# Patent

## Title
Hetero substituted sodium channel blockers

## Abstract

The present invention relates to group of butylphenyl-pyrazinoylguanidine compounds useful as sodium channel blockers. The compounds may be used to promote promoting hydration of mucosal surfaces.

## Background
CONTINUING APPLICATION DATA
This application is a National Stage of international application No. PCT/US04/04451, filed on Feb. 18, 2004.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to sodium channel blockers. The present invention also includes a variety of methods of treatment using these inventive sodium channel blockers.
2. Description of the Background
The mucosal surfaces at the interface between the environment and the body have evolved a number of “innate defense”, i.e., protective mechanisms. A principal form of such innate defense is to cleanse these surfaces with liquid. Typically, the quantity of the liquid layer on a mucosal surface reflects the balance between epithelial liquid secretion, often reflecting anion (Cl− and/or HCO3−) secretion coupled with water (and a cation counter-ion), and epithelial liquid absorption, often reflecting Na+ absorption, coupled with water and counter anion (Cl− and/or HCO3−). Many diseases of mucosal surfaces are caused by too little protective liquid on those mucosal surfaces created by an imbalance between secretion (too little) and absorption (relatively too much). The defective salt transport processes that characterize these mucosal dysfunctions reside in the epithelial layer of the mucosal surface.
One approach to replenish the protective liquid layer on mucosal surfaces is to “re-balance” the system by blocking Na+ channel and liquid absorption. The epithelial protein that mediates the rate-limiting step of Na+ and liquid absorption is the epithelial Na+ channel (ENaC). ENaC is positioned on the apical surface of the epithelium, i.e. the mucosal surface-environmental interface. Therefore, to inhibit ENaC mediated Na+ and liquid absorption, an ENaC blocker of the amiloride class (which blocks from the extracellular domain of ENaC) must be delivered to the mucosal surface and, importantly, be maintained at this site, to achieve therapeutic utility. The present invention describes diseases characterized by too little liquid on mucosal surfaces and “topical” sodium channel blockers designed to exhibit the increased potency, reduced mucosal absorption, and slow dissociation (“unbinding” or detachment) from ENaC required for therapy of these diseases.
Chronic bronchitis (CB), including the most common lethal genetic form of chronic bronchitis, cystic fibrosis (CF), are diseases that reflect the body's failure to clear mucus normally from the lungs, which ultimately produces chronic airways infection. In the normal lung, the primary defense against chronic intrapulmonary airways infection (chronic bronchitis) is mediated by the continuous clearance of mucus from bronchial airway surfaces. This function in health effectively removes from the lung potentially noxious toxins and pathogens. Recent data indicate that the initiating problem, i.e., the “basic defect,” in both CB and CF is the failure to clear mucus from airway surfaces. The failure to clear mucus reflects an imbalance between the amount of liquid and mucin on airway surfaces. This “airway surface liquid” (ASL) is primarily composed of salt and water in proportions similar to plasma (i.e., isotonic). Mucin macromolecules organize into a well defined “mucus layer” which normally traps inhaled bacteria and is transported out of the lung via the actions of cilia which beat in a watery, low viscosity solution termed the “periciliary liquid” (PCL). In the disease state, there is an imbalance in the quantities of mucus as ASL on airway surfaces. This results in a relative reduction in ASL which leads to mucus concentration, reduction in the lubricant activity of the PCL, and a failure to clear mucus via ciliary activity to the mouth. The reduction in mechanical clearance of mucus from the lung leads to chronic bacterial colonization of mucus adherent to airway surfaces. It is the chronic retention of bacteria, the failure of local antimicrobial substances to kill mucus-entrapped bacteria on a chronic basis, and the consequent chronic inflammatory responses of the body to this type of surface infection, that lead to the syndromes of CB and CF.
The current afflicted population in the U.S. is 12,000,000 patients with the acquired (primarily from cigarette smoke exposure) form of chronic bronchitis and approximately 30,000 patients with the genetic form, cystic fibrosis. Approximately equal numbers of both populations are present in Europe. In Asia, there is little CF but the incidence of CB is high and, like the rest of the world, is increasing.
There is currently a large, unmet medical need for products that specifically treat CB and CF at the level of the basic defect that cause these diseases. The current therapies for chronic bronchitis and cystic fibrosis focus on treating the symptoms and/or the late effects of these diseases. Thus, for chronic bronchitis, β-agonists, inhaled steroids, anti-cholinergic agents, and oral theophyllines and phosphodiesterase inhibitors are all in development. However, none of these drugs treat effectively the fundamental problem of the failure to clear mucus from the lung. Similarly, in cystic fibrosis, the same spectrum of pharmacologic agents is used. These strategies have been complemented by more recent strategies designed to clear the CF lung of the DNA (“Pulmozyme”; Genentech) that has been deposited in the lung by neutrophils that have futilely attempted to kill the bacteria that grow in adherent mucus masses and through the use of inhaled antibiotics (“TOBI”) designed to augment the lungs' own killing mechanisms to rid the adherent mucus plaques of bacteria. A general principle of the body is that if the initiating lesion is not treated, in this case mucus retention/obstruction, bacterial infections became chronic and increasingly refractory to antimicrobial therapy. Thus, a major unmet therapeutic need for both CB and CF lung diseases is an effective means of re-hydrating airway mucus (i.e., restoring/expanding the volume of the ASL) and promoting its clearance, with bacteria, from the lung.
R. C. Boucher, in U.S. Pat. No. 6,264,975, describes the use of pyrazinoylguanidine sodium channel blockers for hydrating mucosal surfaces. These compounds, typified by the well-known diuretics amiloride, benzamil, and phenamil, are effective. However, these compounds suffer from the significant disadvantage that they are (1) relatively impotent, which is important because the mass of drug that can be inhaled by the lung is limited; (2) rapidly absorbed, which limits the half-life of the drug on the mucosal surface; and (3) are freely dissociable from ENaC. The sum of these disadvantages embodied in these well known diurectics produces compounds with insufficient potency and/or effective half-life on mucosal surfaces to have therapeutic benefit for hydrating mucosal surfaces.
Clearly, what is needed are drugs that are more effective at restoring the clearance of mucus from the lungs of patients with CB/CF. The value of these new therapies will be reflected in improvements in the quality and duration of life for both the CF and the CB populations.
Other mucosal surfaces in and on the body exhibit subtle differences in the normal physiology of the protective surface liquids on their surfaces but the pathophysiology of disease reflects a common theme, i.e., too little protective surface liquid. For example, in xerostomia (dry mouth) the oral cavity is depleted of liquid due to a failure of the parotid sublingual and submandibular glands to secrete liquid despite continued Na+ (ENaC) transport mediated liquid absorption from the oral cavity. Similarly, keratoconjunctivitis sira (dry eye) is caused by failure of lacrimal glands to secrete liquid in the face of continued Na+ dependent liquid absorption on conjunctional surfaces. In rhinosinusitis, there is an imbalance, as in CB, between mucin secretion and relative ASL depletion. Finally, in the gastrointestinal tract, failure to secrete Cl− (and liquid) in the proximal small intestine, combined with increased Na+ (and liquid) absorption in the terminal ileum leads to the distal intestinal obstruction syndrome (DIOS). In older patients excessive Na+ (and volume) absorption in the descending colon produces constipation and diverticulitis.
Fifty million Americans and, hundreds of millions of others around the world suffer from high blood pressure and the subsequent sequale leading to congestive heart failure and increasing mortality. It is the Western World's leading killer and there is a need there for new medicines to treat these diseases. Thus, in addition, some of the novel sodium channel blockers of this invention can be designed to target the kidney and as such they may be used as diuretics for the treatment of hypertension, congestive heart failure (CHF) and other cardiovascular diseases. These new agents may be used alone or in combination with beta-blockers, ACE inhibitors, HMGCoA reductase inhibitors, calcium channel blockers and other cardiovascular agents.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide compounds that are more potent and/or absorbed less rapidly from mucosal surfaces, and/or are less reversible as compared to known compounds.
It is another aspect of the present invention to provide compounds that are more potent and/or absorbed less rapidly and/or exhibit less reversibility, as compared to compounds such as amilorde, benzamil, and phenamil. Therefore, the compounds will give a prolonged pharmacodynamic half-life on mucosal surfaces as compared to known compounds.
It is another object of the present invention to provide compounds which are (1) absorbed less rapidly from mucosal surfaces, especially airway surfaces, as compared to known compounds and; (2) when absorbed from musosal surfaces after administration to the mucosal surfaces, are converted in vivo into metabolic derivatives thereof which have reduced efficacy in blocking sodium channels as compared to the administered parent compound.
It is another object of the present invention to provide compounds that are more potent and/or absorbed less rapidly and/or exhibit less reversibility, as compared to compounds such as amiloride, benzamil, and phenamil. Therefore, such compounds will give a prolonged pharmacodynamic half-life on mucosal surfaces as compared to previous compounds.
It is another object of the present invention to provide compounds that target the kidney for use in the treatment of cardiovascular disease.
It is another object of the present invention to provide methods of treatment that take advantage of the pharmacological properties of the compounds described above.
In particular, it is an object of the present invention to provide methods of treatment which rely on rehydration of mucosal surfaces.
In particular, it is an object of the present invention to provide methods of treating cardiovascular disease.
The objects of the present invention may be accomplished with a class of pyrazinoylguanidine compounds represented by formula (I):




where

X is hydrogen, halogen, trifluoromethyl, lower alkyl, unsubstituted or substituted phenyl, lower alkyl-thio, phenyl-lower alkyl-thio, lower alkyl-sulfonyl, or phenyl-lower alkyl-sulfonyl;
Y is hydrogen, hydroxyl, mercapto, lower alkoxy, lower alkyl-thio, halogen, lower alkyl, unsubstituted or substituted mononuclear aryl, or —N(R2)2;
R1 is hydrogen or lower alkyl;
each R2 is, independently, —R7, —(CH2)m—OR8, —(CH2)m—NR7R10, —(CH2)n(CHOR8)(CHOR8)n—CH2OR8, —(CH2CH2O)m—R8, (CH2CH2O)m—CH2CH2NR7R10, —(CH2)n—C(═O)NR7R10, —(CH2)n—Zg—R7, —(CH2)m—NR10—CH2(CHOR8)(CHOR8)n—CH2OR8, —(CH2)n—CO2R7, or




where when two —CH2OR8 groups are located 1,2- or 1,3- with respect to each other the R8 groups may be joined to form a cyclic mono- or di-substituted 1,3-dioxane or 1,3-dioxolane;
R3 and R4 are each, independently, hydrogen, a group represented by formula (A), lower alkyl, hydroxy lower alkyl, phenyl, phenyl-lower alkyl, (halophenyl)-lower alkyl, lower-(alkylphenylalkyl), lower (alkoxyphenyl)-lower alkyl, naphthyl-lower alkyl, or pyridyl-lower alkyl, with the proviso that at least one of R3 and R4 is a group represented by formula (A):




where

each RL is, independently, —R7, —(CH2)n—OR8, —O—(CH2)m—OR8, —(CH2)n—NR7R10, —O—(CH2)m—NR7R10, —(CH2)n(CHOR8)(CHOR8), —CH2OR8, —O—(CH2)m(CHOR8)(CHOR8)n—CH2OR8, —(CH2CH2O)m—R8, —O—(CH2CH2O)m—R8, —(CH2CH2O)m—CH2CH2NR7R10, —O—(CH2CH2O)m—CH2CH2NR7R10, —(CH2), —C(═O)NR7R10, —O—(CH2)m—C(═O)NR7R10, —(CH2)n—(Z)g—R7, —O—(CH2)m—(Z)g—R7, —(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,




where when two —CH2OR8 groups are located 1,2- or 1,3- with respect to each other the R8 groups may be joined to form a cyclic mono- or di-substituted 1,3-dioxane or 1,3-dioxolane;

    
    
        each o is, independently, an integer from 0 to 10;
        each p is an integer from 0 to 10;
        with the proviso that the sum of o and p in each contiguous chain is from 1 to 10;
        each x is, independently, O, NR10, C(═O), CHOH, C(═N—R10),
        CHNR7R10, or represents a single bond;
        each R5 is, independently, —(CH2)n—NR12R12, —O—(CH2)m—NR12R12, —O—(CH2)n—NR12R12, —O—(CH2)m—(Z)gR12, —(CH2)nNR11R11, —O—(CH2)mNR11R11, —(CH2)n—N⊕—(R11)3, —O—(CH2)m—N⊕—(R11)3, —(CH2)n—(Z)g—(CH2)m—NR10R10, —O—(CH2)m—(Z)g—(CH2)m—NR10R10, —(CH2CH2O)m—CH2CH2NR12R12, O—(CH2CH2O)m—CH2CH2NR12R12, —(CH2)n—(C═O)NR12R12, —O—(CH2)m—(C═O)NR12R12, —O—(CH2)m—(CHOR8)mCH2NR10—(Z)g—R10, —(CH2)n—(CHOR8)mCH2—NR10—(Z)g—R10, —(CH2)nNR10—O(CH2)m(CHOR8)nCH2NR10—(Z)g—R10, —O(CH2)m—NR10—(CH2)m—(CHOR8)nCH2NR10—(Z)g—R10, -(Het)-(CH2)m—OR8, -(Het)-(CH2)m—NR7R10, -(Het)-(CH2)m(CHOR8)(CHOR8)n—CH2OR8, -(Het)-(CH2CH2O)m—R8, -(Het)-(CH2CH2O)m—CH2CH2NR7R10, -(Het)-(CH2)m—C(═O)NR7R10, -(Het)-(CH2)m—(Z)g—R7, -(Het)-(CH2)m—NR10—CH2(CHOR8)(CHOR8)n—CH2OR8, -(Het)-(CH2)m—CO2R7, -(Het)-(CH2)m—NR12R12, -(Het)-(CH2)n—NR12R12, -(Het)-(CH2)m—(Z)gR12, -(Het)-(CH2)mNR11R11, -(Het)-(CH2)m—N⊕—(R11)3, -(Het)-(CH2)m—(Z)g—(CH2)m—NR10R10, -(Het)-(CH2CH2O)m—CH2CH2NR12R12, -(Het)-(CH2)m—(C═O)NR12R12, -(Het)-(CH2)m—(CHOR8)mCH2NR10—(Z)g—R10, -(Het)-(CH2)m—NR10—(CH2)m—(CHOR8)nCH2NR10—(Z)g—R10, where when two —CH2OR8 groups are located 1,2-or 1,3-with respect to each other the R8 groups may be joined to form a cyclic mono-or di-substituted 1,3-dioxane or 1,3-dioxolane,
        —(CH2)n(CHOR8)(CHOR8), —CH2OR8, with the proviso that two —CH2OR8 groups are located 1,2-or 1,3-with respect to each other and the R8 groups are joined to form a cyclic mono or disubstituted 1,3-dioxane or 1,3-dioxolane,
        —O—(CH2)m(CHOR8)(CHOR8)n—CH2OR8, with the proviso that two —CH2OR8 groups are located 1,2-or 1,3-with respect to each other and the R8 groups are joined to form a cyclic mono or disubstituted 1,3-dioxane or 1,3-dioxolane,
        —(CH2)n—NR10—CH2(CHOR8)(CHOR8)n—CH2OR8, with the proviso that two —CH2OR8 groups are located 1,2-or 1,3-with respect to each other and the R8 groups are joined to form a cyclic mono or disubstituted 1,3-dioxane or 1,3-dioxolane, or
        —O—(CH2)m—NR10—CH2(CHOR8)(CHOR8)n—CH2OR8, with the proviso that two —CH2OR8 groups are located 1,2-or 1,3-with respect to each other and the R8 groups are joined to form a cyclic mono or disubstituted 1,3-dioxane or 1,3-dioxolane;
        each R6 is, independently, —R5, —R7, —OR8, —N(R7)2, —(CH2)m—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, —(CH2CH2O)m—R8, —O—(CH2CH2O)m—R8, —(CH2CH2O)m—CH2CH2NR7R10, —O—(CH2CH2O)m—CH2CH2NR7R10, —(CH2)n—C(═O)NR7R10, —O—(CH2)m—C(═O)NR7R10, —(CH2)n—(Z)g—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,
    
    






where when two R6 are —OR11 and are located adjacent to each other on a phenyl ring, the alkyl moieties of the two R6 may be bonded together to form a methylenedioxy group and
where when two —CH2OR8 groups are located 1,2-or 1,3-with respect to each other the R8 groups may be joined to form a cyclic mono-or di-substituted 1,3-dioxane or 1,3-dioxolane;

    
    
        each R7 is, independently, hydrogen or lower alkyl;
        each R8 is, independently, hydrogen, lower alkyl, —C(═O)—R11, glucuronide, 2-tetrahydropyranyl, or
    
    






    
    
        each R9 is, independently, —CO2R7, —CON(R7)2, —SO2CH3, or —C(═O)R7;
        each R10 is, independently, —H, —SO2CH3, —CO2R7, —C(═O)NR7R9, —C(═O)R7, or —CH2—(CHOH), —CH2OH;
        each Z is, independently, CHOH, C(═O), CHNR7R10, C═NR10, or NR10;
        each R11 is, independently, lower alkyl;
        each R12 is independently, —SO2CH3, —CO2R7, —C(═O)NR7R9, —C(═O)R7, or —CH2—(CHOH)n—CH2OH;
        each Het is independently, —NR7, —NR10, —S—, —SO—, or —SO2—;
        each g is, independently, an integer from 1 to 6;
        each m is, independently, an integer from 1 to 7;
        each n is, independently, an integer from 0 to 7;
        each Q is, independently, C—R5, C—R6, or a nitrogen atom, wherein at most three Q in a ring are nitrogen atoms;
    
    


or a pharmaceutically acceptable salt thereof, and
inclusive of all enantiomers, diastereomers, and racemic mixtures thereof.
The present also provides pharmaceutical compositions which contain a compound described above.
The present invention also provides a method of 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 of restoring mucosal defense, comprising:
topically administering an effective amount of compound represented by formula (I) to a mucosal surface of a subject in need thereof.
The present invention also provides a method of blocking ENaC, comprising:
contacting sodium channels with an effective amount of a compound represented by formula (I).
The present invention also provides a method of promoting mucus clearance in 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 of 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 compound represented by formula (I) to a subject in need thereof.
The present invention also provides a method of treating rhinosinusitis, comprising:
administering an effective amount of a compound represented by a formula (I) to a subject in need thereof.
The present invention also provides a method of treating nasal dehydration, comprising:
administering an effective amount of a compound represented by formula (I) to the nasal passages of a subject in need thereof.
In a more specific embodiment, the nasal dehydration is brought on by administering 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 of 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 by means of a ventilator.
The present invention also provides a method of 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 of 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 of 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 of inducing sputum for diagnostic purposes, comprising:
administering an effective amount of compound represented by formula a) to a subject in need thereof.
The present invention also provides a method of 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 of 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 of 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 of promoting corneal 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 of treating Sjögren's disease, comprising:
administering an effective amount of compound represented by formula (I) to a subject in need thereof.
The present invention also provides a method of treating vaginal dryness, comprising:
administering an effective amount of a compound represented by formula (I) to the vaginal tract of a subject in need thereof.
The present invention also provides a method of treating dry skin, comprising:
administering an effective amount of a compound represented by formula (I) to the skin of a subject in need thereof.
The present invention also provides a method of treating dry mouth (xerostomia), comprising:
administering an effective amount of compound represented by formula (I) to the mouth of the subject in need thereof.
The present invention also provides a method of treating distal intestinal obstruction syndrome, comprising:
administering an effective amount of 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 of 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 method, the compound is administered either orally or via a suppository or enema.
The present invention also provides a method of 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 of treating hypertension, comprising administering the compound represented by formula (I) to a subject in need thereof.
The present invention also provides a method of reducing blood pressure, comprising administering the compound represented by formula (I) to a subject in need thereof.
The present invention also provides a method of treating edema, comprising administering the compound represented by formula (I) to a subject in need thereof.
The present invention also provides a method of promoting diuresis, comprising administering the compound represented by formula (I) to a subject in need thereof.
The present invention also provides a method of promoting natriuresis, comprising administering the compound represented by formula (I) to a subject in need thereof.
The present invention also provides a method of promoting saluresis, comprising administering the compound represented by formula (I) to a subject in need thereof.
A more complete appreciation of the invention and many of the attendant advantages thereof will be readily obtained as the same becomes better understood by reference to the following detailed description.


DETAILED DESCRIPTION OF THE INVENTION
The present invention is based on the discovery that the compounds of formula (I) are more potent and/or, absorbed less rapidly from mucosal surfaces, especially airway surfaces, and/or less reversible from interactions with ENaC as compared to compounds such as amiloride, benzamil, and phenamil. Therefore, the compounds of formula (I) have a longer half-life on mucosal surfaces as compared to these compounds.
The present invention is also based on the discovery that certain compounds embraced by formula (I) are converted in vivo into metabolic derivatives thereof that have reduced efficacy in blocking sodium channels as compared to the parent administered compound, after they are absorbed from mucosal surfaces after administration. This important property means that the compounds will have a lower tendency to cause undesired side-effects by blocking sodium channels located at untargeted locations in the body of the recipient, e.g., in the kidneys.
The present invention is also based on the discovery that certain compounds embraced by formula (1) target the kidney and thus may be used as cardiovascular agents.
In the compounds represented by formula (I), X may be hydrogen, halogen, trifluoromethyl, lower alkyl, lower cycloalkyl, unsubstituted or substituted phenyl, lower alkyl-thio, phenyl-lower alkyl-thio, lower alkyl-sulfonyl, or phenyl-lower alkyl-sulfonyl. Halogen is preferred.
Examples of halogen include fluorine, chlorine, bromine, and iodine. Chlorine and bromine are the preferred halogens. Chlorine is particularly preferred. This description is applicable to the term “halogen” as used throughout the present disclosure.
As used herein, the term “lower alkyl” means an alkyl group having less than 8 carbon atoms. This range includes all specific values of carbon atoms and subranges there between, such as 1, 2, 3, 4, 5, 6, and 7 carbon atoms. The term “alkyl” embraces all types of such groups, e.g., linear, branched, and cyclic alkyl groups. This description is applicable to the term “lower alkyl” as used throughout the present disclosure. Examples of suitable lower alkyl groups include methyl, ethyl, propyl, cyclopropyl, butyl, isobutyl, etc.
Substituents for the phenyl group include halogens. Particularly preferred halogen substituents are chlorine and bromine.
Y may be hydrogen, hydroxyl, mercapto, lower alkoxy, lower alkyl-thio, 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. Particularly preferred are such compounds where each R2 is hydrogen.
R1 may be hydrogen or lower alkyl. Hydrogen is preferred for R1.
Each R2 may be, independently, —R7, —(CH2)m—OR8, —(CH2)m—NR7R10, —(CH2)n(CHOR8)(CHOR8), —CH2OR8, —(CH2CH2O)m—R8, —(CH2CH2O)m—CH2CH2NR7R10, —(CH2)n—C(═O)NR7R10, —(CH2)n—Zg—R7, —(CH2)m—NR10—CH2(CHOR8)(CHOR8)n—CH2OR8, —(CH2)n—CO2R7, or




In the definition of R2 described above, when two —CH2OR8 groups are located 1,2-or 1,3-with respect to each other the R8 groups may be joined to form a cyclic mono-or di-substituted 1,3-dioxane or 1,3-dioxolane.
Hydrogen and lower allyl, particularly C1-C3 alkyl are preferred for R2. Hydrogen is particularly preferred.
R3 and R4 may be, independently, hydrogen, a group represented by formula (A), lower alkyl, hydroxy lower alkyl, phenyl, phenyl-lower alkyl, (halophenyl)-lower alkyl, lower-(alkylphenylalkyl), lower (alkoxyphenyl)-lower alkyl, naphthyl-lower alkyl, or pyridyl-lower alkyl, provided that at least one of R3 and R4 is a group represented by formula (A).
Preferred compounds are those where one of R3 and R4 is hydrogen and the other is represented by formula (A).
In formula (A), the moiety —(C(RL)2)o-x-(C(RL)2)p— defines an alkylene group bonded to the aromatic ring. The variables o and p may each be an integer from 0 to 10, subject to the proviso that the sum of o and p in the chain is from 1 to 10. Thus, o and p may each be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Preferably, the sum of o and p is from 2 to 6. In a particularly preferred embodiment, the sum of o and p is 4.
The linking group in the alkylene chain, x, may be, independently, O, NR10, C(═O), CHOH, C(═N—R10), CHNR7R10, or represents a single bond;
Therefore, when x represents a single bond, the alkylene chain bonded to the ring is represented by the formula —(C(RL)2)o+p—, in which the sum o+p is from 1 to 10.
Each RL may be, independently, —R7, —(CH2), —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, —(CH2CH2O)m—R8, —O—(CH2CH2O)m—R8, —(CH2CH2O)m—CH2CH2NR7R10, —O —(CH2CH2O)m—CH2CH2NR7R10, —(CH2)n—C(═O)NR7R10, —O(CH2)m—C(═O)NR7R10, —(CH2)n—(Z)g—R7, —O—(CH2)m—(Z)g—R7, —(CH2)n—NR10—CH2(CHOR8)(CHOR8)n—CH2OR5, —O—(CH2)m—NR10—CH2(CHOR8)(CHOR8)n—CH2OR8, —(CH2)n—CO2R7, —O—(CH2)m—CO2R7, —OSO3H, —O-glucuronide, —O-glucose,




In the definition of RL above, when two —CH2OR8 groups are located 1,2-or 1,3-with respect to each other the R8 groups may be joined to form a cyclic mono-or di-substituted 1,3-dioxane or 1,3-dioxolane.
The preferred RL groups include —H, —OH, —N(R7)2, especially where each R7 is hydrogen.
In the alkylene chain in formula (A), it is preferred that when one RL group bonded to a carbon atoms is other than hydrogen, then the other RL bonded to that carbon atom is hydrogen, i.e., the formula —CHRL—. It is also preferred that at most two RL groups in an alkylene chain are other than hydrogen, where in the other RL groups in the chain are hydrogens. Even more preferably, only one RL group in an alkylene chain is other than hydrogen, where in the other RL groups in the chain are hydrogens. In these embodiments, it is preferable that x represents a single bond.
In another particular embodiment of the invention, all of the RL groups in the alkylene chain are hydrogen. In these embodiments, the alkylene chain is represented by the formula

—(CH2)o-x-(CH2)p—.

