Title of Invention


Abstract The present invention relates to a process for isolation of 3,l'-didehydro-3 [2" (3'", 3'"- dimethyl - prop - 2 - enyl) - 3"- indolyl methylene]-6 - methylpiperazine-2,5-dione having antibacterial activity extracted from a mangrove associated fungus Penicillium chrysogenum and the gross structure of the compound was elucidated by a detailed analysis of spectroscopic data (IR, NMR, MS), in addition, this invention also assesses the biological activity of the compound which reveals its antibacterial activity against the human pathogen, Vibrio cholerae, demonstrated by the disc diffusion assay.
The present invention relates to the extraction, isolation and identification of a new
compound 3,l'-didehydro-3[2"(3'",3'"-dimethyl-prop-2-enyl)-3"-indolyl
methylene]-6- methylpiperazine-2,5-dione, as shown in Figure 1; containing an indole and a diketopiperazine moiety from a mangrove-associated fungus, Penicillium chrysogenum.
(Figure Removed)
and is designated as chrysogenazine from the specific name of the fungus chrysogenum. This invention also describes the process involved in its isolation and evaluates its antibacterial properties against the human pathogen Vibrio cholerae MCM B-322.
Recent years have seen a growing interest in the discovery of metabolites from associated micro-organisms due to the speculation that a number of metabolites obtained from marine plants and invertebrates may be produced by associated microorganisms.
Penicillium chrysogenum is a known penicillin producer (Ariyo et al, 1998). The antibacterial effect of penicillin was discovered by Alexander Fleming in 1929, which became a "wonder drug" which saved millions of lives. It is still a "front-line" antibiotic, although the development of penicillin-resistance in several pathogenic bacteria now limits its effectiveness. P. Chrysogenum is also 44known to yield hexaketides sorbicillin (Trifonov et al., 1983) and chrysogine, 2-(a-hydroxyethyl -4

(3H) quinazolinone (Bergman and Brynolf, 1990). In lactose containing media it is known to synthesize P-galactosyl oligosaccharides (Ballio and Russi, I960). The increasing incidence of drug resistance in pathogenic microbes as well as the increasing frequency of infectious diseases in immunocompromised individuals necessitates the discovery of new anti-infective agents.
The 2,5-DKP (Diketopiperazine), head-to-tail dipeptide dimers, are a common naturally occurring structural motif. They are known to be frequently generated as unwanted by-products or degradation products in die syntheses of oligopeptides (Dinsmore and Beshore, 2002). Some piperazine derivatives are reported to exhibit activities towards the central nervous systems, such as anti-anxiety activity and anti-convulsive activity, as described in U.S. Patent No. 3,362,956. Piperazine derivatives are also known to possess calmodulin inhibitory activity as reported in Arzneim Forsch., (1987). Some of the compounds with calmodulin inhibitory activity has been revealed to be antihypertensive and vasodilatory in action (U.S. Patent 5,681,954). In view of the above factors, the present invention describes a novel compound, which is a DKP derivative from an associated fungus Penicittium chrysogenum. The present invention also demonstrates its potentials against human pathogen Vibrio chokrae. Natural penicillin obtained from culture filtrates of Penicillium notatum or Penicillium ckrysogenum are penicillin G aad penicillin V. Both these are active against Gram-positive bacteria and not against Gram-negative species. However, our invention has isolated an antibiotic from Penicillium chrysogenum, which is active against Vibrio chokrae which is Gram-negative, rod shaped bacteria causing cholera in humans.
Vibrio chokrae is known to produce cholera toxin, whose action on the mucosal epithelium is responsible for the characteristic diarrhea of the disease cholera. Tetracycline is still the first choice for bacterial infection causing cholera. The emergence of bacterial resistance to tetracycline has limited the use of these agents. In addition, tetracyclines are strong chelating agents. This ability to chelate to metals, such as calcium, results in tooth discoloration when it is administered in children. For the above reasons, chrysogenazine will prove to be a commercially potential alternate source for the above disease in humans.
Since vibrios mostly occur in the surface waters (both marine and fresh water habitats) and are associated with aquatic animals, transmission to humans is by water

or food. Thus cholera can smoulder in an endemic fashion on the subcontinent.
Cholera was reported for the first time in South America (1991), in Peru, the
outbreaks quickly grew to epidemic proportions and spread to other South American
countries, Central American countries, Mexico etc. Out breaks were also reported
from Bangladesh, India etc. Therefore, commercialisation of this drug will have
potential market in all developed and developing countries where cholera epidemic is
a serious problem.
The principal object of the present invention is to isolate a novel compound from the
fermentation broth of Penicillium chrysogenum.
Another object of the present invention is to provide a process for the isolation of the
Yet another object of this invention is to identify the antibacterial activity of the
compound against the human pathogen Vibrio cholerae.
In summary, the present invention provides a process for obtaining substantially pure
and novel chrysogenazine from the fermentation broth of P. chrysogenum as a yellow
solid. This novel compound contains an indole and a diketopiperazine moiety and
shown in Figure 1. In addition, this compound mentioned herein show antibacterial
properties against the human pathogen Vibrio cholerae.
(Figure Removed)

