DIPEPTIDE DERIVATIVE FOR THE TREATMENT OF CANCER
FIELD OF THE INVENTION
This invention relates to a compound of formula (1) having anticancer activity. The
compound may be obtained by fermentation of a microorganism belonging to endophytic
fungal strain (PM0509732/MTCC5544). The invention also includes all isomeric forms and
all tautomeric forms of the compound of formula (1) and pharmaceutically acceptable salts
thereof. The present invention further relates to processes for the production of the anticancer
compound, to the microorganism belonging to endophytic fungal strain
(PM0509732/MTCC5544) and to pharmaceutical compositions containing the compound as
active ingredient and their use in medicines for treatment of cancer.
BACKGROUND OF THE INVENTION
Cancer is a generic term for a large group of diseases caused by uncontrolled growth
and spread of cells that can affect any part of the body. Other terms used for cancer are
malignant tumors and neoplasms. Cancer is a leading cause of death worldwide. As per the
World Health Organization (WHO) report the most frequent types of cancer are lung,
stomach, liver, colorectal, oesophagus and prostate in men, and breast, lung, stomach,
colorectal and cervical in women.
The type of treatment and therapies used are largely determined by the location of the
cancer in the body and also the extent to which the cancer has spread at the time of diagnosis.
Cancer therapy currently falls under the following categories including surgery, radiation
therapy, chemotherapy, bone marrow transplantation, stem cell transplantation, hormonal
therapy, immunotherapy, antiangiogenic therapy, targeted therapy, gene therapy and others.
There are reports of anticancer compounds such as Taxol, Vincristine, Torreyanic
acid, and Camptothecin from natural resources. (Natural Product Communications, 2009,
Vol. 4 (11), 1513). In spite of this, there is still a need for drugs which can effectively treat
various cancers such as lung, prostate, pancreatic and head and neck cancer.
SUMMARY OF THE INVENTION
The present invention relates to a compound designated herein as compound of
formula (1).
The present invention also relates to a purified compound, (designated herein as
compound of formula (1)), isolated from the fermented broth of the microorganism belonging
to endophytic fungal strain (PM0509732/MTCC5544).
The invention also relates to all isomeric forms and tautomeric forms of compound of
formula (1), and pharmaceutically acceptable salts thereof.
The compound of formula (1), isomers, tautomers, or pharmaceutically acceptable
salts thereof, are useful for the treatment of cancer.
The invention further relates to pharmaceutical compositions comprising the
compound of formula (1), an isomer, a tautomer, a pharmaceutically acceptable salt thereof,
as an active ingredient in association with a pharmaceutically acceptable carrier or vehicle for
the treatment of cancer.
The present invention further relates to processes for the production of the compound
of formula (1) and/or its isomers or its tautomers from the microorganism belonging to
endophytic fungal strain (PM0509732/MTCC5544).
The present invention also relates to processes for the isolation of the microorganism
belonging to endophytic fungal strain (PM0509732/MTCC5544), which on cultivation
produces the compound of formula (1), its isomers and its tautomers.
BRIEF DESCRIPTION OF THE DRAWING
Figure 1: Illustrates HNMR (DMSO-d ; 500 MHz; Instrument: Bruker) of the
compound of formula (1).
DETAILED DESCRIPTION OF THE INVENTION
The compound of formula (1) has the molecular formula C 1 H30N2O5S2 (molecular
weight 418). The compound of formula (1) may be characterised by any one or more of the
physico-chemical and spectral properties, such as high performance liquid chromatography
(HPLC), mass spectrum (MS), infra red (IR) and nuclear magnetic resonance (NMR)
spectroscopic data as discussed herein below.
The compound of formula (1) is structurally represented as follows:
Formula (1)
The microorganism, which may be used for the production of the compound of
formula (1), is an endophytic fungal strain of sterile mycelium (PM0509732/MTCC5544),
herein after referred to as culture no. PM0509732, which is isolated from the leaves of
Pongamia pinnata (family Fabaceae) collected from Karnala Bird Sanctuary near Panvel in
Raigad District, Maharashtra, India.
The present invention further provides processes for the production of the compound
of formula (1) from culture no. PM0509732 comprising the steps of:
(a) cultivating the culture no. (PM0509732/MTCC5544) or one of its
variants or mutants under submerged aerobic conditions in nutrient medium containing one or
more sources of carbon and one or more sources of nitrogen and nutrient inorganic salts;
(b) isolating the compound of formula (1) from the culture broth; and
(c) purifying the compound of formula (1).
The step (c) involving purification of the compounds of formula (1) is carried out by
purification procedures generally used in the related art.
The compound of formula (1) produced according to the process of the present
invention is a substantially pure compound. Thus, the compound of formula 1 is an isolated
pure compound having anticancer activity.
As used herein, the term "mutant" refers to an organism or cell carrying a mutation,
which is an alternative phenotype to the wild-type.
As used herein, the term "variant" refers to an individual organism that is
recognizably different from an arbitrary standard type in that species.
The term "mammal" as used herein, refers to a human as well as non-human
mammals, including but not limited to, cows, horses, pigs, dogs and cats. The term
"mammal" may be used interchangeably with the term "patient" or "subject".
