TAN-1251 compounds and their production from penicillium thomii

A compound of the formula ##STR1## wherein R.sup.1 is hydrogen or a hydrocarbon residue which may be substituted; R.sup.2 is oxo or hydrogen plus hydroxy which may be acylated; R.sup.3 is hydrogen or hydroxy which may be acylated; at least one of the dotted lines represents a single bond, or a salt thereof, produced from Penicillum thomii has potent RA-89 muscarinic receptor blocking activity and is of value as therapeutic agent for parkinsonism, ulcer, etc. or as mydriatics.

FIELD OF THE INVENTION 
The present invention relates to a novel compound TAN-1251 (hereinafter 
sometimes referred to briefly as TAN-1251 which, unless otherwise 
indicated, means a series of TAN-1251 compounds) which is a muscarinic 
receptor blocking compound of value as a mydriatic or as an 
antispasmodic/antiulcer agent. 
BACKGROUND OF THE INVENTION 
The parasympathetic nerve inervates a diversity of peripheral tissues to 
manifest multi-facted activity. The receptors of acetylcholine, which is 
its neurotransmitter, are roughly classified into muscarinic receptors 
sensitive to muscarine which is an alkaloid of Amanita muscaria and 
nicotinic receptors sensitive to nicotine which is an alkaloid occurring 
in Nicotiana tabacum. Atropine and scopolamine, which are the alkaloids of 
the belladonna plants, have been utilized for centuries as nonspecific 
antimuscarinic drugs and are in use even today as mydriatics or 
antispasmodics [Goodman and Gilman's the Pharmacological Basis of 
Therapeutics, 7th ed., 130 (1985)]. 
It is known that muscarinic receptors can be classified into subtypes, i.e. 
M1 which has a high affinity for pirenzepine which is an 
antispasmodic/antiulcer agent and M2 which is low in that affinity [Nature 
283, 90 (1980)]. Furthermore, a further ramification of muscarine M2 
receptors according to their affinity for AF-DX 116 has been proposed 
[Life Sciences 38, 1653 (1986) and Clinical and Experimental Pharmacology 
and Physiology 16, 523 (1989)], and much research is in progress on 
methoctoramine and other compounds having subtype specificity [Trends in 
Pharmacological Science 9, 216 (1988)]. 
On the other hand, muscarinic receptor genes have been cloned by genetic 
engineering techniques [FEBS Letters, 235, 257 (1986)] and so far at least 
5 kinds of genes have been reported to exist in man. While the 
pharmacologic correspondence of them to receptors remains yet to be 
elucidated, it will not be long before it is clarified [Trends in 
Pharmacological Science 12, 148 (1989)]. It is expected that muscarinic 
blockers having novel pharmacologic activity will be developed in this 
milieu. 
Under the circumstances, search for muscarinic receptor blocking substances 
in microbial metabolites is also in progress. In fact, Eulissin, Argvalin, 
IJ2702-I & 2702-II and PF6766 [Journal of The Agricultural Chemical 
Society of Japan, 62, 338 (1988)] have already been reported but none of 
them have sufficiently potent activity. 
In the above-mentioned situation, the inventors of the present invention 
explored microbial metabolites for novel compounds which would exhibit 
potent muscarinic blocking activity and discovered a TAN-1251 series of 
compounds having strong antimuscarinic activity. TAN-1251 is comprised of 
4 species which the inventors designated TAN-1251A, B, C and D. Subsequent 
research revealed that these compounds have the following structures: 
##STR2## 
The inventors of the present invention further conducted degradation and 
derivatizing experiments using TAN-1251A, B, C and D as starting compounds 
and examined the biological activity of the degradation products and 
derivatives. As a result, they found that the following compounds were 
promising candidates for antispasmodic/antiulcer agents. The finding 
prompted further study which has resulted in the present invention. 
SUMMARY OF THE INVENTION 
Thus, the present invention is directed to: 
1. A compound of the formula 
##STR3## 
wherein R.sup.1 is hydrogen or a hydrocarbon residue which may be 
substituted; R.sup.2 is oxo or hydrogen and a hydroxy which may be 
acylated; R.sup.3 is hydrogen or hydroxy which may be acylated; at least 
one of the dotted lines represents a single bond, or a salt thereof. 
2. A method of producing a compound of the formula 
##STR4## 
wherein R.sup.3' is hydrogen or hydroxy; at least one of the dotted lines 
represents a single bond; provided that where R.sup.3' is hydroxy, the 
dotted line on the right-hand side represents a single bond and the one on 
the left-hand side represents a double bond, or a salt thereof, 
characterized by culturing a strain of microorganism belonging to the 
genus Penicillium and capable of producing at least one compound having 
the above general formula in a culture medium and harvesting the same from 
the resulting culture broth. 
3. A method of producing a compound of the general formula 
##STR5## 
wherein the respective symbols have the meanings defined below, or a salt 
thereof characterized in that a compound of the general formula 
##STR6## 
wherein R.sup.1' is 3-methyl-2-butenyl or 3-methylbutyl; R.sup.2 is oxo or 
hydrogen and hydroxy which may be acylated; R.sup.3 is hydrogen or hydroxy 
which may be acylated; at least one of the dotted lines represents a 
single bond, or a salt thereof is treated with an acid. 
4. A method of producing a compound of the general formula 
##STR7## 
wherein the respective symbols have the meanings defined below, or a salt 
thereof, characterized by reducing a compound of the general formula 
##STR8## 
wherein R.sup.1 is hydrogen or a hydrocarbon residue which may be 
substituted; R.sup.3 is hydrogen or hydroxy which may be acylated; at 
least one of the dotted lines represents a single bond, or a salt thereof. 
5. A method of producing a compound of the general formula 
##STR9## 
wherein the respective symbols have the meanings defined below 
characterized in that a compound of the general formula 
##STR10## 
wherein R.sup.2 is oxy or hydrogen and hydroxy which may be acylated; 
R.sup.3 is hydrogen or hydroxy which may be acylated, or a salt thereof is 
catalytically reduced. 
6. A method of producing a compound of the general formula 
##STR11## 
wherein the respective symbols have the meanings defined hereinbefore or 
below, characterized in that a compound of general formula (I) is reacted 
with a compound of the general formula 
EQU R.sup.4 -X (IX) 
wherein R.sup.4 is alkyl; X is halogen. 
7. A method of producing a compound of the general formula 
##STR12## 
wherein the respective symbols have the meanings defined below, or a salt 
thereof, characterized in that a culture broth, as it is or as processed, 
of a microorganism of the genus Penicillium is allowed to contact a 
compound of the general formula 
##STR13## 
wherein R.sup.1 is a hydrogen or a hydrocarbon residue which may be 
substituted; R.sup.2 is oxo or a hydrogen and a hydroxy which may be 
acylated; at least one of the dotted lines represents a single bond, or a 
salt thereof. 
8. An antispasmodic/antiulcer composition containing a compound of the 
formula (I) or a pharmacologically acceptable salt thereof. 
9. An antispasmodic composition containing a compound of the formula (I) or 
a pharmacologically acceptable salt thereof. 
10. An antiulcer composition containing a compound of the formula (I) or a 
pharmacologically acceptable salt thereof.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
Referring to the hydrocarbon residue which may be substituted, i.e. R.sup.1 
in the above general formulas, preferred examples of the hydrocarbon group 
are straight-chain or branched alkyl, alkenyl and alkynyl groups of 1 to 6 
carbon atoms. Thus, the alkyl group includes, inter alia, methyl, ethyl, 
propyl, isopropyl, butyl, 1-methylpropyl, 2-methylpropyl, t-butyl, pentyl, 
2-methylbutyl, 3-methylbutyl, hexyl, 4-methylpentyl, etc. The alkenyl 
group includes, inter alia, 2-propyl, 2-butenyl, 3-butenyl, 
2-methyl-2-propenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 
2-methyl-2-butenyl, 3-methyl-2-butenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 
5-hexenyl, 3-methyl-2-pentenyl, 4-methyl-3-pentenyl, etc. The alkynyl 
group includes, inter alia, 2-propyl, 1-methyl-2-propynyl, 2-butynyl, 
3-butynyl, 1-methyl-2-butynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 
2-methyl-3-pentynyl, 2-hexynyl and so on. The substituent or substituents 
thereon include, inter alia, C.sub.3-6 cycloalkyl (e.g. cyclopropyl, 
cyclobutyl, cyclopentyl, cyclohexyl, etc.), phenyl which may be 
substituted (e.g. phenyl, o-hydroxyphenyl, m-hydroxyphenyl, 
p-hydroxyphenyl, etc.), hydroxy, mercapto, C.sub.1-3 alkylthio (e.g. 
methylthio, ethylthio, propylthio, etc.), carboxy, guanidino, amino, 
imidazolyl and so on. 
As examples of such substituted hydrocarbon groups, there may be mentioned 
cyclohexylmethyl, benzyl, p-hydroxybenzyl, hydroxymethyl, mercaptomethyl, 
1-hydroxyethyl, 2-methylthioethyl, carboxymethyl, 2-carboxyethyl, 
3-quanidino-propyl, 4-aminobutyl, 4-imidazolylmethyl, etc. 
