U.S. patent application number 12/600830 was filed with the patent office on 2011-11-03 for novel cyclic pentadepsipeptide derivative and fusarium strain producing the same.
This patent application is currently assigned to Chung-Ang University Industry-Academy Cooperation Foundation. Invention is credited to Chan Lee, Hee-Seok Lee, Hyuk-Hwan Song.
Application Number | 20110269698 12/600830 |
Document ID | / |
Family ID | 42309944 |
Filed Date | 2011-11-03 |
United States Patent
Application |
20110269698 |
Kind Code |
A1 |
Lee; Chan ; et al. |
November 3, 2011 |
Novel Cyclic Pentadepsipeptide Derivative and Fusarium Strain
Producing The Same
Abstract
Disclosed is a Fusarium strain producing novel cyclic
pentadepsipeptides which are of excellent multidrug
resistance-reversing activity and inhibitory activity against
cancer cells. Also, novel cyclic pentadepsipeptides are provided as
active ingredients of the compositions useful in the treatment of
cancer and diseases associated with multidrug resistance.
Inventors: |
Lee; Chan; (Gyeonggi-do,
KR) ; Song; Hyuk-Hwan; (Seoul, KR) ; Lee;
Hee-Seok; (Seoul, KR) |
Assignee: |
Chung-Ang University
Industry-Academy Cooperation Foundation
Seoul
KR
|
Family ID: |
42309944 |
Appl. No.: |
12/600830 |
Filed: |
January 2, 2009 |
PCT Filed: |
January 2, 2009 |
PCT NO: |
PCT/KR09/00005 |
371 Date: |
May 10, 2011 |
Current U.S.
Class: |
514/19.9 ;
435/256.5; 435/71.1; 514/21.1; 530/317 |
Current CPC
Class: |
C12R 1/77 20130101; A61P
35/00 20180101; C07K 11/02 20130101; C12P 21/02 20130101; A61P
43/00 20180101; A61K 38/15 20130101; A61K 38/00 20130101 |
Class at
Publication: |
514/19.9 ;
435/256.5; 435/71.1; 530/317; 514/21.1 |
International
Class: |
A61K 38/15 20060101
A61K038/15; A61P 43/00 20060101 A61P043/00; C07K 11/02 20060101
C07K011/02; A61P 35/00 20060101 A61P035/00; C12N 1/14 20060101
C12N001/14; C12P 21/04 20060101 C12P021/04 |
Claims
1. A Fusarium strain, producing a cyclic pentadepsipeptide,
represented by Chemical Formula 1 or 2: ##STR00004##
2. The Fusarium strain according to claim 1, belonging to Fusarium
solani.
3. The Fusarium strain according to claim 2, being Fusarium solani
KCCM90040 [Accession No. KCCM10881P].
4. A method of producing the cyclic pentadepsipeptide of Chemical
Formula 1 or 2 of claim 1 by culturing the strain of one of claims
1 to 3.
5. The method according to claim 4, wherein the strain is cultured
on a cereal substance.
6. The method according to claim 5, wherein the cereal substance is
rice.
7. The method according to claim 5, wherein the strain is cultured
at 20.about.30.degree. C. and 20.about.50 RH % for 10.about.20
days.
8. A cyclic pentadepsipeptide, represented by the following
Chemical Formula 1: ##STR00005##
9. A cyclic pentadepsipeptide, represented by the following
Chemical Formula 2: ##STR00006##
10. A pharmaceutical composition for reversing multidrug
resistance, comprising as an active ingredient at least one
selected from a group consisting of the cyclic pentadepsipeptides
of the following Chemical Formulas 1 and 2 and pharmaceutically
acceptable salts thereof. ##STR00007##
11. The pharmaceutical composition according to claim 10, wherein
the multidrug resistance comes from tumor cells.
12. A pharmaceutical composition for treatment of cancer,
comprising as an active ingredient at least one selected from a
group consisting of the cyclic pentadepsipeptides of the following
Chemical Formulas 1 and 2 and pharmaceutically acceptable salts
thereof: ##STR00008##
Description
TECHNICAL FIELD
[0001] The present invention relates to medicinally useful
compounds and a microorganism producing the same. More
particularly, the present invention relates to novel cyclic
pentadepsipeptides with excellent multidrug resistance-reversing
activity and inhibitory activity against cancer cells, which are
produced by a soil stain of the genus Fusarium.
BACKGROUND ART
[0002] A fungus of the genus Fusarium distributed in association
with higher marine plants has proven to be a promising source for
the production of sansalvamide, a kind of cytotoxic cyclic
depsipeptide.
[0003] Sansalvamide A was first reported to be produced by Halodule
wrightii, a kind of marine microorganisms [Belofsky G N, Jensen P
R, Fenical W. (1999) Sansalvamide: A new cytotoxic cyclic
depsipeptide produced by a marine fungus of the genus Fusarium.
Tetrahedron Lett. 40, 2913-2916]. It is composed of four
hydrophobic amino acid residues (phenylalanine, two leucines,
valine) and one hydroxy acid ((S)-2-hydroxy-4-methylpentanoic acid;
O-Leu) with five stereogenic centers all having S-stereochemistry.
Sansalvamide A was found to have marked anti-proliferative effects
on 60 cell lines of the National Cancer Institute, with inhibitory
activity against topoisomerase I. Anti-cancer effects of
Sansalvamide A may be, at least in part, mediated by this
mechanism. Further, the analogues formed by N-methylation or
para-bromination of sansalvamide A of FIG. 1 were also found to
exhibit remarkable cytotoxicity against human pancreatic cancer
cells, suggesting that these cyclic compounds may be highly useful
as anti-cancer agents [Ujiki M B, Milam B, Ding X Z, Roginsky A B,
Salabat M R, Talamonti M S, Bell R H, Gu W, Silverman R B, Adrian T
E. (2006) A novel peptide sansalvamide analogue inhibits pancreatic
cancer cell growth through G0/G1 cell-cycle arrest. Biochem. Bioph.
Res. Co. 340, 1224-1228].
[0004] Recently, N-methylsansalvamide, a sansalvamide analogue with
N-methylation, has been produced from different Fusarium species
isolated from green algae. It consists of four amino acid residues
(phenylalanine, leucine, N-methylleucine and valine) and one
hydroxy acid (O-Leu). N-Methylsansalvamide was reported to exhibit
in vitro cytotoxicity in the NCI human tumor cell line screen
[Cueto M, Jensen P R, Fenical W. (2000) N-Methylsansalvamide, a
cytotoxic cyclic depsipeptide from a marine fungus of the genus
Fusarium. Phytochemistry. 55, 223-226].
[0005] Multidrug resistance (MDR) is arising as one of the major
obstacles to successful chemotherapy for human cancer. A variety of
biochemical, pharmacological and clinical strategies for overcoming
MDR have been designed and suggested [Teodori E, Dei S, Scapecchi
S, Gualtieri F. (2002) The medicinal chemistry of multidrug
resistance (MDR) reversing drugs. II Farmaco 57, 385-415]. Although
there are several mechanisms associated with MDR, the
overexpression of P-glycoproteins (P-gp) and multidrug
resistance-associated proteins (MRP) is known to be responsible for
the development of MDR in cancer cells [Thomas H, Coley H M. (2003)
Overcoming multidrug resistance in cancer: an update on the
clinical strategy of inhibiting P-glycoprotein. Cancer Control 10,
159-165; Perez-Tomas R. (2006) Multidrug resistance: retrospect and
prospects in anti-cancer drug treatment. Curr. Med. Chem. 13,
1859-1876].
[0006] Sansalvamide A is a lipophilic, cyclic depsipeptide, which
is of protease resistance and membrane permeability, so that it may
advantageously take an oral route, compared to other drugs. In
addition, forming a cyclic core structure, composed of four amino
acids and one hydroxy acid, in which rotation around C--C bonds is
restricted, sansalvamide A has a firm conformation which is of
excellent compatibility to the body and enjoys a long half
life.
[0007] Although there have been a number of sansalvamide analogues
synthesized to utilize the structural merits and the cytotoxicity
against cancer cells of sansalvamide A or N-methylsansalvamide,
none of them are concerned with separated, cyclic
pentadepsipeptides.
DISCLOSURE
Technical Problem
[0008] It is therefore an object of the present invention to
provide a strain of the genus Fusarium producing novel cyclic
pentadepsipeptides.
