IL107367A - Aglycone of d-serine intermediate for the synthesis of bu-3608 antibiotics - Google Patents

Description

niniDin !?\y mni>o:i O>>J>:I miDiro ]>-ID-D l?\y ii >tP>Nn BU-3608 ni >oi >n>0JN The aglycone of D-Serine intermediate! the synthesis of BU-3608 antibiotics BRISTOL-MYERS SQUIBB COMPANY C: 90682/9 107367/2 BACKGROUND OF THE INVENTION The present invention relates to a benzo[a]naphthacene derivative which is useful as a starting material in the synthesis of antifungal antibiotics.

Few examples of benzo [ a ] naphthacene quinones derived from microbial sources have been reported and these include compounds designated G-2N and G-2A, and S-619-1. While no biological activity was reported for G-2N and G-2A, KS-619-1 is disclosed as inhi hitox o£ calcium ion and calmoduiin-dependent cyclic nucleotide phosphodiesterase. Recently, published European Patent Application 277,621 discloses antifungal antibiotics BU-3608 (la), BU-3608 B (lb), and BU-3608 C (Ic). Antibiotics benanomicins A and B were reported in J. Antibiotics, 1988, 41:807-811; benanomicin B appears to be the same as BU-3608 C whereas benanomicin A has a hydroxy! group in place of the sugar amino group.

SUMMARY OF THE INVENTION The present invention provides a compound of the formula wherein the serine is D-serine; or a salt thereof.

The compound of the invention is useful as a starting material in the synthesis of the antifungal compounds described and claimed in Patent Application No. 92240 from which the present application was divided out.

The following may contain subject matter which is not within the scope of the claims but is retained herein for the sake of completeness of the description.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 represents a 400 MHz proton nuclear magnetic resonance spectrum of BU-3608 FA-1 in DMSO-dc . b Fig. 2 represents a 400 MHz proton nuclear magnetic resonance spectrum of BU-3608 FA-2 in DMSO-d- . 107367/2 DETAILED DESCRIPTION OF THE INVENTION The antibiotics of the invention claimed in the parent application No. 92240 are antifungal agents. Compounds of formula III, i.e.

BU-3608 FA-1 and BU-3608 FA-2, are produced by cultivating a strain of Actinomadura hibisca capable of producing the antibiotics, or a variant thereof, or a mutant thereof, in a medium containing D- or DL-serine. As examples of antibiotic producing strains of Actinomadura hibi sea mention can be made of strain P157-2 and mutant strains derived therefrom designated A2660, A2493 and-B0012. Strain P157-2, which produces BU-3608 FA-1 preferentially in a medium supplemented with a source of D-serine, was isolated from a soil sample collected on Fiji Island in the South Pacific. A biologically pure culture of Actinomadura hibi sea strain P157-2 has been deposited with the American Type Culture Collection in Rockville, MD and added to their permanent collection of microorganism as ATCC 53557. Detailed description of the cultural and physiological characteristics of strain P157-2 is disclosed in our US Patent 4,870,675 which is hereby incorporated by reference. The mutant strains A2660, A2493 and B0012, which are capable of producing both BU-3608 FA-1 and FA-2 in a medium containing a source of D- serine, were obtained from the parent strain P157-2 by exposing the parent strain to N-methyl-N' -nitro-N-nitrosoguanidine (1,000 yg/ml) for an hour; the strains were selected on the basis of their ability to produce BU-3608 antibiotic complex, i.e. BU-3608, or the B, C, D and E components, in the absence of added source of D-serine. Biologically pure cultures of A2660, A2493 and B0012 have been deposited with the ATCC and assigned the accession numbers 53762 (A2660), 53815 (A2493) and 53816 (B0012) .

Cultural characteristics of the above-mentioned antibiotic producing strains of Actinomadura hibisca are provided in Table I .

Table I. Cultural characterization of some prod C Media P157-2 A-26 Yeast extract-malt extract agar (ISP-2) ++ + Glycerol-asparagine agar (ISP-5) + + Sucrose nitrate agar + + Glucose-asparagine agar ++ + D-Ribose ++ + D-Glucose ++ + Sucrose + + Cellobiose ++ + Trehalose ++ + Sol. starch ++ + Inositol + + Salicin + + L-Rhamnose + + D-Mannose + + ++ : Good growth + : Growth No growth It is to be understood that for the production of BU-3608 FA-1 and FA-2 the present invention is not limited to the particular organisms mentioned above, but includes the Use of variants and mutants produced from these strains by various means such as x-ray radiation, UV-radiation and chemical mutagens. Other producers of antibiotics of the BU-3608 family and variants and mutants thereof capable of incorporating D-serine are also included.

