Almiramides A-C: Discovery and development of a new class of leishmaniasis lead compounds

Article abstract of DOI:10.1021/jm100265s

Leishmaniasis is a debilitating disease caused by protozoan parasites of the genus Leishmania, which affects an estimated 12 million people worldwide. The discovery of new lead compounds for leishmaniasis is therefore a pressing concern for global health programs. The organic extract of a Panamanian collection of the marine cyanobacterium Lyngbya majuscula showed strong in vitro activity in two complementary screens against the tropical parasite Leishmania donovani, the causative agent of visceral leishmaniasis. Chromatographic separation of this complex mixture led to the isolation of the highly N-methylated linear lipopeptides, almiramides A-C (1-3). Comparison with the biological activities of a number of related metabolites and semisynthetic derivatives revealed key features required for activity and afforded one new compound (11) with superior in vitro activity. Subsequent synthesis of a library of simplified analogues led to the discovery of several compounds with improved therapeutic indices to the natural products.

Full text of DOI:10.1021/jm100265s

J. Med. Chem. 2010, 53, 4187–4197 4187
DOI: 10.1021/jm100265s
Almiramides A-C: Discovery and Development of a New Class of Leishmaniasis Lead Compounds
Laura M. Sanchez,† Dioxelis Lopez,‡ Brian A. Vesely, Gina Della Togna,‡ William H. Gerwick,§ Dennis E. Kyle, and
Roger G. Linington*,†
†Department of Chemistry and Biochemistry, University of California Santa Cruz, 1156 High Street, Santa Cruz, California 95064, ‡Instituto de
´
´
ꢀ
Investigaciones Cientıficas Avanzados y Servicios de Alta Tecnologıa, INDICASAT, Clayton, Edificio 175, P.O. Box 7250, Panama 5, Panama,
§Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography and Skaggs School of Pharmacy and Pharmaceutical
Sciences, University of California San Diego, La Jolla, California 92093, and Department of Global Health, College of Public Health, University
of South Florida, 3720 Spectrum Boulevard, Suite 304, Tampa, Florida 33612
Received February 26, 2010
Leishmaniasis is a debilitating disease caused by protozoan parasites of the genus Leishmania, which
affects an estimated 12 million people worldwide. The discovery of new lead compounds for
leishmaniasis is therefore a pressing concern for global health programs. The organic extract of a
Panamanian collection of the marine cyanobacterium Lyngbya majuscula showed strong in vitro activity
in two complementary screens against the tropical parasite Leishmania donovani, the causative agent of
visceral leishmaniasis. Chromatographic separation of this complex mixture led to the isolation of the
highly N-methylated linear lipopeptides, almiramides A-C (1-3). Comparison with the biological
activities of a number of related metabolites and semisynthetic derivatives revealed key features required
for activity and afforded one new compound (11) with superior in vitro activity. Subsequent synthesis of
a library of simplified analogues led to the discovery of several compounds with improved therapeutic
indices to the natural products.
Introduction
period and to the relatively high toxicity of this treatment
regimen. More recently, alternative therapeutics have become
prevalent as first line treatments. These include liposomal
amphotericin B, which is highly effective and requires only a
short course of treatment but is too expensive to be a viable
treatment option in most developing nations, and miltefosine,
an alkylphospholipid recently licensed for use against visceral
leishmaniasis in India, Germany, and Colombia that has
shown excellent cure rates but that is already facing instances
of resistance in some areas. This shortfall of affordable and
efficacious treatments has led the World Health Organization
to designate leishmaniasis as a category 1 disease, signifying
that it is an emerging and uncontrolled global health problem.
There is therefore a pressing need for the development of
new drugs to treat leishmaniasis, and it is with this aim that
the Panama International Cooperative Biodiversity Group
(ICBGa) is investigating Panamanian microorganisms for
lead compounds with antileishmanial activity.6,7
Leishmaniasis is a debilitating disease prevalent across
many of the intertropical regions of the world including India,
Sudan, and Brazil.1-3 Caused by over 20 species of related
intracellular parasites from the genus Leishmania, leishma-
niasis can present itself in a number of different clinical
manifestations including cutaneous, mucosal, and visceral
forms of the disease.4 Both the cutaneous and mucosal forms
can cause severe disfigurement to patients, including ulcera-
tive skin lesionsand the destruction of the mucousmembranes
of the nose, mouth, and throat, leading to permanent disfig-
urement and frequently social ostracization.5 However, it is
the visceral form of the disease that represents the greatest
threat to human health, with symptoms ranging from fever
and weight loss in the initial stages to the development of
hepatospleneomegaly (a dramatic enlargement of the liver
and spleen) and ultimately multisystem infection and death in
untreated cases. Visceral leishmaniasis is caused by a small
subgroup of the Leishmania parasites, principally L. donovani,
L. infantum, and L. chagasi. Current treatment is limited to
only a few viable alternatives, each of which suffers from
drawbacks in terms of efficacy, toxicity, or cost. Until re-
cently, the most widely used therapeutic in most regions of the
world was the pentavalent antimony-based drug sodium
stibogluconate.3 Though initially highly efficacious, with an
estimated 90-95% cure rate in most areas after its introduc-
tion in the 1950s, this treatment has suffered from the
increasing emergence of resistance, fueled by the high rate of
patient noncompliance due to the exceedingly long treatment
Discussion
Screening of over 400 prefractions from 40 field collections
of marine cyanobacteria revealed one fraction from a collec-
tion of Lyngbya majuscula from the Bocas del Toro Marine
a Abbreviations: ICBG, International Cooperative Biodiversity
Groups; RP-HPLC, reversed phase high performance liquid chroma-
tography; HRESITOFMS, high resolution electrospray ionization time-
of-flight mass spectrometry; DLVA, NR-(2,4-dinitro-5-fluorophenyl)-L-
valinamide; ER, enoyl reductase; SAM, S-adenosyl methionine; PKS-
NRPS, polyketide synthase-nonribosomal peptide synthetase; SPPS,
solid phase peptide sythesis; DIPEA, diisopropylethylamine; TIPS,
triisopropylsilyl; HBTU, O-benzotriazol-1-yl-N,N,N0,N0-tetramethy-
luronium hexapfluorophosphate; TFA, trifluoroacetic acid; SAR, struc-
ture-activity relationship.
*To whom correspondence should be addressed. Phone: (831) 459-
3014. Fax: (831) 459-2935. E-mail: rogerl@chemistry.ucsc.edu.
r
2010 American Chemical Society
Published on Web 05/04/2010
pubs.acs.org/jmc

4188 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Sanchez et al.
Park on the Caribbean coast of Panama that exhibited a
uniqueprofile againstL. donovaniwithout showingsignificant
cytotoxicity to mammalian Vero cells. This combination of
results indicated the presence of compounds with an intri-
guing biological profile and prompted us to undertake a
further investigation of the mixture. Purification by C18
reversed-phase (RP) solid phase extraction chromatography
(50% MeOH/H2O to100%MeOH) afforded twoconsecutive
active fractions that were further purified by C18 RP-HPLC to
give almiramides A-C (1-3) as optically active white solids.
Figure 1. Subunits a-f and NMR connectivity for 1. Solid arrows
indicate HMBC correlations. Dashed arrows indicate ROESY
correlations.
strongly suggested that the terminal ketone of 1 had been
replaced with a terminal alkyne in 2. Further consideration of
the NMR data for this compound (Table 1) confirmed this
initial assignment, through a combination of COSY and
HMBC correlations.
Initial NMR analysis of almiramide A (1) in CD2Cl2 gave
spectra that contained resonances for 1 in two distinct con-
formations (3:2 ratio). Reacquisition of these spectra in
CD3CN afforded spectra that also contained two conforma-
tions, though in a more acceptable ratio of 6:1. Despite the
complexity of interpreting spectra for compounds that exhibit
multiple stable conformers on the NMR time scale, it was
possible to identify the presence of subunits a-f (Figure 1)
using standard two-dimensional NMR techniques (Support-
ing Information).
HRESITOFMS gave an [M þ H]þ pseudomolecular ion at
743.5062 that was consistent with the molecular formula
C40H66N6O7 (calcd 743.5065), indicating that subunits a-f
contained the required number of double bond equivalents
and that 1 was a linear lipopeptide. Careful consideration
of both HMBC and ROESY data for these subunits as
indicated in Figure 1 allowed for the construction of the
planar structure of 1. This assignment was further supported
by the fragmentation pattern in the atmospheric pressure
chemical ionization (APCI) mass spectrum, which was con-
sistent with the connectivity determined via the NMR data
(data not shown). In particular, the fragmentation data
showed a loss of 17 amu that is a characteristic diagnostic
for terminal amide functional groups due to a neutral loss of
NH3 and gave additional confirmation for the assignment of
subunit f.
Almiramide C (3) gave a HRESITOFMS [M þ H]þ
pseudomolecular ion at 727.5115 that was consistent with
the molecular formula C40H66N6O6 (calcd 727.5116). As with
almiramide B, consideration of the NMR resonances for the
aminoacid residues presentin 3showedthem tobeidentical to
those for almiramide A (1), suggesting that 3 differed from 1
and 2 in the constitution of the lipophilic side chain. The
presence of three new multiplet proton resonances between δH
4.89 and δH 5.83 and new resonances in the carbon spectrum
at δC 115.0 and δC 140.1, coupled with the absence of the
quaternary carbon (δC 85.4) and methyne signals (δH 2.16, δC
69.7) for 2, suggested the presence of a monosubstituted olefin
in place of the terminal alkyne present in 2. This assignment
was confirmed by consideration of the gCOSY, gHSQC, and
gHMBC spectra for 3 that unequivocally identified the fatty
acid terminus as a 2-methyloct-7-enoic acid residue.