[0009] It is another object of the present invention to provide a
method of producing the novel cyclic pentadepsipeptides by
culturing a strain of the genus Fusarium.
[0010] It is a further object of the present invention to provide
novel cyclic pentadepsipeptides.
[0011] It is a still further object of the present invention to
provide a pharmaceutical composition for reversing multidrug
resistance.
[0012] It is still another object of the present invention to
provide a pharmaceutical composition for the treatment of
cancer.
[0013] In accordance with an aspect thereof, the present invention
pertains to a novel cyclic peptadepsipeptide represented by
Chemical Formula 1 or 2:
##STR00001##
[0014] The cyclic pentadepsipeptides, represented by Chemical
Formulas 1 and 2, according to the present invention are
15-membered ring compounds which are proven novel with a difference
from conventionally reported sansalvamide A and
N-methylsansalvamide in the sequence of their constitutional amino
acids and hydroxy acid. As seen in FIG. 1, conventional analogues
synthesized on the basis of the core structure of sansalvamide A
are intended to increase in cytotoxicity as a result of the
bromination of the benzene ring of phenyl alanine or the
methylation of leucine or valine. Nowhere has an attempt to change
the sequence of the constitutional amino acids and hydroxy acid of
sansalvamide A or N-methylsansalvamide been mentioned in previous
reports. Further, organic synthesis for altering the sequence of
the constitutional amino acids and hydroxy acid is accompanied by
the interruption of the ester bond between the hydroxy acid
((S)-2-hydroxy-4-methylpentanoic acid; OLeu) and the phenylalanine
within the core structure.
[0015] Being different from sansalvamide A and N-methylsansalvamide
in the sequence of the constitutional four amino acids and one
hydroxy acid, the novel, cyclic pentadepsipeptides, represented by
Chemical Formulas 1 and 2, according to the present invention have
cytotoxicity over a wide spectrum of types of cancer that is as
potent as that of sansalvamide A. Furthermore, the cyclic
pentadepsipeptides of Chemical Formulas 1 and 2 show the drug
resistance-reversing activity which has not yet been reported in
conventional analogues synthesized on the basis of the sansalvamide
A or N-methylsansalvamide. Excellent drug resistance-reversing
activity is found in the cyclic pentadepsipeptide of Chemical
Formula 1. When pathogens or cells are exposed to chemotherapy,
they may become resistant to the drug and possibly to other
structurally unrelated drugs. The pathogens or cells are said to be
drug-resistant when drugs meant to neutralize them have a reduced
effect. As used herein, the term "drug resistance-reversing
activity" is intended to mean not only suppressing the generation
of drug-resistant cells or keeping the cells sensitive to the
drugs, but also increasing or recuperating the sensitivity of
drug-resistant cells to the drugs.
[0016] Having the ability to make drug-resistant cells sensitive to
drugs or suppress the generation of drug-resistant cells, the
cyclic pentadepsipeptides of Chemical Formulas 1 and 2 according to
the present invention or pharmaceutically acceptable salts thereof
may be useful in the treatment of multidrug-resistant cancer.
[0017] Preferably, the cyclic pentadepsipeptides of Chemical
Formulas 1 and 2 or pharmaceutically acceptable salts thereof may
be used in combination with conventional drug-resistant inhibitors
such as cyclosporine or analogues, phenothiazine, thioxantheres,
verapamil, etc.
[0018] Also, the cyclic pentadepsipeptides of Chemical Formulas 1
and 2 according to the present invention or pharmaceutically
acceptable salts thereof may be used in combination with anticancer
agents, that is, standard chemotherapy agents, in the treatment of
cancer and preferably in the treatment of tumors resistant to drugs
a priori or a posteriori.
[0019] In accordance with another aspect thereof, the present
invention pertains to a Fusarium strain producing the cyclic
pentadepsipeptide of Chemical Formula 1 or 2. Preferably, the
Fusarium strain producing the novel compound is Fusarium solani
KCCM90040 [Accession No.: KCCM10881P].
[0020] In accordance with a further aspect thereof, the present
invention pertains to a method for producing the cyclic
pentadepsipeptide of Chemical Formula 1 or 2 by culturing the
strain. This culturing is preferably conducted on a cereal
substance. The cereal substance useful in the present invention is
selected from among rice, wheat, maize, rye, Indian millet, barley
and a combination thereof, preferably from among rice, wheat, maize
and rye, more preferably from among rice and wheat, and most
preferably rice.
[0021] When cultured in a seawater-based medium, marine Fusarium
strains were reported to produce sansalvamide in an amount of 0.642
g per 17 liters [Belofsky G N, Jensen P R, Fenical W. (1999)
Sansalvamide: A new cytotoxic cyclic depsipeptide produced by a
marine fungus of the genus Fusarium. Tetrahedron Lett. 40,
2913-2916]. As for the Fusarium strain CNL-619, its
N-methylsansalvamide production was reportedly 3.1 mg/L in a
seawater-based medium [Cueto M, Jensen P R, Fenical W. (2000)
N-Methylsansalvamide, a cytotoxic cyclic depsipeptide from a marine
fungus of the genus Fusarium. Phytochemistry. 55, 223-226].
[0022] Being produced from the marine Fusarium strains,
sansalvamide or N-methylsansalvamide requires a seawater-based
medium for the production thereof. Further, the marine strains are
very poor in the productivity of sansalvamide or
N-methylsansalvamide per volume of medium. In contrast, the
Fusarium strain of the present invention is of soil origin and can
produce the compound of Chemical Formula 1 or 2 in high yield on a
solid cereal substance.
[0023] The cereal substance useful for culturing the strain is
preferably rice. In order to produce the compound of Chemical
Formula 1 or 2, the strain of the present invention is preferably
cultured at a temperature of from 20 to 30.degree. C. and at an RH
of from 20 to 50% for 10.about.20 days. Optimally, it is cultured
at 25.84.degree. C. and 37.99% RH for 16.03 days.
[0024] A strain producing a cyclic depsipeptide was isolated from
potatoes grown in Munkyoung Korea and identified as a strain of
Fusarium solani. It was named Fusarium solani KCCM90040 and
deposited with the Korean Culture Center of Microorganisms on Jan.
15, 2008 with accession No. KCCM10881P according to the Budapest
Treaty.
[0025] The compounds of produced by the strain were analyzed to be
15-membered ring compounds different from sansalvamide A and
N-methylsansalvamide in the sequence of constitutional amino acids
and hydroxy acid and identified as cyclic pentadepsipeptides of
Chemical Formulas 1 and 2.
[0026] The cyclic pentadepsipeptides of Chemical Formula 1 or 2 in
accordance with the present invention or pharmaceutically
acceptable salts thereof shows inhibitory activity against
drug-resistant cells, which may be tumor cells or
antibiotic-resistant cells, and thus can be used as an active
ingredient of a pharmaceutical composition for inhibiting the drug
resistance of cells.
[0027] Thanks to their suppressive activity against tumor
proliferation, the pentadepsipeptides of Chemical Formula 1 and 2
in accordance with the present invention may also be used as an
active ingredient of a pharmaceutical composition for the treatment
of cancer.
[0028] The term "pharmaceutically acceptable salt", as used herein,
means non-toxic organic or inorganic acid addition salts of the
compounds of interest. Examples of the inorganic acids useful in
the present invention include hydrochloric acid, hydrobromic acid,
sulfuric acid, acid metal salts (e.g., sodium hydrogen phosphate,
potassium hydrogen sulfate), etc. Examples of the organic acid
useful in the present invention include mono-, di- and
tricarboxylic acid. Among the pharmaceutically acceptable acids,
there may be mentioned, without implying any limitation, acetic
acid, glycolic acid, lactic acid, pyruvic acid, malonic acid,
succinic acid, glutaric acid, fumaric acid, malic acid, tartaric
acid, citric acid, ascorbic acid, maleic acid, hydroxymaleic acid,
benzoic acid, hydroxybenzoic acid, phenylacetic acid, cinnamic
acid, salicylic acid and 2-phenoxybenzoic acid. Other organic acids
may be exemplified by methane sulfonic acid and 2-hydroxyethane
sulfonic acid. Such salts can be either in hydrated or anhydrous
form. The acid addition salts of these compounds are prepared using
a typical method, for example, by dissolving a free base in a
suitable solvent such as an aqueous solvent, an alcohol solvent or
another acid-containing solvent, and then evaporating the solvent,
or by reacting the free base with the organic solvent (in this
case, the salt may be directly separated or obtained via
concentration). Generally, the acid addition salts of the compounds
of the present invention are crystalline materials which can be
dissolved in water or various hydrophilic organic solvents and
generally display higher melting points compared to the free base
forms thereof.
