Discovery of Highly Selective and Potent HDAC3 Inhibitors Based on a 2-Substituted Benzamide Zinc Binding Group

Abstract

The selectivity of histone deacetylase inhibitors (HDACis) is greatly impacted by the zinc binding groups. In an effort to search for novel zinc binding groups, we applied a parallel medicinal chemistry (PMC) strategy to quickly synthesize substituted benzamide libraries. We discovered a series containing 2-substituted benzamides as the zinc binding group which afforded highly selective and potent HDAC3 inhibitors, exemplified by compound 16 with a 2-methylthiobenzamide. Compound 16 inhibited HDAC3 with an IC₅₀ of 30 nM and with unprecedented selectivity of >300-fold over all other HDAC isoforms. Interestingly, a subtle change of the 2-methylthio to a 2-hydroxy benzamide in 20 retains HDAC3 potency but loses all selectivity over HDAC 1 and 2. This significant difference in selectivity was rationalized by X-ray crystal structures of HDACis 16 and 20 bound to HDAC2, revealing different binding modes to the catalytic zinc ion. This series of HDAC3 selective inhibitors served as tool compounds for investigating the minimal set of HDAC isoforms that must be inhibited for the HIV latency activation in a Jurkat 2C4 cell model and potentially as leads for selective HDAC3 inhibitors for other indications.

Histone deacetylases (HDACs) and histone acetyl transferases (HATs) are two enzyme families which regulate histone acetylation level, causing structural changes to chromatin, hence controlling the transcription of DNA. Histone acetylation diminishes the charge–charge interaction between histones and DNA, and this relaxed conformation induces gene expression.^1,2 Eighteen unique isoforms of HDACs have been discovered and are separated into four distinct classes.³ The Zn²⁺-dependent isoforms are class I (HDACs 1, 2, 3, and 8), class II (HDACs 4, 5, 6, 7, 9, and 10), and class IV (HDAC11); the NAD⁺-dependent isoforms are the class III Sirtuins (SirT1–7). HDACs have been the targets for various therapeutics. Small molecule HDAC inhibitors (HDACis) were reported to demonstrate capabilities to regulate gene transcription, regulate cell cycle progression, and induce differentiation and/or apoptosis in cancer cells with a few HDACis being approved for the treatment for oncology.⁴⁻⁷ HDACis have also been broadly explored as HIV latency-reversing agents (LRAs) to activate the latently infected CD4⁺ T cells. Clinically, several HDACi drugs approved for the treatment of cancer have been shown to induce HIV RNA transcription in resting CD4⁺ T cells from the combination antiretroviral therapy (cART) suppressed HIV patients.⁸⁻¹⁰ This HIV latency reversal activity by HDACis was thought to be driven by HDAC3 inhibition,^11,12 which led to the investigation of HDAC3 selective inhibitors.

In the attempt to search for HDAC isoform selective inhibitors, we recently reported two series of HDACs 1, 2, 3 inhibitors: the ethyl ketone series exemplified by compound 1 and the aryl ketone series exemplified by compound 2 (Figure 1).¹³⁻¹⁵ These class I HDAC inhibitors showed efficacy for HIV latency activation in a Jurkat 2C4 cell model and induction of HIV gag p24 protein in patient latent CD4⁺ T cells.¹⁴ In the process of determining the required HDAC subtype inhibition for HIV latency activation, we demonstrated that inhibition of only HDAC1, 2 by compound 3(16) did not show any cellular activity in the HIV latency activation.¹³ We invested our effort to search for highly selective inhibitors of HDAC3. Due to the high homology between HDACs 1 and 2, it has proven very difficult to find a selective inhibitor that distinguishes between these two isoforms. However, there are many reports of HDAC3 selective inhibitors, especially the series of N-(2-aminophenyl)carboxamides represented by compound 4 (RGFP966).¹⁷ A close analog 5 to our reported ethyl ketone and aryl ketone HDAC inhibitors with a methylcarboxamide was described to have improved HDAC3 inhibition compared to 4, with 50-fold selectivity over HDAC1.¹⁸ In this letter, we report our work, taking advantage of a parallel medicinal chemistry (PMC) strategy to quickly synthesize and screen a series of HDAC inhibitors. This effort resulted in the discovery of a series of highly potent and selective HDAC3 inhibitors based on 2-substituted benzamides as the zinc binding group.

Figure 1.

