CA1085391A - Antibacterial agents - Google Patents

Abstract

ABSTRACT
A novel series of potent antibacterial agents designated alkylated-cis-BM123?1 and alkylated-cis-MB123?2 produced by a novel photolytic process whereby alkylated-trans-BM123?1 and alkylated-trans-BM123?2 are transformed to their corresponding cis-isomers. The new antibacterial agents are active against a variety of microorganisms and thus are useful in inhibiting the growth of such bacteria wherever they may be found.

Description

25,612 1~853~

s ~i , This invention relates to new antibacterial agents designated alkylated-c -BM123~1 and alkylated--cis-BM123~2, to their production by photolysis, to meth-ods for their recovery from crude solutions, and to pro-cesses for their purification. The present invention includes within its scope the antibacterial agents in dilute forms, as crude concentrates, and in pure crys-. .
talline form. The effects of the new antibacterialagents on specific microorganisms, together with their chemical and physical properties, differentiate them from previously described antibacterial agents.
The novel antibacterial agents of the present invention are organic bases and thus are capable of form-- ing acid-addition salts with a variety of organic and inorganic salt-forming reagents. Thus, acid-addition salts, formed by admixture of the antibacterial fre~ base

- 2 - ~

~08S391 1 with up to three equivalents of an acid, suitable in a neutral solvent, are formed with such acids as sulfuric, : phosphoric, hydrochloric, hydrobromic, sulfanic citric, maleic, fumaric, tartaric, acetic, benzoic, gluconic, ascorbic, and related acids. The acid-addition salts of the antibacterial agents of the present invention are, in general, crystalline solids relatively soluble in : water, methanol and ethanol but are relatively insoluble in non-polar organic solvents such as diethyl ether, ben-zene, toluene, and the like. For purposes of this inven-tion, the antibacterial free bases are equivalent to their non-toxic acid-addition salts. hereinafter, cis--BM123~ refers to a mixture. in any proportions of cis--BM123~1 and cis-BM123 ~ , and trans-1 3~'refers to a mixture in any proportions of trans-BM123~ and trans--BM123~ .
The present invention provides a process for preparing compounds of the formulae:

R~CH-CH-C-NH- (CH2)3-NH- (CH2)4-NI~

R~3CH-CH-C-NH- ~CH2)3-NH- (CH2)4-NH_CH2_Rl . .

~cH'cH-c-NH-(cH2)3-NH-(cH2)4-N 2 R~3cH=cH-C-NH- (CH2)3-N- (CH2) 4-NH-CH -R

~ _ 3 _ C

- "
~ 1085391 CH-CH-C - NH- (CH2 ) -N- (C H ) - N~ H2 R

or .

R~3C~i-c~-c-NR-(c~2)3-NH-(c~l2)4--N~-cH 2 wherein R is a moiety of the formula:
. 10 ~ H H CH

HO~ H-t-l~
H C-N\ H NH H NH
0 C=O CcO C=NI~
,~ , . . .
N~2 NH2 N~2 20 or >~ ~ ,¦c"~;o-;

C=0 C=0 C=NH
., N"2 N"2 NH2 ~5 Rl is hydrogen, alkyl having up to 8 carbon atoms, phenyl, benzyl and alkenyl having from 2 to 8 carbon atoms; R2 is alkyl having up to three carkon atoms; and R3 is alkyl having up tD 8 carbon atoms, N,N-di-~aLkyl ~1-C4) amino-~aLkyl Cl-C4), chloroalkyl having 3a ~ .

G~
.
.: ., . ~ - ... .. . .
: . . . . ... . .
.. .. . . ..

lQ853~1 1 up to 4 carbon atoms, phenyl, benzyl, ~-phenylethyl, alkenyl having from 2 to 8 carbon atoms, hydroxyalkyl having up to 4 carbon atoms; and R2 and R3 taken together with the associated methylidyne group is cyclobutyl, cyclopentyl, monomethylcyclopentyl, dimethylcyclopentyl, trimethylcyclopentyl, cyclohexyl, monomethylcyclophexyl, dimethylcyclophexyl, trimethylcyclohexyl, cycloheptyl or a moiety of the formula:

~ H~- C~
_C~II N-~
\ CII;~ Cll/ ' ~ ~ ., ., " .

wherein R4 is hydrogen, alkyl having up to 4 carbon atoms, phenyl or benzyl and the pharmaceutically acceptable acid addition salts thereof; characterized by -- :
~a) irradiating an aqueous solution of a compound of the formulae:
~ O
R~) CII=C~-C-I~IJ-~C~2)3-~JIl-(C1l2)~ J1~2 t~
R ~ C~=cl~-c~ -(cl~2)3~Jll(cl2)l~ 2 ;

R~> Cll=cl~-c-Nll-(c~2)3 Nl~ (C 2~4 ~C~J R~

~ - 3b -' ' . ' " , 1~853~1 ~ ans O CH2-Rl R ~ CH=CH-C-NH-(CH2)3-N (CH2)4-NH CH2 trans O C,H2-Rl / OE12 R
R ~ CH=CH-C-NH-(CH2)3-N-(CH2)4-N \

~ O R2 R ~ CH-CH-C-NH-(CH2)3-NH-(CH2)4- NH-CH ~

where m R, Rl, R2 and R3 are as defined above, or (b) where an alkylated compound is required, alkylating an amine of the formula:

R ~ CH=CH-C-NH-(CH2)3-NH-(CH2)4-NH2 wherein R is as defined above with an aldehyde of the formula:
RlCHO or a ketone of the ~rmula R2-CC-R3 wherein Rl, R2 and R3 are as defined above, in the presenoe of a reducing agent and an inert solvent, and where required, converting the compound obtained into the pharmaceutically acoeptable acid addition salts thereof.
The new antibacterial agents which have been designated c -EM123~1 and cis-BMa23 ~ and alkylated-cis-BM123~ and alkylated-c -BM123~ may be prepared by the photochemical transformation of the corresponding trans-isomers. The photolytic conversion of alkylat-ed-trans-BM123 ~ alkylated-trans-BM123~1, and aIkylated--trans-BM123~ to the corresponding alkylated-c -BM123 3c -.
.

1 alkylated-cls-BM123,~ 1~ and aIkylated-cis-BMl23Y~2 is preferably effected by dissolving or dispersing the ; trans-isomer starting material in water and irradiating the solution with light. The concentration of the S trans-isomer starting material in water is not critical.
The light employed in the photolytic process of the present invention is advantageoulsy of a wavelength ~, ' ., .

il .
i, .

- 3d-,~ '' .
~. ...~ . ., , ' ' ' ' . . ~' : ~.
: ;. . : . ~:.
: .. : . . . ..

