Structure-Activity Relationships of Cyanoquinolines with Corrector-Potentiator Activity in delta-F508-Cystic Fibrosis Transmembrane Conductance Regulator Protein

Abstract

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. The most common CF-causing mutation, ΔF508-CFTR, produces CFTR loss-of-function by impairing its cellular targeting to the plasma membrane and its chloride channel gating. We recently identified, cyanoquinolines with both corrector (“Co”; normalizing ΔF508-CFTR targeting) and potentiator (“Po”; normalizing ΔF508-CFTR channel gating) activities. Here, we synthesized and characterized twenty-four targeted cyanoquinoline analogues to elucidate the conformational requirements for corrector and potentiator activities. Compounds with potentiator-only, corrector-only and dual potentiator-corrector activities were found. Molecular modeling studies (conformational search ⇒ force-field lowest energy assessment ⇒ geometry optimization] suggest that (1) a flexible tether and (2) a relatively short bridge between the cyanoquinoline and aryl amide moieties are important cyanoquinoline-based CoPo features. Further, these CoPo’s should adopt two distinct π-stacking conformations to elicit corrector and potentiator activities.

INTRODUCTION

Cystic Fibrosis (CF) is a genetic disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) protein,¹ which is expressed in epithelia in lung, pancreas, intestine, testis, and other tissues.² While there are many CF-causing mutations, the most common CFTR mutation is a deletion of the phenylalanine residue at position 508 (ΔF508-CFTR). Misfolding of ΔF508-CFTR causes folding and conformational defects as a result of its retention in the endoplasmic reticulum and accelerated degradation.^3,4 Additionally, any ΔF508-CFTR that reaches the cell plasma membrane is poorly functional as a chloride channel.² Consequently, the ΔF508-CFTR mutation results in greatly reduced cell membrane chloride permeability.

An ideal small molecule treatment of CF would fully restore ΔF508-CFTR structure to the native wild type state, resulting in normalization of its cellular processing and chloride channel function.⁵ Such compounds have not been identified. Instead, separate compounds, called ‘correctors,’ which partially rescue ΔF508-CFTR cellular misprocessing, and ‘potentiators’, which partially restore ΔF508-CFTR chloride channel function, have been identified (Figure 1). We and others have identified a variety of correctors⁶ (e.g., 1 {N-(2-((5-chloro-2-methoxyphenyl)amino)-4′-methyl-[4,5′-bithiazol]-2′-yl)benzamide; corr-4a}^6d and 2 {4-(cyclohexyloxy)-2-(1-(4-((4-methoxyphenyl)sulfonyl)piperazin-1-yl)ethyl)quinazoline; VRT-325};^6l Figure 1a) and potentiators⁷ (e.g., 3 {2-(2-(1H-indol-3-yl)-N-methylacetamido)-N-(4-isopropylphenyl)-2-phenylacetamide; PG01}^7g and 4 {N-(2,4-di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide; VX-770};^7h Figure 1b). Recently, we identified a dual-acting cyanoquinoline having independent corrector and potentiator activities (5, {N-(2-((3-cyano-5,7-dimethylquinolin-2-yl)amino)ethyl)-3-methoxybenzamide; CoPo-22};⁸ Figure 1c) – such compounds would be advantageous over CF treatments requiring separate corrector and potentiator administration.⁸ Moreover, the development costs for a single drug that treats both defects would be significantly lower than development costs for a two-drug paradigm.

Figure 1.

Structures of (a) correctors 1 and 2, (b) potentiators 3 and 4, and (c) cyanoquinoline 5 with structural subunits identified.

Cyanoquinoline 5 contains three distinct subunits consisting of a cyanoquinoline core and aryl amide moieties linked by a flexible tether (Figure 1). We postulated that this tether allows 5 to adopt two active conformations – an active corrector conformation and an active potentiator conformation – and that altering this tether would modulate CoPo activities by restricting its conformations. The two active conformations of 5 could allow binding to two separate targets (mode #1), two distinct binding sites on the same target (mode #2), or two different forms of a single binding site on ΔF508-CFTR that changes shape during protein transport from the endoplasmic reticulum (ER) to the cell plasma membrane (mode #3; Figure 2). Of these, mode #3 appears the most likely on a statistical basis; i.e., one molecule addressing one binding site that changes shape as it matures is statistically less challenging than one molecule addressing two distinct binding sites (modes #1–#2). The goal of the work reported here was to examine how the tethering moiety in 5 influences its ability to afford both corrector (Co) and potentiator (Po) activities and to assess the feasibility of mode #3 by computational analysis of the proclivities of 5 and tethered analogs for particular conformations.

Figure 2.

Possible CoPo conformational adaptation during the ER ⇒ cell membrane transition.

CHEMISTRY

This study examines the role of the tether and, secondarily, the aryl amide subunits on CoPo activities. Our tether-focused strategy is depicted in Figure 3 where the cyanoquinoline moiety is fixed while the tether and aryl amide moieties are varied. Indeed, quinolines hold privileged heterocycle status⁹ and have received much attention as antibacterial agents¹⁰ and cancer therapeutics.¹¹ Their biological importance has led to the development of several strategies to construct this aromatic ring system.

Figure 3.

Tether-focused CoPo library diversification strategy.

The CoPo cyanoquinoline core was synthesized starting from commercially available 3,5-dimethylaniline 6 (Scheme 1). Acylation of 6 with acetic anhydride gave 7 in near quantitative yield and subsequent Vilsmeier-Haack¹² type reaction gave chloroquinoline carboxaldehyde 8. Neutralization, filtration and drying produced pure 8 in 95 % yield. Aldehyde 8 was then condensed with hydroxylamine to yield oxime 9, which was directly converted to nitrile 10 by dehydration with SOCl₂ in refluxing benzene (93% yield over two steps).

Scheme 1.

Synthesis of chlorocyanoquinoline intermediate 5. Reagents: (a) acetic anhydride, THF (b) POCl₃, DMF (c) NH₂OH·HCl, Et₃N, EtOH (d) SOCl₂, benzene.

Aromatic substitution of 10 with various diamines allowed for ready diversification of the tethering subunit as depicted in Scheme 2. The tethers examined were derived from 1,2-diaminoethane (the tether in original screening hit 5), 1,3-diaminopropane (a longer and more flexible tether), and 1,4-piperazine (a more rigid tether). All three diamino tethers gave their intended products (10 → 11-13) in good yields (61–62%).

Scheme 2.

Tether diversification. Reagents: (e) 1,2-diaminoethylene, dioxane, reflux; (f) 1,3- diaminoproplene, dioxane, reflux; (g) piperazine, dioxane, reflux.

Further diversification of the distal primary or secondary amine in 11-13 was accomplished by EDC-mediated aryl acid coupling (Figure 4). Heteroaromatic amides were not evaluated in our initial screening study. So, as a preliminary test of the effect of heterocycles on CoPo potency, a range of pyridyl acids, an array of arylmethoxy acids (similar to that of initial hit 5), and one methoxypyridyl acid were employed in the acylation of 11-13. The specific aryl acids used in this coupling step are depicted in Figure 4.

