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Preliminary Communication

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Medicinal Chemistry

The search for a common structural moiety among selected pharmacological correctors of the mutant CFTR chloride channel

Background: The F508del mutation impairs the trafficking of cystic fibrosis transmembrane conductance regulator (CFTR) from endoplasmic reticulum to plasma membrane and is responsible of a severe form of cystic fibrosis. Trafficking can be improved by small organic molecules called ‘correctors’. Materials & methods: By different synthetic ways, we prepared 4-chloroanisole and 2-(4-chloroanisol-2-yl) aminothiazole derivatives. Such compounds were ineffective as correctors but we could find a sign of activity in an intermediate. In the meantime, we found a common pharmacophoric moiety present in four known correctors. Results: Following this structural indication, we synthesized a small set of new molecules endowed with a significant, even if not great, F508del-CFTR rescue activity. Conclusion: The cited structural feature seems interesting in the search of new correctors. To corroborate this observation, later on we found a new pyrazine derivative (Novartis) endowed with a potent activity as corrector and having the cited common design.

The chloride channel cystic fibrosis transmembrane conductance regulator (CFTR) is a 1480-amino acid protein belonging to the ABC transporter superfamily. Genetic mutations causing CFTR loss of function are the basis of cystic fibrosis (CF) [1] . There are more than 1900 mutations that have been identified so far in CF patients. Some of them lead to a severe phenotype in 70–80% of patients. In the remaining cases, the disease results in a milder phenotype [2] . The CF disease is caused by the decreased transport of chloride and bicarbonate in the epithelial cells of many organs (e.g., the pancreas, lungs and intestine, among others). Particularly in the lungs, the lack of anion transport causes the formation of dehydrated and thick mucus in which bacteria (e.g., Pseudomonas aeruginosa and Burkholderia cepacia, among others) find an ideal site for their growth, thus triggering severe lung inflammation. The lung disease and the resulting progressive loss of respiratory function is the cause of death in many CF patients [3,4] .

10.4155/FMC.14.118 © 2014 Future Science Ltd

Erika Nieddu1, Benedetta Pollarolo1, Marco T Mazzei1, Maria Anzaldi1, Silvia Schenone1, Nicoletta Pedemonte2, Emanuela Pesce2, Luis JV Galietta2 & Mauro Mazzei*,1 1 Department of Pharmacy, University of Genova, Viale Benedetto XV, 3-16132 Genova, Italy 2 UOC Genetica Medica, Giannina Gaslini Institute, Largo Gerolamo Gaslini, 5-16147 Genova, Italy *Author for correspondence: Tel.: +39 10 3538371 [email protected]

The present CF therapies, although they have made great advances in the improvement of patient quality of life and in the rise of median life survival [5] , do not modify the molecular alteration affecting CFTR, but only relieve the symptoms and control the infection/inflammation [6] . The basic CF defect can be addressed with gene therapy or cell therapy strategies [7,8] . However, a pharmacological approach is also possible [9,10] . In this respect, it must be considered that CF mutations are grouped into five classes according to the mechanism through which they cause the CFTR loss of function. The first three classes lead to a severe CF, whereas the remaining two classes are responsible for milder phenotypes [11] . As the disease is related to many different mutations, it is unreasonable to suppose that a single substance acting on CFTR could be of benefit for all CF patients. To overtake this problem many researchers are performing studies to develop substances acting on targets different from CFTR Na+ [12] . At present, substances that are able to specifically recover the function of mutant

Future Med. Chem. (2014) 6(17), 1857–1868

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ISSN 1756-8919

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Key terms CFTR: A 1480-amino acid membrane protein that acts as a channel for anions. CFTR belongs to the ABC transporter family and is a very complex protein that is made up of two transmembrane domains, two nucleotide binding domains (NBD1 and NBD2) and a regulatory domain. The phenylalanine in position 508 is located in NBD1. Cystic fibrosis: An autosomal recessive genetic disorder that affects the lungs, pancreas, liver and intestine. It is characterized by the abnormal transport of chloride and sodium across the epithelium, leading to thick, viscous secretions. The seriousness of the disease is due to its particular mutation. For instance, the lack of the phenylalanine in position 508 (F508del-CFTR) is the most common mutation and leads to a very heavy phenotype. Correctors F508del-CFTR: Is recognized as incorrect and targeted for degradation by the cellular quality control system. Correctors are small organic molecules that are able to rescue the mutant protein that trafficks it from the endoplasmic reticulum to the plasma membrane. The correctors that are known to date belong to many different chemical classes and have various, although not wellclarified, mechanism of action. Pharmacophore: The smallest structural unit of a drug molecule that is responsible for its biological activity. It consists of a set of functional groups (also called pharmacophoric elements) suitably arranged in the three spatial dimensions that interact specifically with the biological target, giving rise to a biological response.

