U.S. patent application number 16/110878 was filed with the patent office on 2019-03-21 for chloro-pyrazine carboxamide derivatives with epithelial sodium channel blocking activity.
This patent application is currently assigned to Parion Sciences, Inc.. The applicant listed for this patent is Parion Sciences, Inc.. Invention is credited to Michael R. Johnson.
Application Number | 20190084943 16/110878 |
Document ID | / |
Family ID | 49883318 |
Filed Date | 2019-03-21 |
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United States Patent
Application |
20190084943 |
Kind Code |
A1 |
Johnson; Michael R. |
March 21, 2019 |
CHLORO-PYRAZINE CARBOXAMIDE DERIVATIVES WITH EPITHELIAL SODIUM
CHANNEL BLOCKING ACTIVITY
Abstract
This invention provides compounds of the formula I: ##STR00001##
and their pharmaceutically acceptable salts, useful as sodium
channel blockers, compositions containing the same, therapeutic
methods and uses for the same and processes for preparing the
same.
Inventors: |
Johnson; Michael R.; (Chapel
Hill, NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Parion Sciences, Inc. |
Durham |
NC |
US |
|
|
Assignee: |
Parion Sciences, Inc.
Durham
NC
|
Family ID: |
49883318 |
Appl. No.: |
16/110878 |
Filed: |
August 23, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15452387 |
Mar 7, 2017 |
10071970 |
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16110878 |
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14106125 |
Dec 13, 2013 |
9593084 |
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15452387 |
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61738248 |
Dec 17, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/4965 20130101;
A61P 27/16 20180101; A61P 37/06 20180101; A61P 15/02 20180101; A61P
1/02 20180101; A61P 11/12 20180101; A61P 1/10 20180101; C07D 241/26
20130101; A61P 25/02 20180101; A61P 11/06 20180101; A61P 11/08
20180101; A61P 17/16 20180101; A61K 31/047 20130101; A61P 1/04
20180101; A61P 43/00 20180101; A61P 11/00 20180101; A61P 29/00
20180101; C07D 241/32 20130101; A61P 27/04 20180101; A61K 33/14
20130101; A61P 11/02 20180101; A61P 3/12 20180101; A61P 31/00
20180101; A61K 45/06 20130101; A61P 1/00 20180101; A61P 27/02
20180101; A61P 11/14 20180101 |
International
Class: |
C07D 241/28 20060101
C07D241/28; A61K 31/047 20060101 A61K031/047; A61K 31/4965 20060101
A61K031/4965; C07D 241/26 20060101 C07D241/26; A61K 45/06 20060101
A61K045/06; A61K 33/14 20060101 A61K033/14 |
Claims
1-18. (canceled)
19. A method for treating dry mouth (xerostomia), dry skin, vaginal
dryness, sinusitis, rhinosinusitis, nasal dehydration, including
nasal dehydration brought on by administering dry oxygen, dry eye,
Sjogren's disease, otitis media, primary ciliary dyskinesia, distal
intestinal obstruction syndrome, esophagitis, constipation, or
chronic diverticulitis in a subject, or for promoting ocular or
corneal hydration in a subject, the method comprising administering
to the subject a compound of Formula (I): ##STR00063## or a
pharmaceutically acceptable salt thereof, wherein: Ar is selected
from: ##STR00064## n is an integer selected from 0, 1, 2, 3, 4, 5,
and 6; R.sup.1 is selected from hydrogen, C.sub.1-C.sub.8 alkyl,
and polyhydroxylated alkyl groups having from 3 to 8 carbon atoms;
R.sup.2 is hydrogen or a polyhydroxylated alkyl group having from 3
to 8 carbon atoms; and R.sup.3 and R.sup.4 are each, independently,
hydrogen or C.sub.1-C.sub.3 alkyl; or comprising administering to
the subject a compound selected from the group consisting of:
##STR00065## and pharmaceutically acceptable salts thereof.
20. The method of claim 19, wherein the compound is of the formula:
##STR00066## or a pharmaceutically acceptable salt thereof.
21. The method of claim 19, wherein the compound is: ##STR00067##
or a pharmaceutically acceptable salt thereof.
22. The method of claim 19, wherein the compound is of Formula
(II): ##STR00068## or a pharmaceutically acceptable salt thereof,
wherein: n is an integer selected from 1, 2, 3, 4, 5, and 6;
R.sup.1 is selected from hydrogen, C.sub.1-C.sub.8 alkyl, and
polyhydroxylated alkyl groups having from 3 to 8 carbon atoms;
R.sup.2 is hydrogen or a polyhydroxylated alkyl group having from 3
to 8 carbon atoms; and R.sup.3 and R.sup.4 are each, independently,
hydrogen or C.sub.1-C.sub.3 alkyl.
23. The method of claim 19, wherein the compound is selected from
the group consisting of: ##STR00069## ##STR00070## and
pharmaceutically acceptable salts thereof.
24. The method of claim 19, wherein the compound is of Formula
(III): ##STR00071## or a pharmaceutically acceptable salt thereof,
wherein: n is an integer selected from 1, 2, 3, 4, 5, and 6;
R.sup.1 is selected from hydrogen, C.sub.1-C.sub.8 alkyl, and
polyhydroxylated alkyl groups having from 3 to 8 carbon atoms;
R.sup.2 is hydrogen or a polyhydroxylated alkyl group having from 3
to 8 carbon atoms; and R.sup.3 and R.sup.4 are each, independently,
hydrogen or C.sub.1-C.sub.3 alkyl.
25. The method of claim 19, wherein the compound is selected from
the group consisting of: ##STR00072## and pharmaceutically
acceptable salts thereof.
26. The method of claim 19, wherein the compound is of Formula
(IV): ##STR00073## or a pharmaceutically acceptable salt thereof,
wherein: n is an integer selected from 1, 2, 3, 4, 5, and 6;
R.sup.1 is selected from hydrogen, C.sub.1-C.sub.8 alkyl, and
polyhydroxylated alkyl groups having from 3 to 8 carbon atoms;
R.sup.2 is hydrogen or a polyhydroxylated alkyl group having from 3
to 8 carbon atoms; and R.sup.3 and R.sup.4 are each, independently,
hydrogen or C.sub.1-C.sub.3 alkyl.
27. The method of claim 19, wherein the compound is selected from
the group consisting of: ##STR00074## and pharmaceutically
acceptable salts thereof.
28. A method for preventing, mitigating, and/or treating
deterministic health effects to the respiratory tract and/or other
bodily organs caused by respirable aerosols containing
radionuclides in a subject in need thereof, the method comprising
administering to the subject a compound of Formula (I):
##STR00075## or a pharmaceutically acceptable salt thereof,
wherein: Ar is selected from: ##STR00076## n is an integer selected
from 0, 1, 2, 3, 4, 5, and 6; R.sup.1 is selected from hydrogen,
C.sub.1-C.sub.8 alkyl, and polyhydroxylated alkyl groups having
from 3 to 8 carbon atoms; R.sup.2 is hydrogen or a polyhydroxylated
alkyl group having from 3 to 8 carbon atoms; and R.sup.3 and
R.sup.4 are each, independently, hydrogen or C.sub.1-C.sub.3 alkyl.
or comprising administering to the subject a compound selected from
the group consisting of: ##STR00077## and pharmaceutically
acceptable salts thereof
29. The method of claim 28, wherein the compound is of the formula:
##STR00078## or a pharmaceutically acceptable salt thereof.
30. The method of claim 28, wherein the compound is: ##STR00079##
or a pharmaceutically acceptable salt thereof.
31. The method of claim 28, wherein the compound is of Formula
(II): ##STR00080## or a pharmaceutically acceptable salt thereof,
wherein: n is an integer selected from 1, 2, 3, 4, 5, and 6;
R.sup.1 is selected from hydrogen, C.sub.1-C.sub.8 alkyl, and
polyhydroxylated alkyl groups having from 3 to 8 carbon atoms;
R.sup.2 is hydrogen or a polyhydroxylated alkyl group having from 3
to 8 carbon atoms; and R.sup.3 and R.sup.4 are each, independently,
hydrogen or C.sub.1-C.sub.3 alkyl.
32. The method of claim 28, wherein the compound is selected from
the group consisting of: ##STR00081## ##STR00082## and
pharmaceutically acceptable salts thereof.
33. The method of claim 28, wherein the compound is of Formula
(III): ##STR00083## or a pharmaceutically acceptable salt thereof,
wherein: n is an integer selected from 1, 2, 3, 4, 5, and 6;
R.sup.1 is selected from hydrogen, C.sub.1-C.sub.8 alkyl, and
polyhydroxylated alkyl groups having from 3 to 8 carbon atoms;
R.sup.2 is hydrogen or a polyhydroxylated alkyl group having from 3
to 8 carbon atoms; and R.sup.3 and R.sup.4 are each, independently,
hydrogen or C.sub.1-C.sub.3 alkyl.
34. The method of claim 28, wherein the compound is selected from
the group consisting of: ##STR00084## and pharmaceutically
acceptable salts thereof.
35. The method of claim 28, wherein the compound is of Formula
(IV): ##STR00085## or a pharmaceutically acceptable salt thereof,
wherein: n is an integer selected from 1, 2, 3, 4, 5, and 6;
R.sup.1 is selected from hydrogen, C.sub.1-C.sub.8 alkyl, and
polyhydroxylated alkyl groups having from 3 to 8 carbon atoms;
R.sup.2 is hydrogen or a polyhydroxylated alkyl group having from 3
to 8 carbon atoms; and R.sup.3 and R.sup.4 are each, independently,
hydrogen or C.sub.1-C.sub.3 alkyl.
36. The method of claim 28, wherein the compound is selected from
the group consisting of: ##STR00086## and pharmaceutically
acceptable salts thereof.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to novel compounds, including
3,5-diamino-N-(N-(4-(4-((S)-2-amino-3-(4-(3-(bis((2S,3R,4R,5R)-2,3,4,5,6--
pentahydroxyhexyl)amino)propyl)
phenylamino)-3-oxopropyl)naphthalen-1-yl)butyl)carbamimidoyl)-6-chloropyr-
azine-2-carboxamide and related compounds and their
pharmaceutically acceptable salts, useful as sodium channel
blockers, compositions containing the same, therapeutic methods and
uses for the same and processes for preparing the same.
BACKGROUND OF THE INVENTION
[0002] The mucosal surfaces at the interface between the
environment and the body have evolved a number of "innate defense",
i.e., protective mechanisms. A principal form of such innate
defense is to cleanse these surfaces with liquid. Typically, the
quantity of the liquid layer on a mucosal surface reflects the
balance between epithelial liquid secretion, often reflecting anion
(Cr and/or HCO.sub.3.sup.-) secretion coupled with water (and a
cation counter-ion), and epithelial liquid absorption, often
reflecting Na.sup.+ absorption, coupled with water and counter
anion (Cr and/or HCO.sub.3.sup.-). Many diseases of mucosal
surfaces are caused by too little protective liquid on those
mucosal surfaces created by an imbalance between secretion (too
little) and absorption (relatively too much). The defective salt
transport processes that characterize these mucosal dysfunctions
reside in the epithelial layer of the mucosal surface.
[0003] One approach to replenish the protective liquid layer on
mucosal surfaces is to "re-balance" the system by blocking Na.sup.+
channel and liquid absorption. The epithelial protein that mediates
the rate-limiting step of Na.sup.+ and liquid absorption is the
epithelial Na.sup.+ channel ("ENaC"). ENaC is positioned on the
apical surface of the epithelium, i.e. the mucosal
surface-environmental interface. Ideally, to inhibit ENaC mediated
Na.sup.+ and liquid absorption, an ENaC blocker of the amiloride
class will be delivered to the mucosal surface and maintained at
this site to achieve maximum therapeutic benefit.
[0004] The use of ENaC blockers has been reported for a variety of
diseases which are ameliorated by increased mucosal hydration. In
particular, the use of ENaC blockers in the treatment of
respiratory diseases such as chronic bronchitis (CB), cystic
fibrosis (CF), and COPD, which reflect the body's failure to clear
mucus normally from the lungs and ultimately result in chronic
airway infection has been reported. See, Evidence for airway
surface dehydration as the initiating event in CF airway disease,
R. C. Boucher, Journal of Internal Medicine, Vol. 261, Issue 1,
January 2007, pages 5-16; and Cystic fibrosis: a disease of
vulnerability to airway surface dehydration, R. C. Boucher, Trends
in Molecular Medicine, Vol. 13, Issue 6, June 2007, pages
231-240.
[0005] Data indicate that the initiating problem in both chronic
bronchitis and cystic fibrosis is the failure to clear mucus from
airway surfaces. The failure to clear mucus reflects an imbalance
in the quantities of mucus as airway surface liquid (ASL) on airway
surfaces. This imbalance results in a relative reduction in ASL
which leads to mucus concentration, reduction in the lubricant
activity of the periciliary liquid (PCL), mucus adherence to the
airway surface, and failure to clear mucus via ciliary activity to
the mouth. The reduction in mucus clearance leads to chronic
bacterial colonization of mucus adherent to airway surfaces. The
chronic retention of bacteria, inability of local antimicrobial
substances to kill mucus-entrapped bacteria on a chronic basis, and
the consequent chronic inflammatory response to this type of
surface infection, are manifest in chronic bronchitis and cystic
fibrosis.
[0006] There is currently a large, unmet medical need for products
that specifically treat the variety of diseases which are
ameliorated by increased mucosal hydration, including chronic
bronchitis, COPD and cystic fibrosis, among others. The current
therapies for chronic bronchitis, COPD and cystic fibrosis focus on
treating the symptoms and/or the late effects of these diseases.
However, none of these therapies treat effectively the fundamental
problem of the failure to clear mucus from the lung.
[0007] R. C. Boucher, in U.S. Pat. No. 6,264,975, describes the use
of pyrazinoylguanidine sodium channel blockers for hydrating
mucosal surfaces typified by the well-known diuretics amiloride,
benzamil, and phenamil. However, these compounds are relatively
impotent, considering the limited mass of drug that can be inhaled
to the lung; (2) rapidly absorbed, and thereby exhibiting
undesirably short half-life on the mucosal surface; and (3) are
freely dissociable from ENaC. More potent drugs with longer
half-lives on the mucosal surface are needed.
[0008] Too little protective surface liquid on other mucosal
surfaces is a common pathophysiology of a number of diseases. For
example, in xerostomia (dry mouth) the oral cavity is depleted of
liquid due to a failure of the parotid sublingual and submandibular
glands to secrete liquid despite continued Na.sup.+ (ENaC)
transport mediated liquid absorption from the oral cavity.
Keratoconjunctivitis sira (dry eye) is caused by failure of
lacrimal glands to secrete liquid in the face of continued Na.sup.+
dependent liquid absorption on conjunctional surfaces. In
rhinosinusitis, there is an imbalance between mucin secretion and
relative ASL depletion. Failure to secrete Cl-- (and liquid) in the
proximal small intestine, combined with increased Na.sup.+ (and
liquid) absorption in the terminal ileum leads to the distal
intestinal obstruction syndrome (DIOS). In older patients excessive
Na.sup.+ (and volume) absorption in the descending colon produces
constipation and diverticulitis.
[0009] The published literature includes number of patent
applications and granted patents to Parion Sciences Inc., directed
toward pyrazinoylguanidine analogs as sodium channel blockers.
Examples of such publications include PCT Publication Nos.
WO2003/070182, WO2003/070184, WO2004/073629, WO2005/025496,
WO2005/016879, WO2005/018644, WO2006/022935, WO2006/023573,
WO2006/023617, WO2007/018640, WO2007/146869, WO2008/031028,
WO2008/031048, and U.S. Pat. Nos. 6,858,614, 6,858,615, 6,903,105,
7,064,129, 7,186,833, 7,189,719, 7,192,958, 7,192,959, 7,192,960,
7,241,766, 7,247,636, 7,247,637, 7,317,013, 7,332,496, 7,368,447,
7,368,450, 7,368,451, 7,375,102, 7,388,013, 7,399,766, 7,410,968,
7,807,834, 7,842,697, and 7,868,010.
[0010] There remains a need for novel sodium channel blocking
compounds with enhanced potency and effectiveness on mucosal
tissues. There also remains the need for novel sodium channel
blocking compounds that provide therapeutic effect, but minimize or
eliminate the onset or progression of hyperkalemia in
recipients.
SUMMARY OF THE INVENTION
[0011] This invention provides compounds of the formula I:
##STR00002##
[0012] wherein: [0013] Ar is selected from the group of:
[0013] ##STR00003## [0014] n is an integer selected from 0, 1, 2,
3, 4, 5, or 6; [0015] R.sup.1 is selected from hydrogen,
C.sub.1-C.sub.8 alkyl, and a polyhydroxylated alkyl group having
from 3 to 8 carbon atoms; [0016] R.sup.2 is hydrogen or a
polyhydroxylated alkyl group having from 3 to 8 carbon atoms;
[0017] R.sup.3 and R.sup.4 are each, independently, hydrogen or
C.sub.1-C.sub.3 alkyl;
[0018] or a pharmaceutically acceptable salt thereof.
[0019] This invention also provides solvates and hydrates,
individual stereoisomers, including optical isomers (enantiomers
and diastereomers) and geometric isomers cis-/trans-isomerism),
mixtures of stereoisomers, and tautomers of compounds of the
formula (I), or a pharmaceutically acceptable salt thereof, as well
as pharmaceutical compositions comprising the compounds, or a
pharmaceutically acceptable salts thereof, their use in methods of
treatment, and methods for their preparation.
[0020] This invention also provides the compound
3,5-diamino-N-(N-(4-(4-((S)-2-amino-3-(4-(3-(bis((2S,3R,4R,5R)-2,3,4,5,6--
pentahydroxyhexyl)amino)propyl)phenylamino)-3-oxopropyl)
naphthalen-1-yl)butyl)carbamimidoyl)-6-chloropyrazine-2-carboxamide,
or a pharmaceutically acceptable salt thereof, as well as optical
isomers (enantiomers and diastereomers) and geometric isomers
(cis-/trans-isomerism), mixtures of stereoisomers, and tautomers
thereof, as well as pharmaceutical compositions comprising the
compound, or a pharmaceutically acceptable salt thereof, its use in
methods of treatment, and methods for its preparation.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] A more complete appreciation of the invention and many of
the advantages thereof may be readily obtained by reference to the
information herein in conjunction with the following figures:
[0022] FIG. 1 is a plot of the effect of Compound 33 on Sheep MCC
at 4 hours post-dose,
[0023] FIG. 2 is a plot of the effect of Compound 123 on Sheep MCC
at 4 hours post-dose.
[0024] FIG. 3 is a plot of the effect of Compound 48 on Sheep MCC
at 4 hours post-dose.
[0025] FIG. 4 is a plot of the effect of Compound 33 on Sheep MCC
at 8 hours post-dose.
[0026] FIG. 5 is a plot of the effect of Compound 152 on Sheep MCC
at 8 hours post-dose.
[0027] FIG. 6 is a plot of the enhancement of Compound 33 on Sheep
MCC at 8 hours post-dose by hypertonic saline.
[0028] FIG. 7 is a plot of the effect of Comparative Example 1 on
sheep MCC at 4 hrs post-dose.
[0029] FIG. 8 is a plot of the effect of Comparative Example 1 on
sheep plasma potassium levels.
[0030] FIG. 9 is a plot comparing the activity of Comparative
Example 1 and Compound 33 on sheep MCC at 4 h Post-dose.
[0031] FIG. 10 is a plot comparing the effect on sheep Plasma
K.sup.+ levels of Comparative Example 1 and Compound 33.
[0032] FIG. 11 is a plot comparing the activity of Comparative
Example 1 and Compound 123 on sheep MCC at 4 h Post-dose.
[0033] FIG. 12 is a plot comparing the effect on sheep Plasma
K.sup.+ levels of Comparative Example 1 and Compound 123.
[0034] FIG. 13 is a plot comparing the activity of Comparative
Example 1 and Compound 48 on sheep MCC at 4 h Post-dose.
[0035] FIG. 14 is a plot comparing the effect on sheep Plasma
K.sup.+ levels of Comparative Example 1 and Compound 48.
DETAILED DESCRIPTION OF THE INVENTION
[0036] As used herein, the following terms are defined as
indicated.
[0037] "A compound of the invention" means a compound of Formula I
or a salt, particularly a pharmaceutically acceptable salt
thereof.
[0038] "A compound of Formula 1" means a compound having the
structural formula designated herein as Formula I. Compounds of
Formula I include solvates and hydrates (i.e., adducts of a
compound of Formula I with a solvent). In those embodiments wherein
a compound of Formula I includes one or more chiral centers, the
phrase is intended to encompass each individual stereoisomer
including optical isomers (enantiomers and diastereomers) and
geometric isomers (cis-/trans-isomerism) and mixtures of
stereoisomers. In addition, compounds of Formula I also include
tautomers of the depicted formula(s).
[0039] Throughout the description and examples, compounds are named
using standard IUPAC naming principles, where possible, including
the use of the ChemDraw Ultra 11.0 software program for naming
compounds, sold by CambridgeSoft Corp./PerkinElmer.
[0040] In some chemical structure representations where carbon
atoms do not have a sufficient number of attached variables
depicted to produce a valence of four, the remaining carbon
substituents needed to provide a valence of four should be assumed
to be hydrogen. Similarly, in some chemical structures where a bond
is drawn without specifying the terminal group, such bond is
indicative of a methyl (Me, --CH.sub.3) group, as is conventional
in the art.
[0041] In one embodiment, the compound of Formula (I) is
3,5-diamino-N-(N-(4-(4-(2-amino-3-(4-(3-(bis(2,3,4,5,6-pentahydroxyhexyl)-
amino)propyl)phenylamino)-3-oxopropyl)naphthalen-1-yl)butyl)carbamimidoyl)-
-6-chloropyrazine-2-carboxamide, having the structure:
##STR00004##
or a pharmaceutically acceptable salt thereof.
[0042] In another embodiment, the compound of Formula (I) is
3,5-diamino-N-(N-(4-(4-(2-amino-3-(4-(3-(bis(2,3,4,5,6-pentahydroxyhexyl)-
amino)propyl)phenylamino)-3-oxopropyl)-5,6,7,8-tetrahydronaphthalen-1-yl)b-
utyl)carbamimidoyl)-6-chloropyrazine-2-carboxamide, having the
structure:
##STR00005##
or a pharmaceutically acceptable salt thereof.
[0043] In a further embodiment the compound of Formula (I) is
3,5-diamino-N-(N-(4-(6-(2-amino-3-(4-(3-(bis(2,3,4,5,6-pentahydroxyhexyl)-
amino)propyl)phenylamino)-3-oxopropyl)naphthalen-2-yl)butyl)carbamimidoyl)-
-6-chloropyrazine-2-carboxamide, having the structure:
##STR00006##
or a pharmaceutically acceptable salt thereof.
[0044] In another embodiment, the compound of Formula (I) is
3,5-diamino-N-(N-(4-(4-((S)-2-amino-3-(4-(3-(bis((2S,3R,4R,5R)-2,3,4,5,6--
pentahydroxyhexyl)amino)propyl)phenylamino)-3-oxopropyl)naphthalen-1-yl)bu-
tyl)carbamimidoyl)-6-chloropyrazine-2-carboxamide, having the
formula:
##STR00007##
or a pharmaceutically acceptable salt thereof.
[0045] Three independent embodiments comprise compounds of Formula
(II), Formula and Formula (IV), respectively:
##STR00008##
wherein:
[0046] n is an integer selected from 0, 1, 2, 3, 4, 5, or 6;
[0047] R.sup.1 is selected from hydrogen, C.sub.1-C.sub.8 alkyl,
and a polyhydroxylated alkyl group having from 3 to 8 carbon
atoms;
[0048] R.sup.2 is hydrogen or a polyhydroxylated alkyl group having
from 3 to 8 carbon atoms;
[0049] R.sup.3 and R.sup.4 are each, independently, hydrogen or
C.sub.1-C.sub.3 alkyl; or a pharmaceutically acceptable salt
thereof.
[0050] Within each group of compounds independently represented by
Formulas (I), (II), (III), and (IV), there is a further embodiment
wherein:
[0051] n is an integer selected from 1, 2, 3, 4, 5, or 6;
[0052] R.sup.1 is selected from hydrogen, C.sub.1-C.sub.8 alkyl,
and a polyhydroxylated alkyl group having from 3 to 8 carbon
atoms;
[0053] R.sup.2 is hydrogen or a polyhydroxylated alkyl group having
from 3 to 8 carbon atoms;
[0054] R.sup.3 and R.sup.4 are each, independently, hydrogen or
C.sub.1-C.sub.3 alkyl; or a pharmaceutically acceptable salt
thereof.
[0055] Within each group of compounds independently represented by
Formulas (I), (II), (III), and (IV), there is a further embodiment
wherein:
[0056] n is an integer selected from 1, 2, 3, 4, 5, or 6;
[0057] R.sup.1 is selected from hydrogen and C.sub.1-C.sub.8
alkyl;
[0058] R.sup.2 is hydrogen or a polyhydroxylated alkyl group having
from 3 to 8 carbon atoms;
[0059] R.sup.3 and R.sup.4 are each, independently, hydrogen or
C.sub.1-C.sub.3 alkyl; or a pharmaceutically acceptable salt
thereof.
[0060] Within each group of compounds independently represented by
Formulas (I), II), (III), and (IV), there is another embodiment
wherein:
[0061] n is an integer selected from 1, 2, 3, 4, 5, or 6;
[0062] R.sup.1 is selected from hydrogen and C.sub.1-C.sub.8
alkyl;
[0063] R.sup.2 is hydrogen;
[0064] R.sup.3 and R.sup.4 are each, independently, hydrogen or
C.sub.1-C.sub.3 alkyl; or a pharmaceutically acceptable salt
thereof.
[0065] Within each group of compounds independently represented by
Formulas (I), (II), (III), and (IV), there is still another
embodiment wherein:
[0066] n is an integer selected from 1, 2, 3, 4, 5, or 6;
[0067] R.sup.1 and R.sup.2 are each, independently, a
polyhydroxylated alkyl group having from 3 to 8 carbon atoms;
[0068] R.sup.3 and R.sup.4 are each, independently, hydrogen or
C.sub.1-C.sub.3 alkyl; or a pharmaceutically acceptable salt
thereof.
[0069] Within each group of compounds independently represented by
Formulas (I), (II), (III), and (IV), there exists another
embodiment wherein:
[0070] n is an integer selected from 1, 2, 3, 4, 5, or 6;
[0071] R.sup.1 and R.sup.2 are each, independently, a
polyhydroxylated alkyl group having from 3 to 8 carbon atoms;
[0072] R.sup.3 and R.sup.4 are hydrogen; or a pharmaceutically
acceptable salt thereof.
[0073] Within each group of compounds independently represented by
Formulas (I), (II), (III), and (IV), there is another embodiment
wherein:
[0074] n is an integer selected from 1, 2, 3, 4, 5, or 6;
[0075] R.sup.1 and R.sup.2 are each, independently, a
polyhydroxylated alkyl group having from 3 to 8 carbon atoms;
[0076] R.sup.3 and R.sup.4 are each, independently, C.sub.1-C.sub.3
alkyl; or a pharmaceutically acceptable salt thereof.
[0077] Within each group of compounds independently represented by
Formulas (I), (II), (III), and (IV), there is an additional
embodiment wherein:
[0078] n is an integer selected from 1, 2, 3, 4, 5, or 6;
[0079] R.sup.1 and R.sup.2 are each, independently, a
polyhydroxylated alkyl group having from 3 to 8 carbon atoms;
[0080] R.sup.3 is hydrogen; and
[0081] R.sup.4 is C.sub.1-C.sub.3 alkyl; or a pharmaceutically
acceptable salt thereof.
[0082] Polyhydroxylated alkyl groups of this invention are those in
which an alkyl chain of from 3 to 8 carbon atoms substituted by two
or more hydroxyl groups. Examples of polyhydroxylated alkyl groups
are butane-1,4-diol; butane-1,2,2-triol; butane-1,1,2,3,-tetraol;
pentane-1,2,3,4-tetraol; hexane-1,2,3,4,5-pentaol;
heptane-1,2,3,4,5,6-hexaol; and octane-1,2,3,4,5,6,7-heptaol.
[0083] One embodiment within each group of compounds described
herein are those compounds in which the polyhydroxylated alkyl
group has the formula --CH.sub.2--(CHR.sup.5).sub.n--H, wherein n
is an integer selected from 2, 3, 4, 5, 6, or 7, and R.sup.5 is
independently in each instance H or OH, with the proviso that at
least two of the R.sup.5 groups are OH.
[0084] Another embodiment within each group of compounds described
herein are those compounds in which the polyhydroxylated alkyl
group has the formula --CH.sub.2--CHOH--(CHR.sup.6).sub.m--H,
wherein m is an integer selected from 1, 2, 3, 4, 5, or 6, and
R.sup.6 is independently in each instance H or OH, with the proviso
that at least one of the R.sup.6 groups is OH.
[0085] A further embodiment within each group of compounds
described herein comprises compounds in which the polyhydroxylated
alkyl group has the formula --CH.sub.2--(CHOH).sub.n--CH.sub.2OH,
wherein n is an integer selected from 1, 2, 3, 4, 5, or 6. Another
embodiment within each group of compounds described herein
comprises compounds in which n is an integer selected from 2, 3, 4,
or 5. Another embodiment within each group comprises compounds in
which n is an integer selected from 3, 4, or 5.
[0086] In another embodiment within each group of compounds
described herein, the chain represented by the formula
--CH.sub.2--(CHOH).sub.n--CH.sub.2OH is
2,3,4,5,6-pentahydroxyhexane, having the formula:
##STR00009##
[0087] In a further embodiment within each group of compounds
described herein, the chain represented by the formula
--CH.sub.2--(CHOH).sub.n--CH.sub.2OH is of the formula:
##STR00010##
[0088] Three further independent embodiments include compounds of
Formula (V), Formula (VI), and Formula (VII), respectively:
##STR00011##
wherein:
[0089] n is an integer selected from 1, 2, 3, 4, 5, or 6; and
[0090] R.sup.3 and R.sup.4 are each, independently, hydrogen or
C.sub.1-C.sub.3 alkyl; or a pharmaceutically acceptable salt
thereof.
[0091] Within each embodiment represented by Formulas (V), (VI),
and (VII) there is a further embodiment wherein n is an integer
selected from 1, 2, 3, 4, 5, or 6; and R.sup.3 and R.sup.4 are each
hydrogen; or a pharmaceutically acceptable salt thereof. Within
each embodiment represented by Formulas (V), (VI), and (VII) there
is another embodiment wherein n is an integer selected from 1, 2,
3, 4, 5, or 6; and R.sup.3 and R.sup.4 are each C.sub.1-C.sub.3
alkyl; or a pharmaceutically acceptable salt thereof.
[0092] Within each of the embodiments described herein, there is a
further embodiment wherein n is an integer selected from 1, 2, or
3. Within each of the embodiments described herein, there is a
further embodiment wherein n is an integer selected from 4, 5, or
6. Within each of the embodiments described herein, there are six
further independent embodiments wherein n is an integer of,
respectively, 1, 2, 3, 4, 5, and 6.
[0093] The compounds herein, including those of of Formulas (I),
(Ia), (II), (III), (IV), (V), (VI), and (VII), may be in the form
of a free base or a salt, particularly a pharmaceutically
acceptable salt. For a review of pharmaceutically acceptable salts
see Berge et al., J. Pharma Sci. (1977) 66:1-19.
[0094] Pharmaceutically acceptable salts formed from inorganic or
organic acids include for example, hydrochloride, hydrobromide,
hydroiodide, sulfate, bisulfate, nitrate, sulfamate, phosphate,
hydrogen phosphate, acetate, trifluoroacetate, maleate, malate,
fumarate, lactate, tartrate, citrate, formate, gluconate,
succinate, pyruvate, tannate, ascorbate, palmitate, salicylate,
stearate, phthalate, alginate, polyglutamate, oxalate,
oxaloacetate, saccharate, benzoate, alkyl or aryl sulfonates (e.g.,
methanesulfonate, ethanesulfonate, benzenesulfonate,
p-toluenesulfonate or naphthalenesulfonate) and isothionate;
complexes formed with amino acids such as lysine, arginine,
glutamic acid, glycine, serine, threonine, alanine, isoleucine,
leucine and the like. The compounds of the invention may also be in
the form of salts formed from elemental anions such as chlorine,
bromine or iodine.
[0095] For therapeutic use, salts of active ingredients of the
compounds of Formula I will be pharmaceutically acceptable, i.e.
they will be salts derived from a pharmaceutically acceptable acid.
However, salts of acids which are not pharmaceutically acceptable
may also find use, for example, in the preparation or purification
of a pharmaceutically acceptable compound. Trifluoroacetate salts,
for example, may find such use. All salts, whether or not derived
from a pharmaceutically acceptable acid, are within the scope of
the present invention.
[0096] The term "chiral" refers to molecules which have the
property of non-superimposability of the mirror image partner,
while the term "achiral" refers to molecules which are
superimposable on their mirror image partner.
[0097] The term "stereoisomers" refers to compounds which have
identical chemical constitution, but differ with regard to the
arrangement of the atoms or groups in space. "Diastereomer" refers
to a stereoisomer with two or more centers of chirality and whose
molecules are not mirror images of one another. Diastereomers have
different physical properties, e.g. melting points, boiling points,
spectral properties, and reactivities. Mixtures of diastereomers
may separate under high resolution analytical procedures such as
electrophoresis and chromatography. "Enantiomers" refer to two
stereoisomers of a compound which are non-superimposable mirror
images of one another.
[0098] Stereochemical definitions and conventions used herein
generally follow S. P. Parker, Ed., MCGRAW-HILL DICTIONARY OF
CHEMICAL TERMS (1984) McGraw-Hill Book Company, New York; and
Eliel, E. and Wilen, S., STEREOCHEMISTRY OF ORGANIC COMPOUNDS
(1994) John Wiley & Sons, Inc., New York.
[0099] Many organic compounds exist in optically active forms,
i.e., they have the ability to rotate the plane of plane-polarized
light. In describing an optically active compound, the prefixes D
and L or R and S are used to denote the absolute configuration of
the molecule about its chiral center(s). A specific stereoisomer
may also be referred to as an enantiomer, and a mixture of such
isomers is often called an enantiomeric mixture. A 50;50 mixture of
enantiomers is referred to as a racemic mixture or a racemate,
which may occur where there has been no stereoselection or
stereospecificity in a chemical reaction or process. The terms
"racemic mixture" and "racemate" refer to an equimolar mixture of
two enantiomeric species.
[0100] The term "tautomers" refers to a type of stereoisomer in
which migration of a hydrogen atom results in two or more
structures. The compounds of Formula I may exist in different
tautomeric forms. One skilled in the art will recognize that
amidines, amides, guanidines, ureas, thioureas, heterocycles and
the like can exist in tautomeric forms. By way of example and not
by way of limitation, compounds of Formula I can exist in various
tautomeric forms as shown below:
##STR00012##
[0101] All possible tautomeric forms of the amidines, amides,
guanidines, ureas, thioureas, heterocycles and the like of all of
the embodiments of Formula I are within the scope of the instant
invention. Tautomers exist in equilibrium and thus the depiction of
a single tautomer in the formulas provided will be understood by
those skilled in the art to refer equally to all possible
tautomers.
[0102] It is to be noted that all enantiomers, diastereomers, and
racemic mixtures, tautomers, polymorphs, pseudopolymorphs of
compounds within the scope of Formula I and pharmaceutically
acceptable salts thereof are embraced by the present invention. All
mixtures of such enantiomers and diastereomers, including
enantiomerically enriched mixtures and diastereomerically enriched
mixtures are within the scope of the present invention.
Enantiomerically enriched mixtures are mixtures of enantiomers
wherein the ratio of the specified enantiomer to the alternative
enantiomer is greater than 50:50. More particularly, an
enantiomerically enriched mixture comprises at least about 75% of
the specified enantiomer, and preferably at least about 85% of the
specified enantiomer. In one embodiment, the enantiomerically
enriched mixture is substantially free of the other enantiomer.
Similarly, diastereomerically enriched mixtures are mixtures of
diastereomers wherein amount of the specified diastereomer is
greater than the amount of each alternative diastereomer. More
particularly, a diastereomerically enriched mixture comprises at
least about 75% of the specified diastereomer, and preferably at
least about 85% of the specified diastereomer. In one embodiment,
the diastereomerically enriched mixture is substantially free of
all other diastereomers. The term "substantially free of" will be
understood by those skilled in the art to indicate less than a 5%
presence of other diastereomers, preferably less than 1%, more
preferably less than 0.1%. In other embodiments no other
diastereomers will be present or the amount of any other
diastereomers present will be below the level of detection.
Stereoisomers may be separated by techniques known in the art,
including high performance liquid chromatography (HPLC) and
crystallization of chiral salts.
[0103] A single stereoisomer, e.g. an enantiomer, substantially
free of its stereoisomer may be obtained by resolution of the
racemic mixture using a method such as formation of diastereomers
using optically active resolving agents ("Stereochemistry of Carbon
Compounds," (1962) by E. L. Eliel, McGraw Hill; Lochmuller, C. H.,
(1975) J. Chromatogr., 113:(3) 283-302). Racemic mixtures of chiral
compounds of the invention can be separated and isolated by any
suitable method, including: (1) formation of ionic, diastereomeric
salts with chiral compounds and separation by fractional
crystallization or other methods, (2) formation of diastereomeric
compounds with chiral derivatizing reagents, separation of the
diastereomers, and conversion to the pure stereoisomers, and (3)
separation of the substantially pure or enriched stereoisomers
directly under chiral conditions.
[0104] In one embodiment, the present invention provides an
enantiomerically enriched mixture or composition comprising
3,5-diamino-N-(N-(4-(4-((S)-2-amino-3-(4-(3-(bis
((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)propyl)phenylamino)-3-ox-
opropyl)naphthalen-1-yl)butyl)carbamimidoyl)-6-chloropyrazine-2-carboxamid-
e, or a pharmaceutically acceptable salt thereof, as the
predominant isomer.
[0105] Other embodiments comprise the enantiomerically enriched
mixtures or compositions comprising, respectively, the compounds of
Formulas (I), (Ia), (II), (III), (IV), (V), (VI), and (VII), or a
pharmaceutically acceptable salt thereof, as the predominant isomer
in each of their respective mixtures.
[0106] In another embodiment, the present invention provides an
enantiomerically enriched mixture or composition
3,5-diamino-N-(N-(4-(4-((S)-2-amino-3-(4-(3-(bis((2S,3R,4R,5R)-2,3,4,5,6--
pentahydroxyhexyl)amino)propyl)phenylamino)-3-oxopropyl)naphthalen-1-yl)bu-
tyl)carbamimidoyl)-6-chloropyrazine-2-carboxamide, or a
pharmaceutically acceptable salt thereof, substantially free of
other isomers.
[0107] Four other embodiments comprise the enantiomerically
enriched mixtures or compositions comprising, respectively, the
compounds of Formulas (I), (Ia), (II), (III), (IV), (V), (VI), and
(VII), or a pharmaceutically acceptable salt thereof, substantially
free of other isomers in each of their respective mixtures.
[0108] Also provided herein is each of the compounds and groups of
compounds described herein, including those of Formulas (I), (Ia),
(II), (III), (IV), (V), (VI), and (VII), or a pharmaceutically
acceptable salt thereof, for use as a medicament.
[0109] A compound of Formula I and pharmaceutically acceptable
salts thereof may exist as different polymorphs or
pseudopolymorphs. As used herein, crystalline polymorphism means
the ability of a crystalline compound to exist in different crystal
structures. The crystalline polymorphism may result from
differences in crystal packing (packing polymorphism) or
differences in packing between different conformers of the same
molecule (conformational polymorphism). As used herein, crystalline
pseudopolymorphism also includes the ability of a hydrate or
solvate of a compound to exist in different crystal structures. The
pseudopolymorphs of the instant invention may exist due to
differences in crystal packing (packing pseudopolymorphism) or due
to differences in packing between different conformers of the same
molecule (conformational pseudopolymorphism). The instant invention
comprises all polymorphs and pseudopolymorphs of the compounds of
Formula I and pharmaceutically acceptable salts thereof.
[0110] A compound of Formula I and pharmaceutically acceptable
salts thereof may also exist as an amorphous solid. As used herein,
an amorphous solid is a solid in which there is no long-range order
of the positions of the atoms in the solid. This definition applies
as well when the crystal size is two nanometers or less. Additives,
including solvents, may be used to create the amorphous forms of
the instant invention. The instant invention, including all
pharmaceutical compositions, methods of treatment, combination
products, and uses thereof described herein, comprises all
amorphous forms of the compounds of Formula I and pharmaceutically
acceptable salts thereof.
Uses
[0111] The compounds of the invention exhibit activity as sodium
channel blockers. Without being bound by any particular theory, it
is believed that the compounds of the invention may function in
vivo by blocking epithelial sodium channels present in mucosal
surfaces and thereby reduce the absorption of water by the mucosal
surfaces. This effect increases the volume of protective liquids on
mucosal surfaces, and rebalances the system.
[0112] As a consequence, the compounds of the invention are useful
as medicaments, particularly for the treatment of clinical
conditions for which a sodium channel blocker may be indicated.
Such conditions include pulmonary conditions such as diseases
associated with reversible or irreversible airway obstruction,
chronic obstructive pulmonary disease (COPD), including acute
exacerbations of COPD, asthma, bronchiectasis (including
bronchiectasis due to conditions other than cystic fibrosis), acute
bronchitis, chronic bronchitis, post-viral cough, cystic fibrosis,
emphysema, pneumonia, panbronchiolitis, and transplant-associated
bronchiolitis, including lung- and bone marrow-transplant
associated bronchiolitis, in a human in need thereof. The compounds
of the invention may also be useful for treating
ventilator-associated tracheobronchitis and/or preventing
ventilator-associated pneumonia in ventilated patients. The present
invention comprises methods for treating each of the conditions
described herein in a mammal in need thereof, preferably in a human
in need thereof, each method comprising administering to said
mammal a pharmaceutically effective amount of a compound of the
present invention, or a pharmaceutically acceptable salt thereof.
Also provided are (a) a method for reducing exacerbations of COPD
in a mammal in need thereof; (b) a method for reducing
exacerbations of CF in a mammal in need thereof; (c) a method of
improving lung function (FEV1) in a mammal in need thereof, (d) a
method of improving lung function (FEV1) in a mammal experiencing
COPD, (e) a method of improving lung function (FEV1) in a mammal
experiencing CF, (f) a method of reducing airway infections in a
mammal in need thereof.
[0113] Also provided is a method of stimulating, enhancing or
improving mucociliary clearance in a mammal, the method comprising
administering to a mammal in need thereof a pharmaceutically
effective amount of a compound of formula (I), or a
pharmaceutically acceptable salt thereof. Mucociliary clearance
will be understood to include the natural mucociliary actions
involved in the transfer or clearance of mucus in the airways,
including the self-clearing mechanisms of the bronchi. Therefore,
also provided is a method of improving mucus clearance in the
airways of a mammal in need thereof.
[0114] Additionally, sodium channel blockers may be indicated for
the treatment of conditions which are ameliorated by increased
mucosal hydration in mucosal surfaces other than pulmonary mucosal
surfaces. Examples of such conditions include dry mouth
(xerostomia), dry skin, vaginal dryness, sinusitis, rhinosinusitis,
nasal dehydration, including nasal dehydration brought on by
administering dry oxygen, dry eye, Sjogren's disease, otitis media,
primary ciliary dyskinesia, distal intestinal obstruction syndrome,
esophagitis, constipation, and chronic diverticulitis. The
compounds of the invention can also be used for promoting ocular or
corneal hydration.
[0115] The compounds of the present invention may also be useful in
methods for obtaining a sputum sample from a human. The method may
be carried out by administering an effective amount of a compound
of the invention to at least one lung of the patient, and then
inducing and collecting a sputum sample from that human.
[0116] Accordingly, in one aspect, the present invention provides a
method for the treatment of a condition in a mammal, such as a
human, for which a sodium channel blocker is indicated.
[0117] In other embodiments, the present invention provides each of
the methods described herein with the additional benefit of
minimizing or eliminating hyperkalemia in the recipient of the
method. Also provided are embodiments comprising each of the
methods described herein wherein an improved therapeutic index is
achieved.
[0118] The terms "treat", "treating" and "treatment", as used
herein refers to reversing, alleviating, inhibiting the progress
of, or preventing the disorder or condition or one or more symptoms
of such disorder or condition.
[0119] All therapeutic methods described herein are carried out by
administering an effective amount of a compound of the invention, a
compound of Formula I or a pharmaceutically acceptable salt
thereof, to a subject (typically mammal and preferably human) in
need of treatment.
[0120] In one embodiment the invention provides a method for the
treatment of a condition which is ameliorated by increased mucosal
hydration in a mammal, particularly a human in need thereof. In one
embodiment the invention provides a method for the treatment of a
disease associated with reversible or irreversible airway
obstruction in a mammal, particularly a human, in need thereof. In
one particular embodiment the present invention provides a method
for the treatment of chronic obstructive pulmonary disease (COPD)
in a mammal, particularly a human in need thereof. In one
particular embodiment the present invention provides a method for
reducing the frequency, severity or duration of acute exacerbation
of COPD or for the treatment of one or more symptoms of acute
exacerbation of COPD in a mammal, particularly a human in need
thereof. In one embodiment the invention provides a method for the
treatment of asthma in a mammal, particularly a human, in need
thereof. In one embodiment the invention provides a method for the
treatment of bronchiectasis (including bronchiectasis due to
conditions other than cystic fibrosis) in a mammal, particularly a
human, in need thereof. In one embodiment the invention provides a
method for the treatment of bronchitis, including acute and chronic
bronchitis in a mammal, particularly a human, in need thereof. In
one embodiment the invention provides a method for the treatment of
post-viral cough in a mammal, particularly a human, in need
thereof. In one embodiment the invention provides a method for the
treatment of cystic fibrosis in a mammal, particularly a human, in
need thereof. In one embodiment the invention provides a method for
the treatment of emphysema in a mammal, particularly a human in
need thereof. In one embodiment the invention provides a method for
the treatment of pneumonia in a mammal, particularly a human in
need thereof. In one embodiment the invention provides a method for
the treatment of panbronchiolitis in a mammal, particularly a human
in need thereof. In one embodiment the invention provides a method
for the treatment of transplant-associated bronchiolitis, including
lung- and bone marrow-transplant associated bronchiolitis in a
mammal, particularly a human in need thereof. In one embodiment the
invention provides a method for treating ventilator-associated
tracheobronchitis and/or preventing ventilator-associated pneumonia
in a ventilated human in need thereof.
[0121] This invention provides specific methods for treating a
disease selected from the group of reversible or irreversible
airway obstruction, chronic obstructive pulmonary disease (COPD),
asthma, bronchiectasis (including bronchiectasis due to conditions
other than cystic fibrosis), acute bronchitis, chronic bronchitis,
post-viral cough, cystic fibrosis, emphysema, pneumonia,
panbronchiolitis, transplant-associate bronchiolitis, and
ventilator-associated tracheobronchitis or preventing
ventilator-associated pneumonia in a human in need thereof, each
method comprising administering to said human an effective amount
of a compound of formula 1(a), or a pharmaceutically acceptable
salt thereof. In further embodiments for each method of treatment,
the pharmaceutically acceptable salt form is a hydrochloride salt
or a hydroxynaphthoate salt of the compound of formula (1a). In
another embodiment within each method of treatment, the freebase of
the compound of formula (1a) is used.
[0122] In one embodiment the invention provides a method for the
treatment of dry mouth (xerostomia) in a mammal, particularly a
human in need thereof. In one embodiment the invention provides a
method for the treatment of dry skin in a mammal, particularly a
human in need thereof. In one embodiment the invention provides a
method for the treatment of vaginal dryness in a mammal,
particularly a human in need thereof. In one embodiment the
invention provides a method for the treatment of sinusitis,
rhinosinusitis, or nasal dehydration, including nasal dehydration
brought on by administering dry oxygen, in a mammal, particularly a
human in need thereof. In one embodiment the invention provides a
method for the treatment of dry eye, or Sjogren's disease, or
promoting ocular or corneal hydration in a mammal, particularly a
human in need thereof. In one embodiment the invention provides a
method for the treatment of otitis media in a mammal, particularly
a human in need thereof. In one embodiment the invention provides a
method for the treatment of primary ciliary dyskinesia, in a
mammal, particularly a human in need thereof. In one embodiment the
invention provides a method for the treatment of distal intestinal
obstruction syndrome, esophagitis, constipation, or chronic
diverticulitis in a mammal, particularly a human in need
thereof.
[0123] There is also provided a compound of the invention for use
in medical therapy, particularly for use in the treatment of
condition in a mammal, such as a human, for which a sodium channel
blocker is indicated. All therapeutic uses described herein are
carried out by administering an effective amount of a compound of
the invention to the subject in need of treatment. In one
embodiment there is provided a compound of the invention for use in
the treatment of a pulmonary condition such as a disease associated
with reversible or irreversible airway obstruction in a mammal,
particularly a human, in need thereof. In one particular embodiment
there is provided a compound of the invention for use in the
treatment of chronic obstructive pulmonary disease (COPD) in a
mammal, particularly a human in need thereof. In one embodiment,
there is provided a compound of the invention for use in reducing
the frequency, severity or duration of acute exacerbation of COPD
or for the treatment of one or more symptoms of acute exacerbation
of COPD, in a mammal, particularly a human, in need thereof. In one
embodiment there is provided a compound of the invention for use in
the treatment of asthma in a mammal, particularly a human, in need
thereof. In one embodiment there is provided a compound for use in
the treatment of bronchiectasis, including bronchiectasis due to
conditions other than cystic fibrosis, or bronchitis, including
acute bronchitis and chronic bronchitis, in a mammal, particularly
a human, in need thereof. In one embodiment there is provided a
compound for use in the treatment of post-viral cough, in a mammal,
particularly a human, in need thereof. In one embodiment there is
provided a compound for use in the treatment of cystic fibrosis in
a mammal, particularly a human in need thereof. In one embodiment
there is provided a compound of the invention for use in the
treatment of emphysema in a mammal, particularly a human, in need
thereof. In one embodiment there is provided a compound of the
invention for use in the treatment of pneumonia in a mammal,
particularly a human, in need thereof. In one embodiment there is
provided a compound of the invention for use in the treatment of
panbronchiolitis or transplant-associated bronchiolitis, including
lung- and bone marrow-transplant associated bronchiolitis in a
mammal, particularly a human, in need thereof. In one embodiment
there is provided a compound of the invention for use in the
treatment of ventilator-associated tracheobronchitis or preventing
ventilator-associated pneumonia in a ventilated human in need
thereof.
[0124] In one embodiment there is provided a compound of the
invention for use in the treatment of a condition ameliorated by
increased mucosal hydration in mucosal surfaces of a mammal,
particularly a human, in need thereof. In one embodiment there is
provided a compound for use in the treatment of dry mouth
(xerostomia) in a mammal, particularly a human, in need thereof. In
one embodiment there is provided a compound for use in the
treatment of dry skin in a mammal, particularly a human, in need
thereof. In one embodiment there is provided a compound for use in
the treatment of vaginal dryness in a mammal, particularly a human
in need thereof. In one embodiment there is provided a compound of
the invention for use in the treatment of sinusitis,
rhinosinusitis, or nasal dehydration, including nasal dehydration
brought on by administering dry oxygen in a mammal, particularly a
human, in need thereof. In one embodiment there is provided a
compound of the invention for use in the treatment of dry eye, or
Sjogren's disease or promoting ocular or corneal hydration in a
mammal, particularly a human, in need thereof. In one embodiment
there is provided a compound of the invention for use in the
treatment of otitis media in a mammal, particularly a human, in
need thereof. In one embodiment there is provided a compound of the
invention for use in the treatment of primary ciliary dyskinesia in
a mammal, particularly a human, in need thereof. In one embodiment
there is provided a compound of the invention for use in the
treatment of distal intestinal obstruction syndrome, esophagitis,
constipation, or chronic diverticulitis in a mammal, particularly a
human, in need thereof.
[0125] The present invention also provides the use of a compound of
the invention in the manufacture of a medicament for the treatment
of a condition in a mammal, such as a human, for which a sodium
channel blocker is indicated. In one embodiment is provided the use
of a compound of the invention in the manufacture of a medicament
for the treatment of diseases associated with reversible or
irreversible airway obstruction, chronic obstructive pulmonary
disease (COPD), acute exacerbations of COPD, asthma, bronchiectasis
(including bronchiectasis due to conditions other than cystic
fibrosis), bronchitis (including acute bronchitis and chronic
bronchitis), post-viral cough, cystic fibrosis, emphysema,
pneumonia, panbronchiolitis, transplant-associated bronchiolitis,
(including lung- and bone marrow-transplant associated
bronchiolitis), ventilator-associated tracheobronchitis or
preventing ventilator-associated pneumonia.
[0126] In one particular embodiment is provided the use of a
compound of the invention in the manufacture of a medicament for
the treatment of a condition ameliorated by increased mucosal
hydration in mucosal surfaces, treatment of dry mouth (xerostomia),
dry skin, vaginal dryness, sinusitis, rhinosinusitis, nasal
dehydration, including nasal dehydration brought on by
administering dry oxygen, treatment of dry eye, Sjogren's disease,
promoting ocular or corneal hydration, treatment of otitis media,
primary ciliary dyskinesia, distal intestinal obstruction syndrome,
esophagitis, constipation, or chronic diverticulitis
[0127] The terms "effective amount", "pharmaceutically effective
amount", "effective dose", and "pharmaceutically effective dose" as
used herein, refer to an amount of compound of the invention which
is sufficient in the subject to which it is administered, to elicit
the biological or medical response of a cell culture, tissue,
system, or mammal (including human) that is being sought, for
instance by a researcher or clinician. The term also includes
within its scope, amounts effective to enhance normal physiological
function. In one embodiment, the effective amount is the amount
needed to provide a desired level of drug in the secretions and
tissues of the airways and lungs, or alternatively, in the
bloodstream of a subject to be treated to give an anticipated
physiological response or desired biological effect when such a
composition is administered by inhalation. For example an effective
amount of a compound of the invention for the treatment of a
condition for which a sodium channel blocker is indicated is
sufficient in the subject to which it is administered to treat the
particular condition. In one embodiment an effective amount is an
amount of a compound of the invention which is sufficient for the
treatment of COPD or cystic fibrosis in a human.
[0128] The precise effective amount of the compounds of the
invention will depend on a number of factors including but not
limited to the species, age and weight of the subject being
treated, the precise condition requiring treatment and its
severity, the bioavailability, potency, and other properties of the
specific compound being administered, the nature of the
formulation, the route of administration, and the delivery device,
and will ultimately be at the discretion of the attendant physician
or veterinarian. Further guidance with respect to appropriate dose
may be found in considering conventional dosing of other sodium
channel blockers, such as amiloride, with due consideration also
being given to any differences in potency between amiloride and the
compounds of the present invention.
