U.S. patent application number 15/071417 was filed with the patent office on 2016-07-21 for anti-trypanosome compounds and treatments.
This patent application is currently assigned to The Regents of the University of California. The applicant listed for this patent is Roger Linington, Laura Sanchez. Invention is credited to Roger Linington, Laura Sanchez.
Application Number | 20160206680 15/071417 |
Document ID | / |
Family ID | 42740269 |
Filed Date | 2016-07-21 |
United States Patent
Application |
20160206680 |
Kind Code |
A1 |
Linington; Roger ; et
al. |
July 21, 2016 |
ANTI-TRYPANOSOME COMPOUNDS AND TREATMENTS
Abstract
A novel structural class of highly N-methylated linear
lipopeptide compounds useful for the treatment of parasitic
disease.
Inventors: |
Linington; Roger; (Santa
Cruz, CA) ; Sanchez; Laura; (Santa Cruz, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Linington; Roger
Sanchez; Laura |
Santa Cruz
Santa Cruz |
CA
CA |
US
US |
|
|
Assignee: |
The Regents of the University of
California
Oakland
CA
|
Family ID: |
42740269 |
Appl. No.: |
15/071417 |
Filed: |
March 16, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14256254 |
Apr 18, 2014 |
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15071417 |
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13256498 |
Nov 10, 2011 |
8772247 |
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PCT/US10/28071 |
Mar 20, 2010 |
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14256254 |
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61210657 |
Mar 20, 2009 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
Y02A 50/422 20180101;
Y02A 50/401 20180101; Y02A 50/411 20180101; A61P 33/02 20180101;
C07K 7/06 20130101; Y02A 50/423 20180101; A61K 31/155 20130101;
A61K 38/08 20130101; A61K 31/185 20130101; A61K 38/00 20130101;
Y02A 50/491 20180101; A61K 31/555 20130101; Y02A 50/419 20180101;
A61P 33/00 20180101; Y02A 50/415 20180101; Y02A 50/409 20180101;
Y02A 50/414 20180101 |
International
Class: |
A61K 38/08 20060101
A61K038/08; A61K 31/185 20060101 A61K031/185; A61K 31/155 20060101
A61K031/155; A61K 31/555 20060101 A61K031/555 |
Claims
1-13. (canceled)
14. A method for affecting the physiology of a trypanosome, the
method comprising exposing the pathogen to an almiramide compound,
the almiramide compound comprising a linear lipopeptide having the
following structure ##STR00006## but specifically excluding
compounds (1), (2) and (3) wherein for (1), R.sup.1 consists of
##STR00007## R.sup.2 consists of Me, R.sup.3 consists of H, and
R.sup.4 consists of NH2; and wherein for (2), R.sup.1 consists of
##STR00008## R.sup.2 consists of Me, R.sup.3 consists of H, and
R.sup.4 consists of NH2; and wherein for (3), R.sup.1 consists of
##STR00009## R.sup.2 consists of Me, R.sup.3 consists of H, and
R.sup.4 consists of NH2.
15. The method of claim 14 wherein R.sup.1 is selected from one of
the following moieties: ##STR00010## and wherein R.sup.2 is
selected from one of the following moieties: Me or H and wherein
R.sup.3 is selected from one of the following moieties: Me or H and
wherein R.sup.4 is selected from one of the following moieties:
NH.sub.2, NMe.sub.2, OH, OMe.
16. The method of claim 14 wherein the almiramide compound is
selected from the group consisting of compounds 2, 3, 11, 12, 13,
14, 15 and 16 as shown below: TABLE-US-00001 Compound R.sup.1
R.sup.2 R.sup.3 R.sup.4 2 ##STR00011## Me H NH.sub.2 3 ##STR00012##
Me H NH.sub.2 11 ##STR00013## Me Me NH.sub.2 12 ##STR00014## Me Me
NMe.sub.2 13 ##STR00015## H Me NMe.sub.2 14 ##STR00016## H Me
NMe.sub.2 15 ##STR00017## H Me NMe.sub.2 16 ##STR00018## H Me
NMe.sub.2
17-20. (canceled)
21. The method of claim 14 wherein the almiramide possesses
anti-trypanosome properties.
22. The method of claim 21 wherein the almiramide possesses
anti-pathogenic properties against Trypanosoma gambiense.
23. The method of claim 21 wherein the almiramide possesses
anti-pathogenic properties against Trypanosoma rhodesiense.
24. The method of claim 21 wherein the almiramide possesses
anti-pathogenic properties against Trypanosoma brucei.
25. The method of claim 21 wherein the almiramide molecule is
selected from the group consisting of compound 13, compound 14, and
compound 16.
26. The method of claim 22 comprising additionally administering
pentamidine.
27. The method of claim 23 comprising additionally administering
suramin.
28. The method of claim 24 comprising additionally administering
melarsoprol.
Description
RELATIONSHIP TO OTHER APPLICATIONS
[0001] This application claims the benefit of and priority to U.S.
Provisional application No. 61/210,657, filed 20 Mar. 2009, titled
"Novel Anti-Parasitic Compounds" to inventors Drs. Roger Linington
and Laura Sanchez, docket No. SC2009-565PRV. This provisional
application is hereby incorporated by reference for all
purposes.
FIELD OF THE INVENTION
[0002] The invention relates to a new structural class of compounds
for the treatment of diseases, particularly parasitic diseases such
as, but not limited to, leishmaniasis and sleeping sickness. The
invention relates to these new compounds and their derivatives and
analogues that have been shown to demonstrate clinically relevant
levels of therapeutic activity. The invention further relates to
the synthesis and use of such compounds in the treatment of
disease, and to the production of drug formulations containing such
compounds.
BACKGROUND
[0003] Leishmaniasis is a debilitating disease prevalent across
many inter-tropical regions of the world including India, Sudan and
Brazil. Caused by over twenty species of intracellular parasite
from the genus Leishmania, leishmaniasis can present itself in a
number of different clinical manifestations including cutaneous,
mucosal and visceral forms of the disease. Both the cutaneous and
mucosal forms can cause severe disfigurement to patients including
ulcerative skin lesions and the destruction of the mucous membranes
of the nose, mouth and throat leading to permanent disfigurement
and frequent social ostracization. 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 spleneomegaly (a dramatic enlargement
of the spleen) and ultimately multisystem infection and death in
untreated cases. Visceral leishmaniasis is caused by a small
sub-group 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 either in
terms of efficacy, toxicity or cost. Until recently the most widely
used therapeutic in most regions of the world was pentavalent
antimony. Though initially highly efficacious with an estimated
90-95% cure rate in most areas after its introduction in the
1950's, this treatment has suffered from increasing emergence of
resistance fueled by the high rate of patient non-compliance due to
the exceedingly long treatment period. Additionally the relatively
high toxicity of this treatment regimen kills an estimated 2-5% of
patients as a direct effect of drug toxicity. Alternative
therapeutics 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 which has shown excellent cure rates, but which is also
already facing instances of resistance in some areas. This
shortfall of affordable and clinically 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 clearly therefore a
pressing need for the development of new drugs to treat
leishmaniasis, and it is with this aim that the Panama
International Cooperative for Biodiversity Group (ICBG) is
investigating Panamanian microorganisms for lead compounds with
antileishmanial activity.