CFTR are in clinical trials at various stages. For example, ataluren (formerly PTC-124) is under evaluation in order to address the defect caused by class 1 mutations [13] . This molecule favors the readthrough of nonsense mutations, thus allowing the synthesis of full-length CFTR. The most frequent mutation, F508del (i.e., the deletion of phenylalanine 508), which belongs to the class 2 mutations, instead requires a different kind of drug, which are generically called correctors. In this case, the CFTR protein suffers from defective folding and stability, which impairs its trafficking from the endoplasmic reticulum to the plasma membrane. The mutant F508del-CFTR is recognized as an incorrect protein by the cell quality control system and is degraded by the ubiquitin/proteasome system [14] . Correctors may work by stabilizing the CFTR protein or by modifying the quality control system. The most advanced corrector so far is the VX-809 compound [15] . The third class of CF mutations instead causes an intrinsic defect in CFTR channel opening. These mutations require treatment with ‘potentiators’, a class of molecules that have been the most successful in the treatment of CF. The 4-quinolone VX-770, marketed as Kalydeco™ (Vertex Pharmaceuticals Inc., Boston, MA, USA),  was recently approved for the treatment of patients with the G551D mutation [16,17] .

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Because of the high frequency and great severity of the F508del mutation, the search of effective correctors is, at present, one of the most intensive research lines in CF. The majority of correctors were found using highthroughput strategies (i.e., by screening libraries made up of thousands of chemicals) [18,19] . A list of correctors was recently reviewed [9] . Unfortunately, many of the correctors that have been discovered so far show their activity only at high concentrations, thus resulting in toxic side effects at the cellular level. Nevertheless, they represent a proof of principle that small organic molecules may indeed rescue F508del-CFTR. Little is known about the mechanism of action of correctors [20] . Some authors affirm that correctors may bind to the mutant CFTR [21–23] . Other authors conclude that correctors act indirectly by modulating other proteins that are part of the CFTR interactome [24,25] . In the present work, we examine whether, when looking at some already-known correctors, a common chemical group acting as pharmacophores could exist or a shared structural motif that is able to drive the synthesis of new correctors could be found. Our investigation started from the examination of some 4-chloroanisole derivatives, as this group is present in several substances that are capable of rescuing F508delCFTR [18,26] . Then, since the 4-chloroanisole derivatives here tested were found to be inactive, our interest moved to the 2-(4-chloroanisol-2-yl)aminothiazole derivatives, as this moiety is present in some very active correctors [26–28] . Eventually, taking into account the results coming from the analysis of the 2-(4-chloroanisol-2-yl)aminothiazole derivatives, an interesting structural design for the activity of some correctors was found. The development of this structural concern led to a small set of new CFTR correctors endowed with a significant, although not great, activity. Chemistry The synthetic way of obtaining derivatives supporting a 4-chloroanisole group is shown in Figure 1 & Table 1. Briefly, benzylamine 1a (or phenylethylamine 1b) reacted with acetic anhydride in pyridine in order to give the acyl derivatives 2, which in turn were treated with ClSO3H in CHCl3 at 0°C and then refluxed for 30 min, obtaining the sulfonyl chlorides 3. The derivatives 3 were treated with secondary amines at 0°C and then at room temperature for 12 h, giving the amides 4. Then, 4 were hydrolyzed by NaOH at 120°C for 12 h, yielding the amines 5. The derivatives 5 reacted with 2-methoxy-5-chlorobenzoylchloride 7 (obtained from the acid 6 with SOCl2) in order to give the final compounds 8. Then, in order to provide new thiazoles bound through an amino bridge to a 4-chloroanisole moiety,

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Common structural moiety of selected pharmacological correctors of mutant CFTR 

we followed the pattern depicted in Figure 2 & Table 2, obtaining a number of thiazole derivatives. Briefly, ammonium thiocyanate was dissolved in acetone, treated with benzoyl chloride and then refluxed with 5-chloro-2-methoxyaniline in order to obtain 9. Then, 9 was hydrolyzed with 2N NaOH in order to obtain 10 and the latter was treated with ethyl-bromopiruvate in order to yield the thiazole derivative 11. The thiazole 11 was hydrolyzed with 2N NaOH, obtaining the acid 12. Treatment of 12 with a suitable amine in triethylamine in the presence of HBTU yielded the amides 13 and 14. As a consequence of the small, although significant activity found in the thiazole 11 (see the ‘Results’ and ‘Discussion’ section), new derivatives that were structurally related to some known correctors were synthesized. The pattern of the synthesis is outlined in Figure 3 & Table 3. In this case, the esters 17 were obtained from the bromopiruvic acid 15 and the benzyl alcohols 16. Then, by reacting the esters 17 with the intermediate 10 (Figure 2) , the final derivatives 18 were prepared. Experimental details Chemistry