Figure 4.

Cyanoquinoline-based CoPo aryl amide diversity.

PHARMACOLOGY

ΔF508-CFTR correctors were previously identified by screening of a large collection of diverse small molecules.^6d Our screening assay utilized Fischer rat thyroid (FRT) epithelial cells coexpressing ΔF508-CFTR and the yellow fluorescent protein (YFP) halide indicator YFP-H148Q/I152L at 37 °C in a 96-well-plate format. Test compounds at 10 μM concentration (and negative/positive controls) were added to each well for 18–24 h. ΔF508-CFTR–facilitated iodide influx was determined from the kinetics of decreasing YFP fluorescence following addition of extracellular iodide in the presence of the potentiators genistein and forskolin. Active compounds from the primary high-throughput screen were verified and then counterscreened for ΔF508-CFTR potentiator activity. For the potentiator assay ΔF508-CFTR was low-temperature rescued by incubating FRT cells that coexpressing ΔF508-CFTR and YFP at 27 °C for 24 h. Test compounds at 25 μM (and negative/positive control) were added to each well for 10 min in presence of forskolin. Similar to the corrector assay, ΔF508-CFTR–facilitated iodide influx was determined from the kinetics of decreasing YFP fluorescence following addition of extracellular iodide.

COMPUTATIONS

For cyanoquinolines 5 and 26, a 2-step conformational search was performed. First, candidate conformers were identified via a systematic search in Spartan’10¹³ in which each bond between the aryl moieties was given two-fold rotational freedom (sp²-sp² bonds) or three-fold rotational freedom (sp²-sp³ and sp³-sp³ bonds) and cyclic atoms were given 3-fold puckering mobility. This procedure resulted in 36 candidate conformations for 5 and 42 candidate conformations for 26, each within 10 kcal/mol of the lowest energy conformation, as assessed by the MMFF94 force-field.¹⁴ These candidates were then subjected to further geometry optimization using the M06-2X/6-31+G(d,p)¹⁵ density functional theory method as implemented in the GAUSSIAN09 software suite.¹⁶ After this refinement, five conformers within 3 kcal/mol of the lowest energy conformer of 5 and 18 conformers within 3 kcal/mol of the lowest energy conformer of 26 (four within 2 kcal/mol) were found, as assessed by computed electronic energies with solvent (water) modeled by the SMD continuum solvation model¹⁷ (via single-point calculations). Finally, select conformers described below were further refined with full solvent optimization and frequency calculations in order to include entropy contributions to the computed relative free energies. Atomic coordinates and computed energies for all structures can be found in the Supporting Information. As described below, constrained calculations were also performed on some conformers.

RESULTS and DISCUSSION

Corrector and potentiator activities for the twenty-four cyanoquinolines synthesized in this study were assayed separately in ΔF508-CFTR expressing FRT epithelial cells. Table 1 lists corrector (Co-) and potentiator (Po-) activities as EC₅₀ and V_max from concentration-dependence studies. EC₅₀ is a measure of compound potency and V_max is a measure of compound efficacy in increasing cell chloride permeability. Figure 5 shows representative concentration-dependence data for the corrector and potentiator plate reader assays from which EC₅₀ and V_max values were deduced from concentration dependence data by non-linear regression as described in the Experimental Section (plate reader assay). Figure 6 shows representative short-circuit assays of corrector and potentiator activities of compound 15, compared to 5, in which apical membrane chloride current was measured in the ΔF508-CFTR expressing FRT cells after basolateral membrane permeabilization and in the presence of a transepithelial chloride gradient. The corrector assay was done by incubation of cells for 18–24 h with test compound at 37 °C, followed by addition of forskolin and the potentiator genistein. The potentiator assay was done in low temperature-rescued cells by addition of forskolin followed by test compound. Short-circuit data as in Figure 6 confirmed the main findings obtained using the fluorescence plate reader assay.

Table 1.

Corrector and potentiator activities, determined in triplicate, of CoPo analogues as measured by fluorescence plate-reader assay. Concentration–response curves were determined for eight compound concentrations (maximum 50 μM for potentiator assay; 25 μM for corrector assay). EC₅₀ (± S.E.) and V_max were computed as described in the Experimental Section (plate reader assay).

CoPo	Corrector		Potentiator
	EC50 (μM)	Vmax (μM/S)	EC50 (μM)	Vmax (μM/s)
5	2.2 ±0.3	300	5.9 ± 0.5	216
14	11.0 ±0.9	114	2.3 ± 0.4	154
15	3.0 ±0.3	172	4.1 ± 1.0	181
16	inactive	--	1.2 ± 0.4	80
17	2.7 ±0.6	151	13.2 ± 0.6	242
18	7.3 ±0.8	176	10.0 ± 3.1	220
19	inactive	--	10.0 ± 2.0	210
20	4.2 ± 0.4	108	55.0 ± 3.5	426
21	1.5 ± 0.4	380	48.0 ± 5.0	216
22	6.7 ± 1.0	174	inactive	--
23	2.7 ± 0.5	88	3.4 ± 0.5	70
24	3.7 ± 0.6	111	11.5 ± 1.0	119
25	inactive	--	1.0 ± 0.3	129
26	inactive	--	1.2 ± 0.3	200
27	inactive	--	1.3 ± 0.2	281
28	inactive	--	7.4 ± 0.3	102
29	inactive	--	inactive	--
30	inactive	--	inactive	--
31	inactive	--	27.6 ± 2.0	254
32	4.6 ± 0.2	134	4.6 ± 0.4	147
33	4.3 ± 0.2	112	5.0 ± 0.2	231
34	inactive	--	6.1 ± 0.2	233
35	3.0 ± 0.5	140	16.0 ± 0.3	115
36	8.2 ± 0.4	61	13.2 ± 0.4	53

Figure 5.

Concentration-dependence of corrector (left) and potentiator (right) activities of indicated compounds in FRT cells expressing ΔF508-CFTR measured by iodide (I⁻) influx. See Table 1 for summary of deduced EC₅₀ and V_max.

Figure 6.

Short-circuit current measurement of ΔF508-CFTR chloride conductance. Measurements of corrector and potentiator activities of 15 and 5 were done in the presence of an apical-to-basolateral chloride gradient in basolateral membrane-permeabilized cells. Added compounds included forskolin (fsk, 20 μM), genistein (gen, 50 μM) and CFTRinh-172 (10 μM).

Based on these function studies, twelve compounds in this series were identified as having both corrector and potentiator activities (14, 15, 17, 18, 20, 21, 23, 24, 32, 33, 35, and 36), eight compounds were identified as having potentiator-only activity (16, 19, 25-28, 31, and 34), and one compound was identified as having corrector-only activity (22). For compounds having both corrector and potentiator activities, cyanoquinoline 5 is the most active (corrector/potentiator values of 2.2 μM/5.9 μM): the corrector activity EC₅₀ values for cyanoquinolines 15, 17, and 18 were 3.0, 2.7, and 7.3 μM, respectively, and the potentiator activity EC₅₀ values were 4.1, 13, and 10 μM, respectively. The maximal efficacy, V_max, of cyanoquinoline 5 for corrector and potentiator actions are comparable with those of 1 and genistein, respectively.

