U.S. patent application number 11/254743 was filed with the patent office on 2007-04-26 for recognition of oligiosaccaride molecular targets by polycationic small molecule inhibitors and treatment of immunological disorders and infectious diseases.
This patent application is currently assigned to MediQuest Therapeutics, Inc.. Invention is credited to Mark R. Burns, Sunil A. David.
Application Number | 20070093424 11/254743 |
Document ID | / |
Family ID | 37897579 |
Filed Date | 2007-04-26 |
United States Patent
Application |
20070093424 |
Kind Code |
A1 |
Burns; Mark R. ; et
al. |
April 26, 2007 |
RECOGNITION OF OLIGIOSACCARIDE MOLECULAR TARGETS BY POLYCATIONIC
SMALL MOLECULE INHIBITORS AND TREATMENT OF IMMUNOLOGICAL DISORDERS
AND INFECTIOUS DISEASES
Abstract
Small molecule polycationic agents are used to modulate or
interrupt biological processes by binding to oligosaccharide-based
biomolecules. Compounds that inhibit nitric oxide, TNF.alpha. or
other immunomodulators are provided and are useful for treating
immunological disease and disease of an infectious disorder.
Inventors: |
Burns; Mark R.; (Kenmore,
WA) ; David; Sunil A.; (Lawrence, KS) |
Correspondence
Address: |
CONNOLLY BOVE LODGE & HUTZ LLP
P.O. BOX 2207
WILMINGTON
DE
19899-2207
US
|
Assignee: |
MediQuest Therapeutics,
Inc.
Bothell
WA
98021
The University of Kansas
Lawrence
KS
66049
|
Family ID: |
37897579 |
Appl. No.: |
11/254743 |
Filed: |
October 21, 2005 |
Current U.S.
Class: |
514/180 ;
514/1.3; 514/2.1; 514/2.3; 530/326; 530/327; 530/328; 530/329;
530/330 |
Current CPC
Class: |
C07D 231/12 20130101;
A61P 31/04 20180101; C07C 211/08 20130101; C07C 237/22 20130101;
C07D 209/20 20130101; A61P 37/02 20180101; A61P 43/00 20180101 |
Class at
Publication: |
514/014 ;
514/015; 514/016; 514/017; 514/018; 530/326; 530/327; 530/328;
530/329; 530/330 |
International
Class: |
A61K 38/10 20060101
A61K038/10; A61K 38/08 20060101 A61K038/08; C07K 7/08 20060101
C07K007/08; C07K 7/06 20060101 C07K007/06 |
Claims
1-2. (canceled)
3. A compound ##STR22## pharmaceutically acceptable salts thereof;
and prodrugs-thereof.
4. The compound of claim 3 being represented by the formula:
##STR23##
5. The compound of claim 3 being represented by the formula:
##STR24##
6. The compound of claim 3 being represented by the formula:
##STR25##
7. The compound of claim 3 being represented by the formula:
##STR26##
8. A pharmaceutical composition comprising a compound according to
claim 3.
9. A pharmaceutical composition comprising a compound according to
claim 4.
10. A pharmaceutical composition comprising a compound according to
claim 5.
11. A pharmaceutical composition comprising a compound according to
claim 6.
12. A pharmaceutical composition comprising a compound according to
claim 7.
13-32. (canceled)
Description
TECHNICAL FIELD
[0001] The present disclosure relates to certain polycationic
compounds. The present disclosure also relates to methods and
compositions that can be used to define drug targets, setup
screening assays and the design of agents to interrupt pathological
biological processes involving carbohydrate targets. The present
disclosure also relates to drug agents used to treat diseases or
conditions, particularly immunological disorders and infectious
diseases.
[0002] Small molecule polycationic compounds bind and neutralize
bacterial lipopolysaccharides, and along with test results suggest
their suitability for the prevention or treatment of endotoxic
shock states or sepsis.
BACKGROUND
[0003] Carbohydrates represent the bulk of organic matter on
earth..sup.1 It has been noted that roughly 80% of secreted and
cell-surface proteins are glycosylated..sup.2 The scientific field
of glycobiology involves the investigation of the role that
saccharide structure plays in biological function. Given the
generally complex structural nature of glycoconjugates, this has
been the last of the three major biological materials to be
explored in great detail. Unlike proteins and nucleic acids, the
biosynthesis of glycosylated biostructures does not involve a
templated, message-driven production. A diverse set of enzymes
operate on substrates to synthesize three main types of
glycoconjugates: 1) N-linked glycoproteins, 2) O-linked
glycoproteins and 3) glycosaminoglycans..sup.3
[0004] The role of oligosaccharides in biological recognition has
been amply demonstrated in recent scientific literature. This role
extends to cell adhesion, cell-to-cell communications and signal
transduction, route to infection by bacteria and viruses,
development and immunology..sup.4 It has been noted that almost all
of the key molecules involved in the innate and adaptive immune
response are glycoproteins..sup.5
[0005] The specific biological recognition of saccharides is a
tremendous chemical challenge, even for nature, due to their
complex, irregular and multifunctional structures..sup.6 This
challenge is made even more difficult by the ability of the
poly-hydroxylated exteriors to associate well with water. It has
been noted that the binding constant for proteins with
monosaccarides peak at approximately 10.sup.7 M.sup.-1, a
remarkable low value for biological molecular recognition..sup.7
This low molecular affinity for monomeric carbohydrates is
magnified biologically through what has become known as the
"glycoside cluster effect." This effect is manifested when
carbohydrates are oligomerized, thereby maximizing binding
efficiencies through not only an additive manner but also through
entropic means..sup.8
[0006] X-ray structures of oligosaccharide-binding proteins have
revealed that the polar groups of the carbohydrates are involved in
multiple hydrogen bonding interactions with complementary polar
donor and acceptor hydrogen bond sites on the protein. Nature has
used this complementary interaction to a great extent in order to
gain specificity and energy for binding. Furthermore, numerous salt
bridges are observed between charged residues on the protein and
complementary charged carboxylate, phosphate, sulfate or ammonium
functions on the carbohydrate structure. It has been noted that the
involvement of serine, tyrosine and threonine hydroxyl groups is
relatively uncommon..sup.9 It has also been noted that most of the
complementary non-polar interactions with carbohydrates involve
aromatic residues on the protein binding partner..sup.10 Most of
the hydrogen bonds involve planar, multivalent side chain groups
(Asn, Asp, Glu, Gln, Arg, His). An additional insight was the
recognition of the ability of 2-aminopyridine moiety to act as a
heterocyclic mimetic of the asparagines/glutamine amide side
chain..sup.11
[0007] Several examples of the detailed three-dimensional structure
of polybasic protein ligands binding to anionic oligosaccharides
exist. The binding interaction between fibroblast growth factor and
heparin.sup.12 reveals that a significant number of positively
charged protein residues interact with the negatively charged
glycoconjugate receptor. It is important to recognize that many of
the negatively charged species on the receptor are heterogeneously
sulfated on alternating L-iduronic and D-glucosamino sugars..sup.13
X-ray analysis of the glycoprotein hormone follicle-stimulating
hormone interacting with its receptor shows that a large buried
interface (2600 .ANG..sup.2) with a high charge density (1.13
charges per nm.sup.2) defines a universal binding mode where charge
complementarity defines specificity..sup.14 Theoretically, a large
energy barrier must be overcome by desolvating the partners before
binding can occur.
[0008] The carbohydrate-modifying enzymes known as
sulfotransferases represent an intriguing method used by nature to
reversibly create anionic binding sites on biomolecules. Many
literature examples exist of biological phenomena such as
development, differentiation and especially immunology which are
modulated by the presence or absence of sulfated
glyco-conjugates..sup.15 Specifically, the effects of polyamines on
blood coagulation and fibrinolysis in the presence of
glycosaminoglycans (GAGs) has been examined because it is known
that heparin (HP) interacts with polyamines, especially with
spermine..sup.16
[0009] Recent scientific advances have greatly enabled the ability
to delineate the role of specific carbohydrates in biological
processes. Reviews of these advances have appeared..sup.17,18 An
especially exciting development is the automated solid-phase
synthesis of defined oligiosaccarides..sup.19 The interactions of
heparin/heparan sulfate with various proteins have been
reviewed..sup.20 Screening for inhibitors of
oligiosaccharide-mediated biological events has been successfully
applied to the microtiter plate format..sup.21,22 The use of
surface plasmon resonance imaging has been applied to the study of
protein-carbohydrate interactions..sup.23 The general uses of
optical biosensors to drug discovery has also been reviewed..sup.24
Capillary electrophoresis is an additional tool used to define
interactions between sulfated polysaccharides and
proteins..sup.25
[0010] Interruption of carbohydrate-mediated disease processes. A
report by Joosten et al. showed that a series of dendritic
galabiose compounds containing a polyamido core (PAMAM-) had
activity in inhibition of bacterial binding in the subnanomolar
concentration levels..sup.26 A report by Yudovin-Farber showed that
anti-prion agents could be produced using polycationic
oligosaccharides..sup.27 Furthermore, the elimination of prion
particles from infected individuals using polycationic agents has
been shown..sup.28-31 Medicinal chemistry efforts towards
inhibition of integrin-mediated events have been made..sup.32;33
Molecular recognition by these cell adhesive molecules known as
integrin receptors on the cell surface is one of the most important
biological processes not only in cell adhesion but also in
fertilization, organ formation, cell migration, lymphocyte
trafficking, immune response, and cancer metastasis..sup.34
Endotoxins, or lipopolysaccharides (LPS), the predominant
structural component of the outer membrane of Gram-negative
bacteria,.sup.35-37 play a pivotal role in septic shock, a syndrome
of systemic toxicity which occurs frequently when the body's
defense mechanisms are compromised or overwhelmed, or as a
consequence of antibiotic chemotherapy of serious systemic
infections (Gram-negative sepsis)..sup.38-41 Referred to as "blood
poisoning" in lay terminology, Gram-negative sepsis is the
thirteenth leading cause of overall mortality.sup.42 and the number
one cause of deaths in the intensive care unit,.sup.43 accounting
for more than 200,000 fatalities in the US annually..sup.44 Despite
tremendous strides in antimicrobial chemotherapy, the incidence of
sepsis has risen almost three-fold from 1979 through 2000.sup.45
and sepsis-associated mortality has essentially remained unchanged
at about 45%.sup.46, both calling to attention the fact that
aggressive antimicrobial therapy alone is insufficient in
preventing mortality in patients with serious illnesses, and
emphasizing an urgent, unmet need to develop therapeutic options
specifically targeting the pathophysiology of sepsis.