[0029] The pharmaceutical compositions of the present invention for
the treatment of cancer or diseases associated with multidrug
resistance may be administered to subjects in need thereof. As used
herein, the term "subject" is intended to include mammals such as
goats, horses, cow, pigs, dogs, cats, mice, rats, etc. as well as
primates such as humans.
[0030] The effective dosages of the cyclic pentadepsipeptides of
Chemical Formulas 1 and 2 or pharmaceutically acceptable salts
thereof may vary depending on various factors including dosage
units, the duration of treatment, the age and sex of the patient,
traits of the tumor to be treated, drug resistance, etc.
[0031] In combination with other anticancer agents, particularly
chemical agents useful for the treatment of tumors, the cyclic
pentadepsipeptides of Chemical Formulas 1 and 2 or pharmaceutically
acceptable salts thereof may be used. For reversing multidrug
resistance, the cyclic pentadepsipeptides of Chemical Formulas 1
and 2 or pharmaceutically acceptable salts thereof may be
administered at an effective dose of from 15 mg/kg to 500 mg/kg. A
unit daily dosage may contain 25 to 500 mg of the cyclic
pentadepsipeptide of Chemical Formula 1 or 2 or a pharmaceutically
acceptable salt thereof. The cyclic pentadepsipeptides of Chemical
Formulas 1 and 2 or pharmaceutically acceptable salts thereof may
be in an oral or parenteral dosage form formulated with a
pharmaceutically acceptable carrier.
[0032] For use in the treatment of tumors, the cyclic
pentadepsipeptides of Chemical Formulas 1 and 2 or pharmaceutically
acceptable salts thereof are preferably administered at an
effective dose, in combination with an anticancer agent,
particularly, an agent for chemotherapy.
[0033] Among the tumors which can be treated with the compounds of
the present invention are benign or malignant tumors, neoplasms,
melanoma, lymphoma, leukemia and sarcoma. Examples of the tumors
include skin tumors (e.g., malignant melanoma and cutaneous mycosis
fungoides), blood tumors (e.g, acute lymphocytic leukemia, acute or
chronic myelogenous leukemia), lymphoma (e.g. Hodgkin's disease,
malignant lymphoma), gynecologic tumors (ovarian tumor, uterine
tumor), urologic tumors (prostate tumor, bladder tumor, seminoma),
soft tissue sarcoma, osteo- or non-osteosarcoma, breast tumors,
pituitary tumors, thyroid gland tumors, adrenal cortex tumors,
gastrointestinal tumors (esophagus tumor, stomach tumors, intestine
tumor, and colon tumor), pancreatic tumors, liver tumors, larynx
tumors, papilloma and lung tumors. The best therapeutic effects may
be elicited when the compounds of the present invention are applied
to multidrug-resistant tumors or tumors which become multidrug
resistant. Among these tumors are colon tumors, lung tumors,
stomach tumors and liver tumors.
[0034] A cytotoxic agent is typical of the chemical agents which
can be applied together with the cyclic pentadepsipeptides of
Chemical Formulas 1 and 2 or pharmaceutically acceptable salts
thereof. Examples of the agents for chemotherapy include
cyclophosphamide, methotrexate, prednisone, 6-mercaptopurine,
procarbazine, daunorubicin, vincristine, vinblastine, chlorambucil,
cytosine arabinoside, 6-thioguanine, thio TEPA, 5-fluorouracil,
5-fluoro-2-deoxyuridine, 5-azacytidine, nitrogen mustard,
1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU),
(1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea) (CCNU), busulfan,
adriamycin, bleomycin, vindesine, Cycloleucine and methylglyoxal
bis(guanyl hydrazone) (MGBG).
[0035] The effective dosage of the chemical agent useful in the
present invention may vary depending on various factors including
the condition of the patient, the morphology and size of the tumor,
kind of the agents, etc, and may be readily determined by those
skilled in the art. Generally, the chemical agent may be
administered in a smaller dose when administered in combination
with the cyclic pentadepsipeptides of Chemical Formulas 1 and 2 or
pharmaceutically acceptable salts thereof than when administered
alone. The reason is because a large amount of the chemical agent
may cause drug resistance. Also, a cocktail of chemical agents or
surgery or radiotherapy may be used, together with the
administration of the cyclic pentadepsipeptides of Chemical
Formulas 1 and 2 or pharmaceutically acceptable salts thereof.
Although the co-administration of the cyclic pentadepsipeptides of
Chemical Formulas 1 and 2 or pharmaceutically acceptable salts
thereof and the chemical agent is described above, they may not be
in the same dosage form or may not be administered at the same
time. Accordingly, the cyclic pentadepsipeptides of Chemical
Formulas 1 and 2 or pharmaceutically acceptable salts thereof and
the chemical agent may be administered in a mixture or at different
times.
[0036] An oral route is preferable. For use in oral administration,
the cyclic pentadepsipeptides of Chemical Formulas 1 and 2 or
pharmaceutically acceptable salts thereof may be formulated into
solid or liquid forms, such as capsules, pills, tablets, troches,
lozenge, melts, powders, liquids, suspensions and emulsions.
Typical of the solid dosage forms is a capsule. A capsule with a
soft or hard gelatin envelope may contain a surfactant, a
lubricant, and an inert filler such as lactose, sucrose, calcium
phosphate and corn starch. In another embodiment, the compounds of
the present invention may be formulated into a tablet, together
with a tablet base (e.g., lactose, sucrose, corn starch), a binder
(e.g, acacia, corn starch or gelatin), a disintegrant (e.g., potato
starch, alginic acid, corn starch and guar gum) for facilitating
the degradation and dissolution of the tablet after administration,
a lubricant (e.g., talc, stearic acid or magnesium stearate,
calcium stearate or zinc stearate) for enhancing the release of
tablet granules and preventing the attachment of the tablet drug to
the tabletting die or punch, and a dye, a colorant and a fragrant
for improving the color and taste of the tablet. Carriers suitable
for use in oral liquid dosage forms include suspension agents,
water mixed with or without an emulsifier, and a diluent such as an
alcohol (ethanol, benzyl alcohol and polyethylene alcohol).
[0037] Also, the cyclic pentadepsipeptides of Chemical Formulas 1
and 2 or pharmaceutically acceptable salts thereof may be
administered in the form of a solution in a diluent, together with
a pharmaceutically acceptable carrier, through a parenteral route,
that is, a subcutaneous, intravenous, intramuscular or
intraperitoneal route. Examples of the carrier for use in injection
include pharmaceutically acceptable surfactants (e.g., soap or
detergent), suspending agents (pectin, carbomer, methylcellulose,
hydroxypropylmethylcellulose or carboxymethylcellulose), water or
saline mixed with an emulsifier or another pharmaceutical adjuvant,
aqueous solutions of dextrose or corresponding sugars, alcohols
(e.g., ethanol, isopropanol, hexadecylalcohol), glycol (e.g.,
propylene glycol, or polyethylene glycol), glycerol ketal (e.g.,
2,2-dimethyl-1,3-dioxolan-4-methanol), ethers (e.g.,
poly(ethylene-glycol)400), oils, and germ-free solutions or mixture
containing fatty acids, fatty acid esters or glycerides, or
acetylated fatty acid glycerides. Examples of the oil useful in the
formulation of parenteral dosage forms include petroleum, animal
oil, vegetable oil, or synthetic oil, such as peanut oil, soybean
oil, sesame oil, cotton seed oil, corn oil, olive oil, mineral oil
and inorganic oil. Among the fatty acids are oleic acid, stearic
acid and isostearic acid. The fatty acid esters may be exemplified
by oleic acid ethyl and myristic acid isopropyl. Alkaline metal
salts, ammonium salt and triethanolamine salts of fatty acids may
be suitable as the surfactant. Examples of the detergent include
cationic detergents (e.g., dimethyl dialkyl ammonium halide, alkyl
pyridium halide and alkylamine acetate), anionic detergents (e.g.,
alkyl, aryl and olefin sulfonate, alkyl, olefin, ether and
monoglyceride sulfate and sulfosuccinate), non-ionic detergents
(lipid amine oxide, fatty acid alkanolamide and
polyoxyethylenepolypropylene copolymer), and amphiphatic detergents
(e.g., alkyl-.beta.-amino propionate and 2-alkylimidazolin
quarternary ammonium salt) and a combination thereof. Typically,
the parenteral compositions of this invention will contain from
about 0.5% to about 25% by weight of the active ingredient in
solution. Preservatives and buffers may also be used
advantageously. In order to minimize or eliminate irritation at the
site of injection, such compositions may contain a non-ionic
surfactant having a hydrophile-lipophile balance (HLB) of from
about 12 to about 17. The quantity of surfactant in such
formulations ranges from about 5% to about 15% by weight. The
surfactant can be a single component having the above HLB or can be
a mixture of two or more components having the desired HLB.