All details concerning melting points, yields, analytical and spectral data are presented in the Supplementary Materials. AcHN-(CH2)n

H2N-(CH2)n

1a, b

General procedure for the synthesis of amide derivatives (2a, b)

To an ice-cooled solution of 80 mmol of benzylamine (1a) or phenylethylamine (1b) in 5 ml of pyridine, 100 mmol of acetic anhydride were added dropwise. The ice bath was removed and the mixture was allowed to stir at room temperature for 12 h. Then, the mixture was added with 15 ml of ethyl ether and then sequentially washed three times with 10 ml of the following dequence: water, 2N HCl, water, 2N NaOH, water. The organic solution was then dried on anhydrous sodium sulfate and evaporated under reduced pressure, obtaining a quite solid paste. This paste was then crystallized by cyclohexane in order to give the following compounds as white solids: N-benzylacetamide (2a) and N-phenylethylacetamide (2b). General procedure for the synthesis of sulfonyl chloride derivatives (3a, b)

To an ice-cooled solution of 45 mmol of amide 2 in 10 ml of chloroform, 135 mmol of chlorosulfonic acid were added dropwise. The mixture was allowed to stir at 0°C for 5 min, then at room temperature for 10 min and finally refluxed for 30 min. Then, by adding crushed ice, a crude solid was formed. The solid was filtered and crystallized by a suitable solvent in order to give the following compounds AcHN-(CH2)n

2a, b

COOH OCH3 6

AcHN-(CH2)n

SO2R

SO2Cl 3a, b

4a–c

Cl

Cl

Preliminary Communication

H2N-(CH2)n

COCl

OCH3 7

SO2R 5a–c Cl H N (CH2)n

CH3O

O

SO2R

8a–c

Figure 1. Synthetic way of obtaining 4-chloroanisole derivatives. Reagents and conditions: (A) Ac2O, Py, 0°C, then room temperature, 12 h; (B) ClSO3H, CHCl3, 0°C, then reflux, 30 min; (C) amines, ethyl acetate (EtOAc) , 0°C, then room temperature, 12 h; (D) 2N NaOH, reflux, 12 h; (E) SOCl2, 1,2-dichloroethane (DCE), 60°C, 2h; and (F) Py, 110°C, 1 h.

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Table 1. Compound

n

R

1a

1

–

1b

2

–

2a, 3a

1

–

2b, 3b

2

–

4a

1

4b

2

4c

2

5a

1

5b

2

5c

2

8a

1

8b

2

8c

2

N

N CH3

N

O

N

N CH3

N

N CH3

N

O

N

N CH3

N

N CH3

N

O

N

N CH3

as white solids: 4-(acetylaminomethyl)-benzenesulfonyl chloride (3a) and 4-(2-acetylaminoethyl)benzenesulfonyl chloride (3b). General procedure for the synthesis of sulfonamide derivatives (4a, b)

To an ice-cooled solution of 10 mmol of sulfonyl chloride 3 in 10 ml of ethyl acetate, 60 mmol of ammine were added. The mixture was allowed to stir at room temperature for 12 h. After the precipitation of a solid, 20 ml of water and 20 ml of chloroform were added. The organic phase was separated out and extracted three times with 2N HCl, then the acidic phase was neutralized with 2N NaOH until neutrality and extracted again with chloroform. The pooled organic extracts were dried on anhydrous sodium sulfate and evaporated under reduced pressure, giving a crude solid. The solid was then crystallized by ethyl acetate and cyclohexane in order to yield the following compounds as white crystals: N-(4-[4-methylpiperazine-1-sulfonyl]-benzyl)-acetamide (4a), N-(2[4-{morpholine-4-sulfonyl}-phenyl]-ethyl)-acetamide (4b) and N-(2-[4-{4-methyl-piperazine-1-sulfonyl}phenyl]-ethyl)-acetamide (4c).

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To 10 mmol of amide derivatives 4, 100 ml of 2N NaOH were added. The mixture was refluxed for 12 h and then, after cooling, extracted three times with 20 ml of chloroform. The pooled organic extracts were dried on anhydrous sodium sulfate and evaporated under reduced pressure, giving a crude solid. The solid was then crystallized by ethyl acetate in order to yield the following compounds as white crystals: 4-(4-methylpiperazine-1-sulfonyl)-benzylamine (5a), 2-(4-[morpholine-4-sulfonyl]-phenyl)-ethylamine (5b) and 2-(4-[4-methylpiperazine-1-sulfonyl]phenyl)-ethylamine (5c). Synthesis of 5-chloro-2-methoxy-benzoyl chloride (7)