53~1 1 not less than about 2,500 Angstro~s and is preferably of a wave length from about 2,500 to about 4,000 Ang-stroms. In order to conveniently achieve this, the re-action may be carried out in a vessel constructed of a material such as quartz, which filters out substantially ~ all the light passing through the vessel having a wave ; length below about 2,500 Angstroms. The light source is conveniently a high pressure mercury arc lamp of about 450 watts.
The temperature at which the photolysis is car-ried out is not particularly critical for good yields of product, but is conveniently within the range from 5C. to 50C.; for instance, from about 25C. to about 30C. The time required for substantial conversion of the alkylated-trans-isomer to the corresponding alkylated--cis-isomer will naturally vary with the light intensity and the temperature, and is therefore best determined by trial in the individual case. However, a period of time ranging from about 20 minutes to about two hours is generally sufficient.
In like manner, antibiotics trans-BM123~, trans--BM123~1 and trans-BM123~2 may be photochemically trans-formed to the corresponding antibiotics cis-BM123~, Q --BM123~1 and cis-BM123~2 respectively.
After the irradiation step is compIete, the product may be obtained by standard procedures. For ex-ample, the reaction mixture may be lyophilized or evapor-ated to dryness and the residue may be dissolved in a minimal amount of solvent such as ethanol or methanol.
The resulting solution may be diluted with diethyl ether ,:
. :

` 1C~853~1 or acetone, and the resulting precipitated p~oduct may be recovered by filtration. Further purification may then be achieved by standard techniques such as crys-. tallization or chromatography.
me starting materials designated alkylated--trans-BMa23.~ alkylated-trans-BM123 ~, and alkylated--trans-EM123 ~, are derived by the reductive aIkylation of antibiotics trans-EM123 ~, trans-BM123 ~ or trans--BM123 ~ With an aldehyde or ketone of the following general formLla: :

.

: .' or R2 ~ C - R3 wherein ~ is hydrogen, aIkyl having up to 8 carbon atcms, phenyl, benzyl or aIkenyl having from 2 to 8 OE bon atoms, ., R2 is alkyl having up to 3 OE bon atoms; R3 is aLkyl having up to 8 OE bon atcms, N,N-di(alkyl Cl-C4)amino-(aIkyl Cl-C4), chloroalkyl having up to 4 OE bon atoms, phenyl, benzyl, ~-phenylethyl, alkenyl having from 2 to 8 OE bon atoms or hydroxyaIkyl having up to 4 OE b~n atoms, and R2 and R3 . taken together.with ~he associated methylidyne group is ; 20 cyclobutyl, cyclopentyl, monomethylcyclopentyl, dimethyl-cyclopentyl, trimethylcyclopentyl, cyclohexyl, monomethyl-cyclohexyl, dL~ethylcyclohexyl, trimethylcyclohexyl, ?' cycloheptyl or a moiety of the form~la:

~ ~ CH2 \ N
' -CH ~ --R4 ~, CH2--CH2 wherein R4 is hydrogen alkyl having up to 4 carbon atoms, phenyl or benzyl.

: ~ _ 5 _ . .
,, ,- , ~ .

1~853~1 1 The reductive alkylation process whereby the alkylated-trans-isomers may be prepared is carried out as follows: Antibiotic trans-sMl23~ trans-sMl23~l or trans-BM123~ is dissolved in a suitable solvent such as water, methanol, methyl cellosolve, or mixtures there-;~ of, in 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 pl~ of the reaction mixture is maintained at 6.0-8.0 with dilute mineral acid during the course of the reaction. After one to 24 hours at ambient temperature (10-35C.), the reaction mixture is evaporated to dryness _ vacuo and the residue is triturated with methanol and filtered. The filtrate is diluted with acetone and the solid product that pre-cipitates is removed by filtration and dried in vacuo.
The products may also be obtained from the reductive alkylation reaction mixtures by other stand-ard procedures such as precipitation, concentration, solvent extraction or combinations of these procedures.
After isolation, the products may be purified by any of the generally known methods for purification. These include recrystallization from various solvents and mix-ed solvent systems, chromatographic techniques, and coun-ter current distribution, all of which are usually em-^ ployed for this purpose.
In like manner, antibiotics cis-BM123~, cis--BM123~1 and cis-BM123~2 may be reductively alkylated to provide the alkylated-cis-BM123~, alkylated-cls--~M123~1 and alkylated-cis-BM123~2 of the present in-.

:10853~1 l vention, . Aldehydes and ketones which may be so employed ; in the above reductive alkylation process (according to the general procedure of Example 8) are set forth in Table I below. In this table, the deriva~ive name indicates whether antibiotic trans-BMl23~ or antibiotic Q -BMl23~is the starting material employed.

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10853~

1 The antibiotics designated BM123~1 BM123~2 trans-BM123~1 and trans-~M123~2 are formed during the cultivation under controlled conditions of a ~ew strain of an undetermined species of Nocardia. This new anti-biotic producing strain was isolated from a garden soil sample collected at Oceola, Iowa, and is maintained in the culture collection of the Lederle Laboratories Div-ision, American Cyanamid Company, Pearl River, N. Y.
as Culture No. BM123. A viable culture of the new micro-organism has been deposited with the Culture CollectionLaboratory, Northern Utilization Research and Develop-ment Division, United States Department of Agriculture, Peoria, Illinois, and has been added to ist permanent collection. It is freely available to the public in this depository under its accession No. NRRL 5646.
Herein BM123B refers to a mixture in any proportion of BM123~1 and BM123~2.
The following is a general description of the microorganism Nocardia ~., NRRL 5646, based on diag-nostic characteristics observed. Observations were madeof the cultural, physiological, and morphological feat-ures of the organism in accordance with the methods de-tailed by Shirling and Gottlieb, Internat. Journ. of Syst. ~acteriol. 16:213-240 (1966). The chemical compo-25 sition of the culture was determined 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 Harmony Manual, 3rd ed. (1948), Container Corp. of America, Chicago, Illinois. Dexcriptive details , . . .

10853~1 1 are recorded in Tables II through VI below.
Amount of Growth Moderate on yeast extract, asparagine dextrose, Benedict's, Bennett's, potato dextrose and Weinstrein's agars; light on Hickey and Tres-ner's, tomato paste, oatmeal, and pablum agars and a trace of growth on inorganic salts-starch, ~uster's oatflake, Czapek's solution, and rice agars.
0 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 pi~ments not formed on peptone-iron agar; no peptonization or curd formation in purple milk; NaCl toler-ance in yeast extract agar 4% but 7%;
optimal growth temperature 32C. Carbon source utilization, according to the Pridham and Gott]ieb method [J. Bacteriol. 56:107-114 (1948)]
as follows: Good utilization of glycerol, salicin, d-trehalose and dextrose; fair utili-zation of l_inositol; and poor to non utiliza-1~853~1 l tion of d-fructose maltose, adonitol, l-arab-inose, lactose, d-mannitol, d-melibiose, d--raffinose, l-rhamnose, sucrose and d-xylose.
Chemical Composition The organism belongs to cell wall type IV, i.e., contains meso-2,6-diaminopimelic acid and has a type A whole-cell sugar pattern, i.e., contains arabinose and galactose. Meth-ylated whole cell extracts, when subjected to gas chromatography, showed fatty acid patterns similar to those produced by Nocardia asteroides ATCC 3308.
Micromorphology Aerial mycelium arises from substrate mycelium as sparingly branched moderately long flexuous elements that commonly terminate in elongated primitive spirals. The flexuous elements are irregularly segmented into short ellipitical to cyclindrical sections (spores?) which dis-articulate readily. The spiral terminal pro-tions are less conspicuously segmented. Seg-ments generally range 0.8-1.7 ,um x 0.3-0.5 um, averaging 0.4,um x 1.2 um.
Diagnosis The morphological characteristics of Culture No. BM123 are difficult to observe and inter-pret 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 gen-~\
10853~1 1 eric relationship of the or~anism. On the basis of the system proposed 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. A comparison of the gas chromatography pattern of Culture No. BM123 - with that of Nocardia asteroides ATCC 3308 showed the two to be remarkably similar. Other characteristics of Culture No. BM123 that are in keeping with the Nocardia concept, are its fragmenting aerial growth on some media and the total absence of aerial growth on most media. In view of the lack of adequate cri-teria for the characterization of Nocardia to the species level, no attempt has been made to make this determination. Therefore, Cul-ture No. BM123 will be considered an undeter-mined species of Nocardia until such a diag-nosis is feasible.