Two notable observations from these data are that constraining the diamino tether in a piperazine ring abolished corrector activity but, conversely, resulted in excellent potentiator activity (25-27) – better than that of 5. One interpretation of these findings is that that the binding site evolves during the endoplasmic reticulum ⇒ cell membrane transition (mode #3 discussed above; see Figure 2) and that piperazine-constrained cyanoquinolines cannot adequately adapt to the ΔF508- CFTR corrector-binding site when the protein is in the endoplasmic reticulum. Consequently, transport to the cell membrane does not occur with these piperazine-constrained cyanoquinolines. Conversely, an increase in mobility of the chain by lengthening (ethylene → propylene) results in CoPo’s (32-33, 35-36) with comparable, albeit, lower corrector and potentiator activity compared to that of 5. This increased tether chain-length causes increased flexibility and different conformational preferences, which in turn results in a reduced probability of these CoPo’s adopting an active conformation. Thus, the propylene-based tether is slightly less active compared to the ethylene-based tether.

If this model is correct, then the action of cyanoquinoline-based CoPo’s likely involves a slight, but important, conformational change allowing them to adapt to a single binding site that matures as it progresses from the ER to the cell membrane. Indeed, the juxtaposition of Co- and Po-activities with 5 versus only Po-activity with 25-27 implies that, in this cyanoquinoline-based series, the CoPo must be able to access a conformation not precluded by the constraints associated with tethering. In addition, this conformation, which is active for Po-activity must differ from that associated with Co-activity, implying that 5 must also be able to readily access a conformation that is not accessible by 25-27. The active conformation enabling potentiation is satisfied by — indeed, enabled by — piperazine- constrained cyanoquinolines (e.g., 25-27 are all better potentiators than 5). Taken together, the tether-differentiated SAR results delineated in Table 1 suggest that (1) a flexible tether and (2) a relatively short bridge between the cyanoquinoline and aryl amide moieties are vital for these cyanoquinoline-based CoPo’s to adopt the two distinct conformations required for dual Co- and Po- activities.

Are there energetically accessible conformations of 5 and 25-27 that meet these requirements? One such pair of conformers (there may well be others, but these conformers illustrate the point) located by our conformational search calculations are shown in Figure 7 – a conformation possibly associated with the Co-activity of 5 at left and a conformation possibly associated with Po-activity for 26 (the molecule in the 25-27 series that has the same aryl group as 5) at right. First, note that both of these conformers are folded so as to allow for some degree of π-stacking between the quinoline and methoxyphenyl groups, an effect that is perhaps maximized in water. The conformer shown for 26 is predicted to be the lowest energy conformer in water for this molecule (despite the twist-boat conformation of its piperazine core), while that for 5 is predicted to be the seventh-lowest conformer in water, only approximately 3 kcal/mol higher in energy than the lowest energy conformer. Here we focus on a low energy conformer of 5 that could possibly mimic that for 26 by undergoing relatively minor conformational changes; the lowest energy conformers predicted for 5 (see Supporting Information), although still displaying π-stacking interactions, do not orient their NH groups in such a way as to allow for such a conformational exchange to occur. Although strong binding to a target can overcome energy penalties associated with non-optimal conformations of ligands, if the conformer shown for 26 does correspond to the Po-active conformation, no energy penalty associated with changing this conformation from the predominant conformation in water will need to be paid.¹⁸ Since 5is also active, however, a 26-like conformation would need to be energetically accessible. Altering the conformation of 5 such that the isoquinoline and methoxyphenyl groups are in the same relative orientation as in 26 (and allowing the rest of the molecule to relax; see Supporting Information for details) is predicted to be accompanied by an increase in energy of approximately 4 kcal/mol (likely the result of not only disrupting π-stacking, but also disrupting the gauche conformation of the N–C–C–N substructure, which is preferred on both steric and orbital-based grounds over less-staggered conformations;¹⁹ note that this distorted conformation is predicted to be approximately 8 kcal/mol above the lowest energy conformation of 5 in water). Thus, 5 could adopt the shape of 26 if this energy penalty is not enough to cause dissociation or is counterbalanced by favorable interactions with a binding site cavity complementary to 26. We see no reason why this relatively small change to the conformation of 5 could not occur within a binding site (i.e., without dissociation) that switches between a Co- and a Po-form, each complementary to one or the other arrangement of the quinoline and methoxyphenyl groups shown in Figure 7a and 7b. Although this model is speculative, it should be testable through synthesis of, and activity assays on, isoquinolines with further conformational constraints.

Figure 7.

(a) Possible “Co”-active conformer of 5. (b) Possible “Po”-active conformer of 26. Two views of each structure are shown. In the left-hand presentation of each structure, the viewing angle is along the C–C bond of the tether (⇓); this view emphasizes the differing dihedral angles between the C–N bonds. In the right-hand presentation of each structure, the orientation is systematized by viewing down this same C–C bond (↓). Selected through space distances are shown in Å.

Additional structure-activity insight was derived from examination of the aryl amide moiety. Substitution at the ortho position of the aryl amide generally gave worse corrector activity, but better potentiator activity (eg. CoPo’s 14, 16, 25, 28, and 34). The best corrector activity was seen with an ethylene tether employing a methoxy substitution at either the meta (5) or para (CoPo 15) position of the aryl amide and the greatest corrector activity was obtained by placing a methoxy substituent at both (CoPo 21); note that electron-donating methoxy groups may well enhance the preference for π-stacked conformations. A similar trend can be seen with the propylene tether as CoPo’s 32 and 33 are superior correctors compared to 31. In general, hydrogen bond accepting groups at the meta and para positions afforded the best corrector activity as exemplified in the propylene diamine series with CoPo’s 31-36. Methoxy substitution proved to be superior to nitrogen heterocyclic amides in corrector activity. A hybrid pyridyl–methoxy amide (CoPo 23) maintained corrector and improved potentiator activity, while most other modifications giving better corrector or better potentiator activity did so at the expense of the other. These data suggest that dual CoPo activity is not a general feature of cyanoquinolines, but is rather dependent on the particular tether and/or aryl amide substituent.

CONCLUSION

This work provides structure-activity insight around the unique cyanoquinoline-based CoPo scaffold, which has dual, independent ΔF508-CFTR corrector and potentiator activities. The most active diamine tether was shown to be 1,2-diaminoethane–based compared to more constrained (piperazine) and more flexible (1,3-diaminopropylene) counterparts. Additionally, variations of the aryl amide moiety can tune the corrector and/or potentiator activities. This work has lead to the hypothesis that cyanoquinoline-based CoPo’s have dual activity if they can conformationally adapt to a single binding site that evolves during the endoplasmic reticulum ⇒ cell membrane transition. While the target site(s) of CoPo’s remain unknown, this work provides a working hypothesis regarding the corrector and potentiator structural features of the reported cyanoquinoline-based CoPo scaffold.

