Patent Application
20220323425
This disclosure is related to potent potentiators for use in treating cystic fibrosis caused by minimal function fibrosis transmembrane conductance regulator (CFTR) mutants.
Cystic fibrosis (CF) is caused by loss of function mutations in the CFTR gene that affect the production of the CFTR protein, a cAMP-activated chloride channel. More than 2000 CF-causing CFTR gene mutations have been identified (1).
Small-molecule CFTR modulators have been developed that rescue defective cellular processing and cell-surface targeting of mutant CFTRs (correctors) or rescue defective channel gating to restore CFTR anion transport (potentiators). (1-3) The potentiator Kalydeco (ivacaftor/VX-770) has been approved for CF subjects with gating mutations, including G551D-CFTR and now 38 additional mutations (2). The corrector/potentiator combinations Orkambi (VX-770 plus lumacaftor/VX-809) and Symdeko (VX-770 plus tezacaftor/VX-661) have been approved for CF subjects that are homozygous for the most common CF-causing CFTR mutation, F508del, or who have one F508del allele and a residual function CFTR mutation (2). Triple drug combinations, consisting of two correctors and one potentiator, have shown additional benefits in clinical trials and may soon be approved for CF subjects with one F508del allele and a second CFTR allele carrying any mutation (2, 4-6). Current and improved so called “next-generation” therapeutics are promising for treating up to 90% of all CF subjects (2).
However, certain CFTR mutations (about 10% of CF subjects) appear to be refractory to available potentiators and correctors. Non-responsive minimal function CFTR mutations are distributed throughout the CFTR protein and are associated with low CFTR function due to defective channel processing, cell-surface trafficking, and/or channel gating. One such minimal function CFTR mutation is N1303K, a missense point mutation located in nucleotide binding domain 2 (NBD2), which is the 5th most common CFTR mutation worldwide accounting for ˜2.5% of CFTR mutations (www.cftr2.org). Other minimal function missense CFTR mutants are found in membrane spanning domain (MSD) 1, including G85E and R334W, and MSD2, including L1077P and M1101K. These four CFTR mutants are found in ˜1.4% of ˜88,000 CF subjects in the CFTR2 database. Premature termination codon (PTC) mutations also have no available therapy, including G542X located in NBD1 and W1282X located in NBD2, which are the 2nd and 4th most common CFTR mutations (5% and 4% allele frequency in CFTR2 database, respectively).
It has been previously reported that VX-770, when used in combination with a second potentiator (ASP-11), increased chloride channel function of N1303K-CFTR and the truncated W1282X-CFTR protein product by ˜8-fold compared with VX-770 alone (9, 10). This combination potentiator (or co-potentiator) approach was also shown to be effective in increasing the chloride channel function of G551D-CFTR, with ˜50% improvement compared with VX-770 alone (10).
There remains a need in the art for improved therapy for treating cystic fibrosis caused by minimal function CFTR mutations.
Provided herein are methods of using co-potentiators for treating CF subjects having minimal function CFTR mutants and methods of identifying by high-throughput screening, novel co-potentiator scaffolds with nanomolar potency.
As discussed in more detail herein, the co-potentiators are classified into two classes depending on their respective mechanisms of action on the CFTR protein. Briefly speaking, Class I potentiators include the classical potentiators such as VX-770 or GLPG1837 (see
One embodiment thus provides a method of treating cystic fibrosis in a CF subject having at least one missense, nonsense, deletion, or truncation mutation, the method comprising administering at least one Class I potentiator and at least one Class II potentiator to the subject, wherein the Class II potentiator has one of the following structures (A), (B), (C), or (D):
wherein:
m, n, R1a, R2a, R3a, R4a, R1b, R2b, R1c, R2c, R1d, R2d, R3d, R4d, R5d, and R6d are as defined herein.
In the figures, identical reference numbers identify similar elements or acts. The sizes and relative positions of elements in the figures are not necessarily drawn to scale. For example, the shapes of various elements and angles are not drawn to scale and some of these elements are enlarged and positioned to improve figure legibility. Further, the particular shapes of the elements as drawn, are not intended to convey any information regarding the actual shape of the particular elements, and have been solely selected for ease of recognition in the figures.
Disclosed herein are embodiments generally directed to a therapeutic strategy for treating CF subjects, in particular, those with CFTR mutations that do not respond significantly to the available CFTR modulators such as Kalydeco, Orkambi and Symdeko. More specifically, novel co-potentiator scaffolds with drug-like properties and EC50 down to 500 nM or even 300 nM have been identified. It has been observed that compounds of these scaffolds act in synergy with VX-770 or other Class I potentiators to increase CFTR chloride current. The potentiator/co-potentiator paradigm disclosed herein is therefore effective for treating CF subjects having a variety of missense, nonsense, and deletion mutations specifically in NBD2 of CFTR.
According to various embodiments, CF subjects with certain mutations are treated by a combination therapy comprising at least two distinct potentiators. The potentiators are distinct in their mechanisms of actions (e.g., targeting distinct binding sites or regions on CFTR mutants); and the additive or synergistic effects can benefit CF subjects that do not respond significantly to the available drugs. A classification system defining these distinct potentiators is discussed herein.
One of the first reported co-potentiators, arylsulfonamide-pyrrolopyridine (ASP-11) (structure shown in
To investigate the CFTR mutational space specificity for ASP-11, 12 additional minimal function CFTR mutants were studied (
As shown in
Compounds that promote read-through of PTCs to generate full-length CFTR have shown limited efficacy in cell culture studies (14-16). Though the read-through drug ataluren (PTC124) was ineffective in clinical trials (17), newer compounds are in development. The ability of ASP-11 to activate chloride current in predicted W1282X-CFTR read-through products was investigated. G418 action on W1282X was found to insert mainly leucine or cysteine at position 1282 (18), and ataluren to insert arginine (16). FRT cell lines were generated that stably expressing W1282L-, W1282C- and W1282R-CFTR. W1282L-CFTR showed robust forskolin stimulation with little additional VX-770 or ASP-11 effect (
Without wishing to be bound by theory, the applicant hypothesizes that distinct binding sites may be required to bind a potentiator and co-potentiator to rescue channel activity of CFTR mutants. Thus, depending on distinct mechanisms of action or distinct binding sites targeted, CFTR potentiators are classified into Class I and Class II compounds. As an example, potentiator VX-770 is a Class I compound, and co-potentiator ASP-11 is a Class II compound (see also
Studies on FRT cells expressing Q1313X-CFTR produced similar data (
Class I potentiators may include those compounds that thermodynamically alter the closed-open equilibrium of CFTR via binding to the CFTR mutants. VX-770 is a classic example of Class I potentiators. VX-770 is currently approved for CF subjects with one copy of one of 38 mutations located throughout the CFTR sequence, including MSD1 (e.g. Di 10H, E193K), MSD2 (A1067T, R1070W), NBD1 (G551D, D579G), NBD2 (G1244E, G1349D), and other regions (E56K, P67L in the lasso domain) (2, 21, 22). Based on in silico docking and mutagenesis studies, two potential binding sites were identified for the Class I compounds VX-770 and GLPG1837 (23). The putative binding sites are located at the interface of the CFTR transmembrane domains involving residues D924, N1138 and S1141, or residues F229, F236, Y304, F312 and F931 (23). Recently, cryo-electron microscopy confirmed that VX-770 and GLPG1837 bind at the same site within the protein-lipid interface in a pocket formed by transmembrane helices 4, 5 and 8 (24). Evaluation of potential binding sites by alanine substitution revealed that a network of residues (including F236, Y304, F312 and F931, as well as L233, F305 and S308) interact directly with both VX-770 and GLPG1837 (24). Using pharmacological approaches, evidence was found that several previously reported potentiators including P2, P3 and P5 also bind at or near the VX-770 and GLPG1837 binding site. Given the broad efficacy of VX-770 for mutations throughout the CFTR protein, and the absence of large difference in CFTR structure with vs. without bound potentiator, Class I compounds such as VX-770 thermodynamically alter the closed-open equilibrium of CFTR (24).
