U.S. patent application number 13/094789 was filed with the patent office on 2011-08-18 for compounds from mycelium of antrodia cinnamomea and use thereof.
This patent application is currently assigned to SIMPSON BIOTECH CO., LTD.. Invention is credited to MASAO HATTORI, CHIA-CHIN SHEU.
Application Number | 20110201666 13/094789 |
Document ID | / |
Family ID | 40912231 |
Filed Date | 2011-08-18 |
United States Patent
Application |
20110201666 |
Kind Code |
A1 |
SHEU; CHIA-CHIN ; et
al. |
August 18, 2011 |
COMPOUNDS FROM MYCELIUM OF ANTRODIA CINNAMOMEA AND USE THEREOF
Abstract
The present invention relates to compounds from mycelium of
Antrodia cinnamomea. The present invention also relates to a
composition and a method for treating or prophylaxis of hepatitis C
virus (HCV) infection.
Inventors: |
SHEU; CHIA-CHIN; (TAOYUAN
COUNTY, TW) ; HATTORI; MASAO; (TOYAMA, JP) |
Assignee: |
SIMPSON BIOTECH CO., LTD.
Taoyuan County
TW
|
Family ID: |
40912231 |
Appl. No.: |
13/094789 |
Filed: |
April 26, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12811115 |
Jun 29, 2010 |
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PCT/CN2008/000196 |
Jan 28, 2008 |
|
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13094789 |
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Current U.S.
Class: |
514/425 ;
514/473; 514/538; 514/570 |
Current CPC
Class: |
C07D 207/46 20130101;
C07D 207/50 20130101; A61P 31/14 20180101; C07D 207/444 20130101;
A61P 31/20 20180101; C07C 235/34 20130101; C07D 307/60
20130101 |
Class at
Publication: |
514/425 ;
514/570; 514/538; 514/473 |
International
Class: |
A61K 31/4015 20060101
A61K031/4015; A61K 31/192 20060101 A61K031/192; A61K 31/24 20060101
A61K031/24; A61K 31/341 20060101 A61K031/341; A61P 31/14 20060101
A61P031/14 |
Claims
1. A method for treating or prophylaxis hepatitis C virus infection
which comprises administering to a subject in need thereof a
pharmaceutically effective amount of an active agent selected from
(1) a compound having the formula III ##STR00010## wherein R is
--(CH.sub.2).sub.kCOOH or --(CH.sub.2).sub.kCOO(C.sub.mH.sub.2m+1)
wherein k is 0-6 and m is 0-6; (2) a compound having the formula
##STR00011## wherein R.sub.1 is C.sub.1-10 carboxylic acid or
C.sub.1-10 ester; R.sub.2 is C.sub.1-10 carboxylic acid or
C.sub.1-10 ester; R.sub.3 is H, C.sub.1-10 alkyl, C.sub.2-10
alkenyl or C.sub.2-10 alkynyl; and R.sub.4 is H, C.sub.1-10 alkyl,
C.sub.2-10 alkenyl or C.sub.2-10 alkynyl; or (3) a compound having
the formula ##STR00012## wherein X is N or O; R.sub.1 is C.sub.1-10
alkyloxy, C.sub.2-10 alkenyloxy, or C.sub.2-10 alkynyloxy; R.sub.2
is H, C.sub.1-10 alkyl, C.sub.2-10 alkenyl or C.sub.2-10 alkynyl;
and R.sub.3 is absent, H or hydroxy; provided that if X is O,
R.sub.3 is absent.
2. The method of claim 1, wherein the compound is selected from
##STR00013##
3. The method according to claim 1, wherein the subject is a
mammal.
4. The method according to claim 3, wherein the mammal is a human.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to, and is a Divisional of,
U.S. patent application Ser. No. 12/811,115, filed on Jun. 29,
2010, now pending, which is hereby incorporated by reference in its
entirety.
