trabajo jorge medicina tropical

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Maestría en Medicina Tropical

Módulo 10:

Cólera, Leptospirosis, Fiebre Tifoidea yotras virosis

Tema:

Luminally active, nonabsorbable CFTR inhibitorsas

potential therapy to reduce intestinal fluid loss incholera.

Inhibidores CFTR luminalmente activas, noabsorbibles como terapia potencial para reducir

la pérdida intestinal de fluidos en el Cólera.

Nombre:

Md. Jorge Guerrero Jiménez

PROFESOR: DR. EMILIO PEREZ



2010

©2005 FASEB The FASEB Journal express article 10.1096/fj.05-4818fje. Published online Novem ber 29,

2005.

Luminally active, nonabsorbable CFTR inhibitors aspotential therapy to reduce intestinal fluid loss in

cholera N. D. Sonawane, Jie Hu, Chatchai Muan pr asat, and A. S. Verkman

Departments of Medicine and Physiology, Cardiovascular R esearch Institute, University

of Califor nia, San Fr ancisco, Califor nia

Corresponding author: Alan S. Verkman, 1246 Health Sciences East Tower , University

of Califor nia, San Fr ancisco, CA 94143-0521. E-mail: [email protected]

ABSTRACT

Enter otoxin-mediated secretory diarrheas such as choler a involve chloride secretion by

enter ocytes into the intestinal lumen by the cystic f i br osis tr ansmem br ane conductance regulator (CFTR) chloride channel. We previously identif ied glycine hydr azide CFTR

blockers that by electr o physiological studies a ppeared to block the CFTR anion pore at its lumen-facing sur face.

Here, we synthesize highly water -soluble, nonabsor bable malondihydr azides by cou pling 2,4- disulfobenzaldehyde, 4-sulfo phenylisothiocyante, and polyethylene glycol

(PEG) moieties to 2- na phthalenylamino-[(3,5-di br omo-2,4-dihydr oxyphenyl)methylene] pr o panedioic acid dihydr azide, and aminoacethydr azides by cou pling PEG

to [( N -2-na phthalenyl)-2-(2- hydr oxyethyl)]-glycine-2-[(3,5-di br omo-2,4-

dihydr oxyphenyl) methylene] hydr azide.

Compounds r a pidly, fully and reversi bly blocked CFTR-mediated chloride current with

K i of 2±8 M when added to the a pical sur face of epithelial cell monolayers.Compounds did not pass acr oss Caco-2 monolayers, and were absor bed by <2%/hr inmouse intestine. Luminally added compounds blocked by >90% choler a toxin-induced

f luid secretion in mouse intestinal loo ps, without inhi biting intestinal f luid absorption.These or ally administered, nonabsor bable, nontoxic CFTR inhi bitors may reduce

intestinal f luid losses in choler a.

K ey words: diarrhea cystic f i br osis chloride channel dr ug discovery

Intestinal f luid secretion in many types of diarrheas involves active secretion of chloride

into the intestinal lumen by enter ocytes, which creates the driving force for sodium and

water secretion. Cell culture (1±3) and animal (4) studies indicate that CFTR pr ovides

the principle r oute for chloride secretion at the luminal mem br ane in enter otoxin-

mediated secretory diarrheas pr oduced by inf ection with Vi brio choler a and Escherichia

coli (5, 6). Pharmacological CFTR inhi bition has thus been pr o posed as a str ategy to

reduce f luid losses in choler a and other enter otoxin-mediated diarrheas (7, 8), which

remain a major pr oblem in the develo ping world (9, 10).

Our lab has identif ied by high-thr oughput screening two classes of potent CFTR

inhi bitors that blocked choler a toxin-induced intestinal f luid secretion in r odent models



(11, 12). The thiazolidinone CFTR inh-172 (Fig. 1 A) pr oduces a voltage-independent

CFTR chloride channel block with pr olongation of mean channel closed time (13).CFTR inh-172 is r a pidly absor bed acr oss the intestinal wall and under goes enter ohepatic

recirculation (14, 15). Other CFTR inhi bitors also act f r om the cyto plasmic side of CFTR but are much less potent and selective in their action, including gli benclamide,

diphenylamine-2-car boxylate, 5-nitr o-2(3- phenylpr o pylamino) benzoate, and

f luf enamic acid (16±19). In contr ast, the glycine hydr azide GlyH-101 a ppeared by electr o physiological studies to inhi bit CFTR by binding to a site at its exter nal pore (11). GlyH-101 block of CFTR chloride conductance was r a pid and pr oduced inward

rectif ication with reduced mean channel o pen time.

Here, we report the synthesis and char acterization of highly polar , non-absor bable

CFTR inhi bitors based on the glycine hydr azide scaffold. Str ucture-activity analysis

indicated the glycyl methyl gr ou p as the unique site on the glycine hydr azide scaffold

where modif ications could be toler ated with minimal eff ect on CFTR inhi bition activity.

On the basis of this information, we synthesized a series of malonic acid dihydr azides

(MalH) linked to polar moieties as shown in Fig. 1 B, as well as PEG-cou pled butyric

acid hydr azides [GlyH-(PEG)n].

The compounds were highly water -soluble, nontoxic, and eff ective f r om the lumen side in inhi biting CFTR chloride channel function and choler a toxin-induced f luid secretion.

Unlike the parent compound GlyH-101, the new inhi bitors were not absor bed signif icantly by the intestine.

MATERIALS AND METHODS

Synthesis procedures1H and 13C NMR spectr a were obtained in CDCl3 or DMSO-d6 using a 400-MHz Varian

spectr ometer ref erenced to CDCl3 or DMSO. Mass spectr ometry was done using aWaters LC/MS system (Alliance HT 2790+ZQ, HPLC: Waters model 2690, Milford,

MA). Flash chr omatogr a phy was per formed using EM silica gel (230±400 mesh), and

thin-layer chr omatogr a phy was done on Merck silica gel 60 F254 plates.

Diethyl-(2-naphthalenylamino)-propanedioate (2). A mixture of 2-na phthylamine

(compound 1,Fig. 2 A) (10 mmol), diethyl br omomaloante (10 mmol), and sodium

acetate (1.64 g, 20 mmol, dissolved in 4 ml of water ) was stirred at 90°C for 8 h. The

black solid material obtained u pon cooling was f iltered and recrystallized f r om hexane

to yield 2.5 g of 2 (yield 84%); mp:189í190°C; 1H nmr (DMSO-d6): 1.17 (t, 6H,

J=7.33 Hz), 4.17 (q, 4H, J=7.33), 5.10 (d, 1H,

J=8.79 Hz), 6.54 (d, 1H, J=8.79 Hz), 6.75 (d, 1H, J=2.20 Hz), 7.13 (t, 1H, J=7.32 Hz),

7.19 (dd, 1H, J=2.19, 8.79 Hz), 7.28 t, 1H, J=8.06 Hz), 7.51 (d, 1H, J=8.42 Hz), 7.61 (t,2H, J=8.79 Hz).

