Note: Descriptions are shown in the official language in which they were submitted.
WO 96/08266 219 7 2 0 9 pCT~S95/11520
-I-
TITLE OF THE INVENTION
AZA CYCLOHEXAPEPTIDE COMPOUNDS
I~ACKOI~OUND OF THE INVENTION
s The present invention is directed to aza cyclohexapeptide
compounds which may be 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
io 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 susceptibility to the infection of immuno-
compromised patients, such as those suffering from AIDS.
is
SUMMARY OF THE INVENTION
The compound of the present invention, Compound I
(Seq. ID Nos. I-6) is characterized in having a nitrogen attached to the
cyclohexapeptide ring at the 5-carbon of the 4-hydroxyornithine
2o component (hereinafter "C-5-orri') as well as a hydroxy group attached
to the 4-position of the 5-membered ring of the proline component.
The compound may be represented by the formula (I)
30
W O 96108266 219 7 2 0 9 PCT~S95111520
-2-
R~~ _R~
HO O
E~NH O
~1/ NH-C-RI
s N O
R, ~n , ,.,
HO NH ~~ ~OH
O H N
R2 N ~~
. I ~ OH O OH
wherein
is R1 is CH2CH2NH2, CH2CN or CH2CONH2;
R2 is H or OH;
RI is C9-C21 allcyl or C9-C21 alkenyl;
Rn is H, CI-Cq. alkyl, C3-C4 alkenyl, (CH2)2-q.OH,
(CH2)2_qNRNRV, CO(CH2)1-4NH2;
2o RIII is H, CI-C4 alkyl, C3-C4 alkenyl, (CH2)2-40H,
(CH2)2-4~IVRV;
R~ is H or C1-C alkyl;
RV is H or Cl-Cq. alkyl; or a
2s pharmaceutically acceptable acid addition salt and/or hydrate thereof.
This invention also relates to pharmaceutical compositions
containing said compounds and methods of use as antifungal agents and
for the treatment and control of Pneumocystis carinii infections.
Particularly preferred is the compound wherein R 1 is
so CH2CH2NH2; R2 is OH; RI is 9,11-dimethyltridecyl; RB is
CH2CH2NH2; and R~ is hydrogen.
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.
WO 96/08266 Z t 9 T 2 0 9 pCTIUS95/I1520
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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,l-pentene-5-yl and the like.
i o The term alkoxy refers to straight or branched chain
oxyalkyI groups such as, e.g., methoxy, ethoxy, butoxy, heptoxy,
dodecyloxy, and the like.
Pharmaceutically acceptable salts suitable as acid addition
salts include salts of inorganic acid such as hydrochloric, hydrobromic,
i5 sulfuric, nitric, phosphonic and perchloric acids; as well as salts of
organic acids such as tartaric, citric, acetic, succinic, malefic, fumaric
and oxalic acids; as well as other substantially non-toxic acid addition
salts which are known to those skilled in the art.
Representative nuclei for the aza derivatives of the present
2o invention (Compound I) and the sequence m for these compounds may
be seen in the following table. Since the peptide nuclei would be the
same irrespective of substituents RI, RB or Rte, and since the sequence
identification, number is assigned for the nuclear variations, the amines
and salts have the same sequence ID's.
Aza Compound R1 R2 SEQ )D NO.
I-1 CH2CHZNH2 OH 1
I-2 CH2CN OH 2
I-3 CH2CONH2 OH 3
- s o I-4 CH2CH2J~2 H 4
I-5 CH2CN H 5
I-6 CH2CONH2 H 6
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The compounds are soluble in lower alcohols, and polar
aprotic solvents such as dimethylformamide (DMF), dimethyl sulfoxide
(DMSO) and pyridine. They are insoluble in solvents such as diethyl ,
ether and acetonitrile.
s 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
io 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, A. niger. They are
also useful for the treatment and/or prevention of Pneumocystis carinii
pneumonia to which immune-compromised patients are especially
is susceptible as hereinafter described.
The compounds of the present invention may be prepared
from the compound having the formula
OH OH
HO O
ao NH 0
O ~ NH-C-RI
II N o
. H2NCH2C~0 HN CH3
HO NH O~pH
25 O H N
R2 N
OH O OH
(A)
(Seq ID Nos. 1-6)
by a series of reactions in which the oxygen atom at the "C-5-orn" (or
hemiaminal) position is ultimately replaced by nitrogen. The starring
material (Compound A) for the preparation may be a natural product in
which R1 is 9,11-dimethyltridecyl and may be produced by cultivating
CA 02197209 2005-12-13
WO 96/08266 PCT/US95/11520
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Zalerion arboricola ATCC 20868 in a nutrient medium enriched in
mannitol as the primary source of carbon as described in U.S. Patent
No. 5,306,708, issued April 26, 1994.
The sequence IDs of the starting materials are seen in the
following table:
Starting Material
Compound R 1 R2 SEQ ID NO.
A-1 ~2~2NH2 OH 7
. A-2 CH2CN OH 8
A-3 CH2CONH2 OH 9
A-4 CH2CH2NH2 H 10
A-5 CH2CN H 11
A-6 CH2CONH2 H 12
The sequence of reactions is shown in the following
scheme.
25
X197209
WO 96!08266 PCTIUS95111520
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OH OH
H* O
NH-C-RI
HN CH3 H2NCH2CH2SH
OOH H+
Step A
/, OH
io O
HO
(A)
(Seq ID Nos 7-12)
H2NCH2CH2S OH
O 2.2 [o]
.,,~~N~ Step B
(B)
(Seq ID Nos 13-18)
* The position is the "C-5-orn" or the
hemiaminal position.
30
WO 96108266 ~ ~ PCTfITS95111520
_.7 _
HZN OH
H_Z,Pd/C ~~N~_
N OH S p (I)
O 3 (Seq ID Nos 1-6)
N
(D)
LiN3 (Seq ID Nos 1-6)
1o Steo C
H2N--
S02 OFi
O
-NH
,~~~ Ru
is (~) \
(Seq ID Nos 13-18) Rn~H
normal
or epi Rii
R~° \N OH
. ",~"~ N
normal or epi
(I)
(Seq ID Nos 1-6)
In Step A, the starting material Compound A (Seq ID Nos.