Accordingly, exemplary Class I potentiators include, but are not limited to VX-770, P2, P3, P5, and GLPG1837. The structures of Class I potentiators are shown below (see also
VX-770 has the following structure:
P2 has the following structure:
P3 has the following structure:
P5 has the following structure:
GLPG1837 has the following structure:
Class II potentiators may bind to CFTR in a manner that stabilizes partial or misfolded NBD2 structurally or thermodynamically. When used as a co-potentiator with a Class I potentiator, a Class II compound is capable of acting in synergy to increase channel activity or otherwise rescue gating defects in CFTR mutants.
Given the utility of co-potentiators as possible CF therapeutics for several minimal function CFTR mutants, a screen was done to identify novel co-potentiator scaffolds. Screening used FRT cells stably expressing W1282X-CFTR and the halide-sensitive EYFP-H148Q/I152L/F46L (YFP) that were treated for 24 hours with 3 μM VX-809 to increase CFTR1281 cell surface expression (
The most active compounds included spiro[piperidine-4,1pyrido[3,4-b]indole] (CP-A01), phenylazepine (CP-B01), tetrahydroquinoline (CP-C01) and pyrazoloquinoline (CP-D01) (see
To establish structure-activity relationships, 240 commercially available spiro[piperidine-4,1pyrido[3,4-b]indoles]analogs and 160 pyrazoloquinoline analogs were tested in FRT cells expressing W 1282X-CFTR.
In general, as shown in Table 1A, the methoxy substituent on the 4-position (R3) on the pyridoindole ring increased potency (compare A01 vs A534). N-methylation on the pyridoindole ring abolished activity (compare A534 vs A600). For substituent R1 on the piperidine ring, substituted benzyl gave greatest activity (A061 and A662). Other R1 substituents, including sulfonamide (A145), alkyl (A764) and carbocyclic (A714), reduced activity. Changing the benzylic carbon from methylene (CH2) to ketone (C═O) abolished activity (A815, A350 and A956). A061 with R1 being 2,4-difluoro-benzyl was the most potent analog.
Based on the structure activity data, novel spiro[piperidine-4,1pyrido[3,4-b]indoles] compounds were synthesized according to the following Synthetic Scheme (I) and screened for their activities.
Table 1B summarizes certain novel spiro[piperidine-4,1pyrido[3,4-b]indoles] compounds suitable as co-potentiators with 2-17 folds of improvement.
As shown in Table 2, the position of the methoxy on the quinoline ring affected activity as changing from the 4th to 5th position greatly reduced potency (D038 vs D138). Replacing the electron-donating methoxy group to electron-neutral methyl group also reduced activity (D010 vs D136). For R3, pyridine (D003), benzyl (D035) and substituted methyl-pyrazole (D086) abolished activity. Substituted benzenes had a range of potencies with 2,4-disubstituted compounds including 2-chloro-4-fluorobenzene (D018) and 2-chloro-4-nitrobenzene (D038) being the most potent. The D123 pyrazoloquinoline with R3 substituted with thiophene-quinoline heterocycle gave the best potency.
Short-circuit current measurements were done for the most potent spiro[piperidine-4,1pyrido[3,4-b]indole] (CP-A061) and pyrazoloquinoline (CP-D123).
To test the efficacy of new co-potentiators in human airway cell models, short-circuit current was measured in 16HBE14o-human airway epithelial cells in which the endogenous CFTR gene was edited to contain the N1303K mutation (16HBE-N1303Kge, (20)) and in primary cultures of human bronchial epithelial cells from a N1303K homozygous CF subject. Addition of forskolin and then VX-770 to 16HBE-N1303Kge cells gave a limited response (
Thus, according to certain embodiments, the Class II potentiators are spiro[piperidine-4,1pyrido[3,4-b]indole] derivatives represented by Structure (A):
wherein:
m is 0, 1, 2 or 3;
R1a is optionally substituted arylalkyl, optionally substituted heteroaryl or optionally substituted heteroarylalkyl;
R2a is H or C1-C6 alkyl;
R3a is H, halo or C1-C6 alkoxy; and
R4a is H, C1-C6 alkoxy or C1-C6 alkyl.
In more specific embodiments, the Class II potentiators are Compounds A01, A061, A662, A666, A357 or A534 of Table 1A and Compounds 1j, 2d, 2e, 2g, 2i, 5c and 5d of Table 1B.
In other embodiments, the Class II potentiators are phenylazepine derivatives represented by Structure (B):
wherein:
n is 1 or 2;
R1b is a 5- or 6-membered heteroaryl; and
R2b is an optionally substituted arylalkyl.
In a more specific embodiment, the Class II potentiator is Compound CP-B01 (of
In certain other embodiments, the Class II potentiators are tetrahydroquinoline derivatives represented by Structure (C):
wherein:
R1c is a 5- or 6-membered heteroaryl; and
R2c is C1-C6 alkyl.
In a more specific embodiment, the Class II potentiator is Compound CP-C01 (of
In yet other embodiments, the Class II potentiators are pyrazoloquinoline derivatives represented by Structure (D):
wherein:
R1d is H, C1-C6 alkyl, or C1-C6 alkoxy;
R2d is H or C1-C6 alkoxy;
R3d is substituted aryl, or substituted heteroaryl; and
R4d, R5d, and R6d are independently H, C1-C6 alkyl, or C1-C6 alkoxy.
In a more specific embodiment, the Class II potentiator is Compound D01, D018, D038 or D123 of Table 2.
The co-potentiator scaffolds described herein are shown to have nanomolar potency that, in synergy with Class I potentiators such as VX-770, are capable of activating CFTRs with NBD2 mutations including N1303K-CFTR. Thus, potentiator/co-potentiator combination therapy may be effective in a subset of minimal function missense, nonsense and deletion mutations in CFTR that cause cystic fibrosis and are not responsive to current CFTR modulator combinations.
