The present invention is directed to novel carba cyclohexapeptide compounds of the formula ##STR1## where all substituents are defined herein, which are useful as antifungal agents and for the treatment of Pneumocystis carinii infections. Compositions containing the compounds of the invention are also disclosed.

BACKGROUND OF THE INVENTION 
The present invention is directed to novel cyclohexapeptide compounds which 
are useful as antifungal and anti-Pneumocystis agents. 
There presently exists a need for antifungal and anti-Pneumocystis agents 
due to an increase in the number of isolates which are resistant to 
conventional agents. Additionally, conventional agents show somewhat high 
levels of toxicity which limit their usefulness. Lastly, the incidence of 
Pneumocystis carinii pneumonia is increasing, particularly in view of the 
high incidence of immuno-compromised patients susceptible to infection, 
such as those suffering from AIDS. 
SUMMARY OF THE INVENTION 
The compounds of the present invention, Compound I (Seq. ID Nos. 1-6), are 
characterized in having a carbon attached to the cyclohexapeptide ring at 
the 5-carbon of the 4-hydroxyornithine component (hereinafter "C-5-orn") 
and may be represented by the formula: 
##STR2## 
wherein R.sub.1 is H or OH; 
R.sub.2 is H, CH.sub.3 or OH; 
R.sub.3 is H, CH.sub.3, CH.sub.2 CONH.sub.2, CH.sub.2 CN, CH.sub.2 CH.sub.2 
NR.sup.II R.sup.III, CH.sub.2 CH.sub.2 N(R.sup.IV).sub.3.sup.+ X.sup.- or 
CH.sub.2 CH.sub.2 NH(C.dbd.NH)R.sup.VII ; 
R.sub.4 is H or CH.sub.3 ; 
R.sub.5 is H, OH or OSO.sub.3 H; 
R.sub.6 is H or OH; 
R.sup.I is C.sub.9 -C.sub.21 alkyl, C.sub.9 -C.sub.21 alkenyl, C.sub.1 
-C.sub.10 alkoxyphenyl, C.sub.1 -C.sub.10 alkoxynaphthyl, or 
##STR3## 
wherein R.sup.a is C.sub.1 -C.sub.10 alkyl; or (CH.sub.2).sub.q NR.sup.b 
R.sup.c wherein R.sup.b and R.sup.c are independently H, C.sub.1 -C.sub.10 
alkyl or R.sup.b and R.sup.c taken together with the nitrogen atom are 
##STR4## 
wherein R.sup.d is C.sub.1 -C.sub.16 alkyl, cyclohexylmethyl, phenyl or 
benzyl; 
p is 1 or 2; and 
q is 2, 3 or 4; 
R.sup.II is H, C.sub.1 -C.sub.4 alkyl, (CH.sub.2).sub.2-4 OH, 
C.dbd.NH(R.sup.VII), (CH.sub.2).sub.2-4 NR.sup.V R.sup.VI, 
(CH.sub.2).sub.2-4 N(R.sup.IV).sub.3.sup.+ X.sup.-, (CH.sub.2).sub.2-4 
NH(C.dbd.NH)R.sup.VII, (CH.sub.2).sub.1-4 CH(NR.sup.V 
R.sup.VI)(CH.sub.2).sub.1-4 NR.sup.V R.sup.VI, (CH.sub.2).sub.2-4 NR.sup.V 
(CH.sub.2).sub.2-4 NR.sup.V R.sup.VI, CO(CH.sub.2).sub.1-4 NR.sup.V 
R.sup.VI, COCH(NR.sup.V R.sup.VI)(CH.sub.2).sub.1-4 NR.sup.V R.sup.VI ; 
R.sup.III is H, C.sub.1 -C.sub.4 alkyl, (CH.sub.2).sub.2-4 NR.sup.V 
R.sup.VI, (CH.sub.2).sub.2-4 N(R.sup.IV).sub.3.sup.+ X.sup.-, 
(CH.sub.2).sub.2-4 NH(C.dbd.NH)R.sup.VII, (CH.sub.2).sub.1-4 CH(NR.sup.V 
R.sup.VI)(CH.sub.2).sub.1-4 NR.sup.V R.sup.VI, (CH.sub.2).sub.2-4 NR.sup.V 
(CH.sub.2).sub.2-4 NR.sup.V R.sup.VI ; or 
R.sup.II and R.sup.III taken together are --(CH.sub.2).sub.4 --, 
--(CH.sub.2).sub.5 --, --(CH.sub.2).sub.2 O(CH.sub.2).sub.2 --, or 
--(CH.sub.2).sub.2 NH(CH.sub.2).sub.2 --; 
R.sup.IV is C.sub.1 -C.sub.4 alkyl; 
R.sup.V is H or C.sub.1 -C.sub.4 alkyl; 
R.sup.VI is H or C.sub.1 -C.sub.4 alkyl; 
R.sup.VII is H, C.sub.1 -C.sub.4 alkyl or NH.sub.2 ; 
X is Cl, Br or I; or 
a pharmaceutically acceptable salt thereof. 
Additionally, there are disclosed quaternary ammonium salts of the formula 
##STR5## 
where R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sup.I, 
R.sup.IV and X are as previously defined. 
There are also disclosed compounds of the formula 
##STR6## 
wherein R.sub.1 is H or OH; 
R.sub.2 is H, CH.sub.3 or OH; 
R.sub.3 is H, CH.sub.3, CH.sub.2 CONH.sub.2, CH.sub.2 CN, CH.sub.2 CH.sub.2 
NR.sup.II R.sup.III, CH.sub.2 CH.sub.2 N(R.sup.IV).sub.3.sup.+ X.sup.- or 
CH.sub.2 CH.sub.2 NH(C.dbd.NH)R.sup.VII ; 
R.sub.4 is H or CH.sub.3 ; 
R.sub.5 is H, OH or OSO.sub.3 H; 
R.sub.6 is H or OH; 
R.sup.II is H, C.sub.1 -C.sub.4 alkyl, (CH.sub.2).sub.2-4 OH, 
C.dbd.NH(R.sup.VII), (CH.sub.2).sub.2-4 NR.sup.V R.sup.VI, 
(CH.sub.2).sub.2-4 N(R.sup.IV).sub.3.sup.+ X.sup.-, (CH.sub.2).sub.2-4 
NH(C.dbd.NH)R.sup.VII, (CH.sub.2).sub.1-4 CH(NR.sup.V 
R.sup.VI)(CH.sub.2).sub.1-4 NR.sup.V R.sup.VI, (CH.sub.2).sub.2-4 NR.sup.V 
(CH.sub.2).sub.2-4 NR.sup.V R.sup.VI, CO(CH.sub.2).sub.1-4 NR.sup.V 
R.sup.VI, COCH(NR.sup.V R.sup.VI)(CH.sub.2).sub.1-4 NR.sup.V R.sup.VI ; 
R.sup.III is H, C.sub.1 -C.sub.4 alkyl, (CH.sub.2).sub.2-4 NR.sup.V 
R.sup.VI, (CH.sub.2).sub.2-4 N(R.sup.IV).sub.3.sup.+X.sup.-, 
(CH.sub.2).sub.2-4 NH(C.dbd.NH)R.sup.VII, (CH.sub.2).sub.1-4 CH(NR.sup.V 
R.sup.VI)(CH.sub.2).sub.1-4 NR.sup.V R.sup.VI, (CH.sub.2).sub.2-4 NR.sup.V 
(CH.sub.2).sub.2-4 NR.sup.V R.sup.VI ; or 
R.sup.II and R.sup.III taken together are --(CH.sub.2).sub.4 --, 
--(CH.sub.2).sub.5 --, --(CH.sub.2).sub.2 O(CH.sub.2).sub.2 --, or 
--(CH.sub.2).sub.2 NH(CH.sub.2).sub.2 --; 
R.sup.IV is C.sub.1 -C.sub.4 alkyl; 
R.sup.V is H or C.sub.1 -C.sub.4 alkyl; 
R.sup.VI is H or C.sub.1 -C.sub.4 alkyl; 
R.sup.VII is H, C.sub.1 -C.sub.4 alkyl or NH.sub.2 ; 
X is Cl, Br or I; or 
a pharmaceutically acceptable salt thereof, which are useful for the 
preparation of Compounds I and II of the invention. 
Preferred embodiments of the invention are those of Compound I wherein 
R.sub.1 and R.sub.6 are OH 
R.sub.2 and R.sub.5 are H 
R.sub.3 is CH.sub.2 CH.sub.2 NH.sub.2 
R.sub.4 is CH.sub.3 
R.sup.I is 9,11-dimethyltridecyl, 
##STR7## 
R.sup.II is H, CH.sub.2 CH.sub.2 NH.sub.2, COCH.sub.2 NH.sub.2, COCH.sub.2 
CH.sub.2 NH.sub.2 or COCH(NH.sub.2)CH.sub.2 NH.sub.2, and 
R.sup.III is H. 
The compounds of this invention may be formulated into pharmaceutical 
compositions which are comprised of the compounds of formula I or II in 
combination with a pharmaceutically acceptable carrier. 
The compounds of this invention are useful in treating fungal infections 
such as those caused by Candida and Aspergillus and for the treatment and 
prevention of infections caused by Pneumocystis carinii. These infections 
are often found in immunocomprised patients such as those suffering with 
AIDS. 
Throughout the specification and appended claims, a given chemical formula 
or name shall encompass all optical and stereoisomers as well as racemic 
mixtures where such isomers and mixtures exist. 
The term alkyl refers to straight, branched or cyclic chain hydrocarbon 
groups, e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, pentyl, hexyl, 
heptyl, cyclopentyl, cyclohexyl, cyclohexylmethyl and the like. 
The term cycloalkyl refers to a species of alkyl containing from 3 to 15 
carbon atoms without alternating or resonating double bonds between carbon 
atoms. 
The term alkenyl refers to groups such as, e.g., vinyl, 1-propene-2-yl, 
1-butene-4-yl, 2-buten-4-yl, 1-pentene-5-yl and the like. 
The term alkoxy refer to straight or branched chain oxyalkyl groups such 
as, e.g., methoxy, ethoxy, butoxy, heptoxy, dodecyloxy, and the like. 
The compounds of the present invention are generally obtained as mixtures 
of stereoisomeric forms in which one form usually predominates. Conditions 
may be adjusted by means within the normal skill of the skilled artisan to 
obtain predominantly the desired isomer. The compounds with preferred 
stereoisomeric form designated herein as the "normal" form are those in 
which the group at the "C-5-orn" position is below the plane at the said 
position. The designation "epi" has been employed for those compounds in 
which the group at the "C-5-orn" position is above the plane. 
Pharmaceutically acceptable salts suitable as acid addition salts are those 
from acids such as hydrochloric, hydrobromic, phosphoric, sulfuric, 
maleic, citric, acetic, tartaric, succinic, oxalic, malic, glutamic and 
the like, and include other acids related to the pharmaceutically 
acceptable salts listed in Journal of Pharmaceutical Science, 66:2 (1977). 