7-12), alkylthiol or arylthiol and acid are caused to react in an aprotic
ao solvent under anhydrous conditions for time sufficient for reaction to
take place with the formation of Compound B (Seq ID Nos. 13-18), seen
in the following table. Aminoethylthiol has been found to be useful for
this step.
WO 96108266 ~ 19 7 2 0 9 p~~g95111520
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Sulfur Intermediate
Compound R 1 R2 SEQ ID IVO.
B-1 CH2CH2NH2 OH 13
B-2 CHZCN OH 14
s B-3 CH2CONH2 OH 15
B-4 CH2~2~H2 H I6
B-5 CH2CN H 17
B-6 CH2CONH2 H 18
to For Step A, suitable acids include strong organic acid and
mineral acids. Examples of strong organic acids are camphorsulfonic
acid, p-toluenesulfonic acid and methanesulfonic acid. Mineral acids
include hydrochloric acid and hydrobromic acid. Camphorsulfonic acid
is preferred.
is Suitable solvents include DMF, DMSO, 1-methyl-2-
pyrrolidinone and hexamethyl phosphoric triamide (HMPA). DMF or
DMSO is preferred.
'The reaction is generally carned out at ambient
temperature for from 1 to about 10 days.
20 ' 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
a5 (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).
so Appropriate columns for HPLC are commercially available
columns sold under trade mark names or trade names such as
"ZORBAX" (DuPont), "DeltaPak" (Waters), Bio-Rad (Bio-Rad),
"LICHROPREP" RP18 (E. Merck). The specific columns are identified
in the working examples.
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W0 96108266 PGT/US95/11520
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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;' (KHSOS~KHS04~K2S04 2:1:1, Aldrich
Chemicals) metachloroperoxybenzoic acid, and peroxyacetic acid. The
s 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 )Ds of the sulfones are as follows:
Sulfone
io Compound Rl R2 SEQ ID No.
C-1 CH2CHZNH2 OH 13
C-2 CH2CN OH 14
C-3 CH2CONH2 OH 15
C-4 CH2CH2NH2 H 16
i s C-5 CH2CN H 17
C-6 CH2CONH2 H 18
The oxidation of the thioether (Compound B) to the sulfone
(Compound C) is carried out with about two molar amounts of the
20 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 the aza
compounds but the sulfone is preferred. A slight excess over the two
molar amount of the oxidizing agent is employed.
2s The reaction is carned 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
- so 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
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WO 96/08266 PCTIUS95111520
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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
s 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.
Compound C may be converted to a compound having a
nitrogen directly attached to the "C-5-orri'. As seen in the flow
io diagram, reaction of Compound C with an alkali metal azide produces
an azide at that position (Compound D) while reaction with an amine
compound (ammonia or amine) produces an amino group at the "C-5-
orn" position, (Compound I). Compound D is an important
intermediate for most of the compounds of the present invention.
is Although Compound D has nitrogen at "C-5-orn", since it is not a
product, separate sequence ID Nos. are assigned for Compound D.
Sequence ID Nos. for Compound D are found in the following table.
Azide
2o Compound Rl R2 SEQ ID No.
D-1 CH2CH2NH2 OH 19
D-2 CH2CN OH 20
D-3 CH2CONH2 OH 21
D-4 CH2CH2NH2 H 22
z s D-5 CH2CN H 23
D-6 CH2CONH2 _ H 24
The azide may be obtained by adding alkali metal azide
while stirring at ambient temperature to a solution of the sulfone
ao (Compound C, Seq. ID Nos. 13-18) in an aprotic solvent for time
sufficient to complete the reaction with the formation of the azide as
determined by HPLC analysis. The reaction mixture then may be
diluted with aqueous acid such as trifluoroacetic acid and then
chromatographed to separate the desired azide (Compound D) from the
2191209
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reaction mixture. Reverse-phase (C18) flash column chromatography
using 10-25 percent acetonitrile/water (0.1 % TFA) in 5 percent step
gradients is suitable for this procedure.
The azide (Compound D) may then be reduced to a
s compound having a free amino group which is among the products
(Compound I, Seq ID Nos. 1-6) of the present invention.
The reduction may be carned out by mixing the azide
compound (Compound I) with Pd/C in a solvent such as glacial acetic
acid and hydrogenating under balloon pressure for 10 to 20 hours. The
io product then may be recovered by first removing the catalyst by
filtration and the filtrate lyophilized to obtain the amine compound
(Seq ID Nos. 1-6) in which the amine is a primary amine.
The amine thus obtained may be converted into a
substituted amine as subsequently described.
is Compound I in which -NRBR~ is represented by
-NHCH2CH2NH2 or generically by -NH(CH2)2~.NRNRV may be
prepared from the sulfone by a method in which a diamine H~1(CH2)2-
qNKNRV is caused to react with the sulfone (Compound C, Seq 1D
Nos. 13-18).
2o The reaction is carried out in an aprotic solvent such as
those previously named and at ambient temperature. About tenfold
molar excess of the amine compound is employed. The reaction may be
carried out over one to several hours.
In carrying out the reaction, the appropriate amine is added
2s to a solution of the sulfone in anhydrous aprotic solvent and the reaction
mixture stirred at ambient temperature to obtain Compound I (Seq ID
Nos. 1-6) in which the substituent at "C-5-orn" is -NRnR~. The
desired compound may then be recovered ~by diluting with aqueous
trifluoroacetic acid and then chromatographing. Reverse phase (C18)
- ao flash column chromatography eluting with 10 to 25% acetonitrile/water
(0.1 % TFA) in 5 percent step gradients is suitable. The appropriate
fractions may be lyophilized to recover the product as a trifluoroacetate
salt.
VI'O 96/08266 219 7 2 0 9 PCTlUS95I11520
-12-
The trifluoroacetate salt may be converted by dissolving the
salt in water and passing through a Bio-Rad AG2-X8(Cl-) polyprep
column and recovering the product as the hydrochloride salt.
