Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
25~-J~5
~.. O ,~
BRIEF SUMMAR'~ OF THE INVENTION
This invention relates -to a novel group of anti-
~- biotics and, more particularly, is concerned with a novel
series o~ potent antibacterial agents derived by reductive
alkylation of antibiotic BM123~ with an aldehyde or ketone
of the following general formulae:
R -CHO
, , O
.` 11
~ 2 3
.': 10
~ wherein Rl is hydrogen, lower alkyl, halo substituted lower
.; alkyl, lower alkenyl, phenyl, monosubstituted phenyl, phenyl
lower alkyl, 2-furyl, methyl substituted 2-furyl, 2-thienyl,
methyl substituted 2-thienyl, 2-pyrryl, methyl substituted
2-pyrryl, 2-pyridyl or 2-quinolyl; R2 is lower alkyl, halo
. substituted lower alkyl or phenyl lower alkyl; R3 is lower
alkyl, halo substituted lower alkyl, lower alkenyl, lower
. . cycloalkyl, phenyl, monosubstituted phenyl, phenyl lower alkyl
or monosubstituted phenyl lower alkyl; and R2 and R3 taken
~; together with the associated carbonyl group is cyclopentanone,
20
mono-lower alkyl substituted cyclopentanone, di-lower alkyl
substituted cyclopentanone, tri-lower alkyl substituted cyclo-
. pentanone, cyclohexanone, mono-lower alkyl substituted cyclo-
hexanone, di-lower alkyl substituted cyclohexanone or tri-lower
;.; alkyl substituted cyclohexanone. Suitable lower alkyl and halo
: 25
:~:. substituted lower alkyl groups contemplated by the present in-
;; vention are those having up to six carbon atoms wherein halo is
exemplified by chloro, bromo, and iodo such as methyl, ethyl,
; lsopropyl, sec-butyl, _-amyl, dichlorome-thyl, 2-bromoethyl, 2,3-
-diiodopropyl, ~--chloropropyl, etc. Suitable lower alkenyl
: 30
. groups are those having up to four carbon atoms such as vinyl,
allyl, propenyl, lsopropenyl, l-butenyl, 2-butenyl, 3--butenyl,
sobutenyl, e-tc. Suitable lower cycloalkyl groups are cyclo-
- 1 ~
'' :
penyl, cyclohexyl, and cycloheptyl. Suitable monosubstituted phenyl groups
contemplated by the present invention are, for example~ ~-acetamidophenyl,
m~nitrophenyl, m-mercaptophenyl, o-anisyl, E-anisyl, o-tolyl, E-tolyl, and
the like whereas phenyl lower alkyl is exemplified by benzyl, ~-phenylethyl,
and ~-phenylethyl. Suitable monosubstituted phenyl lower alkyl groups may be
o, m, or ~chlorobenzyl, ~--(E-aminophenyl)ethyl, ~-(m-nit~ophenyl)ethyl, etc.
; Suitable methyl substituted 2-furyl, 2-thienyl, and 2-pyrryl groups which may
be employed are, for example~ 5-methyl-2-furyl, 3,4~dimethyl-2-furyl, 4-
methyl-2-thienyl, 3,5-dimethyl-2-thienyl, 5-methyl-2-pyrryl, 1,3,4-trimethyl-
2-pyrryl, and the like.
- Antibiotic BM123~ and its production are the subject of our copend-
- ing patent application Serial No. 254,116, filed on June 4, 1~76.
The present invention provides a process for preparing compounds of
the formulae: 0
trans "
R ~ CH=cH-c-NH-(cH2)3-NH ( 234 2
trans " CH -R~
R ~ cH=cH-c-NH-(cH2)3-NH-(cH2)4-N \ 2 ~~ (II)
0 CH2- ~ 2
trans "
R ~ 0 CH2- ~ 2 4 2 ~ (III)
R ~ ( 2)3( 2)4 \ CH ~ (IV3
trans " / R
R- ~ CH=cH-c-NH-(cH2)3-NH-(cH2)4-NH-cH \ 2 (V)
wherein ~ is hydrogen, lower alkyl, halo substituted lower alkyl, lower
alkenyl, phenyl, E~acetamidophenyl, m-nitrophenyl, m-mercaptophenyl, _-anisyl,
E-anisyl, o-tolyl,E-tolyl, phenyl lower alkyl, 2-furyl, methyl substituted 2-
furyl, 2-thienyl, methyl substituted 2-thienyl, 2-pyrryl, methyl substituted
2-pyrryl, 2-pyridyl or 2-quinolyl; R2 is selected from the group consisting
of lower alkyl, halo substituted lower alkyl and phenyl lower alkyl; R3 is
~3 ; ~ - 2 -
..
selected from the group consisting of lower alkyl, halo substituted lower
alkyl lower alkenyl, lower cycloalkyl, phenyl, E-acetamidophenyl~ m~nitro-
phenyl, m-mercaptophenyl, o-anisyl, ~-anisyl, o-tolyl, ~-tolyl, phenyl lower
alkyl, o, m, or E-chlorobenzyl, ~-(p-aminophenyl) ethyl and ~-(m-nitrophenyl~-
ethyl; or R2 and R3 taken together with the associated methylidyne group is
cyclopentyl, mono-lower alkyl substituted cyclopentyl, di-lower alkyl substi-
tuted cyclohexyl or tri-lower alkyl substituted cyclohexyl; and R is a moiety
of the formula
H
¦ ~ 3
- H ~ H ~ o H
H ~ O V ~ H
\ H /1 ~ OH H / ~ OH H ~ O
HO ~ H ~ ~ ~ H-C-HN ~ (VI)
H IC -N\ ~ NH o H NH
NH2 C=O NH
. NH2
or
: ~ ~I CH3
'1~ ~ `~ ~ ~ o _
O I = O C = O C = NH (VII)
~ NH2 NH2 NH2
: which comprises:
(a) for preparing a compound of formula I, II, III or IV, alkylating an amine
of the formula:
R ~ CH=CH~C-NEI-(CH2)3-NH-(C~2)4--NH2 (A)
~ wherein R is as hereinabove defined with an aldehyde of the formula
'' Rl-C~10
~ a -
7;~
wherein ~ is as hereinabove defined in the presence of a reducing agen~ in
a solvent inert to the reactants for a period of time sufficient for a sub-
stantial degree of reductive alkylation to take place; or (b) for preparing
a compound of formula (V), alkylating an amine of the formula:
O
R ~ CH=CH-C~NH-(CH2)~~NH~(CH2)4-NH2 (B)
wherein R is as hereinabove defined with a ketone of the formula:
It
R -C~R3
- wherein R2 and R3 are as hereinabove defined in the presence of a reducing
agent in a solvent inert to the reactants for a period of time sufficient for
a substantial degree of reductive alkylation to take place.
