Sodium channel blockers

is 2 298 505 T3

DESCRIPTION

Sodium channel blockers.

Background of the invention

Scope of the invention

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

Background description

Mucosal surfaces at the interface between the environment and the body have developed a series of "innate defenses", that is, protection mechanisms. A main form of such innate defense is to clean these surfaces with liquid. Normally, the amount of the liquid layer on the mucosal surface reflects the balance between the epithelial liquid secretion, which often reflects the secretion of anions (Cl^- and / or HCO3^-) coupled with water (and a cation counterion), and epithelial fluid absorption, which often reflects Na absorption⁺, coupled with water and the counternion (C1^-/ HCO3^-). Many diseases of the mucosal surfaces are caused by too little protective fluid on these mucosal surfaces caused by an imbalance between secretion (too little) and absorption (relatively too much). The defective salt transport processes that characterize these mucosal dysfunctions reside in the epithelial layer of the mucosal surface.

One procedure to recharge the protective fluid layer on mucosal surfaces is to "rebalance" the system by blocking the Na channel.⁺ and liquid absorption. The epithelial protein that mediates the rate-limiting stage of Na uptake⁺ and liquid is the epithelial channel of Na⁺ (ENaC). The ENaC is located on the apical surface of the epithelium, that is, at the interface of the mucosa-environment surface. Therefore, to inhibit the absorption of Na⁺ and fluid mediated by ENaC, an amiloride-type ENaC blocker (which blocks from the extracellular domain of ENaC) must be delivered to the mucosal surface, and importantly, maintained at that site to achieve therapeutic utility. The present invention describes diseases characterized by too little fluid on mucosal surfaces, and "topical" sodium channel blockers designed to exhibit higher potency, less mucosal absorption, and slow dissociation ("disbonding" or shedding) of the necessary ENaC. for the therapy of these diseases.

Chronic bronchitis (BC), which includes the most common lethal genetic form of chronic bronchitis, cystic fibrosis (CF), is a disease that reflects the failure of the body to clear mucus normally from the lungs, ultimately leading to chronic infection. of the airways. In the normal lung, the primary defense against chronic intrapulmonary infection of the airways (chronic bronchitis) is mediated by the continuous clearance of mucus from the surface of the bronchial airway. This function in healthy people effectively removes potentially harmful toxins and pathogens from the lungs. Recent data indicate that the initial problem, that is, the "basic defect" in both BC and CF is the failure to clear mucus from the airway surfaces. The failure to clear mucus reflects an imbalance between the amount of fluid and mucin on the airway surfaces. This "airway surface fluid" (ASL) is composed primarily of salt and water in proportions similar to plasma (ie isotonic). Mucin macromolecules are organized into a well-defined "mucus layer" that normally captures inhaled bacteria and is transported out of the lung by cilia churning in a low-viscosity aqueous solution called "periciliary fluid" ( LPC). In the pathological state there is an imbalance in the amounts of mucus such as LSA on the surfaces of the airways. This results in a relative reduction in LSA leading to mucus concentration, reduced lubricating activity of LPC, and failure to clear mucus into the mouth by ciliary activity. Reduced mechanical mucus clearance from the lung leads to chronic bacterial colonization of mucus adhering to airway surfaces. Chronic retention of bacteria, the failure of local antimicrobial substances to kill chronically trapped bacteria in mucus, and the consequent chronic inflammatory responses of the body to this type of surface infection lead to BC and CF syndromes.

The currently affected population in the US is 12,000,000 patients with the acquired form (mainly through exposure to cigarette smoke) of chronic bronchitis, and approximately 30,000 patients with the genetic form, cystic fibrosis. About the same number of both populations are found in Europe. In Asia there is little CF but the incidence of BC is high, and as in the rest of the world, increasing.

There is currently a great unmet need for products that specifically treat BC and CF at the level of the basic defect that produces these diseases. Current therapies for chronic bronchitis and cystic fibrosis focus on treating the symptoms and / or the after effects of these diseases. Therefore, for chronic bronchitis, // - agonists, inhaled steroids, anticholinergic agents, and oral theophyllines and phosphodiesterase inhibitors are being developed. However, neither of these drugs effectively treats the fundamental problem of failure of mucus clearance from the lung. Likewise, in cystic fibrosis, the same spectrum of pharmacological agents is used. These strategies have been complemented by more recent strategies designed to remove from the CF lung DNA (Pulmozyme ”; Genetech) deposited in the lung by neutrophils that have tried in vain to kill bacteria growing in adherent mucus masses, and by the use of is 2 298 505 T3 inhaled antibiotics ("TOBI") designed to augment the lungs' own mechanisms to kill to remove bacteria's adherent mucus plaques. A general principle of the body is that if the injury that begins is not treated, in this case mucus retention / obstruction, bacterial infections become chronic and increasingly refractory to antimicrobial therapy. Therefore, an important unmet therapeutic need, for the 5 lung diseases, both BC and CF, is an effective means of rehydrating airway mucus (i.e., restoring / expanding the volume of the ASL) and promoting their elimination, along with bacteria, from the lung.

RC Boucher in US 6,264,975, describes the use of pyrazinoylguanidine sodium channel blockers for hydration of mucosal surfaces. These compounds, illustrated by the known diuretics amiloride, benzamyl and phenyl, are effective. However, these compounds have the significant disadvantage that they are (1) relatively weak, which is important because the mass of drug that the lungs can inhale is limited; (2) they are absorbed rapidly, which limits the half-life of the drug on the mucosal surface; and (3) can be freely dissociated from ENaC. The sum of these disadvantages manifested in these well-known diuretics produces compounds with insufficient potency and / or effective half-life on mucosal surfaces to have therapeutic benefit in hydrating mucosal surfaces.

Epand et al in British Journal of Cancer vol 63, pages 247-251, 1991, describe derivatives of pyrazinoylguanidines used as reverse sodium transport inhibitors.

International application WO 01/05773 describes covalent sodium channel blocker conjugates which may be dimers of pyrazinoylguanidine sodium channel blocker.

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

Other mucosal surfaces in and on the body show subtle differences in the normal physiology of the protective surface fluids on their surfaces, but the pathophysiology of the disease reflects a common theme, namely, too little surface protective fluid. For example, in xerostomia (dry mouth) fluid in the oral cavity is reduced due to failure of the sublingual and submandibular parotid glands to secrete fluid despite continued Na transport mediated fluid absorption.⁺ (ENaC) from the oral cavity. Similarly, keratoconjunctivitis sicca (dry eye) is caused by a failure of the lacrimal glands to secrete fluid despite continued Na-dependent fluid absorption.⁺ on the conjunctival surfaces. In rhinosinusitis, there is an imbalance, as in BC, between mucin secretion and the relative reduction in LSA. Finally, in 35 the gastrointestinal tract, the failure to secrete Cl^- (and fluid) in the proximal small intestine, combined with increased Na absorption⁺ (and fluid) in the terminal ileum, lead to distal intestinal obstruction syndrome (DOS). In older patients, excessive absorption of Na⁺ (and volume) in the descending colon causes constipation and diverticulitis.

Fifty million Americans and hundreds of millions of people around the world suffer from hypertension and the consequent sequelae that lead to congestive heart failure and increasing mortality. It is the leading cause of death in the Western world and new drugs are needed to treat these diseases. Therefore, in addition, some of the new sodium channel blockers of this invention can be designed to target the kidney and thus can be used as diuretics for the treatment of hypertension, congestive heart failure (CHF) and other diseases. cardiovascular These new agents can 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

An object of the present invention is to provide compounds that are more potent and / or absorb less rapidly from mucosal surfaces, and / or are less reversible compared to known compounds.

Another aspect of the present invention is 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 phenyl. Therefore, the compounds will have a prolonged pharmacodynamic half-life on mucosal surfaces compared to known compounds.

Another object of the present invention is to provide compounds that (1) are less rapidly absorbed from mucosal surfaces, especially airway surfaces, compared to known compounds, and (2) when absorbed from mucosal surfaces after of administration on mucosal surfaces, they are converted in vivo into metabolic derivatives thereof that have a lower efficacy in blocking sodium channels compared to the original administered compound.

Another object of the present invention is to provide compounds that are more potent and / or less rapidly absorbed and / or have less reversibility, compared to compounds such as amiloride, benzamyl and phenyl. Therefore, such compounds will have a prolonged pharmacodynamic half-life on mucosal surfaces compared to previous compounds.

ES 2 298 505 Τ3

Another object of the present invention is to provide kidney-targeting compounds for use in treating cardiovascular disease.

Another object of the present invention is to provide methods of treatment that take advantage of the pharmacodynamic properties of the compounds described above.

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

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

The objects of the present invention can be carried out with a class of pyrazinoylguanidine compounds represented by the formula (I):

<img file="ES2298505T3_D0001.tif" />

in which

X is hydrogen, halogen, trifluoromethyl, lower alkyl, unsubstituted or substituted phenyl, lower alkylthio, phenyl- (lower alkyl) -thio, (lower alkyl) sulfonyl, or phenyl- (lower alkyl) sulfonyl;

Y is hydrogen, hydroxyl, mercapto, lower alkoxy, lower alkylthio, halogen, lower alkyl, unsubstituted or substituted mononuclear aryl, or -N (R²)₂;

R¹ is hydrogen or lower alkyl;

everyR² is, independently, -R⁷, - (CH2) m-OR⁸, - (CH2)_m-NR⁷R¹⁰,

- (CH2)_n(CHOR⁸) (CHOR⁸)_n-CH2OR⁸, - (CH-CH-O). -R⁸,

- (CH₂CH₂OR)_m-CH₂CH₂NR⁷R¹⁰, - (CH2) nC (= O) NR⁷R¹⁰, - (CH2)_n-Z_g-R⁷, - (CH2) m-NR¹⁰-CH2 (CHOR⁸) (CHOR⁸)_nCH2OR⁸, - (CH2) n-CO2R⁷, or

<img file="ES2298505T3_D0002.tif" />

R³ and R⁴ are each, independently, hydrogen, a group represented by the formula (A), lower alkyl, lower hydroxyalkyl, phenyl, phenyl-lower alkyl, (halophenyl) -lower alkyl, (lower alkylphenylalkyl), ((lower alkoxy) phenyl) -alkyl lower, naphthylalkyl, or pyridyl lower alkyl, provided that at least one of R³ and R⁴ let be a group represented by the formula (A):