The amines, prepared as above and having a primary amino
s group -NH2 described, may then be alkylated by conventional means to
obtain a substituted amino group. Briefly, alkylation may be carned
out by causing an appropriately substituted alkyl halide to react with the
amine (Compound I, NRBR~=NH2; Sequence )D Nos. 1-6) in an
aprotic solvent in the presence of a base to obtain the monosubstituted
to amine (Compound I, NRnR~=NHRn wherein Rn is CI-Cq alkyl,
C3-C4 alkenyl, (CH2)2-q.OH, and (CH2)2-qNRNRV). The latter may
be recovered from the reaction mixture by conventional procedures.
The amines, prepared as above described and having a
primary amino group -NH2, may be acylated by conventional means to
is obtain an acylated amino group. The acyl group contemplated is
CO(CH2)I-q.NH2. Since this is a primary amino group, the amino of
the acylating acid is protected such as with a benzyloxycarbonyl group
before the acylation is carried out. An activated ester such as the
pentafluorophenyl ester is preferably used. The acylation may be
2o carried out in an aprotic solvent in the presence of base such as
diisopropylethylamine at ambient temperature for from one to several
hours to obtain the acylation product. The product may be recovered
by diluting the reaction mixture with methanol and purifying by HPLC.
The protecting group may be removed by conventional hydrogenolysis.
2s (Compound I, -NRHR~=-NHCO(CH2)I~NH2).
The amine compounds in which the amino group at the
hemiaminal position is totally substituted, i.e. when neither RB nor RHI
n
3o R pat ~H
~~NH~
W096108266 , ' , PCT/US95111520
~' -13_
hydrogen, are preferably prepared by reacting the sulfone (Compound
B, Seq ID Nos. 19-24) with an appropriately substituted amine
RIIR~NH. The reaction may be carned out by adding the amine to a
stirred solution of the sulfone for time sufficient for reaction to take
s place. The product may be recovered by purifying by preparative
HPLC and lyophilizing the appropriate components.
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 salt. The salt thus
io 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 active against
many fungi and particularly against Candida species. The antifungal
is 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 are solubilized in
20 100% dimethyl sulfoxide (DMSO) at an initial concentration of 5
mg/ml. Once dissolved, the drug stock is brought to a concentration of
512 pg/ml by dilution in water such that the final DMSO concentration
was about 10 percent. The solution is then dispensed via a multichannel
pipetter into the first column of a 96-well plate (each well containing
2s 0.075 mI of YNBD), resulting in a drugconcentration of 256 pg/ml.
Compounds in the first column are diluted two-fold across the rows
yielding final drug concentration ranging from 256 pg/ml to 0.12
pg/ml.
Four-hour broth cultures of organisms to be tested are
ao adjusted using a spectrophotometer at 600 nm to equal a 0.5 McFarland
Standard. This suspension is diluted 1:100 in YNBD to yield a cell
concentration of 1-5 x 104 colony forming units (CFU)/m1. Aliquots of
the suspension (0.075 ml) are inoculated into each well of the microtiter
plate resulting in a final cell inoculum of 5-25 x 103. CFU/ml and final
WO 96/08266 ~ ~ PCTIU895111520
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drug concentrations ranging from 128 pg/ml to 0.06 pg/ml. Each assay
includes one row for drug-free control wells and one row for cell-free
control wells.
After 24 hours of incubation, the microtiter plates are
s shaken gently on a shaker to resuspend the cells. The MIC-2000 _.
inoculator is 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
are incubated for 24 hours at 35°C and then read for minimum
io fungicidal concentration (MFC). MFC is defined as the lowest
concentration of drug showing no growth or less than 4 colonies per
spot.
The in vivo effectiveness of the compounds against fungi
may be demonstrated in the following in vitro assay.
i5 Growth from an overnight SDA culture of C. albicans MY
1055 is suspended in sterile saline and the cell concentration determined
by hemacytometer count and the cell suspension adjusted to 3.75 x 105
cells/ml. Then 0.2 milliliter of this suspension is administered LV. in
the tail vein of mice so that the final inoculum is 7.5 x 104 cells/mouse.
2o The assay then is carried out by administering aqueous
solutions of Compound I at various concentrations intraperitoneally
(LP.), twice daily (b.i.d.) for four consecutive days to 18 to 20 gram
female DBA(Z mice, which previously had been infected with C.
albicans in the manner described above. Distilled water is administered
2s LP. to C. albicans challenged mice as controls. After seven days, the
mice are sacrificed by carbon dioxide gas, paired kidneys are removed
aseptically and placed in sterile polyethylene bags containing 5
milliliters of sterile saline. The kidneys aie homogenized in the bags,
serially diluted in sterile saline and aliquots spread on the surface of
3o SDA plates. The plates are incubated at 35°C for 48 hours and yeast
colonies are enumerated for determination of colony forming units
(CFU) per gram of kidneys.
The compounds of the present invention may also be useful
for inhibiting or alleviating Pneumocystis carinii infections in immune-
W O 96108266 - - ~ ~, 9 7 2 0 9 PCT/US95/I1520
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. compromised patients. The efficacy of the compounds of the present
invention for therapeutic or anti-infective purposes may be
demonstrated in studies on immunosuppressed rats.
Sprague-Dawley rats (weighing approximately 250 grams)
are immunosuppressed 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, rats are sacrificed to confirm the presence of
Pneumocystis carinii pneumonia (PCP). Five rats (weighing
to approximately 150 grams) are injected twice daily for four days
subcutaneously (sc) with Compound in 0.25 ml of vehicle (distilled
water). A vehicle control is also carried out. All animals continue to
receive dexarnethasone in the drinking water and low protein diet
during the treatment period. At the completion of the treaiment, all
is animals were sacrificed, the lungs are removed and processed, and the
extent of disease determined by microscopic analysis of stained slides.
The prevention or reduction of cysts are seen in slides of lungs of
treated rats when compared with the number of cysts in lungs of
untreated or solvent controls.
2o The outstanding properties are most effectively utilized
when the compound is fommlated into novel pharmaceutical
compositions with a pharmaceutically acceptable Garner according to
the conventional pharmaceutical compounding techniques.
The novel compositions contain at least. a therapeutic
25 antifungal or antipneumocystis amount of the active compound.