The term "active ingredient" as used herein, refers to the compound of formula (1) or
to a isomer or tautomer or a pharmaceutically acceptable salt thereof. .
The term "substantially pure" as used herein, means that the compound of formula (1)
and isomers thereof are sufficiently pure such that further purification would not detectably
alter the physical and chemical properties, such as enzymatic and biological activities, of the
substance. Compounds of formula (1) can be purified substantially by following the methods
known to those skilled in the art.
The term "compound of formula (1)" includes compound of formula (1) and isomers,
tautomers, and pharmaceutically acceptable salts thereof.
As used herein the term "therapeutically effective amount" in reference to the
treatment of cancer (as listed herein) using the compound of formula (1) refers to an amount
capable of invoking one or more of the following effects in a subject receiving the compound
of the present invention: (i) inhibition, to some extent, of tumor growth, including, slowing
down and complete growth arrest; (ii) reduction in the number of tumor cells; (iii) reduction
in tumor size; (iv) inhibition (i.e., reduction, slowing down or complete stopping) of tumor
cell infiltration into peripheral organs; (v) inhibition (i.e., reduction, slowing down or
complete stopping) of metastasis; (vi) enhancement of anti-tumor immune response, which
may, but does not have to, result in the regression of the tumor; and/or (vii) relief, to some
extent, of one or more symptoms associated with the cancer being treated.
The term "pharmaceutically acceptable salt(s)", as used herein, means those salts of
compounds of the invention that are safe and effective in mammals and that possess the
desired biological activity. Pharmaceutically acceptable acid addition salts include, but are
not limited to, hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, phosphate, acetate,
lactate, salicylate, citrate, tartrate, ascorbate, succinate, maleate, fumarate, formate, benzoate,
glutamate, methanesulfonate, benzensulfonate, or p-toluenesulfonate salts. Suitable base
addition salts include, but are not limited to, calcium, lithium, magnesium, potassium,
sodium, or zinc, salts.
Preliminary identification of culture no. PM0509732, which is the producer of
compound of formula (1), was performed by examination of its colony characteristics.
Microscopic studies on the strain of isolated culture no. PM0509732 were carried out on
Potato dextrose agar medium. The observations were made after 4, 7, 14, 2 1 and 30 days of
incubation at 25°C. Culture no. PM0509732 has been identified as an endophytic fungal
strain of sterile mycelium.
Growth on Potato dextrose agar medium develops as 64 mm diameter colonies in 5
days at 26°C. The colony is initially white then turned yellowish green (Jaune curry RAL
1027). The back/ reverse surface is initially white then turned to grey brown (RAL 8019)
after 30 days of incubation. Formation of neither pigmentation nor droplet is observed.
Culture no. PM0509732 has been deposited with Microbial Type Culture Collection
(MTCC), Institute of Microbial Technology, Sector 39-A, Chandigarh -160 036, India, a
World Intellectual Property Organization (WIPO) recognized International Depository
Authority (IDA) and has been given the accession number MTCC5544.
In addition to the specific microorganism described herein, it should be understood
that mutants of PM0509732, such as those produced by the use of chemical or physical
mutagens including X-rays, U.V. rays etc. and organisms whose genetic makeup has been
modified by molecular biology techniques, may also be cultivated to produce the compound
of formula (1).
The screening for suitable mutants and variants which can produce the compound
according to the invention can be confirmed by HPLC, NMR, IR, MS determination of
biological activity of the active compounds accumulated in the culture broth, for example by
testing the compounds for anticancer activity or by a combination thereof.
The medium and/or nutrient medium used for isolation and cultivation of culture no.
PM0509732, which produces the compound of formula (1), preferably contains sources of
carbon, nitrogen and nutrient inorganic salts. The carbon sources are, for example, one or
more of starch, glucose, sucrose, dextrin, fructose, molasses, glycerol, lactose, or galactose. A
preferred carbon source is soluble starch and glucose. The sources of nitrogen are, for
example, one or more of soyabean meal, peanut meal, yeast extract, beef extract, peptone,
malt extract, corn steep liquor, gelatin, or casamino acids. Preferred nitrogen source is
soyabean meal and yeast extract. The nutrient inorganic salts are, for example, one or more of
sodium chloride, potassium chloride, calcium chloride, manganese chloride, magnesium
chloride, strontium chloride, cobalt chloride, potassium bromide, sodium fluoride, sodium
hydrogen phosphate, potassium hydrogen phosphate, dipotassium hydrogen phosphate,
disodium phosphate, calcium carbonate, sodium bicarbonate, sodium silicate, sodium nitrate,
ammonium nitrate, potassium nitrate, sodium sulphate, ammonium sulphate, ammonium
heptamolybdate, ferric citrate, copper sulphate, magnesium sulphate, ferrous sulphate, zinc
sulphate or boric acid. Calcium chloride, sodium chloride and sodium nitrate are preferred.
The maintenance of culture no. PM0509732 may be carried out at a temperature
ranging from 21°C to 35°C and a pH of about 6.5 to 8.5. Typically, culture no. PM0509732 is
maintained at 26°C -29°C and a pH of about 6.5. The well-grown cultures may be preserved
in the refrigerator at 6°C -12°C.