13C nmr (DMSO-d6): 14.57, 60.43, 60.49, 62.27, 104.70, 104.75, 119.04, 122.60,126.36, 126.81, 127.79, 128.05, 129.15, 135.21, 144.88, 144.95, 168.26; MS (ES +)

(m/z): [M+1]+calculated for C17H20 NO4, 302.35, found 302.2.

(2-naphthalenylamino)-propanedioic acid dihydrazide (3). A solution of 2 (10 mmol) inethanol (10 ml) was ref luxed with hydr azine hydr ate (12 mmol) for 10 h. Solvent and

excess reagent were distilled under vacuum. The pr oduct was recrystallized f r om ethanol to give 2.5 g of 3 (92%); mp 268-270°C; 1H nmr (DMSO-d6): 4.29 (d, 4H, J =

4.03 Hz), 4.56 (d, 1H, J=8.79 Hz), 6.03 (d, 1H, J=8.79 Hz), 6.62 (d, 1H, J=1.46 Hz),



7.09 (m, 2H), 7.28 (t, 1H, J=8.05 Hz), 7.50 d, 1H, J=8.06 Hz), 7.61 (m, 2H), 9.22 (s,

2H). 13C nmr (DMSO-d6): 59.90, 104.70, 119.51, 122.51, 126.28, 126.81, 127.79,128.07, 129.07, 135.21, 145.11, 167.26; MS (ES +) (m/z): [M+1]+ calculated for

C13H15 N5O2, 273.30, found 274.2.

2-naphthalenylamino-bis[(3,5-dibromo-2,4-dihydroxyphenyl)methylene]propanedioic

acid dihydrazide (MalH-1) . A mixture of 3 (10 mmol) and 3,5-di br omo-2,4-dihydr oxy benzaldehyde (20 mmol) in ethanol (5 ml) was ref luxed for 3 h. The hydr azone that crystallized u pon cooling was f iltered, washed with ethanol, and purif ied

by column chr omatogr a phy (silica gel EtOAc:hexane 2:3) to give 3.2 g of 4 (54%) as an

off-white solid; mp 246±248°C; 1H nmr (DMSO-d6): 4.91, 5.48 (d, 1H, J=7.69, 9.15

Hz, CHCO, D2O exchange), 6.62 (d, 1H, J=7.32 Hz, Ar H), 6.73, 6.84 (s, 1H, NH, D2O

exchange), 7.13í7.32 (m, 3H, Ar H), 7.57 (d, 1H, J=8.06 Hz), 7.61í7.70 (m, 3H, Ar H),

7.80, 7.90 (s, 1H), 8.15, 8.37 (s, 2H, CH=N-NH, CH=N-N=), 10.10±10.40 (br oad s, 2H,

OH, D2O exchange), 11.72, 11.90 (s, 2H, OH, D2O exchange), 12.22, 12. 53 (s, 2H,

CONH, D2O exchange). The MalH-x compounds exist in at least three tautomeric forms

and interpretation of NMR spectr um are done according to other malonic acid hydr azide

derivatives, as descri bed by Zhang et al. ( Z. Anorg . Allg . Chem. 628: 1259±1268, 2002).

MS (ES+) (m/z): [M+1]+ calculated for C27H19Br 4 N5O6, 829.10, found 830.1.

2-naphthalenylamino-[(3,5-dibromo-4-hydroxyphenyl)methylene][(2,4

disodiumdisulfophenyl) methylene] propanedioic acid dihydrazide (MalH-2). A mixture of dihydr azide 4 (5 mmol) and 2,4-disodium-disulfobenzaldehyde (5 mmol) in DMF (5

ml) was ref luxed for 4 h. The reaction mixture, u pon cooling, was added dr o pwise to a stirred solution of

EtOAc:EtOH (1:1), f iltered, washed with ethanol, and further purif ied by columnchr omatogr a phy (silica gel EtOAc:hexane 2:3) to give 2.3 g of 5 (58%) as an off-white

solid; mp >300°C; 1H nmr (DMSOd6): 4.98, 5.63 (d, 1H, J=9.88, 8.51 Hz, COCH),6.33-6.51 (m, 1H, Ar -H), 6.71, 6.84 (m, 1H, Ar -H), 7.03±7.37 (m, 4H, Ar -H and Ar -

NH), 7.42±7.65 (m, 4H, Ar -H), 7.77í7.92 (m, 3H, Ar - H), 7.98±8.11 (m, 1H), 8.93(s,

1H), 9.13, 9.15, 9.21 (three s, 1H), 11.62, 11.70 (two s, 1H), 11.98, 12.00, 12.21 (s, 1H).All signals between 8.93±12.21 and 4.98, 5.63 were D2O exchangeable; MS (ES+)

(m/z): [M+1]+ calculated for C27H21Br 2 N5O10S2, 799.43, found 800.5.

2-na phthalenylamino-[(3,5-di br omo-2,4-dihydr oxyphenyl)methylene][3-(4-

sodiumsulfo phenyl)- thioureido]pr o panedioic acid dihydr azide (MalH-3) and 2-

na phthalenylamino- [(3,5-di br omo-2,4-dihydr oxyphenyl)methylene][3-[4-(3-(PEG)n-

thioureido) phenyl)- thioureido]pr o panedioic acid dihydr azide [MalH-(PEG)n] and

[MalH-(PEG)n-B] were synthesized following similar reaction conditions used for

MalH-2, except that 4-sodiumsulfo phenylisothiocyanate and 6aíd were used in place of

2,4-disodium-disulfobenzaldehyde.

MalH-3, yield 47%, mp >300°C; 1H nmr (DMSO-d6): 4.98, 5.05 (d, 1H, J=8.42, 9.52

Hz, COCH), 6.62-6.91 (m, 2H, Ar H), 7.01í7.21 (m, 2H, Ar H and Ar -NH), 7.25í7.35

(m, 2H, Ar H), 7.34í7.59 (m, 5H, Ar H), 7.64 (d, 2H, J=8.69, Ar H), 7.90, 8.33 (two s,

1H, CH=N-NH, CH=NN=), 9.60 (br oad s, 1H, OH), 9.82 (s, 1H, Ar NHCS), 9.95 (s, 1H,CSNH), 10.54, 10.67 (two s, 1H, OH or CONH), 12.27, 12.45 (two s, 2H, CONH, or

OH), all signals between 9.60 and 12.45 and 4.98, 5.05 were D2O exchangeable; MS(ES+) (m/z): [M-1] ± calculated for C27H22Br 2 N6O7S2, 765.45, found 765.3. MalH-

(PEG)1: yield 40%, mp >300°C; 1H nmr (DMSO-d6): 3.70í4.37 (m, 8H, PEG CH),



4.81, 5.01 (s, 1H, COCH), 5.27 (s, 1H, methyl-OH), 6.60 (d, 1H, J=7.32 Hz, Ar H), 6.75

(s, 1H, Ar NH), 7.19±7.38 (m, 4H, Ar H), 7.59 (d, 2H, 8.00 Hz, Ar H), 7.64í7.76 (m, 3H,Ar H), 7.90 (d, 2H, 8.00 Hz, Ar H), 8.21, 8.30 (s, 1H, CH=N-NH, CH=N-N=), 9.76 (s,

2H, Ar NHCS) 9.83 (s, 1H, CSNH), 10.01 (s, 1H, NH), 10.36 (s, 1H, OH), 11.20, 11. 51(s, 2H, CONH), 11.54í11.62 (s, 1H, OH); MS (ES+) (m/z): [M+1]+ calculated for

C32H32Br 2 N8O6S2, 849.60, found 849.4.