[0002] Although incorporated by reference in its entirety, no
arguments or disclaimers made in the parent application apply to
this divisional application. Any disclaimer that may have occurred
during the prosecution of the above-referenced application(s) is
hereby expressly rescinded. Consequently, the Patent Office is
asked to review the new set of claims in view of all of the prior
art of record and any search that the Office deems appropriate
FIELD OF THE INVENTION
[0003] The present invention relates to compounds from mycelium of
Antrodia cinnamomea. The present invention also relates to a
composition and a method for treating or prophylaxis of hepatitis C
virus (HCV) infection. 2.sup.nh
DESCRIPTION OF PRIOR ART
[0004] It is estimated that approximate 3% of the world's
population is infected with hepatitis C virus (HCV). In developed
countries, chronic hepatitis C is the leading cause for cirrhosis,
hepatocellular carcinoma, and liver transplantation. The protease
of hepatitis C virus is required for the cleavage of viral
nonstructural polyprotein to form the mature virus and represents
one of the attractive therapeutic targets for developing antiviral
agents against HCV (Liu et al., 2004; Hepatitis C NS3 protease
inhibition by peptidyl-a-ketoamide inhibitors: kinetic mechanism
and structure. Arch Biochem Biophys 421: 207-216; Kakiuchi et al.,
1999 A high throughput assay of the hepatitis C virus nonstructural
protein 3 serine proteinase. J Virol 80: 77-84).
[0005] The use of herbal therapy and folk medicines has been known
for thousands of years in China. In fact, records on the use of
herbs date back to biblical times. However, only recently have
scientists begun exploring the possible role for herbs in treatment
of viral infections. For example, extracts from the root of the
Ecballium Elaterium have been used to treat HCV and HBV (EP 0793964
and U.S. Pat. No. 5,648,089). While research in the field of herbal
medicines has increased, much remains to be learned about the
effectiveness of such herbal remedies.
[0006] The fruiting body of Antrodia cinnamomea Chang & W N
Chou (Basidiomycetes, synonym A. camphorate Wu) is a highly valued
folk medicine in Taiwan. It is used as an antidote and for
diarrhea, abdominal pain, hypertension, itchy skin, and liver
cancer. Some bioactive constituents from the fruiting body of
Antrodia cinnamomea have been isolated and characterized as a
series of polysaccharides, steroids, triterpenoids, and
sesquiterpene lactone (Lin et al., 2007, Factors affecting mycelial
biomass and exopolysacharide production in submerged cultivation of
Antrodia cinnamomea using complex media. Bioresource Technology 98:
2511-2517). In previous studies, five new maleic and succinic acid
derivatives (antrodin A-E) are isolated from the mycelium of
Antrodia cinnamomea (Nakamura et al., 2004, Five new maleic and
succinic acid derivatives from the mycelium of Antrodia comphorata
and their cytotoxic effects on LLC tumor cell line. J Nat Prod 67:
46-48).
[0007] U.S. Pat. No. 7,109,232 discloses compounds 1-5 from
Antrodia cinnamomea and their use such as hepatoprotection,
anti-inflammation or anti-tumor activity and preparation.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] FIG. 1 depicts the structure of the compounds 1-10 from the
mycelium of Antrodia cinnamomea.
[0009] FIG. 2 illustrates Lineweaver-Burk plot (1/Vi v.s. 1/[S])
for the inhibition of HCV-NS3 protease by compound 1 in the
presence of various concentrations of substrate (:10 .mu.g/ml,
.quadrature.:5 .mu.g/ml and .smallcircle.:0.mu.).
[0010] FIG. 3 is analytic data of compound 9 of the present
invention.
[0011] FIG. 4 is analytic data of compound 10 of the present
invention.
SUMMARY OF THE INVENTION
[0012] Accordingly, an object of the present invention is to
provide novel compounds from mycelium of Antrodia cinnamomea.
[0013] Another object of the present invention is to provide a
pharmaceutical composition for treating or prophylaxis hepatitis C
virus infection, comprising a compound of the present invention in
an amount effective to attenuate infectivity of said virus, and a
pharmaceutically acceptable carrier.
[0014] Further object of the present invention is to provide a
method for treating or prophylaxis hepatitis C virus infection
which comprises administering to a subject in need thereof a
pharmaceutically effective amount of a composition comprising the
compounds from mycelium of Antrodia cinnamomea.
DETAILED DESCRIPTION OF THE INVENTION
[0015] Since the mycelium of Antrodia cinnamomea was recently
reported to be clinically effective for hepatitis patients infected
with HCV (Akiba T et al. 2007. Clinical study of Shoshi on Japanese
hepatitis patients infected with C type hepatitis virus. The second
Taiwan and Japan Symposium on Antrodia cinnamomea, Abst. pp.
82-98), a SensoLyte.TM. 520 HCV protease assay kit was applied to
investigate the HCV-protease inhibitory activity of the isolated
antrodins, the metabolites of antrodin C as well as one metabolite
analogue. The assay method with a quenched-fluorogenic peptide
substrate can be used for measure the activity of inhibitors as
well as for continuous recording of the progress of the enzyme
reaction. Using this assay method, the mode of inhibition of the
most potent compound was studied by Lineweaver-Burk plot.