The producing organism is grown in a nutrient medium containing a source of D-serine in addition to known nutritional sources for actinomycetes , i.e. assimilable sources of carbon and nitrogen plus optional inorganic salts and other known growth factors. Submerged aerobic conditions are preferably employed for the production of large quantities of antibiotic, although for production of limited amounts, surface cultures and bottles may also be used. The general procedures used for the cultivation of other actinomycetes are applicable to the present specification.

The nutrient medium should contain an appropriate assimilable carbon source such as ribose, glucose, sucrose, cellobiose. As nitrogen source, ammonium chloride, ammonium sulfate, urea, ammonium nitrate, sodium nitrate, etc. may be used either alone or in combination with organic nitrogen sources such as peptone, meat extract, yeast extract, corn steep liquor, soybean powder, cotton seed flour, etc. There may also be added if necessary nutrient inorganic salts to provide sources of sodium, potassium, calcium, ammonium, phosphate, sulfate, chloride, bromide, carbonate, zinc, magnesium, manganese, cobalt, iron, and the like. As a source of D-serine, either D-serine or DL-serine may be used. — ' 107367/2 Production of antibiotics BU-3608 FA-1 and FA-2 may be effected at any temperature conducive to satisfactory growth of the producing organism, e.g. 25 - 40°C and is most conveniently carried out at a temperature of around 27 -32°C. Ordinarily, optimum antibiotic production is obtained in shake flasks after incubation periods of 5 - 8 days although a longer period may be necessary in certain cases. Aeration in shake flasks is achieved by agitation, e.g. shaking on a rotary shaker. If fermentation is to be carried out in tank fermentors, it is desirable to produce a vegetative inoculum in a nutrient broth by inoculating the broth culture from a slant culture or a lyophilized culture of the organism. After obtaining an active inoculum in this manner, it is aseptically transferred to the fermentation tank medium. Antibiotic production in tank fermentors usually reaches the optimum after 3 - 6 days of incubation. Agitation in the tank fermentor is provided by stirring and aeration may be achieved by injection of air or oxygen into the agitated mixture. Antibiotic production may be monitored using chromatographic or spectroscopic techniques, or by a conventional biological assay.

ISOLATION AND PURIFICATION OF ANTIBIOTICS The antibiotics of the parent application No. 92240 may be recovered from the cultivated broth by any suitable method for such recovery. A general scheme for one such method for the isolation and purification of antibiotics BU-3608 FA-1 and FA-2 from the fermentation broth is shown below as Scheme I.

Scheme I . Isolation of BU-3608 FA-1 and FA-2 Fermentation broth I separation supernatant mycelial cake adjust to pH filtration filtrate precipitate 1. adjust to pH 5.0 2. filtration filtrate precipitate 1. dissolve in water adjust solution to pH 9.0 filtration filtrate impurities 1. adjust to pH 2.0 2. HP-20 Diaion column crude BU-3608 HCl complex dissolve in methanol filtration filtrate impurities ethyl acetate filtrate precipitate 1. dissolve in water 2. reversed-phase silica gel chromatography HCl salt of BU-3608 FA-1 HCl BU-3608 FA-2 HCl other components HP-20 HP-20 of BU-3608 complex homogeneous BU-3608 homogeneous BU-3608 FA-2 FA-1 HCl HCl adjust to pH 5.5 adjust to pH5.5 BU-3608 FA-1 BU-3608 FA-2 To elaborate on the flow chart of Scheme I, whole fermentation broth is separated into mycelial cake and supernatant by a conventional method such as centrifugation. The supernatant is acidified and the precipitate thus formed is removed. The filtrate is adjusted to pH 5 to deposit crude antibiotic which is redissolved in alkaline water and filtered to remove impurities. The filtrate is acidified to pH 2 and chromatographed on an adsorption column such as Diaion HP-20 to yield a crude BU-3608 HC1 complex. The crude antibiotic complex may be recrystallized e.g. from ethyl acetate, and the product separated into individual antibiotic components using reversed phase silica gel HPLC. Fractions containing BU-3608 FA-1 and BU-3608 FA-2 may be further purified by Diaion HP-20 chromatography. The hydrochloride salts of the antibiotics may then be converted to the zwitterionic from by adjusting the pH of an aqueous solution of the HC1 salt to pH 5.5.

Antibiotics BU-3608 FA-1 and FA-2 are characterized by the following physico-chemical properties.

