Capped pyrazinoylguanidine sodium channel blockers

New protected pyrazinoylguanidine sodium channel blockers

BACKGROUND OF THE INVENTION

Field of the invention

The present invention relates to sodium channel blockers . The present invention also includes a variety of treatment methods utilizing these sodium channel blockers of the invention.

Description of Priority

Mucosal surfaces at the interface between the environment and the body have developed a number of innate defenses, ie protective mechanisms. A major form of these innate defenses is to clean these surfaces with liquid. Typically, the amount of liquid layer on a mucosal surface reflects the balance between epithelial fluid secretion, often reflecting anion secretion (Cl^- and / or HCO3^-) coupled with water (and a counterion cation) and epithelial absorption of liquid, often reflecting Na absorption⁺, coupled with water and a counter anion (Cl^- and / or HCO3^-). Many diseases of the mucosal surfaces are caused by too little protective fluid on these mucosal surfaces created by an imbalance between secretion (less) and absorption (relatively more). The defective salt transport processes that characterize these mucosal dysfunctions reside in the epithelial layer of the mucosal surface.

One approach to replenishing the protective fluid layer on mucosal surfaces is to rebalance the system by blocking the Na channel.⁺ and the absorption of liquid. The epithelial protein that mediates the rate limiting step of the absorption of liquid and Na⁺ it's Na's channel⁺epithelial (ENaC). ENaC is positioned on the apical surface of the epithelium, ie the surface of the mucosal-environmental interface. Therefore, to inhibit Na absorption⁺ mediated fluid, an ENaC blocker of

66 1 663 235 / ΡΤ amiloride (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 very little fluid on topical sodium channel blocking mucosal surfaces designed to exhibit increased potency, reduced mucosal absorption, and slow dissociation (release or separation) of ENaC required for the therapy of these diseases.

Chronic bronchitis (BC), including the genetic form of the most common lethal chronic bronchitis, cystic fibrosis (CF), is a disease that reflects the body's failure to clear mucus normally from the lungs, which lately produces chronic airway infection. . In the normal lung, the primary defense against chronic intrapulmonary airway infection (chronic bronchitis) is mediated by continuous clearance of mucus from the bronchial surfaces of the airway. This health function effectively removes potentially harmful toxins and pathogens from the lung. Recent data indicate that the initial problem, ie the basic defect in both BC and CF, is the failure of mucus clearance of airway surfaces. Failure to clear mucus reflects an imbalance between the amount of fluid and mucin on airway surfaces. This Airway Surface Liquid (ASL)) is mainly composed of salt and water in similar proportions to plasma (ie, isotonic). Mucin macromolecules are organized in a well-defined mucus layer that normally traps inhaled bacteria and is transported to the outside of the lung through the actions of the eyelashes that hit a watery, low viscosity solution called the periciliary fluid (PCL). In the disease state, there is an imbalance in the amounts of mucus such as ASL on airway surfaces. This results in a relative reduction in ASL leading to mucus concentration, reduced PCL lubricating activity, and 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 adhering to airway surfaces.

bacteria, the insufficiency of

And chronic retention of local antimicrobial substances to kill mucus trapped bacteria on a

1 663 235 / e, and the body's consequent chronic inflammatory responses to this type of surface infection, leading to BC and CF syndromes.

The present affected population in the US is 12,000,000 patients with the acquired form (mainly from cigarette smoke exposure) of chronic bronchitis and approximately 30,000 patients with the genetic form of cystic fibrosis. Approximately equal numbers of both populations are present in Europe. In Asia there is little CF but the incidence of BC is high and, as in the rest of the world, it is increasing.

There is currently a great unmet need for products that specifically treat BC and CF at the level of the underlying defect that causes these diseases. The present therapies for chronic bronchitis and cystic fibrosis focus on treating the symptoms and / or late effects of these diseases. Thus, for chronic bronchitis, β-agonists, inhaled spheroids, anticholinergic agents and oral theophyllines and phosphodiesterase inhibitors are all under development. However, none of these drugs effectively addresses the fundamental problem of insufficient clearance of lung mucus. Similarly, in cystic fibrosis the same spectrum of pharmacological agents is used. These strategies were complemented by more recent strategies designed to purify the DNA CF lung (Pulmozyme; Genentech) that has been deposited in the lung by neutrophils who have futilely attempted to kill bacteria growing in adherent mucus masses and through the use of inhaled antibiotics ( TOBI) designed to enhance the lung's own death mechanisms to rid bacteria of adherent mucus plaques. A general principle of the body is that if the initial lesion is not treated, in this case mucus retention / obstruction, bacterial infections become chronic and antimicrobial increasingly refractory to therapy. Thus, a major unmet therapeutic need for both diseases. BC and CF, is an effective means for rehydrating airway mucus (ie, restoring / expanding ASL volume) and promoting its clearance with bacteria from the lung.

ΕΡ 1 663 235 / ΡΤ

US 3,313,813 refers to disubstituted (3-amino-5,6pyrazinoyl) guanidine compounds having diuretic and natriuretic properties. These compounds are disclosed as selectively increasing sodium ion excretion without causing an increase in potassium ion excretion.

RC Boucher, in US 6,264,975, describes the use of pyrazinoylguanidine sodium channel blockers to hydrate mucosal surfaces. These compounds, typified by the well-known amiloride, benzamyl and fenamyl diuretics, are effective. However, these compounds suffer from the significant disadvantage that (1) are relatively powerless, which is important because the mass of drug that can be inhaled by the lung is limited; (2) they are 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 diuretics produces compounds with insufficient potency and / or effective half-life on mucosal surfaces to have therapeutic benefit for hydrating mucosal surfaces.

Clearly, what is needed are drugs that are most effective in restoring mucus clearance from the lungs of BC / CF patients. The value of these new therapies will be reflected in improvements in quality and life span for both CF and BC populations.

Other mucosal surfaces in the body, and on the body, exhibit subtle differences in the normal physiology of surface protective fluids on their surfaces, but the pathophysiology of the disease reflects a common theme, ie very little surface protective fluid. For example, in dry mouth (dry mouth) the oral cavity is devoid of fluid due to sublingual and submandibular parotid gland insufficiency in fluid secretion despite continued transport-mediated absorption of Na⁺(ENaC) of the oral cavity. Similarly, keratoconjunctivitis sicca (dry eye) is caused by insufficiency of the tear glands in fluid secretion on the Na-dependent continuous fluid absorption face.⁺ on surfaces of the conjunctiva. In rhinosinusitis, there is an imbalance, as in BC, between mucin secretion and relative ASL depletion.

EP 1 663 235 / EN

Finally, in the gastrointestinal tract, insufficient secretion of Cl- (and fluid) in the proximal small intestine, combined with increased Na absorption⁺ (and fluid) in the terminal ileum leads to distal intestinal obstruction syndrome (DIOS). In older patients excessive Na absorption⁺(and volume) in the descending colon produces constipation and diverticulitis.

Fifty million Americans and hundreds of millions of others worldwide suffer from high blood pressure and the subsequent sequelae leading to congestive heart failure and increased mortality. It is the leading cause of death in the western world and there is a need for new medicines to treat these diseases. Thus, in addition, some of the novel sodium channel blockers of the present invention may be designed to target the kidney and as such may be used as diuretics for the treatment of hypertension, congestive heart failure (CHF) and other cardiovascular diseases. These novel 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

It is an object of the present invention to provide compounds which are more potent and / or less rapidly absorbed from mucosal surfaces and / or which are less reversible compared to known compounds.

It is another aspect of the present invention to provide compounds that are more potent and / or less rapidly absorbed and / or exhibit less reversibility compared to compounds such as amiloride, benzamyl and phenamyl. Therefore, the compounds will provide a prolonged pharmacodynamic half-life on mucosal surfaces compared to known compounds.

It is another object of the present invention to provide compounds which (1) are less rapidly absorbed from mucosal surfaces, especially airway surfaces, compared to known compounds and; (2) when

Absorbed from mucosal surfaces after administration to mucosal surfaces, they are converted in vivo to their metabolic derivatives which have reduced efficacy in blocking sodium channels compared to the parent compound administered. It is another object of the present invention to provide compounds that are more potent and / or less rapidly absorbed and / or exhibit less reversibility compared to compounds such as amiloride, benzamyl and phenamyl. Therefore, these compounds will provide a prolonged pharmacodynamic half-life on mucosal surfaces compared to previous compounds.

It is another object of the present invention to provide compounds that target the kidney for use in the treatment of cardiovascular disease.

It is another object of the present invention to provide treatment methods that take advantage of the pharmacological properties of the compounds described above.

In particular, it is an object of the present invention to provide treatment methods based on rehydration of mucosal surfaces.

In particular, it is an object of the present invention to provide methods of treating cardiovascular disease.

The objects of the present invention may be achieved with a class of pyrazinoylguanidine compounds represented by formula (I):

<img file="PT1663235E_D0001.tif" />

on what

X is hydrogen, halogen, trifluoromethyl, C1-C7 alkyl, unsubstituted or halogen-substituted phenyl, C-alkyl₂-Ç₇thio, phenyl C 1 -C 6 alkylthio, C 1 -C 6 alkylsulfonyl or phenyl C 1 -C 6 alkylsulfonyl;

ΕΡ 1 663 235 / ΡΤ

Υ is hydrogen, hydroxyl, mercapto, C1 -C6 alkoxyalkoxy, halogen, alkyl alquilo₂-Ο₇, phenyl substituted or halogen substituted or -N (R²)₂;

C1-C7, no

R¹ is hydrogen or C1-C7 alkyl;

each R² is independently -R⁷, - (CH2) m-0R⁸, - (CH2) _m-NR⁷R¹⁰,

- (CH₂) _no (CHOR⁸) (CHOR⁸) n-CH2OR⁸, - (CH2CH₂O) _m-R⁸, - (CH₂CH₂O) _m-CH₂CH₂NR⁷R¹⁰,

- (CH₂) _no-C (= 0) NR⁷R¹⁰, - (CH2) n-Zg-R⁷, - (CH2) _m-NR¹⁰-CH2 (CHOR⁸) (CHOR⁸) nCH₂OR⁸, - (CH₂) _no-CO₂R⁷ or - (CH₂)_no .0.

<img file="PT1663235E_D0002.tif" />

R '

R³ and R⁴ are each independently hydrogen, a group represented by formula (A), C 1 -C 7 alkyl, hydroxyphenyl, phenyl C 1 -C 7 alkyl, (halophenyl) (alkylphenylalkyl) -C₂-C7, (alkoxyphenyl) Ο₂-Ο₇naphthyl alkyl Ο₂-Ο₇ or pyridyl alkyl Ο₂-Ο₇, is a group represented by formula (A):

C1-C7 alkyl, alkyl Ο₂-Ο₇alkyl Ο₂-Ο₇, provided that at least one of R³ and R⁴ - (C (R^L) 2) the — x— (C (R^l) 2),&gt; -q \ (A) where each R^l is independently -R⁷, - (CH2) n -OR⁸, -0- (CH 2) _m-0R⁸,

- (CH₂) _no-NR⁷R¹⁰, -0- (CH 2) m -NR⁷R¹⁰, - (CH2) _no (CHOR⁸) (CHOR⁸) _no-CH₂OR⁸,

-0- (CH₂) _m (CHOR⁸) (CHOR⁸) n-CH2OR⁸, - (CH2CH₂O) _m-R⁸, -0- (CH₂CH₂O) _m-R⁸,

- (CH₂CH₂O) _m-CH₂CH₂NR⁷R¹⁰, -0- (CH2CH2O) m-CH2CH2NR⁷R¹⁰, - (CH2)_noC (= O) NR⁷R¹⁰, -o- (CH2) mC (= 0) NR⁷R¹⁰, - (CH2) _no- (Z) _g-R⁷, -O- (CH 2) m (Z) g R⁷, - (CH2) _no-NR¹⁰-CH2 (CHOR⁸) (CHOR⁸) n-CH₂OR⁸, -0- (CH 2) m -NR¹⁰CH2 (CHOR⁸) (CHOR⁸) n-CH2OR⁸, - (CH2) _no-CO₂R⁷, -0- (CH₂) _m-CO₂R⁷, -OSO3H, -O-glucuronide, -O-glucose,

<img file="PT1663235E_D0003.tif" />

each is independently an integer from 0 to 10; each p is an integer from 0 to 10;

provided that the sum of oep in each contiguous chain is 1 to 10;

ΕΡ 1 663 235 / ΡΤ each χ is independently 0, NR¹⁰, C (= O), CHOH, C (= NR¹⁰),

CHNR⁷R¹⁰ or represents a single bond;

where each R⁵ is independently Binder- (CH2) n-CAP, Binder- (CH2) n (CHOR⁸) (CHOR⁸) n-CAP, Binder- (CH₂CH₂O) _m-CH₂-CAP, Ligand- (CH₂CH₂O) _m-CH₂CH₂-CAP, Ligand- (CH₂) _no- (Z) _g-CAP, Binder (CH₂) _no (Z) _g- (CH₂)_m-CAP, Ligand- (CH₂) _no-NR¹³-CH2 (CHOR⁸) (CHOR⁸) n-CAP, Binder- (CH₂) _no- (CHOR⁸) mCH2-NR¹³- (Z) g-CAP, Ligand- (CH₂)_noNR¹³(CH2) m (CHOR⁸) nCH₂NR¹³- (Z) _g-CAP, Ligand- (CH₂)_m- (Z)_g- (CH₂)_m-CAP,

NH-C-ligand (= 0) -NH- (CH₂)_m-CAP, Ligand- (CH₂)_m-C (= 0) NR¹³(CH2) mC (= 0) NR¹⁰R¹⁰, Binder- (CH2) _m-C (= 0) NR¹³- (CH2) m-CAP, (CH2) mC Binder (= 0) NR¹¹R¹¹, Binder- (CH2)_no- (Z)_g- (CH₂)_m- (Z) _g-CAP

Ligand-Zg- (CH₂)_m-Het- (CH₂)_m-CAP;

each Binder is independently -0-, - (CH₂)_no-, -O (CH₂)_m-,

-NR¹³-C (= 0) -NR¹³, -NR¹³-C (= 0) - (CH 2) m -, -C (= 0) NR¹³- (CH2) _m-,

- (CH₂) _no-Z_g- (CH₂) _no, -s-, -so-, -so₂-, -so₂nr⁷-, -so₂nr¹⁰- or

-Het-;

each CAP is independently thiazolidinedione, oxazolidinedione, heteroaryl-C (= 0) NR¹³R¹³, heteroaryl-W, -CN, -OC (= S) NR¹³R¹³, - ZgR¹³, -CR¹⁰ (ZgR¹³) (Z_gR¹³), -C (= 0) 0Ar, —C (= 0) NR¹³Air, imidazoline, tetrazole, tetrazoloamide,

-ONLY₂NHR¹³, -SO₂NH-C (R¹³R¹³) - (Z) gR¹³a cyclic amino sugar or oligosaccharide,

<img file="PT1663235E_D0004.tif" />

each Ar is independently phenyl, halogen substituted phenyl, wherein the substituents on halogen substituted phenyl are 1-3 substituents independently selected from the group consisting of OH, OCH₃, NR¹³R¹³, Cl, Fe -CH₃ or heteroaryl;

each W is independently thiazolidinedione, oxazolidinedione, heteroaryl-C (= 0) NR¹³R¹³, -CN, -0-C (= S) NR¹³R¹³, -ZgR¹³, -CR¹⁰ (ZgR¹³) (ZgR¹³), -C (= 0) 0Ar, -C (= 0) NR¹³Ar, imidazoline, tetrazole, tetrazoloamide, -SO₂NHR¹³, -SO 2 NH-C (R¹³R¹³) - (Z) gR¹³a cyclic sugar or oligosaccharide, a cyclic amino sugar or oligosaccharide,