Table 1: NMR data of chrysogenazine (300 MHz, CDC13)
Fig. 1: Structure of chrysogenazine

Fig. 2: H NMR spectrum ofchrysogenazine
Fig. 3:13C NMR spectrum ofchrysogenazine
Fig. 4: IR spectrum of chrysogenazine
Fig. 5: MS data of chrysogenazine
Plate 1: Antibacterial activity of chrysogenazine using simple disc diffusion technique (inhibition
Accordingly, the present invention provides to a 3, 1'-didehydro-3 [2" (3'", 3"'-dimethyl-prop-2enyl)-3"-indolyl methylene]-6-methylpiperazine-2,5-dione having antibacterial activity extracted from a mangrove-associated fungus Penicillium chrysogenum, represented by a general formula
C19H2102N3 and structural formula as shown in Figure 1.

(Formula Removed)

Figure 1 This compound has the NMR assignments as given in Table 1, when recorded in CDCI3 and DMSO.
Table 1:
(Table Removed)

a: Measured in DMSO-d6 *: Exchangeable values
In an embodiment of the present invention the compound has been isolated from a mangrove-associated fungus Penicillium chrysogemm. This fungus was identified from Agharkar Research Institute, Pune, India. The said fungus is known and available in public domain. The specific statin isolated and used in the present invention bears reference number FMB 005. It has also been deposited at Microbial Type Culture Collection & Gene Bank, Institute of Microbial Technology, Sector 39-A, Chandigarh - 160 036 at Accession number MTCC 5108 The organism was obtained from leaves of die mangrove plant Porteresia coarctata (Roxb.). The leaves were collected from Chorao Island along the Mandovi estuary of Goa, India, in sterile polythene bags and transported to die laboratory. In the laboratory, the leaves were rinsed with sterile seawater to remove adherent particles and detritus material. The leaves were next kept in a sterile, moist chamber for 2 weeks to allow the fungus to grow and sporulate. Fungal hyphae were picked and separately subcultured, repeatedly to obtain pure isolate of the culture. The spores of Penicillium chrysogenum are produced in chains from flask-shaped cells, which are found at tips of a brush-like aerial structure. The stalk is called the conidiophore and the spore is called conidium. The spores in Penicillium contain a bluish-green pigment, which gives the culture characteristics bluish-green coloration. In another embodiment, the above culture was initially grown in small Erlenmeyer flask (100 ml) in potato dextrose broth (PDB) prepared in seawater: distilled water (1:1) under shaker conditions. This culture was used as a seed for mass culturing in S litre flasks (4 nos.) containing 1 lit fermentation broth in each flask under stationary conditions. In the present experiment, the fungal strain was cultured at 27-30°C for 15 days. After 15 days, the mycelia were removed by filtration and the broth was separated from the fungal mat.

In yet another embodiment of the present invention, the process for the extraction of
the compound from the fermentation broth is described. Chloroform or ethyl acetate
may be used for extracting the fermentation broth.
In a preferred embodiment, chloroform was used in the present study to extract the
compound of interest. This chloroform filtrate was concentrated under vacuum to
obtain crude chloroform extract (30 mg).
In yet another embodiment of the invention, the isolation of the compound from the
crude chloroform extract is effected by the use of conventional techniques, such as
thin layer chromatography (TLC) and silica gel column chromatography. In a
preferred embodiment the crude chloroform extract was chromatographed over silica
gel first using petroleum ether: ethyl acetate (with gradual increasing percentage of
ethyl acetate) affording fractions yielding impure chrysogenazine.
Further purification of the compound was affected by gel chromatography (Sephadex
LH-20) using chloroform: methanol (1:1), to obtain the pure compound as a yellow
solid (9 mg).
In another embodiment of this invention, antibacterial activity of chrysogenazine was
tested using simple disc diffusion technique (disc containing 5-30 meg/disc of
sample) on agar plated petridishes (Chabbert, 1963; Rinehart et al. 1981). The assay
showed the compound to be active against a human pathogen Vibrio ckolerae.
Degree of sensitivity of chrysogenazine on test organism was determined by
measuring the zone of inhibition in millimetres. In addition, standard discs of
penicillin (lOunits/disc), amphicillin (lOmcg/disc) and streptomycin (10 meg/disc)
were used to compare the sensitivity.
This compound showed an inhibition zone of 4-5 mm, while penicillin showed 0 mm
inhibitions zone, amphicillin showed 0 mm inhibition zone, and streptomycin showed
4-5 mm inhibition zone.
The following examples are given by way of illustrations and should not be construed
to limit the scope of the present invention
The mangrove plant Porteresia coarctata (Roxb.) was collected from Chorao Island
along the Mandovi estuary of Goa. Leaves of the mangrove plant was collected and
transported to the laboratory in sterile polythene bags. In the laboratory the leaves
were rinsed with sterile seawater to remove adhered particles and detritus material.
The leaves were next kept in a moist chamber, using known standard techniques, for 2