Determination of the complete absolute configuration for
1-3 was accomplished using two complementary approaches
(Scheme 1). For the amino acid residues, advanced Marfey’s
analysis was employed8 and the resulting derivatives analyzed
by LC-MS. For the determination of the configuration at the
2-position of the side chain, compounds 2 and 3 were hydro-
genated to form compound 4 and hydrolyzed to afford the
free fatty acid, which was subsequently derivatized with a
chiral derivatizing agent and compared to synthetic standards
by GC-MS.
Almiramide B (2) gave a HRESITOFMS [M þ H]þ
pseudomolecular ion at 725.4964 that was consistent with
the molecular formula C40H64N6O6 (calcd 725.4960). Cursory
examination of the proton spectrum for 2 showed it to possess
the same gross structure as1. A more detailed consideration of
the NMR data showed 2 to possess the same peptidic portion
as found in 1, implying that the structural variation lay in the
lipid side chain. The absence of the methyl singlet and ketone
carbonyl signals from 1 (δH 2.04, δC 29.9, and δC 209.6,
respectively), combined with the presence of new quaternary
carbon (δC 85.4) and methyne signals (δH 2.16, δC 69.7) for 2,
It has been noted that side chains containing a terminal
alkyne moiety are not stable to the strongly acidic conditions
typically employed in the hydrolysis of peptides.9 Conse-
quently, both 2 and 3 were subjected to hydrogenation over
palladium on carbon to form compound 4 prior to hydrolysis.
The hydrolysis products were partitioned between 0.1 N HCl
and EtOAc to afford the amino acids in the aqueous phase
and the lipid chain in the organic phase. The aqueous portions
fromall three samples wereseparatelyconcentratedtodryness
in vacuo. The resulting amino acid residues were derivatized

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 4189
Table 1. NMR Data for Almiramides A-C (1-3) (CD3CN)
1
2
3
a
b
a
b
a
b
residue
Phe
position
δH
mult, J (Hz)
δC
δH
mult, J (Hz)
bs, bs
δC
δH
mult, J (Hz)
bs, bs
δC
NH2
1
5.81, 6.26
bs, bs
5.80, 6.26
5.79, 6.26
172.9
58.5
34.5
173.0
58.6
34.6
173.0
58.6
34.7
2
5.44
2.95, 3.33
dd, 4.4, 12.1
m dd, 4.8, 15.0
5.44
2.95, 3.31
dd, 4.4, 12.1
m dd, 4.8, 15.0
5.44
2.94, 3.31
dd, 4.4, 12.1
m dd, 4.8, 15.0
3
4
138.9
129.9
129.5
127.6
31.4
139.1
130.0
129.5
127.6
31.6
139.1
130.0
129.6
127.6
31.6
5
7.23
7.28
7.22
2.72
m
m
m
s
7.23
7.27
7.21
2.72
m
m
m
s
7.23
7.27
7.21
2.72
m
m
m
s
6
7
NMe
Ala
1
172.5
50.9
14.1
172.7
50.9
14.1
172.7
50.9
14.2
2
5.32
1.03
2.09
q, 6.6
d, 6.6
s
5.32
1.04
2.10
q, 6.6
d, 6.6
s
5.32
10.3
2.09
q, 6.6
d, 6.6
s
3
NMe
29.9
30.0
29.8
Val1
1
171.6
54.3
31.6
171.6
54.3
31.5
171.7
54.3
31.5
2
4.42
1.63
0.69
0.82
6.58
dd, 4.8, 8.8
m
d, 7.0
4.43
1.64
0.69
0.82
6.58
dd, 4.8, 8.8
m
d, 7.0
4.42
1.63
0.69
0.82
6.57
dd, 4.8, 8.8
m
d, 7.0
3
4
17.1
19.9
17.2
20.0
17.2
20.0
5
NH
d, 7.0
bd, 8.8
d, 7.0
bd, 8.8
d, 7.0
bd, 8.8
Val2
Val3
lipid
1
170.4
63.4
26.8
18.8
20.6
31.1
172.6
59.1
27.7
18.7
20.3
30.7
177.4
36.6
34.5
27.8
24.5
43.8
209.6
30.0
18.4
170.5
63.5
26.8
18.9
20.6
31.2
173.0
59.2
27.8
18.9
20.4
30.8
177.5
36.7
34.3
27.5
29.4
18.8
85.5
69.8
18.5
170.4
63.5
26.8
18.9
20.6
31.2
172.7
59.2
27.8
18.8
20.4
30.8
177.6
36.7
34.6
27.8
27.7
34.5
140.1
115.0
18.5
2
4.53
2.16
0.69
0.82
2.91
d, 11.0
m
4.53
2.16
0.69
0.82
2.92
d, 11.0
m
4.53
2.17
0.69
0.82
2.91
d, 11.0
m
3
4
d, 7.0
d, 7.0
s
d, 7.0
d, 7.0
s
d, 7.0
d, 7.0
s
5
NMe
1
2
5.15
2.30
0.77
0.89
2.88
d, 10.6
m
5.16
2.30
0.79
0.89
2.88
d, 10.6
m
5.16
2.29
0.78
0.89
2.88
d, 10.6
m
3
4
d, 6.6
d, 6.2
s
d, 6.6
d, 6.2
s
d, 6.6
d, 6.2
s
5
NMe
1
2
3
4
5
6
7
8
9
2.76
m
m, m
2.77
m
m, m
2.76
m
1.28, 1.64
1.20
1.47
1.32, 1.68
1.32
1.45
1.30, 1.66
1.26
1.36
m, m
m
dt, 7.3, 14.8
dt, 7.5, 14.8
t, 7.5
m
dt, 7.5, 14.8
m
m
2.38
2.14
2.02
5.81
dt, 7.3, 7.0
m
2.04
0.99
s
d, 6.6
n/oc
1.00
4.91, 4,98
1.00
dd, dd
d, 6.6
d, 6.6
a Recorded at 400 MHz. b Recorded at 100 MHz. c n/o = not observed.
with NR-(2,4-dinitro-5-fluorophenyl)-L-valinamide (DLVA)
under standard conditions and analyzed by gradient C18
RP-HPLC in each case. Comparison of the retention times
and mass spectra for these derivatives with commercially
available standards derivatized under identical conditions
allowed assignment of all amino acid residues as L for almir-
amides A-C. These assignments were subsequently con-
firmed by co-injection analyses. The lipid chains of 2 and 3
from the organic phases of the hydrolysis reactions were
separately derivatized with (S)-1-phenylethylamine under
standard amide coupling conditions (Scheme 1) to form com-
pound 9. Commercially available (()-2-methyloctanoic acid
was similarly derivatized to give compounds 9 and 10, which
were separated by flash silica gel column chromatography.
Stereoidentification of these two products was accomplished
by X-ray analysis of compound 9, which defined the struc-
ture as (2R)-2-methyl-N-((S)-1-phenylethyl)octanamide. Com-
parison of the GC retention times of these synthetic materials
by GC analysis with that of the natural product derived
fragments showed that both 2 and 3 possessed 2-R configura-
tions. Comparison of the NMR chemical shifts for 1, 2, and 3,
and consideration of the common biosynthetic origin of these
three compounds, makes it highly likely that this center in
compound 1 is also R, although this was not verified experi-
mentally.
This result was somewhat surprising because a recent total
synthesis of the related metabolite dragonamide A (5) has
shown it to contain the opposite (2S)-2-methyloct-7-ynoic
acid moiety.10 Work from the Panama ICBG has provided
evidence that another related metabolite, dragonamide B (6),
also contains the (2S)-2-methyloct-7-ynoic acid subunit.11
Fortuitously, compound 6 was also isolated as a component
of an inactive fraction from the crude extract investigated in
this study. We therefore analyzed an authentic sample of 6
under identical conditions. GC-MS analysis indicated that
(2S)-2-methyl-N-((S)-1-phenylethyl)octanamide (10) was
produced as the sole product from 6, in line with previous
reports for this class of compounds.
Thus, this strain of L. majuscula possesses the biosynthetic
capability to produce two closely related compounds (2 and 6)
that contain constitutively identical lipid chains with opposite
configurations. A recent study of enoylreductases (ER) in
modular type I polyketide synthases has shown that the
presence or absence of a single tyrosine residue in the active

4190 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Scheme 1. Configurational Analysis Strategy for 2 and 3
Sanchez et al.
dragomabin (8), and dragonamides A (5) and B (6).11 Parallel
testing with almiramides A-C showed compounds 5-8 to be
inactive up to the highest tested concentrations (10 μg/mL),
despite significant structural similarities between these com-
pounds and almiramides B and C (Figure 2). These data
suggest that2 and 3 are highly specific for L. donovani and that
possession of alkyne and primary amide termini for N-methy-
lated peptides is insufficient for activity against this organism.
Almiramides A-C (1-3) were also tested for activity against
both Plasmodium falciparum (W2 chloroquine-resistant
strain) and Trypanasoma cruzi and found to be inactive up
to the highest tested concentrations (13.5 μM) in all cases.
The value of the almiramides as lead molecules for antil-
eishmanial drug development prompted us to further explore
the SAR features of these compounds by the generation of
semisynthetic analogues. Hydrogenation of 2 with H2 and
10% palladium on carbon afforded compound 4 in quantita-
tive yield. Evaluation of 4 in the leishmaniasis assay showed it
to be inactive at the highest tested concentration (13.4 μM),
supporting the hypothesis that the unsaturated terminus plays
an important role in the interaction of these compounds with
their biological target. We next explored the importance of the
primary and secondary amides for activity in the almiramide
series. Methylation of 2 with NaH/MeI afforded a 1:1 mixture
of compounds 11 and 12 (Scheme 2), which exhibited IC50
values of 1.64 and 2.31 μM, respectively. We hypothesize that
the improved activity of 11 over 2 is due to its increased
membrane permeability. Indeed, there have been a number of
studies that recognize the importance of methylation patterns
in determining the degree of membrane permeability for both
cyclic and linear peptidic small molecules,14,15 which is in line
with our observed results.
