The present invention relates to an improved process for the preparation of Caspofungin acetate of a Compound of Formula (II) and Pneumocandin BO of a Compound of Formula (I)3 which is a key intermediate in the synthesis of Caspofungin acetate of a Compound of Formula (II).

BACKGROUND OF THE INVENTION
Caspoftingin acetate (II) is chemically known as 1-[(4R, 5S)-5-[(2-aminoethyl) amino]-N2-(l0)l2-dimethyl-1oxotetradecyl)-4-hydroxy-L-omithine]-5-[(3R)-3-hydroxy-L-ornithine] Pneumocandin BO diacetate salt. Caspofungin acetate is an echinocandin antifungal indicated in adults and pediatric patients for empirical therapy for presumed fungal infections in febrile, neutropenic patients. Caspofungin acetate can be used as the peptide antibiotics for treating invasive aspergillosis, candidosis in esophagus, intra-abdominal abscess, pleurisy, abdominal infection caused by Candida spp, and the fever caused by unidentified pathogens in neutropenia patient. Caspofungin acetate is marketed under the brand name Cancidas®.
Pneumocandin BO of a Compound of Formula (I), is a natural product produced by fermentation or cultivation of the microorganism Glarea lozoyensis or Zahrion arboricola. It is also concerned with methods for enhanced production of the compound. The compound is useful as an antifungal agent and as an antipneumocystis agent. This compound can be used as the key intermediate in the synthesis of approved product Caspofungin acetate of a Compound of Formula (II).
US 5202309 discloses Pneumocandin BO may be produced on cultivation of Zalerion arboricola under aerobic conditions. This patent also discloses methods for producing compositions enriched in the compound of formula (I). This patent discloses purification of Pneumocandin BO using silica gel chromatography wherein methanol as eluent.
Silica gel chromatography exploits the subtle variations in binding affinity of the hydroxy-rich cyclic hexapeplide core of the desired product and the analog impurities to effect a separation.
US 5336756 discloses a process for the preparation of a compound of formula (1) in a crystalline form using aqueous propanol is employed as the crystallizing agent.

In carrying out the crystallization, the compound of formula (I) is dissolved in aqueous n-propanol, employing an amount of from about 25 to 200 milligrams of the compound of formula (I) per milliliter of n-propanol/water. The dissolution is carried out with warming, preferably in the temperature range of about 55°C to 65°C.
US 6610822 discloses a process for purifying a natural product Pneumocandin
BO. after initial isolation by whole broth extraction using a two-phase, multi-solvent
system. Recovery of the product from the back extraction step involves additional :
vacuum concentration and a two-stage precipitation step. Typically three or more solvents are employed since the multi-solvent system can impact the relative polarity balance of the solution in which the compound is being purified.
The above reactions requires higher amounts of solvents and also gives low yields.
US 7241866 discloses a method of purifying a lipopeptide including Pneumocandin BO, by using a mobile phase modifier in a normal phase chromatography system to improve the selectivity and/or productivity of the purification, wherein the mobile phase modifier is selected from a group consisting of an amino acid and an amino acid ester, the normal phase chromatography system includes a mobile phase and a stationary phase, the mobile phase is a solvent system like water, methanol or ethyl acetate, and the stationary phase is selected from silica gel and alumina.
CN 101659693 B discloses purification of Pneumocandin BO using HP-20 resin, wherein methanol or n-butanol as eluent.
CN 102295686 discloses purification of Pneumocandin B0 using HP-20 resin, wherein acetone as eluent.

CN 107778357 discloses purification of Pneumocandin BO using resins like skp-10-4300, D101, HPD300, HPD700, BS-5.
The above purification processes requires higher amounts of solvents and also gives low yields.
US 5936062 discloses two different processes for the preparation of Caspofungin acetate, which are shown below:

The major disadvantages for the above processes, the reaction mass turns hazy after initial formation of product and reaction stops then resulting in lower conversion of product.
Journal of Organic Chemistry 2007; 72(7), 2335-2343 also discloses another process for the preparation of Caspofungin acetate, which is shown below:

This process which uses boronic acid with electron releasing substituents like phenyl gives low conversion.
Considering the importance of Caspofungin acetate (II) and Pneumocandin BO (1) in the pharmaceutical field, there is always a need for an alternative preparative routes, which for example, involve fewer steps, use reagents that are less expensive and/or easier to handle, consume smaller amounts of reagents: provide a higher yield of product, have smaller and/or more eco-friendly waste products, and/or provide a product of higher purity.
OBJECTIVE OF THE INVENTION
The main objective of the present invention is to provide a simple and cost effective process for the preparation of Caspofungin acetate (II) and Pneumocandin BO (I); with high purity and good yield on commercial scale.