[0129] A pharmaceutically effective dose administered topically to
the airway surfaces of a subject (e.g., by inhalation) of a
compound of the invention for treatment of a 70 kg human may be in
the range of from about 10 ng to about 10 mg. In another
embodiment, the pharmaceutically effective dose may be from about
0.1 to about 1000 .mu.g. Typically, the daily dose administered
topically to the airway surfaces will be an amount sufficient to
achieve dissolved concentration of active agent on the airway
surfaces of from about 10.sup.-9, 10.sup.-8, or 10.sup.-7 to about
10.sup.-4, 10.sup.-3, 10.sup.-2, or 10.sup.-1 Moles/liter, more
preferably from about 10.sup.-9 to about 10.sup.-4 Moles/liter. The
selection of the specific dose for a patient will be determined by
the attendant physician, clinician or veterinarian of ordinary
skill in the art based upon a number of factors including those
noted above. In one particular embodiment the dose of a compound of
the invention for the treatment of a 70 kg human will be in the
range of from about 10 nanograms (ng) to about 10 mg. In another
embodiment, the effective dose would be from about 0.1 .mu.g to
about 1,000 .mu.g. In one embodiment, the dose of a compound of the
invention for the treatment of a 70 kg human will be in the range
of from about 0.5 .mu.g to about 0.5 mg. In a further embodiment
the dose will be from about 0.5 .mu.g to about 60 .mu.g. In another
embodiment, the pharmaceutically effective dose will be from about
1 to about 10 .mu.g. In another embodiment, the pharmaceutically
effective dose will be from about 5 .mu.g to about 50 .mu.g.
Another embodiment will have an effective dose of from about 10
.mu.g to about 40 .mu.g. In two further embodiments, the
pharmaceutically effective dose will be from about 15 .mu.g to
about 50 .mu.g from about 15 .mu.g to about 30 .mu.g, respectively.
It will be understood that in each of these dose ranges, all
incremental doses in the range are included. For instance, the
0.5-50 .mu.g range includes individual doses of: 0.5 .mu.g, 0.6
.mu.g, 0.7 .mu.g, 0.8 .mu.g, 0.9 .mu.g, 1.0 .mu.g, 1.1 .mu.g, 1.2
.mu.g, 1.3 .mu.g, 1.4 .mu.g, 1.5 .mu.g, 1.6 .mu.g, 1.7 .mu.g, 1.8
.mu.g, 1.9 .mu.g, 2.0 .mu.g, 2.1 .mu.g, 2.2 .mu.g, 2.3 .mu.g, 2.4
.mu.g, 2.5 .mu.g, 2.6 .mu.g, 2.7 .mu.g, 2.8 .mu.g, 2.9 .mu.g, 3.0
.mu.g, 3.1 .mu.g, 3.2 .mu.g, 3.3 .mu.g, 3.4 .mu.g, 3.5 .mu.g, 3.6
.mu.g, 3.7 .mu.g, 3.8 .mu.g, 3.9 .mu.g, 4.0 .mu.g, 4.1 .mu.g, 4.2
.mu.g, 4.3 .mu.g, 4.4 .mu.g, 4.5 .mu.g, 4.6 .mu.g, 4.7 .mu.g, 4.8
.mu.g, 4.9 .mu.g, 5.0 .mu.g, 5.1 .mu.g, 5.2 .mu.g, 5.3 .mu.g, 5.4
.mu.g, 5.5 .mu.g, 5.6 .mu.g, 5.7 .mu.g, 5.8 .mu.g, 5.9 .mu.g, 6.0
.mu.g, 6.1 .mu.g, 6.2 .mu.g, 6.3 .mu.g, 6.4 .mu.g, 6.5 .mu.g, 6.6
.mu.g, 6.7 .mu.g, 6.8 .mu.g, 6.9 .mu.g, 7.0 .mu.g, 7.1 .mu.g, 7.2
.mu.g, 7.3 .mu.g, 7.4 .mu.g, 7.5 .mu.g, 7.6 .mu.g, 7.7 .mu.g, 7.8
.mu.g, 7.9 .mu.g, 8.0 .mu.g, 8.1 .mu.g, 8.2 .mu.g, 8.3 .mu.g, 8.4
.mu.g, 8.5 .mu.g, 8.6 .mu.g, 8.7 .mu.g, 8.8 .mu.g, 8.9 .mu.g, 9.0
.mu.g, 9.1 .mu.g, 9.2 .mu.g, 9.3 .mu.g, 9.4 .mu.g, 9.5 .mu.g, 9.6
.mu.g, 9.7 .mu.g, 9.8 .mu.g, 9.9 .mu.g. 10.0 .mu.g, 10.1 .mu.g,
10.2 .mu.g, 10.3 .mu.g, 10.4 .mu.g, 10.5 .mu.g, 10.6 .mu.g, 10.7
.mu.g, 10.8 .mu.g, 10.9 .mu.g, 11.0 .mu.g, 11.1 .mu.g, 11.2 .mu.g,
11.3 .mu.g, 11.4 .mu.g, 11.5 .mu.g, 11.6 .mu.g, 11.7 .mu.g, 11.8
.mu.g, 11.9 .mu.g, 12.0 .mu.g, 12.1 .mu.g, 12.2 .mu.g, 12.3 .mu.g,
12.4 .mu.g, 12.5 .mu.g, 12.6 .mu.g, 12.7 .mu.g, 12.8 .mu.g, 12.9
.mu.g, 13.0 .mu.g, 13.1 .mu.g, 13.2 .mu.g, 13.3 .mu.g, 13.4 .mu.g,
13.5 .mu.g, 13.6 .mu.g, 13.7 .mu.g, 13.8 .mu.g, 13.9 .mu.g, 14.0
.mu.g, 14.1 .mu.g, 14.2 .mu.g, 14.3 .mu.g, 14.4 .mu.g, 14.5 .mu.g,
14.6 .mu.g, 14.7 .mu.g, 14.8 .mu.g, 14.9 .mu.g, 15.0 .mu.g, 15.1
.mu.g, 15.2 .mu.g, 15.3 .mu.g, 15.4 .mu.g, 15.5 .mu.g, 15.6 .mu.g,
15.7 .mu.g, 15.8 .mu.g, 15.9 .mu.g, 16.0 .mu.g, 16.1 .mu.g, 16.2
.mu.g, 16.3 .mu.g, 16.4 .mu.g, 16.5 .mu.g, 16.6 .mu.g, 16.7 .mu.g,
16.8 .mu.g, 16.9 .mu.g, 17.0 .mu.g, 17.1 .mu.g, 17.2 .mu.g, 17.3
.mu.g, 17.4 .mu.g, 17.5 .mu.g, 17.6 .mu.g, 17.7 .mu.g, 17.8 .mu.g,
17.9 .mu.g, 18.0 .mu.g, 18.1 .mu.g, 18.2 .mu.g, 18.3 .mu.g, 18.4
.mu.g, 18.5 .mu.g, 18.6 .mu.g, 18.7 .mu.g, 18.8 g, 18.9 .mu.g, 19.0
.mu.g, 19.1 .mu.g, 19.2 .mu.g, 19.3 .mu.g, 19.4 .mu.g, 19.5 .mu.g,
19.6 .mu.g, 19.7 .mu.g, 19.8 .mu.g, 19.9 .mu.g, 20.0 .mu.g, 20.1
.mu.g, 20.2 .mu.g, 20.3 .mu.g, 20.4 .mu.g, 20.5 .mu.g, 20.6 .mu.g,
20.7 .mu.g, 20.8 .mu.g, 20.9 .mu.g, 21.0 .mu.g, 21.1 .mu.g, 21.2
.mu.g, 21.3 .mu.g, 21.4 .mu.g, 21.5 .mu.g, 21.6 .mu.g, 21.7 .mu.g,
21.8 .mu.g, 21.9 .mu.g, 22.0 .mu.g, 22.1 .mu.g, 22.2 .mu.g, 22.3
.mu.g, 22.4 .mu.g, 22.5 .mu.g, 22.6 .mu.g, 22.7 .mu.g, 22.8 .mu.g,
22.9 .mu.g, 23.0 .mu.g, 23.1 .mu.g, 23.2 .mu.g, 23.3 .mu.g, 23.4
.mu.g, 23.5 .mu.g, 23.6 .mu.g, 23.7 .mu.g, 23.8 .mu.g, 23.9 .mu.g,
24.0 .mu.g, 24.1 .mu.g, 24.2 .mu.g, 24.3 .mu.g, 24.4 .mu.g, 24.5
.mu.g, 24.6 .mu.g, 24.7 .mu.g, 24.8 .mu.g, 24.9 .mu.g, 25.0 .mu.g,
25.1 .mu.g, 25.2 .mu.g, 25.3 .mu.g, 25.4 .mu.g, 25.5 .mu.g, 25.6
.mu.g, 25.7 .mu.g, 25.8 .mu.g, 25.9 .mu.g, 26.0 .mu.g, 26.1 .mu.g,
26.2 .mu.g, 26.3 .mu.g, 26.4 .mu.g, 26.5 .mu.g, 26.6 .mu.g, 26.7
.mu.g, 26.8 .mu.g, 26.9 .mu.g, 27.0 .mu.g, 27.1 .mu.g, 27.2 .mu.g,
27.3 .mu.g, 27.4 .mu.g, 27.5 .mu.g, 27.6 .mu.g, 27.7 .mu.g, 27.8
.mu.g, 27.9 .mu.g, 28.0 .mu.g, 28.1 .mu.g, 28.2 .mu.g, 28.3 .mu.g,
28.4 .mu.g, 28.5 .mu.g, 28.6 .mu.g, 28.7 .mu.g, 28.8 .mu.g, 28.9
.mu.g, 29.0 .mu.g, 29.1 .mu.g, 29.2 .mu.g, 29.3 .mu.g, 29.4 .mu.g,
29.5 .mu.g, 29.6 .mu.g, 29.7 .mu.g, 29.8 .mu.g, 29.9 .mu.g, 30.0
.mu.g, 30.1 .mu.g, 30.2 .mu.g, 30.3 .mu.g, 30.4 .mu.g, 30.5 .mu.g,
30.6 .mu.g, 30.7 .mu.g, 30.8 .mu.g, 30.9 .mu.g, 31.0 .mu.g, 31.1
.mu.g, 31.2 .mu.g, 31.3 .mu.g, 31.4 .mu.g, 31.5 .mu.g, 31.6 .mu.g,
31.7 .mu.g, 31.8 .mu.g, 31.9 .mu.g, 32.0 .mu.g, 32.1 .mu.g, 32.2
.mu.g, 32.3 .mu.g, 32.4 .mu.g, 32.5 .mu.g, 32.6 .mu.g, 32.7 .mu.g,
32.8 .mu.g, 32.9 .mu.g, 33.0 .mu.g, 33.1 .mu.g, 33.2 .mu.g, 33.3
.mu.g, 33.4 .mu.g, 33.5 .mu.g, 33.6 .mu.g, 33.7 .mu.g, 33.8 .mu.g,
33.9 .mu.g, 34.0 .mu.g, 34.1 .mu.g, 34.2 .mu.g, 34.3 .mu.g, 34.4
.mu.g, 34.5 .mu.g, 34.6 .mu.g, 34.7 .mu.g, 34.8 .mu.g, 34.9 .mu.g,
35.0 .mu.g, 35.1 .mu.g, 35.2 .mu.g, 35.3 .mu.g, 35.4 .mu.g, 35.5
.mu.g, 35.6 .mu.g, 35.7 .mu.g, 35.8 .mu.g, 35.9 .mu.g, 36.0 .mu.g,
36.1 .mu.g, 36.2 .mu.g, 36.3 .mu.g, 36.4 .mu.g, 36.5 .mu.g, 36.6
.mu.g, 36.7 .mu.g, 36.8 .mu.g, 36.9 .mu.g 37.0 .mu.g, 37.1 .mu.g,
37.2 .mu.g, 37.3 .mu.g, 37.4 .mu.g, 37.5 .mu.g, 37.6 .mu.g, 37.7
.mu.g, 37.8 .mu.g, 37.9 .mu.g, 38.0 .mu.g, 38.1 .mu.g, 38.2 .mu.g,
38.3 .mu.g, 38.4 .mu.g, 38.5 .mu.g, 38.6 .mu.g, 38.7 .mu.g, 38.8
.mu.g, 38.9 .mu.g, 39.0 .mu.g, 39.1 .mu.g, 39.2 .mu.g, 39.3 .mu.g,
39.4 .mu.g, 39.5 .mu.g, 39.6 .mu.g, 39.7 .mu.g, 39.8 .mu.g, 39.9
.mu.g, 40.0 .mu.g, 40.1 .mu.g, 40.2 .mu.g, 40.3 .mu.g, 40.4 .mu.g,
40.5 .mu.g, 40.6 .mu.g, 40.7 .mu.g, 40.8 .mu.g, 40.9 .mu.g, 41.0
.mu.g, 41.1 .mu.g, 41.2 .mu.g, 41.3 .mu.g, 41.4 .mu.g, 41.5 .mu.g,
41.6 .mu.g, 41.7 .mu.g, 41.8 .mu.g, 41.9 .mu.g, 42.0 .mu.g, 42.1
.mu.g, 42.2 .mu.g, 42.3 .mu.g, 42.4 .mu.g, 42.5 .mu.g, 42.6 .mu.g,
42.7 .mu.g, 42.8 .mu.g, 42.9 .mu.g, 43.0 .mu.g, 43.1 .mu.g, 43.2
.mu.g, 43.3 .mu.g, 43.4 .mu.g, 43.5 .mu.g, 43.6 .mu.g, 43.7 .mu.g,
43.8 .mu.g, 43.9 .mu.g, 44.0 .mu.g, 44.1 .mu.g, 44.2 .mu.g, 44.3
.mu.g, 44.4 .mu.g, 44.5 .mu.g, 44.6 .mu.g, 44.7 .mu.g, 44.8 .mu.g,
44.9 .mu.g, 45.0 .mu.g, 45.1 .mu.g, 45.2 .mu.g, 45.3 .mu.g, 45.4
.mu.g, 45.5 .mu.g, 45.6 .mu.g, 45.7 .mu.g, 45.8 .mu.g, 45.9 .mu.g,
46.0 .mu.g, 46.1 .mu.g, 46.2 .mu.g, 46.3 .mu.g, 46.4 .mu.g, 46.5
.mu.g, 46.6 .mu.g, 46.7 .mu.g, 46.8 .mu.g, 46.9 .mu.g, 47.0 .mu.g,
47.1 .mu.g, 47.2 .mu.g, 47.3 .mu.g, 47.4 .mu.g, 47.5 .mu.g, 47.6
.mu.g, 47.7 .mu.g, 47.8 .mu.g, 47.9 .mu.g, 48.0 .mu.g, 48.1 .mu.g,
48.2 .mu.g, 48.3 .mu.g, 48.4 .mu.g, 48.5 .mu.g, 48.6 .mu.g, 48.7
.mu.g, 48.8 .mu.g, 38.9 .mu.g, 49.0 .mu.g, 49.1 .mu.g, 49.2 .mu.g,
49.3 .mu.g, 49.4 .mu.g, 49.5 .mu.g, 49.6 .mu.g, 49.7 .mu.g, 49.8
.mu.g, 39.9 .mu.g, and 50 .mu.g.
[0130] The foregoing suggested doses may be adjusted using
conventional dose calculations if the compound is administered via
a different route. Determination of an appropriate dose for
administration by other routes is within the skill of those in the
art in light of the foregoing description and the general knowledge
in the art.
[0131] Delivery of an effective amount of a compound of the
invention may entail delivery of a single dosage form or multiple
unit doses which may be delivered contemporaneously or separate in
time over a designated period, such as 24 hours. A dose of a
compound of the invention (alone or in the form of a composition
comprising the same) may be administered from one to ten times per
day. Typically, a compound of the invention (alone or in the form
of a composition comprising the same) will be administered four,
three, two, or once per day (24 hours)
[0132] The compounds of formula (I) of the present invention are
also useful for treating airborne infections. Examples of airborne
infections include, for example, RSV. The compounds of formula (I)
of the present invention are also useful for treating an anthrax
infection. The present invention relates to the use of the
compounds of formula (I) of the present invention for prophylactic,
post-exposure prophylactic, preventive or therapeutic treatment
against diseases or conditions caused by pathogens. In a preferred
embodiment, the present invention relates to the use of the
compounds of formula (I) for prophylactic, post-exposure
prophylactic, preventive or therapeutic treatment against diseases
or conditions caused by pathogens which may be used in
bioterrorism.
[0133] In recent years, a variety of research programs and
biodefense measures have been put into place to deal with concerns
about the use of biological agents in acts of terrorism. These
measures are intended to address concerns regarding bioterrorism or
the use of microorganisms or biological toxins to kill people,
spread fear, and disrupt society. For example, the National
Institute of Allergy and Infectious Diseases (NIAID) has developed
a Strategic Plan for Biodefense Research which outlines plans for
addressing research needs in the broad area of bioterrorism and
emerging and reemerging infectious diseases. According to the plan,
the deliberate exposure of the civilian population of the United
States to Bacillus anthracis spores revealed a gap in the nation's
overall preparedness against bioterrorism. Moreover, the report
details that these attacks uncovered an unmet need for tests to
rapidly diagnose, vaccines and immunotherapies to prevent, and
drugs and biologics to cure disease caused by agents of
bioterrorism.
[0134] Much of the focus of the various research efforts has been
directed to studying the biology of the pathogens identified as
potentially dangerous as bioterrorism agents, studying the host
response against such agents, developing vaccines against
infectious diseases, evaluating the therapeutics currently
available and under investigation against such agents, and
developing diagnostics to identify signs and symptoms of
threatening agents. Such efforts are laudable but, given the large
number of pathogens which have been identified as potentially
available for bioterrorism, these efforts have not yet been able to
provide satisfactory responses for all possible bioterrorism
threats. Additionally, many of the pathogens identified as
potentially dangerous as agents of bioterrorism do not provide
adequate economic incentives for the development of therapeutic or
preventive measures by industry. Moreover, even if preventive
measures such as vaccines were available for each pathogen which
may be used in bioterrorism, the cost of administering all such
vaccines to the general population is prohibitive.
[0135] Until convenient and effective treatments are available
against every bioterrorism threat, there exists a strong need for
preventative, prophylactic or therapeutic treatments which can
prevent or reduce the risk of infection from pathogenic agents.
[0136] The present invention provides such methods of prophylactic
treatment. In one aspect, a prophylactic treatment method is
provided comprising administering a prophylactically effective
amount of the compounds of formula (I) to an individual in need of
prophylactic treatment against infection from one or more airborne
pathogens. A particular example of an airborne pathogen is
anthrax.
[0137] In another aspect, a prophylactic treatment method is
provided for reducing the risk of infection from an airborne
pathogen which can cause a disease in a human, said method
comprising administering an effective amount of the compounds of
formula (I) to the lungs of the human who may be at risk of
infection from the airborne pathogen but is asymptomatic for the
disease, wherein the effective amount of a sodium channel blocker
and osmolye are sufficient to reduce the risk of infection in the
human. A particular example of an airborne pathogen is anthrax.
[0138] In another aspect, a post-exposure prophylactic treatment or
therapeutic treatment method is provided for treating infection
from an airborne pathogen comprising administering an effective
amount of the compounds of formula (I) to the lungs of an
individual in need of such treatment against infection from an
airborne pathogen. The pathogens which may be protected against by
the prophylactic post exposure, rescue and therapeutic treatment
methods of the invention include any pathogens which may enter the
body through the mouth, nose or nasal airways, thus proceeding into
the lungs. Typically, the pathogens will be airborne pathogens,
either naturally occurring or by aerosolization. The pathogens may
be naturally occurring or may have been introduced into the
environment intentionally after aerosolization or other method of
introducing the pathogens into the environment. Many pathogens
which are not naturally transmitted in the air have been or may be
aerosolized for use in bioterrorism. The pathogens for which the
treatment of the invention may be useful includes, but is not
limited to, category A, B and C priority pathogens as set forth by
the NIAID. These categories correspond generally to the lists
compiled by the Centers for Disease Control and Prevention (CDC).
As set up by the CDC, Category A agents are those that can be
easily disseminated or transmitted person-to-person, cause high
mortality, with potential for major public health impact. Category
B agents are next in priority and include those that are moderately
easy to disseminate and cause moderate morbidity and low mortality.
Category C consists of emerging pathogens that could be engineered
for mass dissemination in the future because of their availability,
ease of production and dissemination and potential for high
morbidity and mortality. Particular examples of these pathogens are
anthrax and plague. Additional pathogens which may be protected
against or the infection risk therefrom reduced include influenza
viruses, rhinoviruses, adenoviruses and respiratory syncytial
viruses, and the like. A further pathogen which may be protected
against is the coronavirus which is believed to cause severe acute
respiratory syndrome (SARS).
[0139] The present invention also relates to the use of sodium
channel blockers of Formula I, or a pharmaceutically acceptable
salt thereof, for preventing, mitigating, and/or treating
deterministic health effects to the respiratory tract caused by
exposure to radiological materials, particularly respirable
aerosols containing radionuclides from nuclear attacks, such as
detonation of radiological dispersal devices (RDD), or accidents,
such as nuclear power plant disasters. As such, provided herein is
a method for preventing, mitigating, and/or treating deterministic
health effects to the respiratory tract and/or other bodily organs
caused by respirable aerosols containing radionuclides in a
recipient in need thereof, including in a human in need thereof,
said method comprising administering to said human an effective
amount of a compound of Formula (I), or a pharmaceutically
acceptable salt thereof.
[0140] A major concern associated with consequence management
planning for exposures of members of the public to respirable
aerosols containing radionuclides from nuclear attacks, such as
detonation of radiological dispersal devices (RDD), or accidents,
such as nuclear power plant disasters is how to prevent, mitigate
or treat potential deterministic health effects to the respiratory
tract, primarily the lung. It is necessary to have drugs,
techniques and procedures, and trained personnel prepared to manage
and treat such highly internally contaminated individuals.
[0141] Research has been conducted to determine ways in which to
prevent, mitigate or treat potential damage to the respiratory
tract and various organs in the body that is caused by internally
deposited radionuclides. To date, most of the research attention
has focused on strategies designed to mitigate health effects from
internally deposited radionuclides by accelerating their excretion
or removal. These strategies have focused on soluble chemical forms
that are capable of reaching the blood stream and are deposited at
remote systemic sites specific to a given radioelement. Such
approaches will not work in cases where the deposited radionuclide
is in relatively insoluble form. Studies have shown that many, if
not most of the physicochemical forms of dispersed radionuclides
from RDDs, will be in relatively insoluble form.
[0142] The only method known to effectively reduce the radiation
dose to the lungs from inhaled insoluble radioactive aerosols is
bronchoalveolar lavage or BAL. This technique, which was adapted
from that already in use for the treatment of patients with
alveolar proteinosis, has been shown to be a safe, repeatable
procedure, even when performed over an extended period of time.
Although there are variations in procedure, the basic method for
BAL is to anaesthetize the subject, followed by the slow
introduction of isotonic saline into a single lobe of the lung
until the function residual capacity is reached. Additional volumes
are then added and drained by gravity. The results of studies using
BAL on animals indicate that about 40% of the deep lung content can
be removed by a reasonable sequence of BALs. In some studies, there
was considerable variability among animals in the amount of
radionuclide recovered. The reasons for the variability are
currently not understood.
[0143] Further, based on a study on animals, it is believed that a
significant dose reduction from BAL therapy results in mitigation
of health effects due to inhalation of insoluble radionuclides. In
the study, adult dogs inhaled insoluble .sup.144Ce-FAP particles.
Two groups of dogs were given lung contents of .sup.144Ce known to
cause radiation pneumonitis and pulmonary fibrosis (about 2 MBq/kg
body mass), with one group being treated with 10 unilateral lavages
between 2 and 56 days after exposure, the other untreated. A third
group was exposed at a level of .sup.144Ce comparable to that seen
in the BAL-treated group after treatment (about 1 MBq/kg), but
these animals were untreated. All animals were allowed to live
their lifespans, which extended to 16 years. Because there is
variability in initial lung content of .sup.144Ce among the dogs in
each group, the dose rates and cumulative doses for each group
overlap. Nevertheless, the effect of BAL in reducing the risk from
pneumonitis/fibrosis was evident from the survival curves. In the
untreated dogs with lung contents of 1.5-2.5 MBq/kg, the mean
survival time was 370.+-.65 d. For the treated dogs, the mean
survival was 1270.+-.240 d, which was statistically significantly
different. The third group, which received lung contents of
.sup.144Ce of 0.6-1.4 MBq had a mean survival time of 1800.+-.230,
which was not statistically different from the treated group.
Equally important to the increased survival, the dogs in the
high-dose untreated group died from deterministic effects to lung
(pneumonitis/fibrosis) while the treated dogs did not. Instead, the
treated dogs, like the dogs in the low-dose untreated group, mostly
had lung tumors (hemangiosarcoma or carcinoma). Therefore, the
reduction in dose resulting from BAL treatment appears to have
produced biological effects in lung that were predictable based on
the radiation doses that the lungs received.
[0144] Based on these results, it is believed that decreasing the
residual radiological dose further by any method or combination of
methods for enhancing the clearance of particles from the lung
would further decrease the probability of health effects to lung.
However, BAL is a procedure that has many drawbacks. BAL is a
highly invasive procedure that must be performed at specialized
medical centers by trained pulmonologists. As such, a BAL procedure
is expensive. Given the drawbacks of BAL, it is not a treatment
option that would be readily and immediately available to persons
in need of accelerated removal of radioactive particles, for
example, in the event of a nuclear attack. In the event of a
nuclear attack or a nuclear accident, immediate and relatively
easily administered treatment for persons who have been exposed or
who are at risk of being exposed is needed. Sodium channel blockers
administered as an inhalation aerosol have been shown to restore
hydration of airway surfaces. Such hydration of airway surfaces
aids in clearing accumulated mucus secretions and associated
particulate matter from the lung. As such, without being bound by
any particular theory, it is believed that sodium channel blockers
can be used to accelerate the removal of radioactive particles from
airway passages.
[0145] As discussed above, the greatest risk to the lungs following
a radiological attack, such as a dirty bomb, results from the
inhalation and retention of insoluble radioactive particles. As a
result of radioactive particle retention, the cumulative exposure
to the lung is significantly increased, ultimately resulting in
pulmonary fibrosis/pneumonitis and potentially death. Insoluble
particles cannot be systemically cleared by chelating agents
because these particles are not in solution. To date, the physical
removal of particulate matter through BAL is the only therapeutic
regimen shown to be effective at mitigating radiation-induced lung
disease. As discussed above, BAL is not a realistic treatment
solution for reducing the effects of radioactive particles that
have been inhaled into the body. As such, it is desirable to
provide a therapeutic regimen that effectively aids in clearing
radioactive particles from airway passages and that, unlike BAL, is
relatively simple to administer and scalable in a large-scale
radiation exposure scenario. In addition, it is also desirable that
the therapeutic regimen be readily available to a number of people
in a relatively short period of time.
[0146] In an aspect of the present invention, a method for
preventing, mitigating, and/or treating deterministic health
effects to the respiratory tract and/or other bodily organs caused
by respirable aerosols containing radionuclides comprises
administering an effective amount of a sodium channel blocker of
Formula I or a pharmaceutically acceptable salt thereof to an
individual in need. In a feature of this aspect, the sodium channel
blocker is administered in conjunction with an osmolyte. With
further regard to this feature, the osmolyte is hypertonic saline
(HS). In a further feature, the sodium channel blocker and the
osmolyte are administered in conjunction with an ion transport
modulator. With further regard to this feature, the ion transport
modulator may be selected from the group consisting of
.beta.-agonists, CFTR potentiators, purinergic receptor agonists,
lubiprostones, and protease inhibitors. In another feature of this
aspect, the radionuclides are selected from the group consisting of
Cobalt-60, Cesium-137, Iridium-192, Radium-226, Phosphorus-32,
Strontium-89 and 90, Iodine-125, Thallium-201, Lead-210,
Thorium-234, Uranium-238, Plutonium, Cobalt-58, Chromium-51,
Americium, and Curium. In a further feature, the radionuclides are
from a radioactive disposal device. In yet another feature, the
sodium channel blocker or pharmaceutically acceptable salt thereof
is administered in an aerosol suspension of respirable particles
which the individual inhales. In an additional feature, the sodium
channel blocker or a pharmaceutically acceptable salt thereof is
administered post-exposure to the radionuclides.
Compositions
[0147] While it is possible for a compound of the invention to be
administered alone, in some embodiments it is preferable to present
it in the form of a composition, particularly a pharmaceutical
composition (formulation). Thus, in another aspect, the invention
provides compositions, and particularly pharmaceutical compositions
(such as an inhalable pharmaceutical composition) comprising a
pharmaceutically effective amount of a compound of the invention as
an active ingredient, and a pharmaceutically acceptable excipient,
diluent or carrier. The term "active ingredient" as employed herein
refers to any compound of the invention or combination of two or
more compounds of the invention in a pharmaceutical composition.
Also provided are specific embodiments in which a pharmaceutical
composition comprises a pharmaceutically effective amount of a
compound of Formulas (I), (Ia), (II), (III), (IV), (V), (VI), and
(VII), or a pharmaceutically acceptable salt thereof, independently
or in combination, and a pharmaceutically acceptable excipient,
diluent or carrier.
[0148] In some embodiments, the pharmaceutical composition
comprises a pharmaceutically effective amount of a compound of
Formulas (I), (Ia), (II), (III), (IV), (V), (VI), and (VII), or a
pharmaceutically acceptable salt thereof, independently or in
combination, in a diluent. In separate embodiments, the
pharmaceutical composition comprises a pharmaceutically effective
amount of a compound of Formulas (I), (Ia), (II), (III), (IV), (V),
(VI), and (VII), or a pharmaceutically acceptable salt thereof, in
hypertonic saline, sterile water, and hypertonic saline,
respectively, wherein the saline concentration can be as described
herein. In one embodiment the saline concentration is 0.17% w/v and
in another it is 2.8% w/v.
[0149] Also provided is a kit comprising i) a pharmaceutically
effective amount of a compound of Formula (I), (Ia), (II), (III),
(IV), (V), (VI), and (VII), or a pharmaceutically acceptable salt
thereof; ii) one or more pharmaceutically acceptable excipients,
carriers, or diluents; iii) instructions for administering the
compound of group i) and the excipients, carriers, or diluents of
group ii) to a subject in need thereof; and; iv) a container. A
subject in need thereof includes any subject in need of the methods
of treatment described herein, particularly including a human
subject in need thereof. Further embodiments also comprise an
aerosolization device selected from the group of a nebulizer,
including vibrating mesh nebulizers and jet nebulizers, a dry
powder inhaler, including active and passive dry powder inhalers,
and a metered dose inhaler, including pressurized, dry powder, and
soft mist metered dose inhalers.
[0150] In one embodiment a kit comprises i) from about 10 ng to
about 10 mg of a compound of Formula (I), (Ia), (II), (IV), (V),
(VI), and (VII),or a pharmaceutically acceptable salt thereof, per
dose; ii) from about 1 to about 5 mL of diluent per dose; iii)
instructions for administering the compound of group i) and the
diluent of group ii) to a subject in need thereof; and; iv) a
container. In a further embodiment, the diluent is from about 1 to
about 5 mL of a saline solution, as described herein, per dose. In
a further embodiment, the diluent is from about 1 to about 5 mL of
a hypotonic saline solution per dose. In another embodiment, the
diluent is from about 1 to about 5 mL of a hypertonic saline
solution per dose. In a still further embodiment, the diluent is
from about 1 to about 5 mL of sterile water per dose.
[0151] Also provided is a kit comprising i) a solution comprising a
pharmaceutically effective amount of a compound of Formula (I),
(Ia), (II), (III), (IV), (V), (VI), and (VII),or a pharmaceutically
acceptable salt thereof; dissolved in a pharmaceutically acceptable
diluent; iii) instructions for administering the solution of group
i) to a subject in need thereof; and iii) a container.
[0152] Also provided is a kit comprising i) a solution comprising
from about 10 ng to about 10 mg of a compound of Formula (I), (Ia),
(II), (III), (IV), (V), (VI), and (VII), or a pharmaceutically
acceptable salt thereof; dissolved in a pharmaceutically acceptable
diluent; iii) instructions for administering the solution of group
i) to a subject in need thereof; and iii) a container. In a further
embodiment, the diluent is from about 1 to about 5 mL of a saline
solution, as described herein, per dose.
[0153] Another embodiment comprises a kit comprising i) a
pharmaceutically effective amount of a compound of Formula (I),
(Ia), (II), (III), (IV), (V), (VI), and (VII), or a
pharmaceutically acceptable salt thereof; in a dry powder
formulation suitable for inhalation ii) optionally, one or more
pharmaceutically acceptable excipients or carriers suitable for
inhalation; iii) instructions for administering the compound of
group i) and the excipients or carriers of group ii) to a subject
in need thereof; and; iv) a container. In a further embodiment, the
kit also comprises a dry powder inhaler suitable for delivering the
dry powder formulation to a recipient. The dry powder inhaler may
be, in additional embodiments, a single-dose inhaler or a
multi-dose inhaler.
[0154] Further embodiments of each of the kits described herein
includes those in which the concentration of the compound of
Formula (I), (Ia), (II), (III), (IV), (V), (VI), and (VII),or a
pharmaceutically acceptable salt thereof, per dose, is one of the
effective dose ranges described herein, including a) from about 0.1
.mu.g to about 1,000 .mu.g; b) from about 0.5 .mu.g to about 0.5
mg; and c) from about 0.5 .mu.g to about 50 .mu.g.
[0155] For each of the kits described above there is an additional
embodiment in which the diluent is hypertonic saline of the
concentrations described herein. In another embodiment for each kit
the diluent is hypotonic saline of the concentrations described
herein. In a further embodiment for each kit, the diluent is
sterile water suitable for inhalation.
[0156] The pharmaceutically acceptable excipient(s), diluent(s) or
carrier(s) must be acceptable in the sense of being compatible with
the other ingredients of the formulation and not deleterious to the
recipient thereof. Generally, the pharmaceutically acceptable
excipient(s), diluent(s) or carrier(s) employed in the
pharmaceutical formulation are "non-toxic" meaning that it/they
is/are deemed safe for consumption in the amount delivered in the
formulation and "inert" meaning that it/they does/do not
appreciable react with or result in an undesired effect on the
therapeutic activity of the active ingredient(s). Pharmaceutically
acceptable excipients, diluents and carriers are conventional in
the art and may be selected using conventional techniques, based
upon the desired route of administration. See, REMINGTON'S,
PHARMACEUTICAL SCIENCES, Lippincott Williams & Wilkins;
21.sup.st Ed (May 1, 2005). Preferably, the pharmaceutically
acceptable excipient(s), diluent(s) or carrier(s) are Generally
Regarded As Safe (GRAS) according to the FDA.
[0157] Pharmaceutical compositions according to the invention
include those suitable for oral administration; parenteral
administration, including subcutaneous, intradermal, intramuscular,
intravenous and intraarticular; topical administration, including
topical administration to the skin, eyes, ears, etc; vaginal or
rectal administration; and administration to the respiratory tract,
including the nasal cavities and sinuses, oral and extrathoracic
airways, and the lungs, including by use of aerosols which may be
delivered by means of various types of dry powder inhalers,
pressurized metered dose inhalers, softmist inhalers, nebulizers,
or insufflators. The most suitable route of administration may
depend upon, several factors including the patient and the
condition or disorder being treated.
[0158] The formulations may be presented in unit dosage form or in
bulk form as for example in the case of formulations to be metered
by an inhaler and may be prepared by any of the methods well known
in the art of pharmacy. Generally, the methods include the step of
bringing the active ingredient into association with the carrier,
diluent or excipient and optionally one or more accessory
ingredients. In general the formulations are prepared by uniformly
and intimately bringing into association the active ingredient with
one or more liquid carriers, diluents or excipients or finely
divided solid carriers, diluents or excipients, or both, and then,
if necessary, shaping the product into the desired formulation.
[0159] In one preferred embodiment, the composition is an inhalable
pharmaceutical composition which is suitable for inhalation and
delivery to the endobronchial space. Typically, such composition is
in the form of an aerosol comprising particles for delivery using a
nebulizer, pressurized metered dose inhaler (MDI), softmist
inhaler, or dry powder inhaler (DPI). The aerosol formulation used
in the methods of the present invention may be a liquid (e.g.,
solution) suitable for administration by a nebulizer, softmist
inhaler, or MDI, or a dry powder suitable for administration by an
MDI or DPI.
[0160] Aerosols used to administer medicaments to the respiratory
tract are typically polydisperse; that is they are comprised of
particles of many different sizes. The particle size distribution
is typically described by the Mass Median Aerodynamic Diameter
(MMAD) and the Geometric Standard Deviation (GSD). For optimum drug
delivery to the endobronchial space the MMAD is in the range from
about 1 to about 10 .mu.m and preferably from about 1 to about 5
.mu.m, and the GSD is less than 3, and preferably less than about
2. Aerosols having a MMAD above 10 .mu.m are generally too large
when inhaled to reach the lungs. Aerosols with a GSD greater than
about 3 are not preferred for lung delivery as they deliver a high
percentage of the medicament to the oral cavity. To achieve these
particle sizes in powder formulation, the particles of the active
ingredient may be size reduced using conventional techniques such
as micronisation or spray drying. Non-limiting examples of other
processes or techniques that can be used to produce respirable
particles include spray drying, precipitation, supercritical fluid,
and freeze drying. The desired fraction may be separated out by air
classification or sieving. In one embodiment, the particles will be
crystalline. For liquid formulations, the particle size is
determined by the selection of a particular model of nebulizer,
softmist inhaler, or MDI.
[0161] Aerosol particle size distributions are determined using
devices well known in the art. For example a multi-stage Anderson
cascade impactor or other suitable method such as those
specifically cited within the US Pharmacopoeia Chapter 601 as
characterizing devices for aerosols emitted from metered-dose and
dry powder inhalers.
[0162] Dry powder compositions for topical delivery to the lung by
inhalation may be formulated without excipient or carrier and
instead including only the active ingredients in a dry powder form
having a suitable particle size for inhalation. Dry powder
compositions may also contain a mix of the active ingredient and a
suitable powder base (carrier/diluent/excipient substance) such as
mono-, di- or poly-saccharides (e.g., lactose or starch). Lactose
is typically the preferred excipient for dry powder formulations.
When a solid excipient such as lactose is employed, generally the
particle size of the excipient will be much greater than the active
ingredient to aid the dispersion of the formulation in the
inhaler.
[0163] Non-limiting examples of dry powder inhalers include
reservoir multi-dose inhalers, pre-metered multi-dose inhalers,
capsule-based inhalers and single-dose disposable inhalers. A
reservoir inhaler contains a large number of doses (e.g. 60) in one
container. Prior to inhalation, the patient actuates the inhaler
which causes the inhaler to meter one dose of medicament from the
reservoir and prepare it for inhalation. Examples of reservoir DPIs
include but are not limited to the Turbohaler.RTM. by AstraZeneca
and the ClickHaler.RTM. by Vectura.
[0164] In a pre-metered multi-dose inhaler, each individual dose
has been manufactured in a separate container, and actuation of the
inhaler prior to inhalation causes a new dose of drug to be
released from its container and prepared for inhalation. Examples
of multidose DPI inhalers include but are not limited to
Diskus.RTM. by GSK, Gyrohaler.RTM. by Vectura, and Prohaler.RTM. by
Valois. During inhalation, the inspiratory flow of the patient
accelerates the powder out of the device and into the oral cavity.
For a capsule inhaler, the formulation is in a capsule and stored
outside the inhaler. The patient puts a capsule in the inhaler,
actuates the inhaler (punctures the capsule), then inhales.
Examples include the Rotohaler.TM. (GlaxoSmithKline), Spinhaler.TM.
(Novartis), HandiHaler.TM. (IB), TurboSpin.TM. (PH&T). With
single-dose disposable inhalers, the patient actuates the inhaler
to prepare it for inhalation, inhales, then disposes of the inhaler
and packaging. Examples include the Twincer.TM. (U Groningen),
OneDose.TM. (GFE), and Manta Inhaler.TM. (Manta Devices).
[0165] Generally, dry powder inhalers utilize turbulent flow
characteristics of the powder path to cause the excipient-drug
aggregates to disperse, and the particles of active ingredient are
deposited in the lungs. However, certain dry powder inhalers
utilize a cyclone dispersion chamber to produce particles of the
desired respirable size. In a cyclone dispersion chamber, the drug
enters a coin shaped dispersion chamber tangentially so that the
air path and drug move along the outer circular wall. As the drug
formulation moves along this circular wall it bounces around and
agglomerates are broken apart by impact forces. The air path
spirals towards the center of the chamber exiting vertically.
Particles that have small enough aerodynamic sizes can follow the
air path and exit the chamber. In effect, the dispersion chamber
works like a small jet mill. Depending on the specifics of the
formulation, large lactose particles may be added to the
formulation to aid in the dispersion through impact with the API
particles.
[0166] The Twincer.TM. single-dose disposable inhaler appears to
operate using a coin-shaped cyclone dispersion chamber referred to
as an "air classifier." See, U.S. Published Patent Application No.
2006/0237010 to Rijksuniversiteit Groningen. Papers published by
the University of Groningen, have stated that a 60 mg dose of pure
micronized colistin sulfomethate could be effectively delivered as
an inhalable dry powder utilizing this technology.
[0167] In preferred embodiments, the aerosol formulation is
delivered as a dry powder using a dry powder inhaler wherein the
particles emitted from the inhaler have an MMAD in the range of
about 1 .mu.m to about 5 .mu.m and a GSD about less than 2.
[0168] Examples of suitable dry powder inhalers and dry powder
dispersion devices for use in the delivery of compounds and
compositions according to the present invention include but are not
limited to those disclosed in U.S. Pat. No. 7,520,278; U.S. Pat.
No. 7,322,354; U.S. Pat. No. 7,246,617; U.S. Pat. No. 7,231,920;
U.S. Pat. No. 7,219,665; U.S. Pat. No. 7,207,330; U.S. Pat. No.
6,880,555; U.S. Pat. No. 5,522,385; U.S. Pat. No. 6,845,772; U.S.
Pat. No. 6,637,431; U.S. Pat. No. 6,329,034; U.S. Pat. No.
5,458,135; U.S. Pat. No. 4,805,811; and U.S. Published Patent
Application No. 2006/0237010.
[0169] In one embodiment, the pharmaceutical formulation according
to the invention is a dry powder for inhalation which is formulated
for delivery by a Diskus.RTM.-type device. The Diskus.RTM. device
comprises an elongate strip formed from a base sheet having a
plurality of recesses spaced along its length and a lid sheet
hermetically but peelably sealed thereto to define a plurality of
containers, each container having therein an inhalable formulation
containing a predetermined amount of active ingredient either alone
or in admixture with one or more carriers or excipients (e.g.,
lactose) and/or other therapeutically active agents. Preferably,
the strip is sufficiently flexible to be wound into a roll. The lid
sheet and base sheet will preferably have leading end portions
which are not sealed to one another and at least one of the leading
end portions is constructed to be attached to a winding means.
Also, preferably the hermetic seal between the base and lid sheets
extends over their whole width. To prepare the dose for inhalation,
the lid sheet may preferably be peeled from the base sheet in a
longitudinal direction from a first end of the base sheet.
[0170] In one embodiment, the pharmaceutical formulation according
to the invention is a dry powder for inhalation which is formulated
for delivery using a single-dose disposable inhaler, and
particularly the Twincer.TM. inhaler. The Twincer.TM. inhaler
comprises a foil laminate blister with one or more recesses and a
lid sheet hermetically but peelably sealed thereto to define a
plurality of containers. Each container has therein an inhalable
formulation containing a predetermined amount of active
ingredient(s) either alone or in admixture with one or more
carriers or excipients (e.g., lactose). The lid sheet will
preferably have a leading end portion which is constructed to
project from the body of the inhaler. The patient would operate the
device and thereby administer the aerosol formulation by 1)
removing the outer packaging overwrap, 2) pulling the foil tab to
uncover the drug in the blister and 3) inhaling the drug from the
blister.
[0171] In another embodiment, the pharmaceutical formulation
according to the invention is a dry powder for inhalation wherein
the dry powder is formulated into microparticles as described in
PCT Publication No. WO2009/015286 or WO2007/114881, both to NexBio.
Such microparticles are generally formed by adding a counter ion to
a solution containing a compound of the invention in a solvent,
adding an antisolvent to the solution; and gradually cooling the
solution to a temperature below about 25.degree. C., to form a
composition containing microparticles comprising the compound. The
microparticles comprising the compound may then be separated from
the solution by any suitable means such as sedimentation,
filtration or lyophillization. Suitable counterions, solvents and
antisolvents for preparing microparticles of the compounds of the
invention are described in WO2009/015286.
[0172] In another embodiment, a pharmaceutical composition
according to the invention is delivered as a dry powder using a
metered dose inhaler. Non-limiting examples of metered dose
inhalers and devices include those disclosed in U.S. Pat. No.
5,261,538; U.S. Pat. No. 5,544,647; U.S. Pat. No. 5,622,163; U.S.
Pat. No. 4,955,371; U.S. Pat. No. 3,565,070; U.S. Pat. No. 3,361306
and U.S. Pat. No. 6,116,234 and U.S. Pat. No. 7,108,159. In a
preferred embodiment, a compound of the invention is delivered as a
dry powder using a metered dose inhaler wherein the emitted
particles have an MMAD that is in the range of about 1 .mu.m to
about 5 .mu.m and a GSD that is less than about 2.
[0173] Liquid aerosol formulations for delivery to the
endobronchial space or lung by inhalation may for example be
formulated as aqueous solutions or suspensions or as aerosols
delivered from pressurized packs, such as metered dose inhalers,
with the use of suitable liquefied propellants, softmist inhalers,
or nebulizers. Such aerosol compositions suitable for inhalation
can be either a suspension or a solution and generally contain the
active ingredient(s) together with a pharmaceutically acceptable
carrier or diluent (e.g., water (distilled or sterile), saline,
hypertonic saline, or ethanol) and optionally one or more other
therapeutically active agents.
[0174] Aerosol compositions for delivery by pressurized metered
dose inhalers typically further comprise a pharmaceutically
acceptable propellant. Examples of such propellants include
fluorocarbon or hydrogen-containing chlorofluorocarbon or mixtures
thereof, particularly hydrofluoroalkanes, e.g.,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, especially 1,1,1,2-tetrafluoroethane,
1,1,1,2,3,3,3,-heptafluoro-n-propane or a mixture thereof. The
aerosol composition may be excipient free or may optionally contain
additional formulation excipients well known in the art such as
surfactants e.g., oleic acid or lecithin and cosolvents e.g.,
ethanol. Pressurized formulations will generally be retained in a
canister (e.g., an aluminum canister) closed with a valve (e.g., a
metering valve) and fitted into an actuator provided with a
mouthpiece.
[0175] In another embodiment, a pharmaceutical composition
according to the invention is delivered as a liquid using a metered
dose inhaler. Non-limiting examples of metered dose inhalers and
devices include those disclosed in U.S. Pat. Nos. 6,253,762,
6,413,497, 7,601,336, 7,481,995, 6,743,413, and 7,105,152. In a
preferred embodiment, a compound of the invention is delivered as a
dry powder using a metered dose inhaler wherein the emitted
particles have an MMAD that is in the range of about 1.mu.m to
about 5 .mu.m and a GSD that is less than about 2.
[0176] In one embodiment the aerosol formulation is suitable for
aerosolization by a jet nebulizer, or ultrasonic nebulizer
including static and vibrating porous plate nebulizers. Liquid
aerosol formulations for nebulization may be generated by
solubilizing or reconstituting a solid particle formulation or may
be formulated with an aqueous vehicle with the addition of agents
such as acid or alkali, buffer salts, and isotonicity adjusting
agents. They may be sterilized by in-process techniques such as
filtration, or terminal processes such as heating in an autoclave
or gamma irradiation. They may also be presented in non-sterile
form.
[0177] Patients can be sensitive to the pH, osmolality, and ionic
content of a nebulized solution. Therefore these parameters should
be adjusted to be compatible with the active ingredient and
tolerable to patients. The most preferred solution or suspension of
active ingredient will contain a chloride concentration >30 mM
at pH 4.5-7.4, preferably 5.0-5.5, and an osmolality of from about
800-1600 mOsm/kg. The pH of the solution can be controlled by
either titration with common acids (hydrochloric acid or sulfuric
acid, for example) or bases (sodium hydroxide, for example) or via
the use of buffers. Commonly used buffers include citrate buffers,
such as citric acid/sodium citrate buffers, acetate buffers, such
as acetic acid/sodium acetate buffers, and phosphate buffers.
Buffer strengths can range from 2 mM to 50 mM.
[0178] Useful acetate, phosphate, and citrate buffers include
sodium acetate, sodium acetate trihydrate, ammonium acetate,
potassium acetate, sodium phosphate, sodium phosphate dibasic,
disodium hydrogen phosphate, potassium dihydrogen phosphate,
potassium hydrogen phosphate, potassium phosphate, sodium citrate,
and potassium citrate. Other buffers which may be utilized include
sodium hydroxide, potassium hydroxide, ammonium hydroxide,
aminomethylpropanol, tromethamine, tetrahydroxypropyl
ethylenediamine, citric acid, acetic acid, hydroxytricarboxylic
acid or a salt thereof, such as a citrate or sodium citrate salt
thereof, lactic acid, and salts of lactic acid including sodium
lactate, potassium lactate, lithium lactate, calcium lactate,
magnesium lactate, barium lactate, aluminum lactate, zinc lactate,
silver lactate, copper lactate, iron lactate, manganese lactate,
ammonium lactate, monoethanolamine, diethanolamine,
triethanolamine, diisopropanolamine, as well as combinations
thereof, and the like.
[0179] Such formulations may be administered using commercially
available nebulizers or other atomizer that can break the
formulation into particles or droplets suitable for deposition in
the respiratory tract. Non-limiting examples of nebulizers which
may be employed for the aerosol delivery of a composition of the
invention include pneumatic jet nebulizers, vented or
breath-enhanced jet nebulizers, or ultrasonic nebulizers including
static or vibrating porous plate nebulizers. Commercially available
nebulizers include the Aeroneb.RTM. Go nebulizer (Aerogen) and the
eFlow nebulizer (Pari Pharma).
[0180] A jet nebulizer utilizes a high velocity stream of air
blasting up through a column of water to generate droplets.
Particles unsuitable for inhalation impact on walls or aerodynamic
baffles. A vented or breath enhanced nebulizer works in essentially
the same way as a jet nebulizer except that inhaled air passes
through the primary droplet generation area to increase the output
rate of the nebulizer while the patient inhales.
[0181] In an ultrasonic nebulizer, vibration of a piezoelectric
crystal creates surface instabilities in the drug reservoir that
cause droplets to be formed. In porous plate nebulizers pressure
fields generated by sonic energy force liquid through the mesh
pores where it breaks into droplets by Rayleigh breakup. The sonic
energy may be supplied by a vibrating horn or plate driven by a
piezoelectric crystal, or by the mesh itself vibrating.
Non-limiting examples of atomizers include any single or twin fluid
atomizer or nozzle that produces droplets of an appropriate size. A
single fluid atomizer works by forcing a liquid through one or more
holes, where the jet of liquid breaks up into droplets. Twin fluid
atomizers work by either forcing both a gas and liquid through one
or more holes, or by impinging a jet of liquid against another jet
of either liquid or gas.
[0182] The choice of nebulizer which aerosolizes the aerosol
formulation is important in the administration of the active
ingredient(s). Different nebulizers have differing efficiencies
based their design and operation principle and are sensitive to the
physical and chemical properties of the formulation. For example,
two formulations with different surface tensions may have different
particle size distributions. Additionally, formulation properties
such as pH, osmolality, and permeant ion content can affect
tolerability of the medication, so preferred embodiments conform to
certain ranges of these properties.
[0183] In a preferred embodiment, the formulation for nebulization
is delivered to the endobronchial space as an aerosol having an
MMAD between about 1 .mu.m and about 5 .mu.m and a GSD less than 2
using an appropriate nebulizer. To be optimally effective and to
avoid upper respiratory and systemic side effects, the aerosol
should not have a MMAD greater than about 5 .mu.m and should not
have a GSD greater than about 2. If an aerosol has an MMAD larger
than about 5 .mu.m or a GSD greater than about 2 a large percentage
of the dose may be deposited in the upper airways decreasing the
amount of drug delivered to the desired site in the lower
respiratory tract. If the MMAD of the aerosol is smaller than about
1 .mu.m then a large percentage of the particles may remain
suspended in the inhaled air and may then be exhaled during
expiration.
[0184] The compounds of the invention may also be administered by
transbronchoscopic lavage.
[0185] Formulations suitable for oral administration may be
presented as discrete units such as capsules, cachets or tablets,
each containing a predetermined amount of the active ingredient; as
a powder or granules; as a solution or suspension in an aqueous
liquid or a non-aqueous liquid; or as an oil-in-water liquid
emulsion or a water-in-oil liquid emulsion. The active ingredient
may also be presented as a sachet, bolus, electuary or paste.
[0186] A tablet may be made by compression or molding, optionally
with one or more accessory ingredients. Compressed tablets may be
prepared by compressing in a suitable machine the active ingredient
in a free-flowing form such as a powder or granules, optionally
mixed with a binders, lubricant, inert diluent, surface active or
dispersing agent. Molded tablets may be made by molding in a
suitable machine a mixture of the powdered compound moistened with
an inert liquid diluent. The tablets may optionally be coated or
scored and may be formulated so as to provide slow or controlled
release of the active ingredient therein.
[0187] Formulations for topical administration in the mouth, for
example buccally or sublingually, include lozenges, comprising the
active ingredient in a flavored base such as sucrose and acacia or
tragacanth, and pastilles comprising the active ingredient in a
base such as gelatin and glycerin or sucrose and acacia.
[0188] Formulations for parenteral administration include aqueous
and non-aqueous sterile injection solutions which may contain
anti-oxidants, buffers, bacteriostats and solutes which render the
formulation isotonic with the blood of the intended recipient; and
aqueous and non-aqueous sterile suspensions which may include
suspending agents and thickening agents. The formulations may be
presented in unit-dose or multi-dose containers, for example sealed
ampoules and vials, and may be stored in a freeze-dried
(lyophilized) condition requiring only the addition of the sterile
liquid carrier, for example saline or water-for-injection,
immediately prior to use. Extemporaneous injection solutions and
suspensions may be prepared from sterile powders, granules and
tablets of the kind previously described.
[0189] Oral fluids such as solutions, syrups and elixirs can be
prepared in dosage unit form so that a given quantity contains a
predetermined amount of the active ingredient. Syrups can be
prepared by dissolving the active ingredient in a suitably flavored
aqueous solution, while elixirs are prepared through the use of a
pharmaceutically acceptable alcoholic vehicle. Suspensions can be
formulated by dispersing the active ingredient in a
pharmaceutically acceptable vehicle. Solubilizers and emulsifiers
such as ethoxylated isostearyl alcohols and polyoxy ethylene
sorbitol ethers, preservatives, flavor additive such as peppermint
oil or natural sweeteners or saccharin or other artificial
sweeteners, and the like can also be incorporated into oral liquid
compositions.
[0190] Liposome delivery systems such as small unilamellar
vesicles, large unilamellar vesicles and multilamellar vesicles may
also he employed as delivery means for the compounds of the
invention. Liposomes may be formed from a variety of phospholipids
such as cholesterol, stearylamine and phosphatidylcholines.
[0191] Pharmaceutical compositions for topical administration may
be formulated as ointments, creams, suspensions, lotions, powders,
solutions, pastes, gels, sprays, aerosols or oils. Compositions
designed for the treatment of the eyes or other external tissues,
for example the mouth and skin, may be applied as a topical
ointment or cream. When formulated as an ointment, the active
ingredient may be employed with either a paraffinic or a
water-miscible ointment base. Alternatively, the active ingredient
may be formulated in a cream with an oil-in-water cream base or a
water-in-oil base.