[0016] As shown in Table 1, of the 5 constituents (compounds 1-5)
from mycelium of Antrodia cinnamomea, four of them (compounds 1 and
3-5) showed inhibitory activity on HCV protease. Compounds 6-8, the
newly formed in vivo metabolites (or metabolite analogue) of
antrodin C showed activity too. Compound 1, which was isolated from
Antrodia cinnamomea and was also detected in vivo as one of the
major metabolite of compound 3, showed the most potent activity
with an IC5o less than 1 .mu.g/ml.
[0017] The mode of inhibition was kinetically analyzed by plotting
the enzyme activity at different concentrations of the substrate
(10, 50, 100 and 200 times dilution of the substrate stocking
solution) with (5 .mu.g/ml, and 10 .mu.g/ml) and without [0
.mu.g/ml (DMSO)] compound 1. As shown in FIG. 2 the mode of HCV PR
inhibition by compound 1 was found to be competitive.
[0018] Traditionally, the fruit body of Antrodia cinnamomea has
been used for liver cancer (Lin E S, Chen Y H. 2007. Factors
affecting mycelial biomass and exopolysacharide
production in submerged cultivation of Antrodia cinnamomea using
complex media.
[0019] Bioresource Technology 98: 2511-2517). Polysaccharides of
Antrodia cinnamomea has been show to have hepatoprotective effect
(Han et al., 2006b, Protective effects of a neutral polysaccharide
isolated from the mycelium of Antrodia cinnamomea on
Propionibacterium acnes and lipopolysaccharide induced hepatic
injury in mice. Chem Pharm Bull 54: 496-500) and anti-hepatitis B
virus activity (Lee et. al., 2002, Antrodia camphorate
polysaccharides exhibit anti-hepatitis B virus effects. FEMS
Microbiol Lett 209:63-67). Of the maleic and succinic acid
derivatives, antrodin C (compound 3) showed protective activity in
Propionicbacterium acnes and lipopolysaccharide treated mice
(Nakamura N, Five new maleic and succinic acid derivatives from the
mycelium of Antrodia comphorata and their cytotoxic effects on LLC
tumor cell line. J Nat Prod 67: 46-48). Quantitative analysis
showed that compound 3 was the most abundant compound of this
chemical type in the mycelium with a content of ca. 5% of the dry
weight of mycelia (Han et al., 2006a, Protective effects of a
neutral polysaccharide isolated from the mycelium of Antrodia
cinnamomea on Propionibacterium acnes and lipopolysaccharide
induced hepatic injury in mice. Chem Pharm Bull 54: 496-500).
Research on the in vivo metabolism of compound 3 revealed that this
compound was converted to six metabolites, i.e. compounds 1, 2, 6,
7a, 7b and an analogue of compound 8. Pharmacokinetic study on
compound 3 demonstrated that this compound was quickly absorbed
from the gastrointestinal tract followed by rapid and complete
metabolization in liver in such a degree that compound 3 itself
could not be detected in the body after absorption (Masao Hattori,
2007. Metabolism and Disposition of Antrodin C (Hepasim) from the
Mycelium of Antrodia cinnamomea in Rats. The second Taiwan and
Japan Symposium on Antrodia cinnamomea, Abst. pp. 1-9). This
pharmacokinetic property as shown in Table 1 suggested that the
metabolites are responsible for in vivo pharmacological activities
of compound 3 and consequently of the folk medicine. All those
metabolites and the constituents of the mycelium of Antrodia
cinnamomea except for compound 2 showed inhibitory activity on HCV
protease. These results strongly support the use of this folk
medicine for liver cancer which is often caused by long term
infection of hepatitis C virus. The active compounds used in the
present invention could be served as leading compounds for the
development of potent anti-hepatitis C agents through the mechanism
of inhibition against the virus protease.
TABLE-US-00001 TABLE 1 IC.sub.50 values of antrodins and the
metabolites against HCV protease. Sample IC.sub.50 (.mu.g/ml)
1.sup.a,b 0.9 2.sup.a,b >100 3.sup.a 2.9 4.sup.a 20.0 5.sup.a
20.1 6.sup.b 6.6 7.sup.b (7a:7b:1 = 1.2 ca 5:8:4) 8.sup.c 57.5
Embelin 4.1 .sup.aConstituent of the mycelium of Antrodia
cinnamomea; .sup.bin vivo metabolite of antrodin C; .sup.canalogue
of one of the in vivo metabolite of antrodin C.
