Note: Descriptions are shown in the official language in which they were submitted.
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SEL~ v~hY N-ALKYLATED ~LlvOMIMETIC
COMBINATORIA~ T.TRpl~12T~.~ AND COMPOUNDS T~TN
This application claims the benefit of U.S.
Provisional Application No. 60/ , filed March 5,
1996, which was converted from U.S. Serial No.
08/611,390, and is incorporated herein by reference.
FT~T.n OF THE I NV~ ON
The present invention relates generally to
novel, selectively N-alkylated compounds of Formula I
below, as well as novel libraries composed of many such
compounds, methods of synthesizing and screening the
libraries, and methods of using the compounds.
~A~K~RQUND INFO~ATION
The process of discovering new therapeutically
active compounds for a given indication involves the
screening of all compounds from available compound
collections. From the compounds tested, one or more
structure(s) is selected as a promising lead. A large
number of related analogs are then synthesized in order
to develop a structure-activity relationship and select
one or more optimal compounds. With traditional one-at-
a-time synthesis and biological testing of analogs, this
optimization process is long and labor intensive. Adding
significant numbers of new structures to the compound
collections used in the initial screening step of the
discovery and optimization process cannot be accomplished
with traditional one-at-a-time synthesis methods, except
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over a time frame of months or even years. Faster
methods are needed that allow for the preparation of up
to thousands of related compounds in a matter of days or
a few weeks. This need is particularly evident when it
comes to synthesizing more complex compounds, such as the
instant compounds composed of two or more monomers, each
monomer possessing more than one variable substituent.
Solid-phase techniques for the synthesis of
peptides have been extensively developed and
combinatorial libraries of peptides have been generated
with great success. During the past four years there has
been substantial development of chemically synthesized
combinatorial libraries (SCLs) made up of peptides. The
preparation and use of synthetic peptide combinatorial
libraries has been described, for example, by Dooley in
U.S. Patent 5,367,053, Huebner in U.S. Patent 5,182,366,
Appel et al. in W0 PCT 92/09300, Geysen in published
European Patent Application 0 138 855 and Pirrung in U.S.
Patent 5,143,853. Such SCLs provide the efficient
synthesis of an extraordinary number of various peptides
in such libraries and the rapid screening of the library
which identifies lead pharmaceutical peptides.
Substituent limitations have been overcome for
mixtures of peptides and peptidomimetics through the use
of solid phase techniques instead of the more traditional
solution-phase ones. An important step in the
development of solid-phase techniques was the discovery
of methods to identify active individual compounds from
soluble mixtures of large numbers of compounds, as
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described, for example by Rutter in U.S. Patent 5,010,175
and Simon in WO PCT 91/19735. These soluble mixtures,
q however, have never before been applied to compounds with
amide backbones that have different substituent on each
~ 5 amide nitrogen. Until now, it was possible by previously
known methods to add only the same specific substituent
to each and every nitrogen atom of the amide backbone.
Thus, improved methods were needed to synthesize such
selectively N-alkylated amide compounds.
This invention satisfies these needs and
provides related advantages as well. The present
invention overcomes the known limitations to the
shortcomings of combinatorial chemistry with respect to
selective N-alkylation. The present invention combines
the techniques of solid-phase synthesis of peptidomimetic
compounds and the general techniques of synthesis of
combinatorial libraries to prepare new selective
N-alkylated compounds.
SUMMARY OF TU~ ,NV~N~lON
This invention is directed to a single
selectively N-alkylated compound or a library of an
approximately equimolar mixture of two more selectively
N-alkylated compounds of the Formula (I):
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(I)
R,R2R10N ~ (CH2 ~c I y ~ ~ N (CH ~ NR8R~
R3 ~ Rs / R~
Wherein:
Rl and R2 independently are a hydrogen atom, an
amino protecting group, Clto C12acyl, C3 to C10
cycloalkyl, C3 to C6 heterocycle, Clto Cl2alkyl, Clto C12
substituted alkyl, C7 to Cl6 alkylaryl, C7 to Cl6 substitued
alkylaryl, a C6 to Cl5 alkyl heterocycle, or a substituted
C6 to C1s alkyl heterocycle;
R3 Rs and R7 are independently a hydrogen atom,
C1to C12alkyl, C1to C12substituted alkyl, phenyl,
substituted phenyl, C7 to C16 alkylaryl, C7 to C16
substitued alkylaryl, a C6 to C1salkyl heterocycle, or a
substituted C6 to C15 alkyl heterocycle;
R4,R6, and R8 are independently a C1to Cl8
substituent group; with the proviso that all but one of
R4,R6 and R8 can simultaneously be the same group;
Rg is a hydrogen atom or a solid support;
Rlo is optionally present as a Cl to Cl8
substituent group when R1 and R2 are other than a hydrogen
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atom, an amino protecting group or when both Rl and R2 are
Cl to Cl2 acyl group~;
-
AA, BB, and CC are independently 0 to 5;
B is from 0 to 3;
further wherein the stereochemistry at the
- carbons bonded to R3, R5, and R7are independently R or S
or a mixture of the two;
further wherein when B is 2 or 3; each R4and Rs
can be the same or different;
with the proviso that either R1 or R2 can be
taken with R3; R4 can be taken with Rs; and R6 can be
taken with R7; respectively and independently, to form a
subtituted or unsubstituted pyrrolidine ring;
X and Y are either 1) each a hydrogen atom or
2) taken together to represent a carbonyl group;
and a pharmaceutically acceptable salt, solvate
or hydrate thereof.
This invention is also directed to iterative
and positional scanning methods of synthesizing the
libraries of compounds described above as discussed
below. Another aspect of the invention is a method of
selective N-alkylation as set forth below. Furthermore,
the invention comprises methods for affecting analgesia
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in a mammal, effecting a decrease in the postprandial
rise in the blood glucose levels of a mammal after said
~mm~l has ingested a carbohydrate load, and a method for
treating microbial infections, all of which methods
comprise administering a single compound of the above
formula in conjunction with a pharmaceutically acceptable
carrier, as set forth below.
D~T~TT-~n D~ TPTTON OF ~Nv~Nl~ON
The instant invention is directed to a single
compound or an approximately equimolar mixture of two or
more selectively N-alkylated compounds of the Formula
(I):
R1R2R10N ~ (CH ~c N y (CH ~ ~
R~ \ Rs I \~(CH2)AA NR8Rg
Wherein:
R, and R2 independently are a hydrogen atom, an
amino protecting group, C1to Cl2acyl, C3 to C10
cycloalkyl, C3 to C6 heterocycle, Clto C12alkyl, Clto Cl2
substituted alkyl, C7 to C16 alkylaryl, C7 to Cl6 substitued
alkylaryl, a C6 to C15 alkyl heterocycle, or a substituted
C6 to C15 alkyl heterocycle;
R3 Rs and R7 are independently a hydrogen atom,
Clto Cl2alkyl, C1to C12substituted alkyl, phenyl,
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substituted phenyl, C7 to C16 alkylaryl, C7 to C16
substitued alkylaryl, a C6 to C15alkyl heterocycle, or a
~ substituted C6 to C1s alkyl heterocycle;
- R4, R6, and R8 are independently a C1to C18
substituent group; with the proviso that all but one of
R4, R6 and R8 can simultaneoulsy be the same group;
Rg iS a hydrogen atom or a solid support;
R1o is optionally present as a C1 to C18
substituent group when R1 and R2 are other than a hydrogen
atom or an amino protecting group;
AA, BB, and CC are independently 0 to 5;
B is from 0 to 3;
further wherein the stereochemistry at the
carbons bonded to R3, R5, and R7 are independently R or S
or a mixture of the two;
further wherein when B is 2 or 3; each R4 and R5
can be the same or different;
with the proviso that either R1 or R2 can be
taken with R3; R4 can be taken with R5 ; and R6 can be
taken with R7; respectively and independently, to form a
subtituted or unsubstituted pyrrolidine ring;
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X and Y are either 1) each a hydrogen atom or
2) taken together to represent a carbonyl group;
and a pharmaceutically acceptable salt, solvate
or hydrate thereof.
s The terms used in the above Formula I having
the following meanings when used in conjunction with
Formula I and when used in described subse~uent Formulas:
C3 to C10 cycloalkyl - unsubstituted or
substituted mono- or bicyclic saturated rings such as
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,
cycloheptyl, or adamantyl or rings wherein the
substituents are one or more hydroxy, halo, amino,
protected amino, carboxy, protected carboxy, amido,
nitro, trifluoromethyl, phenyl, heterocyclic rings, Cl to
C, acyl, C1 to C7 alkoxy, or Cl to C3 alkyl groups;
C3 to C6 heterocycle - unsubstituted or
substituted mono- or bicyclic rings containing 3 to 6
carbons and from one to three nitrogen, oxygen, sulfur
atoms such as azetidine, pyrrolidine, pyrazolidine,
piperidine, piperazine, perhydroazepine or tropane,
oxazole, thiazole, pyrazole, thiophenyl and pyranyl rings
wherein the substituents are one or more hydroxy,
protected hydroxy, halo, amino, protected amino,
monosubstituted amino, disubstituted amino, carboxy,
2~ protected carboxy, amido, nitro, trifluoromethyl,
phenyl, or C1to C3 alkyl groups;
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C1 to Cl2 Alkyl - straight-chain or branched
carbon chain optionally containing a C3 to C7 saturated or
partially saturated ring, wherein the carbon chain may
also optionally be partially unsaturated, such as methyl,
ethyl, tert-butyl, iso-propyl, 6-~cyclohexyl)-n-hexyl,
allyl, n-octyl, 3-(cyclopentyl)-n-pentyl,
methylcyclopropyl, and the like;
The term C1to C1B Substituent Group indicates a
group of the formula
-CHa-W
wherein W is chosen from the group consisting
of a hydrogen atom, C1to C12alkyl, C3 to ClOcycloalkyl, C1
to C12 substituted alkyl, phenyl, substituted phenyl, C7 to
C16alkylaryl, C7 to Cl6substituted alkylaryl, as those
terms are defined herein;
The term "C1 to C12 substituted alkyl," denotes
the above C1 to Cl2 alkyl groups that are substituted by
one to three halogen, hydroxy, protected hydroxy, amino,
protected amino, guanidino, C1 to C7 acyloxy, C1 to C7
acyl, nitro, carboxy, protected carboxy, carboxamide,
carbonyl, carboxyl, cyano, methylsulfonylamino or C1 to C4
alkoxy groups. The substituted alkyl groups may be
substituted once or twice with the same or with different
substituents;
Examples of the above substituted alkyl groups
include the cyanomethyl, nitromethyl, hydroxymethyl,
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trityloxymethyl, propionyloxymethyl, aminomethyl,
carboxymethyl, allyloxycarbonylmethyl,
allyloxycarbonylaminomethyl, carbamoylmethyl,
methoxymethyl, ethoxymethyl, t-butoxymethyl,
acetoxymethyl, chloromethyl, bromomethyl, iodomethyl, 6-
hydroxyhexyl, 2,4-dichloro(n-butyl), 2-amino(iso-pro-
pyl), 2-carbamoylethyl and the like. A preferred group
of examples within the above "Cl to C6 substituted alkyl"
group includes the substituted methyl group, in other
words, a methyl group substituted by the same
substituents as the "Cl to C6 substituted alkyl" group.
Examples of the substituted methyl group include groups
such as protected hydroxymethyl, (e.g.,
tetrahydropyranyloxymethyl), acetoxymethyl,
carbamoylmethyl, chloromethyl, bromomethyl and
iodomethyl.
The term "substituted phenyl" specifies a
phenyl group substituted with one or more, and preferably
one or two, moieties chosen from the groups consisting of
halogen, hydroxy, protected hydroxy, cyano, nitro,
trifluoromethyl, Cl to C6 alkyl, C1 to C7 alkoxy, Clto C7
acyl, C1 to C, acyloxy, carboxy, protected carboxy,
carboxymethyl, protected carboxymethyl, hydroxymethyl,
protected hydroxymethyl, amino, protected amino,
(monosubstituted)amino, protected (monosubstituted)amino,
(disubstituted)amino, carboxamide, protected carboxamide,
N-(C1 to C6 alkyl)carboxamide, protected N-(Cl to C6
alkyl)carboxamide, N,N-di(C1 to C6alkyl)carboxamide,
trifluoromethyl, N-((Cl to C6 alkyl)sulfonyl)amino, N-
(phenylsulfonyl)amino or phenyl, substituted or
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11
unsubstituted, such that, for example, a biphenyl or
naphthyl group results.
Examples of the term "substituted phenyl"
- includes a mono- or di(halo~phenyl group such as 2, 3 or
4-chlorophenyl, 2,6-dichlorophenyl, 2,5-dichlorophenyl,
3,4-dichlorophenyl, 2, 3 or 4-bromophenyl, 3,4-
dibromophenyl, 3-chloro-4-fluorophenyl, 2, 3 or 4-
fluorophenyl and the like; a mono or di(hydroxy)phenyl
group such as 2, 3 or 4-hydroxyphenyl, 2,4-
dihydroxyphenyl, the protected-hydroxy derivatives
thereof and the like; a nitrophenyl group such as 2, 3 or
4-nitrophenyl; a cyanophenyl group, for example, 2, 3 or
4-cyanophenyl; a mono- or di(alkyl)phenyl group such as
2, 3 or 4-methylphenyl, 2,4-dimethylphenyl, 2, 3 or 4-
lS (iso-propyl)phenyl, 2, 3 or 4-ethylphenyl, 2, 3 or 4-(n-
propyl)phenyl and the like; a mono or di(alkoxy)phenyl
group, for example, 2,6-dimethoxyphenyl, 2, 3 or 4-
methoxyphenyl, 2, 3 or 4-ethoxyphenyl, 2, 3 or 4-
(isopropoxy)phenyl, 2, 3 or 4-(t-butoxy)phenyl, 3-ethoxy-
4-methoxyphenyl and the like; 2, 3 or 4-
trifluoromethylphenyl; a mono- or dicarboxyphenyl or
(protected carboxy)phenyl group such as 2, 3 or 4-
carboxyphenyl or 2,4-di(protected carboxy)phenyl; a mono-
or di(hydroxymethyl)phenyl or (protected
hydroxymethyl)phenyl such as 2, 3, or 4-(protected
- hydroxymethyl)phenyl or 3,4-di(hydroxymethyl)phenyl; a
mono- or di(aminomethyl)phenyl or (protected
aminomethyl)phenyl such as 2, 3 or 4-(aminomethyl)phenyl
or 2,4-(protected aminomethyl)phenyl; or a mono- or di(N-
(methylsulfonylamino))phenyl such as 2, 3 or 4-(N-
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12
(methylsulfonylamino))phenyl. Also, the term
"substituted phenyl" represents disubstituted phenyl
groups wherein the substituents are different, for
example, 3-methyl-4-hydroxyphenyl, 3-chloro-4-
hydroxyphenyl, 2-methoxy-4-bromophenyl, 4-ethyl-2-
hydroxyphenyl, 3-hydroxy-4-nitrophenyl, 2-hydroxy 4-
chlorophenyl and the like.
The terms "halo" and ~'halogen" refer to the
fluoro, chloro, bromo or iodo groups. There can be one
or more halogen, which are the same or different.
Preferred halogens are chloro and fluoro.
The term "C7 to C16 alkylaryl" denotes a C1 to C6
alkyl group substituted at any position by a phenyl or
naphthyl ring. Examples of such a group include benzyl,
2-phenylethyl, 3-phenyl~n-propyl), 4-phenylhexyl, 3-
phenyl(n-amyl), 3-phenyl(sec-butyl) and the like.
Preferred C7 to Cl6 phenylalkyl groups are the benzyl
phenylethyl napth-1-ylmethyl and napth-2-ylmethyl groups.
The term "C7 to C16 substituted alkylaryl"
denotes a C7 to C16 alkylaryl group substituted on the C1
to C6 alkyl portion with one or more, and preferably one
or two, groups chosen from halogen, hydroxy, protected
hydroxy, oxo, protected oxo, amino, protected amino,
(monosubstituted)amino, protected (monosubstituted)amino,
(disubstituted)amino, guanidino, heterocyclic ring,
substituted heterocyclic ring, C1 to C7 alkoxy, C1 to C7
acyl, C1 to C, acyloxy, nitro, carboxy, protected carboxy,
carbamoyl, carboxamide, protected carboxamide, N-(C1 to C6
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13
alkyl)carboxamide, protected N-C1 to C6 alkyl)carboxamide,
N, N-(C1 to C6dialkyl)carboxamide, cyano, N-((C1 to C6
alkylsulfonyl)amino, thiol, C1 to C4 alkylthio, Cl to C4
alkylsulfonyl groups; and/or the phenyl group may be
- 5 substituted with one or more, and preferably one or two,
substituents chosen from halogen, hydroxy, protected
hydroxy, cyano, nitro, C1 to C6 alkyl, Cl to C7 alkoxy, C
to C7 acyl, Cl to C, acyloxy, carboxy, protected carboxy,
carboxymethyl, protected carboxymethyl, hydroxymethyl,
protected hydroxymethyl, amino, protected amino,
(monosubstituted)amino, protected (monosubstituted)amino,
(disubstituted)amino, carboxamide, protected carboxamide,
N-(Cl to C6 alkyl) carboxamide, protected N-(C1 to C6
alkyl) carboxamide, N, N-di(C1 to C6 alkyl)carboxamide,
trifluoromethyl, N-((C1 to C6 alkyl)sulfonyl)amino, N-
(phenylsulfonyl)amino or a phenyl group, substituted or
unsubstituted, for a resulting biphenyl or naphthyl
group. The substituted alkyl or phenyl groups may be
substituted with one or more, and preferably one or two,
substituents which can be the same or different.
Examples of the term "C7 to Cl6 substituted
alkylaryl" include groups such as 2-phenyl-1-chloroethyl,
2-(4-methoxyphenyl)ethyl, 4-(2,6-dihydroxy phenyl)n-
hexyl, 2-(5-cyano-3-methoxyphenyl~n-pentyl, 3-(2,6-
dimethylphenyl)n-propyl, 4-chloro-3-aminobenzyl, 6-(4-
~ methoxyphenyl)-3-carboxy(n-hexyl), 5-(4-
aminomethylphenyl)-3-(aminomethyl)n-pentyl, 5-phenyl-3-
oxo-n-pent-l-yl, (4-hydroxynapth-2-yl)methyl and the
like.
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14
The term "(monosubstituted)amino" refers to an
amino group with one substituent chosen from the group
consisting of phenyl, substituted phenyl, Cl to C6 alkyl,
C1 to C6 substituted alkyl, C1 to C7 acyl, C2 to C7 alkenyl,
C2 to C7 substituted alkenyl, C2 to C7 alkynyl, C7 to C16
alkylaryl, C7 to Cl6 substituted alkylaryl and
heterocyclic ring. The (monosubstituted)amino can
additionally have an amino-protecting group as
encompassed by the term "protected
(monosubstituted)amino."
The term "(disubstituted)amino" refers to amino
groups with two substituents chosen from the group
consisting of phenyl, substituted phenyl, C1 to C6 alkyl,
Cl to C6 substituted alkyl, C1to C7 acyl, C2 to C7 alkenyl,
C2 to C7 alkynyl, C7 to C16 alkylaryl, C7 to C16 substituted
alkylaryl and heterocyclic ring. The two substituents
can be the same or different.