The small quantities of 11 and 12 generated from derivati-
zation of the natural product precluded the use of standard
techniques for their structural characterization. Unequivocal
assignmentof their structures was possible by consideration of
a combination of 1H NMR and MS fragmentation data. 1H
NMR for 11 showed the absence of the Val1-NH proton
doublet at δ 6.58 and the presence of a new methyl singlet at δ
2.94. Coupled with a mass increase of 14 amu in the HRE-
SIMS, this strongly suggested that the Val1-NH proton in 2
had been replaced with a methyl group in 11. This was
confirmed by consideration of the HRESIMS fragmentation
pattern that showed characteristic fragments for the loss of
sequential amino acid residues starting from the C-terminus
and placed the new methyl group on residue Val1 (Supporting
Information). Similarly, 1H NMR for 12 showed the absence
of both the Val1-NH and the C-terminal-NH2 proton reso-
nances (δ 6.58, δ 5.80, and δ 6.26) and the presence of three
additional methyl singlets in the region δ 2.84-2.94 indicating
a permethylation product. As with compound 11, this was
confirmed by the MS fragmentation pattern in the HRESIMS
spectrum, which indicated methylation of residue Val1 and
dimethylation of the phenylalanine residue (Supporting In-
formation). Interestingly the permethylated compound 12
displayed a single conformation on the NMR time scale, in
contrast to compounds 1-4 and 11, which all exhibited major
and minor conformers in their NMR spectra.16 This suggests
that the C-terminal-NH protons play an important role in
hydrogen bonding interactions which leads to these two
conformational states.
site of the ER domain defines the configuration of the final
methyl-branched product as R or S for all systems in which a
propionate unit is incorporated to form the branched methyl
substituent.12 Using site-directed mutagenesis, the authors
were able to validate this hypothesis by generating mutants
that produced compounds bearing opposite configurations at
these positions by replacement or insertion of this key tyrosine
residue. Although pendent methyl groups in cyanobacterial
natural products originate from S-adenosyl methionine
(SAM) rather than incorporation of propionate subunits,13
both of these biosynthetic pathways proceed via a common
R,β unsaturated carbonyl species, which is reduced by the ER
module to form the saturated final product. It is therefore
likely that this organism contains separate PKS-NRPS clus-
ters for the production of the almiramide versus the dragona-
mide series and that differences within the ER modules of
these two clusters give rise to the observed configurational
differences in the two alkyl side chains.
Biological evaluation of these three compounds showed
that compounds 2 and 3 possessed strong in vitro antiparasitic
activity against L. donovani (IC50 = 2.4 and 1.9 μM, re-
spectively), whereas compound 1 was completely inactive up
to 13.5 μM, thus indicating the requirement of an unsaturated
terminus on the side chain for activity. For reference, the
two most widely used treatments against leishmaniasis
(sodium stibogluconate and miltefosine) are active in vitro
against L. donovani with IC50 values of 44.7 and 0.5 μM,
respectively. Previous investigations of cyanobacteria from
the Caribbean by our groups have provided a number of
related secondary metabolites including carmabin A (7),
Generation of semisynthetic derivatives with fully methy-
lated peptide backbones and improved selectivity indices was
particularly encouraging, as this opened the door to a facile

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 4191
Figure 2. Structurally related linear lipopeptides (areas of structural homology outlined with dashed lines).
Scheme 2. Generation of Semisynthetic Derivatives 11 and 12
TIPS for Rink amide resins). The resulting intermediates
were permethylated with NaH/MeI in THF under argon,
and the reactions were monitored by LC-MS. In all cases,
final purification was performed by RP-HPLC (Phenomenex
Jupiter C18 column, 4.6 mm ꢀ 250 mm, 1 mL/min, MeOH/
H2O þ 0.02% HCOOH). Conversion of compounds 17-20
to their corresponding methyl esters (21-24) was accom-
plished by treatment with TMS-diazomethane.
synthetic route for the generation of peptide libraries and the
production of large quantities of material for further in vitro
and in vivo testing. We therefore elected to undertake the
synthesis of a small library of simplified synthetic analogues
(13-24) to probe the viability of this scaffold as a lead for
future medicinal chemistry and drug development. In design-
ing this initial library, we were motivated to explore the SAR
attributes of both the C and N termini in the hope of finding a
suitable synthetic analogue from which to launch the con-
struction of larger, more complex combinatorial libraries.
Fortunately, the peptidic nature of these compounds makes
them amenable to solid phase peptide synthesis (SPPS) to
construct the core framework. This approach permits a
divergent strategy by which different solid supports can be
employed to generate a variety of functional groups at the
C-terminus, and addition of the unsaturated alkyl chain as the
final step on the solid support provides access to structural
diversity at the N-terminus. The library was designed in such a
way that the closest analogue (13) differed from 12 only in the
absence of the methyl group at the R-position of the lipophilic
side chain, while additional library members showed incre-
mentally greater structural divergence.
Biological evaluation of compounds 13-24 in parallel with
the original lead compounds (1-3) identified several new
structures with comparable activities to the original natural
products (Table 2). Specifically, compounds 13-16 contain-
ing the N,N-dimethylamide C-terminus exhibited strong an-
tileishmanial activities. By contrast, the corresponding methyl
ester derivatives (21-24) were completely inactive in this
screen, and the carboxylic acid derivatives (17-20) showed
mixed activities. In addition to screening for antileishmanial
activities, compounds 13-24 were also evaluated for their
cytotoxic properties. Using a standard screening protocol
against mammalian Vero cells, we were able to demonstrate
that several of these synthetic analogues possessed improved
toxicity profiles compared with the natural products. Com-
pound 15 showed particular promise in this regard, with
activityagainstL. donovani of 3.1 μM (IC50) anda cytotoxicity
to Vero cells of 155.7 μM, giving this compound a 50-fold
selectivity index. Given that these initial library members
display potencies that are close to those of the best currently
available therapeutics (e.g., miltefosine potency of 0.5 μM)
and that the therapeutic indices are encouraging, we therefore
consider these compounds promising lead candidates for
further development. Synthesis of second generation libraries
is currently ongoing in our laboratory.
In brief, the linear pentapeptide precursor was constructed
using standard SPPS techniques starting from either Rink
amide or chlorotrityl resins. Addition of the appropriate alkyl
chain to the pentapeptide by formation of the corresponding
amide bond using HBTU and diisopropylethylamine
(DIPEA) was followed by cleavage from the solid support
(1% TFA in CH2Cl2 for chlorotrityl resins, 95% TFA, 5%
The synthesis of compounds 13-16, which exhibit im-
proved selectivity profiles over the initial natural product lead
structures, is an important finding for two reasons. First, this
discovery circumvents one of the most common impediments
tothe developmentof naturalproductdrug leads by providing

4192 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Sanchez et al.
Table 2. Structure and Bioactivities of Almiramide A-C and Synthetic Derivativesa
a nt = not tested.
an efficient route to a renewable supply of material, an
essential requirement for preliminary in vivo studies that
typically requires more material than is available from the
natural source. Second, the peptidic nature of this scaffold
makes it an ideal candidate for the construction of targeted
combinatorial libraries. However, the presence of a standard
(nonmethylated) residue at Val1 and a chiral center at C-2 of
the aglycone makes the large scale synthesis of the natural
product a comparatively laborious process. By demonstrating
that structural variations are tolerated at these positions, we
have been able to design an efficient route to a simplified
family of synthetic analogues that not only proceeds in high
yield but also provides the opportunity to explore the SAR
interdependence of the amino acid residues present in this
framework. For reference, the recent total synthesis of dra-
gonamideA (5) required17stepsinthe longest linearsequence
starting from 1,5-pentanediol. By contrast, the synthesis of 17
was completed in just 1 day, with all but two of the steps
performed on the solid phase.
Conclusion
We have identified a novel family of cyanobacterial natural
products with in vitro activity against the protozoan parasite
Leishmania donovani. Structure elucidation and configurational
determination were accomplished using a combination of ana-
lytical and degradative techniques and revealed the presence of
the (2R)-methyloct-7-ynoic acid lipid terminus, in contrast to
the (2S)-methyloct-7-ynoic acid terminus found in the related
compounds dragonamides A (5) and B (6). Synthesis of a small
library of related structures has identified compounds with
similar potencies against L. donovani and superior cytotoxicity
profiles. These new compounds can be efficiently synthesized
using SPPS methodology and provide incentive for the initiation
of a drug development program based on these lead structures.
Experimental Section
General Experimental Procedures. NMR spectra were ac-
quired on 400, 500, and 600 MHz spectrometers equipped with

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 4193
5 mm inverse detection probe, 5 mm broadband probe, and
5 mm HCN triple resonance cryoprobe, respectively, and refer-
enced to residual solvent proton and carbon signals (δH 1.94, δC
1.34 for CD3CN and δH 3.31, δC 49.15 for CD3OD). HPLC
purifications were performed with a Phenomenex Jupiter C18
(4.6 mm ꢀ 250 mm, 5 μm) RP-HPLC column unless otherwise
stated. All solvents were HPLC grade and were used without
further purification. HPLC analysis data were obtained for all
compounds, and purity over 95% was confirmed for all synthe-
sized compounds.
(43); HRESIMS m/z [M þ H]þ 727.5115 (calcd for C40H67N6O6,
727.5116).
Hydrogenation of 2 and 3. Authentic samples of 2 and 3 (0.1
mg) were independently dissolved in dry CH2Cl2 (10 mL). Then
10% palladium on carbon (2 mg) was added and the suspensions
were stirred under an atmosphere of H2 (balloon) at room
temperature for 16 h. Each sample was concentrated to dryness
under a stream of N2, dissolved in MeOH (1 mL), and filtered
through a 13 mm 0.2 μm nylon filter. The resulting filtrates were
concentrated to dryness under a stream of N2 and purified by
Collection. The cyanobacterium Lyngbya majuscula (46.5 g
dry wt) was collected by hand from a depth of 0.1-0.3 m from
mangrove roots on a small island in the Bocas del Toro National
Marine Park, Bocas del Toro Province on the north coast of
Panama (09° 16.6690 N, 82° 09.8340 W). The cyanobacterium
was strained through a mesh bag to remove excess seawater,
frozen on site, and stored at -4 °C until workup. The taxonomy
was identified by comparison with characteristics described by
Geitler.17 A voucher was deposited at the Smithsonian Tropical
Research Institute, Panama (voucher number PAB-04-NOV-
05-7).