When the acyl substituent at the 2-position on the 4-hydroxyornithine 
nitrogen contains an aromatic chain, it differs from natural products and 
known compounds. The aromatic chain disclosed contains one to three phenyl 
groups further extended by substituents in the para position. 
Representative nuclei for the derivatives of the present invention 
(Compounds I & II) and the sequence ID for these compounds may be seen in 
the following table. Since the peptide nuclei would be the same 
irrespective of substituents R.sub.1, R.sub.2, R.sub.5, R.sub.6, R.sup.I, 
R.sup.II, or R.sup.III and since the sequence identification number is 
assigned for the nuclear variations, the amines and salts have the same 
sequence ID's. 
______________________________________ 
Carba 
Compound R.sub.3 R.sub.4 
SEQ ID NO. 
______________________________________ 
I-1 H CH.sub.3 
1 
I-2 CH.sub.3 CH.sub.3 
2 
I-3 All Others CH.sub.3 
3 
I-4 H H 4 
I-5 CH.sub.3 H 5 
I-6 All Others H 6 
______________________________________ 
The compounds of the present invention are soluble in water, lower 
alcohols, and polar aprotic solvents such as N,N-dimethylformamide (DMF), 
dimethyl sulfoxide (DMSO) and pyridine. They are insoluble in solvents 
such as diethyl ether and acetonitrile. 
The compounds of the present invention are useful as an antibiotic, 
especially as an antifungal agent or as an antiprotozoal agent. As 
antifungal agents they are useful for the control of both filamentous 
fungi and yeasts. They are especially adaptable to be employed for the 
treatment of mycotic infections in mammals, especially those caused by 
Candida species such as C. albicans, C. tropicalis and C. 
pseudotropicalis, Cryptococcus species such as C. neoformans and 
Aspergillus species such as A. fumigatus, A. flavus and A. niger. They are 
also useful for the treatment and/or prevention of Pneumocystis carinii 
pneumonia to which immune-compromised patients are especially susceptible 
as hereinafter described. 
The structural aspect which distinguishes the compounds of the present 
invention from previously disclosed cyclohexapeptides is the carbon 
attached to the cyclohexapeptide ring at the 5-carbon of the 
4-hydroxyornithine residue. 
The most important naturally occurring echinocandins and pneumocandins have 
a labile C--O bond at the C-5 orn position. Other pneumocandins as 
disclosed in U.S. Pat. No. 5,378,804 issued Jan. 3, 1995 have a labile 
C--N bond at the C-5 orn. The compounds disclosed herein have a C--C bond 
at the C-5 orn imparting stability to the compounds while still retaining 
potent antifungal and anti-Pneumocystis activity. 
The compounds of the present invention may be prepared from 
cyclohexapeptides having the formula 
##STR8## 
by a series of reactions in which the oxygen atom at the "C-5-orn" (which 
also may be referred to as the hemiaminal position) is ultimately replaced 
by carbon. The starting materials may be natural products or modified 
natural products as subsequently described. 
The sequence IDs of the starting materials are listed in the following 
table: 
______________________________________ 
Starting 
Material 
Compound R.sub.3 R.sub.4 
SEQ ID NO. 
______________________________________ 
A-1 H CH.sub.3 
7 
A-2 CH.sub.3 CH.sub.3 
8 
A-3 All Others CH.sub.3 
9 
A-4 H H 10 
A-5 CH.sub.3 H 11 
A-6 All Others H 12 
______________________________________ 
A compound where R.sub.1 is OH, R.sub.2 is H, R.sub.3 is CH.sub.2 
CONH.sub.2, R.sub.4 is CH.sub.3, R.sub.5 is H and R.sub.6 is OH and 
R.sup.I is dimethyltridecyl has been identified in the literature as 
pneumocandin B.sub.o ; a similar compound where R.sub.2 is CH.sub.3, has 
been identified as pneumocandin A.sub.o and a third compound where R.sub.2 
is OH and R.sub.6 is H has been identified as pneumocandin C.sub.o (J. 
Antibiotics 45:1855-60, December 1992). A similar compound where R.sub.2 
and R.sub.6 are OH and R.sup.I is dimethyltridecyl has been identified as 
pneumocandin D.sub.o (J. Antibiotics 47:755-764, July 1994). 
When in the starting compound, R.sub.3 is H, CH.sub.3 or CH.sub.2 
CONH.sub.2, they may be directly employed. When R.sub.3 is CH.sub.2 CN, 
CH.sub.2 CH.sub.2 NR.sup.II R.sup.III, CH.sub.2 CH.sub.2 
N(R.sup.IV).sub.3.sup.+ X.sup.- or CH.sub.2 CH.sub.2 
NH(C.dbd.NH)R.sup.VII, the amides must be first converted to CH.sub.2 CN 
or CH.sub.2 CH.sub.2 NH.sub.2 and then modified. 
##STR9## 
In Step A, the starting material, Compound A, an alkylthiol or arylthiol 
and acid are caused to react in an aprotic solvent under anhydrous 
conditions for time sufficient for reaction to take place with the 
formation of Compound B (Seq ID Nos. 13-18), listed in the following 
table. Aminoethanethiol has been found to be especially useful for this 
step. 
______________________________________ 
Sulfur 
Intermediate 
Compound R.sub.3 R.sub.4 
SEQ ID NO. 
______________________________________ 
B-1 H CH.sub.3 
13 
B-2 CH.sub.3 CH.sub.3 
14 
B-3 All Others CH.sub.3 
15 
B-4 H H 16 
B-5 CH.sub.3 H 17 
B-6 All Others H 18 
______________________________________ 
For Step A, suitable acids include strong organic acid and mineral acids. 
Examples of strong organic acids are camphorsulfonic acid, 
p-toluenesulfonic acid, trifluoroacetic acid and methanesulfonic acid. 
Mineral acids include hydrochloric acid and hydrobromic acid. 
Camphorsulfonic acid is preferred. 
Suitable solvents include DMF, DMSO, 1-methyl-2-pyrrolidinone and 
hexamethyl phosphoric triamide (HMPA). DMF or DMSO is preferred. 
The reaction is generally carried out at ambient temperature to 60.degree. 
C. for about 3 hours to about 10 days. 
In carrying out the reaction, the cyclohexapeptide compound, the thiol 
compound and acid are stirred together in a suitable solvent until the 
reaction is substantially complete. The reaction mixture then is diluted 
with water and flash chromatographed on reverse phase resins using 10 to 
40 percent acetonitrile/water (containing 0.1% trifluoroacetic acid) as 
eluant. Trifluoroacetic acid may hereinafter be designated "TFA". The 
fractions containing the desired product may be concentrated and 
lyophilized and the lyophilized material purified by preparative high 
performance liquid chromatography (HPLC). 
Appropriate columns for HPLC are commercially available columns sold under 
trademarks or trade names such as "ZORBAX" (DuPont), "DeltaPak" (Waters), 
"LICHROPREP" RP18 (E. Merck). The specific columns are identified in the 
working examples. 
In Step B, Compound C (Seq ID Nos. 13-18), a sulfone is obtained by the 
oxidation of Compound B. Suitable oxidizing agents or oxidants include 
"OXONE" (KHSO.sub.5.KHSO.sub.4.K.sub.2 SO.sub.4 2:1:1, Aldrich Chemicals), 
metachloroperoxybenzoic acid, and peroxyacetic acid. The sequence ID of 
Compound C is the same as that of Compound B since the atom attached to 
the hemiaminal carbon is still sulfur. Thus, the sequence IDs of the 
sulfones are as follows: 
______________________________________ 
Sulfone 
Compound R.sub.3 R.sub.4 
SEQ ID NO. 
______________________________________ 
C-1 H CH.sub.3 
13 
C-2 CH.sub.3 CH.sub.3 
14 
C-3 All Others CH.sub.3 
15 
C-4 H H 16 
C-5 CH.sub.3 H 17 
C-6 All Others H 18 
______________________________________ 
The oxidation of the thioether (Compound B) to the sulfone (Compound C) is 
carried out with about two molar amounts of the oxidant. When one molar 
amount of oxidant is employed, the product is a sulfoxide which may then 
be converted to the sulfone. The sulfoxides may be employed as an 
intermediate in the formation of the nitrite but the sulfone is preferred. 
A slight excess over the two molar amount of the oxidizing agent is 
employed. 
The reaction is carried out in an aqueous medium, preferably a mixture of 
acetonitrile and water. About equal amounts are preferred although a range 
of 1:9 to 9:1 may be employed. 
In carrying out the reaction, the oxidant is added to a solution of 
Compound B (Seq ID Nos. 13-18) in 1:1 acetonitrile/water and the mixture 
allowed to stand at ambient temperature for time sufficient to complete 
the reaction to obtain Compound C generally from about 30 minutes to one 
hour. 
After completion of the reaction, the compound is recovered from the 
reaction mixture by diluting with water and chromatographing. Reverse 
phase (C18) flash column chromatography is suitable in this purification 
step. The preferred eluting agent is 30-45 percent acetonitrile/water 
(0.1% TFA) in 5 percent step gradients. The appropriate fractions are 
lyophilized to recover the desired sulfone intermediate, Compound C (Seq 
ID Nos. 13-18). The intermediate tends to be labile, thus the isolation 
should be carried out as rapidly as possible. Alternatively, the reaction 
mixture can be lyophilized and the crude sulfone used as is in the 
subsequent step. 
Compound C may be converted to a compound having a carbon directly attached 
to the "C-5-orn". As seen in the flow diagram, reaction of Compound C with 
an alkali metal cyanide produces a nitrile at that position (Compound D). 
The nitrile can subsequently be reacted with sodium borohydride and 
cobaltous chloride to afford the aminoalkyl substituent which may be 
converted into a substituted amine as subsequently described. Compound D 
is an important intermediate for most of the compounds of the present 
invention. Sequence ID Nos. for Compound D, the nitrile, are listed in the 
following table: 
______________________________________ 
Nitrile 
Compound R.sub.3 R.sub.4 
SEQ ID NO. 
______________________________________ 
D-1 H CH.sub.3 
19 
D-2 CH.sub.3 CH.sub.3 
20 
D-3 All Others CH.sub.3 
21 
D-4 H H 22 
D-5 CH.sub.3 H 23 
D-6 All Others H 24 
______________________________________ 
The nitrile may be obtained by adding alkali metal cyanide while stirring 
at ambient temperature to a solution of the sulfone in an aprotic solvent 
for time sufficient to complete the reaction with the formation of the 
cyanide as determined by HPLC analysis. The reaction mixture then may be 
diluted with water and then chromatographed to separate the desired 
nitrile (Compound D) from the reaction mixture. Reverse-phase (C18) flash 
column chromatography using 20-60% acetonitrile/water (0.1% TFA) in 10% 
step gradients is suitable for this procedure. 