[0020] Accordingly, the present invention provides a compound
having the formula
##STR00001##
wherein denotes a single or double bond; R.sub.1 is
--(CH.sub.2).sub.nCOOH or --(CH.sub.2).sub.nCOOC.sub.mH.sub.2m+1
wherein n is 0-6 and m is 1-6; R.sub.2 is absent, H or OH, and
R.sub.3 is absent, H or OH.
[0021] In a preferred compound of the present invention, the
compound having formula I wherein R.sub.1 is COOH; R.sub.2 is
absent and R.sub.3 is OH.
[0022] In an alternative preferred compound of the present
invention, the compound having formula II wherein R.sub.1 is
COOCH.sub.3, R.sub.2 is OH and R.sub.3 is absent.
[0023] The present invention further provides a compound having the
formula III
##STR00002##
wherein R is --(CH.sub.2).sub.kCOOH or
--(CH.sub.2).sub.kCOO(C.sub.mH.sub.2m+1) wherein k is 0-6 and m is
0-6.
[0024] In a preferred compound of formula III, R is
--CH.sub.2COOH.
[0025] Certain compounds exist in one or more particular geometric,
optical, enantiomeric, diasteriomeric, epimeric, stereoisomeric,
tautomeric, conformational, or anomeric forms, including but not
limited to, cis- and trans-forms; E- and Z-forms; c-, t-, and
r-forms; endo- and exo-forms; R-, S-, and meso-forms; D- and
L-forms; d- and l-forms; (+) and (-) forms; keto-, enol-, and
enolate-forms; syn- and anti-forms; synclinal- and
anticlinal-forms; alpha.- and beta.-forms; axial and equatorial
forms; boat-, chair-, twist-, envelope- and halfchair-forms; and
combinations thereof, hereinafter collectively referred to as
"isomers" (or "isomeric forms").
[0026] If the compound is in crystalline form, it may exist in a
number of different polymorphic forms.
[0027] Unless otherwise specified, the compounds of the present
invention include all such isomeric forms, including (wholly or
partially) racemic and other mixtures thereof. Methods for the
preparation (e.g. asymmetric synthesis) and separation (e.g.
fractional crystallization and chromatographic means) of such
isomeric forms are either known in the art or are readily obtained
by adapting the methods taught herein, or known methods, in a known
manner.
[0028] Unless otherwise specified, the compounds of the present
invention include also includes ionic, salt, solvate, and protected
forms of thereof. It may be convenient or desirable to prepare,
purify, and/or handle a corresponding salt of the active compound,
for example, a pharmaceutically-acceptable salt. Examples of
pharmaceutically acceptable salts are discussed in Berge et al.,
1977, "Pharmaceutically Acceptable Salts," J. Pharm. Sci., Vol. 66,
pp. 1-19.
[0029] The pharmaceutically acceptable salts of the compounds are
prepared following procedures which are familiar to those skilled
in the art.
[0030] The present invention provides a pharmaceutical composition
for treating or prophylaxis hepatitis C virus infection, comprising
effective amount of a compound having formula I, II or III, and a
pharmaceutically acceptable carrier.
[0031] The present invention also provides a pharmaceutical
composition for treating or prophylaxis hepatitis C virus
infection, comprising (i) an effective amount of a compound having
formula IV
##STR00003##
wherein R.sub.1 is C.sub.1-10 carboxylic acid or C.sub.1-10 ester;
R.sub.2 is C.sub.1-10 carboxylic acid or C.sub.1-10 ester; R.sub.3
is H, C.sub.1-10 alkyl, C.sub.2-10 alkenyl or C.sub.2-10 alkynyl;
and R.sub.4 is H, C.sub.1-10 alkyl, C.sub.2-10 alkenyl or
C.sub.2-10 alkynyl; or (ii) a compound having formula V
##STR00004##
wherein
X is N or O;
[0032] R.sub.1 is C.sub.1-10 alkyloxy, C.sub.2-10 alkenyloxy, or
C.sub.2-10 alkynyloxy; R.sub.2 is H, C.sub.1-10 alkyl, C.sub.2-10
alkenyl or C.sub.2-10 alkynyl; and R.sub.3 is absent, H or hydroxy;
provided that if X is O, R.sub.3 is absent; and a pharmaceutically
acceptable carrier.
