Microbial transformation of a substituted pyridinone using actinoplanacete sp. MA 6559

Fermentation of the microorganism Actinoplanacete sp. (MA6559), ATTC No. 53771, in the presence of the HIV reverse transcriptase inhibitor ##STR1## yields 3-[2-(benzoxazol-2-yl)ethyl]-5-(1-hydroxyethyl)-6-methyl-2(1H)-pyridinone and 3-[2-(benzoxazol-2-yl)ethyl]-5-ethyl-6-hydroxymethyl-2(1H)-pyridinone, both of which are useful in the prevention or treatment of infection by HIV and the treatment of AIDS.

The present invention relates to a novel process for the preparation of 
compounds (I) and (II) 
##STR2## 
comprising fermentation of compound (III), an inhibitor of the reverse 
transcriptase encoded by human immunodeficiency virus (HIV) 
##STR3## 
with the microorganism Actinoplanacete sp. (MA6559), ATTCC No. 53771. 
Compounds (I) and (II) and the pharmaceutically acceptable salts thereof 
inhibit the reverse transcriptase encoded by HIV and are of value in the 
prevention of infection by HIV, the treatment of infection by HIV and the 
treatment of the resulting acquired immune deficiency syndrome (AIDS). 
BACKGROUND OF THE INVENTION 
A retrovirus designated human immunodeficiency virus (HIV) is the 
etiological agent of the complex disease that includes progressive 
destruction of the immune system (acquired immune deficiency syndrome; 
AIDS) and degeneration of the central and peripheral nervous system. This 
virus was previously known as LAV, HTLV-III, or ARV. A common feature of 
retrovirus replication is reverse transcription of the RNA genome by a 
virally encoded reverse transcriptase to generate DNA copies of HIV 
sequences, a required step in viral replication. It is known that some 
compounds are reverse transcriptase inhibitors and are effective agents in 
the treatment of AIDS and similar diseases, e.g., azidothymidine or AZT. 
Nucleotide sequencing of HIV shows the presence of a pol gene in one open 
reading frame [Ratner, L. et al., Nature, 313, 277(1985)]. Amino acid 
sequence homology provides evidence that the pol sequence encodes reverse 
transcriptase, an endonuclease and an HIV protease [Toh, H. et al., EMBO 
J. 4, 1267 (1985); Power, M. D. et al., Science, 231, 1567 (1986); Pearl, 
L. H. et al., Nature 329, 351 (1987)]. 
The compounds prepared by the process of this invention are inhibitors of 
HIV reverse transcriptase. Furthermore, the compounds of the present 
invention do not require bio-activation to be effective. 
SUMMARY OF THE INVENTION 
Two HIV reverse transcriptase inhibitors, (I) and (II), are produced from 
the fermentation of the microorganism Actinoplanacete sp. (MA 6559), ATCC 
No. 53771, in the presence of substrate compound (III). The 
biotransformations are accomplished under submerged aerobic conditions in 
an aqueous carbohydrate medium containing a nitrogen nutrient at a pH of 
about 7 for a sufficient time to produce compounds (I) and (II). 
DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS 
The novel process of this invention comprises fermentation of the 
microorganism Actinoplanacete sp. (MA6559) in the presence of substrate 
compound (III) 
##STR4## 
in a nutrient medium, and isolation of the resulting biotransformation 
products, compounds (I) and (II), in a conventional manner. 
##STR5## 
A biologically pure sample of Actinoplanacete sp. (MA6559) was deposited 
on May 26, 1988 in the permanent culture collection of the American Type 
Culture Collection, 12301 Parklawn Drive in Rockville, Md., with Accession 
Number ATCC 53771. 
On the basis of the taxonomic analysis performed thus far, the 
microorganism Actinoplanacete sp. has tentatively been assigned in the 
order Actinomycetales, in the family Actinoplanacea, and in the genus 
Streptosporangium, and its characteristics are described below: 
Microscopic observations--Culture grows as branched filaments ranging of 
approximately 6 microns diameter. Spherical to ovoid sporangia are 
detected on glycerolasparagine agar, oatmeal agar, yeast-malt extract agar 
and inorganic salts-starch agar. Sporangia range in size from 2.5-44 
microns in diameter. 
Oat Meal Agar 
Vegetative Growth: Reverse is hyaline 
Aerial Mass: Moderate, off white, powdery 
Soluble Pigment: None. 
Glycerol-Asparagine 
Vegetative Growth: Obverse is mahogany 
Aerial Mycelium: Off white and cottony at periphery turning to dusty rose 
and powdery at colony center 
Soluble Pigment: Very light brown. 
Inorganic Salts-Starch Agar 
Vegetative Growth: Mahogany 
Aerial Mycelium: Off white and cottony at periphery turning to dusty rose 
and powdery at colony center 
Soluble Pigment: Areas of browning around the periphery of growth with 
slight clearing of starch. 
Yeast Extract-Malt Extract Agar 
Vegetative Growth: Mahogany to brown black 
Aerial Mass: Isolated areas of white, cottony growth against a powdery 
dusty rose colored mycelial matte 
Soluble pigment: Yellow-brown. 
