U.S. patent application number 16/110956 was filed with the patent office on 2019-12-26 for quinolone-3-diarylethers.
The applicant listed for this patent is THE UNITED STATES GOVERNMENT AS REPRESENTED BY THE DEPARTMENT OF VETERANS AE, OREGON HEALTH & SCIENCE UNIVERSITY, THE UNITED STATES GOVERNMENT AS REPRESENTED BY THE DEPARTMENT OF VETERANS AE. Invention is credited to Holland Alday, Lisa Bleyle, Igor Bruzual, Rozalia Dodean, J. Stone Doggett, Isaac Forquer, Lisa Frueh, Jane Xu Kelly, Dennis Koop, Yuexin Li, Galen Miley, Aaron Nilsen, Sovitj Pou, Michael Riscoe, Martin Smilkstein, Allison Stickles, Rolf Winter.
Application Number | 20190389802 16/110956 |
Document ID | / |
Family ID | 57834740 |
Filed Date | 2019-12-26 |
![](/patent/app/20190389802/US20190389802A9-20191226-C00001.png)
![](/patent/app/20190389802/US20190389802A9-20191226-C00002.png)
![](/patent/app/20190389802/US20190389802A9-20191226-C00003.png)
![](/patent/app/20190389802/US20190389802A9-20191226-C00004.png)
![](/patent/app/20190389802/US20190389802A9-20191226-C00005.png)
![](/patent/app/20190389802/US20190389802A9-20191226-C00006.png)
![](/patent/app/20190389802/US20190389802A9-20191226-C00007.png)
![](/patent/app/20190389802/US20190389802A9-20191226-C00008.png)
![](/patent/app/20190389802/US20190389802A9-20191226-C00009.png)
![](/patent/app/20190389802/US20190389802A9-20191226-C00010.png)
![](/patent/app/20190389802/US20190389802A9-20191226-C00011.png)
View All Diagrams
United States Patent
Application |
20190389802 |
Kind Code |
A9 |
Riscoe; Michael ; et
al. |
December 26, 2019 |
QUINOLONE-3-DIARYLETHERS
Abstract
Disclosed are derivative compounds of ELQ-300 that include an
ester at position 4. These compounds have enhanced properties
relative to ELQ-300. Also disclosed are pharmaceutical compositions
comprising the compounds and methods of treating and preventing
malaria infections involving administering the pharmaceutical
compositions to the subject.
Inventors: |
Riscoe; Michael; (Tualatin,
OR) ; Nilsen; Aaron; (Portland, OR) ;
Stickles; Allison; (Portland, OR) ; Miley; Galen;
(Portland, OR) ; Winter; Rolf; (Portland, OR)
; Pou; Sovitj; (PORTLAND, OR) ; Li; Yuexin;
(Portland, OR) ; Kelly; Jane Xu; (Portland,
OR) ; Forquer; Isaac; (Portland, OR) ;
Doggett; J. Stone; (Portland, OR) ; Bruzual;
Igor; (Portland, OR) ; Frueh; Lisa; (Portland,
OR) ; Dodean; Rozalia; (Portland, OR) ;
Smilkstein; Martin; (Portland, OR) ; Alday;
Holland; (Portland, OR) ; Koop; Dennis;
(Portland, OR) ; Bleyle; Lisa; (Portland,
OR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
OREGON HEALTH & SCIENCE UNIVERSITY
THE UNITED STATES GOVERNMENT AS REPRESENTED BY THE DEPARTMENT OF
VETERANS AE |
Portland
WASHINGTON |
OR
DC |
US
US |
|
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20180362465 A1 |
December 20, 2018 |
|
|
Family ID: |
57834740 |
Appl. No.: |
16/110956 |
Filed: |
August 23, 2018 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
15215103 |
Jul 20, 2016 |
|
|
|
16110956 |
|
|
|
|
62343319 |
May 31, 2016 |
|
|
|
62194636 |
Jul 20, 2015 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07D 405/12 20130101;
A61P 33/00 20180101; C07D 409/12 20130101; C07D 215/22
20130101 |
International
Class: |
C07D 215/22 20060101
C07D215/22; C07D 409/12 20060101 C07D409/12; C07D 405/12 20060101
C07D405/12 |
Goverment Interests
ACKNOWLEDGEMENT OF GOVERNMENT SUPPORT
[0002] Work resulting in this invention was funded by the United
States government under the terms of a VA Merit Review Grant
awarded to Dr. Michael Riscoe by the United States Veterans
Administration and Grant Numbers R56AI100569, R01AI100569, and
PR130649 awarded by the National Institutes of Health. The United
States government has certain rights in this invention.
Claims
1-12. (canceled)
13. A compound selected from the group of: ##STR00038## or a
pharmaceutically acceptable salt thereof.
14. The compound of claim 13 selected from the group of:
##STR00039## or a pharmaceutically acceptable salt thereof.
15. The compound of claim 13 which is: ##STR00040## or a
pharmaceutically acceptable salt thereof.
16. A pharmaceutical composition comprising one or more
pharmaceutically acceptable vehicles, solubilizing agents, or
carriers and a therapeutically effective amount of a compound
selected from the group of: ##STR00041## or a pharmaceutically
acceptable salt thereof.
17. The pharmaceutical composition of claim 16, wherein the
compound is selected from: ##STR00042## or a pharmaceutically
acceptable salt thereof.
18. The pharmaceutical composition of claim 16, wherein the
compound is: ##STR00043## or a pharmaceutically acceptable salt
thereof.
19. A method of treating, inhibiting, or preventing a disease
selected from the group of malaria, toxoplasmosis, babesiosis,
coccidiosis, cryptosporidiosis, cyclosporiasis, and isosporiasis in
a subject in need thereof, the method comprising administering to
the subject in need thereof a therapeutically effective amount of a
compound selected from the group of: ##STR00044## or a
pharmaceutically acceptable salt thereof.
20. The method of claim 19, wherein the compound is selected from
the group of: ##STR00045## or a pharmaceutically acceptable salt
thereof.
21. The method of claim 19, wherein the compound is: ##STR00046##
or a pharmaceutically acceptable salt thereof.
22. The method of claim 19, wherein the disease is malaria.
23. The method of claim 22, wherein the malaria is caused by an
organism selected from the group of Plasmodium vivax, Plasmodium
ovale, Plasmodium knowlesi, Plasmodium malariae, Plasmodium yoelii,
and Plasmodium berghei.
24. The method of claim 22, wherein the method comprises
administering to the subject in need thereof a therapeutically
effective amount of a compound selected from the group of:
##STR00047## or a pharmaceutically acceptable salt thereof.
25. The method of claim 22, wherein the method comprises
administering to the subject in need thereof a therapeutically
effective amount of a compound selected from the group of:
##STR00048## or a pharmaceutically acceptable salt thereof.
26. The method of claim 22, wherein the method comprises
administering to the subject in need thereof a therapeutically
effective amount of the compound: ##STR00049## or a
pharmaceutically acceptable salt thereof.
Description
RELATED APPLICATIONS
[0001] U.S. Provisional Patent Application 62/194,636, filed on 20
Jul. 2015 and U.S. Provisional Patent Application 62/343,319, filed
on 31 May 2016 are related to this application and are hereby
incorporated by reference in their entireties.
FIELD
[0003] Generally, the field is small molecule therapeutics for use
in treating infectious disease. More specifically, the field is
anti-parasitic compositions derived from
quinolone-3-diarylethers.
BACKGROUND
[0004] Malaria remains an enormous global health problem Malaria
remains one of the deadliest diseases in the world today, as it has
been for thousands of years. For each of the 1 million people
killed each year, hundreds of millions more suffer from severe
illness (1). Spread by mosquitoes from person to person malaria
remains one of the most widespread infectious diseases of our time.
There are five identified species of the parasite responsible for
human malaria all belonging to genus Plasmodium. P. falciparum is
the dominant species in sub-Saharan Africa, and is responsible for
the majority of the malaria-related deaths. P. vivax, known to be
responsible for relapsing malaria, causes as much as 25-40% of the
global malaria burden, whereas P. ovale, and P. malariae represent
a small percentage of infections. A fifth species P. knowlesi, a
species that infects subhuman primates, has Jed to human malaria,
but the exact mode of transmission remains unclear.
[0005] The impact of malaria is particularly devastating in
sub-Saharan Africa where its victims are primarily young children
and pregnant women. This situation is worsened by the growing
emergence of Plasmodium parasites that are resistant to multiple
drugs (2). The list of drugs that are losing potency against
malaria includes the quinolines--chloroquine, quinine, and
mefloquine; the antifolates--pyrimethamine and sulfadoxine; and the
anti-respiratory combination of atovaquone (ATV) and proguanil. In
SE Asia, treatment of multidrug resistant malaria relies solely on
the endoperoxide artesunate, leaving a razor thin wall of
opposition to the total collapse of malaria chemotherapy. One of
the greatest challenges in global health today is the development
of a safe and affordable drug for treatment and prevention of
malaria (3).
