Design, synthesis, and structure-activity relationships of novel 2-substituted pyrazinoylguanidine epithelial sodium channel blockers: Drugs for cystic fibrosis and chronic bronchitis

4098

J. Med. Chem. 2006, 49, 4098-4115

Design, Synthesis, and Structure-Activity Relationships of Novel 2-Substituted

Pyrazinoylguanidine Epithelial Sodium Channel Blockers: Drugs for Cystic Fibrosis and

Chronic Bronchitis

Andrew J. Hirsh,*^,‡,|Bruce F. Molino,^§Jianzhong Zhang,^§Nadezhda Astakhova,^§William B. Geiss,^§Bruce J. Sargent,^§

Brian D. Swenson,^§Alexander Usyatinsky,^§Michael J. Wyle,^§Richard C. Boucher,^|Rick T. Smith,^‡Andra Zamurs,^‡and

M. Ross Johnson*^,‡

Parion Sciences Inc., 2525 Meridian Parkway, Suite 260, Durham, North Carolina 27713, Albany Molecular Research Inc.,

21 Corporate Circle, Albany, New York 12212, and Cystic Fibrosis/Pulmonary Research and Treatment Center, School of Medicine,

UniVersity of North Carolina at Chapel Hill, Thurston Bowles Building, Chapel Hill, North Carolina 27599

ReceiVed NoVember 11, 2005

Amiloride (1), the prototypical epithelial sodium channel (ENaC) blocker, has been administered with limited

success as aerosol therapy for improving pulmonary function in patients with the genetic disorder cystic

fibrosis. This study was conducted to synthesize and identify more potent, less reversible ENaC blockers,

targeted for aerosol therapy and possessing minimal systemic renal activity. A series of novel 2-substituted

acylguanidine analogues of amiloride were synthesized and evaluated for potency and reversibility on bronchial

ENaC. All compounds tested were more potent and less reversible at blocking sodium-dependent short-

circuit current than amiloride. Compounds 30-34 showed the greatest potency on ENaC with IC₅₀values

below 10 nM. A regioselective difference in potency was found (compounds 30, 39, and 40), whereas no

stereospecific (compounds 33, 34) difference in potency on ENaC was displayed. Lead compound 32 was

102-fold more potent and 5-fold less reversible than amiloride and displayed the lowest IC₅₀value ever

reported for an ENaC blocker.

Introduction

clearance, warranted the development of novel more efficacious

ENaC blockers tailored for inhalation therapy.

Sodium channels are ubiquitous in nature and are classified

either as neuronal voltage-gated sodium channels, which are

selectively blocked with neurotoxins (tetrodotoxin or saxitoxin)

or the antiepileptic drug phenytoin,¹or as epithelial/degenerin

sodium channels (ENaC), which are selectively blocked by a

substituted acylguanidine known as amiloride (1).²Amiloride

is a potassium-sparing diuretic that was developed by Cragoe

and his colleagues at Merck Pharmaceuticals in the early 1960s

and has been widely used clinically as an adjunctive treatment

with the earlier developed loop diuretics to help minimize the

hypokalemia commonly experienced with the sulfonamide

agents.^3,4

In the respiratory epithelium, the apical sodium channel helps

regulate the thin layer of airway surface liquid (ASL) that lines

airway surfaces. The ASL provides the necessary milieu for

cilia function and cephalad mucociliary clearance (MC), which

comprises the primary innate defense mechanism of the respira-

tory tract.^5-7Because cystic fibrosis (CF) pathogenisis was

thought to involve defective mucus clearance, amiloride was

tested as an inhalation therapy for enhancing MC and cough

clearance in the respiratory tract of CF patients in 1986.⁸The

rationale for amiloride inhalation as a therapy for CF has been

supported by direct evidence that increasing ASL volume

enhances MC,^9-11which in turn would provide relief from

mucus plugging in CF airways. Although the therapeutic utility

of amiloride in CF patients was inconclusive,^8,12-17the rationale

for an aerosol pharmacotherapy that delivers an active compound

directly to a protein that controls ASL volume and, hence, mucus

Amiloride, which was designed for oral administration, failed

as a form of inhalation therapy because of (1) the restricted

drug delivery associated with inhalation therapy, that is, its

limited solubility restricted the mass that could be delivered by

a conventional nebulizer; (2) limited potency; (3) rapid absorp-

tion by airway epithelia; and (4) rapid wash-off (rapid revers-

ibility) from ENaC.^16,18-21

The proposed ENaC subunit stoichiometry is a heteromeric

protein complex comprised of three subunits (R, â, and γ) with

at least two possible configurations: (1) the 2:1:1 configuration,

where the channel is in an RâRγ complex,²²and (2) the 3:3:3

(R:â:γ) configuration or eight to nine subunits of which a

minimum of two are the γ subunit. ^23,24Amiloride is proposed

to block ENaC at concentrations ranging from approximately

0.2-1 µM via a direct exofacial block, (the invasion hypo-

thesis) proposed by Cuthbert in 1976²⁵and supported by Li et

al.²⁶The model, consists of two steps: first, the positively

charged acylguanidinium side chain of amiloride invades the

channel pore and interacts with an anionic site (a fixed

negatively charged residue, e.g., aspartic or glutamic acid)

forming an encounter complex, and second, the negative charge

from the chlorine atom on the pyrazine ring binds to an

electropositive site (positively charged residue, e.g., arginine,

histidine, or lysine) on the channel to form a stable blocking

complex.²⁶Li et al. further added that the differences in ENaC

blocker potency were due to changes in the on and off rate

constants of the compound to the binding site on the channel.^26,27

Adding to this model, Venanzi et al., using molecular electro-

static potential analysis, found that the distance between the

stable blocking complex formed by the charged acylguanidinium

moiety and the chlorine atom on the pyrazine ring defined an

important spatial requirement that allowed for the stable

amiloride block.²⁸

* To whom correspondence should be addressed. Tel: 919-313-1181.

Fax: 919-313-1190. E-mails: ajhirsh@Parion.com (A.J.H.). E-mail:

mrjohnson@Parion.com (M.R.J).

^‡Parion Sciences Inc.

^§Albany Molecular Research Inc.

^|University of North Carolina at Chapel Hill.

Published on Web 06/13/2006

Drugs for Cystic Fibrosis and Chronic Bronchitis

Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14 4099

afforded compounds 15, 21, 22, and 27. Compound 22 was

further converted to its corresponding acid analogue 25 by

saponification with lithium hydroxide. The methylcarboxylate

in compound 46d was reduced by LiAlH₄to its corresponding

alcohol 46e, which was subsequently converted to compound

27 by coupling with 43b. To study the effect of the chain length

of para-hydroxy-substituted analogues on blocking ENaC, the

demethylation of compounds 46a-c and 46f by treatment with

a 48% hydrobromic acid produced phenols 47a-d, which were

similarly converted to target compounds 13, 14, 16, and 42.

Compound 16 was further converted to its corresponding sulfuric

acid derivative 26 by the treatment of 16 with pyridine sulfur

trioxide complex.

An alternative synthesis for compound 47c is illustrated in

Scheme 3. The carboxylic acid group of the commercially

available 48 was activated by treatment with iso-butylchloro-

formate, the product of which was then treated in situ with

methanolic ammonia to afford amide 49. Amide 49 was reduced

to its corresponding amine by a borane-THF complex. The

reduction product, without purification, was subsequently

subjected to demethylation by hydrobromic acid to afford 47c

as an HBr salt. Compared to a procedure in the literature for

the synthesis of compound 47c,³⁵the synthetic procedure

described herein for the synthesis of 47c is shorter and easier

to scale-up (see Experimental Section).

Figure 1. Tautomeric interconversions of amiloride free base in

solution; 1a acylamino, 1b acylimino, and 1c isoimino.

The potential binding site of amiloride to ENaC was

investigated using site-directed mutagenesis studies. Two sepa-

rate groups identified the serine 583 residue located on the R

subunit to be an important site that maintains ENaC amiloride

activity,^29,30and glycine residues 525 and 537 from the â and

γ subunits, respectively, in an area known as the the pre-M2

segment.³¹Furthermore, residues 278-283 within the R-subunit

ectodomain have been suggested to participate in amiloride

binding (specifically the pyrazine ring moiety).^29,32

To study the effect of the location of the hydroxyl group on

the phenyl ring, compounds 17-19 were synthesized by

employing the Sonogashira reaction³⁶for the preparation of key

intermediates 52a-e (Scheme 4). Compounds 20, 23, and 30

were also synthesized by this method. Thus, the substituted

phenylhalides 50a-e were treated with N-Boc-protected 3-bu-

tynylamine 51 (n ) 2), which was prepared from 3-butynyl

alcohol according to the reported procedure.³⁷The triple bond

in the protected amines 52a-e was saturated by hydrogenation

to afford products 53a-c and e. The 4-nitro group in compound

52d was concomitantly reduced to give 53d. The treatment of

53a-d with 48% aqueous hydrogen bromide affected the

removal of both the Boc and methyl protecting groups present

in these compounds. The resulting amines 54a-d were coupled

with 43b to give desired compounds 17-20. The Boc-protecting

group in 53a and e was separately cleaved by TFA, affording

compounds 55a and e, which were subsequently converted to

target compounds 23 and 30.

Syntheses of the analogues containing an oxygen atom in

the linear chain linker are depicted in Scheme 5. The alkylation

of 56a with 57a and 56b with 57b, mediated by sodium hydride,

afforded 58a and b, which were then treated with methylamine

to remove the phthalimide protecting group. Free amines 59a

and b were then converted to target compounds 24 and 29.

Compound 24 was converted to its phenolic derivative 28 by

treating with aqueous 48% hydrogen bromide.

The synthesis of compound 31 started from intermediate 47c

(Scheme 6). The protection of the terminal amine in 47c by a

carbobenzyloxy protecting group afforded 60a; the allylation

of 60a with allyl bromide provided advanced intermediate 61.

Hydroboration-oxidation of the terminal alkene in 61 afforded

the desired product 62 with the concomitant production of a

10-15% yield of byproduct 63, in which the hydroxy group

was at the 2′- position. Cleavage of the carbobenzyloxy

protecting group in 62 by hydrogenolysis afforded amine 64,

which was converted to target compound 31 by coupling with

43b.

The objective of this study was to identify more potent, less

reversible ENaC blockers than amiloride that would be suitable

for a once or twice daily aerosol therapy in patients suffering

from chronic obstructive pulmonary diseases (COPD) such as

CF. To this end, we synthesized a novel focused library of

2-acylguanidine ENaC blocker analogues and report the intrinsic

activity (potency) and reversibility on airway ENaC. On the

basis of this study, we propose adding three new auxiliary

binding sites to the invasion hypothesis, which rationalizes the

increased potency of the new ENaC analogues.

Chemistry. Amiloride (1a) in solution exists as a mixture

of the three tautomers represented in Figure 1. Previous studies

by Smith et al.³³have shown that the free base exists primarily

as acylimino tautomer 1b, whereas the physiologically active

species exists as the protonated form of the acylamino tautomer

1a²(Figure 1). These structural representations (1a and b) have

been used to represent amiloride and its analogues in both the

patent and scientific literature. We use the acylamino represen-

tation (1a) for convenience throughout this article with the

understanding that the structures are, in reality, a hybrid of the

three forms with the actual amount of each dependent on the

pH and the nature of the substituents.

New compounds 2-4 and 7-42 were all made by coupling

amine precursors with 43a or b. Thus, the synthesis of amiloride

derivatives 2-4 and 7-12 were achieved by coupling guanidines

made in situ from commercial amines with 3,5-diamino-6-

chloropyrazine-2-carboxylic acid methyl ester (43a) (method

A) or by coupling amines made in multisteps with methylthio

pseudourea (43b) which was prepared according to the reported

procedure (Scheme 1).³⁴These methods served as general

procedures for the preparation of all of the novel amiloride

analogues reported in this study.

Syntheses of para-substituted analogues with varied lengths

of linear chain started from the requisite 4′-substituted phenyl

alcohols 44a-e (Scheme 2). The hydroxyl group in compounds

44a-e was activated to the mesylate ester and subsequently

displaced by sodium azide. The resulting azides 45a-e were

reduced to the amines 46a-e by triphenylphosphine and water

in THF. The coupling of amines 46b-e with 43b directly

Syntheses of compounds 32-36 are shown in Scheme 7. The

diol side chain of these structures was established by a reaction

4100 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14

Scheme 1. General Procedure (Method A or B)^a

Hirsh et al.

^aReagents: Method A: (i) RNH₂, 1H-pyrazole-1-carboxamidine hydrochloride, i-Pr₂EtN, DMF; (ii) 43a, 25% NaOMe, MeOH. Method B: 43b, RNH₂,

i-Pr₂EtN, EtOH (or THF).

Scheme 2^a

^aReagents: (i) MsCl, pyr., THF; (ii) NaN₃, DMF; (iii) Ph₃P, THF, H₂O; (iv) Method B (Scheme 1); (v) Method C: concd HCl, MeOH; (vi) 48% HBr,

H₂O; (vii) LiAlH₄, THF; (viii) LiOH, THF. (ix) Pyridine sulfur trioxide complex, pyridine.

Scheme 3^a

^aReagents: (i) iso-butylchloroformate, NMM, THF; (ii) NH₃, MeOH; (iii) BH₃/THF; (iv) 48% HBr.

of the phenol hydroxy group with glycidol in the presence of a

catalytic amount of triethylamine (0.005-0.1 equiv). For the

syntheses of the enantiomerically pure targets 33 (R) and 34

(S), the corresponding enantiomerically pure glycidols R and

S, respectively, were used. The enantiomers of the racemic

compound 32 were separated by chiral HPLC, and their ratio

was found to be 1:1. To ensure stereochemical integrity, the

enantiomeric purity for compounds 33 and 34 was determined

using the same method as that above, and the compounds were

found to possess 100% ee. Compounds 65a-e were subject to

catalytic hydrogenolysis to remove the Cbz protecting group.

The deprotected amines 66a-e were then coupled with com-

pound 43b to afford target compounds 32-36, which were

characterized as either a methanesulfonic acid salt or a

hydrochloride salt.

nylamine 51 followed by the reduction of the triple bond

afforded compounds 69a and b, which underwent alkylation

with THP-protected 2-bromoethanol to give compounds 70a and

b. The deprotection in an acidic medium (TFA) simultaneously

removed both the Boc and THP protecting groups, giving key

intermediates 71a and b (TFA salt). The coupling of 71 with

43b afforded desired products 39 and 40, respectively.

The synthesis of the carboxylic acid derivatives 37 and 38 is

illustrated in Scheme 9. The alkylation of the phenolic hydroxyl

group in 60a with the appropriate bromide afforded compounds

72a and b. The cleavage of the carbobenzyloxy protecting

groups in 72 followed by the coupling of the resulting

deprotected compounds 73a and b with 43b from Scheme 1

using the conventional conditions for this type of reactions

completed the synthesis of compounds 37 and 38.

The synthesis of compounds 39 and 40 is illustrated in

Scheme 8. The Sonogashira coupling of the appropriately

substituted iodophenol 67a and b with N-Boc-protected buty-

The synthesis of compound 41 is described in Scheme 10.

The coupling of 60a with the fully protected sugar 75 (D-

glucuronic acid) mediated by trifluoroborane etherate afforded

Drugs for Cystic Fibrosis and Chronic Bronchitis

Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14 4101

Scheme 4^a

^aReagents: (i) 51, Ph₃P/PdCl₂, CuI, Et₂NH, THF; (ii) H₂, Pd/C, MeOH; (iii) 48% HBr; (iv) Method B (Scheme 1); (v) Method C (Scheme 2); (vi) TFA,

CH₂Cl₂.

Scheme 5^a

inhibition (the channel is in the fully blocked state) followed

by three consecutive mucosal washes. Recovery is reported as

the percent of starting (basal) I_scafter the third and final apical

wash. In general, amiloride and other commercially available

ENaC blockers tested in this study were less active compared

to previously published data, which were derived from multiple

methods and different tissues.²In our in vitro study, (1) all

compounds were tested using the procedure described herein

(i.e., utilizing one method and only one tissue type); (2) the

rank order in potency between amiloride 1a and benzamil 6

was similar (11.8 compared to 9.7-fold) to that in previous

reports; (3) the canine bronchial epithelial cells that were utilized

had bioelectric properties similar to that of human bronchial

epithelia;³⁸and (4) the canine bronchial epithelial cells provided

a robust dynamic range (approximately 65 µA/cm²) with a

dominant sodium-dependent current (>90% of total I_sc), facili-

tating the detection of small shifts in intrinsic blocking activity.

^aReagents: (i) NaH, THF; (ii) MeNH₂, MeOH; (iii) Method B (Scheme

1); (iv) Method C (Scheme 2); (v) 48% Hydrobromic acid.

Using this model, the IC₅₀value for 1a (amiloride) was 776

nM (Table 1). After the full-block (final concentration of the

concentration-effect response) with amiloride, the sodium-

dependent current returned to its basal value (96% recovered)

after three consecutive apical washes.

76, which underwent hydrogenolysis to remove the carboben-

zyloxy protecting group, giving the amine 77. The coupling of

77 with compound 43b followed by a global saponification

reaction with sodium hydroxide to cleave all acetyl protecting

groups as well as the methyl ester afforded the final compound

41 as the sodium salt.

Biological Results. Pharmacology and SAR in Vitro. All

ENaC blockers listed in Table 1 were evaluated using an

electrophysiological assay utilizing bronchial airway epithelia

grown at an air/liquid interface. The short-circuit current (I_sc)

and transepithelial resistance (R_t) were recorded from primary

canine bronchial epithelial cultures mounted in modified Ussing

chambers. The IC₅₀values were calculated from a 12-point

concentration-effect curve, where the IC₅₀value represents the

mucosal concentration required to inhibit 50% of the sodium-

dependent current. Recovery or reversibility is a qualitative

index of drug wash-off and was measured after maximal

The acylguanidinium cation is essential for ENaC activity²

but lacks the potency necessary to achieve our objectives. Our

early SAR focused on the 2-position because it appeared to us

to hold the most promise for increasing potency. Substituting

one of the terminal amino groups by hydrophobic alkyl groups

2-3 or the more hydrophilic ethanol 4 produced compounds

with an approximately 7.5-fold increase in potency and de-

creased reversibility compared to those of amiloride. Substituting

a phenyl group (phenamil 5) or a benzyl group (benzamil 6)

increased potency 1.9- and 11.8-fold respectively and also

decreased reversibility. Discussion on the differences in the

phenamil IC₅₀value reported in this study compared to values

reported in the literature^2,39have been previously addressed.⁴⁰

4102 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14

Hirsh et al.

Scheme 6^a

^aReagents: (i) CbzCl, NaHCO₃, H₂O; (ii) allyl bromide, NaOH, DMF; (iii) BH₃/THF; (iv) H₂O₂, NaOH; (v) Method D: H₂, Pd/C, EtOH or MeOH; (vi)

Method B (Scheme 1); (vii) Method C (Scheme 2).