Generally, the composition contains at least 1% by weight of Compound
I. Concentrate compositions suitable for dilutions prior to use may
contain 90% or more by weight. The coriapositions include
compositions suitable for oral, topical, parenteral (including
3o intraperitoneal, subcutaneous, intramuscular, and intravenous), nasal,
and suppository administration, or insufflation. The compositions may
be prepacked by intimately mixing Compound I with the components
suitable for the medium desired.
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Compositions formulated for oral administration may be a
liquid composition or a solid composition. 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
s capsules and tablets, with solid carriers such as starches, sugars, kaolin,
ethyl cellulose, calcium and sodium carbonate, calcium phosphate,
kaolin, talc, lactose, generally with 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
to advantageous oral dosage form. It is especially advantageous to
formulate the compositions in unit dosage form (as hereinafter defined)
for ease of administraion and uniformity of dosage. Compositions in
unit dosage form constitute an aspect of the present invention.
Compositions may be formulated for injection and may
is 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
ao 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
2s 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 uliits, each unit containing a
predetermined quantity of active ingredient calculated to produce the
so desired therapeutic effect in association with the pharmaceutical Garner
Examples of such unit dosage forms are tablets, capsules, pills, powder
packets, wafers, measured units in ampoules 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.
WO 96!08266 ~~ ~ ~ PCTlIJ595J11520
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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
s 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 nebulisers. The preferred delivery system for inhalation is a
io metered dose inhalation (MDI) aerosol, which may be formulated as a
suspension or solution of Compound I in suitable propellants, such as
fluorocarbons or hydrocarbons.
Although the compounds of the present indention may be
employed as tablets, capsules, topical compositions, insufflation
is powders, suppositories and the like, the solubility of the compounds of
the present inventibn 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
20 ' to be construed as limiting.
2s
';.... . '._ '. . . .
w0 96108266 219 7 2 0 9 PCT~S95111520
-18-
EXAMPLE I
H2N OH
O
N
HO..,. ''~H H
N
HZNOC ~O
~--~ ~~ H
HO NH
1o O H H, N
HO ., N~., ~CF3CO2H
~H O ~OH
(Seq. ID No. 3)
1 s HO
Part A Preparation of Aminoethylthioether Intermediate lSEO ID
No151
20 ~ A solution of 500 milligrams (0.47 mmol) of I-[4,5-
dihydroxy-N2-( 10,12-dimethyl-1-oxotetradecyl)ornithine]-5-(3-
hydroxyglutamine)-6-(4-hydroxyproline)echinocandin B, 5.34 grams
(47 mmol) of 2-aminoethanethiol hydrochloride and 109 milligrams
(0.47 mmol) of (IS)-(+)-10-camphorsulfonic acid in 40 milliliters of
as hydrous N,N-dimethylformamide is stirred at 25°C for 2-6 days or a
period sufficient effect dissapearance of the starting material. The
reaction is diluted with 40 milliliters of water and flash
chromatographed on "LICHROPREP" (E: Merck) RP 18 (40-63 11m, 15
grams) packed in 10 percent acetonitrile/water. The column is eluted
so with 10 to 40 percent acetonitrile/water collecting two 120 milliliter
fractions at each gradient step. The appropriate fractions, as
determined by analytical HPLC (Zorbax Rx C18, 40%
acetonitrile/water/0.1 % trifluoroacetic acid, 210 nrn) are concentrated
and lyophilized. The residue is further purified by preparative HPLC
(ZORBAX C18, 40% acetonitriIe/water/0.1%TFA, 210 nm) to obtain
WO 96/08266 ' , , ~ ~ 9 7 2 0 9 pCT~S95/11520
;:. ,y"
-19-
the desired compound as a trifluroacetate salt with a molecular weight
of 1238.
Part B Oxidation to Sulfone (SEO ID No. 15)
s The mizture of thioethers obtained as described above
(0.358 mmol) is dissolved in 15 mL of 1:1 acetonitriIe/water and
"OXONE" (1.06 mmol equivalents of potassium hydrogen persulfate) is
added. After about an hour, the solution is diluted with an equal
volume of water and rapidly chromatographed using reverse phase C18
to flash chromatography eluting with 35-45% acetonitrile/water containing
0.1 % TFA in 2% step gradients. The product containing fractions are
lyophilized to give the product with a molecular weight of 1270.
Part C I7~i~placement of Sulfones with Azide lSEO ID No. 21)
is -The mixtureof sulfones-(0.257 mmol), prepared as
described above, is dissolved in 10 mL of anhydrous DMF. Lithium
azide (0.257 mmol) is added as a solid and the mixture is stirred for
about a 4-24 hours. The mixture is purified by reverse phase C18 flash
chromatography eluting with 30-65% acetonitrile/water in 5% step
2o gradients. The appropriate fractions, as determined by reverse phase
HPLC (RP:~.8, 40% acetonitrile/water/0.1 % TFA, 210 nm) are pooled,
frozen and lyophilized to give the crude product. Further purificatiorx
by preparative reverse phase HPLC (C18, 40-45%
acetonitrile/water/0.1 % TFA, 210 nm) yields the desired purified
2s compound with a molecular weight of 1090.
Part D Deduction of Azide to Amine lSEO ID No. 3)
A mixture of the azido compound (0.126 mmol) (obtained
as described above) and 10% Pd on charcoal (100-150 mg) is suspended
so in glacial acetic acid (10 mL). The reaction vessel is flushed first with
nitrogen then 'with hydrogen. One atmosphere pressure of hydrogen
gas is maintained for a period of time sufficient to give complete
reduction to the amine product, typically 2 to 24 h. The catalyst is
removed by filtration and the filtrate is lyophilized to obtain the crude
VI'O 96108266 PCTlUS95111520
2197209
-20-
amine. Further purification may be accomplished by preparative
reverse phase chromatography (C18, 35-41 % acetonitrile/water/0.1 %
TFA in 3% step gradients, 210 nm). The product-containing fractions
are lyophilized to give the purified compound with a molecular weight
of 1178.