Seed culture cultivation of culture no. PM0509732 may be carried out at a
temperature ranging from 24°C to 31°C and a pH of about 5.5 to 8.5, for 100-125 hours at
180-220 rpm (revolutions per minute). Typically, culture no. PM0509732 seed is cultivated at
25°C -27°C and a pH of about 5.5, for 115- 120 hours at 180-200 rpm.
The production of the compound of formula (1) may be carried out by cultivating
culture no PM0509732 by fermentation at a temperature ranging from 25°C to 31°C and a pH
of about 5.5 to 8.5, for 60-100 hours at 180- 220 rpm. Typically, culture no. PM0509732 is
cultivated at 26°C-31°C and pH 5.5-7.7 for 66-96 hours at 190- 210 rpm.
The production of the compound of formula (1) can be carried out by cultivating
culture no. PM0509732 in a suitable nutrient broth under conditions described herein,
preferably under submerged aerobic conditions, for example in shake flasks. The progress of
fermentation and production of the compound of formula (1) can be detected by high
performance liquid chromatography (HPLC) and by measuring the bioactivity of the culture
broth by testing against the cancer cell lines.
Fermentation is a process of growing microorganisms for the production of various
chemical or pharmaceutical compounds. Microbes are normally incubated under specific
conditions in the presence of nutrients.
In the resulting culture broth, the compound of formula (1) is present in the culture
filtrate as well as in cell mass and can be isolated using different extraction methods and
chromatographic techniques. Thus, the compound of formula (1) can be recovered from the
culture filtrate by extraction with a water immiscible solvent such as petroleum ether,
dichlorome thane, chloroform, ethyl acetate, diethyl ether or butanol, or by hydrophobic
interaction chromatography using polymeric resins such as "Diaion HP-20 " (Mitsubishi
Chemical Industries Limited, Japan), "Amberlite XAD " (Rohm and Haas Industries, USA)
or adsorption on activated charcoal. These techniques may be used repeatedly, alone or in
combination. The compound of formula (1) can be recovered from the cell mass by extraction
with a water miscible solvent such as methanol, acetone, acetonitrile, n-propanol, or isopropanol
or with a water immiscible solvent such as petroleum ether, dichloromethane,
chloroform, ethyl acetate or butanol. One other option is to extract the whole broth with a
solvent selected from petroleum ether, dichloromethane, chloroform, ethyl acetate, methanol,
acetone, acetonitrile, n-propanol, iso-propanol, or butanol. Typically, the compound of
formula (1) is extracted from the cell mass using acetone and by chromatography using
Diaion HP- 0 from the culture filtrate. Concentration and lyophilization of the extracts gives
the active crude material.
The compound of formula (1) of the present invention can be recovered from the
crude material by fractionation using any of the following techniques: normal phase
chromatography (using alumina or silica gel as stationary phase; eluents such as petroleum
ether, ethyl acetate, dichloromethane, acetone, chloroform, methanol, or combinations
thereof; and additions of amines such as NEt3) ; reverse phase chromatography (using reverse
phase silica gel such as dimethyloctadecylsilylsilica gel, (RP-18) or dimethyloctylsilyl silica
gel (RP-8) as stationary phase; and eluents such as water, buffers (for example, phosphate,
acetate, citrate (pH 2-8)), and organic solvents (for example methanol, acetonitrile, acetone,
tetrahydrofuran, or combinations of these solvents)); gel permeation chromatography (using
resins such as Sephadex LH-20® (Pharmacia Chemical Industries, Sweden), TSKgel®
Toyopearl HW (TosoHaas, Tosoh Corporation, Japan) in solvents such as methanol,
chloroform, acetone, ethyl acetate, or their combinations); or by counter-current
chromatography (using a biphasic eluent system made up of two or more solvents such as
water, methanol, ethanol, iso-propanol, n-propanol, tetrahydrofuran, acetone, acetonitrile,
methylene chloride, chloroform, ethyl acetate, petroleum ether, benzene, and toluene). These
techniques may be used repeatedly, alone or in combination. A typical method is
chromatography over normal phase silica gel and reverse phase silica gel (RP-18).
As used herein, the term "isomer" is a general term used for all isomers of the
compound of formula (1) that differ only in the orientation of their atoms in space. The term
isomer includes mirror image isomers (enantiomers), mixtures of mirror image isomers
(racemates, racemic mixtures) and isomers of compounds with more than one chiral center
that are not mirror images of one another (diastereoisomers). The compound of the present
invention may have asymmetric centers and occur as racemates, racemic mixtures, individual
diastereoisomers, or enantiomers, or may exist as geometric isomers, with all isomeric forms
of said compound being included in the present invention.
As used herein, the term "tautomer" refers to the coexistence of two (or more)
compounds that differ from each other only in the position of one (or more) mobile atoms and
in electron distribution, for example, keto-enol tautomers.
Compound of formula (1), exists as an equilibrium mixture of two compounds which
may be isomers or tautomers.
The compound of formula (1), isomers and tautomers thereof can be converted into
their pharmaceutically acceptable salts which are all contemplated by the present invention.