A preferred reductive alkylation process whereby the novel antibac-
terial agents of the present invention may be prepared is carried out as fol-
; lowsO Antibiotic ~M123Y, B~123~1, or BM123~2 is dissolved in a suitable sol-
;~ vent such as water, mekhanol, methyl cellosolve, or mixtures thereof, an
amount in excess of an equimolar amount of the desired aldehyde or ketone is
-~ then added followed by the addition of a reductive sufficiency of sodium
cyanoborohydride. The pH of the reaction mixture is maintained at 6.0-~.0
with dilute mineral acid during the course of the reaction~ After one to 24
hours at ambient temperature (lO -35 C.), the reaction mixture is evaporated
to dryness in vacuo and the residue is triturated with methanol and filtered.
The filtrate is diluted with acetone and the solid product that precipitates
is removed by filtration and dried in vacuo.
Aldehydes which may be so employed in the above process are~ for
example, acetaldehyde, propionaldehyde, butyraldehyde, _ butyraldehyde, cro-
tonaldehyde, valeraldehyde, ben~aldehydeg ~cyanoben~aldehyde~ salicylalde-
hyde, cinnamaldehyde, trichloroacetaldehyde, etc. Ketones which may be so
employed in the above process are, for example, acetone, 2-butanone,
:
~ ~ ~ - 2b ~
.,.~: . ' "'. ., '' . '' :
,/ztj~
1,3-dibromoacetone, ehloroacetone, acetophenone, m-chloroace-
tophenone, _-bromoacetophenone, _-trifluoromethylaeetophenone,
_-nitroaeetophenone, _-dirr.ethylaminoacetophenone, ete.
The produets are obtained from the reduetive alkyla-
5 tion reaetion mixtures by standard proeedures sueh as preeipi-
tation, eoneentration, solvent extraction or combinations of
these proeedures. After isolation, the products may be puri-
fied by any of the generally known methods for purifieation.
These inelude recrystallization from various solvents and
10 mixed solvent systems, ehromatographic techniques, and counter
current distribution, all of whieh are usually employed for
this purpose.
The novel antibacterial agents of the present inven-
tion are organic bases and thus are capable of forming aeid-
15 -addition salts with a variety of organic and inorganic salt- .:
-forming reagents. Thus, aeid-addition salts, formed by ad-
~ mixture of the antibacterial free base with up to three equi-
- valents of an acid, suitably in a neutrai solvent, are formed
~ with sueh aeids as sulfuric, phosphoric, hydrochlorie, hydro-
- 20 bromie, sulfarnie, citric, maleic, fumaric, tartaric, acetic,
benzoic, glueonic, ascorbie, and related acids. The aeid-
addition salts of the antibacterial agents of the present
invention are, in general, crystalline solids rela-tively
soluble in water, methanol and ethanol but are relatively in-
soluble in non-polar organie solvents such as diethyl ether,
benzene, toluene, and the like. For purposes of this inven-
tion, the antibacterial free bases are equivalen-t to their
non toxic acid addition salts.
DETAILED DESCRIPTION OF THE INVENTION
. 30 The antibiotics designated BM123~1, BM123~2, BM123rl
and BM123~2 are formed during the cultivation under con-trolled
conditions of a new strain of an undetermined species of
Noeardia. This new antibiotie yroducing strain was isolated
-- 3 --
",
:Erom a garden soil sample collected at Oceola, Iowa, and is
maintained in the culture collection of the Lederle Laboratories
Division, Amerlcan Cyanamid Company, Pearl River, N.Y as Culture
No. BM123. A viable culture of the new microorganism has been
deposited with the Culture Collection Laboratory, Northern
Utilization Research and Development Division, United States
Department of Agriculture, Peoria, Illinois, and has been added
to its permanent collection. It is freely available to the
public in this depository under its accession No. NRRL 5646.
Herein BM123~ refers to a mixture in any proportion of BM123~1
and BM123~2, and BM123~ refers to a mixture in any proportion of
BM123~1 and BM123~ .
- The following is a general description of the micro-
organism Nocardia sp., NRRL 5646, based on diagnostic
characteristics observed. Observations were made of -the
cultural, physiological, and morphological features of the
organism in accordance with the methods detailed by Shirling
and Gottlieb, Interna-t. Journ. of Syst. Bacteriol. 16:213-240
(1966). The chemical composition of the culture was deter-
mined by the procedures given by Lechevalier et al., Advan.
Appl. Microbiol. 14:47-72 (1971). The underscored descriptive
colors and color chip designations are taken from Jacobson
et al., Color ~larmony Manual, 3rd ed. (1948), Container Corp.
of America, Chicago, Illinois. Descriptive details are
recorded in Tables I through V below.
~rnount of Growth
Moderate on yeast extract, asparagine dextrose,
Benedict's, Bennett's, potato dextrose and Weins-tein's
` agars; light on ~lickey and Tresner's, tomato paste,
oatmeal, and pablum agars ar~d A trAce of growth
on inorganic sal-ts-starch, Kuster's oatflake, Czapek's
solution,and rice agars.
,
-- 4 --
s~
Aerial Mycelium
; Aerial mycelium whitish when present; produced only on
yeast extract, asparagine dextrose, Benedict's~Bennett's,
and potato dextrose agars.
Soluble Pigments
No soluble pigments produced.
Reverse Color
Colorless to yellowish shades.
Miscellaneous Physiological Reactions
No liquefaction of gelatin; nitrates reduced to nitrites
in 7 days; melanoid pigments not formed on pep-tone-iron
agar; no peptonization or curd formation in purple mil~;
NaCl tolerance in yeast extract agar ~ 4~ but ~ 7~;
optimal growt~ temperature 32C. Carbon source utiliza-
tion, according to the Pridham and Gottlieb method [J.
Bacteriol. 56:107-114 (1948)] as follows: Good utiliza-
tion of glycerol, salicin, _-trehalose and dextrose;
fair utilization of i-inositol; and poor -to non-utiliza-
tion of _-fructose, maltose, adonitol, l-arabinose,
lactose, _-mannitol, _-melibiose, _-raffinose, 1-
-rhamnose, sucrose and _ xylose.
Chemical Composition
The organism belongs -to cell wall type IV, i.e., con-
tains meso-2,6-diaminopimelic acid and has a type A
whole-cell sugar pattern, i.e., con-tains arabinose and
galactose. Methylated whole cell extracts, when sub-
jected to gas chromatography, showed fatty acid patterns
similar to those produced by Nocardia as-teroides ATCC
3308.
Micromorphology
Aerial mycelium arises Erom substrate mycelium as
sparingly branched modera~ely long flexuous elements
-- 5 --
:
that commonly terminate in elongated primitive spirals.
The flexuous elements are irregularly segmented into
short elliptical to cyclindrical sections (spores?)
which disarticulate readily. The spiral terminal por-
tions are less conspicuously segmented. Segments gen-
erally range 0.8-1.7 ~m x 0.3-0.5 ~Im, averaging
0.~ ,um ~ 1.2Jum.