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~3 u~ u~ C~ ~ ~n x ~ ~a . ~ .~ , ~ ~ ~7 ~ u~ ~ ~ P. ~ ~
, ~ . ~ I ~ ~ ~ ~ ~ ~ 0~.~ .
o ~ ~ ~ .~c ~ o ~ ~0 ~ ~ ~
0 C 0 ~ :~ _I N-r~ O 0~ 0 0 la m ~ ~ ~ ~ ~ . ~ ~ E~ o ~
.. . .. - . .....
.

_ 19_ , :

--~ 108~

~o ~ ~ o . QI ~t O u~ O u~ ~
O ~ In ,~ 1 -19a -` 108~;391 TABLE III
Micromorphology of ~ocardia sp NRRL 5h46 Aerial r~ycelium and/or Sporiferous Hedium Structures S . .
.. Yeast Ex- Aerial mvcelium arises from sub-tract Aqar strate mycelium as sparingly . branched, flexuous elements that :~ commonly terminate in elonqated . primitive spirals. The flexuous elèments are irreqularly seqmented . into short sections (spores~) which : disarticulate readily. The spiral ~erminal portions are less-conspi-. cuously segmented. Se~ments ~enerally ran~e 0.8-1.7 ~m x . . 0.3-0.5 ~m, averaqinq 0.4 ~m x 1.2 ~m.
~. '' .
.. . . . .

,~ , .

.
.

.

`` 1~85391 .' ., _ __ , , ~ .
. .0 J~ ~ ~ . ,.
~U ~ .~ o ~D ~. O O
u~ ~ ~ ~a a~
~, ' PC ~ o o ~ a~
~ ~r r~ ~ ~ ~ ~
Z . o ~ o ~ ~ ,~ .,, ~", U~ ~ ~ U~ U~ .
. ~ ~ ~ a) a\
~ ~ ~ ~ ~ ~ _, .,, . ~ .,, .,, .. 1~ rt ~ J~ h ,' ' æ, Z Z 'Z 'Z Z ,-: .-. .
.' o .C . . . ..
~ O 3 . . .
1~1 . ~: ~ . . . . . ', ,~ ~ ~ .c ~ ~a ~ ~
~ t~ O .~ O ~ O O ' .
., .~ O .
.' o~ ~ .
,0 ~ _ I . ' _ ".
~. ,0, . . . .
.C h U~
~ . .~ u) ~q ~n ~n o :~. ~ ~:~ ~ ~ .C :
O' . O ~ ~ ~ ~ ., w . .~ ~ ~ ~a ,~a ,a~
1~1 .a ~ _1 ~.~ ~r 1, 0 H . . ~ .
~ _ _ _ a ,~.
~1 ~ ~ O ~ O a) ~ ~ .
~ .~ .~.~ ~ .
,~ . a~ ~ ~ ~ ~ .c ~ ~ ~ o ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ O
,1 _1~.~ 2 ~.~ 2 P~ .
o z. m o z. m P. I .

~ .
01~ - 21 -)~ ' ' , .

1~)853~1 ~

~ ' O N ~5 ,rl ~ g a) ;, I lzolz :~ ~

O ~ ~

~:
.~ ~ X ~U

~: h ~ h _ - 21a -1~8~3g~ ' TABLE U
Carbon So~lrce U~ilization Pattern of Nocardia ~. N~RL 5646 .
. Incubation: 10 days TemPerature: 32C.
.~ . ' . _ .
Carbon Source hUtilization .__ _ : . Adonitol 0 l-Arabinose 0 :-Glycerol .

d-Fructose 1 . l-Inositol - 2 Lactose 0 . . . d-Mannitol . 0 .
: . Salicin 2 d-Melibiose 0 d-Raffinose 0 Rhamnose .
Maltose 1 . Sucrose . . 0 d-Trehalose 3 d-Xylose '. o . Dextrose 3 Ne~ative Control 0 .
* 3-Good Utilization l-Poor Utilization 2-Fair Utilization 0-No Utilization . - 22 -1~i353~1 1 TAsLE VI
Chemical Composition of Nocardia s~. NRRL 5646 Cell Wall Type Major Constituents Type IV meso-DAP, arabinose, galactose The production of ~M123~ and trans-BM123~ is not limited to this particular organism or to organisms fully answering the above growth and microscopic char-acteristics which are given for illustrative purposes ; only. In fact, mutants produced from this organism by various means such as exposure to X-radiation, ultra--violet radiation, nitrogen mustard, actinophages, and the like, may also be used. A viable culture of a typ-ical such mutant strain has been deposited with the Cul-ture Collection Laboratory, Northern Utilization Research and DevelOpment Division, United States Department of Agriculture, Peoria, Illinois, and has been added to its permanent collection under its accession number NRRL
8050. Although the cultural, physiological, and morpho-; logical features of NRRL 8050 are substantially the same as those of NRRL 5646, it produces enhanced amounts of BM123 during aerobic fermentation. 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 mycelial pig-ment on Bennett's and yeast extract agars.
The novel antibacterial agents of the present invention are, in general, crystalline solids of rela-`" 1085391 1 tively li~ited solubility in non-polar solvents such as diethyl ether and n-hexane, but considerably more soluble in solvents such as water and lower alkanols.
Antibiotics trans-BM123~1 and trans-~M123~2 are struc-.~ . S turai isomers and may be represented by the following structural formulae: .
.
' ;I z .'`~Y
u .
', Z .

. .~
O
.! . I
z .,,, O=u . . 1~ ....

~ ~ .
.; . . ,~, ,, . O . . .. .
z 5 '=~
= O . ..
; o ~
O~ ~ 5~;--U_ mZ
~ m `~ : 30 ] . X

; .
.. . .. . . . . .

108~3~

1 .
Z
r~
X
., o O _ U
tq ~
~ o . .

N
O

D~ z~
~ ' .

= O
, 20 m ~ o N
.~ , o~ z_c_,_ I , , "' I sl ' .
,~7 ' ' ~ ~3~ 3 ~`
O :S~ Z~ Z
Z J :~
o ' , :; . ' ' ' .

.

: - 1085~91 .. .
1 'rhe reductive alkylation of trans-BM1~3r', trans-sM123~ 1,.trans-BM123~ 2~ cis-BM123~', c -~M123~ 1 or cis-BM123~'2 with ketones takes place on the sperma-dlne side-chain to form derivatives of the formula:

R ~ CH=CH- -NH~(CH2)3~H-(CH~)4-NH.CH

wherein R is a moiety of the formulae:

\o ~/b \~
HO\~H ~ H ljl/l~H- Q--HS~ H ljl/Lo b ~ b ~H
Ç=O ¢=O ¢=NII
H2 ~2 ~H2 15 or ~/b \~ /b~\y ~/b \~ ~
(~ H ~ ~ --H~\~H ~/ Lo I!~ H ~1 ~H tl ~IH
=O ¢~0 ¢=NH
'12 ~'12 , ' ' 25 and R2 and R3 are as hereinabove defined. The reductive alkylation of trans-BM123~ , trans-BM123~ 1~ trans-BM123Y 2 cis-BM 123y cis-BM123~1 or cis-BM123 ~2 with aldehydes , takes place on the spermadine side-chain to form mono-, `~ di-, and tri-substituted derivatives of the formulae:

.