Extraction and Isolation. Freshly thawed material was ex-
tracted exhaustively with CH2Cl2/MeOH (2:1, 3 ꢀ 500 mL) and
the combined organic extracts were partitioned against H2O
(200 mL) and concentrated to dryness in vacuo to give 0.57 g of a
dark-brown gum. This material was subjected to flash Si gel CC
(Aldrich, Si gel 60, 230-400 mesh, 40 mm ꢀ 180 mm) eluting
with 100% hexanes (300 mL), 9:1 hexanes/EtOAc (300 mL), 8:2
hexanes/EtOAc (300 mL), 6:4 hexanes/EtOAc (300 mL), 4:6
hexanes/EtOAc (300 mL), 2:8 hexanes/EtOAc (300 mL), 100%
EtOAc (300 mL), 3:1 EtOAc/MeOH (300 mL), 100% MeOH
(300 mL). Two contiguous fractions (100% EtOAc, 3:1 EtOAc/
MeOH) showed antileishmanial activity and possessed similar
LC-MS and NMR features. These were separately subjected to
C18 RP-SPE chromatography, eluting with 1:1 H2O/MeOH (30
mL), 4:6 H2O/MeOH (30 mL), 3:7 H2O/MeOH (30 mL), 2:8
H2O/MeOH (30 mL), 1:9 H2O/MeOH (30 mL), 100% MeOH
(30 mL), 100% EtOAc (30 mL). The active 3:7 H2O/MeOH
fraction was subjected to C18 RP-HPLC (57% MeOH/43%
H2O, 210 nm, 1 mL/min) to give almiramide A (1) as a colorless
glass (3.5 mg, 0.6% of crude extract, tR = 15.4 min) and
almiramide B (2) as a colorless glass (1.9 mg, 0.3% of crude
extract, tR = 39.1 min). HPLC of the 2:8 H2O/MeOH active
fraction (62% MeOH/38% H2O, 210 nm, 1 mL/min) gave
almiramide B (2) as a colorless glass (3.4 mg, 0.6% of crude
extract, tR = 16.9 min) and almiramide C (3) as a colorless glass
(1.2 mg, 0.2% of crude extract, tR = 40.3 min).
C18 RP-HPLC (76% MeOH/24% H2O, 210 nm, 1 mL/min) to
give 4 as a white solid (0.1 mg, >99% yield) in each case.
Tetrahydroalmiramide B (4): white solid; [R]20D -160 (c 0.013,
CH3OH); UV (MeOH) λmax (log ε) 219 (sh) (4.09) nm; IR (NaCl,
cm-1) νmax 3400, 2925, 2855, 1731, 1643, 1463 cm-1; 1H NMR
(CD3OD, 600 MHz) δ 7.31-7.28 (m, 2H), 7.27-7.24 (m, 3H),
5.55 (dd, J = 12.6, 4.8 Hz, 1H), 5.40 (bd, J = 6.6 Hz, 1H), 5.20
(d, J = 10.8 Hz, 1H), 4.60-4.59 (m, 1H), 4.40 (d, J = 6.6 Hz,
1H), 3.66-3.62 (m, 3H), 3.43-3.40 (m, 1H), 3.19-3.18 (m, 1H),
3.05 (s, 3H), 3.00 (s, 3H), 2.84 (s, 3H), 2.36-2.29 (m, 5H), 2.18 (s,
3H), 2.04-2.00 (m, 5H), 1.64-1.57 (m, 2H), 1.09 (d, J = 6.6 Hz,
2H), 1.06 (d, J = 6.6 Hz, 2H), 0.92-0.82 (m, 13H), 0.81-0.74
(m, 6H); HRESIMS m/z [M þ Na]þ 751.5085 (calcd for
C40H68N6O6Na, 751.5093).
Marfey’s Analysis of 1-3. An authentic sample of 1 and
hydrogenated samples of 2 and 3 were independently treated
with 6 N HCl in sealed vials at 120 °C for 18 h. The solutions
were concentrated to dryness in vacuo and treated with a
solution of 1-fluoro-2,4-dinitrophenyl-5-L-valineamide (DLVA)
(0.25 mg, 0.8 μmol) in acetone (50 μL) and a solution of 0.1 M
NaHCO3 (100 μL) in a sealed vial at 90 °C for 5 min. The reaction
mixture was neutralized with 2 N HCl (50 μL) and diluted with
CH3CN (100 μL). The resulting solution was analyzed by C18 RP-
HPLC employing a Phenomenex Jupiter C18 column (4.6 mm ꢀ
250 mm) and a gradient elution profile of 25% CH3CN/75% H2O
(acidified with 0.05% HCOOH) to 55% CH3CN/45% H2O
(acidified with 0.05% HCOOH) over 60 min at a flow of 0.5 mL/
min, monitoring at 340 nm. Retention times in minutes for the
derivatized amino acid standards were as follows: L-N-methylala-
nine 32.2; D-N-methylalanine 32.9; L-valine 35.7; L-N-methylvaline
42.3; L-N-methylphenylalanine 45.7; D-valine and D-N-methylphe-
nylalanine 47.8; D-N-methylvaline 50.3.
Synthesis of 9 and 10. To a stirred solution of (()-2-methyl-
octanoic acid (82 mg, 0.52 mmol) in dry CH2Cl2 (10 mL) at 0 °C
was added (S)-1-phenylethylamine (65 μL, 0.57 mmol), Et3N
(57 μL, 0.78 mmol, freshly distilled over CaH), 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide (109.4 mg, 0.57 mmol),
and 4-di(methylamino)pyridine (6 mg, 0.05 mmol). The reaction
mixture was allowed to warm to room temperature and stirred
for 18 h. The volatiles were removed in vacuo, and the residue
was suspended in EtOAc (5 mL) and filtered through Celite.
The filtrate was diluted with EtOAc (10 mL) and washed
sequentially with 0.2 N HCl (25 mL), H2O (25 mL), 0.1 N
NaHCO3 (25 mL), and H2O (25 mL) and concentrated to
dryness in vacuo to give a white crystalline solid. This material
was purified by two steps of flash silica gel column chromatog-
raphy (26 mm ꢀ 250 mm, 100% hexanes to 7:3 hexanes/EtOAc;
14 mm ꢀ 210 mm, 85:15 hexanes/EtOAc) to give compounds 9
(33 mg, 24%) and 10 (38.4 mg, 28%) as an optically active
crystalline white solid and an optically active colorless glass
respectively.
Purification of the 100% EtOAc fraction from the original
normal phase fractionation by C18 RP-HPLC (Prontosil 120-5-
C
18 4.6 mm ꢀ 150 mm RP-HPLC column, 5 μm, 64% MeOH/
34% H2O, 210 nm, 1 mL/min) afforded dragonamide B (6) as a
colorless glass (1.1 mg, 0.2% of crude extract, tR = 27.1 min)
which was identical to previously isolated material by H and
1
13C NMR spectroscopy and APCI mass spectrometry.
Almiramide A (1): colorless glass; [R]22 -169.1 (c 0.2,
D
MeOH); UV (MeOH) λmax (log ε) 228 (sh) (3.77) nm; IR (film)
ν
max 2960, 1624 cm-1; for 1H and 13C NMR data, see Table 1;
APCIMS m/z 743.3 [M þ H]þ (35), 565.4 (39), 381.3 (100), 268.2
(27); HRESIMS m/z [M þ H]þ 743.5062 (calcd for C40H67N6O7,
743.5065).
Almiramide B (2): colorless glass; [R]22 -148.9 (c 0.1,
D
MeOH); UV (MeOH) λmax (log ε) 230 (sh) (3.76) nm; IR (film)
(2R)-2-Methyl-N-((S)-1-phenylethyl)octanamide (9): white
needles (iPrOH); [R]20 -85.3 (c 0.13, CHCl3); UV (MeOH)
ν
max 2970, 1634 cm-1; for 1H and 13C NMR data, see Table 1;
D
λmax (log ε) 214 (3.71) nm; IR (NaCl, cm-1) νmax 3298, 2966,
APCIMS m/z 727.4 [M þ H]þ (37), 549.4 (63), 365.3 (100), 252.2
(45); HRESIMS m/z [M þ H]þ 725.4964 (calcd for C40H65N6O6,
725.4960).
1
2921, 2851, 1639, 1546 cm-1; H NMR (CDCl3, 300 MHz) δ
7.28-7.16 (5H, m), 5.62 (1H, bd, J = 7.8 Hz), 5.09 (1H, dq, J =
7.3, 7.3 Hz), 2.08 (1H, m), 1.59 (1H, m), 1.43 (3H, d, J = 6.9 Hz),
1.33-1.14 (9H, m), 1.05 (3H, d, J = 6.8 Hz), 0.82 (3H, t, J =
6.7 Hz); 13C NMR (CDCl3, 75 MHz) δ 175.5, 128.6, 127.3,
126.1, 48.3, 41.7, 34.4, 31.7, 29.3, 27.4, 22.6, 21.7, 17.8, 14.0;
Almiramide C (3): colorless glass; [R]22 -136.8 (c 0.1,
D
MeOH); UV (MeOH) λmax (log ε) 228 (sh) (3.69) nm; IR (film)
ν
max 2955, 1630 cm-1; for 1H and 13C NMR data, see Table 1;
APCIMS m/z 725.4 [M þ H]þ (33), 547.4 (67), 363.3 (100), 250.2

4194 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Sanchez et al.