Each R5 is independently, —(CH2)n—NR12R12, —O—(CH2)m—NR12R12, —O—(CH2)n—NR12R12, —O—(CH2)m—(Z)gR12, —(CH2)nNR11R11, —O—(CH2)mNR11R11, —(CH2)n—N⊕—(R11)3, —O—(CH2)m—N⊕—(R11)3, —(CH2)n—(Z)g—(CH2)m—NR10R10, —O(CH2)m—(Z)g—(CH2)m—NR10R10, —(CH2CH2O)m—CH2CH2NR12R12, —O—(CH2CH2O)m—CH2CH2NR12R12, —(CH2)n—(C═O)NR12R12, —O—(CH2)m—(C═O)NR12R12, —O—(CH2)m—(CHOR8)mCH2NR10—(Z)g—R10, —(CH2)n—(CHOR8)mCH2—NR10—(Z)g—R10, —(CH2)nNR10—O(CH2)m(CHOR8)nCH2NR10—(Z)g—R10, —O(CH2)m—NR10—(CH2)m—(CHOR8)nCH2NR10—(Z)g—R10, -(Het)-(CH2)m—OR8, -(Het)-(CH2)m—NR7R10, -(Het)-(CH2)m(CHOR8)(CHOR8)n—CH2OR8, -(Het)-(CH2CH2O)m—R8, -(Het)-(CH2CH2O)m—CH2CH2NR7R10, -(Het)-(CH2)m—C(═O)NR7R10, -(Het)-(CH2)m—(Z)g—R7, -(Het)-(CH2)m—NR10—CH2(CHOR8)(CHOR8)n—CH2OR8, -(Het)-(CH2)m—CO2R7, -(Het)-(CH2)m—NR12R12, -(Het)-(CH2)n—NR12R12-(Het)-(CH2)m—(Z)gR12, -(Het)-(CH2)mNR11R11, -(Het)-(CH2)m—N⊕—(R11)3, -(Het)-(CH2)m—(Z)g—(CH2)m—NR10R10, -(Het)-(CH2CH2O)m—CH2CH2NR12R12, -(Het)-(CH2)m—(C═O)NR12R12, -(Het)-(CH2)m—(CHOR8)mCH2NR10—(Z)g—R10, -(Het)-(CH2)m—NR10—(CH2)m—(CHOR8)nCH2NR10—(Z)g—R10, where when two —CH2OR8 groups are located 1,2-or 1,3-with respect to each other the R8 groups may be joined to form a cyclic mono-or di-substituted 1,3-dioxane or 1,3-dioxolane,
—(CH2)n(CHOR8)(CHOR8)n—CH2OR8 with the proviso that two —CH2OR8 groups are located 1,2-or 1,3-with respect to each other and the R8 groups are joined to form a cyclic mono or disubstituted 1,3-dioxane or 1,3-dioxolane,
—O—(CH2)m(CHOR8)(CHOR8)n—CH2OR8, with the proviso that two —CH2OR8 groups are located 1,2-or 1,3-with respect to each other and the R8 groups are joined to form a cyclic mono or disubstituted 1,3-dioxane or 1,3-dioxolane,
—(CH2)n—NR10—CH2(CHOR8)(CHOR8)n—CH2OR8, with the proviso that two —CH2OR8 groups are located 1,2-or 1,3-with respect to each other and the R8 groups are joined to form a cyclic mono or disubstituted 1,3-dioxane or 1,3-dioxolane, or
—O—(CH2)m—NR10—CH2(CHOR8)(CHOR8)n—CH2OR8, with the proviso that two —CH2OR8 groups are located 1,2-or 1,3-with respect to each other and the R8 groups are joined to form a cyclic mono or disubstituted 1,3-dioxane or 1,3-dioxolane.
Preferred examples of R5 include:
—N(SO2CH3)2,
—CH2—CHNHBocCO2CH3 (α),
—O—CH2—CHNH2CO2H (α),
—O—CH2—CHNH2CO2CH3 (α),
—O—(CH2)2—N+(CH3)3,
—C(═O)NH—(CH2)2—NH2,
—C(═O)NH—(CH2)2—NH—C(═NH)—NH2, and




There are four R6 groups present on the ring in formula (A). Each R6 may be each, independently, R5 as described above, —R7, —OR8, N(R7)2, —(CH2)m—OR8, —O—(CH2)m—OR8, —(CH2), —NR7R10, —O—(CH2)m—NR7R10, —(CH2)n(CHOR8)(CHOR8)n—CH2OR8, —O—(CH2)m(CHOR8)(CHOR8)n—CH2OR8, (CH2CH2O)m—R8, —O—(CH2CH2O)m—R8, (CH2CH2O)m—CH2CH2NR7R10, —O—(CH2CH2O)m—CH2CH2NR7R10, —(CH2)n—C(═O)NR7R10, —O—(CH2)m—C(═O)NR7R10, —(CH2)n—(Z)g—R7, —O—(CH2)m—(Z)g—R7, —(CH2)n—NR10—CH2(CHOR8)(CHOR8), —CH2OR8, O—(CH2)m—NR10—CH2(CHOR8)(CHOR8)n—CH2OR8, —(CH2)n—CO2R7, —O—(CH2)m—CO2R7, —OSO3H, —O-glucuronide, —O-glucose, or




When two R6 are —OR11 and are located adjacent to each other on a phenyl ring, the alkyl moieties of the two R6 groups may be bonded together to form a methylenedioxy group, i.e., a group of the formula —O—CH2—O—. Also, when two —CH2OR8 groups are located 1,2-or 1,3-with respect to each other the R8 groups may be joined to form a cyclic mono-or di-substituted 1,3-dioxane or 1,3-dioxolane.
As discussed above, R6 may be hydrogen. Therefore, 1, 2, 3, or 4 R6 groups may be other than hydrogen. Preferably at most 3 of the R6 groups are other than hydrogen.
Each g is, independently, an integer from 1 to 6. Therefore, each g may be 1, 2, 3, 4, 5, or 6.
Each m is an integer from 1 to 7. Therefore, each m may be 1, 2, 3, 4, 5, 6, or 7.
Each n is an integer from 0 to 7. Therefore, each n maybe 0, 1, 2, 3, 4, 5, 6, or 7.
Each Q in formula (A) is C—R5, C—R6, or a nitrogen atom, where at most three Q in a ring are nitrogen atoms. Thus, there may be 1, 2, or 3 nitrogen atoms in a ring. Preferably, at most two Q are nitrogen atoms. More preferably, at most one Q is a nitrogen atom. In one particular embodiment, the nitrogen atom is at the 3-position of the ring. In another embodiment of the invention, each Q is either C—R5 or C—R6, i.e., there are no nitrogen atoms in the ring.
More specific examples of suitable groups represented by formula (A) are shown in formulas (B)-(F) below:




where o, x, p, R5, and R6, are as defined above;




where n is an integer from 1 to 10 and R5 is as defined above;




where n is an integer from 1 from 10 and R5 is as defined above;




where o, x, p, and R5 are as defined above;
In a preferred embodiment of the invention, Y is —NH2.
In another preferred embodiment, R2 is hydrogen.
In another preferred embodiment, R1 is hydrogen.
In another preferred embodiment, X is chlorine.
In another preferred embodiment, R3 is hydrogen.
In another preferred embodiment, RL 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 is hydrogen.
In another preferred embodiment, at most one Q is a nitrogen atom.
In another preferred embodiment, no Q is a nitrogen atom.
In a preferred embodiment of the present invention:
X is halogen;
Y is —N(R7)2;
R1 is hydrogen or C1-C3 alkyl;
R1 is —R7, CH2OR7, or —CO2R7;
R3 is a group represented by formula (A); and
R4 is hydrogen, a group represented by formula (A), or lower alkyl;
In another preferred embodiment of the present invention:
X is chloro or bromo;
Y is —N(R7)2;
R2 is hydrogen or C1-C3 alkyl;
at most three R6 are other than hydrogen as described above;
at most three RL are other than hydrogen as described above; and
at most 2 Q are nitrogen atoms.
In another preferred embodiment of the present invention:
Y is —NH2;
In another preferred embodiment of the present invention:
R4 is hydrogen;
at most one RL is other than hydrogen as described above;
at most two R6 are other than hydrogen as described above; and
at most 1 Q is a nitrogen atom.
Preferred examples of R5 in the embodiments described above include:
—N(SO2CH3)2,
—CH2—CHNHBocCO2CH3 (α),
—O—CH2—CHNH2CO2H (α),
—O—CH2—CHNH2CO2CH3 (α),
—O—(CH2)2—N+(CH3)3,
—C(═O)NH—(CH2)2—NH2, and
—C(═O)NH—(CH2)2—NH—C(═NH)—NH2.
Examples of compounds of the present invention include the following:







The compounds of formula (I) may be prepared and used as the free base. Alternatively, the compounds may be prepared and used as a pharmaceutically acceptable salt. Pharmaceutically acceptable salts are salts that retain or enhance the desired biological activity of the parent compound and do not impart undesired toxicological effects. Examples of such salts are (a) acid addition salts formed with inorganic acids, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; (b) salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, malonic acid, sulfosalicylic acid, glycolic acid, 2-hydroxy-3-naphthoate, pamoate, salicylic acid, stearic acid, phthalic acid, mandelic acid, lactic acid and the like; and (c) salts formed from elemental anions for example, chlorine, bromine, and iodine.
It is to be noted that all enantiomers, diastereomers, and racemic mixtures of compounds within the scope of formula (I) are embraced by the present invention. All mixtures of such enantiomers and diastereomers are within the scope of the present invention.
Without being limited to any particular theory, it is believed that the compounds of formula (I) function in vivo as sodium channel blockers. By blocking epithelial sodium channels present in mucosal surfaces the compounds of formula (I) reduce the absorption of water by the mucosal surfaces. This effect increases the volume of protective liquids on mucosal surfaces, rebalances the system, and thus treats disease.
The present invention also provides methods of treatment that take advantage of the properties of the compounds of formula (I) discussed above. Thus, subjects that may be treated by the methods of the present invention include, but are not limited to, patients afflicted with cystic fibrosis, primary ciliary dyskinesia, chronic bronchitis, chronic obstructive airway disease, artificially ventilated patients, patients with acute pneumonia, etc. The present invention may be used to obtain a sputum sample from a patient by administering the active compounds to at least one lung of a patient, and then inducing or collecting a sputum sample from that patient. Typically, the invention will be administered to respiratory mucosal surfaces via aerosol (liquid or dry powders) or lavage.
Subjects that may be treated by the method of the present invention also include patients being administered supplemental oxygen nasally (a regimen that tends to dry the airway surfaces); patients afflicted with an allergic disease or response (e.g., an allergic response to pollen, dust, animal hair or particles, insects or insect particles, etc.) that affects nasal airway surfaces; patients afflicted with a bacterial infection e.g., staphylococcus infections such as Staphylococcus aureus infections, Hemophilus influenza infections, Streptococcus pneumoniae infections, Pseudomonas aeuriginosa infections, etc.) of the nasal airway surfaces; patients afflicted with an inflammatory disease that affects nasal airway surfaces; or patients afflicted with sinusitis (wherein the active agent or agents are administered to promote drainage of congested mucous secretions in the sinuses by administering an amount effective to promote drainage of congested fluid in the sinuses), or combined, Rhinosinusitis. The invention may be administered to rhino-sinal surfaces by topical delivery, including aerosols and drops.
The present invention may be used to hydrate mucosal surfaces other than airway surfaces. Such other mucosal surfaces include gastrointestinal surfaces, oral surfaces, genito-urethral surfaces, ocular surfaces or surfaces of the eye, the inner ear and the middle ear. For example, the active compounds of the present invention may be administered by any suitable means, including locally/topically, orally, or rectally, in an effective amount.
The compounds of the present invention are also useful for treating a variety of functions relating to the cardiovascular system. Thus, the compounds of the present invention are useful for use as antihypertensive agents. The compounds may also be used to reduce blood pressure and to treat edema. In addition, the compounds of the present invention are also useful for promoting diuresis, natriuresis, and saluresis. The compounds may be used alone or in combination with beta blockers, ACE inhibitors, HMGCoA reductase inhibitors, calcium channel blockers and other cardiovascular agents to treat hypertension, congestive heart failure and reduce cardiovascular mortality.
The present invention is concerned primarily with the treatment of human subjects, but may also be employed for the treatment of other mammalian subjects, such as dogs and cats, for veterinary purposes.
As discussed above, the compounds used to prepare the compositions of the present invention may be in the form of a pharmaceutically acceptable free base. Because the free base of the compound is generally less soluble in aqueous solutions than the salt, free base compositions are employed to provide more sustained release of active agent to the lungs. An active agent present in the lungs in particulate form which has not dissolved into solution is not available to induce a physiological response, but serves as a depot of bioavailable drug which gradually dissolves into solution.
Another aspect of the present invention is a pharmaceutical composition, comprising a compound of formula (I) in a pharmaceutically acceptable carrier (e.g., an aqueous carrier solution). In general, the compound of formula (I) is included in the composition in an amount effective to inhibit the reabsorption of water by mucosal surfaces.
The compounds of the present invention may also be used in conjunction with a P2Y2 receptor agonist or a pharmaceutically acceptable salt thereof (also sometimes referred to as an “active agent” herein). The composition may further comprise a P2Y2 receptor agonist or a pharmaceutically acceptable salt thereof (also sometimes referred to as an “active agent” herein). The P2Y2 receptor agonist is typically included in an amount effective to stimulate chloride and water secretion by airway surfaces, particularly nasal airway surfaces. Suitable P2Y2 receptor agonists are described in columns 9-10 of U.S. Pat. No. 6,264,975, U.S. Pat. No. 5,656,256, and U.S. Pat. No. 5,292,498, each of which is incorporated herein by reference.
Bronchodiloators 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, albutereol, terbutalin, pirbuterol, bitolterol, metaproterenol, iosetharine, salmeterol xinafoate, as well as anticholinergic agents including but not limited to ipratropium bromide, as well as compounds such as theophylline and aminophylline. These compounds may be administered in accordance with known techniques, either prior to or concurrently with the active compounds described herein.
Another aspect of the present invention is a pharmaceutical formulation, comprising an active compound as described above in a pharmaceutically acceptable carrier (e.g., an aqueous carrier solution). In general, the active compound is included in the composition in an amount effective to treat mucosal surfaces, such as inhibiting the reabsorption of water by mucosal surfaces, including airway and other surfaces.
The active compounds disclosed herein may be administered to mucosal surfaces by any suitable means, including topically, orally, rectally, vaginally, ocularly and dermally, etc. For example, for the treatment of constipation, the active compounds may be administered orally or rectally to the gastrointestinal mucosal surface. The active compound may be combined with a pharmaceutically acceptable carrier in any suitable form, such as sterile physiological or dilute saline or topical solution, as a droplet, tablet or the like for oral administration, as a suppository for rectal or genito-urethral administration, etc. Excipients may be included in the formulation to enhance the solubility of the active compounds, as desired.
The active compounds disclosed herein may be administered to the airway surfaces of a patient by any suitable means, including as a spray, mist, or droplets of the active compounds in a pharmaceutically acceptable carrier such as physiological or dilute saline solutions or distilled water. For example, the active compounds may be prepared as formulations and administered as described in U.S. Pat. No. 5,789,391 to Jacobus, the disclosure of which is incorporated by reference herein in its entirety.
Solid or liquid particulate active agents prepared for practicing the present invention could, as noted above, include particles of respirable or non-respirable size; that is, for respirable particles, particles of a size sufficiently small to pass through the mouth and larynx upon inhalation and into the bronchi and alveoli of the lungs, and for non-respirable particles, particles sufficiently large to be retained in the nasal airway passages rather than pass through the larynx and into the bronchi and alveoli of the lungs. In general, particles ranging from about 1 to 5 microns in size (more particularly, less than about 4.7 microns in size) are respirable. Particles of non-respirable size are greater than about 5 microns in size, up to the size of visible droplets. Thus, for nasal administration, a particle size in the range of 10-500 μm may be used to ensure retention in the nasal cavity.
In the manufacture of a formulation according to the invention, active agents or the physiologically acceptable salts or free bases thereof are typically admixed with, inter alia, an acceptable carrier. Of course, the carrier must be compatible with any other ingredients in the formulation and must not be deleterious to the patient. The carrier must be solid or liquid, or both, and is preferably formulated with the compound as a unit-dose formulation, for example, a capsule, that may contain 0.5% to 99% by weight of the active compound. One or more active compounds may be incorporated in the formulations of the invention, which formulations may be prepared by any of the well-known techniques of pharmacy consisting essentially of admixing the components.
Compositions containing respirable or non-respirable dry particles of micronized active agent may be prepared by grinding the dry active agent with a mortar and pestle, and then passing the micronized composition through a 400 mesh screen to break up or separate out large agglomerates.
The particulate active agent composition may optionally contain a dispersant which serves to facilitate the formulation of an aerosol. A suitable dispersant is lactose, which may be blended with the active agent in any suitable ratio (e.g., a 1 to 1 ratio by weight).
Active compounds disclosed herein may be administered to airway surfaces including the nasal passages, sinuses and lungs of a subject by a suitable means know in the art, such as by nose drops, mists, etc. In one embodiment of the invention, the active compounds of the present invention and administered by transbronchoscopic lavage. In a preferred embodiment of the invention, the active compounds of the present invention are deposited on lung airway surfaces by administering an aerosol suspension of respirable particles comprised of the active compound, which the subject inhales. The respirable particles may be liquid or solid. Numerous inhalers for administering aerosol particles to the lungs of a subject are known.
Inhalers such as those developed by Inhale Therapeutic Systems, Palo Alto, Calif., USA, may be employed, including but not limited to those disclosed in U.S. Pat. Nos. 5,740,794; 5,654,007; 5,458,135; 5,775,320; and 5,785,049, all of which are incorporated herein by reference. The Applicant specifically intends that the disclosures of all patent references cited herein be incorporated by reference herein in their entirety. Inhalers such as those developed by Dura Pharmaceuticals, Inc., San Diego, Calif., USA, may also be employed, including but not limited to those disclosed in U.S. Pat. Nos. 5,622,166; 5,577,497; 5,645,051; and 5,492,112, all of which are incorporated herein by reference. Additionally, inhalers such as those developed by Aradigm Corp., Hayward, Calif., USA, may be employed, including but not limited to those disclosed in U.S. Pat. Nos. 5,826,570; 5,813,397; 5,819,726; and 5,655,516, all of which are incorporated herein by reference. These apparatuses are particularly suitable as dry particle inhalers.
Aerosols of liquid particles comprising the active compound may be produced by any suitable means, such as with a pressure-driven aerosol nebulizer or an ultrasonic nebulizer. See, e.g., U.S. Pat. No. 4,501,729, the contents of which is incorporated herein by reference. Nebulizers are commercially available devices which transform solutions or suspensions of the active ingredient into a therapeutic aerosol mist either by means of acceleration of compressed gas, typically air or oxygen, through a narrow venturi orifice or by means of ultrasonic agitation. Suitable formulations for use in nebulizers consist of the active ingredient in a liquid carrier, the active ingredient comprising up to 40% w/w of the formulation, but preferably less than 20% w/w. The carrier is typically water (and most preferably sterile, pyrogen-free water) or dilute aqueous alcoholic solution. Perfluorocarbon carriers may also be used. Optional additives include preservatives if the formulation is not made sterile, for example, methyl hydroxybenzoate, antioxidants, flavoring agents, volatile oils, buffering agents, and surfactants.
Aerosols of solid particles comprising the active compound may likewise be produced with any solid particulate medicament aerosol generator. Aerosol generators for administering solid particulate medicaments to a subject produce particles which are respirable, as explained above, and generate a volume of aerosol containing predetermined metered dose of medicament at a rate suitable for human administration. One illustrative type of solid particulate aerosol generator is an insufflator. Suitable formulations for administration by insufflation include finely comminuted powders which may be delivered by means of an insufflator or taken into the nasal cavity in the manner of a snuff. In the insufflator, the powder (e.g., a metered dose thereof effective to carry out the treatments described herein) is contained in capsules or cartridges, typically made of gelatin or plastic, which are either pierced or opened in situ and the powder delivered by air drawn through the device upon inhalation or by means of a manually-operated pump. The powder employed in the insufflator consists either solely of the active ingredient or of powder blend comprising the active ingredient, a suitable powder diluent, such as lactose, and an optional surfactant. The active ingredient typically comprises of 0.1 to 100% w/w of the formulation. A second type of illustrative aerosol generator comprises a metered dose inhaler. Metered dose inhalers are pressurized aerosol dispensers, typically containing a suspension or solution formulation of active ingredient in a liquified propellant. During use, these devices discharge the formulation through a valve adapted to deliver a metered volume, typically from 10 to 150 μl, to produce a fine particle spray containing the active ingredient. Suitable propellants include certain chlorofluorocarbon compounds, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane and mixtures thereof. The formulation may additionally contain one of more co-solvents, for example, ethanol, surfactants, such as oleic acid or sorbitan trioleate, antioxidants and suitable flavoring agents.
The aerosol, whether formed from solid or liquid particles, may be produced by the aerosol generator at a rate of from about 10 to 150 liters per minute, more preferable from 30 to 150 liters per minute, and most preferably about 60 liters per minute. Aerosols containing greater amounts of medicament may be administered more rapidly.
The dosage of the active compounds disclosed herein will vary depending on the condition being treated and the state of the subject, but generally may be from about 0.01, 0.03, 0.05, 0.1 to 1, 5, 10 or 20 mg of the pharmaceutic agent, deposited on the airway surfaces. The daily dose may be divided among one or multiple unit dose administrations. The goal is to achieve a concentration of the pharmaceutic agents on lung airway surfaces of between 10−9-104 M.
In another embodiment, they are administered by administering an aerosol suspension of respirable or non-respirable particles (preferably non-respirable particles) comprised of active compound, which the subject inhales through the nose. The respirable or non-respirable particles may be liquid or solid. The quantity of active agent included may be an amount of sufficient to achieve dissolved concentrations of active agent on the airway surfaces of the subject of from about 10−9, 10−8, or 10−7 to about 10−3, 10−2, 10−1 moles/liter, and more preferably from about 10−9 to about 10−4 moles/liter.
The dosage of active compound will vary depending on the condition being treated and the state of the subject, but generally may be an amount sufficient to achieve dissolved concentrations of active compound on the nasal airway surfaces of the subject from about 10−9, 10−8, or 10−7 to about 10−3, 10−2, or 10−1 Moles/liter, and more preferably from about 10−7 to about 10−4 Moles/liter. Depending upon the solubility of the particular formulation of active compound administered, the daily dose may be divided among one or several unit dose administrations. The daily dose by weight may range from about 0.01, 0.03, 0.1, 0.5 or 1.0 to 10 or 20 milligrams of active agent particles for a human subject, depending upon the age and condition of the subject. A currently preferred unit dose is about 0.5 milligrams of active agent given at a regimen of 2-10 administrations per day. The dosage may be provided as a prepackaged unit by any suitable means (e.g., encapsulating a gelatin capsule).
In one embodiment of the invention, the particulate active agent composition may contain both a free base of active agent and a pharmaceutically acceptable salt to provide both early release and sustained release of active agent for dissolution into the mucus secretions of the nose. Such a composition serves to provide both early relief to the patient, and sustained relief over time. Sustained relief, by decreasing the number of daily administrations required, is expected to increase patient compliance with the course of active agent treatments.
Pharmaceutical formulations suitable for airway administration include formulations of solutions, emulsions, suspensions and extracts. See generally, J. Naim, Solutions, Emulsions, Suspensions and Extracts, in Remington: The Science and Practice of Pharmacy, chap. 86 (19th ed 1995), incorporated herein by reference. Pharmaceutical formulations suitable for nasal administration may be prepared as described in U.S. Pat. No. 4,389,393 to Schor; U.S. Pat. No. 5,707,644 to Illum; U.S. Pat. No. 4,294,829 to Suzuki; and U.S. Pat. No. 4,835,142 to Suzuki, the disclosures of which are incorporated by reference herein.
Mists or aerosols of liquid particles comprising the active compound may be produced by any suitable means, such as by a simple nasal spray with the active agent in an aqueous pharmaceutically acceptable carrier, such as a sterile saline solution or sterile water. Administration may be with a pressure-driven aerosol nebulizer or an ultrasonic nebulizer. See e.g. U.S. Pat. Nos. 4,501,729 and 5,656,256, each of which is incorporated herein by reference. Suitable formulations for use in a nasal droplet or spray bottle or in nebulizers consist of the active ingredient in a liquid carrier, the active ingredient comprising up to 40% w/w of the formulation, but preferably less than 20% w/w. Typically the carrier is water (and most preferably sterile, pyrogen-free water) or dilute aqueous alcoholic solution, preferably made in a 0.12% to 0.8% solution of sodium chloride. Optional additives include preservatives if the formulation is not made sterile, for example, methyl hydroxybenzoate, antioxidants, flavoring agents, volatile oils, buffering agents, osmotically active agents (e.g. mannitol, xylitol, erythritol) and surfactants.
Compositions containing respirable or non-respirable dry particles of micronized active agent may be prepared by grinding the dry active agent with a mortar and pestle, and then passing the micronized composition through a 400 mesh screen to break up or separate out large agglomerates.
The particulate composition may optionally contain a dispersant which serves to facilitate the formation of an aerosol. A suitable dispersant is lactose, which may be blended with the active agent in any suitable ratio (e.g., a 1 to 1 ratio by weight).
The compounds of formula (I) may be synthesized according to procedures known in the art. A representative synthetic procedure is shown in the scheme below.