<img file="ES2298505T3_D0003.tif" />

ES 2 298 505 Τ3 in which each R^L is, independently, -R⁷, - (CH2) n-OR⁸, -O- (CH2)_m-OR⁸, - (CH2) n-NR⁷R¹⁰, -O- (CH2)_m-NR⁷R¹⁰, - (CH2) n (CHOR⁸) (CHOR⁸) n-CH₂OR⁸, -O- (CH2) m (CHOR⁸) (CHOR⁸) n-CH2OR⁸, - (CH2CH-Ο). -GO⁸, -O- (CH2CH2O) mR⁸, - (CH2CH2O) m-CH2CH2NR⁷R¹⁰, -O- (CH2CH2O) m-CH2CH2NR⁷R¹⁰, - (CH2) nC (= O) NR⁷R¹⁰, -O- (CH2) mC (= O) NR⁷R¹⁰, - (CH2) n- (Z) gR⁷, -O- (CH2) m- (Z) gR⁷, - (CH2 \ -NR¹⁰-CH2 (CHOR⁸) (CHOR⁸) n -CH2 OR⁸, -O- (CH2) m-NR¹⁰-CH2 (CHOR⁸) (CHOR⁸) n-CH2OR⁸,

- (CH₂)_n-CO₂R⁷, -O- (CH2) m-CO2R⁷, -OSO3H, -O-glucuronide, -O-glucose,

<img file="ES2298505T3_D0004.tif" />

each o is, independently, an integer from 0 to 10;

each p is an integer from 0 to 10;

provided that the sum of o and p in each contiguous string is from 1 to 10;

each x is independently O, NR¹⁰, C (= O), CHOH, C (= NR¹⁰),

CHNR⁷R¹⁰, or represents a single bond;

each R⁶ is, independently, -R⁷, -OH, -OR¹¹, -N (R⁷)₂, - (CH₂)_m-OR⁸,

-O- (CH2) m-OR⁸, - (CH2)_n-NR⁷R¹⁰, -O- (CH2) m-NR⁷R¹⁰,

- (CH₂\ (CHOR⁸) (CHOR⁸) n-CH2OR⁸, -O- (CH2) m (CHOR⁸) (CHOR⁸) n-CH2OR⁸, - (CH2CH2OVR⁸, -O- (CH2CH2O) mR⁸, - (CH2CH2O) m-CH2CH2NR⁷R¹⁰, -O- (CH2CH2O) m-CH2CH2NR⁷R¹⁰, - (CH2)_n-C (= O) NR⁷R¹⁰,

-O- (CH2) mC (= O) NR⁷R¹⁰, - (CH2) n- (Z) gR⁷, -O- (CH2) m- (Z) gR⁷,

- (CH₂\ -NR¹⁰-CH2 (CHOR⁸) (CHOR⁸)_n -CH2 OR⁸,

-O- (CH2) m-NR¹⁰-CH2 (CHOR⁸) (CHOR⁸)_n-CH2OR⁸,

- (CH₂)_n-CO₂R⁷, -O- (CH2) m-CO2R⁷, -OSO3H, -O-glucuronide, -O-glucose,

<img file="ES2298505T3_D0005.tif" />

in which when two R⁶ are -OR¹¹ and are located adjacent to each other on a phenyl ring, the alkyl moieties of the two R⁶ they can be linked together to form a methylenedioxy group;

each R⁷ is, independently, hydrogen or lower alkyl;

ES 2 298 505 Τ3 each R⁸ is independently hydrogen, lower alkyl, -C (= O) -R¹¹, glucuronide, 2-tetrahydropyranyl, or

<img file="ES2298505T3_D0006.tif" />

each R⁹ is, independently, -CO2R⁷, -CON (R⁷) 2, -SO₂CH₃ or -C (= O) R⁷;

everyR¹⁰ is, independently, -H, -SO2CH3, -CO2R⁷, -C (= O) NR⁷R⁹, -C (= O) R⁷ or -CH2- (CHOH)_n-CH₂OH;

each Z is independently CHOH, C (= O), CHNR⁷R¹⁰, C = NR¹⁰ or NR¹⁰;

each R¹¹ is, independently, lower alkyl;

each g is, independently, an integer from 1 to 6;

each m is, independently, an integer from 1 to 7;

each n is, independently, an integer from 0 to 7;

each Q is independently CR⁶ or a nitrogen atom, in which at most three Q's in a ring are nitrogen atoms;

or a pharmaceutically acceptable salt thereof, and including all enantiomers, diastereoisomers and racemic mixtures thereof.

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

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

topically administering an effective amount of a compound represented by formula (I) to a mucosal surface of a subject in need thereof.

The present invention also provides a method for blocking ENaCs, comprising:

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

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

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

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

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

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

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

ES 2 298 505 T3

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

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

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

The present invention also provides a method of treating sinusitis, comprising:

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

The present invention also provides a method of preventing ventilator-induced pneumonia, comprising:

administering an effective compound represented by formula (I) to a subject via respirator.

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

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

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 a subject 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 a subject in need thereof.

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

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

The present invention also provides a method for promoting hydration of the cornea, comprising:

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

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

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

ES 2 298 505 Τ3

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 a subject 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 dry skin of a subject 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) in the mouth of the subject in need.

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

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

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

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

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

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

The present invention also provides a method for treating hypertension, which comprises administering the compound represented by formula (I) to a subject in need thereof.

The present invention also provides a method of reducing blood pressure, which comprises administering the compound represented by formula (I) to a subject in need thereof.

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

The present invention also provides a method of promoting diuresis, which comprises administering the compound represented by formula (I) to a subject in need thereof.

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

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

Brief description of the figures

The invention and many of its advantages will be more fully appreciated when it is better understood by reference to the following detailed description taken in conjunction with the following figures:

fig. 1: Effect of a compound of the present invention on EMC at t = 0 h as described in Example 9 herein.

fig. 2: Effect of a compound of the present invention on the EMC at t = 4 h as described in example 9 of the present document.

Detailed description of the invention

The present invention is based on the discovery that the compounds of formula (I) are more potent and / or less rapidly absorbed from mucosal surfaces, especially airway surfaces, and / or less reversible from the interactions with ENaC compared to compounds such as amiloride, benzamyl, and phenyl. Therefore, the compounds of formula (I) have a longer half-life on mucosal surfaces compared to these compounds.

ES 2 298 505 T3

The present invention is also based on the discovery that some compounds covered by formula (I), in vivo, become metabolic derivatives thereof that have a lower efficacy in blocking sodium channels compared to the original compound administered. , after they are absorbed by mucosal surfaces after administration. This important property means that the compounds will have a lower tendency to produce unwanted side effects by blocking sodium channels at non-target sites in the receptor's body, e.g. eg, in the kidneys.

The present invention is also based on the discovery that some compounds encompassed by formula (I) target the kidney and therefore can be used as cardiovascular agents.

In the compounds represented by formula (I), X can be hydrogen, halogen, trifluoromethyl, lower alkyl, lower cycloalkyl, unsubstituted or substituted phenyl, lower alkylthio, phenyl- (lower alkyl) -thio, (lower alkyl) -sulfonyl or phenyl- (lower alkyl) -sulfonyl. Halogen is preferred.

Examples of halogen include fluorine, chlorine, bromine, and iodine. The preferred halogens are chlorine and bromine. Chlorine is particularly preferred. This description is applicable to the term "halogen" used throughout the present specification.

As used herein, the term "lower alkyl" means an alkyl group having less than 8 carbon atoms. This range includes all specific carbon atom values ​​and the subranges between them, such as 1, 2, 3, 4, 5, 6, and 7 carbon atoms. The term "alkyl" encompasses all types of such groups, eg. eg, linear, branched, and cyclic alkyl groups. This description is applicable to the term "lower alkyl" used throughout the present specification. Examples of suitable lower alkyl groups include methyl, ethyl, propyl, cyclopropyl, butyl, isobutyl, etc.

Substituents on the phenyl group include halogens. Particularly preferred halogen substituents are chlorine and bromine.

Y can be hydrogen, hydroxyl, mercapto, lower alkoxy, lower alkylthio, halogen, lower alkyl, lower cycloalkyl, mononuclear aryl, or -N (R²)₂. The alkyl moiety of the lower alkoxy groups is the same as described above. Examples of mononuclear aryl include phenyl groups. The phenyl group may be unsubstituted or substituted as described above. The preferred identity of Y is -N (R²)2. Those compounds in which each R² it is hydrogen.

R¹ it can be hydrogen or lower alkyl. Hydrogen is preferred for R¹.

<img file="ES2298505T3_D0007.tif" />

·&gt; \^K-'^±±2^^±±2 ^ '7m ^ - ^ 2 ^^ 2 (CHOR⁸) (CHOR⁸)_n-CH2OR⁸, - (CI I) -C (ÍR ', or

<img file="ES2298505T3_D0008.tif" />

Stop² hydrogen and lower alkyl are preferred, in particular C alkyl₁-C₃. Hydrogen is particularly preferred.

R³ and R⁴ may independently be hydrogen, a group represented by formula (A), lower alkyl, lower hydroxyalkyl, phenyl, lower phenylalkyl, (halophenyl) -lower alkyl, (lower alkylphenylalkyl), ((lower alkoxy) phenyl) -lower alkyl , naphthylalkyl, or pyridylalkyl, provided that at least one of R³ and R⁴ be a group represented by formula (A).

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

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

The linking group on the alkylene chain, x, can independently be O, NR¹⁰, C (= O), CHOH, C (= NR¹⁰), CHNR⁷R¹⁰, or represents a single link.

Therefore, when x represents a single bond, the alkylene chain attached to the ring is represented by the formula- (C (R^L)₂)_{o + p}-, where the sum o + p is 1 to 10.

ES 2 298 505 T3

Each R^l can be, independently, -R⁷, - (CH2) n-OR⁸, -O- (CH2)_m-OR⁸, - (CH2) n-NR⁷R¹⁰, -O- (CH2)_m-NR⁷R¹⁰, - (CH2) n (CHOR⁸) (CHOR⁸) n-CH2OR⁸, -O- (CH) m (CHOR⁸) (CHOR⁸) n-CH2OR⁸, - (CH2CH2O) mR⁸, -O- (CH2CH2O) m R⁸, - (CH2CH₂OR)_m-CH₂CH₂NR⁷R¹⁰, -O- (CH2CH2O) m-CH2CH2NR⁷R¹⁰, - (CH2)_n-C (= O) NR⁷R¹⁰, -O- (CH2) mC (= O) NR⁷R¹⁰, - (CH2) n- (Z) „- R⁷, -O- (CH2) m- (Z) gR⁷, - (CH2)_n-NR¹⁰-CH2 (CHOR⁸) (CHOR⁸) n-CH2OR⁸, -O- (CH2) m-NR¹⁰-CH2 (CHOR⁸) (CHOR⁸) n-CH₂OR⁸, - (CH₂)_n-CO₂R⁷, -O- (CH2) m-CO2R⁷, -OSO3H, -O-glucuronide, -O-glucose,

<img file="ES2298505T3_D0009.tif" />

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

In the alkylene chain in formula (A), it is preferred that when a group R^L attached to one carbon atom is other than hydrogen, so the other R^L attached to that carbon atom is hydrogen, that is, the formula -CHR^L-. It is also preferred that at most two R groups^L in an alkylene chain are other than hydrogen, when the other R groups^L in the chain are hydrogens. Even more preferably, only one R group^L in the alkylene chain is other than hydrogen, when the other R groups^L in the chain are hydrogen. In these embodiments, it is preferred that x represents a single bond.