[0004] Human African trypanosomiasis or Sleeping sickness is a
parasitic disease of people and animals, caused by protozoa of the
species Trypanosoma brucei and transmitted by the tsetse fly. The
disease is endemic in some regions of Sub-Saharan Africa, covering
about 36 countries and 60 million people. It is estimated that
50,000 to 70,000 people are currently infected. The current
standard treatment for first stage trypanosomiasis employs
administering intravenous pentamidine (for T.b. gambiense) or
intravenous suramin (for T.b. rhodesiense). Alternative first line
therapies include using intravenous melarsoprol with or without
oral nifurtimox. Intravenous eflornithine may also be used. The
current standard treatment for second stage (later stage) disease
uses intravenous melarsoprol.
BRIEF DESCRIPTION OF THE INVENTION
[0005] The inventors have discovered a new class of compounds
(highly N-methylated linear lipopeptides) that possess novel
structures that are believed to be entirely novel and to have no
precedent in the literature.
[0006] The inventors have proven that these compounds possess
clinically significant anti-parasitic activity, particularly
against protozoan parasites including Leishmania donovani and
Trypanosoma brucei. The anti-parasitic activity experimentally
demonstrated against both Leishmania donovani and Trypanosoma
brucei is better than those of currently available therapeutic
compounds, without showing significant cytotoxicity to mammalian
Vero cells
[0007] Through the research of the inventors it is clear that this
new structural class of compounds may be used for the treatment of
diseases, particularly parasitic diseases such as, but not limited
to, leishmaniasis and sleeping sickness.
[0008] The invention relates to these new compounds (and/or to
their derivatives and analogues, sub-components and fragments
derived therefrom) that demonstrate clinically relevant levels of
therapeutic activity.
[0009] The invention further relates to the synthesis and use of
such compounds in the treatment of disease, and to the production
of drug formulations containing such compounds.
[0010] The invention further encompasses drug targets that bind
specifically to the compounds of the invention (and/or to their
derivatives and analogues, sub-components and fragments derived
therefrom), and methods for identifying drug targets by contacting
a sample containing one or more putative targets with one or more
compounds of the invention and/or to their derivatives and
analogues, sub-components and fragments derived therefrom. A sample
may contain material derived, for example, from a pathogenic
organism, such as a parasite, such as a eukaryotic pathogen, such a
pathogenic amoeba or protozoa.
[0011] In particular, the applicant has discovered that various
linear lipopeptide compounds derived from the marine cyanobacterium
Lyngbya majuscula possess anti-parasitic properties, particularly
against the organisms that cause leishmaniasis and sleeping
sickness. The applicant has performed isolation, analysis,
biological evaluation and synthesis of the highly N-methylated
linear lipopeptides: Almiramides A-C, disclosed herein.
[0012] Bioassay guided fractionation has led to the discovery of a
new structural class of compounds for the treatment of parasitic
diseases, for example, leishmaniasis with clinically relevant
levels of activity. The new compounds are highly N-methylated
linear lipopeptides: Almiramides. 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, one of
the causative agents of leishmaniasis. Chromatographic separation
of this complex mixture led to the isolation of the highly
N-methylated linear lipopeptides--Almiramides A-C (1-3) as the
active constituents of this extract, as well as the known
metabolite dragonamide B (6). Comparison with the biological
activities of a number of related metabolites and semi-synthetic
derivatives revealed key features required for activity and
afforded one new compound (11) with superior in vitro activity
against L. donovani. Derivatization of the hydrolysis products for
almiramide B (2) followed by GC-MS analysis showed the
2-methyloct-7-ynoic acid terminus to possess the R configuration in
contrast to other metabolites from this extract which contained the
closely related (s)-2-methyloct-7-ynoic acid terminus, providing
evidence for an unusual degree of biosynthetic specificity in the
production of closely related compounds from this organism.
Finally, synthesis of a library of simplified synthetic analogues
afforded several compounds with superior potency to the natural
products which paves the way for further drug development.
[0013] Certain specific embodiments appear in the claims, and
additionally include the following: An almiramide molecule, or a
derivative thereof, comprising a linear lipopeptide having the
following structure
##STR00001##
but specifically excluding compounds (1), (2) and (3) [0014]
wherein for (1), R.sup.1 consists of
##STR00002##
[0014] R.sup.2 consists of Me, R.sup.3 consists of H, and R.sup.4
consists of NH2; [0015] and wherein for (2), R.sup.1 consists
of
##STR00003##
[0015] R.sup.2 consists of Me, R.sup.3 consists of H, and R.sup.4
consists of NH2; [0016] and wherein for (3), R.sup.1 consists
of
##STR00004##
[0016] R.sup.2 consists of Me, R.sup.3 consists of H, and R.sup.4
consists of NH2. In some embodiments the R.sup.1 group may include
a terminus or sidechain that includes a lipid and/or fatty acid
group and/or an alkane, alkene or alkyne moiety, being saturated or
having one or more double or triple bonds. Such groups may be
branched or unbranched, substituted or unsubstituted and it may
comprise cyclic moieties. Such groups may be present at one or more
termini or side locations along the length of the molecule. In
certain alternative embodiments the almiramide molecule comprises
at least one unsaturated terminus. One or more unsaturated moieties
may be present on one or more side chains. The almiramide molecule
may be a methyl ester derivative or a carboxylic acid
derivative.
[0017] Other specific embodiments include the following: A molecule
selected from Table 1 or a derivative, analogue, sub-component or
fragment derived therefrom. A molecule selected from Table 1 other
than a molecule that occurs in nature. A molecule selected from
Table 1 other than a molecule that occurs in a cyanobacterium. A
molecule from Table 1 selected from the group consisting of
compounds 13-24 or a derivative, analogue, sub-component or
fragment derived therefrom. A molecule selected from table 1
wherein the molecule comprises a N--N-dimethyl-amide C-terminus
(13-16) or a derivative, analogue, sub-component or fragment
derived therefrom. A molecule selected from table 1 wherein said
molecule is a methyl ester derivative (21-24) or a derivative,
analogue, sub-component or fragment derived therefrom. A molecule
selected from table 1 wherein said molecule is a carboxylic acid
derivative (17-20) or a derivative, analogue, sub-component or
fragment derived therefrom. A molecule described above having
anti-pathogenic properties. A molecule described above having
anti-pathogenic properties against Leishmania or Trypanosoma genus.