MalH-(PEG)15: Yield 51%; mp >300°C; 1H nmr (DMSO-d6): 2.82 (s, 3H, PEG-

OCH3), 3.24í4.10 (m, PEG-H, CH), 4.51 (s, 1H, COCH), 6.63í7.52 (two sets of m,

7H, Ar H), 7.58í8.08 (m, 7H, Ar H), 8.16, 8.21 (s, 1H, CH=N, CH=N-N=), 9.74 (s, 1H,

Ar NHCS) 9.79 (s, 1H, CSNH), 10.05 (s, 1H, CSNH), 10.44 (s, 1H, OH), 11.22, 11. 53

(s, 2H, CONH), 11.59í11.65 (s, 1H, OH); MS (ES+) (m/z): [M+1]+ calculated for

C61H90Br 2 N8O20S2, 1480.38, found 1480.5 ( 44, 88, 132, 176).

MalH-(PEG)1B: Yield 48%; mp >300°C; 1H nmr (DMSO-d6): 3.21í4.47 (m, 10H,

PEG CH2), 4.82, 5.11 (d, 1H, J=7.47, 9.07 Hz, COCH), 5.38 (s, 1H, methyl-OH), 6.71±

7.12 (two sets of multiplate, 8H, Ar H, Ar NH), 7.20í7.57 (m, 4H, Ar H), 7.66í7.89 (m,

5H, Ar H), 8.16, 8.24 (s, 1H, CH=N, CH=N-N=), 9.74 (s, 2H, Ar NHCS) 9.80 (s, 1H,

CSNH), 10.07 (s, 1H, CSNH), 10.25 (s, 1H, OH), 11.12, 11. 47 (s, 2H, CONH), 11.62± 11.67 (s, 1H, OH). MS (ES+) (m/z): [M+1]+ calculated for C39H38Br 2 N8O6S2, 939.7,

found 939.6.

MalH-(PEG)15B: Yield 39%; mp >300°C; 1H nmr (DMSO-d6): 2.92 (s, 3H, CH3),

3.12í4.37 (m, PEG-H, CH2), 4.81 (s, 1H, COCH), 6.68-6.79 (m, 6H, Ar H, Ar NH),

7.15í7.48 (m, 4H, Ar H), 7.57í8.10 (m, 7H, Ar H), 8.24, 8.38 (s, 1H, CH=N-NH,

CH=N-N=), 9.85 (s, 2H, Ar NHCS), 9.89 (s, 1H, CSNH), 10.12 (s, 1H, CSNH), 10.28(s, 1H, OH), 11.22, 11. 54 (s, 2H, CONH), 11.56 (s, 1H, OH); MS (ES+) (m/z): [M+1]+

calculated for C68H96Br 2 N8O20S2, 1570.50, found 1570.2 ( 44, 88, 132, 176).4-[[((2-(2-Hydr oxyethoxy)ethyl)amino)thioxomethyl]amino]phenylisothiocyante (6a).

To a solution of 1,4- phenylene diisothiocyanate (1 mmol, 2 mL DMF) was added 2-

aminoethoxyethanol (0.3 mmol, 2 mL DMF) over 30 min. Af ter stirring for additional

30 min, the DMF was distilled and the pr oduct was purif ied by column chr omatogr a phy

on silica gel using as solvent n-hexane:AcOEt (1:1). Fr actions were eva por ated to give

58 mg of 2 (65%); 1H nmr (DMSO-d6): 3.41 (t, 2H, CH2, J=5.49 Hz), 3.47 (t, 2H,

CH2, J=5.49 Hz), 3.52 (t, 2H, CH2, J=5.49 Hz), 3.59 (t, 2H, CH2,, J=5.86 Hz), 4.55 (t,

1H, OH, J = 5.49 Hz), 7.33 (d, 2H, Ar -H, J=8.42 Hz), 7.52 (d, 2H, Ar -H, J=8.79 Hz),

7.86 (br oad s, 1H, NH), 9.74 (br oad s, 1H, NH); MS (ES+) (m/z): [M+1]+ calculated for

C12H15 N3O2S2, 298.40, found 298.5.

Similarly, the following compounds were synthesized using a ppr o priate amino-PEGs.

105 and 750 are the molecular weight of (PEG)1 and (PEG)15, respectively.

4-[[((Methoxy-(PEG)15)amino)thioxomethyl]amino]phenylisothiocyante (6b). Yield,

58%; 1H nmr (DMSO-d6): 3.21±3.52 (m, PEG-H), 3.91 (s, 3H, OCH3), 7.26 (d, 2H,

Ar -H, 8.46 Hz), 7.47 (d, 2H, Ar -H, 8.46 Hz), 7.92 (br oad s, 1H, Ar -NH), 9.41 (br oad s,

1H, NH); MS (ES+) (m/z): [M+1]+ calculated for C41H73 N3O16S2, 929.18, found 929.2

( 44, 88, 132, 176).



4-[4-[[(2-(2

Hydr oxyethoxy)ethylamino)thioxomethyl]amino]phenylmethyl]phenylisothiocyante (6c). Yield 63%; 1H nmr (DMSO-d6): ~3.28 (t, 2H, CH2, J=5.48 Hz), 3.40 (t, 2H, CH2,

J=5.47 Hz), 3.47 t, 2H, CH2, J=5.46 Hz), 3.52 (t, 2H, CH2, J=5.82 Hz), 4.49 (s, 1H,OH), 7.23±7.29 (m, 4H, Ar -H), 7.29±7.40 (m, 4H, Ar -H), 7.81 (br oad s, 1H, NH), 9.48

(br oad s, 1H, NH); MS (ES+) (m/z): [M+1]+ calculated for C19H21 N3O2S2, 388.53, found

388.3.

4-[4-[[((PEG-750)amino)thioxomethyl]amino]phenylmethyl]phenylisothiocyante (6d).

Yield, 58%; 1H nmr (DMSO-d6): 2.04 (s, 2H, CH2), 3.19±3.48 (m, PEG-CH2), 3.87 (s,

3H, OCH3), 7.10±7.14 (m, 4H, Ar -H), 7.25±7.32 (m, 4H, Ar -H), 7.60 (br oad s, 1H,

NH), 9.49 (br oad s, 1H, NH); MS (ES+) (m/z): [M+1]+ calculated for C48H79 N3O16S2,

1019.30, found 1019.4 ( 44, 88, 132, 176).