Scheme 7^a

^aReagents: (i) glycidol, Et₃N, EtOH, reflux; (ii) Method D (Scheme 6); (iii) Method B (Scheme 1); (iv) Method C (Scheme 2) or CH₃SO₃H, EtOH.

Having established in our hands that benzamil was more potent

than phenamil, we synthesized a homologous series of aralkyl

derivatives, all of which (with the exception of the phenoctyl

analogue 12) significantly increased potency compared to that

of phenamil (Figure 2). Increasing the number of carbon atoms

between the terminal nitrogen atom of the guanidine moiety

and the aromatic ring thus produced compounds 7-11, which

were of greater potency (to a maximum of 27-fold) and were

less reversible than amiloride. Although not statistically sig-

nificant, it appeared that phenylbutyl derivative 9 was the most

active compound of this series.

We, therefore, focused additional SAR studies on phenylbutyl

derivatives. The addition of the 4′-hydroxyl group to the optimal

four-carbon linker (9) increased potency approximately 2-fold,

making the phenol (16) 46-fold more potent than amiloride.

Incorporating the four-carbon linker with the 4′-methoxy group

(21) produced a slight increase in potency with no differences

in reversibility compared to that of 16.

and 21 were significantly more active than their lower and higher

homologues, thus fully establishing the 4-phenyl series as

optimal.

We also confirmed that the lipophilic nature of the four carbon

linker side chain was important. Incorporating an oxygen atom

at position 2 or position 3 in the alkyl chain of the 4′-methoxy

analogues (24 and 29, respectively) decreased potency compared

to that of compound 21. The loss of potency was also found in

phenol 28 compared to that in 16. Reversibility also increased

by substituting oxygen for methylene in the side chain. Thus,

replacing a lipophilic methylene group in the linker with an

oxygen atom was deleterious to our objective.

At this point, we had satisfied our first two design criteria of

increasing potency and making the compounds less reversible.

We approached our third criterion by focusing on incorporating

groups that could become inactive once they traversed the

epithelial lining of the lung and entered the systemic circulation.

Of particular note is compound 16, a phenol that was designed

on the basis of a soft drug/antedrug approach.^41-43The phenol

can be viewed as a metabolite of compound 9 and could also

serve as a substrate for conjugation and/or potential oxidation.

We found that two of its potential metabolites 41(conjugation)

and 42 (oxidation) were indeed less potent and more reversible

Before we continued our SAR around the 4′ position, we

wanted to confirm our original assumption that the 4-phenylbutyl

chain was optimal for potency. We probed the effect of chain

length in the phenol (13, 14, 16, and 17) and anisole (15, 21,

and 23) series. As predicted, the 4-phenylbutyl derivatives 16

Drugs for Cystic Fibrosis and Chronic Bronchitis

Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14 4103

Scheme 8^a

^aReagents: (i) Et₂NH, CuI, PdCl₂, Ph₃P, THF; (ii) H₂, Pd/C, EtOH; (iii) 2-(2-bromoethoxy)tetrahydro-pyran, K₂CO₃, acetone; (iv) TFA, CH₂Cl₂; (v)

Method B (Scheme 1); (vi) Method C (Scheme 2).

Scheme 9^a

^aReagents: (i) NaH, THF; (ii) Method D (Scheme 6); (iii) Method B (Scheme 1); (iv) TFA, MeOH.

Scheme 10^a

^aReagents: (i) BF₃‚OEt₂, CH₂Cl₂; (ii) Method D (Scheme 6); (iii) Method B (Scheme 1); (iv) NaOH, H₂O, THF.

than parent 16. On the basis of these results with compound

16, we then turned our attention to substitutions that could

provide other metabolic pathways toward inactivation. In

addition to being more potent than amiloride by at least an order

of magnitude, we found that these compounds were all

significantly more active than potential metabolites 25 and 41.

Ester 22 and benzyl alcohol 27 did not change potency and

reversibility compared to those of phenol 16. However, the

potential metabolite of 22 and 27, carboxylic acid 25, decreased

in both potency and reversibility. Other groups such as sulfonic

acid 26 and aniline 20 retained good potency. We did not pursue

these analogues further in this study because we had moved on

to other compounds that showed more promise.

We then continued investigating the 4′-substituted position.

We speculated that the potency of primary alcohols 30 and 31

could be decreased by conjugation in plasma or by hepatic

oxidation to the carboxylic acid. The reduced potency of the

respective carboxylic acids 37 and 38 compared to that of the

primary alcohols (30 and 31) supported this hypothesis. Glycol

32 combines the features of both 30 and 31 and was prepared

4104 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14

Hirsh et al.

Table 1. Intrinsic Blocking Activity and Recovery of ENaC by Substituted 2-Acylguanidine Derivative Using Primary Canine Bronchial Epithelial

Cells

IC₅₀( SD

compd

R

(nM)^a

% recovery ( SD^a

1^bamiloride

2

NH₂

776 ( 326 (36)

87 (1)

108 ( 61 (2)

107 (1)

96 ( 22 (35)

24 (1)

7 ( 3 (2)

77 (1)

NH(CH₂)₅CH₃

NH(CH₂)₇CH₃

NH(CH₂)₂)OH

NHC₆H₅

NHCH₂C₆H₅

NH(CH₂)₂C₆H₅

NH(CH₂)₃C₆H₅

NH(CH₂)₄C₆H₅

NH(CH₂)₅C₆H₅

NH(CH₂)₆C₆H₅

NH(CH₂)₈C₆H₅

NH(CH₂)₂C₆H₄4-OH

NH(CH₂)₃C₆H₄4-OH

NH(CH₂)₃C₆H₄4-OCH₃

NH(CH₂)₄C₆H₄4-OH

NH(CH₂)₅C₆H₄4-OH

NH(CH₂)₄C₆H₄3-OH

NH(CH₂)₄C₆H₄2-OH

NH(CH₂)₄C₆H₄4-NH₂

3

4

5^bPhenamil

401 ( 150 (4)

66 ( 33 (56)

66 ( 21 (5)

50 ( 21 (5)

29 ( 5 (6)

68 ( 22 (3)

58 ( 41 (2)

477 ( 365 (4)

62 ( 48 (10)

88 ( 24 (6)

124 ( 64 (2)

17 ( 9 (32)

64 ( 56 (8)

20 ( 8 (3)

30 ( 17 (2)

21 ( 1 (3)

15 ( 12 (7)

22 ( 11 (6)

77 ( 9 (3)

25 (1)

51 ( 27 (6)

21 ( 5 (2)

14 ( 7 (4)

32 (1)

41 (1)

9 ( 4 (10)

7 ( 3 (7)

8 ( 3 (368)

9 ( 3 (12)

7 ( 2 (10)

26 ( 13 (2)

28 ( 0.4 (2)

48 ( 13 (8)

20 ( 4 (4)

17 ( 3 (2)

31 ( 7 (2)

73 ( 50 (2)

42 ( 6 (5)

15 ( 6 (4)

47 ( 24 (52)

21 ( 15 (5)

23 ( 11 (4)

16 ( 10 (5)

6 ( 4 (2)

12 ( 2 (2)

12 ( 10 (4)

65 ( 25 (10)

65 ( 17 (6)

57 ( 29 (2)

24 ( 15 (31)

29 ( 11 (8)

14 ( 0.2 (2)

11 ( 1 (2)

36 ( 21 (3)

19 ( 15 (6)

13 ( 2 (5)

5.5 ( 2 (3)

25 (1)

6^bBenzamil

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

NH(CH₂)₄C₆H₄4-OCH₃

NH(CH₂)₄C₆H₄4-CO₂CH₃

NH(CH₂)₅C₆H₄4-OCH₃

NH(CH₂)₃OC₆H₄4-OCH₃

NH(CH₂)₄C₆H₄4-CO₂H

NH(CH₂)₄C₆H₄4-SO₃H

65 ( 18 (6)

53 ( 7 (2)

20 ( 10 (4)

73 (1)

NH(CH₂)₄C₆H₄4-CH₂OH

NH(CH₂)₃OC₆H₄4-OH

NH(CH₂)₂OCH₂C₆H₄4-OCH₃

NH(CH₂)₄C₆H₄4-O(CH₂)₂OH

NH(CH₂)₄C₆H₄4-O(CH₂)₃OH

NH(CH₂)₄C₆H₄4-OCH₂CHOHCH₂OH

NH(CH₂)₄C₆H₄4-OCH₂CHOHCH₂OH^c

NH(CH₂)₄C₆H₄4-OCH₂CHOHCH₂OH^d

NH(CH₂)₄C₆H₄3-OCH₂CHOHCH₂OH

NH(CH₂)₄C₆H₄2-OCH₂CHOHCH₂OH

NH(CH₂)₄C₆H₄4-OCH₂CO₂H

NH(CH₂)₄C₆H₄4-O(CH₂)₂CO₂H

NH(CH₂)₄C₆H₄3-O(CH₂)₂OH

NH(CH₂)₄C₆H₄2-O(CH₂)₂OH

NH(CH₂)₄C₆H₄4-O-D-glucuronic acid

NH(CH₂)₄C₆H₃3,4-di OH

72 (1)

29 ( 17 (8)

17 ( 13 (7)

20 ( 11 (345)

25 ( 15 (12)

18 ( 16 (10)

37 ( 8 (2)

63 ( 9 (2)

79 ( 25 (8)

65 ( 43 (4)

30 ( 18 (2)

21 ( 4 (2)

86 ( 40 (2)

45 ( 22 (5)

^aAll values are the mean ( SD. The number in parenthesis is the number of individual observations. ^bCommercially available epithelial sodium channel

blockers. ^cThe R-enantiomer of compd 32. ^dThe S-enantiomer of compd 32.

because first, it has a highly oxidized side chain, which may

resemble an already metabolized form, and second, we hypoth-

esized that it would stay on the apical side longer than

compounds 30 and 31. In fact, by placing an oxygen atom at

the 4′ position and incorporating ethanol 30, propanol 31, or

glycol 32 generated the lowest IC₅₀values ever reported (9, 7,

and 8 nM, respectively) for blocking ENaC and were 3.3-, 5.6-,

and 4.9-fold, respectively, less reversible than amiloride.

Having now found a series of compounds that were 2 orders

of magnitude more potent than amiloride, less reversible, and

conceptually antedrugs or soft drugs, we looked at the details

of the regio and stereospecificities of glycol 32. The ortho (36)

and meta (35) analogues were significantly less active than the

para analogue by 3- to 5-fold. The para-position regioselectivity

was similar to that found with compounds 30, 39, and 40.

Furthermore, the regiochemical para preferences for ENaC block

increased with size in the following order: p-OCH₂CHOHCH₂-

OH (32) g p-O(CH₂)₂OH (30) > p-OH (16). We next examined

the stereospecificity of 32 and found that the ENaC blocking

activity of 33 and 34 were the same as that of racemate 32.

Although this was surprising, it is consistent with the finding

that achiral derivatives 30 and 31 were as active as 32 and

indicates a lack of stereochemical requirement in this auxiliary

binding site.

Discussion

Aerosolized amiloride only transiently and marginally in-

creased MC and pulmonary function in humans.^12,16These

shortcomings as an aerosol therapy indicated that the amiloride

lacked sufficient duration and potency to be therapeutically

effective. In addition, because the bronchial and renal epithelial

sodium channel are structurally and functionally similar,⁴⁴

selectivity for this drug class must be achieved by means other

than by channel selectivity, or potential hyperkalemia will result

from systemic absorption of the more potent ENaC blocker.

An effort toward designing more efficacious ENaC blockers

Drugs for Cystic Fibrosis and Chronic Bronchitis

Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14 4105

Figure 2. Proposed binding of compound 32 to the ENaC. (A) Compound 32 represented as a ball-and-stick dot model (color scheme: white, H;

blue, N; gray, C; green, Cl; and red, O); (B) Lipophilic surface (Gaillard MLP) model (color scheme: red (-0.5 to -0.39); purple (-0.39 to

-0.28); orange (-0.28 to -0.17); yellow (-0.17 to -0.06); white (-0.06 to 0.06); cyan (0.06 to 0.17); green (0.17 to 0.28); green blue (0.28 to

0.39); blue (0.39 to 0.50)). (C) Electrophilic surface model; blue is negative, and red is positive. The segments identified by the horizontal dashed

yellow lines correspond to the proposed binding regions. The lipophilicity and electrophilicity are normalized.

selective for COPD such as the life-shortening hereditary disease

CF was therefore warranted. Conceptually, our approach to this

problem included three objectives: first, to increase intrinsic

activity (potency) by finding an auxiliary binding site in the

channel pocket; second, to increase duration of action by slowing

transport across the airway epithelium and/or decreasing channel

reversibility; and third, to achieve selectivity by the conversion

of the drug to a less active metabolite by systemic or epithelial

biotransformation. Alternatively, we could induce a greater

fraction of nonrenal elimination of ENaC blockers by making

them resemble endogenous metabolites and, thus, reduce renal

exposure.

Using a primary bronchial epithelial culture model, we

developed an in vitro testing algorithm and defined the

structure-activity relationship (SAR) for potency and revers-

ibility (a slower rate of basal current recovery after maximal

inhibition) of the new analogues compared to those of amiloride

on ENaC expressed in respiratory epithelia. Our approach was

to achieve our first two objectives of increasing potency and

reducing reversibility. We then sought to identify areas of the

molecule that we could manipulate to induce antedrug⁴³or soft

drug⁴²metabolic features that are sensitive to epithelial or

systemic biotransformation by producing less potent ENaC

blockers and thereby reduce renal side effects. This report

describes the design and synthesis of novel ENaC blockers that

are the most potent and least reversible reported to date. We

found that all of the compounds synthesized in this study were

more potent and less reversible than amiloride 1. The pharma-

cologic blockade of ENaC increases ASL volume and acceler-

ates MC and, therefore to some extent, limits the efficacy by

diluting and physically removing the compound from the

targeted protein. One approach to avoid this limitation of

efficacy was to reduce reversibility (recovery of activity), which

is likely a function of the compound’s affinity on ENaC and

the on-off rate constants. Therefore, we sought to choose a

compound with greatly enhanced potency and better reversibility

parameters. A systematic SAR was used to generate a focused

2-substituted acylguanidine analogue library. The results provide

new insight on the ENaC blocker SAR and putative auxiliary

binding sites for the novel ENaC blockers.

The number of carbon atoms, four being optimum, between

one of the terminal nitrogen atoms of the acylguanidinium

moiety and the aromatic ring increased intrinsic activity. To

extend the invasion hypothesis plug-type model,^25,26we found

that the distance of the acylguanidine moiety and the benzene

ring was critical for good activity. This suggests that a length-

specific hydrophobic lipid cleft in the ENaC pore in close

proximity to the guanidine binding site would support our

finding. This notion is further supported by the observation that

an oxygen atom in the chain is deleterious to activity, for

example, compound 24. Also, the para-substituted aromatic ring

at the end of the straight chain alkyl linker, being essentially

planar, could contribute to active site rigidity in the form of a

π-π interaction (auxiliary binding pocket), providing greater

potency and less reversibility. Consistent with a π-π interaction,

a regiochemical preference was displayed, where the para

position generated the optimal potency for ENaC as the groups

increased in size. Finally, the linked alcohols were the most

potent ENaC blockers reported to date. The hydroxyl groups

in 31-34 likely access an additional secondary binding site in

an area near the proposed pre-M2 segment or extracellular loop

of the R subunit of ENaC. On the basis of these observations,

we propose that the following new auxiliary binding regions

be added to the invasion hypothesis model: a lipophilic binding

region (1) to accommodate the four linear carbon atoms bonded

to an aromatic ring (2) anchored by a π-π interaction followed

by a three-four atom spacer (3) into an electrostatic binding

site (Figure 2). Each of these regional sites cumulatively

contributes about five times in potency relative to that of

amiloride, culminating in the highly potent analogues 30-34.

At this point in our study, it was not clear that designing

antedrug or soft drugs would lead to systemic selectivity.

4106 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14

Hirsh et al.

D: Atlantis C18 5 µm column, 1.5 mL/min; 95:5 (0.02% TFA:

0.02% TFA MeCN) to 56:44 at 6.5 min to 90% (0.02% TFA

MeCN) for 1 min at 40 °C.

Chiral HPLC analysis was performed on a Waters 260 HPLC

instrument using the following method. Column: Chiralcel OD

(Chiral Technologies), 10 µm, 250 × 4.6 mm; detector: λ ) 280

nm; mobile phase ) 60% heptane/40% ethyl alcohol containing

1% diethylamine; flow rate: 1 mL/min; run time: 45 min; sample

concentration: 1 mg/mL; injection volume: 10.00 µL.

However, by designing compounds that resemble endogenous

metabolites (32-34), we provided an alternative means to

diminish or eliminate possible hyperkalemia. Preliminary clinical

data suggest that 32 systemically becomes less selective for renal

elimination (2%) over 24 h⁴⁵than amiloride (87%).²¹This

systemic selectivity is a significant feature of the novel ENaC

blocker (32) and supports the notion of decreasing systemic side

effects by including endogenous metabolic structures in the

design of compounds.

Preparative flash column chromatography was performed on

Biotage’s Horizon autoseparation equipment using a silica gel

cartridge. Preparative HPLC was performed on Gilson CombiChem

or on Waters (600 controller, 484 detector) using methods similar

to those used in the analytical HPLC. Preparative TLC was

performed on Analtech’s UNIPLATE prep TLC plates (20 × 20

cm, 1000 µm) and developed by a mixture of dichloromethane,

methanol, and ammonia hydroxide (CMA). Combustion analysis

was conducted at Quantitative Technologies, Inc. (QTI), White-

house, NJ. High-resolution mass spectroscopy analysis was per-

formed at the Center of Functional Genomics, State University of

New York at Albany, Albany, NY.

Preparation of Stock Solutions. The concentration of a

compound in DMSO was determined using a UV-Vis spectro-

photometer (Hitachi U-3010) and the Beer-Lambert Law; the

relationship between absorbance and concentration is commonly

written as A ) ꢀ*c*l, where A is the absorbance, ꢀ is the millimolar

extinction coefficient or molar absorptivity, c is the concentration

of the analyte, and l is the length of the absorption path. The novel

chemical entities are structurally similar to amiloride, sharing the

same core chromophore (pyrazine ring) as that of amiloride;

therefore, the extinction coefficient for amiloride was used in

calculating the concentration of each compound. When converted

to millimolar units, the extinction coefficient for amiloride at a

wavelength of 362 nm is 18.6 mM^-1cm^-1(i.e., a 1 mM solution

will generate an absorbance value of 18.6 at 362 nm using a 1 cm

cuvette). To determine the concentration of a compound in DMSO,

the absorbance of the solution was measured at a defined

wavelength of 362 nm using a 1 cm cuvette. Concentration is

calculated in millmolar units by substituting the measured absor-

bance value (A), extinction coefficient (ꢀ) for amiloride (18.6 mM^-1

cm^-1), and the path length (1 cm) into the Beer-Lambert Law

equation and solving for c (concentration). Because the absorbance

values must fall within the linear range of the spectrophotometer,

stock solutions were diluted (when appropriate) prior to taking

measurements. The measured concentration was then corrected

using the appropriate dilution factor to determine the concentration

of the compound in the original stock solution. The value obtained

for concentration can be converted to mg/mL free base.