SUMMARY
[0006] The antimalarial drug ELQ-300 is a selective sub-nanomolar
inhibitor of Plasmodium falciparum cytochrome bc1 complex. The
effects of the drug are parasiticidal due to the requirement of
cytochrome bc1 and the coenzyme Q cycle for production of
pyrimidines needed for DNA and RNA synthesis. As a result, ELQ-300
exhibits an excellent parasitological profile with potent activity
against all life cycle stages of P. falciparum including liver,
bloodstream, and vector stages. Unfortunately, the challenging
physical-chemical characteristics of ELQ-300 limit its potential
for clinical development, i.e., a high degree of crystallinity
(e.g., melting point>300.degree. C.) and poor aqueous solubility
limit oral absorption to such a degree that it has been impossible
to establish a therapeutic safety window. To address the issues of
high crystallinity and poor water solubility we initiated a prodrug
effort focusing primarily on carbonate ester prodrugs such as the
ethylcarbonate ester ELQ-337. The degree of crystallinity of the
drug was significantly reduced relative to ELQ-300 (i.e., melting
point for ELQ-337=150.degree. C.) and the oral bioavailability in
mice and rats was also enhanced over ELQ-300. We now wish to
disclose novel alkoxycarbonyloxyalkyl ester prodrugs of ELQ-300
(and similar 4(1H)Quinolone-3-diarylether substituted derivatives
such as ELQ-271, ELQ-316, and ELQ-400) with greatly reduced
crystallinity as well as other features that suggest that they may
be readily formulated for clinical use for treatment and
prophylaxis against malaria as well as for disease eradication.
[0007] Disclosed herein are compounds of the formula:
##STR00001##
[0008] wherein X is halo and wherein R is an ester. In some
examples, X is fluoro or chloro. In other examples, R is a
carbonate ester. In still more examples, X is chloro and R is a
carbonate ester selected from methyl carbonate; ethyl carbonate;
2-methoxyethylcarbonate; 2-(2-methoxyethoxy)ethyl carbonate;
2-(2-(2-methoxyethoxy)ethoxy)ethyl) carbonate; allyl carbonate;
tert-butyl carbonate; ((2,2-dimethyl-1,3-dioxolan-4-yl)methyl)
carbonate; ((2-oxo-1,3-dioxolan-4-yl)methyl carbonate;
2,3-dihydroxypropyl carbonate; or
1,1-dioxidotetrahydrothiophen-3-yl carbonate. In still further
examples, X is fluoro and R is ethyl carbonate or pivalate. In
other examples, X is chloro and R is selected from isobutyrate,
pivalate, or benzoate.
[0009] Disclosed herein are pharmaceutical compositions comprising
a therapeutically effective amount of the compounds described
herein. The composition can further comprise polyethylene glycol or
any other acceptable additive.
[0010] Disclosed herein are uses of the pharmaceutical compositions
described herein for the treatment of malaria, toxoplasmosis,
babesiosis, coccidiosis, cryptosporidiosis, cyclosporiasis, or
isosporiasis in a subject. The pharmaceutical compositions can be
administered prophylactically or therapeutically. The
pharmaceutical compositions can be administered to a subject with a
latent infection of any of the above.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0011] FIG. 1 is a plot of the disappearance of ELQ-331 over time
in the presence of pooled human microsomes (1 mg/ml of
reaction.)
[0012] FIG. 2 is a plot of the conversion of ELQ-331 to ELQ-300 in
the presence of pooled human microsomes. The presence or absence of
NADPH did not affect the conversion rate.
DETAILED DESCRIPTION
[0013] Disclosed herein are ELQ-300 prodrugs comprising ester
derivatives replacing the ketone group at position 4 of the ELQ-300
quinoline.
##STR00002##
Definitions
[0014] Unless specifically defined otherwise, the technical terms,
as used herein, have their normal meaning as understood in the art.
The following explanations of terms and methods are provided to
better describe the present compounds, compositions and methods,
and to guide those of ordinary skill in the art in the practice of
the present disclosure. It is also to be understood that the
terminology used in the disclosure is for the purpose of describing
particular embodiments and examples only and is not intended to be
limiting.
[0015] As used herein, the singular terms "a," "an," and "the"
include plural referents unless context clearly indicates
otherwise. Similarly, the word "or" is intended to include "and"
unless the context clearly indicates otherwise. Also, as used
herein, the term "comprises" means "includes." Hence "comprising A
or B" means including A, B, or A and B.
[0016] "Administration of" and "administering" a compound refers to
providing a compound, (such as a prodrug of a compound), or a
pharmaceutical composition comprising a compound or prodrug thereof
to a subject. The compound or composition can be administered by
another person to the subject or it can be self-administered by the
subject.
[0017] The term "alkyl" refers to a branched or unbranched
saturated hydrocarbon group, such as methyl, ethyl, n-propyl,
isopropyl, n-butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl,
octyl, decyl, tetradecyl, hexadecyl, eicosyl, tetracosyl and the
like. A "lower alkyl" group is a saturated branched or unbranched
hydrocarbon having from 1 to 6 carbon atoms (C.sub.1-6alkyl). The
term "alkyl" also includes cycloalkyl. The alkyl group may be a
"substituted alkyl" wherein one or more hydrogen atoms are
substituted with a substituent such as halogen, cycloalkyl, alkoxy,
amino, hydroxyl, aryl, or carboxyl.
[0018] Alkenyl refers to an unsaturated hydrocarbon group
comprising at least one carbon-carbon double bond.
[0019] The term "alkoxy" refers to an alkyl group attached to an
oxygen atom to form an ether. The alkoxy group may be a
"substituted alkoxy" wherein one or more hydrogen atoms are
substituted with a substituent such as halogen, cycloalkyl, alkoxy,
amino, hydroxyl, aryl, or carboxyl.
[0020] The term "aryl" refers to any carbon-based aromatic group
including, but not limited to, benzene, naphthalene, phenyl, and
oxazole. The term "aryl" also includes heteroaryl, which is defined
as an aromatic group that has at least one heteroatom incorporated
within the ring of the aromatic group. Examples of heteroatoms
include, but are not limited to: nitrogen, oxygen, sulfur, and
phosphorus. The aryl group can be substituted with one or more
groups including, but not limited to, alkyl, alkynyl, alkenyl,
aryl, halide, nitro, amino, ester, ether, ketone, aldehyde,
hydroxy, carboxylic acid, cyano, amido, haloalkyl, haloalkoxy, or
alkoxy, or the aryl group can be unsubstituted.
[0021] The term "cycloalkyl" refers to a non-aromatic carbon-based
ring composed of at least three carbon atoms. Examples of
cycloalkyl groups include, but are not limited to, cyclopropyl,
cyclobutyl, cyclopentyl, and cyclohexyl. The term "heterocycloalkyl
group" is a cycloalkyl group as defined above where at least one of
the carbon atoms of the ring is substituted with a heteroatom such
as nitrogen, oxygen, sulfur, or phosphorus.
[0022] "Derivative" refers to a compound or portion of a compound
that is derived from or is theoretically derivable from a parent
compound.
[0023] The terms "halogenated alkyl" or "haloalkyl group" refer to
an alkyl group as defined above with one or more hydrogen atoms
present on these groups substituted with a halogen (F, Cl, Br, I).
For example, a halomethyl group is a methyl group (--CH.sub.3) with
one or more halogens substituted for the hydrogens. A halomethyl
group may include di- and tri-substituted halogens such as a
trifluoromethyl group. A halogenated ether refers to a group with
one or more hydrogen atoms present on an ether, such as a methyl
ether (--OCH.sub.3), substituted with one or more halogens. A
halogenated ether may also be termed "halomethoxy" and this general
term includes mono, di- and tri-substituted halogens on the ether.
For example, a trifluoromethyl ether has a formula of --OCF.sub.3
and can interchangeably be referred to as "trifluoromethoxy".
Similarly, a difluoromethoxy ether has the formula of
--OCHF.sub.2.
[0024] "Heterocycle" is a term that encompasses both heteroaryls
and heterocycloalkyls. Heterocycles may be monocyclic or polycyclic
rings. Exemplary heterocycles include, but are not limited to,
azepinyl, aziridinyl, azetyl, azetidinyl, diazepinyl,
dithiadiazinyl, dioxazepinyl, dioxolanyl, dithiazolyl, furanyl,
isooxazolyl, isothiazolyl, imidazolyl, morpholinyl, oxetanyl,
oxadiazolyl, oxiranyl, oxazinyl, oxazolyl, piperazinyl, pyrazinyl,
pyridazinyl, pyrimidinyl, piperidyl, piperidino, pyridyl, pyranyl,
pyrazolyl, pyrrolyl, pyrrolidinyl, thiatriazolyl, tetrazolyl,
thiadiazolyl, triazolyl, thiazolyl, thienyl, tetrazinyl,
thiadiazinyl, triazinyl, thiazinyl, thiopyranyl, furoisoxazolyl,
imidazothiazolyl, thienoisothiazolyl, thienothiazolyl,
imidazopyrazolyl, cyclopentapyrazolyl, pyrrolopyrrolyl,
thienothienyl, thiadiazolopyrimidinyl, thiazolothiazinyl,
thiazolopyrimidinyl, thiazolopyridinyl, oxazolopyrimidinyl,
oxazolopyridyl, benzoxazolyl, benzisothiazolyl, benzothiazolyl,
imidazopyrazinyl, purinyl, pyrazolopyrimidinyl, imidazopyridinyl,
benzimidazolyl, indazolyl, benzoxathiolyl, benzodioxolyl,
benzodithiolyl, indolizinyl, indolinyl, isoindolinyl,
furopyrimidinyl, furopyridyl, benzofuranyl, isobenzofuranyl,
thienopyrimidinyl, thienopyridyl, benzothienyl, cyclopentaoxazinyl,
cyclopentafuranyl, benzoxazinyl, benzothiazinyl, quinazolinyl,
naphthyridinyl, quinolinyl, isoquinolinyl, benzopyranyl,
pyridopyridazinyl and pyridopyrimidinyl groups.