The term "pharmaceutically-acceptable salt"
encompasses those salts that form with the carboxylate
anions and includes salts formed with the organic and
inorganic cations such as those chosen from the alkali
and alkaline earth metals, (for example, lithium, sodium,
potassium, magnesium, barium and calcium); ammonium; and
the organic cations (for example, dibenzylammonium,
benzylammonium, 2-hydroxyethylammonium,
bis(2-hydroxyethyl)ammonium, phenylethylbenzylammonium,
dibenzylethylenediammonium, and like cations). Other
cations encompassed by the above term include the
protonated form of procaine, quinine and
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N-methylglucosamine, and the protonated forms of basic
amino acids such as glycine, ornithine, histidine,
phenylglycine, lysine, and arginine, and acetic acid-like
counter-ions such as acetate and trifluoroacetate.
Furthermore, any zwitterionic form of the instant
compounds formed by a carboxylic acid and an amino group
is referred to by this term. A preferred cation for the
carboxylate anion i6 the sodium cation. Furthermore, the
term includes salts that form by standard acid-base
reactions with basic groups (such as amino groups) and
organic or inorganic acids. Such acids include
hydrochloric, sulfuric, phosphoric, acetic, succinic,
citric, lactic, maleic, fumaric, palmitic, cholic,
pamoic, mucic, D-glutamic, D-camphoric, glutaric,
phthalic, tartaric, lauric, stearic, salicyclic,
methanesulfonic, benzenesulfonic, sorbic, picric,
benzoic, c- nn~m; C, and the like acids.
The compounds of Formula I may also exist as
solvates and hydrates. Thus, these compounds may
crystallize with, for example, waters of hydration, or
one, a number of, or any fraction thereof of molecules of
the mother liquor solvent. The solvates and hydrates of
such compounds are included within the scope of this
invention.
- 25 The term "carboxy-protecting group" as used
herein refers to one of the ester derivatives of the
carboxylic acid group commonly employed to block or
protect the carboxylic acid group while reactions are
carried out on other functional groups on the compound.
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Examples of such carboxylic acid protecting groups
include t-butyl, 4-nitrobenzyl, 4-methoxybenzyl, 3,4-
dimethoxybenzyl, 2,4-dimethoxybenzyl, 2,4,6-
trimethoxybenzyl, 2,4,6-trimethylbenzyl,
pentamethylbenzyl, 3,4-methylenedioxybenzyl, benzhydryl,
4,4~-dimethoxytrityl, 4,4',4"-trimethoxytrityl, 2-
phenylpropyl, trimethylsilyl, t-butyldimethylsilyl,
phenacyl, 2,2,2-trichloroethyl, ~-(trimethylsilyl)ethyl,
~-(di(n-butyl)methylsilyl)ethyl, p-toluenesulfonylethyl,
4-nitrobenzylsulfonylethyl, allyl, cinnamyl, 1-
(trimethylsilylmethyl)-propenyl and like moieties. The
species of carboxy-protecting group employed is not
critical so long as the deri~atized carboxylic acid is
stable to the conditions of subse~uent reaction(s) and
can be removed at the appropriate point without
disrupting the remainder of the molecule. Further
examples of these groups are found in C.B. Reese and E.
Haslam, "Protective Groups in Organic Chemistry," J.G.W.
McOmie, Ed., Plenum Press, New York, NY, 1973, Chapter 5,
respectively, and T.W. Greene and P.G.M. Wuts,
"Protective Groups in Organic Synthesis," 2nd ed., John
Wiley and Sons, New York, NY, 1991, Chapter 5, each of
which is incorporated herein by reference. A related
term is '~protected carboxy," which refers to a carboxy
group substituted with one of the above carboxy-
protecting groups.
The term "hydroxy-protecting group" refers to
readily cleavable groups bonded to hydroxyl groups, such
as the tetrahydropyranyl, 2-methoxyprop-2-yl,
1-ethoxyeth-1-yl, methoxymethyl, ~-methoxyethoxymethyl,
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17
methylthiomethyl, t-butyl, t-amyl, trityl,
4-methoxytrityl, 4,4'-dimethoxytrityl,
4,4~,4~-trimethoxytrityl, benzyl, allyl, trimethylsilyl,
~t-butyl)dimethylsilyl, 2,2,2-trichloroethoxycarbonyl
groups and the like.
Further examples of hydroxy-protecting groups
are described by C.B. Reese and E. Haslam, "Protective
Groups in Organic Chemistry,ll J.G.W. McOmie, Ed., Plenum
Press, New York, NY, 1973, Chapters 3 and 4,
respectively, and T.W. Greene and P.G.M. Wuts,
"Protective Groups in Organic Synthesis," Second Edition,
John Wiley and Sons, New York, NY, l99l, Chapters 2 and
3. A preferred hydroxy-protecting group is the tert-
butyl group. The related term "protected hydroxy"
denotes a hydroxy group bonded to one of the above
hydroxy protecting groups.
The term "amino-protecting group" as used
herein refers to substituents of the amino group commonly
employed to block or protect the amino functionality
while reacting other functional groups of the molecule.
The term "protected (monosubstituted)amino" means there
is an amino-protecting group on the monosubstituted amino
nitrogen atom. In addition, the term "protected
carboxamide" means there is an amino-protecting group on
- the carboxamide nitrogen.
Examples of such amino-protecting groups
include the formyl ("For") group, the trityl group, the
phthalimido group, the trichloroacetyl group, the
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trifluoro-acetyl group, the chloroacetyl, bromoacetyl,
and iodoacetyl groups, urethane-type blocking groups,
such as t-butoxycarbonyl ("Boc"), 2-(4-biphenylyl)propyl-
2-oxycarbonyl ("Bpoc"), 2-phenylpropyl-2-oxycarbonyl
("Poc"), 2-(4-xenyl)isopropoxycarbonyl, 1,1-
diphenylethyl-l-oxycarbonyl, 1,1-diphenylpropyl-1-
oxycarbonyl, 2-(3,5-dimethoxyphenyl)propyl-2-oxycarbonyl
("Ddz"), 2-(p-toluyl)propyl-2-oxycarbonyl,
cyclopentanyloxycarbonyl,
l-methylcyclopentanyloxyCarbonyl, cyclohexanyloxy-
carbonyl, l-methylcyclohexanyloxycarbonyl,
2-methylcyclohexanyloxycarbonyl,
2-(4-toluylsulfonyl)ethoxycarbonyl,
2-(methylsulfonyl)ethoxycarbonyl, 2-(triphenylphosphino)-
ethoxycarbonyl, 9-fluorenylmethoxycarbonyl ("Fmoc"),
2-(trimethylsilyl)ethoxycarbonyl, allyloxycarbonyl,
l-(trimethylsilylmethyl)prop-l-enyloxycarbon
5-benzisoxalylmethoxycarbonyl,
4-acetoxybenzyloxycarbonyl,
2,2,2-trichloroethoxycarbonyl, 2-ethynyl-2-
propoxycarbonyl, cyclopropylmethoxycarbonyl,
isobornyloxycarbonyl, l-piperidyloxycarbonyl,
benzyloxycarbonyl ("Cbz"), 4-phenylbenzyloxycarbonyl,
2-methylbenzyloxy-carbonyl, ~-2,4,5,-
tetramethylbenzyloxycarbonyl ("Tmz"),
4-methoxybenzyloxycarbonyl, 4-fluorobenzyloxycarbonyl,
4-chlorobenzyloxycarbonyl, 3-chlorobenzyloxycarbonyl,
2-chlorobenzyloxycarbonyl, 2,4-dichlorobenzyloxycarbonyl,
4-bromobenzyloxycarbonyl, 3-bromobenzyloxycarbonyl,
4-nitrobenzyloxycarbonyl, 4-cyanobenzyloxycarbonyl,
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19
4-(decyloxy)benzyloxycarbonyl and the like; the
benzoylmethylsulfonyl group,
2,2,5,7,8-pentamethylchroman-6-sulfonyl group ("PMC")
dithiasuccinoyl ("Dts"), the 2-(nitro)phenylsulfenyl
- 5 group ("Nps"), the diphenyl-phosphine oxide group and
like amino-protecting groups. The species of amino-
protecting group employed is not critical so long as the
derivatized amino group is stable to the conditions of
the subsequent reaction(s) and can be removed at the
appropriate point without disrupting the remainder of the
compounds. Preferred amino-protecting groups are Boc,
Cbz and Fmoc. Further examples of amino-protecting
groups embraced by the above term are well known in
organic synthesis and the peptide art and are described
by, for example, T.W. Greene and P.G.M. Wuts, "Protective
Groups in Organic Synthesis," 2nd ed., John Wiley and
Sons, New York, NY, 1991, Chapter 7, M. Bodanzsky,
"Principles of Peptide Synthesis," 1st and 2nd revised
ed., Springer-Verlag, New York, NY, 1984 and 1993, and
J.M. Stewart and J.D. Young, "Solid Phase Peptide
Synthesis," 2nd ed., Pierce Chemical Co., Rockford, IL,
1984, E. Atherton and R.C. Shephard, "Solid Phase Peptide
Synthesis - A Practical Approach" IRL Press, Oxford,
England (1989), each of which is incorporated herein by
reference. The related term "protected amino" defines an
amino group substituted with an amino-protecting group
- discussed above.
The term "heterocyclel~ denotes optionally
substituted five-membered or six-membered rings that have
1 to 4 heteroatoms, such as oxygen, sulfur and/or
-
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nitrogen, in particular nitrogen, either alone or in
conjunction with sulfur or oxygen rin~ atoms. These
five-membered or six-membered rings may be saturated,
fully unsaturated or partially unsaturated, with fully
saturated rings bein~ preferred. An "amino-substituted
heterocyclic ring" means any one of the above-described
heterocyclic rings is substituted with at least one amino
group. Preferred heterocyclic rings include morpholino,
piperidinyl, piperazinyl, tetrahydrofurano, pyrrolo, and
tetrahydrothiophenyl.
Furthermore, the above optionally substituted
five-membered or six-membered rings can optionally be
fused to an aromatic 5-membered or 6-membered ring
system, such as a pyridine or a triazole system, and
preferably to a benzene ring.
The following ring systems are examples of the
heterocyclic (whether substituted or unsubstituted)
radicals denoted by the term "heterocyclic ring":thienyl,
furyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl,
isothiazolyl, oxazolyl, isoxazolyl, triazolyl,
thiadiazolyl, oxadiazolyl, tetrazolyl, thiatriazolyl,
oxatriazolyl, pyridyl, pyrimidyl, pyrazinyl, pyridazinyl,
thiazinyl, oxazinyl, triazinyl, thiadiazinyl,
oxadiazinyl, dithiazinyl, dioxazinyl, oxathiazinyl,
tetrazinyl, thiatriazinyl, oxatriazinyl, dithiadiazinyl,
imidazolinyl, dihydropyrimidyl, tetrahydropyrimidyl,
tetrazolo[1,5-b}pyridazinyl and purinyl, as well as
benzo-fused derivatives, for example benzoxazolyl,
benzthiazolyl, benzimidazolyl and indolyl.
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21
Further specific examples of the above
heterocyclic ring systems are 6-membered ring systems
containing one to three nitrogen atoms. Such examples
include pyridyl, such as pyrid-2-yl, pyrid-3-yl and
- 5 pyrid-4-yl; pyrimidyl, preferably pyrimid-2-yl and
pyrimid-4-yl; triazinyl, preferably 1,3,4-triazin-2-yl
and 1,3,5-triazin-4-yli pyridazinyl, in particular
pyridazin-3-yl, and pyrazinyl. The pyridine N-oxides and
pyridazine N-oxides, and the pyridyl, pyrimid-2-yl,
pyrimid-4-yl, pyridazinyl and the 1,3,4-triazin-2-yl
radicals, are a preferred group.
The substituents for the optionally substituted
heterocyclic ring systems, and further examples of the 5-
and 6- membered ring systems discussed above, are found
in W. Durckheimer et al., U.S. Pat. No. 4,278,793, issued
July 14, 1981, columns 9 through 21 and columns 188
through 233, herein incorporated by reference. (In
columns 33 through 188, examples of the term
"heterocyclic ring" are included in the heterocyclic
thiomethyl groups listed under heading "A".)
The term ''Cc to Cl5 alkyl heterocycle" denotes a
C1 to C6 alkyl group substituted at any position by a
heterocycle ring (heterocycle) from as described above,
said heterocycle containing up to 14 carbon atoms, as
long as sum of the carbon atoms of the alkyl chain (up to
6) and the carbon atoms of the heterocycle do not exceed
15. Similarly, the term "substituted C6 to Cls alkyl
heterocycle" refer to a C6 to Cls alkyl heterocycle group
substituted on the alkyl portion with the same
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W O 97/33174 PCT~B97/00349 22
substituents as listed for the C7 to C16substituted
alkylaryl groups and on the heterocycle as defined above
for substituted heterocyle.
The above single compound or library of an
approximately equal molar mixture of two or more
compounds has several preferred embodiments.
Specifically, in the embodiment where a single compound
is indicated, a preferred group of single compounds are
the interior amido compounds, that is, wherein X and Y
are taken together to form a carbonyl moiety. A
preferred group of interior amido single compounds are
the dimers, thus, wherein B, AA, BB and CC are zero,
except that AA can be zero or one when R7 is a hydrogen
atom and that CC can be zero or one when R3 is a hydrogen
1~ atom. In turn, a preferred group of interior amido
dimers are those that are cleaved from the solid support
and are not quaternized, thus, wherein Rg is a hydrogen
atom and Rlo is absent. A preferred group of cleaved
interior amido dimer single compounds are those wherein R3
and R7 are independently chosen from the group consiting
of S- or R-methyl, S- or R-benzyl, a hydrogen atom, S- or
R-(but-2-yl), S- or R-[ 4-(N-methylamino)-n-butyl],S- or
R-[4-(N-ethylamino)-n-butyl], S- or R-[4-(N-allylamino)-
n-butyl], S- or R-[4-(N-benzylamino)-n-butyl], S- or R-
~4-(N-(napth-2-ylmethylamino)-n-butyl], S- or R-[4-
(amino)-n-butyl], S- or R-[sec-butyl], S- or R-
(methylsulfinyl)eth-1-yl, S- or R-acetamido, S- or R -
(N,N-dimethyl)acetamido, S- or R- (N,N-diethyl)acetamido,
S- or R-(N,N-diallyl)acetamido, S- or R-(N-
allyl)acetamido, S- or R-(N,N-dibenzyl)acetamido, S- or
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23
R-(N-benzyl)acetamido, S- or R-(N,N-di(napth-2-
ylmethyl))acetamido, S- or R-(N-(napth-2-
ylmethyl))acetamido, S- or R-propionamido, S-or R- (N,N-
dimethyl)propionamido, S- or R-(N,N-diethyl)propionamido,
~ 5 S- or R-(N,N-diallyl)propionamido, S- or R-(N,N-
dibenzyl)propionamido, S- or R-(N,N-di(napth-2-
ylmethyl)propionamido, S- or R-[3-(guanidino)-n-propyl],
S- or R- [(N,N-diallyl)-3-guanidino-n-propyl], S- or R-
[(N,N,N'-triallyl)-3-guanidino-n-propyl], S- or R-
[(N,N,N'-trimethyl)-3-(guanidino)-n-propyl], S- or R-
[(N,N,N'-triethyl)-3-(guanidino)-n-propyl], S- or R-
hydroxymethyl, S- or R-[l -(hydroxy)ethyl], S-phenyl, S-
or R-[3-(carboxy)-n-propyl], S- or R-[ iso-propyl], S-
or R-[(indol-3-yl)methyl], S- or R-[(N-(methyl)indol-3-
yl)methyl],S- or R-[(N-(ethyl)indol-3-yl)methyl], S- or
R-[(N-(allyl)indol-3-yl)methyl], S- or R-[(N-
(benzyl)indol-3-yl)methyl], S- or R-[(N-(naphth-2-
ylmethyl)indol-3-yl)methyl], S- or R-(4-(methoxy))benzyl,
S- or R-(4-(ethoxy))benzyl, S- or R-(4-(allyloxy))benzyl,
S- or R-[4-hydroxybenzyl], S- or R-(n-butyl), S- or R-(n-
propyl), S- or R-[(napth-2-yl)methyl], AA is zero or one
when R, is a hydrogen atom, CC is zero or one when R3 is
hydrogen atom, S- or R-[cyclohexylmethyl], S- or R-
thiomethyl, or when either Rlor R2 are taken together
with R3 to form an S- or R- pyrrolidine or S-[4-
(hydroxy)pyrrolidine].
An especially preferred group of single
compounds referred to as cleaved interior amido dimers,
hereafter referred to as the "Type I" amido dimers, is
wherein R6and R~ are independently methyl, ethyl, allyl,
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24
benzyl, or napth- 2 -ylmethyl. A preferred group of "Type
I" amido dimers are the "Type II" amido dimers, thus,
wherein either R1or R2 are each a hydrogen atom, or one
of R1or R2 is a hydrogen atom and the other is taken
together with R3 to form an S-pyrrolidine ring. A
preferred group of the Type II interior amido dimers is
the N-terminal monomer as a proline residue, thus,
wherein one of Rlor R2 is a hydrogen atom and the other
is taken together with R3 to form an S-pyrrolidine ring.
A preferred group of N-terminal proline Type II interior
amido dimers occurs when R6 is napth-2-ylmethyl and R8 is
benzyl, and more so when R7 is S- or R-methyl, a hydrogen
atom, S- or R-E3-(guanidino)-n-propyl], S- or R-[4-(N-
benzylamino)-n-butyl], S-[iso-propyl], S-[2-
(methylsulfinyl)ethyl], S- or R-(n-propyl), S- or R-
(hydroxymethyl), S- or R-[n-butyl], R-[(napth-2-
yl)methyl], or S-phenyl, and especially so when the C-
terminal residue is S- or R-alanine, thus, R7is a S- or
R-methyl.
Another preferred group of Type II interior
amido dimers has the N-terminal residue as a
S-phenylalanine, and more so wherein R6 is ethyl and R8 is
(napth-2-yl)methyl, and especially so when R7is S-
methyl, S-(2-(methylsulfinyl)ethyl), a hydrogen atom, S-
(4-(hydroxy)benzyl) or S-[(hydroxy)methyl]. Of a special
note within this preferred group of compounds is the
compound wherein R, is S-methyl.
Another preferred group of Type I interior
amido dimers are wherein Rland R2are each a hydrogen atom
CA 02248078 1998-09-03
WO97133174 PCT~B97/~349
and R3is R-[(N-(napth-2-ylmethyl)indol-3-yl)methyl]. Of
note within this preferred group of Type I compounds are
wherein R6 is napth-2-ylmethyl and R8 is benzyl, and
especially so when R,is S- or R-[3-(guanidino)-n-propyl]
- 5 or S- or R- [4-(benzylamino)-n-butyl].
Yet another preferred group of Type I interior
amido dimers occurs wherein either Rl or R2is a hydrogen
atom or is taken in conjunction with R3 to form a
pyrrolidine ring, and the other is Clto Cl2acyl, C3 to C10
cycloalkyl, C3 to C6 heterocycle, Clto Cl2alkyl, Clto Cl2
substituted alkyl, C,to Cl6 alkylaryl, C7 to Cl6 substitued
alkylaryl, a C6 to Cl5 alkyl heterocycle, or a substituted
C6 to C1salkyl heterocycle.