EXAMPLE 2
H2N~H
N OH
O
N
.~ H
HO~~~~ ~ H
N
H2NOC ~O
HO NH
HO N
O H
~~H O ~°OH
ao rOH
~2HCI
(Seq. ID No. 3)
The sulfone mixture (0.945 mmol), obtained as described
in Part B Example l, is dissolved in 20 mL of anhydrous DMF and
ethylenediamine (9.45 mmol) is added. The mixture is stirred for about
1-12 hours or until analytical HPLC analysis (RP-18, 40%
acetonitrile/water/0.1 % TFA, 210 nm) shows complete disappearance of
so the starting sulfone. The mixture is separated by reverse phase (C18)
flash column chromatography eluting with 10-40%
acetonitrile/water/0.1%TFA in 5% step gradients. The appropriate
fractions are pooled, frozen and lyophilized to give the desired product
with the a-C-5 orn configuration and its (3-C-5 orn epimer. The
WO 96!08266 , ~ ~ ~ PCT/US95/11520
- 21~-
trifluoroacetate salt thus obtained is dissolved in a small volume of
deionized water and passed throu a Bio-Rad AG2-X8 (C!-) polyprep
column washing with additional water. The product-containing eluate is
lyophilized to give the desired product as the dihydrochloride salt with a
s molecular weight of 1180.
EXAMPLE 3
CH3(CH2)3NH OH
O
N
.,~ H
HO~~~~ H
N
H2NOC
c ~~H
O H H, N
HO N~
,'H ~.~~OH
O
OH
ao ~ ~ ~CF3C02H
(Seq. ID No. 3)
2s The sulfone mixture (0.945 mmol), obtained as described
in Part B Example 1, is dissolved in 20 mL of anhydrous DMF and n-
butylamine (9.45 mmol) is added. The mixture is stirred for about 1-12
hours or until analytical HPLC analysis (RP-18, 40%
acetonitrile/water/0.1 % TFA, 210 nm) shows complete disappearance of
~ 30 ~e starting sulfone. The mixture is separated by reverse phase (C18)
flash column chromatography eluting with 10-50%
acetonitriIe/water/0.1 %TFA in 5% step gradients. The appropriate
fractions are pooled, frozen and lyophilized to give the desired product
with the a-C-5 orn configuration and its (3-C-5 orn epimer. The
WO 96108266 ~ ~ .~ ~ PCT/US95I11520
-22-
products, isolated as their trifluoroacetate salts, have a molecular weight
of 1234.
EXAMPLE 4
s
HO
io N
HO~~,.~H
H
HZNOC
~~H
H H N
is N~,
H O 'OH
~CF3C02H
20 (Seq. ID No. 3)
The sulfone mixture (0.945 mmol), obtained as described
in Part B Example 1, is dissolved in 20 mL of anhydrous DMF and
25 ethanolamine (9.45 mmol) is added. The mixture is stirred for about 1-
12 hours or until analytical HPLC analysis (RP-18, 40%
acetonitrile/water/0.1 % TFA, 210 nm) shows complete disappearance of
the starting sulfone. The mixture is separated by reverse phase (C18)
flash column chromatography eluting with 10-40%
so acetonitrile/water/0.1%TFA in 5% step-gradients. The appropriate
fractions are pooled, frozen and lyophilized to give the desired product
with the a-C-5 om configuration and a molecular weight of 1222.
R'O 96108266 ~ ~ ~ PCT1LTS95/II520
- 2~-
EXAMPLE 5
HzN LH
N OH
O
N
HO..,. . .,,~H H
N
H2N '-'~~~H
Io HO NH
O H H N
HO N
,~H ~.~OH
OH
~~ /~
~2HCI
(Seq. ID No. 1)
20 ~ Preparation of Intermediate Nitrile Compound (SEO ID
No~21
A solution of 250 milligrams (0.23 mmol) of 1-[4,5-
dihydroxy-N2-(10,12-dimethyl-I -oxotetradecyl)ornithine]-5-(3-
hydroxyglutamine)-6-(4-hydroxyproline)echinocandin B was prepared
in 3.0 mL of N,N-dimethylformamide. In one portion 64 milligrams of
2s cy~uric chloride was added and the mixture was stirred for 5.5
minutes and immediately quenched with 0.55 mL of 2M sodium acetate
solution. The mixture was purified by preparative HPLC ("ZORBAX"
C8, acetonitrile/water/O.I% TFA, 210 and 277 nm). The appropriate
product-containing fractions as determined by analytical HPLC
so ("ZORBAX" C8, 45% water/acetonitrile/0.1% TFA, 1.5 mL/min, 210
and 277 nm) were pooled and lyophilized to give 45 mg of desired
nitrile compound >98% pure as a white solid. 1H NMR (400 MHz,
CD30D) 87.12 (d, 1 H), 6.74 (d, I H), 5.31 (m, 1 H), 2.83 (dd, 1 H), 2.72
,.. ;
w0 96108266 2 ~ l PCT/US95111520
-24-
(dd, 1H), 2.42 (m, 1H), 1.21 (d, 3H); FAB-MS (Li), m/e 1054
(M+H+Li).
Part B ~duction of Nitrite to Amine (SEO D7 No. 1
s To a solution of 38.5 mg (0.036 mmol) of the nitrite
(prepared above in Part A) in 2.0 mL of methanol, was added 32 mg
(0.25 mmol) of CoCl2~6H20. Next, 46 mg (34 equivalents) of sodium
borohydride was added in several portions. The micture was stirred
under a nitrogen atmosphere at room temperature for 3 hours. The
io reaction mixture was purified by preparative HPLC ("ZORBAX" C8,
45% water/acetonitrile, 210 and 277 nm). The appropriate fractions as
determined by analytical HPLC ("ZORBAX" C8, 45%
water/acetonitrile/0.1 % TFA, 1.5 mL/min, 210 and 277 nm) were
pooled and lyophilized to give 13 mg of the trifluoroacetate salt of the
is desired amine (>98% pure) as a white solid. ~FAB-MS (Li), m/e 1058
(M+H+Li).
