U.S. patent application number 13/011626 was filed with the patent office on 2011-05-19 for hydrazide-containing cftr inhibitor compounds and uses thereof.
This patent application is currently assigned to THE REGENTS OF THE UNIVERSITY OF CALIFORNIA. Invention is credited to Chatchai Muanprasat, Nitin Dattatraya Sonawane, Alan Verkman.
Application Number | 20110119775 13/011626 |
Document ID | / |
Family ID | 35064333 |
Filed Date | 2011-05-19 |
United States Patent
Application |
20110119775 |
Kind Code |
A1 |
Verkman; Alan ; et
al. |
May 19, 2011 |
HYDRAZIDE-CONTAINING CFTR INHIBITOR COMPOUNDS AND USES THEREOF
Abstract
The invention provides compositions, pharmaceutical preparations
and methods for inhibition of cystic fibrosis transmembrane
conductance regulator protein (CFTR) that are useful for the study
and treatment of CFTR-mediated diseases and conditions. The
compositions and pharmaceutical preparations of the invention may
comprise one or more hydrazide-containing compounds, and may
additionally comprise one or more pharmaceutically acceptable
carriers, excipients and/or adjuvants. The methods of the invention
comprise, in certain embodiments, administering to a patient
suffering from a CFTR-mediated disease or condition, an efficacious
amount of a hydrazide-containing compound. In other embodiments the
invention provides methods of inhibiting CFTR that comprise
contacting cells in a subject with an effective amount of a
hydrazide-containing compound. In addition, the invention features
a non-human animal model of CFTR-mediated disease which model is
produced by administration of a hydrazide-containing compound to a
non-human animal in an amount sufficient to inhibit CFTR.
Inventors: |
Verkman; Alan; (San
Francisco, CA) ; Sonawane; Nitin Dattatraya; (San
Francisco, CA) ; Muanprasat; Chatchai; (Nakhonpathom,
TH) |
Assignee: |
THE REGENTS OF THE UNIVERSITY OF
CALIFORNIA
Oakland
CA
|
Family ID: |
35064333 |
Appl. No.: |
13/011626 |
Filed: |
January 21, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12175982 |
Jul 18, 2008 |
7888332 |
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13011626 |
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11093749 |
Mar 29, 2005 |
7414037 |
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12175982 |
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60557930 |
Mar 30, 2004 |
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Current U.S.
Class: |
800/9 ; 435/325;
514/615; 564/150 |
Current CPC
Class: |
A61K 31/195 20130101;
Y02A 50/30 20180101; C07D 215/38 20130101; A01K 67/027 20130101;
A61K 31/7012 20130101; A61K 31/165 20130101; A61K 31/47 20130101;
Y02A 50/471 20180101; A61P 13/00 20180101; A61P 13/12 20180101;
A01K 2227/105 20130101; A01K 2267/0306 20130101 |
Class at
Publication: |
800/9 ; 564/150;
514/615; 435/325 |
International
Class: |
A01K 67/00 20060101
A01K067/00; C07C 243/22 20060101 C07C243/22; A61K 31/165 20060101
A61K031/165; C12N 5/071 20100101 C12N005/071; A61P 13/00 20060101
A61P013/00; A61P 13/12 20060101 A61P013/12 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with government support under grant
numbers HL73854, EB00415, EY13574, DK35124, DK43840, and UC1
AI062530-01 awarded by the National Institutes of Health. The
government has certain rights in the invention.
Claims
1. A compound having the structure of formula (Ic): ##STR00032## or
a pharmaceutically acceptable salt or stereoisomer thereof, wherein
Y is hydrogen or a substituted or unsubstituted, saturated linear
or branched alkyl; R.sub.1 is unsubstituted phenyl, substituted
phenyl wherein phenyl is substituted with one or more of hydroxy,
alkyl and halogen, substituted or unsubstituted anthracenyl, or
substituted or unsubstituted naphthalenyl; R.sub.2 is unsubstituted
phenyl, substituted phenyl, wherein phenyl is substituted with
bromo or carboxy, di(hydroxy)phenyl,
mono(halo)-mono(hydroxy)phenyl, mono(halo)-di(hydroxy)phenyl,
mono(halo)-tri(hydroxy)phenyl, di(halo)-mono(hydroxy)phenyl,
di(halo)-di(hydroxy)phenyl, di(halo)-tri(hydroxy)phenyl,
mono(halo)-mono(hydroxy)-mono(alkoxy)phenyl,
mono(halo)-di(hydroxy)-mono(alkoxy)phenyl,
mono(halo)-mono(hydroxy)-di(alkoxy)phenyl,
mono(halo)-di(hydroxy)-di(alkoxy)phenyl,
di(halo)-mono(hydroxy)-mono(alkoxy)phenyl,
di(halo)-di(hydroxy)-mono(alkoxy)phenyl,
di(halo)-mono(hydroxy)-di(alkoxy)phenyl; and R.sub.3 is hydrogen or
substituted or unsubstituted alkyl.
2. The method of claim 1, wherein R.sub.1 is substituted or
unsubstituted 1-naphthalenyl, substituted or unsubstituted
2-naphthalenyl, 2-chlorophenyl, 4-chlorophenyl, 4-methylphenyl,
2-methylphenyl, or 2-anthracenyl.
3. The compound of claim 1, wherein R.sub.1 is mono-(halo)phenyl;
mono-(alkyl)phenyl; mono-(halo)napthalenyl; di-(halo)napthalenyl;
mono-(hydroxy)napthalenyl; di-(hydroxy)napthalenyl;
mono-(alkoxy)napthalenyl; di-(alkoxy)naphthalenyl;
tri-(alkoxy)naphthalenyl; mono-(alkyl)naphthalenyl;
di-(alkyl)napthalenyl; mono-(hydroxyl)-mono(sulfo)napthalenyl;
mono-(hydroxy)-di(sulfo)napthalenyl;
mono-(alkyl)-mono-(alkoxy)-napthalenyl; or
mono-(alkyl)-di-(alkoxy)-napthalenyl.
4. The compound of claim 1, wherein R.sub.1 is a 2-naphthalenyl
group.
5. The compound of claim 1, wherein R.sub.2 is
3,5-dibromo-2,4-dihydroxyphenyl;
3,5-dibromo-2,4,6-trihydroxyphenyl; 3,5-dibromo-4-hydroxyphenyl;
3,5dibromo-2-dihydroxy-4-methoxyphenyl; 3-bromo-4-hydroxyphenyl;
2,4-dihydroxyphenyl; or 4-bromophenyl.
6. The compound of claim 1, wherein R.sub.2 is chosen from a
3,5-dibromo-2,4-dihydroxyphenyl or a 3,5-dibromo-4-hydroxyphenyl
group.
7. The compound of claim 1, wherein R.sub.3 is chosen from
hydrogen, methyl group, or ethyl.
8. The compound of claim 1, wherein R.sub.3 is hydrogen or
methyl.
9. The compound of claim 1, wherein Y is a substituted alkyl group
comprising a sulfo group, a carboxy group, a substituted or
unsubstituted carboxamide group, a polyoxyalkylether group, a
disaccharide, a polyamine, a substituted or unsubstituted phenyl
group, a polyethyleneimine (PEI), or a dendrimer from 0-10
generation.
10. The compound of claim 1, wherein Y is C.sub.1-C.sub.8
alkyl.
11. The compound of claim 1, wherein Y is hydrogen; R.sub.1 is
mono-(halo)phenyl or naphthalenyl; R.sub.2 is
di-(halo)-mono-(hydroxy)phenyl or di-(halo)-di-(hydroxy)phenyl; and
R.sub.3 is hydrogen or methyl.
12. The compound of claim 1, wherein the compound of formula (Ic)
has the following structure selected from: ##STR00033##
13. A pharmaceutical composition comprising the compound of claim 1
and a pharmaceutically acceptable excipient.
14. A method for inhibiting cystic fibrosis transmembrane
conductance regulator (CFTR) ion transport in a cell, the method
comprising contacting the cell with the compound of claim 1.
15. The method of claim 14, wherein the cell is in a subject who
has aberrantly increased intestinal secretion.
16. The method of claim 15, wherein the aberrantly increased
intestinal secretion is secretory diarrhea.
17. The method of claim 14, wherein the cell is in a subject who
has polycystic kidney disease.
18. A method for inhibiting the activity of cystic fibrosis
transmembrane conductance regulator (CFTR) protein in a cell in an
in vitro assay, comprising contacting the cell with the compound of
claim 1 in an amount effective to inhibit CFTR activity.
19. A method for producing a cystic fibrosis (CF) phenotype in a
non-human animal, wherein the method comprises administering to the
non-human animal the compound of claim 1 in an amount effective to
inhibit CFTR ion transport.
20. A non-human animal having a cystic fibrosis transmembrane
conductance regulator (CFTR) deficiency produced by the method of
claim 19, wherein the deficiency is produced by administration of
the hydrazide-containing compound to the animal.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application patent
Ser. No. 12/175,982, now allowed, which has a filing date of Jul.
18, 2008, which application is a divisional of U.S. patent
application Ser. No. 11/093,749, issued on Aug. 19, 2008 as U.S.
Pat. No. 7,414,037, which has a filing date of Mar. 29, 2005, and
which claims the benefit of U.S. Provisional Application No.
60/557,930, filed Mar. 30, 2004, all of which applications are
incorporated herein by reference in their entireties.
BACKGROUND OF THE INVENTION
[0003] The cystic fibrosis transmembrane conductance regulator
protein (CFTR) is a cAMP-activated chloride (Cl.sup.-) channel
expressed in epithelial cells in mammalian airways, intestine,
pancreas and testis. CFTR is the chloride-channel responsible for
cAMP-mediated Cl.sup.- secretion. Hormones, such as a
.beta.-adrenergic agonist, or a toxin, such as cholera toxin, leads
to an increase in cAMP, activation of cAMP-dependent protein
kinase, and phosphorylation of the CFTR Cl.sup.- channel, which
causes the channel to open. An increase in cell Ca.sup.2- can also
activate different apical membrane channels. Phosphorylation by
protein kinase C can either open or shut Cl.sup.- channels in the
apical membrane. CFTR is predominantly located in epithelia where
it provides a pathway for the movement of Cl.sup.- ions across the
apical membrane and a key point at which to regulate the rate of
transepithelial salt and water transport. CFTR chloride channel
function is associated with a wide spectrum of disease, including
cystic fibrosis (CF) and with some forms of male infertility,
polycystic kidney disease and secretory diarrhea.
[0004] The hereditary lethal disease cystic fibrosis (CF) is caused
by mutations in CFTR. Observations in human cystic fibrosis (CF)
patients and CF mouse models indicate the functional importance of
CFTR in intestinal and pancreatic fluid transport, as well as in
male fertility (Grubb et al., 1999, Physiol. Rev. 79:S193-S214;
Wong, P. Y., 1997, Mol. Hum. Reprod. 4:107-110). However, the
mechanisms remain unclear by which defective CFTR produces airway
disease, which is the principal cause of morbidity and mortality in
CF (Pilewski et al., 1999, Physiol. Rev. 79:S215-S255). Major
difficulties in understanding airway disease in CF include the
inadequacy of CF mouse models, which manifest little or no airway
disease, the lack of large animal models of CF, and the limited
availability of human CF airways that have not been damaged by
chronic infection and inflammation. High-affinity, CFTR-selective
inhibitors have not been available to study airway disease
mechanisms in CF or to create the CF phenotype in large animal
models.
[0005] High-affinity CFTR inhibitors also have clinical
applications in the therapy of secretory diarrheas and cystic
kidney disease, and in inhibiting male fertility. Several CFTR
inhibitors have been discovered, although most of which have a weak
potency and lack CFTR specificity. The oral hypoglycemic agent
glibenclamide inhibits CFTR Cl.sup.- conductance from the
intracellular side by an open channel blocking mechanism (Sheppard
& Robinson, 1997 J. Physiol., 503:333-346; Zhou et al., 2002,
J. Gen. Physiol., 120:647-662) at high micromolar concentrations
where it affects other Cl.sup.- and cation channels (Edwards &
Weston, 1993; Rabe et al., 1995, Br. J. Pharmacol., 110:1280-1281;
Schultz et al., 1999, Physiol. Rev., 79:S109-S144). Other
non-selective anion transport inhibitors including
diphenylamine-2-carboxylate (DPC),
5-nitro-2(3-phenylpropyl-amino)benzoate (NPPB), and flufenamic acid
also inhibit CFTR by occluding the pore at an intracellular site
(Dawson et al., 1999, Physiol. Rev., 79:S47-S75; McCarty, 2000, J.
Exp. Biol., 203:1947-1962).
[0006] There is accordingly a need for CFTR inhibitors,
particularly those that are water-soluble. The present invention
addresses these needs, as well as others, and overcomes
deficiencies found in the background art.
Literature
[0007] Ma et al., 2002, J. Clin. Invest., 110:1651-1658 describes a
thiazolidinone class of CFTR inhibitor.
SUMMARY OF THE INVENTION
[0008] The invention provides compositions, pharmaceutical
preparations and methods for inhibition of cystic fibrosis
transmembrane conductance regulator protein (CFTR) that are useful
for the study and treatment of CFTR-mediated diseases and
conditions. The compositions and pharmaceutical preparations of the
invention may comprise one or more hydrazide-containing compounds,
and may additionally comprise one or more pharmaceutically
acceptable carriers, excipients and/or adjuvants. The methods of
the invention comprise, in certain embodiments, administering to a
patient suffering from a CFTR-mediated disease or condition, an
efficacious amount of a hydrazide-containing compound. In other
embodiments the invention provides methods of inhibiting CFTR that
comprise contacting cells in a subject with an effective amount of
a hydrazide-containing compound. In addition, the invention
features a non-human animal model of CFTR-mediated disease which
model is produced by administration of a hydrazide-containing
compound to a non-human animal in an amount sufficient to inhibit
CFTR.
[0009] These and other objects, advantages, and features of the
invention will become apparent to those persons skilled in the art
upon reading the details of the hydrazide-containing compounds as
more fully described below.
BRIEF DESCRIPTIONS OF THE DRAWINGS
[0010] The invention will be more fully understood by reference to
the following drawings, which are for illustrative purposes
only.
[0011] FIG. 1A is a schematic representation of a screening
technique used for detection of CFTR inhibitors. CFTR was maximally
stimulated by multiple agonists in stably transfected epithelial
cells co-expressing human CFTR and a yellow fluorescent protein
(YFP) having Cl.sup.-/I.sup.- sensitive fluorescence. After
addition of a test compound, I.sup.- influx was induced by adding
an I.sup.- containing solution.
[0012] FIG. 1B shows chemical structures of CFTR inhibitors
identified by the screening technique of FIG. 1A.
[0013] FIG. 1C is a graph representing relative fluorescence versus
time using the screening technique of FIG. 1A for the CFTR
inhibitor
N-2-napthalenyl-[(3,5-dibromo-2,4-dihydroxyphenyl)methylene]glycine
hydrazide (referred to herein as GlyH-101) at several
concentrations.
[0014] FIG. 1D is a graph representing GlyH-101 inhibition of
short-circuit current in permeabilized FRT cells expressing human
CFTR. CFTR was stimulated by 100 .mu.M CPT-cAMP.
[0015] FIG. 2A is a graph representing the time course of
inhibition showing CFTR-mediated I.sup.- transport rates at
different times after addition of 10 .mu.M GlyH-101.
[0016] FIG. 2B is a graph representing the time course of
inhibition reversal showing I.sup.- transport rates at different
times after washout of GlyH-101.
[0017] FIG. 2C is a graph representing iodide influx by GlyH-101
(50 .mu.M) after CFTR stimulation by indicated agonists (50 .mu.M).
Filled bars show agonist, and open bars show agonist with
GlyH-101.
[0018] FIG. 3A provides chemical structures of a class of GlyH-101
analogs with sites of modification indicated with brackets.
[0019] FIG. 3B depicts the reaction scheme for the synthesis of
GlyH-101,
N-(6-quinolinyl)-[(3,5-dibromo-2,4-dihydroxyphenyl)methylene]glycine
hydrazide (referred to herein as GlyH-126),
3,5-dibromo-2,4-di-hydroxy-[2-(2-napthalenamine)aceto]benzoic acid
hydrazide (referred to herein as GlyH-201), and
N-2-napthalenyl-[(3,5-dibromo-2,4-dihydroxyphenyl)methyl]glycine
hydrazide (referred to herein as GlyH-301). Reagents and
conditions: (a) ICH.sub.2COOEt, NaOAc, 95.degree. C.; (b)
N.sub.2H.sub.4.H.sub.2O EtOH/reflux; (c)
3,5-di-Br-2,4-di-OH-Ph-CHO, EtOH/reflux; (d)
3,5-di-Br-2,4-di-OH-Ph-COCl, pyridine, 22.degree. C.; (e)
N.sub.2H.sub.4.H.sub.2O, Pd/C (10%), DMF/reflux; (f) glyoxalic
acid, 10.degree. C.; (g) Na.sub.2BH.sub.3CN/CH.sub.3CN, 48 h; dry
HCl, EtOH.
[0020] FIG. 3C is a graph representing
N-2-napthalenyl-[(3,5-dibromo-2,4-dihydroxyphenyl)methylene]oxamic
acid hydrazide (referred to herein as OxaH-110) inhibition of
short-circuit current premeabilized FRT cells expressing human CFTR
(right panel) and the structure of OxaH-110 (left panel). CFTR was
stimulated by 100 .mu.M CPT-cAMP.
[0021] FIG. 4A is a graph illustrating of GlyH-101 inhibition
measured in whole-cell patch clamp experiments on FRT cells
expressing human CFTR. Whole-cell membrane currents were evoked by
voltages from -100 to +100 mV in 20 mV steps after maximal CFTR
stimulation by 5 .mu.M forskolin. The graph on the left represents
measurements before GlyH-101 was added and the graph on the right
represents measurements after GlyH-101 was added.
[0022] FIG. 4B is a graph representing current-voltage
relationships in the absence of inhibitors (control, open circles),
after addition of 10 .mu.M (filled squares) and 30 .mu.M GlyH-101
(filled circles), after washout of 10 .mu.M GlyH-101 (recovery,
triangles) and after addition of 5 .mu.M CFTR.sub.inh-172 (filled
circles).
[0023] FIG. 4C is a graph illustrating of dose-response
relationships determined for GlyH-101 at the indicated membrane
potentials.
[0024] FIG. 4D is a graph illustrating of representative
cell-attached patch-clamp recordings showing CFTR single channel
activity at GlyH-101 concentrations of 0, 0.4 and 5 .mu.M. Dashed
lines show zero current level (channels closed) with downward
deflections indicating channel openings (Cl.sup.- ions moving from
pipette into the cell). Pipette potential was -60 mV.
[0025] FIG. 5A is a graph illustrating the pH-dependent absorbance
changes (right panel) of the chemical compounds (10 .mu.M)
(corresponding chemical structures, left panel) in NaCl (100 mM)
containing MES, HEPES, boric acid, and citric acid (each 10 mM)
titrated to different pH using HCl/NaOH. Absorbance changes
measured at analytical wavelengths of 346, 348, 346, and 236 nm
(top to bottom).
[0026] FIG. 5B is a representation of deduced ionic equilibria of
GlyH-101 showing pKa values.
[0027] FIG. 6A is a graph illustrating GlyH-101 inhibition in a
nasal potential difference (PD) recording showing responses to
amiloride and low solutions (left panel) or averaged PD values
(right panel, mean.+-.SE, n=5). Where indicated the low Cl.sup.-
solutions contained forskolin without or with GlyH-101.
[0028] FIG. 6B is a graph representing a paired analysis of
experiments as in FIG. 6A showing PD changes (.DELTA.PD) for the
forskolin effect, forskolin and CFTR.sub.inh-172, and forskolin and
GlyH-101.
[0029] FIG. 6C is a graph illustrating of a change in PD
(mean.+-.SE) in a series of low C.sup.- induced hyperpolarization
experiments (left panel) or forskolin induced hyperpolarization
(right panel)in which solutions contained either
4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) or GlyH-101
(*P<0.005 for reduced .DELTA.PD compared to control).
[0030] FIG. 7A is a graph illustrating GlyH-101 inhibition of
short-circuit current after CFTR stimulation in T84 cells (top
panel), human airway cells (middle panel), and isolated mouse ileum
(bottom panel). Following constant baseline current, amiloride (10
.mu.M, apical solution) and CPT-cAMP (0.1 mM, both solutions) were
added, followed by indicated concentrations of GlyH-101 (both
solutions).
[0031] FIG. 7B is a graph representing GlyH-101 inhibition of fluid
secretion in a closed intestinal loop model of cholera
toxin-induced fluid secretion. Intestinal lumenal fluid, shown as
loop weight/length (gm/cm, SE, 6 mice), measured at 4 hours after
injection of saline (control), cholera toxin (1 .mu.g) or cholera
toxin+GlyH-101 (0.25 .mu.g).
[0032] FIG. 8 provides chemical structures of a class of
non-absorbable molonic acid dihydrazide (denoted as MalH-x) analogs
of glycine hydrazide compounds of the invention.
[0033] FIG. 9 depicts the reaction scheme for the synthesis of the
polar non-absorbable CFTR inhibitors
2-naphthalenylamino-bis[(3,5-dibromo-2,4-dihydroxyphenyl)methylene]propan-
edioic acid dihydrazide (MalH-1),
2-naphthalenylamino-[(3,5-dibromo-2,4-dihydroxyphenyl)methylene][(2,4-dis-
odium-disulfophenyl)methylene]propanedioic acid dihydrazide
(MalH-2), and
2-naphthalenylamino-[(3,5-dibromo-2,4-dihydroxyphenyl)methylene][3-(4-sod-
ium-sulfophenyl)-thioureido]propanedioic acid dihydrazide (MalH-3).
Reagents and conditions: (a) diethyl bromomalonate, NaOAc,
90.degree. C., 8 hours, 84%; (b) N.sub.2H.sub.4.H.sub.2O,
EtOH/reflux, 10 hours, 92%; (c), (d)
3,5-di-Br-2,4-di-OH-benzaldehyde (1 equivalnt), EtOH/reflux, 3
hours, 58%; (e) 2,4-di-SO.sub.3Na-benzaldehyde, DMF/reflux, 4
hours, 58%; and (f) 4-sodiumsulfophenyl-isothiocyante, DMF/reflux,
4 hours, 47%.