To a solution of 10 mmol of 5-chloro-2-methoxycarboxylic acid (6) in 20 ml of 1,2-dichloroethane, 20 mmol of thionyl chloride were added. The mixture was allowed to stir at 60°C for 2 h. Then, the solvent was evaporated under reduced pressure, giving a gray precipitate. The solid was washed with diethyl ether and crystallized by cyclohexane to yield 7 as a white solid. General procedure for the synthesis of aryl amide derivatives (8a–c)

To a warm solution (110°C) of 10 mmol of amine 5, 15 mmol of benzoyl chloride 7 in 20 ml of pyridine were added. The mixture was allowed to stir at 110°C for 1 h; then, after cooling, 50 ml of dichloromethane were added. The organic solution was washed three times with water and three times with 2N HCl, then dried on anhydrous sodium sulfate and evaporated under reduced pressure, obtaining an oil. The oil was precipitated with ethyl acetate and the precipitate was then crystallized by ethyl acetate in order to yield the following compounds as white crystals: 5-chloro-2-methoxy-N-(4-[4methylpiperazine-1-sulfonyl]-benzyl)-benzamide (8a), 5-chloro-2-methoxy-N-(2-[4-{morpholine4-sulfonyl}-phenyl]-ethyl)-benzamide (8b) and 5-chloro-2-methoxy-N-(2-[4-{4-methylpiperazine1-sulfonyl}-phenyl]-ethyl)-benzamide (8c). 1-benzoyl-3-(5-chloro-2-methoxyphenyl)thiourea (9)

To a solution of 10 mmol of ammonium thiocianate in 10 ml of acetone, 10 mmol of benzoyl chloride were added dropwise. The mixture was refluxed for 30 min, giving a white suspension. Then, a solution of 10 mmol of 5-chloro-2-methoxyaniline in 10 ml of acetone was added. The mixture was refluxed for 30 min and then poured onto crushed ice, giving a precipitate. The solid

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Common structural moiety of selected pharmacological correctors of mutant CFTR 

was washed with water and methanol (1:1) and crystallized by methanol/acetone in order to yield 9 as a white solid.

OCH3

H3CO

NH2

Preliminary Communication

H N

H N S

N-(5-chloro-2-methoxyphenyl)thiourea (10)

To 10 mmol of amide derivative 9, 100 ml of 2N NaOH were added. The mixture was allowed to stir at 80°C for 3 h and then added with ice and 2N HCl in order to reach an acidic pH. Then the pH was corrected to 8.0 with NaHCO3, obtaining a flaky precipitate. The solid was filtered, washed with water and crystallized from cyclohexane in order to yield 10 as a white solid. 2-(5-chloro-2-methoxy-phenylamino)-thiazole-4carboxylic acid ethyl ester (11)

To a suspension of 10 mmol of thiourea derivative 10 in 40 ml of ethanol, 10 mmol of ethyl bromopyruvate were added. The brown mixture was allowed to stir at 65°C for 12 h, then cooled at room temperature, obtaining a white precipitate. The solid was filtered and crystallized by cyclohexane/acetone, yielding 11 as a white solid. 2-(5-chloro-2-methoxy-phenylamino)-thiazole-4carboxylic acid (12)

To a solution of 10 mmol of ethyl ester derivative 11, 20 ml of 2N NaOH were added. The mixture was allowed to stir at 65°C for 12 h, then evaporated under reduced pressure, diluted with water and acidified with 2N HCl, giving a precipitate. The solid was filtered and dissolved in NaHCO3 solution at 50°C. The mixture was allowed to stir for 1 h, filtered and acidified with 2N HCl in order to reach an acidic pH, obtaining a flaky solid. The solid was filtered and crystallized by ethyl acetate/acetone in order to give 12 as a yellow crystal. General procedure for the synthesis of thiazole derivatives (13, 14)

To a solution of 14.8 mmol of triethylamine in 5 ml of acetonitrile, 10 mmol of carboxylic acid derivative 12 were added and then 10.1 mmol of a suitable amine. When the mixture produced a homogeneous orange solution, 11.1 mmol of HBTU were added. The mixture was allowed to stir for 2 h at room temperature, then the precipitate was filtered and washed with acetonitrile, giving a precipitate. The precipitate was crystallized by acetone in order to obtain the following compounds as white crystals: 2-(5-chloro2-methoxy-phenylamino)-thiazole-4-carboxylic acid(2-[4-{morpholine-4-sulfonyl{-phenyl]-ethyl)-amide (13) and (2-[5-chloro-2-methoxy-phenylamino]thiazol-4-yl)-(3,4-dihydro-1H-isoquinolin-2-yl)methanone (14).