[0192] Other compositions designed for topical administration to
the eyes or ears include eye drops and ear drops wherein the active
ingredient is dissolved or suspended in a suitable carrier, such as
for example an aqueous solvent, including saline.
[0193] Compositions designed for nasal administration include
aerosols, solutions, suspensions, sprays, mists and drops.
Aerosolable formulations for nasal administration may be formulated
in much the same ways as aerosolable formulations for inhalation
with the condition that particles of non-respirable size will be
preferred in formulations for nasal administration. Typically,
particles of about 5 microns in size, up to the size of visible
droplets may be employed. Thus, for nasal administration, a
particle size in the range of 10-500 .mu.m may be used to ensure
retention in the nasal cavity.
[0194] Transdermal patches may also be employed, which are designed
to remain in contact with the epidermis of the patient for an
extended period of time and promote the absorption of the active
ingredient there through.
[0195] Compositions for vaginal or rectal administration include
ointments, creams, suppositories and enemas, all of which may be
formulated using conventional techniques.
[0196] In another aspect, the invention provides a method of
promoting hydration of mucosal surfaces or restoring mucosal
defense in a human in need thereof, comprising administering to the
human a pharmaceutical composition comprising a compound of the
invention, wherein said compound is administered in an effective
amount. In one preferred embodiment, the method comprises
administering the pharmaceutical composition as an inhalable
composition comprising an amount of a compound of the invention
that is sufficient to achieve dissolved concentration of the
compound on the airway surfaces of from about 10.sup.-, 10.sup.-8,
or 10.sup.-7 to about 10.sup.-4, 10.sup.-, 10.sup.-2, or 10.sup.-1
Moles/liter, more preferably from about 10.sup.-9 to about
10.sup.-4 Moles/liter.
[0197] In another aspect, the invention provides a method of
treating any one of: a disease associated with reversible or
irreversible airway obstruction, chronic obstructive pulmonary
disease (COPD), asthma, bronchiectasis (including bronchiectasis
due to conditions other than cystic fibrosis), acute bronchitis,
chronic bronchitis, post-viral cough, cystic fibrosis, emphysema,
pneumonia, panbronchiolitis, transplant-associate bronchiolitis,
and ventilator-associated tracheobronchitis or preventing
ventilator-associated pneumonia in a human in need thereof,
comprising administering to the human a pharmaceutical composition
comprising a compound of the invention, wherein said compound is
administered in an effective amount. In one preferred embodiment,
the method comprises administering the pharmaceutical composition
as an inhalable composition comprising an amount of a compound of
the invention that is sufficient to achieve dissolved concentration
of the compound on the airway surfaces of from about 10.sup.-9,
10.sup.-8, or 10.sup.-7 to about 10.sup.-4, 10.sup.-3, 10.sup.-2,
or 10.sup.-1 Moles/liter, more preferably from about 10.sup.-9 to
about 10.sup.-4 Moles/liter.
[0198] In another aspect, the invention provides a method of
treating any one of dry mouth (xerostomia), dry skin, vaginal
dryness, sinusitis, rhinosinusitis, or nasal dehydration, including
nasal dehydration brought on by administering dry oxygen, dry eye
or Sjogren's disease, promoting ocular or corneal hydration,
treating distal intestinal obstruction syndrome, treating otitis
media, primary ciliary diskinesia, distal intestinal obstruction
syndrome, esophagitis, constipation, or chronic diverticulitis in a
human in need thereof, comprising administering to the human a
pharmaceutical composition comprising a compound of the invention,
wherein said compound is administered in an effective amount.
[0199] Preferred unit dosage formulations for the compounds of the
invention are those containing an effective amount of the active
ingredient or an appropriate fraction thereof.
[0200] It should be understood that in addition to the ingredients
particularly mentioned above, the formulations of this invention
may include other agents conventional in the art having regard to
the type of formulation in question for example those suitable for
oral administration may include flavoring agents.
[0201] The compositions of the present invention may be formulated
for immediate, controlled or sustained release as desired for the
particular condition being treated and the desired route of
administration. For example, a controlled release formulation for
oral administration may be desired for the treatment of
constipation in order to maximize delivery of the active agent to
colon. Such formulations and suitable excipients for the same are
well known in the art of pharmacy. Because the free base of the
compound is generally less soluble in aqueous solutions than the
salt, compositions comprising a free base of a compound of Formula
I may be employed to provide more sustained release of active agent
delivered by inhalation to the lungs. An active agent present in
the lungs in particulate form which has not dissolved into solution
is not available to induce a physiological response, but serves as
a depot of bioavailable drug which gradually dissolves into
solution. As another example, a formulation may employ both a free
base and salt form of a compound of the invention to provide both
immediate release and sustained release of the active ingredient
for dissolution into the mucus secretions of, for example, the
nose.
Combinations
[0202] The compounds of the invention may be formulated and/or used
in combination with other therapeutically active agents. Examples
of other therapeutically active agents which may be formulated or
used in combination with the compounds of the invention include but
are not limited to osmolytes, anti-inflammatory agents,
anticholinergic agents, .beta.-agonists (including selective
.beta..sub.2-agonists), P2Y2 receptor agonists, peroxisome
proliferator-activated receptor (PPAR) delta agonists, other
epithelial sodium channel blockers (ENaC receptor blockers), cystic
fibrosis transmembrane conductance regulator (CFTR) modulators,
kinase inhibitors, antiinfective agents, antihistamines,
non-antibiotic anti-inflammatory macrolides, elastase and protease
inhibitors, and mucus or mucin modifying agents, such as
surfactants. In addition, for cardiovascular indications, the
compounds of the invention may be used in combination with beta
blockers, ACE inhibitors, HMGCoA reductase inhibitors, calcium
channel blockers and other cardiovascular agents.
[0203] The present invention thus provides, as another aspect, a
composition comprising an effective amount of a compound of the
invention and one or more other therapeutically active agents
selected from osmolytes, anti-inflammatory agents, anticholinergic
agents, .beta.-agonists (including selective
.beta..sub.2-agonists), P2Y2 receptor agonists, PPAR delta
agonists, ENaC receptor blockers, cystic fibrosis transmembrane
conductance regulator (CFTR) modulators, kinase inhibitors,
antiinfective agents, antihistamines, non-antibiotic
anti-inflammatory macrolides, elastase and protease inhibitors, and
mucus or mucin modifying agents, such as surfactants. The present
invention thus provides, as another aspect, a composition
comprising an effective amount of a compound of the invention and
one or more other therapeutically active agents selected from beta
blockers, ACE inhibitors, HMGCoA reductase inhibitors, and calcium
channel blockers. Use of the compounds of the invention in
combination with one or more other therapeutically active agents
(particularly osmolytes) may lower the dose of the compound of the
invention that is required to sufficiently hydrate mucosal
surfaces, thereby reducing the potential for undesired side-effects
attributable to systemic blocking of sodium channels such as for
example in the kidneys.
[0204] "Osmolytes" according to the present invention are molecules
or compounds that are osmotically active. "Osmotically active"
molecules and compounds are membrane-impermeable (i.e., essentially
non-absorbable) on the airway or pulmonary epithelial surface. The
terms "airway surface" and "pulmonary surface," as used herein,
include pulmonary airway surfaces such as the bronchi and
bronchioles, alveolar surfaces, and nasal and sinus surfaces.
Suitable osmolytes include ionic osmolytes (i.e., salts), and
non-ionic osmolytes (i.e., sugars, sugar alcohols, and organic
osmolytes). In general, osmolytes (both ionic and non-ionic) used
in combination with the compounds of the invention are preferably
osmolytes that do not promote, or in fact deter or retard bacterial
growth. Osmolytes suitable for use in the present invention may be
in racemic form or in the form of an enantiomer, diastereomer,
tautomer, polymorph or pseudopolymorph.
[0205] Examples of ionic osmolytes useful in the present invention
include any salt of a pharmaceutically acceptable anion and a
pharmaceutically acceptable cation. Preferably, either (or both) of
the anion and cation are osmotically active and not subject to
rapid active transport, in relation to the airway surfaces to which
they are administered. Such compounds include but are not limited
to anions and cations that are contained in FDA approved
commercially marketed salts, see, e.g., Remington: The Science and
Practice of Pharmacy, Vol. II, pg. 1457 (19.sup.th Ed. 1995), and
can be used in any combination as known in the art.
[0206] Specific examples of pharmaceutically acceptable osmotically
active anions include but are not limited to, acetate,
benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide,
calcium edetate, camsylate (camphorsulfonate), carbonate, chloride,
citrate, dihydrochloride, edetate, edisylate
(1,2-ethanedisulfonate), estolate (lauryl sulfate), esylate
(1,2-ethanedisulfonate), fumarate, gluceptate, gluconate,
glutamate, glycollylarsanilate (p-glycollamidophenylarsonate),
hexylresorcinate, hydrabamine
(N,N'-Di(dehydroabietyl)ethylenediamine), hydrobromide,
hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate,
lactobionate, malate, maleate, mandelate, mesylate, methylbromide,
methylnitrate, methylsulfate, mucate, napsylate, nitrate, nitrite,
pamoate (embonate), pantothenate, phosphate or diphosphate,
polygalacturonate, salicylate, stearate, subacetate, succinate,
sulfate, tannate, tartrate, teoclate (8-chlorotheophyllinate),
triethiodide, bicarbonate, etc. Preferred anions include chloride,
sulfate, nitrate, gluconate, iodide, bicarbonate, bromide, and
phosphate.
[0207] Specific examples of pharmaceutically acceptable osmotically
active cations include but are not limited to, organic cations such
as benzathine (N,N'-dibenzylethylenediamine), chloroprocaine,
choline, diethanolamine, ethylenediamine, meglumine (N-methyl
D-glucamine), procaine, D-lysine, L-lysine, D-arginine, L-arginine,
triethylammonium, N-methyl D-glycerol, and the like; and metallic
cations such as aluminum, calcium, lithium, magnesium, potassium,
sodium, zinc, iron, ammonium, and the like. Preferred organic
cations include 3-carbon, 4-carbon, 5-carbon and 6-carbon organic
cations. Preferred cations include sodium, potassium, choline,
lithium, meglumine, D-lysine, ammonium, magnesium, and calcium.
[0208] Specific examples of ionic osmolytes that may be used in
combination with a compound of the invention include but are not
limited to, sodium chloride (particularly hypertonic saline),
potassium chloride, choline chloride, choline iodide, lithium
chloride, meglumine chloride, L-lysine chloride, D-lysine chloride,
ammonium chloride, potassium sulfate, potassium nitrate, potassium
gluconate, potassium iodide, ferric chloride, ferrous chloride,
potassium bromide, and combinations of any two or more of the
foregoing. In one embodiment, the present invention provides a
combination of a compound of the invention and two different
osmotically active salts. When different salts are used, one of the
anion or cation may be the same among the differing salts.
Hypertonic saline is a preferred ionic osmolyte for use in
combination with the compounds of the invention.
[0209] Non-ionic osmolytes include sugars, sugar-alcohols, and
organic osmolytes. Sugars and sugar-alcohols useful as osmolytes in
the present invention include but are not limited to 3-carbon
sugars (e.g., glycerol, dihydroxyacetone); 4-carbon sugars (e.g.,
both the D and L forms of erythrose, threose, and erythrulose);
5-carbon sugars (e.g., both the D and L forms of ribose, arabinose,
xylose, lyxose, psicose, fructose, sorbose, and tagatose); and
6-carbon sugars (e.g., both the D and L forms of altose, allose,
glucose, mannose, gulose, idose, galactose, and talose, and the D
and L forms of allo-heptulose, allo-hepulose, gluco-heptulose,
manno-heptulose, gulo-heptulose, ido-heptulose, galacto-heptulose,
talo-heptulose). Additional sugars useful in the practice of the
present invention include raffinose, raffinose series
oligosaccharides, and stachyose. Both the D and L forms of the
reduced form of each sugar/sugar alcohol are also suitable for the
present invention. For example, glucose, when reduced, becomes
sorbitol; an osmolyte within the scope of the invention.
Accordingly, sorbitol and other reduced forms of sugar/sugar
alcohols (e.g., mannitol, dulcitol, arabitol) are suitable
osmolytes for use in the present invention. Mannitol is a preferred
non-ionic osmolyte for use in combination with the compounds of the
invention.
[0210] "Organic osmolytes" is generally used to refer to molecules
that control intracellular osmolality in the kidney. See e.g., J.
S. Handler et al., Comp. Biochem. Physiol, 117, 301-306 (1997); M.
Burg, Am. J. Physiol. 268, F983-F996 (1995). Organic osmolytes
include but are not limited to three major classes of compounds:
polyols (polyhydric alcohols), methylamines, and amino acids.
Suitable polyol organic osmolytes include but are not limited to,
inositol, myo-inositol, and sorbitol. Suitable methylamine organic
osmolytes include but are not limited to, choline, betaine,
carnitine (L-, D- and DL forms), phosphorylcholine,
lyso-phosphorylcholine, glycerophosphorylcholine, creatine, and
creatine phosphate. Suitable amino acid organic osmolytes include
but are not limited to, the D- and L-forms of glycine, alanine,
glutamine, glutamate, aspartate, proline and taurine. Additional
organic osmolytes suitable for use in the present invention include
tihulose and sarcosine. Mammalian organic osmolytes are preferred,
with human organic osmolytes being most preferred. However, certain
organic osmolytes are of bacterial, yeast, and marine animal
origin, and these compounds may also be employed in the present
invention.
[0211] Osmolyte precursors may be used in combination with the
compounds of the invention An "osmolyte precursor" as used herein
refers to a compound which is converted into an osmolyte by a
metabolic step, either catabolic or anabolic. Examples of osmolyte
precursors include but are not limited to, glucose, glucose
polymers, glycerol, choline, phosphatidylcholine,
lyso-phosphatidylcholine and inorganic phosphates, which are
precursors of polyols and methylamines. Precursors of amino acid
osmolytes include proteins, peptides, and polyamino acids, which
are hydrolyzed to yield osmolyte amino acids, and metabolic
precursors which can be converted into osmolyte amino acids by a
metabolic step such as transamination. For example, a precursor of
the amino acid glutamine is poly-L-glutamine, and a precursor of
glutamate is poly-L-glutamic acid.
[0212] Chemically modified osmolytes or osmolyte precursors may
also be employed. Such chemical modifications involve linking the
osmolyte (or precursor) to an additional chemical group which
alters or enhances the effect of the osmolyte or osmolyte precursor
(e.g., inhibits degradation of the osmolyte molecule). Such
chemical modifications have been utilized with drugs or prodrugs
and are known in the art. (See, for example, U.S. Pat. Nos.
4,479,932 and 4,540,564; Shek, E. et at., J. Med. Chem. 19:113-117
(1976); Bodor, N. et al., J. Pharm. Sci. 67:1045-1050 (1978);
Bodor, N. et al., J. Med. Chem. 26:313-318 (1983); Bodor, N. et
al., J. Pharm. Sci. 75:29-35 (1986).
[0213] Preferred osmolytes for use in combination with the
compounds of the invention include sodium chloride, particular
hypertonic saline, and mannitol.
[0214] For the formulation of 7% and >7% hypertonic saline,
formulations containing bicarbonate anions may be particularly
useful, especially for respiratory disorders with cystic fibrosis
transmembrane conductance regulator (CFTR) dysfunction such as CF
or COPD. Recent findings indicate that, although the relative ratio
of HCO.sub.3.sup.- conductance/Cl.sup.- conductance is between 0.1
and 0.2 for single CFTR channels activated with cAMP and ATP, the
ratio in the sweat duct can range from virtually 0 to almost 1.0,
depending on conditions of stimulation. That is, combining
cAMP+cGMP+.alpha.-ketoglutarate can yield CFTR HCO.sub.3.sup.-
conductance almost equal to that of Cl.sup.- conductance (Quiton et
al. Physiology, Vol. 22, No. 3, 212-225, June 2007). Furthermore,
formulations of 7% and >7% hypertonic saline containing
bicarbonate anions may be particularly useful due to better control
of the pH in the airway surface liquid. First, it has shown that
that airway acidification occurs in CF (Tate et al. 2002) and that
absent CFTR-dependent bicarbonate secretion can lead to an impaired
capacity to respond to airway conditions associated with
acidification of airway surface liquid layer (Coakley et al. 2003).
Second, addition of HS solution without bicarbonate to the surface
of the lung may further dilute the bicarbonate concentrations, and
potentially reduce the pH or the ability to respond to airway
acidification within the airway surface liquid layer. Therefore
addition of bicarbonate anions to HS may help maintain or improve
the pH of airway surface liquid layer in CF patients.
[0215] Due to this evidence, inclusion of bicarbonate anion in the
formulation of 7% or >7% hypertonic saline administered by the
method of this invention would be particularly useful. Formulations
containing up to 30 to 200 mM concentrations of bicarbonate anions
are of particular interest for 7% or >7% HS solutions.
[0216] Hypertonic saline is understood to have a salt concentration
greater than that of normal saline (NS), i.e. greater than 9 g/L or
0.9% w/v, and hypotonic saline has a salt concentration less than
that of normal saline, such as from about 1 g or L/0.1% w/v to
about 8 g/L or 0.8% w/v. Hypertonic saline solutions useful in the
formulations and methods of treatment herein may have a salt
concentration from about 1% to about 23.4% (w/v). In one embodiment
the hypertonic saline solution has a salt concentration from about
60 g/L (6% w/v) to about 100 g/L (10% w/v). In another embodiment,
the saline solution has a salt concentration from about 70 g/L (7%
w/v) to about 100 g/L (10% w/v). In further embodiments, the saline
solution has salt concentrations of a) from about 0.5 g/L (0.05%
w/v) to about 70 g/L (7% w/v); b) from about 1 g/L (0.1% w/v) to
about 60 g/L (6% w/v); c) from about 1 g/L (0.1% w/v) to about 50
g/L (5% w/v); d) from about 1 g/L (0.1% w/v) to about 40 g/L (4%
w/v); e) from about 1 g/L (0.1% w/v) to about 30 g/L (3% w/v); and
f) from about 1 g/L (0.1% w/v) to about 20 g/L (2% w/v).
[0217] Specific concentrations of saline solutions useful in the
formulations and methods of treatment herein include,
independently, those having salt concentrations of 1 g/L (0.1%
w/v), 2 g/L (0.2% w/v), 3 g/L (0.3% w/v), 4 g/L (0.4% w/v), 5 g/L
(0.5% w/v), 6 g/L (0.6% w/v), 7 g/L (0.7% w/v), 8 g/L (0.8% w/v), 9
g/L (0.9% w/v), 10 g/L (1% w/v), 20 g/L (2% w/v), 30 g/L (3% w/v),
40 g/L (4% w/v), 50 g/L (5% w/v), 60 g/L (6% w/v), 70 g/L (7% w/v),
80 g/L (8% w/v), 90 g/L (9% w/v), 100 g/L (10% w/v), 110 g/L (11%
w/v), 120 g/L (12% w/v), 130 g/L (13% w/v), 140 g/L (14% w/v), 150
g/L (15% w/v), 160 g/L (16% w/v), 170 g/L (17% w/v), 180 g/L (18%
w/v), 190 g/L (19% w/v), 200 g/L (20% w/v), 210 g/L (21% w/v), 220
g/L (22% w/v), and 230 g/L (23% w/v).
[0218] Saline concentrations between each of these listed
concentrations/percentages may also be used, such as saline of 1.7
g/L (0.17% w/v), 1.25 g/L (1.25% w/v), 1.5 g/L (1.5% w/v), 25 g/L
(2.5% w/v), 28 g/L (2.8% w/v), 35 g/L (3.5% w/v), 45 g/L (4.5%
w/v), and 75 g/L (7.5% w/v).
[0219] Specific useful concentration of hypotonic saline solutions
include those from about 0.12 g/L (0.012% w/v) to about 8.5 g/L
(0.85% w/v). Any concentration within this range may be used, such
as, on a w/v basis, 0.05%, 0.1%, 0.15%, 0.2%, 0.225% (1/4 NS),
0.25%, 0.3% (1/3 NS), 0.35%, 0.4%, 0.45% (1/2 NS), 0.5%, 0.55%,
0.6% (2/3 NS), 0.65%, 0.675% (3/4 NS), 0.7%, 0.75%, and 0.8%.
[0220] Each of the ranges and specific concentrations of saline
described herein may be used with the formulations, methods of
treatment, regimens, and kits described herein.
[0221] Also intended within the scope of this invention are
chemically modified osmolytes or osmolyte precursors. Such chemical
modifications involve linking to the osmolyte (or precursor) an
additional chemical group which alters or enhances the effect of
the osmolyte or osmolyte precursor (e.g., inhibits degradation of
the osmolyte molecule). Such chemical modifications have been
utilized with drugs or prodrugs and are known in the art. (See, for
example, U.S. Pat. Nos. 4,479,932 and 4,540,564; Shek, E. et al.,
J. Med. Chem. 19:113-117 (1976); Bodor, N. et al., J. Pharm. Sci.
67:1045-1050 (1978); Bodor, N. et al., J. Med. Chem. 26:313-318
(1983); Bodor, N. et al., J. Pharm. Sci. 75:29-35 (1986), each
incorporated herein by reference.
[0222] Suitable anti-inflammatory agents for use in combination
with the compounds of the invention include corticosteroids and
non-steroidal anti-inflammatory drugs (NSAIDs), particularly
phosphodiesterase (PDE) inhibitors. Examples of corticosteroids for
use in the present invention include oral or inhaled
corticosteroids or prodrugs thereof. Specific examples include but
are not limited to ciclesonide, desisobutyryl-ciclesonide,
budesonide, flunisolide, mometasone and esters thereof (e.g.,
mometasone furoate), fluticasone propionate, fluticasone furoate,
beclomethasone, methyl prednisolone, prednisolone, dexamethasone,
6.alpha.,9.alpha.-difluoro-17.alpha.-[(2-furanylcarbonyl)oxy]-11.beta.-hy-
droxy-16.alpha.-methyl-3-oxo-androsta-1,4-diene-17.beta.-carbothioic
acid S-fluoromethyl ester,
6.alpha.,9.alpha.-difluoro-11.beta.-hydroxy-16.alpha.-methyl-3-oxo-17.alp-
ha.-propionyloxy-androsta-1,4-diene-17.beta.-carbothioic acid
S-(2-oxo-tetrahydro-furan-3S-yl) ester, beclomethasone esters
(e.g., the 17-propionate ester or the 17,21-dipropionate ester,
fluoromethyl ester, triamcinolone acetonide, rofleponide, or any
combination or subset thereof. Preferred corticosteroids for
formulation or use in combination with the compounds of the
invention are selected from ciclesonide, desisobutyryl-ciclesonide,
budesonide, mometasone, fluticasone propionate, and fluticasone
furoate, or any combination or subset thereof.
[0223] NSAIDs for use in the present invention include but are not
limited to sodium cromoglycate, nedocromil sodium,
phosphodiesterase (PDE) inhibitors (e.g., theophylline,
aminophylline, PDE4 inhibitors, mixed PDE3/PDE4 inhibitors or mixed
PDE4/PDE7 inhibitors), leukotriene antagonists, inhibitors of
leukotriene synthesis (e.g., 5 LO and FLAP inhibitors), nitric
oxide synthase (iNOS) inhibitors, protease inhibitors (e.g.,
tryptase inhibitors, neutrophil elastase inhibitors, and
metalloprotease inhibitors) .beta.2-integrin antagonists and
adenosine receptor agonists or antagonists (e.g., adenosine 2a
agonists), cytokine antagonists (e.g., chemokine antagonists) or
inhibitors of cytokine synthesis (e.g., prostaglandin D2 (CRTh2)
receptor antagonists). Examples of leukotriene modifiers suitable
for administration by the method of this invention include
montelukast, zileuton and zafirlukast,
[0224] The PDE4 inhibitor, mixed PDE3/PDE4 inhibitor or mixed
PDE4/PDE7 inhibitor may be any compound that is known to inhibit
the PDE4 enzyme or which is discovered to act as a PDE4 inhibitor,
and which are selective PDE4 inhibitors (i.e., compounds which do
not appreciably inhibit other members of the PDE family). Examples
of specific PDE4 inhibitors for formulation and use in combination
with the compounds of the present invention include but are not
limited to roflumilast, pumafentrine, arofylline, cilomilast,
tofimilast, oglemilast, tolafentrine, piclamilast, ibudilast,
apremilast,
2-[4-[6,7-diethoxy-2,3-bis(hydroxymethyl)-1-naphthalenyl]-2-pyridinyl]-4--
(3-pyridinyl)-1(2H)-phthalazinone (T2585),
N-(3,5-dichloro-4-pyridinyl)-1-[(4-fluorophenyl)methyl]-5-hydroxy-.alpha.-
-oxo-1H-indole-3-acetamide (AWD-12-281,
4-[(2R)-2-[3-(cyclopentyloxy)-4-methoxyphenyl]-2-phenylethyl]-pyridine
(CDP-840),
2-[4-[[[[2-(1,3-benzodioxol-5-yloxy)-3-pyridinyl]carbonyl]amino]methyl]-3-
-fluorophenoxy]-(2R)-propanoic acid (CP-671305),
N-(4,6-dimethyl-2-pyrimidinyl)-4-[4,5,6,7-tetrahydro-2-(4-methoxy-3-methy-
lphenyl)-5-(4-methyl-1-piperazinyl)-1H-indol-1-yl]-benzenesulfonamide,
(2E)-2-butenedioate (YM-393059),
9-[(2-fluorophenyl)methyl]-N-methyl-2-(trifluoromethyl)-9H-purin-6-amine
(NCS-613),
N-(2,5-dichloro-3-pyridinyl)-8-methoxy-5-quinolinecarboxamide
(D-4418),
N-[(3R)-9-amino-3,4,6,7-tetrahydro-4-oxo-1-phenylpyrrolo[3,2,1--
][1,4]benzodiazepin-3-yl]-3H-purin-6-amine (PD-168787),
3-[[3-(cyclopentyloxy)-4-methoxyphenyl]methyl]-N-ethyl-8-(1-methylethyl)--
3H-purin-6-amine hydrochloride (V-11294A),
N-(3,5-dichloro-1-oxido-4-pyridinyl)-8-methoxy-2-(trifluoromethyl)-5-quin-
olinecarboxamide (Sch351591),
5-[3-(cyclopentyloxy)-4-methoxyphenyl]-3-[(3-methylphenyl)methyl]-(3S,5S)-
-2-piperidinone (HT-0712),
5-(2-((1R,4R)-4-amino-1-(3-(cyclopentyloxy)-4-methyoxyphenyl)cyclohexyl)
ethynyl)-pyrimidine-2-amine,cis-[4-cyano-4-(3-cyclopropylmethoxy-4-difluo-
romethoxy phenyl)cyclohexan-1-ol], and
4-[6,7-diethoxy-2,3-bis(hydroxymethyl)-1-naphthalenyl]-1-(2-methoxyethyl)-
-2(1H)-pyridinone (T-440), and any combination or subset
thereof.
[0225] Leukotriene antagonists and inhibitors of leukotriene
synthesis include zafirlukast, montelukast sodium, zileuton, and
pranlukast.
[0226] Anticholinergic agents for formulation or use in combination
with the compounds of the invention include but are not limited to
muscarinic receptor antagonists, particularly including pan
antagonists and antagonists of the M.sub.3 receptors. Exemplary
compounds include the alkaloids of the belladonna plants, such as
atropine, scopolamine, homatropine, hyoscyamine, and the various
forms including salts thereof (e.g., anhydrous atropine, atropine
sulfate, atropine oxide or HCl, methylatropine nitrate, homatropine
hydrobromide, homatropine methyl bromide, hyoscyamine hydrobromide,
hyoscyamine sulfate, scopolamine hydrobromide, scopolamine methyl
bromide), or any combination or subset thereof.
[0227] Additional anticholinergics for formulation and use in
combination with the methantheline, propantheline bromide,
anisotropine methyl bromide or Valpin 50, aclidinium bromide,
glycopyrrolate (Robinul), isopropamide iodide, mepenzolate bromide,
tridihexethyl chloride, hexocyclium methylsulfate, cyclopentolate
HCl, tropicamide, trihexyphenidyl CCl, pirenzepine, telenzepine,
and methoctramine, or any combination or subset thereof.
[0228] Preferred anticholinergics formulation and use in
combination with the compounds of the invention include ipratropium
(bromide), oxitropium (bromide) and tiotropium (bromide), or any
combination or subset thereof.
[0229] Examples of .beta.-agonists for formulation and use in
combination with the compounds of the invention include but are not
limited to salmeterol, R-salmeterol, and xinafoate salts thereof,
albuterol or R-albuterol (free base or sulfate), levalbuterol,
salbutamol, formoterol (fumarate), fenoterol, procaterol,
pirbuterol, metaprterenol, terbutaline and salts thereof, and any
combination or subset thereof.
[0230] P2Y2 receptor agonists for formulation and use in
combination with the compounds of the invention may be employed in
an amount effective to stimulate chloride and water secretion by
airway surfaces, particularly nasal airway surfaces. Suitable P2Y2
receptor agonists are known in the art and are described for
example, in columns 9-10 of U.S. Pat. No. 6,264,975, and also U.S.
Pat. Nos. 5,656,256 and 5,292,498.
[0231] P2Y.sub.2 agonists that can be administered by the methods
of this invention include P2Y.sub.2 receptor agonists such as ATP,
UTP, UTP-.gamma.-S and dinucleotide P2Y.sub.2 receptor agonists
(e.g. denufosol or diquafosol) or a pharmaceutically acceptable
salt thereof. The P2Y.sub.2 receptor agonist is typically included
in an amount effective to stimulate chloride and water secretion by
airway surfaces, particularly nasal airway surfaces. Suitable P2Y2
receptor agonists are described in, but are not limited to, U.S.
Pat. No. 6,264,975, U.S. Pat. No.5,656,256, U.S. Pat. No.
5,292,498, U.S. Pat. No. 6,348,589, U.S. Pat. No. 6,818,629, U.S.
Pat. No. 6,977,246, U.S. Pat. No. 7,223,744, U.S. Pat. No.7,531,525
and U.S. Pat.AP.2009/0306009 each of which is incorporated herein
by reference.
[0232] Combination therapies and formulations herein can include
adenosine 2b (A2b) agonists, also, including BAY 60-6583, NECA
(N-ethylcarboxamidoadenosine), (S)-PHPNECA, LUF-5835 and LUF-5845.
A2b agonists that may be used are described by Volpini et al.,
Journal of Medicinal Chemistry 45 (15): 3271-9 (2002); Volpini et
al., Current Pharmaceutical Design 8 (26); 2285-98 (2002); Baraldi
et al., Journal of Medicinal Chemistry 47 (6): Cacciari et al.,
1434-47 (2004); Mini Reviews in Medicinal Chemistry 5 (12): 1053-60
(December 2005); Baraldi et al., Current Medicinal Chemistry 13
(28): 3467-82 (2006); Beukers et al., Medicinal Research Reviews 26
(5): 667-98 (September 2006); Elzein et al., Bioorganic &
Medicinal Chemistry Letters 16 (2): 302-6 (January 2006); Carotti,
et al., Journal of Medicinal Chemistry 49 (1): 282-99 (January
2006); Tabrizi et al., Bioorganic & Medicinal Chemistry 16 (5):
2419-30 (March 2008); and Stefanachi, et al., Bioorganic &
Medicinal Chemistry 16 (6): 2852-69 (March 2008).
[0233] Examples of other ENaC receptor blockers for formulation and
use in combination with the compounds of the invention include but
are not limited to amiloride and derivatives thereof such as those
compounds described in U.S. Pat. No. 6,858,615, and PCT Publication
Nos, WO2003/070182, WO2004/073629, WO2005/018644, WO2006/022935,
WO2007/018640, and WO2007/146869, all to Parion Sciences, Inc.
[0234] Small molecule ENaC blockers are capable of directly
preventing sodium transport through the ENaC channel pore. ENaC
blocker that can be administered in the combinations herein
include, but are not limited to, amiloride, benzamil, phenamil, and
amiloride analogues as exemplified by U.S. Pat. No. 6,858,614, U.S.
Pat. No. 6,858,615, U.S. Pat. No. 6,903,105, U.S. Pat. No.
6,995,160, U.S. Pat. No. 7,026,325, U.S. Pat. No. 7,030,117, U.S.
Pat. No. 7,064,129, U.S. Pat. No. 7,186,833, U.S. Pat. No.
7,189,719, U.S. Pat. No. 7,192,958, U.S. Pat. No. 7,192,959, U.S.
Pat. No. 7,241,766, U.S. Pat. No. 7,247,636, U.S. Pat. No.
7,247,637, U.S. Pat. No. 7,317,013, U.S. Pat. No. 7,332,496, U.S.
Pat. No. 7,345,044, U.S. Pat. No. 7,368,447, U.S. Pat. No.
7,368,450, U.S. Pat. No. 7,368,451, U.S. Pat. No. 7,375,107, U.S.
Pat. No. 7,399,766, U.S. Pat. No. 7,410,968, U.S. Pat. No.
7,820,678, U.S. Pat. No. 7,842,697, U.S. Pat. No. 7,868,010, U.S.
Pat. No. 7,875,619.
[0235] ENaC proteolysis is well described to increase sodium
transport through ENaC. Protease inhibitor block the activity of
endogenous airway proteases, thereby preventing ENaC cleavage and
activation. Protease that cleave ENaC include furin, meprin,
matriptase, trypsin, channel associated proteases (CAPs), and
neutrophil elastases. Protease inhibitors that can inhibit the
proteolytic activity of these proteases that can be administered in
the combinations herein include, but are not limited to, camostat,
prostasin, furin, aprotinin, leupeptin, and trypsin inhibitors.
[0236] Combinations herein may include one or more suitable nucleic
acid (or polynucleic acid), including but not limited to antisense
oligonucleotide, siRNA, miRNA, miRNA mimic, antagomir, ribozyme,
aptamer, and decoy oligonucleotide nucleic acids. See, e.g., US
Patent Application Publication No. 20100316628. In general, such
nucleic acids may be from 17 or 19 nucleotides in length, up to 23,
25 or 27 nucleotides in length, or more. Examples include, but are
not limited to, those described in U.S. Pat. No. 7,517,865 and US
Patent Applications Nos. 20100215588; 20100316628; 20110008366; and
20110104255. In general, the siRNAs are from 17 or 19 nucleotides
in length, up to 23, 25 or 27 nucleotides in length, or more.
[0237] CFTR activity modulating compounds that can be administered
in the combinations of this invention include, but are not limited
to, compounds described in US 2009/0246137 A1, US 2009/0253736 A 1,
US 2010/0227888 A 1, U.S. Pat. No. 7,645,789, US 2009/0246820 A 1,
US 2009/0221597 A1, US 2010/0184739 A1, US 2010/0130547 A1, US
2010/0168094 A1 and issued patent: U.S. Pat. No. 7,553,855; U.S.
Pat. No. 7,772,259 B2, U.S. Pat. No. 7,405,233 B2, US 2009/0203752,
U.S. Pat. No. 7,499,570.
[0238] Mucus or mucin modifying agents useful in the combinations
and methods herein include reducing agents, surfactants and
detergents, expectorants, and deoxyribonuclease agents.
[0239] Mucin proteins are organized into high molecular weight
polymers via the formation of covalent (disulfide) and non-covalent
bonds. Disruption of the covalent bonds with reducing agents is a
well-established method to reduce the viscoelastic properties of
mucus in vitro and is predicted to minimize mucus adhesiveness and
improve clearance in vivo. Reducing agents are well known to
decrease mucus viscosity in vitro and commonly used as an aid to
processing sputum samples. Examples of reducing agents include
sulfide containing molecules or phosphines capable of reducing
protein di-sulfide bonds including, but not limited to, N-acetyl
cysteine, N-acystelyn, carbocysteine, glutathione, dithiothreitol,
thioredoxin containing proteins, and tris (2-carboxyethyl)
phosphine.
[0240] N-acetyl cysteine (NAC) is approved for use in conjunction
with chest physiotherapy to loosen viscid or thickened airway
mucus. Clinical studies evaluating the effects of oral or inhaled
NAC in CF and COPD have reported improvements in the rheologic
properties of mucus and trends toward improvements in lung function
and decreases in pulmonary exacerbations.sup.9. However, the
preponderance of clinical data suggests that NAC is at best a
marginally effective therapeutic agent for treating airway mucus
obstruction when administered orally or by inhalation. A recent
Cochrane review of the existing clinical literature on the use of
NAC found no evidence to support the efficacy of NAC for CF. The
marginal clinical benefit of NAC reflects:
[0241] NAC is a relative inefficient reducing agent which is only
partially active on the airway surface. Very high concentrations of
NAC (200 mM or 3.26%) are required to fully reduce Muc5B, a major
gel-forming airway mucin, in vitro. Furthermore, in the pH
environment of the airway surface (measured in the range of pH 6.0
to 7.2 in CF and COPD airways), NAC exists only partially in its
reactive state as a negatively charge thiolate. Thus, in the
clinic, NAC is administered at very high concentrations. However,
it is predicted that current aerosol devices will not be able to
achieve therapeutic concentrations of even a 20% Mucomyst solution
on distal airway surfaces within the relatively short time domains
(7.5-15 minutes) typically used.
[0242] In non-clinical studies, .sup.14C-labeled NAC, administered
by inhalation, exhibits rapid elimination from the lungs with a
half-life ranging from 6 to 36 minutes.
[0243] NAC is administered as a highly concentrated, hypertonic
inhalation solution (20% or 1.22 molar) and has been reported to
cause bronchoconstriction and cough. In many cases, it is
recommended that NAC be administered with a bronchodilator to
improve the tolerability of this agent.
[0244] Thus, reducing agents such as NAC are not well suited for
bolus aerosol administration. However, it is anticipated that
delivery of reducing agents by pulmonary aerosol infusion would
increase the effectiveness, while allowing for a decrease in the
concentration of reducing agent in the inhalation solution
(predicted to increase tolerability).
[0245] Surfactants and detergents are spreading agents shown to
decrease mucus viscoelasticity, improving mucus clearability.
Examples of surfactants include dipalmitoylphosphatidylcholine
(DPPC), PF, palmitic acid, palmitoyl-oleoylphosphatidylglycerol,
surfactant-associated proteins (e.g. SP-A, B, or C), or may be
animal derived (e.g. from cow or calf lung lavage or extracted from
minced pig lung) or combinations thereof. See, e.g., U.S. Pat. Nos.
7,897,577; 5,876,970; 5,614,216; 5,100,806; and 4,312,860. Examples
of surfactant products include Exosurf.RTM. Neonatal (colfosceril
palmitate), Pumactant.RTM. (DPPC and egg phosphatidylglycerol),
KL-4 surfactant, Venticute.RTM. (lusulptide, rSP-C surfactant),
Alveofact.RTM. (bovactant), Curosurf.RTM. (poractant alfa),
Infasurf.RTM. (calfactant), Newfacten.RTM. (modified bovine
surfactant), Surface.RTM., Natsurf.TM. (nonionic alcohol ethoxylate
surfactant)and Survanta.RTM. (beractant). Examples of detergents
include, but are not limited to, Teen-80 and triton-X 100.
[0246] Any suitable expectorant can be used, including but not
limited to guaifenesin (see, e.g., U.S. Pat. No. 7,345,051). Any
suitable deoxyribonuclease can be used, including but not limited
to Dornase Alpha. (see, e.g., U.S. Pat. No. 7,482,024).
[0247] Examples of kinase inhibitors include inhibitors of NFkB,
PI3K (phosphatidylinositol 3-kinase), p38-MAP kinase and Rho
kinase.
[0248] Antiinfective agents for formulation and use in combination
with the compounds of the invention include antivirals and
antibiotics. Examples of suitable antivirals include Tamiflu.RTM.
(oseltamivir) and Relenza.RTM. (zanamivir). Examples of suitable
antibiotics include but are not limited to aztreonam (arginine or
lysine), fosfomycin, and aminoglycosides such as tobramycin, or any
combination or subset thereof. Additional antiinfective agents that
may be used herein include aminoglycosides, Daptomycin,
Fluoroquinolones, Ketolides, Carbapenems, Cephalosporins,
Erythromycin, Linezolid, Penicillins, Azithromycin, Clindamycin,
Oxazolidinones, Tetracyclines, and Vancomycin.
[0249] Examples of useful carbapenam antibiotics are impenam,
panipenam, meropenam, biapenam, MK-826 (L-749,345), DA-1131,
ER-35786, lenapenam, S-4661, CS-834 (prodrug of R-95867), KR-21056
(prodrug of KR-21012), L-084 (prodrug of LJC 11036) and Ceftolozane
(CXA-101).
[0250] Antihistamines (i.e., H1-receptor antagonists) for
formulation and use in combination with the compounds of the
invention include but are not limited to: ethanolamines such as
diphenhydramine HCl, carbinoxamine maleate, doxylamine, clemastine
fumarate, diphenylhydramine HCl and dimenhydrinate;
ethylenediamines such as pyrilamine maleate (metpyramine),
tripelennamine HCl, tripelennamine citrate, and antazoline;
alkylamines such as pheniramine, chloropheniramine,
bromopheniramine, dexchlorpheniramine, triprolidine and
acrivastine; pyridines such as methapyrilene, piperazines such as
hydroxyzine HCl, hydroxyzine pamoate, cyclizine HCl, cyclizine
lactate, meclizine HCl and cetirizine HCl; piperidines such as
astemisole, levocabastine HCl, loratadine,
descarboethoxyloratadine, terfenadine, and fexofenadine HCl; tri-
and tetracyclics such as promethazine, chlorpromethazine
trimeprazine and azatadine; and azelastine HCl, or any combination
or subset thereof.
[0251] Examples of other classes of therapeutic agents suitable for
use in the combinations and methods herein include antivirals such
as ribavirin, anti-fungal agents such as amphotericin, intraconazol
and voriconazol, anti-rejection drugs such as cyclosporine,
tacrolimus and sirolimus, bronchodilators including but not limited
to anticholinergic agents such as atrovent, siRNAs, gene therapy
vectors, aptamers, endothelin-receptor antagonists,
alpha-1-antitrypsin and prostacyclins.
[0252] In the above-described methods of treatment and uses, a
compound of the invention may he employed alone, or in combination
with one or more other therapeutically active agents. Typically,
any therapeutically active agent that has a therapeutic effect in
the disease or condition being treated with the compound of the
invention may be utilized in combination with the compounds of the
invention, provided that the particular therapeutically active
agent is compatible with therapy employing a compound of the
invention. Typical therapeutically active agents which are suitable
for use in combination with the compounds of the invention include
agents described above.
[0253] In one preferred embodiment, the compounds of the invention
are used in combination with one or more osmolytes, particularly
hypertonic saline or mannitol.
[0254] In another aspect, the invention provides methods for
treatment and uses as described above, which comprise administering
an effective amount of a compound of the invention and at least one
other therapeutically active agent. The compounds of the invention
and at least one additional therapeutically active agent may be
employed in combination concomitantly or sequentially in any
therapeutically appropriate combination. The administration of a
compound of the invention with one or more other therapeutically
active agents may be by administration concomitantly in 1) a
unitary pharmaceutical composition, such as the compositions
described above, or 2) separate pharmaceutical compositions each
including one or more of the component active ingredients. The
components of the combination may be administered separately in a
sequential manner wherein the compound of the invention is
administered first and the other therapeutically active agent is
administered second or vice versa.
[0255] In the embodiments wherein the compound of the invention is
administered in combination with one or more osmolytes, the
administration of each component is preferably concomitant, and may
be in a unitary composition or separate compositions. In one
embodiment, the compound of the invention and one or more osmolytes
are administered concomitantly by transbronchoscopic lavage. In
another embodiment, the compound of the invention and one or more
osmolytes are administered concomitantly by inhalation.
[0256] When a compound of the invention is used in combination with
another therapeutically active agent, the dose of each compound may
differ from that when the compound of the invention is used alone.
Appropriate doses will be readily determined by one of ordinary
skill in the art. The appropriate dose of the compound of the
invention, the other therapeutically active agent(s) and the
relative timings of administration will be selected in order to
achieve the desired combined therapeutic effect, and are within the
expertise and discretion of the attendant physician, clinician or
veterinarian.
[0257] Experimental Procedures The present invention also provides
processes for preparing the compounds of the invention and to the
synthetic intermediates useful in such processes, as described in
detail below.
[0258] Certain abbreviations and acronyms are used in describing
the synthetic processes and experimental details. Although most of
these would be understood by one skilled in the art, the following
table contains a list of many of these abbreviations and
acronyms.
Abbreviation Meaning
[0259] AcOH Acetic Acid [0260] AIBN Azobisisobutyrolnitrile [0261]
DIAD Diisopropyl azidocarboxylate [0262] DIPEA
N,N-Diisopropylethylamine [0263] DCE dichloroethane [0264] DCM
dichloromethane [0265] DMF dimethylformamide [0266] Et Ethyl [0267]
EtOAc or EA ethyl acetate [0268] EtOH Ethanol [0269] ESI
electrospray ionization [0270] HATU
2-(1H-7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium
hexafluorophosphate [0271] HPLC High performance liquid
chromatography [0272] iPrOH Isopropyl alcohol [0273] i.t. or IT
intratracheal [0274] Me Methyl [0275] MeOH methanol [0276] m/z or
m/e mass to charge ratio [0277] MH.sup.+ mass plus 1 [0278]
MH.sup.- mass minus 1 [0279] MIC minimal inhibitory concentration
[0280] MS or ms mass spectrum [0281] rt or r.t. room temperature
[0282] R.sub.f Retardation factor [0283] t-Bu tert-butyl [0284] THF
tetrahydrofuran [0285] TLC or tlc thin layer chromatography [0286]
.delta. parts per million down field from tetramethylsilane [0287]
Cbz Benzyloxycarbonyl, i.e. --(CO)O-benzyl [0288] AUC Area under
the curve or peak [0289] MTBE Methyl tertiary butyl ether
Abbreviation Meaning
[0289] [0290] AcOH Acetic Acid [0291] t.sub.R Retention time [0292]
GC-MS Gas chromatography-mass spectrometry [0293] wt % Percent by
weight [0294] h Hours [0295] min Minutes [0296] MHz megahertz
[0297] TFA Trifluoroacetic acid [0298] UV Ultraviolet [0299] Boc
tert-butyloxycarbonyl [0300] DIAD Diisopropyl azodicarboxylate
[0301] AcOH Acetic Acid [0302] DIPEA N,N-Diisopropylethylamine or
Hunig's base [0303] Ph.sub.3P Triphenylphosine The compounds of
Formula I may be synthesized using techniques known in the art. A
representative synthetic procedure is illustrated in Scheme 1
below.
##STR00013##
[0304] These procedures are described in, for example, E. J.
Cragoe, "The Synthesis of Amiloride and Its Analogs" (Chap 3) in
Amiloride and Its Analogs, pp. 25-36. Other processes for preparing
amiloride analogs are described in, for example, U.S. Pat. No.
3,318,813, to Cragoe, particularly at methods A, B, C, and D of the
'813 patent. Still other processes which may be adapted for the
preparation of the compounds of the invention are described in PCT
Publication Nos. WO2003/07182, WO2005/108644, WO2005/022935, U.S.
Pat. No. 7,064,129, U.S. Pat. No. 6,858,615, U.S. Pat. No.
6,903,105, WO 2004/073629, WO 2007/146869, and WO 2007/018640, all
assigned to Parion Sciences, Inc.
[0305] Preparation of methyl
N'-3,5-diamino-6-chloropyrazine-2-carbonylcarbamimido thioate (2)
can be seen in WO 2009/074575.
[0306] Generally, the compounds of the invention may be
conveniently prepared by treating a compound of Formula II with an
amine of Formula III. More specifically, compounds of Formula 2 are
treated with the amine of Formula 3 in a suitable solvent such as
methanol, ethanol, or tetrahydrofuran, and a base such as
triethylamine (TEA), or di-isoproylethylamine (DIPEA), with heating
to elevated temperature, e.g., 70.degree. C. Further purification,
resolution of stereoisomers, crystallization and/or preparation of
salt forms may be carried out using conventional techniques.
[0307] As will be apparent to those skilled in the art, in certain
instances, the starting or intermediate compounds in the synthesis
may possess other functional groups which provide alternate
reactive sites. Interference with such functional groups may be
avoided by utilization of appropriate protecting groups, such as
amine or alcohol protecting groups, and where applicable,
appropriately prioritizing the synthetic steps. Suitable protecting
groups will be apparent to those skilled in the art. Methods are
well known in the art for installing and removing such protecting
groups and such conventional techniques may be employed in the
processes of the instant invention as well.
[0308] The following specific examples which are provided herein
for purposes of illustration only and do not limit the scope of the
invention, which is defined by the claims.
[0309] Material and methods. All reagent and solvents were
purchased from Aldrich Chemical Corp., Chem-Impex International
Inc. and TCI chemical industry Co. Ltd. NMR spectra were obtained
on either a Bruker AC 400 (.sup.1H NMR at 400 MHz and .sup.13C NMR
at 100 MHz) or a Bruker AC 300 (.sup.1H NMR at 300 MHz and .sup.13C
NMR at 75 MHz). Proton spectra were referenced to tetramethylsilane
as an internal standard and the carbon spectra were referenced to
CDCl.sub.3, CD.sub.3OD, or DMSO-d.sub.6 (purchased from Aldrich or
Cambridge Isotope Laboratories, unless otherwise specified). Flash
chromatography was performed on a Combiflash system (Combiflash Rf,
Teledyne Isco) charged with silica gel column (Redi Sep. Rf,
Teledyne Isco) or reverse phase column (High performance C18 Gold
column). ESI Mass spectra were obtained on a Shimadzu LCMS-2010 EV
Mass Spectrometer. HPLC analyses were obtained using a Waters
XTerra MS C18 5 .mu.m 4.6.times.150 mm Analytical Column detected
at 220 nm (unless otherwise specified) on a Shimadzu Prominence
HPLC system. The following time program was used with a flow rate
of 1.0 mL per minute:
TABLE-US-00001 Time Percent A Percent B (min) (H.sub.2O with 0.05%
TFA) (CH.sub.3CN with 0.05% TFA) 2.50 90 10 20.00 10 90 30.00 10 90
32.50 90 10
UPLC analyses were obtained using a Waters ACQUITY UPLC HSS T3 1.8
.mu.m 2.1.times.100 mm Analytical Column detected at 220 nm (unless
otherwise specified) on a Shimadzu Prominence UFLC system. The
following time program was used with a flow rate of 0.3 mL per
minute:
TABLE-US-00002 Percent B Percent A (CH.sub.3CN/Water 80:20% Time
(H.sub.2O with 0.05% NH.sub.4COOH with 0.05% NH.sub.4COOH (min) and
0.1% HCOOH) and 0.1% HCOOH) 1.00 90 10 4.00 30 70 5.00 30 70 5.50
90 10 6.50 90 10
1. Preparation of the Hydrochloride Salt of
(S)-2-amino-3-(4-(4-(3-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino-
)butyl)naphthalen-1-yl)propanoic acid (16)
##STR00014## ##STR00015##
[0311] Preparation of
4-(tert-Butyldimethylsilyloxy)naphthalene-1-carbaldehyde (2); A
solution of 4-hydroxynaphthalene-1-carbaldehyde (1) (10.0 g, 58.1
mmol) in dry THF (200 mL) was cooled to 0.degree. C., and imidazole
(12.0 g, 174 mmol) and tert-butyldimethylsilyl chloride (TBSCl)
(13.1 g, 87.1 mmol) were added sequentially. After stirring at room
temperature for 16 h the reaction mixture was filtered and the
solvent evaporated. The residue was taken up in EtOAc (500 mL),
washed with saturated aqueous NH4Cl (100 mL), water (100 mL), and
brine (100 mL), and dried over Na.sub.2SO.sub.4. The solvent was
removed under reduced pressure and the residue purified by flash
chromatography on silica gel (2% EtOAc/hexane), yielding 2 (14.8 g,
90%) as a pale yellow solid: .sup.1H NMR (300 MHz, CDCl.sub.3):
.delta. 10.22 (s, 1H), 9.30 (d, J=8.10 Hz, 1H), 8.27 (d, J=8.1 Hz,
1H), 7.86 (d, J=7.8 Hz, 1H), 7.69 (ddd, J=8.4, 7.0, 1.3 Hz, 1H),
7.57 (ddd, J=8.4, 7.0, 1.3 Hz, 1H), 6.95 (d, J=7.5 Hz, 1H), 1.10
(s, 9H), 0.36 (s, 6H)
Preparation of (Z)-Methyl
2-(tert-Butyloxycarbonyl)amino-3-[1-(tert-butyldimethylsilyloxy)naphthale-
n-4-yl]acrylate (4)
[0312] A solution of (MeO).sub.2P(O)CH(NHBoc)CO.sub.2Me, 3 (23.0 g,
52.7 mmol) in dry CH.sub.2Cl.sub.2 (100 mL) was charged with DBU
(10.1 mL, 67.3 mmol), and the mixture was stirred for 30 min at
0.degree. C. A solution of 1 (14.8 g, 51.74 mmol) in dry
CH.sub.2Cl.sub.2 (60 mL) was added slowly via syringe, and the
reaction mixture was warmed to room temperature over 16 h. After
the solvent was removed under reduced pressure, the residue was
dissolved in CH.sub.2Cl.sub.2 (500 mL), quickly washed with
saturated aqueous NH.sub.4Cl (2.times.150 mL) and brine (200 mL),
and dried over Na.sub.2SO.sub.4. The solvent was evaporated and the
crude product purified by flash chromatography on silica gel (20%
EtOAc/hexane with 1% NEt.sub.3), yielding 4 (20.0 g, 85%) as a
yellow solid: .sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 8.23 (dd,
J=8.6, 2.1 Hz, 1H), 7.93 (dd, J=8.6, 2.1 Hz, 1H), 7.67 (s, 1H),
7.57 (d, J=8.4 Hz, 1H), 7.53-7.47 (m, 2H), 6.85 (d, J=7.8 Hz, 1H),
6.05 (brs, 1H), 3.88 (s, 3H), 1.30 (s, 9H), 1.09 (s, 9H), 0.30 (s,
6H).
Preparation of Methyl
2-(tert-butoxycarbonylamino)-3-(4-(tert-butyldimethylsilyloxy)naphthalen--
1-yl)propanoate (5)
[0313] A suspension of 4 (17.2 g, 37.6 mmol) and 10% Pd/C (3.40 g)
in EtOH (200 mL) was degassed and subjected to hydrogenation
conditions (1 atm, balloon) for 16 h at room temperature. The
reaction mixture was filtered through a plug of Celite and the plug
was washed with MeOH. The filtrate was concentrated under vacuum to
afford 5 (17.0 g, 99%) as a white solid: .sup.1H NMR (300 MHz,
CDCl.sub.3): .delta. 823 (d, J=8.2 Hz, 1H), 7.99 (d, J=8.2 Hz, 1H),
7.57-7.44 (m, 2H), 7.10 (d, J=8.2 Hz, 1H), 6.77 (d, J=8.2 Hz, 1H),
5.07-4.94 (brs, 1H), 4.74-4.61 (m, 1H), 3.66 (s, 3H), 3.55-3.17 (m,
2H), 1.40 (s, 9H), 1.18 (s, 9H), 0.30 (s, 6H).