HDAC selective inhibitors with various zinc binding groups.

Parallel medicinal chemistry (PMC) has been widely applied in drug discovery across the industry. Recent technology advancements in automation for synthesis and purification, data analysis, modeling, and in-silico prediction have driven the discovery chemistry effort in our company to become more data guided, knowledge based, and highly collaborative with extensive application of PMC. A PMC library was designed using the common intermediate 11 to carry out an amide coupling using selected carboxylic acids which also contain a potential second zinc binding group in addition to the amide carbonyl. We hypothesized that a zinc binding group capable of bidentate coordination with the HDAC zinc ion might enhance potency; however, it was less clear what, if any, role the zinc binding group may play in HDAC isoform selectivity.

Synthesis of the common intermediate 11 is depicted in Scheme 1, which started by reacting a bromoketone 6 and the protected lysine 7 to form a ketone ester intermediate in DMF with cesium carbonate as base. The imidazole ring in 8 was then prepared by condensation of this ketone ester intermediate with ammonium acetate in toluene under heating. The Boc protection in 8 was selectively removed by the treatment of trifluoroacetic acid in dichloromethane, and the freed amine was then coupled with thiazol-5-carboxylic acid 9 to provide an excellent yield of 10. The benzylcarbamate protection was successfully removed after 10 was stirred in a solution of hydrobromide in acetic acid at room temperature overnight to generate the common intermediate 11. The PMC focus library was designed using the common intermediate 11 with an amide coupling reaction to selected monomer of commercially available or readily prepared benzoic acids with 2-substituents which could potentially coordinate with the HDAC enzyme zinc ion as a bidentate chelation along with the amide carbonyl. These focus library sizes ranged from 24–100 monomers, and the optimization of the structure activity relationship (SAR) was carried out in six rounds of libraries with each round applying different substrates of substituted benzoic acids, pyridinyl carboxylic acids, and 5- and 6-member heteroaryl carboxylic acids.

Scheme 1.

Reagents and conditions: (i) Cs₂CO₃, DMF, rt, 1 h, 97%; (ii) NH₄OAc, toluene, 110 °C, 18 h, 87%; (iii) TFA/DCM, rt, 99%; (iv) HATU, DIEA, DCM, rt, 16 h, 98%; (v) HBr/AcOH, rt, 16 h, 93%; (vi) HATU, TEA, DMF, rt, 16 h.

All compounds from the focus library were tested in in vitro HDAC inhibition assays against HDACs 1, 2, 3, 6, or 8, while at the same time each compound was evaluated for Latency Reversal Activity (LRA) in the Jurkat 2C4 T-cell HIV latency model cell line.¹⁹ The representative compounds are listed in Table 1. We discovered a series of highly selective HDAC3 inhibitors from the PMC libraries. Subtle variation of the substituents led to dramatic changes in subtype selectivity. The 2-methylamino benzamide 13 is a highly HDAC3 selective inhibitor with an IC₅₀ of 41 nM toward HDAC3 and selectivity against other subtypes of over 366-fold (HDAC1/HDAC3). The HDAC3 selective inhibitor 13 showed no LRA activity in the 2C4 Jurkat T-cell model system. When the methyl on the aniline is absent, 14 as a 2-amino-6-fluorobenzamide became a HDAC1, 2, 3 inhibitor with only 4-fold selectivity between HDAC1 and HDAC3 inhibition activity. The HDAC1, 2, 3 inhibitor 14 demonstrated comparable LRA activity to that which we observed in the previous reported class I HDAC inhibitors. However, the 3-aminopyridinyl-2-carboxamide 15 dramatically reduced the inhibition of all HDAC enzymes except HDAC3 with still an IC₅₀ of 1,100 nM. The selectivity of HDAC3 over HDAC1 for 15 is greater than 40-fold, although it is much less active. The most selective HDAC3 inhibitor discovered from this series is 16 with the 2-methylthiobenzamide. This compound afforded an IC₅₀ of 29 nM toward HDAC3 and 690-fold selectivity over HDAC1. The removal of the methyl on the sulfur in 17 significantly reduced the HDAC3 inhibition; however, it also brought back some inhibition activity toward HDAC1 and 2 for much less HDAC3 selectivity. The 2-methyl ether benzamide 18 also showed HDAC3 selectivity with 44-fold over HDAC1, although it is 5-fold less active than the methylthio benzamide 16. On the other hand, when this methyl ether is replaced by a hydroxyl, compound 19 became a HDAC1, 2, 3 inhibitor with high inhibition potencies without any HDAC3 selectivity. The 6-fluoro substituent did not have much impact on the potency and selectivity for both phenol (20) and methyl ether (21). Again interestingly, when the two ortho positions of the benzamide were substituted by hydroxy and methyl ether in 22, it is a HDAC3 selective inhibitor like the methyl ether benzamide 18. Both 4- and 5-methyl ether-2-hydroxybenzamides (23, 24) exhibited the same inhibition pattern of 2-hydroxybenzamide 19 as HDAC1, 2, 3 inhibitors. The 3-methyl ether-2-hydroxybenzamide (25) lost all inhibition activity to all HDACs probably due to unfavorable interactions in the binding pocket. Unlike the aminopyridine compound 15, the 3-hydroxypyridinyl-2-carboxamide compound 26 was a HDAC1, 2, 3 inhibitor. Consistently, all the HDAC3 selective inhibitors in Table 1 displayed no activity in the cellular assay to activate the HIV latency, while all HDAC1, 2, 3 inhibitors showed a certain level of potency and activation in the cellular assay.