ΕΡ 1 663 235 / ΡΤ

<img file="PT1663235E_D0005.tif" />

^ y ^ V-CONR ^ R¹³^L~ NR^H

-R⁷,

-OR '

-OR

-N (R ')₂, each R is independently

- (CH₂) _m-OR⁸, -O- (CH₂) _m-OR⁸,

- (CH₂) _no (CHOR⁸) (CHOR⁸) n-CH2OR⁸, -O- (CH 2) _m (CHOR⁸) (CHOR⁸) _no-CH₂OR⁸,

- (CH₂CH₂O) _m-R⁸, -O- (CH 2 CH 2 O) mR⁸, - (CH2CH₂O) _m-CH₂CH₂NR⁷R¹⁰,

-O- (CH₂)_m- (CH₂) _no-NR⁷R¹⁰,

-O- (CH₂ ) _m-NR⁷R¹⁰,

-0- (CH₂CH₂O) _m-CH₂CH₂NR⁷R¹⁰

- (CH₂) _no-C (= 0) NR⁷R¹⁰,

C (= O) NR⁷R¹⁰

- (CH₂)_no- (Z)_g-R ', CH₂(CHOR⁸) (CHOR⁸) _no-CH₂OR⁸,

CH₂OR⁸, - (CH2) n-CO2R⁷, -0- (CH 2) _m-CO₂R⁷, -O-glucose,

-0- (CH₂) _m- (Z) _g-R ', - (CH₂) _no-NR¹⁰-0- (CH2) m-NR¹⁰-CH2 (CHOR °) (CHOR °) _no-OSO3H, -O-glucuronide,

-ΤΊΒΓ or - (CH₂)"O. R '

<img file="PT1663235E_D0006.tif" />

R 'where when two R⁶ are -OR¹¹ and are located adjacent each other on a phenyl ring, the alkyl moieties of the two R⁶may be linked to form a methylenedioxy group;

provided that when at least two -CH₂OR⁸ are located mutually adjacent, the groups R⁸ may be joined to form a mono- or disubstituted cyclic 1,3-dioxane or 1,3-dioxolane, each R⁷ is independently hydrogen alkyl Ο₂-Ο₇phenyl or halogen substituted phenyl;

each R independently is hydrogen, C1-C7 alkyl, and,

-C (= O) -R¹⁷, glucuronide, 2-tetrahydropyranyl or

<img file="PT1663235E_D0007.tif" />

each R⁹ is independently -CO₂R¹³, -C (= 0) R

-CON (R¹³)₂, -SO₂CH₂R¹³ or each

R and independently

-H,

-ONLY₂CH₃,

-co₂r

-C (= 0) NR¹³R¹³,

-C (= 0) R¹³ or - (CH₂)_m- (CHOH) _no-CH₂OH;

66 1 663 235 / ΡΤ each independentemente is independently -CHOH, C (= O), (CH₂)_no-, CHNR¹³R¹³,

C = NR¹³ or NR¹³;

each R¹¹ is independently C1 -C6 alkyl;

each R¹² is independently -SO 2 CH 3, -CO 2 R¹³, -C (= 0) NR¹³R¹³, -C (= O) R¹³ or -CH2- (CHOH) _no-CH₂OH;

each R¹³ is independently hydrogen, R⁷, R¹⁰, - (CH₂)_m+

NR¹³R¹³, - (CH2) m- NR¹³R¹³R¹³, - (CH2) _m- (CHOR⁸) m- (CH2) mNR¹³R¹³, - (CH2)_m+

NR¹⁰R¹⁰, - (CH2) m- (CHOR⁸) m- (CH₂)_mNR¹³R¹³R¹³,

- (CH₂)_no.

-V

- (CH₂)_noΓ7

I IS

- (CH₂) -V

- (CH₂)_m-tZ \ r¹³ or

- (ch₂) · On the condition that NR¹³R¹³ may be joined over itself to form a ring comprising one of the following:

NR¹³ t / \ | - (CH₂)_m(CKOR)^s[n- (CH₂)_noR ',

N- &lt;CH₂)_m(CHOR)⁸m- (CH₂)_noR¹¹

I v_ /

ΕΡ 1 663 235 / ΡΤ

ΛΛ

CH Ν (CH₂UCHOR) V (CH₂)_rR¹¹R¹¹ each Het is independently -NR¹³-, -S-, -SO- or -S0₂-, -0-, -SO₂NR¹³-, -NHSO2-, -NR¹³CO- or -CONR¹³-;

<td>each</td><td>g is,</td><td>independently an integer</td><td>from 1 to</td><td> 6;</td>

<td>each</td><td>m is</td><td>independently an integer</td><td>from 1 to</td><td> 7;</td>

<td>each</td><td>huh,</td><td>independently an integer</td><td>from 0 to</td><td> 7;</td>

<td colspan="2">each Q is, where in</td><td>independently CR⁵, CR⁶ or a maximum three Q in a ring are atoms of</td><td colspan="2">nitrogen atom nitrogen;</td>

<td>each</td><td>V</td><td>is independently - (CH₂) _m-NR⁷R¹⁰,</td><td>, - (CH</td><td>₂) _m-NR⁷R⁷</td>

<td>- (CH₂</td><td> )_m-N⁺:</td><td>R ^ R¹¹, - (CH2) n- (CHOR⁸) m- (CH₂)_mNR⁷R¹⁰,</td><td>- (CH₂)</td><td>_no-NR¹⁰R¹⁰</td>

<td>- (ch₂</td><td colspan="2"> ) _no- (CHOR⁸) m- (CH2) mNR⁷R⁷, - (CH2) _no- (CHOR⁸)_m- (CH₂</td><td> )_mN⁺R¹¹R^:</td><td>¹¹R¹¹,</td>

provided that when V is directly attached to a nitrogen atom, then V may also be independently R⁷, R¹⁰ or (R^no)₂;

where for each of the above compounds when two groups -CH₂OR⁸ are located 1,2- or 1,3- in relation to each other, the groups R⁸ they may be joined to form a mono- or disubstituted cyclic 1,3-dioxane or 1,3-dioxolane;

wherein any of the above compounds may be a pharmaceutically acceptable salt thereof, and wherein the above compounds are inclusive of all their enantiomers, diastereomers and racemic mixtures.

The present invention also provides pharmaceutical compositions containing a compound described herein above.

The present invention also provides a method for promoting hydration of mucosal surfaces comprising:

administering an effective amount of a compound represented by formula (I) to a mucosal surface of an individual.

The present invention also provides a method for restoring mucosal defense comprising:

1 663 235 / ΡΤ the topical administration of an effective amount of the compound represented by formula (I) to a mucosal surface of an individual in need thereof.

The present invention also provides a method for blocking ENaC comprising:

contacting sodium channels with an effective amount of a compound represented by formula (I).

The present invention also provides a method for promoting mucus clearance on mucosal surfaces, comprising:

administering an effective amount of a compound represented by formula (I) to a mucosal surface of an individual.

The present invention also provides a method for treating chronic bronchitis, comprising:

administering an effective amount of a compound represented by formula (I) to an individual in need thereof.

The present invention also provides a method for treating cystic fibrosis, comprising:

administering an effective amount of the compound represented by formula (I) to an individual in need thereof.

The present invention also provides a method for treating rhinosinusitis, comprising:

administering an effective amount of a compound represented by a formula (I) to an individual in need thereof.

The present invention also provides a method for treating nasal dehydration comprising:

administering an effective amount of a compound represented by formula (I) into the nasal passages of an individual in need thereof.

EP 1 663 235 / EN

In a specific embodiment, nasal dehydration is due to the administration of dry oxygen to the subject.

The present invention also provides a method for treating sinusitis comprising:

administering an effective amount of a compound represented by formula (I) to an individual in need thereof.

The present invention also provides a method for treating pneumonia, comprising:

administering an effective amount of a compound represented by formula (I) to an individual in need thereof.

The present invention also provides a method for the prevention of ventilator-induced pneumonia, comprising:

administering an effective compound represented by formula (I) to an individual by means of a ventilator.

The present invention also provides a method for treating asthma comprising:

administering an effective amount of a compound represented by formula (I) to an individual in need thereof.

The present invention also provides a method for treating primary ciliary dyskinesia, comprising:

administering an effective amount of a compound represented by formula (I) to an individual in need thereof.

The present invention also provides a method for treating otitis media, comprising:

administering an effective amount of a compound represented by formula (I) to an individual in need thereof.

ΕΡ 1 663 235 / ΡΤ

The present invention also provides a method for inducing sputum for diagnostic purposes, comprising:

administering an effective amount of the compound represented by formula (I) to an individual in need thereof.

The present invention also provides a method for treating chronic obstructive pulmonary disease, comprising:

administering an effective amount of a compound represented by formula (I) to an individual in need thereof.

The present invention also provides a method for treating emphysema, comprising:

administering an effective amount of a compound represented by formula (I) to an individual in need thereof.

The present invention also provides a method for treating dry eye, comprising:

administering an effective amount of a compound represented by formula (I) to the eye of the individual in need thereof.

The present invention also provides a method for promoting ocular hydration comprising:

administering an effective amount of a compound represented by formula (I) to the subject's eye.

The present invention also provides a method for promoting corneal hydration comprising:

administering an effective amount of a compound represented by formula (I) to the subject's eye.

The present invention also provides a method for treating Sjogren's disease comprising:

administering an amount represented by formula (I) to the need.

an effective individual compound of that

ΕΡ 1 663 235 / ΡΤ

The present invention also provides a method for treating vaginal dryness, comprising:

administering an effective amount of a compound represented by formula (I) into the vaginal tract of an individual in need thereof.

The present invention also provides a method for treating dry skin, comprising:

administering an effective amount of a compound represented by formula (I) to the skin of an individual in need thereof.

The present invention also provides a method for treating dry mouth (xerostomia) comprising:

administering an effective amount of the compound represented by formula (I) into the mouth of the individual in need thereof.

The present invention also provides a method for treating distal intestinal obstruction syndrome comprising:

administering an effective amount of the compound represented by formula (I) to an individual in need thereof.

The present invention also provides a method for treating esophagitis, comprising:

administering an effective amount of a compound represented by formula (I) to an individual in need thereof.

The present invention also provides a method for treating constipation comprising:

administering an effective amount of a compound represented by formula (I) to an individual in need thereof. In one embodiment of this method the compound is administered orally or via a suppository or enema.

ΕΡ 1 663 235 / ΡΤ

The present invention also provides a method for treating chronic diverticulitis comprising:

administering an effective amount of a compound represented by formula (I) to an individual in need thereof.

The present invention also provides a method for treating hypertension comprising administering the compound represented by formula (I) to an individual in need thereof.

The present invention also provides a method for reducing blood pressure comprising administering the compound represented by formula (I) to an individual in need thereof.

The present invention also provides a method for treating edema, comprising administering the compound represented by formula (I) to an individual in need thereof.

The present invention also provides a method for promoting diuresis, comprising administering the compound represented by formula (I) to an individual in need thereof.

The present invention also provides a method for promoting natriuresis, comprising administering the compound represented by formula (I) to an individual in need thereof.

The present invention also provides a method for promoting saluresis, comprising administering the compound represented by formula (I) to an individual in need thereof.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the finding that the compounds of formula (I) are more potent and / or less rapidly absorbed from mucosal surfaces, especially airway surfaces, and / or less reversible.

Interactions with ENaC compared to compounds such as amiloride, benzamyl and phenamyl. Therefore, the compounds of formula (I) have a longer half-life on mucosal surfaces compared to these compounds.

The present invention is also based on the finding that certain compounds within the formula (I) are converted in vivo to their metabolic derivatives which have reduced sodium channel blocking efficacy compared to the parent compound administered after absorption from the parent compound. mucosal surfaces after administration. This important property means that the compounds will be less likely to cause undesirable side effects due to blockage of sodium channels located at non-target locations in the recipient's body, eg the kidneys.

The present invention is also based on the finding that certain compounds within formula (1) target the kidney and therefore may be used as cardiovascular agents.

In the compounds represented by formula (I), X may be hydrogen, halogen, trifluoromethyl, CN-C7 alkyl, unsubstituted or halogen-substituted phenyl, C1-C7thio alkyl, phenylC-Cv-thio alkyl, Cq-Cv-alkyl. sulfonyl or phenyl C1 -C6 alkylsulfonyl. Halogen is preferred.

Examples of halogen include fluorine, chlorine, bromine and iodine. Chlorine and bromine are the preferred halogens. Chlorine is particularly preferred. This description applies to the term halogen as used throughout the present disclosure.

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 their sub-ranges, such as 1, 2, 3, 4, 5, 6 and 7 carbon atoms. The term alkyl embraces all types of these groups, eg straight, branched and cyclic alkyl groups. This description applies to the term lower alkyl as used throughout

66 1 663 235 / ΡΤ disclosure. Examples of suitable lower alkyl groups include methyl, ethyl, propyl, cyclopropyl, butyl, isobutyl, etc.

The substituents for the phenyl group include halogens. Particularly preferred halogen substituents are chlorine and bromine.

Y may be hydrogen, hydroxy, mercapto, lower alkoxy, C1 -C6 alkylthio, halogen, C1 -C6 alkyl, mononuclear aryl or -N (R²) 2 · The alkyl portion 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 (R²)₂. Particularly preferred are compounds wherein each R² is hydrogen.

R¹ may be hydrogen or preferred C1-C7 alkyl for R¹.

hydrogen is

Each R² can be independently -R⁷

- (CH₂) _m-NR⁷R¹⁰, - (CH2) n (CHOR⁸) (CHOR⁸) n-CH₂OR⁸,

- (CH₂CH₂O) _m-CH₂CH₂NR⁷R¹⁰, - (CH₂) _no-C (= 0) NR⁷R¹⁰,

- (CH₂)_m-NR¹⁰-CH₂ (CHOR⁸) (CHOR⁸) n-CH2OR⁸, - (CH2) _no-CO₂R⁷ OK⁷ .

- (CH_{2) n}-zy_R7 ’

- (CH₂) _m-0R⁸, (CH2CH20) mR⁸, - (CH2) _no-Z_g-R⁷, or

Hydrogen and C-alkyl₂-Ç₇, particularly C alkyl₂-C3, are preferred for R². Hydrogen is particularly preferred.

R 'and R' may represent C1-C7 hydroxyalkyl, (halophenyl) C-alkyl₂-Ç₇hydrogen, C1 -C4 alkyl₂-Ç₇independently be formula (A), phenyl, phenyl (C1-C7) alkyl, (alkylphenylalkyl) -C₂-Ç₇, ie (alkoxyphenyl C₂-Ç₇) -C alkyl₂-Ç₇naphthyl C-alkyl₂-Ç₇ pyridyl C1-C7 alkyl provided that at least one of R³ and R⁴a group represented by formula (A)

Preferred compounds are those wherein one of R³ and R⁴ is hydrogen and the other is represented by formula (A).