In another particular embodiment of the invention, all the R groups^L in the alkylene chain they are hydrogen. In these embodiments, the alkylene chain is represented by the formula

- (CH₂) or - x - (CH₂) p-,

There are four R groups⁶ present on the ring in formula (A), as described above. When two R⁶ are -OR¹¹and are located adjacent to each other on a phenyl ring, the alkyl moieties of the two R groups⁶ they may be linked together to form a methylenedioxy group, that is, a group of formula -O-CH2-O-.

As discussed before, R⁶ it can be hydrogen. Therefore, 1, 2, 3, or 4 R groups⁶ they can be other than hydrogen. Preferably at least 3 of the R groups⁶ they are other than hydrogen.

Each g is, independently, an integer from 1 to 6. Therefore, each g can be 1, 2, 3, 4, 5, or 6.

Each m is an integer from 1 to 7. Therefore, each m can be 1, 2, 3, 4, 5, 6, or 7.

Each n is an integer from 0 to 7. Therefore, each n can be 0, 1, 2, 3, 4, 5, 6, or 7.

Each Q in formula (A) is CR⁶ or a nitrogen atom, where at most three Q's in a ring are nitrogen atoms. Therefore, there can be 1, 2 or 3 nitrogen atoms in a ring. Preferably, at most two Q's are nitrogen atoms. More preferably, at most one Q is a nitrogen atom. In a particular embodiment, the nitrogen atom is in the 3 position of the ring. In another embodiment of the invention, each Q is CR⁶, that is, there are no nitrogen atoms in the ring.

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

In another preferred embodiment, R² it is hydrogen.

In another preferred embodiment, R¹ it is hydrogen.

In another preferred embodiment, X is chlorine.

In another preferred embodiment, R³ it is hydrogen.

In another preferred embodiment, R^L it is hydrogen.

In another preferred embodiment, o is 4.

In another preferred embodiment, p is 0.

In another preferred embodiment, the sum of o and p is 4.

In another preferred embodiment, x represents a single bond.

In another preferred embodiment, R⁶ it is hydrogen.

ES 2 298 505 Τ3

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

In another preferred embodiment, neither Q is a nitrogen atom.

In a preferred embodiment of the present invention:

X is halogen;

Yes -N (R⁷)₂;

R¹ is hydrogen or C alkyl₁-C₃;

R² is -R⁷, -OR⁷, CH2OR⁷, or -CO2R⁷;

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

R⁴ is hydrogen, a group represented by formula (A), or lower alkyl;

In another preferred embodiment of the present invention:

X is chlorine or bromine;

Yes -N (R⁷)2;

R² is hydrogen or C1-C3 alkyl;

at most three Rs⁶ they are other than hydrogen as described above;

at most three Rs^L they are other than hydrogen as described above; and at most 2 Q are nitrogen atoms.

In another preferred embodiment of the present invention:

Yes -NH2;

In another preferred embodiment of the present invention:

R⁴ is hydrogen;

at most an R^L it is other than hydrogen as described above;

at most two Rs⁶ they are other than 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 (I) is represented by the formula:

<img file="ES2298505T3_D0010.tif" />

ES 2 298 505 T3

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

<img file="ES2298505T3_D0011.tif" />

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

<img file="ES2298505T3_D0012.tif" />

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

<img file="ES2298505T3_D0013.tif" />

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

<img file="ES2298505T3_D0014.tif" />

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

<img file="ES2298505T3_D0015.tif" />

The compounds of formula (I) can be prepared and used in the form of the free base. Alternatively, the compounds can be prepared and used as a pharmaceutically acceptable salt. Pharmaceutically acceptable salts are salts that retain or enhance the desired biological activity of the parent compound and do not impart unwanted toxicological effects. Examples of such salts are (a) acid addition salts formed with inorganic acids, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; (b) salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, acid

ES 2 298 505 Τ3 p-toluenesulfonic, naphthalenedisulfonic acid, polygalacturonic acid, malonic acid, sulfosalicylic acid, glycolic acid, 2-hydroxy-3-naphthoate, pamoate, salicylic acid, stearic acid, phthalic acid, mandelic acid, lactic acid and the like ; and (c) salts formed from elemental anions, for example chlorine, bromine and iodine.

It should be noted that the present invention encompasses all enantiomers, diastereoisomers and racemic mixtures of the compounds within the scope of formula (I). All mixtures of said enantiomers and diastereoisomers are within the scope of the present invention.

Without being limited by any particular theory, it is believed that the compounds of formula (I) function in vivo io as sodium channel blockers. By blocking the epithelial sodium channels present on the mucosal surfaces, the compounds of formula (I) reduce the absorption of water by the mucosal surfaces. This effect increases the volume of the protective fluids on the mucosal surfaces, rebalances the system and thus treats the disease.

The present invention also provides treatment methods that take advantage of the properties of the compounds of formula (I) discussed above. Thus, subjects that can be treated by the methods of the present invention include, but are not limited to, patients afflicted with cystic fibrosis, primary ciliary dyskinesia, chronic bronchitis, chronic obstructive airway disease, artificial respiration patients, patients with acute pneumonia, etc. The present invention can be used to obtain a sputum sample from a patient by administering the active compounds to at least one lung of a patient, and then inducing and collecting a sputum sample from the patient. Typically, the invention will be delivered to respiratory mucosal surfaces by aerosol (liquid or dry powder) or washing.

Patients that can be treated by the method of the present invention also include patients who are administered supplemental oxygen nasally (a regimen that tends to dry out the airway surfaces); patients suffering from an allergic disease or response (eg. g., an allergic response to pollen, dust, animal hair or particles, insects or insect particles, etc.) that affect the surfaces of the nasal airway; patients suffering from a bacterial infection (eg. Staphylococcal infections such as Staphylococcus aureus infections, Hemophilus influenza infections, Streptococcus pneumoniae infections, Pseudomonas aeuriginosa infections, etc.) of the nasal airway surfaces; patients afflicted with an inflammatory disease affecting the nasal airway surfaces; o patients afflicted with sinuitis (in which the active agent (s) are administered to promote drainage of congested mucus secretions in the sinuses by administering an amount effective to promote drainage of congested fluid in the sinuses), or combined, rhinosinusitis. The invention can be administered to the rhinonasal surfaces by topical delivery, including aerosols and drops.

The present invention can be used to hydrate mucosal surfaces other than airway surfaces. Such distinct mucosal surfaces include gastrointestinal surfaces, oral surfaces, genitourethral surfaces, ocular surfaces, or surfaces of the eye, inner ear, and middle ear. For example, the active compounds of the present invention can be administered by any suitable means, including local / topical, oral or rectal routes, in an effective amount.

The compounds of the present invention are also useful for treating a variety of functions related to the cardiovascular system. Thus, the compounds of the present invention are useful for use as antihypertensive agents. The compounds can also be used to lower blood pressure and treat edema. Furthermore, the compounds of the present invention are also useful for promoting diuresis, natriuresis, and saluresis. The compounds can 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 is primarily concerned with the treatment of human subjects, but can also be used 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. Because the free base of the compound is generally less soluble in aqueous solutions than salt, compositions of the free base are used to provide the most sustained release of the active agent in the lungs. An active agent present in the lungs in particulate form that has not dissolved in solution is not available to induce a physiological response, but serves as a reservoir of bioavailable drug 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 the reabsorption of water from mucosal surfaces.

The compounds of the present invention can also be used in conjunction with a P2Y2 receptor agonist or a pharmaceutically acceptable salt thereof (sometimes referred to as an "active agent" herein). The composition may further comprise a P2Y2 receptor agonist or a pharmaceutically salt thereof.

Acceptable ES 2 298 505-3 (sometimes also referred to as an "active agent" herein). The P2Y2 receptor agonist is normally included in an amount effective to stimulate chloride and water secretion from 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 can also be used in combination with compounds of the present invention. These bronchodilators include, but are not limited to, β-adrenergic agonists including but not limited to epinephrine, isoproterenol, fenoterol, albuterol, terbutaline, pirbuterol, bitolterol, metaproterenol, iosetharine, salmeterol xinafoate, as well as anticholinergic agents that include but are not limited to to ipatropium bromide, as well as compounds such as theophylline and aminophylline. These compounds can be administered according to known techniques, either before or simultaneously with the active compounds described herein.

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 that it inhibits the reabsorption of water by mucosal surfaces, including airways and other surfaces.

The active compounds described herein can be administered to mucosal surfaces by any suitable means, including topical, oral, rectal, vaginal, ocular, and dermal, etc. For example, for the treatment of constipation, the active compounds can be administered orally or rectally to the gastrointestinal mucosal surface. The active compound can be combined with a pharmaceutically acceptable carrier in any suitable form, such as sterile diluted or physiological solution or topical solution, in the form of drops, tablets or the like for oral administration, in the form of a suppository for rectal administration or genitourethral, ​​etc. Excipients can be included in the formulation to enhance the solubility of the active compounds, as desired.

The active compounds described herein can be administered to the airway surfaces of a patient by any suitable means, including a spray, mist, or drops of the active compounds in a pharmaceutically acceptable carrier such as dilute or physiological saline solutions. or distilled water. For example, the active compounds can be prepared as formulations and administered as described in US Patent No.^or 5,789,391 to Jacobus.

Solid or liquid particulate active agents prepared for the practice of the present invention, as noted above, could include particles of respirable or non-respirable size; that is, for respirable particles, particles of a size small enough to pass through the mouth and larynx after inhalation and to the bronchi 35 and alveoli of the lungs, and for non-respirable particles, particles large enough to be retained in the passages from the nasal airways instead of passing through the larynx and into the bronchi and alveoli of the lungs. In general, particles in the range of about 1 to 5 microns (more particularly, less than about 4.7 microns in size) are respirable. Non-respirable sized particles are greater than about 5 microns in size, down to the size of visible droplets. Therefore, for nasal administration, a particle size in the range of 10-500 pm can be used to ensure retention in the nasal cavity.

In the manufacture of a formulation according to the invention, normally the active agents or the physiologically acceptable salts or their free bases are mixed, inter alia, with an acceptable carrier. Of course, the carrier must be compatible with any other ingredient in the formulation and must not be harmful to the patient. The carrier must be solid or liquid, or both, and is preferably formulated with the compound in the form of a unit dose formulation, eg, a capsule which may contain 0.5% to 99% by weight of the active compound. One or more active compounds can be incorporated into the formulation of the invention, formulations that can be prepared by any of the known pharmacy techniques that essentially consist of mixing the 50 components.