In particular, CF subjects of a variety of missense, nonsense and deletion mutations in NBD2, including N1303K- and I1234del-CFTR, can benefit from two distinct potentiators. In prior studies on the responses of >50 rare CFTR missense mutations to VX-770 and VX-809, N1303K-CFTR was not responsive to VX-770 and showed very limited response to VX-809 (25). This is consistent with the notion that the N1303K mutation causes defective CFTR folding, regulation and gating (26). Han et al. (2018) reported diverse responses to CFTR modulators—some mutations (P5L, G27R, S492F, Y1032C) responded to VX-809 but not VX-770, some (M348V) to VX-770 but not VX-809, and some (G85E, R560T, A561E, Y563N) with no response. In contrast, robust activation of N1303K-CFTR is observed with co-potentiators in the absence of a corrector in a human airway epithelial cell lines expressing endogenous levels of gene-edited CFTR (
It is difficult to estimate the number of CF subjects that might benefit from co-potentiator therapy. The N1303K allele is found in 2,147 subjects in the CFTR2 databases, of which 99 are homozygous and >400 would not be benefitted by VX-770 or therapies that targeting one F508del-CFTR allele. Similarly, c.3700 A>G is found in 28 subjects, of which 5 are homozygous. It is noted that many countries in which N1303K and c.3700 A>G are prevalent do not contribute to the CFTR2 database (27, 28). We previously showed that ASP-11 activates G551D-CFTR, as do the new co-potentiators identified here (not shown). The G551D allele is found in ˜3000 CF subjects in CFTR2, including 69 homozygous subjects. In addition, the co-potentiators were effective in increasing CFTR chloride current for several truncated forms of CFTR resulting from premature termination codons (PTCs) located in NBD2. PTCs result in nonsense-mediated degradation (NMD) of transcript resulting in reduced synthesis of truncated protein products (29, 30). CFTR transcript levels have been reported from ˜10-75% of levels in non-CF cells (31, 32), though one study reported complete absence of W1282X-CFTR transcript in cells from a single CF subject (33). Co-potentiators may thus be therapeutically beneficial for PTCs in NBD2, alone if sufficient transcript is present, or in combination with other drugs such as NMD inhibitors or read-through agents.
Provided herein is a method of treating cystic fibrosis in a CF subject having one or more CFTR missense, nonsense and deletion mutations, the method comprising administering a Class I potentiator and at least one Class II potentiator to the CF subject, wherein the Class II potentiator has one of the structures (A), (B), (C), or (D), as described herein.
In certain embodiments, the missense, nonsense, deletion, or truncation mutation is in NBD2, including for example, N1303K, G542X, W1282X, G551D, I1234del-CFTR, Q1313X, and c.3700 A>G. In certain more specific embodiments, the missense, nonsense, deletion, or truncation mutation is selected from the group consisting of N1303K, W1282X, and G551D. In certain specific embodiments, the missense, nonsense, deletion, or truncation mutation is N1303K.
In some embodiments, the Class I potentiator is selected from the group consisting of VX-770, P2, P3, P5, and GLPG1837.
In certain embodiment, the Class II potentiator is a spiro[piperidine-4,1pyrido[3,4-b]indole] derivative represented by Structure (A):
wherein:
m is 0, 1, 2 or 3;
R1a is optionally substituted arylalkyl, optionally substituted heteroaryl or optionally substituted heteroarylalkyl;
R2a is H or C1-C6 alkyl;
R3a is H, halo or C1-C6 alkoxy; and
R4a is H, C1-C6 alkoxy or C1-C6 alkyl.
In some more specific embodiments, R1a is an optionally substituted arylalkyl (e.g., benzyl). In more specific embodiments, In some embodiments, R1a is unsubstituted benzyl. In some embodiments, R1a is a substituted benzyl. In some specific embodiments, R1a is substituted with one or more substituents selected from the group consisting of halo, and C1-C6 alkoxy. In some embodiments, R1a is substituted with one or more substituents selected from the group consisting of methoxy, and fluoro. In preferred embodiments, R1a is benzyl in which the phenyl moiety is substituted with two or more halo (e.g., fluoro). In some embodiments, R1a is selected from the group consisting of benzyl, 3-methoxy-benzyl, 2,4-difluoro-benzyl, 3,4-difluoro-benzyl, 3-chloro-2,4-difluoro-benzyl, 3,4,5-trifluoro-benzyl, perfluoro-benzyl, 2,3,4-trifluoro-benzyl, 2,4,5-trifluoro-benzyl.
In some embodiments, R1a is an optionally substituted heteroaryl, including for example, optionally substituted thiazolyl.
In other embodiments, R1a is an optionally substituted heteroarylalkyl. In some embodiments, R1a is a heteroarylalkyl substituted with alkyl. In some embodiments, R1a has the following structure:
In certain embodiments, R2a is H. In some embodiments, R2a is C1-C6 alkyl (e.g., methyl, ethyl, propyl). In more specific embodiments, R2a is methyl.
In some embodiments, R3a is H. In some specific embodiments, R3a is C1-C6 alkoxy (e.g., methoxy, ethoxy, propoxy) or halo (e.g., fluoro or chloro). In more specific embodiments, R3a is methoxy or chloro.
In preferred embodiments, m is 0.
In other embodiments, m is 1, and R4a is C1-C6 alkoxy (e.g., methoxy, ethoxy, propoxy).
In some embodiments, the compound of structure (A) has one of the following structures:
In some embodiments, the Class II potentiator has the following structure (B):
wherein:
n is 1 or 2;
R1b is a 5- or 6-membered heteroaryl; and
R2b is an optionally substituted arylalkyl.
In some specific embodiments, R1b is a 5-membered heteroaryl. In certain embodiments, R1b is unsubstituted. In some specific embodiments, R1b comprises sulfur.
In certain embodiments, R1b has the following structure:
In some embodiments, R2b benzyl. In certain embodiments, R2b is substituted. In more specific embodiments, R2b is substituted with chloro. In some embodiments, R2 has the following structure:
In some embodiments, n is 1.
In some specific embodiments, the Class II potentiator has the following structure:
In some embodiments, the Class II potentiator has the following structure (C):
wherein:
R1c is a 5- or 6-membered heteroaryl; and
R2c is C1-C6 alkyl.
In certain embodiments, R1c is a 5-membered heteroaryl. In other embodiments, R1c is unsubstituted. In some specific embodiments, R1c comprises sulfur. In some embodiments, R1c has the following structure:
In more specific embodiments, R2c is n-propyl.
In some specific embodiments, the Class II potentiator has the following structure:
In yet other embodiments, the Class II potentiator is a pyrazoloquinoline derivative represented by Structure (D):
wherein:
R1d is H, C1-C6 alkyl, or C1-C6 alkoxy;
R2d is H or C1-C6 alkoxy;
R3d is substituted aryl, substituted heteroaryl; and
R4d, R5d, and R6d are independently H, C1-C6 alkyl, or C1-C6 alkoxy.
In some embodiments, R1d is H. In some embodiments, R1d is C1-C6 alkyl (e.g., methyl, ethyl, propyl). In some more specific embodiments, R1d is methyl. In preferred embodiments, R1d is C1-C6 alkoxy (e.g., methoxy, ethoxy, propoxy). In more specific embodiments, R1d is methoxy.
In certain embodiments, R2d is H. In other embodiments, R2d is C1-C6 alkoxy (e.g., methoxy, ethoxy, propoxy). In more specific embodiments, R2d is methoxy.