Diagnosis
The morphological characteristics of Culture No. BM123
are difficult to observe and interpret because of the
poor development of aerial mycelium on most media. Hence,
considerable importance is attached, out of necessity, to
the chemical analysis in determining the generic relation-
ship of the organism. On the basis of the system pro-
posed by Lechevalier et al., Culture No. BM123 contains
meso-2,6-diaminopimelic acid in its whole cells and sugar
analysis shows arabinose and galactose to be present.
Therefore, the culture belongs to cell wall type IV.
comparison of the gas chromatography pattern of Culture No.
BM123 with that of Nocardia asteroides ATCC 3308 showed
the two to be rernarkably similar. Other characteristics
- of Culture No. BM123 that are in keeping with the Nocardia
concept, are its fragmenting aerial growth on some media
an~ the total absence of aerial growth on most media.
In view of the lack of adequate criteria for the characteri-
zation of Nocardia to the species level, no attempt has been
made to ~ake this determination. ThereEore, Culture No.
BM123 will be considered an undetermined species of Nocardia
until such a diagnosis is feasib:Le.
3G
i
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E ~cl E o~ aJ ~1 O O Y
C) d O -~ E ~ rl aJ ~ ~ ,/ ~1 >~ ~J ~'
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-- 7
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~1 ,1 E ~1 ~ ,~
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-- 8 --
.
5fl~'i
- TABLE II
Micromorphology of Nocardia sp. NRRL 5646
Aerlal Mycelium and/or Sporlferous
Medium S-tructures
____ _ _
Yeast Extract Aerial mycelium arises from sub-
Agar strate mycelium as sparingly
branced, flexous elements that
commonly terminate in elongated
primi-tive spirals. The flexuous
elements are irregularly segmented
into sho~tsec-tions (spores?)
which disarticulate readily. The
spiral terminal portions are less
consp-icuously segmented. Segments
generally range 0.8-1 7~m x
0.3-0.5 ~m, averaging 0.4 ~m x
1.2 ~m.
.'~ ' , - .
.,
,,~ . .
~ 20
.
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-- I 1- 'T~
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.
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- 10 - .
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.
TABLE IV
Carbon Source Utilization Pattern of Nocardia sp. NRRL 5646
Incubation: 10 days Temperature: 32C.
. _
Carbon Source Utilization*
. .
Adonitol O
l-Arabinose O
Glycerol 3
d-Fructose
l-Inositol 2
Lactose O
. d-Mannitol O
Salicin 2
d-Melibiose O
d-Raffinose O
Rhamnose O
Maltose.
; Sucrose O
d-Trehalose 3
. . d-Xylose O
Dextrose .3
. Negative Control O
. _
*3-Good Utiliza-tion l-Poor Utilization
2-Fair Utilization O-No Utilization
:'
q2~4~
TABLE V
Chemical Composition of Nocardia sp. NRRL 5646
1 Wall Type ~ Major Constituen-ts
Type IV meso-DAP, arabinose,
galactose
.
The production of BMi23~ and BM123~ is not ~imited
to this particular organism or -to organisms fully answering
the above growth and microscopic characteri~stics which are
given for illus-trative purposes only. In fact, mutants pro-
duced from this organism by various means such as exposure~to
~-radiation, ultra-violet radiation, nitrogen mustard, actino-
phages, and the like, may also be used. A viable culture of
a typical such mutant strain has been deposited with the
Culture Collection Laboratory, Northern Utiliza-tion Research
. and Development Division, United States Depar-tment of Agri-
culture, Peoria, Illinois, and has been added to its permanent
collection under its accession number NRRL 8050. Although
the cultural, physioloyical, and morphological features of
- NRRL 8050 are substantially the same as those of NRRL 5646,
it produces enhanced amounts of BM123~ during aerobic fermenta-
tion. Also, NRRL 8050 varies from the parent NRRL 5646 as
follows:
(a~ slower reduction of nitrates -to nitrites; and
(b) production of a rosewood tan mycelialpigment
on sennett's and yeast extract agars.
The novel antibacterial agents of the present inven-
tion are, in general, crystalline solids of relatively limited
solubility in non-polar solvents such as diethyl ether and
hexane, but considerably more soluble in solvents such as
water and lo~er alkanols. Antibiotics BM123~1 and BM123~2 are
structural isomers and may be represented by the following
- 12 -
' '
! '
S~'^J
, --
stru- tural formule~e:
;.
10
.`,'
.
; . ` ' '
~; 20
,
_ 13 --
'r , ,
z ~ :
o=y
h ~
1 ' ~ Z
,. c~ o7Lx
:~ o=o
O ~
R ~
/ Z'
,LI=
~C
, . ~ .
,.
-
: ' , - . ' .
. : .
S~
æ
o
.. , ~'
'
æ
o--o
I
' '' ~l~
~111 . .
,, I ~
~1 ~ . I
; .I I
~,/
` T
.~ o
~:" 7
;. o
.' ,~1 ~, .
$ æ
o=o
/~) 5
0~ æ
~\ ~o~ =o
~ ' ' '
.... . .
~'; ' ,
: 15 -
:
~ 5
The reductive alkylation of BM123~, BM123~1 or ~
BM123~2 with ketones takes place on the spermadine side-chain
to form derivatives of the formula:
R ~ -cR=cH-c-N~-(cH2)3-~3-(c~l2)4~ -c ~R
wherein R is a moie-ty of the formulae:
,
l~o~ lo- :
~ ll N~c=o H N~ ~I rill
r~H2 c=o f=Nil
. NH2 Nil2
- or
H H Çi-13
~ I-C-I ~
Nl~2NH2 2
::
and R2 and R3 are as hereinabove defined. The reductive
alkylation of BM123~, BM123~1 or BM123~2 with aldehydes takes
place on the spermadine side-chain to form mono-, di-, and
tri-substituted derivatives of the formulae:
,. ,
_ 16 -
.L~
R ~ CE~=C~ C-NH-(cll2)3-NH-(c~l2)4 Nll-c~l2
O
~ trans ¦¦ ~ CH2-Rl
R ~ CH=cH-c-NH-(cH2)3-NH-(cll2)4 N~ Cll R
o
~ trans ¦¦
R ~ CH=CH-C-NH-(CH2)3-N-(CH2)4-NH-CH2-R
, CH2-Rl
R ~ CH=CH-C-NH-(C~l2)3-1_(CH2)4 -cH2-
CH2 Rl
wherein R and Rl are as hereinabove deEined.
The usefulness of the alkylated deriva-tives of
BM123~ is demonstrated by their ability to control systemic
lethal infections in mice. These new substances show high
in vlvo antibacterial activity in mice against Escherichia coli
US311 when administered by a single subcu-taneous dose to
groups of Carworth Farms CF-l mice, weigh-t about 20 gm., in-
fected intraperi-toneally with a le-thal dose of -this bacteria
in a 10 3 trypticase soy bro-th TSP dilution of a 5 hour TSP
blood culture. In Table VI below is set forth the ln vlvo
activity of typical products of this invention (prepared from
the indicated carbonyl reagents) against Escherichia coli
US311 in mice. The activity is expressed in tenns of the
ED50 or the dose in mg./kg. oE body weight required to protec-t
50~ of the mice against _. coli.