Egg Albumin Agar 
Vegetative Growth: Pale yellow, flat 
Aerial Mass: Sparse, white and cottony limited to periphery of growth 
Soluble Pigment: None. 
Nutrient Tyrosine Agar 
Vegetative Growth: Transparent to pale yellow 
Aerial Mass: None 
Soluble Pigment: None 
Decomposition of tyrosine: Negative. 
Skim Milk Agar 
Vegetative Growth: Leathery and yellow 
Aerial Mass: Sparse, off white and powdery 
Soluble Pigment: None 
Hydrolysis of casein: Positive. 
Tomato Paste Oatmeal Agar 
Vegetative Growth: Orange-yellow, rugose 
Aerial Mass: Powdery, varying in color from off white to purple-brown. 
Gelatin Stabs 
Vegetative Growth: Orange yellow 
Aerial Mass: None 
Soluble Pigment: None 
Liquification of gelatin: Positive. 
Peptone-Iron-Yeast Extract Agar Slants 
Vegetative Growth: Colorless, leathery 
Aerial Mass: Moderate, off white, powdery 
Soluble Pigment: None 
Melanin: Negative 
H.sub.2 S: Negative. 
Tryptone Yeast Extract Broth 
Soluble Pigment: None. 
______________________________________ 
Carbohydrate utilization pattern 
______________________________________ 
d-glucose ++ l-mannose - 
d-arabinose 
++ d-raffinose ++ 
l-arabinose 
++ l-rhamnose - 
d-fructose ++ sucrose +/- 
l-glucose +/- d-xylose ++ 
inositol + l-xylose - 
d-maltose + alpha d-lactose 
++ 
d-mannitol ++ beta d-lactose 
++ 
d-mannose ++ 
______________________________________ 
Carbon source utilization studies were carried out using Pridham and 
Gottlieb basal medium supplemented with 1% carbon source. Scoring was 
graded according to the methods described in "Methods for Characterization 
of Streptomyces species", IJSB 16: pps 313-340. 
In general, compound (I), the 5-(1-hydroxy)ethyl oxidation product, and 
compound (II), the 6-hydroxymethyl oxidation product, can be produced by 
culturing (fermenting) the above-described microorganism in the presence 
of an appropriate concentration of substrate compound (III) in an aqueous 
nutrient medium containing sources of assimilable carbon and nitrogen, 
preferably under submerged aerobic conditions (e.g. shaking culture, 
submerged culture, etc.). An appropriate concentration of the parent 
compound in the aqueous medium ranges from 0.01 mg/ml to 0.2 mg/ml, 
preferably 0.05 mg/ml; less than 0.01 mg/ml is inefficient and greater 
than 0.2 mg/ml can inhibit the culture. The aqueous medium is incubated at 
a temperature between 26.degree. C. and 29.degree. C., preferably 
27.degree. C.; culture growth will be inhibited below this temperature 
range and culture death will occur above this temperature range. The 
aqueous medium is incubated for a period of time necessary to complete the 
oxidative biotransformations as monitored by HPLC, usually for a period of 
about 24 hours, on a rotary shaker operating at about 220 rpm with a throw 
of about 2 in. The aqueous medium is maintained at a pH between 6 and 8, 
preferably about 7, at the initiation and termination (harvest) of the 
fermentation process. A higher or lower pH will cause the culture to die. 
The desired pH may be maintained by the use of a buffer such as 
morpholinoethanesulfonic acid (MES), morpholinopropanesulfonic acid 
(MOPS), and the like, or by choice of nutrient materials which inherently 
possess buffering properties, such as production media described herein 
below. 
The preferred sources of carbon in the nutrient medium are certain 
carbohydrates such as glucose, xylose, galactose, glycerin, starch, 
dextrin, and the like. Other sources which may be included are maltose, 
rhamnose, raffinose, arabinose, mannose, salicin, sodium succinate, and 
the like. 
The preferred sources of nitrogen are yeast extract, meat extract, peptone, 
gluten meal, cottonseed meal, soybean meal and other vegetable meals 
(partially or totally defatted), casein hydrolysates, soybean hydrolysates 
and yeast hydrolysates, corn steep liquor, dried yeast, wheat germ, 
feather meal, peanut powder, distiller's solubles, etc., as well as 
inorganic and organic nitrogen compounds such as ammonium salts (e.g. 
ammonium nitrate, ammonium sulfate, ammonium phosphate, etc.), urea, amino 
acids, and the like. 
The carbon and nitrogen sources, though advantageously employed in 
combination, need not be used in their pure form because less pure 
materials which contain traces of growth factors and considerable 
quantities of mineral nutrients are also suitable for use. When desired, 
there may be added to the medium mineral salts such as sodium or calcium 
carbonate, sodium or potassium phosphate, sodium or potassium chloride, 
sodium or potassium iodide, magnesium salts, copper salts, cobalt salts, 
and the like. If necessary, especially when the culture medium foams 
seriously, a defoaming agent, such as liquid paraffin, fatty oil, plant 
oil, mineral oil or silicone may be added. 
