U.S. patent application number 14/300191 was filed with the patent office on 2014-09-25 for antileishmanial compositions and methods of use.
This patent application is currently assigned to OHIO STATE INNOVATION FOUNDATION. The applicant listed for this patent is Eric Bachelder, James R. Fuchs, Alan Douglas Kinghorn, Claudio M. Lezama-Davila, Li Pan, Abhay R. Satoskar. Invention is credited to Eric Bachelder, James R. Fuchs, Alan Douglas Kinghorn, Claudio M. Lezama-Davila, Li Pan, Abhay R. Satoskar.
Application Number | 20140287030 14/300191 |
Document ID | / |
Family ID | 47041957 |
Filed Date | 2014-09-25 |
United States Patent
Application |
20140287030 |
Kind Code |
A1 |
Satoskar; Abhay R. ; et
al. |
September 25, 2014 |
ANTILEISHMANIAL COMPOSITIONS AND METHODS OF USE
Abstract
In one aspect, the invention relates methods and compositions
for treating parasitic diseases, for example, leishmaniasis. In a
further aspect, the compounds of the methods and compositions are
isolated from Pentalinon andrieuxii. This abstract is intended as a
scanning tool for purposes of searching in the particular art and
is not intended to be limiting of the present invention.
Inventors: |
Satoskar; Abhay R.; (Upper
Arlington, OH) ; Fuchs; James R.; (Columbus, OH)
; Kinghorn; Alan Douglas; (Columbus, OH) ; Pan;
Li; (Upper Arlington, OH) ; Lezama-Davila; Claudio
M.; (Columbus, OH) ; Bachelder; Eric; (Dublin,
OH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Satoskar; Abhay R.
Fuchs; James R.
Kinghorn; Alan Douglas
Pan; Li
Lezama-Davila; Claudio M.
Bachelder; Eric |
Upper Arlington
Columbus
Columbus
Upper Arlington
Columbus
Dublin |
OH
OH
OH
OH
OH
OH |
US
US
US
US
US
US |
|
|
Assignee: |
OHIO STATE INNOVATION
FOUNDATION
Columbus
OH
|
Family ID: |
47041957 |
Appl. No.: |
14/300191 |
Filed: |
June 9, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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14113379 |
|
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PCT/US2012/034604 |
Apr 22, 2012 |
|
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14300191 |
|
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61478481 |
Apr 22, 2011 |
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Current U.S.
Class: |
424/450 ;
514/171; 514/177; 514/179; 514/182 |
Current CPC
Class: |
A61K 9/2059 20130101;
C07J 9/00 20130101; A61K 9/1271 20130101; A61K 9/1273 20130101;
Y02A 50/414 20180101; A61K 45/06 20130101; A61K 47/34 20130101;
A61K 9/5153 20130101; Y02A 50/411 20180101; A61K 9/2018 20130101;
A61K 47/40 20130101; A61K 47/44 20130101; Y02A 50/30 20180101; Y02A
50/492 20180101; A61K 9/1647 20130101; A61K 9/10 20130101; A61K
9/2054 20130101; Y02A 50/409 20180101; Y02A 50/415 20180101; A61K
31/57 20130101; C07J 7/002 20130101; A61K 31/575 20130101; A61K
9/1075 20130101; A61K 9/0019 20130101 |
Class at
Publication: |
424/450 ;
514/177; 514/171; 514/182; 514/179 |
International
Class: |
C07J 9/00 20060101
C07J009/00; A61K 31/57 20060101 A61K031/57; C07J 7/00 20060101
C07J007/00; A61K 31/575 20060101 A61K031/575; A61K 45/06 20060101
A61K045/06 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] This work was sponsored in part by the National Institutes
of Health through RO1 NIH Research Project grants (AI 076309, AT
004160, and AI 090803) and RC4 Recovery Act Limited Competition:
NIH Director's Opportunity for Research in Five Thematic Areas
grant (RC4 092624). The United States government may have certain
rights in the inventions.
Claims
1. A method for the treatment of a parasitic disease in a mammal
diagnosed with the disease, the method comprising the step of
administering to the mammal a therapeutically effective amount of
at least one compound having a structure represented by a formula:
##STR00106## wherein each ---- is independently an optional
covalent bond, wherein valence is satisfied; wherein R.sup.1, when
present, is selected from C1-C12 alkyl and C1-C12 alkenyl; wherein
R.sup.2, when present, is selected from C1-C12 alkyl and C1-C12
alkenyl; wherein R.sup.7 is selected from hydrogen, hydroxyl,
amino, and halogen; and wherein R.sup.8 is selected from hydrogen
and C1-C6 alkyl; or a pharmaceutically acceptable salt, solvate, or
polymorph thereof.
2. The method of claim 1, wherein the parasitic disease is
associated with infection of the mammal by Leishmania spp.
3. The method of claim 2, wherein Leishmania spp. is selected from
Leishmania donovani, Leishmannia brasiliensis, Leishmania mexicana,
Leishmania amazonensis, Leishmania aethiopica, Leishmania major,
Leishmania chagasi, Leishmania panamensis, Leishmania infantum, and
Leishmania tropica.
4. The method of claim 1, wherein the parasitic disease is a
leishmaniases.
5. The method of claim 4, wherein the leishmaniases is cutaneous
leishmaniasis.
6. The method of claim 4, wherein the leishmaniases is visceral
leishmaniasis.
7. The method of claim 1, wherein the mammal is human.
8. The method of claim 1, wherein R.sup.1 is selected from:
##STR00107##
9. The method of claim 1, wherein R.sup.2 is selected from:
##STR00108##
10. The method of claim 1, wherein the compound has a structure
represented by a formula: ##STR00109## wherein each of R.sup.4a and
R.sup.4b is independently selected from hydrogen and C1-C12 alkyl;
or a pharmaceutically acceptable salt, solvate, or polymorph
thereof.
11. The method of claim 1, wherein the compound has a structure
represented by a formula: ##STR00110## wherein each of R.sup.6a and
R.sup.6b is independently selected from hydrogen and C1-C12 alkyl;
or a pharmaceutically acceptable salt, solvate, or polymorph
thereof.
12. The method of claim 1, wherein the compound is selected from:
##STR00111## ##STR00112##
13. A pharmaceutical composition comprising a pharmaceutically
acceptable carrier and an effective amount of a compound
represented by a formula: ##STR00113## wherein each ---- is
independently an optional covalent bond, wherein valence is
satisfied; wherein R.sup.1, when present, is selected from C1-C12
alkyl and C1-C12 alkenyl; wherein R.sup.2, when present, is
selected from C1-C12 alkyl and C1-C12 alkenyl; wherein R.sup.7 is
selected from hydrogen, hydroxyl, amino, and halogen; and wherein
R.sup.8 is selected from hydrogen and C1-C6 alkyl; or a
pharmaceutically acceptable salt, solvate, or polymorph
thereof.
14. The composition of claim 13, wherein the pharmaceutically
acceptable carrier is a liposome.
15. The composition of claim 14, wherein the liposome comprises a
phospholipid.
16. The composition of claim 14, wherein the liposome comprises one
or more lipids selected from phosphatidylcholine, tocopherol,
cholesterol, and 1,2-distearoyl-phosphatidyl
ethanolamine-methyl-polyethyleneglycol conjugate.
17. The composition of claim 14, wherein the liposome comprises
phosphatidylcholine and tocopherol.
18. A kit comprising at least one compound represented by a
formula: ##STR00114## wherein each ---- is independently an
optional covalent bond, wherein valence is satisfied; wherein
R.sup.1, when present, is selected from C1-C12 alkyl and C1-C12
alkenyl; wherein R.sup.2, when present, is selected from C1-C12
alkyl and C1-C12 alkenyl; wherein R.sup.7 is selected from
hydrogen, hydroxyl, amino, and halogen; and wherein R.sup.8 is
selected from hydrogen and C1-C6 alkyl; or a pharmaceutically
acceptable salt, solvate, or polymorph thereof, and one or more of:
a) at least one agent known to increase the likelihood of a
parasitic disease in a mammal; b) at least one agent known to
decrease the likelihood of a parasitic disease in a mammal; c) at
least one agent known to treat a parasitic disease in a mammal; or
d) instructions for treating a parasitic disease.
19. The kit of claim 18, wherein the at least one compound and the
at least one agent are co-formulated.
20. The kit of claim 18, wherein the at least one compound and the
at least one agent are co-packaged.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Utility
application Ser. No. 14/113,379, filed Apr. 22, 2012, which claims
the benefit of U.S. Provisional Application No. 61/478,481, filed
Apr. 22, 2011, which are incorporated herein by reference in
entirety.
BACKGROUND
[0003] Leishmaniasis is a protozoan vector borne parasitic disease
caused by protozoan parasites of the genus Leishmania and is
transmitted through the bite of certain species of Phlembotominae
sandfly. Of the approximately 30 species of Leishmania known to
infect mammals, 21 of these species are believed to cause
leishmaniasis in humans. Leishmaniasis has been reported on all
continents except Australia and Antarctica, and has been found in
parts of about 88 countries. Leishmaniasis is primarily a disease
of the developing countries, and is more rare in the developed
world. However, cases have been reported in military personnel who
have served in the Persian Gulf. In the Americas, leishmaniasis can
be found in Mexico and South America, but has recently been shown
to be spreading to Texas.
[0004] Leishmaniasis manifests in three distinct forms including;
cutaneous leishmaniasis (CL), visceral leishmaniasis (VL) and
mucocutaneous leishmaniasis (ML). Of the three variants recognized
by the Centers for Disease Control and Prevention (CDC), CL and VL
are considered endemic diseases in tropical and subtropical regions
throughout the world. Leishmaniasis threatens approximately 350
million humans in nearly 90 countries. Currently, approximately 12
million humans are believed to be infected, with over 2 million new
cases being reported each year. Leishmaniasis disproportionally
affects the poorest inhabitants of the world and significantly
hinders the economic development of these developing societies.
[0005] Due to the complex life cycles of the causative parasites,
leishmaniasis is rarely diagnosed in its early stages when
therapeutic intervention is most effective. Leishmaniasis typically
presents as skin sores or ulcers which erupt weeks to months after
the person is bitten. However, if left untreated, the infection can
progress and lead to splenomegaly, liver damage, renal damage,
anemia, and death.
[0006] Therapeutic compounds containing antimony, specifically
pentavalent antimonials (e.g., meglumine antimonate and sodium
stibogluconate) were the first drugs introduced to treat
leishmaniasis, and they remain the first-line therapeutic approach
in many parts of the world. However, use of pentavalent antimonials
is associated with significant adverse effects and are administered
by intravenous or intramuscular injection. Moreover, the use of
these drugs as first-line therapy for over 50 years has resulted in
the emergence of drug-resistant parasites.
[0007] Unfortunately, despite the significant prevalence of this
disease throughout large portions of the world, there remains a
scarcity of therapeutic agents that have potent activity against
Leishmania species with minimal adverse effect on the patient and
an efficient route of administration. Therefore, there remains a
need for methods and compositions that overcome these deficiencies
and that provide a therapeutic alternative for leishmaniasis.
SUMMARY
[0008] In one aspect, the invention relates to sterol compounds
useful as therapeutic agents for the treatment of leishmaniasis and
related diseases including, but not limited to malaria, human
African trypanomiasis, babesiosis, Chagas disease,
microsporidiosis, pneumocystosis, primary ameobic
meningoenchephalitis, and toxoplasmosis.
[0009] Disclosed are methods of treatment of a parasitic disease in
a mammal diagnosed with the disease, the method comprising the step
of administering to the mammal a therapeutically effective amount
of at least one compound having a structure represented by a
formula:
##STR00001##
wherein each ---- is independently an optional covalent bond,
wherein valence is satisfied; wherein R.sup.1, when present, is
selected from C1-C12 alkyl and C1-C12 alkenyl; wherein R.sup.2,
when present, is selected from C1-C12 alkyl and C1-C12 alkenyl;
wherein R.sup.7 is selected from hydrogen, hydroxyl, amino, and
halogen; and wherein R.sup.8 is selected from hydrogen and C1-C6
alkyl; or a pharmaceutically acceptable salt, solvate, or polymorph
thereof.
[0010] Also disclosed are methods for the treatment of a parasitic
disease comprising the steps of: a) identifying a mammal in need of
treatment of a parasitic disease; and b) administering to the
mammal an effective amount of at least one compound having a
structure represented by a formula:
##STR00002##
wherein each ---- is independently an optional covalent bond,
wherein valence is satisfied; wherein R.sup.1, when present, is
selected from C1-C12 alkyl and C1-C12 alkenyl; wherein R.sup.2,
when present, is selected from C1-C12 alkyl and C1-C12 alkenyl;
wherein R.sup.7 is selected from hydrogen, hydroxyl, amino, and
halogen; and wherein R.sup.8 is selected from hydrogen and C1-C6
alkyl; or a pharmaceutically acceptable salt, solvate, or polymorph
thereof.
[0011] Also disclosed are method for treating a parasitic disease,
the method comprising the step of contacting a mammalian cell with
an effective amount of at least one compound having a structure
represented by a formula:
##STR00003##
wherein each ---- is independently an optional covalent bond,
wherein valence is satisfied; wherein R.sup.1, when present, is
selected from C1-C12 alkyl and C1-C12 alkenyl; wherein R.sup.2,
when present, is selected from C1-C12 alkyl and C1-C12 alkenyl;
wherein R.sup.7 is selected from hydrogen, hydroxyl, amino, and
halogen; and wherein R.sup.8 is selected from hydrogen and C1-C6
alkyl; or a pharmaceutically acceptable salt, solvate, or polymorph
thereof.
[0012] Also disclosed are pharmaceutical compositions comprising a
pharmaceutically acceptable carrier and an effective amount of a
compound represented by a formula:
##STR00004##
wherein each ---- is independently an optional covalent bond,
wherein valence is satisfied; wherein R.sup.1, when present, is
selected from C1-C12 alkyl and C1-C12 alkenyl; wherein R.sup.2,
when present, is selected from C1-C12 alkyl and C1-C12 alkenyl;
wherein R.sup.7 is selected from hydrogen, hydroxyl, amino, and
halogen; and wherein R.sup.8 is selected from hydrogen and C1-C6
alkyl; or a pharmaceutically acceptable salt, solvate, or polymorph
thereof.
[0013] Also disclosed are kits comprising at least one compound
represented by a formula:
##STR00005##
wherein each ---- is independently an optional covalent bond,
wherein valence is satisfied; wherein R.sup.1, when present, is
selected from C1-C12 alkyl and C1-C12 alkenyl; wherein R.sup.2,
when present, is selected from C1-C12 alkyl and C1-C12 alkenyl;
wherein R.sup.7 is selected from hydrogen, hydroxyl, amino, and
halogen; and wherein R.sup.8 is selected from hydrogen and C1-C6
alkyl; or a pharmaceutically acceptable salt, solvate, or polymorph
thereof, and one or more of: a) at least one agent known to
increase the likelihood of a parasitic disease in a mammal; b) at
least one agent known to decrease the likelihood of a parasitic
disease in a mammal; c) at least one agent know to treat a
parasitic disease in a mammal; or d) instructions for treating a
parasitic disease.
[0014] Additionally, the invention also relates to a product
comprising a disclosed compound as described herein and an
additional pharmaceutical agent, as a combined preparation for
simultaneous, separate or sequential use in the treatment or
prevention of leishmaniasis and related diseases including, but not
limited to malaria, human African trypanomiasis, babesiosis, Chagas
disease, microsporidiosis, pneumocystosis, primary ameobic
meningoenchephalitis, and toxoplasmosis.
[0015] Also disclosed are methods for manufacturing a medicament
comprising combining at least one disclosed compound, or
pharmaceutically acceptable salt, hydrate, solvate, or polymorph
thereof, with a pharmaceutically acceptable carrier or diluent.
Additionally, the invention relates to a compound as defined
herein, or pharmaceutically acceptable salt, hydrate, solvate, or
polymorph thereof, for use as a medicament, and to a compound as
defined herein for use in the treatment or in the prevention of
leishmaniasis and related diseases including, but not limited to
malaria, human African trypanomiasis, babesiosis, Chagas disease,
microsporidiosis, pneumocystosis, primary ameobic
meningoenchephalitis, and toxoplasmosis.
[0016] Also disclosed are uses of a disclosed compound, or
pharmaceutically acceptable salt, solvate, or polymorph thereof, in
the manufacture of a medicament for the treatment of leishmaniasis
and related diseases including, but not limited to malaria, human
African trypanomiasis, babesiosis, Chagas disease,
microsporidiosis, pneumocystosis, primary ameobic
meningoenchephalitis, and toxoplasmosis.
[0017] While aspects of the present invention can be described and
claimed in a particular statutory class, such as the system
statutory class, this is for convenience only and one of skill in
the art will understand that each aspect of the present invention
can be described and claimed in any statutory class. Unless
otherwise expressly stated, it is in no way intended that any
method or aspect set forth herein be construed as requiring that
its steps be performed in a specific order. Accordingly, where a
method claim does not specifically state in the claims or
descriptions that the steps are to be limited to a specific order,
it is in no way intended that an order be inferred, in any respect.
This holds for any possible non-express basis for interpretation,
including matters of logic with respect to arrangement of steps or
operational flow, plain meaning derived from grammatical
organization or punctuation, or the number or type of aspects
described in the specification.
BRIEF DESCRIPTION OF THE FIGURES
[0018] The accompanying figures, which are incorporated in and
constitute a part of this specification, illustrate several aspects
and together with the description serve to explain the principles
of the invention.
[0019] FIG. 1 shows a schematic illustrating the life cycle of
Leishmania.
[0020] FIG. 2 shows a schematic illustrating the biologically
guided extraction and isolation methods used to produce the
leishmanicidal products.
[0021] FIG. 3 shows .sup.1H-.sup.1H COSY ( ) and HMBC (.fwdarw.)
correlations observed for compounds 1 and 2.
[0022] FIG. 4 shows conformation and selected NOESY (.revreaction.)
correlations observed for compound 2.
[0023] FIG. 5 shows representative data pertaining to in vitro
antileishmanial activities of representative isolated P. andrieuxii
compounds.
[0024] FIG. 6 shows representative data pertaining to in vitro
antileishmanial activities of the isolated P. andrieuxii
compounds.
[0025] FIG. 7 shows representative data pertaining to in vitro
antileishmanial activities of the isolated P. andrieuxii
compounds.
[0026] FIG. 8 shows representative data pertaining to in vitro
antileishmanial activities of the isolated P. andrieuxii
compounds.
[0027] FIG. 9 shows representative data pertaining to in vitro
antileishmanial activities of the isolated P. andrieuxii
compounds.
[0028] FIG. 10 shows representative data pertaining to in vitro
antileishmanial activities of the isolated P. andrieuxii
compounds.
[0029] FIG. 11 shows representative data pertaining to in vitro
antileishmanial activities of the isolated P. andrieuxii
compounds.
[0030] FIG. 12 shows representative data pertaining to in vitro
antileishmanial activities of the isolated P. andrieuxii
compounds.
[0031] FIG. 13 shows data pertaining to in vivo antileishmanial
activities of the P. andrieuxii root extracts.
[0032] FIG. 14 shows representative data pertaining to in vivo
antileishmanial activities of a liposomal formulation of compound
2.
[0033] FIG. 15 shows representative data pertaining to the effect
of a liposomal formulation of compound 2 on T-cell proliferation in
an animal model of Leishmaniasis.
[0034] FIG. 16 shows representative data pertaining the effect of a
liposomal formulation of compound 2 on cytokine release in an
animal model of Leishmaniasis.
[0035] Additional advantages of the invention will be set forth in
part in the description which follows, and in part will be obvious
from the description, or can be learned by practice of the
invention. The advantages of the invention will be realized and
attained by means of the elements and combinations particularly
pointed out in the appended claims. It is to be understood that
both the foregoing general description and the following detailed
description are exemplary and explanatory only and are not
restrictive of the invention, as claimed.
DESCRIPTION
[0036] The present invention can be understood more readily by
reference to the following detailed description of the invention
and the Examples included therein.
[0037] Before the present compounds, compositions, articles,
systems, devices, and/or methods are disclosed and described, it is
to be understood that they are not limited to specific synthetic
methods unless otherwise specified, or to particular reagents
unless otherwise specified, as such may, of course, vary. It is
also to be understood that the terminology used herein is for the
purpose of describing particular aspects only and is not intended
to be limiting. Although any methods and materials similar or
equivalent to those described herein can be used in the practice or
testing of the present invention, example methods and materials are
now described.
[0038] All publications mentioned herein are incorporated herein by
reference to disclose and describe the methods and/or materials in
connection with which the publications are cited. The publications
discussed herein are provided solely for their disclosure prior to
the filing date of the present application. Nothing herein is to be
construed as an admission that the present invention is not
entitled to antedate such publication by virtue of prior invention.
Further, the dates of publication provided herein can be different
from the actual publication dates, which can require independent
confirmation.
A. DEFINITIONS
[0039] As used herein, nomenclature for compounds, including
organic compounds, can be given using common names, IUPAC, IUBMB,
or CAS recommendations for nomenclature. When one or more
stereochemical features are present, Cahn-Ingold-Prelog rules for
stereochemistry can be employed to designate stereochemical
priority, E/Z specification, and the like. One of skill in the art
can readily ascertain the structure of a compound if given a name,
either by systemic reduction of the compound structure using naming
conventions, or by commercially available software, such as
CHEMDRAW.TM. (Cambridgesoft Corporation, U.S.A.).
[0040] As used in the specification and the appended claims, the
singular forms "a," "an" and "the" include plural referents unless
the context clearly dictates otherwise. Thus, for example,
reference to "a functional group," "an alkyl," or "a residue"
includes mixtures of two or more such functional groups, alkyls, or
residues, and the like.
[0041] Ranges can be expressed herein as from "about" one
particular value, and/or to "about" another particular value. When
such a range is expressed, a further aspect includes from the one
particular value and/or to the other particular value. Similarly,
when values are expressed as approximations, by use of the
antecedent "about," it will be understood that the particular value
forms a further aspect. It will be further understood that the
endpoints of each of the ranges are significant both in relation to
the other endpoint, and independently of the other endpoint. It is
also understood that there are a number of values disclosed herein,
and that each value is also herein disclosed as "about" that
particular value in addition to the value itself. For example, if
the value "10" is disclosed, then "about 10" is also disclosed. It
is also understood that each unit between two particular units are
also disclosed. For example, if 10 and 15 are disclosed, then 11,
12, 13, and 14 are also disclosed.
[0042] References in the specification and concluding claims to
parts by weight of a particular element or component in a
composition denotes the weight relationship between the element or
component and any other elements or components in the composition
or article for which a part by weight is expressed. Thus, in a
compound containing 2 parts by weight of component X and 5 parts by
weight component Y, X and Y are present at a weight ratio of 2:5,
and are present in such ratio regardless of whether additional
components are contained in the compound.
[0043] A weight percent (wt. %) of a component, unless specifically
stated to the contrary, is based on the total weight of the
formulation or composition in which the component is included.
[0044] As used herein, the terms "optional" or "optionally" means
that the subsequently described event or circumstance can or can
not occur, and that the description includes instances where said
event or circumstance occurs and instances where it does not.
[0045] As used herein, the term "Leishmaniasis" means a protozoal
infection caused by any member of the genus Leishmania and
characterized by one or any combination of the following symptoms;
skin sores, ulcers, fever, infection, damage to the liver, damage
to the kidneys, anemia, and enlarged spleen.
[0046] As used herein, the terms "protozoa," "protozoan," and
"protozoal" can be used interchangeably and mean any member of a
diverse group of single-celled eukaryotic organisms which display
motile properties.
[0047] As used herein, the terms "antileishmanial agent,"
"antileishmanial compound," and "antileishmanial drug" can be used
interchangeably and means any molecule, therapeutic agent, or
compound that inhibits, eliminates, and/or reduces the expression
of leishmaniasis in infected mammals.
[0048] As used herein, the term "inhibit" means disinfect, inhibit,
damage, eliminate, reduce, kill, or a combination thereof.
[0049] As used herein, the term "sterol" means any molecule from a
group of predominately unsaturated solid alcohols of the steroid
group, such as cholesterol and ergosterol, present in the fatty
tissues of plants and animals.
[0050] As used herein, the term "coumarins" means any chemical
compound characterized by a benzopyrone skeleton, which are
typically found in plants. Coumarins are chemical compounds that
typically have pleasantly fragrant aromas and typically serve as
appetite suppressants when ingested.