The nitrile (Compound D) may then be reduced to a compound having a free 
amino group (Compound E). 
The reduction may be carried out employing either chemical or catalytic 
reduction. When chemical reduction is employed, hydride or hydride 
combinations have been found useful. 
Sodium borohydride with cobaltous chloride in alcoholic solvent has been 
found to be particularly useful. When this combination of reagents is 
used, from about 5 to 50 molar equivalents of sodium borohydride and from 
2 to 20 molar equivalents of cobaltous chloride are used for each molar 
amount of the nitrile. 
Other reducing agents such as Raney nickel, sodium cyanoborohydride, 
aluminum hydride, diborane, diisobutyl aluminum hydride and the like may 
also be used. Frequently these reducing agents are used in combination 
with a Lewis acid such as cobaltous chloride or aluminum chloride as in 
the present combination of sodium borohydride and cobaltous chloride. 
Catalytic hydrogenation also may be carried out over a variety of catalysts 
including palladium on carbon, platinum oxide or rhodium on alumina. Low 
pressure catalytic reduction over Pd/C as the catalyst is especially 
preferred. 
Typical solvents depending on the reagent include alcohols, especially 
methanol and ethanol; dimethylformamide, pyridine, tetrahydrofuran or 
other ethers. 
Compounds containing a selectively derivatized amine at the C5-orn position 
in the presence of an amine at R.sub.3 (i.e. R.sub.3 =CH.sub.2 CH.sub.2 
NH.sub.2) may be prepared by initially introducing the C5-orn amine when 
R.sub.3 =CH.sub.2 CONH.sub.2. The C5-orn amine may then be substituted to 
provide compounds where R.sup.II and R.sup.III are not H. Finally, the 
primary amide at R.sub.3 may be converted to an amine following 
established procedures. Alternatively, the amine of R.sub.3 may be 
protected as a CBZ derivative prior to reduction of the C5-orn nitrile to 
an amine. 
The amine thus obtained may be converted into an acylated amine by 
conventional means using a CBZ protected amino acid to obtain, after 
deprotection, Compound I where R.sup.II is 
COCH(NH.sub.2)(CH.sub.2).sub.1-4 NH.sub.2 and R.sup.III is H. 
Compound I where R.sup.II and/or R.sup.III are alkyl may be prepared using 
any suitable known procedure for preparing secondary or tertiary amines. 
When the desired alkyl group on the nitrogen is methyl, the carbon may be 
introduced by formylating, followed by reduction of the hydroxymethyl 
group with sodium cyanoborohydride or other reducing agent. Alternatively, 
alkylation may be carried out by causing an appropritately substituted 
alkyl halide to react with the amine in an aprotic solvent in the presence 
of a base. 
To prepare compounds in which the R.sub.3 amine (i.e. R.sub.3 =CH.sub.2 
CH.sub.2 NH.sub.2) is selectively derivatized in the presence of an amine 
at C5-orn, the R.sub.3 amine may be substituted prior to reduction of the 
nitrile at C5-orn. 
The invention also embraces quaternary ammonium salts of formula (II). 
These may be prepared by treatment of an amine with an alkyl halide and 
base in a protic or aprotic solvent. A typical procedure would be to add 
excess methyl iodide to a solution of the amine and sodium bicarbonate in 
DMF at room temperature. The product may be isolated by diluting with 
H.sub.2 O followed by C18 HPLC. 
The invention also embraces acid addition salts. The compound in the normal 
course of isolation is obtained as an acid addition salt. Generally, it is 
as a trifluoroacetic acid or acetic acid salt. The salt thus obtained may 
be dissolved in water and passed through an anion exchange column bearing 
the desired anion. The eluate containing the desired salt may be 
concentrated to recover the salt as a solid product. 
The compounds of the present invention are water soluble in their 
protonated or permanently charged quaternary forms. This is an advantage 
over the neutral, uncharged echinocandins which are not water soluble. 
The compounds of the present invention are active against many fungi and 
particularly against Candida species. The antifungal properties may be 
illustrated with the minimum fungicidal concentration (MFC) determination 
against certain Candida organisms in a microbroth dilution assay carried 
out in a Yeast Nitrogen Base (DIFCO) medium with 1% dextrose (YNBD). 
In a representative assay, compounds were solubilized in 100% dimethyl 
sulfoxide (DMSO) at an initial concentration of 5 mg/ml. Once dissolved, 
the drug stock was brought to a concentration of 512 .mu.g/ml by dilution 
in water such that the final DMSO concentration was about 10 percent. The 
solution was then dispensed via a multichannel pipetter into the first 
column of a 96-well plate (each well containing 0.075 ml of YNBD), 
resulting in a drug concentration of 256 .mu.g/ml. Compounds in the first 
column were diluted 2-fold across the rows yielding final drug 
concentration ranging from 256 .mu.g/ml to 0.12 .mu.g/ml. 
Four-hour broth cultures of organisms to be tested were adjusted using a 
spectrophotometer at 600 nm to equal a 0.5 McFarland Standard. This 
suspension was diluted 1:100 in YNBD to yield a cell concentration of 
1-5.times.10.sup.4 colony forming units (CFU)/ml. Aliquots of the 
suspension (0.075 ml) were inoculated into each well of the microtiter 
plate resulting in a final cell inoculum of 5-25.times.10.sup.3 CFU/ml and 
final drug concentrations ranging from 128 .mu.g/ml to 0.06 .mu.g/ml. Each 
assay included one row for drug-free control wells and one row for 
cell-free control wells. 
After 24 hours of incubation, the microtiter plates were shaken gently on a 
shaker to resuspend the cells. The MIC-2000 inoculator was used to 
transfer a 1.5 microliter sample from each well of the 96-well microtiter 
plate to a single reservoir inoculum plate containing Sabouraud dextrose 
agar (SDA). The inoculated SDA plates were incubated for 24 hours at 
35.degree. C. and then read for minimum fungicidal concentration (MFC). 
MFC is defined as the lowest concentration of drug showing no growth or 
less than 4 colonies per spot. Compound I-A where R.sub.1 =OH, R.sub.2 =H, 
R.sub.3 =CH.sub.2 CH.sub.2 NH.sub.2, R.sub.4 =CH.sub.3, R.sub.5 =H, 
R.sub.6 =OH, R.sup.I =dimethyltridecyl, R.sup.II =H and R.sup.III =H as 
the bishydrochloride salt had the following MFCs (1.mu.g/ml): 
Candida albicans (MY1055) &lt;0.06 
Candida tropicalis (MY1012) &lt;0.06 
Candida glabrata (MY1381) 0.25 
The in vivo effectiveness of the compounds against fungi may be seen in the 
following assay. 
Growth from an overnight SDA culture of Candida albicans MY 1055 was 
suspended in sterile saline and the cell concentration determined by 
hemacytometer count and the cell suspension adjusted to 
3.75.times.10.sup.5 cells/ml. Then 0.2 milliliter of this suspension was 
administered I.V. in the tail vein of mice so that the final inoculum is 
7.5.times.10.sup.4 cells/mouse. 
The assay was then carried out by administering aqueous solutions of 
Compound I-A at various concentrations intraperitoneally (I.P.), twice 
daily (b.i.d.) for four consecutive days to 18 to 20 gram female DBA/2 
mice, which previously had been infected with Candida albicans (MY 1055) 
in the manner described above. Distilled water was administered I.P. to C. 
albicans challenged mice as controls. After seven days, the mice were 
sacrificed by carbon dioxide gas, paired kidneys were removed aseptically 
and placed in sterile polyethylene bags containing 5 milliliters of 
sterile saline. The kidneys were homogenized in the bags, serially diluted 
in sterile saline and aliquots spread on the surface of SDA plates. The 
plates were incubated at 35.degree. C. for 48 hours and yeast colonies 
enumerated for determination of colony forming units (CFU) per gram of 
kidneys. Compound I-A gave greater than 90% reduction of recoverable 
Candida CFUs at 0.02 mg/kg i.p. twice daily for four consecutive days. 
The compounds of the present invention are also active against Aspergillus 
species. The in vivo effectiveness of the compounds against Aspergillus 
may be seen in the following assay. 
Conidia of Aspergillus fumigatus MF 5668 were washed from the surface of 
several (3-4) 3-5 day SDA slant cultures with sterile saline plus 0.01% 
Tween 20. The conidia suspension was quantitated by hemacytometer count 
and adjusted to the appropriate concentration in sterile saline. 
Female DBA/2 mice were challenged I.V. with 1.40.times.10.sup.6 
conidia/mouse. Within fifteen minutes after challenge, aqueous solutions 
of Compound I-A were administered intraperitoneally (I.P.) at various 
concentrations twice daily (b.i.d.) for a total of five days. The required 
dose of Compound I-A to increase the 28-day survival rate by at least 50% 
over untreated controls was 0.02 mg/kg. 
A harmful and potentially fatal side reaction of a number of drugs 
including certain antibiotically active echinocandin compounds is red 
blood cell lysis. This is not seen in compounds having the present nuclei 
which is another advantage of the compounds of this invention. 
The compounds of the present invention may also be useful for inhibiting or 
alleviating Pneumocystis carinii infections in immune-compromised 
patients. The efficacy of the compounds of the present invention for 
therapeutic or anti-infection purposes may be demonstrated in studies on 
immunosuppressed rats. 
Sprague-Dawley rats (weighing approximately 250 grams) were 
iunmunosuppressed with dexamethasone in the drinking water (2.0 mg/L) and 
maintained on a low protein diet for seven weeks to induce the development 
of Pneumocystis pneumonia from a latent infection. Before drug treatment, 
two rats were sacrificed to confirm the presence of Pneumocystis carinii 
pneumonia (PCP). Five rats (weighing approximately 150 grams) were 
injected twice daily for four days subcutaneously (sc) with Compound I-A 
in 0.25 ml of vehicle (distilled water). A vehicle control was also 
carried out. All animals continued to receive dexamethasone in the 
drinking water and low protein diet during the treatment period. At the 
completion of the treatment, all animals were sacrificed, the lungs were 
removed and processed, and the extent of disease determined by microscopic 
analysis of stained slides. The prevention or reduction of cysts were seen 
in slides of the lungs of treated rats when compared with the number of 
cysts in the lungs of untreated controls or solvent controls. The results 
of this assay showed that Compound I-A reduced P. carinii cysts in 5 rats 
by at least 90 percent when dosed at 0.02 mg/kg with all rats surviving. 
The outstanding properties are most effectively utilized when the compounds 
are formulated into novel pharmaceutical compositions with a 
pharmaceutically acceptable carrier according to the conventional 
pharmaceutical compounding techniques. 