These procedures are described in, for example, E. J. Cragoe, “The Synthesis of Amiloride and Its Analogs” (Chapter 3) in Amiloride and Its Analogs, pp. 25-36, incorporated herein by reference. Other methods of preparing the compounds are described in, for example, U.S. Pat. No. 3,313,813, incorporated herein by reference. See in particular Methods A, B, C, and D described in U.S. Pat. No. 3,313,813.

Several assays may be used to characterize the compounds of the present invention. Representative assays are discussed below.
In Vitro Measure of Sodium Channel Blocking Activity and Reversibility
One assay used to assess mechanism of action and/or potency of the compounds of the present invention involves the determination of lumenal drug inhibition of airway epithelial sodium currents measured under short circuit current (ISC) using airway epithelial monolayers mounted in Ussing chambers. Cells obtained from freshly excised human, dog, sheep or rodent airways are seeded onto porous 0.4 micron SnapwellTM Inserts (CoStar), cultured at air-liquid interface (ALI) conditions in hormonally defined media, and assayed for sodium transport activity (ISC) while bathed in Krebs Bicarbonate Ringer (KBR) in Using chambers. All test drug additions are to the lumenal bath with half-log dose addition protocols (from 1×10−11 M to 3×10−5M), and the cumulative change in ISC (inhibition) recorded. All drugs are prepared in dimethyl sulfoxide as stock solutions at a concentration of 1×10−2 M and stored at −20° C. Eight preparations are typically run in parallel; two preparations per run incorporate amiloride and/or benzamil as positive controls. After the maximal concentration (5×10−5 M) is administered, the lumenal bath is exchanged three times with fresh drug-free KBR solution, and the resultant ISC measured after each wash for approximately 5 minutes in duration. Reversibility is defined as the percent return to the baseline value for sodium current after the third wash. All data from the voltage clamps are collected via a computer interface and analyzed off-line.
Dose-effect relationships for all compounds are considered and analyzed by the Prism 3.0 program. IC50 values, maximal effective concentrations, and reversibility are calculated and compared to amiloride and benzamil as positive controls.
Pharmacological Assays of Absorption
(1) Apical Disappearance Assay
Bronchial cells (dog, human, sheep, or rodent cells) are seeded at a density of 0.25×106/cm2 on a porous Transwell-Col collagen-coated membrane with a growth area of 1.13 cm2 grown at an air-liquid interface in hormonally defined media that promotes a polarized epithelium. From 12 to 20 days after development of an air-liquid interface (ALI) the cultures are expected to be >90% ciliated, and mucins will accumulate on the cells. To ensure the integrity of primary airway epithelial cell preparations, the transepithelial resistance (Rt) and transepithelial potential differences (PD), which are indicators of the integrity of polarized nature of the culture, are measured. Human cell systems are preferred for studies of rates of absorption from apical surfaces. The disappearance assay is conducted under conditions that mimic the “thin” films in vivo ( ̃25 μl) and is initiated by adding experimental sodium channel blockers or positive controls (amiloride, benzamil, phenamil) to the apical surface at an initial concentration of 10 μM. A series of samples (5 μl volume per sample) is collected at various time points, including 0, 5, 20, 40, 90 and 240 minutes. Concentrations are determined by measuring intrinsic fluorescence of each sodium channel blocker using a Fluorocount Microplate Flourometer or HPLC. Quantitative analysis employs a standard curve generated from authentic reference standard materials of known concentration and purity. Data analysis of the rate of disappearance is performed using nonlinear regression, one phase exponential decay (Prism V 3.0).
2. Confocal Microscopy Assay of Amiloride Congener Uptake
Virtually all amiloride-like molecules fluoresce in the ultraviolet range. This property of these molecules may be used to directly measure cellular update using x-z confocal microscopy. Equimolar concentrations of experimental compounds and positive controls including amiloride and compounds that demonstrate rapid uptake into the cellular compartment (benzamil and phenamil) are placed on the apical surface of airway cultures on the stage of the confocal microscope. Serial x-z images are obtained with time and the magnitude of fluorescence accumulating in the cellular compartment is quantitated and plotted as a change in fluorescence versus time.
3. In Vitro Assays of Compound Metabolism
Airway epithelial cells have the capacity to metabolize drugs during the process of transepithelial absorption. Further, although less likely, it is possible that drugs can be metabolized on airway epithelial surfaces by specific ectoenzyme activities. Perhaps more likely as an ecto-surface event, compounds may be metabolized by the infected secretions that occupy the airway lumens of patients with lung disease, e.g. cystic fibrosis. Thus, a series of assays is performed to characterize the compound metabolism that results from the interaction of test compounds with human airway epithelia and/or human airway epithelial lumenal products.
In the first series of assays, the interaction of test compounds in KBR as an “ASL” stimulant are applied to the apical surface of human airway epithelial cells grown in the T-Col insert system. For most compounds, metabolism (generation of new species) is tested for using high performance liquid chromatography (HPLC) to resolve chemical species and the endogenous fluorescence properties of these compounds to estimate the relative quantities of test compound and novel metabolites. For a typical assay, a test solution (25 μl KBR, containing 10 μM test compound) is placed on the epithelial lumenal surface. Sequential 5 to 10 μl samples are obtained from the lumenal and serosal compartments for HPLC analysis of (1) the mass of test compound permeating from the lumenal to serosal bath and (2) the potential formation of metabolites from the parent compound. In instances where the fluorescence properties of the test molecule are not adequate for such characterizations, radiolabeled compounds are used for these assays. From the HPLC data, the rate of disappearance and/or formation of novel metabolite compounds on the lumenal surface and the appearance of test compound and/or novel metabolite in the basolateral solution is quantitated. The data relating the chromatographic mobility of potential novel metabolites with reference to the parent compound are also quantitated.
To analyze the potential metabolism of test compounds by CF sputum, a “representative” mixture of expectorated CF sputum obtained from 10 CF patients (under IRB approval) has been collected. The sputum has been be solubilized in a 1:5 mixture of KBR solution with vigorous vortexing, following which the mixture was split into a “neat” sputum aliquot and an aliquot subjected to ultracentrifugation so that a “supernatant” aliquot was obtained (neat=cellular; supernatant=liquid phase). Typical studies of compound metabolism by CF sputum involve the addition of known masses of test compound to “neat” CF sputum and aliquots of CF sputum “supernatant” incubated at 37° C., followed by sequential sampling of aliquots from each sputum type for characterization of compound stability/metabolism by HPLC analysis as described above. As above, analysis of compound disappearance, rates of formation of novel metabolities, and HPLC mobilities of novel metabolites are then performed.
4. Pharmacological Effects and Mechanism of Action of the Drug in Animals
The effect of compounds for enhancing mucociliary clearance (MCC) can be measured using an in vivo model described by Sabater et al., Journal of Applied Physiology, 1999, pp. 2191-2196, incorporated herein by reference.
EXAMPLES
Having generally described this invention, a further understanding can be obtained by reference to certain specific examples which are provided herein for purposes of illustration only and are not intended to be limiting unless otherwise specified.
Preparation of Sodium Channel Blockers
Materials and methods. All reagents and solvents were purchased from Aldrich Chemical Corp. and used without further purification. NMR spectra were obtained on either a Bruker WM 360 (1H NMR at 360 MHz and 13C NMR at 90 MHz) or a Bruker AC 300 (1H NMR at 300 MHz and 13C NMR at 75 MHz). Flash chromatography was performed on a Flash EluteTM system from Elution Solution (PO Box 5147, Charlottesville, Va. 22905) charged with a 90 g silica gel cartridge (40M FSO-0110-040155, 32-63 μm) at 20 psi (N2). GC-analysis was performed on a Shimadzu GC-17 equipped with a Heliflex Capillary Column (Alltech); Phase: AT-1, Length: 10 meters, ID: 0.53 mm, Film: 0.25 micrometers. GC Parameters: Injector at 320° C., Detector at 320° C., FID gas flow: H2 at 40 ml/min., Air at 400 ml/min. Carrier gas: Split Ratio 16:1, N2 flow at 15 ml/min., N2 velocity at 18 cm/sec. The temperature program is 70° C. for 0-3 min, 70-300° C. from 3-10 min, 300° C. from 10-15 min.
HPLC analysis was performed on a Gilson 322 Pump, detector LT/Vis-156 at 360 nm, equipped with a Microsorb Mv C8 column, 100 A, 25 cm. Mobile phase: A=acetonitrile with 0.1% TFA, B=water with 0.1% TFA. Gradient program: 95:5 B:A for 1 min, then to 20:80 B:A over 7 min, then to 100% A over 1 min, followed by washout with 100% A for 11 min, flow rate: 1 ml/min.
Example 1
4-{4-[N-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}-N-(2-aminoethyl)benzamide hydrochloride (11698)




4-(4-Aminobutyl)benzoic acid (30)
A solution of sodium hydroxide (0.69 g, 17.37 mmol) in water (30 mL) was added to a solution of 24 (1.2 g, 5.79 mmol) in methanol (30 mL) and stirred at room temperature for 48 h. Then the solvent was removed under reduced pressure. Water (20 mL) was added and pH was adjusted to 7 with HCl. The white solid precipitate was filtered off, washed with water and dried in vacuum. The crude product 30 (1.39 g) was obtained as a white solid and used for the next step without further purification.
4-(4-Benzyloxycarbonylaminobutyl)benzoic acid (31)
Sodium hydrogencarbonate (0.95 g, 11.32 mmol) was added into a suspension of 30 in THF (120 mL), followed by water (10 mL), affording a clear solution. Benzyl chloroformate (1.21 mL, 8.49 mmol) was then added into the reaction mixture at 0° C. The reaction mixture was then stirred at room temperature overnight. After that, the solvent was removed under reduced pressure. Ethyl acetate (70 mL) was added to the residue and the solution was washed with 2N HCl (2×30 mL) and water (2×50 mL), then dried in vacuum. 1.82 g (98%) of 31 was obtained as a white solid. 1H NMR (300 MHz, DMSO-d6) δ 1.11 (m, 2H), 1.28 (m, 2H), 2.33 (m, 2H), 3.02 (m, 2H), 5.01 (m, 2H), 7.15 (m, 7H), 7.93 (d 2H).
4-[4-(2-tert-Butoxycarbonylaminoethylcarbamoyl)phenyl]butylcarbamic acid benzyl ester (32)
N,N′-Dicyclohexylcarbodiimide (DCC) (0.69 g, 3.36 mmol) was added to a cold (0° C.) methylene chloride solution of 31 (1 g, 3.05 mmol) and 1-hydroxybenzotriazole (HOBt) (0.41 g, 3.05 mmol) under a nitrogen atmosphere. The reaction mixture was then stirred at room temperature overnight. A white precipitate was formed. The solvent was removed under reduced pressure and the residue was separated by FlashTM (BIOTAGE, Inc) (90 g silica gel cartridge 40M, 3:2 methylene chloride/ethyl acetate) to provide 32 (0.9 g, 63%) as a white solid. 1H NMR (300 MHz, DMSO-d6) δ 1.70 (m, 11H), 1.94 (m, 2H), 3.23 (m, 2H), 3.72 (m, 2H), 3.82 (m, 2H), 4.08 (m, 2H), 6.19 (s, 2H), 8.61 (m, 1H), 9.04 (m, 7H), 9.61 (d, 2H).
{2-[4-(4-Aminobutyl)benzoylamino]ethyl}carbamic acid tert-butyl ester (33)
A suspension of 32 (0.9 g, 1.92 mmol) with 10% palladium on carbon (0.30 g, wet) in methanol (50 mL) was stirred for 2 h at room temperature under atmospheric pressure of hydrogen. The mixture was then filtered through a silica gel pad. The solvent was evaporated and the residue was purified by FlashTM (BIOTAGE, Inc) (90 g silica gel cartridge 40M, 6:1:0.1 chloroform/ethanol/concentrated ammonium hydroxide) to provide 33 (0.405 g, 63%) as a white solid. 1H NMR (300 MHz, DMSO-d6) δ 1.08 (m, 2H), 1.37 (s, 9H), 1.58 (m, 2H), 2.52 (m, 2H), 3.28 (m, 2H), 6.91 (m, 1H), 7.27 (d, 2H), 7.74 (d, 2H), 8.39 (m, 1H).
[2-(4-{4-[N′-(3,5-Diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}-benzoylamino)ethyl]carbamic acid tert-butyl ester (34)
1-(3,5-Diamino-6-chloropyrazinoyl)-2-methylisothiourea hydriodide (0.32 g, 0.82 mmol) and triethylamine (0.27 mL, 1.64 mmol) were sequentially added into a solution of 33 (0.39 g, 0.82 mmol) in 5 mL of methanol. The reaction mixture was stirred in the boiling solvent for 2 h. The solvent was evaporated and the residue was separated by FlashTM (BIOTAGE, Inc) (90 g silica gel cartridge 40M, 6:1:0.1 chloroform/ethanol/concentrated ammonium hydroxide) to provide 34 (0.39 g, 62%) as a yellow solid. 1H NMR (300 MHz, DMSO-d6) δ 1.07 (t, 2H), 1.38 (s, 9H), 1.52 (m, 2H), 1.65 (m, 2H), 2.66 (m, 2H), 3.10 (m, 2H), 3.28 (m, 2H), 4.39 (m, 1H), 6.65 (br s, 1H), 6.94 (m, 1H), 7.28 (m, 2H), 7.75 (d, 2H), 8.49 (m, 1H).
m/z (APCI)=548 [C24H34ClN9O4+H]+.
N-(2-Aminoethyl)-4-{4-[N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}-benzamide hydrochloride (35 (11698))
The solution of 34 (0.104 g, 0.19 mmol) in a mixture of methanol/HCl (1:1, 8 mL) was stirred at room temperature for 0.5 h; then the solvent was completely evaporated, affording 0.099 g (100%) of 35 as a yellow solid. 1H NMR (300 MHz, DMSO-d6) δ 1.60 (m, 4H), 2.69 (m, 2H), 2.98 (m, 2H), 3.33 (m, 2H), 3.52 (m, 2H), 7.32 (d, 2H), 7.90 (d, 2H), 8.23 (br s, 2H), 8.82 (m, 1H), 8.90 (br s, 1H), 9.02 (br s, 1H), 9.37 (m, 1H), 10.57 (s, 1H). m/z (APCI)=448 [C19H26ClN9O2+H]+.
Example 2
4-{4-[N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}-N-(2-guanidinoethyl)benzamide hydrochloride (11834)




4-{4-[N′-(3,5-Diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}-N-[2-N′′,N′′′-di-(butyloxycarbonylguanidino-ethyl]benzamide (36)
Triethylamine (0.34 mL, 2.44 mmol) was added into a suspension of 35 in methanol (25 mL). The reaction mixture was stirred at room temperature for 20 min; at which time the suspension became a clear solution. N,N′-di-(tert-butoxycarbonyl)-N′′-trifluoromethansulfonylguanidine (Goodman's reagent) (0.193 g, 0.489 mmol) was added into the reaction. The reaction mixture was stirred at room temperature for additional 6 h, after that the solvent was removed under reduced pressure and the residue was purified by FlashTM (BIOTAGE, Inc) (90 g silica gel cartridge 40M, 6:1:0.1 chloroform/ethanol/concentrated ammonium hydroxide) to provide 36 (0.18 g, 80%) as a yellow solid. 1H NMR (300 MHz, DMSO-d6) δ 1.40 (s, 9H), 1.47 (s, 9H), 1.52 (m, 2H), 1.65 (m, 2H), 2.68 (m, 2H), 3.18 (br s, 2H), 3.40 (m, 2H), 3.49 (m, 2H), 6.77 (br s, 2H), 7.30 (d, 2H), 7.75 (d, 2H), 8.50 (br s, 2H).
4-{4-[N′-(3,5-Diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}-N-(2-guanidino-ethyl)benzamide hydrochloride (37 (11834))
The solution of 36 (0.155 g, 0.22 mmol) in a mixture of methanol/HCl (1:1, 4 mL) was stirred at room temperature for 2 h, then the solvent was evaporated and the residue dried in vacuum to provide 0.126 g (100%) of 37 as a yellow solid. 1H NMR (300 MHz, DMSO-d6) δ 1.54 (m, 2H), 1.65 (m, 2H), 2.68 (m, 2H), 3.35 (br.s, 4H), 7.32 (d, 2H), 7.90 (d, 2H), 8.79 (m, 1H), 8.92 (br s, 1H), 8.90 (br s, 1H), 9.02 (br s, 1H), 9.37 (m, 1H), 10.57 (s, 1H). m/z (APCI)=490 [C20H28ClN11O2+H]+.
Example 3
N-(3,5-diamino-6-chloropyrazine-2-carbonyl)-N′-{4-[4-(3-guanidino-2-hydroxypropoxy)phenyl]butyl}guanidine hydrochloride (11975)




