Efficient Regioselective Synthesis of Benzimidazoles and Azabenzimidazoles to Enable the Rapid Development of Structure-Activity Relationships for Activation of SLACK Potassium Channels

Abstract

The sodium activated potassium channel known as SLACK (K_Na1.1 or Slo2.2) is widely expressed in the central nervous system and represents a potential target for the treatment of several neurological disorders. While much recent progress has been made toward the discovery of small molecule inhibitors of these channels, reports regarding small molecule activators have been scant. Having identified such compounds via a high-throughput screen, we were interested in establishing structure-activity relationships that could serve as the foundation for the design of potent activators of SLACK channels. In this Letter, we describe the implementation of an efficient synthetic approach to the regioselective synthesis of a series of benzimidazole and azabenzimidazoles based on one of our hit compounds. The key step utilizes a one-pot reduction/formylation/condensation reaction of 2-nitro-arylamines. Also presented herein is functional activity for 15 new analogs prepared by this approach and obtained via a thallium-flux assay in cells stably expressing human wild-type SLACK channels. Many of these new analogs demonstrated substantially improved potency relative to the initial hit compound and provide valuable new data that can be utilized in the design of additional derivatives.

Graphical Abstract

SLACK, also known as K_Na1.1 or Slo2.2, is a sodium-activated potassium (K⁺) channel that is widely expressed in the central nervous system (CNS) where it plays a pivotal role in the regulation of neuronal excitability.^3–5 SLACK, which stands for Sequence Like A Calcium-activated K⁺ channel, is encoded by the KCNT1 gene. Numerous gain-of-function (GOF) mutations in KCNT1 have been linked to certain childhood epilepsies, most notably epilepsy of infancy with migrating focal seizures (EIMFS).^6–13 While rare, EIMFS is a devastating and pharmacoresistant syndrome that causes severe cognitive impairment, developmental delays, and reduced life expectancy. Thus, the discovery of small molecule inhibitors of SLACK channels represents a potential avenue toward a new therapeutic strategy for these patients.^14–16 In fact, multiple efforts centered on the discovery of novel small molecule SLACK inhibitors have recently been reported in the peer-reviewed literature.^17–24

While progress toward the identification of small molecule inhibitors of SLACK channels has been notable, the discovery of activators of these channels has not been nearly as rapid. Indeed, activators of SLACK channels may also have therapeutic relevance. For example, prior work has shown that SLACK is activated by the binding of fragile X mental retardation protein 1 (FMRP).^25–29 Genetic silencing of FMR1, the gene that encodes FMRP, is the hallmark of fragile X syndrome (FXS), the most common cause of inherited intellectual disability (ID) and monogenic autism spectrum disorder (ASD).^30–35 Studies in neurons isolated from brain slices obtained from Fmr1^−/y mice showed reduced sodium-activated potassium currents despite a level of SLACK expression on par with wild-type (WT) mice.²⁹ Furthermore, behavioral studies in genetically modified mice have demonstrated the importance of SLACK activity for higher cognitive function.²⁶ Thus, a small molecule activator of SLACK channels may be worth investigating as a mechanism to improve the cognitive deficits observed in FXS patients.

The antipsychotic loxapine (Figure 1) is a moderately potent activator of SLACK channels;³⁶ however, it interacts with several other targets more potently. Specifically, loxapine is a potent antagonist of dopamine D₂, serotonin 5-HT_2A, and histamine H₁ receptors.³⁷ Still, studies in mice with loxapine have indicated a potential role for SLACK activation in the treatment of neuropathic pain.³⁸ Additionally, a team of researchers from Goethe University Frankfurt (Germany) recently disclosed the discovery of compound 1 (Figure 1), a loxapine analog with negligible affinity for dopamine receptors D₂ and D₃.³⁹ Functional potency for SLACK activation in a cell-based thallium (Tl⁺)-flux assay⁴⁰ in HEK-293 cells stably expressing human SLACK was similar for both loxapine (EC₅₀ = 20.7 μM; E_max = 100%) and 1 (EC₅₀ = 30.3 μM; E_max = 96.2%). Motivated by studies finding that itch-associated subsets of sensory neurons show enriched expression of SLACK, the authors tested 1 in multiple mouse models of pruritus, observing efficacy at doses of 10–30 mg/kg.³⁹

Figure 1.

Previously reported SLACK activators loxapine and loxapine analog 1.

We became interested in the discovery of small molecule SLACK modulators several years ago and initiated a high-throughput screen (HTS) of approximately 100,000 compounds using a Tl⁺-flux assay⁴¹ in HEK-293 cells stably expressing WT human SLACK.²³ While we have reported our efforts to optimize multiple chemically distinct inhibitor hits identified from this HTS campaign,^{17, 19–21} we identified several validated SLACK activators as well. Recognizing the potential value of a potent and selective SLACK activator tool compound, we turned our attention to HTS hit 2 (Figure 2). Although 2 was only a weak activator of SLACK channels, synthesis of a small library in the western region of the scaffold quickly identified benzimidazole 3 as an analog with improved potency.