General Procedures. Method A: General Procedure for the

Preparation of Compounds 2-12. An appropriate alkylamine (1

equiv) was dissolved in a mixture of anhydrous DMF (3 mL) and

diisopropylethylamine (1.1 mL). To the solution, powdered 1H-

pyrazole-1-carboxamidine hydrochloride (2 equiv) was added, and

the reaction mixture was stirred at room-temperature overnight

followed by the addition of ether (10 mL). The newly formed oil

was washed with ether (3 × 10 mL) and dried under vacuum for

36 h. The resulting oil was further dissolved in anhydrous methanol

(12 mL). To the solution, sodium methoxide (25 wt % solution in

methanol, 1.5 equiv) was added, and the precipitate formed was

filtered off. The mother liquor was then concentrated under vacuum

(up to 1.5 mL). To the residue, 3,5-diamino-6-chloropyrazine-2-

carboxylic acid methyl ester 43a was added, and the mixture was

first stirred at ambient temperature overnight and then heated to

reflux for 12 h. The resulting solvent was removed under vacuum,

and the residue was treated with water (10 mL). The supernatant

was removed, and the resulting oil was washed with water (20 mL)

and ether (2 × 10 mL). The thick oil that was obtained was then

purified by preparative HPLC. Fractions containing the target

compounds were combined and concentrated under reduced pres-

sure. The residue was dissolved in 5 mL of 10% HCl aqueous

Conclusions

In summary, we have synthesized a series of more potent,

less reversible ENaC analogues than amiloride. To demonstrate

that an antedrug or softdrug approach is conceptually possible,

we made potential metabolites (25-41), which are less potent

ENaC blockers and, therefore, pose less of a risk in vivo

(hyperkalemia) than the parent analogue. We have also provided

examples (32-34) of highly oxidized side chains, which for

the most part are not metabolized and are mainly excreted non

renally, thus providing an alternate means of achieving selectiv-

ity. After a considerable amount of in vitro and in vivo testing,

we selected 32 as a clinical candidate. Compound 32 has

increased potency by 2 orders of magnitude, reduced revers-

ibility (five times), and decreased selectivity for renal elimination

than amiloride. Presently, clinical studies (phase I and II) in

CF patients are underway to evaluate 32, and the results will

be published in due course. Our work continues to test our

hypothesis and provide additional compounds for COPD and

CF as well as for ventilated associated pneumonia (VAP) and

provide relief for the dry eyes and dry mouth symptoms

associated with the disease known as Sjo¨gren’s syndrome.

Experimental Section

General Methods. Proton NMR spectra were recorded on a

Bruker WM-360 or 300 UltraShield spectrometer (360 or 300 MHz)

using tetramethylsilane (TMS) as an internal standard. Chemical

shifts were reported in parts per million (ppm) relative to the internal

standard TMS. Melting points were measured in capillary tubes

on Electrothermal’s MEL-TEMP apparatus without correction.

Liquid chromatography (LC)/mass spectroscopy (MS) was per-

formed on a Perkin-Elmer Sciex API 100 using one of the following

methods. Method A: YMC Pro C8 column, 5 µm, 150 × 4.6 mm;

mobile phase A ) water + 0.4% acetic acid, B ) acetonitrile

(MeCN) + 0.4% acetic acid; gradient: 5% B for 1 min, going up

to 80% B in 7 min, followed by 100% B for 5 min. Method B:

YMC Pro C8 column, 5 µm, 150 × 4.6 mm; mobile phase A )

water + 0.4% acetic acid, B ) MeCN + 0.4% acetic acid;

gradient: 5% B for 1 min, going up to 80% B in 5 min. Method

C: Luna C8 (2) column, 5 µm, 150 × 4.6 mm; detector λ ) 360

nm; mobile phase A ) water + 0.4% acetic acid; B) MeCN +

0.4% acetic acid; gradient: 5% B for 1 min, going up to 80% B in

7 min, followed by washout with 100% B for 5 min.

Analytical HPLC Was Performed by One of the Following

Methods. Method A: Shimadzu HPLC 10Avp: Luna C18(2)

column, 5 µm, 250 × 4.6 mm; detector λ ) 360 nm; gradient: A

) water + 0.1% trifluoroacetic acid (TFA); B) MeCN + 0.1%

TFA, concentration of MeCN increases from 10 to 60% during a

0-11 min interval and then 60-100% from 11 to 12 min. Method

B: Shimadzu HPLC 10Avp: Symmetry C8 column, 150 × 4.6

mm; detector λ ) 360 nm; gradient: A ) water + 0.1% TFA; B)

MeCN + 0.1% TFA, concentration of B increases in the A/B

mixture from 10 to 60% during the 0-11 min interval, then B

increases to 60-100% from 11 to 12 min. Method C: Gilson HPLC

(322 pump, 156 UV-Vis detector), polarity dC18 column, 5 µm,

4.6 × 250 mm; detector λ ) 220 nm; 40 °C; gradient: A ) water

+ 0.05% TFA; B ) MeCN + 0.05% TFA, 90:10 B/A for 5 min

and then to 20:80 B/A over 37 min. Diverse HPLC system, Method

Drugs for Cystic Fibrosis and Chronic Bronchitis

Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14 4107

solution and evaporated to dryness to give the desired products

hydrochloride salt (as yellow powder).

N), 7.20-7.50 (m, 8H, Ar + NH₂+ guanidino), 7.90 (br s, 1H,

guanidino), 9.00 (br s, 2H, NH₂), 9.35 (s, 1H, guanidino), 10.60

(s, 1H, guanidino); MS (APCI) m/z 334 (M + H)⁺. Anal. (C₁₄H₁₆-

ClN₇O‚HCl) H. Calcd, C 45.42, N 26.48; found, C 40.67, N 23.28.

N-(3,5-Diamino-6-chloropyrazine-2-carbonyl)-N′-(3-phenyl-

propyl)guanidine Hydrochloride (8). Compound 8 was prepared

Method B: Coupling of Unprotected Amine with 1-(3,5-

Diamino-6-chloropyrazine-2-carbonyl)-2-methylisothiourea Hy-

driodide (43b). The unprotected amine (1 equiv) was dissolved in

anhydrous ethanol (or THF or a mixture of these two solvents;

concentration 3 mL/mmol). To the solution, Hunig’s base (i-Pr₂-

EtN, 3 equiv) was added, and the newly formed solution was heated

at 65 °C for 15 min. Compound 43b (1.1 equiv) was then added.

The reaction mixture was stirred at 65 °C for an additional 2-3 h,

and then cooled to room temperature before being concentrated

under vacuum. The resulting residue was chromatographed on silica

gel by eluting with CMA. The appropriate fractions were collected

and concentrated under vacuum. The desired product (typically a

yellow solid) was characterized by spectroscopic methods.

Method C: HCl Salt Preparation of Final Compounds. The

purified free base of the final compounds was dissolved in a small

amount of methanol (2-3 mL). Concentrated HCl aqueous solution

(36.5wt %, 0.5 mL) was added dropwise. The mixture was stirred

at room temperature for 30 min, concentrated, and further dried

under vacuum. The yellow product was characterized spectroscopi-

cally.

Method D: Hydrogenolysis to Cleave the Carbobenzyloxy

Protecting Group. The substrate to be reduced was dissolved in

methanol or ethanol. The reaction vessel was vacuumed and refilled

with argon. The process was repeated three times before the

palladium catalyst (10% on charcoal, 50% wet) was added. The

reaction was carried out at room temperature under one atmosphere

of hydrogen until no further consumption of hydrogen was observed.

The reaction vessel was vacuumed and refilled with argon a second

time. The catalyst was filtered under vacuum and washed with

methanol or ethanol. The filtrate and washings were combined and

concentrated under vacuum. The residue was chromatographed,

eluting with CMA. The appropriate fractions were collected and

concentrated under vacuum. The dry product was then characterized

by spectroscopic methods.

1

according to method A: mp 192-196 °C; H NMR (360 MHz,

DMSO-d₆) δ 1.90 (m, 2H, 2-CH₂), 2.68 (t, J ) 10 Hz, 2H, 3-CH₂-

Ar), 3.35 (m, 2H, 1-CH₂-N), 7.18-7.30 (m, 5H, phenyl), 7.40-

7.80 (br s, 3H, NH₂+ guanidino), 8.90-9.06 (br s, 2H, NH₂), 9.48

(s, 1H, guanidino), 10.60 (s, 1H, guanidino); MS (APCI) m/z 348

(M + H)⁺. Anal. HRMS (FAB) m/z 348.1330; calcd, C₁₅H₁₈-

ClN₇O: 348.1339 (M + H)⁺. Anal. (C₁₅H₁₈ClN₇O‚HCl). Calcd, C

46.88, H 4.98, N 25.52; found, C 43.43, H 4.43, N 23.29.

N-(3,5-Diamino-6-chloropyrazine-2-carbonyl)-N′-(4-phenyl-

butyl)guanidine Hydrochloride (9). Compound 9 was prepared

1

according to method A: mp 160-162 °C; H NMR (360 MHz,

DMSO-d₆) δ 1.50-1.72 (m, 4H, 2-CH₂-3-CH₂), 2.70 (t, J ) 7.8

Hz, 2H, 4-CH₂-Ar), 3.35 (m, 2H, 1-CH₂-N), 7.18-7.32 (m, 5H,

Ar-), 7.45 (br s, 1H, guanidino), 7.60-7.80 (br s, 2H, NH₂), 9.02-

9.18 (br s, 2H, NH₂), 9.42 (s, 1H, guanidino), 10.65 (s, 1H,

guanidino); MS (APCI) m/z 362 (M + H)⁺. HRMS (FAB) m/z

362.1504; calcd, C₁₆H₂₀ClN₇O: 362.1496 (M + H)⁺. Anal. (C₁₆H₂₀-

ClN₇O‚HCl) H. Calcd, C 48.25, N 24.62; found, C 46.59, N 23.11.

N-(3,5-Diamino-6-chloropyrazine-2-carbonyl)-N′-(5-phenyl-

pentyl)guanidine Hydrochloride (10). Compound 10 was prepared

1

according to method A: mp 240-243 °C (dec); H NMR (300

MHz, DMSO-d₆) δ 1.36 (m, 2H, 3-CH₂-), 1.57-1.63 (m, 4H,

2-CH₂- + 4-CH₂-), 2.58 (t, J ) 7.8 Hz, 2H, 5-CH₂-Ar), 3.34 (m,

2H, 1-CH₂-N), 7.09-7.30 (m, 5H, Ar-), 7.55(br s, 1H, guanidino),

7.60-7.80 (br s, 2H, NH₂), 8.78-8.92 (br s, 2H, NH₂), 9.26 (s,

1H, guanidino), 10.51 (s, 1H, guanidino); MS (APCI) m/z 376 (M

+ H)⁺. Anal. (C₁₇H₂₂ClN₇O‚HCl) C, H, N.

N-(3,5-Diamino-6-chloropyrazine-2-carbonyl)-N′-(6-phenyl-

hexyl)guanidine Hydrochloride (11). Compound 11 was prepared

1

according to method A: mp 126-129 °C; H NMR (360 MHz,

N-(3,5-Diamino-6-chloropyrazine-2-carbonyl)-N′-hexylguani-

dine Hydrochloride (2). Compound 2 was prepared according to

general method A: mp 230-232 °C; ¹H NMR (360 MHz, DMSO-

d₆) δ 0.88 (t, J ) 8 Hz, 3H, 6-CH₃), 1.26-1.30 (m, 6H, 3-CH₂-

4-CH₂-5-CH₂), 1.54 (m, 2H, 2-CH₂), 3.30 (m, 2H, 1-CH₂-N), 7.26-

7.60 (br s, 3H, NH₂+ guanidino), 8.78-9.02 (br s, 2H, NH₂), 9.32

(s, 1H, guanidino), 10.60 (s, 1H, guanidino); MS (APCI) m/z 314

(M + H)⁺; HRMS (FAB) m/z 314.1496; calcd, C₁₂H₂₀ClN₇O‚

HCl: 314.1496 (M + H)⁺. Anal. (C₁₂H₂₀ClN₇O‚HCl). Calcd, C

41.15, H 6.04 N 27.99; found, C 35.87, H 5.55, N 24.26.

N-(3,5-Diamino-6-chloropyrazine-2-carbonyl)-N′-octylguani-

dine Hydrochloride (3). Compound 3 was prepared according to

DMSO-d₆) δ 1.38 (m, 4H, 4-CH₂-5-CH₂), 1.72 (m, 4H, 2-CH₂-3-

CH₂), 2.62 (t, J ) 7.8 Hz, 2H, 6-CH₂-Ar), 3.28 (m, 2H, 1-CH₂-N),

7.18-7.32 (m, 5H, Ar-), 7.45(br s, 1H, guanidino), 7.60-8.00 (br

s, 2H, NH₂), 9.02-9.10 (br s, 2H, NH₂), 9.48 (s, 1H, guanidino),

10.68 (s, 1H, guanidino); MS (APCI) m/z 390 (M + H)⁺. Anal.

HRMS (FAB) m/z 390.1792; calcd, C₁₈H₂₄ClN₇O: 390.1809 (M

+ H)⁺. Anal. (C₁₈H₂₄ClN₇O‚HCl). Calcd, C 50.71, H 5.91, N 23.00;

found, C 49.11, H 6.49, N 21.90.

N-(3,5-Diamino-6-chloropyrazine-2-carbonyl)-N′-(8-phenyloc-

tyl)guanidine Hydrochloride (12). Compound 12 was prepared

1

according to method A: mp 171-173 °C; H NMR (360 MHz,

DMSO-d₆) δ 1.33 (m, 8H, 3-CH₂-4-CH₂-5-CH₂-6-CH₂-); 1.64 (m,

4H, 2-CH₂- and 7-CH₂-); 2.56 (m, 2H, 8-CH₂-Ar); 3.25 (m, 2H,

1-CH₂-N); 7.16-7.27 (m, 5H, Ar-); 7.40 (br s, 2H, NH₂); 8.85 (br

s, 1H, guanidino); 8.95 (br s, 2H, NH₂); 9.31 (s, 1H, guanidino);

10.56 (s, 1H, guanidino); MS (APCI) m/z 418 (M + H)⁺. HRMS

(FAB) m/z 418.2110; calcd, C₂₀H₂₈ClN₇O: 418.2122 (M + H)⁺.

Purity: 99% (method A; t_R) 7.1 min), 97% (method D; t_R) 8.4

min).

1

method A: mp 241-244 °C (dec); H NMR (360 MHz, DMSO-

d₆) δ 0.88 (t, J ) 8 Hz, 3H, 8-CH₃), 1.26-1.30 (m, 8H, 4-CH₂-

5-CH₂-6-CH₂-7-CH₂-), 1.44 (m, 2H, 3-CH₂), 1.60 (m, 2H, 2-CH₂),

3.20 (m, 2H, 1-CH₂-N), 7.26-7.45 (br s, 2H, NH₂), 7.78 (br s,

1H, guanidino), 8.78-9.02 (br s, 2H, NH₂), 9.32 (s, 1H, guanidino),

10.60 (s, 1H, guanidino); MS (APCI) m/z 342 (M + H)⁺. HRMS

(FAB) m/z 342.1805; calcd C₁₄H₂₄ClN₇O: 342.1809 (M + H)⁺.

Anal. (C₁₄H₂₄ClN₇O‚HCl) H. Calcd, C 42.45, N 25.92; found, C

45.73, N 24.06.

N-(3,5-Diamino-6-chloropyrazine-2-carbonyl)-N′-[2-(4-hy-

droxyphenyl)ethyl]-guanidine Hydrochloride (13). Compound 13

was gratefully obtained as a gift from Dr. E. J. Cragoe, Jr.: ¹H

NMR (300 MHz, DMSO-d₆) δ 2.76 (t, 2H, J ) 5.6 Hz, 2-CH₂-

Ar), 3.50 (m, 2H, 1-CH₂-N), 6.72 (d, J ) 7.6 Hz, 2H, Ar-), 7.10

(d, J ) 7.6 Hz, 2H, Ar-), 7.42 (br s, 2H, NH₂), 7.80 (br s, 1H,

guanidino), 8.80 (br s, 2H, NH₂), 9.20 (br s, 1H, 4-OH-Ar), 9.30

(s, 1H, guanidino), 10.50 (s, 1H, guanidino); ¹³C NMR (75 MHz,

DMSO-d₆) δ 34.85, 44.48, 111.19, 117.01 (2×C), 122.14, 129.83,

131.25 (2×C), 155.80, 156.65, 157.96, 158.12, 167.47. MS (APCI)

m/z 350 (M + H)⁺. HRMS (FAB) m/z 350.1142; calcd, C₁₄H₁₆-

ClN₇O₂: 350.1132 (M + H)⁺. Anal. (C₁₄H₁₆ClN₇O_2•HCl). Calcd,

C 43.54, H 4.44, N 25.39; found, C 40.80, H 4.86, N 24.26.

N-(3,5-Diamino-6-chloropyrazine-2-carbonyl)-N′-[3-(4-hy-

droxyphenyl)propyl]-guanidine Hydrochloride (14). Pyridine (15

mL) was added dropwise to a cooled (0 °C) solution of 4-(4-

N-(3,5-Diamino-6-chloropyrazine-2-carbonyl)-N′-(2-hydroxy-

ethyl)guanidine Hydrochloride (4). Compound 4 was prepared

1

according to method A: mp 203-205 °C (dec); H NMR (360

MHz, DMSO-d₆) δ 2.82 (br s, 1H, 2-OH), 3.40 (m, 2H, 1-CH₂-N),

3.62 (m, 2H, 2-O-CH₂), 7.36-7.45 (br s, 3H, guanidino + NH₂),

8.0 (br s, 1H, guanidino), 8.88-9.02 (br s, 2H, NH₂), 9.40 (s, 1H,

guanidino), 10.68 (s, 1H, guanidino); MS (APCI) m/z 274 (M +

H)⁺. HRMS (FAB) m/z 274.0814; calcd, C₈H₁₂ClN₇O₂: 274.0819

(M + H)⁺. Anal. (C₈H₁₂ClN₇O₂‚HCl) H. Calcd, C 30.98, N 31.61;

found, C 30.50, N 27.58.