;'
-' :
_ 17 --
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.
o o o o o o o o o o o o o
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m m
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-- 27
. .
Fermentation Process Selec-ted to Produce Primarily BM123~and
BM123~
Cultivation of Nocardia sp. NRRL 8050 may be carried
out in a wide variety of liquid culture medla. Media which
are useful for the production of the antibiotics include
an assimilable source of carbon such as starch, sugar, molasses,
glycerol, etc.; an assimilable source of nitrogen such as
protein, protein hydrolyzate, polypeptides, amino acids, corn
steep liquor, etc.; and inorganic anions and cations, such as
potassium, magnesium, calcium, ammonium, sulfate, carbonate,
phosphate, chloride, etc. Trace elements such as boron, moyl-
; bdenum, copper, etc.; are supplied as impuri-ties of other
constituents of the media. Aeration in tanks and bottles is
provided by forcing sterile air through or onto the surface of
the fermenting medium. Further agitation in tanks is provided
~` by a mechanical impeller. An antifoaming agent, such as Hodag
FD82 may be added as needed.
Inoculum Preparation for BM1233 and BM123 ~
Primary shaker flask inoculum of Nocardia sp. NRRL
8050 is prepared by inoculating 100 milliliters of sterile
liquid medium in 500 milliliter flasks with scrapings or
washings of spores from an agar slant of the culture. The
following medium is ordinarily used:
Bacto-tryptone~................. 5 gm.
Yeast extract............. O..... 5 gm.
Beef extract................. ... 3 gm.
Glucose...................... . O10 gm.
Water to .................... 1000 ml.
The flasks were incubated at a temperature from 25-
-29C., preferably 28C. and agita-ted vigorously on a rotary
shaker for 30 to 48 hours. The inocula are then transferred
into s-terile screw cap culture tubes and storecl at below 0F.
This bank of vegetative inoculum is used instead of slant
scrapings for inoculation of additional shaker flasks in pre-
paration of this first stage of inoculum.
- 28 -
~ 7Z~'7
.
These first stage flask inocula are used to seed
12 llter batches of the same medium in 20 liter glass fermentors.
The inoculum mash is aerated wi~h sterile air while growth is
continued for 30 to 48 hours.
The 12 liter batches of second stage inocula are
used to seed tank fermentors containing 300 liters of the
following sterile liquid medium to produce the third and final
stage of inoculum:
Meat solub]es....... ..... l5 gm.
Ammonium sulfate~....................... 3 gm.
Potassium phosphate, dibasic... O3 gm.
Calcium carbona-te..O.......... l gm.
Magnesium sulfate hepta-........
hydrate................ . l.5 gm.
Glucose..................... .. 10 gm.
Water to.................... 1000 ml.
The glucose is sterilized separately.
The third stage inoculum is aerated at 0.4 to 0.8
liters of sterile air per liter of broth per minute, and the
fermenting mixture is agitated by an impeller driven at
.. . .
150-300 revolutions per minute. The temperature is maintained
at 25-29C., usually 28C. The growth is continued for 48 to
72 hours, at which time the inoculum is used to seed a 3000
liter tank fermentation.
Tank Fermentation for BM123~ and BM123
. _ . ,
For the production of BM123B and BM123 ~ in tank
fermentors, the following fermentation medium is preferably
used:
Meat solubles...................... 30 gm.
Ammonium sulfate.................... 6 gm.
Potasslum phosphate, dibasic........ 6 gm.
Calcium carbonate............... ~... 2 gm.
Magnesium sulfate heptahydrate..... . 3 gm.
Glucose............................ 20 gm.
Water -to.... ~................ 1000 ml.
The glucose is s-terilized separately.
Each tank is inoculated with 5 to 10~ of third stage
inoculum made as described under inoculum preparation. The
fermenting mash is maintained a-t a temperature of 25-28C.
usually 26C. The mash is aerated with sterile air at a rate
of 0.3-0.5 liters of sterile air per li-ter of mash per minute
- 29 -
:
,,
~^
and agitated by an impeller driven at 70 to 100 revolutions
per minuteO The fermentation is allowed to continue from
65-90 hours and the mash is harvested.
The invention will be described in greater detail
S in conjunction with the following specific examples.
EXAMPLE 1
Inoculum preparation for BM123~ and BM123 ~
A typical medium used to grow the first and second
stages of inoculum was prepared according to the following
formula:
Bacto-tryptonenO.O.. O.~ .5 gm.
Yeast extract.......O................... 5 gm.
Beef extract............................ 3 gm.
Glucose................................ 10 gm.
Water to............................. 1000 ml.
Two 500 milliliter flaskseach containing 100 milli-
liters of the above sterile medium were inoculated with 5
milliliters each of a frozen vegetative inoculum from Nocardia
sp. NRRL 8050. The flasks were placed on a rotary shaker and
agitated vigorously for 48 hours at 28C. The resulting flask
inoculum was transferred to a 5 gallon glass fermentor contain-
ing 12 liters of the above sterile medium. The mash wasaerated with sterile air while growth was carried out for about
48 hours, after which the contents were used to seed a 100
gallon tank fermen-tor containing 300 liters of the following
sterile liquid medium:
Meat solubles.......~.................. 15 gm.
Ammonium sulfate................... ..... 3 gm.
Potassium phosphate, dibasic....... 3 gm.
Calcium carbonate.................. l gm.
Magnesium sulfate heptahydrate... l.5 gm.
Glucose........................... 10 gm.
Water to~....................... 1000 ml.
The glucose is s-terili~ed separately.
The third stage of inoculum mash was aerated wi-th
sterile air sparged into the fermentor a-t 0.4 liters of air
per liter of mash per minute. Agitation was supplied by a
driven impelleF a-t 240 revolutions per mlnute. The mash was
- 30 ~
2~ 7
:
maintained at 28C. and Hodag~ FD82 was used as a defoaming
agent. After 48 hours of growing time the inoculum mash was
used to seed a 3000 liter fermenta-tion.
EXAMPLE 2
Fermentation Employing Nocardia sp. NRRL 8050 and Medium Favor-
ing the Production of BM1233 and BM123~
A fermentation medium was prepared according to the
following formula:
Meat solublesO~........ O.. ~......................... 30 gm.
Ammonium sulfate................. ~.. .6 gm.
Po-tassium phosphate, dibasic....... .6 gm.
Calcium carbonate.~ ...... O......................... .2 gm.
Magnesium sulfate heptahydrate...... .3 gm.
Glucose................ ~...... 20 gm.
Water to.................... l000 ml.
The glucose is sterilized separa-tely.
The fermentation medium was sterilized at 120C. with
steam at 20 pounds pressure for 60 minutes. The pH of the
medium after sterilization was 6.9. Three thousand liters of
sterile medium in a 4000 liter tank fermentor was inoculated
with 300 liters of inoculum such as described in Example 1, and
the fermentation was carried out at 26C. using Hoda ~ FD82
~; 20 as a defoaming agent. Aeration was supplied at the rate of
0.35 liter of sterile air per liter of mash per minute. The
mash was agitated by an impeller driven at 70-72 revolutions
per minute. At the end of 67 hours of fermenta-tion time the
mash was harvested.