[0011] The presence of LPS in the systemic circulation causes a
widespread activation of the innate immune response.sup.47;48
leading to the uncontrolled production of numerous inflammatory
mediators, including tumor necrosis factor-.alpha. (TNF-.alpha.),
interleukin-1.beta. (IL-1.beta.), and interleukin-6 (IL-6),
primarily by cells of the monocyte/macrophage lineage,.sup.49;50 as
well as others, such as nitric oxide produced by the endothelial
cell,.sup.51;52 which, in concert, act to cause a frequently fatal
systemic inflammatory response,.sup.53 termed `septic shock`. The
toxic moiety of LPS is its structurally conserved glycolipid
component called Lipid A,.sup.54 which is composed of a
hydrophilic, bis-phosphorylated diglucosamine backbone, and a
hydrophobic domain of 6 (E. coli) or 7 (Salmonella) acyl
chains.sup.54 (FIG. 1). The pharmacophore necessary for the
neutralization of lipid A.sup.55 by small molecules requires two
protonatable positive charges separated by a distance of .about.14
.ANG., enabling ionic H-bonds between the cationic groups and the
lipid A phosphates; in addition, appropriately positioned pendant
hydrophobic functionalities are required to further stabilize the
resultant complexes via hydrophobic interactions with the polyacyl
domain of lipid A (for a recent review, see Ref..sup.56). These
structural requisites were first identified in certain members of a
novel class of compounds, the lipopolyamines, which were originally
developed, and are currently being used as DNA transfection
(lipofection) reagents. 57-60 In a detailed study of the effect of
the hydrocarbon chain length in a homologous series of
acylhomospermines, it was shown that C.sub.16 is the ideal
lipophilic substituent, corresponding to maximal affinity, optimal
aqueous solubility (and bioavailability), and neutralization
potency..sup.61
SUMMARY
[0012] The present disclosure relates to modulating or interrupting
processes which comprises binding oligosaccharide-based
biomolecules with small molecule polycationic agents.
[0013] The present disclosure also relates to compounds represented
by the formulae: ##STR1##
[0014] wherein x is selected from the group consisting of
--(CH.sub.2).sub.y--; 1, 2-C.sub.6H.sub.4--; 1,3-C.sub.6H.sub.4--;
1,4-C.sub.6H.sub.4--; and --CH.sub.2 OCH.sub.2--;
[0015] y is an integer of 0-10;
[0016] R is selected from the group consisting of --H, --CH.sub.3,
--CH.sub.2C.sub.6H.sub.5, --CH.sub.2-3-indoline,
--CH.sub.2-2-indoline, and --CH.sub.2-4-imidazole
[0017] m is 0-4;
[0018] n is 0-4; and
[0019] R' is selected from the group consisting of
--(CH.sub.2).sub.o--CH.sub.3; --(CH.sub.2).sub.o--CH.sub.2NH.sub.2;
phenyl; 1-naphthyl and 2-naphthyl;
[0020] o is an integer of 0-16;
[0021] pharmaceutically acceptable salts thereof; and prodrugs
thereof.
[0022] The present disclosure also relates to treating diseases of
an immunological disorder such as psoriasis, lupus, Crohn's
disease, inflammatory bowel disease, rheumatoid arthritis, Type 1
diabetes, Type 2 diabetes or sepsis by administering to a patient
in need thereof, an effective amount of a compound disclosed
above.
[0023] A further aspect of this discloses is concerned with
treating diseases of an infectious disorder such as those caused by
bacteria, fungi or viruses by administering to a patient in need
thereof, an effective amount of a compound disclosed above.
SUMMARY OF DRAWINGS
[0024] FIG. 1 shows a schematic and crystal structure of
lipopolysaccharide along with Lipid A.
[0025] FIG. 2 illustrates the bimodal distribution of binding
affinities.
[0026] FIG. 3 illustrates an energy-minimized model of the
disclosed scaffold-lipid A complex.
[0027] FIG. 4 illustrates ability of compounds of the disclosure to
inhibit NO production.
[0028] FIG. 5 illustrates the correlation of binding affirmative to
neutralization patterning.
DESCRIPTION OF BEST AND VARIOUS MODES
[0029] Compounds of the present disclosure are represented by the
following formulae: ##STR2##
[0030] wherein x is selected from the group consisting of
--(CH.sub.2).sub.y--; 1,2-C.sub.6H.sub.4--; 1,3-C.sub.6H.sub.4--;
1,4-C.sub.6H.sub.4--; and --CH.sub.2 OCH.sub.2--;
[0031] y is an integer of 0-10;
[0032] R is selected from the group consisting of --H, --CH.sub.3,
--CH.sub.2C.sub.6H.sub.5, --CH.sub.2-3-indoline,
--CH.sub.2-2-indoline, and --CH.sub.2-4-imidazole
[0033] m is 0-4;
[0034] n is 0-4; and
[0035] R' is selected from the group consisting of
--(CH.sub.2).sub.o--CH.sub.3; --(CH.sub.2).sub.o--CH.sub.2NH.sub.2;
phenyl; 1-naphthyl and 2-naphthyl;
[0036] o is an integer of 0-16;
[0037] pharmaceutically acceptable salts thereof; and prodrugs
thereof.
[0038] Preferred compounds according to the present disclosure are
represented by the following formulae:
[0039] 1. formula for MQTS 1172: ##STR3##
[0040] 2. formula for MQTS 1132: ##STR4##
[0041] 3. The formula for MQTS 1007: ##STR5##
[0042] 4. The formula for MQTS 1242: ##STR6##
[0043] Further preferred compounds, representing
1,3-C.sub.6H.sub.4- molecular arrangement as specified above,
according to the present disclosure are represented by the
following formulae: ##STR7##
[0044] According to the present disclosure a terminally-placed
long-chain aliphatic group is important for effective LPS
neutralization. Furthermore, the chemical space defined by the
described compounds identify novel, non-polyamine scaffolds that
incorporate the LPS-binding pharmacophore described above.
[0045] Prodrug forms of the compounds bearing various nitrogen
functions (amino, hydroxyamino, hydrazino, guanidino, amidino,
amide, etc.) may include the following types of derivatives where
each R group individually may be hydrogen, substituted or
unsubstituted alkyl, aryl, alkenyl, alkynyl, heterocycle,
alkylaryl, aralkyl, aralkenyl, aralkynyl, cycloalkyl or
cycloalkenyl groups as defined above. [0046] Carboxamides,
--NHC(O)R [0047] Carbamates, --NHC(O)OR [0048] (Acyloxy)alkyl
Carbamates, NHC(O)OROC(O)R [0049] Enamines,
--NHCR(.dbd.CHCRO.sub.2R) or --NHCR(.dbd.CHCRONR.sub.2) [0050]
Schiff Bases, --N.dbd.CR.sub.2 [0051] Mannich Bases (from
carboximide compounds), RCONHCH.sub.2NR.sub.2 Preparations of such
prodrug derivatives are discussed in various literature sources
(examples are: Alexander et al., J. Med. Chem. 1988, 31, 318;
Aligas-Martin et al., PCT WO pp/41531, p.30). The nitrogen function
converted in preparing these derivatives is one (or more) of the
nitrogen atoms of a compound of the invention.
[0052] Prodrug forms of carboxyl-bearing compounds of the invention
include esters (--CO.sub.2R) where the R group corresponds to any
alcohol whose release in the body through enzymatic or hydrolytic
processes would be at pharmaceutically acceptable levels.
[0053] Another prodrug derived from a carboxylic acid form of the
invention may be a quaternary salt type ##STR8## of structure
described by Bodor et al., J. Med. Chem. 1980, 23, 469.
[0054] It is of course understood that the compounds of the present
disclosure relate to all optical isomers and stereo-isomers at the
various possible atoms of the molecule.
[0055] The compounds of this disclosure form acid and base addition
salts with a wide variety of organic and inorganic acids and bases
and includes the physiologically acceptable salts which are often
used in pharmaceutical chemistry. Such salts are also part of this
disclosure. Typical inorganic acids used to form such salts include
hydrochloric, hydrobromic, hydroiodic, nitric, sulfuric,
phosphoric, hypophosphoric and the like. Salts derived from organic
acids, such as aliphatic mono and dicarboxylic acids, phenyl
substituted alkonic acids, hydroxyalkanoic and hydroxyalkandioic
acids, aromatic acids, aliphatic and aromatic sulfonic acids, may
also be used. Such pharmaceutically acceptable salts thus include
acetate, phenylacetate, trifluoroacetate, acrylate, ascorbate,
benzoate, chlorobenzoate, dinitrobenzoate, hydroxybenzoate,
methoxybenzoate, methylbenzoate, o-acetoxybenzoate,
naphthalene-2-benzoate, bromide, isobutyrate, phenylbutyrate,
.beta.-hydroxybutyrate, butyne-1,4-dioate, hexyne-1,4-dioate,
cabrate, caprylate, chloride, cinnamate, citrate, formate,
fumarate, glycollate, heptanoate, hippurate, lactate, malate,
maleate, hydroxymaleate, malonate, mandelate, mesylate, nicotinate,
isonicotinate, nitrate, oxalate, phthalate, teraphthalate,
phosphate, monohydrogenphosphate, dihydrogenphosphate,
metaphosphate, pyrophosphate, propiolate, propionate,
phenylpropionate, salicylate, sebacate, succinate, suberate,
sulfate, bisulfate, pyrosulfate, sulfite, bisulfite, sulfonate,
benzene-sulfonate, p-bromobenzenesulfonate, chlorobenzenesulfonate,
ethanesulfonate, 2-hydroxyethanesulfonate, methanesulfonate,
naphthalene-1-sulfonate, naphthalene-2-sulfonate,
p-toleunesulfonate, xylenesulfonate, tartarate, and the like.