A molecule described above that binds specifically to a target
naturally present in a protozoan parasite. A molecule described
above that binds specifically to a target naturally present in
Leishmania or Trypanosoma genus. A method for treating a disease in
a subject the method comprising administering to the subject a
compound selected from Table 1 or a derivative, analogue,
sub-component or fragment derived therefrom. A method for treating
a disease in a subject the method comprising administering to the
subject a compound selected from the group consisting of compounds
13-24 or a derivative, analogue, sub-component or fragment derived
therefrom. The method described above wherein the compound does not
occur in nature. A method for effecting the physiology of a
pathogen, the method comprising contacting the pathogen to a
compound selected from Table 1 or a derivative, analogue,
sub-component or fragment derived therefrom. A method for affecting
the physiology of a pathogen, the method comprising contacting the
pathogen to a compound selected from the group consisting of
compounds 13-24, or a derivative, analogue, sub-component or
fragment derived therefrom. The method described above wherein the
pathogen is a protozoan. The method above wherein the pathogen is
of the genus Leishmania or Trypanosoma. The method described above
wherein compound is selected from the group consisting of compounds
13-24 or a derivative, analogue, sub-component or fragment derived
therefrom. The method described above wherein compound selected
from Table 1 does not occur in the genus Leishmania. A method for
screening a pool of compounds so as to identify a target compound,
the method comprising (i) labelling or immobilizing a first
compound selected from Table 1 or a derivative, analogue,
sub-component or fragment derived therefrom, (ii) providing a pool
of second compounds, (iii) contacting first the labelled or
immobilized compound with the pool of second compounds under
conditions that allow specific binding to occur between the first
compound and at least one second compound, (iv) washing the
labelled or immobilized first compound so as to remove unbound
second compound(s), and (v) purifying and characterising the second
compound(s) that have bound specifically to the first compound. The
method for screening described above wherein the first compound is
selected from the group consisting of compounds 13-24, or a
derivative, analogue, sub-component or fragment derived therefrom.
The method described above wherein the first compound is
immobilized within a chromatography apparatus. The method described
above wherein the first compound is labeled with a radioactive,
fluorescent, chromatological, color-developing, or immunological
marker. A target compound identified by any or the methods
described herein. A drug formulation comprising a drug and a
carrier, wherein the drug is selected from a compound described in
Table 1, or a derivative, analogue, sub-component or fragment
derived therefrom. The drug formulation described above wherein the
drug is selected from the group consisting of compounds 13-24, or a
derivative, analogue, sub-component or fragment derived therefrom.
A drug formulation comprising a drug and a carrier, wherein the
drug comprises a molecule having the structural formula:
##STR00005##
wherein R1, R2, R3, and R4 may be any specie or group. The drug
formulation of claim 30 wherein the R1, R2, R3, and R4 are selected
from the species disclosed in Table 1. The drug formulation
described above wherein the drug binds specifically to a compound
derived from a protozoan pathogen. The drug formulation of claim 30
wherein the drug binds specifically to a compound derived from a
Leishmania. The drug formulation described above wherein the drug
kills or inhibits the growth, reproduction or otherwise interferes
with the life-cycle of a protozoan pathogen. The drug formulation
described above wherein the protozoan pathogen is Leishmania. A kit
for diagnosis of the presence of Leishmania in a sample, the kit
consisting of a compound selected from the group consisting of
compounds 13-24, or a derivative, analogue, sub-component or
fragment derived therefrom, wherein, in use, the compound bound to
a radioactive, fluorescent, chromatological, color-developing, or
immunological marker.
BRIEF DESCRIPTION OF THE FIGURES, SCHEMES AND TABLES
[0018] FIG. 1a. Almiramides A, B and C
[0019] FIG. 1b. Subunits a-f and NMR connectivity for 1. Solid
arrows=HMBC correlations. Dashed arrows=ROESY correlations.
[0020] FIG. 2. Selective semisynthetic methylation of 2 to afford
compounds 11 and 12
[0021] FIG. 3. Structurally related linear lipopeptides (areas of
highest structural homology outlined with dashed lines).
[0022] FIG. 4. Generic structure of Almiramide.
[0023] Scheme 1. Configurational analysis strategy for 2 and 3
[0024] Scheme 2. Formation of semi-synthetic derivatives 11 and
12.
[0025] Table 1: Structure and Bioactivities of Almiramide A-C and
Derivatives.
GENERAL REPRESENTATIONS CONCERNING THE DISCLOSURE
[0026] It should be noted that although the present disclosure
refers frequently to leishmaniasis and Leishmania donovani, the
compounds and methods of the invention are directed towards any
disease, particularly parasitic diseases caused by amoeba or
protozoa or other eukaryotic organisms. Leishmaniasis is given as
just one exemplary embodiment. African sleeping sickness and Lyme
disease are two other diseases against which the compounds of the
invention have a therapeutic effect.
[0027] Other diseases against which the compounds and methods of
the invention include Amoebiasis, Ascariasis, Babesiosis, Chagas
disease, Clonorchiasis, Cryptosporidiosis, Diphyllobothriasis,
Dracunculiasis (caused by the Guinea worm), Echinococcosis,
Elephantiasis, Enterobiasis, Fascioliasis, Fasciolopsiasis,
Filariasis, Giardiasis, Gnathostomiasis, Hymenolepiasis,
Isosporiasis, Katayama fever, Lyme disease, Mange, Malaria
Metagonimiasis, Myiasis, Onchocerciasis, Pediculosis, Scabies,
Schistosomiasis, Strongyloidiasis, Taeniasis (cause of
Cysticercosis), Toxocariasis, Toxoplasmosis, Trichinosis, and
Trichurias. Other organisms against which the compounds and methods
of the invention may be used, either to prevent or treat a disease,
include, for example, the following: Anisakis, Ascaris
lumbricoides, Botfly Balantidium coli, Bedbug, Cestoda (tapeworm),
Chigger, Hookworm, Liver fluke, Loa loa, Paragonimus (lung fluke),
Pinworm, Plasmodia (including falciparum, vivax and ovale),
Schistosoma, Strongyloides stercoralis, Mites, Tapeworm, Toxoplasma
gondii, all Trypanosomes, Whipworm, Wuchereria bancrofti, and
Ixodes pacificus.
