U.S. patent application number 12/049946 was filed with the patent office on 2008-07-24 for capped pyrazinoylguanidine sodium channel blockers.
This patent application is currently assigned to PARION SCIENCES, Inc.. Invention is credited to Michael R. JOHNSON, Bruce F. Molino, Bruce Sargent, Jianzhong Zhang.
Application Number | 20080176863 12/049946 |
Document ID | / |
Family ID | 34215937 |
Filed Date | 2008-07-24 |
United States Patent
Application |
20080176863 |
Kind Code |
A1 |
JOHNSON; Michael R. ; et
al. |
July 24, 2008 |
CAPPED PYRAZINOYLGUANIDINE SODIUM CHANNEL BLOCKERS
Abstract
The present invention relates to sodium channel blockers. The
present invention also includes a variety of methods of treatment
using these inventive sodium channel blockers.
Inventors: |
JOHNSON; Michael R.; (Chapel
Hill, NC) ; Molino; Bruce F.; (Slingerlands, NY)
; Zhang; Jianzhong; (Rensselaer, NY) ; Sargent;
Bruce; (Delmar, NY) |
Correspondence
Address: |
OBLON, SPIVAK, MCCLELLAND MAIER & NEUSTADT, P.C.
1940 DUKE STREET
ALEXANDRIA
VA
22314
US
|
Assignee: |
PARION SCIENCES, Inc.
Durham
NC
|
Family ID: |
34215937 |
Appl. No.: |
12/049946 |
Filed: |
March 17, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11138280 |
May 27, 2005 |
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12049946 |
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10920410 |
Aug 18, 2004 |
7064129 |
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11138280 |
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60495725 |
Aug 18, 2003 |
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Current U.S.
Class: |
514/255.05 ;
514/255.06; 544/405; 544/406 |
Current CPC
Class: |
A61P 17/00 20180101;
C07D 403/12 20130101; A61P 27/02 20180101; A61P 1/00 20180101; A61P
43/00 20180101; C07D 417/12 20130101; A61P 7/10 20180101; C07D
473/00 20130101; A61K 31/4965 20130101; C07D 413/12 20130101; A61P
11/12 20180101; A61P 27/16 20180101; C07D 473/16 20130101; A61P
1/10 20180101; A61P 9/12 20180101; A61P 1/02 20180101; A61P 15/00
20180101; A61P 11/06 20180101; A61K 31/497 20130101; C07D 241/26
20130101; A61P 11/00 20180101; A61P 17/16 20180101; C07D 241/32
20130101 |
Class at
Publication: |
514/255.05 ;
514/255.06; 544/405; 544/406 |
International
Class: |
A61K 31/4965 20060101
A61K031/4965; C07D 405/02 20060101 C07D405/02; C07D 241/02 20060101
C07D241/02 |
Claims
1. A compound represented by formula (I): ##STR00081## wherein X is
hydrogen, halogen, trifluoromethyl, lower alkyl, unsubstituted or
substituted phenyl, lower alkyl-thio, phenyl-lower alkyl-thio,
lower alkyl-sulfonyl, or phenyl-lower alkyl-sulfonyl; Y is
hydrogen, hydroxyl, mercapto, lower alkoxy, lower alkyl-thio,
halogen, lower alkyl, unsubstituted or substituted mononuclear
aryl, or --N(R.sup.2).sub.2; R.sup.1 is hydrogen or lower alkyl;
each R.sup.2 is, independently, --R.sup.7,
--(CH.sub.2).sub.m--OR.sup.8, --(CH.sub.2).sub.m--NR.sup.7R.sup.10,
--(CH.sub.2).sub.n(CHOR.sup.8)(CHOR.sup.8).sub.n--CH.sub.2OR.sup.8,
--(CH.sub.2CH.sub.2O).sub.m--R.sup.8,
--(CH.sub.2CH.sub.2O).sub.m--CH.sub.2CH.sub.2NR.sup.7R.sup.10,
--(CH.sub.2).sub.n--C(.dbd.O)NR.sup.7R.sup.10,
--(CH.sub.2).sub.n-Z.sub.g-R.sup.7,
--(CH.sub.2).sub.m--NR.sup.10--CH.sub.2(CHOR.sup.8)(CHOR.sup.8).sub.n--CH-
.sub.2OR.sup.8, --(CH.sub.2).sub.n, --CO.sub.2R.sup.7, or
##STR00082## R.sup.3 and R.sup.4 are each, independently, hydrogen,
a group represented by formula (A), lower alkyl, hydroxy lower
alkyl, phenyl, phenyl-lower alkyl, (halophenyl)-lower alkyl,
lower-(alkylphenylalkyl), lower (alkoxyphenyl)-lower alkyl,
naphthyl-lower alkyl, or pyridyl-lower alkyl, with the proviso that
at least one of R.sup.3 and R.sup.4 is a group represented by
formula (A): ##STR00083## wherein each R.sup.L is, independently,
--R.sup.7, --(CH.sub.2).sub.n--OR.sup.8,
--O--(CH.sub.2).sub.m--OR.sup.8, --(CH.sub.2), --NR.sup.7R.sup.10,
--O--(CH.sub.2).sub.m--NR.sup.7R.sup.10,
--(CH.sub.2).sub.n(CHOR.sup.8)(CHOR.sup.8).sub.n--CH.sub.2OR.sup.8,
--O--(CH.sub.2).sub.m(CHOR.sup.8)(CHOR.sup.8).sub.n--CH.sub.2OR.sup.8,
--(CH.sub.2CH.sub.2O).sub.m--R.sup.8,
--O--(CH.sub.2CH.sub.2O).sub.m--R.sup.8,
--(CH.sub.2CH.sub.2O).sub.m--CH.sub.2CH.sub.2NR.sup.7R.sup.10,
--O--(CH.sub.2CH.sub.2O).sub.m--CH.sub.2CH.sub.2NR.sup.7R.sup.10,
--(CH.sub.2).sub.n--C(.dbd.O)NR.sup.7R.sup.10,
--O--(CH.sub.2).sub.n--C(.dbd.O)NR.sup.7R.sup.10,
--(CH.sub.2).sub.n-(Z).sub.g-R.sup.7,
--O--(CH.sub.2).sub.m-(Z).sub.g-R.sup.7,
--(CH.sub.2).sub.n--NR.sup.10--CH.sub.2(CHOR.sup.8)(CHOR.sup.8).sub.n--CH-
.sub.2OR.sup.1,
--O--(CH.sub.2).sub.m--NR.sup.10--CH.sub.2(CHOR.sup.8)(CHOR.sup.8).sub.n--
-CH.sub.2OR.sup.8, --(CH.sub.2).sub.n--CO.sub.2R.sup.7,
--O--(CH.sub.2).sub.n, --CO.sub.2R.sup.7--OSO.sub.3H,
--O-glucuronide, --O-glucose, ##STR00084## each o is,
independently, an integer from 0 to 10; each p is an integer from 0
to 10; with the proviso that the sum of o and p in each contiguous
chain is from 1 to 10; each x is, independently, O, NR.sup.10,
C(.dbd.O), CHOH, C(.dbd.N--R.sup.10), CHNR.sup.7R.sup.10, or
represents a single bond; wherein each R.sup.5 is, independently,
Link-(CH.sub.2).sub.n-CAP,
Link-(CH.sub.2).sub.n(CHOR.sup.8)(CHOR.sup.8).sub.n-CAP,
Link-(CH.sub.2CH.sub.2O).sub.n--CH.sub.2--CAP,
Link-(CH.sub.2CH.sub.2O).sub.m--CH.sub.2CH.sub.2-CAP,
Link-(CH.sub.2).sub.n-(Z).sub.g-CAP,
Link-(CH.sub.2).sub.n(Z).sub.g-(CH.sub.2).sub.m-CAP,
Link-(CH.sub.2).sub.n--NR.sup.13--CH.sub.2(CHOR.sup.8)(CHOR.sup.8).sub.n--
-CAP,
Link-(CH.sub.2).sub.n--(CHOR.sup.8).sub.mCH.sub.2--NR.sup.13-(Z).sub-
.g-CAP,
Link-(CH.sub.2).sub.nNR.sup.13--(CH.sub.2).sub.m(CHOR.sup.8).sub.n-
CH.sub.2NR.sup.13-(Z).sub.g-CAP,
Link-(CH.sub.2).sub.m-(Z).sub.g-(CH.sub.2).sub.m-CAP,
Link-NH--C(.dbd.O)--NH--(CH.sub.2).sub.m--CAP,
Link-(CH.sub.2).sub.m--C(.dbd.O)NR.sup.13--(CH.sub.2).sub.m--C(.dbd.O)NR.-
sup.10R.sup.10, Link-(CH.sub.2).sub.m,
--C(.dbd.O)NR.sup.13--(CH.sub.2).sub.m--CAP,
Link-(CH.sub.2).sub.m--C(.dbd.O)NR.sup.11R.sup.11,
Link-(CH.sub.2).sub.m--C(.dbd.O)NR.sup.12R.sup.12,
Link-(CH.sub.2).sub.n-(Z).sub.g-(CH.sub.2).sub.m-(Z).sub.g-CAP,
Link-Z.sub.g-(CH.sub.2).sub.m-Het-(CH.sub.2).sub.m--CAP; each Link
is, independently, --O--, --(CH.sub.2).sub.m--,
--O(CH.sub.2).sub.m--, --NR.sup.13--C(.dbd.O)--NR.sup.3,
--NR.sup.13--C(.dbd.O)--(CH.sub.2).sub.m--,
--C(.dbd.O)NR.sup.13--(CH.sub.2).sub.m,
--(CH.sub.2).sub.n-Z.sub.g-(CH.sub.2).sub.n, --S--, --SO--,
--SO.sub.2--, --SO.sub.2NR.sup.7--, --SO.sub.2NR.sup.10--, or
-Het-; each CAP is, independently, thiazolidinedione,
oxazolidinedione, heteroaryl-C(.dbd.O)N R.sup.13R.sup.13,
heteroaryl-W, --CN, --O--C(.dbd.S)NR.sup.13R.sup.13,
-Z.sub.gR.sup.13, --CR.sup.10(Z.sub.gR.sup.13)(Z.sub.gR.sup.13),
--C(.dbd.O)OAr, --C(.dbd.O)NR.sup.13Ar, imidazoline, tetrazole,
tetrazole amide, --SO.sub.2NHR.sup.13,
--SO.sub.2NH--C(R.sup.13R.sup.13)-(Z).sub.g-R.sup.13, a cyclic
sugar or oligosaccharide, a cyclic amino sugar or oligosaccharide,
##STR00085## each Ar is, independently, phenyl, substituted phenyl,
wherein the substituents of the substituted phenyl are 1-3
substituents independently selected from the group consisting of
OH, OCH.sub.3, NR.sup.13R.sup.13, Cl, F, and CH.sub.3, or
heteroaryl; each W is independently, thiazolidinedione,
oxazolidinedione, heteroaryl-C(.dbd.O)N R.sup.13R.sup.13, --CN,
--O--C(.dbd.S)NR.sup.13R.sup.13, -Z.sub.gR.sup.13,
--CR.sup.10(ZgR.sup.13)(ZgR.sup.13), --C(.dbd.O)OAr,
--C(.dbd.O)NR.sup.13Ar, imidazoline, tetrazole, tetrazole amide,
--SO.sub.2NHR.sup.13,
--SO.sub.2NH--C(R.sup.13R.sup.13)-(Z).sub.g-R.sup.13, a cyclic
sugar or oligosaccharide, a cyclic amino sugar or oligosaccharide,
##STR00086## each R.sup.6 is, independently, --R.sup.7, --OR.sup.7,
--OR.sup.11, --N(R.sup.7).sub.2, --(CH.sub.2).sub.m--OR.sup.8,
--O--(CH.sub.2).sub.m--OR.sup.8,
--(CH.sub.2).sub.m--NR.sup.7R.sup.10,
--O--(CH.sub.2).sub.m--NR.sup.7R.sup.10,
--(CH.sub.2).sub.n(CHOR.sup.8)(CHOR.sup.8).sub.n--CH.sub.2OR.sup.8,
--O--(CH.sub.2).sub.m(CHOR.sup.8)(CHOR.sup.8).sub.n--CH.sub.2OR.sup.8,
--(CH.sub.2CH.sub.2O).sub.m--R.sup.8,
--O--(CH.sub.2CH.sub.2O).sub.m--R.sup.8,
--(CH.sub.2CH.sub.2O).sub.m--CH.sub.2CH.sub.2NR.sup.7R.sup.10,
O--(CH.sub.2CH.sub.2O), --CH.sub.2CH.sub.2NR.sup.7R.sup.10,
--(CH.sub.2).sub.n--C(.dbd.O)NR.sup.7R.sup.10,
--O--(CH.sub.2).sub.m--C(.dbd.O)NR.sup.7R.sup.10,
--(CH.sub.2).sub.n-(Z).sub.g-R.sup.7,
--O--(CH.sub.2).sub.m-(Z).sub.gR.sup.7,
--(CH.sub.2).sub.n--NR.sup.10--CH.sub.2(CHOR.sup.8)(CHOR.sup.8).sub.n--CH-
.sub.2OR.sup.8,
--O--(CH.sub.2).sub.m--NR.sup.10--CH.sub.2(CHOR.sup.8)(CHOR.sup.8).sub.n--
-CH.sub.2OR.sup.8, --(CH.sub.2), --CO.sub.2R.sup.7,
O--(CH.sub.2).sub.m--CO.sub.2R.sup.7, --OSO.sub.3H,
--O-glucuronide, --O-glucose, ##STR00087## wherein when two R.sup.6
are --OR.sup.11 and are located adjacent to each other on a phenyl
ring, the alkyl moieties of the two R.sup.6 may be bonded together
to form a methylenedioxy group; each R.sup.7 is, independently,
hydrogen lower alkyl, phenyl, substituted phenyl or
--CH.sub.2(CHOR).sup.8.sub.m--R.sup.10; each R.sup.8 is,
independently, hydrogen, lower alkyl, --C(.dbd.O)--R.sup.11,
glucuronide, 2-tetrahydropyranyl, or ##STR00088## each R.sup.9 is,
independently, --CO.sub.2R.sup.13, --CON(R.sup.13).sub.2,
--SO.sub.2CH.sub.2R.sup.13, or --C(.dbd.O)R.sup.13; each R.sup.10
is, independently, --H, --SO.sub.2CH.sub.3, --CO.sub.2R.sup.13,
--C(.dbd.O)NR.sup.3R.sup.13, --C(.dbd.O)R.sup.13, or
--(CH.sub.2).sub.m--(CHOH).sub.m--CH.sub.2OH; each Z is,
independently, CHOH, C(.dbd.O), --(CH.sub.2).sub.n--,
CHNR.sup.3R.sup.3, C.dbd.NR.sup.3, or NR.sup.13; each R.sup.11 is,
independently, lower alkyl; each R.sup.12 is independently,
--SO.sub.2CH.sub.3, --CO.sub.2R.sup.13,
--C(.dbd.O)NR.sup.13R.sup.13, --C(.dbd.O)R.sup.13, or
--CH.sub.2--(CHOH).sub.n--CH.sub.2OH; each R.sup.13 is,
independently, hydrogen, R.sup.7, R.sup.10, ##STR00089## with the
proviso that NR.sup.13R.sup.13 can be joined on itself to form a
ring comprising one of the following: ##STR00090## each Het is
independently, --NR.sub.--3, --S--, --SO--, or --SO.sub.2--; --O--,
--SO.sub.2NR.sup.13--, --NHSO.sub.2--, --NR.sup.13CO--, or
--CONR.sup.13; each g is, independently, an integer from 1 to 6;
each m is, independently, an integer from 1 to 7; each n is,
independently, an integer from 0 to 7; each Q is, independently,
C--R.sup.5, C--R.sup.6, or a nitrogen atom, wherein at most three Q
in a ring are nitrogen atoms; each V is, independently,
##STR00091## with the proviso that when V is attached directly to a
nitrogen atom, then V can also be, independently, R.sup.7,
R.sup.10, or (R.sup.11).sub.2; with the proviso that, when any two
--CH.sub.2OR.sup.8 groups are located 1, 2 or 1,3-with respect to
each other, the R.sup.9 groups may be joined to form a cyclic mono-
or di-substituted 1,3-dioxane or 1,3-dioxolane; or a
pharmaceutically acceptable salt thereof; and inclusive of all
enantiomers, diastereomers, and racemic mixtures thereof.
2-114. (canceled)
Description
CONTINUING APPLICATION DATA
[0001] This application claims benefit of U.S. Provisional
Application Ser. No. 60/495,725, filed on Aug. 18, 2003, and
incorporated herein by reference in its entirety
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to sodium channel blockers.
The present invention also includes a variety of methods of
treatment using these inventive sodium channel blockers.
[0004] 2. Description of the Background
[0005] 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
(Cl.sup.- 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 (Cl.sup.- 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.
[0006] 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. Therefore, to inhibit ENaC
mediated Na.sup.+ and liquid absorption, an ENaC blocker of the
amiloride class (which blocks from the extracellular domain of
ENaC) must be delivered to the mucosal surface and, importantly, be
maintained at this site, to achieve therapeutic utility. The
present invention describes diseases characterized by too little
liquid on mucosal surfaces and "topical" sodium channel blockers
designed to exhibit the increased potency, reduced mucosal
abosrption, and slow dissociation ("unbinding" or detachment) from
ENaC required for therapy of these diseases.
[0007] Chronic bronchitis (CB), including the most common lethal
genetic form of chronic bronchitis, cystic fibrosis (CF), are
diseases that reflect the body's failure to clear mucus normally
from the lungs, which ultimately produces chronic airways
infection. In the normal lung, the primary defense against chronic
intrapulmonary airways infection (chronic bronchitis) is mediated
by the continuous clearance of mucus from bronchial airway
surfaces. This function in health effectively removes from the lung
potentially noxious toxins and pathogens. Recent data indicate that
the initiating problem, i.e., the "basic defect," in both CB and CF
is the failure to clear mucus from airway surfaces. The failure to
clear mucus reflects an imbalance between the amount of liquid and
mucin on airway surfaces. This "airway surface liquid" (ASL) is
primarily composed of salt and water in proportions similar to
plasma (i.e., isotonic). Mucin macromolecules organize into a well
defined "mucus layer" which normally traps inhaled bacteria and is
transported out of the lung via the actions of cilia which beat in
a watery, low viscosity solution termed the "periciliary liquid"
(PCL). In the disease state, there is an imbalance in the
quantities of mucus as ASL on airway surfaces. This results in a
relative reduction in ASL which leads to mucus concentration,
reduction in the lubricant activity of the PCL, and a failure to
clear mucus via ciliary activity to the mouth. The reduction in
mechanical clearance of mucus from the lung leads to chronic
bacterial colonization of mucus adherent to airway surfaces. It is
the chronic retention of bacteria, the failure of local
antimicrobial substances to kill mucus-entrapped bacteria on a
chronic basis, and the consequent chronic inflammatory responses of
the body to this type of surface infection, that lead to the
syndromes of CB and CF.
[0008] The current afflicted population in the U.S. is 12,000,000
patients with the acquired (primarily from cigarette smoke
exposure) form of chronic bronchitis and approximately 30,000
patients with the genetic form, cystic fibrosis. Approximately
equal numbers of both populations are present in Europe. In Asia,
there is little CF but the incidence of CB is high and, like the
rest of the world, is increasing.