: ~;, ;.. ;, .

~ .

-` 10853~1 R~3cl~=cli-c-rlH-~c~ )3-Nll-lcll2)~-Nll-cll -Rl S -~CI~=CH- -IIU- (C112) 3-NH- (CH2) 4-~

R 4~_CH=CH~ N~- ~CH ) 3-N- (CH2) -NH-CH2-R

R~--CH=CH-~-NH- (CH2) 3- 1_ (CH2) 4 ~` CI~ -R

wherein R and Rl are as hereinabove defined.
The usefulness of the c -isomers of the al-kylated derivatives of trans-BM123 is demonstrated by their ability to control systemic lethal infections in mice. These new substances show high in vivo antibac-terial activity in mice against Escherichia coli US311 when administered by a single subcutaneous dose to groups ~ of Carworth Farms CF-l mice, weight about 20 gm., in-¦ fected intraperitoneally with a lethal dose of this bac-teria in a 10 3 trypticase soy broth TSP dilution of a 5 hour TSP blood culture. In Table VII below is set forth the in vivo activity of typical products of this invention against Escherichia coli US311 in mice.

10~353~

Alive/Total Mice Dose-mg./kg. of Tested ~7 days Compound body weight post infection) Cis-BM123~ 2 4/5 0.5 1/5 Cis-BM123~ Oral Dose Alive/Total Mice (mg./kg. of body Tested (7 days weight) after infection) Cis-BM123~ Infected, non- 18/20 mice died -treated controls within 3 days after infectiOn 10 Isopropyl- 2.0 5/5 cis-BM123~ 1.0 5/5 0.5 3/5 0.25 1/5 0.12 2/5 l-Propylpently- 2.0 5/5 -cis-BM123~ 1.0 2/5 ~ 0.5 0/5 - 0.25 0/5 0.12 0/5 1,5-Dimethyl- 2.0 5/5

-4-hexenyl-cis- 1.0 5/5 -BM123~ 0.5 5/5 0.25 0/5 0.12 0/5 l-Benzyl-4- 2.0 5/S
-piperidyl- 1.0 3/5 20 -cis-BM123~ 0.5 0/5 0.25 0/5 0.12 0/5 Infected, 5/25 non-treated (3 days post in-controls fection) Fermentation Process Selected to Produce Primarily BM123 ` and trans-BM123~.
;, _ Cultivation of Nocardia ~. NRRL 8050 may be carried out in a wide variety of liquid culture media.
~ Media which are useful for the production of the anti--~ 30 biotic~ include an assimilable source of carbon such ` ` 108539~

1 as starch, sugar, molasses, glycerol, etc.; an assimil-able source of nitrogen sych as protein, protein hydro-lyzate, polypeptides, amino acids, corn steep liquor, etc.; and inorganic anions and cations, such as potas-

5 sium, magnesium, calcium, ammonium, sulfate, carbonate,phosphate, chloride, etc. Trace elements such as boron, molybdenum, copper, etc.; are supplied as impurities of other constituents of the media. Aeration in tanks and bottles is provided by forcing sterile air through or 10 onto the surface of the fermenting medium. Further agi-tation in tanks is provided by a mechanical impeller.
An antifoaming agent, such as Hodag~ FD32 may be added as needed.
Inoculum Preparation for BM123~ and trans- BM 123~' Primary shaker flask inoculum of ~ocardia sp.
NRRL 8050 is prepared by inoculating 100 milliliters of sterile liquid medium in S00 miliiliter flasks with scrapings or washings of spores from an agar slant of the culture. The following kedium is ordinarily used:

Bacto-tryptone.............. 5 gm.
Yeast extract............... 5 gm.
Beef extract................ 3 gm.
Glucose.................... 10 gm.
Water to................. 1000 ml.
The flasks were incubated at a temperature from 25-29C., preferably 28C. and agitated vigorously 25 on a rotary shaker for 30 to 48 hours. The inocula are then transferred into sterile screw cap culture tubes and stored at below 0F. This bank of vegetati~e inoc-ulum is used instead of slant scrapings for inoculation of additional shakes flasks in preparation of this first 30 stage of inoculum.

~ .~, ",. . ~
' - 10853~1 1 These first stage flask inocula are used to seed 12 liter batches of the same medium in 20 liter glass fermentors. The inoculum mash is aerated with 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 solubles................... 15 gm.
10 Ammonium sulfate................. 3 gm.
Potassium phosphate, dibasic..... 3 gm.
Calcium carbonate................ l gm.
Magnesium sulfate hepta-................................. -hydrate................... l.5 gm.
Glucose......................... 10 gm.
Water to...................... 1000 ~1.
The glucose is sterilized separately.
The third stage inoculum is aerated at 0.4 15 to 0.8 liters of sterile air per liter of broth per min-ute, and the fermenting mixture is agltated by an impel-ler driven at 150-300 revolutions per minute. l`he tem-perature is maintained at 25-29C., usually 28C. The growth is continued for 48 to 72 hours, at which time 20 the inoculum is used to seed a 3000 liter tank fermenta-tion.
Tank Fermentation for BM123~ and trans-BM123 ' For the production of BM123~ and trans-BM123 ' in tank fermentors, the following fermentation medium 25 is preferably used:
Meat solubles................... 30 gm.
Ammonium sulfate................. 6 gm.
, Potassium phosphate, dibasic..... 6 gm.
Calcium carbonate................ 2 gm.
Magnesium sulfate heptahydrate... 3 gm.
Glucose......................... 20 gm.
Water to.................. ,.. 1000 ml.
The glucose is sterilized separately.

,.~

1(~853~1 1 Each tank is inoculated with 5 to 10~ of third stage inoculum made as described under inoculum prepara-tion. The fermenting mash is maintained at a temperature ; of 25-28C. usually 26C. The mash is aerated with ster-5 ile air at a rate of 0.3-0.5 liters of sterile air per liter of mash per minute and agitated by an impeller driven at 70 to 100 revolutions per minute. The fermen-tation is allowed to continue from 65-90 hours and the mash is harvested.
The invention will be described in greater detail in conjunction with the following specific exam-, ples.

Inoculum preparation for Brl123~ and trans-BM123~
A typical medium used to grow the first and second stages of inoculum was prepared accordin~ to the following formula:

, Bacto-tryptone...................... S gm.
Yeast extract....................... 5 gm.
Beef extract........................ 5 gm.
Glucose............................ 10 gm.
~later to........................ 1000 ml.
Two 500 milliliter flasks each containing 100 milliliters of the above sterile medium were inocu-lated with 5 milliliters each of a frozen vegitative inoculum from Nocardia ~. NRRL 8050 . The flasks were placed on a rotary shaker and agitated vigorously for 48 hours at 28C. The resulting flask inoculum was trans-ferred to a 5 gallon glass fermentor containing 12 liters of the above sterile medium. The mash was aer-ated with sterile air while growth was carried out for about 48 hours, after which the contents were used to .

