CA1072547A - Alkylated derivatives of antibiotic bm123 - Google Patents

Abstract

25,185 ABSTRACT OF THE DISCLOSURE
This disclosure describes a novel series of potent antibacterial agents derived by reductive alkylation of anti-biotic BM123? with certain classes of aldehydes and ketones.

Description

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, 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 ", :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.
, 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 :

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.

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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.

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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 ' ' ! ' S~'^J
, --stru- tural formule~e:

;.

.`,' .

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o=y h ~

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R ~
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~ 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.
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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.

~ 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 :
,, ~^

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.

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.

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.

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~]

., .

, , .

~ ~ 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 ; 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.

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~.

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.

. .

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.

:
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 ''; . ' , .

':

~ '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:
. ' .

-' 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 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.
' ' ~

' ' 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.

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.

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 .

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.

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.

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.

.` 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-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.

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.

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.

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.

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.

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.

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.

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 :
; 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.

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.

, . ...
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.

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.

' :'.

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.

,~

'' :
, , 25 :
'' .

. . .

Claims (19)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A process for preparing compounds of the formulae:

(I) (II) (III) (IV) (V) wherein R1 is hydrogen, lower alkyl, halo substituted lower alkyl, lower alkenyl, phenyl, p-acetamidophenyl, m-nitro-phenyl, m-mercaptophenyl, o-anisyl, p-anisyl, o-tolyl, p-toly phenyl lower alkyl, 2-furyl, methyl substituted 2-furyl, 2--thienyl, methyl substituted 2-thienyl, 2-pyrryl, methyl sub-stituted 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 selected from the group consisting of lower alkyl, halo substituted lower alkyl lower alkenyl, lower cycloalkyl, phenyl, p-acetamidophenyl, m-nitrophenyl, m-mercaptophenyl, o-anisyl, p-anisyl, o-tolyl, p-tolyl, phenyl lower alkyl, o, m, or p-chlorobenzyl, .alpha.-(p--aminophenyl) ethyl and .beta.-(m-nitrophenyl)ethyl; or R2 and R3 taken together with the associated methylidyne group is cyclopentyl, mono-lower alkyl substituted cyclopentyl, di--lower alkyl substituted cyclohexyl or tri-lower alkyl substituted cyclohexyl; and R is a moiety of the formula (VI) or (VII) which comprises:
(a) for preparing a compound of formula I, II, III or IV, alkylating an amine of the formula:

(A) wherein R is as hereinabove defined with an aldehyde of the formula wherein R1 is 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; or (b) for preparing a compound of formula (V), alkylating an amine of the formula:

(A) wherein R is as hereinabove defined with a ketone of the formula:

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.

2. A compound of the formula:

(I) (II) (III) (V) wherein R1, R2, R3 and R are as defined in claim 1 and pharmaceutically acceptable acid-addition salts thereof, whenever prepared by the process of claim 1 or by an obvious chemical equivalent thereof.

3. A process for preparing a mixture of compounds of the formula I
given in claim 1, wherein R1 is as defined in claim 1 and R is moiety VI
and moiety VII as defined in claim 1, which comprises carrying out the process of claim I(a) using a mixture of amines of formula A in which R
represents moieties VI and VII and recovering a mixture of compounds of formula I.

4. A mixture of compounds of the formula I given in claim 1 wherein R is moiety VI and moiety VII as defined in claim 1, whenever prepared by the process according to claim 3 or by an obvious chemical equivalent thereof.

5. A process for preparing a mixture of compounds of the formula II
given in claim 1, wherein R1 is as defined in claim 1 and R is moiety VI
and moiety VII as defined in claim 1, which comprises carrying out the process of claim 1(a) using a mixture of amines of formula A in which R
represents moieties VI and VII and recovering a mixture of compounds of formula II.

6. A mixture of compounds of the formula II given in claim 1 wherein R is moiety VI and moiety VII as defined in claim 1, whenever prepared by the process according to claim 5 or by an obvious chemical equivalent thereof.

7. A process for preparing a mixture of compounds of the formula III
given in claim 1, wherein R1 is as defined in claim 1 and R is moiety VI and moiety VII as defined in claim 1, which comprises carrying out the process of claim 1(a) using a mixture of amines of formula A in which R represents moieties VI and VII and recovering a mixture of compounds of formula III.

8. A mixture of compounds of the formula III given in claim 1 wherein R is moiety VI and moiety VII as defined in claim 1, whenever prepared by the process according to claim 7 or by an obvious chemical equivalent thereof.

9. A process for preparing a mixture of compounds of the formula IV
given in claim 1, wherein R1 is as defined in claim 1 and R is moiety VI and moiety VII as defined in claim 1, which comprises carrying out the process of claim 1(a) using a mixture of amines of formula A in which R represents moieties Vl and VII and recovering a mixture of compounds of formula IV.

10. A mixture of compounds of the formula IV given in claim 1 wherein R is moiety VI and moiety VII as defined in claim 1, whenever prepared by the process according to claim 9 or by an obvious chemical equivalent thereof.

11. A process for preparing a mixture of compounds of the formula V
given in claim 1, wherein R2 and R3 are as defined in claim 1 and R is moiety VI and moiety VII as defined in claim 1, which comprises carrying out the process of claim 1(b) using a mixture of amines of formula B in which R
represents moieties VI and VII and recovering a mixture of compounds of form-ula V.

12. A mixture of compounds of the formula V given in claim 1 wherein R
is moiety VI and moiety VII as defined in claim 1, whenever prepared by the process according to claim 11 or by an obvious chemical equivalent thereof.

13. A process according to claim 3 wherein R1 is hydrogen.

14. A mixture of compounds of the formula I given in claim 1 wherein R
is moiety VI and moiety VII as defined in claim 1, and wherein R1 is hydrogen, whenever prepared by the process according to claim 13 or by an obvious chemical equivalent thereof.

157 A process according to claim 1, 3 or 5 wherein sodium cyanoborohy-dride is used as the reducing agent.

16. A process according to claim 7, 9 or 11 wherein sodium cyanoborohy-dride is used as the reducing agent.

17. A process according to claim 1 wherein process (b) is used and R2 and R3 are both methyl groups.

18. A process for preparing isopropyl - BM123.gamma. which comprises reacting BM123.gamma. with acetone in the presence of sodium cyanoborohydride.

19. Isopropyl - BM123.gamma. when made by a process according to claim 18 or an obvious chemical equivalent thereof.