Part C ggr paration of the Aminoethylthioether (SEO ID No. 13)
A solution of the amine prepared in Part B above (0.047
ao mmol), 2-aminoethanethiol hydrochloride (4.7 mmol) and (1S)-(+)-10-
camphorsulfonic acid (0.047 mmol) in 4 milliliters of anhydrous N,N-
dimethylformamide is stirred at 25°C for 2-6 days or a period
sufficient
effect dissapearance of the starting material. The reaction is diluted
with 4 milliliters of water and flash chromatographed on
2s "LICHROPREP" (E. Merck) RP 18 (40-63 pm, 1.5 grams) packed in
percent acetonitrile/water. The column is eluted with 10 to 40
percent acetonitrile/water collecting two 12 milliliter fractions at each
gradient step. The appropriate fractions, as determined by analytical
HPLC (Zorbax Rx C18, 30% acetonitrile/water/0.1% trifluoroacetic
3o acid, 210 nm) are concentrated and lyophilized. The residue is further
purified by preparative HPLC (ZORBAX C18, 30%
acetonitrile/water/0.1 %TFA, 210 nm) to obtain the desired isomeric
compounds as ditrifluoroacetate salts both having molecular weights of
1338.
WO 96108266 ~ ~ 9 7 2 0 9 p~.~7S95/11520
Part D Oxidation to Sulfone lSEO ID No 13)
_ . The mixture of thioethers obtained as described above
(0.036 mmol) is dissolved in 1.5 mL of 1:1 acetonitrile/water and
s "OXONE" (0.106 mmol equivalents of potassium hydrogen persulfate)
is added. After about an hour, the solution is diluted with an equal
volume of water and rapidly chromatographed using reverse phase C18
flash chromatography eluting with 35-45% acetonitrile/water containing
0.1 % TFA in 2% step gradients. The product-containing fractions are
to lyophilized to give the product with a molecular weight of 1370.
Part E D~snlacement of Sulfone with Ethylenediami_ne (SEO ID
No-1 )
The sulfone mixture (0.094 mmoI), obtained as described
is in Part D above, is dissolved in 2.0 mL of anhydrous DMF and
ethylenediamine (0.94 mmol) is added. The mixture is stirred for about
1-12 hours or until analytical HPLC analysis (RP-18, 30%
acetonitrile/water/0.1 % TFA, 210 nm) shows complete disappearance of
the starting sulfone. The mixture is separated by reverse phase (C18)
2o flash column chromatography eluting with 10-30%
acetonitrile/water/0.1%TFA in 5% step gradients. The appropriate
fractions are pooled, frozen and lyophilized to give the desired product
with the a-C-5 orn configuration and its [3-C-5 orn epimer. The
trifluoroacetate salt thus obtained is dissolved in a small volume of
2s deionized water and passed throu a Bio-Rad AG2-X8 (CI-) polyprep
column washing with additional water. The product-containing eluate is
lyophilized to give the desired product as the dihydrochloride salt with a
molecular weight of 1166.
~ ~ 9 T 2 ~ 9 rc~rnJS9sms2o
WO 96108266
-26-
RX_AMPLE 6
O
H2N
s
H
HO~~~~ H
N
H2NOC ~O
"H
HO NN
N
O H
H O ~OH
~ OH
~HOAc
(Seq. ID No. 3)
2o Part A Acylation with Protected Gl cy ine
The amino compound (0.10 mmol), obtained in Example 1
Part D, is dissolved in 1 mL of anhydrous N,N-dimethylformamide
under a nitrogen atmosphere. Diisopropylethylamine (0.11 mrnol) and
N-Carbobenzyloxyglycine pentafluorophenyl ester (0.15 mmol) are
2s added and the reaction is stirred at room temperature for 1-12 hours or
until analysis by analytical HPLC ("ZORBAX" C18, 50%
acetonitrile/water/0.1 % TFA, 210 and 277 nm) indicates the reaction is
complete. The mixture is diluted with 1 mL of methanol and purified
by preparative HPLC ("ZORBAX" C18, 70% water/acetonitrile/0.1%
3o TFA to 50% water/acetonitrile/0.1% TFA, 2 step gradient, 210 and 277
nm) to give the desired glycylated compound with a molecular weight
of 1255.
R'O 96!08266 - ~ ' ~ 219 7 2 0 9 pCTlUS95l11520
_27-
Part B Hydrogenolysis of Carbobenzyloxy Protected Glycine
Compound
The pure carbobenzyloxy-protected compound (0.075
mmol) obtained as in Part A above, is dissolved in a mixture of 3 mL of
methanol, 1 mL of water and 0.2 mL of glacial acetic acid. Next, 50
mg of 10% palladium on charcoal is added and the reaction vessel is
flushed first with nitrogen, then hydrogen. The reaction is stirred
rapidly under 1 atmosphere of hydrogen for several hours. The catalyst
is removed by filtration and the volatiles are removed by rotary
io evaporation under reduced pressure. The residue is dissolved in 2 mL
of water, frozen and lyophilized to give the desired deprotected amine
product as a solid. The desired product has a molecular weight of 1181.
EXAMPLE 7
is
O
N
.,,, H
HO~~~~ H
20 N
O
H2N ' .."H
HO NH
O H H
HO _~~(~~N
25 ~2HOAc
~'H O .~'OH
OH
HQ (Seq. ID No. 1)
Fart A Displacement of Sulfones with Azide (SEO ID No. 191
The mixture of sulfones (0.257 mmol), prepared as
described in Part D of Example 5, is dissolved in 10 mL of anhydrous
DMF. Lithium azide (0.257 mmol) is added as a solid and the mixture
__,,.: ,
. rte.
WO 96108266 ~ ~ ~ ~ PCTlUS95111520
-28-
is stirred for about a 4-24 hours. The mixture is purified by reverse
phase C18 flash chromatography eluting with 20-55% acetonitrile/water
in 5% step gradients. The appropriate fractions, as determined by
reverse phase HPLC (RP-18, 40% acetonitrile/water/0.1% TFA, 210
s nm) are pooled, frozen and lyophilized to give the crude product.
Further purification by preparative reverse phase HPLC (C18, 30-40%
acetonitrile/water/0.1 % TFA, 210 nm) yields the desired purified
compound with a molecular weight of 1190.
io ~$ Reduction of Azide to Amine fSEO ID No l~
A mixture of the azido compound (0.126 mmol) (obtained
as described above in Part A) and 10% Pd on charcoal (100-150 mg) is
suspended in glacial acetic acid (10 mL). The reaction vessel is flushed
first with nitrogen then with hydrogen. One atmosphere pressure of
is hydrogen gas is maintained for a period of time sufficient to give
complete reduction to the amine product, typically 2 to 24 h. The
catalyst is removed by filtration and the filtrate is lyophilized to obtain
the desired amine as a diacetate salt with a molecular weight of 1170.