1~853~1 . .
1 seed a 100 gallon tank fermentor containing 300 liters of the following sterile liquid medium:

Meat solubles..................... l5 gm.
Ammonium sulfate................... 3 gm.
Potassium phosphate, dibasic....... 3 gm.
Calcium carbonate.................. l qm.
Magnesium sulfate heptahydrate... l.5 gm.
Glucose........................... 10 gm.
Water to........................ 1000 ml.
The third stage of inoculum mash was aerated with sterile air sparged into the fermentor at 0.4 lit-ers of air per liter of mash per minute. Agitation was supplied by a driven impeller at 240 revolutions per minute. The mash was maintained at 28C. and Hodag~
FD82 was used to seed a 3000 liter fer~entation.

Fermentation Employing Nocardia sp. NRRL 8050 and Medium ~avoring the ~roduction of BM123 and trans-B~123~
A fermentation medium was prepared according ., .
. to the following formula:

;~ Meat solubles..................... 30 gm.
~ Ammonium sulfate................... 6 gm.
i' Potassium phosphate, dibasic....... 6 gm.
Calcium carbonate.................. 2 gm.
Magnesium sulfate heptahydrate..... 3 gm.
Glucose....~.................................................. 20 gm.
Water to.................................................... 1000 ml.
The glucose is sterilezed separately.

The fermentation medium was sterilezed 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 , ermentor was inoculated with 300 liters of inoculum such as described in Example 1, and the fermentation was carried out at 26C. using Hodag~ F~82 as a defoam-ing agent. Aeration was supplied at the rate of 0.35 , .

~0853~1 1 liter of sterile air per liter of mash per minute. The mash was agitated by an impeller driven at 70-72 revo-lutions per minute. At the end of 67 hours of fermenta-tion time the mash was harvested.
EXAMPL~ 3 Isolation of BM123B and trans-BM123 A 3000 liter portion of fermentation mash pre-par~d as described in Example 2, pH 4 . 3, was adjusted to pl~ 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 com-bined filtrate and wash was pumped upward through three parallel 8 1/4" x 48" stainless steel columns each con-taining 15 liters of CM Sephadex~ C-25 [Na ] 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 with 200 liters of 1% aqueous sodium chloride followed by 560 liters of 5% aqueous sodium chloride. The 5% aqueous sodium chloride eluate was clarified by filtration through di-atomaceous 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 50% aqueous methanol and then 120 liters of 50% aqueous acetone. The 15% aqueous meth-anol eluate was concentrated in vacuo to about 7 liters 108S3~

1 of an aqueous phase and the pH adjusted from 4.5 60 6.0 with Amberlite~ ~R-45 (OH ) resin (a weakly basic poly-styrene-polyamine type anion exchange resin). The resin was removed by filtration and the filtrate was concen-5 trated in vacuo to about 1 liter and then lyophilizedto give 38 grams of material consisting primarly of BM123~ along with a small amount of trans-BM123~f(primar-ily trans-BM123 ~2. The 50% aqueous methanol eluate was adjusted from pH 4.65 to 6.0 with Amberlite~ IR-45 10 (OH ) resin. The resin was removed by filtration and the filtrate was concentrated in vacuo to about 6.3 lit-ers and then lyophilized to give 213 grams of material consisting primarily of trans-BM123 ~. The 50% aqueous acetone eluate was adjusted from pH 4.0 to 6.0 with 15 Amberlite~ IR-45 (OH ) resin. The resin was removed by filtration and the filtrate was concentrated in vacuo to about 1.5 liters and then lyophilized to give 56 grams of impure trans-BM123~, Further Purification of trans-BM123 ~

A slurry of CM Sephadex~ C-25 [NH4] in 2% aque-ous 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 25 was drained away and a 5.O gram sample of trans-BM123y~
prepared as described in Example 3 was dissolved in about 10 milliliters of 2% aqueous ammonium chloride and applied to the column. The column was then eluted with a grad- -ient between 6 liters each of 2% and 4% aqueous ammonium 30 chloride. Fractions of about 75 milliliters each were 5~
~ .. ,,, ~

10853~1 1 collected automatically every 15 minutes. Antibiotic trans-BM123~ was located by monitoring the column efflu-ent in the ultra-violet and by bioautography of dipped paper disks on large agar plates seeded with Klebsiella S ~_eumoniae strain AD. The majority of trans-BM123 was located between fractions 71-107 inclusive.
One hundred thirty milliliters of granular Darco~ G-60 (2040 mesh) was suspended in water, trans-ferred to a glass column, allowed to settle and the ex-cess water was allowed to drain away. Fractions 84-96 inclusive from the above CM Sephadex chromatography were combined and passed through the granular carbon column.
The charged column was washed with 600 milliliters of water and then developed with 1 liter of 50% aqueous acetone. The eluates, both of which contained trans--BM123~ , were concentrate~ to aqueous phases in vacuo and lyophilized to give a total of 886 milli~rams of trans-BM123~ as the hydrochloride sAlt. A microanalyt-ical sample was obtained by subjectinq the above material to a repeat of the above process.
Antibiotic trans-BM123~ does not possess a definite melting point, but gradual decomposition starts in the vicinity of 200C. Microanalysis of a sample equilibrated for 24 hours in a 72F. atmosphere contain-ing 23% relative humidity gave C, 39.44%; H, 6.10~, N,16.19%; Cl(ionic), 11.54%; loss on drying, 8.19%. In water trans-BM123~ gave a U.V. absorption maximum at 286 nm with ElCm = 250. The position of this maximum did not change with pH. Trans-BM123~ had a specific rotation f [~]D = +71 (C = 0.97 in water) and exhib-` 108S3~1 1 ited characteristic absorption in the infrared region of the spectrum at the following wavelenaths: 770, 830, 870, 930, 980, 1035, 1105, 1175, 1225, 1300, 13q0, 1370, 1460, 1510, 1555, 1605, 1660, 1740, 2950 and 3350 cm 1, EXA~lPLE 5 Isolation of trans-BM123~
A slurry of CM Sephadex~ C-25 [Na ] in 2% aque-ous sodium chloride was poured into a 2.6 centimeter diameter glass column to a resin hei~ht of approximate-`~ 10 ly 70 cehtimeters. The excess 2% aqueous sodium chlor-ide was drained away and 4.11 gram of a sample contain-ing primarily trans-BM123~1 along with some trans-BM123f2 and other impurities, prepared as described in Example 3, was dissolved in about 10 milliliters of 2~ aqueous sodium chloride and applied to the column. The column was then eluted with a gradient between 4 liters each ; of 2% and 4% aqueous sodium chloride. Fractions of about ' 75 milllliters each were collected automatically every 15 minutes. Antibiotic trans-BM123~ was located by monitoring the column effluent in the ultraviolet and by bioautography of dipped paper disks on large agar ; plates seeded with Klebsiella pneumoniae strain AD.
The majority of trans-BM123~ was located between frac-tions 64-90 inclusive; the initial fractions (64-80) ; 25 contained a mixture of trans-BM123~1 and trans-BM123~2 whereas the later fractions (81-90) contained essential-~A ly puretrans-BM123 One hundred milliliters of granular Darco~
G-60 (20-40 mesh) was suspended in water, transferred to a glass column, allowed to settle and the excess water ,~ . .