Substrate compound (III) can be obtained by synthetic organic procedures, 
as described elsewhere in this application. 
Submerged aerobic cultural conditions are preferred for the production of 
the 5-(1-hydroxy)ethyl and 6-hydroxymethyl oxidation products in massive 
amounts. For the production in small amounts, a shaking or surface culture 
in a flask or bottle is employed. Furthermore, when the growth is carried 
out in large tanks, it is preferable to use the vegetative form of the 
organism for inoculation in the production tanks in order to avoid growth 
lag in the process of production of the 5-(1-hydroxy)ethyl and 
6-hydroxymethyl oxidation products. Accordingly, it is desirable first to 
produce a vegetative inoculum of the organism by inoculating a relatively 
small quantity of culture medium with spores or mycelia of the organism 
produced in a "slant" and culturing said inoculated medium, also called 
the "seed medium", and then to transfer the cultured vegetative inoculum 
aseptically to large tanks. The fermentation medium, in which the inoculum 
is produced, is substantially the same as or different from the medium 
utilized for the production of the 5-(1-hydroxy)ethyl and 6-hydroxymethyl 
oxidation products and is generally autoclaved to sterilize the medium 
prior to inoculation. The fermentation medium is generally adjusted to a 
pH between 6 and 8, preferably about 7, prior to the autoclaving step by 
suitable addition of an acid or base, preferably in the form of a 
buffering solution. Temperature of the seed medium is maintained between 
26.degree. C. and 29.degree. C., preferably 27.degree. C.; culture growth 
will be inhibited below this range and culture death will occur above this 
range. Incubation of the seed medium is usually conducted for a period of 
about 10 to 30 hours, preferably 24 hours, on a rotary shaker operating at 
220 rpm; the length of incubation time may be varied according to 
fermentation conditions and scales. Agitation and aeration of the culture 
mixture may be accomplished in a variety of ways. Agitation may be 
provided by a propeller or similar mechanical agitation equipment, by 
revolving or shaking the fermentor, by various pumping equipment or by the 
passage of sterile air through the medium. Aeration may be effected by 
passing sterile air through the fermentation mixture. 
Preferred culturing/production media for carrying out the fermentation 
include the following media: 
______________________________________ 
g/l 
______________________________________ 
Seed Medium A 
Dextrose 1.0 
Dextrin 10.0 
Beef Extract 3.0 
Ardamine pH 5.0 
NZ Amine Type E 5.0 
MgSO.sub.4.7H.sub.2 O 
0.05 
K.sub.2 HPO.sub.4 0.3 
Adjust pH to 7.1 
Add CaCO.sub.3 0.5 g/l 
Transformation Medium B 
Glucose 10 
Hycase SF 2 
Beef Extract 1 
Corn Steep Liquor 3 
Adjust pH to 7.0 
Transformation Medium C 
Mannitol 5 
Glycerol 5 
Hycase SF 2 
Beef extract 1 
Corn Steep Liquor 3 
Adjust pH t 7.0 
______________________________________ 
The produced 5-(1-hydroxy)ethyl and 6-hydroxymethyl oxidation products can 
be recovered from the culture medium by conventional means which are 
commonly used for the recovery of other known biologically active 
substances. The 5-(1-hydroxyethyl) and 6-hydroxymethyl oxidation products 
are found in the cultured mycelium and filtrate, which are obtained by 
filtering or centrifuging the cultured broth, and accordingly can be 
isolated and purified from the mycelium and the filtrate by a conventional 
method such as concentration under reduced pressure, lyophilization, 
extraction with a conventional solvent, such as methylene chloride and the 
like, pH adjustment, treatment with a conventional resin (e.g. anion or 
cation exchange resin, non-ionic adsorption resin, etc.), treatment with a 
conventional adsorbent (e.g. activated charcoal, silicic acid, silica gel, 
cellulose, alumina, etc.), crystallization, recrystallization, and the 
like. A preferred recovery method is solvent extraction, particularly 
using methylene chloride. A preferred purification method involves the use 
of chromatography, especially HPLC, using a silica gel column and an 
eluant mixture composed of water and an organic solvent such as methanol, 
acetonitrile and the like. A preferred eluant is composed of water and 
acetonitrile and is run through the column in a linear gradient. 
Compounds (I) and (II) of the present invention are useful in the 
inhibition of HIV reverse transcriptase, the prevention or treatment of 
infection by the human immunodeficiency virus (HIV) and the treatment of 
consequent pathological conditions such as AIDS. Treating AIDS or 
preventing or treating infection by HIV is defined as including, but not 
limited to, treating a wide range of states of HIV infection: AIDS, ARC 
(AIDS related complex) both symptomatic and asymptomatic, and actual or 
potential exposure to HIV. For example, the compounds of this invention 
are useful in treating infection by HIV after suspected past exposure to 
HIV by e.g., blood transfusion, organ transplant, accidental needle stick, 
exchange of body fluids, bites or exposure to patient blood during 
surgery. 