[0056] Bases commonly used for formation of salts include ammonium
hydroxide and alkali and alkaline earth metal hydroxides,
carbonates, as well as aliphatic and primary, secondary and
tertiary amines, aliphatic diamines. Bases especially useful in the
preparation of addition salts include sodium hydroxide, potassium
hydroxide, ammonium hydroxide, potassium carbonate, methylamine,
diethylamine, and ethylene diamine.
[0057] The compounds may be utilized alone or in combination with
other agents.
[0058] In another aspect of the disclosure, compositions containing
the above described compounds and derivatives are provided.
Typically, the compositions are formulated to be suitable for
pharmaceutical use by the inclusion of appropriate carriers or
excipients.
[0059] In a further aspect of the disclosure methods for the use of
the above described compounds, as well as compositions, are
provided. These methods include uses of the invention's compounds
to modulate or interrupt biological processes involving the
recognition or binding of oligosaccharides-based biomolecules.
Compounds of the present disclosure are useful for treating a
disease or condition in which the inhibition of NO (nitric oxide)
is desirable. Examples of human diseases and conditions include,
but are not limited to, chronic or acute inflammation, inflammatory
bowel disease (including Crohn's disease), inflammatory bowel
syndrome, autoimmune diseases rheumatoid arthritis, systemic lupus
erythematosus, cutaneous forms of lupus, Type 1 and Type 2
diabetes, multiple sclerosis, psoriasis, spondyloarthropathies
(SpA) including spondylitis, synovitis, psoriatic arthritis and
subclinical gut inflammation and infectious diseases including
sepsis, septic shock, endotoxic shock, HIV and other viral
infections including cytomegalovirus, herpes simplex virus,
influenza virus; infectious disorders caused by bacteria or
fungi.
[0060] As discussed above, the present disclosure describes an
example of a method to selectively interrupt
oligosaccharide-mediated biological phenomenon. The method
recognizes the exhaustive number of potential biological and drug
targets involving oligosaccharide conjugates. The molecular
contributions for binding to lipid A/endotoxin, one of a number of
these anionic oligosaccharide targets, were assessed in a rapid and
detailed fashion through the application of a prospectively
designed, moderately-sized 540-membered example library. Analysis
of the contribution that each individual monomeric library
component made to the most tight-binding analogs was facilitated by
use of "molecular vector analysis." This analysis confirmed the
importance of the lipophilic long chain aliphatic group (typically
C.sub.12 to C.sub.22 lipid chain and more typically C.sub.18 lipid
chain), while also pointing to the contribution made by
heteroaromatic moieties such as indole portion of tryptophan. The
incorporation of techniques such as Synphase lanterns together with
the data transfer/handling software made synthesis of the
multi-hundred membered library much more straightforward.
[0061] The following non-limiting examples are presented to further
illustrate the present disclosure.
[0062] General experimental methods. The sources of all chemical
reagents and starting materials are of the highest grade available
and are used without further purification. Lanterns used are
Mimotopes SynPhase PS D-Series Lanterns.TM. with a trityl alcohol
linker. Thin-layer chromatography analysis and column
chromatography is performed using Merck F.sub.254 silica gel plates
and Baker 40 .mu.m flash chromatography packing, respectively. TLC
analysis used the specified solvent systems with detection by
ninhydrin staining. Data handling is facilitated by the use of
BalanceLink V3.0 software from Mettler-Toledo to enable transfer of
weight values directly into an Excel spreadsheet. Solvents from
resin cleavage are removed through the use of a Savant centrifugal
evaporator operating at 25.degree. C.
[0063] LC/MS analyzes are performed using a Gilson 322 HPLC system
coupled to a 215 liquid handler. Detection was by a Finnigan AQA
operating in ESI.sup.+ mode (m/z range 140 to 1600 amu) together
with an Agilent 1100 series diode array detector (UV range 220 to
320 nm). Gradient elution from 2 to 7 min. at 0.2 mL/min. is
performed using 2% to 100% CH.sub.3CN in H.sub.2O (both with 0.05%
TFA) using a Waters XTerra MS C.sub.18 2.1.times.150 mm (3.5 .mu.m)
column. .sup.1H NMR spectra are recorded at 300 MHz on a Bruker
AV300 spectrometer at the University of Washington, Seattle.
.sup.1H NMR signals are generally multiples unless otherwise noted
as s=singlet, d=doublet, t=triplet or m=multiplet. Chemical shifts
are relative to external 3-(trimethylsilyl)-1-propanesulfonic acid,
sodium salt.
EXAMPLE 1
[0064] Ethyl N-(2-nitrophenylsulfonamide)-glycinate (2a)--To a
solution of 25.11 g (0.180 mole) of ethyl glycinate hydrochloride
and 42.11 g (0.190 mole, 1.06 eq) of 2-nitrophenylsulfonylchloride
in 400 mL of dry CH.sub.2Cl.sub.2 at 0.degree. C. is added 76.6 mL
(0.44 mole, 2.44 eq) of .sup.iPr.sub.2NEt dropwise. The resulting
solution is stirred for 18 h when it is quenched by the addition of
200 mL of H.sub.2O. The organic layer is removed and the aqueous
part is re-extracted by an additional portion of 200 mL
CH.sub.2Cl.sub.2. The combined organic layers are washed with 0.1 N
HCl then brine and dried and evaporated to give the crude product
as an off-white solid. This is crystallized from 400 mL of abs.
EtOH to give 44.69 g (90%) white crystals. .sup.1H NMR (CDCl.sub.3,
.delta.): 8.16 (d, 1H), 7.82 (d, 1H), 7.78 (m, 2H), 5.62 (s, 1H),
4.04 (q, 2H), 3.98 (s, 2H), 1.24 (t, 3H).
EXAMPLE 2
[0065] Ethyl N-(2-nitrophenylsulfonamide)-2-aminopropionate
(2b)--Using the procedure described above this product is produced
in 80% yield. .sup.1H NMR (CDCl.sub.3, .delta.): 8.24 (d, 1H), 7.81
(d, 1H), 7.74 (m, 2H), 5.75 (s, 1H), 4.11 (q, 2H), 3.43 (q, 2H),
3.23 (m, 2H), 2.43 (t, 2H), 1.22 (t, 3H).
EXAMPLE 3
[0066] Ethyl N-(2-nitrophenylsulfonamide)-3-aminobutyrate
(2c)--Using the procedure described for 2a, this product is
produced in 54% yield following crystallization from abs. EtOH.
.sup.1H NMR (CDCl.sub.3, .delta.): 8.21 (d, 1H), 7.84 (d, 1H), 7.77
(m, 2H), 5.65 (s, 1H), 4.09 (q, 2H), 3.42 (q, 2H), 3.23 (m, 2H),
2.43 (t, 2H), 1.91 (m, 2H), 1.25 (t, 3H).
EXAMPLE 4
[0067] General alkylated monomer synthesis - Mitsunobu alkylation:
Ethyl
N-2-(1-naphthyl)ethyl-(2-nitrophenylsulfonamide)-2-aminopropionate
(3b1)--To the solution produced by dissolving 3.02 g (10 mmol) of
2b, 1.72 g (10 mmol) of 2-(1-naphthyl)-ethanol and 3.98 g (15 mmol)
of triphenylphosphine in 50 mL of dry CH.sub.2Cl.sub.2 is added
dropwise at 25.degree. C. a solution of 2.95 mL (15 mmol) of
diisopropylazodicarboxylate in 15 mL of dry CH.sub.2Cl.sub.2. The
resulting yellow colored solution is stirred for 16 h when the
reaction solution is diluted in 75 mL CH.sub.2Cl.sub.2 and 75 mL
0.1N HCl. The aqueous layer is removed and re-extracted by an
additional 75 mL portion of CH.sub.2Cl.sub.2. The combined organic
layers are washed with brine, dried and evaporated to give the
crude product as a yellow oil. Column chromatography over silica
gel uses 3:1 hexane/ethyl acetate to give 2.17 g (48% yield)
colorless crystals.
EXAMPLE 5
[0068] Ester hydrolysis:
N-2-(1-naphthyl)ethyl-(2-nitrophenylsulfonamide)-2-aminopropionic
acid (4b1)--To the clear solution of 3.17 g (6.9 mmol) of 3b1 in
100 mL of THF is added 13.9 mL (2 eq) of 2N LiOH in H.sub.2O. The
resulting two-phase mixture is vigorously stirred for 16 h when the
THF is evaporated in vacuo and the resulting residue is suspended
in 75 mL CH.sub.2Cl.sub.2 and 50 mL of IN HCl. The aqueous part is
re-extracted by an additional portion of 75 mL CH.sub.2Cl.sub.2 and
the combined organic layers are dried and evaporated to give oily
solids. When TLC analysis using 1:1 hex/EtOAc with
I.sub.2-detection shows the presence of diisopropylhydrazine
side-product column chromatography with 8:2 hex/EtOAc can be used
to obtain pure carboxylic acid monomer material.
[0069] 15-Membered test library production. A set of 15 Mimotopes
SynPhase PS D-Series Lanterns.TM. with a trityl alcohol linker
(15.times.35 .mu.mol=0.525 mmol total) are labeled with spindles
and cogs and dried under high vacuum over P.sub.2O.sub.5 for 18 h.