ΕΡ 1 663 235 / ΡΤ

In formula (A), the portion - (C (R^L) 2) 0-x- (C (R^L) 2) _P- defines an alkylene group attached to the aromatic ring. The variables oep can each be an integer from 0 to 10, provided that the sum of oep in the string is 1 to 10. Thus, oep can each be 0, 1, 2, 3 , 4, 5, 6, 7, 8, 9 or 10. Preferably, the sum of epoid2a6. In a particularly preferred embodiment, the sum of oep is 4.

The alkylene chain linking group, x, may independently be O, NR¹⁰, C (= O), CHOH, C (= NR¹⁰), CHNR⁷R¹⁰ or represent a single bond;

Therefore, when x represents a single bond, the ring-bound alkylene chain is represented by the formula

- (C (R^l) 2) _{o + p}- where the sum o + p is from 1 to 10.

Each R^l can be independently -R⁷, - (CH₂)_no-OR⁸,

-0- (CH₂) _m-OR⁸, - (CH2) n-NR⁷R¹⁰, -O- (CH 2) _m-NR⁷R¹⁰, - (CH2) n (CHOR⁸) (CHOR⁸) nCH₂OR⁸, -O- (CH₂)_m (CHOR⁸) (CHOR⁸) n-CH2OR⁸, - (CH2CH₂O) _m-R⁸, -0 (CH₂CH₂O) _m-R⁸, - (CH2CH2O) m-CH2CH2NR⁷R¹⁰, -O- (CH2CH₂O) _m-CH₂CH₂NR⁷R¹⁰,

- (CH₂) _no-C (= 0) NR⁷R¹⁰, -O- (CH 2) m C (= 0) NR⁷R¹⁰, - (CH2) _no- (Z) _g-R⁷,

-0- (CH₂) _m- (Z) _g-R⁷, - (CH₂)_no-NR¹⁰-CH2 (CHOR⁸) (CHOR⁸) n-CH₂OR⁸, -0 (CH₂) _m-NR¹⁰-CH₂ (CHOR⁸) (CHOR⁸) n-CH2OR⁸, - (CH2) _no-CO₂R⁷, -O- (CH₂)_mCO₂R⁷, -OSO3H, -O-glucuronide, -O-glucose,

<img file="PT1663235E_D0008.tif" />

The R groups^L Preferred include -H, -OH, -N (R^Z)₂especially when each R⁷ is hydrogen.

In the alkylene chain in formula (A), it is preferred that when an R group^L bonded to one carbon atom is different from hydrogen so the other R^L attached to that carbon atom is hydrogen, ie the formula -CHR^L-. It is also preferred that at most two R groups^L in an alkylene chain are different from hydrogen, and that the other groups R^L in the chain are hydrogen. Even more preferably, only one group R^l in a chain other groups, simple embodiments.

alkylene is different from hydrogen, and the

R^l in the chain are hydrogen. In these it is preferable that x represents a bond

EP 1 663 235 / EN

In another particular embodiment of the invention all R groups^L in the alkylene chain are hydrogen. In these embodiments, the alkylene chain is represented by the formula - (CH₂) ox- (CH₂)P-,

D⁷

<img file="PT1663235E_D0009.tif" />

Each R⁵ is independently Linker- (CH2) n-CAP, Linker (CH2) n (CHOR⁸) (CHOR⁸) n-CAP, Binder- (CH₂CH₂O) _m-CH₂-CAP, Binder (CH₂CH₂O)_m-CH₂CH₂-CAP, Ligand- (CH₂)_no- (Z)_g-CAP, Binder (CH₂) _no (Z) _g- (CH₂)_m-CAP, Ligand- (CH₂) _no-NR¹³-CH2 (CHOR⁸) (CHOR⁸) n-CAP, Binder- (CH₂) _no- (CHOR⁸) mCH2-NR¹³- (Z) g-CAP, Ligand- (CH₂) _noNR¹³(CH2) m (CHOR⁸) nCH₂NR¹³- (Z) _g-CAP, Ligand- (CH₂)_m- (Z) _g- (CH₂)_m-CAP,

NH-C-ligand (= 0) -NH- (CH₂)_m-CAP, Ligand- (CH₂)_m-C (= 0) NR¹³- (CH2) mC (= O) NR¹⁰R¹⁰, Binder- (CH2) _m-C (= 0) NR¹³- (CH2) m-CAP, Linker- (CH2) mC (= 0) NR¹¹R¹¹, Binder- (CH2) _no- (Z) _g- (CH₂) _m- (Z) _g-CAP, Z-Linker_g(CH₂) _m-Het- (CH₂) _m-CAP.

Each Binder is independently -0-, (CH₂)_no-, -0 (CH₂)_m-,

-NR¹³-C (= 0) -NR¹³, -NR¹³-C (= 0) - (CH 2) m -, -C (= 0) NR¹³- (CH2) _m, - (CH₂)_noZ_g- (CΗ₂) _no, -S -, - S0 -, - S0₂-, S0₂NR⁷-, SO₂NR¹⁰-or -Het-.

Each CAP is independently thiazolidinedione, oxazolidinedione, heteroaryl-C (= 0) NR¹³R¹³, heteroaryl-W, -CN, -OC (= S) NR¹³R¹³, -ZgR¹³, -CR¹⁰ (ZgR¹³) (Z_gR¹³), -C (= 0) 0Ar,

C (= 0) NR¹³Ar, imidazoline, tetrazole, tetrazoloamide, -SO₂NHR¹³, -SO 2 NH-C (R¹³R¹³) - (Z) gR¹³a cyclic amino sugar or oligosaccharide,

O

NR¹³

Ά

CONRⁱ³R^j nr¹³r¹³ or

Each Ar is independently phenyl, halogen substituted phenyl, wherein the substituents on halogen substituted phenyl are 1-3 substituents independently selected from the group consisting of OH, 0CH₃, NR¹³R¹³, Cl, F and CH₃, or heteroaryl.

ΕΡ 1 663 235 / ΡΤ

Each W is independently thiazolidinedione, oxazolidinedione, heteroaryl-C (= 0) NR¹³R¹³, -CN, -0-C (= S) NR¹³R¹³, -ZgR¹³, -CR¹⁰ (ZgR¹³) (Z_gR¹³), -C (= 0) 0Ar, -C (= 0) NR¹³Ar, imidazoline, tetrazole, tetrazoloamide, -SO₂NHR¹³, -SO 2 NH-C (R¹³R¹³) - (Z) gR¹³a cyclic sugar or oligosaccharide, a cyclic amino sugar or oligosaccharide,

T NR ”NR” R ”

<img file="PT1663235E_D0010.tif" />

Examples of heteroaryl include pyrazyl, tinazyl, furyl, furfuryl, thienyl, pyridyl, tetrazyl, furanyl, pyrazolyl, thiazolidinedionyl and thiophenyl, quinolyl, thiazolyl, isoxazolyl, imidazoyl, pyrrolyl, indolyl, adenyl, quinolinyl, benzyl, purolyl isoquinolinyl, pyridazyl, pyrimidyl, pyrazyl, 1,2,3-triazyl, 1,2,4-triazyl, 1,3,5-triazyl, cinolyl, phthalazyl, quinazolyl, quinoxalyl or pterdyl.

Each R⁶ is independently -R⁷, -OR⁷, -OR¹¹, -N (R⁷)₂,

- (CH₂) _m-0R⁸, -0- (CH2) m-0R⁸, - (CH2) _no-NR⁷R¹⁰, -0- (CH₂) _m-NR⁷R¹⁰,

- (CH₂) _no (CHOR⁸) (CHOR⁸) n-CH2OR⁸, -0- (CH2) _m (CHOR⁸) (CHOR⁸) _no-CH₂OR⁸,

- (CH₂CH₂O) _m-R⁸, -0- (CH 2 CH 2 O) mR⁸, - (CH2CH₂O) _m-CH₂CH₂NR⁷R¹⁰, -0 (CH2CH2O) m-CH2CH2NR⁷R¹⁰, - (CH2) _no-C (= 0) NR⁷R¹⁰, -0- (CH₂) _m-C (= 0) NR⁷R¹⁰,

- (CH₂)_no- (Z)_g-R⁷, -0- (CH 2) m - (Z) gR⁷, - (CH2) _no-NR¹⁰CH₂(CHOR⁸) (CHOR⁸) n-CH2OR⁸, -0- (CH2) _m-NR¹⁰-CH2 (CHOR⁸) (CHOR⁸) nCH₂OR⁸, - (CH₂) _no-CO₂R⁷, -0- (CH2) m-CO2R⁷, -OSO3H, -0-glucuronide, -O-glucose,

R⁷

<img file="PT1663235E_D0011.tif" />

where when two R⁶ are -OR¹¹ and are located adjacent to each other on a phenyl ring, the alkyl moieties of the two R⁶may be mutually linked to form a methylenedioxy group;

provided that when at least two -CH₂OR⁸ are located adjacent to each other, the groups R⁸ may be joined to form a mono- or disubstituted cyclic 1,3-dioxane or 1,3-dioxolane.

ΕΡ 1 663 235 / ΡΤ

In addition, one or more of the R groups⁶ can be one of the R groups⁵ that fall in the definition can of R⁶ established above.

When two R⁶ are -OR¹¹ and are located adjacent to each other on a phenyl ring, the alkyl moieties of the two R groups⁶ may be mutually linked to form a methylenedioxy group, ie a group of the formula -O-CH₂-O-.

As discussed above, R⁶ may be hydrogen. So 1, 2, 3 or 4 R groups⁶ may be different from hydrogen.

Preferably at most 3 of the R groups⁶ are different from hydrogen.

<td rowspan="2">Each Therefore,</td><td rowspan="2">g is, each</td><td colspan="2">regardless,</td><td rowspan="2">one 4,</td><td colspan="2">number</td><td rowspan="2">whole of</td><td rowspan="2">1 to 6.</td>

<td>g can be</td><td> 1, 2, 3,</td><td> 5</td><td>or 6</td>

<td>Each</td><td>m is</td><td>a number</td><td>all</td><td>in</td><td> 1</td><td>to 7</td><td>Therefore,</td><td>every m</td>

<td>Can be</td><td> 1, 2,</td><td> 3, 4, 5, 6</td><td>or 7.</td><td></td><td></td><td></td><td></td><td></td>

<td>Each</td><td>huh</td><td>a number</td><td>all</td><td>in</td><td> 0</td><td>to 7</td><td>Therefore,</td><td>every n</td>

<td>Can be</td><td> 0, 1,</td><td> 2, 3, 4, 5</td><td>, 6 or 7.</td><td></td><td></td><td></td><td></td><td></td>

<td>Each</td><td>Q on</td><td colspan="2">formula (A) is CR⁵</td><td>, ç-</td><td>-R⁶</td><td colspan="2">or an atom of</td><td>nitrogen,</td>

<td>where at most</td><td>three</td><td>Q num</td><td>ring are</td><td colspan="2">nitrogen atoms.</td>

<td>there may be 1,</td><td> 2</td><td>or</td><td>3 atoms</td><td>of nitrogen</td><td>on one</td>

<td>Preferably,</td><td>at the</td><td>maximum</td><td colspan="2">two Q are atoms of</td><td>nitrogen</td>

<td>preferably</td><td>at the</td><td>maximum</td><td>a Q is</td><td>an atom of</td><td>nitrogen</td>

ring. In a particular embodiment, the nitrogen atom is at position 3 of the ring. In another embodiment of the invention, each Q is CR⁵ or

CR⁶, ie

there are no nitrogen atoms in the ring

More specific examples of suitable groups represented by formula (A) are given in formulas (B) - (E) below:

Q = Qx^r5 - (CH₂) ox- (CH₂) p— &lt;J&gt; (B)

Q ~ Q4 (r6)₄ where the, x, p, R⁵ and R⁶, are as defined above;

ΕΡ 1 663 235 / ΡΤ

<img file="PT1663235E_D0012.tif" />

<td>where n above;</td><td>is</td><td>a number</td><td>all</td><td>in</td><td> 1</td><td>The</td><td> 10</td><td>and</td><td>R^O</td><td>is</td><td>as defined</td>

<td></td><td></td><td></td><td>- (CH₂)„—</td><td>ç</td><td>y N</td><td>-R⁵</td><td></td><td>(D)</td><td></td><td></td><td></td>

<td>where n above;</td><td>is</td><td>a number</td><td>all</td><td>in</td><td> 1</td><td>The</td><td> 10</td><td>and</td><td>R⁵</td><td>is</td><td>as defined</td>

(CH₂) o χ— (CH₂) p — ν, /) - R^J (Ε) \\ // where ο, χ, ρ and R³ are as defined above.

In a preferred embodiment of the invention, Y is -NH₂.

In another preferred embodiment, R² is hydrogen.

In another preferred embodiment, R¹ is hydrogen.

In another preferred embodiment, X is chlorine.

In another preferred embodiment, R³ is hydrogen.

In another preferred embodiment, R^L is hydrogen.

In another preferred embodiment, o is 4.

In another preferred embodiment, p is 0.

In another preferred embodiment, the sum of oep is 4.

In another preferred embodiment, x represents a single bond.

In another preferred embodiment, R⁶ is hydrogen.

of nitrogen.

In another preferred embodiment, at most one Q is one atom.

ΕΡ 1 663 235 / ΡΤ

In another preferred embodiment, no Q is a nitrogen atom.

In a preferred embodiment of the present invention:

X is halogen;

Y is -N (R⁷)₂;

R¹ is hydrogen or alkyl θ! -θ₃;

R² is -R⁷, -OR⁷, CH2OR⁷ or -CO2R⁷;

R³ is a group represented by formula (A); and

R⁴ is hydrogen, a group represented by formula (A) or C1 -C6 alkyl₇;

In another preferred embodiment of the present invention:

X is chlorine or bromine;

Y is -N (R⁷)₂;

R² is hydrogen or alkyl₂-Ο3, · at most three R⁶ are different from hydrogen as described above;

at most three R^L are different from hydrogen as described above; and at most 2 Q are nitrogen atoms.

In another preferred embodiment of the present invention:

Y is -NH₂;

In another preferred embodiment of the present invention:

R⁴ is hydrogen;

at most one R^L is different from hydrogen as described above;

at most two R⁶ are different from hydrogen as described above; and at most 1 Q is a nitrogen atom.