[0028] The term "parasite" in this disclosure is used broadly to
mean any organism that is known to cause a pathology by infection
of an animal subject (host). Likewise the term "parasitic disease"
is used broadly to mean a disease caused by such a parasite. The
term "disease" is used to mean any state of an animal that deviates
from normal healthy physiology and that is clinically
detectable.
[0029] The term "binds" or "binding" in connection with the
interaction between a one compound or molecule and another compound
or molecule, such as a target and a potential binding compound,
indicates that the potential binding compound associates with the
target to a statistically significant degree as compared to
association with proteins generally. Thus, the term "specific
binding" refers to binding between two molecules or compounds that
is statistically significantly higher than non-specific binding to
another molecule. Preferably a binding compound interacts with a
specified target with a dissociation constant (k.sub.d) of 1 mM or
less, for example 0.1-100 nM. A binding compound can bind with "low
affinity", "very low affinity", "extremely low affinity", "moderate
affinity", "moderately high affinity", or "high affinity" as
described herein. In the context of compounds binding to a target,
the term "greater affinity" indicates that the compound binds more
tightly than a reference compound, or than the same compound in a
reference condition, i.e., with a lower dissociation constant. In
particular embodiments, the greater affinity is at least 2, 3, 4,
5, 8, 10, 50, 100, 200, 400, 500, 1000, or 10,000-fold greater
affinity. Also in the context of compounds binding to a
biomolecular target, the term "greater specificity" indicates that
a compound binds to a specified target to a greater extent than to
another biomolecule or biomolecules that may be present under
relevant binding conditions, where binding to such other
biomolecules produces a different biological activity than binding
to the specified target. Typically, the specificity is with
reference to a limited set of other biomolecules. In particular
embodiments, the greater specificity is at least 2, 3, 4, 5, 8, 10,
50, 100, 200, 400, 500, or 1000-fold greater specificity.
[0030] The term "derivative" or "derivative compound" refers to a
compound having a chemical structure that contains a common core
chemical structure as a parent or reference compound, but differs
by having at least one structural difference, e.g., by having one
or more substituents added and/or removed and/or substituted,
and/or by having one or more atoms substituted with different
atoms. Unless clearly indicated to the contrary, the term
"derivative" does not mean that the derivative is synthesized using
the parent compound as a starting material or as an intermediate,
although in some cases, the derivative may be synthesized from the
parent.
[0031] The term "fragment" refers to a part of a larger whole, for
example a fragment of a molecule may be any dissociated part of
that molecule, regardless of size.
[0032] The term "specie" or "group" when used to describe an "R"
group in a chemical formula, is used to mean any chemical compound,
sub-compound or substituent that may chemically interact with
(covalently, ionically or by Van der Waal's forces) another
molecule or group such as shown on a chemical formula.
[0033] The terms "formulation, "drug formulation or "pharmaceutical
formulation," refers to a drug combined with a non-drug such as a
carrier material designed not to have a pharmaceutical activity,
such as pharmaceutical excipient, filler, or carrier material that
may be used to modify or improve the drug release, improve its
physical and/or chemical stability, dosage form performance,
processing, manufacturing, etc.
[0034] When a "terminus" or "terminal group" is discussed as having
a substituent, side-chain, group or moiety attached, that
substituent, side-chain, group or moiety may equally be present at
one or more termini or at side locations along the length of the
molecule.
[0035] The terms "drug" or "therapeutic agent" mean any substance
meant to affect the physiology of a subject. Examples of drugs are
described in well known literature references such as the Merck
Index and the Physicians Desk Reference.
[0036] The term "therapeutically effective amount" means an amount
of a therapeutic agent, or a rate of delivery of a therapeutic
agent, effective to facilitate a desired therapeutic effect. The
precise desired therapeutic effect will vary according to the
condition to be treated, the formulation to be administered, and a
variety of other factors that are appreciated by those of ordinary
skill in the art.
[0037] The term "diagnostic agent" means any chemical moiety that
may be used for diagnosis or in a diagnostic test. For example,
diagnostic agents include imaging agents containing radioisotopes,
contrasting agents containing for example iodine, enzymes,
fluorescent substances and the like.
[0038] The term "treatment" means the application of a process to
an individual in order to alter a physiological state, whether or
not the process includes a curative element.
[0039] Where substitutions are mentioned, sometimes in connection
with variable "R" groups as shown in the figures, the substituent
groups may be selected from, for example, the following: hydrogen,
hydroxyl, carboxylate, alkane, alkene or alkyne groups, substituted
or unsubstituted heteroatom, alkyl, alkenyl, alkanoyl, aryl, aroyl,
aralkyl, alkylamino cycloalkyl, heterocycloalkyl, heteroaryl, or
halogen, azido, fluorophore or polypeptide. In certain embodiments
the substituent group may comprise branched or un-branched C1-C18
alkyl, C1-C18 substituted alkyl, C1-C18 alkenyl, C1-C18 acyl,
amino, substituted amino, wherein the alkyl, alkenyl or acyl is
linear or branched, and optionally substituted with a hydroxyl, an
ester and its derivatives, 5 a carboxyl and its derivatives. In a
particular embodiment, Any R group may be a lower hydrocarbon
substituted with alkoxy, substituted alkoxy, imidate, arylthio, or
(substituted aryl)thio. In other embodiments, Any R group may be a
lower alkyl selected from methyl, ethyl, propyl, isopropyl, butyl,
isobutyl, terabutyl and pentyl. In other embodiments, Any R group
may be a lower alkenyl selected from vinyl, substituted vinyl,
ethynyl, or substituted ethynyl. In other embodiments, Any R group
may be a lower alkanoyl selected from formyl, acetyl, propionyl,
isopropionyl, butyryl, isobutyryl, tert-butyryl, valeryl, pivaloyl,
caproyl, capryl, lauryl, myristyl, palmityl, stearyl, arachidyl,
stilligyl, palmitoyl, oleyl, linolenyl, and arachidonyl. In other
embodiments, Any R group may be lower aryl selected from phenyl,
p-tolyl, pchlorophenyl, p-aminophenyl, p-nitrophenyl, p-anisyl. In
yet other embodiments, Any R group may be a lower aroyl selected
from benzoyl and naphthoyl. In other embodiments, Any R group may
be a lower aralkyl selected from benzyl, benzhydryl,
p-chlorobenzyl, m-chlorobenzyl, p-nitrobenzyl, benzyloxybenzyl, or
pentaflourobenzyl. In certain other embodiments, Any R group may be
a lower alkylamino is selected from monoalkylamino,
monoaralkylamino, dialkylamino, diaralkylamino, and
benzylamino.