In some embodiments, R3d is substituted aryl. In some specific embodiments, R3d is substituted phenyl having one or more substituents selected from the group consisting of halo, C1-C6 alkoxy, C1-C6 alkyl, nitro, and heteroaryl. In some embodiments, R3d is selected from the group consisting of 2-chlorobenzene, 4-fluorobenzene, 3,4,5-trimethoxybenzene, 2-chloro-4-fluoro-benzene, 3-methyl-4-nitro-benzene, 3-nitro-4-chloro-benzene, 2-chloro-4-nitro-benzene, 4-fluorobenzene, and 2-chloro-4-nitro-benzene.
In some embodiments, R3d is substituted heteroaryl. In further amendments, R3d is substituted bicyclic heteroaryl. In more specific embodiments, R3d is a substituted quinolinyl. In other more specific embodiments, R3d is a substituted thienyl. In some embodiments, R3d is a substituted quinolinyl (e.g., substituted with one or more substituents selected from the group consisting of halo, C1-C6 alkoxy, C1-C6 alkyl, nitro, and heteroaryl). In more specific embodiments, R3d is quinolinyl substituted with one or more substituents selected from the group consisting of chloro, fluoro, methoxy, methyl, nitro, and thiophenyl. In more specific embodiments, R3d is quinolinyl substituted with thiophenyl. In some embodiments, R3d has the following structure:
In some embodiments, the compound of structure (D) has one of the following structures:
In some embodiments, the CFTR co-potentiator therapy disclosed herein can be combined with one or more additional CFTR modulators. Thus, one embodiment provides a method for treating cystic fibrosis in a CF subject having one or more CFTR missense, nonsense and deletion mutations, the method comprising: administering a Class I potentiator and at least one Class II potentiator to the CF subject, wherein the Class II potentiator has one of the Structures (A), (B), (C), or (D), and administering to the CF subject with a further CFTR modulator.
In more specific embodiments, the one or more additional CFTR modulator may be a corrector, an amplifier, a read-through agent, or a combination thereof. As used herein, a CFTR corrector refers to a compound that increases the amount of functional CFTR protein to the cell surface, resulting in enhanced ion transport. Examples of suitable correctors include, without limitation, VX-809, VX-661, VX-983, VX-152, VX-440, VX-445, VX-659, GLPG2222, GLPG3221, GLPG2737, GLPG2851 and/or GLPG2665, PTI-801.
In other embodiments, the additional CFTR modulator is an amplifier, which acts to increase the amount of CFTR protein produced by the cells. With increased production of CFTR protein, potentiators and/or correctors would be able to allow even more chloride to flow across the cell membrane. Examples of suitable amplifier include, without limitation, PTI-428. Additional examples may be found in WO2017/223188, which reference is incorporated herein by reference in its entirety.
In further embodiments, the additional CFTR modulator is a read-through agent, which promotes read-through of PTCs to generate full-length CFTR. Examples of suitable amplifier include, without limitation, ataluren (PTC124).
There is no particular order by which the co-potentiators and the additional CFTR modulators are administered. They can be administers simultaneously (e.g., all agents are combined in a single unit dosage form) or separately.
“Alkyl” means a straight chain or branched, noncyclic, unsaturated or partially unsaturated aliphatic hydrocarbon containing from 1 to 12 carbon atoms. A lower alkyl refers to an alkyl that has any number of carbon atoms between 1 and 6 (i.e., C1-C6 alkyl) Representative saturated straight chain alkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and the like, while saturated branched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl, tert-pentyl, heptyl, n-octyl, isopentyl, 2-ethylhexyl and the like. Alkyl may be optionally substituted by one or more substituents as defined herein.
“Alkoxy” refers to the radical of —O-alkyl. Examples of alkoxy include methoxy, ethoxy, and the like. The alkyl moiety of alkoxy may be optionally substituted by one or more substituents as defined herein.
“Aryl” means an aromatic carbocyclic moiety such as phenyl or naphthyl (i.e., naphthalenyl) (1- or 2-naphthyl) or anthracenyl (e.g., 2-anthracenyl).
“Arylalkyl” (e.g., phenylalkyl) means an alkyl having at least one alkyl hydrogen atom replaced with an aryl moiety, such as —CH2-phenyl (i.e., benzyl), —CH═CH-phenyl, —C(CH3)═CH-phenyl, and the like.
“Heteroaryl” refers to a 5- to 14-membered ring system comprising one to thirteen carbon atoms, one to six heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur, and at least one aromatic ring. For purposes of certain embodiments of this disclosure, the heteroaryl radical may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include fused or bridged ring systems; and the nitrogen, carbon or sulfur atoms in the heteroaryl radical may be optionally oxidized; the nitrogen atom may be optionally quaternized. Examples include, but are not limited to, azepinyl, acridinyl, benzimidazolyl, benzthiazolyl, benzindolyl, benzodioxolyl, benzofuranyl, benzooxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl (benzothiophenyl), benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl, benzoxazolinonyl, benzimidazolthionyl, carbazolyl, cinnolinyl, dibenzofuranyl, dibenzothiophenyl, furanyl, furanonyl, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl, isoindolyl, indolinyl, isoindolinyl, isoquinolyl, indolizinyl, isoxazolyl, naphthyridinyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl, 1-oxidopyridinyl, 1-oxidopyrimidinyl, 1-oxidopyrazinyl, 1-oxidopyridazinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, pteridinonyl, purinyl, pyrrolyl, pyrazolyl, pyridinyl, pyridinonyl, pyrazinyl, pyrimidinyl, pryrimidinonyl, pyridazinyl, pyrrolyl, pyrido[2,3-d]pyrimidinonyl, quinazolinyl, quinazolinonyl, quinoxalinyl, quinoxalinonyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, thiazolyl, thiadiazolyl, thieno[3,2-d]pyrimidin-4-onyl, thieno[2,3-d]pyrimidin-4-onyl, triazolyl, tetrazolyl, triazinyl, and thiophenyl (i.e. thienyl). Unless stated otherwise specifically in the specification, a heteroaryl group is optionally substituted.
“Heteroarylalkyl” (e.g., pyrazolylalkyl) means an alkyl having at least one alkyl hydrogen atom replaced with an heteroaryl moiety, such as —CH2-pyrazolyl, —CH2-pyridinyl, —CH2-quinolinyl and the like.
“Halogen” or “halo” means fluoro, chloro, bromo, and iodo.
All the above groups may be “optionally substituted,” i.e., either substituted or unsubstituted. The term “substituted” as used herein means any of the above groups (i.e., alkyl, alkoxy, alkoxyalkyl, aryl, aralkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl and/or trifluoroalkyl), may be further functionalized wherein at least one hydrogen atom is replaced by a bond to a non-hydrogen atom substituent. Unless stated specifically in the specification, a substituted group may include one or more substituents selected from: ═O, —CO2H, nitrile, nitro, —CONH2, hydroxyl, thiooxy, alkyl, alkylene, alkoxy, alkoxyalkyl, alkylcarbonyl, alkyloxycarbonyl, aryl, aralkyl, arylcarbonyl, aryloxycarbonyl, aralkylcarbonyl, aralkyloxycarbonyl, aryloxy, cycloalkyl, cycloalkylalkyl, cycloalkylcarbonyl, cycloalkylalkylcarbonyl, cycloalkyloxycarbonyl, heterocyclyl, heteroaryl, dialkylamines, arylamines, alkylarylamines, diarylamines, N-oxides, imides, and enamines; a silicon atom in groups such as trialkylsilyl groups, dialkylarylsilyl groups, alkyldiarylsilyl groups, thioalkyl triarylsilyl groups, perfluoroalkyl or perfluoroalkoxy, for example, trifluoromethyl or trifluoromethoxy.