, - 1~8S3~1 1 was allowed to drain away. Fractions 81-90 inclusive from the above CM Sephadex chromatogra~hy were combined and passed through the granular 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 trans-sMl23~l~ was adjusted from pH 5.9 to

6.0 with Amberlite~ IR-45 ~OH ) resin. The resin was removed by filtration and the filtrate was concen-trated in vacuo to an aqueous phase and lyophilized to give 294 milligrams of white amorphous trans-B~123 as the hydrochloride salt.
Antibiotic trans-BM123~1 does not possess a definite melting point, but gradual decomposition starts in the vicinity of 200C. Microanalysis of a sample equilibrated humidity gave C 37.84%; H, 5.73; N, 15.58;
Cl(ionic~, 10.01% loss of drying 10.45%. In methanol trans-BM123~1 gave a U.V. absorption maximum at 286 nm with E1Cm = 225. The position of this maximum did not change with pH. Trans-BM123~1 had a specific rotation of +55 (C=0.803 in water) and exhibited characteristic absorption in the infrared re~ion of the spectrum at the following 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.

Isolation of trans-BM123~2 A 25 grams sample containing primarily trans--BM123~2 and BM123~, prepared as described in Example , . . - ................. . . .. :

~, : ... .

108~3~1 l 3, was dissolved in about 120 milliliters of 2% aqueous sodium chloride and applied to a column containina 1800 ml. of CM Sephadex~ C-25 [Na ] in 2% aqueous sodium chloride. The column was then eluted with a gradient between 20 liters each of 2~ and 4% aqueous sodium chlor-ide. 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. Antibiotic trans-BMl23~ was located by monitoring the column fractions in the ultraviolet.
The majority of trans-BM123~ was located between frac-: . ~
tions 7-18 inclusive; the initial fractions (7-15) con-tained essentially pure trans-BM123~2 and the later frac-tions (16-18) contained a mixture of trans-BMl23~1 and trans-BMl23~2.
Six hundred milliliters of granular Darco~ G-60 (20-40 mesh) was suspended in water, transferred to a glass column, allowed to settle and the excess water was allowed to drain away. Frac~ions 7-15 inclusive from the above CM Sephadex chromatoqraphy were combined and passed through the granular carbon colu~n. The charg-ed 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 adjusted from pH 5.8 to 6.0 with Amberlite~ IR 45 (OH ) resin. The resin was removed by filtration and the filtrate was concentrated ln vacuo to an aqueous phase and lyophilized to give 5~5 milli-grams of white amorphous trans-BMl23~2 as the hydrochlor-ide salt. The 50~ aqueous methanol eluate was adjusted - 3~ -~ :~08~3~

1 from p~ 4.6 to 6.1 with Amberlite~ (OH ) resin. The resin was removed by filtration and the filtrate was concentrated in vacuo to an aqueous phase and lyophil-ized to give 3.645 grams of slightly less pure white amorphous trans-BM123~2 as the hydrochloride salt.
Antibiotic trans-BM123~2 does not possess a definite melting pointl but gradual decomposition starts in the vicinity of 200C. Microanalysis of a sample equilibrated for 24 hours in a 70~F. atmospher contain-ing 60% relative humidity gave C, 36~la%; H, 5.67; N, 15.1~; Cl(ionic), 11.11%, loss on drying 10.87%. In methanol trans-BM123J2 gave a U.V. absorption maximum at 286 nm with ElCm = 220. The position of this max-imum did not change with pH. Trans-BM123~2 had a spe-cific rotation of +60 (C=0.51 in water) and exhibited characteristic absorption in the infrared region of the spectrum at the following wavelengths: 770, %30, 870, 950, 980, 1035, 1110, 1175, 1225, 1285, 1345, 1380, 1470, 1515, 1560, 1605, 1660, 1755, 2950,and 3350 cm .

Paper Partition and Thin Layer Chromatography of BM123 and trans-BM123 The BM123 antibiotics can be distin~uished by paper chromatography. For this purpose Whatman No.
1 strips were spotted with a water or methanol solution of the substances and equilibrated for 1 to 2 hours in the presence of both upper and lower phases. The strips were developed overnight with the lower (organic) phase obtained from mixing 90% phenol:_-cresol:acetic acid:-pyridine:water (100:25:4:4:75 by volume). The developed ., . .
, ~ i , , ~ ,~,. .. .

1~853~

1 strips were removed from the chromatographic chamber, air dried for 1 to 2 hours, washed with ether to re~ove residual phenol and bioautographed on large agar plates seeded with Klebsiella peunmoniae strain AD. Represent-ative Rf values are listed in Table VIII below:
TABLE VIII

Component RF
~ .
trans- 0.85 -BM12 ~

BM123~0.50, 0.70 . . . _ ~ The ~ component was a mixture of two antibiot-; 15 ics using this system. BM123~ was composed of a major antibiotic (Rf = 0.50) called BM123~1 and a minor anti-biotic (Rf - 0.70) called BM123~2.
.~ .
The BM123 antibitics can also be distinnuish-ed 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 Labora-tories Inc., Elmsford, N.Y. were spotted with a water solution of the substance to be chromatoqraphed (about 20-40 micrograms per spot). The 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 chromatographic chamber and air dried for about one hour. The antibiot-ics were detected by using either standard ninhydrin or Sakaguchi spray reagents. Representative Rf values -`` 10853~1 :

, 1 are listed in Table IX below:

TABLE IX

; Component Rf BM123 0.17, 0.23 .':
BM123~ 0.03, 0.14 Both BM 123~ and trans-BM123~ were a mixture 10 of two components using this system. BM123~ was com-posed of a major component (Rf = 0.08) which was BM123 and a minor component (Rf = 0.14) which was BM123~2.
The less polar component of trans-BM123~ (Rf = 0.23) was trans-BM123~1 and the more polar component (Rf =
15 0.17~ was trans-BM123Y 2.

....
General Procedure for Reductive Alkylation of Antibiotic , trans-BM123y or antibiotic cis-BM123~

; To a stirred solution of 100 mg. of antibiotic 20 trans-BM123~ or antibiotic cls-BM123Y in 2~ ml. of meth-'~~ anol is added 5 ml. ~or 5 q.) of the appropriate alde-. , .
, hyde or ketone and 100 mg. of sodium cyanoborohydride.

The pH of the resulting solution is maintained at about

7.0 with O.lN methanolic hydrogen chloride over a 3 to 25 24 hour period. The reaction is monitored by thin layer chromatography to the disappearance of the starting anti-biotic. The reaction mixture is then filtered and the filtrate is evaporated to dryness. The residue i~s trit-urated with 3 ml. of methanol and filtered. The filtrate 30 is diluted with 50 ml. of acetone and the precipitate . .

; ' - i~853~
., 1 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 solu-. ble.

Preparation of methyl-trans-BM123~
; To a solution of 1.0 g. of trans-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 pH of the reaction mixture was maintained at 7.0 with lN hydrochloric acid durin~ this addition. The reaction mixture was stirred an additional ten minutes at room temperature and then evaporated to dryness in 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-trans-BM123~
To a solution of 200 mg. of trans-BM123~ in 30 ml. of methanol was added 5 ml. of acetone. To this solution was added 139 mg. of sodium cyanoborohydride and the reaction mixture was stirred at room temperature for 30 minutes. During this time the pH of the reaction mixture was maintained between 7.4 and 7.8 by the addi-tion 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 .