2-(2-na phthalenylamino)-4-hydr oxy-butyric acid hydr azide (7). This compound was

synthesized following similar reaction conditions used for compounds 2 and 3. Yield

89%; mp 258-260°C; 1H nmr (DMSO-d6): 1.79 (m, 2H, CH2) 3.46 (q, 2H, CH2,

J=3.98) (s, 1H, OH), 4.17 (d, 2H, NNH2, J=4.52) (t, 1H, COCH), 5.94í5.96 (s, 1H,

Ar NH), 6.68 (s, 1H, Ar H), 6.98 (dd, 1H, J=Ar H), 7.05 (t, 1H, J=Ar H), 7.24 (t, 1H, J=Ar H), 7.46 (d, 1H, J=Ar H), 7.52-7.60 (m, 2H, Ar H) 9.17 (s, 1H, CONH). 13C nmr (DMSO-d6): 36.77, 53.32, 58.26, 104.02, 119.24, 121.92, 126.08, 126.64, 127.29,

128.02, 128.84, 135.54, 146.55, 173.06; MS (ES +) (m/z): [M+1]+ calculated for C14H17 N3O2, 260.31, found 260.2.

[2-(2-na phthalenylamino)-4-hydr oxy] butyric acid-2-[(1,1

dimethylethoxy)car bonyl]hydr azide (8). To a solution of hydr azide 7 (10 mM) in THF(10 ml) was added (BOC)2O (20 mM) and heated under ref lux for 5 h. The solvent was

removed, and the residue was purif ied by column chr omatogr a phy on silica gel. Elutionwith dichlor omethane gave 3.1 g of 8 (86%) as a white

solid; mp 235í237°C; ms (ES+): M/Z 360 (M+1)+; 1H nmr (DMSO-d6): 1.33 (s, 9H),

1.92 (m, 2H, CH2), 3.52 (q, J=2H), 4.01 (q, 1H, J=OH), 4.52 (t, 1H, J=COCH), 6.00(d, 1H, J= NH), 6.70 (s, 1H, Ar H), 6.97 (dd, 1H, J=Ar H), 7.06 (t, 1H, J=Ar H), 7.25 (t,

1H, J=Ar H), 7.45 (d, 1H, J=Ar H), 7.52í7.59 (m, 2H, Ar H), 8.73 (s, 1H, CONH), 9.77

(s, 1H, NH-BOC). 13C nmr (DMSO-d6): 28.49, 28.64, 36.56, 53.14, 58.05, 58.17,

79.67, 104.15, 119.19, 121.90, 126.14, 126.58, 127.31, 127.99, 128.81, 135.54, 146.46,170.04, 173.43, 198.42; MS (ES +) (m/z): [M+1]+ calculated for C19H25 N3O4, 360.43,

found 360.5.

[2-(2-na phthalenylamino)-4-( p-tosyl)] butyric acid-2-[(1,1

dimethylethoxy)car bonyl]hydr azide (9). To a solution of hydr azide 8 (1 mmol) in

pyridine (5 ml) was added p-TsCl (1 mmol) in three portions 30 min a part (±15°C). The

reaction mixture was stirred for 8 h at ±15°C, allowed to warm to r oom temper ature,diluted with 1N HCl, and extr acted 3 times with EtOAc. The com bined or ganic extr act

was washed with brine, dried with Na2SO4, and eva por ated to dryness to give 374 mg of

9 (73%) as a pale yellow oil, used without further purif ication for next step; MS (ES+)

(m/z): [M+1]+ calculated for C26H31 N3O6S, 514.62, found 514.3.

[2-(2-na phthalenylamino)-4-(PEG-amino-105)] butyric acid-2-[(1,1

dimethylethoxy)car bonyl] hydr azide (10a). A solution of 2-aminoethoxyethanol (1 mM)

and 9 (1 mM) in DMF (2 ml) was stirred at 80°C for 24 h. The DMF was eva por ated in



vacuo, and the residue was dissolved in minimum quantity of EtOAc and added to a

stirred solution of Et2O. The white powder -like precipitate was f iltered and washed with Et2O to give 170 mg of 10 (38%) as a yellow sticky mass; 1H nmr (DMSO-d6): 1.35

(s, 9H), 1.71 (m, 2H, CH2) 3.40-3.51 (m, 4H, CH2), 3.57 (t, 2H, J=6.52 Hz CH2), 3.62± 3.89 (m, 5H, CH2, NH), 3.97 (s, 1H, OH), 4.42 (t, 1H, J=COCH), 6.04í6.16 (s, 1H,

Ar NH), 6.67 (s, 1H, Ar H), 6.93 (dd, 1H, J=7.21, 2.33, Ar H), 7.03 (t, 1H, J=7.24 Ar H),

7.32 (t, 1H, J=7.24, Ar H), 7.45 (d, 1H, J=2.34, Ar H), 7.50í7.62 (m, 2H, Ar H), 9.35 (s,1H, CONH), 9.89 (s, 1H, NH-BOC); MS (ES+) (m/z): [M+1]+ calculated for

C23H34 N4O5, 447.55, found 447.8.

[2-(2-na phthalenylamino)-4-(PEG-amino-750)] butyric acid-2-[(1,1dimethylethoxy)car bonyl] hydr azide (10b). Yield 44% as a yellow sticky mass; MS

(ES+) (m/z): [M+1]+ calculated for C52H92 N4O19, 1078.33, found 1078.2 ( 44, 88, 132,176).

[2-(2-na phthalenylamino)-4-(PEG-amino-105)] butyric acid hydr azide (11a). Hydr azide

10 (1 mM) was dissolved in a minimal amount of trif luor oacetic acid:CH2Cl2 (1:1) and stirred at r oom temper ature for 30 min. The reaction mixture was diluted with satur ated

aqueous NaHCO3 and extr acted with CH2Cl2. The com bined or ganic layer was washed successively with water and brine, dried (Na2SO4), and concentr ated in vacuo to yield

253 mg of 11a (73%) as yellow semisolid; ms (ES+): M/Z 347 (M+1)+; 1H nmr (DMSO-

d6): 1.71 (m, 2H, CH2) 3.40±3.51 (m, 4H, CH2), 3.57 (t, 2H, J=6.44, CH2), 3.68-3.79

(m, 5H, CH2, NH), 3.93 (s, 1H, OH), 4.26 (d, 2H, NNH2) 4.52 (t, 1H, J=7.01 COCH),

6.02í6.21 (s, 1H, Ar NH), 6.71 (s, 1H, Ar H), 6.85 (dd, J=7.25, 2.34, 1H, Ar H), 7.10 (t,

1H, J=7.30, Ar H), 7.34 (t, 1H, Ar H), 7.51 (d, 1H, J=6.98 Ar H), 7.53±7.76 (m, 2H,

Ar H), 9.27 (s, 1H, CONH); MS (ES+) (m/z): [M+1]+ calculated for C18H26 N4O3, 347.47,

found 347.4.

[2-(2-na phthalenylamino)-4-(PEG-amino-750)] butyric acid hydr azide (11b). Yield

63%; MS (ES+) (m/z): [M+1]+ calculated for C47H84 N4O17, 978.21, found 978.40 ( 44,

88, 132, 176).