Table 1. PMC Library with Various 2-Substituted Benzamide Zinc Binding Groups.

^aReported values are the average of ≥2 independent measurements with standard deviation less than 3-fold of the reported mean.

^bNT = not tested.

The first round PMC focus library led to the discovery of a series of highly HDAC3 selective inhibitors with 2-substituted benzamide. We noticed that when the 2-substituents were good zinc binding groups such as hydroxy and amino, the compounds were mostly likely HDAC1, 2, 3 inhibitors. However, when the 2-substituents were not good zinc binders and even nitrogen, oxygen, and sulfur with substituents, the compounds were HDAC3 selective inhibitors with various potencies and selectivities. To further explore the SAR, PMC focus libraries were designed using benzamide with bulkier 2-substituents to investigate the impact on HDAC3 inhibition and selectivity. Representative compounds are listed in Table 2. HDAC5, 6 inhibition and cellular assay data were not listed since these compounds were not active against those isoforms. The 2-dimethylamino 27 and the 2-N-pyrolyl 28 were much less active in HDAC3 inhibition and less HDAC3 selective compared to the methylamino compound 13. The larger 2-ethylthio substituent (29) also led to a 20-fold loss of HDAC3 inhibition potency relative to the methylthio compound 16, though the HDAC3 selectivity is probably maintained since 29 was completely inactive in HDAC1, 2 inhibition. The electron-withdrawing and bigger substituents trifluoromethyl ether (30) and trifluoroethyl ether (31) led to a significant loss of HDAC3 inhibition activity compared to the methyl ether substituent (18), although 30 and 31 maintained the HDAC3 selectivity. The small electron-withdrawing substituents 2-chloro (33), cyano (34), and sulfinyl (35) also led to a loss of HDAC3 inhibition but still retain certain levels of HDAC3 selectivity. The 2-alkyl substituents such as 2-ethyl (35), 2-cyclopropyl (36), and the fused dihydroindene carboxamide (37) all demonstrated high HDAC3 selective inhibition. The 2-cyclopropylbenzamide 36 had an HDAC3 inhibition activity with an IC₅₀ of 170 nM and 147-fold selectivity over HDAC1. The cyclopropyl (36) may have the shape fits best to the pocket compared to ethyl (35) and fused cyclopentyl (37). This 2-substituted benzamide SAR confirmed that installing nonzinc binding substituents on the 2-position of the benzamide led to HDAC3 selective inhibitors with the methylthio compound 16 as the most potent HDAC3 selective inhibitor.

Table 2. PMC Library with Various 2-Substituted Benzamide Zinc Binding Groups.

^aReported values are the average of ≥2 independent measurements with standard deviation less than 3-fold of the reported mean.

We profiled the HDAC3 selective inhibitor 16 against all subtypes of histone deacetylases 1 to 11. Besides HDAC1 and 2, 16 also showed weak inhibition of HDAC10 (IC₅₀ = 10,000 nM) and HDAC11 (IC₅₀ = 2,000 nM), although this is 69-fold less active relative to HDAC3.