The synthesis of [4-(4-allyloxyphenyl)butyl]carbamic acid benzyl ester (38) was described in the previously provided experimental details (as compound 30).
[4-(4-Oxiranylmethoxyphenyl)butyl]carbamic acid benzyl ester (39)
3-Chloro-peroxybenzoic acid (2.46 g, 14.25 mmol) was added into a methylene chloride solution (100 mL) of 38 (1.86 g, 5.48 mmol), and the reaction was stirred at room temperature overnight. After that, the solvent was removed under reduced pressure and the residue was purified by flash chromatography (silica gel, 8:1:1 methylene chloride/hexane/ethyl acetate). To eliminate the admixture of benzoic acid the methylene chloride solution of the product was sequentially washed with a saturated aqueous solution of sodium hydrogen carbonate and water, then dried over anhydrous sodium sulfate and evaporated to provide 1.4 g (72%) of 39 as a yellow solid. 1H NMR (300 MHz, CDCl3) δ 1.57 (m, 4H), 2.56 (m, 21), 2.78 (m, 1H), 2.91 (m, 1H), 3.21 (m, 2H), 3.36 (m, 1H), 3.97 (m, 1H), 4.19 (m, 1H), 5.08 (s, 21), 6.82 (d, 2H), 7.06 (d, 2H), 7.72 (s, 5H).
{4-[4-(3-Amino-2-hydroxypropoxy)phenyl]butyl}carbamic acid benzyl ester (40)
An ethanol solution of 39 (0.86 g, 2.42 mmol) was saturated with ammonia and stirred overnight at room temperature. The solvent was evaporated and the residue was purified by FlashTM (BIOTAGE, Inc) (90 g silica gel cartridge 40M, 6:1:0.1 chloroform/ethanol/concentrated ammonium hydroxide) to provide 40 (0.75 g, 87%) as a white solid. 1H NMR (300 MHz, DMSO-d6) δ 1.40 (m, 2H), 1.51 (m, 2H), 2.50 (m, 2H), 2.58 (m, 1H), 2.68 (m, 1H), 3.00 (m, 2H), 3.69 (m, 1H), 3.80 (m, 1H), 3.90 (m, 1H), 5.08 (s, 2H), 6.82 (d, 2H), 7.06 (d, 2H), 7.35 (br s, 5H).
{4-[4-(3-tert-Butoxycarbonylamino-2-hydroxpropoxy)-phenyl]butyl}carbamic acid tert-butyl ester (41)
The compound 41 was prepared in a similar manner to the synthesis of compound 25, starting from compound 40 (0.75 g, 2.03 mmol). It was purified by FlashTM (BIOTAGE, Inc) (90 g silica gel cartridge 40M, 2:1 hexane/ethyl acetate) as a white solid (0.8 g, 83%). 1H NMR (300 MHz, CDCl3) δ 1.45 (s, 9H), 1.57 (m, 4H), 2.58 (m, 2H), 3.20 (m, 2H), 3.31 (m, 2H), 3.94 (m, 2H), 4.10 (m, 1H), 4.73 (br s, 1H), 5.00 (br s, 1H), 5.10 (s, 2H), 6.81 (d, 2H), 7.06 (d, 2H), 7.35 (br s, 5H).
{3-[4-(4-Aminobutyl)phenoxy]-2-hydroxypropyl}carbamic acid tert-butyl ester (42)
A suspension of 41 (0.8 g, 1.69 mmol) with 10% palladium on carbon (0.40 g, wet) in methanol (30 mL) was stirred for 3 h at room temperature under atmospheric pressure of hydrogen. The mixture was then filtered through a silica gel pad; the solvent was evaporated to provide 42 (0.705 g, 99%) as a white solid. 1H NMR (300 MHz, CDCl3) δ 1.45 (s, 9H), 1.55 (m, 4H), 2.58 (m, 4H), 2.71 (m, 2H), 3.29 (m, 1H), 3.45 (m, 2H), 3.92 (m, 2H), 4.10 (br s, 1H), 5.10 (br s, 1H), 6.81 (d, 2H), 7.08 (d, 2H).
[3-(4-{4-[N-(3,5-Diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}phenoxy)-2-hydroxypropyl]carbamic acid tert-butyl ester (43)
Compound 43 was prepared in a similar manner to the synthesis of compound 29, starting from compound 42 (0.46 g, 1.36 mmol). It was purified by FlashTM (BIOTAGE, Inc) (90 g silica gel cartridge 40M, 6:1:0.1 chloroform/ethanol/concentrated ammonium hydroxide) as a yellow solid (0.37 g, 57%). 1H NMR (300 MHz, DMSO-d6) δ 1.38 (s, 9H), 1.58 (br s, 4H), 2.55 (m, 2H), 3.00 (m, 2H), 3.08 (m, 2H), 3.33 (m, 2H), 3.82 (m, 3H), 5.13 (br s, 1H), 6.85 (d, 2H), 7.10 (d, 2H), 7.46 (br s, 2H).
N-{4-[4-(4-Amino-2-hydroxypropoxy)phenyl]butyl}-N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidine (44)
The free base of compound 44 was prepared in a similar manner to the synthesis of the compound 28, starting from the compound 43 (0.18 g, 0.33 mmol) and purified by FlashTM (BIOTAGE, Inc) (90 g silica gel cartridge 40M, 2:1:0.1 chloroform/ethanol/concentrated ammonium hydroxide) to afford the product free base as a yellow solid. It was then treated with 3% HCl. The solvent was evaporated, and the residue was dried in vacuum to afford 0.125 g (73%) of the compound 44. 1H NMR (300 MHz, DMSO-d6) δ 1.58 (br s, 4H), 2.55 (m, 2H), 2.84 (br s, 1H), 3.03 (br s, 1H), 3.34 (br s, 2H), 3.94 (m, 2H), 5.13 (br s, 1H), 6.85 (d, 2H), 7.13 (d, 2H), 8.13 (br s, 2H), 8.90 (br s, if), 9.00 (br s, 1H), 9.34 (br s, 1H), 10.56 (s, 1H).
N-(3,5-Diamino-6-chloro-pyrazine-2-carbonyl)-N′-{4-[4-(3-(N′′,N′′′-di-tert-butyl-oxycarbonyl)-guanidino)-2-hydroxy-propoxy)phenyl]-butyl}-guanidine (45)
Triethylamine (0.30 mL, 2.14 mmol) was added into a solution of 44 in methanol (10 mL) followed by the addition of N,N′-di-(tert-butoxycarbonyl)-N′′-trifluoromethansulfonyl-guanidine (Goodman's reagent) (0.169 g, 0.4295 mmol). The reaction mixture was stirred at room temperature for 2 h. After that the solvent was removed under reduced pressure and the residue was purified by FlashTM (BIOTAGE, Inc) (90 g silica gel cartridge 40M, 6:1:0.1 chloroform/ethanol/concentrated ammonium hydroxide) to provide 45 (0.154 g, 78%) as a yellow solid. 1H NMR (300 MHz, CD3OD) δ 1.47 (s, 9H), 1.52 (s, 9H), 1.66 (br s, 4H), 2.58 (m, 2H), 3.23 (br s, 2H), 3.48 (m, 1H), 3.70 (m, 1H), 3.95 (m, 2H), 4.09 (m, 1H), 6.88 (d, 2H), 7.10 (d, 2H).
N-(3,5-Diamino-6-chloropyrazine-2-carbonyl)-N′-{4-[4-(3-guanidino-2-hydroxy-propoxy)phenyl]butyl}guanidine hydrochloride (46)
A solution of 45 (0.134 g, 0.19 mmol) in concentrated hydrochloric acid was stirred at room temperature for 0.5 h. The solvent was then evaporated and the residue dried in vacuum to provide 0.108 g (99%) of 46 as a yellow solid. 1H NMR (300 MHz, DMSO-d6) δ 1.60 (br s, 4H), 2.52 (br s, 1H), 3.28 (m, 1H), 3.36 (m, 2H), 4.91 (s, 2H), 6.88 (d, 2H), 7.12 (d, 2H), 7.79 (m, 1H), 8.92 (br s, 1H), 9.03 (br s, 1H), 9.37 (m, 1H), 10.57 (s, 1H). m/z (APCI)=493 [C20H29ClN10O3+H]+.
Example 4
N-(3,5-Diamino-6-chloropyrazine-2-carbonyl)-N′-[4-(4-bis(methanesulfonyl)aminophenyl)butyl]guanidine (10316)




[4-(4-Nitrophenyl)but-3-ynyl]carbamic acid tert-butyl ester (51)
To a mixture of anhydrous THF and triethylamine (20 mL, 1/1) were sequentially added 1-iodo-4-nitrobenzene (2.0 g, 8.032 mmol) and copper (I) iodide (0.31 g, 1.606 mmol). The mixture was stirred at room temperature for 15 min. The flask was evacuated and re-filled with argon four times to ensure no oxygen remained. The catalyst, dichlorobis(triphenylphosphine)-palladium(II) (0.56 g, 0.803 mmol) was added into the mixture under argon protection, followed by dropwise addition of but-3-ynyl-carbamic acid tert-butyl ester (1.62 g, 9.638 mmol). The newly formed reaction mixture was further stirred at room temperature overnight. The solid in the reaction mixture was vacuum filtered. The filtrate was concentrated. The residue was re-dissolved in methylene chloride and purified by column chromatography, eluting with a mixture of ethyl acetate (0-10%) and hexanes (100-90%) to afford 2.08 g (89%) of the product 51 as an orange solid. 1H NMR (CDCl3) δ 1.46 (s, 9H), 2.66 (t, J=6.5 Hz, 2H), 3.36-3.43 (m, 2H), 4.86 (br s, 1H), 7.53 (d, J=8.7 Hz, 2H), 8.16 (d, J=8.7 Hz, 2H). m/z (APCI)=291 [C15H18N2O4+H]+, 191 [C15H18N2O4−Boc+H]+.
[4-(4-Aminophenyl)butyl]carbamic acid tert-butyl ester (52)
To a solution of compound 51 (2.01 g, 6.923 mmol) in ethanol (50 mL) was added 10% palladium on carbon (737 mg, wet) in one portion under argon protection. The flask was evacuated and re-filled with argon three times (to remove oxygen), and the mixture stirred at room temperature overnight under one atmosphere of hydrogen. The reaction system was then purged with nitrogen, and the catalyst was vacuum filtered and washed with ethanol (2×5 mL). The filtrate and washings were combined and concentrated under reduced pressure. The residue was chromatographed over silica gel, eluting with a mixture of ethyl acetate (0-25%) and hexanes (100-75%), to afford 1.75 g (96%) of the product 52 as a colorless viscous oil. 1H NMR (CDCl3) δ 1.44 (s, 9H), 1.47-1.64 (m, 4H), 2.50 (t, J=7.1 Hz, 2H), 3.08-3.14 (m, 2H), 3.57 (br s, 2H), 4.55 (br s, 1H), 6.62 (d, J=8.2 Hz, 2H), 6.95 (d, J=8.2 Hz, 2H). m/z (APCI)=265 [C15H24N2O2+H]+.
4-{4-[Bis(methanesulfonyl)amino]phenyl}butylcarbamic acid tert-butyl ester (53)
Compound 52 (0.16 g, 0.605 mmol) was dissolved in anhydrous THF (5 mL). To the clear solution were sequentially added triethylamine (0.18 mL, 1.21 mmol) and 4-dimethylaminopyridine (15 mg, 0.121 mmol). The mixture was cooled to about −10° C. for 15 min by a methanol-ice bath. To the cold solution was slowly added methanesulfonyl chloride (51 μL). The solution was further stirred at the temperature (about −10° C.) for an additional 30 min, then allowed to slowly warm up to room temperature by removing the cooling bath. The reaction mixture was concentrated under reduced pressure. The residue was chromatographed over silica gel, eluting with a mixture of ethyl acetate (0-35%) and hexanes (100-65%), to afford 0.212 g (83%) of the product 53 as a white solid. 1H NMR (CDCl3) δ 1.44 (s, 9H), 1.52-1.70 (m, 4H), 2.68 (t, J=7.4 Hz, 2H), 3.13-3.18 (m, 2H), 3.40 (s, 6H), 4.53 (br s, 1H), 7.27 (s, 4H). m/z (APCI)=321 [C17H28N2O6S2−Boc+H]+.
4-[4-Bis(methanesulfonyl)amino)phenyl]butylamine (54)
A solution of compound 53 (0.21 g, 0.499 mmol) dissolved in methylene chloride (10 mL) was treated with trifluoroacetic acid (1 mL) at room temperature for 2 hours, then concentrated under vacuum. The residue was taken into methanol (2 mL), and concentrated again under reduced pressure. The procedure was repeated three times to ensure no residual trifluoroacetic acid remained. The product was completely dried under vacuum, and directly used for the next reaction without further purification. 0.196 g (100%) of the compound 54 was obtained as a colorless viscous oil. 1H NMR (DMSO-d6) δ 1.50-1.72 (m, 4H), 2.65 (t, J=7.4 Hz, 2H), 2.84-2.89 (m, 2H), 3.51 (s, 6H), 7.33 (d, J=8.3 Hz, 2H), 7.42 (d, J=8.3 Hz, 2H). m/z (APCI)=321 [C12H20N2O4S2+H]+.
N-(3,5-Diamino-6-chloropyrazine-2-carbonyl)-N′-[4-(4-bis(methanesulfonyl)-aminophenyl)butyl]guanidine (55, 10316)
Compound 54 (0.095 g, 0.296 mmol) was mixed with ethanol (5 mL). The mixture was heated at 65° C. for 15 min to achieve complete dissolution. To the clear solution were sequentially added diisopropylethylamine (0.26 mL, 1.48 mmol) and 1-(3,5-diamino-6-chloropyrazinoyl)-2-methylisothiourea hydriodide (0.127 g, 0.326 mmol). The mixture was heated at the same temperature for an additional 1.5 hours, and subsequently concentrated under vacuum. The residue was chromatographed over silica gel, eluting with a mixture of concentrated ammonium hydroxide (0-1%), methanol (0-10%), and methylene chloride (100-89%), to afford 0.113 g (72%) of the product 55 as a light yellow solid. mp 174-176° C. (decomposed). 1H NMR (DMSO-d6) δ 1.48-1.68 (m, 4H), 2.64-2.69 (m, 2H), 3.12-3.25 (m, 2H), 3.51 (s, 6H), 6.65-6.78 (br s, 3H), 7.31 (d, J=8.2 Hz, 2H), 7.41 (d, J=8.2 Hz, 2H), 9.05 (br s, 2H). m/z (APCI)=533 [C18H25ClN8O5S2+H]+.
Example 5
N-(3,5-diamino-6-chloropyrazine-2-carbonyl)-N′-{4-[4-(2-chlorotrimethylammonium)ethoxyphenyl]butyl}guanidine hydrochloride (11223)




{4-[4-(2-Dimethylaminoethoxy)phenyl]butyl}carbamic acid benzyl ester (62)
A mixture of [4-(4-hydroxyphenyl)butyl]carbamic acid benzyl ester (1.5 g, 5 mmol), 2-dimethylaminochloroethane hydrochloride (1.4 g, 10 mmol), potassium carbonate (2.76 g, 20 mmol) and 18-crown-6 ether (154 mg, 0.58 mmol) was stirred at 80° C. (oil bath) for 18 h. After this time, the solvent was removed under reduced pressure and the residue was purified by FlashTM (BIOTAGE, Inc) (90 g silica gel cartridge 40M, 10:1:0.1 chloroform/methanol/concentrated ammonium hydroxide) to provide 62 (1.1 g, 61%) as a white solid. 1H NMR (300 MHz, CDCl3) δ 1.56 (m, 4H), 2.32 (s, 6H), 2.56 (m, 2H), 2.71 (t, 2H), 3.18 (m, 2H), 4.04 (t, 2H), 4.78 (br s, 1H), 5.08 (s, 2H), 6.83 (d, 2H), 7.06 (d, 2H), 7.35 (m, 5H).
4-[4-(2-Dimethylaminoethoxy)phenyl]butylamine (63)
The protected amine 62 (0.552 g, 1.5 mmol) was stirred with 10% palladium on carbon (0.127 g, wet) in methanol (50 mL) at room temperature for 3.5 h under hydrogen (1 atm). After this time, the catalyst was filtered off and the solvent was removed under reduced pressure to give the free amine 63 (0.27 g, 77%) as a white powder. 1H NMR (300 MHz, CD3OD) δ 1.55 (m, 4H), 2.32 (s, 6H), 2.54 (m, 2H), 2.75 (m, 2H), 4.02 (m, 2H), 6.78 (d, 2H), 7.08 (d, 2H).
1-tert-Butyloxycarbonyl-3-(3,5-diamino-6-chloropyrazine-2-carbonyl)-2-methyl-isothiourea (64)
4-Dimethylaminopyridine (87 mg, 0.7 mmol) was added to a stirring solution of di-tert-butyl dicarbonate (0.8 g, 3.6 mmol) and 1-(3,5-diamino-6-chloropyrazinoyl)-2-methylisothiourea hydriodide (1.14 g, 0.5 mmol) in THF/triethylamine (62 mL, 30/1). The reaction mixture was then stirred at room temperature for 48 h. After this time, the solvent was removed under reduced pressure. The residue was purified by flash chromatography (silica gel, 1:1 ethyl acetate/hexanes) to give the protected isothiourea 64 (0.34 g, 32%) as a yellow powder. 1H NMR (300 MHz, DMSO-d6) δ 1.51 (s, 9H), 2.30 (s, 3H), 7.40 (br s, 4H).
N-tert-Butyloxycarbonyl-N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)-N′′-{4-[4-(2-dimethylaminoethoxy)phenyl]butyl}guanidine (65)
A suspension of compound 63 (0.22 g, 0.93 mmol) and 64 (0.33 g, 0.92 mmol) in THF/triethylamine (11 mL, 10/1) was stirred at room temperature for 48 h. After this time, a clear solution was formed. The solvent was removed under reduced pressure and the residue was purified by flash chromatography (silica gel, 10:1:0.1 chloroform/methanol/concentrated ammonium hydroxide) to provide the guanidine 65 (0.3 g, 60%) as a yellow solid. 1H NMR (300 MHz, DMSO-d6) δ 1.42 (s, 9H), 1.55 (m, 4H), 2.19 (s, 6H), 2.58 (m, 4H), 3.99 (m, 2H), 6.83 (d, 2H), 7.12 (d, 2H), 7.40 (br s, 2H), 9.02 (m, 2H).
N-tert-Butyloxycarbonyl-N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)-N′′-{4-[4-(2-iodotrimethylammoniumethoxy)phenyl]butyl}guanidine (66)
Iodomethane (30 μL, 0.49 mmol) was added to a suspension of 65 (0.29 g, 0.52 mmol) in THF (50 mL). The mixture was stirred at room temperature overnight. After this time, additional THF (7 mL) was added and stirring was continued for 2 d to give a clear solution. The solvent from the resulting solution was removed under reduced pressure. The residue was washed with THF (2×5 mL) and dried to afford the salt 66 (0.22 g, 32%) as a yellow powder. 1H NMR (300 MHz, DMSO-d6) δ 1.42 (s, 9H), 1.56 (m, 4H), 2.58 (m, 2H), 3.15 (s, 9H), 3.75 (m, 2H), 4.42 (m, 2H), 6.92 (d, 2H), 7.18 (d, 2H), 7.36 (br s, 2H), 9.02 (m, 1H).
[2-(4-{4-[N′-(3,5-Diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}phenoxy)ethyl]-trimethylammonium chloride (67, 11223)
Trifluoroacetic acid (2 mL) was added to the protected guanidine 66 (0.092 g, 0.13 mmol). The reaction mixture was stirred at room temperature for 15 min. The solvent was removed under reduced pressure and the residue was washed with ethyl acetate (2×1 mL) and dried in vacuum. The obtained dry solid was treated with an aqueous solution of ammonium hydroxide (15%, 1 mL). The formed precipitate was collected by centrifugation and washed with cold water (1 mL). The remaining solid was dissolved in 10% hydrochloric acid, and the solvent was then removed under reduced pressure. The resulting yellow solid was dried in vacuum to give compound 67 (0.055 g, 82%). 1H NMR (300 MHz, CD3OD) δ 1.68 (br s, 4H), 2.65 (m, 2H), 3.35 (m, 2H), 3.83 (m, 2H), 4.46 (m, 2H), 4.95 (s, 9H), 6.96 (d, 2H), 7.18 (d, 2H), 9.25 (br s, 1H). m/z (APCI)=499 [C21H32C12N8O2+H]+.
Sodium Channel Blocking Activity
The compounds shown in the Tables below were tested for potency in canine bronchial epithelia using the in vitro assay described above. Amiloride was also tested in this assay as a positive control. The results for the compounds of the present invention are reported as fold-enhancement values relative to amiloride.
Example 6
















 











X =
Y =
Fold Amiloride*






—NHCO2+
—CO2CH3
29.4 ± 10 (5) 



| | | NH2
—CO2H
17.2 ± 5.8 (4)



| | | NH2
—CO2CH3
 21.1 ± 14.1 (2)



| | | | |NH2
—CONH2





*Relative potency for Amiloride = 665 nM.


**Relative potency for CF552 = 100 using IC50 from 552 in same run.


***3rd Wash


(a) Old Database


(b) NA = Not Available


(c) 1 of 5 is high outlier (252)






Example 7
















 










X =
Fold Amiloride*






N(CH3)2
25.3 ± 2.3 (3)



N⊕(CH3)3
39.4 ± 0.3 (2)





*Relative potency for Amiloride = 665 nM.


**Relative potency for CF552 = 100 using IC50 from 552 in same run.


***3rd Wash


a Guanidinine is Acylated






Example 8































    R =\Z =




    NH(C═O)CH2CH2R





NH2




Xamiloride
86.2 ± 3.01
170





    R =\Z =





















 


Xamiloride
182.3 ± 106.3 (12)
139.7 ± 62.4









Example 9







Synthesis of 2-amino-3-(4-{4-[N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)-guanidino]butyl}phenoxy)propionic acid methyl ester (PSA 11041)




3-[4-(4-Benzyloxycarbonylaminobutyl)phenoxy]-2-(tritylamino)propionic acid methyl ester (1)
Commercially available N-trityl-L-serine methyl ester (1.60 g, 5.34 mmol) was combined with triphenylphosphine (1.28 g, 4.88 mmol) and [4-(4-hydroxyphenyl)butyl]carbamic acid benzyl ester (2.0 g, 6.68 mmol) in benzene (40 mL) at room temperature. Diisopropyl azodicarboxylate (0.958 mL, 4.86 mmol) was added dropwise and the reaction was stirred for 14 days. The solvent was removed under reduced pressure and the residue was purified by column chromatography (silica gel, eluent: 6:1, v/v dichloromethane/ethyl acetate) to provide compound 101 (2.22 g, 51%). 1H NMR (300 MHz, CDCl3) δ 7.52 (m, 6H), 7.39-7.14 (m, 14H), 7.06 (d, 2H), 6.79 (d, 2H), 5.09 (s, 2H), 4.72 (m, 1H), 4.24 (m, 1H), 4.01 (m, 1H), 3.72 (m, 1H), 3.22 (s, 3H), 3.18 (m, 2H), 2.88 (d, 1H), 2.57 (m, 2H), 1.66-1.48 (m, 4H). Rf=0.91 (5:1 v/v dichloromethane/ethyl acetate).
3-[4-(4-Benzyloxycarbonylaminobutyl)phenoxy]-2-tert-butoxycarbonylamino-propionic acid methyl ester (102)
Compound 101 (2.22 g, 3.45 mmol) was dissolved in a mixture of dichloromethane/water (25 mL/0.5 mL) then trifluoroacetic acid (0.75 mL, 10.0 mmol) was added and the reaction was stirred for 2 h. The solvent was removed under reduced pressure and the residue was dissolved in dichloromethane (25 mL) and treated with triethylamine (0.72 mL, 5.12 mmol) and di-tert-butyl dicarbonate (0.829 g, 3.79 mmol) for 72 h. Removal of the solvents under reduced pressure followed by column chromatography (silica gel, eluent: 9:1, v/v dichloromethane/ethyl acetate) provided compound 102 (0.90 g, 52%). 1H NMR (300 MHz, CDCl3) δ 7.34 (m, 5H), 7.07 (d, 2H), 6.79 (d, 2H), 5.50 (d, 1H), 5.10 (s, 2H), 4.68 (m, 2H), 4.38 (m, 1H), 4.17 (m, 1H), 3.77 (s, 3H), 3.20 (m, 2H), 2.57 (m, 2H), 1.67-1.48 (m, 4H), 1.45 (s, 9H). m/z (APCI) 401 [C27H36N2O7−Boc+H]+.
3-[4-(4-Aminobutyl)phenoxy]-2-tert-butoxycarbonylaminopropionic acid methyl ester (103)
Compound 102 (505 mg, 1.00 mmol) was dissolved in methanol (20 mL) and 10% palladium on carbon (100 mg) was added. The flask was evacuated, filled with hydrogen gas under balloon pressure and stirred overnight. Filtration through celite to remove the catalyst followed by removal of the solvent under reduced pressure provided compound 103 (366 mg, 98%). 1H NMR (300 MHz, CDCl3) δ 7.08 (d, 2H), 6.80 (d, 2H), 5.51 (d, 1H), 4.66 (m, 1H), 4.38 (m, 1H), 4.17 (m, 1H), 3.78 (s, 3H), 2.73 (m, 2H), 2.58 (m, 2H), 1.90 (br s, 2H), 1.62 (m, 2H), 1.50 (m, 2H), 1.48 (s, 9H).
2-tert-Butoxycarbonylamino-3-(4-{4-[N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)-guanidino]butyl}phenoxy)propionic acid methyl ester (104)
Compound 103 (366 mg, 0.99 mmol) was dissolved in ethanol (8 mL) then diisopropylethyl amine (0.86 mL, 4.9 mmol) and 1-(3,5-diamino-6-chloropyrazine-2-methyl-isothiourea hydriodide (407 mg, 1.04 mmol) were added and the reaction was heated to 65° C. for 1.5 h. The solvents were removed under reduced pressure and the residue was purified by column chromatography (silica gel, eluent: 90:10:1, v/v dichloromethane/methanol/conc. ammonium hydroxide) to provide compound 104 (215 mg, 37%). 1H NMR (300 MHz, DMSO-d6) δ 9.13 (m, 1H), 8.89 (m, 1H), 8.70 (m, 1H), 7.45 (m, 4H), 7.13 (d, 2H), 6.85 (d, 2H), 4.43 (m, 1H), 4.17 (m, 2H), 3.68 (s, 3H), 3.30 (m, 2H), 2.90 (m, 1H), 2.56 (m, 2H), 1.58 (m, 4H), 1.40 (s, 9H). m/z (APCI) 579 [C25H35ClN8O6+H]+. Rf=0.65 (85:15:1 dichloromethane/methanol/conc. ammonium hydroxide).
2-Amino-3-(4-{4-[N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}-phenoxy)propionic acid methyl ester (105, PSA 11041)
Compound 104 (40 mg, 0.069 mmol) was dissolved in dichloromethane (3 mL) and treated with trifluoroacetic acid (1.5 mL) for 30 min. Removal of the solvents under reduced pressure followed by column chromatography (silica gel, eluent: 85:15:1, v/v dichloromethane/methanol/conc. ammonium hydroxide) provided compound 105 (28 mg, 84%). 1H NMR (300 MHz, DMSO-d6) δ 9.06 (m, 1H), 7.11 (d, 2H), 6.82 (m, 4H), 4.06 (m, 2H), 3.74 (m, 1H), 3.65 (br s, 3H), 3.17 (m, 2H), 2.55 (m, 2H), 1.55 (m, 4H). m/z (APCI) 479 [C20H27ClN8O4+H]+. Rf=0.46 (85:15:1 dichloromethane/methanol/conc. ammonium hydroxide).
Example 10




