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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
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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
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(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),
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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),
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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).
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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
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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,
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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.
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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.
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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,
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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.
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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.
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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
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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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