EXAMPLE 3
Isolation of BM123!3 and BM123~
A 3000 liter portion of fermenta-tion mash prepared as
described in Example 2, pH 4.3, was adjusted to pH 7.0 with
sodium hydroxide and filtered using 5~ diatomaceous earth as
a filter aid. The cake was washed with about 100 liters of
water and discarded. The combined filtrate and wash was pumped
; upward through three parallel 8 1/~" x 48" s-tainless s-teel
columns each containing 15 liters of CM Sephadex ~ C-25 [Na~]
- 31 -
., .
, , .
~ ~ 7~
resin (a cross-linked dextran-epichlorohydrin cation exchange
gel available from Pharmacia Fine Chemicals, Inc.). The
charged columns were washed with a total of about 390 liters
of water and then developed wi-th 200 li-ters of 1~ aqueous
sodium chloride followed by 560 liters of 5% aqueous sodium
chloride. The 56 aqueous sodium chloride eluate was clarified
by filtration through diatomaceous earth and the clarified
filtrate passed through a 9" x 60" glass column containing 25
liters of granular Darco~) G-60 (20-40 mesh)(a granular activated
carbon available from Atlas Chemical Industries, Inc.).
The charged column was washed with 120 liters of water and
then developed with 120 liters of 15% aqueous methanol followed
by 340 liters of 50g6 aqueous methanol and then 120 liters of
506 aqueous acetone. The 156 aqueous methanol eluate was
concentrated ln vacuo to about 7 liters of an aqueous phase
and the pH adjusted from 4.5 to 6.0 with Amberlite ~ IR-45
(OH-) resin (a weakly basic polystyrene-polyamine type anion
exchange resin). The resin was removed by filtration and the
filtrate was concentrated ln vacuo to about 1 liter and then
lyophilized to give 38 grams of material consisting primarily
of BM123~ along with a small amount of BM123?~ (primarily
BM123~2). The 506 aqueous methanol eluate was adjusted from
pH 4.65 -to 6.0 with Amberlite (~;)IR-45 (OH ) resin. The resin
was removed by filtration and the filtrate was concer-trated
in vacuo to about 6.3 liters and -then lyophylized to give 213
grams of materlal consisting primarily of BM123~ The 506
aqueous acetone eluate was adjusted from pH 4.0 to 6.0 with
Amberlite (~) IR-45 (OH-) resinn The resin was removed by filtra-
tion and the filtrate was concentrated in vacuo to about 1.5
liters and then lyophylized to give 56 grams of irnpure BM123
EXAMPLE 4
; Further Purification of BM123~
A slurry of CM Sephadex ~ C-25 [NH4] in 2% aqueous
ammonium chloride was poured into a 2.6 centimeter diameter
glass column to a resin height of approximately 62 centimeters.
The excess 2~ aqueous ammonium chloride was drained away and
a 5.0 gram sample of BM123~ prepared as described in Example 3
was dissolved in about 10 millili-ters of 2~ aqueous ammonium
ehloride and applied to the column. The column was then eluted
with a gradient between 6 liters each of 2% and 4~ aqueous
ammonium chloride. E'ractions of about 75 milliliters each were
collected automatically every 15 minutes. Antibiotic BM123~ ' -
was located by monitoring the column effluent in the ultra-
violet and by bioautography of dipped paper disks on large agar
plates seeded with Klebsiella pneumoniae strain A~. The
majority of BM123~was located between fractions 71-107 inclusive.
One hundred thrity milliliters of granular Darco ~
; G-60 (2040 mesh) was suspended in water, transferred to a glass
column, allowed to settle and the excess water was allowed to
drain away. Fractions 84-96 inclusive from the above CM
Sephadex chromatography were combined and passed through the
granular earbon column. The eharged column was washed with 600
millili-ters of water and then developed with 1 litex of 50
aqueous acetone. The eluates, both of which contalned BM123~
were concentrated to aqueous phases ln vacuo and lyophilized to
give a total of 886 milligrams of ~M123~as the hydroehloride
salt. A microanalytical sample was ob-tained by subjecting the
above material to a reyeat of -the above process.
Antibiotic BM123~ does not possess a definite melt-
ing point, but gradual decomposition star-ts in the vicinity
of 200C. Mieroanalysis of a sample equilibrated for 24 hours
in a 72F. atmosphere containing 23% relativè humidity gave C,
39.44%; H, 6.10~; N, 16.19~; Cl(ionic), 11.54~; loss on
~ _
drying, 8.19%. In water BM123 ~ gave a U.V. absorption maxlmum
at 286 nm with El~ = 250. The position of this maximum did
lcm
not ehange with pH. BM123~ had a specific rotation of
[a]D5 = +71 (C = 0.97 in water).
Antibiotic BM123~ exhibited characteristic absorption
in the infrared region of the spectrum at the following wave-
lengths: 770, 830, 870, 930, 980, 1035j 1105, 1175, 1225,
130P, 1340, 1370, 1460, 1510, 1555, 1605, 1660, 1740, 2950
and 3350 cm 1. A standard infrared absorption spectrum of
BM123~ prepared in a KBr pellet is shown in Figure 1 of the
accompanying drawings.
EXAMPLE 5
Isolation of BM123~ -
A slurry of CM Sephadex ~ C-25 [Na+l in 2% aqueous
sodium chloride was poured into a 2.6 centimeter diameter glass
eolumn to a resin heigh of approximately 70 centimeters~ The
excess 2% aqueous sodium chloride was drained away and 4.11
gram of a sample containing primarlly BM123~1 along with some
BM123~2 and other impurities, prepared as described in Example
20- 3, was dissolved in about 10 milliliters of 2% aqueous sodium
ehloride and applied to the column. The column was then
eluted with a gradient be-tween 4 liters each of 2% and 4%
aqueous sodium chloride. Fractions of about 75 milliliters
eaeh were eollee-ted automatieally every 15 minutes. Anti-
biotic BM123~ was loea-ted by monitoring the column effluent
in the ultraviolet and by bioautography of dipped paper disks
on large agar plates seeded with Klebsiella pneulnoniae strain
` AD. The ma~ority of BM123~ was located between fractions
64-90 inclusive; -the initial fractions (64-80) conta:ined a
mixture of BM123~1 and BM123~2 whereas the later fractions
(81-90) contai~ed essentially pure BM123~.