Reverse Transcriptase Assay 
The assay measures the incorporation of tritiated deoxyguanosine 
monophosphate by recombinant HIV reverse transcriptase (HIV RT.sub.R) (or 
other RT) into acid-precipitable cDNA at the Km values of dGTP and poly 
r(C).oligo d(G).sub.12-18. Compounds (I) and (II) of the present invention 
inhibit this incorporation. 
Thirty uL of a reaction mixture containing equal volumes of: 500 mM 
Tris.HCl (pH 8.2), 300 mM MgCl.sub.2, 1200 mM KCl, 10 mM DTT, 400 .mu.g/mL 
poly r(c).oligo d(G) [prepared by dissolving 1.5 mg (25 U) poly r(C).oligo 
d(G) in 1.5 ml sterile distilled H.sub.2 O and diluting to 400 .mu.g/ml], 
0.1 .mu.Ci/.mu.l [.sup.3 H] dGTP, 160 .mu.M dGTP, was added to 10 .mu.l 
sterile distilled H.sub.2 O, 2.5 .mu.l of potential inhibitor. Ten .mu.L 
of 3.2 nM purified HIV RT.sub.R were added to start the reaction. The 
mixture was incubated at 37.degree. C. for 45 minutes. 
After incubation was complete, the tubes were cooled in ice for 5 minutes. 
Ice-cold 13% TCA containing 10 mM NaPP.sub.i (200 .mu.l) was added and the 
mixture incubated on ice for 30 minutes. The precipitated cDNA was removed 
by filtration using presoaked glass filters [TCA, NaPP.sub.i ]. The 
precipitate was then washed with 1N HCl, 10 mM NaPP.sub.i. The filter 
discs were then counted in a Packard scintillation counter. 
Under these conditions [dGTP] and poly r(C).oligo d(G).sub.12-18 each are 
approximately equal to the appropriate Km value. Approximately 5-6,000 cpm 
of [.sup.3 H] GMP are incorporated into acid-precipitable material. The RT 
reaction is concentration- and time-dependent. DMSO (up to 5%) does not 
affect enzyme activity. 
Using the methodology described above, compounds (I) and (II) were 
evaluated. The calculated IC.sub.50 of compound (I) was found to be about 
18.7 .mu.M and that of compound (II) about 44 nM, thereby demonstrating 
and confirming the utility of compounds (I) and (II) as effective HIV 
reverse transcriptase inhibitors.

EXAMPLE 1 
Preparation of Substrate Compound (III) 
3-[2-(Benzoxazol-2-yl)ethyl]-5-ethyl-6-methyl-2-(1H)-pyridinone 
Step A: Preparation of 3-cyano-5-ethyl-6-methyl-2-(1H)-pyridinone 
According to the method described in J. Heterocyclic Chem., 24, 351 (1987), 
a mixture of 2-ethyl-3-oxobutanal, sodium salt (37.5 g, 0.275 mmol), 
cyanoacetamide (25.2 g, 0.30 mol), aqueous piperidinium acetate (22 mL) 
[prepared from glacial acetic acid (4.2 mL), water (10 mL) and piperidine 
(7.2 mL)] in water (775 ml) was refluxed for four hours. Glacial acetic 
acid (30 ml) was added cautiously (much foaming) as the product 
precipitated. Upon cooling to room temperature, the product was collected 
by filtration, washed with cold water and air dried. The product had m.p. 
of 237.degree.-240.degree. C. 
Step B: Preparation of 2-chloro-3-cyano-5-ethyl-6-methylpyridine 
3-Cyano-5-ethyl-6-methyl-2-(1H)-pyridinone (22.9 g, .141 mol) and 
phosphorus pentachloride (33.1 g, 0.159 mol) were intimately mixed and 
heated at 110.degree.-120.degree. C. for one hour. The liquified solids 
were poured onto crushed ice and water and the semi-solid was extracted 
into chloroform. This extract was washed with water, saturated aqueous 
NaHCO.sub.3, dried (Na.sub.2 SO.sub.4), filtered and evaporated. This 
amber oil was dissolved in hexane and the insoluble material was removed 
when filtered through a pad of charcoal. Removal of the solvent gave a 
light yellow oil which solidified (17.7 g). Trituration of this solid with 
cold hexane yielded pure product, m.p. 63.degree.-64.degree. C. 
Step C: Preparation of 2-methoxy-3-cyano-5-ethyl-6-methylpyridine 
Sodium metal (3.25 g, .141 mol) was dissolved in dry methanol (100 mL) 
under a nitrogen atmosphere. When solution was complete, a slurry of 
2-chloro-5-ethyl-6-methylpyridine (17.95 g, 99.4 mmol) in dry methanol (70 
mL) was added and the reaction was warmed at 60.degree. C. for 15-20 
hours. After cooling the reaction mixture, diethyl ether (250 mL) and 
water (200 mL) were added. The ether layer was separated and washed with 
water, dried (Na.sub.2 SO.sub.4), filtered and evaporated to give a light 
yellow solid (17.5 g). This solid was triturated with cold hexane to yield 
pure product, m.p. 59.degree.-61.degree. C. 