N-(3,5-Diamino-6-chloropyrazine-2-carbonyl)-N′-pheneth-

ylguanidine Hydrochloride (7). Compound 7 was prepared

1

according to method A: mp 156-160 °C (dec); H NMR (360

MHz, DMSO-d₆) δ 2.80 (m, 2H, 2-CH₂-Ar), 3.60 (m, 2H, 1-CH₂-

4108 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14

Hirsh et al.

methoxyphenyl)propanol (44b) (10.0 g, 0.06 mol), and methane-

sulfonyl chloride (14.7 g, 0.078 mol) and dry THF (70 mL) was

added with adequate stirring. The reaction mixture was stirred at

room-temperature overnight. After this time, the solvent was

removed under reduced pressure and the residue quenched with

10% HCl (300 mL) and extracted with ethyl acetate. The organic

fraction was washed with saturated aqueous NaHCO₃solution and

water and dried (anhydrous sodium sulfate). The solvent was

removed, and the residual crude ester (8.8 g, 60%, a yellow oil)

was used in the following step without purification: ¹H NMR (300

MHz, CDCl₃) δ 2.08 (m, 2H, 2-CH₂-), 2.60 (t, J ) 6.9 Hz, 2H,

3-CH₂-Ar), 3.58 (t, J ) 6.9 Hz, 2H, 1-CH₂-O), 3.66 (s, 3H, CH₃-

SO₃-), 3.98 (s, 3H, 4-MeO-Ar), 6.85 (d, J ) 7.8 Hz, 2H, Ar-),

7.03 (d, J ) 7.8 Hz, 2H, Ar-). The above oil was dissolved in

anhydrous DMF (10 mL). To the solution, sodium azide (3 g, 0.045

mol) was added, stirred at room temperature overnight, and

quenched with water (10 mL). The organics were extracted with

ethyl ether (3 × 50 mL) and washed with brine, dried (anhydrous

sodium sulfate), and concentrated. The residue was chromato-

graphed, eluting with a mixture of ethyl acetate and hexane (20:

80, v/v) to afford 45b in 75% yield: ¹H NMR (300 MHz, CDCl₃)

δ 1.90 (m, 2H, 2-CH₂-), 2.65 (t, J ) 6.8 Hz, 2H, 3-CH₂-Ar), 3.28

(t, J ) 6.8 Hz, 2H, 1-CH₂-N₃), 3.80 (s, 3H, 4-MeO-Ar), 6.85 (d, J

) 7.8 Hz, 2H, Ar-), 7.10 (d, J ) 7.8 Hz, 2H, Ar-).

The above azide 45b (5.2 g, 0.027 mol) was dissolved in

anhydrous THF (15 mL). To the solution, Ph₃P (11.56 g, 0.0405

mol) and water (0.73 mL) was added, and the reaction mixture

was stirred at room temperature overnight and then concentrated

under vacuum. The residue was purified by flash chromatography

(silica gel, 2:1:0.05 chloroform/ethanol/concentrated ammonium

hydroxide) to provide pure amine 46b (3.2 g, 74%) as a clear oil:

¹H NMR (300 MHz, CDCl₃) δ 1.58 (m, 2H, 2-CH₂-), 2.50 (m, 4H,

1-CH₂-N + 3-CH₂-Ar), 3.72 (s, 3H, 4-MeO-Ar), 6.85 (d, J ) 7.8

Hz, 2H, Ar-), 7.10 (d, J ) 7.8 Hz, 2H, Ar-).

Following the same procedure used for the preparation of

compound 46b, compound 46c was prepared in 94% yield from

compound 45c: ¹H NMR (360 MHz, DMSO-d₆) δ 1.34 (m, 2H,

3-CH₂-), 1.54 (m, 2H, 2-CH₂-), 2.51 (m, 4H, 1-CH₂-N + 4-CH₂-

Ar), 3.70 (s, 3H, 4-MeO-Ar), 6.83 (d, J ) 8.1 Hz, 2H, Ar-), 7.08

(d, J ) 8.1 Hz, 2H, Ar-).

Following the same procedure used for the preparation of

compound 47b, compound 47c was prepared from 46c in 90%

yield: MS (APCI) m/z 166 for C₁₀H₁₅NO (M + H)⁺.

Alternative Synthesis of 47c. 4-(4-Methoxyphenyl)butyric acid

(48) (450 g, 2.32 mol) was combined with anhydrous THF (4 L)

and 4-methylmorpholine (268 mL, 2.43 mol) in a 12 L, three-necked

flask, which was equipped with a mechanical stirrer, and placed in

an ice-methanol cooling bath and protected under nitrogen

atmosphere. Small pieces of dry ice were used to bring the bath

temperature below -20 °C. Iso-butylchloroformate was added at

a rate so as to not to exceed an internal temperature of -10 °C.

After stirring for 30 min at -10 to -20 °C, a 4.7 M solution of

ammonia in methanol (990 mL, 4.64 mol) was added. During the

addition, the reaction temperature rose to 0 °C. The reaction was

allowed to stir for 30 min and stand overnight. The product mixture

was transferred to a 22 L separatory funnel with ethyl acetate (6

L) and 10% aqueous sodium chloride solution (1.5 L). The layers

were separated and the organic solution washed with the 10%

sodium chloride solution (4 × 1 L), followed by brine (3 × 500

mL). The organic layer was dried (anhydrous sodium sulfate),

filtered, evaporated, and placed under high vacuum overnight. This

afforded 432 g (97%) of pure amide 49 as an off white solid: ¹H

NMR (300 MHz, CD₃OD) δ 1.81-1.93 (m, 2H, 3-CH₂-), 2.20 (t,

J ) 7.7 Hz, 2H, 2-CH₂-CONH₂), 2.57 (t, J ) 7.7 Hz, 2H, 4-CH₂-

Ar), 3.74 (s, 3H, 4-MeO-Ar), 6.82 (d, J ) 8.7 Hz, 2H, Ar-), 7.09

(d, J ) 8.7 Hz, 2H, Ar-); MS (ESI) m/z 194 for C₁₁H₁₅NO₂(M +

H)⁺. 4-(4-Methoxyphenyl)butyramide (49) (200 g, 1.0 mol) and

THF (300 mL) were combined in a 12 L, three-necked flask, which

was equipped with a heating mantle, an internal thermometer, and

a reflux condenser. The suspension was slowly mechanically stirred

while a 1 M Borane‚THF (1 L, 1 mol) solution was dripped in via

a pressure equalizing addition funnel over 20 min. Another 2.2 L

(2.2 mol) of 1 M Borane‚THF complex was dripped in over 20

min. The reaction temperature rose to 45 °C during the addition.

The reaction was heated to reflux over 1 h, maintained at reflux

for 2 h, and then allowed to cool for 2 h. Methanol (500 mL) was

slowly and cautiously dripped into the reaction. Copious H₂

evolution was observed. The reaction was heated at reflux for 2 h

and then allowed to cool overnight. The reaction was evaporated

and then coevaporated with toluene (500 mL) to afford a thick oil.

A 48% aqueous HBr solution (3 L) was slowly and cautiously added

to the reaction. Bubbling and foaming were observed during this

addition, which was exothermic. After the addition, the reaction

was less viscous and was stirred at reflux for 7 h. The reaction

was allowed to cool with stirring overnight. The batch was further

chilled in an ice bath and then suction filtered to collect an off

white solid. The solid was coevaporated with toluene/methanol (1:

1, v/v) and then dried under vacuum at 60 °C overnight. This

afforded 197 g (77%) of 47c as an off white crystalline solid: ¹H

NMR (300 MHz, CD₃OD) δ 1.66 (m, 4H, 2-CH₂-3-CH₂-), 2.57 (t,

J ) 6.8 Hz, 2H, 4-CH₂-Ar), 2.92 (m, 2H, 1-CH₂-N), 6.70 (d, J )

8.5 Hz, 2H, Ar-), 7.01 (d, J ) 8.5, Hz, 2H, Ar-); MS (ESI) m/z

166 for C₁₀H₁₅NO (M + H)⁺.

The above compound 46b (2.5 g, 0.015 mol) was dissolved in

48% aqueous HBr solution (5 mL). The solution was heated to

reflux for 3 h and then concentrated and further dried under vacuum

to afford the desired product 47b (1.99 g, 75% yield) as a light

brown solid: ¹H NMR (300 MHz, DMSO-d₆) δ 1.80 (m, 2H,

2-CH₂-), 2.53 (t, J ) 6.9 Hz, 2H, 3-CH₂-Ar), 2.78 (m, 2H, 1-CH₂-

N), 6.70 (d, J ) 7.8 Hz, 2H, Ar-), 7.02 (d, J ) 7.8 Hz, 2H, Ar-),

7.80 (br s, 4H, HO-Ar + NH₃⁺).

Compound 14 (a yellow solid) was made from 47b in 31% yield

1

according to the methods B and C: mp 133 °C (dec); H NMR

(300 MHz, DMSO-d₆) δ 1.80 (m, 2H, 2-CH₂), 2.58 (t, J ) 6.8 Hz,

2H, 3-CH₂-Ar), 3.35-3.65 (m, 3H, 1-CH₂-N + Ar-OH), 6.70 (d,

J ) 7.6 Hz, 2H, Ar-), 7.03 (d, J ) 7.6 Hz, 2H, Ar-), 7.48 (br s,

2H, NH₂), 8.80 (br s, 1H, guanidino), 8.93 (br s, 1H, guanidino),

9.32 (br s, 2H, NH₂), 10.52 (s, 1H, guanidino); MS (APCI) m/z

364 (M + H)⁺. HRMS (FAB) m/z 364.1276; calcd, C₁₅H₁₈-

ClN₇O₂: 364.1289 (M + H)⁺. Anal. (C₁₅H₁₈ClN₇O₂‚HCl). Calcd,

C 45.01, H 4.79, N 24.50; found, C 42.93, H 4.81, N 18.33.

N-(3,5-Diamino-6-chloropyrazine-2-carbonyl)-N′-[3-(4-meth-

oxyphenyl)propyl]-guanidine Hydrochloride (15). Compound 15

was made from compound 46b following methods B and C: mp

1

134 °C (dec); H NMR (300 MHz, DMSO-d₆) δ 1.85 (m, 2H,

2-CH₂), 2.75 (m, 2H, 3-CH₂-Ar), 3.32 (m, 2H, 1-CH₂-N), 3.78 (s,

3H, 4-MeO-Ar), 6.84 (d, J ) 7.6 Hz, 2H, Ar-), 7.20 (d, J ) 7.6

Hz, 2H, Ar-), 7.50 (br s, 1H, guanidino), 8.12 (br s, 2H, NH₂),

8.95 (br s, 2H, NH₂), 9.40 (br s, 1H, guanidino), 10.60 (s, 1H,

guanidino); MS (APCI) m/z 378 (M + H)⁺. HRMS (FAB) m/z

378.1431; calcd, C₁₆H₂₀ClN₇O₂: 378.1445 (M + H)⁺. Anal.

(C₁₆H₂₀ClN₇O₂‚HCl). Calcd, C 46.39, H 5.11, N 23.67; found, C

49.81, H 6.14, N 14.42.

Using methods B and C, compound 16 was prepared from

compound 47c in 41% yield as a yellow solid: mp 160 °C (dec);

¹H NMR (300 MHz, DMSO-d₆) δ 1.56 (m, 4H, 2-CH₂-3-CH₂),

2.48 (m, 2H, 4-CH₂-Ar), 3.35 (m, 2H, 1-CH₂-N), 6.65 (d, J ) 7.8

Hz, 2H, Ar-), 6.95 (d, J ) 7.8 Hz, 2H, Ar-), 7.50 (br s, 2H, NH₂),

8.75 (br s, 1H, guanidino), 9.05 (br s, 1H, guanidino), 9.33 (br s,

2H, NH₂), 10.55 (s, 1H, guanidino); MS (APCI) m/z 378 (M +

H)⁺. HRMS (FAB) m/z 378.1460; calcd, C₁₆H₂₀ClN₇O₂: 378.1445

(M + H)⁺. Anal. (C₁₆H₂₀ClN₇O₂‚HCl). Calcd, C 46.39, H 5.11, N

23.67; found, C 45.21, H 5.92, N 18.85.

N-(3,5-Diamino-6-chloropyrazine-2-carbonyl)-N′-[4-(4-hy-

droxyphenyl)butyl]-guanidine Hydrochloride (16). Compound

45c (a clear oil) was prepared in 66% yield from 44c using a method

similar to that used to obtain compound 45b: ¹H NMR (360 MHz,

CDCl₃) δ 1.61 (m, 4H, 2-CH₂-3-CH₂-), 2.44 (t, J ) 6.8 Hz, 2H,

4-CH₂-Ar), 2.52 (t, J ) 7.2 Hz, 2H, 1-CH₂-N₃), 3.78 (s, 3H, 4-MeO-

Ar), 6.77 (d, J ) 8.1 Hz, 2H, Ar-), 7.05 (d, J ) 8.1 Hz, 2H, Ar-).

N-(3,5-Diamino-6-chloropyrazine-2-carbonyl)-N′-[5-(4-hy-

droxyphenyl)pentyl]-guanidine Hydrochloride (17). 4-Iodoanisol

Drugs for Cystic Fibrosis and Chronic Bronchitis

Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14 4109

50a (10 g, 42 mmol), palladium (II) chloride (0.2 g, 1.1 mmol),

and triphenylphosphine (0.6 g, 2.2 mmol) were dissolved in

diethylamine (100 mL) then copper (I) iodide (0.5 g, 2.2 mmol),

and N-Boc-protected 4-pentynyl-1-amine 51 (n ) 3, 5 mL, 53

mmol) were added. The reaction mixture was stirred at room

temperature overnight, and then the solvent was removed under

reduced pressure. Ethyl acetate (150 mL) was added to the residue,

and the mixture was sequentially washed with 2 N aqueous HCl

solution, brine, and water. The organic fraction was isolated and

dried (anhydrous sodium sulfate). The solvent was removed under

reduced pressure. Product 52a (7.1 g. 87%) was isolated by flash

chromatography (silica gel, 1:2 ethyl acetate/hexanes) as an oily

yellow solid: ¹H NMR (300 MHz, CDCl₃) δ 1.46 (s, 9H, Boc),

1.88 (m, 2H, 2-CH₂-), 2.53 (t, 2H, J ) 5.7 Hz, 3-CH₂-alkyne),

3.51 (m, 2H, 1-CH₂-N), 3.72 (s, 3H, 4-MeO-Ar), 6.83 (d, J ) 8.0

Hz, 2H, Ar-), 7.45 (d, J ) 8.0 Hz, 2H, Ar-).

A solution of 52a (7.1 g, 37 mmol) in dry ethanol (150 mL)

was placed in a 0.5 L Parr flask. Palladium on carbon (0.92 g, 5%

Pd, 50% wet) was added as a suspension in ethanol (25 mL). The

reaction mixture was shaken at 50 psi of hydrogen pressure at room

temperature for 24 h. After this time, the mixture was filtered

through Celite, and the solvent was removed under reduced

pressure. The residue was purified by flash chromatography (silica

gel, 1:3 ethyl acetate/hexanes) to provide 53a (6.7 g, 92%) as a

clear oil: ¹H NMR (300 MHz, CDCl₃) δ 1.48 (m, 2H, 3-CH₂-),

1.60 (m, 4H, 2-CH₂+ 4-CH₂-), 2.58 (m, 2H, 5-CH₂-Ar), 3.63 (m,

2H, 1-CH₂-N), 3.80 (s, 3H, 4-MeO-Ar), 6.83 (d, J ) 8.0 Hz, 2H,

Ar-), 7.10 (d, J ) 8.0 Hz, 2H, Ar-).

MS (APCI) m/z 378 (M + H)⁺. HRMS (FAB) m/z 378.1450; calcd.

C₁₆H₂₀ClN₇O₂: 378.1445 (M + H)⁺. Anal. (C₁₆H₂₀ClN₇O₂‚HCl)

H. Calcd, C 46.39, N 23.67; found, C 45.19, N 19.50.

N-(3,5-Diamino-6-chloropyrazine-2-carbonyl)-N′-[4-(2-hy-

droxyphenyl)butyl]-guanidine Hydrochloride (19). Compound

52c was prepared from 50c in a manner similar to that used to

prepare compound 52a: ¹H NMR (300 MHz, CDCl₃) δ 1.46 (s,

9H, Boc), 2.64 (t, J ) 6.3 Hz, 2H, 2-CH₂), 3.38 (m, 2H, 1-CH₂-

N), 3.89 (s, 3H, 2-MeO-Ar), 5.14 (br s, 1H, NHBoc), 6.85 (m, 2H,

Ar-), 7.25 (m, 1H, Ar-), 7.37 (d, J ) 7.4 Hz, 1H, Ar-).

Compound 53c was prepared from 52c using a method similar

to that used to prepare compound 53a: ¹H NMR (300 MHz, CDCl₃)

δ 1.43 (s, 9H, Boc), 1.56-1.62 (m, 4H, 2-CH₂-3-CH₂-), 2.58 (t, J

) 9.4 Hz, 2H, 4-CH₂-Ar), 3.26 (m, 2H, 1-CH₂-N), 3.83 (s, 3H,

2-MeO-Ar), 4.70 (br s, 1H, NHBoc), 6.87 (m, 2H, Ar-), 7.17 (m,

2H, Ar-).

Compound 54c was prepared from 53c following the same

procedure used for the preparation of compound 47b and directly

used in the next step without purification.

Compound 19 (a yellow solid) was made in 39% yield from

54c following general methods B and C: mp 240-242 °C; ¹H NMR

(300 MHz, DMSO-d₆) δ 1.58 (m, 4H, 2-CH₂-3-CH₂-), 2.50 (m,

2H, 4-CH₂-Ar), 3.28 (m, 2H, 1-CH₂-N), 6.70 (m, 2H, Ar-), 7.06

(m, 2H, Ar-), 7.75 (br s, 2H, NH₂), 7.80 (s, 1H, guanidino), 8.70

(br s, 2H, NH₂), 9.10 (br s, 1H, guanidino), 10.47 (s, 1H, guanidino);

MS (APCI) m/z 378 (M + H)⁺. HRMS (FAB) m/z 378.1440; calcd.

C₁₆H₂₀ClN₇O₂: 378.1445 (M + H)⁺. Anal. (C₁₆H₂₀ClN₇O₂‚HCl)

C. Calcd, H 5.11, N 23.67; found, H 4.42, N 22.36.

The HBr salt of 54a was prepared from 53a following the same

procedure used for the preparation of compound 47a. The free

amine of 54a (a cloudy oil, 2 g, 80%) was obtained after flash

chromatography (silica gel, 6:3:0.1, chloroform/ethanol/concentrated

ammonium hydroxide): ¹H NMR (300 MHz, DMSO-d₆) δ 1.28

(m, 2H, 3-CH₂-), 1.55 (m, 2H, 4-CH₂-), 1.61 (m, 2H, 2-CH₂-), 2.48

(m, 2H, 1-CH₂-N), 2.58 (m, 2H, 5-CH₂-Ar), 6.68 (d, J ) 8.0 Hz,

2H, Ar-), 6.98 (d, J ) 8.0 Hz, 2H, Ar-).