A pharmaceutical composition, as used herein, refers to a mixture of a compound described herein (e.g., co-potentiators) with other chemical components, such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, and/or excipients.
In certain embodiments, compounds described herein are formulated for oral administration. Compounds described herein are formulated by combining the active compounds with, e.g., pharmaceutically acceptable carriers or excipients. In various embodiments, the compounds described herein are formulated in oral dosage forms that include, by way of example only, tablets, powders, pills, dragees, capsules, liquids, gels, syrups, elixirs, slurries, suspensions and the like.
In certain embodiments, therapeutically effective amounts of at least one of the compounds described herein are formulated into other oral dosage forms. Oral dosage forms include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. In specific embodiments, push-fit capsules contain the active ingredients in admixture with one or more filler. Fillers include, by way of example only, lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In other embodiments, soft capsules, contain one or more active compound that is dissolved or suspended in a suitable liquid. Suitable liquids include, by way of example only, one or more fatty oil, liquid paraffin, or liquid polyethylene glycol. In addition, stabilizers are optionally added.
Methods for the preparation of compositions comprising the compounds described herein include formulating the compounds with one or more inert, pharmaceutically acceptable excipients or carriers to form a solid, semi-solid or liquid. Solid compositions include, but are not limited to, powders, tablets, dispersible granules, capsules, cachets, and suppositories. Liquid compositions include solutions in which a compound is dissolved, emulsions comprising a compound, or a solution containing liposomes, micelles, or nanoparticles comprising a compound as disclosed herein. Semi-solid compositions include, but are not limited to, gels, suspensions and creams. The form of the pharmaceutical compositions described herein include liquid solutions or suspensions, solid forms suitable for solution or suspension in a liquid prior to use, or as emulsions. These compositions also optionally contain minor amounts of nontoxic, auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, and so forth.
Useful pharmaceutical compositions also, optionally, include solubilizing agents to aid in the solubility of a compound described herein. The term “solubilizing agent” generally includes agents that result in formation of a micellar solution or a true solution of the agent. Certain acceptable nonionic surfactants, for example polysorbate 80, are useful as solubilizing agents, as can ophthalmically acceptable glycols, polyglycols, e.g., polyethylene glycol 400, and glycol ethers.
Furthermore, useful pharmaceutical compositions optionally include one or more pH adjusting agents or buffering agents, including acids such as acetic, boric, citric, lactic, phosphoric and hydrochloric acids; bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate and tris-hydroxymethylaminomethane; and buffers such as citrate/dextrose, sodium bicarbonate and ammonium chloride. Such acids, bases and buffers are included in an amount required to maintain pH of the composition in an acceptable range.
Additionally, useful compositions also, optionally, include one or more salts in an amount required to bring osmolality of the composition into an acceptable range. Such salts include those having sodium, potassium or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfite anions; suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite and ammonium sulfate.
In some embodiments, the compounds described herein are formulated or administered in conjunction with liquid or solid tissue barriers also known as lubricants. Examples of tissue barriers include, but are not limited to, polysaccharides, polyglycans, seprafilm, interceed and hyaluronic acid.
The co-potentiators described herein can be administered simultaneously or separately. This administration in combination can include simultaneous administration of the two agents in the same dosage form, simultaneous administration in separate dosage forms, and separate administration. That is, a compound described herein and any of the agents described above can be formulated together in the same dosage form and administered simultaneously. Alternatively, a compound of the invention and any of the agents described above can be simultaneously administered, wherein both the agents are present in separate formulations. In another alternative, a compound of the present invention can be administered just followed by and any of the agents described above, or vice versa. In some embodiments of the separate administration protocol, a compound of the invention and any of the agents described above are administered a few minutes apart, or a few hours apart, or a few days apart.
ASP-11: 1-butyl-N-(4-ethylphenyl)-1H-pyrrolo[2,3-b]pyridine-3-sulfonamide
CFTR: cystic fibrosis transmembrane conductance regulator
EC50: half-maximal effective concentration
FRT: Fisher rat thyroid
GLPG1837: N-(3-carbamoyl-5,5,7,7-tetramethyl-5,7-dihydro-4H-thieno[2,3-c]pyran-2-yl)-1H-pyrazole-5-carboxamide
MSD: membrane spanning domain
NBD: nucleotide binding domain
P2: N-methyl-N-[2-[[4-(1-methylethyl)phenyl]amino]-2-oxo-1-phenylethyl]-1H-indole-3-acetamide
P3: 6-(ethyl-phenyl-sulfonyl)-4-oxo-1,4-dihydro-quinoline-3-carboxylic acid 2-methoxy-benzylamide
P5: 2-(2-chloro-benzylamino)-4,5,6,7-tetrahydro-benzo[b]thiophene-3-carboxylic acid amide
VX-770: N-(2,4-ditert-butyl-5-hydroxyphenyl)-4-oxo-1H-quinoline-3-carboxamide
VX-809: 3-[6-[[1-(2,2-difluoro-1,3-benzodioxol-5-yl)cyclopropanecarbonyl]amino]-3-methylpyridin-2-yl]benzoic acid
VX-661: 1-(2,2-difluoro-1,3-benzodioxol-5-yl)-N-[1-[(2R)-2,3-dihydroxypropyl]-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)indol-5-yl]cyclopropane-1-carboxamide
YFP: yellow fluorescent protein
All compounds described in this manuscript have >95% purity. The analytical method used to determine purity was 1H NMR (see the accompanying Supporting Information file which provides the 1H- and 13C-NMR for the thirty-seven compounds assayed) and HPLC/HRMS. For HRMS analysis, samples were analyzed by flow-injection analysis into a Thermo Fisher Scientific LTQ Orbitrap (San Jose, Calif.) operated in the centroided mode. Samples were injected into a mixture of 50% MeOH/H2O and 0.1% formic acid at a flow of 0.2 mL/min. Source parameters were 5.5 kV spray voltage, capillary temperature of 275° C. and sheath gas setting of 20. Spectral data were acquired at a resolution setting of 100,000 FWHM with the lockmass feature, which typically results in a mass accuracy<2 ppm.
VX-809, VX-770, GLPG1837 and CFTRinh-172 were purchased from Selleck Chemicals (Boston, Mass.). Potentiators P2 (PG-01; Pedemonte, N. et al., (2005) Phenylglycine and sulfonamide correctors of defective delta F508 and G551D cystic fibrosis transmembrane conductance regulator chloride-channel gating. Mol. Pharmacol. 67, 1979-1807), P3 (SF-03; Pedemonte, 2005) and P5 (dF508act-02; Yang, H. et al., (2003) Nanomolar-affinity small-molecular potentiators of DF508-CFTR chloride channel gating. J. Biol. Chem. 278, 35079-35085) were obtained from an in-house repository of CFTR modulators. For screening, 120,000 diverse drug-like synthetic compounds (i.e., Structures (A), (B), (C), and (D); ChemDiv Inc., San Diego, Calif.) were tested. Other chemicals were purchased from Sigma unless otherwise stated.