- 34 -
r-)Lt~il
One hundred milliliters of granular Darco ~ G-60
(20-40 mesh) was suspended in water, transferred to a glass
columnr allowed to settle and the excess water was allowed to
drain away. Fractions 81-90 inclusive from the above CM Sepha-
dex chromatography were combined and passed through the granu-
lar carbon column. The charged column was washed with 500
milliliters of water and then developed with 500 milliliters of
10~ aqueous methanol followed by 1 liter of 50~ aqueous
methanol. The 50~ aqueous methanol eluate, which contained the
majority of BM123~1, was adjusted from pH 5O9 to 6.0 with
Amberlite ~ IR-45(OH 1) resinO The resin was removed by filt-
ration and the filtrate was concentrated ln vacuo to an
aqueous phase and lyophilized to give 294 milligrams of white
amorphous BM12341 as the hydrochloride salt.
Antibiotic BM123~1 does not possess a definite
.
- melting poin-t, but gradual decomposition starts ln the vicinity
of 200C. Microanalysis of a sample equilibrated for 24 hours
in a 70F. atmosphere containing 60~ relative humidity gave
C, 37.84~; H, 5.73%; N, 15.58~; Cl(ionic), 10001%; loss on
drying 10.45%. ~In methanol BM1237~1 gave a U.V. absorption
maximum at 286 nm with ElCm = 225O The posi-tion of this max-
; imum did not change with pH. BM123~1 had a specific rotation
.
of -~55 (C-0.803 in water).
Anti~iotic BM123~fl exhibited characteristic ab-
sorption in the infrared region of the spectrum at the follow-
ing wavelengths: 770, 830, 870, 930, 980, 1045, 1080, 1110,
1125, 1175, 1225, 1305, 1345, 1380, 1465, 1515, 1560, 1605, 1660,
1730, 2950 and 3350 cm 1. A standard infrarecd absorption
spectrum of BM123~1 prepared in a KBr pellet is shown in
Figure 2 of the accompanying drawings. A standard proton
magnetic resonance spectrum of BM123~1 determined on a D20
solution in a 100 megacycle spectrometer is shown in Figure 4
of the accompanying drawings.
- 35 -
. .
z~
~XAMPLE 6
Isolation of BM123~
A 25 gram sample containing prlmarily BM123~2 and
BM123,13, prepared as described in Example 3, was dissolved in
about 120 milliliters of 2% aqueous sodium chloride and applied
to a column containing 1800 ml. of CM Sephadex ~ C-25 [Na+]
in 2% aqueous sodium chloride. The column was then eluted
with a gradien-t between 20 liters each of 2% and 4% aqueous
sodium chloride. The initial 12 liters of eluate was collected
in a large bottle and discarded. Thereafter fractions of about
800 milliliters each were collected automatically every 40
~, minutes. Antibio-tic BM1237r was'located by monitoring the
column fractions in the ultraviolet. The majority of BM123?~
,was located between fractions 7-18 inclusive; the initial
fractions (7-15) contained,essentially pure BM123~ and the
- later fractions (16~18) contained a mixture of BM123~ and
BMl 2 37~2 -
Six hundred milliliters of granular Darco ~ G-60
(20-40 mesh) was suspended in water, transferred to a glass
column, allowed to settle ancl the excess water was allowed to
, ' drain away. Fractions 7-1$ inclusive from the above CM
Sephadex chromatography were combined and passed through the
- granular carbon column. The charged column was washed with
`~ 3 liters of water and then developed with 3 liters of 10~
aqueous methanol followed by 6 liters of 50~ aqueous methanol.
;~ The 10~ aqueous methanol eluate was ad~usted from pH 5.8 to
6.0 with Amberlite ~ IR 45'(OH ) resin. The resin was removed
' by filtration and the filtrate was concentrated in vacuo to
`, an aqueous phase and lyophilized to give 595 milligrams of
white amorphous BM123~2 as the hydrochloride salt. The 50~
aqueous methanol eluate was adjusted from pH ~.6 to 6.1 with
Amberlite IR 45 (OH-) resin, The resin was removed by filtra-
tion and the filtrate was concentrated irl vacuo to an aqueous
~ 36 -
~' .
phase and lyophilized to give 3.645 grams of slightly less pure
white amorphous BM123~2 as the hydrochloride salt.
; Antibiotic BM123~ does not possess a definite
melting point, but gradual decomposition starts ln the vicinity
of 200C. Microanalysis of a sample equilibrated for 24 hours
in a 70F. atmosphere containing 60~ rela-tive humidity gave
C, 36.14%; H, 5.67%; N, 15.1%; Cl(ionic), 11.11~; loss on
drying 10.87~. In methanol BM123~ gave a U.V. absorption
maximum at 286 nm with El = 220. The position of this
maximum did not change with pH. BM123~ had a specific
rotation of +60 (C=0.851 in water).
Antibiotic BM123~f2 exhibi-ted characteristic absorp
tion ln the infrared region of the spectrum at the following
wavelengths: 770, 830, 870, 950, 980, 1035, 1110, 1175, 1225,
1285, 1345, 1380, 1470, 1515, 1560, 1605, 1660, 1755, 2950
and 3350 cm 1. A standard infrared absorption spectrum of
BM123~2 prepared in a KBr pellet is shown in Figure 3 of the
accompanying drawings. A standard proton magne-tic resonance
- spectrum of BM123~2 de-termined on a D2O so]ution in a 100 mega-
cycle spec-trometer is shown in Figure 5 of -the accompanying
drawings.
EXAMPLE 7
:
Paper Parti-tion and Thin Layer Chromatography of BM123~ and B~il23~
. .
` The BM123 antibiotics can be distinguished by
paper chromatography. For this purpose Wha-tman No. 1 strips
were spotted with a water or me-thanol solution of the sub-
stances and equilibrated for 1 to 2 hours in -the presence of
both upper and lower phases. The strips were developed over-
; night with the lower (organic) phase obtained from mixing
90% phenol:m-cresol:acetic acid:pyridine:water (100:25:4:4:75
by volume). The developed strips were removed from the
- chromatographic chamber, air dried for 1 to 2 hours, washed
with ether to remove residual phenol and bioautographed on
''; . ' , .
- 37 -
':
~ '7
" '
large agar plates seeded wi-th KlebsielLa peunmoniae strain AD.
Representative Rf values are listed in Table VII below:
TABLE VII
Componen-t RF
_
BM123~ 0.85
BM123~ 0.50, 0.70
The ~ component was a mixture of two antibiotics
using this system. BM123~ was composed of a major antibiotic
(Rf = 0.50) called BM123~1 and a minor antibiotic (Rf =,0.70)
~ called BM123~2.
- The BM123 antibiotics can also be distinguished by
thin,layer chromatography. For this purpose pre-coated
Cellulose F ~ plates (0.10 millimeters thick), a form of thick
layer cellulose supplied by EM Laboratories Inc., Elmsford,
" N.Y. were spotted with a water solution of the substance to
' ' be chromatographed (about 20-40 micrograms per spot). The
20, plates were developed overnight with the solvent obtained by
mixing l-butanol:water:pyridine:acetic acid (15:12:10:1 by
'' volume). The developed plates were removed from the chroma-
'tographic,cha~ber and air dried for about 1 hour. The anti-
biotics were detected by using either standard ninhydrin or
~Sakaguchi spray reagents. Representative Rf values are listed
in Table VIII below:
. ' .
