Process for producing farnesylated dibenzodiazepinone by fermentation

Abstract

The present invention provides a scalable process for producing a concentrate containing a mass of a farnesylated dibenzodiazepinone by fermenting in an aqueous culture medium a strain of a microorganism that is capable of producing the farnesylated dibenzodiazepinone, upon completion of fermentation harvesting the fermentation broth and extracting the fermentation broth to provide an extract, and thereafter treating the extract to form the concentrate. The concentrate so produced may be utilized in downstream processes for producing pharmaceutical compounds. A strain of a Micromonospora species capable of producing a farnesylated dibenzodiazepinone at a high yield rate is provided, together with culture media for culturing microorganisms, and fermentation conditions for production of the farnesylated dibenzodiazepinone of the concentrate.

1 . A process for recovering and concentrating a farnesylated dibenzodiazepinone of structural Formula I  
     
       
         
         
             
             
         
       
       comprising:  
       a) fermenting a strain of a microorganism capable of producing a farnesylated dibenzodiazepinone of structural Formula I in an aqueous culture medium, thereby producing a fermentation broth comprising said farnesylated dibenzodiazepinone;  
       b) adjusting said fermentation broth to allow said farnesylated dibenzodiazepinone to associate with a particulate matter present in said broth;  
       c) harvesting said particulate matter from said broth, thereby obtaining a harvested particulate matter;  
       d) extracting said harvested particulate matter with a volume of a suitable organic solvent in a proportion of about 2:1 to about 5:1 to said harvested particulate matter, thereby forming an extract; and  
       e) treating said extract to form a first concentrate comprising said farnesylated dibenzodiazepinone of Formula I, wherein said first concentrate comprises said farnesylated dibenzodiazepinone at a concentration greater than about 50-fold than said farnesylated dibenzodiazepinone in said fermentation broth of a), and wherein said farnesylated dibenzodiazepinone is recovered from said fermentation broth of a) in an amount of at least about 50% of the amount of said farnesylated dibenzodiazepinone in said fermentation broth.  
     
   
   
       2 . The process of  claim 1 , wherein said farnesylated dibenzodiazepinone is recovered from said fermentation broth of a) in an amount of at least about 60% of the amount of said farnesylated dibenzodiazepinone in said fermentation broth.  
   
   
       3 . The process of  claim 1 , wherein said farnesylated dibenzodiazepinone is recovered from said fermentation broth of a) in an amount of at least about 65% of the amount of said farnesylated dibenzodiazepinone in said fermentation broth.  
   
   
       4 . The process of  claim 1 , wherein the particulate matter comprises the microorganism present in the fermentation broth.  
   
   
       5 . The process of  claim 4 , wherein the particulate matter further comprises an adsorbent resin A.  
   
   
       6 . The process of  claim 4  or  5 , wherein the fermentation broth is adjusted in b) to a pH value of about 2 to about 4.  
   
   
       7 . The process of  claim 6 , wherein the broth is further adjusted to a temperature range of about 2° C. to about 10° C.  
   
   
       8 . The process of  claim 7 , wherein the broth is maintained at the temperature range for a period of about 16 hours to about 72 hours.  
   
   
       9 . The process of  claim 1 , wherein the fermentation is completed in from about 48 hours to about 110 hours.  
   
   
       10 . The process of  claim 1 , wherein the microorganism is a bacterium.  
   
   
       11 . The process of  claim 10 , wherein the bacterium is an Actinomycete.  
   
   
       12 . The process of  claim 11 , wherein the Actinomycete is a  Micromonospora , a  Streptomyces  or a  Rhodococcus  species.  
   
   
       13 . The process of  claim 12 , wherein the Actinomycete is  Micromonospora  sp. [S01]046 having IDAC accession number 231203-01 or  Micromonospora  sp. [S01U02]046 having IDAC accession number 070905-01.  
   
   
       14 . The process of  claim 1 , wherein the suitable organic solvent is selected from the group consisting of i) at least one lower alkyl alcohol; ii) ethyl acetate; and iii) acetonitrile.  
   
   
       15 . The process of  claim 14 , wherein the lower alkyl alcohol is selected from the group consisting of ethanol, propanol, iso-propanol, butanol or methanol, and a mixture thereof.  
   
   
       16 . The process of  claim 15 , wherein the lower alkyl alcohol is methanol.  
   
   
       17 . The process of  claim 1 , wherein the aqueous culture medium is AP, CA, DZ, GP, HI, JA, MI, PI, QI, QP or YB, as shown in Table 1.  
   
   
       18 . The process of  claim 1 , wherein the aqueous culture medium is QB, MA, KH, RM, JA, FA, or HI, as shown in Table 1 and Table 2.  
   
   
       19 . The process of  claim 1 , wherein the harvesting of (c) is performed using an ultrafiltration system.  
   
   
       20 . The process of  claim 19 , wherein the ultrafiltration system has one or more filter elements having a pore size of about 0.2 to about 0.45 micrometers.  
   
   
       21 . The process of  claim 1 , wherein the harvesting of (c) is performed by centrifugation.  
   
   
       22 . The process of  claim 1 , wherein the extracting of (d) comprises one to three rounds of extraction using the suitable organic solvent.  
   
   
       23 . The process of  claim 22 , wherein the extracting further comprises circulating the particulate matter together with the suitable organic solvent in a system at a flow rate of about 30 Hz to about 50 Hz, and at a temperature of about 39° C. to about 45° C.  
   
   
       24 . The process of  claim 23 , wherein the circulation is for a period of about 50 minutes to about 120 minutes.  
   
   
       25 . The process of  claim 22 , wherein the extraction comprises subjecting the particulate matter together with the suitable organic solvent to a centrifugation treatment.  
   