Preparation of Methyl
2-(tert-butoxycarbonylamino)-3-(4-hydroxynaphthalen-1-yl)propanoate
(6)
[0314] A solution of 5 (17.0 g, 37.0 mmol) in dry THF (200 mL) at
0.degree. C. was charged with tetrabutylammonium fluoride (48.1 mL,
48.1 mmol). The resulting solution was stirred for 15 min and
quenched with saturated aqueous NH.sub.4Cl (150 mL). After the
solvent was removed under reduced pressure, the residue was
dissolved in CH.sub.2Cl.sub.2 (500 mL), quickly washed with
saturated aqueous water (2.times.150 mL) and brine (200 mL), and
dried over Na.sub.2SO.sub.4. The solvent was evaporated and the
crude product purified by flash chromatography on silica gel (25%
EtOAc/hexane), yielding rotamer 6 (14.0 g, 94%) as a yellow solid:
.sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 8.23 (d, J=8.2 Hz, 1H),
7.98 (d, J=8.2 Hz, 1H), 7.57-7.44 (m, 2H), 7.07 (d, J=8.0 Hz, 1H),
6.68 (d, J=7.6 Hz, 1H), 6.55 (brs, 1H), 5.14-4.85 (brs, 1H),
4.77-4.51 (m, 1H), 3.78-3.31 (m, 5H), 1.40 (s, 6H), 1.10 (s,
3H).
Preparation of Compounds 7 and 8
[0315] CHIRALPAK AD column 5 cm I.D.times.50 cm L, particle 20 .mu.
was used to separate enantiomers using isocratic system IPA/Heptane
(7.5% with 0.4% DEA). 8.0 g of racemic compound 6 was purified by
the column to afford S-isomer 8 (3.5 g, 44% yield) as a white solid
and R-isomer 7 (2.2 g, 28%) as a white solid.
Preparation of (S)-methyl
2-(tert-butoxycarbonylamino)-3-[4-(trifluoromethylsulfonyloxy)naphthalen--
1-yl]propanoate (9)
[0316] A solution of compound 8 (1.22 g, 3.53 mmol) in pyridine (20
mL) was charged with triflate (0.9 mL, 5.30 mmol) at 0.degree. C.,
and the reaction mixture was stirred at room temperature for 2 h.
After concentration, the reaction mixture was partitioned between
CH.sub.2Cl.sub.2 (100 mL) and water (50 mL). The aqueous layer was
separated and extracted with CH.sub.2Cl.sub.2 (2.times.50 mL). The
combined organic extracts were washed with brine, dried over
Na.sub.2SO.sub.4, and concentrated to afford compound 9 (1.51 g,
89%) as a brown oil: .sup.1H NMR (400 MHz, CDCl.sub.3): .delta.
8.19-8.07 (m, 2H), 7.69-7.64 (m, 2H), 7.38 (d, J=8.1 Hz, 1H), 7.28
(d, J=7.9 Hz, 1H), 5.12-5.06 (brs, 1H), 4.78-4.67 (m, 1H),
3.68-3.46 (m, 5H), 1.39 (s, 8H), 1.25 (s, 1H).
[0317] Preparation of (S)-methyl
3-{4-[4-(benzyloxycarbonylamino)but-1-ynyl]naphthalen-1-yl}-2-(tert-butox-
ycarbonylamino)propanoate (11); A solution of compound 9 (1.50 g,
3.14 mmol) in anhydrous CH.sub.3CN (60 mL) was charged with TEA
(1.27 mL, 12.6 mmol), 10% (t-Bu).sub.3P in hexanes (1.27 mL, 0.62
mmol), benzyl but-3-ynylcarbamate (10, 948 mg, 4.71 mmol), and CuI
(30 mg, 0.16 mmol) at room temperature. The resulting mixture was
degassed with argon for 10 min and Pd(PPh.sub.3).sub.4 (363 mg,
0.31 mmol) was charged rapidly in one portion. After degassing with
argon for 5 min, the resulting mixture was refluxed for 16 h. The
reaction mixture was concentrated under vacuum and the residue was
purified by column chromatography (silica gel, 60:40 ethyl
acetate/hexanes) to afford compound 11 (1.30 g, 78%) as a brown
oil: .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 8.33 (dd, J=7.5,
2.2 Hz, 1H), 8.07 (dd, J=7.5, 2.2 Hz, 1H), 7.58-7.51 (m, 2H), 7.52
(d, J=7.5 Hz, 1H), 7.35-7.29 (m, 5H), 7.19 (d, J=7.5 Hz, 1H),
5.16-5.12 (m, 1H), 5.13 (s, 2H), 5.07-4.99 (m, 1H), 4.74-4.65 (m,
1H), 3.59 (s, 3H), 3.91-3.42 (m, 2H), 3.53 (d, J=6.2 Hz, 2H), 2.79
(t, J=6.4 Hz, 2H), 1.39 (s, 8H), 1.25 (s, 1H).
Preparation of Acetic Acid Salt of (S)-methyl
3-(4-(4-aminobutyl)naphthalen-1-yl)-2-(tert-butoxycarbonylamino)propanoat-
e (12)
[0318] A suspension of 11 (1.00 g, 1.88 mmol) and 10% Pd/C (200 mg)
in a mixture of MeOH (20 mL) and AcOH (2 mL) was degassed and
subjected to hydrogenation conditions (1 atm) for 16 h at room
temperature. The reaction mixture was filtered through a plug of
Celite and the plug was washed with MeOH. The filtrate was
concentrated under vacuum to afford amine salt 12 (820 mg, 95%) as
a white solid: .sup.1H NMR (300 MHz, CD.sub.3OD): .delta. 8.17-8.05
(m, 2H), 7.62-7.48 (m, 2H), 7.27 (brs, 2H), 4.47 (t, J=7.4 Hz, 1H),
3.75-3.51 (m, 5H), 3.13 (t, J=7.5 Hz, 2H), 2.93 (t, J=7.66 Hz, 2H),
1.93 (s, 3H), 1.88-1.65 (m, 4H), 1.34 (s, 7H), 1.01 (s, 2H).
[0319] Preparation of (S)-methyl
2-(tert-butoxycarbonylamino)-3-(4-{4-[3-(3,5-diamino-6-chloropyrazine-2-c-
arbonyl)guanidino]butyl}naphthalen-1-yl)propanoate (14); A solution
of amine salt 12 (815 mg, 1.77 mmol) and methyl
3,5-diamino-6-chloropyrazine-2-carbonylcarbamimidothioate (13, 1.1
g, 2.83 mmol) in EtOH (6.0 mL) was charged with DIPEA (2.50 mL,
14.2 mmol) at room temperature. The reaction mixture was heated at
70.degree. C. in a sealed tube for 2 h, cooled to room temperature,
and concentrated under vacuum. The residue was purified by column
chromatography (silica gel, 80:18:2
CHCl.sub.3/CH.sub.3OH/NH.sub.4OH) to afford guanidine 14 (870 mg,
80%) as a yellow solid: .sup.1H NMR (400 MHz, CD.sub.3OD): .delta.
8.17-8.07 (m, 2H), 7.58-7.48 (m, 2H), 7.26 (q, J=7.4 Hz, 2H),
4.56-3.68 (m, 1H), 3.75-3.68 (m, 1H), 3.64 (s, 2H), 3.58-3.43 (m,
2H), 3.13 (t, J=6.7 Hz, 2H), 2.98 (q, J=7.2 Hz, 2H), 1.86-1.70 (m,
4H), 1.33 (s, 7H), 0.98 (s, 2H).
[0320]
(S)-2-(tert-butoxycarbonylamino)-3-(4-(4-(3-(3,5-diamino-6-chloropy-
razine-2-carbonyl)guanidino)butyl)naphthalen-1-yl)propanoic acid
(15); A solution of methyl ester 14 (510 mg, 0.83 mmol) in a
mixture of THF (3 mL), methanol (3 mL), and water (1 mL) was
charged with solid LiOH (120 mg, 4.99 mmol) and the reaction
mixture was stirred at room temperature for 2 h. When TLC of the
reaction mixture showed completion of the reaction, the pH of the
reaction mixture was brought to 9-10 by addition of 1 N HCl
(aqueous) and the organic solvent was removed. The pH of the
aqueous part was adjusted to 5-6, and the resulting precipitate was
extracted with dichloromethane. The aqueous part was extracted with
DCM (2.times.50 mL). The organic layers were combined, dried over
Na.sub.2SO.sub.4, filtered, and concentrated to afford compound 15
(375 mg, 76%) as a white solid: .sup.1H NMR (300 MHz,
DMSO-d.sub.6): .delta. 8.22-8.02 (m, 2H), 7.59-7.47 (m, 2H),
7.34-7.22 (m, 2H), 6.82 (brs, 2H), 4.19-4.06 (m, 1H), 3.59-3.46 (m,
1H), 3.25-3.13 (m, 2H), 3.09-2.94 (m, 10H), 1.80-1.55 (m, 4H), 1.28
(s, 7H), 0.93 (s, 2H).
[0321] Preparation of the HCl salt of
(S)-2-amino-3-(4-(4-(3-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino-
)butyl)naphthalen-1-yl)propanoic acid (16); 4 N HCl in dioxane (8.0
mL) was added to 15 (258 mg, 0.43 mmol) followed by water (4.0 mL)
and the reaction mixture was stirred at room temperature for 3 h.
The solvent was removed and the residue was lyophilized to give
compound 16 (250 mg, 99%) as a yellow solid: .sup.1H NMR (400 MHz,
DMSO-d.sub.6): .delta. 10.54 (brs, 1H), 9.33 (t, J=5.92 Hz, 1H),
9.03-8.80 (m, 2H), 8.60 (brs, 3H), 8.17 (ddd, J=10.1, 7.6, 4.5 Hz,
2H), 7.59 (ddd, J=9.2, 6.7, 4.5 Hz, 2H), 7.46-7.36 (m, 2H), 7.34
(dd, J=9.9, 7.5 Hz, 2H), 4.13-4.02 (m, 1H), 3.75-3.44 (m, 3H),
3.43-3.33 (m, 2H), 3.09 (t, J=6.4 Hz, 2H), 1.81-1.62 (m, 4H).
2. Preparation of
(S)-3,5-diamino-N-(N-(4-(4-(2-amino-3-(4-(3-(dimethylamino)propyl)phenyla-
mino)-3-oxopropyl)naphthalen-1-yl)butyl)carbamimidoyl)-6-chloropyrazine-2--
carboxamide (23)
##STR00016##
[0322] Preparation of (S)-methyl
3-{4-[4-(benzyloxycarbonylamino)but-1-ynyl]naphthalen-1-yl}-2-(tert-butox-
ycarbonylamino)propanoate (17)
[0323] A solution of methyl ester 11 (1.71 g, 3.22 mmol) in a
mixture of THF (21 mL), methanol (21 mL), and water (7.0 mL) was
charged with solid NaOH (1.29 g, 32.3 mmol) and the reaction
mixture was stirred at room temperature for 3 h. When TLC of the
reaction mixture showed completion of the reaction, the pH of the
reaction mixture was brought to 9-10 by addition of 1 N HCl
(aqueous) and the organic solvent was removed. The pH of the
aqueous part was adjusted to 5-6, and the resulting precipitate was
extracted with dichloromethane. The aqueous part was extracted with
CH.sub.2Cl.sub.2 (2.times.50 mL). The organic layers were combined,
dried over Na.sub.2SO.sub.4, filtered, and concentrated to afford
compound 17 (1.55 g, 93%) as a brown solid: .sup.1HNMR (400 MHz,
DMSO-d.sub.6): .delta. 8.32 (d, J=7.4 Hz, 1H), 8.13-8.05 (m, 1H),
7.58-7.48 (m, 4H), 7.38-7.29 (m, 5H), 5.21-5.15 (m, 1H), 5.12 (s,
2H), 5.07-4.93 (m, 1H), 4.70-4.54 (m, 1H), 3.77-3.62 (m, 1H),
3.57-3.35 (m, 2H), 2.84-2.68 (m, 2H), 1.37 (s, 9H).
[0324] Preparation of Compound 19; The compound 18 (100 mg, 0.56
mmol) in THF (2.5 mL) was charged with DEPBT (218 mg, 0.72 mmol),
17 (289 mg, 0.56 mmol), and DIPEA (0.3 mL, 1.68 mmol) successively
and stirred at room temperature for 16 h. After the solvent was
removed under reduced pressure, the residue was dissolved in
CH.sub.2Cl.sub.2 (100 mL), quickly washed with saturated aqueous
NaHCO.sub.3 (2.times.50 mL) and brine (50 mL), and dried over
Na.sub.2SO.sub.4. The solvent was evaporated and the crude product
purified by flash chromatography on silica gel (8%
methanol/CH.sub.2Cl.sub.2), yielding amide 19 (250 mg, 66%) as a
yellow solid: .sup.1HNMR (400 MHz, CDCl.sub.3): .delta. 8.34 (dd,
J=8.3, 1.4 Hz, 1H), 8.21 (d, J=8.3 Hz, 1H), 7.61-7.47 (m, 4H),
7.39-7.27 (m, 5H), 7.16 (d, J=8.3 Hz, 2H), 7.05 (d, J=8.3 Hz, 2H),
5.36-5.19 (m, 2H), 5.12 (s, 2H), 4.36-4.53 (m, 1H), 3.66-3.42 (m,
4H), 2.79 (t, J=6.6 Hz, 2H), 2.57 (t, J=7.5 Hz, 2H), 2.40 (t, J=7.5
Hz, 2H), 2.32 (s, 6H), 1.86-1.75 (m, 2H), 1.39 (s, 9H).
[0325] Preparation of Compound 20; A suspension of 19 (210 mg, 0.31
mmol) and 10% Pd/C (150 mg) in a mixture of MeOH (3.0 mL) and AcOH
(0.3 mL) was degassed and subjected to hydrogenation conditions (1
atm) for 12 h at room temperature. The reaction mixture was
filtered through a plug of Celite and the plug was washed with
MeOH. The filtrate was concentrated under vacuum to afford amine
salt 22 which was neutralized with triethylamine, and the crude
product was purified by flash chromatography on silica gel (CMA,
80:18:2) yielding free amine 20 (130 mg, 77%) as a white solid:
.sup.1H NMR (300 MHz, CD.sub.3OD): .delta. 8.24 (dd, J=8.1, 2.1 Hz,
1H), 8.08 (dd, J=8.2, 1.5 Hz, 1H), 7.58-7.47 (m, 2H), 7.33-7.20 (m,
4H), 7.07-7.05 (m, 2H), 4.53 (t, J=7.2 Hz, 1H), 3.66-3.55 (m, 2H),
3.09 (t, J=7.5 Hz, 2H), 2.82 (t, J=7.4 Hz, 2H), 2.57 (t, J=7.2 Hz,
2H), 2.35 (dd, J=10.5, 7.5 Hz, 2H), 2.24 (s, 6H), 1.84-1.61 (m,
6H), 1.36 (s, 7H), 1.10 (s, 2H).
[0326] Preparation of 22; A solution of amine 20 (122 mg, 0.22
mmol) and methyl
3,5-diamino-6-chloropyrazine-2-carbonylcarbamimidothioate (21, 139
mg, 0.35 mmol) in EtOH (4.0 mL) was charged with DIPEA (0.31 mL,
1.76 mmol) at room temperature. The reaction mixture was heated at
70.degree. C. in a sealed tube for 2 h, cooled to room temperature,
and concentrated under vacuum. The residue was purified by column
chromatography (silica gel, 80:18:2
CHCl.sub.3/CH.sub.3OH/NH.sub.4OH) to afford guanidine 22 (111 mg,
66%) as a yellow solid: .sup.1H NMR (400 MHz, CD.sub.3OD): .delta.
8.23 (dd, J=7.5, 2.4 Hz, 1H), 8.10 (d, J=8.1 Hz, 1H), 7.57-7.48 (m,
2H), 7.29 (d, J=7.3 Hz, 2H), 7.24 (d, J=8.0 Hz, 2H), 7.13-7.05 (m,
2H), 4.53 (t, J=8.0 Hz, 1H), 3.60-3.37 (m, 2H), 3.23 (t, J=7.3 Hz,
2H), 3.15-3.03 (m, 2H), 2.55 (t, J=7.3 Hz, 2H), 2.29 (dd, J=9.7,
7.6 Hz, 2H), 2.21 (s, 6H), 1.86-1.64 (m, 6H), 1.36 (s, 7H), 1.12
(s, 2H).
Preparation of the HCl salt of Compound 23
(S)-3,5-diamino-N-(N-(4-(4-(2-amino-3-(4-(3-(dimethylamino)propyl)phenyla-
mino)-3-oxopropyl)naphthalen-1-yl)butyl)carbamimidoyl)-6-chloropyrazine-2--
carboxamide
[0327] 4 N HCl in dioxane (3.0 mL) was added to 22 (100 mg, 0.13
mmol) followed by water (1.0 mL) and the reaction mixture was
stirred at room temperature for 3 h. The solvent was removed and
neutralized with 1N NaOH (aqueous), the resulting solid was washed
with water and again treated with 1 N HCl (aqueous), water was
removed, and the residue was lyophilized to afford compound 22 (65
mg, 65%) as a yellow solid: .sup.1H NMR (400 MHz, DMSO-d.sub.6)
10.50 (s, 1H), 10.48 (s, 1H), 10.46-10.40 (m, 1H), 9.26 (t, J=4.9
Hz, 1H), 9.01-8.74 (m, 2H), 8.61 (brs, 1H), 8.35 (dd, J=6.6, 3.4
Hz, 1H), 8.13 (dd, J=6.5, 3.3 Hz, 1H), 7.58 (ddd, J=9.9, 6.6, 3.6
Hz, 2H), 7.42 (brs, 1H), 7.40 (d, J=7.3 Hz, 2H), 7.34 (d, J=7.3 Hz,
1H), 7.28 (d, J=7.3 Hz, 1H), 7.16 (d, J=8.6 Hz, 2H), 4.29-4.20 (m,
1H), 3.64-3.49 (m, 2H), 3.12-3.03 (m, 2H), 3.02-2.94 (m, 2H), 2.72
(s, 3H), 2.70 (s, 3H), 2.56 (t, J=8.1 Hz, 2H), 1.97-1.88 (m, 2H),
1.79-1.61 (m, 4H).
3. Preparation of
3,5-diamino-N-(N-(4-(4-((S)-2-amino-3-(4-(3-(hexyl((2S,3R,4R,5R)-2,3,4,5,-
6-pentahydroxyhexyl)amino)propyl)phenylamino)-3-oxopropyl)naphthalen-1-yl)-
butyl)carbamimidoyl)-6-chloropyrazine-2-carboxamide (28)
##STR00017##
[0328] Preparation of Compound 25
[0329] The compound 24 (165 mg, 0.38 mmol) in THF (10 mL) was
charged with DEPBT (148 mg, 0.48 mmol), 17 (200 mg, 0.38 mmol), and
DIPEA (0.2 mL, 1.14 mmol) successively and stirred at room
temperature for 16 h. After the solvent was removed under reduced
pressure, the residue was dissolved in CH.sub.2Cl.sub.2 (100 mL),
quickly washed with saturated aqueous NaHCO.sub.3 (2.times.50 mL)
and brine (50 mL), and dried over Na.sub.2SO.sub.4. The solvent was
evaporated and the crude product purified by flash chromatography
on silica gel (8% methanol/CH.sub.2Cl.sub.2), yielding amide 25
(210 mg, 60%) as a yellow solid: .sup.1H NMR (300 MHz, CDCl.sub.3):
.delta. 8.35 (d, J=8.2 Hz, 1H), 8.21 (d, J=8.3 Hz, 1H), 7.63-7.52
(m, 2H), 7.51 (d, J=7.3 Hz, 1H), 7.44-7.39 (m, 1H), 7.37-7.27 (m,
6H), 7.16-7.02 (m, 3H), 5.24-5.16 (m, 1H), 5.13 (s, 2H), 4.68 (ddd,
J=11.3, 10.3, 5.1 Hz, 1H), 4.56 (q, J=7.2 Hz, 1H), 4.19-4.09 (m,
1H), 3.90-3.76 (m, 5H), 3.74-3.68 (m, 1H), 3.63-3.46 (m, 5H),
3.45-3.24 (m, 3H), 2.80 (t, J=6.7 Hz, 2H), 2.70-2.35 (m, 8H),
1.81-1.67 (m, 2H), 1.63-1.53 (m, 1H), 1.35-1.20 (m, 6H), 1.21 (s,
9H), 1.31 (d, J=5.1 Hz, 3H), 0.87 (t, J=6.2 Hz, 3H).
Preparation of Compound 26
[0330] A suspension of 25 (280 mg, 0.30 mmol) and 10% Pd/C (560 mg)
in a mixture of EtOH (9.0 mL) and AcOH (1.0 mL) was degassed and
subjected to hydrogenation conditions (1 atm) for 4 h at room
temperature. The reaction mixture was filtered through a plug of
Celite and the plug was washed with MeOH. The filtrate was
concentrated under vacuum to afford amine salt 22, which was
neutralized with NaHCO.sub.3, and the crude product was purified by
flash chromatography on silica gel (CMA, 80:18:2) yielding free
amine 26 (160 mg, 67%) as a yellow solid: .sup.1H NMR (400 MHz,
CD.sub.3OD): .delta. 8.29 (d, J=8.2 Hz, 1H), 8.07 (d, J=8.6 Hz,
1H), 7.60 (t, J=6.9 Hz, 1H), 7.55 (ddd, J=8.2, 6.9, 1.1 Hz, 1H),
7.24 (d, J=7.1 Hz, 1H), 7.18 (d, J=7.1 Hz, 1H), 7.02-7.96 (m, 1H),
7.95-6.88 (m, 2H), 6.77-6.69 (m, 1H), 5.56-5.35 (m, 1H), 4.68 (q,
J=5.1 Hz, 1H), 4.61-4.53 (m, 1H), 4.12 (dd, J=10.8, 5.4 Hz, 1H),
3.89-3.80 (m, 2H), 3.74 (t, J=3.3 Hz, 2H), 3.46 (d, J=3.8 Hz, 1H),
3.39 (t, J=10.7 Hz, 2H), 3.18-3.09 (m, 1H), 3.02-2.92 (m, 1H), 2.68
(t, J=7.1 Hz, 2H), 2.61-2.47 (m, 5H), 2.46-2.37 (m, 4H), 1.77-1.63
(m, 4H), 1.33 (d, J=5.1 Hz, 3H), 1.31-1.20 (m, 8H), 1.21 (s, 9H),
0.88 (t, J=6.7 Hz, 3H).
Preparation of Compound 27
[0331] A solution of amine 26 (155 mg, 0.20 mmol) and methyl
3,5-diamino-6-chloropyrazine-2-carbonylcarbamimidothioate (21, 123
mg, 0.31 mmol) in EtOH (8.0 mL) was charged with DIPEA (0.28 mL,
1.56 mmol) at room temperature. The reaction mixture was heated at
70.degree. C. in a sealed tube for 2 h, cooled to room temperature,
and concentrated under vacuum. The residue was purified by
silica-gel column chromatography (80:18:2
CHCl.sub.3/CH.sub.3OH/NH.sub.4OH) followed by reverse-phase
chromatography (Gold C18) to afford guanidine 27 (100 mg, 51%) as a
yellow solid: .sup.1H NMR (400 MHz, CD.sub.3OD): .delta. 8.23 (dd,
J=8.8, 2.5 Hz, 1H), 8.10 (d, J=8.2 Hz, 1H), 7.56-7.49 (m, 2H), 7.29
(d, J=7.9 Hz, 2H), 7.24 (d, J=7.4 Hz, 2H), 7.08 (d, J=7.9 Hz, 2H),
4.67 (q, J=5.1 Hz, 1H), 4.56-4.50 (m, 1H), 4.04 (dd, J=10.8, 5.4
Hz, 1H), 3.92-3.86 (m, 1H), 3.82-3.74 (m, 2H), 3.51-3.46 (m, 1H),
3.25 (t, J=7.1 Hz, 2H), 3.15-3.06 (m, 2H), 2.71 (dd, J=13.2, 5.2
Hz, 1H), 2.60-2.45 (m, 6H), 1.87-1.63 (m, 6H), 1.48-1.40 (m, 6H),
1.33-1.26 (m, 6H), 1.23 (d, J=5.1 Hz, 3H), 1.20 (s, 9H), 0.89 (t,
J=6.7 Hz, 3H).
[0332] Preparation of the HCl salt of Compound
28-3.5-diamino-N-(N-(4-(4-((S)-2-amino-3-(4-(3-(hexyl((2S,3R,4R,5R)-2,3,4-
,5,6-pentahydroxyhexyl)amino)propyl)phenylamino)-3-oxopropyl)naphthalen-1--
yl)butyl)carbamimidoyl)-6-chloropyrazine-2-carboxamide; 4 N HCl in
water (3.0 mL) was added to 27 (80 mg, 0.08 mmol) in ethanol (0.5
mL) and the reaction mixture was stirred at 40.degree. C. for 6 h.
The solvent was removed, an additional 4 N HCl was added, and the
mixture was heated at 40.degree. C. for another 4 h. The solvent
was removed, water was added, and the residue was lyophilized to
afford compound 28 (78 mg, 99%) as a yellow solid: .sup.1H NMR (400
MHz, DMSO-d.sub.6): .delta. 10.58 (brs, 1H), 10.56 (brs, 1H),
9.70-9.58 (m, 1H), 9.38-9.31 (m, 1H), 9.04-8.84 (m, 2H), 8.70 (brs,
1H), 8.43-8.34(m, 1H), 8.16-8.08 (m, 1H), 7.62-7.52 (m, 2H),
7.46-7.37 (m, 4H), 7.34 (d, J=7.1 Hz, 1H), 7.27 (d, J=7.1 Hz, 1H),
7.17 (d, J=8.1 Hz, 2H), 5.52-5.46 (m, 1H), 4.85-4.76 (m, 1H),
4.68-4.52 (m, 2H), 4.49-4.37 (m, 1H), 4.32-4.22 (m, 1H), 4.05-3.97
(m, 1H), 3.72-3.43 (m, 6H), 3.17-2.97 (m, 8H), 2.02-1.90 (m, 2H),
1.77-1.54 (m, 6H), 1.33-1.21 (m, 6H), 0.86 (t, J=6.6 Hz, 3H).
[0333] .sup.1H NMR (400 MHz, CD.sub.3OD): .delta. 8.23 (d, J=8.3
Hz, 1H), 8.17 (d, J=8.2 Hz, 1H), 7.62-7.53 (m, 2H), 7.41-7.36 (m,
1H), 7.35-7.32 (m, 1H), 7.31-7.25 (m, 2H), 7.21-7.12 (m, 2H),
4.35-4.25 (m, 1H), 4.17-4.02 (m, 1H), 3.86-3.75 (m, 2H), 3.73-3.59
(m, 6H), 3.23-3.08 (m, 9H), 2.73-2.60 (m, 2H), 2.11-1.97 (m, 2H),
1.91-1.75 (m, 4H), 1.74-1.62 (m, 2H), 1.44-1.30 (m, 6H), 0.92 (t,
J=6.6 Hz, 3H)
4. Preparation of
3,5-diamino-N-(N-(4-(4-((S)-2-amino-3-(4-(3-(bis((2S,3R,4R,5R)-2,3,4,5,6--
pentahydroxyhexyl)amino)propyl)phenylamino)-3-oxopropyl)naphthalen-1-yl)bu-
tyl)carbamimidoyl)-6-chloropyrazine-2-carboxamide (33)
##STR00018## ##STR00019##
[0334] Preparation of Compound 30
[0335] Compound 29 (290 mg, 0.54 mmol) in THF (8.0 mL) was charged
with DEPBT (210 mg, 0.70 mmol), 17 (311 mg, 0.60 mmol), and DIPEA
(0.28 mL, 1.62 mmol) successively and stirred at room temperature
for 16 h. After the solvent was removed under reduced pressure, the
residue was dissolved in CH.sub.2Cl.sub.2 (100 mL), quickly washed
with saturated aqueous NaHCO.sub.3 (2.times.50 mL) and brine (50
mL), and dried over Na.sub.2SO.sub.4. The solvent was evaporated
and the crude product purified by flash chromatography on silica
gel (8% methanol/CH.sub.2Cl.sub.2), yielding amide 30 (400 mg, 72%)
as a yellow solid: .sup.1H NMR (400 MHz, CDCl.sub.3): .delta.
8.36-8.26 (m, 1H), 8.20-8.09 (m, 1H), 8.03-7.85 (m, 1H), 7.61-7.46
(m, 1H), 7.49 (d, J=7.2 Hz, 2H), 7.38-7.28 (m, 5H), 7.18-6.96 (m,
4H), 5.51-5.36 (m, 1H), 5.32-5.21 (m, 1H), 5.12 (s, 2H), 4.67 (q,
J=5.1 Hz, 2H), 4.66-4.53 (m, 1H), 4.11 (dd, J=10.4, 5.2 Hz, 2H),
4.06-3.96 (m, 2H), 3.93-3.86 (m, 2H), 3.86-3.77 (m, 2H), 3.68-3.56
(m, 2H), 3.56-3.44 (m, 6H), 3.39 (t, J=10.4 Hz, 2H), 3.05 (q, J=7.6
Hz, 2H), 2.96-2.88 (m, 2H), 2.79 (t, J=6.1 Hz, 2H), 2.64-2.61 (m,
4H), 1.93-1.72 (m, 4H), 1.48-1.40 (m, 2H), 1.35 (s, 9H), 1.29 (d,
J=5.1 Hz, 6H).
Preparation of Compound 31
[0336] A suspension of 30 (400 mg, 0.39 mmol) and 10% Pd/C (210 mg)
in a mixture of EtOH (54 mL) and AcOH (6.0 mL) was degassed and
subjected to hydrogenation conditions (1 atm) for 4 h at room
temperature. The reaction mixture was filtered through a plug of
Celite and the plug was washed with MeOH. The filtrate was
concentrated under vacuum to afford amine salt 31 (333 mg, 84%) as
a yellow solid: .sup.1H NMR (400 MHz, CD.sub.3OD): .delta. 8.25
(dd, J=7.5, 2.5 Hz, 1H), 8.10 (d, J=7.3 Hz, 1H), 7.60-7.51 (m, 2H),
7.36-7.32 (m, 1H), 7.31 (d, J=7.2 Hz, 2H), 7.26 (d, J=7.8 Hz, 1H),
7.15 (d, J=7.8 Hz, 2H), 4.70 (q, J=4.9 Hz, 2H), 4.54 (d, J=7.3 Hz,
1H), 4.18-4.10 (m, 2H), 4.06 (dd, J=10.6, 5.3 Hz, 2H), 3.87-3.82
(m, 2H), 3.81-3.68 (m, 3H), 3.53 (dd, J=9.5, 1.8 Hz, 2H), 3.39 (t,
J=9.2 Hz, 3H), 3.35-3.30 (m, 2H), 3.15-3.08 (m, 2H), 2.92 (t, J=8.0
Hz, 2H), 2.09-2.00 (m, 4H), 2.77-2.58 (m, 2H), 1.95 (s, 6H),
1.88-1.60 (m, 4H), 1.36 (s, 9H), 1.25 (d, J=4.9 Hz, 6H).
[0337] Preparation of 32; A solution of 31 (370 mg, 0.36 mmol) and
methyl 3,5-diamino-6-chloropyrazine-2-carbonylcarbamimidothioate
(21, 226 mg, 0.58 mmol) in EtOH (12 mL) was charged with DIPEA
(0.51 mL, 2.88 mmol) at room temperature. The reaction mixture was
heated at 70.degree. C. in a sealed tube for 2 h, cooled to room
temperature, and concentrated under vacuum. The residue was
purified by silica-gel column chromatography (80:18:2
CHCl.sub.3/CH.sub.3OH/NH.sub.4OH) to afford guanidine 32 (250 mg,
63%) as a yellow solid: .sup.1H NMR (400 MHz, CD.sub.3OD): .delta.
8.23 (d, J=8.6 Hz, 1H), 8.13-8.03 (m, 1H), 7.54-7.49 (m, 2H),
7.30-7.20 (m, 4H), 7.13-7.04 (m, 2H), 4.67 (q, J=4.9 Hz, 2H),
4.54-4.49 (m, 1H), 4.03 (dd, J=10.8, 5.4 Hz, 2H), 3.91-3.84 (m,
2H), 3.82-3.72 (m, 5H), 3.48-3.43 (m, 5H), 3.41-3.34 (m, 2H),
3.13-3.10 (m, 2H), 2.68-2.50 (m, 8H), 1.87-1.67 (m, 6H), 1.36 (s,
9H), 1.23 (d, J=4.9 Hz, 6H).
Preparation of the HCl Salt of
33-3.5-diamino-N-(N-(4-(4-((S)-2-amino-3-(4-(3-(bis((2S,3R,4R,5R)-2,3,4,5-
,6-pentahydroxyhexyl)amino)propyl)phenylamino)-3-oxopropyl)naphthalen-1-yl-
)butyl)carbamimidoyl)-6-chloropyrazine-2-carboxamide
[0338] 4 N HCl in water (6.0 mL) was added to 32 (200 mg, 018 mmol)
in ethanol (2.0 mL) and the reaction mixture was stirred at
40.degree. C. for 8 h. The solvent was removed, an additional 4N
HCl was added, and the mixture was heated at 40.degree. C. for
another 6 h. The solvent was removed, the mixture was purified by
reverse-phase chromatography (Gold column), and the residue was
lyophilized to afford compound 33 (138 mg, 59%) as a yellow solid:
.sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta. 10.48 (brs, 1H),
10.45-10.41 (m, 1H), 9.25-9.19 (m, 1H), 8.95-8.85 (m, 1H),
8.81-8.69 (m, 1H), 8.64-8.46 (m, 4H), 8.36-8.29 (m, 1H), 8.18-8.10
(m, 1H), 7.62-7.55 (m, 2H), 7.46-7.38 (m, 4H), 7.34 (d, J=7.5 Hz,
1H), 7.28 (d, J=7.3 Hz, 1H), 7.18 (d, J=8.7 Hz, 2H), 5.48-5.39 (m,
2H), 4.87-4.75 (m, 2H), 4.68-4.33 (m, 4H), 4.28-4.17 (m, 1H),
4.05-3.93 (m, 2H), 3.72-3.65 (m, 2H), 3.62-3.53 (m, 4H), 3.52-3.35
(m, 8H), 3.27-3.13 (m, 6H), 3.3.10-3.00 (m, 2H), 2.62-2.48 (m, 4H),
2.03-1.90 (m, 2H), 1.78-1.61 (m, 4H).
[0339] .sup.1H NMR (400 MHz, CD.sub.3OD): .delta. 8.24-8.20 (m,
1H), 8.18-8.15 (m, 1H), 7.57 (td, J=4.6, 1.5 Hz, 2H), 7.38 (d,
J=7.4 Hz, 1H), 7.33 (d, J=7.4 Hz, 1H), 7.28 (dd, J=8.4, 2.6 Hz,
2H), 7.16 (dd, J=8.4, 2.0 Hz, 2H), 4.28 (t, J=6.9 Hz, 1H),
4.19-4.13 (m, 1H), 4.12-4.07(m, 1H), 3.85-3.79 (m, 2H), 3.77 (dd,
J=10.4, 5.2 Hz, 2H), 3.73-3.60 (m, 6H), 3.49-3.45 (m, 2H),
3.42-3.34 (m, 6H), 3.26-3.23 (m, 1H), 3.19-3.13 (m, 2H), 3.14-3.11
(m, 1H), 2.74-2.59 (m, 2H), 2.14-2.00 (m, 2H), 1.90-1.72 (m,
4H).
5. Preparation of
3,5-diamino-N-(N-(4-(4-((8)-2-amino-3-oxo-3-(4-(3-((2S,3R,4R,5R)-2,3,4,5,-
6-
pentahydroxyhexylamino)propyl)phenylamino)propyl)naphthalen-1-yl)butyl)-
carbamimidoyl)-6-chloropyrazine-2-carboxamide (38)
##STR00020## ##STR00021##
[0340] Preparation of Compound 35
[0341] Compound 34 (400 mg, 0.91 mmol) in THF (15 mL) was charged
with DEPBT (389 mg, 1.30 mmol), 17 (516 mg, 1.00 mmol), and DIPEA
(0.52 mL, 3.00 mmol) successively and stirred at room temperature
for 16 h. After the solvent was removed under reduced pressure, the
residue was dissolved in CH.sub.2Cl.sub.2 (100 mL), quickly washed
with saturated aqueous NaHCO.sub.3 (2.times.50 mL) and brine (50
mL), and dried over Na.sub.2SO.sub.4. The solvent was evaporated
and the crude product purified by flash chromatography on silica
gel (8% methanol/CH.sub.2Cl.sub.2), yielding amide 35 (700 mg, 83%)
as a yellow solid: .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 8.35
(dd, J=8.2, 1.5 Hz, 1H), 8.22 (d, J=8.1 Hz, 7.64-7.35 (m, 4H),
7.38-7.26 (m, 5H), 7.06 (d, J=7.8 Hz, 2H), 7.17-7.09 (m, 2H),
5.21-5.13 (m, 2H), 5.12 (s, 2H), 4.69 (q, J=5.1 Hz, 1H), 4.55 (q,
J=7.25 Hz, 1H), 4.15 (dd, J=11.4, 5.6 Hz, 1H), 4.11-4.02 (m, 1H),
4.07-3.92 (m, 1H), 3.88-3.77 (m, 1H), 3.73-3.67 (m, 1H), 3.64-3.49
(m, 5H), 3.41 (d, J=10.6 Hz, 2H), 3.37-3.30 (m, 2H), 3.29-3.20 (m,
3H), 2.80 (t, J=6.2 Hz, 2H), 2.52 (t, J=7.8 Hz, 2H), 1.90-1.76 (m,
3H), 1.42 (s, 18H), 1.32 (d, J=5.2 Hz, 3H).
Preparation of Compound 36
[0342] A suspension of 35 (700 mg, 0.74 mmol) and 10% Pd/C (400 mg)
in a mixture of EtOH (90 mL) and AcOH (10 mL) was degassed and
subjected to hydrogenation conditions (1 atm) for 16 h at room
temperature. The reaction mixture was filtered through a plug of
Celite and the plug was washed with MeOH. The filtrate was
concentrated under vacuum to afford amine salt 36 (650 mg, 95%) as
a yellow solid: .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 8.20 (d,
J=8.4 Hz, 1H), 7.96 (d, J=7.3 Hz, 1H), 7.86-7.71 (m, 1H), 7.70-7.63
(m, 1H), 7.58-7.43(m, 2H), 7.36-7.26 (m, 2H), 7.02-6.91 (m, 2H),
4.70-4.63 (m, 1H), 4.61-4.54 (m, 1H), 4.20-4.05 (m, 2H), 4.04-3.90
(m, 1H), 3.89-3.68 (m, 3H), 3.67-3.46 (m, 3H), 3.45-3.27 (m, 5H),
3.29-3.21 (m, 4H), 3.11-2.91 (m, 4H), 2.90-2.76 (m, 2H), 2.48 (d,
J=7.3 Hz, 2H), 2.08 (s, 6H), 1.86-1.61 (m, 6H), 1.41 (s, 15H), 1.32
(d, J=5.1 Hz, 3H), 1.25 (s, 3H).
Preparation of 37
[0343] A solution of 36 (650 mg, 0.70 mmol) and methyl
3,5-diamino-6-chloropyrazine-2-carbonylcarbamimidothioate (21, 436
mg, 1.13 mmol) in EtOH (12 mL) was charged with DIPEA (0.90 mL,
5.60 mmol) at room temperature. The reaction mixture was heated at
70.degree. C. in a sealed tube for 2 h, cooled to room temperature,
and concentrated under vacuum. The residue was purified by
silica-gel column chromatography (80:18:2
CHCl.sub.3/CH.sub.3OH/NH.sub.4OH) to afford guanidine 37 (444 mg,
62%) as a yellow solid: .sup.1H NMR (400 MHz, CD.sub.3OD): .delta.
8.23 (dd, J=7.7, 2.2 Hz, 1H), 8.10 (d, J=8.1 Hz, 1H), 7.57-7.47 (m,
2H), 7.33-7.21 (m, 4H), 7.08 (d, J=8.1 Hz, 2H), 4.68 (q, J=5.0 Hz,
1H), 4.53 (t, J=7.2 Hz, 1H), 4.04 (dd, J=10.8, 5.4 Hz, 1H),
4.03-3.93 (m, 1H), 3.25 (ddd, J=10.3, 9.2, 5.2 Hz, H), 3.71-3.65
(m, 1H), 3.58-3.37 (m, 4H), 3.27-3.20 (m, 4H), 3.20-3.15 (m, 1H),
3.14-3.05 (m, 2H), 2.66 (q, J=7.5 Hz, 1H), 2.53 (t, J=7.2 Hz, 2H),
1.89-1.76 (m, 4H), 1.76-1.64 (m, 2H), 1.36 (s, 6H), 1.42 (s, 9H),
1.25 (d, J=5.0 Hz, 3H), 1.11 (s, 3H).
Preparation of the HCl Salt of 38
[0344] 4 N HCl in water (6.0 mL) was added to 37 (240 mg, 0.23
mmol) in ethanol (3.0 mL) and the reaction mixture was stirred at
40.degree. C. for 8 h. The solvent was removed, an additional 4N
HCl was added, and the mixture was heated at 40.degree. C. for
another 8 h. The solvent was removed, the mixture was purified by
reverse-phase chromatography (Gold column), and the residue was
lyophilized to afford compound 38 (251 mg, 64%) as a yellow solid:
.sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta. 10.50 (brs, 1H), 9.28
(t, J=5.7 Hz, 1H), 9.02-8.87 (m, 1H), 8.86-8.75 (m, 1H), 8.72-8.55
(m, 4H), 8.39-8.33 (m, 1H), 8.16-8.10 (m, 1H), 7.61-7.55 (m, 2H),
7.45-7.40 (m, 1H), 7.40 (d, J=7.4 Hz, 2H), 7.34 (d, J=7.3 Hz, 1H),
7.28 (d, J=7.4 Hz, 1H), 7.14 (d, J=8.5 Hz, 2H), 5.38 (d, J=4.3 Hz,
1H), 4.74 (d, J=4.9 Hz, 1H), 4.64-4.51 (m, 2H), 4.49-4.35 (m, 1H),
4.30-4.20 (m, 2H), 3.94-3.86 (m, 1H), 3.70-3.64 (m, 1H), 3.63-3.52
(m, 3H), 3.51-3.34 (m, 6H), 3.15-2.98 (m, 3H), 2.98-2.81 (m, 3H),
2.58 (t, J=7.6 Hz, 2H), 1.96-1.85 (m, 2H), 1.79-1.61 (m, 4H).
[0345] .sup.1H NMR (400 MHz, CD.sub.3OD): .delta. 8.26-8.20 (m,
1H), 8.19-8.14 (m, 1H), 7.60-7.53 (m, 2H), 7.38 (d, J=7.2 Hz, 1H),
7.33 (d, J=8.4 Hz, 1H), 7.28 (dd, J=8.4, 2.0 Hz, 2H), 7.14 (d,
J=8.4 Hz, 2H), 4.29 (t, J=8.3 Hz, 1H), 4.07-4.00 (m, 1H), 3.83 (dd,
J=9.8, 1.5 Hz, 1H), 3.77 (dd, J=9.8, 2.6 Hz, 1H), 3.73-3.64 (m,
5H), 3.37 (t, J=7.2 Hz, 2H), 3.21-3.11 (m, 4H), 3.06-2.96 (m, 2H),
2.66 (t, J=7.7 Hz, 2H), 2.03-1.94 (m, 2H), 1.90-1.75 (m, 4H).
6. Preparation of
(8)-3,5-diamino-N-(N-(4-(4-(2-amino-3-(4-(6-(dimethylamino)hexyl)phenylam-
ino)-3-oxopropyl)naphthalen-1-yl)butyl)carbamimidoyl)-6-chloropyrazine-2-c-
arboxamide (43)
##STR00022## ##STR00023##
[0346] Preparation of Compound 40
[0347] A solution of acid 17 (880 mg, 1.70 mmol) in THF (30 mL) was
cooled to 0.degree. C. in an ice bath. NMM (0.37 mL, 3.40 mmol) was
added, followed by PivCl (0.20 mL, 1.70 mmol), and the reaction
mixture was stirred at the same temperature for 2 h. 39 (375 mg,
1.70 mmol, 15 mL THF) was added and the reaction mixture was
stirred at the same temperature for a further 10 min. The reaction
mixture was brought to room temperature and stirred for 16 h. The
organic solvent was removed. The residue was charged with water and
extracted with CH.sub.2Cl.sub.2 (3.times.100 mL). The organic
layers were combined, dried over Na.sub.2SO.sub.4, filtered, and
concentrated. The residue was purified by column chromatography (4%
methanol in chloroform) to afford amide 40 (719 mg, 59%) as a light
yellow solid: [M+H].sup.+720.
Preparation of Compound 41
[0348] A suspension of 40 (719 mg, 1.00 mmol) and 10% Pd/C (300 mg)
in a mixture of EtOH (110 mL) and AcOH (20 mL) was degassed and
subjected to hydrogenation conditions (1 atm) for 16 h at room
temperature. The reaction mixture was filtered through a plug of
Celite and the plug was washed with MeOH. The filtrate was
concentrated under vacuum to afford amine salt 41 as a yellow solid
(660 mg, 93%): [M+H].sup.+589.
Preparation of Compound 42
[0349] A solution of amine 41 (660 mg, 0.93 mmol) and methyl
3,5-diamino-6-chloropyrazine-2-carbonylcarbamimidothioate (21, 650
mg, 1.67 mmol) in EtOH (10 mL) was charged with DIPEA (1.66 mL, 9.3
mmol) at room temperature. The reaction mixture was heated at
70.degree. C. in a sealed tube for 2 h, cooled to room temperature,
and concentrated under vacuum. The residue was purified by
silica-gel column chromatography (80:18:2
CHCl.sub.3/CH.sub.3OH/NH.sub.4OH) to afford guanidine 42 (370 ma,
50%) as a yellow solid: [M+H].sup.+801.
Preparation of the HCl salt of Compound 43
(S)-3,5-diamino-N-(N-(4-(4-(2-amino-3-(4-(6-(dimethylamino)hexyl)phenylam-
ino)-3-oxopropyl)naphthalen-1-yl)butyl)carbamimidoyl)-6-chloropyrazine-2-c-
arboxamide
[0350] TFA (10 mL) was added to 42 (370 mg, 0.46 mmol) in
CH.sub.2Cl.sub.2 (10 mL) and the reaction mixture was stirred at
room temperature for 2 h. The solvent was removed, an additional 1N
HCl was added, and solvent was removed. The mixture was purified by
reverse-phase chromatography (Gold column) and the residue was
lyophilized to afford compound 43 (290 mg, 92%) as a yellow solid:
.sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta. 10.39 (brs, 2H), 9.25
(brs, 1H), 9.02-8.87 (m, 1H), 8.86-8.73 (m, 2H), 8.71-8.44 (m, 2H),
8.35 (brs, 1H), 8.13 (dd, J=6.8, 3.8 Hz, 1H), 7.58 (dd, J=6.5, 3.2
Hz, 2H), 7.42 (brs, 2H), 7.35 (d, J=8.6 Hz, 2H), 7.33 (d, J=7.8 Hz,
1H), 7.27 (d, J=7.3 Hz, 1H), 7.11 (d, J=8.4 Hz, 2H), 4.26-4.18 (m,
1H), 3.65-3.48 (m, 2H), 3.39-3.32 (m, 3H), 3.06 (t, J=6.5 Hz, 2H),
2.99-2.91 (m, 2H), 2.69 (s, 6H), 1.77-1.56 (m, 6H), 1.52 (t, J=8.2
Hz, 2H), 1.34-1.21 (m, 4H).
[0351] .sup.1H NMR (400 MHz, CD.sub.3OD): .delta. 8.22-8.17 (m,
1H), 8.16-8.12 (m, 1H), 7.58-7.51 (m, 2H), 7.36 (d, J=7.2 Hz, 1H),
7.30 (d, J=7.4 Hz, 1H), 7.19 (d, J=8.04 Hz, 2H), 7.05 (d, J=8.3 Hz,
2H), 4.24 (t, J=8.2 Hz, 1H), 3.70-3.58 (m, 2H), 3.33 (t, J=6.9 Hz,
2H), 3.14 (t, J=7.4 Hz, 2H), 3.09-3.03 (m, 2H) 2.84 (s, 6H) 2.53
(t, J=8.6 Hz, 2H), 1.88-1.73 (m, 4H), 1.72-1.63 (m, 2H), 1.61-1.52
(m, 2H), 1.41-1.32 (m, 4H).
7. Preparation of
3,5-diamino-N-(N-(4-(4-((S)-2-amino-3-(4-(6-(bis((2S,3R,4R,5R)-2,3,4,5,6--
pentahydroxyhexyl)amino)hexyl)phenylamino)-3-oxopropyl)naphthalen-1-yl)but-
yl)carbamimidoyl)-6-chloropyrazine-2-carboxamide
##STR00024## ##STR00025##
[0352] Preparation of Compound 45
[0353] A solution of acid 17 (900 mg, 1.74 mmol) in THF (40 mL) was
cooled to 0.degree. C. in an ice bath. NMM (0.38 mL, 3.48 mmol) was
added, followed by PivCl (0.21 mL, 1.74 mmol), and the reaction
mixture was stirred at the same temperature for 2 h. 44 (1.21 g,
1.74 mmol, 20 mL THF) was added and the reaction mixture was
stirred at the same temperature for a further 10 min. The reaction
mixture was brought to room temperature and stirred for 16 h. The
organic solvent was removed. The residue was charged with water and
extracted with CH.sub.2Cl.sub.2 (3.times.100 mL). The organic
layers were combined, dried over Na.sub.2SO.sub.4, filtered, and
concentrated. The residue was purified by column chromatography (4%
methanol in chloroform) to afford amide 45 (2.00 g, impure) as a
light yellow solid: [M+H].sup.+1196.
Preparation of Compound 46
[0354] A suspension of 45 (2.00 g, impure) and 10% Pd/C (400 mg) in
a mixture of EtOH (120 mL) and AcOH (20 mL) was degassed and
subjected to hydrogenation conditions (1 atm) for 16 h at room
temperature. The reaction mixture was filtered through a plug of
Celite and the plug was washed with MeOH. The filtrate was
concentrated under vacuum to afford amine salt 46, which was
neutralized with NaHCO.sub.3, and the crude product was purified by
flash chromatography on silica gel (CMA, 80:18:2) yielding free
amine 46 as a yellow solid (500 mg, 27% over two steps):
[M+H].sup.+1067.
Preparation of Compound 47
[0355] A solution of amine 46 (500 mg, 0.47 mmol) and methyl
3,5-diamino-6-chloropyrazine-2-carbonylcarbamimidothioate (21, 330
mg, 0.84 mmol) in EtOH (20 mL) was charged with DIPEA (0.84 mL,
94.70 mmol) at room temperature. The reaction mixture was heated at
70.degree. C. in a sealed tube for 2 h, cooled to room temperature,
and concentrated under vacuum. The residue was purified by
silica-gel column chromatography (80:18:2
CHCl.sub.3/CH.sub.3OH/NH.sub.4OH) to afford guanidine 47 (325 mg,
55%) as a yellow solid: [M+H].sup.+1278.
Preparation of the HCl Salt Compound 48
3,5-diamino-N-(N-(4-(4-((S)-2-amino-3-(4-(6-(bis((2S,3R,4R,5R)-2,3,4,5,6--
pentahydroxyhexyl)amino)hexyl)phenylamino)-3-oxopropyl)naphthalen-1-yl)but-
yl)carbamimidoyl)-6-chloropyrazine-2-carboxamide
[0356] 4 N HCl in water (20 mL) was added to 47 (325 mg, 0.25 mmol)
in EtOH (2.0 mL) and the reaction mixture was stirred at room
temperature for 2 h. The solvent was removed, the mixture was
purified by reverse-phase chromatography (Gold column), and the
residue was lyophilized to afford compound 48 (165 mg, 60%) as a
yellow solid: NMR (400 MHz, DMSO-d.sub.6): .delta. 10.52 (brs, 1H),
10.44 (brs, 1H), 9.28 (t, J=5.2 Hz, 1H), 9.00-8.88 (m, 1H),
8.87-8.75 (m, 1H), 8.63 (brs, 2H), 8.60-8.50 (m, 1H), 8.39-8.33 (m,
1H), 8.17-8.11 (m, 1H), 7.58 (dd, J=6.5, 3.3 Hz, 2H), 7.47-7.35 (m,
2H), 7.36 (d, J=8.7 Hz, 2H), 7.33 (d, J=6.8 hz, 1H), 7.27 (d, J=3.6
Hz, 1H), 7.11 (d, J=8.8 Hz, 2H), 3.72-3.66 (m, 3H), 3.60 (d, J=3.6
Hz, 1H), 3.57 (d, J=2.8 Hz, 1H), 3.53-3.46 (m, 3H), 3.45-3.38 (m,
3H), 3.37-3.27 (m, 4H), 3.26-3.12 (m, 4H), 3.06 (t, J=8.5 Hz, 2H),
1.76-1.60 (m, 6H), 1.58-1.47 (m, 2H), 1.35-1.23 (m, 4H).
[0357] .sup.1H NMR (400 MHz, CD.sub.3OD): .delta. 8.23-8.18 (m,
1H). 8.17-8.12 (m, 1H), 7.59-7.52 (m, 2H), 7.36 (d, J=7.8 Hz, 1H),
7.31 (d, J=7.2 Hz, 1H), 7.18 (d, J=8.2 Hz, 2H), 7.04 (d, J=8.2 Hz,
2H), 4.25 (t, J=7.8 Hz, 1H), 4.18-4.10 (m, 2H), 3.83-3.79 (m, 2H),
3.77 (d, J=3.1 Hz, 1H), 3.74 (d, J=3.5 Hz, 1H), 3.71-3.60 (m, 8H),
3.49-3.41 (m, 2H), 3.40-3.33 (m, 4H), 3.32-3.30 (m, 1H), 3.25-3.19
(m, 1H), 3.18-3.10 (m, 2H), 2.52 (t, J=7.4 Hz, 2H), 1.88-1.69 (m,
6H), 1.62-1.53 (m, 2H), 1.44-1.30 (m, 4H).
8. Preparation of
3,5-diamino-N-(N-(4-(4-((S)-2-amino-3-oxo-3-(4-(6-((2S,3R,4R,5R)-2,3,4,5,-
6- pentahydroxyhexylamino)hexyl)phenylamino)propyl)naphthalen-1-yl)
butyl)carbamimidoyl)-6-chloropyrazine-2-carboxamide
##STR00026## ##STR00027##
[0358] Preparation of Compound 50
[0359] A solution of acid 17 (950 mg, 1.84 mmol) in THF (30 mL) was
cooled to 0.degree. C. in an ice bath. NMM (0.40 mL, 3.68 mmol) was
added, followed by PivCl (0.23 mL, 1.84 mmol), and the reaction
mixture was stirred at the same temperature for 2 h. 49 (800 mg,
1.47 mmol, 10 mL THF) was added and the reaction mixture was
stirred at the same temperature for a further 10 min. The reaction
mixture was brought to room temperature and stirred for 16 h. The
organic solvent was removed. The residue was charged with water and
extracted with CH.sub.2Cl.sub.2 (3.times.100 mL). The organic
layers were combined, dried over Na.sub.2SO.sub.4, filtered, and
concentrated. The residue was purified by column chromatography (4%
methanol in chloroform) to afford amide 50 (1.40 g, impure) as a
light yellow solid: [M+H].sup.+1043.