Illustrative of surfactants used in parenteral formulations are the
class of polyethylene sorbitan fatty acid esters, for example,
sorbitan monooleate and the high molecular weight adducts of
ethylene oxide with a hydrophobic base, formed by the condensation
of propylene oxide with propylene glycol.
[0038] In accordance with a further aspect thereof, the present
invention pertains to a method of producing the cyclic
pentadepsipeptide of Chemical Formula 1 or 2 by culturing a strain
of the genus Fusarium, preferably Fusarium solani, more preferably
Fusarium solani KCCM90040 [accession No.: KCCM10881P].
Alternatively, the compound of the present invention may be
prepared through biosynthesis or organic synthesis.
DESCRIPTION OF DRAWINGS
[0039] FIG. 1 shows the chemical structure of sansalvamide A
analogues.
[0040] FIG. 2 is a photograph showing microconidia of the strain of
the present invention.
[0041] FIG. 3 is a photograph showing the conidiophores of the
Fusarium strain according to the present invention.
[0042] FIG. 4 is a photograph showing the appearances of the
surface and backside of an agar on which the strain of the present
invention is grown.
[0043] FIG. 5 is a photograph showing PCR products amplified from
DNA templates of Fusarium strain (M, 100-bp DNA ladder; F, Fusarium
strain; S-1, Fusarium moniliforme NRRL 13569; S-2, Fusarium
oxysporum KCCT 16909).
[0044] FIG. 6 shows an array of ITS-5.8 rDNA sequences of the
strain of the present invention (sample) and Fusarium solani for
homology comparison therebetween.
[0045] FIG. 7 is an HPLC chromatogram of an extract from a
submerged culture of the strain of the present invention after
incubation in a Fusarium defined medium.
[0046] FIG. 8 is an HPLC chromatogram of an extract from a cereal
culture of the strain of the present invention after incubation on
a cereal culture.
[0047] FIG. 9 is of electrospray ionization mass spectra
illustrating the molecular weight of Compound A produced by the
strain of the present invention.
[0048] FIG. 10 is of electrospray ionization mass spectra
illustrating the molecular weight of Compound B produced by the
strain of the present invention.
[0049] FIG. 11 is of infrared spectra of Compound A, measured by an
FT IR-8400S infrared spectrophotometer.
[0050] FIG. 12 is a diagram showing the HMBC correlations of
compound A.
[0051] FIG. 13 is of infrared spectra of Compound B, measured by an
FT IR-8400S infrared spectrophotometer.
[0052] FIG. 14 is a diagram showing the HMBC correlations of
compound B.
[0053] FIG. 15 is an HPLC chromatogram for determining the
stereochemical structure of amino acids in Compound A.
[0054] FIG. 16 is an HPLC chromatogram for determining the
stereochemical structure of amino acids in Compound B.
[0055] FIG. 17 is a graph showing the cytotoxic effect of the
compound of Chemical Formula 1 on non-multidrug resistant cancer
cell lines.
[0056] FIG. 18 is a graph showing the cytotoxic effect of the
compound of Chemical Formula 1 on multidrug resistant cancer cell
lines.
[0057] FIG. 19 is a graph showing the cytotoxic effect of the
compound of Chemical Formula 2 on non-multidrug resistant cancer
cell lines.
[0058] FIG. 20 is a graph showing the cytotoxic effect of the
compound of Chemical Formula 2 on non-multidrug resistant cancer
cell lines.
[0059] FIG. 21 is a graph showing the multidrug
resistance-reversing activity of the compounds of Chemical Formulas
1 and 2 against the HCT15 cell line.
[0060] FIG. 22 is a graph showing the multidrug
resistance-reversing activity of the compounds of Chemical Formulas
1 and 2 against the HCT15/CL02 cell line.
[0061] FIG. 23 is a graph showing the multidrug
resistance-reversing activity of the compounds of Chemical Formulas
1 and 2 against the MEA-SA cell line.
[0062] FIG. 24 is a graph showing the multidrug
resistance-reversing activity of the compounds of Chemical Formulas
1 and 2 against the MEA-SA/DX5 cell line.
[0063] FIG. 25 is a photograph showing the anti-fungal activity of
the compounds of Chemical Formulas 1 and 2 against Mucor rouxii
grown on PDA, with 10 mM of compounds of Chemical Formulas 1 and 2
placed thereon.
[0064] FIG. 26 is a photograph showing the anti-fungal activity of
the compounds of Chemical Formulas 1 and 2 against Fusarium
oxysporum grown on PDA, with 10 mM of compounds of Chemical
Formulas 1 and 2 placed thereon.
[0065] FIG. 27 is a graph in which the produced amounts of the
compound of Chemical Formula 1 are plotted against the time of
growth for six different cereal media.
[0066] FIG. 28 is a graph in which the produced amounts of the
compound of Chemical Formula 2 are plotted against the time of
growth for six different cereal media.
[0067] FIG. 29 is a graph showing the production of the compounds
of Chemical Formulas 1 and 2 with the time of growth.
[0068] FIG. 30 is a graph showing the production of the compounds
of Chemical Formulas 1 and 2 with culture temperature.
[0069] FIG. 31 is a graph showing the production of the compounds
of Chemical Formulas 1 and 2 with moisture content.
BEST MODE
[0070] A better understanding of the present invention may be
obtained through the following examples which are set forth to
illustrate, but are not to be construed as limiting the present
invention.
EXAMPLE 1
Isolation and Identification of Fusarium Strain
[0071] (1) Isolation and Morphological Identification
[0072] Fusarium strains were isolated from Fusarium-contaminated
potatoes in Munkyeong, Korea and its identification was determined
using the methods of Samson, et al. and the method of Nelson et al.
[Samson R A, Hoekstra ES, Oorschot V, Connie A N. (1981)
Introduction to food-borne fungi. Published and distributed by
Centraalbureau voor Schimmelcultures; Nelson P E, Toussoun T A,
Marasas W F. (1983) Fusarium species: An illustrated manual for
identification. The Pennsylvania State University Press].
[0073] The isolated Fusarium strain was transferred on carnation
leaf agar (CLA) and real potato dextrose agar (RPDA) and analyzed
for morphological characteristics.
[0074] The microconidia were present in abundance generally in the
form of single cells of an oval to kidney shape (FIG. 2).
Conidiophores of the Fusarium strain put out branches as shown FIG.
3. The microconidia and conidiophores of F. solani are
morphologically similar to those of Fusarium oxysporum. The
microconidia of F. oxysporum were observed to be larger in size and
have thicker walls and conidiophores were formed on short
monophialides, compared to those of F. solani (Nelson et al.,
1983). The isolated Fusarium strains grew fast. The slant surface
of the agar was almost covered with white mycelia with the back
being a dark cream (FIG. 4).
[0075] From these morphological characteristics, the Fusarium
strain was identified as F. solani.
[0076] (2) Molecular Biological Identification
[0077] A. DNA Isolation
[0078] The total genomic DNA of the isolated Fusarium strain was
extracted from the mycelia grown on PDA (potato dextrose agar)
using the method of Correll [Correll J C, Klittich C J R, Leslie J
F. (1987) Nitrate nonutilizing mutants of Fusarium oxysporum and
their use in vegetative compatibility tests. Phytopathology, 77,
16401646].