SUMMARY OF THE INVENTION
In one embodiment, a method of multi-stage fermentation processes using Bleed feed technique, wherein at least one of the unit processes comprises: Growing the mutant strain of Glarea lozoyensis producing Pneumocandin BO (I) compound in a seed media, transferring the grown seed into the fermentation medium in a bioreactor till the desired titer of Pneumocandin BO (1) is reached and then partial harvesting of the culture broth to separate cells and spent media, followed by to charge the bioreactor containing the left over culture with fresh fermentation medium to initiate production of Pneumocandin BO (I).
In another embodiment, the present invention provides a method of extraction of Pneumocandin BO (1) from mutant strain of Glarea lozoyensis and purification of Pneumocandin BO (I) using HP-20 resin, eluted with tetrahydrofuran.

In another embodiment, the present invention provides, an improved process for the preparation of Caspofungin acetate (II), comprises:
(a) treating Pneumocandin BO aryl sulfide (III) with 4-halophenyl boronic acid of a Compound of Formula (IV);
followed by reduction to produce Pneumocandin BO amine aryl sulfide (V);

(b) optionally, Pneumocandin BO amine aryl sulfide (V) is stereo selectively converted to Caspofungin acetate (II) by the displacement of the arylthio group.
DETAILED DESCRIPTION OF THE INVENTION
In an embodiment, a method of multi-stage fermentation processes using Bleed feed technique, wherein at least one of the unit processes comprises:
Growing the mutant strain of Glared lozoyensis producing Pneumocandin BO (I) compound using the fermentation medium in a bioreactor till the desired tiler of Pneumocandin BO (I) is reached and then partial harvesting of the culture broth to separate cells and spent media. To charge the bioreactor containing the left over culture with fresh fermentation medium to initiate production of Pneumocandin BO (1).
The production of the Pneumocandin BO (I) of the present invention may be carried out by one of several methods.
Although the production of the Pneumocandin BO (I) are discussed herein below principally with respect to a specific strain, all strains of the genus Glarea lozoyensis and mutants are contemplated within the scope of this invention.

However, one particular mutant AZM-10 of Glarea lozoyensis is particularly useful in the production of Compound I. The use of this mutant in the production of Compound I, hereinafter described, is more fully described, the teachings of which are incorporated by reference.
Pneumocandin BO (I) may be produced by cultivating Glarea lozoyensis in a suitable nutrient medium under conditions hereinafter described and thereafter recovering from the cell mass by extracting the active component from the cell mass obtained with a suitable solvent, concentrating the solution containing the desired component, and then subjecting the concentrated material to chromatographic separation to separate Pneumocandin BO (I) from other metabolites obtained during extraction.
The fermentation medium may be an otherwise conventional medium. Thus, nitrogen sources such as corn steep powder, soya flour, soya peptone, yeast extract etc.. there may be in addition to the complex nitrogen sources amino acids such as L-glycine, L-proline etc.,. Generally, the sources of carbon include mannitol, lactic acid, maltodextrin, glycerol, starch and other carbohydrates, or carbohydrate derivatives such as dextran, cerelose, as well as complex nutrients such as oat flour, corn meal, millet, corn and the like. The exact quantity of the carbon source which is utilized in the medium will depend, in part, upon the other ingredients in the medium, but it is usually found that an amount of carbohydrate between 0.5 and 40 percent by weight of the medium is satisfactory- These carbon sources can be used individually or several such carbon sources may be combined in the same medium.
Nutrient medium includes inorganic salts, which can be incorporated in the culture media are the customary salts capable of yielding sodium, potassium, magnesium, calcium, phosphate, sulfate, chloride, carbonate, ammonium and like ions. Also included are trace metals such as cobalt, manganese, iron, molybdenum, zinc, cadmium, and the like.