[0051] As used herein, the term "triterpenes" means any chemical
compound comprising six isoprene units and belonging to the
oleanane subfamily.
[0052] As used herein, the term "subject" can be a vertebrate, such
as a mammal, a fish, a bird, a reptile, or an amphibian. Thus, the
subject of the herein disclosed methods can be a human, non-human
primate, horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig
or rodent. The term does not denote a particular age or sex. Thus,
adult and newborn subjects, as well as fetuses, whether male or
female, are intended to be covered. In one aspect, the subject is a
mammal. A patient refers to a subject afflicted with a disease or
disorder. The term "patient" includes human and veterinary
subjects. In some aspects of the disclosed methods, the subject has
been diagnosed with a need for treatment of leishmaniasis and
related diseases including, but not limited to malaria, human
African trypanomiasis, babesiosis, Chagas disease,
microsporidiosis, pneumocystosis, primary ameobic
meningoenchephalitis, and toxoplasmosis, prior to the administering
step. In some aspects of the disclosed method, the subject has been
diagnosed with a need for inhibiting replication, growth or
transmission of a parasite associated with leishmaniasis and
related diseases including, but not limited to malaria, human
African trypanomiasis, babesiosis, Chagas disease,
microsporidiosis, pneumocystosis, primary ameobic
meningoenchephalitis, and toxoplasmosis, prior to the administering
step.
[0053] As used herein, the term "treatment" refers to the medical
management of a patient with the intent to cure, ameliorate,
stabilize, or prevent a disease, pathological condition, or
disorder. This term includes active treatment, that is, treatment
directed specifically toward the improvement of a disease,
pathological condition, or disorder, and also includes causal
treatment, that is, treatment directed toward removal of the cause
of the associated disease, pathological condition, or disorder. In
addition, this term includes palliative treatment, that is,
treatment designed for the relief of symptoms rather than the
curing of the disease, pathological condition, or disorder;
preventative treatment, that is, treatment directed to minimizing
or partially or completely inhibiting the development of the
associated disease, pathological condition, or disorder; and
supportive treatment, that is, treatment employed to supplement
another specific therapy directed toward the improvement of the
associated disease, pathological condition, or disorder. In various
aspects, the term covers any treatment of a subject, including a
mammal (e.g., a human), and includes: (i) preventing the disease
from occurring in a subject that can be predisposed to the disease
but has not yet been diagnosed as having it; (ii) inhibiting the
disease, i.e., arresting its development; or (iii) relieving the
disease, i.e., causing regression of the disease. In one aspect,
the subject is a mammal such as a primate, and, in a further
aspect, the subject is a human. The term "subject" also includes
domesticated animals (e.g., cats, dogs, etc.), livestock (e.g.,
cattle, horses, pigs, sheep, goats, etc.), and laboratory animals
(e.g., mouse, rabbit, rat, guinea pig, fruit fly, etc.).
[0054] As used herein, the term "prevent" or "preventing" refers to
precluding, averting, obviating, forestalling, stopping, or
hindering something from happening, especially by advance action.
It is understood that where reduce, inhibit or prevent are used
herein, unless specifically indicated otherwise, the use of the
other two words is also expressly disclosed.
[0055] As used herein, the term "diagnosed" means having been
subjected to a physical examination by a person of skill, for
example, a physician, and found to have a condition that can be
diagnosed or treated by the compounds, compositions, or methods
disclosed herein. For example, "diagnosed with a disorder treatable
by a disclosed compound" means having been subjected to a physical
examination by a person of skill, for example, a physician, and
found to have a condition that can be diagnosed or treated by a
compound or composition that can inhibit replication,
proliferation, growth or transmission of a parasite associated with
leishmaniasis and related diseases including, but not limited to
malaria, human African trypanomiasis, babesiosis, Chagas disease,
microsporidiosis, pneumocystosis, primary ameobic
meningoenchephalitis, and toxoplasmosis. As a further example,
"diagnosed with a need for inhibition of a parasitic growth" refers
to having been subjected to a physical examination by a person of
skill, for example, a physician, and found to have a condition
characterized by the presence of a parasite. Such a diagnosis can
be in reference to a disorder, such as leishmaniasis and related
diseases including, but not limited to malaria, human African
trypanomiasis, babesiosis, Chagas disease, microsporidiosis,
pneumocystosis, primary ameobic meningoenchephalitis, and
toxoplasmosis, and the like, as discussed herein.
[0056] As used herein, the phrase "identified to be in need of
treatment for a disorder," or the like, refers to selection of a
subject based upon need for treatment of the disorder. For example,
a subject can be identified as having a need for treatment of a
disorder (e.g., leishmaniasis and related diseases including, but
not limited to malaria, human African trypanomiasis, babesiosis,
Chagas disease, microsporidiosis, pneumocystosis, primary ameobic
meningoenchephalitis, and toxoplasmosis) based upon an earlier
diagnosis by a person of skill and thereafter subjected to
treatment for the disorder. It is contemplated that the
identification can, in one aspect, be performed by a person
different from the person making the diagnosis. It is also
contemplated, in a further aspect, that the administration can be
performed by one who subsequently performed the administration.
[0057] As used herein, the terms "administering" and
"administration" refer to any method of providing a pharmaceutical
preparation to a subject. Such methods are well known to those
skilled in the art and include, but are not limited to, oral
administration, transdermal administration, administration by
inhalation, nasal administration, topical administration,
intravaginal administration, ophthalmic administration, intraaural
administration, intracerebral administration, rectal
administration, sublingual administration, buccal administration,
and parenteral administration, including injectable such as
intravenous administration, intra-arterial administration,
intramuscular administration, intradermal administration and
subcutaneous administration. Administration can be continuous or
intermittent. In various aspects, a preparation can be administered
therapeutically; that is, administered to treat an existing disease
or condition. In further various aspects, a preparation can be
administered prophylactically; that is, administered for prevention
of a disease or condition.
[0058] The term "contacting" as used herein refers to bringing a
disclosed compound and a cell, a target receptor, or other
biological entity together in such a manner that the compound can
affect the activity of the target (e.g., enzyme, cell, etc.),
either directly; i.e., by interacting with the target itself, or
indirectly; i.e., by interacting with another molecule, co-factor,
factor, or protein on which the activity of the target is
dependent.
[0059] As used herein, the terms "effective amount" and "amount
effective" refer to an amount that is sufficient to achieve the
desired result or to have an effect on an undesired condition. For
example, a "therapeutically effective amount" refers to an amount
that is sufficient to achieve the desired therapeutic result or to
have an effect on undesired symptoms, but is generally insufficient
to cause adverse side affects. The specific therapeutically
effective dose level for any particular patient will depend upon a
variety of factors including the disorder being treated and the
severity of the disorder; the specific composition employed; the
age, body weight, general health, sex and diet of the patient; the
time of administration; the route of administration; the rate of
excretion of the specific compound employed; the duration of the
treatment; drugs used in combination or coincidental with the
specific compound employed and like factors well known in the
medical arts. For example, it is well within the skill of the art
to start doses of a compound at levels lower than those required to
achieve the desired therapeutic effect and to gradually increase
the dosage until the desired effect is achieved. If desired, the
effective daily dose can be divided into multiple doses for
purposes of administration. Consequently, single dose compositions
can contain such amounts or submultiples thereof to make up the
daily dose. The dosage can be adjusted by the individual physician
in the event of any contraindications. Dosage can vary, and can be
administered in one or more dose administrations daily, for one or
several days. Guidance can be found in the literature for
appropriate dosages for given classes of pharmaceutical products.
In further various aspects, a preparation can be administered in a
"prophylactically effective amount"; that is, an amount effective
for prevention of a disease or condition.
[0060] As used herein, "kit" means a collection of at least two
components constituting the kit. Together, the components
constitute a functional unit for a given purpose. Individual member
components may be physically packaged together or separately. For
example, a kit comprising an instruction for using the kit may or
may not physically include the instruction with other individual
member components. Instead, the instruction can be supplied as a
separate member component, either in a paper form or an electronic
form which may be supplied on computer readable memory device or
downloaded from an internet website, or as recorded
presentation.
[0061] As used herein, "instruction(s)" means documents describing
relevant materials or methodologies pertaining to a kit. These
materials may include any combination of the following: background
information, list of components and their availability information
(purchase information, etc.), brief or detailed protocols for using
the kit, trouble-shooting, references, technical support, and any
other related documents. Instructions can be supplied with the kit
or as a separate member component, either as a paper form or an
electronic form which may be supplied on computer readable memory
device or downloaded from an internet website, or as recorded
presentation. Instructions can comprise one or multiple documents,
and are meant to include future updates.
[0062] As used herein, the terms "therapeutic agent" include any
synthetic or naturally occurring biologically active compound or
composition of matter which, when administered to an organism
(human or nonhuman animal), induces a desired pharmacologic,
immunogenic, and/or physiologic effect by local and/or systemic
action. The term therefore encompasses those compounds or chemicals
traditionally regarded as drugs, vaccines, and biopharmaceuticals
including molecules such as proteins, peptides, hormones, nucleic
acids, gene constructs and the like. Examples of therapeutic agents
are described in well-known literature references such as the Merck
Index (14 th edition), the Physicians' Desk Reference (64 th
edition), and The Pharmacological Basis of Therapeutics (12 th
edition), and they include, without limitation, medicaments;
vitamins; mineral supplements; substances used for the treatment,
prevention, diagnosis, cure or mitigation of a disease or illness;
substances that affect the structure or function of the body, or
pro-drugs, which become biologically active or more active after
they have been placed in a physiological environment. For example,
the term "therapeutic agent" includes compounds or compositions for
use in all of the major therapeutic areas including, but not
limited to, adjuvants; anti-infectives such as antibiotics and
antiviral agents; analgesics and analgesic combinations, anorexics,
anti-inflammatory agents, anti-epileptics, local and general
anesthetics, hypnotics, sedatives, antipsychotic agents,
neuroleptic agents, antidepressants, anxiolytics, antagonists,
neuron blocking agents, anticholinergic and cholinomimetic agents,
antimuscarinic and muscarinic agents, antiadrenergics,
antiarrhythmics, antihypertensive agents, hormones, and nutrients,
antiarthritics, antiasthmatic agents, anticonvulsants,
antihistamines, antinauseants, antineoplastics, antipruritics,
antipyretics; antispasmodics, cardiovascular preparations
(including calcium channel blockers, beta-blockers, beta-agonists
and antiarrythmics), antihypertensives, diuretics, vasodilators;
central nervous system stimulants; cough and cold preparations;
decongestants; diagnostics; hormones; bone growth stimulants and
bone resorption inhibitors; immunosuppressives; muscle relaxants;
psychostimulants; sedatives; tranquilizers; proteins, peptides, and
fragments thereof (whether naturally occurring, chemically
synthesized or recombinantly produced); and nucleic acid molecules
(polymeric forms of two or more nucleotides, either ribonucleotides
(RNA) or deoxyribonucleotides (DNA) including both double- and
single-stranded molecules, gene constructs, expression vectors,
antisense molecules and the like), small molecules (e.g.,
doxorubicin) and other biologically active macromolecules such as,
for example, proteins and enzymes. The agent may be a biologically
active agent used in medical, including veterinary, applications
and in agriculture, such as with plants, as well as other areas.
The term therapeutic agent also includes without limitation,
medicaments; vitamins; mineral supplements; substances used for the
treatment, prevention, diagnosis, cure or mitigation of disease or
illness; or substances which affect the structure or function of
the body; or pro-drugs, which become biologically active or more
active after they have been placed in a predetermined physiological
environment.
[0063] As used herein, "IC.sub.50," is intended to refer to the
concentration of a substance (e.g., a compound or a drug) that is
required for 50% inhibition of a biological process, or component
of a process. For example, IC.sub.50 refers to the half maximal
(50%) inhibitory concentration (IC) of a substance as determined in
a suitable assay. For example, an IC.sub.50 can be determined in an
in vitro or cell-based assay system. Frequently, cell-based assays
are used to assay efficacy of therapeutic agents directed to
leishmaniasis and trypanomiasis, e.g. an assy of promastigote
replication or amastigote infection rates of bone marrow-derived
macrophages. Alternatively, an IC.sub.50 value can be determined
from an analysis of expression levels of cytokines such as
IFN-.gamma. or IL-10 following stimulation of isolated T-cells with
L. donovani antigen in the presence of varied concentrations of
therapeutic agent.
[0064] The term "pharmaceutically acceptable" describes a material
that is not biologically or otherwise undesirable, i.e., without
causing an unacceptable level of undesirable biological effects or
interacting in a deleterious manner.
[0065] As used herein, the term "derivative" refers to a compound
having a structure derived from the structure of a parent compound
(e.g., a compound disclosed herein) and whose structure is
sufficiently similar to those disclosed herein and based upon that
similarity, would be expected by one skilled in the art to exhibit
the same or similar activities and utilities as the claimed
compounds, or to induce, as a precursor, the same or similar
activities and utilities as the claimed compounds. Exemplary
derivatives include salts, esters, amides, salts of esters or
amides, pegylated derivatives of a parent compound and N-oxides of
a parent compound.
[0066] As used herein, the term "pharmaceutically acceptable
carrier" refers to sterile aqueous or nonaqueous solutions,
dispersions, suspensions or emulsions, as well as sterile powders
for reconstitution into sterile injectable solutions or dispersions
just prior to use. Examples of suitable aqueous and nonaqueous
carriers, diluents, solvents or vehicles include water, ethanol,
polyols (such as glycerol, propylene glycol, polyethylene glycol
and the like), carboxymethylcellulose and suitable mixtures
thereof, vegetable oils (such as olive oil) and injectable organic
esters such as ethyl oleate. Proper fluidity can be maintained, for
example, by the use of coating materials such as lecithin, by the
maintenance of the required particle size in the case of
dispersions and by the use of surfactants. These compositions can
also contain adjuvants such as preservatives, wetting agents,
emulsifying agents and dispersing agents. Prevention of the action
of microorganisms can be ensured by the inclusion of various
antibacterial and antifungal agents such as paraben, chlorobutanol,
phenol, sorbic acid and the like. It can also be desirable to
include isotonic agents such as sugars, sodium chloride and the
like. Prolonged absorption of the injectable pharmaceutical form
can be brought about by the inclusion of agents, such as aluminum
monostearate and gelatin, which delay absorption. Injectable depot
forms are made by forming microencapsule matrices of the drug in
biodegradable polymers such as polylactide-polyglycolide,
poly(orthoesters), poly(anhydrides) and polyacetals. Depending upon
the ratio of drug to polymer and the nature of the particular
polymer employed, the rate of drug release can be controlled. Depot
injectable formulations are also prepared by entrapping the drug in
liposomes or microemulsions which are compatible with body tissues.
The injectable formulations can be sterilized, for example, by
filtration through a bacterial-retaining filter or by incorporating
sterilizing agents in the form of sterile solid compositions which
can be dissolved or dispersed in sterile water or other sterile
injectable media just prior to use. Alternatively, injectable
formulations can be sterilized, for example, by irradiation such as
gamma irradiation. Suitable inert carriers can include sugars such
as lactose. Desirably, at least 95% by weight of the particles of
the active ingredient have an effective particle size in the range
of 0.01 to 10 micrometers.
[0067] A residue of a chemical species, as used in the
specification and concluding claims, refers to the moiety that is
the resulting product of the chemical species in a particular
reaction scheme or subsequent formulation or chemical product,
regardless of whether the moiety is actually obtained from the
chemical species. Thus, an ethylene glycol residue in a polyester
refers to one or more --OCH.sub.2CH.sub.2O-- units in the
polyester, regardless of whether ethylene glycol was used to
prepare the polyester. Similarly, a sebacic acid residue in a
polyester refers to one or more --CO(CH.sub.2).sub.8CO-- moieties
in the polyester, regardless of whether the residue is obtained by
reacting sebacic acid or an ester thereof to obtain the
polyester.
[0068] As used herein, the term "substituted" is contemplated to
include all permissible substituents of organic compounds. In a
broad aspect, the permissible substituents include acyclic and
cyclic, branched and unbranched, carbocyclic and heterocyclic, and
aromatic and nonaromatic substituents of organic compounds.
Illustrative substituents include, for example, those described
below. The permissible substituents can be one or more and the same
or different for appropriate organic compounds. For purposes of
this disclosure, the heteroatoms, such as nitrogen, can have
hydrogen substituents and/or any permissible substituents of
organic compounds described herein which satisfy the valences of
the heteroatoms. This disclosure is not intended to be limited in
any manner by the permissible substituents of organic compounds.
Also, the terms "substitution" or "substituted with" include the
implicit proviso that such substitution is in accordance with
permitted valence of the substituted atom and the substituent, and
that the substitution results in a stable compound, e.g., a
compound that does not spontaneously undergo transformation such as
by rearrangement, cyclization, elimination, etc. It is also
contemplated that, in certain aspects, unless expressly indicated
to the contrary, individual substituents can be further optionally
substituted (i.e., further substituted or unsubstituted).
[0069] In defining various terms, "A.sup.1," "A.sup.2," "A.sup.3,"
and "A.sup.4" are used herein as generic symbols to represent
various specific substituents. These symbols can be any
substituent, not limited to those disclosed herein, and when they
are defined to be certain substituents in one instance, they can,
in another instance, be defined as some other substituents.
[0070] The term "aliphatic" or "aliphatic group," as used herein,
denotes a hydrocarbon moiety that may be straight-chain (i.e.,
unbranched), branched, or cyclic (including fused, bridging, and
spirofused polycyclic) and may be completely saturated or may
contain one or more units of unsaturation, but which is not
aromatic. Unless otherwise specified, aliphatic groups contain 1-20
carbon atoms. Aliphatic groups include, but are not limited to,
linear or branched, alkyl, alkenyl, and alkynyl groups, and hybrids
thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or
(cycloalkyl)alkenyl.
[0071] The term "alkyl" as used herein is a branched or unbranched
saturated hydrocarbon group of 1 to 24 carbon atoms, such as
methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl,
t-butyl, n-pentyl, isopentyl, s-pentyl, neopentyl, hexyl, heptyl,
octyl, nonyl, decyl, dode cyl, tetradecyl, hexadecyl, eicosyl,
tetracosyl, and the like. It is understand that the alkyl group is
acyclic. The alkyl group can be branched or unbranched. The alkyl
group can also be substituted or unsubstituted. For example, the
alkyl group can be substituted with one or more groups including,
but not limited to, alkyl, cycloalkyl, alkoxy, amino, ether,
halide, hydroxy, nitro, silyl, sulfo-oxo, or thiol, as described
herein. A "lower alkyl" group is an alkyl group containing from one
to six (e.g., from one to four) carbon atoms. The term alkyl group
can also be a C1 alkyl, C1-C2 alkyl, C1-C3 alkyl, C1-C4 alkyl,
C1-05 alkyl, C1-C6 alkyl, C1-C7 alkyl, C1-C8 alkyl, C1-C9 alkyl,
C1-C10 alkyl, and the like up to and including a C1-C24 alkyl.
[0072] Throughout the specification "alkyl" is generally used to
refer to both unsubstituted alkyl groups and substituted alkyl
groups; however, substituted alkyl groups are also specifically
referred to herein by identifying the specific substituent(s) on
the alkyl group. For example, the term "halogenated alkyl" or
"haloalkyl" specifically refers to an alkyl group that is
substituted with one or more halide, e.g., fluorine, chlorine,
bromine, or iodine. Alternatively, the term "monohaloalkyl"
specifically refers to an alkyl group that is substituted with a
single halide, e.g. fluorine, chlorine, bromine, or iodine. The
term "polyhaloalkyl" specifically refers to an alkyl group that is
independently substituted with two or more halides, i.e. each
halide substituent need not be the same halide as another halide
substituent, nor do the multiple instances of a halide substituent
need to be on the same carbon. The term "alkoxyalkyl" specifically
refers to an alkyl group that is substituted with one or more
alkoxy groups, as described below. The term "aminoalkyl"
specifically refers to an alkyl group that is substituted with one
or more amino groups. The term "hydroxyalkyl" specifically refers
to an alkyl group that is substituted with one or more hydroxy
groups. When "alkyl" is used in one instance and a specific term
such as "hydroxyalkyl" is used in another, it is not meant to imply
that the term "alkyl" does not also refer to specific terms such as
"hydroxyalkyl" and the like.
[0073] This practice is also used for other groups described
herein. That is, while a term such as "cycloalkyl" refers to both
unsubstituted and substituted cycloalkyl moieties, the substituted
moieties can, in addition, be specifically identified herein; for
example, a particular substituted cycloalkyl can be referred to as,
e.g., an "alkylcycloalkyl." Similarly, a substituted alkoxy can be
specifically referred to as, e.g., a "halogenated alkoxy," a
particular substituted alkenyl can be, e.g., an "alkenylalcohol,"
and the like. Again, the practice of using a general term, such as
"cycloalkyl," and a specific term, such as "alkylcycloalkyl," is
not meant to imply that the general term does not also include the
specific term.
[0074] The term "alkenyl" as used herein is a hydrocarbon group of
from 2 to 24 carbon atoms with a structural formula containing at
least one carbon-carbon double bond. Asymmetric structures such as
(A.sup.1A.sup.2)C.dbd.C(A.sup.3A.sup.4) are intended to include
both the E and Z isomers. This can be presumed in structural
formulae herein wherein an asymmetric alkene is present, or it can
be explicitly indicated by the bond symbol C.dbd.C. The alkenyl
group can be substituted with one or more groups including, but not
limited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl,
alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino,
carboxylic acid, ester, ether, halide, hydroxy, ketone, azide,
nitro, silyl, sulfo-oxo, or thiol, as described herein.
[0075] The term "cycloalkenyl" as used herein is a non-aromatic
carbon-based ring composed of at least three carbon atoms and
containing at least one carbon-carbon double bound, i.e., C.dbd.C.
Examples of cycloalkenyl groups include, but are not limited to,
cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadienyl,
cyclohexenyl, cyclohexadienyl, norbornenyl, and the like. The
cycloalkenyl group can be substituted or unsubstituted. The
cycloalkenyl group can be substituted with one or more groups
including, but not limited to, alkyl, cycloalkyl, alkoxy, alkenyl,
cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde,
amino, carboxylic acid, ester, ether, halide, hydroxy, ketone,
azide, nitro, silyl, sulfo-oxo, or thiol as described herein.
[0076] The term "alkynyl" as used herein is a hydrocarbon group of
2 to 24 carbon atoms with a structural formula containing at least
one carbon-carbon triple bond. The alkynyl group can be
unsubstituted or substituted with one or more groups including, but
not limited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl,
alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino,
carboxylic acid, ester, ether, halide, hydroxy, ketone, azide,
nitro, silyl, sulfo-oxo, or thiol, as described herein.
[0077] The term "cycloalkynyl" as used herein is a non-aromatic
carbon-based ring composed of at least seven carbon atoms and
containing at least one carbon-carbon triple bound. Examples of
cycloalkynyl groups include, but are not limited to, cycloheptynyl,
cyclooctynyl, cyclononynyl, and the like. The cycloalkynyl group
can be substituted or unsubstituted. The cycloalkynyl group can be
substituted with one or more groups including, but not limited to,
alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl,
cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid,
ester, ether, halide, hydroxy, ketone, azide, nitro, silyl,
sulfo-oxo, or thiol as described herein.
[0078] The term "aromatic group" as used herein refers to a ring
structure having cyclic clouds of delocalized .pi. electrons above
and below the plane of the molecule, where the .pi. clouds contain
(4n+2) .pi. electrons. A further discussion of aromaticity is found
in Morrison and Boyd, Organic Chemistry, (5th Ed., 1987), Chapter
13, entitled "Aromaticity," pages 477-497, incorporated herein by
reference. The term "aromatic group" is inclusive of both aryl and
heteroaryl groups.
[0079] The term "aryl" as used herein is a group that contains any
carbon-based aromatic group including, but not limited to, benzene,
naphthalene, phenyl, biphenyl, anthracene, and the like. The aryl
group can be substituted or unsubstituted. The aryl group can be
substituted with one or more groups including, but not limited to,
alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl,
cycloalkynyl, aryl, heteroaryl, aldehyde, --NH.sub.2, carboxylic
acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl,
sulfo-oxo, or thiol as described herein. The term "biaryl" is a
specific type of aryl group and is included in the definition of
"aryl." In addition, the aryl group can be a single ring structure
or comprise multiple ring structures that are either fused ring
structures or attached via one or more bridging groups such as a
carbon-carbon bond. For example, biaryl refers to two aryl groups
that are bound together via a fused ring structure, as in
naphthalene, or are attached via one or more carbon-carbon bonds,
as in biphenyl.