Compositions containing dry respirable or non-respirable particles of micronized active agent can be prepared by grinding the dry active agent with a mortar and pestle, and then passing the micronized composition through a 400 mesh sieve to break or separate the particles. large agglomerates.

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

The active compounds described herein can be administered to the airway surfaces including the nasal passages, sinuses and lungs of a subject by a suitable means known in the art, such as by drops, nasal sprays, 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 the airway surfaces of the lungs by administration of an aerosol suspension of respirable particles comprising the active compound, which the subject inhales. Respirable particles can be liquid or solid. Numerous inhalers are known to deliver aerosol particles to the lungs of a subject.

ES 2 298 505 Τ3

Inhalers such as those developed by Inhale Therapeutic Systems, Palo Alto, California, USA, including, but not limited to, those described in US Pat.^you 5,740,794; 5,654,007; 5,458,135; 5,775,320 and 5,785,049. Inhalers developed by Dura Pharmaceuticals, Inc., San Diego, California, USA can also be used, including but not limited to those described in US Patent Nos.^you 5,622,166; 5,577,497; 5 5,645,051 and 5,492,112. Additionally, inhalers such as those developed by Aradigm Corp., Hayward,

California, USA, including but not limited to those described in US Patent Nos.^you 5,826,570; 5,813,397; 5,819,726 and 5,655,516. These devices are particularly suitable as dry particle inhalers.

Liquid particulate aerosols comprising the active compound can be produced by any suitable method, such as with a pressure-actuated aerosol nebulizer or an ultrasonic nebulizer. See, for example, US Pat.^or 4,501,729. Nebulizers are commercially available devices that transform solutions or suspensions of the active ingredient into a therapeutic aerosol nebulization by acceleration of compressed gas, usually 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 vehicle, the active ingredient comprising up to 40% w / w of the formulation, but preferably less than 20% w / w. The vehicle is usually water (and more preferably sterile pyrogen-free water) or dilute aqueous alcoholic solution. Perfluorocarbon vehicles can also be used. Optional additives include preservatives if the formulation is not rendered sterile, eg, methyl hydroxybenzoate, antioxidants, flavoring agents, volatile oils, buffering agents, and surfactants.

Solid particulate aerosols comprising the active compound can be produced in the same way with any solid particulate drug aerosol generator. Aerosol generators for delivering solid particulate drugs to a subject produce particles that are respirable, as explained above, and generate an aerosol volume containing a predetermined metered dose of drug at a rate suitable for human administration. An illustrative type of solid particle aerosol generator is an insufflator. Formulations suitable for administration by insufflation include finely divided powders that can be delivered by insufflator or taken into the nasal cavity as a puff. In the insufflator, the powder (p. g., a metered dose thereof effective to carry out the treatments described herein) is contained in capsules or cartridges, usually made of gelatin or plastic, which are opened or punctured in situ and the powder is supplied entrained by the air through the device after inhalation or by means of a manually operated pump. The powder used in the insufflator consists of the active ingredient only or of a powder mixture comprising the active ingredient, a suitable powder diluent, such as lactose, and an optional surfactant. The active ingredient normally comprises 0.1 to 10% w / w of the formulation. A second type of exemplary aerosol generator comprises a metered dose inhaler. Metered dose inhalers are pressurized aerosol dispensers, which typically contain a suspension or solution formulation of the active ingredient in a liquefied propellant. During use, these devices discharge the formulation through a valve adapted to deliver a metered volume, typically 10 to 150 µΐ, to produce a fine particle spray containing the active ingredient. Suitable propellants include some chlorofluorocarbon compounds, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, and mixtures thereof. The formulation may further contain one or more cosolvents, for example ethanol, surfactants, such as oleic acid, sorbitan trioleate, antioxidants, and suitable flavoring agents.

The aerosol, formed from solid or liquid particles, can be produced by the aerosol generator at a rate of about 10 to 150 liters per minute, more preferably 30 to 150 liters per minute, 45 and more preferably about 60 liters per minute. minute. Sprays containing larger amounts of medicine can be given more quickly.

The dosage of the active compounds described herein will vary depending on the condition being treated and the condition of the subject, but in general it can be from about 0.01, 0.03, 0.05, 0.1 to 1 50, 5, 10 or 20 mg of the pharmaceutical agent, deposited on the surfaces of the airways. The daily dose can be divided into one or multiple unit dose administrations. The object is to achieve a concentration of pharmaceutical agents on the surfaces of the pulmonary airway between 10^-9-10⁴ M.

In another embodiment, they are administered by administration of an aerosol suspension of respirable or non-respirable particles (preferably non-respirable particles) composed of active compound, which the subject inhales through the nose. Respirable or non-respirable particles can be liquid or solid. The amount of active agent included may be an amount sufficient to achieve dissolved concentrations of active agent on the subject's airway surfaces of about 10^-9, 10 ⁸ or 10 ⁷ to about 10 ³, 10 ², 10 'moles / liter, and more preferably about 10^-9 to about 10 ⁴ moles / liter.

The dosage of active compound will vary depending on the condition to be treated and the condition of the subject, but in general it may be an amount sufficient to achieve dissolved concentrations of active compound on the subject's nasal airway surfaces of about 10 ^-9, 10 ⁸ or 10 ⁷ to about 10 ^-3, 10 ² or 10 ^-1moles / liter, and more preferably about 10 ⁷ to about 10 ⁴ moles / liter. Depending on the solubility of the particular formulation of the administered active compound, the daily dose can be divided between one or more unit dose administrations. The unit dose by weight may be in the range of about 0.01, 0.03, 0.1, 0.5, or 1.0 to 10 or 20 milligrams of active agent particles for a human subject, depending on age. and condition of the subject. A currently preferred unit dose is about 0.5 milligrams of principle

ES 2 298 505 Τ3 active given with a regimen of 2-10 daily administrations. The dosage can be provided as a prepackaged unit with any suitable medium (eg, encapsulation of a gelatin capsule).

In one embodiment of the invention, the particulate active agent composition may contain both an active agent free base and a pharmaceutically acceptable salt to provide both early release and sustained release of the active agent for dissolution in mucus secretions of nose. Said composition serves to provide both early relief to the patient and sustained relief over time. Sustained relief, by decreasing the number of daily administrations required, is expected to increase patient compliance over the course of active agent treatments.

Pharmaceutical formulations suitable for administration into the airway include formulations of solutions, emulsions, suspensions, and extracts. See generally J. Nairn, "Solutions, Emulsions, Suspensions and Extracts," in Remington: The Science and Practice of Pharmacy, Chapter 86 (19^or ed. 1995), incorporated herein by reference. Pharmaceutical formulations suitable for nasal administration can 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.

Nebulizations or aerosols of liquid particles comprising the active compound can be produced by any suitable means, such as by a simple nasal spray with the active agent in a pharmaceutically acceptable aqueous vehicle, such as sterile saline or sterile water. Administration can be with a pressure-actuated aerosol nebulizer or an ultrasonic nebulizer. See, for example, US Patent Nos. 4,501,729 and 5,656,256.

Formulations suitable for use in a spray bottle or nasal drops or in nebulizers consist of the active ingredient in a liquid vehicle, the active ingredient comprising up to 40% w / w of the formulation, but preferably less than 20% w / weight. Typically, the vehicle is water (and more preferably sterile, pyrogen-free water) or dilute aqueous alcoholic solution, preferably made in 0.12% to 0.8% sodium chloride solution. Optional additives include preservatives if the formulation is not rendered sterile, eg, methyl hydroxybenzoate, antioxidants, flavoring agents, volatile oils, buffering agents, osmotically active agents (eg, mannitol, xylitol, erythritol), and surfactants. .

Compositions containing dry respirable or non-respirable particles of micronized active agent can be prepared by grinding the dry active agent with a mortar and pestle, and then passing the micronized composition through a 400 mesh sieve to break up or separate the agglomerates. large.

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

Compounds of formula (I) can be synthesized according to procedures known in the art. A representative synthetic procedure is shown in the following scheme:

<img file="ES2298505T3_D0016.tif" />

These procedures are described, for example, by EJ Cragoe, "The Synthesis of Amiloride and Its Analogs" (Chapter 3) in Amiloride and Its Analogs, p. 25-36. Other procedures for preparing the compounds are described, for example, in US 3,313,813. See in particular procedures A, B, C and D described in US 3,313,813.

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

In vitro measurement of the activity and reversibility of sodium channel blockade

An assay used to evaluate the mechanism of action and / or potency of the compounds of the present invention involves the determination of the inhibition by the drug in the lumen of the epithelial sodium currents of the airways measured with short-circuit current (I_SC) using airway epithelial monolayers mounted in Ussing chambers. Cells obtained from freshly excised human, dog, sheep, or rodent airways are seeded onto 0.4 micron Sanpwell ™ inserts (CoStar), grown under air-liquid interface (ALI) conditions in hormone-defined medium, and sodium transport activity (I_SC) in a Krebs-Ringer Bicarbonate (KBR) bath in Ussing chambers. All test drug additions are in the lumen bath with

ES 2 298 505 T3 semi-logarithmic dose addition protocols (1 x 10 ¹¹ M to 3 x 10 ⁵ M), and the cumulative change in ISC (inhibition) is recorded. All drugs are prepared in dimethylsulfoxide as stock solutions with a concentration of 1 x 10² M and are stored at -20OC. Typically 8 preparations are run in parallel; two preparations per experiment incorporate amiloride and / or benzamyl as positive controls. After administering the maximum concentration 5 (5 x 10^-5 M), the lumen bath is changed three times with fresh KBR solution without drug, and the resulting ISC is measured after each wash for approximately 5 minutes duration. Reversibility is defined as the percentage return to baseline for the sodium current after the third lift. All voltage clamp data is collected with a computer interface and analyzed outside.

Dose-effect relationships are considered and analyzed for all compounds with the Prism 3.0 program. CI values₅₀Maximum effective concentrations and reversibility are calculated and compared with amiloride and benzamyl as positive controls.

Pharmacological absorption tests

1. Apical disappearance test

Bronchial cells (dog, human, sheep or rodent cells) are seeded at a density of 0.25 x 10⁶/ cm²on a membrane coated with Transwell-Col collagen with a growth area of ​​1.13 cm² in an air-liquid interface in a medium defined with hormones that promotes a polarized epithelium. From 12 to 20 days after growth in 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 (DP), which are indicators of the integrity of the polarized nature of the culture. Human cell systems are preferred for studies of apical surface absorption rates. The disappearance assay is carried out under conditions that mimic "thin" films in vivo (~ 25 gl) and is initiated by adding experimental sodium channel blockers or positive controls (amiloride, benzamyl, phenyl) to the surface. apical with an initial concentration of 10 gM. A series of samples (5 gl of volume per sample) are collected at different time points, including 0, 5, 20, 40, 90, and 240 minutes. Concentrations are determined by measuring the intrinsic fluorescence of each sodium channel blocker 30 using a Fluorocount microplate fluorometer or HPLC. Quantitative analysis uses a standard curve generated from authentic reference standards of known concentration and purity. Disappearance rate data analysis is carried out using non-linear regression, exponential decay in one phase (Prism V 3.0).

