Calcd for C33H55N4O7+ : 619.4066, found 619.4069.
Rf = 0.49 (Silica gel, 6% MeOH in CHCl3); 1H NMR (400 MHz, CDCl3):
δ 7.29 – 7.19 (m, 3H), 7.16 – 7.02 (m, 4H), 6.86 – 6.75 (m, 1H), 6.58
Data for 4D: Scale of reaction 295 mg, 0.48 mmol; yield 218 mg, – 6.45 (m, 1H), 4.89 – 4.83 (m, 1H), 4.44 – 4.35 (m, 2H), 4.28 – 4.21
73%. (m, 1H), 4.08 – 4.00 (m, 2H), 3.69 (s, 3H), 3.60 – 3.57 (m, 1H), 3.50 –
Rf = 0.6 (Silica gel, 10% MeOH in CHCl3); 1H NMR (300 MHz, DMSO- 3.34 (m, 5H), 3.27 (s, 3H), 3.12 –3.03 (m, 2H), 2.25 – 2.19 (m, 1H),
d6): δ 8.21 (d, J = 7.8 Hz, 1H), 7.90 – 7.81 (m, 1H), 7.79 – 7.63 (m, 2.17 – 2.08 (m, 1H), 2.06 – 1.99 (m, 2H), 1.66 – 1.56 (m, 2H), 1.31 –
1H), 7.59 – 7.47 (m, 1H), 7.31 – 7.16 (m, 5H), 4.49 – 4.37 (m, 1H), 1.22 (m, 8H), 0.94 – 0.83 (m, 15H); 13C NMR (100 MHz, CDCl3): δ
4.22 – 4.04 (m, 2H), 3.59 (s, 3H), 3.01 (dd, J = 13.7, 5.4 Hz, 1H), 2.86 173.47, 171.79, 171.57, 171.17, 169.77, 135.77, 129.30, 128.50,
(dd, J = 13.7, 9.5 Hz, 1H), 2.75 – 2.69 (m, 1H), 2.25 – 2.08 (m, 5H), 127.09, 86.85, 85.96, 83.16, 82.66, 58.79, 58.40, 57.41, 57.29,
2.05 – 1.85 (m, 4H), 1.63 – 1.49 (m, 3H), 1.49 – 1.32 (m, 5H), 1.09 – 52.68, 52.28, 41.05, 37.98, 36.58, 31.66, 31.15, 30.61, 29.19, 28.98,
0.93 (m, 1H), 0.89 – 0.72 (m, 18H); 13C NMR (75 MHz, DMSO-d6): δ 25.81, 22.55, 19.25, 19.16, 18.32, 18.06, 14.01; IR νmax (neat, cm–1):
172.62, 172.46, 171.39, 170.99, 137.77, 129.49, 128.61, 126.92, 3276, 2957, 2927, 1745, 1675, 1631, 1544, 1453, 1223, 1111, 1018;
84.79, 71.52, 58.65, 58.23, 53.99, 52.55, 52.21, 38.34, 37.11, 34.97, MS (ESIMS/[M+H]+): m/z(%) 691; HRMS (ESI/[M+Na]+): Calcd for
32.58, 30.82, 30.60, 27.96, 25.23, 25.02, 19.80, 19.59, 18.71, 18.66, C36H58N4O9Na+ : 713.4096, found 713.4101. HPLC data: Linear
17.91, 15.45, 11.83; IR νmax (neat, cm–1): 3279, 2925, 2868, 1740, gradient 50→90% B in 10 mins, total run 30 mins, tR= 10.54 min,
1635, 1542, 1454, 1223, 1019; MS (ESIMS/[M+H]+): m/z(%) 627; 94% purity.
HRMS (ESI/[M+Na]+): Calcd for C35H54N4O6Na+ : 649.3935, found Data for 4O: Scale of reaction 300 mg, 0.45 mmol; yield 266 mg, 82
649.3921. HPLC data: Linear gradient 50→90% B in 10 mins, total %.
run 25 mins, tR= 5.907 min, 98% purity.