EXPERIMENTAL

General Experimental

All purchased starting materials and reagents were used without further purification. Product purification was performed either on an automated flash chromatography system (Combiflash by Teledyne: 35 min of elution with linear gradient from 100% hexane to 100% EtOAc solvent) with silica gel columns or on an HPLC system [Waters: 15 mL/min flow rate, linear gradient elution with 0.1% TFA-containing H₂O/MeCN from 5 to 95% MeCN in 20 min, Xterra Prep MS C18 OBD column (19 mm 100 mm), and dual wavelength absorbance detector]. NMR spectra (¹H at 400 MHz; ¹³C at 100 MHz) were recorded in CDCl₃ solvent on a Varian 600. Chemical shifts are expressed in parts per million relative to solvent. Coupling constants are expressed in units of hertz (Hz). Splitting patterns are designated as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), and bs (broad singlet). LC/MS (Waters Micromass ZQ) specifications are as follows: electrospray (+) ionization, mass ranging from 100 to 900 Da, 20 V cone voltage. LC: Xterra MS C18 column (2.1 mm × 50 mm × 3.5 μm), 0.2 mL/min water/acetonitrile (containing 0.1% TFA), 30 min linear gradient 0–100% acetonitrile. The LC/MS UV detector is a diode array with 200–400 nm wavelength range. Purity is based on integration of peak area as a percentage of the UV diode array signals. Compound purities are determined by RP-HPLC (≥ 95%).

ΔF508-CFTR corrector and potentiator activity assays

Plate reader assay

FRT cells stably coexpressing human ΔF508-CFTR and the halide-sensing fluorescent protein YFP-H148Q/I152L were used as described previously.^6d For corrector assay, cells were grown at 37 °C for 24 h and then incubated for 16–20 h with 200 μL of medium containing the test compound. At the time of the assay, cells were washed with PBS and then incubated with PBS containing forskolin (20 μM) and genistein (50 μM) for 20 min. For potentiator assay, cells were grown at 37 °C for 18–24 h and then for 18–24 h at 27 °C. At the time of the assay, cells were washed with PBS and then incubated for 10 min with PBS (50 μL) containing forskolin (20 μM) and test compound (0–50 μM final concentration). Measurements were carried out using FLUOstar fluorescence plate readers (Optima, BMG LABTECH Gmbh), equipped with 500 ± 10 nm excitation and 535 ± 15 nm emission filters. Each well was assayed individually for I⁻ influx by recording fluorescence for 2 s (baseline) and then for 12 s after rapid addition of 165 μL of PBS in which 137 mM Cl⁻ was replaced by I⁻. I⁻ influx rate was computed by exponential regression. All experiments contained negative control (DMSO vehicle) and positive controls (potentiator assay, genistein; corrector assay, 1). EC₅₀ and V_max, and their associated uncertainties, were determined by 4 parameter logistic nonlinear regression from concentration-activity data using GraphPad Prism Version 5.01. Initial iodide influx rates were fitted to the Hill equation: Activity (initial slope) = A_o + V_maxC_i^H/(C_i^H + IC₅₀^H), where C_i is compound concentration, H is coefficient, and A_o is background signal.

Short-circuit current measurements

ΔF508-CFTR–expressing FRT cells were cultured on Snapwell inserts for 7–9 days. For corrector assay, test compounds were incubated with cells for 18–24 h at 37 °C prior to measurements. For potentiator assay, the FRT cells were incubated for 18–24 h at 27 °C prior to measurements. Short-circuit current was recorded using established procedures.^6d

N-(3,5-Dimethylphenyl)acetamide (7)

Acetic anhydride (2.84 mL, 30 mmol) was dissolved in dry THF (10 mL), purged with N₂ and brought to 0 °C. 3,5-Dimethylaniline (1.25 mL, 10 mmol) was added dropwise and, upon completion of the addition, the reaction was allowed to warm to room temperature and stirred an additional hour. The reaction solution was then poured over ice and 1 M NaOH (aq) was added to adjust the pH to 12–14. The precipitate was collected by filtration, dissolved in DCM, and dried over Na₂SO₄. The drying agent was removed by filtration and the solvent was removed under reduced pressure to afford pure product in 99% yield as a white solid.¹²

2-Chloro-5,7-dimethylquinoline-3-carbaldehyde (8)

Phosphorous oxychloride (6.52 mL, 70 mmol) and dry DMF (1.94 mL, 25 mmol) was refluxed for 2 h under a N₂ atmosphere. Acetamide 2 (1.632 g, 10 mmol) was then added to the reaction solution as a solid and stirred at room temperature for an additional 3 h. The reaction solution was poured slowly over ice, diluted with water (200 mL) and carefully neutralized with solid K₂CO₃. The precipitate was then collected by filtration, dissolved in chloroform, and dried over Na₂SO₄. The drying agent was removed by filtration and the solvent was removed under reduced pressure to afford pure product in 95% yield as an orange solid.¹²

2-Chloro-5,7-dimethylquinoline-3-carbonitrile (10)

Aldehyde 8 (1.095 g, 5 mmol), hydroxylamine hydrochloride (0.365 g, 5.25 mmol) and triethylamine (1.00 mL, 7 mmol) were combined in ethanol (50 mL). The solution was refluxed for 3 h and then the ethanol was removed under reduced pressure. Aqueous HCl (1M, 100 mL) was added to the crude material and product was extracted with DCM (100 mL). The organic layer was separated and dried over Na₂SO₄. The drying agent was filtered and solvent was removed under reduced pressure. The crude product 4 was then dissolved in dry benzene (50 mL). Thionyl chloride (0.73 mL, 10 mmol) was added dropwise to the solution and the reaction was refluxed under a N₂ atmosphere for 4 h. The solution was allowed to cool to room temperature, then the benzene and excess thionyl chloride were removed under reduced pressure to afford pure product 5 in 93% yield as a light brown solid. ¹H NMR (400 MHz, CDCl₃) δ 8.62 (s, 1H), 7.62 (s, 1H), 7.33 (s, 1H), 2.66 (s, 3H), 2.54 (s, 3H).

General Procedure for Aromatic Substitution of 5 with Diamine Tether (11–13)

Carbonitrile 5 (1.083 g, 5 mmol) and diamine (15 mmol) were refluxed in dioxane (50 mL) until judged complete by TLC. The reaction was allowed to cool to room temperature and dioxane was removed under reduced pressure. The crude product was suspended in NH₄Cl (1M, aq) and collected by filtration. The solids were washed with diethylether and allowed to dry on a filter paper under vacuum; yields ranged from 40–80%.