[0080] The term "aldehyde" as used herein is represented by the
formula --C(O)H. Throughout this specification "C(O)" is a short
hand notation for a carbonyl group, i.e., C.dbd.O.
[0081] The terms "amine" or "amino" as used herein are represented
by the formula --NA.sup.1A.sup.2, where A.sup.1 and A.sup.2 can be,
independently, hydrogen or alkyl, cycloalkyl, alkenyl,
cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as
described herein. A specific example of amino is --NH.sub.2.
[0082] The terms "halo," "halogen," or "halide," as used herein can
be used interchangeably and refer to F, Cl, Br, or I.
[0083] The terms "pseudohalide," "pseudohalogen" or "pseudohalo,"
as used herein can be used interchangeably and refer to functional
groups that behave substantially similar to halides. Such
functional groups include, by way of example, cyano, thiocyanato,
azido, trifluoromethyl, trifluoromethoxy, perfluoroalkyl, and
perfluoroalkoxy groups.
[0084] The term "hydroxyl" or "hydroxy" as used herein is
represented by the formula --OH.
[0085] "R.sup.1," "R.sup.2," "R.sup.3," "R.sup.n," where n is an
integer, as used herein can, independently, possess one or more of
the groups listed above. For example, if R.sup.1 is a straight
chain alkyl group, one of the hydrogen atoms of the alkyl group can
optionally be substituted with a hydroxyl group, an alkoxy group,
an alkyl group, a halide, and the like. Depending upon the groups
that are selected, a first group can be incorporated within second
group or, alternatively, the first group can be pendant (i.e.,
attached) to the second group. For example, with the phrase "an
alkyl group comprising an amino group," the amino group can be
incorporated within the backbone of the alkyl group. Alternatively,
the amino group can be attached to the backbone of the alkyl group.
The nature of the group(s) that is (are) selected will determine if
the first group is embedded or attached to the second group.
[0086] As described herein, compounds of the invention may contain
"optionally substituted" moieties. In general, the term
"substituted," whether preceded by the term "optionally" or not,
means that one or more hydrogens of the designated moiety are
replaced with a suitable substituent. Unless otherwise indicated,
an "optionally substituted" group may have a suitable substituent
at each substitutable position of the group, and when more than one
position in any given structure may be substituted with more than
one substituent selected from a specified group, the substituent
may be either the same or different at every position. Combinations
of substituents envisioned by this invention are preferably those
that result in the formation of stable or chemically feasible
compounds. In is also contemplated that, in certain aspects, unless
expressly indicated to the contrary, individual substituents can be
further optionally substituted (i.e., further substituted or
unsubstituted).
[0087] The term "stable," as used herein, refers to compounds that
are not substantially altered when subjected to conditions to allow
for their production, detection, and, in certain aspects, their
recovery, purification, and use for one or more of the purposes
disclosed herein.
[0088] The term "organic residue" defines a carbon containing
residue, i.e., a residue comprising at least one carbon atom, and
includes but is not limited to the carbon-containing groups,
residues, or radicals defined hereinabove. Organic residues can
contain various heteroatoms, or be bonded to another molecule
through a heteroatom, including oxygen, nitrogen, sulfur,
phosphorus, or the like. Examples of organic residues include but
are not limited alkyl or substituted alkyls, alkoxy or substituted
alkoxy, mono or di-substituted amino, amide groups, etc. Organic
residues can preferably comprise 1 to 18 carbon atoms, 1 to 15,
carbon atoms, 1 to 12 carbon atoms, 1 to 8 carbon atoms, 1 to 6
carbon atoms, or 1 to 4 carbon atoms. In a further aspect, an
organic residue can comprise 2 to 18 carbon atoms, 2 to 15, carbon
atoms, 2 to 12 carbon atoms, 2 to 8 carbon atoms, 2 to 4 carbon
atoms, or 2 to 4 carbon atoms.
[0089] A very close synonym of the term "residue" is the term
"radical," which as used in the specification and concluding
claims, refers to a fragment, group, or substructure of a molecule
described herein, regardless of how the molecule is prepared. In
some embodiments the radical (for example an alkyl) can be further
modified (i.e., substituted alkyl) by having bonded thereto one or
more "substituent radicals." The number of atoms in a given radical
is not critical to the present invention unless it is indicated to
the contrary elsewhere herein.
[0090] "Organic radicals," as the term is defined and used herein,
contain one or more carbon atoms. An organic radical can have, for
example, 1-26 carbon atoms, 1-18 carbon atoms, 1-12 carbon atoms,
1-8 carbon atoms, 1-6 carbon atoms, or 1-4 carbon atoms. In a
further aspect, an organic radical can have 2-26 carbon atoms, 2-18
carbon atoms, 2-12 carbon atoms, 2-8 carbon atoms, 2-6 carbon
atoms, or 2-4 carbon atoms. Organic radicals often have hydrogen
bound to at least some of the carbon atoms of the organic radical.
One example, of an organic radical that comprises no inorganic
atoms is a 5,6,7,8-tetrahydro-2-naphthyl radical. In some
embodiments, an organic radical can contain 1-10 inorganic
heteroatoms bound thereto or therein, including halogens, oxygen,
sulfur, nitrogen, phosphorus, and the like. Examples of organic
radicals include but are not limited to an alkyl, substituted
alkyl, cycloalkyl, substituted cycloalkyl, mono-substituted amino,
di-substituted amino, acyloxy, cyano, carboxy, carboalkoxy,
alkylcarboxamide, substituted alkylcarboxamide, dialkylcarboxamide,
substituted dialkylcarboxamide, alkylsulfonyl, alkylsulfinyl,
thioalkyl, thiohaloalkyl, alkoxy, substituted alkoxy, haloalkyl,
haloalkoxy, aryl, substituted aryl, heteroaryl, heterocyclic, or
substituted heterocyclic radicals, wherein the terms are defined
elsewhere herein. A few non-limiting examples of organic radicals
that include heteroatoms include alkoxy radicals, trifluoromethoxy
radicals, acetoxy radicals, dimethylamino radicals and the
like.
[0091] Compounds described herein can contain one or more double
bonds and, thus, potentially give rise to cis/trans (E/Z) isomers,
as well as other conformational isomers. Unless stated to the
contrary, the invention includes all such possible isomers, as well
as mixtures of such isomers.
[0092] Unless stated to the contrary, a formula with chemical bonds
shown only as solid lines and not as wedges or dashed lines
contemplates each possible isomer, e.g., each enantiomer and
diastereomer, and a mixture of isomers, such as a racemic or
scalemic mixture. Compounds described herein can contain one or
more asymmetric centers and, thus, potentially give rise to
diastereomers and optical isomers. Unless stated to the contrary,
the present invention includes all such possible diastereomers as
well as their racemic mixtures, their substantially pure resolved
enantiomers, all possible geometric isomers, and pharmaceutically
acceptable salts thereof. Mixtures of stereoisomers, as well as
isolated specific stereoisomers, are also included. During the
course of the synthetic procedures used to prepare such compounds,
or in using racemization or epimerization procedures known to those
skilled in the art, the products of such procedures can be a
mixture of stereoisomers.
[0093] Many organic compounds exist in optically active forms
having the ability to rotate the plane of plane-polarized light. In
describing an optically active compound, the prefixes D and L or R
and S are used to denote the absolute configuration of the molecule
about its chiral center(s). The prefixes d and 1 or (+) and (-) are
employed to designate the sign of rotation of plane-polarized light
by the compound. For example, a compound prefixed with (-) or 1
meaning that the compound is levorotatory or a compound prefixed
with (+) or d is dextrorotatory. For a given chemical structure,
these compounds, called stereoisomers, are identical except that
they are non-superimposable mirror images of one another. A
specific stereoisomer can also be referred to as an enantiomer, and
a mixture of such isomers is often called an enantiomeric mixture.
A 50:50 mixture of enantiomers is referred to as a racemic mixture.
Many of the compounds described herein can have one or more chiral
centers and therefore can exist in different enantiomeric forms. If
desired, a chiral carbon can be designated with an asterisk (*).
When bonds to the chiral carbon are depicted as straight lines in
the disclosed formulas, it is understood that both the (R) and (S)
configurations of the chiral carbon, and hence both enantiomers and
mixtures thereof, are embraced within the formula. As is used in
the art, when it is desired to specify the absolute configuration
about a chiral carbon, one of the bonds to the chiral carbon can be
depicted as a wedge (bonds to atoms above the plane) and the other
can be depicted as a series or wedge of short parallel lines is
(bonds to atoms below the plane). The Cahn-Inglod-Prelog system can
be used to assign the (R) or (S) configuration to a chiral
carbon.
[0094] Compounds described herein comprise atoms in both their
natural isotopic abundance and in non-natural abundance. The
disclosed compounds can be isotopically-labelled or
isotopically-substituted compounds identical to those described,
but for the fact that one or more atoms are replaced by an atom
having an atomic mass or mass number different from the atomic mass
or mass number typically found in nature. Examples of isotopes that
can be incorporated into compounds of the invention include
isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous,
fluorine and chlorine, such as .sup.2H, .sup.3H, .sup.13C,
.sup.14C, .sup.15N, .sup.18O, .sup.17O, .sup.35S, .sup.18F and
.sup.36Cl, respectively. Compounds further comprise prodrugs
thereof, and pharmaceutically acceptable salts of said compounds or
of said prodrugs which contain the aforementioned isotopes and/or
other isotopes of other atoms are within the scope of this
invention. Certain isotopically-labelled compounds of the present
invention, for example those into which radioactive isotopes such
as .sup.3H and .sup.14C are incorporated, are useful in drug and/or
substrate tissue distribution assays. Tritiated, i.e., .sup.3H, and
carbon-14, i.e., .sup.14C, isotopes are particularly preferred for
their ease of preparation and detectability. Further, substitution
with heavier isotopes such as deuterium, i.e., .sup.2H, can afford
certain therapeutic advantages resulting from greater metabolic
stability, for example increased in vivo half-life or reduced
dosage requirements and, hence, may be preferred in some
circumstances. Isotopically labelled compounds of the present
invention and prodrugs thereof can generally be prepared by
carrying out the procedures below, by substituting a readily
available isotopically labelled reagent for a non-isotopically
labelled reagent.
[0095] The compounds described in the invention can be present as a
solvate. In some cases, the solvent used to prepare the solvate is
an aqueous solution, and the solvate is then often referred to as a
hydrate. The compounds can be present as a hydrate, which can be
obtained, for example, by crystallization from a solvent or from
aqueous solution. In this connection, one, two, three or any
arbitrary number of solvent or water molecules can combine with the
compounds according to the invention to form solvates and hydrates.
Unless stated to the contrary, the invention includes all such
possible solvates.
[0096] The term "co-crystal" means a physical association of two or
more molecules which owe their stability through non-covalent
interaction. One or more components of this molecular complex
provide a stable framework in the crystalline lattice. In certain
instances, the guest molecules are incorporated in the crystalline
lattice as anhydrates or solvates, see e.g. "Crystal Engineering of
the Composition of Pharmaceutical Phases. Do Pharmaceutical
Co-crystals Represent a New Path to Improved Medicines?"
Almarasson, O., et. al., The Royal Society of Chemistry, 1889-1896,
2004. Examples of co-crystals include p-toluenesulfonic acid and
benzenesulfonic acid.
[0097] It is known that chemical substances form solids which are
present in different states of order which are termed polymorphic
forms or modifications. The different modifications of a
polymorphic substance can differ greatly in their physical
properties. The compounds according to the invention can be present
in different polymorphic forms, with it being possible for
particular modifications to be metastable. Unless stated to the
contrary, the invention includes all such possible polymorphic
forms.
[0098] In some aspects, a structure of a compound can be
represented by a formula:
##STR00006##
[0099] which is understood to be equivalent to a formula:
##STR00007##
wherein n is typically an integer. That is, R.sup.n is understood
to represent five independent substituents, R.sup.n(a), R.sup.n(b),
R.sup.n(c), R.sup.n(d), R.sup.n(e). By "independent substituents,"
it is meant that each R substituent can be independently defined.
For example, if in one instance R.sup.n(a) is halogen, then
R.sup.n(b) is not necessarily halogen in that instance.
[0100] Certain materials, compounds, compositions, and components
disclosed herein can be obtained commercially or readily
synthesized using techniques generally known to those of skill in
the art. For example, the starting materials and reagents used in
preparing the disclosed compounds and compositions are either
available from commercial suppliers such as Aldrich Chemical Co.,
(Milwaukee, Wis.), Acros Organics (Morris Plains, N.J.), Fisher
Scientific (Pittsburgh, Pa.), or Sigma (St. Louis, Mo.) or are
prepared by methods known to those skilled in the art following
procedures set forth in references such as Fieser and Fieser's
Reagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons,
1991); Rodd's Chemistry of Carbon Compounds, Volumes 1-5 and
Supplementals (Elsevier Science Publishers, 1989); Organic
Reactions, Volumes 1-40 (John Wiley and Sons, 1991); March's
Advanced Organic Chemistry, (John Wiley and Sons, 4th Edition); and
Larock's Comprehensive Organic Transformations (VCH Publishers
Inc., 1989).
[0101] Unless otherwise expressly stated, it is in no way intended
that any method set forth herein be construed as requiring that its
steps be performed in a specific order. Accordingly, where a method
claim does not actually recite an order to be followed by its steps
or it is not otherwise specifically stated in the claims or
descriptions that the steps are to be limited to a specific order,
it is no way intended that an order be inferred, in any respect.
This holds for any possible non-express basis for interpretation,
including: matters of logic with respect to arrangement of steps or
operational flow; plain meaning derived from grammatical
organization or punctuation; and the number or type of embodiments
described in the specification.
[0102] Disclosed are the components to be used to prepare the
compositions of the invention as well as the compositions
themselves to be used within the methods disclosed herein. These
and other materials are disclosed herein, and it is understood that
when combinations, subsets, interactions, groups, etc. of these
materials are disclosed that while specific reference of each
various individual and collective combinations and permutation of
these compounds can not be explicitly disclosed, each is
specifically contemplated and described herein. For example, if a
particular compound is disclosed and discussed and a number of
modifications that can be made to a number of molecules including
the compounds are discussed, specifically contemplated is each and
every combination and permutation of the compound and the
modifications that are possible unless specifically indicated to
the contrary. Thus, if a class of molecules A, B, and C are
disclosed as well as a class of molecules D, E, and F and an
example of a combination molecule, A-D is disclosed, then even if
each is not individually recited each is individually and
collectively contemplated meaning combinations, A-E, A-F, B-D, B-E,
B-F, C-D, C-E, and C-F are considered disclosed. Likewise, any
subset or combination of these is also disclosed. Thus, for
example, the sub-group of A-E, B-F, and C-E would be considered
disclosed. This concept applies to all aspects of this application
including, but not limited to, steps in methods of making and using
the compositions of the invention. Thus, if there are a variety of
additional steps that can be performed it is understood that each
of these additional steps can be performed with any specific
embodiment or combination of embodiments of the methods of the
invention.
[0103] It is understood that the compositions disclosed herein have
certain functions. Disclosed herein are certain structural
requirements for performing the disclosed functions, and it is
understood that there are a variety of structures that can perform
the same function that are related to the disclosed structures, and
that these structures will typically achieve the same result.
B. COMPOUNDS
[0104] In one aspect, the invention relates to compounds useful as
therapeutic agents for the treatment of leishmaniasis and related
diseases including, but not limited to malaria, human African
trypanomiasis, babesiosis, Chagas disease, microsporidiosis,
pneumocystosis, primary ameobic meningoenchephalitis, and
toxoplasmosis.
[0105] In various aspects, the invention pertains to a new
cholesterol derivative, pentalinonsterol (1), and a new
polyoxygenated pregnane sterol glycoside, pentalinonoside (2),
together with 18 known compounds, including 14 sterols (3-16),
three coumarins (17-19), and a triterpene (20), were isolated from
the roots of Pentalinon andrieuxii. Isolated and purified compounds
were evaluated in vitro for their antileishmanial activity. Among
these compounds, 6,7-dihydroneridienone (15), a known C-21 sterol,
was found to be potent against promastigotes of L. mexicana. The
new cholesterol analogue, pentalinonsterol
(cholest-4,20,24-trien-3-one, 1), together with other two known
sterols, 24-methylcholest-4,24(28)-dien-3-one (3) and neridienone
(16), also exhibited significant leishmanicidal activity. The
intracellular parasites treated with compounds 1, 3, 4, 15, and 16
were further studied by electron microscopy, and morphological
abnormalities and destruction of the amastigotes were observed, as
a result of the treatment with these compounds.
[0106] 1. Structure
[0107] In one aspect, the present invention pertains to compounds
having a structure represented by a formula:
##STR00008##
wherein each ---- is independently an optional covalent bond,
wherein valence is satisfied; wherein R'' is selected from the
group consisting of H, alkyl, alkenyl, aryl, and heteroaryl;
wherein R.sup.12 is selected from the group consisting of
.dbd.CH.sub.2, .dbd.O, and --CH.sub.3; R.sup.13 is selected from
the group consisting of hydrogen, hydroxyl, amino, halogen, and
keto; and wherein R.sup.14 is selected from the group consisting of
hydrogen, hydroxyl, amino, halogen, and keto; or a pharmaceutically
acceptable salt, solvate, or polymorph thereof.
[0108] In a further aspect, the present invention pertains to
compound (1):
##STR00009##
Compound 1 is also referred to herein as pentalinonsterol or
alternatively as
(3S,8S,9S,10R,13S,14S,17R)-10,13-dimethyl-17-(6-methylhepta-1,5-dien-2-yl-
)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phena-
nthren-3-ol, or a pharmaceutically acceptable salt, solvate, or
polymorph thereof.
[0109] In one aspect, the present invention pertains to compounds
having a structure represented by a formula:
##STR00010##
wherein R.sup.21 is selected from the group consisting of H, alkyl,
alkenyl, aryl, heteroaryl, silyl, and sugar; wherein R.sup.22 is
selected from the group consisting of hydrogen, hydroxyl, and keto;
and wherein R.sup.23 is selected from the group consisting of
hydrogen, hydroxyl, and keto; or a pharmaceutically acceptable
salt, solvate, or polymorph thereof.
[0110] In a further aspect, the present invention pertains to
compound (2):
##STR00011##
Compound 2 is a new polyoxygenated pregnane sterol glycoside, and
is also referred to herein as pentalinonoside or alternatively as
(2R,3S,4R,6R)-6-(((1S,3aS,5aR,5bR,9S,11aR,11bS,13aR,13bS)-11a,13a-dimethy-
l-1,2,3a,4,5b,6,8,9,10,11,11a,11b,12,13,13a,13b-hexadecahydro-1,5a-epoxyfu-
ro[2,3-c]naphtho[1,2-h]chromen-9-yl)oxy)-4-methoxy-2-methyltetrahydro-2H-p-
yran-3-ol, or a pharmaceutically acceptable salt, solvate, or
polymorph thereof
[0111] In one aspect, the invention relates to a compound having a
structure represented by a formula:
##STR00012##
wherein R.sup.1 is selected from C1-C12 alkyl and C1-C12 alkenyl;
or a pharmaceutically acceptable salt, solvate, or polymorph
thereof. In a further aspect, R.sup.1 is C1-C12 alkyl. In a still
further aspect, R.sup.1 is C1-C12 alkenyl. In a yet further aspect,
R.sup.1 is C8-C12 alkenyl. In a yet further aspect, R.sup.1 is
C8-C11 alkenyl. In an even further aspect, R.sup.1 is C8-C10
alkenyl. In a still further aspect, R.sup.1 is C8-C9 alkenyl. In a
yet further aspect, R.sup.1 is C8 alkenyl. In an even further
aspect, R.sup.1 is selected from:
##STR00013##
In a still further aspect, R.sup.1 is selected from:
##STR00014##
[0112] In a further aspect, the invention relates to a compound
having a structure represented by a formula:
##STR00015##
wherein R.sup.2 is selected from C1-C12 alkyl and C1-C12 alkenyl;
or a pharmaceutically acceptable salt, solvate, or polymorph
thereof. In a further aspect, R.sup.2 is C1-C12 alkyl. In a still
further aspect, R.sup.2 is C1-C12 alkenyl. In a yet further aspect,
R.sup.2 is C8-C12 alkenyl. In a yet further aspect, R.sup.2 is
C8-C11 alkenyl. In an even further aspect, R.sup.2 is C8-C10
alkenyl. In a still further aspect, R.sup.2 is C8-C9 alkenyl. In a
yet further aspect, R.sup.2 is C8 alkenyl. In an even further
aspect, R.sup.2 is selected from:
##STR00016##
In a still further aspect, R.sup.2 is:
##STR00017##
[0113] In one aspect, the invention relates to a compound having a
structure represented by a formula:
##STR00018##
wherein R.sup.3 is selected from C1-C8 alkyl and C1-C8 alkenyl; or
a pharmaceutically acceptable salt, solvate, or polymorph thereof.
In a further aspect, R.sup.3 is C1-C8 alkyl. In a still further
aspect, R.sup.3 is C1-C8 alkenyl. In a yet further aspect, R.sup.3
is C4-C8 alkenyl. In a yet further aspect, R.sup.3 is C5-C7
alkenyl. In an even further aspect, R.sup.3 is C6 alkenyl.
[0114] In one aspect, the invention relates to a compound having a
structure represented by a formula:
##STR00019##
wherein R.sup.3 is selected from C1-C12 alkyl and C1-C12 alkenyl;
or a pharmaceutically acceptable salt, solvate, or polymorph
thereof. In a further aspect, R.sup.3 is C1-C8 alkyl. In a still
further aspect, R.sup.3 is C1-C8 alkenyl. In a yet further aspect,
R.sup.3 is C4-C8 alkenyl. In a yet further aspect, R.sup.3 is C5-C7
alkenyl. In an even further aspect, R.sup.3 is C6 alkenyl.
[0115] In one aspect, the invention relates to a compound having a
structure represented by a formula:
##STR00020##
wherein each of R.sup.4a and R.sup.4b is independently selected
from hydrogen and C1-C12 alkyl; or a pharmaceutically acceptable
salt, solvate, or polymorph thereof. In a further aspect, each of
R.sup.4a and R.sup.4b is independently selected from hydrogen,
methyl, ethyl, propyl, and isopropyl. In a further aspect, each of
R.sup.4a and R.sup.4b is independently selected from hydrogen,
methyl, and ethyl. In a yet further aspect, each of R.sup.4a and
R.sup.4b is independently selected from hydrogen and methyl.
[0116] In a further aspect, the invention relates to a compound
having a structure represented by a formula:
##STR00021##
wherein R.sup.5 is selected from C1-C8 alkyl and C1-C8 alkenyl; or
a pharmaceutically acceptable salt, solvate, or polymorph thereof.
In a further aspect, R.sup.5 is C1-C8 alkyl. In a still further
aspect, R.sup.5 is C1-C8 alkenyl. In a yet further aspect, R.sup.5
is C4-C8 alkenyl. In a yet further aspect, R.sup.5 is C5-C7
alkenyl. In an even further aspect, R.sup.5 is C6 alkenyl.
[0117] In a further aspect, the invention relates to a compound
having a structure represented by a formula:
##STR00022##
wherein R.sup.5 is selected from C1-C8 alkyl and C1-C8 alkenyl; or
a pharmaceutically acceptable salt, solvate, or polymorph thereof.
In a further aspect, R.sup.5 is C1-C8 alkyl. In a still further
aspect, R.sup.5 is C1-C8 alkenyl. In a yet further aspect, R.sup.5
is C4-C8 alkenyl. In a yet further aspect, R.sup.5 is C5-C7
alkenyl. In an even further aspect, R.sup.5 is C6 alkenyl.
[0118] In a further aspect, the invention relates to a compound
having a structure represented by a formula:
##STR00023##
wherein each of R.sup.6a and R.sup.6b is independently selected
from hydrogen and C1-C12 alkyl; or a pharmaceutically acceptable
salt, solvate, or polymorph thereof. In a further aspect, each of
R.sup.6a and R.sup.6b is independently selected from hydrogen,
methyl, ethyl, propyl, and isopropyl. In a further aspect, each of
R.sup.6a and R.sup.6b is independently selected from hydrogen,
methyl, and ethyl. In a yet further aspect, each of R.sup.6a and
R.sup.6b is independently selected from hydrogen and methyl.