Preparation of Compound 51
[0360] A suspension of 50 (1.40 g, impure) and 10% Pd/C (400 mg) in
a mixture of EtOH (120 mL) and AcOH (20 mL) was degassed and
subjected to hydrogenation conditions (1 atm) for 16 h at room
temperature. The reaction mixture was filtered through a plug of
Celite and the plug was washed with MeOH. The filtrate was
concentrated under vacuum to afford amine salt 51, directly used in
the next step (1.20 g, crude): [M+H].sup.+913.
Preparation of Compound 52
[0361] A solution of amine 51 (1.20 g, 0.47 mmol, crude) and methyl
3,5-diamino-6-chloropyrazine-2-carbonylcarbamimidothioate (21, 723
mg, 1.86 mmol) in EtOH (20 mL) was charged with DIPEA (2.00 mL,
11.6 mmol) at room temperature. The reaction mixture was heated at
70.degree. C. in a sealed tube for 2 h, cooled to room temperature,
and concentrated under vacuum. The residue was purified by
silica-gel column chromatography (80:18:2
CHCl.sub.3/CH.sub.3OH/NH.sub.4OH) to afford guanidine 52 (500 mg,
24% over three steps) as a yellow solid: [M+H].sup.+1125.
Preparation of the HCl Salt of Compound 53
3,5-diamino-N-(N-(4-(4-((S)-2-amino-3-oxo-3-(4-(6-((2S,3R,4R,5R)-2,3,4,5,-
6-pentahydroxyhexylamino)hexyl)phenylamino)propyl)
naphthalen-1-yl)butyl)carbamimidoyl)-6-chloropyrazine-2-carboxamide
[0362] 4 N HCl in water (25 mL) was added to 52 (500 mg, 0.44 mmol)
in EtOH (5.0 mL) and the reaction mixture was stirred at room
temperature for 2 h. The solvent was removed, the mixture purified
by reverse-phase chromatography (Gold column), and the residue was
lyophilized to afford compound 53 (170 mg, 41%) as a yellow solid:
.sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta. 10.52 (brs, 1H),
10.45-10.41 (m, 1H), 9.31-9.24 (m, 1H), 9.02-8.89 (m, 1H),
8.88-8.76 (m, 1H), 8.70-8.58 (m, 3H), 8.57-8.46 (m, 2H), 8.40-8.31
(m, 1H), 8.17-8.10 (m, 1H), 7.62-7.54 (m, 2H), 7.42 (brs, 2H), 7.36
(d, J=8.7 Hz, 2H) 7.33 (d, J=6.7 Hz, 1H), 7.27 (d, J=7.5 Hz, 1H),
7.11 (d, J=8.7 Hz, 2H), 5.41-5.35 (m, 1H), 4.79-4.72 (m, 1H),
4.62-4.53 (m, 2H), 4.47-4.38 (m, 1H), 4.29-4.19 (m, 1H), 3.94-3.87
(m, 1H), 3.63-3.52 (m, 3H), 3.50-3.39 (m, 3H), 3.38-3.32 (m, 2H),
3.12-2.96 (m, 3H), 2.97-2.90 (m, 1H), 2.89-2.80 (m, 2H), 1.77-1.56
(m, 6H), 1.54-1.45 (m, 2H), 1.35-1.20 (m, 4H).
[0363] .sup.1H NMR (400 MHz, CD.sub.3OD): .delta. 8.24-8.20 (m,
1H), 8.19-8.14 (m, 1H), 7.60-7.53 (m, 2H), 7.38 (d, J=7.6 Hz, 1H),
7.33 (d, J=7.2 Hz, 1H), 7.24-7.18 (m, 2H), 7.07 (d, J=8.1Hz, 2H),
4.31-4.22 (m, 1H), 4.08-4.01 (m, 1H), 3.84 (dd, J=4.8, 1.3 Hz, 1H),
3.77 (dd, J=10.1, 2.5 Hz, 1H), 3.71-3.62 (m, 5H), 3.36 (t, J=7.2
Hz, 2H), 3.19-3.12 (m, 4H), 3.03-2.96 (m, 2H), 2.55 (t, J=7.7 Hz,
2H), 1.90-1.74 (m, 4H), 1.73-1.64 (m, 2H), 1.63-1.53 (m, 2H),
1.45-1.31 (m, 4H).
9. Preparation of
3,5-diamino-N-(N-(4-(4-((S)-2-amino-3-(4-(6-(hexyl((2S,3R,4R,5R)-2,3,4,5,-
6-pentahydroxyhexyl)amino)hexyl)phenylamino)-3-oxopropyl)naphthalen-1-yl)b-
utyl)carbamimidoyl)-6-chloropyrazine-2-carboxamide
##STR00028## ##STR00029##
[0365] Preparation of Compound 55; Compound 54 (770 mg, 1.45 mmol)
in THF (50 mL) was charged with DEPBT (564 mg, 1.88 mmol), 17 (752
mg, 1.45 mmol), and DIPEA (0.77 mL, 4.35 mmol) successively and
stirred at room temperature for 16 h. After the solvent was removed
under reduced pressure, the residue was dissolved in
CH.sub.2Cl.sub.2 (100 mL), quickly washed with saturated aqueous
NaHCO.sub.3 (2.times.100 mL) and brine (50 mL), and dried over
Na.sub.2SO.sub.4. The solvent was evaporated and the crude product
purified by flash chromatography on silica gel (5%
methanol/CH.sub.2Cl.sub.2) and by reverse-phase chromatography
(Gold column), yielding amide 55 as a yellow solid (800 mg, 54%):
[M+H].sup.+1027.
Preparation of Compound 56
[0366] A suspension of 55 (800 mg, 0.78 mmol) and 10% Pd/C (400 mg)
in a mixture of EtOH (120 mL) and AcOH (30 mL) was degassed and
subjected to hydrogenation conditions (1 atm) for 16 h at room
temperature. The reaction mixture was filtered through a plug of
Celite and the plug was washed with MeOH. The filtrate was
concentrated under vacuum to afford amine salt 56 as a yellow solid
(780 mg, 99%): [M+H].sup.+897.
Preparation of Compound 57
[0367] A solution of amine salt 56 (780 mg, 0.75 mmol) and methyl
3,5-diamino-6-chloropyrazine-2-carbonylcarbamimidothioate (21, 466
mg, 1.20 mmol) in EtOH (20 mL) was charged with DIPEA (1.37 mL,
7.67 mmol) at room temperature. The reaction mixture was heated at
70.degree. C. in a sealed tube for 2 h, cooled to room temperature,
and concentrated under vacuum. The residue was purified by
silica-gel column chromatography (80:18:2
CHCl.sub.3/CH.sub.3OH/NH.sub.4OH) to afford guanidine 57 (455 mg,
55%) as a yellow solid: [M+H].sup.+1110.
Preparation of the HCl Salt of Compound 58
3,5-diamino-N-(N-(4-(4-((S)-2-amino-3-(4-(6-(hexyl((2S,3R,4R,5R)-2,3,4,5,-
6-pentahydroxyhexyl)amino)hexyl)phenylamino)-3-oxopropyl)naphthalen-1-yl)b-
utyl)carbamimidoyl)-6-chloropyrazine-2-carboxamide
[0368] 4 N HCl in water (25 mL) was added to 57 (455 mg, 0.41 mmol)
in ethanol (10 mL) and the reaction mixture was stirred at room
temperature for 2 h. The mixture was purified by reverse-phase
chromatography (Gold column) and the residue was lyophilized to
afford compound 58 (230 mg, 55%) as a yellow solid: .sup.1H NMR
(400 MHz, DMSO-d.sub.6): .delta. 10.45 (brs, 1H), 9.30 (brs, 1H),
9.09-8.49 (m, 3H), 8.41-8.32 (m, 1H), 8.16-8.08 (m, 1H), 7.62-7.52
(m, 2H), 7.42 (brs, 2H), 7.37 (t, J=8.4 Hz, 2H), 7.32 (d, J=7.8 Hz,
1H), 7.27 (d, J=7.2 Hz, 1H), 7.10 (d, J=8.1 Hz, 2H), 5.52-5.36 (m,
1H), 4.87-4.70 (m, 1H), 4.63-4.51 (m, 2H), 4.47-4.38 (m, 1H), 4.23
(t, J=6.7 Hz, 1H), 4.03-3.94 (m, 1H), 3.71-3.66 (m, 1H), 3.65-3.52
(m, 2H), 3.50-3.34 (m, 5H), 3.21(d, J=3.2 Hz, 1H), 3.12 (d, J=3.2
Hz, 1H), 3.09-2.96 (m, 6H), 1.77-1.58 (m, 8H), 1.57-1.46 (m, 2H),
1.35-1.21 (m, 10H), 0.86 (t, J=6.4 Hz, 3H).
[0369] .sup.1H NMR (400 MHz, CD.sub.3OD): .delta. 8.26-8.20 (m,
1H), 8.19-8.12 (m, 1H), 7.60-7.51 (m, 2H), 7.38 (d, J=7.2 Hz, 1H),
7.32 (d, J=7.4 Hz, 1H), 7.21 (d, J=8.3 Hz, 2H), 7.06 (d, J=8.5 Hz,
2H), 4.26 (t, J=7.4 Hz, 1H), 4.16-4.09 (m, 1H), 3.82 (dd, J=5.0,
1.5 Hz, 1H), 3.78 (dd, J=11.3, 3.2 Hz, 1H), 3.72-3.61 (m, 6H), 3.35
(t, J=6.7 Hz, 2H), 3.24-3.11 (m, 7H), 2.54 (t, J=7.4 Hz, 2H),
1.90-1.67 (m, 8H), 1.64-1.54 (m, 2H), 1.44-1.30 (m, 10H), 0.92 (t,
J=6.7 Hz, 3H).
10. Preparation of
(S)-2-amino-3-(6-(4-(3-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino-
)butyl)naphthalen-2-yl)propanoic acid (80)
##STR00030## ##STR00031## ##STR00032##
[0370] Preparation of Compound 62
[0371] The stable Wittig ylide
carbomthoxymethylenetriphenylphosphorane (Ph.sub.3PCHCO.sub.2Me,
43.0 g, 129 mmol) was added to a solution of the aldehyde 59 (20.0
g, 107 mmol) in CH.sub.2Cl.sub.2 (200 mL) under nitrogen atmosphere
and the reaction mixture was stirred 16 h at ambient temperature.
TLC monitored the completion of the reaction (16 h).
CH.sub.2Cl.sub.2 was removed under reduced pressure and FCC using
10% ethyl acetate-hexanes gave the corresponding
trans-.alpha.,.beta.-unsaturated ester 62 (24.0 g, 92%) as a white
solid: .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.88-7.82 (m, 1H),
8.81 (d, J=15.8 Hz, 1H), 7.73 (d, J=9.0 Hz, 1H), 7.70 (d, J=8.8 Hz,
1H), 7.61 (dd, J=8.8, 2.2 Hz, 1H), 7.15 (dd, J=9.2, 2.2 Hz, 1H),
7.11 (d, J=2.2 Hz, 1H), 6.49 (d, J=15.8 Hz, 1H), 3.82 (s, 3H), 3.82
(s, 3H).
Preparation of Compound 62 (Additional Route)
[0372] To the Trimethyl phosphonoacetate (55.6 mL, 381 mmol) in 250
mL anhydrous CH.sub.2Cl.sub.2 cooled to 0.degree. C. was added DBU
(48.8 mL, 322 mmol) and the mixture was stirred for 15 min.
Aldehyde 59 (40.0 g, 215 mmol) in 50 mL CH.sub.2Cl.sub.2 was added
dropwise. The temperature of the reaction mixture brought to rt and
resulting reaction mixture was stirred at rt for 16 h, and then
quenched with 100 mL of Water. The mixture was partitioned, and the
aq. layer was extracted with CH.sub.2Cl.sub.2 (3.times.150 mL). The
combined organics were washed with brine, dried (Na2SO4), filtered,
concentrated and the residue was purified by silica gel column
chromatography (10:1 hexanes/ethyl acetate) to give the desired
trans-.alpha.,.beta.-unsaturated ester 62 (48.0 g, 92%) as a white
solid.
Preparation of Compound 64
[0373] A suspension of compound 62 (48.0 g, 196 mmol) and 10% Pd/C
(10 g) in EtOAc/THF (600 mL/75 mL) was subjected to hydrogenation
conditions (1 atm) for 16 h at room temperature. The reaction
mixture was filtered through celite and washed with MeOH. The
filtrate was concentrated in vacuum to afford 64 (46.5 g, 96%) as a
white solid: .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.67 (d,
J=9.4 Hz, 2H), 7.57-7.54 (m, 1H), 7.29(dd, J=8.6, 1.8 Hz, 1H), 7.12
(dd, J=8.8, 2.5 Hz, 1H), 7.11-7.09 (m, 1H), 3.90 (s, 3H), 3.66 (s,
3H), 3.07 (t, J=7.7 Hz, 2H), 2.70 (t, J=7.7 Hz, 2H).
Preparation of Compound 66
[0374] To a solution of methyl ester 64 (46.5 g, 191 mmol) in
THF/MeOH/H.sub.2O (500 mL/500 mL/150 mL) was added NaOH (45.6 g,
114 mmol) and the reaction mixture was stirred at room temperature
for 2 h. Solvent was removed and pH value was adjusted to 1 with 1
N aq HCl; white solid precipitated out. Solid was filtered, washed
with water and dried under vacuum to afford acid 66 (42.5 g, 97%)
as a white solid: .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 12.14
(brs, 1H), 7.73 (dd, J=9.5, 2.3 Hz, 2H), 7.64-7.61 (m, 1H), 7.35
(dd, J=8.5, 1.5 Hz, 1H), 7.26 (d, J=2.8 Hz, 1H), 7.12 9 (dd, J=9.1,
2.5 Hz, 1H), 3.85 (s, 3H), 2.94 (t, J=7.6 Hz, 2H), 2.60 (t, J=7.6
Hz, 2H).
Preparation of Compound 67
[0375] To a solution of compound 60 (39.3 g, 222 mmol) in dry THF
(500 mL) was added n-butyl lithium (110 mL, 2M solution in
cyclohexane) drop wise at -78.degree. C. and the reaction mixture
was stirred for 1 h to give a solution of compound 61. To another
solution of compound 66 (42.5 g, 185 mmol) in dry THF (1000 mL) was
added NMM (26.3 mL, 240 mmol) and PivCl (27.3 mL, 222 mmol) drop
wise at -78.degree. C. The reaction mixture was stirred for for 1
min at the same temperature, and then the prepared solution of
compound 66 was added slowly at -78.degree. C. The reaction mixture
was stirred for another 10 min then brought to 0.degree. C. and
stirred for 1 h followed by at room temperature for 30 min,
quenched with satd NH.sub.4Cl, concentrated to remove THF, and
partitioned between CH.sub.2Cl.sub.2 (1000 mL) and water (1000 mL).
The aqueous layer was separated and extracted with CH.sub.2Cl.sub.2
(2.times.1000 mL). The combined organic extracts were dried over
Na.sub.2SO.sub.4 and concentrated. The residue was purified by
column chromatography (silica gel, CH.sub.2Cl.sub.2) to afford
compound 67 (45.0 g, 63%) as a white solid: .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 7.68 (d, J=8.6 Hz, 2H), 7.64-7.61 (m, 1H), 7.31
(dd, J=8.5, 1.8 Hz, 1H), 7.33-7.24 (m, 4H), 7.17-7.12 (m, 2H),
7.11-7.09 (m, 1H), 4.69-4.61 (m, 1H), 4.15 (d, J=2.4 Hz, 1H), 4.13
(s, 1H), 3.90 (s, 3H), 3.46-3.21 (m, 3H), 3.20-3.08 (m, 2H), 2.74
(dd, J=13.6, 9.4 Hz, 1H).
Preparation of Compound 68
[0376] To a solution of compound 67 (45.0 g, 116 mmol) in dry THF
(700 mL) was added KHMDS (34.6 g, 174 mmol) portion wise at
-78.degree. C. After the resulting mixture was stirred for 30 min,
trisyl azide (53.6 g, 174 mmol) was added and the reaction mixture
was stirred for 5 min. Then acetic acid (69.6 mL, 1158 mmol)
followed by tetramethyl ammonium acetate (30.9 g, 232 mmol) was
added slowly at the same temperature. The reaction mixture was
allowed to be warmed to 24.degree. C., stirred for 16 h, quenched
with satd NaHCO.sub.3 (300 mL), concentrated to remove THF and
extracted with CH.sub.2Cl.sub.2 (2.times.500 mL). The combined
organic extracts were dried over Na.sub.2SO.sub.4 and concentrated.
The residue was purified by column chromatography (silica gel,
10:90 EtOAc/Hexane followed by DCM) to afford compound 68 (31.0 g,
62%) as yellow solid: .sup.1H NMR (400 MHz, CDCl.sub.3) .delta.
7.70 (d, J=9.1 Hz, 2H), 7.68-7.65 (m, 1H), 7.40 (dd, J=8.6, 1.8 Hz,
1H), 7.36-7.23 (m, 3H), 7.20 (d, J=1.8 Hz, 1H), 7.19-7.17 (m, 1H),
7.13 (dd, J=9.0, 2.6 Hz, 1H), 7.10 (d, J=2.4 Hz, 1H), 5.36 (dd,
J=9.0, 6.0 Hz, 1H), 4.58-4.50 (m, 1H), 4.11 (dd, J=9.1, 2.6 Hz,
1H), 3.90 (s, 3H), 3.91 (t, J=8.6 Hz, 1H), 3.34 (dd, J=13.8, 6.5
Hz, 1H), 3.30 (dd, J=13.0, 3.5 Hz, 1H), 3.19 (dd, J=13.4, 8.6 Hz,
1H), 2.81 (dd, J=13.4, 9.5 Hz, 1H).
Preparation of Compound 69
[0377] To a solution of compound 68 (31.0 g, 72.1 mmol) in
THF/H.sub.2O (300 mL/100 mL) was added H.sub.2O.sub.2 (49 mL, 433
mmol) followed by LiOH (6.04 g, 144 mmol) portion wise at 0.degree.
C. The reaction mixture was stirred for 10 min at the same
temperature followed by at rt for 1 hr then quenched with satd
Na.sub.2SO.sub.3 (200 mL), concentrated under reduced pressure to
remove THF and washed with CH.sub.2Cl.sub.2 (500 mL). The aqueous
layer was acidified with 1N aq HCl and extracted with
CH.sub.2Cl.sub.2 (2.times.500 mL). The combined organic extracts
were dried over Na.sub.2SO.sub.4, concentrated and washed with MTBE
to afford compound 69 (15.0 g, 82%) as an off-white solid: .sup.1H
NMR (400 MHz, MeOD-d.sub.3) .delta. 7.70 (t, J=8.4 Hz, 2H),
7.66-7.63 (m, 1H), 7.35 (dd, J=8.6, 1.7 Hz, 1H), 7.19 (d, J=2.8 Hz,
1H), 7.10 (dd, J=9.1, 2.6 Hz, 1H), 4.25 (dd, J=8.6, 5.3 Hz, 1H),
3.88 (s, 3H), 3.29 (dd, J=13.9, 5.1 Hz, 1H), 3.10 (dd, J=14.3, 8.6
Hz, 1H).
Preparation of Compound 70
[0378] A suspension of compound 69 (15.0 g, 55.1 mmol) and 10% Pd/C
(3.50 g) in AcOH/H.sub.2O (300 mL/100 mL) was subjected to
hydrogenation conditions (1 atm) for 3 h at room temperature. The
reaction mixture was filtered through Celite and washed with
AcOH/H.sub.2O followed by MeOH. The filtrate was concentrated in
vacuum to afford acetic salt 70 (14.0 g, 83%) as a yellow solid:
.sup.1H NMR (400 MHz, DMSO-d.sub.6, TFA) .delta. 8.38-8.18 (m, 3H),
7.78 (dd, J=11.4, 8.1 Hz, 2H), 7.75-7.70 (m, 1H), 7.41 (dd, J=8.6,
1.6 Hz, 1H), 7.29 (d, J=2.3 Hz, 1H), 7.18 (dd, J=8.8, 2.4 Hz, 1H),
4.33-4.23 (m, 1H), 3.89 (s, 3H), 3.33 (dq, J=14.5, 5.9 Hz, 2H),
1.92 (s, 3H).
Preparation of Compound 71
[0379] To a solution of compound 70 (14.0 g, 45.9 mmol) in acetic
acid (140 mL) was added hydro bromic acid (140 mL) drop wise at
room temperature and the reaction mixture was refluxed for 3 h. The
reaction mixture was cooled to room temperature and concentrated.
The crude brown residue 71 (12.4 g, 87%) was directly used for the
next step without any purification: .sup.1H NMR (400 MHz,
DMSO-d.sub.6) .delta. 13.83 (brs, 1H), 9.71 (brs, 1H), 8.41 (brs,
1H), 8.25 (brs, 2H), 7.67 (dd, J=13.8, 8.7 Hz, 2H), 7.64-7.61 (m,
1H), 7.29 (dd, J=8.6, 1.7 Hz, 1H), 7.13-7.05 (m, 2H), 4.29-4.19 (m,
1H), 3.20 (t, J=5.5 Hz, 2H).
Preparation of Compound 72
[0380] Acetyl chloride (38.4 mL, 540 mmol) was added to dry
methanol (400 mL) at 0.degree. C. and then compound 71 (24.0 g,
77.2 mmol) was added. The reaction mixture was refluxed for 4 h and
concentrated. The residue was partitioned between CH.sub.2Cl.sub.2
(500 mL) and saturated NaHCO.sub.3 (300 mL). The aqueous layer was
separated and extracted with CH.sub.2Cl.sub.2 (2.times.300 mL). The
combined organic extracts were dried over Na.sub.2SO.sub.4 and
concentrated to afford compound 72 (16.6 g, 88%) as white solid:
.sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 9.62 (brs, 1H), 7.67
(d, J=9.4 Hz, 1H), 7.58 (d, J=8.8 Hz, 1H), 7.53 (s, 1H), 7.22 (dd,
J=8.2, 1.4 Hz, 1H), 7.09-7.06 (m, 1H), 7.04 (dd, J=8.8, 2.6 Hz,
1H), 3.67 (t, J=6.5 Hz, 1H), 3.57 (s, 3H), 2.97 (dd, J=13.5, 6.1
Hz, 1H), 2.86 (dd, J=13.2, 7.4 Hz, 1H), 1.90 (brs, 2H).
Preparation of Compound 73
[0381] To a solution of compound 72 (16.6 g, 67.8 mmol) in
MeOH/H.sub.2O (360 mL/120 mL) was added NaHCO.sub.3 (22.8 g, 271
mmol) and Boc.sub.2O (17.7 g, 81.3 mmol) at 0.degree. C. The
resulting mixture was allowed to warm to room temperature and
stirred for 1 h. The reaction mixture was partitioned between
CH.sub.2Cl.sub.2 (200 mL) and water (200 mL). The aqueous layer was
separated and extracted with CH.sub.2Cl.sub.2 (2.times.400 mL). The
combined organic extracts were washed with brine, dried over
Na.sub.2SO.sub.4 and concentrated. FCC using 20% ethyl
acetate-hexanes followed by CH.sub.2Cl.sub.2 gave the compound 73
(17.0 g, 73%) as a white solid: .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. 7.60 (d, J=9.5 Hz, 1H), 7.51 (d, J=8.2 Hz, 1H), 7.49-7.43
(m, 1H), 7.15 (d, J=8.2 Hz, 1H), 7.09-6.99 (m, 2H), 6.31 (brs, 1H),
5.15-4.84 (m, 1H), 4.73-4.46 (m, 1H), 3.71 (s, 3H), 3.23 (dd,
J=13.7, 5.3 Hz, 1H), 3.14 (dd, J=13.7, 5.5 Hz, 1H), 1.39 (s,
9H).
Preparation of Compound 74
[0382] To a solution of compound 73 (7.0 g, 20.3 mmol) in
CH.sub.2Cl.sub.2 (300 mL) was added pyridine (16.5 mL, 203 mmol)
was added inflate (5.11 mL, 30.4 mmol) at 0.degree. C. and stirred
at same temperature for 1 h followed by at room temperature for 2
h. After concentrated, the reaction mixture was partitioned between
CH.sub.2Cl.sub.2 (300 mL) and water (200 mL). The aqueous layer was
separated and extracted with CH.sub.2Cl.sub.2 (2.times.300 mL). The
combined organic extracts were washed with brine, dried over
Na.sub.2SO.sub.4 and concentrated to afford compound 74 (8.80 g,
91%) as a brown oil (pyridine present as confirmed by NMR). The
reaction was monitored by using LC-MS, and product formation was
confirmed by LM-MS data: .sup.1H NMR (400 MHz, CDCl.sub.3) 7.85 (d,
J=9.2 Hz, 1H), 7.80 (d, J=8.7 Hz, 1H), 7.71 (d, J=2.7 Hz, 1H),
7.66-7.63 (m, 1H), 7.37 (ddd, J=10.0, 7.3, 2.0 Hz, 2H), 5.12-5.03
(m, 1H), 4.73-4.61 (m, 1H), 3.72 (s, 3H), 3.32 (dd, 13.3, 5.3 Hz,
1H), 3.20 (dd, J=13.3, 6.2 Hz, 1H), 1.38 (s, 9H).
Preparation of Compound 75
[0383] Compound 74 (16.5 g, 34.6 mmol) and benzyl
but-3-ynylcarbamate (17, 10.4 g, 51.9 mmol) in anhydrous CH.sub.3CN
(450 mL) was degassed with Argon for 10 min at rt, then added TEA
(19.3 mL, 138 mmol), 10% (t-Bu).sub.3P in hexanes (13.9 mL, 6.91
mmol), and CuI (0.33 g, 1.72 mmol) at room temperature. The
resulting mixture was degassed with Argon for 10 min and
Pd(PPh.sub.3).sub.4 (3.99 g, 3.45 mmol) was added rapidly in one
portion. After degassed with Argon for 5 min, the resulting mixture
was refluxed for 18 h. The reaction mixture was concentrated in
vacuum and the residue was purified by column (silica gel, 75:25
hexanes/EA) to afford compound 75 (14.1 g, 77%) as a brown solid:
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.86 (brs, 1H), 7.68 (t,
J=7.8 Hz, 2H), 7.53 (brs, 1H), 7.41 (dd, J=8.5, 1.6 Hz, 1H),
7.38-7.28 (m, 5H), 7.27-7.22 (m, 1H), 5.26-5.17 (m, 1H), 5.13 (s,
2H), 5.06-4.99 (m, 1H), 4.70-4.59 (m, 1H), 3.69 (s, 3H), 3.46 (q,
J=6.7 Hz, 2H), 3.27 (dd, J=14.1, 5.9 Hz, 1H), 3.16 (dd, J=13.2, 6.2
Hz, 1H), 2.67 (t, J=6.6 Hz, 2H), 1.38 (s, 9H).
Preparation of Compound 76
[0384] To a solution of methyl ester 75 (12.1 g, 22.8 mmol) in
THF/MeOH/H.sub.2O (150 mL/150 mL/50 mL) was added NaOH (4.56 g, 114
mmol) and the reaction mixture was stirred at room temperature for
2 h. The pH value was adjusted to 9 with 1 N aq HCl and organic
solvent was removed. The pH value of residue was adjusted to 5-6,
and the suspension was partitioned between CH.sub.2Cl.sub.2 (500
mL) and water (200 mL). The aqueous layer was separated and
extracted with CH.sub.2Cl.sub.2 (2.times.400 mL). The combined
organic extracts were dried over Na.sub.2SO.sub.4 and concentrated
to afford compound 76 (10.50 g, 89%) as a brown solid: .sup.1H NMR
(400 MHz, CD.sub.3OD) .delta. 7.83 (s, 1H), 7.73-7.61 (m, 3H),
7.44-7.19 (m, 7H), 5.10 (s, 2H), 4.42-4.34 (m, 1H), 3.41-3.32 (m,
3H), 3.06 (dd, J=14.3, 9.3 Hz, 1H), 2.64 (t, J=7.0 Hz, 2H), 1.31
(s, 7H), 1.21 (s, 2H).
Preparation of Compound 77; SG-SJL-B-27
[0385] A suspension of 75 (2.0 g, 3.77 mmol) and 10% Pd/C (500 mg)
in a mixture of EtOH (90 mL) and AcOH (10 mL) was degassed and then
subjected to hydrogenation conditions (1 atm) for 16 h at room
temperature. The reaction mixture was filtered through a plug of
Celite and the plug was washed with MeOH. The filtrate was
concentrated in vacuum to afford amine salt 77 (1.60 mg, 93%) as a
white solid: .sup.1H NMR (400 MHz, CD.sub.3OD) 7.73 (d, J=8.5, 1H),
7.72 (d, J=8.7 Hz, 1H), 7.62 ((brs, 2H), 7.73 (ddd, J=10.0, 8.7,
2.7 Hz, 2H), 4.44 (dd, J=8.8, 5.6 Hz, 1H), 3.68 (s, 3H), 3.25 (dd,
J=14.0, 6.6 Hz, 1H), 3.04 (dd, J=13.5, 9.2 Hz, 1H), 2.93 (t, J=7.4
Hz, 2H), 2.83 (t, J=7.4 Hz, 2H), 1.96 (s, 6H), 1.85-1.75 (m, 2H),
1.74-1.68 (m, 2H), 1.33 (s, 7H), 1.26 (s, 2H).
Preparation of Compound 78; SG-SJL-B-30
[0386] To a solution of amine salt 77 (1.60 g, 3.47 mmol) and
methyl 3,5-diamino-6-chloropyrazine-2-carbonylcarbamimidothioate
(13, 2.16 g, 5.56 mmol) in EtOH (40 mL) was added DIPEA (6.20 mL,
34.70 mmol) at room temperature. The reaction mixture was heated at
70.degree. C. in a sealed tube for 1 h, then cooled to room
temperature, and concentrated in vacuum. The residue was purified
by column chromatography (silica gel, 80:18:2
CHCl.sub.3/CH.sub.3OH/NH.sub.4OH) to afford guanidine 78 (1.24 g,
59%) as a yellow solid: .sup.1H NMR (400 MHz, CD.sub.3OD) .delta.
7.71 (dd, J=8.4, 2.8 Hz, 2H), 7.60 (brs, 2H), 7.34 (dd, J=8.5, 1.9
Hz, 1H), 7.30 (dd, J=8.7, 1.7 Hz, 1H), 4.45 (dd, J=8.9, 5.7 Hz,
1H), 3.68 (s, 3H), 3.28-3.26 (m, 1H), 3.25 (t, J=2.4 Hz, 1H), 3.22
(d, J=5.9 Hz, 1H), 3.04 (dd, J=14.0, 9.2 Hz, 1H), 2.82 (t, J=7.2
Hz, 2H), 1.86-1.77 (m, 2H), 1.73-1.63 (m, 2H), 1.32 (s, 7H), 1.23
(s, 2H).
Preparation of Compound 79; SG-SJL-B-32
[0387] A solution of methyl ester 78 (1.24 g, 2.00 mmol) in a
mixture of THF (25 mL), methanol (25 mL) and water (10 mL) was
added solid NaOH (324 mg, 8.00 mmol) and the reaction mixture was
stirred at room temperature for 1 h. TLC of the reaction mixture
showed completion of reaction then pH of the reaction mixture was
brought to pH 9-10 by addition of 1 N HCl. (Aquous) and organic
solvent was removed. The pH of aq. part was adjusted to pH 5-6 and
precipitated came out and extracted with dichloromethane. Aquous
part was extracted with CH.sub.2Cl.sub.2 (2.times.50 mL). Organic
layers were combined, dried over Na.sub.2SO.sub.4, filtered, and
concentrated. Yellow colored solid compound (79, 1.10 g, 92%) was
dried under vacuum: .sup.1H NMR ((400 MHz, CD.sub.3OD) 7.70 (t,
J=9.4, 2H), 7.61 (d, J=5.3 Hz, 2H), 7.33 (dd, J=8.4, 1.4 Hz, 2H),
4.38 (dd, J=8.4, 5.1 Hz, 1H), 3.05 (dd, J=14.1, 9.1 Hz, 1H), 2.84
(t, J=6.9 Hz, 2H), 3.35-3.34 (m, 3H), 1.88-1.79 (m, 2H), 1.76-1.67
(m, 2H), 1.32 (s, 7H), 1.21 (s, 2H).
Preparation of Compound 80 Hydrochloride Salt of
(S)-2-amino-3-(6-(4-(3-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino-
)butyl)naphthalen-2-yl)propanoic acid
[0388] 4 N HCl in dioxane (25 mL) was added to 79 (1.10 g, 1.83
mmol) in EtOH (5.0 mL) and reaction mixture was stirred at room
temperature for 2 h. The solvent was removed, purified by reverse
phase column (gold column) and residue was lyophilized to afford
compound 80 (700 mg, 67%) as a yellow solid: .sup.1H NMR (400 MHz,
DMSO-d6) 10.48 (s, 1H), 9.24 (brs, 1H), 8.99-8.86 (m, 1H),
8.84-8.70 (m, 1H), 8.38 (brs, 3H), 7.80 (t, J=9.2 Hz, 2H), 7.73 (s,
1H), 7.69 (s, 1H), 7.45-7.35 (m, 4H), 4.25 (dd, J=11.4, 5.9 Hz,
1H), 3.34 (q, J=6.6 Hz, 2H), 3.27 (d, J=6.9 Hz, 2H), 2.79 (t,
J=7.70 Hz, 2H), 1.79-1.67 (m, 2H), 1.65-1.54 (m, 2H).
[0389] .sup.1H NMR ((400 MHz, CD.sub.3OD) 7.82 (d, J=8.5 Hz, 1H),
7.78 (d, J=8.7 Hz, 1H), 7.73 (s, 1H), 7.68 (s, 1H), 7.40 (ddd,
J=10.5, 8.6, 1.6 Hz, 2H), 4.33 (dd, J=7.7, 5.2 Hz, 1H), 3.46 (dd,
J=14.9, 6.0 Hz, 1H), 3.37 (t, J=7.5 Hz, 2H), 3.33-3.29 (m, 1H),
2.87 (t, J=7.7 Hz, 2H), 1.90-1.80 (m, 2H), 1.79-1.71 (m, 2H).
11. Preparation of
(S)-3,5-diamino-6-chloro-N-(N-(4-(6-(2,3-diamino-3-oxopropyl)naphthalen-2-
-yl)butyl)carbamimidoyl)pyrazine-2-carboxamide (84)
##STR00033##
[0390] Preparation of Compound 81
[0391] A solution of acid 76 (2.0 g, 3.87 mmol) in THF (80 ML) was
cooled to 0.degree. C. in ice-bath, NMM (0.63 mL, 5.03 mmol) was
added followed by dropwise i-BCF (0.63 mL, 5.80 mmol) and the
reaction mixture was stirred at the same temperature for 2 h.
NH.sub.3 (7.0 N in methanol, 5.52 mL, 38.7 mmol) was added dropwise
and the reaction mixture was stirred at the same temperature for a
further 2 h. Reaction mixture was then brought to rt and stirred
for 16 h. Organic solvent was removed. To this residue was added
water and extracted with CH.sub.2Cl.sub.2 (3.times.100 mL). The
organic layers were combined, dried over Na.sub.2SO.sub.4,
filtered, and concentrated. The residue was purified by column
chromatography (3% methanol in chloroform) to afford amide 81 (1.75
g, 88%) as a light yellow solid: .sup.1H NMR (400 MHz, CD.sub.3OD)
7.83 (s, 1H), 7.69 (d, J=8.1 Hz, 2H), 7.66 (s, 1H), 7.39 (dt,
J=8.8, 1.9 Hz, 2H), 7.35-7.21 (m, 5H), 5.09 (s, 2H), 4.40 (dd,
J=9.6, 5.8 Hz, 1H), 3.37 (t, J=6.9 Hz, 2H), 3.27 (dd, J=13.8, 5.2
Hz, 1H), 2.97 (dd, J=13.7, 9.4 Hz, 1H), 2.63 (t, J=7.0 Hz, 2H),
1.27 (s, 7H), 1.21 (s, 2H).
Preparation of Compound 82
[0392] A suspension of 81 (1.75 mg, 3.39 mmol) and 10% Pd/C (600
mg) in a mixture of EtOH (110 mL) and AcOH (15 mL) was degassed and
then subjected to hydrogenation conditions (1 atm) for 12 h at room
temperature. The reaction mixture was filtered through a plug of
Celite and the plug was washed with MeOH. The filtrate was
concentrated in vacuum to afford amine salt 82 as a white solid
(1.40 g, 93%): .sup.1H NMR (400 MHz, CD.sub.3OD) 7.72 (dd, J=8.3,
5.6 Hz, 2H), 7.66 (s, 1H), 7.61 (s, 1H), 7.38 (dd, J=8.6, 1.3 Hz,
1H), 7.34 (dd, J=8.5, 1.5 Hz, 1H), 4.38 (dd, J=9.0, 5.0 Hz, 1H),
3.27 (dd, J=13.8, 5.0 Hz, 1H), 2.93 (t, J=7.9 Hz, 2H), 2.83 (t,
J=7.5 Hz, 2H), 3.01-2.95 (m, 1H), 1.96 (s, 3H), 1.86-1.75 (m, 2H),
1.74-1.64 (m, 2H), 1.29 (s, 7H), 1.23 (s, 2H).
Preparation of Compound 83
[0393] To a solution of amine salt 82 (1.40 g, 3.15 mmol) and
methyl 3,5-diamino-6-chloropyrazine-2-carbonylcarbamimidothioate
(13, 1.96 g, 5.04 mmol) in EtOH (40 mL) was added DIPEA (5.64 mL,
31.5 mmol) at room temperature. The reaction mixture was heated at
70.degree. C. in a sealed tube for 2 h, then cooled to room
temperature, and concentrated in vacuum. The residue was purified
by column chromatography (silica gel, 80:18:2
CHCl.sub.3/CH.sub.3OH/NH.sub.4OH) to afford guanidine 83 (1.15 g,
61%) as a yellow solid: .sup.1H NMR (400 MHz, CD.sub.3OD) .delta.
7.70 (d, J=8.3 Hz, 2H), 7.64 (s, 1H), 7.60 (s, 1H), 7.34 (dt,
J=8.9, 1.9 Hz, 2H), 4.38 (dd, J=9.0, 5.5 Hz, 1H), 3.28-3.20 (m,
3H), 2.96 (dd, J=9.6, 14.1 Hz, 1H), 2.81 (t, J=7.4 Hz, 2H),
1.85-1.76 (m, 2H), 1.70-1.61 (m, 2H), 1.27 (s, 7H), 1.20 (s,
2H).
Preparation of Compound the HCl Salt of
(S)-3,5-diamino-6-chloro-N-(N-(4-(6-(2,3-diamino-3-oxopropyl)naphthalen-2-
-yl)butyl)carbamimidoyl)pyrazine-2-carboxamide (84)
[0394] 4 N HCl in dioxane (25 mL) was added to 83 (1.15 g, 1.92
mmol) in EtOH (6.0 mL) and reaction mixture was stirred at room
temperature for 2 h. The solvent was removed, purified by reverse
phase column (gold column) and residue was lyophilized lyophilized
to afford compound 84 (310 mg, 28%) as a yellow solid: .sup.1H NMR
(400 MHz, DMSO-d.sub.6) 10.56 (s, 1H), 9.38 (t, J=5.5 Hz, 1H),
9.06-8.83 (m, 2H), 8.31 (brs, 3H), 8.02 (s, 1H), 7.79 (t, J=8.6 Hz,
2H), 7.73 (s, 1H), 7.69 (s, 1H), 7.51 (s, 1H), 7.46-7.36 (m, 4H),
4.06 (dd, J=11.5, 6.0 Hz, 1H), 3.37 (q, J=6.4 Hz, 2H), 3.27 (dd,
J=6.6, 1.4 Hz, 1H), 3.18 (dd, J=13.7, 6.9 Hz, 1H), 2.79 (t, J=7.1
Hz, 2H), 1.79-1.69 (m, 2H), 1.64-1.54 (m, 2H).
[0395] .sup.1H NMR (400 MHz, CD.sub.3OD) 7.82 (d, J=8.4 Hz, 1H),
7.79 (d, J=8.7 Hz, 1H), 7.74 (s, 1H), 7.68 (s, 1H), 7.41 (td,
J=8.1, 1.6 Hz, 2H), 4.18 (dd, J=8.1, 6.3 Hz, 1H), 3.42-3.34 (m,
3H), 3.21 (dd, J=14.1, 8.0 Hz, 1H), 2.86 (t, J=7.4 Hz, 2H),
1.91-1.80 (m, 2H), 1.79-1.71 (m, 2H).
12. Preparation of
3,5-diamino-N-(N-(4-(6-((S)-2-amino-3-(4-(3-(hexyl((2S,3R,4R,5R)-2,3,4,5,-
6-pentahydroxyhexyl)amino)propyl)phenylamino)-3-oxopropyl)naphthalen-2-yl)-
butyl)carbamimidoyl)-6-chloropyrazine-2-carboxamide (89)
##STR00034## ##STR00035##
[0396] Preparation of compound 86
[0397] To the compound 85 (1.10 g, 2.32 mmol) in THF (50 mL) were
added DEPBT (766 mg, 2.56 mmol), 76 (1.00 g, 1.97 mmol) and DIPEA
(1.0 mL, 5.91 mmol) successively and stirred at rt for 16 h. After
the solvent was removed under reduced pressure, the residue was
dissolved in CH.sub.2Cl.sub.2 (100 mL), quickly washed with
saturated aqueous water (2.times.100 mL) and brine (50 mL), and
dried over Na.sub.2SO.sub.4. The solvent was evaporated and the
crude product purified by flash chromatography on silica gel (5%
Methanol/CH.sub.2Cl.sub.2), yielding amide 86 as a yellow solid
product (1.19 g, 57%): .sup.1H NMR (400 MHz, CD.sub.3OD): 7.82 (d,
J=5.8 Hz, 2H), 7.73-7.61 (m, 4H), 7.51-7.43 (m, 2H), 7.39-7.19 (m,
10H), 7.05 (d, J=8.3 Hz, 2H), 5.52 (s, 1H), 5.10 (s, 2H), 4.51 (t,
J=7.8 Hz, 1H), 4.31-4.25 (m, 1H), 4.24 (dd, J=11.0, 5.4 Hz, 1H),
4.01-3.91 (m, 2H), 3.88 (dd, J=5.5, 2.1 Hz, 1H), 3.76 (dd, J=9.3,
2.1 Hz, 1H), 3.61 (t, J=10.6 Hz, 1H), 3.37 (t, J=6.9 Hz, 2H),
3.12-3.00 (m, 1H), 2.74 (dd, J=13.2, 5.3 Hz, 1H), 2.64 (t, J=7.1
Hz, 2H), 2.57-2.37 (m, 7H), 1.74-1.64 (m, 2H), 1.31 (s, 9H),
1.29-1.16 (m, 8H), 0.86 (t, J=6.9 Hz, 3H).
Preparation of Compound 87
[0398] A suspension of 86 (1.19 g, mixture) and 10% Pd/C (220 mg)
in a mixture of EtOH (110 mL) and AcOH (15 mL) was degassed and
then subjected to hydrogenation conditions (1 atm) for 3 h at room
temperature. The reaction mixture was filtered through a plug of
Celite and the plug was washed with MeOH. The filtrate was
concentrated in vacuum to afford amine salt 87 which was then
neutralized with NaHCO.sub.3 and crude product was purified by
flash chromatography on silica gel (CMA, 80:18:2) yielding free
amine 87 as a yellow solid (550 mg, 58%, over two steps): .sup.1H
NMR (400 MHz, CD.sub.3OD) 7.71 (t, J=8.4 Hz, 2H), 7.62 (d, J=1.8
Hz, 1H), 7.49-7.45 (m, 3H), 7.40 (d, J=8.2 Hz, 2H), 7.36-7.28 (m,
5H), 7.09 (d, J=8.2 Hz, 2H), 5.55 (s, 1H), 4.51 (dd, J=15.6, 8.4
Hz, 1H), 4.25 (dd, J=10.6, 5.4 Hz, 1H), 4.17-4.03 (m, 2H),
3.98-3.90 (m, 2H), 3.81-3.74 (m, 1H), 3.63 (t, J=10.4 Hz, 1H),
3.27-3.20 (m, 1H), 3.09-2.98 (m, 5H), 2.93 (t, J=7.6 Hz, 2H), 2.83
(t, J=6.8 Hz, 2H), 2.61-2.54 (m, 2H), 1.95-1.86 (m, 2H), 1.85-1.75
(m, 2H), 1.74-1.65 (m, 2H), 1.57-1.47 (m, 2H), 1.39-1.19 (m, 7H),
1.33 (s, 9H), 0.88 (t, J=6.9 Hz, 3H).
Preparation of 88
[0399] To a solution of amine 87 (550 mg, 0.65 mmol) and methyl
3,5-diamino-6-chloropyrazine-2-carbonylcarbamimidothioate (21, 400
mg, 1.04 mmol) in EtOH (20 mL) was added DIPEA (1.15 mL, 6.44 mmol)
at room temperature. The reaction mixture was heated at 70.degree.
C. in a sealed tube for 2 h, then cooled to room temperature, and
concentrated in vacuum. The residue was purified by silica gel
column chromatography (80:18:2 CHCl.sub.3/CH.sub.3OH/NH.sub.4OH)
followed by reverse phase column (Gold C18) to afford guanidine 88
(333 mg, 48%) as a yellow solid: .sup.1H NMR (400 MHz, CD.sub.3OD)
7.69 (dd, J=8.6, 3.5 Hz, 2H), 7.66 (s, 1H), 7.60 (s, 1H), 7.48-7.44
(m, 2H), 7.35 (ddd, J=10.4, 8.6, 1.6 Hz, 2H), 7.33-7.28 (m, 5H),
7.04 (d, J=8.3 Hz, 2H), 5.52 (s, 1H), 4.52-4.55 (m, 1H), 4.24 (dd,
J=10.6, 5.4 Hz, 1H), 4.00-3.91 (m, 2H), 3.88 (dd, J=5.4, 2.0 Hz,
1H), 3.75 (dd, J=9.6, 2.2 Hz, 1H), 3.60 (t, J=10.6 Hz, 2H),
3.28-3.23 (m, 3H), 3.06 (dd, J=13.5, 8.3 Hz, 1H), 2.82 (t, J=7.0
Hz, 2H), 2.77 (dd, J=13.9, 5.6 Hz, 1H), 2.59-2.40 (m, 7H),
1.86-1.76 (m, 2H), 1.74-1.68 (m, 4H), 1.42-1.60 (m, 7H), 1.33 (s,
9H), 0.86 (t, J=7.1 Hz, 3H).
Preparation of the HCl Salt of
3,5-diamino-N-(N-(4-(6-((S)-2-amino-3-(4-(3-(hexyl((2S,3R,4R,5R)-2,3,4,5,-
6-pentahydroxyhexyl)amino)propyl)phenylamino)-3-oxopropyl)naphthalen-2-yl)-
butyl)carbamimidoyl)-6-chloropyrazine-2-carboxamide Compound
(89)
[0400] 4 N HCl in water (20 mL) was added to 88 (333 mg, 0.31 mmol)
in ethanol (10 mL) and reaction mixture was stirred at rt for 2 h.
Purified by reverse phase column (gold column) and residue was
lyophilized to afford compound 89 (210 mg, 68%) as a yellow solid:
.sup.1H NMR (400 MHz, DMSO-d.sub.6) 10.94 (brs, 1H), 9.29 (brs,
1H), 9.02-8.77 (m, 2H), 8.64-8.17 (m, 2H), 7.80-7.73 (m, 3H), 7.68
(s, 1H), 7.52 (d, J=8.8 Hz, 2H), 7.47 (dd, J=8.2, 1.0 Hz, 1H),
7.44-7.36 (m, 3H), 7.19 (d, J=8.6 Hz, 2H), 5.52-5.41 (m, 1H),
4.86-4.71 (m, 1H), 4.60 (d, J=5.4 Hz, 1H), 4.59-4.53 (m, 1H), 4.42
(t, J=5.8 Hz, 1H), 4.38 (t, J=7.0 Hz, 1H), 4.03-3.95 (m, 1H),
3.71-3.66 (m, 1H), 3.62-3.55 (m, 1H), 3.53-3.34 (m, 5H), 3.27 (d,
J=7.7 Hz, 1H), 3.23 (d, J=7.4 Hz, 1H), 3.16-2.99 (m, 5H), 2.78 (t,
J=7.4 Hz, 2H), 2.58 (t, J=7.9 Hz, 2H), 2.01-1.90 (m, 2H), 1.78-1.68
(m, 2H), 1.66-1.54 (m, 4H), 1.32-1.21 (m, 6H), 0.85 (t, J=6.6 Hz,
3H).
[0401] .sup.1H NMR (400 MHz, CD.sub.3OD) 7.79 (d, J=8.5 Hz, 1H),
7.77-7.73 (m, 2H), 7.67 (s, 1H), 7.47-7.37 (m, 4H), 7.21 (d, J=8.5
Hz, 2H), 4.30 (dd, J=7.7 , 6.7 Hz, 1H), 4.12-4.05 (m, 1H),
3.82-3.74 (m, 1H), 3.71-3.61 (m, 2H), 3.49 (dd, J=14.0, 6.6 Hz,
1H), 3.47(t, J=6.9 Hz, 2H), 3.33-3.27 (m, 3H), 3.26-3.13 (m, 4H),
2.86 (t, J=7.6 Hz, 2H), 2.73-2.64 (m, 2H), 2.10-2.00 (m, 2H),
1.89-1.80 (m, 2H), 1.79-1.72 (m, 2H), 1.71-1.63 (m, 2H), 1.40-1.30
(m, 6H), 0.91 (t, J=6.6 Hz, 3H),
13. Preparation of
3,5-diamino-N-(N-(4-(6-((S)-2-amino-3-(4-(3-(bis((2S,3R,4R,5R)-2,3,4,5,6--
pentahydroxyhexyl)amino)propyl)phenylamino)-3-oxopropyl)naphthalen-2-yl)bu-
tyl)carbamimidoyl)-6-chloropyrazine-2-carboxamide (94)
##STR00036## ##STR00037##
[0402] Preparation of Compound 91
[0403] To the compound 90 (484 mg, 0.91 mmol) in THF (30 mL) were
added DEPBT (300 mg, 1.00 mmol), 19 (400 g, 0.77 mmol) and DIPEA
(0.40 mL, 2.31 mmol) successively and stirred at rt for 16 h. After
the solvent was removed under reduced pressure, the residue was
dissolved in CH.sub.2Cl.sub.2 (100 mL), quickly washed with
saturated aqueous water (2.times.100 mL) and brine (50 mL), and
dried over Na.sub.2SO.sub.4. The solvent was evaporated and the
crude product purified by flash chromatography on silica gel (5%
Methanol/CR,Cb), yielding amide 91 as a yellow solid product (600
mg, 76%, impure). Product formation was confirmed by LCMS.
Preparation of Compound 92
[0404] A suspension of 91 (600 mg, 0.59 mmol) and 10% Pd/C (200 mg)
in a mixture of EtOH (90 mL) and AcOH (10 mL) was degassed and then
subjected to hydrogenation conditions (1 atm) for 16 h at room
temperature. The reaction mixture was filtered through a plug of
Celite and the plug was washed with MeOH. The filtrate was
concentrated in vacuum to afford amine salt 92which was then
neutralized with NaHCO.sub.3 and crude product was purified by
flash chromatography on silica gel (CMA, 80:18:2) yielding free
amine 36 as a yellow solid (350 mg, 66%, impure): .sup.1H NMR (400
MHz, CD.sub.3OD) 7.71 (d, J=8.1 Hz, 2H), 7.68 (s, 1H), 7.61 (s,
2H), 7.43-7.37 (m, 2H), 7.34 (dd, J=8.3, 1.3 Hz, 1H), 7.16 (d,
J=8.2 Hz, 2H), 4.69 (q, J=5.1 Hz, 2H), 4.50 (t, J=7.1 Hz, 1H),
4.13-4.06 (m, 2H), 4.05 (dd, J=11.0, 5.6 Hz, 2H), 3.83 (dd, J=4.8,
2.1 Hz, 2H), 3.81-3.73 (m, 2H), 3.51 (dd, J=9.5, 2.3 Hz, 2H), 3.38
(t, J=10.8 Hz, 2H), 3.13-3.03 (m, 6H), 2.93 (t, J=7.6 Hz, 2H), 2.82
(t, J=7.2 Hz, 2H), 2.74-2.57 (m, 2H), 2.04-1.95 (m, 2H), 1.84-1.75
(m, 3H), 1.74-1.63 (m, 3H), 1.33 (s, 9H), 1.25 (d, J=5.1 Hz,
6H).
Preparation of 93
[0405] To a solution of amine 92 (350 mg, 0.38 mmol) and methyl
3,5-diamino-6-chloropyrazine-2-carbonylcarbamimidothioate (13, 242
mg, 0.62 mmol) in EtOH (10 mL) was added DIPEA (0.67 mL, 3.80 mmol)
at room temperature. The reaction mixture was heated at 70.degree.
C. in a sealed tube for 2 h, then cooled to room temperature, and
concentrated in vacuum. The residue was purified by silica gel
column chromatography (80:18:2 CHCl.sub.3/CH.sub.3OH/NH.sub.4OH)
followed by reverse phase column (Gold C18) to afford guanidine 93
(170 mg, 20% over three steps) as a yellow solid: .sup.1H NMR (400
MHz, CD.sub.3OD) 7.71 (d, J=8.2 Hz, 2H), 7.68 (s, 1H), 7.62 (s,
1H), 7.43 (d, J=8.2 Hz, 2H), 7.26 (ddd, J=10.6, 8.6, 1.3 Hz, 2H),
7.18 (d, J=8.2 Hz, 2H), 4.70 (q, J=0.5 Hz, 2H), 4.49 (t, J=7.8 Hz,
1H), 4.22-4.09 (m, 2H), 4.06 (dd, J=10.4, 5.1 Hz, 2H), 3.89-3.81
(m, 2H), 3.80-3.71 (m, 2H), 3.60-3.49 (m, 2H), 3.43-3.32 (m, 8H),
3.31-3.23 (m, 2H), 3.10-2.98 (m, 2H), 2.85 (t, J=6.9 Hz, 2H),
2.77-2.61 (m, 2H), 2.12-2.02 (m, 2H), 1.89-1.79 (m, 2H), 1.78-1.68
(m, 2H), 1.31 (s, 9H), 1.25 (d, J=5.1 Hz, 6H).
Preparation of the HCl Salt of
3,5-diamino-N-(N-(4-(6-((S)-2-amino-3-(4-(3-(bis((2S,3R,4R,5R)-2,3,4,5,6--
pentahydroxyhexyl)amino)propyl)phenylamino)-3-oxopropyl)naphthalen-2-yl)bu-
tyl)carbamimidoyl)-6-chloropyrazine-2-carboxamide (94)
[0406] 4 N HCl in water (20 mL) was added to 93 (170 mg, 0.15 mmol)
in ethanol (5.0 mL) and reaction mixture was stirred at 40.degree.