Referring to the hydroxy which may be acylated, as represented by R.sup.2 
and R.sup.3 in the above general formulas, the acyl group includes 
aromatic acyl groups such as phthaloyl, p-nitrobenzoyl, 
p-tert-butylbenzoyl, p-tert-butylbenzensulfonyl, benzensulfonyl, 
toluenesulfonyl, etc. and aliphatic acyl groups such as formyl, actyl, 
propionyl, monochloroacetyl, dichloroacetyl, trichloroacetyl, 
methanesulfonyl, ethanesulfonyl, trifluoroacetyl, malonyl, succinyl and so 
on. 
The salts of the above-mentioned compounds include, inter alia, salts of 
the conventional kinds, such as the corresponding hydrochlorides, 
sulfates, phosphates, etc., and the quaternary salts with the nitrogen 
atom in 4-position, and these salts can be produced by the processes 
described hereinafter. 
The microorganism to be employed for the production of TAN-1251A, B, C 
and/or D in accordance with the present invention may be any microorganism 
belonging to the genus Penicillium and capable of producing TAN-1251A, B, 
C and/or D. As an example of such microorganism, there may be mentioned 
Penicillium thomii RA-89 which the inventors isolated from the soil in 
Miyagi Prefecture. The micrological characteristics of this strain are as 
follows. 
(a) Morphological characteristics 
The RA-89 strain shows good growth on malt extract agar, potato glucose 
agar and other media, producing abundant conidia. The hyphae are 
transparent and septate, and conidiophores are adnate from the substrate 
and aerial mycellia. The conidiophores show little branching but occur 
singly and are more than 200 .mu.m long, each having a rough surface with 
spikes and a swollen end. Detected at the end of the conidiophore are more 
than 10 phialides in a brush-like formation (penicilli), with tens of 
conidia arranged in brush-like chains. The conidia are oblong or oval, 
measuring 3.5.about.4.0.times.2.3.about.2.8 .mu.m, and have a rough 
surface. Moreover, the RA-89 strain forms a multiplicity of sclerotia. 
These sclerotia are amorphous, although primarily oval or spherical, 
measure about 300 .mu.m, and although white in an early stage of 
formation, turn brown with a tinge or orange on aging. Further observation 
was continued but the sclerotia did not ripen to form ascospores. 
(b) Cultural characteristics 
The strain was cultured on various media at 28.degree. C. for 2 weeks. The 
results are shown below in Tables 1 and 2. 
TABLE 1 
__________________________________________________________________________ 
Cultural characteristics of P. thomii RA-89 
Agar Growth on 
Reverse color 
Conidium Soluble 
Diameter 
medium 
medium 
of colony 
Formation 
Color 
Sclerotium 
pigment 
of colony 
__________________________________________________________________________ 
Malt Good, not 
Center: Good Gray Abundant 
None 58 
extract 
diffuse, 
red green (mm) 
velvety, 
yellow 
white Margin: 
Light 
yellow 
Potato 
Good, not 
Light scarlet 
Good Gray Abundant 
None 55 
glucose 
diffuse, green 
velvety, 
white 
Czapek 
Good, not 
Light brown to 
Good Gray Abundant 
None 50 
diffuse, 
light purple white 
velvety, Annular 
white green 
Sabouraud 
Good, not 
Yellow Poor White 
Occasion- 
None 40 
diffuse, ally 
felt-like, found 
white 
__________________________________________________________________________ 
TABLE 2 
__________________________________________________________________________ 
Cultural characteristics of P. thomii RA-89 
Agar Growth on 
Reverse color 
Conidium Soluble 
Diameter 
medium 
medium 
of colony 
Formation 
Color 
Sclerotium 
pigment 
of colony 
__________________________________________________________________________ 
Oatmeal 
Moderate, 
White to 
Good Gray 
Moderate 
None 55 
not light yellow green (mm) 
diffuse, 
velvety, 
white 
Synthetic 
Good, not 
White to 
Poor White 
Moderate 
None 45 
mucor 
diffuse, 
light yellow 
felt-like, 
white 
Yeast 
Moderate, 
White Good Gray 
Abundant 
None 55 
extract 
not green 
solble 
diffuse, 
starch 
velvety, 
white 
__________________________________________________________________________ 
(c) Physiological Characteristics 
The conditions for growth of P. thomii RA-89 were investigated using a 
potato glucose agar medium. The optimal temperature and pH for growth were 
25.degree..about.30.degree. C. and pH 4.about.5. The temperature range for 
growth was 5.degree.-32.5.degree. C. and the pH range for growth was pH 
3.about.pH 7. 
Comparing the above data, particularly morphological findings, with the 
descriptions in A Manual of the Penicillia (1949, The Williams and Wilkins 
Company) and The genus Penicillium and its Teleomorphic States 
Eupenicillium and Talaromyces (1979, Academic Press), the inventors 
identified the RA-89 strain as Penicillium thomii Maire. 
This strain Penicillium thomii RA-89 has been deposited with the Institute 
for Fermentation, Osaka (IFO) as of Dec. 25, 1989 under the deposit number 
of IFO-32288 and with Fermentation Research Institute of the Agency for 
Industrial Research and Technology, the Ministry of International Trade 
and Industry as of Feb. 7, 1990 under the deposit number of FERM BP-2753. 
The TAN-1251A, B, C and/or D-producing strains of microorganisms belonging 
to the genus Penicillium, like other fungi in general, can be caused to 
mutate by means of ultraviolet light, X-rays, and other radiations, by 
monospore isolation, or with various chemical mutagens, etc., and even the 
mutants so obtained and any spontaneous mutants can all be exploited for 
purposes of the present invention insofar as they cannot be substantially 
classified as strains of other species in view of the above taxonomical 
characteristics and as long as they retain the property to produce said 
particular compound or compounds. 
The media for culture of such producer strains may be fluid or solid, only 
provided that they contain nutrients which the strains require and can 
utilize. However, fluid media are preferred for mass culture. The media 
should contain sources of carbon and nitrogen which the particular strains 
used may digest and assimilate, minerals and trace nutrients in 
appropriate amounts. The useful carbon source includes, inter alia, 
glucose, lactose, sucrose, maltose, dextrin, starch, glycerin, mannitol, 
sorbitol, oils and fats (e.g. soybean oil, lard oil, chicken oil, etc.) 
n-paraffin and so on. The nitrogen source includes, inter alia, meat 
extract, yeast extract, dried yeast, soybean flour, corn steep liquor, 
peptone, cottonseed flour, spent molasses, urea, ammonium salts (e.g. 
ammonium sulfate, ammonium chloride, ammonium nitrate, ammonium acetate, 
etc.) and so on. In addition, salts containing sodium, potassium, calcium, 
magnesium, etc., salts of metals such as iron, manganese, zinc, cobalt, 
nickel, etc., salts of phosphoric acid, boric acid, etc. and salts of 
organic acids such as acetic acid, propionic acid, etc. are incorporated 
in appropriate amounts. The medium may further contain amino acids (e.g. 
glutamic acid, aspartic acid, alanine, lysine, methionine, proline, etc.), 
peptides (e.g. dipeptides, tripeptides, etc.), vitamins (e.g. B.sub.1, 
B.sub.2, nicotinic acid, B.sub.12, C, etc.) and nucleic acids (e.g. 
purine, pyrimidine and their derivatives). Of course, an inorganic or 
organic acid or base or a buffer for pH adjustment and an oil or fat or a 
surfactant as an antifoam can also be incorporated in suitable amounts. In 
fluid culture, the pH of the medium is preferably controlled near neutral, 
particularly pH 5.5.about.7. The incubation temperature and time are 
preferably about 20.degree..about.30.degree. C. and about 48.about.168 
hours. 
The TAN-1251A, B, C and/or D which is produced with the progress of culture 
can be assayed by the radio receptor assay using a membrane fraction of 
the rat cerebral cortex as a crude receptor and .sup.3 
H-QNB[L-[N-methyl-.sup.3 H]-quinuclidinyl benzilate methyl chloride 
(Amersham, U.K.) as a radioligand. Generally speaking, the production of 
TAN-1251A, B, C and/or D reaches a maximum in 4 to 5 days of culture. 
TAN-1251, in general, being a basic and liposoluble substance, the product 
TAN-1251A, B, C and/or D can be harvested from the culture broth by 
utilizing these properties. Thus, since the respective component of 
TAN-1251 are accumulated in the filtrate of the broth, these compounds can 
be recovered by adjusting the filtrate to pH 7.about.11, preferably pH 
8.about.10 and extracting them with a water-immiscible organic solvent 
such as dichloromethane, ethyl acetate, methyl isobutyl ketone or isobutyl 
alcohol. On the other hand, if the filtrate is adjusted to pH 1.5.about.6, 
preferably pH 2.about.4, and said organic solvent is added thereto, the 
active substance or substances will be brought into the aqueous layer. By 
taking advantage of this principle, one may advantageously recover the 
active substances by solvent redistribution or chromatography using an 
adsorbent resin such as Amberlite XAD-II (Rohm and Haas Co., U.S.A.), 
Diaion HP-20 (Mitsubishi Kasei, Japan) or Diaion SP-207 (Mitsubishi Kasei, 
Japan). For elution of the activity from a column packed with such an 
adsorbent resin, water or a hydrous solvent such as aqueous methanol, 
aqueous acetone, etc. can be employed. By concentrating the resulting 
extract or eluate under reduced pressure, a crude product containing the 
various species of TAN-1251 is obtained. 