The novel compositions contain at least a therapeutic antifungal or 
antipneumocystis amount of the active compound. Generally, the composition 
contains at least 1% by weight of Compound I or II. Concentrate 
compositions suitable for dilutions prior to use may contain 90% or more 
by weight. The compositions include compositions suitable for oral, 
topical, parenteral (including intraperitoneal, subcutaneous, 
intramuscular, and intravenous), nasal, and suppository administration, or 
insufflation. The compositions may be prepacked by intimately mixing 
Compound I or II with the components suitable for the medium desired. 
Compositions formulated for oral administration may be liquid or solid 
compositions. For liquid preparation, the therapeutic agent may be 
formulated with liquid carriers such as water, glycols, oils, alcohols, 
and the like, and for solid preparations such as capsules and tablets, 
with solid carriers such as starches, sugars, ethyl cellulose, calcium and 
sodium carbonate, calcium phosphate, kaolin, talc, lactose, generally with 
a lubricant such as calcium stearate, together with binders disintegrating 
agents and the like. Because of their ease in administration, tablets and 
capsules represent the most advantageous oral dosage form. It is 
especially advantageous to formulate the compositions in unit dosage form 
for ease of administration and uniformity of dosage. Compositions in unit 
dosage form constitute an aspect of the present invention. 
Compositions may be formulated for injection and may take such forms as 
suspensions, solutions or emulsions in oily or aqueous vehicles such as 
0.85 percent sodium chloride or 5 percent dextrose in water and may 
contain formulating agents such as suspending, stabilizing and/or 
dispersing agents. Buffering agents as well as additives such as saline or 
glucose may be added to make the solutions isotonic. The compound may also 
be solubilized in alcohol/propylene glycol or polyethylene glycol for drip 
intravenous administration. These compositions also may be presented in 
unit dosage form in ampoules or in multidose containers, preferable with 
added preservative. Alternatively, the active ingredients may be in powder 
form for reconstituting with a suitable vehicle prior to administration. 
The term "unit dosage form" as used in the specification and claims refers 
to physically discrete units, each unit containing a predetermined 
quantity of active ingredient calculated to produce the desired 
therapeutic effect in association with the pharmaceutical carrier. 
Examples of such unit dosage forms are tablets, capsules, pills, powder 
packets, wafers, measured units in ampules or in multidose containers and 
the like. A unit dosage of the present invention will generally contain 
from 100 to 200 milligrams of one of the compounds. 
When the compound is for antifungal use, any method of administration may 
be employed. For treating mycotic infections, oral or intravenous 
administration is usually employed. 
When the compound is to be employed for control of Pneumocystis infections, 
it is desirable to directly treat lung and bronchi. For this reason 
inhalation methods are preferred. For administration by inhalation, the 
compounds of the present inventions are conveniently delivered in the form 
of an aerosol spray presentation from pressurized packs or nebulizers. The 
preferred delivery system for inhalation is a metered dose inhalation 
(MDI) aerosol, which may be formulated as a suspension or solution of 
Compound I or II in suitable propellants, such as fluorocarbons or 
hydrocarbons. Preferred propellants are those which do not damage the 
ozone layer. 
Although the compounds of the present invention may be employed as tablets, 
capsules, topical compositions, insufflation powders, suppositories and 
the like, the solubility of the compounds of the present invention in 
water and aqueous media render them adaptable for use in injectible 
formulations and also in liquid compositions suitable for aerosol sprays.

The following examples illustrate the invention but are not to be construed 
as limiting. All temperatures are in degrees centigrade (.degree.C.) 
unless indicated otherwise. 
EXAMPLE 1 
##STR10## 
Part A: Preparation of Thioether Intermediate 
Trifluoroacetic acid (0.4 ml, 5.3 mmol) was added to a solution of 
##STR11## 
(22.9 g, 21.1 mmol) and 2-aminoethanethiol hydrochloride (47.9 g, 422 
mmol) in 100 ml of anhydrous N,N-dimethylformamide at 60.degree. C. After 
a period of 4 h, the reaction mixture was cooled to room temperature and 
diluted with 400 ml of H.sub.2 O. Filtration of the resulting solution was 
followed by pump-injection of the filtrate onto a Waters Delta Pak C18-100 
.ANG. radial cartridge (47 mm.times.30 cm) at a rate of 50 ml/min. Elution 
with 25-30% CH.sub.3 CN/H.sub.2 O (0.1% CF.sub.3 COOH) in one 5% step 
gradient gave, after lyophilization of the appropriate fractions, 6.5 g of 
the nor-thioether and 6.8 g of the epi-thioether as bistrifluoroacetate 
salts. By analytical HPLC (Zorbax RX-C18, 40% CH.sub.3 CN/H.sub.2 O (0.1% 
CF.sub.3 COOH), uv at 210 nm), the thioethers were sufficiently pure 
(&gt;80%) for conversion to sulfone as described below. Rechromatography of 
the individual isomers followed by ion exchange on a Bio-Rad AG2-X8 
(Cl.sup.-) column eluting with H.sub.2 O provided, after lyophilization, 
pure bishydrochlorides as amorphous solids. Nor-thioether: .sup.1 H NMR 
(400 MHz, CD.sub.3 OD) d 1.17 (d, J=6.2 Hz, 3H), 2.9 (m, 2H), 3.06 (t, 
J=7.2 Hz, 2H), 3.20 (t, J=6.7 Hz, 2H), 4.91 (d, J=5.8 Hz, 2H), 4.99 (d, 
J=3.4 Hz), 5.27 (d, J=2.1 Hz, 1H), 6.74 (d, J=8.6 Hz, 2H), 7.11 (d, J=8.6 
Hz, 2H); FAB-MS (Li) m/z 1117 (MH+Li).sup.+. Epi-thioether: .sup.1 H NMR 
(400 MHz, CD.sub.3 OD) d 2.23 (m, 3H), 2.41 (dd, J=7.4 and 13.1 Hz, 1H), 
2.92 (m, 2H), 3.11 (m, 2H), 3.06-3.29 (m, 4H), 4.01 (m, 1H), 4.10 (d, 
J=4.6 Hz, 1H), 4.64 (dd, J=7.2 and 10.9 Hz, 1H), 4.69 (br s, 1H), 4.95 (d, 
J=3.9 Hz, 1H), 6.76 (d, J=8.6 Hz, 2H), 7.14 (d, J=8.6 Hz, 2H); FAB-MS (Li) 
m/z 1117 (MH+).sup.+. 
Part B: Preparation of Sulfone 
To a stirred solution of the epi-thioether from Part A (6.5 g, .about.70% 
pure) in 55 ml of 1:1 acetonitrile/water at 25.degree. C. was added 
OXONE.RTM. (3.1 g). After a period of 15 min, analysis by C18-HPLC showed 
the conversion to a more polar product to be complete. The reaction 
mixture was lyophilized to provide the crude sulfone which was used in the 
subsequent step without purification. 
Part C: Preparation of Nitrile 
A solution of the epi-sulfone bistrifluoroacetate from Part B (2.3 g, 73% 
pure, 1.23 mmol corrected for purity) in 123 ml of 0.5M lithium cyanide in 
N,N-dimethylformamide was stirred at 25.degree. C. for a period of 15 
minutes. HPLC analysis RP-C18, 45% CH.sub.3 CN/H.sub.2 O (0.1% CF.sub.3 
COOH)! of the reaction mixture indicated complete conversion to two less 
polar products. The reaction mixture was diluted with water (400 ml) and 
the resulting heterogeneous mixture was loaded onto a reverse-phase flash 
column (C18, 30 g) packed in 20% CH.sub.3 CN/H.sub.2 O. Elution with 
H.sub.2 O (200 ml) was followed by 20-70% CH.sub.3 CN/H.sub.2 O (0.1% 
CF.sub.3 COOH) in 10% step-gradients collecting 100 ml at each step. The 
insoluble cake remaining at the top of the column was removed and 
dissolved in 70% CH.sub.3 CN/H.sub.2 O (0.1% CF.sub.3 COOH). This solution 
was combined with the product-containing fractions and lyophilized to give 
1.5 g of crude nitriles. Reverse-phase HPLC of this material C18, 30-45% 
CH.sub.3 CN/H.sub.2 O (0.1% CF.sub.3 COOH) in 5% step-gradients! gave, 
after lyophilization of the appropriate fractions, 220 mg (21%) of the 
nor-nitrile and 270 mg (36%) of the epi-nitrile as the trifluoroacetate 
salts. Nor-nitrile: .sup.1 H NMR (500 MHz, CD.sub.3 OD) d 1.16 (d, J=6.2 
Hz, 3H), 1.60 (m, 2H) 1.81 (m, 1H), 2.44 (dd, J=7.0 and 13.0 Hz, 1H), 3.06 
(m, 2H), 3.83 (m, 3H), 3.95 (dd, J=3.2 and 11.2 Hz, 1H), 4.03 (m, 1H), 
4.43 (m, 1H), 4.54 (dd, J=7.1 and 11.7 Hz, 1H), 4.60 (dd, J=3.4 and 6.2 
Hz, 1H), 4.80 (d, J=2.3 Hz, 1H), 4.97 (d, J=3.2 Hz, 1H), 6.75 (d, J=8.5 
Hz, 2H), 7.11 (d, J=8.5 Hz, 2H); ESI-MS (M+H).sup.+ =1060.7. Epi-nitrile: 
.sup.1 H NMR (500 MHz, CD.sub.3 OD) d 1.23 (d, J=6.0 Hz), 2.05 (m, 1H), 
2.42 (dd, J=6.6 and 13.5 Hz, 1H), 3.10 (t, J=7.4 Hz, 2H), 3.76 (m, 3H), 
3.94 (dd, J=3.2 and 11.2 Hz, 1H), 4.00 (d, J=5.5 Hz, 1H), 4.11 (m, 1H), 
4.62 (dd, J=3.9 and 11.4 Hz, 1H), 4.81 (d, J=6.9 Hz, 1H), 4.88 (d, J=2.8 
Hz, 1H), 6.75 (d, J=8.5 Hz, 2H), 7.11 (d, J=8.5 Hz, 2H); ESI-MS 
(M+H).sup.+ =1060.7. 