Complementary DNAs (cDNAs) for the I1234del-, W1282C/L/R and Q1313X-mutants CFTRs were generated using standard techniques. In brief, gBLOCK gene fragment (Integrated DNA Technology, Coralville, Iowa) were synthesized and introduced into full-length CFTR cDNA in the vector pcDNA3.1/Zeo (+) (Invitrogen). For subcloning, I1234del-CFTR was generated using a HindIII site at position 3171-3176 of the CFTR cDNA; for W1282C/L/R and Q1313X-CFTR a BstXI site at position 3801-3812 of CFTR cDNA was used. The mutated CFTR cDNAs were subcloned into vector pIRESpuro3 (Clontech, Mountain View, Calif.) using NheI and NotI restriction sites. All constructs were confirmed by sequencing.
Fischer rat thyroid (FRT) cells were cultured in Kaign's modified Ham's F-12 medium supplemented with 10% FBS, 2 mM L-glutamine, 100 units/mL penicillin, 100 μg/mL streptomycin, 18 μg/mL myoinositol, and 45 μg/mL ascorbic acid. To generate FRT cells stably expressing I1234del-, W1282C/L/R and Q1313X-CFTR, cells were transfected with pIRESpuro3-based vectors and clonal cell lines were isolated after inclusion of 0.15 μg/mL puromycin (Invitrogen) in cell culture medium. FRT cell lines expressing wild type, W1282X- and N1303K-CFTR were cultured as reported (Pranke, I. et al., (2019) Emerging therapeutic approaches for cystic fibrosis. From gene editing to personalized medicine. Front. Pharmacol. 10, 121; Phuan, P.-W. et al., (2018) Combination potentiator (‘co-potentiator’) therapy for CF caused by CFTR mutants, including N1303K, that are poorly responsive to single potentiators. J. Cyst. Fibros. 17, 595-606; Cil, O. et al., (2016) CFTR activator increases intestinal fluid secretion and normalizes stool output in a mouse model of constipation. Cell. Mol. Gastroentrol. 2, 317-327). FRT cells lines expressing G85E-, R334W-, R347P-, S492F-, V520F-, R560T-, A561E-, L1077P-, M1101K- and R1162X-CFTR were a generous gift from Dr. Eric Scorcher (Emory University) and cultured as described (Han, S. T. et al., (2018) Residual function of cystic fibrosis mutants predicts response to small molecule CFTR modulators. JCI Insight 3, (14):e121159). Gene edited 16HBE14o-cells expressing N1303K-CFTR were provided by the CFFT Lab, and were cultured as described (Valley, H. C. et al, (2018) Isogenic cell modules of cystic-fibrosis-causing variants in natively expressing pulmonary epithelial cells. J. Cyst. Fibros. 18, 476-483).
Human bronchial epithelial cells isolated from a lung transplant from a N1303K homozygous CF subject were provided by Scott H. Randell (Marsico Lung Institute, The University of North Carolina at Chapel Hill, USA). The cells were obtained under protocol #03-1396 approved by the University of North Carolina at Chapel Hill Biomedical Institutional Review Board. Cells were isolated, conditionally reprogrammed, and expanded as described (Haggie, 2017; Fulcher, M. L., and Randell, S. H. (2013) Human nasal and treacho-bronchial respiratory epithelial cell culture. Methods Mol. Biol. 945, 109-121).
High-throughput screening used a semi-automated screening platform (Beckman, Fullerton, Calif.) as described (Haggie, 2017). FRT cells expressing W1282X and YFP were plated in 96-well black-walled, clear-bottom tissue culture plates (Corning) at a density of 20,000 cells/well and grown for 24 h at 37° C. to ˜90% confluency. Cells were treated with 3 μM VX-809 for 24 hours. Cells were then washed twice with PBS, and incubated in 100 μl of PBS containing forskolin (10 μM), VX-770 (15 nM) and test compounds (25 μM) for 10 min prior to assay of CFTR activity. All plates contained wells with positive (5 μM VX-770+20 μM ASP-11) and negative (5 μM VX-770) controls. Assays were done using a BMG Labtech FLUOstar OMEGA plate reader (Cary, N.C.) over 12 s with initial fluorescence intensity recorded for 2 s prior to addition of 100 μl of NaI-substituted PBS (137 mM NaCl replaced with NaI). Initial iodide influx was computed from fluorescence intensity by single exponential regression.
Short-circuit current was measured on cells cultured on permeable supports (Corning) as described (Haggie, 2017; Phuan, 2018). For FRT cells, the basolateral membrane was permeabilized with 250 μg/mL amphotericin B, and experiments were done using a HCO3−-buffered system (in mM: 120 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2), 5 Hepes, 25 NaHCO3, 10 glucose, pH 7.4) with a basolateral to apical chloride gradient (60 mM NaCl replaced by sodium gluconate in the apical solution). For human airway epithelial cells, symmetrical HCO3−-buffered solutions (containing 120 mM NaCl) were used. Cells were equilibrated with 95% O2, 5% CO2 and maintained at 37° C. Hemichambers were connected to a DVC-1000 voltage clamp (World Precision Instruments Inc., Sarasota, Fla.) via Ag/AgCl electrodes and 3 M KCl agar bridges for recording of the short-circuit current.
GraphPad Prism software (GraphPad Inc., San Diego, Calif., USA) was used for statistical analysis. EC50 values were determined by non-linear regression to a single-site inhibition model. Statistical significance was determined using unpaired Student's t test, with P<0.05 considered significant.
Synthesis of 2′,3′,4′,9′-tetrahydrospiro[piperidine-4,1′-pyrido[3,4-b]indoles] (8→9): Tryptamine (8, 0.6 mmol) was mixed with glacial acetic acid (3 mL) in a 10 mL vial and stirred with a magnetic stirrer. Corresponding ketone (12, 0.5 mmol) was added and the vial was sealed with a plastic cap. The vial was heated at 100° C. for 16 h in an oil bath. After cooling, the solution was diluted with water (˜20 mL) and neutralized by adding 4 M HCl. The product was extracted with dichloromethane, and the organic solution was washed with water, brine and dried over magnesium sulfate. The solvent was removed in vacuo, and the product was purified by using flash column chromatography (2.5% MeOH/DCM).
Synthesis of N-alkylated piperidin-4-one analogs (11→12): 4-Piperidone hydrochloride (11, 5 mmol) was mixed with 25 mL of dichloromethane in an Erlenmeyer flask. Small amount of methanol (5 drops) was added and benzyl bromide (2.5 mmol) and potassium carbonate (5 mmol) was added. The mixture was stirred at RT for 16 h. Water was added to the reaction mixture and the product was extracted with dichloromethane. The extracted organic solution was washed with water, brine, and dried over magnesium sulfate. The solvent was removed in vacuo, and the product was purified by using flash column chromatography (2.5% MeOH/DCM).