For isolation of the respective components of TAN-1251 by purification of 
the above crude product, a variety of chromatographic techniques can be 
employed with advantage. The stationary phase or packing material may be 
silica gel, cellulose, Sephadex LH-20 (Pharmacia, Sweden) or the like, and 
such packing material can be used in the manner of conventional column 
chromatography. Elution of the activity from such a column can be carried 
out using an appropriate organic solvent such as hexane, toluene, 
chloroform, ethyl acetate, dichloroethane, acetone, methanol, etc., as 
used independently or as a suitable mixture. The eluate thus obtained is 
concentrated to dryness or freeze-dried, or the concentrate is dissolved 
in an appropriate solvent such as diethyl ether, ethyl acetate or methanol 
or a mixture of such solvents and allowed to stand in the cold, whereby 
the respective species of TAN-1251 can be isolated as powders or crystals. 
If the powder obtained in the above manner is not sufficiently high in 
purity, the technique of high performance liquid chromatography (HPLC) can 
be advantageously employed for further purification. The stationary phase 
for HPLC may for example be a reverse-phase column octadecylsilane such as 
YMC Gel (YMC, Japan) or TSK Gel (Toso, Japan), while the mobile phase may 
for example be a mixture of acetonitrile or methanol with either an acid, 
an inorganic salt-containing solution or a buffer solution. 
Since each species of TAN-1251 is a basic substance, it can be treated with 
a suitable mineral acid to give the corresponding salt. The salt can be 
prepared in the per se known manner. Examples of such salt are the 
hydrochloride, sulfate, phosphate and so on. 
Below presented as the physicochemical properties of the respective species 
of TAN-1251 prepared in the Examples given hereinafter. 
TAN-1251A 
(1) Appearance: Colorless crystals 
(2) Melting point: 118.5.degree.-120.degree. C. 
(3) Specific rotation: -8.1.degree. (D line, c 0.42, methanol) 
(4) Molecular weight: m/z 381 (M+H).sup.+, (SI-mass spectrum) 
(5) Elemental analysis: (%)(for the hemihydrate) Found: C, 73.83; H, 8.56; 
N, 6.92 Calcd.: C, 74.00; H, 8.54; N, 7.19 
(6) Molecular formula: C.sub.24 H.sub.32 N.sub.2 O.sub.2 
(7) UV spectrum: Methanol (FIG. 1) Absorption maxima: 265.+-.3 nm 
(.epsilon.23,800.+-.3,000) 304.+-.3 nm (.crclbar.1,600.+-.400, shoulder) 
(8) IR spectrum: KBr disk (FIG. 2); dominant absorptions (wave-number, 
cm.sup.-1) 3420, 2980, 2940, 2800, 1720, 1600, 1500, 1450, 1380, 1300, 
1250, 1180, 1130, 1030, 920, 890, 830, 780, 750, 690, 620, 530, 510. 
(9) .sup.13 C NMR spectrum: 75 MHz, CDCl.sub.3, .epsilon. ppm; (FIG. 3) 
211.95(Q), 157.90(Q), 141.38(Q), 138.14(Q), 130.75(CH), 128.50(Q), 
123.01(CH), 119.69(CH), 114.09(CH), 64.65(CH.sub.2), 64.07(Q), 61.15(CH), 
58.41(CH.sub.2), 55.62(CH.sub.2), 42.44(CH.sub.3), 38.62(CH.sub.2), 
38.56(CH.sub.2), 38.22(CH.sub.2), 34.62(CH.sub.2), 32.23(CH.sub.2), 
25.82(CH.sub.3), 18.21(CH.sub.3). [CH.sub.3 =methyl, CH.sub.2 =methylene, 
CH=methine, Q=quaternary carbon] 
(10) Color reactions: 
Positive: Dragendorff, phosphomolybdic acid and ninhydrin reactions 
Negative: Greig-Liebach reaction 
(11) HPLC: 
Stationary phase: ODS, YMC-Pack A-312 (YMC) 
Mobile phase: 35% Acetonitrile-0.05M sodium phosphate solution (pH3.0) 
Flow rate: 2 ml/min 
Detection: UV spectrophotometry (214 & 254 nm) 
Elution time: 6.9 min. 
(12) TLC: 
Stationary phase: Silica gel 60 F.sub.254 (E. Merck, W. Ger.) 
Developing solvent: Chloroform-methanol (19:1) 
Rf: 0.30 
(13) Properties: Basic and liposoluble 
TAN-1251B 
(1) Appearance: Solid 
(2) Specific rotation: +65.degree. (D line, C 0.41, methanol) 
(3) Molecular weight: m/z 397 (M+H).sup.+, (SI-mass spectrum) 
(4) Elemental analysis: (%) (for the hemihydrate) Found: C, 71.35; H, 8.03; 
N, 6.84 Calcd.: C, 71.08; H, 8.20; N, 6.91 
(5) Molecular formula: C.sub.24 H.sub.32 N.sub.2 O.sub.3 
(6) UV spectrum: Methanol, (FIG. 4) Absorption maxima: 265.+-.3 nm 
(.epsilon.22,700.+-.3,000) 304.+-.3 nm (.delta.1,700.+-.400, shoulder) 
(7) IR spectrum: KBr disk (FIG. 5); dominant absorptions (wave-number, 
cm.sup.-1) 3430, 2940, 2780, 1720, 1600, 1500, 1440, 1380, 1290, 1240, 
1170, 1110, 1060, 1000, 920, 880, 850, 820, 580, 520. 
(8) .sup.13 C NMR spectrum: 75 MHz, CDCl.sub.3, .delta. ppm; (FIG. 6) 
211.74(Q), 157.96(Q), 141.17(Q), 138.24(Q), 130.67(CH), 128.22(Q), 
123.21(CH), 119.59(CH), 114.21(CH), 72.40(CH), 65.30(Q), 64.68(CH.sub.2), 
60.97(CH), 58.76(CH.sub.2), 55.41(CH.sub.2), 47.13(CH.sub.2), 
42.44(CH.sub.3), 36.25(CH.sub.2), 34.86(CH.sub.2), 33.07(CH.sub.2), 
24.82(CH.sub.3), 18.22(CH.sub.3). 
(9) Color reactions: 
Positive: Dragendorff, phosphomolybdic acid and ninhydrin reactions 
Negative: Greig-Liebach reaction 
(10) HPLC: 
Stationary phase: ODS, YMC-Pack A-312 
Mobile phase: 35% Acetonitrile-0.05M sodium phosphate solution (pH 3.0) 
Flow rate: 2 ml/min 
Detection: UV spectrophotometry (214 & 254 nm) 
Elution time: 3.8 min. 
(11) TLC: 
Stationary phase: Silica gel 60 F254 
Developing solvent: Chloroform-methanol (19:1) 
Rf: 0.24 
(12) Properties: Basic and liposoluble 
TAN-1251C 
(1) Appearance: Oil 
(2) Specific rotation: +24.degree. (D line, c 0.44, methanol) 
(3) Molecular weight: m/z 380 M.sup.+ (EI-mass spectrum) 
(4) Elemental analysis: (%) (for the hemihydrate) Found: C, 74.01; H, 8.40; 
N, 7.28 Calcd.: C, 74.00; H, 8.54; N, 7.19 
(5) Molecular formula: C.sub.24 H.sub.32 N.sub.2 O.sub.2 
(6) UV spectrum: Methanol Absorption maxima: 225.+-.3 nm 
(.epsilon.7,800.+-.500) 278.+-.3 nm (.epsilon.1,400.+-.400, shoulder) 
285.+-.3 nm (.epsilon.1,100.+-.300, shoulder) 
(7) IR spectrum: KBr disk; dominant absorptions (wave-number, cm.sup.-1) 
3430, 2950, 2880, 1720, 1680, 1640, 1610, 1510, 1450, 1370, 1320, 1300, 
1240, 1170, 1120, 1050, 1000, 860, 840, 810, 790, 730, 630, 510. 
(8) .sup.13 C NMR spectrum: 75 MHz, CDCl.sub.3, .delta. ppm; 221.54(Q), 
157.16(Q), 137.84(Q), 131.95(Q), 129.82(CH), 128.12(Q), 127.80(CH), 
119.94(CH), 114.40(CH), 71.42(Q), 64.72(CH.sub.2), 59.05(CH), 
52.20(CH.sub.2), 42.94(CH.sub.2), 41.44(CH.sub.2), 40.29(CH.sub.3), 
39.50(CH.sub.2), 37.80(CH.sub.2), 37.25(CH.sub.2), 34.59(CH.sub.2), 
25.81(CH.sub.3), 18.19(CH.sub.3) 
(9) Color reactions: 
Positive: Dragendorff, phosphomolybdic acid and ninhydrin reactions 
Negative: Greig-Liebach reaction 
(10) HPLC: 
Stationary phase: ODS, YMC-Pack A-312 
Mobile phase: 35% Acetonitrile-0.05M sodium phosphate solution (pH 3.0) 
Flow rate: 2 ml/min 
Detection: UV spectrophotometry (214 & 254 nm) 
Elution time: 9.3 min. 