They are then suspended in a solution of 18 mL dry CH.sub.2Cl.sub.2
and 2 mL of acetyl chloride. Following gentle shaking for 3.5 h the
lanterns are washed three times with dry CH.sub.2Cl.sub.2 to give
P1. While still in their CH.sub.2Cl.sub.2 swollen form they are
suspended in 20 mL of dry CH.sub.2Cl.sub.2 and 5 mL of
3-aminopropanol is added. The vessel is shaken for 18 h then washed
three times each with CH.sub.2Cl.sub.2, DMF, .sup.iPrOH, THF and
CH.sub.2Cl.sub.2 (standard washing sequence) then is dried under
high vacuum giving P2.
[0070] The lanterns are next transformed into their amine form (P4)
by the following two-step sequence. Suspension in 20 mL dry
CH.sub.2Cl.sub.2 is followed by the addition of 0.39 g (2.63 mmol,
5 eq) of phthalimide and 0.69 g (5 eq) of triphenylphosphine as
solids. The reaction vessel is shaken to dissolve these reagents
then treated portionwise with a solution of 0.52 mL of
diisopropyldiazodicarboxylate dissolved in 10 mL of dry
CH.sub.2Cl.sub.2. This vessel is shaken for 3 h when standard
washing and drying give lanterns P3. These are suspended in 10 mL
of abs. EtOH and treated with 10 mL of hydrazine hydrate. The
vessel is tightly capped then heated to 60.degree. C. in a rotating
oven for 18 h. Following cooling to room temperature the lanterns
are washed and dried in standard manner to give lantern form
P4.
[0071] Fmoc-amino acid couplings use the following standard
conditions for production of lanterns P5. The amino lanterns P4 are
suspended and swelled in 5 mL of dry DMF. A solution containing
1.02 g (2.63 mmol, 5 eq) of Fmoc-Phe-OH, 1.0 g (5 eq) of HBTU, 0.2
g (2.5 eq) of HOBt and 0.92 mL (10 eq) of .sup.iPr.sub.2NEt is
prepared and shaken for 10 min prior to addition to the lanterns
suspended above. The resulting reaction mixture is shaken gently
for 2 h when standard washing and drying give the product lanterns
P5. The peptide coupling and resin loading of the lanterns is
measured by dilution of the solution from next, Fmoc-group removal
reaction. The lanterns are suspended in 15 mL 20% piperidine in DMF
and shaken for 15 min. A 0.10 mL aliquot is removed and diluted to
10 mL in the same solvent mixture. Following solvent zeroing a UV
measurement of the absorbance at 301 nm gives a value of 1.186.
Using the .epsilon..sub.a value for Fmoc-piperidine adduct of 14102
L mol.sup.-1 cm.sup.-1 a loading efficiency of 16.7 .mu.mole or 48%
is calculated (1.186.times.14102=16.7) (Manufacturer's stated
loading was 35 .mu.mole). Standard washing and drying of the
lanterns following a 1.5 h reaction time gives lanterns P6.
[0072] These lanterns are now used to couple, individually, to each
of the 15 monomers synthesized through the process described above.
Fifteen 4 mL dried vials are loaded with 0.066 g (0.14 mmol, 4 eq
based on an avg MW of 474.4) of each monomer. One mL of a solution
that is prepared containing 0.80 g (2.1 mmol, 4.times.15 eq) of
HBTU, 0.16 g (1.05 mmol, 2.times.15 eq) of HOBt and 0.732 mL (4.2
mmol, 8 eq) of .sup.iPr.sub.2NEt in 15 mL of dry DMF is added to
each monomer containing vial. These vials are gently shaken while
the lanterns are pre-swelled together in 15 mL of dry DMF. After 10
min each labeled lantern is placed into its respective vial
containing the activated monomer ester. The lanterns are shaken
overnight then the reaction solution is decanted. They are combined
and washed in standard fashion. Drying gaies the protected lanterns
P7. The NPS group is removed by treating the combined set of
lanterns with 5 mL of 2-mercaptoethanol, 5 mL of DBU and 15 mL of
DMF for 18 h. Standard washing and drying gives the loaded resins
P8 ready for cleavage.
[0073] Each lantern is placed in an individually marked and
pre-tared 4 mL vial and treated with 2.0 mL of cleavage cocktail
consisting of 80:18:2 CH.sub.2Cl.sub.2/TFA/.sup.iPr.sub.3SiH for 1
h. The lanterns are extracted with tweezers and washed with
CH.sub.2Cl.sub.2 and the cleavage solutions are evaporated. The
resulting yellow oil residues are each dissolved in 0.50 mL MeOH
and 20 .mu.L removed and diluted to 200 .mu.L in H.sub.2O for LC/MS
analysis. LC/MS is performed on all fifteen analogs. The
concentrated stock MeOH solutions are also used for TLC analysis in
two solvent systems: a) 8:2 CH.sub.3CN/concd NH.sub.4OH; b) 90:8:2
CHCl.sub.3/MeOH/concd NH.sub.40H. .sup.1H NMR is performed on two
analogs below.
[0074] MQTS 1093T--LC/MS calcd [M+H] for
C.sub.27H.sub.32N.sub.4O.sub.2: m/z 445; obsd 445 at 13.6 min.
.sup.1H NMR (D.sub.2O, .delta.): 7.73-7.16 (m, 14H), 4.38 (t, 1H),
4.22 (m, 2H), 3.84 (m, 2H), 3.32-2.83 (m, 2H), 2.60 (m, 2H), 2.02
(m, 2H), 1.60 (m, 2H).
[0075] MQTS 1095T--LC/MS calcd [M+H] for
C.sub.17H.sub.29N.sub.5O.sub.2: m/z 336; obsd 336 at 12.9 min.
.sup.1H NMR (D.sub.2O, .delta.): 7.38-7.20 (m, 5H), 4.51 (t, 1H),
3.86 (m, 2H), 3.30-3.15 (m, 2H), 3.06 (t, 8H), 2.02 (m, 2H), 1.62
(m, 2H).
[0076] 540-Membered example library production. Synthesis of the
complete 540-membered library follows the same sequence as that for
the 1 5-membered test library. 540 lanterns are labeled with
spindles and cogs and are activated to their P1 forms using the
procedure above. Six sets of 90 lanterns are sorted into individual
vessels and are treated with 10 g (or 10 mL) of the amino alcohol
shown in Chart 1. Following reaction and washing to give their P2
forms the 540 lanterns are re-combined and converted to their free
amine form via the sequence outlined above (P2 to P4). The lanterns
are then split into six groups with 90 members and coupled to the
requisite Fmoc-amino acid using the procedure outlined above. UV
analysis of the Fmoc-loading of six randomly selected lanterns
showed respectable 60-125% loading efficiencies. Following
couplings the re-combined lanterns are treated with 20%
piperidine/DMF as above to give the free-amine form P6 lanterns.
The lanterns are then split (36.times.15) for their final coupling
reaction to the 15 monomers 4 using the standard coupling
conditions. NPS-protecting group cleavage readies the lanterns for
sorting into 540 individual pre-tared 4 mL vials. Final cleavage
gives the crude analogs MQTS 1001 to 1540 in their TFA salt forms.
An average yield of 84% is calculated based on the expected
structure and excluding those with >200% yield (n=36).
[0077] The entire library is characterized by TLC and LC/MS. The
crude material is mostly dissolved in 1.0 mL of MeOH and spotted
onto TLC plates. If insoluble particles remain they are removed by
filtration prior to chemical or biological characterization.
Elution of the plates use the solvent system CHCl.sub.3/MeOH/concd
NH.sub.4OH 85:13:2 with ninhydrin detection. The above concentrated
stock solutions are diluted 20.times. into 1% TFA in H.sub.2O for
LC/MS analysis. The MeOH sample solutions are treated with 1.0 mL
of 6N HCl then evaporated to give the HCl salts of the final
products. These are dissolved in the amount of 20% DMSO/H.sub.2O
that is required to give 20 mM solutions based on the crude yields
that are obtained. The use of an Excel spreadsheet with the
pretared and final vial weights together with MQTS number,
structures, molecular formula, and molecular weights of free bases
imported from ISIS base greatly facilitate the calculations of salt
molecular weights, percent yields and amount of solvent necessary
to give bioscreening solutions.
[0078] Rationale and design of molecular scaffold and library
monomers. The design of the example 540-membered library is to: i)
to confirm and validate the lipid A binding pharmacophore in
compounds with non-polyamine scaffolds; ii) to maximize diversity
of library members within this context; iii) to systematically test
the hypotheses that the introduction of aromatic groups and/or
H-bond donor/acceptor atoms in the scaffold enhance binding
affinity. Several potential strategies to enhance
carbohydrate-binding affinity are used by targeting additional
interactions with the diglucosamine backbone of lipid A. Both
covalent (such as by using boronates which form esters with the
vicinal cis diols).sup.62;63 as well as noncovalent
interactions.sup.64;65 are considered. An examination of the
Protein Data Bank for lectin-sugar complexes.sup.66;67 as well as
relevant literature.sup.68-70 point to (a) multiple H-bond
donor/acceptor pairs contributing to the enthalpy of binding and
(b) an unusual preponderance of aromatic side chains around the
sugar binding site,.sup.66 suggesting either multiple
CH-.pi..sup.71;72 or OH-.pi. weak H-bonds..sup.73;74 Indeed, a
lipid A receptor with a oligocyclopentane backbone substituted with
amino and indole functionalities has been described..sup.75 A
recent report described LPS-targeting peptoids isolated from a
positional scanning library which incorporated various aromatic
constituents along its backbone..sup.76 Furthermore, the crystal
structure of LPS indicates a range of inter-atomic distances
between 2.4-4 .ANG. between H-bond donor/acceptor atoms on the
lipid A backbone (see FIG. 1)..sup.77 Library members are therefore
designed with an intervening distance of 2-3 carbon bonds between
H-bond donor/acceptor atoms in order to favor complementarity with
the anionic carbohydrate target.