EXAMPLE 2 
##STR12## 
Sodium borohydride (91.2 mg, 2.41 mmol) was added in portions to a solution 
of CoCl.sub.2.6H.sub.2 O (115 mg, 0.482 mmol) and the nor-nitrile (Example 
1, 283 mg, 0.241 mmol) in MeOH (9 ml). The ensuing exothermic reaction 
produced a precipitate while evolving copious quantities of hydrogen. HPLC 
analysis (Zorbax RX-C18, 4.6 mm.times.25 cm; 45% CH.sub.3 CN/H.sub.2 O 
(0.1% CF.sub.3 COOH) at 1.5 ml/min; uv detection at 210 and 277 nm) after 
10 minutes indicated 70% conversion to a more polar product (T.sub.R =3.0 
min). 2N CF.sub.3 COOH (7.8 ml) was added to the reaction mixture and 
stirring was continued for a period of 30 minutes, resulting in the 
dissolution of the precipitate. The mixture was diluted with H.sub.2 O (40 
ml) and then filtered through a packed bed of diatomaceous earth. The 
filtrate was pump-injected onto a Zorbax RX-C18 HPLC column (21.2 
mm.times.25 cm) in 30% CH.sub.3 CN/H.sub.2 O (0.1% CF.sub.3 COOH). Elution 
with 30-45% CH.sub.3 CN/H.sub.2 O (0.1% CF.sub.3 COOH) in 5% 
step-gradients at a flow rate of 10 ml/min followed by lyophilization of 
the appropriate fractions gave 93 mg of the bisamine as the 
trifluoroacetate. The bistrifluoroacetate was dissolved in H.sub.2 O (10 
ml) and the solution loaded onto a Bio-Rad AG2-X8 (Cl.sup.-) polyprep 
column (2 ml resin bed). Gravity elution with water (3.times.10 ml) 
followed by lyophilization of the eluate gave 80 mg of the above compound. 
Yield (corrected)=34%. .sup.1 H NMR (500 MHz, CD.sub.3 OD) d 1.18 (d, 
J=6.2 Hz, 3H), 1.80 (m, 1H) 1.92-2.12 (m, 4H), 2.18-2.36 (m, 4H), 2.43 
(dd, J=6.5 and 12.9 Hz, 1H), 3.07 (m, 2H), 3.16 (dd, J=5.4 and 13.2 Hz, 
1H), 3.23 (dd, J=3.9 and 13.2 Hz, 1H), 3.80 (m, 3H), 3.99 (dd, J=3.1 and 
11.1 Hz, 1H), 4.02-4.10 (m, 2H), 4.15 (m, 1H), 4.19 (dd, J=1.5 and 8.1 Hz, 
1H), 4.51-4.65 (m, 4H), 4.97 (d, J=3.2 Hz, 1H), 6.75 (d, J=8.6 Hz, 2H), 
7.11 (d, J=8.6 Hz, 2H); ESI-MS m/z 1064.6 (M+H).sup.+, 532.9 (M+H).sup.++. 
EXAMPLE 3 
##STR13## 
Part A: Preparation of Thioether Intermediate 
(1S)-(+)-10-Camphorsulfonic acid (2.39 g, 10.3 mmol) was added to a 
solution of 
##STR14## 
(11.0 g, 10.3 mmol) and 2-aminoethanethiol hydrochloride (53 g, 467 mmol) 
in 200 ml of anhydrous N,N-dimethylformamide at 25.degree. C. After a 
period of 72 h, the reaction mixture was diluted with H.sub.2 O (400 ml) 
and loaded onto a reverse-phase flash column (C18, 110 g) packed in 10% 
CH.sub.3 CN/H.sub.2 O. Elution with 10-60% CH.sub.3 CN/H.sub.2 O in 10% 
step-gradients followed by lyophilization of the product-containing 
fractions (40-50% CH.sub.3 CN/H.sub.2 O) gave 8.7 g of impure thioethers. 
Preparative HPLC of this mixture (Waters Delta Pak C18-100 .ANG. radial 
cartridge, 47 mm.times.30 cm) eluting with 20-40% CH.sub.3 CN/H.sub.2 O 
(0.1% CF.sub.3 COOH) at 50 ml/min in 10% step gradients gave, after 
lyophilization of the appropriate fractions, 2.0 g of the nor-thioether 
(yield=16%, HPLC purity &gt;95%) and 5.2 g of the epi-thioether (yield=40%, 
HPLC purity ca. 85%) as the trifluoroacetate salts. Nor-thioether: .sup.1 
H NMR (400 MHz, CD.sub.3 OD) d 1.14 (d, J=6.2 Hz, 3H), 2.83 (m, 2H), 5.44 
(d, J=1.8 Hz, 1H); FAB-MS (Li) m/z 1131 (M+H+Li ).sup.+. Epi-thioether: 
.sup.1 H NMR (400 MHz, CD.sub.3 OD) d 1.34 (d, J=6.3 Hz, 3H), 2.89 (m, 
2H), 4.72 (d, J=4.9 Hz, 1H); FAB-MS (Li) m/z 1131 (M+H+Li).sup.+. 
Part B: Preparation of Sulfone 
In a manner similar to that described in Example 1, Part B, the epi-sulfone 
was prepared from the epi-thioether. 
Part C: Preparation of Nitrile 
A solution of the epi-sulfone (1.0 g) in 79 ml of 0.5M lithium cyanide in 
N,N-dimethylformamide was stirred at 25.degree. C. for a period of 10 
minutes. HPLC analysis RP-C18, 50% CH.sub.3 CN/H.sub.2 O (0.1% CF.sub.3 
COOH)! of the reaction mixture indicated complete conversion to two less 
polar products. The reaction mixture was diluted with water (240 ml) and 
the resulting solution was loaded onto a reverse-phase flash column (C18, 
20 g) packed in 10% CH.sub.3 CN/H.sub.2 O. Elution with 20-70% CH.sub.3 
CN/H.sub.2 O in 10% step-gradients collecting 100 ml at each step followed 
by lyophilization of the product-containing fractions gave 610 mg of crude 
nitriles. Reverse-phase HPLC of this mixture (C18, 45-55% CH.sub.3 
CN/H.sub.2 O in 5% step-gradients) gave, after lyophilization of the 
appropriate fractions, 87 mg (yield=10%, HPLC purity @ 210 nm=97%) of the 
nor-nitrile and 190 mg (yield=22%, HPLC purity @ 210 nm=99%) of the 
epi-nitrile as white amorphous solids. Nor-nitrile: .sup.1 H NMR (500 MHz, 
CD.sub.3 OD) d 1.14 (d, J=6.2 Hz, 3H), 1.59 (m, 2H) 1.93-2.08 (m, 3H), 
2.15-2.27 (m, 5H), 2.43 (m, 1H), 2.47 (dd, J=9.5 and 15.4 Hz, 1H), 2.74 
(dd, J=3.8 and 15.4 Hz, 1H), 3.78 (m, 2H), 3.97 (m, 2H), 4.18 (m, 1H), 
4.39 (dd, J=4.4 and 12.8 Hz, 1H), 4.55 (m, 3H), 4.98 (m, 2H), 5.07 (d, 
J=4.1 Hz, 1H), 6.74 (d, J=8.5 Hz, 2H), 7.12 (d, J=8.5 Hz, 2H); ESI-MS 
(M+H).sup.+ =1074.5. Epi-nitrile: .sup.1 H NMR (500 MHz, CD.sub.3 OD) d 
1.59 (m, 2H), 1.76 (m, 1H), 1.98 (m, 1H), 2.07 (m, 2H), 2.22 (m, 3H), 2.39 
(dd, J=7.4 and 13.2 Hz, 1H), 2.45 (dd, J=8.0 and 15.1 Hz, 1H), 2.57 (dd, 
J=5.4 and 15.1 Hz, 1H), 4.02 (m, 1H), 4.07 (d, J=4.6 Hz, 1H), 4.31 (dd, 
J=1.9 and 7.9 Hz, 1H), 4.36 (m, 4H), 4.55 (m, 3H), 4.63 (dd, J=7.4 and 
10.6 Hz), 5.04 (d, J=3.4 Hz), 6.76 (d, J=8.5 Hz, 2H), 7.14 (d, J=8.5 Hz, 
2H); ESI-MS (M+H).sup.+ =1074.4. 
EXAMPLE 4 
##STR15## 
In a manner similar to that described in Example 2, the nor-nitrile from 
Example 3 was reduced to the amine shown above. Yield=44% (CF.sub.3 COOH 
salt). .sup.1 H NMR of hydrochloride salt (500 MHz, CD.sub.3 OD) d 1.16 
(d, J=6.2 Hz, 3H), 1.9-2.1 (m, 3H), 2.23 (m, 4H), 2.42 (dd, J=6.6 and 12.6 
Hz, 1H), 2.48 (dd, J=9.4 and 15.4 Hz, 1H), 2.75 (dd, J=3.7 and 15.4 Hz, 
1H), 3.15 (d, J=5.7 Hz, 2H), 3.80 (m, 2H), 3.96 (m, 2H), 4.06 (m, 1H), 
4.17 (m, 1H), 4.23 (dd, J=1.4 and 7.8 Hz, 1H), 4.57 (m, 4H), 5.00 (d, 
J=3.4 Hz, 1H), 5.06 (d, J=5.0 Hz, 1H), 6.74 (d, J=8.6 Hz, 2H), 7.12 (d, 
J=8.6 Hz, 2H); ESI-MS m/z 1078.7 (M+H).sup.+, 531.1 (M--H.sub.2 
O+H).sup.++. 
EXAMPLE 5 
##STR16## 
To a stirred solution of the amine trifluoroacetate from Example 4 (153 mg, 
0.128 mmol) and 1N sodium hydroxide (130 .mu.l, 0.130 mmol) in water (5 
ml) and N,N-dimethylformamide (5 ml) is added ethylacetimidate 
hydrochloride (160 mg, 1.29 mmol). After a period of 18 h at pH 8.5, 
trifluoroacetic acid is added to pH 7. Reverse-phase (C18) flash column 
chromatography of the neutralized reaction mixture, eluting with 
acetonitrile/water, is followed by lyophilization of the 
product-containing fractions. Preparative reverse-phase (C18) HPLC of this 
material, eluting with acetonitrile/water (0.1% CF.sub.3 COOH), is 
followed by lyophilization of the product-containing fractions to give the 
acetamidine as the trifluoroacetate salt: C.sub.55 H.sub.87 F.sub.3 
N.sub.10 O.sub.18, formula weight=1233.36. 
EXAMPLE 6 
##STR17## 
In a manner similar to that described in Example 5, bisamine from Example 2 
is converted to the bisacetamidine shown above: C.sub.59 H.sub.93 F.sub.6 
N.sub.11 O.sub.19, formula weight=1374.45. 
EXAMPLE 7 
##STR18## 
To a stirred solution of the amine trifluoroacetate from Example 4 (163 mg, 
0.137 mmol) and 1M sodium bicarbonate (150 .mu.l, 0.150 mmol) in absolute 
methanol (5 ml) is added aminoiminomethanesulfonic acid (30 mg, 0.242 
mmol). After a period of 1.5 h, the solvent is removed in vacuo. 
Preparative reverse-phase HPLC (C18) of the residue, eluting with 
acetonitrile/water (0.1% trifluoroacetic acid), is followed by 
lyophilization of the product-containing fractions to give the guanidine 
trifluoroacetate: C.sub.54 H.sub.86 F.sub.3 N.sub.11 O.sub.18, formula 
weight=1234.35. 
EXAMPLE 8 
##STR19## 
In a manner similar to that described in Example 7, bisamine from Example 2 
is converted to the bisguanidine shown above: C.sub.57 H.sub.91 F.sub.6 
N.sub.13 O.sub.19, formula weight=1376.43. 