[0040] It should be noted that the invention encompasses compounds,
methods and treatments wherein compounds of the invention, and
their derivatives and analogues, sub-components and fragments
derived therefrom, may be employed to treat any disease of any
organism, either animal or plant.
[0041] Although the disclosure refers frequently to leishmaniasis
and Leishmania donovani, the compounds and methods of the invention
are directed towards any disease, particularly parasitic diseases
caused by pathogenic eukaryotic organisms such as protozoa.
Leishmaniasis is given as just one exemplary embodiment. Other
pathogens and diseases are disclosed herein and still further
pathogens and diseases may be treated with the compounds of the
invention, such as those pathogens and diseases mentioned in the
text book "Parasitic Diseases (5th Edition) by Despommier et al.,
Apple Trees Productions LLC, Pub., which is hereby incorporated by
reference for all purposes.
[0042] In this specification, reference is made to particular
features of the invention (including for example components,
ingredients, elements, devices, apparatus, systems, groups, ranges,
method steps, test results, etc). It is to be understood that the
disclosure of the invention in this specification includes all
appropriate combinations of such particular features. For example,
where a particular feature is disclosed in the context of a
particular embodiment or a particular claim, that feature can also
be used, to the extent appropriate, in the context of other
particular embodiments and claims, and in the invention generally.
The embodiments disclosed in this specification are exemplary and
do not limit the invention. Other embodiments can be utilized and
changes can be made. As used in this specification, the singular
forms "a", "an", and "the" include plural reference unless the
context clearly dictates otherwise. Thus, for example, a reference
to "a part" includes a plurality of such parts, and so forth.
[0043] The term "comprises" and grammatical equivalents thereof are
used in this specification to mean that, in addition to the
features specifically identified, other features are optionally
present. Where reference is made in this specification to a method
comprising two or more defined steps, the defined steps can be
carried out in any order or simultaneously. Where reference is made
herein to "first" and "second" features, this is generally done for
identification purposes; unless the context requires otherwise, the
first and second features can be the same or different, and
reference to a first feature does not mean that a second feature is
necessarily present (though it may be present).
[0044] A number of references exist that contain relevant subject
matter useful in the understanding of the invention. These include:
(1) Linington, R G, J Gonzalez, L-D Urena, L I Romero, E
Ortega-Barria, W H Gerwick: Venturamides A and B: Antimalarial
Constituents of the Panamanian Marine Cyanobacterium Oscillatoria
sp. J. Nat. Prod. 70, 397-401 (2007); (2) McPhail, K L, J Correa, R
G Linington et al.: Antimalarial Linear Lipopeptides from a
Panamanian Strain of the Marine Cyanobacterium Lyngbya majuscula.
J. Nat. Prod. 70, 984-988 (2007); and (3) Balunas, M. J.;
Linington, R. G.; Tidgewell, K.; Fenner, A. M.; Urena, L.-D.; Della
Togna, G.; Kyle, D. E.; Gerwick, W. H. "Dragonamide E, a Modified
Linear Lipopeptide from Lyngbya majuscula with Antileishmanial
Activity" J. Nat. Prod., 2010, 73, 60; and (4) Sarath P. Gunasekera
et al., "Dragonamides C and D, Linear Lipopeptides from the Marine
Cyanobacterium Brown Lyngbya polychroa" J. Nat. Prod., 2008, 71
(5), pp 887-890 These references and all documents and publications
referred to in this disclosure are hereby incorporated by
reference, in their entirety, for all purposes.
[0045] The entirely of the "definitions" sections of the following
applications are hereby incorporated by reference for all purposes:
WO2009114325 and US20060281914.
DETAILED DESCRIPTION OF THE INVENTION
[0046] The Panama International Cooperative for Biodiversity Group
(ICBG) is investigating Panamanian microorganisms for lead
compounds with antileishmanial activity. Initial screening of over
400 pre-fractions from cyanobacterial extracts revealed one
fraction from a collection of Lyngbya majuscula from Bocas del Toro
Marine Park on the Caribbean coast of Panama that exhibited a
unique profile against L. donovani without showing significant
cytotoxicity to mammalian Vero cells. This combination of results
indicated the presence of compounds with clinically relevant
activity against the target parasite and prompted us to undertake a
further investigation of the mixture. Purification of this fraction
by C.sub.18 reversed-phase solid phase extraction chromatography
(50% MeOH/H.sub.2O to 100% MeOH) afforded two consecutive active
fractions that were further purified by C.sub.18 RP HPLC to give
Almiramides A-C (1-3) as optically active white solids.
[0047] Initial NMR analysis of almiramide A (1) in CD.sub.2Cl.sub.2
gave spectra that contained two observable conformations in a 3:2
ratio. Reacquisition of these spectra in CD.sub.3CN afforded
spectra that also contained two stable conformations, though in a
more acceptable ratio of 6:1. Despite the added complexity of
interpreting spectra that contain multiple conformers it was
possible to identify the presence of subunits a-f (FIG. 1) without
significant difficulty. HRESITOFMS gave an [M+H].sup.+
pseudomolecular ion at 743.5062 that was consistent with the
molecular formula C.sub.40H.sub.66N.sub.6O.sub.7 (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 FIG. 1 allowed for the construction of the
planar structure of 1 which was further supported by fragmentation
patterns in the mass spectrum.
[0048] Almiramide B (2) gave a HRESITOFMS [M+H].sup.+
pseudomolecular ion at 725.4964 that was consistent with the
molecular formula C.sub.40H.sub.64N.sub.6O.sub.6 (calcd 725.4960).
Cursory examination of the proton spectrum for 2 showed it to
possess the same gross structure as 1. 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 sidechain. The absence of the methyl singlet and
ketone carbonyl signals from 1 (.delta..sub.H 2.04 .delta..sub.C
29.9 and .delta..sub.C 209.6 respectively) combined with the
presence of new quaternary carbon (.delta..sub.C 85.4) and methyne
signals (.delta..sub.H 2.16, .delta..sub.C 69.7) for 2 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, thus
defining the structure of this new metabolite as
NH2-Phe-NMe-Ala-NMe-Val-Val-NMe-Val-NMe-COCH(CH.sub.3)(CH.sub.2).sub.5C.i-
dent.CH.
[0049] Almiramide C (3) gave a HRESITOFMS [M+H].sup.+
pseudomolecular ion at 727.5115 that was consistent with the
molecular formula C.sub.40H.sub.66N.sub.6O.sub.6 (calcd 727.5116).