[0079] The scaffold and elements (PORTIONS 1-3) of the
combinatorial library are shown in Chart 1. The distance between
the terminal amines are `dialed in` by varying intervening elements
in both PORTION 1 as well as the Gly/Ala/GABA amino acids in
PORTION 3. As can be seen in Chart 1, PORTION2 contains a
preponderance of aromatic groups. In PORTION 3.y, both aliphatic
and aromatic substituents are incorporated in order to meet the
requirement of a long-chain aliphatic group for optimal
activity..sup.61
[0080] Synthesis of library monomers. Using a Mitsunobu-mediated
alkylation of solid-phase 2-nitrophenylsulfonamides.sup.78;79 is
initially considered, but during the formation of the requisite
resin-bound sulfonamides to completion is not achieved. Similar
difficulties using this approach on solid-phase have been
previously reported in the literature..sup.80 A solution-phase
alkylation of the esters of amino-acid sulfonamides for the
synthesis of the fifteen PORTION 3 monomers en route to the
synthesis of the 540-membered library by the route depicted in
Scheme 1 is instead employed. Modification of the conditions by the
use of the more hindered base .sup.iPr.sub.2NEt enables the desired
sulfonamides to be prepared in good yield following
crystallization.
[0081] Alkylation of each of the three sulfonamides by the five
primary alcohols corresponding to the PORTION 3 substituents shown
in Chart 1 proceeds in straightforward fashion. Following column
chromatography purification some of these alkylated
ester-sulfonamides show the presence of various amounts of an
impurity corresponding to diisopropylhydrazinedicarboxylate, a
side-product from the Mitsunobu alkylation. This material can be
eliminated either at this ester material step (3a-c) or in the
next, carboxylic acid step (4a-c) by column chromatography. In
either case, the impurity is readily detected by TLC using I.sub.2
staining or by .sup.1H NMR, ensuring its complete removal in the
products. Hydrolysis of the esters is accomplished in a
straightforward manner. All molecules show high purity by TLC and
.sup.1H NMR with their identities being confirmed by LC/MS
analysis.
[0082] Chemical route to library: Fifteen-membered test library. A
test of the solid-phase synthetic route is carried out using each
of the fifteen monomers produced above attached to fifteen
identical lanterns containing the 1,3-diaminopropane-Phe PORTION
1:PORTION 2 resin partner (Scheme 2). Prior experience with
symmetrical diamine attachment to tritylchloride solid-phase resin
shows that significant crosslinking occurs leading to substantial
diamine contamination in the cleaved products. For this reason a
three-step sequence is used involving attachment of an amino
alcohol followed by --OH to --NH.sub.2 conversion.
Mitsunobu-mediated phthalimide group attachment followed by
hydrazine liberation of the free amine gives the desired lanterns.
This process completely eliminates the formation of the diamine
side-product while substantially increasing the loading efficiency
of the desired product.
[0083] Standard peptide coupling conditions are used to add the Phe
residue to this set of lanterns. UV analysis of the liberated Fmoc
group from the next step shows a loading efficiency of 48% at this
stage. The lanterns are then individually attached to each of the
fifteen monomers using HBTU coupling conditions. The NPS-groups are
removed using 2-mercaptoethanol/DBU/DMF. The products are then
cleaved using 80:18:2 CH.sub.2Cl.sub.2/TFA/.sup.iPr.sub.3SiH
directly into individual pre-tared 4 mL vials. Data handling is
facilitated by direct data acquisition from the weighing balance
into a spreadsheet program. In this way, data associated with the
samples including vial tare weight and net crude weight can be
coordinated with the sample ID number, structure, molecular weight,
theoretical yield, and crude percent yield. The samples are
dissolved in MeOH and sampled for TLC and LC/MS analyses as
described below. These crude samples are then treated with an equal
volume of 6N HCl and evaporated to give their per-HCl salts. For
the fifteen test samples an average weight percent yield of 70% is
calculated represented by an average crude weight of 18.4 mg.
[0084] TLC and LC/MS analysis of the crude samples support the
viability of the 8-step process. Several informative observations
are made following this analysis: 1) Side-products with masses at
147 amu lower m/z values are observed. Two major spots are seen in
the TLC analysis of most of these test analogs and a side-peak
showed up at a shorter retention times in the LC/MS chromatograms.
It is deduced that these side-products are generated from the
incomplete coupling of the Phe-PORTION 2 residue. It is concluded
that this lower than desired Phe loading gives rise to substantial
amounts of truncated products in the samples and may explain the
lower than expected 48% loading efficiency measured following this
step. 2) Alkene addition products are observed with the unsaturated
PORTION 3.x4 monomers. A mixture of un- and mono-substituted
TFA-adducts are seen. Subsequent analysis of the HCl salts showed
complete exchange of .sup.-OTFA by .sup.-Cl. By carrying out a test
library synthesis it is reasoned that PORTION 2 loading conditions
should be modified to decrease truncated side-product formation.
Furthermore, it is determined that the cleavage conditions do not
completely eliminate side-product formation involving acid-mediated
alkene addition.
[0085] Synthesis of 540-membered example library. A coding system
is devised to label the lanterns and the entire library's
structures are enumerated into an ISIS.TM. database. A spreadsheet
is configured for handling the data generated. Library production
follows the route outlined in Scheme 2 and utilizes the components
shown in Chart 1. A large excess of the six amino alcohols is used
to elaborate 90 labeled lanterns in six individual vessels. The
lanterns are then recombined for the next two-step --OH to
--NH.sub.2 conversion. Splitting and sorting enable the next
PORTION2 components to be added. The number of equivalents of
Fmoc-amino acid used in this step is increased from 4 to 5 in order
to decrease the amount of incomplete addition products. The loading
is improved by UV analysis of the liberated Fmoc-piperidine adduct
from a selection of individual lanterns with different PORTION 1:
PORTION2 components. An average loading of 109% (relative to the
manufacturer's value of 35 .mu.mole) is obtained.
[0086] The Fmoc-groups are removed from the entire library and the
monomer set is coupled to fifteen sets of 36 lanterns each. The
NPS-grouping is removed and the lanterns are sorted into individual
pre-tared vials in preparation for final cleavage. Cleavage occurs
in similar fashion to that described above and gives an average of
84% yield of the hydrochloride salt of the crude products. TLC and
LC/MS analysis of the entire set of compounds show adequate purity
for the majority of the library and confirmed that each contain the
desired product as the major component. Side-products due to
truncated PORTION 2 addition are absent but acid-mediated addition
products to some alkene containing products is still observed. The
crude library is dissolved in 20% DMSO/H.sub.2O at 20 mM and is
screened in the assays described below.
[0087] Quantitative Estimation of LPS Binding Affinity. The
relative binding affinities of the entire library of analogs with a
recently-described high-throughput fluorescence based displacement
assay, is examined using BODIPY-TR cadaverine (BC)..sup.81;82
Results are reported as half-maximal effective displacement of
probe (ED.sub.50). In all experiments, Polymyxin B (PMB), a
decapeptide antibiotic, known to bind and neutralize LPS,.sup.83-86
is used as a reference compound.
[0088] As shown in FIG. 2, a distinct biphasic distribution of
binding affinities can be observed, with a clear demarcation of
high- and low-affinity compounds. A particularly instructive method
of graphical evaluation of library screening results is shown in
FIG. 3. This methodology is referred to herein as `molecular vector
analysis`; and it involves counting the number of occurrences of
each individual monomer in the subset of analogs in the top binders
(52 analogs with ED.sub.50<10 .mu.M), and the frequency of
monomers in weak-binding compounds (488 analogs with
ED.sub.50>10 .mu.M). The resulting histograms are easy to
interpret, and simple statistical analyses (.chi.-square) can be
employed to verify the importance of those building blocks that
contribute most to the resulting binding.
[0089] The most profound effects on activity appear with the
selection of long chain hydrophobic C.sub.18 chain (PORTION 3.x.2)
and by the selection of an indole moiety (Trp) in the PORTION 2
position. More subtle, albeit no less important, insights can be
gleaned from the observations made concerning selection of PORTION
1 and PORTION 3 monomers. It would appear that the original concept
of "distance dialing".sup.61;82;87;88 designed into the library
play a role in the results observed. The incorporation of PORTION 1
monomer 1,3-diaminopropane gives an unexpectedly high population of
members in the top 52 samples. Likewise, selection of
2-aminopropionic acid for PORTION 3.B lead to a higher number of
tight binders than the other two monomer components. Based on prior
work,.sup.61;82;88-90 this is attributed to a better congruence in
the distance between the two terminal protonatable nitrogen atoms
in this subset of analogs and that between the anionic phosphates
on the lipid A backbone, enabling effective ionic H-bonds between
the charged groups. It is therefore instructive to construct the
"best" scaffold using the optimal PORTION 1 "vector" component
1,3-diaminopropane, Trp in PORTION 2, and a PORTION 3 bearing a
C.sub.18 alkyl group. The backbone of this molecule (without the
C.sub.18 alkyl chain) is then docked on a crystal
structure-derived.sup.77;91 model of lipid A using
AutoDock.sup.92;93. The alkyl chain is omitted in the modeling
since it has previously been observed that the force fields within
AutoDock do not adequately reflect hydrophobic interactions for
glycolipids, such as lipid A. In the energy-minimized model of the
docked scaffold-lipid A complex, (FIG. 3), a distance of 14.7 .ANG.
is observed between the terminal amines, matching very closely the
previously determined optimized distance between protonatable amine
groups in LPS binders..sup.55;82;88;90 The O atoms on the lipid A
phosphates are also found to be within H-bonding distances of the
amines (FIG. 3). Other PORTION 1 monomer components such as those
composed of 1,5-diaminopentane, significantly diverge from the
optimal value and consequently do not bind LPS as well (FIG.