2. Assay by confocal microscopy of the absorption of the congener amiloride

Virtually all amiloride-type molecules fluoresce in the ultraviolet range. This property of these molecules can be used to directly measure cell update using xz confocal microscopy. Equimolar concentrations of experimental compounds and positive controls including amiloride and 40 compounds that demonstrate rapid absorption into the cell compartment (benzamyl and phenyl) are placed on the apical surface of airway cultures on the confocal microscope stage. Serial xz images are obtained over time and the magnitude of fluorescence accumulating in the cell compartment is quantified and plotted as a change in fluorescence versus time.

3. In vitro assays of compound metabolism

Airway epithelial cells have the ability to metabolize drugs during the transepithelial absorption process. In addition, although less likely, drugs can be metabolized on the epithelial surfaces of the airways by specific ectoenzymatic activities. Perhaps more possibly as an ectosurface event, the compounds can be metabolized by infected secretions occupying the airway lumens of patients with pulmonary diseases, e.g. ex. cystic fibrosis. Therefore, a series of assays are carried out to characterize the metabolism of the compound resulting from the interaction of the test compounds with human airway epithelia and / or human airway epithelial lumen products.

In the first series of tests, the interaction of test compounds in KBR as an "LSA" stimulant was applied to the apical surface of human airway epithelial cells grown in the T-Col insert system. For most compounds, metabolism (generation of new species) is assayed using high performance liquid chromatography (HPLC) to resolve the chemical species and endogenous fluorescence properties of these compounds to calculate the relative amounts of the test compound and the new metabolites. For a typical assay, a test solution (KBR 25 gl, containing 10 gM test compound) is placed on the surface of the epithelial lumen. Sequential samples of 5 to 10 gl are obtained from the lumen and serous compartments for HPLC analysis of (1) the mass of test compound that permeates from the lumen bath to the serous bath and (2) the potential formation of metabolites to starting from the parent compound. In cases where the fluorescence properties of the test molecule are not suitable for such characterizations, radiolabeled compounds are used for these tests. From the HPLC data, the rate of disappearance and / or formation of new metabolite compounds on the surface of the lumen and the appearance of test compound and / or new metabolite in the basolateral solution are quantified. The data related to the chromatographic mobility of potential new metabolites are also quantified with reference to the parent compound.

ES 2 298 505 T3

To analyze the potential metabolism of test compounds by CF sputum, a “representative” mixture of expectorated CF sputum obtained from 10 CF patients (with IRB approval) has been collected. The sputum has been solubilized in a 1: 5 mixture of KBR solution with vigorous vortex mixing, after which the mixture was divided into an aliquot of "clean" sputum and an aliquot subjected to ultracentrifugation so that a aliquot of "supernatant liquid" (clean = cell; supernatant liquid = liquid phase). Typical CF sputum compound metabolism studies involve the addition of known masses of test compound to "clean" CF sputum and incubation at 37^ORC of the CF sputum "supernatant fluid" aliquots, followed by sequential sampling of aliquots of each sputum type for compound stability / metabolism characterization by HPLC analysis as described above. As before, analysis of compound disappearance, rates of new metabolite formation, and HPLC mobilities of new metabolites is then carried out.

Four. Pharmacological effects and mechanism of action of the drug in animals

The effect of compounds to enhance mucociliary clearance (EMC) can be measured using an in vivo model described by Sabater et al., Journal of Applied Physiology, 1999, pp. 2191-2196.

Examples

Having described this invention generally, a further understanding can be obtained by reference to some specific examples which are provided herein for the purpose of illustration only and are not intended to be limiting unless otherwise specified.

Preparation of sodium channel blockers

Materials and procedures. All reagents and solvents are obtained from Aldrich Chemical Corp. and used without further purification. NMR spectra were obtained on a Bruker WM 360 spectrometer (NMR¹360 MHz H and NMR ¹³C at 90 MHz) or a Bruker AC 300 (NMR of ¹H at 300 Mhz and NMR of ¹³C at 75 MHz). Flash chromatography was carried out on an Elution Solution Flash Elute ™ system (PO Box 5147, Charlottesville, Virginia 22905) loaded with a 90 g silica gel cartridge (40M FSO-0110-040155, 32-63mm) at 1.4 kg / cm² (N₂). GC analysis was carried out on a Shimadzu GC-17 equipped with a Heliflex capillary column (Alltech); Phase: AT-1, Length: 10 meters, ID: 0.53mm, Film: 0.25 microns. GC Parameters: 320OC Injector, 320OC Detector, FID Gas Flow: H₂ at 40 ml / min, Air at 400 ml / min. Vehicle gas: 16: 1 split ratio, N flow₂at 15 ml / min, N rate₂ at 18 cm / sec. The temperature program is 70OC for 0-3 min, 70-300OC for 3-10 min, 300OC for 10-15 min. HPLC analysis was carried out with a Gilson 322 pump, UV / Vis-156 detector at 360 nm, equipped with a C8 Microsorb MV, 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 20:80 B: A over 7 min, then 100% A over 1 min, followed by washing with 100% A for 11 min, flow rate: 1 ml / min.

Example 1

4- (4-Hydroxyphenyl) butylamidino-3,5-diamino-6-chloropyrazinecarboxamide hydrochloride (V)

The title compound was prepared as shown in Scheme 1 below. 4- (4-Hydroxyphenylbutyl) amine was prepared by routine organic transformations described in the following procedures. Coupling was done according to the procedure described by Cragoe, EJ Jr., Oltersdorf, OW Jr., and delSolms. SJ (1981) US Patent 4,246,406. The treatment and purification were modified according to the physical properties of compound V.

4-Methylphenylsulfonic acid (I) 4- (4-methoxyphenyl) butyl ester

Pyridine (15 ml) was added dropwise to a cooled solution (0^orC) 4- (4-methoxyphenyl) butane1 (10.0 g, 0.055 mol) and p-toluenesulfonyl chloride (13.6 g, 0.072 mol) in dry chloroform (100 ml) with stirring. The reaction mixture was stirred overnight at room temperature. After this, the reaction was quenched with 10% HCl (300 ml) and extracted with chloroform. The organic fraction was washed with saturated NaHCO3 solution, water and dried over magnesium sulfate. The solvent was removed under reduced pressure and the residue was purified by flash chromatography (eluent: hexane, ethyl acetate = 15: 1) affording 12.9 g (66%) of compound I as a clear oil. NMR¹H (360 MHZ, CDCl3) 1.61 (m, 4H), 2.44 (s, 3H), 2.52 (m, 2H), 3.78 (s, 3H), 4.05 (m, 2H ),

6.77 (d, J = 12.5 Hz, 2H), 7.05 (d, J = 12.5 Hz, 2H), 7.34 (d, J = 10.5 Hz, 2H), 7, 78 (d, J = 10.5 Hz, 2H).

ES 2 298 505 T3

Scheme 1

<img file="ES2298505T3_D0017.tif" />

4- (4-Methoxyphenyl) butylazide (II)

Sodium azide (3.07 g, 0.047 mol) was added to a solution of compound II (12.9 g, 0.04 mol) in anhydrous DMF (70 ml) and the reaction mixture was stirred 12 h at 80^orC (oil bath). Then the solvent was removed under reduced pressure and the residual oil was treated with a mixture of CH₂Cl₂: ether = 3: 1 (100 ml). The resulting solution was washed with water (2

ES 2 298 505 T3 x 100 ml), brine and dried over magnesium sulfate. The solvent was removed under reduced pressure and 7.6 g (95%) of compound II were obtained. The purity of compound II (99%) was determined by GC and TLC (eluent: hexane, ethyl acetate = 1: 1), R_F = 0,84.

4- (4-Methoxyphenyl) butylamine (III)

Lithium aluminum hydride (55 ml of a 1 M solution in THF, 0.055 mol) was added dropwise to a solution of compound II (7.6 g, 0.037 mol) in dry THF (70 ml) at 0 ° C and stirred overnight at room temperature under argon atmosphere. The reaction mixture was treated with water (1.5 ml), then 15% NaOH (1.5 ml), then with more water (3 ml) and filtered. The solid precipitate was washed with THF. The combined organic fractions were dried over magnesium sulfate and the solvent was removed under reduced pressure to give 6.2 g (94%) of compound III. The purity of compound III (99%) was determined by cG. RmN¹H (360 MHz, DMSO-d₆) 1.34 (m, 2H), 1.54 (m, 2H), 2.51 (m, 4H), 3.70 (s, 3H), 6.83 (d, J = 8.6 Hz, 2H), 7.08 (d, J = 8.3 Hz, 2H); ¹³C (90 MHz, DMSO-de) 28.6, 330, 34.1, 41.5, 54.8, 113.1, 129.1, 132.2, 157.3.

4- (4-Hydroxyphenyl) butylamine hydrobromide (IV)

Amine III (2.32 g, 0.012 mol) was stirred in boiling 48% HBr (50 ml) for 3 h. After completion of the reaction, argon was bubbled through the solution and the solvent was evaporated under reduced pressure. The solid residue was dried over KOH affording 3.1 g (90%) of compound IV. API MS m / z = 166 [C₁₀H₁₅NO + H]⁺.

4- (4-Hydroxyphenyl) butylamidino-3,5-diamino-6-chloropyrazinecarboxamide hydrochloride (V)

1- (3,5-diamino-6-chloropyrazinoyl-2-methyl-pseudothiourea hydroiodide (0.4 g, 1.03 mmol) was added to a suspension of 4- (4-hydroxyphenyl) butylamine hydrobromide (IV) in a mixture of THF (35 ml) and triethylamine (3 ml). The reaction mixture was stirred at reflux temperature for 3 h, then the supernatant was removed and the solvent was removed under reduced pressure. The oily residue was washed. with water (2 x 30 ml), ether (3 x 30 ml) and then 10% HCl (40 ml) was added. The mixture was vigorously stirred for 10 min and then the yellow solid was filtered, dried and recrystallized twice from ethanol to give 181 mg (41%) of compound V as a yellow solid. Purity is 98% by HPLC, retention time is 9.77 min; NMR¹H (300 MHz, DMSO-d6) 1.56 (s broad, 4H), 2.48 (s broad, 2H), 3.35 (m, 2H), 6.65 (d, J = 8.5 Hz , 2h), 6.95 (d, J = 8.6 Hz, 2H), 7.50 (s broad, 2H), 8.75 (s broad, 1H), 9.05 (s broad, 1H), 9.33 (s broad, 2H), 10.55 (s, 1H); NMR¹³C (75 MHz, CD3OD) 28.7, 29.8, 35.4.42.4, 111.2, 116.1, 122.0, 130.0, 134.0, 155.0, 156.1 , 156.8, 157.5, 167.0; APCIMS m / z = 378 [C16H₂₀C1N₇OR₂ + H]⁺.