2-((2-Aminoethyl)amino)-5,7-dimethylquinoline-3-carbonitrile (11)

¹H NMR (400 MHz, CDCl3) δ 8.31 (s, 1H), 7.32 (s, 1H), 6.92 (s, 1H), 5.64 (t, J = 4.8, 1H), 3.65 (q, J = 5.7, 2H), 3.01 (t, J = 6.0, 2H), 2.52 (s, 3H), 2.42 (s, 3H).⁸

2-(3-aminopropylamino)-5,7-dimethylquinoline-3-carbonitrile (12)

Yellow solid. Yield: 61%. ¹H NMR (600 MHz, CDCl₃) δ = 8.30 (s, 1H), 7.32 (s, 1H), 6.91 (s, 1H), 6.18 (s, 1H), 3.72 (q, J=6.2, 2H), 2.89 (t, J=6.2, 2H), 2.52 (s, 3H), 2.42 (s, 3H), 1.82 (quint., J=6.4, 2H), 1.64 (s, 2H); ¹³C NMR (150 MHz, CDCl₃) δ = 154.56, 150.37, 143.73, 140.41, 135.13, 126.48, 124.52, 118.89, 117.41, 94.14, 40.40, 40.05, 32.52, 22.20, 18.53. ESI-MS m/z [M+H]⁺: 255.33. Purity: 99%.

5,7-dimethyl-2-(piperazin-1-yl)quinoline-3-carbonitrile (13)

Orange solid. Yield: 62%. ¹H NMR (600 MHz, CDCl₃) δ = 8.35 (d, J=4.1, 1H), 7.35 (s, 1H), 6.95 (s, 1H), 3.60 (m, 4H), 3.02 (m, 4H), 2.49 (s, 3H), 2.40 (s, 3H), 1.87 (s, 1H); ¹³C NMR (150 MHz, CDCl₃) δ = 158.00, 149.29, 143.62, 142.63, 134.79, 127.89, 125.21, 120.08, 118.47, 97.37, 50.30, 46.16, 22.23, 18.47. ESI-MS m/z [M+H]⁺: 267.28. Purity: 99%.

General procedure for amide coupling reactions

EDC hydrochloride (0.192 g, 1 mmol), aryl acid (1 mmol), and triethylamine (0.14 mL, 2.5 mmol) were dissolved in dry DCM (10 mL). The reaction was stirred at room temperature for 30 min. Carbonitrile (1 mmol) dissolved in dry DCM (5 mL) was added dropwise to the solution and reaction stirred for 18 h. The reaction was diluted with DCM (50 mL) and washed with 1M NaHSO₄ (aq, 2 × 100 mL). Organics were dried over Na₂SO₄, filtered, and the solvent was removed under reduced pressure. The crude product was purified by flash chromatography. Characterization data can be found in the supporting information for all final compounds.

N-(2-((3-Cyano-5,7-dimethylquinolin-2-yl)amino)ethyl)-2-methoxybenzamide (14)

Yellow solid. Yield 72%. ¹H NMR (600 MHz, CDCl₃) δ 8.31 (s, 1H), 8.24-8.17 (m, 2H), 7.41 (t, J = 7.4, 1H), 7.30 (s, 1H), 7.06 (t, J = 7.4, 1H), 6.96 – 6.88 (m, 2H), 5.85 (s, 1H), 3.90 – 3.78 (m, 9H), 2.52 (s, 3H), 2.41 (s, 3H); ¹³C NMR (150 MHz, CDCl₃) δ 166.69, 157.65, 154.31, 143.81, 140.49, 135.15, 133.00, 132.45, 131.07, 128.98, 126.76, 124.66, 121.49, 119.08, 117.09, 111.45, 94.37, 56.13, 42.05, 39.91, 22.31, 18.82. ESI-MS m/z [M+H]⁺: 375.18. Purity: 98%.

N-(2-((3-Cyano-5,7-dimethylquinolin-2-yl)amino)ethyl)-4-methoxybenzamide (15)

Yellow solid. Yield: 72% ¹H NMR (600 MHz, CDCl₃) δ 8.26 (s, 1H), 7.87 (s, 1H), 7.61 (d, J = 8.0, 2H), 7.24 (s, 1H), 6.90 (s, 1H), 6.67 (d, J = 8.7, 2H), 5.80 (s, 1H), 3.83 (q, J = 5.2, 3H), 3.70 (s, 3H), 3.67 (q, J = 5.2, 2H), 2.46 (s, 3H), 2.37 (s, 3H). 13C NMR (150 MHz, CDCl₃) δ 167.82, 162.18, 155.18, 149.70, 144.14, 140.93, 135.45, 132.48, 129.16, 127.12, 124.50, 119.39, 116.87, 113.66, 94.49, 55.43, 43.09, 41.29, 22.15, 18.47. ESI-MS m/z [M+H]⁺: 375.18. Purity: 98%.

N-(2-((3-Cyano-5,7-dimethylquinolin-2-yl)amino)ethyl)picolinamide (16)

Yellow solid. Yield: 60%. ¹H NMR (600 MHz, CDCl₃) δ 8.87 (s, 1H), 8.43 – 8.41 (m, 1H), 8.32 (s, 1H), 8.20 (d, J = 7.8, 1H), 7.81 (td, J = 7.7, 1.7, 1H), 7.49 (s, 1H), 7.40 – 7.35 (m, 1H), 6.95 (s, 1H), 5.78 (s, 1H), 3.91 (dd, J = 11.3, 5.7, 2H), 3.81 (dd, J = 11.2, 5.6, 2H), 2.53 (s, 3H), 2.47 (s, 3H); ¹³C NMR (150 MHz, CDCl₃) δ 165.40, 154.49, 150.13, 148.14, 143.59, 140.55, 137.44, 135.08, 126.82, 126.28, 125.02, 122.49, 119.24, 117.07, 94.17, 41.66, 40.86, 22.30, 18.53. ESI-MS m/z [M+H]⁺: 346.16. Purity: 96%.

N-(2-((3-Cyano-5,7-dimethylquinolin-2-yl)amino)ethyl)nicotinamide (17)

Yellow solid. Yield: 66%. ¹H NMR (600 MHz, CDCl₃) δ 8.93 (s, 1H), 8.66 (d, J = 4.6, 1H), 8.38 (s, 1H), 8.34 (s, 1H), 8.00 (d, J = 7.8, 1H), 7.24 – 7.20 (m, 2H), 7.00 (s, 1H), 5.79 (s, 1H), 3.94 (q, J = 5.6, 2H), 3.77 (q, J = 4.3, 2H), 2.56 (s, 3H), 2.43 (s, 3H); ¹³C NMR (150 MHz, CDCl₃) δ 166.28, 155.17, 152.18, 148.59, 144.58, 141.05, 135.54, 135.18, 130.46, 127.41, 124.24, 123.35, 119.45, 116.77, 94.37, 43.57, 41.25, 22.26, 18.55. ESI-MS m/z [M+H]⁺: 346.16. Purity: 97%.