2).
[0090] Re-synthesis of active molecules. Based on the combination
of results from the binding and preliminary NO inhibition assays, a
series of 25 analogs is selected for re-synthesis and purification
(Table 1). In order to provide enough material for purification, we
used two lanterns are used for each individual analog. As
previously seen in the fifteen test-analog series, minor amounts of
truncated (-PORTION 2 amino-acid) species are seen with several of
these examples. It is possible to isolate 9 molecules representing
these truncated analogs (Table 1). The same synthetic route is used
as before and the crude products are purified over 900 mg disposal
SiO.sub.2 solid-phase extraction columns. These purified analogs
show greater than 90% purity when analyzed by TLC and LC/MS
methods. Table 1 shows the MQTS numbers, structures and BC-binding
data (ED.sub.50 values) together with NO inhibition data (IC.sub.50
values).
[0091] Assessment of neutralization of LPS toxicity: NO inhibition
activity. Murine monocytes (J774.A1 cells) produce measurable
quantities of NO upon exposure to LPS and provide a high-throughput
and validated model for the rapid and quantitative assessment of
compounds in neutralizing the toxicity of LPS..sup.81;82;94
Compounds that neutralize LPS inhibit NO production in a
dose-dependent manner from which 50% inhibitory concentrations
(IC.sub.50) were determined (FIG. 4). The analogs determined to
have the highest affinity in the BC-binding assay are then assayed
in this NO inhibition assay (Table 1). Results in this assay
parallel those in the binding assay (FIG. 5).
[0092] Hit re-synthesis and characterization. Two lanterns per
analog are used to resynthesize the analogs shown in Table 1.
Synthesis follow the procedures given above. The resulting crude
products in their TFA salt forms are purified over disposable
Alltech SPE cartridges containing 900 mg of SiO.sub.2.
Chromatography uses 5 to 20% MeOH in CH.sub.2Cl.sub.2 with 1% concd
NH4OH. TLC solvent is 80:18:2 CH.sub.2Cl.sub.2/MeOH/concd NH4OH
with detection by ninhydrin. The product containing fractions are
pooled and evaporated then converted to their per-HCl salt forms by
treatment with 6N HCl in MeOH and re-evaporation. In several cases
shown in Table 1, truncated analogs (MQTS 2322-2330) without the
internal amino acid portion are also isolated from these products.
Purified samples are analyzed by .sup.1H NMR, TLC and LC/MS using
the methods described above. A selection of LC/MS and NMR data is
given here. Yields for these products range from 6 to 25% following
the 8-step solid-phase route and all show over 90% purity by the
methods noted.
[0093] MQTS 1002--2.9 mg (6% yield) white solid is obtained. LC/MS
calcd [M+H] for C.sub.33H.sub.60N.sub.4O.sub.3: m/z 562; obsd 562
at 14.2 min. .sup.1H NMR (D.sub.2O, .delta.): 7.35-6.92 (m, 5H),
3.86 (m, 1H), 3.68 (m, 2H), 3.57 (m, 2H), 3.39 (m, 4H), 3.14 (m,
4H), 2.98 (m, 2H), 1.63 (m, 2H), 1.17 (s, 30H), 0.80 (s, 3H).
[0094] MQTS 1007--5.3 mg (12% yield) white solid is obtained. LC/MS
calcd [M+H] for C.sub.34H.sub.62N.sub.4O.sub.3: m/z 575; obsd 575
at 14.7 min. .sup.1H NMR (D.sub.2O, .delta.): 7.38-6.97 (m, 5H),
4.56 (m, 1H), 3.63 (t, 2H), 3.48 (m, 2H), 3.31 (m, 2H), 3.14 (m,
4H), 2.83 (m, 2H), 2.60 (m, 4H), 1.42 (m, 2H), 1.18 (s, 30H), 0.82
(t, 3H)
[0095] MQTS 1012--3.6 mg (8% yield) white solid is obtained. LC/MS
calcd [M+H] for C.sub.35H.sub.64N.sub.4O.sub.3: m/z 590; obsd 590
at 14.7 min. .sup.1H NMR (D2O, .delta.): 7.36-6.94 (m, 5H), 4.58
(m, 1H), 3.63 (m, 2H), 3.52 (m, 2H), 3.32 (m, 2H), 3.11 (m, 4H),
2.55 (m, 2H), 2.28 (m, 2H), 1.63 (m, 2H), 1.50 (m, 2H), 1.17 (s,
32H), 0.80 (t, 3H).
[0096] MQTS 1032--6.3 mg (14% yield) white solid is obtained. LC/MS
calcd [M+H] for C.sub.30H.sub.58N.sub.6O.sub.3: m/z 552; obsd 552
at 13.0 min. .sup.1H NMR (D.sub.2O, .delta.): 8.53 (s, 1H), 7.28
(s, 1H), 3.93 (s, 1H), 3.63 (t, 2H), 3.48 (m, 2H), 3.32 (m, 2H),
3.11 (m, 4H), 3.01 (m, 2H), 1.66 (m, 2H), 1.20 (s, 32H), 0.79 (t,
3H).
[0097] MQTS 1037--5.7 mg (13% yield) white solid is obtained. LC/MS
calcd [M+H] for C.sub.31H.sub.60N.sub.6O.sub.3: m/z 566; obsd 566
at 12.9 min. .sup.1H NMR (D.sub.2O, .delta.): 8.58 (s, 1H), 7.26
(s, 1H), 4.58 (t, 1H), 3.63 (s, 2H), 3.47 (t, 2H), 3.28 (m, 2H),
3.31 (m, 2H), 3.12 (m, 4H), 2.98 (m, 2H), 2.78 (m, 2H), 1.65 (m,
2H), 1.20 (s, 30H), 0.78 (t, 3H).
[0098] MQTS 1042--8.1 mg (18% yield) white solid is obtained. LC/MS
calcd [M+H] for C.sub.32H.sub.62N.sub.6O.sub.3: m/z 580; obsd 580
at 12.9 min. .sup.1H NMR (D20, 6): 8.61 (s, 1H), 7.27 (s, 1H), 4.58
(t, 1H), 3.63 (t, 2H), 3.49 (m, 2H), 3.32 (m, 2H), 3.13 (m, 4H),
2.95 (m, 4H), 2.38 (m, 2H), 1.60 (m, 2H), 1.64 (m, 2H), 1.22 (s,
30H), 0.78 (t, 3H).
[0099] MQTS 1137--6.8 mg (19% yield) white solid is obtained. LC/MS
calcd [M+H] for C.sub.25H.sub.52N.sub.4O.sub.2: m/z 441; obsd 441
at 13.3 min. .sup.1H NMR (D.sub.2O, .delta.): 3.96 (m, 2H), 3.28
(m, 4H), 3.04 (m, 4H), 1.48 (m, 2H), 1.70 (m, 2H), 1.22 (s, 30H),
0.82 (t, 3H).
[0100] MQTS 1142--3.2 mg (9% yield) white solid is obtained. LC/MS
calcd [M+H] for C.sub.26H.sub.54N.sub.4O.sub.2: m/z 455; obsd 455
at 13.3 min. .sup.1H NMR (D.sub.2O, .delta.): 3.88 (m, 2H), 3.26
(m, 4H), 2.96 (m, 4H), 2.78 (m, 2H), 1.83 (m, 2H), 1.65 (m, 2H),
1.20 (s, 30H), 0.78 (t, 3H).
[0101] MQTS 1147--5.9 mg (16% yield) white solid is obtained. LC/MS
calcd [M+H] for C.sub.27H.sub.56N.sub.4O.sub.2: m/z 470; obsd 470
at 13.4 min. .sup.1H NMR (D.sub.2O, .delta.): 3.81 (s, 2H), 3.24
(t, 2H), 3.02 (m, 2H), 2.94 (t, 4H), 2.42 (t, 2H), 1.96 (m, 2H),
1.82 (t, 2H), 1.66 (m, 2H), 1.15 (s, 30H), 0.76 (t, 3H).
[0102] MQTS 1227--6.1 mg (16% yield) white solid is obtained. LC/MS
calcd [M+H] for C.sub.27H.sub.56N.sub.4O.sub.2: m/z 469; obsd 469
at 13.3 min. .sup.1H NMR (D.sub.2O, .delta.): 3.88 (s, 2H), 3.12
(m, 2H), 2.92 (m, 4H), 1.63 (m, 4H), 1.43 (m, 2H), 1.18 (s, 32H),
0.78 (t, 3H).
[0103] MQTS 1232--3.4 mg (9% yield) white solid is obtained. LC/MS
calcd [M+H] for C.sub.28H.sub.58N.sub.4O.sub.2: m/z 483; obsd 483
at 13.3 min. .sup.1H NMR (D.sub.2O, .delta.): 3.82 (s, 2H), 3.26
(t, 2H), 3.14 (t, 2H), 2.94 (m, 4H), 2.88 (t, 2H), 1.67 (m, 4H),
1.47 (m, 2H), 1.23 (s, 32H), 0.79 (t, 3H).
[0104] MQTS 1237--5.3 mg (13% yield) white solid is obtained. LC/MS
calcd [M+H] for C.sub.29H.sub.60N.sub.4O.sub.2: m/z 497; obsd 497
at 13.0 min. .sup.1H NMR (D.sub.2O, .delta.): 3.83 (s, 2H), 3.17
(m, 2H), 2.94 (m, 4H), 2.43 (m, 2H), 1.96 (m, 2H), 1.64 (m, 4H),
1.51 (m, 2H), 1.34 (m, 4H), 1.24 (s, 30H), 0.80 (t, 3H).