Table II. Physico-chemical properties of BU-3608 FA-1 and FA-2 BU-3608 FA-1 BU-3608 FA-2 Nature : Dark red amorphous powder Dark red amorphous powder M.P. (dec.) : 215 - 220°C 186 - 190°C 24 + 919° (c 0.1, 0.1N HC1) + 79° (c 0.1, 0.1N HC1) SIMS m/z 857 (M+H) 843 (M+H) Molecular formula C40H44N2°19 C39H42N2°19 ½ NMR substantially as shown in substantially as shown in Figure 1 Figure 2 13 C NMR (100MHz DMSO-dg) 16.3(q) 20.3(q), 36.4(q), 16.4(q), 20.4(q), 54.9(d) 56.1(q), 61.6(t), 55.0(d), 56.2(q), 63.1(d) 65.9(q), 67.8(d), 61.7(t), 54.3(d), 69.3(d) 69.9(d), 71.7(d), 65.9(q), 67.3(d), 73.6(d) 75.8(d), 80.5(d), 69.4(d), 69.7(q), 82.1(d) 104.1(d), 71.8(d), 73.5(d), 104.2(d 105.2(d) 75.9(d), 79.1(d), 106.0(d 110.3(s) 82.5(d), 104.2(d), 111.4(d 117.1(d) 104.3(d) 105.1(d) 119.0(s 119.2(s) 106.1(d) 110.4(s) 126.3(s 132. l(s) 111.5(d) 117.2(d) 133.0(s 136.8(s) 119. l(s) 119.3(s) 137.6(s 137.9(s) 126.3(s) 132. l(s) 143.5(s 158.0(s) 133. l(s) 136.8(s) 163.6(s 165.8(s) 137.8(s) 138.0(s) 166.2(s 168.5(s) 143.5(s) 158.2(s) 172.2(s 180.2(s) 163.7(s) 165.8(s) 187.3(s 166.2(s) 168. (s) 172.3(e) 180. l(s) 187.4(s) in 50% MeOH 221(32,100), 276(27,400) 223(27,700), 277(25,400) 499(12,900) 499(12,200) in 0.01N HCl-50% MeOH 234(37,400), 299(31,100) 234(34,500), 296(28,900) 460(12,900) 460(12,000) in 0.0IN NaOH-50% MeOH 244(34,100), 320(15,700) 233(37,200), 319(16,600) 498(14,900) 498(15,100) IR (KBr)cm 3400, 2920, 1605, 1385, 3400, 2920, 1600, 1385, 1295, 1260, 1160, 1040 1295, 1255, 1160, 1040 TLC Si02 Rf 0.26 0.22 (S-114, MeOAc-n-PrOH-28% NH^OH = 45:105:60, v/v) HPLC Rt (min) : 8.61 CODS, CH3CN-0.15 KH2P04> pH 3.5 (25:75) Antibiotics BU-3608 FA-1 arid FA-2 may be subjected to further chemical modification. Heating BU-3608 FA-1, FA-2, or a mixture thereof in an acidic medium for a period sufficient to cleave the xylosyl group provides the corresponding desxylosyl derivatives and small amounts of the aglycone IV. The solvent used may be for example dioxane, tetrahydrofuran, water, lower alkanol, or mixtures thereof; acid catalyst may be for example hydrochloric acid, sulfuric acid, and trifluoroacetic acid. The temperature may be from about 60 to about 100°C, or the reflux temperature of the solvent. Reaction time may be from about 0.5 to about 10 hrs and will depend on the reaction conditions employed. Ν,Ν-dimethyl BU-3608 FA-2, prepared by reductive alkylation procedure described below, may be converted to its corresponding desxylosyl compound in a similar fashion. It has been discovered that acid hydrolysis of Ν,Ν-dimethyl BU-3608 FA-2 results in a practical yield of the aglycone IV.

The amino group of BU-3608 FA-1, FA-2 or their corresponding desxylosyl derivatives may be alkylated by reductive alkylation which comprises first reacting the antibiotic starting material with an aldehyde or a ketone to form an imine, and subsequently reducing the imine thus formed. The condensation and reduction may be conducted in the same reaction vessel in one step, or in two separate steps. The primary amine group of BU-3608 FA-2 or its desxylosyl derivatives may be converted into a tertiary amine having two identical alkyl groups by treatment with at least two equivalents of the carbonyl compound relative to the antibiotic, followed by reduction; or a tertiary amine having two different alkyl substituents may be obtained by using a controlled amount of a first carbonyl reactant to convert the primary amine into a secondary amine which is 107367/2 then reacted with a second different carbonyl compound to give the product tertiary amine. If the second carbonyl compound is not added, a secondary amine is obtained.

The carbonyl reactant may be an aldehyde or a ketone having one to six carbon atoms, for example, formaldehyde, acetaldehyde , propionaldehyde , and acetone. Reduction of the imine may be accomplished by using reducing agents such as metal hydrides, for example, sodium borohydride, sodium cyanoborohydride , lithium aluminum hydride. The reaction is carried out in a polar organic solvent or a mixture thereof such as water, acetonitri le , lower alkanols, and dimethyl sulfoxide. The reaction temperature is not particularly restricted and may be from room temperature to about 100°C. In our experience, the alkylation reaction carried out at room temperature is usually complete within 24 hours.