(11) TLC: 
Stationary phase: Silica gel 60 F254 
Developing solvent: Chloroform-methanol (19:1) 
Rf: 0.80 
(12) Properties: Basic and liposoluble 
TAN-1251D 
(1) Appearance: Oil 
(2) Specific rotation: +24.degree. (D line, c 0.47, methanol) 
(3) Molecular weight: m/z 382 M.sup.+ (EI-mass spectrum) 
(4) Elemental analysis: (%) (for the hemihydrate) Found: C, 73.66; H, 8.92; 
N, 7.28 Calcd.: C, 73.62; H, 9.01; N, 7.15 
(5) Molecular formula: C.sub.24 H.sub.34 N.sub.2 O.sub.2 
(6) UV spectrum: Methanol 
Absorption maxima: 226.+-.3 nm (.epsilon.7,800.+-.1,500) 275.+-.3 nm 
(.epsilon.1,600.+-.400, shoulder) 284.+-.3 nm (.epsilon.1,200.+-.300, 
shoulder) 
(7) IR spectrum: KBr disk; dominant absorptions (wave-number, cm.sup.-1) 
3420, 2970, 2940, 2880, 2800, 1720, 1610, 1510, 1450, 1380, 1340, 1300, 
1240, 1180, 1150, 1110, 1060, 1020, 1000, 950, 910, 850, 830, 810, 770, 
730, 680, 640, 510. 
(8) .sup.13 C NMR spectrum: 75 MHz, CDCl.sub.3, .delta. ppm; 210.86(Q), 
158.43(Q), 137.96(Q), 131.89(Q), 129.76(CH), 119.82(CH), 114.70(CH), 
65.78(CH), 64.98(Q), 64.78(CH.sub.2), 61.88(CH.sub.2), 61.31(CH), 
52.30(CH.sub.2), 42.42(CH.sub.3), 41.39(CH.sub.2), 39.63(CH.sub.2), 
39.22(CH.sub.2), 37.97(CH.sub.2), 33.27(CH.sub.3), 33.02(CH.sub.2), 
25.81(CH.sub.3), 18.18(CH.sub.3) 
(9) Color reactions: 
Positive: Dragendorff, phosphomolybdic acid and ninhydrin reactions 
Negative: Greig-Liebach reaction 
(10) HPLC: 
Stationary phase: ODS, YMC-Pack A-312 
Mobile phase: 35% Acetonitrile-0.05M sodium phosphate solution (pH 3.0) 
Flow rate: 2 ml/min 
Detection: UV spectrophotometry (214 & 254 nm) 
Elution time: 3.2 min. 
(11) TLC: 
Stationary phase: Silica gel 60 F254 
Developing solvent: Chloroform-methanol (19:1) 
Rf: 0.18 
(12) Properties: Basic and liposoluble 
The compound numbers and chemical structures of the compounds mentioned in 
the description and examples are as follows. 
______________________________________ 
Compound 
No. Chemical structure 
______________________________________ 
1 
##STR14## 
2 
##STR15## 
3 
##STR16## 
4 
##STR17## 
5 
##STR18## 
6 
##STR19## 
7 
##STR20## 
8 
##STR21## 
9 
##STR22## 
10 
##STR23## 
11 
##STR24## 
12 
##STR25## 
13 
##STR26## 
14 
##STR27## 
15 
##STR28## 
16 
##STR29## 
______________________________________ 
(Compounds 9 and 10 and Compounds 11 and 12 are stereoisomers in the 
position *) 
Compounds of general formula (I) can be synthesized from TAN-1251A, B, C 
and D by conducting the ether bond cleaving reaction with an acid, 
carbonyl reduction reaction, catalytic double bond reduction, 
quaternization reaction by alkylation of tertiary amine, hydroxyl 
acylation reaction, and/or reaction for introduction of a hydrocarbon 
group into the OH group of phenol in the per se conventional manner. 
The above procedures are now described in detail taking the above-mentioned 
Compounds 1 through 16 as examples. 
The conversion of Compound 1 to Compound 7, or Compound 2 to Compound 8, is 
best performed under acidic conditions. Thus, the starting compound is 
dissolved in 0.1.about.2.0N, preferably 0.2.about.1.0N, hydrochloric acid 
or sulfuric acid at a final concentration of 2.about.50 mg/ml, preferably 
5.about.30 mg/ml, and allowed to react at a temperature of 
4.degree..about.80.degree. C., preferably 10.degree..about.40.degree. C., 
for 30 minutes to 2 days, preferably 1.about.8 hours. 
The conversion of Compound 1 to Compounds 9 and 10, or Compound 2 to 
Compounds 11 and 12, is most advantageously carried out using sodium 
borohydride. Thus, the starting compound is dissolved in methanol, ethanol 
or tetrahydrofuran at a final concentration of 5.about.100 mg/ml, 
preferably 10.about.50 mg/ml, and after addition of 0.2.about.10 
equivalents, preferably 1.about.5 equivalents, of sodium borohydride, the 
reaction is carried out at 4.degree..about.80.degree. C., preferably 
10.degree..about.40.degree. C., for 30 seconds.about.5 hours, preferably 5 
minutes to 1 hour. Other reducing agents such as sodium cyanoborohydride, 
lithium aluminum hydride, etc. can also be employed in lieu of sodium 
borohydride. 
The conversion of Compound 1 to Compound 13, or Compound 2 to Compound 14, 
can be effectively carried out by catalytic reduction. Thus, the starting 
compound is dissolved in methanol or ethanol at a final concentration of 
2.about.50 mg/ml, preferably 5.about.20 mg/ml and, after addition of a 
catalytic amount (2.about.60%, preferably 10.about.50% by weight) of 
palladium black, palladium-on-carbon, platinum black or platinum dioxide, 
the reaction is carried out in a hydrogen gas atmosphere at 
4.degree..about.80.degree. C., preferably 10.degree..about.40.degree. C., 
for 1 hour.about.2 days, preferably 2.about.8 hours. 
The conversion of Compound 2 to Compound 15 can be carried out most 
advantageously using methyl iodide. Thus, the starting compound is 
dissolved in methanol, ethanol or propanol at a final concentration of 
5.about.200 mg/ml, preferably 10.about.100 mg/ml and, after addition of 
1.about.10 equivalents, preferably 1.1.about.6 equivalents, of methyl 
iodide, the reaction is carried out at a temperature of 
20.degree..about.100.degree. C., preferably 60.degree..about.80.degree. 
C., for 30 minutes to 5 hours, preferably 1.about.2 hours. 
The conversion of Compound 2 or 6 to Compound 16 is most effectively 
carried out using acetic anhydride and pyridine. Thus, the starting 
compound is dissolved in pyridine at a final concentration of 
10.about.1000 mg/ml, preferably 20.about.500 mg/ml and, after addition of 
1 or more equivalents of acetic anhydride, the reaction is conducted at 
4.degree..about.80.degree. C., preferably 10.degree..about.40.degree. C., 
for 1 hour.about.3 days, preferably 5 hours.about.2 days. 
Compound 7 or 8 has a phenolic hydroxyl group(s) and, as such, gives an 
ether derivative as described hereinafter. Thus, for the introduction of 
an alkyl group into the acidic group of phenol or the like, the following 
procedures, for instance, are known and can be adequately applied to the 
production of compounds of the invention. 
1) The starting compound is reacted with a diazoalkane (e.g. diazomethane) 
in a solvent (e.g. ethyl ether, tetrahydrofuran, dioxane, methanol, etc.) 
at a temperature between about 0.degree. C. and the reflux temperature for 
a period of about 2 minutes.about.10 hours. 
2) The starting compound is reacted with an active alkyl halide (e.g. 
methyl iodide, n-butyl chloride, etc.). Referring to suitable conditions, 
the reaction is conducted in a solvent (e.g. dimethylformamide, 
dimethylacetamide, etc.) at a temperature of about 
0.degree..about.60.degree. C. for about 2 minutes.about.20 hours. The 
presence of an alkali metal salt (e.g. sodium carbonate, potassium 
carbonate, etc.), ammonia, triethylamine or the like in the reaction 
system does not interfere with the reaction. 
3) The starting compound is reacted with an alcohol (e.g. methanol, 
n-butanol, etc.). Preferably this reaction is conducted in a solvent (e.g. 
dimethylformamide etc.) in the presence of a condensing agent (e.g. 
dicyclohexylcarbodiimide) at a temperature of about 
0.degree..about.60.degree. C. for about 2 hours.about.2 days. The reaction 
system may contain an auxiliary condensing agent (e.g. 
1-hydroxy-1H-benzotriazole etc.). 