As with almiramide B, consideration of the NMR resonances for the
amino acid residues present in 3 showed them to be identical to
those for almiramide A (1), suggesting that 3 differed from 1 and 2
in the constitution of the side chain. The presence of three new
multiplet proton resonances between .delta..sub.H 4.89 and
.delta..sub.H 5.83 and new resonances in the carbon spectrum at
.delta..sub.C 115.0 and .delta..sub.C 140.1 coupled with the
absence of the quaternary carbon (.delta..sub.C 85.4) and methyne
signals .delta..sub.H 2.16, .delta..sub.C 69.7) for 2 suggested the
presence of a mono-substituted 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 which unequivocally
identified the fatty acid terminus as a 2-methyloct-7-enoic acid
residue.
[0050] Determination of the stereoconfiguration for 1-3 was
accomplished using two complimentary approaches (scheme 1). For the
amino acid residues standard Marfey's analysis was employed, and
the resulting derivatives analyzed by LC-MS. For the determination
of the configuration at the 2 position of the side chain the fatty
acid was derivatized with a chiral derivatizing agent and the
retention time on GC-MS compared to that of known synthetic
standards.
[0051] 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. Consequently both
2 and 3 were subjected to hydrogenation over palladium on carbon to
form compound 4 prior to hydrolysis in order to preserve the
integrity of the side chains. Hydrolysis products were partitioned
against 0.1 N HCl and EtOAc to afford the amino acids in the
aqueous phase and the side chain in the organic phase. The aqueous
portions from all three samples were separately concentrated to
dryness in vacuo. The resulting residues were derivatized with
N.sub..alpha.-(2,4-dinitro-5-fluorophenyl)-L-valinamide (DLVA)
under standard conditions and analyzed by gradient C.sub.18 RP-HPLC
in each case. Comparison of the retention times and mass spectra
for these derivatives with commercially available standards that
had been derivatized with DLVA in an identical fashion
unequivocally assigned all amino acid residues as L for almiramides
A-C. These assignments were all subsequently confirmed by
co-injection analyses.
[0052] To determine the configuration of the side chains for 2 and
3 the organic phases from the hydrolysis partitions were separately
derivatized with (S)-1-phenylethylamine under standard amide
coupling conditions (scheme 1) to form compound 9. Commercially
available (.+-.)-2-methylocatanoic acid was similarly derivatized
to give compounds 9 and 10 which were separated by flash silica gel
column chromatography. Differentiation of these two products was
accomplished by X-ray analysis of compound 9 which defined the
structure as (2R)-2-methyl-N--((S)-1-phenylethyl)octanamide.
Comparison of the retention times of these synthetic materials by
GC analysis with the retention time of the natural product
derivatives showed that both 2 and 3 possessed the R configuration
at the 2 position of the side chain. This result was somewhat
surprising as a recent total synthesis of the related metabolite
dragonamide A (5) has shown it to contain the
(2S)-2-methyloct-7-ynoic acid moiety. 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. 6 was
also isolated as a component of an inactive fraction from the crude
extract investigated in this study and 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 the derivatization of 6. This
result confirmed the previous configurational assignment and
indicated that this organism possesses the biosynthetic capability
to produce two closely related compounds (2 and 6) that contain
constitutively identical side chains with opposite configurations,
likely through a divergent biosynthetic pathway.
[0053] By extension of the biosynthetic capability of this organism
in the production of the almiramides and comparison of the NMR
chemical shifts for 1, 2 and 3 we consider it highly likely that
the stereoconfiguration of this center in compound 1 is also R,
though this was not verified experimentally.
[0054] Biological evaluation of these three compounds showed that
compounds 2 and 3 possessed strong in vitro antiparasitic activity
against L. donovani (2.38 and 1.91 .mu.M respectively) whereas
compound 1 was completely inactive up to 13.5 .mu.M suggesting the
requirement of an unsaturated terminus on the side chain for
activity. For reference the two most widely used treatments against
leishmaniasis (Pentostam.RTM. and Miltefosine.RTM.) are active in
vitro against L. donovani at 44.7 and 0.5 .mu.M respectively.
Previous investigations of cyanobacteria from this region by our
group have provided a number of related secondary metabolites
including carmabin A (7), dragomabin (8) and dragonamides A (5) and
B (6). Parallel testing with almiramides A-C showed compounds 5-8
to be inactive up to the highest tested concentrations (10
.mu.g/mL) despite significant structural similarities between these
compounds and almiramides B and C (FIG. 3). These data suggest that
the active site for 2 and 3 exhibits a high degree of substrate
specificity, and show that possession of alkyne and primary amide
termini for N-methylated peptides is insufficient for activity
against this target organism.
[0055] The value of the almiramides as lead molecules for
antileishmanial drug development prompted us to further explore the
structure activity relationship (SAR) features of these compounds
by the generation of semi-synthetic analogues. Hydrogenation of 2
with H.sub.2 and 10% palladium on carbon afforded compound 4 in
quantitative yield. Biological evaluation showed 4 to be inactive
at the highest tested concentration (13.4 .mu.M), supporting the
hypothesis that the unsaturated terminus plays an important role in
the interaction of these compounds with their biological target. We
were next prompted to explore the importance of the primary and
secondary amides for activity in 1-3. Methylation of 2 with NaH/MeI
afforded a 1:1 mixture of compounds 11 and 12 (FIG. 2) which were
active at 1.64 and 2.31 .mu.M respectively. We hypothesize that the
improved activity of 11 over 2 is due to the improved
bioavailability of 11 due to its increased membrane permeability.
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.
Given that the addition of the methyl group at Val1-N has little
effect on the calculated conformation of 11 versus 2 it is
reasonable to suggest that membrane permeability is an important
factor in explaining the comparative efficacy of these two closely
related compounds. The small quantities of 11 and 12 generated from
derivatization of the natural product precluded the use of standard
techniques for structure determination. The unequivocal assignment
of these structures was however possible by consideration of a
combination of .sup.1H NMR and MS fragmentation data. .sup.1H NMR
for 11 showed the absence of the Val1-NH proton doublet at .delta.
6.58 and the presence of a new methyl singlet at .delta. 2.94.
Coupled with a mass increase of 14 amu in the low resolution
electrospray ionisation mass spectrometry (LRESIMS) 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 MS
fragmentation pattern which showed characteristic fragments for the
loss of sequential amino acid residues starting from the
C-terminus. Similarly, .sup.1H NMR for 12 showed the absence of
both the Val1-NH and the C-terminal-NH.sub.2 proton resonances
(.delta. 6.58; .delta. 5.80 and .delta. 6.26) and the presence of
three additional methyl singlets in the region .delta. 2.84-2.94
indicating permethylation at these three positions. As with
compound 11 this was confirmed by the MS fragmentation pattern in
the LRESIMS spectrum. Interestingly the permethyl species displayed
a single conformation on the NMR timescale in contrast to compounds
1-3 and 11, all of which exhibit major and minor conformers in
their NMR spectra, suggesting that the C-terminal NH protons play
an important role in the hydrogen bonding interactions leading to
these two conformational states.