Optimum reaction conditions will of course depend on the nature and reactivity of the particular reactants used. It will be appreciated that Ν,Ν-dimethyl BU-3608 FA-2 may be obtained from BU-3608 FA-2, FA-1, or a mixture thereof without having to separate the two components. Similarly, Ν,Ν-dimethyl desxylosyl BU-3608 FA-2 may be obtained from desxylosyl BU-3608 FA-2, FA-1, or a mixture thereof.

BIOLOGICAL ACTIVITY Antifungal activities of representative compounds of the parent application No. 92240 were evaluated both in vitro and in vivo. The minimum inhibitory concentrations (MICs) against various fungi were determined by serial agar dilution method using Sabouraud dextrose agar. Thus, approximately 0.003 ml of fungal suspension containing 106 cells/ml was applied to the surface of agar plates containing the test antibiotics. The MIC values recorded after the cultures had been incubated for 40 hours at 28°C are set forth below in Table III.

Table III. In vitro antifungal activity in Sabouraud dextrose agar ( MIC (yg/ml) Test organism B Candida albicans IAM4888 6.3 6.3 3.1 3.1 C. albicans A9540 12.5 6.3 6.3 3.1 Cryptococcus neoformans D49 0.8 0.8 0.8 1.6 C. neoformans IAM4514 0.8 0.8 0.8 1.6 Aspergillus fumigatus IAM2530 3.1 3.1 3.1 6.3 A. fumigatus IAM2034 6.3 6.3 3.1 6.3 A. flavus FA21436 6.3 6.3 25.0 50 Fusarium moniliforme A2284 6.3 6.3 25.0 >100 Trichophyton mentagrophytes D155 6.3 6.3 12.5 12.5 T. mentagrophytes #4329 6.3 6.3 6.3 12.5 Blastomyces dermatitidis D40 3.1 3.1 6.3 6.3 Sporothrix schenckii IF08158 1.6 1.6 1.6 6.3 Petriellidium boydii IFO8073 12.5 25 D >100 Mucor spinosus IF05317 >100 >100 D 12.5 ND: Not determined In vivo antifungal activities were evaluated against intravenous infections with Candida albicans A9540, Cryptococcus neoformans IAM4514 and Aspergillus fumigatus IAM2034 in mice. Candida albicans and Cryptococcus neoformans were cultured for 18 and 48 hours, respectively, at 28°C in YGP medium [yeast extract ( 0.2%) , glucose ( 1.5%) , peptone(0.5%) , K2HP04( 0.05%) and MgS04 ( 0.05%) ] and suspended in saline. Aspergillus fumigatus was cultured for 7 days at 28°C on YGP agar slant and suspended in saline. Spores were collected by filtering fungal suspension through gauze.

Male ICR mice weighing 20 to 24 g were infected intravenously with about 10 times the median lethal dose of the fungi .

Test compounds were administered at various doses to groups of 5 mice each intravenously either once just after fungal challenge (day 0) or once daily for 5 days from day 0 to day 4 (qd X 5). The 50% protective dose (PD50) was calculated from survival rates recorded on the 20th day after fungal challenge. All control animals died within 7 to 15 days after fungal infection. The results of in vivo studies are presented in Table IV.

Table IV. n vivo activity against Candida, Cryptococcus and Aspergillus intravenous infections PD50 (mg/kg/inj. iv) C. albicans C. neofortnans A. fumigatus A9540 IAM4514 IAM2034 Single qd x 5 Single qd x5 Single qd x 5 BU-3608 FA-1 18 - - BU-3608 FA-2 7.4 - - - -N,N-dimethyl BU-3608 FA-2 9.0 7.5 11 2.8 36 15 The LD50 for Ν,Ν-dimethyl BU-3608 FA-2 was determined in mice after a single intravenous dose. At 600 mg/kg, the highest dose test, neither lethal toxicity nor any significant toxic sign was observed.

For treatment of fungal infections in animals and human beings, the antibiotics of the present invention may be given in an antifungally effective amount by any accepted routes of administration; these include, but are not limited to, intravenous, intramuscular, oral, intranasal, and for superficial infections, topical administration. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions or emulsions. They may also be manufactured in the form of sterile solid compositions which can be dissolved in sterile water, physiological saline, or some other sterile injectable medium immediately before use. Oral formulation may be in the form of tablets, gelatin capsules, powders, lozenges, syrups, and the like. For topical administration, the compound may be incorporated into lotions, ointments, gels, creams, salves, tinctures, and the like. Unit dosage forms may be prepared using methods generally known to those skilled in the art of pharmaceutical formulations.