[0033] In the pharmaceutical composition of the present invention,
the preferred compound is selected from [0034]
3-isobutyl-4-[4-(3-methyl-2-butenyloxy)phenyl]furan-2,5-dione,
[0035]
3-isobutyl-4-[4-(3-methyl-2-butenyloxy)phenyl]-1H-pyrrol-2,5-dione,
[0036]
3-isobutyl-4-[4-(3-methyl-2-butenyloxy)phenyl]-1H-pyrrol-1-ol-2,5--
dione, [0037]
3R*,48*-1-hydroxy-3-isobutyl-4-[4-(3-methyl-2-butenyloxy)phenyl]pyrrolidi-
ne-2,5-dione, [0038]
3R*,4R*-1-hydroxy-3-isobutyl-4-[4-(3-methyl-2-butenyloxy)phenyl]pyrrolidi-
ne-2,5-dione. [0039]
(2Z)-2-isobutyl-3-{4-[(3-methylbut-2-en-1-yl)oxy]phenyl}but-2-enedioic
acid, [0040]
(2Z)-2-isobutyl-3-{4-[(3-methylbut-2-en-1-yl)oxy]phenyl}but-2-enedioic
acid 4-methyl ester; or [0041]
(2Z)-2-isobutyl-3-{4-[(3-methylbut-2-en-1-yl)oxy]phenyl}but-2-enedioic
acid 1-methyl ester.
[0042] The composition may be prepared in various forms for
administration, including tablets, caplets, pills or dragees, or
can be filled in suitable containers, such as capsules, or, in the
case of suspensions, filled into bottles. As used herein,
"pharmaceutically acceptable carrier" includes any and all
solvents, diluents, or other liquid vehicle, dispersion or
suspension aids, surface active agents, isotonic agents, thickening
or emulsifying agents, preservatives, solid binders, lubricants and
the like, as suited to the particular dosage form desired.
Remington's Pharmaceutical Sciences, Fifteenth Edition, E. W.
Martin (Mack Publishing Co., Easton, Pa., 1975) discloses various
carriers used in formulating pharmaceutical compositions and known
techniques for the preparation thereof. Except insofar as any
conventional carrier medium is incompatible with the anti-viral
compounds of the invention, such as by producing any undesirable
biological effect or otherwise interacting in a deleterious manner
with any other component(s) of the pharmaceutical composition, its
use is contemplated to be within the scope of this invention. In
the pharmaceutical compositions of the invention, the active agent
may be present in an amount of at least 0.5% and not more than 90%
by weight based on the total weight of the composition, including
carrier medium and/or auxiliary agent(s). Preferably, the
proportion of active agent varies between 5%-50% by weight of the
composition. Pharmaceutical organic or inorganic solid or liquid
carrier media suitable for enteral or parenteral administration can
be used to make up the composition. Gelatine, lactose, starch,
magnesium, stearate, talc, vegetable and animal fats and oils, gum,
polyalkylene glycol, or other known excipients or diluents for
medicaments may all be suitable as carrier media.
[0043] The present invention further provides a method for treating
or prophylaxis hepatitis C virus infection which comprises
administering to a subject in need thereof a pharmaceutically
effective amount of an active agent selected from
(1) a compound having the formula
##STR00005##
wherein denotes a single or double bond; R.sub.1 is
--(CH.sub.2).sub.nCOOH or --(CH.sub.2).sub.nCOOC.sub.mH.sub.2m+1
wherein n is 0-6 and m is 1-6; R.sub.2 is absent, H or OH, and
R.sub.3 is absent, H or OH; (2) a compound having the formula
III
##STR00006##
wherein R is --(CH.sub.2).sub.kCOOH or
--(CH.sub.2).sub.kCOO(C.sub.mH.sub.2m+1) wherein k is 0-6 and m is
0-6; (3) a compound having the formula
##STR00007##
wherein R.sub.1 is C.sub.1-10 carboxylic acid or C.sub.1-10 ester;
R.sub.2 is C.sub.1-10 carboxylic acid or C.sub.1-10 ester; R.sub.3
is H, C.sub.1-10 alkyl, C.sub.2-10 alkenyl or C.sub.2-10 alkynyl;
and R.sub.4 is H, C.sub.1-10 alkyl, C.sub.2-10 alkenyl or
C.sub.2-10 alkynyl; or (4) a compound having the formula
##STR00008##
wherein
X is N or O;
[0044] R.sub.1 is C.sub.1-10 alkyloxy, C.sub.2-10 alkenyloxy, or
C.sub.2-10 alkynyloxy; R.sub.2 is H, C.sub.1-10 alkyl, C.sub.2-10
alkenyl or C.sub.2-10 alkynyl; and R.sub.3 is absent, H or hydroxy;
provided that if X is O, R.sub.3 is absent.