[0056] Generation of semi-synthetic derivatives with fully
methylated peptide backbones and improved pharmacological
properties was particularly gratifying as this opened the door to a
facile 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 drug development. In designing 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 construction 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 frameworks. 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 a-position of the lipophilic sidechain
(Moya-2) while additional library members showed incrementally
greater structural divergence.
[0057] 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 DIPEA was followed by cleavage from the solid support (1%
TFA in CH2Cl2 for chlorotrityl resins, 95% TFA 5% TIPS for Rink
amide resins). The resulting intermediates were permethylated with
NaH/MeI in THF under Argon starting at -78.degree. C. with warming
to room temperature, and the reactions were monitored by LC-MS. In
all cases, final purification was performed by reversed-phase HPLC
(Phenomenex Jupiter C18 column, 4.6.times.250 mm, 1 mL/min,
MeOH/H2O+0.02% HCOOH). Conversion of compounds 17-20 to their
corresponding methyl esters (21-24) was accomplished by treatment
with TMS-diazomethane in 3:1 C6D6/MeOH.
[0058] Gratifyingly, biological evaluation of compounds 13-24 in
parallel with the original lead compounds (1-3) identified several
compounds with improved potency over the natural products.
Specifically, compounds 13-16 containing the N--N-dimethyl-amide
C-terminus exhibited strong antileishmanial 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.
[0059] The synthesis of compounds 13-16 which exhibit improved
activity over the initial natural product lead structures is an
important finding for two reasons. Firstly this discovery
circumvents one of the most common impediments to the development
of natural product drug leads by providing an efficient route to a
renewable supply of material; an essential requirement for
preliminary in vivo studies which typically requires more material
than is available from the natural source. Secondly, the peptidic
nature of this scaffold makes it an ideal candidate for the
construction of targeted combinatorial libraries however the
presence of a standard (non-methylated) residue at Val-1 and a
chiral center at C-2 of the aglycon 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 which not only proceeds in
high yield but also provides the opportunity for exploring the SAR
interdependence of the amino acid residues present in this
framework.
[0060] For reference, the recent total synthesis of dragonamide A
(5) required 17 steps in the longest linear sequence starting from
1,5 pentanediol with an overall yield of 1.92%. By contrast the
synthesis of 17 was completed in just 15 steps, all but 2 of which
were performed on the solid phase in a total yield of 2.25%.
[0061] As an additional benefit, the replacement of the sole
secondary amide linkage in the natural products with a tertiary
N-methyl-amide removes the possibility of proteolytic degradation
in the host system, thus improving the predicted pharmacokinetic
properties of this compound class in murine model systems.
[0062] Experimental Section
[0063] General Experimental Procedures. Optical rotations were
measured with a Jasco P1010 polarimeter. UV spectra were acquired
on a Shimadzu UV2401-PC spectrophotometer. NMR spectra were
acquired on a JEOL Eclipse 400 MHz spectrometer and referenced to
residual solvent proton and carbon signals (.delta..sub.H 1.94,
.delta..sub.C 1.34 for CD.sub.3CN). Low resolution APCI mass
spectra were acquired on a JEOL LC-mate mass spectrometer
(INDICASAT). Accurate mass ESI mass spectra were acquired on an
Agilent ESI-TOF mass spectrometer (Scripps Center from Mass
Spectrometry, San Diego). HPLC purifications were performed on an
Agilent 1100 series HPLC system employing a G1312A binary gradient
pump, a G1322A degasser, a G1314A variable wavelength detector
tuned to 210 nm and a Phenomenex Jupiter C.sub.18 (4.6.times.250
mm) RP-HPLC column All solvents were HPLC grade and were used
without further purification. X-ray analysis was performed. GC-MS
analysis was performed on a Thermo Trace GC equipped with an AI3000
series autosampler and a DSQ EIMS.
[0064] 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.degree.
16.669' N 82.degree. 09.834' W). The cyanobacterium was strained
through a mesh bag to remove excess seawater, frozen on site, and
stored at -4.degree. C. until workup. The taxonomy was identified
by comparison with characteristics described by Geitler. A voucher
was deposited at the Smithsonian Tropical Research Institute,
Panama (voucher number PAB-04-NOV-05-7).
[0065] Extraction and Isolation. Freshly thawed material was
extracted exhaustively with CH.sub.2Cl.sub.2/MeOH (2:1, 3.times.500
mL) and the combined organic extracts partitioned against H.sub.2O
(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.times.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 C.sub.18 RP-SPE chromatography eluting
with: 1:1 H.sub.2O/MeOH (30 mL), 4:6 H.sub.2O/MeOH (30 mL), 3:7
H.sub.2O/MeOH (30 mL), 2:8 H.sub.2O/MeOH (30 mL), 1:9 H.sub.2O/MeOH
(30 mL), 100% MeOH (30 mL), 100% EtOAc (30 mL). The active
fractions from these separations were separately subjected to
C.sub.18 RP-HPLC to give from the more polar fraction (Phenomenex
Jupiter C.sub.18 4.6.times.250 mm RP-HPLC column, 5 .mu.m, 57%
MeOH/43% H.sub.2O, 210 nm, 1 mL/min, 24.5 min) almiramide A (1) as
a colorless glass (3.5 mg, 0.6% of crude extract, t.sub.R=15.4
mins) and almiramide B (2) as a colorless glass (1.9 mg, 0.3% of
crude extract, t.sub.R=39.1 mins). HPLC of the less polar active
fraction (Phenomenex Jupiter C.sub.18 4.6.times.250 mm RP-HPLC
column, 5 .mu.m, 62% MeOH/38% H.sub.2O, 210 nm, 1 mL/min) gave
almiramide B (2) as a colorless glass (3.4 mg, 0.6% of crude
extract, t.sub.R=16.9 mins) and almiramide C (3) as a colorless
glass (1.2 mg, 0.2% of crude extract, t.sub.R=40.3 mins).
[0066] Almiramide A (1) was obtained as a colorless glass;
[a]22D-169.1 (c 0.002, MeOH); UV (MeOH) .lamda.max (log .epsilon.)