Step D: Preparation of 2-methoxy-5-ethyl-6-methylnicotinaldehyde 
To a solution of 2-methoxy-3-cyano-5-ethyl-6-methylpyridine (1.0 g, 5.68 
mmol) in dry tetrahydrofuran (50 mL) under a nitrogen atmosphere and 
cooled to -70.degree. C., was added 1.3M diisobutyl aluminum hydride/THF 
(17.4 mL, 22.7 mmol). The resulting mixture was allowed to warm to room 
temperature and stir for 15-20 hours. The reaction mixture was acidified 
with 1N hydrochloric acid and then neutralized with aqueous sodium 
bicarbonate. Water was then added and the product extracted into diethyl 
ether. The etheral extract was dried (Na.sub.2 SO.sub.4), filtered and the 
solvent evaporated. This residue was flash chromatographed on silica gel 
eluting with 10% diethyl ether/pentane to give the product. 
Step E: Preparation of 
2-[2(R/S)-hydroxy-2-(2-methoxy-5-ethyl-6-methyl-pyridin-3-yl)ethyl]benzoxa 
zole 
To a solution of 2-methylbenzoxazole (226 mg, 1.7 mmol) in anhydrous THF (4 
mL), cooled to -100.degree. C. under an argon atmosphere, was added 1.6M 
n-butyllithium/hexane (1.05 mL) slowly over 35 minutes. After 0.5 hour a 
solution of 2-methoxy-5-ethyl-6-methylnicotinaldehyde (300 mg, 1.7 mmol) 
in dry THF (1 mL) was added dropwise. The reaction was allowed to warm to 
room temperature and poured onto crushed ice. This mixture was extracted 
with diethyl ether. The combined extracts were dried (MgSO.sub.4) and the 
solvent removed to give an oil which was flash chromatographed over silica 
gel. Elution with ethyl acetate/hexane (1:19) gave analytically pure 
racemic product, mp 102.degree.-103.degree. C. 
Anal. Calcd for C.sub.18 H.sub.20 N.sub.2 O.sub.3. 0.1 H.sub.2 O: C, 68.81; 
H, 6.48; N, 8.92. Found: C, 68.80; H, 6.76; N, 8.95. 
Step F: Preparation of 
3-[2-(benzoxazol-2-yl)ethenyl]-5-ethyl-6-methyl-2-(1H)-pyridinone. 
A mixture of 
2-[2(R/S)-hydroxy-2-(2-methoxy-5-ethyl-6-methylpyridin-3-yl)ethyl]benzoxaz 
ole (72 mg, 0.23 mmol) and pyridine hydrochloride (133 mg, 1.2 mmol), under 
a nitrogen atmosphere, was placed in a preheated oil bath (165.degree. C.) 
for 5 minutes. The reaction flask was removed, cooled, and water added to 
give a solid. This crude product was extracted into chloroform, dried 
(MgSO.sub.4) and the solvent evaporated to yield pure product. 
Recrystallization from methanol gave analytically pure product, mp 
262.degree.-264.degree. C. 
Anal. Calcd for C.sub.17 H.sub.16 N.sub.2 O.sub.2 : C, 72.83; H, 5.75; N, 
10.00. Found: C, 72.93; H, 5.95; N, 9.99. 
Step G: Preparation of 
3-[2-(benzoxazol-2-yl)ethyl]-5-ethyl-6-methyl-2-(1H)-pyridinone 
A solution of 80% pure 
3-[2-(benzoxazol-2-yl)ethenyl]-5-ethyl-6-methyl-2-(1H)-pyridinone (200 mg) 
in methanol/ethanol/THF (25 mL, 1:1:1) was hydrogenated at atmospheric 
pressure over 5% palladium/charcoal for four hours. After filtering off 
the catalyst, the solvents were evaporated and the residue flash 
chromatographed over silica gel. Elution with 2% methanol-98% chloroform 
gave analytically pure product, mp 155.degree.-156.5.degree. C. 
Anal. Calcd. for C.sub.17 H.sub.18 N.sub.2 O.sub.2 : C, 72.31; H, 6.43; N, 
9.92. Found: C, 72.45; H, 6.52; N, 9.99. 
EXAMPLE 2 
Microorganism and Culture Conditions 
A frozen vial (2.0 ml) of culture (MA 6559) ATCC No. 53771 was used to 
inoculate a 250 ml baffled shake flask containing 50 ml of an autoclaved 
(sterilized) seed medium consisting of (in units of grams/liter) dextrin 
10.0, dextrose 1.0, beef extract 3.0, ardamine pH (Yeast Products, Inc.) 