EXAMPLE 9 
##STR20## 
To a stirred solution of the amine trifluoroacetate from Example 4 (149 mg, 
0.125 mmol) in N,N-dimethylformamide (10 ml) and 1M sodium bicarbonate (2 
ml, 2 mmol) is added iodomethane (2 ml, 32.1 mmol). The reaction mixture 
is stirred for a period of 18 h. The mixture is diluted with water 
(2.times.) and chromatographed. Reverse-phase (C18) flash column 
chromatography eluting with acetonitrile/water is followed by 
lyophilization of the product-containing fractions to provide the 
trimethylammonium iodide: C.sub.54 H.sub.90 IN.sub.9 O.sub.16, formula 
weight=1248.27. 
EXAMPLE 10 
##STR21## 
In a manner similar to that described in Example 9, bisamine from Example 2 
is converted to the bistrimethylammonium iodide shown above: C.sub.57 
H.sub.99 I.sub.2 N.sub.9 O.sub.15, formula weight=1404.28. 
EXAMPLE 11 
##STR22## 
Part A: Preparation of CBZ-Glyamide 
The amine trifluoroacetate from Example 4 (215 mg, 0.180 mmol) is dissolved 
in N,N-dimethylformamide (2 ml). To this solution 1M sodium bicarbonate 
(200 .mu.l, 0.200 mmol) and pentafluorophenyl N-benzyloxycarbonylglycinate 
(106 mg, 0.270 mmol) is added. After 1 h, the reaction mixture is diluted 
with water (2.times.). Isolation by reverse-phase (C18) flash column 
chromatography eluting with acetonitrile/water gives, after lyophilization 
of the product-containing fractions, the N-CBZ glyamide: C.sub.61 H.sub.92 
N.sub.10 O.sub.19, formula weight=1269.47. 
Part B: Deprotection 
A solution of the N-CBZ glyamide from Part A in glacial acetic acid is 
hydrogenated under balloon pressure in the presence of 10% Pd/C for a 
period of 1.5 hours. The reaction mixture is filtered to remove the 
catalyst and the filtrate is lyophilized to give the glyamide as the 
acetate salt: C.sub.55 H.sub.90 N.sub.10 O.sub.19, formula weight=1195.39. 
EXAMPLE 12 
##STR23## 
Part A. Preparation of the Deacylating Enzyme 
P. acidovorans ATCC.sub.53942, maintained on Luria-Bertani medium agar 
slants was used to produce the deacylation enzyme. 
A seed culture was prepared by inoculating a 50-ml portion of Luria-Bertani 
medium in a 250 ml flask with a loopful of the bacterium and the culture 
was incubated for about 24 hours at 27.degree. C. with constant shaking. 
Cells for the deacylation were grown by inoculating 15 liters of 
Luria-Bertani medium in a stirred fermentor with 30 ml of the seed culture 
and incubating with agitation of 400 rpm and aeration at 7.5 liters/min. 
at 28.degree. C. for 20 to 24 hours. The cells were washed with 50 mM 
potassium phosphate buffer, pH 7.5 and resuspended in about 4 liters of 
the same buffer. The suspension was equilibrated to 37.degree. C. to 
obtain the deacylating enzyme. 
Part B. Deacylation 
The bisamine from Example 2.(3.5 g) is dissolved in 900 ml of distilled 
water and added slowly over a 1 hour period to 2 liters of the suspension 
of P. acidovorans cells from Part A. The resulting mixture is maintained 
at 37.degree. C. while stirring at about 300 rpm without aeration. After 
24 hours, the deacylation mixture is cleared of P. acidovorans cells by 
centrifugation and the nucleus is isolated from the supernatant by 
C18-high pressure liquid chromatography. Elution with 0-2% CH.sub.3 
CN/H.sub.2 O containing 0.1% CF.sub.3 COOH in 0.5% step gradients is 
followed by lyophilization of the nucleus-containing fractions to give the 
deacylated product shown above as the tristrifluoroacetate salt: C.sub.41 
H.sub.58 F.sub.9 N.sub.9 O.sub.20, formula weight=1167.95. 
EXAMPLE 13 
##STR24## 
Part A: Selective Protection and Reacylation of the Nucleus 
To a stirred solution of the nucleus (102 mg, 0.087 mmol) from Example 12 
and benzyl 4-nitrophenylcarbonate (47.4 mg, 0.173 mmol) in anhydrous 
N,N-dimethylformamide (3.5 ml) is added triethylamine (48.4 .mu.l, 0.347 
mmol). The reaction mixture is stirred for a period of 1 hour. 
4-(n-Pentoxyphenyl)-4'-pentafluorophenoxycarbonylbiphenyl (46 mg, 0.087 
mmol) prepared as described in Preparation of Starting Materials is added 
and stirring is continued for a period of 60 hours. The reaction mixture 
is diluted with water (3.5 ml) and the product is isolated by C18 
solid-phase extraction eluting initially with CH.sub.3 CN/H.sub.2 O and 
then CH.sub.3 OH. Concentration of the product-containing CH.sub.3 OH 
fractions as determined by analytical HPLC gives crude bis-CBZ 
pentoxyterphenyl intermediate: C.sub.75 H.sub.89 N.sub.9 O.sub.20, 
molecular weight=1436.59. 
Part B. Deprotection 
A solution of the crude bis-CBZ terphenyl intermediate from Part A in 
methanol (10 ml) and glacial acetic acid (4 ml) is hydrogenated under 
balloon pressure in the presence of 10% Pd/C for a period of 1.75 hours. 
The reaction mixture is filtered through a bed of diatomaceous earth to 
remove the catalyst, rinsing with MeOH. The filtrate is concentrated in 
vacuo. Preparative C18-HPLC of the residue, loaded in mobil phase 
containing sufficient CH.sub.3 OH to fully solubilize, eluting with 
CH.sub.3 CN/H.sub.2 O containing 0.1% CF.sub.3 COOH is followed by 
lyophilization of the product-containing fractions as determined by 
analytical HPLC to give the pentoxyterphenyl compound shown above as the 
bistrifluoroacetate salt: C.sub.63 H.sub.79 F.sub.6 N.sub.9 O.sub.20, 
formula weight=1396.37. 
EXAMPLE 14 
##STR25## 
Part A: Selective Protection and Reacylation of the Nucleus 
To a stirred solution of the nucleus (102 mg, 0.087 mmol) from Example 12 
and benzyl 4-nitrophenylcarbonate (47.4 mg, 0.173 mmol) in anhydrous 
N,N-dimethylformamide (3.5 ml) is added triethylamine (48.4 .mu.l, 0.347 
mmol). The reaction mixture is stirred for a period of 1 hour. 
Pentafluorophenyl 6-octyloxy-2-naphthoate (39 mg, 0.087 mmol) prepared as 
described in Preparation of Starting Materials is added and stirring is 
continued for a period of 60 hours. The reaction mixture is diluted with 
water (3.5 ml) and the product is isolated by C18 solid-phase extraction 
eluting initially with CH.sub.3 CN/H.sub.2 O and then CH.sub.3 OH. 
Concentration of the product-containing CH.sub.3 OH fractions as 
determined by analytical HPLC gives crude bis-CBZ octyloxynaphthoyl 
intermediate: C.sub.70 H.sub.89 N.sub.9 O.sub.20, molecular 
weight=1376.54. 
Part B. Deprotection 
A solution of the crude bis-CBZ octyloxynaphthoyl intermediate from Part A 
in methanol and glacial acetic acid (2.5:1) is hydrogenated under balloon 
pressure in the presence of 10% Pd/C for a period of 1.75 hours. The 
reaction mixture is filtered through a bed of diatomaceous earth to remove 
the catalyst, rinsing with MeOH. The filtrate is concentrated in vacuo. 
Preparative C18-HPLC of the residue, loaded in mobil phase containing 
sufficient CH.sub.3 OH to fully solubilize, eluting with CH.sub.3 
CN/H.sub.2 O containing 0.1% CF.sub.3 COOH is followed by lyophilization 
of the product-containing fractions as determined by analytical HPLC to 
give the octyloxynaphthoyl compound shown above as the bistrifluoroacetate 
salt: C.sub.58 H.sub.79 F.sub.6 N.sub.9 O.sub.20, formula weight=1336.32. 
EXAMPLE 15 
##STR26## 
Part A: Alkylation 
To a vigorously stirred solution of the amine trifluoroacetate from Example 
4 (149 mg, 0.125 mmol) and N,N-diisopropylethylamine (16.2 mg, 0.125 mmol) 
in N,N-dimethylformamide (10 ml) is added dropwise a solution of 
2-(benzyloxycarbonyl)-aminoethyl bromide (32.3 mg, 0.125 mmol) in 
N,N-dimethylformamide (5 ml). The reaction mixture is stirred until C18 
HPLC analysis with CH.sub.3 CN/H.sub.2 O indicates complete consumption of 
starting material. The mixture is diluted with water and chromatographed. 
Reverse-phase (C18) flash column chromatography eluting with CH.sub.3 
CN/H.sub.2 O (0.1% CF.sub.3 COOH) is followed by lyophilization of the 
product-containing fractions to provide the (benzyloxycarbonyl)-aminoethyl 
intermediate: C.sub.61 H.sub.94 N.sub.10 O.sub.18, formula weight=1255.49. 
Part B: Deprotection 
A solution of the CBZ protected intermediate from Part A in glacial acetic 
acid is hydrogenated under balloon pressure in the presence of 10% Pd/C 
for a period of 1.5 hours. The reaction mixture is filtered to remove the 
catalyst and the filtrate is lyophilized. Preparative C18 HPLC of the 
lyophilizate eluting with CH.sub.3 CN/H.sub.2 O (0.1% CF.sub.3 COOH) 
provides the bisamine shown above as the ditrifluoroacetate salt: C.sub.57 
H.sub.90 F.sub.6 N.sub.10 O.sub.20, formula weight=1349.40. 
EXAMPLE 16 
##STR27## 
A suspension of the bisamine ditrifluoroacetate from Example 15 (169 mg, 
0.125 mmol) in anhydrous tetrahydrofuran (10 ml) is cooled to 
0.degree.-4.degree. C. Neat BH.sub.3.S(CH.sub.3).sub.2 (107 mg, 1.41 mmol) 
is added slowly. The resulting reaction mixture is stirred at ca. 
0.degree. C. for a period of 4 h. The mixture is slowly quenched with 2N 
HCl (352 .mu.l) and diluted with water. Preparative C18 HPLC of this 
solution eluting with CH.sub.3 CN/H.sub.2 O (0.1% CF.sub.3 COOH) followed 
by lyophilization of the product-containing fractions provides the 
trisamine shown above as the tritrifluoroacetate salt: C.sub.59 H.sub.93 
F.sub.9 N.sub.10 O.sub.21, formula weight=1449.44. 