Data for 4F: Scale of reaction 300 mg, 0.45 mmol; yield 251 mg, 7.28 – 7.16 (m, 4H), 7.15 – 7.10 (m, 3H), 6.86 (t, J = 5.2 Hz, 1H), 6.61
84%. (d, J = 8.8 Hz, 1H), 4.89 – 4.83 (m, 1H), 4.44 – 4.37 (m, 2H), 4.28 –
Rf = 0.6 (Silica gel, 6% MeOH in CHCl3); 1H NMR (400 MHz, CDCl3): δ
Rf = 0.6 (Silica gel, 10% MeOH in CHCl3); 1H NMR (400 MHz, CDCl3): 4.21 (m, 1H), 4.08 – 4.00 (m, 2H), 3.69 (s, 3H), 3.61 – 3.57 (m, 1H),
δ 7.28 – 7.18 (m, 4H), 7.16 – 7.10 (m, 3H), 6.93 – 6.83 (m, 1H), 6.66 3.47 – 3.36 (m, 5H), 3.26 (s, 3H), 3.13 –3.03 (m, 2H), 2.27 – 1.96 (m,
– 6.56 (m, 1H), 4.89 – 4.82 (m, 1H), 4.44 – 4.37 (m, 2H), 4.28 – 4.22 4H), 1.67 – 1.55 (m, 2H), 1.33 – 1.18 (m, 15H), 0.94 – 0.83 (m, 12H);
(m, 1H), 4.07 – 4.02 (m, 2H), 3.69 (s, 3H), 3.60 – 3.58 (m, 1H), 3.45 – 13C NMR (100 MHz, CDCl3): δ 173.50, 171.89, 171.55, 171.24,
3.38 (m, 5H), 3.26 (s, 3H), 3.09 – 3.05 (m, 2H), 2.25 – 2.17 (m, 3H), 169.77, 135.75, 129.30, 128.49, 127.09, 86.78, 85.91, 83.16, 82.63,
2.17 – 1.95 (m, 2H), 1.67 – 1.54 (m, 4H), 1.33 – 1.25 (m, 4H), 0.95 – 58.80, 58.35, 57.41, 57.28, 52.66, 52.29, 41.03, 37.97, 36.56, 31.81,
0.82 (m, 12H); 13C NMR (100 MHz, CDCl3): δ 173.48, 171.89, 171.55, 31.18, 30.59, 29.44, 29.34, 29.25, 29.23, 25.83, 22.61, 19.24, 19.15,
171.29, 169.79, 135.77, 129.29, 128.49, 127.07, 86.86, 85.92, 83.17, 18.33, 18.09, 14.06; IR νmax (neat, cm–1): 3280, 2957, 2926, 1746,
82.65, 58.83, 58.37, 57.39, 57.24, 52.67, 52.26, 41.01, 37.97, 31.36, 1676, 1633, 1545, 1453, 1224, 1113, 1019; MS (ESIMS/[M+Na]+):
31.19, 30.60, 29.64, 25.48, 22.33, 19.23, 19.14, 18.36, 18.11, 13.88; m/z(%) 741; HRMS (ESI/[M+Na]+): Calcd for C38H62N4O9Na+
:
10 | J. Name., 2012, 00, 1-3
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741.4409, found 741.4402. HPLC data: Linear gradient 50→90% B in Rf = 0.47 (Silica gel, 5% MeOH in CHCl3); 1H NMR (400 MHz, DMSO-
View Article Online
DOI: 10.1039/C6OB02610A
d6): δ 8.01 – 7.81 (m, 3H), 7.74 – 7.52 (m, 2H), 7.47 (bs, 1H), 7.29 –
10 mins, total run 20 mins, tR= 13.599 min, 96% purity.
Data for 4R: Scale of reaction 250 mg, 0.37 mmol; yield 197 mg, 73 7.12 (m, 6H), 4.48 – 4.37 (m, 1H), 4.32 – 4.26 (m, 1H), 4.21 – 4.07
%. (m, 2H), 3.94 – 3.78 (m, 2H), 3.71 – 3.54 (m, 2H), 3.35 – 3.25 (m,
Rf = 0.5 (Silica gel, 5% MeOH in CHCl3); 1H NMR (400 MHz, CDCl3): δ 8H), 3.24 – 3.11 (m, 5H), 2.98 – 2.91 (m, 1H), 2.40 (t, J = 6.9 Hz, 2H),
7.28 – 7.21 (m, 3H), 7.16 – 7.10 (m, 3H), 7.05 – 6.96 (m, 1H), 6.79 – 2.17 – 1.88 (m, 9H), 1.72 – 1.61 (m, 3H), 0.92 – 0.74 (m, 18H); 13C
6.71 (m, 1H), 6.49 – 6.42 (m, 1H), 5.84 – 5.72 (m, 1H), 5.01 – 4.83 NMR (100 MHz, DMSO-d6): δ 208.52, 172.82, 172.76, 172.71,
(m, 3H), 4.43 – 4.40 (m, 1H), 4.39 – 4.34 (m, 1H), 4.26 – 4.21 (m, 172.35, 171.99, 170.92, 169.79, 137.97, 129.75, 128.59, 128.45,
1H), 4.07 – 4.01 (m, 2H), 3.69 (s, 3H), 3.59 – 3.56 (m, 1H), 3.52 – 126.69, 87.84, 85.53, 82.65, 57.22, 57.03, 53.59, 42.87, 42.44,
3.34 (m, 5H), 3.27 (s, 3H), 3.13 –3.03 (m, 2H), 2.24 – 1.95 (m, 4H), 34.65, 34.59, 30.17, 20.09, 20.02, 19.87, 19.72, 19.62, 18.85, 18.69,
1.66 – 1.56 (m, 2H), 1.39 – 1.23 (m, 10H), 0.95 – 0.86 (m, 12H); 13C 18.13, 15.44, 14.86, 11.89, 11.77; IR νmax (neat, cm–1): 3397, 3293,
NMR (100 MHz, CDCl3): δ 173.42, 171.76, 171.58, 171.13, 169.75, 2960, 2923, 1641, 1535, 1455, 1369, 1264, 1227, 1157, 1116, 1018;
139.02, 135.76, 129.31, 128.51, 127.11, 114.17, 86.83, 85.97, 83.16, MS (ESIMS/[M+H]+): m/z(%) 803; HRMS (ESI/[M+H]+): Calcd for
+
82.66, 58.78, 58.42, 57.42, 57.30, 52.67, 52.29, 41.07, 37.98, 36.57, C41H67N6O10 : 803.4913, found 803.4900. HPLC data: Isocratic
33.69, 31.13, 30.61, 29.18, 29.16, 28.92, 28.80, 25.77, 19.26, 19.17, (50%), total run 25 mins, tR= 7.350 min, 86% purity.