[0119] In a further aspect, the invention relates to a compound
having a structure represented by a formula:
##STR00024##
wherein each ---- is independently an optional covalent bond,
wherein valence is satisfied; wherein R.sup.7 is selected from
hydrogen, hydroxyl, amino, and halogen; and wherein R.sup.8 is
selected from hydrogen and C1-C6 alkyl; or a pharmaceutically
acceptable salt, solvate, or polymorph thereof. In a still further
aspect, R.sup.7 is hydroxyl. In a yet further aspect, R.sup.8 is
selected from hydrogen, methyl, ethyl, propyl, and isopropyl. In an
even further aspect, R.sup.8 is methyl. In a still further aspect,
R.sup.7 is hydroxyl and R.sup.8 is methyl.
[0120] In a further aspect, the invention relates to a compound
having a structure represented by a formula:
##STR00025##
wherein ---- is an optional covalent bond, wherein valence is
satisfied; wherein R.sup.7 is selected from hydrogen, hydroxyl,
amino, and halogen; and wherein R.sup.8 is selected from hydrogen
and C1-C6 alkyl; or a pharmaceutically acceptable salt, solvate, or
polymorph thereof.
[0121] In a further aspect, the invention relates to a compound
having a structure represented by a formula:
##STR00026##
wherein R.sup.7 is selected from hydrogen, hydroxyl, amino, and
halogen; and wherein R.sup.8 is selected from hydrogen and C1-C6
alkyl; or a pharmaceutically acceptable salt, solvate, or polymorph
thereof.
[0122] In a further aspect, the invention relates to a compound
having a structure represented by a formula:
##STR00027##
wherein R.sup.7 is selected from hydrogen, hydroxyl, amino, and
halogen; and wherein R.sup.8 is selected from hydrogen and C1-C6
alkyl; or a pharmaceutically acceptable salt, solvate, or polymorph
thereof.
[0123] In a further aspect, the invention relates to a compound
having a structure represented by a formula:
##STR00028##
wherein R.sup.7 is selected from hydrogen, hydroxyl, amino, and
halogen; and wherein R.sup.8 is selected from hydrogen and C1-C6
alkyl; or a pharmaceutically acceptable salt, solvate, or polymorph
thereof.
[0124] In various aspects, disclosed herein is the isolation and
structure elucidation of new sterols 1 and 2, as well as the in
vitro leishmanicidal activity of all isolates obtained when
evaluated against promastigotes and amastigotes of L. mexicana.
[0125] In a further aspect, disclosed herein is a new cholesterol
derivative, pentalinonsterol (1), and a new polyoxygenated pregnane
sterol glycoside, pentalinonoside [(2), FIG. 1].
[0126] In a further aspect, disclosed herein is the spectroscopic
characterization (FIGS. 3 and 4; Table 4) and biological evaluation
of 1-20 (Table 5) in an in vitro model of Leishmania mexicana for
leishmaniasis.
[0127] 2. Example Compounds
[0128] In one aspect, a compound can be present as:
##STR00029## ##STR00030## ##STR00031## ##STR00032##
or a subgroup thereof.
[0129] In one aspect, a compound can be present as:
##STR00033## ##STR00034## ##STR00035##
or a subgroup thereof.
[0130] In one aspect, a compound can be present as:
##STR00036## ##STR00037## ##STR00038##
or a subgroup thereof.
[0131] In one aspect, a compound can be present as:
##STR00039## ##STR00040## ##STR00041## ##STR00042##
or a subgroup thereof
[0132] In one aspect, a compound can be present as:
##STR00043##
or a subgroup thereof
[0133] In one aspect, a compound can be present as:
##STR00044## ##STR00045## ##STR00046##
or a subgroup thereof.
[0134] In one aspect, a compound can be present as:
##STR00047##
or a subgroup thereof
[0135] In one aspect, a compound can be present as:
##STR00048## ##STR00049##
or a subgroup thereof
[0136] In one aspect, a compound can be present as:
##STR00050##
or a subgroup thereof
[0137] In one aspect, a compound can be present as:
##STR00051##
or a subgroup thereof
[0138] In one aspect, a compound can be present as:
##STR00052##
or a subgroup thereof
[0139] In one aspect, a compound can be present as:
##STR00053##
[0140] In one aspect, a compound can be present as:
##STR00054##
or a subgroup thereof
[0141] In one aspect, a compound can be present as:
##STR00055##
or a subgroup thereof
[0142] In one aspect, a compound can be present as:
##STR00056##
[0143] In one aspect, a compound can be present as:
##STR00057##
or a subgroup thereof.
[0144] In one aspect, a compound can be present as:
##STR00058##
[0145] In one aspect, a compound can be present as:
##STR00059##
[0146] In one aspect, a compound can be present as:
##STR00060##
[0147] In one aspect, a compound can be present as:
##STR00061##
[0148] In one aspect, a compound can be present as:
##STR00062##
[0149] In one aspect, a compound can be present as:
##STR00063##
[0150] In one aspect, a compound can be present as:
##STR00064##
[0151] In one aspect, a compound can be present as:
##STR00065##
[0152] In one aspect, a compound can be present as:
##STR00066##
[0153] In one aspect, a compound can be present as:
##STR00067##
[0154] It is contemplated that one or more compounds can optionally
be omitted from the disclosed invention.
C. METHODS OF ISOLATING THE COMPOUNDS FROM P. ANDRIEUXII
[0155] Pentalinon andrieuxii [(Muell.-Arg.); syn.: Urechites
andrieuxii (B. F. Hansen & Wunderlin); Apocynaceae
(Muell.-Arg.)], is a native plant in the Yucatan Peninsula of
Mexico known as "bejuco guaco", "cantibteac", or "contrayerba".
This plant is used in Mayan folk medicine to treat cutaneous
leishmaniasis lesions (Chiclero's ulcer) in the states of Campeche
and Quintana Roo, Mexico (Pulido, M. T.; Serralta, L. Centro de
Investigaciones de Quintana Roo: Chetumal, Quintana Roo, Mexico,
1993, 6; Argueta, A.; Cano, L.; Rodarte, M. Instituto Nacional
Indigenista: Mexico, D. F., 2, 204). Treatment of Chiclero's ulcer
in Mayan traditional medicine uses the innermost part of the roots,
which is fixed tightly to the skin lesions. This procedure is
repeated each day after careful cleaning of lesions with an
infusion of the roots, until visible healing is observed. In
addition, this plant may be used also to treat snakebites as well
as to alleviate headaches and nervous disturbances (Pulido and
Serralta, op. cit.; Argueta, et al., op. cit.). Previous biological
studies on extracts of this plant have led to reports of
antiatherogenic, anti-inflammatory, antileishmanial, and depressant
activities (Jiu, J. Lloydia 1966, 29, 250; Lezama-Davila, C. M.;
Isaac-Marquez, A. P. Muell.-Arg. Divulg. Biomed., 1994, 2, 13;
Chan-Bacab, M. J.; Balanza, E.; Deharo, E.; Munoz, V.;
Duran-Garcia, R.; Pena-Rodriguez, L. M. Journal of
Ethnopharmacology 2003, 86, 243; and Lezama-Davila, C. M.;
Isaac-Marquez, A. P.; Zamora-Crescencio, P.; c-Encalada, M. R.;
Justiniano-Apolinar, S. Y.; Angel-Robles, R.; Satoskar, A.;
Hernandez-Rivero, L. Fitoterapia 2007, 78, 255).
[0156] Among these activities, the antileishmanial effect of this
plant is of great interest. It has been reported that both aqueous
and organic-solvent soluble extractives of P. andrieuxii roots
showed in vitro antileishmanial activity, and the most active
extract was ascribed to the hexane-soluble partition. A 10 .mu.g/mL
extract was effective in killing 1 million L. mexicana
promastigotes cultured in vitro, with even more potent
leishmanicidal activity than that of the control compound,
meglumine antimoniate (Lezama-Davila, C. M.; Isaac-Marquez, A. P.;
Zamora-Crescencio, P.; c-Encalada, M. R.; Justiniano-Apolinar, S.
Y.; Angel-Robles, R.; Satoskar, A.; Hernandez-Rivero, L.
Fitoterapia 2007, 78, 255). Thus far, the phytochemical
investigation of this plant is very limited. Cardenolides,
flavonoids, and two trinosesquiterpenoids were isolated as
secondary metabolites from the roots of P. andrieuxii (Yam-Puc, A.;
Escalante-Erosa, F.; Pech-Lopez, M.; Chan-Bacab, M. J.;
Arunachalampillai, A.; Wendt, O. F.; Sterner, O.; Pena-Rodriguez,
L. M. J. Nat. Prod. 2009, 72, 745), but there are no active
principles reported corresponding to the antileishmanial activity
of this plant to date. The roots of P. andrieuxii were selected for
an activity-guided fractionation following an initial screen for
lethal effect on the protozoa of Leishmania mexicana.
[0157] In various aspects, the invention relates to methods of
isolating compounds useful as therapeutic agents, which can be
useful in the treatment of leishmaniasis and related diseases
including, but not limited to malaria, human African trypanomiasis,
babesiosis, Chagas disease, microsporidiosis, pneumocystosis,
primary ameobic meningoenchephalitis, and toxoplasmosis. The
compounds of this invention can be prepared by employing isolation
methods as shown in the following schemes (e.g. see FIG. 2 and
discussion in "Examples"), in addition to other standard
manipulations that are known in the literature, exemplified in the
experimental sections or clear to one skilled in the art.
[0158] In one aspect, the disclosed compounds comprise the products
of the isolation methods described herein. In a further aspect, the
disclosed compounds comprise a compound isolated by a method
described herein. In a still further aspect, the invention
comprises a pharmaceutical composition comprising a therapeutically
effective amount of the product of the disclosed isolation methods
and a pharmaceutically acceptable carrier. In a still further
aspect, the invention comprises a method for manufacturing a
medicament comprising combining at least one compound of any of
disclosed compounds or at least one product of the disclosed
methods with a pharmaceutically acceptable carrier or diluent.
[0159] It is contemplated that each disclosed methods can further
comprise additional steps, manipulations, and/or components. It is
also contemplated that any one or more step, manipulation, and/or
component can be optionally omitted from the invention. It is
understood that a disclosed methods can be used to provide the
disclosed compounds. It is also understood that the products of the
disclosed methods can be employed in the disclosed methods of
using.
D. HEMISYNHESIS OF DISCLOSED COMPOUNDS
[0160] The compounds of the present invention can be obtained by
isolation from Pentalinon andrieuxii as described herein.
Alternatively, the compounds can be prepared by synthetic methods,
e.g. hemisynthetic methods beginning with the appropriate
commercially available sterol precursor. For example, an analogue
of compound 1 can be prepared as shown in the synthetic scheme
below.
##STR00068##
[0161] The reaction conditions shown above can be used to prepare
additional analogues with alkyl substituents at the carbonyl. The
above reaction with exemplary alkyl substituents is illustrated
below.
##STR00069##
[0162] Alternatively, compounds can be prepared as described in the
reaction scheme shown below.
##STR00070##
[0163] Alternatively, compounds of the invention can be prepared as
described in the reaction scheme shown below.
##STR00071##
E. PHARMACEUTICAL COMPOSITIONS
[0164] In one aspect, the invention relates to a pharmaceutical
composition comprising a pharmaceutically acceptable carrier and an
effective amount of a compound represented by a formula:
##STR00072##
wherein each ---- is independently an optional covalent bond,
wherein valence is satisfied; wherein R.sup.1, when present, is
selected from C1-C12 alkyl and C1-C12 alkenyl; wherein R.sup.2,
when present, is selected from C1-C12 alkyl and C1-C12 alkenyl;
wherein R.sup.7 is selected from hydrogen, hydroxyl, amino, and
halogen; and wherein R.sup.8 is selected from hydrogen and C1-C6
alkyl; or a pharmaceutically acceptable salt, solvate, or polymorph
thereof.
[0165] In a further aspects, the pharmaceutical composition
comprises oral administration of compound 1, 2, derivatives
thereof, and any combination of 1, 2, and related compounds to a
mouse model, related biological models, human subjects, clinical
subjects, and patients being treated for leishmaniasis and related
diseases including, but not limited to malaria, human African
trypanomiasis, babesiosis, Chagas disease, microsporidiosis,
pneumocystosis, primary ameobic meningoenchephalitis, and
toxoplasmosis.
[0166] In a further aspects, the pharmaceutical composition
comprises intraperitoneal ("IP") injection, intravenous
administration, or both methods of administering compound 1, 2,
derivatives thereof, and any combination of 1, 2, and related
compounds to a mouse model, related biological models, human
subjects, clinical subjects, and patients being treated for
leishmaniasis and related diseases including, but not limited to
malaria, human African trypanomiasis, babesiosis, Chagas disease,
microsporidiosis, pneumocystosis, primary ameobic
meningoenchephalitis, and toxoplasmosis.
[0167] In a further aspect, the pharmaceutical composition
comprises oral, topical, or both methods of administering compound
1, 2, derivatives thereof, and any combination of 1, 2, and related
compounds to a mouse model, related biological models, human
subjects, clinical subjects, and patients being treated for
leishmaniasis and related diseases including, but not limited to
malaria, human African trypanomiasis, babesiosis, Chagas disease,
microsporidiosis, pneumocystosis, primary ameobic
meningoenchephalitis, and toxoplasmosis.
[0168] In a further aspects, the pharmaceutical composition
comprises intranasal administration, pulmonary delivery (e.g. using
a metered dose dry powder inhaler), or both methods of
administering compound 1, 2, derivatives thereof, and any
combination of 1, 2, and related compounds to a mouse model,
related biological models, human subjects, clinical subjects, and
patients being treated for leishmaniasis and related diseases
including, but not limited to malaria, human African trypanomiasis,
babesiosis, Chagas disease, microsporidiosis, pneumocystosis,
primary ameobic meningoenchephalitis, and toxoplasmosis.
[0169] In various aspects, the invention relates to pharmaceutical
compositions comprising the disclosed compounds. That is, a
pharmaceutical composition can be provided comprising a
therapeutically effective amount of at least one disclosed compound
or at least one product of a disclosed method and a
pharmaceutically acceptable carrier.
[0170] In a further aspect, the pharmaceutical composition further
comprises an effective amount of a therapeutic agent selected from
pentavalent antimonial, pentamidine, amphotericin B, allopurinol,
ketaconazole, suramin, melarsoprol, paramomycin, miltefosine,
sitamaquine, imiquimod, eflornitine, nifurtimox, benznidazole,
crystal violet, amiodarone, ethidium, isomethamidium, and
berenil.
[0171] In various aspects, the pharmaceutical composition further
comprises a therapeutic agent, wherein the therapeutic agent is
selected from eflornitine, ethidium, isomethamidium, and berenil.
In a further aspect, the pharmaceutical composition further
comprises a therapeutc agent, wherein the therapeutic agent is a
pentavalent antimonial and wherein the pentavalent antimonial is
selected from sodium stibogluoconate and meglumine antimoniate. In
a still further aspect, the pharmaceutical composition further
comprises a pentavalent antimonial, wherein the pentavalent
antimonial is sodium stibogluoconate. In a yet further aspect, the
pharmaceutical composition further comprises a pentavalent
antimonial, wherein the pentavalent antimonial is meglumine
antimoniate. In an even further aspect, the pharmaceutical
composition further comprises a therapeutc agent, wherein the
therapeutic agent is amphotericin B. In a still further aspect, the
pharmaceutical composition further comprises a therapeutc agent,
wherein the therapeutic agent is pentamidine. In a yet further
aspect, the pharmaceutical composition further comprises a
therapeutc agent, wherein the therapeutic agent is miltefosine.
[0172] In a further aspect, the pharmaceutical composition
comprises an effective amount of the compound. In a still further
aspect, the effective amount is a therapeutically effective amount.
In a yet further aspect, the effective amount is a prophylatically
effective amount.
[0173] In a further aspect, the pharmaceutically acceptable carrier
is selected from a liposome, nanoparticle, microparticle,
cyclodextrins, nanoemulsion, microemulsion, polymersome,
surfactant, biocompatible organic solvent, and micelle. In a still
further aspect, the surfactant is selected from a phospholipid, a
poloxamer, and a polysorbate. In a yet further aspect, the
biocompatible organic solvent is selected from propylene glycol,
polyethylene glycols, ethanol), dimethyl sulfoxide,
N-methyl-2-pyrrolidone, glycofurol, Solketal.TM., glycerol formal,
acetone tetrahydrofurfuryl alcohol, diglyme, dimethyl isosorbide,
cremophor, and ethyl lactate.
[0174] In a further aspect, the pharmaceutically acceptable carrier
is a liposome. In a still further aspect, the liposome comprises a
phospholipid. In a yet further aspect, the liposome comprises one
or more lipids selected from phosphatidylcholine, tocopherol,
cholesterol, and 1,2-distearoyl-phosphatidyl
ethanolamine-methyl-polyethyleneglycol conjugate. In an even
further aspect, the liposome comprises phosphatidylcholine and
tocopherol. In a still further aspect, the liposome further
comprises an effective amount of a therapeutic agent is selected
from pentavalent antimonial, pentamidine, amphotericin B,
allopurinol, ketaconazole, suramin, melarsoprol, paramomycin,
miltefosine, sitamaquine, imiquimod, eflornitine, nifurtimox,
benznidazole, crystal violet, amiodarone, ethidium, isomethamidium,
and berenil.
[0175] In various aspects, the pharmaceutically acceptable carrier
is a liposome, wherein the liposome further comprises a therapeutic
agent, and wherein the therapeutic agent is selected from
eflornitine, ethidium, isomethamidium, and berenil. In a further
aspect, the pharmaceutically acceptable carrier is a liposome,
wherein the liposome further comprises a therapeutic agent, wherein
the therapeutic agent is a pentavalent antimonial and wherein the
pentavalent antimonial is selected from sodium stibogluoconate and
meglumine antimoniate. In a still further aspect, the
pharmaceutically acceptable carrier is a liposome, wherein the
liposome further comprises a therapeutic agent, wherein the
therapeutic agent is a pentavalent antimonial and wherein the
pentavalent antimonial is sodium stibogluoconate. In a yet further
aspect, the pharmaceutically acceptable carrier is a liposome,
wherein the liposome further comprises a therapeutic agent, wherein
the therapeutic agent is a pentavalent antimonial and wherein the
pentavalent antimonial is meglumine antimoniate. In an even further
aspect, the pharmaceutically acceptable carrier is a liposome,
wherein the liposome further comprises a therapeutic agent, wherein
the therapeutic agent is amphotericin B. In a still further aspect,
the pharmaceutically acceptable carrier is a liposome, wherein the
liposome further comprises a therapeutic agent, wherein the
therapeutic agent is pentamidine. In a yet further aspect, the
pharmaceutically acceptable carrier is a liposome, wherein the
liposome further comprises a therapeutic agent, wherein the
therapeutic agent is miltefosine.
[0176] In certain aspects, the disclosed pharmaceutical
compositions comprise the disclosed compounds (including
pharmaceutically acceptable salt(s) thereof) as an active
ingredient, a pharmaceutically acceptable carrier, and, optionally,
other therapeutic ingredients or adjuvants. The instant
compositions include those suitable for oral, rectal, topical, and
parenteral (including subcutaneous, intradermal, intramuscular,
intraperitoneal, and intravenous) administration, although the most
suitable route in any given case will depend on the particular
host, and nature and severity of the conditions for which the
active ingredient is being administered. The pharmaceutical
compositions can be conveniently presented in unit dosage form and
prepared by any of the methods well known in the art of
pharmacy.
[0177] In various aspects, the invention also relates to a
pharmaceutical composition comprising a pharmaceutically acceptable
carrier or diluent and, as active ingredient, a therapeutically
effective amount of a disclosed compound, a product of a disclosed
method of making, a pharmaceutically acceptable salt thereof, a
hydrate thereof, a solvate thereof, a polymorph thereof, or a
stereochemically isomeric form thereof. In a further aspect, a
disclosed compound, a product of a disclosed method of making, a
pharmaceutically acceptable salt thereof, a hydrate thereof, a
solvate thereof, a polymorph thereof, or a stereochemically
isomeric form thereof, or any subgroup or combination thereof may
be formulated into various pharmaceutical forms for administration
purposes.
[0178] As used herein, the term "pharmaceutically acceptable salts"
refers to salts prepared from pharmaceutically acceptable non-toxic
bases or acids. When the compound of the present invention is
acidic, its corresponding salt can be conveniently prepared from
pharmaceutically acceptable non-toxic bases, including inorganic
bases and organic bases. Salts derived from such inorganic bases
include aluminum, ammonium, calcium, copper (-ic and -ous), ferric,
ferrous, lithium, magnesium, manganese (-ic and -ous), potassium,
sodium, zinc and the like salts. Particularly preferred are the
ammonium, calcium, magnesium, potassium and sodium salts. Salts
derived from pharmaceutically acceptable organic non-toxic bases
include salts of primary, secondary, and tertiary amines, as well
as cyclic amines and substituted amines such as naturally occurring
and synthesized substituted amines. Other pharmaceutically
acceptable organic non-toxic bases from which salts can be formed
include ion exchange resins such as, for example, arginine,
betaine, caffeine, choline, N,N'-dibenzylethylenediamine,
diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol,
ethanolamine, ethylenediamine, N-ethylmorpholine,
N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine,
isopropylamine, lysine, methylglucamine, morpholine, piperazine,
piperidine, polyamine resins, procaine, purines, theobromine,
triethylamine, trimethylamine, tripropylamine, tromethamine and the
like.
[0179] As used herein, the term "pharmaceutically acceptable
non-toxic acids", includes inorganic acids, organic acids, and
salts prepared therefrom, for example, acetic, benzenesulfonic,
benzoic, camphorsulfonic, citric, ethanesulfonic, fumaric,
gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic,
maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic,
pantothenic, phosphoric, succinic, sulfuric, tartaric,
p-toluenesulfonic acid and the like. Preferred are citric,
hydrobromic, hydrochloric, maleic, phosphoric, sulfuric, and
tartaric acids.
[0180] For therapeutic use, salts of the disclosed compounds are
those wherein the counter ion is pharmaceutically acceptable.
However, salts of acids and bases which are non-pharmaceutically
acceptable may also find use, for example, in the preparation or
purification of a pharmaceutically acceptable compound. All salts,
whether pharmaceutically acceptable or not, are included within the
ambit of the present invention.
[0181] The pharmaceutically acceptable acid and base addition salts
as mentioned hereinabove or hereinafter are meant to comprise the
therapeutically active non-toxic acid and base addition salt forms
which the disclosed compounds are able to form. The
pharmaceutically acceptable acid addition salts can conveniently be
obtained by treating the base form with such appropriate acid.
Appropriate acids comprise, for example, inorganic acids such as
hydrohalic acids, e.g. hydrochloric or hydrobromic acid, sulfuric,
nitric, phosphoric and the like acids; or organic acids such as,
for example, acetic, propanoic, hydroxyacetic, lactic, pyruvic,
oxalic (i.e. ethanedioic), malonic, succinic (i.e. butanedioic
acid), maleic, fumaric, malic, tartaric, citric, methanesulfonic,
ethanesulfonic, benzenesulfonic, p-toluenesulfonic, cyclamic,
salicylic, p-aminosalicylic, pamoic and the like acids. Conversely
said salt forms can be converted by treatment with an appropriate
base into the free base form.
[0182] The disclosed compounds containing an acidic proton may also
be converted into their non-toxic metal or amine addition salt
forms by treatment with appropriate organic and inorganic bases.
Appropriate base salt forms comprise, for example, the ammonium
salts, the alkali and earth alkaline metal salts, e.g. the lithium,
sodium, potassium, magnesium, calcium salts and the like, salts
with organic bases, e.g. primary, secondary and tertiary aliphatic
and aromatic amines such as methylamine, ethylamine, propylamine,
isopropylamine, the four butylamine isomers, dimethylamine,
diethylamine, diethanolamine, dipropylamine, diisopropylamine,
di-n-butylamine, pyrrolidine, piperidine, morpholine,
trimethylamine, triethylamine, tripropylamine, quinuclidine,
pyridine, quinoline and isoquinoline; the benzathine,
N-methyl-D-glucamine, hydrabamine salts, and salts with amino acids
such as, for example, arginine, lysine and the like. Conversely the
salt form can be converted by treatment with acid into the free
acid form.