EXAMPLE 17 
##STR28## 
Part A: Preparation of CBZ Protected Amine 
To a stirred solution of the starting amine trifluoroacetate from Example 1 
(92 mg, 0.087 mmol) and benzyl 4-nitrophenyl carbonate (26.1 mg, 0.095 
mmol) in anhydrous N,N-dimethylformamide (3.5 ml) is added triethylamine 
(24.3 .mu.l, 0.174 mmol). The reaction mixture is stirred until C18-HPLC 
analysis indicates complete consumption of starting material. The reaction 
mixture is diluted with water (3.5 ml) and the product is isolated by C18 
solid-phase extraction eluting initially with CH.sub.3 CN/H.sub.2 O and 
then CH.sub.3 OH. Concentration of the product-containing CH.sub.3 OH 
fractions as determined by analytical HPLC gives crude CBZ intermediate: 
C.sub.59 H.sub.87 N.sub.9 O.sub.17, formula weight=1194.4. 
Part B: Reduction of Nitrile 
In a manner similar to that described in Example 2, the nitrile from Part A 
is reduced and the amine product is isolated as the trifluoroacetate salt: 
C.sub.61 H.sub.92 F.sub.3 N.sub.9 O.sub.19, formula weight=1312.46. 
Part C: Preparation of the CBZ-Protected Glyamide 
In a manner similar to that described in Example 11, Part A, the amine from 
Part B above is converted to a CBZ-protected glyamide derivative: C.sub.69 
H.sub.100 N.sub.10 O.sub.20, formula weight=1389.62. 
Part D: Deprotection 
In a manner similar to that described in Example 11, Part B, deprotection 
of the intermediate from Part C provides the glyamide derivative shown 
above as the diacetate salt. Purification by preparative C18-HPLC eluting 
with CH.sub.3 CN/H.sub.2 O (0.1% CF.sub.3 COOH) provides the 
ditrifluoroacetate salt: C.sub.57 H.sub.90 F.sub.6 N.sub.10 O.sub.20, 
formula weight=1349.4. 
EXAMPLE 18 
##STR29## 
Part A: Preparation of Guanidine 
To a stirred solution of the starting amine trifluoroacetate from Example 1 
(145 mg, 0.137 mmol) and 1M sodium bicarbonate (150 .mu.l, 0.150 mmol) in 
absolute methanol (5 ml) is added aminoiminomethanesulfonic acid (30 mg, 
0.242 mmol). After a period of 1.5 h, the solvent is removed in vacuo. 
Preparative reverse-phase HPLC (C18) of the residue, eluting with 
acetonitrile/water (0.1% trifluoroacetic acid), is followed by 
lyophilization of the product-containing fractions to give the guanidine 
trifluoroacetate salt: C.sub.54 H.sub.84 F.sub.3 N.sub.11 O.sub.17, 
formula weight=1216.33. 
Part B: Reduction of Nitrile 
In a manner similar to that described in Example 2, the nitrile from Part A 
is reduced and the product shown above is isolated as the 
bistrifluoroacetate salt: C.sub.56 H.sub.89 F.sub.6 N.sub.11 O.sub.19, 
formula weight=1334.39. 
The following non-limiting examples illustrate representative compositions 
containing the compounds of the invention. 
COMPOSITION EXAMPLE A 
1000 compressed tablets each containing 500 mg of the compound of Example 4 
are prepared from the following formulation: 
______________________________________ 
Compound Grams 
______________________________________ 
Compound of Example 4 500 
Starch 750 
Dibasic calcium phosphate, hydrous 
5000 
Calcium stearate 2.5 
______________________________________ 
The finely powdered ingredients are mixed well and granulated with 10 
percent starch paste. The granulation is dried and compressed into 
tablets. 
EXAMPLE B 
1000 hard gelatin capsules, each containing 500 mg of the compound are 
prepared from the following formulation: 
______________________________________ 
Compound Grams 
______________________________________ 
Compound of Example 4 
500 
Starch 250 
Lactose 750 
Talc 250 
Calcium stearate 10 
______________________________________ 
A uniform mixture of the ingredients is prepared by blending and used to 
fill two-piece hard gelatin capsules. 
EXAMPLE C 
An aerosol composition may be prepared having the following formulation: 
______________________________________ 
Per Canister 
______________________________________ 
Compound of Example 4 24 mg 
Lecithin NF Liquid Concd. 
1.2 mg 
Trichlorofluoromethane, NF 
4.026 g 
Dichlorodifluoromethane, NF 
12.15 g 
______________________________________ 
EXAMPLE D 
250 milliliters of an injectible solution may be prepared by conventional 
procedures having the following formulation: 
Dextrose 12.5 g 
Water 250 ml 
Compound of Example 4 400 mg 
The ingredients are blended and thereafter sterilized for use. 
PREATION OF STARTING MATERIALS 
Compounds where R.sup.I is dimethyltridecyl and R.sub.1 is OH, R.sub.2 is 
H, R.sub.3 is CH.sub.2 CONH.sub.2, R.sub.4 is CH.sub.3 and R.sub.6 is OH 
may be produced by cultivating Zalerion arboricola ATCC 20868 in a 
nutrient medium containing mannitol as the primary source of carbon as 
described in U.S. Pat No. 5,021,341 issued Jun. 4, 1991. 
Compounds in which R.sub.3 is H and R.sup.I is 11-methyltridecyl may be 
produced by cultivating Aspergillus sydowi in nutrient medium as descirbed 
in J. Antibiotics XL (No. 3) p.28 (1987). 
Compounds in which R.sub.3 is CH.sub.3 and R.sup.I is linoleyl may be 
produced by cultivating Aspergillus nidulans NRRL 11440 in nutrient medium 
as described in U.S. Pat No. 4,288,549 issued Sep. 8, 1981. 
Compounds in which R.sub.3 is CH.sub.2 CN may be produced by the reaction 
of a compound having a carboxamide group in the corresponding position 
with excess cyanuric chloride in an aprotic solvent. Molecular sieves may 
be employed in this reaction. After completion of the reaction, the 
sieves, if employed, are removed, and the filtrate concentrated to obtain 
the nitrile compound as more fully described in U.S. Pat No. 5,348,940 
issued Sep. 20, 1994. 
Compounds in which R.sub.3 is CH.sub.2 CH.sub.2 NH.sub.2 may be produced by 
either a chemical or catalytic reduction of the nitrile. It is 
conveniently carried out employing large molar excess of sodium 
borohydride with cobaltous chloride as more fully described in copending 
application Ser. No. 936,558 filed Sep. 3, 1992. 
Compounds in which R.sub.3 is CH.sub.2 CH.sub.2 NH.sub.2 may also be 
directly prepared from the carboxamide employing a large molar excess of 
diborane. 
Compounds in which R.sub.5 is OH or OSO.sub.3 H are described in European 
Patent Applications 0 431 350 and 0 462 531 by Fujisawa Pharmaceutical 
Co., Ltd. 
Starting materials in which R.sup.I is a different group from that of the 
natural product may be obtained by deacylating the lipophilic group of the 
natural product by subjecting the natural product in a nutrient medium to 
a deacylating enzyme until substantial deacylation occurs, said enzyme 
having first been obtained by cultivating a microorganism of the family 
Pseudomondaceae or Actinoplanaceae, as described in Experentia 34, 1670 
(1978) or U.S. Pat No. 4,293,482, recovering the deacylated cyclopeptide, 
and thereafter acylating the deacylated cyclopepetide by mixing together 
with an appropriate active ester R.sup.I COX to obtain Compound A with the 
desired acyl group. 
The active esters R.sup.I COX may be prepared by methods known to the 
skilled chemist as illustrated in the following examples. Although any 
active ester is appropriate, the compounds are illustrated with 
pentafluorophenyl esters. 
Preparation of Alkoxy Terphenyl Side Chains 
The terphenylcarboxylic acid esters may be prepared through the following 
sequence of reactions, illustrated with a specific example as follows: 
A. Preparation of pentyloxy-substituted-terphenyl-carboxylic acid 
##STR30## 
Part A: 4-(4-n-Pentyloxyphenyl)bromobenzene 
To a stirred solution of 25.5 g of 4-(4-bromophenyl)phenol (Compound (a)) 
in 400 mL of dimethylsulfoxide was added 40.9 mL of 2.5N NaOH, followed by 
12.7 mL of n-pentyl bromide, and the resulting mixture heated at 
70.degree. C. for 18 hours to obtain in the mixture, compound (b). The 
mixture was partitioned between 1000 mL of ethyl acetate and 500 mL water 
and from the organic phase after washing with water and brine, and drying 
was obtained 30.9 grams of Compound (b) as a white solid. 
.sup.1 H NMR (400 MHz, DMSO-d.sub.6) .delta. 0.93 (t, J=7.2 Hz, 3H), 1.41 
(m, 4H), 1.79 (m, 2H), 3.97 (t, J=6.6 Hz, 2H), 6.94 (d, J=8.8 Hz, 2H), 
7.39 (d, J=8.6 Hz, 2H), 7.45 (d, J=8.8 Hz, 2H), 7.51 (d, J=8.6 Hz, 2H). 
Part B: 4-(4-n-Pentyloxyphenyl)phenylboronic acid 
To a stirred suspension of 1.0 grams of Compound (b) in 20 mL anhydrous 
tetrahydrofuran at -78.degree. C. under a nitrogen atmosphere was added 
1.32 mL of 2.5M n-butyl lithium in hexanes. After 15 minutes 0.760 mL of 
tri-isopropyl borate was added and the stirring continued at -78.degree. 
C. for 15 minutes and then at 25.degree. C. for 40 minutes. The mixture 
was acidified and partitioned between ether and water to obtain the 
boronic acid compound (c) in the reaction mixture. The compound was 
recovered by washing with water and brine and drying to obtain 750 mg of 
4-(4-n-pentyloxyphenyl)phenylboronic acid as white solid with the 
following .sup.1 H NMR. 
.sup.1 H NMR (400 MHz, DMSO-d.sub.6) .delta. 0.89 (t, J=7.2 Hz, 3H), 1.38 
(m, 4H), 1.72 (m, 2H), 3.99 (t, J=6.5 Hz, 2H), 6.99 (d, J=8.8 Hz, 2H), 
7.57 (d, J=8.2 Hz, 2H), 7.60 (d, J=8.8 Hz, 2H), 7.83 (d, J=8.2 Hz, 2H). 
Part C: Pentafluorophenyl 
4"-(n-pentyloxy)-1,1':4',1"-terphenyl!-4-carboxylate 
To a stirred mixture of 1.0 g of the boronic acid and 0.0874 mL of 
4-iodobenzoic acid in 11 mL ethanol and 30 mL toluene was added 5.3 mL of 
a 2M aqueous solution of sodium carbonate followed by 204 mg 
tetrakis(triphenylphosphine)palladium and the reaction mixture heated 
under reflux (100.degree. C.) for 18 hours. Thereafter, the mixture was 
cooled, acidified and partitioned between ethyl acetate and water. The 
organic phase was washed with water and brine and dried, then filtered 
through a bed of celite to obtain after removal of solvent and 
purification with flash silica gel chromatography to obtain 
4"-(n-pentyloxy)-1,1':4',1"-terphenyl!-4-carboxylic acid. 