The HDAC3 selective inhibitor 16 was characterized in a global counter screen and pharmacokinetic (PK) tests (Table 3). Compound 16 showed very strong activity toward the potassium ion channel with an hERG IC₅₀ of 16 nM.²⁰ In the pharmacokinetic profiling when dosed at 0.05 mg/kg intravenously (IV) in rats, 16 did not show any drug concentration above the detectable level, presumably due to its high clearance. A similar SAR strategy to the other imidazole core HDAC inhibitors we reported^13−15,21 was applied here to the HDAC3 selective inhibitor series, to improve the off-target selectivity and PK profile (Table 4). Replacing the 2-naphthalenyl with 2-methoxy-3-quinolinyl in compound 38 retained the HDAC3 selectivity and inhibition and at the same time, significantly reduced the hERG activity²² with an IC₅₀ of 1,329 nM. HDAC3 inhibitor 38 also showed limited drug exposure of AUC 0.049 μM·h in the PK test²³ when dosed IV at 0.05 mg/kg. Upon further modification by replacing the imidazole with a 1,3,4-oxodiazole in 39, the HDAC3 inhibition was weaker with an IC₅₀ of 857 nM. The hERG off target activity is also much weaker for 39. Compound 39 also demonstrated a similar PK profile to 38 in the IV PK test in rats. The SAR here proved that this HDAC3 inhibitor lead can be optimized to improve the off-target selectivity and the pharmacokinetic profile.

Table 3. HDAC Screen for HDAC3 Selective Inhibitor 16.

Comp#	HDAC1 IC50 (nM)	HDAC2 IC50 (nM)	HDAC3 IC50 (nM)	HDAC4 IC50 (nM)	HDAC5 IC50 (nM)	HDAC6 IC50 (nM)	HDAC7 IC50 (nM)	HDAC8 IC50 (nM)	HDAC9 IC50 (nM)	HDAC10 IC50 (nM)	HDAC11 IC50 (nM)
16	20,000	31,000	29	>40,000	>40,000	>45,000	>40,000	0.45,000	>40,000	10,000	2,000

Table 4. SAR of HDAC3 Selective Inhibitors to Improve hERG Inhibition and Pharmacokinetic Profile.

^aWinstar Han rats were the PK test species, dosing vehicle = DMSO/PEG400/water: 20/60/20.

^bBLQ = Below the lower limit of quantification of 0.00220 μM (i.e., 1.00 ng/mL).

To further investigate the observed HDAC isoform selectivity of 2-substituted benzamide HDACis, we determined the X-ray crystal structures of human HDAC2 bound to compounds 16 and 20 at resolutions of 2.7 and 1.3 Å, respectively (Figure 2).²⁴ Crystals of the wild-type human HDAC2 were used for inhibitor 20, while crystals of human HDAC2 with a single amino acid insertion of residue Tyr205, designed to better reflect the amino acid insertion in HDAC3, were used for inhibitor 16. Interestingly, the interactions of 16 and 20 with HDAC2 are quite similar on the surface and both inhibitors bind with their hydrophobic alkyl linkers in the same conformation, consistent with the interactions previously reported for the crystal structures of HDAC2 bound to 1 and 2.^13,14 However, there are significant differences in the binding mode of the 2-substituted benzamides with HDAC2 at the zinc binding site. The HDAC2 enzyme with compound 20 has a similar conformation as reported before for other HDAC1, 2, 3 inhibitors.¹³⁻¹⁵ The phenol oxygen and the benzamide carbonyl chelate the zinc ion in a bidentate mode. Unexpectedly, the HDAC3 selective inhibitor 16 bound to HDAC2 with the amide carbonyl making a monodentate contact to the zinc ion, and the methylthiophenyl group rotated in a manner that induced a rotamer change of the conserved catalytic site tyrosine (Tyr305) from an “in” to “out” position, significantly expanding the volume of the inhibitor binding pocket (Figure 2B). The energetic cost of the conformational rearrangement of the HDAC2 binding pocket to accommodate inhibitor 16 with only monodentate zinc binding may explain why the methylthiobenzamide is much less active against HDAC2 and HDAC1. The structural data therefore suggests that inhibitor 16 maintains potency against HDAC3 either by allowing a high affinity bidentate zinc binding mode in the HDAC3 active site or possibly by other structural features of HDAC3 that would stabilize the flipped out conformation of the catalytic site tyrosine. Interestingly, we note the previously reported HDAC3 crystal structure (Figure 2C) was determined as a complex with inositol tetraphosphate (IP4) and the DAD domain of the SMRT corepressor (PDB 4A69).²⁵ The catalytic pocket of HDAC3 is quite similar to HDAC2 with the equivalent tyrosine side chain phenol at the “in” position. However, the HDAC3 crystal structure also showed the proximity of Tyr298 and the nearby Arg265 to IP4. If compound 16 binds HDAC3 in the monodentate zinc binding mode, the induced conformation of Tyr298 and concomitant changes to Arg265 would be expected to be influenced by the presence of IP4 in the HDAC3/IP4/SMRT complex. While a crystal structure of HDAC3 bound to inhibitor 16 will be required to resolve these possibilities, these results nonetheless reveal unexpected conformational plasticity and an induced subpocket dependent on a rotamer change of the catalytic site tyrosine that is exploited by exquisitely selective HDAC3 inhibitors. In the meantime, SAR of 2-substitution on the benzamide demonstrated that several substituents unable to coordinate the zinc ion, such as cyclopropyl (36), remain as potent HDAC3 selective inhibitors. Compound 36 must be monodentate and therefore should prefer the induced pocket to fit the 2-substituents. The SAR is more consistent with and supports the hypothesis that this series of 2-substituted benzamide HDAC3 selective inhibitors bind to zinc in a monodentate fashion in the induced pocket.