[0034] FIG. 10 depicts the reaction scheme for the synthesis of the
PEG-ylated CFTR inhibitor MalH-(PEG).sub.n (Panel A) and
MalH-(PEG).sub.nB (Panel B).
[0035] FIG. 11 depicts the reaction scheme for the synthesis of the
PEG-ylated CFTR inhibitor GlyH-(PEG).sub.n. Reagents and
conditions: (i) Br-buterolactone, NaOAc, 90.degree. C., 8 hours,
89%; (j) N.sub.2H.sub.4.H.sub.2O, EtOH/reflux, 10 hours, 89%; (k)
(BOC).sub.2O, THF, rt, 86%; (1) TsCl, pyridine, -15.degree. C., 8
hours, 73%; (m) NH.sub.2-PEG, DMF, 80.degree. C., 24 hours, 38%;
(n) TFA, CH.sub.2Cl.sub.2, rt 30 min, 73%; and (o)
3,5-di-Br-2,4-di-OH-benzaldehyde, EtOH/reflux, 3 hours, 58%.
[0036] FIG. 12 is a series of graphs showing inhibition of apical
membrane chloride current in FRT epithelial cells expressing human
wildtype CFTR. Chloride current was measured by short-circuit
current analysis in cells subjected to a chloride ion gradient and
after permeabilization of the basolateral membrane. CFTR was
stimulated by 100 .mu.M CPT-cAMP. Increasing concentrations of MalH
compounds were added as indicated.
[0037] FIG. 13 is a series of graphs showing intestinal absorption
and antidiarrheal efficacy of CFTR inhibitors. Panel A is a graph
showing absorption over 2 hours of indicated MalH compounds in
closed jejunal loops in living mice (SD, n=4-6 mice). For
comparison absorption of CFTR.sub.inh-172 show as measured by same
method. Panel B is a graph showing inhibition of cholera
toxin-induced fluid secretion in closed jejunal loops. Loops were
injected with saline (PBS) or saline containing 1 .mu.g cholera
toxin (CT) with indicated amounts of MalH compounds. Loops
weight-to-length ratio measured at 6 hours (SD, n=3-5 mice).
[0038] Before the present invention is described, it is to be
understood that this invention is not limited to particular
embodiments described, as such may, of course, vary. It is also to
be understood that the terminology used herein is for the purpose
of describing particular embodiments only, and is not intended to
be limiting, since the scope of the present invention will be
limited only by the appended claims.
[0039] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limit of that range and any other stated or intervening
value in that stated range is encompassed within the invention. The
upper and lower limits of these smaller ranges may independently be
included in the smaller ranges is also encompassed within the
invention, subject to any specifically excluded limit in the stated
range. Where the stated range includes one or both of the limits,
ranges excluding either both of those included limits are also
included in the invention.
[0040] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can also be used in the practice or testing of the present
invention, the preferred methods and materials are now described.
All publications mentioned herein are incorporated herein by
reference to disclose and describe the methods and/or materials in
connection with which the publications are cited.
[0041] It should be noted that as used herein and in the appended
claims, the singular forms "a", "and", and "the" include plural
referents unless the context clearly dictates otherwise. Thus, for
example, reference to "an inhibitor" includes a plurality of such
inhibitors and reference to "the cell" includes reference to one or
more cells and equivalents thereof known to those skilled in the
art, and so forth.
[0042] The publications discussed herein are provided solely for
their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an admission that
the present invention is not entitled to antedate such publication
by virtue of prior invention. Further, the dates of publication
provided may be different from the actual publication dates which
may need to be independently confirmed.
DETAILED DESCRIPTION OF THE INVENTION
[0043] The invention is based on the discovery of
hydrazide-containing compounds that are high-affinity CFTR
inhibitors. The structure of these compounds having CFTR inhibitory
activity disclosed herein, and derivatives thereof, as well as
pharmaceutical formulations and methods of use are described in
more detail below.
Definitions
[0044] A "cystic fibrosis transmembrane conductance regulator
protein-mediated condition or symptom" or "CFTR-mediated condition
or symptom" means any condition, disorder or disease, or symptom of
such condition, disorder, or disease, that results from activity of
cystic fibrosis transmembrane conductance regulator protein (CFTR),
e.g., activity of CFTR in ion transport. Such conditions,
disorders, diseases, or symptoms thereof are treatable by
inhibition of CFTR activity, e.g., inhibition of CFTR ion
transport. CFTR activity has been implicated in, for example,
intestinal secretion in response to various agonists, including
cholera toxin (see, e.g., Snyder et al. 1982 Bull. World Health
Organ. 60:605-613; Chao et al. 1994 EMBO J. 13:1065-1072; Kimberg
et al. 1971 J. Clin. Invest. 50:1218-1230).
[0045] A "CFTR inhibitor" as used herein is a compound that reduces
the efficiency of ion transport by CFTR, particularly with respect
to transport of chloride ions by CFTR. Preferably CFTR inhibitors
of the invention are specific CFTR inhibitors, i.e., compounds that
inhibit CFTR activity without significantly or adversely affecting
activity of other ion transporters, e.g., other chloride
transporters, potassium transporters, and the like. Preferably the
CFTR inhibitors are high-affinity CFTR inhibitors, e.g., have an
affinity for CFTR of at least about one micromolar, usually about
one to five micromolar.
[0046] The term "isolated compound" means a compound which has been
substantially separated from, or enriched relative to, other
compounds with which it occurs in nature. Preferably, the compound
is at least about 80%, more preferably at least 90% pure, even more
preferably at least 98% pure, most preferably at least about 99%
pure, by weight. The present invention is meant to comprehend
diastereomers as well as their racemic and resolved,
enantiomerically pure forms and pharmaceutically acceptable salts
thereof.
[0047] "Treating" or "treatment" as used herein covers the
treatment of a disease, condition, disorder or symptom in a
subject, wherein the disease, condition, disorder or symptom is
mediated by the activity of CFTR, and includes: (1) preventing the
disease, condition, or disorder, i.e. causing the clinical symptoms
of the disease not to develop in a subject that may be exposed to
or predisposed to the disease, condition, or disorder, but does not
yet experience or display symptoms thereof, (2) inhibiting the
disease, condition or disorder, i.e., arresting or reducing the
development of the disease, condition or disorder, or its clinical
symptoms, or (3) relieving the disease, condition or disorder,
i.e., causing regression of the disease, condition or disorder, or
its clinical symptoms.
[0048] A "therapeutically effective amount" or "efficacious amount"
means the amount of a compound of the invention that, when
administered to a mammal or other subject in need thereof, is
sufficient to effect treatment, as defined above, for diseases,
conditions, disorders or symptoms mediated by the activity of CFTR.
The amount of a compound of the invention that constitutes a
"therapeutically effective amount" will vary depending on the
compound, the disease and its severity and the age, weight, etc.,
of the subject to be treated, but can be determined routinely by
one of ordinary skill in the art having regard to his own knowledge
and to this disclosure.
[0049] The terms "subject" and "patient" mean a member or members
of any mammalian or non-mammalian species that may have a need for
the pharmaceutical methods, compositions and treatments described
herein. Subjects and patients thus include, without limitation,
primate (including humans), canine, feline, ungulate (e.g., equine,
bovine, swine (e.g., pig)), avian, and other subjects. Humans and
non-human animals having commercial importance (e.g., livestock and
domesticated animals) are of particular interest.
[0050] "Mammal" means a member or members of any mammalian species,
and includes, by way of example, canines; felines; equines;
bovines; ovines; rodentia, etc. and primates, particularly humans.
Non-human animal models, particularly mammals, e.g., primate,
murine, lagomorpha, etc. may be used for experimental
investigations.
[0051] The term "unit dosage form," as used herein, refers to
physically discrete units suitable as unitary dosages for human and
animal subjects, each unit containing a predetermined quantity of
compounds of the present invention calculated in an amount
sufficient to produce the desired effect in association with a
pharmaceutically acceptable diluent, carrier or vehicle. The
specifications for the novel unit dosage forms of the present
invention depend on the particular compound employed and the effect
to be achieved, and the pharmacodynamics associated with each
compound in the host.
[0052] The term "physiological conditions" is meant to encompass
those conditions compatible with living cells, e.g., predominantly
aqueous conditions of a temperature, pH, salinity, etc. that are
compatible with living cells.
[0053] A "pharmaceutically acceptable excipient" means an excipient
that is useful in preparing a pharmaceutical composition that is
generally safe, non-toxic and neither biologically nor otherwise
undesirable, and includes an excipient that is acceptable for
veterinary use as well as human pharmaceutical use. "A
pharmaceutically acceptable excipient" as used in the specification
and claims includes both one and more than one such excipient.
[0054] As used herein, a "pharmaceutical composition" is meant to
encompass a composition suitable for administration to a subject,
such as a mammal, especially a human. In general a "pharmaceutical
composition" is sterile, and preferably free of contaminants that
are capable of eliciting an undesirable response within the
subject. Pharmaceutical compositions can be designed for
administration to subjects or patients in need thereof via a number
of different routes of administration including oral, buccal,
rectal, parenteral, intraperitoneal, intradermal, intracheal and
the like. In some embodiments the composition is suitable for
administration by a transdermal route, using a penetration enhancer
other than DMSO. In other embodiments, the pharmaceutical
compositions are suitable for administration by a route other than
transdermal administration.
[0055] As used herein, "pharmaceutically acceptable derivatives" of
a compound of the invention include salts, esters, enol ethers,
enol esters, acetals, ketals, orthoesters, hemiacetals, hemiketals,
acids, bases, solvates, hydrates or prodrugs thereof. Such
derivatives may be readily prepared by those of skill in this art
using known methods for such derivatization. The compounds produced
may be administered to animals or humans without substantial toxic
effects and either are pharmaceutically active or are prodrugs.
[0056] A "pharmaceutically acceptable salt" of a compound of the
invention means a salt that is pharmaceutically acceptable and that
possesses the desired pharmacological activity of the parent
compound. Such salts include: (1) acid addition salts, formed with
inorganic acids such as hydrochloric acid, hydrobromic acid,
sulfuric acid, nitric acid, phosphoric acid, and the like; or
formed with organic acids such as acetic acid, propionic acid,
hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic
acid, lactic acid, malonic acid, succinic acid, malic acid, maleic
acid, fumaric acid, tartaric acid, citric acid, benzoic acid,
3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid,
methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic
acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid,
4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid,
4-toluenesulfonic acid, camphorsulfonic acid,
4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, glucoheptonic
acid, 4,4'-methylenebis-(3-hydroxy-2-ene-1-carboxylic acid),
3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic
acid, lauryl sulfuric acid, gluconic acid, glutamic acid,
hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid,
and the like; or (2) salts formed when an acidic proton present in
the parent compound either is replaced by a metal ion, e.g., an
alkali metal ion, an alkaline earth ion, or an aluminum ion; or
coordinates with an organic base such as ethanolamine,
diethanolamine, triethanolamine, tromethamine, N-methylglucamine,
and the like.
[0057] A "pharmaceutically acceptable ester" of a compound of the
invention means an ester that is pharmaceutically acceptable and
that possesses the desired pharmacological activity of the parent
compound, and includes, but is not limited to, alkyl, alkenyl,
alkynyl, aryl, heteroaryl, aralkyl, heteroaralkyl, cycloalkyl and
heterocyclyl esters of acidic groups, including, but not limited
to, carboxylic acids, phosphoric acids, phosphinic acids, sulfonic
acids, sulfinic acids and boronic acids.
[0058] A "pharmaceutically acceptable enol ether" of a compound of
the invention means an enol ether that is pharmaceutically
acceptable and that possesses the desired pharmacological activity
of the parent compound, and includes, but is not limited to,
derivatives of formula C.dbd.C(OR) where R is hydrogen, alkyl,
alkenyl, alkynyl, aryl, heteroaryl, aralkyl, heteroaralkyl,
cycloalkyl or heterocyclyl.
[0059] A "pharmaceutically acceptable enol ester" of a compound of
the invention means an enol ester that is pharmaceutically
acceptable and that possesses the desired pharmacological activity
of the parent compound, and includes, but is not limited to,
derivatives of formula C.dbd.C(OC(O)R) where R is hydrogen, alkyl,
alkenyl, alkynyl, aryl, heteroaryl, aralkyl, heteroaralkyl,
cycloalkyl or heterocyclyl.
[0060] A "pharmaceutically acceptable solvate or hydrate" of a
compound of the invention means a solvate or hydrate complex that
is pharmaceutically acceptable and that possesses the desired
pharmacological activity of the parent compound, and includes, but
is not limited to, complexes of a compound of the invention with
one or more solvent or water molecules, or 1 to about 100, or 1 to
about 10, or one to about 2, 3 or 4, solvent or water
molecules.
[0061] A "pro-drug" means any compound that releases an active
parent compound of formula (I) in vivo when the prodrug is
administered to a mammalian subject. Prodrugs of the compounds of
formula (I) contain functional groups that, under standard
physiological conditions, are hydrolyzed into the corresponding
carboxy, hydroxy, or amino group. Examples of such functional
groups include, but are not limited to, esters (e.g., acetate,
formate and benzoate derivatives) and carbamates (e.g.,
N,N-dimethylaminocarbonyl) of hydroxy groups in compounds of
formula (I), and the like. Additional examples include dipeptide or
tripeptide esters of hydroxy or carboxy groups in compounds of
formula (I), and the like. The preparation of such functional
groups is well known in the art. For example, a compound of formula
(I) having a hydroxy group attached thereto may be treated with a
carboxylic acid or a dipeptide having a free carboxy terminus under
esterification conditions well known in the art to yield the
desired ester functional group. Likewise, a compound of formula (I)
having a free carboxy group attached thereto may be treated with an
alcohol or a tripeptide containing a hydroxy group such as a serine
residue (e.g., --N(H)--C(H)(CH.sub.2OH)--C(O)--) under
esterification conditions well known in the art to produce the
desired ester functional group. In addition, compounds of formula
(I) having a carboxylic ester group attached thereto may be treated
with a different carboxylic ester under standard
transesterification conditions to produce compounds of formula (I)
with the desired functional ester group attached thereto. All such
functional groups are considered to be within the scope of this
invention.
[0062] The term "organic group" and "organic radical" as used
herein means any carbon-containing group, including hydrocarbon
groups that are classified as an aliphatic group, cyclic group,
aromatic group, functionalized derivatives thereof and/or various
combination thereof. The term "aliphatic group" means a saturated
or unsaturated linear or branched hydrocarbon group and encompasses
alkyl, alkenyl, and alkynyl groups, for example. The term "alkyl
group" means a substituted or unsubstituted, saturated linear or
branched hydrocarbon group or chain (e.g., C.sub.1 to C.sub.8)
including, for example, methyl, ethyl, isopropyl, tert-butyl,
heptyl, n-octyl, dodecyl, octadecyl, amyl, 2-ethylhexyl, and the
like. Suitable substituents include carboxy, protected carboxy,
amino, protected amino, halo, hydroxy, protected hydroxy, mercapto,
lower alkylthio, nitro, cyano, monosubstituted amino, protected
monosubstituted amino, disubstituted amino, C.sub.1 to C.sub.7
alkoxy, C.sub.1 to C.sub.7 acyl, C.sub.1 to C.sub.7 acyloxy, and
the like. The term "substituted alkyl" means the above defined
alkyl group substituted from one to three times by a hydroxy,
protected hydroxy, amino, protected amino, cyano, halo,
trifloromethyl, mono-substituted amino, di-substituted amino, lower
alkoxy, mercapto, lower alkylthio, carboxy, protected carboxy, or a
carboxy, amino, and/or hydroxy salt. As used in conjunction with
the substituents for the heteroaryl rings, the terms "substituted
(cycloalkyl)alkyl" and "substituted cycloalkyl" are as defined
below substituted with the same groups as listed for a "substituted
alkyl" group. The term "alkenyl group" means an unsaturated, linear
or branched hydrocarbon group with one or more carbon-carbon double
bonds, such as a vinyl group. The term "alkynyl group" means an
unsaturated, linear or branched hydrocarbon group with one or more
carbon-carbon triple bonds. The term "cyclic group" means a closed
ring hydrocarbon group that is classified as an alicyclic group,
aromatic group, or heterocyclic group. The term "alicyclic group"
means a cyclic hydrocarbon group having properties resembling those
of aliphatic groups. The term "aromatic group" or "aryl group"
means a mono- or polycyclic aromatic hydrocarbon group, and may
include one or more heteroatoms, and which are further defined
below. The term "heterocyclic group" means a closed ring
hydrocarbon in which one or more of the atoms in the ring are an
element other than carbon (e.g., nitrogen, oxygen, sulfur, etc.),
and are further defined below.
[0063] "Organic groups" may be functionalized or otherwise comprise
additional functionalities associated with the organic group, such
as carboxyl, amino, hydroxyl, and the like, which may be protected
or unprotected. For example, the phrase "alkyl group" is intended
to include not only pure open chain saturated hydrocarbon alkyl
substituents, such as methyl, ethyl, propyl, t-butyl, and the like,
but also alkyl substituents bearing further substituents known in
the art, such as hydroxy, alkoxy, mercapto, alkylthio,
alkylsulfonyl, halo, cyano, nitro, amino, carboxyl, etc. Thus,
"alkyl group" includes ethers, esters, haloalkyls, nitroalkyls,
carboxyalkyls, hydroxyalkyls, sulfoalkyls, etc.
[0064] The terms "halo group" or "halogen" are used interchangeably
herein and refer to the fluoro, chloro, bromo or iodo groups.
[0065] The term "haloalkyl" refers to an alkyl group as defined
above that is substituted by one or more halogen atoms. The halogen
atoms may be the same or different. The term "dihaloalkyl " refers
to an alkyl group as described above that is substituted by two
halo groups, which may be the same or different. The term
"trihaloalkyl" refers to an alkyl group as describe above that is
substituted by three halo groups, which may be the same or
different. The term "perhaloalkyl" refers to a haloalkyl group as
defined above wherein each hydrogen atom in the alkyl group has
been replaced by a halogen atom. The term "perfluoroalkyl" refers
to a haloalkyl group as defined above wherein each hydrogen atom in
the alkyl group has been replaced by a fluoro group.
[0066] The term "cycloalkyl" means a mono-, bi-, or tricyclic
saturated ring that is fully saturated or partially unsaturated.
Examples of such a group included cyclopropyl, cyclobutyl,
cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, cyclooctyl, cis-
or trans-decalin, bicyclo[2.2.1]hept-2-ene, cyclohex-1-enyl,
cyclopent-1-enyl, 1,4-cyclooctadienyl, and the like.
[0067] The term "(cycloalkyl)alkyl" means the above-defined alkyl
group substituted for one of the above cycloalkyl rings. Examples
of such a group include (cyclohexyl)methyl,
3-(cyclopropyl)-n-propyl, 5-(cyclopentyl)hexyl, 6-(adamantyl)hexyl,
and the like.
[0068] The term "substituted phenyl" specifies a phenyl group
substituted with one or more moieties, and in some instances one,
two, or three moieties, chosen from the groups consisting of
halogen, hydroxy, protected hydroxy, cyano, nitro, mercapto,
alkylthio, trifluoromethyl, C.sub.1 to C.sub.7 alkyl, C.sub.1 to
C.sub.7 alkoxy, C.sub.1 to C.sub.7 acyl, C.sub.1 to C.sub.7
acyloxy, carboxy, oxycarboxy, protected carboxy, carboxymethyl,
protected carboxymethyl, hydroxymethyl, protected hydroxymethyl,
amino, protected amino, (monosubstituted)amino, protected
(monosubstituted)amino, (disubstituted)amino, carboxamide,
protected carboxamide, N--(C.sub.1 to C.sub.6 alkyl)carboxamide,
protected N--(C.sub.1 to C.sub.6 alkyl)carboxamide, N,N-di(C.sub.1
to C.sub.6 alkyl)carboxamide, trifluoromethyl, N--((C.sub.1 to
C.sub.6 alkyl)sulfonyl)amino, N-(phenylsulfonyl)amino or phenyl,
substituted or unsubstituted, such that, for example, a biphenyl or
naphthyl group results.
[0069] 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-(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-(isopropoxy)phenyl,
2-, 3- or 4-(t-butoxy)phenyl, 3-ethoxy-4-methoxyphenyl and the
like; a mono- or di(halo)-, mono-, di- or tri-(hydroxyl)phenyl such
as 3,5-dibromo-2,4,6-trihydroxyphenyl
3,5-dibromo-2,4-dihydroxyphenyl, 3,5-dibromo-4-hydroxyphenyl, and
3-bromo-4-hydroxyphenyl and the like; a mono- or di(halo)-mono- or
di-(hydroxyl)-mono- or di-(alkoxy)phenyl such as
3,5-dibromo-2-hydroxyl-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-(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.
[0070] The term "(substituted phenyl)alkyl" means one of the above
substituted phenyl groups attached to one of the above-described
alkyl groups. Examples include such groups 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)propyl, 4-chloro-3-aminobenzyl,
6-(4'-methoxyphenyl)-3-carboxyhexyl,
5-(4'-aminomethylphenyl)-3-(aminomethyl)pentyl,
5-phenyl-3-oxopent-1-yl, (4-hydroxynapth-2-yl)methyl and the
like.
[0071] As noted above, the term "aromatic" or "aryl" refers to five
and six membered carbocyclic rings. Also as noted above, the term
"heteroaryl" denotes optionally substituted five-membered or
six-membered rings that have 1 to 4 heteroatoms, such as oxygen,
sulfur and/or nitrogen atoms, in particular nitrogen, either alone
or in conjunction with sulfur or oxygen ring atoms. These
five-membered or six-membered rings may be fully unsaturated.
[0072] 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. For example, the rings can be
optionally 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.
[0073] The following ring systems are examples of the heterocyclic
(whether substituted or unsubstituted) radicals denoted by the term
"heteroaryl": thienyl, furyl, pyrrolyl, pyrrolidinyl, imidazolyl,
isoxazolyl, triazolyl, thiadiazolyl, oxadiazolyl, tetrazolyl,
thiatriazolyl, oxatriazolyl, pyridyl, pyrimidyl, pyrazinyl,
pyridazinyl, oxazinyl, triazinyl, thiadiazinyl tetrazolo,
1,5-[b]pyridazinyl and purinyl, as well as benzo-fused derivatives,
for example, benzoxazolyl, benzthiazolyl, benzimidazolyl and
indolyl.