N-(2-((3-Cyano-5,7-dimethylquinolin-2-yl)amino)ethyl)isonicotinamide (18)

Yellow solid. Yield: 69%. ¹H NMR (600 MHz, CDCl₃) δ 8.53 – 8.46 (m, 3H), 8.33 (s, 1H), 7.45 (d, J = 4.5, 2H), 7.17 (s, 1H), 6.96 (s, 1H), 5.71 (s, 1H), 3.88 (q, J = 5.4, 2H), 3.68 (q, J = 5.0, 2H), 2.51 (s, 3H), 2.38 (s, 3H); 13C NMR (151 MHz, cdcl3) δ 166.03, 155.37, 150.49, 144.68, 141.89, 141.18, 135.74, 127.49, 124.18, 121.36, 119.54, 116.72, 113.58, 94.48, 77.43, 77.22, 77.01, 44.14, 41.00, 22.27, 18.56. ESI-MS m/z [M+H]⁺: 346.16. Purity: 98%.

N-(2-((3-Cyano-5,7-dimethylquinolin-2-yl)amino)ethyl)benzamide (19)

Yellow solid. Yield: 84%. ¹H NMR (600 MHz, CDCl₃) δ 8.35 (s, 1H), 8.14 (s, 1H), 7.71 (d, J = 8.0, 2H), 7.41 (t, J = 7.4, 1H), 7.28 (s, 1H), 7.26 (t, J = 5.5, 1H), 6.98 (s, 1H), 5.82 (t, J = 5.5, 1H), 3.91 (q, J = 5.7, 2H), 3.75 (q, J = 4.6, 2H), 2.54 (s, 3H), 2.43 (s, 3H); 13C NMR (151 MHz, cdcl3) δ 168.16, 155.19, 149.55, 144.15, 140.96, 135.46, 131.40, 130.30, 128.49, 127.40, 127.15, 124.46, 119.40, 116.89, 94.37, 43.40, 41.25, 22.24, 18.60. ESI-MS m/z [M+H]⁺: 345.12. Purity: 99%.

N-(2-((3-Cyano-5,7-dimethylquinolin-2-yl)amino)ethyl)-2,4-dimethoxybenzamide (20)

Yellow solid. Yield: 52%. ¹H NMR (600 MHz, CDCl₃) δ 8.28 (s, 1H), 8.18 (d, J = 8.7, 1H), 8.04 (s, 1H), 7.29 (s, 1H), 6.91 (s, 1H), 6.57 (dd, J = 8.8, 2.2, 1H), 6.40 (d, J = 2.1, 1H), 5.87 (s, 1H), 3.86 – 3.76 (m, 10H), 2.51 (s, 3H), 2.40 (s, 3H); ¹³C NMR (150 MHz, CDCl₃) δ 166.26, 163.58, 159.00, 154.31, 150.21, 143.68, 140.37, 135.08, 134.17, 126.67, 124.71, 119.05, 117.09, 114.68, 105.41, 98.69, 94.18, 56.06, 55.70, 42.11, 39.57, 22.21, 18.52. ESI-MS m/z [M+H]⁺: 405.15. Purity: 98%.

N-(2-((3-Cyano-5,7-dimethylquinolin-2-yl)amino)ethyl)-3,4-dimethoxybenzamide (21)

Yellow solid. Yield: 41%. ¹H NMR (600 MHz, CDCl₃) δ 8.37 (s, 1H), 7.89 (s, 1H), 7.36 (d, J = 1.8, 1H), 7.29 (s, 1H), 7.18 (dd, J = 8.3, 1.9, 1H), 6.99 (s, 1H), 6.65 (d, J = 8.3, 1H), 5.78 (s, 1H), 3.92 (q, J = 5.8, 2H), 3.86 (s, 3H), 3.78 (s, 3H), 3.75 (q, J = 5.3, 2H), 2.56 (s, 3H), 2.43 (s, 3H); ¹³C NMR (150 MHz, CDCl₃) δ 168.07, 155.20, 151.72, 149.68, 149.04, 144.18, 140.94, 135.52, 127.57, 127.11, 124.58, 119.87, 119.40, 116.89, 110.89, 110.24, 94.37, 56.11, 56.09, 43.14, 41.37, 22.25, 18.59. ESI-MS m/z [M+H]⁺: 405.15. Purity: 98%.

N-(2-((3-Cyano-5,7-dimethylquinolin-2-yl)amino)ethyl)pyrazine-2-carboxamide (22)

Yellow solid. Yield: 38%. ¹H NMR (600 MHz, CDCl₃) δ 9.36 (s, 1H), 8.94 (s, 1H), 8.66 – 8.65 (m, 1H), 8.32 (s, 1H), 8.32 – 8.30 (m, 1H), 7.48 (s, 1H), 6.96 (s, 1H), 5.76 (s, 1H), 3.92 (q, J = 5.0, 2H), 3.80 (q, J = 5.2, 2H), 2.53 (s, 3H), 2.47 (s, 3H); ¹³C NMR (150 MHz, CDCl₃) δ 163.98, 154.64, 149.96, 147.22, 144.85, 144.53, 143.61, 142.56, 140.67, 135.19, 126.93, 124.93, 119.32, 116.98, 94.15, 41.66, 41.17, 22.35, 18.53. ESI-MS m/z [M+H]⁺: 376.12. Purity: 97%.

N-(2-((3-Cyano-5,7-dimethylquinolin-2-yl)amino)ethyl)-5-methoxypicolinamide (23)

Yellow solid. Yield: 78%. ¹H NMR (600 MHz, CDCl₃) δ 8.69 (s, 1H), 8.29 (s, 1H), 8.13 (d, J = 8.6, 1H), 8.05 (d, J = 2.8, 1H), 7.47 (s, 1H), 7.22 (dd, J = 8.6, 2.9, 1H), 6.93 (s, 1H), 5.81 (t, J = 5.5, 1H), 3.88 (q, J = 5.5, 2H), 3.85 (s, 3H), 3.78 (q, J = 5.6, 2H), 2.51 (s, 3H), 2.46 (s, 3H); ¹³C NMR (150 MHz, CDCl₃) δ 165.32, 157.93, 154.51, 150.12, 143.50, 142.88, 140.49, 136.52, 135.06, 126.74, 125.02, 123.58, 120.18, 119.19, 117.08, 94.16, 55.88, 41.73, 40.79, 22.31, 18.52. ESI-MS m/z [M+H]⁺: 376.12. Purity: 99%.

N-(2-((3-Cyano-5,7-dimethylquinolin-2-yl)amino)ethyl)-2,6-dimethoxybenzamide (24)

¹H NMR (600 MHz, CDCl₃) δ 8.30 (s, 1H), 7.26 (t, J = 4.3, 1H), 7.22 (t, J = 8.4, 1H), 6.98 (s, 1H), 6.90 (s, 1H), 6.49 (d, J = 8.4, 2H), 5.69 (t, J = 5.6, 1H), 3.85 (q, J = 5.6, 2H), 3.74 (q, J = 4.9, 2H), 3.69 (s, 6H), 2.51 (s, 3H), 2.33 (s, 3H); ¹³C NMR (150 MHz, CDCl₃) δ 166.38, 157.60, 154.45, 149.77, 143.85, 140.52, 135.05, 130.63, 126.84, 124.35, 119.10, 116.95, 116.35, 104.15, 94.19, 56.15, 41.58, 41.35, 22.17, 18.47. ESI-MS m/z [M+H]⁺: 405.15. Purity: 99%.