18.30, 18.03; IR νmax (neat, cm–1): 3278, 2958, 2926, 1746, 1675, Data for 4E: Scale of reaction 60 mg, 0.096 mmol; yield 32 mg, 55%.
1
1632, 1545, 1448, 1222, 1114, 1018; MS (ESIMS/[M-1]+): m/z(%) Rf = 0.5 (Silica gel, 8% MeOH in CHCl3); H NMR (300 MHz, DMSO-
715; HRMS (ESI/[M+Na]+): Calcd for C38H60N4O9Na+ : 739.4252, d6): δ 7.88 – 7.76 (m, 2H), 7.70 (d, J = 8.6 Hz, 1H), 7.48 (d, J = 8.8
found 739.4258. HPLC data: Linear gradient 50→90% B in 10 mins, Hz, 1H), 7.37 (bs, 1H), 7.29 – 7.13 (m, 5H), 6.99 (bs, 1H), 4.48 – 4.33
total run 25 mins, tR= 13.682 min, 93% purity.
Data for 4U: Scale of reaction 280 mg, 0.42 mmol; yield 261 mg, 84 1H), 2.82 – 2.65 (m, 2H), 2.25 – 2.08 (m, 4H), 2.06 – 1.85 (m, 4H),
%.
1.65 – 1.29 (m, 8H), 1.11 – 0.94 (m, 1H), 0.93 – 0.69 (m, 18H); 13C
(m, 1H), 4.23 – 4.03 (m, 2H), 3.64 – 3.47 (m, 1H), 3.08 – 2.93 (m,
Rf = 0.6 (Silica gel, 3% MeOH in CHCl3); 1H NMR (400 MHz, CDCl3): δ NMR (100 MHz, DMSO-d6): δ 173.75, 172.46, 172.32, 171.42,
7.28 – 7.21 (m, 6H), 7.18 – 7.11 (m, 6H), 6.89 – 6.82 (m, 1H), 6.56 170.95, 138.69, 129.58, 128.38, 126.58, 84.80, 71.57, 58.67, 58.24,
(d, J = 8.6 Hz, 1H), 4.90 – 4.83 (m, 1H), 4.45 – 4.38 (m, 2H), 4.27 – 54.19, 52.52, 38.37, 38.02, 34.97, 30.79, 30.60, 27.97, 25.26, 25.03,
4.21 (m, 1H), 4.07 – 4.02 (m, 2H), 3.69 (s, 3H), 3.60 – 3.57 (m, 1H), 19.83, 19.59, 18.72, 18.68, 17.92, 15.45, 11.86; IR νmax (neat, cm–1):
3.48 – 3.35 (m, 5H), 3.26 (s, 3H), 3.10 –3.06 (m, 2H), 2.57 (t, J = 7.7 3381, 3277, 2923, 2855, 1633, 1541, 1456, 1372, 1223, 1019; MS
Hz, 2H), 2.25 – 1.97 (m, 4H), 1.70 – 1.56 (m, 4H), 1.39 – 1.29 (m, (ESIMS/[M+Na]+): m/z(%) 634; HRMS (ESI/[M+H]+): Calcd for
2H), 0.96 – 0.86 (m, 12H); 13C NMR (100 MHz, CDCl3): δ 173.29, C34H54N5O5+ : 612.4120, found 612.4153. HPLC data: Isocratic (50%),
171.79, 171.57, 171.18, 169.76, 142.41, 135.76, 129.29, 128.49, total run 20 mins, tR= 13.910 min, 100% purity.
128.29, 128.21, 127.08, 125.62, 86.81, 85.94, 83.16, 82.63, 58.79, Data for 4G: Scale of reaction 211 mg, 0.32 mmol; yield 163 mg,
58.32, 57.40, 57.27, 52.66, 52.28, 41.02, 37.96, 36.41, 35.72, 31.21, 79%.
1
31.13, 30.62, 28.85, 25.63, 19.24, 19.13, 18.33, 18.11; IR νmax (neat, Rf = 0.5 (Silica gel, 8% MeOH in CHCl3); H NMR (400 MHz, DMSO-
cm–1): 3275, 2958, 2928, 1745, 1675, 1631, 1543, 1220, 1115, 1018; d6): δ 8.19 – 8.11 (m, 1H), 7.99 (d, J = 8.7 Hz, 1H), 7.83 (d, J = 8.8
MS (ESIMS/[M+Na]+): m/z(%) 761 ; HRMS (ESI/[M+Na]+): Calcd for Hz, 1H), 7.65 (t, J = 8.8 Hz, 1H), 7.57 (d, J = 8.4 Hz, 1H), 7.46 (bs, 1H),
C40H58N4O9Na+ : 761.4096, found 761.4102. HPLC data: Linear 7.27 – 7.15 (m, 5H), 4.47 – 4.37 (m, 1H), 4.30 (d, J = 1.9 Hz, 1H), 4.22
gradient 50→90% B in 10 mins, total run 30 mins, tR= 10.542 min, – 4.13 (m, 2H), 3.93 – 3.88 (m, 1H), 3.83 (t, J = 1.8 Hz, 1H), 3.66 –
93% purity.