[0183] In practice, the compounds of the invention, or
pharmaceutically acceptable salts thereof, of this invention can be
combined as the active ingredient in intimate admixture with a
pharmaceutical carrier according to conventional pharmaceutical
compounding techniques. The carrier can take a wide variety of
forms depending on the form of preparation desired for
administration, e.g., oral or parenteral (including intravenous).
Thus, the pharmaceutical compositions of the present invention can
be presented as discrete units suitable for oral administration
such as capsules, cachets or tablets each containing a
predetermined amount of the active ingredient. Further, the
compositions can be presented as a powder, as granules, as a
solution, as a suspension in an aqueous liquid, as a non-aqueous
liquid, as an oil-in-water emulsion or as a water-in-oil liquid
emulsion. In addition to the common dosage forms set out above, the
compounds of the invention, and/or pharmaceutically acceptable
salt(s) thereof, can also be administered by controlled release
means and/or delivery devices. The compositions can be prepared by
any of the methods of pharmacy. In general, such methods include a
step of bringing into association the active ingredient with the
carrier that constitutes one or more necessary ingredients. In
general, the compositions are prepared by uniformly and intimately
admixing the active ingredient with liquid carriers or finely
divided solid carriers or both. The product can then be
conveniently shaped into the desired presentation.
[0184] It is especially advantageous to formulate the
aforementioned pharmaceutical compositions in unit dosage form for
ease of administration and uniformity of dosage. Unit dosage form
as used herein refers to physically discrete units suitable as
unitary dosages, each unit containing a predetermined quantity of
active ingredient calculated to produce the desired therapeutic
effect in association with the required pharmaceutical carrier.
Examples of such unit dosage forms are tablets (including scored or
coated tablets), capsules, pills, powder packets, wafers,
suppositories, injectable solutions or suspensions and the like,
and segregated multiples thereof.
[0185] Thus, the pharmaceutical compositions of this invention can
include a pharmaceutically acceptable carrier and a compound or a
pharmaceutically acceptable salt of the compounds of the invention.
The compounds of the invention, or pharmaceutically acceptable
salts thereof, can also be included in pharmaceutical compositions
in combination with one or more other therapeutically active
compounds.
[0186] The pharmaceutical carrier employed can be, for example, a
solid, liquid, or gas. Examples of solid carriers include lactose,
terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium
stearate, and stearic acid. Examples of liquid carriers are sugar
syrup, peanut oil, olive oil, and water. Examples of gaseous
carriers include carbon dioxide and nitrogen.
[0187] In order to enhance the solubility and/or the stability of
the compounds of Formula (I) in pharmaceutical compositions, it can
be advantageous to employ .alpha.-, .beta.- or
.gamma.-cyclodextrins or their derivatives, in particular
hydroxyalkyl substituted cyclodextrins, e.g.
2-hydroxypropyl-.beta.-cyclodextrin or
sulfobutyl-.beta.-cyclodextrin. Also co-solvents such as alcohols
may improve the solubility and/or the stability of the compounds
according to the invention in pharmaceutical compositions.
[0188] Because of the ease in administration, oral administration
is preferred, and tablets and capsules represent the most
advantageous oral dosage unit forms in which case solid
pharmaceutical carriers are obviously employed. In preparing the
compositions for oral dosage form, any convenient pharmaceutical
media can be employed. For example, water, glycols, oils, alcohols,
flavoring agents, preservatives, coloring agents and the like can
be used to form oral liquid preparations such as suspensions,
elixirs and solutions; while carriers such as starches, sugars,
microcrystalline cellulose, diluents, granulating agents,
lubricants, binders, disintegrating agents, and the like can be
used to form oral solid preparations such as powders, capsules and
tablets. Because of their ease of administration, tablets and
capsules are the preferred oral dosage units whereby solid
pharmaceutical carriers are employed. Optionally, tablets can be
coated by standard aqueous or nonaqueous techniques
[0189] A tablet containing the composition of this invention can be
prepared by compression or molding, optionally with one or more
accessory ingredients or adjuvants. Compressed tablets can be
prepared by compressing, in a suitable machine, the active
ingredient in a free-flowing form such as powder or granules,
optionally mixed with a binder, lubricant, inert diluent, surface
active or dispersing agent. Molded tablets can be made by molding
in a suitable machine, a mixture of the powdered compound moistened
with an inert liquid diluent.
[0190] The pharmaceutical compositions of the present invention
comprise a compound of the invention (or pharmaceutically
acceptable salts thereof) as an active ingredient, a
pharmaceutically acceptable carrier, and optionally one or more
additional therapeutic agents or adjuvants. The instant
compositions include compositions suitable for oral, rectal,
topical, and parenteral (including subcutaneous, intramuscular,
intradermal, intraperitoneal, and intravenous) administration,
although the most suitable route in any given case will depend on
the particular host, and nature and severity of the conditions for
which the active ingredient is being administered. The
pharmaceutical compositions can be conveniently presented in unit
dosage form and prepared by any of the methods well known in the
art of pharmacy.
[0191] Pharmaceutical compositions of the present invention
suitable for parenteral administration can be prepared as solutions
or suspensions of the active compounds in water. A suitable
surfactant can be included such as, for example,
hydroxypropylcellulose. Dispersions can also be prepared in
glycerol, liquid polyethylene glycols, and mixtures thereof in
oils. Further, a preservative can be included to prevent the
detrimental growth of microorganisms.
[0192] Pharmaceutical compositions of the present invention
suitable for injectable use include sterile aqueous solutions or
dispersions. Furthermore, the compositions can be in the form of
sterile powders for the extemporaneous preparation of such sterile
injectable solutions or dispersions. In all cases, the final
injectable form must be sterile and must be effectively fluid for
easy syringability. The pharmaceutical compositions must be stable
under the conditions of manufacture and storage; thus, preferably
should be preserved against the contaminating action of
microorganisms such as bacteria and fungi. The carrier can be a
solvent or dispersion medium containing, for example, water,
ethanol, polyol (e.g., glycerol, propylene glycol and liquid
polyethylene glycol), vegetable oils, and suitable mixtures
thereof.
[0193] Injectable solutions, for example, may be prepared in which
the carrier comprises saline solution, glucose solution or a
mixture of saline and glucose solution. Injectable suspensions may
also be prepared in which case appropriate liquid carriers,
suspending agents and the like may be employed. Also included are
solid form preparations that are intended to be converted, shortly
before use, to liquid form preparations.
[0194] Pharmaceutical compositions of the present invention can be
in a form suitable for topical use such as, for example, an
aerosol, cream, ointment, lotion, dusting powder, and the like.
Further, the compositions can be in a form suitable for use in
transdermal devices. These formulations can be prepared, utilizing
a compound of the invention, or pharmaceutically acceptable salts
thereof, via conventional processing methods. As an example, a
cream or ointment is prepared by mixing hydrophilic material and
water, together with about 5 wt % to about 10 wt % of the compound,
to produce a cream or ointment having a desired consistency.
[0195] In the compositions suitable for percutaneous
administration, the carrier optionally comprises a penetration
enhancing agent and/or a suitable wetting agent, optionally
combined with suitable additives of any nature in minor
proportions, which additives do not introduce a significant
deleterious effect on the skin. Said additives may facilitate the
administration to the skin and/or may be helpful for preparing the
desired compositions. These compositions may be administered in
various ways, e.g., as a transdermal patch, as a spot-on, as an
ointment.
[0196] Pharmaceutical compositions of this invention can be in a
form suitable for rectal administration wherein the carrier is a
solid. It is preferable that the mixture forms unit dose
suppositories. Suitable carriers include cocoa butter and other
materials commonly used in the art. The suppositories can be
conveniently formed by first admixing the composition with the
softened or melted carrier(s) followed by chilling and shaping in
moulds.
[0197] In addition to the aforementioned carrier ingredients, the
pharmaceutical formulations described above can include, as
appropriate, one or more additional carrier ingredients such as
diluents, buffers, flavoring agents, binders, surface-active
agents, thickeners, lubricants, preservatives (including
anti-oxidants) and the like. Furthermore, other adjuvants can be
included to render the formulation isotonic with the blood of the
intended recipient. Compositions containing a compound of the
invention, and/or pharmaceutically acceptable salts thereof, can
also be prepared in powder or liquid concentrate form.
[0198] The exact dosage and frequency of administration depends on
the particular disclosed compound, a product of a disclosed method
of making, a pharmaceutically acceptable salt thereof, a hydrate
thereof, a solvate thereof, a polymorph thereof, or a
stereochemically isomeric form thereof; the particular condition
being treated and the severity of the condition being treated;
various factors specific to the medical history of the subject to
whom the dosage is administered such as the age; weight, sex,
extent of disorder and general physical condition of the particular
subject, as well as other medication the individual may be taking;
as is well known to those skilled in the art. Furthermore, it is
evident that said effective daily amount may be lowered or
increased depending on the response of the treated subject and/or
depending on the evaluation of the physician prescribing the
compounds of the instant invention.
[0199] Depending on the mode of administration, the pharmaceutical
composition will comprise from 0.05 to 99% by weight, preferably
from 0.1 to 70% by weight, more preferably from 0.1 to 50% by
weight of the active ingredient, and, from 1 to 99.95% by weight,
preferably from 30 to 99.9% by weight, more preferably from 50 to
99.9% by weight of a pharmaceutically acceptable carrier, all
percentages being based on the total weight of the composition.
[0200] In the treatment conditions which require inhibition of
parasitic activity in a cell, an appropriate dosage level will
generally be about 0.01 to 1000 mg per kg patient body weight per
day and can be administered in single or multiple doses. In various
aspects, the dosage level will be about 0.1 to about 500 mg/kg per
day, about 0.1 to 250 mg/kg per day, or about 0.5 to 100 mg/kg per
day. A suitable dosage level can be about 0.01 to 1000 mg/kg per
day, about 0.01 to 500 mg/kg per day, about 0.01 to 250 mg/kg per
day, about 0.05 to 100 mg/kg per day, or about 0.1 to 50 mg/kg per
day. Within this range the dosage can be 0.05 to 0.5, 0.5 to 5.0 or
5.0 to 50 mg/kg per day. For oral administration, the compositions
are preferably provided in the from of tablets containing 1.0 to
1000 miligrams of the active ingredient, particularly 1.0, 5.0, 10,
15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 750,
800, 900 and 1000 milligrams of the active ingredient for the
symptomatic adjustment of the dosage of the patient to be treated.
The compound can be administered on a regimen of 1 to 4 times per
day, preferably once or twice per day. This dosing regimen can be
adjusted to provide the optimal therapeutic response.
[0201] Such unit doses as described hereinabove and hereinafter can
be administered more than once a day, for example, 2, 3, 4, 5 or 6
times a day. In various aspects, such unit doses can be
administered 1 or 2 times per day, so that the total dosage for a
70 kg adult is in the range of 0.001 to about 15 mg per kg weight
of subject per administration. In a further aspect, dosage is 0.01
to about 1.5 mg per kg weight of subject per administration, and
such therapy can extend for a number of weeks or months, and in
some cases, years. It will be understood, however, that the
specific dose level for any particular patient will depend on a
variety of factors including the activity of the specific compound
employed; the age, body weight, general health, sex and diet of the
individual being treated; the time and route of administration; the
rate of excretion; other drugs that have previously been
administered; and the severity of the particular disease undergoing
therapy, as is well understood by those of skill in the area.
[0202] A typical dosage can be one 1 mg to about 100 mg tablet or 1
mg to about 300 mg taken once a day, or, multiple times per day, or
one time-release capsule or tablet taken once a day and containing
a proportionally higher content of active ingredient. The
time-release effect can be obtained by capsule materials that
dissolve at different pH values, by capsules that release slowly by
osmotic pressure, or by any other known means of controlled
release.
[0203] It can be necessary to use dosages outside these ranges in
some cases as will be apparent to those skilled in the art.
Further, it is noted that the clinician or treating physician will
know how and when to start, interrupt, adjust, or terminate therapy
in conjunction with individual patient response.
[0204] The present invention is further directed to a method for
the manufacture of a medicament for inhibiting parasitic activity
(e.g., treatment of one or more parasitic diseases) in mammals
(e.g., humans) comprising combining one or more disclosed
compounds, products, or compositions with a pharmaceutically
acceptable carrier or diluent. Thus, in one aspect, the invention
relates to a method for manufacturing a medicament comprising
combining at least one disclosed compound or at least one disclosed
product with a pharmaceutically acceptable carrier or diluent.
[0205] The disclosed pharmaceutical compositions can further
comprise other therapeutically active compounds, which are usually
applied in the treatment of the above mentioned pathological
conditions.
[0206] It is understood that the disclosed compositions can be
prepared from the disclosed compounds. It is also understood that
the disclosed compositions can be employed in the disclosed methods
of using.
[0207] As already mentioned, the invention relates to a
pharmaceutical composition comprising a therapeutically effective
amount of a disclosed compound, a product of a disclosed method of
making, a pharmaceutically acceptable salt thereof, a hydrate
thereof, a solvate thereof, a polymorph thereof, and a
pharmaceutically acceptable carrier. Additionally, the invention
relates to a process for preparing a such pharmaceutical
composition, characterized in that a pharmaceutically acceptable
carrier is intimately mixed with a therapeutically effective amount
of a compound according to the invention.
[0208] As already mentioned, the invention also relates to a
pharmaceutical composition comprising a disclosed compound, a
product of a disclosed method of making, a pharmaceutically
acceptable salt thereof, a hydrate thereof, a solvate thereof, a
polymorph thereof, and one or more other drugs in the treatment,
prevention, control, amelioration, or reduction of risk of diseases
or conditions for a disclosed compound or the other drugs may have
utility as well as to the use of such a composition for the
manufacture of a medicament. The present invention also relates to
a combination of disclosed compound, a product of a disclosed of
isolating a compound from Pentalinon andrieuxii, a pharmaceutically
acceptable salt thereof, a hydrate thereof, a solvate thereof, a
polymorph thereof, and an anti-parasitic compound that is not a
disclosed compound. The present invention also relates to such a
combination for use as a medicine. The present invention also
relates to a product comprising (a) disclosed compound, a product
of a disclosed method of making, a pharmaceutically acceptable salt
thereof, a hydrate thereof, a solvate thereof, a polymorph thereof,
and (b) an anti-parasitic compound known to treat an infection with
a hemoflagellated protozoa, as a combined preparation for
simultaneous, separate or sequential use in the treatment or
prevention of a condition in a mammal, including a human, the
treatment or prevention of which is affected or facilitated by the
anti-parasitic compounds, in particular compounds which treat a
disease associated with infection by a with a hemoflagellated
protozoa such as Leishmania spp or Trypanosoma spp.. The different
drugs of such a combination or product may be combined in a single
preparation together with pharmaceutically acceptable carriers or
diluents, or they may each be present in a separate preparation
together with pharmaceutically acceptable carriers or diluents.
F. METHODS OF USING THE COMPOUNDS AND COMPOSITIONS
[0209] Leishmaniasis is a protozoan vector borne parasitic disease
caused by protozoan parasites of the genus Leishmania and is
transmitted through the bite of certain species of Phlembotominae
sandfly. Of the approximately 30 species of Leishmania known to
infect mammals, 21 of these species are believed to cause
leishmaniasis in humans. Leishmaniasis manifests in three distinct
forms including; cutaneous leishmaniasis ("CL"), visceral
leishmaniasis ("VL") and mucocutaneous leishmaniasis ("ML").
Leishmaniasis typically presents as skin sores or ulcers which
erupt weeks to months after the person is bitten. However, if left
untreated, the infection can progress and lead to splenomegaly,
liver damage, renal damage, anemia, and death. Due to the complex
life cycles (FIG. 2) of the causative parasites, leishmaniasis is
rarely diagnosed in its early stages when therapeutic intervention
is most effective. The parasite life cycle typically begins when
sandflies transfer metacyclic promastigotes during blood meals.
Metacyclic promastigotes that reach the puncture wound are ingested
or phagocytized by macrophages. Inside the macrophages, the
promastigotes transform into amastigotes. Here, the amastigotes
multiply in infected cells and affect different tissues. These
differing tissue specificities cause the differing clinical
manifestations of the various forms of leishmaniasis as described
above. The cycle is continued when sandflies become infected during
blood meals from infected hosts when they ingest macrophages
infected with amastigotes. In the sandfly's midgut, the parasites
differentiate into promastigotes, then multiply, and differentiate
into metacyclic promastigotes to repeat the cycle. Leishmaniasis
typically presents as skin sores or ulcers which erupt weeks to
months after the person is bitten. However, if left untreated, the
infection can progress and lead to splenomegaly, liver damage,
renal damage, anemia, and death.
[0210] Like fungi, the predominant endogenous sterols found in
Leishmania species are ergosterol and its derivatives, and these
sterols are constituents of cell membranes, which are essential for
the normal structure and function of these parasites (de Souza, W.;
Rodrigues, J. C. F. Interdiscip. Perspect. Infect. Dis. 2009, doi:
10.1155/2009/642502). Thus, the sterol biosynthesis pathway is
considered as a promising target in the development of new
therapeutic agents for the treatment of parasitic diseases
(Werbovetz, K. A. Exert Opin. Ther. Targets. 2002, 6, 407-422). In
recent years, studies have shown that a number of sterol-derived
compounds exhibit antileishmanial activities, and some of them such
as azasterols are reported as inhibitors of certain enzymes
involved in the sterol biosynthesis pathway of these parasites
(Haughan, P. A.; Chance, M. L.; Goad, L. J. Biochem. J. 1995, 308,
31-38; Magaraci, F.; Jimenez, C. J.; Rodrigues, C.; Rodrigues, J.
C.; Braga, M. V.; Yardley, V.; de Luca-Fradley, K.; Croft, S. L.;
de Souza, W.; Ruiz-Perez, L. M.; Urbina, J.; Gonzalez, J. J. Med.
Chem. 2003, 46, 4714-4727; Bazin, M.-A.; Loiseau, P. M.; Bories,
C.; Letourneux, Y.; Rault, S.; El Kihel, L. Eur. J. Med. Chem.
2006, 41, 1109-1116; and Sartorelli, P.; Andrade, S. P.; Melhem, M.
S. C.; Prado, F. O.; Tempone, A. G. Phytother. Res. 2007, 21,
644-647).
[0211] The structure of the disclosed compounds, e.g.
pentalionsterol, closely mimics Leishmania-synthesized sterols
which are critical for lipid synthesis in the parasite. Without
wishing to be bound by a particular theory, the active sterols
isolated from P. audriexii in the present invention can exert their
activity by acting as antagonists of endogenous sterols to
interfere or inhibit sterol biosynthesis and lead to organism
death. Thus, in one aspect, the disclosed sterols can have
therapeutic anti-Leishmania activity via dysregulation of the
synthesis of parasite lipids which are required for membrane
integrity. Further, and without wishing to be bound by a particular
theory, since similar pathways are also present in other protozoan
parasites, it is believed that the disclosed compounds can display
antiparasitic activity against other pathogens, such as
Trypanosoma, Plasmodium, and Toxoplasma.
[0212] Hence, the present invention relates to compounds and
compositions disclosed herein for use as a medicament, as well as
to the use of a compound or composition disclosed herein or a
pharmaceutical composition according to the invention for the
manufacture of a medicament, including, for example, the
manufacture of a medicament for treating or preventing, in
particular treating, a condition in a mammal, including a human,
the treatment or prevention of which is affected or facilitated by
anti-protozoal activity, e.g. by anti-leishmanial activity. The
present invention also relates to a compound disclosed herein or a
pharmaceutical composition according to the invention for use in
the treatment or prevention of a condition in a subject such as a
mammal, including a human, the treatment or prevention of which is
affected or facilitated by anti-protozoal activity, e.g. by
anti-leishmanial activity.
[0213] a. Treatment of a Parasitic Disease: Administering a
Compound
[0214] In one aspect, the invention relates to a method for the
treatment of a parasitic disease in a mammal diagnosed with the
disease, the method comprising the step of administering to the
mammal a therapeutically effective amount of at least one compound
having a structure represented by a formula:
##STR00073##
wherein each ---- is independently an optional covalent bond,
wherein valence is satisfied; wherein R.sup.1, when present, is
selected from C1-C12 alkyl and C1-C12 alkenyl; wherein R.sup.2,
when present, is selected from C1-C12 alkyl and C1-C12 alkenyl;
wherein R.sup.7 is selected from hydrogen, hydroxyl, amino, and
halogen; and wherein R.sup.8 is selected from hydrogen and C1-C6
alkyl; or a pharmaceutically acceptable salt, solvate, or polymorph
thereof.
[0215] In a further aspect, the invention relates to a method for
the treatment of a parasitic disease in a mammal comprising the
step of administering to the mammal an effeasctive amount of at
least one compound; or a pharmaceutically acceptable salt, hydrate,
solvate, or polymorph thereof; wherein the compound is a disclosed
compound or a product of a disclosed method of isolating a compound
from P. andrieuxii.
[0216] In a further aspect, the invention relates to a method for
the treatment of a parasitic disease in a mammal comprising the
step of administering to the mammal at least one disclosed compound
or a product of a disclosed method of isolating a compound from P.
andrieuxii, in a dosage and amount effective to treat the disorder
in the mammal.
[0217] In a further aspect. the parasitic disease is associated
with a hemoflagellated protozoa.
[0218] In a further aspect, the parasitic disease is associated
with infection of the mammal by Leishmania spp. In a still further
aspect, the Leishmania spp. is selected from Leishmania donovani,
Leishmannia brasiliensis, Leishmania mexicana, Leishmania
amazonensis, Leishmania aethiopica, Leishmania major, Leishmania
chagasi, Leishmania panamensis, Leishmania infantum, and Leishmania
tropica.
[0219] In a further aspect, the parasitic disease is associated
with infection of the mammal by Trypanosoma spp. In a still further
aspect, the Trypanosoma spp. is selected from Trypanosoma brucei,
Trypanosoma cruzi, Trypanosoma brucei gambiense, Trypanosoma brucei
rhodesiense, Trypanosoma congolense, Trypanosoma equinum,
Trypanosoma equiperdum, Trypanosoma melophagium, Trypanosoma
theileri, and Trypanosoma vivax.
[0220] In a further aspect, the parasitic disease is a
trypanosomiases. In a still further aspect, the trypanosomiases is
Chaga's disease.
[0221] In a further aspect, the parasitic disease is a
leishmaniases. In a still further aspect, the leishmaniases is
visceral leishmaniasis, cutaneous leishmaniasis, mucocutaneous
leishmaniasis, diffus cutaneous leishmaniasis, recidivans
leishmaniasis, and post-kala-azar dermal leishmaniasis. In a yet
further aspect, the leishmaniases is cutaneous leishmaniasis. In an
even further aspect, the leishmaniases is visceral
leishmaniasis.
[0222] In a further aspect, an effective amount is a
therapeutically effective amount. In a still further aspect, an
effective amount is a prophylatically effective amount. In a yet
further aspect, treatment is symptom amelioration or prevention,
and wherein an effective amount is a prophylatically effective
amount.
[0223] In a further aspect, the mammal that the compound is
administered to is a human. In a still further aspect, the mammal
that the compound is administered to is a bovine. In a yet further
aspect, the mammal that the compound is administered to is a
canine. In a further aspect, the mammal has been diagnosed with a
need for treatment of the disorder prior to the administering step.
In a further aspect, the method further comprises the step of
identifying a mammal in need of treatment of the disorder.
[0224] In a further aspect, the parasitic disease is leishmaniasis
and related diseases including, but not limited to malaria, human
African trypanomiasis, babesiosis, Chagas disease,
microsporidiosis, pneumocystosis, primary ameobic
meningoenchephalitis, and toxoplasmosis.