Synthesis of 2′,3′,4′,9′-tetrahydrospiro[piperidine-4,1′-pyrido[3,4-b]indoles] (10→9): Compound 3a (0.5 mmol) was mixed with 5 mL of dichloromethane in a small vial. Small amount of methanol (1 drop) was added for better solubility, and benzyl bromide (0.5 mmol) and potassium carbonate (1.5 mmol) was added. The vial was capped, and the mixture was stirred at RT for 16 h. Water was added to the reaction mixture and the product was extracted with dichloromethane. The extracted organic solution was washed with water, brine, and dried over magnesium sulfate. The solvent was removed in vacuo, and the product was purified by using flash column chromatography (2.5% MeOH/DCM).
Synthesis of 2′,3′,4′,9′-tetrahydrospiro[piperidine-4,1′-pyrido[3,4-b]indoles] (8→10): 5-methoxytryptamine (8, 25 mmol) was mixed with glacial acetic acid (20 mL) in a round bottom flask. 4-piperidone hydrochloride (11, 25 mmol) was added and the solution was heated at 100° C. for 16 h in an oil bath with stirring. The solution was cooled to RT and diluted with water (100 mL) and neutralized with 4 M NaOH. Tan precipitate formed upon standing within an hour. The precipitate was filtered and washed with water and air dried. Yield=54%.
Synthesis of tryptamines (7→8): 1-Dimethylamino-2-nitroethylene (3 mmol) was mixed in TFA (4 mL) in a vial. Substituted indole (7, 3.6 mmol) was dissolved in dichloromethane (3 mL) separately and added. The mixture was stirred at RT for 2 h and the solution was diluted with dichloromethane. The organic solution was washed with water, brine and dried over magnesium sulfate. Solvent was removed in vacuo and purified by using flash column chromatography (50% EtOAc/hexane). LiAlH4 (12 mmol) was mixed with THE (75 mL) and cooled at −78° C. and stirred. The product from the previous step dissolved in small amount of THE was added dropwise and the mixture was stirred overnight with slowly warming to RT. The reaction mixture was cooled in an ice bath and quenched with water and 4 M NaOH solution. Organic solvent was removed in vacuo and the product was extracted with dichloromethane. The extracted solution was washed with water, brine, and dried over magnesium sulfate. The solvent was removed in vacuo, and the product was directly used.
1-(3-Chloro-2,4-difluorobenzyl)-6′-methoxy-2′,3′,4′,9′-tetrahydrospiro[piperidine-4,1′-pyrido[3,4-b]indole] (1j). Yield=124 mg (58%) 1H NMR (400 MHz, CDCl3) δ 8.10 (s, 1H), 7.36-7.24 (m, 1H), 7.17 (s, 1H), 7.01-6.87 (m, 2H), 6.83 (dd, J=8.7, 2.5 Hz, 1H), 3.88 (s, 3H), 3.65 (s, 2H), 3.15 (t, J=5.7 Hz, 2H), 2.80-2.66 (m, 4H), 2.60 (td, J=11.9, 2.6 Hz, 2H), 2.10 (td, J=13.4, 4.6 Hz, 2H), 1.87-1.67 (m, 2H). 13C NMR (101 MHz, CDCl3) δ 158.08 (dd, JC-F=250.1, 2.9 Hz), 157.44 (d, JC-F=251.6, 2.9 Hz), 154.01, 140.77, 130.62, 129.34 (dd, JC-F=9.0, 5.7 Hz), 127.74, 121.95 (dd, JC-F=15.0, 4.0 Hz), 111.50, 111.46, 111.44 (dd, JC-F=20.6, 4.4 Hz), 109.77 (t, JC-F=21.3 Hz), 108.58, 100.56, 56.06, 55.41, 50.51, 48.65, 39.10, 36.27, 23.21. HRMS (ESI) m/z for C23H25C1F2N30 [M+H]. calcd 432.1654, found 432.1646.
6′-Methoxy-1-(3,4,5-trifluorobenzyl)-2′,3′,4′,9′-tetrahydrospiro[piperidine-4,1′-pyrido[3,4-b]indole] (2d). Yield=159 mg (77%).1H NMR (400 MHz, CDCl3) δ 7.76 (s, 1H), 7.23 (d, J=8.6 Hz, 1H), 7.04 (t, J=7.5 Hz, 2H), 6.96 (d, J=2.4 Hz, 1H), 6.84 (dt, J=8.7, 1.8 Hz, 1H), 3.87 (s, 3H), 3.52 (s, 2H), 3.16 (t, J=5.7 Hz, 2H), 2.71 (q, J=5.3 Hz, 4H), 2.56 (t, J=11.8 Hz, 2H), 2.10 (td, J=13.2, 4.3 Hz, 2H), 1.81 (d, J=13.7 Hz, 2H). 13C NMR (201 MHz, CDCl3) δ 154.07, 151.14 (ddd, JC-F=249.5, 10.1, 3.7 Hz), 140.68, 139.34-138.02 (m), 135.07, 130.58, 127.74, 112.54 (dd, JC-F=17.1, 3.7 Hz), 111.53, 111.50, 108.64, 100.53, 61.90, 56.05, 50.55, 48.79, 39.08, 36.34, 23.19. HRMS (ESI) m/z for C23H24F3N30 [M+H]. calcd 416.1950, found 416.1939.
6′-Methoxy-1-((perfluorophenyl)methyl)-2′,3′,4′,9′-tetrahydrospiro[piperidine-4,1′-pyrido[3,4-b]indole] (2e). Yield=202 mg (90%).1H NMR (400 MHz, CDCl3) δ 7.73 (s, 1H), 7.19 (d, J=8.7 Hz, 1H), 6.95 (t, J=1.8 Hz, 1H), 6.83 (dt, J=8.7, 1.9 Hz, 1H), 3.87 (d, J=1.4 Hz, 3H), 3.79 (d, J=2.4 Hz, 2H), 3.13 (t, J=5.7 Hz, 2H), 2.76 (d, J=11.1 Hz, 2H), 2.72-2.61 (m, 4H), 2.06 (td, J=13.1, 4.5 Hz, 2H), 1.80 (d, J=13.6 Hz, 2H). 13C NMR (201 MHz, CDCl3) δ 154.03, 145.63 (d, JC-F=248.0 Hz), 140.70, 140.59 (d, JC-F=254.2 Hz), 137.39 (d, JC-F=251.6 Hz), 130.54, 127.73, 111.47, 111.46, 110.75 (t, JC-F=18.7 Hz), 108.66, 100.50, 56.01, 50.26, 48.92, 48.07, 39.04, 36.34, 23.20. HRMS (ESI) m/z for C23H23F5N30 [M+H]. calcd 452.1761, found 452.1753.