5.0, N-Z Amine type E 5.0, MgSO.sub.4.7H.sub.2 O 0.05, K.sub.2 HPO.sub.4 
0.3, and CaCO.sub.3 0.5. The pH of the seed medium was adjusted to 7.1 
before autoclaving. The seed was incubated in the seed medium at 
27.degree. C. for 24 hours on a rotary shaker operating at 220 rpm. A 2.5 
ml aliquot of the resulting seed medium was used to inoculate a 250 ml 
non-baffled shake flask containing 50 ml of the following previously 
autoclaved (sterilized) transformation medium B..sup.1 A DMSO solution of 
substrate compound (III) was added to the fermentation at zero hour to 
achieve a final concentration of 0.05 mg/ml. The shake flask contents were 
subsequently incubated for 24 hours at 27.degree. C. on a rotary shaker 
operating at 220 rpm. This procedure was followed three times and the 
three resultant broths were combined for isolation and purification. 
1. Transformation medium B consisted of (in grams/liter) glucose 10.0; 
Hycase SF 2.0; beef extract 1.0; corn steep liquor 3.0; where the pH was 
adjusted to 7.0 before autoclaving. 
Isolation and Purification Procedure for the Broth 
The whole broth (150 ml) of transformation media B was extracted three 
times with methylene chloride (3.times.150 ml). Methylene chloride 
extracts were combined, dried over sodium sulfate, and concentrated under 
vacuum to an oily residue. The residue was dissolved in methanol and 
subjected to high performance liquid chromatography (HPLC) purification. 
HPLC was carried out on Whatman Partisil 10 ODS-3, 9.4 mm.times.25 cm 
column at room temperature and monitored at 250 nm. The column was 
developed at 3 ml/min with linear gradient from H.sub.2 O--CH.sub.3 CN, 
80:20, to H.sub.2 O--CH.sub.3 CN, 20:80 in 30 minutes. The compounds were 
collected during repeated injections of the above described extract. The 
fractions at retention time 9.1 and 12.6 minutes were pooled respectively, 
and evaporated to remove solvents to yield 2.5 mg. of compound (I) 
characterized as the 5-(1-hydroxy)ethyl oxidation product, and 150 .mu.g 
of compound (II) characterized as the 6-hydroxymethyl oxidation product, 
respectively. 
EXAMPLE 3 
Preparation of Compound (II) 
3-[2-(benzoxazol-2-yl)ethyl]-5-ethyl-6-hydroxymethyl-2(1H)-pyridinone 
Step A: Preparation of 4-benzyloxy-3-oxo-2-ethylbutanal 
A solution of 90% pure 1-(N-morpholino)-1-butene (9.3 g, 60 mmol) and 
triethylamine (8.4 mL, 60 mmol) in tetrahydrofuran (85 mL), under a 
nitrogen atmosphere, was warmed to 70.degree. C. and benzyloxyacetyl 
chloride (9.45 mL, 60 mmol) was added dropwise via syringe. This cloudy 
yellow solution was warmed for 45 minutes and then cooled to room 
temperature. An aqueous solution of 10% HCl (75 mL) was added and the two 
phase mixture was stirred for 1 hour. This mixture was partitioned into 
methylene chloride and the organic layer was separated, dried (Na.sub.2 
SO.sub.4), filtered and the solvent evaporated to give a yellow oil (14.7 
g) which contained 80% desired product. This oil was used directly in the 
next step. 
Step B: Preparation of 3-cyano-5-ethyl-6-benzyloxymethyl-2(1H)-pyridinone 
Acetic acid (2.0 mL, 35 mmol) was added to a solution of crude 
4-benzyloxy-3-oxo-2-ethylbutanal (6.4 g, .about.24 mmol) and malononitrile 
(2.22 g, 33 mmol) in ethanol (30 mL) followed by dropwise addition of 
piperidine (2.37 mL, 24 mmol) to give a dark reddish brown solution. After 
stirring for 14 hours, a copious precipitate formed. This mixture was 
warmed at 70.degree. C. for 15 hours and then allowed to cool to room 
temperature. This blackish mixture was diluted with ethanol and the 
precipitated product was filtered, rinsed with ethanol and diethyl ether 
to give off-white product with m.p. of 141.degree.-143.degree. C. 
Additional material was obtained by evaporation of the filtrate. The 
residue was extracted into chloroform, and after washing the extract with 
saturated aqueous NaHCO.sub.3, filtered through a pad of charcoal. This 
amber solution was evaporated and the residue triturated with diethyl 
ether to give additional product. 
Step C: Preparation of 
2-benzyloxy-3-cyano-5-ethyl-6-benzyloxymethylpyridine 
To a partial suspension of 
3-cyano-5-ethyl-6-benzyloxymethyl-2(1H)-pyridinone (1.36 g, 5.08 mmol) in 
dry benzene (20 mL) was added benzyl bromide (0.80 mL, 6.7 mmol) and then 
silver carbonate (1.43 g, 5.2 mmol). This mixture was covered with 
aluminum foil and allowed to stir at room temperature. After 24 hours, the 
reaction was estimated to be .about.80% complete. Additional silver 
carbonate (0.40 g, 1.45 mmol) was added and the mixture was stirred for 
another 24 hours until complete. The silver salts were removed by 
filtration, rinsed with benzene and the combined benzene washings were 
evaporated to give the pure product as an oil. 