Compound 17 as a yellow solid was made in 39% yield from

54a following methods B and C: mp 188 °C; ¹H NMR (300 MHz,

DMSO-d₆) δ 1.32 (m, 2H, 3-CH₂), 1.55 (m, 4H, 2-CH₂and 4-CH₂),

2.45 (m, 2H, 5-CH₂-Ar), 3.29 (m, 2H, 1-CH₂-N), 6.68 (d, J ) 8.0

Hz, 2H, Ar-), 6.97 (d, J ) 8.0 Hz, 2H, Ar-), 7.46 (s, 1H, guanidino),

8.00 (br s, 2H, NH₂), 8.97 (br s, 1H, guanidino), 9.46 (br s, 2H,

NH₂), 10.55 (s, 1H, guanidino); MS (APCI) m/z 392 (M + H)⁺.

HRMS (FAB) m/z 392.1588; calcd. C₁₇H₂₂ClN₇O₂: 392.1602 (M

+ H)⁺. Anal. (C₁₇H₂₂ClN₇O₂‚HCl). Calcd, C 47.67, H 5.41, N

22.89; found, C 45.21, H 5.92, N 18.85.

N-(3,5-Diamino-6-chloropyrazine-2-carbonyl)-N′-[4-(5-hy-

droxyphenyl)butyl]-guanidine Hydrochloride (18). Compound

52b was prepared in a manner similar to that used to prepare

compound 52a: ¹H NMR (300 MHz, CDCl₃) δ 1.45 (s, 9H, Boc),

2.65 (t, J ) 6.6 Hz, 2H, 2-CH₂), 3.34 (m, 2H, 1-CH₂-N), 3.72 (s,

3H, 5-MeO-Ar), 4.65 (br s, 1H, NHBoc), 6.83 (d, J ) 7.6 Hz, 1H,

Ar), 6.96 (m, 1H, Ar), 7.03 (d, J ) 7.6 Hz, 1H, Ar), 7.26 (m, 1H,

Ar).

N-[4-(4-Aminopentyl)butyl]-N′-(3,5-diamino-6-chloropyrazine-

2-carbonyl)-guanidine Hydrochloride (20). Compound 52d was

prepared from compound 50d in a manner similar to that used to

prepare compound 52a: ¹H NMR (300 MHz, CDCl₃) δ 1.46 (s,

9H, Boc), 2.66 (t, J ) 6.5 Hz, 2H, 2-CH₂), 3.40 (m, 2H, 1-CH₂-

N), 4.83 (br s, 1H, NHBoc), 7.54 (d, J ) 9.0 Hz, 2H, Ar-), 8.18

(d, J ) 9.1 Hz, 2H, Ar-).

Compound 53d was prepared from 52d in a method similar to

that used to prepare compound 53a: ¹H NMR (300 MHz, CDCl₃)

δ 1.49 (s, 9H, Boc), 1.56-1.68 (m, 4H, 2-CH₂-3-CH₂-), 2.53 (t, J

) 7.1 Hz, 2H, 4-CH₂-Ar), 3.17 (m, 2H, 1-CH₂-N), 3.47 (br s, 2H,

4-NH₂-Ar), 4.46 (br s, 1H, NHBoc), 6.62 (d, J ) 8.3 Hz, 2H, Ar-),

6.95 (d, J ) 8.3 Hz, 2H, Ar-).

Compound 54d was prepared from 53d following the same

procedure used for the preparation of compound 47b and directly

used in the next step without purification.

Compound 20 as a yellow solid was made from 54d in 39%

yield following methods B and C: mp 195-200 °C (dec); ¹H NMR

(300 MHz, DMSO-d₆) δ 1.56-1.64 (m, 4H, 2-CH₂-3-CH₂-), 2.62

(t, J ) 9.6 Hz, 2H, 4-CH₂-Ar), 3.39 (m, 2H, 1-CH₂-N), 4.99 (br s,

2H, 4-NH₂-Ar), 7.34 (m, 4H, Ar-), 7.66 (br s, 2H, NH₂), 8.06 (s,

1H, guanidino), 8.90 (br s, 2H, NH₂), 9.38 (br s, 1H, guanidino),

10.56 (s, 1H, guanidino); MS (APCI) m/z 377 (M + H)⁺. HRMS

(FAB) m/z 377.1607; calcd, C₁₆H₂₁ClN₈O: 377.1605 (M + H)⁺.

Anal. (C₁₆H₂₁ClN₈O‚2HCl). Calcd, C 42.73, H 5.15, N 24.91; found

C 38.20, H 5.31, N 21.08.

Compound 53b was prepared from 52b using a method similar

to that used to prepare compound 53a: ¹H NMR (300 MHz, CDCl₃)

δ 1.45 (s, 9H, Boc), 1.56-1.78 (m, 4H, 2-CH₂-3-CH₂-), 2.56 (t, J

) 6.4 Hz, 2H, 4-CH₂-Ar), 3.32 (m, 2H, 1-CH₂-N), 3.72 (s, 3H,

5-MeO-Ar), 4.65 (br s, 1H, NHBoc), 6.76 (m, 3H, Ar-), 7.20 (m,

1H, Ar-).

Compound 54b was prepared from 53b following the same

procedure used for the preparation of compound 54a: ¹H NMR (300

MHz, DMSO-d₆) δ 1.52 (m, 2H, 3-CH₂-), 1.68 (m, 2H, 2-CH₂-),

2.62 (t, J ) 10.0 Hz, 2H, 4-CH₂-Ar), 2.72 (m, 2H, 1-CH₂-N), 3.34

(br s, 2H, NH₂), 6.74 (m, 3H, Ar-), 7.20 (m, 1H, Ar-).

N-(3,5-Diamino-6-chloropyrazine-2-carbonyl)-N′-[4-(4-meth-

oxyphenyl)butyl]-guanidine Hydrochloride (21). Compound 21

was prepared as a yellow powder from compound 40c following

methods B and C: mp 108-111 °C; ¹H NMR (360 MHz, DMSO-

d₆) δ 1.56 (m, 4H, 2-CH₂-3-CH₂); 2.56 (m, 2H, 4-CH₂-Ar); 3.32

(m, 2H, 1-CH₂-N); 3.74 (s, 3H, 4-MeO-Ar), 6.85 (d, J ) 7.8 Hz,

2H, Ar-); 7.10 (d, J ) 7.8 Hz, 2H, Ar-); 7.46 (br s, 2H, NH₂), 7.85

(br s, 1H, guanidino), 8.98 (br s, 2H, NH₂); 9.46 (s, 1H, guanidino);

10.60 (s, 1H, guanidino); MS (APCI) m/z 392 (M + H)⁺. HRMS

(FAB) m/z 392.1604; calcd, C₁₇H₂₂ClN₇O₂: 392.1602 (M + H)⁺.

Purity: 99% (method A; t_R) 6.4 min), 98% (method D; t_R) 6.0

min).

4-{4-[N′-(3,5-Diamino-6-chloropyrazine-2-carbonyl)guanidino]-

butyl}benzoic Acid Methyl Ester Hydrochloride (22). Compound

45d was prepared as a clear oil from 44d in 85% yield following

the same method used for the preparation of compound 45a: ¹H

NMR (300 MHz, CDCl₃) δ 1.68 (m, 4H, 2-CH₂-3-CH₂-), 2.22 (t,

Compound 18 was made as a yellow solid from 54b in 39%

1

yield following methods B and C: mp >215 °C (dec); H NMR

(300 MHz, DMSO-d₆) δ 1.52 (m, 4H, 2-CH₂-3-CH₂), 2.51 (m, 2H,

4-CH₂-Ar), 3.30 (m, 2H, 1-CH₂-N), 6.60 (m, 3H, Ar-), 7.10 (m,

1H, Ar-), 7.40 (br s, 2H, NH₂), 7.90 (s, 1H, guanidino), 8.40 (br s,

2H, NH₂), 9.30 (br s, 1H, guanidino), 10.60 (s, 1H, guanidino);

4110 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14

Hirsh et al.

1

J ) 6.4 Hz, 2H, 4-CH₂-Ar), 3.29 (t, J ) 6.5 Hz, 2H, 1-CH₂-N₃),

3.92 (s, 3H, 4-MeO₂C-Ar), 7.28 (d, J ) 7.8 Hz, 2H, Ar-), 7.98 (d,

J ) 7.8 Hz, 2H, Ar-).

make the HCl salt: mp 123-126 °C (dec); H NMR (300 MHz,

DMSO-d₆) δ 1.60 (m, 4H, 2-CH₂-3-CH₂), 2.74 (m, 2H, 4-CH₂-

Ar), 3.36 (m, 2H, 1-CH₂-N), 7.30 (d, J ) 7.8 Hz, 2H, Ar-), 7.48

(br s, 2H, NH₂), 7.60 (br s, 1H, guanidino), 7.90 (d, J ) 7.8 Hz,

2H, Ar-), 8.90 (br s, 2H, NH₂), 9.10 (br s, 1H, guanidino), 9.50 (br

s, 1H, guanidino), 12.80 (s, 1H, 4-CO₂H); MS (APCI) m/z 406 (M

+ H⁺). HRMS (FAB) m/z 406.1395; calcd, C₁₇H₂₀ClN₇O₃: 406.1394

(M + H)⁺. Anal. (C₁₇H₂₀ClN₇O₃‚HCl) H. Calcd, C 46.16, N 22.17;

found C 41.08, N 19.42.

Compound 46d was made from 45d in 53% yield using the same

procedure used for the preparation of compound 46b.

Following methods B and C, compound 22 was made as a yellow

1

solid from 46d in 25% yield: mp 210 °C (dec); H NMR (300

MHz, DMSO-d₆) δ 1.59 (m, 4H, 2-CH₂-3-CH₂), 2.71 (m, 2H,

4-CH₂-Ar), 3.23 (m, 2H, 1-CH₂-N), 3.83 (s, 3H, 4-CO₂Me-Ar),

7.40 (d, J ) 7.8 Hz, 2H, Ar-), 7.48 (br s, 2H, NH₂), 7.80 (d, J )

7.8 Hz, 2H, Ar-), 8.92 (br s, 2H, NH₂), 9.00 (br s, 1H, guanidino),

9.48 (br s, 1H, guanidino), 10.55 (s, 1H, guanidino); MS (APCI)

m/z 420 (M + H)⁺. HRMS (FAB) m/z 420.1544; calcd, C₁₈H₂₂-

ClN₇O₃: 420.1551 (M + H)⁺. Anal. (C₁₈H₂₂ClN₇O₃‚HCl) H. Calcd,

C 47.38, N 21.49; found C 46.83, N 18.33.

N-(3,5-Diamino-6-chloropyrazine-2-carbonyl)-N′-[5-(4-meth-

oxyphenyl)pentyl]-guanidine Hydrochloride (23). Compound 53a

was dissolved in dichloromethane. TFA was added to the solution.

The reaction mixture was stirred at room temperature for 1 h,

concentrated, and further dried under vacuum. The product was

directly used in the next step without purification.

Sulfuric Acid Mono-(4-{4-[N-(3,5-diamino-6-chloropyrazine-

2-carbonyl)guanidine]butyl}phenyl)ester (26). To a solution of

16 (0.2 g, 0.5 mmol) dissolved in dry pyridine (5 mL) was added

the pyridine sulfur trioxide complex (0.45 g, 2.5 mmol). The

reaction mixture was stirred at room-temperature overnight. The

precipitate formed was isolated by vacuum filtration and washed

with ethyl acetate (2 × 25 mL) to give crude 26 (0.18 g, 39%

yield, 87% purity by HPLC). An aliquot of crude 26 (67 mg) was

purified by flash chromatography (silica gel, dichloromethane/

methanol/concentrated ammonium hydroxide, 6:3:0.1, v/v) to give

26 as a yellow solid (9.3 mg, 4% overall yield): ¹H NMR (300

MHz, DMSO-d₆) δ 1.59 (br s, 4H), 2.58 (m, 2H), 3.28 (m, 2H),

7.08 (s, 4H), 7.1-7.9 (m, 6H). MS (ESI) m/z 456 [C₁₆H₂₀ClN₇O₅S-

H]^-. Purity: 95% (method B; t_R) 8.7 min), 88% (method D; t_R

) 6.6 min).

The above crude material was reacted with 43b using methods

B and C to produce desired compound 23 as a yellow solid in 56%

1

yield: mp 132 °C (dec); H NMR (300 MHz, DMSO-d₆) δ 1.32

N-(3,5-Diamino-6-chloropyrazine-2-carbonyl)-N′-[4-(4-hy-

droxymethylphenyl)-butyl]guanidine Hydrochloride (27). Lithium

aluminum hydride (35 mL of a 1.0 M solution in THF, 0.035 mol)

was added dropwise to a vigorously stirred solution of 46d (2.4 g,

0.010 mol) in dry THF (120 mL) cooled at 0 °C. Stirring was

continued overnight under a nitrogen atmosphere. To break up the

complex, water (6 mL) and 15% NaOH (1.5 mL) were sequentially

added dropwise to the cold reaction mixture. The white solid

precipitate was filtered off and washed with THF. All organic phases

were combined and evaporated. The material was purified by

column chromatography (silica gel, 2:1:0.05 chloroform/ethanol/

concentrated ammonium hydroxide) to provide 46f (1.17 g, 64%)

as a white solid: ¹H NMR (300 MHz, CDCl₃) δ 1.15 (br, 2H, NH₂),

1.54 (m, 2H, 2-CH₂-), 1.70 (m, 2H, 3-CH₂-), 2.60 (m, 4H, 1-CH₂-N

+ 4-CH₂-Ar), 4.67 (s, 2H, 4-HO-CH₂-Ar), 7.47 (s, 2H, Ar-), 7.60

(s, 2H, Ar-).

(m, 2H, 3-CH₂), 1.55 (m, 4H, 2-CH₂and 4-CH₂), 2.45 (m, 2H,

5-CH₂-Ar), 3.29 (m, 2H, 1-CH₂-N), 6.68 (d, J ) 8.0 Hz, 2H, Ar-),

6.97 (d, J ) 8.0 Hz, 2H, Ar-), 7.46 (s, 1H, guanidino), 8.00 (br s,

2H, NH₂), 8.97 (br s, 1H, guanidino), 9.46 (br s, 2H, NH₂), 10.55

(s, 1H, guanidino); MS (APCI) m/z 406 (M + H)⁺. HRMS (FAB)

m/z 406.1742; calcd, C₁₇H₂₂ClN₇O₂: 406.1758 (M + H)⁺. Anal.

(C₁₇H₂₂ClN₇O₂‚HCl) H. Calcd, C 48.84, N 22.17; found, C 44.19,

N 19.53.

N-(3,5-Diamino-6-chloropyrazine-2-carbonyl)-N′-[3-(4-meth-

oxyphenoxy)propyl]-guanidine Hydrochloride (24). 4-Methox-

yphenol (56a) (10 g, 0.081mol) was dissolved in a mixture of

anhydrous THF and DMF (200 mL, 1/1, v/v). To the solution,

sodium hydride (60% dispersion in mineral oil, 4.2 g) was added

in one portion. The mixture was stirred at room temperature for 1

h before N-(3-bromopropyl)phthalimide (57a) (23.9 g, 0.089 mol)

was added. Stirring was continued overnight. The reaction was

quenched with water (50 mL) and partitioned between water and

dichloromethane (500 mL, 1/1, v/v). The organic layer was

separated, washed with brine, and dried (anhydrous sodium sulfate).

After concentration under vacuum, the material was purified by

column chromatography (silica gel, 3:1 hexanes/ethyl acetate) to

provide 58a (12 g, 49%): ¹H NMR (300 MHz, CDCl₃) δ 2.12 (m,

2H, 2-CH₂-), 3.78 (s, 3H, 4-MeO-Ar), 3.92 (m, 2H, 1-CH₂-N), 3.98

(m, 2H, 3-O-CH₂-), 6.77 (s, 4H, phthalimide), 7.73 (m, 2H, Ar-),

7.85 (m, 2H, Ar-).

Following general methods B and C, compound 27 was prepared

1

as a yellow solid from 46f in 98% yield: mp >140 °C (dec); H

NMR (300 MHz, DMSO-d₆) δ 1.57 (m, 4H, 2-CH₂-3-CH₂), 2.62

(m, 2H, 4-CH₂-Ar), 3.35 (m, 2H, 1-CH₂-N), 3.73 (br s, 1H, OH-

Bn), 4.45 (s, 2H, 4-O-CH₂-Ar), 7.12 (d, J ) 7.8 Hz, 2H, Ar-),

7.24 (d, J ) 7.8 Hz, 2H, Ar-), 7.67 (br s, 2H, NH₂), 8.85 (br s, 2H,

NH₂), 9.98 (br s, 1H, guanidino), 9.32 (br s, 1H, guanidino), 10.55

(s, 1H, guanidino); MS (APCI) m/z 392 (M + H)⁺. HRMS (FAB)

m/z 392.1588; calcd. C₁₇H₂₂ClN₇O₂: 392.1602 (M + H)⁺. Anal.

(C₁₇H₂₂ClN₇O₂‚HCl) H. Calcd, C 47.67, N 22.89; found, C 44.51,

N 19.28.

Compound 58a (6 g, 0.019 mol) was dissolved in ethanol (150

mL). Methylamine (2.0 M solution in methanol, 50 mL, 0.1 mol)

was added to the solution, and the mixture was stirred at room

temperature for 4 h and then concentrated under vacuum. The crude

material was purified by column chromatography (silica gel, 3:1:

0.1 chloroform/ethanol/concentrated ammonium hydroxide) to

provide 59a (2.0 g, 58%): ¹H NMR (300 MHz, DMSO-d₆) δ 1.66

(m, 2H, 2-CH₂-), 2.70 (m, 2H, 1-CH₂-N), 3.68 (s, 3H, 4-MeO-Ar),

3.93 (m, 2H, 3-O-CH₂-), 6.85 (s, 4H, Ar-).

N-(3,5-Diamino-6-chloropyrazine-2-carbonyl)-N′-[3-(4-hy-

droxyphenoxy)propyl]-guanidine Hydrobromide (28). The vig-

orously stirred solution of 24 (80 mg, 0.19 mmol) in 48% HBr (15

mL) was refluxed for 2 h and then cooled to room temperature.

The precipitate formed was collected by vacuum filtration, washed

with water, and dried under vacuum overnight to provide 28 (52

1

mg, 52%) as a yellow solid: mp >150 °C (dec); H NMR (300

Following methods B and C, compound 24 was prepared as a

MHz, DMSO-d₆) δ 1.99 (m, 2H, 2-CH₂), 3.30 (m, 2H, 1-CH₂-N),

3.96 (t, J ) 10.6 Hz, 2H, 3-O-CH₂), 6.77 (d, J ) 8.0 Hz, 2H, Ar-),

6.79 (d, J ) 8.0 Hz, 2H, Ar-), 7.45 (br s, 2H, NH₂), 8.74 (br s, 1H,

guanidino), 8.87 (br s, 2H, NH₂), 9.30 (s, 1H, guanidino), 10.48

(s, 1H, guanidino); MS (APCI) m/z 380 (M + H)⁺. HRMS (FAB)

m/z 380.1239; calcd, C₁₅H₁₈ClN₇O₃: 380.1238 (M + H)⁺. Anal.

(C₁₅H₁₈ClN₇O₃‚HBr). Calcd, C 39.1, H 4.16, N 21.28; found, C

26.65, H 2.92, N 14.11.