I
I
Thus as sources of nitrogen there may be employed ammonium salts, amino acids such as glycine, arginine, threonine, methionine and the like as well as complex nutrients such as yeast hydrolysates, yeast autolysates, inactivated yeast cells, soybean meal, casein hydrolysates, yeast extracts, corn steep liquor, distillers solubles, cottonseed meal, meat extract, and the like. The various sources of nitrogen can be used alone or in combination in amounts ranging from 0.2 to 10 percent by weight of the medium.
As sources of carbon, materials previously detailed may be employed, although compositions containing lactic acid or mannitol or maltodextrin are preferred in combinations.

SAG 471 is an antifoam and fermentation is carried out by maintaining the '
temperature at 24±2°C, airflow at 0.5 VVM to 1.5 VVM, backpressure at 0.5 bar, agitation 200-1000 RPM, pH at 5.4±0.2 and DO level at 0-100%5 till the desired titer of Pneumocandin B0 is achieved.
Bleed feed is performed by taking out a part of the grown broth and replacing similar amounts of the taken out broth with the freshly prepared sterile fermentation medium. The fermentation is carried out as per the earlier mentioned processes.
Moreover, it has been discovered that by use of certain mannitol containing media, the production of the desired product may be enhanced. The improved production may be manifest in the increased yield of Compound I.
The inventions are as a result of screening the mutant strains of Glarea lozoyensis AZM-10 using different media while extracting the Pneumocandin BO highly selectively.

The microorganism used for the present invention is Glarea lozoyensis AZM-10, synthesizing the Pneumocandin BO in the fungal body. Although, the strain may be mutated by an artificial variation means to use ultraviolet rays, y-rays, or known chemical mutagens, even if it is such a variant, as long as it has the capability to synthesize the Pneumocandin BO, which are made into the object of the present invention.
In another embodiment, the present invention, the fermentation reaction can be carried out in an aqueous medium over wide ranges of pH and temperature. To dissolve the substrate, a polar solvent such as water, methanol, ethanol, acetonitrile or tetrahydrofuran may be employed at a ratio which will not affect the recovery. The reaction may be carried out at a pH of about 5.0 to 8.5, but typically at about 6.0 to 7.5. The pH can be controlled by the addition of alkali or acid. The pH can also be controlled internally by using suitable buffer.
In still another embodiment, the buffer solution, includes phosphate buffer, tris buffer, phosphate-buffered saline, acetic acid buffer solution, etc.
At the end of this period, cells are separated from the fermentation broth using a filter press. The supernatant is discarded and the cells are washed with water to remove water soluble components. The cells are mixed with methanol and product is extracted into the methanol. Other suitable solvents include ethyl acetate and acetone. The solvent is then removed under reduced pressure to obtain a residue which may be solubilized with a known volume of methanol, dried and assayed by HPLC. and the remaining material subjected to isolation procedures.
The present invention Bleed feed advantages:
Avoids batch to batch cleaning step which further reduces the process time (40-50%), down time and also cost of utility.
Target yield can achieved by half of the original process time using less feed than the original feed volume.

Number of unit operations and also Man-hours are reduced as there is no need of generating fresh seed for bleed feed process.
In one embodiment, the present invention provides a method of extraction of
Pneumocandin BO (I) from mutant strain of Glarea lozoyensis and purification of
Pneumocandin BO (I) using HP-20 resin, eluted with tetrahydrofuran. •
The methanol product solution is concentrated using a membrane, then short
path distillation. The crude residue is purified using HP20 resin, eluted with
tetrahydrofuran, which is subsequently removed by evaporator. The crude product is
then slurry washed sequentially with di-isopropyl ether, methyl t-butyl ether, ethyl
acetate and acetonitrile solvents. The afford Pneumocandin BO crude for subsequent
purification by prep HPLC. i
The reaction is usually carried out at low temperature or room temperature. The temperature is preferably 5 to 30°C, and more preferably 25 to 30°C for a time period of 1 -5 hours.
The obtained Pneumocandin BO is a key intermediate for the preparation of Caspofungin acetate (II).
In another embodiment, the present invention provides, an improved process for the preparation of Caspofungin acetate (II), comprises;

(a) reacting Pneumocandin BO (I) with aryl thiol (Ar-SH) to produce Pneumocandin BO aryl sulfide (III);
(b) reacting Pneumocandin BO aryl sulfide (III) with 4-halophenyl boronic acid of a Compound of Formula (IV);
followed by reduction to produce Pneumocandin BO amine aryl sulfide (V);
(c) Pneumocandin BO amine aryl sulfide (V) is stereo selectively converted to Caspofungin acetate (II) by the displacement of the aryl ihio group.