[0079] In this regard, mycelium-covered agar was placed in a tube
filled with liquid nitrogen, followed by evaporating the liquid
nitrogen at room temperature. This procedure of liquid nitrogen
filling and evaporation was repeated again. After evaporation of
the liquid nitrogen, 0.5 mL of a lysis buffer [50 mM Tris pH 8.0,
50 mM ethylenediaminetetraacetic acid(EDTA), 3% sodium
dodecylsulfate (SDS), 1% 2-mercaptoethanol and 0.1 m/ml proteinase
K] warmed to 65.degree. C. was added into the tube and incubated
for 1 hr at 65.degree. C. After incubation, 0.5 mL of a phenol
solution was added and then spun for 5 min at 8,000 rpm in a
microcentrifuge to separate a phenol phase and an aqueous phase
from each other. The aqueous phase was transferred to a new tube.
The phenol extraction was repeated again. Phenol residues in the
aqueous phase were removed with 0.4 mL of a mixture of chloroform:
isoamyl alcohol (24:1). Ammonium acetate (0.05 ml, 7.5 M) was mixed
gently with the aqueous phase. DNA was precipitated with 0.88 mL of
95% ethanol cold to -20.degree. C. The DNA pellet was spun down for
20 min at 13,000 rpm. The pellet was rinsed with 70% ethanol, spun
down, and dried. The DNA was resuspended in TE buffer (10 mM Tris,
1 mM EDTA, pH 8.0) and stored until use.
[0080] B. Identification by DNA Electrophoresis and Homology
Comparison
[0081] Fusarium specific primers, P28SL (5'-ACA AAT TAC AAC TCG GGC
CCG AGA-3') of SEQ ID NO: 1 and P58SL (5'-AGT ATT CTG GCG GGC ATG
CCT GT-3') of SEQ ID NO. 2, designed as described by Hue et al.
[Hue, F. X., M. Huerre, M. A. Rouffault, and C. D. Bievre.
Specified detection of Fusarium species in blood and tissues by a
PCR technique. Journal of Clinical Microbiology, 37: 2434-2438.
1999] were used for a control PCR assay. A pair of the Fusarium
specific primers amplified a fragment of DNA coding for the
ribosomal DNA (rDNA) of Fusarium strains by PCR. The binding sites
of the P28SL and P58SL primers, corresponding to the ITS2 region
and a portion of 5.8s and 28s rDNA, are conserved among Fusarium
strains.
[0082] For PCR, 1 ng of the DNA isolated in A was used together
with a primer pair of the primers P28SL and P58SL, and a PCR
pre-mixture purchased from Promega. Starting by denaturation at
94.degree. C. for 10 min, PCR was performed with 40 cycles of
denaturation at 94.degree. C. for 1 min, annealing at 60.degree. C.
for 1 min and extension at 72.degree. C. for 1 min, followed by a
final extension at 72.degree. C. for 10 min.
[0083] The PCR product thus obtained was run on a 2% agarose gel in
Tris-acetate-EDTA buffer in the presence of an electric field.
After completion of the electrophoresis, the gel was stained with
ethidium bromide and visualized under UV light.
[0084] The PCR product (F) amplified from Fusarium stains was
detected at a position between 300 and 400 bp, which matched with
the sizes of PCR products from two controls Fusarium moniliforme
NRRL 13569 and Fusarium oxysporum KCTC 16909 (FIG. 5).
[0085] The PCR product obtained with the Fusarium-specific primers
was purified and sequenced commercially (Macrogen Inc. Seoul,
Korea). BLAST search for homology in the GenBank database showed
that the ITS-5.8 rDNA sequence of the Fusarium strain of the
present invention shared more than 98% homology with those of F.
solani (FIG. 6). The ITS-5.8 rDNA sequence of the Fusarium strain
is given in SEQ ID NO. 3.
[0086] This Fusarium strain was named Fusarium solani KCCM90040 and
deposited with the Korean Culture Center of Microorganisms on Jan.
15, 2008 with accession No. KCCM10881P, according to the Budapest
Treaty.
EXAMPLE 2
Production and Separation of Cyclic Depsipeptide
[0087] (1) Cultivation in Fusarium Defined Medium
[0088] Fusarium solani KCCM90040 was inoculated at a density of
1.times.10.sup.5 spores/mL into 100 mL of a Fusarium defined medium
(FDM) broth (25 g of sucrose, 4.25 g of NaNO.sub.3, 5 g of NaCl,
2.5 g of MgSO.sub.47H2O, 1.36 g of KH.sub.2PO.sub.4, 0.01 g of
FeSO.sub.47H2O, and 0.0029 g of ZnSO.sub.47H2O per liter) and
incubated at 25.degree. C. for 7 days.
[0089] (2) Cultivation on Cereal Substrate
[0090] Fusarium solani KCCM90040 was inoculated at a density of
1.times.10.sup.5 spores/mL to a rice medium which was prepared from
50 g of autoclaved rice with the water content thereof adjusted to
40 wt % with sterile distilled water, followed by culturing at
25.degree. C. for 7 days.
[0091] (3) Extraction of Cyclic Depsipeptide from the Culture
[0092] To the Fusarium strain culture including the mycelia of (1)
was added two volumes of chloroform. It was vigorously stirred and
the bottom layer was evaporated to dryness. The residue was
resuspended in methanol.
[0093] The cereal medium in which the Fusarium of (2) was cultured
was dried at room temperature for 12 hrs. The mycelia thus dried
were homogenized, extracted overnight with 75 ml of a solvent
mixture of 16:3:1 (v/v/v) acetonitrile: MeOH: water, and filtered
through sterile filter paper. The filtrate was defatted twice with
25 mL of n-heptane and the bottom layer was evaporated to dryness.
The residue was dissolved in 50 mL of a solvent mixture of 55:45
(v/v) MeOH: water and extracted twice with 25 mL of
CH.sub.2Cl.sub.2. The CH.sub.2Cl.sub.2 layer was evaporated to
dryness, and the residue was resuspended in methanol.
[0094] (4) Purification of Cyclic Depsipeptide from the
Extracts
[0095] Each of the extracts from the cultures of (1) and (2) was
flowed for 40 min at a constant flow rate (1 mL/min) along a
Shiseido pack C18 column (0.46.times.25 cm) (Shiseido co., Japan)
with a mixture of 70:30 (v/v) acetonitrile: water serving as an
eluent. Peaks were read at 210 nm.
[0096] No secondary metabolites were detected in the extract of the
culture in FDM of (1) (FIG. 7). On the other hand, two main peaks
could be eluted with a retention time of 9.7 and 13.4 minutes from
the extract from the culture on the cereal substance of (2) (FIG.
8). The compounds detected at 9.7 and 13.4 min were named Compound
A and B, respectively.
[0097] The compounds were separated using a GROM-sil pack ODS
preparative column (1.0.times.25 cm) with a mixture of
acetonitrile: water solution (65:35, v/v) serving as a mobile phase
at a flow rate of 3 ml/min for 60 min, followed by further
purification through a Shiseido pack C18 column (0.46.times.25 cm)
with a mixture of acetonitrile: water (70:30, v/v) at a flow rate
of 1 mL/min.
[0098] (5) Determination of Molecular Weight
[0099] Compounds A and B were found to have molecular weights of
586.36 and 600.36 m/z, respectively, as measured by electrospray
ionization mass spectrometry (ESI-MS) (FIGS. 9 and 10).
[0100] These mass data of the secondary metabolites (Compounds A
and B) were very similar to previously reported ones for cyclic
depsipeptides. A summarization of previously reported cyclic
depsipeptides for origin, molecular weight, and reference is given
in Table 1, below.
TABLE-US-00001 TABLE 1 Cpd. Mw (m/z) Origin Reference Enniatin H
653.6 F. oxysporum KFCC1169P Song et al..sup.1 Enniatin I 667.6 F.
oxysporum KFCC1169P Song et al..sup.1 Enniatin MK1688 681.6586 F.
oxysporum KFCC1169P Song et al..sup.1 Sansalvamide Marine Fusarium
sp. Belofsky.sup.2 N-Methylsan- 600 Marine Fusarium sp.
Cueto..sup.3 salvamide Zygosporamide 634.38 Marine Fusarium sp.
Oh.sup.4 (Zygosporium masonii) .sup.1Song H H, Ahn J H, Lim Y H,
Lee C, (2006) Analysis of beauvericin and unusual enniatins
co-produced by Fusarium oxysporum FB1501 (KFCC 11363P). J.
Microbiol. Biotechnol. 16, 1111-1119 .sup.2Belofsky G N, Jensen P
R, Fenical W. (1999) Sansalvamide: A new cytotoxic cyclic
depsipeptide produced by a marine fungus of the genus Fusarium.