In another preferred embodiment of the present invention the compound of formula (1) is represented by the formula:

ΕΡ 1 663 235 / ΡΤ

<img file="PT1663235E_D0013.tif" />

In another preferred embodiment of the present invention the compound of formula (1) is represented by the formula:

<img file="PT1663235E_D0014.tif" />

In another preferred embodiment of the present invention the compound of formula (1) is represented by the formula:

<img file="PT1663235E_D0015.tif" />

In another preferred embodiment of the present invention the compound of formula (1) is represented by the formula:

<img file="PT1663235E_D0016.tif" />

In another preferred embodiment of the present invention the compound of formula (1) is represented by the formula:

<img file="PT1663235E_D0017.tif" />

In another preferred embodiment of the present invention the compound of formula (1) is represented by the formula:

ΕΡ 1 663 235 / ΡΤ

<img file="PT1663235E_D0018.tif" />

In compound another preferred embodiment of formula (1) is represented by the present invention by formula

<img file="PT1663235E_D0019.tif" />

In another preferred embodiment of the present compound of formula (1) is represented by formula

<img file="PT1663235E_D0020.tif" />

In another preferred embodiment of the present compound of formula (1) is represented by formula

<img file="PT1663235E_D0021.tif" />

In another preferred embodiment of the present compound of formula (1) is represented by formula

<img file="PT1663235E_D0022.tif" />

In another preferred embodiment of the present invention the

1 663 235 / ΡΤ compound of formula (1) is represented by formula

<img file="PT1663235E_D0023.tif" />

In another preferred embodiment of the present compound of formula (1) is represented by formula

<img file="PT1663235E_D0024.tif" />

In another preferred embodiment of the present compound of formula (1) is represented by formula

<img file="PT1663235E_D0025.tif" />

In another preferred embodiment of the present compound of formula (1) is represented by formula

<img file="PT1663235E_D0026.tif" />

In another preferred embodiment of the present compound of formula (1) is represented by formula

<img file="PT1663235E_D0027.tif" />

In another preferred embodiment of the present invention the

1 663 235 / ΡΤ compound of formula (1) is represented by formula

<img file="PT1663235E_D0028.tif" />

In another preferred embodiment of the present compound of formula (1) is represented by formula

<img file="PT1663235E_D0029.tif" />

In another preferred embodiment of the present compound of formula (1) is represented by formula

<img file="PT1663235E_D0030.tif" />

In another preferred embodiment of the present compound of formula (1) is represented by the formula

invention the

<img file="PT1663235E_D0031.tif" />

In another preferred embodiment of the present compound of formula (1) is represented by formula

<img file="PT1663235E_D0032.tif" />

In another preferred embodiment of the present compound of formula (1) is represented by formula

ΕΡ 1 663 235 / ΡΤ

<img file="PT1663235E_D0033.tif" />

In another preferred embodiment of the present invention the compound of formula (1) is represented by the formula:

<img file="PT1663235E_D0034.tif" />

In another preferred embodiment of the present invention the compound of formula (1) is represented by the formula:

The NH h₂nn nh₂ ___XAp

2HC1

KNOW

In another preferred embodiment of the present invention the compound of formula (1) is represented by the formula:

The NH

Cl &lt;.Ν. XX

<img file="PT1663235E_D0035.tif" />

CONH, h₂nn nh₂

2HC1

In another preferred embodiment of the present invention the compound of formula (1) is represented by the formula:

<img file="PT1663235E_D0036.tif" />

CO, H

H, N

2HCI

The compounds of formula (I) may be prepared and used in free base form. 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 confer no undesirable toxicological effects. Examples of these salts

66 1 663 235 / ΡΤ (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, poly (glutamic acid), naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalene disulfonic acid, poly (galacturonic 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 such as chlorine, bromine and iodine.

It should be noted that all enantiomers, diastereomers and racemic mixtures of compounds within the scope of formula (I) are encompassed by the present invention. All mixtures of these enantiomers and diastereomers are within the scope of the present invention.

Without limitation to any particular theory, the compounds of formula (I) are believed to function in vivo as sodium channel blockers. By blocking epithelial sodium channels present on mucosal surfaces the compounds of formula (I) reduce water absorption by mucosal surfaces. This effect increases the volume of protective liquids on mucosal surfaces, rebalances the system, and thereby treats disease.

The present invention also provides treatment methods that take advantage of the properties of the compound of formula (I) discussed above. Thus, individuals who may be treated with the methods of the present invention include, but are not limited to, patients affected 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 sample of

1 663 235 / ΡΤ a patient's sputum 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 by aerosol (liquid or dry powders) or washing.

Individuals who may be treated with the method of the present invention also include patients who are administered nasal supplemental oxygen (a regimen that tends to dry airway surfaces); patients affected with an allergic disease or response (eg, an allergic response to pollen, dust, animal hair or to particles, insects or insect particles, etc.) affecting nasal airway surfaces; patients affected with a bacterial infection eg, staph infections such as Staphylococcus aureus infections, Hemophilus influenza infections, Streptococcus pneumoniae infections, Pseudomonas aeuriginosa infections, etc.) of nasal airway surfaces; patients affected with an inflammatory disease affecting nasal airway surfaces; or patients affected with sinusitis (wherein the active agent or agents are administered to promote drainage of congested sinus mucous secretions by administering an effective amount to promote drainage of congested fluid in the sinuses), or combined, rhinosinusitis. The invention may be administered to rhino-signal surfaces by topical delivery, including aerosols and drops.

The present invention may be used to hydrate mucosal surfaces other than airway surfaces. These other mucosal surfaces include gastrointestinal surfaces, oral surfaces, genitourethral surfaces, ocular surfaces, or eye, inner ear, and middle ear surfaces. 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.

ΕΡ 1 663 235 / ΡΤ

The compounds of the present invention are also useful for treating a variety of cardiovascular system related functions. 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.

The present invention relates primarily to 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.

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. As the free base of the compound is generally less soluble in aqueous solutions than salt, free base compositions are employed to provide more sustained release of the active agent to the lungs. An active agent present in the lungs in the form of particles that did not dissolve in solution is not physiologically available, but serves as an induce a bioavailable drug deposition response that gradually dissolves in solution.

Another aspect of the present invention is a pharmaceutical composition comprising a compound of formula (I) in a pharmaceutically acceptable carrier (eg, an aqueous carrier solution). In general, the compound of formula (I) is included in the composition in an amount effective to inhibit water resorption by mucosal surfaces.

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 herein).

(1 663 235 / ΡΤ referred to as an active agent). The composition may further comprise a P2Y2 receptor agonist or a pharmaceutically acceptable salt thereof (also sometimes referred to herein as an active agent). 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 US 6,264,975, US 5,656,256 and US 5,292,498.

Bronchodilators may also be used in

These combinations with compounds of the present invention, bronchodilators include, but are not limited to, β-adrenergic agonists including, but not limited to, epinephrine, isoproterenol, fenoterol, albutereol, terbutaline, bitolterol, metaproterenol, iosetarin, 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 according to known techniques, prior to or simultaneously with the active compounds described herein. Additional procedures useful for preparation are in USUSUS especially for the preparation of various

Another aspect of the present invention is a pharmaceutical formulation comprising an active compound as described above in a pharmaceutically acceptable carrier (eg, an aqueous carrier solution). In general, the active compound is included in the composition in an amount effective to treat mucosal surfaces, such as for inhibiting water resorption by mucosal surfaces, including the airways and other surfaces.

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 surface.

66 1 663 235 / gastr gastrointestinal mucosa. The active compound may be combined with a pharmaceutically acceptable carrier in any suitable form, such as sterile or diluted physiological saline or topical solution, in the form of droplets, tablets or the like for oral administration, as a rectal administration suppository. or genitourethral, ​​etc. Excipients may be included in the formulation to increase the solubility of the active compounds as desired.

The active compounds disclosed herein may be administered to a patient's airway surfaces 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 or distilled water. . For example, the active compounds may be prepared as formulations and administered as described in Jacobus US Patent 5,789,391.

breathable, retained in

Particulate active agents, solid or liquid, prepared for the practice of the present invention may include, as noted above, breathable or non-breathable particles; i.e. for breathable particles, particles small enough to pass through the mouth and larynx by inhalation and to the lung bronchi and alveoli, and for non-particle particles large enough to be passages of the nasal airway rather than passing 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 breathable. Non-breathable particles 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 µm may be used to ensure retention in the nasal cavity.

In the manufacture of a formulation according to the invention the active agents or their free salts or bases

Physiologically acceptable, are typically mixed with, inter alia, an acceptable carrier. It is evident that the carrier must be compatible with any other ingredients of the formulation and cannot be harmful 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, which may contain 0.5% to 99% by weight of active compound. . One or more active compounds may be incorporated into the formulations of the invention, which formulations may be prepared by any well-known pharmacy techniques consisting essentially of mixing the components.

Compositions containing breathable or non-breathable 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 or separate large agglomerates.

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 combined with the active agent in any suitable ratio (eg, a 1 to 1 weight ratio).

The active compounds disclosed herein may be administered to airway surfaces including an individual's nasal passages, sinuses and lungs by a suitable means known in the art, such as nasal drops, nebulisations, etc. In one embodiment of the invention, the active compounds of the present invention are administered by transbronchoscopic lavage. In a preferred embodiment of the invention, the active compounds of the present invention are deposited on pulmonary airway surfaces by administering an aerosol suspension of breathable particles comprised of the active compound which the subject inhales. Breathable particles may be liquid or solid. Numerous inhalers are known to

66 1 663 235 / ΡΤ administering aerosol particles to the lungs of an individual.

Inhalers such as those developed by Inhale Therapeutical Systems, Palo Alto, California, USA may be employed, including but not limited to those disclosed in US Patent 5,740,794; 5,654,007; 5,458,135; 5,775,320; and 5,785,049. Inhalers such as those developed by Dura Pharmaceuticals, Inc., San Diego, California, USA may also be employed, including, but not limited to, those disclosed in US Patents 5,622,166; 5,577,497; 5,645,051; and 5,492,112. Additionally, inhalers such as those developed by Aradigm Corp., Hayward, California, USA may be employed, including, but not limited to, those disclosed in US Patents 5,826,570; 5,813,397; 5,819,726; and 5,655,516. These devices are particularly suitable as dry particle inhalers.

per

eg

are pressure or a patent devices solutions or

Liquid particle aerosols comprising the active compound may be produced by any means, such as with an ultrasonic nebulizer driven aerosol nebulizer. See if,

US 4,501,729. Commercially available nebulizers which transform suspensions of the active ingredient into a therapeutic aerosol nebulization either by accelerating compressed gas, typically air or oxygen, through a narrow venturi orifice or by ultrasonic agitation. Formulations suitable for use in nebulizers consist of the active ingredient in a liquid carrier, the active ingredient constituting up to 40% w / w of the formulation, but preferably less than 20% w / w. The carrier is typically water (and more preferably sterile, free water or perfluorocarbon alcoholic solution 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.

pyrogen) aqueous carriers may be diluted. The also be

ΕΡ 1 663 235 / ΡΤ

Solid particulate aerosols comprising the active compound may likewise be produced with any solid particulate medicament generator. Aerosol generators for delivering solid particulate medicaments to an individual produce particles that are breathable, as explained above, and generate an aerosol volume containing predetermined metered doses of medicament at a rate suitable for human administration. An illustrative type of solid particulate aerosol generator is an insufflator. Formulations suitable for administration by insufflation include finely comminuted powders which may be delivered by means of an insufflator or taken into the nasal cavity as an inspiration. In the insufflator, the powder (eg, its effective metered dose to perform the treatments described herein) is contained in capsules or cartridges, typically made of gelatin or plastic, which are perforated or opened in situ and the powder is delivered by air drained through. device by inhalation or by means of a manually operated pump. 0 The powder employed in the insufflator consists of either only the active ingredient or a mixture of powders comprising the active ingredient, a suitable powder diluent such as lactose, and an optional surfactant. The active ingredient typically constitutes from 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 formulation or active solution in a liquefied propellant. During these devices the formulation discharges a valve adapted to deliver a metered volume, typically from 10 to 150 μΐ, 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 or more co-solvents, for example ethanol, surfactants such as oleic acid or sorbitan trioleate, antioxidants and suitable flavoring agents.

ingredient use through

Aerosol, whether formed of solid or liquid particles, can be produced by the aerosol generator at a

A speed of about 10 to 150 liters per minute, more preferably 30 to 150 liters per minute, and most preferably about 60 liters per minute. The aerosols containing larger amounts administered faster.

medication can be

<td>The</td><td colspan="2">treat and</td><td>state of</td>

<td>to be</td><td>in</td><td>fence</td><td>of 0.01, 0.03,</td>

<td>mg</td><td>of</td><td>agent</td><td>pharmaceutical,</td>

The dosage of active compounds disclosed herein will vary depending on the individual condition, but generally may 0.05, 0.1 to 1, 5, 10 or 20 deposited on airway surfaces. The daily dose may be divided between one or multiple unit dose administrations. The aim is to achieve a concentration of pharmaceutical agents on lung airway surfaces of between 10 ”⁹ - 10⁴ M.

In another aerosol particle administration administered by the embodiment, a breathable or non-breathable suspension (preferably non-breathable particle) comprised of active compound, which the subject inhales through the nose. Breathable or non-breathable particles may be liquid or solid. The amount of active agent included may be sufficient to achieve dissolved concentrations of active agent on an individual's airway surfaces of about 1CT.⁹, 10”⁸ or

10 ~ about 10 ~

10~

10 ~ moles / liter, more preferably about 10 ⁹ about 10 ^what moles / liter.

The dosage of active compound will vary depending upon the condition to be treated and the condition of the subject, but may generally be sufficient to achieve dissolved concentrations of active compound on the subject's nasal airway surfaces of about ΙΟ ”.⁹, ΙΟ ”⁸, 10”⁷ about ² or 10 ¹ moles / liter, and more preferably about 10 ”⁷ at about 10 ”moles / liter. Depending on the solubility of the particular active compound formulation administered, the daily dose may be divided between one or more 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 milligrams of active agent particles for a human subject, depending on the age and condition of the subject. individual. An

ΕΡ 1 663 235 / ΡΤ unit dose 0.5 milligrams 2-10 administrations per day. In the form of a presently preferred unit is about active agent given in a regimen. The dosage may be provided prepackaged by any suitable means (eg, encapsulation in a gelatin capsule).

In one embodiment of the invention, the particulate active agent composition may contain both a free base of the active agent and a pharmaceutically acceptable salt to provide both initial release and sustained release of active agent for dissolution in mucus secretions of the nose. This composition serves to provide both initial 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.

Pharmaceutical formulations suitable for airway administration include solution, emulsion, suspension and extract formulations. See generically, J. Nairn, Solutions, Emulsions, Suspensions and Extracts, in Remington: The Science and Practice of Pharmacy, chap. 86 (19th ed. 1995). Pharmaceutical formulations suitable for nasal administration may be prepared as described in US Patent Nos. 4,389,393 to Schor; 5,707,644 to Illum; 4,294,829 to Suzuki; and 4,835,142 to Suzuki.

Liquid particulate nebulizations or aerosols comprising the active compound may be produced by any suitable means, such as by a simple nasal spray with the active agent in a pharmaceutically acceptable aqueous carrier, such as a sterile saline or sterile water. Administration may be with a pressure-driven aerosol nebulizer or with an ultrasonic nebulizer. See eg US Patents 4,501,729 and 5,656,256. Formulations suitable for use in a nasal droplet or spray bottle or nebulizers consist of the active ingredient in a liquid carrier, the active ingredient constituting up to

40% w / w of the formulation, but preferably less than 20%

ΕΡ 1 663 235 / ΡΤ p / p. Typically, the carrier is water (and more preferably sterile, pyrogen-free water) or dilute aqueous alcoholic solution, preferably prepared in 0.12% to 0.8% sodium chloride solution. 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 (eg mannitol, xylitol, erythritol) and surfactants.

Compositions containing breathable or non-breathable 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 or separate large agglomerates.

The particulate composition may optionally contain a dispersant which serves to facilitate aerosol formation. A suitable dispersant is lactose, which may be combined with the active agent in any suitable ratio (eg, a 1 to 1 weight ratio).

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:

<img file="PT1663235E_D0037.tif" />

NHR Ν 1 '' N = C — S-CH₃

NHR²

HNR³R⁴-► (i)

These procedures are described, for example, in EJ Cragoe, The Synthesis of Amiloride and Its Analogs (Chapter 3) in Amiloride and Its Analogs, p. 25-36. Other methods of preparing the compounds are described, for example, in US 3,313,813. In particular, see Methods A, B, C and D described in US 3,313,813. Other methods useful for the preparation of these compounds, especially for the preparation of the novel HNR3R4 fragment, are described, for example, in 229929US, 233377US and

ΕΡ 1 663 235 / ΡΤ

234105US. Schemes 1 to 11 are representative, but not limited to, of procedures used to prepare the sodium channel blockers described herein.