228 (sh) (3.77) nm; IR (film) vmax 2960, 1624 cm-1; for 1H and 13C
NMR data, see Table 2; HRESIMS m/z [M+H]+743.5062 (calcd for
C40H67N6O7, 743.5065)
[0067] Almiramide B (2) was obtained as a colorless glass;
[a]22D-148.9 (c 0.001, MeOH); UV (MeOH) .lamda.max (log .epsilon.)
230 (sh) (3.76) nm; IR (film) vmax 2970, 1634 cm-1; for 1H and 13C
NMR data, see Table 2; HRESIMS m/z [M+H]+725.4964 (calcd for
C40H65N6O6, 725.4960)
[0068] Almiramide C (3) was obtained as a colorless glass;
[a]22D-136.8 (c 0.001, MeOH); UV (MeOH) .lamda.max (log .epsilon.)
228 (3.69) nm; IR (film) vmax 2955, 1630 cm-1; for 1H and 13C NMR
data, see Table 2; HRESIMS m/z [M+H]+727.5115.???? (calcd for
C40H67N6O6, 727.5116)
[0069] Hydrogenation of 2 and 3: Authentic samples of 2 and 3 (0.1
mg) were independently dissolved in dry CH.sub.2Cl.sub.2 (10 mL).
10% palladium on carbon (2 mg) was added and the suspensions
stirred under an atmosphere of H.sub.2 (balloon) at room
temperature for 16 hours. Each sample was concentrated to dryness
under a stream of N.sub.2, dissolved in MeOH (1 mL) and filtered
through a 13 mm 0.2 .mu.m nylon filter. The resulting filtrates
were concentrated to dryness under a stream of N.sub.2 and purified
by C.sub.18 RP-HPLC (Phenomenex Jupiter C.sub.18 4.6.times.250 mm
RP-HPLC column, 5 .mu.m, 76% MeOH/24% H.sub.2O, 210 nm, 1 mL/min)
to give pure 4 in each case.
[0070] Marfey's Analysis of 1-3: An authentic sample of 1 and
hydrogenated samples of 2 and 3 were independently treated with 6N
HCl in sealed vials at 120.degree. C. for 18 hr. The solutions were
concentrated to dryness in vacuo and treated with a solution of
1-fluoro-2,4-dinitrophenyl-S-L-valine-amide (DLVA) (0.25 mg, 0.8
.mu.mol) in acetone (50 .mu.L) and a solution of 0.1 M NaHCO.sub.3
(100 .mu.L) in a sealed vial at 90.degree. C. for 5 min. The
reaction mixture was neutralized with 2N HCl (50 .mu.L) and diluted
with CH.sub.3CN (100 .mu.L). The resulting solution was analyzed by
RP-HPLC employing a Phenomenex Jupiter C.sub.18 column
(4.6.times.250 mm) and a gradient elution profile of 25%
CH.sub.3CN/75% H.sub.2O (acidified with 0.05% HCOOH) to 55%
CH.sub.3CN/45% H.sub.2O (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-methylalanine 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-methylphenylalanine 47.8; D-N-methylvaline 50.3.
[0071] Synthesis of 9 and 10: To a stirred solution of
(.+-.)-2-methyloctanoic acid (82 mg, 0.52 mmol) in dry
CH.sub.2Cl.sub.2 (10 mL) at 0.degree. C. was added
(S)-1-phenylethylamine (65 .mu.L, 0.57 mmol), Et.sub.3N (57 .mu.L,
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
hrs. The volatiles were removed in vacuo and the residue suspended
in EtOAc (5 mL) and filtered through Celite. The filtrate was
diluted with EtOAc (10 mL) and washed sequentially with 0.2N HCl
(25 mL), H.sub.2O (25 mL), 0.1N NaHCO.sub.3 (25 mL) and H.sub.2O
(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 chromatography (26.times.250 mm, 100% hexanes to
7:3 hexanes/EtOAc; 14.times.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.
[0072] (2R)-2-methyl-N--((S)-1-phenylethyl)octanamide (9): white
needles (.sup.iPrOH); [.alpha.].sup.20.sub.D-85.3 (c 1.3,
CHCl.sub.3); UV (MeOH); .sup.1H NMR (CDCl.sub.3, 300 MHz) .delta.
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);
.sup.13C NMR (CDCl.sub.3, 75 MHz) .delta. 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.
[0073] (2S)-2-methyl-N--((S)-1-phenylethyl)octanamide (10):
colorless glass; [.alpha.].sup.20.sub.D-66.1 (c 1.4, CHCl.sub.3);
UV (MeOH); .sup.1H NMR (CDCl.sub.3, 300 MHz) .delta.; .sup.13C NMR
(CDCl.sub.3, 75 MHz) .delta. 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.
[0074] GC-MS Analysis of 9 and 10: GC-MS analyses were performed on
an Altech Chirasil Val column (25 m.times.0.25 mm, 0.16 .mu.m ID)
employing a temperature gradient from 90 to 220.degree. C. at a
ramp rate of 4.degree. C./min followed by 5 minutes at 220.degree.
C. Retention times in minutes for compounds 9 and 10 were as
follows: 9 25.61, 10 25.05.
[0075] 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 stirred under argon at room temperature
for 2 hrs. MeI (50 .mu.L, 0.80 mmol) was added dropwise and the
resulting solution stirred for a further 21 hours. The reaction
mixture was quenched with water (0.5 mL) and partitioned with EtOAc
(2 mL). The phases were separated and the aqueous phase washed with
EtOAc (2 mL). The combined organics were concentrated to dryness
under a stream of N.sub.2 gas and purified by C.sub.18 RP-HPLC
(Phenomenex Jupiter C.sub.18 4.6.times.250 mm RP-HPLC column, 5
.mu.m, 72% MeOH/28% H.sub.2O, 210 nm, 0.8 mL/min) to give 11 (0.2
mg, 13%) and 12 (0.6 mg, 38%) as white solids.
[0076] Bioassays. All bioassays were performed in duplicate,
testing at 10, 2, 0.4, 0.08 and 0.016 .mu.g/mL. Malaria bioassays
were performed as previously reported by our program, using
chloroquine as a positive control (IC.sub.50=80-100 nM). Chagas
bioassays were performed following the protocol of Buckner et al,
and using nifurtimox as a positive control (IC.sub.50 3-5
.mu.g/mL). Leishmaniasis bioassays were performed using a method
previously employed in our laboratory, based on parasite DNA
fluorescence. In this latter assay, amphotericin-B was used as the
positive control and had an IC.sub.50 value of 80 ng/mL.
Cytotoxicity bioassays were performed following an MTT cell
proliferation assay protocol with green monkey Vero kidney
cells.
* * * * *