Another preferred group of compounds of the
above Formula I wherein a single compound is indicated
is the interior amine compounds, in other words, wherein
X and Y are each a hydrogen atom. A preferred group of
these interior amine compounds is dimers, wherein B, AA,
BB and CC are zero, except that AA can be zero or one
when R, is a hydrogen atom and that CC can be zero or one
when R3 is a hydrogen atom. In turn, a preferred group of
interior amine dimers is those cleaved from the solid
support, thus, wherein Rgis a hydrogen atom and Rlo is
absent. A preferred group of cleaved interior amine
- 25 dimers is those wherein R6and R8are independently methyl,
benzyl or 4-hydroxybenzyl. A preferred group of these
preferred cleaved interior dimers are referred to
hereafter as "Type I cleaved interior amine dimers", that
is, wherein R3and R,are independently benzyl or 4-
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26
hydroxybenzyl. A preferred group of the Type I cleaved
interior amine dimers are those wherein R8is benzyl, R7is
4-hydroxybenzyl, R6is methyl, and R3is 4-hydroxybenzyl.
A more preferred group of these Type I compounds are
wherein a)R1and R2are the same and are methyl or a
hydrogen atom; b)either Rlor R2is a hydrogen atom and the
other is chosen from the group consisting of methyl, iso-
propyl, cyclopropylmethyl, 4-hydroxymethyl, N-
methylpiperidin-4-yl, and 3-(N,N-dimethylamino)-2-methyl-
prop-2-en-1-yl. Another preferred group of Type I
cleaved interior amine dimers are wherein Rais methyl, R7
is benzyl, R6is 4-hydroxybenzyl, and R3is 4-
hydroxybenzyl, and especially so wherein R1and R2are the
same and are either a hydrogen atom or methyl, or one of
Rlor R2is a hydrogen atom and the other is methyl.
Yet another preferred group of Type I cleaved
interior amine dimers are wherein R8is methyl, R7is 4-
hydroxymethyl, R6 is benzyl, and R3 is 4-hydroxybenzyl,
and especially so wherein R1and R2are the same and are
either a hydrogen atom or methyl, or one of Rlor R2is a
hydrogen atom and the other is methyl.
Another preferred class of compounds within the
invention encompassed by Formula I is a library of an
approximately equimolar mixture of two or more compounds.
A preferred group of this library of compounds are the
interior amido compounds, thus, wherein X and Y are taken
together to form a carbonyl group, and especially the
interior amido dimers, wherein B, AA, BB and CC are zero,
except that AA can be zero or one when R7 is a hydrogen
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27
atom and that CC can be zero or one when R3 is a hydrogen
atom.
A preferred group of library of interior amido
~ dimers are the resin-bound interior amido dimers, wherein
Rgis a solid support and R1~is absent. A preferred group
of these resin bound dimers are wherein R3and R7 are
independently chosen from the group consisting of S- or
R-methyl, S- or R-benzyl, a hydrogen atom, S- or R-~but-
2-yl), S- or R-[4-(t-butoxycarbonylamino)-n-butyl], S- or
R-[sec-butyl], S- or R-(methylsulfinyl)eth-l-yl, S- or
R-[ 3-(guanidino)-n-propyl], S- or R- [(N-PMC)-3-
(guanidino)-n-propyl], S- or R-(t-butyloxy)methyl, S- or
R-[2-(t-butyloxy)ethy], S-phenyl, S- or R-[2-(t-
butoxycarbonyl)ethyl], S- or R-[iso-propyl], S- or R-[(N-
(t-butoxycarbonyl)indol-3-yl)methyl], S- or R-[4-
hydroxybenzyl], S- or R-[ (4-(t-butyloxy))benzyl], S- or
R-(n-propyl), S- or R-(n-butyl), S- or R-[(napth-2-
yl)methyl], S- or R-(2-carboxyethyl), S- or R-
(cyclohexylmethyl), S-[(4-methoxybenzylthio)methyl], S-or
R-[(4- methylbenzylthio)methyl], S- or R-thiomethyl, S-
or R-[ 4-(N-methyl-N-(t-butoxycarbonyl)amino)-n-butyl],
S- or R-[ 4-(N-ethyl(N-(t-butoxycarbonyl)amino)-n-butyl],
S- or R-[4-(N-allyl(N-(t-butoxycarbonyl)amino)-n-butyl],
S- or R-[4-(N-benzyl(N-(t-butoxycarbonyl)amino)-n-butyl~,
S- or R-[4-(N-(naphth-2-yl)(N-(t-butoxycarbonyl)amino)-n-
- butyl], S- or R-acetamido, S- or R-[2-(N,N-
dimethylamino)ethyl], S- or R-(N, N-diethyl)acetamido, S-
or R-(N,N-diallyl)acetamido, S- or R-(N-allyl)acetamido,
S- or R-(N,N-dibenzyl)acetamido, S- or R-(N-
benzyl)acetamido, S- or R-(N,N-di(napth-2-
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28
ylmethyl))acetamido, S- or R-(N-(napth-2-
ylmethyl))acetamido, S- or R- propionamido, S- or R-(N,N-
dimethyl)propionamido, S- or R-(N,N-diethyl)propionamido,
S- or R-(N,N-diallyl)propionamido, S- or R-(N,N-
dibenzyl)propionamido, S- or R-(N,N-di(napth-2-
ylmethyl)propionamido, S- or R-[(N,N'-diallyl-N-PMC)-3-
guanidino-n-propyl], S- or R-[(N,N',N''-trimethyl-N-PMC)-
3-(guanidino)-n-propyl], S- or R-[(N,N',N''-triethyl-N-
PMC)-3-(guanidino)-n-propyl], S- or R-[N,N',N'' triallyl-
N-PMC-3-(guanidino)-n-propyl], S- or R-[(indol-3-
yl)methyl], S- or R-[(N-(methyl)indol-3-yl)methyl], S-
or R-[(N-(ethyl)indol-3-yl)methyl], S- or R-[(N-
(allyl)indol-3-yl)methyl], S- or R-[(N-(benzyl)indol-3-
yl)methyl], S- or R-[(N-(naphth-2-ylmethyl)indol-3-
yl)methyl], S- or R-(4-(methoxy))benzyl, S- or R-(4-
(allyloxy))benzyl, S- or R-(4-(ethoxy))benzyl, S- or R-
(4-(benzoxy))benzyl, S- or R-(4-(naphth-2-
ylmethoxy))benzyl, AA is one or zero when R7 is a hydrogen
atom, CC is one or zero when R3 is a hydrogen atom, or
when either Rlor R2 are taken together with R3 to form an
S- or R- pyrrolidine or S-[4-(hydroxy)pyrrolidine].
A more preferred group of the library of resin-
bound interior amido dimers, referred to hereafter as the
"Type I bound amido dimers (Library)", occurs wherein R6
and R~ are independently methyl, ethyl, allyl, benzyl, or
napth-2-ylmethyl. A preferred group of the Type I bound
amido dimers (Library) is wherein either Rl or R2is a
hydrogen atom or is taken in conjunction with R3 to form a
pyrrolidine ring, and the other is C1to Cl2acyl, C3 to C10
cycloalkyl, C3 to C6 heterocycle, C1to Cl2alkyl, C1to C12
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29
substituted alkyl, C7to C~6 alkylaryl, C7 to Cl6 substltued
alkylaryl, a C6 to Clsalkyl heterocycle, or a substituted
C6 to Cl5alkyl heterocycle.
- Another preferred group of the Type I bound
amido dimers ~Library) is wherein either Rlor R2 are each
a hydrogen atom, or one of Rlor R2 is a hydrogen atom and
the other is taken together with R3 to form an S-
pyrrolidine ring.
Another preferred group of interior amido
dimers (Library) are those that have been cleaved from
the solid support, hereinafter referred to as "cleaved
interior amido dimers ~Library)", and in the above
Formula I corresponds to wherein Rgis hydrogen and Rlois
absent. A preferred group of cleaved interior amido
dimers are wherein R3 and R7 are independently chosen
from the group consisting of S- or R-methyl, S- or R-
benzyl, a hydrogen atom, S- or R-(but-2-yl), S- or R-[ 4-
(N-methylamino)-n-butyl], S- or R-[4-(N-ethylamino)-n-
butyl], S- or R-[4-(N-allylamino)-n-butyl], S- or R-[4-
(N-benzylamino)-n-butyl], S- or R-[4-(N-(napth-2-
ylmethylamino)-n-butyl], S- or R-[ 4-(amino)-n-butyl], S-
or R-[sec-butyl], S- or R-(methylsulfinyl)eth-l-yl, S- or
R- acetamido, S- or R-[2-(N,N-dimethyl)acetamido], S- or
R-(N, N-diethyl)acetamido, S- or R-(N,N-
- 25 diallyl)acetamido, S- or R-(N-allyl)acetamido, S- or R-
(N,N-dibenzyl)acetamido, S- or R-(N-benzylacetamido, S-
and R-(N,N-di(napth-2-ylmethyl)acetamido, S- and R-(N-
(napth-2-ylmethyl)acetamido, S- or R-(N,N-
dimethyl)propionamido, S- or R-(N,N-diethyl)propionamido,
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S- or R-(N,N-dially)propionamido, S- or R-(N,N-
dibenzyl)propionamido, S- or R-(N,N-di(napth-2-
ylmethyl)propionamido, S- or R-
[3-(guanidino)-n-propyl], S- or R-[(N,N-diallyl)-3-
guanidino-n-propyl], S- or R-[(N,N,N'-triallyl)-3-
guanidino-n-propyl], S- or R-[(N,N,N'-trimethyl)-3-
(guanidino)-n-propyl], S- or R-[(N,N,N'-triethyl)-3-
(guanidino)-n-propyl], S- or R-hydroxymethyl, S- or R-[1
-(hydroxy)ethyl], S-phenyl, S- or R- [2-(carboxy)ethyl],
10 S- or R- [ iso-propyl], S- or R-[(indol-3-yl)methyl], S-
or R-[(N-(methyl)indol-3-yl)methyl], S- or R-[(N-
(ethyl)indol-3-yl)methyl], S- or R- [ (N-(allyl)indol-3-
yl)methyl], S- or R- [ (N-(benzyl)indol-3-yl)methyl], S- or
R-[(N-(naphth-2-ylmethyl)indol-3-yl)methyl], S- or R-(4-
(methoxy))benzyl, S- or R- (4- (ethoxy))benzyl, S- or R-(4-
(allyloxy))benzyl, S- or R- [4-hydroxybenzyl], S- or R- (n-
butyl), S- or R-(n-propyl), S- or R- [ (napth-2-yl)methyl],
AA is zero when R7 is hydrogen, CC is zero or one when R3
is a hydrogen atom, S- or R- (cyclohexylmethyl), S- or R-
thiomethyl, or when either R1or R2 are taken togetherwith R3 to form an S- or R- pyrrolidine or S-[4-
(hydroxy)pyrrolidine].
A more preferred group of library of cleaved
interior dimers, hereinafter referred to as "Type I
2S cleaved amido dimers", wherein R6and R8 are independently
methyl, ethyl, allyl, benzyl, or napth-2-ylmethyl.
Specific examples of the Type I cleaved amido dimers
occur when:
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31
(1) wherein R6 is napth-2-ylmethyl, R3 is R-
[(N-(naphth-2-ylmethyl)indol -3-yl) methyl],
and R1and R2are the same and are each a
hydrogen atom;
(2) wherein R6is ethyl, R3 iS benzyl and Rland
R2are the same and are each a hydrogen
atom;
~3) wherein R6is naphth-2-ylmethyl, R3 iS S-
methyl, Rland R2are the same and are each
a hydrogen atom; and
(4) wherein either R1or R2is a hydrogen atom
and the other is taken in conjunction with
R3 to form an S-pyrrolidine ring, and R6is
napth-2-ylmethyl.
Another preferred group of compounds within the
library of an approximately equimolar mixture of two or
more compounds of Formula I are the interior amine
compounds, thus, wherein in the above Formula I, X and Y
are the same and are each a hydrogen atom. A preferred
library of interior amine compounds are those that are
dimers, that is wherein B, AA, BB and CC are zero, except
that AA can be zero or one when R7 is a hydrogen atom and
that CC can be zero or one when R3 iS a hydrogen atom.
Preferred interior amine dimers (Library) are those that
have been cleaved from the solid support, wherein R9 is a
hydrogen atom and R1ois absent. A preferred group of
such cleaved interior amine dimers are wherein R3 and R7
-
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W O 97/33174 PCT~B97/00349 32
are independently chosen from the group consisting of S-
or R-methyl, S- or R-benzyl, a hydrogen atom, S- or R-
(but-2-yl~, S- or R-[4-(N-methylamino)-n-butyl], S- or R-
[4-(N-ethylamino)-n-butyl], S- or R-[4-(N-allylamino)-n-
butyl], S- or R-[4-(N-benzylamino)-n-butyl], S- or R-[4-
(N-(napth-2-ylmethylamino)-n-butyl], S- or R-[4-(amino)-
n-butyl], S- or R-[sec-butyl], S- or R-(2-aminoethyl), S-
or R-(methylsulfinyl)eth-1-yl, S- or R-[2-(N,N-
dimethylamino)ethyl], S- or R-(N, N-diethylamino)ethyl,
S- or R-(N,N-diallylamino)ethyl, S- or R-(N-
allylamino)ethyl, S- or R-(N,N-dibenzylamino)ethyl, S- or
R-(N-benzylamino)ethyl, S- and R-(N,N-di(napth-2-
ylmethylamino))ethyl, S- and R-(N-(napth-2-
ylmethylamino))ethyl, S- or R-(N-propyl)amine, S- or R-
(N,N-dimethylamino)propyl, S- or R-(N,N-
diethylamino)propyl, S- or R-(N,N-diallylamino)propyl, S-
or R-(N,N-dibenzylamino)propyl, S- or R-(N,N-di(napth-2-
ylmethylamino)propyl, S- or R-[3-(guanidino)-n-propyl],
S- or R- [(N,N-diallyl)-3-guanidino-n-propyl}, S- or R-
[(N,N,N'-triallyl)-3-guanidino-n-propyl], S- or R-
[(N,N,N'-trimethyl)-3-(guanidino)-n-propyl], S- or R-
[(N,N,N'-triethyl)-3-~guanidino)-n-propyl], S- or R-
hydroxymethyl, S- or R-~1 -(hydroxy)ethyl], S-phenyl, S-
or R-[3-(hydroxy)-n-propyl], S- or R-[ iso-propyl], S-
or R-~(indol-3-yl)methyl], S- or R-[(N-(methyl)indol-3-
yl)methyl],S- or R-[(N-(ethyl)indol-3-yl)methyl], S- or
R-[(N-(allyl)indol-3-yl)methyl], S- or R-[(N-
(benzyl)indol-3-yl)methyl], S- or R-[(N-(naphth-2-
ylmethyl)indol-3-yl)methyl], S- or R-(4-(methoxy))benzyl,
S- or R-(4-(ethoxy))benzyl, S- or R-(4-
(allyloxy))benzyl, S- or R-(4-hydroxybenzyl), S- or R-(n-
CA 02248078 1998-09-03
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33
butyl), S- or R-(n-propyl), S- or R-[(napth-2-yl)methyl],
AA is zero or one when R7 is a hydrogen atom, CC is zero
or one when R3 is a hydrogen atom, S- or R-
(cyclohexylmethyl), S- or R-thiomethyl, or when either R
- 5 or R2 are taken together with R3 to form an S- or R-
pyrrolidine or S-[4-(hydroxy)pyrrolidine]. A preferred
group of dimers within the immediately preceeding
preferred group occurs when R6and R8 are independently
- methyl, ethyl, allyl, benzyl, or napth-2-ylmethyl.
Another preferred group within the library of
interior amine dimers are the resin-bound compounds,
thus, wherein Rg is a solid support and R1ois absent.
A preferred group of the resin-bound interior amine
dimers (Library) occurs wherein R3 and R7 are
independently chosen from the group consisting of S- or
R-methyl, S- or R-benzyl, a hydrogen atom, S- or R-(but-
2-yl), S- or R-[4-(N-methylamino)-n-butyl], S- or R-[4-
(N,N-dimethylamino)-n-butyl], S- or R-[4-(N-ethylamino)-
n-butyl], S- or R-[4-(N-methyl-N-ethylamino)-n-butyl], S-
or R-[4-(N-allylamino)-n-butyl], S- or R- [4- ~N-methyl-N-
alkylamino)-n-butyl), S- or R- [4- (N-benzylamino)-n-
butyl], S- or R- [4- (N-methyl-N-benzylamino)-n-butyl], S-
or R- [4- (N-(napth-2-ylmethylamino)-n-butyl], S- or R-[4-
(N-methyl-N-naphth-2-ylmethylamino)-n-butyl], S- or R-
25 [4- (amino)-n-butyl], S- or R-[sec-butyl], S- or R-(2-
- aminoethyl), S- or R- (methylsulfinyl)eth-1-yl, S- or R-
acetamido, S- or R-[2-(N,N-dimethylamino)ethyl], S- or R-
(N, N-diethylamino)ethyl, S- or R-(N,N-
diallylamino)ethyl, S- or R-(n-allylamino)ethyl, S- or R-
(N,N-dibenzylamino)ethyl, S- or R-~N-benzylamino)ethyl,
CA 02248078 1998-09-03
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34
S- or R-(N,N-di(napth-2-ylmethylamino))ethyl, S- or R-(N-
(napth-2-ylmethylamino))ethyl, S- or R-(N-propylamine),
S- or R-propionamido, S- or R-(N,N-dimethylamino)propyl,
S- or R-(N,N-diethylamino)propyl, S- or R-(N,N-
diallylamino)propyl, S- or R-(N,N-dibenzylamino)propyl,
S- or R-(N,N-di(napth-2-ylmethylamino)propyl, S- or R-[3-
(N-PMC-guanidino)-n-propyl], S- or R-[(N,N'-diallyl-N-
PMC)-3-guanidino-n-propyl], S- or R-[(N,N',N''-triallyl-
N-PMC)-3-guanidino-n-propyl], S- or R-[(N,N',N''-
trimethyl-N-PMC)-3-(guanidino)-n-propyl], S- or R-
[(N,N',N''-triethyl-N-PMC)-3-(guanidino)-n-propyl], S- or
R-hydroxymethyl, S- or R-[1 -(hydroxy)ethyl], S-phenyl,
S- or R-[3-(hydroxy)-n-propyl], S- or R-[ iso-propyl],
S- or R-[(indol-3-yl)methyl], S- or R-[(N-(methyl)indol-
3-yl)methyl],S- or R-[(N-(ethyl)indol-3-yl)methyl], S- or
R-[(N-(allyl)indol-3-yl)methyl], S- or R-[(N-
(benzyl)indol-3-yl)methyl], S- or R-[(N-(naphth-2-
ylmethyl)indol-3-yl)methyl], S- or R-(4-(methoxy))benzyl,
S- or R-(4-(ethoxy))benzyl, S- or R-(4-(allyloxy))benzyl,
S- or R-[4-hydroxybenzyl], S- or R-[n-butyl], S- or R-(n-
propyl), S- or R-[(napth-2-yl)methyl], AA is zero or one
when R7 is a hydrogen atom, CC is zero or one when R3 is a
hydrogen atom, S- or R-[cyclohexylmethyl], S- or R-
[thiomethyl], or when either R1or R2 are taken together
with R3 to form an S- or R- pyrrolidine or S-[4-
(hydroxy)pyrrolidine]. A still more preferred group
within the library of resin bound interior amine dimers
occurs when R6and RB are independently methyl, ethyl,
allyl, benzyl, or napth-2-ylmethyl.