[0025] The terms "treatment", "treat" and "treating" refer to a
therapeutic intervention that ameliorates a sign or symptom of a
disease or pathological condition. As used herein, the terms
"treatment", "treat" and "treating," with reference to a disease,
pathological condition or symptom, also refers to any observable
beneficial effect of the treatment. The beneficial effect can be
evidenced, for example, a reduction in severity of some or all
clinical symptoms of the disease, a slower progression of the
disease, a reduction in the number of relapses of the disease, an
improvement in the overall health or well-being of the subject, or
by other parameters well known in the art that are specific to the
particular disease.
[0026] A "prophylactic" treatment is a treatment administered to a
subject who does not exhibit signs of a disease or exhibits only
early signs, for the purpose of decreasing the risk of developing
pathology.
[0027] A "therapeutic" treatment is a treatment administered to a
subject who has already begun to exhibit signs of a disease for the
purpose of slowing or reversing the pathology.
[0028] "Coadminister" means that each of at least two compounds are
administered during a time frame wherein the respective periods of
biological activity overlap. Thus, the term includes sequential as
well as coextensive administration of two or more drug
compounds.
[0029] The terms "pharmaceutically acceptable salt" or
"pharmacologically acceptable salt" refers to salts prepared by
conventional methods, and include basic salts of inorganic and
organic acids, such as hydrochloric acid, hydrobromic acid,
sulfuric acid, phosphoric acid, methanesulfonic acid,
ethanesulfonic acid, malic acid, acetic acid, oxalic acid, tartaric
acid, citric acid, lactic acid, fumaric acid, succinic acid, maleic
acid, salicylic acid, benzoic acid, phenylacetic acid, and mandelic
acid.
[0030] Pharmaceutically acceptable salts of the presently disclosed
compounds also include those formed from cations such as sodium,
potassium, aluminum, calcium, lithium, magnesium, zinc, and from
bases such as ammonia, ethylenediamine, N-methyl-glutamine, lysine,
arginine, ornithine, choline, N,N'-dibenzylethylenediamine,
chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine,
diethylamine, piperazine, tris(hydroxymethyl)aminomethane, and
tetramethylammonium hydroxide. These salts may be prepared by
standard procedures, for example by reaction of the free acid with
a suitable organic or inorganic base. Any chemical compound recited
in this specification may alternatively be administered as a
pharmaceutically acceptable salt thereof.
[0031] Pharmaceutically acceptable salts are also inclusive of the
free acid, base, and zwitterionic forms. Descriptions of exemplary
pharmaceutically acceptable salts can be found in Stahl and
Wermuth, Eds., Handbook of Pharmaceutical Salts; Properties,
Selection and Use, Wiley VCH (2008). When compounds disclosed
herein include an acidic function such as a carboxy group, then
suitable pharmaceutically acceptable cation pairs for the carboxy
group are well known to those skilled in the art and include
alkaline, alkaline earth, ammonium, and quaternary ammonium
cations. Such salts are known to those of skill in the art. For
additional examples of "pharmacologically acceptable salts," see
Berge et al., J. Pharm. Sci. 66:1 (1977).
[0032] The term "subject" includes both human and veterinary
subjects.
[0033] A "therapeutically effective amount" refers to a quantity of
a specified agent sufficient to achieve a desired effect in a
subject being treated with that agent. For example, this may be the
amount of a compound disclosed herein useful in treating malaria in
a subject. Ideally, a therapeutically effective amount of an agent
is an amount sufficient to inhibit or treat the disease without
causing substantial toxicity in the subject. The therapeutically
effective amount of an agent will be dependent on the subject being
treated, the severity of the affliction, and the manner of
administration of the therapeutic composition. Methods of
determining a therapeutically effective amount of the disclosed
compound sufficient to achieve a desired effect in a subject
infected with a malaria parasite will be understood by those of
skill in the art in light of this disclosure.
Synthesis of ELQ-300 Prodrugs
[0034] The term "prodrug" refers to any active or inactive compound
that is modified chemically through an in vivo physiological
action, such as hydrolysis or metabolism, into an active compound
following administration of the prodrug to a subject. The
suitability and techniques involved in making and using prodrugs
are well known by those skilled in the art. For a general
discussion of prodrugs involving esters see Svensson and Tunek,
Drug Metabolism Reviews 165 (1988), and Bundgaard, Design of
Prodrugs, Elsevier (1985).
[0035] The synthesis processes described herein can be monitored
according to any suitable method known in the art. For example,
product formation can be monitored by spectroscopy, such as nuclear
magnetic resonance spectroscopy (e.g., .sup.1H or .sup.13C NMR),
infrared spectroscopy, spectrophotometry (e.g., UV-visible), mass
spectrometry, or by chromatography such as high performance liquid
chromatograpy (HPLC) or thin layer chromatography.
[0036] Earlier attempts to develop a prodrug of ELQ-300 were
unsuccessful--the prodrugs proved to be too unstable in
physiological media to result in sufficient bioavailability or too
stable to be metabolized into active ELQ-300. For example, the
4-position acetyl ester (ELQ-370) is chemically unstable to mildly
acidic conditions and even decomposes rapidly in methanol while the
corresponding 4-oxo linked dimethylcarbamate analog (ELQ-301) is
stable to metabolism and displays inferior in vivo efficacy
compared to the parent compound ELQ-300.
Synthesis of ELQ-300 Carbonate Esters
[0037] Disclosed herein are O-linked esters and carbonates that are
effective prodrugs of ELQ-300. ELQ-337 is the O-linked
ethyl-carbonate of ELQ-300. O-linked carbonate esters of ELQs
enhance oral delivery and efficacy against murine malaria.
Placement of the promoiety at the 4-oxo-position removes the H-atom
from the ring nitrogen thereby upsetting crystal lattice formation.
This is evidenced by a reduction of the melting point from
314.degree. C. for ELQ-300 to 160.degree. C. for ELQ-337.
[0038] ELQ-337 is produced from ELQ-300 in one step, using sodium
hydride in tetrahydrofuran. Ethyl chloroformate is then added
dropwise. The reaction goes to completion in minutes upon the
addition of the chloroformate forming one regioisomer in very high
yield. ELQ-337 is chemically stable in 50/50 mixtures of methanol
and water at pH 3 and 8 overnight. Results are summarized in Scheme
1
##STR00003##
[0039] Ester derivatives of ELQ-300, including carbonate esters are
disclosed.
Ester Formation:
##STR00004##
[0041] The R.sub.1 group selected can result in the formation of
any ester. Esters generally have the structure:
##STR00005##
Carbonate Esters Generally have the Structure:
##STR00006##
[0042] wherein R.sub.2 or R.sub.3 can be any alkyl, substituted
alkyl, alkenyl, substituted alkenyl, ether, substituted ether,
aryl, substituted aryl, heterocycloalkyl, substituted
heterocycloalkyl, heteroaryl, or substituted heteroaryl.
[0043] The compounds disclosed herein have the general structure of
Formula I:
##STR00007##
[0044] wherein X is halo and R is ester. In particular examples X
is chloro or fluoro. In still other examples, R is a carbonate
ester. The carbonate can be any carbonate ester including ethyl
carbonate; 2-methoxyethylcarbonate; 2-(2-methoxyethoxy)ethyl
carbonate; 2-(2-(2-methoxyethoxy)ethoxy)ethyl) carbonate; allyl
carbonate; tert-butyl carbonate;
((2,2-dimethyl-1,3-dioxolan-4-yl)methyl) carbonate;
((2-oxo-1,3-dioxolan-4-yl)methyl carbonate; and 2,3-dihydroxypropyl
carbonate. In still further examples, R is a non-carbonate ester.
The non-carbonate ester can be any non-carbonate ester including
isobutyrate, pivalate, and benzoate groups.
[0045] As described herein, the definition of ester, particularly
with regard to the R group of Formula I above does not encompass
carbamates. Carbamates have the general structure:
##STR00008##
Pharmaceutical Compositions
[0046] The compounds disclosed herein may be included in
pharmaceutical compositions (including therapeutic and prophylactic
formulations). Pharmaceutical compositions as described herein
include one or more compounds according to the present description.
In addition to one or more compounds as described herein,
pharmaceutical compositions according to the present disclosure may
include one or more additional therapeutic agents, including, for
example, one or more additional antimalarial or anti-infective
agents, antibiotics, anti-inflammatory agents, or drugs that are
used to reduce pruritus, such as an antihistamine. In preparing the
pharmaceutical compositions, the one or more compounds as described
herein and, optionally, the one or more additional active agents,
may be combined together with one or more pharmaceutically
acceptable vehicles, salts, solubilizing agents (e.g., co-crystals,
lipids, or hydrophilic polymers) or carriers. The pharmaceutical
compositions described herein may be combined with or used
simultaneously with one or more other therapeutic regimens or
compositions. Where one or more additional antimalarial or
anti-infective agent is included in a pharmaceutical composition
according to the present invention, such agent(s) may be selected
from, for example, quinolines, such as chloroquine, quinine, and
mefloquine; the antifolates, such as pyrimethamine and sulfadoxine;
the anti-respiratory agents atovaquone and/or proguanil, as well as
inhibitors of parasite dihydro-orotate dehydrogenase (DHOD) such as
DSM265.