- 38 -
-'
4')
.
- TABLE VIII
Component Rf
.
BM123~ 0.17, 0.23
BM123~ 0.08, 0.14 ,
Both BM123~ and ~ were a mixture of two components
using this system. BM123~ was composed of a major component
(Rf = 0.08~ which was BM123~1 and a minor component (Rf =
0.14) which was BM123~2. The less polar component of BM123
(Rf = 0.23) was BM123~1 and the more polar component (Rf =~
; 0.17) was BM123~2.
~ EXAMPLE 8
General Procedure for Reductive Alkylation of Antibiotic BM123
To a stirred solution of~100 mg. oE antibiotic
BM123~ in 20 ml. of methanol is added 5 ml. (or 5 g.) of the
appropriate aldehyde or Ketone and 100 mg. of sodium cyano-
borohydride. The pH of the resulting solution is maintained
at about 7.0 with O.lN methanolic hydrogen chloride over a
3 to 24 hour period. The reaction is monitored by thin layer
chromatograpy to the disappearance of the BM123~. The reactlon
mixture is then filtered and the filtrate is evaporated to
dryness. The residue is triturated wi-th 3 ml. of methanol
; and filtered. The filtrate is diluted with 50 ml. of acetone
- 25
and the precipitate which forms is removed by filtration and
dried. The methanol solvent may be replaced by 20 ml. of
water wherever the starting aldehyde or ketone is water soluble.
' ' ~
39 -
' '
EXAMPLE 9
Preparation of methyl-BM123
-
To a solution of 1.0 g. of BM123'~ and 2.5 ml. of a
37~ aqueous formaldehyde solution in 50 ml. of water was added,
portionwise, 400 mg. of sodium cyanoborohydride. The pE~ of
the reaction mixture was maintained at 7.0 with lN hydrochloric
acid during this additionO The reaction mixture was stirred
an additional ten minutes at room temperature and then'evapor-
.ated to dryness ln vacuo. The residue was triturated with 20
ml. of methanol, filtered and -the filtrate diluted with 250 ml.
of acetone. The product which precipitated was removed by
filtration and dried; yield, 667 mg.
EXAMPLE 10
Preparation of isopropyl-BM123 ~
To a solution of 200 mg. of BM123'~ in 30 ml. of
methanol was added 5 ml. of ace-tone. To this solution was
added 139 mg. of sodium cyanoborohydride and the reaction mix-
ture was s-tirred at room temperature for 30 minutes~ During
this time the pEI of the reaction mixture was maintained be-
tween 7.4 and 7.8 by the addition of O.lN methanolic hydrogen
chloride. The small amount of precipitate which had formed
was removed by filtration and the filtrate was evaporated to
dryness in vacuo. The residue was -triturated with two ml. of
methanol and filtered. The filtrate was diluted with 100 ml.
of acetone and the solid produc-t that separated was removed
by filtration and dried; yield, 184 mg.
EXAMPLE 11
Preparation of ~-phenylethyl-BMl23
_ _
To a solution of 200 mg. of BM123'~ in 15 ml. of
water and 25 ml. of acetonitrile was added a solution of 2 ml.
of phenylacetaldehyde in 4 ml. of ethanol. To this was added
103 mg. of sodium cyanoborohydride. The reaction mixture was
stirred at room temperature for thir-ty minutes during which
- 40 -
.
time the pH of the mixture was maintained at 7 with 0.2N hydro-
chloric acid. The reaction mixture was then filtered and the
~ filtrate was evaporated to dryness in vacuo. The residue was
triturated with two ml. of methanol and Eiltered. The filtrate
was diluted with 100 ml. of acetone and the product that
separated was removed by filtration and dried; yield, 1~0 mg.
EXAMPLE 12
Preparation of 1,3,3-trimethylbutY1-BM123~
To a solution of 200 mg. of BM123~ hydrochloride in
50 ml. of methanol was added 3 ml. of 4,4-dimethyl-2-pentanone
and 106 mg. of sodium cyanoborohydride. The reaction solution
was maintained at pH 7 by the dropwise ~ddition of me-thanolic
hydrogen chloride. The reaction was stirred at room temperature
for 18 hours and filtered. The filtrate was evaporated to
dryness ln vacuo. The residue was dissolved in 3 ml. of methanol,
diluted with 50 ml. of acetone and filtered, yield 125 mg.
EXAMPLE 13
Pre ration of l-methylphenethyl-B~1123
.
; To a solution of 200 mg. of BM123 ~in 50 ml. of
methanol was added 5 ml. of phenylacetone. To this solution was
added 170 mg. of sodium cyanoborohydride and the reaction mix-
ture stirred at room tempera-ture for 3 and a half hours. During
this time the pH of the reaction mixture was maintained at 7.0
with me-thanol saturated with hydrogen chloride gas. Reaction
mixture was concentrated to about 5 ml. volume, diluted with
two ml. of methanol, and fil-tered. Filtrate was poured into
100 ml. of acetone and the solid product that separated was re-
moved by filtratiorl and dried; yield,233 mg.
EXAMPLE 1
.` 3b . Preparation of l-methyJnony]-BM123~~
Sodium cyanoborohydride (100-mg.) was added to a
solution of BM123~ (200 mg.) and 2-decanone (1 ml.) in 40 ml. of
methanol. The p~l of the solution was adjusted to 7.0 and main-
- 41 -
tained at 7.0 ~ 0.2 by the addition of O.lN methanolic hyd~ogen
chloride as necessary. After 19.5 hours the reac-tion~mixture
was filtered and the filtrate was concentrated in vacuo at 35C.
The residue was slurried in 5 ml. of methanol and filtered.
The filtrate was added to 5O ml. of acetone. ,The off white
solid which precipitated was collected'by fil-tration, washed
wi-th acetone, and dried ln vacuo. The yield of crude
l-methylnonyl-BM123~ was 167 mg.
EXAMPLE 15
Preparation of 1,3-dimethylbu-tyl-B~123 ~
To a solution of 210 mg. of BM123~ in 50 ml. of
methanol was added 5 ml. of methyl isobutyl ketone. To this
solution was added 166 mg. of sodium cyanoborohydride and the
reaction mixture stirred at room temperature for five hours.
During this time the pH of -the reaction mixture was maintained
at 7.0 with methanol saturated with hydrogen chloride gas.
Reaction mixture was evaporated to dryness, ln vacuo. The
residue was triturated with two ml. of methanol and filtered.
The filtrate was diluted with 100 ml. of acetone and the solid
' product that separated was removed by filtration and dried;
yield, 210 mg.
EXAMPL~ 16
Preparation of 2-norbornyl-B L ~
" 25 , Sodium cyanoborohydride (100 mg.) was added to a
~ solution of BM123~ (200 mg.) and 2-norbornanone (400 my.) in
; 40 ml. of methanol. The pH of the solution was adjusted to
7.0 with O.lN methanolic hydrogen chloride. The p~l was main-
tained at 7.0 ~ 0.2 by the addition of 0.1l~ hydrogen chloride
as necessary. After 21.5 hours the reaction mixture was
filtered and the filtrate was concentrated ln vacuo at 35C.