The salts can be prepared by standard procedures known to one skilled in the art, for
example, salts like sodium and potassium salts, can be prepared by treating the compound of
formula (1), isomers, and tautomers thereof, with a suitable sodium or potassium base, for
example sodium hydroxide, potassium hydroxide. Similarly, salts like hydrochloride and
sulphate salts, can be prepared by treating the compound of formula (1), isomers, and
tautomers thereof, with a suitable acid, for example hydrochloric acid, and sulphuric acid.
The compound of formula (1) has anticancer activity against a wide range of cancer
cells.
The compound of formula (1), isomers, tautomers or pharmaceutically acceptable
salts thereof, can be administered to animals, such as mammals, including humans, as
pharmaceuticals and in the form of a pharmaceutical composition. The compound of formula
(1), isomers, tautomers, pharmaceutically acceptable salts thereof, can be administered to a
patient who is diagnosed with cancer.
Accordingly, the present invention also relates to the compound of formula (1),
isomers, tautomers or pharmaceutically acceptable salts thereof for use as pharmaceuticals
and to the use of the compound of formula (1), isomers, tautomers or pharmaceutically
acceptable salts thereof for the production of medicaments having anticancer activity.
The present invention further relates to pharmaceutical composition, which contain an
effective amount of the compound of formula (1) and/or isomers and/or tautomers and/or one
or more pharmaceutically acceptable salts thereof, together with a pharmaceutically
acceptable carrier. The effective amount of the compound of formula (1), or its stereoisomer,
or its tautomer or its pharmaceutically acceptable salt as the active ingredient in the
pharmaceutical preparations normally is from about 0.01 mg to 1000 mg.
The present invention also relates to a method for the manufacture of a medicament
containing the compound of formula (1) and/or isomers and/or tautomers and/or one or more
pharmaceutically acceptable salts thereof, for the treatment of cancer.
The compounds of the present invention are particularly useful as anti-cancer agents.
The present invention accordingly relates to the use of the compound of formula (1) and/or
isomers and/or tautomers and/or one or more pharmaceutically acceptable salts thereof, for
the manufacture of a medicament for the treatment of cancer.
The compounds of the present invention i.e. the compound of formula (1) and/or
isomers and/or tautomers and/or one or more pharmaceutically acceptable salts find use in the
treatment of cancers. Compounds of the present invention are used to reduce, inhibit, or
diminish the proliferation of tumor cells, and thereby assist in reducing the size of a tumor.
Representative cancers that may be treated by such compounds include but are not limited to
bladder cancer, breast cancer, colorectal cancer, endometrial cancer, gastric cancer, head &
neck cancer, kidney cancer, melanoma, non-small-cell lung cancer, ovarian cancer, pancreatic
cancer, prostate cancer, renal cancer, soft tissue sarcoma, oesophageal cancer, cancer of
uterus, testicular and germ cell cancer, thyroid cancer, cervix cancer, pleuramesothelioma,
brain tumors (glioblastoma, cerebellar astrocytoma, cerebral astrocytoma, ependymoma,
meduUoblastoma, neuroblastoma, retinoblastoma, supratentorial primitive neuroectodermal
and pineal tumors, visual pathway and hypothalamic glioma, brain stem glioma), liver cancer,
Ewing's sarcoma family of tumors, osteosarcoma, malignant fibrous histiocytoma of bone,
rhabdomyosarcoma, skin cancer, small-cell lung cancer, Wilms' tumors, leukemias (acute
lymphoblastic leukemia, adult acute myeloid leukemia, chronic lymphocytic leukemia,
chronic myeloid leukemia), and lymphomas (Hodgkin's disease, non-Hodgkin's lymphoma,
hairy cell leukemia, multiple myeloma, primary central nervous system lymphoma), among
others.
According to an embodiment, the present invention provides a method for the
treatment of cancer by administering to a mammal in need thereof a therapeutically effective
amount of a compound of formula (1).
The compound of the present invention can be administered orally, nasally, topically,
subcutaneously, intramuscularly, intravenously, or by other modes of administration.
Pharmaceutical compositions which contain compound of formula (1) or isomers or
tautomers or a pharmaceutically acceptable salt thereof, an be prepared by mixing the
compound of formula (1), with one or more pharmacologically tolerated auxiliaries and/or
excipients such as, wetting agents, solubilisers such as surfactants, vehicles, tonicity agents,
fillers, colorants, masking flavors, lubricants, disintegrants, diluents, binders, plasticizers,
emulsifiers, ointment bases, emollients, thickening agents, polymers, lipids, oils, cosolvents,
complexation agents, or buffer substances, and converting the mixture into a suitable
pharmaceutical form such as, for example, tablets, coated tablets, capsules, granules,
powders, creams, ointments, gels, syrup, emulsions, suspensions, or solutions suitable for
parenteral administration.