[0225] b. Treatment of a Parasitic Disease: Identifying a Need for
Treatment and Administering a Compound
[0226] In various aspects, the invention relates to a method for
the treatment of a parasitic disease comprising the steps of: a)
identifying a mammal in need of treatment of a parasitic disease;
and b) administering to the mammal an effective amount of at least
one compound having a structure represented by a formula:
##STR00074##
wherein each ---- is independently an optional covalent bond,
wherein valence is satisfied; wherein R.sup.1, when present, is
selected from C1-C12 alkyl and C1-C12 alkenyl; wherein R.sup.2,
when present, is selected from C1-C12 alkyl and C1-C12 alkenyl;
wherein R.sup.7 is selected from hydrogen, hydroxyl, amino, and
halogen; and wherein R.sup.8 is selected from hydrogen and C1-C6
alkyl; or a pharmaceutically acceptable salt, solvate, or polymorph
thereof.
[0227] In a further aspect, the mammal that the compound is
administered to is a human. In a still further aspect, the mammal
that the compound is administered to is a bovine. In a yet further
aspect, the mammal that the compound is administered to is a
canine.
[0228] In a further aspect, the identifying step is performed prior
to the administering step. In a still further aspect, the mammal
has been diagnosed with a parasitic disease prior to the
administering step.
[0229] In a further aspect, the effective amount is a
prophylatically effective amount. In a still further aspect, the
effective amount is a therapeutically effective amount.
[0230] c. Treating a Parasitic Disease in a Cell
[0231] In one aspect, the invention relates to a method for
treating a parasitic disease, the method comprising the step of
contacting a mammalian cell with an effective amount of at least
one compound having a structure represented by a formula:
##STR00075##
wherein each ---- is independently an optional covalent bond,
wherein valence is satisfied; wherein R.sup.1, when present, is
selected from C1-C12 alkyl and C1-C12 alkenyl; wherein R.sup.2,
when present, is selected from C1-C12 alkyl and C1-C12 alkenyl;
wherein R.sup.7 is selected from hydrogen, hydroxyl, amino, and
halogen; and wherein R.sup.8 is selected from hydrogen and C1-C6
alkyl; or a pharmaceutically acceptable salt, solvate, or polymorph
thereof.
[0232] In a further aspect, the invention relates to a method for
treating a parasitic disease in at least one cell, comprising the
step of contacting the at least one cell with an effective amount
of at least one compound, or a pharmaceutically acceptable salt,
hydrate, solvate, or polymorph thereof; wherein the compound is a
disclosed compound or a product of a disclosed method of isolating
a compound from P. andrieuxii.
[0233] In a further aspect, the invention relates to a method for
treating a parasitic disease in a mammal by contacting at least one
cell in a mammal, comprising the step of contacting the at least
one cell with at least one disclosed compound or a product of a
disclosed method of isolating a compound from P. andrieuxii, in an
amount effective to inhibit growth of the parasite in the at least
one cell.
[0234] In a further aspect, the effective amount is an amount
sufficient to inhibit replication of the parasite. In a still
further aspect, the effective amount is an amount sufficient to
inhibit proliferation of the parasite. In a yet further aspect, the
effective amount is an amount sufficient to be cytostatic to the
parasite. In an even further aspect, the effective amount is an
amount sufficient to kill the parasite.
[0235] In a further aspect, the cell is human. In a still further
aspect, the cell is bovine. In a yet further aspect, the cell is
canine.
[0236] In a further aspect, the cell has been isolated from a
mammal prior to the contacting step. In a still further aspect, the
contacting is via administration to the mammal.
[0237] In a further aspect, an effective amount is a
therapeutically effective amount. In a yet further aspect, an
effective amount is a prophylatically effective amount. In an even
further aspect, treatment is symptom amelioration or prevention,
and wherein an effective amount is a prophylatically effective
amount.
[0238] In a further aspect, contacting is via administration to a
mammal. In a still further aspect, the mammal has been diagnosed
with a need for inhibiting parasitic activity prior to the
administering step. In a yet further aspect, the mammal has been
diagnosed with a parasitic infection prior to the administering
step. In an even further aspect, the mammal has been diagnosed with
a need for inhibiting parasitic replication prior to the
administering step. In a still further aspect, the mammal has been
diagnosed with a need for inhibiting parasitic proliferation prior
to the administering step. In a yet further aspect, the mammal has
been diagnosed with a need for a cytostatic effect on parasite
prior to the administering step. In a further aspect, the mammal
has been diagnosed with a need for treatment of a parasitic disease
prior to the administering step.
[0239] In one aspect, the parasitic disease is leishmaniasis and
related diseases including, but not limited to malaria, human
African trypanomiasis, babesiosis, Chagas disease,
microsporidiosis, pneumocystosis, primary ameobic
meningoenchephalitis, and toxoplasmosis
[0240] 2. Manufacture of a Medicament
[0241] In one aspect, the invention relates to a method for the
manufacture of a medicament for treating a parasitic disease in a
mammal comprising combining a therapeutically effective amount of a
disclosed compound or a product of a disclosed method of isolating
a compound from P. andrieuxii with a pharmaceutically acceptable
carrier or diluent.
[0242] In various aspect, the invention relates methods for the
manufacture of a medicament for treatment of a parasitic disease
(e.g., treatment of leishmaniasis and related diseases including,
but not limited to malaria, human African trypanomiasis,
babesiosis, Chagas disease, microsporidiosis, pneumocystosis,
primary ameobic meningoenchephalitis, and toxoplasmosis) in mammals
(e.g., humans) comprising combining one or more disclosed
compounds, products, or compositions or a pharmaceutically
acceptable salt, solvate, hydrate, or polymorph thereof, with a
pharmaceutically acceptable carrier. It is understood that the
disclosed methods can be performed with the disclosed compounds,
products, and pharmaceutical compositons. It is also understood
that the disclosed methods can be employed in connection with the
disclosed methods of using.
[0243] 3. Use of Compounds
[0244] In one aspect, the invention relates to the use of a
disclosed compound or a product of a disclosed method of isolating
a compound from P. andrieuxii. In a further aspect, the use relates
to the manufacture of a medicament for the treatment of a parasitic
disease. In a further aspect, the parasitic disease is
leishmaniasis and related diseases including, but not limited to
malaria, human African trypanomiasis, babesiosis, Chagas disease,
microsporidiosis, pneumocystosis, primary ameobic
meningoenchephalitis, and toxoplasmosis. In a further aspect, a use
relates to treatment of leishmaniasis and related diseases
including, but not limited to malaria, human African trypanomiasis,
babesiosis, Chagas disease, microsporidiosis, pneumocystosis,
primary ameobic meningoenchephalitis, and toxoplasmosisin a mammal.
In a further aspect, a use relates to treatment of a parasitic
disease in a cell.
[0245] In one aspect, a use is treatment of leishmaniasis and
related diseases including, but not limited to malaria, human
African trypanomiasis, babesiosis, Chagas disease,
microsporidiosis, pneumocystosis, primary ameobic
meningoenchephalitis, and toxoplasmosis.
[0246] In one aspect, the invention relates to the use of a
disclosed compound or a product of a disclosed method of isolating
a compound from P. andrieuxii in the manufacture of a medicament
for the treatment in a mammal of leishmaniasis and related diseases
including, but not limited to malaria, human African trypanomiasis,
babesiosis, Chagas disease, microsporidiosis, pneumocystosis,
primary ameobic meningoenchephalitis, and toxoplasmosis.
[0247] In one aspect, the invention relates to the use of a
disclosed compound or a product of a disclosed method of isolating
a compound from P. andrieuxii, or a pharmaceutically acceptable
salt, solvate, or polymorph thereof, or a pharmaceutical
composition for use in treating or preventing leishmaniasis and
related diseases including, but not limited to malaria, human
African trypanomiasis, babesiosis, Chagas disease,
microsporidiosis, pneumocystosis, primary ameobic
meningoenchephalitis, and toxoplasmosis.
[0248] In one aspect, the invention relates to the use of a
disclosed compound or a product of a disclosed method of isolating
a compound from P. andrieuxii, or a pharmaceutically acceptable
salt, solvate, or polymorph thereof, or a pharmaceutical
composition, in combination with an additional pharmaceutical agent
for use in the treatment of leishmaniasis and related diseases
including, but not limited to malaria, human African trypanomiasis,
babesiosis, Chagas disease, microsporidiosis, pneumocystosis,
primary ameobic meningoenchephalitis, and toxoplasmosis.
[0249] In one aspect, the invention relates to a process for
preparing a pharmaceutical composition comprising a therapeutically
effective amount of a disclosed compound or a product of a
disclosed method of isolating a compound from P. andrieuxii, or a
pharmaceutically acceptable salt, solvate, or polymorph thereof,
characterized in that a pharmaceutically acceptable carrier is
intimately mixed with a therapeutically effective amount of the
compound or the product of a disclosed method of making.
[0250] In a further aspect, the invention relates to a process for
preparing a pharmaceutical composition comprising a therapeutically
effective amount of a disclosed compound or a product of a
disclosed method of isolating a compound from P. andrieuxii, or a
pharmaceutically acceptable salt, solvate, or polymorph thereof,
for use as a medicament.
[0251] 4. Kits
[0252] In one aspect, the invention relates to a kit comprising at
least one compound represented by a formula:
##STR00076##
wherein each ---- is independently an optional covalent bond,
wherein valence is satisfied; wherein R.sup.1, when present, is
selected from C1-C12 alkyl and C1-C12 alkenyl; wherein R.sup.2,
when present, is selected from C1-C12 alkyl and C1-C12 alkenyl;
wherein R.sup.7 is selected from hydrogen, hydroxyl, amino, and
halogen; and wherein R.sup.8 is selected from hydrogen and C1-C6
alkyl; or a pharmaceutically acceptable salt, solvate, or polymorph
thereof, and one or more of: a) at least one agent known to
increase the likelihood of a parasitic disease in a mammal; b) at
least one agent known to decrease the likelihood of a parasitic
disease in a mammal; c) at least one agent known to treat a
parasitic disease in a mammal; or d) instructions for treating a
parasitic disease.
[0253] In a further aspect, the at least one compound and the at
least one agent are co-formulated.
[0254] In a further aspect, the at least one compound and the at
least one agent are co-packaged.
[0255] In a further aspect, the at least one agent of the kit is
selected from pentavalent antimonial, pentamidine, amphotericin B,
allopurinol, ketaconazole, suramin, melarsoprol, paramomycin,
miltefosine, sitamaquine, imiquimod, eflornitine, nifurtimox,
benznidazole, crystal violet, amiodarone, ethidium, isomethamidium,
and berenil. In a still further aspect, the at least one agent of
the kit is selected from eflornitine, ethidium, isomethamidium, and
berenil. In a yet further aspect, the at least one agent of the kit
is selected from sodium stibogluoconate and meglumine antimoniate.
In an even further aspect, the at least one agent of the kit is a
pentavalent antimonial. In a still further aspect, the at least one
agent of the kit is a pentavalent antimonial selected from is
sodium stibogluoconate and meglumine antimoniate. In a yet further
aspect, the at least one agent of the kit is a pentavalent
antimonial, wherein the pentavalent antimonial is meglumine
antimoniate. In a still further aspect, the at least one agent of
the kit is amphotericin B. In a yet further aspect, the at least
one agent of the kit is pentamidine. In an even further aspect, the
at least one agent of the kit is miltefosine.
[0256] The kits of the present invention can also comprise
compounds and/or products co-packaged, co-formulated, and/or
co-delivered with other components. For example, a drug
manufacturer, a drug reseller, a physician, a compounding shop, or
a pharmacist can provide a kit comprising a disclosed compound
and/or product and another component for delivery to a patient.
[0257] It is contemplated that the disclosed kits can be used in
connection with the disclosed methods of making, the disclosed
methods of using, and/or the disclosed compositions.
[0258] 5. Non-Medical Uses
[0259] Also provided are the uses of the disclosed compounds and
products as pharmacological tools in the development and
standardization of in vitro and in vivo test systems for the
evaluation of the effects of a hemoflagellated protozoa activity in
laboratory animals such as cats, dogs, rabbits, monkeys, rats and
mice, as part of the search for new therapeutic agents for the
treatment of a with a hemoflagellated protozoa infection, e.g.
Leishmania spp. or Trypanosoma spp.
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H. EXPERIMENTAL
[0309] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how the compounds, compositions, articles, devices
and/or methods claimed herein are made and evaluated, and are
intended to be purely exemplary of the invention and are not
intended to limit the scope of what the inventors regard as their
invention. Efforts have been made to ensure accuracy with respect
to numbers (e.g., amounts, temperature, etc.), but some errors and
deviations should be accounted for. Unless indicated otherwise,
parts are parts by weight, temperature is in .degree. C. or is at
ambient temperature, and pressure is at or near atmospheric.
[0310] Several methods for preparing the compounds of this
invention are illustrated in the following Examples. Starting
materials and the requisite intermediates are in some cases
commercially available, or can be prepared according to literature
procedures or as illustrated herein.
[0311] The following exemplary compounds of the invention were
synthesized. The Examples are provided herein to illustrate the
invention, and should not be construed as limiting the invention in
any way. The Examples are typically depicted in free base form,
according to the IUPAC naming convention. However, some of the
Examples were obtained or isolated in salt form.
[0312] 1. General Methods.
[0313] Optical rotations were obtained on a Perkin-Elmer 343
automatic polarimeter. UV spectra were measured with a Perkin-Elmer
Lambda 10 UV/vis spectrometer. IR spectra were run on a Thermo
Scientific Nicolet 6700 FT-IR spectrometer. NMR spectroscopic data
were obtained on a Bruker Avance DRX-400 or 600 MHz spectrometer
(with TMS as an internal standard). Column chromatography was
performed with 65-250 or 230-400 mesh silica gel (Sorbent
Technologies, Atlanta, Ga.). Analytical thin-layer chromatography
was conducted on precoated 250 .mu.m thickness silica gel plates
(UV.sub.254, glass backed, Sorbent Technologies, Atlanta, Ga.), and
preparative thin-layer chromatography was performed on precoated 20
cm.times.20 cm, 500 .mu.m thickness silica gel plates (UV.sub.254,
glass backed, Sorbent Technologies, Atlanta, Ga.). Analytical HPLC
was conducted on a 150 mm.times.4.6 mm i.d. Sunfire PrepC.sub.18
column (Waters, Milford, Mass.), and semi-preparative HPLC was
conducted on a 150 mm.times.10 mm i.d. or a 150 mm.times.19 mm
i.d., 5 .mu.m Sunfire PrepC.sub.18 column (Waters, Milford, Mass.),
along with a Waters system equipped with a 600 controller, a 717
Plus autosampler, and a 2487 dual wavelength absorbance detector.
Electron micrographs were generated on JEOL JEM-1400 TEM (JEOL,
Ltd. Tokyo, Japan), operating at 80K equipped with a Veleta digital
camera (Olympus Soft Imaging Solutions GmbH, Munster, Germany).
[0314] 2. Isolation of Compounds from Pentalinon andrieuxii
[0315] a. Harvesting of Plant Material.
[0316] The roots of P. andrieuxii Mueller-Argoviensis (syn.
Urechites andrieuxii, Apocynaceae; hereinafter "P. andrieuxii")
were collected from a region of riparian forest at coordinates
96.degree. 16'N 90.degree. 36W in Campeche, Mexico (muncipalities
of Campeche and Escarcega) located in the Yucatan Peninsula of
Mexico. The plant was identified in the Herbarium of the Autonomous
University of Campeche (Universidad Autonoma de Campeche; UACAM),
Campeche, Mexico, under voucher no. 6921 (Zamora-Crescencio &
Lezama-Davila) and in the Herbarium of the Autonomous University of
Yucatan (Universidad Autonoma de Yucatan), Mexico, under voucher
no. 1 (Viscencio de la Rosa & Lezama-Davila). The collection
and positive identification of this plant was supervised by the
Director of the Herbarium of UACAM.
[0317] b. Extraction and Isolation of Compounds from Roots.
[0318] The air-dried roots of P. andrieuxii (900 g) were milled and
then extracted with methanol overnight at room temperature
(3.times.4 L). The macerate was concentrated in vacuo (80 g) and
partitioned to afford a hexane-soluble extract (7.0 g), and a
CHCl.sub.3-soluble extraction (20 g). The hexane-soluble extract
was found to be active against promastigotes of L. mexicana
(IC.sub.50=35 .mu.g/mL). Part of the hexane-soluble partition (6.5
g) was subjected to separation over a silica gel column using pure
hexane initially, followed in turn by a gradient of increasing
polarity using hexane-ethyl acetate and pure acetone to yield eight
pooled fractions (F01-F08). FIG. 2 provides a schematic
representation of the biologically guided extraction and isolation
methods used to produce the leishmanicidal products from Pentalinon
andriuxii.
[0319] Fraction F01 (900 mg) was chromatographed by passage over a
fine silica gel (3.0.times.45 cm) column using a gradient solvent
of hexane-EtOAc, with nine pooled fractions obtained (F01F1-F01F9).
TLC detection showed that fraction F01F03 (120 mg) to contain
compounds with similar TLC behavior to terpenoids or steroids. This
fraction was further purified repeatedly by HPLC on a
semi-preparative RP18 column (150 mm.times.10 mm i.d.), using
MeOH--H.sub.2O (95:5; 5 mL/min) as the mobile phase, to yield
compounds 1 (1.2 mg, t.sub.R=11.2 min), 3 (4.0 mg, t.sub.R=16.7
min), 4 (2.0 mg, t.sub.R=19.2 min), 5 (6.0 mg, t.sub.R=22.3 min),
and 6 (8.0 mg, t.sub.R=25.2 min).
[0320] Fraction F02 (980 mg) was subjected to separation over a
silica gel column and eluted with a hexane-EtOAc gradient solvent
system. Of the subfractions obtained, F0203 was determined by TLC
to be a sterol-rich fraction, and was further chromatographed on a
semi-preparative RP18 column (150 mm.times.10 mm i.d.) by HPLC
using MeOH--H.sub.2O (90:10; 5 mL/min) as the solvent system, to
yield compounds 20 (4.0 mg, t.sub.R=7.2 min), 7 (4.0 mg,
t.sub.R=12.0 min), 8 (6.0 mg, t.sub.R=15.7 min), 9 (6.0 mg,
t.sub.R=17.8 min), 10 (2.0 mg, t.sub.R=20.2 min), 11 (4.0 mg,
t.sub.R=21.7 min), and 12 (24.0 mg, t.sub.R=27.4 min). Compound 17
(2.0 mg) was purified from subfraction F0202 by repeated
chromatography on silica gel columns, using hexane-acetone as
eluent.
[0321] Fractions F06 and F07 were combined (380 mg) and
chromatographed on a silica gel column and eluted with a
CH.sub.2Cl.sub.2-acetone gradient solvent system (30:1 to pure
acetone) to yield 14 subfractions. Compound 2 (5.0 mg) was purified
from subfraction F06F12 by repeated chromatography using silica gel
columns with CH.sub.2Cl.sub.2-acetone gradient solvent system (15:1
to 4:1). Subfraction F06F03 was chromatographed on a
semi-preparative RP18 column (150 mm.times.10 mm i.d.) by HPLC
using CH.sub.3CN--H.sub.2O (35:65; 5 mL/min) as mobile phase, to
give compounds 15 (1.0 mg, t.sub.R=13.6 min), and 16 (0.8 mg,
t.sub.R=15.8 min).
[0322] In order to find certain common flavonoids or coumarins as
potential marker compounds to provide a basis for extract
standardization, fraction F04, which was found to be rich in
compounds showing a fluorescence under UV light at 365 nm on TLC
plates, was also investigated. Compounds 14 (1.5 mg, t.sub.R=17.2
min) and 13 (4.0 mg, t.sub.R=19.5 min) were purified from a less
polar subfraction of F04 by preparative HPLC on a semi-preparative
RP18 column (150 mm.times.10 mm i.d.), using MeOH--H.sub.2O (92:8,
5 mL/min) as eluting solvent. Compounds 18 (3.0 mg, t.sub.R=6.9
min) and 19 (2.0 mg, t.sub.R=8.1 min) were purified from the more
polar subfraction F04 by preparative HPLC on a semi-preparative
RP18 column (150 mm.times.10 mm i.d.), using MeOH--H.sub.2O (70:30,
5 mL/min) as the eluting solvent.
[0323] c. Extraction and Isolation of Compounds from Stems.
[0324] The stems of P. andrieuxii were also processed with a
similar extraction and isolation method used for the roots (see
above and FIG. 2 for description of root extraction procedure).
Nine compounds (Table 3) were isolated and identified from pooled
subfractions (PASD3F2, PASD3F3 and PASD3F4) of a CHCl.sub.3-soluble
extract (PASD3) of the stem of P. antrieuxii. The activity of the
organic partition samples isolated from the stem of are shown below
in Table 1. The activity is the IC50 determined in the promastigote
assay described below.
TABLE-US-00001 TABLE 1 Stem Promastigote Fraction IC.sub.50
(.mu.g/mL) PASD3F1 768 PASD3F2 21.5 PASD3F3 22.0 PASD3F4 37.0
PASD3F5 178 PASD3F6 703 PASD3F7 700
[0325] 3. Structure Elucidation of Isolated Compounds.
[0326] The hexane-soluble extract of P. andrieuxii led to the
isolation, identification, structural elucidation and biological
characterizatino of 16 sterol derivatives (1-16), as well as three
coumarins (17-19), and a triterpenoid (20). Among these compounds,
1 was determined to be a novel cholestane derivative, and 2 a novel
sterol glycoside bearing a rare polyoxygenated 14,15-secopregane
skeleton. The structures of these two novel compounds were
elucidated based on the analysis of their physical and
spectroscopic data ([.alpha.].sub.D, CD, .sup.1H NMR, .sup.13C NMR,
DEPT, 2D-NMR, and HRESIMS). The remaining compounds isolated were
identified as 24-methylcholesta-4,24(28)-dien-3-one (3),
cholest-4-en-3-one (4), stigmast-4,22-dien-3-one (5),
stigmast-4-en-3-one (6), cholest-5,20,24-trien-3.beta.-ol (7),
cholest-5,24-dien-3.beta.-ol (demosterol, 8),
24-methylcholest-5,24(28)-dien-3.beta.-ol (9), cholesterol (10),
isofucosterol (11), .beta.-sitosterol (12), 7-ketositosterol (13),
7-ketostigmasterol (14), 6,7-dihydroneridienone (15), neridienone
(16), serborosin (17), xanthoxyletin (18), xanthyletin (19), and
oleanolic acid 3-acetate (20), using comparison of their physical
and spectroscopic data with published values. Table 2 below lists
the specific compound isolated from the root of P. antrieuxii.
TABLE-US-00002 TABLE 2 Compound Number Stucture 1 ##STR00077## 2
##STR00078## 3 ##STR00079## 4 ##STR00080## 5 ##STR00081## 6
##STR00082## 7 ##STR00083## 8 ##STR00084## 9 ##STR00085## 10
##STR00086## 11 ##STR00087## 12 ##STR00088## 13 ##STR00089## 14
##STR00090## 15 ##STR00091## 16 ##STR00092## 17 ##STR00093## 18
##STR00094## 19 ##STR00095## 20 ##STR00096##
[0327] Nine compounds (Table 3) were isolated and identified from
pooled active subfractions (PASD3F2, PASD3F3 and PASD3F4) of a
CHCl.sub.3-soluble extract (PASD3) of the stem of P. antrieuxii.
The structures of these compounds were elucidated as
pentalinonoside (2, novel compound, also found from root extract),
6,7-dihydroneridienone A (15), neridienone A (16), cybisterol (21),
12.beta.-hydroxypregn-4-ene-3,20-dione (22), fraxidin (23),
fraxetol (fratexin, 24), betulinic acid (25), and (+)-pinoresinol
(26). Table 4 below lists the specific compound isolated from the
stem of P. antrieuxii.
TABLE-US-00003 TABLE 3 Compound Number Stucture 2 ##STR00097## 15
##STR00098## 16 ##STR00099## 21 ##STR00100## 22 ##STR00101## 23
##STR00102## 24 ##STR00103## 25 ##STR00104## 26 ##STR00105##
[0328] 4. Structure Elucidation of Novel Compounds.