[0074] Substituents for the above optionally substituted heteroaryl
rings are from one to three halo, trihalomethyl, amino, protected
amino, amino salts, mono-substituted amino, di-substituted amino,
carboxy, protected carboxy, carboxylate salts, hydroxy, protected
hydroxy, salts of a hydroxy group, lower alkoxy, mercapto, lower
alkylthio, alkyl, substituted alkyl, cycloalkyl, substituted
cycloalkyl, (cycloalkyl)alkyl, substituted (cycloalkyl)alkyl,
phenyl, substituted phenyl, phenylalkyl, and (substituted
phenyl)alkyl. Substituents for the heteroaryl group are as
heretofore defined, or in the case of trihalomethyl, can be
trifluoromethyl, trichloromethyl, tribromomethyl, or triiodomethyl.
As used in conjunction with the above substituents for heteroaryl
rings, "lower alkoxy" means a C.sub.1 to C.sub.4 alkoxy group,
similarly, "lower alkylthio" means a C.sub.1 to C.sub.4 alkylthio
group.
[0075] The term "(monosubstituted)amino" refers to an amino group
with one substituent chosen from the group consisting of phenyl,
substituted phenyl, alkyl, substituted alkyl, C.sub.1 to C.sub.4
acyl, C.sub.2 to C.sub.7 alkenyl, C.sub.2 to C.sub.7 substituted
alkenyl, C.sub.2 to C.sub.7 alkynyl, C.sub.7 to C.sub.16 alkylaryl,
C.sub.7 to C.sub.16 substituted alkylaryl and heteroaryl group. 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, alkyl, substituted alkyl, C.sub.1 to
C.sub.7 acyl, C.sub.2 to C.sub.7 alkenyl, C.sub.2 to C.sub.7
alkynyl, C.sub.7 to C.sub.16 alkylaryl, C.sub.7 to C.sub.16
substituted alkylaryl and heteroaryl. The two substituents can be
the same or different.
[0076] The term "heteroaryl(alkyl)" denotes an alkyl group as
defined above, substituted at any position by a heteroaryl group,
as above defined.
[0077] "Optional" or "optionally" means that the subsequently
described event, circumstance, feature or element may, but need
not, occur, and that the description includes instances where the
event or circumstance occurs and instances in which it does not.
For example, "heterocyclo group optionally mono- or disubstituted
with an alkyl group" means that the alkyl may, but need not, be
present, and the description includes situations where the
heterocyclo group is mono- or disubstituted with an alkyl group and
situations where the heterocyclo group is not substituted with the
alkyl group.
[0078] The term "electron-withdrawing group" refers to the ability
of a functional group on a molecule to draw electrons to it self
more than a hydrogen atom would if the hydrogen atom occupied the
same position in the molecule. Examples of electron-withdrawing
groups include, but are not limited to, halogen groups, --C(O)R
groups (where R is alkyl); carboxylic acid and ester groups; --NR3+
groups (where R is alkyl or hydrogen); azo; nitro; --OR and --SR
groups (where R is hydrogen or alkyl); and organic groups (as
defined herein) containing such electron-withdrawing groups, such
as haloalkyl groups (including perhaloalkyl groups), and the
like.
[0079] Compounds that have the same molecular formula but differ in
the nature or sequence of bonding of their atoms or the arrangement
of their atoms in space are termed "isomers." Isomers that differ
in the arrangement of their atoms in space are termed
"stereoisomers." Stereoisomers that are not mirror images of one
another are termed "diastereomers" and those that are
non-superimposable mirror images of each other are termed
"enantiomers." When a compound has an asymmetric center, for
example, it is bonded to four different groups, a pair of
enantiomers is possible. An enantiomer can be characterized by the
absolute configuration of its asymmetric center and is described by
the R- and S-sequencing rules of Cahn and Prelog, or by the manner
in which the molecule rotates the plane of polarized light and
designated as dextrorotatory or levorotatory (i.e., as (+) or
(-)-isomers respectively). A chiral compound can exist as either an
individual enantiomer or as a mixture of thereof. A mixture
containing equal proportions of the enantiomers is called a
"racemic mixture."
[0080] The compounds of this invention may possess one or more
asymmetric centers; such compounds can therefore be produced as
individual (R)- or (S)-stereoisomers or as mixtures thereof. Unless
indicated otherwise, the description or naming of a particular
compound in the specification and claims is intended to include
both individual enantiomers and mixtures, racemic or otherwise,
thereof. The methods for the determination of stereochemistry and
the separation of stereoisomers are well-known in the art (see,
e.g., the discussion in Chapter 4 of "Advanced Organic Chemistry",
4th edition J. March, John Wiley and Sons, New York, 1992).
Overview
[0081] The invention provides hydrazide-containing compounds,
derivative compositions and methods of their use in high affinity
inhibition of cystic fibrosis transmembrane conductance regulator
protein (CFTR) and for the study and treatment of CFTR-mediated
diseases and conditions. The discovery of the subject
hydrazide-containing compounds and derivatives was based on
screening of numerous potential candidate compounds using an assay
designed to identify CFTR inhibitors that interact directly with
CFTR. Without being held to any particular theory or mode of
operation, since multiple CFTR activators that work on different
activating pathways were included in the studies leading to
identification of the subject compounds, the inhibitory compounds
of the invention likely effect inhibition by acting at or near the
CFTR Cl.sup.- transporting pathway. A screening of 100,000 diverse
compounds identified several compounds and derivatives as effective
CFTR inhibitors (FIG.1B). These compounds and derivatives are
unrelated chemically and structurally to previously known CFTR
activators or to the previously known CFTR inhibitors DPC, NPPB
glibenclamide, or thiazolidinone. The most potent CFTR inhibitor
identified from screening had a K.sub.1 of .about.2 .mu.M for
inhibition of Cl.sup.- current in human airway cells. Inhibition
was rapid, reversible and CFTR-specific.
[0082] The compositions and methods of the invention will now be
described in more detail.
Hydrazide-Containing Compounds
[0083] The hydrazide-containing compounds described herein comprise
an aromatic- or heteroaromatic-substituted nitrogen, a hydrazide
(which can be a glycine or oxamic hydrazide), and a substituted or
substituted aryl group. In specific embodiments, the subject
compounds are generally described by Formula (I) as follows:
##STR00001##
wherein X is independently chosen from an alkyl group, or a
carbonyl group; Y is independently chosen from an alky group; an
alkyl group having polar substitutions, such as a sulfo group, or a
carboxyl group, or a linker, such as an amide bond or an ether
linker to provide for attachment of one or more larger polar
molecules, such as a polyoxyalkyl polyether (such as a polyethylene
glycol (PEG),polypropylene glycol, polyhydroxyethyl glycerol),
disaccharides, a substituted or unsubstituted phenyl group,
polyalkylimines, a dendrimer from 0-10 generation and the like,
where Y can further include such an attached polar molecule(s);
R.sub.1 is independently chosen from a substituted or unsubstituted
phenyl group, a substituted or unsubstituted heteroaromatic group
such as a substituted or unsubstituted quinolinyl group, an
substituted or unsubstituted anthracenyl group, and a substituted
or unsubstituted naphthalenyl group; R.sub.2 is a substituted or
unsubstituted phenyl group; and R.sub.3 is independently chosen
from hydrogen and an alkyl group; or a pharmaceutically acceptable
derivative thereof, as an individual stereoisomer or a mixture
thereof. In one embodiment, R.sub.1 is chosen from a substituted
phenyl group, an unsubstituted quinolinyl group, an unsubstituted
anthracenyl group, and an unsubstituted naphthalenyl group; R.sub.2
is a substituted phenyl group; and R.sub.3 is independently chosen
from hydrogen and an alkyl group. Exemplary substituents for
R.sub.1, R.sub.2, and R.sub.3, are described in more detail
below.
[0084] In certain embodiments, the hydrazide-containing compounds
are generally described by Formula (I), wherein X is an alkyl
group. Such compounds are generally described by Formula (Ia) as
follows:
##STR00002##
wherein Y is a hydrogen or an alkyl group such as a substituted or
unsubstituted, saturated linear or branched hydrocarbon group or
chain (e.g., C.sub.1 to C.sub.8) including, e.g., methyl, ethyl,
isopropyl, tert-butyl, heptyl, n-octyl, dodecyl, octadecyl, amyl,
2-ethylhexyl; X.sub.1 is independently chosen from a hydrogen or an
alkyl group such as a substituted or unsubstituted, saturated
linear or branched hydrocarbon group or chain (e.g., C.sub.1 to
C.sub.8) including, e.g., methyl, ethyl, isopropyl, tert-butyl,
heptyl, n-octyl, dodecyl, octadecyl, amyl, 2-ethylhexyl, or an
alkyl group comprising a polar molecule chosen from a sulfo group,
a carboxy group, a carboxamide group, a polyoxyalkyl polyether, a
disaccharide, a substitute or unsubstituted phenyl group, or a
polyethylene imine (PEI), or a dendrimer from 0-10 generation;
R.sub.1 is independently chosen from a substituted or unsubstituted
phenyl group, a substituted or unsubstituted heteroaromatic group
such as a quinolinyl group, a substituted or unsubstituted
anthracenyl group, and a substituted or unsubstituted naphthalenyl
group; R.sub.2 is a substituted or unsubstituted phenyl group; and
R.sub.3 is independently chosen from hydrogen and an alkyl group.
In some embodiments, when X.sub.1 is hydrogen, R.sub.1 is a
substituted or unsubstituted anthracenyl group, or a heteroaromatic
group. In still other embodiments, when X.sub.1 is hydrogen, Y is
not hydrogen.
[0085] In specific embodiments, R.sub.1 is independently chosen
from a mono-(halo)phenyl group such as 2-, 3-, or 4-chlorophenyl; a
mono-(alkyl)phenyl such as a 2-, 3-, or 4-methylphenyl; a
naphthalenyl group such as 1- or 2-naphthalenyl; a mono-or
di(halo)naphthalenyl, such as 1-, 3-, 4-, 5-, 6-, 7-, or
8-chloronaphthalenyl, 3,4- or 5,6- or 5,7-or
5,8-dichloronaphthalenyl; a mono- or di(hydroxy)naphthalenyl, such
as 1-, 3-, 4-, 5-, 6-, 7-, or 8-hydroxynaphthalenyl, 1,8-, 3,4-,
dihydroxynaphthalenyl; a mono-or di or tri(alkoxy)naphthalenyl,
such as 1-, 3-, 5-, 6-, 7-, or 8-methoxynaphthalenyl,
5,8-dimethoxynaphthalenyl, 1,4,8-trimethoxynaphthalenyl; a mono- or
di(alkyl)naphthalenyl, such as 1-, 3-, 4-, 5-, or
6-methylnaphthalenyl, 4,5-, 4,6-dimethynaphthalenyl; a
mono-(hydroxy)-mono or di(sulfo)naphthalenyl such as
4-hydroxy-2-sulfo-naphthalenyl, 8-hydroxy-3,6-disulfo-naphthalenyl;
mono(alkyl)-mono- or di (alkoxy)naphthalenyl, such as
1methyl-5,6-dimethoxynaphthalenyl; or a quinolinyl group such as
6-quinolynyl; R.sub.2 is independently chosen from the group
consisting of substituted phenyl groups such as: a
mono-(halo)phenyl group such as 2-, 3-, or 4-bromophenyl; a mono or
di(hydroxyl)phenyl group such as 2, 3, 4-hydroxyphenyl and
2,4-dihydroxyphenyl; a mono- or di(halo)-mono-, di-, or
tri-(hydroxyl)phenyl such as 3,5-dibromo-2,4,6-trihydroxyphenyl,
3,5-dibromo-2,4-dihydroxyphenyl, 3,5-dibromo-4-hydroxyphenyl, and
3-bromo-4-hydroxyphenyl; a mono- or di(halo)-mono- or
di-(hydroxyl)-mono- or di-(alkoxy)phenyl such as
3,5-dibromo-2-hydroxy-4-methoxyphenyl; and R.sub.3 is independently
chosen from hydrogen or an alkyl group.
[0086] In further embodiments, the hydrazide-containing compounds
and derivatives of Formula (Ia) may comprise of compounds, wherein
Y is a hydrogen; X is a hydrogen, methyl or ethyl group; R.sub.1 is
independently chosen from a mono-(halo)phenyl group, such as a 2-,
3-, or 4-chlorophenyl group, a naphthalenyl group, such as a
2-naphthalenyl or a 1-naphthalenyl; R.sub.2 is independently chosen
from a di-(halo)-mono- or di(hydroxyl)phenyl group such as a
3,5-di-bromo-2,4-di-hydroxyphenyl group,
3,5-di-bromo-4-hydroxyphenyl group; and R.sub.3 is a hydrogen or a
methyl group.
[0087] In other embodiments, the hydrazide-containing compounds are
generally described by Formula (I) wherein X is CH.sub.2. Such
compounds are generally described by Formula (Ib) as follows:
##STR00003##
wherein Y is a hydrogen or an alkyl group such as a substituted or
unsubstituted, saturated linear or branched hydrocarbon group or
chain (e.g., C.sub.1 to C.sub.8) including, e.g., methyl, ethyl,
isopropyl, tert-butyl, heptyl, n-octyl, dodecyl, octadecyl, amyl,
2-ethylhexyl; R.sub.1 is independently chosen from a substituted or
unsubstituted phenyl group, a substituted or unsubstituted
heteroaromatic group such as a quinolinyl group, a substituted or
unsubstituted anthracenyl group, and a substituted or unsubstituted
naphthalenyl group; R.sub.2 is a substituted or unsubstituted
phenyl group; and R.sub.3 is independently chosen from hydrogen and
an alkyl group.
[0088] In some embodiments, Y is an alkyl group such as a
substituted or unsubstituted, saturated linear or branched
hydrocarbon group or chain (e.g., C.sub.1 to C.sub.8) including,
e.g., methyl, ethyl, isopropyl, tert-butyl, heptyl, n-octyl,
dodecyl, octadecyl, amyl, 2-ethylhexyl; and R.sub.1 is
independently chosen from a substituted or unsubstituted phenyl
group, a substituted or unsubstituted heteroaromatic group such as
a quinolinyl group, a substituted or unsubstituted anthracenyl
group, and a substituted or unsubstituted naphthalenyl group;
R.sub.2 is a substituted or unsubstituted phenyl group; and R.sub.3
is independently chosen from hydrogen and an alkyl group.
[0089] In specific embodiments, R.sub.1 is independently chosen
from a mono-(halo)phenyl group such as 2-, 3-, or 4-chlorophenyl; a
mono-(alkyl)phenyl such as a 2-, 3-, or 4-methylphenyl; a
naphthalenyl group such as 1- or 2-naphthalenyl; a mono-or
di(halo)naphthalenyl, such as 1-, 3-, 4-, 5-, 6-, 7-, or
8-chloronaphthalenyl, 3,4- or 5,6- or 5,7-or
5,8-dichloronaphthalenyl; a mono- or di(hydroxy)naphthalenyl, such
as 1-, 3-, 4-, 5-, 6-, 7-, or 8-hydroxynaphthalenyl, 1,8-, 3,4-,
dihydroxynaphthalenyl; a mono-or di or tri(alkoxy)naphthalenyl,
such as 1-, 3-, 5-, 6-, 7-, or 8-methoxynaphthalenyl,
5,8-dimethoxynaphthalenyl, 1,4,8-trimethoxynaphthalenyl; a mono- or
di(alkyl)naphthalenyl, such as 1-, 3-, 4-, 5-, or
6-methylnaphthalenyl, 4,5-, 4,6-dimethynaphthalenyl; a
mono-(hydroxy)-mono or di(sulfo)naphthalenyl such as
4-hydroxy-2-sulfo-naphthalenyl, 8-hydroxy-3,6-disulfo-naphthalenyl;
mono(alkyl)-mono- or di(alkoxy)naphthalenyl, such as
lmethyl-5,6-dimethoxynaphthalenyl; or a quinolinyl group such as
6-quinolynyl; R.sub.2 is independently chosen from the group
consisting of substituted phenyl groups such as: a
mono-(halo)phenyl group such as 2-, 3-, or 4-bromophenyl; a mono or
di(hydroxyl)phenyl group such as 2, 3, 4-hydroxyphenyl and
2,4-dihydroxyphenyl; a mono- or di(halo)-mono-, di-, or
tri-(hydroxyl)phenyl such as 3,5-dibromo-2,4,6-trihydroxyphenyl,
3,5-dibromo-2,4-dihydroxyphenyl, 3,5-dibromo-4-hydroxyphenyl, and
3-bromo-4-hydroxyphenyl; a mono- or di(halo)-mono- or
di-(hydroxyl)-mono- or di-(alkoxy)phenyl such as
3,5-dibromo-2-hydroxy-4-methoxyphenyl; and R.sub.3 is independently
chosen from hydrogen or an alkyl group. Compounds described by
Formula (Ib) are generally described as glycine hydrazides.
[0090] In further embodiments, the hydrazide-containing compounds
and derivatives of Formula (Ib) may comprise of compounds, wherein
Y is a hydrogen; R.sub.1 is independently chosen from a
mono-(halo)phenyl group, such as a 2-, 3-, or 4-chlorophenyl group,
a naphthalenyl group, such as a 2-naphthalenyl or a 1-naphthalenyl;
R.sub.2 is independently chosen from a di-(halo)-mono- or
di(hydroxyl)phenyl group such as a
3,5-di-bromo-2,4-di-hydroxyphenyl group,
3,5-di-bromo-4-hydroxyphenyl group; and R.sub.3 is a hydrogen or a
methyl group.
[0091] In yet other embodiments, the hydrazide-containing compounds
are generally described by Formula (I) wherein X is a carbonyl.
Such compounds are generally described by Formula (Ic) as
follows:
##STR00004##
wherein Y is a hydrogen or an alkyl group such as a substituted or
unsubstituted, saturated linear or branched hydrocarbon group or
chain (e.g., C.sub.1 to C.sub.8) including, e.g., methyl, ethyl,
isopropyl, tert-butyl, heptyl, n-octyl, dodecyl, octadecyl, amyl,
2-ethylhexyl; R.sub.1 is independently chosen from a substituted or
unsubstituted phenyl group, a substituted or unsubstituted
heteroaromatic group such as a quinolinyl group, a substituted or
unsubstituted anthracenyl group, and a substituted or unsubstituted
naphthalenyl group; R.sub.2 is a substituted or unsubstituted
phenyl group; and R.sub.3 is independently chosen from hydrogen and
an alkyl group.
[0092] In some embodiments, Y is an alkyl group such as a
substituted or unsubstituted, saturated linear or branched
hydrocarbon group or chain (e.g., C.sub.1 to C.sub.8) including,
e.g., methyl, ethyl, isopropyl, tert-butyl, heptyl, n-octyl,
dodecyl, octadecyl, amyl, 2-ethylhexyl; R.sub.1 is independently
chosen from a substituted or unsubstituted phenyl group, a
substituted or unsubstituted heteroaromatic group such as a
quinolinyl group, a substituted or unsubstituted anthracenyl group,
and a substituted or unsubstituted naphthalenyl group; R.sub.2 is a
substituted or unsubstituted phenyl group; and R.sub.3 is
independently chosen from hydrogen and an alkyl group.
[0093] In specific embodiments, R.sub.1 is independently chosen
from a mono-(halo)phenyl group such as 2-, 3-, or 4-chlorophenyl; a
mono-(alkyl)phenyl such as a 2-, 3-, or 4-methylphenyl; a
naphthalenyl group such as 1- or 2-naphthalenyl; a mono- or
di(halo)naphthalenyl, such as 1-, 3-, 4-, 5-, 6-, 7-, or
8-chloronaphthalenyl, 3,4- or 5,6- or 5,7-or
5,8-dichloronaphthalenyl; a mono- or di(hydroxy)naphthalenyl, such
as 1-, 3-, 4-, 5-, 6-, 7-, or 8-hydroxynaphthalenyl, 1,8-, 3,4-,
dihydroxynaphthalenyl; a mono-or di or tri(alkoxy)naphthalenyl,
such as 1-, 3-, 5-, 6-, 7-, or 8-methoxynaphthalenyl,
5,8-dimethoxynaphthalenyl, 1,4,8-trimethoxynaphthalenyl; a mono- or
di(alkyl)naphthalenyl, such as 1-, 3-, 4-, 5-, or
6-methylnaphthalenyl, 4,5-, 4,6-dimethynaphthalenyl; a
mono-(hydroxy)-mono or di(sulfo)naphthalenyl such as
4-hydroxy-2-sulfo-naphthalenyl, 8-hydroxy-3,6-disulfo-naphthalenyl;
mono(alkyl)-mono- or di(alkoxy)naphthalenyl, such as
lmethyl-5,6-dimethoxynaphthalenyl; or a quinolinyl group such as
6-quinolynyl; R.sub.2 is independently chosen from the group
consisting of substituted phenyl groups such as: a
mono-(halo)phenyl group such as 2-, 3-, or 4-bromophenyl; a mono or
di(hydroxyl)phenyl group such as 2, 3, 4-hydroxyphenyl and
2,4-dihydroxyphenyl; a mono- or di(halo)-mono-, di-, or
tri-(hydroxyl)phenyl such as 3,5-dibromo-2,4,6-trihydroxyphenyl,
3,5-dibromo-2,4-dihydroxyphenyl, 3,5-dibromo-4-hydroxyphenyl, and
3-bromo-4-hydroxyphenyl; a mono- or di(halo)-mono- or
di-(hydroxyl)-mono- or di-(alkoxy)phenyl such as
3,5-dibromo-2-hydroxy-4-methoxyphenyl; and R.sub.3 is independently
chosen from hydrogen or an alkyl group. Compounds described by
Formula (Ic) are generally described as oxamic acid hydrazides.
[0094] In further embodiments, the hydrazide-containing compounds
and derivatives of Formula (Ic) may comprise of compounds, wherein
Y is hydrogen; R.sub.1 is a naphthalenyl group, such as a
2-naphthalenyl or a 1-naphthalenyl; R.sub.2 is a di-(halo)-mono- or
di(hydroxyl)phenyl group such as a
3,5-di-bromo-2,4-di-hydroxyphenyl group,
3,5-di-bromo-4-hydroxyphenyl group; and R.sub.3 is a hydrogen or a
methyl group.