In another embodiment, aryl comprises phenyl and/or substituted phenyl group and Aryl thiol selected from phenylthiol.
In another embodiment, step (a) is carried out in the presence of solvent comprises polar protic' solvent or polar aprotic solvent or non-polar solvent and/or mixtures thereof.
In another embodiment, polar protic solvent comprises water, methanol, ethanol, isopropyl alcohol, n-butanol, and/or mixtures thereof; polar aprotic solvent comprises dimethylformamide (DMF), dimethylsulfoxide (DMSO), tetrahydrofuran (THF), acetonitrile, acetone, ethyl acetate, N-methylpyrrolidoneand/or mixtures thereof; and non-polar solvents comprises hexane, benzene, toluene, 1,4-dioxane, chloroform, diethyl ether, methylene chloride (CH2CI2) and/or mixtures thereof.
In still another embodiment, preferably the solvent is acetonitrile.
In still another embodiment, after completion of the reaction slowly added an acid selected from acetic acid or trifluoro acetic acid.
In one more embodiment, the reaction is carried out at room temperature depending upon the solvent used in the reaction for a time period of 2-15 hours.
In still another embodiment, after completion of the reaction cooling die reaction mixture to 0°C, and added water and acetonitrile for isolation.
In another embodiment, in step (b) halogen is selected from chloro, fluoro. bromo or iodo, preferable chloro and 4-halophenylboronic acid is selected from 4-chlorophenyl boronicacid.
After treating Pneumocandin BO aryl sulfide (III) with 4-chlorophenyl boronic acid; the reaction was charged with Bis(trimethyl silyl) trifluoro acetamide (BSTFA) and then followed by reduction and hydrolysis.

In another embodiment, reduction is carried out using a borane complex comprises borane with tetrahydrofuran (THF), dimethylsulfide, diphenylsulfide, dibenzylsulfide, 1,4-oxathiane or BH2 CI with dimethylsuifide or a metal boride comprises ZrCU /NaBHj or TiCl^ /NaBH4 in THF or other suitable solvent. The reduction may also be carried out using borane complexes with ammonia, dimethylamine, pyridine or piperazine.
Preferred reduction agents include the borane complexes with tetrahydrofuran (THF). Any amide unconverted by this reduction is separated using reverse phase chromatography. Following the reduction, Step 2 also involves the removal of the arylboronate groups during workup with aqueous acid.
The advantage of using chlorophenylboronic acid is having electron withdrawing substituent is more reactive towards reaction with dihydroxy groups. This lead to strong dihydroxy protection and increased conversion.
Finally, Step (c) involves the displacement of the phenylthio group. The Pneumocandin BO amine phenyl sulfide (V) is reacted in neat ethylenediamine (1:3) at ambient temperature to provide Caspofungin acetate (II). The reaction may take place at a temperature of about 10° C. to about 40° C. for about 0.5 to about 12 hours. Preferably the reaction takes place at room temperature for about 12-15 hours. The reaction can also be conducted using ethylenediamine dissolved in a suitable solvent such as water, methanol; ethanol, isopropanol, tetrahydrofuran. trifluoroethanol, dichloroethane or acetonitrile.
The following examples illustrate the nature of the invention and are provided for illustrative purposes only and should not be construed to limit the scope of the invention.
EXAMPLES:
EXAMPLE 1: PRODUCTION OF PNEUMOCANDIN BO FROM GLAREA LOZOYENSfS AZM-10:

A seed culture (seed stage-1) from the master cell bank of Glarea Lozoyensis AZM-10 was inoculated into 50 ml of seed media containing sucrose (lOOg/L), yeast extract (5g/L), soya flour (40g/L), soya peptone (lOg/L), magnesium sulphate hepta hydrate (lg/L), ammonium sulphate (5g/L) and SAG 471 (lg/L) the seed flask was incubated at 24°C at 200 RPM, on reaching 30% WCW and confirming the culture purity, similar seed flask (seed stage-2) with 250 ml of seed media was inoculated with seed stage 1 culture and incubated at the earlier mentioned conditions.
Production fermenter was prepared with mannitol (100 g/L), potassium dihydrogen phosphate (2.5g/L), lactic acid (5.6g/L), corn steep powder (1.4g/L), soya flour (14.4g/L), maltodextrin (7.5g/L) and SAG 471 lg/L was used to prepare a production fermenter with 12 L of the above mentioned media. The seed stage -2 on reaching a WCW of 30% and confirming the culture purity was inoculated into the production media containing fermenter.
The initial parameters set were Temperature: 24 °C, pH: 5.4, Agitation: 125 RPM, Airflow: 15 LPM and Back pressure: 0.3 bar. However, based on the DO requirements i.e., to maintain 30% DO levels, the airflow, agitation and back pressure were set to vary up to 1000 rpm, 40 LPM and 0.8 bar respectively. Nitrogen feed (2L) comprising of glycine (12.8 g/L), L-proline (96 g/L), soya peptone (90 g/L), magnesium sulphate hepta hydrate (40 g/L) and SAG 471 (0.8g/L) was dosed at a rate of 50 ml every 4 h after 24 h after inoculation till 120 h and post 120 h, 100 ml of nitrogen feed was added till feed exhaustion.
Carbon feed (as per requirement) comprised of mannitol (250g/L). Residual mannitol content was estimated every 24 h after 72 h after inoculation. Required amounts of mannitol was dosed to maintain the residual mannitol content at 30±10g/L. A sample was withdrawn every day since inoculation to estimate the Pneumocandin B0 content. 11 L i.e.. approximately 85±5% of the batch was withdrawn in a sterile manner after 497h (Pneumocandin B0 litre of 1470 mg/L. 10 L of sterile earlier mentioned production media was transferred to the fermenter. Feeding of nitrogen and

carbon feeds were as described earlier and the fermentation was continued for 206 h before complete harvest of the broth (Pneumocandin BO titre of 1227 mg/L).
EXAMPLE 2: PREPARATION OF CRUDE PNEUMOCAND1N BO:
Cells (21.5kg) are separated from the fermentation broth (860L) using a filter press. The supernatant is discarded and the cells are washed with purified water (2400L) to remove water soluble components. The cells are lysed in methanol (1680L) and product is extracted into the methanol.
The methanol product solution is concentrated using a membrane, then short path distillation. The crude residue taken in tetrahedron /water and it is purified using HP20 resin (40kg). product adsorbed on HP20, impurities removed using 10% water in acetone (240L) finally eluted with tetrahydrofuran (THF) (100L), which is subsequently removed by evaporator. The crude product is then slurry washed sequentially with di-isopropyl ether (15L), ethyl acetate (15L) solvent. The obtained Pneumocandin BO crude is subjected for subsequent purification by prep HPLC.
EXAMPLE 3: PURIFICATION OF CRUDE PNEUMOCANDIN BO:
40 g of Pneumocandin Bo crude (Assay 40%) was dissolved in 4 L of ethyl acetate: methanol: water (86:9:7) The solution was purified by preparative HPLC using Silica -100 A-lOu stationary phase (column dimension 8 X 2.5 cm) and ethyl acetate: methanol: water (86:9:7) as mobile phase. Fractions containing Pneumocandin Bo with HPLC purity more than 85% were combined and concentrated to 200 mL at 40°C under reduced pressure resulting precipitation. The suspension was stirred for lh and filtered. The product was dried under vacuum at 40°C to afford pure Pneumocandin Bo (13 g).
EXAMPLE 4: PREPARATION OF PNEUMOCANDIN B0 PHENYL SULFIDE:
A solution of Pneumocandin Bo (5.0 g) was added to acelonolrile (160 mL). cooled to -10 lo-5 °C and thiophenol was added (0.97 g) drop wise over a period of 30 min. Trifluoroacelic acid (40 mL) was added slowly drop wise over a period of 30 min at -10 to 0 °C. The reaction mixture was stirred at -10 to 0 °C for 4-8 h. After