[0329] Compound 1 was obtained as a colorless gum. The molecular
formula was assigned as C.sub.27H.sub.40O, based on the ion peak at
m/z 381.3154 [M+H].sup.+ (calcd for C.sub.27H.sub.41O for
C.sub.27H.sub.41O, 381.3157) in the HRESIMS. The typical IR
absorptions supported the presence of methyl (2953 and 2873
cm.sup.-1), methylene (2925 and 2852 cm.sup.-1), ketone (1733
cm.sup.-1), and double bond (1676 cm.sup.-1) moieties. The UV
maximum at 239 nm (log .epsilon. 3.99) suggested the presence of an
.alpha.,.beta.-unsaturated ketone group. The .sup.1H NMR
spectroscopic data of 1 (Table 2) showed two characteristic broad
singlets at .delta..sub.H 4.80 (1H) and 4.89 (1H), consistent with
the presence of a vinylic group. A singlet at .delta..sub.H 5.73
(1H, H-4) and a broad triplet at 5.11 (1H, br t, J=7.0 Hz, H-24)
indicated the occurrence of two trisubstituted double bonds. In the
high-field region, proton signals of two tertiary methyl groups at
.delta..sub.H 0.62 (3H, s, H-18) and 1.18 (3H, s, H-19), together
with signals of two tertiary methyl groups linked to the double
bond at .delta..sub.H 1.61 (3H, s, H-26) and 1.69 (3H, s, H-27),
could be recognized. Besides these methyl group protons, the highly
overlapped signals distributed in the high field region from 0.8 to
2.4 ppm suggested the presence of a group of alkyl m' ethylene and
methine signals. Consistent with the .sup.1H NMR data, the .sup.13C
NMR spectroscopic data of 1, which were sorted using DEPT and HSQC
spectra, showed signals of a vinylic group at .delta..sub.C 149.4
(C-20) and 110.1 (C-21), two trisubstituted double bonds at
.delta..sub.C 124.2 (C-4), 172.0 (C-5), 124.7 (C-24), and 131.3
(C-25), and four methyl carbons at .delta..sub.C 13.3 (C-18), 17.8
(C-19 and C-26), and 26.2 (C-27). Furthermore, the other carbon
signals in the .sup.13C NMR spectrum could be classified into ten
methylene carbons, four methine carbons, two quaternary carbons,
and a ketone group. The NMR information combined with the molecular
formula obtained from the HRESIMS, suggests that compound 1 is a
cholestane steroid with an .alpha.,.beta.-unsaturated ketone
feature within the tetracyclic ring system and two double bonds in
the side chain. In the HMBC spectrum of 1, correlations from H-4 to
C-6 and C-10, and from H.sub.3-19 to C-1, C-5, and C-9, confirmed
the 3-keto-4-ene functionality of the A ring. The key HMBC
correlations of the vinylic protons (H-21) with C-17 and C-22,
H.sub.3-26 and H.sub.3-27 with C-24 and C-25, led to the placement
of the two double bonds at C-20 and C-24 of the side chain,
respectively (FIG. 3). Observed key NOE effects of H-8/H.sub.3-19,
H-8/H.sub.3-18, H-14/H-9, H-14/H-17, and H-21/H-18 were used to
establish the relative configuration of compound 1. The absolute
configuration of 1 was determined from the CD spectrum, and 1
showed a weak negative band around 317 nm (.DELTA..epsilon.=-1.44),
corresponding to an .alpha.,.beta.-unsaturated carbonyl
n-.pi.*excitation, and an intensive positive band around 238 nm
(.DELTA..epsilon.=+7.14), representing a .pi.-.pi.*transition.
These observed Cotton effects were very similar to those of the
known compound, (8S,9S,10R,13R,14S,17R,20R)-cholest-4-en-3-one.
Thus, the structure of the new compound 1 was proposed as
(8S,9S,10R,13R,14S,17R)-cholest-4,20,24-trien-3-one, and was
accorded the trivial name, pentalinonsterol.
[0330] The specific characterization data of pentalinonsterol (1):
colorless gum; [.alpha.].sup.20.sub.D+56.0 (c .sub.0.1,
CH.sub.2Cl.sub.2); UV (MeOH) .lamda..sub.max (log .epsilon.) 239
(3.99), 316 (0.90) nm; CD (c 1.31.times.10.sup.-5 M,
CH.sub.2Cl.sub.2/MeOH) .lamda..sub.max (.DELTA..epsilon.) 238
(+7.14), 317 (-1.44) nm; IR (film) .nu..sub.max 2952, 2925, 2873,
2852, 1733, 1676, 1456, 1376, 1230, 1170 cm.sup.-1; .sup.1H NMR
(400 MHz, CDCl.sub.3) and .sup.13C NMR (100 MHz, CDCl.sub.3) data,
see Table 2; HRESIMS m/z m/z 381.3154 [M+H].sup.+ (calcd for
C.sub.27H.sub.41O, 381.3157).
[0331] Compound 2 was obtained as a colorless resin. The HRESIMS of
2 afforded a sodiated molecular ion peak at m/z 513.2817,
corresponding to an elemental formula of C.sub.28H.sub.42O.sub.7Na
(calcd m/z 518.2828). The IR spectrum exhibited typical absorptions
of hydroxy (3477 cm.sup.-1), alkyl methyl (2949 and 2889
cm.sup.-1), and methylene (2933 and 2870 cm.sup.-1) moieties. In
the UV spectrum, no obvious absorption was observed within the
200-400 nm region. In the .sup.1H NMR spectrum of 2, a 2,6-deoxy
sugar unit was evident from signals at .delta..sub.H 4.84 (1H, dd,
J=9.6, 2.0 Hz, H-1', the anomeric proton), 1.73 and 2.10 (each 1H,
m, H-2'), 3.43 (1H, ddd, J=12.0, 5.0, 2.0 Hz, H-3'), 3.91 (1H, brs,
H-4'), 3.60 (1H, q, J=6.5 Hz, H-5'), 1.55 (1H, d, J=6.5 Hz, H-6'),
and 3.40 (3H, s, OCH.sub.3-3'). The chemical shifts and the
coupling pattern of these proton signals were found to be
comparable with those of diginose, and the large Jvalue (9.6 Hz) of
the anomeric proton indicated the 13 configuration of this
2,6-deoxyhexose sugar unit (Zhao et al., 2007; Kuroda et al.,
2010). The corresponding .sup.13C NMR data of this monosaccharide
were observed at .delta..sub.C 98.8 (C-1', anomeric carbon), 33.3
(C-2'), 79.3 (C-3'), 67.1 (C-4'), 71.5 (C-5'), 17.8 (C-6'), and
55.4 (OCH.sub.3-3'). Besides the signals of the sugar unit, the
.sup.1H NMR spectrum also displayed signals of seven oxygenated
protons at .delta..sub.H 3.90 (1H, m, H-3), 4.02 (dd, J=10.0, 4.4
Hz, H.sub.a-15), 4.23 (1H, d, J=10 Hz, H.sub.b-15), 4.56 (1H, d,
J=4.5 Hz, H-16), 4.49 (1H, ddd, J=7.7, 6.2, 1.5 Hz, H-20), 3.79
(dd, J=12.4, 1.3 Hz, H.sub.a-21), 4.06 (dd, J=12.0, 6.0 Hz,
H.sub.b-21), and 4.02 (dd, J=10.0, 4.4 Hz, H.sub.a-15), a signal
for an olefinic proton at .delta..sub.H 5.52 (1H, t, J=2.5 Hz,
H-6), and the presence of two tertiary methyl groups at
.delta..sub.H 0.97 (3H, s, H-19) and 1.08 (3H, s, H-18), as well as
a number of highly overlapped signals distributed in the high-field
region from 0.8 to 2.4 ppm for alkyl methylenes and methines, all
of which could be attributed to a highly oxygenated steroidal
aglycone moiety. The 21 skeletal signals in the .sup.13C NMR
spectrum were classified by the DEPT and HSQC spectra into two
methyls, six methylene groups, two alkyl methines, three quaternary
alkyl carbons, six oxygenated carbons (including two primary, three
secondary and one tertiary), and a trisubstituted double bond. This
suggested that compound 2 is a 3-oxy-4-en-14,15-secopregnane
derivative (Plaza et al., 2003 and 2005; Perrone et al, 2006 and
2008). In the COSY spectrum, the oxygenated methine proton at
.delta..sub.H 4.56 (1H, t, J=4.5 Hz, H-16) showed correlations with
an alkyl methine proton at .delta..sub.H 2.60 (1H, dd, J=7.6, 4.4
Hz, H-17) and one geminal proton of an oxygenated methylene at
.delta..sub.H 4.02 (1H, dd, J=10.0, 4.4 Hz, H.sub.a-15). Another
oxygenated methine proton at .delta..sub.H 4.49 (1H, ddd, J=7.6,
6.0, 1.5 Hz, H-20) also showed correlations with H-17, and two
geminal protons of an oxygenated methylene at .delta..sub.H 4.06
(1H, dd, J=12.4, 6.0 Hz, H.sub.a-21) and 3.79 (1H, d, J=12.4, 1.3
Hz, H.sub.b-21), respectively. Thus, an important spin system
including H-15, H.sub.2-16, H-17, H-20 and H.sub.2-21 in compound 2
was deduced as shown (FIG. 3). Furthermore, the key HMBC
correlations from H-16, H-17 and H-21 to C-14, and from H-15 and
H-16 to C-20, were used to construct a highly oxygenated unit. This
unusual moiety in 2 involved the opening of the ring D of the
pregnane skeleton between C-14 and C-15, and the connection through
oxygen bridges of C-14 with C-16, C-15 with C-20, as well as C-21
with C-14 to generate a six-membered ring and two tetrohydrofuran
rings with a ketal group on C-14.
[0332] The occurrence of 14,15-secopregnane derivatives with
similar polyoxygenated features to 2 is rare in the plant kingdom,
and other naturally occurring compounds of this class have been
found from a limited number of plants such as species in the genera
Cynanchum and Solenostemma of the family Asclepiadaceae, and the
genus Mandevilla of the family Apocynaceae (Kennard et al., 1968;
Lavault et al., 1999; Chen et al., 2008; Plaza et al., 2003 and
2005; Perrone et al, 2006 and 2008; Niero et al., 1999 and 2002;
Yunes et al., 1993). These compounds are characterized by the
cleavage of the D ring between C-14 and C-15, a
hexahydrofuro[3,4-b]furan ring on ring C, with C-20 connected to
C-14 or C-18 through an oxygen atom, to generate ketal groups at
C-14 and/or C-20. A structurally unusual feature of compound 2 is
that it exhibits for the first time a C-21 methyl group in the
pregnane skeleton incorporated into two tetrohydrofuran rings to
form a ketal; group at C-14. The relative configuration of 2 was
deduced from a NOESY NMR experiment. The key NOE effects between
H-17 with H-16 suggested a cis junction of the two tetrohydrofuran
rings of the highly oxygenated ring system, and the NOE effects
between H-18 with H-20 and H.sub.a-21 indicated the
.beta.-orientation of the C-21 oxygenated methylene group. The
relative configurations of the remaining portion of the aglycone
were consistent with those of known pregnane steroids, based on an
analysis of the NOE spectrum. The 13 position of the diginosyl
group on C-3 was deduced by the NOE correlations of H-3 with
H.sub..alpha.-4 and H.sub..alpha.-2. The configuration of the
.beta.-diginose unit was confirmed by NOE correlations between
H-1'/H.sub..alpha.-2', H-3', and H-5', H-4'/H-5,
OCH.sub.3-3'/H.sub..beta.-2'. The sugar unit obtained by acid
hydrolysis of compound 1 exhibited a positive optical rotation
value, which demonstrated that the absolute configuration of the
sugar is in the D form. Furthermore, an energy-minimized model
generated by Chem3D Ultra 10.0 based on a presumed configuration
matched well with all the observed NOESY correlations and served to
explain the observed coupling patterns for H.sub.2-15, H-16, H-17,
H-20, and H.sub.2-21 of the polyoxygenated ring system (FIG. 4).
Thus, the structure of compound 2 (pentalinonside) was determined
as
14,16-14,21-15,20-triepoxy-14,15-secopregnan-5-en-3-O-.beta.-D-diginopyra-
noside.
[0333] The specific characterization data of pentalinonside (2):
colorless gum; [.alpha.].sup.20.sub.D-30.0 (c 0.1, MeOH); UV (MeOH)
end absorption; IR (film) .nu..sub.max 3477, 2949, 2933, 2889,
2870, 1640, 1460, 1381, 1258, 1168, 1098, 1067 1025, and 977
cm.sup.-1; .sup.1H NMR (400 MHz, CDCl.sub.3) and .sup.13C NMR (100
MHz, CDCl.sub.3) data, see Table 2; HRESIMS m/z 513.2817 m/z
[M+Na].sup.+ (calcd for C.sub.28H.sub.42O.sub.7Na, 518.2828).
[0334] For acid hydrolysis of compound 2, 2.0 mg dissolved in MeOH
(0.8 mL) was added to 0.05 M H.sub.2SO.sub.4 (0.8 mL), and the
solution was then heated at 70.degree. C. for 3 h. The reaction
mixture was neutralized by passage through a Dowex.RTM. 1.times.2
column (2.0.times.2.0 cm), and was then chromatographed on an ODS
column (1.5.times.2.0 cm) with MeOH--H.sub.2O (40:60 to 70:30) as
eluent, to give diginose (0.2 mg). This 2,6-deoxyhexose sugar unit
was assigned in the D-form based on its optical rotation value.
D-diginose: [.alpha.].sub.20.sup.D+21.0 (c 0.02, 24 h after
dissolution in H.sub.2O) [lit. [.alpha.].sup.27.sub.D+16.4 (c 0.05,
H.sub.2O)].
[0335] .sup.1H and .sup.13C NMR data of compounds 1 and 2 is
provided in Table 4. .sup.1H NMR spectrum was measured at 400 MHz
and .sup.13C NMR was measured at 100 MHz. NMR data of 1 were
obtained in CDCl.sub.3 and the NMR data of 2 were obtained in
pyrdine-d.sub.5. Assignments are based on HSQC and HMBC NMR
spectra. J values (Hz) are given in parentheses, and some geminal
protons are indicated with the .alpha.- or .beta.-orientation based
on a NOESY experiment. Multiplicity was obtained from the DEPT
spectra. For overlapped signals, only chemical shift values are
given.
TABLE-US-00004 TABLE 4 Compound 1 Compound 2 position .delta..sub.H
(mult. J, Hz).sup.b .delta..sub.C, mult..sup.c .delta..sub.H (mult.
J, Hz).sup.b .delta..sub.C, mult..sup.c 1 1.73.sup.d 36.1, CH.sub.2
1.05 .alpha.,.sup.d 37.5, CH.sub.2 2.02.sup.d 1.75 .beta.,.sup.d 2
2.40.sup.d 34.4, CH.sub.2 2.13 .alpha.,.sup.d 30.4, CH.sub.2 1.68
.beta.,.sup.d 77.3, CH 3 200.1, qC 3.90,.sup.d 39.4, CH.sub.2 4
5.73, s 124.2, CH 2.59 .alpha.,.sup.d 2.40 .beta.,.sup.d 5 172.0,
qC 139.7, qC 6 2.30.sup.d 33.4, CH.sub.2 5.52 t (2.5) 122.6, CH
2.40.sup.d 7 1.02.sup.d 32.4, CH.sub.2 1.67.sup.d 25.5, CH.sub.2
1.84.sup.d 2.50.sup.d 8 1.58.sup.d 36.4, CH 1.85.sup.d 36.2, CH 9
1.00.sup.d 54.4, CH 1.42.sup.d 46.1, CH.sub.2 10 39.0, qC 37.0, qC
11 1.43.sup.d 21.5, CH.sub.2 1.43.sup.d 20.6, CH.sub.2 1.59.sup.d
12 1.25 .alpha.,.sup.d 38.9, CH.sub.2 1.38 .alpha.,.sup.d 34.9,
CH.sub.2 1.88 .beta.,.sup.d 2.40 .beta.,.sup.d 13 43.5, qC 41.5, qC
14 1.15.sup.d 56.2, CH 108.2, qC 15 1.22.sup.d 24.5, CH.sub.2 4.02
a, dd 74.3, CH.sub.2 (10.0, 4.4) 1.73.sup.d 4.23 b, d (10.0) 16
1.76.sup.d 26.2, CH.sub.2 4.56 t (4.5) 80.9, CH 1.80.sup.d 17
2.10.sup.d 56.3, CH 2.60, dd 54.6, CH (7.6, 4.4) 18 0.62, s 13.3,
CH.sub.3 1.08, s 17.3, CH.sub.3 19 1.18, s 17.8, CH.sub.3 0.97, s
19.6, CH.sub.3 20 149.4, qC 4.49, ddd 76.3, CH (7.6, 6.0, 1.5) 21
4.80, brs 110.1, CH.sub.2 4.05 a, dd 66.3, CH.sub.2 (12.4, 6.0)
4.89, br s 3.79 b, dd (12.4, 1.3) 22 2.06.sup.d 38.0, CH.sub.2 23
2.10.sup.d 27.5, CH.sub.2 24 5.11, br t (7.0) 124.7, CH 25 131.3,
qC 26 1.61, s 17.8, CH.sub.3 27 1.69, s 26.2, CH.sub.3 1' 4.84, dd
98.8, CH (9.6, 2.0) 2' 1.73, m 33.3, CH.sub.2 2.10, m 3' 3.43, ddd
79.3 , CH (12.0, 5.0, 2.0) 4' 3.91, brs 67.1, CH 5' 3.60, q (6.5)
71.5, CH 6' 1.55, d (6.4) 17.8, CH.sub.3 OCH.sub.3-3' 3.40, s 55.4,
CH.sub.3 .sup.a1H NMR measured at 400 MHz, .sup.13C NMR measured at
100 MHz; NMR data of 1 were obtained in CDCl.sub.3, and those of 2
in pyridine-d.sub.5; assignments based on HSQC and HMBC NMR
spectra. .sup.bJ values (Hz) are given in parentheses. Some geminal
protons were indicated as .alpha.- or .beta.-oriented based on a
NOESY experiment. .sup.cMultiplicity obtained from the DEPT
spectra. .sup.dFor overlapped signals, only chemical shift values
are given.
[0336] 5. Leishmania mexicana Parasite Strain.
[0337] a. Promastigote Assay.
[0338] Leishmania mexicana (MNYC/BZ/62/M379) was maintained by
serial passage of amastigotes inoculated subcutaneously into the
shaven rumps of 129SvE mice and was obtained by in vitro culture of
amastigotes in RPMI-1640 supplemented with 10% fetal calf serum
(FCS, Sigma-Aldrich Corporation, St. Louis, Mo.) and incubated at
28.degree. C. All work including experimental animals was approved
by the Institutional Animal Care and Use Committee (IACUC) of The
Ohio State University.
[0339] 6. In Vitro Antilieshmanial Activity Studies.
[0340] a. Promastigote Assay.
[0341] One million log phase promastigotes of L. mexicana were
seeded in 1 mL of complete RPMI-1640 medium, and parasite numbers,
and their mobility and morphology were measured from 24 and 48 h.
This was performed by flow cytometry measurements using
experimental and control groups of parasites stained with propidium
iodide and with Quillaja saponaria saponin as positive control, as
reported in the literature. The test compounds isolated from P.
andrieuxii were compared with a positive control: sodium
stibogluconate, a pentavalent antimonial drug.
[0342] b. Amastigote Assay.
[0343] Bone marrow derived macrophages were obtained from C57BL/6
mice. Bone marrow-derived macrophages (BMDM) (0.5.times.10.sup.6)
were adhered on top of glass rounded coverslips deposited at the
bottom of individual plate's well (24 well tissue culture plate,
Corning, Inc.). Next, macrophages were infected overnight with
2.5.times.10.sup.6 stationary phase promastigotes of the L.
mexicana (ratio 5:1). After this period, cells were extensively
washed with Hank's balanced salt solution (HBSS) to eliminate
non-phagocytosed parasites, and, after treatment with test
compounds for 48 hours, cells were stained with Giemsa stain
(Sigma). In each experiment, infection rates were recorded by
counting the number of parasites in 100 macrophages on each slide
in triplicate in a blinded fashion.
[0344] 7. Cytotoxicity Assay in Non-Infected Macrophages.
[0345] Bone marrow derived-macrophages (0.5.times.10.sup.6) from
C57BL/6 mice were seeded into 24-well tissue culture plates
(Corning, Inc.) and co-cultured (48 h) with a 100 .mu.M
concentration of each test compound, including non-treated sham
controls. Cells were stained with trypan blue for viability
determination (Ferreira et al., 2011). Viability of treated cells
was similar to sham controls (more than 80%) in experiments ran in
triplicate.
[0346] 8. In Vitro Assay and Cell Sample Preparation for Electron
Microscopy (EM).
[0347] Intracellular parasites were co-cultured, as previously
described herein, by treating a 100 .mu.M concentration of each
pure compound for 2 h and suspending them in glutaraldehyde.
Samples were further processed in an electron microscopy unit as
follows: Incubation steps were carried out on a Lab-Line orbital
shaker operating at 700 rpm (Barnstead/Thermolyne, Melrose Park,
Ill.). After the initial fixation in 3% buffered glutaraldehyde,
cell pellets were washed twice with sodium cacodylate buffer (pH
7.4, 10 minutes each) and spun down after each wash at 1500 rpm for
5 min. Cell samples were then post-fixed in 1% osmium tetroxide in
sym-collidine buffer (pH 7.6) for 1 h at room temperature.
Following two washes with sym-collidine buffer (10 min each) the
cell pellet was stained with a saturated aqueous uranyl acetate
solution (pH 3.3) for 1 h. Cell pellets were dehydrated in a graded
EtOH series up to absolute (10 min each). Acetone was used as the
transitional solvent, two changes for 10 min each. The cell
suspensions were infiltrated overnight with a 1:1 mixture of
acetone and Spurr's epoxy resin (Electron Microscopy Sciences, Fort
Washington, Pa.). Finally, cell pellets were placed into BEEM.TM.
embedding capsules containing 100% Spurr's resin. Polymerization of
epoxy blocks was carried out at 70.degree. C. overnight.
Polymerized blocks were sectioned with a Leica Ultraut UCT
ultramicrotome (Leica Microsystem GmbH, Wein, Austria). Methylene
Blue-Basic Fuschin stained semi-thin (750 nm) sections were
evaluated and two representative areas were thin-sectioned for
ultrastructural examination. Ultrathin (80 nm) sections were
collected on 200 mesh copper grids (Electron Microscopy Sciences)
and post-stained with lead citrate (3 min).
[0348] 9. In Vitro Activity of Compounds.
[0349] All the isolated compounds (1-20) from the roots of P.
antrieuxii were evaluated for their antileishmanial activity on
both the promastigote and amastigote stages of L. mexicana. After
48 h of exposure against L. mexicana promastigotes, five sterols
(1, 3, 4, 15, and 16) were found to show inhibitory effects and
were more active than that observed for the reference compound,
pentostam. Of these compounds, 6,7-dihydroneridienone (15) (FIG. 5,
panel D) was the most potent principle, with an IC.sub.50 value of
9.2 .mu.M. The novel cholestrol analogue,
cholest-4,20,24-trien-3-one (pantalinonsterol, 1) (FIG. 6, panel B)
together with three additional sterols, cholest-4-en-3-one (4)
(FIG. 7, panel B), 24-methylcholest-4,24(28)-dien-3-one (3) (FIG.
6, panel D) and neridienone (16) (FIG. 5, panel B), exhibited
antileishmanial activity against promastigotes of L. mexicana. The
sham control and pentosam positive control are shown in FIG. 8,
panel B and D, respectively.
[0350] All five compounds with leishmanicidal activity against L.
mexicana promastigotes, together with compound 7, which was
considered inactive in the extracellular stage (FIG. 7, panel D),
were observed to show significant activity against the amastigote
stage. Among these compounds, 1 (FIG. 6, panel A), 3 (FIG. 6, panel
C), 15 (FIG. 5, panel C), and 16 (FIG. 5, panel A), gave IC.sub.50
values of 3.3, 3.5, 1.4 and 3.5 .mu.M, respectively, and exhibited
comparable potency to the positive control, pentostam (IC.sub.50
2.7 .mu.M). Compound 7 (FIG. 7, panel C) was observed to be less
active than pentostam (FIG. 8, panel C), with an IC.sub.50 value of
14.5 .mu.M. Compound 4 (FIG. 7, panel A) showed the most potent
leishmanicidal activity on this intracellular stage, with an
IC.sub.50 value of 0.03 .mu.M, nearly 100 times more potent than
pentostam.