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Another aspect of the instant invention is a
method for effecting analgesia in a mammal, which
comprises administering an effective amount of a single
compound of Formula I in conjunction with a
- 5 pharmaceutically-acceptable carrier. A preferred method
of effecting analgesia in a mammal occurs when a single
compound that is an interior amido dimer and further
wherein B, AA, BB and CC are zero, Rgis a hydrogen atom,
RB is napth-2-ylmethyl, R7is S-methyl, R6is ethyl, R3is
S-benzyl, and R1and R2are each a hydrogen atom is used.
Another preferred method of effecting analgesia in
mammals utilizes a single interior amine dimer wherein
further Rgis a hydrogen atom, R~is benzyl, R,is S-methyl,
R6is naphth-2-ylmethyl, R3is taken in conjunction with
either Rlor R2to form an S-pyrrolidine ring and the other
of R1and R2a hydrogen atom.
Another aspect of the instant invention is a
method of effecting a decrease in the postprandial rise
in blood glucose of a m~m~l after ingestion of a
carbohydrate load by said mammal, which comprises
administering an effective amount of a single compound
of Formula I in conjunction with a pharmaceutically-
acceptable carrier. A preferred method of affecting a
decrease in the postprandial rise in the blood glucose of
a mammal occurs wherein the single compound has X and Y
taken together to form a carbonyl group, B, AA, BB and CC
are zero, Rg is a hydrogen atom, Re is benzyl, R6 is
naphth-2-ylmethyl, R3 is R-(N-(naphth-2-ylmethyl)indol-3-
ylmethyl), R1 and R2 are each hydrogen, Rlo is absent, and
R, is chosen from the group consisting of S-(4-(N-
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36
benzylamino)-n-butyl), R-(4-(N-benzylamino)-n-butyl),
S-(3-guanidino)-n-propyl), and R-(3-guanidino)-n-propyl).
Yet another aspect of the instant invention is
a method of treating microbial infections in mammals,
which comprises administering an effective amount of a
single compound of Formula I in conjunction with a
pharmaceutically-acceptable carrier. A preferred method
of treating microbial infections in mammals occurs when
wherein the single compound has X and Y taken together
to form a carbonyl group, B, AA, BB and CC are zero, Rg is
a hydrogen atom, R8 is benzyl, R6 is naphth-2-ylmethyl, R3
is R-(N-(naphth-2-ylmethyl)indol-3-ylmethyl), Rl and R2
are each hydrogen, Rlo is absent, and R~ is chosen from
the group consisting of S-(4-(N-benzylamino)-n-butyl), R-
(4-~N-benzylamino)-n-butyl), S-(3-guanidino)-n-propyl),
and R-(3-guanidino)-n-propyl).
Another aspect of the instant invention is a
method of step-wise N-alkylation of the amide bond of the
N-terminal residue of a compound of the Formula (II):
(II) R11 ~
Trityl N 1(CH2,Z NH--R12
Wherein:
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W O 97133174 PCT~B97100349 37
Rll is independently a hydrogen atom, Clto Cl2
alkyl, Clto Cl2substituted alkyl, phenyl, substituted
phenyl, C7 to C16 alkylaryl, C7 to C16 substitued alkylaryl,
a C6 to C1s alkyl heterocycleal, or a substituted C6 to Cls
- 5 alkyl heterocycle;
ZZ is from zero to five;
And Rl2 is a solid support or a group of the
Formula (III~:
(III)
~ \
R13 ~
/~(CH~ NR14R15
/ W
Wherein Rl4 is a C1 to C1B substituent group;
Wherein W is O to 4;
Rl3is independently a hydrogen atom, C1to Cl2
alkyl, C1to Cl2substituted alkyl, phenyl, substituted
phenyl, C,to Cl6 alkylaryl, C7 to Cl6 substitued alkylaryl,
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38
a C6 to Cl5 alkyl heterocycle, or a substituted C6 to Cls
alkyl heterocycle;
Rls is a solid support (when W is one) or a bond
to the preceeding methylene group (when W is from two to
four);
YY is from zero to five;
Wherein the compound of the above formula is
a)first reacted under anhydrous conditions in an inert
atmosphere with an excess amount non-nucleophilic base
having a pKa between about 18 to about 40; then
b)reacting the resulting anion under anhydrous conditions
in an inert atmosphere in a polar aprotic solvent with an
excess amount of an alkylting agent of the formula
(LG) -Q
Wherein LG is leaving group;
Q is a Cl to Cl8substituent groups as defined
above for Formula (I);
and repeating steps a) and b) as necessary to
drive the alkylation to completion;
with the proviso that all previous internal
backbone amide bonds have been previously alkylated with
a C1to Cl8substituent group and, when W is from 2 to 4,
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39
all of the Rl4 groups are not the same C1 to Cl8
substituent group.
A preferred method of step-wise N-alkylation
occurs when LG is iodo or bromo and the -C~2-Q moiety is
methyl, ethyl, allyl, benzyl or napth-2-ylmethyl. A
further preferred method of step-wise N-alkylation occurs
wherein Rlland Rl3 are indepently chosen from the group
consisting of S- or R-methyl, S- or R-benzyl, a hydrogen
atom, S- or R-tbut-2-yl), S- or R-[4-~t-
butoxycarbonylamino)-n-butyl3, S- or R-[4-(amino)-n-
butyl], S- or R-[sec-butyl], S- or R-(methylsulfinyl)eth-
l-yl, S- or R-[ 3-(guanidino)-n-propyl], S- or R-[(N-
PMC)-3-(guanidino)-n-propyl], S- or R-(t-butoxy)methyl,
S- or R-[2-(t-butoxy)ethy], S-phenyl, S- or R-(3-(2-
butoxycarbonyl)ethyl), S- or R-[iso-propyl], S- or R-[(N-
(t-butoxycarbonyl)indol-3-yl)methyl], S- or R-[4-
hydroxybenzyl], S- or R-[(4-(t-butoxy))benzyl], S- or R-
[n-propyl], S- or R-(n-butyl), S- or R-[(napth-2-
yl)methyl], S- or R-(cyclohexylmethyl), S-[(4-
methoxybenzylthio)methyl], S-[(4-
methylbenzylthio)methyl], S- or R-thiomethyl, S- or R-[4-
(N-methyl-(N-(t-butoxycarbonyl))amino)-n-butyl], S- or R-
[ 4-(N-ethyl-(N-(t-butoxycarbonyl))amino)-n-butyl], S- or
R-[4-(N-allyl-(N-(t-butoxycarbonyl))amino)-n-butyl], S-
or R-[4-(N-benzyl-(N-(t-butoxycarbonyl))amino)-n-butyl],
S- or R-[4-(N-(naphth-2-yl)-(N-(t-butoxycarbonyl))amino)-
n-butyl], S- or R-[2-(N,N-dimethyl)acetamido], S- or R-
acetamido, S- or R-(N, N-diethyl)acetamido, S- or R-
(N,N-diallyl)acetamido, S- or R-(n-allyl)acetamido, S- or
R-(N,N-dibenzyl)acetamido, S- or R-(N-benzyl)acetamido,
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S- or R-(N,N-di(napth-2-ylmethyl))acetamido, S- or R-(N-
(napth-2-ylmethyl))acetamido, S- or R-n-propylamine, S-
or R-propionamido, S- or R-(N,N-dimethyl)propionamido, S-
or R-(N,N-diethyl)propionamido, S- or R-(N,N-
diallyl)propionamido, S- or R-(N,N-dibenzyl)propionamido,
S- or R-(N,N-di(napth-2-ylmethyl)propionamido, S- or R-
[(N,N'-diallyl-N-PMC)-3-guanidino-n-propyl], S- or R-
[(N,N',N''-trimethyl-N-PMC)-3-(guanidino)-n-propyl], S-
or R-[(N,N',N''-triethyl-N-PMC)-3-(guanidino)-n-propyl],
S- or R-[N,N',N''-triallyl-N-PMC)-3-guanidino-n-propyl],
S- or R-[(indol-3-yl)methyl], S- or R-[(N-(methyl)indol-
3-yl)methyl], S- or R-[(N-lethyl)indol-3-yl)methyl], S-
or R-[(N-(allyl)indol-3-yl)methyl], S- or R-[(N-
(benzyl)indol-3-yl)methyl], S- or R-[(N-(naphth-2-
ylmethyl)indol-3-yl)methyl], S- or R-(4-(methoxy))benzyl,
S- or R- (4-(ethoxy))benzyl, S- or R- (4-(allyloxy))benzyl,
S- or R-(4-(benzoxy)benzyl, S- or R- (4- (naphth-2-
ylnethoxy)benzyl, ZZ is one or zero when R1l is a hydrogen
atom, YY is one or zero when R13 is a hydrogen atom, or
when either R13 is taken together with R14to form an S- or
R- pyrrolidine or S-[4-(hydroxy)pyrrolidine].
Another aspect of the instant invention
utilizes the positional scanning method and is a method
of synthesizing and testing for biological activity a
library of an approximately equimolar amount of compounds
of the following ~ormula (IV):
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41
(IV)
R~9 \ Rzl /B ~
Wherein in the above Formula (IV):
Rlg, R2l and R23 independently are a hydrogen
atom, Clto Cl2alkyl, Clto Cl2substituted alkyl, phenyl,
substituted phenyl, C7 to Cl6 alkylaryl, C7 to Cl6
substitued alkylaryl, a C6 to Cl5 alkyl heterocycle, or a
substituted C6 to Cl5 alkyl heterocycle;
R25 is a hydrogen atom or a solid support;
R20, R22 and R24 are independently a Clto Cl8
substituent group;
AA, BB and CC are independently 0 to 5;
B is from 0 to 3;
X and Y are taken together to form a carbonyl
group or are separate and are each a hydrogen atom;
R16, Rl7 and Rla independently are a hydrogen
atom, an amino protecting group, Clto Cl2acyl, C3 to ClO
cycloalkyl, C3 to C6 heterocycle, Clto Cl2alkyl, C1to C12
substituted alkyl, C7 to Cl6 alkylaryl, C7 to C16 substitued
alkylaryl, a C6 to Cl5alkyl heterocycle, or a substituted
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42
C6 to Cl5alkyl heterocycle; R16 is optionally present as a
Cl to Cl8 substituent group when R1 and R2 are other than a
hydrogen atom or an amino protecting group;
Wherein said library of compounds is composed
of SL physically separate sublibraries; wherein SL is
equal to (2~ + ~);
Further wherein each sublibrary is composed of
physically separate mixtures, wherein the number of said
mixtures is equal to the number of different substituents
incorporated at RfiX, which RfiX can be any one of Rlg, R20,
R2l, R22, R23, or R24 in the above Formula IV;
Wherein the compounds of the above Formula IV
are synthesized and tested as follows:
(a) For each sublibrary SL, choosing RfiX
from Rlg, R20, R2l, R22, R23, or R24;
(b) Dividing a solid support into
approximately equal separate portions with the number of
portions equal to the number of substituents to be
incorporated at R23, and couple each physically separate
portions of solid support to one of the monomers
containing a single substituent at R23, then mixing all of
said physically separate portions;
(c) Dividing the mixed solid support from
step (a) into approximately equal separate portions in a
number equal to the number of different substituents to
CA 02248078 1998-09-03
WO97t33174 PCT~B97/~349
43
be incorporated at R24 by alkylation, alkylating each
physically separate solid support mixtures with one alkyl
group, then mixing said resins;
(d) When B is 1 through 3, dividing each
of said solid support portions into a number of
approximately equal separate portions, said number equal
to the number of substituents at R21, coupling one of the
monomers containing a single substituent R23 to each
separate solid support portion then mixing said portions;
(e) When B is 1 to 3, separating said
mixture of solid support portions into a number of
approximately equal separate portions, said number equal
to the number of alkyl substituents at R20, alkylating
each physically separate portion with one such alkylating
agent, and mixing all the resultant solid support
portions;
(f) Optionally repeating steps (d) and
(e) one or two times when B is two or three,
respectively;
(g) Dividing the mixture of solid support
portions from either step (c), (e), or step (f) into
approximately equal separate portions equal to the number
of substituents to be placed at R19, coupling one such
monomer containing a single Rlg to each physically
separate solid support portion, and mixing said portions;
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W O 97133174 PCTAB97/00349 44
(h) Dividing the mixture of portions from
step (g) into a number of approximately equal separate
portions, said number equal to the number of alkyl
substituents at R22 to be utilized, alkylating each said
separate portion with a single alkyl group R22;
(i) Optionally adding R17 and/or R1B by
reductive alkylation;
(j) Optionally adding the quaternary
substituent Rl6 i
(k) Optionally reducing the interior
amides, thus converting X and Y taken together are a
carbonyl oxygen to wherein each X and Y is a hydrogen
atom; and
(l) Cleaving said molecules from the
solid support;
(m) Testing each portion of each SL
sublibraries in the appropriate biological screen or
screens; and determining from the results of said screens
which substitutent at RfiX is the best.
(n) Optionally synthesizing the molecule
of Formula (I) containing the best (RfiX) substitutent at
R19, R20, R2l, R22, R23, or R24;
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With the proviso that for each sublibrary SL
the first solid support mixing step immediately following
the introduction of RfiX is omitted;
- Further wherein:
(1) each coupling step in the above series
of steps ((b), (d), (f) and (g)) involves a substrate of
the Formula (V):
(V)
X ~Y \
R26N ><
\~(CH ) NR28 SS
R27
/ C
With an excess of an active acylating form of
the monomer of the Formula (VI):
(VI) PG- HN (CH2~ A
RVAR ~
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46
Wherein in the above Formulas (V) and (VI):
SS is a solid support;
R26 are two hydrogen atoms each bound to the
nitrogen atom;
R2B is a C1to C18 substituent group;
R27 is independently a hydrogen atom, C1to C12
alkyl, C1to C12substituted alkyl, phenyl, substituted
phenyl, C7 to C16 alkylaryl, C7 to C16 substitued alkylaryl,
a C6 to C1s alkyl heterocycle, or a substituted C6 to C1s
alkyl heterocycle;
Rv~ can be the same or different as R27 and is
chosen from the same group of substituents as R27;
DD and EE are independently 0 to 5;
X and Y are either taken together to form a
carbonyl oxygen;
PG is an amino protecting group other than
trityl;
A is a group, when taken with the preceeding
carbonyl group; that forms an active acylating agenti and
C is from 0 to 4;
(2) Each alkylating step in the above
steps (c), (e), (f) and (h) requires reacting a substrate
of the ~ormula (VII):
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47
(VII)
X >,~Y
R2gHN~(CH2)FF NR3,--SS
With an excess of an alkylating agent of the
Formula (VIII):
5 (VIII) ( LG) - Q
Under anhydrous conditions, and an inert
atmosphere in a polar, aprotic solvent;
Wherein in the above Formulas (VII) and (VIII):
LG is a leaving group under the conditions of
the alkylation;
Q is a C1to Cl8substituent group as defined
- above in Formula (I);
FF is O to 5;
X and Y are taken together to form a carbonyl
oxygen;
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48
R31 is a hydrogen atom when R29 is a trityl group
or is a Clto C18 substituent group;
R30 is independently a hydrogen atom, C1to Cl2
alkyl, C1to Cl2substituted alkyl, phenyl, substituted
phenyl, C7 to C16 alkylaryl, C, to Cl6 substitued alkylaryl,
a C6 to Cl5 alkyl heterocycle, or a substituted C6 to Cls
alkyl heterocycle; and
R29 is a trityl group when R3l is a hydrogen atom
or is a group of the Formula (IX~:
(IX)
R32
/\
Trityl N~(CH2)GG
R33 / C
Wherein in the above Formula (IX):
X and Y are as X and Y above;
GG is O to 5;
C is from 1 to 4;
R33 is independently a hydrogen atom, Clto C12
alkyl, Clto Cl2substituted alkyl, phenyl, substituted
phenyl, C7 to Cl6 alkylaryl, C7 to Cl6 substitued alkylaryl,
a C6 to Clsalkyl heterocycle, or a substituted C6 to Cls
alkyl heterocycle; and
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49
R32 is a hydrogen atom if R32 is bonded to the N-
terminal amino group or otherwise it is a Cl to C18
substituent wherein one such Cl to Cl8 substituent differs
from the other substituents;
-
(3) Reductive alkylation of the N-
terminal nitrogen group as described above in step (i) of
a compound of the Formula (X):
~X)
\~ RX~ ~ ~(CH2~R~2 SS
Under mildly acidic conditions with a ketone or
aldehyde containing the R34and/or R35 groups followed by
the treatment of a reducing agent;
Wherein in the above Formula (X):
X and Y are taken together to form a carbonyl
oxygen;
HH, II, and JJ are independently 0 to 5;
B is from 0 to 3;
R39 , R41 and R37 are the same or different and
are chosen from the group consisting of independently a
hydrogen atom, C1to C12alkyl, C1to C12substituted alkyl,
phenyl, substituted phenyl, C7to Cl6 alkylaryl, C7 to C16
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substitued alkylaryl, a C6 to Cl5alkyl heterocycle, or a
substituted C6 to Cl5 alkyl heterocycle;
R40 and R42are different and are a Cl to C1a
substituent group;
R34 or R35 when B is 0, is optionally one or two
hydrogen atoms attached to the nitrogen atom, or is a
optionally one or more, same or different groups, chosen
from the group consisting of a hydrogen atom, an amino
protecting group, Clto Cl2acyl, C3 to Cl0 cycloalkyl, C3
to C6 heterocycle, Clto Cl2alkyl, Clto Cl2 substituted
alkyl, C,to Cl6 alkylaryl, C7 to Cl6 substituted alkylaryl,
a C6 to Cl5alkyl heterocycle, or a substituted C6 to Cl5
alkyl heterocycle;
R36 is a hydrogen or a bond to a R34 or R35 before
the reduction occurs, and when B is from 1 to 3; R38 is a
C1to Cl8substituent group different from at least one
other R40, R42 or R38 groups;
(4) Optional reduction of the amide bonds
as described above in step (j) of a compound of Formula
(X) wherein X and Y are taken together to form a carbonyl
groups, before or after it is cleaved from the solid
support, using a boron reducing agent.
(5) Optionally quaternization of the
terminal amino groups with an excess amount of alkylating
agent of above of the formula:
(LG) -Q
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51
Where (LG) and Q have the same meanings as
above in a polar, aprotic solvent.
It will be obvious to one skilled in the art
that, in the above positional scanning method, the
substituents at R34, R3s and R36 can be designated as "RfiX",
such that the number of sublibraries SL would be
increased by one, two or three, respectively, depending
on how many of these three "R" groups are varied.