[2-(2-na phthalenylamino)-4-(PEG-amino-105)] butyric acid-2-[(3,5-di br omo-2,4-dihydr oxyphenyl) methylene] hydr azide (GlyH-(PEG)1). A mixture of 11a (1 mmol)

and 3,5- di br omo-2,4-dihydr oxy benzaldehyde (1 mmol) in ethanol (2 ml) was ref luxed for 3 h. The reaction mixture was concentr ated and added to a stirred solution of Et2O,

and the precipitated hydr azone was f iltered and washed with Et2O to yield 362 mg of GlyH-(PEG)1 (58%); 1H nmr (DMSO-d6): 1H nmr (DMSO-d6): 1.75 (m, 2H, CH2)

3.43-3.48 (m, 4H, CH2), 3.59 (t, 2H, J=6.52, CH2), 3.72í3.81 (m, 5H, CH2, NH), 3.97

(s, 1H, OH), 4.59 (t, 1H, COCH), 6.12, 6.26 (s, 1H, Ar NH), 6.75 (s, 1H, Ar H),

6.85í6.96 (m, 2H, Ar H), 7.15í7.51 (m, 3H, Ar H), 7.53í7.76 (m, 2H, Ar H), 8.87, 8.93

(s, 1H, CH=N), 9.27 (s, 1H, CONH), 10.68 (s, 1H, OH), 11.92 (s, 1H, OH); MS (ES+)(m/z): [M+1]+ calculated for C25H28Br 2 N4O5, 625.33, found 625.2.

[2-(2-na phthalenylamino)-4-(PEG-amino-750)] butyric acid-2-[(3,5-di br omo-2,4-

dihydr oxyphenyl) methylene] hydr azide (GlyH-(PEG)15). Yield 46%; 1H nmr (DMSO-d6): 2.82 (s, 3H, OMe) 3.16±3.94 (m, PEG-H, 2CH2, NH), 4.56 (s, 1H, J=COCH),

6.19, 6.32 (s, 1H, Ar NH), 6.79í6.98 (m, 3H, Ar H), 7.13±7.62 (m, 3H, Ar H), 7.53±7.76(m, 2H, Ar H), 8.87±9.16 (m, 2H, CH=N, CONH), 10.51 (s, 1H, OH), 11.62 (s, 1H,



OH); MS (ES+) (m/z): [M+1]+ calculated for C54H86Br 2 N4O19, 1256.11, found 1256.1

( 44, 88, 132, 176).

Short-circuit current measurements

Fischer r at thyr oid (FRT) cells (stably expressing human wild-type CFTR) were

cultured on Sna pwell f ilters with 1 cm2 sur face area (Cor ning-Costar ) to resistance >1,000 ·cm2 as descri bed by Muan pr asat and colleagues (11). Filters were mounted inan Easymount Cham ber System (Physiologic Instr uments, San Diego, CA). For a pical

Cl ± current measurements, the basolater al hemicham ber contained (in mM): 130 NaCl,

2.7 KCl, 1.5 KH2PO4, 1 CaCl2, 0.5 MgCl2, 10 Na-HEPES, 10 glucose ( pH 7.3). The

basolater al mem br ane was permeabilized with amphotericin B (250 g/ml) for 30 min.

In the a pical solution 65 mM NaCl was replaced by sodium gluconate, and CaCl2 was

increased to 2 mM. Solutions were bubbled with 95% O2/5% CO2 and maintained at

37°C. Current was recorded using a DVC-1000 voltage-clamp (World Precision

Instr uments) using Ag/AgCl electr odes and 1 M KCl agar bridges.

Cholera model

Mice (CD1 str ain, 28±34 g) were deprived of food for 24 h (but given 5% sucr ose inwater ) and then anesthetized with 2.5% Avertin intr a pertitoneally. Body temper ature

was maintained at 36± 38°C using a heating pad. Af ter a small abdominal incision, three closed mid je junal loo ps (length 15±20 mm) were isolated by sutures. Loo ps were

injected with 100 l of PBS or PBS containing choler a toxin (1 g) without or with test compounds. The abdominal incision was closed with a suture, and mice were allowed to

recover f r om anesthesia. At 6 h, the mice were anesthetized, intestinal loo ps were removed, and loo p length and weight were measured to quantif y net f luid secretion. In

some experiments, intestinal f luid absorption (without choler a toxin) was measured by injection of loo ps with 100 l phosphate-buff ered saline containing glucose (10 mM),

with or without test compounds, and the f luid remaining at 20 and 30 min was measured

by the diff erence in weight of intact and empty loo p. Mice were killed by an overdose

of Avertin. All pr otocols were a ppr oved by the UCSF Committee on Animal R esearch.

Inhibitor absorption

Caco-2 cells were cultured on Sna pwell f ilters (1 cm2, Cor ning-Costar ) to resistance

~1000 ·cm2. For permeability measurements, the culture medium was replaced with

an equal volume of Hank¶s buff ered salt solution (HBSS) containing 15 mM glucose

and 25 mM HEPES ( pH 7.3). Af ter 1 h, compounds (20±100 M) were added to the

donor (u pper ) cham ber , and plates were gently r ocked at 37°C. Af ter 4 h, solution f r om the lower cham ber was assayed for inhi bitor concentr ation. For measurements of

intestinal absorption, mid je junal loo ps were injected with 100 l of phosphate buff ered-saline containing 10±20 g test compound and 5 g FITC-dextr an (40 k Da), in which

100 mM NaCl was replaced by 200 mM r aff inose. The added r aff inose prevented

intestinal f luid absorption. Af ter 0 or 2 h, loo p f luid was withdr awn for assay of

compound concentr ation f r om the r atio of o ptical absor bance of test compound vs.

FITC-dextr an

(OD342/OD494nm), which was assumed to be impermeant. In some experiments, f luid

samples were also analyzed by LC/MS.



Inhibitor stabilityInhi bitors were tested for stability at diff erent pHs and in the presence of intestinal

contents (je junal f luid). Solutions of inhi bitors (100 M) at pH 1.0 (0.1 N HCl), 4.4 (20mM AcONa), 6.1 (20 mM MES), 7.4 (20 mM HEPES), and 8.4 (20 mM TRIS) were

incubated at 37°C for 2 h, and samples were analyzed by LC/MS af ter ad justing pH to

7.0. Similarly, inhi bitors (100 M) added to je junal stool (1:1 diluted with PBS) were incubated at 37°C for 2 h, solids were removed by centrifugation at 20,000 g for 10 min,and the su per natant was analyzed. For LC/MS analysis, reversed- phase HPLC

separ ations were carried out using a Su pelco C18 column (2.1 × 100 mm, 3 m particle

size) connected to a solvent delivery system (Waters model 2690, Milford, MA). The

solvent system consisted of a linear gr adient f r om 15% CH3CN/10 mM KH2PO4, pH

6.95 to 95% CH3CN/10mM KH2PO4, pH 6.95 r un over 15 min, followed by 6 min at

95% CH3CN/10mM KH2PO4 (0.2 ml/min f low r ate). Inhi bitors were detected at 256 nm

with linear standard cali br ation cur ve for the r ange of 20±5000 nM r ange. Mass spectr a

were acquired on a mass spectr ometer (Alliance HT 2790 + ZQ) using negative and

positive (for MalH-1) ion detection, scanning f r om 200 to 2000 Da, as descri bed

Sonawane and colleagues (15).