2-(4-(2-Methoxybenzoyl)piperazin-1-yl)-5,7-dimethylquinoline-3-carbonitrile (25)

Yellow solid. Yield: 40%. ¹H NMR (600 MHz, CDCl₃) δ = 8.47 (s, 1H), 7.44 (s, 1H), 7.37 (t, J=7.9, 1H), 7.28 (m, 1H), 7.07 (s, 1H), 7.01 (t, J=7.4, 1H), 6.94 (d, J=8.3, 1H), 4.03 (m, 2H), 3.85 (s, 3H), 3.76 (dd, J=6.4, 10.8, 2H), 3.54 (m, 4H), 2.58 (s, 3H), 2.47 (s, 3H); ¹³C NMR (150 MHz, CDCl₃) δ = 168.23, 157.75, 155.51, 149.20, 144.02, 142.73, 134.97, 130.82, 128.44, 128.29, 125.72, 125.36, 121.24, 120.47, 118.22, 111.13, 97.67, 55.80, 49.62, 48.95, 46.90, 41.69, 22.29, 18.54. ESI-MS m/z [M+H]⁺: 401.13. Purity: 93%.

2-(4-(3-Methoxybenzoyl)piperazin-1-yl)-5,7-dimethylquinoline-3-carbonitrile (26)

Yellow solid. Yield: 50%. ¹H NMR (600 MHz, CDCl₃) δ = 8.49 (s, 1H), 7.44 (s, 1H), 7.33 (td, J=1.4, 7.4, 1H), 7.08 (s, 1H), 6.98 (m, 3H), 3.83 (s, 3H), 3.76 (m, 8H), 2.58 (s, 3H), 2.47 (s, 3H); ¹³C NMR (150 MHz, CDCl₃) δ = 170.51, 159.92, 157.76, 149.15, 144.05, 142.69, 137.10, 134.99, 129.90, 128.54, 125.40, 120.55, 119.30, 118.14, 115.93, 112.64, 97.77, 55.59, 49.59, 49.18, 47.71, 42.20, 29.91, 22.29, 18.54. ESI-MS m/z [M+H]⁺: 401.26. Purity: 99%.

2-(4-(4-Methoxybenzoyl)piperazin-1-yl)-5,7-dimethylquinoline-3-carbonitrile (27)

Yellow solid. Yield: 45%. ¹H NMR (600 MHz, CDCl₃) δ = 8.47 (s, 1H), 7.44 (m, 3H), 7.07 (s, 1H), 6.94 (d, J=8.8, 2H), 3.84 (s, 8H), 3.83 (m, 8H), 2.57 (s, 3H), 2.47 (s, 3H); ¹³C NMR (150 MHz, CDCl₃) δ = 170.48, 161.10, 157.75, 144.02, 142.69, 134.98, 129.43, 128.49, 127.82, 125.38, 120.51, 118.18, 114.00, 97.72, 55.62, 49.33, 29.91, 22.29, 21.60, 18.86, 18.53. ESI-MS m/z [M+H]⁺: 401.13. Purity: 93%.

5,7-Dimethyl-2-(4-picolinoylpiperazin-1-yl)quinoline-3-carbonitrile (28)

Yellow Solid. Yield: 40%. ¹H NMR (600 MHz, CDCl₃) δ = 8.62 (d, J=4.8, 1H), 8.48 (s, 1H), 7.83 (t, 7=8.4, 1H), 7.71 (d, J=7.8, 1H), 7.43 (s, 1H), 7.38 (dd, J=5.2, 7.2, 1H), 7.07 (s, 1H), 3.85 (m, 8H), 2.58 (s, 3H), 2.47 (s, 3H); ¹³C NMR (150 MHz, CDCl₃) δ = 167.89, 157.64, 154.06, 149.20, 148.56, 143.99, 142.74, 137.36, 134.94, 128.41, 125.38, 124.86, 124.28, 120.46, 118.25, 97.55, 49.58, 48.82, 47.23, 42.46, 22.28, 18.53. ESI-MS m/z [M+H]⁺: 360.24. Purity: 99%.

5,7-Dimethyl-2-(4-nicotinoylpiperazin-1-yl)quinoline-3-carbonitrile (29)

Yellow solid. Yield: 49%. ¹H NMR (600 MHz, CDCl₃) δ = 8.73 (s, 1H), 8.70 (d, J=3.7, 1H), 8.50 (s, 1H), 7.81 (d, J=7.8, 1H), 7.45 (s, 1H), 7.40 (dd, J=4.9, 7.8, 1H), 7.09 (s, 1H), 4.02 (s, 2H), 3.70 (m, 6H), 2.59 (s, 3H), 2.48 (s, 3H); ¹³C NMR (150 MHz, CDCl₃) δ = 168.14, 157.66, 151.16, 149.09, 148.25, 144.13, 142.70, 135.29, 135.01, 131.64, 128.66, 125.42, 123.73, 120.62, 118.07, 97.80, 49.42, 49.15, 47.79, 42.35, 22.29, 18.54. ESI-MS m/z [M+H]⁺: 360.31. Purity: 99%.

2-(4-Isonicotinoylpiperazin-1-yl)-5,7-dimethylquinoline-3-carbonitrile (30)

Yellow solid. Yield: 50%. ¹H NMR (600 MHz, CDCl₃) δ = 8.74 (d, J=5.6, 2H), 8.50 (s, 1H), 7.45 (s, 1H), 7.34 (d, J=5.8, 2H), 7.10 (s, 1H), 4.01 (s, 2H), 3.76 (s, 2H), 3.61 (s, 4H), 2.59 (s, 3H), 2.48 (s, 3H); ¹³C NMR (150 MHz, CDCl₃) δ = 168.10, 157.67, 150.62, 149.08, 144.16, 143.40, 142.70, 135.03, 128.71, 125.42, 121.45, 120.65, 118.04, 97.85, 49.45, 49.12, 47.45, 42.08, 22.30, 18.55. ESI-MS m/z [M+H]⁺: 360.31. Purity: 99%.

N-(3-(3-Cyano-5,7-dimethylquinolin-2-ylamino)propyl)-2-methoxybenzamide (31)

Yellow solid. Yield: 88%. ¹H NMR (600 MHz, CDCl₃) δ = 8.28 (s, 1H), 8.21 (t, J=5.3, 1H), 8.18 (dd, J=1.8, 7.8, 1H), 7.44 (td, J=1.8, 8.4, 1H), 7.20 (s, 1H), 7.07 (t, J=7.9, 1H), 6.97 (d, J=8.2, 1H), 6.89 (s, 1H), 5.76 (t, J=5.7, 1H), 3.92 (s, 4H), 3.74 (q, J=6.3, 2H), 3.59 (q, J=6.2, 2H), 2.51 (s, 3H), 2.36 (s, 3H), 1.98 (m, 2H); ¹³C NMR (150 MHz, CDCl₃) δ = 166.01, 157.51, 154.46, 150.24, 143.66, 140.43, 135.16, 132.75, 132.37, 126.51, 124.41, 122.32, 121.44, 118.93, 117.23, 111.40, 94.16, 56.11, 38.60, 37.11, 30.17, 22.25, 18.55. ESI-MS m/z [M+H]⁺: 389.32. Purity: 99%.