Yet another aspect of the invention is an
iterative synthetic approach wherein the method for the
iterative synthesis and screening of a library of an
approximately equimolar amount of compounds of the
Formula (XI):
(XI)
X X y X X y
R43N ~ (CH2)KK I ~/ (CH2)LL NR47R48
R45 ~
R44 R46
Wherein in the above Formula (XI):
R48 is a hydrogen atom or a solid support;
R4sand R47 are different and are each a Cl to Cl8
substituent group;
KK and LL are independently 0 to 5;
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52
R44 and R46 are independently chosen from the
group consisting of independently a hydrogen atom, Clto
Cl2alkyl, Clto Cl2substituted alkyl, phenyl, substituted
phenyl, C7 to Cl6 alkylaryl, C7 to Cl6 substitued alkylaryl,
a C6 to Cls alkyl heterocycle, or a substituted C6 to Cls
alkyl heterocycle;
X and Y are either taken together to form a
carbonyl group or are separate and are each a hydrogen
atom;
R43is one or two hydrogen atoms, or groups of
the formula Ra~ Rb and Rc, wherein Ra and Rb independently
are a hydrogen atom, an amino protecting group, Clto Cl2
acyl, C3 to ClO cycloalkyl, C3 to C6 heterocycle, Clto Cl2
alkyl, Clto Cl2 substituted alkyl, C7 to Cl6 alkylaryl, C7
to Cl6 substitued alkylaryl, a C6 to Cls alkyl heterocycle,
or a substituted C6 to Clsalkyl heterocycle; Rc is
optionally present as a Cl to Cl8 substituent group when Ra
and Rb are other than a hydrogen atom or an amino
protecting group; and
Wherein the method comprises:
(a) Splitting a solid support into a
number of approximately equal, separate portions, the
number of said portions being equal to the number of
monomers containing different substituent groups at R46;
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53
(b) Coupling a monomer containing one of
the number of substituent groups at R46 to a separate
portion of the solid support;
(c) Mixing all of the separate portions
of solid support;
(d) Splitting the solid support mixture
into approximately equal, separate portions, the number
of portions equal to the number of different substituents
to be added at R47;
(e) Alkylating each separate portion of
solid support with a single alkylating agent, each agent
containing a unique alkyl group at R47, thus adding a
single alkyl group at R47 to the plurality of the
compounds bonded to each separate portion of solid
support;
(f) Mixing all of the separate portions
of solid support ;
(g) Splitting the solid support mixture
into a number of approximately equal separate portions,
the number of said portions equal to the number of
different substituents to be added at R44;
(h) Coupling each monomer containing a
single substituent group at R44 to a separate portion of
the solid support, thus coupling a single different
CA 02248078 1998-09-03
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monomer to the plurality of the compounds bonded to each
separate portion of the resin;
(i) Splitting each of the separate
portions of solid support into a number of approximately
equal physically-separate portions, wherein the number of
portions is equal to the number of different substituents
to be added by alkylation at R4s;
(j) Alkylating each separate portion of
solid support with a separate alkylating agent containing
a single different R45 group, thus adding a single
different alkyl group at R4s to the plurality of the
compound bonded to each separate portion of the resin;
(k) Cleaving the generated compound
mixtures of Formula (XI) from each separate portion of
solid support and testing each separate mixture from each
separate portion of solid support in the appropriate
biological screen or screens, and determining from the
results of said screens which mixture contains the best
combination of substituents at R44 and R4s;
(1~ Repeating steps (a) through (e),
wherein the substituents at R46 and R47 are the same used
in said original steps (a) through (e);
(m) Coupling the monomer containing the
most active R44 substituent to each of the separate
2S portions of resin from step (l);
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(n) Alkylating each of the portions from
step (m) with the best alkyl group at R45 determined in
step ~k);
~ o) Cleaving each separate mixture of
compounds of the above Formula (XI) from the solid
support, testing each separate mixture of compounds in
the same biological screens as in step (k), and
determining the most active substituent at R47 in those
screens;
(p) Repeating steps (a) and (b), wherein
the same group of monomers containing the various
substituent R46 are used as in the original step (a);
(q) Alkylating each separate resin
portion from step (p) with an alkylating agent placing
the best alkyl group at Rq7 as such alkyl group was
determined in step (o);
(r) Coupling to each separate portion of
resin the monomer containing the best R44 substituent as
such substituent was determined in step (k);
(s) Alkylating each separate portion of
resin with a group that was the best alkyl group R4s as
such group determined in step (k);
(t) Cleaving each separate compound from
the solid support, and testing each separate mixture of
compound separately in the same screens as in steps (o)
CA 02248078 l998-09-03
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56
and (k) in order to determine the best substituents at
R46;
(aa) Optionally reductively alkylating and
quaternizing the N-terminal amino group (R43) , either
before or after cleavage of the compound from the solid
support; and
(bb) Optionally reducing the interior
amide groups before or after cleavage of the compound
from the solid support such that X and Y in Formula (XI)
are each a hydrogen atom;
Further wherein:
(1) each of the above coupling steps (b),
(h), (1~, (m), (p) or (r), involves a substrate of the
Formula (XII):
15 (XII) / X Y \
R4gN ~
\~(CH2)MM
\ R50
/ C
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57
With an excess of an active acylating form of
the monomer of the Formula (XIII):
(XIII)
PG--HN~ (CH2~ A
RVAR2 ~
Wherein in the above Formulas (XII) and (XIII):
SS is a solid support;
R49 is two hydrogen atomsi
R5l is a Clto Cl8 substituent group
R50 is independently a hydrogen atom, Clto Cl2
alkyl, Clto Cl2substituted alkyl, phenyl, substituted
phenyl, C7 to Cl6 alkylaryl, C7 to Cl6 substitued alkylaryl,
a C6 to Cl5 alkyl heterocycle, or a substituted C6 to Cls
alkyl heterocycle;
RV~2 can be the same or different as R50 and is
chosen from the same group of substituents as R50;
MM and NN are independently 0 to 5;
X and Y are either taken together to form a
carbonyl group or are separate and are each a hydrogen
atom;
PG is an amino protecting group other than
trityl;
A is a group, when taken with the preceeding
carbonyl group; that forms an active acylating agent; and
CA 02248078 1998-09-03
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58
C is O or 1;
(2) Each of the above alkylating steps
(e), (j), (1), (m~,(q) and (s), requires reacting a
substrate of the Formula (XIV):
(XIV)
X ><~Y
Rs2HN~ (CH2)00
53
With an excess of an alkylating agent of the
Formula (XV):
(xv) (LG) - Q
Under anhydrous conditions, and an inert
atmosphere in a polar, aprotic solvent;
Wherein in the above Formulas (XIV) and (XV):
LG is a leaving group under the conditions of
the alkylation;
CA 02248078 1998-09-03
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59
Q is a Clto Cl~substituent group;
OO is 0 to 5;
X and Y are taken together to form a carbonyl
group; or are separate and are each a hydrogen atom;
Rs4 is a hydrogen atom if Rs2 is a trityl group
or is a Clto Cl~ substituent group;
R53 is independently a hydrogen atom, Clto Cl2
alkyl, Clto Cl2substituted alkyl, phenyl, substituted
phenyl, C7 to Cl6 alkylaryl, C7 to Cl6 substitued alkylaryl,
a C6 to Cls alkyl heterocycle, or a substituted C6 to C15
alkyl heterocycle;
R52 is a trityl group if R54 is a hydrogen atom
or is a group of the Formula (XVI):
(XVI)
X X Y
R55HN ~ (CH2)PP
R56
Wherein in the above Formula (XVI):
X and Y are as X and Y above
PP is 0 to 5;
R55 is a trityl group;
Rs6 is independently a hydrogen atom, Clto Cl2
alkyl, Clto C12substituted alkyl, phenyl, substituted
phenyl, C7 to C16 alkylaryl, C7 to Cl6 substitued alkylaryl,
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a C6 to C15 alkyl heterocycle, or a substituted C6 to C15
alkyl heterocycle; and
(3) Optional reductive alkylation of
the N-terminal nitrogen group of a compound of the
Formula (XVII):
(XVII)
XXY XXY
R57N ~ (CH2)QQ I ~ (CH2)RR NR6
R58 R60
Under mildly acidic conditions with a ketone or
aldehyde containing the Rland/or R2 groups followed by
treatment with a reducing agent;
Wherein in the above Formula (XVII):
X and Y are taken together to form a carbonyl
group;
QQ and RR are independently 0 to 5;
Rs8 and R60 are the same or different and are
chosen from the group consisting of independently of a
hydrogen atom, C1to C12alkyl, C1to C12substituted alkyl,
phenyl, substituted phenyl, C7 to Cl6 alkylaryl, C7 to C16
substitued alkylaryl, a C6 to C1s alkyl heterocycle, or a
substituted C6 to C1s alkyl heterocycle;
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61
R59 and R6,are the same or different and are a C
to C1~ substituent group;
Rs7 is either two hydrogen atoms attached to
the nitrogen atom, or is a single hydrogen atom and
another group bonded to the nitrogen atom which groups is
selected from the group consisting of a hydrogen atom, an
amino protecting group, C,to C,2acyl, C3 to C,0
cycloalkyl, C3 to C6 heterocycle, Clto C,2alkyl, Clto C12
substituted alkyl, C7 to C,6 alkylaryl, C7 to C,6 substitued
alkylaryl, a C6 to Cl5alkyl heterocycle, or a substituted
C6 to C,5 alkyl heterocycle;
(4) Optional reduction of the amide bonds
of a compound of Formula (XI), before or after it is
cleaved from the solid support, using a boron-based
reducing agent, such as borane or sodium borohydride, and
the like.
In the above iterative synthetic approach, it
would be obvious to one skilled in the art the method
could easily be extended to encompass a library of all of
the compounds of Formula I, in other words, such that
repeating the coupling; alkylation and testing steps
described above be applied to compounds within the scope
of Formula I, wherein B is 1, 2, or 3. Furthermore, it
would be obvious to one skilled in the art that
substitutent at R" R2 and Rlo would be separate variables
that could be synthesized and screened by the above
iterative method. Due to their location in the molecule,
these substituents, if present, would be screened to find
,
CA 02248078 1998-09-03
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62
the best substituent before the N-terminal monomer and
the attendant N-alkyl group could be determined.
Finally, one skilled in the art would be able
to combine the above iterative and positional scanning
approaches in order to conserve resources and time. For
example, in libraries where B in the above Formula I is
1, 2, or 3, the iterative approach could be used to
determine the optimum substituents on the last two
variable substituents and the optimum substituents at the
rem~;n;ng positions could be determined by the positional
scanning approach.
Simultaneous multiple solid phase methodology
(Merrifield, R.B., J. Am. Chem. Soc., 85:2149 (1963)) was
the basic technology used to synthesize and design the
peptidomimetic library set forth in Formula I. A solid
phase-based synthetic method was developed to
successively alkylate each amide bond following its
formation. In this library, different alkylating agents
were used to create increased molecular diversity and to
eliminate the hydrogen bonding potential of the amide
functionality. Optionally, the N-terminal nitrogen can
be reductively alkylated and quarternized and the
interior amide bonds can be reduced (i.e., X and Y are
each a hydrogen atom). In Formula I, when B is 2 or 3, R4
and R5 do not have to be the same as the other R4 and R5
groups present in the molecule. Cleavage from the solid
support led to peptidomimetics of the Formula I (wherein
Rgis hydrogen, each having diversity positions at the
amino acid side chain positions (R3, Rs and R7), at the
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63
amide alkyl groups (R4, R6, RA)~ and at the N-terminal
groups (Rl, R2 and Rlo)
Although a number of methods for the
permethylation of peptides in solution (Hakomori, S.-I.,
J. E~10chem 55:205 (1964); Vilkas, E., et al., Tetrahedron
T.etters, 26:3089 (1968); Challis, B.C., et ~1., 1 ~
Chemistry of Aml (les, Zabicky, J. Ed.; Interscience: New
York, 1970, PP. 731-857; are known, the permethylation
of resin-bound peptides, using sodium hydride for the
formation of amide anions, was reported only recently
(ostresh et al., Proc. N~tl. Acad. Sci. USA
91:11138-11142 (1994)). For the purpose of a stepwise
alkylation following each amino acid coupling on the
solid support, lithium t-butoxide was found to be more
effective for the successive formation of the amide
anions.
As an important prerequisite for the synthesis
of this library, reproducible conditions for the N-amide
alkylations had to be established for the base treatment
of solid phase-bound amino acids or peptides. The
reactions were carried out under an anhydrous nitrogen
atmosphere, and the amino acid or peptide resin of
interest was treated with excess lithium t-butoxide in
tetrahydrofuran. Following removal of excess base, the
alkylating agent in an aprotic, polar solvent such as
dimethyl sulfoxide was reacted with the resin-bound
compound. The alkylation reaction mixture was then
removed and the base and alkylation treatments were
repeated to drive the alkylation reaction to completion.
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64
Potential racemization during alkylation was studied
using analytical reversed-phase hi~h performance liquid
chromatography (RP-HPLC); the four possible permethylated
stereoisomers of Phe-Leu-NH2 were used as reference
standards. (Ostresh, J.M., et al., Peptides 94:
Procee~;ngs of the ~3r~ Furope~n Pepti~e Sy~os;l~m. Maia,
H.L.S. Ed.; Escom: Leiden, 1995, pp. 416-417). The
maximum percentage of racemization found following
repeated base and methylation treatments was c 1%.
The techniques for the synthesis of the
selectively N-alkylated compounds of Formula I are well
known in the art, with the exception of the selective N-
alkylation procedures discussed above. These techniques
can be conveniently di~cussed in conjunction with Scheme
1 :
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W O 97/33174 PCT~B97/00349
sr~
H2N-M BHA-resin
R7 H,
Trt~N~N-MBHA-re8in
R7 R"~
Trt~N~N-MBHA-resin
H O
iii
T 'N~N~N-MBHA-resin
R3 H O iv
T t'N~N~N-MBHA-resin
R3 R6 0 v
~N, ~ R5
R3 R6 ~
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66
In the above Scheme l, Reaction "i" represents
the coupling of the C-terminus of peptide-like residue to
an amino-derivatized solid support. For instance, an
peptide-like residue with amino terminus protected by a
base or weak-acid labile protecting group, such as Fmoc,
is converted to a good acylating agent ; n situ using
known reagents and conditions. Such reagents include
carbodiimide reagents (eg. N,N'-dicyclohexylcarbodiimide
(DCC) and N,N'-diiso-propylcarbodiimide in conjunction
with l-hydroxybenzotriazole) in an aprotic, polar solvent
such as DMF. (Such couplings were repeated if necessary.)
In the reaction labelled as "ii" in Scheme 1, the N-
terminal protecting group was removed and replaced with a
trityl group, which was found to protect such group
during the next interior, selective N-alkylation step.
Under anhydrous conditions and an inert atmosphere, the
tritylated residue was treated with an excess amount of
lithium t-butoxide in a polar, aprotic solvent (such as
tetrahydrofuran), followed by the addition of an excess
of an alkylating agent of the formula
(LG) -Q
wherein LG is a good leaving group under the Sn2
conditions of the reaction (such as bromo, iodo, tosyl,
triflate and the like), and Q forms a C1to Cl8linear,
2~ branched, cyclic, saturated, partially or fully
unsaturated alkyl group, as described above in conjuction
with the "C1to C18substituent group" of R4 R6 and R8 of
Formula I. Multiple repetitions of such alkylating
CA 02248078 1998-09-03
W O 97/33174 rCT~B97tO0349 67
conditions are often necessary. The reaction denoted as
"iii" in the above Scheme 1 denotes the steps necessary
to couple a second (and any additional) monomer (as
defined in Formula II) the recently N-alkylated resin-
bound residue. Thus, the trityl protecting group isremoved under weakly acidic conditions (2~
trifluoroacetic acid), the deprotected molecule
neutralized then coupled with and FMOC-protected amino
acid (or one bearing an equivalent protecting group)
using the same conditions for coupling the first residue
to the amino-deriviatized resin. The reaction labelled
"iv" in Scheme 1 is a repeat of the selective N-
alkylation procedure, including the preliminary N-
protection steps, of reaction "ii". Reaction "v" shows
the removal of the trityl group from the amino-terminus
of the bound residue as before followed by the removal of
the selectively-N-alkylated molecule from the amino-
derivatized residue with hydrogen fluoride. One skilled
in the art would recognize that subsequent residues could
be added then selectively alkylated before removal of
the amino-derivatized according to the steps set forth in
Scheme 1, thus yielding compounds of Formula I wherein B
is 1, 2 or 3. Furthermore, while still attached to the
resin, it is advantages to derivatize the N-terminal
amino group to form compounds wherein at least one of R
and R2is other than hydrogen, and where possibly R1o is
present. Rland R2groups are most frequently added by the
process of reductive alkylation, as set forth (Borch,
R.F., et al., J. Am. Chem. Soc., 93:2897 (1971); Coy,
D.H., et al., Tetrahedron, 44:835 (1988); Sta~kova, M.,
et ~l., Dru~ Development Research, 33:146 (1994)(herein
CA 02248078 1998-09-03
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68
incorporated by reference). Thus, appropriate aldehyde or
ketone is added to the resin-bound compounds under mildly
acidic conditions to effect Schiff-base (imine)
formation, which imine is reduced to the substituted
amine by sodium cyanoborohydride, or other mild reducing
agent. Additionally, the free amino terminus can be
acylated with a C1to C12acyl group, using well-known
conditions as described in Sta~kova, M., et al., ~L~g
Develo~ment Research, 33:146 (1994) (herein incorporated
by reference). It is preferable, however, to add the Rl~
(alkyl) substituent before reductive alkylation. Such an
alkylation proceeds under the same alkylation conditions
used for the R4, R6, and RB groups. Finally, while still
resin-bound,or after cleavage from the resin, the
interior amide groups can be reduced (i.e. X and Y taken
together form a carbonyl group to X and Y are each a
hydrogen atom.) Such a reduction is known in the art
(see, for instance, Dooley, C.T., et al., ~nalges;a, INRC
Procee~-~gs, 1:400 (1995)). Thus, for both situations
(i.e.,when R9 is a hydrogen atom or a solid (resin))
mild,soluble hydrogenation catalysts such as a boric
acid/trimethylborate/borane-tetrahydrofuran combination
can be used.
Individual model compounds were used to study
the modification of amino acid side chains during the
alkylation conditions. Fifty N-trityl (triphenylmethyl;
Trt) dipeptide resins, designated Trt-O-Leu-MBHA resin
(MBHA = p-methylbenzhydrylamine) were alkylated where O
represents a single proteinogenic L-amino acid, their D-
counterparts, or 11 other individual "unnatural" amino
CA 02248078 l998-09-03
WO97/33174 PCTnB97/00349 69
acids. Aspartic acid was excluded from the 20
proteinogenic amino acids, since multiple products were
formed following base treatment and alkylation. Methyl
iodide, allyl bromide and benzyl bromide were used
initially as alkylating agents. The individual crude
alkylated products were analyzed by RP-HPLC and matrix
assisted laser desorption ionization-mass spectroscopy
(MALDI-MS) to determine their purity and identity.