Examples of auxiliaries and/or excipients that may be mentioned for use in
preparation of pharmaceutical composition are cremophor, poloxamer, benzalkonium
chloride, sodium lauryl sulphate, dextrose, glycerin, magnesium stearate, polyethylene
glycol, starch, dextrin, lactose, cellulose, carboxymethylcellulose sodium, talc, agar-agar,
mineral oil, animal oil, vegetable oil, organic and mineral waxes, paraffin, gels, propylene
glycol, benzyl alcohol, dimethylacetamide, ethanol, polyglycols, Tween 80, solutol HS 15,
and water. It is also possible to administer the active ingredient as such, without vehicles or
diluents, in a suitable form, for example, in capsules.
As is customary, the galenic formulation and the method of administration as well as
the dosage range which are suitable in a specific case depend on the species to be treated and
on the state of the respective condition or disease, and can be optimized using methods
known in the art. On average, the daily dose of active compound in a patient is 0.05 mg to
100 mg per kg, typically 1 mg to 50 mg per kg. If required, higher or lower daily doses can
also be administered. Actual dosage levels of the active ingredient in the pharmaceutical
compositions of this invention may be varied so as to obtain an amount of the active
ingredient, which is effective to achieve the desired therapeutic response for a particular
patient, composition, and mode of administration without resulting in unacceptable side
effects to the patient.
The following are provided as illustrative examples of the present invention and do
not limit the scope thereof.
Examples:
The following terms/symbol/abbreviations/chemical formulae are employed in the
examples:
1 Litre
ml Millilitre
kg Kilogram
g Gram
mg : Milligram
mm : Millimeter
cm : Centimeter
m : Micron
nm : Nanometer
hrs : Hours
min : Minute
mg/1 : Milligram per litre
mg/ml : Milligram per Millilitre
g/ml : Microgram per Millilitre
mΐ : Microlitre
ml/min : Millilitre per Minute
mM : Millimole
mM : Micromole
fhi : Revolutions per Minute
l :Wavelength
IC50 : 50 % Inhibitory concentration
ATCC : American Type Culture Collection
NCI : National Cancer Institute
DSMZ :Deutsche Sammlung von. Mikroorganismen und Zellkulturen
GmbH :(German Collection of Microorganisms and Cell Cultures)
PAA : PAA Laboratories GmbH
RPMI : Roswell Park Memorial Institute
v/v : Volume (of solute) per volume (of solvent).
PCR : Polymerase Chain Reaction
DNA : Deoxy ribose Nucleic Acid
NSCLC : Non-Small Cell Lung Carcinoma
DMSO : Dimethyl sulphoxide
Example 1
Isolation of culture no. PM0509732 collected from plant source
a) Composition of the isolation medium:
Potato dextrose agar (PDA) media (Hi Media, Mumbai, India) supplemented with 50
mg/1 chloramphenicol was used for isolation purpose. While preparing the medium
chloramphenicol was added after dissolving the same in 10 ml of 95% ethanol and mixing
thoroughly with the hot medium. The medium was autoclaved at 121 °C for 15 min. The pH
prior to autoclaving was adjusted to 6.5 (at 25°C).
b) Procedure
The leaves of the plant Pongamia pinnata (family Fabaceae) were collected from
Karnala Bird Sanctuary near Panvel in Raigad District, Maharashtra, India in the month of
March 2005. These samples were collected in polythene bags and brought to the laboratory
and the fresh leaves of the samples were processed for isolation of fungi.
The undamaged fresh leaves of plant Pongamiapinnata were washed thoroughly with
water, air dried, surface sterilized by treating with 4% sodium hypochlorite (NaOCl,
Qualigens) in water for 1 min and subsequently washed with sterilized demineralised water to
remove traces of the disinfectants. These were cut into segments of about 5 mm with sterile
surgical blade. Five segments of leaves were aseptically placed equidistant on 90 mm Petri
plate containing above media (Potato dextrose agar) so that they were separated from each
other by 2-3 cm. The plates were incubated in alternative 12 hrs dark/light chamber at 26°C +
1°C. After 6 days an elongating hypha over the medium was picked up at its tips with thin
needle, and it was transferred to new medium for purification purpose. The isolates were
repeatedly sub-cultured on to Potato dextrose agar slants to get pure culture no. PM0509732.
The slants were incubated for 14 days at 26°C.
Example 2
Purification of culture no. PM0509732
a) Composition of the isolation medium:
Potato dextrose agar (PDA) media (Hi Media, Mumbai, India) supplemented with 50
mg/1 chloramphenicol was used for isolation purpose. While preparing the medium
chloramphenicol was added after dissolving the same in 10 ml of 95% ethanol and mixing
thoroughly with the hot medium. The medium was autoclaved at 121 °C for 15 min. The pH
prior to autoclaving was adjusted to 6.5 (at 25°C).
b) Procedure:
The culture was available on Potato dextrose agar medium in 90 mm diameter Petri
plate. Another plate with Potato dextrose agar medium was inoculated with the growing
mycelial tips of the culture under binocular microscope. As and when the growth was
observed on the plate it was then transferred to fresh slant. The slants were incubated for 10
days at 25°C. These were then used for shake flask fermentation.