   
       26 . The process of  claim 1 , wherein the volume of the suitable organic solvent is calculated in proportion to the harvested particulate matter mass (volume:mass).  
   
   
       27 . The process of  claim 1 , wherein the volume of the suitable organic solvent is calculated in proportion to the harvested particulate matter volume (volume:volume).  
   
   
       28 . The process of claims  1 , wherein the treating of (e) is an evaporation treatment.  
   
   
       29 . The process of  claim 28 , wherein the evaporation treatment is conducted under a reduced pressure.  
   
   
       30 . The process of  claim 28 , wherein the evaporation treatment is performed while incubating the extract with an adsorbent resin B.  
   
   
       31 . The process of  claim 30 , wherein the evaporation treatment is conducted under a reduced pressure.  
   
   
       32 . The process of  claim 30  or  31 , wherein the adsorbent resin B is HP20.  
   
   
       33 . The process of  claim 1 , wherein the treating of (e) comprises incubating the extract with an absorbent resin C to form a mixture, followed by an addition of water to the mixture to displace the farnesylated dibenzodiazepinone of Formula I onto the resin C.  
   
   
       34 . The process of  claim 33 , wherein the adsorbent resin C mixed with the extract is provided in a ratio (W/W) of about 10 times to about 40 times to the farnesylated dibenzodiazepinone of Formula I in the extract.  
   
   
       35 . The process of  claim 33  or  34 , wherein the water is added to the mixture at a volume flow rate (V/V) of about 0:5% to about 20% of the volume of the mixture.  
   
   
       36 . The process of  claim 35 , wherein the volume flow rate is about 0.5% to about 5% of the volume of the mixture.  
   
   
       37 . The process of  claim 35 , wherein the water added to the mixture is of a total volume of about 1.0 to about 1.5 times the volume of the mixture.  
   
   
       38 . The process of  claim 37 , wherein the total volume of water added to the mixture is of about 1.2 to about 1.5 times the volume of the mixture.  
   
   
       39 . The process of  claim 1 , wherein said fermentation broth of a) comprises about 10 mg/L to about 465 mg/L of said farnesylated dibenzodiazepinone.  
   
   
       40 . The process of  claim 1 , further comprising processing said first concentrate obtained by the method of  claim 1  to reduce a level of an impurity in said first concentrate to thereby produce a second concentrate.  
   
   
       41 . The process of  claim 40 , further comprising the step of crystallizing from said second concentrate the farnesylated dibenzodiazepinone of Formula I to thereby produce a crystal of the farnesylated dibenzodiazepinone of Formula I suitable for use in the preparation of a pharmaceutical formulation.  
   
   
       42 . A concentrate obtained by the method of  claim 1 .  
   
   
       43 . The concentrate of  claim 42 , wherein said farnesylated dibenzodiazepinone is recovered from said fermentation broth of a) in an amount of at least about 60% of the amount of said farnesylated dibenzodiazepinone in said fermentation broth.  
   
   
       44 . The concentrate of  claim 42 , wherein said farnesylated dibenzodiazepinone is recovered from said fermentation broth of a) in an amount of at least about 65% of the amount of said farnesylated dibenzodiazepinone in said fermentation broth.  
   
   
       45 . The concentrate of  claim 42 , wherein said fermentation broth comprises a microorganism capable of producing said farnesylated dibenzodiazepinone.  
   
   
       46 . The concentrate of  claim 45 , wherein said microorganism is a bacterium.  
   
   
       47 . The concentrate of  claim 46 , wherein said bacterium is an Actinomycete.  
   
   
       48 . The concentrate of  47 , wherein said Actinomycete is a  Micromonospora , a  Streptomyces  or a  Rhodococcus  species.  
   
   
       49 . The concentrate of  claim 48 , wherein the Actinomycete is  Micromonospora  sp. [S01]046 having IDAC accession number 231203-01 or  Micromonospora  sp. [S01U02]046 having IDAC accession number 070905-01.  
   
   
       50 . The concentrate of  claim 42 , wherein the extract is mixed with an adsorbent resin D to form a mixture, wherein the resin D is added to the extract in a ratio (W/W) of about 10 times to about 40 times of the farnesylated dibenzodiazepinone of Formula I present in said extract, and wherein water is added to the mixture at a volumetric rate (V/V) of about 0.5% to about 20% of the volume of the mixture.  
   
   
       51 . A microorganism capable of producing, during a fermentation period in a volume of an aqueous culture medium, a farnesylated dibenzodiazepinone of structural Formula I  
     
       
         
         
             
             
         
       
     
     at a yield rate of about 0.073 mg/Uh to about 5.06 mg/Uh as averaged over the fermentation period.  
   
   
       52 . The microorganism of  claim 51 , wherein the yield rate is of about 2.15 mg/Uh to about 5.06 mg/Uh as averaged over the fermentation period.  
   
   
       53 . The microorganism of  claim 51 , wherein the yield rate is of about 3.00 mg/Uh to about 5.06 mg/L/h as averaged over the fermentation period.  
   
   
       54 . The microorganism of  claim 51 , wherein said microorganism is a bacterium.  
   
   
       55 . The microorganism of  claim 54 , wherein said bacterium is an Actinomycete.  
   
   
       56 . The microorganism of 55, wherein said Actinomycete is a  Micromonospora , a  Streptomyces  or a  Rhodococcus  species.  
   
   
       57 . The microorganism of  claim 56 , wherein the Actinomycete is  Micromonospora  sp. [S01]046 having IDAC accession number 231203-01 or  Micromonospora  sp. [S01U02]046 having IDAC accession number 070905-01.  
   
   
       58 . The microorganism  Micromonospora  sp. [S01U02]046 having IDAC accession number 070905-01.