The following microbial transformation technique may be advantageously 
employed in combination with the various transformation reactions 
described above. 
The hydroxylation of a compound of general formula (I) wherein R.sup.3 H to 
a compound wherein R.sup.3 is OH can be carried out with the aid of a 
microorganism belonging to the genus Penicillum. This reaction can be 
conducted in a medium favoring growth of the microorganism or in the 
presence of a processed culture broth such as washed cells, immobilized 
cells and so on. A specific example is presented in Example 13, where the 
microbial transformation of TAN-1251A into TAN-1251B was carried out using 
Penicillium thomii RA-89. 
The biological activity of TAN-1251 is shown below. The assay of activity 
was carried out by the following two methods. 
(1) Muscarinic receptor-radio receptor assay 
This assay was carried out in accordance with the method of R.F.T. Gilbert 
et al. [British Journal of Pharmacology 65, 451 (1979)]. A Wistar rat 
(male, 8 weeks old, Clea Japan, Inc.) was decapitated and the brain was 
isolated. The cerebral cortex was then separated and using a Teflon 
homogenizer, the whole cerebral cortex (0.8.about.1.0 g) was homogenized 
in 30 ml of 0.32M sucrose solution. The homogenate was centrifuged at 
1,000G for 10 minutes and the supernatant was re-centrifuged at 20,000G 
for 20 minutes. The pellet was used as a crude receptor membrane fraction 
(P2 fraction). In the binding assay, the P2 fraction was suspended in 30 
ml of 0.1M sodium potassium phosphate buffer (protein concentration: 0.5 
mg/ml) and diluted 50.about.80-fold with the same buffer. A 200 .mu.l 
portion of this dilution was used in the assay. As a radioligand, .sup.3 
H-QNB (1.63 TBq/mmol, Amersham, U.K.) was added at the level of 0.148 KBq 
and the sample was added at the same time. The reaction was carried out at 
room temperature for 60 minutes. Then, using a cell harvester (290 PHD, 
Cambridge Technology, Inc., U.K.), the reaction mixture was subjected to 
rapid filtration through a glass filter (GF/B, Whatman, U.S.A.) to 
terminate the reaction and after washing with three 300 .mu.l portions of 
the same buffer as above, the residual radioactivity on the filter was 
measured with a liquid scintillation counter. The activity was expressed 
in the number of units representing the reciprocal of the sample size (ml) 
required for 50% inhibition or in the corresponding concentration (M), 
viz. IC.sub.50. 
(b) Antagonizing activity against acetylcholine-induced contraction of the 
isolated Guinea pig ileum 
A Guinea pig (Std Hartley, male, 250 g, Japan SLC) deprived of food for 24 
hours was subjected to brain concussion and after exsanguination from the 
carotid artery, the ileum was isolated. An ileal strip, about 3 cm long, 
was prepared and suspended in a Magnus tube containing 20 ml of Tyrode 
solution. At a constant bath temperature of 37.degree. C., a mixed gas 
(95% O.sub.2 -5% CO.sub.2) was bubbled through the bath. The sample was 
added and after 5 minutes of equillibration, acetylcholine (Daiichi 
Seiyaku, Japan) was added at a final concentration of 1.times.10.sup.-7 M 
to induce contraction of the ileal strip. The contraction was recorded 
through an isotonic transducer (ME-4013, Suruga Electronics, Japan) on a 
recorder (Rectiholy 8K, Japan Electronics Sanei, Japan). The activity of 
the same was expressed in ED.sub.50 representing 50% inhibition of the 
maximal contraction. 
The results of the above tests with TAN-1251A and B are shown in Table 3. 
TABLE 3 
______________________________________ 
Biological activity of TAN-1251 
b. Guinea pig ileal 
a. Radio receptor assay 
contractility 
Compound IC.sub.50 (M) ED.sub.50 (M) 
______________________________________ 
1 5.7 .times. 10.sup.-9 
8.0 .times. 10.sup.-9 
2 1.3 .times. 10.sup.-10 
1.3 .times. 10.sup.-8 
______________________________________ 
It will be apparent from Table 3 that TAN-1251 showed very high inhibitory 
activity. This anti muscarinic activity was comparable to that of 
atropine. 
For assessment of the acute toxicity of TAN-1251, an oral administration 
experiment using TAN-1251A and B was performed in mice. As a result, no 
toxic signs were observed at the dose level of 100 mg/kg for either 
substance. 
It will be apparent from the physiochemical and biological properties 
described above that TAN-1251 is a novel compound and that, as an 
antimuscarinic agent, it is of value as a therapeutic drug for various 
diseases such as gastric and duodenal ulcers, spastic pain of the 
gastrointestinal tract, parkinsonism, etc. or as a mydriatic. 
TAN-1251 or a salt thereof is administered orally or parenterally as an 
injectable preparation. The oral dosage for humans is generally 
0.05.about.50 mg/kg/day, preferably 0.1.about.10 mg/kg/day, and the 
parenteral dosage is 0.01.about.10 mg/kg/day, preferably 0.05.about.5 
mg/kg. 
The dosage form for oral administration includes, inter alia, capsules, 
tablets, granules, syrups, powders, etc. and may contain, in addition to 
TAN-1251 or a salt thereof, such additives as various excipients, binders, 
disintegrators, lubricants, colorants, corrigents, stabilizers and so on. 
For parenteral administration, the active compound can be dissolved or 
suspended in the common diluent (an aqueous or non-aqueous vehicle) and 
provided in a variety of pertinent dosage forms such as solutions, eye 
drops, emulsions, suspensions, suppositories and so on. In the manufacture 
of such pharmaceutical compositions, there may be employed a diversity of 
additives such as emulsifiers, suspending agents, cosolvents, stabilizers, 
preservatives, soothing agents, isotonizing agents, buffers, pH adjusting 
agents, colorants, coating agents and so on. These pharmaceutical 
compositions or preparations can be manufactured by the established 
pharmaceutical procedures. 
EXAMPLES 
The following examples are merely intended to illustrate the invention in 
further detail and should by no means be construed as defining the metes 
and bounds of the invention. It should be noted that all percents (%) 
relating to media are weight/volume percents unless otherwise indicated. 
EXAMPLE 1 
Penicillium thomii RA-89 (IFO-32288, FERM BP-2753) grown on a slant agar 
medium composed of 24 g potato dextrose broth (Difco, U.S.A.), 20 g agar 
and 1 l water at 28.degree. C. for 7 days was inoculated into 40 ml of a 
seed medium (pH 6.5) composed of 2% glucose, 3% maltose, 1% raw soybean 
flour (SBF), 0.5% corn steep liquor (CSL), 0.25% peptone, 0.15% yeast 
extract and 0.15% NaCl. The inoculated medium in a 200 ml conical flask 
was incubated on a rotary shaker at 24.degree. C. for 48 hours to provide 
a preculture. The whole amount of this preculture was transferred to a 
2000 ml Sakaguchi flask containing 500 ml of the seed culture medium and 
incubated on a reciprocating shaker at 24.degree. C. for 24 hours to 
provide a seed culture. A 1000 ml portion of the seed culture obtained in 
the above manner was transferred to a 200 liter stainless steel tank 
containing 120 l of a fermentation medium (pH 6.7) containing 5% of 
glycerol, 2.5% of sucrose, 1% of SBF, 0.5% of peptone, 0.2% of malt 
extract, 0.1% of yeast extract, 0.2% of ammonium sulfate, 0.5% of calcium 
carbonate and 0.05% of Aktocol (Takeda Chemical Industries, Ltd., Japan). 
The inoculated medium was incubated at 24.degree. C., with aeration at a 
rate of 150 l/min. and agitation at 200 rpm and under an internal pressure 
of 1 kg/cm.sup.2 for 90 hours. As found by radio receptor assay of 
TAN-1251 in the culture supernatant, its output was 45,000 units. 
EXAMPLE 2 
To the culture broth (100 l) was added Hyflo Super-Cel (Johns-Manville 
Corporation, U.S.A.) as a filter aid and the mixture was filtered. After 
the filtrate was adjusted to pH 8.0, the bioactive substance was extracted 
with ethyl acetate (70 l). The organic layer was extracted with 0.01N 
hydrochloric acid (50 l) and the aqueous solution was adjusted to pH 8.0 
and re-extracted with ethyl acetate (33 l). The organic layer was washed 
with water (23 l) and concentrated to give a crude oil (1.09 g). It was 
combined with the crude oil (0.99 g) obtained in the same manner and the 
pooled oil was dissolved in chloroform and subjected to silica gel (100 g) 
column chromatography. The bioactive components were eluted successively 
with chloroform-methanol mixtures, (99.1, 3.5 l) (98.2, 2.0 l) and (95:5, 
0.5 l), the respective fractions were analyzed by HPLC, and the fractions 
containing either TAN-1251A or TAN-1251B only were respectively pooled and 
concentrated to dryness. 
The oil containing TAN-1251A (0.27 g) was combined with the oil containing 
TAN-1251A (1.8 g) obtained in the same manner and the pooled oil was 
subjected to preparative HPLC [stationary phase YMC-Pack S-363 I-15 (YMC, 
Japan), mobile phase 32% acetonitrile/0.01M sodium phosphate (pH 3.0)]. 