[0047] Pharmaceutical compositions according to the present
invention can be administered to subjects by a variety of mucosal
administration modes, including by oral, rectal, intranasal,
intrapulmonary, or transdermal delivery, or by topical delivery to
other surfaces. Optionally, the compositions can be administered by
non-mucosal routes, including by intramuscular, subcutaneous,
intravenous, intra-arterial, intra-articular, intraperitoneal,
intrathecal, intracerebroventricular, or parenteral routes. In an
embodiment, the compound can be administered ex vivo by direct
exposure to cells, tissues or organs originating from a subject. To
formulate the pharmaceutical compositions, the one or more
compounds can be combined with various pharmaceutically acceptable
additives, as well as a base or vehicle for dispersion of the
compound. Such additives include, but are not limited to, pH
control agents, such as arginine, sodium hydroxide, glycine,
hydrochloric acid, and citric acid. In addition, local anesthetics
(for example, benzyl alcohol), isotonizing agents (for example,
sodium chloride, mannitol, sorbitol), adsorption inhibitors (for
example, Tween 80 or medium chain triacylglycerols such as myglyol
812), solubility enhancing agents (for example, cyclodextrins and
derivatives thereof), stabilizers (for example, serum albumin), and
reducing agents (for example, glutathione) can be included.
[0048] In preparing a pharmaceutical composition according to the
present description, the one or more compounds can be dispersed in
a base or vehicle which can include a hydrophilic compound having a
capacity to disperse the disclosed compound and any additives. The
base can be selected from a wide range of suitable compounds,
including but not limited to, copolymers of polycarboxylic acids or
salts thereof; carboxylic anhydrides (for example, maleic
anhydride); with other monomers (for example, methyl(meth)acrylate
and acrylic acid); hydrophilic vinyl polymers, such as polyvinyl
acetate, polyvinyl alcohol, polyvinylpyrrolidone, cellulose
derivatives such as hydroxymethylcellulose and
hydroxypropylcellulose; natural polymers, such as chitosan,
collagen, sodium alginate, gelatin, hyaluronic acid; and nontoxic
metal salts thereof.
[0049] A biodegradable polymer may be selected as a base or
vehicle, such as, for example, polylactic acid, poly(lactic
acid-glycolic acid) copolymer, polyhydroxybutyric acid,
poly(hydroxybutyric acid-glycolic acid) copolymer and mixtures
thereof. Alternatively or additionally, synthetic fatty acid esters
such as polyglycerin fatty acid esters and sucrose fatty acid
esters may be employed as vehicles. Hydrophilic polymers and other
vehicles can be used alone or in combination, and enhanced
structural integrity can be imparted to the vehicle by, for
example, partial crystallization, ionic bonding, or cross-linking.
The vehicle may be provided in a variety of forms, including fluid
or viscous solutions, gels, pastes, powders, microspheres, and
films for direct application to a mucosal surface.
[0050] The one or more compounds may be combined with the base or
vehicle according to a variety of methods, and release of the
compound can be via diffusion, disintegration of the vehicle, or
associated formation of water channels. In some embodiments, the
compound can be dispersed in microcapsules (microspheres) or
nanoparticles prepared from a suitable polymer, for example,
5-isobutyl-2-cyanoacrylate (see, for example, Michael et al, J.
Pharmacy Pharmacol 43, 1-5, 1991), and dispersed in a biocompatible
dispersing medium, which may provide sustained delivery and
biological activity over a protracted time.
[0051] In certain embodiments, the pharmaceutical compositions of
the disclosure can contain as pharmaceutically acceptable vehicles,
substances as required to approximate physiological conditions,
such as pH adjusting and buffering agents, tonicity adjusting
agents, and wetting agents, for example, sodium acetate, sodium
lactate, sodium chloride, potassium chloride, calcium chloride,
sorbitan monolaurate, and triethanolamine oleate.
[0052] For solid compositions, conventional nontoxic
pharmaceutically acceptable vehicles may be used which include, for
example, pharmaceutical grades of mannitol, lactose, starch,
magnesium stearate, sodium saccharin, talcum, cellulose, glucose,
sucrose, and magnesium carbonate.
[0053] Pharmaceutical compositions for administering the one or
more compounds can also be formulated as a solution, microemulsion,
or other ordered structure suitable for a high concentration of
active ingredients. The vehicle may be a solvent or dispersion
medium containing, for example, water, ethanol, polyol (for
example, glycerol, propylene glycol, and liquid polyethylene
glycol), and suitable mixtures thereof. Proper fluidity for
solutions may be maintained, for example, by the use of a coating
such as lecithin, by the maintenance of a desired particle size in
the case of dispersible formulations, and by the use of
surfactants.
[0054] In an embodiment, it may be desirable to include isotonic
agents, for example, sugars, polyalcohols such as mannitol and
sorbitol, or sodium chloride in the composition. Prolonged
absorption of the one or more compounds may be obtained by
including in the composition an agent which delays absorption, for
example, monostearate salts and gelatin.
[0055] In certain embodiments, the one or more compounds can be
administered in a time release formulation, for example in a
composition which includes a slow release polymer. These
compositions may be prepared with vehicles that will protect
against rapid release, for example, a controlled release vehicle
such as a polymer, microencapsulated delivery system or bioadhesive
gel. Controlled release binders suitable for use in accordance with
the disclosure include any biocompatible controlled release
material which is inert to the active agent and which is capable of
incorporating the compound and/or other biologically active agent.
Numerous such materials are known in the art. Controlled-release
binders may be materials that are metabolized slowly under
physiological conditions following their delivery (for example, at
a mucosal surface, or in the presence of bodily fluids).
[0056] Exemplary binders include, but are not limited to,
biocompatible polymers and copolymers well known in the art for use
in sustained release formulations. Such biocompatible compounds are
non-toxic and inert to surrounding tissues, and do not trigger
significant adverse side effects, such as nasal irritation, immune
response, or inflammation. They are metabolized into metabolic
products that are also biocompatible and easily eliminated from the
body.
[0057] Exemplary polymeric materials for use in the present
disclosure include, but are not limited to, polymeric matrices
derived from copolymeric and homopolymeric polyesters having
hydrolyzable ester linkages. A number of these are known in the art
to be biodegradable and to lead to degradation products having no
or low toxicity.
[0058] Exemplary polymers include polyglycolic acids and polylactic
acids, poly(DL-lactic acid-co-glycolic acid), poly(D-lactic
acid-co-glycolic acid), and poly(L-lactic acid-coglycolic acid).
Other useful biodegradable or bioerodable polymers include, but are
not limited to, poly(epsilon-caprolactone),
poly(epsilon-caprolactone-CO-lactic acid),
poly(epsilon-caprolactone-CO-glycolic acid), poly(beta-hydroxy
butyric acid), poly(alkyl-2-cyanoacrylate), hydrogels such as
poly(hydroxyethyl methacrylate), polyamides, poly(amino acids) such
as L-leucine, glutamic acid, L-aspartic acid, poly(ester urea),
poly(2-hydroxyethyl DL-aspartamide), polyacetal polymers,
polyorthoesters, polycarbonate, polymaleamides, polysaccharides,
and copolymers thereof.
[0059] Methods for preparing such formulations are well known to
those skilled in the art (see, for example, Sustained and
Controlled Release Drug Delivery Systems, J. R. Robinson, ed.,
Marcel Dekker, Inc., New York, 1978). Other useful formulations
include controlled-release microcapsules (U.S. Pat. Nos. 4,652,441
and 4,917,893), lactic acid-glycolic acid copolymers useful in
making microcapsules and other formulations (U.S. Pat. Nos.
4,677,191 and 4,728,721) and sustained release compositions for
water-soluble peptides (U.S. Pat. No. 4,675,189).
[0060] The pharmaceutical compositions of the disclosure typically
are sterile and stable under conditions of manufacture, storage and
use. Sterile solutions can be prepared by incorporating the
compound in the required amount in an appropriate solvent with one
or a combination of ingredients enumerated herein, as required,
followed by filtered sterilization. Dispersions may be prepared by
incorporating the compound and/or other biologically active agent
into a sterile vehicle that contains a basic dispersion medium and
the required other ingredients from those enumerated herein. In the
case of sterile powders, methods of preparation include vacuum
drying and freeze-drying which yields a powder of the compound plus
any additional desired ingredient from a previously
sterile-filtered solution thereof. The prevention of the action of
microorganisms can be accomplished by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
sorbic acid, and thimerosal.
Methods of Treatment
[0061] The compounds and pharmaceutical compositions disclosed
herein can be used for treating, inhibiting or preventing parasitic
diseases, such as malaria, caused by organisms such as Plasmodium
sp., including Plasmodium falciparum. Other examples of human or
animal parasitic diseases that may be treated using the compounds
and pharmaceutical compositions disclosed herein include
toxoplasmosis, amebiasis, giardiasis, leishmaniasis,
trypanosomiasis, coccidiosis, and schistosomiasis, caused by
organisms such as Toxoplasma sp., Eimeria sp., Babesia sp, or
Theileria sp. Additional parasites that cause malaria include
Plasmodium vivax, Plasmodium ovale, Plasmodium knowlesi, Plasmodium
malariae, Plasmodium yoelii, and Plasmodium berghei.