weeks, to allow the fungi to grow and sporulate. Repeated subculturing resulted in
pure fungal isolate.
The growth conditions of the fungal isolate was optimised and grown on potato
dextrose agar (PDA) slants (HiMedia Industries Ltd.) and later grown in small
Erlenmeyer flasks (100 ml) in potato dextrose broth prepared in seawater: distilled
(1:1) under shaker conditions. The culture obtained at the end of 4-5 days was used to
seed 5 lit. Erlenmeyer flasks containing 1 lit of the same medium prepared similarly
in replicates of four at room
temperature (28-30°C). The flasks were kept stationary for 15 days. At the end of 15
days fungal mycelia were removed by filtration and fermentation broth was extracted
with chloroform.
The chloroform extract (30 mg), after removal of the solvent in vacuum, was
fractionated through a column of silica gel using petroleum ether: ethyl acetate
mixture. Initially, 200ml of ethyl acetate: petroleum ether in the ratio (1:99%) was
used. This was followed by elution with 200ml of a mixture of ethyl acetate:
petroleum ether (2:98%). The next percentage of ethyl acetate used was 5% and
petroleum ether was 95%. Subsequently, ethyl acetate percentage was increased by
5%. The sub-fractions obtained were spotted on silica gel TLC plates, combined and
concentrated after developing and spraying with cerric sulphate.
The final purification of the compound was obtained by chromatography using
sephadex LH-20 as adsorbent and eluting the compound with chloroform: methanol
(1:1). Approximately, 9 mg of chrysogenazine was purified as a yellow solid.
In determining the structure of the compound, correlation spectroscopy (COSY),
heteronuclear multiple quantum correlation (HMQC), heteronuclear multiple bond
correlations (HMBC), distortionless enhancement by polarization transfer (DEFT),
IH and I3C NMR spectral data were obtained using a Brucker Avance 300
Spectrometer. *H and 13C NMR spectra was recorded at 300 MHz. All the chemical
shifts were recorded using TMS as internal standard, at 5 7.24 for proton resonance
and § 77.0 for the carbon spectra. Mass spectral data (ESI-MS) was obtained on a