C. for 2 h. The solvent was removed again 4N HCl was added and
heated at 40.degree. C. for another 2 h. This addition repeated two
more times. Solvent was removed and purified by reverse phase
column (gold column) and residue was lyophilized to afford compound
94 (80 mg, 50%) as a yellow solid: .sup.1H NMR (400 MHz,
DMSO-d.sub.6) 10.74 (brs, 1H), 928-9.19 (m, 1H), 9.03-8.60 (m, 2H),
8.58-8.04 (m, 1H), 7.81-7.73 (m, 3H), 7.68 (s, 1H), 7.50 (d, J=8.4
Hz, 2H), 7.48-7.34 (m, 4H), 7.19 (d, J=9.0 Hz, 2H), 5.39-5.35 (m,
1H), 4.87-4.63 (m, 1H), 4.62-4.47 (m, 3H), 4.45-4.35 (m, 2H),
4.32-4.23 (m, 1H), 4.01-3.85 (m, 1H), 3.67 (d, J=4.6 Hz, 1H),
3.62-3.55 (m, 2H), 3.53-3.38 (m, 5H), 3.37-3.29 (m, 2H), 3.24-3.09
(m, 2H), 2.78 (t, J=7.2 Hz, 2H), 2.62-2.53 (m, 2H), 2.01-1.86 (m,
2H), 1.79-1.68 (m, 2H), 1.64-1.55 (m, 2H).
[0407] .sup.1H NMR (400 MHz, CD.sub.3OD) 7.78 (d, J=8.4 Hz, 1H),
7.75 (d, J=7.8 Hz, 1H), 7.73 (s, 1H), 7.66 (s, 1H), 7.42 (d, J=8.8
Hz, 2H), 7.39 (d, J=8.4 Hz, 2H), 7.21 (d, J=8.7 Hz, 2H), 4.16 (t,
J=7.0 Hz, 1H), 4.13-4.05 (m, 2H), 3.81 (dd, J=4.7, 1.9 Hz, 2H),
3.77 (dd, J=10.6, 3.0 Hz, 2H), 3.72-3.61 (m, 6H), 3.44-3.30 (m,
10H), 2.86 (t, J=7.0 Hz, 2H), 2.76-2.61 (m, 2H), 2.11-2.01 (m, 2H),
1.89-1.80 (m, 2H), 1.79-1.72 (m, 2H).
14. Preparation of
3,5-diamino-N-(N-(4-(6-((S)-2-amino-3-oxo-3-(4-(3-((2S,3R,4R,5R)-2,3,4,5,-
6-
pentahydroxyhexylamino)propyl)phenylamino)propyl)naphthalen-2-yl)butyl)-
carbamimidoyl)-6-chloropyrazine-2-carboxamide (99)
##STR00038## ##STR00039##
[0408] Preparation of Compound 96
[0409] A solution of acid 19 (1.17 g, 2.27 mmol) in THF (60 mL) was
cooled to 0.degree. C. in ice-bath, NMM (0.30 mL, 2.95 mmol) was
added followed by PivCl (0.30 mL, 2.49 mmol) and the reaction
mixture was stirred at the same temperature for 2 h. 34 (1.0 g,
2.27 mmol, 10 mL THF) of aniline 171 was added and the reaction
mixture was stirred at the same temperature for a further 10 mih.
Reaction mixture was then brought to rt and stirred for 16 h.
Organic solvent was removed. To this residue was added water and
extracted with CH.sub.2Cl.sub.2 (3.times.100 mL). The organic
layers were combined, dried over Na.sub.2SO.sub.4, filtered, and
concentrated. The residue was purified by column chromatography (4%
methanol in chloroform) to afford amide 96 (1.40 g, 66%, impure) as
a light yellow solid. Product formation was confirmed by LCMS.
Preparation of Compound 97
[0410] A suspension of 96 (1.40 g, 1.50 mmol) and 10% Pd/C (300 mg)
in a mixture of EtOH (120 mL) and AcOH (12 mL) was degassed and
then subjected to hydrogenation conditions (1 atm) for 16 h at room
temperature. The reaction mixture was filtered through a plug of
Celite and the plug was washed with MeOH. The filtrate was
concentrated in vacuum to afford amine salt 97 which was then
neutralized with NaHCO.sub.3 and crude product was purified by
flash chromatography on silica gel (CMA, 80:18:2) yielding free
amine 97 as a yellow solid (550 mg, 30%, over two steps): .sup.1H
NMR (400 MHz, CD.sub.3OD) 7.74-7.65 (m, 3H), 7.59 (s, 1H),
7.41-7.29 (m, 4H), 7.11 (d, J=8.4 Hz, 2H), 4.69 (q, J=4.9 Hz, 1H),
4.50 (t, J=7.9 Hz, 1H), 4.04 (dd, J=10.4, 5.2 Hz, 1H), 4.02-3.94
(m, 1H), 3.79-3.71 (m, 1H), 3.70-3.63 (m, 1H), 3.54-3.39 (m, 3H),
3.26-(dd, J=13.6, 6.8 Hz, 1H), 3.07 (dd, J=13.1, 8.3 Hz, 1H), 2.79
(t, J=7.5 Hz, 2H), 2.75-2.67 (m, 2H), 2.55 (t, J=7.3 Hz, 2H),
1.91-1.80 (m, 2H), 1.79-1.69 (m, 2H), 1.62-1.52 (m, 2H), 1.50-1.37
(m, 12H), 1.33 (s, 9H), 1.25 (d, J=4.9 Hz, 3H).
Preparation of 98
[0411] To a solution of amine 97 (550 mg, 0.68 mmol) and methyl
3,5-diamino-6-chloropyrazine-2-carbonylcarbamimidothioate (13, 423
mg, 0.62 mmol) in EtOH (20 mL) was added DIPEA (1.21 mL, 6.80 mmol)
at room temperature. The reaction mixture was heated at 70.degree.
C. in a sealed tube for 2 h, then cooled to room temperature, and
concentrated in vacuum. The residue was purified by silica gel
column chromatography (80:18:2 CHCl.sub.3/CH.sub.3OH/NH.sub.4OH)
followed by reverse phase column (Gold C18) to afford guanidine 98
(500 mg, 72%) as a yellow solid: .sup.1H NMR (400 MHz, CD.sub.3OD)
7.73-7.64 (m, 3H), 7.61 (s, 1H), 7.40-7.30 (m, 4H), 7.11 (d, J=8.5
Hz, 2H), 4.68 (d, J=4.9 Hz, 1H), 4.49 (t, J=7.2 Hz, 1H), 4.04 (dd,
J=10.9, 5.5 Hz, 1H), 4.02-3.93 (m, 1H), 3.78-3.70 (m, 1H),
3.69-3.64 (m, 1H), 3.54-3.38 (m, 4H), 3.30-3.20 (m, 2H), 3.15-3.01
(m, 1H), 2.83 (t, J=7.4 Hz, 2H), 2.54 (t, J=7.3 Hz, 2H), 1.90-1.78
(m, 4H), 1.73-1.64 (m, 2H), 1.53-1.37 (m, 12H), 1.32 (s, 9H), 1.25
(d, J=4.9 Hz, 3H).
Preparation of the HCl Salt of
3,5-diamino-N-(N-(4-(6-((S)-2-amino-3-oxo-3-(4-(3-((2S,3R,4R,5R)-2,3,4,5,-
6-pentahydroxyhexylamino)propyl)phenylamino)
propyl)naphthalen-2-yl)butyl)carbamimidoyl)-6-chloropyrazine-2-carboxamid-
e (99)
[0412] 4 N HCl in water (20 mL) was added to 98 (500 mg, 0.15 mmol)
in ethanol (5.0 mL) and reaction mixture was stirred at 40.degree.
C. for 2 h. The solvent was removed again 4N HCl was added and
heated at 40.degree. C. for another 2 h. This addition repeated two
more times. Solvent was removed purified by reverse phase column
(gold column) and residue was lyophilized to afford compound 99
(206 mg, 50%) as a yellow solid: .sup.1H NMR (400 MHz,
DMSO-d.sub.6) 11.0 (brs, 1H), 9.34 (brs, 1H), 9.09-8.25 (m, 6H),
7.82-7.73 (m, 2H), 7.68 (s, 1H), 7.53 (d, J=8.5 Hz, 2H), 7.49 (d,
J=9.2 Hz, 1H), 7.41 (s, 2H), 7.39 (d, J=8.2 Hz, 2H), 7.17 (d, J=8.2
Hz, 2H), 5.39 (d, J=3.7 Hz, 1H), 4.80-4.70 (m, 1H), 4.62 (d, J=4.3
Hz, 1H), 4.60-4.54 (m, 1H), 4.46-4.36 (m, 2H), 3.96-3.88 (m, 1H),
3.71-3.65 (m, 1H), 3.62-3.54 (m, 1H), 3.51-3.35 (m, 5H), 3.09 (d,
J=13.3 Hz, 1H), 2.94 (d, J=10.9 Hz, 1H), 2.87 (t, J=9.1 Hz, 2H),
2.78 (t, J=6.7 Hz, 2H), 2.60 (t, J=7.7 Hz, 2H), 2.00-1.86 (m, 2H),
1.85-1.67 (m, 2H), 1.65-1.53 (m, 2H).
[0413] .sup.1H NMR (400 MHz, CD.sub.3OD) 7.80 (d, J=9.5 Hz, 1H),
7.78-7.73 (m, 2H), 7.68 (s, 1H), 7.45-7.37 (m, 4H), 7.19 (d, J=7.1
Hz, 2H), 4.31 (t, J=6.3 Hz, 1H), 4.09-4.00 (m, 1H), 3.87-3.81 (m,
1H), 3.78 (d, J=11.4 Hz, 1H), 3.73-3.61 (m, 3H), 3.46 (dd, J=13.6,
6.3 Hz, 1H), 3.37 (t, J=6.8 Hz, 2H), 3.30-3.25 (m, 1H), 3.22-3.12
(m, 2H), 3.03 (t, J=7.9 Hz, 2H), 2.86 (t, J=6.8 Hz, 2H), 2.69 (t,
J=7.4 Hz, 2H), 2.07-1.95 (m, 2H), 1.91-1.81 (m, 2H), 1.80-1.69 (m,
2H).
15. Preparation of
(S)-3,5-diamino-N-(N-(4-(6-(2-amino-3-(4-(3-(dimethylamino)propyl)phenyla-
mino)-3-oxopropyl)naphthalen-2-yl)butyl)carbamimidoyl)-6-chloropyrazine-2--
carboxamide (103)
##STR00040##
[0414] Preparation of Compound 100
[0415] A solution of acid 19 (1.75 g, 3.39 mmol) THF (70 mL) was
cooled to 0.degree. C. in ice-bath, NMM (0.74 mL, 6.78 mmol) was
added followed by PivCl (0.41 mL, 3.39 mmol) and the reaction
mixture was stirred at the same temperature for 2 h. 18 (825 mg,
4.61 mmol, 10 mL THF) was added and the reaction mixture was
stirred at the same temperature for a further 10 mih. Reaction
mixture was then brought to rt and stirred for 16 h. Organic
solvent was removed. To this residue was added water and extracted
with CH.sub.2Cl.sub.2 (3.times.100 mL). The organic layers were
combined, dried over Na.sub.2SO.sub.4, filtered, and concentrated.
The residue was purified by column chromatography (4% methanol in
chloroform) to afford amide 100 (1.60 g, 71%) as a light yellow
solid: .sup.1H NMR (400 MHz, CDCl.sub.3) 7.87 (s, 1H), 7.71 (d,
J=8.5 Hz, 1H), 7.67 (d, J=8.5 Hz, 1H), 7.65-7.62 (m, 2H), 7.42 (dd,
J=8.4, 1.9 Hz, 1H), 7.40-7.29 (m, 5H), 7.22 (d, J=8.6 Hz, 2H), 7.08
(d, J=8.4 Hz, 2H), 5.21-5.10 (m, 2H), 5.13 (s, 2H), 4.51 (q, J=7.6
Hz, 1H), 3.47 (q, J=6.5 Hz, 2H), 3.29 (d, J=6.9 Hz, 2H), 2.68 (t,
J=6.7 Hz, 2H), 2.57 (t, J=7.9 Hz, 2H), 2.26 (ddt, J=11.5, 9.3, 2.5
Hz, 2H), 2.21 (s, 6H), 2.22-2.19 (m, 1H), 1.78-1.69 (m, 3H), 1.39
(s, 9H).
Preparation of Compound 101
[0416] A suspension of 100 (1.60 g, 2.30 mmol) and 10% Pd/C (400
mg) in a mixture of EtOH (130 mL) and AcOH (20 mL) was degassed and
then subjected to hydrogenation conditions (1 atm) for 16 h at room
temperature. The reaction mixture was filtered through a plug of
Celite and the plug was washed with MeOH. The filtrate was
concentrated in vacuum to afford amine salt 101 as a yellow solid
(1.60 g, 99%): .sup.1H NMR (400 MHz, CD.sub.3OD) 7.71 (d, J=8.5 Hz,
2H), 7.68 (s, 1H), 7.61 (s, 1H), 7.42 (d, J=8.5 Hz, 2H), 7.39 (dd,
J=8.5, 1.3 Hz, 1H), 7.33 (dd, J=8.5, 1.3 Hz, 1H), 7.16 (d, J=8.6
Hz, 2H), 4.50 (t, J=7.6 Hz, 1H), 3.28 (dd, J=14.0, 6.3 Hz, 1H),
3.07 (dd, J=13.3, 8.7 Hz, 1H), 3.05-2.98 (m, 2H), 2.93 (t, J=7.6
Hz, 2H), 2.82 (t, J=7.3 Hz, 2H), 2.78 (s, 6H), 2.65 (t, J=7.5 Hz,
2H), 2.06-1.96 (m, 2H), 1.93 (s, 6H), 1.86-1.75 (m, 2H), 1.74-1.64
(m, 2H), 1.33 (s, 9H).
Preparation of 102
[0417] To a solution of amine 101 (1.60 g, 2.30 mmol) and methyl
3,5-diamino-6-chloropyrazine-2-carbonylcarbamimidothioate (21, 1.60
g, 4.14 mmol) in EtOH (25 mL) was added DIPEA 4.1 mL, 23.0 mmol) at
room temperature. The reaction mixture was heated at 70.degree. C.
in a sealed tube for 2 h, then cooled to room temperature, and
concentrated in vacuum. The residue was purified by silica gel
column chromatography (80:18:2 CHCl.sub.3/CH.sub.3OH/NH.sub.4OH) to
afford guanidine 102 (645 mg, 37% and 640 mg, 37% impure) as a
yellow solid: .sup.1H NMR (400 MHz, CD.sub.3OD) 7.70 (dd, J=9.0,
4.3 Hz, 2H), 7.66 (s, 1H), 7.61 (s, 1H), 7.39-7.31 (m, 4H), 7.11
(d, J=8.4 Hz, 2H), 4.48 (t, J=7.6 Hz, 1H), 3.30-3.22 (m, 3H), 3.06
(dd, J=13.8, 8.9 Hz, 1H), 2.83 (t, J=7.2 Hz, 2H), 2.57 (t, J=7.9
Hz, 2H), 2.32 (dd, J=10.5, 7.6 Hz, 2H), 2.23 (s, 6H), 1.86-1.74 (m,
4H), 1.73-1.64 (m, 2H), 1.32 (s, 9H).
Preparation of the Hcl Salt of
(S)-3,5-diamino-N-(N-(4-(6-(2-amino-3-(4-(3-(dimethylamino)propyl)phenyla-
mino)-3-oxopropyl)naphthalen-2-yl)butyl)carbamimidoyl)-6-chloropyrazine-2--
carboxamide (103)
[0418] TFA (10 mL) was added to 47 (545 mg, 0.71 mmol) in
CH.sub.2Cl.sub.2 (15 mL) and reaction mixture was stirred at rt for
1 h. The solvent was removed again 1N HCl was added and solvent was
removed, purified by reverse phase column (gold column) and residue
was lyophilized to afford compound 48 (206 mg, 50%) as a yellow
solid: .sup.1H NMR (400 MHz, DMSO-d.sub.6) 11.02 (brs, 1H),
10.81-10.58 (m, 1H), 10.53 (s, 1H), 9.32 (s, 1H), 9.04-8.72 (m,
2H), 8.50 (brs, 3H), 7.82-7.73 (m, 3H), 7.68 (s, 1H), 7.53 (d,
J=8.5 Hz, 2H), 7.48 (d, J=9.2 Hz, 1H), 7.45-7.35 (m, 3H), 7.18 (d,
J=8.4 Hz, 2H), 4.45-4.35 (m, 1H), 3.74-3.45 (m, 1H), 3.27 (dd,
J=14.7, 8.3 Hz, 1H), 3.03-2.93 (m, 2H), 2.78 (t, J=7.3 Hz, 2H),
2.70 (s, 6H), 2.58 (t, J=7.3 Hz, 2H), 2.02-1.88 (m, 2H), 1.79-1.66
(m, 2H), 1.64-1.54 (m, 2H).
[0419] .sup.1H NMR (400 MHz, CD.sub.3OD) 7.80 (d, J=9.2 Hz, 1H),
7.78-7.73 (m, 2H), 7.67 (s, 1H), 7.47-7.38 (m, 4H), 7.20 (d, J=8.9
Hz, 2H), 4.33 (t, J=7.5 Hz, 1H), 3.46 (dd, J=13.6, 6.6 Hz, 1H),
3.37 (t, J=6.8 Hz, 2H), 3.36-3.26 (m, 3H), 2.87-2.83 (m, 2H), 2.87
(s, 6H), 2.68 (t, J=7.6 Hz, 2H), 2.07-1.97 (m, 2H), 1.89-1.80 (m,
2H), 1.80-1.71 (m, 2H).
16. Preparation of
3,5-diamino-N-(N-(4-(4-((S)-2-amino-3-(4-(3-(hexyl((2S,3R,4R,5R)-2,3,4,5,-
6-pentahydroxyhexyl)amino)propyl)phenylamino)-3-oxopropyl)-5,6,7,8-tetrahy-
dronaphthalen-1-yl)butyl)carbamimidoyl)-6-chloropyrazine-2-carboxamide
(123)
##STR00041## ##STR00042##
[0420] Preparation of Compound 105
[0421] A solution of 104 (100 g, 0.675 mmol) in dry THF (800 mL)
was charged with NaOH (32.0 mg, 0.809 mmol) and dimethylsulfate
(102 g, 0.809 mmol) dropwise at 0.degree. C. The reaction mixture
was stirred for 2 h at room temperature. THF was removed under
reduced pressure, and the mixture was partitioned between
CH.sub.2Cl.sub.2 (1.0 L) and water (1.0 L). The aqueous layer was
separated and extracted with CH.sub.2Cl.sub.2 (2.times.1.0 L). The
combined organic extracts were dried over Na.sub.2SO.sub.4 and
concentrated. The residue was purified by column chromatography
(silica gel, 100% CH.sub.2Cl.sub.2) to afford compound 105 (108.0
g, 90%) as a yellow liquid: .sup.1H NMR (400 MHz, DMSO-d.sub.6):
.delta. 7.06 (t, J=7.85 Hz, 1H), 6.71 (d, J=7.25, 1H), 6.64 (t,
J=7.7 Hz, 1H), 3.80 (s, 3H), 2.74 (t, J=2.75 Hz, 2H), 2.65 (t,
J=2.65 Hz, 2H), 1.81-1.71 (m, 4H).
Preparation of Compound 106
[0422] A solution of dry DMF (71.45 ml, 0.923 mmol) was charged
with POCl.sub.3 (57.40 ml, 0.616 mmol) dropwise under nitrogen
atmosphere at 0.degree. C. The reaction mixture was stirred for 30
min at 0.degree. C. A solution of 105 (50.0 g, 0.308 mmol) in dry
1,2-dichloromethane (500 mL) was added to the reaction mixture
dropwise under nitrogen atmosphere at 0.degree. C. After the
addition was complete, the reaction mixture was heated at
80.degree. C. for 6 h. The reaction mixture was quenched with cold
H.sub.2O and partitioned between CH.sub.2Cl.sub.2 (1.0 L) and water
(1.0 L). The aqueous layer was separated and extracted with
CH.sub.2Cl.sub.2 (2.times.1.0 L). The combined organic extracts
were dried over Na.sub.2SO.sub.4 and concentrated. The residue was
purified by column chromatography (silica gel, 5% EA/Hexane) to
afford compound 106 (35.0 g, 61%) as a yellow solid: .sup.1H NMR
(400 MHz, DMSO-d.sub.6): .delta. 10.10 (s, 1H), 7.65 (d, J=7.81,
1H), 6.78 (d, J=7.47 Hz, 1H), 3.89 (s, 3H), 3.18 (t, J=5.80 Hz,
2H), 2.70 (t, 4.64 Hz, 2H), 1.82-1.73 (m, 4H).
Preparation of Compound 107
[0423] A solution of trimethylphosphonoacetate (55.0mL, 0.378 mmol)
in 100 mL anhydrous CH.sub.2Cl.sub.2 cooled to 0.degree. C. was
charged with DBU (58.0 g, 0.380 mmol) and the mixture was stirred
for 15 min. Aldehyde 106 (16.0 g, 0.084 mmol) in 50 mL
CH.sub.2Cl.sub.2 was added dropwise. The reaction mixture was
brought to room temperature, stirred for 16 h, and quenched with
100 mL of water. The mixture was partitioned, and the aqueous layer
was extracted with CH.sub.2Cl.sub.2 (3.times.150 mL). The combined
organics were washed with brine, dried (Na.sub.2SO.sub.4),
filtered, and concentrated, and the residue was purified by
silica-gel column chromatography (10:1 hexanes/ethyl acetate) to
give the cis & trans-.alpha.,.beta.-unsaturated ester 107 (15.0
g, 72%) as a white solid: .sup.1H NMR (400 MHz, DMSO-d.sub.6):
.delta. 7.83 (d, J=14.7 Hz, 1H), 7.58 (d, J=8.3 Hz, 1H), 6.82 (d,
J=8.4 Hz, 1H), 6.35 (d, J=15.2 Hz, 1H), 3.80 (s, 3H), 3.70 (s, 3H),
2.76 (t, J=5.7 Hz, 2H), 2.55 (t, J=5.4 Hz, 2H), 1.80-1.60 (m,
4H).
Preparation of Compound 108
[0424] A suspension of 107 (33.0 g, 0.134 mmol) and 10% Pd/C (15 g,
0.127) in EtOH (300 mL) was subjected to hydrogenation conditions
(1 atm) for 3 h at room temperature. The reaction mixture was
filtered through Celite and washed with MeOH. The filtrate was
concentrated under vacuum to afford 108 (28.0 g, 90%) as a white
solid: .sup.1HNMR (400 MHz, CDCl.sub.3): .delta. 7.65 (d, J=7.62,
1H), 6.78 (d, J=7.96 Hz, 1H), 4.06-4.11 (m, 1H), 3.78 (s, 3H), 2.86
(t, J=7.79 Hz, 2H), 2.69-2.64 (m, 4H), 2.57-2.51 (m, 2H), 1.79-1.74
(m, 4H).
Preparation of Compound 109
[0425] A solution of methyl ester 108 (28.0 g, 0.106 mmol) in
THF/MeOH/H.sub.2O (200 mL/200 mL/60 mL) was charged with NaOH (25.0
g, 0.625 mmol) and the reaction mixture was stirred at room
temperature for 3 h. The solvent was removed and the pH was
adjusted to 1 with 1 N aqueous HCl; a white solid precipitated and
was filtered, washed with water, and dried under vacuum to afford
acid 109 (25.5 g, 92%) as a white solid: .sup.1H NMR (400 MHz,
CDCl.sub.3): .delta. 6.96 (d, J=7.29, 1H), 6.63 (d, J=6.86 Hz, 1H),
3.78 (s, 3H), 2.88 (t, J=7.29 Hz, 2H), 2.69-2.66 (m, 4H), 2.63-2.59
(m, 2H), 1.80-1.73 (m, 4H).
Preparation of Compound 110
[0426] A solution of 60 (13.70 g, 77.31 mmol) in dry THF (200 mL)
was charged with n-butyllithium (45.07 mL, 90.08 mmol, 2M solution
in cyclohexane) dropwise at -78.degree. C., and the reaction
mixture was stirred for 1 h to give a solution of lithium salt 61.
Another solution of 109 (15.0 g, 64.37 mmol) in dry THF (200 mL)
was charged with NMM (9.30 mL, 83.64 mmol) and PivCl (10.30 mL,
83.64 mmol) dropwise at -78.degree. C. The reaction mixture was
stirred for 30 min and warmed to -20.degree. C. for 1 h, and the
prepared solution of lithium salt was added slowly at -78.degree.
C. The reaction mixture was stirred for another 10 min, brought to
0.degree. C. and stirred for 1 h, brought to room temperature and
stirred for 30 min, quenched with saturated NH.sub.4Cl,
concentrated to remove THF, and partitioned between
CH.sub.2Cl.sub.2 (300 mL) and water (100 mL). The aqueous layer was
separated and extracted with CH.sub.2Cl.sub.2 (150 mL). The
combined organic extracts were dried over Na.sub.2SO.sub.4 and
concentrated. The residue was purified by column chromatography
(silica gel, CH.sub.2O.sub.2) to afford compound 110 (15.0 g, 60%)
as a white solid.
Preparation of Compound 111
[0427] A solution of 110 (15.0 g, 38.14 mmol) in dry THF (250 mL)
was charged with KHMDS (13.70 g, 68.67 mmol) portionwise at
-78.degree. C. After the resulting mixture was stirred for 30 min,
trisyl azide (19.0 g, 61.40 mmol) was added and the reaction
mixture was stirred for 5 min. Acetic acid (15.0 mL, 228 mmol) and
tetramethylammonium acetate (30.9 g, 76.28 mmol) were added slowly
at the same temperature. The reaction mixture was warmed to
24.degree. C., stirred for 16 h, quenched with saturated
NaHCO.sub.3 (100 mL), concentrated to remove THE, and extracted
with CH.sub.2Cl.sub.2 (300 mL). The combined organic extracts were
dried over Na.sub.2SO.sub.4 and concentrated. The residue was
purified by column chromatography (silica gel, 90:10 hexanes/EtOAc
followed by DCM) to afford compound 111 (8.80 g, 54%) as a yellow
solid: .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 7.36-7.30 (m,
3H), 7.23 (m, 1H), 7.20 (m, 1H), 7.16 (m, 1H), 7.01 (d, J=7.79 Hz,
1H), 6.60 (d, J=7.59 Hz, 2H), 5.35 (t, J=7.99, 2H), 4.89 (s, 1H),
4.58-4.51 (m, 1H), 4.13-4.10 (m, 3H), 3.93 (t, J=7.54, 1H), 3.77
(s, 3H), 3.33-3.27 (m, 3H), 2.71 (m, 2H), 2.63 (m, 2H), 1.78-1.75
(m, 5H), 1.58 (m, 2H).
Preparation of Compound 112
[0428] A solution of 111 (31.0 g, 72.1 mmol) in THF/H.sub.2O (300
mL/100 mL) was charged with H.sub.2O.sub.2 (49 mL, 433 mmol)
followed by LiOH (6.04 g, 144 mmol) portionwise at 0.degree. C. The
reaction mixture was stirred for 10 min at 0.degree. C. and at room
temperature for 1 h, quenched with saturated Na.sub.2SO.sub.3 (200
mL), concentrated under reduced pressure to remove THF, and washed
with CH.sub.2Cl.sub.2 (500 mL). The aqueous layer was acidified
with 1 N aqueous HCl and extracted with CH.sub.2Cl.sub.2
(2.times.500 mL). The combined organic extracts were dried over
Na.sub.2SO.sub.4, concentrated, and washed with MTBE to afford
compound 112 (15.0 g, 82%) as an off-white solid: .sup.1H NMR (400
MHz, CD.sub.3OD): .delta. 6.92 (d, J=7.7 Hz, 1H), 6.63 (d, J=8.0
Hz, 1H), 3.75 (s, 3H), 2.81 (t, J=7.8 Hz, 2H), 2.67 (t, J=6.0 Hz,
2H), 2.61 (t, J=5.7 Hz, 2H), 2.49-2.47 (m, 2H), 1.84-1.70 (m,
6H).
Preparation of Compound 113
[0429] A suspension of 112 (15.0 g, 55.1 mmol) and 10% Pd/C (3.50
g) in AcOH/H.sub.2O (300 mL/100 mL) was subjected to hydrogenation
conditions (1 atm) for 3 h at room temperature. The reaction
mixture was filtered through Celite and washed with AcOH/H.sub.2O
followed by MeOH. The filtrate was concentrated under vacuum to
afford acetic salt 113 (14.0 g, 83%) as a yellow solid.
Preparation of Compound 114
[0430] A solution of 113 (11.0 g, 44.1 mmol) in acetic acid (120
mL) was charged with hydrobromic acid (120 mL) dropwise at room
temperature and the reaction mixture was refluxed for 3 h. The
reaction mixture was cooled to room temperature and concentrated.
The crude brown residue 114 (8.90g, 80%) was directly used for the
next step without any purification: .sup.1H NMR (400 MHz,
CDCl.sub.3): .delta. 6.80 (d, J=7.85, 1H), 6.57 (d, J=7.21 Hz, 1H),
3.92-3.91 (m, 1H), 3.04-2.98 (m, 1H), 2.91-2.86 (m, 1H), 2.61 (m,
2H), 2.54-2.53 (m, 2H), 1.69-1.68 (m, 5H).
Preparation of Compound 115
[0431] Acetyl chloride (17.0 mL, 243 mmol) was added to dry
methanol (300 mL) at 0.degree. C. and 114 (8.90g, 28.2 mmol) was
added. The reaction mixture was refluxed for 4 h and concentrated.
The residue was partitioned between CH.sub.2Cl.sub.2 (200 mL) and
saturated NaHCO.sub.3 (100 mL). The aqueous layer was separated and
extracted with CH.sub.2Cl.sub.2 (200 mL). The combined organic
extracts were dried over Na.sub.2SO.sub.4 and concentrated to
afford compound 115 (7.30 g, 90%) as a white solid: .sup.1H NMR
(400 MHz, CDCl.sub.3): .delta. 6.81 (d, J=7.51, 1H), 6.59 (d,
J=7.21 Hz, 1H), 4.12-4.11 (m, 1H), 3.75 (s, 1H), 3.31-3.30 (m, 2H),
2.70-2.67 (m, 2H), 2.63 (t, J=6.16 Hz, 2H).
Preparation of Compound 116
[0432] A solution of 115 (7.30 g, 25.60 mmol) in MeOH/H.sub.2O (100
mL/60 mL) was charged with NaHCO.sub.3 (12.0 g, 145 mmol) and
Boc.sub.2O (10.0 g, 45.8 mmol) at 0.degree. C. The resulting
mixture was warmed to room temperature and stirred for 1 h. The
reaction mixture was partitioned between CH.sub.2Cl.sub.2 (100 mL)
and water (50 mL). The aqueous layer was separated and extracted
with CH.sub.2Cl.sub.2 (100 mL). The combined organic extracts were
washed with brine, dried over Na.sub.2SO.sub.4, and concentrated.
Flash-column chromatography using 20% ethyl acetate/hexanes
followed by CH.sub.2Cl.sub.2 gave compound 116 (7.1 g, 81%) as a
white solid: .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 6.77 (d,
J=7.36, 1H), 6.55 (d, J=7.86 Hz, 1H), 4.96-4.94 (m, 1H), 4.71 (s,
1H), 4.96-4.94 (m, 1H), 4.71 (s, 1H), 4.50-4.48 (m, 3.69 (s, 3H),
3.07-3.01 (m, 1H), 2.89-2.84 (m, 1H), 2.86 (m, 2H), 2.63 (m, 2H),
1.80-1.78 (m, 4H), 1.39 (s, 9H).
Preparation of Compound 117
[0433] A solution of 116 (7.0 g, 20.05 mmol) in CH.sub.2Cl.sub.2
(80 mL) was charged with pyridine (100 ml) and triflate (4.64 mL,
24.0 mmol) at 0.degree. C., stirred for 1 h, and stirred at room
temperature for 2 h. After concentration, the reaction mixture was
partitioned between CH.sub.2Cl.sub.2 (150 mL) and water (70 mL).
The aqueous layer was separated and extracted with CH.sub.2Cl.sub.2
(100 mL). The combined organic extracts were washed with brine,
dried over Na.sub.2SO.sub.4, and concentrated to afford compound
117 (8.00 g, 83%) as a brown oil: .sup.1H NMR (400 MHz,
CD.sub.3OD): .delta. 8.81 (d, J=4.63 Hz, 5H), 8.56-8.51 (m, 2H),
8.02-7.99 (m, 4H), 7.11 (d, J=7.98 Hz, 1H), 7.03 (d, J=7.98, 1H),
4.39-4.35 (m, 1H), 3.68 (s, 3H), 3.19-3.14 (dd, 1H), 2.90-2.77 (m,
5H), 1.86-1.81 (m, 4H), 1.35 (s, 9H), 1.32-1.28 (m, 4H).
Preparation of Compound 118
[0434] Compound 117 (8.0 g, 16.6 mmol) and benzyl
but-3-ynylcarbamate (10, 5.00 g, 24.9 mmol) in anhydrous CH.sub.3CN
(100 mL) were degassed with argon for 10 min at room temperature
and charged with TEA (9.34 mL, 66.50 mmol), 10% (t-Bu).sub.3P in
hexanes (7.0 mL, 3.32 mmol), and CuI (0.16 g, 0.84 mmol). The
resulting mixture was degassed with argon for 10 min and Pd
(PPh.sub.3).sub.4 (2.00 g, 1.73 mmol) was added rapidly in one
portion. After degassing with argon for 5 min, the resulting
mixture was refluxed for 16 h. The reaction mixture was
concentrated under vacuum and the residue was purified by column
chromatography (silica gel, 75:25 hexanes/ethyl acetate) to afford
compound 118 (4.50 g, 52%) as a brown solid: .sup.1H NMR (400 MHz,
CD.sub.3OD): .delta. 7.36-7.34 (m, 4H), 7.33-7.29 (m, 2H), 7.16 (d,
J=7.63 Hz, 1H), 6.82 (d, J=7.02 Hz ,1H), 5.12-5.08 (m, 2H), 4.95
(d, J=7.88 Hz, 1H), 4.52-4.51 (m, 1H), 3.67 (s, 3H), 3.48-3.34 (m,
2H), 3.10-3.05 (dd, 1H), 2.84-2.83 (m, 2H), 2.68-2.65 (m, 4H),
1.81-1.76 (m, 4H), 1.39 (s, 9H).
Preparation of Compound 119
[0435] A solution of methyl ester 118 (4.50 g, 8.42 mmol) in
THF/MeOH/H.sub.2O (30 mL/30 mL/10 mL) was charged with NaOH (3.60
g, 90 mmol) and the reaction mixture was stirred at room
temperature for 3 h. The pH value was adjusted to 9 with 1 N
aqueous HCl and the organic solvent was removed. The pH value of
the residue was adjusted to 5-6, and the suspension was partitioned
between CH.sub.2Cl.sub.2 (100 mL) and water (50 mL). The aqueous
layer was separated and extracted with CH.sub.2Cl.sub.2 (100 mL).
The combined organic extracts were dried over Na.sub.2SO.sub.4 and
concentrated to afford compound 119 (3.66 g, 85%) as a brown solid:
.sup.1H NMR (400 MHz, CD.sub.3OD): .delta. 7.28-7.24 (m, 5H),
7.05-7.03 (d, J=7.67 Hz, 1H), 6.89-6.87 (d, J=7.55 Hz, 1H), 5.04
(brs, J=7.02 Hz, 1H), 5.12-5.08 (m, 2H), 4.95 (d, J=7.88 Hz, 1H),
4.52-4.51 (m, 1H), 3.67 (s, 3H), 3.48-3.34 (m, 2H), 4.27-4.26 (m,
1H), 3.38-3.30 (m, 2H), 3.15-3.10 (m, 1H), 2.78-2.71 (m, 4H), 2.59
(d, J=5.95, 2H), 1.73-1.71 (m, 4H), 1.31 (s, 9H).
Preparation of Compound 120
[0436] Compound 119 (800 mg, 1.53 mmol) in THF (30 mL) was charged
with DEPBT (845 mg, 2.56 mmol), 24 (700 mg, 2.33 mmol), and DIPEA
(1.0 mL, 4.65 mmol) successively and stirred at room temperature
for 16 h. After the solvent was removed under reduced pressure, the
residue was dissolved in CH.sub.2Cl.sub.2 (50 mL), quickly washed
with saturated aqueous water (50 mL) and brine (50 mL), and dried
over Na.sub.2SO.sub.4. The solvent was evaporated and the crude
product purified by flash chromatography on silica gel (6%
methanol/CH.sub.2Cl.sub.2), yielding amide 120 (1.0 g) as a yellow
solid: .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 7.46-7.44 (m,
3H), 7.36-7.30 (m, 7H), 7.17 (d, J=7.2 Hz, 2H), 7.07 (d, J=7.5 Hz,
1H), 6.99-6.92 (m, 1H), 5.49 (s, 1H), 5.10 (s, 2H), 4.35-4.31 (m,
1H), 4.05-3.90 (m, 2H), 3.80-3.82 (m, 1H), 3.75-3.72 (m, 1H), 3.62
(t, J=9.9 Hz, 1H), 3.43 (t, J=5.7 Hz, 2H), 3.18-3.16 (m, 1H),
3.01-3.08 (m, 1H), 2.83-2.82 (m, 2H), 2.68-2.48 (m, 8H), 1.86-1.78
(m, 3H), 1.71-1.62 (m, 10H), 1.44 (s, 9H), 0.87 (t, J=6.3 Hz,
3H).
Preparation of Compound 121
[0437] A suspension of 120 (1.00 g, 1.01 mmol) and 10% Pd/C (600
mg) in a mixture of EtOH (50 mL) and AcOH (2 mL) was degassed and
subjected to hydrogenation conditions (1 atm) for 12 h at room
temperature. The reaction mixture was filtered through a plug of
Celite and the plug was washed with MeOH. The filtrate was
concentrated under vacuum to afford amine salt 121 as a white solid
(700 mg, 80%):.sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 7.49-7.41
(m, 2H), 7.34-7.30 (m, 5H), 7.12-6.78 (m, 5H), 4.30-4.27 (m, 2H),
4.19-4.18 (m, 1H), 3.98-391 (m, 2H), 3.78-3.58 (m, 2H), 3.19-3.08
(m, 3H), 3.02-2.89 (m, 6H), 2.75-2.73 (m, 2H), 2.65-2.62 (m, 3H),
2.55-2.52 (m, 3H), 1.98-1.92 (m, 2H), 1.73-1.68 (m, 3H), 1.60-1.52
(m, 7H), 1.41 (s, 9H), 1.29-1.20 (m, 7H), 0.88-0.84 (m, 3H), 0.87
(t, J=6.4 Hz, 3H).
Preparation of Compound 122
[0438] A solution of amine salt 121 (700 mg, 0.81 mmol) and methyl
3,5-diamino-6-chloropyrazine-2-carbonylcarbamimidothioate (13, 680
mg, 1.75 mmol) in EtOH (20 mL) was charged with DIPEA (1.60 mL,
9.26 mmol) at room temperature. The reaction mixture was heated at
70.degree. C. in a sealed tube for 2 h, cooled to room temperature,
and concentrated under vacuum. The residue was purified by column
chromatography (silica gel, 80:18:2
CHCl.sub.3/CH.sub.3OH/NH.sub.4OH) to afford guanidine 122 (380 g,
48%) as a yellow solid: .sup.1H NMR (400 MHz, DMSO-d.sub.6):
.delta. 7.43-7.39 (m, 3H), 7.33-7.31 (m, 3H), 7.05 (d, J=6.69 Hz,
2H), 6.99-6.95 (m, 2H), 6.86 (d, J=7.59 Hz, 1H), 5.47 (s, 1H),
4.33-4.31 (m, 1H), 4.14-4.10 (m, 1H), 3.79-3.72 (m, 4H), 3.68-3.65
(m, 2H), 2.69-2.66 (m, 6H), 2.56-2.53 (m, 3H), 2.45-2.36 (m, 7H),
1.70 (m, 4H), 1.56 (m, 6H), 1.32 (s, 9H), 0.86 (t, J=7.0 Hz,
3H).
Preparation of the HCl Salt of
3,5-diamino-N-(N-(4-(4-((S)-2-amino-3-(4-(3-(hexyl((2S,3R,4R,5R)-2,3,4,5,-
6-pentahydroxyhexyl)amino)propyl)phenylamino)-3-oxopropyl)-5,6,7,8-tetrahy-
dronaphthalen-1-yl)butyl)carbamimidoyl)-6-chloropyrazine-2-carboxamide
(Compound 123)
[0439] 4 N HCl in dioxane (15 mL) was added to 122 (350 g, 0.35
mmol) in EtOH (5.0 mL) and the reaction mixture was stirred at room
temperature for 2 h. The solvent was removed, the mixture was
purified by reverse-phase chromatography (Gold column), and the
residue was lyophilized to give 110 mg (45%) of compound 123 as a
yellow solid: .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta. 10.16
(s, 1H), 9.16 (brs, 1H), 8.51-8.34 (brs, 2H), 7.41 (t, J=8.1 Hz,
4H), 7.20 (d, J=8.6 Hz, 2H), 6.95-6.89 (q, 2H), 5.42 (brs, 1H),
4.42 (m, 1H), 4.53 (d, J=5.3 Hz, 2H), 4.42 (m, 1H), 4.01 (m, 1H),
3.93 (m, 1H), 3.60 (m, 1H), 3.50-3.38 (m, 4H), 3.08-3.03 (m, 6H),
2.72 (brs, 2H), 2.66-2.65 (m, 2H), 2.57 (m, 2H), 1.91-1.90 (m, 2H),
1.72-1.69 (m, 4H), 1.61-1.54 (m, 6H), 1.26 (s, 6H), 0.86 (t, J=7.0
Hz, 3H).
[0440] .sup.1H NMR (400 MHz, D.sub.2O): .delta. 7.08 (d, J=8.4 Hz,
2H), 7.04-6.98 (q, 2H), 6.92 (d, J=8.3 Hz, 2H), 4.06-4.02 (m, 2H),
3.78-3.69 (m, 3H), 3.62-3.54 (m, 2H), 3.25 (t, J=5.3 Hz, 1H),
3.19-3.14 (m, 3H), 3.10-3.04 (m, 4H), 2.66-2.54 (m, 7H), 1.90-1.86
(m, 2H), 1.65-1.58 (m, 5H), 1.50-1.40 (m, 4H), 1.19-1.18 (m, 6H),
0.78 (t, J=6.62).
17. Preparation of
3,5-diamino-N-(N-(4-(4-((S)-2-amino-3-(4-(3-(bis((2S,3R,4R,5R)-2,3,4,5,6--
pentahydroxyhexyl)amino)propyl)phenylamino)-3-oxopropyl)-5,6,7,8-tetrahydr-
onaphthalen-1-yl)butyl)carbamimidoyl)-6-chloropyrazine-2-carboxamide
(127)
##STR00043## ##STR00044##
[0441] Preparation of Compound 124
[0442] The compound 119 (1.0 g, 1.92 mmol) in THF (30 mL) was
charged with DEPBT (845 mg, 2.82 mmol), 29 (1.25 g, 1.91 mmol), and
DIPEA (1.0 mL, 5.73 mmol) successively and stirred at room
temperature for 16 h. After the solvent was removed under reduced
pressure, the residue was dissolved in CH.sub.2Cl.sub.2 (50 mL),
quickly washed with saturated aqueous water (50 mL) and brine (50
mL), and dried over Na.sub.2SO.sub.4. The solvent was evaporated
and the crude product purified by flash chromatography on silica
gel (5% methanol/CH.sub.2Cl.sub.2), yielding amide 124 [900 mg
(mixture)] as a yellow solid.
Preparation of Compound 125
[0443] A suspension of 124 [900 mg (mixture), 0.77 mmol] and 10%
Pd/C (600 mg) in a mixture of EtOH (50 mL) and AcOH (1.5 mL) was
degassed and subjected to hydrogenation conditions (1 atm) for 12 h
at room temperature. The reaction mixture was filtered through a
plug of Celite and the plug was washed with MeOH. The filtrate was
concentrated under vacuum to afford crude 125 (800 mg) as a
colorless oil.
Preparation of Compound 126
[0444] A solution of crude 125 (800 mg) and methyl
3,5-diamino-6-chloropyrazine-2-carbonylcarbamimidothioate (13, 400
mg, 1.02 mmol) in EtOH (40 mL) was charged with DIPEA (1.10 mL,
6.38 mmol) at room temperature. The reaction mixture was heated at
70.degree. C. in a sealed tube for 2 h, cooled to room temperature,
and concentrated under vacuum. The residue was purified by column
chromatography (silica gel, 80:18:2
CHCl.sub.3/CH.sub.3OH/NH.sub.4OH) to afford guanidine 126 (285 mg,
12% over 3 steps) as a yellow solid: .sup.1H NMR (400 MHz,
DMSO-d.sub.6): .delta. 7.44-7.42 (m, 4H), 7.30-7.28 (m, 6H), 7.22
(d, J=7.27 Hz, 2H), 6.96 (d, J=7.11 Hz, 2H), 6.91 (d, J=7.0 Hz,
2H), 6.86 (d, J=7.61 Hz, 2H), 5.47 (s, 2H), 4.33 (m, 1H), 4.23-4.19
(m, 2H), 3.97-3.91 (m, 4H), 3.84-3.82 (m, 2H), 3.71 (d, J=2.29 Hz,
1H), 3.69 (d, J=2.2 Hz, 1H), 3.58 (t, J=10.08 Hz, 2H), 3.06-3.00
(m, 1H), 2.91-2.86 (m, 1H), 2.76 (m, 2H), 2.71-2.68 (m, 4H),
2.61-2.55 (m, 4H), 2.44-2.35 (m, 4H), 1.74-1.60 (m, 10H), 1.38 (s,
9H).
Preparation of the HCl Salt
of3,5-diamino-N-(N-(4-(4-((S)-2-amino-3-(4-(3-(bis((2S,3R,4R,5R)-2,3,4,5,-
6-pentahydroxyhexyl)amino)propyl)phenylamino)-3-oxopropyl)-5,6,7,8-tetrahy-
dronaphthalen-1-yl)butyl)carbamimidoyl)-6-chloropyrazine-2-carboxamide
(Compound 127)
[0445] 4 N HCl in dioxane (10 mL) was added to 126 (1.15 g, 0.23
mmol) in EtOH (3.0 mL) and the reaction mixture was stirred at room
temperature for 2 h. The solvent was removed, the mixture was
purified by reverse-phase chromatography (Gold column), and the
residue was lyophilized to give 62 mg (32%) of compound 127:
.sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta. 10.39 (brs, 1H), 10.03
(brs, 1H), 8.91-8.82 (brs, 2H), 8.48 (brs, 2H), 7.42 (d, J=7.6 Hz,
4H), 7.18 (d, J=7.6 Hz, 2H), 6.96 (d, J=7.1, 1H), 6.89 (d, J=7.4,
1H), 5.44 (d, J=10.8, 2H), 4.81 (br, 2H), 4.59 (d, J=4.2, 2H), 4.55
(d, J=5.4 Hz, 2H), 4.42 (t, J=4.4, 2H), 4.11 (br, 1H), 4.00 (brs,
2H), 3.69-3.65 (m, 2H), 3.58 (m, 2H), 3.47 (m, 4H), 3.43-3.39 (m,
4H), 3.25-3.22 (m, 4H), 3.04 (d, J=6.3, 2H), 2.73 (m, 2H) 2.64 (m,
2H), 2.58-2.56 (m, 2H), 1.98 (m, 2H), 1.97 (m, 2H), 1.70-1.67 (m,
4H), 1.61-1.59 (m, 2H), 1.54-1.52 (m, 2H), 1.70-1.67 (m, 4H),
1.61-1.59 (m, 2H), 1.54-1.52 (m, 2H).
[0446] .sup.1H NMR (400 MHz, D.sub.2O): .delta. 7.10 (d, J=8.30 Hz,
2H), 7.02-6.90 (m, 2H), 6.91 (d, J=7.42 Hz, 2H), 4.07-3.92 (m, 5H),
3.77-3.70 (m, 8H), 3.62-3.55 (m, 5H), 4.07-3.95 (m, 5H), 3.74-3.56
(m, 8H), 3.60-3.55 (m, 5H), 3.30 (d, J=8.2 Hz, 5H), 3.20-3.16 (m,
7H), 2.60-2.51 (m, 10H), 1.97-1.95 (m, 3H), 1.61-1.59 (m, 7H),
1.49-1.45 (m, 2H).
18. Preparation of
3,5-diamino-N-(4-(4-((S)-2-amino-3-oxo-3-(4-(3-((2S,3R,4R,5R)-2,3,4,5,6-p-
entahydroxyhexylamino)propyl)phenylamino)propyl)-
5,6,7,8-tetrahydronaphthalen-1-yl)butylcarbamoyl)-6-chloropyrazine-2-carb-
oxamide (131)
##STR00045##
[0447] Preparation of Compound 128
[0448] The compound 119 (1.00 g, 1.92 mmol) in THF (30 mL) was
charged with DEPBT (862 mg, 2.88 mmol), 34 (1.50 g, 2.98 mmol), and
DIPEA (1.0 mL, 5.76 mmol) successively and stirred at room
temperature for 16 h. After the solvent was removed under reduced
pressure, the residue was dissolved in CH.sub.2Cl.sub.2 (50 mL),
quickly washed with saturated aqueous water (30 mL) and brine (20
mL), and dried over Na.sub.2SO.sub.4. The solvent was evaporated
and the crude product purified by flash chromatography on silica
gel (6% methanol/CH.sub.2Cl.sub.2), yielding amide 128 (780 mg,
42%) as a yellow solid: .sup.1H NMR (400 MHz, CDCl.sub.3): .delta.
7.49 (m, 3H), 7.31-7.29 (m, 1.0H), 7.00-7.08 (m, 3H), 6.94 (d,
J=7.4 Hz, 1H), 5.54 (m, 1H), 5.50-5.49 (m, 1H), 5.08 (s, 2H), 4.36
(m, 1H), 4.26-4.22 (m, 2H), 4.05 (m, 2H), 3.95-3.91 (m, 1H), 3.80
(m, 2H), 3.64-3.59 (m, 1H), 3.52-3.48 (m, 1H), 3.14-3.06 (m, 1H),
2.94-2.89 (m, 1H), 2.79 (d, J=16.12 Hz, 4H), 2.63 (t, J=5.98 Hz,
1H), 2.51 (t, J=6.9 Hz, 1H), 1.82-1.75 (m, 7H), 1.41 (s, 18H).
Preparation of Compound 129
[0449] A suspension of 128 (780 mg, 0.776 mmol) and 10% Pd/C (300
mg) in a mixture of EtOH (30 mL) and AcOH (1.0 mL) was degassed and
subjected to hydrogenation conditions (1 atm) for 12 h at room
temperature. The reaction mixture was filtered through a plug of
Celite and the plug was washed with MeOH. The filtrate was
concentrated under vacuum to afford amine salt 129 (720 mg, 85%) as
a white solid: .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 7.49-7.46
(m, 2H), 7.32-7.30 (m, 5H), 7.08-7.06 (d, J=7.2 Hz, 1H), 6.88 (d,
J=7.4 Hz, 1H), 5.53 (s, 1H), 4.34-4.33 (m, 1H), 4.25-4.21 (m, 1H),
4.03-4.02 (m, 1H), 3.96-3.89 (m, 1H), 3.78-3.76 (m, 1H), 3.71-3.69
(m, 1H), 3.60 (t, J=9.9 Hz, 1H), 3.48-3.46 (m, 1H), 3.09-3.04 (m,
1H), 2.89 (t, J=7.3 Hz, 3H), 2.79 (m, 2H), 2.69 (m, 2H), 2.58 (t,
J=6.5 Hz, 2H), 2.51 (t, J=6.8 Hz, 2H), 1.84-1.77 (m, 6H), 1.67-1.66
(m, 2H), 1.61-1.57 (m, 2H), 1.41 (s, 18H).
Preparation of Compound 130
[0450] A solution of amine salt 129 (720 mg, 0.77 mmol) and methyl
3,5-diamino-6-chloropyrazine-2-carbonylcarbamimidothioate (13, 456
mg, 1.17 mmol) in EtOH (20 mL) was charged with DIPEA (1.12 mL,
6.24 mmol) at room temperature. The reaction mixture was heated at
70.degree. C. in a sealed tube for 2 h, cooled to room temperature,
and concentrated under vacuum. The residue was purified by column
chromatography (silica gel, 80:18:2
CHCl.sub.3/CH.sub.3OH/NH.sub.4OH) to afford guanidine 130 (380 mg,
45%) as a yellow solid: .sup.1H NMR (400 MHz, CDCl.sub.3): .delta.
7.48-7.46 (m, 2H), 7.30 (t, J=2.70 Hz, 5H), 7.08-7.06 (d, J=7.6 Hz,
2H), 6.93 (d, J=7.1 Hz, 1H), 6.88 (d, J=7.3 Hz, 1H), 5.53 (s, 1H),
4.34 (m, 1H), 4.25-4.21 (m, 1H), 4.04 (m, 1H), 3.96-3.90 (m, 1H),
3.79 (m, 2H), 3.60 (t, J=10.0 Hz, 1H), 3.50-3.46 (m, 1H), 3.25 (t,
J=5.9 Hz, 3H), 3.07-3.02 (m, 1H), 2.92-2.87 (m, 1H), 2.77 (m, 2H),
2.69-2.67 (m, 2H), 2.58 (t, J=6.0 Hz, 2H), 2.48 (t, J=6.8 Hz, 2H),
1.82-1.74 (m, 6H), 1.67-1.64 (m, 5H), 1.40 (s, 18H).
Preparation of the HCl Salt of
3,5-diamino-N-(4-(4-((S)-2-amino-3-oxo-3-(4-(3-((2S,3R,4R,5R)-2,3,4,5,6-p-
entahydroxyhexylamino)propyl)phenylamino)propyl)-5,6,7,8-tetrahydronaphtha-
len-1-yl)butylcarbamoyl)-6-chloropyrazine-2-carboxamide (131)
[0451] 4 N HCl in dioxane (25 mL) was added to 130 (350 mg, 0.35
mmol) in EtOH (5.0 mL) and the reaction mixture was stirred at room
temperature for 2 h. The solvent was removed, the mixture was
purified by reverse-phase chromatography (Gold column) and the
residue was lyophilized to give compound 131 (125 mg, 48%) as a
yellow solid: NMR (400 MHz, CD.sub.3OD): .delta. 7.35 (d, J=7.6,
2H), 7.18 (d, J=7.3, 2H), 6.99-6.98 (m, 2H), 4.07-4.03 (m, 2H),
3.83 (d, J=1.30, 1H), 3.82 (d, J=1.40 Hz, 1H), 3.78-3.75 (m, 1H),
3.68-3.66 (m, 3H), 3.36 (t, J=6.3, 2H), 3.18-3.15 (m, 4H),
3.04-3.00 (m, 2H), 2.76 (t, J=5.3 Hz, 2H), 2.69-2.61 (m, 5H),
2.00-1.97 (m, 2H), 1.77-1.73 (m, 5H), 1.69-1.65 (m, 3H).