Example 3
Maintenance of producer strain - Culture no. PM0509732
a) Composition of isolation medium
Potato dextrose agar (PDA) media (Hi Media, Mumbai, India) supplemented with 50
mg/1 chloramphenicol was used for isolation purpose. While preparing the medium
chloramphenicol was added after dissolving the same in 10 ml of 95% ethanol and mixing
thoroughly with the hot medium. The pH prior to autoclaving was adjusted to 6.5. After
dissolving the ingredients thoroughly by heating, the resultant solution was distributed in test
tubes and sterilized by autoclaving at 121 °C for 15 min. The test tubes with media were
allowed to solidify in a slanting position.
The agar slants were inoculated with the growing culture of PM0509732 aseptically
by a wire loop and incubated at 26-29°C until a good growth was observed. The well-grown
cultures were stored in the refrigerator at 6 - 12°C.
Example 4
Fermentation of the culture no. PM0509732 in shake flasks
a) Composition of seed culture medium:
Soluble Starch 15.0 g; Soya Bean Meal 15.0 g; Glucose 5.0 g; Calcium Chloride
(CaCl2) 2.0 g; Sodium Chloride (NaCl) 5.0 g; Yeast Extract 2.0 g; Corn Steep Liquor 1.0 g;
Glycerol 11.6 g; Ammonium Heptamolybdate ((NH4)6Mq7q 24 .4H20 ) 0.16 mg; Zinc Sulphate
(ZnS0 4.7H20 ) 0.22 mg; Manganese Chloride (MnCl2 -4H20 ) 0.5 mg; Ferrous Sulphate
(FeS0 4.7H20 ) 0.5 mg; Copper Sulphate (CuS0 4.5H20 ) 0.16 mg; Cobalt Chloride
(CoCl2.6H20 ) 0.16 mg; Demineralised Water 1.0 1, pH 5.5 prior to autoclaving.
b) The above medium was distributed in 100 ml amount in 500 ml Erlenmeyer flasks
and autoclaved at 121°C for 20 min. The flasks were cooled and then inoculated with the
above mentioned (Example 3) well producing strain (culture no. PM0509732) on the slant
and fermented on shaker at 200 rpm for 120 hrs at 26°C ± 1°C to give the seed. This was
used as the seed culture for inoculating the production with the following composition:
c) Composition of the production medium:
Glucose 30.0 g; Sodium Nitrate (NaNOs) 3.0 g; Dipotassium Hydrogen Phosphate
(K2HP0 4) 1.0 g; Potassium Chloride (KC1) 0.5 g; Magnesium Sulphate (MgS0 4.7H20 ) 0.5 g;
Ferrous Sulphate (FeS0 4.7H20 ) 0.01 g; Demineralized Water 1.0 1, pH 5.5 prior to
autoclaving.
d) The production medium was distributed in 200 ml in each 1000 ml Erlenmeyer
flask and autoclaved at 121°C for 20 min. The flasks were cooled to 25-28°C and then
inoculated with the seed culture (1% v/v) obtained in step (b).
e) Fermentation parameters
Temperature 26-30°C; agitation 190-210 rpm; harvest time 66 -96 hrs.
The progress of fermentation and production of the compound of formula (1) can be
detected by high performance liquid chromatography (HPLC) and by measuring the
bioactivity of the culture broth by testing against the cancer cell lines.
Example 5
Isolation and characterization of compound of formula (1)
20.0 1 of whole broth was filtered to separate biomass and culture filtrate. The
biomass (1.0 kg) was sequentially extracted using 7.0 1 acetone followed by 2.0 1 of
methanol, and filtered and concentrated. The residue obtained was suspended in water (1.0 1)
and, extracted using petroleum ether followed by chloroform. Organic layer was concentrated
under reduced pressure to get crude extract.
The crude extract was subjected to silica gel column chromatography. The active
compound was eluted using step gradient mode with chloroform and methanol mixture. The
final purification was performed on preparative RP-18 silica gel column chromatography.
Preparative HPLC conditions:
Column : Eurospher RP-18 (250 mm X 20 mm), 10 m
Flow rate : 20 ml / min
Detection : UV 220 nm
Solvent system : An isocratic run of acetonitrile and water (40:60) for
16 min, followed by additional 4 min gradient of 40 - 100 % acetonitrile in water.
The active compounds were eluted between 11 - 16 min and the solvents evaporated
to obtain a white powder which was analyzed by analytical HPLC.
Analytical HPLC conditions:
Column : Lichrospher RP-18 (125 mm X 4.6 mm), 5 m
Flow rate : 0.9 ml/min
Solvent system : Acetonitrile and water (45:55)
Detection : UV 220 nm
Retention time : 8.01 min (Tl) & 9.15 min (T2).
The sample contains two compounds Tl & T2, and it has been observed that even
after separation, these compounds exist as a mixture. This was confirmed by analytical
HPLC. The peaks Tl and T2 were collected separately as two fractions and dried under
vacuum and reinjected. Both the fractions eluted again as two peaks with retention time 8.01
(Tl) and 9.15 (T2). This was further indicated by LCMS and UV data of the compounds.
Culture filtrate (18 1) separated from whole broth was passed through a column of HP-
20 resin. The column was washed with water and eluted using methanol (2 1). Active
methanol eluate was concentrated to obtain crude extract. The extract was suspended in
water. The aqueous suspension was extracted using pet ether followed by chloroform.