Scheme 1: Synthesis of PSA 17926:

<img file="PT1663235E_D0038.tif" />

OH

CbzHN

<img file="PT1663235E_D0039.tif" />

CbzHN

NaN_3&gt;NH₄Cl | DMF

<img file="PT1663235E_D0040.tif" />

<img file="PT1663235E_D0041.tif" />

<img file="PT1663235E_D0042.tif" />

<img file="PT1663235E_D0043.tif" />

5, PSA 17926

ΕΡ 1 663 235 / ΡΤ

Scheme 2: Synthesis of PSA 17846:

<img file="PT1663235E_D0044.tif" />

MeNH₂^HiC'n^XO ¹¹

CH, 10

MeOH

<img file="PT1663235E_D0045.tif" />

NH,

The SMe ^c1-'^no'AAnh₂.hi

ΪΪ¹' H₂nn nh₂^H’^ç'n^XO

CH,

<img file="PT1663235E_D0046.tif" />

Et₃N, THF

H₂NN NH₂

NH O'Cl

11, PSA 17846

ΕΡ 1 663 235 / ΡΤ

Scheme 3: Synthesis of PSA 19008:

O

<img file="PT1663235E_D0047.tif" />

CISOjH, 0 ° C

<img file="PT1663235E_D0048.tif" />

MeNH₂

<img file="PT1663235E_D0049.tif" />

16, PSA 19008

ΕΡ 1 663 235 / ΡΤ

Scheme 4: Synthesis of PSA 17482

CbzHN

<img file="PT1663235E_D0050.tif" />

OH

Aniline, EDCI DMAP / CH₂CI₂

<img file="PT1663235E_D0051.tif" />

The fjçu

H

<img file="PT1663235E_D0052.tif" />

CbzHN

<img file="PT1663235E_D0053.tif" />

H

20, PSA 17482

ΕΡ 1 663 235 / ΡΤ

Scheme 5: Synthesis of PSA 23022:

CbzHN

<img file="PT1663235E_D0054.tif" />

<img file="PT1663235E_D0055.tif" />

22, PSA 23022

ΕΡ 1 663 235 / ΡΤ

Scheme 6:

Summary of

PSA 16826:

OH

CbzHN

<img file="PT1663235E_D0056.tif" />

O + HN

OH

EtOH

CbzHN

<img file="PT1663235E_D0057.tif" />

N s

OH

H₂, Pd / C _r EtOH / AcOH

Β, Ν

<img file="PT1663235E_D0058.tif" />

OH

<img file="PT1663235E_D0059.tif" />

OH

27, PSA 16826

ΕΡ 1 663 235 / ΡΤ

Scheme 7: PSA 16313 Synthesis:

<img file="PT1663235E_D0060.tif" />

<img file="PT1663235E_D0061.tif" />

30, PSA 16313

ΕΡ 1 663 235 / ΡΤ

Scheme 8: Synthesis of PSA 16437:

OH

SoeHN

<img file="PT1663235E_D0062.tif" />

BrCH₂CO₂Me

Bochn

Nal, K₂CO_3s^dmf

4?

<img file="PT1663235E_D0063.tif" />

OMe

Bochn

Hn

KOH MeOH c

<img file="PT1663235E_D0064.tif" />

OH

Η₂2 ^ Ύ&gt; V2H₂ONLY₄

Bochn

<img file="PT1663235E_D0065.tif" />

HCl

Dioxane

The HN ^

H

EDC, HOAt, DMAP Pr₂NEt, CH₂C1₂/ THF / CH₃CN

<img file="PT1663235E_D0066.tif" />

H₂n • 2HCi

The SMe

Z-pr₂NEt, EtOH / MeOH

The NH

Τ / Λ

H, NN NH,

<img file="PT1663235E_D0067.tif" />

(2) HCl, MeOH

Ο HN — λ

O ^ Jk /

• NH • 2HC1

36, PSA 16437

ΕΡ 1 663 235 / ΡΤ

Scheme 9: PSA 16314 Synthesis:

OH

CbzHN

CbzHN

<img file="PT1663235E_D0068.tif" />

NH,

<img file="PT1663235E_D0069.tif" />

<img file="PT1663235E_D0070.tif" />

39, PSA 16314

ΕΡ 1 663 235 / ΡΤ

Figure 10: Synthesis of PSA 16208:

<img file="PT1663235E_D0071.tif" />

<img file="PT1663235E_D0072.tif" />

<img file="PT1663235E_D0073.tif" />

42, PSA 16208

ΕΡ 1 663 235 / ΡΤ

Scheme 11: Synthesis of PSA 15143:

OH

CbzHN

EtOH / TEA

OH OH

X / 0 ^ A .OH

CbzHN

II₂/ Pd / C

EtOH

<img file="PT1663235E_D0074.tif" />

45, PSA 15143

ΕΡ 1 663 235 / ΡΤ

Various assays may be used to characterize the compounds of the present invention. Representative assays are discussed below.

In Vitro Measurement of Activity and Reversibility of

Sodium Channel Blockage

One assay used to determine the mechanism of action and / or potency of the compounds of the present invention involves the determination of luminal inhibition by short-circuit current (Isc) measured epithelial airway sodium currents using airway epithelial monolayers. aerials in Ussing chambers. Cells obtained from newly excised human, dog, sheep or rodent airways are seeded onto Snapwell ™ 0.4 micron porous inserts (CoStar), cultured under air-liquid interface (ALI) conditions in hormonally defined media, and assayed for sodium transport activity (Isc) while in a Krebs-Ringer Bicarbonate (KBR) bath in Ussing chambers. All test drug additions are to the luminal bath with semi-logar dose addition protocols (1 x 1CU¹¹ M at 3 x 1CU⁵ Μ), and the cumulative change in Isc (inhibition) is recorded. All drugs are prepared in dimethyl sulfoxide as stock solutions at a concentration of 1 x 1CU.² M and stored at -20 ° C. Eight preparations in parallel are typically used per operation; two preparations per operation incorporate amiloride and / or benzamyl as positive controls. After maximum concentration (5 x 1CT⁵ M) be administered, the luminal bath is exchanged three times for fresh drug-free KBR solution, and the resulting Isc was measured after each wash for approximately 5 minutes. Reversibility is defined as the percentage that returns to the baseline value for the sodium stream after the third wash. All voltage controlled data is collected through a computer interface and offline analyzer.

Dose-effect ratios for all compounds are considered and analyzed with the Prism 3.0 program. CI values₅o, maximum effective concentrations, and reversibility are

66 1 663 235 / ΡΤ calculated and compared with amiloride and benzamyl as positive controls.

Pharmacological Absorption Assays (1) Apical Disappearance Assay

Bronchial cells (dog, human, sheep or rodent cells) are seeded at a density of 0.25 x 10⁶/ cm² over a Transwell-Col collagen-coated porous membrane with a growth area of ​​1.13 cm² grown at an air-liquid interface in hormonally defined media that promote a polarized epithelium. From 12 to 20 days after the development of an air-liquid interface (ALI) cultures are expected to be&gt; 90% ciliated, and mucins will accumulate in cells. To ensure the integrity of primary airway epithelial cell preparations, transepithelial resistance (R_t) and transepithelial potential differences (PD), which are indicators of the polarized integrity of the culture. Human cell systems are preferred for studies of absorption rates from apical surfaces. The disappearance test is conducted under conditions that mimic the thin films in vivo (~ 25 μΐ) and is initiated by the addition of experimental sodium channel blockers or positive controls (amiloride, benzamyl, phenamyl) to the apical surface at an initial concentration of 10 µM. μΜ. A series of samples (5 μ volume per sample) are taken at various time points, including 0, 5,

20, 40, 90 and 240 minutes. Concentrations are determined by measuring the intrinsic fluorescence of each sodium channel blocker using a Fluorocount or HPLC microplate fluorometer. Quantitative analysis employs a standard curve generated from authentic reference standard materials of known concentration and purity. Disappearance rate data analysis is performed using nonlinear regression, one-phase exponential decay (Prism V 3.0).

2. Confocal Microscopy Assay for the Assimilation of

Amiloride counterparts

Virtually all amiloride-like molecules fluoresce in the ultraviolet range. This property of these

66 1,663,235 / ΡΤ molecules can be used to directly measure cell assimilation using xz confocal microscopy. Equimolar concentrations of experimental compounds and positive controls including amiloride and compounds demonstrating rapid assimilation in the cell compartment (benzamyl and phenamyl) are placed on the apical surface of airway cultures on the confocal microscope support. Xz series of images are obtained over time and the magnitude of the fluorescence that accumulates in the cell compartment is quantified and represented as a change in fluorescence versus time.

3 In vitro Compound Metabolism Assays

Airway epithelial cells have the ability to metabolize drugs during the transepithelial absorption process. Additionally, although less likely, it is possible that drugs may be metabolised to epithelial airway surfaces by specific ectoenzyme activities. Perhaps most likely as an ecto-surface event, the compounds may be metabolised by infected secretions that occupy the airway lumens of patients with lung disease, eg. cystic fibrosis. Thus, a series of assays are performed to characterize compound metabolism that results from the interaction of test compounds with human airway epithelia and / or human airway epithelial lumen products.

In the first series of tests, the interaction of KBR test compounds as ASL stimulants are applied to the apical surface of human airway epithelial cells grown in the T-Col insertion system. For most compounds, metabolism (generation of new species) is tested using high performance liquid chromatography (HPLC) to resolve chemical species and the endogenous fluorescence properties of these compounds to estimate the relative amounts of compound metabolites. For a typical assay, one (25 μΐ KBR containing 10 μΜ test compound) is placed on the epithelial luminal surface. Sequential samples of 5 to 10 μΐ from luminal and serosal compartments are obtained for HPLC analysis of (1) the mass of test compound that permeates the luminal to serosal and (2) potential test and new test solution.

ΕΡ 1 663 235 / ΡΤ formation of metabolites from the parent compound. In cases where the fluorescence properties of the test molecule are not suitable for these characterizations, radiolabelled compounds are used for these assays. From the HPLC data, the rate of disappearance and / or formation of new metabolite compounds on the luminal surface and the appearance of test compound and / or new metabolites in the basolateral solution are quantified. Data related to chromatographic mobility of potential new metabolites with reference to the parent compound are also quantified.

To analyze the potential metabolism of test compounds for CF sputum, a representative mixture of expelled CF sputum obtained from 10 CF patients (IRB approved) was collected. Sputum was solubilized in a 1: 5 mixture of vigorous vortex KBR solution, after which the mixture was divided into a crude sputum aliquot and an aliquot subjected to ultracentrifugation so that a supernatant aliquot was obtained (crude = cellular; supernatant = liquid phase). Typical CF sputum compound metabolism studies involve adding known masses of test compound to crude CF sputum and CF sputum supernatant aliquots incubated at 37 ° C, followed by sequential sampling of aliquots of each sputum type. for characterization of compound stability / metabolism by HPLC analysis as described above. As above, the disappearance analysis of the compound, rates of new metabolite formation, and HPLC mobilities of new metabolites are then performed.

4 Pharmacological Effects and Mechanism of Action of the Drug in

Animals

The effect of the compounds on increasing mucociliary clearance (MCC) can be measured using an in vivo model described by Sabater et al., Journal of Applied Physiology, 1999, p. 2191-2196.

ΕΡ 1 663 235 / ΡΤ

EXAMPLES

Having generally described the present invention, further understanding may be obtained by reference to certain specific examples which are provided herein for illustration purposes only and are not intended to be limiting unless otherwise specified.

Preparation of Sodium Channel Blockers

Materials and methods. All reagents and solvents were purchased from Aldrich Chemical Corp. and used without further purification. NMR spectra were obtained on a Bruker WM 360 (³H NMR 360 MHz and ¹³90 MHz NMR) or a Bruker AC 300 (³300 MHz NMR and ¹³75 MHz NMR). Flash chromatography was performed on a Flash Elute ™ Elution Solution system (PO Box 5147, Charlottesville, Virginia 22905) loaded with a 90 g silica gel cartridge (40M FSO-0110-040155,

32-63 pm) at 20 psi (N₂). 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: 320 ° C Injector, 320 ° C Detector, FID Gas Flow: H₂ at 40 ml / min., Ar at 400 ml / min. Carrier gas: 16: 1 split ratio, N flow₂ at 15 ml / min. N rate₂ at 18 cm / s. The temperature program is 70 ° C for 0-3 min, 70-300 ° C for 3-10 min, 300 ° C for 10-15 min.

HPLC analysis was performed on a Gilson 322 Pump, UV / Vis-156 detector at 360 nm equipped with a Microsorb MV C8, 100 A, 25 cm column. Mobile Phase: A = acetonitrile with 0.1% TFA, B = water with 0.1% TFA. Gradient Program: 95: 5 B: A for 1 min, then up to 20:80 B: A over 7 min, then up to 100% A over 1 min, followed by washing with 100% A during 11 min, flow rate: 1 ml / min.

ΕΡ 1 663 235 / ΡΤ

Example 1

Synthesis of N- (3,5-Diamino-6-chloropyrazine-2carbonyl) -N '- (4- {4- [3- (1H-tetrazol-5-yl) propoxy] phenyl} butyl) guanidine hydrochloride (PSA 17926)

<img file="PT1663235E_D0075.tif" />

Butyl} carbamic benzyl ester (2).

{4- [4- (3-cyanopropoxy) phenyl] 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 ° C for 16 h. The 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 product purified by crude flash column chromatography was silica gel eluting with ethyl acetate / CH₂Cl 2 (1: 9, v / v) to afford the desired product 2 75% yield) 2.15 (m, 2H), 2.55 as a white solid (1.80 g, ⁴1H NMR (300 MHz, CDCl3)₃) δ 1.56 (m, 4H), (m, 4H), 3.15 (m, 2H), 4.00 (m, 2H), 4.70 (sg, 1H), 5.10 (s , 2H), 6.80 (d, 2H), 7.05 (d, 2H), 7.30 (m,

5H). m / z (ESI): 367 [C₂₂H₂₆N₂O₃⁺ H] ⁺ .

(4- {4- [3- (1H-Tetrazol-5yl) propoxy] phenyl} butyl) carbamic acid benzyl ester (3).

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 ° C for 16 h. Inorganic materials were removed by vacuum filtration. The filtrate was diluted with ethyl acetate and washed with water and brine. The organic solution was dried over Na₂ONLY₄, filtered and concentrated. The residue was taken up in ethyl acetate (5 mL) and diluted with hexanes (10 mL). The solid precipitates were collected by suction filtration and purified by

66 1,663,235 / flash silica gel flash 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). %). B NMR (300 MHz, CD₃OD) δ 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 22 H 27 N 5 O 3 + H]⁺.

4- {4- [3- (1H-Tetrazol-5-yl) propoxy] phenyl} butylamine (4).

(4- {4- [3- (1H-Tetrazol-5-yl) propoxy] phenyl} butyl) carbamic acid benzyl ester solution 3 (0.30 g,

0.73 mmol) in methanol (20 mL) and dichloromethane (5 mL) was stirred at room temperature overnight under a 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 afford the desired product 4 as a white solid (200 mg, 99% yield) which was used in the next step without further purification. m / z (ESI): 276 [C₁₄H₂₁N₅O + H]⁺.