Figure 2.

X-ray crystal structures of HDAC inhibitors binding to the HDAC2 enzyme with the pocket shown in gray: (A) zinc binding region of HDAC1, 2, 3 selective inhibitors 20; (B) zinc binding region of HDAC3 selective inhibitor 16; (C) HDAC3 X-ray crystal structure as a complex with the IP4/SMRT catalytic pocket (PDB code 4a69).

We also investigated binding kinetics for the two representative compounds—HDAC1, 2, 3 inhibitor 20 and HDAC3 selective inhibitor 16. We applied the global progress curve analysis (GPCA) method²⁶ to measure the on rate for both inhibitors to associate with HDAC3. Interestingly, the HDAC3 selective inhibitor 16 had a fast on rate, although it needs to induce a pocket with quick conformational change of the protein, with a k_on of 1.5 × 10⁴ M^–1 S^–1 while the HDAC1, 2, 3 inhibitor 20 also had a fast on rate, but relatively slower than 16 with k_on of 1.7 × 10³ M^–1 S^–1 (detailed measurement can be found in the Supporting Information). The k_off of both compounds can be calculated based on the enzyme binding equation K_D = k_off/k_on, in which K_D is usually the same as the IC₅₀ of the inhibitors against HDAC3. k_off for the HDAC1, 2, 3 inhibitor 20 is calculated to be 4.0 × 10^–6 s^–1 which results in a calculated residence time = 1/k_off of 69.4 h. However, the calculated k_off for the HDAC3 selective inhibitor 16 is 4.4 × 10^–4 s^–1 which results in a much shorter calculated residence time of 38 min. Furthermore, this significant difference of off rate and residence time for 20 and 16 was also confirmed by a jump dilution assay of both compounds in HDAC3 (Figure 3). The HDAC inhibitors with a concentration of 10 × IC₅₀, 0.1 × IC₅₀, and DMSO were treated with HDAC3 enzyme and the enzyme activity was monitored by the relative fluorescence units. The jump dilution of the assay solutions by 100 folds were performed on the 10 × IC₅₀ assay solution which resulted in the inhibitor concentration to 0.1 × IC₅₀ concentration. The HDAC1, 2, 3 inhibitor 20 had a very slow off rate with quite long residence time. Although the inhibitor concentration is diluted to a 0.1 × IC₅₀ concentration of 0.7 nM, the inhibitor still binds to the HDAC3 enzyme within the experimental time, which resulted in the continuous inhibition of enzyme activity. However, the HDAC3 selective inhibitor 16 has a fast-off rate, when jump dilution was performed on the assay solution with 10 × IC₅₀ concentration to 0.1 × IC₅₀ concentration of 3 nM, inhibitor 16 was released off the HDAC3 enzyme. Thus, the HDAC3 enzyme activity is recovered to a similar rate to that of the assay with 3 nM inhibitor. The off rate for 16 was measured from this jump dilution to be k_off = 3.6 × 10^–04 s^–1 which matches very well with that calculated from the assay with the GPCA method. Since the off rate of 20 is very slow, the k_off could not be obtained from the jump dilution assays. This also confirmed the calculated slow off rate for 20 from data with the GPCA method.