1

yellow solid from 59a in 85% yield: mp >98 °C (dec); H NMR

(300 MHz, DMSO-d₆) δ 2.03 (m, 2H, 2-CH₂), 3.32 (m, 2H, 1-CH₂-

N), 3.52 (m, 2H, 3-O-CH₂), 3.70 (s, 3H, 4-MeO-Ar), 6.91 (m, 4H,

Ar-), 7.46 (br s, 3H, NH₂+ guanidino), 8.93 (br s, 2H, NH₂), 9.48

(s, 1H, guanidino), 10.57 (s, 1H, guanidino); MS (APCI) m/z 395

(M + H)⁺. Anal. (C₁₆H₂₀ClN₇O₃‚HCl) H. Calcd, C 44.66, N, 22.79,

found, C 41.11, N 18.99.

4-{4-[N′-(3,5-Diamino-6-chloropyrazine-2-carbonyl)guanidino]-

butyl}benzoic Acid Hydrochloride (25). Compound 25 was made

in 65% yield from compound 22 by saponification with lithium

hydroxide in THF and purified by chromatography eluting with

CMA. The free base was then treated with HCl using method C to

N-(3,5-Diamino-6-chloropyrazine-2-carbonyl)-N′-[2-(4-meth-

oxybenzyloxy)ethyl]-guanidine (29). Compound 58b was synthe-

sized from 56b in 72% yield in a manner similar to that used to

prepare compound 58a: ¹H NMR (300 MHz, CDCl₃) δ 3.32 (m,

2H, 1-CH₂-N), 3.78 (s, 3H, 4-MeO-Ar), 3.92 (m, 2H, 2-CH₂-O),

Drugs for Cystic Fibrosis and Chronic Bronchitis

Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14 4111

4.48 (s, 2H, O-CH₂-Ar), 6.77 (d, J ) 8.0 Hz, 2H, Ar-), 7.20 (d, J

) 8.0 Hz, 2H, Ar-), 7.72 (m, 2H, phthalimide), 7.83 (m, 2H,

phthalimide).

followed by the addition of allyl bromide in one portion. The newly

formed mixture was further stirred at room temperature for an

additional 4 h and quenched by water (5 mL). The mixture was

partitioned between water (50 mL) and dichloromethane (50 mL).

The organic layer was separated, washed with brine, dried

(anhydrous sodium sulfate), and then concentrated under vacuum.

The residue was subject to chromatographic purification, eluting

with a mixture of ethyl acetate (0-17%, gradient) and hexanes

(100-87%) to afford desired product 61 (0.38 g, 94% yield) as a

white solid: ¹H NMR (300 MHz, CDCl₃) δ 1.45-1.63 (m, 4H,

2-CH₂-3-CH₃-), 2.56 (t, J ) 6.9 Hz, 2H, 4-CH₂-Ar), 3.20 (q, J )

6.9 Hz, 2H, 1-CH₂-N), 4.50 (d, J ) 7.3 Hz, 2H, O-CH₂-ally), 4.78

(br s, 1H, NHCbz), 5.09 (s, 2H, Cbz), 5.28 (dd, J ) 1.2 Hz, 10.5

Hz, 1H, H₂CdC), 5.43 (dd, J ) 1.2 Hz, 10.5 Hz, H₂CdC), 6.08

(m, 1H, CdCH), 6.82 (d, J ) 8.5 Hz, 2H, Ar-), 7.08 (d, J ) 8.5

Hz, 2H, Ar-), 7.34 (m, 5H, Cbz); MS (ESI) m/z 340 (M + H)⁺.

Compound 61 (0.38 g, 1.12 mmol) was dissolved in anhydrous

THF (5 mL). The solution was cooled to 0 °C in an ice bath, to

which the BH₃-THF complex (1.23 mL, 1.0 M solution) was added

dropwise over 2 min. The mixture was stirred at 0 °C for 1 h and

then at room temperature for 4 h. Water (2 mL) was added to the

reaction mixture, followed by aqueous 3 N NaOH solution (2 mL),

and finally H₂O₂(30% aqueous solution, 2 mL). The reaction

mixture was further stirred at room temperature overnight and then

partitioned between water (20 mL) and dichloromethane (20 mL).

The organic layer was separated, washed with brine, dried

(anhydrous sodium sulfate), and concentrated under vacuum. The

residue was subjected to chromatographic purification, eluting with

a mixture of ethyl acetate (25-50%, gradient) and hexanes (75-

50%), affording desired product 62 (0.18 g, 45% yield) as an off-

white solid: ¹H NMR (300 MHz, CDCl₃) δ 1.48-1.70 (m, 4H,

2-CH₂-3-CH₂-), 2.06 (m, 2H, 2′-CH₂-), 2.56 (t, J ) 7.4 Hz, 2H,

4-CH₂-Ar), 3.22 (q, J ) 6.4 Hz, 2H, 1-CH₂-N), 4.12 (m, 2H, 3′-

CH₂-OH), 4.39 (t, J ) 5.9 Hz, 2H, Ar-O-1′-CH₂), 4.74 (br s, 1H,

NHCbz), 5.11 (s, 2H, Cbz), 6.84 (d, J ) 8.5 Hz, 2H, Ar-), 7.06 (d,

J ) 8.5 Hz, 2H, Ar-), 7.34 (m, 5H, Cbz); MS (ESI) m/z 358 (M +

H)⁺. A 2-hydroxy isomer 63 was also isolated in 14% yield (55

mg, off-white solid): ¹H NMR (300 MHz, CDCl₃) δ 1.27 (d, J )

6.4 Hz, 3H, 3′-CH₃), 1.50-1.65 (m, 4H, 2-CH₂-3-CH₂-), 2.38 (d,

J ) 5.4 Hz, 1H, 2′-HO), 2.52 (t, J ) 7.4 Hz, 2H, 4-CH₂-Ar), 3.20

(q, J ) 6.4 Hz, 2H, 1-CH₂-N), 3.76 (dd, J ) 2.6 Hz, 8.6 Hz, 1H,

O-1′-CH₂), 3.94 (dd, J ) 2.9 Hz, 9.2 Hz, 1H, 1′-O-CH₂), 4.20 (m,

1H, 2′-CH-O-), 4.72 (br s, 1H, NHCbz), 5.10 (s, 2H, Cbz), 6.84

(d, J ) 8.5 Hz, 2H, Ar-), 7.08 (d, J ) 8.5 Hz, 2H, Ar-), 7.34 (m,

5H, Cbz); MS (ESI) m/z 358 (M + H)⁺.

Compound 62 (180 mg, 0.504 mmol) was subject to hydro-

genolysis following method D to afford desired product 64 (79 mg,

70% yield) as an off-white solid: ¹H NMR (300 MHz, DMSO-d₆)

δ 1.35 (m, 2H, 2-CH₂), 1.52 (m, 2H, 3-CH₂-), 1.82 (m, 2H, 2′-

CH₂-), 2.46-2.56 (m, 4H, 1-CH₂-Ar + 1-CH₂-N), 2.92 (br s, 1H,

3′-OH), 3.56 (m, 2H, 3′-CH₂-OH), 3.98 (t, J ) 6.4 Hz, 2H, Ar-

O-1′-CH₂-), 4.56 (m, 2H, NH₂), 6.82 (d, J ) 8.5 Hz, 2H, Ar-),

7.08 (d, J ) 8.5 Hz, 2H, Ar-); MS (ESI) m/z 324 (M + H)⁺.

Compound 31 was prepared as a yellow solid in 50% yield from

64 using methods B and C: mp 211-213 °C (dec); ¹H NMR (300

MHz, DMSO-d₆) δ 1.55 (m, 2H, 2-CH₂-3-CH₂-), 1.84 (m, 2H, 2′-

CH₂-), 2.52 (m, 2H, 4-CH₂-Ar), 3.30 (m, 2H, 1-CH₂-N), 3.56 (m,

2H, 3′-CH₂-OH), 3.98 (t, J ) 6.4 Hz, 2H, Ar-O-1′-CH₂-), 4.58 (t,

J ) 5.1 Hz, 1H, OH), 6.82 (d, J ) 8.5 Hz, 2H, Ar-), 7.12 (d, J )

8.5 Hz, 2H, Ar-), 7.45 (br s, 2H, NH₂), 7.95 (br s, 1H, guanidino),

8.20-8.45 (br s, 2H, NH₂), 9.22 (br, 1H, guanidino), 10.50 (br s,

1H, guanidino); MS (ESI) m/z 436 (M + H)⁺. HRMS (FAB) m/z

436.1852; calcd, C₁₉H₂₄ClN₇O₃: 436.1864 (M + H)⁺. Anal.

(C₁₉H₂₄ClN₇O₃‚HCl) H. Calcd, C 48.31, N 20.76; found, C 47.41,

N 19.89.

Compound 59b was prepared from 58b in 56% yield using a

method similar to that used to prepare 59a: ¹H NMR (300 MHz,

CDCl₃) δ 1.78 (m, 2H, NH₂), 2.90 (m, 2H, 1-CH₂-N), 3.60 (m,

2H, 2-CH₂-O), 3.82 (s, 3H, 4-MeO-Ar), 4.48 (s, 2H, O-CH₂-Ar),

6.88 (d, J ) 8.0 Hz, 2H, Ar-), 7.27 (d, J ) 8.0 Hz, 2H, Ar-).

Using general method B, compound 29 was prepared as a yellow

solid in 60% yield from compound 59b: mp 99-102 °C; ¹H NMR

(300 MHz, DMSO-d₆+ CDCl₃) δ 3.46 (m, 2H, 1-CH₂-N), 3.58 (t,

J ) 11 Hz, 2H, O-2-CH₂), 3.77 (s, 3H, 4-MeO-Ar), 4.49 (s, 2H,

ArCH₂-O), 6.95 (d, J ) 7.8 Hz, 2H, Ar-), 7.31 (d, J ) 7.8 Hz, 2H,

Ar-), 7.18 (s, 1H, guanidino), 8.90 (br s, 2H, NH₂); MS (APCI)

m/z 394 (M + H)⁺. HRMS (FAB) m/z 394.1376; calcd for C₁₆H₂₀-

ClN₇O₃: 394.1394 (M + H)⁺. Anal. (C₁₆H₂₀ClN₇O₃‚HCl) H. Calcd,

C 44.66, N 22.79; found, C 43.43, N, 20.22.

N-(3,5-Diamino-6-chloropyrazine-2-carbonyl)-N′-{4-[4-(2-hy-

droxyethoxy)phenyl]-butyl}guanidine Hydrochloride (30). Com-

pound 52e was prepared as a brown oil from 50e in 43% yield in

a manner similar to that used to prepare compound 52a: ¹H NMR

(300 MHz, CDCl₃) δ 1.46 (s, 9H, Boc), 2.80 (m, 2H, 2-CH₂-),

3.95 (m, 4H, 1-CH₂-N + HO-2′-CH₂-), 4.08 (m, 2H, 1′-CH₂-O-

Ar), 6.83 (d, J ) 7.8 Hz, 2H, Ar-), 7.35 (d, J ) 7.8 Hz, 2H, Ar-).

Compound 53e was prepared from 52e as a brown oil using a

method similar to that used to prepare compound 53a and used in

the next step without purification.

Compound 55e was prepared as a clear oil from 53e in 35%

yield using a method similar to that used to prepare compound

55a: ¹H NMR (300 MHz, DMSO-d₆) δ 1.58 (m, 4H, 2-CH₂-3-

CH₂-), 2.55 (m, 2H, 4-CH₂-Ar), 2.73 (m, 2H, 1-CH₂-N), 3.83 (m,

2H, 2′-CH₂-OH), 3.97 (m, 2H, 1′-CH₂-Ar), 6.83 (d, J ) 7.8 Hz,

2H, Ar), 7.07 (d, J ) 7.8 Hz, 2H, Ar).

Following general methods B and C, compound 30 was prepared

1

as a yellow solid from 55e in 73% yield: mp 218 °C (dec); H

NMR (300 MHz, DMSO-d₆) δ 1.56 (m, 4H, 2-CH₂-3-CH₂), 2.57

(m, 2H, 4-CH₂-Ar), 3.32 (m, 2H, 1-CH₂-N), 3.70 (t, J ) 10.6 Hz,

2H, HO-1′-CH₂-Ar), 3.93 (t, J ) 10.8 Hz, 2H, 2′-CH₂-OAr), 4.90

(t, J ) 10.8 Hz, 1H, 1′-OH), 6.84 (d, J ) 8.0 Hz, 2H, Ar-), 7.12

(d, J ) 8.0 Hz, 2H, Ar-), 7.45 (br s, 2H, NH₂), 8.70 (br s, 1H,

guanidino), 8.88 (br s, 2H, NH₂), 9.12 (br s, 1H, guanidino) 10.45

(s, 1H, guanidino); MS (APCI) m/z 422 (M + H)⁺. Anal. (C₁₈H₂₄-

ClN₇O₃‚HCl) H. Calcd, C 47.17, N 21.39; found, C 43.53, N 19.90.

N-(3,5-Diamino-6-chloropyrazine-2-carbonyl)-N′-{4-[4-(3-hy-

droxypropoxy)phenyl]-butyl}guanidine Hydrochloride (31). 4-(4-

Aminobutyl)phenol hydrobromide (47c) (197 g, 0.80 mol), water

(1 L), 1,4-dioxane (1 L), and sodium bicarbonate (336 g, 4 mol)

were combined and stirred while cooled in an ice-methanol cooling

bath. Benzyl chloroformate (141 mL, 0.96 mol) was dripped in

over 5 min at -2 °C with no appreciable exotherm observed. This

was stirred and allowed to warm to room temperature as the cooling

bath thawed overnight. An additional quantity of benzyl chloro-

formate (8 mL, 0.54 mol) was dripped in and allowed to stir for 2

h. The product mixture was then evaporated to approximately 500

mL and transferred to a 2 L separatory funnel with ethyl acetate

(decanting the solids away). The aqueous layer was extracted with

ethyl acetate (3 × 1 L). The extracts were combined, washed with

brine, dried (anhydrous sodium sulfate), filtered, and evaporated

to afford 265 g of the crude product. The crude material was

crystallized from a mixture of toluene/heptane (1:1, v/v). The

crystallized product was suction filtered and washed with toluene/

heptane (1:1, v/v). This material was vacuum-dried at 45 °C for 2

h to afford compound 60a (150 g, 62% yield) as a white crystalline

1

solid. H NMR (300 MHz, CDCl₃) δ 1.43-1.65 (m, 4H, 2-CH₂-

3-CH₂-), 2.52 (t, J ) 7.4 Hz, 2H, 4-CH₂-Ar), 3.19 (q, J ) 6.4 Hz,

2H, 1-CH₂-N), 4.78 (br s, 1H, NHCbz), 5.09 (s, 2H, Cbz), 5.77 (s,

1H, 4-HO-Ar), 6.74 (d, J ) 8.5 Hz, 2H, Ar-), 6.98 (d, J ) 8.5 Hz,

2H, Ar-), 7.34 (s, 5, Cbz). MS (ESI) m/z 300 (M + H)⁺.

N-(3,5-Diamino-6-chloropyrazine-2-carbony)-N′-{4-[4-(2,3-di-

hydroxypropoxy)-phenyl]butyl}guanidine Methanesulfonate (32).

A mixture of 4-(4-hydroxyphenyl)butylcarbamic acid benzyl ester

(60) (30 g, 0.10 mol), glycidol (8.0 mL, 0.12 mol), ethanol (30

mL), and triethylamine (0.7 mL, 0.005 mol) was stirred at reflux

for 2 h under the protection of argon. The product mixture was

Compound 60a (0.3 g, 1.00 mmol) was dissolved in anhydrous

DMF (5 mL). To the solution, granular NaOH (48 mg, 1.20 mmol)

was added. The mixture was stirred at room temperature for 1.5 h

4112 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14

Hirsh et al.

evaporated, taken up in hot ethyl acetate, and suction filtered

through a plug of silica gel, eluting with ethyl acetate. After

evaporating to a white solid, the solid was re-crystallized from

toluene to afford 21.8 g (58%) of compound 65a: ¹H NMR (300

MHz, CD₃OD) δ 1.42-1.65 (m, 4H, 2-CH₂-3-CH₂-), 2.54 (t, J )

7.5 Hz, 2H, 4-CH₂-Ar), 3.11 (t, J ) 6.4 Hz, 2H, 1-CH₂-N), 3.58-

3.71 (m, 2H, 3′-CH₂-), 3.88-4.04 (m, 3H, O-1′-CH₂-2′-CH-O-),

5.05 (s, 2H, Cbz), 6.84 (d, J ) 8.7 Hz, 2H, Ar-), 7.06 (d, J ) 8.5

Hz, 2H, Ar-), 7.32 (s, 5H, Cbz).

Compound 66a was prepared from 65a using method D. The

crude product was used directly in the following step without

purification. A sample of the reaction mixture was dried and

characterized by ¹H NMR (300 MHz, CD₃OD): δ 1.42-1.55 (m,

2H, 2-CH₂-), 1.55-1.68 (m, 2H, 3-CH₂-), 2.56 (t, J ) 7.5 Hz, 2H,

4-CH₂-Ar), 2.65 (t, J ) 7.2 Hz, 2H, 1-CH₂-N), 3.58-3.72 (m, 2H,

3′-CH₂-O), 3.89-4.05 (m, 3H, O-1′-CH₂-2′-CH-O), 6.85 (d, J )

8.7 Hz, 2H, Ar-), 7.08 (d, J ) 8.7 Hz, 2H, Ar-). Following general

method B, the free base of compound 32 was prepared from 66a

in 71% yield.

Hz, 2H, Ar-); MS (APCI) m/z 452 (M + H)⁺. Anal. (C₁₉H₂₆-

ClN₇O_4•CH₃SO₃H•H₂O) C, N. Calcd, H 5.70; found H 5.13.

N-(3,5-Diamino-6-chloropyrazine-2-carbony)-N′-{4-[4-(2(S)-

2,3-dihydroxypropoxy)-phenyl]butyl}guanidine Methanesulfonate

(34). Compound 65c was prepared from (S)-glycidol in 55% yield

as a bright white solid in a manner similar to that used to prepare

compound 65a: [R]₂₅^D: +6.4° (c 1.0, MeOH); ¹H NMR (300 MHz,

CD₃OD) δ 1.44-1.62 (m, 4H, 2-CH₂-3-CH₂-), 2.55 (t, J ) 8.9

Hz, 2H, 4-CH₂-Ar), 3.12 (t, J ) 8.7 Hz, 2H, 1-CH₂-N), 3.65 (m,

2H, 3′-CH₂-OH), 3.93-4.07 (m, 3H, 2′-CH- + 1′-CH₂-O-Ar), 4.73

(br s, 1H, NHCbz), 5.08 (s, 2H, CBz), 6.83 (d, J ) 7.8 Hz, 2H,

Ar-), 7.08 (d, J ) 7.8 Hz, 2H, Ar-), 7.42 (m, 5H, Cbz).