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Cl

H3CO

O

9

Cl

H3CO

H N

N

H N

COOC2H5

NH2 S

S

Cl

Cl

10

H N

N

11

H3CO

H3CO

H N

N

COOH

O R

S

S

Cl

Cl

13, 14

12

Figure 2. Synthetic way of obtaining 2-(5-chloro-2methoxy-phenylamino)thiazole derivatives. Reagents and conditions: (A) NH4CNS, PhCOCl, acetone, reflux, 30 min; (B) 2N NaOH, 80°C, 3 h; (C) C2H5OCOCOCH2Br, ethanol (EtOH) , 65°C, 12 h; (D) 2N NaOH, 65°C, 12 h; and (E) amines, triethylamine (TEA) , CH3CN, HBTU, room temperature, 2 h.

General procedure for the synthesis of bromoderivatives (17a–c)

To a warm solution (50°C) of 9.3 mmol of bromopyruvic acid 15 in 25 ml of toluene, 10 mmol of the suitable alcohol 16 and a catalytic quantity of p-toluensulfonic acid were added. The mixture was refluxed with a Dean–Stark apparatus for 1 h, then evaporated under reduced pressure, yielding a brown oil of the following compounds: 3-bromo-2-oxopropionic acid benzyl ester (17a), 3-bromo-2-oxopropionic acid 4-chlorobenzyl ester (17b) and 3-bromo-2-oxopropionic acid 4-nitrobenzyl ester (17c). General procedure for the synthesis of thiazole derivatives (18a–c)

To a suspension of 10 mmol of thiourea derivative 10 in 40 ml of ethanol, 10 mmol of bromoderivatives Table 2. Compound

R

13

O

HNCH2CH2

S

N

O

O

14

N

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O

O OH

Br

+

R

HOH2C

OCH2

Br

O 15

16a–c

17a–c

Cl

Cl S

H3CO

R

O

N H

+

O

S 17a–c

NH2

H3CO

10

N H

N

O

18a–c

R

Figure 3. Synthetic way of obtaining new thiazoles that are structurally related to some known correctors. Reagents and conditions: (A) p-toluensulfonic acid, toluene, reflux, 1 h; and (B) ethanol (EtOH) , 65°C, 12 h.

Table 3. Compound

R

16a, 17a, 18a

H

16b, 17b, 18b

Cl

16c, 17c, 18c

NO2

17 were added. The brown mixture was allowed to stir at 65°C for 12 h, then cooled at room temperature, obtaining a white precipitate. The solid was filtered and crystallized by cyclohexane/acetone, yielding white crystals of the following compounds: 2- (5-chloro-2-methox yphenylamino) -thia zole4-carboxylic acid benzyl ester (18a), 2-(5-chloro2 -me t hox y phe ny l a m i no ) -t h i a z ole - 4 - c a rboxylic acid 4-chlorobenzyl ester (18b) and 2- (5-chloro-2-methox yphenylamino) -thia zole4-carboxylic acid 4-nitrobenzyl ester (18c).

Biology CFTR assays

For CFTR assays, the CF bronchial epithelial cell line CFBE41o- was used [29] . All details concerning cell transfection [30] , cell culture, sample preparations and the measurement of CFTR activity by a fluorescence assay are in the Supplementary Materials. Molecular modeling

The images have been created with the molecular modeling software Chimera 1.7 running on Linux. Chimera

Table 4. Evaluation of compounds 8, 9, 11, 13, 14, 18, 19 and 22 on CFBE41o-F508del-CFTR cells. Compound Activity (QR with respect to DMSO)

Activity with respect to compound 19 (%)

8a

0.98 ± 0.12

0 ± 0.2

8b

0.97 ± 0.06

0 ± 0.1

8c

0.96 ± 0.05

0 ± 0.1

9

0.95± 0.07

0 ± 0.1

11

1.12 ± 0.09

14 ± 0.1

13

0.89 ± 0.11

0 ± 0.2

14

1.00 ± 0.14

0 ± 0.2

18a

1.31 ± 0.10

48 ± 0.2

18b

1.41 ± 0.06

63 ± 0.1

18c

1.08 ± 0.15

12 ± 0.2

19

1.65 ± 0.05

100 ± 0.1

22

1.29 ± 0.09

45 ± 0.1

Activity values are the mean ± standard error of the mean of five to ten experiments. DMSO: Dimethylsulfoxide; QR: Quenching rate.