[0045] In a preferred embodiment of the present invention, active
agent is selected from
##STR00009##
[0046] The compounds of the invention may be administered using any
amount and any route of administration effective for attenuating
infectivity of the hepatitis C virus. Thus, the term "effective
amount" used herein refers to a nontoxic but sufficient amount of
the antiviral agent to provide the desired treatment of viral
infection. The exact amount required will vary from subject to
subject, depending on the species, age, and general condition of
the subject, the severity of the infection, the particular
antiviral agent and its mode of administration, and the like. The
anti-hepatitis C compounds are preferably formulated in dosage unit
form for ease of administration and uniformity of dosage. Dosage
unit form as used herein refers to a physically discrete unit of
anti-viral agent appropriate for the patient to be treated. Each
dosage should contain the quantity of active material calculated to
produce the desired therapeutic effect either as such, or in
association with the selected pharmaceutical carrier medium.
[0047] The compounds of the invention may be administered orally,
parenterally, such as by intramuscular injection, intraperitoneal
injection, intravenous infusion or the like, depending on the
severity of the infection being treated.
[0048] Although the compounds of the present invention can be
administered to any patient which is susceptible to hepatitis C
infection, the compounds are intended for the treatment of
mammalian hosts, and especially humans.
[0049] In view of the inhibitory effect on enzyme activity produced
by the compounds of the invention, it is anticipated that these
compounds will be useful not only for therapeutic treatment of
infection, but for hepatitis C viral prophylaxis, as well. The
above-noted dosages will be essentially the same whether for
treatment or prophylaxis of hepatitis C infection.
[0050] The term "organic solvent" used herein includes but is not
limited to alcohol (such as CH.sub.3OH, C.sub.2H.sub.5OH,
C.sub.3H.sub.7OH), ester (such as acetyl acetate), alkane (such as
hexane) and halogenated alkane (such as CH.sub.3Cl,
C.sub.2H.sub.2Cl.sub.2). The preferred organic solvent is ethanol
or alcoholic solvent without causing any side effect of human.
Especially, the compounds 9 and 10 are prepared from organic
solvent extract from Antrodia cinnamomea.
[0051] The following examples are provided to describe the
invention in further detail. These examples, which set forth the
best mode presently contemplated for carrying out the invention,
are intended to illustrate and not to limit the invention.
EXAMPLE
[0052] The examples below are non-limiting and are merely
representative of various aspects and features of the present
invention.
[0053] Apparatus.
[0054] NMR spectra were obtained on a Varian Unity Plus 500
.sup.1H, 500 MHz; .sup.13C, 125 MHz) spectrometer. MS spectrum was
measured on an electrospray ionization mass spectrometer (ESI-MS,
Esquire 3000.sup.Plus, Bruker Daltonik GmbH, Bremen, Germany).
[0055] Materials for HCV protease assay.
[0056] HCV NS3/4A protease (lot# Lot 046-047 for the screening and
lot# Lot 046-079 for the mechanism study) and SensoLyte.TM. 520 HCV
Protease Assay Kit *Fluorimetric* (lot# AK 71147-1005) were
purchased from AnaSpec, San Jose, Calif., USA. The substrate was a
5-FAM/QXL.TM. 520 FRET peptide based on the sequence of
Ac-Asp-Glu-Asp(EDANS)-Glu-Glu-Abu-.PSI.-[COO]Ala-Ser-Lys(DABCYL)-NH2.
The assay was carried out on BD Falcon.TM. Microtest.TM.384-well
120 .mu.l black assay plates (lot#05391155). Fluorescence was
measured by TECAN GENios plate reader at excitation/emission
485/530 nm Chemical compounds. Antrodins A-E (compounds 1-5) were
isolated from the mycelium of Antrodia cinnamomea as reported
(Nakamura et al., 2004. Five new maleic and succinic acid
derivatives from the mycelium of Antrodia comphorata and their
cytotoxic effects on LLC tumor cell line. J Nat Prod 67: 46-48).
Embelin used as a positive control was isolated in our laboratory
in previous work (Hussein et al., 2000, Inhibitory effects of
Sudanese medicinal plant extracts on hepatitis C virus (HCV)
Protease. Phytother Res 14: 510-516). Compounds 6, 7a and 7b were
detected as the metabolites of antrodin C in vivo. Compounds 7a and
7b were un-separable due to the equilibrium of these two compounds
through compound 1. It was thus used for the assay as a mixture of
compounds 1, 7a and 7b and named 7. Compound 8 was an analogue of
another metabolite whose glycine group was supposed to connect to
the other carboxyl group in the structure.