[0009] There is currently a large, unmet medical need for products
that specifically treat CB and CF at the level of the basic defect
that cause these diseases. The current therapies for chronic
bronchitis and cystic fibrosis focus on treating the symptoms
and/or the late effects of these diseases. Thus, for chronic
bronchitis, .beta.-agonists, inhaled steroids, anti-cholinergic
agents, and oral theophyllines and phosphodiesterase inhibitors are
all in development. However, none of these drugs treat effectively
the fundamental problem of the failure to clear mucus from the
lung. Similarly, in cystic fibrosis, the same spectrum of
pharmacologic agents is used. These strategies have been
complemented by more recent strategies designed to clear the CF
lung of the DNA ("Pulmozyme"; Genentech) that has been deposited in
the lung by neutrophils that have futilely attempted to kill the
bacteria that grow in adherent mucus masses and through the use of
inhaled antibiotics ("TOBI") designed to augment the lungs' own
killing mechanisms to rid the adherent mucus plaques of bacteria. A
general principle of the body is that if the initiating lesion is
not treated, in this case mucus retention/obstruction, bacterial
infections became chronic and increasingly refractory to
antimicrobial therapy. Thus, a major unmet therapeutic need for
both CB and CF lung diseases is an effective means of re-hydrating
airway mucus (i.e., restoring/expanding the volume of the ASL) and
promoting its clearance, with bacteria, from the lung.
[0010] R. C. Boucher, in U.S. Pat. No. 6,264,975, describes the use
of pyrazinoylguanidine sodium channel blockers for hydrating
mucosal surfaces. These compounds, typified by the well-known
diuretics amiloride, benzamil, and phenamil are effective. However,
these compounds suffer from the significant disadvantage that they
are (1) relatively impotent, which is important because the mass of
drug that can be inhaled by the lung is limited; (2) rapidly
absorbed, which limits the half-life of the drug on the mucosal
surface; and (3) are freely dissociable from ENaC. The sum of these
disadvantages embodied in these well known diurectics produces
compounds with insufficient potency and/or effective half-life on
mucosal surfaces to have therapeutic benefit for hydrating mucosal
surfaces.
[0011] Clearly, what is needed are drugs that are more effective at
restoring the clearance of mucus from the lungs of patients with
CB/CF. The value of these new therapies will be reflected in
improvements in the quality and duration of life for both the CF
and the CB populations.
[0012] Other mucosal surfaces in and on the body exhibit subtle
differences in the normal physiology of the protective surface
liquids on their surfaces but the pathophysiology of disease
reflects a common theme, i.e., too little protective surface
liquid. 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.
Similarly, 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, as in CB, between mucin
secretion and relative ASL depletion. Finally, in the
gastrointestinal tract, 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.
[0013] Fifty million Americans and hundreds of millions of others
around the world suffer from high blood pressure and the subsequent
sequale leading to congestive heart failure and increasing
mortality. It is the Western World's leading killer and there is a
need there for new medicines to treat these diseases. Thus, in
addition, some of the novel sodium channel blockers of this
invention can be designed to target the kidney and as such they may
be used as diuretics for the treatment of hypertension, congestive
heart failure (CHF) and other cardiovascular diseases. These new
agents may be used alone or in combination with beta-blockers, ACE
inhibitors, HMGCoA reductase inhibitors, calcium channel blockers
and other cardiovascular agents.
SUMMARY OF THE INVENTION
[0014] It is an object of the present invention to provide
compounds that are more potent and/or absorbed less rapidly from
mucosal surfaces, and/or are less reversible as compared to known
compounds.
[0015] It is another aspect of the present invention to provide
compounds that are more potent and/or absorbed less rapidly and/or
exhibit less reversibility, as compared to compounds such as
amilorde, benzamil, and phenamil. Therefore, the compounds will
give a prolonged pharmacodynamic half-life on mucosal surfaces as
compared to known compounds.
[0016] It is another object of the present invention to provide
compounds which are (1) absorbed less rapidly from mucosal
surfaces, especially airway surfaces, as compared to known
compounds and; (2) when absorbed from musosal surfaces after
administration to the mucosal surfaces, are converted in vivo into
metabolic derivitives thereof which have reduced efficacy in
blocking sodium channels as compared to the administered parent
compound. It is another object of the present invention to provide
compounds that are more potent and/or absorbed less rapidly and/or
exhibit less reversibility, as compared to compounds such as
amiloride, benzamil, and phenamil. Therefore, such compounds will
give a prolonged pharmacodynamic half-life on mucosal surfaces as
compared to previous compounds.
[0017] It is another object of the present invention to provide
compounds that target the kidney for use in the treatment of
cardiovascular disease.
[0018] It is another object of the present invention to provide
methods of treatment that take advantage of the pharmacological
properties of the compounds described above.
[0019] In particular, it is an object of the present invention to
provide methods of treatment which rely on rehydration of mucosal
surfaces.
[0020] In particular, it is an object of the present invention to
provide methods of treating cardiovascular disease.
[0021] The objects of the present invention may be accomplished
with a class of pyrazinoylguanidine compounds represented by
formula (I):
##STR00001##
wherein
[0022] X is hydrogen, halogen, trifluoromethyl, lower alkyl,
unsubstituted or substituted phenyl, lower alkyl-thio, phenyl-lower
alkyl-thio, lower alkyl-sulfonyl, or phenyl-lower
alkyl-sulfonyl;
[0023] Y is hydrogen, hydroxyl, mercapto, lower alkoxy, lower
alkyl-thio, halogen, lower alkyl, unsubstituted or substituted
mononuclear aryl, or --N(R.sup.2).sub.2;
[0024] R.sup.1 is hydrogen or lower alkyl;
[0025] each R.sup.2 is, independently, --R.sup.7,
--(CH.sub.2).sub.m--OR.sup.8, --(CH.sub.2).sub.m--NR.sup.7R.sup.10,
--(CH.sub.2).sub.n(CHOR.sup.8)(CHOR.sup.8).sub.n--CH.sub.2OR.sup.8,
--(CH.sub.2CH.sub.2O).sub.m--R.sup.8,
--(CH.sub.2CH.sub.2O).sub.m--CH.sub.2CH.sub.2NR.sup.7R.sup.10,
--(CH.sub.2).sub.n--C(.dbd.O)NR.sup.7R.sup.10,
--(CH.sub.2).sub.n-Z.sub.g-R.sup.7,
--(CH.sub.2).sub.m--NR.sup.10--CH.sub.2(CHOR.sup.8)(CHOR.sup.8).sub.n--CH-
.sub.2OR.sup.8, --(CH.sub.2).sub.n--CO.sub.2R.sup.7, or
##STR00002##
[0026] R.sup.3 and R.sup.4 are each, independently, hydrogen, a
group represented by formula (A), lower alkyl, hydroxy lower alkyl,
phenyl, phenyl-lower alkyl, (halophenyl)-lower alkyl,
lower-(alkylphenylalkyl), lower (alkoxyphenyl)-lower alkyl,
naphthyl-lower alkyl, or pyridyl-lower alkyl, with the proviso that
at least one of R.sup.3 and R.sup.4 is a group represented by
formula (A):
##STR00003##
wherein [0027] each R.sup.L is, independently, --R.sup.7,
--(CH.sub.2).sub.n--OR.sup.8, --O--(CH.sub.2).sub.m--OR.sup.8,
--(CH.sub.2).sub.n--NR.sup.7R.sup.10,
--O--(CH.sub.2).sub.m--NR.sup.7R.sup.10,
--(CH.sub.2).sub.n(CHOR.sup.8)(CHOR.sup.8).sub.n--CH.sub.2OR.sup.8,
--O--(CH.sub.2).sub.m(CHOR.sup.8)(CHOR.sup.8).sub.n--CH.sub.2OR.sup.8,
--(CH.sub.2CH.sub.2O).sub.m--R.sup.8,
--O--(CH.sub.2CH.sub.2O).sub.m--R.sup.8,
--(CH.sub.2CH.sub.2O).sub.m--CH.sub.2CH.sub.2NR.sup.7R.sup.10,
--O--(CH.sub.2CH.sub.2O).sub.m--CH.sub.2CH.sub.2NR.sup.7R.sup.10,
--(CH.sub.2).sub.n--C(.dbd.O)NR.sup.7R.sup.10,
--O--(CH.sub.2).sub.m--C(.dbd.O)NR.sup.7R.sup.10,
--(CH.sub.2).sub.n-(Z).sub.g-R.sup.7,
--O(CH.sub.2).sub.m-(Z).sub.g-R.sup.7,
--(CH.sub.2).sub.n--NR.sup.10--CH.sub.2(CHOR.sup.8)(CHOR.sup.8).sub.n--CH-
.sub.2OR.sup.8,
--O--(CH.sub.2).sub.m--NR.sup.10--CH.sub.2(CHOR.sup.8)(CHOR.sup.8).sub.n--
-CH.sub.2OR.sup.8, --(CH.sub.2).sub.n--CO.sub.2R.sup.7,
--O--(CH.sub.2).sub.m--CO.sub.2R.sup.7, --OSO.sub.3H,
--O-glucuronide, --O-glucose,
##STR00004##
[0028] each o is, independently, an integer from 0 to 10;
[0029] each p is an integer from 0 to 10;
[0030] with the proviso that the sum of o and p in each contiguous
chain is from 1 to 10;
[0031] each x is, independently, O, NR.sup.10, C(.dbd.O), CHOH,
C(.dbd.N--R.sup.10), CHNR.sup.7R.sup.10, or represents a single
bond;
[0032] wherein each R.sup.5 is, independently, Link
--(CH.sub.2).sub.n--CAP, Link
--(CH.sub.2).sub.n(CHOR.sup.8)(CHOR.sup.8).sub.n-CAP, Link
--(CH.sub.2CH.sub.2O).sub.m--CH.sub.2-CAP, Link
--(CH.sub.2CH.sub.2O).sub.m--CH.sub.2CH.sub.2-CAP, Link
--(CH.sub.2).sub.n-(Z).sub.g-CAP, Link
--(CH.sub.2).sub.n(Z).sub.g-(CH.sub.2).sub.m--CAP, Link
--(CH.sub.2).sub.n--NR.sup.13--CH.sub.2(CHOR.sup.8)(CHOR.sup.8).sub.n-CAP-
, Link
--(CH.sub.2).sub.n--(CHOR.sup.8).sub.mCH.sub.2--NR.sup.13-(Z).sub.g-
-CAP, Link
--(CH.sub.2).sub.nNR.sup.13--(CH.sub.2).sub.m(CHOR.sup.8).sub.n-
CH.sub.2NR.sup.13-(Z).sub.g-CAP, Link
--(CH.sub.2).sub.m-(Z).sub.g-(CH.sub.2).sub.m-CAP, Link
--NH--C(.dbd.O)--NH--(CH.sub.2).sub.m-CAP, Link
--(CH.sub.2).sub.m--C(.dbd.O)NR.sup.13--(CH.sub.2).sub.m--C(.dbd.O)NR.sup-
.10R.sup.10, Link
--(CH.sub.2).sub.m--C(.dbd.O)NR.sup.13--(CH.sub.2).sub.m-CAP, Link
--(CH.sub.2).sub.m--C(.dbd.O)NR.sup.11R.sup.11, Link
--(CH.sub.2).sub.m--C(.dbd.O)NR.sup.12R.sup.12, Link
--(CH.sub.2).sub.n-(Z).sub.g-(CH.sub.2).sub.m-(Z).sub.g-CAP,
Link-Z.sub.g-(CH.sub.2).sub.m-Het-(CH.sub.2).sub.m-CAP.
[0033] each Link is, independently, --O--, --(CH.sub.2).sub.n--,
--O(CH.sub.2).sub.m--, --NR.sup.13--C(.dbd.O)--NR.sup.13,
--NR.sup.13--C(.dbd.O)--(CH.sub.2).sub.m--,
--C(.dbd.O)NR.sup.13--(CH.sub.2).sub.m,
--(CH.sub.2).sub.n-Z.sub.g-(CH.sub.2).sub.n, --S--, --SO--,
--SO.sub.2--, --SO.sub.2NR.sup.7--, --SO.sub.2NR.sup.10, or
-Het-;
[0034] each CAP is, independently, thiazolidinedione,
oxazolidinedione, heteroaryl-C(.dbd.O)N R.sup.13R.sup.13,
heteroaryl-W, --CN, --O--C(.dbd.S)NR.sup.13R.sup.13,
-Z.sub.gR.sup.13, --CR.sup.10(Z.sub.gR.sup.13)(Z.sub.gR.sup.13),
--C(.dbd.O)OAr, --C(.dbd.O)NR.sup.13Ar, imidazoline, tetrazole,
tetrazole amide, --SO.sub.2NHR.sup.13,
--SO.sub.2NH--C(R.sup.13R.sup.13)-(Z).sub.gR.sup.13, a cyclic sugar
or oligosaccharide, a cyclic amino sugar or oligosaccharide,
##STR00005##
each Ar is, independently, phenyl, substituted phenyl, wherein the
substituents of the substituted phenyl are 1-3 substituents
independently selected from the group consisting of OH, OCH.sub.3,
NR.sup.13R.sup.13, Cl, F, and CH.sub.3, or heteroaryl,
[0035] each W is independently, thiazolidinedione,
oxazolidinedione, heteroaryl-C(.dbd.O)NR.sup.13R.sup.13, --CN,
--O--C(.dbd.S)NR.sup.13R.sup.13, -Z.sub.gR.sup.13,
--CR.sup.10(Z.sub.gR.sup.13)(Z.sub.gR.sup.13), --C(.dbd.O)OAr,
--C(.dbd.O)NR.sup.13Ar, imidazoline, tetrazole, tetrazole amide,
--SO.sub.2NHR.sup.13,
--SO.sub.2NH--C(R.sup.13R.sup.13)-(Z).sub.g-R.sup.13, a cyclic
sugar or oligosaccharide, a cyclic amino sugar or
oligosaccharide,
##STR00006##
each R.sup.6 is, independently, --R.sup.7, --OR.sup.7, --OR.sup.11,
--N(R.sup.7).sub.2, --(CH.sub.2).sub.m--OR.sup.8,
--O--(CH.sub.2).sub.m--OR.sup.8,
--(CH.sub.2).sub.n--NR.sup.7R.sup.10,
--O--(CH.sub.2).sub.m--NR.sup.7R.sup.10,
--(CH.sub.2).sub.n(CHOR.sup.8)(CHOR.sup.8).sub.n--CH.sub.2OR.sup.8,
--O--(CH.sub.2).sub.m(CHOR.sup.8)(CHOR.sup.8).sub.n--CH.sub.2OR.sup.8,
--(CH.sub.2CH.sub.2O).sub.m--R.sup.8,
--O--(CH.sub.2CH.sub.2O).sub.m--R.sup.8,
--(CH.sub.2CH.sub.2O).sub.m--CH.sub.2CH.sub.2NR.sup.7R.sup.10,
--O--(CH.sub.2CH.sub.2O).sub.m--CH.sub.2CH.sub.2NR.sup.7R.sup.10,
--(CH.sub.2).sub.n--C(.dbd.O)NR.sup.7R.sup.10,
--O--(CH.sub.2).sub.m--C(.dbd.O)NR.sup.7R.sup.10,
--(CH.sub.2).sub.n-(Z).sub.g-R.sup.7,
--O--(CH.sub.2).sub.m-(Z).sub.g-R.sup.7,
--(CH.sub.2).sub.n--NR.sup.10--CH.sub.2(CHOR.sup.8)(CHOR.sup.8).sub.n--CH-
.sub.2OR.sup.8,
--O--(CH.sub.2).sub.m--NR.sup.10--CH.sub.2(CHOR.sup.8)(CHOR.sup.8).sub.n--
-CH.sub.2OR.sup.8, --(CH.sub.2).sub.n--CO.sub.2R.sup.7,
--O--(CH.sub.2).sub.m--CO.sub.2R.sup.7, --OSO.sub.3H,
--O-glucuronide, --O-glucose,
##STR00007##
[0036] where when two R.sup.6 are --OR.sup.11 and are located
adjacent to each other on a phenyl ring, the alkyl moieties of the
two R.sup.6 may be bonded together to form a methylenedioxy group;
with the proviso that when at least two --CH.sub.2OR.sup.8 are
located adjacent to each other, the R.sup.8 groups may be joined to
form a cyclic mono- or di-substituted 1,3-dioxane or
1,3-dioxolane,
[0037] each R.sup.7 is, independently, hydrogen lower alkyl,
phenyl, substituted phenyl or
--CH.sub.2(CHOR).sup.8.sub.m--R.sup.10;
[0038] each R.sup.8 is, independently, hydrogen, lower alkyl,
--C(.dbd.O)--R.sup.11, glucuronide, 2-tetrahydropyranyl, or
##STR00008##
[0039] each R.sup.9 is, independently, --CO.sub.2R.sup.13,
--CON(R.sup.13).sub.2, --SO.sub.2CH.sub.2R.sup.13, or
--C(.dbd.O)R.sup.13;
[0040] each R.sup.10 is, independently, --H, --SO.sub.2CH.sub.3,
--CO.sub.2R.sup.13, --C(.dbd.O)NR.sup.13R.sup.13,
--C(.dbd.O)R.sup.13, or
(CH.sub.2).sub.m--(CHOH).sub.n--CH.sub.2OH;
[0041] each Z is, independently, CHOH, C(.dbd.O),
--(CH.sub.2).sub.n--, CHNR.sup.13R.sup.13, C.dbd.NR.sup.13, or
NR.sup.13;
[0042] each R.sup.11 is, independently, lower alkyl;
[0043] each R.sup.12 is independently, --SO.sub.2CH.sub.3,
--CO.sub.2R.sup.13, --C(.dbd.O)NR.sup.13R.sup.13,
--C(.dbd.O)R.sup.13, or --CH.sub.2--(CHOH).sub.n--CH.sub.2OH;
[0044] each R.sup.13 is, independently, hydrogen, R.sup.7,
R.sup.10,
##STR00009##
[0045] with the proviso that NR.sup.13R.sup.13 can be joined on
itself to form a ring comprising one of the following:
##STR00010##
each Het is independently, --NR.sup.13, --S--, --SO--, or
--SO.sub.2--; --O--, --SO.sub.2NR.sup.13--, --NHSO.sub.2--,
--NR.sup.13CO--, or --CONR.sup.13--;
[0046] each g is, independently, an integer from 1 to 6;
[0047] each m is, independently, an integer from 1 to 7;
[0048] each n is, independently, an integer from 0 to 7;
[0049] each Q is, independently, C--R.sup.5, C--R.sup.6, or a
nitrogen atom, wherein at most three Q in a ring are nitrogen
atoms;
[0050] each V is, independently,
##STR00011##
with the proviso that when V is attached directly to a nitrogen
atom, then V can also be, independently, R.sup.7, R.sup.10, or
(R.sup.11).sub.2;
[0051] wherein for any of the above compounds when two
--CH.sub.2OR.sup.8 groups are located 1,2- or 1,3- with respect to
each other the R.sup.8 groups may be joined to form a cyclic mono-
or di-substituted 1,3-dioxane or 1,3-dioxolane;
[0052] wherein any of the above compounds can be a pharmaceutically
acceptable salt thereof, and wherein the above compounds are
inclusive of all enantiomers, diastereomers, and racemic mixtures
thereof.
[0053] The present invention also provides pharmaceutical
compositions which contain a compound described above.
[0054] The present invention also provides a method of promoting
hydration of mucosal surfaces, comprising:
[0055] administering an effective amount of a compound represented
by formula (I) to a mucosal surface of a subject.
[0056] The present invention also provides a method of restoring
mucosal defense, comprising:
[0057] topically administering an effective amount of compound
represented by formula (I) to a mucosal surface of a subject in
need thereof.
[0058] The present invention also provides a method of blocking
ENaC, comprising:
[0059] contacting sodium channels with an effective amount of a
compound represented by formula (I).
[0060] The present invention also provides a method of promoting
mucus clearance in mucosal surfaces, comprising:
[0061] administering an effective amount of a compound represented
by formula (I) to a mucosal surface of a subject.
[0062] The present invention also provides a method of treating
chronic bronchitis, comprising:
[0063] administering an effective amount of a compound represented
by formula (I) to a subject in need thereof.
[0064] The present invention also provides a method of treating
cystic fibrosis, comprising:
[0065] administering an effective amount of compound represented by
formula (I) to a subject in need thereof.
[0066] The present invention also provides a method of treating
rhinosinusitis, comprising:
[0067] administering an effective amount of a compound represented
by a formula (I) to a subject in need thereof.