Example 2

4- (4-Hydroxyphenyl) butylamidino-3,5-diamino-6-chloropyrazinecarboxamide hydrochloride

<img file="ES2298505T3_D0018.tif" />

4-Methylphenylsulfonic acid 4- (4-methoxyphenyl) butyl ester (1)

Pyridine (15 ml) was added dropwise to a cooled (OC) solution of 4- (4-methoxyphenyl1) butane1 (10.0 g, 0.055 mol) and p-toluenesulfonyl chloride (13.6 g, 0.072 mol) in dry chloroform (100 ml) with stirring. The reaction mixture was stirred overnight at room temperature. After this time, the reaction was quenched with 10% HCl (300 ml) and extracted with chloroform. The organic fraction was washed with saturated NaHCO solution₃, water and dried over magnesium sulfate. The solvent was removed under reduced pressure and the residue was purified by flash chromatography (eluent: hexane / ethyl acetate 15: 1) affording 12.9 g (66%) of compound 1 as a clear oil. NMR¹H (360 MHz, CDCI3) 1.61 (m, 4H), 2.44 (s, 3H), 2.52 (m, 2H), 3.78 (s, 3H), 4.05 (m, 2H ), 6.77 (d, 2H), 7.05 (d, 2H), 7.34 (d, 2H), 7.78 (d, 2H).

4- (4-Methoxyphenyl) butylazide (2)

A solution of sodium azide (3.07 g, 0.047 mol) was added to a solution of compound 1 (12.9 g, 0.04 mol) in anhydrous DMF (70 ml) and the reaction mixture was stirred 12 h at 80OC (oil bath). The solvent was removed under reduced pressure and the residual oil was treated with a mixture of CH₂C1₂/ ether 3: 1 (100 ml). The resulting solution was washed with water (2 x 100 ml), brine, and dried over magnesium sulfate. The solvent was removed under reduced pressure and 7.6 g (95%) of compound 2 were obtained. The purity of compound 2 (99%) was determined by GC and TLC (eluent: hexane / ethyl acetate 1: 1), R_F = 0,84.

ES 2 298 505 T3

4- (4-Methoxyphenyl) butylamine (3). Typical procedure A

Lithium aluminum hydride (LAH) (55 mL of a 1.0 M solution in THF, 0.055 mol) was added dropwise to a solution of compound 2 (7.6 g, 0.037 mol) in dry THF (70 ml ) at 0oC. The mixture was stirred overnight at room temperature under an argon atmosphere and then the mixture was treated with water (1.5 ml), then with 15% NaOH (1.5 ml), then with more water (3 ml ) and leaked. The solid precipitate was washed with THF. The combined organic fractions were dried over magnesium sulfate and the solvent was removed under reduced pressure to give 6.2 g (94%) of compound 3. The purity of compound 3 (99%) was determined by GC. NMR¹H (360 MHz, DMSO-de) 1.34 (m, 2H), 1.54 (m, 2H), 2.51 (m, 4H), 3.70 (s, 3H), 6.83 (d , 2H), 7.08 (d, 2H). ¹³C (90 MHz, DMSO-d₆) 28,6, 33,0, 34,1, 41,5, 54,8, 113,1, 129,1, 132,2, 157,3.

4- (4-hydroxyphenyl) butylamine hydrobromide (4). Typical procedure B

Amine 3 (2.32 g, 0.012 mol) was stirred in boiling 48% HBr (50 ml) for 3 h. After completion of the reaction, argon was bubbled through the solution and the solvent was evaporated under reduced pressure. The solid residue was dried over KOH affording 3.1 g (90%) of compound 4. APCI mS m / z = 166 [C₁₀H₁₅NO + H]⁺.

4- (4-Hydroxyphenyl) butylamidino-3,5-diamino-6-chloropyrazinecarboxamide hydrochloride (5)

1- (3,5-Diamino-6-chloropyrazinoyl-2-methyl-pseudothiourea hydroiodide (0.4 g, 1.03 mmol) was added to a suspension of 4- (4-hydroxyphenyl) butylamine hydrobromide (4) (0.8 g, 32 mmol) in a mixture of THF (35 ml) and triethylamine (3 ml) The reaction mixture was stirred in the boiling solvent for 3 h, then the supernatant was removed and the solvent was removed under reduced pressure The oily residue was washed with water (2 x 30 ml), ether (3 x 30 ml) and then 10% HCl (40 ml) was added. The mixture was stirred vigorously for 10 min and then the yellow solid was filtered, dried and recrystallized twice from ethanol to give compound 5 (0.18 g, 41%) as a yellow solid. Purity is 98% by HPLC, retention time is 9.77 min. NMR¹H (300 MHz, DMSO-d6) 1.56 (s broad, 4H), 2.48 (s broad, 2H), 3.35 (m, 2H), 6.65 (d, 2H), 6.95 (d, 2H), 7.50 (s broad, 2H), 8.75 (s broad, IH), 9.05 (s broad, IH), 9.33 (s broad, 2H), 10.55 ( Yes H). NMR¹³C (75 MHz, CD3OD) 28,7,29,8, 35,4,42,4,111,2,116,1,122,0,130.0, 134.0, 155.0, 156.1, 156.8, 157.5 , 167.0. APCI MS m / z = 378 [C16H20CIN7O2 + H]⁺.

Example 3

3- (4-Hydroxyphenyl) propylamidino-3,5-diamino-6-chloropyrazinecarboxamide hydrochloride

<img file="ES2298505T3_D0019.tif" />

Methanesulfonic acid 3- (4-methoxyphenyl) propyl ester (11). Typical procedure E

Pyridine (15 ml) was added dropwise to a cooled (0OC) solution of 4- (4-methoxyphenyl) propanol (10.0 g, 0.06 mol) and methanesulfonyl chloride (14.7 g, 0.078 mol) in dry THF (70 ml) with stirring. The reaction mixture was stirred overnight at room temperature. After this time, the solvent was removed under reduced pressure and the residue was quenched with 10% HCl (300 ml) and extracted with ethyl acetate. The organic fraction was washed with saturated NaHCO solution₃, water and dried over sodium sulfate. The solvent was removed and the residual crude ester 11 was used in the next step without further purification. Compound 11 was obtained as a yellow oil (8.8 g, 60%). NMR¹H (300 MHz, CDCI3) 2.08 (m, 2H), 2.60 (m, 2H), 2.98 (m, 2H) 3.98 (s, 3H), 3.66 (s, 3H) , 6.85 (d, 2H), 7.03 (d, 2H).

3- (4-Methoxyphenyl) propylazide (12)

Azide 12 was prepared according to procedure C from compound 11 (8.8 g, 0.036 mol) and sodium azide (3 g, 0.045 mol) in 75% yield. NMR¹H (300 MHz, CDC1₃) 1.90 (m, 2H), 2.65 (t, 2H), 3.28 (m, 2H), 3.80 (s, 3H), 6.85 (d, 2H), 7.10 ( d, 2H).

3- (4-Methoxyphenyl) propylamine (13)

Amine 13 was prepared as described in procedure A from azide 12 (5.2 g, 0.027 mol) and LAH (26 ml of 1 M solution in THF).

ES 2 298 505 T3

The crude compound 13 was purified by flash chromatography (silica gel, chloroform / ethanol / concentrated ammonium hydroxide 2: 1: 0.05) to provide pure amine 13 (3.2 g, 74%) as a clear oil. NMR¹H (300 MHz, CDCl₃) 1.58 (m, 2H), 2.50 (m, 4H), 3.72 (s, 3H), 6.85 (d, 2H), 7.10 (d, 2H).

3- (4-Hydroxyphenyl) propylamine hydrobromide (14)

Compound 14 was synthesized according to procedure B from compound 13 (2.5 g, 0.015 mol) in 75% yield as a light brown solid. NMR¹H (3θ0 MHz, DMS0-d₆) 1.80 (m, 2H), 2.53 (m, 2H),

2.78 (m, 2H), 6.70 (d, 2H), 7.02 (d, 2H), 7.80 (bs, 4H).

3- (4-Hydroxyphenyl) propylamidino-3,5-diamino-6-chloropyrazinecarboxamide hydrochloride (15)

Triethylamine (8 ml) was added to a suspension of compound 14 (0.470 g, 2 mmol) in THF (40 ml) and the mixture was stirred at room temperature for 15 min. After this time the 1- (3,5-diamino-6-chloropyrazinoyl-2-methyl-pseudothioureaiohydrate (0.15 g, 0.4 mmol) was added and the mixture was stirred under reflux for 3 h. The solution was it was then cooled to room temperature and the supernatant was isolated. The solvent was evaporated and the residual oil was washed with ether (2 x 50 ml), ethyl acetate (50 ml) and treated with 20 ml of 10% HCl. The solid obtained was isolated by filtration and dissolved in MeOH (approx. 50 ml). The addition of ethyl acetate (20 ml) to the solution caused the precipitation of a yellow solid, which was isolated by centrifugation, washed with ethyl acetate and dried in vacuo to give compound 15 (48 mg, 31%) in form of a yellow solid. NMR¹H (300 MHz, DMSO-d₆) 1.80 (s broad, 2H), 2.58 (m, 2H), 3.95 (s broad, 4H), 6.70 (d, 2H), 7.03 (d, 2H), 7, 48 (bs, 2H), 8.80 (bs, 1H), 8.93 (bs, 1H), 9.32 (bs, 2H), 10.52 (s, 1H). APCI MS m / z = 364 [C₁₅H₁₈ClN₇OR₂ + H]⁺.

Example 4

5- (4-Hydroxyphenyl) pentylamidino-3,5-diamino-6-chloropyrazinecarboxamide hydrochloride

<img file="ES2298505T3_D0020.tif" />

5- (4-Methoxyphenyl) pent-4-in-1-ol (16)

4-iodoanisole (10 g, 42 mmol), palladium (II) chloride (0.2 g, 1.1 mmol) and triphenylphosphine (0.6 g, 2.2 mmol) were dissolved in diethylamine (100 ml) and then copper (I) iodide (0.5 g, 2.2 mmol) and 4-pentin-1-ol (5 ml, 53 mmol) were added. The reaction mixture was stirred overnight at room temperature, and then the solvent was removed under reduced pressure. Ethyl acetate (150 ml) was added to the residue and the mixture was washed with 2N HCl, brine, and water. The organic fraction was isolated, dried over sodium sulfate, and the solvent was removed under reduced pressure. Product 16 (7.1 g, 87%) was isolated by flash chromatography (silica gel, 1: 2 ethyl acetate / hexanes) as a yellow oily solid. NMR¹H (300 MHz, CDCL) 1.88 (m, 2H), 2.53 (m, 2H), 3.72 (s, 3H), 3.74 (m, 2H), 6.83 (d, 2H ), 7.45 (d, 2H).