[0095] In some embodiments of the invention, the
hydrazide-containing compound may comprise a formula of the
following:
##STR00005## ##STR00006## ##STR00007## ##STR00008##
[0096] The hydrazide-containing compounds described herein may be
modified, for example, to provide for a desired characteristic.
Preferably, modification of the compounds does not significantly or
undesirably adversely affect the desirable characteristics of the
hydrazide-containing compounds, e.g., ability to inhibit CFTR
function and water solubility of the compound. For example, the
compounds described herein can be modified so as decrease the
ability of the compound to cross a cell membrane, e.g., a cell
membrane of a cell lining a mucosal surface, e.g., a
gastrointestinal cell. Membrane impermeance of the compounds
disclosed herein can be increased by, for example, increasing the
size or other physical characteristics of the compound.
[0097] In such embodiments, the membrane permeability of the
compounds generally described by Formula I, are decreased by the
addition of polar groups, such as sulfo and alkyl-carboxyl groups.
Such compounds are generally described by Formula (I) as
follows:
##STR00009##
wherein Y is independently chosen from an alky group; an alkyl
group having polar substitutions, such as a sulfo group, or a
carboxyl group; or a linker, such as an amide bond or an ether
linker to provide for attachment of one or more larger polar
molecules, such as a polyoxyalkyl polyether (such as a polyethylene
glycol (PEG), polypropylene glycol, polyhydroxyethyl glycerol),
disaccharides, polyalkylimines, and the like, where Y can further
include such an attached polar molecule(s); X is independently
chosen from an alkyl group, or a carbonyl group; R.sub.1 is
independently chosen from a substituted or unsubstituted phenyl
group, a substituted or unsubstituted heteroaromatic group such as
a quinolinyl group, a substituted or unsubstituted anthracenyl
group, and a substituted or unsubstituted naphthalenyl group;
R.sub.2 is a substituted or unsubstituted phenyl group; and R.sub.3
is independently chosen from hydrogen and an alkyl group.
[0098] In specific embodiments Y is independently chosen from a
substituted or unsubstituted alkyl group, such as a substituted or
unsubstituted, saturated linear or branched hydrocarbon group or
chain (e.g., C.sub.1 to C.sub.8) including, e.g., methyl, ethyl,
isopropyl, tert-butyl, heptyl, n-octyl, dodecyl, octadecyl, amyl,
2-ethylhexyl; an alkyl group carrying polar groups such as hydroxy,
sulfo, carboxylate, or a substituted or unsubstituted carboxamide
groups (where exemplary groups include 3-sulfopropyl, 4-sulfobutyl,
carboxymethyl, 2-carboxypropyl, 2-methoxy-2-oxoethyl,
3-methoxy-3-oxopropyl); or a linker such as an amide bond or ether
linker to provide for attachment of one or more larger polar
molecules, such as a polyoxyalkyl polyether (such as polyethylene
glycol (PEG), polypropylene glycol, polyhydroxyethyl glycerol),
polyethyleneimines, disaccharides, trisaccharides, polyalkylimines,
small amino dextrans and the like, where Y can further include such
an attached polar molecule(s).
[0099] In some embodiments, the nitrogen of the unsaturated amide
bond of the compound may be substituted as exemplified below:
##STR00010##
wherein X is independently chosen from an alkyl group, or a
carbonyl group; R.sub.1 is independently chosen from a substituted
or unsubstituted phenyl group, a substituted or unsubstituted
heteroaromatic group such as a quinolinyl group, a substituted or
unsubstituted anthracenyl group, and a substituted or unsubstituted
naphthalenyl group; R.sub.2 is a substituted or unsubstituted
phenyl group; R.sub.3 is independently chosen from hydrogen and an
alkyl group; and Y' is independently chosen from an substituted or
unsubstituted alkyl group, such as a substituted or unsubstituted,
saturated linear or branched hydrocarbon group or chain (e.g.,
C.sub.1 to C.sub.8) including, e.g., methyl, ethyl, isopropyl,
tert-butyl, heptyl, n-octyl, dodecyl, octadecyl, amyl,
2-ethylhexyl; an alkyl group carrying polar groups such as hydroxy,
sulfo, carboxylate, and a substituted or unsubstituted carboxamide
groups (where exemplary groups include, such as 3-sulfopropyl,
4-sulfobutyl, carboxymethyl, 2-carboxypropyl, 2-methoxy-2-oxoethyl,
3-methoxy-3-oxoproplyl); or a linker such as an amide bond or ether
linker to provide for attachment of one or more to larger polar
molecules, such as a polyoxyalkyl polyether (such as polyethylene
glycol (PEG), polypropylene glycol, polyhydroxyethyl glycerol),
polyethyleneimines, disaccharides, trisaccharides, polyalkylimines,
small amino dextrans and the like, where Y' can further include
such an attached polar molecule(s).
[0100] In some embodiments, X is independently chosen from a
carbonyl group; an alkyl group, such as a substituted or
unsubstituted, saturated linear or branched hydrocarbon group or
chain (e.g., C.sub.1 to C.sub.8) including, methylene, substituted
alkyl groups, such as propene; substituted or unsubstituted phenyl
groups, such as a phenyl group carrying polar groups; or a linker
to carry polar groups; R.sub.1 is independently chosen from a
mono-(halo)phenyl group such as 2-, 3-, or 4-chlorophenyl; a
mono-(alkyl)phenyl such as a 2-, 3-, or 4-methylphenyl; a
naphthalenyl group such as 1- or 2-naphthalenyl; a mono-or
di(halo)naphthalenyl, such as 1-, 3-, 4-, 5-, 6-, 7-, or
8-chloronaphthalenyl, 3,4- or 5,6- or 5,7-or
5,8-dichloronaphthalenyl; a mono- or di(hydroxy)naphthalenyl, such
as 1-, 3-, 4-, 5-, 6-, 7-, or 8-hydroxynaphthalenyl, 1,8-, 3,4-,
dihydroxynaphthalenyl; a mono-or di or tri(alkoxy)naphthalenyl,
such as 1-, 3-, 5-, 6-, 7-, or 8-methoxynaphthalenyl,
5,8-dimethoxynaphthalenyl, 1,4,8-trimethoxynaphthalenyl; a mono- or
di(alkyl)naphthalenyl, such as 1-, 3-, 4-, 5-, or
6-methylnaphthalenyl, 4,5-, 4,6-dimethynaphthalenyl; a
mono-(hydroxy)-mono or di(sulfo)naphthalenyl such as
4-hydroxy-2-sulfo-naphthalenyl, 8-hydroxy-3,6-disulfo-naphthalenyl;
mono(alkyl)-mono- or di (alkoxy)naphthalenyl, such as
lmethyl-5,6-dimethoxynaphthalenyl; or a quinolinyl group such as
6-quinolynyl; R.sub.2 is independently chosen from the group
consisting of substituted phenyl groups such as a mono-(halo)phenyl
group such as 2-, 3-, or 4-bromophenyl; a mono or
di(hydroxyl)phenyl group such as 2,3,4-hydroxyphenyl and
2,4-dihydroxyphenyl; a mono- or di(halo)-mono- or di- or
tri-(hydroxyl)phenyl such as 3,5-dibromo-2,4,6-trihydroxyphenyl,
3,5-dibromo-2,4-dihydroxyphenyl, 3,5-dibromo-4-hydroxyphenyl, and
3-bromo-4-hydroxyphenyl; a mono- or di(halo)-mono- or
di-(hydroxyl)-mono- or di-(alkoxy)phenyl such as
3,5-dibromo-2-hydroxy-4-methoxyphenyl; and R.sub.3 is independently
chosen from hydrogen or an alkyl group.
[0101] In some embodiments, X of the compound may be substituted as
exemplified below:
##STR00011##
wherein X is an alkyl group; R.sub.1 is independently chosen from a
substituted or unsubstituted phenyl group, a substituted or
unsubstituted heteroaromatic group such as a quinolinyl group, a
substituted or unsubstituted anthracenyl group, and a substituted
or unsubstituted naphthalenyl group; R.sub.2 is a substituted or
unsubstituted phenyl group; R.sub.3 is independently chosen from
hydrogen and an alkyl group; and Y'' is independently chosen from
an substituted or unsubstituted alkyl group, such as a substituted
or unsubstituted, saturated linear or branched hydrocarbon group or
chain (e.g., C.sub.1 to C.sub.8) including, e.g., methyl, ethyl,
isopropyl, tert-butyl, heptyl, n-octyl, dodecyl, octadecyl, amyl,
2-ethylhexyl; an alkyl group carrying polar groups such as hydroxy,
sulfo, carboxylate, and a substituted or unsubstituted carboxamide
groups (where exemplary groups include, such as 3-sulfopropyl,
4-sulfobutyl, carboxymethyl, 2-carboxypropyl, 2-methoxy-2-oxoethyl,
3-methoxy-3-oxoproplyl); or a linker such as an amide bond or ether
linker to provide for attachment of one or more to larger polar
molecules, such as substituted or unsubstituted phenyl group, a
polyoxyalkyl polyether (such as polyethylene glycol (PEG),
polypropylene glycol, polyhydroxyethyl glycerol),
polyethyleneimines, disaccharides, trisaccharides, polyalkylimines,
small amino dextrans, a dendrimer from 0-10 generation, and the
like, where Y'' can further include such an attached polar
molecule(s).
[0102] In some embodiments, X is a substituted alkyl group, such as
a methyl group carrying polar groups or a linker to carry polar
groups; R.sub.1 is independently chosen from a mono-(halo)phenyl
group such as 2-, 3-, or 4-chlorophenyl; a mono-(alkyl)phenyl such
as a 2-, 3-, or 4-methylphenyl; a naphthalenyl group such as 1- or
2-naphthalenyl; a mono-or di(halo)naphthalenyl, such as 1-, 3-, 4-,
5-, 6-, 7-, or 8-chloronaphthalenyl, 3,4- or 5,6- or 5,7-or
5,8-dichloronaphthalenyl; a mono- or di(hydroxy)naphthalenyl, such
as 1-, 3-, 4-, 5-, 6-, 7-, or 8-hydroxynaphthalenyl, 1,8-, 3,4-,
dihydroxynaphthalenyl; a mono-or di or tri(alkoxy)naphthalenyl,
such as 1-, 3-, 5-, 6-, 7-, or 8-methoxynaphthalenyl,
5,8-dimethoxynaphthalenyl, 1,4,8-trimethoxynaphthalenyl; a mono- or
di(alkyl)naphthalenyl, such as 1-, 3-, 4-, 5-, or
6-methylnaphthalenyl, 4,5-, 4,6-dimethynaphthalenyl; a
mono-(hydroxy)-mono or di(sulfo)naphthalenyl such as
4-hydroxy-2-sulfo-naphthalenyl, 8-hydroxy-3,6-disulfo-naphthalenyl;
mono(alkyl)-mono- or di(alkoxy)naphthalenyl, such as
lmethyl-5,6-dimethoxynaphthalenyl; or a quinolinyl group such as
6-quinolynyl; R.sub.2 is independently chosen from the group
consisting of substituted phenyl groups such as a mono-(halo)phenyl
group such as 2-, 3-, or 4-bromophenyl; a mono or
di(hydroxyl)phenyl group such as 2,3,4-hydroxyphenyl and
2,4-dihydroxyphenyl; a mono- or di(halo)-mono- or di- or
tri-(hydroxyl)phenyl such as 3,5-dibromo-2,4,6-trihydroxyphenyl,
3,5-dibromo-2,4-dihydroxyphenyl, 3,5-dibromo-4-hydroxyphenyl, and
3-bromo-4-hydroxyphenyl; a mono- or di(halo)-mono- or
di-(hydroxyl)-mono- or di-(alkoxy)phenyl such as
3,5-dibromo-2-hydroxy-4-methoxyphenyl; R.sub.3 is independently
chosen from hydrogen or an alkyl group; and Y'' is independently
chosen from an alky group; an alkyl group having polar
substitutions, such as a sulfo group, or a carboxyl group; or a
linker, such as an amide bond or an ether linker, to provide for
attachment of one or more larger polar molecules, a polyoxyalkyl
polyether (such as polyethylene glycol (PEG), polypropylene glycol,
polyhydroxyethyl glycerol), disaccharides, polyalkylimines, and a
substituted or unsubstituted phenyl group, such as a
2,4-dihydroxy-3,5-di-bromophenyl group, a
2,4-disodium-disulfophenyl group, and a
3-monosodium-monosulfophenyl group, where Y can further include
such an attached polar molecule(s).
[0103] In some embodiments of the invention, the
hydrazide-containing compound may comprise a formula of the
following:
##STR00012## ##STR00013## ##STR00014## ##STR00015##
[0104] In further embodiments, the hydrazide containing compounds
are dimerized by using a bifunctional linker with varied chain
lengths. Such compounds are cell impermeant due to their large,
bulky nature and steric hindrance. In specific embodiments, the
subject compounds are generally described by Formula (Id) as
follows:
##STR00016##
wherein Z is a monomeric or polymeric unit, such as a polyoxyalkyl
polyether (such as a polyethylene glycol, polypropylene glycol,
polyhydroxyethyl glycerol), a linear polyamine, or a bifunctional
polysaccharide; and n is in the range of 0 to 500, 1 to 450, 2 to
400, 5 to 300, 10 to 250, 20 to 200, 30 to 150, 40 to 100, 50 to
90, and the like. In certain embodiments N has a range of 0 to 100,
1 to 95, 10 to 90, 20 to 80, 30 to 70, 40 to 60, and the like. In
specific embodiments, X is independently chosen from an alkyl
group, or a carbonyl group; Y is independently chosen from an alky
group; an alkyl group having polar substitutions, such as a sulfo
group, or a carboxyl group; or a linker, such as an amide bond or
an ether linker, to provide for attachment of one or more larger
polar molecules, a polyoxyalkyl polyether (such as polyethylene
glycol (PEG), polypropylene glycol, polyhydroxyethyl glycerol),
disaccharides, polyalkylimines, and the like, where Y can further
include such an attached polar molecule(s); R.sub.1, is
independently chosen from a substituted phenyl group, a quinolinyl
group, an anthracenyl group, and a naphthalenyl group; R.sub.2 is a
substituted phenyl group; and R.sub.3 is independently chosen from
hydrogen and an alkyl group; or a pharmaceutically acceptable
derivative thereof, as an individual stereoisomer or a mixture
thereof.
[0105] In some embodiments of the invention, the
hydrazide-containing compound may comprise a formula of the
following:
##STR00017##
Pharmaceutical Preparations
[0106] Also provided by the invention are pharmaceutical
preparations of the subject hydrazide-containing compounds
described above. The subject compounds can be incorporated into a
variety of formulations for therapeutic administration by a variety
of routes. More particularly, the compounds of the present
invention can be formulated into pharmaceutical compositions by
combination with appropriate, pharmaceutically acceptable carriers,
diluents, excipients and/or adjuvants, and may be formulated into
preparations in solid, semi-solid, liquid or gaseous forms, such as
tablets, capsules, powders, granules, ointments, solutions,
suppositories, injections, inhalants and aerosols. Preferably, the
formulations are free of detectable DMSO (dimethyl sulfoxide), or
are formulated with a penetration enhancer other than DMSO. The
formulations may be designed for administration to subjects or
patients in need thereof via a number of different routes, which
may be parenteral or enteral. Exemplary routes of administration
include oral, buccal, rectal, parenteral, intraperitoneal,
intradermal, transdermal, intracheal, etc., administration.
[0107] In pharmaceutical dosage forms, the subject compounds of the
invention may be administered in the form of their pharmaceutically
acceptable derivative, such as a salt, or they may also be used
alone or in appropriate association, as well as in combination,
with other pharmaceutically active compounds. The following methods
and excipients are merely exemplary and are in no way limiting.
[0108] In one embodiment of particular interest, the compounds of
the invention are administered to the gastrointestinal tract of the
subject, so as to provide for decreased fluid secretion. Suitable
formulations for this embodiment of the invention include any
formulation that provides for delivery of the compound to the
gastrointestinal surface, particularly an intestinal tract
surface.
[0109] For oral formulations, the subject compounds can be used
alone or in combination with appropriate additives to make tablets,
powders, granules or capsules, for example, with conventional
additives, such as lactose, mannitol, corn starch or potato starch;
with binders, such as starch, gelatin, natural sugars such as
glucose or beta-lactose, corn sweeteners, natural and synthetic
gums such as acacia, tragacanth, or sodium alginate,
carboxymethylcellulose, polyethylene glycol, waxes, crystalline
cellulose, cellulose derivatives, and acacia; with disintegrators,
such as corn starch, potato starch or sodium
carboxymethylcellulose, methyl cellulose, agar, bentonite, or
xanthan gum; with lubricants, such as talc, sodium oleate,
magnesium stearate sodium stearate, sodium benzoate, sodium
acetate, or sodium chloride; and if desired, with diluents,
buffering agents, moistening agents, preservatives, coloring
agents, and flavoring agents. Of particular interest is formulation
of the subject hydrazide-containing compounds with a buffering
agent, to provide for protection of the compound from low pH of the
gastric environment. It may also be preferable to provide an
enteric coating. In one embodiment, the compounds are formulated
for oral delivery with a flavoring agent, e.g., in a liquid, solid
or semi-solid formulation.
[0110] Oral formulations can be provided as gelatin capsules, which
may contain the active substance and powdered carriers, such as
lactose, starch, cellulose derivatives, magnesium stearate, stearic
acid, and the like. Similar carriers and diluents may be used to
make compressed tablets. Tablets and capsules can be manufactured
as sustained release products to provide for continuous release of
active ingredients over a period of time. Compressed tablets can be
sugar coated or film coated to mask any unpleasant taste and
protect the tablet from the atmosphere, or enteric coated for
selective disintegration in the gastrointestinal tract. Liquid
dosage forms for oral administration may contain coloring and/or
flavoring agents to increase patient acceptance.
[0111] Other suitable oral formulations include those that provide
for sustained release, which may be controlled release, of the
compound. Such formulations include hydrogels, microparticles, and
other dosage forms and formulations known in the art.
[0112] Water, a suitable oil, saline, aqueous dextrose, and related
sugar solutions and glycols such as propylene glycol or
polyethylene glycols, may be used as carriers for parenteral
solutions. Such solutions can also contain a water soluble salt of
the active ingredient, suitable stabilizing agents, and if
necessary, buffer substances. Suitable stabilizing agents include
antioxidizing agents such as sodium bisulfate, sodium sulfite, or
ascorbic acid, either alone or combined, citric acid and its salts
and sodium EDTA. Parenteral solutions may also contain
preservatives, such as benzalkonium chloride, methyl- or
propyl-paraben, and chlorobutanol.
[0113] The subject compounds of the invention can be formulated
into preparations for injection by dissolving, suspending or
emulsifying them in an aqueous or nonaqueous solvent, such as
vegetable or other similar oils, synthetic aliphatic acid
glycerides, esters of higher aliphatic acids or propylene glycol;
and if desired, with conventional additives such as solubilizers,
isotonic agents, suspending agents, emulsifying agents, stabilizers
and preservatives. Where desired, solubilizers for use can include
vitamin E TPGS (d-.alpha.-tocopheryl polyethylene glycol 1000
succinate), cyclodextrins, and the like.
[0114] Furthermore, the subject compounds can be made into
suppositories by mixing with a variety of bases such as emulsifying
bases or water-soluble bases. The compounds of the present
invention can be administered rectally via a suppository. The
suppository can include vehicles such as cocoa butter, carbowaxes
and polyethylene glycols, which melt at body temperature, yet are
solidified at room temperature.
[0115] Unit dosage forms for oral or rectal administration such as
syrups, elixirs, and suspensions may be provided wherein each
dosage unit, for example, teaspoonful, tablespoonful, tablet or
suppository, contains a predetermined amount of the composition
containing one or more inhibitors. Similarly, unit dosage forms for
injection or intravenous administration may comprise the
inhibitor(s) in a composition as a solution in sterile water,
normal saline or another pharmaceutically acceptable carrier.
[0116] The compounds of the invention can be utilized in aerosol
formulation to be administered via inhalation. The compounds of the
present invention can be formulated into pressurized acceptable
propellants such as dichlorodifluoromethane, propane, nitrogen and
the like.
[0117] In one embodiment, topical administration (e.g., by
transdermal administration) is of interest. Topical formulations
can be in the form of a transdermal patch, ointment, paste, lotion,
cream, gel, and the like. Topical formulations may include one or
more of a penetrating agent, thickener, diluent, emulsifier,
dispersing aid, or binder. Where the compound is formulated for
transdermal delivery, the compound may be formulated with or for
use with a penetration enhancer. Penetration enhancers, which
include chemical penetration enhancers and physical penetration
enhancers, facilitate delivery of the compound through the skin,
and may also be referred to as "permeation enhancers"
interchangeably. Physical penetration enhancers include, for
example, electrophoretic techniques such as iontophoresis, use of
ultrasound (or "phonophoresis"), and the like. Chemical penetration
enhancers are agents administered either prior to, with, or
immediately following compound administration, which increase the
permeability of the skin, particularly the stratum corneum, to
provide for enhanced penetration of the drug through the skin.