[0062] In particular embodiments, the compounds and compositions
disclosed herein can be administered to a subject to prevent or
inhibit drug-resistant malaria such as chloroquine-resistant
malaria or multidrug-resistant malaria that is caused by organisms
harboring resistance to chloroquine, quinine, mefloquine,
pyrimethamine, dapsone, atovaquone, P. falciparum DHOD inhibitors
such as DSM265 (Coteron J M et al, J Med Chem 54, 5540-5561 (2011);
incorporated by reference herein) or any other available
anti-malarial drug.
[0063] In further embodiments, the compounds and pharmaceutical
compositions disclosed herein can be coadministered with another
pharmaceutically active compound. For example, the compounds may be
coadministered with quinine, chloroquine, atovaquone, proguanil,
primaquine, amodiaquine, mefloquine, piperaquine, artemisinin,
artesunate, endoperoxidases, methylene blue, pyrimethamine,
sulfadoxine, artemether-lumefantrine (Coartem.RTM.),
dapsone-chlorproguanil (LAPDAP.RTM.), artesunate, quinidine,
clopidol, pyridine/pyridinol analogs, 4(1H)-quinolone analogs,
dihydroartemisinin, a mixture of atovaquone, proguanil, an
endoperoxide, an acridone as disclosed in WO 2008/064011, another
3-aryl quinoline as disclosed in WO 2010/059633, or any combination
or mixtures of these, whether administered separately or in a
single pharmaceutical composition.
[0064] In accordance with the various treatment methods of the
disclosure, the compound can be delivered to a subject in a manner
consistent with conventional methodologies associated with
management of the disorder for which treatment or prevention is
sought. In accordance with the disclosure herein, a
prophylactically or therapeutically effective amount of the
compound and/or other biologically active agent is administered to
a subject in need of such treatment for a time and under conditions
sufficient to prevent, inhibit, and/or ameliorate a selected
disease or condition or one or more symptom(s) thereof.
[0065] Typical subjects intended for treatment with the compounds,
compositions and methods of the present disclosure include humans,
as well as non-human primates and other animals such as companion
animals, livestock animals, animals used in models of parasitic
infection, or animals used in pharmaceutical testing, such as
pharmacokinetics and toxicological testing, including mice, rats,
rabbits, and guinea pigs.
[0066] To identify subjects for prophylaxis or treatment according
to the methods of the disclosure, accepted screening methods are
employed to determine risk factors associated with a parasitic
infection to determine the status of an existing disease or
condition in a subject. These screening methods include, for
example, preparation of a blood smear from an individual suspected
of having malaria. The blood smear is then fixed in methanol and
stained with Giemsa and examined microscopically for the presence
of Plasmodium infected red blood cells. These and other routine
methods allow a clinician to select patients in need of therapy
using the methods and pharmaceutical compositions of the
disclosure.
[0067] The administration of the disclosed compounds and
pharmaceutical compositions can be for prophylactic or therapeutic
purposes or to block transmission of disease. When provided
prophylactically, the compound is administered to a subject in
advance of a symptom. The prophylactic administration of the
compound serves to prevent or ameliorate subsequent disease process
or to achieve disease eradication. When provided therapeutically,
the compound is administered to a subject at or after the onset of
a symptom of disease or infection.
[0068] For prophylactic and therapeutic purposes, the compound or
pharmaceutical composition may be administered to the subject
orally or in a single bolus delivery, via continuous delivery (for
example, continuous transdermal, mucosal or intravenous delivery)
over an extended time period, or in a repeated administration
protocol (for example, by an hourly, daily or weekly, repeated
administration protocol). The therapeutically effective dosage of
the compound may be provided as repeated doses within a prolonged
prophylaxis or treatment regimen to yield clinically significant
results to alleviate one or more symptoms or detectable conditions
associated with a targeted disease or condition as set forth
herein.
[0069] Determination of effective dosages in this context may be
based on animal model studies followed up by human clinical trials
and may be guided by administration protocols that significantly
reduce the occurrence or severity of targeted disease symptoms or
conditions in the subject. Suitable models in this regard include,
for example, murine, rat, avian, porcine, feline, non-human
primate, and other accepted animal model subjects known in the art.
Alternatively, effective dosages may be determined using in vitro
models (for example, immunologic and histopathologic assays). Using
such models, calculations and adjustments can be required to
determine an appropriate concentration and dose to administer a
therapeutically effective amount of the compound (for example,
amounts that are effective to elicit a desired immune response or
alleviate one or more symptoms of a targeted disease). In certain
embodiments, an effective amount or effective dose of the compound
may simply inhibit or enhance one or more selected biological
activities correlated with a disease or condition, as set forth
herein, for either therapeutic or diagnostic purposes.
[0070] The actual dosage of the compound may vary according to
factors such as the disease indication and particular status of the
subject (for example, the subject's age, size, fitness, extent of
symptoms, and susceptibility factors), time and route of
administration, other drugs or treatments being administered
concurrently, as well as the specific pharmacology of the compound
for eliciting the desired activity or biological response in the
subject. Dosage regimens can be adjusted to provide an optimum
prophylactic or therapeutic response.
[0071] A therapeutically effective amount may be one in which any
toxic or detrimental side effects of the compound and/or other
biologically active agent is outweighed in clinical terms by
therapeutically beneficial effects. A non-limiting range for a
therapeutically effective amount of a compound and/or other
biologically active agent within the methods and compositions of
the disclosure is about 0.01 mg/kg body weight to about 100 mg/kg
body weight, such as about 0.05 mg/kg to about 50 mg/kg body
weight, or about 0.5 mg/kg to about 5 mg/kg body weight.
[0072] The dosage may be varied to maintain a desired concentration
at a target site (for example, the lungs or systemic circulation).
Higher or lower concentrations can be selected based on the mode of
delivery, for example, trans-epidermal, rectal, oral, pulmonary, or
intranasal delivery versus intravenous or subcutaneous delivery.
Dosage can also be adjusted based on the release rate of the
administered formulation, for example, of an intrapulmonary spray
versus powder or sustained release oral versus injected particulate
or transdermal delivery formulations.
[0073] The instant disclosure also includes kits, packages and
multi-container units containing the herein described
pharmaceutical compositions, active ingredients, and/or devices and
consumables that facilitate the administration the same for use in
the prevention and treatment of diseases and other conditions in
mammalian subjects.
[0074] In an embodiment, the compound may be formulated in a
pharmaceutical composition for delivery to a subject. In such
embodiments, pharmaceutical compositions according to the present
description may be used. The compound or composition within which
it is formulated may be contained in a bulk dispensing container or
unit or multiunit dosage form. Optional dispensers can be provided,
for example, a pulmonary or intranasal spray applicator. Packaging
materials optionally include a label or instruction indicating for
what treatment purposes and/or in what manner the pharmaceutical
agent packaged therewith can be used.
[0075] In an embodiment, the method of treating a Plasmodium
infection comprises administering a therapeutically effective
amount of a compound. The compound may be administered orally,
subcutaneously, intravenously, or intramuscularly to a subject
suffering from or at risk of suffering from a Plasmodium
infection.
Mechanism of Action of ELQ-300 Prodrugs
[0076] The chemical structures of the prodrugs ELQ-330 and ELQ-331
are shown in Scheme 1 below along with the structure of the parent
drug ELQ-300. It is well established that alkoxycarbonyloxyalkyl
ester prodrugs serve as neutral lipophilic prodrugs of
pharmaceutical agents. Consider for example the clinical drug
Tenofovir Disoproxil (FIG. 2) which contains two
alkoxycarbonyloxyalkyl ester promoieties attached to a central
phosphonate residue. ELQ-330 and ELQ-331 were formed by reaction of
ELQ-300 with either chloro-methyl-isopropylcarbonate (ELQ-330) or
chloro-methyl-ethylcarbonate (ELQ-331).
##STR00009##
[0077] The development of ELQ-300 for clinical use is hindered by
relatively poor water solubility that is linked to a high degree of
crystallinity. One simple measure that can be used to compare
crystal lattice strength is the melting point. Pure ELQ-300 has a
melting point of >300.degree. C. Disclosed herein are compounds
with an alkoxycarbonate ester at position 4 of the quinoline ring
system have significantly reduced crystal lattice energy compared
to ELQ-300 as evidenced by an impressive drop in the melting point:
99.7-99.9/ELQ-330; 103.5-103.7.degree. C./ELQ-331; and
135.0-135.7.degree. C./ELQ-387. Without being bound by theory,
reducing crystallinity means that the alkoxycarbonate ester
prodrugs can have a reduced tendency for re-crystallization in the
intestines prior to absorption. As a consequence, this subclass of
ELQ prodrugs, i.e., the alkoxycarbonate esters, can be used to
improve oral bioavailability and bloodstream exposures to the
active parent ELQs (e.g., ELQ-271, ELQ-300, ELQ-316, and
ELQ-400).
[0078] The potential for hepatic P450-dependent metabolism of
ELQ-331 at 11 .mu.M was assessed using pooled human liver
microsomes in the presence of an NADPH regenerating system. The
loss of substrate was monitored by LC/MS/MS. Midazolam was
monitored as a well characterized positive control. Midazolam
represents a drug with comparatively low metabolic stability.