[0068] The present invention also provides a method of treating
nasal dehydration, comprising:
[0069] administering an effective amount of a compound represented
by formula (I) to the nasal passages of a subject in need
thereof.
[0070] In a specific embodiment, the nasal dehydration is brought
on by administering dry oxygen to the subject.
[0071] The present invention also provides a method of treating
sinusitis, comprising:
[0072] administering an effective amount of a compound represented
by formula (I) to a subject in need thereof.
[0073] The present invention also provides a method of treating
pneumonia, comprising:
[0074] administering an effective amount of a compound represented
by formula (I) to a subject in need thereof.
[0075] The present invention also provides a method of preventing
ventilator-induced pneumonia, comprising:
[0076] administering an effective compound represented by formula
(I) to a subject by means of a ventilator.
[0077] The present invention also provides a method of treating
asthma, comprising:
[0078] administering an effective amount of a compound represented
by formula (I) to a subject in need thereof.
[0079] The present invention also provides a method of treating
primary ciliary dyskinesia, comprising:
[0080] administering an effective amount of a compound represented
by formula (I) to a subject in need thereof.
[0081] The present invention also provides a method of treating
otitis media, comprising:
[0082] administering an effective amount of a compound represented
by formula (I) to a subject in need thereof.
[0083] The present invention also provides a method of inducing
sputum for diagnostic purposes, comprising:
[0084] administering an effective amount of compound represented by
formula (I) to a subject in need thereof.
[0085] The present invention also provides a method of treating
chronic obstructive pulmonary disease, comprising:
[0086] administering an effective amount of a compound represented
by formula (I) to a subject in need thereof.
[0087] The present invention also provides a method of treating
emphysema, comprising:
[0088] administering an effective amount of a compound represented
by formula (I) to a subject in need thereof.
[0089] The present invention also provides a method of treating dry
eye, comprising:
[0090] administering an effective amount of a compound represented
by formula (I) to the eye of the subject in need thereof.
[0091] The present invention also provides a method of promoting
ocular hydration, comprising:
[0092] administering an effective amount of a compound represented
by formula (I) to the eye of the subject.
[0093] The present invention also provides a method of promoting
corneal hydration, comprising:
[0094] administering an effective amount of a compound represented
by formula (I) to the eye of the subject.
[0095] The present invention also provides a method of treating
Sjogren's disease, comprising:
[0096] administering an effective amount of compound represented by
formula (I) to a subject in need thereof.
[0097] The present invention also provides a method of treating
vaginal dryness, comprising:
[0098] administering an effective amount of a compound represented
by formula (I) to the vaginal tract of a subject in need
thereof.
[0099] The present invention also provides a method of treating dry
skin, comprising:
[0100] administering an effective amount of a compound represented
by formula (I) to the skin of a subject in need thereof.
[0101] The present invention also provides a method of treating dry
mouth (xerostomia), comprising:
[0102] administering an effective amount of compound represented by
formula (I) to the mouth of the subject in need thereof.
[0103] The present invention also provides a method of treating
distal intestinal obstruction syndrome, comprising:
[0104] administering an effective amount of compound represented by
formula (I) to a subject in need thereof.
[0105] The present invention also provides a method of treating
esophagitis, comprising:
[0106] administering an effective amount of a compound represented
by formula (I) to a subject in need thereof.
[0107] The present invention also provides a method of treating
constipation, comprising:
[0108] administering an effective amount of a compound represented
by formula (I) to a subject in need thereof. In one embodiment of
this method, the compound is administered either orally or via a
suppository or enema.
[0109] The present invention also provides a method of treating
chronic diverticulitis comprising:
[0110] administering an effective amount of a compound represented
by formula (I) to a subject in need thereof.
[0111] The present invention also provides a method of treating
hypertension, comprising administering the compound represented by
formula (I) to a subject in need thereof.
[0112] The present invention also provides a method of reducing
blood pressure, comprising administering the compound represented
by formula (I) to a subject in need thereof.
[0113] The present invention also provides a method of treating
edema, comprising administering the compound represented by formula
(I) to a subject in need thereof.
[0114] The present invention also provides a method of promoting
diuresis, comprising administering the compound represented by
formula (I) to a subject in need thereof.
[0115] The present invention also provides a method of promoting
natriuresis, comprising administering the compound represented by
formula (I) to a subject in need thereof.
[0116] The present invention also provides a method of promoting
saluresis, comprising administering the compound represented by
formula (I) to a subject in need thereof.
DETAILED DESCRIPTION OF THE INVENTION
[0117] The present invention is based on the discovery that the
compounds of formula (I) are more potent and/or, absorbed less
rapidly from mucosal surfaces, especially airway surfaces, and/or
less reversible from interactions with ENaC as compared to
compounds such as amiloride, benzamil, and phenamil. Therefore, the
compounds of formula (I) have a longer half-life on mucosal
surfaces as compared to these compounds.
[0118] The present invention is also based on the discovery that
certain compounds embraced by formula (I) are converted in vivo
into metabolic derivatives thereof that have reduced efficacy in
blocking sodium channels as compared to the parent administered
compound, after they are absorbed from mucosal surfaces after
administration. This important property means that the compounds
will have a lower tendency to cause undesired side-effects by
blocking sodium channels located at untargeted locations in the
body of the recipient, e.g., in the kidneys.
[0119] The present invention is also based on the discovery that
certain compounds embraced by formula (I) target the kidney and
thus may be used as cardiovascular agents.
[0120] In the compounds represented by formula (I), X may be
hydrogen, halogen, trifluoromethyl, lower alkyl, lower cycloalkyl,
unsubstituted or substituted phenyl, lower alkyl-thio, phenyl-lower
alkyl-thio, lower alkyl-sulfonyl, or phenyl-lower alkyl-sulfonyl.
Halogen is preferred.
[0121] Examples of halogen include fluorine, chlorine, bromine, and
iodine. Chlorine and bromine are the preferred halogens. Chlorine
is particularly preferred. This description is applicable to the
term "halogen" as used throughout the present disclosure.
[0122] As used herein, the term "lower alkyl" means an alkyl group
having less than 8 carbon atoms. This range includes all specific
values of carbon atoms and subranges there between, such as 1,2, 3,
4, 5, 6, and 7 carbon atoms. The term "alkyl" embraces all types of
such groups, e.g., linear, branched, and cyclic alkyl groups. This
description is applicable to the term "lower alkyl" as used
throughout the present disclosure. Examples of suitable lower alkyl
groups include methyl, ethyl, propyl, cyclopropyl, butyl, isobutyl,
etc.
[0123] Substituents for the phenyl group include halogens.
Particularly preferred halogen substituents are chlorine and
bromine.
[0124] Y may be hydrogen, hydroxyl, mercapto, lower alkoxy, lower
alkyl-thio, halogen, lower alkyl, lower cycloalkyl, mononuclear
aryl, or --N(R.sup.2).sub.2. The alkyl moiety of the lower alkoxy
groups is the same as described above. Examples of mononuclear aryl
include phenyl groups. The phenyl group may be unsubstituted or
substituted as described above. The preferred identity of Y is
--N(R.sup.2).sub.2. Particularly preferred are such compounds where
each R.sup.2 is hydrogen.
[0125] R.sup.1 may be hydrogen or lower alkyl. Hydrogen is
preferred for R.sup.1.
[0126] Each R.sup.2 may be, independently, --R.sup.7,
(CH.sub.2).sub.m--OR.sup.8, --(CH.sub.2).sub.m--NR.sup.7R.sup.10,
--(CH.sub.2).sub.n(CHOR.sup.8)(CHOR.sup.8).sub.n--CH.sub.2OR.sup.8,
--(CH.sub.2CH.sub.2O).sub.m--R.sup.8,
--(CH.sub.2CH.sub.2O).sub.m--CH.sub.2CH.sub.2NR.sup.7R.sup.10,
--(CH.sub.2).sub.n--C(.dbd.O)NR.sup.7R.sup.10,
--(CH.sub.2).sub.n-Z.sub.g-R.sup.7,
--(CH.sub.2).sub.m--NR.sup.10--CH.sub.2(CHOR.sup.8)(CHOR.sup.8).sub.n--CH-
.sub.2OR.sup.8, --(CH.sub.2).sub.n--CO.sub.2R.sup.7, or
##STR00012##
[0127] Hydrogen and lower alkyl, particularly C.sub.1-C.sub.3 alkyl
are preferred for R.sup.2. Hydrogen is particularly preferred.
[0128] R.sup.3 and R.sup.4 may be, independently, hydrogen, a group
represented by formula (A), lower alkyl, hydroxy lower alkyl,
phenyl, phenyl-lower alkyl, (halophenyl)-lower alkyl,
lower-(alkylphenylalkyl), lower (alkoxyphenyl)-lower alkyl,
naphthyl-lower alkyl, or pyridyl-lower alkyl, provided that at
least one of R.sup.3 and R.sup.4 is a group represented by formula
(A).
[0129] Preferred compounds are those where one of R.sup.3 and
R.sup.4 is hydrogen and the other is represented by formula
(A).
[0130] In formula (A), the moiety
--(C(R.sup.L).sub.2).sub.o-x-(C(R.sup.L).sub.2).sub.p-- defines an
alkylene group bonded to the aromatic ring. The variables o and p
may each be an integer from 0 to 10, subject to the proviso that
the sum of o and p in the chain is from 1 to 10. Thus, o and p may
each be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Preferably, the sum of
o and p is from 2 to 6. In a particularly preferred embodiment, the
sum of o and p is 4.
[0131] The linking group in the alkylene chain, x, may be,
independently, O, NR.sup.10, C(.dbd.O), CHOH, C(.dbd.N--R.sup.10),
CHNR.sup.7R.sup.10, or represents a single bond;
[0132] Therefore, when x represents a single bond, the alkylene
chain bonded to the ring is represented by the formula
--(C(R.sup.L).sub.2).sub.o+p--, in which the sum o+p is from 1 to
10.
[0133] Each R.sup.L may be, independently, --R.sup.7,
--(CH.sub.2).sub.n--OR.sup.8, --O--(CH.sub.2).sub.m--OR.sup.8,
--(CH.sub.2).sub.n--NR.sup.7R.sup.10,
--O--(CH.sub.2).sub.m--NR.sup.7R.sup.10,
--(CH.sub.2).sub.n(CHOR.sup.8)(CHOR.sup.8).sub.n--CH.sub.2OR.sup.8,
--O--(CH.sub.2).sub.m(CHOR.sup.8)(CHOR.sup.8).sub.n--CH.sub.2OR.sup.8,
--(CH.sub.2CH.sub.2O).sub.m--R.sup.8,
O--(CH.sub.2CH.sub.2O).sub.m--R.sup.8,
--(CH.sub.2CH.sub.2O).sub.m--CH.sub.2CH.sub.2NR.sup.7R.sup.10,
--O--(CH.sub.2CH.sub.2O).sub.m--CH.sub.2CH.sub.2NR.sup.7R.sup.10,
--(CH.sub.2).sub.n--C(.dbd.O)NR.sup.7R.sup.10,
--O--(CH.sub.2).sub.m--C(.dbd.O)NR.sup.7R.sup.10,
--(CH.sub.2).sub.n-(Z).sub.g-R.sup.7,
--O--(CH.sub.2).sub.m-(Z).sub.g-R.sup.7,
--(CH.sub.2).sub.n--NR.sup.10--CH.sub.2(CHOR.sup.8)(CHOR.sup.8).sub.n--CH-
.sub.2OR.sup.8,
--O--(CH.sub.2).sub.m--NR.sup.10--CH.sub.2(CHOR.sup.8)(CHOR.sup.8).sub.n--
-CH.sub.2OR.sup.8, --(CH.sub.2).sub.n--CO.sub.2R.sup.7,
--O--(CH.sub.2).sub.m--CO.sub.2R.sup.7, --OSO.sub.3H,
--O-glucuronide, --O-glucose,
##STR00013##
[0134] The preferred R.sup.L groups include --H, --OH,
--N(R.sup.7).sub.2, especially where each R.sup.7 is hydrogen.
[0135] In the alkylene chain in formula (A), it is preferred that
when one R.sup.L group bonded to a carbon atoms is other than
hydrogen, then the other R.sup.L bonded to that carbon atom is
hydrogen, i.e., the formula --CHR.sup.L. It is also preferred that
at most two R.sup.L groups in an alkylene chain are other than
hydrogen, where in the other R.sup.L groups in the chain are
hydrogens. Even more preferably, only one R.sup.L group in an
alkylene chain is other than hydrogen, where in the other R.sup.L
groups in the chain are hydrogens. In these embodiments, it is
preferable that x represents a single bond.
[0136] In another particular embodiment of the invention, all of
the R.sup.L groups in the alkylene chain are hydrogen. In these
embodiments, the alkylene chain is represented by the formula
--(CH.sub.2).sub.o-x-(CH.sub.2).sub.p--.
##STR00014##
[0137] Each R.sup.5 is, independently, Link-(CH.sub.2).sub.n-CAP,
Link-(CH.sub.2).sub.n(CHOR.sup.8)(CHOR.sup.8).sub.n-CAP,
Link-(CH.sub.2CH.sub.2O).sub.m--CH.sub.2-CAP,
Link-(CH.sub.2CH.sub.2O).sub.m--CH.sub.2CH.sub.2-CAP,
Link-(CH.sub.2).sub.n-(Z).sub.g-CAP,
Link-(CH.sub.2).sub.n(Z).sub.g-(CH.sub.2).sub.m-CAP,
Link-(CH.sub.2).sub.n--NR.sup.13--CH.sub.2(CHOR.sup.8)(CHOR.sup.8).sub.n--
CAP,
Link-(CH.sub.2).sub.n--(CHOR.sup.8).sub.nCH.sub.2--NR.sup.13-(Z).sub.-
g-CAP,
Link-(CH.sub.2).sub.nNR.sup.13--(CH.sub.2).sub.m(CHOR.sup.8).sub.nC-
H.sub.2NR.sup.13-(Z).sub.g-CAP,
Link-(CH.sub.2).sub.m-(Z).sub.g-(CH.sub.2).sub.m-CAP,
Link-NH--C(.dbd.O)--NH--(CH.sub.2).sub.m-CAP,
Link-(CH.sub.2).sub.m--C(.dbd.O)NR.sup.13--(CH.sub.2).sub.m--C(.dbd.O)NR.-
sup.10R.sup.10,
Link-(CH.sub.2).sub.m--C(.dbd.O)NR.sup.13--(CH.sub.2).sub.m-CAP,
Link-(CH.sub.2).sub.m--C(.dbd.O)NR.sup.11R.sup.11,
Link-(CH.sub.2).sub.m--C(.dbd.O)NR.sup.12R.sup.12,
Link-(CH.sub.2).sub.n-(Z).sub.g-(CH.sub.2).sub.m-(Z).sub.g-CAP,
Link-Z.sub.g-(CH.sub.2).sub.m-Het-(CH.sub.2).sub.m-CAP.
[0138] Each Link is, independently, --O--, (CH.sub.2).sub.n--,
--O(CH.sub.2).sub.m--, --NR.sup.13--C(.dbd.O)--NR.sup.13,
--NR.sup.13--C(.dbd.O)--(CH.sub.2).sub.m--,
--C(.dbd.O)NR.sup.13--(CH.sub.2).sub.m,
--(CH.sub.2).sub.n-Z.sub.g-(CH.sub.2).sub.n, --S--, --SO--,
--SO.sub.2--, SO.sub.2NR.sup.7--, SO.sub.2NR.sup.10--, or
-Het-.
[0139] each CAP is, independently, thiazolidinedione,
oxazolidinedione, heteroaryl-C(.dbd.O)NR.sup.13R.sup.13,
heteroaryl-W, --CN, --O--C(.dbd.S)NR.sup.13R.sup.13,
-Z.sub.gR.sup.13, --CR.sup.10(Z.sub.gR.sup.13)(Z.sub.gR.sup.13),
--C(.dbd.O)OAr, --C(.dbd.O)NR.sup.13Ar, imidazoline, tetrazole,
tetrazole amide, --SO.sub.2NHR.sup.13,
--SO.sub.2NH--C(R.sup.13R.sup.13)-(Z).sub.g-R.sup.13, a cyclic
sugar or oligosaccharide, a cyclic amino sugar or
oligosaccharide,
##STR00015##
[0140] Each Ar is, independently, phenyl, substituted phenyl,
wherein the substituents of the substituted phenyl are 1-3
substituents independently selected from the group consisting of
OH, OCH.sub.3, NR.sup.13R.sup.13, Cl, F, and CH.sub.3, or
heteroaryl.
[0141] Each W is independently, thiazolidinedione,
oxazolidinedione, heteroaryl-C(.dbd.O)NR.sup.13R.sup.13--CN,
--O--C(.dbd.S)NR.sup.13R.sup.13, -Z.sub.gR.sup.13,
--CR.sup.10(Z.sub.gR.sup.13)(Z.sub.gR.sup.13), --C(.dbd.O)OAr,
--C(.dbd.O)NR.sup.13Ar, imidazoline, tetrazole, tetrazole amide,
--SO.sub.2NHR.sup.13,
--SO.sub.2NH--C(R.sup.13R.sup.13)-(Z).sub.g-R.sup.13, a cyclic
sugar or oligosaccharide, a cyclic amino sugar or
oligosaccharide,
##STR00016##
[0142] Examples of heteroaryl include pyridyl, pyrazyl, tinazyl,
furyl, furfuryl, thienyl, tetrazyl, thiazolidinedionyl and
imidazoyl, pyrrolyl, furanyl, thiophenyl, quinolyl, indolyl,
adenyl, pyrazolyl, thiazolyl, isoxazolyl, indolyl, benzimidazolyl,
purinyl, quinolinyl, isoquinolinyl, pyridazyl, pyrimidyl, pyrazyl,
1,2,3-triazyl, 1,2,4-triazyl, 1,3,5-triazyl, cinnolyl, phthalazyl,
quinazolyl, quinoxalyl or pterdyl groups.
[0143] Each R.sup.6 is, independently, --R.sup.7, --OR.sup.7,
--OR.sup.11, --N(R.sup.7).sub.2, --(CH.sub.2).sub.m--OR.sup.8,
--O--(CH.sub.2).sub.m--OR.sup.8,
--(CH.sub.2).sub.n--NR.sup.7R.sup.10,
--O--(CH.sub.2).sub.m--NR.sup.7R.sup.10,
--(CH.sub.2).sub.n(CHOR.sup.8)(CHOR.sup.8).sub.n--CH.sub.2OR.sup.8,
--O--(CH.sub.2).sub.m(CHOR.sup.8)(CHOR.sup.8).sub.n--CH.sub.2OR.sup.8,
--(CH.sub.2CH.sub.2O).sub.m--R.sup.8,
--O--(CH.sub.2CH.sub.2O).sub.m--R.sup.8--(CH.sub.2CH.sub.2O).sub.m--CH.su-
b.2CH.sub.2NR.sup.7R.sup.10,
--O--(CH.sub.2CH.sub.2O).sub.m--CH.sub.2CH.sub.2NR.sup.7R.sup.10,
--(CH.sub.2).sub.n--C(.dbd.O)NR.sup.7R.sup.10,
--O--(CH.sub.2).sub.m--C(.dbd.O)NR.sup.7R.sup.10,
--(CH.sub.2).sub.n-(Z).sub.g-R.sup.7,
--O--(CH.sub.2).sub.m-(Z).sub.g-R.sup.7,
--(CH.sub.2).sub.n--NR.sup.10--CH.sub.2(CHOR.sup.8)(CHOR.sup.8).sub.n--CH-
.sub.2OR.sup.8,
--O--(CH.sub.2).sub.m--NR.sup.10--CH.sub.2(CHOR.sup.8)(CHOR.sup.8).sub.n--
-CH.sub.2OR.sup.8, --(CH.sub.2).sub.n--CO.sub.2R.sup.7,
--O--(CH.sub.2).sub.m--CO.sub.2R.sup.7, --OSO.sub.3H,
--O-glucuronide, --O-glucose,
##STR00017##
[0144] where when two R.sup.6 are --OR.sup.11 and are located
adjacent to each other on a phenyl ring, the alkyl moieties of the
two R.sup.6 may be bonded together to form a methylenedioxy
group;
[0145] with the proviso that when at least two --CH.sub.2OR.sup.8
are located adjacent to each other, the R.sup.8 groups may be
joined to form a cyclic mono- or di-substituted 1,3-dioxane or
1,3-dioxolane.