It will be appreciated that when treating a host infected with a fungus susceptible to the antibiotics of this invention, the actual preferred route of administration and dosage used will be at the discretion of the attending clinician skilled in the treatment of fungal infections, and will vary according to the causative organism, its sensitivity to the antibiotic, severity and site of the infection, and patient characteristics such as age, body weight, rate of excretion, concurrent medications, and general physical condition.

The following examples are illustrative without limiting the scope of the present invention.

Example 1. Fermentation Production Of BU-3608 FA-1 (a) Agar Slant. Actinomadrua hibisca P157-2 (ATCC 53557) was grown on an agar slant of pH 7.0 having the composition 0.5% soluble starch 0.5% glucose 0.1% fish meat extract 0.1% yeast extract 0.2% NZ-case 0.2% NaCl 0.1% CaC03 1.6% agar The culture was incubated at 28°C for 10 days. (b) Seed Culture. A portion of the microbial growth from the slant culture was transferred to a 500-ml Erlenmeyer flask containing 100 ml of vegetative medium pH 7.0 composed of 3% glucose 3% soybean meal 0.5% Pharmamedia 0.1% yeast extract 0.3% CaC03 The culture was incubated at 32 °C for 6 days on a rotary shaker set at 200 rpm. (c) Production Culture. Five ml of the microbial growth was transferred from the seed culture to a 500-ml Erlenmeye flask containing 100 ml of sterile production medium composed of 3% glucose 3% soybean meal 0.5% Pharmamedia 0.1% yeast extract 0.3% CaC03 0.25% D-serine The culture was incubated at 28°C for 6 days on a rotary shaker set at 200 rpm. Antibiotic production reached 443 yg/ml and 72.9% of which was BU-3608 FA-1, 26.0% BU-3608 and 1.1% BU-3608C. Antibiotic production was determined by measuring the optical density of the liquid from the fermentation broth at 500 and 600 nm. Genuine optical density was obtained by substracting the OD value at 600 nm from that at 500 nm. Antibiotic concentration is expressed as equivalent amount of BU-3608 free base. The antibiotics were identified using the HPLC procedure described in Example 5.

Example 2. Fermentation Production Of BU-3608 FA-1 and FA-2 Using Strain A-2493 Agar slant culture and seed culture of an arginine auxotroph mutant of Actinomadura hibisca P157-2 designated as strain A-2493 (ATCC 53815) were grown using media having the same compositions as those described in Example 1 and under conditions given in Example 1.

Production A. Five ml of the microbial growth was transferred from the seed culture to each 500-ml Erlenmeyer flask (100 flasks) containing 100 ml of the same production medium described in Example 1(c). The culture was incubated at 28°C for 6 days on a rotary shaker set at 200 rpm.

Antibiotic production reached 261 yg/ml and was composed of 29.8% BU-3608 FA-1, 28.9% BU-3608 FA-2, 19.5% BU-3608C and 21.8% BU-3608.

Production Β. Production of antibiotics by strain A2493 was also effected in a medium (pH 7.0) composed of 3% glucose 3% Protein S (soybean flour, Ajinomoto) 0.3% CaC03 0.5% DL-serine Antibiotic production reached 890 pg/ml after the culture had been incubated for 11 days at 28°C. The ratio of components was BU-3608 FA-2 17.7%, FA-1 17.0%, BU-3608 33.5% and BU-3608 C 31.8%.

Example 3. Fermentation Production Of BU-3608 FA-1 And FA-2 Using Strain B-0012.

Production A. The conditions and culture media described in Example 1 parts (a), (b) and (c) were followed using the variant strain designated B-0012 (ATCC 53816) instead of the parent strain P157-2. Antibiotic production reached 1,150 vg/ml after 6 days and the ratio of components was BU-3608 FA-1 33.7%, FA-2 21.2%, BU-3608 24.0% and BU-3608 C 21.1%.

Production B. A portion of microbial growth from the slant culture of strain B-0012 was also transferred to a 500-ml Erlenmeyer flask containing 100 ml of a medium pH 7.0 having the following composition 1% soluble starch 1% glucose 0.5% yeast extract 0.5% peptone 0.3% NaCl 0.2% CaC03 The seed culture was grown at 32°C for 6 days and 5 ml of the microbial growth was transferred to a 500-ml Erlenmeyer flask containing 100 ml of a production medium pH 7.0 composed of 3% glucose 3% Protein S (soybean flour, Ajinomoto) 0.3% CaC03 0.25% D-serine » The culture was incubated at 28°c for 11 days.

Antibiotic production reached 1,970 ug/ml and the ratio of components was BU-3608 FA-2 20.0%, FA-1 10.0%, BU-3608 C 39.0% and BU-3608 31.0%.

Example 4. Fermentation Production Of BU-3608 FA-1 and FA-2 Using Strain A-2660.