5- (4-Methoxyphenyl) pentan-1-ol (17)

A solution of compound 16 (7.1 g, 37 mmol) in dry ethanol (150 ml) was placed in a 0.5 liter Parr flask and palladium on charcoal (0.92 g, 5% wet, Pd / C) as a suspension in ethanol (25 ml). The reaction mixture was stirred at 3.5 kg / cm² of hydrogen pressure at room temperature for 24 hours. After this time, the mixture was filtered through a pad of silica gel and the solvent was removed under reduced pressure. The residue was purified by flash chromatography (silica gel, 1: 3 ethyl acetate / hexanes) to provide compound 17 (6.7 g, 92%) as a clear oil. NMR¹H (300 MHz, CDCl₃) 1.48 (m, 2H), 1.60 (m, 4H), 2.58 (m, 2H), 3.63 (m, 2H) 3.80 (s, 3H), 6.83 (d , 2H), 7.10 (d, 2H).

Methanesulfonic acid 4- (4-methoxyphenyl) pentyl ester (18)

Ester 18 was prepared following procedure E from alcohol 17 (6.7 g, 34.5 mmol) and methanesulfonyl chloride (4.5 mL, 50 mmol). The crude product 18 (9.0 g) was obtained as a brown oil and was used in the next step without purification.

5- (4-Methoxyphenyl) pentylazide (19)

Compound 19 was synthesized according to procedure C from crude product 18 (9.0 g) and sodium azide (2.7 g, 40 mmol). Azide 19 (6 g, 79% from compound 17) was isolated by flash chromatography, (gel

ES 2 298 505 T3 of silica, ethyl acetate / hexanes 1: 1). NMR¹H (300 MHz, CDC1₃) 1.40 (m, 2H), 1.62 (m, 4H), 2.56 (m, 2H), 3.35 (m, 2H) 3.80 (s, 3H), 6.85 (d , 2H), 7.10 (d, 2H).

5- (4-Methoxyphenyl) pentylamine (20)

Amine 20 was made following procedure A from compound 19 (6 g, 27 mmol) and LAH (26 ml of 1.0 M solution in THF) in 70% yield. NMR¹H (300 MHz, CDC1₃) 1.35 (m, 2H), 1.48 (m, 2H) 1.61 (m, 2H), 2.55 (m, 2H), 2.70 (m, 2H) 3.80 (s, 3H), 6.85 (d, 2H), 7.10 (d, 2H).

5- (4-Hydroxyphenyl) pentylamine (21)

The HBr salt of compound 21 was prepared according to procedure B from amine 20 (2.8 g, 14 mmol). Free amine 21 (2 g, 80%) was obtained after flash chromatography (silica gel, chloroform / ethanol / concentrated ammonium hydroxide 6: 3: 0.1) as a cloudy oil. NMR¹H (300 MHz, DMSO-d₆) 1.28 (m, 2H), 1.55 (m, 2H), 1.61 (m, 2H), 2.48 (m, 2H), 2.58 (m, 2H), 6.68 ( d, 2H), 6.98 (d, 2H).

5- (4-Hydroxyphenyl) pentylamidino-3,5-diamino-6-chloropyrazinecarboxamide hydrochloride (22)

1- (3,5-Diamino-6-chloropyrazinoi1-2-methi1-pseudothiourea hydrochloride (0.25 g, 0.65 mmol) was added to a solution of compound 21 (0.6 g, 3.4 mmol ) in THF (5θ ml) The reaction mixture was stirred under reflux for 2 h, then the solvent was removed under reduced pressure and the resulting oil was washed with ether (2 x 50 ml) and treated with ethyl acetate until that a yellow powder was formed. The yellow solid was dissolved in methanol (70 ml) and the volume was slowly reduced until precipitation started (approximately 25 ml). The solution was cooled to 0 ° C and the precipitate was collected by centrifugation. Dilute HCl (20 ml of a 10% solution) was added and the mixture was stirred vigorously for 20 min, and then the precipitate was filtered, washed with cold water and dried to give compound 22 (183 mg, 39%) in the form of a yellow solid. NMR¹H (300 MHz, DMSO-d₆) 1.32 (s broad, 2H), 1.55 (m, 4H), 2.45 (m, 2H), 3.29 (m, 2H), 6.68 (d, 2H), 6.97 (d, 2H), 7.46 (s, 1H), 8.00 (s broad, 1H), 8.83 (s broad, 1H), 8.97 (s broad, 1H), 9.46 (d , 2H), 10.55 (s, 1H). APCI MS m / z = 392 [C17H22CIN7O2 + H]⁺.

Example 5

4- (3,4-Dihydroxyphenyl) butylamidino-3,5-diamino-6-chloropyrazinecarboxamide hydrochloride

<img file="ES2298505T3_D0021.tif" />

4- (3,4-Dimethoxyphenyl) butanol (23)

4- (3,4-Dimethoxyphenyl) butyric acid (13 g, 58 mmol) was dissolved in dry THF (150 ml) and then BH was added dropwise₃THF (110 ml, 1 M solution, 110 mmol) with vigorous stirring under argon atmosphere. The reaction mixture was then stirred overnight at room temperature. After this time, the reaction was quenched with water and 10% HCl solution at 0OC and extracted with ethyl acetate. The organic fraction was dried over sodium sulfate and passed through a pad of silica gel. The solvent was removed under reduced pressure to give compound 23 (12.0 g, 99%) as a clear oil. NMR¹H (300 MHz, CDC1₃) 1.62 (m, 4H), 2.0 (s, 1H), 2.62 (m, 2H), 3.65 (m, 2H), 3.82 (s, 3H), 3.84 ( s, 3H) 6.67-6.82 (m, 3H).

Methanesulfonic acid 4- (3,4-dimethoxyphenyl) butyl ester (24)

Ester 24 was prepared following procedure E from alcohol 23 (12.0 g, 57 mmol) and methanesulfonyl chloride (8.4 g, 74 mmol) in 78% yield. NMR¹H (300 MHz, CDC1₃) 1.65 (m, 4H), 2.62 (m, 2H), 3.05 (s, 3H), 3.88 (s broad, 6H), 4.38 (m, 2H) 6.70- 6.88 (m, 3H).

4- (3,4-Dimethoxyphenyl) butyl-azide (25)

Compound 25 was synthesized according to procedure C from compound 24 (14.1 g, 51 mmol) and sodium azide (4.0 g, 66 mmol) in 96% yield. NMR¹H (300 MHz, CDC1₃) 1.65 (m, 4H), 2.60 (m, 2H), 3.30 (m, 2H), 3.86 (bs, 6H), 6.70 (m, 2H), 6.78 (m, 1H).

ES 2 298 505 T3

4- (3,4-Dimethoxyphenyl) butylamine (26)

Amine 26 was prepared as described in procedure A from azide 25 (11.0 g, 49 mol) and LAH (26 ml of 1 M solution in THF). The crude compound 26 was purified by flash chromatography (silica gel, chloroform / ethanol / concentrated ammonium hydroxide 93: 7: 1) to give pure compound 26 (4.8 g, 42%) as a clear oil. NMR¹H (300 MHz, CDCl₃) 1.42 (m, 2H), 1.60 (m, 2H), 2.55 (m, 2h), 2.74 (m, 2H), 3.82 (s, 6H), 3.84 ( s, 3H), 6.70 (m, 2H), 6.78 (m, 1H).

4- (3,4-Dihydroxyphenyl) butylamine hydrobromide (27)

Compound 27 was synthesized according to procedure B from compound 26 (2.5 g, 11 mmol) in 62% yield as a pink solid. NMR¹H (300 MHz, DMSO-d₆) 1.52 (s broad, 4H), 2.40 (m, 2H),

2.78 (m, 2H), 6.42 (m, 1H), 6.60 (m, 2H), 7.80 (bs, 4H).

4- (3,4-Dihydroxyphenyl) butylamidino-3,5-diamino-6-chloropyrazinecarboxamide hydrochloride (28)

1- (3,5-diamino-6-chloropyrazinoi1-2-methi1-pseudothiourea) hydroiodide (0.2 g, 0.51 mmol) was added to a suspension of compound 27 in a mixture of THF (35 ml) and triethylamine (3 ml). The reaction was stirred under reflux for 3 h, then the supernatant was removed and the solvent was removed under reduced pressure. The brown residue was washed with ether (2 x 30 ml) followed by the addition of HCl 10% (5 ml). The solid material was collected, dissolved in methanol, and precipitated by the addition of ethyl acetate. The precipitate was washed with 10% HCl and dried to give compound 28 (131 mg, 51%) as a beige solid. NMR¹H (300 MHz, DMSO-d₆) 1.52 (s broad, 4H), 2.42 (m, 2H), 3.31 (m, 2H), 6.43 (m, 1H), 6.61 (m, 2H), 7.42 (s broad, 2H), 7.90 (s broad, 1h), 8.82 (s broad, 1H), 8.98 (s broad, 1H), 9.25 (s, 1H) 10.52 (s , 1 HOUR). APCI MS m / z = 394 [C16H20ClN7O₃ + H]⁺.

Example 6

4- (4-Hydroxyphenyl) -4-oxabutylamidino-3,5-diamino-6-chloropyrazinecarboxamide hydrobromide

<img file="ES2298505T3_D0022.tif" />

4- (4-Hydroxyphenyl) -4-oxabutylamidino-3,5-diamino-6-chloropyrazine-carboxamide hydrobromide (63)

The vigorously stirred solution of compound 62 (80 mg, 0.19 mmol) in 48% HBr (15 ml) was heated under reflux for 2 h and then cooled. The precipitate that formed was separated, washed with water, and dried overnight to provide compound 63 (52 mg, 52%). NMR¹H (300 MHz, DMSO-d₆) 1.99 (m, 2H), 3.96 (m, 2H), 6.77 (m, 2H), 6.79 (m, 2H), 7.45 (s, 2H), 8.74 ( s broad, 1H), 8.87 (s broad, 1H), 9.30 (s, 1H), 10.48 (s, 1H). APCI MS m / z 380 [C₁₅H₁₈ClN₇OR₃ + H]⁺.