Synthesis of 2-amino-3-(4-{4-[N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)-guanidino]butyl}phenoxy)propionic acid (60, PSA 10941)




Compound 105 (175 mg, 0.302 mmol) was dissolved in a mixture of tetrahydrofuran/water (5 mL/2.5 mL) and treated with 1 N NaOH (0.75 mL, 0.75 mmol) for 1 h. The solvents were removed under reduced pressure and the residue was dissolved in a minimum amount of water, treated with 2 N HCl to precipitate the product then filtered to collect the product. The solid was suspended in dichloromethane (3 mL) and treated with trifluoroacetic acid (3 mL) for 1 h. Removal of the solvents under reduced pressure followed by column chromatography (silica gel, eluent: 80:20:1, v/v dichloromethane/methanol/conc. ammonium hydroxide to start then change to 6:3:1, v/v to obtain the product) provided compound 106 (70 mg, 49%). 1H NMR (300 MHz, DMSO-d6) δ 7.78 (br m, 4H), 7.13 (d, 2H), 6.88 (d, 2H), 6.67 (m, 2H), 4.28 (m, 1H), 4.11 (m, 1H), 3.53 (m, 1H), 3.16 (m, 2H), 2.56 (m, 2H), 1.55 (m, 4H). m/z (APCI) 465 [C19H25ClN8O4+H]+.
Example 11




Synthesis of 2-amino-4-(4-{4-[N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)-guanidino]butyl}phenoxy)butyric acid methyl ester hydrochloride (PSA 17587)




2-Amino-4-hydroxybutyric acid methyl ester hydrochloride (70)
A suspension of DL-homoserine (1.00 g, 8.39 mmol) in methanol (40 mL) was placed in an ice bath. Trimethylsilyl chloride (2.34 mL, 18.5 mmol) was added dropwise via syringe. The reaction mixture gradually became homogenous and was further stirred at rt for 14 h, concentrated by rotary evaporation, and further dried under high vacuum. The crude oil thus obtained was used for the next step without further purification. 1H NMR (300 MHz, CD3OD) δ 2.00-2.24 (m, 2H), 3.70-3.80 (m, 2H), 3.85 (s, 3H), 4.12-4.22 (m, 1H). m/z (ESI) 134 [C5H11NO3+H]+.
2-tert-Butoxycarbonylamino-4-hydroxybutyric acid methyl ester (80)
2-Amino-4-hydroxybutyric acid methyl ester hydrochloride (70) was suspended in anhydrous THF (15 mL) and placed in an ice bath. Diisopropylethylamine (2.92 mL, 16.8 mmol) was added via syringe, followed by the addition of DMAP (205 mg, 1.68 mmol) and Boc2O (3.85 g, 17.6 mmol). The mixture was stirred at 0° C. for 10 min and at room temperature for 14 h. Solvent was removed under reduced pressure and residue was taken up by ethyl acetate (100 mL), washed with water (30 mL×2) and brine (40 mL), dried over sodium sulfate, and concentrated. The colorless oil (1.96 g) was used for the next step without further purification. 1H NMR (300 MHz, CDCl3) δ 1.45 (s, 9H), 2.05-2.28 (m, 2H), 3.68-3.75 (m, 2H), 3.80 (s, 3H), 4.08-4.15 (m, 1H), 5.30-5.41 (m, 1H). m/z (ESI) 234 [C10H19NO5+H]+.
4-Bromo-2-tert-butoxycarbonylaminobutyric acid methyl ester (90)
A solution of triphenylphosphine (2.20 g, 8.39 mmol) in dry CH2Cl2 (20 mL) was added dropwise via syringe to a solution of N-Boc homoserine methyl ester 80 (8.39 mmol) and carbon tetrabromide (4.18 g, 12.60 mmol) in dry CH2Cl2 (20 mL). The resulting dark solution was stirred at room temperature for 16 h. Hexanes was added and precipitates were removed by suction filtration. The filtrate was concentrated under reduced pressure and subject to flash silica gel column chromatography using ethyl acetate/hexanes (1:10, v/v then 1:6, v/v) to give the desired product 90 as a yellow oil (501 mg, 20% overall yield from homoserine). 1H NMR (300 MHz, CDCl3) δ 1.45 (s, 9H), 2.12-2.48 (m, 2H), 3.39-3.47 (m, 2H), 3.78 (s, 3H), 4.33-4.48 (m, 1H), 5.10-5.22 (m, 1H). m/z (ESI) 296 [C10H18BrNO4+H]+.
4-[4-(4-Benzyloxycarbonylaminobutyl)phenoxy]-2-tert-butoxycarbonylamino-butyric acid methyl ester (100)
Potassium carbonate (935 mg, 6.77 mmol) was added in one portion to a solution of 4-[4-(benzyloxycarbonylamino)butyl]phenol (506 mg, 1.69 mmol) and N-Boc bromide 90 (501 mg, 1.69 mmol) in anhydrous DMF (10 mL). The reaction mixture was stirred at 70° C. (oil bath) for 14 h, cooled to room temperature, and diluted with ethyl acetate (100 mL) and hexanes (20 mL). The mixture was washed with water (4×20 mL) and brine (40 mL) and concentrated under reduced pressure. Flash silica gel column chromatography using ethyl acetate/CH2Cl2 (1:25, 1:20, v/v) gave the desired product 100 as a thick yellow oil (718 mg, 83% yield). 1H NMR (300 MHz, CDCl3) δ 1.44 (s, 9H), 1.48-1.68 (m, 4H), 2.12-2.38 (m, 2H), 2.48-2.60 (m, 2H), 3.10-3.24 (m, 2H), 3.75 (s, 3H), 3.97-4.06 (m, 2H), 4.41-4.52 (m, 1H), 4.70 (br s, 1H), 5.09 (s, 2H), 5.25-5.37 (m, 1H), 6.70-6.80 (m, 2H), 6.95-7.09 (m, 2H), 7.30-7.38 (m, 5H). m/z (ESI) 515 [C28H38N2O7+H]+.
{4-[4-(3-tert-Butoxycarbonylamino-3-carbamoylpropoxy)phenyl]butyl}carbamic acid benzyl ester (110)
Ammonia (7 M in methanol, 20 mL) was added to a solution of N-Boc methyl ester 100 (718 mg, 1.40 mmol) in methanol (5 mL) and the mixture was stirred at room temperature in a sealed tube for 40 h. The mixture was concentrated by rotary evaporation and purified by flash silica gel column chromatography using methanol/dichloromethane (1:30, 1:20, v/v) to give the desired amide 110 as a white solid (436 mg, 63% yield). 1H NMR (300 MHz, CDCl3) δ 1.45 (s, 9H), 1.48-1.68 (m, 4H), 2.09-2.32 (m, 2H), 2.48-2.61 (m, 2H), 3.08-3.25 (m, 2H), 3.97-4.20 (m, 2H), 4.30-4.45 (m, 1H), 4.75 (br s, 1H), 5.09 (s, 2H), 5.48-5.58 (m, 1H), 5.62 (br s, 1H), 6.38 (br s, 1H), 6.75-6.85 (m, 2H), 6.99-7.10 (m, 2H), 7.28-7.40 (m, 5H). m/z (ESI) 500 [C27H37N3O6+H]+.
{3-[4-(4-Aminobutyl)phenoxy]-1-carbamoylpropyl}carbamic acid tert-butyl ester (120)
A mixture of N-Cbz amide 110 (436 mg, 0.873 mmol) and Pearlman's catalyst (100 mg) in methanol (10 mL) and dichloromethane (10 mL) was stirred under atmospheric hydrogen for 18 h. Catalyst was removed by suction filtration over Celite and the filtrate was concentrated to give a colorless oil (318 mg, 100% crude yield). Crude product 120 was used for next step without further purification. m/z (ESI) 366 [C19H31N3O4+H]+.
[1-Carbamoyl-3-(4-{4-[NA-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino]-butyl}phenoxy)propyl]carbamic acid tert-butyl ester (130)
A solution of N-Boc amine 120 (318 mg, 0.873 mmol) and diisopropylethylamine (0.30 mL, 1.75 mmol) in absolute ethanol (8 mL) was stirred at 70° C. for 5 min, after which 1-(3,5-diamino-6-chloropyrazine-2-carbonyl)-2-methylisothiourea hydriodide (441 mg, 1.13 mmol) was added in one portion. The reaction mixture was stirred at that temperature for 2.5 h and cooled to room temperature. It was concentrated by rotary evaporation and purified by flash silica gel column chromatography using dichloromethane/methanol/concentrated ammonium hydroxide (200:10:0, 200:10:1, 150:10:1, and 100:10:1, v/v) to give the desired product 130 as a yellow solid (470 mg, 93% yield). m/z (ESI) 578 [C25H36ClN9O5+H]+.
2-Amino-4-(4-{4-[N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}-phenoxy)butyric acid methyl ester dihydrochloride (140, PSA 17587) and 2-amino-4-(4-{4-[N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}phenoxy)-butyramide dihydrochloride (150, PSA 17588)
A solution of N-Boc amide 130 and HCl (4 M in dioxane, 3 mL) in methanol (6 mL) was stirred at room temperature for 14 h, and then evaporated to dryness in vacuo. Flash silica gel column chromatography using dichloromethane/methanol/concentrated ammonium hydroxide (200:10:0, 200:10:1, 150:10:1, 100:10:1, and 100:10:2, v/v) to give 2-amino-4-(4-{4-[N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}-phenoxy)butyric acid methyl ester (fast eluting, 313 mg, 78% yield) and 2-amino-4-(4-{4-[N-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}phenoxy)butyramide (slow eluting, 70 mg, 18% yield).
2-Amino-4-(4-{4-[N-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}-phenoxy)butyric acid methyl ester was dissolved in methanol and treated with 3 drops of 4N aqueous HCl and the mixture was then concentrated in vacuo immediately. It was further co-evaporated with water and dried at 40° C. under high vacuum for 16 h to give 140. 1H NMR (300 MHz, CD3OD) δ 1.62-1.78 (m, 4H), 2.35-2.45 (m, 2H), 2.59-2.69 (m, 2H), 3.33-3.40 (m, 2H), 3.84 (s, 3H), 4.09-4.18 (m, 2H), 4.24-4.32 (m, 1H), 6.80 (d, J=8.6 Hz, 2H), 7.08 (d, J=8.6 Hz, 2H). m/z (ESI) 493 [C21H29ClN8O4+H]+. mp 88-90° C.
2-Amino-4-(4-{4-[N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}-phenoxy)butyramide was dissolved in methanol and treated with 3 drops of 4N aqueous HCl and the mixture was then concentrated in vacuo immediately. It was further co-evaporated with water and dried at 40° C. under high vacuum for 16 h to give 150. 1H NMR (300 MHz, CD3OD) δ 1.60-1.78 (m, 4H), 2.00-2.30 (m, 2H), 2.55-2.65 (m, 2H), 3.31-3.38 (m, 2H), 3.70-3.78 (m, 1H), 4.05-4.12 (m, 2H), 6.86 (d, J=8.6 Hz, 2H), 7.12 (d, J=8.6 Hz, 2H). m/z (ESI) 478 [C20H28ClN9O3+H]+. mp 128-130° C.
Example 12




Synthesis of 2-amino-4-(4-{4-[N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)-guanidino]butyl}phenoxy)butyric acid (PSA 17666)




2-Amino-4-(4-{4-[N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}-phenoxy)butyric acid methyl ester (140) (110 mg, 0.223 mmol) was dissolved in a mixture of methanol (1 mL)/THF (1 mL)/water (1 mL). Lithium hydroxide monohydrate (14 mg, 0.334 mmol) was added and the reaction mixture was stirred at room temperature for 16 h before it was concentrated in vacuo. The resulting residue was diluted with water (1 mL), acidified to pH 2 with 1N aqueous HCl, and purified through a DOWEX 50WX 8-200 ion exchange resin eluting with 10% aqueous ammonium hydroxide. The desired product was obtained as a yellow solid (75 mg, 70% yield). 1H NMR (300 MHz, CD3OD) δ 1.55-1.78 (m, 4H), 2.10-2.45 (m, 2H), 2.52-2.65 (m, 2H), 3.30-3.35 (m, 2H), 3.65-3.75 (m, 1H), 4.05-4.15 (m, 2H), 6.85 (d, J=8.5 Hz, 2H), 7.10 (d, J=8.5 Hz, 2H). m/z (ESI) 479 [C20H27ClN8O4+H]+. mp 178-180° C.
Example 13




Synthesis of 2-amino-3-[2-(4-{4-[N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)-guanidino]butyl}phenoxy)ethylamino]propionamide tri-hydrochloride (PSA 18704)




3-{2-[4-(4-Benzyloxycarbonylaminobutyl)phenoxy]ethylamino}-2-N,N′-di-(tert-butoxycarbonyl)aminopropionic acid methyl ester (160)
A mixture of {4-[4-(2-aminoethoxy)phenyl]butyl}carbamic acid benzyl ester hydrochloride (141 mg, 0.372 mmol), 2-[N,N′-di(tert-butoxycarbonyl)]aminoacrylic acid methyl ester (102 mg, 0.338 mmol), and triethylamine (0.16 mL, 1.11 mmol) in methanol (3 mL) was stirred at 55° C. (oil bath) for 16 h. It was then cooled to room temperature. The solvent was removed by rotary evaporation. The residue was taken up in ethyl acetate and washed with saturated sodium bicarbonate solution and brine. The organic layer was concentrated in vacuo and purified by flash silica gel column chromatography using ethyl acetate/hexanes (gradient 1:10, 1:6, 1:4, and 1:2, v/v) to give the desired Michael adduct 160 (110 mg, 51% yield). 1H NMR (300 MHz, CDCl3) δ 1.42 (s, 9H), 1.43-1.50 (m, 9H), 1.50-1.65 (m, 4H), 2.50-2.60 (m, 2H), 3.12-3.25 (m, 2H), 3.49-3.72 (m, 4H), 3.75 (s, 3H), 3.98-4.10 (m, 2H), 4.45-4.65 (m, 1H), 4.79 (br s, 1H), 5.09 (s, 2H), 5.50 (m, 1H), 6.81 (d, J=8.5 Hz, 211), 7.06 (d, J=8.5 Hz, 2H), 7.28-7.38 (m, 5H). m/z (ESI) 644 [C34H49N3O9+H]+.
(4-{4-[2-(2-Di-(tert-butoxycarbonyl)amino-2-carbamoylethylamino)ethoxy]phenyl}-butyl)carbamic acid benzyl ester (170)
Methanolic ammonia (7M, 10 mL, 70 mmol) was added to a solution of methyl ester 160 (110 mg, 0.171 mmol) in methanol (3 mL). The mixture was stirred in a sealed tube at room temperature for 3 days, concentrated under reduced pressure, and further dried under high vacuum for 16 h. The crude solid product 170 (94 mg, 87% crude yield) was used for next step without further purification. m/z (ESI) 629 [C33H48N4O8+H]+.
(2-{2-[4-(4-Aminobutyl)phenoxy]ethylamino}-1-carbamoylethyl)dicarbamic acid di-tert-butyl ester (180)
N-Cbz amide 170 (94 mg, 0.150 mmol) was dissolved in methanol (5 mL) and dichloromethane (5 mL) and the solution was purged with nitrogen. Pearlman's catalyst (palladium hydroxide on carbon, 26 mg) was added and the reaction mixture was stirred under a hydrogen atmosphere at room temperature for 16 h. Catalyst was filtered off over Celite and the filtrate was concentrated to give the desired product 180 as a white solid (73 mg, 99% crude yield) and was used for next step without further purification. m/z (ESI) 495 [C25H42N4O6+H]+.
2-(N,N′-Di-tert-butoxycarbonyl)amino-3-{4-{4-[N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}phenoxy)ethylamino}propionamide (190)
A solution of primary amine 180 (73 mg, 0.15 mmol) and diisopropylethylamine (52 μL, 0.298 mmol) in absolute ethanol (3 mL) was stirred at 70° C. for 5 min, after which 1-(3,5-diamino-6-chloropyrazine-2-carbonyl)-2-methylisothiourea hydriodide (69 mg, 0.18 mmol) was added in one portion. The reaction mixture was stirred at that temperature for 2 h and cooled to room temperature, and concentrated by rotary evaporation. The residue was purified by silica gel column chromatography using dichloromethane/methanol/concentrated ammonium hydroxide (200:10:1, 150:10:1, and 100:10:1, v/v) to give the desired product 190 as a yellow solid (68 mg, 65% yield). m/z ESI 707 [C31H47ClN10O7+H]+.
2-Amino-3-[2-(4-{4-[N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino]-butyl}phenoxy)ethylamino]propionamide trihydrochloride (200, PSA 18704)
A solution of N,N′-bis(Boc) amide 190 and HCl in dioxane (4 M, 3 mL) was stirred at room temperature for 14 h and then concentrated by rotary evaporation. The residue was re-dissolved in methanol, evaporated to dryness and further dried under high vacuum at 40° C. for 16 h. Product 200 was obtained as a yellow solid (58 mg, 98% yield). 1H NMR (300 MHz, CD3OD) δ 1.65-1.75 (m, 4H), 2.60-2.69 (m, 2H), 3.31-3.37 (m, 2H), 3.55-3.68 (m, 4H), 3.71-3.76 (m, 1H), 4.32-4.35 (m, 2H), 4.45-4.48 (m, 1H), 6.98 (d, J=8.5 Hz, 2H), 7.18 (d, J=8.5 Hz, 2H). m/z (ESI) 507 [C21H31ClN10O3+H]+. mp 160-160° C.
Example 14







Synthesis of 2-(R)-amino-5-(4-{4-[N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)-guanidino]butyl}phenyl)pentanoic acid methyl ester (PSA 19603)