3.61 (m, 1H), 3.33 (s, 3H), 3.17 – 3.14 (m, 5H), 2.99 – 2.91 (m, 2H),
2.22 – 1.88 (m, 5H), 1.54 – 1.44 (m, 4H), 1.31 – 1.17 (m, 4H), 0.88 –
0.79 (m, 12H); 13C NMR (100 MHz, DMSO-d6): δ 172.79, 172.67,
171.54, 171.48, 169.72, 137.94, 129.77, 128.44, 126.68, 87.67,
85.22, 83.36, 82.82, 82.70, 58.31, 58.03, 57.21, 57.04, 53.57, 35.60,
31.28, 30.48, 25.58, 22.34, 19.76, 19.61, 18.67, 18.52, 14.31; IR νmax
(neat, cm–1): 3396, 3293, 2959, 2928, 1643, 1535, 1456, 1224, 1108,
1018; MS (ESIMS/[M+Na]+): m/z(%) 670; HRMS (ESI/[M+H]+): Calcd
General Procedure for the Synthesis of C-terminal Primary
Amide: First the C-terminal ester group was hydrolyzed to the
corresponding acid derivative by following the general procedure as
discussed earlier. Then the resulted acid was dissolved in THF (6
mL/mmol) and cooled to -20 °C. Et3N (1.1 eq) and ClCO2Et (1.1 eq)
were added successively and the reaction mixture was stirred for 30
min at the same temperature. Then 30% aq. NH3 (5 eq) was added
to it and further stirred for 1.5 h at 0 °C. After that it was quenched
with saturated aqueous NH4Cl solution. The mixture was extracted
with EtOAc and the combined organic extracts were washed with
water, brine, dried over Na2SO4. Solvent was concentrated in vacuo
and purified by flash column chromatography using 100-200 mesh
silica gel (1-2% MeOH in chloroform as eluant) to give the amide.
Data for 4B: Scale of reaction 250 mg, 0.30 mmol; yield 150 mg,
61%.
+
for C33H54N5O8 : 648.3967, found 648.3964. HPLC data: Isocratic
(50%), total run 25 mins, tR= 10.480 min, 92% purity.
Data for 4J: Scale of reaction 63 mg, 0.1 mmol; yield 52 mg, 84%.
1
Rf = 0.4 (Silica gel, 8% MeOH in CHCl3); H NMR (400 MHz, DMSO-
d6): δ 8.16 (t, J = 5.4 Hz, 1H), 7.91 (d, J = 8.8 Hz, 1H), 7.70 (d, J = 8.8
Hz, 1H), 7.59 (d, J = 8.3 Hz, 1H), 7.47 (bs, 1H), 7.25 – 7.15 (m, 6H),
4.47 – 4.39 (m, 1H), 4.31 (bs, 1H), 4.22 – 4.12 (m, 2H), 3.93 – 3.86
(m, 1H), 3.83 (bs, 1H), 3.64 (bs, 1H), 3.31 – 3.23 (m, 4H), 3.21 – 3.12
(m, 4H), 2.99 – 2.91 (m, 2H), 2.77 (t, J = 2.6 Hz, 1H), 2.32 – 2.10 (m,
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4H), 2.01 – 1.89 (m, 2H), 1.71 – 1.61 (m, 2H), 0.87 – 0.79 (m, 12H); 1H), 3.68 – 3.58 (m, 1H), 3.40 – 3.04 (m, 7H), 3.01 –Vi2e.w8A7rt(icmle,O2nHlin)e,
13C NMR (100 MHz, DMSO-d6): δ 172.71, 172.16, 171.52 (2C), 2.23 – 1.89 (m, 4H), 1.54 – 1.40 (m, 2H), 1.37 – 1.18 (m, 10H), 0.87 –
DOI: 10.1039/C6OB02610A
169.74, 137.96, 129.81, 128.48, 126.73, 87.65, 85.19, 84.59, 82.84, 0.79 (m, 12H); 13C NMR (100 MHz, DMSO-d6): δ 172.76, 172.66,
82.73, 71.93, 58.34, 58.08, 57.24, 57.07, 53.59, 37.83, 34.54, 31.23, 171.52, 169.70, 139.27, 137.93, 129.76, 128.44, 126.68, 115.09,
30.53, 24.99, 19.79, 19.65, 18.69, 18.60, 17.85; IR νmax (neat, cm–1): 87.65, 85.20, 82.80, 82.67, 58.23, 58.02, 57.21, 57.04, 53.55, 37.83,
3295, 2923, 2853, 1644, 1529, 1447, 1219, 1112, 1018; MS 35.61, 33.62, 31.21, 30.48, 29.05, 28.99, 28.88, 28.67, 25.87, 19.76,
(ESIMS/[M+Na]+): m/z(%) 666; HRMS (ESI/[M+Na]+): Calcd for 19.61,18.66, 18.52; IR νmax (neat, cm–1): 3396, 3291, 2926, 2856,
C33H49N5O8Na+ : 666.3473, found 666.3480. HPLC data: Linear 1639, 1535, 1450, 1221, 1114, 1018; MS (ESIMS/[M-1]+): m/z(%)
gradient 50→90% B in 10 mins, total run 25 mins, tR= 9.890 min, 700; HRMS (ESI/[M+Na]+): Calcd for C37H59N5O8Na+ : 724.4256,
92% purity.
Data for 4M: Scale of reaction 170 mg, 0.25 mmol; yield 130 mg, total run 20 mins, tR= 10.590 min, 94% purity.