Example 7

4- (2,4-Dihydroxyphenyl) butylamidino-3,5-diamino-6-chloropyrazinecarboxamide hydrochloride

<img file="ES2298505T3_D0023.tif" />

4- (2,4-Dimethoxyphenyl) -but-3-in-1-ol (75)

1-Bromo-2,4-dimethoxybenzene (10 g 0.046 mol), palladium chloride (0.2 g 0.11 mmol) and triphenylphosphine (0.6 g 0.0023 mol) were dissolved in diethylamine (100 ml) in nitrogen atmosphere. Copper (I) iodide (0.44 g,

ES 2 298 505 T3

0.0023 mol) and 3-butyn-l-ol (7 ml, 0.092 mol) to the reaction mixture at once. The mixture was stirred overnight at 55 ° C under a nitrogen atmosphere. The catalyst was filtered from the reaction mixture and the same amounts of palladium chloride, triphenylphosphine, copper (I) iodide, and 3-butyn-l-ol were added. The reaction mixture was stirred and heated at 85 ° C for 48 hours. Then the solvent was removed under reduced pressure and water (approximately 100 ml) was added to the residue. The mixture was extracted with ethyl acetate (350 ml), passed through a pad of silica gel, and concentrated. The product was purified by flash chromatography (silica gel, hexanes / ethyl acetate 1: 1). Compound 75 (4.2 g, 24%) was isolated as a brown oil. NMR '11 (300 MHz, CDC1₃) 2.24 (t, 1H), 2.73 (t, 2H), 3.80 (s broad, 5H), 3.87 (s, 3H), 6.43 (m, 2H), 7.30 (m, 1H).

4- (2,4-Dimethoxyphenyl) -butan-1-ol (76)

To a solution of compound 75 (4.2 g, 0.022 mol) in ethanol (approximately 200 ml) was added palladium (5% wet on activated carbon, 1 g). The mixture was then hydrogenated at 2.8 kg / cm² overnight at room temperature. The mixture was filtered through a pad of silica gel and the solvent was evaporated to give compound 76 (4.15 g, 97%) as a yellow oil. NMR 'H (300 MHz, CDC1₃) 1.59 (m, 4H), 2.55 (m, 2H), 3.79 (s, 6H), 6.43 (m, 2H), 7.00 (m, 1H).

Methanesulfonic acid 4- (2,4-dimethoxyphenyl) butyl ester (77)

Ester 77 was prepared by typical procedure E from alcohol 76 (4.15 g, 0.021 mol), methanesulfonyl chloride (2.4 ml, 0.03 mol) and triethylamine (20 ml). Crude compound 77 (4.6 g, 80%) was isolated as a yellow oil.

4- (2,4-Dimethoxyphenyl) butyl-azide (78)

Azide 78 was prepared by typical procedure C from ester 77 (4.6 g, 0.015 mol) and sodium azide (1.5 g, 0.023 mol). Compound 77 (4.06 g, 75%) was isolated as a yellow oil. NMR¹H (3θθ MHz, CDCl₃) 1.62 (m, 4H), 2.58 (m, 2H), 3.30 (m, 2H), 3.80 (s, 6H), 6.43 (m, 2H), 7.00 ( m, 1H).

4- (2,4-Dimethoxyphenyl) butylamine (79)

Amine 79 was prepared by typical procedure A from azide 78 (4.06 g, 0.017 mol) and LiAlH₄ (13 ml of a 1.0 M solution in THF). The material was purified by column chromatography (silica gel, chloroform / ethanol / concentrated ammonium hydroxide 2: 1: 0.1) to provide compound 79 (2.3 g, 64%) as a white solid. NMR (300 MHz, CDCl₃) 1.53 (m, 4H), 2.53 (m, 2H), 2.73 (m, 2H), 3.80 (s, 6H), 6.43 (m, 2H), 7.52 ( m, 1H).

4- (2,4-Dimethoxyphenyl) butylamidino-3,5-diamino-6-chloropyrazine-carboxamide hydrochloride (80)

1- (3,5-Diamino-6-chloropyrazinoyl-2-methyl-pseudothiourea iohydrate (0.3 g, 0.77 mmol) was added to a solution in anhydrous THF (30 ml) of compound 79 (0.4 g, 1.9 mmol). The reaction mixture was stirred at reflux temperature for 3 h and then the solvent was evaporated. The residue was washed with ethyl acetate (2 x 20 ml) and then treated with 3 HCl. % (15 ml). The yellow solid that formed was separated, washed with water and dried overnight to provide compound 80 (0.32 g, 90%). 'H NMR (300 MHz, DMSO-O6) 1.57 (s, 4H), 2.50 (s broad, 2H), 3.35 (s broad, 2H), 3.73 (s, 3H), 3 , 76 (s, 3H), 6.43 (m, 1H), 6.52 (s, 1H), 7.02 (m, 1H), 7.45 (s broad, 2H), 8.86 (s broad, 1H), 8.99 (s, 1H), 9.03 (m, 1H), 10.56 (s, 1H). APCI MS m / z 422 [Ci₈H24ClN₇O3 + H]⁺.

4- (2,4-Dihydroxyphenyl) butylamidino-3,5-diamino-6-chloropyrazine-carboxamide hydrochloride (81)

A vigorously stirred solution of compound 80 (290 mg, 0.63 mmol) in 48% HBr (20 ml) was refluxed for 4 h and then cooled. The solvent was removed under reduced pressure and the material was purified by column chromatography (silica gel, chloroform / ethanol / concentrated ammonium hydroxide 4: 1: 0.1). The product fractions were collected and the solvent was removed under reduced pressure. The residue was treated with 3% HCl, washed with water (2 x 5 ml) and dried to provide compound 81 (79 mg, 32%) as a yellow solid. NMR¹H (300 MHz, DMSO-O6) 1.54 (s, 4H), 2.43 (s broad, 2H), 3.31 (s broad, 2H), 6.12 (d, 1H), 6.32 (s, 1H), 6.78 (d, 1H), 8.86 (s, 1H), 8.99 (s, 1H), 9.28 (s, 1H), 10.56 (s, 1 HOUR). APCI MS m / z 394 [C ^ oClN ^ + H]⁺.

References

1. Tailor, EC; Harrington, PM; Schin, C. Heterocycles, 1989, 28, 1169, incorporated herein by reference.

2. Widsheis et al, Synthesis, 1994, 87-92, incorporated herein by reference.

ES 2 298 505 T3

Example 8

Sodium channel blocking activity

The compounds shown in the following Tables 1-5 were tested for potency in canine bronchial epithelia using the in vitro assay described above. In this assay amiloride was tested as a positive control. The results for the compounds of the present invention are reported as values ​​of number of times of enhancement relative to amiloride.

TABLE 1

<img file="ES2298505T3_D0024.tif" />

TABLE 2

<img file="ES2298505T3_D0025.tif" />

ES 2 298 505 T3

<img file="ES2298505T3_D0026.tif" />

Example 9

Effect of 4- (4-hydroxyphenyl) -butylamidino-3,5-diamino-6-chloropyrazinecarboxamide (V) hydrochloride on EMC

This experiment was carried out with 4- (4-hydroxyphenyl) butylamidino-3,5-diamino-6-chloro-pyrazinecarboxamide hydrochloride (V) and vehicle as control. The results are shown in Figures 1 and 2.

Procedures

Animal Preparation: The Mount Sinai Animal Research Committee approved all procedures for the in vivo evaluation of mucociliary clearance. Adult female sheep (in the weight range 25 to 35 kg) are⁵⁰ kept in an upright position in a special harness adapted to a modified shopping cart. The heads of the animals were immobilized and local anesthesia of the nasal passage was induced with 2% lidocaine. The animals were then intubated through the nose with a 7.5 mm internal diameter endotracheal tube (ETT). 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 before starting the measurements of ⁵⁵ mucociliary elimination.

Administration of radioaerosol: Human serum albumin aerosols containing ^99mTc (3.1 mg / ml; containing approximately 20 mCi) were generated using a Raindrop nebulizer that produces a droplet with a mean aerodynamic diameter of 3.6 pm. The nebulizer was connected with a dosimetric system consisting of a valve⁶⁰ solenoid and a source of compressed air (1.4 kg / cm²). The nebulizer outlet went to a plastic T-connector; one end was connected to the endotracheal tube and the other end was connected to a piston respirator. The system was activated for one second at the start of the respirator inspiration cycle. The ventilator was set to a total breath volume of 500 ml, an inspiration-to-expiration ratio of 1: 1, and a rate of 2θ breaths per minute to maximize deposition in the central airway. The sheep breathed the radiolabeled aerosol for ⁶⁵ 5 min. A gamma camera was used to measure the removal of albumin from human serum with^99mTc of the airways. The camera was placed on top of the animal's back with the sheep in a natural upright position held on a cart so that the image field was perpendicular to the animal's spinal cord. They got

ES 2 298 505 T3 external radiolabeled markers in sheep to ensure proper alignment under gamma camera. All images were stored on a computer integrated with the gamma camera. A region of interest was drawn on the image corresponding to the sheep's right lung and the counts were recorded. The counts were corrected for decay and expressed as the percentage of radioactivity present in the baseline image. The left lung was excluded from analysis because its outline overlaps with the stomach and counts may be radiolabeled mucus swallowed.

Treatment protocol (Evaluation of the activity at zero t): A deposition image of the initial value was obtained immediately after the administration of the radioaerosol. At time zero, after acquiring the baseline image, the vehicle control (distilled water), the positive control (amiloride) or the experimental compounds were aerosolized from a volume of 4 ml using a Pari LC JetPlus nebulizer. , in freely breathing animals. The nebulizer was operated with 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 delivery of the full dose in order to prevent spurious increases in counts caused by aspiration of excess radiotracer from ETT. Serial images of the lung were obtained at 15 minute intervals for the first 2 hours after dosing and every hour for the next 6 hours after dosing for a total observation period of 8 hours. A washout period of at least 7 days separated the dosing sessions with different experimental agents.

Treatment protocol (Activity evaluation at t-4 hours): The following variation of the standard protocol was used to evaluate 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 research compounds from a volume of 4 ml were aerolyzed using a Pari LC JetPlus nebulizer, onto the free-breathing animals. The nebulizer was operated with 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 kept in an upright position in a special body harness for 4 hours. At the end of the 4 hour period the animals received a single aerolized dose of human serum albumin with ^99mTc (3.1 mg / ml; containing approximately 20 mCi) from a Raindrop nebulizer. Animals were extubated immediately after giving the full dose of radiotracer. A baseline deposition image was obtained immediately after administration of the radioaerosol. Serial images of the lung were obtained at 15 minute intervals during the first 2 hours after radiotracer administration (representing hours 4 to 6 after drug administration) and every hour for the next 2 hours after dosing during a total observation period of 4 hours. A washout period of at least 7 days separated the dosing sessions with different experimental agents.

Statistics: Data were analyzed using SYSTAT for Windows version 5. Data were analyzed using a repeated two-factor ANOVA (to assess effects), followed by a paired data t-test to identify differences between specific pairs. Significance was accepted when P was less than or equal to 0.05. Slope values ​​(calculated from data collected during the initial 45 minutes after dosing in the at-zero evaluation) for the mean NDE curves were calculated using least squares linear regression to evaluate differences in initial velocities during the rapid elimination phase.