Incubations were conducted in the absence of the NADPH regenerating
system to monitor for potential P450-independent metabolism. In the
absence of NADPH we did not observe significant metabolism of the
midazolam control however it was rapidly metabolized in the
presence of NADPH with a t112 value of 2.6 minutes. In the presence
of 1 mg/ml of pooled human microsomes and NADPH regenerating system
we observed a linear conversion of ELQ-331 to ELQ-300 throughout
the first 20 minutes of reaction time. The t.sub.1/2 value recorded
for ELQ-331 was 37.7 minutes in the presence or absence of NADPH.
That the conversion rate of the prodrug ELQ-331ELQ-300 did not vary
between the samples with and without NADPH indicates that host
esterases are primarily responsible for enzymatic production of
ELQ-300 from ELQ-331 (FIGS. 1 and 2)
EXAMPLES
[0079] The following examples are for illustration only. In light
of this disclosure, those of skill in the art will recognize that
variations of these examples and other examples of the disclosed
invention be possible without undue experimentation.
Example 1--ELQ-331 Synthesis
[0080] ELQ-300 (0.85 g, 1.8 mmol), tetrabutylammonium iodide (1.33
g, 3.6 mmol) and potassium carbonate (0.50 g, 3.6 mmol) were
dissolved anhydrous dimethylformamide (8 ml) in a flame-dried round
bottom flask at 60.degree. C. under inert atmosphere. Chloromethyl
ethyl carbonate (0.5 g, 3.6 mmol) was added dropwise and the
reaction stirred under inert atmosphere at 60.degree. C. for two
hours, at which point reaction completion was confirmed by thin
layer chromatography. After cooling to room temperature, the
reaction solvent was removed under reduced pressure and the mixture
taken up in water (10 ml) and extracted with dichloromethane
(3.times.20 ml). Combined organic layers were washed with brine (10
ml), dried over MgSO.sub.4, and the dichloromethane evaporated
under reduced pressure. The resulting crude product was purified by
flash chromatography (EtOAc/DCM) to yield the title compound,
ELQ-331, as a white crystalline solid (560 mg, 54%). .sup.1H NMR
(400 MHz, DMSO-d.sub.6): .delta.=7.98 (s, 1H), 7.57 (s, 1H), 7.44
(m, 4H), 7.21 (m, 4H), 5.76 (s, 2H), 5.35 (s, 2H), 4.03 (s, 3H),
2.44 (s, 3H), 1.11 (t, 3H, J=7.1 Hz); M.P. (.degree. C.):
103.5-103.7.
Example 2--Characterization of ELQ-331 by Gas Chromatography-Mass
Spectrometry (Gc-Ms)
[0081] ELQ-331 was characterized by gc-ms on an Agilent 5977A
MSD/5890B gas chromatography system. The instrument was equipped
with an Agilent J&W GC column with stationary phase HP-5MS with
overall dimensions of 30 m.times.0.250 mm.times.0.25 micrometers
with helium as the inert carrier gas. The temperature gradient was
200-300.degree. C. at 30.degree. C./min.
Example 3--ELQ-387 Synthesis
[0082] Tetrabutyl-ammonium iodide (0.15 g, 0.42 mmol), potassium
carbonate (0.06 g, 0.42 mmol), and 1-chloroethyl ethyl carbonate
(0.06 mL, 0.42 mmol) were dissolved in anhydrous dimethylformamide
(5 mL) in a flame-dried round bottom flask at 70.degree. C. under
inert atmosphere. ELQ-300 (0.10 g, 0.21 mmol) was added and the
reaction stirred under inert atmosphere at 70.degree. C. for four
hours, until complete by thin layer chromatography. The reaction
was cooled to room temperature and the reaction solvent evaporated
under temperature and the reaction solvent evaporated under reduced
pressure. The mixture was taken up in water (10 ml) and extracted
with dichloromethane (3.times.15 mL). Combined organic layers were
washed with brine (15 mL) and concentrated. Purification by silica
column chromatography (EtOAc/DCM) yielded the title compound,
ELQ-387, as a white crystalline solid (39 mg, 32%). .sup.1H NMR
(400 MHz, DMSO-d.sub.6): .delta.=8.03 (s, 1H), 7.54 (s, 1H), 7.44
(m, 3H), 7.25 (m, 2H), 7.20 (m, 2H), 5.83 (q, 1H, J=5.4 Hz), 4.02
(s, 3H), 3.79 (m, 2H), 2.44 (s, 3H), 1.19 (d, 3H, J=5.3 Hz), 0.88
(t, 3H, J=7.1 Hz); M.P. (.degree. C.): 135.0-135.7.
Example 4--In Vitro Antlplasmodial Activity of ELQ-330, ELQ-331,
and ELQ-387 vs. Chloroquine Sensitive (D6) and Resistant (Dd2,
Tm90.C2B) Strains of Plasmodium falciparum
[0083] ELQ-330, ELQ-331 and ELQ-387 were evaluated for
anti-plasmodial activity by the fluorescence based SYBR green assay
developed in our lab and published in 2004. Briefly, experiments
were set up in triplicate in 96-well plates (Costar, Corning) with
2-fold dilutions of each drug across the plate in a total volume of
100 .mu.L and at a final red blood cell concentration of 2% (v/v).
The dilution series was initiated at a concentration of 1 .mu.M and
the experiment was repeated beginning with a lower initial
concentration for those compounds in which the IC.sub.50 value was
below 10 nM. Automated pipetting and dilution was carried out with
the aid of a programmable Precision 2000 robotic station (BioTek,
Winooski, Vt.). An initial parasitemia of 0.2% was obtained by
addition of normal uninfected red cells to a stock culture of
asynchronous parasite infected red cells (PRBC). The plates were
incubated for 72 h at 37.degree. C. in an atmosphere of 5%
C0.sub.2, 5% 0.sub.2, and 90% N.sub.2. After this period, the SYBR
Green I dye-detergent mixture (1 00 !JL) was added and the plates
were incubated at room temperature for an hour in the dark and then
placed in a 96-well fluorescence plate reader (Spectramax
Gemini-EM, Molecular Diagnostics) for analysis, with excitation and
emission wavelength bands centered at 497 and 520 nm, respectively.
The fluorescence readings were plotted against the logarithm of the
drug concentration, and curve fitting by nonlinear regression
analysis (GraphPad Prism software) yielded the drug concentration
that produced 50% of the observed decline relative to the maximum
readings in drug-free control wells (IC.sub.50). Chloroquine was
used as an internal control to establish zero percent viability and
cross-resistance.
[0084] IC.sub.50 values are presented in Table 1. As shown the
IC.sub.50 values for alkoxycarbonyloxyalkyl ester prodrugs ELQ-330
and ELQ-387 are significantly higher than for the parent molecule
ELQ-300 against all three tested strains. These results indicate
that while P. falciparum infected red cells apparently have the
enzymic capacity to break down the prodrugs to release ELQ-300 it
would appear that for these alkoxycarbonyloxyalkyl ester are poorly
processed by parasite encoded esterases. It is both interesting and
significant that the IC.sub.50 values for ELQ-331 are quite similar
to the ELQ-300 values, thereby indicating that this prodrug is more
effectively converted to ELQ-300 by P. falciparum esterases. Taken
together and because it appears that ELQ-331 is more efficiently
converted to ELQ-300 by both host as well as parasite esterases we
hypothesized that this drug would have superior efficacy in
vivo.
TABLE-US-00001 TABLE 1 Antiplasmodial activities for standard
antimalarials (Chloroquine and Atovaquone) and ELQ-300 and the
esterase sensitive prodrug ELQ-337 against drug sensitive (06) and
multidrug resistant (Dd2 and Tm90:C2B) strains of P. falciparum.
IC.sub.50, nM, IC.sub.50, nM, IC.sub.50, nM, P. falciparum P.
falciparum P. falciparum strain Drug strain D6.sup.a strain
Dd2.sup.a Tm90-C2B.sup.a Chloroquine 10 137 98 Atovaquone 0.2 0.2
>250 ELQ-300 6 6 2 ELQ-330 62 38 47 ELQ-331 6 8 4 ELQ-387 326
141 231 .sup.8IC.sub.50 = The drug concentration that decreases
parasite proliferation by 50% relative to control (no-drug) values.
D6 is sensitive to chloroquine while Dd2 and Tm90-C2B are resistant
to chloroquine. Tm90-C2B Is also resistant to the antirespiratory
drug atovaquone.
Example 5--In Vivo Efficacy of Alkoxycarbonyloxyalkyl Ester
Prodrugs ELQ-330 and ELQ-331 Against the Blood Stage of Murine
Malaria Infection
[0085] Typically, antimalarial drugs are provided over the course
of a 3 to 4 day regimen. Such multi-dose schedules are sub-optimal
because it simply may not be feasible in the field where resources
are often limited and dosing schedules may vary. Ideally drugs
could be delivered in a single dose regimen that can be directly
monitored to ensure compliance. Currently there are no drugs in
clinical use for treatment of malaria with sufficient potency and
safety to deliver cures following a single oral dose.