HRESIMS m/z [M þ H]þ 262.2171 (calcd for C17H28NO,
262.2165).
(2S)-2-Methyl-N-((S)-1-phenylethyl)octanamide (10): color-
Cytotoxicity bioassays were performed following an 3-[4,5-
dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)
cell proliferation assay protocol with green monkey Vero kidney
cells.
less glass; [R]20 -66.1 (c 0.14, CHCl3); UV (MeOH) λmax
D
(log ε) 215 (3.71) nm; IR (NaCl, cm-1) νmax 3304, 2961, 2928,
Representative Library Member Synthesis. Fmoc-L-phenyla-
lanine (3.246 g, 8.37 mmol) and DIPEA (2.7 mL, 16.7 mmol)
were added to a stirred suspension of 2-chlorotrityl chloride
resin (1.047 g, 200-400 mesh, 1% DBV) in dichloromethane (50
mL) under argon, and the resulting suspension was stirred at
room temperature for 3 h. The solution was transferred to a
polypropylene vessel containing a fritted disk and fitted with a
Teflon stopcock, drained, and washed (3 ꢀ 20 mL of CH2Cl2,
3 ꢀ 20 mL of DMF, 3 ꢀ 20 mL of CH2Cl2). The resulting pale-
yellow resin was air-dried for 24 h and analyzed to determine the
loading value of the phenylalanine substrate as outlined below.
Two aliquots (7.5 mg and 6.6 mg) of the resin were transferred
to Eppendorf tubes, dissolved in 1% DBU in DMF (500 μL),
and agitated for 1 h. Then 50 μL aliquots of these solutions were
diluted with DMF (6 mL), and absorbance was measured at λ =
301 nm to determine the loading value of the Fmoc-L-phenyl-
alanine as 2.56 mmol per gram of resin.
Subsequent amino acids were added using a standard proto-
col. The dried resin was allowed to swell for 5 min in CH2Cl2
(10 mL), drained, and washed with DMF (10 mL). The washed
resin was deprotected using 20% piperidine in DMF (20 mL)
with agitation for 20 min. After deprotection, the resin was
drained and washed (3 ꢀ 20 mL of DMF, 3 ꢀ 20 mL of CH2Cl2,
3 ꢀ 20 mL of DMF). To this washed resin was added a solution
of HBTU (4.983 g, 13.1 mmol), the appropriate amino acid (13.4
mmol), and DIPEA (4.4 mL, 26.8 mmol). The resulting suspen-
sion was gently agitated for 1 h, drained, and washed, as
described above. This process was repeated with successive
amino acids as appropriate to form resin-bound Phe-Ala-Val-
Val-Val-Fmoc.
2853, 1640, 1540 cm-1 1H NMR (CDCl3, 600 MHz) δ
;
7.33-7.24 (5H, m), 5.57 (1H, bd, J = 7.8 Hz), 5.12 (1H, q,
J = 7.2 Hz), 2.11 (1H, m), 1.59 (1H, m), 1.47 (3H, d, J = 6.6 Hz),
1.34 (1H, m), 1.24-1.19 (9H, m), 1.12 (3H, d, J = 6.6 Hz), 0.83
(3H, t, J = 6.6 Hz); 13C NMR (CDCl3, 75 MHz) δ 175.6, 128.6,
127.2, 126.1, 48.3, 41.6, 34.4, 31.7, 29.2, 27.4, 22.5, 21.6, 17.8,
14.0; HRESIMS m/z [M þ H]þ 262.2173 (calcd for C17H28NO,
262.2165).
GC-MS Analysis of 9 and 10. GC-MS analyses were
performed on an Altech Chirasil Val column (25 m ꢀ 0.25
mm, 0.16 μm i.d.) employing a temperature gradient from 90 to
220 °C at a ramp rate of 4 °C/min followed by 5 min at 220 °C.
Retention times in minutes for compounds 9 and 10 were as
follows: 9 25.61, 10 25.05.
Methylation of 2. To a stirred solution of 2 (2 mg, 0.002 mmol)
in dry THF (1 mL) was added NaH (60% dispersion in oil, 2 mg,
0.05 mmol), and the solution was stirred under argon at room
temperature for 2 h. MeI (50 μL, 0.80 mmol) was added
dropwise and the resulting solution stirred for a further 21 h.
The reaction mixture was quenched with water (0.5 mL) and
partitioned with EtOAc (2 mL). The phases were separated, and
the aqueous phase was washed with EtOAc (2 mL). The
combined organics were concentrated to dryness under a stream
of N2 gas and purified by C18 RP-HPLC (72% MeOH/28%
H2O, 210 nm, 0.8 mL/min) to give 11 (0.2 mg, 13%) and 12 (0.6
mg, 38%) as white solids.
Monomethylalmiramide B (11): white amorphous solid; [R]22
D
-80 (c 0.03, MeOH); UV (MeOH) λmax (log ε) 228 (sh) (3.77)
1
nm; IR (NaCl) νmax 3400, 2926, 1634, 1463 cm-1; H NMR
(CD3CN, 600 MHz) δ 7.30-7.28 (m, 2H), 7.23-7.21 (m, 3H),
5.39-5.36 (m, 2H), 5.16 (d, J = 10.8 Hz, 1H), 5.08 (d, J = 10.8
Hz, 1H), 4.80 (d, J = 10.8 Hz, 1H), 3.29 (dd, J = 15, 4.8 Hz, 1H),
2.93 (s, 3H), 2.92 (s, 3H), 2.84 (s, 3H), 2.75 (s, 3H), 2.29-2.21 (m,
3H), 2.10 (s, 3H), 1.46 (q, J = 7.2 Hz, 2H), 1.35-1.28 (m, 4H),
1.00 (d, J = 6.6 Hz, 7H), 0.84-0.81 (m, 8H), 0.77 (d, J = 6.6 Hz,
5H), 0.76 (d, J = 6.6 Hz, 3H), 0.69 (dd, J = 6.6, 2.4 Hz, 7H);
HRESIMS m/z [M þ Na]þ 761.4938 (calcd for C41H66N6O6Na,
761.4936).
From this common pentapeptide precursor, library members
were synthesized by addition of appropriate alkyl acids. For
compound 22, loaded resin (82.5 mg) was deprotected by
treatment with 20% piperidine in DMF (5 mL) with agitation
for 20 min. 7-Octenoic acid (32 μL, 0.21 mmol), HBTU (77.4
mg, 0.20 mmol), and DIPEA (70 μL, 0.42 mmol) were added,
and the resulting suspension was agitated for 1 h, drained, and
washed as previously descibed. The resulting linear lipopeptide
was cleaved from the resin by treatment with 1% TFA in
CH2Cl2 for 20 min and the solvent removed by evaporation
under a stream of N2 gas. The resulting glossy white solid was
triturated with tert-butyl methyl ether and centrifuged at 1000
rpm for 20 min. Removal of the tert-butyl methyl ether afforded
the lipopentapeptide as a white solid (12.7 mg) which was
carried to the next step without further purification. To a
solution of this lipopentapeptide in THF (10 mL) under argon
at -78 °C was added 0.585 g of NaH (60% dispersed in mineral
oil, 0.585 g, 14.6 mmol). After the mixture was stirred for 5 min,
MeI (0.55 mL, 8.88 mmol) was added dropwise over 2 min and
the reaction mixture allowed to warm to room temperature with
stirring over 3 h. Water was added dropwise until effervescence
subsided, and the resulting suspension was concentrated to
dryness in vacuo. The resulting slurry was diluted with water
(50 mL) and acidified to pH 1 using 1 N HCl. The acidified
mixture was partitioned against EtOAc (50 mL), the phases were
separated, and the aqueous phase was washed with EtOAc (2 ꢀ
50 mL). The combined organics were concentrated to dryness in
vacuo, and the resulting yellow oil was partitioned between
MeOH (50 mL) and pentane (3 ꢀ 100 mL) to remove residual
mineral oil. The MeOH phase was concentrated to dryness in
vacuo to give a yellow solid and purified by C18 RP-HPLC
(Agilent Eclipse XDB-C18 4.6 mm ꢀ 150 mm column, 80%
MeOH/20% H2O (acidified with 0.002% formic acid), 1 mL/
min, tR = 14.4 min) to give 18 as an amorphous white solid (2.4
mg, 4.7%). To a solution of 18 (0.5 mg, 1 mmol) in dry C6H6 (600
μL) and dry MeOH (150 μL, 3.71 mmol) was added a solution of
Permethylalmiramide B (12): white amorphous solid; [R]22
D
-240 (c 0.08, MeOH); UV (MeOH) λmax (log ε) 225 (sh) (3.93)
nm; IR (NaCl) νmax 3400, 2962, 2929, 1637, 1463, 1401 cm-1; 1H
NMR (CD3CN, 600 MHz) δ 7.27 (t, J = 7.8 Hz, 2H), 7.24 (d,
J = 6.6 Hz, 2H), 7.19 (t, J = 7.2 Hz, 1H), 5.75 (dd, J = 9.6, 6.6
Hz, 1H), 5.39 (q, J = 13.2, 6.6 Hz, 1H), 5.16 (d, J = 10.8 Hz,
1H), 5.09 (d, J = 10.8 Hz, 1H), 4.91 (d, J = 10.8 Hz, 1H), 3.07
(dd, J = 14.4, 6 Hz, 1H), 2.93 (s, 3H), 2.92 (s, 6H), 2.86 (s, 3H),
2.84 (s, 3H), 2.83 (s, 3H), 2.28-2.22 (m, 3H), 2.21 (s, 3H), 1.46
(q, J = 6.6 Hz, 2H), 1.35-1.27 (m, 3H), 1.00 (dd, J = 6.6, 4.2
Hz, 6H), 0.84 (t, J = 7.2 Hz, 6H), 0.78 (t, J = 6.6 Hz, 6H), 0.70
(t, J = 6.6 Hz, 6H); 13C NMR (CD3OD, 125 MHz) δ 14.6, 18.4,
18.5, 19.0, 19.1, 19.7, 20.4, 27.9, 28.4, 28.6, 29.8, 30.4, 31.1, 31.2,
31.3, 34.7, 35.3, 36.1, 36.3, 37.5, 37.8, 51.5, 55.7, 59.8, 59.9, 60.0,
69.7, 84.9, 128.1, 129.7, 130.6, 138.6, 170.6, 171.8, 172.0, 172.6,
172.7, 179.6; HRESIMS m/z [M þ Na]þ 789.5258 (calcd for
C43H70N6O6Na, 789.5249).