[0452] .sup.1H NMR (400 MHz, D.sub.2O): .delta. 10.46 (s, 1H), 9.31
(br, 1H), 8.55 (br, 4H), 7.45 (d, J=6.6, 4H), 7.20 (d, J=7.62 Hz,
2H), 7.00 (d, J=6.6, 1H), 6.93 (d, J=6.6 Hz, 1H), 5.43 (d, J=3.8
Hz, 1H), 4.79 (d, J=5.38 1H), 4.64-4.63 (m, 2H), 4.46 (t, J=4.9 Hz,
1H), 4.15 (t, J=4.6 Hz, 1H), 3.96-3.94 (m, 1H), 3.71 (m, 1H),
3.64-3.61 (m, 1H), 3.51-3.45 (m, 3H), 2.96-2.92 (m, 3H), 2.78-2.77
(m, 2H), 2.68-2.65 (m, 2H), 2.62 (t, J=6.6 Hz, 2H), 1.95-1.94 (m,
2H), 1.76-1.15 (m, 8H).
19. Preparation of
(S)-3,5-diamino-N-(N-(4-(4-(2-amino-3-(4-(3-(dimethylamino)propyl)phenyla-
mino)-3-oxopropyl)-5,6,7,8-tetrahydronaphthalen-1-yl)butyl)carbamimidoyl)--
6-chloropyrazine-2-carboxamide (135)
##STR00046##
[0453] Preparation of Compound 132
[0454] The compound 119 (700 mg, 1.34 mmol) in THF (30 mL) was
charged with DEPBT (600 mg, 2.00 mmol), 18 (360 mg, 1.51 mmol), and
DIPEA (0.80 mL, 4.03 mmol) successively and stirred at room
temperature for 16 h. After the solvent was removed under reduced
pressure, the residue was dissolved in CH.sub.2Cl.sub.2 (50 mL),
quickly washed with saturated aqueous water (50 mL) and brine (50
mL), and dried over Na.sub.2SO.sub.4. The solvent was evaporated
and the crude product purified by flash chromatography on silica
gel (6% methanol/CH.sub.2O.sub.2), yielding amide 132 [800 mg
(mixture)] as a yellow solid product: .sup.1H NMR (400 MHz,
DMSO-d.sub.6): .delta. 8.13 (d, J=7.54 Hz, 1H), 8.03 (d, J=7.7 Hz,
1H), 7.89-7.85 (m, 1H), 7.71 (t, J=7.52 Hz, 1H), 7.64-7.59 (m, 2H),
7.44 (d, J=7.7 Hz, 2H), 7.33-7.30 (m, 6H), 7.12-7.06 (m, 3H), 7.0
(d, J=7.6 Hz, 1H), 5.02 (s, 2H), 2.70 (m, 4H), 2.63-2.61 (m, 5H),
2.45 (m, 5H), 1.83 (s, 6H), 1.69-1.65 (m, 3H), 1.33 (s, 9H).
Preparation of Compound 133
[0455] A suspension of 132 [800 mg (mixture), 1.01 mmol] and 10%
Pd/C (350 mg) in a mixture of EtOH (30 mL) and AcOH (1 mL) was
degassed and subjected to hydrogenation conditions (1 atm) for 12 h
at room temperature. The reaction mixture was filtered through a
plug of Celite and the plug was washed with MeOH. The filtrate was
concentrated under vacuum and purified by column chromatography
(silica gel, 80:18:2 CHCl.sub.3/CH.sub.3OH/NH.sub.4OH) to afford
compound 233 (500 mg, 67% over 2 steps) as a yellow solid: .sup.1H
NMR (400 MHz, DMSO-d.sub.6): .delta. 7.31 (d, J=7.54 Hz, 2H), 7.12
(d, 7.1 Hz, 2H), 6.93 (d, J=7.2 Hz, 1H), 6.88 (d, J=6.8 Hz, 1H),
4.32 (m, 1H), 3.08-3.03 (m, 1H), 2.91-2.86 (m, 1H), 2.77-2.76 (m,
4H), 2.69 (m, 2H), 2.60-2.55 (m, 4H), 2.35-2.31 (m, 2H), 1.82 (s,
6H), 1.58-1.57 (m, 4H), 1.40 (s, 9H).
Preparation of Compound 134
[0456] A solution of amine salt 133 (500 mg, 0.90 mmol) and methyl
3,5-diamino-6-chloropyrazine-2-carbonylcarbamimidothioate (13, 530
mg, 1.36 mmol) in EtOH (20 mL) was charged with DIPEA (1.30 mL,
7.25 mmol) at room temperature. The reaction mixture was heated at
70.degree. C. in a sealed tube for 2 h, cooled to room temperature,
and concentrated under vacuum. The residue was purified by column
chromatography (silica gel, 80:18:2
CHCl.sub.3/CH.sub.3OH/NH.sub.4OH) to afford guanidine 134 (285 mg,
42%) as a yellow solid: .sup.1H NMR (400 MHz, DMSO-d.sub.6):
.delta. 7.29 (d, J=7.5 Hz, 2H), 7.10 (d, J=8.1 Hz, 2H), 6.94-6.87
(m, 2H), 4.33 (m, 1H), 3.27-3.24 (m, 2H), 3.07-3.00 (m, 1H),
2.92-2.87 (m, 1H), 2.76 (m, 2H), 2.70 (m, 2H), 2.61-2.54 (m, 4H),
2.35-2.31 (m, 2H), 2.22 (s, 6H), 1.80-1.72 (m, 5H), 1.69-1.62 (m,
4H), 1.39 (s, 9H).
Preparation of the HCl Salt of
(S)-3,5-diamino-N-(N-(4-(4-(2-amino-3-(4-(3-(dimethylamino)propyl)phenyla-
mino)-3-oxopropyl)-5,6,7,8-tetrahydronaphthalen-1-yl)butyl)carbamimidoyl)--
6-chloropyrazine-2-carboxamide Compound 135
[0457] 4 N HCl in dioxane (10 mL) was added to 134 (380 g, 0.35
mmol) in EtOH (5.0 mL) and the reaction mixture was stirred at room
temperature for 2 h. The solvent was removed, the mixture was
purified by reverse-phase chromatography (C18 Gold column), and the
residue was lyophilized to afford compound 135 (125 mg, 49%) as a
yellow solid: .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta. 10.69
(brs, 1H), 10.54-10.50 (d, J=16.7 Hz, 2H), 9.32 (t, J=4.8 Hz, 1H),
8.96 (brs, 1H), 8.86 (brs, 1H), 8.58 (brs, 3H), 7.42 (d, J=8.0 Hz,
4H), 7.18 (d, J=8.2 Hz, 2H), 6.96 (d, J=7.3 Hz, 1H), 6.88 (d, J=7.4
Hz, 1H), 4.15 (t, J=4.4 Hz, 1H), 3.36-3.32 (m, 2H), 3.09-3.06 (m,
2H), 3.00-2.95 (m, 2H), 2.74-2.73 (m, 1H), 2.22 (s, 6H), 2.64 (m,
2H), 2.57-2.56 (m, 2H), 1.94-1.90 (m, 2H), 1.70-1.67 (m, 3H),
1.62-1.58 (m, 2H), 1.54-1.52 (m, 2H).
[0458] .sup.1H NMR (400 MHz, D.sub.2O): .delta. 7.08 (d, J=7.7 Hz,
2H), 7.00-6.97 (q, 2H), 6.91 (d, J=8.1 Hz, 2H), 4.12-4.08 (q, 1H),
3.25 (t, J=5.2 Hz, 3H), 3.21-3.17 (m, 1H), 3.10 (t, J=9.8 Hz, 1H),
3.0-2.96 (m, 2H), 2.77 (s, 6H), 2.60-2.58 (m, 5H), 2.50-2.50 (m,
4H), 1.91-1.87 (m, 2H), 1.60-1.58 (m, 6H), 1.45-1.43 (m, 2H).
20. Preparation of
(S)-2-amino-3-(4-(4-(3-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino-
)butyl)-5,6,7,8-tetrahydronaphthalen-1-yl)propanoic acid (139)
##STR00047##
[0459] Preparation of Compound 136
[0460] A suspension of 118 (800 mg, 1.49 mmol) and 10% Pd/C (350
mg) in a mixture of EtOH (50 mL) and AcOH (1.0 mL) was degassed and
subjected to hydrogenation conditions (1 atm) for 12 h at room
temperature. The reaction mixture was filtered through a plug of
Celite and the plug was washed with MeOH. The filtrate was
concentrated under vacuum and purified by column chromatography
(silica gel, 80:18:2 CHCl.sub.3/CH.sub.3OH/NH.sub.4OH) to afford
compound 136 (700 mg, 93%) as a yellow solid.
Preparation of Compound 137
[0461] A solution of amine salt 136 (700 mg, 1.50 mmol) and methyl
3,5-diamino-6-chloropyrazine-2-carbonylcarbamimidothioate (13, 880
mg, 2.26 mmol) in EtOH (30 mL) was charged with DIPEA (2.15 mL,
12.03 mmol) at room temperature. The reaction mixture was heated at
70.degree. C. in a sealed tube for 2 h, cooled to room temperature,
and concentrated under vacuum. The residue was purified by column
chromatography (silica gel, 80:18:2
CHCl.sub.3/CH.sub.3OH/NH.sub.4OH) to afford guanidine 137 (560 mg,
60%) as a yellow solid: .sup.1H NMR (400 MHz, CD.sub.3OD); .delta.
6.95-6.85 (m, 2H), 4.32-4.28 (m, 1H), 3.72-3.67 (m, 2H), 3.34 (m,
3H), 3.22-3.16 (m, 2H), 3.08-3.03 (m, 1H), 2.73 (m, 4H), 2.62 (t,
J=7.0 Hz, 1H), 1.81-1.78 (m, 4H), 1.74-1.72 (m, 2H), 1.68-1.60 (m,
2H), 1.36 (s, 9H), 1.34 (s, 5H).
Preparation of Compound 138
[0462] A solution of methyl ester 137 (560 mg, 0.907 mmol) in
THF/MeOH/H.sub.2O (30 mL/30 mL/10 mL) was charged with NaOH (3.60
g, 7.25 mmol) and the reaction mixture was stirred at room
temperature for 3 h. The pH value was adjusted to 9 with 1 N
aqueous HCl and the organic solvent was removed. The pH value of
the residue was adjusted to 5-6, and the suspension was partitioned
between CH.sub.2Cl.sub.2 (100 mL) and water (50 mL). The aqueous
layer was separated and extracted with CH.sub.2Cl.sub.2 (100 mL).
The combined organic extracts were dried over Na.sub.2SO.sub.4 and
concentrated to afford compound 138 (420 mg, 78%) as a brown solid:
.sup.1H NMR (400 MHz, DMSO-d.sub.6); .delta. 6.93 (d, J=6.7 Hz,
1H), 6.84 (d, J=7.35 Hz, 1H), 6.70 (s, 3H), 3.93 (m, 1H), 3.16 (m,
5H), 2.98-2.94 (m, 1H), 2.74-2.64 (m, 6H), 1.70 (m, 5H), 1.55 (m,
5H), 1.31 (s, 9H), 1.16-1.06 (m, 2H).
Preparation of the HCl Salt of
(S)-2-amino-3-(4-(4-(3-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino-
)butyl)-5,6,7,8-tetrahydronaphthalen-1-yl)propanoic acid Compound
139
[0463] 4 N HCl in dioxane (10 mL) was added to 138 (420 mg, 0.69
mmol) in EtOH (5.0 mL) and the reaction mixture was stirred at room
temperature for 2 h. The solvent was removed, the mixture was
purified by reverse-phase chromatography (C18 Gold column), and the
residue was lyophilized to afford compound 139 as a yellow solid
(200 mg, 49%): .sup.1H NMR (400 MHz, DMSO-d.sub.6); .delta. 10.56
(brs, 1H), 9.36 (t, J=4.7 Hz, 1H), 8.9-8.8 (brs, 2H), 6.98-6.93 (m,
2H), 3.95-3.92 (m, 2H), 3.38-3.35 (m, 2H), 3.04 (d, J=7.0 Hz, 2H),
2.67-2.66 (m, 4H), 2.56-2.55 (m, 2H), 1.72-1.70 (m, 4H), 1.63-1.56
(m, 4H).
[0464] .sup.1H NMR (400 MHz, D.sub.2O); .delta. 7.43 (brs, 2H),
6.94 (d, J=7.2 Hz, 1H), 6.87 (d, J=7.1 Hz, 1H), 3.41 (t, J=6.0 Hz,
2H), 3.28-3.26 (m, 4H), 3.11 (d, 1H), 3.08 (m, 1H), 2.66-2.64 (m,
6H), 1.67-1.57 (m, 8H).
21. Chiral Synthesis of
3,5-diamino-N-(N-(4-(4-((S)-2-amino-3-(4-(3-(bis((2S,3R,4R,5R)-2,3,4,5,6--
pentahydroxyhexyl)amino)propyl)phenylamino)-3-oxopropyl)naphthalen-1-yl)bu-
tyl)carbamimidoyl)-6-chloropyrazine-2-carboxamide (33)
##STR00048##
[0465] Preparation of Compound 141
[0466] To a solution of 1-naphthol (140, 10.0 g, 69.4 mmol) in
acetonitrile (70.0 mL) was added several portions of NBS (142, 12.3
g, 69.4 mmol) over a period of 30 min. The resulting mixture was
stirred at room temperature for 4 h, concentrated under vacuum,
followed by addition of water (200 mL) and ethyl acetate (200 mL).
The aqueous layer was separated and extracted with ethyl acetate
(2.times.200 mL). The combined organic extracts were washed with
brine, dried over Na.sub.2SO.sub.4 and concentrated. The residue
was purified by column chromatography (silica gel, 4:1
hexanes/EtOAc) to afford desired compound 141 (9.50 g, 61%) as a
white solid: .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 10.49 (s,
1H), 8.20 (dd, J=8.3, 0.5 Hz, 1H), 8.02 (d, J=8.3 Hz, 1H), 7.66
(dd, J=8.4, 1.4 Hz, 1H), 7.64 (d, J=8.1 Hz, 1H), 7.55 (ddd, J=8.2,
7.7, 1.1 Hz, 1H), 6.83 (d, J=8.2 Hz, 1H).
Preparation of Compound 7
[0467] Zinc dust (7.03 g, 107.6 mmol) was added to a flame-dried,
nitrogen-purged side arm round-bottomed flask. Anhydrous DMF (50.0
mL) was added via syringe, followed by a catalytic amount of iodine
(1.00 g, 3.94 mmol). The resulting mixture was observed to undergo
a colour change from colorless to yellow and back to colourless.
Protected iodoalanine 143 (11.8 g, 35.9 mmol) was added in one
portion, followed by a catalytic amount of iodine (1.00 g, 3.94
mmol) and stirred at room temperature for 30 min; successful zinc
insertion is accompanied by a mild exotherm. The solution of
organozinc reagent was allowed to cool to room temperature before
Pd.sub.2dba.sub.3 (821 mg, 0.89 mmol), SPhos (736 mg, 1.79 mmol),
and aryl bromide 141 (8.00 g, 35.9 mmol) were added and the mixture
was heated at 50.degree. C. for 16 h, under a positive pressure of
nitrogen. The reaction mixture was allowed to cool to room
temperature. Saturated NH.sub.4Cl solution (300 mL) and EtOAc (300
mL) were added, and then the mixture was filtered through Celite
and washed with EtOAc (100 mL). The aqueous layer was separated and
extracted with EtOAc (2.times.300 mL). The combined organic
extracts were washed with brine, dried over Na.sub.2SO.sub.4 and
concentrated under vacuum. Crude product was purified by column
chromatography (silica gel, 4:1 hexanes/EtOAc) to afford desired
compound 7 (4.60 g, 37%) as a yellow solid: .sup.1H NMR (400 MHz,
CDCl.sub.3, mixture of rotamers) .delta. 8.23 (d, J=8.3 Hz, 1H),
7.99 (d, J=8.6 Hz, 1H), 7.54 (t, J=8.04 Hz, 1H), 7.48 (ddd, J=8.3,
6.9, 1.3 Hz, 1H), 7.08 (d, J=7.8 Hz, 1H), 6.70 (d, J=7.6 Hz, 1H),
6.57 (br s, 0.2H), 6.45 (br s, 0.2H), 5.91 (br s, 0.65H), 5.05 (d,
J=7.7 Hz, 0.75H), 4.89 (br s, 0.25H), 4.68 (q, J=6.8 Hz, 0.7H),
4.56 (br s, 0.2H), 3.73 (s, 0.7H), 3.62 (s, 2.3H), 3.49 (dd,
J=14.0, 5.9 Hz, 0.8H), 3.89 (dd, J=14.0, 7.2 Hz, 0.7H), 3.05 (br s,
0.2H), 1.39 (s, 7.5H), 1.09 (s, 2.5H).
Preparation of Compound 9
[0468] To a solution of compound 7 (7.60 g, 21.8 mmol) in
CH.sub.2Cl.sub.2 (150 mL) was added pyridine (18.0 mL) and
Tf.sub.2O (9.19 g, 32.6 mmol) at 0.degree. C. The resulting mixture
was stirred at room temperature for 2 h, concentrated under vacuum
and partitioned between CH.sub.2Cl.sub.2 (100 mL) and water (50
mL). The aqueous layer was separated and extracted with
CH.sub.2Cl.sub.2 (2.times.50 mL). The combined organic extracts
were washed with brine, dried over Na.sub.2SO.sub.4 and
concentrated to afford compound 9 (11.0 g, crude) as a brown oil.
The crude product was directly used for the next step without
further purification: .sup.1H NMR (400 MHz, CDCl.sub.3, mixture of
rotamers) .delta. 8.19-8.07 (m, 2H), 7.69-7.64 (m, 2H), 7.38 (d,
J=8.1 Hz, 1H), 7.28 (d, J=7.9 Hz, 1H), 5.12-5.06 (br s, 1H),
4.78-4.67 (m, 1H), 3.68-3.46 (m, 5H), 1.39 (s, 8H), 1.25 (s,
1H).
Preparation of Compound 11
[0469] The solution of compound 9 (11.0 g, 21.8 mmol) and benzyl
but-3-ynylcarbamate 10 (6.56 g, 32.6 mmol) in anhydrous
acetonitrile (100 mL) was degassed for 10 min under Argon
atmosphere followed by addition of TEA (11.9 mL, 87.0 mmol), 10%
(i-Bu).sub.3P in hexanes (8.80 mL, 4.35 mmol) and CuI (207 mg, 1.08
mmol) at room temperature. The resulting mixture was degassed with
Argon for another 10 min and Pd(PPh.sub.3).sub.4 (2.51 g, 2.17
mmol) was added in one portion. After degassing with argon for 5
min, the resulting mixture was refluxed for 16 h. The reaction
mixture was concentrated in vacuum and the residue was purified by
column chromatography (silica gel, 2:3 hexanes/EtOAc) to afford
compound 11 (7.00 g, 61% over two steps) as a brown oil: .sup.1H
NMR (400 MHz, CDCl.sub.3, mixture of rotamers) .delta. 8.33 (dd,
J=8.9, 1.9 Hz, 1H), 8.07 (dd, J=9.0, 1.7 Hz, 1H), 7.59-7.49 (m,
3H), 7.39-7.27 (m, 5H), 7.19 (d, J=7.3 Hz, 1H), 5.24-5.16 (m, 1H),
5.12 (s, 2H), 5.08-4.99 (m, 1H), 4.69 (q, J=6.7 Hz, 1H), 3.59 (s,
3H), 3.57-3.40 (m, 4H), 2.79 (t, J=6.4 Hz, 2H), 1.39 (s, 7.5H),
1.11 (s, 1.5H).
Preparation of Compound 17
[0470] To a solution of methyl ester 11 (7.00 g, 13.2 mmol) in THF
(200 mL), methanol (200 mL) and water (75.0 mL) was added solid
NaOH (16.0 g, 79.2 mmol). The resulting mixture was stirred at room
temperature for 1 h until TLC showed the reaction was completed. 1
N hydrochloric acid was added to adjust pH of reaction mixture to
10. After concentrated; water (100 mL) was added and pH was
adjusted to 5-6. The resulting precipitate was extracted with
CH.sub.2Cl.sub.2 (2.times.250 mL). Organic layers were combined,
dried over Na.sub.2SO.sub.4, filtered, concentrated and triturated
with MTBE to afford compound 17 (5.00 g, 75%) as a white solid:
.sup.1H NMR (400 MHz, CD.sub.3OD: mixture of rotamers) .delta. 8.33
(d, J=8.2 Hz, 1H), 8.28-8.20 (m, 1H), 7.59-7.45 (m, 3H), 7.38-7.21
(m, 6H), 5.09 (s, 2H), 4.55-4.45 (m, 1H), 3.76-3.66 (m, 1H), 3.44
(t, J=6.7 Hz, 2H), 3.28-3.20 (m, 1H), 2.76 (t, J=6.7 Hz, 2H), 1.29
(s, 6H), 0.82 (s, 3H).
Preparation of Compound 30
[0471] To a solution of compound 17 (4.60 g, 8.91 mmol) in THF (160
mL) were added T.sub.3P (50% in ethyl acetate, 10.7 mL) and NMM
(4.89 mL, 44.5 mmol) successively. After stirring at room
temperature for 10 min, amine 29 (6.11 g, 9.33 mmol) was added and
the reaction mixture was stirred at room temperature for 16 h.
After the solvent was removed, the residue was dissolved in
CH.sub.2Cl.sub.2 (100 mL), quickly washed with saturated
NH.sub.4Cl, saturated NaHCO.sub.3 and brine, dried over
Na.sub.2SO.sub.4 and concentrated. The residue was purified by
column chromatography (silica gel, 9:1 CH.sub.2Cl.sub.2/MeOH) to
afford amide 30 (6.60 g, 64%) as an off-white solid: .sup.1H NMR
(400 MHz, CDCl.sub.3) .delta. 8.33 (dd, J=9.0, 1.7 Hz, 1H), 8.17
(d, J=7.3 Hz, 1H), 7.62-7.47 (m, 4H), 7.42 (dd, J=7.7, 4.1 Hz, 4H),
7.37-7.28 (m, 11H), 7.09-6.95 (m, 4H), 5.46 (s, 2H), 5.33 (br s,
1H), 5.22 (t, J=5.8 Hz, 1H), 5.11 (s, 2H), 4.63-4.51 (m, 1H), 4.27
(dd, J=10.8, 5.4 Hz, 2H), 4.02-3.84 (m, 6H), 3.71 (t, J=4.5 Hz, 6H
impurities), 3.57 (t, J=10.6 Hz, 2H), 3.54-3.45 (m, 4H), 2.82-2.60
(m, 6H), 2.59-2.45 (m, 3H), 2.44-2.36 (m, 4H), 1.82-1.69 (m, 2H),
1.38 (s, 9H).
Preparation of Compound 31
[0472] A suspension of 30 (7.26 g, 6.20 mmol) and 10% Pd/C (1.50 g)
in EtOH/AcOH (240 mL/40.0 mL) was degassed by bubbling with Argon
using syringe for 10 min and then subjected to hydrogenation
conditions (1 atm) for 16 h at room temperature. The reaction
mixture was filtered through Celite and washed with MeOH. The
filtrate was concentrated in vacuum and triturated with MTBE to
afford amine salt 31 (7.06 g, 98%) as a brown solid: .sup.1H NMR
(400 MHz, CD.sub.3OD, mixture of rotamers) .delta. 8.24 (dd, J=7.2,
2.0 Hz, 1H), 8.09 (d, J=7.0 Hz, 1H), 7.59-7.22 (m, 2H), 7.49-7.41
(m, 4H), 7.39-7.22 (m, 10H), 6.95 (d, J=8.5 Hz, 2H), 5.51 (s, 2H),
4.55 (t, J=7.2 Hz, 1H), 4.24 (dd, J=10.7, 5.4 Hz, 2H), 4.19-4.10
(m, 2H), 3.99-3.88 (m, 4H), 3.83-3.73 (m, 8H, impurities), 3.61 (t,
J=10.5, Hz, 2H), 3.59-3.52 (m, 1H), 3.45-3.36 (m, 1H), 3.19-3.02
(m, 4H), 2.93-2.81 (m, 8H), 2.54.2.39 (m, 2H), 1.95 (s, 6H),
1.88-1.80 (m, 2H), 1.80-1.65 (m, 4H), 1.36 (s, 7H), 1.09 (s,
2H).
Preparation of 32
[0473] To a solution of 31 (7.06 g, 6.18 mmol) in EtOH (50.0 mL)
was added DIPEA (8.80 mL, 49.4 mmol) followed by methyl
3,5-diamino-6-chloropyrazine-2-carbonylcarbamimidothioate (13, 3.84
g, 9.88 mmol) at room temperature. The reaction mixture was heated
at 70.degree. C. for 2 h, cooled to room temperature and
concentrated under vacuum. The residue was purified twice by column
chromatography (silica gel, 80:18:2
CHCl.sub.3/CH.sub.3OH/NH.sub.4OH) to afford compound 32 (2.50 g,
33%) as a yellow solid: NMR (400 MHz, CD.sub.3OD, mixture of
rotamers) .delta. 8.22 (d, J=9.3 Hz, 8.08 (d, J=7.8 Hz, 1H),
7.56-7.47 (m, 4H), 7.43 (dd, J=7.4, 3.6 Hz, 4H), 7.33-7.14 (m,
10H), 6.94 (d, J=8.0 Hz, 2H), 5.47 (s, 2H), 4.53 (t, J=7.7 Hz, 1H),
4.22 (dd, J=10.8, 5.4 Hz, 2H), 3.99-3.89 (m, 4H), 3.84 (dd, J=5.5,
2.3 Hz, 2H), 3.70 (dd, J=9.2, 2.2 Hz, 2H), 3.59 (t, J=10.8 Hz, 2H),
3.54-3.46 (m, 1H), 3.47-3.38 (m, 1H), 3.22 (t, J=6.4 Hz, 2H),
3.11-3.02 (m, 2H), 2.70 (dd, J=13.5, 4.6 Hz, 2H), 2.61 (dd, J=13.6,
8.9, 2H), 2.57-2.47 (m, 2H), 2.46-2.34 (m, 2H), 1.84-1.73 (m, 2H),
1.72-1.61 (m, 4H), 1.36 (s, 7H), 1.12 (s, 2H).
Preparation of the HCl Salt of
3,5-diamino-N-(N-(4-(4-((S)-2-amino-3-(4-(3-(bis((2S,3R,4R,5R)-2,3,4,5,6--
pentahydroxyhexyl)amino)propyl)phenylamino)-3-oxopropyl)naphthalen-1-yl)bu-
tyl)carbamimidoyl)-6-chloropyrazine-2-carboxamide (33)
[0474] To a solution of 32 (2.50 g, 2.02 mmol) in EtOH (30.0 mL)
was added 4 N hydrochloric acid (80.0 mL). The resulting mixture
was stirred at room temperature for 2 h. The solvent was removed,
purified by reverse phase column and lyophilized to afford compound
33 (1.82 g, 85%) as a yellow hygroscopic solid: .sup.1H NMR (400
MHz, DMSO-d.sub.6) .delta. 10.61 (s, 1H), 10.59 (s, 1H), 9.41 (t,
J=5.2 Hz, H), 9.01 (br s, 1H), 8.96 (br s, 1H), 8.81 (br s, 2H),
8.77 (br s, 2H), 8.44-8.37 (m, 1H), 8.16-8.10 (m, 1H), 7.61-7.52
(m, 2H), 7.41 (d, J=8.6 Hz, 2H), 7.35 (d, J=7.5 Hz, 1H), 7.27 (d,
J=7.3 Hz, 1H), 7.17 (d, J=8.5 Hz, 2H), 4.28 (q, J=7.4 Hz, 1H),
4.09-3.99 (m, 2H), 3.75-3.65 (m, 3H), 3.58 (dd, J=11.0, 2.6 Hz,
2H), 3.55-3.31 (m, 10H), 3.30-3.13 (m, 4H), 3.32-3.00 (m, 2H),
2.63-2.53 (m, 2H), 2.05-1.92 (m, 2H), 1.78-1.61 (m, 4H).
[0475] .sup.1H NMR (400 MHz, CD.sub.3OD): .delta. 9.25 (t, J=5.9
Hz, 0.5H), 8.26-8.21 (m, 1H), 8.17-8.12 (m, 1H), 7.60-7.54 m, 2H),
7.38 (d, J=7.2 Hz, 1H), 7.32 (d, J=7.2 Hz, 1H), 7.25 (d, J=8.6 Hz,
2H), 7.15 (d, J=8.6 Hz, 2H), 4.31 (t, J=8.1 Hz, 1H), 4.21-4.14 (m,
1H), 4.13-4.08 (m, 1H), 3.85-3.80 (m, 2H), 3.79(d, J=2.9 Hz, 1H),
3.76 (d, J=3.2 Hz, 1H), 3.73-3.62 (m, 8H), 3.51-3.34 (m, 8H), 3.15
(t, J=6.8 Hz, 2H), 2.73-2.57 (m, 2H), 2.15-1.98 (m, 2H), 1.91-1.73
(m, 4H).
22. Preparation of
(2R,2'R,3R,3'R,4R,4'R,5S,5'S)-6,6'-(3-(4-aminophenyl)propylazanediyl)dihe-
xane-1,2,3,4,5-pentaol (29)
##STR00049##
[0476] Preparation of Compound 145
[0477] To a solution of compound 144 (8.80 g, 154.1 mmol) in
CH.sub.2Cl.sub.2 (150 mL) was added TEA (32.2 mL, 231.2 mmol) and
Boc.sub.2O (40.4 g, 185.3 mmol) at 0.degree. C. The reaction
mixture was continued to be stirred at 0.degree. C. for 0.5 h,
allowed to be warmed to room temperature and stirred for 5 h. Then
the mixture was partitioned between CH.sub.2Cl.sub.2 (150 mL) and
water (150 mL). The aqueous layer was separated and extracted with
CH.sub.2Cl.sub.2 (2.times.150 mL). The combined organic extracts
were washed with brine, dried over Na.sub.2SO.sub.4, concentrated
to afford desired compound 145 (22.0 g, 91%) as a colorless oil.
.sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 5.90-5.77 (m, 1H), 5.17
(dq, J=17.1, 1.7 Hz, 1H), 5.10 (dq, J=10.4, 1.4 Hz, 1H), 4.64 (brs,
1H), 3.74 (t, J=5.2 Hz, 2H), 1.45 (s, 9H).
Preparation of Compound 147
[0478] To a solution of compound 145 (14.0 g, 89.12 mmol) in
anhydrous THF (150 mL) was added 9-BBN (0.5 M in THF, 270 mL, 133.8
mmol) under argon. After the reaction mixture was stirred for 2 h
at room temperature, compound 146 (17.7 g, 71.3 mmol),
Pd(PPh.sub.3).sub.2Cl.sub.2 (3.12 g, 4.45 mmol), and 1 N aq NaOH
(150 mL) were added at room temperature. The resulting mixture was
stirred for additional 1 h. After solvent removed; the residue was
partitioned between EtOAc (200 mL) and water (200 mL). The aqueous
layer was separated and extracted with EtOAc (2.times.200 mL). The
combined organic extracts were washed with brine, dried over
Na.sub.2SO.sub.4 and concentrated under vacuum. The crude product
was purified by column chromatography (silica gel, 4:1
hexanes/EtOAc) to afford compound 147 (8.00 g, 43%) as a brown
solid: .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 8.14 (d, J=8.9 Hz,
2H), 7.34 (d, J=8.9 Hz, 2H), 4.56 (br s, 1H), 3.17 (q, J=6.2 Hz,
2H), 2.75 (t, J=7.7 Hz, 2H), 1.89-1.79 (m, 2H), 1.44 (s, 9H).
Preparation of Compound 148
[0479] Compound 147 (8.00 g, 28.6) was dissolved in 4 N HCl in
dioxane (50.0 mL) at room temperature and the solution was stirred
for 1 h. The reaction mixture was concentrated under vacuum and the
residue was triturated with MTBE to afford compound 148 (4.00 g,
65%) as a brown solid: .sup.1H NMR (400 MHz, CD.sub.3OD) .delta.
8.19 (d, J=8.7 Hz, 2H), 7.50 (d, J=8.7 Hz, 2H), 2.98 (t, J=7.4 Hz,
2H), 2.86 (t, J=7.6 Hz, 2H), 2.07-1.97 (m, 2H).
Preparation of Compound 150
[0480] To a solution of compound 148 (4.00 g, 18.5 mmol) and triol
149 (24.8 g, 92.5 mmol) in MeOH (150 mL) was added AcOH (11.1 mL,
185 mmol) and the reaction mixture was stirred at room temperature
for 10 min. After NaCNBH.sub.3 (5.83 g, 92.5 mmol) was added, the
solution was continued to be stirred at room temperature for 24 h.
Additional compound 149 (4.0 equiv), AcOH (4.0 equiv) and
NaCNBH.sub.3 (4.0 equiv) were added over 4 days. Then hexanal (2.0
equiv), AcOH (2.0 equiv) and NaCNBH.sub.3 (2.0 equiv) were added.
The solution was further stirred at room temperature for 1 h. After
removal of solvent, the residue was neutralized with saturated
NaHCO.sub.3 and the residue was partitioned between EtOAc (200 mL)
and water (200 mL). The aqueous layer was separated and extracted
with CH.sub.2Cl.sub.2 (2.times.300 mL). The combined organic
extracts were dried over Na.sub.2SO.sub.4 and concentrated under
vacuum. The residue was purified by column chromatography (silica
gel, 9:1 CH.sub.2Cl.sub.2/MeOH, 80:18:2 CHCl.sub.3/MeOH/NH.sub.4OH)
to afford compound 150 (6.50 g, 52%) as an off-white solid.
Additional 4.00 g of material from impure fractions was isolated
and purified by reverse phase column to afford 1.50 g (12%) of pure
compound 150 (total 7.70 g, 64%): .sup.1H NMR (400 MHz, CD.sub.3OD)
.delta. 8.03 (d, J=8.7 Hz, 2H), 7.50-7.41 (m, 4H), 7.35-7.23 (m,
8H), 5.48 (s, 2H), 4.22 (dd, J=10.6, 5.3 Hz, 2H), 3.99-3.91 (m,
4H), 3.85 (dd, J=5.5, 2.4 Hz, 2H), 3.70 (dd, J=9.5, 2.4 Hz, 2H),
3.59 (t, J=10.6 Hz, 2H), 2.73 (dd, J=13.6, 4.5 Hz, 2H), 2.67-2.50
(m, 6H), 1.83-1.71 (m, 2H).
Preparation of
(2R,2'R,3R,3'R,4R,4'R,5S,5'S)-6,6'-(3-(4-aminophenyl)propylazanediyl)
dihexane-1,2,3,4,5-pentaol (Compound 153)
[0481] A suspension of compound 150 (6.50 g, 9.50 mmol) and 10%
Pd/C (1.30 g) in EtOH (150 mL) was degassed by bubbling with Argon
using syringe for 10 min then stirred at rt under hydrogen
atmosphere (balloon, 1 atm) for 6 h at room temperature. The
reaction mixture was filtered through celite and washed with MeOH.
The filtrate was concentrated in vacuum to afford 153 (6.01 g, 97%)
as an off-white solid: .sup.1H NMR (400 MHz, CD.sub.3OD): .delta.
7.49-7.42 (m, 4H), 7.35-7.26 (m, 6H), 6.82 (d, J=8.4 Hz, 2H), 6.60
(d, J=8.4 Hz, 2H), 5.48 (s, 2H), 4.22 (dd, J=10.8, 5.9 Hz, 2H),
3.98-3.89 (m, 4H), 3.83 (dd, J=5.7, 2.3 Hz, 2H), 3.69 (dd, J=13.2,
3.4 Hz, 2H), 3.62-3.55 (m, 3H), 2.71 (dd, J=13.2, 3.4 Hz, 2H),
2.65-2.48 (m, 3H), 2.45-2.29 (m, 2H), 1.74-1.63 (m, 2H).
23. Preparation of
3,5-diamino-N-(N-(4-(4-((R)-2-amino-3-(4-(3-(bis((2S,3R,4R,5R)-2,3,4,5,6--
pentahydroxyhexyl)amino)propyl)phenylamino)-3-oxopropyl)naphthalen-1-yl)bu-
tyl)carbamimidoyl)-6-chloropyrazine-2-carboxamide (152)
##STR00050##
[0482] Preparation of Compound 14
[0483] To a solution of 1-naphthol (1, 10.0 g, 69.4 mmol) in
acetonitrile (70.0 mL) was added several portions of NBS (142, 12.3
g, 69.4 mmol) over a period of 30 min. The resulting mixture was
stirred at room temperature for 4 h, concentrated under vacuum,
followed by addition of water (200 mL) and ethyl acetate (200 mL).
The aqueous layer was separated and extracted with ethyl acetate
(2.times.200 mL). The combined organic extracts were washed with
brine, dried over Na.sub.2SO.sub.4 and concentrated. The residue
was purified by crystallization (heptane/EtOAc) to afford desired
compound 14 (6.0 g, 39%) as a white solid. .sup.1H NMR (400 MHz,
DMSO-d.sub.6): .delta. 10.49 (s, 1H), 8.20 (dd, J=8.3, 0.5 Hz, 1H),
8.02 (d, J=8.3 Hz, 1H), 7.66 (dd, J=8.4, 1.4 Hz, 1H), 7.64 (d,
J=8.1 Hz, 1H), 7.55 (ddd, J=8.2, 7.7, 1.1 Hz, 1H), 6.83 (d, J=8.2
Hz, 1H).
Preparation of Compound 145
[0484] Zinc dust (4.76 g, 72.9 mmol) was added to a flame-dried,
nitrogen-purged side arm round-bottomed flask. Anhydrous DMF (25.0
mL) was added via syringe, followed by a catalytic amount of iodine
(677 mg, 2.67 mmol). The resulting mixture was observed to undergo
a colour change from colorless to yellow and back to colourless.
Protected iodoalanine 114 (8.00 g, 24.3 mmol) was added in one
portion, followed by a catalytic amount of iodine (677 mg, 2.67
mmol) and stirred at room temperature for 30 min; successful zinc
insertion is accompanied by a mild exotherm. The solution of
organozinc reagent was allowed to cool to room temperature before
Pd2(dba).sub.3 (556 mg, 0.60 mmol), SPhos (498 mg, 1.21 mmol), and
aryl bromide 14 (5.40 g, 24.3 mmol) were added and the mixture was
heated at 50.degree. C. for 16 h, under a positive pressure of
nitrogen. The reaction mixture was allowed to cool to room
temperature. Saturated NH.sub.4Cl solution (300 mL) and EtOAc (300
mL) were added, and then the mixture was filtered through Celite
and washed with EtOAc (100 mL). The aqueous layer was separated and
extracted with EtOAc (2.times.300 mL). The combined organic
extracts were washed with brine, dried over Na.sub.2SO.sub.4 and
concentrated under vacuum. Crude product was purified by column
chromatography (silica gel, 4:1 hexanes/EtOAc) to afford desired
compound 145 (3.10 g, 37%) as a yellow solid. .sup.1H NMR (400 MHz,
CDCl.sub.3, mixture of rotamers): .delta. 8.23 (d, J=8.3 Hz, 1H),
7.99 (d, J=8.6 Hz, 1H), 7.54 (t, J=8.04 Hz, 1H), 7.48 (ddd, J=8.3,
6.9, 1.3 Hz, 1H), 7.08 (d, J=7.8 Hz, 1H), 6.70 (d, J=7.6 Hz, 1H),
5.98 (brs, 0.3H), 5.59 (br s, 0.7H), 5.03 (d, J=7.7 Hz, 0.85H),
4.84 (br s, 0.15H), 4.68 (q, J=6.8 Hz, 1H), 3.76-3.68 (m, 1H), 3.62
(s, 3H), 3.54-3.33 (m, 2H), 1.39 (s, 7H), 1.09 (s, 2H).
Preparation of Compound 146
[0485] To a solution of compound 145 (3.07 g, 8.90 mmol) in
CH.sub.2Cl.sub.2 (75.0 mL) was added pyridine (7.25 mL, 88.9 mmol)
and Tf.sub.2O (2.24 mL, 13.3 mmol) at 0.degree. C. The resulting
mixture was stirred at room temperature for 2 h, concentrated under
vacuum and partitioned between CH.sub.2Cl.sub.2 (100 mL) and water
(50 mL). The aqueous layer was separated and extracted with
CH.sub.2CL (2.times.50 mL). The combined organic extracts were
washed with brine, dried over Na.sub.2SO.sub.4 and concentrated to
afford compound 146 (4.20 g, crude) as a brown oil. The crude
product was directly used for the next step without further
purification. .sup.1H NMR (400 MHz, CDCl.sub.3, mixture of
rotamers): .delta. 8.19-8.07 (m, 2H), 7.69-7.64 (m, 2H), 7.38 (d,
J=8.1 Hz, 1H), 7.28 (d, J=7.9 Hz, 1H), 5.12-5.06 (br s, 1H),
4.78-4.67 (m, 1H), 3.68-3.46 (m, 5H), 1.39 (s, 8H), 1.25 (s,
1H).
Preparation of Compound 147
[0486] The solution of compound 6 (4.20 g, 8.80 mmol, crude) and
benzyl but-3-ynylcarbamate 7 (2.65 g, 13.2 mmol) in anhydrous
acetonitrile (50.0 mL) was degassed for 10 min under Argon
atmosphere followed by addition of TEA (4.81 mL, 35.2 mmol), 10%
(t-Bu).sub.3P in hexanes (3.56 mL, 1.76 mmol) and CuI (84 mg, 0.44
mmol) at room temperature. The resulting mixture was degassed with
Argon for another 10 min and Pd(PPh.sub.3).sub.4 (1.01 g, 0.88
mmol) was added in one portion. After degassing with argon for 5
min, the resulting mixture was refluxed for 18 h. The reaction
mixture was concentrated in vacuum and the residue was purified by
column chromatography (silica gel, 2:3 hexanes/EtOAc) to afford
compound 147 (3.20 g, 67% over two steps) as a brown oil. .sup.1H
NMR (400 MHz, CDCl.sub.3, mixture of rotamers): .delta. 8.33 (dd,
J=8.9, 1.9 Hz, 1H), 8.07 (dd, J=9.0, 1.7 Hz, 1H), 7.59-7.49 (m,
3H), 7.39-7.27 (m, 5H), 7.19 (d, J=7.3 Hz, 1H), 5.24-5.16 (m, 1H),
5.12 (s, 2H), 5.08-4.99 (m, 1H), 4.69 (q, J=6.7 Hz, 1H), 3.59 (s,
3H), 3.57-3.40 (m, 4H), 2.79 (t, J=6.4 Hz, 2H), 1.39 (s, 7.5H),
1.11 (s, 1.5H).
Preparation of Compound 148
[0487] To a solution of methyl ester 147 (3.10 g, 5.84 mmol) in THF
(60 mL), methanol (60 mL) and water (20.0 mL) was added solid NaOH
(1.40 g, 35.09 mmol). The resulting mixture was stirred at room
temperature for 2 h until TLC showed the reaction was completed. 1
N hydrochloric acid was added to adjust pH of reaction mixture to
10. After concentrated; water (100 mL) was added and pH was
adjusted to 5-6. The resulting precipitate was extracted with
CH.sub.2Cl.sub.2 (2.times.200 mL). Organic layers were combined,
dried over Na.sub.2SO.sub.4, filtered, concentrated and triturated
with MTBE to afford compound 148 (3.00 g, 99%) as a white solid.
.sup.1H NMR (400 MHz, CD.sub.3OD; mixture of rotamers): .delta.
8.33 (d, J=8.2 Hz, 1H), 8.28-8.20 (m, 1H), 7.59-7.45 (m, 3H),
7.38-7.21 (m, 6H), 5.09 (s, 2H), 4.55-4.45 (m, 1H), 3.76-3.66 (m,
1H), 3.44 (t, J=6.7 Hz, 2H), 3.28-3.20 (m, 1H), 2.76 (t, J=6.7 Hz,
2H), 1.29 (s, 6H), 0.82 (s, 3H).
Preparation of Compound 149
[0488] To a solution of compound 148 (800 mg, 1.55 mmol) in THF (30
mL) were added T.sub.3P (50% in ethyl acetate, 1.86 mL) and NMM
(0.85 mL, 7.75 mmol) successively. After stirring at room
temperature for 10 min, amine 29 (1.01 g, 1.55 mmol) was added and
the reaction mixture was stirred at room temperature for 1 h. After
the solvent was removed, the residue was dissolved in
CH.sub.2Cl.sub.2 (100 mL), quickly washed with saturated
NH.sub.4Cl, saturated NaHCO.sub.3 and brine, dried over
Na.sub.2SO.sub.4 and concentrated. The residue was purified by
column chromatography (silica gel, 9:1 CH.sub.2Cl.sub.2/MeOH) to
afford amide 149 (1.20 g, 67%) as an off-white solid. .sup.1H NMR
(400 MHz, CDCl.sub.3): .delta. 8.35 (d, J=8.0, 1.7 Hz, 1H), 8.19
(d, J=8.5 Hz, 1H), 7.60-7.52 (m, 2H), 7.50 (d, J=7.3 Hz, 2H),
7.45-7.39 (m, 5H), 7.37-7.28 (m, 1.1H), 7.08-6.96 (m, 3H), 5.47 (s,
2H), 5.33-5.17 (m, 2H), 5.12 (s, 2H), 4.59-4.48 (m, 1H), 4.29 (dd,
J=10.8, 5.4 Hz, 2H), 4.07-4.00 (m, 2H), 3.99-3.91 (m, 4H),
3.78-3.68 (m, 3H), 3.59 (t, J=10.6 Hz, 2H), 3.55-3.46(m, 4H),
2.95-2.82 (m, 2H), 2.81-2.69 (m, 4H), 2.68-2.57 (m, 1H), 2.56-2.44
(m, 3H), 2.43-2.38 (m, 1H), 1.85-1.69 (m, 2H), 1.38 (s, 9H).
Preparation of Compound 150
[0489] A suspension of 149 (1.15 g, 1.00 mmol) and 10% Pd/C (230
mg) in EtOH/AcOH (80.0 mL/20.0 mL) was degassed by bubbling with
Argon using syringe for 10 min and then subjected to hydrogenation
conditions (1 atm) for 16 h at room temperature. The reaction
mixture was filtered through Celite and washed with MeOH. The
filtrate was concentrated in vacuum and triturated with MTBE to
afford amine salt 150 (1.12 g, 97%) as a brown solid. .sup.1H NMR
(400 MHz, CD.sub.3OD, mixture of rotamers): .delta. 8.25 (dd,
J=7.2, 2.0 Hz, 1H), 8.09 (d, J=7.0 Hz, 1H), 7.59-7.51 (m, 2H),
7.48-7.41 (m, 4H), 7.37-7.21 (m, 10H), 6.94 (d, J=8.5 Hz, 2H), 5.52
(s, 2H), 4.54 (t, J=7.2 Hz, 1H), 4.24 (dd, J=10.7, 5.4 Hz, 2H),
4.16-4.08 (m, 2H), 3.97-3.88 (m, 4H), 3.75-3.70 (m, 2H), 3.62 (t,
J=10.5, Hz, 2H), 3.60-3.51 (m, 1H), 3.28-3.15 (m, 2H), 3.14-2.95
(m, 4H), 2.89 (t, J=7.4 Hz, 2H), 2.73-2.67 (m, 1H), 2.54-2.39 (m,
2H), 1.95 (s, 6H), 1.88-1.64 (m, 8H), 1.36 (s, 7.5H), 1.09 (s,
1.5H).
Preparation of 151
[0490] To a solution of 150 (1.05 g, 0.92 mmol) in EtOH (15.0 mL)
was added DIPEA (1.30 mL, 7.35 mmol) followed by methyl
3,5-diamino-6-chloropyrazine-2-carbonylcarbamimidothioate (13, 573
mg, 1.47 mmol) at room temperature. The reaction mixture was heated
at 70.degree. C. for 2 h, cooled to room temperature and
concentrated under vacuum. The residue was purified twice by column
chromatography (silica gel, 80:18:2
CHCl.sub.3/CH.sub.3OH/NH.sub.4OH) to afford compound 151 (410 mg,
36%) as a yellow solid. .sup.1H NMR (400 MHz, CD.sub.3OD, mixture
of rotamers): .delta. 8.22 (d, J=8.4 Hz, 1H), 8.09 (d, J=8.2 Hz,
1H), 7.56-7.48 (m, 2H), 7.47-7.40 (m, 4H), 7.33-7.25 (m, 6H), 7.22
(d, J=7.5 Hz, 2H), 7.16 (d, J=7.8 Hz, 2H), 6.94 (d, J=8.1 Hz, 2H),
5.47 (s, 2H), 4.53 (t, J=8.1 Hz, 1H), 4.22 (dd, J=10.8, 5.4 Hz,
2H), 3.99-3.89 (m, 4H), 3.84 (dd, J=5.5, 2.1 Hz, 2H), 3.70 (dd,
J=9.1, 2.0 Hz, 2H), 3.59 (t, J=10.8 Hz, 2H), 3.53-3.47 (m, 1H),
3.46-3.39 (m, 1H), 3.26-3.17 (m, 2H), 3.12-3.04 (m, 2H), 2.70 (dd,
J=13.2, 4.0 Hz, 2H), 2.60 (dd, J=13.0, 8.2, 2H), 2.57-2.49 (m, 2H),
2.47-2.33 (m, 2H), 1.84-1.73 (m, 2H), 1.72-1.61 (m, 4H), 1.37 (s,
7H), 1.12 (s, 2H).
Synthesis of
3,5-diamino-N-(N-(4-(4-((R)-2-amino-3-(4-(3-(bis((2S,3R,4R,5R)-2,3,4,5,6--
pentahydroxyhexyl)amino)propyl)phenylamino)-3-oxopropyl)naphthalen-1-yl)bu-
tyl)carbamimidoyl)-6-chloropyrazine-2-carboxamide (152)
[0491] To a solution of 151 (480 mg, 0.42 mmol) in EtOH (5.0 mL)
was added 4 N hydrochloric acid (25.0 mL). The resulting mixture
was stirred at room temperature for 2 h. The solvent was removed,
purified by reverse phase column and lyophilized to afford compound
152 (300 mg, 71%) as a yellow hygroscopic solid. .sup.1H NMR (400
MHz, DMSO-d.sub.6): .delta. 10.57 (brs, 1H), 10.55 (brss, 1H), 9.35
(t, J=6.0 Hz, 1H), 9.04-8.84 (m, 2H), 8.81-8.66 (m, 4H), 8.42-8.36
(m, 1H), 8.16-8.10 (m, 1H), 7.61-7.53 (m, 2H), 7.41 (d, J=8.6 Hz,
2H), 7.35 (d, J=7.5 Hz, 1H), 7.28 (d, J=7.8 Hz, 1H), 7.17 (d, J=9.0
Hz, 2H), 4.32-4.23 (m, 1H), 4.08-3.97 (m, 2H), 3.75-3.30 (m, 13H),
3.29-3.15 (m, 4H), 3.14-2.97 (m, 2H), 2.64-2.53 (m, 2H), 2.05-1.92
(m, 2H), 1.79-1.60 (m, 4H).
[0492] .sup.1H NMR (400 MHz, CD.sub.3OD): .delta. 8.25-8.21 (m,
1H), 8.18-8.13 (m, 1H), 7.59-7.53 (m, 2H), 7.38 (d, J=7.3 Hz, 1H),
7.32 (d, J=7.3 Hz, 1H), 7.26 (d, J=8.8 Hz, 2H), 7.15 (d, J=8.5 Hz,
2H), 4.30 (t, J=7.3 Hz, 1H), 4.20-4.14 (m, 1H), 4.13-4.08 (m, 1H),
3.84-3.80 (m, 2H), 3.79-3.75(m, 2H), 3.72-3.61 (m, 8H), 3.51-3.34
(m, 8H), 3.15 (t, J=7.3 Hz, 2H), 2.74-2.58 (m, 2H), 2.13-1.98 (m,
2H), 1.91-1.73 (m, 4H).
[0493] HRMS calculated for
C.sub.44H.sub.64ClN.sub.10O.sub.12[M+Na].sup.+, 959.4418 and found
959.4394.
24. Preparation of Intermediate 18
##STR00051##
[0494] Preparation of Compound 155
[0495] To a solution of compound 154 (500 mg, 9.00 mmol) in
CH.sub.2Cl.sub.2 (50 mL) was added TEA (1.63 mL, 11.7 mmol) and
Boc.sub.2O (2.16 g, 9.90 mmol) at 0.degree. C. The reaction mixture
was continued to be stirred at 0.degree. C. for 0.5 h, allowed to
be warmed to room temperature and stirred for 3 h. Then the mixture
was partitioned between CH.sub.2Cl.sub.2 (50 mL) and water (50 mL).
The aqueous layer was separated and extracted with CH.sub.2Cl.sub.2
(2.times.50 mL). The combined organic extracts were washed with
brine, dried over Na.sub.2SO.sub.4, concentrated, the residue was
purified by column chromatography (silica gel, 2:3 hexanes/EtOAc)
to afford desired compound 155 (1.20 g, 86%) as a colorless oil.
.sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 4.70 (br s, 1H), 3.91
(dd, J=5.3, 2.2 Hz, 2H), 2.21 (t, J=2.7 Hz, 1H), 1.45 (s, 9H).
Preparation of Compound 157
[0496] The solution of compound 155 (1.00 g, 6.45 mmol) and 156
(1.30 g, 6.45 mmol) in anhydrous THF (15 mL) was degassed for 10
min under Argon atmosphere followed by addition of TEA (3.53 mL,
25.8 mmol), PPh.sub.3 (424 mg, 1.61 mmol) and CuI (246 mg, 1.29
mmol) at room temperature. The resulting mixture was degassed with
Argon for another 10 min and Pd(PPh.sub.3).sub.4 (7.45 g, 6.45
mmol) was added in one portion. After degassing with argon for 5
min, the resulting mixture was refluxed for 16 h. The reaction
mixture was concentrated in vacuum and the residue was purified by
column chromatography (silica gel, 2:3 hexanes/EtOAc) to afford
compound 157 (750 mg, 42%) as a brown oil. .sup.1H NMR (400 MHz,
CDCl.sub.3): .delta. 8.17 (d, J=9.2 Hz, 2H), 7.55 (d, J=9.2 Hz,
2H), 4.79 (brs, 1H), 4.18 (d, J=6.0 Hz, 2H), 1.47 (s, 9H).
Preparation of Compound 158
[0497] Compound 157 (2.00 g, 7.24) was dissolved in 4 N HCl in
dioxane (20.0 mL) at room temperature and the solution was stirred
for 2 h. The reaction mixture was concentrated under vacuum and the
residue was triturated with MTBE to afford compound 158 (1.25 g,
82%) as a brown solid. .sup.1H NMR (300 MHz, CD.sub.3OD): .delta.
8.26 (d, J=9.2 Hz, 2H), 7.72 (d, J=9.2 Hz, 2H), 4.09 (s, 2H).