Figure 2.

Initial SLACK activators: HTS hit 2 and benzimidazole derivative 3. E_max defined as the amplitude of response in the presence of 30 μM test compound as a percentage of the maximum response for loxapine.

Benzimidazole 3 was attractive as a launch point for additional optimization for several reasons. First, standard calculated properties (calculated via ChemDraw^® Professional v. 16.0) were generally encouraging. For instance, values were consistent with Lipinski’s guidelines; molecular weight (423.45 Da), cLogP (3.49), H-bond donors (0), and H-bond acceptors (5) all satisfied criteria.⁴² Likewise, other parameters such as topological polar surface area (53.0 Ų) and the number of rotatable bonds (4) were in line with values associated with CNS-penetrant molecules.^{43, 44} Finally, the recently described blood-brain barrier (BBB) score, which defines values between 4 and 6 as indicative of good passive CNS permeability,⁴⁵ was calculated and found to be 4.48.

Synthesis of analog 3 was quite straightforward, allowing access to the analog in only two steps (Scheme 1). Reaction of 4-piperidin-4-ylmethanol 4 with 3-(trifluoromethyl)benzene-sulfonyl chloride under basic conditions provided intermediate 5 in good yield. While the new sulfonamide bond was quite stable, the new sulfonate ester of 5 represented a good leaving group. Thus, treatment of 5 with benzimidazole and base under microwave irradiation provided the target analog 3 in moderate yield. Unfortunately, were this route to be used with substituted benzimidazoles or azabenzimidazoles, a mixture of regioisomers would have resulted.

Scheme 1.

Synthesis of benzimidazole 3. Reaction conditions: (a) 3-(trifluoromethyl)-benzenesulfonyl chloride, DMAP, NEt₃, CH₂Cl₂, 74%; (b) benzimidazole, Cs₂CO₃, DMF, μwave, 150 °C, 30 min, 38%.

Having already generated structure-activity relationships (SAR) in the eastern sulfonamide region of the chemotype that failed to identify significant potency improvements over 3, we were eager to evaluate the western region. To do so efficiently, we envisioned use of an approach that would employ a one-pot reduction/formylation/condensation reaction of an intermediate such as 6 (Scheme 2). Such an approach would allow incorporation of a nitrogen atom at four different locations (W-Z), allowing access to azabenzimidazole analogs. Likewise, the approach would work equally well with substituted carbons at any of the same locations (W-Z). Critically, this approach would ensure regioselectivity. By utilizing a nucleophilic addition of a primary amine to a 2-halonitroarene to prepare 6, we could establish which nitrogen atom of the resultant benzimidazole or azabenzimidazole would be alkylated and avoid the regioisomers that would have resulted from using an N-alkylation approach. Our plan was to utilize a palladium-catalyzed transfer hydrogenation of 6 where formic acid served as both the source of hydrogen and the requisite formyl group. Searching of the literature revealed support for this general concept.^46–48

Scheme 2.

General strategy for the regioselective synthesis

Synthesis of analogs 7 began with commercial Boc-protected piperidine monomer 8 (Scheme 3). Reaction of 8 with 3-(trifluoromethyl)benzenesulfonyl chloride as before provided intermediate 9 in excellent yield. Removal of the tert-butyl carbamate protecting group was accomplished via treatment with trifluoracetic acid to afford primary amine salt 10. Reaction of 10 with the appropriate nitroaryl halide in a nucleophilic aromatic substation gave the key penultimate intermediates 6 in moderate to good yields. As expected, nitroaryl fluorides were generally more reactive than nitroaryl chlorides in this reaction. Gratifyingly, heating of 6 with 10% palladium on carbon and sodium bicarbonate in formic acid provided the desired analogs 7 in low to moderate yield with an average yield of 40 ± 11% (see Supporting Information for individual results). The reaction conditions were not optimized further. It may very well be possible to further enhance the yields through systematic screening of alternative catalysts, catalyst loading, hydrogen sources, solvents, and/or temperature. While the yields were low at times, we were pleased that all three transformations could be carried out in one-pot, allowing us to isolate sufficient quantities of pure compounds for pharmacological testing.

Scheme 3.

Regioselective synthesis of analogs 7. Reaction conditions: (a) 3-(trifluoromethyl)benzenesulfonyl chloride, DMAP, NEt₃, CH₂Cl₂, 100%; (b) CF₃CO₂H, CH₂Cl₂, 99%; (c) Nitroaryl halide, DIEA, CH₃CN, 47–85%; (d) 10% Pd/C, NaHCO₃, HCO₂H, 110 °C, 20–66%.