Example 1
Synthesis of Compound 8
[0057] A pyridine (5 mL) solution of 1 (314 mg, 1 mmol),
4-(dimethylamino) pyridine (122 mg, 1 mmol) and glycine (113 mg,
1.5 mmol) was heated at 40.degree. C. for 12 h and then kept at
room temperature overnight. The product mixture was partitioned
between EtOAc and 0.2N HCl solution. The EtOAc layer was washed
with water and concentrated to dryness. The residue was
chromatographed over ODS eluted with CH.sub.3CN--H.sub.2O (30-100%)
to obtain compound 8 from the 60% CH.sub.3CN eluted part (200 mg,
51%). .sup.1H NMR (DMSO-d.sub.6, 500 MHz): .delta.0.81, 0.82 (3H
each, s, H-3',4'), 1.71, 1.75 (3H each, s, H-4''',5'''), 1.93 (1H,
m, H-2'), 2.50 (overlapped with NMR solvent, H-1'), 4.20 (2H, s,
H-a), 4.58 (2H, d. =6.5 Hz, H-1'''), 5.45 (1H, m, H-2''), 7.07 (2H,
d, J=9.0 Hz, H-3'', 5''), 7.52 (2H, d, J=9.0 Hz, H-2'', 6'').
.sub.13C NMR (DMSO-d.sub.6, 125 MHz): .delta.18.1 (5'''), 22.5
(3',4'), 25.5 (4''), 27.5 (2'), 32.2 (1'), 39.5 (a), 64.5 (1''),
114.9 (3'',5''), 119.6 (2''), 120.9 (1''), 130.9 (2'', 6''), 137.4
(3), 137.6 (3''), 137.9 (2), 159.5 (4''), 169.1 (4), 170.4
(.beta.), 171.1 (1). ESI-MS (negative): m/z 370.0
([M-H.sub.20-H]--, 100%).
Assay Procedure:
[0058] Compound 6 was dissolved in H.sub.2O and other compounds
were dissolved in DMSO for the assay. To each well were added 2
.mu.l of respective compound solution and 8 .mu.l of freshly
diluted enzyme (0.5 .mu.g/ml). The reaction was started by adding
10 .mu.l of freshly diluted substrate (100 times dilution of a DMSO
stocking solution). After being incubated at room temperature
(28.degree. C.) for 30 min, the fluorescence intensities were
measured at Ex/Em=485 nm/535 nm. Inhibition percentages were
calculated as 100.times.(F.sub.vehicle-F.sub.sample)
F.sub.vehicle=% inhibition, where F is the fluorescence value of
vehicle control or of compound minus the fluorescence of the
substrate control.
Example 2
Synthesis of Compounds 9 and 10
[0059] Antrodia camphorata mycelia powder (ACM) (200 g), from
Simpson Biotech Co. Ltd., Taiwan, were four times extracted with
hot EtOH (4 l) per each. After removal of residues by filtration,
the EtOH extract was transferred to separatory funnels. Water and
CH.sub.2Cl.sub.2 (1:1) were added and was mixed for approximately
1-5 minutes. The addition and mixing were repeated by four times.
The aqueous was separated and the CH.sub.2Cl.sub.2 layer was
subjected to Diaion HP20 column chrotomatography, and then eluted
with from 70%, 80%, 90% to 100% MeOH to give fourteen fractions
(Fr. 1-14). Fraction 3 was chromatographed on OPN-75 Packing Column
(resin, Merck) and eluted with MeOH to give three fractions (Fr.
15-17). Fraction 16 was chromatographed on Sephadex LH.sub.2O
column and eluted with 100% MeOH to give six fractions (Fr. 18-23).
Fraction 20 was separated by preparative HPLC [column: Cosmosil
5C18-AR-II (20.times.250 mm)] to give compound 9.