Step D: Preparation of 2-benzyloxy-5-ethyl-6-benzyloxymethylnicotinaldehyde 
To a solution of 2-benzyloxy-3-cyano-5-ethyl-6-benzyloxymethylpyridine 
(2.57 g, 6.2 mmol) in dry toluene (10 mL), under a nitrogen atmosphere in 
an ice/acetone bath, was added dropwise a solution of 1.5M 
diisobutylaluminum hydride/toluene (4.6 mL, 6.9 mmol). After stirring at 
room temperature for 15 hours, the reaction was cautiously poured into 10% 
HCl (30 mL), stirred for 0.5 hours and the product extracted into diethyl 
ether. The ethereal solution was dried, filtered through a pad of charcoal 
and evaporated to give a pale yellow oil. This oil was further purified by 
passing a benzene solution through a plug of silica gel to give pure 
product upon evaporation. 
Step E: Preparation of 
3-[2-(benzoxazol-2-yl)ethenyl]-5-ethyl-6-benzyloxymethyl-2-benzyloxypyridi 
ne 
To a suspension of [(benzoxazol-2-yl)methyl]triphenylphosphonium chloride 
(1.77 g, 4.12 mmol) (prepared using substantially the same procedures 
described in Example 5, but substituting 2-aminophenol and the 
intermediate thereof for the 2,5-dimethyl-6-aminophenol and its 
corresponding intermediate used therein), in dry tetrahydrofuran (20 mL), 
under a nitrogen atmosphere, was added 60% NaH/mineral oil (0.42 g, 10 
mmol). After 0.5 hour a solution of 
2-benzyloxy-5-ethyl-6-benzyloxymethylnicotinaldehyde (1.44 g, 4.0 mmol) in 
tetrahydrofuran (10 mL) was added and the reaction mixture was refluxed 
for 23 hours. The reaction was cooled, neutralized with acetic acid and 
partitioned between water and chloroform. The chloroform layer was washed 
with NaHCO.sub.3 solution, dried, filtered through a pad of charcoal and 
evaporated. This material was dissolved in chloroform and flash 
chromatographed through silica gel to give pure product as a yellow oil. 
The product was a mixture of cis and trans olefins. 
Step F: Preparation of 
3-[2-(benzoxazol-2-yl)ethyl]-5-ethyl-6-benzyloxymethyl-2(1H)-pyridinone 
A solution of cis/trans 
3-[2-(benzoxazol-2-yl)ethenyl]-5-ethyl-6-benzyloxymethyl-2-benzyloxypyridi 
ne (1.40 g, 2.94 mmol) in dry tetrahydrofuran (20 mL) and methanol (15 mL) 
containing 10% Pd on charcoal (215 mg) as a catalyst was hydrogenated at 
atmospheric pressure for 20 hours. The catalyst was filtered off and the 
solution evaporated to give a white residue. This residue was triturated 
with diethyl ether to give pure product with m.p. of 
140.degree.-142.degree. C. 
Step G: Preparation of 
3-[2-(benzoxazol-2-yl)ethyl]-5-ethyl-6-hydroxymethyl-2(1H)-pyridinone 
A solution of 
3-[2-(benzoxazol-2-yl)ethyl]-5-ethyl-6-benzyloxymethyl-2(1H)-pyridinone 
(198 mg, 0.51 mmol) in dry methylene chloride (6 mL) was cooled in an 
ice/acetone bath and 1M boron tribromide/hexane (1.5 mL, 1.5 mmol) was 
added dropwise to give a white precipitate. This suspension was stirred 
for one hour and then the reaction was quenched by addition of saturated 
aqueous NaHCO.sub.3 (10 mL). After stirring for 0.5 hours, the product was 
extracted into CH.sub.2 Cl.sub.2, dried, filtered and the solvent 
evaporated to give a solid. Trituration with diethyl ether gave product as 
a tan solid. Recrystallization from ethyl acetate afforded a light yellow 
solid with m.p. of 155.degree.-156.degree. C. 
Anal. Calcd. for C.sub.17 H.sub.18 N.sub.2 O.sub.3 : C, 68.44; H, 6.08; N, 
9.39. Found: C, 68.15; H, 6.03; N, 9.07. 
EXAMPLE 4 
Preparation of Compound 
(I):(+/-)-3-[2-(benzoxazol-2-yl)ethyl]-5-(1-hydroxyethyl)-6-methyl-2(1H)-p 
yridinone 
Step A: 2-Benzyloxy-5-acetyl-6-methylpyridin-3-carbonitrile 
A mixture of 3-cyano-5-acetyl-6-methyl-2(1H)-pyridinone (1.76 g, 0.01 mol) 
(L. Mosti et al, J. Het. Chem. 22, 1503 (1985)), benzyl bromide (2.12 g, 
0.012 mol), silver carbonate (3.06 g, 0.011 mol) in benzene was stirred at 
room temperature under a nitrogen atmosphere overnight, while protected 
from light. The following day the mixture was filtered, and the solids 
washed well with benzene. Evaporation of the filtrate gave the desired 
intermediate, mp 94.degree.-99.degree. C. 