77%. Data for 4V: Scale of reaction 200 mg, 0.27 mmol; yield 155 mg,
found 724.4279. HPLC data: Linear gradient 50→90% B in 10 mins,
1
Rf = 0.5 (Silica gel, 8% MeOH in CHCl3); H NMR (400 MHz, DMSO- 79%.
d6): δ 8.15 (t, J = 5.3 Hz, 1H), 7.84 (d, J = 8.8 Hz, 1H), 7.69 (d, J = 8.7 Rf = 0.6 (Silica gel, 5% MeOH in CHCl3); 1H NMR (400 MHz, CDCl3): δ
Hz, 1H), 7.58 (d, J = 8.3 Hz, 1H), 7.47 (bs, 1H), 7.25 – 7.13 (m, 6H), DMSO-d6): δ 8.16 (t, J = 5.6 Hz, 1H), 7.85 (d, J = 8.8 Hz, 1H), 7.69 (d,
4.47 – 4.39 (m, 1H), 4.30 (bs, 1H), 4.22 – 4.13 (m, 2H), 3.93 – 3.86 J = 8.7 Hz, 1H), 7.59 (d, J = 8.4 Hz, 1H), 7.47 (bs, 1H), 7.29 – 7.12 (m,
(m, 1H), 3.82 (bs, 1H), 3.63 (bs, 1H), 3.31 – 3.23 (m, 4H), 3.22 – 3.11 11H), 4.47 – 4.40 (m, 1H), 4.32 – 4.29 (m, 1H), 4.22 – 4.15 (m, 2H),
(m, 4H), 2.99 – 2.91 (m, 2H), 2.21 – 2.07 (m, 2H), 2.00 – 1.88 (m, 3.92 – 3.87 (m, 1H), 3.84 – 3.81 (m, 1H), 3.65 – 3.62 (m, 1H), 3.31 –
2H), 1.52 – 1.42 (m, 2H), 1.29 – 1.16 (m, 8H), 0.87 – 0.79 (m, 15H); 3.24 (m, 4H), 3.21 – 3.13 (m, 4H), 3.00 – 2.89 (m, 2H), 2.54 (t, J = 7.6
13C NMR (100 MHz, DMSO-d6): δ 172.79, 172.69, 171.57, 171.50, Hz, 2H), 2.22 – 2.07 (m, 2H), 2.00 – 1.89 (m, 2H), 1.59 – 1.47 (m,
169.73, 137.96, 129.80, 128.47, 126.71, 87.65, 85.19, 82.83, 82.71, 4H), 1.30 – 1.21 (m, 2H), 0.87 – 0.79 (m, 12H); 13C NMR (100 MHz,
58.23, 58.03, 57.23, 57.07, 53.58, 37.83, 35.64, 31.69, 31.24, 30.53, DMSO-d6): δ 172.75, 172.69, 171.54, 171.49, 169.73, 142.73,
29.03, 28.94, 25.93, 22.54, 19.79, 19.64, 18.69, 18.56, 14.44; IR νmax 137.95, 129.79, 128.72, 128.67, 128.46, 126.70, 126.06, 87.67,
(neat, cm–1): 3283, 2957, 2926, 1670, 1634, 1542, 1106, 1018; MS 85.21, 82.83, 82.71, 58.24, 58.04, 57.22, 57.06, 53.58, 37.84, 35.58,
(ESIMS/[M+H]+): m/z(%) 676; HRMS (ESI/[M+H]+): Calcd for 35.55, 31.25, 30.52, 28.70, 25.75, 19.79, 19.64, 18.68, 18.56; IR νmax
C35H58N5O8+ : 676.4280, found 676.4286. HPLC data: Linear gradient (neat, cm–1): 3395, 3292, 2926, 2855, 1640, 1535, 1455, 1112, 1018;
50→90% B in 10 mins, total run 20 mins, tR= 10.272 min, 97% MS (ESIMS/[Na]+): m/z(%) 746; HRMS (ESI/[M+Na]+): Calcd for
purity.
Data for 4P: Scale of reaction 210 mg, 0.29 mmol; yield 161 mg, gradient 50→90% B in 10 mins, total run 20 mins, tR= 10.272 min,
C39H57N5O8Na+ : 746.4099, found 746.4100. HPLC data: Linear
78%.
96% purity.
Rf = 0.45 (Silica gel, 8% MeOH in CHCl3); 1H NMR (400 MHz, DMSO-
d6): δ 8.15 (t, J = 5.8 Hz, 1H), 7.84 (d, J = 8.8 Hz, 1H), 7.68 (d, J = 8.7
Hz, 1H), 7.58 (d, J = 8.4 Hz, 1H), 7.47 (bs, 1H), 7.24 – 7.15 (m, 6H),
4.46 – 4.39 (m, 1H), 4.32 – 4.28 (m, 1H), 4.21 – 4.13 (m, 2H), 3.92 –
3.86 (m, 1H), 3.85 – 3.79 (m, 1H), 3.66 – 3.60 (m, 1H), 3.31 – 3.23
(m, 4H), 3.22 – 3.11 (m, 4H), 2.99 – 2.89 (m, 2H), 2.21 – 2.06 (m,
2H), 2.01 – 1.89 (m, 2H), 1.52 – 1.42 (m, 2H), 1.25 – 1.20 (m, 12H),
0.88 – 0.79 (m, 15H); 13C NMR (75 MHz, DMSO-d6): δ 172.77,
172.65, 171.51, 171.45, 169.69, 137.92, 129.75, 128.43, 126.67,
87.66, 85.21, 82.79, 82.67, 79.62, 58.24, 58.00, 57.19, 57.02, 53.54,
37.83, 35.62, 31.72, 31.19, 30.45, 29.36, 29.24, 29.09, 29.02, 25.88,
22.53, 19.75, 19.59, 18.64, 18.49, 14.38; IR νmax (neat, cm–1): 3285,
2958, 2925, 2854, 1638, 1541, 1455, 1219, 1115, 1019; MS
(ESIMS/[M+Na]+): m/z(%) 726; HRMS (ESI/[M+Na]+): Calcd for
C37H61N5O8Na+ : 726.4412, found 726.44415. HPLC data: Linear
gradient 50→90% B in 10 mins, total run 20 mins, tR= 10.316 min,
98% purity.