The eluate was analyzed by HPLC and the fractions containing TAN-1251A 
were pooled. A portion (1.4 l) of this solution was concentrated to 0.3 l, 
adjusted to pH 8.0 and extracted with ethyl acetate (200 ml). The 
resulting organic layer was washed with water and concentrated to dryness 
to recover a powder of TAN-1251A (300 mg). This powder (130 mg) was 
recrystallized from ethyl acetate to provide colorless crystals of 
TAN-1251A (51 mg). 
The oil containing TAN-1251B (0.49 g) was combined with the oil containing 
TAN-1251B (0.33 g) obtained in the same manner and the pooled oil was 
subjected to preparative HPLC [stationary phase YMC-Pack S-363 I-15, 
mobile phase 25% acetonitrile/0.01M sodium phosphate (pH 3.0)]. The eluate 
was analyzed by HPLC and the fractions containing TAN-1251B were pooled. A 
portion (500 ml) of this solution was concentrated to 100 ml, adjusted to 
pH 8.0 and extracted with ethyl acetate (150 ml). The extract was washed 
with water and concentrated to dryness to provide a powder of TAN-1251B 
(132 mg). 
EXAMPLE 3 
The solution containing TAN-1251A (1.4 l) obtained by preparative HPLC in 
Example 2 was concentrated and passed through a column of Amberlite 
IRA-402 (SO.sub.4.sup.2- form, 0.3 l, Rohm and Haas Company, U.S.A.). The 
effluent and the aqueous wash were chromatographed on Amberlite XAD-II (60 
ml) and the activity was eluted with 50% aqueous methanol (240 ml) and 70% 
aqueous acetone (300 ml). The eluate was concentrated and freeze-dried to 
provide a white powder of TAN-1251A sulfate (667 mg). 
Similarly, the solution containing TAN-1251B (280 ml) obtained by 
preparative HPLC in Example 2 was chromatographed using Amberlite IRA-402 
(SO.sub.4.sup.2- form, 50 ml) and Amberlite XAD-II (20 ml) and the eluate 
was concentrated and freeze-dried to provide a white powder of TAN-1251B 
sulfate (114 mg). 
TAN-1251A sulfate 
Specific rotation: -14.degree. (D line, c 0.23, 50% aqueous methanol, 
22.degree. C.) 
UV: Absorption maxima in H.sub.2 O: 266 nm (.epsilon.24,200), 303 nm 
(.epsilon.1,900, shoulder) IR: KBr disk, dominant absorptions 
(wave-number, cm.sup.-1): 3430, 2960, 1720, 1600, 1500, 1450, 1240, 1120, 
1000, 830, 620 
Elemental analysis (for C.sub.24 H.sub.32 N.sub.2 O.sub.2.0.5H.sub.2 
SO.sub.4.2H.sub.2 O) Calcd.: C, 61.91: H, 8.01; N, 6.02; S, 3.44 Found: C, 
62.10; H, 7.97; N, 5.85; S, 3.19 
TAN-1251B sulfate 
Specific rotation: +67.degree. (D line, c 0.24, 50% aqueous methanol, 
22.degree. C.) 
UV: Absorption maxima in H.sub.2 O: 264 nm (.epsilon.25,200), 303 nm 
(.epsilon.1,400, shoulder 
LR: KBr disk, dominant absorptions (wave-number, cm.sup.-1): 3430, 2950, 
1720, 1600, 1500, 1450, 1240, 1120, 1010, 620 
Elemental analysis (for C.sub.24 H.sub.32 N.sub.2 O.sub.3.0.5H.sub.2 
SO.sub.4.1.5H.sub.2 O) Calcd.: C, 61.00; H, 7.68; N, 5.93; S, 3.39 Found: 
C, 61.30; H, 7.85; N, 5.92; S, 3.12 
EXAMPLE 4 
The spores formed on the same slant as used in Example 1 were suspended in 
10 ml of water and the whole amount was transferred to a 2 l Sakaguchi 
flask containing 500 ml of a seed medium and incubated on a reciprocating 
shaker at 24.degree. C. for 48 hours to give a preculture. One liter 
portion of the preculture was transferred to a 200 l stainless steel tank 
containing 100 l of a seed medium (with 0.05% Aktocol added) and incubated 
at 24.degree. C. with aeration at a rate of 120 l/min. and agitation at 
150 rpm under an internal pressure of 1 kg/cm.sup.2 for 48 hours. A 50 l 
portion of the resulting seed culture was transferred to a 6,000 l 
stainless steel thank containing 3,600 l of the same fermentation medium 
as used in Example 1 and incubated at 24.degree. C. with aeration at a 
rate of 3,600 l/min. and agitation at 200 rpm under an internal pressure 
of 1 kg/cm.sup.2 for 90 hours. The culture supernatant contained 110,000 
units of TAN-1251. 
The culture broth (3,480 l) was subjected to Oliver filtration using 
Radiolite (Showa Chemical Industry, Japan) as a filter aid. The filtrate 
was adjusted to pH 6.5 and passed through a column of Diaion HP-20 (70 l, 
Mitsubishi Kasei, Japan). The column was washed with water (210 l) and 30% 
aqueous methanol (210 l) in that order and elution was carried out with 
60% acetone/0.01N sulfuric acid (280 l). The eluate was adjusted to pH 4.2 
and concentrated to remove the acetone. The resulting aqueous solution (80 
l) was adjusted to pH 8.4 and extracted with ethyl acetate (40 
l.times.2). The extract was washed with water (25 l.times.2) and 
concentrated to 5 l and the concentrate was extracted with 0.02N 
hydrochloric acid (2 l.times.2). The solution was adjusted to pH 3.4, 
concentrated and subjected to Diaion HP-20 (50.about.100 mesh, 0.7 l) 
column chromatography. The column was washed with water (2 l) and 20% 
aqueous methanol (2 l), followed by elution with 50% aqueous methanol (2.1 
l), 60% aqueous acetone (2.1 l) and 70% acetone/0.01N hydrochloric acid 
(2.1 l) in the order mentioned. The fraction eluted by 70% acetone/0.01N 
hydrochloric acid was concentrated to 400 ml for removal of the acetone 
and the concentrate was adjusted to pH 8.2 and extracted with ethyl 
acetate (200 ml.times.2). The extract was washed with water (150 
ml.times.2) and concentrated to dryness. The residue was subjected to 
silica gel (100 ml; solvent system: chloroform-methanol) column 
chromatography. The fractions containing TAN-1251A were pooled and 
concentrated to dryness and the resulting powder was crystallized from 
ethyl acetate-hexane to provide crystals of TAN-1251A (925 mg). 
The eluate obtained with 50% aqueous methanol and that obtained with 60% 
aqueous acetone from the Diaion HP-20 column and concentrated and the 
concentrate was adjusted to pH 8.2 and extracted with ethyl acetate. The 
extract was washed with water, concentrated to dryness and subjected to 
silica gel (200 ml; solvent system: chloroform-methanol) column 
chromatography. The fractions rich in TAN-1251B, C and D, respectively, 
were taken independently and concentrated to dryness to provide an oil 
containing TAN-1251C (1.3 g) and an oil containing TAN-1251D (2.0 g). The 
fraction containing TAN-1251B was concentrated to dryness and crystallized 
to give crystals of TAN-1251B (1.2 g). 
The oil containing TAN-1251C (1.3 g) and the oil containing TAN-1251C 
obtained in the same manner (4.3 g) were combined and purified by silica 
gel (300 ml; solvent system: dichloroethane-methanol) column 
chromatography to give TAN-1251C (5.1 g). The oil containing TAN-1251D 
(2.0 g) and the oil containing TAN-1251D (8.1 g) obtained in the same 
manner were combined and purified by silica gel (500 ml; solvent system: 
dichloroethane-methanol) column chromatography to provide TAN-1251D (7.6 
g). 
EXAMPLE 5 
Compound 1 (109 mg) was dissolved in 0.5N hydrochloric acid (10 ml) and the 
solution was allowed to stand at room temperature for 5 hours. The 
reaction mixture was then adjusted to pH 8.5 and extracted with ethyl 
acetate. The insoluble matter which separated out in this extraction step 
was recovered by filtration to provide Compound 7 (49 mg). The organic 
layer was washed with water, dehydrated over anhydrous sodium sulfate and 
concentrated to dryness to give a further crop of Compound 7 (36 mg). 
Specific rotation: -8.1.degree. (D line, c 0.41, methanol, 25.degree. C.) 