Anal. Calc'd. for C.sub.16 H.sub.14 N.sub.2 O.sub.2, MW 266.299 C, 72.17; 
H, 5.30; N, 10.52. Found: C, 71.97; H, 5.30; N, 10.19. 
Step B: 2-Benzyloxy-5-(1-hydroxyethyl)-6-methylpyridin-3-carbonitrile 
Sodium borohydride (0.35 g, 0.0092 mol) was added at room temperature to 
2-benzyloxy-5-acetyl-6-methylpyridin-3-carbonitrile (2.44 g, 0.0092 mol) 
in ethanol (60 mL). After 2 hours chloroform was added plus a few drops of 
acetic acid. After washing with water and brine, the chloroform solution 
was evaporated to give product which was not purified further. 
Step C: 2-Benzyloxy-5-(1-hydroxyethyl)-6-methylpyridin-3-carboxaldehyde 
To a mixture of 
2-benzyloxy-5-(1-hydroxyethyl)-6-methylpyridin-3-carbonitrile (1.00 g, 
0.0037 mol) in toluene (25 mL), cooled in an ice-acetone bath, was added 
dropwise diisobutylaluminum hydride (1.5M in toluene) (5.22 mL, 0.0078 
mol). After stirring for one hour under nitrogen the mixture was worked up 
by quenching into a mixture of excess 1N hydrochloric acid and ice. 
Extraction with chloroform followed by washing with water, saturated 
sodium bicarbonate solution, and then brine, and evaporation, gave the 
desired product. 
Step D: 
3-[2-(benzoxazol-2-yl)ethenyl]-5-(1-hydroxyethyl)-6-methyl-2-benzyloxypyri 
dine 
Following substantially the same procedure described in Example 3, Step E, 
but substituting 
2-benzyloxy-5-(1-hydroxyethyl)-6-methylpyridin-3-carboxaldehyde for the 
benzyloxynicotinaldehyde compound used therein, the title compound is 
obtained. 
Step E: 
(+/-)-3-[2-(Benzoxazol-2-yl)ethyl]-5-(1-hydroxyethyl)-6-methyl-2(1H)-pyrid 
inone 
Following substantially the same procedure described in Example 3, Step F, 
but substituting 
3-[2-(benzoxazol-2-yl)ethenyl]-5-(1-hydroxyethyl)-6-methyl-2-benzyloxypyri 
dine for the benzyloxypyridine compound used therein, the title compound is 
obtained. 
EXAMPLE 5 
Preparation of [(4,7-dimethylbenzoxazol-2-yl)methyl]triphenylphosphonium 
chloride 
Step A: Preparation of 2-chloromethyl-4,7-dimethylbenzoxazole 
To a solution of 2,5-dimethyl-6-aminophenol (0.67 g, 4.9 mmol) in methylene 
chloride, solid ethyl 2-chloroiminoacetate hydrochloride (0.85 g, 4.9 
mmol) was added. The resultant slurry was stirred at room temperature for 
18 hours, then filtered through a plug of diatomaceous earth and 
concentrated under reduced pressure (15 torr). The solid residue was 
subjected to column chromatography on silica gel (50 g, eluted with 1% 
methanol in chloroform). Collection and concentration of appropriate 
fractions yielded the title benzoxazole. 
Step B: Preparation of 
[(4,7-dimethylbenzoxazol-2-yl)methyl]-triphenylphosphonium chloride 
The product of Step A above was heated with an equimolar amount of 
triphenylphosphine in refluxing toluene for 15-25 hours to obtain the 
title compound. 
EXAMPLE 6 
Characterization 
The structures of the two biotransformation products, compounds (I) and 
(II), were determined by proton NMR run in CD.sub.3 OD. Compound (II) was 
shown to result from hydroxylation of the methyl group, and compound (I) 
from hydroxylation at --CH.sub.2 -- of the ethyl side chain. 
______________________________________ 
##STR6## 
Compound R.sup.1 
R.sup.2 
______________________________________ 
(I) OH H 
(II) H OH 
(III) H H 
______________________________________ 
Hydroxylation of the methyl group in (II) was indicated by the 
disappearance of the methyl singlet near 2.2 ppm of substrate compound 
(III) and the appearance instead of a new singlet at .about.4.45 ppm 
ascribable to the CH.sub.2 OH protons. 
Hydroxylation of C-l of the ethyl side chain in compound (I) was evident 
from the appearance of the terminal methyl group as a doublet (rather than 
a triplet), and the appearance of a CHOH quartet near 4.8 ppm instead of 
the methylene quartet at .about.2.25 ppm in the parent compound. 
While the foregoing specification teaches the principles of the present 
invention, with examples provided for the purpose of illustration, it will 
be understood that the practice of the invention encompasses all of the 
usual variations, adaptations, and modifications, as come within the scope 
of the following claims and its equivalents.