Using the approach outlined above, we were able to prepare and test 11 different substituted benzimidazoles for their ability to activate SLACK channels (Table 1). We chose to systematically target each position (4–7) on the benzimidazole with methyl, methoxy, and trifluoromethyl substituents, each common moieties in drugs and with varied electronic character. Only the 4-trifluoromethyl analog proved elusive as low conversion prevented our isolation of a sample with sufficient purity for testing. Analysis of the SAR revealed that substitution improved potency in many cases. Notably, methyl (7a-d) and methoxy (7e-h) substituents were preferred to the strong electron withdrawing trifluoromethyl group (7i-k). Perhaps this result is an indication that the N³-nitrogen of the benzimidazole makes a key hydrogen bond contact within the binding site that is weakened by an electron withdrawing group on the ring. The 4- and 6-position analogs showed similar potencies with both methyl (7a and 7c) and methoxy (7e and 7g) substituents. Potency and maximum response for 5-substituted analogs 7b (Me) and 7-f (OMe) were modestly reduced compared to 4- and 6-position analogs. While the 7-methyl analog 7d showed good potency, the maximum response was considerably reduced. On the other hand, 7-methoxy analog 7h showed reduced potency but a maximum response on par with more potent analogs. Understanding how potency and maximum response values translate to an impact on the function of SLACK in a native system would require several additional studies; however, this exercise successfully identified multiple analogs that might serve as useful tools in that regard. Investigation of whether such results are indicative of differences in binding kinetics, allosteric effects, or partial activation of the channel could prove illuminating.

Table 1.

Substituted benzimidazole analogs

Entry	R	SLACK EC50 (μM)1	SLACK Emax (%)1,2
3	H	4.3	86
7a	4-Me	1.9	90
7b	5-Me	2.4	52
7c	6-Me	1.7	67
7d	7-Me	1.3	30
7e	4-OMe	1.3	62
7f	5-OMe	2.1	59
7g	6-OMe	1.6	88
7h	7-OMe	6.2	74
7i	5-CF3	>10	<10
7j	6-CF3	3.0	82
7k	7-CF3	6.6	40

¹Concentration-response curve (CRC) from Tl⁺-flux assay in HEK-293 cells expressing WT human SLACK

²Amplitude of response in the presence of 30 μM test compound as a percentage of the max response for loxapine

Reducing lipophilicity can be a viable strategy for the optimization of multiple important parameters relevant to a safe and effective drug, including the reduction of protein binding, cytochrome P450 inhibition, metabolism, and hERG inhibition.^49–53 Thus, we were keen to learn whether azabenzimidazole analogs were tolerated in this chemotype to evaluate its potential to allow for solving these types of common challenges down the road. Gratifyingly, we were able to prepare all four azabenzimidazole derivatives 7l-7o (Table 2). Only analog 7n demonstrated reduced potency relative to unsubstituted benzimidazole 3. Analogs 7m and 7o both exhibited potency on par with 3, and analog 7l showed a modest enhancement in activity. In theory, the introduction of another nitrogen atom creates the potential for additional binding interactions within the pocket that could lead to enhanced potency; however, we do not observe such to be the case here, with the possible exception of analog 7l. Still, such SAR may indeed prove valuable in the continued optimization of other parameters within this scaffold in the future.

Table 2.

Azabenzimidazole analogs

Entry	SLACK EC50 (μM)1	SLACK Emax (%)1,2
7l	2.9	87
7m	4.3	90
7n	5.7	93
7o	4.0	88

¹Concentration-response curve (CRC) from Tl⁺-flux assay in HEK-293 cells expressing WT human SLACK

²Amplitude of response in the presence of 30 μM test compound as a percentage of the max response for loxapine

In conclusion, we have successfully employed a one-pot reduction/formylation/condensation reaction to efficiently and regioselectively prepare 11 substituted benzimidazole and 4 azabenzimidazole analogs of SLACK activator 3. This approach allowed for the rapid development of SAR in the western region of the chemotype and represents the first report of such work in a non-loxapine scaffold. Notably, one azabenzimidazole analog and multiple substituted benzimidazole analogs exhibited enhanced potency relative to initial lead 3, and each of these are likewise substantial improvements over the original hit 2, which was only weakly active at the channel. Immediate future work for the scaffold will include synthesis of compounds that investigate additional substituents and combine optimal substituents into single analogs. We hope to identify analogs that may be used as tools for studying the potential therapeutic applications of SLACK activation in a variety of diseases and disorders. Key next steps toward that end will include assessment of ancillary pharmacology and evaluation of optimized analogs in native cell cultures.