UV: Absorption maxima in methanol: 264 mm (.epsilon.22,000), 303 nm 
(.epsilon.1,700, shoulder) 
IR: KBr disk, dominant absorptions (wave-number, cm.sup.-1): 3520, 2970, 
2940, 1705, 1610 
EI-MS: 312 (M.sup.+) 
Elemental analysis (for C.sub.19 H.sub.24 N.sub.2 O.sub.2.H.sub.2 O) 
Calcd.: C, 69.07; H, 7.93; N, 8.48 Found: C, 69.46; H, 7.92; N, 8.09 
.sup.13 C NMR spectrum (75 MHz, CD.sub.3 OD, .delta. ppm): 214.58(Q), 
157.75(Q), 141.69(Q), 132.07(CH.times.2), 128.73(Q), 124.66(CH), 
115.72(CH.times.2), 65.20(Q), 62,46(CH), 58.75(CH.sub.2), 56.21(CH.sub.2), 
42.43(CH.sub.3), 39.34(CH.sub.2 .times.2), 38.65(CH.sub.2), 
35.63(CH.sub.2), 32.94(CH.sub.2) 
EXAMPLE 6 
Command 2 (578 mg) was dissolved in 0.5N hydrochloric acid (30 ml) and the 
solution was stirred at room temperature for 2.5 hours. The reaction 
mixture was then adjusted to pH 8.5, diluted with a saturated aqueous 
solution of sodium chloride, and extracted with ethyl acetate. The organic 
layer was washed with a saturated aqueous solution of sodium chloride, 
dehydrated over anhydrous sodium sulfate and concentrated to dryness. The 
residue was tritulated with ethyl acetate-hexane to provide Compound 8 
(446 mg). 
Specific rotation: +81.9.degree. (D line, c 0.42, methanol, 25.degree. C.) 
UV: Absorption maxima in methanol: 262 nm (.epsilon.22,500), 303 nm 
(.epsilon.1,600, shoulder) 
IR: KBr disk, dominant absorptions (wave-number, cm.sup.-1): 3430, 2950, 
1725, 1610 EI-MS: 328(M.sup.+) 
Elemental analysis (for C.sub.19 H.sub.24 N.sub.2 O.sub.3) Calcd.: C, 
69.49; H, 7.37; N, 8.53 Found: C, 68.75; H, 7.25; N, 8.42 
.sup.13 C NMR spectrum (75 MHz, CDCl.sub.3, .delta. ppm): 211.63(Q), 
156.08(Q), 139.73(Q), 130.98(CH.times.2), 127.23(Q), 123.97(CH), 
115.35(CH.times.2), 72.40(CH), 65.26(Q), 60.86(CH), 57.96(CH.sub.2), 
55.05(CH.sub.2), 46.84(CH.sub.2), 42.13(CH.sub.3), 36.28(CH.sub.2), 
35.13(CH.sub.2), 33.00(CH.sub.2). 
EXAMPLE 7 
Compound 1 (180 mg) was dissolved in ethanol (6 ml) followed by addition of 
sodium borohydride (60 mg) and the mixture was stirred at room temperature 
for 30 minutes. The reaction mixture was adjusted to pH 2.5 with diluted 
hydrochloric acid and washed with ethyl acetate. The aqueous layer was 
adjusted to pH 8.5 and extracted with ethyl acetate. The organic layer was 
washed with a saturated aqueous solution of sodium chloride, dehydrated 
over anhydrous sodium sulfate and concentrated to dryness to provide a 
crude powder containing Compounds 9 and 10 (184 mg). This crude powder was 
subjected to preparative HPLC [stationary phase: YMC-Pack D-ODS-5 (YMC), 
mobile phase: 28% acetonitrile-0.02M sodium phosphate (pH 3)]. The eluate 
was analyzed by HPLC and the fractions containing 9 and 10, respectively, 
were independently pooled. Each of the solutions thus obtained was 
concentrated, adjusted to pH 8.5 and extracted with ethyl acetate. The 
organic layer was washed with a saturated aqueous solution of sodium 
chloride, dehydrated over anhydrous sodium sulfate and concentrated to 
dryness to provide Compound 9 (92 mg) and Compound 10 (47 mg). 
Compound 9 
UV: Absorption maxima in methanol: 265 nm (.epsilon.29,000), 304 
nm(.epsilon.1,900, shoulder) 
Elemental analysis (for C.sub.24 H.sub.34 N.sub.2 O.sub.2) Calcd.: C, 
75.35; H, 8.96; N, 7.32 Found: C, 75.05; H, 9.22; N, 7.05 
.sup.13 C NMR spectrum (75 MHz, CDCl.sub.3, .delta. ppm): 157.65(Q), 
141.91(Q), 137.99(Q), 131.18(CH.times.2(, 129.13(Q), 122.29(CH), 
119.79(CH), 113.87(CH.times.2), 69.31(CH), 64.67(CH.sub.2), 64.37(Q), 
61.02(CH), 58.10(CH.sub.2), 55.82(CH.sub.2), 42.44(CH.sub.3), 
35.84(CH.sub.2), 34.76(CH.sub.2), 32.16(CH.sub.2), 32.05(CH.sub.2), 
29.68(CH.sub.2), 25.81(CH.sub.3), 18.20(CH.sub.3) 
HPLC: 
Stationary phase: ODS, YMC-Pack A-312 
Mobile phase: 35% Acetonitrile-0.05M sodium phosphate solution (pH 3) 
Flow rate: 2 ml/min 
Detection: UV spectrophotometry (214 & 254 nm) 
Elution time: 3.6 min. 
Compound 10 
UV: Absorption maxima in methanol: 265 nm (.epsilon.26,400), 304 nm 
(.epsilon.2,000, shoulder) 
Elemental analysis (for C.sub.24 H.sub.34 N.sub.2 O.sub.2.1/2H.sub.2 O) 
Calcd.: C, 73.62; H, 9.01; N, 7.15 Found: C, 73.46; H, 9.06; N, 6.78 
.sup.13 C NMR spectrum (75 MHz, CDCl.sub.3, .delta. ppm): 157.61(Q), 
141.92(Q), 138.00(Q), 130.90 (CH.times.2), 129.22(Q), 122.10(CH), 
119.81(CH), 113.98(CH.times.2), 68.83(CH), 64.72(CH.sub.2), 64.70(Q), 
61.12(CH), 57.61(CH.sub.2), 55.55(CH.sub.2), 42.42(CH.sub.3), 
35.47(CH.sub.2), 33.41(CH.sub.2), 32.61(CH.sub.2), 31.70(CH.sub.2), 28.90 
(CH.sub.2), 25.82(CH.sub.3), 18.19(CH.sub.3) 
HPLC: The same conditions for Compound 9. 
Elution time: 4.4 min. 
EXAMPLE 8 
Compound 2 (178 mg) was dissolved in ethanol (6 ml) followed by addition of 
sodium borohydride (60 mg) and the mixture was stirred at room temperature 
for 15 minutes. The reaction mixture was extracted in the same manner as 
in Example 7 to give a crude powder containing Compounds 11 and 12 (173 
mg). This crude powder was subjected to preparative HPLC [stationary 
phase: YMC-Pack D-ODS-5; mobile phase 22% acetonitrile-0.02M sodium 
phosphate (pH 3)]. The fractions were treated in the same manner as in 
Example 7 to provide Compound 11 (127 mg) and Compound 12 (25 mg). 
Compound 11 
Specific rotation: +45.0.degree. (D line, c 0.46, methanol, 25.degree. C.) 
UV: Absorption maxima in methanol: 264 nm (.epsilon.27,000), 303 nm 
(.epsilon.1,900, shoulder) 
Elemental analysis (for C.sub.24 H.sub.34 N.sub.2 O.sub.3.H.sub.2 O) 
Calcd.: C, 69.20; H, 8.71; N, 6.72 Found: C, 69.03; H, 8.36; N, 6.64 
.sup.13 C NMR spectrum (75 MHz, CDCl.sub.3, .delta. ppm): 157.73(Q), 
141.27(Q), 137.98(Q), 131.09(CH.times.2), 128.81(Q), 122.68(CH), 
119.74(CH), 113.94(CH.times.2), 76.05(CH), 73.08(CH), 65.88(Q), 
64.69(CH.sub.2), 60.87(CH), 58.52(CH.sub.2), 55.72(CH.sub.2), 
44.06(CH.sub.2), 42.37(CH.sub.3), 35.89(CH.sub.2), 30.61(CH.sub.2), 
29.77(CH.sub.2), 25,81(CH.sub.3), 18.21(CH.sub.3) 
HPLC: 
Stationary phase: ODS, YMC-Pack A-312 
Mobile phase: 35% Acetonitrile-0.02 sodium phosphate solution (pH 3) 
Flow rate: 2 ml/min 
Detection: UV spectrophotometry (214 & 254 nm) 
Elution time: 2.6 min. 
Compound 12 
Specific rotation: +81.3.degree. (D line, c 0.29, methanol, 25.degree. C.) 
UV: Absorption maxima in methanol: 264 nm (.epsilon.24,600), 303 nm 
(.epsilon.1,800, shoulder) 
Elemental analysis (for C.sub.24 H.sub.34 N.sub.2 O.sub.3.1/2H.sub.2 O) 
Calcd.: C, 70,73; H, 8.66; N, 6.87 Found: C, 71.06; H, 8.74; N, 6.70 
.sup.13 C NMR spectrum (75 MHz, CDCl.sub.3, .delta. ppm): 157.64(Q), 
141.65(Q), 138.03(Q), 130.84(CH.times.2), 128.91(Q), 122.49(CH), 
119.74(CH), 113.94(CH.times.2), 69.43(CH), 68.47(CH), 65.95(Q), 
64.69(CH.sub.2), 60.89(CH), 58.31(CH.sub.2), 55.71(CH.sub.2), 
42.34(CH.sub.3), 40.28(CH.sub.2), 36.34(CH.sub.2), 27.93(CH.sub.2), 
25.82(CH.sub.3), 25.44(CH.sub.2), 18.21CH.sub.3) 
HPLC: The same conditions for Compound 11. 