Toxicity

For cell cultures studies, compounds (50±100 M) or vehicle were added to the mediafor 2 days in FRT cells, and then quantitative cell counting was done. For mouse

studies, compounds (4±8 mg/k g) were administered by intr avenous or or al r outes every 12 h to adult mice for three days.

Contr ol mice were administered vehicle (PBS or PBS containing 1% DMSO). Body

weight was measured daily, and af ter 4 days, blood was collected for analysis of ser um chemistries.

RESULTS

Design and synthesis of nonabsorbable CFTR pore-blockers

We synthesized a series of highly polar , nonabsor bable CFTR inhi bitors based on the

glycine hydr azide scaffold (Fig. 1 B). Prior str ucture-activity analysis indicated the

glycyl methylene gr ou p as the unique site on the glycine hydr azide scaffold where

modif ications could be toler ated (11). One str ategy for design of water -soluble and

mem br ane impermeant CFTR inhi bitors was modif ication of GlyH-101 by linking it

with bulky moieties containing polar gr ou ps such as sulfonic acid or car boxy, or hydr oxy with p K a < 7. A second str ategy was conjugation of GlyH- 101 to the water -

soluble polymer polyethylene glycol. In initial studies to explore the types of substitutions that could be toler ated without loss of activity, we synthesized a series of

derivatives containing substituted phenyl, hydr oxyethyl, ethoxycar bonyl, car boxyl, and

ethyl at the glycyl methyl position of GlyH-101 (data not shown). Most derivatives

retained CFTR inhi bitory activity, and it was found that malonic acid hydr azides had

greatest CFTR inhi bitory potency, even better than the parent compound, GlyH-101. A

series of GlyH-101 analogs in which na phthalenyl moiety was substituted with more

polar /functional gr ou ps were substantially less active than the malonic acid hydr azides.



Therefore, we devised eff icient syntheses of water -soluble, nonabsor bable inhi bitors by

using diethyl br omomalonate intermediate (Scheme 1, Fig. 2). R eaction of 2-na phthalenamine with diethyl br omomalonate followed by subsequent reaction with

hydr azine gener ated a versatile malonic acid dihydr azide intermediate 3. Condensationof this dihydr azide with 3,5-di br omo- 2,4-dihydr oxy benzaldehyde pr oduced a key

intermediate, compound 4, which on further condensation with the same aldehyde gave

MalH-1. Similarly, 2,4-disulfobenzaldehyde and 4- sulfo phenyl-isothiocyanate were reacted with 4 to gener ate MalH-2 and MalH-3, respectively.

MalH-1 is str uctur ally similar to GlyH-101 except for an additional (3,5-di br omo-2,4-

dihydr oxyphenyl)methylene]hydr azide moiety, making it doubly char ged, bulkier , and

more hydr o philic, which conf erred greater water solubility (~5 mM) compared with

GlyH-101 and decreased mem br ane permeability. MalH-2 carries two disulfonic acid

gr ou ps, whereas MalH-3 contains one sulfonic acid moiety with a hydr o philic thiourea

link . MalH-2 and MalH-3 are f reely water soluble, exceeding 50% wt/vol at 20°C in

saline.

Intermediate 4 was also used to gener ate MalH-(PEG)n and MalH-(PEG)nB by

condensation with PEG-containing phenylisothiocyanates 6a± d (Scheme 2).Intermediates 6aíd were synthesized by reaction of 1,4- phenylenediisothiocyanate 5a or

bis[(4-isothiocyanato)] phenylmethane 5b with a ppr o priate amino-PEGs. The

conjugated PEG moiety increased compound water solubility to 5±10 mM. MalH-(PEG)nB has a slightly longer hydr o phobic chain than MalH-(PEG)n between the

inhi bitor and PEG moieties, potentially impr oving CFTR accessi bility by reducingintr amolecular inter actions. Another a ppr oach taken for synthesis of PEG-ylated

compounds involved incorpor ation of hydr oxyethyl moiety onto the glycyl methylene,in which br omobuter olactone was reacted with 2-na phthalenamine and subsequently

reacted with hydr azine to give hydr azide 7 (Scheme 3), which was PEG-ylated thr ough its hydr oxyl gr ou p using standard pr otection-depr otection Boc chemistry. The PEG-

ylated hydr azide 11 was condensed with ar omatic aldehyde to pr oduce GlyH-(PEG)n.

CFTR inhibition in cell monolayers by apically added compounds

CFTR inhi bition was assayed by short-circuit current analysis in FRT cells expressing

human wild-type CFTR. A pical mem br ane chloride current was measured af ter

permeabilization of the cell basolater al mem br ane in the presence of a tr ansepithelial

chloride gr adient, and CFTR chloride channel stimulation by the cell- permeant cAMP

agonist CPT-cAMP. Under these conditions the measured current is a CFTR-dependent

chloride current. R epresentative data shown in Fig. 3 A indicate pr ompt reduction in

chloride current by the various compounds when added to the a pical bathing solution,with inhi bitory potencies (K i) in the r ange 2±8 M and near complete inhi bition at

higher compound concentr ations. CFTR inhi bition for each of the compounds was reversed u pon compound washout, as shown for MalH-3 in Fig. 3 B.

Compound absorption and antidiarrheal properties

Tr ansepithelial permeability of the CFTR inhi bitors was assessed by the Caco-2 assay,

in which a ppear ance of compounds in the basal solution was assayed at 4 h af ter compound addition to the a pical solution. For each of the compounds in Fig. 1 B,

a ppear ance in the basal solution could not be detected at 4 h. In this Caco-2 assay,



CFTR inh-172 was ~45% equili br ated at 4 h. Intestinal absorption was measured in living

mice f r om compound disa ppear ance f r om the lumens of closed mid je junal loo ps over 2h. In these experiments, r aff inose was included in the infusate

solutions to prevent f luid absorption. Figure 4 A shows less than 2% compound absorption per hour , whereas >90% of the thiazolidinone CFTR inh-172 was absor bed

over 2 h, and ~65% of GlyH-101.

Antidiarrheal eff icacy was assayed in closed mid je junal loo ps in mice. Loo ps were injected with saline or solutions of choler a toxin containing diff erent inhi bitor amounts.

Intestinal f luid secretion at 6 h was measured. The data summary inFig. 4 B shows a

loo p weight-to-length r atio (corresponding to 100% inhi bition) of ~0.09 in saline-

injected loo ps, and 0.28 (corresponding to 0% inhi bition) in choler a toxin-injected

loo ps. Each compound inhi bited loo p secretion in a dose-dependent manner with

essentially complete inhi bition at the higher amounts.

To determine whether the compounds aff ected intestinal f luid absorption, measurements

of remaining f luid were done in closed intestinal loo ps at 20 and 30 min af ter injection

of 100 l saline containing glucose (without or with compounds). Figure 4C shows no

signif icant eff ect of the CFTR inhi bitors on intestinal f luid absorption.