[0118] Compounds that have been used to enhance skin permeability
include: the sulfoxides dimethylsulfoxide (DMSO) and
decylmethylsulfoxide (C.sub.10 MSO); ethers such as diethylene
glycol monoethyl ether, dekaoxyethylene-oleylether, and diethylene
glycol monomethyl ether; surfactants such as sodium laurate, sodium
lauryl sulfate, cetyltrimethylammonium bromide, benzalkonium
chloride, Poloxamer (231, 182, 184),
[0119] Tween (20, 40, 60, 80) and lecithin; the 1-substituted
azacycloheptan-2-ones, particularly
1-n-dodecylcyclazacycloheptan-2-one; alcohols such as ethanol,
propanol, octanol, benzyl alcohol, and the like; petrolatums, such
as petroleum jelly (petrolatum), mineral oil (liquid petrolatum),
and the like; fatty acids such as C.sub.8-C.sub.22 and other fatty
acids (e.g., isostearic acid, octanoic acid, oleic acid, lauric
acid, valeric acid); C.sub.8-C.sub.22 fatty alcohols (e.g., oleyl
alcohol, lauryl alcohol); lower alkyl esters of C.sub.8-C.sub.22
fatty acids and other fatty acids (e.g., ethyl oleate, isopropyl
myristate, butyl stearate, methyl laurate, isopropyl myristate,
isopropyl palmitate, methylpropionate, ethyl oleate);
monoglycerides of C.sub.8-C.sub.22 fatty acids (e.g., glyceryl
monolaurate); tetrahydrofurfuryl alcohol polyethylene glycol ether;
2-(2-ethoxyethoxy)ethanol; diethylene glycol monomethyl ether;
alkylaryl ethers of polyethylene oxide; polyethylene oxide
monomethyl ethers; polyethylene oxide dimethyl ethers; di-lower
alkyl esters of C.sub.6-C.sub.8 diacids (e.g., diisopropyl
adipate); ethyl acetate; acetoacetic ester; polyols and esters
thereof such as propylene glycol, ethylene glycol, glycerol,
butanediol, polyethylene glycol, and polyethylene glycol
monolaurate; amides and other nitrogenous compounds such as urea,
dimethylacetamide (DMA), dimethylformamide (DMF), 2-pyrrolidone,
N-alkylpyrrolidone, e.g., 1-methyl-2-pyrrolidone; ethanol amine,
diethanol amine and triethanolamine; terpenes; alkanones, and
organic acids, particularly salicylic acid and salicylates, citric
acid and succinic acid. Additional chemical and physical
penetration enhancers are described in, for example, Transdermal
Delivery of Drugs, A. F. Kydonieus (ED) 1987 CRL Press;
Percutaneous Penetration Enhancers, eds. Smith et al. (CRC Press,
1995); Lenneruas et al., J Pharm. Pharmacol. 2002; 54(4):499-508;
Karande et al., Pharm. Res. 2002; 19(5):655-60; Vaddi et al., J.
Pharm. Sci. 2002 July; 91(7):1639-51; Ventura et al., J. Drug
Target 2001; 9(5):379-93; Shokri et al., Int. J. Pharm.
2001;228(1-2):99-107; Suzuki et al., Biol. Pharm. Bull. 2001;
24(6):698-700; Alberti et al., J. Control Release
2001;71(3):319-27; Goldstein et al., Urology 2001; 57(2):301-5;
Kiijavainen et al., Eur. J. Pharm. Sci. 2000; 10(2):97-102; and
Tenjarla et al., Int. J. Pharm. 1999; 192(2):147-58.
[0120] Where the compound is formulated with a chemical penetration
enhancer, the penetration enhancer is selected for compatibility
with the compound, and is present in an amount sufficient to
facilitate delivery of the compound through skin of a subject,
e.g., for delivery of the compound to the systemic circulation. In
one embodiment, the compound is formulated with a penetration
enhancer other than DMSO.
[0121] In one embodiment, the compound is provided in a drug
delivery patch, e.g., a transmucosal or transdermal patch, and can
be formulated with a penetration enhancer.
[0122] The patch generally includes a backing layer, which is
impermeable to the compound and other formulation components, a
matrix in contact with one side of the backing layer, which matrix
provides for sustained release, which may be controlled release, of
the compound, and an adhesive layer, which is on the same side of
the backing layer as the matrix. The matrix can be selected as is
suitable for the route of administration, and can be, for example,
and can be a polymeric or hydrogel matrix.
[0123] Depending on the subject and condition being treated and on
the administration route, the subject compounds may be administered
in dosages of, for example, 0.1 .mu.g to 10 mg/kg body weight per
day. The range is broad, since in general the efficacy of a
therapeutic effect for different mammals varies widely with doses
typically being 20, 30 or even 40 times smaller (per unit body
weight) in man than in the rat. Similarly the mode of
administration can have a large effect on dosage.
[0124] A typical dosage may be a solution suitable for intravenous
administration; a tablet taken from two to six times daily, or one
time-release capsule or tablet taken once a day and containing a
proportionally higher content of active ingredient, etc. The
time-release effect may be obtained by capsule materials that
dissolve at different pH values, by capsules that release slowly by
osmotic pressure, or by any other known means of controlled
release.
[0125] For use in the subject methods, the subject compounds may be
formulated with other pharmaceutically active agents, including
other CFTR-inhibiting agents or agents that block intestinal
chloride channels.
[0126] Pharmaceutically acceptable excipients usable with the
invention, such as vehicles, adjuvants, carriers or diluents, are
readily available to the public. Moreover, pharmaceutically
acceptable auxiliary substances, such as pH adjusting and buffering
agents, tonicity adjusting agents, stabilizers, wetting agents and
the like, are readily available to the public.
[0127] Those of skill in the art will readily appreciate that dose
levels can vary as a function of the specific compound, the
severity of the symptoms and the susceptibility of the subject to
side effects. Preferred dosages for a given compound are readily
determinable by those of skill in the art by a variety of
means.
[0128] Kits with unit doses of the subject compounds, usually in
oral or injectable doses, are provided. In such kits, in addition
to the containers containing the unit doses will be an
informational package insert describing the use and attendant
benefits of the drugs in treating pathological condition of
interest. Preferred compounds and unit doses are those described
herein above.
Conditions Amenable to Treatment Using the CFTR Inhibitors of the
Invention
[0129] The CFTR inhibitors disclosed herein are useful in the
treatment of a CFTR-mediated condition, i.e., any condition,
disorder or disease, or symptom of such condition, disorder, or
disease, that results from activity of CFTR, e.g., activity of CFTR
in ion transport. Such conditions, disorders, diseases, or symptoms
thereof are amenable to treatment by inhibition of CFTR activity,
e.g., inhibition of CFTR ion transport.
[0130] In one embodiment, the CFTR inhibitors of the invention are
used in the treatment of conditions associated with aberrantly
increased intestinal secretion, particularly acute aberrantly
increased intestinal secretion. CFTR activity has been implicated
in intestinal secretion in response to various agonists, including
cholera toxin (see, e.g., Snyder et al. 1982 Bull. World Health
Organ. 60:605-613; Chao et al. 1994 EMBO J. 13:1065-1072; Kimberg
et al. 1971 J. Clin. Invest.50:1218-1230). Thus CFTR inhibitors of
the invention can be administered in an amount effective to inhibit
CFTR ion transport and thus decrease intestinal fluid secretion. In
such embodiments, CFTR inhibitors according to the invention are
generally administered by administration to a mucosal surface of
the gastrointestinal tract (e.g., by an enteral route, e.g., oral,
intraintestinal, rectal, and the like) or to a mucosal surface of
the oral or nasal cavities, or (e.g., intranasal, buccal,
sublingual, and the like). In certain embodiments administration of
a CFTR inhibitor of the invention that is relatively membrane
impermeant (e.g., having decreased membrane permeance
characteristics (e.g., due to modification by PEGylation and the
like as described above)) is of particular interest.
[0131] Thus, CFTR inhibitors can be used in the treatment of
intestinal inflammatory disorders and diarrhea, particularly
secretory diarrhea. Secretory diarrhea is the biggest cause of
infant death in developing countries, with about 5 million deaths
annually (Gabriel et al., 1994 Science 266: 107-109). Several
studies, including those using CF mice, indicate that CFTR is the
final common pathway for intestinal chloride ion (and thus fluid)
secretion in response to various agonists (Snyder et al., 1982,
Bull. World Health Organ. 60: 605-613; Chao et al., 1994 EMBO. J.
13: 1065-1072; and Kimberg et al., 1971, J. Clin. Invest. 50:
1218-1230).
[0132] Diarrhea amenable to treatment using the CFTR inhibitors of
the invention can result from exposure to a variety of pathogens or
agents including, without limitation, cholera toxin (Vibrio
cholera), E. coli (particularly enterotoxigenic (ETEC)), Shigella,
Salmonella, Campylobacter, Clostridium difficile, parasites (e.g.,
Giardia, Entamoeba histolytica, Cryptosporidiosis, Cyclospora),
diarrheal viruses (e.g., rotavirus), food poisoning, or toxin
exposure that results in increased intestinal secretion mediated by
CFTR.
[0133] Other diarrheas include diarrhea associated with AIDS (e.g.,
AIDS-related diarrhea), diarrheas caused by anti-AIDS medications
such as protease inhibitors, and inflammatory gastrointestinal
disorders, such as ulcerative colitis, inflammatory bowel disease
(IBD), Crohn's disease, and the like. It has been reported that
intestinal inflammation modulates the expression of three major
mediators of intestinal salt transport and may contribute to
diarrhea in ulcerative colitis both by increasing transepithelial
Cl.sup.- secretion and by inhibiting the epithelial NaCl absorption
(see, e.g., Lohi et al., 2002, Am. J. Physiol. Gastrointest. Liver
Physiol. 283(3):G567-75).
[0134] CFTR inhibitors of the invention can also be used in
treatment of conditions such as polycystic kidney disease, and find
further use as male infertility drugs, by inhibition of CFTR
activity in the testis.
[0135] CFTR inhibitors of the invention can be further screened in
larger animal models (e.g., the rabbit model described in Spira et
al., 1981, Infect. Immun. 32:739-747.). In addition, analysis of
stool output using live Vibrio cholerae can also be examined to
further characterize the CFTR inhibitors of the invention.
Non-Human Animal Models and Human Tissue Models of
CFTR-Deficiencies
[0136] The CFTR inhibitors of the invention can also be used to
generate non-human animal models of disease, where the disease is
associated with decreased CFTR function (e.g., decreased ion
transport). There is increasing evidence that defective fluid and
macromolecular secretion by airway submucosal glands leads to
impaired mucociliary and bacterial clearance in CFTR-deficient
subjects, particularly in those affected with cystic fibrosis (CF);
however, functional studies in human airway glands have been
restricted to severely diseased airways obtained at the time of
lung transplantation (Jayaraman et al. 2001 Proc. Natl. Acad. Sci.
USA 98:8119-8123). Acute CFTR inhibition permits determination of
the role of CFTR in water, salt and macromolecule secretion by
submucosal glands. High-affinity CFTR inhibitors permit the
pharmacological creation of non-human animal models that mimic
CFTR-deficiency in humans, e.g., mimics the human CF phenotype. In
particular, large animal models of CFTR deficiency (e.g., CF) find
particular use in elucidating the pathophysiology of initiation and
progression of airway disease in CF, and in evaluating the efficacy
of CF therapies, e.g., screening candidate agents for treatment of
CFTR-deficiencies or symptoms thereof.
[0137] Inhibition of CFTR ion transport can be manifested in airway
and pancreatic disorders, as well as infertility in males. For
example, inhibition of CFTR channels in the lungs and airways
influences airway surface fluids leading to accumulation of mucus,
which in turn plugs airways and collects heavily on the lung walls,
providing a prime environment for infection to occur, which in turn
can lead to chronic lung disease. This same phenomenon occurs in
the pancreas, where the accumulated mucus disrupts the exocrine
function of the pancreas and prevents essential food-processing
enzymes from reaching the intestines.
[0138] Such non-human animal models can be generated by
administration of an amount of a CFTR inhibitor effective to
decrease CFTR activity in ion transport. Of particular interest is
the use of the CFTR inhibitors of the invention to induce the
cystic fibrosis (CF) phenotype in a non-human animal.
Administration of an amount of a CFTR inhibitor effective to
inhibit CFTR in, for example, lung effectively mimics the CFTR
defect found in CF. Routes of delivery for CFTR inhibitor are
discussed in detail above. Depending on the non-human animal used,
the subject compounds may be administered in dosages of, for
example, 50 to 500 .mu.g/kg body weight one to three times a day by
an intraperitoneal, subcutaneous, or other route to generate the
non-human animal models. Oral dosages may be up to about ten times
the intraperitoneal or subcutaneous dose.
[0139] Non-human animal models of CFTR-associated disease can be
used as models of any appropriate condition associated with
decreased CFTR activity. Such conditions include those that are
associated with CFTR mutations, which mutations result in
abnormalities in epithelial ion and water transport. These
abnormalities can in turn be associated with derangements in airway
mucociliary clearance, as well as in other mucosal epithelia and
ductal epithelia. Conditions that can be pharmacologically modeled
by inducing a CFTR-deficient phenotype in a non-human animal
include, without limitation, cystic fibrosis (including atypical
CF), idiopathic chronic pancreatitis, vas deferens defects, mild
pulmonary disease, asthma, and the like. For a review of disorders
associated with impaired CFTR function, see, e.g., Noone et al.
Respir Res 2 328-332 (2001). CFTR inhibitor-generated non-human
animal models can also serve as models of microbial infection
(e.g., bacterial, viral, or fungal infection, particularly
respiratory infections) in a CFTR-deficient subject. In one
embodiment of particular interest, the CFTR inhibitors of the
invention are used to pharmacologically induce the cystic fibrosis
(CF) phenotype.
[0140] Animals suitable for use in the production of the animal
models of the invention include any animal, particularly a mammal,
e.g., non-human primates (e.g., monkey, chimpanzee, gorilla, and
the like), rodents (e.g., rats, mice, gerbils, hamsters, ferrets,
and the like), lagomorphs, swine (e.g., pig, miniature pig),
equine, canine, feline, and the like. Large animals are of
particular interest.
[0141] The CFTR inhibitors can also be contacted with isolated
human tissue to create ex vivo models of disease. Such tissue is
contacted with an amount of a CFTR inhibitor effective to decrease
CFTR activity in the tissue, which may be for as little as 15
minutes, or as much as two hours or more. Human tissues of interest
include, without limitation, lung (including trachea and airways),
liver, pancreas, testis, and the like. Physiological, biochemical,
genomic or other studies can be carried out on the
inhibitor-treated tissue to identify novel therapeutic target
molecules that are important in the pathophysiology of a disease.
For example, isolated tissue from humans without CF can be exposed
to inhibitor sufficient to induce the CF phenotype and such studies
can be carried out to identify novel therapeutic target molecules
that are important in the pathophysiology of CF.
Synthesis of the Compounds of the Invention
[0142] Compounds of the invention may be prepared according to
methods known to one skilled in the art, or by methods similar to
the method described below.
[0143] It is understood that in the following description,
combinations of substituents and/or variables of the depicted
formulae are permissible only if such contributions result in
stable compounds.
[0144] It will also be appreciated by those skilled in the art that
in the process described below the functional groups of
intermediate compounds may need to be protected by suitable
protecting groups. Such functional groups include hydroxy, amino,
mercapto and carboxylic acid. Suitable protecting groups for
hydroxy include trialkylsilyl or diarylalkylsilyl (e.g.,
t-butyldimethylsilyl, t-butyldiphenylsilyl or trimethylsilyl),
tetrahydropyranyl, benzyl, and the like. Suitable protecting groups
for amino, amidino and guanidino include t-butoxycarbonyl,
benzyloxycarbonyl, and the like. Suitable protecting groups for
mercapto include --C(O)--R (where R is alkyl, aryl or aralkyl),
p-methoxybenzyl, trityl and the like. Suitable protecting groups
for carboxylic acid include alkyl, aryl or aralkyl esters.
[0145] Protecting groups may be added or removed in accordance with
standard techniques, which are well-known to those skilled in the
art and as described herein.
[0146] The use of protecting groups is described in detail in
Theodora W. Greene, Peter G. M. Wuts, Protective Groups in Organic
Synthesis (1999), 3rd Ed., Wiley-Interscience. The protecting group
may also be a polymer resin such as a Wang resin or a
2-chlorotrityl chloride resin.
[0147] It will also be appreciated by those skilled in the art,
although such protected derivatives of compounds of formula (I), as
described above (e.g., in the Overview and in Hydrazide-Containing
Compounds and Derivatives), may not possess pharmacological
activity as such, they may be administered to a mammal and
thereafter metabolized in the body to form compounds of the
invention which are pharmacologically active. Such derivatives may
therefore be described as "prodrugs". All prodrugs of compounds of
formula (I) are included within the scope of the invention.
[0148] The following Reaction Schemes illustrate methods to make
compounds of the invention. It is understood that one of ordinary
skill in the art would be able to make the compounds of the
invention by similar methods or by methods known to one skilled in
the art. In general, starting components may be obtained from
sources such as Aldrich, or synthesized according to sources known
to those of ordinary skill in the art (see, e.g., Smith and March,
March's Advanced Organic Chemistry: Reactions, Mechanisms, and
Structure, 5th edition (Wiley Interscience, New York)). Moreover,
the various substituted groups (e.g., R.sub.1, R.sub.2, R.sub.3,
and X, etc.) of the compounds of the invention may be attached to
the starting components, intermediate components, and/or final
products according to methods known to those of ordinary skill in
the art.
[0149] The following Reaction Scheme 1 is directed to the
preparation of compounds of formula (1), which are compounds of the
invention as described above (e.g., in the Overview and in
Hydrazide-Containing Compounds and Derivatives), where R.sub.1,
R.sub.2, and R.sub.3 are as described above (e.g., in the Overview
and in Hydrazide-Containing Compounds and Derivatives).
##STR00018##
[0150] In general, compounds of Formula (I) are prepared by first
combining an R.sub.i-group containing a terminal amine containing a
Y group with diethyl oxalate or an X containing compound such as
X-substituted ethyl iodoacetate, where X is as described above,
each at 10 mmol. The resulting reaction mixture is then stirred
overnight at elevated temperature. Upon cooling, the solid material
is filtered and recrystallized from hexane to yield compound of
formula (A). A solution of the compound of formula (A) in ethanol
is then refluxed with 12 mmol hydrazine hydrate for a period of
time of about 10 hours. The solvent and excess reagent are then
distilled under vacuum. The product is then recrystallized from
ethanol to yield the compound of formula (B). The compound of
formula (B) is then combined with a R.sub.2, R.sub.3-group
containing carbonyl group (e.g., a ketone or an aldehyde) in
ethanol and then refluxed for a period of time of about 3 hours to
yield the desired product of Formula (I).
[0151] Alternatively, compounds of Formula (I), where and X is an
alkyl group containing X.sub.1, wherein X.sub.1 is an alkyl group
such as a substituted or unsubstituted, saturated linear or
branched hydrocarbon group or chain (compounds of Formula Ia) can
be prepared according to the following Reaction Scheme 2 wherein
R.sub.1, R.sub.2, and R.sub.3 are as described above (e.g., in the
Overview and in Hydrazide-Containing Compounds and
Derivatives).
##STR00019##
[0152] In general, compounds of Formula (Ia) are prepared by first
combining an R.sub.1-group containing a terminal amine containing
an Y group with ethyl iodoacetate containing an X.sub.1 group,
where X.sub.1 is as described above, each at 10 mmol with 20 mmol
sodium acetate. The resulting reaction mixture is then stirred at
elevated temperature for a period of time of about 3 hours. Upon
cooling, the solid material is filtered and recrystallized from
hexane to yield compound of formula (C). A solution of the compound
of formula (C) in ethanol is then refluxed with 12 mmol hydrazine
hydrate for a period of time of about 10 hours. The solvent and
excess reagent are then distilled under vacuum. The product is then
recrystallized from ethanol to yield the compound of formula (D).
The compound of formula (D) is then combined with a R.sub.2,
R.sub.3-group containing carbonyl group (e.g., a ketone or an
aldehyde) in ethanol and then refluxed for a period of time of
about 3 hours to yield the desired product of Formula (Ia).
[0153] The following Reaction Scheme 3 is directed to the
preparation of compounds of Formula (Ib) wherein X is CH.sub.2,
which are compounds of the invention as described above (e.g., in
the Overview and in Hydrazide-Containing Compounds and
Derivatives), where R.sub.1, R.sub.2, and R.sub.3 are as described
above (e.g., in the Overview and in Hydrazide-Containing Compounds
and Derivatives).
##STR00020##
[0154] In general, compounds of Formula (Ib) are prepared by first
combining an R.sub.1-group containing a terminal amine with ethyl
iodoacetate each at 10 mmol with 20 mmol sodium acetate. The
resulting reaction mixture is then stirred at an elevated
temperature for a period of time of about 3 hours. Upon cooling,
the solid material is filtered and recrystallized from hexane to
yield compound of formula (E). A solution of the compound of
formula (E) in ethanol is then refluxed overnight with 12 mmol
hydrazine hydrate for a period of time of about 10 hours. The
solvent and excess reagent are then distilled under vacuum. The
product is then recrystallized from alcohol to yield the compound
of formula (F). The compound of formula (F) is then combined with a
R.sub.2, R.sub.3-group containing carbonyl group (e.g., a ketone or
an aldehyde) in ethanol and then refluxed for a period of time of
about 3 hours to yield the desired product of Formula (Ib).
[0155] Alternatively, compounds of Formula (I), where X is a
carbonyl group (compounds of Formula Ic) can be prepared according
to the following Reaction Scheme 4 wherein R.sub.1, R.sub.2, and
R.sub.3 are as described above in the Overview.
##STR00021##
[0156] In general, compounds of Formula (Ic) are prepared by first
combining an R.sub.1-group containing a terminal amine containing a
Y group with diethyl oxalate each at 10 mmol in toluene. The
resulting reaction mixture is then stirred at an elevated
temperature for a period of time of about 3 hours. Upon cooling,
the solid material is filtered and recrystallized from hexane to
yield compound of formula (G). A solution of the compound of
formula (G) in ethanol is then refluxed with 12 mmol hydrazine
hydrate for a period of time of about 10 hours. The solvent and
excess reagent are then distilled under vacuum. The product is then
recrystallized from ethanol to yield the compound of formula (H).
The compound of formula (H) is then combined with a R.sub.2,
R.sub.3-group containing a carbonyl group in ethanol and then
refluxed for a period of time of about 3 hours to yield the desired
product of Formula (Ic).
[0157] Alternatively, compounds of Formula (I), where X is an alkyl
group containing Y'' (compounds of Formula Ie) can be prepared
according to the following Reaction Schemes 5-8 wherein R.sub.1,
R.sub.2, and R.sub.3 are as described above (e.g., in the Overview
and in Hydrazide-Containing Compounds and Derivatives), and wherein
Y'' is independently chosen from an substituted or unsubstituted
alkyl group, such as a substituted or unsubstituted, saturated
linear or branched hydrocarbon group or chain (e.g., C.sub.1 to
C.sub.8) including, e.g., methyl, ethyl, isopropyl, tert-butyl,
heptyl, n-octyl, dodecyl, octadecyl, amyl, 2-ethylhexyl; an alkyl
group carrying polar groups such as hydroxy, sulfo, carboxylate,
and a substituted or unsubstituted carboxamide groups (where
exemplary groups include, such as 3-sulfopropyl, 4-sulfobutyl,
carboxymethyl, 2-carboxypropyl, 2-methoxy-2-oxoethyl,
3-methoxy-3-oxoproplyl); or a linker such as an amide bond or ether
linker to provide for attachment of one or more to larger polar
molecules, such as substituted or unsubstituted phenyl group, a
polyoxyalkyl polyether (such as polyethylene glycol (PEG),
polypropylene glycol, polyhydroxyethyl glycerol),
polyethyleneimines, disaccharides, trisaccharides, polyalkylimines,
small amino dextrans and the like, where Y'' can further include
such an attached polar molecule(s).