[0105] MQTS 2326--8.5 mg (25% yield) white solid is obtained. LC/MS
calcd [M+H] for C.sub.24H.sub.51N.sub.3O.sub.2: m/z 414; obsd 414
at 13.5 min. .sup.1H NMR (D.sub.2O, .delta.): 3.90 (s, 2H), 3.69
(m, 2H), 3.61 (m, 2H), 3.40 (m, 2H), 3.16 (m, 2H), 3.0 (m, 2H),
1.70 (m, 2H), 1.21 (s, 30H), 0.78 (t, 3H).
[0106] MQTS 2328--4.2 mg (12% yield) white solid is obtained. LC/MS
calcd [M+H] for C.sub.25H.sub.53N.sub.3O.sub.2: m/z 428; obsd 428
at 13.6 min. .sup.1H NMR (D.sub.2O, .delta.): 3.71 (m, 2H), 3.58
(m, 2H), 3.37 (m, 2H), 2.23 (m, 2H), 3.16 (m, 2H), 3.00 (m, 2H),
2.72 (m, 2H), 1.64 (m, 2H), 1.22 (s, 30H), 0.82 (t, 3H).
[0107] MQTS 2330--4.9 mg (16% yield) white solid is obtained. LC/MS
calcd [M+H] for C.sub.26H.sub.550N.sub.3O.sub.2: m/z 442; obsd 442
at 14.7 min. .sup.1H NMR (D.sub.2O, .delta.): 3.70 (m, 2H), 3.58
(m, 2H), 3.49 (m, 2H), 3.22 (m, 2H), 3.18 (m, 4H), 2.96 (m, 2H),
2.73 (m, 2H), 1.68 (m, 2H), 1.23 (m, 30H), 0.81 (t, 3H).
[0108] Rapid-throughput Fluorescence Displacement Assay for
quantifying binding affinities to LPS. The BODIPY-TR-cadaverine
(BC;
(5-(((4-(4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)
phenoxy)acetyl)amino)pentylamine, hydrochloride; obtained from
Molecular probes, Inc., Eugene, OR) displacement assay to quantify
the affinities of binding of compounds to LPS has been described in
detail recently..sup.81 This assay is performed in a
rapid-throughput format as follows: the first column (16 wells) of
a Corning Nonbinding Surface 384-well flat-bottom black
fluorescence microplate contain 15 test compounds plus polymyxin B,
all at 5 mM in DMSO, and are serially diluted two-fold in 50 mM
Tris buffer, pH 7.4, across the remaining 23 columns, achieving a
final dilution of 0.596 nM in a volume of 40 .mu.. Polymyxin B
(PMB), a peptide antibiotic known to bind and neutralize LPS.sup.95
serve as the positive control and reference compound for every
plate, enabling the quantitative assessment of repeatability and
reproducibility (CV and Z' factors) for the assay. Automated liquid
handling is performed on a Precision 2000 automated microplate
pipetting system, programmed using the Precision Power software,
Bio-Tek Instruments Inc., VT, USA.
[0109] Nitric Oxide Assay. Nitric oxide production is measured as
total nitrite in murine macrophage J774A.1 cells using the Griess
assay96 as described previously..sup.94 J774A.1 cells are plated at
.about.10.sup.5/ml in a volume of 40 .mu.l/well, in 384-well,
flat-bottomed, cell culture treated microtiter plates and
subsequently stimulated with 10 ng/ml lipopolysaccharide (LPS).
Concurrent to LPS stimulation, serially diluted concentrations of
test compounds are added to the cell medium and left to incubate
overnight for 16 h. Polymyxin B is used as reference compound in
each plate. Positive-(LPS stimulation only) and negative-controls
(J774A. 1 medium only) are included in each experiment. Nitrite
concentrations are measured by adding 40 .mu.l of supernatant to
equal volumes of Griess reagents (50 .mu.l/well; 0.1% NED solution
in ddH.sub.2O and 1% sulfanilamide, 5% phosphoric acid solution in
ddH.sub.2O) and incubating for 15 minutes at room temperature in
the dark. Absorbance at 535 run is measured using a Molecular
Devices Spectramax M2 multifunction plate reader (Sunnyvale,
Calif.). Nitrite concentrations are interpolated from standard
curves obtained from serially diluted sodium nitrite standards.
TABLE-US-00001 TABLE 1 Binding affinity (BC displacement;
ED.sub.50) and biological activity (NO inhibition in murine J774
cells; IC.sub.50) of leads following re-synthesis. ##STR9## MQTS X
m = R = n = ED.sub.50 value (.mu.M) IC.sub.50 value (.mu.M) 1002
--CH.sub.2OCH.sub.2-- 1 --CH.sub.2Ph 0 12.4 17.6 1007
--CH.sub.2OCH.sub.2-- 1 --CH.sub.2Ph 1 2.54 2.79 1012
--CH.sub.2OCH.sub.2-- 1 --CH.sub.2Ph 2 7.68 3.78 1032
--CH.sub.2OCH.sub.2-- 1 --CH.sub.2-imid 0 13.1 1.64 1037
--CH.sub.2OCH.sub.2-- 1 --CH.sub.2-imid 1 3.17 1.88 1042
--CH.sub.2OCH.sub.2-- 1 --CH.sub.2-imid 2 5.38 1.86 1047
--CH.sub.2OCH.sub.2-- 1 --H 0 14.0 11.5 1052 --CH.sub.2OCH.sub.2--
1 --H 1 14.2 1.84 1057 --CH.sub.2OCH.sub.2-- 1 --H 2 10.8 3.33 1092
--CH.sub.2-- 1 --CH.sub.2Ph 0 8.80 6.74 1097 --CH.sub.2-- 1
--CH.sub.2Ph 1 4.13 8.70 1102 --CH.sub.2-- 1 --CH.sub.2Ph 2 5.75
3.42 1122 --CH.sub.2-- 1 --CH.sub.2-imid 0 4.87 6.90 1127
--CH.sub.2-- 1 --CH.sub.2-imid 1 6.86 8.06 1132 --CH.sub.2-- 1
--CH.sub.2-imid 2 3.01 1.83 1137 --CH.sub.2-- 1 --H 0 6.61 7.94
1142 --CH.sub.2-- 1 --H 1 2420 5.26 1147 --CH.sub.2-- 1 --H 2 6.14
6.57 1187 --CH.sub.2CH.sub.2CH.sub.2-- 1 --CH.sub.2Ph 1 3850 4.88
1192 --CH.sub.2CH.sub.2CH.sub.2-- 1 --CH.sub.2Ph 2 7.51 4.80 1212
--CH.sub.2CH.sub.2CH.sub.2-- 1 --CH.sub.2-imid 0 12.1 2.04 1222
--CH.sub.2CH.sub.2CH.sub.2-- 1 --CH.sub.2-imid 2 18.7 0.90 1227
--CH.sub.2CH.sub.2CH.sub.2-- 1 --H 0 28.2 3.27 1232
--CH.sub.2CH.sub.2CH.sub.2-- 1 --H 1 11.2 4.61 1237
--CH.sub.2CH.sub.2CH.sub.2-- 1 --H 2 9.77 3.22 2322 --CH.sub.2-- 0
-- 0 3.80 5.56 2323 --CH.sub.2-- 0 -- 1 9.92 7.49 2324 --CH.sub.2--
0 -- 2 6.21 4.87 2325 --CH.sub.2CH.sub.2CH.sub.2-- 0 -- 0 8.74 3.96
2326 --CH.sub.2OCH.sub.2-- 0 -- 0 12.15 9.73 2327
--CH.sub.2CH.sub.2CH.sub.2-- 0 -- 1 4.03 1.08 2328
--CH.sub.2OCH.sub.2-- 0 -- 1 9.16 6.54 2329
--CH.sub.2CH.sub.2CH.sub.2-- 0 -- 2 7.61 2.07 2330
--CH.sub.2OCH.sub.2-- 0 -- 2 5.73 5.00
[0110] TABLE-US-00002 CHART 1 Solid-phase Lantern .TM.-based
scaffold and combinatorial elements. ##STR10## PORTION 1 PORTION 2
PORTION 3.x (N = 6) (N = 6) (N = 3) ##STR11## ##STR12## ##STR13##
PORTION 3.y (N = 5) ##STR14## ##STR15## ##STR16## ##STR17##
##STR18## ##STR19##
[0111] ##STR20## ##STR21##
[0112] The pharmaceutically acceptable carriers described herein,
for example, vehicles, adjuvants, excipients, or diluents, are
well-known to those who are skilled in the art. Typically, the
pharmaceutically acceptable carrier is chemically inert to the
active compounds and has no detrimental side effects or toxicity
under the conditions of use. The pharmaceutically acceptable
carriers can include polymers and polymer matrices.
[0113] The compounds of this disclosure can be administered by any
conventional method available for use in conjunction with
pharmaceuticals, either as individual therapeutic agents or in a
combination of therapeutic agents.
[0114] The dosage administered will, of course, vary depending upon
known factors, such as the pharmacodynamic characteristics of the
particular agent and its mode and route of administration; the age,
health and weight of the recipient; the nature and extent of the
symptoms; the kind of concurrent treatment; the frequency of
treatment; and the effect desired. A daily dosage of active
ingredient can be expected to be about 0.001 to 1000 milligrams
(mg) per kilogram (kg) of body weight, with the preferred dose
being 0.1 to about 30 mg/kg.
[0115] Dosage forms (compositions suitable for administration)
contain from about 1 mg to about 500 mg of active ingredient per
unit. In these pharmaceutical compositions, the active ingredient
will ordinarily be present in an amount of about 0.5-95% weight
based on the total weight of the composition.
[0116] The active ingredient can be administered orally in solid
dosage forms, such as capsules, tablets, and powders, or in liquid
dosage forms, such as elixirs, syrups and suspensions. It can also
be administered parenterally, in sterile liquid dosage forms. The
active ingredient can also be administered intranasally (nose
drops) or by inhalation of a drug powder mist. Other dosage forms
are potentially possible such as administration transdermally, via
patch mechanism or ointment. The active ingredient can be
administered employing a sustained or delayed release delivery
system or an immediate release delivery system.