TABLE-US-00002 TABLE 1 .sup.1H-NMR Spectral Data of Compound 9 2
166.2 (s) 2.07; 2.35(weak) 2 3 88.4 (s) 1.67; 2.07; 2.35 11 4 131.8
(s) 2.07(weak); 2.35; 8.01 5 5 135.6 (s) 6 1' 43.9 (t) 2.07(1H, m);
2.35(1H, m) 0.82; 0.84; 1.67 13 2' 24.1 (d) 1.67(1H, m) 0.82; 0.84;
2.07; 2.35 16 3' 24.1 (q) 0.82(3H, d, 6.5 Hz) 0.84; 1.67; 2.07;
2.35 15 4' 23.5.5 (q) 0.84(3H, d, 6.5 Hz) 0.82; 1.67 17 1'' 121 (s)
6.92 8 2'', 6'' 130.9 (d) 8.01(2H, d, 9.0 Hz) 8.01 7 3'', 5'' 114.6
(d) 6.92(2H, d, 9.0 Hz) 6.92 10 4'' 159.7 (s) 4.51; 6.92; 8.01 3
1''' 64.8 (t) 4.51(2H, d, 7.0 Hz) 12 2''' 119.2 (d) 5.46(1H, bro,
6.5 Hz) 1.72; 1.78; 4.51 9 3''' 138.7 (s) 1.72; 1.78; 4.51 4 4'''
25.8 (q) 1.78(3H, s) 1.72 14 5''' 18.2 (q) 1.72(3H, s) 1.78; 5.46
18 --COOCH.sub.2 168.9 (s) 1
[0060] Fraction 21 was subsequently separated by preparative HPLC
[column: Cosmosil 5C18-AR-II (20.times.250 mm)] to give compound
10.
TABLE-US-00003 TABLE 2 .sup.1H-NMR Spectral Data of Compound 10 2
172.1 (s) 2.33 1 3 133.7 (s) 2.00; 2.33 6 4 148.5 (s) 2.33; 7.22 4
5 90.4 (s) 11 1' 33.1 (t) 2.33(2H, d, 0.79; 0.81; 2.00 14 1.5; 7.0
Hz) 2' 27.2 (d) 2.00(1H, m) 0.79; 0.81; 2.33 15 3' 22.5 (q)
0.81(3H, d, 6.5 Hz) 0.79; 2.00; 2.33 17 4' 22.3 (q) 0.79(3H, d, 6.5
Hz) 0.81; 2.00; 2.33 18 1'' 123.1 (s) 6.89 8 2'', 6'' 129.9 (d)
7.22(2H, d, 8.5 Hz) 7.22 7 3'', 5'' 114.8 (d) 6.89(2H, d, 8.5 Hz)
6.89 10 4'' 159.6 (s) 4.50; 6.89; 7.22 3 1''' 64.79 (t) 4.50(2H, d,
6.5 Hz) 12 2''' 119.2 (d) 5.46(1H, tt,, 1.72; 1.78; 4.50 9 1.5; 5.5
Hz) 3''' 138.7 (s) 1.72; 1.78; 4.50 5 4''' 25.8 (q) 1.78(3H, s)
1.72; 5.46 16 5''' 18.2 (q) 1.72(3H, s) 1.78; 5.46 19 --COOCH.sub.2
169.2 (s) 4.29 2 --OCH.sub.2CH.sub.3 64.0 (t) 4.29(2H, m) 1.22 13
--OCH.sub.2CH.sub.3 14.0 (q) 1.22(3H, t, 7.5 Hz) 4.29 20
Sample Treating Before HPLC:
[0061] 1. Powder 0.2217 g mycelium of Antrodia cinnamomea in 5 mL
alcohol. [0062] 2. After 30 minutes for extraction of ultrasonic
vibration, centrifuge it in 9500 rpm for 5 minutes, then
supernatant liquid filter through 0.45 .mu.m screen filter. [0063]
3. HPLC analyze sample.
HPLC Assay Condition:
[0064] 1. Mobile phase: 0.1% H.sub.3PO.sub.4, CH.sub.3CN, MeOH
2. Column: Agilent, Zorbax SB-C18, 4.6.times.250 mm
[0065] 3. Rate: 1 mL/min
4. Wavelength: 254 nm
5. Temperature: 30.degree. C.
6. Injection: 20 .mu.L
TABLE-US-00004 [0066] HPLC assay result: (HPLC analyzing spectrum
in FIGS. 3 & 4) Retention Concentration Sample Time Area in ACM
Compound 9 69.253 19797906 -- Compound 10 102.34 28403438 --
Simpson Compound 9 -- -- 2.3 mg/g ACM Compound 10 -- -- 0.15
mg/g
[0067] While the invention has been described and exemplified in
sufficient detail for those skilled in this art to make and use it,
various alternatives, modifications, and improvements should be
apparent without departing from the spirit and scope of the
invention.
[0068] One skilled in the art readily appreciates that the present
invention is well adapted to carry out the objects and obtain the
ends and advantages mentioned, as well as those inherent therein.
The animals, and processes and methods for producing them are
representative of preferred embodiments, are exemplary, and are not
intended as limitations on the scope of the invention.
Modifications therein and other uses will occur to those skilled in
the art. These modifications are encompassed within the spirit of
the invention and are defined by the scope of the claims.
* * * * *