Compound 66c was prepared as a white solid from 65c in

quantitative yield in a fashion similar to the synthesis of compound

66a: [R]₂₅^D: +12.0° (c 0.26, MeOH); ¹H NMR (300 MHz, DMSO-

d₆) δ 1.32 (m, 2H, 2-CH₂-), 1.54 (m, 2H, 3-CH₂-), 2.55 (m, 2H,

4-CH₂-Ar), 3.45 (m, 2H, 1-CH₂-N), 3.75 (m, 2H, 3′-CH₂-OH), 3.94

(m, 3H, 1′-O-CH₂- + 2′-CH-), 6.83 (d, J ) 8.0 Hz, 2H, Ar-), 7.08

(d, J ) 8.0 Hz, 2H, Ar-).

Following general method B, compound 34 was prepared from

66c in 76% yield as a yellow solid. Its methanesulfonic acid salt

was also prepared in 46% yield in the same manner as that used to

prepare compound 32: mp 160-162 °C (dec); [R]₂₅^D: +12.0° (c

1.0, MeOH); ee: 100% (chiral HPLC: retention time: 26.60 min);

¹H NMR (300 MHz, CD₆OD) δ 1.64-1.76 (m, 4H, 2-CH₂-3-CH₂-

), 2.62 (m, 2H, 4-CH₂-Ar), 2.70 (s, 3H, CH₃SO₃H), 3.33 (m, 2H,

1-CH₂-N), 3.68 (m, 2H, 3′-CH₂-OH), 3.94 (m, 2H, 1′-O-CH₂-), 4.02

(m, 1H, 2′-CH-), 6.86 (d, J ) 8.8 Hz, 2H, Ar-), 7.14 (d, J ) 8.0

Hz, 2H, Ar-); MS (APCI) m/z 452 (M + H)⁺. Anal. (C₁₉H₂₆-

ClN₇O₄‚CH₃SO₃H‚H₂O) C, N. Calcd, H 5.7; found H 5.27.

N-(3,5-Diamino-6-chloropyrazine-2-carbony)-N′-{4-[3-(2,3-di-

hydroxypropoxy)-phenyl]butyl}guanidine Hydrochloride (35).

Compound 65d was prepared from 60b in 58% yield in a manner

similar to that used to prepare compound 65a: ¹H NMR (300 MHz,

CDCl₃) δ 1.48-1.70 (m, 4H, 2-CH₂-3-CH₂-), 2.32 (t, J ) 3.4 Hz,

1H, 3′-OH), 2.60 (t, J ) 7.5 Hz, 2H, 4-CH₂-Ar), 2.86 (d, J ) 4.1

Hz, 1H, 2′-OH), 3.20 (t, J ) 6.4 Hz, 2H, 1-CH₂-N), 3.70-3.85

(m, 2H, 3′-CH₂-OH), 4.02 (m, 3H, 1′-O-CH₂- + 2′-CH-), 4.30 (br

s, 1H, NHCbz), 5.10 (s, 2H, Cbz), 6.76 (m, 3H, Ar-), 7.20 (m, 1H,

Ar-), 7.35 (m, 5H, Cbz).

Preparation of Methanesulfonic Acid Salt. The free base of

32 (10.1 g, 0.022 mol) was suspended in absolute ethanol (200

mL). To the suspension, methanesulfonic acid (1.45 mL, 0.022 mol)

was added dropwise to a point where the suspension turned to a

practically clear light brown solution. Stirring was continued for

an additional 20 min before the undissolved solid was filtered under

vacuum. The filter cake was washed with ethanol (2 × 5 mL), and

the combined washings and filtrate were slowly added into methyl

tert-butyl ether (MTBE) (200 mL), which was cooled in a wet ice-

methanol bath (at -10 °C). After the addition was complete, the

precipitate was stirred for an additional 1 h at the above temperature.

The precipitate was filtered under vacuum, washed with MTBE (3

× 50 mL), and dried under vacuum at room temperature overnight

to afford desired product 32 (10.3 g, 84% yield) as a yellow solid:

1

mp 92-95 °C; H NMR (300 MHz, DMSO-d₆) δ 1.52-1.64 (m,

4H, 2-CH₂-3-CH₂-), 2.36 (s, 3H, CH₃SO₃H), 2.56 (m, 2H, 4-CH₂-

Ar), 3.31 (m, 2H, 1-CH₂-N), 3.42 (m, 2H, 3′-CH₂-O), 3.74-3.82

(m, 2H, O-1′-CH₂-), 3.92-3.98 (m, 1H, 2′-CH₂-O), 4.70 (br s, 1H,

OH), 4.92 (br s, 1H, OH), 6.84 (d, J ) 8.0 Hz, 2H, Ar-), 7.10 (d,

J ) 8.0 Hz, 2H, Ar-), 7.45 (br s, 2H, NH₂), 7.90 (br s, 1H,

guanidino), 8.88 (br s, 2H, NH₂), 9.16 (br, 1H, guanidino), 10.44

(s, 1H, guanidino); MS (APCI) m/z 452 (M + H)⁺. Anal. (C₁₉H₂₆-

ClN₇O₄‚CH₃SO₃H‚H₂O) C, H, N.

Compound 66d was prepared from 65d in a manner similar to

that used to prepare compound 66a. The crude product was used

directly in the next step without purification. A sample of the

N-(3,5-Diamino-6-chloropyrazine-2-carbony)-N′-{4-[4-(2(R)-

2,3-dihydroxy-propoxy)phenyl]butyl}guanidine Methanesulfonate

(33). Compound 65b was prepared from 60a in 83% yield as a

bright white solid from (R)-glycidol in a manner similar to that

used to prepare compound 65a: [R]₂₅^D: -5.7° (c 1.0, MeOH); ¹H

NMR (300 MHz, DMSO-d₆) δ 1.40 (m, 2H, 2-CH₂-), 1.54 (m,

2H, 3-CH₂-), 2.52 (m, 2H, 4-CH₂-Ar), 3.00 (m, 2H, 1-CH₂-N), 3.44

(m, 2H, 3′-CH₂-OH), 3.78 (t, J ) 6.7 Hz, 2H, 1′-CH₂-O-Ar), 3.94

(m, 1H, 2′-CH-), 4.68 (br s, 2H, 2×OH), 4.93 (s, 1H, NHCbz),

5.00 (s, 2H, CBz), 6.83 (d, J ) 7.8 Hz, 2H, Ar-), 7.08 (d, J ) 7.8

Hz, 2H, Ar-), 7.35 (m, 5H, Cbz).

1

reaction mixture was dried and characterized by H NMR (300

MHz, DMSO-d₆): δ 1.38 (m, 2H, 2-CH₂-), 1.58 (m, 2H, 3-CH₂-),

2.60 (t, J ) 7.5 Hz, 2H, 4-CH₂-Ar), 2.72 (t, J ) 7.2 Hz, 2H, 1-CH₂-

N), 3.72-3.88 (m, 2H, 3′-CH₂-OH), 4.08 (m, 3H, 1′-O-CH₂- +

2′-CH-), 6.78 (m, 3H, Ar-), 7.20 (t, J ) 2.8 Hz, 1H, Ar-).

Following general methods B and C, compound 35 (HCl salt)

was prepared from 66d in 71% yield as a yellow solid: mp 91-

93 °C; ¹H NMR (300 MHz, DMSO-d₆) δ 1.52-1.70 (m, 4H,

2-CH₂-3-CH₂-), 2.60 (t, 2H, J ) 6.4 Hz, 4-CH₂-Ar), 3.34 (m, 2H,

1-CH₂-N), 3.42 (m, 2H, 3′-OH + 2′-OH), 3.74-3.86 (m, 3H, 3′-

CH₂-2′-CH-), 3.98 (m, 2H, 1′-O-CH₂-), 6.76 (m, 3H, Ar-), 7.18 (t,

J ) 2.8 Hz, 1H, Ar-), 7.46 (br s, 2H, NH₂), 7.90 (br s, 1H,

guanidino), 8.92 (br s, 2H, NH₂), 9.30 (br s, 1H, guanidino), 10.55

(br s, 1H, guanidino); MS (APCI) m/z 452 (M + H)⁺. Anal. (C₁₉H₂₆-

ClN₇O₄‚HCl) H, N. Calcd. C 45.07; found: C 44.85.

N-(3,5-Diamino-6-chloropyrazine-2-carbony)-N′-{4-[2-(2,3-di-

hydroxypropoxy)-phenyl]butyl}guanidine Hydrochloride (36).

Compound 65e was prepared from 60c in 84% yield in a manner

similar to that used to prepare compound 65a, except the protecting

group in compound 65e was Boc: ¹H NMR (300 MHz, CDCl₃) δ

1.44 (s, 9H, Boc), 1.49-1.66 (m, 4H, 2-CH₂-3-CH₂-), 2.34 (t, J )

3.4 Hz, 1H, 3′-OH), 2.62 (t, J ) 7.5 Hz, 2H, 4-CH₂-Ar), 3.05 (d,

J ) 4.1 Hz, 1H, 2′-OH), 3.13 (m, 2H, 1-CH₂-N), 3.85 (m, 2H,

3′-CH₂-OH), 4.10 (m, 2H, 1′-O-CH₂-), 4.18 (m, 1H, 2′-CH-), 4.61

(br s, 1H, NHBoc), 6.89 (m, 2H, Ar-), 7.15 (m, 2H, Ar-).

Compound 66b was prepared from compound 65b in quantitative

yield as a white solid in a manner similar to that used to prepare

compound 66a: [R]₂₅^D: -5.1° (c 0.98, MeOH); H NMR (300

1

MHz, DMSO-d₆) δ 1.32 (m, 2H, 2-CH₂-), 1.54 (m, 2H, 3-CH₂-),

2.55 (m, 2H, 4-CH₂-Ar), 2.87 (m, 2H, 1-CH₂-N), 3.05 (m, 1H, 3′-

OH), 3.45 (t, J ) 6.8 Hz, 2H, 3′-CH₂-OH), 3.82 (m, 3H, 1′-CH₂-

O- + 2′-OH), 3.94 (m, 2H, 1′-CH₂-O-Ar), 6.83 (d, J ) 7.8 Hz,

2H, Ar-), 7.08 (d, J ) 7.8 Hz, 2H, Ar-).

Following general method B, compound 33 was prepared from

66b in 58% yield as a yellow solid. Its methansulfonic acid salt

was also prepared in 78% yield in a manner similar to that used to

prepare compound 32: mp 169-172 °C (dec); [R]₂₅^D: -3.73° (c

0.43, MeOH); ee: 100% (chiral HPLC: retention time: 22.21 min);

¹H NMR (300 MHz, CD₆OD) δ 1.60-1.76 (m, 4H, 2-CH₂-3-CH₂-

), 2.62 (m, 2H, 4-CH₂-Ar), 2.70 (s, 3H, CH₃SO₃H), 3.35 (m, 2H,

1-CH₂-N), 3.66 (m, 2H, 3′-CH₂-OH), 3.92 (m, 2H, 1′-O-CH₂-), 4.03

(m, 1H, 2′-CH-), 6.84 (d, J ) 8.8 Hz, 2H, Ar-), 7.10 (d, J ) 8.0

The Boc protecting group in 65e was cleaved by TFA using the

same procedure used for the preparation of compound 55a. Crude

product 66e was used directly in the next step without purifica-

Drugs for Cystic Fibrosis and Chronic Bronchitis

Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14 4113

tion: ¹H NMR (300 MHz, CD₃OD) δ 1.67 (m, 4H, 2-CH₂-3-

CH₂-), 2.70 (t, J ) 7.5 Hz, 2H, 4-CH₂-Ar), 2.90 (t, J ) 7.2 Hz,

2H, 1-CH₂-N), 3.78 (m, 2H, 3′-CH₂-OH), 4.08 (m, 3H, 1′-O-CH₂-

+ 2′-CH-), 6.88 (m, 2H, Ar-), 7.20 (m, 2H, Ar-); MS (APCI) m/z

240 (M + H)⁺.

Following general methods B and C, compound 36 was prepared

from 66e in 38% yield as a yellow solid: mp 85-86 °C; ¹H NMR

(300 MHz, DMSO-d₆) δ 1.60 (m, 4H, 2-CH₂-3-CH₂-), 2.59 (m,

2H, 4-CH₂-Ar), 3.34 (m, 2H, 1-CH₂-N), 3.47 (m, 2H, 3′-OH +

2′-OH), 3.86-4.06 (m, 5H, 3′-CH₂-2′-CH- + 1′-O-CH₂-), 6.88 (m,

2H, Ar-), 7.12 (m, 2H, Ar-), 7.46-7.80 (br s, 3H, guanidino +

NH₂), 8.80 (br s, 2H, NH₂), 9.25 (br s, 1H, guanidino), 10.51 (br

s, 1H, guanidino); MS (APCI) m/z 452 (M + H)⁺. HRMS (FAB)

m/z 452.1816; calcd. C₁₉H₂₆ClN₇O₄: 452.1813 (M + H)⁺. Anal.

(C₁₉H₂₆ClN₇O₄‚HCl). Calcd, C 46.73, H 5.57, N 20.08; found, C

45.34, H 6.02, N 19.61.

(4-{4-[N-(3,5-Diamino-6-chloropyrazine-2-carbonyl)guanidino]-

butyl}phenoxy)acetic Acid Hydrochloride (37). Using a method

similar to that used for the preparation of compound 70a, compound

72a was prepared in 99% yield by the alkylation of 60 (1.00 g,

4.30 mmol) with tert-butyl 2-bromoacetate: ¹H NMR (300 MHz,

CDCl₃) δ 1.46 (s, 9H, t-Bu), 1.52-1.68 (m, 4H, 2′-CH₂-3′-CH₂),

2.55 (t, J ) 6.38 Hz, 2H, 1′-CH₂), 3.20 (m, 2H, 4′-CH₂-N), 4.44

(s, 2H, Ar-O-CH₂-), 4.66 (br s, 1H, NH-CBz), 5.08 (s, 2H, N-CBZ),

6.78 (d, J ) 7.80 Hz, 2H, 2-ArH, 6-ArH), 7.04 (d, J ) 7.78 Hz,

2H, 3-ArH, 5-ArH).

Compound 73a was prepared in 93% yield from 72a using

method D: ¹H NMR (300 MHz, CDCl₃) δ 1.48 (s, 9H, t-Bu), 1.52-

1.66 (m, 4H, 2′-CH₂-3′-CH₂), 2.54 (t, J ) 6.38 Hz, 2H, 1′-CH₂),

2.74 (t, J ) 6.46 Hz, 2H, 4′-CH₂-N), 4.49 (s, 2H, Ar-O-CH₂), 6.82

(d, J ) 7.80 Hz, 2H, 2-ArH, 6-ArH), 7.10 (d, J ) 7.78 Hz, 2H,

3-ArH, 5-ArH).

Compound 73b was prepared in 96% yield from 72b using

method D: ¹H NMR (300 MHz, CDCl₃) δ 1.60-1.78 (m, 4H, 2′-

CH₂-3′-CH₂), 2.60 (m, 4H, Ar-1′-CH₂, 4′-CH₂-N), 2.88 (t, J ) 6.21

Hz, 2H, HO₂C-2′′-CH-), 4.20 (t, J ) 6.18 Hz, 2H, Ar-O-1′′-CH₂-),

6.82 (d, J ) 7.80 Hz, 2H, 2-ArH, 6-ArH), 7.10 (d, J ) 7.78 Hz,

2H, 3-ArH, 5-ArH).

Using method B, compound 38, a yellow solid, was prepared

from compound 73b in 52% yield: mp 198-200 °C (dec); ¹H NMR

(300 MHz, DMSO-d₆) δ 1.55-1.72 (m, 4H, 2-CH₂-3-CH₂), 2.52

(m, 2H, Ar-4-CH₂), 2.88 (t, J ) 6.21 Hz, 2H, HO₂C-2′′-CH-), 3.33

(m, 2H, 1-CH₂-N), 4.18 (t, J ) 6.18 Hz, 2H, Ar-O-1′′-CH₂-), 6.84

(d, J ) 7.80 Hz, 2H, 2′-Ar-H, 6′-ArH), 7.10 (d, J ) 7.78 Hz, 2H,

3′-ArH, 5′-ArH), 7.40-7.68 (br s, 3H, CO₂H, NH₂), 7.96 (br s,

1H, guanidino), 8.88-9.06 (br s, 2H, NH₂), 9.36 (s, 1H, guanidino),

10.58 (s, 1H, guanidino); MS (APCI) m/z 450 (M + H)⁺. Anal.

HRMS (FAB) m/z 450.1651; calcd, C₁₉H₂₄ClN₇O₄‚HCl: 450.1656

(M + H)⁺. Anal. (C₁₉H₂₄ClN₇O₄‚HCl) N. Calcd, C 46.92, H 5.18;

found, C 44.90, H 5.63.

N-(3,5-Diamino-6-chloropyrazine-2-carbonyl)-N′-{4-[3-(2-hy-

droxyethoxy)phenyl]-butyl}guanidine Hydrochloride (39). Com-

pound 68a was prepared via a Sonogashira coupling reaction

between compound 67a and 51, a reaction similar to that used for

the preparation of compound 52e: ¹H NMR (300 MHz, CDCl₃) δ

1.50 (s, 9H, t-Bu), 2.58 (t, J ) 6.33 Hz, 2H, 3′-CH₂), 3.36 (m, 2H,

4′-CH₂-N), 4.96 (br s, 1H, NHBoc), 6.38 (br s, 1H, 3-OH), 6.82

(dd, J ) 8.55 Hz, 3.12 Hz, 1H, 5-ArH), 6.90 (s, 1H, 2-ArH), 6.94

(d, J ) 7.56 Hz, 1H, 6-ArH), 7.16 (d, J ) 7.8 Hz, 1H, 4-ArH).

The above compound 68a underwent the hydrogenation reaction

in a manner similar to that used for compound 53e to give

compound 69a: ¹H NMR (300 MHz, CDCl₃) δ 1.48 (s, 9H, t-Bu),

1.50-1.68 (m, 4H, 2′-CH₂-3′-CH₂), 2.56 (t, J ) 1.83 Hz, 2H, 1′-

CH₂), 3.11 (m, 2H, 4′-CH₂-N), 4.56 (br s, 1H, NHBoc), 6.18 (br s,

1H, 3-OH), 6.66-6.72 (m, 3H, 2-ArH, 4-ArH, 6-ArH), 7.14 (dd,

J ) 8.33 Hz, 2.82 Hz, 1H, 5-ArH).

A suspension of compound 69a (3.34 g, 12.60 mmol) and NaH

(60% in mineral oil, 0.76 g, 19.00 mmol) in anhydrous THF was

cooled by an ice bath and stirred at ∼0 °C for 30 min. Tetrabu-

tylammonium iodide (TBAI, 0.46 g, 1.30 mmol) and THP-protected

2-bromoethyl alcohol (3.16 g, 15.00 mmol) were sequentially added

to the suspension. The mixture was slowly warmed to room

temperature, while stirring continuously overnight and then heated

to 50 °C for an additional 3 h. It was then quenched by the slow

addition of water (20 mL). The mixture was concentrated under

vacuum, and the residue was taken up in dichloromethane (50 mL).