##STR00022##
[0158] In general, in some embodiments, compounds of Formula (Ie)
are prepared by first combining an R.sub.1-group containing a
terminal amine containing a Y group with diethyl bromomalonate each
at 10 mmol. The resulting reaction mixture is then stirred at an
elevated temperature for a period of time of about 8 hours. Upon
cooling, the solid material is filtered and recrystallized from
hexane to yield compound of formula (I). A solution of the compound
of formula (I) in ethanol is then refluxed with 12 mmol hydrazine
hydrate for a period of time of about 10 hours. The solvent and
excess reagent are then distilled under vacuum. The product is then
recrystallized from ethanol to yield the compound of formula (J).
The compound of formula (J) is then combined with a R.sub.2,
R.sub.3-group containing a carbonyl group in ethanol and then
refluxed for a period of time of about 3 hours to yield the desired
product of Formula (K). The compound of formula (K) is then
combined with a substituted or unsubstituted phenyl group as
described in greater detail above (e.g., in the Overview and in
Hydrazide-Containing Compounds and Derivatives) and refluxed for a
period of time. The product is then recrystallized from ethanol to
yield the compound of formula (L).
##STR00023##
[0159] In general, in some embodiments, compounds of Formula (Ie)
are prepared by first combining an R.sub.1-group containing a
terminal amine containing a Y group with diethyl bromomalonate each
at 10 mmol. The resulting reaction mixture is then stirred at an
elevated temperature for a period of time of about 8 hours. Upon
cooling, the solid material is filtered and recrystallized from
hexane to yield compound of formula (I). A solution of the compound
of formula (I) in ethanol is then refluxed with 12 mmol hydrazine
hydrate for a period of time of about 10 hours. The solvent and
excess reagent are then distilled under vacuum. The product is then
recrystallized from ethanol to yield the compound of formula (J).
The compound of formula (J) is then combined with a R.sub.2,
R.sub.3-group containing a carbonyl group in ethanol and then
refluxed for a period of time of about 3 hours to yield the desired
product of Formula (K). The compound of formula (K) is then
combined with a thiocyanate substituted phenyl group as described
in greater detail above (e.g., in the Overview and in
Hydrazide-Containing Compounds and Derivatives) and refluxed for a
period of time. The product is then recrystallized from ethanol to
yield the compound of formula (M).
##STR00024##
[0160] In general, in some embodiments, compounds of Formula (Ie)
are prepared by first combining a thiocyanate containing a phenyl
group with Amino-PEG in DMF and stirred at an elevated temperature
for a period of time of about 24 hours. The DMF is then evaporated
in vacuo, and the residue is dissolved in minimal quantity EtOAc
and added to a stirred solution of Et.sub.2O. The resulting
precipitate is then filtered and washed in Et.sub.2O to give the
PEG-containing compound. The PEG-containing compound is then
combined with the compound of formula (K) and refluxed for a period
of time. The product is then recrystallized from ethanol to yield
the compound of formula (N).
##STR00025##
[0161] In general, in some embodiments, compounds of Formula (Ie)
are prepared by first combining a thiocyanate containing a phenyl
group with Amino-PEG in DMF and stirred at an elevated temperature
for a period of time of about 24 hours. The DMF is then evaporated
in vacuo, and the residue is dissolved in minimal quantity EtOAc
and added to a stirred solution of Et.sub.2O. The resulting
precipitate is then filtered and washed in Et.sub.2O to give the
PEG-containing compound. The PEG-containing compound is then
combined with the compound of formula (K) and refluxed for a period
of time. The product is then recrystallized from ethanol to yield
the compound of formula (O).
##STR00026## ##STR00027##
[0162] In general, in some embodiments, compounds of Formula (Ie)
are prepared by first combining an R.sub.1-group containing a
terminal amine containing a Y group with diethyl
bromobuterolacetone or bromobuterolactone each at 10 mmol. The
resulting reaction mixture is then stirred at an elevated
temperature for a period of time of about 8 hours. Upon cooling,
the solid material is filtered and recrystallized from hexane to
yield compound of formula (P). A solution of the compound of
formula (P) in ethanol is then refluxed with 12 mmol hydrazine
hydrate for a period of time of about 10 hours. The solvent and
excess reagent are then distilled under vacuum. The product is then
recrystallized from ethanol to yield the compound of formula (Q).
The compound of formula (Q) (10 mM) is then combined with 20 mM
(BOC).sub.2O in 10 mL of THF and heated under reflux conditions for
a period of time of about 5 hours. The solvent is then removed, and
the residue is purified by column chromatography on silica gel and
eluted with dichloromethane to give the compound of formula (R).
The compound of formula (R) (1 mmol) is then combined with TsCl (1
mmol) in pyridine (5 ml) in three portion a period of time of about
30 min apart form one another. The reaction mixture is then stirred
at a low temperature of about -15.degree. C. for a period of time
of about 8 hours. The reaction mixture is then allowed to warm to
room temperature, diluted with 1N HCl, and then extracted three
times with EtOAc. The combined organic extract is then washed with
brine, dried with NaSO4 and evaporated to dryness to give the
compound of formula (S). The compound of formula (S) is then
combined with amino-PEG, such as 2-aminoethoxyethanol, in DMF and
stirred at an elevated temperature for a period of time of about 24
hours. The DMF is then evaporated in vacuo, and the residue is
dissolved in minimal quantity EtOAc and added to a stirred solution
of Et.sub.2O. The resulting precipitate is then filtered and washed
in Et.sub.2O to give the compound of formula (T). The compound of
formula (T) is then dissolved in minimal amount of trifluoroacetic
acid:Ch.sub.2Cl.sub.2 (1:1) and stirred at room temperature for a
period of time of about 30 minutes. The reaction mixture is the
diluted with saturated aqueous NaHCO.sub.3 and extracted with
CH.sub.2Cl.sub.2. The combined organic layer is then washed
successively with water and brine, dried and concentrated in vacuo
to yield the compound of formula (U). The compound of formula (U)
is then combined with a R.sub.2, R.sub.3-group containing a
carbonyl group in ethanol and then refluxed for a period of time of
about 3 hours to yield the desired product of Formula (V).
[0163] Structures were confirmed by .sup.1H-NMR and Mass
spectrometry. Purity was >98% as judged by thin layer
chromatography and HPLC.
EXAMPLES
[0164] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to make and use the present invention, and are
not intended to limit the scope of what the inventors regard as
their invention nor are they intended to represent that the
experiments below are all or the only experiments performed.
Efforts have been made to ensure accuracy with respect to numbers
used (e.g. amounts, temperature, etc.) but some experimental errors
and deviations should be accounted for. Unless indicated otherwise,
parts are parts by weight, molecular weight is weight average
molecular weight, temperature is in degrees Centigrade, and
pressure is at or near atmospheric.
Method and Materials
[0165] The following materials and methods were used in the
examples that follow.
[0166] High-Throughput Screening for Identification of CFTR
Inhibitors
[0167] Screening was performed using an integrated system (Beckman)
consisting of a 3-meter robotic arm, CO.sub.2 incubator, plate
washer, liquid handling work station, barcode reader, delidding
station, plate sealer and two fluorescence plate readers (Optima,
BMG Lab Technologies), each equipped with two syringe pumps and
HQ500/20X (500.+-.10 nm) excitation and HQ535/30M (535.+-.15 nm)
emission filters (Chroma).
[0168] One hundred thousand small molecules (most 350-550 daltons)
were selected for screening from commercial sources (ChemBridge and
ChemDiv, both of San Diego, Calif.) using algorithms designed to
maximize chemical diversity and drug-like properties. The compounds
were obtained as a dried powder and solutions were made in DMSO
just before testing, stored frozen as 2.5 mM stock solutions for
further use.
[0169] Fisher Rat Thyroid (FRT) cells stably expressing wildtype
human CFTR and YFP-H148Q were cultured on 96-well black wall plates
as described previously (Ma et al., J. Biol. Chem.,
277:37235-37241, 2002). For screening, cells in 96-well plates were
washed three times and then CFTR halide conductance was activated
by incubation for 15 minutes with an activating cocktail containing
10 .mu.M forskolin, 20 .mu.M apigenin and 100
isobutylmethyl-xanthine (IBMX). Test compounds (final 25 .mu.M)
were added 5 minutes prior to assay of iodide influx in which cells
were exposed to a 100 mM inwardly-directed iodide gradient. YFP
fluorescence was recorded for 2 seconds prior to and 12 seconds
after creation of the iodide gradient. Initial rates of iodide
influx were computed from the time course of decreasing
fluorescence after the iodide gradient (Yang et al., J. Biol.
Chem., 35079-35085, 2003).
[0170] Short-Circuit Current Measurements
[0171] FRT, T84 colon epithelial cells and human airway epithelial
cells were cultured on Snapwell filters with 1 cm.sup.2 surface
area (Corning-Costar) to resistances >1,000 cm.sup.2 as
described previously (Ma et al., J. Biol. Chem., 277:37235-37241,
2002). Filters were mounted in an Easymount Chamber System
(Physiologic Instruments, San Diego). For apical Cl.sup.- current
measurements on FRT cells, the basolateral hemichamber was filled
with buffer containing (in mM): 130 NaCl, 2.7 KCl, 1.5
KH.sub.2PO.sub.4, 1 CaCl.sub.2, 0.5 MgCl.sub.2, 10 Na-HEPES, 10
glucose (pH 7.3). The basolateral membrane was permeabilized with
amphotericin B (250 .mu.g/ml) just prior to measurements. In the
apical solution 65 mM NaCl was replaced by sodium gluconate, and
CaCl.sub.2 was increased to 2 mM. For short-circuit current
measurements in (non-permeabilized) T84 and human airway cells,
both hemichambers contained Kreb's solution (in mM): 120 NaCl, 25
NaHCO.sub.3, 3.3 KH.sub.2PO.sub.4, 0.8 K.sub.2HPO.sub.4, 1.2
MgCl.sub.2, 1.2 CaCl.sub.2 and 10 glucose (pH 7.3). Solutions were
bubble with 95% O.sub.2 and 5% CO.sub.2 and maintained at
37.degree. C. For studies in mouse intestine, ileal segments were
isolated, washed with ice-cold Kreb's buffer, opened longitudinally
through the mesenteric border, and mounted in a micro-Ussing
chamber (0.7 cm.sup.2 aperture area, World Precision Instruments).
Hemichambers were filled with Kreb's solutions containing 10 .mu.M
indomethacin. Apical Cl.sup.-/short-circuit current were recorded
using a DVC-1000 voltage-clamp (World Precision Instruments) with
Ag/AgCl electrodes and 1 M KCl agar bridges.
[0172] Patch-Clamp Analysis
[0173] Patch-clamp experiments were carried out at room temperature
on FRT cells stably expressing wildtype CFTR. Cell-attached and
whole-cell configurations were used (Hamill et al., Pflugers Arch.
391:85-100, 1981). The cell membrane was clamped at specified
voltages using an EPC-7 patch-clamp amplifier (List Medical). Data
were filtered at 500 Hz and digitized at 2000 Hz. For whole-cell
experiments the pipette solution contained (in mM): 120 CsCl, 10
TEA-Cl, 0.5 EGTA, 1 MgCl.sub.2, 40 mannitol, 10 Cs-HEPES and 3 mM
MgATP (pH 7.3). For cell attached experiments EGTA was replaced
with 1 mM CaCl.sub.2. The bath solution for whole-cell experiments
contained (in mM): 150 NaCl, 1 CaCl.sub.2, 1 MgCl.sub.2, 10
glucose, 10 mannitol, 10 Na-TES (pH 7.4). In cell-attached
experiments the bath solution contained (in mM): 130 KCl, 2 NaCl, 2
CaCl.sub.2, 2 MgCl.sub.2, 10 glucose, 20 mannitol, and 10 K-Hepes
(pH 7.3) Inhibitors were applied by extracellular perfusion. CFTR
channel activity in cell-attached patches was analyzed as described
previously (Taddei et al., FEBS Lett. 558:52-56, 2004).
[0174] Nasal Potential Difference Measurements in Mice
[0175] Following anesthesia with intraperitoneal ketamine (90-120
mg/kg) and xylazine (5-10 mg/kg) the airway was protected by
orotracheal intubation with a 21-gauge angiocatheter as described.
A PE-10 cannula pulled to a tip diameter of 0.3 mm was inserted
into one nostril 5 mm distal to the anterior nares and connected
though a 1M KCl agar bridge to a Ag/AgCl electrode and
high-impedance digital voltmeter (IsoMillivolt Meter, World
Precision Instruments). The nasal cannula was perfused at 50
.mu.L/min using dual microperfusion pumps serially with PBS, low
chloride PBS (chloride replaced by gluconate), low chloride PBS
containing forskolin (10 .mu.M) without and then with GlyH-101 (10
.mu.M), and then PBS. In some studies GlyH-101 (10 .mu.M) or
4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) (100 .mu.M)
was present in all solutions. The reference electrode was a
PBS-filled 21-gauge needle inserted in the subcutaneous tissue in
the abdomen and connected to a second Ag/AgCl electrode by a 1M KCl
agar bridge.
[0176] Intestinal Fluid Secretion Measurements
[0177] Mice (CD1 strain, 25-35 g) were deprived of food for 24 hr
and anaesthetized with intraperinoneal ketamine (40 mg/kg) and
xylazine (8 mg/kg). Body temperature was maintained at
36-38.degree. C. using a heating pad. Following a small abdominal
incision 3 closed ileal loops (length 20-30 mm) proximal to the
cecum were isolated by sutures. Loops were injected with 100 .mu.l
of PBS or PBS containing cholera toxin (1 .mu.g) without or with
GlyH-101 (2.5 .mu.g). The abdominal incision was closed with suture
and mice were allowed to recover from anesthesia. At 4 hours, the
mice were anesthestized, intestinal loops were removed, and loop
length and weight were measured to quantify net fluid
secretion.
[0178] Cholera Models
[0179] For closed loop studies, mice (CD1 strain, 28-34 g) were
deprived of food for 24 hours and then anaesthetized with
intraperinoneal ketamine (40 mg/kg) and xylazine (8 mg/kg). Body
temperature was maintained at 36-38.degree. C. using a heating pad.
Following a small abdominal incision three closed mid jejunal loops
(length 15-20 mm) were isolated by sutures. Loops were injected
with 100 .mu.l of PBS or PBS containing cholera toxin (1 .mu.g)
without or with test compounds. The abdominal incision was closed
with suture and mice were allowed to recover from anesthesia. At 4
hours the mice were anesthestized, intestinal loops were removed,
and loop length and weight were measured to quantify net fluid
secretion. Mice were sacrificed by an overdose of ketamine and
xylazine. All protocols were approved by the UCSF Committee on
Animal Research.
[0180] Intestinal Absorption Studies
[0181] Absorption studies were performed using mid-jejunal loops
created as described above. Loops were injected separately with
MalH-1, MalH-2, MalH-3, MalH-(PEG).sub.n, and GlyH-(PEG).
containing 10-20 .mu.g of test compounds together with 5 .mu.g
FITC-dextran (40 kDa). After 2 hours loop fluid was withdrawn and
optical absorbance of test compound and FITC were measured
(OD.sub.342/OD.sub.494 nm). Percentage intestinal absorption was
computed assuming zero absorption of FITC-dextran.
[0182] Synthesis of Compounds
[0183] The synthesis of compounds of the invention are exemplified
with but not limited to the following examples. All synthesized
compounds were >98% pure (TLC/HPLC) and were confirmed by mass
and .sup.1H nmr spectrometry.
Synthesis of
N-2-napthalenyl-[(3,5-dibromo-2,4-dihydroxyphenyl)methylene]glycine
hydrazide (GlyH-101) and related glycine hydrazides (GlyH-102-109,
114-127)
[0184] A mixture of 2-napthylamine (compound I, FIG. 3B) (1.43 g,
10 mmol), ethyl iodoacetate (2.14 g, 10 mmol), and sodium acetate
(1.64 g, 20 mmol, dissolved in 2 ml of water) was stirred at
90.degree. C. for 3 hours. The solid material obtained upon cooling
was filtered and recrystallized from hexane to yield 1.5 g ethyl
N-(2-naphthalenyl)glycinate (compound II, FIG. 3B) (yield, 65%, mp
83-84.degree. C.) (Ramamurthy and Bhatt, J. Med. Chem.
32:2421-2426, 1989). A solution of above product (2.29 g, 10 mmol)
in ethanol (10 ml) was refluxed with hydrazine hydrate (0.6 g, 12
mmol) for 10 hours. Solvent and excess reagent were distilled under
vacuum. The product was recrystallized from ethanol to yield 1.8 g
of N-(2-naphthalenyl) glycine hydrazide (compound III, FIG. 3B)
(yield 82%, mp 147-148.degree. C.). A mixture of compound III (2.15
g, 10 mmol) and 3,5-dibromo-2,4-dihydroxybenzaldehyde (3 g, 10
mmol) in ethanol (5 ml) was refluxed for 3 hours. The hydrazone
that crystallized upon cooling was filtered, washed with ethanol,
and recrystallized from ethanol to give 3.8 g (78%) of GlyH-101.
Melting point (mp) >300.degree. C., ms (ES): M/Z 492 (M);
.sup.1H nmr (DMSO-d.sub.6): .delta. 4.1(s, 2H, CH.sub.2),
6.5-7.5(m, 9H, aromatic, NH), 8.5 (s, 1H, CH.dbd.N), 10.4 (s, 1H,
NH--CO), 11.9 (s, 1H, OH), 12.7 (s, 1H, OH). Compounds
GlyH-102-109, GlyH-114-127 and AceH401-404 were synthesized
similarly by condensing appropriate hydrazides with substituted
benzaldehydes.
Synthesis of
N-(6-quinolinyl)-[(3,5-dibromo-2,4-dihydroxyphenyl)methylene]glycine
hydrazide (GlyH-126) and related quinolinyl-glycine hydrazides
[0185] To a stirred solution of 6-aminoquinoline (compound IV, FIG.
3B) (0.72 g, 5 mmol) in acetonitrile (20 ml) was added 33% aqueous
glyoxylic acid (1.85 g, 20 mmole) solution. A solution of
NaBH.sub.3CN (0.64 g, 10.2 mmol) in acetonitrile (20 ml) was then
added at 3.degree. C. over 20 minutes and the reaction mixture was
warmed to room temperature and stirred for 48 hours. Acetonitrile
was evaporated under vacuum, water (20 ml) was added to the
residue, the solution was alkalinized to pH 9.5, and unreacted
amine was extracted with ether. Concentrated HCl (25 ml) was added
to the aqueous solution and the mixture was stirred at 25.degree.
C. for 1 hour. Solvent was evaporated under vacuum. The resultant
residue of N-(6-quinolinyl)glycine was dissolved in dry ethanol (50
ml) saturated with dry HCl, stirred overnight and then refluxed for
3 hours. Ethanol was evaporated, the ester hydrochloride was
suspended in dry ether, and ammonia gas was bubbled. The ammonium
chloride was filtered and ether was removed by evaporation to give
ethyl N-(6-quinolinyl)glycinate (0.5 g, 87%, mp 122-123.degree.
C.). N-(6-quinolinyl)glycine hydrazide (compound VI, FIG. 3B),
synthesized by hydrazinolysis of the above ester, was reacted with
3,5-dibromo-2,4-dihydroxybenzaldehyde to give GlyH-126. Similar
procedures were used for synthesis of GlyH-127.
Synthesis of Oxamic hydrazides (OxaH-110-113)
[0186] The oxamic hydrazides were synthesized by heating a mixture
of 2-napthaleneamine with diethyl oxalate in toluene. The resultant
N-substituted oxamic acid ethyl ester was treated with hydrazine
hydrate followed by condensation with substituted benzaldehydes to
yield compounds OxaH-110-113.
Synthesis of
3,5-dibromo-4-hydroxy-[2-(2-napthalenamine)aceto]benzoic acid
hydrazide (GlyH-202) and related GlyH-201 and Oxa-203-204
[0187] N-(2-naphthalenyl)glycine hydrazide (compound III, FIG. 3B)
(2.15 g, 10 mmole) was reacted with 3,5-dibromo-4-hydroxybenzoyl
chloride (3.14 g, 10 mmole) (Gilbert et. al., Eur. J. Med. Chem.,
17:581-588, 1982) in pyridine (10 ml) for 5 hours. Pyridine was
removed and the residue was diluted with water. The product was
recrystallized from ethanol to yield a gray powder 3.8 g (77%), mp
>300.degree. C. Compounds GlyH-201 and Oxa-203-204 were
synthesized by similar procedure.
Synthesis of
N-2-napthalenyl-[(3,5-dibromo-2,4-dihydroxyphenyl)methyl]glycine
hydrazide (GlyH-301) and related glycine hydrazides (GlyH-302,
OxaH-303-304)
[0188] A mixture of GlyH-101 (1.5 g, 3 mmole), hydrazine hydrate
(0.15 ml, 3 mmol) and Pd/C catalyst (0.1 g, 10% Pd) in 5 ml of
dimethylformamide was refluxed for 6-8 hours (Verma et al., Arch.
Pharm. 317:890-894, 1984). The reaction mixture was filtered,
diluted with cold water, and extracted with diethyl ether. GlyH-301
was crystallized from ether to yield 0.9 g (60%), mp
258-260.degree. C. Compounds GlyH-302 and OxaH-303-304 were
prepared similarly.
[0189] Synthesis of Analogs
[0190] The synthesis of analog of the compounds of the invention
are exemplified with but not limited to the following examples. All
synthesized compounds were >98% pure (TLC/HPLC) and were
confirmed by mass and .sup.1H nmr spectrometry. .sup.1H NMR spectra
were obtained in CDCl.sub.3 or DMSO-d.sub.6 using a 400 MHz Varian
Spectrometer referenced to CDCl.sub.3 or DMSO. Mass spectrometry
was done using a Waters LCMS system (Alliance HT 2790+ZQ, HPLC:
Waters model 2960, Milford, Mass.). Flash chromatography was
performed using EM silica gel (230-400 mesh), and thin layer
chromatography was done on Merk silica gel 60 F254 plates.