Tetrahedron Lett. 40, 2913-2916 .sup.3Cueto M, Jensen P R, Fenical
W. (2000) N-Methylsansalvamide, a cytotoxic cyclic depsipeptide
from a marine fungus of the genus Fusarium. Phytochemistry. 55,
223-226 .sup.4Oh D C, Jensen P R, Fenical W. (2006) Zygosporamide,
a cytotoxic cyclic depsipeptide from the marine-derived fungus
Zygosporium masonii. Tetrahedron Lett. 47, 8625-8628
EXAMPLE 3
Structural Analysis of Cyclic Depsipeptides
[0101] (1) Compound A
[0102] The functional group of compound 1 was investigated by an FT
IR-8400S infrared spectrophotometer (Shimadzu, Japan). IR analysis
of Compound A showed amide (1654.42 cm.sup.-1) and ester (1745.52
cm.sup.-1) bonds (FIG. 10). The maximum UV spectrum of compound 1
was determined at 287 nm in methanol. Compound A was found to have
a melting point of 82.degree. C. as measured by a melting point
apparatus (Thermo Fisher scientific Inc. Waltham. USA).
[0103] 1D-NMR CH NMR, .sup.13C NMR, and DEPT) was analyzed on a
Bruker DMX 600 spectrometer system while 2D-NMR (COSY, HMQC, and
HMBC) was measured on a Bruker AVANCE 800 spectrometer system. The
1D and 2D NMR spectra were collected in methanol (CD.sub.3OD).
[0104] The .sup.1H and .sup.13C NMR spectral data acquired for
Compound A accounted for typical resonances for a cyclic
depsipeptide are shown in Table 2.
TABLE-US-00002 TABLE 2 Position 13C 1H J in brackets HMBC
Correlations O-Leu 1 172.254 (C) H2, H3, H27 2 75.407 (CH) 5.191 q
(4.59, 4.81) H3 3 42.436 (CH2) 1.950 m, 1.695 m H4 4 25.939 (CH)
1.695 m H5, H6 5 23.253 (CH3) 0.997 t (6.62) H3, H4, H6 6 23.205
(CH3) 0.997 t (6.62) H3, H4, H5 Leu 7 171.175 (C) H2, H3, H8, H9 8
52.895 (CH) 4.743 q (6.01, 5.90) H9 9 42.740 (CH2) 1.876 m, 1.630 m
H8, H11, H12 10 26.065 (CH) 1.379 t (7.09) H9, H11, H12 11 23.436
(CH3) 0.854 d (6.59) H9, H12 12 22.049 (CH3) 0.787 d (6.51) H9,
H10, H11 Phe 13 173.887 (C) H8, H9, H14, H15 14 57.905 (CH) 4.685 q
(4.85, 4.78) H15 15 39.514 (CH2) 3.157 m H14, H17 16 138.803 (C)
H19, H15 17 130.696 (CH .times. 2) 7.345 d (7.38) H18, H19 18
127.811 (CH) 7.185 t (7.28) H17 19 129.528 (CH .times. 2) 7.257 t
(7.55) H17, H18 Val 20 173.392 (C) H14, H21 21 62.408 (CH) 4.151 d
(10.17) H22, H23, H24 22 31.968 (CH2) 2.289 m H23, H24 23 20.028
(CH3) 0.956 t (6.86) H24 24 19.528 (CH3) 0.956 t (6.86) H23 Leu 25
174.247 (C) H21, H26, H28, H29 26 55.289 (CH) 4.115 d (10.11) H27
27 30.903 (CH2) 1.290 s H28 28 26.065 (CH) 1.630 m H29, H30 29
22.317 (CH3) 0.905 q (7.14, 6.06) H28, H30 30 22.411 (CH3) 0.956 t
(6.86) H28, H29
[0105] According to the DEPT and 2D NMR spectral data (COSY, HMQC,
and HMBC), Compound A was composed of five units: leucic acid
(OLeu), leucine (Leu), valine (Val), phenylalanine (Phe) and
leucine (Leu). The sequence of the five units in Compound A was
determined by analyzing HMBC correlation data (FIG. 12).
[0106] The data identified Compound A as the cyclic
pentadepsipeptide of Chemical Formula 2.
##STR00002##
[0107] Differing from sansalvamide in the sequence of four amino
acid residues and one hydroxy acid, the compound of Chemical
Formula 2 was a novel cyclic pentadepsipeptide and named
neo-sansalvamide.
[0108] (2) Compound B
[0109] IR spectra of Compound B showed amide (1653.24 cm.sup.-1)
and ester (1742.65 cm.sup.-1) bonds (FIG. 13). The maximum UV
spectrum was determined at 213 nm in methanol and the melting point
was measured to be about 82.degree. C.
[0110] 1D-NMR CH NMR, .sup.13C NMR, and DEPT) was analyzed on a
Bruker DMX 600 spectrometer system while 2D-NMR (COSY, HMQC, and
HMBC) was measured on a Bruker 600 spectrometer system. The 1D and
2D NMR spectra were collected in CDCl.sub.3.
[0111] The .sup.1H and .sup.13C NMR spectral data acquired for
Compound B accounted for typical resonances for a cyclic
depsipeptide (Table 3).
TABLE-US-00003 TABLE 3 Position 13C 1H J in brackets HMBC
Correlations OLeu 1 170.313 H-29, H-30 2 75.347 4.970 q (3.63,
4.03) H-3 3 40.814 1.671 m, 1.729 m H-2, H-4 4 25.122 1.729 m H-3 5
23.402 0.937 m H-2, H-6 6 23.453 0.937 m H-3, H-5 Phe 7 170.949
H-2, H-3, H-8, H-9 8 54.435 4.803 q (5.68, 6.41) H-9 9 37.359 3.075
q (6.01, 5.97), H-8, H-10, H-11, H-12 3.453 q (6.59, 6.60) 10
136.553 H-8, H-9, H-11 11 128.975 7.284 m H-12, 12 129.496 7.284 m
H-11 13 127.393 7.190 d (6.79), H-12, 14 (NH) 6.530 d (8.78) Leu 15
170.290 NH-14, H-16, H-17 16 51.222 4.694 q (8.15, 8.25) H-17,
H-18, H-21 17 39.694 1.644 t (7.66, 7.26), H-16, H-18 1.644 t
(7.66, 7.26) 18 24.637 1.388 m H-16, H-17 19b 21.809 0.937 m H-17,
H-18, H-20 20b 21.809 0.937 m H-17, H-18, H-20 21 (NH) 7.465 d
(9.07) Val 22 171.129 NH-21, H-23 23 54.416 4.590 q (5.61, 5.62)
H-24, H-25, H-26 24 30.855 1.991 m H-23, H-25, H-26 25 17.127 0.833
d (6.75) H-22, H-23, H-26 26 20.029 0.976 q (2.76, 3.21) H-22,
H-23, H-24 27 (NH) 6.805 d (8.78) NMeLeu 28 173.143 H-23, NH-27,
H-29, N--Me-34 29 66.762 4.803 q (6.41, 6.31) H-30, H-31 30 37.493
1.579 m, 1.772 m H-29, H-31 31 25.528 1.472 m H-29, H-30, H-32,
H-33 32b 22.390 0.937 m H-30, H-31 33b 23.143 0.937 m H-31, H-32 34
40.331 3.180 s H-29 (N--Me)
[0112] According to the DEPT and 2D-NMR spectral data (COSY, HMQC,
and HMBC), Compound B was composed of five units: leucic acid
(OLeu), N-methylleucine (N-MeLeu), valine (Val), phenylalanine
(Phe) and leucine (Leu). The sequence of the five units in Compound
B was determined by analysis of the HMBC correlation data (FIG.
14).
[0113] The data identified Compound B as the cyclic
pentadepsipeptide of Chemical Formula 1.
##STR00003##
[0114] Differing from N-methylsansalvamide in the sequence of four
amino acid residues and one hydroxy acid, the compound of Chemical
Formula 1 was a novel cyclic pentadepsipeptide and was named
neo-Nmethylsansalvamide.
[0115] (3) Determination of Stereo-Chemical Structures
[0116] The absolute stereo-chemistries of amino acids in Compounds
A and B were determined by acid hydrolysis, followed by the
derivation of the amino acids with Marfeys' Reagent and HPLC
analysis. The amino acids were identified by co-injection with
authentic amino acid standards. The results are given in FIGS. 15
(Compound A) and 16 (Compound B). The amino acids in Compounds A
and B were all shown to possess L configurations.