N- (3,5-Diamino-6-chloropyrazine-2-carbonyl) -AU (4- {4- [3- (1H-tetrazol-5-yl) propoxy] phenyl} butyl) guanidine hydrochloride (5,

PSA 17926).

A solution of 4- {4- [3- (1H-tetrazol-5-yl) propoxy] phenyl} butylamine (0.39 mmol) for 5 (100 mg, 0.36 mmol) and triethylamine (0.15 mL in Absolute ethanol (2 mL) was stirred at 60 ° C after which time sodium hydroxide iodide was added.

1- (3,5-diamino-6-chloropyrazine-2-carbonyl) -2-methylisothiourea (150 mg, 0.39 mmol) 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 slurry of dark yellow solid (140 mg, 80% yield) thus obtained was formed in a mixture of methanol and dichloromethane (5/95, v / v). The solid was collected by suction filtration, and 40 mg of this 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 ') 1 663 235/1 carbonyl) -N' - (4- {4- [3- (1H- tetrazol-5-yl) propoxy] phenyl} butyl) -

<td>guanidine (5,</td><td>PSA</td><td> 17926)</td><td>under the form</td><td>in</td><td>a solid</td><td>yellow.</td>

<td>Mp 125-127 ° C</td><td colspan="2">(decomposed)</td><td> . ⁴H NMR (300</td><td>MHz</td><td>, CD₃OD) δ</td><td>1.70 (m,</td>

<td>4H), 2.22 (m,</td><td>2H),</td><td> 2,60</td><td>(m, 2H), 3.10</td><td>(m,</td><td>2H), 4.00</td><td>(m, 2H),</td>

<td>6.70 (d, 2H),</td><td> 7, 09</td><td>(d, 2H</td><td>). m / z (ESI):</td><td> 488</td><td>[CzoHseClNn</td><td>O₂ + H]⁺.</td>

Example 2

Synthesis of dimethylthiocarbamic acid O- (4- {4- [AU- (3,5-diamino-6-chloropyrazine-2carbonyl) guanidino] butyl} phenyl ester (PSA 17846)

<img file="PT1663235E_D0076.tif" />

2- [4- (4-Hydroxyphenyl) butyl] isoindole-1,3-dione (8).

A mixture of 4- (4-aminobutyl) phenol 6 hydrobromide (8.2 g, 33.5 mmol), phthalic anhydride 7 (5.0 g, 33.8 mmol), and triethylamine (4.6 mL, 33 mL). 0.5 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 ° C for 3 h. The solvent was evaporated and the resulting residue was purified by flash column chromatography on silica gel eluting with CtbCU / EtOAc / hexanes (8: 1: 1, v / v) to give the desired product 8 as a white powder. (4.1 g, 41% yield). ⁴1 H NMR (300 MHz, DMSO-cA) δ 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 [Ci₈H₁₇AT THE₃ + H] ⁺ .

Dimethylthiocarbamic acid 0- {4- [4- (1,3-dioxo-1,3-dihydroisoindol-2-yl) butyl] phenyl} ester (9).

A suspension of sodium hydride (60% in mineral oil, 0.44 g, 0.11 mmol) in anhydrous DMF (10 mL) was cooled to 0 ° 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 ° C for 30 min and then at room temperature for a further hour. A solution of dimethylthiocarbamic acid chloride was then added.

ΕΡ 1,663,235 / ΡΤ (1.35 g, 11 mmol) in DMF (10 mL). The reaction mixture was stirred at room temperature first for 16 h and then at 50 ° 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 column chromatography on silica gel eluting with CH₂Cl 2 / hexanes / EtOAc (10: 1: 0.2, v / v) to give the desired product 9 as a yellowish solid (2.27 g,

<td>income from</td><td> 59%). ⁴H</td><td>NMR</td><td>(300 MHz,</td><td>CDC1₃)</td><td>δ 1.72 (m, 4H),</td>

<td>2.67 (m, 2H),</td><td>3.33 (s,</td><td>3H),</td><td>3.45 (s,</td><td>3H), 3,</td><td>71 (m, 2H), 6.95</td>

<td>(d, 2H), 7.18</td><td>(d, 2H),</td><td> 7, 70</td><td>(m, 2H),</td><td>7.84 (m,</td><td>2H). m / z (ESI):</td>

<td>383 [C₂₃H₂₂N₂O₃s</td><td>+ H] ⁺.</td><td></td><td></td><td></td><td></td>

Dimethylthiocarbamic acid O- [4- (4-aminobutyl) phenyl] ester (10).

A mixture of dimethylthiocarbamic acid 0- {4- [4- (1,3-dioxo-1,3-dihydroisoindol-2-yl) butyl] phenyl} ester (0.30 g, 0.80 mmol) and methylamine (2M in methanol, mL, 20 mmol) was stirred at room temperature overnight. The solvent was removed by rotary evaporation and the residue was purified by flash column chromatography on silica gel (Biotage) eluting with chloroform / methanol / concentrated ammonium hydroxide (10: 1: 0.1, v / v) to give the solvent. dimethylthiocarbamic acid 0 [4- (4-aminobutyl) phenyl] ether (10) as a clear colorless oil (118 mg, 46% yield). ⁴1H NMR (300 MHz, CD₃OD) δ 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 [C₁₃H₂₀N₂OS + H] ⁺ .

Dimethylthiocarbamic acid 0- (4- {4- [N '- (3,5-diamino-6-chloropyrazine-2-carbonyl) guanidino] butyl} phenyl ester (11,

PSA 17846).

A solution of dimethylthiocarbamic acid O- [4- (4-aminobutyl) phenyl] ester 10 (115 mg, 0.45 mmol), triethylamine (0.30 mL, 2.2 mmol) and 1- (3, 5-Diamino-6-chloropyrazine-2-carbonyl) -2-methylisothiourea (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 column chromatography on silica gel (Biotage) eluting with chloroform / methanol / concentrated ammonium hydroxide (15: 1: 0.1, v / v) to give the desired product.

ΕΡ 1 663 235 / ΡΤ

<td>11 under</td><td>form of</td><td>one</td><td>yellow solid</td><td>(180 mg,</td><td>Yield</td><td>in</td>

<td>86%). Federal Police</td><td>102-105 ° C.</td><td>⁴H</td><td>NMR (300 MHz,</td><td>CD3OD) δ</td><td>1.70 (m, 4H</td><td> ) ,</td>

<td>2.65 (m,</td><td>2H), 3.20</td><td>(m,</td><td>2H), 3.30 (s,</td><td>3H), 3.40</td><td>(s, 3H), 6,</td><td> 95</td>

<td>(d, 2H),</td><td>7.20 (d, 2H</td><td> ) ·</td><td>m / z (ESI): 465</td><td colspan="2">[Ç₁₉H₂₅C1N₈O₂S + H]⁺.</td><td></td>

Example 3

Synthesis of (2S) - (4- {4- [Ν '- (3,5-diamino-6-chloropyrazine-2carbonyl) guanidino] butyl} benzenesulfonylamino) -3methylbutyramide (PSA 19008).

<img file="PT1663235E_D0077.tif" />

4- [4- (1,3-dioxo-1,3-dihydroisoindol-2-yl) butyl] benzenesulfonyl chloride (13).

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 ° C and the mixture was stirred for 1 h the temperature. After storage in a refrigerator at -5 ° C overnight, the reaction mixture was poured onto crushed ice (100 g) and the precipitates were collected by suction filtration and dried under high vacuum to yield the desired product 13 (2 48 g, 99% yield).⁴1H NMR (300 MHz, CDCl3)₃) δ 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).

4- [4- (1,3-dioxo-1,3-dihydroisoindol-2yl) 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. The solvent was removed by rotary evaporation and the residue was purified by flash chromatography on silica gel eluting with chloroform / methanol / concentrated ammonium hydroxide (15: 1: 0.1,

(1 663 235 / v / v) to give the desired product 14 as a white powder (0.41 g, 73% yield). ⁴1H NMR (300 MHz, DMSO-d6) δ 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).

A mixture of (2S) - {4- [4- (1,3-dioxo-1,3-dihydroisoindol-2-yl) butyl] benzenesulfonylamino} -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. The solvent was removed by rotary evaporation and the residue was purified by flash column chromatography on silica gel eluting with chloroform / methanol / concentrated ammonium hydroxide (3: 1: 0.1, v / v) to give (2Sj). [4- (4aminobutyl) benzenesulfonylamino] -3-methylbutyramide (15) as a white powder (156 mg, 54% yield). ⁷1H NMR (300 MHz, CD₃OD) δ 0.85 (d, 3H), 0.87 (d, 3H), 1.66 (m, 4H), 1.90 (m, 1H), 2.69 (m, 4H), 3, (D, 1H), 7.35 (d, 2H) 7.75 (d, 2H). m / z (ESI): 328 [C15H25N3O3S + H]⁺ .

(2S) - (4- {4- [Ν '- (3,5-Diamino-6-chloropyrazine-2-carbonyl) guanidino] butyl} benzenesulfonylamino) -3-methylbutyramide (16, PSA 19008).

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- (1-iodide) 3,5-Diamino-6-chloropyrazine-2-carbonyl) -2-methylisothiourea (230 mg, 0.61 mmol) in absolute ethanol (8 mL) was stirred at 70 ° 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 column chromatography on silica gel 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 analytically pure sample which was then coevaporated with 5% aqueous HCl to give the hydrochloride salt. 16. mp 154 ​​ΕΡ 1 663 235 / ΡΤ

156 ° C (decomposed). ²Η NMR (300 MHz, CD₃OD) δ 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 [C₂iH₃oC1N₉0₄S + H] ⁺ . [The]_D²⁵ + 5.2 ° (c 0.50, MeOH).

Example 4

Synthesis of 2- (4- {4- [AU- (3,5-diamino-6-chloropyrazine-2carbonyl) guanidino] butyl} phenoxy) -Î ”-phenylacetamide (PSA 17482)

<img file="PT1663235E_D0078.tif" />

Phenyl) butyl] carbamic acid benzyl ester (18).

[4- (4-phenylcarbamoylmethoxy) 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 CH₂C1₂ HCl (30 mL) was stirred at room temperature for 66 h. The reaction mixture was concentrated under vacuum and the residue was subjected to silica gel flash column chromatography eluting with methanol / CH₂Cl₂ (1:99, v / v) to give the desired amide 18 as a white solid (360 mg, 99% yield). ²1H NMR (300 MHz, CDCl3)₃) δ 1.55 (m, 4H), 2.60 (m, 2H), 3.20 (m,

2H), 4.58 (s, 2H), 4.70 (sg, 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 [C₂₆H₂₈N₂O₄ + H] ⁺ .

2- [4- (4-Aminobutyl) phenoxy] -N-phenylacetamide (19).

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 a hydrogen atmosphere in the presence of 10% Pd / C catalyst (0.2 g, wet 50%). 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 CH₂Cl₂/ methanol / concentrated ammonium hydroxide (30: 1: 0,

ΕΡ 1 663 235 / ΡΤ

30: 1: 0.3, v / v) to give the desired amine 19 as a white solid (200 mg, 97% yield). ²1H NMR (300 MHz, CD₃OD) δ 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 18 H 22 N 2 O 2 + H]⁺ .

2- (4- {4- [N- (3,5-Diamino-6-chloropyrazine-2-carbonyl) guanidino] butyl} phenoxy) -N-phenylacetamide (20, PSA 17482).

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 ° C for 30 min, after which 1- (3,5-diamino-6-chloropyrazine-2-carbonyl) -2-methylisothiourea iodide (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 ground with water and filtered. The filter cake was purified by flash column chromatography on silica gel eluting with dichloromethane / methanol / concentrated ammonium hydroxide (500: 10: 0, 500: 10: 1, 200: 10: 1, v / v) to give 2- (4- {4- [AU - (3,5-diamino-6-chloropyrazine-2-carbonyl) guanidino] butyl} phenoxy) -N-phenylacetamide (20, PSA 17482) as a yellow solid (120 mg, 67% yield). mp 168-170 ° C. % NMR (300 MHz, DMSO-d₆) δ 1.55 (m, 4H), 2.55 (m, 2H), 3.16 (m, 2H), 4.65 (s, 2H), 6.60 (s lg, 2H), 6, 90 (d, 2H), 7.08 (m, 2H), 7.15 (d, 2H), 7.30 (m, 5H), 7.60 (d, 2H), 9.00 (sg, 1H), 10.00 (sg, 1H). m / z (ESI): 511 [CziXCINgOg + H]⁺ .

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 )

<img file="PT1663235E_D0079.tif" />

4- {4- [3- (1H-Imidazol-2-yl) propoxy] phenyl} butylamine (21).

ΕΡ 1 663 235 / ΡΤ

Compound 2 (0.156 g, 0.425 mmol) was dissolved in anhydrous ethanol (10 mL). Anhydrous HCl gas was bubbled into the solution 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, the temperature was raised to reflux which was continued for a further 3 h before the mixture was cooled to room temperature. The solvent was removed under vacuum and the residue was treated with 1.2 N aqueous HCl solution at 80 ° C for 2 hours. The mixture was then cooled back to room temperature and neutralized to pH ~ 9 with powdered K 2 CO 3. Water was completely removed under vacuum and the residue was dissolved in methanol. The methanolic solution was charged over silica gel, and the product was eluted with a concentrated ammonium hydroxide / MeOH / CH mixture.₂C12 (1.8: 18: 81.2, v / v), yielding product 21 (27 mg, 23% overall yield) as an off-white solid. ⁴1 H NMR (300 MHz, CD 3 OD): δ 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 (A-PCI): 274 [C₁₆H₂3N₃O + H] ⁺ .

N- (3,5-Diamino-6-chloropyrazine-2-carbonyl) -N '- (4- {4- [3- (1-Himidazol-2-yl) propoxy] phenyl} butyl) guanidine (22, PSA 23022) .

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 ° C over 15 min. 1- (3,5-Diamino-6-chloropyrazine-2-carbonyl) -2-methylisothiourea iodide (43 mg, 0.109 mmol) was added to the solution and the resulting mixture was stirred at previous temperature for a further 3 h before stirring. liquid is removed under vacuum. The residue was chromatographed on 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 as a solid. yellow.

(300 MHz, CD 3 OD): δ 1.62 (m,

2.88 (t, 2H), 3.21 (t, 2H), (s, 2H), 7.06 (d, 2H). m / z (34 mg, 83% yield) mp 123-126 ° C (decomposed),⁴H NMR

4H), 2.14 (m, 2H), 2.58 (t, 2H),

3.94 (t, 2H), 6.77 (d, 2H), 6.90 (APCI): 486 [C22H28CIN9O2 + H] ⁺ .

ΕΡ 1 663 235 / ΡΤ

Example 7

Synthesis of 2- (4- {4- [N '- (3,5-diamino-6-chloropyrazine-2-carbonyl) guanidino] butyl} phenoxy) -N, N-dimethylacetamide hydrochloride (PSA 16313)

<img file="PT1663235E_D0080.tif" />

[4- (4-Dimethylcarbamoylmethoxyphenyl) butyl] carbamic acid benzyl ester (28).