[0146] In addition, one of more of the R.sup.6 groups can be one of
the R.sup.5 groups which fall within the broad definition of
R.sup.6 set forth above.
[0147] When two R.sup.6 are --OR.sup.11 and are located adjacent to
each other on a phenyl ring, the alkyl moieties of the two R.sup.6
groups may be bonded together to form a methylenedioxy group, i.e.,
a group of the formula --O--CH.sub.2--O--.
[0148] As discussed above, R.sup.6 may be hydrogen. Therefore, 1,
2, 3, or 4 R.sup.6 groups may be other than hydrogen. Preferably at
most 3 of the R.sup.6 groups are other than hydrogen.
[0149] Each g is, independently, an integer from 1 to 6. Therefore,
each g may be 1, 2, 3, 4, 5, or 6.
[0150] Each m is an integer from 1 to 7. Therefore, each m may be
1, 2, 3, 4, 5, 6, or 7.
[0151] Each n is an integer from 0 to 7. Therefore, each n may be
0, 1, 2, 3, 4, 5, 6, or 7.
[0152] Each Q in formula (A) is C--R.sup.5, C--R.sup.6, or a
nitrogen atom, where at most three Q in a ring are nitrogen atoms.
Thus, there may be 1, 2, or 3 nitrogen atoms in a ring. Preferably,
at most two Q are nitrogen atoms. More preferably, at most one Q is
a nitrogen atom. In one particular embodiment, the nitrogen atom is
at the 3-position of the ring. In another embodiment of the
invention, each Q is either C--R.sup.5 or C--R.sup.6, i.e., there
are no nitrogen atoms in the ring.
[0153] More specific examples of suitable groups represented by
formula (A) are shown in formulas (B)-(E) below:
##STR00018##
[0154] where o, x, p, R.sup.5, and R.sup.6, are as defined
above;
##STR00019##
[0155] where n is an integer from 1 to 10 and R.sup.5 is as defined
above;
##STR00020##
[0156] where n is an integer from 1 from 10 and R.sup.5 is as
defined above;
##STR00021##
where o, x, p, and R.sup.5 are as defined above.
[0157] In a preferred embodiment of the invention, Y is
--NH.sub.2.
[0158] In another preferred embodiment, R.sup.2 is hydrogen.
[0159] In another preferred embodiment, R.sup.1 is hydrogen.
[0160] In another preferred embodiment, X is chlorine.
[0161] In another preferred embodiment, R.sup.3 is hydrogen.
[0162] In another preferred embodiment, R.sup.L is hydrogen.
[0163] In another preferred embodiment, o is 4.
[0164] In another preferred embodiment, p is 0.
[0165] In another preferred embodiment, the sum of o and p is
4.
[0166] In another preferred embodiment, x represents a single
bond.
[0167] In another preferred embodiment, R.sup.6 is hydrogen.
[0168] In another preferred embodiment, at most one Q is a nitrogen
atom.
[0169] In another preferred embodiment, no Q is a nitrogen
atom.
[0170] In a preferred embodiment of the present invention:
[0171] X is halogen;
[0172] Y is --N(R.sup.7).sub.2;
[0173] R.sup.1 is hydrogen or C.sub.1-C.sub.3 alkyl;
[0174] R.sup.2 is --R.sup.7, --OR.sup.7, CH.sub.2OR.sup.7, or
--CO.sub.2R.sup.7;
[0175] R.sup.3 is a group represented by formula (A); and
[0176] R.sup.4 is hydrogen, a group represented by formula (A), or
lower alkyl;
[0177] In another preferred embodiment of the present
invention:
[0178] X is chloro or bromo;
[0179] Y is --N(R.sup.7).sub.2;
[0180] R.sup.2 is hydrogen or C.sub.1-C.sub.3 alkyl;
[0181] at most three R.sup.6 are other than hydrogen as described
above;
[0182] at most three R.sup.L are other than hydrogen as described
above; and
[0183] at most 2 Q are nitrogen atoms.
[0184] In another preferred embodiment of the present
invention:
[0185] Y is --NH.sub.2;
[0186] In another preferred embodiment of the present
invention:
[0187] R.sup.4 is hydrogen;
[0188] at most one R.sup.L is other than hydrogen as described
above;
[0189] at most two R.sup.6 are other than hydrogen as described
above; and
[0190] at most 1 Q is a nitrogen atom.
[0191] In another preferred embodiment of the present invention the
compound of formula (1) is represented by the formula:
##STR00022##
[0192] In another preferred embodiment of the present invention the
compound of formula (1) is represented by the formula:
##STR00023##
[0193] In another preferred embodiment of the present invention the
compound of formula (1) is represented by the formula:
##STR00024##
[0194] In another preferred embodiment of the present invention the
compound of formula (1) is represented by the formula:
##STR00025##
[0195] In another preferred embodiment of the present invention the
compound of formula (1) is represented by the formula:
##STR00026##
[0196] In another preferred embodiment of the present invention the
compound of formula (1) is represented by the formula:
##STR00027##
[0197] In another preferred embodiment of the present invention the
compound of formula (1) is represented by the formula:
##STR00028##
[0198] In another preferred embodiment of the present invention the
compound of formula (1) is represented by the formula:
##STR00029##
[0199] In another preferred embodiment of the present invention the
compound of formula (1) is represented by the formula:
##STR00030##
[0200] In another preferred embodiment of the present invention the
compound of formula (1) is represented by the formula:
##STR00031##
[0201] In another preferred embodiment of the present invention the
compound of formula (1) is represented by the formula:
##STR00032##
[0202] In another preferred embodiment of the present invention the
compound of formula (1) is represented by the formula:
##STR00033##
[0203] In another preferred embodiment of the present invention the
compound of formula (1) is represented by the formula:
##STR00034##
[0204] In another preferred embodiment of the present invention the
compound of formula (1) is represented by the formula:
##STR00035##
[0205] In another preferred embodiment of the present invention the
compound of formula (1) is represented by the formula:
##STR00036##
[0206] In another preferred embodiment of the present invention the
compound of formula (1) is represented by the formula:
##STR00037##
[0207] In another preferred embodiment of the present invention the
compound of formula (1) is represented by the formula:
##STR00038##
[0208] In another preferred embodiment of the present invention the
compound of formula (1) is represented by the formula:
##STR00039##
[0209] In another preferred embodiment of the present invention the
compound of formula (1) is represented by the formula:
##STR00040##
[0210] In another preferred embodiment of the present invention the
compound of formula (1) is represented by the formula:
##STR00041##
[0211] In another preferred embodiment of the present invention the
compound of formula (1) is represented by the formula:
##STR00042##
[0212] In another preferred embodiment of the present invention the
compound of formula (1) is represented by the formula:
##STR00043##
[0213] In another preferred embodiment of the present invention the
compound of formula (1) is represented by the formula:
##STR00044##
[0214] In another preferred embodiment of the present invention the
compound of formula (1) is represented by the formula:
##STR00045##
[0215] In another preferred embodiment of the present invention the
compound of formula (1) is represented by the formula:
##STR00046##
[0216] The compounds of formula (I) may be prepared and used as the
free base. Alternatively, the compounds may be prepared and used as
a pharmaceutically acceptable salt. Pharmaceutically acceptable
salts are salts that retain or enhance the desired biological
activity of the parent compound and do not impart undesired
toxicological effects. Examples of such salts are (a) acid addition
salts formed with inorganic acids, for example, hydrochloric acid,
hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and
the like; (b) salts formed with organic acids such as, for example,
acetic acid, oxalic acid, tartaric acid, succinic acid, maleic
acid, fumaric acid, gluconic acid, citric acid, malic acid,
ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic
acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic
acid, p-toluenesulfonic acid, naphthalenedisulfonic acid,
polygalacturonic acid, malonic acid, sulfosalicylic acid, glycolic
acid, 2-hydroxy-3-naphthoate, pamoate, salicylic acid, stearic
acid, phthalic acid, mandelic acid, lactic acid and the like; and
(c) salts formed from elemental anions for example, chlorine,
bromine, and iodine.
[0217] It is to be noted that all enantiomers, diastereomers, and
racemic mixtures of compounds within the scope of formula (I) are
embraced by the present invention. All mixtures of such enantiomers
and diastereomers are within the scope of the present
invention.
[0218] Without being limited to any particular theory, it is
believed that the compounds of formula (I) function in vivo as
sodium channel blockers. By blocking epithelial sodium channels
present in mucosal surfaces the compounds of formula (I) reduce the
absorption of water by the mucosal surfaces. This effect increases
the volume of protective liquids on mucosal surfaces, rebalances
the system, and thus treats disease.
[0219] The present invention also provides methods of treatment
that take advantage of the properties of the compounds of formula
(I) discussed above. Thus, subjects that may be treated by the
methods of the present invention include, but are not limited to,
patients afflicted with cystic fibrosis, primary ciliary
dyskinesia, chronic bronchitis, chronic obstructive airway disease,
artificially ventilated patients, patients with acute pneumonia,
etc. The present invention may be used to obtain a sputum sample
from a patient by administering the active compounds to at least
one lung of a patient, and then inducing or collecting a sputum
sample from that patient. Typically, the invention will be
administered to respiratory mucosal surfaces via aerosol (liquid or
dry powders) or lavage.
[0220] Subjects that may be treated by the method of the present
invention also include patients being administered supplemental
oxygen nasally (a regimen that tends to dry the airway surfaces);
patients afflicted with an allergic disease or response (e.g., an
allergic response to pollen, dust, animal hair or particles,
insects or insect particles, etc.) that affects nasal airway
surfaces; patients afflicted with a bacterial infection e.g.,
staphylococcus infections such as Staphylococcus aureus infections,
Hemophilus influenza infections, Streptococcus pneumoniae
infections, Pseudomonas aeuriginosa infections, etc.) of the nasal
airway surfaces; patients afflicted with an inflammatory disease
that affects nasal airway surfaces; or patients afflicted with
sinusitis (wherein the active agent or agents are administered to
promote drainage of congested mucous secretions in the sinuses by
administering an amount effective to promote drainage of congested
fluid in the sinuses), or combined, Rhinosinusitis. The invention
may be administered to rhino-sinal surfaces by topical delivery,
including aerosols and drops.
[0221] The present invention may be used to hydrate mucosal
surfaces other than airway surfaces. Such other mucosal surfaces
include gastrointestinal surfaces, oral surfaces, genito-urethral
surfaces, ocular surfaces or surfaces of the eye, the inner ear and
the middle ear. For example, the active compounds of the present
invention may be administered by any suitable means, including
locally/topically, orally, or rectally, in an effective amount.
[0222] The compounds of the present invention are also useful for
treating a variety of functions relating to the cardiovascular
system. Thus, the compounds of the present invention are useful for
use as antihypertensive agents. The compounds may also be used to
reduce blood pressure and to treat edema. In addition, the
compounds of the present invention are also useful for promoting
diuresis, natriuresis, and saluresis. The compounds may be used
alone or in combination with beta blockers, ACE inhibitors, HMGCoA
reductase inhibitors, calcium channel blockers and other
cardiovascular agents to treat hypertension, congestive heart
failure and reduce cardiovascular mortality.
[0223] The present invention is concerned primarily with the
treatment of human subjects, but may also be employed for the
treatment of other mammalian subjects, such as dogs and cats, for
veterinary purposes.
[0224] As discussed above, the compounds used to prepare the
compositions of the present invention may be in the form of a
pharmaceutically acceptable free base. Because the free base of the
compound is generally less soluble in aqueous solutions than the
salt, free base compositions are employed to provide more sustained
release of active agent 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.
[0225] Another aspect of the present invention is a pharmaceutical
composition, comprising a compound of formula (I) in a
pharmaceutically acceptable carrier (e.g., an aqueous carrier
solution). In general, the compound of formula (I) is included in
the composition in an amount effective to inhibit the reabsorption
of water by mucosal surfaces.
[0226] The compounds of the present invention may also be used in
conjunction with a P2Y2 receptor agonist or a pharmaceutically
acceptable salt thereof (also sometimes referred to as an "active
agent" herein). The composition may further comprise a P2Y2
receptor agonist or a pharmaceutically acceptable salt thereof
(also sometimes referred to as an "active agent" herein). The P2Y2
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 columns 9-10 of U.S. Pat. No. 6,264,975, U.S. Pat.
No. 5,656,256, and U.S. Pat. No. 5,292,498, each of which is
incorporated herein by reference.
[0227] Bronchodiloators can also be used in combination with
compounds of the present invention. These bronchodilators include,
but are not limited to, .beta.-adrenergic agonists including but
not limited to epinephrine, isoproterenol, fenoterol, albutereol,
terbutalin, pirbuterol, bitolterol, metaproterenol, iosetharine,
salmeterol xinafoate, as well as anticholinergic agents including
but not limited to ipratropium bromide, as well as compounds such
as theophylline and aminophylline. These compounds may be
administered in accordance with known techniques, either prior to
or concurrently with the active compounds described herein.
Additional procedures useful for the preparation are found in
USUSUS especially for the preparation of various
[0228] Another aspect of the present invention is a pharmaceutical
formulation, comprising an active compound as described above in a
pharmaceutically acceptable carrier (e.g., an aqueous carrier
solution). In general, the active compound is included in the
composition in an amount effective to treat mucosal surfaces, such
as inhibiting the reabsorption of water by mucosal surfaces,
including airway and other surfaces.
[0229] The active compounds disclosed herein may be administered to
mucosal surfaces by any suitable means, including topically,
orally, rectally, vaginally, ocularly and dermally, etc. For
example, for the treatment of constipation, the active compounds
may be administered orally or rectally to the gastrointestinal
mucosal surface. The active compound may be combined with a
pharmaceutically acceptable carrier in any suitable form, such as
sterile physiological or dilute saline or topical solution, as a
droplet, tablet or the like for oral administration, as a
suppository for rectal or genito-urethral administration, etc.
Excipients may be included in the formulation to enhance the
solubility of the active compounds, as desired.
[0230] The active compounds disclosed herein may be administered to
the airway surfaces of a patient by any suitable means, including
as a spray, mist, or droplets of the active compounds in a
pharmaceutically acceptable carrier such as physiological or dilute
saline solutions or distilled water. For example, the active
compounds may be prepared as formulations and administered as
described in U.S. Pat. No. 5,789,391 to Jacobus, the disclosure of
which is incorporated by reference herein in its entirety.
[0231] Solid or liquid particulate active agents prepared for
practicing the present invention could, as noted above, include
particles of respirable or non-respirable size; that is, for
respirable particles, particles of a size sufficiently small to
pass through the mouth and larynx upon inhalation and into the
bronchi and alveoli of the lungs, and for non-respirable particles,
particles sufficiently large to be retained in the nasal airway
passages rather than pass through the larynx and into the bronchi
and alveoli of the lungs. In general, particles ranging from about
1 to 5 microns in size (more particularly, less than about 4.7
microns in size) are respirable. Particles of non-respirable size
are greater than about 5 microns in size, up to the size of visible
droplets. 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.
[0232] In the manufacture of a formulation according to the
invention, active agents or the physiologically acceptable salts or
free bases thereof are typically admixed with, inter alia, an
acceptable carrier. Of course, the carrier must be compatible with
any other ingredients in the formulation and must not be
deleterious to the patient. The carrier must be solid or liquid, or
both, and is preferably formulated with the compound as a unit-dose
formulation, for example, a capsule, that may contain 0.5% to 99%
by weight of the active compound. One or more active compounds may
be incorporated in the formulations of the invention, which
formulations may be prepared by any of the well-known techniques of
pharmacy consisting essentially of admixing the components.
[0233] Compositions containing respirable or non-respirable dry
particles of micronized active agent may be prepared by grinding
the dry active agent with a mortar and pestle, and then passing the
micronized composition through a 400 mesh screen to break up or
separate out large agglomerates.
[0234] The particulate active agent composition may optionally
contain a dispersant which serves to facilitate the formulation of
an aerosol. A suitable dispersant is lactose, which may be blended
with the active agent in any suitable ratio (e.g., a 1 to 1 ratio
by weight).
[0235] Active compounds disclosed herein may be administered to
airway surfaces including the nasal passages, sinuses and lungs of
a subject by a suitable means know in the art, such as by nose
drops, mists, etc. In one embodiment of the invention, the active
compounds of the present invention and administered by
transbronchoscopic lavage. In a preferred embodiment of the
invention, the active compounds of the present invention are
deposited on lung airway surfaces by administering an aerosol
suspension of respirable particles comprised of the active
compound, which the subject inhales. The respirable particles may
be liquid or solid. Numerous inhalers for administering aerosol
particles to the lungs of a subject are known.
[0236] Inhalers such as those developed by Inhale Therapeutic
Systems, Palo Alto, Calif., USA, may be employed, including but not
limited to those disclosed in U.S. Pat. Nos. 5,740,794; 5,654,007;
5,458,135; 5,775,320; and 5,785,049, each of which is incorporated
herein by reference. The Applicant specifically intends that the
disclosures of all patent references cited herein be incorporated
by reference herein in their entirety. Inhalers such as those
developed by Dura Pharmaceuticals, Inc., San Diego, Calif., USA,
may also be employed, including but not limited to those disclosed
in U.S. Pat. Nos. 5,622,166; 5,577,497; 5,645,051; and 5,492,112,
each of which is incorporated herein by reference. Additionally,
inhalers such as those developed by Aradigm Corp., Hayward, Calif.,
USA, may be employed, including but not limited to those disclosed
in U.S. Pat. Nos. 5,826,570; 5,813,397; 5,819,726; and 5,655,516,
each of which is incorporated herein by reference. These
apparatuses are particularly suitable as dry particle inhalers.
[0237] Aerosols of liquid particles comprising the active compound
may be produced by any suitable means, such as with a
pressure-driven aerosol nebulizer or an ultrasonic nebulizer. See,
e.g., U.S. Pat. No. 4,501,729, which is incorporated herein by
reference. Nebulizers are commercially available devices which
transform solutions or suspensions of the active ingredient into a
therapeutic aerosol mist either by means of acceleration of
compressed gas, typically air or oxygen, through a narrow venturi
orifice or by means of ultrasonic agitation. Suitable formulations
for use in nebulizers consist of the active ingredient in a liquid
carrier, the active ingredient comprising up to 40% w/w of the
formulation, but preferably less than 20% w/w. The carrier is
typically water (and most preferably sterile, pyrogen-free water)
or dilute aqueous alcoholic solution. Perfluorocarbon carriers may
also be used. Optional additives include preservatives if the
formulation is not made sterile, for example, methyl
hydroxybenzoate, antioxidants, flavoring agents, volatile oils,
buffering agents and surfactants.