Agar slant and seed cultures of Actino adura hibisca mutant strain A-2660 (ATCC 53762) were produced using the same conditions and media given in Example 1 parts (a) and (b). Five ml of the seed culture was transferred to a 500-ml Erlenmeyer flask containing 100 ml of a production medium composed of 3% glucose 3% soybean meal 0.5% Pharmamedia 0.1% yeast extract 0.3% CaC03 0.5% DL-serine The culture was incubated at 28 eC for 7 days.

Antibiotic production reached 620 μς/ml with the ratio of components as BU-3608 FA-1 17.0%, FA-2 15.6%, BU-3608 23.9%, BU-3608 C 24.7%, D 8.3% and E 10.5%.

Example 5. Isolation And Purification of BU-3608 FA-1 And FA-2 From Fermentation Broth.

Ten liters of fermentation broth obtained from the procedure described in Example 2, Production A, was separated into mycelial cake and supernatant by centrifugation. The supernatant was acidified to pH 2.0 using 6N HC1 and the amorphous precipitate deposited was removed by filtration. The clear filtrate was adjusted to pH 5.0 using 6N NaOH, and kept at 5°C for two hours. The dark red precipitate deposited was collected by filtration. The precipitate was dissolved in 4.1 L of water adjusted to pH 9.0 with 6N NaOH and the solution was filtered to remove insoluble impurities. The filtrate was adjusted to pH 2.0 and applied on a column of Diaion HP-20 (2.0 L) . The column was washed with water and eluted with 60% aqueous acetone (pH 3.0). Concentration of the red eluate afforded amorphous solid of BU-3608 complex hydrochloride (3.1 g) . The complex solid (3.0 g) was dissolved in methanol (120 ml) and filtered. To the stirred filtrate, 720 ml of ethyl acetate was added dropwise and the resulting solution was kept at 5°C for 15 hours. The precipitate deposited was collected by filtration and dried (1.28 g) .

The solid (1.28 g) was dissolved in water (100 ml) and subjected to reversed phase chromatography on a column of YMC GEL ODS A 60 (10 L, Yamamura Chemical Lab.) which had been equilibrated with a mixture of CH.-CN-0.15% KE^ O^ pH 3.5 (21:79). Elution was carried out with the same solvent mixture, and the eluate was collected in lL-fractions . The fractions were analyzed by HPLC (column : YMC A-301-3, 4.6 mm I.D. x 100 mm, 3ym, ODS Yamamura Chemical Lab., mobile phase : CH3CN-0.15% KH2P04, pH 3.5 (25:75), flow rate : 0.8 ml/min, detection : UV absorption at 254 ran, retention time : BU-3608 FA-2, 7.65 min; BU-3608 FA-1, 8.61 min ; BU-3608A, 19.11 min) . The fractions containing homogeneous BU-3608 FA-2 or BU-3608 FA-1, were pooled and concentrated in vacuo to remove CH^CN. Each concentrate was desalted by Diaion HP-20 chromatography to yield nearly homogeneous BU-3608 FA-2 hydrochloride (75 mg) and BU-3608 FA-1 hydrochloride (50 mg) .

In order to convert the hydrochloride salt to its free form and to remove contaminated inorganic salts, an aqueous solution of each salt was adjusted to pH 5.5 with 0.1 N NaOH to deposit pure zwitterionic form of BU-3608 FA-2 (48 mg) and BU-3608 FA-1 (11 mg) .

Example 6. Preparation Of Ν,Ν-dimethyl BU-3608 FA-2 A mixture of BU-3608 FA-1 and BU-3608 FA-2 (45:55, 510 mg) was dissolved in 50 ml of water and the solution was adjusted to pH 7.9 by addition of IN sodium hydroxide and diluted with 50 ml of acetonitrile . To this solution were added sequentially aqueous formaldehyde (>35%, 1.6 ml) and sodium cyanoborohydride (240 mg) at room temperature. The solution was stirred for 1 hour at room temperature and the progress of reaction monitored by HPLC. The organic solvent was removed in vacuo and the aqueous residue was adjusted to pH 10.9. The solution (40 ml) was added dropwise to 240 ml of acetone with stirring and kept at 5°C for 2 hours. The resulting precipitate was collected by centrifugation (3000 rpm) and redissolved in 40 ml of water. After removal of traces of the acetone in vacuo , the solution was adjusted to pH 5.0 and allowed to stand at 5°C for 24 hours. The precipitate deposited was collected by centrifugation, washed sequentially with water and acetone, and vacuum-dried at 60°C to afford 80 mg of zwitterionic Ν,Ν-dimethyl FA-2. _, n /I 0, oo^ ,j \ ,0.01N NaOH M.p. 214 - 218°C (dec); UV ^ Q (32#900)# 320.0(15,500), 498.4(15,200).