completion of the reaction, saturated sodium carbonate solution (180 mL) was slowly added at 0°C. The resulting suspension was stirred for 30 min at 0°C. Product was filtered and washed with water (50 mL). The wet product was dried under vacuum to afford 5 g (with 82% HPLC purity) of Pneumocandin Bo phenyl thioether.
EXAMPLE 5: PREPARATION OF PNEUMOCANDIN BO AMINE PHENYL SULFIDE:
A suspension of Pneumocandin Bo phenyl thioether (lg) and 4-chiorophenyl boronic acid (0.20 g) in THF (30 mL) was refluxed at 40-45°C under vacuum passing the condensate through a column of molecular sieves. During reflux the reaction mass turns to clear solution. After 16 h. the mass was cooled to 20°C and bis(trimethyl silyl)trifluoro acetamide (0.67 g) was added. The resulting solution was stirred at 20°C. After 90 minutes, the mass was cooled to -10 to 0°C and 1.0 M borane in THF (8.6 mL) was added over a period of 5-10 seconds at -10 to 0°C. The reaction was stirred at -10 to 0°C for 2-6 hours. After completion of the reaction, 6N HC1 (1 mL) at -5° to 0 °C was added and stirred for 2 h at 0°C. The reaction mixture was diluted with DM water (90 mL) and purified through a CI8 silica column chromatography using acetonitrile- water as eluent. Fractions containing product were combined and striped out solvent to obtain the amine compound (0.4 g).
EXAMPLE 6: PREPARATION OF CASPOFUNGIN ACETATE:
A solution of the above amine (1 g) was added to methanol (3.0 mL), cooled to 0 to 10°C and ethylenediamine (3.0 mL) was added drop wise over a period of 30 min at 0 to 10°C. The mixture was warmed to 20-25°C and stirred for 6h. After completion of the reaction the reaction mass was purified by preparative HPLC using C-18 silica column and aq. acetic acid-acetonitrile as mobile phase. The fractions containing pure product were combined and lyophilized to produce Caspofungin acetate (0.6g).

WE CLAIM:
1. An improved process for the preparation of Caspofungin acetate of a Compound of Formula (II), comprises:
(a) treating Pneumocandin BO aryl sulfide of a Compound of Formula (111);
with 4-halophcnyl boronic acid of a Compound of Formula (IV);

followed by reduction to produce Pneumocandin BO amine aryl sulfide of a Compound of Formula (V);
(b) optionally, Pneumocandin BO amine aryl sulfide of a Compound of Formula (V) is stereo selectively converted to Caspofungin acetate of a Compound of Formula (II) by the displacement of the arylthio group.
2. The process as claimed in claim 1, aryl comprises phenyl and/or substituted phenyl group and halogen is selected from chloro, fluoro, bromo or iodo.
3. The process as claimed in claim 1. wherein the reduction in Step (a) is carried out using a borane complex comprises borane complexed with dimethylsulfide, dibenzylsulfide, diphenylsuifide, THF or 1,4-oxathiane or BH2 CI with dimethylsulfide or metal boride comprises ZrCI4 /NaBH4 or TiCI4 /NaBH4 and/or mixtures thereof.
4. The process as claimed in claim 1. wherein the step (b). Pneumocandin BO amine phenyl sulfide of a Compound of Formula (V) is treated with ethylenediamine followed by acetic acid to produce Caspofungin acetate of a Compound of Formula (11).
5. The process as claimed in claim 1, Pneumocandin BO aryl sulfide of a Compound of Formula (111) is produced by reacting Pneumocandin BO of a Compound of Formula (I);

6. A fermentation process of Pneumocandin BO of a Compound of Formula (1),
wherein method of multi-stage fermentation processes using Bleed feed
technique, wherein at least one of the unit processes comprises:
Growing the mutant strain of Glarea lozoyensis producing Pneumocandin BO (1), using the production medium in a bioreactor till the desired titer of Pneumocandin BO (I) is reached, further partial harvesting of the culture broth to separate cells and spent media followed by feeding the left over culture in the fermenter with fresh production media and repeating the partial harvest for a limited number of times to yield high Pneumocandin BO (1) titers.
7. The process as claimed in claim 6, mutant strain is selected from AZM-10 of Glarea lozoyensis and wherein said mutant is cultivated in a Sporulation media.
8. The process as claimed in claim 7. wherein said spore suspension is inoculated in seed medium followed by production medium.
9. The process as claimed in claim 7, wherein said seed medium comprises Sucrose, Soya flour. Soya peptone. Yeast extract. Magnesium sulphate heplahydrate. Ammonium sulphate, SAG 471 and production medium comprises Mannitol, Potassium phosphate Monobasic. Lactic acid, Com steep powder. Soya flour, Maltodextrin, SAG 471.

10. A method of extraction of Pneumocandin BO of a compound of formula (I) from mutant strain of Glarea lozoyensis and purification using HP-20 resin, eluted using tetrahydrofuran.