Synthesis of Diethyl-(2-naphthalenylamino)-propanedioate (compound
2, FIG. 9)
[0191] A mixture of 2-naphthylamine (compound 1, FIG. 9) (10 mmol),
diethyl bromomaloante (10 mmol), and sodium acetate (1.64 g, 20
mmol, dissolved in 4 ml of water) was stirred at 90.degree. C. for
8 hours. The black solid material obtained upon cooling was
filtered and recrystallized from hexane to yield 2.5 g of 2 (yield
84%); mp, 189-190.degree. C.; ms (ES.sup.+): M/Z 302 (M+1).sup.+;
.sup.1H nmr (DMSO-d.sub.6): .delta. 1.17 (t, 6H, 7.33 Hz), 4.17 (q,
4H, 7.33), 5.10 (d, 1H, 8.79 Hz), 6.54 (d, 1H, 8.79 Hz), 6.75 (d,
1H, 2.20 Hz), 7.13 (t, 1H, 7.32 Hz), 7.19 (dd, 1H, 2.19, 8.79 Hz),
7.28 (t, 1H, 8.06 Hz), 7.51 (d, 1H, 8.42 Hz), 7.61 (t, 2H, 8.79
Hz).
Synthesis of (2-naphthalenylamino)-propanedioic acid dihydrazide
(Compound 3, FIG. 9).
[0192] A solution of compound 2 (FIG. 9) (10 mmol) in ethanol (10
ml) was refluxed with hydrazine hydrate (12 mmol) for 10 hours.
Solvent and excess reagent were distilled under vacuum. The product
was recrystallized from ethanol to give 2.5 g of compound 3 (92%);
mp 268-270.degree. C.; ms (ES.sup.+): M/Z 274 (M+1).sup.+; .sup.1H
nmr (DMSO-d.sub.6): .delta. 4.29 (d, 4H, 4.03), 4.56 (d, 1H, 8.79
Hz), 6.03 (d, 1H, 8.79 Hz), 6.62 (d, 1H, 1.46 Hz), 7.09 (m, 2H),
7.28 (t, 1H, 8.05 Hz), 7.50 (d, 1H, 8.06 Hz), 7.61 (m, 2H), 9.22
(s, 2H).
Synthesis of 2-naphthalenylamino-bis
[(3,5-dibromo-2,4-dihydroxyphenyl)methylene]propanedioic acid
dihydrazide (MalH-1)
[0193] A mixture of compound 3 (FIG. 9) (10 mmol) and
3,5-dibromo-2,4-dihydroxybenzaldehyde (20 mmol) in ethanol (5 ml)
was refluxed for 3 hours. The hydrazone that crystallized upon
cooling was filtered, washed with ethanol, and purified by column
chromatography (silica gel EtOAc:hexane 2:3) to give 3.2 g of
compound 4 (58%) as an off-white solid; mp 246-248.degree. C.; ms
(ES.sup.+): M/Z 830 (M+1).sup.+; .sup.1H nmr (DMSO-d.sub.6):
.delta. 4.91, 5.48 (d, 1H, 7.69, 9.15 Hz), 6.62 (d, 1H, 7.32 Hz,),
6.73, 6.84 (s, 1H), 7.13-7.32 (m, 3H), 7.57 (d, 1H, 8.06 Hz),
7.61-7.70 (m, 3H), 7.80, 7.90 (s, 1H), 8.15, 8.37 (s, 2H),
10.10-10.40 (broad s, 2H), 11.72, 11.90 (s, 2H), 12.22, 12. 53 (s,
2H).
Synthesis of
2-naphthalenylamino-[(3,5-dibromo-2,4-dihydroxyphenyl)methylene][(2,4-dis-
odium-disulfophenyl)methylene]propanedioic acid dihydrazide
(MalH-2)
[0194] A mixture of dihydrazide 4 (FIG. 9) (5 mmol) and
2,4-disodium-disulfobenzaldehyde (5 mmol) in DMF (5 ml) was
refluxed for 4 hours. The reaction mixture, upon cooling, was added
dropwise to a stirred solution of EtOAc:EtOH (1:1), filtered,
washed with ethanol, and further purified by column chromatography
(silica gel EtOAc:hexane 2:3) to give 2.3 g of compound MalH-2
(58%) as an off-white solid; mp >300.degree. C.; ms (ES.sup.+):
M/Z 800 (M+1).sup.+; .sup.1H nmr (DMSO-d.sub.6): .delta. 4.95, 5.44
(d, 1H, 7.63, 9.16 Hz), 6.64 (d, 1H, 7.31 Hz), 6.70, 6.81 (s, 1H),
7.12-7.44 (m, 4H), 7.59 (d, 1H, 8.00 Hz), 7.64-7.76 (m, 4H), 7.80,
7.90 (s, 1H), 8.25, 8.37 (s, 2H), 10.36 (broad s, 1H), 11.62, 11.82
(s, 1H), 12.11, 12. 43 (s, 2H).
[0195]
2-naphthalenylamino-[(3,5-dibromo-2,4-dihydroxyphenyl)methylene][3--
(4-sodium-sulfophenyl)-thioureido]propanedioic acid dihydrazide
(MalH-3) and
2-naphthalenylamino-[(3,5-dibromo-2,4-dihydroxyphenyl)methylene][3-[4-
-(3-(PEG).sub.n-thioureido)phenyl)-thioureido]propanedioic acid
dihydrazide (MalH-(PEG).sub.n) were synthesized following similar
reaction conditions used for MalH-2 except that
4-sodium-sulfophenylisothiocyanate and compound 6 (FIG. 10) were
used respectively, in place of
2,4-disodium-disulfobenzaldehyde.
[0196] MalH-3: mp>300.degree. C.; ms (ES): M/Z 765 (M-1).sup.+;
.sup.1H nmr (DMSO-d.sub.6): .delta. 4.90, 5.31 (d, 1H, 7.61, 9.12
Hz), 6.54 (d, 1H, 7.31 Hz), 6.70, 6.81 (s, 1H), 7.12-7.44 (m, 4H),
7.59 (d, 1H, 8.00 Hz), 7.64-7.76 (m, 4H), 7.90 (d, 2H), 8.25, 8.37
(s, 1H), 9.88 (s, 1H) 10.05 (s, 1H, CSNH), 10.36 (s, 1H, OH),
11.11, 11.43 (s, 2H, CONH), 11.62, 11.82 (s, 1H, OH).
[0197] MalH-(PEG).sub.1: mp>300.degree. C.; ms (ES.sup.+): M/Z
849 (M+1).sup.+; .sup.1H nmr (DMSO-d.sub.6): .delta. 3.70-4.37 (m,
8H), 4.81, 5.01 (d, 1H, 7.51, 9.13 Hz), 5.27 (s, 1H), 6.60 (d, 1H,
7.31 Hz), 6.75 (s, 1H), 7.19-7.38 (m, 4H), 7.59 (d, 2H, 8.00 Hz),
7.64-7.76 (m, 3H), 7.90 (d, 2H, 8.00 Hz), 8.21, 8.30 (s, 1H), 9.76
(s, 2H) 9.83 (s, 1H), 10.01 (s, 1H), 10.36 (s, 1H), 11.20, 11.51
(s, 2H), 11.54, 11.62 (s, 1H).
Synthesis of
2-[3-(4-isothiocyanato-phenyl)-thioureido]ethyl-(PEG).sub.1
(compound 6a, FIG. 10).
[0198] To a solution of 1,4-phenylene diisothiocyanate (1 mmol, 2
mL DMF) was added 2-aminoethoxyethanol (0.3 mmol, 2 mL DMF) over 30
minutes. After stirring for additional 30 minutes, the DMF was
distilled off and product was purified by column chromatography on
silica gel using as solvent n-hexane:AcOEt (1:1). Fractions were
evaporated to give 58 mg of compound 2 in (65%); ms (ES.sup.+): M/Z
298 (M+1).sup.+; .sup.1H nmr (DMSO-d.sub.6): .delta. 2.84 (t, 2H,
6.46 Hz), 2.95 (t, 2H, 6.31 Hz), 3.12 (t, 2H, 6.38 Hz), 3.58 (q,
2H, 5.98 Hz), 5.63 (s, 1H), 7.15 (d, 2H, 8.62 Hz), 7.44 (d, 2H,
8.62 Hz), 7.97 (s, 2H, NH). Similarly, compound 6b was synthesized
using appropriate amino-PEG; yield, 58%; ms (ES.sup.+): M/Z 736
(+/-44, 88, 132, 176) (M+1).sup.+; .sup.1H nmr (DMSO-d.sub.6):
.delta. 3.24 (s, 3H), 3.31-3.82 (m), 7.21 (d, 2H, 8.60 Hz), 7.47
(d, 2H, 8.60 Hz), 7.92 (s, 2H).
Synthesis of 2-(2-naphthalenylamino)-4-hydroxy-butyric acid
hydrazide (compound 7, FIG. 11)
[0199] This compound was synthesized following similar reaction
conditions used for compounds 2 and 3. 89%; mp 258-260.degree. C.;
ms (ES.sup.+): M/Z 260 (M+1).sup.+; .sup.1H nmr (DMSO-d.sub.6):
.delta. 1.79 (m, 2H) 3.46 (q, 2H) 3.98 (s, 1H), 4.17 (d, 2H) 4.52
(t, 1H), 5.94-5.96 (s, 1H), 6.68 (s, 1H), 6.98 (dd, 1H), 7.05 (t,
1H), 7.24 (t, 1H), 7.46 (d, 1H), 7.52-7.60 (m, 2H) 9.17 (s,
1H).
Synthesis of [2-(2-naphthalenylamino)-4-hydroxy]butyric
acid-2-[(1,1-dimethylethoxy)carbonyl]hydrazide (compound 8, FIG.
11)
[0200] To a solution of hydrazide 7 (10 mM) in THF (10 ml) was
added (BOC).sub.2O (20 mM) and heated under reflux for 5 hours. The
solvent was removed, and the residue was purified by column
chromatography on silica gel. Elution with dichloromethane gave 3.1
g of compound 8 (86%) as a white solid; mp 235-237.degree. C.; ms
(ES.sup.+): M/Z 360 (M+1).sup.+; .sup.1H nmr (DMSO-d.sub.6):
.delta. 1.33 (s, 9H), 1.92 (m, 2H), 3.52 (q, 2H), 4.01 (q, 1H),
4.52 (t, 1H), 6.00 (d, 1H), 6.70 (s, 1H), 6.97 (dd, 1H), 7.06 (t,
1H), 7.25 (t, 1H), 7.45 (d, 1H), 7.52-7.59 (m, 2H), 8.73 (s, 1H),
9.77 (s, 1H).
Synthesis of [2-(2-naphthalenylamino)-4-(p-tosyl)lbutyric acid-2-8
(1,1-dimethylethoxy)carbonyl]hydrazide (compound 9, FIG. 11)
[0201] To a solution of hydrazide 7 (1 mmol) in pyridine (5 ml) was
added p-TsCl (1 mmol) in three portions 30 min apart (-15.degree.
C.). The reaction mixture was stirred for 8 hours at -15.degree.
C., allowed to warm to room temperature, diluted with 1N HCl, and
extracted three times with EtOAc. The combined organic extract was
washed with brine, dried with Na.sub.2SO.sub.4 and evaporated to
dryness to give 374 mg of compound 9 (73%) as a pale yellow oil,
used without further purification for next step; ms (ES.sup.+): M/Z
514 (M+1).sup.+.
Synthesis of [2-(2-naphthalenylamino)-4-(PEG-amino)]butyric
acid-2-[(1,1-dimethylethoxy)carbonyl]hydrazide (compound 10, FIG.
11)
[0202] A solution of 2-aminoethoxyethanol (1 mM) and compound 9 (1
mM) in DMF (2 ml) was stirred at 80.degree. C. for 24 hours. The
DMF was evaporated in vacuo, and the residue was dissolved in
minimum quantity of EtOAc and added to a stirred solution of
Et.sub.2O. The white powder-like precipitate was filtered and
washed with Et.sub.2O to give 170 mg of compound 9 (38%) as a
yellow sticky mass; ms (ES.sup.+): M/Z 447 (M+1).sup.+; .sup.1H nmr
(DMSO-d.sub.6): .delta. 1.35 (s, 9H), 1.71 (m, 2H) 3.40-3.51 (m,
4H), 3.57 (t, 2H), 3.68-3.79 (m, 5H, CH2), 3.93 (s, 1H), 4.52 (t,
1H), 6.04, 6.16 (s, 1H), 6.67 (s, 1H), 6.93 (dd, 1H), 7.03 (t, 1H),
7.32 (t, 1H), 7.45 (d, 1H), 7.50-7.62 (m, 2H), 9.27 (s, 1H), 9.89
(s, 1H).
Synthesis of [2-(2-naphthalenylamino)-4-(PEG-amino)]butyric acid
hydrazide (compound 11, FIG. 11)
[0203] Hydrazide 10 (1 mM) was dissolved in a minimal amount of
trifluoroacetic acid:CH.sub.2Cl.sub.2 (1:1) and stirred at room
temperature for 30 minutes. The reaction mixture was diluted with
saturated aqueous NaHCO.sub.3 and extracted with CH.sub.2Cl.sub.2.
The combined organic layer was washed successively with water and
brine, dried (Na.sub.2SO.sub.4), and concentrated in vacuo to yield
253 mg of compound 11 (73%) as yellow semisolid; ms (ES.sup.+): M/Z
347 (M+1).sup.30 ; .sup.1H nmr (DMSO-d.sub.6): .sup.1H nmr
(DMSO-d.sub.6): .sup.1H nmr (DMSO-d.sub.6): .delta. 1.71 (m, 2H)
3.40-3.51 (m, 4H), 3.57 (t, 2H), 3.68-3.79 (m, 5H, CH2), 3.93 (s,
1H), 4.26 (d, 2H) 4.52 (t, 1H), 6.02, 6.21 (s, 1H), 6.71 (s, 1H),
6.85 (dd, 1H), 7.10 (t, 1H), 7.34 (t, 1H), 7.51 (d, 1H), 7.53-7.76
(m, 2H), 9.27 (s, 1H).
Synthesis of [2-(2-naphthalenylamino)-4-(PEG-amino)]butyric
acid-2-[3,5-dibromo-2,4-dihydroxyphenyl)methylene]hydrazide
(compound 12, FIG. 11)
[0204] A mixture of compound 11 (1 mmol) and
3,5-dibromo-2,4-dihydroxybenzaldehyde (1 mmol) in ethanol (2 ml)
was refluxed for 3 hours. The reaction mixture was concentrated and
added to a stirred solution of Et.sub.2O, and the precipitated
hydrazone was filtered and washed with Et.sub.2O to yield 362 mg of
compound 12 (58%); ms (ES.sup.+): M/Z 625 (M+1).sup.30 ; .sup.1H
nmr (DMSO-d.sub.6): .sup.1H nmr (DMSO-d.sub.6): .delta. 1.75 (m,
2H) 3.43-3.48 (m, 4H), 3.59 (t, 2H), 3.72-3.81 (m, 5H, CH2), 3.97
(s, 1H), 4.59 (t, 1H), 6.12, 6.26 (s, 1H), 6.75 (s, 1H), 6.85-6.96
(m, 2H), 7.15-7.51 (t, 3H), 7.53-7.76 (m, 2H), 8.87 (s, 1H), 9.27
(s, 1H), 10.68 (s, 1H), 11.92 (s, 1H).
Example 1
Discovery of Novel Classes of CFTR Inhibitors
[0205] A collection of 100,000 small, drug-like compounds was
screened to identify new CFTR inhibitors. As diagrammed in FIG. 1A,
compounds were screened at 25 .mu.M in a cell-based assay of iodide
influx after CFTR activation by an agonist mixture containing
forskolin, IBMX and apigenin. Initial rates of iodide influx were
computed from the kinetics of fluorescence decrease following
chloride replacement by iodide. Four compounds (FIG. 1B) reducing
iodide influx by greater than 50% were identified, which were not
related structurally to known CFTR activators or inhibitors. Twelve
compounds reduced iodide influx by 25-50%, most of which were
related structurally to the compounds in FIG. 1B or to the
thiazolidinones.
[0206] To select inhibitor(s) for further evaluation, dose-response
measurements were done for the compounds in FIG. 1B, and CFTR
inhibition was confirmed electrophysiologically by short-circuit
current analysis. K.sub.i was .about.7, 5, 5 and 5 .mu.M for
compounds a-d, respectively. FIG. 1C shows representative
fluorescence and FIG. 1D shows a representation of short-circuit
current data for compound d. 100-250 commercially available analogs
of each compound class were screened to determine whether active
structural analogs exist, an important prerequisite for follow-up
compound optimization by synthesis of targeted analogs. Whereas few
or no active analogs of compounds a, b and c were found, initial
screening of 285 analogs of compound d (substituted glycine
hydrazides, GlyH) revealed 34 analogs that inhibited CFTR-mediated
iodide influx by >25% at 25 .mu.M.
[0207] The structure-activity analysis and characterization of
inhibition mechanism, as well as the time course of action and
reversibility of action of synthesized GlyH analogs was determined.
In addition, the effectiveness of the analogs for different CFTR
activating mechanisms was also analyzed. FIG. 2A shows prompt
inhibition of iodide influx in the fluorescence and short-circuit
current assays upon GlyH-101 addition. Interestingly .about.50% of
the inhibition occurred within the .about.1 second addition/mixing
time, with further inhibition over .about.1 minute. FIG. 2B
indicates complete reversal of inhibition after GlyH-101 washout
with >75% reversal over 5 minutes. FIG. 2C shows effective CFTR
inhibition by GlyH-101 after activation by different types of
agonists, including potent direct activators of CFTR that do not
elevate cytosolic cAMP or inhibit phosphatase activity
(CFTR.sub.act-01, 08, and 10; Ma et al., J. Clin. Invest.
110:1651-1658, 2002).
Example 2
Chemistry and Structure-Activity Relationships of Glycin
Hydrazides
[0208] The GlyH-101 structure was modified systematically to
establish structure-activity relationships and to identify analogs
with improved CFTR inhibitory activity. FIG. 3A shows the various
classes of structural analogues that were synthesized and tested
for CFTR inhibition. Structural modifications were performed on
both ends of the glycine hydrazide backbone (FIG. 3A, left, top and
middle). Replacing the glycine methylene group by a carbonyl group
and replacing nitrogen by oxygen generated oxamic acid hydrazides
(OxaH, right, top) and acetic acid hydrazides (AceH, right,
middle), respectively. The hydrazone group modification produced
two important series of compounds (middle, bottom and right,
bottom). Also shown are compounds containing an additional methyl
group at the hydrazone bond (top, middle), and containing a
6-qunolinyl group replacing the naphthalenyl group (left,
bottom).
[0209] FIG. 3B shows the reaction schemes developed for synthesis
of the different classes of glycine hydrazide analogs. Synthesis of
GlyH-101 involves reaction of 2-naphthalemine with ethyl
iodoacetate followed by reactions with hydrazine hydrate and
2,4-dihydroxy-3,5-dibromobenzaldehyde. A similar procedure was used
for most of the remaining glycine hydrazide derivatives (listed in
Table 1). The heteroaromatic analogues containing a 6-qunolinium
group required different synthetic route in which 6-aminoquinoline
was condensed with glyoxalic acid, and reduced using sodium
cyanoborohydride (yielding N-6-quinolineglycine, Ramamurthy et al.,
1989), which was further esterified and reacted with hydrazine
hydrate and benzaldehyde. The oxamic acid hydrazides were
synthesized starting from aromatic amines and diethyl oxalate.
[0210] Modifications were made initially on the N-aryl (R.sub.1)
and benzaldehyde (R.sub.2) positions (see Tables 1-4 for R- and
X-group definitions and CFTR inhibition). Good CFTR inhibition was
found when R.sub.2 contained 3,5-dibromo and at least one hydroxyl
substituent at the 4-position (GlyH-102, 105, 114); addition of a
second hydroxyl group increased inhibition (GlyH-101, 104,
115-116). Inhibition was reduced when R.sub.2 contained
4-bromophenyl or 4-carboxyphenyl substituents (GlyH-120-121). In
addition, the 4-hydroxyl group in GlyH-101 was important for
inhibition since its 4-methoxy analogue GlyH-103 had little
activity. Similar structure-activity results were found for
GlyH-115 and GlyH-122.
[0211] R.sub.1 group modifications were carried out, maintaining
R.sub.2 as 2,4-dihydroxy-3,5-dibromophenyl and
3,5-dibromo-4-hydroxyphenyl. Analogues with R.sub.1 as
2-naphthalenyl were much better inhibitors than R.sub.1 as
4-chlorophenyl or 4-methylphenyl. Replacement of the 2-napthalenyl
of GlyH-101 by 1-napthalenyl (GlyH-104) decreased inhibition
activity ten-fold, supporting the requirement of the 2-naphthalenyl
substituent. GlyH-124-125, containing a 2-anthacenyl group, were
less active. Replacement of 2-naphthalenyl group in GlyH-101 and
GlyH-102 by more polar heteroaromatic rings such as 6-qunolinyl
gave compound with little activity (Gly-126-127), as did the
2-naphthoxy analogues AceH-401 and AceH-402.
[0212] X was next modified (replacing methylene), keeping
2-naphthalenyl as R.sub.1 and dibromo-dihydroxyphenyl as R.sub.2.
Introduction of a carbonyl group in GlyH-101 and GlyH-102 at X,
giving OxaH-110 and OxaH-111, gave two-three fold greater
inhibitory potency. FIG. 3C shows short-circuit current analysis of
CFTR inhibition for the most active analog OxaH-110, with an
apparent K.sub.i.about.2 .mu.M. Replacement of CH.sub.2 by
CHCH.sub.3 (GlyH-106-107) also improved CFTR inhibition. In another
structural variation, addition of a methyl group at R.sub.3 to
GlyH-102, yielding GlyH-109, gave improved CFTR inhibition.
Modification of the N.dbd.C group in GlyH-101 and GlyH-102 to
NH--CH.sub.2 in GlyH-301 and GlyH-302, or to NH--CO in GlyH-201 and
GlyH-202, reduced CFTR inhibitory potency.