N-(3-(3-Cyano-5,7-dimethylquinolin-2-ylamino)propyl)-3-methoxybenzamide (32)

Yellow solid. Yield: 87%. ¹H NMR (600 MHz, CDCl₃) δ = 8.30 (s, 1H), 7.66 (t, J=6.0, 1H), 7.42 (m, 2H), 7.35 (t, J=7.9, 1H), 7.11 (s, 1H), 7.06 (dd, J=2.5, 8.1, 1H), 6.90 (s, 1H), 5.65 (t, J=6.3, 1H), 3.83 (s, 2H), 3.80 (dd, J=6.3, 12.2, 2H), 3.51 (dd, J=6.1, 12.0, 2H), 2.51 (s, 3H), 2.30 (s, 3H), 1.92 (m, 2H); ¹³C NMR (150 MHz, CDCl₃) δ = 168.26, 159.97, 154.81, 149.96, 144.07, 140.81, 137.26, 135.35, 129.67, 126.72, 124.17, 119.32, 119.01, 117.62, 117.14, 112.70, 94.10, 55.60, 37.89, 36.37, 30.43, 30.42, 22.12, 18.54. ESI-MS m/z [M+H]⁺: 389.32. Purity: 99%.

N-(3-(3-Cyano-5,7-dimethylquinolin-2-ylamino)propyl)-4-methoxybenzamide (33)

Yellow solid. Yield: 81%. ¹H NMR (600 MHz, CDCl₃) δ = 8.30 (s, 1H), 7.83 (d, J=8.6, 2H), 7.50 (s, 1H), 7.20 (s, 1H), 6.92 (m, 3H), 5.77 (s, 1H), 3.84 (s, 3H), 3.80 (dd, J=5.9, 11.8, 2H), 3.51 (dd, J=6.1, 11.9, 2H), 2.51 (s, 3H), 2.34 (s, 3H), 1.93 (m, 2H); ¹³C NMR (150 MHz, CDCl₃) δ = 167.96, 162.21, 154.61, 135.47, 130.31, 129.31, 128.52, 127.87, 127.25, 126.78, 124.01, 118.85, 117.05, 113.91, 94.27, 55.61, 38.14, 36.46, 30.32, 22.22, 18.55. ESI-MS m/z [M+H]⁺: 389.32. Purity: 99%.

N-(3-(3-Cyano-5,7-dimethylquinolin-2-ylamino)propyl)picolinamide (34)

Yellow solid. Yield: 42%. ¹H NMR (600 MHz, CDCl₃) δ = 8.72 (s, 1H), 8.58 (d, J=4.2, 1H), 8.27 (s, 1H), 8.18 (d, J=7.8, 1H), 7.80 (td, J=1.7, 7.7, 1H), 7.59 (s, 1H), 7.39 (ddd, J=1.1, 4.7, 7.5, 1H), 6.87 (s, 1H), 5.56 (t, J=5.9, 1H), 3.70 (q, J=6.2, 2H), 3.51 (q, J=6.4, 2H), 2.48 (s, 3H), 2.36 (s, 3H), 1.89 (quint., J=6.2, 2H); ¹³C NMR (150 MHz, CDCl₃) δ = 167.44, 164.92, 154.65, 150.40, 148.15, 143.70, 140.54, 137.55, 135.13, 126.65, 126.32, 124.84, 122.67, 119.08, 117.24, 94.09, 38.25, 36.50, 30.60, 22.33, 18.55. ESI-MS m/z [M+H]⁺: 372.24. Purity: 99%.

N-(3-(3-Cyano-5,7-dimethylquinolin-2-ylamino)propyl)nicotinamide (35)

Yellow solid. Yield: 40%. ¹H NMR (600 MHz, CDCl₃) δ = 9.09 (s, 1H), 8.77 (d, J=6.3, 1H), 8.34 (s, 1H), 8.15 (d, J=7.9, 1H), 7.70 (s, 2H), 7.41 (dd, J=4.8, 7.8, 1H), 7.03 (s, 1H), 6.92 (s, 1H), 5.51 (t, J=6.4, 1H), 3.82 (s, 2H), 3.53 (s, 2H), 2.53 (s, 3H), 2.31 (s, 3H), 1.94 (quint., J=5.5, 2H); ¹³C NMR (150 MHz, CDCl₃) δ = 166.37, 154.83, 152.30, 149.84, 148.46, 144.32, 140.91, 135.50, 135.49, 131.46, 126.91, 124.03, 123.64, 119.11, 117.03, 94.19, 37.89, 36.43, 30.41, 22.18, 18.53. ESI-MS m/z [M+H]⁺: 372.24. Purity: 99%.

N-(3-(3-Cyano-5,7-dimethylquinolin-2-ylamino)propyl)isonicotinamide (36)

Yellow solid. Yield: 42%. ¹H NMR (600 MHz, CDCl₃) δ = 8.70 (d, J=4.4, 2H), 8.28 (s, 1H), 7.73 (s, 1H), 7.61 (dd, J=1.6, 4.3, 2H), 6.92 (s, 1H), 6.87 (s, 1H), 5.43 (d, J=6.0, 1H), 3.76 (q, J=6.3, 2H), 3.46 (q, J=5.8, 2H), 2.47 (s, 3H), 2.23 (s, 3H), 1.87 (m, 2H); ¹³C NMR (150 MHz, CDCl₃) δ = 166.36, 154.88, 150.74, 149.78, 144.33, 142.93, 140.96, 135.62, 126.97, 123.86, 121.50, 119.15, 116.95, 94.26, 37.84, 36.40, 30.40, 22.20, 18.54. ESI-MS m/z [M+H]⁺: 372.24. Purity: 99%.

Constrained Calculation for 5

The conformer of structure 5 that could easily interconvert to a conformation similar to the lowest energy structure 26 conformer (see Fig. 7 and associated text in manuscript) was modified to place the two aryl moieties in the same relative disposition as in 26. The two rings were then held in this configuration with a series of distance constraints while the intervening tether was allowed to relax to best accommodate the rings. The figure below illustrates these constraints via long bond lines drawn between each set of atoms for which the distances were constrained. The resulting computed electronic energy (constrained optimization at the M062X/6-31+G(d,p) level with SMD water continuum model) was then compared to the electronic energy computed at the same level of theory for both the starting (shown in Fig. 7) and lowest energy conformers of 5. These relative energies were found to be 4.48 and 8.35 kcal/mol, respectively.

ABBREVIATIONS USED

CF

cystic fibrosis

CFTR

cystic fibrosis transmembrane conductance regulator

ΔF508-CFTR

deletion of phenylalanine-508 in CFTR

CoPo

corrector and potentiator

THF

tetrahydrofuran

DMF

dimethylformamide

DCM

dichloromethane

EDC

1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride

LCMS

liquid chromatography mass spectrometry