[4-(4-Iodophenyl)but-3-ynyl]carbamic acid benzyl ester (210)
To a mixture of anhydrous THF and triethylamine (24 mL, 2/1, v/v) were sequentially added 1,4-diiodobenzene (2.03 g, 6.15 mmol) and copper (1) iodide (0.094 g, 0.246 mmol). The mixture was stirred at room temperature for 15 min. The flask was then evacuated and re-filled with Argon. The procedure was repeated three more times to ensure no oxygen remained. The catalyst, dichlorobis(triphenylphosphine)palladium(II) (0.173 g, 0.246 mmol) was added into the mixture under Argon protection. The other starting material, but-3-ynylcarbamic acid benzyl ester (0.50 g, 2.46 mmol), dissolved in TBF (8 mL) was added dropwise over 6 hours. The newly formed reaction mixture was further stirred at room temperature overnight. The solid in the reaction mixture was vacuum filtered. The filtrate was concentrated. The residue was re-dissolved in dichloromethane and purified by column chromatography, eluting with a mixture of ethyl acetate (0-12%) and hexanes (100-88%) to afford the product 210 (0.852 g, 86%) as an off-white solid. 1H NMR (300 MHz, CDCl3): δ 2.61 (t, J=6.4 Hz, 2H), 3.44 (t, J=6.4 Hz, 2H), 5.07 (br s, 1H), 5.12 (s, 2H), 7.10 (d, J=8.3 Hz, 2H), 7.35 (m, 5H), 7.63 (d, J=8.3 Hz, 2H). m/z (APCI) 405 [C18H16NO2+H]+.
2-(R)-tert-Butoxycarbonylaminopent-4-ynoic acid methyl ester (220)
The commercially available compound, 2-(R)-tert-butoxycarbonylaminopent-4-ynoic acid (0.321 g, 1.50 mmol) was dissolved in anhydrous DMF (5 mL). To the solution was added cesium carbonate (0.54 g, 1.65 mmol) in one portion. The mixture was stirred at room temperature for 45 min before methyl iodide (0.20 mL, 3.00 mmol) was added, and further stirred for three hours. The reaction was quenched with water (10 mL). The organics was extracted with dichloromethane (2×30 mL), washed with water (3×50 mL), and dried over anhydrous sodium sulfate. The solvent was completely removed under vacuum. The residue was further dried under high vacuum over night and used in the next reaction without further purification. The product 220 was obtained as a colorless viscous oil (0.326 g, 95% yield). 1H NMR (300 MHz, CDCl3): δ 1.48 (s, 9H), 2.08 (t, J=2.6 Hz, 1H), 2.75 (t, J=6.3 Hz, 2H), 3.78 (s, 3H), 4.46 (m, 1H), 5.32 (br s, 5H).
5-[4-(4-Benzyloxycarbonylaminobut-1-ynyl)phenyl]-2-(R)-tert-butoxycarbonylaminopent-4-ynoic acid methyl ester (230)
Compound 210, [4-(4-iodophenyl)-but-3-ynyl]carbamic acid benzyl ester (0.52 g, 1.283 mmol) was dissolved in a mixture of anhydrous THF and triethylamine (8 mL, 1/1, v/v). To the solution was added copper (I) iodide (0.025 g, 0.128 mmol). The mixture was stirred at room temperature for 15 min. The flask was then evacuated and re-filled with Argon. The procedure was repeated three more times to ensure no oxygen remained. The catalyst, dichlorobis(triphenylphosphine)palladium(II) (0.09 g, 0.128 mmol) was added into the mixture under Argon protection. The mixture was further stirred at room temperature for 30 min. The other starting material 220, 2-tert-butoxycarbonylamino-pent-4-ynoic acid methyl ester (0.32 g, 1.412 mmol), dissolved in THF (4 mL) was added dropwise over 15 min. The newly formed reaction mixture was further stirred overnight at room temperature. The solid in the reaction mixture was vacuum filtered. The filtrate was concentrated. The residue was re-dissolved in dichloromethane and purified by column chromatography, eluting with a mixture of ethyl acetate (0-25%) and hexanes (100-75%) to afford the product 230 (0.64 g, 99%) as a gummy, yellowish solid. 1H NMR (300 MHz, CDCl3): δ 1.46 (s, 9H), 2.64 (t, J=6.3 Hz, 2H), 2.95 (t, J=4.1 Hz, 2H), 3.42 (m, 2H), 3.79 (s, 3H), 4.56 (m, 1H), 5.12 (s, 2H), 5.38 (br s, 11), 7.29 (s, 4H), 7.35 (m, 5H). m/z (APCI) 502 [C29H32N2O6—H]+.
5-[4-(4-Aminobutyl)phenyl]-2-(R)-tert-butoxycarbonylaminopentynoic acid methyl ester (240)
Compound 230, 5-[4-(4-benzyloxycarbonylaminobut-1-ynyl)phenyl]-2-(R)-tert-butoxycarbonylaminopent-4-ynoic acid methyl ester (0.36 g, 0.713 mmol) was dissolved in a mixture of ethanol and methanol (50 mL, 1/1, v/v) and placed in a Parr shaker bottle. To the solution was added 10% Palladium on carbon (0.20 g, wet) in one portion under Argon protection. The flask was evacuated and re-filled with Argon. The procedure was repeated three more times. The mixture was then stirred at room temperature over night under 35 psi hydrogen atmosphere. The flask was then evacuated and re-filled with nitrogen. The procedure was repeated three times. The catalyst was then filtered under vacuum and washed with ethanol (2×5 mL). The filtrate and washings were combined and concentrated under reduced pressure. The residue was chromatographed over silica gel, eluting with a mixture of methanol (0-12%), ammonium hydroxide (0-1.2%) and di-chloromethane (100-86.8%), to afford two products, the desired product 240 (0.045 g, 17%, a colorless, glass-like solid) and its protected form 250 (0.218 m, 60%, a yellowish solid). 1H NMR (300 MHz, CD3OD) for compound 240: δ 1.43 (s, 9H), 1.63-1.76 (m, 8H), 2.58-2.64 (m, 4H), 2.79 (t, J=7.0 Hz, 2H), 3.69 (s, 3H), 4.10 (m, 1H), 7.09 (s, 4H). m/z (APCI) for compound 7: 379 [C12H34N2O4+H]+.
1H NMR (300 MHz, CD3OD) for compound 250: δ 1.42 (s, 9H), 1.61-1.78 (m, 8H), 2.54-2.68 (m, 4H), 2.92 (t, J=7.0 Hz, 2H), 3.14 (t, J=6.9 Hz, 2H), 3.70 (s, 3H), 4.13 (m, 1H), 5.18 (s, 2H), 7.08 (s, 4H), 7.35 (m, 5H).
The compound 250 was resubmitted to hydrogenation to remove the benzyloxycarbonyl protecting group. The procedure was performed as follows: the compound 250 (0.218 g, 0.425 mmol) was dissolved in ethanol (10 mL). The solution was purged with nitrogen both before and after the palladium catalyst (0.10 g, 10% on charcoal, 50% wet) was added, and subjected to hydrogenation for two hours under atmospheric hydrogen. The catalyst was vacuum filtered and washed with ethanol (2×5 mL). The filtrate and washings were combined and concentrated under vacuum. The residue was chromatographed over silica gel, eluting with a mixture of methanol (0-14%), ammonium hydroxide (0-1.4%) and dichloromethane (100-84.6%), to afford the compound 240 (0.131 g, 81%).
2-(R)-tert-Butoxycarbonylamino-5-(4-{4-[N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}phenyl)pentanoic acid methyl ester (270, PSA 19603)
Compound 240 (0.127 g, 0.336 mmol) was mixed with ethanol (4 mL). The mixture was heated at 65° C. for 30 min to achieve complete dissolution. To the clear solution were sequentially added diisopropylethylamine (0.29 mL, 1.68 mmol) and the 1-(3,5-diamino-6-chloropyrazinoyl)-2-methylisothiourea hydriodide (0.157 g, 0.403 mmol). The mixture was heated at the same temperature for an additional three hours, and subsequently concentrated under vacuum. The residue was chromatographed over silica gel, eluting with a mixture of concentrated ammonium hydroxide (0-1.5%), methanol (0-15%), and dichloromethane (100-83.5%), to afford the product 260 (0.169 g, 85%) as a light yellow solid. A part of the product 260 (0.06 g) was dissolved in methanol (10 mL) and treated with concentrated hydrochloric acid (2 mL) for one hour. All liquid was completely removed under vacuum, and the residue was chromatographed again over silica gel, eluting with a mixture of concentrated ammonium hydroxide (0-1.1%), methanol (0-11%) and dichloromethane (100-87.9%) to afford the desired product 270 (0.043 g, 86%) as a yellow solid. mp 76-79° C. [α]25D −5.6° C. (c 0.31, MeOH). 1H NMR (300 MHz, CD3OD) δ 1.54-1.78 (m, 8H), 2.58-2.68 (m, 4H), 3.26 (t, J=6.8 Hz, 2H), 3.46 (t, J=6.4 Hz, 1H), 3.70 (s, 3H), 7.10 (s, 4H). m/z (APCI) 491 [C22H31ClN8O3+H]+.
Example 15




Synthesis of 2-(R)-amino-5-(4-{4-[N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)-guanidino]butyl}phenyl)pentanoic acid (PSA 23184)




5-[4-(4-Aminobutyl)phenyl]-2-(R)-tert-butoxycarbonylaminopentanoic acid (280)
Compound 240 (0.40 g, 0.780 mmol) was dissolved in a mixture of methanol and THF (10 mL, 1/1, v/v). To the solution was added lithium hydroxide monohydrate (0.063 g, 1.561 mmol) dissolved in water (1 mL). The reaction mixture was stirred at room temperature for two hours, and then neutralized with 2N aqueous HCl solution to pH 5. The mixture was then concentrated under vacuum. The residue was taken into ethanol (3 mL) and the resulting solution was concentrated again under vacuum. The procedure was repeated two more times to ensure no aqueous solvent remained. The residue was chromatographed over silica gel, eluting with a mixture of concentrated ammonium hydroxide (0-10%), methanol (0-30%), and dichloromethane (100-60%), to afford the product 280 (0.29 g, 75%) as a white solid. 1H NMR (300 MHz, CD3OD): δ 1.46 (s, 9H), 1.58-1.78 (m, 8H), 2.54-2.68 (m, 4H), 2.85 (t, J=6.6 Hz, 2H), 3.98 (t, J=6.5 Hz, 1H), 7.08 (s, 4H), 8.50 (s, 1H). m/z (APCI) 365 [C20H32N2O4+H]+.
2-(R)-tert-Butoxycarbonylamino-5-(4-{4-[N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}phenyl)pentanoic acid (290)
The compound 280 (0.077 g, 0.211 mmol) was mixed with ethanol (5 mL). The mixture was heated at 65° C. for 15 min to achieve complete dissolution. To the clear solution were sequentially added diisopropylethylamine (0.184 mL, 1.056 mmol) and 1-(3,5-diamino-6-chloropyrazinoyl)-2-methylisothiourea hydriodide (0.99 g, 0.254 mmol). The mixture was heated at 65° C. for an additional three hours. It was then concentrated under vacuum. The residue was chromatographed over silica gel, eluting with a mixture of concentrated ammonium hydroxide (0-2.2%), methanol (0-22%) and dichloromethane (100-75.8%) to afford the product 290 (0.091 g, 75%) as a yellow solid. 1H NMR (300 MHz, CD6OD): δ 1.44 (s, 9H), 1.58-1.84 (m, 8H), 2.54-2.68 (m, 4H), 3.34-3.42 (m, 2H), 4.02 (m, 1H), 7.08 (s, 4H). m/z (APCI) 577 [C26H37ClN8O5+H]+.
2-(R)-Amino-5-(4-{4-[N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino]-butyl}phenyl)pentanoic acid (300, PSA 23184)
Compound 290 (0.031 g, 0.0538 mmol) was dissolved in methanol (3 mL) and treated with concentrated hydrochloric acid (0.5 mL) for one hour. All liquid was completely removed under vacuum, and the residue was chromatographed over silica gel, eluting with a mixture of concentrated ammonium hydroxide (0-8%), methanol (0-30%) and dichloromethane (100-62%) to afford the desired product 300 (0.024 g, 94%) as a yellow solid. mp 190-192° C. (decomposed). [α]25D −4.3° C. (c 0.25, MeOH). 1H NMR (300 MHz, CD3OD): δ 1.68-1.83 (m, 8H), 2.52-2.62 (m, 4H), 3.27 (t, J=6.8 Hz, 2H), 3.47 (t, 3=6.4 Hz, 1H), 7.09 (s, 4H). m/z (APCI) 477 [C21H29ClN8O3+H]+.
Example 16







Synthesis of 3-(4-{4-[N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)-guanidino]butyl}phenylamino)propionamide dihydrochloride (PSA 14568)




[2-(4-Iodophenylcarbamoyl)ethyl]carbamic acid tert-butyl ester (310)
A mixture of 4-iodoaniline (2.0 g, 9.17 mmol), 3-tert-butoxycarbonylaminopropionic acid (1.61 g, 8.49 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.95 g, 10.19 mmol), 1-hydroxy-7-azabenzotriazole (0.12 g, 0.85 mmol), and DMF (20 mL), was stirred at room temperature for 72 hours. The solvent was evaporated in vacuo. The residue was dissolved in methylene chloride (100 mL) and washed with 10% citric acid (2×50 mL), then 10% aqueous K2CO3 (2×50 mL). The organic layer was dried over sodium sulfate and evaporated in vacuo. The residue was purified by flash chromatography (silica gel, 3:1, v/v hexanes/ethyl acetate, then 1:1 hexanes/ethyl acetate) to provide 310 (2.44 g, 69%). 1H NMR (300 MHz, CDCl3) δ 1.44 (s, 9H), 2.60 (t, 2H), 3.49 (q, 2H), 7.36 (d, 2H), 7.60 (d, 2H).
{4-[4-(3-tert-Butoxycarbonylaminopropionylamino)phenyl]-but-3-ynyl}carbamic acid benzyl ester (320)
To a solution of [2-(4-iodophenylcarbamoyl)ethyl]carbamic acid tert-butyl ester 310 (1.5 g, 3.84 mmol), and triethylamine (40 mL) was added trans-dichlorobis-(triphenylphosphine)palladium (II) (Ph3P, PdCl2, 0.27 g, 0.38 mmol). The reaction mixture was stirred for 20 minutes when it became a clear solution. But-3-ynyl-carbamic acid benzyl ester (0.94 g, 4.6 mmol) and copper iodide (0.14 g, 0.768 mmol) were then added and the reaction was further stirred at room temperature for 18 hours. The solvent was evaporated in vacuo. The residue was dissolved in dichloromethane (100 mL) and washed with water (3×150 mL). The organic layer was dried over sodium sulfate and then concentrated in vacuo. The residue was purified by flash chromatography (silica gel, 1:1, v/v hexanes/ethyl acetate) to provide 320 (1.91 g, still a mixture, but used as it was without further purification). 1H NMR (300 MHz, CDCl3) δ 1.44 (s, 9H), 2.60 (m, 4H), 3.47 (m, 4H), 5.12 (s, 2H), 7.37 (m, 7H), 7.49 (d, 2H).
{4-[4-(3-tert-Butoxycarbonylaminopropionylamino)phenyl]butyl}carbamic acid benzyl ester (330)
To a degassed solution of {4-[4-(3-tert-butoxycarbonylaminopropionylamino)phenyl]-but-3-ynyl}carbamic acid benzyl ester 320 (1.91 g, 4.1 mmol) and 1,2-dimethoxyethane (DME, 35 mL) was added 10% palladium on activated carbon (0.5 g, 50% wet). The mixture was hydrogenated overnight at 50 psi pressure. The catalyst was filtered through a pad of Celite and the solvent was evaporated in vacuo to provide 330 (1.78 g, 99%). 1H NMR (300 MHz, CDCl3) δ 1.42 (s, 9H), 1.58 (m, 4H), 2.59 (t, 4H), 3.17 (q, 2H), 3.47 (q, 2H), 5.18 (m, 3H), 5.58 (m, 1H), 5.70 (m, 1H), 7.07 (d, 2H), 7.33 (m, 5H), 7.49 (d, 2H).
{2-[4-(4-Aminobutyl)phenylcarbamoyl]ethyl}carbamic acid tert-butyl ester (340)
To a degassed solution of {4-[4-(3-tert-butoxycarbonylaminopropionylamino)phenyl]-butyl}carbamic acid benzyl ester 330 (1.78 g, 3.79 mmol) and methanol (50 mL) was added 10% palladium on activated carbon (0.5 g, 50% wet). The mixture was hydrogenated for 4 hours under atmospheric hydrogen. The catalyst was filtered through a pad of Celite and the solvent was evaporated in vacuo. The residue was purified by flash chromatography (silica gel, 6:1:0.1, v/v chloroform/methanol/concentrated ammonium hydroxide) to provide 340 (0.74 g, 58%). 1H NMR (300 MHz, CH3OD) δ 1.44 (s, 9H), 1.50 (m, 2H), 1.66 (m, 2H), 2.60 (m, 6H), 3.38 (m, 2H), 7.14 (d, 2H), 7.44 (d, 2H).
[2-(4-{4-[N′-(3,5-Diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}-phenylcarbamoyl)ethyl]carbamic acid tert-butyl ester (350)
1-(3,5-Diamino-6-chloropyrazine-2-carbonyl)-2-methylisothiourea hydriodide (0.32 g, 0.82 mmol) and triethylamine (0.42 mL) were added to a solution of {2-[4-(4-amino-butyl)phenylcarbamoyl]ethyl}carbamic acid tert-butyl ester 340 (0.25 g, 0.74 mmol) in ethanol (6 mL). The reaction mixture was stirred at 65° C. for 3 hours, and cooled to room temperature. The solvent was evaporated in vacuo. The residue was purified by flash chromatography (silica gel, 10:1:0.1 to 5:1:0.1, v/v chloroform/methanol/concentrated ammonium hydroxide). The product was washed with water (2×25 mL) and dried in a vacuum oven overnight to provide 350 (0.32 g, 78%) as a yellow solid. 1H NMR (300 MHz, CD3OD) δ 1.44 (s, 9H), 1.69 (m, 4H), 2.57 (t, 2H), 2.66 (t, 2H), 3.27 (t, 2H), 3.40 (m, 2H), 7.15 (d, 2H), 7.47 (d, 2H).
(4-{4-[N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}-phenylamino)propionamide dihydrochloride (360, PSA 14568)
To a solution of [2-(4-{4-[N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino]-butyl}phenylcarbamoyl)ethyl]carbamic acid tert-butyl ester 350 (0.32 g, 0.58 mmol) in methanol (5 mL) was added dropwise concentrated HCl (7 mL). The reaction was stirred for 30 minutes. The solvent was evaporated in vacuo to provide 360 (0.30 g, 99%) as a yellow solid. 1H NMR (300 MHz, DMSO-d6) δ 1.57 (m, 4H), 2.58 (t, 2H), 2.75 (t, 2H), 3.04 (m, 2H), 3.34 (m, 2H), 7.15 (d, 2H), 7.42 (m, 1H), 7.52 (d, 2H), 8.11 (s, 2H), 8.89 (s, 1H), 9.00 (s, 1H), 9.32 (s, 1H), 10.33 (s, 1H), 10.55 (s, 1H).
Example 17




Synthesis of N-(4-{4-[N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino]-butyl}phenyl)-3-guanidinopropionamide dihydrochloride (PSA 14984)




N-(4-{4-[N′-(3,5-Diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}phenyl)-3-[N′′,N′′′-bis-(tert-butoxycarbonyl)guanidino]propionamide (370)
To a solution of 3-amino-N-(4-{4-[N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)-guanidino]butyl}phenyl)propionamide dihydrochloride 360 (0.2 g, 0.36 mmol) in methanol (10 mL) was added 1,3-di-(tert-butoxycarbonyl)-2-(trifluoromethanesulfonyl)guanidine (0.22 g, 0.58 mmol) and triethylamine (0.4 mL, 2.88 mmol). The reaction mixture was stirred at room temperature for 18 hours. The solvent was evaporated in vacuo. The residue was purified by flash chromatography (silica gel, 10:1:0.1 chloroform/methanol/concentrated ammonium hydroxide, v/v). The product was washed with water and sonicated to provide 370 (0.20 g, 77%) as a yellow solid. 1H NMR (300 MHz, CD3OD) δ 1.46 (s, 9H), 1.49 (s, 9H), 1.67 (m, 4H), 2.63 (m, 4H), 3.25 (m, 2H), 3.71 (t, 2H), 7.13 (d, 2H), 7.42 (d, 2H).
N-(4-{4-[N′-(3,5-Diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}phenyl)-3-guanidinopropionamide dihydrochloride (380, PSA 14984)
To a solution of N-(4-{4-[N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino]-butyl}phenyl)-3-[N′′,N′′′-bis-(tert-butoxycarbonyl)guanidino]propionamide 370 (0.20 g, 0.29 mmol) in methanol (4 mL) was added dropwise concentrated HCl (4 mL). The reaction was stirred at room temperature for 4 hours. The solvent was evaporated in vacuo. The residue was purified by flash chromatography (silica gel, 2:1:0.15 to 1:1:0.4, chloroform/methanol/concentrated ammonium hydroxide, v/v). Purity of the product was 84%. The product was further purified by preparative HPLC to provide the desired product as a free base (0.05 g, 35%). The dihydrochloride salt was prepared by adding 12N HCl (40 μL) to a solution of the product in methanol (1 mL) which was subsequently poured into ethyl acetate (10 mL). The resulting precipitate was filtered and dried under vacuum to provide 380 as a yellow solid. 1H NMR (300 MHz, CD3OD) δ 1.71 (m, 4H), 2.69 (m, 4H), 3.32 (m, 2H), 3.53 (m, 2H), 7.16 (d, 2H), 7.48 (d, 2H).
Example 18




Synthesis of 2-(4-{4-[N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino]-butyl}phenylamino)acetamide dihydrochloride (PSA 15104)




{4-[4-(Carbamoylmethylamino)phenyl]butyl}carbamic acid tert-butyl ester (390)
A mixture of [4-(4-aminophenyl)butyl]carbamic acid tert-butyl ester (0.8 g, 3.03 mmol), 2-bromoacetamide (0.46 g, 3.33 mmol), triethylamine (0.2 mL, 3.33 mmol), and DMF (30 mL) was stirred at 80° C. for 20 hours. TLC indicated that the reaction was not complete. Silver carbonate (0.92 g, 3.33 mmol) was thus added. The reaction was further stirred at 80° C. for 24 hours. The reaction mixture was passed through a silica plug. The solvent was evaporated in vacuo. The residue was purified by flash chromatography (silica gel, 3:1 ethyl acetate/dichloromethane, v/v) to provide 390 (0.20 g, 22%) as a white solid. 1H NMR (300 MHz, CD3OD) δ 1.42 (s, 9H), 1.55 (m, 4H), 2.50 (t, 2H), 3.03 (t, 2H), 3.70 (s, 2H), 6.52 (d, 211), 7.00 (d, 2H).
2-[4-(4-Aminobutyl)phenylamino]acetamide (400)
12N HCl (5 mL) was added to {4-[4-(carbamoylmethylamino)phenyl]butyl}carbamic acid tert-butyl ester 390 (0.2 g, 0.62 mmol). The reaction was stirred at room temperature for 1 hour. The solvent was evaporated in vacuo. The residue was purified by flash chromatography (silica gel, 4:1:0.1 chloroform/methanol/concentrated ammonium hydroxide, v/v) to provide 400 (0.10 g, 70%) as a white solid. 1H NMR (300 MHz, CD3OD) δ 1.55 (m, 4H), 2.50 (t, 2H), 2.68 (t, 2H), 3.70 (s, 2H), 6.52 (d, 2H), 6.97 (d, 2H).
2-(4-{4[(3,5-Diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}-phenylamino)acetamide dihyrochloride (410, PSA 15104)
2-[4-(4-Aminobutyl)phenylamino]acetamide 400 (0.19 g, 0.86 mmol), 1-(3,5-diamino-6-chloropyrazine-2-carbonyl)-2-methylisothiourea hydriodide (0.33 g, 0.86 mmol), and triethylamine (0.50 mL) were combined in ethanol (10 mL). The mixture was stirred at 72° C. for 5 hours. The solvent was evaporated in vacuo. The residue was purified by flash chromatography (silica gel, 4:1:0.1, chloroform/methanol/concentrated ammonium hydroxide, v/v). The chromatography was repeated to provide the freebase (0.12 g, 32%) as a yellow solid. The salt was prepared by adding 1 drop of concentrated HCl to a solution of 2-(4-{4-[N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)-uanidino]butyl}-phenylamino)acetamide (40 mg) in methanol (0.5 mL). The solution was then poured into ethyl acetate (20 mL). The resulting precipitate was filtered, and then taken up in water and evaporated to provide 410 (0.031 g) as a yellow solid. 1H NMR (300 MHz, CD3OD) δ 1.71 (m, 4H), 2.75 (t, 2H), 3.38 (t, 2H), 4.19 (s, 2H), 7.47 (s, 4H).
Example 19




Synthesis of 4-(4-(4-[N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino]-butyl}phenylamino)butyramide dihydrochloride (PSA 18150)