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Preliminary Communication

Common structural moiety of selected pharmacological correctors of mutant CFTR 

is developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (CA, USA; supported by NIGMS 9P41GM103311). Results In Table 4, the effectiveness of compounds 8, 9, 11, 13, 14, 18, 19 and 22 are reported. Compounds 19 and 22 are already-known correctors named Corr. 4a and RDR-1, respectively [18,22] , but, due to its greater activity, 19 was used as the reference compound (100% activity). Note that in this first approach to the activity of a family of correctors similar to 19 (Corr. 4a), we did not use VX-809 (a corrector that is more potent than Corr. 4a) as the reference because VX-809 has a structure that is completely different from the structures of our concern, whereas in this case, we were interested in collecting data among homogeneous compounds. Whereas all tested compounds were administered at 20 μM, corrector 19 was administered at 10 μM, since at 20 μM, signs of cellular toxicity may be initiated. After incubation with correctors, the functional assay evaluating F508del-CFTR in the membrane was conducted in the presence of genistein as a potentiator. Indeed, the rescued F508del-CFTR also has a partial gating defect that requires treatment with a potentiator in order to generate full anion transport. Activity was calculated as the variation of fluorescence over time due to the influx of iodide through CFTR and the quenching of YFP fluorescence (fluorescence quenching rate). Data were normalized with respect to control DMSO; the compound activity in Table 4 shows how many times the compound is more efficient than DMSO. In addition, the percentage of Cl

OCH3

RDR-1: A phenylhydrazone compound that is reported to act as a corrector by binding to NBD1 in BHK and CFBE41ocells. RDR-1 acts as a pharmacological chaperone. YFP: Is the yellow fuorescent variant of GFP showing improved sensitivity to halides by a mechanism involving halide binding. It is suitable as a halide indicator for cellular applications such as the measurement of intracellular chloride concentrations and cell membrane halide transport.

activity with respect to the positive control corrector 19 was calculated. Discussion Correctors that have been discovered until now belong to various chemical classes. This fact may be related to the many mechanisms through which these molecules can act. Consequently, we desired to investigate whether, in some thiazoles able to rescue mutant CFTR, there could exist a moiety acting as a potential pharmacophore. In this approach to the question, our attention was pointed at the corrector 19 and its derivative 20 (Figure 4) . They belong to the class of bithiazoles and bear a 4-chloroanisole group at the end of the molecule. This class encompasses several very active correctors and was subjected to many studies [18,27,28] . Interestingly, other correctors that come from studies by the same research team also possess the 4-chloroanisole moiety. For example, the derivative 21 (Figure 4)

O

N

S S

N

Corr. 4a: Is a cell-permeable bisaminomethylbithiazole compound that is reported to rescue the folding defect of F508del-CFTR via direct interaction and promote proper trafficking and surface expression in both transfected cells and primary airway epithelial cells from F508del-CFTRhomozygous patients.

Cl

H3C

N H

Key terms

N

S

N H

OCH3

S

N

N H

O

CH3 C N CH3 H H3C

20

19

H3CO N

NH O

S

N N

O

CH3

Cl

C N CH3 H H3C

C H3CO

H3CO

Cl 21

H N

O

S O

H N

C

H N

O O

Glybenclamide

Figure 4. Structure of F508del-CFTR correctors.

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N

NH

N

O

N

H3C

H3C

responsible for acidity, consequently blocking the channel. Indeed, some glibenclamide derivatives 8 seemed to us to be very promising for the presence of a nonacidic phenylsulfonamide. In fact, such a substituent is present in other published correctors (e.g., VRT-422, VRT-325 [19] and sildenafil and its derivatives [32]). In particular, the compounds 8a and 8c contain moieties from glibenclamide (the left part of the molecule) and sildenafil (the right part of the molecule) (Figure 5) . Since glibenclamide-like nonacidic derivatives 8 were found to be devoid of activity as correctors, we started a synthesis program targeted on new compounds possessing a thiazole ring in addition to the 4-chloroanisole group. For this purpose, we based our syntheses on the structure of correctors (see formulae 19 and 20) in which the 4-chloroanisole moiety is connected to a thiazole via an amino bridge. The compounds so obtained possessed the following structures (Figure 6) . Unfortunately, the derivatives 13 and 14 were also ineffective as F508del-CFTR correctors. Although our interest was primarily addressed to more elongated molecules such as 8, 13 and 14, which have better possibilities for forming bonds with the target, we also tested the intermediates 9 and 11 as potential F508del-CFTR correctors, both of which contain the 4-chloroanisole and thiourea moieties. While the open intermediate 9 was inactive, we found that the heterocyclic intermediate 11 was the only derivative that was endowed with an activity slightly higher than the blank sample (cells plus DMSO). As it is easy to ascertain, the thiazole derivative 11 is very similar to the left part of corrector 19. Therefore, wanting to speculate about some new correctors that could maintain the 4-chloroanisole moiety and the

O

H 3C

S

O N N

O

CH3

8a, X = CH2 8c, X = CH2CH2 Cl

O H N

H3CO

S

O N N

X

CH3

O Sildenafil

Figure 5. Structure of sildenafil and its derivative.