[0117] Formulations suitable for oral administration can consist of
(a) liquid solutions, such as an effective amount of the compound
dissolved in diluents, such as water, saline, or orange juice; (b)
capsules, sachets, tablets, lozenges, and troches, each containing
a predetermined amount of the active ingredient, as solids or
granules; (c) powders; (d) suspensions in an appropriate liquid;
and (e) suitable emulsions. Liquid formulations may include
diluents, such as water and alcohols, for example, ethanol, benzyl
alcohol, propylene glycol, glycerin, and the polyethylene alcohols,
either with or without the addition of a pharmaceutically
acceptable surfactant, suspending agent, or emulsifying agent.
Capsule forms can be of the ordinary hard- or soft-shelled gelatin
type containing, for example, surfactants, lubricants, and inert
fillers, such as lactose, sucrose, calcium phosphate, and corn
starch. Tablet forms can include one or more of the following:
lactose, sucrose, mannitol, corn starch, potato starch, alginic
acid, microcrystalline cellulose, acacia, gelatin, guar gum,
colloidal silicon dioxide, croscarmellose sodium, talc, magnesium
stearate, calcium stearate, zinc stearate, stearic acid, and other
excipients, colorants, diluents, buffering agents, disintegrating
agents, moistening agents, preservatives, flavoring agents, and
pharmacologically compatible carriers. Lozenge forms can comprise
the active ingredient in a flavor, usually sucrose and acacia or
tragacanth, as well as pastilles comprising the active ingredient
in an inert base, such as gelatin and glycerin, or sucrose and
acadia, emulsions, and gels containing, in addition to the active
ingredient, such carriers as are known in the art.
[0118] The compounds of the present disclosure, alone or in
combination with other suitable components, can be made into
aerosol formulations to be administered via inhalation. These
aerosol formulations can be placed into pressurized acceptable
propellants, such as dichlorodifluoromethane, propane, and
nitrogen. They also may be formulated as pharmaceuticals for
non-pressured preparations, such as in a nebulizer or an
atomizer.
[0119] Formulations suitable for parenteral administration include
aqueous and non-aqueous, isotonic sterile injection solutions,
which can contain anti-oxidants, buffers, bacteriostats, and
solutes that render the formulation isotonic with the blood of the
intended recipient, and aqueous and non-aqueous sterile suspensions
that can include suspending agents, solubilizers, thickening
agents, stabilizers, and preservatives. The compound can be
administered in a physiologically acceptable diluent in a
pharmaceutical carrier, such as a sterile liquid or mixture of
liquids, including water, saline, aqueous dextrose and related
sugar solutions, an alcohol, such as ethanol, isopropanol, or
hexadecyl alcohol, glycols, such as propylene glycol or
polyethylene glycol such as poly(ethyleneglycol) 400, glycerol
ketals, such as 2,2-dimethyl-1,3-dioxolane-4-methanol, ethers, an
oil, a fatty acid, a fatty acid ester or glyceride, or an
acetylated fatty acid glyceride with or without the addition of a
pharmaceutically acceptable surfactant, such as a soap or a
detergent, suspending agent, such as pectin, carbomers,
methylcellulose, hydroxypropylmethylcellulose, or
carboxymethylcellulose, or emulsifying agents and other
pharmaceutical adjuvants.
[0120] Oils, which can be used in parenteral formulations include
petroleum, animal, vegetable, or synthetic oils. Specific examples
of oils include peanut, soybean, sesame, cottonseed, corn, olive,
petrolatum, and mineral. Suitable fatty acids for use in parenteral
formulations include oleic acid, stearic acid, and isostearic acid.
Ethyl oleate and isopropyl myristate are examples of suitable fatty
acid esters. Suitable soaps for use in parenteral formulations
include fatty alkali metal, ammonium, and triethanolamine salts,
and suitable detergents include (a) cationic detergents such as,
for example, dimethyldialkylammonium halides, and alkylpyridinium
halides, (b) anionic detergents such as, for example, alkyl, aryl,
and olefin sulfonates, alkyl, olefin, ether, and monoglyceride
sulfates, and sulfosuccinates, (c) nonionic detergents such as, for
example, fatty amine oxides, fatty acid alkanolamides, and
polyoxyethylene polypropylene copolymers, (d) amphoteric detergents
such as, for example, alkyl .beta.-aminopropionates, and
2-alkylimidazoline quaternary ammonium salts, and (e) mixtures
thereof.
[0121] The parenteral formulations typically contain from about
0.5% to about 25% by weight of the active ingredient in solution.
Suitable preservatives and buffers can be used in such
formulations. In order to minimize or eliminate irritation at the
site of injection, such compositions may contain one or more
nonionic surfactants having a hydrophile-lipophile balance (HLB) of
from about 12 to about 17. The quantity of surfactant in such
formulations ranges from about 5% to about 15% by weight. Suitable
surfactants include polyethylene sorbitan fatty acid esters, such
as sorbitan monooleate and the high molecular weight adducts of
ethylene oxide with a hydrophobic base, formed by the condensation
of propylene oxide with propylene glycol.
[0122] Pharmaceutically acceptable excipients are also well-known
to those who are skilled in the art. The choice of excipient will
be determined in part by the particular compound, as well as by the
particular method used to administer the composition. Accordingly,
there is a wide variety of suitable formulations of the
pharmaceutical composition of the present invention. The following
methods and excipients are merely exemplary and are in no way
limiting. The pharmaceutically acceptable excipients preferably do
not interfere with the action of the active ingredients and do not
cause adverse side-effects. Suitable carriers and excipients
include solvents such as water, alcohol, and propylene glycol,
solid absorbants and diluents, surface active agents, suspending
agent, tableting binders, lubricants, flavors, and coloring
agents.
[0123] The formulations can be presented in unit-dose or multi-dose
sealed containers, such as ampules and vials, and can be stored in
a freeze-dried (lyophilized) condition requiring only the addition
of the sterile liquid excipient, for example, water, for
injections, immediately prior to use. Extemporaneous injection
solutions and suspensions can be prepared from sterile powders,
granules, and tablets. The requirements for effective
pharmaceutical carriers for injectable compositions are well known
to those of ordinary skill in the art. See Pharmaceutics and
Pharmacy Practice, J. B. Lippincott Co., Philadelphia, Pa., Banker
and Chalmers, Eds., 238-250 (1982) and ASHP Handbook on Injectable
Drugs, Toissel, 4th ed., 622-630 (1986).
[0124] Formulations suitable for topical administration include
lozenges comprising the active ingredient in a flavor, usually
sucrose and acacia or tragacanth; pastilles comprising the active
ingredient in an inert base, such as gelatin and glycerin, or
sucrose and acacia; and mouth washes comprising the active
ingredient in a suitable liquid carrier; as well as creams,
emulsions, and gels containing, in addition to the active
ingredient, such carriers as are known in the art.
[0125] Additionally, formulations suitable for rectal
administration may be presented as suppositories by mixing with a
variety of bases such as emulsifying bases or water-soluble bases.
Formulations suitable for vaginal administration may be presented
as pessaries, tampons, creams, gels, pastes, foams, or spray
formulas containing, in addition to the active ingredient, such
carriers as are known in the art to be appropriate.
[0126] Suitable pharmaceutical carriers are described in
Remington's Pharmaceutical Sciences, Mack Publishing Company, a
standard reference text in this field.
[0127] The dose administered to an animal, particularly a human, in
the context of the present invention should be sufficient to affect
a therapeutic response in the animal over a reasonable time frame.
One skilled in the art will recognize that dosage will depend upon
a variety of factors including a condition of the animal, the body
weight of the animal, as well as the condition being treated.
[0128] A suitable dose is that which will result in a concentration
of the active agent in a patient which is known to affect the
desired response.
[0129] The size of the dose also will be determined by the route,
timing and frequency of administration as well as the existence,
nature, and extent of any adverse side effects that might accompany
the administration of the compound and the desired physiological
effect.
[0130] Usefull pharmaceutical dosage forms for administration of
the compounds according to the present invention can be illustrated
as follows:
[0131] Hard Shell Capsules
[0132] A large number of unit capsules are prepared by filling
standard two-piece hard gelatine capsules each with 100 mg of
powdered active ingredient, 150 mg of lactose, 50 mg of cellulose
and 6 mg of magnesium stearate.
[0133] Soft Gelatin Capsules
[0134] A mixture of active ingredient in a digestible oil such as
soybean oil, cottonseed oil or olive oil is prepared and injected
by means of a positive displacement pump into molten gelatin to
form soft gelatin capsules containing 100 mg of the active
ingredient. The capsules are washed and dried. The active
ingredient can be dissolved in a mixture of polyethylene glycol,
glycerin and sorbitol to prepare a water miscible medicine mix.
[0135] The foregoing description illustrates and describes the
present disclosure. Additionally, the disclosure shows and
describes only the preferred embodiments of the disclosure, but, as
mentioned above, it is to be understood that it is capable of
changes or modifications within the scope of the concept as
expressed herein, commensurate with the above teachings and/or the
skill or knowledge of the relevant art. The embodiments described
hereinabove are further intended to explain best modes known of
practicing the invention and to enable others skilled in the art to
utilize the disclosure in such, or other, embodiments and with the
various modifications required by the particular applications or
uses disclosed herein. Accordingly, the description is not intended
to limit the invention to the form disclosed herein. Also, it is
intended that the appended claims be construed to include
alternative embodiments.
[0136] The term "comprising" (and its grammatical variations) as
used herein is used in the inclusive sense of "having" or
"including" and not in the exclusive sense of "consisting only
of".
[0137] All publications, patents and patent applications cited in
this specification are herein incorporated by reference, and for
any and all purposes, as if each individual publication, patent or
patent application were specifically and individually indicates to
be incorporated by reference. In the case of inconsistencies, the
present disclosure will prevail.
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