In toxicity studies, none of the compounds reduced cell pr olif er ation when added to

FRT cells cultures at 50±100 M for 2 days (data not shown). In vivo toxicity was evaluated at 4 days af ter twice daily administr ation of MalH-1, MalH-2, MalH-3, or

MalH-(PEG)n in PBS (4±8 mg/k g) by intr avenous or or al r outes (3 mice per r oute per compound). No abnormalities were seen in mouse activity, and no signif icant

diff erences were found in test vs. contr ol mice in body weight, urine output, or ser um electr olyte, urea, creatinine, amylase or tr ansaminase concentr ations (Table 1).

Compound stability was assessed in solutions of diff erent pH (simulating diff erent

regions of the gastr ointestinal tr act) and in the presence of intestinal contents. LC/MS

analysis showed that compounds were stable (<5% loss) when incubated at 37°C for 2 h

in saline solutions ( pH 4.4± 8.5). However , moder ate degr adation was found when

incubated at pH 1.0, with reductions of 20±30% over 2 h of intact MalH-2, MalH-3, and

MalH-(PEG)1. A ppr o priate compound formulation to bypass the gastric envir onment

may thus be required. LC/MS analysis also showed that the compounds were stable

when incubated at 37°C for 2 h in a 1:1 saline dilution of small intestinal f luid contents.

DISCUSSION

CFTR is a unique tar get for antidiarrheal ther a py because of its location at the lumen-facing sur face of enter ocytes and its r ole as the r ate-limiting step in ion secretion caused

by sever al enter otoxins, including choler a toxin. The glycine hydr azide-based CFTR inhi bitors synthesized here under go little intestinal absorption and are eff ective in

preventing choler a toxin-induced f luid secretion in a r odent model of intestinal f luid

secretion. The potential advantages of antidiarrheal ther a py using a nonabsor bable

compound are that high concentr ations can be achieved in the gut lumen with minimal

concer ns about toxicity and off-tar get eff ects related to cellular u ptake and systemic

absorption. The nonabsor bable glycine hydr azide inhi bitors can be synthesized at

relatively low cost, which is an important consider ation for third-world antidiarrheal

a pplications.



Short-circuit current analysis in CFTR-expressing epithelial cell monola yers showed pr ompt inhi bition of chloride current in response to compound addition to the luminal

solution. Near 100% block of chloride current was achieved at high inhi bitor concentr ations. Inhi bitor tr ansport acr oss Caco-2 monolayers was undetectable and <2%

disa ppear ance f r om the intestinal lumen per hour was found in closed intestinal loo ps in

mice in vivo. Also, the inhi bitors were chemically stable in the presence of intestinal contents, and no toxicity was seen when the inhi bitors were present at high concentr ation in cell cultures or when administered systemically to mice.

The eff icacy of the luminally active CFTR inhi bitors in preventing choler a toxin-

induced f luid secretion in je junal loo ps indicates that the inhi bitors have adequate access

to the luminal sur face of crypt epithelial cells where f luid secretion occurs. The small-

molecule CFTR inhi bitors are thus able to overcome the potential barrier imposed by

cryptal f luid secretion, in which convection away f r om the crypt might impair inhi bitor

access. Sever al factors may contri bute to this, including the r a pid diffusion of small

molecules of the size of the CFTR inhi bitors (20), the relatively shallow crypts in small

intestine vs. colon, and the intermittent nature of cryptal f luid secretion. Similar

mechanism may account for the ability of the relatively lar ger choler a toxin molecule toaccess crypt epithelial cells.

The mainstay of current ther a py in choler a is or al rehydr ation solution (ORS) ther a py to prevent the consequences of massive volume and electr olyte depletion (21). Choler a

toxin and other enter otoxins pr oduce diarrhea by binding to ganglioside receptors at the enter ocyte lumen, resulting in elevated cyclic nucleotide concentr ations, pr otein kinase

activation, and CFTR phosphorylation (22). Vaccines and anti biotic ther a py aimed at reducing bacterial load are under clinical evaluation (23±25). Alter native str ategies to

reduce intestinal f luid losses in choler a have been pr o posed, including enkephalinase inhi bition to reduce cyclic nucleotide concentr ation (26) and inhi bition of toxin binding

to cell sur face receptors (27). CFTR inhi bition is predicted to reduce intestinal f luid

losses in choler a and other enter otoxin-mediated diarrheas and may be of particular

benef it in young and elderly subjects where mor bidity and mortality remain high despite

ORS ther a py, as well as where ORS ther a py is not available or pr actical. Also, since

ORS ther a py does not reduce stool volume or the dur ation of diarrhea (28±30), CFTR

inhi bitors may have a r ole in reducing the dur ation and clinical severity of choler a.

Lar ge animal testing and clinical studies will be needed to determine the utility of the

nonabsor bable CFTR inhi bitors develo ped here.

ACKNOWLEDGMENTS

This work was su pported by gr ants DK-72517, AI-062530, HL-73854, EB-00415, EY-13574, DK35124, and DK-43840 f r om the National Institutes of Health, and Dr ug

Discovery and R esearch Develo pment Pr ogr am gr ants f r om the Cystic Fi br osis

Foundation.

REFERENCES

1. Chao, A. C., de Sauvage, F. J., Dong, Y. J., Wagner , J. A., Goeddel, D. V., and

Gardner , P. (1994) Activation of intestinal CFTR Cl ± channel by heat-stable



14. Thiagar ajah, J. R., Br oad bent, T., Hsieh, E., and Verkman, A. S. (2004)Prevention of toxininduced intestinal ion and f luid secretion by a small-molecule

CFTR inhi bitor . Gastroenterology 126, 511±519

15. Sonawane, N. D., Muan pr asat, C., Nagatani, R., Song, Y., and Verkman, A.

S. (2005) In vivo pharmacology and antidiarrheal eff icacy of a thiazolidinone CFTR inhi bitor in r odents. J . Pharm. S ci. 94, 134±143

16. Sheppard, D. N., and Robinson, K. A. (1997) Mechanism of gli benclamide

inhi bition of cystic f i br osis tr ansmem br ane conductance regulator Cl ± channels

expressed in a murine cell line. J . Physiol . 503, 333±346

17. Zhou, Z., Hu, S., and Hwang, T. C. (2002) Pr obing an o pen CFTR pore with

or ganic anion blockers. J . Gen. Physiol . 120, 647±662

18. Dawson, D. C., Smith, S. S., and Mansour a, M. K. (1999) CFTR :

mechanism of anion conduction. Physiol . Rev. 79, S47±S75

19. McCarty, N. A. (2000) Permeation thr ough the CFTR chloride channel. J . Exp. Biol . 203, 1947±1962

20. Thiagar ajah, J. R., Pedley, K. C., and Naf talin, R. J. (2002) Evidence of

amiloride-sensitive f luid absorption in r at descending colonic crypts f r om f luorescence recovery of FITClabelled bdextr an af ter photobleaching. J .

Physiol . 536, 541±553

21. Guerr ant, R. L., Car neir o-Filho, B. A., and Dillingham, R. A. (2003)Choler a, diarrhea, and or al rehydr ation ther a py: triumph and indictment. Clin.