EXAMPLE 4
Cytotoxicity
[0117] Known cyclic hexadepsipeptides (beauvericin, enniatin H, I,
and MK1688) and the compounds of Chemical Formulas 1 and 2 were
evaluated for in vitro cytotoxicity against various
non-multidrug-resistant human cancer cell lines and
multidrug-resistant cancer cell lines using the SRB method.
[0118] Human non-small-cell lung cancer cell line (A549) ovarian
cancer cell line (SK-OV-3), skin cancer cell line (SK-MEL-2), and
uterine sarcoma cell line MES-SA and its multi-drug resistant
subline (MES-SA/DX5) were purchased from the American Type Culture
Collection (USA). Colorectal carcinoma cancer cell line (HCT-15)
was provided by the National Cancer Institute (NCI). HCT15/CL02
cell lines were established from HCT15 cells by continuous and
stepwise exposure of the cells to doxorubicin in the Korea Research
Institute of Chemical Technology (Korea). The inhibitory activities
were quantified as the concentration required for inhibiting cell
growth in vitro by 50% (EC.sub.50) under the assay conditions, with
doxorubicin serving as a control.
TABLE-US-00004 TABLE 4 EC50 (.mu.M) Sample A549 SK-OV-3 SK-MEL-2
BEA 1.43 .+-. 0.16 1.39 .+-. 0.09 1.47 .+-. 0.09 EN H 1.84 .+-.
0.11 1.71 .+-. 0.03 1.77 .+-. 0.03 EN I 0.50 .+-. 0.04 0.49 .+-.
0.03 0.53 .+-. 0.06 EN MK1688 0.45 .+-. 0.04 0.46 .+-. 0.03 0.63
.+-. 0.01 Com. 1 11.70 .+-. 0.55 10.38 .+-. 0.64 13.99 .+-. 1.32
Com. 2 10.73 .+-. 0.15 11.24 .+-. 1.23 10.02 .+-. 0.53 Doxorubicin
0.03 .+-. 0.01 0.06 .+-. 0.01 0.04 .+-. 0.01
TABLE-US-00005 TABLE 5 EC50 (.mu.M) Sample MES-SA MES-SA/DX5 HCT15
HCT15/CL02 BEA 1.29 .+-. 0.02 1.34 .+-. 0.03 1.53 .+-. 0.09 1.66
.+-. 0.08 EN H 12.94 .+-. 0.23 14.89 .+-. 0.59 16.74 .+-. 0.96
17.71 .+-. 0.49 EN I 3.94 .+-. 0.03 4.90 .+-. 0.27 5.56 .+-. 0.22
5.88 .+-. 0.21 EN MK1688 13.62 .+-. 1.05 13.59 .+-. 0.79 14.02 .+-.
0.54 16.35 .+-. 0.84 Com. 1 11.75 .+-. 0.13 19.45 .+-. 1.10 9.95
.+-. 1.00 22.08 .+-. 2.45 Com. 2 13.96 .+-. 0.74 11.42 .+-. 0.30
12.46 .+-. 0.71 13.50 .+-. 1.19 Doxorubicin 0.01 .+-. 0.0001 1.03
.+-. 0.31 0.01 .+-. 0.001 4.48 .+-. 2.15
[0119] Belofsky et al. reported that sansalvamide was responsible
for the majority of the cancer cell cytotoxicity present in the
crude extract, exhibiting an in vitro IC.sub.50 value of 9.8
.mu.g/ml toward HCT-116 colon carcinoma [Belofsky et al., 1999].
Ceuto et al. (2000) reported that N-Methylsansalvamide exhibited in
vitro cytotoxicity in the NCI human tumor cell line screen
(GI.sub.50 8.3 .mu.M) [Ceuto et al., 2000]. As is apparent from the
data of Tables 4 and 5, the cyclic pentadepsipeptides of Chemical
Formulas 1 and 2 exhibit cytotoxicity against most cell lines as
potent as that of sansalvamide A or N-methylsansalvamide
irrespective of the possession of multidrug resistance. The cyclic
pentadepsipeptide of Chemical Formula 1 was of more potent
inhibitory activity against MDR cancer lines than was that of
Chemical Formula 2 (FIGS. 17 to 20).
EXAMPLE 5
Multidrug Resistance-Reversing Activity
[0120] The cyclic pentadepsipeptides of Chemical Formulas 1 and 2
were analyzed for MDR-reversing activity by comparing inhibitory
activities against multidrug-resistant cancer cell lines
(MES-SA/DX5 and HCT15/CL02) with those against
non-multidrug-resistant cancer cell lines (MES-SA and HCT15). In
this regard, the effects of the cyclic pentadepsipeptides of
Chemical Formulas 1 and 2 on paclitaxel's cytotoxicity against MDR
tumor cells were measured (Table 6). Verapamil (VER), an
MDR-reversing agent with inhibitory activity against
P-glycoprotein, was used as a control.
TABLE-US-00006 TABLE 6 EC50 (nM) Sample MES-SA MES-SA/DX5 HCT15
HCT15/CL02 TAX 1.00 .+-. 0.20 10.00 .+-. 0.53 0.85 .+-. 0.63
>1,000 Com. 1 1.00 .+-. 0.30 6.31 .+-. 0.91 0.39 .+-. 0.08
>1,000 (3 .mu.M) Com. 2 1.00 .+-. 0.30 1.58 .+-. 0.12 0.10 .+-.
0.02 288.40 .+-. 21.02 (3 .mu.M) VER 1.00 .+-. 0.10 1.78 .+-. 0.33
0.11 .+-. 0.05 33.89 .+-. 8.42 (10 .mu.M)
[0121] The cyclic pentadepsipeptide of Chemical Formula 2 increased
the cytotoxicity of paclitaxel against MDR cell lines, but only
slightly. On the other hand, the cyclic pentadepsipeptide of
Chemical Formula 1 remarkably enhanced the cytotoxicity of
paclitaxel (FIGS. 21 to 24). Therefore, the N-methyl group in the
cyclic pentadepsipeptide may be a factor crucial for the expression
of the MDR reversal activity. The MDR-reversing activity of the
cyclic pentadepsipeptide of Chemical Compound 1 was similar to that
of the positive control verapamil.
EXAMPLE 6
Anti-Bacterial and Anti-Fungal Activities
[0122] (1) Anti-Bacterial Activity
[0123] Assays for antibacterial activity were performed with
Gram-positive (Listeria monocytogenes ATCC 14028, Staphylococcus
aureus ATCC 35556 and Bacillus cereus ATCC 13061) and 3
Gram-negative bacteria (Escherichia coli ATCC 8739, Pseudomonas
aeruginosa ATCC 9026 and Salmonella typhimurium ATCC 14028). Each
of the compounds of Chemical Formulas 1 and 2 was dissolved in
different concentrations (0.1, 0.5, 1, and 2 mM) in dimethyl
sulfoxide (DMSO). The solutions were applied to a sterile paper
disc (5 mm diameter), followed by evaporating the DMSO solvent.
Bacteria was inoculated at a density of 1.times.10.sup.7 CFU/ml on
Tryptic soy agar (TSA) and then, the paper disc was placed on the
bacteria-inoculated agars. After incubation at 37.degree. C. for 24
hr, the clear zone around each disc was observed and its diameter
was measured.
[0124] Neither the compound of Chemical Formula 1 nor Chemical
Formula 2 showed inhibitory activity against all of the bacteria
tested.
[0125] (2) Anti-Fungal Activity
[0126] Inhibitory activity was performed against four fungus
strains (Mucor rouxii, Penicillium citrinum, Fusarium oxysporum,
and Aspergillus oryzae).
[0127] Drops of 1 and 10 mM solutions of compounds of Chemical
Formulas 1 and 2 in methanol were loaded onto sterile paper discs
(8 mm diameter) and the methanol solvent was evaporated under a
hood. The paper discs were placed on fungus mycelia grown on potato
dextrose agar. After incubation at 25.degree. C. for 48 hr, clear
zones around the discs were observed and their diameters were
measured. A filter treated with pure methanol was used as a
negative control.