Micro Mass spectrometer; IR spectral data was recorded on FTIR-8201 PC, Shimadzu speedometer.
Chrysogenazine has the molecular formula of C19 H21 O2 N3. Its molecular ion (M*) was 323 from (M+ + Na*) and (2M* + Na*) signals at m/z 346 and 669 respectively. A close inspection of the *H and 13C NMR spectra of "1" by DEPT and 1H-13C COSY experiments disclosed signals for 19 carbons: These included one secondary methyl (C-7), two tertiary methyls (C-4"\ C-5'"), one sp3 quarternary carbon (C-3"'), one sp2 hybridized methylene (C-l'"), one sp3 hybridized methine (C-6), six sp2 methines (C-F, C-4", C-5", C-6", C-7" and C-2'") and seven sp2 quarternary carbons including amide carbonyls (C-2, C-3, C-5, C-2", C-3", C-3"a and C-T'a). The presence of two secondary amide groups were inferred from signals at 165.5 and 159.6 ppm from its 13C NMR spectra (CDC13), sharp and strong IR absorptions at 3350 cm"1 and 1676 cm"1, and also from the presence of two D2O exchangeable protons at 5 6.4 and 7.4 (these signals appeared at 5 8.2 and 8.6 respectively in DMSO). The IR absorption at 1676 cm'1 was also indicative of cc-P unsaturated carbonyl functionality. The presence of a third exchangeable proton at SI 1.15 in DMSO spectrum and at § 8.27 in CDCb spectrum along with the pattern of lB NMR signals in DMSO (7.47, 7.21, 7.14, 7.06 and 6.96) was suggestive of a conjugated indole nuecleus, as present in dipodazine, (Sorensen et al., 1999) a metabolite from Peniciltium dipodomis. The only exception observed was that olefinic methine proton signal at 7.93 of the indole nucleus in dipodazine was absent in chrysogenazine indicating that C-2" position was also substituted in the latter.
The I3C NMR spectrum of dipodazine and chrysogenazine (Figure 1) are virtually identical with the following changes. The C-2" carbon at 143 ppm in chrysogenazine is a singlet and has undergone ~17.0 ppm downfield shift appropriate for tertiary alkyl group substitution (Stothers, 1972). Four new signals (27.2, 39, 113 and 1442 ppm) have appeared in chrysogenazine spectrum. The intensity of the signal at S27.2 is suggestive of two similar carbons. These new carbon signals are attributed to an a, a-dimethyl (reversed isopentenyl) substituent which must be attached to the C-2" of the indole moiety. The cross peaks originating form the vtiiyue proton 2J c-3-, H-2- and 3J c-2", H-2" and 2J c-i", H-2>M in HMfiC spectrum confirmed the position and the nature of the isopentenyl substituted (mis substituent may also be taken as 1,1 dimethyl-2-propenyl unit).

Considering the formula, the conjugated moiety, isopentenyl substituent and the presence of two secondary amide groups, it was suggestive of tryp-alanine derived cyclic dipeptide. The cross peaks, in the HMBC spectrum, 3J c-r% u-V, 3J c-r\ H-V and 3J c-2, H-r connected C-l' to die indole and diketopiperazine moieties. HMBC connectivities is also observed with the C-7 secondary methyl and the C-6 methine with the C-5 and C-2 carbonyls of diketopiperazine moiety respectively. All the above data indicated mat chrysogenazine is dipodazine extended by a reversed isopentynyl or 1,1 dimethyl 2-propenyl moiety attached at position 2" of the pyrazole ring of indole moiety, as shown in 1, and dipodazine is tryp-glycine derived cyclic dipeptide whereas chrysogenazine is tryp-alanine derived cyclic depeptide. EXAMPLES
This example demonstrates antibacterial activity of chrysogenazine. Antibacterial activity was determined using a Gram negative bacterial strain, Vibrio cholerae, in a agar diffusion assay, essentially as described by Chabbert, (1963) and Rinehart et.al.,(1981). Briefly, nutrient-containing agar plates were seeded with the selected target microorganisms and the disc (loaded with 5-10 meg/disc of chrysogrnazine) was placed on the surface of the medium. Following an appropriate incubation interval, microbial growth inhibition was visualized and quantified by measuring the clear zone around each disc (Plate 1.)- Comparison of mis was made with the standard antibiotics (penicillin, amphicillin, and streptomycin) Advantages of the present invention
> The process for the extraction and isolation of chrysogenazine is simple and
requires minimum purification steps.
> P. chrysogenum was associated with the mangrove leaves of the plant
Porteresia coarctata, collected from Goa Coast, and is well known to produce
several antibiotics, active against Gram-positive bacteria. However, in the
present invention, chrysogenazine is reported to be active against Gram-
negative bacteria Vibrio cholerae, causing cholera in humans.
> Another advantage is mat the yield of the compound may be enhanced by
modifying the carbon and nitrogen source in the fermentation broth as well by
modifying the laboratory conditions, so as to make it economical/profitable if
found suitable for use against pathogens.