Bioassays. All bioassays were performed in duplicate, testing
at 10, 2, 0.4, 0.08, and 0.016 μg/mL. Malaria bioassays were
performed as previously reported by our program, using chloro-
quine as a positive control (IC50 = 80-100 nM).18 Chagas
bioassays were performed following the protocol of Buckner
et al. and using nifurtimox as a positive control (IC50 = 3-5
μg/mL).19 Leishmaniasis bioassays were performed using a
method previously employed in our laboratory, based on para-
site DNA fluorescence.20 In this latter assay, amphotericin B
was used as the positive control and had an IC50 of 80 ng/mL.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 4195
3501, 2963, 2931, 2873, 1639, 1470, 1400 cm-1 1H NMR
TMS-diazomethane (2 M, 150 μL, 0.30 mmol) dropwise over
1 min, and the reaction mixture was stirred at room temperature
for 12 h. The reactants were evaporated under a stream of N2
gas, and the product was azeotroped with MeOH (2 ꢀ 5 mL).
The solvent was evaporated under a stream of N2 gas and the
resulting solid purified by C18 RP-HPLC (Agilent Eclipse XDB-
C18 4.6 mm ꢀ 150 mm column, 70-100% MeOH with H2O/
0.002% formic acid over 20 min, 1 mL/min, tR = 15.5 min) to
give 22 as an amorphous white solid (0.28 mg, 56% yield).
Compound 13: white solid; [R]20D -245 (c 0.64, MeOH); UV
(MeOH) λmax (log ε) 228 (sh) (3.97) nm; IR (NaCl, cm-1) νmax
;
(CD3CN, 600 MHz) δ 7.27 (t, J = 7.2 Hz, 2H), 7.23, (d, J =
7.2 Hz, 2H), 7.19 (t, J = 7.2 Hz, 1H), 5.82 (dddd, J = 13.8, 10.8,
6.6, 6.6 Hz, 1H), 5.74 (dd, J = 9.6, 6 Hz, 1H), 5.38 (q, J = 13.4,
6.6 Hz, 1H), 5.12 (d, J = 10.8 Hz, 1H), 5.09 (d, J = 10.8 Hz, 1H),
5.01 (ddt, J = 17.1, 2.2 Hz, 1.6 Hz, 1H), 4.93 (ddt, J = 10.2, 2.2,
1.2 Hz, 1H), 4.91 (d, J = 10.8 Hz, 1H), 3.06 (dd, J = 14.2, 6 Hz,
1H), 2.92 (s, 6H), 2.86 (s, 3H), 2.85 (s, 3H), 2.84 (s, 3H), 2.83 (s,
3H), 2.33 (dt, J = 10.8, 7.8, 4.2 Hz, 2H), 2.26-2.23 (m, 3H), 2.21
(s, 3H), 2.07-2.04 (m, 2H), 1.56 (q, J = 7.2 Hz, 2H), 1.39 (q, J =
7.2 Hz, 2H), 1.01 (d, J = 7.2 Hz, 3H), 0.84 (dd, J = 12, 6.6 Hz,
7H), 0.76 (dd, J = 9.5, 6.6 Hz, 6H), 0.70 (dd, J = 6.6, 3.6 Hz,
6H); 13C NMR (CD3CN, 125 MHz) δ 14.6, 18.2, 18.3, 18.5, 19.7,
19.9, 20.3, 25.3, 27.8, 27.9, 28.0, 29.3, 29.7, 30.7, 30.8, 30.9, 33.9,
34.2, 35.8, 35.9, 37.2, 50.4, 54.8, 58.9, 59.0, 59.2, 115.0, 127.4,
129.3, 130.4, 139.0, 139.9, 169.5, 170.5, 170.5, 171.1, 171.6,
174.2; HRESIMS m/z [M þ Na]þ 763.5092 (calcd for
C41H68N6O6Na, 763.5093).
3255, 2963, 2934, 2873, 1635, 1470, 1400 cm-1 1H NMR
;
(CD3CN, 600 MHz) δ 7.27 (t, J = 7.2 Hz, 2H), 7.24 (d, J =
7.2 Hz, 1H), 7.19 (t, J = 7.2 Hz, 2H), 5.75 (dd, J = 9.6, 6 Hz,
1H), 5.39 (q, J = 13.5, 6.6 Hz, 1H), 5.13 (d, J = 10.8 Hz, 1H),
5.09 (d, J = 10.2 Hz, 1H), 4.91 (d, J = 10.8 Hz, 1H), 3.07 (dd,
J = 14.4, 6 Hz, 1H), 2.93 (s, 6H), 2.87 (s, 3H), 2.86 (s, 3H), 2.85
(s, 3H), 2.84 (s, 3H), 2.33 (dt, J = 12.6, 7.2, 1.8 Hz, 2H),
2.29-2.24 (m, 3H), 2.22 (s, 3H), 2.19-2.13 (m, 6H), 1.58 (q,
J = 7.8 Hz, 2H), 1.51 (q, J = 8.4 Hz, 2H), 1.42-1.38 (m, 2H),
1.01 (d, J = 6 Hz, 3H), 0.84 (dd, J = 11.7, 6.4 Hz, 6H), 0.77 (q,
J = 9, 6.6 Hz, 6H), 0.70 (dd, J = 6.8, 2 Hz, 6H); 13C NMR
(CD3CN, 125 MHz) δ 14.7, 18.2, 18.3, 18.5, 18.8, 19.7, 19.9,
20.3, 25.4, 27.8, 27.9, 28.1, 29.2, 29.3, 29.8, 30.7, 30.7, 30.8, 33.9,
35.8, 35.9, 37.3, 50.4, 54.9, 59.0, 59.0, 59.2, 69.7, 85.5, 127.5,
129.3, 130.5, 139.0, 169.6, 170.5, 170.9, 171.1, 171.6, 174.2;
HRESIMS m/z [M þ Na]þ 775.5094 (calcd for C42H68N6O6Na,
775.5093).
Compound 17: white solid; [R]20 -200 (c 0.2, MeOH); UV
D
(MeOH) λmax (log ε) 228 (sh) (3.85) nm; IR (NaCl, cm-1) νmax
1
3307, 2963, 1738, 1639, 1470, 1403 cm-1; H NMR (CD3OD,
600 MHz) δ 7.32-7.28 (m, 2H), 7.24-7.21 (m, 3H) 5.35 (q, J =
13.2, 6.6 Hz, 1H), 5.12 (d, J = 10.8 Hz, 1H), 5.08 (d, J = 10.8
Hz, 1H), 4.85 (d, J = 10.8 Hz, 1H), 3.01 (s, 3H), 2.96 (s, 3H), 2.93
(s, 3H), 2.81 (s, 3H), 2.44-2.42 (m, 3H), 2.35-2.26 (m, 3H),
2.19-2.18 (m, 3H), 2.14 (s, 3H), 1.62 (q, J = 7.8 Hz, 2H), 1.52
(q, J = 7.8 Hz, 2H), 1.46 (q, J = 7.8 Hz, 2H), 1.07 (d, J = 7.8 Hz,
2H), 0.90-0.86 (m, 6H), 0.84-0.81 (m, 7H), 0.77-0.73 (m, 6H);
13C NMR (CD3OD, 125 MHz) δ 14.3, 18.4, 18.6, 19.0, 19.7,
20.0, 20.4, 20.6, 25.8, 28.3, 28.5, 28.6, 28.7, 29.6, 30.0, 31.0, 31.1,
31.2, 31.3, 34.4, 36.3, 50.2, 51.3, 59.8, 60.0, 69.7, 84.9, 120.1,
128.1, 129.8, 130.2, 130.5, 138.7, 129.3, 170.2, 171.7, 171.8,
172.5, 172.6, 176.2; HRESIMS m/z [M þ Na]þ 748.4614
(calcd for C40H63N5O7Na, 748.4620).
Compound 14: white solid; [R]20D -234 (c 0.73, MeOH); UV
(MeOH) λmax (log ε) 224 (sh) (3.94) nm; IR (NaCl, cm-1) νmax
3501, 2963, 2932, 2873, 1727, 1644, 1470, 1400 cm-1; 1H NMR
(CD3CN, 600 MHz) δ 7.27 (t, J = 7.6 Hz, 2H), 7.23 (d, J = 8.3
Hz, 2H), 7.19 (t, J = 7.1 Hz, 1H), 5.83 (dddd, J = 16.8, 10.2, 6.6,
6.6 Hz, 1H), 5.75 (dd, J = 9.5, 6.1 Hz, 1H), 5.39 (q, J = 13.8, 6.6
Hz, 1H), 5.12 (dd, J = 10.8, 2 Hz, 1H), 5.09 (d, J = 10.8 Hz, 1H),
5.01 (ddt, J = 16.8, 2.4, 1.6 Hz, 1H), 4.92 (ddt, J = 10.8, 2.4 Hz,
1.3 Hz, 1H), 4.91 (d, J = 10.8 Hz, 1H), 3.07 (dd, J = 14.4, 6 Hz,
1H), 2.92 (s, 6H), 2.86 (s, 3H), 2.85 (s, 3H), 2.84 (s, 3H), 2.83 (s,
3H), 2.33-2.14 (m, 2H), 2.27-2.23 (m, 3H), 2.21 (s, 3H),
2.05-2.02 (m, 1H), 1.61 (dt, J = 4.7, 2.7, 1.2 Hz, 1H),
1.57-1.52 (m, 3H), 1.40-1.30 (m, 3H), 1.01 (d, J = 6.6 Hz,
3H), 0.84 (dd, J = 12, 6.6 Hz, 7H), 0.77 (dd, J = 9.6, 6.7 Hz,
6H), 0.70 (dd, J = 7.2, 3.6 Hz, 6H); 13C NMR (CD3CN, 125
MHz) δ 14.7, 18.2, 18.2, 18.3, 18.5, 19.7, 19.8, 19.9, 20.3, 25.7,
27.8, 27.9, 28.0, 29.5, 29.6, 29.7, 29.9, 30.7, 30.8, 33.0, 33.9, 34.0,
34.4, 35.8, 35.9, 37.2, 50.4, 54.8, 58.9, 59.0, 59.2, 114.9, 125.8,
127.4, 129.3, 130.4, 132.2, 139.0, 140.1, 169.5, 170.5, 170.8,
171.1, 171.6, 174.2; HRESIMS m/z [M þ Na]þ 777.5256
(calcd for C42H70N6O6Na, 777.5249).