A mixture of [4- (4-benzyloxycarbonylaminobutyl) phenoxy] acetic acid ethyl ester (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 ° C for 48 h. The solvent was evaporated in vacuo. The residue was purified by flash chromatography (silica gel, ethyl acetate / CH₂Cl 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 . ³1H NMR (300 MHz, CDCl3) δ 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).

the atmospheric temperature. O

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 overnight under hydrogen 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 afford 2- [4- (4-aminobutyl) phenoxy] -N, N- dimethylacetamide 29 (100 mg, 60% yield) as a white solid. ³1 H NMR (300 MHz, CD 3 OD) δ 1.55 (m, 4H), 2.55 (m, 2H), 2.66 (m, 2H), 2.90 (s,

ΕΡ 1 663 235 / ΡΤ

3Η), 3.05 (s, 3Η), 4.70 (s, 2Η), 6.80 (d, 2Η), 7.05 (d, 2Η).

m / z (ESI): 251 [C₁₄H22N₂O2 + H] ⁺ .

2- (4- {4- [N '- (3,5-diamino-6-chloropyrazine-2carbonyl) guanidino] butyl} phenoxy) -N, N-dimethylacetamide hydrochloride (30,

PSA 16313).

A solution of 2- [4- (4-aminobutyl) phenoxy] -Ν, N-dimethylacetamide 29 (67 mg, 0.27 mmol) in absolute ethanol (1 mL) was stirred at 65 ° C for 30 min, after which time. 1- (3,5-diamino-6-chloropyrazine-2carbonyl) -2-methylisothiourea iodide (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 ground with water and filtered. The filter cake was purified by flash column chromatography on silica gel 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} phenoxy) -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 afforded 2- (4- {4- [N- (3,5-diamino-6-chloropyrazine-2-carbonyl) guanidino] butyl} phenoxy) β, β-dimethylacetamide hydrochloride (30, PSA 16313 ). mp 130-132 ° C (decomposed).³1H NMR (300 MHz, CD₃OD) δ 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 [C₂₀H₂₇Cln₈O₃ + H] ⁺ .

Example 8

Synthesis of 2- (4- {4- [N '- (3,5-diamino-6-chloropyrazine-2-carbonyl) guanidino] butyl} phenoxy) -N- (1H-imidazol-2yl) acetamide dihydrochloride (PSA 16437)

<img file="PT1663235E_D0081.tif" />

Aminobutyl) phenoxy] acetic acid methyl ester (32).

[4- (4-tert-Butoxycarbonyl] 1 663 235 / ΡΤ

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 x 4) and brine (30 mL), and was concentrated under reduced pressure to yield the desired product 32 under as a yellow oil (1.28 g, 100% yield) which was used in the next step without further purification. ⁴1H NMR (300 MHz, CDCl3) δ

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 [C₁₈H₂7NO₅ + H]⁺.

[4- (4-tert-Butoxycarbonylaminobutyl) phenoxy] acetic acid (33).

To a solution of [4- (4-tertbutoxycarbonylaminobutyl) phenoxy] acetic acid methyl ester 32 (1.28 g, 3.78 mmol) in methanol (80 mL) was added milled potassium hydroxide (2.50 g, 85 mL). %, 37.8 mmol) and the mixture was stirred at room temperature for 5 h. The solvent was removed by rotary evaporation. The residue was taken up in water and acidified to pH ~ 1 with 6N aqueous HCl, and extracted with dichloromethane. The combined organic materials were washed with brine, dried over Na₂ONLY₄ and concentrated to complete dryness to afford the desired product 33 as a white solid (1.19 g, yield 97%). ⁴1H NMR (300 MHz, CD₃OD) δ 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 [C₁₇H₂₅NO5 - M] h

(4- {4 - [(1H-Imidazol-2ylcarbamoyl) methoxy] phenyl} butyl) carbamic acid tert-butyl ester (34).

[4- (4-tert-Butoxycarbonylaminobutyl) phenoxy] acetic acid 33 (1.19 g, 3.68 mmol) was dissolved in anhydrous THF (10 mL), CH₂C1₂ (10 mL) and CH₃CN (5 mL). HOAt (200 mg, 1.47 mmol), DMAP (135 mg, 1.10 mmol) and diisopropylethylamine (3.2 mL, 18.40 mmol) were added sequentially, followed by the addition of EDC. HCl (1.03 g, 5.35 mmol). The reaction mixture was stirred at room temperature for 15 min. Aminoimidazole sulfate (583 mg,

4.41 mmol) and stirring was continued for 48 h. the

1 663 235 / ΡΤ solvents were removed by rotary evaporation. The residue was taken up in CH₂Cl 2 (250 mL), washed with water and brine, and concentrated under reduced pressure. Flash column chromatography on silica gel eluting with methanol / dichloromethane (1:30, 1:20, v / v) afforded the desired amide as a white solid (0.95 g, 66% yield).³1H NMR (300 MHz, CD₃OD) δ 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 [C₂₀H₂₈N₄O₄ + H] ⁺ .

2- [4- (4-Aminobutyl) phenoxy] -N- (1H-imidazol-2yl) acetamide dihydrochloride (35).

(4- {4 - [(1H-Imidazol-2-ylcarbamoyl) 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 was further coevaporated with dichloromethane and methanol, and dried under high vacuum. The desired product was obtained as a white solid (779 mg, 98%) and was used directly without further purification.³1H NMR (300 MHz, CD₃OD) δ 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 [C₁₅H₂₀N₄O₂ + H] ⁺ .

2- (4- {4- [N '- (3,5-Diamino-6-chloropyrazine-2carbonyl) guanidino] butyl} phenoxy) -N- (1H-imidazol-2-yl) acetamide dihydrochloride (36, PSA 16437).

A solution of 2- [4- (4aminobutyl) 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 ° C for 30 min, after which 1% iodide was added. (3,5-diamino-6-chloropyrazine-2-carbonyl) -2-methylisothiourea (130 mg, 0.34 mmol) in one portion. The reaction mixture was stirred for 3 h and then cooled to room temperature. The yellow insoluble materials were removed by suction filtration and the liquid filtrate was concentrated by rotary evaporation. The liquid residue was purified by flash column chromatography dichloromethane / methanol / hydroxide (200: 10: 0, 200: 10: 1, 150: 10: 1 and by eluting with concentrated ammonium silica gel 100: 10: 1, v / v) to originate

2- (4- {4- [N '- (3,5-diamino-6-chloropyrazine-2-carbonyl) guanidino] butyl} phenoxy) -Ν' (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 ° C.⁴1H NMR (300 MHz, CD₃OD) δ 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 [C₂iH25C1N₁₀O₃ + H]⁺.

Example 9

Synthesis of N-carbamoylmethyl-2- (4- {4- [N '- (3,5-diamino-6-chloropyrazine-2-carbonyl) guanidino] butyl} phenoxy) acetamide (PSA 16314)

<img file="PT1663235E_D0082.tif" />

NR,

Carbamoyl) methoxy] phenyl] butyl) carbamic acid benzyl ester (4- {4 - [(carbamoylmethyl) (37).

Compound 1 (0.50 g, 1.77 mmol) (10 mL). 2.66 mmol) was added to the solution. The mixture was dissolved in milled DMF NaOH (0.107 g, stirred at room temperature for 30 min.) 2-Bromoacetamide (0.367 g,

2.66 mmol). The reaction mixture was further stirred at room temperature overnight, quenched with water (2 mL) and partitioned between water and dichloromethane (50 mL each). The organic layer was separated, washed with water (2 x 50 mL), dried over Na₂Anhydrous SO 4 and concentrated under vacuum. The residue was purified on silica gel, eluting with a methanol / dichloromethane mixture (7:93, v / v) to afford the desired product 37 (0.131 g, 18% yield) as a white solid.⁴1H NMR (300 MHz, CDCl3)₃): δ 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 [C₂₂H₂₇N₃O₅+ H] ⁺.

ΕΡ 1 663 235 / ΡΤ

2- [4- (4-Aminobutyl) phenoxy] -N-carbamoylmethylacetamide (38) 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 before and after catalyst (100 mg,

<td colspan="2">10% Pd / C,</td><td>50% wet).</td><td>The mix</td><td>was</td><td>stirred under</td>

<td>atmosphere</td><td>in</td><td>hydrogen (1 atm)</td><td>during the</td><td>night</td><td>. After purge</td>

<td>with nitrogen,</td><td>O</td><td colspan="2">catalyst was filtered under</td><td>vacuum</td><td>and washed with</td>

<td>ethanol (3</td><td>x 5</td><td>mL). The filtrates</td><td>combined</td><td>were</td><td>concentrates</td>

<td>under vacuum</td><td> . 0</td><td colspan="2">residue was chromatographed</td><td>about</td><td>silica gel,</td>

<td>eluting</td><td>with</td><td>a mix</td><td colspan="2">hydroxide</td><td>ammonium</td>

concentrate / methanol / dichloromethane (2:20:88, v / v) to afford the desired product 38 (80 mg, 91% yield) as a white solid. ⁴1H NMR (300 MHz, CD₃OD): δ 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-2carbonyl) guanidino] butyl} phenoxy) acetamide (39, PSA 16314).

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 ° C over 10 min. 1- (3,5-Diamino-6-chloropyrazine-2-carbonyl) -2-methylisothiourea iodide (132 mg, 0.34 mmol) was added in one portion. The resulting reaction mixture was stirred continuously for a further 2h before being cooled to room temperature and subsequently concentrated under vacuum. The resulting residue was purified by chromatography eluting with concentrated methanol / dichloromethane / ammonium hydroxide (10/2/88, v / v) to yield the free base (93 mg, 67% yield) as a yellow solid. The HCl salt was prepared 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 yield 39 (47 mg). mp 178-180 ° C (decomposed). ⁴1H NMR (300 MHz, DMSO-

<td>d₆) :</td><td>δ 1.61 (m,</td><td>4H), 2.58</td><td>(t, 2H)</td><td> , 3,32</td><td>(m,</td><td>2H), 3.70 (s,</td><td>2H),</td>

<td> 4, 48</td><td>(s, 2H), 6,</td><td>93 (d, 2H)</td><td> ), 7,08</td><td>dg,</td><td>1H),</td><td>7.13 (d, 2H),</td><td> 7,36</td>

<td>(IG,</td><td>1H), 7.44</td><td>(lg, 2H),</td><td> 8,17</td><td>(t, 1H</td><td> ) , 8,</td><td>-74 (br. 1H),</td><td> 8,90</td>

<td>(IG,</td><td>2H), 9.18</td><td>(t, 1H),</td><td> 10, 48</td><td>(IG,</td><td>1H)</td><td>. m / z (APCI):</td><td> 492</td>

<td>[Ç₂₀H</td><td>₂6ClNgO₄ + H]</td><td> +</td><td></td><td></td><td></td><td></td><td></td>

ΕΡ 1 663 235 / ΡΤ

Example 10

Synthesis of Ν- [4- (4-cyanomethoxyphenyl) butyl] -Ν '- (3,5-diamino-6chloropyrazine-2-carbonyl) guanidine (PSA 16208)

<img file="PT1663235E_D0083.tif" />

acid

Tert-Butyl] carbamic ester (40).

[4- (4-cyanomethoxyphenyl) mixture of a tert-butyl acid hydroxyphenyl) butyl] carbamic ester mixture [4- (4-31 (0.365 g, 1.37 mmol) and Cs₂CO₃ (0.672 g, 2.06 mmol) in anhydrous DMF (8 mL) was heated at 65 ° 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 ° C overnight, and then cooled to room temperature. The precipitated solid was filtered, and the filtrate was partitioned between water and dichloromethane (50 mL each). The organic layer was separated, washed with brine (3 x 50 mL), dried over Na₂ONLY₄ anhydrous 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 viscous oil. . NMR (300 MHz, CDCl3)₃): δ

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).

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.²1H NMR (300 MHz, CD₃OD): δ 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 ° C₁₂H₁₆N₂O + H] ⁺ .

ΕΡ 1 663 235 / ΡΤ

Β- [4- (4-Cyanomethoxyphenyl) butyl] -β '- (3,5-diamino-6-chloropyrazine-2-carbonyl) guanidine (42, PSA 16208).

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 ° C for 20 min. 1- (3,5-Diamino-6-chloropyrazine-2-carbonyl) -2-methylisothiourea iodide (0.148 g, 0.379 mmol) was added in one portion. Heating was continued for a further 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 (10/1/89, v / v) to yield the desired product 42 (0.031 g, 22%). ) as a yellow solid, mp 129-132 ° C. ⁴1H NMR (300 MHz, CD₃OD): δ 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 [C₁₈H₂iC1N₈O₂ + H]⁺.

Reference Example 11

Synthesis of N- (3,5-diamino-6-chloropyrazine-2-carbonyl) -N '- (4- {4- [3- (2,3-dihydroxypropoxy) -2-hydroxypropoxy] phenyl} butyl) guanidine (PSA 15143)

<img file="PT1663235E_D0084.tif" />

PSA 15143

(4- {4- [3- (2,3-Dihydroxypropoxy) -2-hydroxypropoxy] phenyl} butyl) carbamic acid benzyl ester (43).

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 ° C for 1 h. Oxiranyl methanol (0.5 mL, 6.68 mmol) was added hourly for a total of 4 h (total amount of oxiranyl methanol added was 2.0 mL, 26.72 mmol). The reaction mixture was concentrated under vacuum. The residue was chromatographed on silica gel eluting with a methanol / dichloromethane mixture (3:97, v / v) to afford 168 mg (4.6% yield) of the desired product 43. m / z (APCI) : 448 [C24H33NO7 + H] ⁺ .

ΕΡ 1 663 235 / ΡΤ

3- {3- [4- (4-Aminobutylphenoxy] -2-hydroxypropoxy} propane-1,2-diol (44).

A solution containing compound 43 (0.15 g, 0.34 mmol) in ethanol (1.5 mL) was purged with nitrogen before and after catalyst (0.15 g, 10% Pd / C, 50% wet).

The reaction mixture was placed under hydrogenation atmosphere for 45 min. The catalyst was filtered under vacuum through diatomaceous earth and washed with ethanol (3 x 2 mL). The combined filtrates were concentrated under vacuum. The residue was chromatographed on silica gel, eluting with methanol / dichloromethane / concentrated ammonia (25 / 2.5 / 73.5, v / v) to afford the desired product 44 (0.053 g, yield of

51%) as a viscous colorless oil. NMR (300 MHz, CD₃OD): δ 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 [C₁₆H₂7NO₅ + H] ⁺.

N- (3,5-Diamino-6-chloropyrazine-2-carbonyl) -N '- (4- {4- [3- (2,3dihydroxypropoxy) -2-hydroxypropoxy] phenyl} butyl) guanidine (45, PSA 15143).

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 ° C over 15 min. 1- (3,5-Diamino-6-chloropyrazine-2-carbonyl) -2-methylisothiourea iodide (74 mg, 0.191 mmol) was added to the solution. The reaction mixture was stirred at previous temperature for an additional 50 min, cooled to room temperature and subsequently concentrated under vacuum. The residue was chromatographed on silica gel, eluting with concentrated methanol / dichloromethane / ammonium hydroxide (10/1/40, v / v) to afford the desired product 45 (53 mg, 36% yield) as a solid. yellow solid, mp 73-82 ° C (decomposed). ³1 H NMR (300 MHz, CD 3 OD): δ 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 [C₂2H₃2C1N₇O₆ + H] ⁺ .

Example 12

Using the procedures set forth above, the following protected pyrazinoylguanidine was prepared.