[0128] The inhibitory effects of compounds purified from F. solani
KCCM 90040 on the mycelial growth of four fungal strains (Mucor
rouxii, Penicillium citrinum, Fusarium oxysporum, and Aspergillus
oryzae) are shown in FIG. 28. The results of the antifungal tests
revealed that compound 1 and 2 were inhibitory against Mucor
rouxii, and Fusarium oxysporum, weakly. No inhibitory activities
were detected at 1 mM of the compound of Chemical Formula 1 or 2
against all of the four fungal strains. At 10 mM of the compounds
of Chemical Formulas 1 and 2, no clear zones were observed on the
agar in which Mucor rouxii was grown, but the hyphal growth of
Mucor rouxii was reduced near the purified compounds containing
paper disc (FIG. 25). While the compound of Chemical Formula 2 did
not produce a clear zone on the mycelium of Fusarium oxysporum at
10 mM, the compound of Chemical Formula 1 exhibited an inhibitory
effect at 10 mM, weakly (FIG. 26).
EXAMPLE 7
Selection of Cereal Medium
[0129] Fusarium solani KCCM90040 was inoculated at a density of
1>10.sup.5 spores/mL on a cereal medium which was prepared from
50 g of an autoclaved cereal substance with the water content
thereof adjusted to 40 wt % with sterile distilled water. The
microorganism was grown at 25.degree. C. during which the medium
was shaken once a day. The production of the compounds of Chemical
Formulas 1 and 2 by F. solani KCCM 90040 upon culturing on six
different solid cereal substrates is quantitatively depicted in
FIGS. 27 and 28, respectively.
[0130] The production of the compound of Chemical Formula 2 peaked
on the 2.sup.th week after incubation on rice (Avg. 0.375 g/kg).
The use of maize, wheat or Indian millet, instead of rice,
decreased the productivity of the compound of Chemical Formula 2 by
about 80%. Particularly low production (0.112 g/kg) was observed on
barley.
[0131] As for the compound of Chemical Formula 1, its maximal
production was obtained on the 2.sup.th week after incubation on
rice (Avg. 0.689 g/kg). On the 3.sup.rd week after the incubation
on wheat, its production peaked to 0.672 g/kg. Replacement of rice
by maize or rye decreased the production of the compound of
Chemical Formula 1 by about 25%. The final production of the
compound of Chemical Formula 1 on rice was twice as great as that
on Indian millet or barley.
EXAMPLE 8
Determination of Culture Conditions
[0132] The experimental data for the production of the compounds of
Chemical Formulas 1 and 2 from F. solani KCCM 90040 in different
treatment conditions are given in Table 7, below.
TABLE-US-00007 TABLE 7 Moisture com. 1 com. 2 Run Temp. % Time
(g/kg) (g/kg) 1 15 30 20 0.005 0.010 2 35 40 15 0.136 0.262 3 35 30
20 0.115 0.230 4 35 30 20 0.124 0.248 5 25 40 20 0.300 0.522 6 15
50 10 0.001 0.005 7 25 40 15 0.376 0.580 8 25 40 15 0.400 0.772 9
25 50 15 0.332 0.480 10 25 40 15 0.380 0.628 11 25 40 15 0.432
0.702 12 25 40 15 0.416 0.802 13 35 50 10 0.024 0.068 14 15 30 10
0.002 0.003 15 25 40 15 0.350 0.624 16 25 30 15 0.320 0.508 17 35
50 20 0.005 0.080 18 25 40 10 0.250 0.200 19 15 40 15 0.008 0.050
20 15 50 20 0.005 0.009
[0133] When cultured in a seawater-based medium, marine Fusarium
strains were reported to produce sansalvamide in an amount of about
0.642 g/17 L [Belofsky G N, Jensen P R, Fenical W. (1999)
Sansalvamide: A new cytotoxic cyclic depsipeptide produced by a
marine fungus of the genus Fusarium. Tetrahedron Lett. 40,
2913-2916]. It was also reported that N-methylsansalvamide produced
by the Fusarium strain CNL-619 reached 3.1 mg/L in a seawater-based
medium [Cueto M, Jensen P R, Fenical W. (2000)
N-Methylsansalvamide, a cytotoxic cyclic depsipeptide from a marine
fungus of the genus Fusarium. Phytochemistry. 55, 223-226].
[0134] Variables for the production of the novel compounds of
Chemical Formulas 1 and 2 upon incubation on rice were determined
to specify 20.about.30.degree. C. for culture temperature,
20.about.50% for RH, and 10.about.20 days for growth period,
preferably 23.about.28.degree. C. for culture temperature,
35.about.45% for RH, and 13.about.18 days.
[0135] Optimally, the microorganisms were cultured at 25.84.degree.
C. and 37.99 RH % for 16.03 days for optimal production of the
cyclic pentadepsipeptide of Chemical Formula 1 (FIGS. 29 to 31) and
at 25.87.degree. C. and 33.87 RH % for 15.58 days for the optimal
production of the cyclic pentadepsipeptide of Chemical Formula 2.
Under the optimal conditions, the production was measured to be
about 0.40 g/kg for the cyclic pentadepsipeptide of Chemical
Formula 1 and about 0.70 g/kg for the cyclic pentadepsipeptide of
Chemical Formula 2.
FORMULATION EXAMPLE 1
Preparation of Tablet
[0136] Ingredients and Amounts
[0137] Cyclic pentadepsipeptide of Chemical Formula 1 or 2
TABLE-US-00008 Cyclic pentadepsipeptide of Chemical Formula 1 or 2
100 mg Corn Starch 68 mg Lactose 90 mg Microcrystalline Cellulose
40 mg Mg Stearate 2 mg
[0138] The ingredients were added in the given amounts,
homogeneously mixed together and granulized. The granules were
prepared into tablets, each containing 100 mg of the cyclic
pentadepsipeptide of Chemical Formula 1 or 2, according to a
typical method.
Formulation Example
Preparation of Injection
[0139] Ingredients and Amounts
[0140] Cyclic pentadepsipeptide of Chemical Formula 1 or 2
TABLE-US-00009 Cyclic pentadepsipeptide of Chemical Formula 1 or 2
50 mg Na metabisulfite 1.5 mg Methyl paraben 1.0 mg Propyl paraben
0.1 mg Pure water for injection q.s.
[0141] The ingredients were dissolved in the given amounts in
boiling water with stirring, cooled and loaded into 2 mL sterile
vials and supplemented with injection water to form a total volume
of 2 mL to afford injections, each containing 50 mg of the cyclic
pentadepsipeptide of Chemical Formula 1 or 2.
FORMULATION EXAMPLE 3
Preparation of Syrup
[0142] Ingredients and Amounts
[0143] Cyclic pentadepsipeptide of Chemical Formula 1 or 2
TABLE-US-00010 Cyclic pentadepsipeptide of Chemical Formula 1 or 2
200 mg Concentrated fruit juice 2 g Sucrose 5 g Sodium citrate 100
mg Fragrant 70 mg Water q.s.
[0144] According to a typical method, the ingredients were mixed in
the given amounts, dissolved in water with heating, cooled and
loaded into vessels, each containing 200 mg of Cyclic
pentadepsipeptide of Chemical Formula 1 or 2, to afford syrups.
INDUSTRIAL APPLICABILITY
[0145] Exhibiting cytotoxicity against a wide range of cancer cell
lines, as described hitherto, the cyclic pentadepsipeptides of the
present invention can be used as therapeutics for tumors. Further,
they are useful as multidrug resistance inhibitors. These novel
cyclic pentadepsipeptides are produced by the Fusarium strains of
the present invention.
Sequence CWU 1
1
3124DNAArtificial SequenceFusarium specific primer P28SL
1acaaattaca actcgggccc gaga 24223DNAArtificial SequenceFusarium
specific primer P58SL 2agtattctgg cgggcatgcc tgt 233305DNAFusarium
solani 3gggcctggcg ttggggatcg gcggagcccc ctgtgggcac acgccgtccc
tcaaatacag 60tggcggtccc gccgcagctt ccattgcgta gtagctaaca cctcgcaact
ggagagcggc 120gcggccatgc cgtaaaacac ccaacttctg aatgttgacc
tcgaatcagg taggaatacc 180cgctgaactt aagcatatca ataagcggag
gaaaagaaac caacagggat tgccccagta 240acggcgagtg aagcggcaac
agctcaaatt tgaaatctgg ctctcgggcc cgagttgtaa 300tttgt 305
* * * * *