[0238] Aerosols of solid particles comprising the active compound
may likewise be produced with any solid particulate medicament
aerosol generator. Aerosol generators for administering solid
particulate medicaments to a subject produce particles which are
respirable, as explained above, and generate a volume of aerosol
containing predetermined metered dose of medicament at a rate
suitable for human administration. One illustrative type of solid
particulate aerosol generator is an insufflator. Suitable
formulations for administration by insufflation include finely
comminuted powders which may be delivered by means of an
insufflator or taken into the nasal cavity in the manner of a
snuff. In the insufflator, the powder (e.g., a metered dose thereof
effective to carry out the treatments described herein) is
contained in capsules or cartridges, typically made of gelatin or
plastic, which are either pierced or opened in situ and the powder
delivered by air drawn through the device upon inhalation or by
means of a manually-operated pump. The powder employed in the
insufflator consists either solely of the active ingredient or of
powder blend comprising the active ingredient, a suitable powder
diluent, such as lactose, and an optional surfactant. The active
ingredient typically comprises of 0.1 to 100% w/w of the
formulation. A second type of illustrative aerosol generator
comprises a metered dose inhaler. Metered dose inhalers are
pressurized aerosol dispensers, typically containing a suspension
or solution formulation of active ingredient in a liquified
propellant. During use, these devices discharge the formulation
through a valve adapted to deliver a metered volume, typically from
10 to 150 .mu.l, to produce a fine particle spray containing the
active ingredient. Suitable propellants include certain
chlorofluorocarbon compounds, for example, dichlorodifluoromethane,
trichlorofluoromethane, dichlorotetrafluoroethane and mixtures
thereof. The formulation may additionally contain one of more
co-solvents, for example, ethanol, surfactants, such as oleic acid
or sorbitan trioleate, antioxidants and suitable flavoring
agents.
[0239] The aerosol, whether formed from solid or liquid particles,
may be produced by the aerosol generator at a rate of from about 10
to 150 liters per minute, more preferable from 30 to 150 liters per
minute, and most preferably about 60 liters per minute. Aerosols
containing greater amounts of medicament may be administered more
rapidly.
[0240] The dosage of the active compounds disclosed herein will
vary depending on the condition being treated and the state of the
subject, but generally may be from about 0.01, 0.03, 0.05, 0.1 to
1, 5, 10 or 20 mg of the pharmaceutic agent, deposited on the
airway surfaces. The daily dose may be divided among one or
multiple unit dose administrations. The goal is to achieve a
concentration of the pharmaceutic agents on lung airway surfaces of
between 10.sup.-9-10.sup.4 M.
[0241] In another embodiment, they are administered by
administering an aerosol suspension of respirable or non-respirable
particles (preferably non-respirable particles) comprised of active
compound, which the subject inhales through the nose. The
respirable or non-respirable particles may be liquid or solid. The
quantity of active agent included may be an amount of sufficient to
achieve dissolved concentrations of active agent on the airway
surfaces of the subject of from about 10.sup.-9, 10.sup.-8, or
10.sup.-7 to about 10.sup.-3, 10.sup.-2, 10.sup.-1 moles/liter, and
more preferably from about 10.sup.-9 to about 10.sup.-4
moles/liter.
[0242] The dosage of active compound will vary depending on the
condition being treated and the state of the subject, but generally
may be an amount sufficient to achieve dissolved concentrations of
active compound on the nasal airway surfaces of the subject from
about 10.sup.-9, 10.sup.-8, 10.sup.-7 to about 10.sup.-3,
10.sup.-2, or 10.sup.-1 moles/liter, and more preferably from about
10.sup.-7 to about 10.sup.-4 moles/liter. Depending upon the
solubility of the particular formulation of active compound
administered, the daily dose may be divided among one or several
unit dose administrations. The daily dose by weight may range from
about 0.01, 0.03, 0.1, 0.5 or 1.0 to 10 or 20 milligrams of active
agent particles for a human subject, depending upon the age and
condition of the subject. A currently preferred unit dose is about
0.5 milligrams of active agent given at a regimen of 2-10
administrations per day. The dosage may be provided as a
prepackaged unit by any suitable means (e.g., encapsulating a
gelatin capsule).
[0243] In one embodiment of the invention, the particulate active
agent composition may contain both a free base of active agent and
a pharmaceutically acceptable salt to provide both early release
and sustained release of active agent for dissolution into the
mucus secretions of the nose. Such a composition serves to provide
both early relief to the patient, and sustained relief over time.
Sustained relief, by decreasing the number of daily administrations
required, is expected to increase patient compliance with the
course of active agent treatments.
[0244] Pharmaceutical formulations suitable for airway
administration include formulations of solutions, emulsions,
suspensions and extracts. See generally, J. Naim, Solutions,
Emulsions, Suspensions and Extracts, in Remington: The Science and
Practice of Pharmacy, chap. 86 (19.sup.th ed. 1995), incorporated
herein by reference. Pharmaceutical formulations suitable for nasal
administration may be prepared as described in U.S. Pat. Nos.
4,389,393 to Schor; 5,707,644 to Illum; 4,294,829 to Suzuki; and
4,835,142 to Suzuki, the disclosures of which are incorporated by
reference herein in their entirety.
[0245] Mists or aerosols of liquid particles comprising the active
compound may be produced by any suitable means, such as by a simple
nasal spray with the active agent in an aqueous pharmaceutically
acceptable carrier, such as a sterile saline solution or sterile
water. Administration may be with a pressure-driven aerosol
nebulizer or an ultrasonic nebulizer. See e.g. U.S. Pat. Nos.
4,501,729 and 5,656,256, both of which are incorporated herein by
reference. Suitable formulation is for use in a nasal droplet or
spray bottle or in nebulizers consist of the active ingredient in a
liquid carrier, the active ingredient comprising up to 40% w/w of
the formulation, but preferably less than 20% w/w. Typically the
carrier is water (and most preferably sterile, pyrogen-free water)
or dilute aqueous alcoholic solution, preferably made in a 0.12% to
0.8% solution of sodium chloride. Optional additives include
preservatives if the formulation is not made sterile, for example,
methyl hydroxybenzoate, antioxidants, flavoring agents, volatile
oils, buffering agents, osmotically active agents (e.g. mannitol,
xylitol, erythritol) and surfactants.
[0246] Compositions containing respirable or non-respirable dry
particles of micronized active agent may be prepared by grinding
the dry active agent with a mortar and pestle, and then passing the
micronized composition through a 400 mesh screen to break up or
separate out large agglomerates.
[0247] The particulate composition may optionally contain a
dispersant which serves to facilitate the formation of an aerosol.
A suitable dispersant is lactose, which may be blended with the
active agent in any suitable ratio (e.g., a 1 to 1 ratio by
weight).
[0248] The compounds of formula (I) may be synthesized according to
procedures known in the art. A representative synthetic procedure
is shown in the scheme below:
##STR00047##
[0249] These procedures are described in, for example, E. J.
Cragoe, "The Synthesis of Amiloride and Its Analogs" (Chapter 3) in
Amiloride and Its Analogs, pp. 25-36, incorporated herein by
reference. Other methods of preparing the compounds are described
in, for example, U.S. Pat. No. 3,313,813, incorporated herein by
reference. See in particular Methods A, B, C, and D described in
U.S. Pat. No. 3,313,813. Other methods useful for the preparation
of these compounds, especially for the preparation of the novel
HNR3R4 fragment are described in, for example, 229929US, 233377US,
and 234105US, incorporated herein by reference. Schemes 1 to 11 are
representative, but limited to, of procedures used to prepare the
Sodium Channel Blockers described herein.
##STR00048##
##STR00049##
##STR00050##
##STR00051##
##STR00052##
##STR00053##
##STR00054##
##STR00055##
##STR00056##
##STR00057##
##STR00058##
[0250] Several assays may be used to characterize the compounds of
the present invention. Representative assays are discussed
below.
In Vitro Measure of Sodium Channel Blocking Activity and
Reversibility
[0251] 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 Ussing 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 ISc (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.
[0252] 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.
Pharmacological Assays of Absorption
(1) Apical Disappearance Assay
[0253] Bronchial cells (dog, human, sheep, or rodent cells) are
seeded at a density of 0.25.times.10.sup.6/cm.sup.2 on a porous
Transwell-Col collagen-coated membrane with a growth area of 1.13
cm.sup.2 grown at an air-liquid interface in hormonally defined
media that promotes a polarized epithelium. From 12 to 20 days
after development of an air-liquid interface (ALI) the cultures are
expected to be >90% ciliated, and mucins will accumulate on the
cells. To ensure the integrity of primary airway epithelial cell
preparations, the transepithelial resistance (R.sub.t) and
transepithelial potential differences (PD), which are indicators of
the integrity of polarized nature of the culture, are measured.
Human cell systems are preferred for studies of rates of absorption
from apical surfaces. The disappearance assay is conducted under
conditions that mimic the "thin" films in vivo (.about.25 .mu.l)
and is initiated by adding experimental sodium channel blockers or
positive controls (amiloride, benzamil, phenamil) to the apical
surface at an initial concentration of 10 .mu.M. A series of
samples (5 .mu.l volume per sample) is collected at various time
points, including 0, 5, 20, 40, 90 and 240 minutes. Concentrations
are determined by measuring intrinsic fluorescence of each sodium
channel blocker using a Fluorocount Microplate Fluorometer or HPLC.
Quantitative analysis employs a standard curve generated from
authentic reference standard materials of known concentration and
purity. Data analysis of the rate of disappearance is performed
using nonlinear regression, one phase exponential decay (Prism V
3.0).
[0254] 2. Confocal Microscopy Assay of Amiloride Congener
Uptake
[0255] Virtually all amiloride-like molecules fluoresce in the
ultraviolet range. This property of these molecules may be used to
directly measure cellular update using x-z confocal microscopy.
Equimolar concentrations of experimental compounds and positive
controls including amiloride and compounds that demonstrate rapid
uptake into the cellular compartment (benzamil and phenamil) are
placed on the apical surface of airway cultures on the stage of the
confocal microscope. Serial x-z images are obtained with time and
the magnitude of fluorescence accumulating in the cellular
compartment is quantitated and plotted as a change in fluorescence
versus time.
[0256] 3. In Vitro Assays of Compound Metabolism
[0257] Airway epithelial cells have the capacity to metabolize
drugs during the process of transepithelial absorption. Further,
although less likely, it is possible that drugs can be metabolized
on airway epithelial surfaces by specific ectoenzyme activities.
Perhaps more likely as an ecto-surface event, compounds may be
metabolized by the infected secretions that occupy the airway
lumens of patients with lung disease, e.g. cystic fibrosis. Thus, a
series of assays is performed to characterize the compound
metabolism that results from the interaction of test compounds with
human airway epithelia and/or human airway epithelial lumenal
products.
[0258] In the first series of assays, the interaction of test
compounds in KBR as an "ASL" stimulant are applied to the apical
surface of human airway epithelial cells grown in the T-Col insert
system. For most compounds, metabolism (generation of new species)
is tested for using high performance liquid chromatography (HPLC)
to resolve chemical species and the endogenous fluorescence
properties of these compounds to estimate the relative quantities
of test compound and novel metabolites. For a typical assay, a test
solution (25 .mu.l KBR, containing 10 .mu.M test compound) is
placed on the epithelial lumenal surface. Sequential 5 to 10 .mu.l
samples are obtained from the lumenal and serosal compartments for
HPLC analysis of (1) the mass of test compound permeating from the
lumenal to serosal bath and (2) the potential formation of
metabolites from the parent compound. In instances where the
fluorescence properties of the test molecule are not adequate for
such characterizations, radiolabeled compounds are used for these
assays. From the HPLC data, the rate of disappearance and/or
formation of novel metabolite compounds on the lumenal surface and
the appearance of test compound and/or novel metabolite in the
basolateral solution is quantitated. The data relating the
chromatographic mobility of potential novel metabolites with
reference to the parent compound are also quantitated.
[0259] To analyze the potential metabolism of test compounds by CF
sputum, a "representative" mixture of expectorated CF sputum
obtained from 10 CF patients (under IRB approval) has been
collected. The sputum has been be solubilized in a 1:5 mixture of
KBR solution with vigorous vortexing, following which the mixture
was split into a "neat" sputum aliquot and an aliquot subjected to
ultracentrifugation so that a "supernatant" aliquot was obtained
(neat=cellular; supernatant=liquid phase). Typical studies of
compound metabolism by CF sputum involve the addition of known
masses of test compound to "neat" CF sputum and aliquots of CF
sputum "supernatant" incubated at 37.degree. C., followed by
sequential sampling of aliquots from each sputum type for
characterization of compound stability/metabolism by HPLC analysis
as described above. As above, analysis of compound disappearance,
rates of formation of novel metabolities, and HPLC mobilities of
novel metabolites are then performed.
[0260] 4. Pharmacological Effects and Mechanism of Action of the
Drug in Animals
[0261] 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.
EXAMPLES
[0262] Having generally described this invention, a further
understanding can be obtained by reference to certain specific
examples which are provided herein for purposes of illustration
only and are not intended to be limiting unless otherwise
specified.
Preparation of Sodium Channel Blockers
[0263] Materials and methods. All reagents and solvents were
purchased from Aldrich Chemical Corp. and used without further
purification. NMR spectra were obtained on either a Bruker WM 360
(.sup.1H NMR at 360 MHz and .sup.13C NMR at 90 MHz) or a Bruker AC
300 (.sup.1H NMR at 300 MHz and .sup.13C NMR at 75 MHz). Flash
chromatography was performed on a Flash Elute.TM. system from
Elution Solution (PO Box 5147, Charlottesville, Va. 22905) charged
with a 90 g silica gel cartridge (40M FSO-0110-040155, 32-63 .mu.m)
at 20 psi (N.sub.2). GC-analysis was performed on a Shimadzu GC-17
equipped with a Heliflex Capillary Column (Alltech); Phase: AT-1,
Length: 10 meters, ID: 0.53 mm, Film: 0.25 micrometers. GC
Parameters: Injector at 320.degree. C., Detector at 320.degree. C.,
FID gas flow: H.sub.2 at 40 ml/min, Air at 400 ml/min. Carrier gas:
Split Ratio 16:1, N.sub.2 flow at 15 ml/min, N.sub.2 velocity at 18
cm/sec. The temperature program is 70.degree. C. for 0-3 min,
70-300.degree. C. from 3-10 min, 300.degree. C. from 10-1 15
min.
[0264] HPLC analysis was performed on a Gilson 322 Pump, detector
U/Vis-156 at 360 nm, equipped with a Microsorb MV C8 column, 100 A,
25 cm. Mobile phase: A=acetonitrile with 0.1% TFA, B=water with
0.1% TFA. Gradient program: 95:5 B:A for 1 min, then to 20:80 B:A
over 7 min, then to 100% A over 1 min, followed by washout with
100% A for 11 min, flow rate: 1 ml/min.
Example 1
Synthesis of
N-(3,5-diamino-6-chloropyrazine-2-carbonyl)-N'-(4-{4-[3-(1H-tetrazol-5-yl-
)propoxy]phenyl}butyl)guanidine hydrochloride (PSA 17926)
##STR00059##
[0265] {4-[4-(3-Cyanopropoxy)phenyl]butyl}carbamic acid benzyl
ester (2)
[0266] A mixture of [4-(4-hydroxyphenyl)butyl]carbamic acid benzyl
ester 1 (2.00 g, 6.70 mmol), 4-bromobutyronitrile (0.70 mL, 6.70
mmol), and potassium carbonate (1.00 g, 7.4 mmol) in DMF (10 mL),
was stirred at 65.degree. C. for 16 h. Solvent was removed by
rotary evaporation and the residue was taken up in ethyl acetate,
washed with water and brine, and concentrated under vacuum. The
crude product was purified by flash silica gel column
chromatography eluting with ethyl acetate/CH.sub.2Cl.sub.2 (1:9,
v/v) to give the desired product 2 as a white solid (1.80 g, 75%
yield). .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 1.56 (m, 4H),
2.15 (m, 2H), 2.55 (m, 4H), 3.15 (m, 2H), 4.00 (m, 2H), 4.70 (br s,
1H), 5.10 (s, 2H), 6.80 (d, 2H), 7.05 (d, 2H), 7.30 (m, 5H). m/z
(ESI): 367 [C.sub.22H.sub.26N.sub.2O.sub.3+H].sup.+.
(4-{4-[3-(1H-Tetrazol-5-yl)propoxy]phenyl}butyl)carbamic acid
benzyl ester (3)
[0267] A mixture of {4-[4-(3-cyanopropoxy)phenyl]butyl}carbamic
acid benzyl ester 2 (0.90 g, 2.5 mmol), sodium azide (0.50 g, 7.5
mmol), and ammonium chloride (0.40 g, 7.5 mmol) in DMF (7 mL), was
stirred at 120.degree. C. for 16 h. Inorganics were removed by
vacuum filtration. The filtrate was diluted with ethyl acetate, and
washed with water and brine. The organic solution was dried over
Na.sub.2SO.sub.4, filtered and concentrated. The residue was taken
up in ethyl acetate (5 mL) and diluted with hexanes (10 mL). Solid
precipitates were collected by suction filtration and purified by
flash silica gel column chromatography eluting with
methanol/dichloromethane (1:50, v/v) to give the desired product 3
as a white solid (0.78 g, 76% yield). .sup.1H NMR (300 MHz,
CD.sub.3OD) .delta. 1.51 (m, 4H), 2.20 (m, 2H), 2.50 (m, 2H), 3.10
(m, 4H), 4.00 (m, 2H), 5.00 (s, 2H), 6.75 (d, 2H), 7.05 (d, 2H),
7.30 (m, 5H). m/z (ESI): 410
[C.sub.22H.sub.27N.sub.5O.sub.3+H].sup.+.
4-{4-[3-(1H-Tetrazol-5-yl)propoxy]phenyl}butylamine (4)
[0268] A solution of
(4-{4-[3-(1H-tetrazol-5-yl)propoxy]phenyl}butyl)carbamic acid
benzyl ester 3 (0.30 g, 0.73 mmol) in methanol (20 mL) and
dichloromethane (5 mL) was stirred at room temperature overnight
under hydrogen atmosphere in the presence of 10%
palladium-on-carbon catalyst (0.1 g, 50% wet). The catalyst was
removed by suction filtration, and the filtrate was concentrated in
vacuo to give the desired product 4 as a white solid (200 mg, 99%
yield) which was used for the next step without further
purification. m/z (ESI): 276
[C.sub.14H.sub.21N.sub.5O+H].sup.+.
N-(3,5-Diamino-6-chloropyrazine-2-carbonyl)-N'-(4-{4-[3-(1H-tetrazol-5-yl)-
-propoxy]phenyl}butyl)guanidine hydrochloride (5, PSA 17926)
[0269] A solution of
4-{4-[3-(1H-tetrazol-5-yl)propoxy]phenyl}butylamine 4 (100 mg, 0.36
mmol) and triethylamine (0.15 mL, 0.39 mmol) in absolute ethanol (2
mL) was stirred at 60.degree. C. for 5 min, after which
1-(3,5-diamino-6-chloropyrazine-2-carbonyl)-2-methyl-isothiourea
hydriodide (150 mg, 0.39 mol) was added in one portion. The
reaction mixture was stirred at that temperature for 4 h and then
cooled to room temperature. The reaction mixture was concentrated
by rotary evaporation. The crude residue was washed with water and
filtered. The filter cake was further washed with dichloromethane.
A dark yellow solid (140 mg, 80% yield) thus obtained was slurried
in a mixture of methanol and dichloromethane (5/95, v/v). The solid
was collected by suction filtration, and 40 mg of such solid was
mixed with 3% aqueous HCl (4 mL). The mixture was sonicated,
stirred at room temperature for 15 min and filtered. The filter
cake was dried under high vacuum to give
N-(3,5-diamino-6-chloropyrazine-2-carbonyl)-N'-(4-{4-[3-(1H-tetrazol-5-yl-
)propoxy]phenyl}butyl)guanidine hydrochloride (5, PSA 17926) as a
yellow solid. mp 125-127.degree. C. (decomposed). .sup.1H NMR (300
MHz, CD.sub.3OD) .delta. 1.70 (m, 4H), 2.22 (m, 2H), 2.60 (m, 2H),
3.10 (m, 2H), 4.00 (m, 2H), 6.70 (d, 2H), 7.09 (d, 2H). m/z (ESI):
488 [C.sub.20H.sub.26ClN.sub.11O.sub.2+H].sup.+.