ΕΡ 1 663 235 / ΡΤ

<img file="PT1663235E_D0085.tif" />

<td>TEST</td><td>RESULT / REFERENCE</td>

<td>description</td><td>Yellow solid</td>

<td>Identification: Spectrum of ^XH NMR a 30 0 MHz (DMSO-dg)</td><td>Consistent</td>

<td>Fusion point</td><td>108-110 ° C dec</td>

<td>HPLC analysis</td><td>96.5% (area percent), Polarity dC18 column, Detector @ 220 nm</td>

<td>Miscellaneous Tests: ESI Mass Spectrum</td><td>m / z 527 [C₂₁H₃₁C1N₈O₄S + H] ⁺</td>

Example 13

Using the procedures set forth above, the following protected pyrazinoylguanidine was prepared.

<img file="PT1663235E_D0086.tif" />

ch₃

PSA18211

<td>TEST</td><td>RESULT / REFERENCE</td>

<td>description</td><td>Yellow solid</td>

<td>Identification: Spectrum of ^XH NMR a 30 0 MHz (DMSO-dg)</td><td>Consistent</td>

<td>Fusion point</td><td>153-155 ° C dec</td>

<td>HPLC analysis</td><td>96.3% (area percent), Polarity dC18 Column, Detector @ 220 nm</td>

<td>Miscellaneous Tests: ESI Mass Spectrum</td><td>m / z 465 [C₁₉H₂₅C1N₈O₂S + H] ⁺</td>

ΕΡ 1 663 235 / ΡΤ

Example 14

Using the procedures set forth above, the following protected pyrazinoylguanidine was prepared.

<img file="PT1663235E_D0087.tif" />

PSA18212

<td>TEST</td><td>RESULT / REFERENCE</td>

<td>description</td><td>Yellow solid</td>

<td>Identification: NMR Spectrum: 500 MHz (CD₃OD)</td><td>Consistent</td>

<td>Fusion point</td><td>115-116 ° C</td>

<td>HPLC analysis</td><td>97.1% (area percent), Polarity dC18 Column, Detector @ 220 nm</td>

<td>Miscellaneous Tests: ESI Mass Spectrum</td><td>m / z 639 [C₃oh₃5C1N₈0₆ + H] ⁺</td>

Reference Example 15

Using the procedures set forth above, the following protected pyrazinoylguanidine was prepared.

<img file="PT1663235E_D0088.tif" />

PSA18229

<td>TEST</td><td>RESULT / REFERENCE</td>

<td>description</td><td>Yellow solid</td>

<td>Identification: Spectrum of ^X300 nmr MHz (CD₃OD)</td><td>Consistent</td>

<td>Fusion point</td><td>190-192 ° C</td>

<td>HPLC analysis</td><td>97.9% (area percent), Polarity dC18 Column, Detector @ 220 nm</td>

<td>Miscellaneous Tests: ESI Mass Spectrum</td><td>m / z 476 [C₂oh₂6C1N₉0₃ + H] ⁺</td>

ΕΡ 1 663 235 / ΡΤ

Example 16

Using the procedures set forth above, the following protected pyrazinoylguanidine was prepared.

O

II

<img file="PT1663235E_D0089.tif" />

PSA18361

<td>TEST</td><td>RESULT / REFERENCE</td>

<td>description</td><td>Yellow solid</td>

<td>Identification: Spectrum of ^X300 MHz NMR (CD₃OD)</td><td>Consistent</td>

<td>Fusion point</td><td>124-126 ° C dec</td>

<td>HPLC analysis</td><td>95.2% (area percent), Polarity dC18 Column, Detector @ 220 nm</td>

<td>Miscellaneous Tests: ESI Mass Spectrum</td><td>m / z 441 [C₁₆H₂₁C1N₈O₃S + H] ⁺</td>

Example 17

Using the procedures set forth above, the following protected pyrazinoylguanidine was prepared.

O

<img file="PT1663235E_D0090.tif" />

PSA18592

<td>TEST</td><td>RESULT / REFERENCE</td>

<td>description</td><td>Yellow solid</td>

<td>Identification: Spectrum of ^XH NMR a 500 MHz (CD3OD)</td><td>Consistent</td>

<td>Fusion point</td><td>189 ° C dec</td>

<td>HPLC analysis</td><td>95.0% (area percent), Polarity dC18 Column, Detector @ 220 nm</td>

<td>Miscellaneous Tests: ESI Mass Spectrum</td><td>m / z 503 [C 21 H₂₇Cln₁₀O₃ + H] ⁺</td>

ΕΡ 1 663 235 / ΡΤ

Reference Example 18

Using the procedures set forth above, the following protected pyrazinoylguanidine was prepared.

<img file="PT1663235E_D0091.tif" />

<td>TEST</td><td>RESULT / REFERENCE</td>

<td>description</td><td>Pale yellow solid</td>

<td>Identification: Spectrum of ^XH NMR a 300 MHz (CD₃OD)</td><td>Consistent</td>

<td>Fusion point</td><td>195-197 ° C</td>

<td>HPLC analysis</td><td>97.4% (area percent), Polarity dC18 Column, Detector @ 220 nm</td>

<td>Miscellaneous Tests: ESI Mass Spectrum</td><td>m / z 4ΊΊ [C₂oH25C1N₈0₄ + H] ⁺</td>

Example 19

Using the procedures set forth above, the following protected pyrazinoylguanidine was prepared.

<img file="PT1663235E_D0092.tif" />

PSA 19007

<td>TEST</td><td>RESULT / REFERENCE</td>

<td>description</td><td>Yellow solid</td>

<td>Identification: Spectrum of ^XH NMR a 300 MHz (CD₃OD)</td><td>Consistent</td>

<td>Fusion point</td><td>210-212 ° C dec</td>

<td>HPLC analysis</td><td>95.5% (area percent), Polarity dC18 Column, Detector @ 220 nm</td>

<td>Miscellaneous Tests: APCI Mass Spectrum</td><td>m / z 486 [C₂2H28C1N₉O₂ + H] ⁺</td>

ΕΡ 1 663 235 / ΡΤ

Example 20

Using the procedures set forth above, the following protected pyrazinoylguanidine was prepared.

<img file="PT1663235E_D0093.tif" />

PSA 19912

<td>TEST</td><td>RESULT / REFERENCE</td>

<td>description</td><td>Yellow solid</td>

<td>Identification: Spectrum of ²H NMR a 300 MHz (CD₃OD)</td><td>Consistent</td>

<td>Optical Rotation</td><td>[Hi]²⁵d -7.8 ° (c 0.46, methanol)</td>

<td>Fusion point</td><td>178-180 ° C</td>

<td>HPLC analysis</td><td>97.0% (area percent), Polarity dC18 Column, Detector @ 220 nm</td>

<td>Miscellaneous Tests: ESI Mass Spectrum</td><td>m / z 490 [C 21 H₂8ClN₉O₃ + H] ⁺</td>

Reference Example 21

Using the procedures set forth above, the following protected pyrazinoylguanidine was prepared.

<img file="PT1663235E_D0094.tif" />

2HC1

KNOW

PSA 24406

<td>TEST</td><td>RESULT / REFERENCE</td>

<td>description</td><td>Yellow solid</td>

<td>Identification: Spectrum of ²300 MHz 1 H NMR (CD 3 OD)</td><td>Consistent</td>

<td>Optical Rotation</td><td>[The]²⁵D + 0.5 ° (c 0.35, Methanol)</td>

<td>Fusion point</td><td>215 ° C dec</td>

<td>HPLC analysis</td><td>96.1% (area percent), Polarity dC18 Column, Detector @ 220 nm</td>

<td>Miscellaneous Tests: ESI Mass Spectrum</td><td>m / z 462 [C₂oH28C1N₉0₂ + H] ⁺</td>

ΕΡ 1 663 235 / ΡΤ

Reference Example 22

Using the procedures set forth above, the following protected pyrazinoylguanidine was prepared.

<img file="PT1663235E_D0095.tif" />

PSA 24407

<td>TEST</td><td>RESULT / REFERENCE</td>

<td>description</td><td>Yellow solid</td>

<td>Identification: 1 H NMR Spectrum: 300 MHz (CD₃OD)</td><td>Consistent</td>

<td>Optical Rotation</td><td>[The]²⁵D + 4.1 ° (c 0.30, Methanol)</td>

<td>Fusion point</td><td>230 ° C dec</td>

<td>HPLC analysis</td><td>95.3% (area percent), Polarity dC18 Column, Detector @ 220 nm</td>

<td>Miscellaneous Tests: ESI Mass Spectrum</td><td>m / z 463 [C₂oh₂₇C1N₈03 + H] ⁺</td>

ΕΡ 1 663 235 / ΡΤ

Example 23

Blocking Activity of Selected Protected Pyrazinoylguanidine Sodium Channels.

<td>PSA</td><td>EC50 (nM)</td><td>N.^s times Amiloride ** (PSA 4022 = 100)</td>

<td> 15143</td><td>7 ± 3 (n = 3)</td><td>107 + 11 (n = 3)</td>

<td> 16208</td><td>11 ± 4 (n = 6)</td><td>52 + 21 (n = 6)</td>

<td> 16314</td><td>13 ± 2 (n = 4)</td><td>41 + 6 (n = 4)</td>

<td> 16313</td><td>15 ± 4 (n = 4)</td><td>41 + 7 (n = 4)</td>

<td> 16437</td><td>13 ± 7 (n = 7)</td><td>77 + 53 (n = 7)</td>

<td> 17482</td><td>16 ± 4 (n = 3)</td><td>39 + 6 (n = 3)</td>

<td> 17846</td><td>11 ± 6 (n = 4)</td><td>104 + 49 (n = 4)</td>

<td> 17926</td><td>25 ± 9 (n = 6)</td><td>29 + 12 (n = 6)</td>

<td> 17927</td><td>13 ± 4 (n = 3)</td><td>83 + 26 (n = 3)</td>

<td> 18211</td><td>10 ± 4 (n = 3)</td><td>112 + 52 (n = 2)</td>

<td> 18212</td><td>27 ± 17 (n = 4)</td><td>32 + 16 (n = 4)</td>

<td> 18229</td><td>15 ± 6 (n = 3)</td><td>49 + 15 (n = 3)</td>

<td> 18361</td><td>11 ± 4 (n = 3)</td><td>76 + 25 (n = 3)</td>

<td> 18592</td><td>8 ± 4 (n = 2)</td><td>136 + 58 (n = 2)</td>

<td> 18593</td><td>48 ± 16 (n = 6)</td><td>13 + 4 (n = 4)</td>

<td> 19007</td><td>18 ± 13 (n = 4)</td><td>42 + 17 (n = 4)</td>

<td> 19008</td><td>9 ± 1 (n = 4)</td><td>54 + 6 (n = 4)</td>

<td> 19912</td><td>26 ± 8 (n = 4)</td><td>32 + 10 (n = 4)</td>

<td> 23022</td><td>12 ± 3 (n = 4)</td><td>79 + 15 (n = 4)</td>

<td> 24406</td><td>8 ± 3 (n = 6)</td><td>107 + 38 (n = 6)</td>

<td> 24407</td><td>32 ± 11 (n = 10)</td><td>23 + 4 (n = 10)</td>

<td> 24851</td><td>28 ± 13 (n = 8)</td><td>25 + 10 (n = 8)</td>

** Relative power for PSA 4022 = 100 using PSA EC50

4022 in the same operation

ΕΡ 1 663 235 / ΡΤ

Methods

Animal Preparation: Adult sheep (weighing 25 to 35 kg) were immobilized in an upright position in a specialized body harness fitted to a modified shopping cart. Animal 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 (TTE). 0 The ETT cuff 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 the commencement of mucociliary clearance measurements.

(3.1 mg / ml; containing aerosols generated using a

Radio Aerosol Administration ^99mHuman serum Tc-albumin (approximately 20 mCi) was Raindrop nebulizer which produces a droplet with a median aerodynamic diameter of 3.6 µm. The nebulizer was connected to a dosimetry system consisting of a solenoid valve and a compressed air source (20 psi). The nebulizer outlet was directed to a plastic T-connector; one end was connected to the endotracheal tube, the other was connected to a piston one. The system was activated during initiation of the respirator respirator inspiratory cycle and was set to a tidal volume of 500 mL, an inspiratory to expiratory ratio of 1: 1, and a rate of 20 breaths per minute to maximize central airway deposition. The sheep breathed the radiolabeled aerosol for 5 minutes. A gamma camera was used to measure the clearance of^99mHuman serum airway tc-alburnin. The camera was positioned above the back of the animal with the sheep in a natural upright position supported on a cart so that the image field was perpendicular to the respirator according to no.

The animal's spine

External radiolabelled markers were placed on the sheep to ensure proper alignment under the gamma chamber. All images were scored on a computer integrated with the gamma camera. A region of interest was plotted over the image corresponding to the sheep's right lung and counts were recorded. At

ΕΡ 1,663,235 / ΡΤ counts were corrected for decay and expressed as a percentage of the radioactivity present in the initial baseline image. The left lung was excluded from the analysis because its contours are overlapping the stomach and the counts can be swallowed and enter the stomach in the form of radiolabelled mucus.

Treatment Protocol (Determination of activity at t-zero): An image of the baseline deposition was obtained immediately after administration of the radio aerosol. At time zero, after baseline imaging, vehicle control (distilled water), positive control (amiloride), or experimental compounds were aerosolized from a 4 ml volume using a Pari LC JetPlus nebulizer. to animals with free breathing. 0 The nebulizer was powered by compressed air at a flow rate of 8 liters per minute. The time to deliver the solution was 10 to 12 minutes. Animals were extubated immediately after total dose delivery to avoid false elevations in counts caused by aspiration of excess tracer by ETT. Serial images of the lung were taken at 15 minute intervals during the first 2 hours after dosing and hourly for the next 6 hours after dosing over a total observation period of 8 hours. A washout period of at least 7 days separated dosing sessions with different experimental agents.

Treatment Protocol (Activity Determination at t-4 hours): The following variation from the standard protocol was used to determine the durability of the response after a single exposure to vehicle control (distilled water), positive control compounds (amiloride or benzamyl). ), or investigating agents. At time zero, vehicle control (distilled water), positive control (amiloride) or investigational compounds were aerosolized from a volume of 4 ml using a Pari LC JetPlus nebulizer to free-breathing animals. The nebulizer was powered by compressed air at a flow rate of 8 liters per minute. The time to deliver the solution was 10 to 12 minutes. The animals were immobilized in an upright position in a specialized body harness for 4 hours. At the end of the 4 hour period the animals received a single dose of ^99mTc-alburnin serum

Aerosolized human 66 1,663,235 / ((3.1 mg / ml; containing approximately 20 mCi) from a Raindrop Nebulizer. The animals were extubated immediately after delivery of the full dose of tracer. A baseline deposition image was obtained immediately after administration of the radio aerosol. Serial images of the lung were taken at 15-minute intervals for the first 2 hours after radiotracer administration (representing hours 4 to 6 after drug administration) and hourly for the next 2 hours after administration. dosing over 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 repeated two-way ANOVA (to assess overall effects), followed by a paired t-test to identify differences between specific pairs. Significance was accepted when P was less than or equal to 0.05. The slope values ​​(calculated from data collected during the initial 45 minutes after dosing at t-zero assessment) for mean MCC curves were calculated using linear least squares regression to determine differences in initial velocities during the rapid clearance phase. .

Of course, 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.