25
W O 96!08266 - 2 I 9 7 2 0 9 PCT/US95/11520
.r.
_29_
CH3
~N, OH
O
N
HOu,. ~,"H H
N
O
H2N ' .,~~H
. HO niN
O H H ,. N
HO N ~2CF3C02H
.'H O .~OH
OH
is ~
HO (Seq. ID No. 1 )
The sulfone mixture (0.094 mmol); obtained as described
~ part D Example 5, is dissolved in 2.0 mL of anhydrous DMF and N-
methyl-N-allylamine (0.945 mmol) is added. The mixture is stirred for
about 1-12 hours or until analytical HPLC analysis (RP-18, 40%
acetonitrile/water/0.1 % TFA, 210 nm) shows complete disappearance of
the starting sulfone. ~'he mixture is separated by reverse phase (C18)
2s Eash column chromatography eluting with 10-40%
acetonitrile/water/0.1%TFA in 5% step gradients. The appropriate
fractions are pooled, frozen and lyophilized to give the desired product
with the a-C-5 orn configuration and a molecular weight of 1332.
' 30
VI'O 96108266 ~ O 9 PCT/US95111520
-30-
EXAMPLE 9
(HsC)2N
~N OH
O
N
.,~ H
HO~~~~ H
N
. HZN ., H
H H N
-I ~.~~OH ~2CF3C02H
O
is
H (Seq. ID No. 1)
2o The sulfone mixture (0.094 mmol), obtained as described
in Part D Example 5, is dissolved in 2.0 mL of anhydrous DMF and
N,N-dimethylethyl-1,3-diaminopropane (0.945 mmol) is added. The
mixture is stirred for about 1-12 hours or until analytical HPLC
analysis (RP-18, 40% acetonitrile/water/0.1 % TFA, 210 nm) shows
2s complete disappearance of the starting sulfone. The mixture is
separated by reverse phase (C18) flash column chromatography eluting
with 10-40% acetonitrile/water/0.1%TFA.in 5% step gradients. The
appropriate fractions are pooled, frozen and lyophilized to give the
desired product with the a-C-5 orn configuration and a molecular
ao weight of 1363.5.
The following examples illustrate representative
compositions containing the compounds of the invention.
WO 96!08266 219 7 2 0 9 PCT/US95/11520
- 31~ -
1000 compressed tablets each containing 500 mg of the
compound of Example 5 are prepared from the following formulation:
s
Cod Grams
Compound of Example 5 500
Starch 750
Dibasic calcium phosphate, hydrous 5000
i o 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.
is
EXAMPLE B
1000 hard gelatin capsules, each containing 500 mg of the
same compound are prepared from the following formulation:
Compound of Example 5 500
Starch 250
Lactose . 750
2s Talc . 250
Calcium stearate 10
A uniform mixture of the ingredients is prepared by
blending and used to fill two-piece hard gelatin capsules.
w0 96/08266 g ~ 2 ~ g PCT/US95111520
-32-
EXAMPLE C
An aerosol composition may be prepared having the
following formulation:
s
Per Canister
Compound of Example 5 24 mg
Lecithin NF Liquid Concd. 1..2 mg
Trichlorofluoromethane, NF 4.026 g
io Dichlorodifluoromethane, NF 12.15 g
EXAMPLE D
250 milliliters of an injectible solution may be prepared by
is conventional procedures having the following formulation:
Dextrose 12.5 g
Water 250 ml
Compound of Example 5 400 mg
The ingredients are blended and thereafter sterilized for
use.
Preparation Of Starting Materials:
2s The starting material, Compound A-3 Seq. ID No. 3, in
which R1 is 9,11-dimethyltridecyl may be produced by cultivating
Zalerion arboricola ATCC 20868 in a nutrient medium enriched with
mannitol as the primary source of carbon as described in U.S. Patent
No. 5,021,341 issued June 4, 1991.
W096I08266 ~ ~ pCT/U895111520
-33-
Starting-materials in which RI 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, the 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. Patent No. 4,293,482.
The deacylated cyclopeptide is recovered and thereafter reacylated by
mixing together with an appropriate active ester RCOZ where Z is
io halogen,pentachlorophenoxy,pentafluorophenoxy,p-nitrophenoxy and
the like, to obtain compound a with the desired acyl group.
i5 -
2n
30
R'O 96/08266 2 ~ 9 PCTIi1S95111520
-34-
SEQUENCE LISTING -.
(1) GENERAL INFORMATION:
(i) APPLICANT: BALKOVEC, JAMES M
BOUFFARD, FRANCES A
HAMMOND, MILTON L -
(ii) TITLE OF-INVENTION: AZA CYCLOHEXAPEPTIDE COMPOUNDS-
(iii) NUMBER OF SEQUENCES: 24 -
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: ELLIOTT KORSEN
(B) STREET: P.O. BOX 2000, 12b EAST LINCOLN AVENUE
(C) CITY: RAHWAY
(D) STATE: NJ
(E) COUNTRY: USA -
(F) ZIP: 07065 _
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible -
(C) OPERATING SYSTEM:.PC-DOS/MS-DOS -
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION: -
(A) NAME: KORSEN, ELLIOTT - _
(B) REGISTRATION NUMBER: 32,705
(C) REFERENCE/DOCKET NUMBER: 19305 -
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 908-594-5493 - -
(B) TELEFAX: 908-594-4720 ...
(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) HYPOTF~TICAL-: NO - _. - __
(iv) ANTI-SENSE: NO
WO 96/08266 /~ ~ ~ g PC17US95/11520
-35-
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
Xaa Thr Xaa Xaa Xaa Xaa
(2) INFORMATION FOR SEQ ID N0:2:
(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:Z:. ,
Xaa Thr Xaa Xaa Xaa Xaa
1 5
(2) INFORMATION FOR BEQ ID N0:3: -
(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 N0:3:
Xaa Thr Xaa Xaa Xaa Xaa
1 5
(2) INFORMATION FOR SEQ ID N0:4:-
(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 N0:4:
.. v.:;; :. .