Organic layer was concentrated and crude extract was obtained, which contain compounds
Tl and T2 (analysed by HPLC).
Physical and Spectral properties:
Appearance White powder
Solubility Soluble in pyridine, acetic acid, dimethyl sulfoxide;
sparingly soluble in chloroform and methanol; and
insoluble in water
UV Detected by HPLC - PDA in acetonitrile and water at
220 nm for both the peaks
Mass ESI LCMS: Tl 418. 9 ( M+H), T2 418.9 (M+H)
ESI HRMS: 419.1640 (M+H), 441.1466 (M+Na)
Molecular formula C18H30N2O5S2
Elemental analysis Obtained: C 51.80 , H 6.71 , N 6.46 % and S
15.33 ;
Calculated for Ci H oN O S : C 51.67 , H 7.17 ,
N 6.69 % and S 15.31 %
IR (KBr) 3345, 1646, 1671, 1715, 1459, 1399 cm 1
H NMR d 8.8 (bs, 1H), 8.1 (bs, 1H), 4.9 (bs, 1H), 4.8 (bs, 1H),
(500 MHz, DMSO-d ) 4.5 (bs, 1H), 4.0 (s, 1H), 3.9 (s, 1H), 3.6 (m, 2H), 3.2
(m, 1H), 3.1 (d, 1H), 1.6-1.0 (m, 16H, methylene
groups), 0.8 (t, 3H) (as given in Figure 1)
(bs refers to broad singlet)
" C NMR d 174.17, 173.91, 169.42, 74.50, 70.30, 52.07, 51.26,
(75 MHz, DMSO-de) 43.25, 42.10, 36.20, 35.60, 34.30, 32.90, 27.90, 26.70,
22.00, 18.30 and 13.70
Biological evaluation of the compound of formula (1):
Example 6
In-vitro assays
Monolayer assay
Step 1
Maintenance of the cell lines
Oncotest GmbH, Germany's proprietary tumor cell line panel comprises 40 cell lines.
These cell lines were derived from 15 different tumor histotypes, each represented by 1 to 6
different cell lines. Cell lines were established from cancer of the bladder (3), colon (4), head
and neck (1), lung (6), breast (3), pancreas (3), prostate (4), ovary (2), kidney (3), liver (1),
stomach (1) and the uteri body (1), as well as from melanoma (3), sarcoma (2) and
pleuramesothelioma (3). Out of these 40 cell lines, 24 cell lines were established at Oncotest,
from patient-derived tumor xenografts. The other 16 cell lines were either provided by the
NCI (Bethesda; MD, USA) or were purchased from ATCC (Rockville, MD, USA) or DSMZ
(Braunschweig, Germany).
Cell lines were routinely passaged once or twice weekly and maintained in culture for
upto 20 passages. All the cells were grown at 37 °C in a humidified atmosphere with 5 %
(Carbon Dioxide) CO2 in RPMI 1640 medium supplemented with 10 % (v/v) fetal calf serum
(FCS) and 0.1 mg/ml gentamicin (medium and all other components from PAA, Colbe,
Germany). Authenticity of all cell lines was proven by STR (short tandem repeat) analysis, a
PCR based DNA-fingerprinting methodology.
Step 2
Sample preparation
Stock solutions of the compound of formula (1) (3.3mM in DMSO) were prepared
and stored in small aliquots (200 mΐ ) at -20 °C. An aliquot of the stock solution was thawed
on the day of use and stored at room temperature prior to and during dosing. The subsequent
dilutions were done with complete RPMI 1640 cell culture medium as follows: the stock
solution was diluted 1:22 followed by serial dilutions finally resulting in a (compared to the
test concentration) 15-fold higher concentration. For the final dilution step (1:15), 10 mΐ / well
of the solution was directly added to 140 mΐ/ well culture medium. Compound of formula (1)
[also referred as test compound] was tested at 0.00032, 0.001, 0.0032, 0.01, 0.032, 0.1, 0.32,
1, 3.2 and 10 mM. Each concentration was evaluated in triplicate.
Step 3
Assay
A modified Propidium Iodide assay was used to assess the effect of the compound of
formula (1) on the growth of the human tumor cell lines and was designed as in reference,
Anti-cancer Drugs, 6, 522-532, (1995), the disclosure of which is incorporated by reference
for the teaching of the assay.
Cells were plated in 96-well flat-bottom microtiter plates at a cell density of 4,000 to
20,000 cells/well. After a 24 hr recovery period to allow the cells to resume exponential
growth, the compound of formula (1) was applied at 10 concentrations in half-log increments
in triplicates and treatment continued for 4 days. After 4 days of treatment, cells were next
washed with 200 mΐ phosphate buffer solution (PBS) to remove dead cells, then 200 mΐ of a
solution containing 7 mg/ml propidium iodide (PI) and 0.1%(v/v) Triton X-100 were added to
the wells. After an incubation period of 1-2 hours at room temperature, fluorescence (FU)
was measured using the Cytofluor 4000 microplate reader (excitation l= 530 nm, emission
l= 620 nm) to quantify the amount of attached viable cells. Pharmacological effects on cell
proliferation and survival were expressed as Test/Control x 100 (%T/C) values, with T and C
representing the fluorescence read-outs for wells with and without test compound,
respectively, i . e. T/C represents the ratio of viable cell numbers following incubation of cell
line with and without test compound. Based on the T/C values, relative IC50 values were
determined by non-linear regression (log[conc. of inhibitor] versus response ( T/C)) using
the GraphPad Prism® analysis software (Prism 5 for Windows, version 5.01, GraphPad
Software Inc., CA). For calculation of mean IC50 values over the 40 cell lines as tested, the
geometric mean was selected. If Graph Pad Prism could not calculate reliable IC50 values by
non-linear regression, the IC50 value was estimated by visual inspection of the concentrationeffect
curve.