General Procedure for Permethylation: To a stirring solution of
compound in dry DMF (3 mL/mmol) at 0 °C Ag2O (2 eq) and MeI (2.5
eq) were sequentially added. After stirring for 12 h at room
temperature, the reaction mixture was diluted with EtOAc, filtered
to remove the silver oxide, washed with saturated aqueous Na2S2O3
solution, water, brine, dried (Na2SO4), filtered and concentrated in
vacuo. Purification by silica gel (100-200 mesh) column
chromatography (0.5-1.5% MeOH in chloroform as eluant) afforded
the permethylated compound.
Data for 4C: Scale of reaction 80 mg, 0.1 mmol; yield 66 mg, 73%.
Rf = 0.51 (Silica gel, 4% MeOH in CHCl3); 1H NMR (400 MHz, DMSO-
d6): δ 7.34-7.13 (m, 5H), 5.56-5.37 (m, 1H), 5.14-4.9 (m, 2H), 4.67-
4.47 (m, 1H), 4.41-4.29 (m, 1H), 4.28-4.12 (m, 1H), 3.77-3.48 (m,
2H), 3.39-3.26 (m, 6H), 3.25-3.07 (m, 5H), 3.05-2.56 (m, 24H), 2.34-
2.14 (m, 6H), 2.13-2.04 (m, 4H), 1.96-1.62 (m, 4H), 0.94-0.63 (m,
18H); 13C NMR (100 MHz, DMSO-d6): δ 211.99, 174.87, 172.79,
172.68, 170.27, 170.13, 168.92, 137.89, 129.61, 128.49, 126.73,
85.07, 84.34, 70.24, 58.22, 58.01, 54.95, 47.04, 45.85, 36.58, 35.76,
31.94, 31.72, 30.21, 30.03, 29.44, 28.46, 26.97, 26.81, 22.51, 19.66,
19.48, 18.52, 18.48, 18.39, 17.85, 16.40, 16.34, 14.34, 10.67, 8.09;
IR νmax (neat, cm–1): 3406, 2923, 1637, 1454, 1365, 1265, 1219,
1018; MS (ESIMS/[M+Na]+): m/z(%) 923; HRMS (ESI/[M+Na]+):
Calcd for C48H80N6O10Na+ : 923.5828, found 923.5834. HPLC data:
Data for 4S: Scale of reaction 140 mg, 0.19 mmol; yield 111 mg,
81%.
Rf = 0.49 (Silica gel, 5% MeOH in CHCl3); 1H NMR (300 MHz, DMSO-
d6): δ 8.24 – 8.10 (m, 1H), 7.85 (d, J = 8.8 Hz, 1H), 7.72 – 7.64 (m,
1H), 7.58 (d, J = 8.4 Hz, 1H), 7.47 (bs, 1H), 7.31 – 7.12 (m, 6H), 5.85 –
5.70 (m, 1H), 5.03 – 4.89 (m, 2H), 4.78 – 4.35 (m, 1H), 4.32 – 4.25
(m, 1H), 4.24 – 4.09 (m, 3H), 3.93 – 3.86 (m, 1H), 3.85 – 3.78 (m,
12 | J. Name., 2012, 00, 1-3
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Linear gradient 50→90% B in 10 mins, total run 20 mins, tR= 7.753 1718, 1641, 1462, 1403, 1103, 1018; MS (ESIMS/[VNieaw]+A)r:ticmle /Ozn(l%ine)
+
+
DOI: 10.1039/C6OB02610A
min, 100% purity.
Data for 4H: Scale of reaction 90 mg, 0.14 mmol; yield 75 mg, 74%.
Rf = 0.5 (Silica gel, 4% MeOH in CHCl3); H NMR (400 MHz, DMSO- 13.923 min, 100% purity.
811; HRMS (ESI/[M+Na] ): Calcd for C43H73N5O8Na : 810.5351,
found 810.5356. HPLC data: Isocratic (50%), total run 20 mins, tR=
1
d6): δ 7.29-7.14 (m, 5H), 5.59-5.40 (m,1H), 5.08-4.94 (m, 2H), 4.73- Data for 4T: Scale of reaction 60 mg, 0.08 mmol; yield 52 mg, 77 %.