2~9T209
VVO 96108266 PCTIU595111520
-36-
Xan Thr Xaa Xaa Xaa Xaa -- - ,
1 5
(2) INFORMATION FOR SEQ ID NO: S:
(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:S:
Xaa Thr Xaa Xaa Xaa Xaa - -
1 5
(2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids -
(B) TYPE: amine acid
(C) STRANDEDNESS:.unknown
(D) TOPOLOGY: circular - _
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6: -
Xaa Thr Xaa Xaa Xaa Xaa
1 5
(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid -
(C) STRANDEDNESS: unknown
(D) TOPOLOGY:.circular -
(ii7 MOLECULE TYPE: peptide -.
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
Xaa Thr Xaa Xaa Xaa Xaa
1 5 __
(2) INFORMATION FOR SEQ ID N0:8:
X197209
WO 96108266 PCT/US95/11520
s
-37-
(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: B:
Xas Thr Xaa Xaa Xaa Xaa
1 5
(2) INFORMATION FOR SEQ ID N0:9:
(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 N0:9:
Xaa Thr Xaa Xaa Xaa Xaa
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNE$S:-unknown
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Xaa Thr Xaa Xaa Xaa Raa
1 5
(2) INFORMATION FOR SEQID N0:11:
' (iJ SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: circular
,"
WO 96108266 2 I 9 7 2 0 9 1'CT~S95111520
-38-
(ii) MOLECULE TYPE: peptide -.
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:11: .-
Xaa Thr Xaa Xaa Xaa Xaa
1 5 -
(2) INFORMATION FOR SEQ ID N0:12.. - -....
( i ) SEQUENCECHARACTERISTICS. - _ _ _-. _. - __. _.. -.
(A) LENGTH: 6 amino acids - ~ -.
(B) TYPE: amino acid -
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: circular _ ..._. _- .. ...::.._...... ....._... __- . . _
(ii) MOLECULE TYPE: peptide -... - -.
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:12:
Xaa Thr Xaa Xaa Xaa Xaa
1 5 - ._
(2) INFORMATION FOR SEQ ID N0:13:
(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 N0:13:
Xaa Thr Xaa Xaa Xaa Xaa
1 5 -. _-
(2) INFORMATION FOR SEQ ID N0:14: - ' -
(i) SEQUENCE CHARACTERISTICS:.. -.
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid --. -
(C) STRANDEDNESS: unknown
(D) TOPOLOGY. circular- - -
(ii) MOLECULE TYPE: peptide
W O 96108266 2 ~ ~ PC1'/US95/I 1520
-39-
(xi) SEQUENCE DESCRIPTION. SEQ ID N0:14:
Xaa Thr Xaa Xaa Xaa Xaa
1 S
(2) INFORMATION FOR SEQ ID N0:15:
(i) SEQUENCE.CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPEa amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: circular -
(ii) MOLECULE-TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:15: -
Xaa Thr Xaa Xaa Xaa Xaa
1 5._.._ _. _ _ _ _._
(2) INFORMATION FOR SEQ ID N0:16:
(i) SEQUENCE CHARACTERISTICS: -
(A) LENGTH: 6 amino acids
(B) TYPE: aittino acid
(C) STRANDEDNESS: unknown
(D) TOFOLOGY: circular -
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:15:
Xaa Thr Xaa Xaa Xaa Xaa
1 5
(2) INFORMATION FOR-SEQ ID N0:17: -
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: circular
(ii) 220LECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTLON: SEQ ID N0:17:
Xaa Thr Xaa Xaa Xaa Xaa
1 -. _. _ 5 .. _
_; w..~::;a .~,: :.
WO 96108266 219 7 2 0 9 PCT~S95111520
0
-40-
(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 N0:18:
Xaa Thr Xaa Xaa Xaa Xaa
1 5 _ . __ _ .. .
(2) INFORMATION FOR SEQ ID N0:19:
(i) SEQUENCE CHARACTERISTICS: - ._ .- .. ,
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown -
(D) TOPOLOGY: circular -
tii) MOL~CC7LE TYPE: peptide - -
(xi) SEQUENCE.DESCRIPTION: S~Q ID N0:19:
Xaa Thr Xaa Xaa-Xaa Xaa
(2) INFORMATION FOR SEQ ID N0:20:
( i ) SEQUENCE CHARACTERISTICS: - - -.
(A) LENGTH: 6 amino acids -
(B) TYPE: amino acid
(C) STRANDEDNESS~--unknown -
(D) TOPOLOGY: circular - -.
(ii) MOLECULE TYPE: peptide - ~.
(xi) SEQUENCE DESCRIPTIDN: S~Q ID N0:20:
Xaa Thr Xaa Xaa Xaa Xaa
(2) TNFORMATION FOR SEQ ID N0:21: -. '
(i) SEQUENCE CHARACTERISTICS.: - -.-.. -.--. --
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid -
W096108266 ~g PCT/US95l11520
-41 -
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: peptide -
(xi) 5EQ'UFNCE DESCRIPTION: SEQ-ID N0:21: -
Xaa Thr Xaa Xaa Xaa Xaa
1 - 5
(2) INFORMATION FOR SEQ-ID N0:22:
(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 N0:22:
Xaa Thr Xaa Xaa Xaa Xaa
1 5
(2) INFORMATION FOR SEQ ID N0:23:
(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 N0:23: -
Xaa Thr Xaa Xaa Xaa Xaa
(2) INFORMATION FOR SEQ ID N0:24:
' (i) SEQUENCE CHARACTERISTICS: -
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
~ (C) STRANDEDNESS: unknown
fD) TOPOLOGY:-circular
(iii MOLECULE TYPE: peptide
ka-. . , . - .-..: '.~... _.:
2 ~ 97209
WO 96108266 PCTlUS95111520
- 42 -
(xi) SEQiTENCE DESCRIPTION: SEQ ID N0:24:
Xaa Thr Xaa Xaa Xaa Xaa
1 5