Figure 3.

Jump dilution assays to measure off rates: (A) jump dilution for HDAC1, 2, 3 inhibitor 20; (B) jump dilution for HDAC3 inhibitor 16. RFU = relative fluorescence units.

In the HIV latency in the Jurkat 2C4 cell model, we reported that HDAC1, 2, 3 inhibitors such as compounds 1 and 2 can induce HIV transcription.^13,14 However, the HDAC1, 2 selective inhibitor 3 and the HDAC3 selective inhibitor 16 are all inactive in this cellular assay. To further check if the inhibition of all HDAC1, 2, and 3 are required for the activation of the cellular HIV latency model, we carried out a test with the combinations of HDAC3 selective inhibitor 16 and HDAC1, 2 selective inhibitor 3 (Figure 4). Three titration curves were obtained with a constant concentration of HDAC1, 2 selective inhibitor 3 at 0 (red), 20000 (purple), and 40000 nM (green), combining with various concentrations of HDAC3 selective inhibitor 16 at 0, 78, 156, 1250, 2500, 5000, 10000, and 20000 nM. No cellular activity was observed when there is no HDAC1, 2 inhibitor 3 in the test. There is enhanced activation with a combination of 16 at low concentration, especially at 156 nM of 16 when the activation percentage reached the maximum. However, with an increasing concentration of 16, the percentage of activation dropped due to the cytotoxicity of the combination of high concentrations of both 3 and 16. This experiment confirmed again that inhibition of HDAC1, 2, and 3 is sufficient to cause activation of HIV latency in the Jurkat 2C4 cell model. The combination of the HDAC1, 2 inhibitor (inactive in 2C4 cells alone) and HDAC3 inhibitor (inactive in 2C4 cells alone) can lead to HIV latency activation. These data demonstrate that HDAC1/2 and 3 inhibition is required for HIV latency reversal and that inhibition of HDAC3 alone is not sufficient.

Figure 4.

Cellular 2C4 activation by combinations of 16 (HDAC3 inhibitor) and 3 (HDAC1, 2 inhibitor).

In summary, taking advantage of a parallel medicinal chemistry strategy, we discovered a series of HDAC inhibitors with 2-substituted benzamide as the zinc binding group. When the 2-substituents are involved in the zinc bidentate coordination, the inhibitors are HDAC1, 2, and 3 selective represented by 20. However, if the 2-substituents do not chelate with the zinc ion, it could induce a binding pocket by flipping the Tyr305 side chain from an “in” to an “out” conformation which was observed in the X-ray crystal structure of 16 bound to the HDAC2 enzyme. The selectivity of 16 with inhibition to HDAC3 is rationalized by this induced pocket protein conformation of HDAC3 which is speculated to be stabilized by the HDAC3/IP4/SMRT complex while no comparable stabilization exists for HDAC1 and 2. This highly HDAC3 selective inhibitor 16 served as the tool compound to confirm that inhibition alone on HDAC3 does not activate HIV latency in the Jurkat 2C4 cells, but combination of 16 with HDAC1,2 inhibitor 3 is sufficient to activate HIV latency. The HDAC3 selective inhibitor 16 and HDAC1,2,3 inhibitor 20 demonstrated different binding kinetic with dramatically distinctive length of residence time. The off target and pharmacokinetic profile of this series of HDAC3 inhibitors can be optimized by SAR of the surface binding groups. Overall, we discovered a series of highly HDAC3 selective inhibitors with 2-substituted benzamides as the zinc binding groups which can serve as tool compounds to explore therapeutic indications.

Glossary

Abreviations

HDAC

histone deacetylase

HATs

histone acetyl transferases

HDACi

histone deacetylase inhibitor

HIV

human immunodeficiency viruses

LRA

latency reversing agency

PMC

parallel medicinal chemistry

cART

combined antiretroviral therapy

SAR

structure activity relationship

DMF

dimethylformaldehyde

THF

tetrahydrofuran

DCM

dichloromethane

HATU

(1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate

TFA

trifluoroacetic acid

DIEA

diisopropylethylamine

hERG

human ether-à-go-go-related gene

GPCA

global progress curve analysis

PK

pharmacokinetic.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.0c00462.

• Synthetic methods, characterization of key compounds, biology assay protocols, and data (PDF)

The authors declare no competing financial interest.