[0086] We evaluated ELQ-330 and ELQ-331 for their potential to cure
mice of a patent malaria infection in a single dose, i.e., single
dose cure (SDC), and compared our findings to the direct
administration of the parent drug ELQ-300. As described above, mice
(female, CF1, Charles River Labs) were infected intravenously with
10.sup.5 P. yoelii (Kenya strain, MR4 MRA-428/Murine LDH Elevating
Virus-Free) parasitized erythrocytes from a donor animal. Drug
administration commenced the day after the animals were inoculated
(Day 1). The test compounds were dissolved in PEG-400 and
administered by oral gavage once. On the 5th day blood films were
prepared and the extent of parasitemia was determined by
microscopic examination of Giemsa stained smears. Animals remaining
parasite free 30 days after the last drug dose were considered
cured of their infection.
[0087] In vivo studies were carried out as described above with
ELQ-300 as an internal control. As previously published, while
ELQ-300 is highly effective in low multi-dose regimens its poor
aqueous solubility and high crystallinity prevent it from being
useful as a single dose curative agent. In this experiment carried
out at oral doses in the range of 1 to 20 mg/kg, ELQ-300 suppressed
parasitemia completely however recrudescence occurred within two
weeks of dosing. In comparison both ELQ-330 and ELQ-331 proved
highly effective against murine malaria and superior to the parent
drug ELQ-300. The lowest fully protective single-dose cure for
ELQ-330 was achieved with an oral dose of 5 mg/kg and for ELQ-331
the lowest observable SDC was 2.5 mg/kg. Evaluation of ELQ-387 is
currently being evaluated in this model. Taken together It is clear
that the 4-position alkoxycarbonate ester derivatives of ELQ-300
are highly effective prodrugs that may be formulated for clinical
use as antimalarial agents. Similar prodrug variants of other ELQs
with clinical or veterinary potential should be more effective than
the corresponding parent molecule, e.g., ELQ-271, ELQ-300, ELQ-316,
and ELQ-400 etc., for treatment of malaria and other parasitic
diseases including malaria (falciparum, vivax, ovale, knowlesi, and
malariae), toxoplasmosis, babesiosis, coccidiosis, theileria, and
other diseases caused by Apicomplexan parasites.
Example
6--6-chloro-7-methoxy-2-methyl-3-(4-(4-(trifluoromethoxy)phenoxy)p-
henyl)quinolin-4-yl ethyl Carbonate (ELQ-337)
##STR00010##
[0089] To a flame dried 50 mL round bottom flask was added 0.5 g
ELQ-300 (1.05 mmol, 1 eq), 84 mg sodium hydride (60% disp., 2.1
mmol, 2 eq) and anhydrous THF 5 mL. The resulting suspension was
heated and stirred at 60.degree. C. under argon atmosphere for 30
mins or until a clear solution was obtained. The reaction was
removed from heat and 200 .mu.L Ethyl chloroformate (2.1 mmol, 2
eq) was added dropwise via a syringe resulting in an immediate
precipitation of white solids. The suspension was stirred for 5
mins and then quenched by dropwise addition of water. The reaction
mixture was diluted with water (5 mL) and extracted with ethyl
acetate (3.times.5 mL). The organic layer was washed with brine (5
mL) and dried over MgSO.sub.4. The residue after evaporation was
recrystallized (DCM, hexanes) to give 0.558 g ELQ-337
(6-chloro-7-methoxy-2-methyl-3-(4-(4-(trifluoromethoxy)phenoxy)phenyl)qui-
nolin-4-yl ethyl carbonate) (97%) as white microcrystals. .sup.1H
NMR (400 MHz, CDCl.sub.3) .delta. 7.89 (s, 1H), 7.50 (s, 1H),
7.32-7.27 (m, 2H), 7.23 (d, J=8.4 Hz, 2H), 7.15-7.01 (m, 4H), 4.16
(q, J=7.1 Hz, 2H), 4.06 (s, 3H), 2.54 (s, 3H), 1.22 (t, J=7.1 Hz,
3H).
Example
7--6-chloro-7-methoxy-2-methyl-3-(4-(4-(trifluoromethoxy)phenoxy)
phenyl)quinolin-4-yl(2-methoxyethyl) carbonate (ELQ-354)
##STR00011##
[0091] ELQ-354 was prepared according to the method of Example 6
except that 2 eq. 2-methoxyethyl chloroformate was used in place of
ethyl chloroformate. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.91
(bs, 1H), 7.52 (s, 1H), 7.29 (d, J=8.7 Hz, 2H), 7.24 (d, J=9.0 Hz,
2H), 7.14-7.04 (m, 4H), 4.31-4.20 (m, 2H), 4.07 (s, 4H), 3.57-3.49
(m, 2H), 3.37 (s, 3H), 1.25 (s, 3H).
Example
8--6-chloro-7-methoxy-2-methyl-3-(4-(4-(trifluoromethoxy)phenoxy)
phenyl)quinolin-4-yl (2-(2-methoxyethoxy)ethyl) carbonate
(ELQ-362)
##STR00012##
[0093] ELQ-362 was prepared according to the method of Example 6
except that 2 eq. 2-(2-methoxyethoxy)ethyl chloroformate was used
in place of ethyl chloroformate. .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. 7.90 (s, 1H), 7.52 (s, 1H), 7.33-7.27 (m, 2H), 7.23 (s,
2H), 7.15-7.05 (m, 4H), 4.30-4.19 (m, 2H), 4.06 (d, J=9.9 Hz, 4H),
3.68-3.58 (m, 4H), 3.57-3.50 (m, J=6.0, 3.0 Hz, 2H), 3.37 (s, 3H),
1.25 (s, 3H).
Example
9--6-chloro-7-methoxy-2-methyl-3-(4-(4-(trifluoromethoxy)phenoxy)
phenyl)quinolin-4-yl (2-(2-(2-methoxyethoxy)ethoxy)ethyl) carbonate
(ELQ-363)
##STR00013##
[0095] ELQ-363 Was prepared according to the method of Example 6
above except that 2 eq. 2-(2-(2-methoxyethoxy)ethoxy)ethyl
chloroformate was used in place of ethyl chloroformate. .sup.1H NMR
(400 MHz, CDCl.sub.3) .delta. 7.89 (s, 1H), 7.49 (bs, 1H), 7.28 (d,
J=8.7 Hz, 2H), 7.24 (d, J=8.5 Hz, 2H), 7.13-7.06 (m, 4H), 4.27-4.22
(m, 2H), 4.06 (s, 3H), 3.68-3.60 (m, 8H), 3.54 (dd, J=5.7, 3.6 Hz,
2H), 3.37 (s, 3H), 1.25 (s, 3H).
Example 10--allyl
(6-chloro-7-methoxy-2-methyl-3-(4-(4-(trifluoromethoxy)phenoxy)
phenyl)quinolin-4-yl) carbonate (ELQ-359)
##STR00014##
[0097] ELQ-359 was prepared according to the method of Example 6
above except that 2 eq. allyl chloroformate in was used place of
ethyl chloroformate. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.89
(s, 1H), 7.52 (s, 1H), 7.28 (d, J=8.7 Hz, 2H), 7.23 (d, J=8.4 Hz,
2H), 7.14-7.02 (m, 4H), 5.79 (ddd, J=22.7, 11.0, 5.7 Hz, 1H),
5.31-5.26 (m, 1H), 5.23 (dd, J=10.6, 1.2 Hz, 1H), 4.58 (dt, J=5.7,
1.2 Hz, 2H), 4.06 (s, 3H), 2.54 (s, 3H).
Example 11--6-chloro-7-methoxy-2-methyl-3-(4-(4-(trifluoromethoxy)
phenoxy)phenyl)quinolin-4-yl isobutyrate (ELQ-375)
##STR00015##
[0099] ELQ-375 was prepared according to the method of Example 6
above except that 2 eq. isobutyryl chloride was used in place of
ethyl chloroformate. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 8.50
(s, 1H), 7.86 (s, 1H), 7.25-7.02 (m, 8H), 4.19 (s, 3H), 2.88 (s,
3H), 2.71 (dq, J=13.8, 7.0 Hz, 1H), 1.08 (d, J=7.0 Hz, 6H).
Example
12--6-chloro-7-methoxy-2-methyl-3-(4-(4-(trifluoromethoxy)phenoxy)-
phenyl)quinolin-4-yl pivalate (ELQ-357)
##STR00016##
[0101] ELQ-357 was prepared according to the method of Example 6
above except that 2 eq. trimethylacetyl chloride was used in place
of ethyl chloroformate. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta.
8.35 (s, 1H), 7.78 (s, 1H), 7.25-7.01 (m, 8H), 4.17 (s, 3H), 2.80
(s, 3H), 1.15 (s, 9H).
Example
13--6-chloro-7-methoxy-2-methyl-3-(4-(4-(trifluoromethoxy)phenoxy)
phenyl)quinolin-4-yl benzoate (ELQ-379)
##STR00017##
[0103] ELQ-379 was prepared according to the method of Example 6
above except 2 eq. Benzoyl chloride was used in place of ethyl
chloroformate. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 8.00 (dd,
J=8.3, 1.2 Hz, 2H), 7.81 (s, 1H), 7.66 (dd, J=10.6, 4.4 Hz, 1H),
7.53 (s, 1H), 7.49 (t, J=7.8 Hz, 2H), 7.30 (d, J=8.7 Hz, 2H), 7.03
(dd, J=31.9, 8.5 Hz, 4H), 6.83-6.70 (m, 2H), 4.07 (s, 3H), 2.58 (s,
3H).