Example 7. Preparation Of Desxylosyl BU-3608 FA-1 A solution of BU-3608 FA-1 (54 mg) in dioxane (5.4 ml) and IN HC1 (5.4 ml) was refluxed on a steam bath for 8 hr. The reaction mixture was diluted with water (30 ml) and charged on a short column of Diaion HP-20 (Mitsubishikasei , 1.8 x 25 cm). The column was washed with water and then eluted with acidic 80% acetone (pH 3, acidified with IN HC1). The reddish orange eluate was collected and evaporated to give a deep red powder. The powder was dissolved in 35% acetonitrile/phosphate buffer (pH 3.5) and chromatographed on an ODS column (YMC-ODS, 2.1 x 25 cm, eluted with the same solvent) . The fractions containing the desired compound were combined and passed through an HP-20 column. The column was washed with water and eluted with 80% acetone (pH 3). Evaporation of the eluate gave a dark red powder, which was dissolved in water (8 ml) and the solution was adjusted to pH 5.3 with 0.1 N NaOH. The resulting precipitate was collected by centrifugation, washed with acetone and dried to give a dark red powder (23.3 mg, 51%). MP > 180°C (dec). Purity by HPLC: >95%.

IR: v max ( Br) cm"1: 3400, 1605, 1290, 1255, 1060.

UV: λ max (1/lOON NaOH) nm (ε): 212(35,400), 319(15,300), 498(14,500) .

½ NMR: (400 MHz, DMSO-dg) : 1.26 (3H, d, J=6Hz, 6'-CH3), 2.32 (3H, s, Ph-CH3), 2.65 (3H, s, CH3 ) , 3.75 (2H, m, OCH2), 3.91 (3H, s, 0CH3 ) , 4.68 (1H, d, J=8Hz, l'-H), 6.72 (1H, d, J=2Hz, 10-H) , 6.87 (1H, s, 4-H) , 7.12 (1H, d, J=2Hz, 12-H) , 7.71 (1H, s, 7-H) .

Example 8. Preparation Of Desxylosyl BU-3608 FA-2 A solution of BU-3608 FA-2 (54 mg) in dioxane (5.4 ml) and IN HC1 (5.4 ml) was refluxed on a steam bath for 8 hr. The reaction mixutre was diluted with water (30 ml), adsorbed on a short column of Diaion HP-20 ( itsubishikasei , 1.8 x 25 cm), washed with water and eluted with 80% acetone (pH 3). The vermilion eluate was pooled and concentrated and the residue was dissolved in water (8 ml). The solution was adjusted to pH 5.3 by 0.1N NaOH and the resulting precipitate was collceted by centrifugation, washed with acetone and dried in vacuo to give a dark red powder (37.7 mg 85%). MP >180°C dec. Purity by HPLC:>95%.

IR: vmax ( Br) cm"1: 3400, 1605, 1290, 1265, 1035 UV: Xmax (1/100 N NaOH) m (ε): 214(33,000), 234(32,300), 319(14,900), 498(14,100) ½ NMR (DMSO-d5): 1.15 (3H, d, J=7Hz, 6'-CH3), 2.32 (3H, s, PhCH3), 3.75 (2H, m, OCH2 ) , 3.91 (3H, s, OCH3), 4.67 (1H, d, J=8Hz, , l'-H), 6.72 (1H, d, J=3Hz, 10-H), 6.93 (1H, s, 4-H), 7.12 (1H, d, J=3Hz, 12-H) , 7.71 (1H, s, 7-H) .

Example 9. Preparation Of Desxylosyl Ν,Ν-dimethyl BU-3608 FA-2 Method A A solution of Ν,Ν-dimethyl BU-3608 FA-2 (product of Example 6, 50 mg) in dioxane (5 ml) and IN HC1 (5 ml) was refluxed on a steam bath for 8 hr . The reaction mixutre was cooled to ambient temperature and filtered to give 7.2 mg of a precipitate. The filtrate was charged on a dry silica gel column (Merck ieselgel 60, 4 x 30 cm) and eluted with n-BuOH-AcOH-H20(3 : 1 : 1) . The eluate was collected in 10-ml fractions. Fractions 2-14 and the precipitate earlier obtained were worked-up as described in Example 10.

Fractions 21-32 were combined and charged on an HP-20 column (1.8 x 25 cm). The column was washed with water and eluted with 80% acetone (pH 3). Concentration of the vermilion eluate gave a solid, which was successively purified by ODS column chromatorgraphy (YMC-ODS, 2 x 37 cm, eluted with 20% CH3CN/pH 3.5 phosphate buffer) and Diaion HP-20 chromatography (1.8 x 25 cm, eluted with 80% acetone at pH 3) to give 7.8 mg (17%) of the title compound as a dark red powder.