Example 2
Synthesis of dimethylthiocarbamic acid
O-(4-{4-[N'-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}phen-
yl) ester (PSA 17846)
##STR00060##
[0270] 2-[4-(4-Hydroxyphenyl)butyl]isoindole-1,3-dione (8)
[0271] A mixture of 4-(4-aminobutyl)phenol hydrobromide 6 (8.2 g,
33.5 mmol), phthalic anhydride 7 (5.0 g, 33.8 mmol), and
triethylamine (4.6 mL, 33.5 mmol) in chloroform (50 mL) was stirred
at reflux for 18 h, cooled to room temperature and concentrated by
rotary evaporation. The residue was dissolved in acetic acid (50
mL) and stirred at 100.degree. C. for 3 h. Solvent was evaporated
and the resulting residue was purified by flash silica gel column
chromatography eluting with CH.sub.2Cl.sub.2/EtOAc/hexanes (8:1:1,
v/v) to give the desired product 8 as a white powder (4.1 g, 41%
yield). .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 1.57 (m, 4H),
2.46 (m, 2H), 3.58 (m, 2H), 6.64 (d, 2H), 6.95 (d, 2H), 7.82 (m,
4H), 9.12 (s, 1H). m/z (ESI): 296
[C.sub.18H.sub.17NO.sub.3+H].sup.+.
Dimethylthiocarbamic acid
O-{4-[4-(1,3-dioxo-1,3-dihydroisoindol-2-yl)butyl]-phenyl}ester
(9)
[0272] A suspension of sodium hydride (60% in mineral oil, 0.44 g,
0.11 mmol) in anhydrous DMF (10 mL) was cooled to 0.degree. C. and
added to a solution of
2-[4-(4-hydroxyphenyl)-butyl]isoindole-1,3-dione 8 (2.95 g, 10
mmol) in DMF (15 mL). The mixture was stirred at 0.degree. C. for
30 min and then at room temperature for an additional one hour. A
solution of dimethylthiocarbamic acid chloride (1.35 g, 11 mmol) in
DMF (10 mL) was then added. The reaction mixture was stirred at
room temperature first for 16 h and then at 50.degree. C. for 1 h,
cooled back to room temperature and quenched with methanol (10 mL).
The mixture was concentrated under vacuum and the residue was
purified by flash silica gel column chromatography eluting with
CH.sub.2Cl.sub.2/hexanes/EtOAc (10:1:0.2, v/v) to give the desired
product 9 as a yellowish solid (2.27 g, 59% yield). .sup.1H NMR
(300 MHz, CDCl.sub.3) .delta. 1.72 (m, 4H), 2.67 (m, 2H), 3.33 (s,
3H), 3.45 (s, 3H), 3.71 (m, 2H), 6.95 (d, 2H), 7.18 (d, 2H), 7.70
(m, 2H), 7.84 (m, 2H). m/z (ESI): 383
[C.sub.21H.sub.22N.sub.2O.sub.3S+H].sup.+.
Dimethylthiocarbamic acid O-[4-(4-aminobutyl)phenyl]ester (10)
[0273] A mixture of dimethylthiocarbamic acid
O-{4-[4-(1,3-dioxo-1,3-dihydroisoindol-2-yl)-butyl]phenyl}ester 9
(0.30 g, 0.80 mmol) and methylamine (2M in methanol, 10 mL, 20
mmol) was stirred at room temperature overnight. Solvent was
removed by rotary evaporation and the residue was purified by flash
silica gel column chromatography (Biotage) eluting with
chloroform/methanol/concentrated ammonium hydroxide (10:1:0.1, v/v)
to give dimethylthiocarbamic acid O-[4-(4-aminobutyl)phenyl]ester
(10) as a clear colorless oil (118 mg, 46% yield). .sup.1H NMR (300
MHz, CD.sub.3OD) .delta. 1.70 (m, 4H), 2.70 (m, 4H), 3.34 (s, 3H),
3.46 (s, 3H), 6.96 (d, 2H), 7.20 (d, 2H). m/z (ESI): 253
[C.sub.13H.sub.20N.sub.2OS+H].sup.+.
Dimethylthiocarbamic acid
O-(4-{4-[N'-(3,5-diamino-6-chloropyrazine-2-carbonyl)-guanidinio]butyl}ph-
enyl) ester (11, PSA 17846)
[0274] A solution of dimethylthiocarbamic acid
O-[4-(4-aminobutyl)phenyl]ester 10 (15 mg, 0.45 mmol),
triethylamine (0.30 mL, 2.2 mmol), and
1-(3,5-diamino-6-chloropyrazine-2-carbonyl)-2-methylisothiourea
hydriodide (175 mg, 0.45 mmol) in anhydrous THF (6 mL) was stirred
at reflux for 3 h and then cooled to room temperature. The reaction
mixture was concentrated by rotary evaporation. The crude residue
was purified by flash silica gel column chromatography (Biotage)
eluting with chloroform/methanol/concentrated ammonium hydroxide
(15:1:0.1, v/v) to give the desired product 11 as a yellow solid
(180 mg, 86% yield). mp 102-105.degree. C. .sup.1H NMR (300 MHz,
CD.sub.3OD) .delta. 1.70 (m, 4H), 2.65 (m, 2H), 3.20 (m, 2H), 3.30
(s, 3H), 3.40 (s, 3H), 6.95 (d, 2H), 7.20 (d, 2H). m/z (ESI): 465
[C.sub.19H.sub.25ClN.sub.8O.sub.2S+H].sup.+.
Example 3
Synthesis of
(2S)-(4-{4-[N'-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino]-butyl}-
benzenesulfonylamino)-3-methylbutyramide (PSA 19008)
##STR00061##
[0275]
4-[4-(1,3-Dioxo-1,3-dihydroisoindol-2-yl)butyl]benzenesulfonyl
chloride (13)
[0276] 2-(4-Phenylbutyl)isoindole-1,3-dione 12 (1.9 g, 6.8 mmol)
was added to chlorosulfonic acid (10 mL, 138 mmol) at 0.degree. C.
and the mixture was stirred for 1 h at the temperature. After
storing in refrigerator at -5.degree. C. overnight, the reaction
mixture was poured onto crushed ice (100 g) and precipitates were
collected by a suction filtration and dried under high vacuum to
afford the desired product 13 (2.48 g, 99% yield). .sup.1H NMR (300
MHz, CDCl.sub.3) .delta. 1.70 (m, 4H), 2.78 (m, 2H), 3.70 (m, 2H),
7.40 (d, 2H) 7.70 (d, 2H), 7.85 (d, 2H), 7.95 (d, 2H).
(2S)-{4-[4-(1,3-Dioxo-1,3-dihydroisoindol-2-yl)butyl]benzenesulfonylamino}-
-3-methylbutyramide (14)
[0277]
4-[4-(1,3-Dioxo-1,3-dihydroisoindol-2-yl)butyl]benzenesulfonyl
chloride 13 (0.45 g, 1.19 mmol) was dissolved in dry DMF (5 mL),
and added to a solution of N-methylmorpholine (3 mL) and
(2S)-amino-3-methylbutyramide (0.18 g, 1.19 mmol) in DMF (10 mL).
The reaction mixture was stirred at room temperature for 66 h.
Solvent was removed by rotary evaporation and the residue was
purified by flash silica gel chromatography eluting with
chloroform/methanol/concentrated ammonium hydroxide (15:1:0.1, v/v)
to give the desired product 14 as a white powder (0.41 g, 73%
yield). .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 0.72 (d, 3H),
0.76 (d, 3H), 1.77 (m, 4H), 1.79 (m, 1H), 2.68 (m, 2H), 3.40 (m,
1H), 3.60 (m, 2H), 6.92 (s, 1H), 7.21 (s, 1H), 7.34 (d, 2H) 7.50
(d, 1H), 7.65 (d, 2H), 7.82 (m, 4H).
(2S)-[4-(4-Aminobutyl)benzenesulfonylamino]-3-methylbutyramide
(15)
[0278] A mixture of
(2S)-{4-[4-(1,3-dioxo-1,3-dihydroisoindol-2-yl)butyl]-benzenesulfonylamin-
o}-3-methylbutyramide 14 (0.40 g, 0.87 mmol) and methylamine (2 M
in methanol, 20 mL, 40 mmol) was stirred at room temperature
overnight. Solvent was removed by rotary evaporation and the
residue was purified by flash silica gel column chromatography
eluting with chloroform/methanol/concentrated ammonium hydroxide
(3:1:0.1, v/v) to give
(2S)-[4-(4-aminobutyl)benzenesulfonylamino]-3-methyl-butyramide
(15) as a white powder (156 mg, 54% yield). .sup.1H NMR (300 MHz,
CD.sub.3OD) .delta. 0.85 (d, 3H), 0.87 (d, 3H), 1.66 (m, 4H), 1.90
(m, 1H), 2.69 (m, 4H), 3.51 (d, 1H), 7.35 (d, 2H) 7.75 (d, 2H). m/z
(ESI): 328 [C.sub.15H.sub.25N.sub.3O.sub.3S+H].sup.+.
(2S)-(4-{4-[N'-(3,5-Diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}-b-
enzenesulfonylamino)-3-methylbutyramide (16, PSA 19008)
[0279] A solution of
(2S)-[4-(4-aminobutyl)benzenesulfonylamino]-3-methylbutyramide 15
(156 mg, 0.47 mmol), diisopropylethylamine (0.60 mL, 3.0 mmol), and
1-(3,5-diamino-6-chloropyrazine-2-carbonyl)-2-methylisothiourea
hydriodide (230 mg, 0.61 mmol) in absolute ethanol (8 mL) was
stirred at 70.degree. C. for 5 h and then cooled to room
temperature. The reaction mixture was concentrated by rotary
evaporation. The crude residue was washed with water, filtered and
the crude solid product was purified by flash silica gel column
chromatography eluting with chloroform/methanol/concentrated
ammonium hydroxide (5:1:0.1, v/v) to give the desired product as a
yellow solid (137 mg, 54% yield). Part of the solid (86 mg) was
further purified by semi-preparative HPLC (acetonitrile/water/0.1%
TFA) to give the analytical pure sample which was then
co-evaporated with 5% aqueous HCl to give the hydrochloride salt
16. mp 154-156.degree. C. (decomposed). .sup.1H NMR (300 MHz,
CD.sub.3OD) .delta. 0.85 (d, 3H), 0.86 (d, 3H), 1.70 (m, 4H), 1.90
(m, 1H), 2.75 (m, 2H), 3.32 (m, 2H), 3.52 (d, 1H), 7.35 (d, 2H),
7.75 (d, 2H). m/z (ESI): 540
[C.sub.21H.sub.30ClN.sub.9O.sub.4S+H].sup.+.
[.alpha.].sub.D.sup.25+5.2.degree. (c 0.50, MeOH).
Example 4
Synthesis of
2-(4-{4-[N'-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino]-butyl}phe-
noxy)-N-phenylacetamide (PSA 17482)
##STR00062##
[0280] [4-(4-Phenylcarbamoylmethoxyphenyl)butyl]carbamic acid
benzyl ester (18)
[0281] A mixture of
[4-(4-benzyloxycarbonylaminobutyl)phenoxy]acetic acid (300 mg, 0.84
mmol), aniline (0.15 mL, 1.70 mmol), DMAP (60 mg, 0.50 mmol) and
EDC.HCl (320 mg, 1.70 mmol) in CH.sub.2Cl.sub.2 (30 mL) was stirred
at room temperature for 66 h. The reaction mixture was concentrated
under vacuum and the residue was subjected to flash silica gel
column chromatography eluting with methanol/CH.sub.2Cl.sub.2 (1:99,
v/v) to give the desired amide 18 as a white solid (360 mg, 99%
yield). .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 1.55 (m, 4H),
2.60 (m, 2H), 3.20 (m, 2H), 4.58 (s, 2H), 4.70 (br s, 1H), 5.10 (s,
2H), 6.88 (d, 2H), 7.15 (m, 3H), 7.35 (m, 7H), 7.58 (d, 2H), 8.25
(s, 1H). m/z (ESI): 433
[C.sub.26H.sub.28N.sub.2O.sub.4.sup.+H].sup.+.
2-[4-(4-Aminobutyl)phenoxy]-N-phenylacetamide (19)
[0282] A solution of
[4-(4-phenylcarbamoylmethoxyphenyl)butyl]carbamic acid benzyl ester
18 (0.30 g, 0.69 mmol) in ethanol (10 mL), THF (6 mL), and acetic
acid (2 mL) was stirred at room temperature for 2 h under hydrogen
atmosphere in the presence of 10% Pd/C catalyst (0.2 g, 50% wet).
The catalyst was removed by suction filtration and the filtrate was
concentrated in vacuo. The residue was purified by flash silica gel
column chromatography eluting with
CH.sub.2Cl.sub.2/methanol/concentrated ammonium hydroxide (30:1:0,
30:1:0.3, v/v) to give the desired amine 19 as a white solid (200
mg, 97% yield). .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 1.60 (m,
4H), 2.55 (m, 2H), 2.70 (m, 2H), 4.60 (s, 2H), 6.88 (d, 2H), 7.15
(m, 3H), 7.35 (m, 2H), 7.58 (d, 2H), 8.25 (s, 1H). m/z (ESI): 299
[C.sub.18H.sub.22N.sub.2O.sub.2+H].sup.+.
2-(4-{4-[N'-(3,5-Diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}pheno-
xy)-N-phenylacetamide (20, PSA 17482).
[0283] A solution of 2-[4-(4-aminobutyl)phenoxy]-N-phenylacetamide
19 (100 mg, 0.35 mmol) and triethylamine (0.14 mL, 1.00 mmol) in
absolute ethanol (2 mL) was stirred at 60.degree. C. for 30 min,
after which
1-(3,5-diamino-6-chloropyrazine-2-carbonyl)-2-methyl-isothiourea
hydriodide (140 mg, 0.37 mmol) was added in one portion. The
reaction mixture was stirred at that temperature for 4 h, cooled to
room temperature, and concentrated by rotary evaporation. The crude
residue was triturated with water and filtered. The filter cake was
purified by flash silica gel column chromatography eluting with
dichloromethane/methanol/concentrated ammonium hydroxide (500:10:0,
500:10:1, 200:10:1, v/v) to give
2-(4-{4-[N'-(3,5-diamino-6-chloro-pyrazine-2-carbonyl)guanidino]butyl}phe-
noxy)-N-phenylacetamide (20, PSA 17482) as a yellow solid (120 mg,
67% yield). mp 168-170.degree. C. .sup.1H NMR (300 MHz,
DMSO-d.sub.6) .delta. 1.55 (m, 4H), 2.55 (m, 21), 3.16 (m, 21),
4.65 (s, 2H), 6.60 (br s, 2H), 6.90 (d, 21), 7.08 (m, 2H), 7.15 (d,
2H), 7.30 (m, 5H), 7.60 (d, 2H), 9.00 (br s, 1H), 10.00 (br s, 1H).
m/z (ESI): 511 [C.sub.24H.sub.27ClN.sub.8O.sub.3+H].sup.+.
Example 5
Synthesis of
N-(3,5-diamino-6-chloropyrazine-2-carbonyl)-N'-(4-{4-[3-(1H-imidazol-2-yl-
)propoxy]phenyl}butyl)guanidine (PSA 23022)
##STR00063##
[0284] 4-{4-[3-(1H-Imidazol-2-yl)propoxy]phenyl}butylamine (21)
[0285] Compound 2 (0.156 g, 0.425 mmol) was dissolved in anhydrous
ethanol (10 mL). To the solution was bubbled anhydrous HCl gas for
3 min. The reaction vessel was sealed and the mixture was stirred
at room temperature for 48 h, and then concentrated to dryness
under vacuum. The resulting residue was dissolved in anhydrous
methanol (5 mL). To the newly formed solution was added
2,2-dimethoxyethylamine (0.097 mL, 0.891 mmol) in one portion.
After stirring at room temperature overnight, temperature was
raised to reflux which was maintained for another 3 h before the
mixture was cooled to ambient temperature. Solvent was removed
under vacuum and the residue was treated with 1.2 N HCl aqueous
solution at 80.degree. C. for 2 hours. The mixture was then cooled
to ambient temperature again and neutralized to pH .about.9 with
powder K.sub.2CO.sub.3. Water was completely removed under vacuum
and the residue was dissolved in methanol. The methanol solution
was loaded onto silica gel, and the product was eluted with a
mixture of concentrated ammonium hydroxide/MeOH/CH.sub.2Cl.sub.2
(1.8:18:81.2, v/v), affording the product 21 (27 mg, 23% overall
yield) as an off-white solid. .sup.1H NMR (300 MHz, CD.sub.3OD):
.delta. 1.60 (m, 4H), 2.14 (m, 2H), 2.56 (t, 2H), 2.76 (t, 2H),
2.86 (t, 2H), 3.94 (t, 2H), 6.79 (d, 2H), 6.91 (s, 2H), 7.08 (d,
2H). m/z (APCI): 274 [C.sub.16H.sub.23N.sub.3O+H].sup.+.
N-(3,5-Diamino-6-chloropyrazine-2-carbonyl)-N'-(4-{4-[3-(1H-imidazol-2-yl)-
propoxy]phenyl}butyl)guanidine (22, PSA 23022)
[0286] Compound 21 (23 mg, 0.084 mmol) was dissolved in a mixture
of ethanol (3 mL) and Hunig's base (0.074 mL, 0.421 mmol) at
65.degree. C. over 15 min. To the solution was added
1-(3,5-diamino-6-chloropyrazine-2-carbonyl)-2-methylisothiourea
hydriodide (43 mg, 0.109 mmol) and the resulting mixture was
stirred at the above temperature for an additional 3 h before all
liquid was removed under vacuum. The residue was chromatographed
oil silica gel, eluting with a mixture of concentrated ammonium
hydroxide/methanol/dichloromethane (1.5:15:63.5, v/v), to afford
the desired product 22 (34 mg, 83% yield) as a yellow solid. mp
123-126.degree. C. (decomposed), .sup.1H NMR (300 MHz, CD.sub.3OD):
.delta. 1.62 (m, 4H), 2.14 (m, 2H), 2.58 (t, 2H), 2.88 (t, 2H),
3.21 (t, 2H), 3.94 (t, 2H), 6.77 (d, 2H), 6.90 (s, 2H), 7.06 (d,
2H). m/z (APCI): 486
[C.sub.22H.sub.28ClN.sub.9O.sub.2+H].sup.+.
Example 6
Synthesis of
2-(4-{4-[N'-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino]-butyl}phe-
noxy)-N,N-bis-(2-hydroxyethyl)acetamide (PSA 16826)
##STR00064##
[0287]
[4-(4-{[N,N-Bis-(2-hydroxyethyl)carbamoyl]methoxy}phenyl)butyl]carb-
amic acid benzyl ester (25)
[0288] A solution of
[4-(4-benzyloxycarbonylaminobutyl)phenoxy]acetic acid ethyl ester
23 (0.3 g, 0.78 mmol), 2-(2-hydroxyethylamino)ethanol 24 (0.15 mL,
1.6 mmol), and ethanol (20 mL) was heated at 70.degree. C. for 72
hours. Solvent was evaporated in vacuo. The residue was purified by
flash chromatography (silica gel, dichloromethane/methanol, 100:5,
v/v) to provide
[4-(4-{[N,N-bis-(2-hydroxyethyl)carbamoyl]methoxy}phenyl)-butyl]carbamic
acid benzyl ester 25 [0.19 g, 100% based on the recovered starting
material (0.13 g)] as a pale yellow solid. .sup.1H NMR (300 MHz,
CD.sub.3OD) .delta. 1.65 (m, 4H), 2.50 (m, 2H), 3.20 (m, 2H), 3.55
(m, 4H), 3.75 (m, 4H), 4.80 (s, 2H), 5.10 (s, 2H), 6.85 (d, 2H),
7.10 (d, 2H), 7.40 (m, 5H). m/z (ESI): 445
[C.sub.24H.sub.32N.sub.2O.sub.6+H].sup.+.