Elution time: 3.2 min. 
EXAMPLE 9 
Compound 1 (64 mg) was dissolved in methanol (10 ml) followed by addition 
of palladium black (20 mg) and the mixture was stirred under a hydrogen 
atmosphere at room temperature for 4 hours. The reaction mixture was then 
filtered and the filtrate was concentrated to dryness. The residue was 
purified by preparation HPLC [stationary phase YMC-Pack D-ODS-5; mobile 
phase 33% acetonitrile-0.05M sodium phosphate (pH 3)] to give fractions 
containing 7 and 13, respectively. Each of these fractions was 
concentrated, adjusted to pH 8.5 and extracted with ethyl acetate. The 
organic layer was washed with water, dehydrated over anhydrous sodium 
sulfate and concentrated to dryness to recover Compound 7 (15 mg) and 
Compound 13 (25 mg). 
Compound 13 
UV: Absorption maxima in methanol: 264 nm (.epsilon.21,000), 304 nm 
(.epsilon.1,900, shoulder) 
.sup.1 H NMR spectrum (300 MHz, CDCl.sub.3, .delta. ppm): 7.75(2H, d, J=8.8 
Hz), 6.78(2H, d, J=8.8 Hz), 6.02(1H, d, J=1.3 Hz), 3.95 (2H, t, J=6.7 Hz), 
3.25-3.35 (3H, m), 3.03 (1H, dd, J=1.5, 13.0 Hz), 2.95 (1H, dd, J=1.3, 
13.7 Hz), 2.77 (1H, m), 2.20 (3H, s), 1.90-2.24 (5H, m), 1.60-1.90 (6H, 
m), 1.49 (1H, dd, J=5.4, 14.0 Hz), 0.95 (6H, d, J=6.6 Hz) 
EXAMPLE 10 
Compound 2 (80 mg) was dissolved in methanol (10 ml) followed by addition 
of palladium black (30 mg) and the mixture was stirred under a hydrogen 
atmosphere at room temperature for 4 hours. The reaction mixture was then 
filtered and the filtrate was concentrated to dryness. The residue was 
subjected to preparative HPLC [stationary phase YMC-Pack D-ODS-5; mobile 
phase 30% acetonitrile-0.05M sodium phosphate (pH 3)]. The eluate was 
treated in the same manner as in Example 9 to provide Compound 8 (33 mg) 
and Compound 14 (21 mg). 
Compound 14 
UV: Absorption maxima in methanol: 263 nm (.epsilon.22,700), 303 nm 
(.epsilon.1,600, shoulder) 
.sup.1 H NMR spectrum (300 MHz, CDCl.sub.3, .delta. ppm): 7.67 (2H, d, 
J=8.8 Hz), 6.78 (2H, d, J=8.8 Hz), 6.05 (1H, d, J=1.2 Hz), 4.53 (1H, dd, 
J=6.5, 12.1 Hz), 3.95 (2H, t, J=6.7 Hz), 3.43 (1H, dd, J=3.4, 12.1 Hz), 
3.30 (1H, m), 3.28 (1H, d, J=13.7 Hz), 3.10 (1H, dd, J=1.6, 12.1 Hz), 2.94 
(1H, dd, J=1.2, 13.7 Hz), 2.61 (1H, dd, J=4.0, 6.5, 13.0 Hz), 2.26 (1H, 
m), 2.18 (3H, s), 2.04-2.16 (2H, m), 1.70-1.90 (3H, m), 1.60-1.70 (2H, m), 
0.95 (6H, d, J=6.6 Hz) 
EXAMPLE 11 
Compound 2 (23 mg) was dissolved in ethanol (0.5 ml) followed by addition 
of methyl iodide (25 .mu.l) and the mixture was refluxed for 1 hour. After 
cooling, ether was added and the resulting precipitate was recovered by 
filtration. To the powder thus obtained was added water (5 ml) and the 
insoluble matter was filtered off. The filtrate was concentrated and 
freeze-dried to provide Compound 15 (24 mg). 
UV: Absorption maxima in H.sub.2 O: 266 nm (.epsilon.22,900), 303 nm 
(.epsilon.1,700, shoulder) 
Elemental analysis (for C.sub.25 H.sub.35 N.sub.2 O.sub.3 I.H.sub.2 O) 
Calcd.: C, 53.96; H, 6.70; N, 5.03 Found: C, 54.20; H, 6.70; N, 4.86 
.sup.1 H NMR spectrum (300 MHz, D.sub.2 O, .delta. ppm): 7.85 (2H, d, J=8.7 
Hz), 6.93 (2H, d, J=8.7 Hz), 6.51 (1H, s), 5.43 (1H, t, J=7.0 Hz), 4.63 
(1H, dd, J=7.0, 13.0 Hz), 4.50 (2H, d, J=7.0 Hz), 4.06-4.20 (2H, m), 3.97 
(1H, d, J=15.0 Hz), 3.78 (1H, d, J=14.0 Hz), 3.64 (1H, d, J=14.0 Hz), 3.31 
(3H, s), 3.17 (3H, s), 2.63 (1H, m), 2.45 (1H, d, J=16.0 Hz), 2.28 (1H, 
dd, J=6.0, 16.0 Hz), 1.74-2.22 (5H, m), 1.74 (3H, s), 1.67 (3H, s) 
EXAMPLE 12 
Compound 6 (100 mg) was dissolved in pyridine (1 ml) followed by addition 
of acetic anhydride (1 ml) and the mixture was allowed to stand at room 
temperature for 15 hours. The reaction mixture was then concentrated, 
diluted with water, adjusted to pH 8.5 and extracted with ethyl acetate. 
The organic layer was washed with a saturated aqueous solution of sodium 
chloride, dehydrated over anhydrous sodium sulfate and concentrated to 
dryness. The residue was purified by silica gel (6 g) column 
chromatography [solvent system: chloroform-methanol=98:2.about.96.4)] to 
provide Compound 16 (79 mg). 
Specific rotation: +122.5.degree. (D line, c 0.45, methanol, 25.degree. C.) 
UV: Absorption maxima in methanol: 265 nm (.epsilon.22,600), 303 nm 
(.epsilon.2,000, shoulder) 
Elemental analysis (for C.sub.26 H.sub.34 N.sub.2 O.sub.4.1/2H.sub.2 O) 
Calcd.: C, 69.77; H, 7.88; N, 6.26 Found: C, 69.2; H, 7.87; N, 6.16 
.sup.13 C NMR spectrum (75 MHz, CDCl.sub.3, .delta. ppm): 204.68(Q), 
169.47(Q), 158.27(Q), 139.81(Q), 138.00(Q), 130.67(CH.times.2), 127.79(Q), 
123.96(CH), 119.73(CH), 114.48(CH.times.2), 73.91(CH), 65.61(Q), 
54.70(CH.sub.2), 60.88(CH), 58.22(CH.sub.2), 54. 94(CH.sub.2), 
43.60(CH.sub.2), 41.96CH.sub.3), 37.39 (CH.sub.2), 35. 27(CH.sub.2), 
32.62(CH.sub.2), 25.80(CH.sub.3), 20.77(CH.sub.3), 18.19(CH.sub.3) 
EXAMPLE 13 
A 200 ml creased Erlenmyer's flask containing 30 ml of a fermentation 
medium (pH 6.7) composed of 5% glycerol, 2.5% sucrose, 0.5% peptone, 0.2% 
yeast extract, 0.3% ammonium sulfate and 0.5% calcium carbonate was 
inoculated with a preculture (inoculum size 1 ml) of Penicillium thomii 
RA-89 prepared in the same manner as Example 1. Separately, a solution of 
TAN-1251A in dimethyl sulfoxide was diluted 10-fold with methanol, and 0.3 
ml of the dilution was added concurrently with inoculation. The flask was 
incubated on a rotary shaker at 24.degree. C. for 72 hours. The assay of 
TAN-1251A and B was carried out by HPLC under the Conditions set forth 
under the heading of Physicochemical Properties. The results are shown in 
Table 4. It is apparent that approximately 60% of TAN-1251A added was 
hydroxylated to TAN-1251B. 
TABLE 4 
______________________________________ 
Incu- 
Cultural bation 
conditions 
time TAN-1251A .mu.g/ml 
TAN-1251B .mu.g/ml 
______________________________________ 
Without 0 0 0 
TAN-1251A 
24 0 0 
48 0.3 1.8 
72 0.6 2.7 
With 0 28.0 0 
TAN-1251A 
24 25.4 0.2 
48 16.2 10.4 
72 11.1 17.2 
______________________________________