(Ostresh, J.M., et ~1., Pept;~es 94: Procee~'ngs of the
23r~ ~urope~n PeDt;~le S ~ OS;l~m, Maia, H.L.S. Ed.; Escom:
Leiden, 1995, pp. 463-464). During the alkylation
procedure, the functional groups of the amino acid side
chains were reproducibly modified. Based on preliminary
evidence the following modifications were observed: 1)
theE-monoalkyl amine was formed during alkylation when
the e-amino group of lysine was protected with Boc; 2)
the unprotected amide functionality of the side chains of
L-asparagine and L-glutamine, when alkylated with any of
the three alkylating agents, yielded dialkyl amides,
whereas allylation and benzylation of the D-isomers led
to mono and dialkyl amides, indicating stereochemical
hindrance of the diastereomers; 3) the 2,2,5,7,8-
pentamethylchroman-6-sulfonyl (Pmc) protected arginine
side chain yielded the trimethyl derivative following
permethylation and di- and triallyl derivatives following
perallylation, but was negligibly alkylated following
perbenzylation; 4) when unprotected, the reactive indole
nitrogen of tryptophan was alkylated; 5) the use of 2-
bromo-Cbz protection for tyrosine resulted in formation
of the methyl and allyl ether analogs and any O-benzyl
products formed using benzyl bromide in the alkylation
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were cleaved during the hydrogen fluoride treatment; and
6) when tyrosine hydroxyl was t-Bu protected, the side
chain was unmodified. Although not studied in detail,
glutamic acid t-Bu ester led to multiple products
following repeated alkylations. Other amino acid
derivatives having side chains with potentially reactive
functionalities, including serine, threonine,
hydroxyproline (all protected as their t-Bu ether),
methionine (sulfoxide), and tryptophan (Boc), did not
undergo any modification during the alkylation step.
Repetitive alkylations of trityl-protected N-terminal
glycine and ~-alanine led to side products containing
additional alkyl groups as detected by MALDI-MS.
The combinatorial library of compounds of
lS Formula I (wherein B=0) has an OOXX format, where O
represents a defined position and X represents a mixture
position. Forty-six different amino acids (cysteine and
histidine were excluded since analogs containing these
amino acids were found to have significant side reactions
and/or incomplete reaction during the alkylation
procedure) were incorporated into the first X position
(R3), and 50 different amino acids were incorporated into
the first O position (R7). The amide alkyl groups in the
second X (R8) and second O positions (R6) were: methyl,
ethyl, allyl, benzyl or naphthylmethyl. This
combinatorial library consists of 250 mixtures (50 amino
acids x 5 alkyl groups), each of which is composed of 230
compounds (46 amino acids x 5 alkyl groups), and was
prepared applying the divide, couple and recombine
process, also independently reported as the "mixing and
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71
portioning" and "split synthesis" approaches (Lam, K.S.,
et al., Nature, 354: 82 (1991); Furka, A., et a]., Int. J.
Pept. Protein Res., 37:487 (1991)). The stepwise
synthesis was carried out on the solid phase by
alternating amino acid attachment and alkylation of the
previously formed amide bond as outlined in ~igure 1.
Standard Fmoc chemistry for the incorporation of amino
acids was used with MBHA resin as the solid support.
Alkylation of the amide bond between the C-terminal amino
acid and the MBHA linker was found to significantly
decrease the stability of the amide-resin linkage to
acidolytic conditions. (Kornreich, W., et al., I~t. J.
Pept. Prote'n Res., 25:414 (1985)). The amino groups
were protected with the bulky trityl group to avoid
modification of the N-terminal amine during the
manipulation of the amide groups of the resin-bound
compounds. The five alkyl halides [methyl iodide, ethyl
iodide, allyl bromide, benzyl bromide, and 2-
(bromomethyl)naphthalene] were reacted with the
previously formed amide anions using repeated treatments
of the alkylation method described above. Replicates of
control resins Trt-Leu-MBHA and Trt-Trp-MBHA were added
during each of the five separate alkylation treatments on
the solid phase resins. The Ra residues were introduced
at the same time, enabling the completeness of each of
these reactions to be determined. A second amino acid
derivative was then coupled to these control resins
following removal of the trityl group with 2~
trifluoroacetic acid in dichloromethane. This resulted
in the generation of individual compounds having the
formulas H2N-Phe-Leu-NHR and H2N-Ala-Trp(R)-NHR (R =
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methyl, ethyl, allyl, benzyl, or naphthylmethyl). No
starting material was detected by RP-HPLC for the crude
compounds following three treatments with methyl iodide
and ethyl iodide. Allylation, benzylation and
naphthylmethylation required six repetitions of the
alkylation procedure, with generally less than 10~
starting material re~;n'ng (as determined by RP-HPLC).
In case of the Ala-Trp controls up to 40~ of
monoalkylated material was seen (using RP-HPLC). The 230
resins containing the first two library positions were
then combined, thoroughly mixed and divided into 250
equal portions (50 x 5 library resin packets). Following
trityl removal, the second group of protected amino acids
was added (cysteine and histidine included), the Fmoc
1~ group was removed, and the free amino groups were again
reacted with trityl chloride. The newly formed amide
bond was then alkylated as described above, with the
exception that five repetitions of the alkylation
procedure were carried out. For this second alkylation
step, control resins were prepared having the formula
Trt-Phe-Leu-NHMe-MBHA and Trt-Ala-Trp-NHMe-MBHA. These
control resins were permethylated at the first amide
position to determine the completeness of the second
alkylation. Following trityl removal, starting material
was not detected by RP-HPLC or MALDI-MS for any of the
five crude alkylation control products. The highly acid
labile amide linkage between the peptidomimetic and the
MBHA resin linker does not permit the acid labile side
chain protecting groups to be removed prior to final
cleavage from the resin. Thus, the mixtures were cleaved
from the resin under standard high hydrogen fluoride
CA 02248078 1998-09-03
WO97t33174 PCTAB97/00349
73
cleavage conditions (Houghten, R.A., et al., Int. J. Pept.
Prote;n Res., 27:673 ~1986)) and obtained as lyophilized
powders following extraction with 50~ aqueous
acetonitrile. The yields of some of the crude control
compounds were found to be sequence-dependent. During
the final acidic Trt removal, compounds having bulky
alkyl residues in position R2 were partially cleaved from
the resin. (Gisin, B.F., et ~l., J. ~m. ~hem. Soc.,
94:3102 (1972)).
Compounds of the formula H2N-Phe-N~R)-Leu-NHMe
(R = methyl, ethyl, allyl, benzyl or naphthylmethyl)
were individually synthesized using the described method
to provide material as analytical controls. Following
purification by preparative RP-HPLC, the identity of each
compound was confirmed by RP-HPLC, MALDI-MS, HR-MS,
microanalysis, and NMR.
The nonsupport-bound library mixtures were
screened in solution in radio-receptor, antimicrobial and
enzyme inhibition assays. Deconvolution of the highly
active mixtures was carried out by both iterative and
positional scanning methods. The iterative method is set
forth in Dooley, et ~l., Sc;ence, 266:2019-2022 (1994)
and the positional scanning method is set forth in U.S.
Patent Application Serial No. 07/943,709, herein
incorporated by reference.
The immediately preceeding description sets
forth in general the reaction techniques utilized in
synthesizing the selectively N-alkylated compounds of
CA 02248078 1998-09-03
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74
Formula I. These techniques can be utilize in one of two
strategic approaches for finding the most active
compound; the iterative approach or the positional
scanning approach. The iterative approach is well-known
and is set forth in general in Houghten et al., Nature,
354, 84-86 (1991); and Dooley et al ., Science 266, 2019-
2022 (1994); herein incorporated by reference. In the
iterative approach, for example, sublibraries of a
molecule having six variable groups are made wherein the
first two variables are defined. (With reference to
Figure, an example for this discussion would have B is 1,
Rlois absent, Rland R2are each a hydrogen atom, with R8
through R3as the six variable groups.) Each of the
compounds with the two defined variable groups is
reacted with all of the other possibilites at the other
four variable groups. These sub-libraries are each
tested to define the identity of the third varible in the
sub-library having the highest activity in the screen of
choice is determined. A new sub-library with the first
three variable poisitions defined is reacted again with
all the other possibilities at the remaining three
undefined variable positions. As before, the identity of
the fourth variable position in the sub-library having
the highest activity is determined, and a new set of sub-
libraries, with four defined variable regions, issynthesized. This process is repeated for all six
variables, yielding the compound with each variable
contributing to the highest desired activity in the
screening pocess. Promising compounds from this process
can then be synthesized on larger scale in traditional
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single-compound synthetic methods for further biological
investigation.
The positional-scanning approach has been
~ 5 described for various organic libraries and for various
peptide libraries (see,for example, R. Houghten et al .
PCT/US91/08694, S.P.A. Fodor and L. Stryer and U.S.
Patent Application No. 07/876,792, herein incorporated by
reference). However, the positional-scanning approach
has never been applied to the selectively N-alkylated
compounds of the instant Formula I. In the positional
scanning approach sublibraries are made defining only one
variable with each set of sublibraries- and all possible
sublibraires with each single variable defined (and all
other possibilities at all of the other variable
positions) is made and tested. From the instant
description one skilled in the art could synthesize
libraries wherein 2 fixed positions are defined at a
time. From the testing of each single-variable defined
library, the optimum substituent at that position is
determined, pointing to the optimum or at least a series
of compounds having a maximum of the desired biological
activity. As this approach is applied to the selectively
N-alkylated compounds of Formula I, sublibraries where
each possibility of any one R group (e.g. R8) is defined
and all other possibilities of the remaining R groups are
synthesized and screened for the desired activity. Thus,
the number of sublibraries for compounds with a single
position defined will be the number of different
substituents desired at that position, and the number of
all the compounds in each sublibrary will be the product
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76
of the number of substituents at each of the other
varibles. Thus, the instant invention is directed to
screening sublibraries of the selectively N-alkylated
componds of Formula I wherein each sublibrary has an R
group defined, and all other R groups are synthesized
with the desired subtstituents, and defining each single
variable in a similar grouping of sublibrairies and
screening for biological activity, until all such
variable positions have been defined and screened for the
desired activity. One skilled in the art would realize
that this approach could also be applied in the situation
wherein each sublibrary has to R groups defined, using a
modification of the above techniques.
lS The reduction of the interior amide of the
compounds of Formula I is another means for the chemical
transformation of such compounds which adds stability and
can enhance activity. A number of reagents are
available and well known for the reduction of amides to
amines such as those disclosed in Wann et al., JOC,
46:257 (1981) and Raucher et al., Tett.T,et., 21:14061
(1980), both of which are incorporated herein by
reference. Diborane has the advantage that
trimethylborate, the only by-product in the reaction
workup, is volatile and is therefore readily removed by
evaporation in solution phase reduction. The use of
excess diborane in refluxing tetrahydrofuran permits
simple aliphatic and aromatic amides to be rapidly, and
often quantitative be reduced into their corresponding
amines.
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A newly synthesized compound can be purified
using a method such as reverse phase high performance
liquid chromatography (RP-HPLC) or other methods of
separation based on the size or charge of the compound.
~ 5 Furthermore, the purified compound can be characterized
using these and other well known methods such as amino
acid analysis and mass spectrometry.
After manufacture, the compounds can be assayed
for receptor binding activity using the radioreceptor
assay (Examples III and IV) or other assays outlined
below, including the glycosidase assay (Example V).
Because some of the compounds of the present
invention bind to the ~ receptor, they can be used in }n
vitro assays to study the opiate receptor subtypes. For
example, in a sample receptor of unknown type or origin,
the compounds, after being labeled with a detectable
marker such as a radioisotope, can be contacted with the
receptor sample under conditions which specifically favor
binding to a particular receptor subtype. Unbound
receptor and compound can be removed, for example, by
washing with a saline solution, and bound receptor can
then be detected using methods well known to those
skilled in the art. Therefore, the compounds of the
present invention are useful m vitro for the diagnosis
of relevant opioid receptor subtypes, and in particular
the ~ type, in brain and other tissue samples.
In addition to their utility in in vitro
screening methods, the compounds are also useful n v vo.
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For example, certain of the instant compounds can be used
n vivo diagnostically to localize opioid receptor
subtypes. The compounds are also useful as drugs to
treat pathologies associated with other compounds which
interact with the opioid receptor system. It can be
envisioned that these compounds can be used for
therapeutic purposes to block the peripheral effects of a
centrally acting pain killer. For instance, morphine is
a centrally acting pain killer. Morphine, however, has a
number of deleterious effects in the periphery which are
not required for the desired analgesic effects, such as
constipation and pruritus ~itching). While it is known
~hat the many peptides do not readily cross the blood-
brain barrier and, therefore, elicit no central effect,
the subject peptides can have value in blocking the
periphery effects of morphine, such as constipation and
pruritus.
This invention provides pharmaceutical
compositions comprising the compounds of Formula I in a
pharmaceutically acceptable carrier. As used herein, the
term "pharmaceutically acceptable carrier" encompasses
any of the standard pharmaceutical carriers, such as a
phosphate buffered saline solution, water, and emulsions,
such as an oil/water or water/oil emulsion, and various
types of wetting agents.
Suitable pharmaceutical carriers and their
formulations are described in Martin, REMINGTON'S
PHARMACEUTICAL SCIENCES, 15th Ed. (Mack Publishing Co.,
Easton 1975). Such compositions will, in general,
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79
contain an effective amount of the active reagent
together with a suitable amount of carrier so as to
prepare the proper dosage form for proper administration
to the subject.
Useful pharmaceutical carriers for the
preparation of the pharmaceutical compositions can be
solids, liquids or gases. Thus, the compositions can
take the form of tablets, pills, capsules, powders,
enterically coated or other protected formulations (such
as by binding on ion exchange resins or other carriers,
or packaging in lipid protein vesicles or adding
additional terminal amino acids), sustained release
formulations, solutions (e.g. ophthalmic drops),
suspensions, elixirs, aerosols, and the like. Water,
saline, aqueous dextrose, and glycols are preferred
liquid carriers, particularly (when isotonic) for
injectable solutions. The carrier can be selected from
various oils including those of petroleum, animal,
vegetable or synthetic origin, for example, peanut oil,
soybean oil, mineral oil, sesame oil, and the like.
Suitable pharmaceutical excipients include starch,
cellulose. talc, glucose, lactose, sucrose, gelatin,
malt, rice, flour, chalk, silica gel, magnesium stearate,
sodium stearate, glycerol monostearate, sodium chloride,
dried skim milk, glycerol, propylene glycol, water,
ethanol, and the like.
The compositions may be subjected to
conventional pharmaceutical procedures such as
sterilization and may contain conventional pharmaceutical
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additives such as preservatives, stabilizing agents,
wetting or emulsifying agents, salts for adjusting
osmotic pressure, buffers, and the like.
This invention provides methods of effecting
treating a mammal comprising the step of administering a
therapeutically effective amount of a pharmaceutical
composition of this invention to a subject. As used
herein, the term "therapeutically effective amount" is
that amount necessary to alleviate the condition from
which the mammal suffers.
In the practice of the therapeutic methods of
the present invention, an effective amount of a
pharmaceutical composition of a compound of Formula I is
administered via any of the usual and acceptable methods
known in the art, either singly or in combination with
another compound of the present invention. These
compounds or compositions can thus be administered
orally, sublingually, topically (e.g., on the skin or in
the eyes), parenterally (e.g., intramuscularly,
intravenously, subcutaneously or intradermally), or by
inhalation, and in the form of either solid, liquid or
gaseous dosage including tablets, suspensions, and
aerosols, as is discussed in more detail above. The
administration can be conducted in single unit dosage
form with continuous therapy or in single dose therapy ad
lib;tum.
In one embodiment, the therapeutic methods of
the present invention are practiced when the relief of
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81
symptoms is specifically required or perhaps imminently
so. In another embodiment, the method is effectively
practiced as continuous or prophylactic treatment.
In the practice of the therapeutic methods of
the invention, the particular dosage of pharmaceutical
composition to be administered to the subject will depend
on a variety of considerations including the nature of
the disease, the severity thereof, the schedule of
administration, the age and physical characteristics of
the subject, and so forth. Proper dosages may be
established using clinical approaches familiar to the
medicinal arts. It is presently believed that dosages in
the range 0.1 of 100 mg of a compound of this invention
per kilogram of subject body weight will be useful, and a
range of 1 to 100 mg per kg generally preferred where the
administration is by injection or ingestion. Topical
dosages may utilize formulations containing active
peptides and a liquid carrier or excipient, with multiple
daily applications being appropriate.
Fluorenylmethoxycarbonyl (Fmoc) amino acid
derivatives were purchased from Calbiochem-Novabiochem
Corp. (San Diego, CA, USA), Bachem Bioscience Inc.
(Philadelphia, PA, USA) and Bachem California (Torrance,
CA, USA). MBHA resin, (1~ divinylbenzene, 100 - 200
mesh, 0.9 mmol/g substitution), was received from
Peninsula Laboratories, Inc (Belmont, CA, USA). N,N'-
Diisopropylcarbodiimide (DIC) and 1-hydroxybenzotriazole
(HOBT) were purchased from Chem Impex International (Wood
Dale, IL, USA), trifluoroacetic acid from Halocarbon
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82
(River Edge, NJ, USA) and hydrogen fluoride from Air
Products ~San Marcos, CA, USA). A11 other reagents and
anhydrous solvents (DMSO, THF) were purchased from
Aldrich Chemical Company (Milwaukee, WI, USA). The
solvents dichloromethane (DCM), dimethylformamide (DMF),
isopropanol (IPA), and methanol were obtained from Fisher
Scientific (Fair Lawn, NJ, USA). All reagents and
solvents were used without further purification. MALDI-
MS analyses were carried out on a Kratos Analytical
Compact MALDI II (Ramsey, NJ, USA). HR-FAB-MS were
recorded at the University of California Riverside Mass
Spectrometry Facility, Department of Chemistry
(Riverside, CA, USA) on a ZAB mass spectrometer.
Analytical RP-HPLC was performed on a Beckman System Gold
instrument (Beckman Instruments, Fullerton, CA, USA).
Samples were analyzed using Vydac 218TP54 C1a columns
(0.46 x 25 cm). Preparative RP-HPLC purification was
performed on a Waters Delta Prep 3000 instrument
(Millipore, Waters Division, San Francisco, CA, USA).
Samples were purified using Waters Delta-Pak C18 columns
(2.5 x 10 cm). All gradients reported were linear in
eluent A (0.05% TFA aqueous) and eluent B (0.05~ TFA in
acetonitrile); flow rates were 1 mL/min (analytical) and
20 mL/ min (preparative); the eluent was monitored at 214
nm. Routine lH NMR and 13C NMR spectra were recorded on
a Varian Gemini 200 (200 MHz). Microanalyses were
performed at Galbraith Laboratories, Inc. (Knoxville, TN,
USA).
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Library synthesis
Amino aci d deri va ti ves
The following amino acid derivatives were used
- in synthesizing a combinatorial library according to
Formula I above: Fmoc-Ala-OH, Fmoc-Phe-OH, Fmoc-Gly-OH,
Fmoc-Ile-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-
Met(O)-OH, Fmoc-Asn-OH, Fmoc-Pro-OH, Fmoc-Gln-OH, Fmoc-
Arg(Pmc)-OH, Fmoc-Ser(t-Bu)-OH, Fmoc-Thr(t-Bu)-OH, Fmoc-
Val-OH, Fmoc-Trp(Boc)-OH, Fmoc-Trp-OH, Fmoc-Tyr(2BrCbz)-
OH, Fmoc-Tyr(t-Bu)-OH, Fmoc-D-Ala-OH, Fmoc-D-Phe-OH,
Fmoc-D-Ile-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-D-Leu-OH, Fmoc-D-
Asn-OH, Fmoc-D-Pro-OH, Fmoc-D-Gln-OH, Fmoc-D-Ser(t-Bu)-
OH, Fmoc-D-Thr(t-Bu)-OH, Fmoc-D-Val-OH, Fmoc-D-Trp(Boc)-
OH, Fmoc-D-Trp-OH, Fmoc-D-Tyr(t-Bu)-OH, Fmoc-D-Arg(Pmc)-
OH, Fmoc-L-Nle-OH, Fmoc-D-Nle-OH, Fmoc-L-Nve-OH, Fmoc-D-
Nve-OH, Fmoc-L-Nal-OH, Fmoc-D-Nal-OH, Fmoc-L-Phg-OH,
Fmoc-L-Glu(t-Bu)-OH, Fmoc-D-Glu(t-Bu)-OH, Fmoc-~-Ala-OH,
Fmoc-L-Cha-OH, Fmoc-D-Cha-OH, and Fmoc-Hyp(t-Bu)-OH.