2-[4-(4-Aminobutyl)phenoxy]-N,N-bis-(2-hydroxyethyl)acetamide
(26)
[0289] To a degassed solution of
[4-(4-{[N,N-bis-(2-hydroxyethyl)carbamoyl]methoxy}phenyl)-butyl]carbamic
acid benzyl ester 25 (0.19 g, 0.43 mmol) in ethanol (4 mL) was
added 10% palladium on activated carbon (0.1 g, 50% wet). The
mixture was hydrogenated overnight at atmospheric hydrogen. The
catalyst was filtered through a pad of diatomaceous earth and the
solvent was evaporated in vacuo. The residue was purified by flash
chromatography (silica gel, 20-5:1:0.1-1
dichloromethane/methanol/concentrated ammonium hydroxide, v/v) to
provide 26 (0.09 g, 72%) as a colorless oil. .sup.1H NMR (300 MHz,
CD.sub.3OD) .delta. 1.56 (m, 4H), 2.56 (t, 2H), 2.65 (t, 1H), 3.29
(m, 1H), 3.55 (m, 4H), 3.72 (m, 4H), 4.90 (s, 2H), 6.86 (d, 2H),
7.09 (d, 2H). m/z (ESI): 311
[C.sub.16H.sub.26N.sub.2O.sub.4.sup.+H].sup.+.
2-(4-{4-[N'-(3,5-Diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}pheno-
xy)-N,N-bis-(2-hydroxyethyl)acetamide (27, PSA 16826)
[0290]
1-(3,5-Diamino-6-chloropyrazine-2-carbonyl)-2-methylisothiourea
hydriodide (0.13 g, 0.33 mmol) was added to a solution of
2-[4-(4-aminobutyl)phenoxy]-N,N-bis-(2-hydroxyethyl)acetamide 26
(0.09 g, 0.3 mmol), triethylamine (0.12 mL), and ethanol (1.7 mL).
The reaction mixture was stirred at 60.degree. C. for 3 h. The
solvent was evaporated in vacuo. The residue was triturated with
water and then purified by flash chromatography (silica gel,
20-10:1:0-0.2 CH.sub.2Cl.sub.2/methanol/concentrated ammonium
hydroxide, v/v) to provide
2-(4-{4-[N'-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino]bu-
tyl}-phenoxy)-N,N-bis-(2-hydroxyethyl)acetamide 27 (0.1 g, 64%) as
a yellow solid. mp 114-116.degree. C. .sup.1H NMR (300 MHz,
CD.sub.3OD) .delta. 1.70 (m, 4H), 2.60 (m, 2H), 3.32 (m, 2H), 3.50
(m, 4H), 3.70 (m, 4H), 4.81 (s, 2H), 6.85 (d, 2H), 7.10 (d, 2H).
m/z (ESI): 523 [C.sub.22H.sub.31ClN.sub.8O.sub.5+H].sup.+.
Example 7
Synthesis of
2-(4-{4-[N'-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino]-butyl}phe-
noxy)-N,N-dimethylacetamide hydrochloride (PSA 16313)
##STR00065##
[0291] [4-(4-Dimethylcarbamoylmethoxyphenyl)butyl]carbamic acid
benzyl ester (28)
[0292] A mixture of
[4-(4-benzyloxycarbonylaminobutyl)phenoxy]acetic acid ethyl ester
23 (0.50 g, 1.3 mmol) and dimethylamine (2.0 M in THF, 10 mL, 20
mmol) in a sealed tube was heated at 55.degree. C. for 48 h. The
solvent was evaporated in vacuo. The residue was purified by flash
chromatography (silica gel, ethyl acetate/CH.sub.2Cl.sub.2, 1:4,
1:3, v/v) to provide
[4-(4-dimethylcarbamoylmethoxyphenyl)butyl]carbamic acid benzyl
ester 28 (0.26 g, 52% yield) as a white solid. .sup.1H NMR (300
MHz, CDCl.sub.3) .delta. 1.55 (m, 4H), 2.55 (m, 2H), 2.90 (s, 3H),
3.05 (s, 3H), 3.20 (m, 2H), 4.65 (s, 2H), 5.08 (s, 2H), 6.80 (d,
2H), 7.05 (d, 2H), 7.35 (m, 5H).
2-[4-(4-Aminobutyl)phenoxy]-N,N-dimethylacetamide (29)
[0293] To a degassed solution of
[4-(4-dimethylcarbamoylmethoxyphenyl)butyl]carbamic acid benzyl
ester (28) (0.26 g, 0.68 mmol) in ethanol (10 mL) was added 10%
palladium on activated carbon (0.1 g, 50% wet). The mixture was
stirred at room temperature overnight under atmospheric hydrogen.
The catalyst was filtered through a pad of diatomaceous earth and
the solvent was evaporated in vacuo. The residue was purified by
flash chromatography (silica gel,
dichloromethane/methanol/concentrated ammonium hydroxide, 100:5:1,
v/v) to provide 2-[4-(4-aminobutyl)phenoxy]-N,N-dimethylacetamide
29 (100 mg, 60% yield) as a white solid. .sup.1H NMR (300 MHz,
CD.sub.3OD) .delta. 1.55 (m, 4H), 2.55 (m, 2H), 2.66 (m, 2H), 2.90
(s, 3H), 3.05 (s, 3H), 4.70 (s, 2H), 6.80 (d, 2H), 7.05 (d, 2H).
m/z (ESI): 251 [C.sub.14H.sub.22N.sub.2O.sub.2+H].sup.30 .
2-(4-{4-[N'-(3,5-Diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}pheno-
xy)-N,N-dimethylacetamide hydrochloride (30, PSA 16313)
[0294] A solution of
2-[4-(4-aminobutyl)phenoxy]-N,N-dimethylacetamide 29 (67 mg, 0.27
mmol) in absolute ethanol (1 mL) was stirred at 65.degree. C. for
30 min, after which
1-(3,5-diamino-6-chloropyrazine-2-carbonyl)-2-methylisothiourea
hydriodide (110 mg, 0.29 mmol) was added in one portion. The
reaction mixture was stirred at that temperature for 3 h and then
cooled to room temperature. The reaction mixture was concentrated
by rotary evaporation. The crude residue was triturated with water
and filtered. The filter cake was purified by flash silica gel
column chromatography eluting with
dichloromethane/methanol/concentrated ammonium hydroxide (200:10:0,
200:10:1, v/v) to give
2-(4-{4-[N'-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}-phe-
noxy)-N,N-dimethylacetamide as a yellow solid (35 mg, 28% yield).
This solid was dissolved in methanol (2 mL) and added to 4 N
aqueous HCl (4 drops). Concentration in vacuo gave
2-(4-{4-[N'-(3,5-diamino-6-chloropyrazine-2-carbonyl)-guanidino]butyl}phe-
noxy)-N,N-dimethylacetamide hydrochloride (30, PSA 16313). mp
130-132.degree. C. (decomposed). .sup.1H NMR (300 MHz, CD.sub.3OD)
.delta. 1.69 (m, 4H), 2.60 (m, 2H), 2.95 (s, 3H), 3.10 (s, 3H),
3.35 (m, 2H), 4.75 (s, 2H), 6.80 (d, 2H), 7.10 (d, 2H). m/z (ESI):
463 [C.sub.20H.sub.27ClN.sub.8O.sub.3+H].sup.+.
Example 8
Synthesis of
2-(4-{4-[N'-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino]-butyl}phe-
noxy)-N-(1H-imidazol-2-yl)acetamide dihydrochloride (PSA 16437)
##STR00066##
[0295] [4-(4-tert-Butoxycarbonylaminobutyl)phenoxy]acetic acid
methyl ester (32)
[0296] A mixture of [4-(4-hydroxyphenyl)butyl]carbamic acid
tert-butyl ester 31 (1.00 g, 3.78 mmol), potassium carbonate (0.627
g, 4.54 mmol), sodium iodide (0.567 g, 3.78 mmol), and methyl
bromoacetate (0.40 mL, 4.21 mmol) in anhydrous DMF (8 mL) was
stirred at room temperature for 14 h. The reaction mixture was then
diluted with ethyl acetate (100 mL) and hexanes (20 mL), washed
with water (20 mL.times.4) and brine (30 mL), and concentrated
under reduced pressure to afford the desired product 32 as a yellow
oil (1.28 g, 100% yield) which was used for the next step without
further purification. .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.
1.40 (s, 9H), 1.41-1.65 (m, 4H), 2.49-2.60 (m, 2H), 3.02-3.16 (m,
2H), 3.79 (s, 3H), 4.45 (br s, 1H), 4.59 (s, 2H), 6.79 (d, 2H),
7.05 (d, 2H). m/z (ESI): 338
[C.sub.18H.sub.27NO.sub.5+H].sup.+.
[4-(4-tert-Butoxycarbonylaminobutyl)phenoxy]acetic acid (33)
[0297] A solution of
[4-(4-tert-butoxycarbonylaminobutyl)phenoxy]acetic acid methyl
ester 32 (1.28 g, 3.78 mmol) in methanol (80 mL) was added with
crushed potassium hydroxide (2.50 g, 85%, 37.8 mmol) and the
mixture was stirred at room temperature for 5 h. Solvent was
removed by rotary evaporation. The residue was taken up in water
and acidified to pH .about.1 with 6N aqueous HCl, and extracted
with dichloromethane. The combined organics were washed with brine,
dried over Na.sub.2SO.sub.4, and concentrated to complete dryness
to afford the desired product 33 as a white solid (1.19 g, 97%
yield). .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 1.41 (s, 9H),
1.42-1.70 (m, 4H), 2.45-2.60 (m, 2H), 3.00-3.20 (m, 2H), 4.60 (s,
2H), 6.80 (d, 2H), 7.08 (d, 2H). m/z (ESI): 322
[C.sub.17H.sub.25NO.sub.5--H].sup.-.
(4-{4-[(1H-Imidazol-2-yl-carbamoyl)methoxy]phenyl}butyl)carbamic
acid tert-butyl ester (34)
[0298] [4-(4-tert-Butoxycarbonylaminobutyl)phenoxy]acetic acid 33
(1.19 g, 3.68 mmol) was dissolved in anhydrous THF (10 mL),
CH.sub.2Cl.sub.2 (10 mL) and CH.sub.3CN (5 mL). To the solution
were sequentially added HOAt (200 mg, 1.47 mmol), DMAP (135 mg,
1.10 mmol), and diisopropylethylamine (3.2 mL, 18.40 mmol),
followed by the addition of EDC.HCl (1.03 g, 5.35 mmol). The
reaction mixture was stirred at room temperature for 15 min. Amino
imidazole sulfate (583 mg, 4.41 mmol) was then added and stirring
was continued for 48 h. Solvents were removed by rotary
evaporation. The residue was taken up in CH.sub.2Cl.sub.2 (250 mL),
washed with water and brine, and concentrated under reduced
pressure. Flash silica gel column chromatography eluting with
methanol/dichloromethane (1:30, 1:20, v/v) gave the desired amide
as a white solid (0.95 g, 66% yield). .sup.1H NMR (300 MHz,
CD.sub.3OD) .delta. 1.40 (s, 9H), 1.42-1.70 (m, 4H), 2.48-2.60 (m,
2H), 3.00-3.20 (m, 2H), 4.65 (s, 2H), 6.79-6.89 (m, 4H), 7.10 (d,
2H). m/z (ESI): 389
[C.sub.20H.sub.28N.sub.4O.sub.4.sup.+H].sup.+.
2-[4-(4-Aminobutyl)phenoxy]-N-(1H-imidazol-2-yl)acetamide
dihydrochloride (35)
[0299]
(4-{4-[(1H-Imidazol-2-yl-carbamoyl)methoxy]phenyl}butyl)carbamic
acid tert-butyl ester 34 (950 mg, 2.45 mmol) was treated with HCl
(4 M in dioxane, 24 mL, 96 mmol) at room temperature for 12 h. The
reaction mixture was concentrated in vacuo and further
co-evaporated with dichloromethane and methanol, and dried under
high vacuum. The desired product was obtained as a white solid (779
mg, 98%) and used directly without further purification. .sup.1H
NMR (300 MHz, CD.sub.3OD) .delta. 1.59-1.74 (m, 4H), 2.55-2.67 (m,
2H), 2.85-2.98 (m, 2H), 4.80 (s, 2H), 7.00 (d, 2H), 7.18 (d, 2H),
7.19 (s, 2H). m/z (ESI): 289
[C.sub.15H.sub.20N.sub.4O.sub.2+H].sup.+.
2-(4-{4-[N'-(3,5-Diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}pheno-
xy)-N-(1H-imidazol-2-yl)acetamide dihydrochloride (36, PSA
16437)
[0300] A solution of
2-[4-(4-aminobutyl)phenoxy]-N-(1H-imidazol-2-yl)acetamide
dihydrochloride 35 (99 mg, 0.27 mmol) and diisopropylethylamine
(0.27 mL, 1.53 mmol) in absolute ethanol (4 mL) and anhydrous
methanol (3 mL) was stirred at 70.degree. C. for 30 min, after
which
1-(3,5-diamino-6-chloropyrazine-2-carbonyl)-2-methylisothiourea
hydriodide (130 mg, 0.34 mmol) was added in one portion. The
reaction mixture was stirred for 3 h and then cooled to room
temperature. The yellow insolubles were removed by suction
filtration and the liquid filtrate was concentrated by rotary
evaporation. The crude residue was purified by flash silica gel
column chromatography eluting with
dichloromethane/methanol/concentrated ammonium hydroxide (200:10:0,
200:10:1, 150:10:1, and 100:10:1, v/v) to give
2-(4-{4-[NA-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}phen-
oxy)-N-(1H-imidazol-2-yl)-acetamide as a yellow solid (44 mg, 29%
yield). The free base thus obtained was dissolved in methanol and
treated with 4 drops of 4 N aqueous HCl. The solution was
concentrated under reduced pressure and further dried under vacuum
to give the final compound 36. mp 172-174.degree. C. .sup.1H NMR
(300 MHz, CD.sub.3OD) .delta. 1.61-1.77 (m, 4H), 2.58-2.70 (m, 2H),
3.32-3.40 (m, 2H), 4.80 (s, 2H), 7.00 (d, 2H), 7.18 (d, 2H), 7.20
(s, 2H). m/z (ESI): 501
[C.sub.21H.sub.25ClN.sub.10O.sub.3+H].sup.+.
Example 9
Synthesis of
N-carbamoylmethyl-2-(4-{4-[N'-(3,5-diamino-6-chloropyrazine-2-carbonyl)gu-
anidino]butyl}phenoxy)acetamide (PSA 16314)
##STR00067##
[0301]
(4-{4-[(Carbamoylmethylcarbamoyl)methoxy]phenyl}butyl)carbamic acid
benzyl ester (37)
[0302] Compound 1 (0.50 g, 1.77 mmol) was dissolved in DMF (10 mL).
To the solution was added crushed NaOH (0.107 g, 2.66 mmol). The
mixture was stirred at room temperature for 30 min.
2-Bromoacetamide (0.367 g, 2.66 mmol) was added. The reaction was
further stirred at room temperature overnight, quenched with water
(2 mL) and partitioned between water and dichloromethane (each 50
mL). The organic layer was separated, washed with water (2.times.50
mL), dried over anhydrous Na.sub.2SO.sub.4 and concentrated under
vacuum. The residue was purified on silica gel, eluting with a
mixture of methanol/dichloromethane (7:93, v/v), to afford the
desired product 37 (0.131 g, 18% yield) as a white solids. .sup.1H
NMR (300 MHz, CDCl.sub.3): .delta. 1.58 (m, 4H), 2.60 (t, 2H), 3.20
(m, 2H), 4.04 (d, 2H), 4.54 (s, 2H), 4.75 (br, 2H), 5.12 (s, 2H),
5.43 (br, 1H), 5.80 (br, 1H), 6.85 (d, 2H), 7.12 (d, 2H), 7.36 (m,
5H). m/z (APCI): 414 [C.sub.22H.sub.27N.sub.3O.sub.5+H].sup.+.
2-[4-(4-Aminobutyl)phenoxy]-N-carbamoylmethylacetamide (38)
[0303] Compound 37 (130 mg, 0.314 mmol) was dissolved in EtOH and
THF (14 mL, 1/1 ratio). The reaction vessel was purged with
nitrogen both before and after the catalyst (100 mg, 10% Pd/C, 50%
wet) was added. The mixture was stirred under hydrogen atmosphere
(1 atm) overnight. After purging with nitrogen, the catalyst was
vacuum filtered and washed with ethanol (3.times.5 mL). The
combined filtrates were concentrated under vacuum. The residue was
chromatographed on silica gel, eluting with a mixture of
concentrated ammonium hydroxide/methanol/dichloromethane (2:20:88,
v/v), to afford the desired product 38 (80 mg, 91% yield) as a
white solid. .sup.1H NMR (300 MHz, CD.sub.3OD): .delta. 1.62 (m,
4H), 2.60 (t, 2H), 2.75 (t, 2H), 3.92 (s, 2H), 4.54 (s, 2H), 6.92
(d, 2H), 7.14 (d, 2H).
N-Carbamoylmethyl-2-(4-{4-[N'-(3,5-diamino-6-chloropyrazine-2-carbonyl)-gu-
anidino]butyl}phenoxy)acetamide (39, PSA 1634)
[0304] Compound 38 (79 mg, 0.283 mmol) was dissolved in a mixture
of absolute ethanol (5 mL) and Hunig's base (0.25 mL, 1.41 mmol) at
65.degree. C. over 10 min. To the solution was added
1-(3,5-diamino-6-chloropyrazine-2-carbonyl)-2-methylisothiourea
hydriodide (132 mg, 0.34 mmol) in one portion. The newly resulting
reaction mixture was continuously stirred for an additional 2 h
before it was cooled down to ambient temperature and subsequently
concentrated under vacuum. The resulting residue was purified by
chromatography eluting with methanol/dichloromethane/concentrated
ammonium hydroxide (Oct. 2, 1988, v/v) to afford the free base (93
mg, 67% yield) as a yellow solid. The HCl salt was made using the
following procedure: 45 mg of the free base was suspended in
ethanol (2 mL) and treated with concentrated HCl (12 N, 0.5 mL) for
10 min. All liquid was then completely removed under vacuum to
afford 39 (47 mg). mp 178-180.degree. C. (decomposed). .sup.1H NMR
(300 MHz, DMSO-d.sub.6): .delta. 1.61 (m, 4H), 2.58 (t, 2H), 3.32
(m, 2H), 3.70 (s, 2H), 4.48 (s, 2H), 6.93 (d, 2H), 7.08 (br, 1H),
7.13 (d, 2H), 7.36 (br, 1H), 7.44 (br, 2H), 8.17 (t, 1H), 8.74 (br,
1H), 8.90 (br, 2H), 9.18 (t, 1H), 10.48 (br, 1H). m/z (APCI): 492
[C.sub.20H.sub.26ClN.sub.9O.sub.4+H].sup.+.
Example 10
Synthesis of
N-[4-(4-cyanomethoxyphenyl)butyl]-N'-(3,5-diamino-6-chloro-pyrazine-2-car-
bonyl)guanidine (PSA 16208)
##STR00068##
[0305] [4-(4-Cyanomethoxyphenyl)butyl]carbamic acid tert-butyl
ester (40)
[0306] A mixture of [4-(4-hydroxyphenyl)butyl]carbamic acid
tert-butyl ester 31 (0.365 g, 1.37 mmol) and Cs.sub.2CO.sub.3
(0.672 g, 2.06 mmol) in anhydrous DMF (8 mL) was heated at
65.degree. C. for 30 min. Iodoacetonitrile (0.276 g, 1.651 mmol)
was then added to the mixture in one portion. The mixture was
stirred at 65.degree. C. ovenight, and then cooled to room
temperature. The precipitated solid was filtered, and the filtrate
was partitioned between water and dichloromethane (each 50 mL). The
organic layer was separated, washed with brine (3.times.50 mL),
dried over anhydrous Na.sub.2SO.sub.4 and concentrated under
vacuum. The residue was chromatographed on silica gel, eluting with
a mixture of diethyl ether/dichloromethane (6:94, v/v), to afford
the desired product 40 (0.109 g, 38% yield) as a colorless viscous
oil. .sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 1.43 (s, 9H), 1.57
(m, 4H), 2.60 (t, 2H), 3.15 (m, 2H), 4.49 (br, 1H), 4.75 (s, 2H),
6.91 (d, 2H), 7.13 (d, 2H).