Example 14--tert-butyl
(6-chloro-7-methoxy-2-methyl-3-(4-(4-(trifluoromethoxy)
phenoxy)phenyl)quinolin-4-yl) carbonate (ELQ-358)
##STR00018##
[0105] ELQ-358 was prepared according to the method of Example 6
above except that 2 eq. di-tert-butyl dicarbonate was used in place
of ethyl chloroformate. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta.
7.90 (s, 1H), 7.49 (s, 1H), 7.32-7.28 (m, 2H), 7.25-7.20 (m, 2H),
7.13-7.03 (m, 4H), 4.06 (s, 3H), 2.54 (s, 3H), 1.37 (s, 9H)
Example 15--6-chloro-7-methoxy-2-methyl-3-(4-(4-(trifluoromethoxy)
phenoxy)phenyl)quinolin-4-yl
((2,2-dimethyl-1,3-dioxolan-4-yl)methyl) carbonate (ELQ-374)
##STR00019##
[0107] ELQ-374 was prepared according to the method of Example 6
above except that 2 eq. (2,2-dimethyl-1,3-dioxolan-4-yl)methyl
chloroformate was used in place of ethyl chloroformate. .sup.1H NMR
(400 MHz, CDCl.sub.3) .delta. 7.90 (s, 1H), 7.52 (s, 1H), 7.28 (d,
J=8.6 Hz, 2H), 7.24 (d, J=9.3 Hz, 2H), 7.10 (dd, J=8.8, 1.7 Hz,
4H), 4.23-4.08 (m, 3H), 4.06 (s, 3H), 4.01 (dd, J=8.6, 6.3 Hz, 1H),
3.64 (dd, J=8.6, 5.3 Hz, 1H), 2.54 (s, 3H), 1.40 (s, 3H), 1.37 (s,
3H).
Example 16--6-chloro-7-methoxy-2-methyl-3-(4-(4-(trifluoromethoxy)
phenoxy)phenyl)quinolin-4-yl (2,3-dihydroxypropyl) carbonate
(ELQ-376)
##STR00020##
[0109] ELQ-376 was prepared according to the method of Example 6
above, except that 2 eq. (2-oxo-1,3-dioxolan-4-yl)methyl
chloroformate was used in place of ethyl chloroformate. .sup.1H NMR
(400 MHz, CDCl.sub.3) .delta. 7.85 (s, 1H), 7.50 (s, 1H), 7.27 (t,
J=8.0 Hz, 4H), 7.17-7.05 (m, 4H), 4.79 (ddt, J=8.4, 6.2, 4.1 Hz,
1H), 4.48 (t, J=8.7 Hz, 1H), 4.31 (qd, J=12.4, 4.0 Hz, 2H), 4.07
(t, J=7.5 Hz, 1H), 4.06 (s, 3H), 2.54 (s, 3H).
Example 17--6-chloro-7-methoxy-2 methyl-3-(4-(4-(trifluoromethoxy)
phenoxy)phenyl)quinolin-4-yl ((2-oxo-1,3-dioxolan-4-yl)methyl)
carbonate (ELQ-373)
##STR00021##
[0111] ELQ-373 was prepared from
6-chloro-7-methoxy-2-methyl-3-(4-(4
(trifluoromethoxy)phenoxy)phenyl) quinolin-4-yl
((2,2-dimethyl-1,3-dioxolan-4-yl)methyl) carbonate upon stirring in
2M HCl for 12h. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.79 (s,
1H), 7.44 (s, 1H), 7.27-7.11 (m, 4H), 7.11-7.00 (m, 4H), 4.72 (ddt,
J=8.3, 6.2, 4.1 Hz, 1H), 4.41 (t, J=8.7 Hz, 1H), 4.24 (qd, J=12.3,
3.9 Hz, 2H), 4.00 (s, 3H), 4.04-3.93 (m, 1H), 2.48 (s, 3H).
Example
18--6-chloro-7-methoxy-2-methyl-3-(4-(4-(trifluoromethoxy)phenoxy)-
phenyl)quinolin-4-yl (1,1-dioxidotetrahydrothiophen-3-yl) carbonate
(ELQ-355)
##STR00022##
[0113] ELQ-355 was prepared according to the method of Example 6
above, except that 2 eq. 1,1-dioxidotetrahydrothiophen-3-yl
chloroformate was used in place of ethyl chloroformate. .sup.1H NMR
(400 MHz, CDCl.sub.3) .delta. 7.88 (s, 1H), 7.52 (s, 1H), 7.39-7.27
(m, J=2.6 Hz, 4H), 7.12 (dd, J=13.0, 8.9 Hz, 4H), 5.36-5.22 (m,
1H), 4.07 (s, 3H), 3.30 (dd, J=14.7, 6.5 Hz, 1H), 3.12 (dd, J=9.6,
5.8 Hz, 2H), 2.91 (d, J=14.6 Hz, 1H), 2.56 (s, 3H), 2.50-2.38 (m,
1H), 2.35-2.22 (m, 1H).
Example
19--6-chloro-7-methoxy-2-methyl-3-(4-(4-(trifluoromethoxy)phenoxy)-
phenyl)quinolin-4-yl methyl carbonate (ELQ-336)
##STR00023##
[0115] ELQ-336 was prepared according to the method of Example 6
above, except that 2 eq. methyl chloroformate was used in place of
ethyl chloroformate. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.88
(s, 1H), 7.51 (s, 1H), 7.30-7.27 (m, 2H), 7.24 (d, J=8.4 Hz, 2H),
7.13-7.05 (m, 4H), 4.06 (s, 3H), 3.75 (s, 3H), 2.54 (s, 3H).
Example
20--6-fluoro-7-methoxy-2-methyl-3-(4-(4-(trifluoromethoxy)phenoxy)-
phenyl)quinolin-4-yl ethyl carbonate (ELQ-334)
##STR00024##
[0117] ELQ-334 was prepared according to the method of Example 6
above from
6-fluoro-7-methoxy-2-methyl-3-(4-(4-(trifluoromethoxy)phenoxy)phenyl-
)quinolin-4-ol and ethyl chloroformate. .sup.1H-NMR (400 MHz;
CDCl.sub.3): .delta. 7.52 (d, J=8.0 Hz, 1H), 7.48 (d, J=11.2 Hz,
1H), 7.30-7.27 (m, 2H), 7.24-7.22 (m, 2H), 7.12-7.06 (m, 4H), 4.15
(q, J=7.1 Hz, 2H), 4.04 (s, 3H), 2.53 (s, 3H), 1.22 (t, J=7.1 Hz,
3H). .sup.19F-NMR (376 MHz; CDCl.sub.3): .delta.-58.26 (s, 1F),
-131.68 (t, J=9.9 Hz,).
Example
21--6-fluoro-7-methoxy-2-methyl-3-(4-(4-(trifluoromethoxy)phenoxy)-
phenyl)quinolin-4-yl pivalate (ELQ-377)
##STR00025##
[0119] ELQ-377 was prepared according to the method of Example 6
above from
6-fluoro-7-methoxy-2-methyl-3-(4-(4-(trifluoromethoxy)phenoxy)phenyl-
)quinolin-4-ol and trimethylacetyl chloride .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 7.51 (d, J=8.0 Hz, 1H), 7.28 (s, 1H), 7.25-7.19
(m, 4H), 7.12-7.00 (m, 4H), 4.04 (s, 3H), 2.49 (s, 3H), 1.14 (s,
9H).
Example 22--Efficacy of ELQ-337 In Vitro and In Vivo
[0120] In vitro experiments show that the intrinsic antiplasmodial
activity of ethylcarbonate ester ELQ-337 is indistinguishable from
ELQ-300, with IC.sub.50 values against all test strains in the low
to sub-nanomolar range. In vivo experiments using the 4-day
suppression test protocol (dosing on 4 sequential days with smears
on Day 5) vs. P. yoelii infected mice show that the action profile
is unchanged at lower doses needed for ED.sub.50 (0.02 mg/kg/d),
ED.sub.90 (0.05 mg/kg/d), ED.sub.99 (0.075 mg/kg/d), and
non-recrudescence dose remains impressive (0.3 to 1 mg/kg/d).
Importantly and unlike ELQ-300 (at any dose), ELQ-337 provided 4/4
single dose cures (SDC) at doses as low as 3 mg/kg (1 mg/kg failed
in 4/4 animals on Day 12).
TABLE-US-00002 TABLE 1 Efficacy of exemplary compounds. Lowest
fully P. effective falciparum single dose strain D6, cure P. yoelii
Code Chemical Structure c.sub.logP IC.sub.50, nM (mg/kg) ELQ-300
##STR00026## 5.66 3.1 >20* ELQ-336 ##STR00027## 8.16 2.5 4
ELQ-337 ##STR00028## 8.5 2.5 3 ELQ-354 ##STR00029## 7.2 3.0 3
ELQ-355 ##STR00030## 5.8 3.9 4 ELQ-357 ##STR00031## 8.5 3.5 4
ELQ-358 ##STR00032## 8.3 6.0 >4 ELQ-359 ##STR00033## 7.9 2.5 4
ELQ-373 ##STR00034## 6.2 3.1 4 ELQ-374 ##STR00035## 8.0 2.7 4
ELQ-375 ##STR00036## 8.1 8.2 4 ELQ-379 ##STR00037## 9.4 2.5 ND
* * * * *