Infect . Dis. 37, 398±405

22. Barrett, K. E., and K eely, S. J. (2000) Chloride secretion by the intestinal

epithelium: molecular basis and regulatory aspects. Annu. Rev. Physiol . 62, 535±

572

23. Svennerholm, A. M., and Steele, D. (2004) Micr obial-gut inter actions in

health and disease. Pr ogress in enteric vaccine develo pment. Best Pract . Res.

Clin. Gastroenterol . 18, 421±445

24. Boedeker , E. C. (2005) Vaccines for enter otoxigenic Escherichia coli: current status. Curr . O pin. Gastroenterol . 21, 15±19

25. K han, W. A., Saha, D., Rahman, A., Salam, M. A., Bogaerts, J., and

Bennish, M. L. (2002) Comparison of single-dose azithr omycin and 12-dose, 3-

day erythr omycin for childhood choler a: a r andomised, double-blind trial.

Lancet 360, 1722±1727

26. Alam, N. H., Ashr af, H., K han, W. A., Karim, M. M., and Fuchs, G. J.

(2003) Eff icacy and toler ability of r acecadotril in the treatment of choler a in

adults: a double-blind, r andomised, contr olled clinical trial. Gut 52, 1419±1423



27. Pickens, J. C., Mitchell, D. D., Liu, J., Tan, X., Zhang, Z., Verlinde, C. L.,Hol, W. G., and Fan, E. (2004) Nonspanning bivalent ligands as impr oved

sur face receptor binding inhi bitors of the choler a toxin B pentamer . Chem. Biol . 11, 1205±1215

28. Guir aldes, E., Trivino, X., Hodgson, M. I., Quintana, J. C., and Quintana, C.(1995) Treatment of acute infantile diarrhoea with a commercial rice-based or al rehydr ation solution. J . Diarrhoeal Dis. Res. 13, 207±211

29. CHOICE Study Gr ou p (2001) Multicenter , r andomized, double-blind

clinical trial to evaluate the eff icacy and saf ety of a reduced osmolarity or al

rehydr ation salts solution in children with acute watery diarrhea. Pediatrics 107,

613±618

30. Santosham, M., Fayad. I., Abu. Zikri. M., Hussein, A., Amponsah, A.,

Duggan, C., Hashem, M., el Sady, N., Abu, Zikri, M., and Fontaine, O. (1996) A

double-blind clinical trial comparing World Health Or ganization or al

rehydr ation solution with a reduced osmolarity solution containing equal amounts of sodium and glucose. J . Pediatr . 128, 45±51 Received J uly 26, 2005;accepted S eptember9, 2005

1 Table 1

Serum chemistries in control and inhibitor-treated miceControl Mice MalH-3 MalH-(PEG)15

Oral IV Oral IV Oral IV

Na+ (mM) 155 ± 3 154 ± 3 150 ± 1 148 ± 3 147 ± 2 148 ± 2Cl ± (mM) 115 ± 2 113 ± 2 110 ± 1 108 ± 1 110 ± 1 108 ± 3HCO3

± (mM) 20 ± 1 22 ± 2 19 ± 3 21 ± 1 21 ± 1 21 ± 1ALT (U/L) 35 ± 5 35 ± 3 33 ± 6 31 ± 6 29 ± 7 37 ± 6AST (U/L) 88 ± 19 124 ± 14 156 ± 17 92 ± 15 60 ± 7 120 ± 16Amylase (U/L) 1157 ± 37 1338 ± 24 1145 ± 69 1035 ± 50 1130 ± 55 1195 ± 65

BUN (mM) 17 ± 0.9 15 ± 2 19 ± 0.5 16 ± 0.4 16 ± 3 21 ± 4Creatinine (mM) 0.30 ± 0.05 0.20 ± 0.04 0.27 ± 0.02 0.27 ± 0.05 0.23 ± 0.05 0.17 ± 0.03Data are presented as means ± SE for 5 mice per gr ou p.

Fig. 1

Figure 1. Small-molecule CFTR inhi bitors. A) Str uctures of the thiazolidinone CFTR inh-172and the glycine hydr azide GlyH-101. B) Nonabsor bable CFTR inhi bitors: malonic acid dihydr azides (MalH-x) and

glycine hydr azides [GlyH-

(PEG)n].

Fig. 2

Figure 2. Synthesis of polar nonabsor bable CFTR inhi bitors. Scheme 1) Synthesis of polar

CFTR inhi bitors, MalH-1,MalH-2, and MalH-3: a. diethyl br omomalonate, NaOAc, 90°C, 8 h, 84%; b. N2H4. H2O,

EtOH/ref lux, 10 h, 92%; c, d.3,5-di-Br -2,4-di-OH-benzaldehyde (1 eq), EtOH/ref lux, 3 h, 54%; e. 2,4-di-SO3 Na- benzaldehyde, DMF/ref lux, 4 h, 58%. f.

4-sodiumsulfo phenyl-isothiocyante, DMF/ref lux, 4 h, 47%. Scheme 2) Synthesis of PEG-ylated

compounds, MalH-(PEG)n



and MalH-(PEG)nB: g. Amino-PEG (0.3 eq), DMF, rt, 1 h, 65%. 4, DMF/ref lux, 3 h, 63 and

58%. Scheme 3) Synthesis of

PEG-ylated GlyH-(PEG)n. i. Br -buter olactone, NaOAc, 90°C, 8 h, 89%; j. N2H4. H2O,EtOH/ref lux, 10 h, 89%; k .

(BOC)2O, THF, rt, 86%; l. TsCl, pyridine, ±15°C, 8 h, 73%; m. NH2-PEG, DMF, 80°C, 24 h,38%; n. TFA, CH2Cl2, rt, 30min, 73%; o. 3,5-di-Br -2,4-di-OH-benzaldehyde, EtOH/ref lux, 3 h, 58 and 46%.

Fig. 3

Figure 3. Inhi bition of a pical mem br ane chloride current in FRT epithelial cells expressinghuman wild-type CFTR.

Chloride current was measured by short-circuit current analysis in cells subjected to a chloride ion gr adient and af ter

permeabilization of the basolater al mem br ane (see Methods). CFTR was stimulated by 100 M

CPT-cAMP. A) Increasing

concentr ations of MalH compounds were added as indicated. B) R epresentative wash-out study showing reversi ble CFTR

inhi bition by MalH-3.

Fig. 4Figure 4. Antidiarrheal pr o perties and intestinal absorption of CFTR inhi bitors. A) Compound absorption over 2 h in

closed je junal loo ps (SD, n=4í6 mice). For comparison absorption of CFTR inh-172 and GlyH-101 is shown. B) Inhi bitionof choler a toxin-induced f luid secretion. Loo ps were injected with saline or saline containing 1

g choler a toxin (CT) with

indicated compound amounts. Loo p weight-to-length r atio was measured at 6 h (SD, n=3í5

mice). C ) Intestinal f luid absorption. Mid je junal loo ps were injected with 100 l of saline containing 10 mM glucose,without (contr ol) or with

indicated compounds (100 M). Percentage f luid absorption measured at 20 and 30 min (SD,

3í6 mice per time point).

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