4-[4-(4-tert-Butoxycarbonylaminobutyl)phenylamino]butyric acid ethyl ester (420)
A solution of [4-(4-aminophenyl)butyl]carbamic acid tert-butyl ester (1.7 g, 6.43 mmol), 4-bromo-n-butyric acid ethyl ester (1.88 g, 9.64 mmol), 4-methylmorpholine (1.0 mL, 9.64 mmol), and DMF (10 mL) was stirred at 85° C. for 3 hours under a nitrogen atmosphere. The solvent was evaporated in vacuo. The residue was purified by flash chromatography (silica gel, 4:1, hexanes/ethyl acetate, v/v) to provide 420 (0.44 g, 18%) as a colorless oil. 1H NMR (300 MHz, CDCl3) δ 1.23 (t, 3H), 1.47 (s, 9H), 1.55 (m, 4H), 1.96 (m, 2H), 2.43 (t, 2H), 2.51 (t, 2H), 3.19 (m, 4H), 4.14 (q, 2H), 6.54 (d, 2H), 7.00 (d, 2H). m/z (ESI) 379.
{4-[4-(3-Carbamoylpropylamino)phenyl]butyl}carbamic acid tert-butyl ester (430)
4-[4-(4-tert-Butoxycarbonylaminobutyl)phenylamino]butyric acid ethyl ester 420 (0.44 g, 1.16 mmol) was added to 7N NH3 in methanol (40 mL). The reaction was stirred at 30° C. for 18 hours, at 45° C. for 25 hours, and finally at 50° C. for 84 hours. The solvent was evaporated in vacuo. The residue was purified by flash chromatography (silica gel, 20:1:0.1, chloroform/methanol/concentrated ammonium hydroxide, v/v) to provide 430 (0.23 g, 56%) as a white solid. 1H NMR (300 MHz, CDCl3) δ 1.47 (s, 9H), 1.55 (m, 4H), 1.96 (m 2H), 2.37 (t, 2H), 2.50 (t, 2H), 3.11 (m, 4H), 6.52 (d, 2H), 6.97 (d, 2H). m/z (ESI) 350.
{4-[4-(4-Aminobutyl)phenylamino]butyramide (440)
{4-[4-(3-Carbamoylpropylamino)phenyl]butyl}carbamic acid tert-butyl ester 430 (0.23 g, 0.66 mmol) was cooled to 0° C. in an ice bath. A cold solution of TFA/dichloromethane (10 mL) was added. The reaction was slowly warmed to room temperature and stirred for 30 minutes. The solvent was evaporated in vacuo to provide 440. 1H NMR (300 MHz, CD3OD) δ 1.69 (m, 4H), 1.95 (m, 2H), 2.47 (m, 2H), 2.71 (m, 2H), 2.93 (m, 2H), 3.39 (m, 2H), 7.39 (s, 4H). m/z (ESI) 250.
4-(4-{4-[N′-(3,5-Diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}-phenylamino)butyramide dihydrochloride (450, PSA 18150)
{4-[4-(4-Aminobutyl)phenylamino]butyramide 440 (0.16 g, 0.64 mmol), 1-(3,5-diamino-6-chloropyrazine-2-carbonyl)-2-methylisothiourea hydriodide (0.27 g, 0.71 mmol), and triethylamine (0.90 mL) were combined in ethanol (5 mL). The reaction was stirred at 50° C. for 2 hours. The solvent was evaporated in vacuo. The residue was sequentially washed with diethyl ether (2×5 mL) and water (5 mL), and then purified by flash chromatography (silica gel, 8:1:0.1 chloroform/methanol/concentrated ammonium hydroxide, v/v). The obtained product was further washed with ethanol (5 mL) to provide pure freebase of 450 (0.15 g, 51%). The salt was prepared by adding 12N HCl (18.4 μL) to a solution of 4-(4-{4-[N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}-phenylamino)butyramide (50 mg) in methanol (1.0 mL) which was then poured into diethyl ether. The solid was separated by decanting off the clear liquid, and then taken up in water. Removal of all liquid under reduced pressure provided 450 (0.05 g) as a yellow solid. 1H NMR (300 MHz, DMSO-d6) δ 1.55 (m, 4H), 1.91 (t, 2H), 2.15 (t, 2H), 2.71 (m, 2H), 3.33 (m, 2H), 6.92 (s, 1H), 7.35 (s, 2H), 7.49 (s, 2H), 8.93 (m, 2H), 9.31 (s, 1H), 10.45 (s, 1H). m/z (ESI) 462.
Example 20




Synthesis of 4-(4-{4-[N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino]-butyl}phenylamino)butyric acid (PSA 18345)




4-[4-(4-Aminobutyl)phenylamino]butyric acid (460)
2.5N aqueous NaOH (1.2 mL) was added to a solution of 4-[4-(4-tert-butoxycarbonylaminobutyl)phenylamino]butyric acid ethyl ester 420 (0.45 g, 1.18 mmol) dissolved in a mixture of water (25 mL) and THF (30 mL). The reaction was stirred for 2 h. 12 N HCl was added dropwise until pH 3. The solvent was evaporated in vacuo. The residue was cooled to 0° C. and a solution of TFA/dichloromethane (10 mL) was added. The reaction was stirred for 30 minutes. The solvent was evaporated in vacuo. The residue was purified by flash chromatography (silica gel, 4:1:0.1, chloroform/methanol/concentrated ammonium hydroxide, v/v) to provide 460 (0.38 g, 100%). 1H NMR (300 MHz, CD3OD) δ 1.61 (m, 4H), 1.82 (m, 2H), 2.29 (t, 2H), 2.50 (m, 2H), 2.88 (m, 2H), 3.09 (t, 2H), 6.58 (d, 2H), 6.92 (d, 2H). m/z (ESI) 251.
4-(4-{4-[N′-(3,5-Diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}-phenylamino)butyric acid (470, PSA 18345)
4-[4-(4-Aminobutyl)phenylamino]butyric acid 460 (0.24 g, 0.94 mmol), 1-(3,5-diamino-6-chloropyrazine-2-carbonyl)-2-methylisothiourea hydriodide (0.37 g, 0.94 mmol), and triethylamine (1.0 mL) were combined in ethanol (5 mL). The reaction was stirred at 50° C. for 4 hours. The solvent was evaporated in vacuo. The residue was purified twice by flash chromatography (silica gel, 4:1:0.1, 2:1:0.1, chloroform/methanol/concentrated ammonium hydroxide, v/v) to provide 470 (0.12 g, 27%) as a yellow solid. 1H NMR (300 MHz, CD3OD) δ 1.67 (m, 4H), 1.89 (m, 2H), 2.30 (t, 2H), 2.59 (m, 2H), 3.09 (t, 2H), 3.30 (m, 2H), 6.58 (d, 2H), 6.92 (d, 2H). m/z (ESI) 463.
Example 21




Synthesis of 2-(4-{4-[N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino]-butyl}phenoxy)-N,N-bis-(2-hydroxyethyl)acetamide (PSA 16826)




[4-(4-Benzyloxycarbonylaminobutyl)phenoxy]acetic acid ethyl ester (480)
A solution of [4-(4-hydroxyphenyl)butyl]carbamic acid benzyl ester (2.0 g, 6.7 mmol), potassium carbonate (1.0 g, 7.3 mmol), sodium iodide (0.4 g, 2.7 mmol), and DMF (10 mL) was stirred for 30 minutes. A solution of ethyl bromoacetate (0.8 mL, 7.4 mmol) in DMF (10 mL) was added dropwise to the reaction. The reaction was further stirred at room temperature for 3 days, and then poured into water (200 mL). The product was extracted with ethyl acetate. The organic layer was dried over sodium sulfate and concentrated in vacuo. The residue was purified by flash chromatography (silica gel, 1:5 ethyl acetate/hexanes, v/v) to provide the desired product 480 (2.3 g, 89%) as a white solid. 1H NMR (300 MHz, CDCl3) δ 1.34 (t, 3H), 1.57 (m, 4H), 2.56 (t, 2H), 3.20 (q, 2H), 4.25 (q, 2H), 4.59 (s, 2H), 5.10 (s, 2H), 6.85 (d, 2H), 7.06 (d, 2H), 7.38 (m, 5H).
[4-(4-{[N,N-Bis-(2-hydroxyethyl)carbamoyl]methoxy}phenyl)butyl]carbamic acid benzyl ester (490)
A solution of [4-(4-benzyloxycarbonylaminobutyl)phenoxy]acetic acid ethyl ester 480 (0.3 g, 0.78 mmol), 2-(2-hydroxyethylamino)ethanol (0.15 m-L, 1.6 mmol), and ethanol (20 mL) was heated at 70° C. for 72 hours. The solvent was evaporated in vacuo. The residue was purified by flash chromatography (silica gel, dichloromethane/methanol, 100:5-1, v/v) to provide 490 (0.19 g, 100% based on the recovered starting material (0.13 g)) as a pale yellow solid. 1H NMR (300 MHz, CDCl3) δ 1.55 (m, 4H), 2.56 (t, 2H), 3.20 (q, 2H), 3.60 (m, 4H), 3.90 (m, 2H), 3.96 (m, 2H), 4.85 (s, 2H), 5.09 (s, 2H), 6.85 (d, 2H), 7.06 (d, 2H), 7.40 (m, 5H). m/z (ESI) 445.
2-[4-(4-Aminobutyl)phenoxy]-N,N-bis-(2-hydroxyethyl)acetamide (500)
To a degassed solution of [4-(4-{[N,N-bis-(2-hydroxyethyl)carbamoyl]methoxy}phenyl)-butyl]carbamic acid benzyl ester 490 (0.19 g, 0.43 mmol) in ethanol (4 mL) was added 10% palladium on activated carbon (0.1 g, 50% wet). The mixture was hydrogenated at atmospheric hydrogen overnight. The catalyst was filtered through a pad of Celite and the solvent was evaporated in vacuo. The residue was purified by flash chromatography (silica gel, 20-5:1:0.1-1 dichloromethane/methanol/concentrated ammonium hydroxide, v/v) to provide 500 (0.09 g, 72%) as a colorless oil. 1H NMR (300 MHz, CD3OD) δ 1.56 (m, 4H), 2.56 (t, 2H), 2.65 (t, 1H), 3.29 (m, 1H), 3.55 (m, 4H), 3.72 (m, 4H), 4.90 (s, 2H), 6.86 (d, 2H), 7.09 (d, 2H). m/z (ESI) 311.
2-(4-{4-[N′-(3,5-Diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}phenoxy)-N,N-bis-(2-hydroxyethyl)acetamide (510, PSA 16826)
1-(3,5-Diamino-6-chloropyrazine-2-carbonyl)-2-methylisothiourea hydriodide (0.13 g, 0.33 mmol) was added to a solution of 2-[4-(4-aminobutyl)phenoxy]-N,N-bis-(2-hydroxyethyl)acetamide 500 (0.09 g, 0.3 mmol), triethylamine (0.12 mL), and ethanol (1.7 mL). The reaction mixture was stirred at 60° C. for 3 h. The solvent was evaporated in vacuo. The residue was washed with water and then purified by flash chromatography (silica gel, 20-10:1:0-0.2 dichloromethane/methanol/concentrated ammonium hydroxide, v/v) to provide 510 (0.1 g, 64%) as a yellow solid. The dihydrochloride salt was prepared by adding 4N HCl (3 drops) to a solution of the product (40 mg) in methanol (2 mL). Some decomposition was observed by HPLC analysis. The product was thus re-purified by a preparative TLC plate (developed with 10:2:0.2 dichloromethane/methanol/concentrated ammonium hydroxide, v/v) to provide 510 (20 mg) as a yellow solid. 1H NMR (300 MHz, CD3OD) δ 1.65 (m, 4H), 2.58 (t, 2H), 3.33 (m, 2H), 3.54 (m, 4H), 3.72 (m, 4H), 4.89 (s, 2H), 6.86 (d, 2H), 7.09 (d, 2H). m/z (ESI) 523.
Example 22







Synthesis of 3-(4-{4-[N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}phenylthio)propionamide (PSA 19605)




N-{4-[4-(Dimethylthiocarbamoyloxy)phenyl]butyl}phthalimide (520)
A suspension of sodium hydride in mineral oil (0.44 g of 60%) in anhydrous DMF (10 mL) was cooled to 0° C. N-[4-(4-hydroxyphenyl)butyl]phthalimide (2.95 g, 10 mmol) dissolved in dry DMF (15 ml) was added into the mixture which was then stirred for 30 min at 0° C. and allowed to warm to room temperature over 1 h. To the mixture was added portionwise N,N-dimethylthiocarbamoyl chloride (1.35 g, 11 mmol) dissolved in DMF (10 ml). The newly formed mixture was stirred at room temperature overnight, then at 50° C. for 1 h, and was cooled back to room temperature, at which point methanol (10 mL) was added into the mixture. The solvent was removed under reduced pressure. The residue was purified by flash chromatography over silica gel (dichloromethane/hexane/ethyl acetate, 10:1:0.2, v/v) to give N-{4-[4-(dimethythiocarbamoyloxy)phenyl]butyl}phthalimide 520 (2.27 g, 59%) as a slightly yellow solid. 1H NMR (300 MHz, CDCl3) δ 1.72 (m, 4H), 2.66 (m, 2H), 3.33 (s, 3H), 3.45 (s, 31), 3.70 (m, 2H), 6.96 (d, 2H), 7.18 (d, 2H), 7.71 (m, 2H), 7.84 (m, 2H). m/z (ESI) 383 [C21H22N2O3S+H]+.
N-{4-[4-(Dimethylcarbamoylthio)phenyl]butyl}phthalimide (530)
N-{4{4-[(Dimethythiocarbamoyloxy)phenyl]butyl}phthalimide 520
(2.1 g, 5.4 mmol) was placed in preheated sand bath at 230° C. The temperature was raised to 280° C. and the melted compound was kept at this temperature for 2 h in argon atmosphere. The mixture was cooled and the residue was purified by flash chromatography over silica gel (dichloromethane/hexane/ethyl acetate, 10:1:0.2, v/v) to give 530 (1.2 g, 57%) as a white powder. 1H NMR (300 MHz, CDCl3) δ 1.70 (m, 4H), 2.66 (t, 2H), 3.05 (br s, 3H), 3.71 (t, 2H), 6.96 (d, 2H), 7.19 (d, 2H), 7.38 (d, 2H), 7.70 (m, 2H), 7.83 (m, 2H). m/z (ES) 383 [C21H22N2O3S+H]+.
{4-[4-(Dimethylcarbamoylthio)phenyl]butylamine (540)
Phthalimide 530 (1.1 g, 2.8 mmol) was dissolved in a solution containing 6.6% methylamine in ethanol (100 mL) and allowed to stir at room temperature overnight. The solvent was removed under reduced pressure and the residue was purified by flash chromatography (silica gel, chloroform/methanol/ammonium hydroxide, 10:1:0.1, v/v) to afford the free amine 540 (0.31 g, 42%) as a white powder. 1H NMR (300 MHz, CD3OD) δ 1.51 (m, 2H), 1.65 (m, 2H), 2.66 (m, 4H), 3.02 (m, 6H), 7.23 (d, 2H), 7.35 (d, 2H).
N-tert-Butoxycarbonyl-{4-[4-(dimethylcarbamoylthio)phenyl]butylamine (550)
Di-tert-butyldicarbonate (0.35 g, 1.6 mmol) was added to a solution of 540 (0.31 g, 1.22 mmol), 4-Dimethylaminopyridine (DMAP), and dichloromethane (50 mL). The mixture was stirred at room temperature for 26 h. The solvent was removed under reduced pressure and the residue was purified by flash chromatography over silica gel (hexane/ethyl acetate, 3:1, v/v) to give 540 (0.36 g, 83%) as a white powder. 1H NMR (300 MHz, CDCl3) δ 1.44 (s, 9H), 1.51 (m, 2H) 1.64 (m, 2H), 3.08 (m, 8H), 4.48 (br s, 1H), 7.18 (d, 2H), 7.39 (d, 2H).
N-[4-(4-Mercaptophenyl)butyl]carbamic acid tert-butyl ester (560)
N-tert-Butoxycarbonyl-{4-[4-(dimethylcarbamoylthio)phenyl]butylamine 550 (0.35 g, 1.02 mmol) was dissolved in MeOH (8 mL). KOH (0.19 g, 3.4 mmol) dissolved in water (2 ml) was added. The mixture was stirred under reflux for 6 h and cooled to room temperature. The solvent was removed under reduced pressure. The residue was dissolved in water and acidified with 5% aqueous HCl to pH  ̃5. The solvent was removed again under reduced pressure and the residue was purified by flash chromatography over silica gel (hexane/ethyl acetate, 3:1, v/v) to give the desired thiophenol 560 (0.13 g, 45%) as a clear oil. 1H NMR (300 MHz, CDCl3) δ 1.43 (s, 9H), 1.49 (m, 2H) 1.59 (m, 2H), 2.57 (t, 2H), 3.12 (m, 2H), 3.38 (s, 1H), 4.47 (br s, 1H), 7.04 (d, 2H), 7.19 (d, 2H).
{4-[4-(2-Carbamoylethylthio)phenyl]butyl}carbamic acid tert-butyl ester (570)
A solution of 3-Bromopropionic acid amide (0.075 g, 0.5 mmol) in THF (5 ml) was added to a solution of [4-(4-mercaptophenyl)butyl]carbamic acid tert-butyl ester 560 (0.13 g, 0.46 mmol) dissolved in THF (15 mL) and 4-methylmorpholine (NMM) (0.3 mL). The reaction mixture was stirred at 50° C. for 4 h and then cooled to room temperature. Two more equivalents of 3-bromoprionic acid amide (0.15 g, 1 mmol) were added. The mixture was further stirred at room temperature for 2 days. The solvent was removed under reduced pressure and the residue was purified by flash chromatography (silica gel, chloroform/methanol/ammonium hydroxide, 15:1:0.1, v/v) to afford the desired product {4-[4-(2-carbamoylethylsulfanyl)phenyl]butyl}carbamic acid tert-butyl ester 570 (0.13 g, 80%) as a white powder. 1H NMR (300 MHz, CD3OD) δ 1.42 (s, 9H), 1.48 (m, 2H) 1.60 (m, 2H), 2.46 (m, 2H), 2.58 (t, 2H), 3.09 (t, 2H), 3.13 (m, 2H), 7.14 (d, 2H), 7.29 (d, 2H). m/z (ESI) 353 [C18H28N2O3S+H]+.
3-[4-(4-Aminobutyl)phenylthio]propionamide dihydrochloride (580)
The protected amine 570 was dissolved in THF (5 mL) and 4N HCl in dioxane (5 mL) was added. The mixture was stirred at room temperature for 6 h. The solvent was removed under reduced pressure and the residue was dried in vacuum to give the dihydrochloride salt 580 (0.11 g, 100%) as a white powder. 1H NMR (300 MHz, DMSO-d6) δ 1.56 (m, 4H), 2.35 (t, 2H), 2.56 (t, 2H), 2.47 (m, 2H), 3.08 (t, 2H), 6.68 (br s, 1H), 7.15 (d, 2H), 7.27 (d, 2H), 7.37 (br s, 1H), 7.78 (br s, 3H). m/z (ESI) 253 [C13H20N2OS+H]+.
3-(4-{4-[N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}phenylthio)propionamide (590, PSA 19605)
Diisopropylethylamine (0.4 mL, 2.2 mmol) and [1-(3,5-diamino-6-chloropyrazine)-2-methylisothiourea hydriodide (0.17 g, 0.44 mmol) were sequentially added into a solution of 580 (0.11 g, 0.31 mmol) in ethanol (8 mL). The reaction mixture was stirred at 60° C. for 6 h. The solvent was removed under reduced pressure and the residue was treated with water (10 mL). The formed precipitate was collected by centrifugation. A portion of the product was purified by flash chromatography (silica gel, chloroform/methanol/ammonium hydroxide, 15:1:0.1, v/v) to give the target compound 590 (0.027 g, 18%) as a yellow powder. (300 MHz, CD3OD) δ 1.6 (m, 4H), 2.47 (m, 2H), 2.62 (t, 2H), 3.12 (t, 2H), 3.23 (m, 2H), 7.14 (d, 2H), 7.29 (d, 2H). m/z (APCI) 465 [C19H25ClN8O2S+H]+.
Example 23




Synthesis of N-{4-[4-(2-aminoethylamino)phenyl]butyl}-N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidine (PSA 16436)




N-{4-[4-(2-tert-Butoxycarbonylaminoethylamino)phenyl]butyl}carbamic acid benzyl ester (600)
A solution of N-tert-butoxycarbonylaminoacetaldehyde (0.28 g, 1.76 mmol) in 1,2-dichloroethane (DCE, 5 mL) was added to a solution of [4-(4-aminophenyl)butyl]carbamic acid benzyl ester (0.48 g, 1.6 mmol) in DCE (10 mL) and acetic acid (0.108 mL, 1.6 mmol). The mixture was stirred at room temperature for 20 min. NaB(OAc)3H (0.48 g, 2.26 mmol) was then added into the mixture which was further stirred at room temperature overnight. Water (10 mL) was added and the mixture was acidified to pH 7 by 10% aqueous HCl. The organic fraction was isolated and dried over sodium sulfate. The solvent was removed under reduced pressure and the residue was purified by FlashTM (BIOTAGE, Inc) (90 g silica gel cartridge 40M, ethyl acetate/hexane, 1:2, v/v) to afford the protected diamine 600 (0.33 g, 47%) as a white powder. 1H NMR (300 MHz, CDCl3) δ 1.45 (s, 9H), 1.54 (m, 2H), 2.51 (m, 2), 3.18 (m, 4H), 3.36 (m, 2H), 3.89 (br s, 1H), 4.69 (br s, 1H), 4.78 (br s, 1H), 5.51 (s, 2H), 6.54 (d, 2), 6.96 (d, 2H), 7.33 (m, 5H). m/z (ESI) 442 [C25H35N3O4+H]+.
N-2-[4-(4-Aminobutyl)phenylamino]ethyl}carbamic acid tert-butyl ester (610)
The protected diamine 600 (0.315 g, 0.71 mmol) was stirred at room temperature for 4 h in methanol (25 mL) with 10% Pd/C (137 mg, 50% wet) under atmospheric hydrogen. The catalyst was filtered off and the solvent was removed under reduced pressure to give amine 610 (0.21 g, 96%) as a clear oil. 1H NMR (300 MHz, CDCl3) δ 1.46 (m, 11H), 1.56 (m, 2H), 2.51 (m, 2H), 2.68 (m, 2H), 3.22 (m, 2H), 3.54 (m, 2H), 3.82 (br s, 1H), 4.80 (br s, 1H), 6.54 (d, 2H), 6.98 (d, 2H).
[2-(4-{4-[N′-3,5-Diamino-6-chloro-pyrazine-2-carbonylguanidino]butyl}phenylamino)ethyl]carbamic acid tert-butyl ester (620)
Diisopropylethylamine (0.3 mL, 1.65 mmol) and [1-(3,5-diamino-6-chloropyrazine)-2-methylisothiourea hydriodide (0.254 g, 0.67 mmol) were sequentially added into a solution of 610 (0.20 g, 0.67 mmol) in ethanol (10 mL). The reaction mixture was stirred at 60° C. for 4 h. The solvent was removed under reduced pressure and the residue was washed with ethyl acetate and water and dried under vacuum. A portion of the residue was purified by FlashTM (BIOTAGE, Inc) (90 g silica gel cartridge 40M, chloroform/methanol/ammonium hydroxide, 10:1:0.1, v/v) to give 620 (0.206 g, 60%) as a yellow powder. 1H NMR (300 MHz, CD3OD) δ 1.42 (s, 9H), 1.62 (m, 4H), 2.52 (m, 2H), 3.15 (m, 2H), 3.22 (m, 4H), 6.57 (d, 2H), 6.95 (d, 2H). m/z (ESI) 520 [C23H34ClN9O3+H]+.
N-{4-[4-(2-Aminoethylamino)phenyl]butyl}-N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidine trihydrochloride (630, PSA 16346)
The protected guanidine 620 was dissolved in a mixture of methanol (7 mL) and 4N HCl in dioxane (12 mL). The mixture was stirred at room temperature for 2.5 h. The formed precipitate was collected by centrifugation and dried under vacuum. The dry yellow powder was dissolved in aqueous HCl (5%) and the solvent was evaporated to give the dihydrochloride salt 630 (0.14 g, 75%) as a yellow powder. 1H NMR (300 MHz, CD3OD) δ 1.74 (m, 4H), 2.77 (m, 2H), 3.39 (m, 4H), 3.73 (m, 2H), 7.47 (d, 2H), 7.58 (d, 2H). m/z (ESI) 420 [C18H26ClN9O+H]+.
Example 24