MP >180°C (dec.) Purity by HPLC:>95%.

IR: vmax (KBr) cm"1: 3400, 1730, 1610, 1380, 1260, 1070. UV: Xmax (1/lOON NaOH)nm (ε): 211(38,100), 318(14,200), 496(12,500).

½ NMR (DMSO-d6): 1.23 (3H, d, J=7Hz , 6*-CH3), 2.29 (3H, s, PhCH3), 2.75 (6H, s, NCH3 ) , 3.74 (2H, m, OCH2), 3.91 (3H, s, OCH3 ) , 4.60 (1H, d, J=8Hz, l'-H), 6.73 (1H, d, J=3Hz, 10-H) , 6.89(1H, s, 4-H), 7.12 (1H, d, J=3Hz, 12-H), 7.77 (1H, s, 7-H) .

Method B A solution of desxylosyl BU-3608 FA-2 (product of Example 8, 71 mg) in water (7 ml) and acetonitrile (7 ml) was adjusted to pH 7 using 0.1N NaOH. To this solution was added aqueous formaldehyde (35%, 0.3 ml) and sodium cyanoborohydride (45 mg) at room temperature. The reaction mixutre was stirred overnight and the organic solvent was removed in vacuo . The aqueous residue was adjusted to pH 10 with NaOH and then added dropwise to acetone (70 ml). The precipitate was filtered off and dissolved in water at pH 2.5. The solution was charged on a column of Diaian HP-20 (1.8 x 25 cm), washed with water, and developed with acidic acetone (pH 3, acidified with IN HC1). The deep red eluate was pooled, concentrated to about 5 ml, adjusted to pH 5.3 with dilute NaOH, and then added dropwise to acetone (70 ml). The resulting precipitate was collected by filtration, and re-precipitated from, aqueous acetone to give 66 mg (90%) of of the title compound as a dark red powder which was identical in all respects to that obtained by Method A.

Purity by HPLC: >95%.

Example 10. BU-3608 FA Aglycone Fractions 2-14 of the eluate from the silica gel column in Example 9 (Method A) were combined and concentrated to dryness to give a powder. The powder and the precipitate of Example 9 (Method A) were dissolved in 0.01N NaOH, charged on a column of Diaion HP-20 (1.8 x 25 cm), washed with water and eluted with 80% acetone (pH 3). The vermilion eluate was pooled and acetone was evaporated in vacuo to give a suspension. The suspension was acidified with IN HC1 (pH 3) and extracted with butanol. Concentration of the butanol extract gave 11.5 mg of the aglycone (33%) as a dark red amorphous powder.

MP >200°C (dec). Purity by HPLC:>95%.

IR: vmax (KBr) cm"1: 3240, 1720, 1605, 1340, 1305, 1165. UV: Xmax (1/lOON NaOH) nm (ε): 212(34,500), 319(15,200), 498(14,000) .

½ MR (DMSO-d6): 2.34 (3H, s, PhCH3 ) , 3.73 (2H, m, 0 ≡2 3.91 (3H, s, OCH3), 4.24 (2H, AB-q, J=llHz, 5-H & 6-H), 4.46 ( 1H, m, N-CH-COOH), 6.92 (1H, d, J=2Hz, 10-H) , 7.06 (1H, s, 4-H), 7.28 (1H, d, J=2Hz, 12-H), 8.08 (1H, s, 7-H) .

Example 11 The general procedure described in Example 6 is followed using the reactants listed below to provide the corresponding alkylated analogs of BU-3608 FA-1 and FA-2 A + B > II.

B II BU-3608 FA-1 acetaldehyde (1 equiv)X propionaldehyde (1 equiv) acetone (1 equiv) BU-3608 FA-2 acetaldehyde (2 equiv) CH CH - 3 1 acetone CI equiv) R -β-D-xylosyl ; R =H, R2=CHCCCH,)„ 3 3 2 1 2 propionaldehyde (2 equiv) R =p-D-xylosyl; R =R = CHTCH„CH - 3 2 2 butyraldehyde CI equiv) Desxylosy BU-3608 acetaldehyde CI equiv) FA-1 propionaldehyde CI equiv) R3=H; R^CH,; R2= CHTCH„CH - 3 2 2 II acetone (1 equiv) R3=H; R^CH ; R2=-CH(CH3)2 Desxylosy BU-3608 acetaldehyde (2 equiv) R =H; R =R = CHjCh FA-2 acetone CI equiv) R3=H; RX=H, R2=-CH(CH3)2 propionaldehyde (2 equiv) R3=H; R =R2= CHjCh^Ch^- butyraldehyde (1 equiv) R3=H; RX=H; R2=CH3

Claims (1)

1. C L A I M A compound of the formula: COR wherein R is a D-serine residue; or a salt thereof.