is endowed with notable correction activity against the F508del-CFTR [26] . Starting from the above reports and wanting to employ a new scaffold for our derivatives in order to prove whether the presence of the 4-chloroanisole group was intrinsically important for the F508delCFTR correction, our interest was driven by the observation that such a group is also present in glibenclamide, a known CFTR blocker. Glibenclamide produces a CFTR blockade as its acidic moiety (the hydrogen of the sulfonylurea group) and binds the basic amino acids of the channel [31] . Therefore, there was a strong interest in testing the role of the 4-chloroanisole group in terms of modifying a molecule endowed with an already-verified tropism for the CFTR. Accordingly, we first prepared some compounds that were similar to glibenclamide and ending with a 4-chloroanisole moiety, but removing the ureic group

Cl

N H

N

H N CH2CH3

OCH2CH3

OCH3

N H

11

O S N

O

O

N

O

S

OCH3

O

Cl

S

13

O

Cl

N

N

OCH3

N H

S 14

Figure 6. Structure of thiazole analogs.

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Common structural moiety of selected pharmacological correctors of mutant CFTR 

thiazole ring, we reconsidered the structures of some correctors. In particular, we focused our attention on the already-mentioned correctors 19, 20 and 21 and the furan-based corrector 22 (Figure 7), since it is possible to note that the superimposition of those molecules leads to a common structural motif (Figure 8) . On this ground, our final purpose was to modify the structure of 11 in order to produce a suitable moiety that was capable of mimicking the common motif of the above correctors. Thus, by a new synthetic route, a small set of compounds 18a–c was realized. Compounds 18a–18c maintain in the left part a structural moiety that is identical to corrector 19 and in the right part a moiety that is similar to the corrector 22. Figure 9 shows the superimposition of 19, 20, 21, 22 and 18a. As it is possible to note from Table 4, 18 possesses a clear, although not very high, action in the rescuing of F508del-CFTR. Such an action in the best compound of the series (18b) is more than 60% with respect to the reference corrector 19. Conclusion The attempt to produce F508del-CFTR correctors from glibenclamide-like nonacidic derivatives 8, depending on the presence of the 4-chloroanisole group, was unsuccessful. In addition, when the 4-chloroanisole group was linked to a thiazole, as in derivatives 13 and 14, there was no evidence of a positive action on mutant CFTR. Consequently, even if we can rely on a small set of derivatives, the 4-chloroanisole group does not seem to be endowed per se with significant activity as a potential pharmacophore for the F508del-CFTR correction. The appearance of a small mark of activity in the small molecule 11 is, in our opinion, a sign of the inherent value of the thiazole ring in such a type of activity, as extensively demonstrated by other authors [18,26–28] . If this assumption is correct, we must deduce that, in derivatives 13 and 14, the right-hand section of the molecule (namely, the part of the molecule over the thiazole) is completely wrong for fitting into the still-unknown binding site of correctors and, therefore, the little activity present in 11 is missed. These results prompted us to reconsider our initial thoughts and look at the thiazole derivative 11 as a compound worthy of theoretical concern: if the far left-hand section of 11 (the 4-chloroanisole) has little importance for its activity, great attention must be devoted to the thiazole and to the extension of the right-hand part of the molecule. Thus, when looking at some CFTR correctors, a structural relationship was found, as shown in Figure 8. From this common structural design, some new thiazoles were easily synthesized, and their biological tests gave a sig-

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H N

N

O

Preliminary Communication

NO2 22

Figure 7. Structure of furan corrector.

Figure 8. Superimposition of 19 (green), 20 (blue), 21 (magenta) and 22 (yellow). For color images please see online www.future-science. com/doi/full/10.4155/FMC.14.118.

Figure 9. Superimposition of 19 (green), 20 (blue), 21 (magenta), 22 (yellow) and 18a (red). For color images please see online www.future-science. com/doi/full/10.4155/FMC.14.118.

nificant, although not great, correction of the F508delCFTR basic defect. Therefore, it is possible to make the following remarks regarding the correctors presented herein: in order to provide a correction for F508del-CFTR, a molecule must possess a central part with a structural design as in Figures 8 & 9. The two heterocycles (as in 19, 20 and 21) are not strictly necessary since the presence of one ring is sufficient (as in 18 and 22), although from our biological results, the monocyclic compounds do not attain the same level of activity with respect to the bicyclic corrector 19. The hydrophobic ending parts may be highly variable and probably contribute to a better Key term Glibenclamide: Glibenclamide is also named ‘glyburide’. It is an oral antidiabetic drug belonging to the class of sulfonylureas. Glibenclamide is an ATP-sensitive potassium channel inhibitor. Glibenclamide is also a tool in cystic fibrosis research; in fact, it causes a concentrationdependent decrease in the open time of the CFTR chloride channel, resulting in the blockage of the channel.

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Ar

N

Y

X

R

23

Figure 10. Theoretical structure of F508del-CFTR corrector.