[0351] With the exception of compound 7, all the other active
sterols with leishmanicidal activity in the present study were
observed to share a common 4-ene-3-oxo functionality in the
steroidal ring system, while most of sterols (8-14) with a
3-ol-5-ene moiety, no obvious antileishmanial activity was
observed. Without wishing to be bound by a particular theory, it is
believed that variation of the side chain on the five-membered D
ring of these 4-ene-3-oxo sterols also influenced the resultant
activity. Thus, compounds 5 and 6, two stigmasterol derivatives
with an extra ethyl group at C-24 of the side chain when compared
with the active cholestrol derivatives 1 and 4, were both inactive
in the bioassays used. The novel compound, pentalinosterol (1),
with double bonds at C-20 and C-24, was observed to be almost three
times more active in the promastigote bioassay than
cholest-4-en-3-one (4), for which the side chain is saturated. None
of the 3-ol-5-ene sterols was found active in the promastigote
assay. Compound 7, the only agent isolated with a 3-ol-5-ene
fuctionality active in the amastigote assay, possesses the same
side chain on the D ring as that in compound 1. For the two active
C.sub.21 sterols, 6,7-dihydroneridienone (15) and neridienone (16),
both have an acetyl group on the D ring instead of a long alkyl
chain that occurs in other active compounds. Compound 15 was
observed to be more active than 16 in both the promastigote and
amastigote bioassay, with the latter possessing an extra double
bond at C-5 in the B ring.
[0352] The in vitro antileishmanial activities of the active
compounds isolated from the roots of Pentalinon andrieuxiia were
compared to sodium stibogluconate (Table 5). Compounds with
IC.sub.50 100 .mu.g/mL are not shown in the table below and the
compound number corresponds to the numbering used in Table 2 above.
The IC.sub.50 values (.mu.M) were calculated by linear regression
analysis from the K.sub.C values at the concentrations used (1, 10,
50 and 100 .mu.g/mL) at 48 hours in culture.
TABLE-US-00005 TABLE 5 Promastigote Amastigote Compound IC.sub.50
(.mu.M) IC.sub.50 (.mu.M) 1 30.0 3.3 3 24.0 3.5 4 81.0 0.03 7
>262 14.5 15 26.2 3.5 16 9.2 1.4 Sodium 346.1 2.7
stibogluconate
[0353] All the isolates were also evaluated for cytotoxicity in
non-infected bone marrow derived macrophages (FIG. 9, panel A) from
C57BL16 mice, the host cells. None of the compounds were found
active in this bioassay (IC.sub.50>100 .mu.g/mL), suggesting the
compounds of the present invention are selective for the protozoal
cell.
[0354] Intracellular parasites treated with compounds 1, 3 (FIG.
10, panel A), 4 (FIG. 10, panel C), 15 (FIG. 7, panel C), and 16
were further studied by electron microscopy. The intracellular
parasites were treated for 2 h at a 100 .mu.M concentration of the
individual bioactive molecules (compounds 1, 3, 4, 15, and 16).
Electron microscopy shows morphological abnormalities (FIG. 12,
panel C) and destruction as compared to sham control (FIG. 12,
panel A) or reference drug-treated parasites (100 .mu.M) (FIG. 12,
panel C). Membrane alterations in the parasites were observed after
treating the organisms with the active sterols. Without wishing to
be bound by a particular theory, the morphological changes seen in
the EM images suggests the active sterols can replace cholesterol
during the membrane biosynthesis of the parasites and induce
membrane instability and disruption.
[0355] 10. Preparation of Liposomes for Use in In Vivo Studies.
[0356] Liposomes were made by rehydration of a lipid cake. First,
hydrogenated soy phosphatidylcholine (Avanti Polar Lipids, Inc.,
Alabaster, Ala.), cholesterol (Avanti Polar Lipids),
1,2-distearoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] ("DSPG";
Avanti Polar Lipids,), and .alpha.-tocopherol (Sigma-Aldrich
Corporation, St. Louis, Mo.) were dissolved in a chloroform and
methanol (9:1, v/v). Compound 1 was dissolved in the lipid solution
at a weight ratio of compound 1 to lipid of 1:9 (i.e. for clarity,
the solution comprised 10% by weight of compound 1 and 90% by
weight of the lipid solution). The solution was placed on at rotary
evaporator for about 30 minutes and the liquid was completely
evaporated. The lipids were reconstituted into liposomes in
deionized water (3 mL) for 30 minutes in a 60.degree. C. water
bath, followed by 3 freeze-thaw cycles. The liposomes were extruded
through an 80 nm polycarbonate filter (Avanti Polar Lipids). The
extrusion step was repeated about 10-11 times. Liposomes containing
compound 1 were passed over a PD-10 column (GE Healthcare, USA)
with a deionized water mobile phase in order to remove any
unencapsulated drug. Sucrose (300% of lipid weight) was added to
the liposomes and the liposome-sucrose solution was lyophilized.
Loading of liposomes was verified and quantified by HPLC.
[0357] ii. In Vivo Activity of Pentalinon andrieuxii Root Extract
and an Isolated Compound.
[0358] a. Antilieshmanial Activity of a Pentalinon andrieuxii root
Extract in L. mexicana.
[0359] A preliminary in vivo experiment with Pentalinon andrieuxii
root extract (PARE), was performed to evaluate potential topical
leishmanicidal activity. 10 week old male C57BL/6 mice were
infected with L. mexicana promastigotes in the ear dermis.
Following innoculation with L. mexicana promastigotes, the infected
ear was treated topically once daily for 21 days with either drug
(10 .mu.g of PARE dissolved in 50 .mu.l of DMSO/PBS) or control
(DMSO). Lymphocytes and macrophages (white cells) from mice treated
with PARE (FIG. 13, panel A) did not show the presence of
parasites. In contrast, the lymphocytes and macrophages (white
cells) from mice treated with control showed the intracellular
presence of parasite (FIG. 13, Panel B). In conjunction with the
overall bioassay testing results observed in this disclosure, this
data suggest that PARE, and its sterol constituents, have
leishmanicidal activity.
[0360] b. Antilieshmanial Activity of Liposomal Preparation of
Compound 1 on L. donovani.
[0361] In vivo studies were carried out with a liposomal
formulation of compound 1 in order to evaluate parenteral
leishmanicidal activity. Briefly, 8-10 week old
Leishmania-susceptible BALB/c mice were injected with
2.times.10.sup.7 Leishmania donovani (Strain LV9) amastigotes via
the tail vein. The injection volume was 100 .mu.L. Two weeks after
infection, the mice were administered a liposomal preparation of
compound 1 via injection of the tail vein. The liposomal
preparation used in these studies was prepared as described above.
Dose groups (n=3) were administered 100 .mu.L of a liposomal
suspension with or without compound. The liposomal preparation with
compound (100 .mu.L; 3.525 mg liposome preparation) comprised
415.53 .mu.g lipids, 46.1 .mu.g compound 1, and 3063.3 .mu.g
sucrose; the liposomal preparation without compound (100 .mu.L;
3.525 mg liposome preparation) comprised 461.7 .mu.g lipids and
3063.3 .mu.g sucrose.
[0362] One week after the treatment, the mice were euthanized and
parasite loads in the liver and spleen were determined. Parasite
loads in the spleen and liver were quantified as previously
described ((Murray. H. W. 2000. Infect. Immun. 68: 6294-6299).
Briefly, the liver and spleen impression smears were stained using
Giemsa and parasite loads we quantified microscopically. LDU is
determined by the number of amastigotes per 1000 nuclei multiplied
by the weight (gm) of the liver or spleen. At this time, spleen
cell suspensions were prepared and these cells were stimulated in
vitro with 20 microgram/ml of Leishmania donovani antigen for 48-72
hrs to stimulate lymphocyte release. Proliferation of spleen cells
were determined by Alamar blue assays and levels of cytokines
including IFN-gamma in supernatant were quantified by ELISA using
commercially available reagents from Biolegend and BD Biosciences.
The parasite load detected in splenic cells (FIG. 14, panel A) and
hepatic cells (FIG. 14, panel B) samples taken from the
drug-treated mice showed a decreased parasite load, suggesting that
compound 1 has leishmanicidal activity in vivo.
[0363] T cell proliferation was determined as previously described
(Rosas, L. E., et al., 2006. Am. J. Pathol. 168: 158-169.).
Briefly, 5.times.10.sup.5 cells were added in quadruplicate to the
wells of sterile 96-well, flat-bottom tissue culture plates and
stimulated with freeze-thawed L. donovani Ag (20 .mu.g/ml). The
proliferation responses were measured by Alamar blue assay
(Ansar-Ahmed, A., et al., 1994. J. Immunol. Methods 170: 211-214.).
Supernatants were collected after 72 hours of incubation at
37.degree. C. and analyzed for the production of IFN-.gamma. and
IL-10 by standard ELISA methods (BD Pharmingen, Inc., San Diego,
Calif.).
[0364] The data (FIG. 15) show that splenocytes from animals that
were treated with compound 1 had a significant increase (P
value<0.0001) in T cell proliferation upon stimulation with
Leishmanial antigen compared to a parallel group that did not
receive compound 1. The data show that splenocytes from animals
that were treated with compound 1 had a significant increase (P
value<0.01) in the secretion of IFN-.gamma. upon with
Leishmanial antigen stimulation (FIG. 16, panel A), whereas there
was no significant change in the levels of IL-10 secreted from the
same splenocyte samples (FIG. 16, panel B). Without wishing to be
bound by a particular theory, increased levels of IFN-gamma and
T-cell proliferation are believed to be a critical hallmarks for an
effective protective immunity response to VL.
[0365] 12. Prophetic Pharmaceutical Composition Examples
[0366] "Active ingredient" as used throughout these examples
relates to one or more disclosed compounds, a product of a
disclosed method of making, or a pharmaceutically acceptable salt,
solvate, polymorph, hydrate or stereochemically isomeric form
thereof. The following examples of the formulation of the compounds
of the present invention in tablets and injectable formulations are
prophetic. Typical examples of recipes for the formulations of the
invention are as given below.
[0367] In the examples below, active ingredient can be replaced
with the same amount of any of the compounds according to the
present invention, in particular by the same amount of any of the
exemplified compounds.
[0368] The amount of a disclosed compound in a pharmaceutical
composition in terms of dosage unit, e.g. an ampule for single use
administration or a table, for human use is determined from both
toxicological and pharmacokinetic data obtained in suitable animal
models, e.g. rat and at least one non-rodent species, and adjusted
based upon human clinical trial data. For example, it could be
appropriate that a disclosed compound is present at a level of
about 10 to 1000 mg per dosage unit.
[0369] a. Pharmaceutical Composition for Oral Administration
[0370] A tablet can be prepared as follows:
TABLE-US-00006 Component Amount Active ingredient 10 to 500 mg
Lactose 100 mg Crystalline cellulose 60 mg Magnesium stearate 5
Starch (e.g. potato starch) Amount necessary to yield total weight
indicated below Total (per capsule) 1000 mg
[0371] Alternatively, about 100 mg of a disclosed compound, 50 mg
of lactose (monohydrate), 50 mg of maize starch (native), 10 mg of
polyvinylpyrrolidone (PVP 25) (e.g. from BASF, Ludwigshafen,
Germany) and 2 mg of magnesium stearate are used per tablet. The
mixture of active component, lactose and starch is granulated with
a 5% solution (m/m) of the PVP in water. After drying, the granules
are mixed with magnesium stearate for 5 min. This mixture is
moulded using a customary tablet press (e.g. tablet format:
diameter 8 mm, curvature radius 12 mm). The moulding force applied
is typically about 15 kN.
[0372] Alternatively, a disclosed compound can be administered in a
suspension formulated for oral use. For example, about 100-5000 mg
of the desired disclosed compound, 1000 mg of ethanol (96%), 400 mg
of xanthan gum, and 99 g of water are combined with stirring. A
single dose of about 10-500 mg of the desired disclosed compound
according can be provided by 10 ml of oral suspension.
[0373] In these Examples, active ingredient can be replaced with
the same amount of any of the compounds according to the present
invention, in particular by the same amount of any of the
exemplified compounds. In some circumstances it may be desirable to
use a capsule, e.g. a filled gelatin capsule, instead of a tablet
form. The choice of tablet or capsule will depend, in part, upon
physicochemical characteristics of the particular disclosed
compound used.
[0374] Examples of alternative useful carriers for making oral
preparations are lactose, sucrose, starch, talc, magnesium
stearate, crystalline cellulose, methyl cellulose, hydroxypropyl
cellulose, hydroxypropylmethyl cellulose, carboxymethyl cellulose,
glycerin, sodium alginate, gum arabic, etc. These alternative
carriers can be substituted for those given above as required for
desired dissolution, absorption, and manufacturing
characteristics.
[0375] The amount of a disclosed compound per tablet for use in a
pharmaceutical composition for human use is determined from both
toxicological and pharmacokinetic data obtained in suitable animal
models, e.g. rat and at least one non-rodent species, and adjusted
based upon human clinical trial data. For example, it could be
appropriate that a disclosed compound is present at a level of
about 10 to 1000 mg per tablet dosage unit.
[0376] b. Pharmaceutical Composition for Injectable Use: Pegylated
Liposomes.
[0377] The compounds of the present invention can be used to
produce pegylated liposomal formulations. Pegylated liposomes can
be made using lipids that are covalently attached to polyethylene
glycol (PEG). The size (or molecular weight) of the PEG chains can
be varied to optimize the desired pharmacokinetic properties. By
adding these lipids in small percentages to existing formulations,
the circulation half life of the drug can be enhanced. Without
wishing to be bound by a particular theory, this formulation can
potentially allow for increased duration in blood circulation, and
can allow potentially better penetration into the bone marrow for
increased efficacy.
[0378] Liposomes are prepared by a simple film hydration method.
For example, required quantities of soyaphosphotidylcholine,
cholesterol (1:1, 2:1), MPEG 2000-DSPE (5 mol %)[14] and a
disclosed compound, e.g. compound 1, (0.2 mg/ml) in appropriate
molar ratios are dissolved in chloroform. Glass beads (10 g) are
added to increase surface area available for film formation.
Chloroform is evaporated under reduced pressure on a rotary
evaporator to form a thin film on the inner surface of the flask.
Lipid film is hydrated using above gel to liquid crystalline phase
transition temperature)(65.degree. of the lipids and cholesterol
for two minutes and flask manually shaken vigorously for 5 min for
formation of liposomes followed by heating it again for 2 min for
annealing of lipsomes. The dispersion is sonicated using bath
sonicator for 15 or 45 s to get the liposomes of smaller size.
[0379] c. Pharmaceutical Composition for Injectable Use:
Nanoparticles and Microparticles.
[0380] The compounds of the present invention can be used to
produce nanoparticles and microparticles. Nanoparticles and
microparticles can be made using hydrophobic degradable polymers to
encapsulate hydrophobic drugs, such as pentalinosterol. By using
emulsion processes, a disclosed compound, e.g. compound 1, can be
incorporated into the polymeric matrix. Additionally, PEG can be
incorporated into these particles to increase the circulation half
life.
[0381] Microparticles containing pentalinosterol are prepared using
an oil-in-water emulsion method. For example, a disclosed compound,
e.g. compound 1, is dissolved in chloroform, and this solution is
then used to dissolve (polylactide acid) PLA (100 mg). The
resulting solution is added to an aqueous solution (2 mL, 3% w/w in
PBS) of poly(vinyl alcohol) ("PVA," e.g. MW=13,000-23,000 g/mol,
87-89% hydrolyzed) and sonicated for 30 seconds on ice using a
probe sonicator (Branson Sonifier 450, with a 0.5 in. flat tip)
with an output setting of 3 and a duty cycle of 10%. The resulting
single emulsion is immediately poured into a second PVA solution
(10 ml, 0.3% w/w in PBS) and stirred for 4 hours to allow the
organic solvent to evaporate. The particles are isolated by
centrifugation (14,800.times.g, 15 min, 4.degree. C.) and washed
with H.sub.2O (e.g. double-distilled). The washed particles are
re-suspended in H.sub.2O (2 mL, pH 9) and lyophilized.
[0382] In a further example, calculated amounts of
poly(lactic-co-glycolic acid) (PLGA) and pentalinosterol are
dissolved in acetone and injected in DSPEmPEG2000 emulsifier
dissolved in water or PBS followed by immediate rigorous
emulsification by a high power sonicator. This result in the
synthesis of PEGylated nanoparticles (PNPs) of PLGA dispersed in
the aqueous solution, with pentalinosterol entrapped in the
hydrophobic PLGA matrix. The acetone can be removed acetone by
rotary vacuum evaporation and purified drug-loaded nanoparticles by
ultracentrifugation followed by rigorous washing (3.times.) with
water or PBS and resuspension in PBS.
[0383] d. Pharmaceutical Composition for Injectable Use:
Cyclodextrin Complexes.
[0384] The compounds of the present invention can be used to
produce cyclodextrin complexes. Cyclodextrins (CD) are capable of
forming complexes with hydrophobic drugs and have been used for
drug delivery. Depending on the size of the cyclodextrin (usually a
six, seven or eight member ring) and the size of the hydrophobic
drug, the complex will be formed. For example, a 1:1 or a 1:2 molar
ratio of drug and CD is prepared by freeze drying an aqueous
solution containing drug and various CDs (e.g. varying size and
hydrophobicity characteristics). The solution is filtred, frozen
and then freeze-dried at -52.degree. C. for 48 h, thereby yielding
a powder comprising the complex.
[0385] e. Pharmaceutical Composition for Injectable Use:
Microemulsions and Nanoemulsions.
[0386] The compounds of the present invention can be used to
produce microemulsion and nanoemulsions. Microemulsion droplets are
composed of a lipid core comprising a mixture of oil (e.g., soybean
oil) stabilized by a surfactant shell comprising a mixture of
PEG-surfactants, and dispersed in an aqueous phase (e.g. saline).
The microemulsion comprises the hydrophobic drug residing in the
lipid core, and such emulsions can be readily used for the
injection of the drug intravenously. Surfactants commonly used in
this technique are Tween and Span. Nanoemulsions can be produced in
a method similar to microemulsions, but high energy is required to
decrease the size of the emulsion
[0387] For example, a disclosed compound, e.g. compound 1 (30 mg),
is dissolved in an oil such as castor oil (about 20 g). The
resulting oil phase is mixed with polyethylene glycol (about 10.8
g), then mixed with Tween 80 (about 7.2 g), and water added so that
it is about 60% by volume, and then the mixture is passed through
high-pressure homogenizer.
[0388] Alternatively, a nanoemulsion can be prepared by mixing
about 80 mg of drug with about 2.8 g of a solution of castor oil
and middle-chain triglycerides (1:1, w/w), and then adding this
mixture to about 200 ml of acetone and ethanol (1:1, v/v)
containing about 2.0 g of soy lecithin. The oily phase is slowly
added under magnetic stirring into about 400 ml of aqueous phase
containing poloxamer 188 (about 600 mg) and glycerol (about 900
mg), thereby forming a nanoemulsion. Solvents and most water are
removed under reduced pressure resulting in about 40 ml of a
nanoemulsion formulation.
[0389] Alternatively, a nanoemulsion can also be prepared by
dissolving a mixture of caprylic/capric triglyceride (70%), soya
bean lecithin (27%), and cholesterol (3%) in chloroform-methanol
(2:1, v/v). To this liquid mixture is added a disclosed compound
(about 100 mg), e.g. compound 1. The mixture is dried under
nitrogen flow and kept under vacuum overnight in order to remove
organic solvent. Following removal of organic solvent, about 40 ml
of tris(hydroxymethyl)aminomethane (TRIS)-HCl buffer (0.01 M, pH
8.05) is added and emulsified with a probe-type sonicator for 30
min at 50-60.degree. C. The final dispersion is obtained after
centrifugation at 150,000 g for 30 min to precipitate
non-incorporated drug. The nanoemulsion is filtrated through a 0.22
.mu.m membrane and stored at 4-25.degree. C. protected from
light.
[0390] f. Pharmaceutical Composition for Injectable Use:
Polyersomes.
[0391] In various aspects, the compounds of the present invention
can be used to produce polymersomes. Polymersomes are made from
diblock or triblock co-polymers that form artificial vesicles. In
general they are made in a method similar to that of liposomes
described above.
[0392] g. Pharmaceutical Composition for Injectable Use:
Micelles.
[0393] The compounds of the present invention can be used to
produce micelles with the use of a surfactant wherein a disclosed
compound is incorporated into the core of the micelles. Using the
methods described herein, the micelle can self assemble and allow
the drug to be solubilized by incorporation within the micelle.
[0394] A co-solvent evaporation method can be used for the
self-assembly of MePEO-b-PCL block copolymers and drug
encapsulation. "PEO" is polyethylene oxide and "PCL" is
polycapralactone. The type of applied organic solvent, the ratio of
organic to the aqueous phase, and the order of addition of the
phases in the co-solvent evaporation method can be selected to
optimize the micelle formed in terms of carrier size and
encapsulation efficiency. For example, MePEO-b-PCL (about 30 mg) is
dissolved in a suitable solvent such as acetone, tetrahydrofuran
(THF) or acetonitrile. For this amount of MePEO-b-PCL, the volume
of organic solvent can be either 0.5 or 1.5 mL, corresponding to a
final 1:6 or 1:2 organic:aqueous phase ratio, respectively. The
solution can be added drop-wise to water (3 mL), or alternatively
water can be added drop-wise to this solution. The mixture is then
stirred at room temperature for a suitable time, e.g. about 4 h.
Vacuum is applied to remove the remainder of the organic solvent.
Drug encapsulation is accomplished by dissolving about 3 mg of a
disclosed compound, e.g. compound 1, in the organic solvent and
following an identical procedure to the self-assembly condition. At
the end of encapsulation process, the colloidal solution is
centrifuged at 11,600.times.g for 5 min, to remove any precipitated
disclosed compound.
[0395] h. Pharmaceutical Composition for Injectable Use:
Suspensions.
[0396] In various aspects, the compounds of the present invention
can be used to produce parenteral suspensions. In a preferred
suspension formulation, insoluble particles should be uniformly
dispersed and should redisperse uniformly in the continous phase,
upon moderate shaking, for a sufficient period of time. This allows
the withdrawal of the correct amount of medication with minimal
dose variation. The rate of settling can be decreased by using
viscosity improving agents, and ease of redispersibility can be
controlled by using flocculating agents. For example, surfactants
can be used to stabilize the suspensions and serve as flocculating
agents. However, there are only a limited numbers of nonionic and
anionic surfactants that have been approved by regulatory agencies
such as the FDA for use as excipients for parenteral use. Examples
of approved excipients for parenteral use include phospholipids,
polysorbate 80, and poloxamers.
[0397] For example, a parenteral suspension comprising a disclosed
compound, e.g. compound 1, at a final concentration of about 50
mg/ml in seasame oil with about 20 mg/ml aluminum monostearate and
0.1% propylparaben.
[0398] Alternatively, a parenteral suspension comprising a
disclosed compound, e.g. compound 1, at a final concentration of
about 3 mg/ml in a physiological sodium phosphate solution (pH
6.8-7.2) with 0.1 mg/ml Na.sub.z-EDTA and 0.2 mg/ml benzalkonium
chloride.
[0399] Alternatively, a parenteral suspension comprising a
disclosed compound, e.g. compound 1, at a final concentration of
about 8 mg/ml in an aqueous solution comprising the following
excepients: sodium carboxymethyl cellulose (CMC), 5 mg/ml;
polysorbate 80, 0.75 mg/ml; sodium chloride, 6.7 mg/ml; creatinine,
5 mg/ml; sodium bisulfate, 1 mg/ml; and Na.sub.z-EDTA, 0.5 mg/ml,
pH 5-7.5.
[0400] i. Pharmaceutical Composition for Injectable Use:
Solutions.
[0401] In various aspects, a biocompatible solution of a disclosed
compound, e.g. compound 1, can be prepared using an organic solvent
that is biocompatible. For example, propylene glycol, polyethylene
glycols, ethanol), dimethyl sulfoxide, N-methyl-2-pyrrolidone,
glycofurol, Solketal.TM., glycerol formal, acetone
tetrahydrofurfuryl alcohol, diglyme, dimethyl isosorbide,
cremophor, and ethyl lactate.
[0402] It will be apparent to those skilled in the art that various
modifications and variations can be made in the present invention
without departing from the scope or spirit of the invention. Other
embodiments of the invention will be apparent to those skilled in
the art from consideration of the specification and practice of the
invention disclosed herein. It is intended that the specification
and examples be considered as exemplary only, with a true scope and
spirit of the invention being indicated by the following
claims.
* * * * *
References