Preparation of Compound 159
[0498] To a solution of compound 158 (100 mg, 0.47 mmol) and
formaldehyde solution in water (30%, 1.40 mL, 1.41 mmol) in MeOH
(3.0 mL) was added AcOH (0.09 mL, 1.41 mmol) and the reaction
mixture was stirred at room temperature for 30 min. After
NaCNBH.sub.3 (88 mg, 1.41 mmol) was added, the solution was
continued to be stirred at room temperature for 16 h. Additional
formaldehyde solution in water (30%, 0.92 mL, 0.94 mmol), AcOH
(0.09 mL, 1.41 mmol) and NaCNBH.sub.3 (88 mg, 1.41 mmol) were added
and stirred for another 16 h. After removal of solvent, the residue
was neutralized with saturated NaHCO.sub.3 and the residue was
partitioned between EtOAc (30 mL) and water (30 mL). The aqueous
layer was separated and extracted with CH.sub.2Cl.sub.2 (2.times.40
mL). The combined organic extracts were dried over Na.sub.2SO.sub.4
and concentrated under vacuum. The residue was purified by column
chromatography (silica gel, 9:1 CH.sub.2Cl.sub.2/MeOH, 80:18:2
CHCl.sub.3/MeOH/NH.sub.4OH) to afford compound 159 (50 g, 52%) as
an off-white oil. .sup.1H NMR (300 MHz, CD.sub.3OD): .delta. 8.17
(d, J=9.0 Hz, 2H), 7.57 (d, J=9.0 Hz, 2H), 3.50 (s, 2H), 2.37 (s,
6H).
Preparation of Compound 18
[0499] A suspension of compound 159 (100 mg, 0.49 mmol) and 10%
Pd/C (40 mg) in MeOH (3.0 mL) was degassed with Argon for 10 min
then stirred under hydrogen atmosphere (balloon, 1 atm) for 3 h at
room temperature. The reaction mixture was filtered through celite
and washed with MeOH. The filtrate was concentrated in vacuum and
triturated with CH.sub.2Cl.sub.2/hexane to afford 18 (48 mg, 55%)
as a white crystal .sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 6.96
(d, J=8.3 Hz, 2H), 6.60 (d, J=8.3 Hz, 2H), 3.47 (br s, 2H), 2.53
(t, J=7.8 Hz, 2H), 2.26 (dd, J=8.7, 7.2 Hz, 2H), 2.22 (s, 6H),
1.77-1.67 (m, 2H).
Preparation of Intermediate 29
##STR00052##
[0500] Preparation of Compound 161
[0501] To a solution of compound 158 (4.00 g, 18.9 mmol) and triol
160 (11.7 g, 56.6 mmol) in MeOH (50 mL) was added AcOH (3.40 mL,
56.6 mmol) and the reaction mixture was stirred at room temperature
for 30 min. After NaCNBH.sub.3 (3.55 g, 56.6 mmol) was added, the
solution was continued to be stirred at room temperature for 16 h.
Additional compound 160 (11.7 g, 56.6 mmol), AcOH (3.40 mL, 56.6
mmol) and NaCNBH.sub.3 (3.55 g, 56.6 mmol) were added the solution
was continued to be stirred at room temperature for 16 h. After
removal of solvent, the residue was neutralized with saturated
NaHCO.sub.3 and the residue was partitioned between CH.sub.2O.sub.2
(10 mL) and water (10 mL). The aqueous layer was separated and
extracted with CH.sub.2Cl.sub.2 (2.times.10 mL). The combined
organic extracts were dried over Na.sub.2SO.sub.4 and concentrated
under vacuum. The residue was purified by column chromatography
(silica gel, 9:1 CH.sub.2Cl.sub.2/MeOH, 80:18:2
CHCl.sub.3/MeOH/NH.sub.4OH) to afford compound 29 (700 mg, 7.0%) as
an off-white solid, .sup.1H NMR (300 MHz, CD.sub.3OD): .delta. 8.21
(d, J=8.8 Hz, 2H), 7.66 (d, J=8.8 Hz, 2H), 4.68 (q, J=5.1 Hz, 2H),
4.04 (dd, J=10.8, 5.4 Hz, 2H), 3.99-3.93 (m, 2H), 3.86-3.74 (m,
6H), 3.54 (dd, J=9.8, 2.3 Hz, 2H), 3.36 (t, J=10.7 Hz, 2H), 2.87
(dd, J=13.3, 4.9 Hz, 2H), 2.74 (dd, J=13.3, 7.8 Hz, 2H), 1.25 (d,
J=5.1 Hz, 6H).
Preparation of Compound 29
[0502] A suspension of compound 161 (500 mg, 0.90 mmol) and 10%
Pd(OH).sub.2/C (215 mg) in EtOH (230 mL) was degassed by bubbling
with Argon using syringe for 10 min then stirred under hydrogen
atmosphere (balloon, 1 atm) for 2 h at room temperature. The
reaction mixture was filtered through celite and washed with MeOH.
The filtrate was concentrated in vacuum and residue was purified by
column chromatography (silica gel, 9:1 CH.sub.2Cl.sub.2/MeOH,
80:18:2 CHCl.sub.3/MeOH/NH.sub.4OH) to afford compound 29 (264 mg,
55%) as an off-white solid. .sup.1H NMR (400 MHz, CD.sub.3OD):
.delta. 6.97 (d, J=8.6 Hz, 2H), 6.67 (d, J=8.6 Hz, 2H), 4.71 (q,
J=5.1 Hz, 2H), 4.06 (dd, J=10.6, 5.3 Hz, 2H), 4.13-4.05 (m, 2H),
3.81 (dd, J=5.0, 2.3 Hz, 2H), 3.80-3.72 (m, 2H), 3.51 (dd, J=9.6,
2.4 Hz, 2H), 3.33-3.23 (m, 2H), 3.38 (t, J=10.7 Hz, 2H), 2.83-2.54
(m, 6H), 1.85-1.69 (m, 2H), 1.26 (d, J=5.1 Hz, 6H).
Preparation of Intermediate 24
##STR00053##
[0503] Preparation of Compound 162
[0504] To a solution of compound 158 (200 mg, 0.94 mmol) and triol
160 (194 mg, 0.94 mmol) in MeOH (2.0 mL) was added AcOH (0.17 mL,
2.82 mmol) and the reaction mixture was stirred at room temperature
for 30 min. After NaCNBH.sub.3 (148 mg, 2.35 mmol) was added, the
solution was continued to be stirred at room temperature for 16 h.
Additional compound 160 (0.2 equiv), AcOH (3.0 equiv) and
NaCNBH.sub.3 (1.0 equiv) were added the solution was continued to
be stirred at room temperature for 16 h. After removal of solvent,
the residue was neutralized with saturated NaHCO.sub.3 and the
residue was partitioned between CH.sub.2Cl.sub.2 (10 mL) and water
(10 mL). The aqueous layer was separated and extracted with
CH.sub.2Cl.sub.2 (2.times.10 mL). The combined organic extracts
were dried over Na.sub.2SO.sub.4 and concentrated under vacuum. The
residue was purified by column chromatography (silica gel, 9:1
CH.sub.2Cl.sub.2/MeOH, 80:18:2 CHCl.sub.3/MeOH/NH.sub.4OH) to
afford compound 162 (95 mg, 28%) as an off-white solid. .sup.1H NMR
(400 MHz, CD.sub.3OD): .delta. 8.24 (d, J=9.1 Hz, 2H), 7.69 (d,
J=9.1 Hz, 2H), 4.70 (q, J=5.1 Hz, 1H), 4.09-4.02 (m, 2H), 4.00 (d,
J=2.1 Hz, 2H), 3.83 (dd, J=5.1, 2.3 Hz, 1H), 3.81-3.71 (m, 1H),
3.53 (dd, J=9.3, 2.3 Hz, 1H), 3.38 (t, J=11.0 Hz, 1H), 3.21-3.07
(m, 2H), 1.25 (d, J=5.1 Hz, 3H).
[0505] Preparation of Compound 164; To a solution of compound 162
(95 mg, 0.26 mmol) and hexanal 163 (52 mg, 0.51 mmol), AcOH (0.05
mL, 0.78 mmol) and NaCNBH.sub.3 (41 mg, 0.65 mmol) were added. The
solution was stirred at room temperature for 16 h. After removal of
solvent, the residue was neutralized with saturated NaHCO.sub.3 and
the residue was partitioned between EtOAc (10 mL) and water (10
mL). The aqueous layer was separated and extracted with
CH.sub.2Cl.sub.2 (2.times.10 mL). The combined organic extracts
were dried over Na.sub.2SO.sub.4 and concentrated under vacuum. The
residue was purified by column chromatography (silica gel, 9:1
CH.sub.2Cl.sub.2/MeOH, 80:18:2 CHCl.sub.3/MeOH/NH.sub.4OH) to
afford compound 164 (70 mg, 59%) as an off-white solid. .sup.1H NMR
(400 MHz, CDCl.sub.3): .delta. 8.18(d, J=8.9 Hz, 2H), 7.56 (d,
J=8.9 Hz, 2H), 4.70 (q, J=5.0 Hz, 1H), 4.15 (dd, J=10.4, 5.2 Hz,
1H), 4.01-3.89 (m, 2H), 3.83 (dd, J=3.8, 2.7 Hz, 1H), 3.77 (brs,
1H), 3.70 (brs, 1H), 3.64 (t, J=6.2 Hz, 1H), 3.56 (dd, J=9.2, 4.0
Hz, 1H), 3.41 (t, J=10.8 Hz, 1H), 2.87 (dd, J=13.2, 4.3 Hz, 1H),
2.78-2.68 (m, 2H), 2.63-2.55 (m, 1H), 1.75-1.43 (m, 4H), 1.34 (d,
J=5.0 Hz, 3H), 1.32-1.25 (m, 6H), 0.89 (t, J=6.6 Hz, 3H).
Preparation of Compound 24
[0506] A suspension of compound 164 (1.70 g, 3.77 mmol) and 10%
Pd/C (200 mg) in MeOH (40 mL) was degassed with Argon for 10 min
then stirred under hydrogen atmosphere (balloon, 1 atm) for 2 h at
room temperature. The reaction mixture was filtered through celite
and washed with MeOH. The filtrate was concentrated in vacuum and
residue was purified by column chromatography (silica gel, 9:1
CH.sub.2Cl.sub.2/MeOH, 80:18:2 CHCl.sub.3/MeOH/NH.sub.4OH) to
afford compound 24 (1.20 g, 76%) as an off-white solid. .sup.1H NMR
(300 MHz, CDCl.sub.3): .delta. 6.96 (d, J=8.9 Hz, 2H), 6.62 (d,
J=8.9 Hz, 2H), 4.68 (q, J=5.0 Hz, 1H), 4.14 (dd, J=11.0, 5.5Hz,
1H), 3.92-3.81 (m, 2H), 3.72 (dd, J=3.8, 2.4 Hz, 1H), 3.50 (dd,
J=9.1, 4.0 Hz, 1H), 3.40 (t, J=10.5 Hz, 1H), 2.76-2.38 (m, 10H),
1.81-1.64 (m, 3H), 1.48-1.36 (m, 2H), 1.33 (d, J=5.0 Hz, 3H),
1.30-1.20 (m, 6H), 0.88 (t, J=6.6 Hz, 3H),
Preparation of Intermediate 85
##STR00054##
[0507] Preparation of Compound 166
[0508] To a solution of compound 148 (4.60 g, 21.3 mmol) and triol
165 (17.1 g, 63.9 mmol) in MeOH (100 mL) was added AcOH (12.1 mL,
63.9 mmol) and the reaction mixture was stirred at room temperature
for 10 min. After NaCNBH.sub.3 (4.00 g, 63.9 mmol) was added, the
solution was continued to be stirred at room temperature for 6 h.
Then hexanal 163 (5.10 mL, 42.6 mmol) and NaCNBH.sub.3 (2.60 g,
42.6 mmol) were added. The solution was further stirred at room
temperature for 2 h. After removal of solvent, the residue was
neutralized with saturated NaHCO.sub.3 and the residue was
partitioned between EtOAc (200 mL) and water (200 mL). The aqueous
layer was separated and extracted with CH.sub.2Cl.sub.2
(2.times.300 mL). The combined organic extracts were dried over
Na.sub.2SO.sub.4 and concentrated under vacuum. The residue was
purified by column chromatography (silica gel, 9:1
CH.sub.2Cl.sub.2/MeOH, 80:18:2 CHCl.sub.3/MeOH/NH.sub.4OH) to
afford compound 166 (6.90 g, 64%) as an off-white solid. .sup.1H
NMR (400 MHz, CD.sub.3OD): .delta. 8.12 (d, J=8.6 Hz, 2H),
7.51-7.43 (m, 2H), 7.38 (d, J=8.6 Hz, 2H), 7.37-7.27 (m, 3H), 5.55
(s, 1H), 4.24 (dd, J=11.5, 5.5 Hz, 1H), 4.18-4.01 (m, 1H),
4.00-3.94 (m, 1H), 3.93-3.89 (m, 1H), 3.77 (dd, J=9.3, 1.8 Hz, 1H),
3.61 (t, J=10.7 Hz, 1H), 3.13-2.77 (m, 6H), 2.71 (t, J=7.5 Hz, 2H),
1.99-1.85 (m, 2H), 1.55-1.42 (m, 2H), 1.38-1.18 (m, 6H), 0.87 (t,
J=7.0 Hz, 3H).
Preparation of Compound 85
[0509] A suspension of compound 166 (800 mg, 1.55 mmol) and 10%
Pd/C (300 mg) in EtOH (40 mL) was degassed by bubbling with Argon
using syringe for 10 min then stirred at room temperature under
hydrogen atmosphere (balloon, 1 atm) for 2 h at room temperature.
The reaction mixture was filtered through celite and washed with
MeOH. The filtrate was concentrated in vacuum to afford 85 (700 mg,
93%) as an off-white solid. .sup.1H NMR (400 MHz, CD.sub.3OD):
.delta. 7.52-7.42 (m, 2H), 7.38-7.25 (m, 3H), 6.88 (d, J=8.4 Hz,
2H), 6.63 (d, J=8.4 Hz, 2H), 5.53 (s, 1H), 4.24 (dd, J=10.8, 5.5
Hz, 1H), 4.05-3.84 (m, 3H), 3.76 (dd, J=9.6, 1.8 Hz, 1H), 3.61 (t,
J=10.8 Hz, 1H), 2.93 (dd, J=13.6, 5.0 Hz, 1H), 2.79 (dd, J=13.4,
9.0 Hz, 1H), 2.73-2.60 (m, 4H), 2.42 (t, J=8.0 Hz, 2H), 1.88-1.68
(m, 2H), 1.48-1.36 (m, 2H), 1.33-1.14 (m, 6H), 0.87 (t, J=7.0 Hz,
3H).
Preparation of Intermediate 34
##STR00055##
[0510] Preparation of Compound 168
[0511] A solution of 162 (534 mg, 1.45 mmol) in MeOH (30 mL) and
was charged with saturated NaHCO.sub.3 solution in water (5.0 ml)
at 0.degree. C. and stirred for 10 min. (Boc).sub.2O (350 mg, 1.60
m mmol) was then added and the reaction mixture was stirred for 3 h
at the same temperature,brought to room temperature, and stirred
for another 30 min. The mixture was concentrated, the residue was
dissolved in CH.sub.2Cl.sub.2 (100 mL), and the solution was washed
with water (100 mL) and brine (50 mL). The organic layer was dried
over Na.sub.2SO.sub.4, filtered, concentrated and residue was
purified by column chromatography (silica gel, 9:1
CH.sub.2Cl.sub.2/MeOH, 8:2 CHCl.sub.3/MeOH) to afford compound 168
(435 mg, 64%) as an off-white solid. .sup.1H NMR (400 MHz,
CDCl.sub.3): .delta. 8.18 (d, J=8.7 Hz, 2H), 7.56 (d, J=8.7 Hz,
2H), 4.72 (q, J=5.1 Hz, 1H), 4.41-4.35 (m, 2H), 4.16 (dd, J=10.8,
5.5 Hz, 1H), 4.15-4.04 (m, 1H), 3.93-3.83 (m, 1H), 3.81-3.76 (m,
1H), 3.66-3.53 (m, 4H), 3.40 (t, J=11.0 Hz, 1H), 3.25-3.12 (m, 1H),
3.08-2.96 (m, 1H), 1.49 (s, 9H), 1.32 (d, J=5.1 Hz, 3H).
Preparation of Compound 34
[0512] A suspension of compound 168 (80 mg, 0.21 mmol) and 10% Pd/C
(40 mg) in EtOH (10 mL) was degassed by bubbling with Argon using
syringe for 10 min then stirred under hydrogen atmosphere (balloon,
1 atm) for 2 h at room temperature. The reaction mixture was
filtered through celite and washed with MeOH. The filtrate was
concentrated in vacuum to afford 34 (82 mg, 89%) as an off-white
solid. .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 6.96 (d, J=8.1
Hz, 2H), 6.62 (d, J=8.1 Hz, 2H), 4.69 (q, J=5.1 Hz, 1H), 4.15 (dd,
J=10.8, 5.5 Hz, 1H), 4.13-4.09 (m, 1H), 4.01-3.93 (m, 1H),
3.89-3.78 (m, 1H), 3.75-3.68 (m, 1H), 3.62-3.43 (m, 4H), 3.40 (t,
J=11.3 Hz, 1H), 3.35 (dd, J=13.5, 4.0 Hz, 1H), 3.26 (t, J=7.9 Hz,
1H), 3.23-3.13 (m, 1H), 2.48 (t, J=7.8 Hz, 2H), 1.86-1.76 (m, 2H),
1.43 (s, 9H), 1.33 (d, J=5.1 Hz, 3H).
Preparation of Intermediate 171
##STR00056##
[0514] To a solution of compound 148 (6.40 g, 29.6 mmol) and triol
165 (11.9 g, 44.5 mmol) in MeOH (300 mL) was added AcOH (5.32 mL,
88.8 mmol) and the reaction mixture was stirred at room temperature
for 30 min. After NaCNBH.sub.3 (3.73 g, 59.2 mmol) was added, the
solution was continued to be stirred at room temperature for 16 h.
Additional compound 165 (11.9 g, 44.5 mmol), AcOH (5.32 mL, 88.8
mmol) and NaCNBH.sub.3 (3.73 g, 59.2 mmol) were added the solution
was continued to be stirred at room temperature for 14 h.
Additional compound 165 (7.93 g, 29.6 mmol), AcOH (3.55 mL, 59.2
mmol) and NaCNBH.sub.3 (2.80 g, 44.4 mmol) were added the solution
was continued to be stirred at room temperature for 10 h. After
removal of solvent, the residue was neutralized with saturated
NaHCO.sub.3 and the residue was partitioned between
CH.sub.2Cl.sub.2 (100 mL) and water (100 mL). The aqueous layer was
separated and extracted with CH.sub.2Cl.sub.2 (2.times.100 mL). The
combined organic extracts were dried over Na.sub.2SO.sub.4 and
concentrated under vacuum. Challenging purification encountered by
column chromatography (silica gel, 9:1 CH.sub.2Cl.sub.2/MeOH,
80:18:2 CHCl.sub.3/MeOH/NH.sub.4OH) to afford compound 150 and 169
(20 g, mixture). The mixture was directly used for next step.
Preparation of Compound 170
[0515] A solution of 150 and 169 (20.0 g, mixture) in MeOH (120 mL)
and water (40 mL) was charged with saturated NaHCO.sub.3 (9.99 g,
118.4 mmol) at 0.degree. C. and stirred for 10 min. (Boc).sub.2O
(9.69 g, 44.4 mmol) was added and the reaction mixture was stirred
for 10 min at the same temperature, brought to room temperature,
and stirred for another 2 h. The mixture was concentrated, the
residue was dissolved in CH.sub.2Cl.sub.2 (100 mL), and the
solution was washed with water (100 mL) and brine (50 mL). The
organic layer was dried over Na.sub.2SO.sub.4, filtered,
concentrated and residue was purified by column chromatography
(silica gel, 9:1 CH.sub.2O.sub.2/MeOH, 8:2 CHCl.sub.3/MeOH) to
afford compound 150 (1.50 g) and 170 (4.50 g) as an off-white
solid. ESI-MS m/z. 529
[C.sub.27H.sub.32N.sub.2O.sub.9+H].sup.+.
Preparation of Compound 171
[0516] A suspension of compound 170 (4.20 g, 7.92 mmol) and 10%
Pd/C (500 mg) in EtOH (100 mL) and AcOH (10 mL) was degassed with
Argon for 10 min then stirred under hydrogen atmosphere (balloon, 1
atm) for 16 h at room temperature. The reaction mixture was
filtered through celite and washed with MeOH. The filtrate was
concentrated in vacuum, neutralized with Na.sub.2CO.sub.3 and
residue was purified by column chromatography (silica gel, 9:1
CH.sub.2Cl.sub.2/MeOH, 8:2 CHCl.sub.3/MeOH) to afford compound 172
(2.70 g, 68%) as an off-white solid. .sup.1H NMR (400 MHz,
CD.sub.3OD): .delta. 7.52-7.44 (m, 2H), 7.36-7.29 (m, 3H), 6.89 (d,
J=8.3 Hz, 2H), 6.64 (d, J=8.3 Hz, 2H), 5.54 (s, 1H), 4.23 (dd,
J=11.9, 5.9 Hz, 1H), 4.10-3.97 (m, 1H), 3.97-3.89 (m, 1H),
3.81-3.75 (m, 1H), 3.74-3.69 (m, 1H), 3.60 (t, J=10.9 Hz, 1H), 3.48
(dd, J=14.1, 4.6 Hz, 1H), 3.28-3.22 (m, 3H), 2.41 (t, J=7.5 Hz,
2H), 1.83-1.71 (m, 2H), 1.41 (s, 9H).
Preparation of Intermediate 39
##STR00057##
[0518] The solution of compound 17 (30.0 g, 121 mmol) and 173 (14.2
g, 145 mmol) in anhydrous acetonitrile (300 mL) was degassed for 10
min under Argon atmosphere followed by addition of TEA (67 mL, 484
mmol), 10% (t-Bu).sub.3P in hexanes (49.0 mL, 24.2 mmol) and CuI
(1.15 g, 6.05 mmol) at room temperature. The resulting mixture was
degassed with Argon for another 10 min and Pd(PPh.sub.3).sub.4
(14.0 g g, 12.1 mmol) was added in one portion. After degassing
with argon for 5 min, the resulting mixture was heated at
50.degree. C. for 16 h. The reaction mixture was concentrated in
vacuum and the residue was purified by column chromatography
(silica gel, 2:3 hexanes/EtOAc) to afford compound 174 (15.0 g,
58%) as a brown oil. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 8.14
(d, J=8.8 Hz, 2H), 7.50 (d, J=8.8 Hz, 2H), 3.71 (t, J=6.4 Hz, 2H),
2.50 (t, J=6.8 Hz, 2H), 1.80-1.70 (m, 4H), 1.70-1.65 (m, 1H).
Preparation of Compound 175
[0519] To a solution of compound 174 (15.0 g, 67.9 mmol) in
anhydrous CH.sub.2Cl.sub.2 (50 mL) was added Et.sub.3N (28.0 mL,
203.7 mmol) and DMAP (4.12 g, 33.9 mmol) under argon at 0.degree.
C. After the reaction mixture was stirred for 5 min at same
temperature, TsCl (32.5 g, 170 mmol), was added at 0.degree. C. The
resulting mixture was stirred for additional 4 h at room
temperature. After solvent removed; the residue was partitioned
between CH.sub.2Cl.sub.2 (250 mL) and water (150 mL). The aqueous
layer was separated and extracted with CH.sub.2Cl.sub.2
(2.times.250 mL). The combined organic extracts were washed with
brine, dried over Na.sub.2SO.sub.4 and concentrated under vacuum.
The residue was purified by column chromatography (silica gel,
hexanes/EtOAc) to afford compound 175 (15.0 g, 60%) as a brown oil.
.sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 8.15 (d, J=8.8 Hz, 2H),
7.79 (d, J=8.8 Hz, 2H), 7.50 (d, J=8.8 Hz, 2H), 7.34 (d, J=8.8 Hz,
2H), 4.10 (t, J=6.4 Hz, 2H), 2.44 (t, J=7.0 Hz, 2H), 2.44 (s, 3H),
1.90-1.79 (m, 2H), 1.75-1.61 (m, 2H).
Preparation of Compound176
[0520] To a solution of compound 175 (5.00 g, 12.9 mmol, crude) in
THF (10 mL) was added NHMe.sub.2 in water (30%, 50.0 mL) and then
stirred at rt in seal tube for 3 h. After solvent removed; the
residue was partitioned between CH.sub.2Cl.sub.2 (100 mL) and water
(100 mL). The aqueous layer was separated and extracted with
CH.sub.2Cl.sub.2 (2.times.100 mL). The combined organic extracts
were washed with brine, dried over Na.sub.2SO.sub.4 and
concentrated under vacuum. The crude product was purified by column
chromatography (silica gel) to afford compound 176 (400 mg, 13%) as
a yellow sticky solid. .sup.1H NMR (400 MHz, CDCl.sub.3): .delta.
8.15 (d, J=7.3 Hz, 2H), 7.51 (d, J=7.3 Hz, 2H), 2.48 (t, J=6.6 Hz,
2H), 2.30 (t, J=5.7 Hz, 2H), 2.23 (s, 6H), 1.70-1.61 (m, 4H).
Preparation of Compound 39
[0521] A suspension of compound 176 (400 mg, 1.62 mmol) and 10%
Pd/C (50 mg) in EtOH (50 mL) was degassed by bubbling with Argon
using syringe for 10 min then stirred at room temperature under
hydrogen atmosphere (balloon, 1 atm) for 16 h at room temperature.
The reaction mixture was filtered through celite and washed with
MeOH. The filtrate was concentrated in vacuum to afford 39 (300 mg,
84%) as a brown sticky solid. .sup.1H NMR (400 MHz, CD.sub.3OD):
.delta. 6.91 (d, J=7.5 Hz, 2H), 6.65 (d, J=7.5 Hz, 2H), 2.47 (t,
J=7.0 Hz, 2H), 2.30 (dd, J=8.4, 6.5 Hz, 2H), 2.23 (s, 6H),
1.60-1.52 (m, 2H), 1.51-1.41 (m, 2H), 1.38-1.27 (m, 4H).
31 Preparation of Intermediate 44
##STR00058##
[0522] Preparation of Compound 177
[0523] Solution of compound 175 (6.00 g, 16.0 mmol) in 7 N NH.sub.3
in methanol (150 mL) was heated at 30.degree. C. in seal tube for 5
h. Temperature raised to 40.degree. C. and stirred for 16 h then
again temperature raised to 60.degree. C. and stirred for 4 h.
After solvent removed; the residue was partitioned between
CH.sub.2Cl.sub.2 (100 mL) and water (100 mL). The aqueous layer was
separated and extracted with CH.sub.2Cl.sub.2 (2.times.100 mL). The
combined organic extracts were washed with brine, dried over
Na.sub.2SO.sub.4 and concentrated under vacuum. The crude product
was purified by column chromatography (silica gel, 9:1
CH.sub.2Cl.sub.2/MeOH) to afford compound 177 (1.48 g, 43%) as
yellow oil. .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 8.16 (d,
J=8.4 Hz, 2H), 7.39 (d, J=8.4 Hz, 2H), 3.61 (t, J=5.6 Hz, 2H),
2.08-2.05 (m, 2H), 1.65-1.53 (m, 4H).
Preparation of Compounds 178 and 179
[0524] To a solution of compound 177 (1.38 g, 6.33 mmol) and triol
165 (2.03 g, 7.59 mmol) in MeOH (10 mL) was added AcOH (0.6 mL,
9.49 mmol) and the reaction mixture was stirred at room temperature
for 30 min. After NaCNBH.sub.3 (800 mg, 12.7 mmol) was added, the
solution was continued to be stirred at room temperature for 16 h.
Additional compound 165 (2.55 g, 9.49 mmol), AcOH (0.80 mL, 12.7
mmol) and NaCNBH.sub.3 (1.19 g, 18.9 mmol) were added the solution
was continued to be stirred at room temperature for 16 h.
Additional compound 165 (2.55 g, 9.49 mmol), AcOH (0.80 mL, 12.7
mmol) and NaCNBH.sub.3 (1.19 g, 18.9 mmol) were added the solution
was continued to be stirred at room temperature for 16 h. After
removal of solvent, the residue was neutralized with saturated
NaHCO.sub.3 and the residue was partitioned between
CH.sub.2Cl.sub.2 (10 mL) and water (10 mL). The aqueous layer was
separated and extracted with CH.sub.2Cl.sub.2 (2.times.10 mL). The
combined organic extracts were dried over Na.sub.2SO.sub.4 and
concentrated under vacuum. The residue was purified by column
chromatography (silica gel, 9:1 CH.sub.2Cl.sub.2/MeOH, 80:18:2
CHCl.sub.3/MeOH/NH.sub.4OH) to afford compound 179 (2.28 g, 51%) as
an off-white solid. .sup.1H NMR (300 MHz, CD.sub.3OD): .delta. 8.14
(d, J=9.0 Hz, 2H), 7.54 (d, J=9.0 Hz, 2H), 7.47-7.44 (m, 4H),
7.34-7.30 (m, 6H), 5.48 (s, 2H), 4.24-4.19 (m, 2H), 3.99-3.94 (m,
4H), 3.86-3.84 (m, 2H), 3.73-3.69 (m, 2H), 3.57 (t, J=10.8 Hz, 4H),
3.35-3.25 (m, 4H), 2.33 (d, J=6.9 Hz, 2H) 1.61-1.51 (m, 4H).
[0525] A mixture of 178/179 (900 mg) was isolated as well and was
directly used for next step (SG-GHC-G-106).
Preparation of Compound 44
[0526] A suspension of compound 179 (2.26 g, 3.11 mmol) and 10%
Pd/C (100 mg) in a mixture of EtOH (50 mL) and AcOH (10 mL) was
degassed with Argon for 10 min then stirred under hydrogen
atmosphere (balloon, 1 atm) for 16 h at room temperature. The
reaction mixture was filtered through celite and washed with MeOH.
The filtrate was concentrated in vacuum to afford 44 (1.90 g, 80%)
as a brown solid. .sup.1H NMR (400 MHz, CD.sub.3OD): .delta.
7.46-7.44 (m, 4H), 7.33-7.31 (m, 6H), 6.89 (d, J=8.4 Hz, 2H), 6.65
(d, J=8.4 Hz, 2H), 5.51 (s, 2H), 4.26-4.14 (m, 2H), 3.93-3.90 (m,
2H), 3.76-3.73 (m, 4H), 3.63-3.58 (m, 4H), 3.35-3.25 (m, 2H),
3.10-3.00 (m, 2H), 2.41 (t, J=7.2 Hz, 2H), 1.47-1.45 (m, 4H),
1.16-1.12 (m, 4H).
Preparation of Intermediate 49
##STR00059##
[0527] Preparation of Compound 180
[0528] A solution of 178 (900 mg, mixture, apox, 2.0 mmol) in a
mixture of MeOH (20 mL) and water (10 mL) a and was charged with
NaHCO.sub.3 (672 mg, 4.0 mmol) at 0.degree. C. and stirred for 10
min. (Boc).sub.2O (524 mg, 2.40 mmol) was added and the reaction
mixture was stirred for 1 h at the same temperature, brought to
room temperature, and stirred for another 4 h. The mixture was
concentrated, the residue was dissolved in CH.sub.2Cl.sub.2 (100
mL), and the solution was washed with water (100 mL) and brine (50
mL). The organic layer was dried over Na.sub.2SO.sub.4, filtered,
concentrated and residue was purified by column chromatography
(silica gel, 9:1 CH.sub.2Cl.sub.2/MeOH, 8:2 CHCl.sub.3/MeOH) to
afford compound 180 (780 mg, 64%) as an off-white solid. .sup.1H
NMR (300 MHz, CD.sub.3OD): .delta. 8.16 (d, J=9.0 Hz, 2H), 7.55 (d,
J=9.0 Hz, 2H), 7.50-7.47 (m, 2H), 7.34-7.30 (m, 3H), 5.53 (s, 1H),
4.25-4.20 (m, 1H), 4.10 (br s, 1H), 3.94-3.91 (m, 1H), 3.80-3.48
(m, 4H), 3.35-3.25 (m, 3H), 2.46 (t, J=6.9 Hz, 2H), 1.70-1.49 (m,
4H), 1.43 (s, 9H).
Preparation of Compound 49
[0529] A suspension of compound 180 (780 mg, 1.36 mmol) and 10%
Pd/C (50 mg) in a mixture of EtOH (10 mL) and AcOH (2.0 mL) was
degassed by bubbling with Argon using syringe for 10 min then
stirred at rt under hydrogen atmosphere (balloon, 1 atm) for 4 h at
room temperature. The reaction mixture was neutralized with
Na.sub.2CO.sub.3, filtered through celite and washed with MeOH. The
filtrate was concentrated in vacuum to afford 49 (625 g, 84%) as a
white solid. .sup.1H NMR (300 MHz, CD.sub.3OD): .delta. 7.50-7.46
(m, 2H), 7.32-7.30 (m, 3H), 6.90 (d, J=8.4 Hz, 2H), 6.66 (d, J=8.4
Hz, 2H), 5.53 (s, 1H), 4.25-4.20 (m, 1H), 4.04 (br s, 1H),
3.94-3.89 (m, 1H), 3.77-3.43 (m, 4H), 3.35-3.25 (m, 3H), 2.45 (t,
J=7.5 Hz, 2H), 1.52-1.47 (m, 4H), 1.42 (s, 9H), 1.27-1.24 (m,
4H).
Preparation of Intermediate 54
##STR00060##
[0530] Preparation of Compound 182
[0531] To a solution of compound 181 (1.60 g, 16.00 mmol) in
anhydrous THF (40 mL) was added 9-BBN (0.5 M in THF, 80 mL, 40.0
mmol) under argon. After the reaction mixture was stirred for 2 h
at room temperature, compound 172 (3.17 g, 12.8 mmol),
Pd(PPh.sub.3).sub.2Cl.sub.2 (561 mg, 0.80 mmol), and 1 N aq NaOH
(24 mL) were added at room temperature. The resulting mixture was
stirred for additional 1 h. After solvent removed; the residue was
partitioned between EtOAc (100 mL) and water (100 mL). The aqueous
layer was separated and extracted with EtOAc (2.times.100 mL). The
combined organic extracts were washed with brine, dried over
Na.sub.2SO.sub.4 and concentrated under vacuum. The crude product
was purified by column chromatography (silica gel, 4:1
hexanes/EtOAc) to afford compound 182 (1.20 g, 34%) as a brown
solid. .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 8.13 (d, J=9.0
Hz, 2H), 7.31 (d, J=9.0 Hz, 2H), 3.64 (t, J=6.7 Hz, 2H), 2.71 (t,
J=7.8 Hz, 2H), 1.73-1.46 (m, 4H), 1.43-1.31 (m, 4H).
Preparation of Compound 183
[0532] To a solution of compound 182 (1.20 g, 5.38 mmol) in
anhydrous CH.sub.2Cl.sub.2 (20 mL) was added Et.sub.3N (7.32 mL,
53.8 mmol) under argon at 0.degree. C. After the reaction mixture
was stirred for 5 min at same temperature, Mesyl chloride (0.62 mL,
8.07 mmol), was added at 0.degree. C. The resulting mixture was
stirred for additional 2 h at rt. After solvent removed; the
residue was partitioned between CH.sub.3Cl.sub.2 (50 mL) and water
(50 mL). The aqueous layer was separated and extracted with
CH.sub.2Cl.sub.2 (2.times.50 mL). The combined organic extracts
were washed with brine, dried over Na.sub.2SO.sub.4 and
concentrated under vacuum. The crude product183 (3.00 g, crude) was
directly used for the next step.
Preparation of Compound 184
[0533] Solution of compound 183 (3.00 g, 5.38 mmol, crude) in 7 N
NH.sub.3 in methanol (30.0 mL) was heated at 60.degree. C. in seal
tube for 2 h. After solvent removed; the residue was partitioned
between CH.sub.2Cl.sub.2 (100 mL) and water (100 mL). The aqueous
layer was separated and extracted with CH.sub.2Cl.sub.2
(2.times.100 mL). The combined organic extracts were washed with
brine, dried over Na.sub.2SO.sub.4 and concentrated under vacuum.
The crude product was purified by column chromatography (silica
gel) to afford compound 184 (390 mg, 33%, over two steps) as a
yellow oil. .sup.1H NMR (400 MHz, CD.sub.3OD): .delta. 8.14 (d,
J=9.0 Hz, 2H), 7.42 (d, J=9.0 Hz, 2H), 2.75 (t, J=7.8 Hz, 2H), 2.67
(t, J=7.3 Hz, 2H), 1.72-1.63 (m, 2H), 1.53-1.46 (m, 2H), 1.42-1.35
(m, 4H).
Preparation of Compound 185
[0534] To a solution of compound 184 (620 mg, 2.79 mmol) and triol
165 (938 mg, 3.49 mmol) in MeOH (30 mL) was added AcOH (1.16 mL,
27.8 mmol) and the reaction mixture was stirred at room temperature
for 10 min. After NaCNBH.sub.3 (526 mg, 8.37 mmol) was added, the
solution was continued to be stirred at room temperature for 16 h.
Additional compound 165 (0.3 equiv), AcOH (10 equiv) and
NaCNBH.sub.3 (1.0 equiv) were added over 16 h. Then hexanal 163
(0.96 mL, 8.37 mmol), AcOH (1.00 mL) and NaCNBH.sub.3 (526 mg, 8.37
mmol) were added. The solution was further stirred at room
temperature for 2 h. After removal of solvent, the residue was
neutralized with saturated NaHCO.sub.3 and the residue was
partitioned between EtOAc (100 mL) and water (100 mL). The aqueous
layer was separated and extracted with CH.sub.2Cl.sub.2
(2.times.100 mL). The combined organic extracts were dried over
Na.sub.2SO.sub.4 and concentrated under vacuum. The residue was
purified by column chromatography (silica gel, 9:1
CH.sub.2Cl.sub.2/MeOH, 80:18:2 CHCl.sub.3/MeOH/NH.sub.4OH) to
afford compound 185 (950 g, 61%) as an off-white oil. .sup.1H NMR
(400 MHz, CDCl.sub.3): .delta. 8.02 (d, J=8.7 Hz, 2H), 7.48-7.42
(m, 3H), 7.37-7.34 (m, 2H), 7.31 (d, J=8.7 Hz, 2H), 5.54 (s, 1H),
4.46-4.40 (m, 1H), 4.30 (dd, J=11.6, 6.6 Hz, 1H), 4.03 (t, J=4.0
Hz, 1H), 3.97 (dd, J=10.5, 5.4 Hz, 1H), 3.88 (dd, J=9.4, 4.0 Hz,
1H), 3.65 (t, J=10.4 Hz, 1H), 3.11-3.00 (m, 4H), 2.69 (t, J=7.8 Hz,
2H), 2.00 (s, 1H), 1.70-1.55 (m, 6H), 1.37-1.30 (m, 4H), 1.29-1.20
(m, 8H), 0.87 (t, J=7.1 Hz, 3H).
Preparation of Compound 54
[0535] A suspension of compound 185 (950 g, 1.70 mmol) and 10% Pd/C
(300 mg) in EtOH (100 mL) was degassed with Argon for 10 min then
stirred under hydrogen atmosphere (balloon, 1 atm) for 3 h at room
temperature. The reaction mixture was filtered through celite and
washed with MeOH. The filtrate was concentrated in vacuum to afford
54 (790 mg, 88%) as yellow oil. .sup.1H NMR (400 MHz, CD.sub.3OD):
.delta. 7.51-7.44 (m, 2H), 7.35-7.29 (m, 3H), 6.90 (d, J=8.5 Hz,
2H), 6.65 (d, J=8.5 Hz, 2H), 5.54 (s, 1H), 4.24 (dd, J=10.8, 5.4
Hz, 1H), 4.08-4.02 (m, 1H), 4.00-3.92 (m, 1H), 3.91 (dd, J=5.6, 1.8
Hz, 1H), 3.78 (dd, J=9.6, 1.8 Hz, 1H), 3.61 (t, J=10.9 Hz, 1H),
3.01 (dd, J=13.7, 5.4 Hz, 1H), 2.91 (dd, J=12.1, 8.1 Hz, 1H),
2.82-2.71 (m, 4H), 2.45 (t, J=7.5 Hz, 2H), 1.59-1.42 (m, 6H),
1.37-1.13 (m, 10H), 0.89 (t, J=7.1 Hz, 3H).
[0536] Several assays may be used to characterize the compounds of
the present invention. Representative assays are discussed
below.
[0537] In Vitro Measure of Sodium Channel Blocking Activity and
Reversibility
[0538] One assay used to assess mechanism of action and/or potency
of the compounds of the present invention involves the
determination of lumenal drug inhibition of airway epithelial
sodium currents measured under short circuit current (I.sub.SC)
using airway epithelial monolayers mounted in Using chambers. Cells
obtained from freshly excised human, dog, sheep or rodent airways
are seeded onto porous 0.4 micron Snapwell.TM. Inserts (CoStar),
cultured at air-liquid interface (ALI) conditions in hormonally
defined media, and assayed for sodium transport activity (I.sub.SC)
while bathed in Krebs Bicarbonate Ringer (KBR) in Using chambers.
All test drug additions are to the lumenal bath with half-log dose
addition protocols (from 1.times.10.sup.-11 M to 3.times.10.sup.-5
M), and the cumulative change in I.sub.SC (inhibition) recorded.
All drugs are prepared in dimethyl sulfoxide as stock solutions at
a concentration of 1.times.10.sup.-2 M and stored at -20.degree. C.
Eight preparations are typically run in parallel; two preparations
per run incorporate amiloride and/or benzamil as positive controls.
After the maximal concentration (5.times.10.sup.-5 M) is
administered, the lumenal bath is exchanged three times with fresh
drug-free KBR solution, and the resultant I.sub.SC measured after
each wash for approximately 5 minutes in duration. Reversibility is
defined as the percent return to the baseline value for sodium
current after the third wash. All data from the voltage clamps are
collected via a computer interface and analyzed off-line.
[0539] Dose-effect relationships for all compounds are considered
and analyzed by the Prism 3.0 program. IC.sub.50 values, maximal
effective concentrations, and reversibility are calculated and
compared to amiloride and benzamil as positive controls. The
potency of the sodium channel blocking activity of representative
compounds relative to amiloride in freshly excised cell from canine
airways is shown in Table 1.
TABLE-US-00003 TABLE 1 Inhibition of Short-Circuit Current by
Compound (Ia) in canine bronchial epithelial cells (IC.sub.50 nM)
Potency of Sodium Compound Channel Blockade Number IC.sub.50 nM
Amiloride 773 23 20.7 38 25.4 28 7.4 33 21.8 16 79.6 103 17.9 99
7.6 94 21.2 80 19.4 135 5.2 131 6.0 123 2.3 127 8.6 139 73.7 43
50.1 53 15.5 58 10.6 48 47
Assay 2. Mucociliary Clearance (MCC) Studies in Sheep
[0540] The animal model that has been used most often to measure
changes in MCC is the sheep model. The effect of compounds for
enhancing mucociliary clearance (MCC) can be measured using an in
vivo model described by Sabater et al., Journal of Applied
Physiology, 1999, pp. 2191-2196, incorporated herein by
reference.
[0541] In these studies, adult sheep were restrained and nasally
intubated with an endotracheal tube. Aerosolized test articles were
administered over 10-15 minutes to sheep. Radiolabeled
.sup.99mTc-sulfur colloid (TSC, 3.1 mg/mL; containing approximately
20 mCi) was then administered at a specified time four or eight
hours after test article. The radiolabeled aerosol was administered
through the endotracheal tube for about 5 minutes. The sheep were
then extubated, and total radioactive counts in the lung were
measured every 5 minutes for a 1-hour observation period. The rate
of radiolabel clearance from the lung is representative of the MCC
rate in the animal. The advantage of this system is that it closely
simulates the human lung environment. The model also allows for the
collection of simultaneous PK/PD information through plasma and
urine sampling over the test period. There are also several
techniques to measure the drug concentrations on the airway surface
during the MCC measurements. These include the collection of
exhaled breath condensates or a filter paper method to obtain ASL
via bronchoscopy.
[0542] The ovine model described above was used to evaluate the in
vivo effects (efficacy/durability) of aerosol-delivered test agent
on MCC. Treatments consisting of either 4 mL of test agent or test
agent in combination with HS were tested. To determine if combining
HS with test agent enhanced MCC, HS was administered immediately
following test agent administration. Test solutions were
aerosolized using a Raindrop nebulizer at a flowrate of eight
liters per minute and connected to a dosimetry system consisting of
a solenoid valve and a source of compressed air (20 psi). The
deposited dose of drug in sheep lungs after an aerosol
administration using the Raindrop nebulizer is estimated to be
8-15% of the dose. Using a Raindrop nebulizer, radiolabeled TSC was
administered over approximately 3 minutes either 4 or 8 hours after
drug treatment to evaluate efficacy/durability. Radioactive counts
were measured in a central region in the right lung at 5 min
intervals for one hour with a gamma camera. Three methods of
analysis were utilized, 1) initial rate of clearance (slope) over
the first 30 min fitted using linear regression 2) area under the
curve for % clearance over time over one hour, and 3) the maximum
clearance obtained in one hour.
[0543] The effect of Compound 33 at 0.24 nmol/kg (3 .mu.M) was
tested and compared to vehicle (4 mL sterile H.sub.2O) on sheep MCC
four hour post-dosing (FIG. 1). The analyses of effects are shown
in Table A. Compound 33 enhanced MCC compared to vehicle
control.
TABLE-US-00004 TABLE A MCC in Sheep at 4 h Post-dose of Compound 33
or Vehicle Initial Slope Maximum Compound 33 Dose (4.0-4.5 h) AUC
(% Cl-h) Clearance 0.24 nmol/kg (3 .mu.M) 37.5* (4) 17.4* (4) 30.0*
(4) Vehicle (H.sub.2O) 4 mL 17.2 .+-. 6.8 (8) 7.3 .+-. 1.5 (8) 12.2
.+-. 2.9 (8)
[0544] Tables B and C Along With FIGS. 2 and 3 Demonstrate That
Other Compounds of This Invention Similarly Enhance MCC Compared to
Vehicle (see e.g., Compounds 123 and 48)
TABLE-US-00005 TABLE B MCC in Sheep at 4 h Post-dose of Compound
123 or Vehicle Compound 123 Initial Slope Maximum Dose (4.0-4.5 h)
AUC (% Cl-h) Clearance 0.24 nmol/kg (3 .mu.M) 29.2* (2) 14.4* (2)
22.8* (2) Vehicle (H.sub.2O) 4 mL 17.2 .+-. 6.8 (8) 7.3 .+-. 1.5
(8) 12.2 .+-. 2.9 (8)
TABLE-US-00006 TABLE C MCC in Sheep at 4 h Post-dose of Compound 48
or Vehicle Initial Slope Maximum Compound 48 Dose (4.0-4.5 h) AUC
(% Cl-h) Clearance 0.24 nmol/kg (3 .mu.M) 29.8* (2) 15.4* (2) 26.7*
(2) Vehicle (H.sub.2O) 4 mL 17.2 .+-. 6.8 (8) 7.3 .+-. 1.5 (8) 12.2
.+-. 2.9 (8)
[0545] To determine whether compounds of this invention have
enhance duration of action, they were tested at 8 hours post dose.
Tables D and E along with FIGS. 4 and 5 clearly show enhanced
duration action of MCC vs. vehicle for Compounds 33 and 152.
TABLE-US-00007 TABLE D MCC in Sheep at 8 h Post-dose of Compound 33
or Vehicle Initial Slope Maximum Compound 33 Dose (8.0-8.5 h) AUC
(% Cl-h) Clearance 0.24 nmol/kg (3 .mu.M) 25.8* (4) 11.7* (4) 21.4*
(4) Vehicle (H.sub.2O) 4 mL 17.2 .+-. 6.8 (8) 7.3 .+-. 1.5 (8) 12.2
.+-. 2.9 (8)
TABLE-US-00008 TABLE E MCC in Sheep at 8 h Post-dose of Compound
152 or Vehicle Compound 152 Initial Slope Maximum Dose (8.0-8.5 h)
AUC (% Cl-h) Clearance 0.24 nmol/kg (3 .mu.M) 37.5* (4) 17.4* (4)
30.0* (4) Vehicle (H.sub.2O) 4 mL 17.2 .+-. 6.8 (8) 7.3 .+-. 1.5
(8) 12.2 .+-. 2.9 (8)
[0546] To determine whether HS increases the MCC effect of Compound
33, 7% HS was dosed immediately following 0.24 nmol/kg of Compound
33 and MCC was assessed eight hours after the combined dosing (FIG.
6). HS increased the effect of Compound 33 on MCC as shown in FIG.
6.
Assay 3. Airway Surface Liquid Drug (ASL) Clearance and Metabolism
by Human Airway Epithelium
[0547] The disappearance of 33 from the apical surface and airway
epithelial metabolism were assessed in human bronchial epithelial
(HBE) cells (Table 3). In these experiments 25 .mu.L of a 25 .mu.M
solution of ENaC blocker was added to the apical surface of HBE
cells grown at an air/liquid interface, and the drug and metabolite
concentration in the apical and basolateral compartment was
measured over 2 h by UPLC.
TABLE-US-00009 TABLE G Apical Disappearance and Metabolism of
Compound 33 % of Initial % of Initial % on Drug Mass on % of Apical
Apical Mass Basolateral Apical Side Mass as on Side as (Parent and
Metabolites Basolateral Metabolites Compound metabolite, 2 h) (2 h)
Side (2 h) (2 h) 33 44.8 .+-. 18% 4% 1.1 .+-. 0.45% 32% Values
represent the mean .+-. SD
COMPARATIVE EXAMPLES
[0548] The present compounds of formula (I) are more potent and/or
absorbed less rapidly from mucosal surfaces, especially airway
surfaces, compared to known sodium channel blockers, such as
amiloride and third generation compounds such as Comparative
Example 1 described below. Therefore, the compounds of formula (I)
have a longer half-life on mucosal surfaces compared to these know
compounds as evidenced by the data shown in Table G. The
disappearance of Compound 33 from the apical surface and airway
epithelial metabolism were assessed in HBE and compared to
Comparative Example 1 (Table H). In these experiments 25 .mu.L of a
25 .mu.M solution of ENaC blocker was added to the apical surface
of HBE cells grown at an air/liquid interface, and the drug
concentration in the apical and basolateral compartment was
measured over 2 h by UPLC. After 2 h incubation of the compounds of
this present invention on the apical surface (37.degree. C.),
Compound 33 was mostly unmetabolized on the apical side.
Conversely, most of Comparative Example 1 was eliminated from the
apical side with 83% metabolized to the less active carboxylic
acid,
(S)-2-amino-3-(4-(4-(3-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino-
)butyl) phenoxy)propanoic acid, structure below.
##STR00061##
TABLE-US-00010 TABLE H Apical Disappearance and Metabolism of
Compound 33 vs. Comparative Example 1 in HBE % of Initial % of
Initial % on Drug Mass on % of Apical Apical Mass Basolateral
Apical Side Mass as on Side as (Parent and Metabolites Basolateral
Metabolites Compound metabolite, 2 h) (2 h) Side (2 h) (2 h) 33
44.8 .+-. 18% 4% 1.1 .+-. 0.45% 32% Comparative 41.6 .+-. 7.6% 83.0
.+-. 3.5% 8.3 .+-. 0.2 94.7 .+-. 1.0% Example 1 (8% Parent) (1%
Parent) Values represent the mean .+-. SD
[0549] Comparative Example 1 is claimed, described or within the
disclosures of WO 2003/070182 (U.S. Pat. Nos. 6,858,615; 7,186,833;
7,189,719; 7,192,960; and 7,332,496), as sodium channel blockers
having useful medicinal properties and can be prepared by methods
described therein and others known in the art.
Comparative Example 1
##STR00062##
[0551] The compound of Comparative Example 1 can be seen on page 15
of US 2005/0080093 and as Compound 2 on page 90 of WO 2008/031048,
and as Compound 2 on pages 42-43 of WO 2008/031028. In order to
have useful activity in treating Cystic Fibrosis and C.O.P.D a
compound must have properties that will cause enhancement of
mucociliary clearance (MCC) at doses that do not elevate plasma
potassium which will eventually lead to hyperkalemia, a serious and
dangerous condition, upon multiple dosing. It must therefore be
avoided in this class of compounds, which are known to elevate
plasma potassium if they are significantly excreted by the kidney.
In order to evaluate this potential, it is beneficial to have MCC
activity in vivo and not cause elevation of plasma potassium at the
useful dose. One model to assess this is the sheep MCC model
described below.
[0552] As can be seen from the Table I and FIG. 7 the ED.sub.50 for
Comparative Example 1 in the sheep MCC model is approximately 240
nmol/kg (3 mM) using three different measures (slope, AUC and
Maximum Clearance). At this dose, which would be a clinically
active dose, Comparative Example 1 causes a rise in plasma
potassium (FIG. 8) which on repeat dosing will lead to
hyperkalemia. Thus, Comparative Example I is unacceptable for human
use while Compound (Ia) produces a safe and effective MCC with a
benefit to risk ratio greater than 1000 in this model.
TABLE-US-00011 TABLE I MCC in Sheep at 4 h Post-dose of vehicle,
Comparative Example 1 or Compound 33 Initial Slope Maximum Dose
(4.0-4.5 h) AUC (% Cl .times. h) Clearance Comparative 32.2 .+-.
7.3* (6) 14.1 .+-. 2.2* (6) 22.9 .+-. 2.1* (6) Example 1 240
nmol/kg (3 mM) Comparative 14.5 .+-. 1.3 (3) 6.9 .+-. 1.0 (3) 14.6
.+-. 0.9 (3) Example 1 24 nmol/kg (300 .mu.M) Compound 33 37.5* (4)
17.4* (4) 30.0* (4) 0.240 nmol/kg (30 .mu.M) Vehicle H.sub.2O 17.2
.+-. 6.8 (8) 7.3 .+-. 1.5 (8) 12.2 .+-. 2.9 (8) (4 mL)
[0553] FIG. 1 graphs the percentage mucus clearance over time by
Compound 33 and Comparative Example 1, as described in the MCC
model above. An even greater percentage mucus clearance was
provided by Compound 33 at a 1000-fold lower dose than seen with
Comparative Example 1. Thus, Compound 33 provided a maximal effect
in a clinically relevant dose range free of potassium
elevations.
[0554] FIG. 10 illustrates the significant increase in plasma
potassium levels at an efficacious dose seen in the plasma of the
sheep receiving Comparative Example 1 in the MCC study. Compound 33
is more than 1000 times more potent in sheep MCC than Comparative
Example 1 with no elevation of Plasma K at doses as high as 24
nmol/kg (1000 times the ED50 dose), whereas Comparative Example 1
has elevations of plasma K at the approximate ED50 dose of 3 mM
(FIGS. 7 and 8). This again demonstrates the unique and unexpected
potency and safety advantage of Compound 33 as seen in Table J with
a Therapeutic Index of more than 1,000 times greater renal safety
than Comparative Example 1.
TABLE-US-00012 TABLE J Therapeutic Ratio (Benefit/Risk) Top Dose in
MCC Sheep with no Highest Elevation of Plasma Therapeutic
Submaximal Dose Potassium Ratio Comparative 240 nmol/kg (3 mM) 24
nmol/kg 0.1 Example 1 (300 .mu.M) 33 <0.24 nmol/kg (3 .mu.M) 24
nmol/kg >100 (300 .mu.M) Ratio >1,000 1 >1,000
[0555] Other compounds of this invention have similar safety and
efficacy advantages over know compounds as exemplified in FIGS. 11,
12, 13 and 14.
* * * * *