[4-(4-Aminobutyl)phenoxy]acetonitrile (41)
[0307] Compound 40 (0.105 g, 0.345 mmol) was dissolved in
dichloromethane (10 mL). Trifluoroacetic acid (2 mL) was added in
one portion. The mixture was stirred at room temperature for 2 h,
and then concentrated under vacuum to dryness. The crude residue
was used directly without further purification. .sup.1H NMR (300
MHz, CD.sub.3OD): .delta. 1.60-1.75 (m, 4H), 2.65 (t, 2H), 2.92 (t,
2H), 4.38 (s, 2H), 6.96 (d, 2H), 7.20 (d, 2H). m/z (APCI): 205
[C.sub.12H.sub.16N.sub.2O+H].sup.+.
N-[4-(4-Cyanomethoxyphenyl)butyl]-N'-(3,5-diamino-6-chloropyrazine-2-carbo-
nyl)guanidine (42, PSA 16208)
[0308] A mixture of compound 41 (0.070 g, 0.345 mmol) and Hunig's
base (0.3 mL, 1.72 mmol) in anhydrous ethanol was heated at
65.degree. C. for 20 min. To the solution was added
1-(3,5-diamino-6-chloropyrazine-2-carbonyl)-2-methylisothiourea
hydriodide (0.148 g, 0.379 mmol) in one portion. The heating was
continued for another 2 h. The reaction mixture was then
concentrated under vacuum. The residue was chromatographed by flash
column chromatography and further purified by preparative TLC,
eluting with methanol/dichloromethane/concentrated ammonium
hydroxide (Oct. 1, 1989, v/v), to afford the desired product 42
(0.031 g, 22%) as a yellow solid. mp 129-132.degree. C. .sup.1H NMR
(300 MHz, CD.sub.3OD): .delta. 1.72 (m, 4H), 2.68 (t, 2H), 3.32 (m,
2H), 4.92 (s, 2H), 6.95 (d, 2H), 7.22 (d, 2H); m/z (APCI): 417
[C.sub.18H.sub.21ClN.sub.8O.sub.2+H].sup.+.
Example 11
Synthesis of
N-(3,5-diamino-6-chloropyrazine-2-carbonyl)-N'-(4-{4-[3-(2,3-dihydroxypro-
poxy)-2-hydroxypropoxy]phenyl}butyl)guanidine (PSA 15143)
##STR00069##
[0310]
(4-{4-[3-(2,3-Dihydroxypropoxy)-2-hydroxypropoxy]phenyl}butyl)carba-
mic acid benzyl ester (43).
[0311] A solution containing compound 1 (2.0 g, 6.68 mmol),
triethylamine (0.093 mL, 0.668 mmol) and anhydrous ethanol (2.2 mL)
was heated at 70.degree. C. for 1 h. Oxiranylmethanol (0.5 mL, 6.68
mmol) was added every hour for a total of 4 h (the total amount of
oxiranylmethanol added was 2.0 ml, 26.72 mmol). The reaction was
concentrated under vacuum. The residue was chromatographed on
silica gel with the elution of a mixture of
methanol/dichloromethane (3:97, v/v) to provide 168 mg (4.6% yield)
of the desired product 43. m/z (APCI): 448
[C.sub.24H.sub.33NO.sub.7+H].sup.+.
3-{3-[4-(4-Aminobutylphenoxy]-2-hydroxypropoxy}propane-1,2-diol
(44)
[0312] A solution containing the compound 43 (0.15 g, 0.34 mmol) in
ethanol (1.5 mL) was purged with nitrogen before and after the
catalyst (0.15 g, 10% Pd/C, 50% wet) was added. The reaction
mixture was placed under hydrogenation atmosphere for 45 min. The
catalyst was vacuum filtered through diatomaceous earth and washed
with ethanol (3.times.2 mL). The combined filtrates were
concentrated under vacuum. The residue was chromatographed on
silica gel, eluting with methanol/dichloromethane/concentrated
ammonium (25/2.5/73.5, v/v), to afford the desired product 44
(0.053 g, 51% yield) as a colorless, viscous oil. .sup.1H NMR (300
MHz, CD.sub.3OD): .delta. 1.52 (m, 4H), 2.55 (t, 2H), 2.65 (t, 2H),
3.61 (m, 10H), 6.85 (d, 2H), 7.09 (d, 2H). m/z (APCI): 314
[C.sub.16H.sub.27NO.sub.5+H].sup.+.
N-(3,5-Diamino-6-chloropyrazine-2-carbonyl)-N'-(4-{4-[3-(2,3-dihydroxyprop-
oxy)-2-hydroxypropoxy]phenyl}butyl)guanidine (45, PSA 15143)
[0313] Compound 44 (50 mg, 0.159 mmol) was dissolved in a mixture
of absolute ethanol (0.5 mL) and triethylamine (0.076 mL, 0.541
mmol) at 65.degree. C. over 15 min. To the solution was added
1-(3,5-diamino-6-chloropyrazine-2-carbonyl)-2-methylisothiourea
hydriodide (74 mg, 0.191 mmol). The reaction mixture was stirred at
the above temperature for an additional 50 min, cooled down to
ambient temperature and subsequently concentrated under vacuum. The
residue was chromatographed on silica gel, eluting with
methanol/dichloromethane/concentrated ammonium hydroxide (Oct. 1,
1940, v/v) to afford the desired product 45 (53 mg, 36% yield) as a
yellow solid. mp 73-82.degree. C. (decomposed). .sup.1H NMR (300
MHz, CD.sub.3OD): .delta. 1.70 (m, 4H), 2.55 (m, 2H), 3.22 (m, 2H),
3.65 (m, 7H), 3.98 (m, 3H), 6.86 (d, 2H), 7.08 (d, 2H). m/z (APCI):
526 [C.sub.22H.sub.32ClN.sub.7O.sub.6+H].sup.+.
Example 12
[0314] Utilizing the procedures set forth above, the following
capped pyrazinoylguanidine was prepared.
TABLE-US-00001 ##STR00070## TEST RESULT/REFERENCE Description
Yellow solid Identification: 300 MHz .sup.1H NMR Spectrum
(DMSO-d.sub.6) Consistent Melting Point 108-110.degree. C. dec HPLC
Analysis 96.5% (area percent), Polarity dC18 Column, Detector @ 220
nm Miscellaneous Tests: ESI Mass Spectrum m/z 527
[C.sub.21H.sub.31ClN.sub.8O.sub.4S + H].sup.+
Example 13
[0315] Utilizing the procedures set forth above, the following
capped pyrazinoylguanidine was prepared.
TABLE-US-00002 ##STR00071## TEST RESULT/REFERENCE Description
Yellow solid Identification: 300 MHz .sup.1H NMR Spectrum
(DMSO-d.sub.6) Consistent Melting Point 153-155.degree. C. dec HPLC
Analysis 96.3% (area percent), Polarity dC18 Column, Detector @ 220
nm Miscellaneous Tests: ESI Mass Spectrum m/z 465
[C.sub.19H.sub.25ClN.sub.8O.sub.2S + H].sup.+
Example 14
[0316] Utilizing the procedures set forth above, the following
capped pyrazinoylguanidine was prepared.
TABLE-US-00003 ##STR00072## TEST RESULT/REFERENCE Description
Yellow solid Identification: 500 MHz .sup.1H NMR Spectrum
(CD.sub.3OD) Consistent Melting Point 115-116.degree. C. HPLC
Analysis 97.1% (area percent), Polarity dC18 Column, Detector @ 220
nm Miscellaneous Tests: ESI Mass Spectrum m/z 639
[C.sub.30H.sub.35ClN.sub.8O.sub.6 + H].sup.+
Example 15
[0317] Utilizing the procedures set forth above, the following
capped pyrazinoylguanidine was prepared.
TABLE-US-00004 ##STR00073## TEST RESULT/REFERENCE Description
Yellow solid Identification: 300 MHz .sup.1H NMR Spectrum
(CD.sub.3OD) Consistent Melting Point 190-192.degree. C. HPLC
Analysis 97.9% (area percent), Polarity dC18 Column, Detector @ 220
nm Miscellaneous Tests: ESI Mass Spectrum m/z 476
[C.sub.20H.sub.26ClN.sub.9O.sub.3 + H].sup.+
Example 16
[0318] Utilizing the procedures set forth above, the following
capped pyrazinoylguanidine was prepared.
TABLE-US-00005 ##STR00074## TEST RESULT/REFERENCE Description
Yellow solid Identification: 300 MHz .sup.1H NMR Spectrum
(CD.sub.3OD) Consistent Melting Point 124-126.degree. C. dec HPLC
Analysis 95.2% (area percent), Polarity dC18 Column, Detector @ 220
nm Miscellaneous Tests: ESI Mass Spectrum m/z 441
[C.sub.16H.sub.21ClN.sub.8O.sub.3S + H].sup.+
Example 17
[0319] Utilizing the procedures set forth above, the following
capped pyrazinoylguanidine was prepared.
TABLE-US-00006 ##STR00075## TEST RESULT/REFERENCE Description
Yellow solid Identification: 500 MHz .sup.1H NMR Spectrum
(CD.sub.3OD) Consistent Melting Point 189.degree. C. dec HPLC
Analysis 95.0% (area percent), Polarity dC18 Column, Detector @ 220
nm Miscellaneous Tests: ESI Mass Spectrum m/z 503
[C.sub.21H.sub.27ClN.sub.10O.sub.3 + H].sup.+
Example 18
[0320] Utilizing the procedures set forth above, the following
capped pyrazinoylguanidine was prepared.
TABLE-US-00007 ##STR00076## TEST RESULT/REFERENCE Description Pale
yellow solid Identification: 300 MHz .sup.1H NMR Spectrum
(CD.sub.3OD) Consistent Melting Point 195-197.degree. C. HPLC
Analysis 97.4% (area percent), Polarity dC18 Column, Detector @ 220
nm Miscellaneous Tests: ESI Mass Spectrum m/z 477
[C.sub.20H.sub.25ClN.sub.8O.sub.4 + H].sup.+
Example 19
[0321] Utilizing the procedures set forth above, the following
capped pyrazinoylguanidine was prepared.
TABLE-US-00008 ##STR00077## TEST RESULT/REFERENCE Description
Yellow solid Identification: 300 MHz .sup.1H NMR Spectrum
(CD.sub.3OD) Consistent Melting Point 210-212.degree. C. dec HPLC
Analysis 95.5% (area percent), Polarity dC18 Column, Detector @ 220
nm Miscellaneous Tests: APCI Mass Spectrum m/z 486
[C.sub.22H.sub.28ClN.sub.9O.sub.2 + H].sup.+
Example 20
[0322] Utilizing the procedures set forth above, the following
capped pyrazinoylguanidine was prepared.
TABLE-US-00009 ##STR00078## TEST RESULT/REFERENCE Description
Yellow solid Identification: 300 MHz .sup.1H NMR Spectrum
(CD.sub.3OD) Consistent Optical Rotation [.alpha.].sup.25.sub.D
-7.8.degree. (c 0.46, Methanol) Melting Point 178-180.degree. C.
HPLC Analysis 97.0% (area percent), Polarity dC18 Column, Detector
@ 220 nm Miscellaneous Tests: ESI Mass Spectrum m/z 490
[C.sub.21H.sub.28ClN.sub.9O.sub.3 + H].sup.+
Example 21
[0323] Utilizing the procedures set forth above, the following
capped pyrazinoylguanidine was prepared.
TABLE-US-00010 ##STR00079## TEST RESULT/REFERENCE Description
Yellow solid Identification: 300 MHz .sup.1H NMR Spectrum
(CD.sub.3OD) Consistent Optical Rotation [.alpha.].sup.25.sub.D
+0.5.degree. (c 0.35, Methanol) Melting Point 215.degree. C. dec
HPLC Analysis 96.1% (area percent), Polarity dC18 Column, Detector
@ 220 nm Miscellaneous Tests: ESI Mass Spectrum m/z 462
[C.sub.20H.sub.28ClN.sub.9O.sub.2 + H].sup.+
Example 22
[0324] Utilizing the procedures set forth above, the following
capped pyrazinoylguanidine was prepared.
TABLE-US-00011 ##STR00080## TEST RESULT/REFERENCE Description
Yellow solid Identification: 300 MHz .sup.1H NMR Spectrum
(CD.sub.3OD) Consistent Optical Rotation [.alpha.].sup.25.sub.D
+4.1.degree. (c 0.30, Methanol) Melting Point 230.degree. C. dec
HPLC Analysis 95.3% (area percent), Polarity dC18 Column, Detector
@ 220 nm Miscellaneous Tests: ESI Mass Spectrum m/z 463
[C.sub.20H.sub.27ClN.sub.8O.sub.3 + H].sup.+
Example 23
[0325] Sodium Channel Blocking Activity of Selected Capped
Pyrazinoylguanidines.
TABLE-US-00012 PSA EC.sub.50(nM) Fold Amiloride** (PSA 4022 = 100)
15143 7 .+-. 3 (n = 3) 107 .+-. 11 (n = 3) 16208 11 .+-. 4 (n = 6)
52 .+-. 21 (n = 6) 16314 13 .+-. 2 (n = 4) 41 .+-. 6 (n = 4) 16313
15 .+-. 4 (n = 4) 41 .+-. 7 (n = 4) 16437 13 .+-. 7 (n = 7) 77 .+-.
53 (n = 7) 17482 16 .+-. 4 (n = 3) 39 .+-. 6 (n = 3) 17846 11 .+-.
6 (n = 4) 104 .+-. 49 (n = 4) 17926 25 .+-. 9 (n = 6) 29 .+-. 12 (n
= 6) 17927 13 .+-. 4 (n = 3) 83 .+-. 26 (n = 3) 18211 10 .+-. 4 (n
= 3) 112 .+-. 52 (n = 2) 18212 27 .+-. 17 (n = 4) 32 .+-. 16 (n =
4) 18229 15 .+-. 6 (n = 3) 49 .+-. 15 (n = 3) 18361 11 .+-. 4 (n =
3) 76 .+-. 25 (n = 3) 18592 8 .+-. 4 (n = 2) 136 .+-. 58 (n = 2)
18593 48 .+-. 16 (n = 6) 13 .+-. 4 (n = 4) 19007 18 .+-. 13 (n = 4)
42 .+-. 17 (n = 4) 19008 9 .+-. 1 (n = 4) 54 .+-. 6 (n = 4) 19912
26 .+-. 8 (n = 4) 32 .+-. 10 (n = 4) 23022 12 .+-. 3 (n = 4) 79
.+-. 15 (n = 4) 24406 8 .+-. 3 (n = 6) 107 .+-. 38 (n = 6) 24407 32
.+-. 11 (n = 10) 23 .+-. 4 (n = 10) 24851 28 .+-. 13 (n = 8) 25
.+-. 10 (n = 8) **Relative potency for PSA 4022 = 100 using
EC.sub.50 from PSA 4022 in same run
Methods
[0326] Animal Preparation Adult ewes (ranging in weight from 25 to
35 kg) were restrained in an upright position in a specialized body
harness adapted to a modified shopping cart. The animals' heads
were immobilized and local anesthesia of the nasal passage was
induced with 2% lidocaine. The animals were then nasally intubated
with a 7.5 mm internal diameter endotracheal tube (ETT). The cuff
of the ETT was placed just below the vocal cords and its position
was verified with a flexible bronchoscope. After intubation the
animals were allowed to equilibrate for approximately 20 minutes
prior to initiating measurements of mucociliary clearance.
[0327] Administration of Radio-aerosol: Aerosols of
.sup.99mTc-Human serum albumin (3.1 mg/ml; containing approximately
20 mCi) were generated using a Raindrop Nebulizer which produces a
droplet with a median aerodynamic diameter of 3.6 .mu.m. The
nebulizer was connected to a dosimetry system consisting of a
solenoid valve and a source of compressed air (20 psi). The output
of the nebulizer was directed into a plastic T connector; one end
of which was connected to the endotracheal tube, the other was
connected to a piston respirator. The system was activated for one
second at the onset of the respirator's inspiratory cycle. The
respirator was set at a tidal volume of 500 mL, an inspiratory to
expiratory ratio of 1:1, and at a rate of 20 breaths per minute to
maximize the central airway deposition. The sheep breathed the
radio-labeled aerosol for 5 minutes. A gamma camera was used to
measure the clearance of .sup.99mTc-Human serum albumin from the
airways. The camera was positioned above the animal's back with the
sheep in a natural upright position supported in a cart so that the
field of image was perpendicular to the animal's spinal cord.
External radio-labeled markers were placed on the sheep to ensure
proper alignment under the gamma camera. All images were stored in
a computer integrated with the gamma camera. A region of interest
was traced over the image corresponding to the right lung of the
sheep and the counts were recorded. The counts were corrected for
decay and expressed as percentage of radioactivity present in the
initial baseline image. The left lung was excluded from the
analysis because its outlines are superimposed over the stomach and
counts can be swallowed and enter the stomach as radio-labeled
mucus.
[0328] Treatment Protocol (Assessment of activity at t-zero): A
baseline deposition image was obtained immediately after
radio-aerosol administration. At time zero, after acquisition of
the baseline image, vehicle control (distilled water), positive
control (amiloride), or experimental compounds were aerosolized
from a 4 ml volume using a Pari LC JetPlus nebulizer to
free-breathing animals. The nebulizer was driven by compressed air
with a flow of 8 liters per minute. The time to deliver the
solution was 10 to 12 minutes. Animals were extubated immediately
following delivery of the total dose in order to prevent false
elevations in counts caused by aspiration of excess radio-tracer
from the ETT. Serial images of the lung were obtained at 15-minute
intervals during the first 2 hours after dosing and hourly for the
next 6 hours after dosing for a total observation period of 8
hours. A washout period of at least 7 days separated dosing
sessions with different experimental agents. Treatment Protocol
(Assessment of Activity at t-4 hours): The following variation of
the standard protocol was used to assess the durability of response
following a single exposure to vehicle control (distilled water),
positive control compounds (amiloride or benzamil), or
investigational agents. At time zero, vehicle control (distilled
water), positive control (amiloride), or investigational compounds
were aerosolized from a 4 ml volume using a Pari LC JetPlus
nebulizer to free-breathing animals. The nebulizer was driven by
compressed air with a flow of 8 liters per minute. The time to
deliver the solution was 10 to 12 minutes. Animals were restrained
in an upright position in a specialized body harness for 4 hours.
At the end of the 4-hour period animals received a single dose of
aerosolized .sup.99mTc-Human serum albumin (3.1 mg/ml; containing
approximately 20 mCi) from a Raindrop Nebulizer. Animals were
extubated immediately following delivery of the total dose of
radio-tracer. A baseline deposition image was obtained immediately
after radio-aerosol administration. Serial images of the lung were
obtained at 15-minute intervals during the first 2 hours after
administration of the radio-tracer (representing hours 4 through 6
after drug administration) and hourly for the next 2 hours after
dosing for a total observation period of 4 hours. A washout period
of at least 7 days separated dosing sessions with different
experimental agents.
Statistics: Data were analyzed using SYSTAT for Windows, version 5.
Data were analyzed using a two-way repeated ANOVA (to assess
overall effects), followed by a paried t-test to identify
differences between specific pairs. Significance was accepted when
P was less than or equal to 0.05. Slope values (calculated from
data collected during the initial 45 minutes after dosing in the
t-zero assessment) for mean MCC curves were calculated using linear
least square regression to assess differences in the initial rates
during the rapid clearance phase.
[0329] Obviously, numerous modifications and variations of the
present invention are possible in light of the above teachings. It
is therefore to be understood that within the scope of the appended
claims, the invention may be practiced otherwise than as
specifically described herein.
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