Compound 18: white solid; [R]20 -243 (c 0.7, MeOH); UV
D
(MeOH) λmax (log ε) 225 (sh) (3.95) nm; IR (NaCl, cm-1) νmax
3445, 2964, 2932, 2874, 1737, 1640, 1470, 1403 cm-1; 1H NMR
(CD3CN, 600 MHz) δ 7.32-7.29 (m, 2H), 7.24-7.19 (m, 3H),
5.82 (dddd, J = 16.2 Hz, 10.2 Hz, 6.6 Hz, 6.6 Hz, 1H), 5.37-5.33
(m, 1H), 5.12 (d, J = 10.8 Hz, 1H), 5.08 (d, J = 10.8 Hz, 1H),
5.01-4.97 (m, 1H), 4.93-4.91 (m, 1H), 4.85 (d, J = 10.2 Hz,
1H), 2.91 (s, 3H), 2.85 (s, 6H), 2.72 (s, 3H), 2.33-2.30 (m, 2H),
2.28-2.21 (m, 4H), 2.05-2.02 (m, 3H), 1.55 (q, J = 7.2 Hz, 2H),
1.38 (q, J = 7.2 Hz, 2H), 1.32 (q, J = 7.2 Hz, 2H), 1.01 (d, J = 6
Hz, 2H), 0.84-0.81 (m, 6H), 0.76 (bd, J = 6.6 Hz, 7H), 0.69 (bd,
J = 6.6 Hz, 6H); 13C NMR (CD3CN, 125 MHz) δ 14.4, 18.1,
18.3, 18.5, 19.7, 19.8, 19.9, 20.2, 25.7, 27.7, 27.8, 27.9, 28.0, 29.6,
30.3, 30.7, 30.8, 30.9, 33.0, 34.0, 34.4, 34.9, 35.6, 49.9, 50.5, 59.0,
59.1, 59.6, 114.9, 127.7, 129.5, 130.0, 130.2, 138.8, 140.1, 169.4,
170.8, 171.6, 172.1, 172.1, 174.3; HRESIMS m/z [M þ Na]þ
750.4771 (calcd for C40H65N5O7Na, 750.4776).
Compound 15: white solid; [R]20D -274 (c 0.86, MeOH); UV
(MeOH) λmax (log ε) 228 (sh) (4.00) nm; IR (NaCl, cm-1) νmax
3501, 3255, 2964, 2934, 2874, 1633, 1470, 1402 cm-1; 1H NMR
(CD3CN, 600 MHz) δ 7.27 (t, J = 7.2 Hz, 2H), 2.24 (d, J = 7.2
Hz, 2H), 7.19 (t, J = 7.2 Hz, 1H), 5.75 (dd, J = 9.6, 6 Hz, 1H),
5.38 (q, J = 13.8, 6.6 Hz, 1H), 5.12 (d, J = 10.8 Hz, 1H), 5.09 (d,
J = 10.8 Hz, 1H), 4.91 (d, J = 10.8 Hz, 1H), 3.06 (dd, J = 14.4, 6
Hz, 1H), 2.93 (s, 3H), 2.87 (s, 3H), 2.86 (s, 3H), 2.84 (s, 3H), 2.83
(s, 3H), 2.35 (dt, J = 13.2, 7.2, 1.8 Hz, 2H), 2.27-2.23 (m, 3H),
2.21 (s, 3H), 2.19 (dt, J = 11.4 Hz, 7.7, 2.4 Hz, 2H), 2.15 (t, J =
2.6 Hz, 1H), 1.64 (q, J = 7.2 Hz, 2H), 1.51 (q, J = 7.8 Hz, 2H),
1.01 (d, J = 6.6 Hz, 3H), 0.84 (dd, J = 10.8, 6.6 Hz, 7H), 0.77
(dd, J = 7.8, 6.6 Hz, 7H), 0.70 (dd, J = 7.2, 2.1 Hz, 7H); 13C
NMR (CD3CN, 125 MHz) δ 14.6, 18.2, 18.3, 18.5, 18.6, 19.7,
19.9, 20.3, 25.0, 27.8, 27.9, 28.1, 28.9, 29.7, 30.7, 30.8, 30.9, 33.5,
35.8, 35.9, 37.2, 50.4, 54.8, 59.0, 59.1, 59.2, 69.8, 85.2, 127.4,
129.3, 130.4, 139.0, 169.5, 170.5, 170.8, 171.1, 171.6, 174.0;
HRESIMS m/z [M þ Na]þ 761.4935 (calcd for C41H66N6O6Na,
761.4936).
Compound 19: white solid; [R]20D -160 (c 0.18, MeOH); UV
(MeOH) λmax (log ε) 226 (sh) (3.90) nm; IR (NaCl, cm-1) νmax
3472, 3257, 2964, 2874, 1733, 1639, 1470, 1403 cm-1; 1H NMR
(CD3CN, 600 MHz) δ 7.32-7.29 (m, 2H), 7.28-7.22 (m, 3H),
5.36 (q, J = 12, 6.6 Hz, 1H), 5.26 (bs, 1H), 5.12 (d, J = 10.8 Hz,
1H), 5.08 (d, J = 10.8 Hz, 1H), 4.85 (d, J = 10.2 Hz, 1H), 2.92 (s,
3H), 2.85 (s, 6H), 2.72 (s, 3H), 2.35 (dt, J = 13.2, 7.2, 1.8 Hz,
2H), 2.28-2.22 (m, 4H), 2.20-2.17 (m, 2H), 2.15 (t, J = 2.4 Hz,
1H), 2.13 (s, 3H), 1.64 (q, J = 7.2 Hz, 2H), 1.51 (q, J = 7.2 Hz,
2H), 1.01 (d, J = 6.6 Hz, 2H), 0.84-0.78 (m, 6H), 0.77 (bd, J =
6.6 Hz, 7H), 0.70-0.66 (m, 8H); 13C NMR (CD3CN, 125 MHz)
δ 14.4, 18.2, 18.3, 18.6, 19.7, 19.8, 19.9, 20.2, 25.1, 27.7, 27.9,
28.1, 29.6, 30.4, 30.7, 32.9, 33.5, 35.0, 49.6, 50.6, 59.0, 59.1, 59.5,
69.8, 85.2, 127.6, 127.8, 129.5, 130.0, 130.2, 138.5, 138.9, 169.4,
169.9, 170.8, 171.5, 172.1, 174.0; HRESIMS m/z [M þ Na]þ
734.4456 (calcd for C39H61N5O7Na, 734.4463).
Compound 20: white solid; [R]20D -200 (c 0.20, MeOH); UV
(MeOH) λmax (log ε) 223 (sh) (3.93) nm; IR (NaCl, cm-1) νmax
3401, 2963, 2932, 2873, 1733, 1639, 1470, 1403 cm-1; 1H NMR
Compound 16: white solid; [R]20D -227 (c 0.22, MeOH); UV
(MeOH) λmax (log ε) 225 (sh) (4.00) nm; IR (NaCl, cm-1) νmax

4196 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Sanchez et al.
(CD3OD, 600 MHz) δ 7.33-7.28 (m, 2H), 7.26-7.21 (m, 3H),
5.81 (dddd, J = 17.4, 10.2, 6.6, 6.6 Hz, 1H), 5.43 (q, J = 12.6, 6.6
Hz, 1H), 5.17 (d, J = 10.8 Hz, 1H), 5.13 (d, J = 10.8 Hz, 1H),
5.02-4.98 (m, 1H), 4.95-4.92 (m, 1H), 4.88 (s, 1H), 4.84 (s, 1H),
3.01 (s, 3H), 2.96 (s, 6H), 2.80 (s, 3H), 2.44-2.41 (m, 2H),
2.34-2.26 (m, 3H), 2.11 (s, 3H), 2.09-2.06 (m, 4H), 1.62 (q, J =
7.2 Hz, 2H), 1.45 (q, J = 7.2 Hz, 2H), 1.06 (d, J = 6 Hz, 2H),
5.09 (d, J = 10.8 Hz, 1H), 5.02-4.98 (m, 1H), 4.94-4.92 (m,
1H), 4.87 (d, J = 10.2 Hz, 1H), 3.68 (s, 3H), 2.92 (s, 3H), 2.85 (s,
6H), 2.70 (s, 3H), 2.34-2.31 (m, 3H), 2.17 (s, 3H), 2.07-2.04 (m,
3H), 1.56 (q, J = 7.2 Hz, 3H), 1.39 (q, J = 7.2 Hz, 3H), 1.01 (d,
J = 6.6 Hz, 2H), 0.84-0.79 (m, 6H), 0.78-0.75 (m, 7H), 0.69
(dd, J = 6.6, 2.4 Hz, 6H); 13C NMR (CD3CN, 125 MHz) δ 14.4,
18.1, 18.3, 18.5, 19.7, 19.9, 20.2, 23.7, 24.6, 25.3, 27.7, 27.9, 28.1,
29.3, 30.7, 30.8, 31.2, 33.8, 34.9, 39.7, 50.4, 52.8, 59.0, 59.1, 59.6,
61.6, 63.3, 68.7, 115.0, 127.7, 129.5, 129.7, 130.0, 132.3, 139.9,
170.8, 170.9, 171.6, 171.9, 171.9, 174.2; HRESIMS m/z [M þ
Na]þ 750.4773 (calcd for C40H65N5O7Na, 750.4776).