TABLE-US-00001 TABLE 1 Structure-activity relationships of Group 1
hydrazide-containing compounds Group I (I) ##STR00028## %
inhibition Compound R.sub.1 X R.sub.2 R.sub.3 K.sub.i (.mu.M) at 50
.mu.M GlyH-101 2-naphthalenyl CH.sub.2 3,5-di-Br-2,4-di-OH--Ph H 5
95 GlyH-102 2-naphthalenyl CH.sub.2 3,5-di-Br-4-OH--Ph H 5 98
GlyH-103 2-naphthalenyl CH.sub.2 3,5-di-Br-2-OH-4-OMe--Ph H 20 56
GlyH-104 1-naphthalenyl CH.sub.2 3,5-di-Br-2,4-di-OH--Ph H 12 86
GlyH-105 1-naphthalenyl CH.sub.2 3,5-di-Br-4-OH--Ph H 15 87
GlyH-106 2-naphthalenyl CHCH.sub.3 3,5-di-Br-2,4-di-OH--Ph H 6 91
GlyH-107 2-naphthalenyl CHCH.sub.3 3,5-di-Br-4-OH--Ph H 10 80
GlyH-108 2-naphthalenyl CH.sub.2 3,5-di-Br-2,4-di-OH--Ph CH.sub.3
10 81 GlyH-109 2-naphthalenyl CH.sub.2 3,5-di-Br-4-OH--Ph CH.sub.3
2.5 100 OxaH-110 2-naphthalenyl CO 3,5-di-Br-2,4-di-OH--Ph H 2 86
OxaH-111 2-naphthalenyl CO 3,5-di-Br-4-OH--Ph H 2.5 52 OxaH-112
2-naphthalenyl CO 3,5-di-Br-2,4-di-OH Ph CH.sub.3 3 95 OxaH-113
2-naphthalenyl CO 3,5-di-Br-4-OH--Ph CH.sub.3 3 90 GlyH-114
4-Cl--Ph CH.sub.2 3,5-di-Br-4-OH--Ph H 5 95 GlyH-115 4-Cl--Ph
CH.sub.2 3,5-di-Br-2,4-di-OH Ph H 5 91 GlyH-116 4-Me--Ph CH.sub.2
3,5-di-Br-2,4-di-OH Ph H 10 79 GlyH-117 2-Me--Ph CH.sub.2
3,5-di-Br-2,4-di-OH Ph H GlyH-118 1-naphthalenyl CH.sub.2
3-Br-4-OH--Ph H GlyH-119 2-naphthalenyl CH.sub.2 2,4-di-OH--Ph H
GlyH-120 2-naphthalenyl CH.sub.2 4-Br--Ph H GlyH-121 2-naphthalenyl
CH.sub.2 4-carboxy-Ph H GlyH-122 4-Cl--Ph CH.sub.2
3,5-di-Br-2-OH-4-OMe--Ph H GlyH-123 4-Cl--Ph CH.sub.2 2,4-di-OH--Ph
H GlyH-124 2-anthracenyl CH.sub.2 3,5-di-Br-2,4-di-OH Ph H GlyH-125
2-anthracenyl CH.sub.2 3,5-di-Br-4-OH--Ph H GlyH-126 6-quinolinyl
CH.sub.2 3,5-di-Br-2,4-di-OH Ph H GlyH-127 6-quinolinyl CH.sub.2
3,5-di-Br-4-OH--Ph H
TABLE-US-00002 TABLE 2 Structure-activity relationships of Group 2
hydrazide-containing compounds Group II (II) ##STR00029## % Com-
Inhibition pound R.sub.1 X R.sub.2 K.sub.i (.mu.M) at 50 .mu.M
GlyH-201 2-naphthalenyl CH.sub.2 3,5-di- 20 65 Br-2,4-di-OH Ph
GlyH-202 2-naphthalenyl CH.sub.2 3,5-di- 22 57 Br-4-OH--Ph OxaH-203
2-naphthalenyl CO 3,5-di- >50 Br-2,4-di-OH Ph OxaH-204
2-naphthalenyl CO 3,5-di- >50 Br-4-OH--Ph
TABLE-US-00003 TABLE 3 Structure-activity relationships of Group 3
hydrazide-containing compounds Group III (III) ##STR00030## % Com-
Inhibition pound R.sub.1 X R.sub.2 K.sub.i (.mu.M) at 50 .mu.M
GlyH-301 2-naphthalenyl CH.sub.2 3,5-di-Br- ~50 50 2,4-di-OH Ph
GlyH-302 2-naphthalenyl CH.sub.2 3,5-di-Br- ~50 55 4-OH--Ph
OxaH-303 2-naphthalenyl CO 3,5-di-Br- 10 70 2,4-di-OH Ph OxaH-304
2-naphthalenyl CO 3,5-di-Br- 12 78 4-OH--Ph
TABLE-US-00004 TABLE 4 Structure-activity relationships of Group 4
hydrazide-containing compounds Group IV (IV) ##STR00031## %
Inhibition Compound R.sub.1 R.sub.2 K.sub.i (.mu.M) at 50 .mu.M
AceH-401 2-naphthoxy 3,5-di-Br-2,4-di-OH Ph 21 84 AceH-402
2-naphthoxy 3,5-di-Br-4-OH--Ph 17 86 AceH-403 4-Me--Ph
3,5-di-Br-2,4-di-OH Ph 10 54 AceH-404 4-Me--Ph 3,5-di-Br-4-OH--Ph
15 63
(Tables 1-4: K.sub.1 indicates the concentration giving 50%
inhibition of CFTR Cl.sup.- conductance by short-circuit current
analysis on CFTR-expressing FRT cells.)
Example 3
Patch-Clamp Analysis of CFTR Inhibition Mechanism
[0213] The mechanism of CFTR block by GlyH-101 was studied using
the whole-cell configuration of the patch-clamp technique. After
maximal activation of CFTR in stably transfected FRT cells by 5
.mu.M forskolin, current-voltage relationships were measured at
GlyH-101 concentrations from 0 to 50 .mu.M. Representative original
current recordings are shown in FIG. 4A. In the absence of
inhibitor (left panel), membrane current increased linearly with
voltage and did not show relaxation phenomena, as expected for pure
CFTR Cl.sup.- currents. Extracellular perfusion with 10 .mu.M
GlyH-101 produced an immediate reduction in current that was
strongly dependent on membrane potential (FIG. 4A, right panel). At
more positive membrane potentials outward positive currents
(Cl.sup.- movement into the cell) were reduced compared to inward
currents. FIG. 4B shows current-voltage relationships for GlyH-101
concentrations of 0 (control), 10 and 30 .mu.M, and after washout
of 30 .mu.M GlyH-101 (recovery). Data for the thiazolidinone
3-[(3-trifluoromethyl)phenyl]-5-[(4-carboxyphenyl)methylene]-2-thioxo-4-t-
hiazolidinone (referred to herein as CFTR.sub.inh-172) (5 .mu.M) is
shown for comparison. The current-voltage relationship was linear
in the absence of inhibitor, after GlyH-101 washout, and after
inhibition by CFTR.sub.inh-172, whereas GlyH-101 inhibition at
submaximal concentrations produced inward rectification. FIG. 4C
summarizes percentage CFTR current block as a function of GlyH-101
concentration at different membrane voltages. GlyH-101 inhibitory
potency was reduced at more negative voltages, with apparent
K.sub.1 of 1.4, 3.8, 5.0, and 5.6 .mu.M for voltages of +60, +20,
-20 and -60 mV, respectively (Hill coefficients, n.sub.H=0.5, 0.7,
1.3, 1.8).
[0214] Cell-attached patch-clamp experiments were carried out to
investigate the mechanism of GlyH-101 block of CFTR Cl.sup.-
current at the single-channel level. FIG. 4D shows a GlyH-101
concentration-dependent reduction in CFTR channel activity without
a change in single channel conductance. Mean channel open time was
remarkably reduced with the appearance of brief closures during the
open bursts whose frequency increased with GlyH-101 concentration.
In the absence of the inhibitor, mean channel open time was
264.+-.11 ms (SE, n=10). Mean channel open times at +60 mV at 0.4,
1, and 5 .mu.M GlyH-101 were reduced to 181.+-.29, 38.+-.5, and
13.+-.2 ms, respectively (n=5; p<0.01 for all concentrations vs.
control).
[0215] The kinetic and electrophysiological data indicate that
hydrazide-containing compounds block CFTR Cl.sup.- conductance by
occluding the CFTR anion pore at or near the external membrane
surface. Unlike all other CFTR inhibitors, including the
thiazolidinone CFTR.sub.inh-172, CFTR block by the
hydrazide-containing GlyH-101 produced inwardly rectifying CFTR
Cl.sup.- currents. Compared to CFTR.sub.inh-172, GlyH-101 is
.about.50-fold more water soluble and rapidly acting/reversible
when added to or removed from the extracellular solution,
consistent with its action at the external-facing surface of CFTR.
Structure-activity analysis of a series of targeted
hydrazide-containing analogs defined the structural determinants
for CFTR inhibition and provided analogs with greater CFTR
inhibitory potency, the best being OxaH-110 with Ki.about.2 .mu.M.
Although the most potent thiazolidinone CFTR.sub.inh-172 has Ki of
0.2-0.3 .mu.M in permeabilized cell preparations, its Ki is 2-5
.mu.M in most intact epithelial cells because of the interior
negative membrane potential which reduces its concentration in
cytoplasm. Thus, the hydrazide-containing compounds are as or more
potent than the thiazolidinones, and like the thiazolidinones they
block CFTR in nasal and intestinal epithelia in vivo.
[0216] Patch-clamp studies indicated that CFTR inhibition by
GlyH-101 is sensitive to membrane potential. At sub-maximal
concentrations of GlyH-101 there was marked inward rectification in
the CFTR current-voltage relationship indicating that Cl.sup.- flux
from the extracellular to the intracellular side of the membrane is
more strongly blocked than that in the opposite direction. The
apparent Ki increased approximately four-fold as applied potential
was varied from +60 to -60 mV. Since GlyH-101 is negatively charged
at pH 6-8, the simplest interpretation of these data is that
GlyH-101 inhibition involves direct interaction with the channel
pore at the extracellular side of the membrane. Accordingly,
negative membrane potentials reduce the inhibitory efficacy of the
negatively charged GlyH-101 by electrostatic repulsion, which
drives the compound outside of the pore. In contrast, the open
channel blocker glibenclamide, which is thought to act from the
intracellular side of the CFTR pore (Sheppard & Robinson, 1997
J. Physiol., 503:333-346), produces outward rectification of CFTR
current-voltage relationship (Zhou et al., 2002, J. Gen. Physiol.,
120:647-662).
[0217] Analysis of GlyH-101 dose-response data also revealed an
increase in apparent Hill coefficient at more negative membrane
potentials, demonstrating the possibility of more than one
inhibitor binding site within the pore and/or cooperative
interaction between inhibitor molecules, as reported previously for
other ion channels (Pottosin et al., 1999, Biophys. J.,
77:1973-1979; Brock et al., 2001, J. Gen. Physiol. 118:113-134). In
support of the hypothesis that GlyH-101 is an open channel blocker,
cell-attached patch-clamp experiments revealed fast closures within
bursts of channel openings. The frequency of fast closures
increased with GlyH-101 concentration, producing a reduction in
mean channel open time as found for glibenclamide (Sheppard &
Robinson, 1997 J. Physiol., 503:333-346). The appearance of closure
events on the millisecond time scale classifies GlyH-101 as an
"intermediate"-type channel blocker, similar to glibenclamide; in
contrast, "fast" blockers reduce apparent single channel
conductance, and "slow" blockers that cause closures of many
seconds duration. In whole-cell patch-clamp and short-circuit
current experiments, CFTR Cl.sup.- conductance was fully inhibited
at high concentrations (.gtoreq.30 .mu.M) of GlyH-101. Together
these results demonstrate that the GlyH-101 inhibition mechanism
involves direct CFTR pore occlusion at a site at or near the
extracellular-facing pore surface.
Example 4
Physical Properties of Glycine Hydrazides
[0218] Interpretation of the voltage-dependent inhibition mechanism
requires knowledge of the GlyH-101 ionic species that interacts
with CFTR. Short-circuit studies indicated that the K.sub.i for
GlyH-101 inhibition of CFTR Cl.sup.- current was independent of pH
in the range 6-8 (not shown), where the compound is highly water
soluble (0.8-1.3 mM in water, 22.degree. C.). The possible titrable
groups on GlyH-101 in the pH range 3-10 include the secondary
glycinyl amine and the resorcinolic hydroxyls. Spectrophotometric
titration of GlyH-101 indicated at least two
protonation/deprotonations at pH between 4 and 9 (FIG. 5A, top
panel). To assign pKa values, GlyH-101 analogs that lacked one or
more titrable groups were synthesized. Removal of the secondary
amine (AccH-403) had little effect on the titration, with only a
minor left-shift of the ascending portion of the curve, suggesting
a pKa of .about.5.5 for titration of the first phenolic hydroxyl.
Removal of one ortho hydroxyl (GlyH-102) eliminated the descending
portion of the curve, confirming the pKa of .about.5.5 for the
first para hydroxyl and .about.8.5 for the second ortho hydroxyl.
Removal of the aromatic ring containing the resorcinolic hydroxyls
(ethyl N-(2-napthalenyl)glycinate, FIG. 5A, bottom panel) indicated
a pKa .about.4.7 for the residual secondary amine. From these data
the deduced equilibria among the ionic forms of GlyH-101 is shown
in FIG. 5B. GlyH-101 exists primarily as a singly charged anion at
pH between 6 and 8.
Example 5
CFTR Inhibition in Mice In Vivo
[0219] Inhibition of CFTR-dependent airway epithelial Cl.sup.-
current in vivo was demonstrated by nasal potential difference (PD)
measurements in mice. Nasal PDs were measured continuously in
response to serial solution exchanges in which amiloride was added
(to block ENaC Na.sup.+ channels) followed by Cl.sup.- replacement
by gluconate (to induce Cl.sup.- dependent hyperpolarization),
forskolin addition (to activate CFTR) and GlyH-101 addition (to
inhibit CFTR). The representative PD recording in FIG. 6A (left
panel) shows hyperpolarizations (more negative PDs) following low
Cl.sup.- and forskolin solutions, representing CFTR-independent and
dependent Cl.sup.- currents, respectively. Topical application of
GlyH-101 in the perfusate rapidly reversed the forskolin-induced
hyperpolarization. Averaged results from a series of measurements
are summarized in FIG. 6A (right panel). Paired analysis of PD
changes (.DELTA.PD, FIG. 6B) indicated .about.4 mV
hyperpolarization after forskolin with depolarization of similar
magnitude after GlyH-101; for comparison data are shown for
CFTR.sub.inh-172 from a previous study. In a separate series of
experiments, nasal PDs were measured as in A except that all
solutions contained DIDS or GlyH-101. FIG. 6C shows partial
inhibition by DIDS of the (CFTR-independent) hyperpolarization
produced by low C.sup.- (left panel), and substantial inhibition by
GlyH-101 of the forskolin-induced hyperpolarization (right panel).
Together these results indicate rapid inhibition of upper airway
CFTR C.sup.- conductance by topical GlyH-101.
[0220] The efficacy of GlyH-101 in inhibiting cAMP/cholera
toxin-induced intestinal fluid secretion was also evaluated.
Short-circuit current experiments were done in different cell types
and in intact mouse ileum under non-permeabilized conditions and in
the absence of a C.sup.- gradient. In each case CFTR was activated
by CPT-cAMP after ENaC inhibition by amiloride. FIG. 7A shows
similar K.sub.i.about.5 .mu.M for inhibition of cAMP-stimulated
short-circuit current by GlyH-101 in T84 cells (top panel), primary
human bronchial cell cultures (middle panel), and intact mouse
ileum (bottom panel). Inhibition was .about.100% at higher GlyH-101
concentrations. Cholera toxin-induced intestinal fluid secretion
was measured in an in vivo closed-loop model in which loops for
each mouse were injected with saline (control), cholera toxin (1
.mu.g), or cholera toxin (1 .mu.g)+GlyH-101 (0.25 .mu.g). GlyH-101
was added to the lumen (rather than systemically) based on initial
studies showing poor intestinal absorption and little effect of
systemically administered compound. Compared to the saline control,
the cholera toxin-induced increase in fluid secretion over 4 hours,
quantified from loop weight-to-length ratio, was 80% reduced by
GlyH-101.
Example 6
Synthesis of Highly Water Soluble CFTR Pore-Blocking Compounds
[0221] The strategy for design of highly water-soluble CFTR
inhibitor compounds with minimal intestinal absorption was to
modify the structure of GlyH-101 by addition of polar, bulky groups
as shown in FIG. 8. From analysis of structure-activity
relationship of glycine hydrazides compounds it was found that the
minor modifications at the glycyl methyl position did not affect
CFTR inhibition activity. Efficient synthesis of highly water
soluble CFTR inhibitors were devised by utilizing a
diethylbromomalonate intermediate (FIGS. 9-11). Reaction of
2-naphthalenamine with diethylbromomalonate followed by subsequent
reaction with hydrazine generated a versatile malonic acid
dihydrazide intermediate (FIG. 9). Condensation of this dihydrazide
with 3,5-dibromo-2,4-dihydroxybenzaldehyde produced a key
intermediate compound 4 which on further condensation with same
aldehyde produced the compound MalH-1. Similarly,
2,4-disodium-disulfobenzaldehyde and
4-sodium-sulfophenylisothiocyanate were condensed with compound 4
to generate the compounds MalH-2 and MalH-3, respectively.
[0222] MalH-1 is structurally similar to GlyH-101 except for an
additional benzaldehyde moiety that makes it doubly charged,
bulkier and more hydrophilic. MalH-1 is water soluble to >5 mM.
MalH-2 carries two disulfonic acid groups, and MalH-3 contains one
sulfonic acid moiety with hydrophilic thiourea linker. Both
compounds are freely soluble (>50% wt/volume, 20.degree. C.) in
water and saline.
[0223] Intermediate compound 4 was also used to generate
MalH-(PEG).sub.n and MalH-(PEG).sub.n B by condensation with
various phenylisothiocyantes 6a and 6b carrying PEG (FIG. 10). The
intermediate compounds 6a and 6b were synthesized by reaction of
1,4-phenylenediisothiocyante 5a and
bis[(4-isothiocyanato)phenyl]methane 6b with appropriate
amino-PEGs. The PEG moiety increased water solubility to .about.10
mM. Another approach for synthesis of PEG-ylated compounds involved
incorporation of hydroxyethyl moiety onto glycyl methyl and further
manipulating hydroxyl group to link PEG chain (FIG. 11). Reaction
of bromobuterolactone with 2-naphthalenamine and subsequent
reaction with hydrazine produced hydrazide 7. Using standard
protection-deprotection Boc chemistry, this hydrazide was
PEG-ylated by utilizing its hydroxyl group. The PEG-ylated
hydrazide 11 was condensed with aromatic aldehyde to produce
GlyH-(PEG).sub.n, which have similar was solubility as
MalH-(PEG).sub.n.
Example 7
CFTR Inhibition with Highly Water Soluble CFTR Pore-Blocking
Compounds
[0224] CFTR inhibition by MalH compounds was assayed by
short-circuit current analysis using FRT cells expressing human
wildtype CFTR. Apical membrane chloride current was measured after
permeabilization of the cell basolateral membrane in the presence
of a transepithelial chloride gradient. As shown in FIG. 12, CFTR
was activated by the cell permeant cAMP agonist CPT-cAMP and then
increasing MalH compound concentrations were added. The results
show that inhibition was rapid and nearly complete at high MalH
concentrations. In addition, the results also show that inhibitory
potencies (K.sub.i) were in the range 2-8 .mu.M.
[0225] Short-circuit current analysis in CFTR-expressing epithelial
cell monolayers showed prompt inhibition of chloride current in
response to compound addition to the luminal solution. Importantly,
near 100% block of chloride current was achieved at high inhibition
concentrations. Also, the inhibitors were chemically stable in the
presence of intestinal contents, and no toxicity was seen when the
inhibitors were present at high concentration in cell cultures or
when administered systemically to mice. The effective CFTR block of
these water soluble impermeant compounds when added externally
provides direct evidence that the site of the block is at the
external-facing surface of CFTR.
Example 8
Intestinal Absorption and Antidiarrheal Efficacy Studies with
Highly Water Soluble CFTR Pore-Blocking Compounds
[0226] Intestinal absorption was measured in mice in vivo from the
disappearance of MalH compounds from the lumens of closed
mid-jejunal loops over 2 hours. In these experiments mannitol was
included in the MalH-containing solutions to prevent fluid
absorption. Absorption rates were referenced against a large
FITC-dextran, which was assumed to undergo no absorption over the 2
hour study. The summarized data in FIG. 13, panel A, shows under 5%
absorption of the MalH compounds in 2 hours, whereas the >90% of
the thiazolidinone CFTR.sub.inh-172 was absorbed over this
time.
[0227] Antidiarrheal efficacy was assayed in closed mid jejunal
loops in mice. Loops were injected with saline or solutions of
cholera toxin containing different concentrations of MalH
compounds. Intestinal fluid secretion was determined at 6 hours by
measurements of loop length and weight. The data summary in FIG.
13, panel B, shows a loop weight-to-length ratio (corresponding to
100% inhibition) of .about.0.09 in saline-injected loops, and 0.28
(corresponding to 0% inhibition) in cholera toxin-injected loops.
The results show that each of the MalH compounds inhibited loop
secretion in a dose-dependent manner with essentially complete
inhibition at the higher concentrations.
[0228] The results show that the glycine hydrazide-based CFTR
inhibitors undergo little intestinal absorption and are effective
in preventing cholera toxin-induced fluid secretion in a rodent
model of cholera toxin-induced fluid secretion. The advantages of
antidiarrheal therapy using a non-absorbable compound are that high
concentrations can be achieved in the gut with minimal concerns
about toxicity and off-target effects related to cellular uptake
and systems absorption.
[0229] While the present invention has been described with
reference to the specific embodiments thereof, it should be
understood by those skilled in the art that various changes may be
made and equivalents may be substituted without departing from the
true spirit and scope of the invention. In addition, many
modifications may be made to adapt a particular situation,
material, composition of matter, process, process step or steps, to
the objective, spirit and scope of the present invention. All such
modifications are intended to be within the scope of the claims
appended hereto.
* * * * *