- 10853~1 1 100 ml. of acetone and the solid product that separated was removed by filtration and dried; yield, 1~4 mg.

Preparation of ~-phenylethyl-trans-BM123~
To a solution of 200 mg. of trans-BM123~ in 15 ml. of water and 2S ml. of acetonitrile was added a solutin of 2 ml. of phenylacetaldehyde in 4 ml. of ethanol. To this was added 103 mg. of sodium cyanobor-ohydride. The reaction mixture was stirred at room tem-perature for thirty minutes during which time the pH
of the mixture was maintained at 7 with 0.2N hydrochloric acid. The reaction mixture was then filtered and the filtrate was evaporated to dryness in vacuo. The res-idue was triturated with two ml. of methanol and filt-15 ered. The filtrate was diluted with 100 ml. of acetone and the product that separated was removed by filtration - and dried; yield, 180 mg.

; Preparation of 1,3,3-trimethylbutyl-trans-BM123 .
To a solution of 200 mg. of trans-BM123~ hydro-chloride in 50 ml. of methanol was added 3 ml. of 4,4--dimethyl-2-pentanone and 106 mg. of sodium cyanoborohy-dride. The reaction solution was maintained at pH 7 by the dropwise addition of methanolic hydrogen chloride.
25 The reaction was stirred at room temperature for 18 hours and filtered. The filtrate was evaporated to dryness in vacuo. The residue was dissolved in 3 ml. of meth-anol, diluted with 50 ml. of acetone and filtered, yield 125 mg.

. , ' :

1~853~1 -Preparation of l-methylphenethyl-trans-sMl23 . .
To a solution of 200 mg. of trans-BM123~ in 50 ml. of methanol was added 5 ml. of phenylacetone.
To this solution was added 170 mg. of sodium cyanoborhy-dride and the reaction mixture stirred at room temper-ature for 3 and a half hours. During this time the p~
of the reaction mixture was maintained at 7.0 with meth-anol saturated with hydrogen chloride gas. Reaction mixture was concentrated to about 5 ml. volume, diluted with two ml. of methanol, and flltered. Filtrate was poured into 100 ml. of acetone and the solid product that separated was removed by filtration and dried; yield 233 mg.

Preparation of l-methylnonyl-trans-BM123~
Sodium cyanoborohydride (100 mg.) was added to a solution of trans-BM123~ ~200 mg.) and 2-decanone (1 ml.) in 40 ml. of methanol. The pH of the solution was adjusted to 7.0 and maintained at 7.0 + 0.2 by the addition of O.lN methanolic hydrogen chloride as neces-sary. After 19.5 hours the reaction mixture was filt-ered and the filtrate was concentrated in vacuo at 35C.
The residue was slurried in 5 ml. of methanol and filt-ered. The filtrate was added to 50 ml. of acetone.The off white solid which precipitated was collected by filtration, washed with acetone, and dried in vacuo.
The yield of crude l-methylnonyl-trans-BM123~ was 167 mg.

, , 1~853~1 Preparation of 1,3-dimethylbutyl-trans-BM123`~
To a solution of 210 mg. of trans-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 mi~ture 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. Reac-tion mixture was evaporated to dryness, in vacuo. Theresidue was triturated with two ml. of methanol and fil-tered. The filtrate was diluted with 100 ml. of ace-tone and the solid product that separated was removed by filtration and dried; yield, 210 mg.

-Preparation of lsopropyl-trans-BM123~1 A mixture of 50 mg. of trans-BM123~1, 5 ml.
of acetone and 60 mg. of sodium cyanoborohydride in 35 ml. of methanol was stireed at room temperature for 40 minutes. The pH of the solution was maintained at 7 by the dropwise addition of methanolic hydrogen chlor-ide solution. The mixture was evaporated to dryness ln vacuo. The residue was triturated with 5 ml. of meth-anol and the resulting solution was diluted with 50 ml.
of acetone; yield, 49 mg.
By following the above procedure but substi-tuting for acetone the carbonyl compound starting ma-terials set forth in the following table, there is ob-tained the corresponding alkylated products.

1~853~1 Starting Material Product .
formaldehyde* methyl-trans-BMl23j acetaldehyde ethyl-trans-BM123 propionaldehyde n-propyl-trans-BM123 n-butyraldehyde n-butyl-trans-sMl23 isobutyraldehyde _ butyl-trans-BM123 2-butanone sec-butyl-trans-~123 3-pentanone 1-ethylbutyl-trans-BM123 methyl isobutyl 1,3-~imethylbutyl-trans-ketone -BM123~1 _ *As 37~ aqueous solution in water as solvent in-stead of methanol.

Preparation of lsopropyl-trans-BM123 ~
A mixture of 41 mg. of trans-BM123~2, 5 ml.
of acetone and S0 mg. of sodium cyanoborohydride in 35 ml. of ~ethanol was stirred at room temperature for 40 minutes. The pH of the solution was maintained at 7 by the dropwise addition of a methanolic hydrogen chlor-ide solution (satureated). The mixture was filtered and evaporated to dryness in vacuo. The residue was triturated with 5 ml. of methanol and the resulting so-lution was diluted with 50 ml. of acetone; yield, 46 mg.
By following the above procedure but substi-tuting for acetone the carbonyl compound starting ma-terials set forth in the following table, there is ob-tained the corresponding alkylated products.

Starting Material Product formaldehyde* methyl-trans-BMl23~2 acetaldehyde ethyl-trans-BMl23~2 propionaldehyde _-propyl-trans-BMl23~2 n-butyraldehyde n-butyl-trans-BMl23~2 isobutyraldehyde isobutyl-trans-BM123~2 2-butanone sec-butyl-trans-BM123~2 3-pentanone l-ethylbutyl-trans- --BM123,~2 methyl isobutyl 1,3-dimethylbutyl-trans-ketone -BMl23C2 _ *As 37% aqueous solution in water as solvent instead of methanol.

Preparation of l=methyl-2-phenyl-ethyl-trans-BMl23~?
A mixture of 200 mg. of trans-BMl23~2, 5 ml.
of phenylacetone and 170 mg. of sodium cyanoborohydride in 50 ml. of methanol was stirred at room temperature for 3 hours and 45 minutes. During this time the pH
of the reaction mixture was maintained at 7 with drop-wise addition of a methanolic hydrogen chloride solution (saturated). The mixture was evaporated to dryness in vacuo. The residue was triturated with 5 ml. of meth-anol and the resulting methanol solution was dilutedwith approximately 50 ml. of acetone, yield 233 mg.