The aqueous layer was separated and washed with dichloromethane

(3 × 20 mL). All organic layers were combined, dried (anhydrous

sodium sulfate), and concentrated under vacuum. The residue was

purified by column chromatography, eluting with 10-20% ethyl

acetate in hexane to afford compound 70a (3.83 g, 77%) as a light

green solid: ¹H NMR (300 MHz, CDCl₃) δ 1.48 (s, 9H, t-Bu),

1.50-1.70 (m, 10H, 2′-CH₂-3′-CH₂, THP: 2-CH₂-3-CH₂-4-CH₂),

2.60 (t, J ) 6.78 Hz, 2H, 1′-CH₂), 3.12 (m, 2H, 4′-CH₂-N), 3.84

(m, 2H, 2′′-CH₂-O-THP), 4.18 (m, 4H, 3-Ar-O-1′′-CH₂, THP:

5-CH₂), 4.50 (br s, 1H, NHBoc), 4.78 (t, J ) 2.65 Hz, 1H, THP:

1-CH), 6.78-6.86 (m, 3H, 2-ArH, 4-ArH, 6-ArH), 7.20 (dd, J )

8.36 Hz, 2.84 Hz, 1H, 5-ArH); MS (ESI) m/z 394 (M + H)⁺.

Using method B, compound 74, a yellow solid, was prepared

from compound 73a in 16% yield: ¹H NMR (300 MHz, DMSO-

d₆) δ 1.42 (s, 9H, t-Bu), 1.50-1.66 (m, 4H, 2-CH₂-3-CH₂), 2.56

(t, J ) 6.38 Hz, 2H, 4-CH₂), 3.34 (m, 2H, 1-CH₂-N), 4.62 (s, 2H,

Ar-O-CH₂), 6.82 (d, J ) 7.80 Hz, 2H, 2′-ArH, 6′-ArH), 7.14 (d, J

) 7.78 Hz, 2H, 3′-ArH, 5′-ArH). 7.40-7.58 (br s, 2H, NH₂), 7.96

(br s, 1H, guanidino), 8.88-9.06 (br s, 2H, NH₂), 9.36 (s, 1H,

guanidino), 10.58 (s, 1H, guanidino); MS (APCI) m/z 492 (M +

H)⁺.

A mixture of compound 74 (160 mg, 0.30 mmol) in dichlo-

romethane (10 mL) and trifluoroacetic acid (5 mL) was stirred at

room temperature for 1 h. The mixture was concentrated under

vacuum to almost dryness. The residue was subjected to column

chromatography, eluting with a mixture of methanol and dichlo-

romethane (gradient 0-30% methanol, v/v). The product fraction

was collected and concentrated under vacuum. The resulting residue

was treated with 3% aqueous hydrochloric acid (5 mL) for 15 min

with stirring, and concentrated again. This procedure was repeated

two more times. The residue was then dried under high vacuum to

afford desired product 37 (98 mg, 69%) as a yellow solid: mp 225

1

°C (dec); H NMR (300 MHz, DMSO-d₆) δ 1.48-1.66 (m, 4H,

2-CH₂-3-CH₂), 2.55 (t, J ) 6.38 Hz, 2H, 4-CH₂), 3.32 (m, 2H,

1-CH₂-N), 4.64 (s, 2H, Ar-O-CH₂), 6.84 (d, J ) 7.80 Hz, 2H, 2′-

ArH, 6′-ArH), 7.14 (d, J ) 7.78 Hz, 2H, 3′-ArH, 5′-ArH). 7.38-

7.58 (br s, 2H, NH₂), 7.94 (br s, 1H, guanidino), 8.82-9.04 (br s,

2H, NH₂), 9.30 (s, 1H, guanidino), 10.54 (s, 1H, guanidino); MS

(APCI) m/z 434 (M + H)⁺. Anal. (C₁₄H₂₂ClN₇O₄‚HCl) H. Calcd,

C 45.77, N 20.76; found, C 41.04, N 18.41.

Both Boc and THP protecting groups in 70a were cleaved by

TFA using a method similar to that used for the preparation of

compound 55e. Crude 71a was used directly without purification.

MS (ESI) m/z 210 (M + H)⁺.

(4-{4-[N-(3,5-Diamino-6-chloropyrazine-2-carbonyl)guanidino]-

butyl}phenoxy)propionic Acid Hydrochloride (38). Compound

72b was prepared in 3% yield by the alkylation of 60 (4.17 g, 13.91

mmol) with 3-bromopropionic acid in a method similar to that used

to prepare compound 72a: ¹H NMR (300 MHz, CD₃OD) δ 1.47-

1.66 (m, 4H, 2′-CH₂-3′-CH₂), 2.56 (t, J ) 6.38 Hz, 2H, 1′-CH₂),

2.71 (t, J ) 6.21 Hz, 2H, HO₂C-2′′-CH-), 3.15 (m, 2H, 4′-CH₂-N),

4.18 (t, J ) 6.18 Hz, 2H, Ar-O-1′′-CH₂-), 5.05 (s, 2H, N-CBZ),

6.79 (d, J ) 7.80 Hz, 2H, 2-ArH, 6-ArH), 7.04 (d, J ) 7.78 Hz,

2H, 3-ArH, 5-ArH), 7.34 (m, 5H, Cbz).

Compound 39, a yellow solid, was prepared in 23% yield from

compound 71a using methods B and C: mp 161-163 °C (dec); ¹H

NMR (300 MHz, DMSO-d₆) δ 1.51-1.68 (m, 4H, 2-CH₃-3-CH₂),

2.60 (t, J ) 6.33 Hz, 2H, 4-CH₂), 3.36 (m, 2H, 1-CH₂-N), 3.70 (t,

J ) 5.68 Hz, 2H, 2′′-CH₂-OH), 3.96 (t, J ) 8.56 Hz, 2H, Ar-O-

1′′-CH₂), 6.78 (m, 3H, 2′-ArH, 4′-ArH, 6′-ArH), 7. 20 (m, 1H, 5′-

ArH), 7.36-7.50 (br s, 2H, NH₂), 8.02 (br s, 1H, guanidino), 8.78-

9.02 (br s, 2H, NH₂), 9.32 (s, 1H, guanidino), 10.58 (s, 1H,

guanidino); MS (APCI) m/z 422 (M + H)⁺. Anal. (C₁₈H₂₄ClN₇O₃‚

HCl) H. Calcd, C 47.17, N 21.39; found, C 46.18, N 20.20.

4114 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14

Hirsh et al.

N-(3,5-Diamino-6-chloropyrazine-2-carbonyl)-N′-{4-[2-(2-hy-

droxyethoxy)phenyl]-butyl}guanidine Hydrochloride (40). Com-

pound 40, a yellow solid, was prepared using the same method as

that used for to prepare compound 39: mp 115 °C (dec); ¹H NMR

(300 MHz, DMSO-d₆) δ 1.52-1.71 (m, 4H, 2-CH₃-3-CH₂), 2.58

(t, J ) 6.33 Hz, 2H, 4-CH₂), 3.26 (m, 2H, 1-CH₂-N), 3.76 (m, 2H,

2′′-CH₂-OH), 3.99 (t, J ) 5.04 Hz, 2H, Ar-O-1′′-CH₂), 4.81 (t, J

) 5.37 Hz, 1H, OH), 6.83 (d, J ) 7.29 Hz, 1H, 3′-ArH), 6.94 (d,

J ) 8.01 Hz, 1H, 4′-ArH), 7.16 (m, 2H, 5′-ArH, 6′-ArH), 7.36-

7.50 (br s, 2H, NH₂), 8.02 (br s, 1H, guanidino), 8.78-9.02 (br s,

2H, NH₂), 9.32 (s, 1H, guanidino), 10.58 (s, 1H, guanidino); MS

(APCI) m/z 422 (M + H)⁺. Anal. HRMS (FAB) m/z 422.1719;

calcd. C₁₈H₂₄ClN₇O₃: 422.1707 (M + H)⁺. Anal. (C₁₈H₂₄ClN₇O₃‚

HCl) H. Calcd, C 47.17, N 21.39; found, C 45.70, N 20.12.

6-(4-{4-[N′-(3,5-Diamino-6-chloropyrazine-2-carbonyl)-

guanidino]butyl}phenoxy)-3,4,5-trihydroxytetrahydropyran-2-

sodium Carboxylate (41). 2,3,4-Tri-O-acetyl-R-D-glucuronic acid

methyl ester trichloroimidate (75) (1.60 g, 3.3 mmol) was added

under an argon atmosphere to 60 (1.50 g, 6.60 mmol) and dissolved

in anhydrous dichloromethane (40 mL). The mixture was cooled

at -25 °C for 10 min and BF₃‚OEt₂(0.045 mL, 0.33 mmol) was

added. The reaction mixture was stirred at -25 °C for 1.5 h and

then allowed to warm to -10 °C. The stirring was continued at

-10 °C for an additional 1 h before the temperature was raised to

25 °C and continuously stirred for one more hour. The mixture

was then quenched with saturated ammonium chloride (25 mL).

The product was extracted with dichloromethane (3 × 30 mL). The

combined extracts were washed with water (3 × 50 mL) and dried

(anhydrous sodium sulfate). The solvent was evaporated and the

residue was purified by flash chromatography (silica gel, 1:2 ethyl

acetate/hexanes, v/v) to provide 76 (1.50 g, 72%) as a white solid:

¹H NMR (300 MHz, DMSO-d₆) δ 1.41-1.51 (m, 4H, 2′-CH₂-3′-

CH₂), 1.99 (s, 9H, 3 x AcO), 2.54 (m, 2H, 1′-CH₂-Ar), 3.10 (m,

2H, 4′-CH₂-N), 3.63 (s, 3H, MeO₂C-), 4.56 (br s, 1H, NHCbz),

4.69 (m, 1H, 6′′-CH-CO₂Me), 4.99 (m, 2H, 2′′-CH-OAc, 4′′-CH-

OAc), 5.06 (s, 2H, Cbz), 5.46 (m, 1H, 3′′-CH-OAc), 5.60 (m, 1H,

1′′-O-CH-O), 6.88 (d, J ) 7.82 Hz, 2H, 2-ArH, 4-ArH), 7.12 (d, J

) 7.82 Hz, 2H, 3-ArH, 5-ArH), 7.33 (m, 5H, Cbz); MS (APCI)

m/z 616 (M + H)⁺.

Using method D, compound 77 was prepared from 76 in 84%

yield as a white solid: ¹H NMR (300 MHz, CDCl₃) δ 1.41-1.51

(m, 4H, 2′-CH₂-3′-CH₂), 2.02 (s, 9H, 3 × AcO), 2.54-2.68 (m,

4H, 1′-CH₂-Ar, 4′-CH₂-N), 3.67 (s, 3H, MeO₂C-), 4.69 (m, 1H,

6′′-CH-CO₂Me), 4.99 (m, 2H, 2′′-CH-OAc, 4′′-CH-OAc), 5.46 (m,

1H, 3′′-CH-OAc), 5.60 (m, 1H, 1′′-O-CH-O), 6.93 (d, J ) 7.82

Hz, 2H, 2-ArH, 6-ArH), 7.18 (d, J ) 7.82 Hz, 2H, 3-ArH, 5-ArH).

Following method B, compound 78, a yellow solid, was prepared

in 48% yield from compound 77: ¹H NMR (300 MHz, CDCl₃) δ

1.61 (m, 4H, 2-CH₂-3-CH₂-), 2.05 (s, 9H, 3 × AcO), 2.55 (m, 2H,

4-CH₂-Ar), 3.49 (m, 2H, 1-CH₂-N), 3.71 (s, 3H, MeO₂C-), 4.22

(m, 1H, 6′′-CH-CO₂Me), 5.12 (m, 1H, 2′′-CH-OAc), 5.34 (m, 3H,

1′′-CH-O, 3′′-CH-OAc, 5′′-CH-OAc), 6.88 (d, J ) 7.80 Hz, 2H,

2′-ArH, 4′-ArH), 7.04 (d, J ) 7.80 Hz, 2H, 3′-ArH, 5′-ArH); MS

(APCI) m/z 694 (M + H)⁺.

Compound 78 (0.31 g, 0.44 mmol) was added to a mixed solvent

of THF and water (1:1, 40 mL). The suspension was cooled to

-10 °C. To the suspension was added dropwise an aqueous NaOH

solution (1.24 N, 4 mL). The stirring was continued at -10 °C for

1.5 h. After this time, the reaction mixture was allowed to warm

to room temperature and THF was removed under reduced pressure.

The pH of the remaining solution was adjusted to 6 by 1 N aqueous

HCl solution. The precipitate formed upon neutralization was

collected by centrifugation and washed with cold water (3 × 20

mL). Compound 41 (0.18 g, 75%) was isolated as a yellow powder

after drying under vacuum for 48 h: mp >184 °C (dec); ¹H NMR

(300 MHz, DMSO-d₆) 1.56 (m, 4H, 2-CH₂-3-CH₂-), 2.56 (m, 2H,

4-CH₂-Ar), 3.19 (m, 2H, 1-CH₂-N), 3.15-3.40 (br s, 3H, 3 × OH),

3.25 (m, 2H, 6′′-CH-CO₂H, 3′′-CH-OH), 3.57 (m, 1H, 4′′-CH-OH),

4.87 (m, 1H, 5′′-CH-OH), 5.10-5.40 (m, 1H, 1′′-CH-O), 6.89 (d,

J ) 7.80 Hz, 2H, 2′-ArH, 6′-ArH), 7.06 (d, 2H, 3′-ArH, 5′-ArH),

7.38-7.58 (br s, 2H, NH₂), 8.82-9.04 (br s, 2H, NH₂) (some peaks

for pyrazine and acylguanidine moiety were not shown); MS (APCI)

m/z 554 (M + H)⁺. HRMS (FAB) m/z 554.1757 (Na lost during

analysis); calcd. C₂₂H₂₈ClN₇O₈: 554.1765 (M + H - Na)⁺. Anal.

(C₂₂H₂₇ClN₇NaO₈) H. Calcd, C 45.88, N 17.02; found, C 39.74, N

13.79.

N-(3,5-Diamino-6-chloropyrazine-2-carbonyl)-N′-[4-(3,4-dihy-

droxyphenyl)butyl]-guanidine (42). Using the same method as

that used to prepare compound 13, compound 42 was prepared in

1

51% yield as a beige solid: mp >154 °C; H NMR (300 MHz,

DMSO-d₆) δ 1.52 (m, 4H, 2-CH₂-3-CH₂-), 2.42 (m, 2H, 4-CH₂-

Ar), 3.31 (m, 2H, 1-CH₂-N), 6.43 (d, J ) 1.86 Hz, 1H, 2′-ArH),

6.61 (m, 2H, 5′-ArH, 6′-ArH), 7.42 (br s, 2H, NH₂), 7.90 (br s,

1H, guanidino), 8.82 (br s, 2H, NH₂), 9.25 (s, 1H, guanidino) 10.52

(s, 1H, guanidino); MS (APCI) m/z 394 (M + H)⁺; HRMS (FAB)

m/z 394.1410; calcd, C₁₆H₂₀ClN₇O₃: 394.1394 (M + H)⁺. Anal.

(C₁₆H₂₀ClN₇O₃‚HCl). Calcd, C 44.66, H 4.92, N 22.79; found, C

43.53, H 5.39, N 18.52.

Materials. Amiloride, benzamil, and phenamil were purchase

from Sigma (St. Lois, MO). Compound 13, was supplied by Dr.

E. J. Cragoe, Jr. All other chemicals were of analytical grade, used

without purification, and purchased from VWR International or

other commercial vendors.

Biological Evaluation and ENaC Assays. The canine bronchial

tissue for primary culture was provided by Marshall Bioresources

(North Rose, NY) Veterinarian Committee to ensure the humane

care and treatment of experimental animals. Briefly, to isolate and

culture primary bronchial epithelial cells, canine bronchi were

incubated in an MEM medium containing 0.1% protease (Sigma

Type XIV) and 50 µg/mL DNase at 4 °C for a minimum of 24 h.

Fetal bovine serum (10%) was added to the medium and the

epithelial layer scraped and rinsed to improve cell yield. Cells were

then centrifuged for 5 min at 500g. Re-suspended cells were seeded

at a density of 0.4 × 10⁶/cm²on 0.4 µm porous collagen coated

(human placenta type VI Sigma) Snapwell or Transwell (Corning

Costar Corp., Cambridge, MA) membranes (1.13 cm²) and main-

tained at an air-liquid interface in a hormonally defined medium

supplemented with penicillin and streptomyocin.¹⁰Bronchial epi-

thelial monolayers grown from 6 to 12 days on permeable

membrane supports were mounted in modified Ussing chambers

(Physiologic Instruments Inc., San Diego, CA). All experiments

were performed in Krebs-Ringer bicarbonate solution (KRB) at

pH 7.4 containing 140 mM Na⁺, 120 mM Cl^-, 5.2 mM K⁺, 1.2

mM Ca²⁺, 1.2 mM Mg²⁺, 2.4 mM HPO₄²⁺, 0.4 mM H₂PO₄^-, 25

mM HCO₃^-, and 5 mM glucose. The epithelium was bathed on

both sides with warmed (37 °C) KRB circulated by gas lift with

95% O₂-5% CO₂, maintaining the pH at 7.4. The transepithelial

voltage was clamped to 0 mV, except for 0.2-s pulses (+5 mV)

every 20 s to calculate R_t. The I_scand R_tvalues were digitized and

recorded on a computer. Data were acquired and analyzed using

Acquire and Analysis (V. 1.2) software (Physiological Instruments).

The 50% inhibition of I_scconcentration (IC₅₀) was calculated from

apical drug additions ranging from 10^-11to 10^-4M (∼ half log

increments) and analyzed using nonlinear regression (Prism V.3,

Graphpad software Inc). Stocks of ENaC blockers were dissolved

in DMSO at a concentration of 10 mM and stored at -20 °C until

use.

The percent recovery of I_scfrom the apical sodium channel

blocker exposure was measured 3 min after the third consecutive

mucosal bath replacements (KRB), following a full concentration-

effect study.

Acknowledgment. We gratefully acknowledge the contribu-

tions of Jacquelyn Fleegle, Betty DiMassimo, Pam Rusnak, and

Sam Hopkins from Parion Sciences to this project.

Supporting Information Available: Analytical results (Table

2), elemental analysis (Table 3), and high-resolution mass spec-

trometry for compounds. This material is available free of charge

via the Internet at http://pubs.acs.org.

Drugs for Cystic Fibrosis and Chronic Bronchitis

Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14 4115

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JM051134W

Article Doi

Design, synthesis, and structure-activity relationships of novel 2-substituted pyrazinoylguanidine epithelial sodium channel blockers: Drugs for cystic fibrosis and chronic bronchitis

DOI: 10.1021/jm051134w

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