Fmoc-Cys(MeOBn)-OH (MeOBn = 4-methoxybenzyl),
Fmoc-Cys(MeBn)-OH (MeBn = 4-methylbenzyl), Fmoc-His(Trt)-
OH and Fmoc-D-His(Trt)-OH were also used in the N-
terminal position of the library.
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~ rPT.~ T
A. Synthesis of the combinatorial library
1. Coupling of the first amino acid derivative
The library described below was synthesized
using simultaneous multiple peptide synthesis (Houghten,
R.A., Proc. Natl. Acad. Sci. USA, 82:5131 (1985). The
solid support (MBHA resin) was contained in 230
polypropylene mesh packets (250 mg resin per packet;
packet size 3 cm x 3 cm). For use in the synthesis of
control compounds, 40 additional polypropylene mesh
packets were prepared containing MBHA resin (100 mg).
After the common wash and neutralization steps
were carried out (1 x DCM, 2 x 5~ DIEA, 2 x DCM, 2 x DMF;
approximately 8 mL per packet; all resin packets were
completely covered with solvent) on all of the resin
packets, the individual resin packets were separated into
46 groups, each containing five packets for the addition
of the 46 amino acid derivatives used in the first
coupling step. Fmoc-Leu-OH and Fmoc-Trp-OH were added to
two groups of 20 control resin packets. Amino acid
couplings were carried out on each of the 46 groups of
five library resin packets by vigorously shaking 44
groups in a solution ( 67. 5 mL) of 0.1 M Fmoc amino acid
derivative (6.75 mmol)/DIC/HOBt in DMF overnight (Fmoc-
Gly coupling required only 75 min); for the other twogroups, library resin packets and control packets were
vigorously shaken in a solution (175.5 mL) of 0.1 M Fmoc-
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L-Leu (17.55 mmol)/DIC/HOBt and 0.1 M of Fmoc-L-Trp
(17.55 mmol)/DIC/HOBT in DMF overnight. The resin
packets were washed (2 x DMF, 1 x DCM, 1 x MeOH;
approximately 8 mL per packet) and the completeness of
amino acid coupling was verified using the ninhydrin test
(Kaiser, E.T., et ~ nAl. B;ochem, 34:595 (1970)).
The only amino acids which required repetitive couplings
were Fmoc-L-Gln-OH, Fmoc-D-Gln-OH, Fmoc-L-Arg(Pmc)-OH and
Fmoc-D-Lys(Boc)-OH. Removal of the Fmoc protecting group
was accomplished by shaking the resin packets in 20%
piperidine/DMF (1 x 3 min, 1 x 10 min; 2 L) followed by a
wash cycle (5 x DMF, 2 x IPA, 3 X DCM; approximately 8 mL
per packet).
2. Tritylation of the N-terminal amino group
Following removal of the Fmoc group, the 270
resin packets (a total of 55 mmol of free N-c~-amino
groups) were shaken for 3 h in a 0. 077 M solution of
trityl chloride (276.75 mmol) in DCM/DMF (9:1, 3.6 L)
containing diisopropylethylamine (DIEA, 1. 6 mol, 280 mL).
After a short wash procedure (1 x DMF, 1 x 5% DIEA, 1 x
DCM; approximately 8 mL per packet), the tritylation
procedure was repeated twice more by shaking overnight in
a 0. 05 M solution of trityl chloride in DCM (5.S L)
containing the same amount of base and washed ( 2 x DMF,
x 5% DIEA, 3 x DCM, 1 x MeOH; approximately 8 mL per
packet). The completeness of the trityl coupling was
verified for each of the 46 different amino acid resins
using the bromophenol blue color test (Krchnak, V., et
al., Coll. Czech. t~hem. Comm., 53:2542 (1988)).
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86
3 . Alkylation of the first amide posi tion
All manipulations were performed under a
nitrogen atmosphere and strictly anhydrous conditions.
The 270 resin packets were dried overnight at 50 mTorr.
Each of five groups, containing 46 amino acid resin
packets plus control resin packets (including four Trt-
Leu-MBHA packets and four Trt-Trp-MBHA packets), were
placed in one of five separate round-bottom flasks - one
for each of the five alkylation reactions. Each flask
contained the same amount of available amide groups
(11.07 mmol each). 1 M lithium t-butoxide in THF (220
mmol, 220 mL) and THF (220 mL) were added to each of the
five reaction vessels and shaken at room temperature for
15 min. Excess base solution was removed by cannulation.
Following addition of DMSO (440 mL), the individual
alkylating agent was added (665 mmol i.e., 41.4 mL methyl
iodide; 53.1 mL ethyl iodide; 57.5 mL allyl bromidei 79.0
mL benzyl bromide). 2-(Bromomethyl)naphthalene (665
mmol, 1~7 g) was dissolved in DMSO (440 mL) and
transferred as a solution to the reaction vessel. The
reaction mixture was vigorously shaken for 2 h at room
temperature. The alkylation solution was removed by
cannulation and the entire procedure repeated twice more.
The resulting resin packets were washed (3 x DMF, 2 x
IPA, 3 x DCM, 1 x MeOH; approximately 8 mL per packet)
and dried. Following complete drying of the resin
packets overnight at 50 mTorr, the process described
above was repeated three times for allylation,
benzylation and naphthylmethylation (each alkylation, 2 x
2 h and 1 x 5 h).
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4. Recombine, mix and divide the resin
The resin of the 230 library packets was
combined, mixed in DCM (2 L; 15 h shaking), and dried.
- The resin was divided into 250 polypropylene mesh packets
(packet size 3 cm x 3cm; each containing 310 mg resin).
5. Removal of the trityl protecting group
The resin packets, prepared as described above,
were washed (1 x DCM; approximately 8 mL per packet),
treated twice with 2~ TFA in DCM (1 x 2 min, 1 x 30 min;
2 L), and washed (1 x DCM, 2 x IPA, 2 x DCM, 1 x MeOH;
approximately 8 mL per packet).
6. Coupling of the second amino acid derivative and
second alkylation
The amino acid coupling (using the 50 different
amino acid derivatives), Fmoc removal, tritylation of the
free amino groups, alkylation of the previously formed
amide bond and trityl removal were performed as described
above. Trt-Phe-Leu-NMe-MBHA resin packets and Trt-Ala-
Trp(Me)-NMe-MBHA resin packets were added as control
resins during alkylation. The second amide position was
treated five times for alkylation (methylation and
ethylation, each 5 x 2 h; allylation, benzylation and
naphthylmethylation, each 3 x 2 h and 2 x 3 h).
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7. HF cleavage
The 250 mixture resin packets were cleaved 24
at a time with hydrogen fluoride (5 mL per resin packet
with 0.35 mL anisole added as scavenger) using a multiple
vessel cleavage apparatus (Kornreich, W., et al., Int.
J. Pe~t. Protein Res., 25:414 (1985)). The resulting
mixtures were extracted by sonicating with 50~ aqueous
acetonitrile (3 x 5 ml). The resulting solutions were
lyophilized and relyophilized twice more from 50~ aqueous
acetonitrile.
8. Individual compounds
Individual compounds were prepared in the same
manner as described for the library synthesis. The
alkylations were generally performed with repetitions.
Following HF cleavage, the crude individual compounds
were purified by preparative RP-HPLC. Condition for
preparative HPLC:vydac C18; linear gradient 25-55%B, in
30 min; eluent A:0.05~ TFA aqueous;eluent B:0.05~ TFA in
acetonitrile; flow rate:20 mL/min; the eluent was
monitored at 214 nm.
Phenylalanyl-N-methyl-leucinemethylamide. Yield after
preparative HPLC (TFA salt): 59.8~. lH NMR (200 MHz,
CDCl3; mixture of conformers; selected data for the major
conformer; ratio 78 : 22): ~ = 0.85 (m; 6H), 1.23 - 1.57
(m; 2H), 1.71 - 1.85 (m; lH), 2.65 (s; 3H), 2.7 (d; 3H),
3.01 - 3.28 (m; 2H), 4.51 - 4.58 (m; 2H), 6.85 (m; lH),
7.15 - 7.28 (m; 5H), 8.5 (br; 2H).
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89
13C NMR (200 MHz, CDCl3; selected data for major
conformer): ~ = 22.0, 22.7, 24.7, 26.1, 32.6, 36.8,
37.4, 51.7, 57.8, 128.1, 129.1, 129.4, 133.4, 168.9,
170.1. MALDI-MS: 307 (M+2), 329 (M+Na). Anal. calcd.
- 5 for ClgH28F3N3O4 (TFA salt): C, 54.39; H, 6.73; N, 10.02.
Found: C, 54.19; H, 7.03; N, 9.99. HR-FA~3-MS calcd. for
Cl7H2BN3O2 (MH+) 306.2175, found 306.2165.
Phenylalanyl-N-ethyl-leucinemethylamide. Yield after
preparative HPLC (TFA salt): 18.8~. lH NMR (200 MHz,
CDCl3; selected data for major conformer; ratio 72 : 28):
~= 0.75 - 1.15 (m; 9H), 1.35 - 1.60 (m; 2H), 1.95 - 2.20
(m; lH), 2.66 (d; 3H), 2.85 - 3.45 (m; 4H), 3.97 (m; lH),
4.41 (m; lH), 6.99 (m; lH), 7.18 - 7.40 (m; 5H), 8.2 -
9.2 (br; 2H). 13C NMR (200 MHz, CDC13; selected data for
major conformer): ~ = 14.3, 22.9, 23.1, 25.7, .26.6,
38.3, 43.5, 52.2, 58.3, 128.6, 129.6, 130.1, 134.1,
169.3, 171.2. MALDI-MS: 321 (M+l), 343 (M+Na). Anal.
calcd. for C20H30F3N3O4 (TFA salt): C, 55.399; H, 6.9789; N,
9.697. Found: C, 54.39; H, 6.97; N, 9.47. HR-FA~3-MS
calcd. for ClBH30N3O2 (MHt) m/z = 320.2331, found m/z =
320.2335.
Phenylalanyl-N-allyl-leucinemethylamide. Yield after
preparative HPLC (TFA salt): 20.05~. lH NMR (200 MHz,
CDCl3; selected data for major conformer; ratio 71 : 29):
~ = 0.76 - 0.90 (m; 6H), 1.27 - 1.50 (m; 2H), 1.98 - 2.08
(m; lH), 2.68 (d; 3H), 3.02 - 3.60 (m; 4H), 4.18 (m; lH),
4.41 - 4.49 (m; lH), 5.12 - 5.24 (m; 2H), 5.56 - 5.78 (m;
lH), 6.99 (m; lH), 7.21 - 7.36 (m; aromatic protons),
8.35 - 9.35 (br; 2H). 13C NMR (200 MHz, CDCl3; selected
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data for major conformer): ~ = 22.3, 25.0, 26.0, 37.8,
49.9, 51.8, 58.0, 119.9, 128.1, 129.1, 129.5, 132.1,
133.5, 169.1, 170.4. MALDI-MS: 333 (M+1), 355 (M+Na).
Anal. calcd. for C21H30F3N3O4 (TFA salt): C, 56.60; H, 6.79;
N, 9.44. Found: C, 56.00; H, 6.83; N, 9.23. HR-FAB-MS
calcd. for ClgH30N3O2 (MH+)m/z = 332.2331, found m/z =
332.2335.
Phenylalanyl-N-benzyl-leucinemethylamide. Yield after
preparative HPLC (TFA salt): 20.34~. lH NMR (200 MHz,
CDCl3; selected data for major conformer; ratio 65 : 35):
~=0.72 - 0.89 (m; 6H), 1.02 - 1.53 (m; 2H), 1.87 - 2.11
(m; lH), 2.52 (d; 3H), 2.98 - 3.45 (m; 2H), 4.05 - 4.79
(m; 4H), 6.92 (m; lH), 7.08 - 7.38 (m; lOH), 8.20 - 9.20
(br; 2H). 13C NMR (200 MHz, CDCl3; selected data for
major conformer): ~ = 22.0, 22.9, 25.5, 26.0, 37.7,
38.1, 47.3, 52.5, 57.9, 127.2, 127.7, 128.2, 129.3,
129.8, 133.5, 134.9, 169.7, 170.6. MALDI-MS: 383 (M+1),
405 (M+Na). Anal. calcd. for C2sH32F3N3O4 (TFA salt): C,
60.58; H, 6.512; N, 8.48. Found: C, 60.33; H, 6.41; N,
8.43. HR-FAB-MS calcd. for C23H32N3O2 (MH+) m/z = 382.2487,
found m/z = 382.2511.
Phenylalanyl-N-naphthylmethyl-leucinemethylamide. Yield
after preparative HPLC (TFA salt): 18.54~. lH NMR (200
MHz, CDCl3 selected data for major conformer; ratio 67 :
33): ~ = 0.70 - 0.90 (m; 6H), 1.15 - 1.58 (m; 2H), 1.94 -
2.13 (m; lH), 2.44 (d; 3H), 3.04 - 3.49 (m; 2H), 4.20 -
4.90 (m; 4H), 6.90 - 6.99 (m; lH), 7.08 - 7.89 (m; 12H),
8.1 - 9.5 (br; 2H). 13C NMR (200 MHz, CDCl3; selected
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91
data for major conformer): ~ = 22.0, 23.0, 25.5, 25.7,
37.7, 38.1, 52.6, 57.7, 124.6 - 134.2 (aromatic carbons),
- 169.8, 170.5. MALDI-MS: 433 (M+1), 455 (M+Na). Anal.
calcd. for C29H34F3N3O4 (TFA salt): C, 63.~2; H, 6.28; N,
~ 5 7.70. Found: C, 63.96; H, 6.27; N, 7.76. HR-F~3-MS
calcd. for C27H34N3O2 (MH+) m/z = 432.2643, found m/z =
432.2663.
E~PT.R T T
A. Synthesis of the resin bound peptidomimetic compound
H,N-Tyr(tBu)-N(Me)-Tyr(t~u)-N(Bzl)-resin
1. Coupling of the first amino acid derivative
The peptidomimetic compound was synthesized
using simultaneous multiple peptide synthesis (Merrifield,
R. B. J. Am. Chem. Soc. 1963, ~, 2149; Houghten, R. A.
15 Proc. Natl. Acad. Sci. USA 1985, 82, 5131) and Fmoc
strategy. The solid support (MBHA resin) was contained
in a polypropylene mesh packet (100 mg resin per packet;
packet size 3 cm x 3 cm).
After the neutralization and wash steps were
carried out [1 x DCM, 2 x 5% N,N-diisopropylethylamine
(DIEA), 2 x DCM, 2 x DMF; approximately 5mL for each
- washing step] the resin packet was vigorously shaken in a
solution (5.4 mL) of 0.1 M Fmoc-L-Tyr(tBu)-OH (0.54
mmol)/DIC/HOBt in DMF overnight. The resin packet was
washed (2 x DMF, 1 x DCM, 1 x MeOH) and the completeness
of amino acid coupling was verified using the ninhydrin
CA 02248078 1998-09-03
W O 97133174 PCT~B97/00349 92
test (Kaiser, E. T.; Colescott, R. L.; Blossinger, C. D.;
Cook, P. I. Anal. Biochem. 1970, 34, 595). One
repetitive coupling was required. Removal of the Fmoc
protecting group was accomplished by shaking the resin
5 packet in 20~ piperidine/DMF (1 x 3 min, 1 x 10 min; 2 L)
followed by a wash cycle (5 x DMF, 2 x IPA, 3 x DCM).
2. Tritylation of the N-terminal amino group
The resin packet (0.09 mmol of free N-~-amino
groups) was shaken for 2 h in a 0.077 M solution of
trityl chloride (0.45 mmol) in DCM/DMF (9:1, 5.84 mL)
containing DIEA (2.61 mmol, 0. 45 m~). After a short wash
procedure (1 x DMF, 1 x 5~ DIEA, 1 x DCM), the
tritylation procedure was repeated three more times by
shaking overnight in a 0.077 M solution of trityl
chloride in DCM (5. 84 mL), for 3 h in a 0.077 M solution
of trityl chloride in DCM/DMF (9:1, 5.84 mL~ and again
overnight in a 0.05 M solution of trityl chloride in DCM
(9 mL), containing the same amount of base. The resin
packet was washed (2 x DMF, 1 x 5~ DIEA, 3 x DCM, 1 x
MeOH) and a small resin sample was tested for the
completeness of the trityl coupling using the bromophenol
blue color test (Krch~ak, V.; Vagner, J.; Safa~, P.;
Lebl, M. Coll . Czech . Chem. Comm. 1988, 53, 2542).
3. Alkylation of the first amide posi tion
All manipulations were performed under a
nitrogen atmosphere and strictly anhydrous conditions.
The resin packet was dried overnight at 50 mTorr. 1 M
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lithium t-butoxide in THF (1.8 mmol, 1.8 mL) and THF (1.8
mL) were added to the reaction vessel containing the
resin packet (0.09 mmol amide groups) and it was shaken
at room temperature for 15 min. Excess base solution was
- 5 removed by cannulation. Following addition of DMSO (3.6
mL), benzyl bromide (5.4 mmol, 0. 64 mL) was added. The
reaction mixture was vigorously shaken for 2 h at room
temperature. The alkylation solution was removed by
cannulation and the entire procedure repeated twice more.
The resulting resin packet was washed (3 x DMF, 2 x IPA,
3 x DCM, 1 x MeOH; approximately 5 mL) and dried.
Following complete drying of the resin packet overnight
at 50 mTorr, the process described above was repeated
again two times.
4. Removal of the trityl protecting group
The resin packet was washed (l x DCM;
approximately 5 mL), treated twice with 2~ TFA in DCM (1
x 3 min, 1 x 30 min), and washed (1 x DCM, 2 x IPA, 2 x
DCM, 1 x MeOH; approximately 5 mL).
5. Coupling of the second amino acid derivative and
second alkyla tion
The coupling of Fmoc-L-Tyr(tBu)-OH to the resin
bound compound, the Fmoc removal, and the tritylation
(only three treatments were required) of the free amino
groups were performed as described above.
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6 . Alkyla tion of the second amide posi tion
All manipulations and the base treatment were
performed as described above. For the alkylation DMSO
~3.6 mL) and methyl iodide (5.4 mmol, 0.34 mL) were
added. The reaction mixture was vigorously shaken for 2
h at room temperature. The alkylation solution was
removed by cannulation and the entire procedure repeated
twice more. The resulting resin packet was washed (3 x
DMF, 2 x IPA, 3 x DCM, 1 x MeOH; approximately 5 mL) and
the trityl protecting group removed as described above.
B. Synthesis of (CH3)2CH-NH-Tyr(tBu)-N(Me)-Tyr(tBu)-
N(Bzl)-resin
1. Reductive alkylation
This procedure was adapted from those known in
the art: Borch, R.F., et al., J. Am. Chem. Soc., 93:2897
(1971); Coy, D.H., et ~l., Tetrahedron, 44:835 (1988);
Sta~kova, M., et ~l., Drug Development Research, 33:146
(1994)(herein incorporated by reference).