.sup.1 H NMR (400 MHz, DMSO-d.sub.6) .delta. 0.89 (t, 3H), 1.37 (m, 4H), 
1.72 (m, 2H), 3.98 (t, 2H), 7.01 (d, 2H). 
To a mixture of 4"-(n-pentyloxy)-1,1':4',1"-terphenyl!-4-carboxylic acid 
(10.5 mmol) and dicyclohexylcarbodiimide (10.5 mmol) in ethyl acetate at 
0.degree. C. is added pentafluorophenol (11.5 mmol). The mixture is 
stirred at 25.degree. C. for a period of 18 h, producing a precipitate. 
The mixture is filtered. The filtrate is washed with water and brine and 
dried with magnesium sulfate. The solvent is removed in vacuo to obtain 
pentafluorophenyl 4"-(n-pentyloxy)-1,1':4',1"-terphenyl!-4-carboxylate, 
C.sub.30 H.sub.23 F.sub.5 O.sub.3, M.W.=526.5. 
Preparation of Alkoxy Biphenyl Side Chains 
The biphenylcarboxylic acid esters may be obtained through the following 
sequence of reactions illustrated as follows: 
A. Preparation of Octyloxybiphenylcarboxylic acid 
##STR31## 
The acid is prepared as described in EP 462531 by Fujisawa Pharmaceutical 
Co., Ltd. 
B. Preparation of pentafluorophenyl Ester 
Pentafluorophenol (11.5 mmol) is added at 0.degree. to a mixture of 10.5 
mmol 4'-n-octyloxy1,1'-biphenyl!-4-ylcarboxylic acid and 10.5 mmol of 
dicyclohexylcarbodiimide in ethyl acetate. The mixture is stirred at 
25.degree. C. for a period of 18 hours whereupon a precipitate is formed. 
The reaction mixture is filtered, the filtrate washed with water and brine 
and dried, the solvent removed in vacuo to obtain pentafluorophenyl 
4'-n-octyloxy1,1'-biphenyl!-4-ylcarboxylate, C.sub.27 H.sub.25 F.sub.5 
O.sub.3, M.W. 492.5. 
Preparation of AminoethyloxyBiphenyl Side chains 
Preparation of 
4'-(2-4-Cyclohexylmethylpiperidin-1-yl!ethoxy)-1,1'-biphenyl!-4-ylcarbox 
ylic acid, Pentafluorophenyl Ester 
##STR32## 
Part A: Preparation of 4-Cyclohexylmethylpiperidine 
4-Benzylpiperidine is dissolved in glacial acetic acid containing PtO.sub.2 
(approximately 50 wt percent). A Paar hydrogenator is used and the 
reaction vessel is flushed with H.sub.2 and pressurized to 3 atm. The 
mixture is shaken for sufficient time to give reduction of the aromatic 
ring to the fully saturated product which is determined by the uptake of 3 
molar equivalents of H.sub.2. The black solid is filtered and the acetic 
acid removed by evaporation under reduced pressure to obtain the product 
as an acetate salt. 
Part B: Preparation of 1-(2-Hydroxyethyl)-4-cyclohexylmethylpiperidine 
The product from Part A (1.0 eq) is dissolved in dichloromethane containing 
an equimolar amount of diisopropylethyl amine. Ethylene oxide (10 eq) is 
added and the mixture is stirred until starting material is consumed. The 
desired product is obtained by removal of the solvent in vacuo followed by 
purification by column chromatography. 
Part C: Preparation of 
4'-(2-4-cyclohexylmethylpiperidine-1-yl!ethoxy)-1,1'-biphenyl!-4-ylcarbo 
xylic acid 
4'-Hydroxy-1,1'-biphenyl-4-ylcarboxylic acid methyl ester (1.0 eq) is 
dissolved in dichloromethane and triphenylphosphine (1.3 eq) and the 
hydroxyethyl compound (1.0 eq) from Part B is added. Next, diethyl 
azodicarboxylate (1.3 eq) is added and the mixture is stirred until 
starting material is consumed. The mixture is diluted with dichloromethane 
and washed with water. The organic layer is dried with MgSO.sub.4 and 
filtered. The solvent is removed in vacuo and the residue is dissolved in 
ethanol. An excess of 3N sodium hydroxide is added and the mixture stirred 
for several hours. The reaction is neutralized with 2N HCl and is 
extracted with ethyl acetate. The ethyl acetate layer is dried with 
MgSO.sub.4, filtered and the solvent vaporized under reduced pressure. The 
desired product is obtained in substantially pure form by column 
chromatography. 
Part D: Preparation of the Pentafluorophenyl Ester 
The carboxylic acid (1.0 eq) and dicyclohexylcarbodiimide (1.0 eq) are 
dissolved in ethyl acetate and the solution is cooled to 0.degree. C. 
Pentafluorophenol (1.05 eq) is added, the ice bath then is removed and the 
reaction stirred at ambient temperature for 18-24 h. An equal volume of 
ether is added, the mixture is filtered and the solvent removed in vacuo. 
The product (MW=587.64) may be obtained in a sufficiently pure form to be 
utilized for nucleus acylation. 
Preparation of 
4'-(2-4-Undecylpiperizin-1-yl!-ethoxy)1,1'-biphenyl!-4-ylcarboxylic 
acid, Pentafluorophenyl Ester 
##STR33## 
Part A: Preparation of 4-Undecylpiperazine 
Excess piperazine (5 eq) and 1-bromoundecane (1.0 eq) are dissolved in 
dichloromethane and allowed to react overnight. The mixture is extracted 
with aqueous sodium bicarbonate and the organic layer dried with sodium 
sulfate. The mixture is filtered, the solvent removed in vacuo and the 
residue purified by column chromatography. 
Part B: Preparation of 1-(2-Hydroxyethyl)-4-undecylpiperazine 
The substituted piperazine above (1.0 eq) is dissolved in n-propanol and 
bromoethanol (1.0 eq) is added along with diisopropylethyl amine (1,1 eq). 
After several hours, the solvent is removed in vacuo and the residue 
dissolved in dichloromethane. The organic layer is washed with water and 
then aqueous sodium bicarbonate. The organic layer is dried with 
MgSO.sub.4 and filtered. Removal of the solvent in vacuo is followed by 
purification by column chromatography. 
Part C: Preparation of the Carboxylic Acid 
The procedure is essentially the same as describe in Part C above except 
that the hydroxyethyl piperazine from above is substituted for the 
hydroxyethyl piperidine. 
Part D: Preparation of the Pentafluorophenyl Ester 
The procedure is identical to Part D from above except that 
piperazinyl-substituted-biphenyl carboxylic acid is used. The product 
(MW=646.75) may be obtained in a sufficiently pure form to be utilized "as 
is" in nucleus acylation. 
Preparation of Pentafluorophenyl 6-Octyloxy-2-naphthoate 
##STR34## 
To a suspension of 6-octyloxy-2-naphthoic acid (3.15 g, 10.5 mmol) and 
dicyclohexylcarbodiimide in ethyl acetate (25 ml) at 0.degree. C. was 
added pentafluorophenol (2.12 g, 11.5 mmol). The mixture was stirred at 
25.degree. C. for a period of 18 h. The precipitate was removed by 
filtration. The filtrate was washed with water (2.times.150 ml) and brine 
and dried with magnesium sulfate. Removal of the ethyl acetate in vacuo 
gave 5.4 g of pentafluorphenyl 6-octyloxy-2-naphthoate as a solid: .sup.1 
H NMR (400 MHz, CD.sub.3 OD) .delta. 0.88 (t, 3, J=6.9 Hz), 4.10 (t, 2, 
J=6.6 Hz), 7.16 (d, 1), 7.21 (d, 1), 7.80 (d, 1), 7.87 (d,1), 8.08 (dd, 
1), 8.69 (d, 1). 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 24 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: circular 
(ii) MOLECULE TYPE: peptide 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
XaaThrXaaXaaSerXaa 
15 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: circular 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
XaaThrXaaXaaThrXaa 
15 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: circular 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
XaaThrXaaXaaXaaXaa 
15 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: circular 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
XaaSerXaaXaaSerXaa 
15 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: circular 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
XaaSerXaaXaaThrXaa 
15 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: circular 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
XaaSerXaaXaaXaaXaa 
15 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: circular 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
XaaThrXaaXaaSerXaa 
15 
(2) INFORMATION FOR SEQ ID NO:8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: circular 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
XaaThrXaaXaaThrXaa 
15 
(2) INFORMATION FOR SEQ ID NO:9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: circular 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
XaaThrXaaXaaXaaXaa 
15 
(2) INFORMATION FOR SEQ ID NO:10: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: circular 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
XaaSerXaaXaaSerXaa 
15 
(2) INFORMATION FOR SEQ ID NO:11: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: circular 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
XaaSerXaaXaaThrXaa 
15 
(2) INFORMATION FOR SEQ ID NO:12: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: circular 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
XaaSerXaaXaaXaaXaa 
15 
(2) INFORMATION FOR SEQ ID NO:13: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: circular 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: 
XaaThrXaaXaaSerXaa 
15 
(2) INFORMATION FOR SEQ ID NO:14: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: circular 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: 
XaaThrXaaXaaThrXaa 
15 
(2) INFORMATION FOR SEQ ID NO:15: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: circular 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: 
XaaThrXaaXaaXaaXaa 
15 
(2) INFORMATION FOR SEQ ID NO:16: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: circular 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: 
XaaSerXaaXaaSerXaa 
15 
(2) INFORMATION FOR SEQ ID NO:17: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: circular 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: 
XaaSerXaaXaaThrXaa 
15 
(2) INFORMATION FOR SEQ ID NO:18: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: circular 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: 
XaaSerXaaXaaXaaXaa 
15 
(2) INFORMATION FOR SEQ ID NO:19: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: circular 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: 
XaaThrXaaXaaSerXaa 
15 
(2) INFORMATION FOR SEQ ID NO:20: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: circular 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: 
XaaThrXaaXaaThrXaa 
15 
(2) INFORMATION FOR SEQ ID NO:21: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: circular 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: 
XaaThrXaaXaaXaaXaa 
15 
(2) INFORMATION FOR SEQ ID NO:22: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: circular 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: 
XaaSerXaaXaaSerXaa 
15 
(2) INFORMATION FOR SEQ ID NO:23: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: circular 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: 
XaaSerXaaXaaThrXaa 
15 
(2) INFORMATION FOR SEQ ID NO:24: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: circular 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: 
XaaSerXaaXaaXaaXaa 
15 
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