1
4.53 (m, 1H), 4.08-3.99 (m, 1H), 3.56-3.42 (m, 2H), 3.35-3.31 (m, Rf = 0.7 (Silica gel, 5% MeOH in CHCl3); H NMR (400 MHz, DMSO-
5H), 3.30-3.26 (m, 3H), 3.25-3.20 (m, 3H), 3.01-2.94 (m, 3H), 2.93- d6): δ 7.34-7.10 (m, 5H), 5.84-5.72 (m, 1H), 5.52-5.38 (m, 1H), 5.08-
2.88 (m, 2H), 2.87-2.76 (m, 12H), 2.35-2.14 (m, 5H), 1.56-1.44 (m, 4.89 (m, 4H), 4.75-4.59 (m, 1H), 4.22-3.88 (m, 2H), 3.58-3.44 (m,
2H), 1.31-1.20 (m, 6H), 0.9-0.61 (m, 12H ); 13C NMR (100 MHz, 2H), 3.36-3.21 (m, 9H), 3.03-2.75 (m, 15H), 2.36-2.13 (m, 5H), 2.04-
DMSO-d6): δ 173.14, 173.12, 170.13, 169.11, 168.63, 138.17, 1.96 (m, 2H), 1.55-1.43 (m, 2H), 1.38-1.19 (m, 10H), 0.89-0.61 (m,
129.60, 128.48, 126.61, 87.42, 87.03, 81.65, 8144, 80.36, 58.07, 12H); 13C NMR (100 MHz, DMSO-d6): δ 173.17, 170.13, 169.60,
57.84, 57.24, 57.21, 54.69, 36.33, 35.70, 32.99, 31.32, 30.59, 30.30, 169.14, 168.66, 139.29, 138.24, 129.69, 128.56, 126.69, 115.13,
30.02, 26.91, 24.73, 22.34, 20.03, 19.72, 19.64, 18.40, 17.92, 14.26 ; 87.43, 87.04, 79.67, 58.14, 57.88, 57.29, 54.77, 36.49, 36.39, 35.77,
IR νmax (neat, cm–1): 3410, 2959, 2927, 1640, 1463, 1404, 1098, 35.04, 33.65, 33.03, 30.66, 30.36, 30.07, 29.14, 29.10, 28.92, 28.69,
1018; MS (ESIMS/[M+Na]+): m/z(%) 754; HRMS (ESI/[M+Na]+): 27.01, 2508, 20.12, 19.81, 18.47, 18.00; IR νmax (neat, cm–1): 3403,
Calcd for C39H65N5O8Na+ : 754.4725, found 754.4739. HPLC data: 2928, 2855, 1717, 1640, 1462, 1405, 1103, 1018; MS (ESIMS/[Na]+):
Isocratic (50%), total run 20 mins, tR= 13.798 min, 100% purity.
m/z(%) 808; HRMS (ESI/[M+Na]+): Calcd for C43H71N5O8Na+
808.5195, found 808.5199. HPLC data: Isocratic (50%), total run 20
:
Data for 4K: Scale of reaction 20 mg, 0.03 mmol; yield 16 mg, 73%.
1
Rf = 0.6 (Silica gel, 6% MeOH in CHCl3); H NMR (400 MHz, DMSO- mins, tR= 10.831 min, 100% purity.
d6): δ 7.32-7.09 (m, 5H), 5.09-4.89 (m, 1H), 4.78-4.49 (m, 2H), 4.48- Data for 4W: Scale of reaction 80 mg, 0.11 mmol; yield 67 mg, 74%.
1
4.27 (m, 1H), 4.21-3.99 (m, 1H), 3.74-3.51 (m, 2H), 3.44-3.19 (m, Rf = 0.8 (Silica gel, 4% MeOH in CHCl3); H NMR (400 MHz, DMSO-
14H), 3.07-2.64 (m, 14H), 2.56-2.52 (m, 1H), 2.38-2.08 (m, 4H), 2.07- d6): δ 7.29-7.13 (m, 10H), 5.53-5.38 (m, 1H), 5.07-4.94 (m, 2H), 4.74-
1.84 (m, 2H), 1.32-1.16 (m, 2H), 0.92-0.55 (m, 12H); IR νmax (neat, 4.58 (m, 1H), 4.07-3.99 (m, 1H), 3.58-3.45 (m, 2H), 3.36-3.22 (m,
cm–1): 3396, 2958, 2924, 2853, 1722, 1642, 1456, 1413, 1261, 1219, 17H), 3.00-2.89 (m, 2H), 2.83-2.78 (m, 9H), 2.57-2.52 (m, 2H), 2.35-
1100, 1018; MS (ESIMS/[M]+): m/z(%) 727; HRMS (ESI/[M]+): Calcd 2.27 (m, 2H), 2.25-2.14 (m, 2H), 1.60-1.48 (m, 4H), 1.33-1.25 (m,
+
for C39H61N5O8 : 727.4515, found 727.4524. HPLC data: Linear 2H), 0.87-0.61 (m, 12H); 13C NMR (100 MHz, DMSO-d6): δ 173.06,
gradient 50→90% B in 10 mins, total run 20 mins, tR= 13.640 min, 170.09, 169.59, 169.12, 168.63, 142.62, 138.18, 129.61, 128.66,
91% purity.
128.61, 128.49, 126.62, 126.02, 87.42, 87.03, 81.65, 80.36, 58.09,
57.85, 57.25, 57.22, 54.70, 36.45, 36.33, 35.72, 35.56, 35.02, 32.94,
Data for 4N: Scale of reaction 60 mg, 0.09 mmol; yield 52 mg, 76%.