Cl S

N

OCH3

N

S

N H

24a

O

CH3 C N CH3 H H3C

24

Cl

H3C H3C

O C

CH3

S

N N

N H

S 24b

N H

OCH3

Figure 11. Theoretical structure of F508del-CFTR corrector.

F3C

NH2

N

N N

N CH3

O

C F3C

OH

Figure 12. Pyrazine derivative patented by Novartis.

Figure 13. Superimposition of 19 (green), 20 (blue), 21 (magenta), 22 (yellow), 18a (red) and pyrazine derivative (black). For color images please see online www.future-science. com/doi/full/10.4155/FMC.14.118.

fitting of the whole molecule at the binding site. These results enable the opportunity of identifying a theoretically smallest molecular structure for this type of correction. Such a structure is represented by the formula ofr 23 in which the position of the nitrogen and that of a second hetero atom (X = O or S) seems to be mandatory, whereas Y is represented by a nitrogen that is

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sometimes located outside of the cycle (as in 18, 19, 20 and 21) or inside of the cell (as in 22) (Figure 10) . In this connection, a final observation seems to us to be very valuable for corroborating our structural suggestion for this type of F508del-CFTR correction activity. In the paper in which the locked corrector 20 was presented, the authors very brilliantly also described 24, with the heterocyclic atoms inverted [28] . They found that 24 was devoid of correction activity. For our convenience, we call the same molecule 24a and 24b only to show the two spatial dispositions, which the structure 24 can be assumed to bind to a putative receptor (Figure 11). Following our proposal, it is evident that if we consider the disposition as in 24a, nitrogen and sulfur are in an uncorrected position in order to produce the expected action, whereas in the case of the disposition 24b, although nitrogen and sulfur are in a corrected position, the molecule is lacking the aromatic section, which must be present at the left-hand end. The substituents in the phenyl group (Cl and OCH3) are also probably unsuitable for an appropriate binding to the receptor, as the group located in this part of the more active correctors (19 and 20) is an unsubstituted phenyl or alkyl. In conclusion, the correction activity in this series of compounds derived from the corrector 19 seems to be due to a quite fixed central section and more variable ending sections. The left-hand ending part of the template 23 probably accepts larger aromatic groups than a simply substituted phenyl, such as the 4-chloroanisole, whereas the right-hand ending part probably also accepts alkyl groups. In order to corroborate our findings, during the assembly of this work, an additional collection of correctors was patented by Novartis (Switzerland) [33] . Many correctors are picolinamide-based derivatives, but the most active compound of this family is a pyrazine derivative (Figure 12) endowed with relevant potency (EC50 = 1.4 nM). As is easy to observe, this corrector is composed of a five-membered cycle (oxadiazole) and a six-membered cycle (pyrazine) and therefore presents a structural difference from the aforementioned correctors. Nevertheless, as shown in Figure 13, the position of nitrogen and oxygen matches our previous suggestions, confirming that this central design could be a possible signature for F508del-CFTR correction activity. Future perspective All of the compounds here show a molecular signature that is probably able to confer F508del-CFTR correction activity. Future research in this field, together with growing knowledge of binding sites inside CFTR or chaperone/co-chaperone systems, will pro-

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Common structural moiety of selected pharmacological correctors of mutant CFTR 

vide proof of whether the structural design outlined in this article could contribute to the synthesis of new substances that are clinically useful for CF patients. In this regard, we are now synthesizing new and more potent phenylhydrazones derived from RDR-1 [34] . Supplementary data To view the supplementary data that accompany this paper please visit the journal website at www.future-science.com/ doi/full/10.4155/FMC.14.118.

Preliminary Communication

Financial & competing interests disclosure This work was supported by Italian Cystic Fibrosis Foundation (Project FFC#5/2010) with the financial support of P Watch–Morellato and Sector Group. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

Executive summary Background • The F508del-CFTR is a mutation that leads to a very dangerous form of cystic fibrosis. • Small organic molecules (correctors) may rescue the mutant protein from degradation. Some correctors bear a 4-chloroanisole group.

Chemistry & biology • First, by taking in account the structure of glibenclamide (a CFTR blocker carrying the 4-chloroanisole group) as a model, many glibenclamide-like nonacidic derivatives were synthesized. Such derivatives were found to be inactive as correctors. • Second, the 2-(4-chloroanisol-2-yl)aminothiazole moiety was thought to have pharmacophoric features for F508del-CFTR correction, but the derivatives obtained by such a design were also inactive.

Molecular modeling • The investigation was targeted towards some correctors (possessing or not possessing the 4-chloroanisole moiety) in which a common structure could be recognized. • The superimposition of such correctors showed a clear common moiety. This structural design may help with the search for new correctors.

Results • On these grounds, it was possible to synthesize a small set of new CFTR correctors endowed with a significant, although not great, activity. • Such correctors possess the 4-chloroanisole group, but in our opinion, this group is not essential.

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