Ariyo B., C. Tamerier, C. Bucke, T. Keshavarz, Enhanced penicillin production by
oligosaccharides from batch culture of Penicllium chrysogenum in the stirred tank
reactors. FEMS Letter Microbiology, 166, (1998), 165-170.
Arzneim Forseh, Vol. 37(4), (1987), 498-502.
Ballio A. and S. Russi, Chromatographic fractionation and chemical characterization
of some oligosaccharides synthesized from lactose by P. chrosogenum.
Bergman J. and A. Brynolf. Synthesis of chrysogine, a metabolite of Penicillium
chrysogenum and some related 2-substituted -4-(3H)-QuinazoHnones. Tetrahedron,
46,(1990), 1295-1310.
Chabbert Y. A. L'antibiogramme coll Technique de base, Ed Touralle, Saint Mande,
(1963), 257.
Dinsmore C.J. and D.C. Beshore. Recent advances in the synthesis of
diketopiperazine. Tetrahedron 58, (2002), 3297-3312.
Rinehart K. L. Jr., P.D. Shaw, L.S. Schield, J.B. Bloer, G.C. arbour, E.S. Koker,
D. Samain, R.E. Schwartz, A.A. Tymiak, D.L. Welier, G.T. Carter, M. H.G. Munro,
R.G. Hughes Jr., H.E. Renis, E.B. Swynenberg, DA. Stringfellow, JJ. Vavra, J.H.
Coats, G.E. Zurenko, S.L. Kuentzel, H.Li, GJ.Bakus, R.C. Brusca, L.L. Craft, D.N.
Young and J.L. Connor. Marine natural products as sources of antiviral,
Antimicrobial and antineoplastic agents. Pure and Applied Chem., 53, (1981), 795-
Sorensen D., T. O. Larsen, C. Christophersen, P. H. Nielsen, U. Aumoni.
Phytochemistry, 51,(1999), 1181-1183.
Stothers J.B. In Carbon-13 NMR Spectroscopy, Academic Press, New York, 1972,
Thykaer J., B. Christensen and J. Nielsen. Metabolic network analysis of an
Adipoyl-7-ADCA-Producing strain of P. chrysogenum. Elucidation of Adipate
Degradation. Metabolic Engineering 4, (2002) 151-158.
Trifonov L.S., J.H. Bieri, R. Prewo and D. L. Hoesch and D.M. Rast. Isolation and
structural elucidation of three metabolites from VertieUlium intertextum, Sorbicittin,
dihydrosorbieillin and bisvertinoquinol. Tetrahedron 39, (1983) 4243-4256.
Whiteman P.A. and E.P. Abraham. Phenoxymethyl penicillin amidohydrolases from
Penicillium chrysogenum FEBS Letters, 394, (1996), 31-33.
Patent No. 3,362,956, Jan 1968, Archer, 260/268
Patent No. 5,681,954, Oct 1997, Yamamoto et al., 544/114.

We claim:
1. A process for isolation of 3,l'-didehydro-3 [2" (3'", 3"'- dimethyl - prop - 2 -
enyl) - 3"- indolyl methylene]-6 - methylpiperazine-2,5-dione having antibacterial
activity extracted from a mangrove associated fungus Penicillium chrysogenum,
said process comprising the steps:
a) growing Penicillium chrysogenum in a fermentation broth comprising potato dextrose agar, sea water and distilled water;
b) extracting the fermentation broth with chloroform to obtain the filtrate;
c) evaporating the filtrate of step (b) to obtain a crude extract;
d) isolating the impure chrysogenazine from the crude extract of step (c) by chromatographic fractionation, such as herein described and
e) purifying the impure chrysogenazine of step (d) using gel chromatography to obtain the pure chrysogenazine.

2. A process as claimed in claim 1, wherein in step (a), seawater and distilled water is mixed in 1:1 ratio.
3. A process as claimed in claim 1, wherein in step (c), the evaporation is performed under vacuum.
4. A process as claimed in claim 1, wherein in step (d), the chromatographic fractionation is performed by column chromatography and thin layer chromatography.
5. A process as claimed in claim 4, wherein silica gel chromatography is used for fractionation.
6. A process as claimed in claim 5, wherein in silica gel chromatography the eluent used is mixture of petroleum ether and ethyl acetate.
7. A process as claimed in claim 6, wherein in the chromatography the adsorbent used is silica gel with a pore size of 60-120A.
8. A process as claimed in claim 1, wherein in step (e), the adsorbent used in gel chromatography is Sephadex LH-20.
9. A process as claimed in claim 1, wherein in step (e), chloroform and methanol mixture is used as an eluent in gel chromatography.
10. A process as claimed in claim 9, wherein the chloroform and methanol are mixed in 1:1 ratio.
11. A process as claimed in claim 1, wherein Penicillium chrysogenum is Penicillium chrysogenum,. bearing accession No. MTCC 5108.







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Patent Number 241482
Indian Patent Application Number 581/DELNP/2004
PG Journal Number 29/2010
Publication Date 16-Jul-2010
Grant Date 07-Jul-2010
Date of Filing 08-Mar-2004
Applicant Address Rafi Marg, New Delhi-110001
# Inventor's Name Inventor's Address
PCT International Classification Number C07D 493/00
PCT International Application Number PCT/IN03/00435
PCT International Filing date 2003-12-31
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 PCT/IN03/00435 2003-12-31 PCT