Rf = 0.55 (Silica gel, 3% MeOH in CHCl3); 1H NMR (400 MHz, DMSO- 31.20, 30.61, 30.28, 30.02, 28.76, 26.96, 26.90, 24.87, 22.51, 20.04,
d6): δ 7.29-7.12 (m, 5H), 5.53-5.38 (m, 1H), 5.09-4.93 (m, 2H), 4.75- 19.74, 18.40, 17.92; IR νmax (neat, cm–1): 3479, 2959, 2930, 1716,
4.54 (m, 1H), 4.08-3.99 (m, 1H), 3.59-3.45 (m, 2H), 3.37-3.19 (m, 1640, 1460, 1405, 1257, 1102, 1019; MS (ESIMS/[Na]+): m/z(%)
9H), 3.19-3.03 (m, 2H), 3.02-2.88 (m, 6H), 2.87-2.74 (m, 11H), 2.37- 830; HRMS (ESI/[M+Na]+): Calcd for C45H69N5O8Na+ : 830.5038,
2.13 (m, 4H), 1.56-1.44 (m, 2H), 1.30-1.19 (m, 8H), 0.89-0.62 (m, found 830.5036. HPLC data: Isocratic (50%), total run 30 mins, tR=
15H); 13C NMR (100 MHz, DMSO-d6): δ 173.16, 170.12, 169.54, 13.663 min, 92% purity.
168.65, 138.24, 129.69, 128.55, 126.67, 87.43, 81.74, 80.42, 79.67,
58.12, 57.87, 57.28, 54.76, 36.49, 36.39, 35.76, 33.06, 31.71, 30.65,
30.35, 30.06, 29.11, 28.99, 26.96, 25.11, 22.53, 20.12, 19.79, 18.46,
17.99, 14.43; IR νmax (neat, cm–1): 3427, 2927, 2856, 1642, 1463,
1403, 1218, 1099, 1018; MS (ESIMS/[Na]+): m/z(%) 782; HRMS
(ESI/[M+H]+): Calcd for C41H69N5O8Na+ : 782.5038, found 782.5043.
HPLC data: Isocratic (50%), total run 20 mins, tR= 13.872 min, 94%
purity.
Data for 4Q: Scale of reaction 70 mg, 0.1 mmol; yield 58 mg, 74%.
Rf = 0.66 (Silica gel, 5% MeOH in CHCl3); 1H NMR (400 MHz, DMSO-
d6): δ 7.32-7.12 (m, 5H), 5.52-5.41 (m, 1H), 5.10-4.94 (m, 2H), 4.73-
4.58 (m, 1H), 4.17-4.00 (m, 1H), 3.57-3.38 (m, 2H), 3.36-3.21 (m,
15H), 3.01-2.89 (m, 4H), 2.86-2.78 (m, 9H), 2.35-2.14 (m, 4H), 1.55-
1.44 (m, 2H), 1.29-1.18 (m, 12H), 0.89-0.62 (m, 15H); 13C NMR (100
MHz, DMSO-d6): δ 173.09, 170.10, 169.11, 168.63, 167.93, 138.17,
129.60, 128.47, 126.59, 87.43, 87.04, 82.38, 81.65, 58.08, 57.84,
57.24, 57.21, 54.69, 36.46, 36.44, 36.33, 35.69, 35.03, 33.01, 31.70,
30.59, 30.29, 30.01, 29.35, 29.26, 29.08, 29.06, 25.04, 22.51, 20.03,
19.72, 18.38, 17.92, 14.32; IR νmax (neat, cm–1): 3407, 2926, 2856,
Assays for In vitro Activity against Intra-macrophagic
Amastigotes: Murine macrophages (J-774A.1 cell line) were
seeded in 96-well plates at a density of 4 × 104/mL/100µL/well and
incubated at 37 °C in a CO2 incubator. After 24 h, cells were infected
with WHO reference strain (MHOM/IN/80/Dd8) of promastigotes
(expressing luciferase firefly reporter gene) at 1:10 ratio (1
macrophage: 10 promastigotes). After incubation for 24 h, the
plates were washed with fresh media to remove un-internalized
extracellular promastigotes. The infected macrophages were
incubated with the synthetic peptides at two-fold dilutions up to
seven concentrations starting from 100 µM and the plates were
incubated for 72 h. Miltefosine was used as a reference drug. After
incubation, the compounds containing medium was aspirated and
50 µL of phosphate buffer saline (PBS) was added in each well and
mixed with an equal amount of Steady Glo® reagent. The reading
was taken in a luminometer after gentle shaking of plates for 2 min.
After gentle shaking for 2 min, the reading was taken in a
luminometer.25 The values are expressed as relative luminescence
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Journal Name
3
R. D. Pearson, A. de Queiroz Sousa, Clin. Infect. Dis.,
unit (RLU) and data were transformed into a graphic program (MS
Excel). 50% inhibitory concentration (IC50) value of each tested
compound was calculated by nonlinear regression analysis of the
concentration response curve using the four parameter Hill
equations.
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mg/mL) was added to each well and incubated at 37 °C for 2 h. At
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µL of pure DMSO was added to each well for solubilising the
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Acknowledgements
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and IISc, Bangalore (H.P.A.K.) for research fellowships. The
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