For mode-of-action analysis (MoAs), a Compare Analysis was performed. The
individual IC50 values of the test compound as obtained in 40 cell lines in the monolayer
assay were correlated by a Spearman rank test to the corresponding IC50 values for 94
standard agents as determined for these 40 cell lines. These standard agents represent the
main MoAs of approved and experimental anti-cancer drugs. Similarities between the activity
pattern of a test compound and those of standard drugs are expressed quantitatively as
Spearman correlation coefficients. High correlations (p>0.6, p<0.05) between the activity
patterns of two compounds are indicative of a similar MoAs.
In vitro antitumor activity of compounds in a panel of 40 human tumor cell lines:
Compound of formula (1) showed concentration-dependent activity in all cell lines as
tested, i.e. cell lines derived from bladder, colon, gastric, head & neck, liver, lung (NSCLC),
mammary, ovarian, pancreatic, prostate, renal and uterus cancer, as well as melanoma,
pleuramesothelioma and sarcoma (Table 1). For all the 40 cell lines an ICso<0.33 mM was
achieved. The overall very strong antitumor potency was evident from a mean IC50 value of
0.089 mM. The compound displayed a remarkable level of tumor selectivity (Table 2). Cell
lines derived from bladder (2 out of 3 tested bladder cancer cell lines showed above-average
sensitivity), colon (3/4), lung (5/6) and pancreatic cancer (3/3) were particularly sensitive.
Mainly the strong selective activity towards the 5/6 cell lines derived from lung carcinoma
(individual IC50 £ 0.05 mM) and towards the 3/3 pancreatic tumor cell lines (individual
IC5o < 0.06 mM) is to be highlighted. The most sensitive cell lines towards compound of
formula (1) were found to be the pancreatic cancer cell line PAXF 546L (IC50=0.016 mM)
and the lung cancer cell line LXFA 526L (IC5o=0.021 mM) . Compare Analysis revealed no
significant correlations to any of the reference compounds (Spearman correlation coefficient
p<0.4) indicating that the mode-of-action of compound of formula (1) is not represented by
the standard agents used for Compare Analysis. The results are indicated in Table 1 and
Table 2.
Table 1: In vitro anti-tumor activity of compound of formula (1) in a panel of 40 human
tumor cell lines
Sr Cell line Histotype ICso
No. [mM]
1 BXF 1218L Bladder 0.046
2 BXF 1352L Bladder 0.103
3 BXF T 24 Bladder 0.050
4 CXF 269L Colon 0.055
5 CXF HCT 116 Colon 0.048
6 CXF HT 29 Colon 0.137
7 CXF RKO Colon 0.074
8 GXF 251 L Gastric 0.093
9 HNXF CAL 27 Head and Neck 0.039
10 LIXF 575L Liver 0.295
1 1 LXFA 289L Lung 0.041
12 LXFA 526L Lung 0.021
13 LXFA 629L Lung 0.040
14 LXFL 1121L Lung 0.047
15 LXFL 529L Lung 0.050
16 LXFL H 460 Lung 0.146
17 MAXF 401NL Mammary 0.064
18 MAXF MCF 7 Mammary 0.276
19 MAXF MDA 231 Mammary 0.090
20 MEXF 1341L Melanoma 0.102
2 1 MEXF 276L Melanoma 0.090
22 MEXF 462NL Melanoma 0.149
23 OVXF 899L Ovarian 0.301
24 OVXF OVCAR3 Ovarian 0.195
25 PAXF 1657L Pancreas 0.039
26 PAXF 546L Pancreas 0.016
27 PAXF PANC 1 Pancreas 0.059
28 PRXF 22RV1 Prostate 0.159
29 PRXF DU 145 Prostate 0.052
30 PRXF LNCAP Prostate 0.122
3 1 PRXF PC3M Prostate 0.047
32 PXF 1118L Pleuramesothelioma 0.054
33 PXF 1752L Pleuramesothelioma 0.187
34 PXF 698L Pleuramesothelioma 0.178
35 RXF 1781L Renal 0.260
36 RXF 393NL Renal 0.323
37 RXF 486L Renal 0.159
38 SXF SAOS2 Sarcoma 0.185
39 SXF TE671 Sarcoma 0.082
40 UXF 1138L Uterus 0.126
Mean 0.089
Table 2 : In vitro anti-tumor activity of compound of formula (1) towards cell lines
derived from selected tumor histotypes.
Number of cell lines among the respective histotype with individual IC50