0.92-0.87 (m, 6H), 0.84-0.80 (m, 8H), 0.76-0.73 (m, 6H); 13
C
NMR (CD3OD, 125 MHz) δ 14.3, 18.4, 18.6, 18.6, 19.7, 19.8,
20.0, 20.4, 20.6, 25.8, 28.3, 28.4, 28.6, 28.7, 29.8, 29.9, 30.5, 31.1,
31.2, 34.4, 34.7, 50.2, 51.4, 59.8, 59.9, 115.2, 128.1, 129.8, 130.2,
130.5, 138.7, 129.4, 139.7, 170.2, 120.3, 171.8, 172.5, 173.0,
176.3; HRESIMS m/z [M þ Na]þ 736.4610 (calcd for
C39H63N5O7Na, 736.4620).
Acknowledgment. We thank the Government of Panama
€
for support of the ICBG program, H.-E. Hogberg for the
Compound 21: white solid; [R]20D -132 (c 0.43, MeOH); UV
(MeOH) λmax (log ε) 225 (sh) (3.90) nm; IR (NaCl, cm-1) νmax
3309, 2961, 2931, 2873, 1738, 1639, 1462, 1403 cm-1; 1H NMR
(CD3CN, 600 MHz) δ 7.12-7.28 (m, 2H), 7.25-7.20 (m, 3H),
5.37 (q, J = 13.2, 6.6 Hz, 1H), 5.22 (dd, J = 12, 7.2 Hz, 1H), 5.12
(d, J = 10.2 Hz, 1H), 5.09 (d, J = 10.8 Hz, 1H), 4.87 (d, J = 10.2
Hz, 1H), 3.68 (s, 3H), 2.92 (s, 3H), 2.85 (s, 6H), 2.70 (s, 3H), 2.33
(tt, J = 7.2, 1.8 Hz, 3H), 2.28-2.22 (m, 4H), 2.17 (s, 3H), 1.57 (q,
J = 7.8 Hz, 2H), 1.50 (q, J = 7.2 Hz, 2H), 1.39 (q, J = 7.2 Hz,
2H), 1.01 (d, J = 7.2 Hz, 2H), 0.83-0.75 (m, 13H), 0.70-0.68
(m, 6H); 13C NMR (CD3CN, 125 MHz) δ 14.4, 18.2, 18.4, 18.5,
18.8, 19.7, 19.9, 20.3, 23.8, 24.6, 25.4, 27.8, 28.0, 28.1, 29.2, 29.3,
29.8, 30.7, 30.8, 31.2, 33.1, 34.0, 35.0, 39.7, 50.5, 52.8, 59.0, 59.2,
69.7, 85.5, 127.7, 129.6, 130.0, 138.7, 169.5, 170.9, 171.6, 171.9,
172.0, 174.2; HRESIMS m/z [M þ Na]þ 762.4765 (calcd for
C41H65N5O7Na, 762.4776).
provision of authentic standards of (2R)- and (2S)-methyl-
octanoic acid, A. Lawrence and the staff of the Smithsonian
Tropical Research Institute Bocas del Toro Field Station for
assistance with field collections, and R. Okuda for assistance
with optical rotation measurements. X-ray analysis was per-
formed by A. Rheingold at the UCSD Department of Chem-
istry Small Molecule X-ray Facility. Financial support was
provided by the Fogarty International Center’s International
Cooperative Biodiversity Group (ICBG) program in Panama
(Grant ICBG TW006634), UCSC startup funds, and the
Panamanian Ministry of Science (SENACYT, Grant
EST06-O-022). L.M.S. is supported by an NSF Graduate
Research Fellowship.
Supporting Information Available: 1H and 13C NMR spectra
of 1-4 and 9-24, structure determination of subunits a-f,
HRESIMS fragmentation interpretation for 11 and 12, and
ORTEP diagram for 9; CIF data for 9. This material is available
free of charge via the Internet at http://pubs.acs.org.
Compound 22: white solid; [R]20D -157 (c 0.69, MeOH); UV
(MeOH) λmax (log ε) 227 (sh) (4.00) nm; IR (NaCl, cm-1) νmax
3473, 2962, 2931, 2873, 1741, 1640, 1463, 1403 cm-1; 1H NMR
(CD3CN, 600 MHz) δ 7.31-7.28 (m, 2H), 7.25-7.20 (m, 3H),
5.83 (dddd, J = 17.4, 10.2, 6.6, 6.6 Hz, 1H), 5.38 (q, J = 13.2, 6.6
Hz, 1H), 5.24 (dd, J = 12, 4.8 Hz, 1H), 5.12 (d, J = 10.8 Hz, 1H),
5.09 (d, J = 10.8 Hz, 1H), 5.02-4.96 (m, 1H) 4.94-4.92 (m, 1H),
4.87 (d, J = 10.8 Hz, 1H), 3.68 (s, 3H), 2.92 (s, 3H), 2.85 (s, 6H),
2.70 (s, 3H), 2.33-2.30 (m, 2H), 2.28-2.21 (m, 4H), 2.17 (s, 3H),
2.05-2.03 (m, 2H), 1.55 (q, J = 7.2 Hz, 2H), 1.38 (q, J = 7.2 Hz,
2H), 1.31 (q, J = 7.2 Hz, 2H), 1.01 (d, J = 6.6 Hz, 2H), 0.82 (dd,
J = 9, 6.6 Hz, 6H), 0.79-0.75 (m, 7H), 0.69 (dd, J = 6.6, 2.4 Hz,
6H); 13C NMR (CD3CN, 125 MHz) δ 14.4, 18.1, 18.3. 18.5, 19.7,
19.9, 20.2, 23.7, 24.6, 25.7, 27.7, 27.9, 28.1, 29.5, 29.6, 30.5, 30.7,
31.2, 33.0, 34.0, 34.4, 34.9, 39.7, 50.4, 52.8, 58.9, 59.1, 59.6, 68.7,
114.9, 127.7, 129.5, 130.0, 132.3, 140.1, 169.4, 170.8, 171.6,
171.9, 171.9, 174.2; HRESIMS m/z [M þ Na]þ 764.4930
(calcd for C41H67N5O7Na, 764.4933).
References
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Target-Directed Ligands in the Search for Novel Lead Candidates
against Trypanosoma and Leishmania. J. Med. Chem. 2009, 52,
7339–7359.
(2) Croft, S. L.; Sundar, S.; Fairlamb, A. H. Drug Resistance in
Leishmaniasis. Clin. Microbiol. Rev. 2006, 19, 111–126.
(3) Chappuis, F.; Sundar, S.; Hailu, A.; Ghalib, H.; Rijal, S.; Peeling,
R. W.; Alvar, J.; Boelaert, M. Visceral Leishmaniasis: What Are
the Needs for Diagnosis, Treatment and Control? Nat. Rev.
Microbiol. 2007, 5, 873–882.
(4) Bern, C.; Maguire, J. H.; Alvar, J. Complexities of Assessing the
Disease Burden Attributable to Leishmaniasis. PLoS Neglected
Trop. Dis. 2008, 2, e313.
(5) Reithinger, R. Leishmaniases’ Burden of Disease: Ways Forward
for Getting from Speculation to Reality. PloS Neglected Trop. Dis.
2008, 2, e285.
Compound 23: white solid; [R]20D -145 (c 0.34, MeOH); UV
(MeOH) λmax (log ε) 225 (sh) (3.93) nm; IR (NaCl, cm-1) νmax
3308, 2962, 2873, 1739, 1639, 1462, 1403 cm-1 1H NMR
;
(CD3CN, 600 MHz) δ 7.34-7.28 (m, 2H), 7.25-7.20 (m, 3H),
5.36 (q, J = 13.2, 6.6 Hz, 1H), 5.23 (dd, J = 12, 4.7 Hz, 1H), 5.12
(d, J = 10.8 Hz, 1H), 5.09 (d, J = 10.6 Hz, 1H), 4.87 (d, J = 10.2
Hz, 1H), 3.68 (s, 3H), 2.92 (s, 3H), 2.85 (s, 6H), 2.70 (s, 3H), 2.35
(dt, J = 13.2, 7.2 Hz, 1.8 Hz, 2H), 2.27-2.23 (m, 4H), 2.22-2.18
(m, 3H), 2.17 (s, 3H), 1.64 (q, J = 7.8 Hz, 2H), 1.51 (q, J = 7.8
Hz, 2H), 1.01 (d, J = 7.2 Hz, 2H), 0.83-0.80 (m, 6H), 0.79-0.76
(m, 7H), 0.70-0.68 (m, 6H); 13C NMR (CD3CN, 125 MHz) δ
14.4, 18.3, 18.5, 18.6, 19.7, 19.9, 20.2, 23.7, 24.6, 25.0, 27.7, 27.9,
28.1, 28.9, 29.7, 30.7, 30.8, 31.2, 33.5, 34.9, 39.7, 50.4, 52.8, 59.0,
59.6, 69.8, 85.2, 116.1, 127.7, 129.5, 130.0, 130.3, 132.3, 138.7,
169.4, 170.8, 171.6, 171.9, 171.9, 174.0; HRESIMS m/z [M þ
Na]þ 748.4614 (calcd for C40H63N5O7Na, 748.4620).
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