Resin packets containing resin bound compounds
with free N-terminal amino groups were shaken in a
solution of methanol (MeOH) 20~/dichloromethane (DCM)
79~/acetic acid 1~ (for one resin packet containing 0.05
mmol amine 4 ml of solvent were used - enugh to cover the
resin packet) and 2 - 10 equivalents of the aldehyde or
ketone (depending on their reactivity). After 20
minutes, 2-10 equivalents of a 1 M solution of sodium
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cyanoborohydride in N,N-dimethylformamide (DMF) were
added and the reaction mixture shaken for 60 min. The
resin packets were washed using the following washing
sequence: 5 x DMF, 1 x DCM, 1 x MeOH (for one resin
- 5 packet of the size mentioned above approximately 5 ml of
solvent for each step). The completeness of the
formation of secondary amines can be tested using hte
Kaiser test. If necessary the reaction can be repeated,
also by using a different solvent system like DMF
containing 1~ acetic acid.
The applicability of this reaction to all amino
acid derivatives used in the peptidomimetic library was
applied to sets of 50 model dipeptide resins (OL-resins).
Following HF cleavage, the model compounds were analyzed
by HPLC and MALDI-MS. This procedure was carried out as
described in R.F. Borch, et al., J. Am. Chem. Soc.,
93:2897-2904 (1971); D.H. Coy, et al., Tetrahe~ron,
44:835-841(1988); and M. Stankova, et ~l., Er~
Developm~nt Research, 33:146-156 (1994)(all of which are
herein incorporated by reference).
Following neutralization (3 x 5~ DIEA, 2 x DCM)
and a washing step (1 x DMF/ 2 ~ acetic acid), the resin
packet was shaken in a solution of MeOH 20~/DCM 79~/
acetic acid 1~ (4 mL) and acetone (0.9 mmol; 66.6 ~L).
After 20 min 0.9 mL (0.9 mmol) of a 1 M solution of
sodium cyanoborohydride in DMF were added and the
reaction mixture was shaken for 60 min. The resin packet
was washed using the following washing sequence: 5 x
DMF, 1 x DCM, 1 x MeOH, approximately 5 ml of solvent
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96
for each step. The completeness of the formation of the
secondary amines was tested using the Kaiser test [if
necessary the reaction can be repeated, also by using a
different solvent system like DMF containing 1~ acetic
acid].
C. Synthesis of red~(CH3)2CH-NH-Tyr(tBu)-N(Me)-Tyr(tBu)-
NH(Bz1)3; (red = reduced)
1. Reduction
Reduction can either be performed on solid
support [procedure A] or in solution [procedure B].
Procedure A:
Into a 50 ml glass tube ~teflon-lined cap) were
added the resin packet and 310 mg boric acid ~5.014
mmol). Under nitrogen atmosphere, 0.5 ml
trimethylborate ~0.0042 mmol) were added, followed by the
addition of 15 ml of 1 M borane-tetrahydrofuran complex
~15 mmol). Following cessation of hydrogen evolution,
the tube was sealed and heated at 65~C for 100 hr. The
tubes were then removed, cooled to room temperature and
2ml methanol were added to quench excess reducing agent.
The resin packet was washed with THF ~1 x lmin x 10 ml)
and MeOH ~4 x 1 min x 10 ml). After drying the resin
packet, it was covered with 15 ml piperidine and heated
at 65~C for 18 hr. The resin packet was washed with DMF
~2 x 1 min x 5ml), DCM ~2 x 1 min x 5 ml), MeOH (1 x 1
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97
min x 5 ml), DMF (2 x 1 min x 5ml), DCM (2 x 1 min x 5
ml) and MeOH (1 x 1 min x 5 ml).
Procedure B:
The following procedure was adapted from
Dooley, C.T., et al., An~lgesia, INRC Procee~ings, 1:400
(1995)(herein incorporated by reference). Into a 50 ml
glass tube (teflon-lined cap) were added the compound
(0.09 mmol; two backbone carbonyl groups) and 310 mg
boric acid (5.014 mmol). Under nitrogen atmosphere, 0.5
ml trimethylborate (0.0042 mmol) were added, followed by
the addition of 15 ml of 1 M borane-tetrahydrofuran
complex (15 mmol). Following cessation of hydrogen
evolution, the tube was sealed and heated at 60~C for 90
hr. The tubes were cooled to room temperature and 5ml
methanol were added dropwise to remove excess reducing
agent. Excess solvent was removed by immersion of the
tube in a 55~C water bath under a constant nitrogen flow
(10-15 psi). The compound subsequently underwent
successive washes and evaporations with methanol (2 x 5
ml). After addition of 2 N hydrogen chloride (3 mL; 6
mmol) in water/MeOH (1:3) the glass tube was sealed and
heated at 60~C for 18 h to hydrolyze boron-nitrogen
complexes. The tube was removed from heat, MeOH (2 ml)
was added and the solvent evaporated.
C. HF cleavage - Soluble Compounds
The compound was cleaved from the resin with
hydrogen fluoride (5 mL per resin packet with 0.35 mL
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98
anisole added as scavenger) using a multiple vessel
cleavage apparatus (Houghten, R. A., et ~1., Int. J.
Pept. Prote;n Res., 27:673 (1986)). If the compound has
been reduced on the solid support, the cleavage time was
9 h at 0~C; the nonreduced compound was cleaved in 90 min
at 0~C. The resulting compound was extracted by
sonicating with 50~ aqueous acetonitrile (3 x 5 ml).
The resulting solution was lyophilized and relyophilized
twice more from 50~ a~ueous acetonitrile.
~Y~MPLE TTI
A. Identification Of Mu Selective Opioid Peptides By A
Radioreceptor Assay
This example describes the identification of
individual compounds, either contained within a synthetic
combinatorial library mixture or prepared separately, as
inhibitors of the ~-selective opioid peptide [3H]-[D-Ala2,
MePhe4, Gly-015 enkephalin ([3H]-DAMGO). Individual
peptides were identified as capable of inhibiting [3H]-
DAMGO by a radioreceptor assay.
As detailed below, the compound libraries of
the instant invention were screened at a single
concentration (0.0~ mg/ml~ in a radioreceptor assay using
rat brain homogenates and [3H]-DAMGO as radioligand. IC50
values were determined for mixtures in the library which
significantly inhibited the binding of [3H]-DAMGO.
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B. Radioreceptor Assays Selective For The Mu Receptor
Rat and guinea pig brains, frozen in liquid
nitrogen, were obtained from Harlan Bioproducts for
- Science (Indianapolis, IN). Frozen brains were thawed,
the cerebella removed and the remaining tissue weighed.
Each brain was individually homogenized in 40 ml Tris-HCl
buffer (50 mM, pH 7.4, 4~C) and centrifuged (39000 x g)
(Model J2-HC; Beckman Instruments, Fullerton, CA) for 10
min at 4~C. The pellets were resuspended in fresh Tris-
HCl buffer and incubated at 37~C for 40 min. Followingincubation, the su~pensions were centrifuged as above,
the resulting pellets resuspended in 100 volumes of Tris
buffer and the suspensions combined. Membrane
suspensions were prepared and used in the same day.
Protein content of the crude homogenates ranged from
0.15-0.2 mg/ml as determined using the method described
by Bradford (Bradford, Anal. Biochem. 72:248-254 (1976),
which is incorporated herein by reference).
Binding assays were carried out in
polypropylene tubes. Each tube contained 0.5 ml of
membrane suspension, 3 nM of the ~-selective opioid
peptide [3H]-DAMGO (specific activity 36 Ci/mmol), 0.08
mg/ml compound mixture and Tris-HCl buffer in a total
volume of 0.65 ml. Assay tubes were incubated for 60 min
- 25 at 25~C. The reaction was terminated by filtration
through GF-B filters (Wallac, Inc., Gaithersburg, MD).
The filters were subsequently washed with 6 ml Tris-HCl
buffer at 4~C. Bound radioactivity was counted on a
Beta-plate Liquid Scintillation Counter (Life
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Technologies, Gaithersburg, M~) and expressed in counts
per minute (cpm). Inter- and intra-assay variation
standard curves were determined by incubation of [3H]-
DAMGO in the presence of 0.13-3900 nM of unlabeled DAMGO.
Competitive inhibition assays were performed as above
using serial dilutions of the peptide mixtures. IC50
values were then calculated using the software GRAPHPAD
(ISI, San Diego, CA). IC50 values of less than 1000 nM
are indicative of highly active opioid compounds which
bind to the ~ receptor, with particularly active
compounds having IC50 values of 100 nM or less and the
most active compounds with values of less than 10 nM.
In the following Table, the only variable
occurs at R7. Thus, all of the following compounds, in
reference to Formula I, have the following structure: X
and Y are taken together to form a carbonyl group, Rl and
R2 are each a hydrogen atom, Rlo is absent, B is zero, AA,
BB, and CC are zero, R6 is ethyl, R8 is napth-2-ylmethyl,
and Rg is a hydrogen atom.
TABLE 1
Mu Receptor Assay
R7 IC50(nM)
S-methyl 2
S-(2-(methylsulfinyl)ethyl) 7
25 hydrogen atom 13
S-(4-hydroxy)benzyl (2-BrZ)1 31
S-(Hydroxy~methyl 40
S-(4-hydroxybenzyl) (t-butyl)1 74
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TABLE 1
Mu Receptor A~say
R7 ICsO(nM)
S-(indol-3-yl)methyl (Boc)l 92
S-(1-methyl)prop-1-yl 129
S-~3-(N,N,N-triethyl)guanidino)-N-propyl) 138
S-(4-(N-(naphth-2-ylmethyl)amino)-n-butyl 237
R-methyl 246
S-Cyclohexylmethyl 265
S-Phenyl 384
S-Benzyl 471
R-(4-(N-(naphth-2-ylmethyl)amino)-n-butyl) 476
S-(2-carboxy)eth-1-yl 476
S-(N-(napth-2-ylmethyl)indol-3-ylmethyl) 494
R-4-(hydroxy)benzyl 542
S-(N,N-di(napth-2-ylmethyl)amidoethyl 585
R-(2-methyl)prop-1-yl 666
S-Pyrrolidine (taken in conjunction with 891
R8)
R-(n-butyl) 1056
S-((N,N-di(naphth-2-ylmethyl)amidomethyl)) 1106
R-(Hydroxy)methyl 1106
R-Pyrrolidine (takin in conjunction with 1115
R8)
S-(n-propyl) 1133
S-(Napth-2-yl)methyl 1206
R-(n-propyl) 1343
25 S-(2-methyl)prop-1-yl 1493
S-(n-butyl) 1560
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2 0 TABLE
Mu Receptor Assay
R7 IC50 ~nM)
R-(indol-3-yl)methyl (Boc)1 1593
R-(3-(N,N,N-triethyl)guanidino)-N-propyl) 1630
Protecting group removed before testing
EXAMPLE IV
A. Assay for Kappa Opiate Receptor Inhibition
This example demonstrates the specificity of
the novel selectively N-alkylated compounds of Formula I
for the kappa opiate receptors.
Assays demonstrating selective inhibition of
binding to kappa oppiate receptors for K receptors were
carried out using [3H]-U69,593 (3 nM, specific activity 62
Ci/mmol) as the radioligand and tissue homogenates
prepared from guinea pig brains (cortex and cerebellum)
using Tris buffer containing 100 ~M PMSF, 5 mM MgCl2 and 1
mg/ml BSA, pH 7.4. Sample tubes were incubated for 2.5
hr. Standard curves were prepared using 0.05-6300 nM
naloxone.
Tritiated ligands, [3H]-DAMGo, ~3H]-DPDPE and
[3H]-[D-Ser2, Leu5, Thr6]enkephalin ([3H]-DSLET), Abuse
(NIDA) repository, as prepared by Multiple Peptide
Systems (San Diego, CA), [3H]-U69,593 from Amersham
(Arlington Heights, IL) and [3H]-naltrindole from DuPont
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103
NEN Research Products (Los Angeles, CA). The average
standard deviation for IC50 values was +20~.
.,
In the following Table 2, all compounds have
either R1 or R2 as a hydrogen atom and the other taken
together with R~ to form a pyrrolidine group. X and Y are
taken together to form a carbonyl group, B is zero, AA,
BB, and CC are zero except where noted, R6 is benzyl and
R9 is a hydrogen atom. Thus, only R7 is varied as the
compounds are tested in the assay.
TABLE 2
Kappa Receptor Assay
R7 IC5~(nM)
S-methyl
R-methyl
hydrogen atom
S-(3-guanidino)-N-propyl) 2
S-~4-(N-benzylamino)-n-butyl 4
S-(iso-propyl) 7
S-(2-(methylsulfinyl)ethyl) 11
20 S-(n-propyl) 14
R-(n-propyl) 15
R-(hydroxy)methyl 16
R-(n-butyl) 21
R-(3-guanidino)-N-propyl 22
25 R-(Naphth-2-yl)methyl 23
S-(hydroxy)methyl 28
R-(n-butyl) 31
R-(4-(N-benzylamino)-n-butyl 42
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TABLE 2
Kappa Receptor Assay
R7 IC50(nM)
S-Phenyl 50
S-Benzyl 53
S-(4-hydroxybenzyl) 56
R-(1-methyl)propyl 67
5 R-(4-hydroxybenzyl) (t-butyl)1 69
S-(l-methyl)propyl 74
S-(4-hydroxybenzyl) (t-butyl)l 85
S-(2-methyl)propyl 99
S-(Cyclohexylmethyl) 102
10 S-(1-hydroxy)ethyl 108
R-benzyl 146
R-(iso-propyl) 203
S-(Naphth-2-ylmethyl) 222
R-pyrrolidine (taken in conjunction with 247
R~)
R-Cyclohexylmethyl 2B4
R-(2-methyl)propyl 291
S-(indol-3-yl)methyl (Boc)l 306
S-(N,N-dibenzylamido)ethyl 313
20 hydrogen atom (AA=1) 487
R-(N'(t-butoxycarbonyl)indol-3-ylmethyl) 496
R-(propionamide) 642
1-(hydroxy)ethyl 650
2-(carboxy)ethyl 898
25 R-(indol-3-ylmethyl) 931
S-(indol-3-ylmethyl) 1277
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TABLE 2
Kappa Receptor Assay
R, IC50(nM)
R-~N,N-dibenzylamido)methyl 2430
R-(2-(carboxy)ethyl) 4688
S-(4-hydroxy-pyrrolidine) taken in 8735
conjunction with R&
S-pyrrolidine (taken in conjunction with 13533
R~)
Protecting group removed before testing
MPT~ V
A. ~-Glucosidase Inhibitor
~-Glucosidases are not only essential to
carbohydrate metabolism, but also vital for the
processing of various glycoproteins and glycolipids.
Inhibitors of these enzymes, in particular of ~-
glucosidase, are therefore of high therapeutic potential.
~-glucosidase inhibitors are potent oral anti-diabetics
(Lebovitz, H.E. Drugs, 44(3):21-28 (1992)), and have been
implicated in the blocking of microbial infection
(Fischer,P.B., et al. J.Virol. 69(9):5791-5797 (1995);
Rademacher, T.W., IN: Sandler, M. and Smith, J.H. (Eds.),
Enzymes as Drugs Vol.2, Oxford University Press, Oxford,
333-343 (1994)) and tumor growth (Pili, R. et al., Cancer
~es., 55:2920-2926 (1995)) . Most of the known natural
and synthetic ~-glucosidase inhibitors are sugar analogs,
such as pseudooligosaccharides ( Bischoff, H.,
CA 02248078 1998-09-03
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106
Eur.J.Cl;n.Investig. 24(3):3-10 (1994)), azasugars
(Wong, C.H.,et ~l., J.Org.~hem.. 60:1492-1501 (1995)), or
indolizidine alkaloids (Elbein, A.D., Ann .Rev.Biochem.,
56:497-534 (1987)). Glycosidase inhibitors often inhibit
more than one glycolytic enzyme (Kajimoto, T., et al.,
J.~m.Chem.Soc. , 113:6187-6196 (1990)).
The results set forth below in Table 3 are
obtained from an ~-glucosidase inhibition assay performed
in a 96-well format using p-nitrophenyl-~-D-
glucopyranoside as chromogenic substrate and ~-
glucosidase from bakers yeast, essentially as described
by Haslvorson and Ellias (Biochem. Biophys. Acta, 30:28-
40 (1958)). The ICso values represent the concentration
necessary for 50~ enzyme inhibition. The most active
inhibitors are compounds of Formula I wherein X and Y are
taken together to form a carbonyl group, B is zero, AA,
BB, and CC are zero except were noted, Rg is a hydrogen
atom, R8 is benzyl, R6 is naphth-2-ylmethyl, R3 is S-(N-
(naphth-2-ylmethyl)indol-3-ylmethyl), Rl and R2 are each
hydrogen, Rlo is absent, and R, is as set forth in the
following Table 3:
TABLE 3
~-Glucosidase Inhibition Assay
R7 IC50(~M)
25 R-(4-(N-benzylamino)-n-butyl) 17
S-(4-(N-benzylamino)-n-butyl) 19
S-(3-guanidino)-n-propyl) 38
R-(3-guanidino)-n-propyl) 38
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TA~E 3
~-Glucosidase Inhibition Assay
S-pyrrolidine (ta~en in 141
conjunction with R8)
S-methyl 16 7
Hydrogen atom 16 7
R-(2-methyl)propyl 170
S-(l-hydroxymethyl) 176
S-(phenyl) 184
S-(4-hydroxybenzyl) 190
R-methyl 199
10 S-benzyl 328
S-(2-methyl)propyl 356
S-(indol-3-ylmethyl) 356
S-(iso-propyl) 356
R-(2-methyl)prop-1-yl 398
S-4-hydroxyprrolidine (in 43 7
conjunction with R8)
S-(l-hydroxyethyl) 460
S-[N',N'-dibenzylamido)ethyl]) 529
R-(4-hydroxybenzyl) 540
R-(iso-propyl) 552
R-(N'-benzyl indol-3-ylmethyl) 552
S-(2-(methylsulfinyl)ethyl) 564
S-(l-methyl)prop-l-yl 610
S-(N'-benzyl indol-3-ylmethyl) 632
S-(n-propyl) 632
R-(indol-3-ylmethyl) 667
S-(cyclohexylmethyl) 667
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108
TABLE 3
~-Glucosidase Inhibition Assay
R'-(1-hydroxyethyl) 678
R-pyrrolidine (taken in 702
conjunction with R8)
S-[N',N'-dibenzylamido)ethyl 713
5 R-(n-butyl) 724
hydrogen atom, AA=1 770
R-(n-propyl) 828
S-(n-butyl) >1000
S-(napth-2-ylmethyl) >1000
R-(napth-2-ylmethyl) >1000
R-(1-hydroxyethyl) >1000
S-(2-carboxyethyl) >1000
R-(2-carboxyethyl) >1000
N',N-dibenzyl R-propionamide >1000
15 R-(cyclohexylmethyl) >1000
R-benzyl >1000
S-[(N'N'-dibenzylamido)ethyl >1000
Numerous modifications and variations are
possible in light of the above teachings and, therefore,
within the scope of the appended claims, the invention
may be practiced otherwise than as particulary described
above.