The residue was slurried in 5 ml. of methanol and filtered.
The filtrate was added to 50 ml. of acetone. The ofE white
- ~2 -
-
solid which precipitated was collected by fil-tration, washed
with acetone and dried in vacuo. The yield of crude 2-norbornyl-
BM123~ was 175 mg.
EXAMPLE 17
Preparation of isopropyl-BM123~1
A mixture of 50 mg. of BM123~1, 5 ml. of acetone and
60 mg. of sodium cyanoborohydride in 35 ml. of methanol was
stirred at room -temperature for 40 minu,tes. The pH o the
solution was main-tained at 7 by -the dropwise addition of a
10 methanolic hydrogen chloride solution. The mixture was evap-
, orated to dryness ln'vacuo. The residue was triturated with
5 ml. of methanol and the resulting solution was diluted with
50 ml. of acetone; yield, 49 mg.
;~ EXAMPLE 18
Preparation of isopropyl-BM123~2
A mixture of 41 mg. of BM123~2, 5 ml. of acetone and
50 mg. of sodium cyanoborohydride in 35 ml. of'methanol was
stirred at room temperature for 40 minutes. The pll of the
' solution was maintained at 7 by the dropwise addition of a
20 methanolic hydrogen chloride solu-tion ~saturated). The mixture
- , was filtered and evaporated to dryness in vacuo. The residue
` was triturated wi-th 5 ml. of me-thanol and the resul-ting solu-
tion was diluted with 50 ml. of acetone; yield, 46 mg.
EXAMPLE 19
Preparation of l-me-thyl-2-phenyl-ethyl-BM123~2
~, A mixture of 200 mg. of BM123~2, 5 ml. of phenyl-
acetone and 170 mg. of sodiurn cyanoborohydride in 50 ml. of
'~ methanol was stirred at room temperature for 3 hours and 45
i, minutes. During this -time the pll of the reaction mixture
was maintained at 7 with dropwise addi-tion of a methanolic
hydrogen chloride solution (saturated). The mixture was
evaporated to dryness in vacuo. The residue was triturated
with 5 ml. of methanol and the resulting methanol solution was
diluted with approxima-tely 50 ml. of ace-tone; yield 233 mg.
- 43 -
EXAMPLE 20
Preparation of_(2-ethylcyclopentyl) BM123~
A solution of 200 mg. of BM123 ~ 3 ml. of 2-ethyl-
cyclopentanone and 101 mg. of sodium cyanoborohydride in 50 ml.
of methyl alcohol was stored at room temperature for 18 hours.
During this time the pH of -the solution was maintained at 7 with
the addition of a satura-ted solu-tion of hydrogen chloride in
methanol. The reaction mixture was e~aporated to dryness.
! The residue was tri-turated with 3 ml. of methanol, filtered and
the filtrate was diluted with 40 ml. of acetone, yield, 157 mg.
EXAMPLE 21
Preparation of 3,5-dimethylcyclohexyl BM123~
A solution of 200 mg. of BM123~, 5 ml. of 3,5-dimethyl-
cyclohexanone and 200 mg. of sodium cyanoborohydride in 50 ml.
of methanol was stored at room temperature for 1 hour. During
this time the pH of the solution was maintained at 7 with the
addition of a saturated solution of hydrogen chloride in methanol.
The reaction was triturated with 3 ml. of methanol, filtered and
the filtrate was diluted with 40 ml. of acetone, yield 200 mg.
FXAMPLE 22
Preparation of 2,4-dimethylcyclopentyl BM123 ~
A solution of 206 mg. of BM123~ 3 ml. of 2,4-dimethyl-
cyclopentanone and 104 mg. of sodium cyanoborohydride in 50 ml.
of methanol was stored at room temperature for 6 hours. During
this time the pH of the solution was maintained at 7 with the
addition of a saturated solution of hydrogen chloride in methanol.
The reaction was triturated with 3 ml. of methanol, filtered and
the filtrate was diluted with 40 ml. of acetone, yield 101 mg.
EXAMPLE 23
Preparation of 2-ethylcyclohexyl BM123 ~
A solution of 200 mg. of BM123 ~ 5 ml. of 2-e-thylcyclo-
hexanone and 213 mg. of sodium cyanoborohydride in 50 ml. of
methanol was stored at room -temperature for 3 hours. During this
- 44 -
:
; time -the pH of the solution was maintained at 7 with the addi-
tion of a saturated solution of hydrogen chloride in methanol.
The reaction was triturated with 3 ml. of methanol, filtered
and the filtrate was diluted with 40 ml. of acetone, yield 200 mg.
EXAMPLE 24
A solution of 200 mg. of BM123 ~ 1.5 ml. of 3-methyl-
cyclohexanone and 200 mg. of sodium cyanoborohydride in 50 ml.
of methanol was stored at room temperature for 2 hours. During
this time the pH of the solution was maintained at 7 with the
addition of a saturated solution of hydrogen chloride in
methanol. The reaction was triturated with 3 ml. of methanol,
filtered and the filtrate was diluted with 40 ml. of acetone,
yield 200 mg.
EXAMPLE 25
, . ...
Preparat~on of 2,4,4-trimethylcyclopentyl BM123~
A solution of 200 mg. of BM123~ 5 ml. of 2,4,4-tri-
methylcyclopentanone and 179 mg. of sodium cyanoborohydride in
50 ml. of methanol was stored at room temperature for 24 hours.
During this time the pH of the solution was maintained at 7 with
the addition of a saturated solution of hydrogen chloride in
methanol. The reaction was triturated with 3 ml. of methanol,
; filtered and the filtrate was diluted with 40 ml. of acetone,
yield 176 mg.
EXAMPLE 26
PreParation of 2-propylcyclohexyl BM123~
A solution of 200 mg. of BM123~ 3 ml. of 2-propy]cyclo-
hexanone and 157 mg. of sodium cyanoborohydride in 50 ml. of
methanol was stored a-t room -temperature for 4 hours. During
this time the pH of the solution was maintained a-t 7 with the
addition of a saturated solution of hydrogen chloride in methanol.
The reaction was triturated with 3 ml. of methanol, filtered
and the filtrate was diluted with 40 ml. of ace-tone, yield 75 mg.
- 45 -
'
:'.
EXAMPLE 27
Preparation of 2-methylcyclopentyl BM123~
A solution of 211 mgO of BM123 ~ 3 ml. of 2-methyl-
cyclopentanone and 98 mg. of sodium cyanoborohydride in 50 ml.
of methanol was stored at room temperature for 3.5 hours. Dur-
ing this time the pH of the solution was maintained at 7 with
the addition of a saturated solution of hydrogen chloride in
methanol. The reaction was triturated with 3 ml. of methanol,
filtered and the filtrate was diluted with 40 ml. of acetone,
yield 157 mg.
,~
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