Preparation of isopropyl-c -BM123 , A solution of 200 mg. of isopropyl-trans-BMl23 in 200 ml. of water is photolyzed with a Hanovia light lG853~1 1 in a water-jacketed, three-necked, round bottom flask for a period of one hour, during which time the maximum ultraviolet absorption of the reaction solution shifts from 290 m~ to 275 m~. The produc~ ls then recovered from the reaction solution by lyophilization and wei~hs 160 mg.
This photolytic reaction is best monitored by taking aliquots at various time intervals and meas-uring the ultraviolet absorption. The reaction is com-plete when the maximum absorption shifts from 290 m~to 275 ~.
By following the above procedure but substi-tuting for lsopropyl-trans-BM123~ the alkylated deriv-atives listed in Examples 16 and 17, there is obtained methyl-cis-BM123~1, ethyl-cis-BM123~1, n-propyl-cls--BM123~1, isopropyl-cis-BMl23~1, n-butyl-cis-BM123~1, l butyl-cis-BM123fl, sec-butyl-cls-BM123~1, l-ethyl--butyl-cis-BM123~1, 1,3-dimethylbutyl-cis-BM123~1, methyl-cis-BM123~2, ethyl-cis-BM123~2, n-propyl-cis--BM123~2, isopropyl-cis-BMl23~2,_-butyl-cis-BMl23 ~, l butyl-cis-BM123~2, sec-butyl-cis-BM123 ~, l-ethyl-butyl-cis-BM123~2, 1, 3-dimethylbutyl-cis-BM123~2.
ExAMPLE ?
Preparation of l-propylpentyl-cis-BM123 ;
A solution of 175 mg. of l-propylpentyl-trans-BM123~ is 175 ml. of water and 45 ml. of methanol was irradiated with a high pressure ultraviolet lamp for one hour and 15 minutes. The solution was the evapor-ated to dryness in vacuo to yield 169 mg. of product.

1C~853~1 :

Preparation of 1,5-dimethyl-4-hexenyl-cis-BM123V
A solution of 200 mg. of 1,5-dimethyl-4-hex-enyl-trans-sM123~ in 200 ml. of water was irradiated with a high pressure ultraviolet lamp for one hour.
The solution was then freeze dried to yield 182 mg. of product.
ExAMPLE 22 Preparation of l-benzyl-4-piperidyl-cis-sM123~

; 10A solution of 200 mg. of 1-benzyl-4-piperidyl--trans-BM123~ in 200 ml. of water and 20 ml. of methanol ; was irradiated with a high pressure ultraviolet lamp for one hour. The solution was then freeze dried to yield 125 mg. of product.

Preparation of 3,5-dimethylcyclohexyl-trans-BM123 A solution of 200 mg. of trans-BM123~, S ml.
of 3,5-dimethylcyclohexanone and 200 mg. of sodium cy-anoborohydride 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 sat-urated 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, 25 !yield 200 mg.

I Preparation of 2,4-dimethylcyclopentyl-trans-BM123 I
~A solution of 206 mg. of trans-BM123 ~, 3 ml.
¦of 2,4-dimethylcyclopentanone and 104 mg. of sodium cy-anoborohydride in 50 ml. of methanol was stored at room : ' ' ' ` `

853~L

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, filt-5 ered and the filtrate was diluted with 40 ml. of acetone,yield 101 mg.

Preparation of 3-methylcyclohexyl-trans-BM123~
A solution of 200 mg. of ~rans-BM123~, 1.5 10 m~. of 3-methylcyclohexanone and 200 mg. of sodium cy-anoborohydride 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.
15 The reaction was triturated with 3 ml. of methanol, fil-tered and the filtrate was diluted with 40 ml. of ace-tone, yield 200 mg.
EXA~IPLE 26 Preparation of 2,4,4-trimethylcyclopentyl-trans-BM123 A solution of 200 mg. of trans-BM123~, 5 ml.
of 2,4,4-trimethylcyclopentanone 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 25 Of a saturated solution of hydrogen chloride in methanol.
The reaction was trituratecl with 3 ml. of methanol, fil-tered and the filtrate was diluted with 40 ml. of ace-tone, yield 176 mg.

Preparation of 2-methylcyclopentyl-trans-BM12 5o .. ' ~ ' ~ 853~1 1 A solution of 211 mg. of trans-BM123~, 3 ml.
of 2-methylcyclopentanone and 98 mg. of sodium cyanoboro-hydride in 50 ml. of methanol was stored at room temper-ature for 3.5 hours. During this time the pH of the S solution was maintained at 7 with the addition of a sat-urated 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.

i Preparation of cis-BM123~
A solution of 200 mg. of trans-sM123~in 200 ml. of water is photolyzed with a Hanovia~ light in a water-jacketed, three-necked, round bottom flask for a period of time of half an hour, during which time the maximum U.V. absorption of the reaction solution shifts from 290 m~ to 275 my. The reaction is best monitored by taking aliquots at various time intervals and meas-uring the ultraviolet absorption. The reaction is com-plete when the maximum absorption shifts from 290 m~to 275 m~. The product is then recovered by lyophili-; zation.
The above procedure is repeated but with thesolution being photolyzed for 1.5 hours. Lyophilization yields 170 mg. of cis-BM123~.

'`

.

Claims (6)

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

1. A process for the preparation of a compound of the following formulae:

or wherein R is a moiety of the formulae:

or ;

R1 is hydrogen, alkyl having up to 8 carbon atoms, phenyl, benzyl and alkenyl having from 2 to 8 carbon atoms; R2 is alkyl having up to three carbon atoms; and R3 is alkyl having up to 8 carbon atoms, N,N-di(alkyl C1-C4) amino--(alkyl C1-C4), chloroalkyl having up to 4 carbon atoms, phenyl, benzyl, .beta.-phenylethyl, alkenyl having from 2 to 8 carbon atoms, hydroxyalkyl having up to 4 carbon atoms;
and R2 and R3 taken together with the associated methylidyne group is cyclobutyl, cyclopentyl, monomethylcyclopentyl, dimethylcyclopentyl, trimethylcyclopentyl, cyclohexyl, monomethylcyclohexyl, dimethylcyclohexyl, trimethylcyclohexyl, cycloheptyl or a moiety of the formula:

wherein R4 is hydrogen, alkyl having up to 4 carbon atoms, phenyl or benzyl and the pharmaceutically acceptable acid-addition salts thereof; characterized by (a) irradiating an aqueous solution of a compound of the formula:

or wherein R, R1, R2 and R3 are as defined above, or (b) where an alkylated compound is required, alkylating an amine of the formula wherein R is as defined above with an aldehyde of the formula: R1CHO or a ketone of formula R2-CO-R3 wherein R1, R2 and R3 are as defined above, in the presence of a reducing agent and an inert solvent, and where required, converting the compound obtained into the pharmaceutically acceptable acid addition salts thereof.

2. A compound of the formulae:

or wherein R, R1, R2 and R3 are as defined above; and the pharmaceutically acceptable addition salts thereof; whenever prepared by the process of claim 1 or by an obvious chemical equivalent thereof.

3. A process for the preparation of the compound antibacterial cis BM123? 1 of the formula:

and the pharmaceutically acceptable addition salts thereof; characterized by irradiating an aqueous solution of a corresponding trans compound, and where required, converting the compound obtained into the pharmaceutically acceptable acid addition salts thereof.

4. The compound antibacterial Cis-BM123?1 of the formula:

and the pharmaceutically acceptable addition salts thereof whenever prepared by the process of claim 3 or by an obvious chemical equivalent thereof.

5. A process for the preparation of the compound antibacterial cis-BM123?2 of the formula:

and the pharmaceutically acceptable addition salts thereof; characterized by irradiating an aqueous solution of a corresponding trans compound, and where required, converting the compound obtained into the pharmaceutically accept-able acid addition salts thereof.

6. The compound antibacterial cis-BM123?2 of the formula:

and the pharmaceutically acceptable addition salts thereof; whenever prepared by the process of claim 5 or by an obvious chemical equivalent thereof.