6′-Methoxy-1-(2,3,4-trifluorobenzyl)-2′,3′,4′,9′-tetrahydrospiro[piperidine-4,1′-pyrido[3,4-b]indole] (2g). Yield=200 mg (96%) 1H NMR (400 MHz, CDCl3) δ 8.24 (s, 1H), 7.18 (d, J=8.8 Hz, 1H), 7.15-7.03 (m, 1H), 6.99-6.86 (m, 2H), 6.85-6.67 (m, 1H), 3.87 (d, J=2.9 Hz, 3H), 3.68-3.59 (m, 2H), 3.13 (t, J=5.7 Hz, 2H), 2.77-2.65 (m, 4H), 2.65-2.50 (m, 2H), 2.16-1.98 (m, 2H), 1.85-1.71 (m, 2H). 13C NMR (101 MHz, CDCl3) δ 153.99, 150.40 (ddd, JC-F=250.2, 9.9, 2.6 Hz), 150.20 (d, JC-F=250.4, 9.5, 3.0 Hz), 140.80, 139.88 (dt, JC-F=251.8, 15.6 Hz), 130.65, 127.72, 124.92 (ddd, JC-F=8.5, 6.3, 3.2 Hz), 122.42 (dd, JC-F=12.1, 2.8 Hz), 111.71 (dd, JC-F=17.1, 3.9 Hz), 111.51, 111.42, 108.52, 100.53, 56.05, 55.14, 50.49, 48.58, 39.08, 36.21, 23.20. HRMS (ESI) m/z for C23H24F3N30 [M+H]. calcd 416.1950, found 416.1941.
6′-Methoxy-1-(2,4,5-trifluorobenzyl)-2′,3′,4′,9′-tetrahydrospiro[piperidine-4,1′-pyrido[3,4-b]indole] (2i). Yield=193 mg (93%) 1H NMR (400 MHz, CDCl3) δ 8.06 (s, 1H), 7.30 (ddd, J=10.7, 8.9, 6.6 Hz, 1H), 7.19 (d, J=8.6 Hz, 1H), 6.98 (d, J=2.4 Hz, 1H), 6.95-6.86 (m, 1H), 6.84 (dd, J=8.7, 2.5 Hz, 1H), 3.89 (s, 3H), 3.59 (d, J=1.3 Hz, 2H), 3.16 (t, J=5.7 Hz, 2H), 2.81-2.66 (m, 4H), 2.60 (dd, J=23.9, 2.7 Hz, 2H), 2.10 (td, J=13.7, 4.6 Hz, 2H), 1.80 (dd, J=14.1, 2.7 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 156.20 (ddd, JC-F=247.5, 10.1, 2.2 Hz), 154.03, 149.1 (ddd, JC-F=251.7, 14.9, 14.0), 146.8 (ddd, JC-F=245.3, 12.5, 4.0), 140.79, 130.64, 127.76, 121.72 (dt, JC-F=16.9, 4.6 Hz), 118.72 (dd, JC-F=18.9, 5.7 Hz), 111.52, 111.47, 108.60, 105.31 (dd, JC-F=28.6, 20.5 Hz), 100.57, 56.06, 54.76, 50.52, 48.70, 39.09, 36.34, 23.21. HRMS (ESI) m/z for C23H24F3N30 [M+H]. calcd 416.1950, found 416.1940.
6′-Chloro-1-(2,4-difluorobenzyl)-2′,3′,4′,9′-tetrahydrospiro[piperidine-4,1′-pyrido[3,4-b]indole](5c). Yield=134 mg (67%).1H NMR (400 MHz, CDCl3) δ 8.86-8.49 (m, 1H), 7.43 (d, J=9.3 Hz, 2H), 7.21 (d, J=8.7 Hz, 1H), 7.08 (d, J=8.5 Hz, 1H), 6.82 (q, J=9.0 Hz, 2H), 3.69 (s, 2H), 3.11 (d, J=5.8 Hz, 2H), 2.81 (d, J=11.3 Hz, 2H), 2.67 (q, J=8.5, 8.0 Hz, 4H), 2.18 (q, J=8.0, 5.3 Hz, 2H), 1.76 (d, J=13.6 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 163.25 (dd, JC-F=100.2, 12.4 Hz), 160.77 (dd, JC-F=100.6, 12.3 Hz), 141.11, 133.91, 132.90 (dd, JC-F=9.8, 5.8 Hz), 128.44, 124.90, 121.74, 119.48 (d, JC-F=15.4 Hz), 117.69, 111.85, 111.29 (d, JC-F=20.7 Hz), 108.51, 103.82 (t, JC-F=25.7 Hz), 54.96, 50.36, 48.43, 39.00, 35.72, 22.99. HRMS (ESI) m/z for C22H23C1F2N3 [M+H]. calcd 402.1549, found 402.1539.
1-(2,4-Difluorobenzyl)-7′-methoxy-2′,3′,4′,9′-tetrahydrospiro[piperidine-4,1′-pyrido[3,4-b]indole] (5d). Yield=90 mg (45%).1H NMR (400 MHz, CDCl3) δ 8.24 (s, 1H), 7.48-7.32 (m, 2H), 6.94-6.68 (m, 4H), 3.82 (s, 3H), 3.67 (s, 2H), 3.13 (t, J=5.7 Hz, 2H), 2.83-2.76 (m, 2H), 2.72-2.54 (m, 4H), 2.17 (td, J=13.5, 13.1, 4.4 Hz, 2H), 1.77 (d, J=13.7 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 163.17 (dd, JC-F=91.6, 11.9 Hz), 160.70 (dd, JC-F=92.1, 12.1 Hz), 156.28, 138.34, 136.25, 132.75 (dd, JC-F=9.5, 5.9 Hz), 121.77, 119.96 (dd, JC-F=14.8, 3.8 Hz), 118.65, 111.20 (dd, JC-F=20.8, 3.8 Hz), 108.93, 108.49, 103.74 (t, JC-F=25.7 Hz), 94.99, 55.78, 54.99, 50.38, 48.59, 39.15, 35.97, 23.14. HRMS (ESI) m/z for C23H26F2N30 [M+H]. calcd 398.2044, found 398.2033.
Additional biological studies were performed for most potent Compounds 2i and 2e. Co-potentiator efficacy was initially determined by short-circuit current measurements in FRT cells expressing N1303K-CFTR in the presence of a transepithelial chloride gradient and with permeabilization of the basolateral cell membrane such that measured current directly reports CFTR channel activity. Representative data in
Compound 2i was also tested on a second minimal function CFTR mutation, I1234del-CFTR, which is generated by the c.3700A>G mutation that results in deletion of 6 amino acids from the CFTR polypeptide (p.Ile1234_Arg1239del-CFTR, hereafter termed I1234del-CFTR) due to introduction of a cryptic splice site in the CFTR transcript. As seen in
The efficacy of Compound 2i was also tested in 16HBE14o-human bronchial epithelial cell models in which the endogenous CFTR gene was edited to contain the N1303K mutation (16HBEge-N1303K) or the I1234del mutation (16HBEge-I1234del). As shown for N1303K- (
The various embodiments described above can be combined to provide further embodiments. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications referred to in this specification and/or listed in the Application Data Sheet are incorporated herein by reference, in their entirety. Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, applications, and publications to provide yet further embodiments.
These and other changes can be made to the embodiments in light of the above-detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.
This application claims the benefit of priority to U.S. Provisional Application No. 62/893,107 filed Aug. 28, 2019, the entirety of which is incorporated by reference herein.
This invention was made with government support under Grant No. P30 DK072517 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2020/048645 | 8/28/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62893107 | Aug 2019 | US |