Synthesis of N-(3,5-diamino-6-chloropyrazine-2-carbonyl)-N′-{4-[(2-guanidinoethylamino)phenyl]butyl}guanidine trihydrochloride (PSA 17339)




N-(4-{4-[2-(N′,N′-Di-tert-butoxycarbonylguanidino)ethylamino]phenyl}butyl)-N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidine (640)
Diisopropylethylamine (0.12 mL, 0.6 mmol) and N,N′-di-(tert-butoxycarbonyl)-N′′-trifluoromethansulfonylguanidine (Goodman's reagent) (0.12 g, 0.30 mmol) in methanol (3 mL) were sequentially added into a suspension of 630 in methanol (5 mL). The reaction mixture was stirred at room temperature for 2 days. The solvent was removed under reduced pressure and the residue was purified by flash chromatography (silica gel, chloroform/methanol/ammonium hydroxide, 10:1:0.1, v/v). The obtained yellow solid was washed with water and dried under vacuum to give 640 (0.076 g, 57%) as a yellow powder. 1H NMR (300 MHz, CD3OD) δ 1.48 (s, 9H), 1.50 (s, 91), 1.67 (m, 4H), 2.57 (m, 2H), 3.26 (m, 4H), 3.55 (m, 2H), 6.63 (d, 2), 6.95 (d, 2H). m/z (ESI) 662 [C29H44ClN11O5+H].
N-(3,5-Diamino-6-chloropyrazine-2-carbonyl)-N′-{4-[(2-guanidinoethylamino)phenyl]butyl}guanidine trihydrochloride (650, PSA 17339)
Compound 640 (0.072 g, 0.11 mmol) was stirred in 33% aqueous HCl (10 mL) at room temperature for 3 h. The solvent was removed under reduced pressure. The residue was dissolved in 0.1% aqueous TFA and purified by preparative HPLC (C 8 Spherosorb column S5C8, isocratic method, 0.1% TFA water/acetonitrile, 75%:25%, v/v). The fractions containing the target compound were combined and the solvent was removed under reduced pressure. The residue was dissolved in 5% HCl and the solvent was removed again under reduced pressure. This procedure was repeated twice. The obtained compound was dissolved in water and lyophylized to give 650 (0.012 g, 20%) as a yellow solid. 1H NMR (300 MHz, CD3OD) δ 1.74 (m, 4H), 2.77 (m, 2H), 3.37 (m, 2H), 3.63 (m, 4H), 7.40-7.56 (m, 4H). m/z (ESI) 462 [C19H28ClN11O5+H].
Example 25




Synthesis of N-(4{4-[{2-[N,N-bis-((2S)-2,3-dihydroxypropyl)amino]ethyl}-N-((2S)-2,3-dihydroxypropyl)amino]phenyl}butyl)-N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidine (PSA 16311)




N-{4-[4-(2-Aminoethyl)aminophenyl]butyl}carbamic acid tert-butyl ester (660)
A mixture of N-[4-(4-aminophenyl)butyl]carbamic acid tert-butyl ester (4.5 g, 17 mmol) and 2-bromoethylamine hydrobromide (1.75 g, 8.5 mmol) in toluene was stirred at 100° C. for 3 days. The solvent was removed under reduced pressure and the residue was purified by flash chromatography (silica gel, chloroform/methanol/ammonium hydroxide, 8:1:0.1, v/v) to give 660 (1.47 g, 56%) as a white powder. 1H NMR (300 MHz, CD3OD) δ 1.42 (s, 9H), 1.51 (m, 4H), 2.48 (t, 2H), 2.85 (t, 2H), 3.03 (t, 2H), 3.17 (m, 2H), 6.59 (d, 2H), 6.94 (d, 2H). m/z (ESI) 308 [C17H29lN3O2+H].
N-tert-Butoxycarbonyl-4-{4-[{2-[N,N-bis-((2S)-2,3-dihydroxypropyl)amino]ethyl}-N-((2S)-2,3-dihydroxypropyl)amino]phenyl}butylamine (670)
A mixture of (R)-(+)-glycidol (0.22 g, 3 mmol) and amine 660 in ethanol (6 mL) were stirred at 70° C. (oil bath) overnight. The solvent was removed under reduced pressure and the residue was purified by FlashTM (BIOTAGE, Inc) (90 g silica gel cartridge 40M, chloroform/methanol/ammonium hydroxide, 7:1:0.1, v/v) to give 670 (0.27 g, 60%) as a clear oil. 1H NMR (300 MHz, CD3OD) δ 1.42 (s, 9H), 1.44 (m, 2H), 1.56 (m, 2H), 2.45-2.78 (br m, 6H), 3.0 (m, 2H), 3.32 (m, 8H), 3.52 (m, 8H), 3.72 (m, 2H), 3.85 (m, 1H), 6.70 (d, 2H), 6.98 (d, 2H).
4-{4-[{2-[N,N-Bis-((2S)-2,3-dihydroxypropyl)amino]ethyl}-N-((2S)-2,3-dihydroxypropyl)amino]phenyl}butylamine bis-(trifluoroacetate) (680)
Dichloromethane (2 mL) was added to the protected amine 670 (0.27 g, 0.5 mmol) followed by addition of TFA (5 mL). The mixture was stirred at room temperature for 25 min. The solvent was removed under reduced pressure. The residue was dissolved in methanol and the solvent was removed again under reduced pressure. The residue was dried under vacuum overnight to afford the compound 680 (0.31 g, 80%) as a clear oil. 1H NMR (300 MHz, CD3OD) δ 1.66 (m, 4H), 2.59 (m, 2H), 2.92 (m, 2H), 3.15-4.10 (m, 20H), 6.78 (d, 2H), 6.98 (d, 2H).
N-(4{4-[{2-[N,N-Bis-((2S)-2,3-dihydroxypropyl)amino]ethyl}-N-((2S)-2,3-dihydroxypropyl)amino]phenyl}butyl)-N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidine (690, PSA 16311)
Triethylamine (0.5 mL, 3.6 mmol) and [1-(3,5-diamino-6-chloropyrazine)-2-methylisothiourea hydriodide (0.20 g, 0.52 mmol) were sequentially added into a solution of 680 (0.31 g, 0.40 mmol) in ethanol (10 mL). The reaction mixture was stirred at 70° C. (oil bath) for 2.5 h and then at room temperature overnight. The solvent was removed under reduced pressure and the residue was purified by FlashTM (BIOTAGE, Inc) (90 g silica gel cartridge 40M, chloroform/methanol/ammonium hydroxide, 3:1:0.3, v/v) to give 690 (0.127 g, 17%) as a yellow powder. 1H NMR (300 MHz, CD3OD) δ 1.64 (m, 4H), 2.49-2.81 (m, 8H), 3.25 (m, 4H), 3.51 (m, 9H), 3.70 (m, 2H), 3.85 (m, 1H), 6.71 (d, 2H), 7.00 (d, 2H). m/z (ESI) 642 [C27H44ClN9O7+H]+, 768 [C27H44ClN9O7+I]−. [α]25D=−23.4° (c=0.5, MeOH).
Example 26




Synthesis of N-(3,5-diamino-6-chloropyrazine-2-carbonyl)-N′-{4-[4-((2R)-2,3-dihydroxypropylamino)phenyl]butyl}guanidine (PSA 16722)




{4-[4-((2R)-2,3-Dihydroxypropylamino)phenyl]butyl}carbamic acid benzyl ester (700)
A solution of (S)-(−)-glycidol (0.16 mL, 2.3 mmol) in ethanol (5 mL) was added dropwise to a stirring solution of N-[4-(4-aminophenyl)butyl]carbamic acid benzyl ester (0.59 g, 1.97 mmol) in ethanol (15 mL) at 70° C. The reaction mixture was stirred at 70° C. (oil bath) overnight. The solvent was removed under reduced pressure and the residue was purified by FlashTM (BIOTAGE, Inc) (90 g silica gel cartridge 40M, chloroform/methanol/ammonium hydroxide, 15:1:0.1, v/v) to give 700 (0.29 g, 40%) as a white solid. 1H NMR (300 MHz, CD3OD) δ 1.51 (m, 4H), 2.48 (t, 2H), 3.07 (m, 1H), 3.26 (t, 2H), 3.30 (m, 1 if), 3.57 (m, 2H), 3.80 (m, 1H), 5.00 (s, 2H), 6.59 (d, 2H), 6.99 (d, 2H), 7.33 (m, 5H). m/z (ESI) 373 [C21H28N2O4+H]+.
4-[4-((2R)-2,3-Dihydroxypropylamino)phenyl]butylamine (710)
The protected amine 700 (0.29 g, 0.77 mmol) was stirred at room temperature for 6 h in methanol (20 mL) with 10% Pd/C (129 mg, 50% wet) under hydrogen (1 atm). The catalyst was filtered off and the solvent was removed under reduced pressure to give amine 710 (0.165 g, 90%) as a clear oil. 1H NMR (300 MHz, CD3OD) δ 1.56 (m, 4H), 2.51 (t, 2H), 2.71 (t, 2H), 3.02 (m, 1H), 3.27 (m, 1H), 3.30 (m, 2H), 3.56 (m, 2H), 3.80 (m, 1H), 6.60 (d, 2H), 6.95 (d, 2H). m/z (ESI) 239 [C13H22N2O2+H]+.
N-(3,5-Diamino-6-chloropyrazine-2-carbonyl)-N′-{4-[4-((2R)-2,3-dihydroxypropylamino)phenyl]butyl}guanidine (720, PSA 16722)
Diisopropylethylamine (0.5 mL, 2.75 mmol) and [1-(3,5-diamino-6-chloropyrazinoyl)-2-methylisothiourea hydriodide (0.272 g, 0.70 mmol) were sequentially added into a solution of 710 (0.165 g, 0.70 mmol) in ethanol (10 mL). The reaction mixture was stirred at 70° C. (oil bath) for 4 h. The solvent was removed under reduced pressure and the residue was sequentially washed with ethyl acetate and water (5 mL, once each time) and dried under vacuum. The dried crude material was purified by flash chromatography (silica gel, chloroform/methanol/ammonium hydroxide, 4:1:0.15, v/v) to give the target compound 720 (0.168 g, 54%) as a yellow powder. 1H NMR (300 MHz, CD3OD) δ 1.63 (m, 4H), 2.53 (t, 2H), 3.02 (m, 1H), 3.22 (m, 2H), 3.27 (m, 1H), 3.57 (m, 2H), 3.81 (m, 1H), 6.60 (d, 2H), 6.96 (d, 2H). m/z (ESI)=451 [C19H27ClN8O3+H]+. [α]25D=+6.9° (c=0.5, MeOH).
Example 27




Synthesis of N-(4-{4-[{2-[((2R)-2,3-dihydroxypropyl)amino]ethylamino}phenyl)butyl-N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidine trihydrochloride (PSA 16721)




N-tert-Butoxycarbonyl-4-{4-[{2-[((2R)-2,3-dihydroxypropyl)amino]ethylamino]phenyl}butylamine (730)
A mixture of (S)-(−)-glycidol (0.30 g, 4 mmol) and triethylamine (0.5 mL, 3.6 mmol) in ethanol (5 mL) was added portionwise into a solution of amine 660 (0.61 g, 2 mmol) in ethanol (15 mL) over 30 min at room temperature. The reaction mixture was stirred at room temperature overnight. Extra (S)-(−)-glycidol (0.080 g, 1 mmol) in 10 mL ethanol was added dropwise and the mixture was stirred for an additional 18 h. The solvent was removed under reduced pressure and the residue was purified by FlashTM (BIOTAGE, Inc) (90 g silica gel cartridge 40M, chloroform/methanol/ammonium hydroxide, 8:1:0.1, v/v) to afford the crude compound 730 (0.176 g).
A second batch of the same reaction was run by adding dropwise a solution of (S)-(−)-glycidol (0.090 g, 1.2 mmol) in ethanol (5 mL) into a solution of amine 660 (0.36 g, 1.17 mmol) in ethanol (10 mL) over 3 h at 70° C. The reaction mixture was stirred at 70° C. The solvent was removed under reduced pressure and the residue was purified by FlashTM (BIOTAGE, Inc) (90 g silica gel cartridge 40M, chloroform/methanol/ammonium hydroxide, 7:1:0.1, v/v) to afford the crude compound 730 (0.22 g).
The crude products from both batches were combined and purified again by FlashTM (BIOTAGE, Inc) (90 g silica gel cartridge 40M, chloroform/methanol/ammonium hydroxide, 9:1:0.1, v/v) to afford the pure 730 (0.206 g, 1% overall yield) as a white paraffin. 1H NMR (300 MHz, CD3OD) δ 1.44 (s, 9H), 1.47 (m, 2H), 1.54 (m, 2H), 2.48 (t, 2H), 2.61 (m, 1H), 2.75 (m, 1H), 2.83 (m, 2H), 3.02 (t, 2H), 3.22 (t, 2H), 3.49 (t, 2H), 3.72 (m, 1H), 6.59 (d, 2H), 6.93 (d, 2H).
4-{4-[{2-[((2R)-2,3-Dihydroxypropyl)amino]ethylamino]phenyl}butylamine dihydrochloride (740)
4N HCl in dioxane (10 mL) was added into the protected amine 730. The mixture was stirred at room temperature for 3 h. The solvent was removed under reduced pressure. Dichloromethane (15 mL) was added into the resulting white solid and subsequently evaporated. This procedure was repeated three times. The resulting solid was further dried under vacuum to afford the dihydrochloride salt 740 (0.209 g, 96%) as a white powder. 1H NMR (300 MHz, CD3OD) δ 1.43 (m, 4H), 2.72 (m, 2H), 2.93 (m, 2H), 3.12 (m, 1H), 3.49 (m, 2H), 3.58 (m, 2H), 3.75 (m, 2H), 3.97 (m, 1H), 6.70 (m, 4H). m/z (ESI) 282 [C15H27N3O2+H]+.
N-(4{4-[{2-[((2R)-2,3-Dihydroxypropyl)amino]ethylamino}phenyl)butyl-N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidine trihydrochloride (750, PSA 16721)
Diisopropylethylamine (0.5 mL, 2.75 mmol) and [1-(3,5-diamino-6-chloropyrazine)-2-methylisothiourea hydriodide (0.230 g, 0.60 mmol) were sequentially added into a solution of 740 (0.165 g, 0.70 mmol) in ethanol (10 mL). The reaction mixture was stirred at 70° C. (oil bath) for 2.5 h. and then at room temperature overnight. The solvent was removed under reduced pressure and the residue was purified by FlashTM (BIOTAGE, Inc) (90 g silica gel cartridge 40M, chloroform/methanol/ammonium hydroxide, 3:1:0.1, v/v) to give the crude free base N-(4{4-[{2-[((2R)-2,3-dihydroxypropyl)amino]ethylamino}phenyl)butyl-N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidine (0.176 g, 58%, 80% purity by HPLC) as a yellow solid. It was then dissolved in methanol (5 mL). To the solution was added 4N HCl in dioxane (10 mL). The solvent was removed under reduced pressure and the residue was dissolved in methanol (5 mL) again. The solution was poured into 2-propanol (20 mL). The resulting precipitate was collected and purified by flash chromatography (silica gel, chloroform/methanol/ammonium hydroxide, 3:1:0.3, v/v) to give the free base 750 (0.042 g, 12%). The compound 750 was dissolved in 3% aqueous HCl. The solvent was removed under reduced pressure to give the final compound as a trihydrochloride salt (0.050 g, 9.3%, a yellow powder). 1H NMR (300 MHz, CD3OD) δ 1.74 (m, 4H), 2.76 (t, 2H), 3.13 (m, 1H), 3.37 (m, 2H), 3.56 (m, 4H), 3.80 (m, 1H), 7.44 (d, 2H), 7.53 (d, 2H). m/z (ESI) 494 [C21H32ClN9O3+H]+. [α]25D=+6.7° (c=0.5, MeOH).
Example 28




Synthesis of N-[4-(4-{2-[bis-((2S,3R)-2,3,4-trihydroxybutyl)amino]ethylamino}phenyl)butyl]-N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidine trihydrochloride (PSA 17218)




N-tert-Butoxycarbonyl-4-{2-[N,N-bis-((2S,3R)-2,3,4-trihydroxybutyl)amino]ethylamino}phenylbutylamine (760)
Acetic acid (0.09 mL, 1.4 mmol) and a solution of D-(−)-erythrose (0.52 g, 4.2 mmol) in methanol (2.5 mL) were sequentially added into a solution of amine 660 (0.44 g, 4.2 mmol) in methanol (20 mL). The reaction mixture was stirred at room temperature for 20 minutes under nitrogen protection. The reaction was cooled to −78° C. Sodium cyanoborohydride (0.39 g, 6.16 mmol) was then added into the mixture. The reaction was allowed to warm up to room temperature and stirred for 18 h. The solvent was evaporated and the residue was purified by FlashTM (BIOTAGE, Inc) (90 g silica gel cartridge 40M, 4:1:0.15 chloroform/methanol/concentrated ammonium hydroxide, v/v) to provide 760 (0.12 g, 16%) as a white solid. 1H NMR (300 MHz, CD3OD) δ 1.42 (s, 9H), 1.45 (m, 2H), 1.47 (m, 2H), 2.49 (m, 2H), 2.61-2.98 (m, 4H), 3.02 (m, 2H), 3.22 (m, 2H), 3.45-3.85 (m, 10H), 6.64 (d, 2H), 6.94 (d, 2H). m/z (APCI) 516 [C25H45N3O8+H]+.
4-{2-[N,N-Bis-((2S,3R)-2,3,4-trihydroxybutyl)amino]ethylamino}phenylbutylamine trifluoroacetate (770)
The protected amine 760 (0.12 g, 0.22 mmol) was stirred in a mixture of dichloromethane/TFA (10 mL, 1:1) for 2 h. The solvent was removed under reduced pressure and the residue was purified by flash chromatography (silica gel, chloroform/methanol/ammonium hydroxide, 3:1:0.3, v/v) to provide 770 (0.12 g, 98%) as a colorless oil. (300 MHz, CD3OD) δ δ 1.64 (m, 4H), 2.55 (m, 2H), 2.90 (m, 2H), 3.45 (m, 4H), 3.60 (m, 10H), 3.97 (m, 2H), 6.67 (d, 2H), 7.00 (d, 2H). m/z (APCI) 416 [C20H37N3O6+H]+.
N-[4-(4-{2-[Bis-((2S,3R)-2,3,4-trihydroxybutyl)amino]ethylamino}phenyl)butyl]-N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidine trihydrochloride (780, PSA 17218)
1-(3,5-Diamino-6-chloropyrazine)-2-methylisothiourea hydriodide (0.121 g, 0.31 mmol) and diisopropylethylamine (0.20 mL, 1.2 mmol) were sequentially added into a solution of 770 (0.12 g, 0.22 mmol) in ethanol (10 mL). The reaction mixture was stirred at 70° C. for 3.5 h and then at room temperature overnight. The solvent was evaporated. The residue was purified by flash chromatography (silica gel, chloroform/methanol/ammonium hydroxide, 3:1:0.3, v/v) to provide the crude free base (0.070 g) as a yellow solid. The free base was taken up in 3% aqueous HCl (2 mL). The undissolved solid was filtered off and the solvent in the filtrate was removed under reduced pressure. The product was dried under vacuum to afford the trihydrochloride salt 780 (0.057 g, 34%) as a yellow powder. 1H NMR (300 MHz, CD3OD) δ 1.72 (m, 4H), 2.74 (m, 2H), 3.36 (m, 2H), 3.49 (m, 2H), 3.65 (m, 8H), 3.89 (m, 4H), 4.07 (m, 2H), 7.41 (d, 2H). m/z (APCI) 629 [C26H42ClN9O7+H]+. [α]25D −13.8° (c=0.5, MeOH).
Sodium Channel Blocking Activity
The compounds listed in the Table below were tested for potency in canine bronchial epithelia using the in vitro assay described above. PSA 4022 was also tested in each assay as a positive control. The results for the compounds of the present invention are reported as fold-enhancement values relative to amiloride, setting PSA 4022=100× Amiloride.
Example 29














EC50 (nM)
Fold Amiloride** (PSA 4022 = 100)













PSA
N
Mean
S.D.
N
Mean
S.D.
















10316
6
9.56
5.09
6
132
71


10735
5
24.2
6.0
5
46.5
22.4


10940
1
26.2

1
63.6


10941
10
32.1
15.4
10
30.0
7.7


11041
3
32.0
24.4
3
38.6
21.4


11180
3
14.6
7.9
3
66.8
18.2


11223
4
14.7
3.5
4
79.6
22.2


11698
4
5.82
2.35
4
136
51


11833
2
12.3
1.3
2
58.6
29.0


11834
6
3.68
2.33
6
276
133


11975
6
4.85
2.45
6
287
134


11976
2
401
158
2
3.0
1.3


12218
3
312
268
3
5.6
5.6


14568
2
3.92
0.00
2
131
10


14984
2
3.27
0.68
2
268
56


15104
3
19.1
7.0
3
26.4
10.6


16311
4
33.2
14.7
4
25.6
11.2


16436
2
15.4
1.6
2
35.6
3.8


16721
2
25.4
1.8
2
44.7
3.2


16722
2
22.8
3.9
2
50.5
8.7


16826
3
12.7
6.2
3
64.9
43.6


17218
3
32.4
5.0
3
24.7
5.4


17339
2
6.83
3.61
2
108
56


17587
2
19.4
6.9
2
31.3
18.3


17666
2
29.3
3.8
2
34.8
23.7


18150
3
23.8
6.3
3
44.4
19.5


18345
3
69.0
3.5
3
12 .4
4.0


18704
2
14.1
3.9
2
90.2
18.4


19603
3
32.3
7.7
3
39.4
12.6


19605
3
4.70
1.17
3
145
37


23184
3
17
4
3
43.3
12.0





**Relative potency for PSA 4022 = 100 using EC50 from PSA 4022 in same run






REFERENCES


    1. Rappoport, D. A.; Hassid, Z.; J. Amer. Chem. Soc., 1951, 73, 5524-5525, incorporated herein by reference.


Obviously, numerous modifications and variations of the present invention are possible in light of the above teachings. It is therefore to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described herein.

## Claims

1. A compound represented by the formula:









or a pharmaceutically acceptable salt thereof.

2. A compound represented by formula:



or a pharmaceutically acceptable salt thereof.

3. The compound of claim 1, which is in the form of a free base.

4. The compound of claim 1, which is a pharmaceutically acceptable salt.

5. The compound of claim 1, which is an acid addition salt of an inorganic acid or an organic acid.

6. The compound of claim 1, which is an acid addition salt of an inorganic acid or an organic acid selected from the group consisting of hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, furmaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalensulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, malonic acid, sulfosalicylic acid, glycolic acid, 2-hydroxy-3-naphthoate, pamoate, salicylic acid, stearic acid, phthalic acid, mandelic acid, and lactic acid.

7. A pharmaceutical composition, comprising the compound of claim 1 and a pharmaceutically acceptable carrier.

8. A method of promoting hydration of mucosal surfaces, comprising:
administering an effective amount of the compound of claim 1 to a mucosal surface of a subject in need thereof.

9. A method of treating chronic bronchitis, treating cystic fibrosis, treating ventilator-induced pneumonia, treating asthma, treating chronic obstructive pulmonary disease, treating emphysema, treating pneumonia, comprising:
administering an effective amount of the compound of claim 1 to a subject in need thereof.